Vitiligo 2nd Edition PDF

Vitiligo
Mauro Picardo
Alain Taïeb
Editors
Second Edition
123
Vitiligo
Mauro Picardo • Alain Taïeb
Editors
Vitiligo
Second Edition 2019
ERRNVPHGLFRVRUJ
Editors
Mauro Picardo Alain Taïeb
Cutaneous Physiopathology & CIRM Hôpital Saint André, Service de
San Gallicano Dermatological Institute Dermatologie Adulte et Pédiatrique
Rome INSERM U 1035, Université de Bordeaux
Italy Bordeaux
France
ISBN 978-3-319-62958-2 ISBN 978-3-319-62960-5 (eBook)

https://doi.org/10.1007/978-3-319-62960-5
© Springer-Verlag Berlin, Heidelberg 2010 First Edition

© Springer Nature Switzerland AG 2019 Second Edition
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Preface to the First Edition
Vitiligo has been, until recently, a rather neglected area in dermatology and
medicine. Patients complain about this situation, which has offered avenues
to quacks, and has led to the near orphan status of the disease. The apparently,
simple and poorly symptomatic presentation of the disease has been a strong
disadvantage to its study, as compared to other common chronic skin disor-
ders such as psoriasis and atopic dermatitis. Vitiligo is still considered by
doctors as a non disease, a simple aesthetic problem. A good skin-based angle
of attack is also lacking because generalized vitiligo is clearly epitomizing
the view of skin diseases as simple targets of a systemic unknown dysregula-
tion (diathesis), reflecting the Hippocratic doctrine. This view has mostly
restricted vitiligo to the manifestation of an auto-immune diathesis in the past
30 years. Thus, skin events, which are easily detected using skin biospies in
most other situations, have not been precisely recorded, with the argument
that a clinical diagnosis was sufficient for the management (or most com-
monly absence of management) of the patient.
This book is an international effort to summarize the information gathered
about this disorder at the clinical, pathophysiological and therapeutic levels.
Its primary aim is to bridge current knowledge at the clinical and investigative
level, to point to the many unsolved issues, and to delineate future priorities
for research. Its impetus was also to provide the best guidelines for integrated
patient care, which is currently possible at a very limited number of places
around the world, especially for surgical procedures.
A striking feature in the vitiligo field was, until recently, the absence of
consensus on definitions, nomenclature, and outcome measures. With a group
of European dermatologists, who had a strong interest in vitiligo and pigment
cell research, we had launched some years ago, the Vitiligo European Task
Force (VETF). The VETF has addressed those issues as a priority. This group,
joined by other colleagues from the rest of the world also involved in the
vitiligo research community, has communicated its experience in this book.
We have tried to pilot the editing of the book according to consistent princi-
ples based on discussions held at VETF meetings and international IPCC
(international pigment cell conference) workshops. However, some areas
remain controversial and we have highlighted the existing conflicting issues
and uncertainties.
After reviewing the field, much needs to be done. In particular, besides
basic research based on the many hypotheses raised, new unbiased epidemio-
logical, clinical, histopathological, natural history, and therapeutic data are
v
vi Preface to the First Edition
clearly needed. They should be confronted by genetics and other investigative

variables to better define the disease and its subsets. We hope that the com-
bined efforts of all participating authors will prove useful to bring more atten-
tion to this field, and we are confident that both the research community (the
mystery of melanocyte loss in vitiligo is a true scientific challenge) and the
drug industry (the potential market is large) will be stimulated to bring in new
treatment strategies to this large number of patients with unmet needs.
Bordeaux, France Alain Taïeb

Rome, Italy Mauro Picardo
Preface to the Second Edition
Nearly 10 years after the first edition, it was necessary to update but also to
reassess the field more globally. What has changed in our vision of vitiligo in
about a decade?
Developments in the field have included classification, identification of
new identities, pathogenic mechanisms, investigation of normal appearing
skin, and new approaches for diagnosis, treatment, and maintenance of treat-
ment, just to mention a few. To reflect the expansion of the field, we have
benefited of the expertise of a larger community of scientists and physicians,
but in parallel we also tried to improve the book plan with the underlying
challenging endeavor to simplify and limit redundancies.
For disease definition, clinical aspects have been better delineated, and
international efforts conducted under the auspices of the Vitiligo Global
Issues Consensus Conferences have simplified nomenclature and classifica-
tion as well as scoring and paved the way to more uniform outcome
research tools. Considering disease expression, the epidemic of vitiligoid
depigmentation caused by immunotherapies has questioned the immune
mechanisms of melanocyte loss in common vitiligo, a central part of disease
pathophysiology.
The basic understanding of the disease for its immune/inflammatory com-
ponent has undergone significant progresses and seems at work in all subsets
of the disease, with immediate practical consequences but also more durable
translational perspectives for therapy. Melanocyte stability, melanocyte
regeneration, and epigenetics are relatively new fields in terms of basic
research explored in this new edition with also important potential added
value for therapy.
For therapy, our perspective has clearly changed over the last decade for
segmental vitiligo which was considered previously as poorly or not at all
responsive to medical treatment. Given the efficacy of early medical interven-
tion now reported by several investigators, vitiligo of any subtype should bet-
ter be considered as a therapeutic emergency to avoid irreversible immune/
inflammatory losses in pigment cells. The advantage of vitiligo over type 1
diabetes is the visibility and early detection of tissue damage in most patients,
allowing more reactivity. However, patients do still complain, and this has not
sufficiently changed over the last decade, of the absence of interest of doctors
for their disease. Patients’ voices are probably better heard with the more
coordinated action of patients’ advocacies, but much more education is
needed for nonspecialist and specialist physicians to use the current medical
vii
viii Preface to the Second Edition
and surgical armamentarium for vitiligo. Fortunately, things should change

for the availability of registered treatments, since the next decade should
clearly be the era of drug development for vitiligo, following for dermatology
significant progresses already accomplished for other major skin disorders
such as psoriasis, atopic dermatitis, and alopecia areata.
Finally, we want to thank warmly the large group of international col-
leagues who accepted to participate in this second edition of the book.
Bordeaux, France Alain Taïeb

Rome, Italy Mauro Picardo
Contents
Part I Defining the Disease
1 Historical Aspects of Vitiligo �� 3

Yvon Gauthier and Laila Benzekri
2 Definitions and Classification�� 11
Alain Taïeb and Mauro Picardo
3 Vitiligo: Histopathology, Including Electron Microscopy�� 25
Carlo Cota and Daniela Kovacs
4 Introduction to Clinical Aspects Chapters�� 39
5 Vitiligo/nonsegmental Vitiligo Including Acrofacial
and Universalis �� 41
Thierry Passeron, Jean-Paul Ortonne, Prasad Kumarasinghe,
and Alain Taïeb
6 Segmental Vitiligo�� 53
Seung-Kyung Hann, Hsin-Su Yu, Cheng-Che Eric Lan,
Ching-Shuang Wu, Yvon Gauthier, Laïla Benzekri, and
Alain Taïeb
7 Mixed Vitiligo �� 73
Alain Taïeb
8 Clinically Inflammatory Vitiligo and Rare Variants�� 81
Alain Taïeb, Khaled Ezzedine, Julien Seneschal, and
Ratnam Attili
9 Halo Nevus, Leucotrichia and Mucosal Vitiligo�� 93
Anuradha Bishnoi and Davinder Parsad
10 Extra-Cutaneous Melanocytes�� 103
Tag S. Anbar, Rehab A. Hegazy, and Suzan Shalaby
11 Koebner Phenomenon�� 115
Nanja van Geel and Reinhart Speeckaert
ix
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x Contents
12 Environmental Triggers and Occupational/Contact

Vitiligo �� 121
Charlotte Vrijman
13 Vitiligo, Associated Disorders and Comorbidities
(Autoimmune-Inflammatory Disorders,
Immunodeficiencies, Rare Monogenic Diseases)�� 125
Julien Seneschal, Fanny Morice-Picard, and Alain Taïeb
14 Age and Vitiligo: Childhood, Pregnancy and
Late-Onset Vitiligo �� 141
Steven Thng, Sai Yee Chuah, and Emily Yiping Gan
15 Vitiligo and Skin of Color�� 153
Onyeka Obioha, Candrice Heath, and Pearl E. Grimes
16 Vitiligo-Like Lesions in Patients with Metastatic
Melanoma Receiving Immunotherapies�� 163
Katia Boniface and Julien Seneschal
17 Evaluation, Assessment, and Scoring �� 169
18 Quality of Life�� 177
19 Defining the Disease: Editor’s Synthesis�� 181
Part II Understanding the Disease
20 Pathophysiology Overview�� 189

Mauro Picardo
21 Methods to Study Vitiligo: Noninvasive Techniques and
In Vivo Reflectance Confocal Microscopy �� 193
Hee Young Kang and Marco Ardigò
22 Animal Models �� 205
Gisela F. Erf and I. Caroline Le Poole
23 In Vitro Study of Vitiligo �� 225
Maria Lucia Dell’Anna and Muriel Cario-André
24 Genetics�� 237
Richard A. Spritz
25 Epigenetics�� 253
Li Zhou, Henry W. Lim, and Qing-Sheng Mi
26 Melanocyte Homeostasis in Vitiligo�� 265
Véronique Delmas and Lionel Larue
27 Oxidative Stress and Intrinsic Defects �� 277
Mauro Picardo and Maria Lucia Dell’Anna
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Contents xi
28 Immunity/Immunopathology�� 285
Kirsten C. Webb, Steven W. Henning,
and I. Caroline Le Poole
29 Cytokines, Growth Factors, and POMC Peptides�� 303
Markus Böhm, Katia Boniface, and Silvia Moretti
30 Regenerating Melanocytes: Current Stem
Cell Approaches with Focus on Muse Cells �� 313
Mari Dezawa, Kenichiro Tsuchiyama, Kenshi Yamazaki,
and Setsuya Aiba
31 Other Defects/Mechanisms �� 329
Maria Lucia Dell’Anna and Mauro Picardo
32 Pathophysiology of Segmental Vitiligo �� 333
Nanja van Geel, Carole Van Haverbeke,
and Reinhart Speeckaert
33 Editor’s Synthesis�� 337
Mauro Picardo and Alain Taïeb
Part III Treating the Disease
34 Management Overview�� 345

35 Medical Therapies�� 353
Alain Taïeb
36 Surgical Therapies �� 381
Boon Kee Goh
37 Depigmenting Therapies�� 399
Abdulrahman Aljamal, Mohammed Aljamal, Sanjeev
Mulekar, and Aleissa Ahmed
38 Combined/Sequential/Integrated Therapies for Vitiligo�� 411
Thierry Passeron
39 Camouflage�� 421
Alida DePase and Thomas Jouary
40 Photoprotection Issues�� 429
Alessia Pacifico, Giovanni Leone, and Mauro Picardo
41 Treating the Disease: Age, Gender, Ethnic Skin, and
Specific Locations�� 437
Savita Yadav and M. Ramam
42 Psychological Interventions�� 451
Panagiota Kostopoulou and Alain Taïeb
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xii Contents
43 Patients’ Perspectives�� 455

Jean-Marie Meurant
44 Discussion on Empirical, Traditional, and Alternative
Treatments�� 463
Mauro Picardo
45 Beyond Guidelines�� 469
Tag S. Anbar, Rehab A. Hegazy, and Amira A. Eid
46 Editor’s Synthesis and Perspectives �� 481
Index�� 485
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Contributors
Setsuya Aiba Department of Dermatology, Tohoku University Graduate

School of Medicine, Sendai, Japan
Aleissa Ahmed The National Center of Vitiligo and Psoriasis, Riyadh,
Kingdom of Saudi Arabia
Abdulrahman Aljamal The National Center of Vitiligo and Psoriasis,
Riyadh, Kingdom of Saudi Arabia
Mohammed Aljamal The National Center of Vitiligo and Psoriasis, Riyadh,
Tag S. Anbar (in memory) Department of Dermatology and Andrology,
Faculty of Medicine, Al-Minya University, Al-Minya, Egypt
Marco Ardigò San Gallicano Dermatological Institute, IRCCS, Rome, Italy
Ratnam Attili Visakha Institute of Skin and Allergy, Visakhapatnam, India
Markus Böhm Department of Dermatology, University of Münster,
Münster, Germany
Laïla Benzekri Department of Dermatology, CHU Ibn Sina, Mohammed V
University, Rabat, Morocco
Anuradha Bishnoi Department of Dermatology, Postgraduate Institute of
Medical Education and Research, Chandigarh, India
Katia Boniface INSERM U 1035, University of Bordeaux, France
INSERM U1035, BMGIC, Immunodermatology team, ATIP-AVENIR,
Université de Bordeaux, Bordeaux, France
Muriel Cario-André INSERM U 1035, Centre de référence des maladies
rares de la peau, Université de Bordeaux, Bordeaux, France
Sai Yee Chuah National Skin Centre, Singapore, Singapore
Carlo Cota Dermatopathology Unit, San Gallicano Dermatologic Institute
(IRCCS), Rome, Italy
Dipankar De Department of Dermatology, Postgraduate Institute of Medical
Education and Research, Chandigarh, India
xiii
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xiv Contributors
Maria Lucia Dell’Anna Cutaneous Physiopathology, San Gallicano

Dermatological Institute, IFO, Rome, Italy
Véronique Delmas INSERM U1021, Normal and Pathological Development
of Melanocytes, Institut Curie, PSL Research University, Orsay, France
Alida DePase Associazione Ricerca Informazione per la Vitiligine (ARIV),
Cernusco Lombardone, LC, Italy
Mari Dezawa Department of Stem Cell Biology and Histology, Tohoku
University Graduate School of Medicine, Sendai, Japan
Amira A. Eid Department of Dermatology, Venereology and Andrology,
Faculty of Medicine, Alexandria University, Alexandria, Egypt
Gisela F. Erf Division of Agriculture, Center of Excellence for Poultry
Science, University of Arkansas, Fayetteville, AR, USA
Khaled Ezzedine Service de dermatologie, Hôpital Henri Mondor, France
Emily Yiping Gan National Skin Centre, Singapore, Singapore
Yvon Gauthier Former Consultant, Department of Dermatology, CHU de
Bordeaux, France
Boon Kee Goh Skin Physicians Pte Ltd, Mount Elizabeth Medical,
Singapore, Singapore
Pearl E. Grimes Vitiligo & Pigmentation Institute of Southern California,
Los Angeles, CA, USA
Seung-Kyung Hann Korea Institute of Vitiligo Research, Drs. Woo &
Hann’s skin clinic, Seoul, South Korea
Candrice Heath Vitiligo & Pigmentation Institute of Southern California,
Rehab A. Hegazy Department of Dermatology, Faculty of Medicine, Cairo
University, Cairo, Egypt
Steven W. Henning Oncology Research Institute, Loyola University
Chicago, Maywood, IL, USA
Thomas Jouary Service de Dermatologie, Hôpital Saint André, CHU de
Bordeaux, Bordeaux, France
Hee Young Kang Department of Dermatology, Ajou University School of
Medicine, Suwon, Korea
Panagiota Kostopoulou Service de Dermatologie, Hôpital Saint André,
Bordeaux, France
Daniela Kovacs Cutaneous Physiopathology and Integrated Center of
Metabolomics Research, San Gallicano Dermatologic Institute (IRCCS),
Rome, Italy
Prasad Kumarasinghe Department of Dermatology, Royal Perth Hospital,
Perth, WA, Australia
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Contributors xv
Cheng-Che Eric Lan Department of Dermatology, Kaohsiung Medical

University, Kaohsiung, Taiwan
Lionel Larue INSERM U1021, Normal and Pathological Development of
Melanocytes, Institut Curie, PSL Research University, Orsay, France
I. Caroline Le Poole Professor of Dermatology, Microbiology and
Immunology Northwestern University at Chicago, IL, USA
Giovanni Leone San Gallicano Dermatological Institute IRCCS, Rome,
Italy
Henry W. Lim Henry Ford Immunology Program, Department of
Dermatology, Henry Ford Hospital, Detroit, MI, USA
Jean-Marie Meurant Association Française du Vitiligo, Paris, France
Qing-Sheng Mi Henry Ford Immunology Program, Department of
Dermatology, Henry Ford Hospital, Detroit, MI, USA
Silvia Moretti Division of Clinical Preventive and Oncologic Dermatology,
University of Florence, Florence, Italy
Fanny Morice-Picard Department of Dermatology, Hôpital Pellegrin-
Enfants, Bordeaux, France
Sanjeev Mulekar The National Center of Vitiligo and Psoriasis, Riyadh,
Onyeka Obioha Vitiligo and Pigmentation Institute of Southern California,
Jean-Paul Ortonne Department of Dermatology, Archet-2 hospital, Nice,
France
Alessia Pacifico San Gallicano Dermatological Isitute, IRCCS, Rome, Italy
Davinder Parsad Department of Dermatology, Postgraduate Institute of
Medical Education and Research, Chandigarh, India
Thierry Passeron Department of Dermatology, University Hospital of Nice,
Nice Cedex 3, France
Mauro Picardo Cutaneous Physiopathology and CIRM, San Gallicano
Dermatological Institute, IRCCS, Rome, Italy
M. Ramam Department of Dermatology and Venereology, All India Institute
of Medical Sciences, New Delhi, India
Julien Seneschal Service de dermatologie, Hôpital St André, CHU de
Suzan Shalaby Department of Dermatology, Faculty of Medicine, Cairo
University, Cairo, Egypt
Reinhart Speeckaert Department of Dermatology, Ghent University
Hospital, Ghent, Belgium
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xvi Contributors
Richard A. Spritz Human Medical Genetics and Genomics Program,

University of Colorado School of Medicine, Aurora, CO, USA
Department of Pediatrics, University of Colorado School of Medicine,
Aurora, CO, USA
Alain Taïeb Service de Dermatologie, Hôpital St André, CHU de Bordeaux,
Bordeaux, France
Department of Dermatology and Pediatric Dermatology, Bordeaux University
Hospitals, and INSERM U 1035, University of Bordeaux, Bordeaux, France
Steven Thng National Skin Centre, Singapore, Singapore
Kenichiro Tsuchiyama Department of Dermatology, Tohoku University
Graduate School of Medicine, Sendai, Japan
Wietze Van der Veen Department of Dermatology, Institute for Pigment
Cell Disorders, AMC, Amsterdam, The Netherlands
Nanja van Geel Department of Dermatology, Ghent University Hospital,
Ghent, Belgium
Carole Van Haverbeke Department of Dermatology, Ghent University
Charlotte Vrijman Department of Dermatology, Ziekenhuisgroep Twente,
Hengelo, Netherlands
Kirsten C. Webb Division of Dermatology, Department of Medicine,
Loyola University Chicago, Maywood, IL, USA
Ching-Shuang Wu Faculty of Biomedical Laboratory Science, Kaohsiung
Medical University, Kaohsiung, Taiwan
Savita Yadav Department of Dermatology and Venereology, All India
Institute of Medical Sciences, New Delhi, India
Kenshi Yamazaki Department of Dermatology, Tohoku University Graduate
School of Medicine, Sendai, Japan
Hsin-Su Yu Department of Dermatology, Kaohsiung Medical University,
Kaohsiung, Taiwan
Li Zhou Henry Ford Immunology Program, Department of Dermatology,
Henry Ford Hospital, Detroit, MI, USA
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Part I
Defining the Disease
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Historical Aspects of Vitiligo
1
Yvon Gauthier and Laila Benzekri
Contents
1.1 Before Vitiligo: Understanding Old Terms Meaning White Skin Spots 4
1.2 From Celsus to the Modern Period 6
1.3 Social Status of Vitiligo Patients Across the Ages 6
1.4 Early History of the Medical Treatment of Vitiligo 7
1.5 Modern History of the Medical Treatment of Vitiligo 8
1.6 Modern History of Surgical Treatment of Vitiligo 9
1.7 Conclusions 9
References 10
Abstract word “Zoorat” in the bible corresponds to a

Vitiligo was recognized long ago under differ- group of achromic diseases including vitiligo.
ent names. Firstly, the Ebers Papyrus (circa “Baras and alabras” were the Arabic names
1500 BC) mentions two types of diseases used to describe vitiligo. The term “vitiligo”
affecting the colour of the skin, one being itself was introduced in the first century of our
probably leprosy and the other vitiligo. In the era. The confusion of leprosy with vitiligo in
ancient Vedic scripture of India (circa 1400 the Old Testament under “Zoorat” is an impor-
BC), Sanskrit words “kilas” and “svitra” tant cause for the social stigma attached to
(white patches on the skin) can be found. The white spots on the skin. Detailed and effective
early classics of the Far East (1200 BC) men- treatments for vitiligo are found in different
tion “shirabito” (white man). The Hebrew sacred books. The modern photochemotherapy
is an improvement of a photochemotherapy
practised in the ancient world with herbals
containing furocoumarins (mainly Ammi
Y. Gauthier (*)
Former Consultant, Department of Dermatology,
majus Linnaeus, Psoralea corylifolia) and sun.
CHU de Bordeaux, Bordeaux, France In the mid-1960s the synthetic furocoumarin
L. Benzekri
(trioxsalen and trimethylpsoralen) were devel-
Department of Dermatology, CHU Ibn Sina, oped. The effectiveness of PUVA (pso-
Mohammed V University, Rabat, Morocco ralen + UVA) for the treatment of some patients
© Springer Nature Switzerland AG 2019 3

M. Picardo, A. Taïeb (eds.), Vitiligo, https://doi.org/10.1007/978-3-319-62960-5_1
ERRNVPHGLFRVRUJ
4 Y. Gauthier and L. Benzekri
with vitiligo was confirmed during 1974–1982. with swellings and recommended to be left alone
More recently, narrowband UVB therapy, local “Thou shall anything to-do it” is probably leprosy.
microphototherapy, excimer laser, topical The other, manifested with only changes of colour,
treatments with corticosteroids and calcineurin is likely to be vitiligo, as this is said to have been
inhibitors and melanocyte transplantation have treated (Fig. 1.1).
been successively introduced. Hundreds of years before Christ, vitiligo was
present in ancient Indian sacred books such as the
Atharva Veda (1400 BC) and the Buddhist sacred
Key Points book Vinay Pitak (224–544 BC). Atharva Veda
• The term vitiligo was introduced in the gives a description of vitiligo but also of many
first century of our era. achromic or hypochromic diseases under differ-
• The mistaking of leprosy for vitiligo in ent names “Kilasa”, “SvetaKhista” and “Charak”.
the Old Testament under Zoorat or The Sanskrit word “kilas” is derived from kil
Zaraath is an important cause for the meaning white. So “kilas” means “which throw
social stigma attached to white spots on away colour”; the term “SvetaKhista” was used
the skin. meaning white leprosy, and vitiligo was probably
• Modern photochemotherapy (PUVA) confused with macular leprosy. The term
was an improvement of photochemo- “Charak” used by villagers means “which spreads
therapy practised in the ancient world or is secret”. In the Buddhist “Vinay Pitak”, the
with herbals containing furocoumarins. term “kilas” was also used to describe the white
• The stigma historically associated with spots on the skin [4].
the disease has not disappeared, and Descriptions of vitiligo and other leukodermas
information and educational pro- can also be found in other ancient Indian writings
grammes should be implemented to such as the Charaka Samhita (800 BC), Manusmriti
fight this problem, especially the fear of (200 BC) and Amorkasha (600 AC). There are also
contagion. references to disease with a whitening of the skin in
the early classics of the Far East. In
“Makataminoharai”, a collection of Shinto prayers
The physician must know what his predecessors (dating back to about 1200 BC), there is mention of
have known, if he does not want to deceive both a disease “shirabito” which literally means white
himself and others. man. This also could have been vitiligo.
—Hippocrates
Though vitiligo, the disease with white spots,
was recognized in the ancient times, it was fre-
quently confused with leprosy. Even Hippocrates
1.1 Before Vitiligo: (460–355 BC) did not differentiate vitiligo and
Understanding Old Terms leprosy and included lichen, leprosy, psoriasis
Meaning White Skin Spots and vitiligo under the same category. In the Old
Testament of the Holy Bible, the Hebrew word
Even though the term vitiligo appeared in the first “Zoorat” is referring to a group of skin diseases
century of our era, descriptions of the disease now that where classified into five categories [1]:
known as vitiligo can be found in the ancient med-
ical classics of the second millennium BC [1, 2]. 1. White spots per se interpreted as vitiligo or
The earliest reference to the disease was found in post-inflammatory leukoderma
2200 BC in the period of Aushooryan according to 2. White spots associated with inflammation
the ancient literature of Iran “Tarkh-e-Tibble”. In 3. White spots associated with scaling
the Ebers Papyrus (circa 1500 BC) [3], there is a 4. White spots associated with atrophy
mention of two types of diseases affecting the 5. White spots associated with the regrowth of
colour of the skin. One of them which is associated white hairs
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1 Historical Aspects of Vitiligo 5
Fig. 1.1 Papyrus

(Published under
Creative Common
Licence CCO 1.0,
Leipzig, University
Library, Papyrus Ebers,
Table XXIII, Column
LXXVII-LXXIX,
https://papyri.uni-
leipzig.de/receive/
UBLPapyri_
text_00038150)
Ptolemy II (250 BC) demanded the translation “white skin”. In the Koran (Surah the family of
of the Hebrew Bible into Greek so that more peo- Imran chapter 3, verse 48, and Surah the table
ple could understand the Bible. The word spread chapter 5 verse 109), we can read (Fig. 1.2):
“Zoorat’” was unfortunately translated in Greek
In accord with God’ will Jesus was able to cure
versions of the Bible as “white leprosy” (Leviticus patients with vitiligo
Chap. XIII). According to modern dermatologists
and theologians, biblical leprosy represented not “Alabras” was translated as leprosy in many
a specific illness but psoriasis or leukoderma and languages. However, Tahar Ben Achour [7] in
other disorders perceived to be associated with a 1973 explained that “alabras” meant vitiligo. For
spiritual uncleanliness. Consequently, persons this theologian, the aetiology of vitiligo in the
with white spots, independent of the cause, were Koran is unknown, but it is inherited and is not
isolated from the healthy ones [1, 2, 5, 6]. contagious.
“Bohak”, “baras” and “alabras” are the Arabic The name vitiligo was first used by the famous
names used to describe vitiligo. “Baras” means Roman physician Celsus at the second century AC
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Fig. 1.2 From the

Koran, Surah “The
Family of Imran”,
Chapter 3 verse 48
in his medical classic De Medicina. The word vit- progressive, melanocyte loss of still mysterious
iligo has often been said to have derived from aetiology, clinically characterized by circum-
“vitium” (defect or blemish) rather than “vitellus” scribed achromic macules often associated with
meaning calf [2]. leukotrichia and progressive disappearance of
melanocytes in the involved skin”. However, con-
troversies still remain about achromic macules
1.2 rom Celsus to the Modern
F occurring during the evolution of malignant mel-
Period anoma and lymphomas. Are they “vitiligoid
lesions” or true vitiligo? (see Chap. 16).
Over the last century and up to now, the alterna-
tive use of the terms of “vitiligo” and “leuko-
derma” has perpetuated confusion in the medical 1.3 ocial Status of Vitiligo
S
literature. Pearson, just after the end of the nine- Patients Across the Ages
teenth century, described under the name leuko-
derma a disease which seems to be vitiligo. In the The history of vitiligo reveals much information
late twentieth century, the elusive nature of vitil- regarding the social stigma of patients suffering
igo has led to prudent definitions excluding disor- from this disease. In the Buddhist literature (624–
ders of established aetiology. “Vitiligo can be 544 BC), we can read “men and women suffering
described as an acquired primary, usually from the disease named Kilasa were not eligible to
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get ordainment”. The social implication of this dis- clean. The Hebrew term used does not refer to
ease is also well documented in Rgveda (Indian Hansen’s disease, currently called leprosy” [1, 2].
book) “persons suffering from switra and their Islamic theologians consider that baras is a
progeny are disqualified from marrying others” [4]. defect in the couple. Thus, the husband or the
Herodotus (484–425 BC) in his book Clio wife has the choice to divorce. Arabic kings
(1138) has written “if a Persian has leprosy or talked to vitiligo patients only behind a screen as
white sickness he is not allowed to enter into a reported by the poet Harith bin Hilliza [7].
city or to have dealings with other Persians, he Therefore the patient and their parents should
must have sinned against the sun. Foreigners suf- be reassured about the non-infectious origin of vit-
fering from this disorder are forced to leave the iligo. Unfortunately, patients with vitiligo are still
country; even white pigeons are often driven regarded as social outcasts in some countries.
away, as guilty of the same offence”.
In Leviticus XIII, 34, white spot diseases are
considered as a punishment sent by God: “Anyone 1.4 arly History of the Medical
E
with these skin affections must wear torn clothes Treatment of Vitiligo
and have his hair dishevelled, he must conceal his
upper lip, and call out “unclean, unclean”. So long The Ebers Papyrus (circa 1500 BC), the Atharva
the disease persists, he is to be considered virtu- Veda (circa 1400 BC), other Indian and Buddhist
ally unclean and live alone outside the camp”. medical literature (circa 200 AC) and Chinese
During many centuries the stigma of leprosy was manuscript from the Sung period (circa 700 AC)
strengthened by old edicts and cruel laws [1]. make reference to the treatment of vitiligo with
In 1943, according to the initiative of Pope Pius black seeds from the plant Bawachee or Vasuchika
XII and the American Catholic office, the church which is now called Psoralea corylifolia.
added the following note with reference to Photochemotherapy was practised in the ancient
Leviticus XIII “Various kinds of skin blemishes world by physicians and herbalists who used boiled
are treated here which were not contagious but extracts of leaves, seeds or the roots of Ammi majus
simply disqualified their subjects from associa- Linnaeus in Egypt (Fig. 1.3) or Psoralea corylifo-
tions with others, until they were declared ritually lia in India (Fig. 1.4) which contain psoralens and
Fig. 1.3 Ammi majus

Linnaeus (AML) a
weed which grows
throughout the Nile
Valley. The seeds of
AML were used in
ancient Egypt as a
therapy for leucoderma
(Published under
Creative Commons
Attribution ShareAlike
3.0 License, https://
commons.wikimedia.
org/wiki/File:Ammi_
majus_001.JPG)
ERRNVPHGLFRVRUJ
Born by night art thou, O plant

Dark, black, sable, do thou
That art rich in colour, stain
This leprosy and white grey spots.
Even colour is the name of thy mother,
Even colour is the name of thy father.
Thou, O plant produced even colour,
Render this spot to even colour
According to Indian medical books, the two most

commonly applied and effective herbs were “
Malapu” (Ficus hispida) and “Bawachee” (Psoralea
corylifolia). These herbs were administered orally or
topically followed by exposure to sunlight until
sweating was observed. Blisters were then produced
and after their rupture, repigmentation occurred. In
Ayurvedic, the association of internal treatment con-
sisting of dried ginger, black pepper, pippali and
leadwort root fermented in cow’s urine and local
ointment with a paste made of several medicinal
herbs including Psoralea corylifolia was proposed
to induce the repigmentation of vitiligo [4].
Another important plant Ammi majus Linnaeus
[4, 8, 9], which grows throughout the Nile Valley
as a weed, has been used many centuries BC as a
treatment for leukoderma. Ibn el Bitar in his
thirteenth-century book Mafradat Al Adwiya
Fig. 1.4 Psoralea corylifolia grows in India. Preparations
for the treatment of vitiligo were made from seeds of this described the treatment of vitiligo “baras” with the
plant (Photo by Biswarup Ganguly, published under seeds of aatrillal (Ammi majus) and sunlight [10].
Creative Commons Attribution ShareAlike 3.0 License, Aatrillal seemed to be a common therapy for leu-
https://commons.wikimedia.org/w/index. koderma in “Ben Shoerb”, a Berberian tribe living
php?curid=23839722)
in the north-western African desert. Egyptian
herbalists used these herbs in the form of a powder
several furocoumarins [6]. These preparations
[11]. Through the ages, in Egypt, topical or oral
made from seeds obtained from herbal stores were
use of seeds from the plant Ammi majus followed
either applied to the skin or ingested before solar
by sunlight exposure as mentioned earlier in India
exposure. In the ancient “Ayurvedic system of
was the mainstay of treatments.
medicine”, the use of Psoralea corylifolia
Natural psoralens have been found in more
(Leguminosae family) for inducing the repigmen-
than 30 plants, including lime, lemon, bergamot,
tation of vitiligo is carefully recorded. In Charaka
celery, fig and clove [12].
Samhita both topical and systemic uses of figs are
also recommended before sun exposure. Figs are
known to contain both psoralens and glucosides of
psoralens. In Atharva Veda (2000–1400 BC) and 1.5 Modern History
many other writings of this period, the treatment of of the Medical Treatment
vitiligo with “Bawachee” (Psoralea corylifolia) is of Vitiligo
described to be associated with exposure to solar
radiation and worshipping prayer. This is illus- In 1938 Kuske investigated phytophotodermati-
trated in Atharva Veda by the following poem to tis occurring on areas of the skin which have
one of the plant used. been in contact with certain plants and also
ERRNVPHGLFRVRUJ
exposed to sunlight [13]. Extensive research 1.6 odern History of Surgical

M
began in 1941 in Cairo, Egypt, in the laboratory Treatment of Vitiligo
of Fahmi and his group. Three crystalline com-
pounds were obtained from Ammi majus L. and Many investigators have tried to induce repig-
named ammoidin, ammidin and majudin [11]. mentation in patients with vitiligo not respond-
El Mofty [14] was the first, in the early 1940s, ing to medical therapies with grafting and
to use crystalline methoxsalen followed by transplantation techniques. Successively the
exposure to sunlight for the treatment of vitil- dermoepidermal grafts were introduced by Behl
igo. The results were found good, and shortly in 1964 [19]. Epidermal grafting with suction
afterwards two of the compounds, 8-methoxyp- blisters was successfully used for the first time in
soralen and 8- isoamyleneoxypsoralen, were 1971 by Falabella [20] on segmental vitiligo and
produced by an Egyptian firm in Cairo. In 1957 post-burn depigmentation. Minigrafting was
Harris and Lerner isolated a melanocyte-stimu- proposed in 1983 by Falabella in three patients
lating hormone or MSH which they obtained with segmental vitiligo [21]. In 1989 epidermis
from the pituitary glands of pigs and studied its with melanocytes was grown for the first time for
skin-darkening effects in hypopituitarism skin repigmentation purposes by Falabella et al. [22].
and vitiligo skin [10]. No changes were seen in The use of epidermal suspensions for repigmen-
the vitiliginous areas after alpha-MSH treat- tation of stable vitiligo macules was initially
ment. Furthermore microscopic examination of described in 1992 by Gauthier [23]. This method
DOPA-stained skin sheets from the injected sites was improved in 1998 by Olsson and Juhlin [8].
and from normal skin did not reveal any signifi- In 1992 pure melanocyte suspensions were cul-
cant increase in the number of melanocytes [15]. tured and implanted in patients with vitiligo by
However more recently, afamelanotide, an ana- Olsson and Juhlin [24]. A new approach to sim-
logue of alpha-melanocyte-stimulating hormone, plify the delivery of cultured melanocytes to
was shown to promote melanoblast differentia- patients via a cell delivery carrier which could
tion and proliferation of melanocytes in vitiligo also be used for transport of cells over consider-
skin [16]. In 1974 a new high-intensity UVA light able distances has been proposed by Mac Neil
source was initially used in combination with and Eves [25]. Because melanocyte stem cells
either oral 8 MOP or 4,5,8-trimethylpsoralen for reside in the outer root sheath of hair follicles,
the treatment of vitiligo. It was the beginning of suspension of outer root sheath cells has been
PUVA therapy which permitted to induce a good used as a source of melanocytes when trans-
repigmentation on hairy skin using the melano- planted into patients with vitiligo [26, 27].
cytes from the follicular reservoir. The period
from 1974 to 1988 was the period of photomedi-
cine that established the therapeutic effective- 1.7 Conclusions
ness of psoralens in combination with UVA in
the treatment of vitiligo, psoriasis and various Vitiligo, under different names, was recognized
other skin diseases [17]. and feared over several centuries. Even though
More recently several strategies were used modern phototherapies and photochemotherapies
[18] including topical and systemic corticoste- represent an improvement of the herbal and sun
roids, topical calcineurin inhibitors, photoche- ancient world techniques, our current therapies,
motherapy (oral and topical) such as psoralen including surgical techniques, fail in a large num-
plus UVA (PUVA), psoralen plus sunlight ber of cases. The stigma historically associated
(PUVA sol), broad- and narrowband UVB with the disease has not disappeared, and infor-
phototherapy, 308 nm microphototherapy [2], mation and educational programmes should be
excimer light and combination phototherapy implemented to fight this problem, especially the
(see Chap. 38). fear of contagion.
ERRNVPHGLFRVRUJ
References 16. Lim HW, Grimes PE, Agbai O, et al. Afamelanotide

and narrowband UVB phototherapy for the treatment
of vitiligo: a randomized multicenter trial. JAMA
1. Goldman L, Richard S, Moraites R. White spots in
Dermatol. 2015;151(1):42–50.
Biblical Times. Arch Dermatol. 1966;93:744–53.
17. Pathak M, Fitzpatrick TB. The evolution of photoche-
2. Ortonne JP, Mosher DP, Fitzpatrick TB. Vitiligo and
motherapy with psoralens and UVA (PUVA) 2000BC
other hypomelanoses of hair and skin. New York, NY:
to 1992 AD. J Photochem Photobiol. 1992;14:3–22.
Plenum medical school; 1983. p. 129–32.
18. Lotti TM, Menchini G, Andreassi L. UV-B radia-
3. Ebbel B. The papyrus Ebers. Copenhagen: Levin and
tion microphototherapy. An elective treatment for
Munksgaard; 1963.
segmental vitiligo. J Eur Acad Dermatol Venereol.
4. Donata SR, Kesavan M, Austin SR. Clinical trial of
1999;12:102–8.
certain Ayurveda medicines indicated in vitiligo.
19. Behl PN. Treatment of vitiligo with homologous thin
Ancient Sci Life. 1990;4:202–6.
Thiersch skin grafts. Curr Med Pract. 1964;8:218–21.
5. Nair BK. Vitiligo: a retrospect. Int J Dermatol.
20. Falabella R. Epidermal grafting: an original technique
1978;17:755–7.
and its application in achromic areas. Arch Dermatol.
6. Panda AK. The medicohistorical perspective of vit-
1971;104:592–600.
iligo. Bull Ind Hist Med. 2005;25:41–6.
21. Falabella R. Repigmentation of leucoderma by autol-
7. Ben Achour T. TafsirAt Tahir Wat-Tanwir. Edn Dar
ogous epidermal grafting. J Dermatol Surg Oncol.
SahouneHannadi, city, 1973.
1984;10:136–44.
8. Olsson M, Juhlin M. Leucoderma treated by trans-
22. Falabella R, Escobar C, Borrero L. Treatment of

plantation of a basal cell layer enriched suspension.
refractory and stable vitiligo by transplantation of
Br J Dermatol. 1998;138:644–8.
in vitro cultured epidermal autografts bearing mela-
9. Prasad PV, Bhatnagar VK. Medico-historical study of
nocytes. J Am Acad Dermatol. 1992;262(1):230–6.
“Kilasa” (vitiligo/leucoderma) a common skin disor-
23. Gauthier Y, Surleve-Bazeille JE. Autologous grafting
der. Bull Ind Inst Hist Med. 2003;33:113–27.
with non cultured melanocytes: a simplified method
10. Harris JL, Lerner AB. Amino acid sequence of

for treatment of depigmented lesions. J Am Acad
alpha-melanocyte stimulating hormone. Nature.
Dermatol. 1992;26:191–4.
1957;179:1346–7.
24. Olsson M, Juhlin M. Melanocyte transplantation in
11. Fahmy IR, Abn-Shady H. Pharmacological study and
vitiligo. Lancet. 1992;34(8825):981.
isolation of crystalline constituents: Amoïdin. Quart J
25. Mac Neil S, Eves P. Simplifying the delivery of cul-
Pharm and Pharmacol. 1947;20:281–6.
tured melanocytes. In: Gupta S, Olsson MJ, Kanwar
12. Pathak M, Daniels F, Fitzpatrick TB. The presently
A, editors. Surgical management of vitiligo. Oxford:
known distribution of furocoumarins (psoralens) in
Blackwell; 2007. p. 191–201.
plants. J Invest Dermatol. 1962;39:225–39.
26. Singh C, Parsad D, Kanwar AJ, Dogras AS, Kumar
13.
Kuske H. Experimentelle untershunger-
R. Comparison between autologous non cultured
sur Photosensibiliste der Haut durch pflanzli-
extracted hair follicle outer root sheath cell suspen-
che Wisrktoffe. Arch F Dermatol et Syphil.
sion and autologous non cultured epidermal cell
1938;178:112–4.
suspension in the treatment of stable vitiligo: a ran-
14. El Mofty AM. A preliminary clinical report on the
domized study. Br J Dermatol. 2013;169(2):287–93.
treatment of leukodermias with Ammi Majus Linn. J
27. Vanscheidt W, Hunziker T. Repigmentation by

Roy Egypt. 1948;31:651–6.
outer-root sheath-derived melanocytes: proof of
15. Lerner AB, McGuire JS. Vitiligo and sympathec-

concept in vitiligo and leucoderma. Dermatology.
tomy: the effect of sympathectomy and alpha-
2009;218(4):342–3.
melanocyte stimulating hormone. Arch Dermatol.
1966;94:269–78.
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Definitions and Classification
2
Contents
2.1 VETF Position Paper Definition and Classification 12
2.2 International Consensus on Nomenclature 13
2.3 Key Points of Consensus and Definitions/Glossary 13
2.3.1 Vitiligo/NSV, A Consensus Umbrella Term for All Forms of Generalized
Vitiligo 13
2.3.2 Definitions 13
2.4 Pending Classification Issues/Rare Variants 14
2.5 Differential Diagnosis 14
2.5.1 Conditions to Exclude from the Definition of Vitiligo/NSV 14
2.5.2 Conditions to Exclude from the Definition of SV 22
References 23
Abstract (VGICC). Vitiligo/nonsegmental vitiligo

Since the first edition of this textbook, the (NSV) is a consensus umbrella term for all
nomenclature has been revised after an forms of generalized vitiligo. It is an acquired
extensive international work within the chronic pigmentation disorder characterized
Vitiligo Global Issues Consensus Conference by white patches, often symmetrical, which
usually increases in size with time, corre-
sponding to a substantial loss of functioning
epidermal and sometimes hair follicle mela-
A. Taïeb (*)
Department of Dermatology and Pediatric nocytes. The two other subsets of vitiligo
Dermatology, Bordeaux University Hospitals, defined by the international consensus are seg-
and INSERM U 1035, University of Bordeaux, mental vitiligo and unclassified/undetermined
Bordeaux, France
e-mail: alain.taieb@chu-bordeaux.fr
vitiligo which correspond to focal disease and
rare variants. A series of hypopigmented dis-
M. Picardo
Cutaneous Physiopathology and CIRM, San Gallicano
orders may masquerade as vitiligo, and some
Dermatological Institute, IRCCS, Rome, Italy of them need to be ruled out by specific proce-
e-mail: mauro.picardo@ifo.gov.it dures including a skin biopsy.

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12 A. Taïeb and M. Picardo
meetings and workshops a set of consensus state-

Key Points ments in 2007, including descriptive definitions,
• An international consensus has been classification, and scoring [1]. For common gen-
reached for the classification of vitiligo in eralized vitiligo, the definition was as follows:
three major categories, namely, vitiligo or “Vitiligo (nonsegmental or NSV) is an acquired
nonsegmental vitiligo, segmental vitiligo, chronic pigmentation disorder characterized by
and unclassified/undetermined vitiligo. white patches, often symmetrical, which usually
• Vitiligo/NSV is an acquired chronic increases in size with time, corresponding to a
pigmentation disorder characterized by substantial loss of functioning epidermal and
white patches, often symmetrical, which sometimes hair follicle melanocytes.” As it, the
usually increases in size with time, cor- VETF definition (1) was not specific enough and
responding to a substantial loss of func- needed to be completed by a list of disorders
tioning epidermal and sometimes hair which may clinically resemble vitiligo but which
follicle melanocytes. are clearly attributable to known etiologic fac-
• A series of hypopigmented disorders tors; (2) it intentionally made no reference to
may masquerade as vitiligo, and some either a single disease or to a syndrome of various
of them need to be ruled out by specific causes.
procedures including a skin biopsy. Concerning classification, the simplest one, pro-
posed by Koga [2], distinguishing segmental (SV)
and nonsegmental (NSV) forms of disease, served
as a pragmatic framework in the 2007 VETF clas-
2.1 ETF Position Paper
V sification, which in addition introduced the recently
Definition and Classification identified subset mixed vitiligo (MV) [3, 4].
In 2011, the Vitiligo Global Issues Consensus
Although vitiligo is clinically identified without Conference (VGICC) proposed a novel classifi-
much difficulty in most instances, difficulties cation and nomenclature, based on a revision of
arise when searching for the vitiligo literature the 2007 VETF classification [Table 2.1]. For the
due to a lack of consensus in definitions. In order sake of consistency, the definitions used through-
to address this problem, the Vitiligo European out the book are those proposed by the VGICC
Task Force (VETF) proposed after several and reviewed thereafter.
Table 2.1 Bordeaux VGICC classification of vitiligo (2011)

Type of vitiligo Subtypes Consensus statements and other remarks
Vitiligo (V)/ Acrofacial The term “vitiligo” is the recommended umbrella term for all
nonsegmental Mucosal (more than nonsegmental forms of vitiligo. As a transition, vitiligo/NSV can be
vitiligo (NSV) one mucosal site) used
Generalized Mixed vitiligo, being the coexistence of SV + V, is a subgroup of V
Universal Subtyping may not reflect a distinct nature but is a useful information
Mixed (associated for epidemiologic studies
with SV)
Rare variants
Segmental vitiligo Uni-, bi-, or Segmental vitiligo refers to a clinically unambiguous segmental
(SV) plurisegmental distribution of depigmented lesions, typically associated with rapid
onset and with leukotrichia. There is no consensus concerning the
mechanism underlying lesion distribution in SV
Undetermined/ Focal Focal vitiligo, a term that applies to localized macules characterized by
unclassified vitiligo Mucosal (one site in loss of melanocytes, is assigned to the category UnV until more
(UnV) isolation) definitive classification can be made on clinical grounds (generally
after 1–2 years of follow-up). Cases with long-lasting focal lesions or
of pure mucosal vitiligo, if not classified as SV, may remain
“unclassifiable”
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2 Definitions and Classification 13
2.2 International Consensus “vulgaris,” as synonymous with “common,” con-

on Nomenclature veys a pejorative connotation to patients and the
general public and should not be used. The vali-
The VETF, which includes medical experts, scien- dation of the term vitiligo (alone) as a synonym
tists, and representatives of patient support groups, of nonsegmental vitiligo was agreed upon,
drafted a preliminary list of issues of global impor- because it is the most common presentation and
tance for clinical research and patient management that for nonspecialists, it is difficult to understand
which was sent to even regional working groups the negative naming of the disease; however, a
were established: North and South Africa, North test period with the double name vitiligo/NSV
and South/Central America, Europe, Middle East, was suggested to avoid confusion.
Continental Asia/Singapore, Japan/Taiwan, and
the Pacific. Classification and nomenclature were
the first items selected besides definition of stable 2.3.2 Definitions
disease, definition of Koebner’s phenomenon
(KP), and significance/individualization of “auto- The following descriptive consensus statements
immune vitiligo.” were proposed in the accompanying glossary of
In January 2011, each group submitted a pre- the VGICC consensus [6]:
liminary contribution addressing the four selected
topics. A first round of discussion of the VGICC 2.3.2.1 Vitiligo/Nonsegmental
was held in Seoul, Korea, in May 2011, prior to Vitiligo Subsets
the 22nd World Congress of Dermatology • Generalized common vitiligo (formerly
attended by representatives from all seven referred to as vitiligo vulgaris): This most
regional groups. Following this first meeting, a common form of vitiligo is characterized by
draft consensus document was prepared and cir- asymptomatic, well-circumscribed, milky-white
culated for further comments. The final round of macules involving multiple parts of the body,
discussion was held in Bordeaux, France, in usually in a symmetrical pattern. The disease can
September 2011 at the 21st IPCC, the draft start at any site of the body, but the fingers,
revised accordingly and published in 2012 [5]. hands, and face are frequently the initial sites.
• Acrofacial vitiligo: In acrofacial vitiligo, the
involved sites are usually limited to the face,
2.3 ey Points of Consensus
K head, hands, and feet. A distinctive feature is
and Definitions/Glossary depigmentation of the distal fingers and facial
orifices, and a common genital involvement. It
2.3.1 Vitiligo/NSV, A Consensus may later include other body sites, resulting in
Umbrella Term for All Forms typical generalized vitiligo.
of Generalized Vitiligo • Vitiligo universalis: Vitiligo universalis is the
most extensive form of the disease and gener-
Although not fully satisfactory, the term “non- ally occurs in adulthood. “Universalis” is gen-
segmental vitiligo” is currently used to describe erally used when depigmentation is virtually
different clinical subtypes of vitiligo that are all universal (80–90% of the body surface), but
clearly distinct from SV including acrofacial, some pigmentation may be still present. Hairs
generalized, mucosal (multifocal), and universal may also be partially spared. Whereas this
vitiligo. The terms “bilateral” and “unilateral” diagnosis is easy in dark-skinned individuals,
vitiligo have been used as alternative approaches it may be difficult in very fair-skinned indi-
to classification but raised possible confusion viduals. Usually, vitiligo universalis is pre-
with mixed vitiligo and multisegmental vitiligo ceded by generalized vitiligo that gradually
(see respective definitions in next sections). evolves to complete or near-complete depig-
The VGICC participants agreed that the term mentation of the skin and hair.
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2.3.2.2 Segmental Vitiligo p igmentation. The relation to true vitiligo comes

Mono-segmental vitiligo is the most common from pathology and coexistence with conven-
form of SV, referring to the presence of one or tional vitiligo macules. The differential diagno-
more white depigmented macules distributed on sis from early-stage cutaneous lymphoma is of
one side of the body, usually respecting the mid- primary importance, and repeated biopsies with
line (although some lesions may partly cross the molecular studies of clonality may be needed.
midline), early follicular involvement (leuko- A recent case series has been reported under the
trichia), and rapid development over a few name “hypochromic vitiligo” [7].
weeks or months and overall protracted course. • Follicular vitiligo: This refers to a form of
Rarely, segmental vitiligo may refer to multiple generalized vitiligo seen in a young black
segmental lesions distributed either unilaterally patient that primarily involved the follicular
or bilaterally. The onset may be simultaneous or reservoir with limited skin involvement, con-
not. A clear segmental distribution of the lesions trasting with marked generalized hair whiten-
with midline demarcation, together with the ing, and melanocyte loss in hair follicles [5].
associated features described in mono-segmen- A recent case series has been published which
tal cases (leukotrichia, protracted course), dis- documents in all patients’ significant whiten-
tinguishes this subset versus vitiligo/NSV in ing of their body and, in some, scalp hairs
bilateral cases. before cutaneous depigmentation. Examination
revealed classic generalized depigmented
lesions of vitiligo and an impressive presence
2.4 Pending Classification of leukotrichia, not only in vitiliginous areas
Issues/Rare Variants but also in areas with clinically normal-
(see Chap. 1.5.4) appearing skin. Punch biopsy specimen of the
leukotrichia and vitiligo lesions demonstrated
The Bordeaux VGICC classification suggests to loss of melanocytes and precursors in the basal
leave focal cutaneous or mucosal vitiligo within epidermis and hair follicles [8].
the category undetermined/unclassified vitiligo
(UnV) and discusses some rare variants:
2.5 Differential Diagnosis
• Focal vitiligo: Focal vitiligo refers to a small
isolated patch that does not fit a segmental dis- 2.5.1 Conditions to Exclude
tribution and which has not evolved into vitil- from the Definition
igo/NSV after a period of at least 2 years. This of Vitiligo/NSV
form of vitiligo may evolve into either SV or
vitiligo/NSV. 2.5.1.1 Inherited or Genetically
• Vitiligo punctata: Lesions present as sharply Induced Hypomelanoses
demarcated depigmented punctiform 1–1.5 mm Usually, contrary to vitiligo, hypopigmented
macules involving any area of the body, differ- patches are present at birth, but in patients of low
ent from hypomelanosis guttata and the heredi- phototype, hypopigmented patches are usually
tary dyschromatoses. discovered after the first sun exposure, some-
• Vitiligo minor/hypochromic vitiligo: This times in the second or third year of life. The
form of NSV is rarely reported. The disease group of genetic disorders to exclude is detailed
seems to be limited to dark-skinned individuals. in Table 2.2 and Figs. 2.1, 2.2, 2.3, and 2.4.
The term “minor” does not strictly refer to the Piebaldism may be mistaken for vitiligo when
limitation of the disease to a restricted surface the patient without informative family history
area but rather to the partial defect in comes with symmetrical limb patches without
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Table 2.2 Monogenic hypomelanoses

Disorder/OMIM
number Clinical presentation Transmission/diagnosis
Piebaldism/172800 White forelock, anterior body midline Autosomal dominant. Skin biopsy: usually, the
depigmentation, bilateral shin absence of c-Kit protein immunostaining in
depigmentation melanocytes
Molecular analysis of c-KIT gene or SNA1 gene
Tuberous Small or larger (ash-leaf) white spots, Autosomal dominant, brain imaging, cardiac and
sclerosis/191100 seizures; other usually later cutaneous renal imaging
symptoms (shagreen patches, Molecular analysis of TSC1 and 2
angiofibromas, etc.)
Ito’s Blaschkolinear distribution, uni- or Sporadic
hypomelanosis/300337 bilateral of hypopigmented streaks Chromosomal or genetic mosaicism (blood or
skin cells)
Waardenburg’s White forelock, hypertelorism, deafness Autosomal dominant
syndrome/193500 (variable according to genotype) Genetic testing according to phenotype (several
(type I) Possible association to congenital clinical variants and six possible causative genes
megacolon (Hirschsprung’s disease) mutated)
Hermansky-Pudlak Diffuse depigmentation pattern, eye Autosomal dominant, genetic heterogeneity
syndrome/203300 pigment dilution, hemorrhagic diathesis (eight possible causative genes)
(type 1) Specific ethnic background Molecular testing possible according to ethnic
background and phenotype
Menkès Hair and body diffuse pigment dilution, X-linked recessive
syndrome/300011 pili torti, neurodegenerative changes ATP7A gene mutations; affects cupper-dependent
enzymes
Molecular testing possible
Ziprkowski-Margolis Iris heterochromia, depigmentation Described in Israel in one family
syndrome/300700 (diffuse) + hyperpigmented macules X-linked recessive
can remain, neurosensorial deafness Mapped to Xq26.3-q27.1
Griscelli’s syndrome Silvery hairs usually diffuse liver Autosomal recessive, three types corresponding
214450 (type 1) enlargement and symptoms related to to three different causative genes
immunodeficiency Molecular testing possible
a b
Fig. 2.1 Piebaldism: diffuse form without spontaneous an adult (courtesy of Dr Y Gauthier) (c); repigmentation
repigmentation (a); limited form (white forelock) in the the two first years of life (d–f), becoming clearly tri-
mother (b); typical spontaneous repigmentation pattern in chrome in (f)
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c d
Fig. 2.1 (continued)
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a b
Fig. 2.2 Tuberous sclerosis (a) ash-leaf macules and bromas (c) and large vitiligoid patch (d) of the abdominal
shagreen patch on the dorsum of a young patient; Koenen’s belt in a 25-year-old female patient
periungual tumor in the same patient (b); facial angiofi-
midline anomalies. The hyperpigmented rim at On the opposite, vitiligo universalis is some-
the interface of depigmented and normally pig- times misdiagnosed as albinism when the history
mented skin is characteristic of v itiligo after sun cannot be obtained properly (Chap. 1.5.1).
exposure, but may be also seen in piebaldism, in Classic oculocutaneous albinism with hair depig-
addition with perifollicular central hyperpigmentation and nystagmus at birth is not a consid-
mented patches. Piebaldism shows however a eration, but milder syndromic albinisms should
ventral distribution of lesions and early onset. As be mentioned (Table 2.2). Some rare vitiligo-like
usual for monogenetic disorders, information or true vitiligo conditions ( “syndromic vitiligo”)
about specific ethnic background/consanguinity are seen in the context of monogenic disorders.
and a detailed family tree are mandatory. They are reviewed separately in Chap. 1.5.8.
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Fig. 2.3 Hypomelanosis of Ito (courtesy of Dr O Fig. 2.4 Waardenburg’s syndrome

Enjolras, France). Note the striking blaschkolinear pattern
(thin bands of depigmentation following the dorsoventral
pattern of skin development) may occur during the acceleration phase of depig-
mentation, in the case of mild subclinical lichen-
2.5.1.2 Post-inflammatory oid inflammation (Chap. 1.4). In the majority of
Hypomelanoses (Fig. 2.5) cases, a progressive pigment dilution accompa-
There are two main processes for melanin pig- nies the disappearance of pigment cells.
ment to be eliminated following inflammation. Distinguishing NSV from postinflammatory
First, in inflammatory disorders accompanied by hypomelanoses is usually made on clinical
an increased epidermal turnover (e.g., psoriasis, grounds rather than on histopathology, when the
atopic dermatitis), pigment is lost upwards within primary skin inflammatory disease can be diag-
eliminated cells, resulting in focal pigment dilu- nosed clinically (e.g., scalp or plaque psoriasis,
tion until cessation of inflammation and recovery flexural dermatitis for atopic dermatitis, sclero-
of melanin production/distribution within the epi- derma plaques, lichen sclerosus “white spot dis-
dermis. Second, when an acute lichenoid/cyto- ease,” etc.). However, true association may occur
toxic infiltrate attacks the epidermal basal layer [9] and sometimes in similar locations, suggesting
(e.g., lichen planus, toxic drug reactions), there is pathophysiological links, such as koebnerization
a leakage of epidermal pigment into the superfi- in the case of pruritic dermatoses followed by viti-
cial dermis (“pigment incontinence”), which is liginous patches (see Chap. 1.5.7). In difficult
removed when the inflammation has stopped by cases, a biopsy can be useful to make an accurate
macrophages (“melanophages”), a process which diagnosis, e.g., showing spongiosis (eczema) or
takes months or years. In vitiligo/NSV there is psoriasiform changes with neutrophils in the stra-
limited evidence of pigment incontinence, which tum corneum (psoriasis).
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a b
Fig. 2.5 Dermatitis with post-inflammatory hypopigmentation: pityriasis alba in a child with mild atopic dermatitis
(a); psoriasis (b)
2.5.1.3 Para-malignant 2.5.1.4 Para-malignant Hypomelanoses/

Hypomelanoses/Mycosis Melanoma-Associated
Fungoides (Fig. 2.6) Depigmentation (Fig. 2.7)
In dark-skinned patients, skin whitening may cor- The vitiligoid changes associated with melanoma
respond to an early stage of epidermotropic T cell may result from a halo of depigmentation around a
lymphoma. When signs of inflammation and skin cutaneous melanoma (malignant Sutton’s phenom-
infiltration are lacking, this presentation can be enon) to more widespread vitiligoid changes. The
misleading [10]. A biopsy is able to show diag- margins of such vitiligoid lesions under Wood’s
nostic changes (large size and atypia of epidermo- lamp are usually less distinct than those of com-
tropic lymphocytes especially in the basal layer, mon vitiligo, and depigmentation is usually incom-
Chap. 1.4). The mechanism of pigment dilution in plete [11]. Koebner’s phenomenon is usually
mycosis fungoides has been studied, and a absent. The prognostic value of depigmentation in
reduced expression of c-Kit has been shown. The the context of melanoma treated with interferon
loss of c-Kit, and subsequent downstream effects has been established [12]. Chapter 16 discusses the
on melanocyte survival, might be initiated by vitiligoid changes after immune checkpoint inhibi-
cytotoxic effects of melanosomal-antigen-specific tors, a frequent side effect since the introduction of
CD8-positive neoplastic T lymphocytes [10]. these molecules in metastatic melanoma.
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Fig. 2.6 Mycosis fungoides can present as hypopig-

mented patches in dark-skinned individuals.
Histopathology is diagnostic
Fig. 2.8 Indeterminate leprosy (a elbow, b forehead)

should be suspected in individuals living in endemic
areas. Loss of sensitivity is usually associated
Fig. 2.7 Melanoma-associated depigmentation: Melanoma-
associated leukoderma is associated with spontaneous or
vaccine-induced regression of a primary melanoma untreated lesions allow a definite diagnosis.
Indeterminate leprosy (Fig. 2.8) is manifested by
2.5.1.5 Para-infectious hypochromic patches which are hypoesthetic under
Hypopigmentation light touch. In both cases, the infectious process
Tinea versicolor can cause vitiligoid changes, gen- can inhibit melanogenesis through largely unknown
erally after treatment in the absence of re-exposure mechanisms. For tinea versicolor, toxic effects on
to UV light. However, distribution, shape of the pigment synthesis by fungal metabolites, espe-
lesion, and some scaling and green fluorescence of cially tryptophan-derived metabolites of M. furfur,
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a b
Fig. 2.9 Hypopigmented vitiligoid sequel of pityriasis versicolor (a) (upper back) and acquired macular hypomelano-
sis (b) (abdomen) (Guillet-Westerhof syndrome)
a b
Fig. 2.10 Posttraumatic depigmentation (a): note the unusual square limits; (b) definitive depigmentation following
toxic epidermal necrolysis
have been discussed [13] (Fig. 2.9a). Acquired contains melanocyte precursors is destroyed, the
macular
hypomelanosis (Guillet-Westerhof resulting wound healing process will not recapit-
disease) (Fig. 2.9b) is seen in young adults and fre- ulate pigmentation from the center, and marginal
quently referred to as a “recalcitrant pityriasis ver- repigmentation fails to compensate the loss. It
sicolor” [14–16]. The white macules are present on may sometimes be difficult to distinguish some
the trunk and more marked on the lower back and aspects from true vitiligo, when scarring is not
axillae. The role of Propionibacterium acnes has obvious. This is the case in depigmenting sequels
been based on red spots centering the macules of toxic epidermal necrolysis (Fig. 2.10) [17].
under Wood’s lamp, but this finding is not constant.
This disease remains largely unknown by derma- 2.5.1.7 Melasma
tologists and is usually not diagnosed but responds This common hypermelanotic disorder is surpris-
well to UVB therapy. ingly a not so rare diagnostic pitfall in the vitiligo
clinic when the hyperpigmented facial lesions
2.5.1.6 Posttraumatic Leukoderma surround normal but hypochromic-looking skin
When the melanocyte reservoir is depleted, as (Fig. 2.11). Usually the pattern is different from
after deep burns or scars, which remove the hair vitiligo, and the examination of other body sites
follicles entirely or when the bulge area which allows a definitive diagnosis.
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Fig. 2.12 Vitiligoid depigmentation of dorsum of hands

in a patient with hand eczema. Note the persistence of pig-
ment on knuckle pads
via c-Kit signaling downregulation [20], as well

as topically induced vitiligoid lesions following
imiquimod therapy [21]. The long-term use of
potent topical corticosteroids may cause vitiligoid
depigmentation (Fig. 2.12).
2.5.2 Conditions to Exclude

from the Definition of SV
Fig. 2.11 Melasma: a complete skin examination helped
by Wood’s lamp examination is mandatory in this setting Although several disorders may pose problems,
congenital hypomelanoses of segmental distri-
2.5.1.8 Occupational and Drug- bution named collectively naevus depigmento-
Induced Depigmentation sus or achromic nevus [22, 23], which might
(Chap. 1.5.7) correspond to somatic mosaic defects of cutane-
The so-called occupational vitiligo is a subset of ous pigmentary genes, are the most common dif-
vitiligo triggered by an occupational exposure, ferential diagnoses. Naevus depigmentosus is
which evolves from contact depigmentation usually congenital or detectable in the first year
caused generally by a phenolic-catecholic deriva- of life and stable in size in proportion to the
tive to a generalized phenomenon. When depig- child’s growth (Fig. 2.13). The lesion usually
mentation is widespread, the limits between contains a normal or subnormal number of mela-
drug-induced depigmentation and true vitiligo are nocytes compared with control perilesional skin,
not easy to delineate. An occupational medicine but the production of melanin pigment is
advice can be helpful. For systemic drugs, except reduced. Sun exposure may attenuate the differ-
the depigmentation occurring after toxic epider- ence in pigmentation from the normal skin. In
mal necrolysis, which is related to a kind of super- difficult cases, a biopsy is needed to differentiate
ficial burn-like injury but also a rare type of naevus depigmentosus from SV. Segmental or
universal depigmentation [18], other drugs may hemicorporeal hypomelanosis of Ito (Fig. 2.3),
rarely cause vitiligoid depigmentation (chloro- which corresponds to another type of cutaneous
quine, fluphenazine, physostigmine) [19]. More mosaicism made of narrow depigmented streaks
recently vitiligoid depigmentations following following Blaschko’s lines, is not in practice a
imatinib treatment for chronic myeloid leukemia real differential diagnosis problem for multiseg-
have been reported and are thought to be mediated mental v itiligo (Table 2.2).
ERRNVPHGLFRVRUJ
Conference Panelists. Revised classification/nomen-

clature of vitiligo and related issues: the Vitiligo
Global Issues Consensus Conference. Pigment Cell
Melanoma Res. 2012;25:E1–13.
7. Ezzedine K, Mahé A, van Geel N, Cardot-Leccia
N, Gauthier Y, Descamps V, Al Issa A, Ly F,
Chosidow O, Taïeb A, Passeron T. Hypochromic
vitiligo: delineation of a new entity. Br J Dermatol.
2015;172:716–21.
8. Gan EY, Cario-André M, Pain C, Goussot JF, Taïeb A,
Seneschal J, Ezzedine K. Follicular vitiligo: a report
of 8 cases. J Am Acad Dermatol. 2016;74:1178–84.
9. Berger TG, Kiesewetter F, Maczek C, et al. Psoriasis
confined strictly to vitiligo areas--a Koebner-like
phenomenon? J Eur Acad Dermatol Venereol.
2006;20:178–83.
10. Singh ZN, Tretiakova MS, Shea CR, Petronic-Rosic
VM. Decreased CD117 expression in hypopig-
mented mycosis fungoides correlates with hypomel-
anosis: lessons learned from vitiligo. Mod Pathol.
2006;19:1255–60.
11. Hartmann A, Bedenk C, Keikavoussi P, et al.

Vitiligo and melanoma-associated hypopigmentation
(MAH):shared and discriminative features. J Dtsch
Dermatol Ges. 2008;6:1053–9.
12. Gogas H, Ioannovich J, Dafni U, et al. Prognostic sig-
nificance of autoimmunity during treatment of mela-
noma with interferon. N Engl J Med. 2006;354:709–18.
13. Thoma W, Krämer HJ, Mayser P. Pityriasis versicolor
alba. J Eur Acad Dermatol Venereol. 2005;19:147–52.
14. Guillet G, Helenon R, Gauthier Y, et al. Progressive
Fig. 2.13 Nevus depigmentosus, a common pitfall for macular hypomelanosis of the trunk: primary acquired
the diagnosis of segmental vitiligo (see text) hypopigmentation. J Cutan Pathol. 1988;15:286–9.
15. Relyveld GN, Dingemans KP, Menke HE, et al.

Ultrastructural findings in progressive macular
hypomelanosis indicate decreased melanin produc-
References tion. J Eur Acad Dermatol Venereol. 2008;22:568–74.
16. Westerhof W, Relyveld GN, Kingswijk MM, et al.
1. Taïeb A, Picardo M, VETF Members. The defini- Propionibacterium acnes and the pathogenesis of
tion and assessment of vitiligo: a consensus report of progressive macular hypomelanosis. Arch Dermatol.
the Vitiligo European Task Force. Pigment Cell Res. 2004;140:210–4.
2007;20:27–35. 17. Oplatek A, Brown K, Sen S, et al. Long-term follow-
2. Koga M. Vitiligo: a new classification and therapy. Br up of patients treated for toxic epidermal necrolysis. J
J Dermatol. 1977;97:255–61. Burn Care Res. 2006;27:26–33.
3. Gauthier Y, Cario-Andre M, Taieb A. A critical 18. Smith DA, Burgdorf WH. Universal cutaneous depig-
appraisal of vitiligo etiologic theories. Is melano- mentation following phenytoin-induced toxic epider-
cyte loss a melanocytorrhagy? Pigment Cell Res. mal necrolysis. J Am Acad Dermatol. 1984;10:106–9.
2003;16:322–32. 19. Boissy RE, Manga P. On the etiology of contact/occu-
4. Mulekar SV, Al Issa A, Asaad M, et al. Mixed vitiligo. pational vitiligo. Pigment Cell Res. 2004;17:208–14.
J Cutan Med Surg. 2006;10:104–7. 20. Cario-André M, Ardilouze L, Pain C, et al. Imatinib
5. Ezzedine K, Amazan E, Séneschal J, Cario-André mesilate inhibits melanogenesis in vitro. Br J
M, Léauté-Labrèze C, Vergier B, Boralevi F, Taieb Dermatol. 2006;155:493–4.
A. Follicular vitiligo: a new form of vitiligo. Pigment 21. Mashiah J, Brenner S. Possible mechanisms in the
Cell Melanoma Res. 2012;25:527–9. induction of vitiligo-like hypopigmentation by topical
6. Ezzedine K, Lim HW, Suzuki T, Katayama I, imiquimod. Clin Exp Dermatol. 2008;33:74–6.
Hamzavi I, Lan CC, Goh BK, Anbar T, Silva de 22. Hann SK, Lee HJ. Segmental vitiligo: clinical findings
Castro C, Lee AY, Parsad D, van Geel N, Le Poole IC, in 208 patients. J Am Acad Dermatol. 1996;35:671–4.
Oiso N, Benzekri L, Spritz R, Gauthier Y, Hann SK, 23. Lee HS, Chun YS, Hann SK. Nevus depigmentosus:
Picardo M, Taieb A. Vitiligo Global Issue Consensus clinical features and histopathologic characteristics in
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Vitiligo: Histopathology, Including
Electron Microscopy
3
Carlo Cota and Daniela Kovacs
Contents
3.1 Introduction 26
3.2 Histopathology of Lesional, Marginal, and Perilesional Skin 26
3.3 Histopathology of Normal-Appearing Skin 30
3.4 Special Stains and Immunohistochemistry in Vitiligo 31
3.5 Electron Microscopy: Lesional, Marginal, and Perilesional Skin 32
3.6 Melanocytes 32
3.7 Keratinocytes 32
3.8 Langerhans Cells 34
3.9 Basal Membrane 34
3.10 Dermis 34
3.11 Normally Pigmented Skin 35
References 36
Abstract ered. However, the increasing interest in

The diagnosis of vitiligo is mainly based on predicting the stability of the disease has
clinical features; thus the role of the histo- reawakened awareness of the histopathologi-
logical evaluation is generally poorly consid- cal features of vitiligo. Histopathologically,
both epidermal and dermal changes charac-
terize vitiligo, mainly in relation to the activ-
C. Cota (*) ity and duration of the disease. Microscopical
Dermatopathology Unit, San Gallicano Dermatologic alterations of vitiligo may be subtle and over-
Institute (IRCCS), Rome, Italy looked in routine practice; thus a clinico-
e-mail: carlo.cota@ifo.gov.it pathological correlation is crucial for a
D. Kovacs proper diagnosis.
Laboratory of Cutaneous Physiopathology and
Integrated Center of Metabolomics Research, San
Gallicano Dermatologic Institute (IRCCS),
Rome, Italy

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26 C. Cota and D. Kovacs
increasing interest in predicting the stability of

Key Points the disease has reawakened awareness of the his-
• Lack of melanocytes and reduction/ topathological features of vitiligo. Several mor-
absence of melanin in long-lasting phological abnormalities of lesional and
lesions of stable disease perilesional areas have been highlighted by rou-
• Clear cells in semi-thin sections in long- tine histology, histochemical/immunohistochem-
lasting disease (25 years), associated ical stainings, and electron microscopy. In
with positive DOPA reaction (residual addition, the presence of widespread cutaneous
melanocytes in depigmented lesional alterations has also been described in normally
skin) pigmented areas of vitiligo patients, focusing
• Progressive reduction of melanocytes interest on normally appearing skin.
along the lesional borders, with residual Histopathologically, both epidermal and der-
melanocytes at the edges of the lesions mal changes characterize vitiligo, mainly in rela-
• Focal spongiosis at the basal layer or the tion to the activity and duration of the disease.
Malpighian layers of the epidermis, along Indeed, early-stage/unstable progressive vitiligo
with epidermal mononuclear cell infiltra- is expected to show more prominent morphologi-
tion and dermal perivascular lymphocytic cal changes than the stable disease [2]. In this
infiltrates, in advancing edge of actively context, biopsy site selection is pivotal. If the dis-
spreading or inflammatory vitiligo ease is stable, the biopsy should be performed on
• Increased number/activity of melano- the margin of a representative macula, including
cytes and reduction of degenerative fea- adjacent normal-appearing skin in order to com-
tures in both melanocytes and pare the two areas histologically. In the case of
keratinocytes after phototherapy unstable spreading vitiligo, a biopsy including
• “Microdepigmentation” (loss of melano- the edges of a newly appeared macula or of a
cytes and T cell epidermal infiltration) in lesion with a recently increased size will be pre-
areas of macroscopically uninvolved ferred [2, 3].
skin of active vitiligo patients One should remember that microscopical alter-
• Perilesional melanocytes well con- ations of vitiligo may be subtle and overlooked in
nected to the neighboring cells by long routine practice; thus a clinico-pathological corre-
dendrites in stable disease vs. shorter lation is crucial for a proper diagnosis.
dendrites and weak connection to sur-
rounding keratinocytes in active form
(electron microscopy) 3.2 Histopathology of Lesional,
• Keratinocytes with focal desmosome Marginal, and Perilesional
interruptions, few melanosomes and Skin
extracellular granular material, and
swollen mitochondria in the active dis- The loss of melanocytes in the basal layer of the
ease (electron microscopy) lesional epidermis is the hallmark of vitiligo,
varying on the life of the lesion [3].
Long-lasting lesions of stable disease com-
monly show a lack of melanocytes associated with
3.1 Introduction a reduction or absence of melanin (Fig. 3.1). If
basal melanocytes of normally pigmented epider-
The diagnosis of vitiligo is mainly based on clini- mis typically appear as clear cells due to fixation
cal features; thus the role of the histological eval- by routine histology, depigmented vitiliginous
uation is generally discussed. In some instances, lesions display a deficit of clear cells and an
a skin biopsy is useful to differentiate a vitiligi- absence of DOPA reaction, which allows for the
nous lesion from other hypopigmented disorders, detection of functional melanin-producing melano-
especially in its early spreading stage [1]. The cytes (Table 3.1). Fontana-Masson silver staining
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3 Vitiligo: Histopathology, Including Electron Microscopy 27
Fig. 3.1 Histopathological
examination of stable
lesional vitiligo shows the
lack of melanocytes and
melanin in the epidermis
and the absence of dermal
inflammatory infiltrate (HE
100×)
Table 3.1 Special stains and antibodies to detect melanocytes

Special stains Staining target Staining pattern References
Fontana-Masson Melanin Black silver [10]
precipitates
DOPA reaction Melanocytes (enzymatically active) Brown black, [47]
cytoplasmic
Antibodies
Microphtalmia transcription Melanocytes: it may be expressed by macrophages, Nuclear [48, 49]
factor (MITF) lymphocytes, fibroblasts, Schwann cells, and
smooth muscle cells
Tyrosinase Melanocytes, melanocytic differentiation marker Cytoplasmic [50, 51]
(T311, MAT-1)
MART1/melanA Melanocytes, melanosomal component Cytoplasmic [52, 53]
Melanoma antigen recognized
by T cells-1 (A103, M2-7C10)
S100 protein Melanocytes, family of calcium-binding proteins Nuclear and [47]
Low specificity: Langerhans cells, histiocytes, cytoplasmic
Schwann cells, and sweat glands
c-Kit, stem cell factor receptor Melanocytes. Cell surface [54]
Not specific, it recognizes also germ cells, epithelial
cells (breast and kidney), mast cells, and several
other hematopoetic cell types
Tyrosinase-related protein 1 Melanocytes [50]
(TRP1, TYRP1) Melanocytic differentiation marker
Dopachrome tautomerase-/ Melanocytes and melanoblasts [50, 55]
tyrosinase-related protein 2
(DCT, TRP2)
HMB-45 glycoprotein 100 Fetal, neonatal, and stimulated adult melanocytes, Cytoplasmic [56, 57]
(gp100) associated to immature, pre-melanosomes
It may react with cells of angiomyolipoma and
lesions of the PEComa group of neoplasms
NKI-beteb PMEL17/gp100 Melanocytes Cytoplasmic [58]
melanosome-associated
antigen
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Fig. 3.2 Cutaneous
biopsy of lesional skin
shows the absence of
melanin in the basal
layer of the epidermis,
as assessed by Fontana-
Masson stain (FM
100×)
corroborates the loss of melanin in the epidermal ual melanocytes at the edges of the lesions
basal layer of these patients (Fig. 3.2) (Table 3.1). (Fig. 3.3). The latter are described as larger, with
However, the identification of clear cells in semi- low pigmentation and with longer, pronouncedly
thin sections of biopsies from patients with even arborized, dendrites [10]. Degenerative changes
long-lasting disease (25 years) associated with and fragmentation of these cells in marginal skin
positive DOPA reaction has suggested the pres- have also been reported by Abdel-Naser et al. and
ence of residual melanocytes in depigmented Van den Wijngaard et al. [9, 11]. Additional epi-
lesional skin, although no reactivity for melano- dermal changes appear scant in established lesions,
cyte markers has been detected [4]. In line with whereas they are most frequently correlated with
these observations, Anbar et al. found scanty mela- an unstable, clinically dynamic, disease, being
nocytes by H&E in the center of vitiligous areas in more evident in the active margin of the lesions.
3 out of 30 biopsies evaluated [5]. These cells did Sparse to mild lymphocytic infiltration may be
not stain with DOPA or HMB45 marker, suggest- seen in the papillary dermis, sometimes with a
ing the inactivity of these cells. In the same work, lichenoid interface dermatitis pattern.
most of marginal melanocytes, stained with DOPA Lymphocytes at the basal layer of the epidermis
or HMB45, show retracted dendrites, whereas per- may be arranged as “small lymphocytic nests” or
ilesional ones display normal dendrites [5]. By “focal lichenoid infiltrates,” and, in many cases,
studying 16 patients with progressive and stable they surround or are in close vicinity to melano-
disease, Kubanov et al. demonstrated melanocytes cytes [12]. Epidermal-clustered lymphocytes
and melanin granules in lesional skin, and Kim may also show epidermotropism and mimic
et al. found melanocytes in lesions lasting 5 or small Pautrier microabscess observed in mycosis
more years [6, 7]. If the follicular melanocytes are fungoides, thus prompting a misdiagnosis.
not usually involved in the early stages of the dis- Focal spongiosis involving the basal layer or
ease, they appear to be the first target in follicular the Malpighian layers of the epidermis, along
vitiligo, reinforcing the hypothesis that follicular with epidermal mononuclear cell infiltration and
and epidermal melanocytes probably exhibit dif- dermal perivascular lymphocytic infiltrates, may
ferent antigenic profiles [8]. The progressive be observed in biopsy specimens taken from the
reduction in the number of melanocytes has been advancing edge of actively spreading or
observed along the lesional borders [9], with resid- inflammatory vitiligo (stage I lesions—onset

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Fig. 3.3 Skin biopsy of

lesional/marginal and
perilesional skin shows
the progressive reduction
in the number of
melanocytes with
residual melanocytic
cells at the edges of the
lesions, as demonstrated
by immunohistochemical
reactivity for Melan-A
(Melan-A 25×).
Courtesy of Prof.
L. Cerroni,
“Dermatopathology
Unit, Department of
Dermatology, Medical
University of Graz”
Graz, Austria
Fig. 3.4 Cutaneous biopsy of the advancing edge of Exocytosis of mononuclear cells may also be seen (HE
actively spreading or inflammatory vitiligo shows focal 200×). Courtesy of Prof. L. Cerroni, “Dermatopathology
spongiosis of the basal layer of the epidermis, as well as Unit, Department of Dermatology, Medical University of
dermal, mainly perivascular, lymphocytic infiltrates. Graz” Graz, Austria
3–13 weeks) (Fig. 3.4). This has been demon- reported in the central area of vitiliginous patches
strated by evaluating epon-embedded sections as opposed to the periphery, as a possible
from 25 patients [13]. The inflammatory pattern reparative/protective phenomenon in a study of
can be interpreted as non-specific, with no proper 33 Greek patients [16]. A recent study has dem-
clinical information, and easily misdiagnosed as onstrated an increased thickening of the stratum
spongiotic or lichenoid disorders. corneum of viable epidermis (defined as the
In thinner sections, a focal but severe vacuolar thickness measured from the stratum granulosum
degeneration of basal keratinocytes mainly to the basal layer) and of the total epidermis in
observed around dermal papillae as well as a vitiliginous lesions compared to adjacent normal-
thickening of the basement membrane have been appearing skin [17]. These features were statisti-
described [14, 15]. Thickening of the stratum cally significant in sun-exposed areas, suggesting
corneum and underlying epidermis associated a protective role against recurrent UV exposure
with an elongation of the basement membrane [17]. Although we usually observe an increased
(defined as the result of an increased number and/ number of Langerhans cells in the lesional area
or length of epidermal rete ridges) has been of vitiligo, conflicting observations regarding the
ERRNVPHGLFRVRUJ
presence of these dendritic cells have been melanocytes as well as a reduction of degenera-
reported. A reduced [18], an increased [17], or an tive features in both melanocytes and keratino-
unmodified [19, 20] amount have been observed cytes [5]. In our experience, the repigmentation
in different studies. A tendency to a higher num- observed after punch grafts is due to the horizon-
ber of Langerhans cells along the basement mem- tal migration of activated melanocytes from
brane has been confirmed by Le Poole et al. [9], donor sites through the enlargement of the inter-
and the hypothesis that these cells may replace cellular spaces and the release of pro-melanogenic
lacking basal epidermal melanocytes has been mediators. The regular function of these melano-
put forth [21]. cytes has been proven by the reactivity for MiTF,
As concerns the dermal compartment, peri- c-Kit, MART1, and TRP1 as well as by the pres-
vascular lymphocytic infiltrate in the upper der- ence of melanin in the neighboring keratinocytes
mis has been described in recently spreading [24].
lesions, whereas no inflammatory infiltrate is sel-
dom noticed in established depigmented skin
areas [13]. Melanophages as well as inflamma- 3.3 Histopathology of Normal-
tory and degenerative changes in sweat glands, Appearing Skin
dermal nerves, and nerve endings have also been
observed [1, 22]. An increased number of dermal In our experience, we have not observed signifi-
blood vessels with angioneogenesis accompa- cant differences in the amount and distribution of
nied the epidermal hyperplasia as a possible melanocytes between normal-appearing vitiligo
reparative/protective phenomenon in a study of skin and healthy control, in agreement with other
33 Greek patients [16]. studies [9, 25]. However, “microdepigmenta-
In evaluating the histopathological features of tion,” consisting of the loss of melanocytes in the
66 patients with stable and unstable vitiligo, Yadav presence of a parallel T cell epidermal infiltra-
et al. recently showed that five parameters—spon- tion, has been identified in areas of macroscopi-
giosis, basal layer vacuolization, epidermal/der- cally uninvolved skin of active vitiligo patients
mal lymphocytes, and melanophages—were more [26]. Analyzing histological changes in skin dis-
recurrent in the unstable disease compared to the tant from depigmented areas, Aslanian and co-
stable disease, suggesting these variables to be workers also showed a decreased pigmentation of
potential values of the disease activity [2]. In an the epidermal basal layer as well as dermal pig-
attempt to correlate the clinical aspect to the histo- ment incontinence with the presence of melano-
logical features of vitiligo, Benzekri et al. distin- phages [27].
guished hypomelanotic lesions with poorly defined The presence of melanocytes located supra-
borders and amelanotyc lesions with sharply basally is occasionally detected in uninvolved
demarcated borders [23]. The former were the skin areas (Fig. 3.5). This observation supports
expression of active spreading vitiligo character- the melanocytorrhagy theory, which has been
ized histopathologically by a progressive loss of proposed as one of the mechanisms responsible
melanocytes, epidermal spongiosis, vacuolar for melanocyte loss in vitiligo. Based on this
degeneration of basal cells, clustered lymphocytic hypothesis, melanocyte disappearance is caused
infiltration, and dermal melanophages. On the by chronic detachment and transepidermal loss
contrary, amelanotic lesions with sharply demar- due to abnormal responses to mechanical stresses,
cated borders were the clinical picture of a stable such as friction or other traumas [28]. Besides the
disease, featured by the normal epidermis, in the presence of alteration related to melanocytes and
majority of cases, with a well-defined depigmenta- pigmentation, keratinocytes may be involved. In
tion and scant inflammatory infiltrates. semi-thin sections, mostly at the basal layer,
The histopathological evaluation of skin biop- keratinocytes showed degenerative changes in
sies following therapies, such as phototherapy, areas distant from depigmented skin [14]. On the
shows an increase in the number and activity of other hand, Gokhale and Mehta did not observe
ERRNVPHGLFRVRUJ
Fig. 3.5 Normal-
appearing skin shows
the presence of
melanocytes located
suprabasally, as
observed with Melan-A
immunostaining
(Melan-A 200×,
enlarged view 400×).
Observation from
Dermatopatology
Laboratory, San
Gallicano
Dermatological Institute,
Rome
any degenerative or inflammatory modifications HMB-45 was detected [4]. Kubanov et al.
or the presence of suprabasal clear cells in observed Melan-A-positive cells and melanin
normal-appearing vitiligo skin [22]. granules in depigmented skin of patients with a
disease duration ranging from 21 months to
40 years [6]. The hypothesis that these residual
3.4 Special Stains melanocytes may be functional has also been
and Immunohistochemistry proposed [7]. Anbar et al. detected a few melano-
in Vitiligo cytes in lesional areas of 3 out of 30 biopsies ana-
lyzed by routine E&E staining and electron
The detection of melanocytes and the nature of microscopy [5]. However, these cells did not
inflammatory infiltrates have been studied by react with DOPA or HMB-45 antibody by immu-
immunohistochemistry. nohistochemistry, thus suggesting their inactiv-
Several authors performed immunohisto- ity. Melanocytes positive for DOPA and HMB-45
chemical analyses using antibodies directed at are instead detected in marginal and perilesional
different target antigens, with the aim of identify- areas [5]. A decrease of positive melanocytes was
ing and possibly characterizing melanocytes of also observed in perilesional normal skin com-
vitiligo skin. Employing a large panel of antibod- pared to that of healthy controls, when using the
ies directed at distinct melanocyte-related anti- Melan-A antibody [6]. Melanocyte-specific
gens (one polyclonal and 17 monoclonal markers have also been employed to analyze the
antibodies), Le Poole and co-workers showed a number and distribution of melanocytes in
loss of melanocytes in lesional areas and no sig- normal-appearing skin. Van den Wijngaard and
nificant differences in the number of cells co-workers demonstrated a similar amount and
between non-lesional and control skin [25]. distribution of melanocytes in non-lesional vitil-
Norris et al. showed an absence of melanocytes igo skin compared to normal skin, using NKI-
positively stained for the stem cell factor receptor beteb antibody by immunohistochemistry [9].
c-KIT in vitiliginous skin [29]. Other studies Similarly, Wagner et al. did not observe any dif-
show that melanocytes may be sporadically pres- ferences in the overall number of melanocytes in
ent in some depigmented skin, even in stable, vitiligo normal-appearing skin compared to the
long-lasting lesions, as reported by Tobin et al. normal control utilizing the Trp2 melanocyte
who found rare clear cells and positive DOPA marker [30]. However, the authors report the
reaction in the epidermis of vitiligo patients with presence of melanocytes located in the upper epi-
stable disease lasting 25 years. However, no reac- dermal layers and demonstrate more basal kerati-
tivity for the melanocyte markers NKI-beteb or nocytes between two melanocytes, pointing to an
ERRNVPHGLFRVRUJ
irregular ratio in the number of cells forming the these cells in the central region of lesional skin
epidermal melanin unit [30]. [35–39]. However, Tobin et al. hypothesized the
The inflammatory infiltrate, as shown by his- presence of residual altered functioning melano-
topathological examination, depends on the life cytes as the source of pigment granules detected
of the lesions. Immunohistochemical analysis of in basal and suprabasal keratinocytes of lesional
the lymphocytic infiltrate by Al Badri et al. areas [4]. Residual melanocytes with poorly mel-
showed, in keeping with the histology, a greater anized melanosomes, vacuolization, and dilated
number of epidermal and dermal CD3+, CD4+, rough endoplasmic reticulum have also been
and CD8+ cells at the margins of depigmented demonstrated by Kim et al. [7]. In early active
skin [31]. Later studies have focused on the dif- spreading vitiligo, residual melanocytes in the
ferences between stable and unstable diseases. center and in the edges of the macular lesions
Ahn et al. demonstrated the higher expression of may show cytoplasm vacuolization, fatty degen-
ICAM1 and CD4 in the epidermis of actively eration, pyknosis, and atypical/small melano-
spreading vitiligo compared to the stable disease somes. Sometimes, apparently normal
[32]. No differences were found regarding CD8 melanocytes have been observed at the margins
and HLA-DR. However, several studies have of the lesions [36]. Breathnach et al. have
shown an increase in the CD8 T lymphocytes in described many melanocytes on the pigmented
active perilesional skin of unstable disease [33, side of the margin with minimal melanogenic
34]. These cells are located in the epidermis/pap- activity and more evident filamentous structures
illary dermis, often juxtaposed to the remaining in the cytoplasm [35]. In addition, melanocytes
melanocytes [33]. Clustered CLA+ lymphocytes located in the perilesional skin have been reported
at the epidermal/dermal junction may express to show electron-lucent vacuoles [39, 40]
cytotoxic markers, such as perforin and gran- (Fig. 3.6).
zyme-B at the site of melanocyte destruction [9]. In contrast to stable disease, where perile-
In addition, Le Poole et al. also found a high sional melanocytes appeared well connected to
number of CD68+ macrophages in the papillary the neighboring cells by long dendrites, melano-
dermis of perilesional and lesional skin [9]. cytes from active spreading disease showed
Finally, Kim et al. measured the dermal shorter dendrites and a weak connection to sur-
inflammation, by quantitative analysis, observing rounding keratinocytes, as demonstrated by Ding
that CD3+ cells accounted for 65.38% of the and co-workers. Mitochondria appeared swollen
inflammatory cells, whereas CD20+, CD68+, with obscure cristae, irregular and vacuolated,
and CD1a+ cells accounted for 5.67%, 13.5%, mainly in active lesions [41]. While Prignano
and 1.89%, respectively [7]. et al. observed well-preserved perilesional mela-
nocytes (with abundant organelles, well-
preserved melanosomes mainly at the second,
3.5 Electron Microscopy: third, and fourth stages of differentiation but
Lesional, Marginal, enlarged mitochondria), Anbar et al. reported
and Perilesional Skin degenerative changes in melanocytes and kerati-
nocytes located in marginal and perilesional viti-
As for light microscopy, ultrastructural features liginous areas. These cells showed cytoplasmic
of vitiligo skin may differ according to the active vacuolization, pyknotic nuclei, and peripheral
progressing or stable stage of the disease. margination of nuclear chromatin [5, 37].
3.6 Melanocytes 3.7 Keratinocytes
Divergent conclusions have been reported regard- Regarding the ultrastructural features of lesional
ing the presence of melanocytes in depigmented keratinocytes, Galadari et al. did not find any
areas. Several studies have described the loss of alterations in the center of the lesions [36]. In line
ERRNVPHGLFRVRUJ
Fig. 3.6 Ultrastructural
analysis of lesional
vitiligo skin showing the
absence of melanosomes
inside keratinocytes and
mild spongiosis.
Courtesy of Prof.
M.R. Torrisi and Dr.
S. Raffa, Cellular
Diagnostics Unit,
Sant’Andrea Hospital;
Dept. of Clinical and
Molecular Medicine,
Sapienza University of
Rome, Italy
with these observations, Prignano et al. described

lesional keratinocytes with no cytoskeletal altera-
tions or organelle rearrangement, except for some
lighter areas possibly representing vacuoles [37].
On the contrary, other authors noticed a varied
level of degeneration in lesional keratinocytes,
including irregular nuclei, alterations in the cyto-
plasm consisting of dilated Golgi complex and/or
endoplasmic reticulum, vacuolar degeneration,
and fragmented tonofilaments [7, 42]. Ding et al.
also described keratinocytes with focal desmo-
some interruptions, few melanosomes associated
with the presence of extracellular granular mate-
rial and swollen mitochondria, mainly in the Fig. 3.7 Ultrastructural analysis of normal-appearing
active disease [41] (Fig. 3.7). vitiligo skin. The presence of melanosomes inside kerati-
As demonstrated by Anbar et al., lesional, mar- nocytes is evident. Courtesy of Prof. M.R. Torrisi and Dr.
ginal, and perilesional keratinocytes exhibited S. Raffa, Cellular Diagnostics Unit, Sant’Andrea Hospital;
Dept. of Clinical and Molecular Medicine, Sapienza
dilatation of the rough endoplasmic reticulum, University Rome, Italy
cytoplasmic vacuoles, peripheral margination of
the fragmented tonofilaments, pyknosis, periph-
eral margination of the nuclear chromatin, and an the presence of EGM deposits, associated with
absence of nucleoli. Extracellular granular mate- focal vacuolar degeneration of keratinocytes [14].
rial (EGM) located in the dilated intercellular Fawzy and co-workers confirmed the existence of
space between keratinocytes has also been vacuolar degeneration in the basal and suprabasal
observed [5]. Moellmann et al., who observed lesional cells, extensive intercellular areas show-
these abnormalities more abundantly in rapidly ing extracellular granular material [39]. The
progressing or stable disease compared to treated authors have highlighted two types of keratinocyte
and repigmenting patients, had already described modifications: (1) electron-lucent degeneration
ERRNVPHGLFRVRUJ
with cytoplasmic vacuolizations with both large suprabasal layers in perilesional skin [37, 38].
indented and small electron-dense nuclei and (2) They show rarefied chromatin in the nuclei, mito-
dark degeneration with electron-dense cytoplasm, chondria alterations with rarefaction, or destruc-
margination of clumped tonofilaments, electron- tions of their membranes [38].
dense melanosomes, and electron-dense nuclei
with dispersed chromatin and distinct nucleoli.
Perilesional keratinocytes show vacuoles, focally 3.9 Basal Membrane
interrupted desmosomes, swollen mitochondria,
and rearranged cristae, as well as granular mate- The basal membrane has been reported to be nor-
rial in the cytoplasm and in the extracellular mal in many cases of vitiligo. However, both a
spaces, especially in the active disease [37, 38]. thicker lamina densa and a focal disappearance of
Bartosik et al. have described the presence of the basement membrane with a passage of granu-
melanosomes and, more recurrently, melanosome lar material and vesicles from the epidermis into
complexes in basal keratinocytes located in the the dermis have been described in lesional skin
center of lesions of long-lasting stable vitiligo, [39, 42]. Prignano et al. also observed interrup-
suggesting the possible persistence of melanin in tions in the basal membrane of perilesional areas,
keratinocytes some time after the appearance of with no significant alterations in the hypopig-
the disease [43]. mented vitiliginous skin [37]. On the other hand,
early reports described multiple focal replication
or “layering” of the basement membrane beneath
3.8 Langerhans Cells melanocytes in the perilesional pigmented mar-
gin [35].
Langerhans cells have been observed to appar-
ently replace melanocytes in central regions of
vitiligo lesions [35]. These cells are character- 3.10 Dermis
ized by the presence of many Birbeck granules,
intracytoplasmatic vacuoles, dilated Golgi com- The cellularity of lesional and marginal areas
plexes, and sometimes, irregular nuclei and appears greater than normal with an increase of
dilated endoplasmic reticulum, damaged mito- small vessels, Schwann cells, and in some cases,
chondria, and small indentations in the cyto- mast cells. Lymphocytes and active fibroblasts
plasm of the adjacent keratinocytes [7, 42]. are more copious at the dermo-epidermal junc-
Prignano et al. have observed numerous tion compared to the control [35]. The presence
Langerhans cells in both basal and suprabasal of lymphocytes with cytoplasmic protrusions
layers of lesional skin. They present an indented both in the dermis and in contact with degener-
nucleus, rough endoplasmic reticulum and Golgi ated keratinocytes has been observed in the cen-
apparatus in the cytoplasm, typical Birbeck tral areas of vitiligous lesions [42]. Nerve
granules, and membrane alterations in the mito- alterations, including axon swelling; axon mem-
chondria. The authors have also described an brane discontinuity; reduplication or multiplicity
additional group of clear cells, resembling less of Schwann cell basement membrane; increase of
differentiated Langerhans cells, characterized by Schwann cell cytoplasmic RNP granules, endo-
the absence of typical Birbeck granules but plasmic reticulum, and mitochondria; extrusion
abounding in structures with a similar electron of degenerate axons from Schwann cytoplasm;
density [37, 38]. and regenerating axons, have been observed in
The basal layer of pigmented marginal areas central and marginal areas of vitiligo lesions
presents more abundant Langerhans cells com- [35]. Changes consisting of regeneration and
pared to normal control skin, as demonstrated by degeneration of dermal nerves have also been
Breathnach [35]. Prignano et al. have described reported by Al’Abadie et al., who observed an
an inclination of these cells to be localized in the increased thickening, often with reduplication, of
ERRNVPHGLFRVRUJ
the basement membrane of Schwann cells sur- Differential diagnosis of vitiligo mainly
rounding the nerve fibers in both lesional and includes nevus depigmentosus, hypopigmented
marginal areas [44]. mycosis fungoides, lichen sclerosus, and pityria-
sis alba [45].
Morphologic abnormalities of melanosomes
3.11 Normally Pigmented Skin and functional defects of melanocytes that
appeared normal in number, compared to the nor-
Ultrastructurally, several studies were unable to mal epidermis, featured nevus depigmentosus, a
detect significant differences in healthy skin of congenital hypopigmented nonprogressive iso-
vitiligo patients compared to normal skin. lated macule. In a study, melanocyte counts were
Normal-appearing melanocytes in size and num- significantly decreased in nevus depigmentosus
ber, with melanosomes at all stages of differenti- with GP-100 and MART-1 stain for melanocytes,
ation, structured rough endoplasmic reticulum, whereas no significant differences in the number
and large but well-preserved mitochondria were of these cells were identified with S100 [46].
detected. Occasionally, some melanocytes Compared to nevus depigmentosus, vitiligo
showed intracellular edema and vacuoles. shows a significant decrease in the number of
Keratinocytes appeared as normal cells with only melanocytes and mild inflammatory dermal
two cases showing reduced distribution of the infiltrate.
cytoskeleton. Langerhans cells show the typical Hypopigmented mycosis fungoides may
indented nucleus, rough endoplasmic reticulum, mimic both clinical and histopathological
mitochondria, and Golgi complex [38]. The basal inflammatory vitiligo. Epidermotropism,
membrane looked well developed without degen- hydropic degeneration of basal cells, partial loss
erative signs [37, 38]. These findings differ from of pigment, preservation of some basal melano-
the observations of Moellmann et al. who cytes, increased density of dermal lymphocytic
observed that the accumulation of extracellular infiltrate, and wiry fibrosis of the papillary der-
granular material (EGM) and foci of vacuolar mis are all clues significantly associated with the
degeneration of basal keratinocytes (consisting histopathological diagnosis of hypopigmented
of swollen mitochondria, dilated endoplasmic mycosis fungoides [15]. On the other hand, focal
reticulum, cytoplasmic vacuoles, loss of desmo- thickening of the basal membrane, a complete
somes and hemidesmosomes) were more evident loss of melanocytes, an absence of melanin, and
in normally pigmented skin of patients with rap- an absence or sparseness of a mild lymphocytic
idly progressing or stable disease [14]. infiltrate in the dermal papillae characterized vit-
iligo and differentiated it from mycosis fungoi-
des [15].
3.12 Differential Diagnosis Microscopical features allow for an easy dif-
ferentiation between vitiligo and lichen sclero-
Diagnosis of vitiligo may be challenging, espe- sus, which is histopathologically characterized
cially in the early spreading inflammatory phase. by vacuolar interface changes, lichenoid inflam-
Although in some cases, an accurate medical his- matory infiltrate, and papillary dermal sclerosis.
tory associated with a careful clinical inspection Pityriasis alba is a common benign dermatoses
leads to the correct diagnosis. A histological with a predilection for the upper part of the body
examination is useful to rule out other hypopig- in atopic preadolescent and adolescent individu-
mented disorders that may be present in a differ- als. Notwithstanding the paucity of morphologic
ential diagnosis [3]. In most cases, an analysis of studies in literature, the latter seems to be charac-
melanocytes and melanin distribution as well as terized by an irregular pigmentation of the basal
the inflammatory infiltrate features represent layer with a normal melanocytic number associ-
clues that allow for a histopathological confirma- ated with additional skin changes, such as follicu-
tion of the diagnosis of vitiligo. lar spongiosis, mild acanthosis of epidermis, and
ERRNVPHGLFRVRUJ
sebaceous gland atrophy. However, the clinical eration of keratinocytes in the normally pigmented
presentation is quite characteristic and allows it to epidermis of patients with vitiligo. J Invest Dermatol.
1982;79(5):321–30.
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vs. hypopigmented mycosis fungoides (histopatho-
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analysis). Eur J Dermatol. 2006;16(1):17–22.
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123.2016.1218987. 55. Steel KP, Davidson DR, Jackson IJ. TRP-2/DT, a new
40. Hann SK, Park YK, Lee KG, et al. Epidermal changes early melanoblast marker, shows that steel growth
in active vitiligo. J Dermatol. 1992;19(4):217–22. factor (c-kit ligand) is a survival factor. Development.
41. Ding GZ, Zhao WE, Li X, et al. A comparative study 1992;115(4):1111–9.
of mitochondrial ultrastructure in melanocytes from 56. Gown AM, Vogel AM, Hoak D, et al. Monoclonal
perilesional vitiligo skin and perilesional halo nevi antibodies specific for melanocytic tumors distin-
skin. Arch Dermatol Res. 2015;307(3):281–9. https:// guish subpopulations of melanocytes. Am J Pathol.
doi.org/10.1007/s00403-015-1544-4. 1986;123(2):195–203.
42. Panuncio AL, Vignale R. Ultrastructural stud-
57. Smoller BR, Hsu A, Krueger J. HMB-45 monoclo-
ies in stable vitiligo. Am J Dermatopathol. nal antibody recognizes an inducible and reversible
2003;25(1):16–20. melanocyte cytoplasmic protein. J Cutan Pathol.
43. Bartosik J, Wulf HC, Kobayasi T. Melanin and
1991;18(5):315–22.
melanosome complexes in long standing stable vit- 58. Vennegoor C, Hageman P, Van Nouhuijs H, et al. A
iligo—an ultrastructural study. Eur J Dermatol. monoclonal antibody specific for cells of the melano-
1998;8(2):95–7. cyte lineage. Am J Pathol. 1988;130(1):179–92.
ERRNVPHGLFRVRUJ
Introduction to Clinical Aspects
Chapters
4
Abstract In addition, specific clinical aspects are

The following chapters cover the entities highlighted such as subtypes of mucocutaneous
delineated in the current nosology of vitiligo pigment cell involved by vitiligo: mucosal, hair
(vitiligo/NSV and its variants Chap. 5), follicle, and nevus cells Chap. 9; involvement of
segmental vitiligo Chap. 6, mixed vitiligo extracutaneous melanocytes Chap. 10; environ-
Chap. 7, and rare variants Chap. 8, in accor- mental triggers such as Koebner’s phenomenon
dance with the recent VGICC international and occupational vitiligo Chaps. 11 and 12
consensus summarized in Chap. 2. The clini- associated disorders and comorbidities including
cal aspects and natural history are treated in autoimmune/inflammatory disorders, immuno-
more detail and illustrated. deficiencies, and rare monogenic diseases
Chap. 13; age and vitiligo, childhood, pregnancy,
and late-onset disease Chap. 14; skin colour
Chap. 15; and vitiligoid changes occurring dur-
ing immunotherapies of melanoma Chap. 16.
A. Taïeb (*)
Service de Dermatologie, Hôpital St André, CHU de
M. Picardo
Cutaneous Physiopathology and CIRM, San
Gallicano Dermatological Institute, IRCSS, Rome,
Italy
e-mail: mauro.picardo@ifo.gov.it

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Vitiligo/nonsegmental Vitiligo
Including Acrofacial
5
and Universalis
Thierry Passeron, Jean-Paul Ortonne,
Prasad Kumarasinghe, and Alain Taïeb
Contents
5.1 Introduction 42
5.2 Generalized Vitiligo 42
5.2.1 Distribution 43
5.2.2 Natural Course 44
5.2.3 Clinical Variants 44
5.3 Acrofacial Vitiligo 45
5.4 Vitiligo Universalis 45
5.4.1 Clinical Features 46
5.4.2 Precipitating Factors and Progression 46
5.4.3 Influence of Previous Treatments 48
5.4.4 Autoimmune Diseases Associated with Extensive Vitiligo and Application
to VU 48
5.4.5 Diagnosis 48
5.4.6 Management 49
5.5 Conclusions 50
References 50
Abstract
T. Passeron
Department of Dermatology, University Hospital Based on the VGICC classification, vitiligo/
of Nice, Nice, France NSV, a consensus umbrella term for all forms
e-mail: passeron@unice.fr of generalized vitiligo, is meant to describe
J.-P. Ortonne different clinical subtypes of vitiligo that are
Department of Dermatology, Archet-2 hospital, all clearly distinct from SV including acrofa-
Nice, France
cial, generalized, mucosal (multifocal), and
P. Kumarasinghe universal. According to the VETF definition,
Department of Dermatology, Royal Perth Hospital,
generalized vitiligo is characterized by asymp-
Perth, WA, Australia
tomatic well-circumscribed milky-white mac-
A. Taïeb (*)
ules involving both sides of the body with
Service de Dermatologie, Hôpital St André,
CHU de Bordeaux, Bordeaux, France usually a symmetrical pattern. In acrofacial
e-mail: alain.taieb@chu-bordeaux.fr vitiligo (AFV), the involved sites are by

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42 T. Passeron et al.
d efinition limited to the face, head, hands, and 5.1 Introduction

feet with typically depigmentation of the
distal fingers and facial orifices. Vitiligo Based on the VGICC classification [1], vitiligo/
universalisis a rapidly progressive form of the NSV, a consensus umbrella term for all forms
disease, which needs a more in-depth investi- of generalized vitiligo, is meant to describe dif-
gation especially for associated autoimmunity. ferent clinical subtypes of vitiligo that are all
clearly distinct from SV including acrofacial,
generalized, mucosal (multifocal), and univer-
sal (Chap. 1.2). Mixed vitiligo and mucosal vit-
iligo (more than one mucosal site) are also
Key Points
included under the vitiligo/NSV umbrella but
• The existence of various phenotypes
discussed separately (respectively, Chaps. 1.5.3
among vitiligo / NSV (e.g. acrofacial or
and 1.5.5). Generalized vitiligo (formerly vul-
vulgaris) is suggested by clinical obser-
garis) is the most common form of vitiligo/
vation and possibly by genetic
NSV corresponding to several achromic patches
associations.
symmetrically distributed on the body [2]. In
• Half of initially “focal” vitiligo patients
the largest series of patients (from China), it
evolve into vitiligo / NSV.
accounted for 41% of cases [3].
• The fingers, hands, and face are fre-
Although large clinicogenetic databases are
quently reported to be the initial sites by
currently lacking, different phenotypes of vitiligo
the patients.
may have different genetic backgrounds [4].
• Palms and soles are frequently involved
if a systematic Wood’s lamp examina-
tion is performed.
• Most of the ‘spontaneous’ repigmenta-
5.2 Generalized Vitiligo
tion reported by the patients is corre-
Generalized vitiligo is characterized by asymp-
lated to sun exposure.
tomatic well-circumscribed milky-white mac-
• Multichrome vitiligo refers to various
ules involving both sides of the body with
degrees of depigmentation within a vit-
usually a symmetrical pattern [5]. The shape of
iligo macule, a phenomenon noted in
individual macules is round to oval with slightly
dark skin.
brushed to fairly distinct margins (Fig. 5.1a).
• Vitiligo universalis is the most uncom-
Initial spot size varies from a few to several cen-
mon form of vitiligo and is probably the
timetres in diameter. The depigmentation is
extreme severity end of the spectrum of
macular, and the epidermis shows no sign of
vitiligo / NSV.
atrophy, telangiectasias, or any other signs.
• For vitiligo universalis, patients have to
The presence of epidermal atrophy should sug-
be informed that associated autoimmune
gest some other disorders, especially lichen
diseases may manifest months or years
sclerosus (Chap. 1.2). A transient erythema,
after being diagnosed with vitiligo.
which can be clinically misleading, is fre-
• For vitiligo universalis, sun protection is
quently observed after ultraviolet (UV) expo-
important to avoid sunburns and other
sure, but the history is usually contributive
sequelae.
(Fig. 5.1b). Hyperpigmented lesional borders
• For vitiligo universalis, depigmenting
are not uncommon, especially in dark skin
creams such as monobenzyl ether of
patients and after UV exposure. Hairs are usu-
hydroquinone or laser depigmentation
ally spared and remain pigmented, but in some
may be used to depigment small areas of
cases hair depigmentation may also occur
breakthrough pigmentation.
simultaneously. In the scalp, vitiligo usually
leads to localized patches of grey or white hairs,
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5 Vitiligo/nonsegmental Vitiligo Including Acrofacial and Universalis 43
a b
Fig. 5.1 Extensive generalized vitiligo with mostly symmetrical lesions in a child with fuzzy borders indicative of
progressive disease (a) and erythema in sun-exposed areas in an adult (b)
but total d epigmentation of the scalp hair may 5.2.1 Distribution

occur. Depigmented body hairs within vitiligo
macules are considered as markers of poor Generalized vitiligo can start at any site of the
repigmentation prognosis [6]. Follicular vitiligo body, but the fingers, hands, and face are fre-
has been recently delineated as a variant with quently reported to be the initial sites by the
initial and prominent hair depigmentation [7]. patients. It has been reported that when the
Vitiligo patches are easy to recognize on hands are the initial site, vitiligo most com-
darker phototypes, but depigmentation is some- monly progresses to the face, explaining the fre-
times difficult to detect in patients with very fair quency of acrofacial vitiligo in those patients
skin. Wood’s lamp or 365 nm monochromatic [8]. The same study suggests that when the pos-
lamp examination is very helpful to delineate the terior trunk, hands, or feet are the initial sites,
areas involved in light-coloured individuals and vitiligo tends to have a more widespread pro-
also to assess the remaining reservoir of melano- gression. The extensor surfaces are commonly
cytes. Even in darker skin individuals, palms and affected including interphalangeal joints, meta-
soles are light coloured and need to be examined carpal/metatarsal interphalangeal joints, elbows,
with Wood’s lamp. Systematic examination of and knees. Other surfaces involved include volar
palms and soles suggest that palmoplantar vitil- wrists, malleoli, umbilicus, lumbosacral area,
igo is not uncommon, like in psoriasis. Wood’s anterior tibia, and axillae. The role of the
lamp examination can also show the earliest Koebner phenomenon in the distribution pattern
signs of repigmentation at the border of the is discussed in Chap. 1.5.7. Most of the time, the
lesion or in perifollicular areas (Fig. 5.2). distribution of the lesions is clearly symmetri-
ERRNVPHGLFRVRUJ
a b
Fig. 5.2 Aspect of vitiligo lesion of the knee in Wood’s melanocyte reservoir; (b) same lesion during after photo-
lamp examination (a). Note the persistence of a grey pat- therapy. Note the perifollicular repigmentation
tern in the centre of the lesion, suggesting of a remaining
cal. Vitiligo macules may also be periorificial by another period of more rapid evolution.
and involve the skin around the eyes, nose, ears, Periods of rapid progression last often less than
mouth, and anus. Periungual involvement may a year, after which there is little extension or
occur alone or with certain cutaneous or muco- regression. The evolution to vitiligo universalis
sal surfaces (lip-tip vitiligo-, distal penis, nip- is rare (Sect. 5.4). The most common course is
ples). Vitiligo can affect genital areas and in one of gradual extension of existing macules and
some cases almost exclusively. This location periodic development of new ones. Vitiligo can
should be discussed with the patients because it repigment spontaneously, but this phenomenon
causes a great concern. is rare and mostly localized to some lesions.
Most of the ‘spontaneous’ repigmentation
reported by the patients is correlated to sun
5.2.2 Natural Course exposure.
The evolution of generalized vitiligo is unpre-

dictable. Focal vitiligo, although stable for a 5.2.3 Clinical Variants
time, may be a precursor of generalized vitiligo.
In a large series of vitiligo patients, about 5.2.3.1 Multichrome Vitiligo
three-quarters of the patients occurred as focal This form of vitiligo is mostly seen in darker pho-
vitiligo, but more than half of those patients totypes. Within a vitiligo lesion, areas of depig-
evolved to a generalized form [3]. The natural mentation coexist with hypopigmented areas and
course of common vitiligo is often one of abrupt with normal colour as in the surrounding skin
onset, followed by progression for a time; then a (Fig. 5.3). In the hypopigmented area, a partial
period of stability follows and may last for some loss of melanocyte is observed. Trichrome vitiligo
time, even decades. This may be followed later is commonly used to describe this pattern, but
ERRNVPHGLFRVRUJ
AFV. There are no series looking specifically at

the natural history of AFV. The association with
autoimmune thyroid disease seems less strong
when compared with generalized vitiligo [13].
The Northeastern Chinese series showed an
association with mucosal vitiligo [12]. This
association is also strongly suggested by the
study of Attili, which indicates that lichen scle-
rosus needs in addition to be also actively
researched in this context and could be under-
estimated because a thorough examination of
genital areas is frequently omitted [15]. Of 217
cases of AFV recorded over a period of 12 years
Fig. 5.3 Multichrome vitiligo of the leg by the authors, 116 (53%) had associated oral/
genital lesions. Among these, 15 patients
demonstrated typical clinical as well as histo-
various degrees of hypopigmentation can be logical features of lichen sclerosus. Interestingly
observed leading to trichrome, quadrichrome, or one female patient had cutaneous lichen sclero-
pentachrome vitiligo [9, 10]. Thus, the term “mul- sus guttata among acral lesions of vitiligo [15].
tichrome vitiligo” should be preferred.
5.2.3.2 Blue Vitiligo 5.4 Vitiligo Universalis

Postinflammatory hyperpigmentation leading to
a blue discolouration of vitiligo patches has been Acquired complete depigmentation or nearly
reported after inflammatory vitiligo [11]. complete depigmentation of the skin (and some-
time hairs) is termed vitiligo universalis (VU),
which is the most extensive form of vitiligo/
5.3 Acrofacial Vitiligo NSV. This form can start as common non-
segmental vitiligo (NSV) and advance to com-
In acrofacial vitiligo (AFV), the involved sites plete depigmentation of the skin and hair. True
are by definition (Chap. 2) limited to the face, segmental vitiligo (SV) does not progress to vit-
head, hands, and feet with typically depigmen- iligo universalis. Within the group of vitiligo/
tation of the distal fingers and facial orifices. NSV, it is not clear what factors trigger the cas-
Since it may later include other body sites, cade of complete or near-complete depigmenta-
resulting in typical generalized vitiligo, sub- tion of the integument in VU.
stantial variation in estimates of this subtype There appears to be some minor differences
exists in the literature, according to stage and in the prevalence of vitiligo among different
more or less strict application of the definition, populations, but no specific data is available on
in particular involvement of only acral sites VU [16]. VU is certainly the most uncommon
without periorificial involvement, or mixed clinical manifestation of vitiligo and appears to
forms with coexisting non-acrofacial lesions. occur worldwide. It is not clear whether VU
According to recent large series, 6.8% of has the same prevalence among fair-skinned
Chinese Han and non-Han [12], 5.8% of French Caucasians as compared to Asians or Africans,
[1], 25.6% of Brazilian [13], and 44% of Indian where it is easily recognizable. Although vitil-
[14] vitiligo patients received a diagnosis of igo may occur at any age, VU is uncommon in
ERRNVPHGLFRVRUJ
childhood. The population prevalence of VU is

not known. In a study by Song et al. [17], it has
been reported that there was only one case of
VU among 1315 vitiligo patients. In a retro-
spective study we carried out at the National
Skin Centre, Singapore [18], 1.4% (4 patients
out of 282 vitiligo patients) had vitiligo univer-
salis. Male to female ratio of vitiligo universalis
appears to be the same. More data are needed to
confirm this observation.
5.4.1 Clinical Features
In VU, nearly all the skin becomes completely

depigmented. However, in sun-exposed areas
there can be minor perifollicular, discrete, or
coalescent pigmentation (Figs. 5.4, 5.5, 5.6, and
5.7). Areolae and genitalia may depigment either
early in the disease process or later. Some VU
patients may show areas of partial depigmenta-
tion, appearing clinically as trichrome vitiligo.
However, these apparently resisting areas may
also lose colour and become uniformly
depigmented. Fig. 5.4 A Singaporean woman of Indian origin with vit-
iligo universalis and breakthrough repigmentation on
Some patients retain the dark hairs on the exposed areas (Photograph, courtesy, National Skin
scalp at least in the early course of disease. Centre, Singapore)
Pubic and axillary hairs may or may not be
depigmented. Body hairs often get depigmented
in affected areas. Generally, in established VU, 5.4.2 Precipitating Factors
the hairs in the depigmented skin also get and Progression
depigmented.
Iris pigmentation is usually unaffected, but It is uncertain what factors (genetic or environ-
exceptions exist. In some VU patients, associated mental) precipitate common NSV to progress to
uveitis may even lead to blindness. Mucosae, VU. Often the progression is symmetrical. There
particularly the lips, gums, and genitalia too, may is no evidence that vitiligo universalis increases
be partially or completely depigmented [19]. the risk of another vitiligo universalis case among
According to a Turkish study [20], minor family members. No sufficient genetic data is
hearing defects are not uncommon in NSV available which would help determining a risk of
patients. These authors have reported that 14% vitiligo universalis in a new case of NSV in a
of NSV patients have at least mild hyperacusis. vitiligo family.
Eye and ear symptoms in extensive vitiligo Extensive NSV can evolve into VU. Sometimes
patients are likely to be due to the loss of mela- this evolution can be rather rapid after a period of
nocytes in these organs. This is a prominent apparent stability (personal observation).
feature in Vogt-Koyanagi-Harada syndrome Depigmentation may spread from the edges of
(Chaps. 8 and 10). existing lesion or by occurrence of new macules.
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Fig. 5.7 An Indian woman with vitiligo universalis and

Fig. 5.5 A 65-year-old Indian male with vitiligo univer-
photo-induced partial repigmentation, with a squamous
salis and breakthrough repigmentation in a lupus pattern
cell carcinoma on the cheek. Carcinomas on vitiliginous
(Photograph courtesy of Prof Binod Khaitan, All India
skin are rare although sunburns are common (Photograph
Institute of Medical Science, New Delhi, India)
courtesy of Prof Binod Khaitan, All India Institute of
Medical Science, New Delhi, India)
Occasionally before complete depigmentation,

surrounding borders of the pigmented skin may
show signs of inflammation, such as erythema.
After the inflammation recedes, these areas also
become depigmented. In one case of vitiligo (per-
sonal observation PK), a patient with limited, sta-
ble generalized vitiligo rapidly progressed to
vitiligo universalis after an episode of diarrhoea
and vomiting, following food poisoning. Similarly,
pregnancy, physical injury, malignant melanoma,
and emotional stress have been incriminated as
‘triggers’ for progression of vitiligo. But it is not
clear whether these associations are true associa-
tions or chance occurrences [19]. Occasionally,
Fig. 5.6 A 58-year-old Sri Lankan woman with vitiligo
universalis with an acute sunburn on the shoulders after chemical leukoderma, which starts focally, can
exposure to strong sun while working in a paddy field spread all over the body. Following the topical use
ERRNVPHGLFRVRUJ
of diphencyprone for extensive alopecia areata, tis, but it is uncommon. Scalp hair, as well as
extensive vitiligo has been reported to appear in beard or moustache ones, may have been dyed.
areas beyond the contact areas [21]. Perifollicular Contact dermatitis due to PPD (p-phenylenedi-
breakthrough pigmentation on sun-exposed areas, amine) containing hair dyes is not uncommon.
especially the upper cheeks and exposed areas of
forearms, is not uncommon (Figs. 5.4 and 5.5).
Pigmentation around nipples may be lost or 5.4.4 Autoimmune Diseases
retained. The author (PK) has observed several Associated with Extensive
cases of localized repigmentation in VU cases, Vitiligo and Application to VU
after a secondary event, such as lichen planus or
insect bite reactions. Dogra et al. have reported Many autoimmune/autoinflammatory disorders
partial repigmentation of vitiligo universalis in a and several other conditions have been reported
patient who was given dexamethasone and cyclo- in association with vitiligo (Chap. 13). These
phosphamide pulse therapy for pemphigus vul- conditions may be found in patients with VU as
garis [22]. This coincidental finding addresses an well. These patients may have a family history of
important aspect. Indeed, even in established VU, other autoimmune disorders, as well as a family
inactive but viable melanocytes are probably pres- history of NSV.
ent and can be reactivated by adequate signals of Some patients with Vogt-Koyanagi-Harada
the cellular environment. This confirms previous syndrome may also evolve into VU, and the lim-
studies, where some viable melanocytes were its between the two conditions are somewhat
found within established vitiliginous macules [23, blurred. In this condition, visual defects, alope-
24]. Most of these melanocytes or precursors of cia, and auditory and CNS symptoms may pre-
melanocytes appear to be associated with hair fol- cede skin depigmentation. Often they present to
licles. This may be a reason why the perifollicular the neurologist or ophthalmologist first, due to
pigmentation is the most common type of break- eye or meningeal symptoms.
through repigmentation in VU. In VU, other autoimmune disorders may evolve
after the onset of vitiligo; therefore these patients
have to be followed up with this in mind. In the
5.4.3 Influence of Previous author’s experience, autoimmune thyroiditis and
Treatments alopecia areata are the commonest associations.
When a patient with VU presents to a given der-

matologist, he or she may have already tried 5.4.5 Diagnosis
several medications. There may be skin atrophy
and striae due to prolonged use of potent topical Usually there is no diagnostic difficulty in vitil-
steroids. Monobenzyl ether of hydroquinone igo universalis. Diagnosis can be made based on
(MBEHQ) may have been used to depigment the the history and the clinical features. Skin biopsy
residual pigmented spots (Chap. 37). This may is not required unless for a research purpose. In
sometimes cause a contact dermatitis. some situations, some forms of oculocutaneous
Breakthrough pigmentation may be a problem if albinism may be confused with vitiligo, espe-
proper sun protection has not been adhered to. cially if the onset of disease or history is not
The skin may appear yellowish if high doses of known. Nystagmus and photophobia are not
oral beta-carotene have been taken. Some may associations of vitiligo, but of oculocutaneous
have applied artificial tanning substances (dyes) albinism. A skin biopsy is helpful in this setting
on the skin (Chap. 39). A few of these tanning to show the presence of non-functioning melano-
substances may cause allergic contact dermati- cytes in albinism.
ERRNVPHGLFRVRUJ
5.4.6 Management Q-switched alexandrite lasers have proven to be

useful in destroying residual pigmented macules
In simple NSV detailed investigations are not in patients where topical bleaching agents have
essential, but in VU several investigations are failed to depigment (Chap. 3.2.3).
recommended (Table 5.1). Many patients with Sun protection is important to avoid sunburns
extensive vitiligo have serum antibodies against as well as to prevent unwanted breakthrough,
melanocytes [25], as well as antithyroid, anti- spotty repigmentation. Some studies have revealed
parietal cell, and anti-nuclear ones. Anti- that wild-type p53 gene is upregulated in vitiligo
melanocyte antibodies are not found in all cases patients [27]. This may have a protective effect
of vitiligo, and these antibodies may be found in with regard to sun-induced skin malignancy.
some other conditions as well (e.g. mucocutane- Tumours, such as squamous cell carcinomas, on
ous candidiasis). Therefore, this test is not indi- vitiliginous skin are very uncommon in dark-
cated as a routine test. skinned individuals. However, it can occur rarely.
Once vitiligo universalis has fully developed, Furthermore, as melanomas are a known associa-
it is unrealistic to expect complete repigmentation of vitiligoid depigmentation, if a suspicious
tion. Management of VU patients is mostly new pigmented lesion appears, particularly in
directed at treating the residual patches of the association with a pre-existing nevus, it should be
pigmented skin, preventing breakthrough pig- excised and sent for histological evaluation.
mentation and protecting from sun damage while Vitiligo universalis causes a huge psychologi-
addressing camouflage issues and psychosocial cal impact in dark-skinned individuals, and some
aspects (Table 5.2). Counselling is important patients may show signs of depression at the time
before implementing complete depigmentation of presentation [28]. In certain communities and
procedures [17, 26]. Occasionally, some patients cultures, VU would be even more problematic,
are keen to attempt to retain pigmentation in patients being ostracized with regard to marriage
some areas, notwithstanding extensive depig- and social contacts. Counselling and sometimes
mentation in other areas. Q-switched ruby and even psychiatric help may be needed, depending
on emotional impact, particularly in dark-skinned
Table 5.1 Recommended investigations in vitiligo individuals (Chaps. 15 and 42). Counselling the
universalis spouse and the family members may also be nec-
Basic haematology Full cell blood count essary in some situations. Occasionally, even in
Fasting blood sugar the fair-skinned individuals, vitiligo may cause
Endocrinology Free thyroxine major psychological problems, because sun-
TSH
induced pigmentation occurs only in the unaf-
PTH
Immunology Antithyroid antibodies
fected areas. After understanding the disease
Antinuclear factor process and after realization that no treatment is
Others Ophthalmological tests 100% effective in VU, some patients accept it
Audiological tests and continue with their lives well.
Table 5.2 Management of residual pigmented areas and avoidance of breakthrough pigmentation
Chemical Physical
depigmentation depigmentation Camouflage Others
MBEHQ Q-switched ruby Oral beta-carotene Counselling patient and/or family
laser
Alexandrite laser Topical dyes for the skin Psychiatric help
NdYag laser Topical dyes for scalp hair Sun protection
and eyebrows
Surveillance versus autoimmune and
neoplastic diseases
ERRNVPHGLFRVRUJ
5.5 Conclusions 9. Fargnoli MC, Bolognia JL. Pentachrome vitiligo. J

Am Acad Dermatol. 1995;33:853–6.
10. Sehgal VN, Srivastava G. Vitiligo: compendium of
VU is probably the extreme severity end of the clinico-epidemiological features. Indian J Dermatol
spectrum of NSV, and differentiation from VKH Venereol Leprol. 2007;73:149–56.
syndrome is not always easy. Genetic studies of 11. Ivker R, Goldaber M, Buchness MR. Blue vitiligo. J
families with vitiligo universalis in different eth- 12. Mchepange UO, Gao XH, Liu YY, Liu YB, Ma L,
nic groups would be helpful. Future research Zhang L, Chen HD. Vitiligo in North-Eastern China:
should focus on identifying and modifying the an association between mucosal and acrofacial
triggering factors which start the cascade of lesions. Acta Derm Venereol. 2010;90:136–40.
13. Silva de Castro CC, do Nascimento LM, Olandoski
depigmentation process all over the skin. M, Mira MT. A pattern of association between clini-
cal form of vitiligo and disease-related variables in a
Acknowledgements Dr. Prasad Kumarasinghe is grate- Brazilian population. J Dermatol Sci. 2012;65:63–7.
ful to Prof. Roy Chan, Medical Director, and Dr. Goh 14. Agarwal S, Ojha A, Gupta S. Profile of vit-

Boon Kee, Consultant Dermatologist, National Skin iligo in Kumaun region of Uttarakhand, India.
Centre, Singapore, for providing some of the VU Indian J Dermatol. 2014;59:209. https://doi.
photographs. org/10.4103/0019-5154.127706.
15. Attili VR, Attili SK. Acral vitiligo and lichen scle-
rosus - association or a distinct pattern?: a clinical
and histopathological review of 15 cases. Indian J
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indicates atopic diathesis association in pre-pubertal vitiligo: comparative study of Type A and Type B vit-
onset vitiligo. Br J Dermatol. 2012;167:490–5. iligo. Ann Dermatol. 1994;6:22–30.
2. Le Poole C. Vitiligo vulgaris. In: Nordlund J, edi- 18. Tan WP, Goh BK, Tee SI, Kumarasinghe SPW. Clinical
tor. The pigmentary system. Malden, MA: Blackwell Profile of vitiligo in Singapore. 2nd Meeting of
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3. Liu JB, Li M, Yang S, et al. Clinical profiles of vit- 2007, Singapore. Pigment Cell Res. 2007;20:253..
iligo in China: an analysis of 3742 patients. Clin Exp (abstract).
Dermatol. 2005;30:327–31. 19. Hann SK, Nordlund JJ. Clinical features of general-
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genetic epidemiology and genetic models for vitiligo. Vitiligo. Oxford: Blackwell; 2000. p. 81–8.
J Am Acad Dermatol. 2004;51:383–90. 20. Aydogan K, Turan OF, Onart S, et al. Audiological
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other hypomelanoses of hair and skin. In: Parrish Dermatol. 2006;31:110–3.
JA, Fitzpatrick TB, editors. Monographs in topics in 21.
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dermatology. New York, NY: Plenum Press; 1983. SPW. Vitiligo as a reaction to topical treatment with
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Seneschal J, Ezzedine K. Follicular vitiligo: a report 23. Husain I, Vijayan E, Ramaiah A, et al. Demonstration
of 8 cases. J Am Acad Dermatol. 2016;74:1178–84. of tyrosinase in the vitiligo skin of human beings by
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mental vitiligo: clinical analysis of 318 patients. Int J L tyrosine incorporation into melanin. J Invest
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ERRNVPHGLFRVRUJ
Segmental Vitiligo
6
Seung-Kyung Hann, Hsin-Su Yu, Cheng-
Che Eric Lan, Ching-Shuang Wu, Yvon Gauthier,
Laïla Benzekri, and Alain Taïeb
Contents
6.1 Historical Background and Controversies on Patterning 54
6.2 Clinical and Pathological Features 58
6.3 Preferential Melanocytic Follicular Reservoir Involvement in SV 60
6.4 Classification 61
6.4.1 Classification of Cephalic SV 61
6.4.2 Classification of SV of the Trunk 62
6.5 Diagnosis, Course, and Special Locations 62
6.6 Repigmentation in Segmental Vitiligo and Phototherapies 63
6.7 Treatment Overview and Perspectives 68
References 69
S.-K. Hann Abstract

Korea Institute of Vitiligo Research, Drs. Woo & Segmental vitiligo (SV) has been historically
Hann’s skin clinic, Seoul, South Korea
opposed to vitiligo/nonsegmental vitiligo
e-mail: skhann@paran.com
(NSV). However, evidence that the two forms
H.-S. Yu · C.-C. E. Lan
are not mutually exclusive (delineation of
Department of Dermatology, Kaohsiung Medical
University, Kaohsiung, Taiwan mixed vitiligo) and share similar inflamma-
e-mail: dermyu@kmu.edu.tw; laneric@kmu.edu.tw tory features has challenged classical views
C.-S. Wu over the last decade. SV belongs to the clinical
Faculty of Biomedical Laboratory Science, spectrum of vitiligo, including inflammatory
Kaohsiung Medical University, Kaohsiung, Taiwan skin features. SV has usually an early onset
e-mail: m785034@kmu.edu.tw
and spreads rapidly in the affected segment.
Y. Gauthier · A. Taïeb (*) The hair follicle melanocytic compartment is
Service de Dermatologie, Hôpital Saint-André, CHU
frequently involved. Distribution in SV paral-
de Bordeaux, Bordeaux, France
e-mail: alain.taieb@chu-bordeaux.fr lels that of other acquired pigmentation disor-
ders such as nevus spilus, pointing out to some
L. Benzekri
Department of Dermatology, CHU Ibn Sina, underlying developmental defect. Neurogenic
Rabat, Morocco influences have been highlighted by striking

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54 S.-K. Hann et al.
clinical observations and experimental stud- mental type (type A) and segmental type (type
ies. Classification of SV is needed to establish B). He proposed that the nonsegmental type
a prognosis, and two recent classifications for results from immunologic mechanisms, while
cephalic and truncal SV have been proposed. segmental type results from dysfunction of the
Segmental vitiligo is a perfect model for sympathetic nervous system in the affected skin
studying repigmentation since it is a relative and less prone to Koebner’s phenomenon (Koga
stable disease with limited active melanocyte et al. 1977).
loss. Early aggressive medical treatment of The controversy on the separation of SV from
SV is currently recommended. vitiligo/NSV has been fuelled by the interpreta-
tion of SV patterning, with two major options,
namely, dermatomal/neural vs. blaschkolinear/
Key Points developmental.
• SV belongs to the clinical spectrum of
vitiligo, including inflammatory skin • For the dermatomal/neural hypothesis, it has
features. been considered that SV follows partially or
• SV has usually an early onset and completely one or more dermatomes, similar
spreads rapidly in the affected segment. to that commonly observed in herpes zoster for
• The hair follicle melanocytic compart- the trigeminal nerve dermatome [1–7]
ment is frequently involved. (Fig. 6.1a, b). However, in 64% of cases, depig-
• Distribution in SV parallels that of other mented macules do not follow a true dermato-
acquired pigmentation disorders, such mal pattern. The depigmentation involves
as nevus spilus, pointing out to some either partially each dermatome or overlaps
underlying developmental defect. two or three dermatomes especially when SV
• Classification of SV is needed to estab- is located on the face. Many clinical observa-
lish a prognosis. tions of leukoderma have been reported in
• Segmental vitiligo is a perfect model for areas corresponding to local neurologic dam-
studying repigmentation since it is a age such as unilateral vitiligo, and poliosis
relative stable disease with limited located on the area innervated by both trigemi-
active melanocyte loss. nal and upper cervical nerves was reported in a
• Early aggressive medical treatment of child with viral encephalitis [8]; ipsilateral
SV is currently recommended. macules of vitiligo at the level of the upper
arm, chest, buttocks, and sacrum were observed
in a 31-year-old patient following trauma of the
right brachial plexus [9], grayer hair on the left
6.1 Historical Background than on the right side after right cervical sym-
and Controversies pathectomy [10–12], and localized depigmen-
on Patterning tations similar to SV in patients with a spinal
cord tumor or following nerve injury [13, 14].
Segmental vitiligo (SV) has been historically • In addition to Koga’s experiments, an abnor-
opposed to vitiligo/nonsegmental vitiligo (NSV). mal effect of neurohormones and neuropep-
However, evidence that the two forms are not tides has been speculated to explain the
mutually exclusive (Chap. 7) and share similar chronic melanocyte loss in generalized vitil-
inflammatory features has challenged classical igo [10]. Electron microscopy has established
views over the last decade. that direct contact occurs between intraepider-
In 1977, Koga performed a sweat secretion mal nerve endings and melanocytes [15].
stimulation test using physostigmine and Dystrophic changes in nerve trunks and nerve
accordingly reclassified vitiligo into nonseg- endings, as well as degenerative and regenera-
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6 Segmental Vitiligo 55
a b
Fig. 6.1 Similar segmental distribution of SV and herpes zoster. SV corresponding strictly to V2 dermatome (a), her-
pes zoster with the same distribution (used with permission of JJ Morand) (b)
tive changes in terminal cutaneous nerves, tion has been followed by the development of
have been found in vitiligo [15, 16]. Using melanin pigment aggregation.
antibodies against neuropeptide Y and calcito- • Embryologically, melanocytes have the
nin gene-related peptide (CGRP), several same origin as neural cells. They can react
authors have been able to show an increased to many compounds that affect the nervous
expression of these peptides in lesional and system. In frogs, acetylcholine, norepineph-
perilesional skin and frequently an increased rine, epinephrine, and melatonin lighten
number of CGRP-positive nerve fibers in dermal melanocytes [19]. In guinea pig skin
involved skin [17]. Abnormalities in neuro- [20], the spread of grafted melanocytes in
peptides or other neurochemical mediators denervated skin is statistically better than in
secreted by nerve endings could theoretically nonoperated control sites. This could sug-
harm nearby melanocytes. The existence of a gest that peripheral nerves could play a role
dermal adrenergic innervation to melano- in the regulation of melanocyte function
phores of teleost fish has been demonstrated. especially for migration. A single subcuta-
Electrical stimulation of nerve fibers of the neous injection of epinephrine into rats
skin causes lightening in the European turbot causes local depigmentation of the hairs,
[18]. Application of epinephrine similarly but the interpretation of this phenomenon is
leads to aggregation of melanin within mela- not straightforward, since it seems likely
nophores in some fishes accompanied by that vasoconstriction can induce ischemic
lightening of skin color. Sympathetic denerva- depigmentation.
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• In patients with vitiligo/NSV, local abnormal- gin (like melanocytes) [36], but several
ities related to possible neural-mediated aber- observations of purely dermal diseases fol-
rations have been reported. However, data are lowing this pattern mitigate this view [37].
discordant. An increased adrenergic nerve- The two types of lines (thin or large, see
ending activity in vitiligo macules was sug- Fig. 6.2a, b) may somewhat correspond to the
gested by Lerner [21]. Other authors came to underlying cellular origin of somatic mosa-
the conclusion that there was a dominant cho- icism, dermal disorders tending to form broad
linergic influence in vitiliginous skin com- bands [38]. Other types of patterns which do
pared to normal skin [22, 23]. Koga’s not fit Blaschko’s lines (checkerboard, phyl-
classification proposes that SV could result loid, garment-type) are also observed in the
from the dysfunction of sympathetic nerves dermatology clinic [39] and may concern the
in the affected area. On the basis of physo- melanocytic system or other types of cells.
stigmine-induced sweating response, a Some blaschkolinear distribution patterns in
decreased responsiveness of SV was inter- SV are strikingly similar to that of epidermal
preted to be due to an increased cholinergic nevi [40]. Mosaicism may sometimes also
influence [24–26]. In SV lesions, the cutane- involve germline cell mutations, a fact which
ous blood flow was shown to be increased explains the coexistence of segmental and
almost threefold compared to control normal generalized patterns in some rare pedigrees
skin, and a similar anomaly was shown to a [41]. The dermatomal distribution has been
lesser extent in vitiligo/NSV. A significant historically considered as reflecting better the
increase in cutaneous alpha- and beta-adreno- distribution of SV and the underlying neural
receptor response was found in the segmental theory, but cases intersecting dermatomes
type suggesting that a dysfunction of the without “filling” their theoretical territory of
sympathetic nerves could exist [26–31]. As distribution were difficult to relate to this
noted before, the catecholamine stimulation etiologic background. The sympathetic
of adrenoreceptors may cause severe vaso- anomalies noted in favor of the neurogenic
constriction. It is thus possible that hypoxia- theory of SV may also be a confounding fac-
induced oxidative stress at the epidermal tor related to the absence of melanocytes
level could lead to partial or total depigmen- which have been considered as “neurons of
tation of one or several dermatomes. However the skin” [42] and can release several neuro-
no changes in plasma catecholamine level or mediators. The large blaschkolinear pattern,
adrenoreceptor densities on blood cells have when isolated on a limb, can mimic a derma-
been so far reported [32]. tomal distribution (Fig. 6.2c, d). On the face,
• For the blaschkolinear/developmental the lines of Blaschko have been drawn more
hypothesis, the lines described by Alfred recently [43], and some cases of SV fit clearly
Blaschko in 1901 (Blaschko’s lines, BL) better Blaschko’s bands than dermatomal ter-
were based on drawings of cutaneous nevoid ritories [38, 40] (Fig. 6.3). It has also been
lesions [33]. They have been revisited exten- suggested that segmental vitiligo might fol-
sively by Happle [34] and considered to follow acupuncture lines [44]. Another possibil-
low the dorsoventral development of cellular ity is that the distribution of segmental
components of the skin. When visible, BL vitiligo follows a so far unknown develop-
reflect an underlying somatic mosaicism mental pathway corresponding to a group of
demonstrated first in monogenic keratin dis- identical clonal cells. Differing characteris-
orders [35]. BL have been postulated to cor- tics of melanocyte survival and melanogene-
respond to a migration pattern restricted to sis may influence the location/course of the
cells of ectodermal or neuroectodermal ori- lesions. In Fig. 6.4a, the 14-year-old male
ERRNVPHGLFRVRUJ
a b
c d
Fig. 6.2 Blaschko’s bands vs. Blaschko’s lines. lare (b) (reported by Morice-Picard et al. [37]); (c) shows
Hypomelanosis of Ito (a) demonstrates nicely the thin same patient as in (b); note the difficulty to relate the pat-
blaschkolinear hypopigmented streaks originally tern to Blaschko’s bands (compare to d); segmental vitil-
described by Alfred Blaschko (courtesy of Dr Odile igo of the thigh considered as following a dermatomal
Enjolras) and large band blaschkolinear granuloma annu- distribution (d)
patient had vitiligo around the left eye, while in different diseases—segmental vitiligo
nevus spilus occurred in a symmetrical fash- (Fig. 6.4b) and nevus spilus (Fig. 6.4c). These
ion. Both conditions appeared almost simul- findings suggest that clones of melanocytes
taneously several months before, suggesting with different functional characteristics
a variant twin-spotting phenomenon [45]. exist in acquired pigmentary disorders.
The same cutaneous pattern may also occur Melanocytes in nevus spilus can produce
ERRNVPHGLFRVRUJ
Fig. 6.3 Facial distribution of some cases of SV fits better Blaschko’s lines than dermatomes. Two examples with the
corresponding summary of lines by Happle and Assim, [43], From Taieb et al, [38], with permission
more melanin than normal and may have a Chap. 32 for a more in-depth review of patho-
normal lifespan. The type of melanocytes physiology of SV and a modern convergent
found in segmental vitiligo of the same loca- theory for SV).
tion can be easily lost by an unknown mecha-
nism. The exact nature of this phenomenon
has not been fully elucidated but might reflect 6.2 Clinical and Pathological
the embryonic migration pathway of a mela- Features
nocyte colony with an inherited defect, which
is not readily observable at birth or in the first Koga and Tango reported that SV affects more
months of life, thus suggesting an associated commonly the young and indicated a higher
environmental trigger. (27.9%) incidence in children SV develops before
• The hypothesis of cutaneous mosaicism in SV 30 years of age in 87.0% of cases and 41.3%
favoring underlying developmental defect before 10 years of age. The earliest reported onset
does not rule out other triggering factors, was immediately after birth, whereas the latest
especially neurogenic ones. It does not also was 54 years [3].
exclude the possibility of an inflammatory The typical lesion is not that different from
component extending locally beyond the area the macule observed in nonsegmental vitiligo.
demarcated by the developmental lines, which The most common form is a totally amelanotic
has received recent confirmation [46–48] (see macule surrounded by the normal skin. The color
ERRNVPHGLFRVRUJ
a b
Fig. 6.4 (a) Segmental vitiligo and nevus spilus coexist symmetrically; same facial pattern in segmental vitiligo (b)
and nevus spilus (c) in two different patients better to have b and c on the same horizontal level for comparison
of the macule is usually pure white or chalk involving several dermatomes occurs, and
white. However, as in vitiligo/NSV, a multi- lesions may sometimes cross the midline
chrome variation of hypopigmentation can be (Fig. 6.5). In the majority of cases, depigmenta-
observed, and overall a less uniform depigmen- tion spreads within the segment in a short period
tation pattern was seen in SV compared to vitil- of time and then stops. It leaves the skin segment
igo/NSV when a decision was needed for grading partially or totally depigmented. It is rare for a
a patch [49]. In some cases, such as on fair skin, patient with SV to progress to the generalized
the lesions are not easy to see under normal light form. Such a phenomenon is now best recog-
but can be distinguishable with Wood’s light nized as the association of SV and NSV called
examination. A dermatomal or pseudo dermato- “mixed vitiligo” [Chap. 1.5.3]. In this case the
mal distribution has been suggested and fuelled increased severity of SV versus vitiligo/NSV in
the neural theory of SV, but common overlap response to therapy suggests that the dosing of
ERRNVPHGLFRVRUJ
a b
Fig. 6.5 Typical aspect of SV I-b of the face with poliosis of the eyebrow/eyelids crossing either slightly (a) or more
markedly (b) the midline
the predisposing skin anomaly is augmented in

the SV area (Chap. 7).
The head is involved in more than 50% of
cases. In decreasing order of frequency, the trunk,
the limbs (Fig. 6.6), the extremities, and the neck
are common sites of involvement. In females, the
neck is more frequently involved than the extrem-
ities. Lerner [10] reported that segmental vitiligo
occurs as a single lesion in 75% of patients, a
finding confirmed by Hann and Lee [50] who
found that 87% of patients had a single lesion and
Fig. 6.6 SV of the left shoulder and upper limb
more recently by Van Geel et al., who found 88%
unilateral SV, 2% bilateral, and 15% mixed vitil-
igo cases in their series [51]. other diseases. Similarly, Hann and Lee [50]
There is usually no preferential distribution reported 6.7% of patients with associated disease
between right and left sides of the body. Some either allergic or autoimmune. Looking at associ-
patients have lesions in two different unilateral ated features to SV and vitiligo/NSV, including
cutaneous segments. The most commonly involved those pertaining to autoimmune diathesis, the
is that sharing in part the distribution of the trigem- global impression is more a continuum than two
inal nerve, a finding which was given as a major completely separate diseases [54]. Histopathology
support of the neural theory of SV. Recent data of recent progressing SV confirms the existence
indicate that no clinically recognizable pattern— of infiltrating T cells [47] (Shin et al. 2016) - see
non-dermatomal and non-blaschkoid—is common also Chap. 32 [54].
(77% of cases) and that facial involvement for SV
is usually of later onset than extrafacial involve-
ment (median 19 years vs. 6.5 years) [51]. 6.3 Preferential Melanocytic
A family history of vitiligo is present in Follicular Reservoir
approximately 12% of SV cases [51]. El Mofty & Involvement in SV
El Mofty [52] and Koga [1] suggested that SV is
not significantly associated with other autoim- Body leukotrichia varies from 10% to more than
mune disease. However, Park et al. [53] showed 60% in the literature for all types of vitiligo
that about 9.5% of SV cases were associated with (Chap. 9). It is striking that the follicular com-
ERRNVPHGLFRVRUJ
partment of the melanocyte organ may be spared, This particular tropism for hair melanocytes
while vitiligo affects the epidermal compart- has therapeutic consequences. Repigmentation of
ment. This dissociated behavior of epidermal and hair is difficult to address with UV and other
follicular melanocytes is very common in vitil- medical treatments. Transplantation of melano-
igo/NSV. NSV affects both epidermal and fol- cytes is required for rapid and complete repig-
licular melanocytes in its early phase of disease mentation (Chap. 36).
as compared to vitiligo/NSV (Fig. 6.5, simulta-
neous involvement of the skin and eyelids/
lashes). 6.4 Classification
Depigmentation of hairs (poliosis, leuko-
trichia) occurs more specifically in vitiliginous Knowledge about the exact spreading pattern and
SV macules. Poliosis has been shown to occur prognosis is important for both patients and doc-
in around half of cases of SV. If cases of body tors. So far cephalic and truncal distribution of
involvement of SV (for which this item is less SV have been systematically studied.
accurately documented) had been excluded, the
incidence of leukotrichia-associated SV would
be even higher. Involvement is variable; a few to 6.4.1 Classification of Cephalic SV
many hairs of a single macule may be depig-
mented. The eyebrow and eyelashes are com- Based on a large (257 unambiguous cases) and
monly involved in SV of the upper face. Other careful Korean study, the distribution of segmen-
involved hairy sites include the scalp, pubis, and tal vitiligo on the face is classified into six sub-
axillae. types (Fig. 6.7) [55].
Fig. 6.7 Hann and

coworkers revised
I II III
classification of
segmental vitiligo of the
face
IV V
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Typical lesions of type I-a, the most common six patterns: (1) midline (3.8%), (2) shoulder
type (28.8%), start on one side of the middle area of (3.8%), (3) V-shape high on the thorax (2.6%),
the forehead, cross the midline of the face around (4) V-shape low on the thorax 7 (6.6%), (5) band-
the glabella, and extend downward and laterally to like (17.9%), and (6) checkerboard (13.2%);
the eyelid, nose, philtrum, and cheek of the contra- 32.1% of cases could not be classified (Fig. 6.8).
lateral side of the face without involving scalp hair. Type 1 is a mid-central linear subtype that seems
Spread to the neck can be seen rarely. Surprisingly, to be an extension of type III SV pattern on the
the left side of the face is more frequently involved face (previous section). It can cross the midline
(89% of cases). In type I-b (10.5%), lesions appear of the trunk.
on the right or left side of the forehead with fre- Types 2, 3, and 4 can extend to the ipsilateral
quent involvement of scalp hair, with rare spread arm, while type 6 may affect the upper leg. Type
down to the periorbital area and eyebrows. 3 features a V shape to the lower midline, compa-
In type II (16%), the lesions begin at the angle rable with the down movement of the Blaschko’s
of the mouth and then arch to the auricular area lines toward the ventral part of the trunk. A simi-
across the cheek or mandible. The degrees of cur- lar bowlike V pattern can sometimes be observed
vature and lesion widths vary widely, and involve- on the back, although in our cases this was always
ment of the upper neck area is common. associated with an accompanying V-shaped pat-
In type III (14.4%), lesions begin below the tern on the ventral side of the trunk. Type 5 has a
lower lip and spread down to include the chin and more horizontal band-shape distribution, mostly
neck. Unlike type II, type III lesions usually overlapping the lateral side of the trunk. Type 6
involve medial rather than lateral aspects of the is located on the lower part of the trunk under the
neck and rarely the upper chest. waist, similar to the checkerboard pattern
In type IV (10.9%), all the lesions originate on described by Happle [57]. In half of the cases, a
the right side of the forehead and extend to the eye- combination of SV on both the front and back of
lids, nose, and cheek areas without crossing the the lower trunk was found, but the lateral side
midline at the forehead and glabella, unlike type I-a. was spared in most instances.
In type V (8.6%), lesions are confined to the In summary, truncal SV is frequently located
right orbital area, although they could spread at the ventral or ventral-lateral side of the trunk
contiguously to the temporal area. and rarely exclusively on the back. In contrast to
For mixed types (10.9%), the coincidence of the interpretation of SV patterning of the face
type II and type III (15 cases) is the most com- [55], there is no left or right preferred distribution
mon combination. of certain subtypes. However, lesions of the left
A suggestion to simplify this classification at side of the trunk are observed in a younger age
the international level would be to combine in one group.
type I-a and IV on the basis that crossing the mid-
line is common in other developmental somatic
mosaics affecting pigmentation, which would 6.5 Diagnosis, Course,
give four basic patterns for cephalic vitiligo. and Special Locations
Another item to study further is the distribution of
lesions in the lateral and posterior cephalic area, Most often SV patches remain unchanged for the
which is so far not taken sufficiently into account. rest of the patient’s life after rapid initial spread-
ing in the affected segment [50]. However, rarely
it can progress again after being quiescent for
6.4.2 Classification of SV several years (Fig. 6.9). When segmental vitiligo
of the Trunk progresses, it usually spreads over the predicted
segment. Early SV most often appears as a soli-
The data were collected by the group of Ghent in tary oval-shaped white macule or as a patch that
106 patients [56] and are summarized thereafter. is difficult to differentiate from “focal” vitiligo
The distribution on the trunk was classified into until proven by a subsequent typical distribution
ERRNVPHGLFRVRUJ
Type 1 Type 2 Type 3
Type 4 Type 5 Type 6
Fig. 6.8 Van Geel and coworkers classification of segmental vitiligo of the trunk
ally, following the same contralateral (or differ-

ent) dermatomes, it may cause difficulties in
defining vitiligo type (Fig. 6.11). It may at some
point be confused with NSV or, for some loca-
tions, such as white patches of both legs, to pie-
baldism (Chap. 2). Lee and Hann [58] reported
that 5 out of 240 patients, who had SV, exhibited
two different depigmented segments on the same
or opposite site of the body. The clinical course
of bilateral SV seems to be the same as unilateral
SV.
Fig. 6.9 Recurrence of vitiliginous lesions at the periph-
ery of an autologously grafted SV site. The graft was done
10 years before the recurrence (corresponds to Hann’s 6.6 Repigmentation
type III, see Fig. 6.7)
in Segmental Vitiligo
and Phototherapies
pattern. A white macule on the nipple or areola
appearing as the initial lesion can be assumed to Previous studies on vitiligo have focused on vit-
be an early manifestation of SV (Fig. 6.10a) [3]. iligo/NSV, and very few studies have addressed
Nipple or areolar involvement as the initial lesion specifically SV [1, 3, 40, 50, 52, 59]. In order to
in nonsegmental vitiligo is very rare and becomes gain further insights on how to treat this disorder
bilateral later (Fig. 6.10b). If SV occurs bilater- more effectively, a careful analysis of how differ-
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Fig. 6.10 (a) A white

macule on the areola or a
nipple often progresses
to segmental vitiligo
(left panel early stage,
difficult to predict; right
panel, clear segmental
involvement, different
patient). (b) Bilateral
involvement of the
nipple in vitiligo/NSV
ent modalities induce vitiligo repigmentation the nearby epidermis to form the follicular pig-
would be invaluable. Segmental vitiligo is ment islands [62]. During this process, the mela-
regarded as a stable disease once the initial melanoblasts mature morphologically and functionally
nocyte loss has ceased. Since the active destruc- and subsequently begin normal transfer of melanin
tion of melanocyte is no longer present, the to keratinocytes [63]. On the other hand, perile-
recovery scheme of segmental vitiligo depends sional or diffuse patterns of repigmentation involve
on (1) the activation, migration, and functional probably the migration of perilesional melano-
development of melanoblasts, (2) the prolifera- cytes toward the vitiligo macules or activation of
tion and migration of functional melanocytes, inactive melanocytes within vitiligo lesions,
and (3) the impact of epidermal keratinocytes on respectively [64, 65]. Figure 6.12 summarizes the
pigment cells [60]. different repigmentation processes described
The pattern for vitiligo repigmentation may above.
take two forms: the follicular pattern and the dif- Epidermal melanocytes and follicular melano-
fuse pattern. The melanoblasts in the outer root cytes express different antigens [66] providing
sheath (ORS) of the hair follicle serve as the source further evidence of intrinsic differences between
for follicular repigmentation [61]. In the follicular segmental and generalized vitiligo. Regardless of
pattern, recovery from vitiligo is initiated by the the initial damaging process, once the disease
activation and proliferation of immature melano- becomes stable, the most important cells involved
blasts, followed by their upward migration onto in segmental vitiligo repigmentation besides
ERRNVPHGLFRVRUJ
a b
Fig. 6.11 Bilateral segmental vitiligo of the same distribution in Asian (a) and black (b) patients; bilateral facial vit-
iligo (c) bilateral facial SV I + IV
melanoblasts and melanocytes are keratinocytes, lished SV lesions [68]. The biological impact of
which play a crucial role in modulating epider- PUVA on epidermal cells includes (1) limited stim-
mal pigmentation. Through their growth factors/ ulatory effect on keratinocytes; (2) increased matrix
mitogens, keratinocytes can modulate the bio- metalloproteinase (MMP)-2 activity, an important
logical behavior of neighboring melanocytes. modulator for pigment cell motility, from melano-
Stem cell factor, basic fibroblast growth factor, cytes [69]; and (3) increased tyrosinase activity of
endothelins, and nerve growth factor are among melanocytes [70]. In addition, the photosensitizer
the documented factors that participate in this 8-methoxypsoralen by itself can stimulate MMP-2
delicate relationship. expression from melanoblasts [71]. These biologi-
PUVA treatment has been shown to be more cal effects imparted by PUVA on epidermal cells
effective for treating vitiligo/NSV as compared to provide a reasonable explanation for the therapeu-
SV in a Saudi study [67]. The immunomodulating tic effect of PUVA on SV. Westerhof and
effects of PUVA are probably more important for Nieuweboer-Krobotova [72] reported that vitiligo
active vitiligo/NSV and play a limited role in SV as patients receiving PUVA and NBUVB experi-
active melanocyte loss is not a feature of estab- enced a repigmentation rate of 46% and 67%,
ERRNVPHGLFRVRUJ
Fig. 6.12 Repigmentation
scheme of vitiligo. The
process of follicular
pigmentation is shown in
blue color. This
repigmentation route
involves activation,
proliferation, migration,
and functional Functional melanocytes Inactivated melanocytes
development of immature
melanoblasts residing on
the outer root sheath of the
hair follicle. Diffuse
(green) and perilesional
(red) patterns of
repigmentation require
activation of inactive
melanocytes and migration
Differentiated
of perilesional melanoblasts
melanocytes, respectively.
All three forms of
pigmentation pattern may
be observed in
repigmenting vitiliginous
skin
Inactive
melanoblasts
Follicular repigmentation
Perilesional repigmentation
Diffuse repigmentation
respectively, after 4 months of phototherapy. lation of melanoblasts in terms of melanin forma-

NBUVB is known to have immune modulatory tion and cell migration (Yu et al, unpublished
effects including depleting skin infiltrating T cells data).
and arresting maturation of epidermal dendritic Comparing the in vitro effects of NBUVB and
cells [73]. In terms of biological effects, NBUVB PUVA on epidermal cells, it is clear that both
irradiation affects several cellular targets: (1) it modalities have significant immunomodulatory
causes a direct stimulation of melanocyte locomo- effects, but NBUVB has more direct biological
tion via increased expressions of phosphorylated effects on epidermal cells in terms of supporting
focal adhesion kinase, (2) it increases matrix pigment cell migration, proliferation, and devel-
metalloproteinase (MMP)-2 activity from melano- opment. Therefore, according to the in vitro
cytes, (3) it causes indirect stimulation of melano- repigmentation model, NBUVB may provide
cyte proliferation by increasing the production of better therapeutic efficacy for inducing repig-
basic fibroblast growth factor and endothelin from mentation in SV [74], as compared to PUVA
keratinocytes [69], and (4) it causes a direct stimu- therapy.
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Direct effects on pigmented cells
Proliferation Migration Differentiation Immune

suppression
PUVA
NBUVB
He-Ne laser
Indirect effect on pigment cells via keratinocytes
Proliferation Migration Differentiation Immune

suppression
PUVA
NBUVB
He-Ne laser
Fig. 6.13 Schematic diagram summarizing the mecha- cytes. More specifically, NBUVB directly or vicariously
nisms involved in PUVA, NBUVB, and laser red light- via keratinocyte increases the proliferation and locomo-
induced vitiligo repigmentation. PUVA treatment has tion of melanocytes. In addition, NBUVB also stimulates
significant immune modulatory effects on both melano- melanogenesis and migration of immature melanoblasts.
cytes and cytotoxic immune cells. It also directly pro- The Ne-He laser red light has also direct effects on mela-
motes melanocyte melanin formation and migrations but noblasts. Abbreviations: NBUVB narrowband UVB, KC
has limited biological effects on keratinocytes. Besides keratinocyte, MC melanocyte, CTL cytotoxic T lympho-
immune suppressive effects, NBUVB treatment has sig- cyte, MB melanoblast
nificant biological effects on pigment cells and keratino-
More recently, helium-neon (He-Ne) laser, Tacrolimus ointment is a promising topical

which emits within visible red light, has been agent for treating SV. In vitro studies using epi-
shown as a novel phototherapeutic modality for dermal cells have shown that tacrolimus (1)
treating SV [75]. He-Ne laser promotes vitiligo directly stimulates keratinocytes to release stem
repigmentation through (1) direct stimulation of cell factor, (2) induces keratinocytes to promote
melanocyte and melanoblast migration [75, 76], the growth of both melanoblasts and melano-
(2) direct stimulation of melanoblasts to undergo cytes, (3) induces keratinocytes to upregulate
functional development [76], (3) increased secre- MMP activities [77], and (4) directly induces
tion of nerve growth factor and basic fibroblast pigmentation and migration of melanocytes [78].
growth factor from keratinocytes, and (4) indirect Therefore, besides immunomodulatory effects of
stimulation of melanocyte growth via keratino- tacrolimus on immune cells, the direct biological
cytes [75]. A summary of direct vs. indirect effect effects of tacrolimus on epidermal cells probably
of phototherapies in repigmentation is shown in account for its therapeutic effects on SV. Of
Fig. 6.13. interest is the effect of different therapies on the
ERRNVPHGLFRVRUJ
cutaneous nervous system. Currently, in vitro activation and migration of epidermal melano-
data regarding this topic is lacking. However, it cytes to the hair follicle. Overall, stable SV is
has been shown that He-Ne laser irradiation leads a good indication for epidermal grafting and
to improvement in nerve injury [79, 80] and topi- can be cured almost completely without recur-
cal tacrolimus induces neuropeptide release [81] rence (for a discussion of surgical therapies,
and may lead to neuronal cell differentiation see Chap. 3.2.3).
[82]. These reports suggest that neural modula- Although new strides have been made on treat-
tory effects may have a previously unrecognized ment options for segmental vitiligo, the underlying
role in the treatment of segmental vitiligo. molecular mechanisms inducing repigmentation
must be elucidated for optimizing treatment out-
comes. Another issue, which requires further
6.7 Treatment Overview attention, is the involvement of neurocutaneous
and Perspectives interactions in the pathogenesis of vitiligo. Since
skin homeostasis may be regulated by the periph-
SV was previously known to be resistant to eral neuropeptidergic nerve fibers [84], further
treatment. However, recent studies reported work focusing on modulation of neurocutaneous
surprisingly good results. SV at an early stage unit may provide a key to more effective vitiligo
has an excellent prognosis. Since the most fre- treatment.
quently involved site of SV is the face, it can Segmental vitiligo is a perfect model for
be easily detected and treated. Effective treat- studying repigmentation since it is a relative
ment modalities at an early stage are not stan- stable disease with limited active melanocyte
dardized but include topical steroids, topical loss. Moreover, the cells involved in the repig-
calcineurin inhibitors, or UV therapy, isolated mentation scheme of segmental vitiligo are
or in combination (Fig. 6.14). Because SV readily available for in vitro experimentation.
often causes leukotrichia, it may resist stan- With recent advances in regenerative medicine,
dard medical therapies. Stable SV with leuko- the use of melanocyte stem cells [85] may
trichia can be cured successfully with evolve from in vitro experimentations to in vivo
epidermal grafting and subsequent UV treat- reality as suggested by Yonetani et al. [86] (see
ment [83]. There may be also a possibility of Chap. 2.3.7).
a b
Fig. 6.14 Almost complete repigmentation of SV after 4 months of 308 nm excimer laser treatment (a, before; b, after)
(corresponds to Hann’s type I)
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Mixed Vitiligo
7
Alain Taïeb
Contents
7.1 Historical Background 74
7.2 Clinical Features and Differential Diagnosis 74
7.3 Risk Factors for Generalized Vitiligo in SV Patients 77
7.4 Interpretations and Current Issues 77
7.4.1 Genetic Link SV-Vitiligo/NSV 77
7.4.2 Halo Nevi as Markers of Vitiligo Diathesis and Progression to MV in SV 78
7.4.3 Leukotrichia and MV, Consequence of Loss of Immune Privilege? 78
7.4.4 Bilateral Mosaicism Hypothesis in Vitiligo/NSV 78
References 79
Abstract SV to MV. In addition, this progression of SV

By definition, mixed vitiligo (MV) is the asso- to MV carries a stronger link if the segment is
ciation of a characteristic segmental involve- initially situated on the trunk. Combined
ment associated usually in a second step with somatic mosaicism and immune dysregula-
the onset of bilateral vitiligo patches. The tion may explain the continuum SV-vitiligo/
association of segmental vitiligo (SV) to vit- NSV in the case of MV.
iligo/nonsegmental vitiligo (vitiligo/NSV)
suggests a continuum between the two subsets
which were formerly opposed. A common Key Points
mode of revelation is a segment resistant to • Mixed vitiligo (MV) is the association
phototherapy in a patient diagnosed previ- of SV to vitiligo/NSV.
ously as vitiligo/NSV. Halo nevi and leuko- • It suggests a continuum between the two
trichia at first consultation in segmental subsets which were formerly opposed.
vitiligo are risk factors for the progression of • A common mode of revelation of MV is
a segment resistant to phototherapy in a
patient diagnosed previously as vitiligo/
NSV.
A. Taïeb (*)
Service de Dermatologie, Hôpital Saint-André, CHU
• Presence of halo nevi and leukotrichia at
de Bordeaux, Bordeaux, France first consultation in segmental vitiligo is

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74 A. Taïeb
the diagnosis of vitiligo/NSV localized to the

a risk factor for the progression of SV to trunk and the extremities was first made in three
MV. patients, and narrowband (NB) UVB photother-
• Progression of SV to MV carries a apy revealed the presence of a pre-existing thora-
stronger link if initial segmental involve- coabdominal SV undetected at the first visit that
ment is situated on the trunk. had poorly or not responded to the treatment. The
• Combined somatic mosaicism and patients further acknowledged the primary onset
immune dysregulation may explain the of this segmental involvement. A clear segmental
continuum SV-vitiligo/NSV in the case blaschkolinear pattern was noted in two patients.
of MV. A relevant history of axial skeletal abnormality
(i.e., scoliosis) was found in four patients. In all
patients SV preceded NSV with a delay ranging
from 6 months to more than 24 months (Fig. 7.1).
7.1 Historical Background For the Belgian series of van Geel et al. [10],
14 out of 141 patients with SV received a final
Until the last decade, the opposition of SV and diagnosis of MV. The aim of the authors was
vitiligo/NSV was considered rather dogmatically. mostly to describe the pattern of segmental
The association of SV and NSV was first sug- lesions. However, they made interesting addi-
gested in 2003 by the description of a pediatric tional observations concerning their MV cases:
NSV case treated with ultraviolet (UV) B that left (1) rather late apparition of the nonsegmental
a recalcitrant segmental portion compatible with involvement (3 years, 6 years, and 12 years,
pre-existing SV [1]. Other observations [2–4] respectively, after the segmental lesion); (2) most
were subsequently reported, and the term “mixed generalized vitiligo lesions in the mixed vitiligo
vitiligo” (MV) proposed by Mulekar et al. to des- group were very mild (12/14 patients), and in two
ignate this particular form of the disease, followed of these patients, differentiation from fully depig-
by case series [5–11] was officially endorsed by mented halo nevi was difficult; and (3) the age of
international consensus [5]. By definition, MV is onset was higher in patients with MV (median
the association of a characteristic segmental 22 years) compared with pure segmental vitiligo
involvement associated usually in a second step (median 13 years).
with the onset of bilateral vitiligo patches. For the Italian pediatric study of Neri et al., 13
patients with MV were enrolled among 92 chil-
dren affected by vitiligo in 5 years (59 NSV, 20
7.2 Clinical Features SV, 13 MV), with contrary to the Ezzedine et al.
and Differential Diagnosis series a preponderance of male cases
(M/F = 10:3). The mean age of onset was
Four case series have been published [3, 7, 10, 7.5 years, and all cases showed a primary seg-
12] and case reports with therapeutic intervention mental involvement. Three patients showed sco-
[13–15] which permit to better delineate mixed liosis at clinical examination. Only two children
vitiligo. had a segmental blaschkolinear pattern.
For the French Ezzedine et al. series [7], For the German study of Schallreuter et al. [3],
which comprises 19 children and adults extracted extracted from an international group of 2411
from a prospective cohort of 498 vitiligo (all patients (1033 male, 1378 female), 57 patients
types) in 3.5 years, the sex ratio is surprisingly were diagnosed with SV (29 males/28 females;
skewed (male/female ratio, 1:4) but may just age of onset 1–47 years, mean age 15.4 years),
reflect that in hospital consultations for vitiligo in and 76 patients had mixed vitiligo (mean age
Europe, females predominate. Most patients had 16.0 years, range 1–61 years), a prevalence of
a disease onset before the age of 18 years and a 3.2% for MV. The mean age of the patients with
segmental involvement of the trunk. Interestingly, SV was 15 years (range 9–19 years), while in the
ERRNVPHGLFRVRUJ
7 Mixed Vitiligo 75
1 2 3 4
5 6 7 8
9 10 11 12
13 14 15 16
17 18 19
Fig. 7.1 Pattern of segmental (red) and nonsegmental (blue) involvement in 19 patients with mixed vitiligo
ERRNVPHGLFRVRUJ
76 A. Taïeb
Before UV
After UV
Fig. 7.2 Mixed vitiligo: better efficacy of NB UVB on nonsegmental involvement
MV group, the mean age was 25.5 years (range (Fig. 7.2). In the case of grafted MV, the segmen-
7–57 years). This study shows in SV and MV tal type showed an excellent response after epi-
accumulation of epidermal biopterin together dermal grafting, but it recurred in the grafted area
with significantly decreased epidermal catalase repeatedly, suggesting that the segmental type
and presence of H2O2 in the skin as in vitiligo/ may behave differently [14]. Concerning associ-
NSV, as well as a response to the combination of ated scoliosis, a possible neurogenic trigger in a
low-dose NB-UVB-activated pseudocatalase. topographically compatible dermatomal segment
Based on these cumulated data, it appears that is possible but may also be coincidental.
MV is a significant subset, especially in pediatric Limited data exists concerning the pathology
recruitments, and has most probably been under- of segmental vs. nonsegmental lesions in MV. A
diagnosed previously. A notable difference recent study in a Japanese patient with hepatitis C
between SV in MV and SV in isolation is the lim- and MV showed more prominent inflammation
ited number of cases of SV of the face and neck. in the nonsegmental more recent lesion [13].
The segment pattern may be blaschkolinear but Other diagnoses should be ruled out before
only in a minority of cases. Response to UVB accepting the diagnosis of MV. This is particu-
phototherapy is better in nonsegmental lesions larly true for the other segmental hypomelanoses
ERRNVPHGLFRVRUJ
7 Mixed Vitiligo 77
Passage from segmental vitiligo to mixed vitiligo
Antithyroid
5.9% antibodies 23.1%
44.1% Percentage of body involvement, > 1% 80.0%
Initial trunk
32.0% segmental involvement 69.2%
8.3% Koebner 44.0%

12.0% Halo nevi 65.4%
36.0% Leukotrichia 92.0%
Odds ratio for univariate analysis

Segmental vitiligo (n = 101) Mixed vitiligo (n = 26)
Fig. 7.3 Distribution and weighted contribution of selected features (odds ratios) according to the univariate analysis
for segmental and mixed vitiligo
such as nevus depigmentosus that are generally respectively) than those with segmental vitiligo
present at birth or discovered in the first years of (12.0%, 5.9%, and 36.0%, respectively). Finally,
life but may be misdiagnosed at that time in trunk involvement was confirmed as more fre-
patients with fair skin. Wood’s lamp examination quent in mixed vitiligo compared with segmental
is usually sufficient to make a difference between vitiligo (69.2% and 32.0%, respectively). In the
SV and nevus depigmentosus (Chap. 1.2). Van univariate analysis, segmental vitiligo located on
Geel et al. noted that fully depigmented halo nevi the trunk, Koebner phenomenon, and the pres-
may pose a problem if interpreted as a mild non- ence of circulating antithyroid antibodies were
segmental involvement [11]. strongly linked with the progression of SV to
MV, arguing for a more marked autoimmune
background in MV. In multivariate analysis, ini-
7.3 isk Factors for Generalized
R tial percentage of body surface involvement of
Vitiligo in SV Patients the segment of more than 1% and the presence of
halo nevi and leukotrichia were found to be inde-
Given the established sequence SV first vitiligo/ pendent factors associated with the evolution of
NSV later in MV patients, a study by Ezzedine patients’ disease from SV to MV [7] (Fig. 7.3).
et al. has compared 101 patients with SV and 26
who had disease which evolved from SV into
MV. The age at disease onset in patients with seg- 7.4 Interpretations and Current
mental vitiligo was comparable in the two groups, Issues
as opposed to the van Geel study (9.2 ± 8.3 years
for SV vs. 8.1 ± 7.3 years for MV). The presence 7.4.1 Genetic Link SV-Vitiligo/NSV
of halo nevi, circulating antithyroid antibodies,
and leukotrichia was noted more often in patients The hypothesis that the difference in response to
with mixed vitiligo (65.4%, 23.0%, and 92.0%, therapy of SV may be linked to the dosage of
ERRNVPHGLFRVRUJ
78 A. Taïeb
underlying predisposing cutaneous defect iligo [19] and furthermore that halo nevi could be
involved in the SV area has previously been a clinical sign of vitiligo. However, some cases of
raised [16, 17]. This may relate to a possible extensive vitiligo clearly spare melanocytic nevi
mosaic expression in the skin of a major predis- (Chap. 5). Whether or not SV with associated
posing gene, following the model of monogenic halo nevi can be classified as a special mixed type
disorders, where a loss of heterozygosity is found of vitiligo and whether or not the mild variant of
in type II mosaicism, whereas type I mosaicism nonsegmental lesions appearing in mixed vitiligo
results from a single dominant mutation [18]. is based on the same pathomechanism determin-
The sequence (early SV, late NSV) may also ing generalized vitiligo/NSV need to be further
reflect the role of a first cutaneous gene defect investigated [11].
causing SV triggering a generalized immune
response against cutaneous melanocytes sup-
ported by another immune-related gene defect. 7.4.3 Leukotrichia and MV,
However, pedigrees showing the presence of Consequence of Loss
cases with established SV in families with previ- of Immune Privilege?
ous cases of NSV [17] reinforce the possibility of
a mechanism close to type II mosaicism already The presence of leukotrichia at the time of first
demonstrated in monogenic skin disorders. consultation of SV is one of the predictors of the
evolution to MV. In vitiligo/NSV, repigmentation
depends on available melanocytes from three pos-
7.4.2 H
alo Nevi as Markers sible sources, including the hair follicle, the bor-
of Vitiligo Diathesis der of vitiligo lesions, and unaffected melanocytes
and Progression to MV in SV within depigmented areas ([20]; see Chap. 45).
Among these sources, the hair follicle is the main
The strong link between the progression of SV to source of melanocytes. In most cases of vitiligo/
MV and the initial presence of halo nevi needs to NSV, there is a preferential targeting of epidermal
be taken into consideration [6, 11]. Specifically, but not of follicular melanocytes arguing for dif-
halo nevi may be considered as a clinical marker ferent antigenic profiles between these two types
of cellular immune response against nevocytes, of melanocytes. Some of this immunological dif-
which may indirectly pertain to the process tar- ference is likely to reflect the fact that the follicu-
geting normal melanocytes in vitiligo [6]. In MV, lar melanin unit resides in the immune-privileged
there might be a stronger natural cutaneous proximal anagen hair bulb in contrast to the
immunosurveillance as shown by the presence of immunocompetent nature of the epidermal mela-
halo nevi. The generalization of SV to MV might nin unit [21] (see Chap. 9 and [11]).
be initiated by an immune response first directed
against nevocellular targets with collateral dam-
age to surrounding melanocytes belonging to the 7.4.4 Bilateral Mosaicism
same territory of lymphatic circulation, espe- Hypothesis in Vitiligo/NSV
cially for truncal SV, as hypothesized for seg-
mental vitiligo [9]. This may inversely suggest Mixed patterns of vitiligo are documented as the
that the majority of SV without halo nevi patients progression from localized SV to vitiligo/
have some counteracting protective mechanisms NSV. However, a phenomenon of segmentation
respective to the development of vitiligo/NSV of lesions (mirror images) occurring in vitiligo/
and which explains the usual good take of autolo- NSV was observed in the Indian study of Attili
gous grafts in SV. and Attili [22] (Fig. 7.4) suggesting that mosa-
Some authors have proposed that halo nevi icism may be an underlying factor explaining this
could be a risk factor for the development of vit- bilateral mirror pattern. The authors suggest that
ERRNVPHGLFRVRUJ
7 Mixed Vitiligo 79
a b c
d e f
Fig. 7.4 Mirror patterns in vitiligo/NSV (from 22) The authors suggest that in vitiligo/NSV the sometimes striking
symmetrical patterns correspond to an underlying developmental defect revealed by vitiligo
the multiplicity of underlying mosaic segments ment with pseudocatalase PC-KUS in 71 children
with vitiligo. Int J Dermatol. 2008;47:743–53.
affected in vitiligo/NSV may not be appreciated 5. Ezzedine K, Lim HW, Suzuki T, Katayama I,
when lesions do not extend up to the predeter- Hamzavi I, Lan CC, Goh BK, Anbar T, Silva de
mined cutoff lines and may simply give an Castro C, Lee AY, Parsad D, van Geel N, Le Poole IC,
impression of bilateralism. Oiso N, Benzekri L, Spritz R, Gauthier Y, Hann SK,
Picardo M, Taieb A, Vitiligo Global Issue Consensus
Conference Panelists. Revised classification/nomen-
clature of vitiligo and related issues: the Vitiligo
References Global Issues Consensus Conference. Pigment Cell
Melanoma Res. 2012a;25:E1–13.
1. Gauthier Y, Cario André M, Taïeb A. A critical 6. Ezzedine K, Diallo A, Léauté-Labrèze C, Seneschal
appraisal of vitiligo etiologic theories. Is melano- J, Mossalayi D, AlGhamdi K, Prey S, Bouchtnei S,
cyte loss a melanocytorrhagy? Pigment Cell Res. Cario-André M, Boralevi F, Jouary T, Taieb A. Halo
2003;16:322–32. nevi association in nonsegmental vitiligo affects age
2. Mulekar SV, Al Issa A, Asaad M, et al. Mixed vitiligo. at onset and depigmentation pattern. Arch Dermatol.
J Cutan Med Surg. 2006;10:104–7. 2012b;148:497–502.
3. Schallreuter KU, Krüger C, Rokos H, et al. 7. Ezzedine K, Diallo A, Léauté-Labrèze C, Séneschal
Basic research confirms coexistence of acquired J, Prey S, Ballanger F, Alghamdi K, Cario-André M,
Blaschkolinear Vitiligo and acrofacial Vitiligo. Arch Jouary T, Gauthier Y, Taieb A. Halo nevi and leuko-
Dermatol Res. 2007;299:225–30. trichia are strong predictors of the passage to mixed
4. Schallreuter KU, Krüger C, Würfel BA, et al. From vitiligo in a subgroup of segmental vitiligo. Br J
basic research to the bedside: efficacy of topical treat- Dermatol. 2012c;166:539–44.
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8. Schallreuter KU, Salem MA, Holtz S, Panske 15. Oiso N, Kawada A. Superimposed segmental vit-

A. Basic evidence for epidermal H2O2/ONOO(-)- iligo (mixed vitiligo) with non-segmental vitiligo
mediated oxidation/nitration in segmental vitiligo is and segmental vitiligo along the narrow Blaschko
supported by repigmentation of skin and eyelashes lines (type 1a). J Dermatol. 2016;43:1388. https://doi.
after reduction of epidermal H2O2 with topical org/10.1111/1346-8138.13405.
NB-UVB-activated pseudocatalase PC-KUS. FASEB 16. Taieb A. Intrinsic and extrinsic pathomechanisms in
J. 2013;27:3113–22. vitiligo. Pigment Cell Res. 2000;13:41–7.
9. Van Geel N, Mollet IG, De Schepper S, et al. First 17. Taïeb A, Morice-Picard F, Jouary T, et al. Segmental
histopathological and immunophenotypic analysis vitiligo as the possible expression of cutaneous
of early dynamic events in a patient with segmen- somatic mosaicism: implications for common non-
tal vitiligo associated with halo nevi. Pigment Cell segmental vitiligo. Pigment Cell Melanoma Res.
Melanoma Res. 2010;23:375–84. 2008;21:646–52.
10. Van Geel N, De Lille S, Vandenhaute S, et al. Different 18. Koga M. Vitiligo: a new classification and therapy. Br
phenotypes of segmental vitiligo based on a clinical J Dermatol. 1977;97:255–61.
observational study. J Eur Acad Dermatol Venereol. 19. Barona MI, Arrunategui A, Falabella R, Alzate A. An
2011a;25:673–8. epidemiologic case-control study in population with
11. Van Geel N, Speeckaert R, Lambert J, et al. Halo naevi vitiligo. J Am Acad Dermatol. 1995;33:621–5.
with associated vitiligo-like depigmentations: patho- 20. Parsad D, Pandhi R, Dogra S, Kumar B. Clinical
genetic hypothesis. J Eur Acad Dermatol Venereol. study of repigmentation patterns with different treat-
2011b;25:673–8. ment modalities and their correlation with speed and
12. Neri I, Russo T, Piccolo V, Patrizi A. Mixed vitiligo in stability of repigmentation in 352 vitiliginous patches.
childhood: a study on 13 italian patients. J Eur Acad J Am Acad Dermatol. 2004;50:63–7.
Dermatol Venereol. 2013;27:e136–47. 21. Ito T, Meyer KC, Ito N, Paus R. Immune privilege and
13. Hashimoto N, Tanemura A, Yamada M, Itoi S,
the skin. Curr Dir Autoimmun. 2008;10:27–52.
Katayama I. Hepatitis C-related mixed type vitiligo 22. Attili VR, Attili SK. Anatomical segmentations in
in a patient with Ivemark syndrome. J Dermatol. all forms of vitiligo: a new dimension to the etio-
2014;41:185–6. pathogenesis. Indian J Dermatol Venereol Leprol.
14. Lee DY, Kim PS, Lee JH. Mixed vitiligo treated by 2016;82:379–88.
suction blister epidermal grafting: long-term follow
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ERRNVPHGLFRVRUJ
Clinically Inflammatory Vitiligo
and Rare Variants
8
Alain Taïeb, Khaled Ezzedine, Julien Seneschal,
and Ratnam Attili
Contents
8.1 General Background 82
8.2 Isolated Clinically Inflammatory Vitiligo 82
8.3 Clinically Inflammatory Vitiligo Associated with Other Disorders 83
8.5 istological Features of Clinically Inflammatory Vitiligo vs. Common
H
Clinically Noninflammatory Vitiligo 84
8.6 Rare Variants 85
8.6.1 Follicular Vitiligo 85
8.6.2 Hypopigmented Vitiligo/Vitiligo Minor 88
8.6.3 Vitiligo Guttata/Punctata, Leukoderma Punctata, and Confetti Leukoderma 89
8.7 Summary and Concluding Remarks 90
References 90
Abstract oid infiltrates in the margins of progressing

A particular form of vitiligo of clinically lesions. In pigmented progressing borders of
inflammatory vitiligo has been reported as common vitiligo/NSV and SV, similar fea-
“inflammatory vitiligo with raised borders” or tures of lesser intensity have been demon-
marginal vitiligo and is associated with lichen- strated suggesting that most subtypes of
vitiligo belong to the spectrum of a clinically
silent chronic inflammatory skin disorder
A. Taïeb (*) · K. Ezzedine · J. Seneschal responsible for melanocyte loss. Clinically
Service de dermatologie, Hôspital St André, CHU de inflammatory vitiligo (CIV) has been some-
Bordeaux, Bordeaux, France times associated with infectious or inflamma-
e-mail: alain.taieb@chu-bordeaux.fr; khaled. tory diseases. Other rare variants considered
ezzedine@chu-bordeaux.fr
in this chapter include follicular vitiligo, vit-
R. Attili iligo/leukoderma guttata, and hypopigmented
Visakha Institute of Skin and Allergy,
Visakhapatnam, India vitiligo/vitiligo minor. For both CIV and

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82 A. Taïeb et al.
hypopigmented vitiligo, biopsies (sometimes also with psoriasis, which shows a predominant
repeated) are needed to establish a firm diag- TH1-TH17 cytokine imbalance and where
nosis, in particular to exclude cutaneous T-cell lesions are clinically very inflammatory, redness
lymphoma, a major differential diagnosis. being mandatory in disease expression, with
milder pruritus if present.
However, a particular form of vitiligo defined as
Key Points “inflammatory vitiligo with raised borders” has been
• A rare form of clinically inflammatory noticed long ago [2–4]. The clinical presentation of
vitiligo (CIV) reported as “inflamma- this particular form of vitiligo consists in depig-
tory vitiligo with raised borders” or mented patches with an erythematous micropapular
marginal vitiligo is associated with edge. This condition is associated with inflamma-
lichenoid infiltrates at the margins of tory infiltrates in the margin of progressing lesions.
progressing lesions. In general, clinically inflammatory vitiligo (CIV)
• Histopathological findings in still pig- may occur in isolation but has unfrequently been
mented progressing borders of vitiligo/ associated with various disorders including infec-
NSV and SV may show less intense but tious diseases [5–8], lichen sclerosus [9], atopic der-
similar features, suggesting that vitiligo matitis [10], or Vogt-Koyanagi-Harada (VKH)
could be considered as a clinically silent disease [11, 12]. Several histological studies indicate
chronic inflammatory skin disorder. that common vitiligo can also be microinflamma-
• Clinically inflammatory vitiligo has tory at the progressing edge of depigmented lesions
been sometimes associated with other (Chap. 3). Other rare variants characterized by spe-
infectious or inflammatory diseases. cial clinical features most of them associated with
• Rare vitiligo variants include follicular microscopic inflammation are also discussed in this
vitiligo, vitiligo guttata, and hypopig- chapter (hypopigmented vitiligo, follicular vitiligo,
mented vitiligo/vitiligo minor. vitiligo/leukoderma guttata/punctata).
• For both CIV and hypopigmented vitiligo,
biopsies (sometimes repeated) are needed
to establish a firm diagnosis, in particular 8.2 Isolated Clinically
to exclude cutaneous T-cell lymphoma. Inflammatory Vitiligo
A few cases of CIV without documented asso-

ciation with other diseases have been reported
[2–4, 13–20] (Fig. 8.1). In all reported cases,
8.1 General Background progressive lesions with erythema and fine
scaling, with or without pruritus, are common
One of the striking features of vitiligo, when features. Duration at diagnosis varied from
compared with other chronic skin conditions, is
the absence of symptoms and signs of inflamma-
tion. In contrast with atopic dermatitis, which is
characterized locally by a TH2 cytokine imbal-
ance, where erythema and edema are common
during flares, pruritus is not a classic vitiligo
symptom. However, since this item has been
included in the VETF evaluation form [1], some
patients definitely report a mild pruritus preced-
ing flares of depigmentation. Vitiligo contrasts Fig. 8.1 Vitiligo with raised borders
ERRNVPHGLFRVRUJ
8 Clinically Inflammatory Vitiligo and Rare Variants 83
2 months to 2 years. An erythematous raised oid lesions which result in complete pigment
border has been documented, coexisting or not loss. Interestingly in this setting, the histopath-
with non-clinically inflammatory depigmented ological sequence shows a loss of melanocytic
macules. Marginal hyperpigmentation sur- antigens preceding the definitive loss of pig-
rounding hypopigmented patches has also been ment cells. Contrarily, in two cases of vitiligo
noticed. Inflammatory vitiligo is generally occurring, respectively, in the setting of chronic
considered as a progressive (unstable) form hepatitis associated, respectively, with hepati-
since extensive involvement seems to be the tis C infection [8] and atopic dermatitis [10],
rule, with improvement using topical cortico- clinical features were close to that of inflam-
steroids, UV, or both. It seems to affect both matory vitiligo with raised borders. In these
sexes at any age, but most described cases con- case reports, patients presented both inflamma-
cern adults. Interestingly, in almost half the tory patches with raised border in recent
patients, concomitant inflammatory and nonin- lesions and noninflammatory older patches.
flammatory patches are observed. Based on Interestingly, Tsuruta et al. reported [11] a
histopathological studies, this unusual clinical VKH patient with a 1-year history of a super-
manifestation can be interpreted as disease imposed thin inflammatory raised erythema
progression due to an inflammatory phase. CIV and plaque-type inflammatory erythema occur-
in segmental vitiligo was described in only one ring in the setting of vitiligo/NSV of 20 years
case [17]. duration. Two particular features were noted:
first, within patches of vitiligo with raised
inflammatory borders, erythema was separated
8.3 Clinically Inflammatory from the totally depigmented areas by an
Vitiligo Associated incompletely vitiliginous area; second, the
with Other Disorders presence of plaque-type erythema, which is
uncommonly seen in the so-called inflamma-
Inflammatory vitiligo has also been associated tory vitiligo [11]. Weisberg et al. [9] reported
with specific disorders (Table 8.1). The clini- the case of a 41-year- old African-American
cally inflammatory stage of HIV-associated woman who had a 6-month history of inflam-
cases is different from CIV with raised borders matory segmental vitiligo that started on the
(Chap. 1.5.8). It consists in nummular lichen- left foot, then spread proximally up her leg to
Table 8.1 Inflammatory vitiligo in the setting of infectious or inflammatory diseases

Age/ Presence of other
gender of Type of Associated Duration of noninflammatory
Reference patient vitiligo disease Pruritus disease patches Evolution
Tsuruta 48/M NSV Vogt-Koyanagi- Yes 1 year Yes Extensive
Harada disease
Sugita 31/M ? Atopic dermatitis Yes 5 years ? Extensive
Tsuboi 38/M NSV Hepatitis C virus Yes 2 years Yes Extensive
infection
Weisberg 41/W SV Lichen sclerosus Yes 6 months No Extensive
Shin 45/W NSV Sjögren’s No 1 month ? Regressive
syndrome
Tanioka 53/W NSV Sjögren’s No ? Yes Regressive
syndrome 5 years for under NBUVB
Sjögren’s therapy
syndrome
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84 A. Taïeb et al.
her thigh, and then to her buttocks with an

a
involvement of pubic hairs that turned white.
Concomitantly, the patient had severe burning
and itching in a clinically whitish pink skin of
the vaginal area consistent with the diagnosis
of lichen sclerosus [9]. Two reports point to a
possible link with Sjögren’s syndrome, with
the presence of leukocytoclasis in addition to
the T-cell lichenoid infiltrate [18, 21].
8.4 Differential Diagnosis

b
The diagnosis of annular inflammatory dermato-
ses, such as lupus and cutaneous T-cell lym-
phoma, needs to be ruled out in all cases by
appropriate testing. For Sjögren’s syndrome, the
overlap needs clarification, and SSA/SSB testing
should be performed.
A clinical presentation similar to that noted in
HIV infection-associated inflammatory vitiligo
may occur in patients with graft versus host disease
(GVHD) following allogeneic hematopoietic cell
transplantation (Fig. 8.2). The depicted GVHD
patient presented with erythroderma followed by
generalized depigmented macules affecting the
whole body, clinically consistent with the diagno-
sis of vitiligo. In addition, he developed alopecia
areata. One hypothesis is that GVHD may have
triggered skin-targeted autoimmunity toward mela-
nocytes (epidermal and follicular).
A peculiar annular lichenoid dermatitis
named annular lichenoid dermatitis of youth,
the clinical appearance of which can initially
suggest diagnoses of morphea, mycosis fungoi-
des, or annular erythema, may be mistaken for
inflammatory vitiligo [22] (Fig. 8.3). Lesions Fig. 8.2 Graft versus host disease at erythrodermic (a)
consist of persistent asymptomatic erythema- and vitiligoid (b) phases (Courtesy M. Beylot-Barry)
tous macules and round annular patches with a
red-brownish border and central hypopigmenta-
tion, mostly distributed on the groin and flanks. 8.5 istological Features
H
Histology reveals a lichenoid dermatitis with of Clinically Inflammatory
massive necrosis/apoptosis of the keratinocytes Vitiligo vs. Common Clinically
limited to the tips of rete ridges, in the absence Noninflammatory Vitiligo
of dermal sclerosis and epidermotropism of
atypical lymphocytes. The infiltrate is com- Histopathological examination in clinically
posed mainly of memory CD4(+) CD30(−) T inflammatory vitiligo should include biopsies
cells with few B cells and macrophages [22]. taken from the inflammatory border. Whatever
ERRNVPHGLFRVRUJ
pigmented skin have shown inflammatory

a
changes in the pigmented border with decreasing
inflammation from the marginal pigmented areas
to the fully depigmented vitiligo patches [20,
23–25]. The biopsy needs to be taken from
hypopigmented fuzzy, confetti-like borders to
maximize yield. Our experience based on rapidly
progressing pediatric cases biopsied on clinically
pigmented skin 0.5 cm of the visible border of
depigmentation confirms this finding, and the
intensity of lichenoid inflammation and some-
b times epidermotropism may simulate mycosis
fungoides (Chap. 3) (Fig. 8.4). Early biopsies
taken in clinically noninflammatory SV show
similar pathological features (Fig. 8.5) [23, 26].
8.6 Rare Variants
8.6.1 Follicular Vitiligo
Leukotrichia is considered as a marker of segmen-

tal vitiligo (Chap. 1.5.2). In vitiligo/NSV body
hairs are usually spared although hair depigmenta-
tion may occur with disease progression. In 2011,
a case of vitiligo with primary follicular involve-
ment was presented at the VGICC in Bordeaux. It
was named follicular vitiligo and classified after
discussion as a rare variant within the spectrum of
vitiligo/NSV (Chap. 2). The index case was a
young black patient, who had primary involve-
Fig. 8.3 ALDY (a) clinical features (b) histopathology ment of the hair follicle melanocytic reservoir but
limited interfollicular compartment involvement,
manifesting in marked generalized hair whitening
the case, vitiligo being associated or not with preceding the appearance of depigmented skin
another disease, the perivascular mononuclear patches [27, 28] (Fig. 8.6a, b). A case series
infiltrate with an associated lichenoid pattern is extracted from a prospective cohort of 1243 vitil-
almost always present. In addition, degeneration igo patients was recently published to better delin-
of melanocytes and basal keratinocytes may be eate the clinical and histological characteristics of
observed by ultrastructure. Immunohistochemistry follicular vitiligo [29]. Concomitant alopecia
may reveal CD8 cells and sparse CD4 cells. areata of the scalp with leukotrichial regrowth was
Table 8.2 summarizes published reports in case noted in one patient, and three patients had halo
of associated conditions. Interestingly, Shin et al. nevi. Histologic examination of a punch biopsy
have shown evidence of leukocytoclasis in two specimen taken from an area with both depig-
cases from Korea, one being associated with mented skin and leukotrichia demonstrated the
Sjögren’s syndrome [18]. presence of a discrete perifollicular infiltrate in the
Histopathological studies of clinically nonin- infundibular region of the hair follicle and an
flammatory vitiligo/NSV that include adjacent absence of melanocytes in both the basal layer of
ERRNVPHGLFRVRUJ
86
Table 8.2 Histological and immunohistochemistry findings in patients with clinically inflammatory vitiligo and associated diseases
Age/ Duration/progression Type of Associated
Reference sex of disease vitiligo Localization Histological findings Immunohistochemistry disease Treatment/evolution
Tsuruta 48/M 20 years/12 months NSV Entire body Perivascular LFA-1, CD3, CD8, and rare Vogt- NA
except face mononuclear infiltrate, CD4 and CD20 Koyanagi-
lichenoid pattern Harada
disease
Sugita 31/M 5 years/5 years NSV Limbs, trunk Perivascular No melanocytes, CD4 and Atopic Topical corticosteroid/
mononuclear infiltrate, CD8 in raised border/CD8 dermatitis improvement of erythema
lichenoid pattern outside the border
Tsuboi 38/M 24 months/24 months NSV Buttock, axilla, Degeneration of basal NA Hepatitis Prednisolone 20 mg
inguinal, layer, perivascular virus C daily + PUVA/resolution
extremities mononuclear infiltrate infection of erythema + partial
repigmentation
Weisberg 41/W 6 months/6 months SV Left foot, thigh, Papular dermal NA Lichen Topical corticosteroid/
ERRNVPHGLFRVRUJ
and sclerosis, Perivascular sclerosus involvement of
buttock + Left mononuclear infiltrate erythema + partial
genital area lichenoid pattern repigmentation
Shin 45/W 1 month/? NSV Neck and arm Basal vacuolization Melan-A and Fontana-Masson Sjögren’s Recovery with topical
leukocytoclasia no melanocyte or melanin syndrome steroids
pigment CD8 and
myeloperoxidase stains positive
A. Taïeb et al.
Fig. 8.4 Microscopic
inflammation in
progressive vitiligo/
NSV, biopsy taken at
the pigmented margin
of a clinically
noninflammatory lesion
(HES ×400). The infiltrate
is mostly composed of
CD8+ cells (Chap. 1.4)
a b
Fig. 8.5 Segmental vitiligo: recent onset lesion (3 months across the midline (chin), at the margin of the lesion at
in a) and 4 months later (b). Microscopic lichenoid stage depicted in (a) (Photographs Dr R Attili)
inflammation (c), on biopsy taken on pigmented skin
ERRNVPHGLFRVRUJ
88 A. Taïeb et al.
a b
Fig. 8.6 Follicular vitiligo (a) leg with a patch of skin and hair depigmentation (b) scalp with a large patch of leuco-
trichia but normally pigmented underlying skin
the epidermis and the hair follicle with Melan-A mainly cutaneous T-cell lymphoma (CTCL) and
staining. Presence of cells with concordant mark- various postinflammatory dermatoses (Chap. 1.2).
ers of mast cells in the perifollicular infiltrate was Hypochromic vitiligo/vitiligo minor is consid-
a surprising finding. It was speculated that follicu- ered as a rare form of vitiligo, so far described
lar vitiligo might bridge vitiligo and alopecia only in dark-skinned individuals, characterized
areata and that the absence of follicular melano- by the presence of hypopigmented lesions alone
cytes and precursors may affect prognosis [29]. or associated with suggestive achromic macules,
which arise without any preceding clinical
inflammation. A recent international series of 24
8.6.2 Hypopigmented Vitiligo/ patients has been published [30].
Vitiligo Minor In this series, the diagnosis was made in
adulthood, but one-third of patients had disease
Hypopigmented macules occur at onset of vitiligo/ onset before the age of 20 years. The mean dis-
NSV or when vitiligo is unstable and spreading. ease duration was 12.3 years (range 1–42). All
Wood’s lamp examination allows discrimination patients had skin types V and VI. The pattern of
between hypopigmentation and completely depig- distribution was highly similar in most of the
mented lesions, which are highly suggestive of patients with involvement of seborrhoeic areas,
vitiligo. However, hypopigmentation should associated with multiple isolated hypopig-
prompt consideration of differential diagnoses, mented macules predominantly involving the
ERRNVPHGLFRVRUJ
systematic review of the biopsy material could be

done. No atypical lymphocytes located at the
dermo-epidermal interface were observed.
Of note, one of the published patients fol-
lowed at Bordeaux University hospitals, who had
serial biopsies, developed full-blown mycosis
fungoides in the year following the publication of
the paper (Fig. 8.7). The diagnosis of hypopig-
mented vitiligo remains thus very challenging
and should be restricted to cases with long-term
observation (more than 5 years) and serial nega-
tive biopsies for CTCL. Several recent observa-
tions point the difficulty of the differential
[31–34].
8.6.3 Vitiligo Guttata/Punctata,

Leukoderma Punctata,
and Confetti Leukoderma
This entity was described by Falabella et al. in

13 patients with vitiligo (one segmental, four
focal, eight generalized), most of them female
children, who developed numerous punctate
Fig. 8.7 Case diagnosed as hypopigmented vitiligo with hypopigmented and achromic spots, 0.5–
a diagnosis of mycosis fungoides after longer follow-up 1.5 mm in diameter located primarily on the
sun-exposed areas of the extremities, following
treatment with PUVASOL [35]. Two of these
scalp, the limbs, and/or the trunk. None of the patients improved subsequently, whereas the
patients had clinical signs of inflammation, scal- remaining patients showed a stable clinical
ing or hypoesthesia, or a clear/pronounced skin course. Immunohistochemistry showed
infiltration. Leukotrichia was absent. Completely decreased numbers of functional melanocytes,
depigmented isolated macules were observed in marked reduction of melanin at Fontana-
only five patients (21%). Wood’s lamp examina- Masson stain, and ultrastructural studies dem-
tion confirmed the hypopigmentation and the onstrated degenerative damage of keratinocytes
absence of fluorescence due to fungi. Twenty- and melanocytes of the type already reported
one patients were treated with conventional vit- in vitiligo. The phototoxic effect of PUVASOL
iligo treatments, including narrowband therapy was suggested as a possible etiologic
ultraviolet B, PUVA, topical corticosteroids, factor in these patients. The link vitiligo-gut-
and tacrolimus. Response to treatment was tata hypomelanosis needs further investigation
overall very poor. especially if the association occurs [36], and
Histopathology was performed in 18 patients. biopsies are needed to make a definitive diag-
In all cases the patterns were similar, with an irreg- nosis. In particular cases of amyloidosis with
ular decrease of melanin associated with a decrease confetti-like leukoderma have been described
in the number of melanocytes in hypopigmented [37]. In addition, difficult to classify true punc-
areas, compared with the surrounding normal- tate vitiligo lesions on photoexposed areas may
looking skin. Inflammation was said not promi- occur even without a context of immunother-
nent in pathology reports, but unfortunately no apy for melanoma or use of psoralens.
ERRNVPHGLFRVRUJ
90 A. Taïeb et al.
8.7 Summary and Concluding References

Remarks
1. Taïeb A, Picardo M, VETF Members. The defini-
tion and assessment of vitiligo: a consensus report of
Clinically inflammatory vitiligo is a rare clinical the Vitiligo European Task Force. Pigment Cell Res.
presentation of common vitiligo, described so far 2007;20:27–35.
mostly in the context of vitiligo/NSV. When asso- 2. Buckley W, Lobitz W. Vitiligo with raised inflamma-
ciated with other cutaneous diseases, it may cor- tory border. Arch Derm Syph. 1953;67:316–20.
3. Garb J, Wise F. Vitiligo with raised borders. Arch
respond to a particular mode of onset of Derm Syphilol. 1948;58:149–53.
depigmentation triggered by local inflammation 4. Wise F. Leukoderma with inflammatory borders. Arch
different from that of Sutton’s (Chap. 9) or even Derm Syph. 1942;45:218–9.
Koebner’s phenomena (Chap. 11). Overlap with 5. Duvic M, Rapini R, Hoots WK, et al. Human immu-
nodeficiency virus-associated vitiligo: expression of
Sjögren’s syndrome cutaneous involvement or autoimmunity with immunodeficiency? J Am Acad
subacute lupus needs to be investigated. When Dermatol. 1987;17:656–62.
associated with HIV infection, it has a clearly dis- 6. Grandhe NP, Dogra S, Kumar B. Spontaneous
tinct clinical presentation for which the outcome repigmentation of vitiligo in an untreated HIV-
positive patient. J Eur Acad Dermatol Venereol.
of severe cutaneous inflammation leads to a vitil- 2006;20:234–5.
igo-like disorder, difficult to distinguish from true 7. Niamba P, Traoré A, Taieb A. Vitiligo sur peau noire
vitiligo/NSV. However, besides such rare observa- associée au VIH et repigmentation lors du traitement
tions, consistent histopathological findings in still antiretroviral. Ann Dermatol Venereol. 2007;134:272–6.
8. Tsuboi H, Yonemoto K, Katsuoka K. Vitiligo with
pigmented progressing borders in NSV and SV inflammatory raised borders with hepatitis C virus
[26] suggest that common vitiligo could be con- infection. J Dermatol. 2006;33:577–8.
sidered as a clinically silent chronic microinflam- 9. Weisberg EL, Le LQ, Cohen JB. A case of simul-
matory skin disorder, clinically visible forms taneously occurring lichen sclerosus and segmen-
tal vitiligo: connecting the underlying autoimmune
being the emerged tip of an iceberg [38]. New pathogenesis. Int J Dermatol. 2008;47:1053–5.
basic science information has given clues to 10. Sugita K, Izu K, Tokura Y. Vitiligo with inflammatory
inflammation triggers in vitiligo such as inflamma- raised borders, associated with atopic dermatitis. Clin
some activation and iHSP 70 production [39–41]. Exp Dermatol. 2006;31:80–2.
11. Tsuruta D, Hamada T, Teramae H, et al. Inflammatory
Why the inflammation is usually silent remains a vitiligo in Vogt-Koyanagi-Harada disease. J Am Acad
mystery. This could eventually relate either to a Dermatol. 2001;44:129–31.
particular inflammatory cytokine profile of vitil- 12. Wong SS, Ng SK, Lee HM. Vogt-Koyanagi-Harada
igo, which would provoke minimal inflammation Disease: extensive vitiligo with prodromal general-
ized erythroderma. Dermatology. 1999;198:65–8.
and pruritus, or to the loss of melanocyte as pro- 13.
Gunasekera N, Murphy GF, Sheth VM.
viders of mediators of an inflammatory response Repigmentation of extensive inflammatory vitiligo
which would be lost in vitiligo, or to both phenom- with raised borders using early and aggressive treat-
ena. In this respect, the “neuron of the skin” view ment. Dermatology. 2015;230(1):11–5.
14. Michaëlsson G. Vitiligo with raised borders. Report
of the melanocyte needs probably to be re- of two cases. Acta Derm Venereol. 1968;48:158–61.
investigated more closely in terms of production 15. Ortonne JP, Baran R, Civatte J. Vitiligo with an

of cytokines and neuropeptides mediating inflam- inflammatory border. A propos of 2 cases with review
matory skin responses. of the literature (18 cases). Ann Dermatol Venereol.
1979;106:613–5.
For the rare variants discussed in this chapter, 16. Petit T, Cribier B, Bagot M, Wechsler J. Inflammatory
there is still a significant gap of knowledge to fill vitiligo-like macules that simulate hypopigmented
with new investigations. The links vitiligo-guttata mycosis fungoides. Eur J Dermatol. 2003;17:410–2.
hypomelanosis-UV exposure, vitiligo-CTCL, 17. Swick BL, Walling HW. Depigmented patches with
an annular inflammatory border. Clin Exp Dermatol.
follicular vitiligo-alopecia areata are clearly 2008;33:671–2.
important to consider in future studies.
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18. Shin J, Lee JS, Kim MR, Kim do Y, Hann SK, Oh vitiligo: delineation of a new entity. Br J Dermatol.
SH. New suggestive clue to the mechanism of vit- 2015;172(3):716–21.
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kocytoclasis at the erythematous rim. J Dermatol. A. Childhood hypopigmented mycosis fungoides:
2013;40(6):488–90. a commonly delayed diagnosis. BMJ Case Rep.
19. Trikha R, McCowan N, Brodell R. Marginal vitiligo: 2014;2014:pii: bcr2014208306.
an unusual depigmenting disorder. Dermatol Online J. 32.
Ranawaka RR, Abeygunasekara PH, de Silva
2014;21(3):pii: 13030. MV. Hypopigmented mycosis fungoides in type v
20. Yagi H, Tokura Y, Furukawa Y, Takigawa M. Vitiligo skin: a report of 5 cases. Case Rep Dermatol Med.
with raised inflammatory borders: involvement of T cell 2011;2011:190572.
immunity and keratinocytes expressing MHC class II 33. Soro LA, Gust AJ, Purcell SM. Inflammatory vit-
and ICAM-1 molecules. Eur J Dermatol. 1997;7:19–22. iligo versus hypopigmented mycosis fungoides in a
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with raised borders associated with Sjögren’s syn- 34. Tolkachjov SN, Comfere NI. Hypopigmented myco-
drome. Clin Exp Dermatol. 2009;34(3):418–20. sis fungoides: a clinical mimicker of vitiligo. J Drugs
22. Annessi G, Paradisi M, Angelo C, et al. Annular
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2003;49:1029–36. JA. Leukoderma punctata. J Am Acad Dermatol.
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Int J Dermatol. 2008;47:663–9. M. Confetti-like lesions with hyperkeratosis: a novel
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Halo Nevus, Leucotrichia
and Mucosal Vitiligo
9
Contents
9.1 Clinical Features 94
9.1.1 Halo Nevi 94
9.1.2 Leukotrichia 95
9.2 Pathogenesis 96
9.2.1 Halo Nevus 96
9.3 Histopathology 97
9.3.1 Halo Nevus 97
9.4 Association with Vitiligo 98
9.4.1 Halo Nevus 98
9.5 Diagnosis 100
9.5.1 Halo Nevus and Leukotrichia 100
9.6 Treatment 100
9.6.1 Halo Nevus 100
References 101
Abstract general, the diameter of halo correlates with

Halo nevus is defined as a well-defined, sym- the diameter of the nevus.
metrical, round or oval, hypopigmented, or Leukotrichia refers to whitening of the hair
depigmented rim/halo around a central mela- associated with a depigmented vitiligo patch.
nocytic nevus. The size of nevus and ring can Leukotrichia, poliosis, and canities are all
vary from few millimeters to centimeters. In characterized by whitening of the hair. Poliosis
represents a circumscribed collection of white
hair. It may or may not be associated with
whitening of the underlying skin. Vitiligo may
A. Bishnoi · D. Parsad (*)
Department of Dermatology, Postgraduate Institute of
present with both leukotrichia and poliosis,
Medical Education and Research, Chandigarh, India and midline frontal hair poliosis identical to

ERRNVPHGLFRVRUJ
94 A. Bishnoi and D. Parsad
piebaldism can be seen in vitiligo. Canities

refers to premature and generalized graying of
hair in the scalp and beard.
The incidence of halo nevus and leuko-
trichia has been reported to be 4.4% and
12.3%, respectively.
In this chapter, we shall discuss about halo
nevus, mucosal vitiligo and leucotrichia; and
their significance in relation to vitiligo.
Key Points Fig. 9.1 Depigmented patch on shin. Some of the hair is
• Halo nevi commonly develop in chil- still pigmented, while majority are depigmented
dren or young adults having multiple
melanocytic nevi. 15 years [2]. Approximately 20% of those having
• Careful examination of the central nevus halo nevi have vitiligo. Association with other
is required to rule out melanoma, espe- autoimmune diseases like alopecia areata,
cially in elderly. Hashimoto’s thyroiditis, celiac disease, and intense
• Leukotrichia is considered to be a sig- sun exposure has been reported [2]. Familial ten-
nificant marker of segmental vitiligo. dency to develop halo nevi has also been reported.
• Presence of halo nevi and leukotrichia is They commonly appear on the trunk, espe-
considered to be a significant predictor cially the upper back. It can occur in several types
of evolution from segmental vitiligo to of melanocytic lesions, including acquired mela-
mixed one. nocytic nevus (junctional or dermal), congenital
• Presence of premature hair graying has melanocytic nevus (CMN), dysplastic nevus,
been found to be an important risk fac- blue nevus, Spitz nevus, melanoma [3], and
tor for development of vitiligo in subse- dermatofibroma.
quent generations. Careful examination of the central nevus is
therefore required to rule out melanoma, espe-
cially in elderly. Halo nevus around melanoma is
generally irregular and asymmetrical as com-
9.1 Clinical Features pared to other nevi [3].
In a recent study [4] evaluating the incidence
Halo nevus refers to a well-defined, symmetrical, of depigmentation in the presence of large CMN,
round or oval, hypopigmented or depigmented it was observed that 8 of 92 patients developed
ring/halo around a central melanocytic nevus. depigmentation associated with nevus, with six
The size of nevus and the depigmented ring can developing depigmentation inside nevus, one
vary from few millimeters to centimeters. In gen- developing halo nevi around a satellite nevus, and
eral, the diameter of depigmented halo correlates one developing vitiligo-like depigmentation.
with the diameter of the nevus. In another report [5], the authors observed
spontaneous repigmentation of periorbital vitil-
igo in a girl after excision of her CMN with a
9.1.1 Halo Nevi halo nevus around it.
Halo nevi have been found to be significantly
Halo nevi [1] (Fig. 9.1) commonly develop in chil- associated with Turner’s syndrome and
dren or young adults having multiple melanocytic HLA-Cw6 locus [6].
nevi. Incidence in individuals below 20 years of Four stages in its natural history have been
age is around 1% with mean age of onset being described:
ERRNVPHGLFRVRUJ
9 Halo Nevus, Leucotrichia and Mucosal Vitiligo 95
1. Development of depigmented/hypopigmented
halo around the nevus
2. Development of hypopigmentation/depig-

mentation in the central nevus which becomes
pinkish in color
3. Disappearance of the nevus
4. Disappearance of halo and subsequent repig-
mentation, which may take years to complete
Multiple nevi in different stages can be seen in

the same individual.
Aouthmany M et al. [7] observed that 51% of
the halo nevi demonstrate no change in the halo
or nevus after an average of 4.2 years; 14.3%
demonstrated partial nevus regression with per- Fig. 9.2 Segmental vitiligo involving face. Leucotrichia
sistence of the halo after an average of 6.7 years; and poliosis both are visible
4.1% demonstrated complete involution of the
nevus with persistent halo depigmentation after
an average of 7.7 years; 8.2% demonstrated com- affected quite early and prominently in segmen-
plete nevus involution with some repigmentation tal vitiligo (Fig. 9.2). A clinic-epidemiological
of the halo after an average of 11.8 years; and study conducted in North India assessed 762 vit-
22.4% demonstrated complete resolution of the iligo patients and found the prevalence of leuko-
nevus with complete repigmentation of the halo trichia to be 33.5% [8]. Another study carried out
after an average of 7.8 years. The authors demon- in Turkey found incidence of leukotrichia to be
strate that natural history of halo nevi is long, and 3.38% in 148 patients [9].
patients should be educated about the same in Leukotrichia was found to be a significant
order to avoid undue apprehension and surgical marker of segmental vitiligo in a study assessing
procedures for halo nevi. the characteristics of segmental vitiligo in 188
patients, with 162 patients (86.1%) having leuko-
trichia, and 70 of these had leukotrichia in all hair
9.1.2 Leukotrichia of the lesion, whereas 92 had both white and
black hair in the lesion. In the same study, 12
Leucotrichia refers to whitening of hair associ- patients did not have leukotrichia, and 122
ated with a depigmented vitiligo patch. patients had leukotrichia extending beyond the
Leucotrichia, poliosis and canities are all char- margins of vitiligo patch [10].
acterized by whitening of hair. Poliosis repre- A study evaluated the percentage of leuko-
sents a circumscribed collection of white hair. trichia in 82 patients with segmental vitiligo and
It may or may not be associated with whitening found that all 82 patients had leukotrichia, though
of underlying skin. Vitiligo may present with the percentage of white hair in the patch varied in
both leucotrichia and poliosis, and midline different patients. While evaluating, the authors
frontal hair poliosis identical to piebaldism can used digital microscope with 30× magnification
be seen in vitiligo. Canities refers to premature for patches where they could not identify leuko-
and generalized graying of hair in scalp and trichia with the naked eye/magnifier. They also
beard. observed that due to underlying depigmentation
Incidence varies in different studies, and over- in vitiligo patch, leukotrichia can be difficult to
all, leukotrichia has been observed significantly notice, especially if hair are vellus. But leuko-
more commonly in segmental vitiligo than non- trichia in segmental vitiligo involving terminal
segmental vitiligo (NSV). Also, the hair is hair of the scalp, eyelashes, and eyebrows is
ERRNVPHGLFRVRUJ
u sually readily apparent and causes significant resemble epidermal melanocytes and are poly-
psychosocial distress by virtue of its location on dendritic, pigmented, and DOPA (dihydroxyphe-
exposed areas [11]. nylalanine) positive, whereas those in bulge are
bipolar, nonpigmented, and DOPA negative.
Melanocytes in bulb near the dermal papilla are
9.2 Pathogenesis responsible for the pigmentation of the hair.
Melanosomes produced by these melanocytes are
9.2.1 Halo Nevus taken up by keratinocytes of the hair shaft, result-
ing in pigmented hair fiber.
It was initially believed that halo nevus occurs due Follicular melanogenesis is strongly coupled
to immune response against dysplastic melano- to hair cycle, in contrast to epidermal melanogen-
cytes developing within the nevus. But histopathol- esis, which is continuous. Anagen phase is asso-
ogy did not reveal dysplasia in the nevus component. ciated with pigmentation of the hair, whereas
It is now believed that it arises because of the initiation of catagen marks a decline in the func-
destruction of nevus cells by cytotoxic T cells that tion of melanocytes in the follicle. The regenera-
get activated against specific antigens of normal tion of the follicular melanocytes in the next
nevus melanocytes. This cytotoxic T cell response cycle results from migration of amelanotic mela-
can even eliminate the existing nevus as seen in nocyte precursor stem cells located in the outer
stage 3 of normal evolution. A recent case report root sheath that have high regenerative potential
described the disappearance of a medium-sized [20–22]. It is generally presumed that melano-
CMN after formation of halo nevus around it [18]. cytes present in hair follicles are protected from
Halo around the nevus is suggested to develop immune damage by virtue of their protected
because of two mechanisms: niche inside hair follicles. But they can get tar-
geted as well, resulting in white hair in a vitiligo
1. IFN-γ-mediated apoptosis of surrounding patch, referred to as leukotrichia.
skin melanocytes Gan and colleagues have reported eight cases
2. Direct destruction of surrounding skin mela- of follicular vitiligo. These patients had classical
nocytes by T lymphocytes after they get acti- depigmented vitiligo patches on the body, and in
vated against the antigens of nevus addition, there was depigmentation involving
melanocytes hair follicles without involvement of the skin
underneath, resulting in leukotrichia on clinically
There are reports of partial regression of pig- normal skin. Histopathology showed loss of
mented nevi without the formation of halo, but melanocytes from hair follicles, and the authors
associated with generalized vitiligo [19]. proposed that the entity “follicular vitiligo”
might serve as the pathological link between alo-
pecia areata and vitiligo [23].
9.2.2 Leukotrichia Four patients developed vitiligo-like leuko-
derma with one having leukotrichia also, follow-
Melanocytes are the dendritic cells derived from ing psoralen and ultraviolet A (PUVA)
the neural crest that form melanin and provide phototherapy treatment for mycosis fungoides.
the skin and hair with color. They are located in Vitiligo patches had developed at the sites of
the basal layer of the epidermis and matrix of hair mycosis fungoides plaques, and authors proposed
follicles. Melanocytes residing in hair follicles that induction of cell-mediated immunity against
are larger, more dendritic, and metabolically tumor cells had resulted in cytotoxicity against
more active than those present in the interfollicu- melanocytes as well as causing vitiligo-like
lar area. depigmentation [24].
Melanocytes in hair follicle are present in the Leukoderma and leukotrichia have been
infundibulum, bulge and sub-bulge, and follicu- reported in two patients following PUVA treat-
lar bulb. The ones in the infundibulum and bulb ment for GVHD. The authors proposed that
ERRNVPHGLFRVRUJ
immune-mediated destruction of cells was the occurrence of white hair in an individual

responsible for both GVHD and associated leu- younger than 20 years in Whites, 25 years in
koderma and leukotrichia [25]. Leukotrichia has Asians, and 30 years in Africans. Premature
been reported with nevus depigmentosus and dis- graying usually causes low self-esteem in the
coid lupus erythematosus [26, 27]. The use of affected individual. Premature canities can be
chloroquine has been reported to result in leuko- seen in some autoimmune disorders and prema-
trichia totalis [28]. ture aging syndromes, and can be isolated or
Two patients having halo nevi were evaluated idiopathic. Some studies have seen the associa-
using portable digital microscope, and leuko- tion between canities and osteopenia and car-
trichia was visible in both, though it was not vis- diovascular disorders [32]. Others have tried to
ible with the naked eye. The authors proposed identify underlying biochemical abnormalities
that hair follicle melanocytes are also targeted in like decreased ferritin and vitamin B12 and
halo nevi formation, in association with epider- high-density lipoprotein cholesterol levels in
mal melanocytes [29]. individuals with premature canities [33].
Poliosis in alopecia areata: Pigmented hair is Smoking, obesity, and family history of prema-
preferentially affected in alopecia areata, and ture canities were seen to be associated with
regrowing hair is initially white. It can result in premature graying of the hair in another study
circumscribed patch with sparse white hair caus- [34].
ing poliosis. Various morphological changes have
been observed that suggest active involvement of
melanocytes in alopecia areata [20]. 9.3 Histopathology
Colocalization of vitiligo and alopecia areata in a
patch on the frontal scalp has been reported. The 9.3.1 Halo Nevus
patch had depigmented base, suggestive of vitil-
igo; and the hair was relatively decreased in Histology of the central nevus in a halo nevus
patch. Biopsy and immunohistochemistry reveals dense infiltration by antigen-presenting
revealed loss of melanocytes in the epidermis and cells and lymphocytes around and inside the
melanocyte stem cells in the outer root sheath dermal component of the nevus (sometimes
suggestive of vitiligo and peribulbar infiltrate even inside the junctional component) and
suggestive of alopecia areata [30]. degenerative changes in the melanocytes like
Poliosis can be idiopathic or can be seen in vit- vacuolar changes and pyknotic nuclei. Histology
iligo, alopecia areata, halo nevi, Vogt-Koyanagi- of halo component reveals very few or absent
Harada syndrome, piebaldism, Waardenburg lymphocytes.
syndrome, and many other hereditary and acquired Most of the infiltrating lymphocytes are CD8+
conditions that result in abnormal melanocyte cytotoxic T lymphocytes [35]. Humoral response
mobilization during embryogenesis or their in the form of circulating antibodies against
destruction later on. It is usually described as a melanocytes has been observed, but this seems
forelock of white hair on the frontal scalp, but can secondary to destruction of melanocytes by T
occur virtually anywhere on the body, resulting in cells, rather than a primary response.
a circumscribed collection of white hair. Leukoderma associated with melanoma can
Canities is defined as generalized graying of be of three types [36]:
the hair. The pathogenesis of canities has been
linked to oxidative stress that causes depletion 1. Associated with regression of melanoma,

of melanocytes and their precursor stem cells. which is considered to be a poor prognostic
The process is usually gradual and correlated factor since it implies increase in depth of
well with chronological aging that results in a melanoma. Depigmentation and white-red
combination of depigmented and pigmented areas developing within a melanoma lesion
hair on scalp resulting in the so-called graying characterize this type of leukoderma.
of the hair [31]. Premature canities is defined as 2. Halo nevus around melanoma.
ERRNVPHGLFRVRUJ
3. Generalized leukoderma/vitiligo associated

ferent prevalence of vitiligo developing in a
with primary melanoma elsewhere. patient having halo nevi or vice versa.
Development of halo nevi is associated with less
Halo nevus associated with nevus is character- extensive vitiligo, although halo nevi have been
istically more inflammatory and minimally described in both segmental and non-segmental
fibrotic than regressing stage of melanoma, vitiligo.
which is associated with significant fibrosis, Presence of halo nevi and leukotrichia was
more activated melanocytes, and rich cytokine found to be a significant predictor of evolution
milieu including more fibrogenic cytokines in from segmental vitiligo to mixed vitiligo
melanoma component [37]. [12–14].
A retrospective study assessing 208 pediatric
vitiligo patients observed that 55 (26%) had halo
9.4 Association with Vitiligo nevus, and presence of halo nevus was associated
with male gender, generalized vitiligo, develop-
Presence of halo nevi and leukotrichia was found to ment of vitiligo at a later age, and statistically
be a significant predictor of evolution from segmen- nonsignificant yet increased chances of repig-
tal vitiligo to mixed vitiligo in a study [12–14]. mentation; but it was not found to be associated
Another study assessing the prognostic factors with increased rate of progression of the disease
for progression of vitiligo observed that [38].
Koebner’s phenomenon, positive family history, A case series of nine patients reported devel-
and mucosal involvement are poor prognostic opment of both halo nevi and vitiligo in patients
factors and associated with significant progres- having CMN [39].
sion of the disease. Leukotrichia, late age at A study [40] assessing the role of CMN in
onset, and longer duration of disease were not 1004 vitiligo patients found that presence of these
found to be associated significantly with progres- nevi was associated with significantly increased
sion of the disease [15]. incidence of halo phenomena, earlier age of onset
Presence of premature hair graying was found of vitiligo, development of acral/joint lesions, and
to be an important risk factor for the develop- Koebner’s phenomenon. This study found CMN
ment of vitiligo in subsequent generations [16]. in 3.3% of vitiligo patients and 1% of control
In another study assessing antithyroid hormone patients. Halo nevi were seen in 30.3% of the
autoantibodies in vitiligo, it was observed that cases and were positively associated with the
anti-T3 antibodies are significantly associated diameter of the nevus. Presence of CMN was not
with leukotrichia, and anti-T4 antibodies are asso- found to be associated with progression of dis-
ciated with vitiligo duration and activity [17]. ease, family history, or autoimmune diseases.
Since repigmentation in a vitiligo patch Another case report [41] demonstrated the
depends predominantly on the activity of hair fol- development of localized sacral and ankle vit-
licle melanocytes, presence of leukotrichia is iligo in a patient having halo nevus associated
considered a bad prognostic marker by virtue of with CMN and spontaneous partial repigmenta-
deficient melanocyte reservoir and decreased tion in both halo nevi and vitiligo a few years
potential of repigmentation. later.
In a latent class analysis, it was observed that
prepubertal onset of vitiligo (<12 years of age)
9.4.1 Halo Nevus was significantly associated with halo nevi,
Koebner’s phenomenon, atopic dermatitis, previ-
Relationship between halo nevus and vitiligo is ous spontaneous repigmentation, family history
unclear. Vitiligo and halo nevi can appear sepa- of vitiligo, and early onset graying of hair [12,
rately or together. Different studies report dif- 42].
ERRNVPHGLFRVRUJ
Van Geel et al. [43] propose that analogous nevi does not correlate with subtype, extent,
to generalized depigmentation associated with and progression/activity of NSV. Halo nevi
melanoma, halo nevi can also trigger additional alone group had significantly less association
leukoderma-/vitiligo-like lesions, and this is an with Koebner’s phenomenon, associated auto-
entity distinct from vitiligo with respect to immune diseases and family history of vitiligo,
lesion distribution, extent, and prognosis. They and significant positive association with multi-
characterize this halo nevi-associated leuko- ple nevi.
derma and point out that these patients are Patrizi et al. [45] studied the difference
younger and have asymmetric, limited-sized between 98 patients having halo nevi alone and
subtle lesions with ill-defined borders that show 27 patients having halo nevi associated with
lack of progression and absence of Koebner’s vitiligo. They observed that two patients in halo
phenomenon, other autoimmune diseases and nevi alone group developed vitiligo at follow-
family history of vitiligo, have significantly up period of >5 years (both had multiple halo
larger number of halo nevi (>3) and temporal nevi). While in those having both halo nevus
correlation of development of vitiligo-like and vitiligo, 11 patients developed both simul-
lesions with halo nevi, and hence, proposed that taneously, seven developed vitiligo first, and
halo nevi-associated leukoderma is a separate nine developed halo nevi first followed by vit-
entity and has a distinct pattern from vitiligo iligo later (all of these nine patients had multi-
per se. ple halo nevi). They also observed that presence
In a prospective study evaluating 553 patients of both halo nevi and vitiligo was associated
with non-segmental vitiligo (NSV), 130 patients with multiple nevi and associated autoimmune
with halo nevi (NSV-HN) and 423 without halo diseases in patients and families. At follow-up
nevi (NSV) were compared. It was observed period of 5 years, 18 of 52 available halo nevi-
that presence of halo nevus is positively associ- only patients developed multiple nevi. In vitil-
ated with family history of premature graying of igo and halo nevi group, all nine patients where
the hair; trunk involvement; age of onset vitiligo was preceded by halo nevi had multiple
<18 years; phototypes I, II, and III; and depig- nevi.
mentation pattern (lesser staging, lesser leuko- HLA typing was compared between NSV
trichia), whereas no association was seen with alone and that associated with halo nevi, and
hand and feet involvement and total affected it was observed that both subsets have distinct
area [13]. The authors proposed strong associa- HLA associations and might represent differ-
tion between NSV-HN and premature hair gray- ent entities with distinct pathogenesis [46].
ing and concluded that NSV-HN most commonly A study [47] assessed the ultrastructural dif-
occurred on the trunk, had a central distribution, ferences between mitochondria in the perile-
and spared the hand and feet, implying that sional skin of halo nevi and vitiligo and found
NSV in isolation preferentially affects interfol- significant differences and thus concluded that
licular epidermal melanocytes, whereas both achromic lesions might actually have differ-
NSV-HN involves hair follicular melanocytes ent pathogenic mechanisms.
preferentially. Another study evaluated hydrogen peroxide
In another study by van Geel et al. [44] com- concentration in halo of halo nevi and vitiligo
paring halo nevi alone, NSV alone, and halo and concluded on biochemical basis that these
nevi with NSV, it was observed that presence of entities are distinct [48].
halo nevi in NSV is significantly associated In a study comparing NSV and SV, it was
with younger age of onset and reduced risk of observed that halo nevi are significantly more
autoimmune diseases. They also observed that associated with NSV than SV [49].
in 61% of NSV cases, halo nevi preceded the Halo nevus has been associated with Vogt-
development of vitiligo, and presence of halo Koyanagi-Harada syndrome [50].
ERRNVPHGLFRVRUJ
9.5 Diagnosis ing vellus hair using dermatoscopy, before initi-

ating treatment, because of inherent poor
9.5.1 Halo Nevus and Leukotrichia response in such patients by virtue of minimum
repigmentation potential [53].
Diagnosis of halo nevus and leukotrichia is usu- Surgical treatment options like tissue grafts
ally straightforward, and primarily clinical. Is and follicular unit transplants have been used in
usually straightforward, but Wood’s lamp exami- treating leukotrichia. Eight patients with stable
nation and dermatoscopy may be required to vitiligo patches having leukotrichia affecting the
detect if clinical features are subtle, especially in scalp, beard, and eyebrows were treated with
lighter skin tones. split-thickness skin grafting, and it was observed
that response was earlier (by 3 months) and
greater (75–90% repigmentation) in eyebrows as
9.6 Treatment compared to the scalp and beard, where it was
late and lesser (6–9 months, 50–60%). Hair fol-
9.6.1 Halo Nevus licle melanocytes are responsible for perifollicu-
lar pattern of repigmentation observed in vitiligo.
Patient education about natural history of halo Narrowband ultraviolet B phototherapy results
nevi and reassurance is required in majority of primarily in this pattern of repigmentation, by
cases. activation and migration of hair follicle melano-
Non-cultured epidermal cell grafting has been cytes (anterograde repigmentation). But after
successfully used in the treatment of 11 patients split-thickness grafting, the epidermal repigmen-
with halo nevi, and repigmentation thus achieved tation is followed by subsidence of leukotrichia,
was maintained after a mean follow-up of 4 years. implying reverse migration of melanocytes from
Excellent repigmentation was achieved in halo the repigmented epidermis to follicular ostia,
nevi [51]. outer root sheath, and bulb [54]. Follicular unit
Excimer laser has been successfully used in transplantation was done in stable vitiligo
patients having facial halo nevi [52]. patients, and 11 of 46 patients with leukotrichia
Individuals developing halo nevus after responded well [55].
40 years of age should be screened for melanoma Non-cultured cellular grafts were successfully
(ocular, cutaneous, and mucosal). employed in the treatment of leukotrichia in gen-
eralized and segmental vitiligo with good to
excellent repigmentation (>50%) achieved after a
9.6.2 Leukotrichia period of 9–12 months, though initial response at
3 months was poor. Out of 11 cases in this study,
Leukotrichia is difficult to treat. When affecting nine had eyebrow leukotrichia and two had leg
vellus hair, it might be difficult to appreciate leuko- and scalp leukotrichia. The authors concluded
trichia on top of depigmented vitiligo patches. But that good to excellent repigmentation can be
when terminal hair of the scalp, beard, eyebrows, achieved in patients having leukotrichia with cel-
and eyelashes are affected, camouflage is also dif- lular grafts, thus obviating the need for hair trans-
ficult. Even after repigmentation of a vitiligo patch plantation [56, 57].
with medical treatment, overlying leukotrichia Eyebrow leukotrichia usually responds well to
might not improve and require transplantation of the treatment of vitiligo patch underneath, but
melanocytes. response of eyelash leukotrichia is not that well
A study employing xenon chloride excimer to the treatment of periocular vitiligo and is a
laser in vitiligo observed that presence of leuko- source for considerable psychological morbidity
trichia was associated with poor treatment out- for the patient. Fifteen patients having eyelash
comes in both segmental and non-segmental vitiligo were treated with follicular unit trans-
vitiligo alike. The authors also proposed that it plantation from temporal scalp, and good to
might be beneficial to rule out leukotrichia affect- excellent response (>50% repigmentation) was
ERRNVPHGLFRVRUJ
seen in 13 patients, whereas fair and poor 12. Ezzedine K, Diallo A, Leaute-Labreze C, Seneschal J,
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Extra-Cutaneous Melanocytes
10
Tag S. Anbar, Rehab A. Hegazy,
and Suzan Shalaby
Contents
10.1 Introduction 104
10.2 Ocular Melanocytes 105
10.2.1 he Uveal Tract
T 105
10.2.2 Retinal Pigment Epithelium 106
10.2.3 Eye Changes in Vitiligo 106
10.3 O tic Melanocytes 107
10.3.1 Ear Changes in Vitiligo 108
10.4 C ephalic Melanocytes 108
10.4.1 Cephalic Changes in Vitiligo 108
10.5 Other Extra-Cutaneous Melanocyte Populations 109
10.6 Extra-Cutaneous Melanocytes in Pigmentation-Related Syndromes 109
10.7 Conclusion 110
References 111
Abstract leus of the brain, heart, and lungs. In vitiligo,

Besides their being in the skin, melanin and although melanocyte loss has been recog-
melanocytes are detected in the eye, stria nized as being mainly restricted to the skin,
vascularis of the cochlea in the ear, lepto- extra-cutaneous melanocyte involvement and
meninges, substantia nigra, and locus coeru- subsequent extra-cutaneous site alterations
have been detected. The knowledge of extra-
cutaneous involvement may point toward
considering vitiligo as a systemic disorder
T. S. Anbar (*)
rather than just a cosmetic problem. Still, no
Department of Dermatology and Andrology, Faculty
of Medicine, Al Minya University, Al Minya, Egypt clear guidelines on who deserve screening of
their extra-cutaneous melanocyte sites in
R. A. Hegazy · S. Shalaby
Department of Dermatology, Faculty of Medicine, cases of vitiligo exist, an aim yearning to be
Cairo University, Cairo, Egypt fulfilled.

ERRNVPHGLFRVRUJ
104 T. S. Anbar et al.
10.1 Introduction
Key Points
• The extra-cutaneous melanocytes are Even though melanocyte function is well estab-
getting more under the spotlight, with lished for pigmentation and photoprotection in
the ocular, otic, and cephalic melano- cutaneous location, the existence and functions of
cytes being still on the top list. pigment cells in extra-cutaneous sites should not be
• Their entanglement in vitiligo acquired overlooked. Besides their being in the skin, mela-
more proof over the years and may point nin and melanocytes are detected in the eye [1],
toward considering vitiligo as a sys- stria vascularis of the cochlea in the ear [2], lepto-
temic disorder rather than just a cos- meninges [3], substantia nigra, and locus coeruleus
metic problem. of the brain [4], heart [5, 6], and lungs [7], and there
• Clear guidelines on who deserve screen- is evidence that they operate even in unsuspected
ing of their extra-cutaneous melanocyte territories such as adipose tissue [8, 9] (Fig. 10.1).
sites in cases of vitiligo need to be In such sites, melanocytes have been mostly recog-
established. nized for providing physiologic functions impor-
tant in organ development and maintenance [1].
Heart Eye
RPE and uveal tract
Adipose tissue Cochlea
Lung Epidermis and hair bulge C.N.S
Fig. 10.1 Diagram showing the distribution of melanocytes in various human tissues, including the skin (the main
site), eye, ear, CNS (well established), heart, adipose tissue, and lung (more recently suggested)
ERRNVPHGLFRVRUJ
10 Extra-Cutaneous Melanocytes 105
In vitiligo, although melanocyte loss has been dark brown, mainly owing to the amount of mela-
recognized as being mainly restricted to the skin, nin in the iris melanocytes [14]. Originating from
extra-cutaneous melanocyte involvement and the neural crest, these melanocytes immigrate
subsequent extra-cutaneous site alterations have during embryonic development to the uveal tract.
been detected, particularly in the eyes and ears, The microenvironment of uveal melanocytes
resulting in a possible compromise to the func- differs significantly from that of cutaneous melano-
tion of those sensory organs [10]. cytes. The uveal tract is very highly vascularized,
and the melanocytes of the choroid have ready
access to blood-borne factors. This peculiar micro-
10.2 Ocular Melanocytes environment allows the uveal melanocytes to get in
physical contact with several cellular populations,
The presence of two totally separate populations including the fibroblasts that form the sclera and the
of pigment-producing cells in one organ is unique vascular endothelial cells that form the choriocapil-
to the eye [11]. The first population is present in laris, and are typically attached to other melano-
the uveal tract, while the second exists in the reti- cytes. This comes in contrast with the cutaneous
nal pigment epithelium (RPE) [12] (Fig. 10.2). interfollicular melanocytes that are distributed sin-
gly among clusters of keratinocytes [15].
Furthermore, the anterior structures of the eye
10.2.1 The Uveal Tract that overlay the choroidal melanocytes absorb or
scatter the majority of UV, visible, and infrared
The main contingent of the ocular melanocytes radiation found in sunlight, and the melanocytes
resides in the uveal tract [1], which also happens of the choroid and ciliary body receive virtually
to be the site of most ocular melanomas [13]. The no exposure to UV or visible light. Thus, the
uveal tract is comprised of the choroid, ciliary potential for direct photodamage is greatest for
body, and iris. The latter is responsible for the interfollicular cutaneous melanocytes, followed
coloration of the eye which ranges from blue to in decreasing order by follicular cutaneous
Fig. 10.2 Anatomy of Pars plana Retina

the eye. Ocular
melanocytes reside in Choroid
Conjunctiva
two totally separate
populations, namely, the Anterior
chamber Sclera
uveal tract and the
retinal pigment
epithelium [25]
Macula
Cornea Lens
Viterous
humor
Optic
disc
lris
Ciliary body
ERRNVPHGLFRVRUJ
melanocytes and iris melanocytes [15–17]. ment membranes[26], which will make it
Further discrepancies exist in the fact that unlike clearer and still serves the meaning of its
cutaneous melanocytes which distribute pigment importance in vision [27]. Furthermore, its
to adjacent keratinocytes, uveal melanocytes melanin—similar to the function of the rest of
retain the melanosomes they produce [18]. the ocular melanin—absorbs incident light
Until recently it was believed that melanin that enters through the retina and prevents
synthesis occurred continuously in interfollicular photons from bouncing about and restimulat-
cutaneous melanocytes, cyclically during the ing or overstimulating the rods and cones
anagen phase of the hair cycle in follicular cuta- [25].
neous melanocytes, but only during prenatal
development by uveal melanocytes [19].
However, because optic treatments with the pros- 10.2.3 Eye Changes in Vitiligo
taglandin analog latanoprost result in heterochro-
mia and increased pigmentation of the iris, it Ocular pigmentary changes have been reported
became apparent that melanin synthesis can be in patients with vitiligo; in fact, 18–50% of
induced in adult eyes [20, 21]. Further evidence vitiligo patients exhibit focal hypopigmented
that uveal melanocytes retain the potential for lesions involving the iris, anterior chamber,
melanin synthesis in adults is apparent in the and RPE/choroid, particularly the fundus, on
observations that (1) induction of tyrosinase ophthalmoscopic evaluation [27–31]. These
activity by latanoprost in cultured adult human pigmentary changes are frequently associated
uveal melanocytes is blocked by the tyrosinase with vitiligo patches and macules involving
inhibitor 1-methyltyrosine, (2) the artificial mela- the eyelids, in addition to poliosis of the
nin precursor [3H]-methimazole labels the uveal eyebrows and eyelashes [27, 28]. Ocular find-
tract of pigmented adult DBA mice, and (3) ings in vitiligo are, however, not a constant
tyrosinase activity is evident in the uveal tract of finding [32].
bovine eyes [22–24]. Thus, the potential for mel- In addition, recent studies demonstrated
anin synthesis appears to be present in adult ocu- that patients with vitiligo may have more len-
lar melanocytes. ticular and retinal findings than normal [33]
and can be more prone to the dry eye syn-
drome [33, 34]. The uveal tract has been also
10.2.2 Retinal Pigment Epithelium reported to be affected in vitiligo patients in
the form of uveitis [10]. The relation between
Retinal pigment epithelium (RPE) cells are also vitiligo and primary open-angle glaucoma has
derived from the neural tube; however, this uni- recently been investigated [35], and despite
cellular layer does not migrate as neural crest the small sample size, the authors concluded
cells, but rather develops directly from the neuro- that the correlation between both diseases is
ectoderm neural tube of the developing forebrain not random. Furthermore, negative ocular
[25]. RPE is the brown monolayer of cells situ- electrophysiologic findings were demon-
ated between the choroid and the retina proper, strated in vitiligo patients, a finding that posi-
composed of cells joined by tight junctions and tively correlated with the disease severity and
filled with pigment mainly melanin and lipofus- duration [36].
cin. Pigment production in adult RPE cells Owing to the growing evidence linking
remains a matter of debate. Some authors have between ocular disorders and vitiligo, a rec-
postulated melanin continuous production ommendation of complete ophthalmologic
throughout life; others suggest that it ceases at screening of patients treated for vitiligo, irre-
birth [11]. spective of their age, sex, affected area, local-
The RPE plays a critical role in the active ization, and duration of the disease, was
phagocytosis of the photoreceptor outer seg- issued [37].
ERRNVPHGLFRVRUJ
10.3 Otic Melanocytes that are necessary for normal hearing and endo-
lymph maintenance [43, 44], and their loss results
Melanocytes are found in the stria vascularis of in deafness [2].
the cochlea. The stria vascularis forms the lateral The melanin in the otic melanocytes acts as a
wall of the scala media, which is an endolymph- free radical scavenger and a cation exchanger.
filled chamber housing the principal sound- Thereby, it has the ability to protect the cochlea by
detecting component of the inner ear, i.e., the binding to ototoxic drugs, such as cisplatin and
organ of Corti (Fig. 10.3). Otic melanocytes are aminoglycosides, which may be deleterious to the
of neural crest origin [38], and their embryonic sensory function of the cochlea [45]. Furthermore,
development is under the regulation of Kit [39] several researches have highlighted the melanin
and Sox 10 [40, 41]. Once established in the ear, involvement in the maintenance of calcium homeo-
otic melanocytes synthesize pigmented melano- stasis, via calcium ion regulation in the inner ear,
somes until congested and then halt melanin syn- which may improve hearing [46–49]. Another
thesis [42]. interesting function is the role played by the mela-
The melanocytes of the stria vascularis are nin granules produced by the melanocytes of the
termed intermediate cells and provide functions inner ear in maintaining the body balance [50].
Reissner’s membrane Scala vestibuli
Stria vascularis
Cochlear
duct
Tectorial
membrane
Spiral
limbus
Cochlear
Spiral
nerve
ligament
Basilar
membrane Scala tympani
Organ of Corti ©1997 Encyclopaedia Britannica, Inc.
Fig. 10.3 Section of cochlea. Melanocytes are situated in the stria vascularis and modiolus of the cochlea [25]
ERRNVPHGLFRVRUJ
10.3.1 Ear Changes in Vitiligo [3]. They may have several neuroendocrine
functions, through generating prostaglandin D2
Melanin—as shown above—may play a sig- (PGD2) and β-endorphin (endogenous opioid)
nificant role in the establishment and/or main- [62]. Their involvement in sleep regulation has
tenance of the structure and function of the been suggested based on the facts that PGD2 is
auditory system and may modulate the trans- a potent sleep-inducing substance [63], and
duction of the auditory stimuli by the inner ear opioid receptors are located in the nuclei that
[45–51]. Thereby, it has been hypothesized are active in sleep regulation [50]. Moreover,
that the loss of melanocytes and the subse- there are indications that a certain melanocyte-
quent decrease in melanin production, which derived factor might be involved in controlling
occurs in vitiligo, could lead to cochlear dys- the central chemosensor that generates the
function and subsequently to sensorineural respiratory rhythm [50].
hearing loss [2]; this hypothesis was the focus The melanin in the cephalic melanocytes is
of multiple studies. Although Escalente- formed from oxidation products of dopamine and
Ugalde et al. [52] failed to find a significant cysteinyl-dopamine [64] and occurs only in two
association between vitiligo and hearing loss, forms: neuromelanin and melanosomic melanin
other studies reported an incidence of 12.5– [65]. The distribution of these two types of mela-
18.9% sensorineural hearing loss in vitiligo nin is different. Neuromelanin distribution occurs
patients as evident in conventional pure tone in the zona compacta of the substantia nigra,
audiograms [51, 53]. Moreover, vitiligo locus coeruleus, dorsal motor nucleus of the
patients with normal hearing sensitivity vagus, tegmentum of the brain stem, and scat-
showed evidence of subclinical cochlear tered neurons in the roof of the fourth ventricle.
pathology, evident by absent or abnormal oto- Melanosomic melanin is normally found only in
acoustic emissions [54–56]. the leptomeninges, mostly concentrated in the
Brain stem auditory response abnormalities leptomeninges over the ventral aspect of the
have been reported in 13–16% of vitiligo medulla oblongata [66].
patients [57–59]. Significantly lower pure tone Melanosomic or true melanin is the same
thresholds to high-frequency sounds were type of melanin as that seen in the skin. However,
detected in vitiligo patients, particularly males, neuromelanin is chemically different from
compared to the normal population [60]. eumelanin and pheomelanin [67]. The functions
Furthermore, a recent study [61] showed bilat- of both melanosomic melanin and neuromelanin
eral cochlear dysfunction in 60% of vitiligo are not fully understood but have been consid-
patients with no significant difference between ered to be involved in the protection of the neu-
segmental and non-segmental types. rological tissues against metals, such as iron
Interestingly, in cases with segmental vitiligo, [68], and potentially toxic organic and inorganic
both ears were significantly affected compared cations and free radical species [69, 70], in addi-
to controls. Those findings opened new horizons tion to their well-known role as light-absorbing
toward the vital role of melanocytes and mela- photoprotectors [1]. A selective loss of dopami-
nin in cochlear functions. nergic neurons containing neuromelanin has
been reported to be associated with Parkinson’s
disease [4].
10.4 Cephalic Melanocytes
Cephalic melanocytes originate from the neural 10.4.1 Cephalic Changes in Vitiligo
crest, and get distributed through the meninges
covering the central nervous system (CNS), It is unknown whether this population of melano-
particularly within the ventrolateral leptomen- cytes is lost in vitiligo. There have been very few
inges overlying the pons and medulla oblongata anecdotal cases of patients with active vitiligo
ERRNVPHGLFRVRUJ
developing concurrent severe headaches. It is addition, adipocytic melanin has been also sug-
conceivable that the destructions of the lepto- gested to suppress the secretion of proinflamma-
meningeal melanocytes, probably by an acceler- tory molecules [8].
ated immunologic response, may induce such a
presentation [25]. Nevertheless, this finding is in
need of further confirmation. 10.6 Extra-Cutaneous
Melanocytes
in Pigmentation-Related
10.5 Other Extra-Cutaneous Syndromes
Melanocyte Populations
Hereditary pigmentary disorders, in addition to
Other melanocyte populations attracted less involving the skin, are associated with ocular pig-
attention, compared to the previously discussed mentary abnormalities, such as iris heterochro-
ones. Nevertheless, their existence points to mia or pigmentary changes in the fundus.
potential melanocyte and melanin functions that However, ocular motility defects such as strabis-
remain to be unraveled. mus and nystagmus are more commonly encoun-
First are the cardiac melanocytes, which are tered, pointing to a possible concomitant
located in the valves and septa [5, 6]. Cardiac neurologic developmental defect. Meanwhile in
melanocytes may originate from the same pre- albinos, the lack of melanin in the pigment epi-
cursor population as skin melanocytes as they thelium during development is believed to be
depend on the same signaling molecules directly responsible for both the neurologic
known to be required for proper skin melano- abnormality in the visual pathway and foveal
cyte development such as endothelin 3 [5], but hypoplasia [37].
their function in this location in humans so far Vogt-Koyanagi-Harada (VKH) syndrome,
remains obscure. They have been postulated to also known as uveo-meningo-encephalitic syn-
be involved in anti-inflammatory functions drome, is one of the acquired disorders of pig-
and in scavenging reactive oxygen species mentation [71] that is characterized by
(ROS) [9]. involvement of cutaneous, ocular, otic, and cen-
There are no melanocytes recognized in the tral nervous system melanocytes. Its frequent
lungs; however melanin is seen in lymphangi- association with uveitis suggests an inflamma-
oleiomyomatosis (LAM), a rare disease in the tory cause for the cutaneous pigmentary changes
lungs, in which muscle cells revert toward their [72]. Furthermore, an aberrant T cell-mediated
developmental origins and express some melano- immune response directed against self-antigens
cyte markers, such as tyrosinase, Pmel17, etc. present in the melanocytes has been incriminated
The resulting production and accumulation of in the pathogenesis of the disease [73].
melanin in lung tissues are eventually lethal [7], Two other rare syndromes exist in which the
exposing a dark side for melanin. involvement of the extra-cutaneous melanocytes
Melanin biosynthesis also takes place in the in pigmentary disorders is evident, namely,
visceral adipose tissue of morbidly obese Waardenburg syndrome (WS) and Alezzandrini
humans [8]. Hypothetically, the ectopic synthe- syndrome. WS was first described by an ophthal-
sis of melanin in the cytosol of obese adipocytes mologist [74] who described the association
may serve as a compensatory mechanism to act between depigmentation, deafness, neurological
as an anti-inflammatory factor and to reduce symptoms, and dysmorphology.
oxidative damage. During increases of cellular Alezzandrini syndrome, first described by
fat deposition, adipocytes become more exposed Alezzandrini and Casala in 1959, is characterized
to endogenous apoptotic signals, especially by unilateral facial vitiligo and partial poliosis
reactive oxygen species, which could be coun- with ipsilateral retinal detachment that may be
teracted by ectopically produced melanin. In combined with hypoacusis [75].
ERRNVPHGLFRVRUJ
10.7 Conclusion functions, autoimmunity, or malignancy war-

ranted this attention. Their entanglement in vit-
By all means are the cutaneous melanocytes the iligo acquired more proof over the years and
most studied among their family members. may point toward considering vitiligo as a sys-
However, the extra-cutaneous ones are getting temic disorder rather than just a cosmetic prob-
more under the spotlight, with the ocular, otic, lem. Still, no clear guidelines on who deserve
and cephalic melanocytes being still on the top screening of their extra-cutaneous melanocyte
list (Table 10.1). Their involvement in disor- sites in cases of vitiligo exist, an aim yearning
ders that may involve pigmentation, sensory to be fulfilled.
Table 10.1 Extra-cutaneous melanocytes: types, differences, and changes in cases of vitiligo
Extra-
cutaneous MC Ocular Otic Cephalic
Site of – Uveal tract – Retinal pigmented – Cochlea: stria vascularis – Meninges overlying
melanocytes epithelium (RPE) (inner ear) CNS
Melanocyte – Neural crest – Neural tube – Neural crest under – Neural crest [3]
origin Immigrate during (Directly from the regulation of Kit and Sox
embryonic neuroectoderm of the 10 [38, 39]
development [1] developing forebrain)
Melanin – Synthesis during – Debatable whether – Once established, – Neuromelanin first
synthesis prenatal life only continuous synthesis melanocytes of cochlea appears in 2–3-year-
[19] of melanin start synthesis of old human brains
– Recent evidence throughout life or it melanosomes till histologically and
show it might be ceases at birth congestion then stop [42] accumulates with
inducible in adult aging [27]
life [20, 21]
Function of – Color of the eye – Phagocytosis – Hearing sensation [2] – Neuroendocrine
MC according to iris – Retinoid metabolism – Endolymph maintenance function: PGD2,
melanocytes [14] – Phototransduction [2] β-endorphins [62]
– Free radical scavenger, – Sleep regulation [50]
protect cochlea against – Protection of neural
harmful drugs such as tissue against toxins
aminoglycosides [45] [68]
– Body balance
mechanisms [50]
Unique – Two separate populations of melanocytes Melanocytes of cochlea are – Two types of melanin:
characters in the same organ [12] known as the intermediate 1. Neuromelanin
– Melanocytes are closely attached to each cells [43, 44] and are 2. Melanosomic
other unlike cutaneous melanocytes [1] scattered between the (only in leptomeninges)
– Melanocytes retain their melanosomes, no marginal cells (epithelial in – Only melanosomic
distribution to adjacent cells [18] origin) and basal cells melanin similar to
(mesodermal in origin) cutaneous melanin
[65]
Changes in – Focal hypopigmented lesions in the iris, – Cochlear dysfunctions – Headaches in vitiligo
vitiligo anterior chamber, and RPE/choroid leading to sensorineural patients due to
[27–31] hearing loss [2, 51] immune response
– Eyelid vitiligo lesions [30] – Subclinical cochlear against
– Poliosis (eyebrows and eyelashes) [27, pathology [54–56] leptomeningeal
28], dry eye syndrome [33, 34] – Absent or abnormal melanocytes [25]
– Uveitis [10] otoacoustic emission
– Negative ocular electrophysiologic [54–56]
findings correlating with disease severity
and duration [36]
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Koebner Phenomenon
11
Nanja van Geel and Reinhart Speeckaert
Contents
11.2 Etiopathogenesis 117
11.3 Clinical Presentation 117
References 120
Abstract 11.1 Introduction

Koebner phenomenon (KP) is a well-known
entity in dermatology. It was first described in The reported incidence of KP in vitiligo ranges
1876 by Heinrich Koebner, a German derma- between 21% and 62% [2–4]. This wide range
tologist, as “the development of lesions at probably reflects different assessment methods to
sites of specifically traumatized uninvolved evaluate KP. In the majority of clinical trials, KP
skin of patients with cutaneous diseases”. is assessed only by medical history, while a phys-
Since then, this isomorphic response has been ical examination can give additional clini-
reported in a several skin diseases, such as vit- cal information. KP is reported to occur more
iligo, lichen planus, and psoriasis. In vitiligo a frequently in the non-segmental type of vitiligo
relation with disease activity has been sug- than in the segmental form of vitiligo [5].
gested, as well as its possible negative influ- The Vitiligo European Task Force (VETF)
ence on surgical treament results [1, 2]. group introduced a new assessment and classifi-
cation method for the evaluation of KP in vitiligo.
This classification includes three different sub
Key Points types of Koebner’s phenomenon: assessment by
• Vitiligo, Koebner phenomenon, Triggers, patient history (type 1), by clinical examination
Classification, Friction areas. (type 2), and by lesions resulting from experi-
mental induction (type 3) (Table 11.1) [2, 5].
In KP type 2, clinical differentiation can be
made between types 2A and 2B. In type 2A, leu-
N. van Geel, MD, PhD (*) · R. Speeckaert koderma is present on any of the following areas
Department of Dermatology, Ghent University corresponding either to areas of repeated pres-
e-mail: Nanja.vanGeel@UGent.be sure or friction (e.g., elbows and knees) or areas

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116 N. van Geel and R. Speeckaert
Table 11.1 Proposed classification by the Vitiligo European Task Force group of Koebner phenomenon [2]
Koebner
Type1 Type2 Type3

History Clinically Experimentally induced
Depigmentation after trauma during last A. Depigmentation corresponding Eliciting trauma

year? either to areas of repeated -I: repeated pressure/friction
Type of trauma: pressure or friction (elbows and -II: Superficial (epidermal) trauma
1. Physical (wound, cuts, scratching) kness) or areas of chronic friction -III: Dermo-epidermal trauma
2. Mechanical (friction) related to cloths/accessory.
3. Chemical/thermal (burns) B. Depigmentation clearly induced by
4. Allergic (contact dermatiitis or trauma (liner, punctiform, crenate)
irritant reactions (vaccination,
tattoos...)
5. Chronic pressure
6. Inflammatory dermatoses
7. Therapeutics (radiotherapy,
phototherapy,....)
van Geel N, Speeckaert R, Taieb A et al. Koebner’s phenomenon in vitiligo: European position paper. Pigment Cell
Melanoma Res 2011; 24: 564–73. Permission still needed from Pigm Mel Research
of chronic friction related to clothes or accesso- observed in KP1- and KP2B-positive patients.
ries (e.g., waistband, hips due to jeans, dorsal site Patients with KP will also be more prone to
of feet due to shoes, wrist site of a watch, etc.). In develop further depigmentation [5].
type 2B, the presence of linear, punctiform, or Furthermore, the response on topical therapy
crenate depigmentations can be seen and is sug- was evaluated. In this observational study patients
gestive of previous injury [2]. with any subtype of KP showed significantly less
With this new classification system, our group response to treatment than patients without
assessed the clinical significance of KP with KP. This negative relationship might be due to
respect to clinical profile, future disease course, the external triggering event of KP, maintaining
and treatment response [5]. The results of this the pathogenesis of vitiligo [2, 5]. In a previous
study suggested that KP, when assessed both by study, Njoo et al. already concluded that experi-
history and by clinical examination, can be used mentally induced KP may function as a clinical
as a clinical parameter. Several significant differ- indicator for assessing present disease activity
ences were found between patients with and and may predict responsiveness to therapy with
without signs of KP. It was established that the topical steroids and UVA [6]. Experimentally
affected body surface area (BSA) was signifi- induced Koebner phenomenon is however an
cantly higher when any KP subtype was present invasive procedure and may worsen further evo-
and that active disease was more frequently lution, making it difficult to perform in routine
ERRNVPHGLFRVRUJ
11 Koebner Phenomenon 117
clinical practice [5]. Moreover, our previous integrated: cytotoxic melanocyte elimination
study results suggested that KP on friction (possibly enhanced by activated pDCs sensing
areas (Type 2A) was associated with an increased self-DNA and LL37), increased oxidative stress,
prevalence of autoimmune disease [5]. defective melanocyte adhesion, and deficiency of
Whether or not the incidence of KP is linked to melanocyte growth factors [such as stem cell fac-
other factors (e.g. ethnicity, skin phototype) [5]. tor (SCF) and basic fibroblast growth factor
Several investigators described its negative influ- (bFGF)]. In the first step, common inflammatory
ence on the outcome of surgical treatments. signals are released, and in the second step, mul-
Surgical treatment are suggested when lesions tiple mechanisms are involved in the specific tar-
become therapy resistant and are clinically stable. geting of melanocytes.
However, the assessment of disease activity in vit- Skin injuries, responsible for KP in vitiligo,
iligo is still a matter of discussion [5, 7]. Attempts may be of any kind. Physical, mechanical, chem-
have been made to determine the possibility of ical, allergic, or irritant reactions and even thera-
success by using a mini-grafting test before a sur- peutics like radiotherapy or phototherapy can
gical intervention. Koebnerization at the donor cause this phenomenon. These stimuli are non-
site during this test is considered to be a warning specific and commonly induce inflammatory
sign about possible repigmentation failure after a reactions. Sometimes the distinction between
surgical procedure and thus a contraindication for koebnerization and post-inflammatory hypopig-
surgical treatment. Some researchers, however, mentation can be difficult, but in doubtful
noted depigmentation at the donor site but with cases Wood’s light examinationand and long-
appreciable perigraft pigment spread [7]. term follow-up can be helpful [2].
The possibility to actively induce koebneriza-
tion on a localized area makes it suitable and
11.2 Etiopathogenesis interesting for experimental models. In a pilot
study, our group described the proof of concept
To date, the exact etiopathogenesis of Koebner of a new experimental vitiligo induction model,
phenomenon in vitiligo is still unknown. Most which allows both to investigate the pathophysi-
clinical trials and experiments on KP have been ology of traumatic-induced depigmentation and
performed in patients with psoriasis, although to compare adequately the efficacy of treatments
some research has been performed in vitiligo. in a standardized and reproducible way [9].
Multiple mechanisms including immunological,
neural, and vascular factors leading to depigmen-
tation have been suggested to play a role [2, 5]. 11.3 Clinical Presentation
Ueki [8] classified the pathophysiology of KP in
different skin diseases in two particular steps, the In patients with vitiligo, the lesions due to this
first being the release of several common inflam- Koebner phenomenon are clinically and histo-
matory factors (e.g., TNF-α, IL1, IL6, Hsp70, logically indistinguishable from vitiligo. These
Hsp72, Hsp90, and ICAM-1). This release is trig- post-traumatic depigmented lesions can be
gered by environmental stimuli such as skin present in different clinical forms. In some
trauma. In the second step, a local flare of the cases the depigmented macules are easy to rec-
skin disease is induced by disease-specific auto- ognize because of their artifactual or elongated
antigens [8]. In our European position paper with linear shape. They correspond exactly to the
respect to Koebner phenomenon, we proposed a traumatized areas. In areas of friction (type 2A)
model of a two-step hypothesis (2) (Fig. 11.1). In these lesions may occur with a border of inter-
this model multiple etiological mechanisms are mediate pigmentation (trichrome vitiligo) [2].
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Fig. 11.1 Proposed model of the two-step hypothesis of the Koebner phenomenon in vitiligo (adapted from [2])
In a subgroup of patients, the lesions slightly tible to koebnerization are in accordance with the
exceed the dimension of trauma but still pre- typical distribution of KP observed in other
serve the morphology of a Koebner reaction. inflammatory dermatoses, such as psoriasis.
Other patients state that depigmented lesions Other areas of chronic friction, pressure, or
occur after trauma, while the depigmented area repeating movement (e.g., belt, tight underwear,
corresponds to classic vitiligo. It is suggested perioral, etc.) are predominantly affected in
that the location of vitiligo lesions is related to patients with vitiligo [5].
areas of repeated friction, for example, due to Koebner phenomenon in patients with vitil-
washing, dressing, sports, and other occupa- igo should be differentiated from post-inflam-
tional activity (Fig. 11.2). The chronicity of vit- matory hypopigmentation, a hypopigmentation
iligo can probably be explained by these following cutaneous trauma or inflammation.
koebnerization factors [2]. Post-inflammatory hypopigmentation is a com-
Manifestations of Koebner phenomenon can mon physiological phenomenon which may be
occur on the classical areas of vitiligo. The typi- caused by a partial loss of melanocytes or a
cal sites of involvement however seem to be deficient melanosome production and transfer.
related to the possibility of external injury to the Moreover, in contrary to vitiligo, this skin con-
skin. In this way locations including the hands, dition is characterized by its temporary appear-
the lower arms and legs, are often involved. Areas ance. Wood’s light examination will show a
of protected or shock-absorbing skin (e.g., haired hypomelanotic skin because melanocytes are
scalp, palmar side of hands) are less prone to koe- not totally absent in the affected area, whereas
bnerization. In vitiligo, some of the areas suscep- in vitiligo an amelanotic skin can be seen [2].
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11 Koebner Phenomenon 119
Fig. 11.2 Friction areas

on the body
Eye rubbing
Shaving
Washing, sport,
shaving
Sport, running
Watch
Friction of
clothing
Friction of
walking/running
Friction of clothing,
falling during playing
(children)
Sport activities,
friction
Friction of shoes
Although Koebner phenomenon in vitiligo has advances in our current knowledge on vitiligo but
been defined and documented a lot, many unan- also help to create novel therapies against this
swered questions still remain. More active research chronic and often psychologically devastating skin
focusing on KP in vitiligo may not only lead to disease.
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References 6. Njoo MD, Das PK, Bos JD, et al. Association of
the Koebner phenomenon with disease activity and
therapeutic responsiveness in vitiligo vulgaris. Arch
1. Sagi L, Trau H. The Koebner phenomenon. Clin
Dermatol. 1999;135:407–13.
Dermatol. 2011;29:231–6.
7. Sanjeev V, Mulekar MD, Marwan Asaad MD,
2. van Geel N, Speeckaert R, Taieb A, et al. Koebner’s
et al. Koebner phenomenon in vitiligo: not always
phenomenon in vitiligo: European position paper.
an indication of surgical failure. Arch Dermatol.
Pigment Cell Melanoma Res. 2011;24:564–73.
2007;143:799–816.
3. Mazereeuw-Hautier J, Bezio S, Mahe E, et al.
8. Ueki H. Koebner phenomenon in lupus erythema-
Segmental and non-segmental childhood vitiligo has
tosus with special consideration of clinical findings.
distinct clinical characteristics: a prospective observa-
Autoimmun Rev. 2005;4:219–23.
tional study. J Am Acad Dermatol. 2010;62:945–9.
9. van Geel N, Speeckaert R, Mollet I, et al. In vivo vit-
4. Barona MI, Arrunategui A, Falabella R, et al. An epi-
iligo induction and therapy model: double-blind, ran-
demiologic case-control study in a population with
domized clinical trial. Pigment Cell Melanoma Res.
vitiligo. J Am Acad Dermatol. 1995;33:621–5.
2012;25(1):57–65.
5. van Geel N, Speeckaert R, De Wolf S, et al. Clinical
significance of Koebner phenomenon in vitiligo. Br J
Dermatol. 2012;167:1017–24.
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Environmental Triggers
and Occupational/Contact Vitiligo
12
Charlotte Vrijman
Content
References 123
Abstract unawareness of patients. Therefore, patients

Different factors, including the genetic ones, should be educated how to avoid these known
predispose an individual to developing vitil- risk factors.
igo, but a trigger event initiates the actual
depigmentation. The triggers are environ-
mental factors that are encountered in every- Key Points
day life. When the development of vitiligo is • The triggers for vitiligo are environmen-
influenced by occupational exposures like tal factors that are encountered in every-
chemical exposure, frequent physical trauma, day life.
or sun exposure, it is called occupational or • Epidemiological studies on provoking
contact vitiligo. Phenolic/catecholic deriva- factors of vitiligo show percentages of
tives are major chemicals known to be asso- work-related factors up to 11.5%.
ciated with vitiligo since they interfere in the • Phenolic/catecholic derivatives are
melanin synthesis and induce oxidative major chemicals known to be associated
stress. Other chemicals mentioned as caus- with vitiligo. Monobenzone (monoben-
ative agents are nickel, chrome, cobalt, zyl ether of hydroquinone) is used as
leather, hair dye, cosmetics, and cleaning bleaching cream.
products, all allergens that also cause allergic • 4-TBP is a resin in neoprene adhesives
contact dermatitis by contact hypersensitiv- used in glues, rubber, and isolation
ity (CHS). Identification of provoking factors material.
and therefore risk factors for vitiligo are • Contact hypersensitivity might play a
important in preventing disease progression, role in the initiation of contact vitiligo.
although they are often not recognised due to
C. Vrijman (*)
Department of Dermatology, Ziekenhuisgroep
Twente, Hengelo, Netherlands
e-mail: c.vrijman@amc.uva.nl

ERRNVPHGLFRVRUJ
122 C. Vrijman
Vitiligo is thought to develop in persons with a could be seen as a non-segmental vitiligo with a
genetic predisposition that determines melano- known provoking factor rather than a distinctive
cyte fragility and susceptibility to precipitating type of vitiligo.
environmental factors. Hence, genetic factors Phenolic/catecholic derivatives are major
predispose an individual to developing vitiligo, chemicals known to be associated with vitiligo
but a trigger event initiates the actual depigmen- [4, 7, 8]. There is experimental evidence that cer-
tation. These triggers are environmental factors tain environmental chemicals, like phenolic/cat-
that are encountered in everyday life. However, echolic derivatives, are melanocytotoxic and can
in many cases these initiation or aggravating fac- cause skin depigmentation [4, 9]. Phenolic com-
tors have not been identified [1, 2]. In a Dutch pounds have structural similarities to tyrosine,
cohort study, the majority of the patients with vit- the substrate for tyrosinase which plays an
iligo did not mention provoking factors, probably important role in the melanin synthesis [10].
because of unawareness of patients [2]. Of the These phenolic compounds serve as alternate
ones who did mention a provoking factor, 25% substrates for the enzyme tyrosinase to be enzy-
was work related. Other studies on provoking matically converted into cytotoxic quinones and
factors of vitiligo show percentages of work- thus interfere in the melanin synthesis and
related factors up to 11.5% [1, 3]. These rates are induce oxidative stress [11]. Monobenzone
probably underestimated due to difficulty of (monobenzyl ether of hydroquinone) is such a
detection and also of unawareness of patients. phenolic derivate and is used as bleaching cream.
Unfortunately, the epidemiological data of pro- Monobenzone can induce vitiligo in susceptible
voking factors, especially on the role of contact persons by specifically inducing melanocyte-
with chemicals, is scarce. directed autoimmunity [7]. Another phenolic
When the development of vitiligo is influ- derivate is 4-tertiarybutylphenol (4-TBP) also
enced by occupational exposures like chemical known as para-tertiarybutylphenol (PTBP).
exposure, frequent physical trauma, or sun expo- 4-TBP is a resin in neoprene adhesives used in
sure, it is called occupational vitiligo [1, 4]. glues, rubber, and isolation materials and often
However, in literature other terms are also used mentioned as a causative agent for vitiligo [1, 4,
for occupational vitiligo. It is frequently called 12–14]. 4-TBP is known to be melanocytotoxic
contact vitiligo, chemically induced vitiligo, or as it interferes with melanin synthesis as it acts
chemical leukoderma which creates some confu- as a competitive inhibitor of tyrosinase-related
sion regarding terminology [4, 5]. Besides, the protein-1 (Tyrp1), a melanocyte-specific enzyme
term occupational vitiligo is often used when the [15, 16]. In addition 4-TBP is able to induce oxi-
disease is initiated after exposure to chemicals dative stress in melanocytes and apoptotic cell
that are toxic to melanocytes [1, 4]. The Vitiligo death; it increases the immunogenicity of pig-
Global Issues Consensus Conference (VGICC) mented cells and can trigger melanocyte-specific
of 2011 concluded that the term contact vitiligo autoimmunity [9, 15, 17–20]. Therefore, it is
or chemically induced vitiligo requires clearer very likely that 4-TBP could induce vitiligo in
definition by epidemiological investigations in susceptible persons. Other chemicals mentioned
at-risk populations [6]. For this definition it is as causative agents are nickel, chrome, cobalt,
important to distinguish chemically induced vit- leather, hair dye, cosmetics, and cleaning prod-
iligo, with spread of leukodermic lesions beyond ucts, all allergens that also cause allergic contact
the original contact site even after cessation of dermatitis by contact hypersensitivity (CHS) [1,
the use of chemicals, from chemical leukoderma 3, 21]. CHS upon skin contact with haptens is
which remains confined to the site of contact [2, characterized by local infiltration of CD4+
4]. Contact vitiligo or chemically induced vitil- T-cells, followed by dampening of the response
igo has been suggested as a separate type of vit- by the activation of regulatory T-cells producing
iligo [6]. However, commonly it behaves and IL-10 [22]. Though CD8+ T-cells play an impor-
reacts like non-segmental vitiligo and therefore tant role in vitiligo, CD4+ T-cells are also found
ERRNVPHGLFRVRUJ
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4. Boissy RE, Manga P. On the etiology of con-
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1. Jeon IK, Park CJ, Lee MH, et al. A multicenter collab- 18. Kroll TM, Bommiasamy H, Boissy RE, et al. 4-Tertiary
orative study by the Korean Society of Vitiligo about butyl phenol exposure sensitizes human melanocytes
patients’ occupations and the provoking factors of vit- to dendritic cell-mediated killing: relevance to vit-
iligo. Ann Dermatol. 2014;26:349–56. iligo. J Invest Dermatol. 2005;124:798–806.
2. Vrijman C, Hosseinpour D, Bakker JG, et al. Provoking 19. Mosenson JA, Eby JM, Hernandez C, et al. A central
factors, including chemicals, in Dutch patients with role for inducible heat-shock protein 70 in autoim-
vitiligo. Br J Dermatol. 2013;168:1003–11. mune vitiligo. Exp Dermatol. 2013;22:566–9.
ERRNVPHGLFRVRUJ
124 C. Vrijman
20. van den Boorn JG, Konijnenberg D, Dellemijn TA, 22. Lecart S, Boulay V, Raison-Peyron N, et al. Phenotypic
et al. Autoimmune destruction of skin melanocytes characterization of human CD4+ regulatory T cells
by perilesional T cells from vitiligo patients. J Invest obtained from cutaneous dinitrochlorobenzene-
Dermatol. 2009;129:2220–32. induced delayed type hypersensitivity reactions. J
21. Ghosh S, Mukhopadhyay S. Chemical leucoderma: Invest Dermatol. 2001;117:318–25.
a clinico-aetiological study of 864 cases in the per- 23. Ezzedine K, Eleftheriadou V, Whitton M, et al.

spective of a developing country. Br J Dermatol. Vitiligo. Lancet. 2015;386:74–84.
2009;160:40–7.
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Vitiligo, Associated
Disorders and Comorbidities
13
(Autoimmune-Inflammatory
Disorders, Immunodeficiencies,
Rare Monogenic Diseases)
Julien Seneschal, Fanny Morice-Picard,
and Alain Taïeb
Contents
13.2 ommon Autoinflammatory/Autoimmune Diseases Associated
C
with Vitiligo 126
13.3 I mmunodeficiencies 128
13.3.1 V
itiligo, Acquired Immunodeficiency and Idiopathic CD4+ T-Cell
Lymphocytopenia 130
13.3.2 CVID 131
13.3.3 Complement Deficiencies 132
13.3.4 Concluding Remarks 132
13.4 R are Inherited Diseases 132
13.4.1 The Interest of Studying Monogenic Disorders for the Understanding
of Common NSV 132
13.4.2 Discussion of Some Selected Monogenic Disorders 134
References 138
Abstract the leading hypothesis, and most vitiligo sus-

Vitiligo involves complex combinatorial fac- ceptibility loci identified studies encode
tors, namely genetic predisposition, environ- immunomodulatory proteins. Therefore, vitil-
mental triggers, metabolic abnormalities, and igo could be associated with other chronic
altered inflammatory and immune responses. autoimmune/inflammatory diseases, but also
The autoimmune and inflammatory theory is could occur in patients with inherited or
acquired immunodeficiencies or patients with
monogenic disorders that could impact
immune response.
J. Seneschal (*) · F. Morice-Picard · A. Taïeb

Department of Dermatology and Pediatric
Dermatology, National Reference Center for Rare
Skin Diseases, Saint-André Hospital,
University of Bordeaux, Bordeaux, France
e-mail: julien.seneschal@chu-bordeaux.fr;
alain.taieb@chu-bordeaux.fr

ERRNVPHGLFRVRUJ
126 J. Seneschal et al.
symptoms of vitiligo. One interesting point is

Key Points that some of them can be linked to autoimmunity.
• Vitiligo pathogenesis involves complex For example, patients with breakage disorders
combinatorial mechanisms with promi- such as ataxia-telangiectasia and the Nijmegen
nent autoimmune component. breakage syndrome present immunodeficiency
• Vitiligo is associated with other autoim- due to reduced immunoglobulin production and
mune/inflammatory chronic disorders T-cell anomalies and symptoms of autoimmune
such as thyroid disorders. disorders as vitiligo.
• Inherited or acquired immunodeficien-
cies are often associated with autoim-
mune disorders and vitiligo. 13.2 Common Autoinflammatory/
• Monogenic disorders including organ- Autoimmune Diseases
specific autoimmune, mitochondrial, Associated with Vitiligo
and breakage disorders can be associ-
ated with vitiligo suggesting potential Vitiligo involves complex combinatorial patho-
mechanisms involved in vitiligo. genic effects leading to the loss of melanocytes.
Results of three large genome-wide association
studies (GWAS, two in Caucasians and one in
Chinese) show that vitiligo is involving numer-
ous different susceptibility genes and that the
13.1 Introduction great majority of these genes encode proteins
associated with the immune system supporting
Factors involved in the initiation of vitiligo the hypothesis of a deregulated immune response
remain still largely unknown, despite recent proin vitiligo [4–10]. Many vitiligo susceptibility
gresses. However, the most common hypothesis genes are shared with other autoimmune dis-
is that vitiligo is an autoimmune disorder, with eases, usually with the same high-risk alleles. For
involvement of both innate and adaptive immuni- example, susceptibility loci HLA classes I and II,
ties. Genome-wide associations (GWAS) have PTPN22, CTLA4, and IL2RA are also shared by
shown that 90% of vitiligo susceptibility loci type 1 diabetes, thyroid diseases, rheumatoid
encode components of the immune system, and arthritis, alopecia areata, systemic lupus disease,
some of them are shared with other autoimmune/ and inflammatory bowel disease. Other suscepti-
inflammatory conditions [1, 2]. bility loci identified in vitiligo have also been
A contrario, a break in immune competence identified in coeliac disease, psoriasis, or
associated with inherited or acquired immunode- Addison’s disease. Moreover other susceptibility
ficiencies can lead to autoimmunity and vitiligo. genes such as vitamin D receptor or TSLP [11,
HIV infection, the most common cause of 12], found to be associated with atopic diseases,
acquired immunodeficiency, can be associated have also been associated with vitiligo.
with vitiligo at onset or during the reconstitution Recent epidemiological studies have been
of the immune system after antiviral therapies conducted to identify comorbid autoinflamma-
[3]. Among primary immunodeficiencies com- tory/autoimmune diseases associated with vitil-
mon variable immunodeficiency (CVID), which igo (Table 13.1). One was conducted in the US
is associated with longer survival, could be asso- population [18] and analyzed results from a
ciated with other autoimmune conditions and 10-year follow-up. This study showed that nearly
vitiligo. 20% of patients with vitiligo have at least one
Besides the association of vitiligo with inher- comorbid autoimmune disease. The most com-
ited immunodeficiencies, monogenic disease monly associated diseases were autoimmune thy-
involving organ-specific autoimmune, mitochon- roiditis (Hashimoto’s thyroiditis or Graves’
drial, and breakage disorders can present clinical diseases) and alopecia areata. 12.3% of their
ERRNVPHGLFRVRUJ
13 Vitiligo, Associated Disorders and Comorbidities (Autoimmune-Inflammatory Disorders… 127
Table 13.1 Frequencies of autoimmune/inflammatory disorders associated with vitiligo

Sheth VM Silverberg JI
Alkhateeb Liu J.B et al. [15] et al. [16] Chen YT Gill L et al.
Comorbid autoimmune et al. [13] et al. [14] n = 2441 n = 2273 et al. [17] [18]
disorders (%) n = 2078 n = 3742 ns ns n = 14,883 n = 1098
Thyroid disease 17a 1.36a 11.8 23.8 1.56a 12.3a
Alopecia areata 1.1 0.32a 2.4 2.7 1.81a 3.8a
Psoriasis 1.0 0.11 7.6 2.6 2.75a 2.2
Inflammatory bowel 0.67a NR 2.3 1.1 NR 0.7a
disease
Coeliac disease NR NR NR 0.8 0 NR
Pernicious anemia 1.78a NR NR 3.5 0.06 0.5a
Rheumatoid arthritis 0.67 0.32a 2.9 3.6 0.40 0.5
Systemic lupus disease 0.19a 0.05 2.2 0.7 0.28a 0.3a
Atopic dermatitis NR NR NR NR 7.98a NR
Sjögren syndrome 0 NR NR 0.8 0.36a 0.2a
Myasthenia gravis 0 NR NR NR 0.15a 0.2a
Diabetes mellitus type I 0.48 0.43 0.8 1.2 0.10 0.7
Frequency found significant compared to controls or the frequency in the general population, NR None Reported
a
patients had thyroid disease. This study demon-

strated a 15-fold increase in the prevalence of
clinical thyroid disease in patients with vitiligo
compared to the general population. This obser-
vation was associated with an increased percent-
age of patients with abnormal TSH level (18.6%)
and positive antithyroglobulin or anti-
thyroperoxydase antibodies. Another US study
based on medical records in Massachusetts also
identified thyroid disease as the most commonly
associated condition in 11.8% of the patients
[15]. These results confirm data previously Fig. 13.1 Patches of alopecia areata with regrowing
obtained from a large survey of more than 2000 white hairs, which is common in this setting and not
Caucasian patients with generalized vitiligo from related to vitiligo. However, true vitiligo may coexist with
North America and the UK [13] that found thy- alopecia areata
roid disease in 17% of their patients. However,
data from Chinese populations showed different 1.609 (95% CI 1.437–1.802)) and Graves’ dis-
results. A large retrospective survey of 3742 ease (OR 2.610 (95% CI 2.319–2.938)) in vitil-
patients with vitiligo in Mainland China [14] igo patients [19]. However despite this
could not confirm the association of vitiligo with discrepancy between Caucasian and Asian popu-
thyroid disease. Moreover, Cheng et al. investi- lations, the need to screen patients with vitiligo
gated comorbid autoimmune disease in vitiligo for thyroid abnormalities remains clinically
patients in a Taiwanese population with a follow- important.
up period of 4 years [17]. This study observed Gill et al. confirmed alopecia areata as another
that thyroid disease was associated with vitiligo inflammatory disorder associated with vitiligo
in only 1.56% of the population. More recently, a [18]. They found a prevalence of 3.8% corre-
nationwide population-based study conducted in sponding to a 31-fold increase in the frequency of
Korea that includes 73.336 vitiligo patients found alopecia areata in patients with vitiligo compared
a significant increased risk of autoimmune thy- to the general population (Fig. 13.1). A similar
roid disorders as Hashimoto’s thyroiditis (OR but weaker association was found in the
ERRNVPHGLFRVRUJ
Taiwanese population (1.81%) [17] and in the in familial probands [21]. Moreover in the
Chinese population [14]. These results suggest Taiwanese study, 7.8% of the patients with vitil-
similar pathomechanisms between these two dis- igo reported atopic dermatitis association [4].
eases. In line with this hypothesis, we have Taken together, these studies indicate that vit-
recently reported a new entity called “follicular iligo is epidemiologically associated with other
vitiligo” in which hair reservoir involvement gen- common chronic diseases, such as autoimmune
erally precedes interfollicular involvement. These thyroiditis or alopecia areata, suggesting that
data suggest that this entity could be the missing pathologic variants in specific genes predispose
link between alopecia areata and vitiligo [20]. to all these disorders. However, good genetic epi-
Psoriasis, the most common chronic inflam- demiology studies are still lacking to understand
matory skin disorder, has also been associated the major discrepancies noted across populations
with vitiligo. Besides numbers of clinical cases of various ethnic backgrounds, including ade-
published in the literature, reporting the coexis- quate control populations to check the validity of
tence of psoriasis and vitiligo, the recent reported associations, which may reflect the sim-
Taiwanese observational study made by Chen ple coexistence of two disorders, vitiligo itself
et al. demonstrated a significant association of being not rare.
this condition with vitiligo [17]. However this
association was not confirmed in other epidemio-
logical studies. 13.3 Immunodeficiencies
Other chronic autoinflammatory or autoim-
mune diseases could also be linked to vitiligo but Combined or T-cell immunodeficiencies, inher-
remain still in debate and need larger studies to ited or acquired, are commonly associated with
confirm such association: inflammatory bowel autoimmunity [22]. During frank losses of immu-
diseases, systemic lupus disease, pernicious ane- nocompetence, autoimmune diseases, which are
mia, rheumatoid arthritis, type 1 diabetes, predominantly CD8 T-cell driven, predominate.
Guillain-Barré syndrome, morphea, myasthenia Isolated reports of vitiligo associated with cellu-
gravis, and Sjögren syndrome. lar or combined immunodeficiencies, especially
A number of epidemiological studies reported during HIV infection, suggest a possible link to
the association of atopic diseases with vitiligo. pathomechanisms involved in vitiligo. However,
The largest study made in the US population the initial clinical presentation, which leads to a
found higher prevalence of atopic diseases in vit- vitiligoid condition, can be strikingly different
iligo compared with previously established prev- from that of vitiligo. For example, Figs. 13.2 and
alence in the general adult population for atopic 13.3 show a patient who presented with vitiligoid
dermatitis, asthma, hay fever, or food allergy condition in the context of HIV infection. In this
[16]. Based on a survey including 2645 adults patient, depigmented lesions followed an actinic
and pediatric patients with vitiligo, 61.8% of the lichen planus-like condition on sun-exposed
patients reported a history of at least one atopic areas before spreading to the rest of the body.
disease. Moreover atopic disease (including Among primary immunodeficiencies, the
atopic dermatitis) was associated with vitiligo most severe forms are usually lethal in the first
involving large body surface area, suggesting years of life if cell or gene therapy is not available
common genetic predisposition as already shown and a vitiligo phenotype is not clearly established
for vitamin D receptor or TSLP [11, 12]. in association. Worth of mention are ataxia-
Moreover the pro-inflammatory state of atopic telangiectasia and the Nijmegen breakage syn-
dermatitis may predispose toward melanocyte drome which are leading indirectly to reduced
loss, while scratching can influence the Koebner immunoglobulin production and T-cell anoma-
phenomenon important in the initiation of vitil- lies. Common variable immunodeficiency
igo disease. These results are consistent with (CVID) is compatible with longer survival and is
other studies. A Chinese study of 6516 patients the most commonly primary inherited immuno-
with vitiligo found a higher prevalence of asthma deficiency encountered in clinical practice [23].
ERRNVPHGLFRVRUJ
a b
Fig. 13.2 HIV-associated vitiligo. (a) lichenoid annular dermatitis evolving into vitiligoid lesions undistinguishable
from common vitiligo (b)
First biopsy One year later
Non lesional skin Lesional skin Non lesional skin Lesional skin
HES
Melanin
Vimentin
S100
HMB45
Melan A
1 2 1 2
Fig. 13.3 Histopathologic sequence corresponding to the immunostaining for proteins S100, HMB45, Melan A was
patient depicted in Fig. 13.2. Two biopsies were obtained positive. Pigmentary incontinence was observed, and
from lesional and nonlesional skin. The procedure was there was no melanin within keratinocytes. Second biop-
repeated after a 1-year interval. First biopsies (correspond sies, 1 year later (correspond to Fig. 13.2b): In nonle-
to Fig. 13.2a): In nonlesional skin (1): melanocytes were sional skin (1), no changes; in lesional skin (2), pigmentary
present and expressed protein S100, HMB45, Melan incontinence. Melanocytes were still present as shown by
A. Melanin was present in basal keratinocytes; in lesional vimentin staining although they did not express protein
skin (2): perivascular mononuclear infiltrate and lichenoid S100, HMB45, Melan A, and there was a loss of func-
pattern. Melanocytes were present in the basal layer, and tion (no melanin in keratinocytes)
Vitiligo could also result from anomalies of the this context. Oxidative stress dysregulation has
innate immunity, but so far no inherited or been associated with immunosuppression, as in
acquired condition fitting a well-established granulomatous disease [24], but also a link to vit-
innate immunity disturbance has been detected in iligo is not established.
ERRNVPHGLFRVRUJ
Besides the commonest cause, HIV infection, Concerning vitiligo presenting as a manifesta-
other causes of secondary immunodeficiencies tion of HIV infection, all patients reported by
which can be associated with vitiligo include Duvic et al. in the 1980s were in an advanced
chronic undernutrition and other conditions AIDS stage [25]. The CD4 cell count available
including protein-losing enteropathy, nephrotic for three patients was less than 400 cells/mm3.
syndrome, and hematological malignancies. There was no information on the viral load.
Concerning the improvement of previous vit-
iligo, spontaneous repigmentation of vitiligo has
13.3.1 Vitiligo, Acquired been reported in an untreated HIV-positive
Immunodeficiency patient. This patient had seroconverted for HIV
and Idiopathic CD4+ T-Cell infection 4 years before [26]. He noticed sponta-
Lymphocytopenia neous and significant repigmentation of vitiligo
lesions of 15-year duration. His CD4 cell count
Human immunodeficiency virus (HIV) infection is was at 390/mm3 when repigmentation occurred.
a common cause of immunodeficiency leading to Contrary to the previous scenario, pigmentation
CD4 T-cell depletion. Clinical consequences asso- has occurred following HAART. A first patient
ciate opportunistic infections and specific manifes- had generalized vitiligo onset associated with
tations defining the acquired immunodeficiency photosensitivity 2 years after the diagnosis of
syndrome (AIDS) stage. HIV infection is also HIV infection [27]. Two years later, he was com-
associated with an early immune dysregulation. A menced on HAART, and the skin began to repig-
few cases of vitiligo associated with HIV infection ment. At vitiligo onset, the CD4 cell count was at
have been reported. The circumstances of occur- 72/mm3. When vitiligo began to repigment, the
rence of vitiligo are however variable if disease his- CD4 cell count was at 149. The improvement was
tory, immune status, and history of exposition to attributed to the change in CD4 cell count.
highly active a ntiretroviral treatment (HAART) are Another case reported in 2014 showed the discov-
taken into account. Three different situations have ery of HIV infection in the context of the develop-
been observed, namely, (1) vitiligo revealing ment of severe alopecia areata and vitiligo in an
immunosuppression, (2) improvement of previous 18-year-old man [29]. CD4 T cells count was as
vitiligo during the course of immunodeficiency, low as 9 cells/mm3 and HIV viremia at
and (3) modification of vitiligo presentation with 280,000 copies/ml. Interestingly under HAART, a
antiretroviral treatment and/or immune restoration complete response of the alopecia areata was
with inflammatory signs (described as “punctuate observed as well as progressive repigmentation of
advanced erythematous margins”) (Table 13.2). the previously depigmented macules, concomi-
Table 13.2 HIV infection and vitiligo

Delay from HIV infection
Patient Reference, year diagnosis to vitiligo onset Vitiligo course
Patient 1 Duvic M et al. [25] 3 years Repigmentation following ribavirin
Patient 2 Duvic M et al. [25] Simultaneous Unknown
Patient 3 Duvic M et al. [25] 14 months No improvement following ribavirin
Patient 4 Duvic M et al. [25] 7 months Unknown
Patient 5 Duvic M et al. [25] Vitiligo preceded HIV Unknown
infection for 14 years
Patient 6 Grandhe NP et al. [26] Vitiligo preceded HIV Spontaneous repigmentation 2 years
infection for 11 years following HIV infection diagnosis
Patient 7 Antony FC et al. [27] 1 year Repigmentation following HAART
Patient 8 Niamba P et al. [28] Simultaneous Repigmentation following HAART
Patient 9 Nikolic DS et al. [29] Simultaneous Repigmentation following HAART
Patient 9 Personal data Simultaneous No repigmentation following HAART
Patient 10 Personal data Simultaneous No repigmentation following HAART
ERRNVPHGLFRVRUJ
tantly with the control of HIV viremia and restitu-

tion of the immune system. The role of the
immune reconstitution inflammatory syndrome
(IRIS) was also discussed in this context.
The relation of vitiligo to IRIS which may
occur in HIV-infected patients initiating antiret-
roviral therapy is intriguing. The mechanisms of
these reactions have not been studied in detail but
likely reflect an even broader spectrum of immu-
nologic events that differ on the basis of not only
the underlying antigenic trigger but also the spe-
cific nature of the immunosuppressive state. It
may involve uncoupling of innate and acquired
immune responses, restoration of exuberant
pathogen-specific cellular responses, and defec-
tive or delayed regulatory responses. An excess
of pro-inflammatory cytokines has also been
associated with IRIS. It is probable that the pres-
ence of high self or nonself antigen in IRIS drives
pro-inflammatory cytokine responses directly
through stimulation of innate immune responses
and indirectly when adaptive immunity recovers,
leading to autoimmune response or excessive
inflammatory responses to pathogens [3].
A patient with a long history of vitiligo associ- Fig. 13.4 CVID-associated vitiligo in a 34-year-old
ated with idiopathic CD4+ T-cell lymphocytope- man. Lesions began at age 1 and expanded progressively
nia (ICTL) has been reported. ICTL is defined as over more than 30% of the body surface. Other clinical
a persistent depletion of peripheral blood CD4 manifestations included Hashimoto’s thyroiditis, alopecia
areata, and recurrent nasopharyngeal and pulmonary
cell count of less than 300 cells/mm3 in the infections
absence of either HIV infection or other known
causes of immunodeficiency. ICTL has a variable
clinical spectrum that ranges from patients exhib-
iting minimal symptoms to those who have died autoimmune diseases associated include autoim-
from opportunistic infections. In the Yamauchi mune thrombocytopenic purpura and autoim-
et al. case report, in addition to the depletion of mune hemolytic anemia (frequently) and less
CD4+ T lymphocytes, the patient presented a commonly rheumatoid arthritis, vasculitis, and
depletion of CD8+ T lymphocytes. Vitiligo asso- vitiligo. These autoimmune manifestations have
ciated with ICTL could be tentatively explained suggested a genetic dysregulation provoking
as a shift in the balance of self-tolerance versus autoreactivity during B-cell development, with
autoimmunity [30]. the production of multiple autoantibodies against
various antigenic targets [31]. CVID can be
caused by mutation in the TNFRSF13B gene
13.3.2 CVID located on chromosome 17, which encodes the
transmembrane activator and CAML interactor
Common variable immunodeficiency (CVID) is (TACI). Figure 13.4 shows a patient with a very
the most common primary immunodeficiency. early onset of vitiligo (before the age of 1) that
Among patients with CVID, up to 25% present was further diagnosed with CVID and various
with autoimmune events. The clinical phenotype autoimmune disorders including alopecia areata
of the condition is broad and heterogeneous. The and Hashimoto’s thyroiditis.
ERRNVPHGLFRVRUJ
13.3.3 Complement Deficiencies common pathogenetic pathways or disease mark-

ers. For vitiligo, even though the phenotype is easily
The arguments for a role of the complement cas- recognizable in humans, there are limited human
cade are mostly based on in vitro studies and monogenic disease models (Table 13.3).
theoretical speculations. However, heterozygous Unfortunately, the “vitiligo” phenotype is usually
C4 deficiency has been linked to an increased risk rarely well documented in genetics publications.
for vitiligo [32]. Complement-activating anti- We have included only monogenic disorders with a
melanocyte antibodies have been implicated in confirmed ascertainment of this phenotype based
vitiligo pathogenesis, with complement compo- on personal experience or literature review.
nents directly involved in cell killing. Along this Animal models may be misleading because
line, it has been speculated a role in vitiligo the human pigmentation system is the result of a
pathogenesis for a plasmatic protein called very specific evolutional maturation. The discus-
mannose-binding lectin (MBL). This protein rec- sion around separate human cutaneous melano-
ognizes mannose terminal residues of microbial cyte compartments (hair follicule and
glycoproteins or glycolipids and then activates interfollicular glabrous skin) which underlies
the classic complement pathway without antibod- clinical differences between SV and NSV is
ies, through an associated serine protease [33]. probably relevant for the analysis of the various
vitiligo-related phenotypes in monogenic disor-
ders. Along the same line, the heritability of pre-
13.3.4 Concluding Remarks mature graying genes has been already addressed
in some studies [37], but the definitive evidence
The link between immunodeficiencies and vitil- of a link between the two phenotypes (premature
igo remains poorly documented and understood. graying and vitiligo) is still missing.
T-cell deficiencies and combined deficiencies Some clearly distinct monogenic disorders
such as CVID can be associated with vitiligo. have marked phenotypic overlap with vitiligo,
The role of pure B-cell deficiencies and of innate which raise the question of a common pathogenic
immunity deficiencies is not as well established. background. Piebaldism is an autosomal domi-
From the clinical standpoint, it is important to nant disorder of melanocyte development charac-
consider—especially in the case of widespread terized by ventral leucoderma/trichia due to KIT
vitiligo following a clinically inflammatory mutations which impair melanocyte precursor
phase—an underlying T-cell immunosuppression migration. Given the importance of its encoded
and that triggered by HIV infection should be protein c-Kit in the maintenance of viable mela-
investigated in priority (Figs. 13.2 and 13.3). nocytes, the hypothesis of a direct genetic link to
common vitiligo had to be raised but was
excluded in genome-wide scans. An uncommon
13.4 Rare Inherited Diseases variant, the Val620Ala (1859T>C) mutation of
the KIT gene, has been found in so-called pro-
13.4.1 The Interest of Studying gressive piebaldism leading to progressive loss of
Monogenic Disorders pigmentation as well as the progressive appear-
for the Understanding ance of hyperpigmented macules also noted in
of Common NSV trichrome vitiligo [38]. Another phenotype which
is clinically related to vitiligo has been observed
The candidate gene approach has not been success- in a large Canadian family. Vitiligoid patches
ful for vitiligo. However, the observation of relevant occurred at adolescence and progressed toward
common features in monogenic disorders and com- diffuse depigmentation. This “autosomal domi-
plex multigenic ones may offer interesting clues, nant vitiligo” was associated with a heterozygous
either for extracting a major genetic component in a -639G-T transversion identified in a highly con-
complex disorder [34] or for looking at possible served area in the promoter region of the FOXD3
ERRNVPHGLFRVRUJ
Table 13.3 Monogenic disorders and NS vitiligo

Name
OMIM entry
Reference if not
detailed in text Inheritance/population Gene/protein/antigen Clinical symptoms/remarks
APS1 (autoimmune Autosomal recessive AIRE (autoimmune regulator Vitiligo not mandatory;
polyglandular (most cases) gene) autoimmunity to NALP5
syndrome, type I) / Finns, Iranian Jews, frequent in case of
APECED Sardinians hypoparathyroidism
(autoimmune
polyendocrinopathy-
candidiasis-ectodermal
dystrophy)
240300
APS 2/Schmidt Autosomal recessive or Unknown No straightforward marker,
syndrome autosomal dominant Linked to MHC class I on vitiligo not mandatory
269200 with incomplete chromosome 6
penetrance?
Ataxia-telangiectasia X-linked recessive ATM/atm Skin: vitiligo
208900 Spontaneous chromosomal telangiectasias
instability with multiple Granulomas
rearrangements especially Nonskin
chromosomes 7 and 14 Elevated serum alpha
Chromosomal hypersensitivity fetoprotein
to ionizing radiation and
alkylating agents
Nijmegen breakage Autosomal recessive 8q21 NBS 1/nibrin Skin: progressive vitiligo,
syndrome Eastern European Spontaneous chromosomal cafe au lait spots,
251260 origin instability with multiple photosensitivity (?)
rearrangements, especially Nonskin: facial dysmorphy,
chromosomes 7 and 14 microcephaly++, mental
Chromosomal hypersensitivity retardation, immunodeficiency,
to ionizing radiation and autoimmune disorders,
alkylating agents neoplasias
Radioresistant DNA synthesis
MELAS Mitochondrial Genetically heterogeneous: Vitiligo associated in 11% of
540000 mutations in MTTL1 (most cases studied with common Mt
common) and MTTQ, MTTH, mutation 3243
MTTK, MTTS1, MTND1,
MTND5, MTND6, and MTTS2
Vitiligo-spasticity Autosomal recessive Mapped to 1q24-q32 Skin: vitiligo and lentigines of
syndrome/spastic inbred Arab families exposed areas (apparent at birth
paraplegia with or in infancy); premature
pigmentary graying of body hair, canitia
abnormalities Nonskin: microcephaly, thin
270750 face, micrognathia, retrognathia
[35] Severe spastic paraplegia in
childhood +++; mild cognitive
impairment
Combined Autosomal recessive or Unknown Skin: vitiligo and
immunodeficiency dominant hyperpigmented macules
with autoimmunity Nonskin: Spondylometaphyseal
and dysplasia + combined humoral
spondylometaphyseal and cellular immunodeficiency:
dysplasia recurrent infections (pneumonia,
607944 sinusitis, fulminant varicella)
[36] autoimmune disorders
ERRNVPHGLFRVRUJ
Fig. 13.5 The role of O2 H2O

TPH, TH, and PAH in
the biosynthesis of
serotonin and dopamine.
All three enzymes are TPH
dependent on Tryptophan 5-OH-Tryptophan Serotonin
tetrahydrobiopterin
(BH4) as a cofactor TH
(NAD = nicotinamide Tyrosine L-DOPA Dopamine
adenine dinucleotide)
PAH
Phenylalanine Tyrosine
BH4 BH2
NAD+ NADH+H+
gene [39]. Functional expression studies of the where it catalyzes the conversion of phenylala-
gene variant indicated that it increased transcrip- nine into tyrosine. Mutations in PAH are the most
tion in neural crest melanoblast precursors, pos- common defect responsible for phenylketonuria,
sibly altering the differentiation profile of the which is characterized by hair and skin pigment
melanoblast lineage. dilution. However, against this hypothesis, the
Another interest of monogenic diseases which vitiligo phenotype is not clearly associated in
include organ-specific autoimmunity is the use of APECED patients with one subset of antibodies
the monogenic disease as a model to identify auto- to tetrahydrobiopterin-dependent hydroxylases
antigens of importance in other disorders, such as [41], and these have not been tested in common
vitiligo. APECED/APS1 is a relevant example. NSV or its autoimmune subsets. Similar to glu-
The gene causing APECED has been named auto- tamic acid decarboxylase and AADC which are
immune regulator (AIRE). It is predominantly autoantigens in APECED, as well as enzymes of
expressed in some cells of the immune system and great importance in the synthesis of GABA, sero-
is thought to be involved in transcriptional regula- tonin, and dopamine, it remains possible that the
tion. Autoantibodies seem able to enter in target group of tetrahydrobiopterin-dependent hydrox-
cells and neutralize enzymatic activities. APECED ylases has shared immunogenic properties or
patients have autoantibodies against several may play a role in the pathogenesis of vitiligo as
enzymes involved in the biosynthesis of neu- in APECED (Fig. 13.5).
rotransmitters. Autoantibodies against aromatic
l-amino acid decarboxylase (AADC) in APS I
patients are associated with the presence of auto- 13.4.2 Discussion of Some Selected
immune hepatitis and vitiligo [40]. Monogenic Disorders
TPH catalyzes the hydroxylation of trypto-
phan into 5-OH tryptophan and is the rate- • Autoimmune Polyendocrine Syndromes
limiting enzyme in the synthesis of serotonin. TH Autoimmune polyendocrine syndromes
is the rate-limiting enzyme in the biosynthesis of (APS), also known as polyglandular autoim-
catecholamines, where it converts tyrosine into mune syndromes (PGA), are associated with
l-DOPA. PAH is mainly expressed in the liver, an increased incidence of NS vitiligo. APS are
ERRNVPHGLFRVRUJ
clinically and genetically heterogeneous, but not been analyzed comprehensively to

they reflect multiple endocrine gland insuffi- bring specific and useful information in the
ciency caused by mainly humoral autoimmu- vitiligo field.
nity. APS-1 and APS-2 are the two major The analysis of autoantibodies in APS I
autoimmune polyendocrine syndromes. The patients is a useful tool for establishing
APS-1/APECED gene AIRE is on 21q. The autoimmune manifestations of the disease
gene for Schmidt syndrome or APS-2 is not as well as providing diagnosis in patients
yet cloned. According to linkage studies, the with suspected disease. For hypoparathy-
AIRE gene is not associated with vitiligo roidism, the most common autoimmune
coexisting with other autoimmune diseases in endocrinopathy of APECED, autoantibod-
European-descent families. This does not ies specific for NACHT leucine-rich repeat
exclude some common pathophysiological protein-5 (NALP5), has been shown in
background (see discussion above). 49% of patients with APS1 and hypopara-
–– APS1/APECED thyroidism [43]. NALP5 which is predomi-
Autoimmune polyglandular syndrome nantly expressed in the cytoplasm of
type I or APECED is characterized by the parathyroid chief cells appears to generate
following major clinical symptoms: tissue-specific autoantibodies. Its role is
Addison’s disease, hypoparathyroidism, under close investigation because of a sus-
and chronic mucocutaneous candidiasis. pected role of the innate immune system in
The spectrum of associated minor clinical triggering autoinflammation and autoim-
diseases includes other autoimmune endo- munity [44]. Since NALP1 variants are
crinopathies (hypergonadotropic hypogo- associated with autoimmune vitiligo [45],
nadism, insulin-dependent diabetes this could be relevant to the vitiligo field.
mellitus, autoimmune thyroid diseases, and –– Schmidt (APS2) Syndrome
pituitary defects), autoimmune or immuno- Schmidt syndrome is characterized by
mediated gastrointestinal diseases, chronic the presence of autoimmune Addison’s dis-
active hepatitis, skin diseases (vitiligo and ease in association with either autoimmune
alopecia areata), ectodermal dystrophy thyroid disease or type 1 diabetes mellitus
including enamel and nail defects, kerato- or both [42]. Chronic candidiasis is not
conjunctivitis, immunologic defects (cellu- present. APS2 may occur at any age and in
lar and humoral), asplenia, and both sexes but is most common in middle-
cholelithiasis. The first manifestations aged females and is very rare in childhood.
occur commonly in childhood with the Alopecia areata, vitiligo, myasthenia gra-
three main diseases developing in the first vis, pernicious anemia, and Graves’ disease
20 years of life, but other accompanying are frequently associated. Schmidt syn-
diseases continue to appear until at least drome can be more specifically associated
the fifth decade. In a majority of cases, can- with interstitial myositis [46]. An associa-
didiasis is the first clinical manifestation to tion of HLA-B8 with APS2 has been found
appear, usually before the age of 5 years, in three generations of a family [47].
followed by hypoparathyroidism (usually –– Immunodysregulation Polyendocrinopathy
before the age of 10 years), and later by and Enteropathy X-Linked (IPEX; 304790)
Addison’s disease (usually before the age Interestingly, this more recently
of 15 years). Overall, the three main com- described X-linked recessive disease due to
ponents of APECED occur in chronologic mutations in FOXP3 which block the
order, but they are present together in only development of regulatory T cells has not
about one-third to one-half of the cases yet been linked to vitiligo but induces mul-
[42]. Vitiligo is more frequent in the tiple immune-related diseases, including
APECED population but had until recently TH2 predominant (atopic dermatitis) and
ERRNVPHGLFRVRUJ
TH1 predominant (alopecia areata, type 1 used in that study was the S100 protein which
diabetes) phenotypes. Further observations stains also Langerhans cells. The mitochon-
are necessary to exclude the alteration of drial mutation was not studied in epidermal or
this important pathway in the pathogenesis skin cells [50]. In skeletal muscle, the bio-
of immune vitiligo. chemical defect is often segmental [51], sug-
• Mitochondrial Disorders: MELAS (Myopathy, gesting a nonrandom distribution of mutant
Encephalopathy, Lactic Acidosis, and Stroke- and wild-type mtDNAs within a muscle cell.
Like Episodes) and MERRF (Myoclonic A decreased activity of the mitochondrial
Epilepsy Associated with Ragged-Red Fibers) respiratory chain may lead to a metabolic
Syndromes impairment within the epidermal melanin
These disorder belongs to the mitochon- unit, which is consistent with previous hypoth-
drial encephalomyopathies which include eses concerning the altered redox status in vit-
Leigh syndrome (LS; 256000), Kearns-Sayre iligo [50]. Another possible molecular target
syndrome (KSS; 530000), and Leber optic is the transport of melanosomes via microtu-
atrophy (535000) [48]. The most common bules, which requires adenosine triphosphate
clinical manifestation in these disorders is (ATP). This is a possibility since all the muta-
encephalomyopathy, because the nerve and tions in mtDNA affect the respiratory chain
muscle cells depend more heavily on aerobic and oxidative phosphorylation and may thus
energy production. However, any tissue or lead to a decreased amount of ATP in the cell.
organ may be affected. Mutations of mtDNA accumulate during nor-
The most frequent MELAS symptom is mal aging. The most frequent mutation is a
episodic sudden headache with vomiting and deletion, which is increased in photoaged
convulsions, which is found in 80% of cases skin. Oxidative stress in turn may play a major
in patients aged 5–15 years. Laboratory evi- role in the generation of large-scale mtDNA
dence of myopathy related to abnormal mito- deletions. Reactive oxygen species have been
chondrial metabolism includes elevated shown to be involved in the generation of
resting serum lactate increased with exercise aging-associated mtDNA lesions in human
ragged-red fibers on muscle biopsy and sub- cells [52].
sarcolemmal pleomorphic mitochondria on • Breakage Disorders: Ataxia-Telangiectasia
electron microscopy [49]. The common mito- and Nijmegen Breakage Syndrome
chondrial causative mutations are mostly –– Ataxia-Telangiectasia (AT)
found in the muscle mtDNA molecules but Patients present in early childhood with
can be found in other cell type responsible for progressive cerebellar ataxia and usually
the main symptoms. The variability of the later develop conjunctival telangiectasias,
clinical phenotype is partly due to mutation progressive neurologic degeneration, recur-
heteroplasmy, a condition where the normal rent infections, and mostly lymphoid malig-
genome and the mutant variant coexist in nancies. Lymphomas in AT patients tend to
mitochondria [48]. An A to G transition at be of B-cell origin, whereas the leukemias
base pair 3243 in the tRNALeu(UUR) gene in tend to be of the T-cell type. Oculocutaneous
mtDNA is the most common molecular etiol- telangiectasias typically develop between 3
ogy of the MELAS syndrome, but other muta- and 5 years of age (Fig. 13.6). Vitiligo is a
tions have been described (Table 13.3). feature already mentioned [53, 54] but not
Vitiligo was associated in 11% of cases of emphasized in the context of major neuro-
MELAS bearing the common bp 3243 point logical problems.
mutation [50]. Interestingly, there is no Elevated levels of alpha-fetoprotein and
reported evidence of melanocyte loss in this cytogenetic analysis may help confirm the
form of vitiligo but only decreased melano- diagnosis (7;14 translocations). Ataxia-
genesis. Unfortunately, the melanocyte marker telangiectasia mutated (ATM), the product
ERRNVPHGLFRVRUJ
a nocytes in AT- related vitiligo. ATM

interactions with beta-adaptin in the cyto-
plasm might mediate axonal transport and
vesicle trafficking in the central nervous
system and so account for the neuronal
dysfunction and eventual neurodegenera-
tion seen in ataxia-telangiectasia.
The previous pathways may be interest-
ing for the immune and neural theories of
vitiligo, but of considerable interest is that
the AT phenotype seems to be a conse-
quence, at least in part, of an inability to
respond appropriately to oxidative damage
{3} and that the absence of the ATM pro-
tein affects stem cells. Ionizing radiation
oxidizes macromolecules and causes tissue
damage through the generation of reactive
oxygen species (ROS). When compared to
normal human fibroblasts, AT dermal fibro-
blasts exhibit increased sensitivity to
t-butyl hydroperoxide toxicity. These cells
b fail to show G1 to G2 phase checkpoint
functions or to induce p53 in response to
oxidative challenge. Cells derived from
ATM-deficient mice, grow poorly in cul-
ture, are genetically instable and appear to
undergo apoptosis more readily than con-
trol cells. In a mouse AT model, hair pre-
mature graying has been observed in
heterozygous mice after a sublethal dose of
ionizing radiation. Furthermore, ATM pro-
tein deficiency and telomere dysfunction
seem to act together to impair cellular via-
Fig. 13.6 Ataxia-telangiectasia manifested by vitiligo in bility and cause adverse effects on stem/
the napkin area (a) and equatorial conjunctival telangiec- progenitor cell reserves.
tasias (b) –– Nijmegen Breakage Syndrome (NBS)
This syndrome has been related to AT as
of the mutated gene, is a 370-kDa protein a variant until more recent clear clinical
member of the phospatidyl inositol and molecular evidence of a distinct etiol-
3-kinases superfamily. Increased recombi- ogy {34}. The gene product, nibrin, is
nation is a component of genetic instability member of the hMre11/hRad50 protein
in AT and may contribute to the cancer risk. complex, suggesting that the gene is
The phenotypic pleiotropy of AT may involved in DNA double- strand break
result from different tissue expressions of repair. The immunological, cytogenetic,
ATM targets. ATM plays an important role and cell biological findings in NBS closely
in the activation of the tumor suppressor resemble those described in AT (see
gene product p53. In the skin, this may pre- Table 13.3). For the vitiligo phenotype, it is
cipitate the apoptotic commitment of mela- better documented (14/21 cases studied in
ERRNVPHGLFRVRUJ
the international NBS study group). ized vitiligo supports XBP1, FOXP3, and TSLP. J
Telangiectasias are rare and café au lait 12. Li K, Shi Q, Yang L, et al. The association of vita-
spots common. Concerning the non-skin min D receptor gene polymorphisms and serum
clinical aspects, however, NBS is more 25-hydroxyvitamin D levels with generalized vitiligo.
similar to Bloom syndrome (BS), which Br J Dermatol. 2012;167:815–21.
13. Alkhateeb A, Fain PR, Thody A, et al. Epidemiology
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Age and Vitiligo: Childhood,
Pregnancy and Late-Onset Vitiligo
14
Steven Thng, Sai Yee Chuah, and Emily Yiping Gan
Contents
14.2 Vitiligo in Childhood 142
14.2.1 pidemiology of Vitiligo in Childhood
E 142
14.2.2 Clinical Characteristics of Vitiligo in Childhood 143
14.2.3 Familial Background and Associated Diseases of Vitiligo in Childhood 144
14.2.4 Differential Diagnosis of Vitiligo in Childhood 145
14.2.5 Psychological Sequelae of Vitiligo in Childhood 145
14.2.6 Therapeutic Considerations of Vitiligo in Childhood 146
14.3 V itiligo During Pregnancy 146
14.3.1 Clinical Characteristics of Vitiligo During Pregnancy 146
14.3.2 Therapeutic Considerations of Vitiligo During Pregnancy 147
14.4 Late-Onset Vitiligo 147
14.4.1 efinition and Epidemiology of Late-Onset Vitiligo
D 147
14.4.2 Clinical Features, Treatment Response and Course of Disease 148
14.4.3 Therapeutic Considerations of Late-Onset Vitiligo 149
References 149
Abstract of life (QOL) impairment in the patient and in the

Vitiligo usually begins in childhood or in young parent. Prepubertal onset of vitiligo associates
adults with some studies recording around half of with atopic dermatitis, halo naevi, family history
patients with disease onset before the age of of vitiligo and premature hair greying. When
20 years old. Childhood vitiligo tends to present treating children with vitiligo, special consider-
more with segmental vitiligo and is associated ations would include the need to coordinate treat-
with higher incidences of thyroid dysfunction as ments with school schedules and the significant
well as family history of autoimmune disease, psychosocial impact of the disease on children
significant psychological morbidity and quality especially if they have visible lesions. Patients
with vitiligo have possibly a higher incidence of
worsening during pregnancy and 6 months’ post-
S. Thng (*) · S. Y. Chuah · E. Y. Gan
National Skin Centre, Singapore, Singapore partum. Treatment of vitiligo during pregnancy is
e-mail: steventhng@nsc.com.sg largely influenced by how treatment may affect

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142 S. Thng et al.
the foetus in utero, and treatment has to be bal- characteristics, treatment response as well as
anced with possible adverse effects to foetus in disease progression of this disfiguring disease

utero. Late-onset vitiligo is an interesting sub- among different age groups and during preg-
group of patients with vitiligo, and its prevalence nancy. This chapter will therefore examine the
ranges from 6.8% in Northern India to 14.7% in clinical characteristics of vitiligo through the
Singapore; they seem to progress faster than ages, paying special attention to clinical presen-
childhood vitiligo, and the disease progression is tation, disease progression, treatment response
significantly associated with a stressful event. and considerations of childhood vitiligo, late-
onset vitiligo and vitiligo in pregnancy.
Key Points
• Segmental vitiligo and associated thy- 14.2 Vitiligo in Childhood
roid dysfunction are frequent in child-
hood patients. Childhood vitiligo is not uncommonly encoun-
• Significant psychological morbidity and tered in the paediatric dermatology clinic. While
quality of life (QOL) impairment are recent reports show that the worldwide preva-
reported in the patient and in the lence of childhood/adolescent vitiligo does not
parent. differ from adult vitiligo, ranging between 0.5%
• Prepubertal onset of vitiligo associates and 2% [3], from a clinical characteristic and
with atopic dermatitis, halo naevi, fam- treatment response perspective, vitiligo in child-
ily history of vitiligo and premature hair hood differs from adult-onset cases. In addition,
greying. there are specific considerations one needs to
• In childhood treatment must take into bear in mind when managing paediatric patients
account school schedules and psychoso- with vitiligo. These will be discussed in this
cial impact of the disease. segment.
• Possible worsening of vitiligo has been
noted during pregnancy and 6 months’
post-partum. 14.2.1 Epidemiology of Vitiligo
• The prevalence of late-onset vitiligo in Childhood
ranges from 6.8% in Northern India to
14.7% in Singapore. Nearly half of all cases of vitiligo begin before
• The progress of late-onset vitiligo is the age of 20 years [2] and one quarter before the
faster than childhood vitiligo, and the age of 10 years [4]. The age at onset of vitiligo
disease progression is significantly varies between studies, although many have
associated with a stressful event. reported that most cases are acquired early in life,
between 4 and 12 years of age [5, 6]. There have
been reports of patients with vitiligo lesions since
birth [7, 8]; however the entity “congenital vitil-
14.1 Introduction igo” still remains controversial, as it is not clear
whether these are patients with piebaldism or
Many epidemiological studies around the world true vitiligo. In most reported paediatric case
have surmised that vitiligo usually begins in series, the majority of reported cases tend to be in
childhood or in young adults [1] with some stud- girls [9, 10], with some studies reporting a preva-
ies recording around half of patients with disease lence of up to 60% in girls versus 40% in boys [6,
onset before the age of 20 years old [2]. While 11–13]. However, there have also been other
there are many studies looking at vitiligo in child- studies in the literature which do not show a gen-
hood and vitiligo in general, there are few publi- der predilection [14]. In a local study in Singapore
cations that evaluate the differences in clinical examining 369 cases of childhood vitiligo over
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14 Age and Vitiligo: Childhood, Pregnancy and Late-Onset Vitiligo 143
5 years [52], 52.3% of our childhood vitiligo

patients were girls, while 47.7% were boys with
a mean age of onset of 8.9 years.
14.2.2 Clinical Characteristics

of Vitiligo in Childhood
14.2.2.1 Classification of Vitiligo

Non-segmental (NSV) vitiligo is the most com-
mon type of vitiligo in children, of which the Fig. 14.2 Segmental Vitiligo in a paediatric patient
subtype generalised vitiligo (Fig. 14.1) occurs
most frequently [6, 9, 11, 14, 15]. This is fol-
lowed by focal and then segmental vitiligo [6,
9, 11, 14] (Fig. 14.2). Notwithstanding, seg-
mental vitiligo tends to be more common in
children compared to adults [5, 16] with a prev-
alence ranging from 4.6% to 32.5%. In our
local study comprising 369 patients [52], 35%
of patients (n = 129) had segmental vitiligo, of
which two patients progressed to develop vitil-
Fig. 14.3 Vitiligo vulgaris on the trunk and groin
igo vulgaris (mixed vitiligo) sometime later in

the course of their disease.
14.2.2.2 Distribution of Vitiligo

The head and neck are the most common sites of
onset of vitiligo [6, 9, 11, 15] in children. The
perineum, especially the buttocks and perianal
area, is a relatively frequent site of onset of non-
segmental vitiligo in toddlers (Fig. 14.3), sug-
gesting a role for koebnerisation triggered by use
of nappies and hygiene care. In a large French
study analysing children with non-segmental ver-
sus segmental vitiligo, Mazereeuw-Hautier et al.
reported that children with non-segmental vitil-
igo had a greater number of lesions, a larger body
surface area of involvement, higher incidence of
Koebner phenomenon and more frequent pro-
gression of disease [17].
14.2.2.3 Disease Onset in Childhood

In a study comparing 679 patients [18] with pre-
(before 12 years old) versus post-pubertal onset
Fig. 14.1 Unstable vitiligo vulgaris in a 7 years old child (after 12 years old) of vitiligo, atopic dermatitis
ERRNVPHGLFRVRUJ
144 S. Thng et al.
was often associated with or preceding prepuber- Although children tend to scrape or scratch
tal onset (odds ratio 2.42, p = 0.006), whereas their elbows, knees and shins often, it is not
thyroid disease or presence of thyroid antibodies clear whether koebnerisation occurs more fre-
was more frequent in post-pubertal onset (odds quently in children. In a series of 625 children
ratio 0.31, p < 0.003). Other factors associated with vitiligo, Handa and Dogra reported koeb-
with prepubertal onset include the presence of nerisation in 11% of patients [6]. In another
halo naevi, family history of vitiligo, premature study by Kayal et al., 83 children and 26 adult
hair greying and previous episode of spontaneous patients who had onset of vitiligo before
repigmentation. In contrast, factors associated 12 years of age were categorised as having
with post-pubertal onset include stress as an initi- childhood-onset vitiligo (COV). The Koebner
ating factor, personal history of thyroid disease phenomenon was found to occur in 37% of the
and the acrofacial type of vitiligo [18]. A separate COV group as compared to 29% of those with
study by Nicolaidou et al., which also used adult-onset vitiligo (186 patients) [11].
12 years old as the cut-off value for age at onset,
found that the group with childhood-onset vitil-
igo had the head as the most frequent site of ini- 14.2.2.5 Treatment Response
tial presentation, more cases of segmental Characteristics and Disease
vitiligo, a higher prevalence of allergic diseases Progression
and a lower prevalence of thyroid diseases com- In paediatric vitiligo, the repigmentation pat-
pared to the later-onset group [19]. terns differ from that in adults. Gan et al.
In a separate study of purely paediatric vitil- reported that the most common pattern seen in
igo patients, Mu et al. divided 208 children into children is the combined pattern (62%), fol-
early-onset (less than 3 years old) and later-onset lowed by diffuse, marginal, perifollicular and
(3–18 years old) groups. Early-onset vitiligo was lastly medium spotted repigmentation [23]. This
associated with more extensive and progressive is in contrast to adults, where the most common
disease during the average 1.9 years of follow- pattern is perifollicular. A possible explanation
up. There were no significant differences is that in children the melanocytic reservoirs
between the two groups in terms of repigmenta- have not yet been depleted or affected by other
tion, vitiligo type, halo naevi, gender ratio or factors such as ageing, senescence of melano-
personal and family history of autoimmune dis- cyte precursors, cumulative sun exposure and
eases [20]. sun-induced changes.
Longitudinal studies evaluating the evolu-
14.2.2.4 Other Clinical Features tion of childhood vitiligo are lacking. In one
Halo naevi occur more commonly in paediat- series, after approximately 1 year of follow-up,
ric vitiligo patients than in adults with vitiligo it was noted that vitiligo was clinically unde-
[21]. Cohen et al. analysed 208 children with tectable in only 5.5% of patients. More progres-
vitiligo and found 26% of them had halo naevi. sion was noted in the group of children with
Children with vitiligo and halo naevi were non-segmental (23%) compared to segmental
more likely to present with generalised vitiligo (6%) [17].
vitiligo. There was
however no significant
association with percentage of body surface
area affected or family history of vitiligo or 14.2.3 Familial Background
autoimmune diseases [22]. Leukotrichia has and Associated Diseases
been reported in 4–12% of patients with vitil- of Vitiligo in Childhood
igo [5, 6, 10]. Handa and Dogra reported it
occurring most commonly in patients with 14.2.3.1 Family History
generalised vitiligo (84%), followed by focal A positive family history of vitiligo is elicited
and segmental vitiligo [6]. more often in children with vitiligo, although this
ERRNVPHGLFRVRUJ
incidence varies considerably, from 3% up to levels of thyroid-stimulating hormone (TSH),

46% in different studies [5, 9, 24]. The presence free thyroxine, anti-thyroglobulin and antithy-
of autoimmune disease within the family also roid peroxidase antibodies [28]. Antinuclear
tends to occur more frequently [5, 6]. Pajvani antibodies (ANA) have been detected in children
et al. reported that patients with vitiligo and an with vitiligo, occurring in up to 5%, although
extended family history of vitiligo were signifi- other studies have not been typically consistent
cantly more likely to have an earlier age of onset in finding positive ANA levels [24, 30]. Other
of disease compared to those with a negative autoimmune diseases which have been reported
family history (odds ratio 3.7) [13]. Knowledge in children with vitiligo include alopecia areata,
of this association can allow for closer monitor- diabetes mellitus, Addison’s disease and autoim-
ing of siblings, earlier detection and initiation of mune polyglandular syndrome [6, 9].
treatment.
14.2.3.2 Associated Conditions 14.2.4 Differential Diagnosis

Children with vitiligo are generally healthy; of Vitiligo in Childhood
however, as with adult vitiligo, other autoim-
mune diseases can be associated, the most com- In children, other common causes of hypomela-
mon being autoimmune thyroid disorders, noses are postinflammatory hypopigmentation,
which may be associated with thyroid dysfunc- naevus depigmentosus (for segmental vitiligo)
tion [25]. and piebaldism. For cases with vulvar depigmen-
The risk for autoimmune thyroid disease tation, lichen sclerosus is also an important dif-
increases with age and duration of disease, con- ferential diagnosis that should be excluded. A
sistent with the findings of a systematic review thorough history and clinical examination includ-
where the median prevalence of autoimmune ing Wood’s lamp examination would assist in
thyroid disease in children was 6.9%, with making the right diagnosis.
one-third found in adults (18.6%) [26].

Notwithstanding, it has been reported that
Hashimoto’s thyroiditis is 2.5 times more fre- 14.2.5 Psychological Sequelae
quent among children and adolescents with vit- of Vitiligo in Childhood
iligo than in a healthy age- and sex-matched
population. Iacovelli et al. showed that 11% of Childhood vitiligo is associated with significant
paediatric patients with vitiligo, particularly psychological morbidity and quality of life
16% of patients with non-segmental vitiligo, had (QOL) impairment, both of which are present
thyroid abnormalities on testing [27]. In another not only in the patient but also in the parent [31].
study, Halder et al. found antithyroid antibodies An affected body surface area of more than 25%
in 12% of the 33 children with vitiligo who were is associated with greater impairment in QOL,
screened [5]. Thyroid dysfunction has been self-consciousness, difficulty with friendships
reported to correlate with the specific type of vit- and schoolwork and teasing and bullying [32].
iligo, namely, non-segmental as opposed to seg- Teenagers aged 15–17 years seem to have the
mental vitiligo, but not with extension of lesions greatest QOL burden from vitiligo [32], and
and localisation [27]. Vitiligo usually appears their emotional development may be affected
before the development of thyroid disease [28], especially if they have lesions in visible areas
and cases of subclinical hypo- or hyperthyroid- [33]. Cosmetic camouflage has been shown to
ism have been detected with thyroid testing [29]; have a positive impact on the QOL of this group
therefore, it is prudent to screen children and of patients [34]. It is essential to screen for QOL
adolescents with non-segmental vitiligo for thy- impairment in both paediatric vitiligo patients
roid dysfunction at presentation and during sub- and their parents and to incorporate this informa-
sequent follow-up. Screening should include tion into therapeutic decision-making.
ERRNVPHGLFRVRUJ
146 S. Thng et al.
14.2.6 Therapeutic Considerations adolescents and adults, respectively, with a mean

of Vitiligo in Childhood extent of repigmentation of 81%, 79% and 77%
in the three respective groups [38]. Punch mini-
The approach to the treatment of childhood vitil- grafting has also been carried out with success in
igo largely mirrors that in an adult. First-line children [39]. With appropriate pre-surgical
treatment includes topical corticosteroids, topical counselling of parents and child and willingness
calcineurin inhibitors and topical vitamin D ana- of the child to cooperate, surgical intervention for
logues. Other options are narrowband ultraviolet vitiligo is indeed a viable treatment option.
B phototherapy, targeted phototherapy such as
308 nm excimer light, surgical grafting and cos-
metic camouflage. The use of systemic cortico- 14.3 Vitiligo During Pregnancy
steroids for unstable disease may be considered
on a case-by-case basis. Specific considerations While the epidemiology and clinical characteris-
to make when treating children with vitiligo tics of vitiligo in pregnant patients should not
include the need to coordinate treatments with differ much from general population, the prog-
school schedules and the significant psychosocial nosis of vitiligo might be affected by the preg-
impact of the disease on children especially if nancy, as pregnancy worsens certain autoimmune
they have visible lesions. disease like systemic lupus erythematosus [40].
Koh et al. reviewed 71 Asian patients aged More importantly, there are important therapeu-
5–15 years who had received various modes of tic considerations one has to be mindful of when
phototherapy for vitiligo and found that the treating pregnant patients with vitiligo, so as to
response rates, defined as at least 50% repigmen- ensure that the treatment does not affect the
tation, with narrowband UVB (74%) and com- unborn foetus.
bined UVA1 and UVB phototherapy (67%) were
marginally higher than those with the 308 nm
excimer lamp phototherapy (53%) and paint 14.3.1 Clinical Characteristics
psoralen-UVA photochemotherapy (52%) [35]. of Vitiligo During Pregnancy
Paediatric patients with generalised vitiligo
respond better to phototherapy than those with There has been a paucity of studies examining the
segmental vitiligo [35], consistent with results prognosis of vitiligo during pregnancy as well as
also reported in adult patients [36]. the pregnancy outcome in patients with vitiligo.
Vitiligo surgery is generally not preferred in In a study of 28 patients with vitiligo while preg-
children because the techniques are time con- nant [41], 17.54% noted a worsening of vitiligo
suming and require cooperation from the child in during pregnancy with about 66.6% of patients
order to be successful. Mulekar et al. reported on remaining stable. In the same study, it was noted
a series of 25 children aged 4–16 years with seg- that 28% of patients experienced worsening of
mental or focal vitiligo who underwent noncul- vitiligo in the 6 months after delivery [41].
tured cellular grafting under sedation and local A similar descriptive study was carried out by
anaesthesia. Good to excellent repigmentation the authors at the Vitiligo Clinic in the National
(≥65%) was reported in 77% and 83% of patients Skin Centre, Singapore, from January 2016 till
with segmental and focal vitiligo, respectively April 2016 (unpublished data). The study
[37]. In a separate study comparing the results of included women who had been diagnosed with
children, adolescents and adults who underwent vitiligo before or during their pregnancies.
transplantation of autologous cultured melano- A standardised questionnaire was employed to
cytes, Hong et al. reported no statistically signifi- determine the clinical characteristics of vitiligo
cant difference in repigmentation among the during pregnancy and in the 6 months post-
three groups. Repigmentation of 50% or more partum. A total of 22 vitiligo patients participated
was obtained in 83%, 95% and 84% of children, in the survey. Of these, 70% recorded either
ERRNVPHGLFRVRUJ
worsening of their vitiligo (40%) or onset of vit- in pregnant vitiligo patients undergoing NBUVB
iligo during pregnancy (30%). All the patients phototherapy. NBUVB phototherapy has also
who had vitiligo onset during pregnancy were been shown to be safe during breastfeeding and
primigravida. During the 6 months post-partum, should be offered to patients, especially since up
about 36% of patients experienced worsening of to 36% of patients may experience worsening of
vitiligo, while 10% noted an improvement. their condition in the 6 months post-partum.
The observed worsening of vitiligo during
pregnancy and in the 6 months post-partum is
interesting and should be studied further. 14.4 Late-Onset Vitiligo
Pregnancy is associated with considerable physi-
ological stress and significant hormonal changes; There is limited published data on late-onset vit-
in addition, most patients would stop their first- iligo. Furthermore, there is no standardised defi-
line topical therapies during pregnancy. Therefore, nition of late-onset vitiligo. Several authorities
a confluence of these factors could account for the have placed the cut-off point of late-onset vitiligo
worsening of vitiligo during pregnancy. to be after the age of 12 [19, 44], 20 [45], 30 [46],
With regard to effects of vitiligo on pregnancy 40 [47] and 50 [48, 49]. Therefore, it is challeng-
outcomes, a study by Horev et al. [42] concluded ing to analyse the published data due to the het-
that vitiligo is not associated with adverse preg- erogeneity of the study population.
nancy outcomes, and hence no special antenatal
management considerations are required for this
group of patients. 14.4.1 Definition and Epidemiology
of Late-Onset Vitiligo
14.3.2 Therapeutic Considerations In this chapter, we will arbitrarily adopt the defi-
of Vitiligo During Pregnancy nition by Dogra et al. [48], where late-onset vit-
iligo is defined as vitiligo that appears after the
The approach to the treatment of vitiligo during age of 50 years. In that particular case series,
pregnancy is largely influenced by how the treat- 2672 vitiligo patients were seen over a period of
ment may affect the unborn foetus. The benefits 10 years, and of these, a total of 182 patients
of any treatment have to be weighed against pos- (6.8%) were found to have late-onset vitiligo.
sible adverse effects on the unborn foetus. First- The mean age at onset of disease was 55 years
line treatment like topical corticosteroids, topical [48]. In a recent retrospective review done in the
calcineurin inhibitors and topical vitamin D ana- National Skin Centre, Singapore, out of 3134
logues are all classified by the FDA as Class C. vitiligo patients seen over a period of 5 years,
(Risk cannot be ruled out. Human studies are 461 (14.7%) patients had late-onset vitiligo
lacking, and animal studies are either positive for [53]. The mean age at onset was 59 years, simi-
foetal risk or lacking as well.) As such, most lar to that reported by Dogra et al. Although a
pregnant ladies are not keen to continue with the slight female preponderance has been reported
usual first-line topical treatment. The only treat- [48], there is no significant gender difference in
ment that has been deemed safe for use during most studies including Singapore’s series [48,
pregnancy is NBUVB, and patients with vitiligo, 49, 53].
especially those with unstable vitiligo, should be Childhood-onset vitiligo has more f requently
treated with NBUVB. However, a recent study on been associated with a family history of vitil-
use of NBUVB in psoriasis patients seems to igo, as compared to late-onset vitiligo [19, 44,
suggest that high cumulative NBUVB doses can 47]. Notwithstanding, Dogra et al. reported that
decrease serum folate levels in patients with pso- up to 16% of late-onset vitiligo patients had a
riasis treated with NBUVB [43]. In light of this, family history of vitiligo, with first-degree rela-
it is important to enforce folate supplementation tives affected in 11.5% and second-degree
ERRNVPHGLFRVRUJ
148 S. Thng et al.
relatives affected in 4.4% of patients [48]. In

our Singapore study, only 5.4% of late-onset
patients had a family history of vitiligo [53]. It
has been suggested that there may be two coex-
isting modes of inheritance for vitiligo, depend-
ing on the age at onset [50]. In early-onset
vitiligo, the mode of inheritance has been
reported to be dominant with incomplete pene-
trance, whereas in late-onset vitiligo, it is reces-
sive and, hence, exposure to certain
environmental factors is needed to trigger the
Fig. 14.4 Acral vitiligo in late onset vitiligo patient
presentation.
elbows at work and those who engage in house-

14.4.2 Clinical Features, Treatment hold-related activities would tend to traumatise
Response and Course their hands and wrists more often [45]. Facial
of Disease vitiligo has not been linked to specific age
groups; however vitiligo in the perioral and
14.4.2.1 Clinical Features beard areas in men and vitiligo in the axillae in
Vitiligo vulgaris is the most common subtype of women have been seen more often in older
late-onset vitiligo [48, 49]. It is reported to patients [45]. This may be accounted for by
account for 83.5% of cases of late-onset vitiligo Koebner’s phenomenon secondary to shaving in
seen by Dogra et al. [48] and 94.6% in the these areas [45, 48]. While the Koebner’s phe-
Singapore series [53]. Focal vitiligo was reported nomenon may explain the predilection for cer-
to be the second most commonly occurring sub- tain locations, it neither accounts for all age- and
type in the series by Dogra et al. [48], and this gender-related differences [45], nor does it
has been observed in our case series too; explain the occurrence of vitiligo in locations
Esfandiarpour et al. [49], on the other hand, without a history of trauma. Genome-wide asso-
reported that the acrofacial subtype was the sec- ciation studies have given us more information
ond most common presentation followed by focal on the genetic background of vitiligo, and the
vitiligo. Universal and pure mucosal vitiligo were difference in the distribution pattern may reflect
the least common subtypes [48, 49]. Leukotrichia the underlying genetic predisposition rather than
was found to be common in late-onset vitiligo age at onset or gender factors [45].
with reports ranging from 47.3% [50] up to Besides koebnerisation, psychological
77.8% [49]. Halo nevus is not commonly seen in stress has been associated with vitiligo onset
late-onset vitiligo [48]. and progression in both childhood and late-
Although the head and neck was reported by onset vitiligo [19, 46, 49]. However, patients
Dogra et al. [48] to be the most common site of with late-onset vitiligo seem to progress faster
early involvement, other studies have found the than childhood vitiligo, and the disease pro-
main site of presentation for late-onset vitiligo to gression is significantly associated with a
be the upper extremities [45, 47, 49] (Fig. 14.4). stressful event [19, 46].
This is in contrast to childhood vitiligo where
the lower extremities were affected more often 14.4.2.2 Associated Conditions
[19, 45]. It has been postulated that such site pre- Vitiligo is associated with autoimmune diseases,
dilection occurs because younger patients are and it was reported to be present in up to 21.4%
more susceptible to lower extremity trauma sus- of late-onset vitiligo patients [48, 49]. The most
tained during play, whereas older patients hold- common associated autoimmune disease reported
ing desk jobs would exert pressure on their was diabetes mellitus followed by thyroid disease
ERRNVPHGLFRVRUJ
and alopecia areata [47–49]. Other minor associ- The association of late-onset vitiligo with dia-
ated conditions included rheumatoid arthritis, betes mellitus and thyroid disease has been
pernicious anaemia and Addison’s disease [47, reported to be significant [19, 47]. Screening for
48]. The association of diabetes mellitus with these conditions should be done appropriately.
late-onset vitiligo was reported to be between
15% [48] and as high as 69% [47]. Both diabetes
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Vitiligo and Skin of Color
15
Onyeka Obioha, Candrice Heath,
and Pearl E. Grimes
Contents
15.2 Quality of Life (QOL) 154
15.3 Etiology and Pathogenesis 155
15.4 Types of Vitiligo 156
15.5 Treatment 157
15.6 Depigmentation 158
15.7 Conclusion 159
References 159
Abstract emotional impact on the female population

The prevalence of vitiligo does not differ with and those with darker skin tones may be more
respect to different racial and ethnic groups; profound. In some geographic areas, social
however the prevalence has been shown to stigma of having vitiligo is very high, and psy-
vary in different regions of the world. Given chiatric morbidity is reported in nearly 75% of
the stark contrast between white patches and affected individuals. According to the psycho-
normal skin tone in black patients, vitiligo logical and social impact, even the manage-
may have distressing psychological impacts in ment should be planned and discussed
affected individuals and may adversely affect considering combinatory therapies as well as
quality of life. Its impact on self-esteem and depigmenting approaches not frequently
self-image may be profound. Additionally, the asked by fair-skin patients.
O. Obioha · C. Heath · P. E. Grimes (*)

Vitiligo & Pigmentation Institute of Southern
California, Los Angeles, CA, USA

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154 O. Obioha et al.
skin tone of subjects was uniquely associated

Key Points with a few factors, as patients with darker skin
• The prevalence does not differ with tones were more likely to think that vitiligo had
respect to different racial and ethnic repercussions on physical appearance and felt
groups. burdened by the daily management of their vitil-
• Vitiligo may adversely affect quality of igo [13]. This is in contrast to the subjects with
life mainly in dark-skin patients consid- fair skin tones who worried more about a per-
ering the stark contrast with unaffected ceived increase risk of skin cancer [13]. A recent
skin. study assessed the psychosocial impact of vitil-
• The path mechanism is the same in the igo and acne compared to normal controls [14].
fair- and dark-skin patients. Irrespective of age, sex, and severity of their vit-
• Higher incidence of trichrome vitiligo iligo or acne, quality of life was negatively
and hypochromic and follicular vitiligo impacted based upon the Dermatology Life
in darker racial ethnic groups. Quality Index (DLQI) score [14]. Both groups
• NB-UVB combined with afamelanotide had higher levels of social anxiety, depression,
appears to be highly effective in and general anxiety [14].
Fitzpatrick V–VI skin phototypes. Commonly utilized dermatologic quality of
life assessments, such as the Skindex-16 and
Dermatology Life Quality Index, may not accu-
rately capture all of the relevant information
15.1 Introduction needed to further assess the true impact of vitil-
igo on quality of life [15]. Some studies have
Vitiligo affects 0.5–2% of the general population demonstrated little to no impact, which is a dis-
worldwide [1–3]. The prevalence does not differ connect from what is expressed during patient
with respect to different racial and ethnic groups; interactions [15]. The questions on traditional
however the prevalence has been shown to vary generalized quality of life assessments contain
in different regions of the world. An isolated vil- symptom-focused questions about pruritus, scal-
lage in Gujarat, India, has the highest reported ing, and pain, all of which are usually absent in
prevalence of vitiligo at 8.8% [4]. In contrast, vitiligo [15]. Thus, several vitiligo-specific
large population studies in China and Denmark impact scoring systems have been published
report the prevalence to be much lower at 0.093% [16]. Investigators in India have also developed a
and 0.38%, respectively [5]. To date, there is no vitiligo-specific quality of life measurement
data that demonstrates there is a higher or lower (VIS-22) that includes culturally specific topics
incidence of vitiligo in skin of color. by including questions which address social
stigma, issues with marriage, the impact of others
avoiding physical contact, and concerns about
15.2 Quality of Life (QOL) contagiousness [17, 18]. Such topics are crucial
in addressing QOL in the Indian culture, since
Given the stark contrast between white patches the social stigma of having vitiligo is very high
and normal skin tone, vitiligo may have distress- and psychiatric morbidity is reported in nearly
ing psychological impacts in affected individuals 75% of affected individuals [19]. Although many
and may adversely affect quality of life [6, 7]. Its of these questions may not be relevant in other
impact on self-esteem and self-image may be cultures, they are an example of conducting
profound [8, 9]. Additionally, the emotional research and delivering patient care through the
impact on the female population and those with prism of cultural competence [20].
darker skin tones may be more profound [6, Lilly et al. developed and validated a new
10–12]. Ezzedine et al. concluded that regardless vitiligo-
specific quality of life instrument
of skin type, dark or light, patients are equally (VitiQol) [21]. They studied 90 subjects, 65% of
stressed about having vitiligo [13]. However, the whom had Fitzpatrick photo skin types IV–
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15 Vitiligo and Skin of Color 155
VI. VitiQol has also been validated in Brazilian matched controls [27]. Both groups were screened
Portuguese patients with vitiligo [22]. Despite for thyroid, antinuclear, parietal cell, smooth mus-
the validation of this scale, other investigators cle, and mitochondrial antibodies. Antithyroidal
continue to emphasize the need for an interna- antibodies were significantly increased in the pop-
tional vitiligo impact instrument consensus [15]. ulation of affected black patients compared to
From a global perspective, regardless of race controls. Other studies in Indian patients have
or ethnicity, patients with vitiligo are signifi- documented an increase of thyroglobulin, thyroid
cantly impacted by their disease. In patients with peroxidase, and antinuclear antibodies in vitiligo
dark skin tones, the contrast between their nor- patients [28, 36]. The presence of such antibodies
mally pigmented skin and depigmented skin may in patients further substantiates the concept that
be dramatic, but in the senior author’s opinion, autoimmune mechanisms contribute to vitiligo
even patients without a striking contrast may still regardless of race and ethnicity.
have the same level of emotional disturbance Recent reports have investigated the role of
which prompts them to seek care. Vitamin D in the pathogenesis of vitiligo. Vitamin
D deficiency has been associated with other auto-
immune disorders including multiple sclerosis,
15.3 Etiology and Pathogenesis systemic lupus erythematosus, undifferentiated
connective tissue disease, and rheumatoid arthri-
There is no data in the literature to suggest or tis [29]. Vitamin D has been shown to influence
support that the etiology of vitiligo differs in dif- melanocyte activation, differentiation, and prolif-
ferent racial and ethnic groups. eration, as well as modulation of T-cell activation
Genetic studies support a non-Mendelian, resulting in the suppression of proinflammatory
multifactorial, polygenic inheritance pattern [23, cytokines [30]. Vitamin D exerts its effect via its
24]. Twenty-five percent to 50% of patients with nuclear hormone, Vitamin D receptor (VDR).
vitiligo have affected relatives, and 6% of siblings To date, many of the studies assessing the
have the disorder [24]. Thirty-six susceptibility association between vitiligo and Vitamin D defi-
loci have been identified for nonsegmental vitil- ciency have been performed in people of color. In
igo [25]. Results from genetic studies demonstrate a Chinese population, Li et al. suggested that
that there is significant genetic h eterogeneity in VDR polymorphisms may influence the risk of
susceptibility loci in different racial groups. In a developing vitiligo by affecting the formation of
genome-wide linkage analysis of 71 multiplex 25-hydroxyvitamin D (25 (OH) D) [31]. In this
Caucasian families with generalized vitiligo in the study of 749 patients, a significantly decreased
United States and the United Kingdom, Fain and risk of vitiligo was found to be associated with
colleagues found a highly significant linkage of VDR polymorphisms in the BsmI-B, ApaI-A, and
vitiligo with an autoimmune susceptibility locus TaqI-t alleles [31]. They also found significantly
on chromosome 1p31, suggesting that it repre- lower levels of 25 (OH) D in patients with vitil-
sents a major susceptibility locus in the Caucasian igo, as well as a dose-response relationship
population [23]. In contrast, a genome-wide anal- between decreased vitiligo risk and increased
ysis of 57 multiplex Chinese families identified serum 25 (OH) D levels in subjects with the ApaI
the presence of a major susceptibility locus on allele [31].
chromosome 4q13-q21 [26]. In this Chinese sam- In a pilot study, Silverberg et al. assessed
ple, the authors found no overlap in linkage serum 25 (OH) D levels in 45 vitiligo patients
between the major susceptibility loci identified in and determined that vitiligo subjects with comor-
white populations in previous genome-wide link- bid autoimmune disease and increasing
age studies [26]. Further studies are needed to Fitzpatrick phototype were more likely to have
fully elucidate the difference in susceptibility loci low serum levels of Vitamin D [32]. In another
in different racial and ethnic groups. case-control study of 40 vitiligo subjects with
The frequency of autoantibodies was compared Fitzpatrick skin phototypes III–IV, the authors
between 70 black patients with vitiligo and 70 reported lower serum 25 (OH) D levels in vitiligo
ERRNVPHGLFRVRUJ
patients with autoimmune disease than in those

without a comorbid autoimmune disorder [33].
Serum levels of Vitamin D and Vitamin D
receptor expression in lesional and non-lesional
skin were assessed in 30 Egyptian patients and
30 age- and gender-matched controls. Forty
percent of the patients had insufficient levels
and 27% had deficient levels. VDR-mRNA
expression was significantly decreased in
lesional versus non-lesional skin, further sug-
gesting a role for Vitamin D as a contributor in
the pathogenesis or progression of vitiligo [34].
Hence, Vitamin D may be a useful screening
tool in patients with vitiligo.
15.4 Types of Vitiligo
The clinical subtypes of vitiligo are determined

by the distribution and appearance of lesions on
the skin. Common subtypes include generalized
(vitiligo vulgaris), acral, acrofacial, and segmen-
tal vitiligo (Table 15.1). Studies suggest a higher
incidence of trichrome vitiligo and hypochromic
and follicular vitiligo in darker racial ethnic
Fig. 15.1 Trichrome vitiligo axillary region. Note shades
groups [35–40] (Figs. 15.1 and 15.2). of white, tan, and normal skin
Table 15.1 Clinical subtypes of vitiligo

Subtypes of vitiligo Distribution Skin of color highlights
Generalized (vitiligo vulgaris) Symmetric, generalized No distinct features compared to
Acral vitiligo Extremities other groups
Acrofacial vitiligo Extremities and face
• Lip-tip subtype affects the lips and distal
digits only
Segmental vitiligo Dermatomal pattern
Trichrome vitiligo Color of patches include white, brown, and Trichrome vitiligo most commonly
normal skin color occurs in those with darker skin hues
Follicular vitiligo Primarily involves the hair follicles; Common in skin of color
leukotrichia of the hairs overlying vitiligo
patches and normal skin
Hypochromic vitiligo Hypopigmented patches in seborrheic Reported cases have been
distribution on the face, neck, and exclusively in those with Fitzpatrick
hypopigmented scalp macules; few or no skin types V and VI
depigmented macules or patches; no or poor
response to standard vitiligo treatments;
leukotrichia is absent
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Patients with skin of color have been found to

exhibit enhanced treatment responses to vitiligo
therapy compared to patients with lighter skin
types. Silverberg and Silverberg conducted a ret-
rospective chart review of adults and children
with vitiligo treated using tacrolimus 0.003% and
0.1%. While tacrolimus was effective in all
Fitzpatrick skin types, maximal repigmentation
of body lesions occurred in individuals with skin
types III and IV [41]. Studies show that photo-
chemotherapy with PUVA achieves maximal
repigmentation in patients with darker skin types
compared to lighter skin types and in patients
who reach erythema grade 2 [42, 43]. However,
the risk of hyperpigmentation with repigmenta-
tion induced by PUVA is more common in darker
skin types. In the senior author’s experience, the
hyperpigmentation fades either during or upon
cessation of therapy.
Nicolaidou and colleagues found that darker
skin (phototypes III–V) is a predictor of good
Fig. 15.2 Hypochromic vitiligo. Multiple hypopig- response to narrowband UVB (NB-UVB) in
mented lesions of the back patients with vitiligo [44]. In a study of 70 vitil-
igo patients, those with skin types III–V were
15.5 Treatment significantly more likely to reach cosmetically
acceptable repigmentation on the face after
The therapeutic objectives for vitiligo are stabili- NB-UVB compared to patients with skin types I
zation of disease and repigmentation of vitiligi- and II [44]. This enhanced treatment response
nous skin lesions. Treatment requires an was not seen with treatment of nonfacial sites.
individualized approach based on patient expec- The response to NB-UVB among different
tations, disease severity, and progression of the darker racial ethnic groups may also vary. In
disease. Current medical therapies include topi- studies performed by Kanwar et al. evaluating the
cal and systemic steroids, topical calcineurin efficacy of NB-UVB in two different samples of
inhibitors, narrowband UVB phototherapy, Indian patients (all Fitzpatrick skin types IV to
psoralen with ultraviolet A (PUVA), targeted
VI), the rates of cosmetically acceptable repig-
phototherapy, nutritional vitamin supplementa- mentation were found to be 71.4% and 75% [45,
tion, and calcipotriol. Of the aforementioned 46]. Alternatively, results from a sample of
therapies, maximum repigmentation has been Chinese subjects, skin types III and IV, reported a
achieved with topical steroids, calcineurin much lower rate of repigmentation, 12.5% [47].
inhibitors, and narrowband UVB phototherapy This difference may be explained by skin
(Figs. 15.3 and 15.4). Emerging therapies phototype.
include afamelanotide, a synthetic analog of Grimes et al. and Lim et al. assessed the effi-
naturally occurring alpha-melanocyte-stimulating cacy and safety of afamelanotide implants (a syn-
hormone (α-MSH), bimatoprost, a prostaglandin thetic α-MSH analog) combined with NB-UVB
F2-alpha analog, and prostaglandin E2. phototherapy compared to NB-UVB monother-
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Before After
Fig. 15.3 Narrowband UVB phototherapy. Note repigmentation of the arms
apy for vitiligo [48, 49]. Fifty-five patients (28 discussions regarding depigmentation are
combination group, 27 NB-UVB monotherapy) broached. Some providers suggest or require
were included in this 6-month randomized multi- patients to visit a psychologist or psychiatrist
center trial [49]. Maximal repigmentation in the before depigmentation is started. The senior
combination group was achieved in patients with author has no patients of color who regretted
Fitzpatrick SPT V and VI. their decision to depigment their skin.
Depigmentation can be achieved using topical
phenolic compounds, laser therapy, and cryother-
15.6 Depigmentation apy [50, 51]. However, monobenzyl ether of
hydroquinone (MBEH), a phenol derivative, is
Depigmentation therapy can be a viable thera- the most commonly used depigmenting agent for
peutic alternative in patients with extensive vitil- vitiligo. The mechanism of action is not com-
igo affecting greater than 30–40% of body pletely understood, but it has been shown to
surface areas. In skin of color patients, many cause selective necrosis of non-follicular human
physicians may delay discussing depigmentation melanocytes [52]. This may be due to its struc-
due to sensitivity and concerns for the perceived tural homology with tyrosine. Full depigmenta-
culturally driven self-image issues. In the senior tion may require 4–12 months of therapy and
author’s opinion, patients with skin of color usu- generally takes longer in patients with darker
ally develop feelings of acceptance by the time skin [53, 54].
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Before After
Fig. 15.4 Narrowband UVB phototherapy. Note significant repigmentation of the back
Depigmentation with MBEH is permanent 15.7 Conclusion

in most cases and is histologically associated
with loss of melanosomes and melanocytes; Our current database documents an equal inci-
however spontaneous repigmentation can dence of vitiligo in all racial ethnic groups.
occur within weeks to years after discontinua- However, the condition is most disfiguring in
tion of therapy [54]. The mechanism of sponta- darker skin types given the contrast between the
neous repigmentation after therapy is not well normal skin and white patches. The disorder is
characterized but may occur due to activation often psychologically devastating for darker skin
of the follicular melanocytes which are unaf- types. No studies have reported differences in
fected by MBEH [51]. pathogenesis. However, multiple studies docu-
In a retrospective study of 53 patients at a ment enhanced repigmentation responses in skin
London hospital undergoing depigmentation, the of color.
majority had olive or darker skin [55]. The high-
est concentration MBEH tolerated was 20%.
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Vitiligo-Like Lesions in Patients
with Metastatic Melanoma
16
Receiving Immunotherapies
Katia Boniface and Julien Seneschal
Contents
16.2 General Background 164
16.3 pecial Considerations in Vitiligo-Like Lesions in Patients Receiving
S
Anti-PD-1 Therapies 164
References 166
Abstract This review aims to discuss special consider-

The development of immunotherapies has ations in vitiligo-like lesions occurring in
remarkably improved the efficacy of treatment patients receiving anti-PD-1 therapies from a
of patients with metastatic melanoma, as clinical and physiopathological perspective.
observed for example with immune check-
point therapies targeting cytotoxic
T-lymphocyte-associated antigen 4 (CTLA-4)
or programmed cell death-1 (PD-1). Key Points
Nonetheless, reinstatement of an effective • Vitiligo-like lesions are one of the most
antitumor immune response also contributes frequent immune-related adverse events
to the development of immune-related adverse in melanoma patients receiving anti-
events, including vitiligo-like depigmentation. PD-1 therapies.
• Vitiligo-like lesions occurring under
anti-PD-1 are associated with clinical
K. Boniface benefit and overall survival in patients
1INSERM U1035, BMGIC, Immuno-dermatology with metastatic melanoma.
team, University of Bordeaux, Bordeaux, France • Vitiligo-like lesions occurring in meta-
J. Seneschal (*) static melanoma patients receiving anti-
1INSERM U1035, BMGIC, Immuno-dermatology PD-1 harbor a strong type 1 skewed T
team, University of Bordeaux, Bordeaux, France
cell phenotype.
Department of Dermatology and Pediatric • Vitiligo-like lesions in patients receiv-
Dermatology, National Reference Center for Rare
Skin Diseases, Saint-André Hospital, University of
ing anti-PD-1 clinically differ from
Bordeaux, Bordeaux, France vitiligo.
e-mail: julien.seneschal@chu-bordeaux.fr

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164 K. Boniface and J. Seneschal
16.1 Introduction same responses that underlie autoimmunity.

Among these autoimmune side effects, one is
Immunotherapy is poised to play a more central of particular interest in the context of mela-
role in the treatment of metastatic melanoma. noma: the occurrence of vitiligo-like lesions. It
These therapies have shown variable success has been recently shown in a systemic review
rated in the antitumor response. However, the use published by Teulings et al. [1] that while
of these therapies is also associated with the occurring in a low percentage of patients with
development of several autoimmune side effects, melanoma treated with immunotherapy, vitil-
including vitiligo-like lesions. Interestingly, igo-like lesions were significantly associated
development of vitiligo-like lesions (a clinically with overall survival (HR 0.25; 95% CI 0.10–
visible immune-related adverse event) has been 0.61). It has been estimated that the cumulative
correlated with increased survival in patients incidence of vitiligo-like lesions was 3.4%
receiving immunotherapies [1]. Vitiligo-like under immunotherapies. Richards et al.
lesions seem to appear frequently in patients described first a positive correlation between
receiving anti-programmed cell death-1 (PD-1) vitiligo-like lesions with tumor regression in
therapies, and recent data suggest that the pattern patients treated with sequences of chemother-
of depigmentation is different from spontane- apy (carmustine, dacarbazine, and cisplatin)
ously occurring vitiligo [2, 3]. and immunotherapy (IL-2 and IFN alfa-2) [8].
Boasberg et al. reported an incidence of vitiligo-
like depigmentations of 43% in 49 patients with
16.2 General Background metastatic melanoma who were treated with
injections of IL-2 after the induction of chemo-
Several immunotherapies have been evaluated therapy [9]. A positive correlation of autoim-
in the treatment of metastatic melanoma includ- mune manifestations and clinical response in
ing (1) immune stimulation with interferon patients with metastatic melanoma who were
(IFN)-alpha or interleukin (IL)-2; (2) vaccina- treated with anti-CTLA4 antibodies has also
tion strategies using vaccines based on dendritic been reported [10]. Of interest, vitiligo-like
cells, tumor cells, tumor antigenic peptides, lesions in patients with metastatic melanoma
and/or gene transfers; or (3) more recently, neu- treated with anti-PD-1 neutralizing antibodies
tralizing antibodies directed against immuno- occur more frequently than with other immuno-
suppressive checkpoints, such as cytotoxic therapies (further detailed below) [1]. Moreover,
T-lymphocyte- associated antigen 4 (CTLA-4) vitiligoid lesions are a much more common
or PD-1 or its ligand PD-L1 [4, 5]. Some of adverse event in melanoma immunotherapy
these strategies have shown clear antitumor compared with immunotherapy for other solid
activity in the treatment of advanced melanoma, tumors [11, 12].
suggesting a promising role for these novel
immunotherapies in the treatment of metastatic
melanoma. This was exemplified with the use of 16.3 Special Considerations
selective inhibitors of PD-1 (pembrolizumab, in Vitiligo-Like Lesions
nivolumab), which have shown impressive clini- in Patients Receiving
cal results in the treatment of metastatic mela- Anti-PD-1 Therapies
noma, leading to an accelerated development
and authorization [6, 7]. Vitiligo-like depigmentation is one of the most
However, the use of immunotherapies is frequent dermatologic adverse events observed
often associated with increased burden of toxic- with the use of anti-PD-1 therapies in cancer
ity, and some of them are immune-related patients, together with rash and pruritus [13, 14].
adverse effects. Indeed, restoring effective In patients with metastatic melanoma receiving
tumor immunity requires the induction of the anti-PD-1, occurrence of vitiligo-like depigmen-
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16 Vitiligo-Like Lesions in Patients with Metastatic Melanoma Receiving Immunotherapies 165
tations ranges from 8% to 25%, which is about signal that drives MITF is UV-induced DNA
tenfold higher than the occurrence of vitiligo in damage through p53, important for the promo-
the general population [7, 13, 15–18]. In line tion of melanocytes survival after UV exposure
with these observations, clinical studies on [22, 23]. Some of the MITF-regulated genes may
patients with metastatic melanoma receiving contain tumor-specific neoantigens, and others
anti-PD-1 therapies reported an objective may represent melanocyte lineage-specific anti-
response and overall survival benefit that were gens (such as melan A, gp100, and tyrosinase-
associated with development of vitiligo-like related protein-2) [21, 24–26]. Therefore,
lesions in patients [2, 15, 19]. activation of the immune system against MITF-
PD-1 is a well-known negative immune check- associated epitopes with anti-PD-1 could result
point receptor expressed by T cells. PD-L1/PD1 in destruction of melanoma and sometimes
pathway causes T-cell apoptosis, anergy, and destruction of normal melanocytes.
exhaustion. Therefore, PD-1 targeting on T cells Of interest is our recent observation that
with pembrolizumab or nivolumab will restore vitiligo-like depigmentation in patients receiving
T-cell activation that will be involved in immune anti-PD-1 therapies occurs mainly on photoex-
responses to tumor but will also lead to cytotoxic posed areas associated with pre-existing solar
effects responsible for development of adverse lentigines, with a specific depigmentation pattern
events, as vitiligo-like lesions. Interestingly, all consisting of multiple flecked lesions, without
the patients receiving anti-PD-1 therapies that the involvement of a Koebner phenomenon
developed vitiligo-like lesions have been treated (Fig. 16.1) [3]. A case report also described the
with anti-PD-1 for metastatic melanoma [13]. disappearance of pigmented skin lesions with
Histological analysis of vitiligo-like lesions in anti-PD-1 in a patient with metastatic melanoma,
patients receiving anti-PD-1 showed a prominent including naevi, seborrheic keratoses, and solar
CD8 T-cell infiltration and that the majority of lentigines [27]. These clinical observations con-
these cells expressed the chemokine receptor trast with spontaneously occurring vitiligo char-
CXCR3 and produced high levels of interferon-γ acterized by white patches symmetrically
and tumor necrosis factor-α [3]. In line with these distributed, and most patients develop lesions on
observations, PD-1 blockade induces the expres- repetitive friction sites. In addition, no history of
sion of the IFN-γ-inducible chemokine CXCL10 vitiligo of other autoimmune diseases were
at the tumor site in mice [20], leading to the hom- reported by patients receiving anti-PD-1 thera-
ing of CXCR3+ T cells. Interestingly, it was pies, suggesting that vitiligo-like depigmentation
recently shown in a mouse model of melanoma in these patients is clinically distinct from
that CD8 T-cell responses to melanoma initiate vitiligo.
autoimmune vitiligo and melanocyte destruction Therefore, melanocyte loss in vitiligo-like
was required for robust CD8 T-cell-mediated depigmentation occurring in patients receiving
antitumor response [21]. Therefore, the mecha- anti-PD-1 therapies appears to mainly result from
nistic correlation between vitiligo-like lesions melanocyte destruction, while in vitiligo two
and response to checkpoint therapies may shed main mechanisms have been involved: destruc-
light on the mechanisms of successful immune tion by the immune system and detachment from
attack of melanoma cells and incidental attack of the basal layer of the epidermis [28, 29].
normal melanocytes. Microphthalmia-associated Moreover, the occurrence of vitiligo-like lesions
transcription factor (MITF) that induces the tran- mainly on previously sun-exposed areas supports
scription of many genes important in pigment the possible link between MITF and the immune
production and the maintenance of the melano- response reinforced by immune checkpoint ther-
cyte, both in melanoma and normal cells, could apies. Hence, the mechanisms involved in mela-
also play an important role. MITF is important nocyte loss in spontaneously occurring vitiligo
for promoting survival of melanocytes after UV and vitiligo-like lesions in patients receiving anti-
exposure and DNA damage. Indeed, the original PD- 1 therapies might be different, and future
ERRNVPHGLFRVRUJ
166 K. Boniface and J. Seneschal
Fig. 16.1 Clinical pattern of depigmentation in patients receiving anti-PD-1 therapies
in patients receiving anti-programmed cell death-1

studies should in depth characterize and compare therapies are clinically and biologically distinct from
the molecular pathways involved in melanocyte vitiligo. J Am Acad Dermatol. 2017;76(5):863–70.
disappearance in these two conditions. 4. Achkar T, Tarhini AA. The use of immunotherapy
in the treatment of melanoma. J Hematol Oncol.
2017;10(1):88.
5. Anguille S, Smits EL, Lion E, van Tendeloo VF,
Berneman ZN. Clinical use of dendritic cells for can-
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receiving immunotherapy and its association with sur- 7. Robert C, Schachter J, Long GV, Arance A, Grob
vival: a systematic review and meta-analysis. J Clin JJ, Mortier L, et al. Pembrolizumab versus ipi-
Oncol. 2015;33(7):773–81. limumab in advanced melanoma. N Engl J Med.
2. Hua C, Boussemart L, Mateus C, Routier E, Boutros 2015;372(26):2521–32.
C, Cazenave H, et al. Association of vitiligo with 8. Richards JM, Mehta N, Ramming K, Skosey
tumor response in patients with metastatic mela- P. Sequential chemoimmunotherapy in the treat-
noma treated with pembrolizumab. JAMA Dermatol. ment of metastatic melanoma. J Clin Oncol.
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3. Larsabal M, Marti A, Jacquemin C, Rambert J, Thiolat 9. Boasberg PD, Hoon DS, Piro LD, Martin MA,
D, Dousset L, et al. Vitiligo-like lesions occurring Fujimoto A, Kristedja TS, et al. Enhanced survival
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associated with vitiligo expression during mainte- patients treated with nivolumab: a multi-institutional
nance biotherapy for metastatic melanoma. J Invest retrospective study. J Dermatol. 2017;44(2):117–22.
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Evaluation, Assessment,
and Scoring
17
Contents
17.2 Step-by-Step Evaluation 170
17.3 Associated Disorders and Laboratory Workup 172
17.4 Skin Biopsy 172
17.5 Scoring 172
17.6 Interobserver Variability 174
17.7 Correlations Between Assessment Variables 175
17.8 Subjective Items 175
References 176
Key Points • For scoring, extent, staging, and progres-

• At the first visit enough time is needed sion dimensions need to be covered.
for a clinical evaluation. • Wood’s lamp examination is important
• A checklist of important items is useful. in light skin for all scoring dimensions.
• The VETF assessement and scoring • Subjective items need to be considered, a
system covers most important items. global visual analog scale as used in VETF
is usually sufficient in clinical routine.
A. Taïeb
Service de Dermatologie, Hôpital St André, 17.1 Introduction
CHU de Bordeaux, Bordeaux, France
The first contact with a patient coming with a pre-
M. Picardo (*) vious diagnostic of vitiligo is of major impor-
Dermatological Institute, IFO, Rome, Italy tance. He/she says generally that previous doctors
e-mail: mauro.picardo@ifo.gov.it have not been keen to engage on a c onventional

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doctor-patient relationship about their disease, 17.2 Step-by-Step Evaluation

which they said was benign but out of therapeutic
reach. This common approach denies the status It is necessary to take enough time for a first visit,
of patient and explains the long search for alter- which includes a comprehensive clinical evalua-
native sources of cure. Secondly, it is important tion. The time required is roughly 20 min (an
to check the accuracy of the diagnosis (Chap. 2), identical extra time is needed for counseling and
because in a subset of patient, the depigmenting replying to questions). For lightly pigmented
disease has been labelled loosely “vitiligo” for patients, a dark room is needed for Wood’s lamp
some reason, and patients are grateful to obtain a examination (Fig. 17.1). The help of a standard-
diagnosis, if not a cure. This is commonly the ized sheet as available in the appendix allows not
case in children consulting for segmental to miss some issues and to secure the relevant
hypopigmentation (Chap. 14). information for routine clinical work or research,
There is a current lack of consensus in which is summarized in Table 17.1. It includes
methods of assessment of vitiligo, which the analysis of the Koebner phenomenon
makes it generally impossible to compare the (Chap. 11) is of particular interest for prevention
outcomes of different studies of the same treat- [5]. A question about vitiligo on genitals is on the
ment and to perform meta-analyses [1]. In VETF checklist because it causes a strong embar-
order to address these problems, the Vitiligo rassment to patients. Itch preceding lesions may
European Task Force (VETF) was founded in correspond to the micro-inflammatory nature of
2003 during the ESPCR meeting in Ghent with the disease. This symptom was mentioned by the
three initial goals: (1) the proposal of a consen- patient’s support groups. However, since com-
sus definition of the disease (Chap. 2), (2) the mon disorders such as atopic dermatitis may
design of biometric tools to assess disease coexist, especially in children, the exact value of
severity/stability, and if possible (3) the deriva- this symptom needs more in-depth investiga-
tion of a consensus scoring system. The second tions, and questioning should address a topo-
and third step are still ongoing, but following a graphic relation to the vitiligo patches.
method already used for the SCORAD index in Family history of vitiligo and/or premature
atopic dermatitis [2], an evaluation form was hair graying is currently routinely part of the
discussed and gradually improved at several assessment, as well as personal and family history
meetings which included the input of patient’s of thyroid disease and the presence of thyroid
support groups and tested in patients with gen- autoantibodies and autoimmune diseases. Hair
eralized vitiligo in a dozen of academic medi- graying is defined as more than 50% of white/
cal centers. Secondly, the most relevant gray hairs before the age of 40. This item is gener-
descriptive variables were selected for severity ally more difficult to assess in the female ancestry.
assessment and prognosis. Following this step, It is commonly found in pedigrees of vitiligo
the VETF held a workshop in Rome to test and patients [3, 6] and suggests the role of an aging
validate the evaluation sheet on a series of defect in the pigmentary system (Chap. 9). For
patients [3]. This chapter is written following the personal/family history of chronic possible
the remarks and criticism, which have followed autoimmune/inflammatory disorders, Table 17.2
the publication of this report [3]. An updated indicates some hints to ask questions, because the
and simplified version has been produced more related diseases are not always known by nonpro-
recently [4].1 fessionals. Clinical examination should guide the
need of specialized investigations (see below).
Halo nevi, considered as a marker of cellular
autoimmunity (Chap. 9), are assessed by history
The last version is also available online at http://content.
1 and at examination. A global quality of life assess-
nejm.org/cgi/content/full/360/2/160/DC1. ment is recommended (“how does vitiligo cur-
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17 Evaluation, Assessment, and Scoring 171
a b
Fig. 17.1 Wood’s lamp examination: it is best performed Wood’s lamp (b) and details on intermediate pigment
in a completely dark room. The examiner should adapt to tones to compare with (a). A magnifying lens incorpo-
the darkness for at least 30 s before starting examining the rated to the lamp is useful to analyze (terminal and vellus)
patient. Note the bright reflection of white patches under hair pigmentation
Table 17.1 Evaluation checklist for NSV (adapted from Taieb and Picardo [3])
Subject features Disease features Family Interventions
Phototype (Fitzpatrick’s skin Duration Premature hair Type and duration of
type) graying previous treatments
including opinion of patient
on previous treatments
(list): useful/not useful
Ethnic origin Activity, based on patient’s Vitiligo (detail if Current treatment(s)
opinion (progressive, regressive, needed in family
stable over the last 6 months) tree)
Age at onset Previous episodes of Other diseases and
repigmentation, and if yes, treatments (list)
spontaneous or not (details)
Stress/anxiety levels Koebner phenomenon on scars
or following mechanical trauma
Halo nevus Itch before flares
History of autoimmune diseases: Thyroid disease, if yes detail,
if yes, which type including the presence of thyroid
autoantibodies
Global QoL assessment (10 cm Vitiligo on genitals History of
analog scale) “how does vitiligo autoimmune
currently (last week) affect your diseases (detail
everyday life” if needed in
family tree)
Clinical photographs and if
possible UV light photographs
ERRNVPHGLFRVRUJ
rently (last week) affects your everyday life” or mune polyendocrine syndrome (APS),
“how much have you been bothered by the white anti-21-hydroxylase autoantibodies (Addison’s)
spots last week” assessed on a visual analog and anti-NALP 5 antibodies (hypoparathyroid-
scale). The impact of stress on disease onset/pro- ism) [10] can be looked for. For precursors to
gression is also taken into account. This form has diabetes, antibodies can be searched against
been designed for NSV but can serve also for seg- GAD65 and ICA512. A specialized advice is
mental or unclassified forms of the disease. strongly suggested.
17.3 Associated Disorders 17.4 Skin Biopsy

and Laboratory Workup
A skin biopsy is usually not requested for com-
Because of the magnitude of the association with mon NSV, except when another diagnosis cannot
autoimmune thyroid disease with NSV (25% in be ruled out (Chap. 2). However, this point of
our recruitment), the assessment of thyroid func- view needs probably to be challenged. Since skin
tion (thyrotropin, thyroid hormones) and pres- inflammation in vitiligo seems clinically not
ence of antibodies to thyroid peroxidase (TPO)/ detectable (Chap. 8), this simple test might allow
thyroglobulin should be routinely performed. A a more precise “inflammatory” staging of vitiligo
specific follow-up is needed when Hashimoto’s in the close future (pending the implementation
thyroiditis and Graves’ disease are diagnosed of less invasive tools). The finding of a
with referral to an endocrinologist. Associated micro-inflammatory border in active NSV
autoimmune disease frequencies are apparently (Chap. 3)—and/or of other local markers of
not similar according to ethnic background [7, disease activity—might become useful to make
8]. Some data emphasize the need of a larger more appropriate therapeutic decisions.
preventive screening because vitiligo may pre-
cede the onset of organ-specific autoimmune
diseases [9]. If a personal and/or family history 17.5 Scoring
of autoimmune/auto-inflammatory disorders is
detected (Table 17.2), broadening the etiologic Vitiligo treatments have been previously analyzed
investigations more specifically should be using the proportion of treated patients who
considered. In the context of familial autoim- achieve a specified degree of repigmentation, usu-
mune syndromes (Chap. 13) such as autoim- ally >50% for a “good” response [11], which is
much lower than patient’s expectations. A quanti-
tative score has been recently proposed [12]. The
Table 17.2 Assessment of chronic autoimmune/inflam- VETF has partially validated a clinical method of
matory diseases
assessment which combines analysis of extent
Ask by symptoms or (rule of nines), grading of depigmentation, and
Ask by name treatments
Alopecia areata Chronic diarrhea
progression. Figure 17.2 details the scoring sys-
Psoriasis, psoriatic arthritis Arthritis tem. Several problems have been raised during the
Lupus Hair loss patient’s session [3]. For staging, color of the
Scleroderma Chronic bowel disease selected patch is clearly not homogeneous, espe-
Lichen planus, lichen Chronic skin disease cially in SV. Staging chosen by investigators gen-
sclerosus erally reflects the worst stage and not the most
Rheumatoid arthritis
representative stage. This poses problems when a
Ulcerative colitis Insulin
Crohn’s disease Thyroxin few white hairs are present in association with
Biermer’s (pernicious) (Hydro)cortisone skin repigmentation, and more than 30% is
anemia required for the worst stage in the revised scoring
Addison’s disease Vitamin B12 system [4]. Another difficulty was related to
Celiac disease the need to magnify the lesions to assess hairs,
ERRNVPHGLFRVRUJ
a Area % Area
Staging* Spreading*
(0–4) (–1+1)
Head and neck (0–9%)
Trunk (0–36%)
Arms (0–18%)
Legs (0–36%)
Hands and feet
Totals (0–100%) 0–20 (–5 +5)
*Largest patch in each area
b 4.5%
4.5%
4.5%
4.5%
18% 4.5%
18% 4.5%
1% 9% 9%
9% 9%
c No or Hair
partial whitening
hair more
whitening than 30%
0 1 2 3
Fig. 17.2 Scoring system (adapted from Taieb and and either marginal (from the border of the patch) or peri-
Picardo [4]). The system assesses three dimensions of the follicular (melanocyte precursors migrating from the hair
disease (extent, staging, spreading/progression), which follicle). Based on this, four stages (0–3) (Part c) are dis-
are summarized in a table for practical purposes (Part a of tinguished from normal pigmentation (0) to complete
panel). To assess extent (Part b), it is useful to refer to the depigmentation (3), based on the assessment of the largest
patient’s palm including digits, which averages 1% of patch in each territory. Intermediate stages are defined as
body surface area. We recommend to draw the patches follows: stage 1 means incomplete depigmentation; stage
and mark the evaluated patches on figure, if necessary 2 means complete depigmentation (may include hair whit-
with a higher magnification for detail; if child under 5, the ening in a minority of hairs, less than 30%). If stage 3
head and neck total 18% and legs 13.5% each and no persists after attempts of medical therapy, this would indi-
change in other parts. If any, indicate halo nevi on the cate a need for surgical treatment. Spreading is intro-
graph. Staging of vitiligo is based on the assumption that duced to include a dynamic dimension, since rapidly
repigmentation requires melanocytes to be still present progressive vitiligo needs urgent intervention to stabilize
either in the epidermis or in the hair follicle (reservoir) to the disease. +1 means additional patches in a given area or
repigment the skin. Repigmentation patterns reflect this demonstrated ongoing repigmentation using Wood’s lamp
assumption: homogeneous diffuse repigmentation occurs in light skin-colored patients: 0 means stable disease, and
when melanocytes remain in the interfollicular epidermis −1 means observed ongoing depigmentation
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especially vellus hairs. Wood’s lamp equipment The concordance between investigators using
for vitiligo assessment should include a magnify- the same assessment grid was measurable, but it
ing lens. Analysis of spreading (progressive/sta- seems reasonable to predict that the results can be
ble/regressive) was the most difficult item in a improved if some interactive training is available.
blind (non-patient influenced) test. The view There are probably, as noted for atopic dermatitis
already expressed in textbooks that concavity ver- workshops [13], intrinsic high and low scorer pro-
sus convexity as related to the general shape of a files which can be minimized when the causes of
patch may predict progression versus regression variability between observers have been identified.
is probably an indication to take with caution.
Perifollicular repigmentation may occur with pro-
gressing marginal depigmentation. Partial depig- 17.6 Interobserver Variability
mentation in a border of a patch may be interpreted
as repigmentation. Surprisingly, at the Rome As tested at a workshop with patients [3], the
workshop [3], the investigator’s opinion was right interobserver agreement was acceptable for the
in the majority of cases if the patient’s opinion three items, namely, extent, staging, and
was chosen as the gold standard. Overall, it was spreading, and individual scorer’s profiles could
felt that this item should be graded more accu- be defined. Black and white digitized photo-
rately using the patient’s opinion. graphs (Fig. 17.3) were chosen as references for
Visible light
UV light
UV light
+
Contrast
Patient 1 Patient 2 Patient 3*

Extent 0.8% Extent 1.5% Extent 6.9%
Staging 1 Staging 3 Staging 2
Spreading –1 Spreading –1 Spreading –1
*For patient 3 phototherapy was ongoing, explaining erythema. The area scored comprised
only the neck and upper thorax lesion on this patient
Fig. 17.3 Examples of assessments in three patients and spreading variables. The underlying assumption is
using the “ideal observer” method. This “ideal observer” that the value expressed by the majority of specialists is
is a virtual investigator, assigning the reference mean most likely to be the true value, as there is no gold
area involved and the modal value (mode) for the staging standard
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the discussion of data. However, it was noted close, because 92% of the evaluations were
that erythema (in patients treated with photother- within a range of 1% of the mean value.
apy) will appear gray in digitized black and Denominators were important to make a good
white images. judgment, as larger areas such as trunk and legs
To check the validity of the data gathered, are more difficult to score. Image analysis may
concordance analysis was carried out based on help in different settings (clinical research
the “ideal observer” method (Table 17.3). The mostly) to reduce interobserver variability.
investigators tended to underestimate staging 2
for staging 1, which resulted in being overesti-
mated (27 indications against 10 in part a of 17.7 Correlations Between
Table 17.3). The frequency of assigning incorrect Assessment Variables
values decreased with staging: 40% for stage 1,
34% for stage 2, and 30% for stages 3 and 4, Possible correlations between the three main
respectively. Table 17.3 (part b) shows a wide assessment variables (extent, staging, and
dispersion of data for spreading. Based on these spreading) have been investigated. Some associ-
data, it seems easier to identify regression (iden- ation was suggested between high staging and
tified in 72.5% of cases) even if one investigator limited extent of vitiligo, when assessment was
assigned progression instead of regression than limited to only one patch. Similarly, larger
stability and progression. For extent (Table 17.3 patches tended to be assumed by investigators to
part c), the investigators’ findings were very spread less than smaller patches, but the differ-
ence was not significant here, and a wide vari-
Table 17.3 Interobserver variability ability in spreading assessment makes an
Panelist scores interpretation of this item difficult. Stage 2
1 2 3 4 Tot increased with extent, and stages 3 and 4 were
Staging mainly assigned to areas >1% [3].
1 6 1 3 0 10
2 17 46 6 1 70
3 4 7 28 1 40
17.8 Subjective Items
4 0 0 3 7 10
Total 27 54 40 9 130
Panelist scores Response to disfiguring diseases is affected by basic
−1 0 1 ego strength [14]. Thus, psychological factors
Spreading should be taken seriously into account in the global
−1 29 10 1 40 care of vitiligo patients. Quality of life impact
0 11 27 12 50 (Chap. 18) is generally considered overall as
1 11 8 21 40 moderate in vitiligo, but the patient’s phototype,
Total 51 45 34 130
cultural background, and gender may influence the
Extent ±0.2% ±0.5% ±1%
Head and 17 19 20 20 differences in data reported [15]. The perceived
neck (85%) (95%) (100%) severity of vitiligo is explained mainly by the
Arms 11 13 20 20 patients’ personality and their psychological fea-
(55%) (65%) (100%) tures and less significantly by the clinical objective
Leg 13 17 29 (97%) 30
criteria. Perceived severity of the illness and
(43%) (57%)
Trunk 23 36 51 (85%) 60 patient’s psychological features such as trait depres-
(38%) (60%) sion are predictors of the patients’ QoL. Patients
64 85 120 130 who tend to be more anxious in their daily life and
(49%) (65%) (92%) who have a poor self-esteem perceive their illness as
The ideal observer method was used for staging, K ¼ more severe even if vitiligo is less extensive [15].
0.499; SE ¼ 0.059 (P < 0.00001); spreading, K ¼ 0.388; A higher prevalence of alexithymia and depression
SE ¼ 0.064 (P < 0.0001); and extent. The reference values
correspond to the modal (staging and spreading) or mean or anxiety was found in vitiligo patients as com-
(extent) score of the 10 panelists pared with the general population [16].
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There are now some specific arguments for 6. Halder RM, Grimes PE, Cowan CA, et al. Childhood
including a psychological support in the care of vitiligo. J Am Acad Dermatol. 1987;16:948–54.
7. Alkhateeb A, Fain PR, Thody A, et al. Epidemiology
vitiligo patients including consideration of their of vitiligo and associated autoimmune diseases in
personality and their difficulty of living with Caucasian probands and their families. Pigment Cell
this disease (Chap. 18). Consequently, some Res. 2003;16:208–14.
factors which are easy to assess such as per- 8. Liu JB, Li M, Yang S, et al. Clinical profiles of vit-
iligo in China: an analysis of 3742 patients. Clin Exp
ceived severity and patient’s personality (trait Dermatol. 2005;30:327–31.
anxiety, trait depression, trait self-esteem) could 9. Betterle C, Caretto A, De Zio A, et al. Incidence and
be included in the assessment and management significance of organ-specific autoimmune disorders
of this chronic disfiguring disease. A simple (clinical, latent or only autoantibodies) in patients
with vitiligo. Dermatologica. 1985;171:419–23.
perceived severity scale is clearly useful in clin- 10. Alimohammadi M, Björklund P, Hallgren A, et al.
ical practice as a screening tool. However, the Autoimmune polyendocrine syndrome type 1 and
development of a vitiligo specific QoL scale NALP5, a parathyroid autoantigen. N Engl J Med.
would probably be helpful. 2008;358:1018–28.
11. Westerhof W, Nieuweboer-Krobotova L. Treatment of
vitiligo with UV-B radiation vs topical psoralen plus
UV-A. Arch Dermatol. 1997;133:1525–8.
12. Hamzavi I, Jain H, McLean D, et al. Parametric

References modeling of narrowband UV-B phototherapy for vit-
iligo using a novel quantitative tool: the Vitiligo Area
1. Whitton ME, Ashcroft DM, Barrett CW, Gonzalez Scoring Index. Arch Dermatol. 2004;140:677–83.
U. Interventions for vitiligo. Cochrane Database Syst 13. Kunz B, Oranje AP, Labreze L, et al. Clinical vali-
Rev. 2006;(1):CD003263. dation and guidelines for the SCORAD index: con-
2. Anonymous. Severity scoring of atopic dermatitis: the sensus report of the European Task Force on Atopic
SCORAD index. Consensus Report of the European Dermatitis. Dermatology. 1997;195:10–9.
Task Force on Atopic Dermatitis. Dermatology. 14. Porter JR, Beuf AH, Lerner A, Nordlund J. Psychological
1993;186:23–31. reaction to chronic skin disorders: a study of patients
3. Taïeb A, Picardo M, VETF Members. The defini- with vitiligo. Gen Hosp Psychiatry. 1979;1:73–7.
tion and assessment of vitiligo: a consensus report of 15. Kostopoulou P, Jouary T, Quintard B, et al. Objective
the Vitiligo European Task Force. Pigment Cell Res. vs subjective factors in the psychological impact of
2007;20:27–35. vitiligo: the experience from a French referral center.
4. Taieb A, Picardo M. Clinical practice. Vitiligo. N Engl Br J Dermatol. 2009;161(1):128.
J Med. 2008;360:160–9. 16.
Sampogna F, Raskovic D, Guerra L, et al.
5. Gauthier Y. The importance of Koebner’s phenom- Identification of categories at risk for high qual-
enon in the induction of vitiligo vulgaris lesions. Eur ity of life impairment in patients with vitiligo. Br J
J Dermatol. 1995;5:704–8. Dermatol. 2008;159:351–9.
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Quality of Life
18
Contents
18.1 Introduction and Historical Perspective 177
18.2 Modern Psychological Studies 178
18.3 Socio-economic and Educational Consequences 178
18.4 Quality of Life Evaluation 179
References 179
Abstract
Key Points
Vitiligo can induce considerable psychosocial
stress and psychiatric comorbidity. Then, it is • Vitiligo can have a staggering effect on
important to recognize and manage the psy- the psychosocial well-being of an indi-
chological component not only to improve vidual, especially in those with darker
coping but also to obtain a better treatment phototypes and lesions on exposed sites.
response. • Various health-related quality of life
The training in assertiveness, relaxation measurement tools are now available,
skills and help in building self-confidence with those specifically designed to mea-
would have substantial effects on quality of sure the impact of vitiligo.
life as well as treatment outcomes. In fact, • An enhanced awareness amongst the
social and psychological well-being increase masses about this common disease shall
when patients with facial disfigurement are help to bring down the stigmatization
helped to develop social skills and to confront associated with vitiligo.
their difficulties. There is need for a standard-
ized quality of life evaluation tool which can
quantify the psychosocial stress of vitiligo
subject and which could be used for treatment 18.1 Introduction and Historical
evaluation. Perspective
Vitiligo has a major impact on the quality of life

A. Bishnoi · D. Parsad (*)
of patients, many of whom feel distressed and
Department of Dermatology, Postgraduate Institute of
Medical Education and Research, Chandigarh, India stigmatized by their condition [1–3].

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In ancient Indian texts, vitiligo was referred and stigmatization is the impossibility of con-
by the name kilas (in Sanskrit, kil - white, as - cealing the affected skin parts and consequent
throw or cast away), meaning one who throws visibility. In a recent study, stigmatization experi-
away the colour. Since ancient times, patients of enced by vitiligo patients was found psychologi-
vitiligo have suffered the same physical and cally relevant since patients with visible lesions
mental abuses as lepers of that age, and they experienced a higher level of stigmatization [10].
were considered to have ‘shweta kustha’. Vitiligo Appearance of skin can condition an individu-
is particularly disfiguring for people with dark al’s self-image, and any pathological alteration
skin and carries such a social stigma in Indian can have psychological consequences [11].
society that patients are sometimes considered Patients often develop negative feelings about
unmarriageable [4]. Society greets vitiligo their skin, which are reinforced by their experi-
patients in as much the same way as it does any- ences over a number of years. Most patients with
one else who appears to be different. In some vitiligo report embarrassment, which can lead to a
parts of India, they are still stared at or subjected low self-esteem and social isolation [12]. Facial
to whispered comments, antagonism, insult or vitiligo lesions may be particularly embarrassing,
isolation. A woman with vitiligo may face and the frustration of resistant lesions over
numerous social problems and experience great exposed part of hands and feet can lead to anger
difficulties in getting married. If vitiligo devel- and disillusionment. Particularly in teenagers,
ops after marriage, it grounds for divorce by the mood disturbances including irritability and
husband. In some parts of India, disease is often depression are common. Patients with vitiligo are
considered as a punishment by God, presumably extensively sensitive to the way others perceive
caused by unconscious feelings of guilt. Patients them, and they will often withdraw, because they
are subjected to various dietary restrictions like anticipate being rejected. Sometimes, strangers
avoidance of milk, fish, citrus fruits, etc. [5]. and even close friends can make extremely hurtful
and humiliating comments. The impact of such
factors is profound subjecting them to emotional
18.2 Modern Psychological distress, interference with their employment or
Studies use of tension-lessening, oblivion-producing sub-
stances such as alcohol [13]. Severe depression
Porter et al. [6, 7] in the late 1970s brought the has been known to lead to suicide attempts [14].
psychosocial effects of vitiligo to the attention of Vitiligo can also result in problems in interper-
dermatologists. A questionnaire survey among sonal relations and induce depression and frustra-
62 vitiligo patients in a hospital-based outpatient tion. In a study from India, vitiligo has been
setting indicated that two-thirds felt embarrassed shown to be associated with high psychiatric mor-
by the disease, more than half of them felt ill at bidity [12]. One fourth of vitiligo patients attend-
ease, and a majority of patients felt anxious, con- ing a specialized clinic was found to have
cerned and worried about the disease itself. They psychiatric morbidity, and a diagnosis of adjust-
also studied the effect of vitiligo on sexual rela- ment disorder was made in the majority of cases.
tionship and found that embarrassment during Psychiatric morbidity was significantly correlated
sexual relationship was especially frequent for with dysfunction arising out of illness [12].
men with vitiligo [8]. Many patients chose
adapted clothing and used large amounts of cos-
metics in order to hide the skin lesions. Although 18.3 Socio-economic
80% perceived their friends and family as sup- and Educational
portive, strangers expressed less understanding, Consequences
and patients felt uncomfortable meeting them.
Salzer and Schallreuter [9] reported that 75% Patients often suffer financial loss because they
found their disfigurement moderately or severely have to take time off from work to attend hospital
intolerable. An important component of stigma appointments to perform treatments such as photo-
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18 Quality of Life 179
therapies. Lesions in exposed sites can adversely logical and psychotherapeutic interventions may
affect a person’s chances of getting a job at inter- be helpful. In a study of 150 vitiligo patients, we
view and so restrict career choices. Vitiligo begin- assessed the nature and extent of the social and
ning in childhood can be associated with significant psychological difficulties associated with the
psychological trauma that may have long lasting disease and their impact on treatment outcome
effects on personal self-esteem [15]. Children with by using Dermatology Life Quality Index [21].
vitiligo usually avoid sport or restrict such activi- Our study clearly demonstrated that patients
ties and often lose vital days from school. with high DLQI scores responded less favour-
ably to a given therapeutic modality. Recently,
many vitiligo-specific quality of life scoring sys-
18.4 Quality of Life Evaluation tems have been developed like vitiligo impact
scale (VIS) [22], VIS-22 [23, 24], vitiligo spe-
Most of the studies have used Dermatology Life cific quality of life (vitiQoL) [25, 26] and vitil-
Quality Index (DLQI), a widely validated ques- igo impact patient scale [27]. All these are well
tionnaire that is easy to use and allows compari- validated and correlate well with other general
son between several skin disorders. However, skin disease quality of life scores (like DLQI,
there is a need for a uniformly acceptable scale Skindex), while catering specifically to the need
which can more specifically quantify psychoso- of measuring quality-of-life impact of vitiligo,
cial stress associated with this disease. Moreover, which extends much beyond the visible
this type of quality of life measures shall be use- depigmentation.
ful as tool to assess treatment effectiveness or to These results suggest that additional psycho-
compare treatment outcome. logical approaches may be particularly helpful in
Although a limited number of studies have these patients. In a preliminary study by
paid attention to the psychosocial effects of vitil- Papadopoulos et al. [28], it has been shown that
igo, they point towards an appreciable psychoso- counselling can help to improve the body image,
cial impact on those afflicted. Kent and Al’ self-esteem and quality of life of patients with
Abadie [16] found that the stigmatization experi- vitiligo as well as it may have a positive effect on
ence accounted for 39% of the variance in the course of the disease. To conclude, vitiligo can
quality of life of vitiligo patients. On the other have a tremendous effect on all spheres of one’s
hand, self-esteem, a number of symptoms on the lives. A major part of this effect arises because of
distress checklist, race and general health the lack of knowledge about this disease in
accounted for only 12% of the variance in quality masses and the resultant stigmatization. Through
of life. initiatives like world vitiligo day (celebrated
There may be a relationship between stress each year on 25th June), and other information
and vitiligo since psychological stress can and education activities, the knowledge regard-
increase levels of neuroendocrine hormones, ing vitiligo should be actively spread amongst
leading to a damage of melanocytes in the skin, the general population. That shall bring a posi-
and affect the immune system altering the level tive change in the manner vitiligo is perceived by
of neuropeptides [17]. Recently, increased levels laymen.
of neuropeptide-Y were shown in the plasma and
skin tissue fluids of patients with vitiligo [18].
Liu et al. [19] studied the occurrence of cutane-
ous nerve endings and neuropeptides in vitiligo References
vulgaris and suggested that emotional trauma and
stressful life events can cause large adrenal secre- 1. Bolognia JL, Pawelek JM. Biology of hypopigmenta-
tions and this can result in acute onset of tion. J Am Acad Dermatol. 1988;19:217–55.
2. Lerner AB. Vitiligo. J Invest Dermatol.
vitiligo. 1959;32:285–310.
Because of the possible connection between 3. Lerner AB, Nordlund JJ. Vitiligo. What is it? Is it
stress and exacerbation of vitiligo [20], psycho- important? JAMA. 1978;239:1183–7.
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4. Fitzpatrick TB. The scourge of vitiligo. Fitzpatrick’s J 19. Liu PY, Bondesson L, Löntz W, Johansson O. The
Clin Dermatol. 1993;1:68–9. occurrence of cutaneous nerve endings and neuropep-
5. Parsad D, Dogra S, Kanwar AJ. Quality of life in tides in vitiligo vulgaris: a case-control study. Arch
patients with vitiligo. Health Qual Life Outcomes. Dermatol Res. 1996;288:670–5.
2003;1:58. 20. Picardi A, Pasquini P, Cattaruzza MS, et al. Stressful
6. Porter JR, Beuf AH, Nordlund JJ, Lerner life events, social support, attachment security
AB. Personal responses to vitiligo. Arch Dermatol. and alexithymia in vitiligo. A case-control study.
1978;114:1348–85. Psychother Psychosom. 2003;72:150–8.
7. Porter JR, Beuf AH, Nordlund JJ, Lerner 21. Parsad D, Pandhi R, Dogra S, et al. Dermatology
AB. Psychological reaction to chronic skin disorders. Life Quality Index score in vitiligo and its impact
A study of patients with vitiligo. Gen Hosp Psychiatry. on the treatment outcome. Br J Dermatol. 2003;148:
1979;1:73–7. 373–4.
8. Porter J, Beuf A, Lerner A, et al. The effect of vit- 22. Krishna GS, Ramam M, Mehta M, Sreenivas V, Sharma
iligo on sexual relationship. J Am Acad Dermatol. VK, Khandpur S. Vitiligo impact scale: an instrument
1990;22:221–2. to assess the psychosocial burden of vitiligo. Indian J
9. Salzer B, Schallreuter K. Investigations of the per- Dermatol Venereol Leprol. 2013;79:205–10.
sonality structure in patients with vitiligo and a pos- 23. Gupta V, Sreenivas V, Mehta M, Khaitan BK, Ramam
sible association with catecholamine metabolism. M. Measurement properties of the Vitiligo Impact
Dermatology. 1995;190:109–15. Scale-22 (VIS-22), a vitiligo-specific quality-of-life
10. Schmid-Ott G, Kunsebeck HW, Jecht E, Shimshoni instrument. Br J Dermatol. 2014;171:1084–90.
R, et al. Stigmatization experience, coping and sense 24. Gupta V, Sreenivas V, Mehta M, Ramam M. What do
of coherence in vitiligo patients. J Eur Acad Dermatol Vitiligo Impact Scale-22 scores mean? Studying the
Venereol. 2007;21:456–61. clinical interpretation of scores using an anchor-based
11. Savin J. The hidden face of dermatology. Clin Exp approach. Br J Dermatol. 2018.
Dermatol. 1993;18:393–5. 25. Lilly E, Lu PD, Borovicka JH, Victorson D, Kwasny
12. Mattoo SK, Handa S, Kaur I, et al. Psychiatric mor- MJ, West DP, et al. Development and validation of a
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13. Ginsburg IH. The psychological impact of skin dis- 26. Catucci Boza J, Giongo N, Machado P, Horn R,

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14. Cotterill JA, Cunliffe WJ. Suicide in dermatological children and adults with vitiligo: a cross-sectional
patients. Br J Dermatol. 1997;137(2):246–50. study based on dermatology-specific and disease-
15. Hill-Beuf A, Porter JDR. Children coping with
specific quality of life instruments. Dermatology.
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16. Kent G, Al’ Abadie M. Factors affecting responses on Meurant J-M, Jouary T, et al. The Vitiligo Impact
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Defining the Disease: Editor’s
Synthesis
19
Contents
19.1 Clinical Assessment Is Important 182
19.2 A Need for Epidemiological Studies and Deep Phenotyping 182
19.3 Variable Melanocytic Targets According to Clinical Subtypes 182
19.4 Lessons from Associated Diseases and Rare Syndromic Cases 183
19.5 Predictive assessment of Vitiligo 183
19.6 Suboptimal Use of Pathology to Assess Vitiligo 183
19.7 Summary 184
References 185
Abstract data do not support a significant involvement

The initial clinical evaluation of a vitiligo of non-skin melanocytes in vitiligo/NSV.
patient is a step frequently neglected. Some Major initial determinants are probably sit-
new variants have been described only recently uated directly in the skin and activated in a sub-
such as mixed and follicular vitiligo, and set of patients by largely unknown stressors.
the frontiers of the disease challenge an Vitiligo/NSV can be considered as a marker of
unambiguous delineation of the disease (e.g. an auto-inflammatory/autoimmune diathesis,
vitiligoid depigmentation after cancer immu- and microinflammation is central to its patho-
notherapies, hypochromic vitiligo...). Major physiology. The skewing of the immune sys-
progresses have been made to understand her- tem towards vitiligo seems of benefit to fight
itability, but more epidemiological data are skin cancer and possibly internal cancer. If
needed to address in particular environmental inflammation and autoimmunity are pivotal in
factors, natural history, and comorbidities to all subset of vitiligo, other factors are involved
confirm on a large scale a protection against predisposing to premature melanocyte ageing.
cancer. At this time point, available clinical
Key Points
A. Taïeb (*) • A good clinical assessment is important
Service de Dermatologie, Hôpital St André, for optimal management.
CHU de Bordeaux, Bordeaux, France
• Epidemiology is largely uncovered, but
convergent evidence suggests that
M. Picardo
increased immunosurveillance in vitil-
Dermatological Institute, IRCCS, Rome, Italy igo limits the risk of cancer..

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19.2 A
Need for Epidemiological
• Clinical data suggest possible variable Studies and Deep
melanocyte targets according to subtype Phenotyping
of vitiligo.
• Associated diseases/comordidities need The spectrum of clinical manifestations in vitil-
to be assessed and may provide patho- igo is limited, but simple items such as variations
physiological insight. in age at onset and extension or pattern of dis-
• Mapping of segmental vitiligo has ease and natural course with possible spontane-
begun and may help predicting affected ous repigmentation suggest a complex and
territories. mysterious interplay between host and environ-
• Histopathology is needed for difficult ment. Incidence or prevalence rates seem stable,
cases. without major in changes observable in a short
time span, as noted for some chronic either Th1
or Th2 predominant diseases, which have been
the basis of the hygiene hypothesis, suggesting
19.1 C
linical Assessment Is that a westernized life style changes our micro-
Important bial environment and influence the population
risk for chronic inflammatory disorders [1].
The initial evaluation of a vitiligo patient is a step However, informative studies such as repeated
which is frequently neglected, as witnessed by twin studies have not been performed in vitiligo
the only recent delineation of a mixed form of as in atopic dermatitis [2]. Contrary to the large
vitiligo combining SV and vitiligo/NSV, of fol- data collected to understand the genetics of vit-
licular vitiligo and of frontier disorders such as iligo, more epidemiological data are clearly
vitiligoid depigmentation under cancer immuno- needed to better delineate the role of environ-
therapies and hypochromic vitiligo. It should mental factors, natural history, as well as comor-
include a thorough history taking and examina- bidities. The access to large databases is now
tion including Wood’s lamp or a monochromatic empowering research with new resources, and
365 nm lamp in a dark cabinet for all low photo- the possibility to access to deep phenotyping
type individuals. Clinical evaluation yields with standardized digital photographic systems
important information for routine management may fill some of the current gaps in disease defi-
purposes, such as detection of disease progres- nition. Very recent evidence using insurance
sion or stabilization, but also for asking relevant claims database suggest that vitiligo is protective
clinical research questions. Assessing coping and against cancer.
quality of life issues should not be forgotten. In
particular, the role of stress has been emphasized
by some studies and patients’ support groups. 19.3 Variable Melanocytic Targets
There is a need to clarify this issue of stress act- According to Clinical
ing as a trigger factor or more importantly as Subtypes
influencing disease progression, because it could
open new perspectives for management. The Clinical aspects suggest different cellular targets/
evaluation of the impact of the disease on non- territories according to subtypes of vitiligo as
skin melanocytes (eye, ear, heart, central nervous shown for the marked preference of leukotrichia
system) is not necessary for common vitiligo/ in SV. The mucosal pigmentary system seems to
NSV. For vitiligo universalis, there is a frontier be involved more frequently in patients of dark
with Vogt-Koyanagi-Harada (VKH) syndrome, complexion, but this aspect needs more accurate
which is unclear, and those patients should be studies in other populations.
evaluated more carefully for associated autoim- Data gathered in vitiligo studies worldwide
munity in this respect. do not support the concept of a common
ERRNVPHGLFRVRUJ
19 Defining the Disease: Editor’s Synthesis 183
involvement of non-skin melanocytes (with the 19.5 Predictive assessment

exception of the oral and genital mucosae and of Vitiligo
vitiligo universalis), contrary to what is found in
Vogt Koyanagi Harada syndrome which targets For the common and disfiguring facial involve-
ocular, auditory and central nervous system ment in segmental vitiligo, the possible predic-
melanocytes [3]. However some fulminant tion of territories involved after the detection of
forms of vitiligo which are classified universalis the initial macule is important. There is clearly a
may involve more generalized melanocytic tar- need of establishing a good database including
gets and other types of organ autoimmunity. patients of various ethnic backgrounds to settle
Vitiligo/NSV is in most instances a chronic skin this important issue. Similar studies are needed
condition with major initial determinants situ- for other body locations. The patterns of exten-
ated directly in the skin and which might be sion in vitiligo/NSV, if predictable, deserve also a
activated in a subset of patients by better attention for prognosis and management
largely unknown stressors. issues. The speed of progression is also an impor-
tant parameter of assessment which needs a bet-
ter quantitative validation of clinical symptoms
19.4 Lessons from Associated such as Koebner phenomenon, fuzzy borders of
Diseases and Rare patches and pinpoint depigmentation around
Syndromic Cases lesions. Noninvasive techniques of assessment
such as in vivo confocal microscopy may help the
History assessment of patients underlines two clinician, if more widely available. The use of
facts: the association in 20% of cases with a per- new scoring systems [5] combined with standard-
sonal or familial history of autoimmune/auto- ized photography may help acquiring robust data
inflammatory disorders where vitiligo is a marker for deep phenotyping and predictive assessment.
of an autoimmune diathesis, and the association
in 11% of patients with a family history of pre-
mature hair greying, which witnesses a visible 19.6 Suboptimal Use of Pathology
premature senescence of the pigmentary system to Assess Vitiligo
[4]. Syndromic vitiligo cases occurring in mono-
genic heritable diseases underline possible simi- Pathology has been considered as providing lim-
lar pathophysiological pathways which belong ited information on the nature and course of the
either to the immune diathesis, with more empha- disease, because diagnosis was easy to make on
sis on the humoral responses (APECED), or clinical grounds. Vitiligo, based on the micro-
defects in the oxidative stress and ageing process scopic examination of long lasting NSV depig-
(MIDAS syndrome, ataxia telangiectasia and mented macules, was synonym of an epidermis
Nijmegen breakage syndrome). The without pigment cells. As discussed in this sec-
immune-
mediated destruction of naevus cells tion, the study of the progressing but still pig-
(halo naevus) and the rare clinically inflamma- mented edges of vitiligo/NSV lesions (or with
tory onset of vitiligo, as well as vitiligo changes pinpoint depigmentation) and of distant normal-
in VKH, indicate clearly that inflammation and looking skin has provided evidence of microde-
cell-mediated immunity are important in a subset pigmentation and microinflammation. The
of patients. Interestingly, the recent emphasis on systematic pathology assessment of SV for evi-
pruritus in vitiligo may correspond to a far more dence of microinflammation is still lacking, but
common micro-inflammatory component which evidence has accumulated over the last decade
is not clinically symptomatic. Microinflammation that the onset of SV has histologically inflamma-
might be related to increased immune surveil- tory features. This new vision raises several ques-
lance with the benefit of a decreased risk for skin tions: (1) If vitiligo is inflammatory pathologically,
cancer. why is it usually not clinically? Erythema and
ERRNVPHGLFRVRUJ
Possible overlap
SV-NSV (mixed vitiligo)
Koebner’s phenomenon
Interfollicular skin melanocytic target
Hair melanocytic target
Hair graying** NSV

SV
Naevus
target*
?
Auto-inflammation/immunity**
Skin microinflammation
Syndromic
vitiligo
* Halo nevus ** Personal of family history
Fig. 19.1 Possible research angles of attack based on larger studies). The Koebner phenomenon and personal/
clinical findings in vitiligo. SV and NSV can overlap familial history of auto-inflammation/autoimmunity seem
(mixed vitiligo). The epidermal melanocytic target is mostly associated with NSV. Syndromic vitiligo may
equally shared between SV and NSV, but the hair melano- share some pathways influencing either survival or
cytic target is skewed towards SV. The history of hair immune loss of melanocyte with common nonsyndromic
greying and the presence of halo naevus seem equally vitiligo (SV and NSV). The question of microinflamma-
shared between the two main types (to be confirmed by tion for SV needs additional studies
pruritus are uncommon findings in the setting of least at onset, be inflammatory as it is the rule in
even rapidly progressing vitiligo, suggesting a vitiligo/NSV? Some simple clinical and
unique pattern of responsiveness or a so far unex- histological studies are needed to address the
pected active role of melanocytes in symptomatic problem.
inflammation in nondepigmenting disorders. Is This section has provided clinical definitions,
microinflammation primary, or secondary to classification and methods of assessment based
some basic imbalance in pigment cell homeosta- whenever possible on the largest consensus. The
sis? Whatever the answers, the growing evidence clinically based definitions given have been
of microinflammation in vitiligo which is now intended to be useful for the practitioner and the
considered as a memory T cell disease has con- clinical researcher. We clearly need also better
siderably changed our conception of the disease definitions and monitoring of disease a ctivity/sta-
and of its management [6]. bility, which may include evidence of nonclini-
cally graded inflammation with skin biopsies or
less invasive skin/blood markers. More accurate
19.7 Summary and standardized methods of assessment need to
be developed for specific needs including clinical
In summary, Fig. 19.1 proposes a synthetic vision trials. A basic science understanding of vitiligo
of the vitiligo clinical data. There are several should represent soon an available alternative to
points which need clarification. Is there a com- this clinically based approach in the context of
mon predisposition shared for developing cellu- developing translational research. The next sec-
lar immune response against naevus cells in SV tion will thus review in vivo and in vitro clinical
and NSV, halo naevus being a distinct phenome- and experimental data which form the basis of
non [7] associated to the two forms? Can SV, at our current understanding of vitiligo.
ERRNVPHGLFRVRUJ
19 Defining the Disease: Editor’s Synthesis 185
References 5. van Geel N, Bekkenk M, Lommerts JE, Ezzedine

K, Harris J, Hamzavi I, Eleftheriadou V, Picardo M,
Taieb A, Prinsen CAC, Wolkerstorfer A, Speeckaert
1. Bach JF. The effect of infections on susceptibility
R. The Vitiligo Extent Score (VES) and the VESplus
to autoimmune and allergic diseases. N Engl J Med.
are responsive instruments to assess global and
2002;347:911–20.
regional treatment response in patients with vitiligo.
2. Schultz Larsen F. Atopic dermatitis: a genetic-
J Am Acad Dermatol. 2018;79(2):369–71. https://doi.
epidemiologic study in a population-based twin
org/10.1016/j.jaad.2017.12.070. Epub 2018.
sample. J Am Acad Dermatol. 1993;28:719–23.
6. Boniface K, Jacquemin C, Darrigade A-S,
3. Bae JM, Chung KY, Yun SJ, Kim H, Park BC, Kim
Dessarthe B, Martins C, Boukhedouni N, Vernisse
JS, Seo SH, Ahn HH, Lee DY, Kim YC, Park HJ, Kim
C, Grasseau A, Thiolat D, Rambert J, Lucchese
M. Markedly reduced risk of internal malignancies in
F, Bertolotti A, Ezzedine K, Taieb A, Seneschal
patients with Vitiligo: a nationwide population-based
J. Vitiligo skin is imprinted with resident memory
cohort study. J Clin Oncol. 2019;37(11):903–11.
CD8 T cells expressing CXCR3. J Invest Dermatol.
4. Taïeb A, Picardo M, Members VETF. The definition
2018;138(2):355–64.
and assessment of vitiligo: a consensus report of the
7. Schallreuter KU, Kothari S, Elwary S, et al. Molecular
Vitiligo European Task Force. Pigment Cell Res.
evidence that halo in Sutton’s naevus is not vitiligo.
2007;20:27–35.
Arch Dermatol Res. 2003;295:223–8.
ERRNVPHGLFRVRUJ
Part II
Understanding the Disease
ERRNVPHGLFRVRUJ
Pathophysiology Overview
20
Mauro Picardo
Contents
References 191
Thanks to the advances in technology over the play a more important role. Environmental
past few years, there has been an ongoing and factors can induce epigenetic alterations in

in-depth study of the genes that make up the patients, which can affect gene expression
human being as a whole and more specifically involved in inflammation, immune tolerance and
those involved in pathologies and diseases melanocyte homeostasis, eventually leading to an
affecting the general population. So far, over 50 active immune response and melanocyte destruc-
different vitiligo susceptible genes have been tion [4].
identified [1–3]. Gaining a better understanding A unified approach to the understanding of the
of the genes involved in vitiligo may in the future pathophysiology of vitiligo has not yet been
lead to a better approach in treating vitiligo and completely defined, but melanocyte loss is cer-
might even allow for disease prevention in tainly the pathological hallmark of vitiligo.
genetically susceptible individuals. The loss of functional melanocytes may be
Although the advances in genetics are ongo- attributed to multiple mechanisms including met-
ing and have definitely shed new light on the abolic abnormalities, oxidative stress, generation
pathology of this disorder, many of the genes of inflammatory mediators, cell detachment, and
associated with vitiligo are also associated with autoimmune response.
other autoimmune disorders. There is increasing It seems that in subjects with a genetic predis-
evidence showing a combination of environmen- position to develop autoimmune, there is an
tal and genetic factors involved in the pathogen- intrinsic defect within melanocytes which trig-
esis of vitiligo. Studies on twins have clearly gers the phenomena. The intracellular oxidative
demonstrated that there is only a 23% concor- stress generated can lead to a local inflammatory
dance of vitiligo susceptible genes in monozy- reaction with the activation of an innate immune
gotic twins, suggesting that epigenetics might process and subsequent generation of cell-
specific cytotoxic immune responses. The final
step is a progressive loss of melanocytes with
M. Picardo (*)
skin depigmentation [5].
Dermatological Institute, IRCCS, Rome, Italy The role of oxidative stress is supported by the
e-mail: mauro.picardo@ifo.gov.it evidence of increased levels of reactive oxygen

ERRNVPHGLFRVRUJ
190 M. Picardo
species (ROS) in lesional and non-lesional skin melanocyte. Melanocyte-specific, cytotoxic

both in vitro and in vivo, which lead to an CD8+ T cells will then act in the destruction of
impaired expression and/or activity of the anti- melanocytes. CD8+ lesional T cells from patient
oxidant system. Moreover, epidermis appears to skin biopsies produce IFN-γ and TNF-α, among
be overall characterized by a deregulation of the other cytokines. IFN-γ is also expressed within
biopterin metabolism [6]. lesions, suggesting the importance of this cyto-
The redox alteration of membrane lipid may kine in driving autoreactive T-cell responses
affect lipid rafts with subsequent modification involved in the destruction of melanocytes.
of the membrane receptor function as well as Functional studies in a mouse model of vitiligo
the housing of the proteins ensuring the elec- confirmed the critical role of the IFN-γ-CXCL10-
tron transfer and the ATP production in the CXCR3 axis in both the progression and mainte-
mitochondria. nance of depigmentation in vitiligo [11].
The imbalance of the prooxidant/antioxidant The role of T regulatory cells in controlling
status has been indicated as the basis of the autoimmune responses is likely based on func-
increased sensitivity of vitiligo melanocytes to tional experiments in mouse models as well as
external prooxidant stimuli and, on a long-term observations made in vitiligo patients; however,
exposure, as the cause of generation of a pre- clear evidence of their function is still lacking [12].
senescent status [7]. A large number of genes found to confer risk
The possible detachment of melanocytes, for vitiligo implicate both innate and adaptive
observed at the borders of lesions, may be related immune pathways, confirming the role of auto-
to the stress-mediated damage possibly explain- immunity in vitiligo pathogenesis. However,
ing the Koebner phenomenon (KP). Mitochondrial polymorphisms in nonimmune genes have also
dysfunction, reported in vitiligo cells, has been been identified, including the melanocyte-
related to cell damage observed in other degen- specific genes tyrosinase and the melanocortin 1
erative diseases and may possibly account for receptor96. While these may serve as antigens
metabolic stress. recognized by T cells, they also participate in
Some of the alterations observed in melano- melanin production, which may contribute to the
cytes have been described in other cells of vitil- generation of stress within the cells.
igo skin suggesting that the degenerative The pathogenic mechanisms underlying seg-
phenomenon has a more generalized involve- mental and non-segmental vitiligo were thought
ment. Keratinocytes and fibroblasts present an to be distinct due to their different clinical pat-
intracellular oxidative stress that could be the terns. However, recent data indicate overlapping
basis for an altered secretion of soluble growth inflammatory pathogenesis for segmental and
factors supporting melanocyte survival and non-segmental vitiligo. Both are caused by a
homeostasis [8, 9]. multistep process, which includes an initial
Finally, stressed melanocytes may initiate release of proinflammatory cytokines and neuro-
immune responses through several mechanisms. peptides, triggered by external or internal injury,
The role of autoimmunity in vitiligo is sup- with a subsequent vascular dilatation and immune
ported by the association with other autoim- response. Conventionally, the possible pathoge-
mune diseases, the presence of antibodies netic mechanisms for segmental vitiligo include
against melanocytes, the association with poly- neuronal mechanisms, somatic mosaicism, and
morphisms at immune loci, the presence of microvascular skin homing of immune cells.
prominent T-cell perilesional infiltrates, and Immunohistochemical data are rather scarce, and
cytokine expression [10]. significant lymphocytic infiltrates have only
The mechanisms that initiate autoimmunity occasionally been observed. However, increasing
likely begin with the activation of innate immune evidence has been published on a possible
populations that sense exogenously or endoge- autoimmune/auto-inflammatory destruction of

nously induced stress signals released from the segmental vitiligo. Currently, it remains
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20 Pathophysiology Overview 191
a mbiguous whether the inflammatory process is models. In vitro models are useful in the investi-
based on a deregulated immune system or can be gation of the differences between normal cells
regarded as a secondary event to cellular abnor- and vitiligo cells. The in vitro approach has
malities in the epidermis. evolved over time, and now new techniques
A cell-mediated immune response, character- involving a limited use of biological material are
ized by CD8 and CD4 infiltrates around dermo- proving very interesting. Furthermore, through
epidermal junction, is involved in the early phases in vitro studies, one can generate melanocyte-
of segmental vitiligo with associated halo nevi. As specific silencing or overexpression of modified
regards the immune involvement, it has been sug- genes. The possible development of vitiligo
gested that the midline delineation in unilateral models using normal cells will bypass the diffi-
lesion could represent the migration pattern of culty of culturing vitiligo cells which is currently
cytotoxic T cells from specific lymph nodes along the main limiting factor in studying vitiligo
the microvascular system via homing receptors. in vitro.
According to the literature, these homing recep- The use of animal models has been essential
tors can have a unique unilateral homing code. in understanding the importance of the different
Similarly to that hypothesized for non- elements affecting vitiligo such as genetic sus-
segmental vitiligo, a combinatory three-step the- ceptibility, immune system, and environmental
ory was proposed. The theory of cutaneous factors. Studies have been carried out on both
mosaicism does not exclude the neuronal theory, spontaneous autoimmune vitiligo (Smyth line
as a neurogenic factor can still be one of the ini- chicken) and induced autoimmune vitiligo (in
tial triggers to induce the whole process in its mice). The first includes all the same clinical
early stage. The first step includes the release of and biological manifestations seen in human
inflammatory factors, neuropeptides, and cate- vitiligo, while the latter provides defined in vivo
cholamines, which can be considered as the trig- models useful in examining the loss of melano-
gering factor. This triggering factor leads to a cytes and developing new treatments. Together,
small inflammatory response at a site where the study of these two models is necessary to
melanocytes are more vulnerable to immune- delineate the complex mechanisms involved in
mediated destruction (possibly due to somatic vitiligo [15–17]. The results from these studies
mosaicism) (step 2). Subsequently, an can provide new insights into the prevention,
antimelanocyte-specific response develops in the treatment, and management of vitiligo in
draining lymph node, and specific T cells migrate humans.
by binding to their vascular adhesion receptors
toward the segmental vitiligo area, causing the
characteristic skin depigmentations [13]. References
Some of the alterations observed in melano- 1. Spritz RA, Andersen GH. Genetics of vitiligo.
cytes have also been described in other skin cells, Dermatol Clin. 2017;35(2):245–55. https://doi.
suggesting that vitiligo leads to generalized org/10.1016/j.det.2016.11.013.
degeneration. Keratinocytes and fibroblasts also 2. Shen C, Gao J, Sheng Y, Dou J, Zhou F, Zheng
X, Ko R, Tang X, Zhu C, Yin X, Sun L, Cui Y,
exhibit oxidative stress, phosphorylation of p38, Zhang X. Genetic susceptibility to vitiligo: GWAS
an overexpression of p53, and a senescent pheno- approaches for identifying vitiligo susceptibility
type. These could be the basis for altered secre- genes and loci. Front Genet. 2016;7:3. https://doi.
tion of soluble growth factors supporting org/10.3389/fgene.2016.00003.
3. Spritz RA. Six decades of vitiligo genetics: genome-
melanocyte survival and homeostasis. wide studies provide insights into autoimmune patho-
Reported keratinocytes and fibroblasts could genesis. J Invest Dermatol. 2012;132(2):268–73.
be altered in vitiligo skin showing some aging https://doi.org/10.1038/jid.2011.321.
phenotypes [9, 14]. 4. Alkhateeb A, Fain PR, Thody A, Bennett DC, Spritz
RA. Epidemiology of vitiligo and associated autoim-
The evolution of the experimental approach mune diseases in Caucasian probands and their fami-
to vitiligo has provided new in vivo and in vitro lies. Pigment Cell Res. 2003;16(3):208–14.
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5. Picardo M, Dell’Anna ML, Ezzedine K, Hamzavi 12. Dwivedi M, Kemp EH, Laddha NC, Mansuri MS,
I, Harris JE, Parsad D, Taieb A. Vitiligo. Nat Rev Weetman AP, Begum R. Regulatory T cells in vit-
Dis Primers. 2015;1:15011. https://doi.org/10.1038/ iligo: implications for pathogenesis and therapeutics.
nrdp.2015.11. Autoimmun Rev. 2015;14(1):49–56.
6. Schallreuter KU, Moore J, Wood JM, Beazley WD, 13. van Geel N, Speeckaert R. Segmental vitiligo.

Gaze DC, Tobin DJ, Marshall HS, Panske A, Panzig E, Dermatol Clin. 2017;35(2):145–50. https://doi.
Hibberts NA. In vivo and in vitro evidence for hydro- org/10.1016/j.det.2016.11.005.
gen peroxide (H2O2) accumulation in the epidermis 14. Becatti M, Prignano F, Fiorillo C, Pescitelli L, Nassi
of patients with vitiligo and its successful removal by P, Lotti T, Taddei N. The involvement of Smac/
a UVB-activated pseudocatalase. J Investig Dermatol DIABLO, p53, NF-kB, and MAPK pathways in
Symp Proc. 1999;4(1):91–6. apoptosis of keratinocytes from perilesional vitiligo
7. Bellei B, Pitisci A, Ottaviani M, Ludovici M, Cota C, skin: protective effects of curcumin and capsaicin.
Luzi F, Dell’Anna ML, Picardo M. Vitiligo: a pos- Antioxid Redox Signal. 2010;13(9):1309–21. https://
sible model of degenerative diseases. PLoS One. doi.org/10.1089/ars.2009.2779.
2013;8(3):e59782. https://doi.org/10.1371/journal. 15. Shi F, Kong BW, Song JJ, Lee JY, Dienglewicz

pone.0059782. RL, Erf GF. Understanding mechanisms of vitiligo
8. Lee AY. Role of keratinocytes in the development of development in Smyth line of chickens by transcrip-
vitiligo. Ann Dermatol. 2012;24(2):115–25. https:// tomic microarray analysis of evolving autoimmune
doi.org/10.5021/ad.2012.24.2.115. lesions. BMC Immunol. 2012;13:18. https://doi.
9. Kovacs D, Bastonini E, Ottaviani M, Cota C, org/10.1186/1471-2172-13-18.
Migliano E, Dell’Anna ML, Picardo M. Vitiligo 16. Rashighi M, Agarwal P, Richmond JM, Harris TH,
skin: exploring the dermal compartment. J Invest Dresser K, Su MW, Zhou Y, Deng A, Hunter CA, Luster
Dermatol. 2018;138:394. https://doi.org/10.1016/j. AD, Harris JE. CXCL10 is critical for the progression
jid.2017.06.033.. pii: S0022-202X(17)33029-4. and maintenance of depigmentation in a mouse model
10.
Boniface K, Seneschal J, Picardo M, Taïeb of vitiligo. Sci Transl Med. 2014;6(223):223ra23.
A. Vitiligo: focus on clinical aspects, immu- https://doi.org/10.1126/scitranslmed.3007811.
nopathogenesis, and therapy. Clin Rev Allergy 17. Eby JM, Kang HK, Klarquist J, Chatterjee S,

Immunol. 2018;54:52. https://doi.org/10.1007/ Mosenson JA, Nishimura MI, Garrett-Mayer E,
s12016-017-8622-7. Longley BJ, Engelhard VH, Mehrotra S, Le Poole
11. Harris JE, Harris TH, Weninger W, Wherry EJ, Hunter IC. Immune responses in a mouse model of vitiligo
CA, Turka LA. A mouse model of vitiligo with with spontaneous epidermal de- and repigmentation.
focused epidermal depigmentation requires IFN-γ for Pigment Cell Melanoma Res. 2014;27(6):1075–85.
autoreactive CD8+ T-cell accumulation in the skin. J https://doi.org/10.1111/pcmr.12284.
Invest Dermatol. 2012;132(7):1869–76. https://doi.
org/10.1038/jid.2011.463.
ERRNVPHGLFRVRUJ
Methods to Study Vitiligo:
Noninvasive Techniques
21
and In Vivo Reflectance Confocal
Microscopy
Hee Young Kang and Marco Ardigò
Contents
21.2 Wood’s Light Examination 194
21.3 Clinical Scoring/Grading Methods 195
21.4 Photograph (Light/UV) and Digital Image Analysis 197
21.5 Reflectance Spectroscopy 198
21.6 Dermoscopy 199
21.7 In Vivo Reflectance Confocal Microscopy 199
21.8 Conclusion 202
References 203
Abstract dermoscopy are methods to characterize and

Standardized methodologies for describing study vitiligo lesion. The Vitiligo European
and classifying vitiligo and for assessing the Task Force (VETF) recommends Wood’s
effect of treatments are needed to be devel- light examination as a diagnostic tool in
oped. Currently, there are many noninvasive evaluating vitiligo to assess staging and
techniques available for diagnosis and spreading in a selected area. More reliable
assessment of vitiligo. Objective measure- and quantitative measures to augment clini-
ments of vitiligo area using digital image cal judgment of vitiligo assessment include
analysis are available. Noninvasive instru- image analysis of digital photographs. The
ments such as reflectance spectroscopy or digital image analysis system can overcome
the inevitable differences between observ-
H. Y. Kang (*)
ers, which are intrinsic to a visual grading
Department of Dermatology, Ajou University School method, and is advisable for clinical trials on
of Medicine, Suwon, Korea vitiligo to objectively assess repigmentation
e-mail: hykang@ajou.ac.kr in limited lesions (Linthorst Homan MW
M. Ardigò et al. J Eur Acad Dermatol Venereol
San Gallicano Dermatological Institute, IRCCS, 27(2):e235–238, 2013). However, this tech-
Rome, Italy
e-mail: marco.ardigo@ifo.gov.it
nique needs to be properly validated.

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194 H. Y. Kang and M. Ardigò
Limitations of this method include as the 21.1 Introduction

technique is complex and laborious, it is only
feasible for monitoring limited areas of vitil- Standardized methodologies for describing
igo and less feasible in daily practice. and classifying vitiligo and for assessing the
Mexameter measurement provides a clini- effect of treatments are needed to be devel-
cally accessible and straightforward means oped. Currently, there are many noninvasive
of increasing diagnostic accuracies in techniques available for diagnosis and assess-
hypopigmentary disorders. In vivo reflec- ment of vitiligo. Wood’s light examination has
tance confocal microscopy (RCM) is a real- been used to measure the extent and progres-
time, repetitive imaging tool that provides sion of the disease. Visual comparison of vit-
noninvasive images at a nearly histological iligo lesion using photographs and the
resolution. RCM could be used in the thera- parametric measurement of vitiligo severity
peutic monitoring and evaluation of the evo- such as VASI (vitiligo area scoring index) or
lution of vitiligo. Limitations of RCM VIDA (vitiligo disease activity) score were
include that the instrument is expensive and introduced to measure the extent of depigmen-
the RCM imaging can still be time-consum- tation both at baseline and following therapeu-
ing especially for the systematic examination tic interventions. Objective measurements of
of lesion area. Also, this machine requires a vitiligo area using digital image analysis are
group of technicians to operate it and an available. Noninvasive instruments such as
expert to analyze the images. reflectance spectroscopy or dermoscopy are
methods to characterize and study vitiligo
lesion. Reflectance confocal microscopy offers
a great potential for noninvasive assessment of
vitiligo. All these techniques have been
Key Points reported to be useful in the diagnosis or assess-
• The extension and severity of vitiligo ment of vitiligo lesions.
guide prognosis and therapeutic choices.
However, wide variations exist both in
assessment rules and interpretation of 21.2 Wood’s Light Examination
their use, making intra- and inter-
observer variations unavoidable. Wood’s lamp emits long-wave UV radiation in a
• Wood’s light examination remains a tra- band between 320 and 400 nm with a peak at
ditional tool in measuring the extent and 365 nm [1] (Fig. 21.1a). The light source is a
progression of the disease. high-pressure mercury arc fitted with a com-
• A variety of clinical scoring systems pound filter made of barium silicate and 9%
and objective assessment with image nickel oxide, the “Wood’s filter.” The UV radia-
analysis of digital photographs is used tion penetrates the epidermis, where it is attenu-
to evaluate the treatment outcome in ated by melanin; upon entering the dermis, it
terms of repigmentation. stimulates fluorescence emission by collagen
• Several instruments such as reflectance bundles (Fig. 21.1b). Part of the emitted fluores-
spectroscopy or dermoscopy could be cence is directed toward the surface of the skin,
useful in characterizing and studying but it is attenuated by hemoglobin in the capillar-
the vitiligo lesion. ies and melanin in the epidermis. This procedure
• In vivo reflectance confocal microscopy enhances the epidermal pigmentation variations
is a promising tool for characterizing that are not apparent under visible light and
melanocytes loss in untreated and improves the assessment of the extent of pig-
treated vitiligo lesions. ment abnormalities. Ideally, the lamp should be
allowed to warm up for about 1 min and the
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21 Methods to Study Vitiligo: Noninvasive Techniques and In Vivo Reflectance Confocal Microscopy 195
examination should be performed in a very dark valuable in dark-skinned patients in whom

room. In depigmented skin lesions of vitiligo, hypopigmented lesions can be easily perceived
there is reduced or no epidermal melanin, and by the naked eye.
hence there is a window through which a light-
induced autofluorescence of dermal collagen can
be seen. The lesion appears bright blue-white 21.3 Clinical Scoring/Grading
due to autofluorescence (Fig. 21.1c, d). The Methods
results of Wood’s light examination can be
recorded through UV photography. The Vitiligo Reliable outcome measures are important to com-
European Task Force (VETF) recommends pare studies and to assess the changes over time.
Wood’s light examination as a diagnostic tool in A variety of scoring systems is used to evaluate
evaluating vitiligo to assess staging and spread- the treatment outcome in terms of repigmentation
ing in a selected area [2]. All patients with vitil- [3]. Most methods used in clinical practice are
igo of lower phototypes should be examined subjective, as they involve subjective assessment
under both visible and Wood’s light. On the by a clinician to grade the degree of repigmenta-
other hand, the technique examination is less tion. Commonly, serial photographs either with
b UVA
340-380 nm
UVA
Fluorescence
Visible light >380nm <750nm
stratum corneum
melanin granules
melanocytes
epidermis
Collagen
dermis Elastin
Artero-venular capillaries
Fig. 21.1 (a) Wood’s lamp. The envelope of the bulb is ing the dermis, it stimulates fluorescence emission by col-
composed of a deep bluish-purple glass called Wood’s lagen bundles. The light emitted, even if absorbed by the
glass, a nickel oxide-doped glass, which blocks almost all epidermal melanin, exits the skin and will reach the eye of
visible light above 400 nm. In this instrument a magnify- the observer. (c, d) Wood’s light examination of depig-
ing glass is present. (b) The UV radiation penetrates the mented patches on the hands of a patient with vitiligo.
epidermis, where it is attenuated by melanin; upon enter- Standard lighting (c) and Wood’s light lighting (d)
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Fig. 21.1 (continued)
visible light or a Wood’s lamp is used to compare Several parametric methods such as vitiligo
the current lesion with its size in the past. A global area severity index (VASI) and vitiligo disease
assessment scale (GAS) is currently one of the activity (VIDA) score were introduced as quanti-
most commonly used methods for evaluating tative methods to standardize outcome measure-
treatment response. It is a four or five ordinal ments in vitiligo. The VASI is a standardized,
scoring system based on repigmentation percent- sensitive method to measure the extent and per-
age within a lesion over time categories (e.g., 1% centage of de-/repigmentation [4]. In the VASI,
to 25%, 26% to 50%, 51% to 75%, >75%). The the patient’s body is divided into five separate
“rule of nines” developed for calculating percent- and mutually exclusive regions: hands, upper
age surface area burns has been proposed by the extremities, trunk, lower extremities, and feet.
VETF [4]. The “rule of nines” assumes that the For each body region, the VASI is determined by
head/neck, each arm, each leg, and the four trunk the product of the area of vitiligo in hand units
quadrants each compose 9% of the total body sur- (which was set at 1% per unit) and the extent of
face area, leaving 1% for the genitalia. In the “flat depigmentation within each hand unit-measured
hand = 1%” method, a flat hand represents 1% of patch (possible values of 0%, 10%, 25%, 50%,
the total body surface area. Both methods are sub- 75%, 90%, or 100%). The total body VASI is
jective and are based on visual assessments [3]. then calculated using the following formula by
ERRNVPHGLFRVRUJ
considering the contributions of all body regions lesion. The reliability of the point-counting
(possible range, 0–100): VASI = P (all body size) method has been tested against image analysis
(hand units) · (depigmentation). It was suggested and has shown to be statistically significant in
that VASI is a valid quantitative clinical tool that measuring surface areas [6].
can be used to evaluate vitiligo parametrically.
However, this method has a subjective compo-
nent as it involves the physician deciding the 21.4 Photograph (Light/UV)
amount of pigmentation and the area of involve- and Digital Image Analysis
ment. The VIDA is a six-point scale for assessing
vitiligo stability over time. It depends on patient’s More reliable and quantitative measures to aug-
own reports of disease activity [5]. Active vitiligo ment clinical judgment of vitiligo assessment
involves either the expansion of existing lesions include image analysis of digital photographs.
or the appearance of new lesions. Grading is as Objective measurements of vitiligo area using
follows: VIDA score +4, activity lasting 6 weeks digital image analysis have been introduced. Van
or less; +3, activity lasting 6 weeks to 3 months; Geel et al. [7] first published a computerized dig-
+2, activity lasting 3–6 months; +1, activity last- ital image analysis system for the objective
ing 6–12 months; 0, stable for 1 year or more; assessment of vitiligo lesions in transplantation
and −1, stable with spontaneous repigmentation studies, followed by other groups that have used
for 1 year or more. A low VIDA score indicates a digital image analysis system in evaluating vit-
less vitiligo activity. iligo treatments. Digital photograph images are
Point-counting method has been proposed and taken under standardized conditions for lighting,
may provide a simple alternative in measuring position, and exposure time. The images are ana-
the surface area of vitiligo lesions [6]. In this lyzed using a commercial image analysis soft-
method, the lesion borders are marked with an ware such as Image-Pro Plus 4.5 (Media
ordinary ballpoint pen, and a piece of paper is Cybernetics, Rockville, MD) [6], AutoCAD 2000
immediately placed over the lesion. For each (Autodesk, Inc, San Rafael, CA, USA) [8], or
lesion, the copied borders of the projection areas Corel Draw 9.0 (Corel Corporation, Ottawa,
are enhanced by redrawing the contours with a Ontario, Canada) [9] or other novel software pro-
pen. To estimate the number of points, a transpar- grams that combine standard image-processing
ent sheet that has a point (+) printed on it is ran- techniques which can compute the surface area of
domly superimposed on the lesion projection a pre-outlined depigmented lesion or by planim-
area (Fig. 21.2). The number of intersections hit- etry (manual tracing of lesions onto transparent
ting the area of interest is counted. The total area graph paper) [10–14]. The technique is based on
of each lesion is estimated by multiplying the principal component analysis and independent
representative area of a point on a grid according component analysis which converts three differ-
to the total number of points counted for the ent spectral bands in a skin image into an image
that represents skin area based only on melanin
and hemoglobin. This technique allows the effec-
tive segmentation of vitiligo skin lesion areas and
normal skin and consistently maintains high
sensitivity, specificity, and accuracy values for all
images. Whereas the results of the digital and
clinical assessments with a global assessment
scale (GAS) were comparable, patients’ ratings
diverged [10]. The digital image analysis system
Fig. 21.2 Point-counting method. The total area of each can overcome the inevitable differences between
lesion is estimated by multiplying the representative area observers, which are intrinsic to a visual grading
of a point on grid by total number of points counted for the
lesion method, and is advisable for clinical trials on vit-
ERRNVPHGLFRVRUJ
iligo to objectively assess repigmentation in lim- meters using the erythema/melanin indices. It
ited lesions [10]. However, this technique needs was reported that moderate to high significant
to be properly validated. linear correlations could be established between
Limitations of this method include as the tech- the L*, a*, b* color parameters and the erythema/
nique is complex and laborious, it is only feasible melanin indices [16]. Reflectance tristimulus col-
for monitoring limited areas of vitiligo and less orimeter converts reflectance data into three color
feasible in daily practice. There also exists poten- values, expressed in the L*, a*, b* color system.
tial difficulty in assessing larger lesions or lesions L* indicates luminance and describes the rela-
over curved surface as any photograph is a two- tive lightness ranging from 0 (black) to 100
dimensional (2D) projection of a three- (white). The a* indicates the balance between
dimensional (3D) surface. Therefore, it is subject green (negative value) and red (positive value),
to errors due to the inability to estimate accu- and b* represents the balance between blue
rately the area from a photograph. Furthermore, (negative value) and yellow (positive value).
perifollicular repigmentation or other complex Different handheld tristimulus reflectance
patterns are difficult to catch using boundary colorimeters are commercially available for
tracing system. The emerging technique of taking the measurement of skin color, and the
whole-body 3D images followed by 3D image Chroma Meter CR 200 or CR 300 (Minolta
analysis to quantify the vitiligo-affected body Osaka, Japan) has become popular (Fig. 21.3a).
surface area (BSA) of the subjects was recently Colorimetric examination of vitiligo patients
introduced [15]. In this study, for the known sur- revealed that the perilesional skin near to the vit-
face area reference, 2D image analysis resulted in iligo spot is not normal [17] (Fig. 21.3b). L* val-
lesion area of 14.98 cm2, whereas 3D image anal- ues decreased significantly in relation to
ysis resulted in a lesion area of 19.50 cm2. It was increasing distance from the vitiligo spot, and the
concluded that 2D image analysis of contoured skin near vitiligo spot was lighter than normal
surfaces can underestimate lesion area by more skin as far as 5 cm from the vitiligo spot. In con-
than 20%, whereas 3D image analysis accounts trast, the pigmentation index (b*) gradually
for the changes in relative contour and has an increased from lesional to perilesional to normal
associated error of <1%. skin. Furthermore, the comparison of the b*
value between the normal skin as far as 5 cm
from the nearest vitiligo spot was significantly
21.5 Reflectance Spectroscopy higher than perilesional skin. This finding sug-
gested that the use of colorimetric method in vivo
Skin color is predominantly determined by pig- makes perceivable slight color variations that the
ments such as hemoglobin, melanin, bilirubin, eyes are not able to distinguish differences where
and carotene. Oxyhemoglobin and deoxyhemo- there are gradual changes as in the perilesional
globin absorb specifically between 540 and skin of vitiligo lesion. In another study, skin color
547 nm. Melanin heavily absorbs all wavelengths measurement was performed with colorimeter
but demonstrates a monotonic increase toward after excimer laser treatment [18]. The L* and a*
shorter wavelengths. Objective skin color- values of the vitiligo lesions before laser treat-
measuring instruments have been reported to pro- ment were significantly different from those after
vide a reproducible and sensitive means of laser treatment. The repigmentation scores were
quantifying small skin color differences [16]. It correlated significantly with the vitiligo a* values
determines color by measuring the intensity of after treatments.
reflected light of specific wavelengths. Two types Narrowband simple reflectance meters mea-
of skin reflectance instruments are available cur- sure “melanin index” (MI) and an “erythema
rently for the determination of skin color: a tris- index” (EI). Each index increases as the skin
timulus colorimeter using the L*, a*, b* color becomes more pigmented or erythematous,
system and the narrowband simple reflectance respectively. There are several commercially
ERRNVPHGLFRVRUJ
clinically accessible and straightforward means

a
of increasing diagnostic accuracies in hypopig-
mentary disorders.
21.6 Dermoscopy
Digital epiluminescence dermoscopy is employed

to examine pigmented lesions. Dermoscopy is
useful for finding leukotrichia that is clinically
common in patients with segmental vitiligo
(Fig. 21.4). In a study using digital microscopy to
b detect tiny and thin vellus hairs unnoticeable with
the naked eye, all 82 patients with segmental vit-
iligo had leukotrichia [20]. The examination of
white hairs with a digital microscope might be
useful for the prediction of treatment outcome
Vitiligo spot
and decision of treatment modalities. Confirming
Perilesional skin
the presence of leukotrichia in patients with vit-
iligo before excimer laser treatment with dermos-
copy was thought to be helpful in predicting the
response to treatment [21]. In this study, 92.9%
of patients with leukotrichia achieved <50%
repigmentation. Various dermoscopic findings
are associated with stability of vitiligo [22].
These include reduced pigmentary network,
Normal skin
absent pigmentary network, reversed pigmentary
network, perifollicular hyperpigmentation, and
perilesional hyperpigmentation in the evolving
Fig. 21.3 (a) Minolta CR-200 Chroma Meter. (b)
Colorimetric examination was performed with the lesional vitiligo lesions; a white glow was present in 90%
skin, the perilesional skin, and the normal skin as far as of patients.
5 cm from the nearest vitiligo spot in a young patient. L*,
a*, b* color parameters were measured
21.7 In Vivo Reflectance Confocal
available instruments that measure MI and EI Microscopy
such as DermaSpectrometer from Cortex
(Denmark) and Mexameter from Courage- In vivo reflectance confocal microscopy (RCM)
Khazaka (Germany). The usefulness of a is a real-time, repetitive imaging tool that pro-
Mexameter for discriminating vitiligo with vides noninvasive images at a nearly histological
nevus depigmentosus was shown [19]. The mean resolution [23]. The basic concept of confocal
relative melanin index (RMI) was calculated by microscopy is the selective collection of light
% melanin index (MI) of an affected lesion/MI from the focused spot of the skin according to the
of a symmetrically located normally pigmented different reflectance indexes of the different skin
area ×100. The RMI of nevus depigmentosus structures (Fig. 21.5). A confocal microscope
lesions was significantly higher than that of vit- consists of a light source, condenser and objec-
iligo lesions. No nevus depigmentosus lesions tive lenses, and a detector. The light source, near-
had an RMI of <50%. These results suggested infrared wavelength laser beam, illuminates a
that the Mexameter measurement provides a specific point within skin, and reflected light is
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POINT SOURCE
OF LIGHT
BEAMSPLITTER
INCIDENT
LIGHT
REFLECTED
LIGHT
PINHOLE,
PHOTODETECTOR
CONDENSOR,
OBJECTIVE LENS
Fig. 21.4 The digital microscopy detects tiny and thin vellus hairs unnoticeable with the naked eye in segmental vit-
iligo (Courtesy of Pr. DY Lee)
a b
Fig. 21.5 Reflectance confocal microscope optical function (a) and commercial device Vivascope® (b)
collected through a pinhole in the detector. The ducing black and white images from the stratum
illuminated spot is then scanned horizontally pro- corneum to the upper dermis with an imaging
ERRNVPHGLFRVRUJ
Vitiligo (forearm) Perilesional normal skin Distant normal skin

(abdomen)
Fig. 21.6 In vivo reflectance confocal microscope (RCM) at the level of basal layer. The brightness of the basal cells in
imaging of vitiligo. (a) RCM images of lesional skin show no perilesional skin is lighter than distant normal skin. (b)
bright cells and the disappearance of the normal papillary ring So-called half rings or scalloped border-like rings (arrows)
depth of up to a maximum of 250 μm. Highly normal skin shows intact papillary rings, but the
reflective skin components including melanin, brightness of cells was significantly reduced
collagen, and keratin appear bright (white) in compared to distant normal skin. Distant normal
RCM images. Because melanin is the strongest skin showed no difference with controls.
endogenous contrast in the skin, RCM is particu- So-called half rings or scalloped border-like rings
larly suitable to analyze pigmented lesions and were observed in distant normal skin but also
consequently pigmentary disorders. normal skin of control (Fig. 21.6d–f). Ardigo
The RCM examination allows the identifica- et al. [25] suggested that this change could derive
tion of characteristic features of vitiligo and from an incomplete distribution of pigment along
repigmented skin during treatment [24] the basal layer because of an initial and
(Fig. 21.6). In normally pigmented skin, RCM progressive disappearing of melanocytes or to a
images from the dermo-epidermal junction congenital defective melanocyte distribution.
appear as bright cells grouped around dermal Another group suggested that the incomplete
papilla defining characteristic ring structures papillary rings are observed in the active lesions
composed of keratinocytes and melanocytes sur- of vitiligo [26]. During UVB treatment, RCM
rounding the upper dermis and “papillary rings.” examination shows large dendritic melanocytes
RCM examination of vitiligo lesions shows a dis- located close to hair follicles, in the epidermis of
appearance of papillary rings at the basal layer perilesional skin, and of repigmentation islands
level and no evidence of melanocytes. Perilesional (Fig. 21.7). These findings suggested that RCM
ERRNVPHGLFRVRUJ
a b
Fig. 21.7 RCM imaging of vitiligo during UVB therapy showing bright dendritic melanocytes (arrows) (a) around a
hair follicle, (b) at the basal layer of perilesional skin
could be used in the therapeutic monitoring and iligo which may show part of the dermal papil-
evaluation of the evolution of vitiligo. lary rings disappear or the brightness of melanin
RCM might be useful to determine stability of in the part of dermal papillary rings decrease
vitiligo [27]. In the active stage of vitiligo, the [26].
RCM examination reveals that the bright dermal Limitations of RCM include that the instru-
papillary rings lose their integrity or totally dis- ment is expensive and the RCM imaging can still
appear; border between vitiligo lesion and nor- be time-consuming especially for the systematic
mal skin becomes unclear, and highly refractile examination of lesion area. Also, this machine
cells that referred to infiltrated inflammatory requires a group of technicians to operate it and
cells could be seen within the papillary dermis at an expert to analyze the images [3].
the edge of the lesions. Differently, in the stable
stage of vitiligo, RCM shows a complete loss of
melanin in lesional skin and a clear border 21.8 Conclusion
between lesional and normal skin. In the cited
study [27], the RCM images were comparable There are many noninvasive techniques available
with vitiligo staging based on vitiligo disease for the diagnosis and assessment of vitiligo.
activity (VIDA) scoring. Wood’s light examination remains a traditional
RCM might serve as a useful instrument in tool in measuring the extent and progression of
noninvasively differentiating vitiligo and other the disease. A variety of clinical scoring systems
hypopigmentary disorders such as nevus depig- and objective assessment with image analysis of
mentosus, nevus anemicus, or postinflammatory digital photographs is used to evaluate the treat-
hypopigmentation in vivo [28, 29]. In the lesional ment outcome in terms of repigmentation.
skin of nevus depigmentosus, the content of mel- However, to date, there is no standardized method
anin and the brightness of dermal papillary rings for measuring vitiligo lesions. The lack of stan-
decrease, and the melanin in adjacent normal dardization is responsible for the high variability
appearing skin of nevus depigmentosus shows no in vitiligo assessment. Objective, noninvasive
change. This differs from the active stage of vit- methods for measuring and monitoring the extent
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of vitiligo skin compared to the surrounding nor- 13. Shamsudin N, Hussein SH, Nugroho H, et al.

mal skin are needed. Several instruments such as Objective assessment of vitiligo with a computerised
digital imaging analysis system. Australas J Dermatol.
reflectance spectroscopy or dermoscopy could be 2015;56(4):285–9.
useful in characterizing and studying the vitiligo 14. Sheth VM, Rithe R, Pandya AG, et al. A pilot study
lesion. In vivo reflectance confocal microscopy is to determine vitiligo target size using a computer-
a promising tool for characterizing the nature of based image analysis program. J Am Acad Dermatol.
2015;73(2):342–5.
melanocytes loss in untreated and treated vitiligo 15. Kohli I, Isedeh P, Al-Jamal M, et al. Three-

lesions. dimensional imaging of vitiligo. Exp Dermatol.
2015;24(11):879–80.
16. Clarys P, Alewaeters K, Lambrecht R, et al.

Skin color measurements: comparison between
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Animal Models
22
Gisela F. Erf and I. Caroline Le Poole
Contents
22.1 he Multifactorial Nature of Autoimmune Disorders:
T
Application to Vitiligo 206
22.2 N aturally Occurring Animal Models of Vitiligo 207
22.2.1 The Smyth Line Chicken Model of Spontaneous
Autoimmune Vitiligo 207
22.2.2 Water Buffalo 213
22.3 E xperimental Mouse Models of Induced Autoimmune Vitiligo 213
22.3.1 Induction of Vitiligo in Mice Through Generation of Endogenous
Melanocyte-Specific Immune Responses 214
22.3.2 Induction of Vitiligo in TCR Transgenic Hosts or by Adoptive
Transfer of Transgenic T Cells 215
22.3.3 Summary on Induced Mouse Models 219
22.4 Concluding Remarks 220
References 220
Abstract
Vitiligo is a non-communicable, multifacto-
rial pigmentation disorder. Autoimmunity
has been identified as a major etiological fac-
tor in the postnatal loss of epidermal melano-
cytes in the skin of vitiligo patients. As with
other tissue-specific autoimmune diseases,
G. F. Erf (*)
genetic predisposition to vitiligo develop-
Division of Agriculture, Center of Excellence for ment may be manifested in altered respon-
Poultry Science, University of Arkansas, siveness of tissue cells and immune system
Fayetteville, AR, USA components to endogenous and exogenous
e-mail: gferf@uark.edu
environmental factors, leading to immuno-
I. C. Le Poole recognition and immune system-mediated
Robert H. Lurie Comprehensive Center, Department
of Microbiology and Immunology, North Western
loss of melanocytes. To understand the etiol-
University, Chicago, IL, USA ogy and pathogenic mechanisms driving dis-
e-mail: caroline.lepoole@northwestern.edu ease onset and progression, and to develop

ERRNVPHGLFRVRUJ
206 G. F. Erf and I. C. Le Poole
effective treatment and prevention strategies

for autoimmune vitiligo, appropriate animal • Induction of autoimmune vitiligo in the
models are required. In this context, experi- mouse by generation of endogenous
mental animal models that naturally develop melanocyte-specific immune responses,
vitiligo would more closely reflect the com- adoptive transfer of melanocyte-specific
plex nature of the disorder in humans than T cells, or introduction of antigen-spe-
experimental models where the autoimmune cific T cell receptor genes provides
disease was induced. Animal models with defined in vivo models to examine mela-
truly naturally occurring autoimmune dis- nocyte loss and develop vitiligo treat-
ease are rare. However, for autoimmune vit- ment strategies.
iligo, the Smyth line of chicken was
established as a highly relevant, spontaneous
model for both basic and translational
research, as it displays the entire spectrum of 22.1 T
he Multifactorial Nature
clinical and biological manifestations of of Autoimmune Disorders:
autoimmune vitiligo in humans. While there Application to Vitiligo
is no natural vitiligo mouse model, induction
of vitiligo in mice by generation of melano- Autoimmunity has been identified as a major etio-
cyte-specific immune responses proved to be logical factor in vitiligo, although many other fac-
an invaluable approach to dissect cellular and tors including infections, stress, neural
molecular mechanisms involved in the auto- abnormalities, aberrant melanocyte function, and
immune depigmentation and repigmentation genetic susceptibility have been implicated [1–8].
processes. Together, research using the natu- This multifactorial nature of vitiligo involving
ral and induced animal models will provide genetic susceptibility and disease-precipitating
the critical knowledge needed to understand, factors in addition to immunopathology is in itself
treat, and prevent autoimmune vitiligo. in accordance with the general concepts described
for tissue-specific autoimmune disease. In tissue-
specific autoimmune diseases such as vitiligo, the
genetic susceptibility is frequently associated
with an inherent target cell defect that predisposes
Key Points the target cell to immunorecognition and may
• Chronic autoinflammatory/autoimmune include aberrant immune activity at various levels
disorders typically are multifactorial in (e.g., dendritic cells/macrophages, T cells, and B
nature, requiring several components cells). The autoimmune destruction of cells has
such as genetic susceptibility, immune been found to be associated with a lack of regula-
system components, and environmental tory function within the immune system, height-
factors for expression. Appropriate ened immune activity, and altered responsiveness
experimental animal models have of immune components and target cells to endog-
become an essential tool to delineate enous and exogenous factors. The role of environ-
and dissect the relative contributions of mental factors in the development of autoimmune
these components. disease is also multifaceted and may include
• The spontaneous autoimmune vitiligo infections by microbes, exposure to chemicals,
described in the Smyth line chicken and a wide array of other stress factors that can
recapitulates the entire spectrum of clin- provoke an autoimmune response to the target
ical and biological manifestations of cells [1, 5, 9–11]. Unfortunately, the relative con-
human vitiligo, providing a unique tributions of these components in organ-specific
opportunity to examine etiology, pro- autoimmune disease cannot be easily delineated
gression, prevention, and treatment of and dissected, especially in human patients when
the disease. they become apparent only after the clinical mani-
festation of the disease.
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22 Animal Models 207
22.2 N
aturally Occurring Animal 22.2.1 The Smyth Line Chicken
Models of Vitiligo Model of Spontaneous
Autoimmune Vitiligo
In order to understand the initial etiology and
pathogenic events leading to the onset and pro- The Smyth line (SL) chicken is characterized by a
gression of autoimmune disease, appropriate spontaneous, vitiligo-like, post-hatch loss of mel-
animal models are required. In this context, anin-producing pigment cells (melanocytes) in
experimental animal models that naturally feather and choroidal tissue (Fig. 22.1, Table 22.1).
develop the autoimmune disease would reflect SL vitiligo (SLV) occurs in approximately
the situation in humans more closely than exper- 80–95% of hatch mates, with about 70% of those
imental models where the autoimmune disease affected expressing complete depigmentation in
was induced. In biomedical research, the mouse adulthood (>20 weeks of age). There are many
has become the most studied animal model, due similarities between SL and human vitiligo. Both
in part to its short-generation time, its small are characterized by autoimmune destruction of
size, and the extensive availability of geneti- melanocytes, usually first seen during adoles-
cally defined strains of mice, research reagents, cence and early adulthood. In both SL chickens
and research procedures. Murine models for and humans, pigmentation loss may be either par-
spontaneous autoimmune disease are, however, tial or complete. Remelanization of amelanotic
rare, and there is currently no mouse model that tissue occurs, although severe pigment loss and
develops spontaneous autoimmune vitiligo [9, remelanization are more frequent in the chicken.
10, 12]. In addition to vitiligo, SL chickens exhibit uveitis,
For vitiligo research, several animal models often resulting in blindness (5–15%), and have
of naturally occurring vitiligo were identified associated autoimmune diseases such as hypothy-
and reviewed [9, 13–15]. These included the roidism (4–8%) and an alopecia areata-like feath-
Smyth line of chicken, the water buffalo, the ering defect (2–3%) [9, 12, 16, 32]. Similarly, in
Sinclair pig, the gray horse, the barred rock humans it is not uncommon to find thyroidal and
chicken, and the vitiligo mouse. Of these mod- other autoimmune diseases associated with vitil-
els, the Smyth line chicken [9, 14, 16] and the igo. Moreover, SL vitiligo, like human vitiligo, is
water buffalo [17, 18] best reflect naturally a multifactorial disorder involving a genetic
occurring vitiligo; vitiligo in the Sinclair pig component (manifested in part as an inherent
[19–21] and the gray horse [22, 23] is generally melanocyte defect; e.g., abnormal melanosome
preceded by melanoma and is the result of membranes), an immune system component
immune system-mediated melanoma regres- (melanocyte-specific cell-mediated immunity),
sion; the barred rock chicken [24–26] represents and environmental triggers (e.g., herpesvirus of
a model of natural melanocyte loss associated turkey; HVT) [9, 12].
with cellular stress; and the vitiligo mouse Continuous research on SL autoimmune vitil-
(C57Bl/J6-vit/vit) [27–29] is no longer studied igo for over more than 35 years has not only estab-
as a model for vitiligo because the progressive lished the many similarities between human and
loss of fur pigmentation was found to be due to SL vitiligo but also has positioned the SL chicken
a mutation of the microphthalmia- associated as a highly suitable intermediate model for transla-
transcription factor gene, a mutation not associ- tional research on vitiligo treatment and preven-
ated with human vitiligo [30]. Of all the natu- tion strategies. Key characteristics of the SL
rally occurring animal models for vitiligo, only chicken that make it a highly relevant and suitable
the Smyth line chicken continues to be studied model for fundamental studies on the etiology,
extensively, and, as outlined later, it is currently progression, and pathology of autoimmune vitil-
the only animal model for spontaneous autoim- igo as well as for development of treatment and
mune vitiligo that has been demonstrated to prevention therapies are summarized in Table 22.1.
recapitulate the entire spectrum of clinical and The origin, development, and characteristics of
biological manifestations of the human this animal model have been reviewed extensively
disease. [9, 12, 16]. Highlights and recent developments
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a d
e f g h
Fig. 22.1 Spontaneous autoimmune vitiligo in the Smyth side and top of which are enclosed by an epidermal layer;
line (SL) chicken model. (a) SL (yellow) and parental BL (f) the epidermis (E) of the bottom 2–3 mm (barb ridge) of
(blue) chicks hatch with normal pigmentation; (b) a GF is modified containing melanocytes with their cell
6-week-old juvenile SL male and female with normal pig- bodies facing the dermis (D) and dendrites extending
mentation; (c) young adult males, (d) females, and (e) along columns of keratinocytes; (g) normal barb ridge
growing feathers (GF) with various extents of depigmen- with melanocytes and pigmented keratinocytes; and (h)
tation; (e) GF consist of a column (8–10 mm by 2 mm) of barb ridge in active vitiligo; brown cells are CD8+ T cells
living tissue (pulp) surrounded by a sheath from which the associated with damaged melanocyte cell bodies and den-
barbs emerge; the pulp consists of an inner dermis, the drites; note: almost no pigment in keratinocytes
regarding this animal model that support its rele- mutant SL (previously known as the delayed-
vance for both basic and translational research on amelanotic (DAM) chicken) was developed
autoimmune vitiligo will be provided below. from one female hatched in 1971 from a non-
pedigreed mating of the Massachusetts Brown
22.2.1.1 T he Genetics Basis of Smyth line (BL) [33]. The ability to develop a line of
Line Autoimmune Vitiligo chickens with a predictably high incidence of
The SL and control lines of chicken were devel- vitiligo starting with one vitiliginous hen
oped by Dr. J. Robert Smyth, Jr. at the University clearly demonstrates the heritability of this
of Massachusetts, Amherst, MA [16]. The disorder.
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Table 22.1 Spontaneous autoimmune vitiligo in the Smyth line chicken: unique opportunities for fundamental and
translational research
Production Robert Smyth, Poultry Geneticist, University of Massachusetts, Amherst, MA
Maintenance Gisela F. Erf, Avian Immunologist, Division of Agriculture, University of Arkansas, Fayetteville, AR,
USA
Features Vitiligo-like, autoimmune, post-hatch loss of pigmentation in feathers and eye
Onset 6–14 weeks of age
Incidence 80–95% by 20 weeks (young adult)
Severity Erratic to complete loss of pigmentation
Model Availability of MHC-matched (B101/101) control lines of chickens, including the parental Brown line,
from which the Smyth line was developed (<2% incidence of vitiligo), and the Light-brown Leghorn
chicken (vitiligo resistant)
Unique Recapitulates the entire spectrum of clinical and biological manifestations of the human disease
features
Autoimmune vitiligo development is truly spontaneous and involves immune system mechanisms
that parallel those established in human vitiligo
Spontaneously occurring, associated autoimmune diseases (e.g., autoimmune thyroiditis, uveitis,
alopecia) observed in some SL individuals also reflect the complexity and true nature of this
autoimmune disease
The target tissue (growing feather) is easily accessible and can be sampled prior to and throughout
the development of SL vitiligo in the same individual
The growing feather is a skin derivative with dermal and epidermal layers; melanocyte is located in
the epidermis in close association with keratinocytes. Unlike hair, the growing feather is an
immunologically active tissue (Fig. 22.1)
The growing feather is an accepted skin test site (in vivo test tube) to monitor cellular/tissue
responses to injected test materials [31]
The progenitor pool of melanocytes is not affected by the melanocyte-specific autoimmune response,
and repigmentation has been demonstrated
The strong direct association of herpesvirus of turkey (HVT) administration at hatch and the
incidence of vitiligo expression (incidence: >80% with HVT, <20% without HVT) allow for
examination of disease-precipitating/protective factors in genetically susceptible individuals
The genetic basis of autoimmune vitiligo susceptible, only a few (<2%) BL chickens
and line-associated traits has long been develop the disorder compared to 80–95% of
described as being under the control of multi- SL chickens. The incidence of vitiligo in the
ple autosomal genes [16, 34]. Next-generation BL however dramatically increased to 71%
whole-genome resequencing identified vari- when B101/101 BL chickens were treated with the
ous potential genetic markers with amino acid DNA methylation inhibitor 5-azacytidine [37].
changes that may play a role in SLV develop- These observations confirmed genetic suscep-
ment. Based on bioinformatic analysis of the tibility in the BL and epigenetic regulation of
genome data, SNPs were found to include vitiligo expression. It should be noted that the
genes involved in dermatological diseases/ 5-azacytidine treatment did not trigger vitiligo
conditions. Furthermore, intermolecular gene in Light-brown Leghorn (LBL) chickens,
network analysis revealed that candidate genes which supports their use as the vitiligo-resis-
identified in SLV play a role in networks cen- tant pigmentation control [37].
tered on protein kinases, phosphatase, ubiqui-
tinylation, and amyloid production [35]. These 22.2.1.2 Immune System
studies provide new insight and direction in Involvement in Smyth Line
identifying genetic components underlying Autoimmune Vitiligo
the susceptibility of SL chickens to develop Several studies have provided evidence support-
SLV. ing the role of the immune system in the pathol-
While SL and parental BL are closely ogy of SLV. Destruction of melanocytes is
related [36] and both are considered vitiligo preceded by increased levels of circulating
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140 18
CD4+ T helper cells IL-21
16
120 Apoptotic cells IFN-γ
Fold change in gene expression

Number of cells per 0.25 mm2
CD8+ cytotoxic cells 14

100 MHCII+ cells
12
80 10
60 8
6
40
4
20
2
0 0
control –8 –6 –4 –2 onset 2 4 6 –8 –6 –4 –2 onset 2 4 6
Time (weeks) with respect to SL vitiligo onset
Fig. 22.2 T cell infiltration, MHC class II expression, and tunnel staining and tissues analyzed by bright-field
melanocyte apoptosis, and IFN-γ and IL-21 mRNA microscope. Right: GF were collected twice per week and
expression in Smyth line growing feathers (GF) prior to RNA isolated for qRT-PCR (mean ± SEM). Because age
and throughout the development of vitiligo. Left: growing of onset differed among birds, data were expressed with
feathers were collected from Smyth line chickens every respect to visible onset of vitiligo for both studies
2 weeks. GF were snap-frozen for immunohistochemical
inflammatory cells and the infiltration of the autoimmune diseases [45–47]. Additionally, as
feather pulp and barb ridge by macrophages and shown by microarray transcriptome analysis
lymphocytes. Prior to onset of vitiligo, the infil- [48], IL-21 receptor expression is similarly
trate consisted primarily of T-helper cells increased in growing feathers within 2 weeks
(CD4+), but with onset of vitiligo and active before SLV onset and throughout active autoim-
melanocyte destruction, cytotoxic T cells (CD8+) mune loss of melanocytes.
predominated (Fig. 22.2) [9, 38–40]. Melanocyte Differential gene expression analysis at the
death was shown to occur by apoptosis, appar- transcriptome level using a 44K microarray also
ently induced by cytotoxic T cells [41]. Overall, confirmed the complex nature of SLV [48]. For
the immunopathology of SLV described earlier is this study, gene expression comparisons were
very similar to observations in affected skin of made between growing feather samples col-
vitiligo patients [6] and supports the involvement lected from the parental BL control chickens,
of cell-mediated immune mechanisms in melano- from SL chickens that did not develop vitiligo,
cyte destruction. In SL chickens, direct evidence and from SL chickens at various stages of SLV
for a role of cell-mediated immunity in the devel- development (within 2 weeks before visible SLV
opment of SLV was provided by immunosup- onset, during active SLV, and 2 weeks after com-
pression studies and by in vivo demonstration of plete depigmentation). Functional and network
anti-melanocyte cell-mediated immunity in viti- analyses of differently expressed genes high-
liginous but not in non-vitiliginous SL and con- lighted innate and adaptive immunity (both cell-
trol chickens [42, 43]. mediated and humoral), as well as neuronal
Targeted cytokine gene expression (RNA) involvement, apoptosis, cellular stress, and
analysis of growing feathers collected prior to melanocyte function. Moreover, the time course
and throughout SLV development revealed a Th1 approach used in this study led to important new
cytokine signature, whereby IFN-γ expression insights into events leading to SLV onset and
was accompanied by high expression of IL-10 provided a more precise window in time to study
and IL-21 (Fig. 22.2) [44]. The observation of the etiology of SLV [44, 48].
IL-21 expression near onset and during SLV pro- Vitiliginous SL chickens have melanocyte-
gression is particularly interesting, especially specific autoantibodies that have been shown to
since IL-21 has been implicated in a number of bind to chicken as well as human melanocytes
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a b c d 3.5
3
2.5 a
SLOPE
2
1.5
1 b b b
0.5
0
SL SLG BL LBL
SL 0 h SL 1 h pi BL 1 h pi
Fig. 22.3 Response of SL and BL melanocytes to 4-TBP fluorescein diacetate assay) by pigmented feather tips cul-
(4-tertiary butyl phenol; 10 μL of 0.15 mg/mL) injection tured with 200 μM 4-TBP (n = 8; mean ± SEM; SL,
into the feather pulp. (a) Vehicle-injected SL feather. (b) Smyth line; SLG, SL with gray shanks; SLG rarely
1-h postinjection (pi) SL melanocytes retracted their den- express vitiligo; BL, Brown line parental control; LBL,
drites, whereas those of BL controls (c) did not. (d) Light-brown Leghorn normal control)
Production of oxidative radicals (kinetic 2′,7′-dichloro-
and to be specific to tyrosine-related protein-1 full restoration of melanocyte morphology 3 days

(TRP1) and heat-shock proteins (HSP), but their after 4-TBP injection. Exposure of melanocyte
role in the etiology of SLV has not been estab- containing tissue sections (feather tips), as well
lished [14, 49, 50]. Maternal autoantibodies to as feather-derived and embryo-derived melano-
melanocytes have been shown to be transferred cytes to 4-TBP, resulted in increased reactive
from the hen to her chick via the egg but disap- oxygen species generation in SL compared to BL
peared from the chick’s circulation within cultures (Fig. 22.3).
10 days of hatch, and their transfer was not cor- These in vivo and ex vivo observations on the
related with vitiligo expression [51]. responses of melanocytes to 4-TBP in the chicken
model further supports aberrant ability of SL
22.2.1.3 Inherent Melanocyte melanocytes to respond to cellular stress.
Alterations in Smyth Line However, as shown through immunosuppression
Autoimmune Vitiligo studies, the inherent melanocyte sensitivity alone
Previous studies by Smyth and coworkers is not sufficient to cause SLV without a function-
describe the presence of a competent pigment ing immune system, but it appears to play a role
system in SL chicks at hatch (Fig. 22.1a). The in provoking a melanocyte-specific autoimmune
earliest abnormalities within SL melanocytes, response [53, 60–63]. Morphological and bio-
prior to visible onset of SLV, were irregularly logical melanocyte defects/alterations have also
shaped melanosomes containing pigmented been reported in vitiligo patients and non-SL ani-
membrane extensions, hyperactive melanization, mal models for vitiligo. In both humans and ani-
and selective autophagocytosis of melanosomes mals, cultured vitiligo melanocytes grow more
[52–55]. Similar degenerative processes were slowly than normal melanocytes, are more depen-
also observed in vitro in embryo-derived SL dent on antioxidants in the medium, and present
melanocytes, including heightened lipid peroxi- structural alterations [7, 24, 25, 55, 64–68].
dation and catalase activity [55, 56]. Exposure of
SL and control melanocytes to 4-TBP, a phenolic 22.2.1.4 R ole of Environmental Factor
compound known to initiate stress in human in Smyth Line Vitiligo
melanocytes [57], also revealed heightened sen- Expression
sitivity of SL melanocytes [58, 59]. Specifically, In addition to heightened sensitivity of SL mela-
in vivo injection of 4-TBP into the dermis of pig- nocytes to stress, we reported the role of environ-
mented growing feathers resulted in melanocyte mental factors, specifically vaccination with live
detachment and dendrite retraction at 1 and 3.5 h turkey herpesvirus (HVT) at hatch, in the expres-
postinjection, with signs of recovery by 6 h and sion of SLV [69]. Without HVT, the incidence of
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SLV is <20%, but with HVT, the incidence is and uveitis/blindness associated with melanocyte
generally >80%. HVT is an alpha-herpesvirus destruction in the choroid [16, 32].
commonly used in commercial chicken produc- Autoimmune hypothyroiditis: Hypothroidism
tion as a vaccine to protect chickens from Marek’s is consistently observed in every hatch of SL
disease caused by serotype 1 Marek’s disease chickens. The observation of autoimmune thy-
viruses (MDV-1). HVT is a non-oncogenic sero- roiditis in the current Smyth line population var-
type 3 MDV isolated from turkeys that causes ies from year to year with an average incidence of
only minor inflammatory lesions. And like other early (3–6 weeks) severe hypothyroidism in
MDV, it exhibits strong tropism for feather folli- approximately 5% of SL hatch mates. The inci-
cles, where it infects the feather follicle epithe- dence of thyroiditis can be increased by selec-
lium [70, 71]. Additional studies on the role of tion. Moreover, extensive mononuclear cell
HVT in SLV revealed that killed HVT had no infiltration can be observed in thyroids of fully
effect on the expression of SLV. Therefore, the developed adult SL chickens with vitiligo [9].
ability of HVT to cause infection must be of Uveitis, impaired vision and blindness:
importance. Administration of other live virus Impaired vision and blindness, like hypothyroid-
vaccines at hatch (i.e., Newcastle disease virus, ism, is also observed in every SL population,
NDV; infectious bronchitis virus, IBV), instead even with the exclusion of vision-impaired
of HVT, did not trigger the expression of SLV, breeders. Unlike hypothyroidism, ocular impair-
suggesting that viral infection and associated ment is strongly associated with vitiligo devel-
antiviral immune activity, as such, are not respon- opment [16]. In an ongoing study [73, 74], the
sible for triggering expression of SLV. Unlike incidence of blindness increased from the usual
HVT, NDV and IBV do not translocate to the 3–5% to nearly 40% among offspring from
feather; hence the presence of HVT where mela- vision-impaired or blind parents. To examine the
nocytes are located may be the key to its effect on phenotype of choroid-infiltrating leukocytes,
SLV expression [9]. Lastly, comparison of HVT- eyes were collected from SL chickens and age-
vaccinated and non-HVT-vaccinated SL and matched parental BL and LBL controls at 1 and
parental control BL chicks revealed heightened 4 weeks of age (before SLV and impaired vision)
cell-mediated immune activity to HVT in SL and between 8 and 12 weeks of age, when SL
compared to BL chicks [72]. chickens exhibited vitiligo and partial (PB) or
Based on these observations, we hypothesize complete (B) blindness. Immunohistochemical
that the translocation of the HVT infection to staining of frozen eye sections revealed that
the feather epithelium brings antiviral immune melanocyte loss in choroids was associated with
activity to the feather where the melanocytes mononuclear leukocyte infiltration similar to
are present. The resulting local antiviral cell- that observed in vitiliginous growing feathers,
mediated immune activity in the melanocytes’ consisting of T cells (CD4+, CD8+, gamma-
environment causes alterations in the already delta), IgM, B cells, macrophages, and MHC II+
inherently defective melanocytes that result in cells. In eyes from birds with impaired vision,
their recognition by the immune system leading gross morphology of the retinal pigmented epi-
to the development of melanocyte-specific thelium (RPE) was intact, whereas in birds
immune activity and autoimmune destruction judged to be blind, the RPE was damaged and
of melanocytes. replaced by the inflammatory infiltrate [74].
Target gene expression profile revealed the same
22.2.1.5 Associated Autoimmune signature cytokines as those reported for vitiligi-
Diseases in Smyth Line nous growing feathers, with the exception of a
Vitiligo more prominent increase in IL-6 in eye tissue
Like in humans, SLV can be associated with other [73]. Our observations further confirm those
autoimmune diseases, including Hashimoto’s thy- summarized by Smyth [75] that impaired vision
roiditis, an alopecia areata-like feathering defect, in SLV resembles induced uveitis and other ocu-
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lar diseases in humans, especially Vogt-

Koyanagi-Harada syndrome and sympathetic
ophthalmia (result of penetrating eye injury),
both of which are autoimmune diseases associ-
ated with vitiligo.
Alopecia areata-like feathering defect: The
alopecia areata-like feathering defect is rarely
seen before adult hood in the current populations,
but patchy loss and/or abnormal feather develop-
ment can be observed on the neck and back in
20–25% of SL adults with vitiligo.
22.2.1.6 O ther Useful Characteristics

of the Avian Model Fig. 22.4 Water buffalo with vitiligo (Courtesy of Dr
F. Roperto, University of Naples Federico II, Naples,
of Naturally Occurring
Italy)
Vitiligo
The avian model has many other useful charac-
teristics for biomedical research, including tion in skin of vitiliginous water buffalo also par-
access to embryos and embryo-derived target alleled observations in human vitiligo [18]. These
cells, availability of age-matched multiple sib- observations together with the similarities
lings from multiple families, relatively short- between human and buffalo epidermis, and the
generation time, ease of maintenance, and fact that spontaneous vitiligo is observed in an
convenient body size (large enough for easy outbred population, make the water buffalo a
blood sampling, small enough for convenient suitable intermediate model for treatment testing,
housing and handling). Additionally, research particularly when test material will be topically
tools, reagents, and advances in chicken immu- applied [17, 18].
nology continue to be generated at a fast pace,
especially since the chicken genome has been
sequenced [76]. 22.3 E
xperimental Mouse Models
of Induced Autoimmune
Vitiligo
22.2.2 Water Buffalo
While no truly spontaneous mouse model for
Vitiligo is also known to occur spontaneously in human vitiligo has been identified, several
water buffalos (Fig. 22.4). Histological, histo- approaches have been used in mice to induce
chemical, and ultrastructural analyses of biopsies depigmentation that models human vitiligo.
taken from involved and uninvolved skin from These induced mouse models for vitiligo are
two female buffalos revealed cytological aberra- important in addressing mechanistic aspects of
tions of melanocytes similar to those reported in the role of cellular stress and inflammation and
humans and other vitiligo models [17]. A recent the nature of autoimmune responses identified in
field survey of water buffalo in India found 286 the human system. Moreover, the ability to effec-
out of 45,043 buffalo examined had depigmented tively dissect the contribution of individual com-
patches (0.64% prevalence). Histological analy- ponents, pathways, and mechanisms underlying
sis of biopsies taken from five adult female viti- depigmentation and melanocyte loss is essential
liginous buffalos confirmed the presence of for the development of treatment and prevention
patchy leukocyte infiltration in perilesional skin strategies for the disease. In fact, some of the
but not in non-lesional skin. The distribution of induced vitiligo mouse models emerged from
lesions and the progressive nature of depigmenta- efforts to induce effective immune responses to
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melanoma where vitiligo was observed as a side antigens contributed to their immunogenicity were
effect of immunotherapy. Methods of vitiligo also made by others [75, 80–83]. In addition to
induction in mice focus to a large extent on tar- finding the most immunogenic forms of the mela-
geting melanocyte differentiation antigens by nocyte self-antigens, these studies further empha-
inducing endogenous melanocyte differentiation sized the importance of local tissue/cellular stress
antigen-specific immune responses, adoptive in targeting the autoimmune response to the skin.
transfer of melanocyte antigen-specific T cells, Melanocytes may be completely ignored by acti-
introduction of melanocyte antigen-specific T vated TRP2-specific CD8+ cytotoxic T lympho-
cell receptor (TCR) genes into the genome of cytes unless an inflammatory environment is
mice, or various combinations thereof. established in the target tissue.
In vitiligo, a wide variety of stress factors
have been reported to provoke an autoimmune
22.3.1 Induction of Vitiligo in Mice response to melanocytes ranging from chemi-
Through Generation cals, overexposure to sunlight (UV), skin injury,
of Endogenous Melanocyte- infection, and emotional stress [2, 84–86]. Cells
Specific Immune Responses under stress will produce stress proteins such as
HSP which serve a protective function during
Experimental mouse models of melanoma have the cell-stress episode. HSP70 was shown to
provided insight into the mechanisms regulating enhance dendritic cell uptake of antigen and to
immunity and tolerance to pigment cells. be an ideal adjuvant for antitumor vaccines, and
Successful approaches to generate an immune HSP70 has been implicated in precipitating vit-
response to melanoma in mice (e.g., C57BL/6 iligo in susceptible individuals [2, 87]. Based on
with black fur) included vaccination with viruses these observations, we [88] developed a repro-
or plasmids that carry DNA coding for melanocyte ducible in vivo mouse model of autoimmune vit-
differentiation antigens. Depending on immuniza- iligo by combining gene gun vaccination with
tion protocols, tumor (melanoma) immunity was eukaryotic expression plasmids encoding mela-
often accompanied by autoimmunity manifested nocyte differentiation antigens (e.g., hTRP2)
in vitiligo-like depigmentation of the fur. Ensuing with human- or mouse-derived HSP70i (induc-
efforts to uncouple tumor immunity and autoim- ible HSP70 isoform). Inclusion of HSP70i, inde-
munity formed the basis for the development of pendent of origin, was found to accelerate
murine vitiligo models. One promising approach depigmentation in this model. Interestingly, the
to achieving active immunity to melanocyte anti- progressive depigmentation induced by HSP70i
gens and vitiligo-like hair depigmentation in mice involved areas not directly exposed to the stress.
involved preparation of microscopic gold particles Depigmentation was accompanied by the induc-
coated with plasmid DNA and bombardment of tion of prolonged antibody responses to HSP70i
the skin with these particles using a gene gun [77, and correlated with T cell-mediated cytotoxicity
78]. However, while this approach was effective in toward targets loaded with TRP2 peptide [88]. In
breaking tolerance and developing self-reactive follow-up studies, the observation that HSP70i
immune system components, reliable induction of administration alone was sufficient to induce vit-
autoimmune vitiligo in the mouse model involved iligo in vitiligo-prone mice (described below)
additional considerations. For example, in genetic was perhaps the most important finding in sup-
immunization studies, TRP2-specific CD8+ T port of HSP70i as a critical player in vitiligo
cells and antibodies, as well as, vitiligo were suc- expression [89]. Considering that HSP70i has
cessfully produced with cDNA encoding xenoge- since been established as an important link
neic human TRP2 or modified murine TRP2 between cellular stress and development of a
peptide, but not with cDNA encoding murine melanocyte-specific autoimmune attack and that
TRP2 [79]. Similar observations that xenogenicity HSP70i overexpression is also observed in the
and/or alteration of melanocyte differentiation skin of vitiligo patients [89–91], this mouse
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model provides a relevant in vivo system to accumulation of CD8+ cells. The application of
examine the role of cellular stress in immuno- the same treatment to Rag-1 knockout mice,
recognition of different melanocyte proteins. which lack mature lymphocytes required for spe-
Moreover, this model can be extended to assess cific immune responses, resulted in hair depig-
the ability of other environmental factors to pre- mentation at the application site, but not at distant
cipitate or protect from the induction of a mela- sites. Therefore in addition to direct cytotoxic
nocyte-specific autoimmune attack. In fact, it effects of MBEH on melanocytes, MBEH is able
was shown that mutant HSP70i interferes with to trigger endogenous melanocyte-specific
immune activation and vitiligo development in immune responses resulting in progressive vitil-
this mouse model [90]. igo. Similar observations were made in melanoma
You et al. [92] reported another mouse vitiligo studies where administration of MBEH together
model based on the induction of an endogenous with imiquimod and CpG induced a robust mela-
immune response to melanocyte differentiation noma antigen-specific response and vitiligo-like
antigens. Addressing the previously established depigmentation of the fur [94]. Considering that
importance of skin inflammation, they adminis- MBEH was shown to make melanocytes more
tered immunogenic TRP2-related peptide 180– vulnerable to cytotoxic lysis and promote devel-
188 together with lipopolysaccharide (LPS) and opment of melanocyte antigen-specific immunity
cytosine-phosphate-guanosine oligodeoxynucleo- in a variety ways [91, 95], the MBEH model may
tides (CpG), which are microbial products with be more reflective of activation mechanisms lead-
known immunostimulatory activity. TRP2 pep- ing to autoimmunity in human vitiligo than the
tide, LPS, and CpG were injected subcutaneously vaccination models.
into the rear footpad for primary immunizations
and intradermally into the tail skin for booster vac-
cinations. Immunized mice developed epidermal 22.3.2 Induction of Vitiligo in TCR
depigmentation in the tail skin without hair Transgenic Hosts or by
involvement. CD8+ IFN-γ+ T cells dominated the Adoptive Transfer
leukocyte infiltration into the tail skin, and the of Transgenic T Cells
development of depigmented skin lesions coin-
cided with TRP2 peptide-specific CD8+ T cell Gene-transfer technology has been applied to
effector functions. Interestingly, priming vaccina- generate mice with increased frequency of mela-
tions were sufficient to break tolerance, but melanocyte reactive T cells. When genes encoding
nocyte killing did not occur without the intradermal genetic information for a T cell receptor are
booster injections into the tail. While it is not clear inserted into the genome of a mouse, all mature T
why skin depigmentation occurred in the absence cells in the periphery will be expressing the sin-
of tail hair pigmentation when self-antigen was gle transgenic TCR. Hence, T cells in TCR trans-
introduced together with microbial products, this genic mice only recognize one antigen peptide in
phenomenon distinguishes this model from other association with self-MHC molecules. The need
vaccination models of vitiligo [92]. for TCR to be specific to both, portions of the
A mouse model of induced vitiligo that is antigen peptide and polymorphic region of MHC
solely based on melanocyte stress employed the molecules, and the stringent endogenous selec-
well-known FDA-approved phenolic bleaching tion procedures imposed on developing T cells to
compound monobenzone (MBEH) [93]. When ensure tolerance to self-antigens further compli-
MBEH was topically applied to the skin, depig- cate the design of the gene-transfer approach.
mentation was not only observed at the applica- However, several T cell clones with a melanocyte
tion sites but also at nonexposed sites (Fig. 22.5). differentiation antigen-specific TCR have been
The severity of lesions was dependent on drug isolated from mice subjected to melanoma immu-
dosage. Histological examination revealed loss notherapy. The use of the TCR genes from these
of epidermal melanocytes and perilesional T cell clones in the generation of TCR transgenic
ERRNVPHGLFRVRUJ
a b
Fig. 22.5 Induced vitiligo mouse models. (a) C57BL/6 Vitesse mouse with vitiligo; (d) Vitesse mouse with repig-
mice before and after 21 days of treatment with topical mentation (Courtesy of Dr. Jonathan Eby and Dr. Caroline
1 M MBEH treatment; (b) h3T-A2 vitiligo mouse with Le Poole, Loyola University, Chicago, IL)
normal (left) and cloudy eye; (c) 9.5-week-old male
mice has led to several mouse strains with trans- various levels of immunodeficiency, gene dele-
genic T cells that survive and function in the tions, or normal, diverse immune systems. By
periphery of the host. These autoreactive, trans- crossing the TCR transgenic mice with other
genic T cells provide important insight into mouse strains with desired genetic characteris-
mechanisms involved in activating the autoim- tics, mouse models can be engineered to address
mune response and melanocyte loss in the trans- a multitude of scientific questions [96].
genic host. Additionally, when used in adoptive In recent years the TCR transduction approach
transfer experiments, their contributions to auto- has led to various TCR transgenic mouse models
immune vitiligo can be explored in hosts with that reflect T cell-mediated melanocyte-specific
ERRNVPHGLFRVRUJ
autoimmunity. T cells from these mouse strains autoimmunity 5 weeks after adoptive transfer.
differ in TCR specificity, affinity, MHC restric- Hair depigmentation started periorbitally and
tion, co-receptors, phenotype, and stimulatory spread in random fashion. Uveitis was also noted
requirements, but all are able to induce hair and found to be dependent on IL-2 or Th pres-
depigmentation [82, 97–99]. The hair depigmen- ence. They concluded that naturally occurring Th
tation observed in murine models has been a dis- cells can help bring self-reactive CD8+ T cells
tinguishing feature between mouse and human out of their functionally tolerant state through
vitiligo, whereby human vitiligo involves loss of an IL-2-dependent mechanism but require the
epidermal melanocytes in the skin and is only absence of naturally occurring Tregs to be
rarely accompanied by hair depigmentation. effective.
Incorporation of the Krt14-Kitl* (aka as the K14- Krt14-Kitl* mouse-pmel-1 T cell adoptive
SCF) mouse strain [100], which has black skin transfer model: Harris et al. [102] have taken the
and black hair, helped address the hair versus optimized adoptive transfer protocol for pmel-1
skin depigmentation concerns in mouse vitiligo T cells a step further and successfully applied it
studies [101, 102]. In the Krt14-kitl* mouse, to the development of a mouse model of vitiligo
localization of melanocytes in the skin is due to with focused skin depigmentation while sparing
induced keratinocyte expression of transgenic the hair. In this model, pmel-1 mice were crossed
stem cell factor (SCF or Kit ligand, kitl). Targeted with mice that express GFP in both CD4+ and
expression of SCF to epidermal keratinocytes in CD8+ T cells to be able to isolate and track CD8+
mice was achieved by introducing a SCF trans- pmel-1 cells. Purified naïve GFP+CD8+ pmel-1
gene that codes for membrane-bound SCF (kitl*) T cells were then transferred into sublethally irra-
and is under the control of the human keratin 14 diated Krt14-Kitl* hosts which have black skin
(K14) promoter. Expression of the membrane- and black hair [100]. On the day of transfer,
bound SCF resulted in the maintenance of a pop- Krt14-Kitl* hosts were also infected i.p. with 106
ulation of melanocytes within the interadnexal pfu of recombinant vaccinia virus (rVV) that
epidermis in mice, the same location where expressed human pmel17, a potent antigenic
melanocytes and melanin are found in human stimulus for the murine CD8+ pmel-1 T cells
skin [100]. [82]. With all three treatments, vitiligo appeared
Pmel-1 mouse: One of the first TCR trans- 4–5 weeks after transfer [102]. Depigmentation
genic mouse strains used for studies on autoim- was observed in the skin of ears, rear footpads,
mune vitiligo is the pmel-1 mouse, which has tail, and, less commonly, the nose; occasionally
CD8+ T cells with TCR specific for melanocyte the trunk skin under the hair was affected, as
differentiation antigen Pmel17 (gp100) [75, 82]. were the front footpads and genitals.
Despite large numbers of gp100-specific T cells Depigmentation was initially patchy but often
in the pmel-1 mouse, B16 melanoma grew nor- progressed to confluence, involving the entire
mally, and there was no loss of normal melano- epidermal surface, while depigmentation of
cytes. Even adoptive transfer of large numbers of the hair was not observed, even months after
transgenic pmel-1 T cells was not effective in transfer. Cutaneous depigmentation was associ-
reducing tumor growth without additional T cell ated with mononuclear cell infiltration into the
stimulation. As shown by Antony et al. [103], epidermis, including aggregates and single pmel-1
optimal results were obtained when CD8+ pmel-1 (GFP+CD8+) T cells. IFN-γ from CD8+ pmel-1
T cells were transferred into Rag-1-deficient or T cells was observed within 2 weeks.
sublethally irradiated mice and provided with All aspects of vitiligo described above for the
in vivo activation signals in the form of gp100 induced Krt14-Kitl*-pmel-1 adoptive transfer
expressing viruses and IL-2 or CD4 T cells that model paralleled observations in the skin from
had been depleted of regulatory T cells (Tregs). human vitiligo patients. To demonstrate the util-
Rag-1-deficient mice treated with pmel-1 T cells, ity of this model in the development of vitiligo
vaccine and IL-2 or Th cells, developed profound treatments, the role of IFN-γ expression was
ERRNVPHGLFRVRUJ
more closely examined. Blocking IFN-γ with expression. Ablation of these genes also resulted
neutralizing antibody significantly abrogated in scarce CD8+ T cell infiltration into the skin.
depigmentation, even when treatment was initi- TRP1 transgenic mouse: The transgenic
ated 2 weeks after vitiligo development and skin- mouse generated by Muranski et al. [99] is
specific accumulation of autoreactive CD8+ T characterized by CD4+ T cells expressing a
cells was greatly diminished [102]. Based on fur- MHC class II-restricted TCR specific for
ther explorations in both humans and the mouse endogenous melanocyte differentiation antigen
model [104], the IFN-γ/CXCL10/CXCR3 path- TRP1 (or gp75). While the role of CD4+ mela-
way was identified as promising treatment strat- nocyte differentiation antigen-specific T cells
egy for vitiligo, which is actively being pursued in autoimmune vitiligo is not clear, the transfer
using this mouse model [105–107]. of TRP1-specific CD4+ T cells (TRP1 T cells)
FH mouse: The FH TCR transgenic mouse from these mice into Rag-1-deficient mice or
vitiligo model was developed by Engelhard and sublethally irradiated TRP1-expressing mice
collaborators [97]. It is based on the recognition resulted in rapid depigmentation of the hair. A
of a melanocyte protein tyrosinase, specifically combination of ex vivo and in vivo differentia-
HLA-A*0201-restricted epitope Tyr 369, by tion and activation studies using the TRP1 T
CD8+ T cells. To engineer a mouse that can pro- cells revealed that Th subsets with different
cess and present the murine homologue of this polarization defining cytokine profiles and dif-
Tyr369 epitope, transgenic C57BL/6 mice were ferent abilities to migrate and infiltrate tissues
produced that express recombinant class I MHC could be generated. In melanoma studies,
molecule AAD, which contains the peptide- Th17-polarized TRP1 T cells were found to be
binding region of human HLA-A*0201 linked to more effective than Th1 TRP1 T cells in elicit-
the CD8-binding domain of murine H-2Dd. ing tumor rejection. However, effects of the
Subsequent transfer of genes for a tyr369- Th17-polarized cells were completely abro-
specific, ADD-restricted TCR (called FH) into gated when IFN-γ-depleting antibodies were
ADD transgenic mice resulted in a double- included in this model, suggesting that Th17-
transgenic mouse with tyr369-specific CD8+ T mediated responses of the TRP1 transgenic T
cells capable of recognizing the tyr369 melano- cells were highly dependent on IFN-γ-based
cyte peptide in the context of endogenous mechanisms.
ADD. The FH T cells did not undergo central TrpHEL mouse: The TrpHEL model devel-
tolerance, and the FH TCR was expressed on oped by Lambe et al. [108] also involves CD4+ T
most peripheral CD8+ T cells of both albino and cells that mediate autoimmune disease with strik-
tyrosinase+ ADD+ mice. All AAD+, tyrosinase+ ing similarities to human vitiligo and Vogt-
FH transgenic mice developed progressive depig- Koyanagi-Harada syndrome. In this model a hen
mentation of epidermis and hair follicles. The egg lysozyme (HEL) peptide is expressed as a
spatial and temporal development of vitiligo dis- neoantigen in melanocytes under the control of
played a depigmentation pattern similar to that the TRP2 promoter, and T cells are MHC class
observed in the human disease, with bilateral II-restricted CD4+ T cells with transgenic TCR
head and facial areas affected in juveniles and specific for HEL peptide. Advantages of using
depigmentation extending over the rest of the the well-defined HEL model antigen as a mela-
body later in adults. Vitiligo was entirely depen- nocyte neoantigen is the enhanced ability to track
dent on CD8+ T cells, while CD4+ T cells exerted autoreactive CD4+ T cells from their develop-
a negative regulatory effect, presumably due to ment in the thymus to their involvement in spon-
the presence of Tregs. Importantly, CD8+ T cells taneous autoimmune disease.
were pervasively present in the skin in the steady h3T-A2 mouse: Mehrotra et al. [98] developed
state without inducing vitiligo in most areas. transgenic mice with a high-affinity tyrosine pep-
Both IFN-γ and T cell chemokine receptor tide (368–376)-specific TCR isolated from an
CXCR3 were found to be necessary for disease unusual MHC class I (HLA-A2)-restricted
ERRNVPHGLFRVRUJ
tumor-infiltrating human CD4+ T cell (h3T). T exogenous cytokine or antigen administration

cells expressing the h3T TCR transgenes were and was again found to be associated with IFN-γ
positively selected whether they expressed CD8 and to a lower but significant extent with IL-17
or CD4, but, when allowed to grow up in mice responses. Gradually appearing patches of
with the HLA-A2 transgene, the transgenic TCR repigmenting skin were also observed
was primarily expressed on CD3+CD4-CD8- (Fig. 22.5d), and repigmentation was associated
double-negative T cells. However, in the h3T with increased presence of Tregs. The signifi-
single- and the h3T-HLA-A2 double-transgenic cant Th17 responses observed in the epidermal
(h3T-A2) mice, all three types of T cells (CD4+, depigmentation in the Vitesse mouse are partic-
CD8+, or double-negative) were functional with- ularly interesting considering the pure Th1
out vaccination or cytokine support. h3T-A2 responses observed in the h3T-A2 model, which
transgenic mice spontaneously developed hair has the same TCR as the Vitesse mouse but no
depigmentation from an early age and exhibited epidermal pigmentation [101]. Hence, the
eye pathology that progressed with age Vitesse mouse offers unique opportunity to dis-
(Fig. 22.5b). Similar to depigmentation in the sect autoimmune activities involved in depig-
skin, visual impairment and interruption of the mentation and repigmentation processes in
RPE were associated with infiltration by trans- cutaneous vitiligo.
genic T cells and macrophages. Addressing the
mechanism regulating autoimmunity in this
model in combination with gene-knockout mice, 22.3.3 Summary on Induced Mouse
Chatterjee et al. [109] confirmed a central role of Models
IFN-γ in the observed vitiligo development. In
the absence of IFN-γ (i.e., h3T-A2-IFN-γ-/- Collectively, the induced mouse models of vitil-
mice), increased presence of Tregs was noted. igo each confirm observations in human vitiligo
When Tregs were deleted by employing anti- and can contribute to our understanding of vari-
CD25 antibody, the depigmentation phenotype ous aspects of autoimmune vitiligo. Opportunities
was fully restored in h3T-A2-IFN-γ-/- mice. In exist to apply the strength of each model to
the absence of IFN-γ and Tregs, development of answer different questions, although there are
depigmentation appeared to be mediated in part clearly overlapping opportunities between the
by other inflammatory cytokines such as IL-17 models. Mechanisms of breaking tolerance to
and IL-22. Efforts to increase the Treg population melanocyte antigens are best pursued in models
in the skin of the h3T-A2 mouse model resulted of endogenously induced immune response,
in lasting remission of vitiligo. while adoptive transfer of autoreactive cells
Vitesse mouse: We used the h3T-A2 mouse yields important insight into mechanisms of
strain to develop the Vitesse mouse [101]. The recruitment and activation of cells with known
Vitesse mouse is a triple transgenic carrying the antigen specificity. Models with T cells
inter-epidermal pigmentation transgene Krt14- expressing a transgenic TCR with specificity for
Kitl* in addition to the h3T-A2 genes. In the one melanocyte differentiation antigen peptide
Vitesse model, spontaneous skin depigmenta- provide insight into T cell development, selec-
tion preceded symmetrical and sharply demar- tion, differentiation, and regulation of events
cated patches of graying hair by 5–7 weeks, at leading to spontaneous depigmentation. All of
which time pigment loss from ears and extremi- these mouse model systems have the potential to
ties was essentially complete (Fig. 22.5c). find answers to mechanisms of melanocyte kill-
Vitesse mice reached maximum depigmentation ing in vitiligo. Studying the autoimmune loss of
earlier and at higher levels than h3T-A2 mice melanocytes in the skin of mice, rather than the
(30 weeks vs. 40 weeks; 83% vs. 65% depig- hair, may more faithfully reflect mechanisms
mentation, respectively). Depigmentation underlying the cutaneous depigmentation
occurred spontaneously and predictably without observed in humans, although membrane-bound
ERRNVPHGLFRVRUJ
SCF also is known to exert immune- and mela- References

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In Vitro Study of Vitiligo
23
Maria Lucia Dell’Anna and Muriel Cario-André
Contents
23.1 ell Isolation and Culture
C 226
23.1.1 Isolation and Culture of Skin Melanocytes and Keratinocytes 226
23.1.2 Isolation and Culture of Skin Fibroblasts 227
23.1.3 Isolation and Culture of Hair Follicle Melanocytes and Keratinocytes 227
23.1.4 Next-Generation Cultures 228
23.1.5 Isolation and Freezing of Peripheral Blood Mononuclear Cells (PBMC) 228
23.2 I n Vitro Reconstructed Epidermis 228
23.2.1 Preparation of Dead De-epidermized Dermis 229
23.2.2 Epidermal Reconstruction 229
23.3 F unctional Studies 229
23.3.1 F unctional Studies of NSV Cells Using Monolayers: Melanocytes,
Keratinocytes, and Fibroblasts 229
23.3.2 Functional Studies Using Genetically Modified Cell Culture in Monolayer 230
23.3.3 Functional Studies of NSV Cells by Next-Generation Approach 230
23.3.4 Functional Studies Using Reconstructed Epidermis 231
23.4 Analytic Techniques 232
23.4.1 Fluorescence-Based Assays 232
23.4.2 Proteomic 233
23.4.3 Metabolomics/Lipidomics 234
23.4.4 Transcriptomic 235
References 235
Abstract
M. L. Dell’Anna (*)
The study of vitiligo has been approached by
Laboratory of Cutaneous Physiopathology, San
Gallicano Dermatological Institute, IFO, Rome, Italy several different perspectives. Accordingly to
e-mail: marialucia.dellanna@ifo.gov.it the progressive improvement of the technologi-
M. Cario-André cal support to the research and discovery of
Inserì U876, Centre de référence des maladies rares new fields in the biological world, even
de la peau, Université V Segalen Bordeaux 2, researchers who focused on vitiligo gained new
Bordeaux, France
opportunities. An unsolved question regards
e-mail: muriel.cario-andre@dermatol.u-bordeaux2.fr

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226 M. L. Dell’Anna and M. Cario-André
how and where to analyze the possible

pathomechanisms underlying the vitiligo onset • The complexity of cell culture models
and progression. A clear-cut position against varies from monolayers for adherent
the best in vitro approach is aleatory. It is surely cells or suspensions for nonadherent
customary to use the primary cell cultures to cells to 2D or 3D hydrogel-supported
perform functional studies; however, the cultures, to cocultures, or to epidermal
in vitro assays with primary cell cultures are reconstructs (3D model).
indissolubly affected by the intrinsic selection • 3D hydrogel cultures, cocultures, and
of the more “aggressive” or best cells among epidermal reconstructs allow the study
the overall tissue bulk. Moreover, the media of paracrine or contact cell-cell effects.
usually used to select and grow the primary • The tests on nonepidermal nonadherent
cells are optimized to burst their proliferative or cells, such as peripheral blood mononu-
differentiative ability, probably overcoming the clear cells (PBMC), expand the view,
intrinsic features of the isolated cells. The first going beyond melanogenesis-associated
requirement of a researcher is to maximize metabolisms.
yield of a bioptic sample, but this requirement • The in vitro studies on hair follicle
affects per se the quality of the resulting cul- melanocytes help understand the matu-
tures that may no longer reflect the initial cel- ration and differentiation of melano-
lular asset of an individual. This initial reflection cytes and to unveil the defective steps of
did not intend to attack the studies currently growth and migration in melanocyte
performed with primary cell cultures, but it just precursors during the repigmentation.
aims to alert about the possible intrinsic bias.
Key Points 23.1 Cell Isolation and Culture

• Vitiligo cells are obtained from lesional
or non-lesional skin of vitiligo patients. Melanocytes and keratinocytes can be cultured from
The specific composition of the growth both the skin and hair follicle. Indeed, epidermal
factor cocktail will affect the prolifera- stem cells occur in the basal layer of the epidermis
tive or differentiative signature as well and within hair follicles, where keratinocyte stem
as growth rate and the delay between cells are situated in the bulge area, whereas those for
isolation and standard growth. melanocytes are found in the sub-bulge area.
• Considering that physiologically the
cells aren’t seeded on plastic or glass but
they grow in a 3D space, biomaterials, 23.1.1 Isolation and Culture of Skin
including hydrogels, have been devel- Melanocytes
oped to investigate the complex world and Keratinocytes
of the live cells.
• The flow cytometric approach permits a Vitiligo cells are obtained from lesional or non-
quali-quantitative multiparametric and lesional skin of vitiligo patients. The method for
time-dependent analysis. the isolation of primary melanocytes or keratino-
• Metabolomic research will provide a fin- cytes is similar for both vitiligo and normal skin
gerprinting of the metabolic activities samples. Split-thickness skin samples are cut in
related to immune deregulation and degen- small pieces and trypsinized. Trypsin disrupts the
erative process, possibly merging the vitil- epidermis above the basal layer, and it is neutral-
igo profile with those known and described ized with fetal calf serum or trypsin-soybean
in other diseases and health population. inhibitor. The epidermis is removed, and the basal
layer is scraped to dissociate melanocytes and
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23 In Vitro Study of Vitiligo 227
Table 23.1 Cocktails of growth factors used for melanocyte culture

Cario-
Boissy Kroll Richmond Medrano Andrè Smith Greatens
commercial 1991 [2] 2005 [3] 2005 [4] 1990 [1] 2007 [5] 2005 [6] 2005 [7]
BPE 0.2% 30 μg/mL 13 μg/mL 30 μg/mL 30 μg/mL 140 μg/mL 30 μg/mL 30 μg/mL
FBS 0.5% 5% 4% 10% 5% 3% 5% 4%
bFGF 3 ng/mL 0.6 ng/mL 0.6 ng/mL 0.6 ng/mL 0.3 ng/mL – 0.6 ng/mL 0.6 ng/mL
Eparin 3 μg/mL – – – – – – –
Hydrocrtisone 0.18 μg/mL 0.5 μg/mL – 0.5 μg/mL 0.5 μg/mL 1.75 μM 0.5 μg/mL 0.5 μg/mL
Insulin 5 μg/mL 5 μg/mL 5 μg/mL 5 μg/mL 5 μg/mL 20 μg/mL 5 μg/mL 5 μg/mL
Vitamin E – 1 μg/mL 1 μg/mL 1 μg/mL 1 μg/mL – 1 μg/mL 1 μg/mL
Transferrin 5 μg/mL 5 μg/mL – 5 μg/mL 5 μg/mL – 5 μg/mL 5 μg/mL
PMA 10 ng/mL 8 nM 8 nM 8 nM – – 8 nM 8 nM
Catalase – 20 μg/mL – 20 μg/mL – – – –
MSH – – – – – – 10−8 M –
Endothelin1 – – – – – – 10−9 M –
Medium M254 MCDB153 M154 MCDB153 MCDB153 MCDB153 M154 MCDB153
basal keratinocytes. Cells are seeded at a density 23.1.3 Isolation and Culture of Hair
of 200,000/cm2 for melanocytes and 100,000/cm2 Follicle Melanocytes
for keratinocytes culture [1]. Usually, the culture and Keratinocytes
media for melanocytes are M2 medium
(PromoCell), M254 (Gibco), or MCDB153 Scalp specimens are cut into small pieces, and the
(Sigma), MGM4 BulletKit (Lonza) added with epidermis and upper 1 mm of the dermis are care-
specific growth factors. Several different hand- fully removed with a scalpel [2]. Hair follicles are
made growth factor cocktails for culture are used isolated by incubating the tissue in Eagle’s mini-
(Tables 23.1 and 23.2). The specific composition mal essential medium (EMEM) containing dis-
of the growth factor cocktail will affect the prolif- pase and then collagenase. The released hair
erative or differentiative signature as well as follicles are washed repeatedly with PBS until
growth rate and the delay between isolation and hair follicles appear pure by microscopic exami-
standard growth. Moreover, the presence of nation. Single-cell suspensions are then obtained
α-MSH analog or precursor could influence the by treatment with trypsin-EDTA [3]. Hair follicle
effect of some in vitro treatments. The culture melanocyte (HFM) cultures are established using
media used for keratinocytes are CellnTechn-07 media supplemented with either artificial mito-
(Chemicon), MCDB153 (Sigma), and M154 gens or natural melanocyte mitogens as follows.
(Gibco) with the appropriate growth factors. Cells Contaminating fibroblasts, when present, are
at passage 2–3 can be used to perform functional eliminated by treating the cultures with 150 μg/
studies, to reconstruct the epidermis, or to start mL Geneticin (G418) sulfate [4]. Follicular kera-
cocultures for studies regarding cellular network. tinocytes are separated from the HFM cultures by
differential trypsinization. The identity of the iso-
lated cells is confirmed by immunophenotyping
23.1.2 Isolation and Culture of Skin with the melanocyte lineage-specific marker NKI/
Fibroblasts beteb against glycoprotein100 (gp100) [5, 6].
Hair follicle keratinocytes (HFK) are established
After obtaining keratinocyte and melanocyte sus- by preparing single-cell suspensions from iso-
pensions, the scraped dermis is cut in small lated hair follicles. At the primary culture stage,
pieces and incubated with collagenase allowing follicular melanocytes are first selectively trypsin-
the fibroblasts to exit from extracellular matrix. ized from the culture. This step is carried out
Fibroblasts are cultured in DMEM supplemented under microscopic observation. Remaining kera-
with 10% fetal calf serum. tinocytes are then switched to the keratinocyte-
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specific medium, which does not support mixed), characterized by specific physical and
melanocyte growth. chemical properties. BD Biosciences, Baxter,
Johnson & Johnson, Sigma, ProNova, and
23.1.3.1 Transgenesis BioTime, Inc. are the major vendors of the different
Since it is difficult to have enough cells from vit- hydrogels for both 2D and 3D cultures. Accordingly
iligo patients to test all hypothesis, we can, accord- to the different physical features, each hydrogel is
ing to genetic data, modulate susceptible genes more or less suitable, based on epitope accessibility
using overexpressing or silencing vectors such as or stability and protein/RNA recovery, for micros-
CCN3 (homeostasis of melanocyte), DDR1 (adhe- copy, flow cytometry, or molecular studies [9].
sion of melanocyte to basal membrane), and cad-
herin (cell adhesion) [7, 8]. After the first or the
second passage, cells are incubated with viral par-
ticles at MOI (multiplicity of infection) 10 in a 23.1.5 Isolation and Freezing
small volume of serum-free medium. After 6–16 h, of Peripheral Blood
fresh SVF-containing medium is added to com- Mononuclear Cells (PBMC)
plete the volume to usual one (e.g., in 25 cm flasks,
transduction is made in 0.5 mL serum-free The peripheral blood mononuclear cells (PBMC)
medium, and then 3 SVF medium is added). have been analyzed in vitiligo in order to character-
Medium is changed after 48 h, and percentage of ize their immunological status, the capability to
transduction if fluorescent reporter gene (RFP, recognize specific melanocyte antigens, the cyto-
GFP, YFP) is present in the vector is estimated toxic effects toward melanocyte or melanoma cell
72 h after transduction to avoid overestimation due lines, redox status, and response to DNA damage
to pseudo-transduction. To obtain 100% transduc- [10–12]. The isolation of vitiligo PBMC is per-
tion, an antibiotic resistance gene such as puromy- formed through the stratification onto Ficoll den-
cin-resistance gene can be added in the vector sity gradient, which allows separating mononuclear
construct. Otherwise cells can be selected by flow from polynuclear and red cells. The PBMC local-
cytometry to obtain around 95% transduction. ize at interface between serum and Ficoll, whereas
the polynucleates, after centrifugation, go to the
bottom of the tube. After recovery, PBMC are
23.1.4 Next-Generation Cultures washed twice with saline solution (NaCl 0.9%) and
used as planned. The procedure should be carefully
Growing estimation is recently gained by the extra- performed (short time, less than 30 min, between
cellular environment role on cellular behavior. The blood withdrawal and PBMC isolation; gentle
network between mechanical, chemical, and struc- manipulation) in order to avoid any physical stress
tural aspects of the environment heavily affects the able to affect vitiligo PBMC independently of
cell function. Considering that physiologically the in vitro test. Theoretically, vitiligo PBMC may be
cells aren’t seeded on plastic or glass but they grow used even after freezing in DMSO/serum mixture.
in a 3D space, biomaterials, including hydrogels, However, our experience suggests avoiding freez-
have been developed to investigate the complex ing when ROS generation or some functional
world of the live cells. Most of the functional data membrane-dependent parameters will be assayed.
on cultured cells arise from 2D support, such as flat On the other hand, the cellular pellet, stored at
stiff materials (polystyrene and glass), determining −80 °C, can be used for enzymatic analysis.
per se flattened shape, aberrant polarization, loss of
differentiation, and altered response to drugs.
Hydrogels, that are water-swollen networks of 23.2 In Vitro Reconstructed
polymers, are developed to overcome these prob- Epidermis
lems by mirroring, more than standard stiff sup-
ports, the physiological extracellular matrix. As early as 1979, Prunieras et al. [13] have demon-
Currently, several different types of hydrogel are strated that it is possible to obtain a fully differenti-
available by market, both natural and synthetic (or ated epidermis in vitro by simply raising
ERRNVPHGLFRVRUJ
keratinocytes up to the air–liquid interface. sion [22, 23]. Twenty-four hours after seeding,
Apparently, the interface with air stimulates syn- the incubation chamber is removed, and the
thesis of profilaggrin by keratinocytes and thus the DDD is immersed for 3 days. The DDD are
appearance of the granular phenotype when kera- shifted to the air–liquid interface for 8 days
tohyalin granules develop [14]. The epidermis can before functional studies (Fig. 23.1). The model
be reconstructed using various supports: dead de- can be improved by seeding fibroblasts in an
epidermized dermis (DDD) [13], gel of collagen incubation chamber placed on the dermal side of
(EpiSkin®), lattices including fibroblasts and col- DDD 72 h before seeding keratinocytes and
lagen [15], lattices including fibroblasts and colla- melanocytes.
gen-glycosaminoglycan-chitosan [16], human
fibrous sheet [17], and basal inert substrates such
as porous filters [18]. Otherwise fibroblasts can 23.3 Functional Studies
colonize dermal matrix used in clinic as dermal
regeneration template such as Integra® and 23.3.1 Functional Studies of NSV
MatriDerm™ (European patent EP 3072535 A1). Cells Using Monolayers:
The reconstructed epidermis has been per- Melanocytes, Keratinocytes,
fected first by adding melanocytes [19] and sec- and Fibroblasts
ondly Langerhans cells [20], but models with
Langerhans cells have not been tested in vitiligo Cultures can be used to characterize the pheno-
experiments. The reconstructed epidermis with type of vitiligo melanocytes as compared to
keratinocytes and melanocytes on DDD repro- control pigment cells under various treatments,
duces the epidermal melanin unit (EMU) and is such as UV or pharmacological agents. The
suitable to study pigmentation [21]. cells are usually seeded the day before to obtain
60–70% confluency on the day of treatment.
Various techniques can be used to observe mela-
23.2.1 Preparation of Dead nocyte behavior. Direct observation by micros-
De-epidermized Dermis copy on fixed and specifically stained
melanocytes (melan-A, DOPA, c-kit, S-100,
Human dermis is obtained from plastic surgery HMB-45) gives information on shape, dendrit-
specimen from normal adults, mostly breast reduc- icity, and pigmentation. DOPA staining often is
tion specimen. Skin samples are thinned, cut into used as alternative method to overcome the poor
very small pieces, and incubated at 37 °C in Hank’s melanocyte number and not enough to allow
balanced salt solution until epidermis can be spectrophotometric melanin content measure.
removed without excessive scraping. After removal, Vitiligo melanocyte and keratinocyte cultures
the dermis is rinsed in 70 °C ethanol and submitted can be tested in vitro in order to determine their
to two cycles of freezing-thawing and stored in specific susceptibility to noxious stimuli or to
Hank’s balanced salt solution at −20 °C until use. physiological growth factors. UVB, cumene
hydroperoxide, and tert-butylphenol are the
most used stimuli [19, 24–31]. Cell prolifera-
23.2.2 Epidermal Reconstruction tion and mortality can be assessed through MTT
test, manual cell count, annexin V/propidium
Melanocytes and keratinocytes (from normal or staining, DNA ladder, or caspase profile. In
non-lesional vitiligo skin) at passages 2 or 3 are addition, the morphology and modality of mela-
seeded in an incubation chamber placed on the nosome transfer in vitro can be studied using
epidermal side of DDD at 4 × 105 cells/cm2 at a atomic force microscopy, which allows to esti-
melanocyte/keratinocyte ratio of 1:20 (5%) for mate and quantize the measure and the distribu-
normal melanocytes [21] or transduced melano- tion of the dendrites including internal
cytes (Ricard et al. 2012, Wagner et al. 2015) and melanosome distribution and arrangement [31–
1:20 or 1:10 (5–10%) for vitiligo melanocytes 33]. Culture on 3T3 feeder layer induces the for-
since vitiligo melanocytes have a defective adhe- mation of colonies of keratinocytes which vary
ERRNVPHGLFRVRUJ
Control vitiligo
or genetically modified fibroblasts
Dead dermis
72 h
95% control, vitiligo or genetically

modified keratinocytes
+ 5% control, vitiligo or genetically

modified melanocytes
24 h
72 h
8 days
Fig. 23.1 Schematic protocol for epidermal reconstruction
in size according to the state of cell prolifera- dendricity for melanocytes) but sometimes in
tion, differentiation, or senescence. Lesional monolayer cells do not express protein they
NSV keratinocytes are characterized by a lower express in tissue. For example, monolayer mela-
proliferative potential, as indicated by a shorter nocytes do not express cadherin; thus silencing of
in vitro life span. Moreover, the expression of cadherin has no effect on their behavior in cul-
p16, PCNA, p53, and p63 markers differs ture. Several genes are implicated in cell adhe-
between lesional and non-lesional cells. sion, but their silencing may have adverse effect;
Lesional keratinocytes show a lower level of the silencing of DDR1 (collagen IV receptor) has no
senescence marker p16 and a higher level of the effect on plastic adhesion, whereas silencing of
melanocyte growth factor SCF [34]. CCN3 (melanocyte’s homeostasis) is lethal for
melanocytes (Fig. 23.2).
23.3.2 Functional Studies Using

Genetically Modified Cell 23.3.3 Functional Studies of NSV
Culture in Monolayer Cells by Next-Generation
Approach
Genetically modified cells in monolayer can be
useful to verify if putative gene can reproduce Cultures can be also used to perform functional
some key features of diseased cells (adhesion, studies based on epigenome editing. This branch
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a b
c d
Fig. 23.2 Effect of inhibition of CCN3 on melanocytes. at day 12 of untransduced melanocytes (c) and corre-
Expression of GFP in melanocytes transduced with a vec- sponding melanocytes transduced with vector coding
tor coding shCCN3 and GFP at day 5 post-transduction shCCN3 and puromycin resistance and treated with puro-
(a) and day 12 post-transduction (b). Brigthfield analysis mycin to obtain 100% transduction (d)
of biology refers to directed alteration of chroma- modifications have a causal role in governing pro-
tin marks at specific genomic loci by Epi effectors cesses such as transcription or alternative splic-
aiming to durable gene regulation with applica- ing? Which modifications lead to stable and
tion in basic research and clinics. Targeted deposi- heritable changes in chromatin status? Which
tion or removal of chromatin modifications is a chromatin behavior affects the maintenance of
powerful approach for functional studies. It such modifications? Is the specificity per se able
includes histone posttranslational modifications, to provide clinical applications [35]?
DNA methylation, and hydroxymethylation, Recently, a single-cell epigenomics approach
which in concert regulate the gene expression. was developed by combining several different
The available profile of chromatin modifications high-throughput techniques allowing studies on
yields thousands of global-scale epigenomic cytosine modification, protein-DNA interaction,
maps. In preclinical studies, epigenome editing chromatin structure, and 3D organization [36].
will give rise to a fine mapping of the regulatory
mechanisms leading to health/diseased process. It
is integrating part of interdisciplinary field of syn- 23.3.4 Functional Studies Using
thetic biology. It represents new and interesting Reconstructed Epidermis
avenues to treat metabolic diseases, possibly
including vitiligo, characterized by aberrant sig- Monolayer cultures and coculture [37] are useful
naling pathways through a causative therapy. to study vitiligo melanocytes and keratinocyte
However, some questions are still open: which direct (cell–cell contact) or indirect interaction
ERRNVPHGLFRVRUJ
(soluble factors/culture insert), but they do not cantly reduced number of basal layer melanocytes,
reproduce the tridimensional interactions of cells whereas the presence of vitiligo keratinocytes
of the EMU. Epidermal reconstructs, which repro- enhances this effect. Similarly, reconstructs made
duce the EMU and basal membrane attachment, with melanocytes silenced for CCN3 have less
are thus a handful “in vivo-like” model. Indeed, basal m elanocytes and more detached melano-
reconstructions of different levels of complexity cytes than their normal counterpart (Ricard et al.
can be prepared (reconstructs with keratinocytes 2012). Vitiligo sera may induce melanocyte
alone, keratinocytes and melanocytes, keratino- detachment independently of the disease activity
cytes, melanocytes, and fibroblasts), including chi- or extent. Hydrogen peroxide induces melanocyte
meric reconstructs (normal vs. pathological cells detachment in reconstructs containing vitiligo
or genetically modified cells). According to the melanocytes or melanocytes silenced for
initial reflection, the analysis of the behavior of E-cadherin (Wagner et al. 2015) and normal kera-
melanocytes and keratinocytes is done in a more tinocytes, but not in normal controls. Finally, epi-
physiological environment than that of monolayer nephrine, but not norepinephrine, allows
cultures and cocultures and allows to test conve- melanocyte detachment. Epidermal reconstructs
niently compounds which are suspected to be are useful to address several research questions
implicated in vitiligo etiology or susceptible to such as: Is the melanocyte primarily affected? Is
improve the attachment and survival of melano- the cellular environment important (keratinocytes,
cytes upon the basal layer. An example of epider- fibroblasts)? Are other (soluble) factors implicated
mal reconstructs tested with epinephrine, in the development of vitiligo? However, this
norepinephrine, dopamine, hydrogen peroxide, or model has some limitation since we have not yet
vitiligo sera is illustrated in Fig. 23.3. Our main been able to introduce, for instance, Langerhans
results using this model [23] were the following: cells or other immune cells to study their implica-
reconstructs made with melanocytes from non- tion in vitiligo etiology; the study of topical mole-
lesional generalized vitiligo skin have a signifi- cules to improve vitiligo treatment is less easy than
that of soluble factors and that long-term studies
(more than 3 weeks) are not possible, since there is
a no renewal of the basal layer. Moreover, the analy-
sis of intracellular signaling and membrane assess-
ment per cell can be just on single-cell-type
culture, when cell sorting is not available.
23.4 Analytic Techniques

b
23.4.1 Fluorescence-Based Assays
Fluorochrome-conjugated monoclonal or poly-

clonal antibodies are widely applied to visualize
and quantify the expression of some surface or
internal melanocyte markers [20, 38–40]. The
Fig. 23.3 Detection of melanocyte in genetically modi- first data were derived by immunohistochemistry
fied reconstructed epidermis. (a) Detachment of melano-
methods on frozen and paraffin-embedded sec-
cyte (red) in reconstructed epidermis made with
melanocytes silenced for E-cadherin. Double staining of tions, followed by fluorescence and confocal
cadherin (green) and melanocyte (red). Detached melano- microscopy on slide-cultured cells. Phenotypic
cyte (arrow). (b) Detachment of melanocyte silenced for characterization of cultured vitiligo melanocytes
CCN3 (green with absence of red nuclear staining)
with respect to control cells was carried out. A
(arrow) and basal location of CCN3-positive melanocyte
(green with red nuclear staining) (arrow head). Double reduced expression of c-Kit and ET-1 receptors,
staining of CCN3 (red) and melanocyte (green) tyrosinase, and MITF was found in vitiligo
ERRNVPHGLFRVRUJ
melanocytes with pattern progressively varying FNS further reduces the amount of sample needed
from the edge of the white spot to the non-lesional for the analysis. The SBC is a significant advance
area [39]. The flow cytometric approach permits a research in the measurement of short-lived pro-
quali-quantitative multiparametric and time- cesses in adherent cells and small samples, two
dependent analysis. Membrane and intracellular crucial features of vitiligo samples. New light
staining together with the analysis of the physical microscope architecture is provided by iMIC,
and time parameters permits the structural and which integrates time-lapse studies, FRET mea-
functional characterization of the melanocytes surements, laser microdissection, and slit-scan
and their sorting for further cultures. The current confocal measurements within coherent illumina-
flow cytometers can analyze up to 16 parameters. tion (340–680 nm).
Besides the antibody-based approach, the cytomic
one has been used to detect in vitiligo melano-
cytes the intracellular ROS production (DCFH-DA 23.4.2 Proteomic
or dhRho123 staining), the membrane lipo-perox-
idation [41] (BODIPY581/591 staining), and the Mass spectrometry in conjunction with free-flow
content and the transmembrane cardiolipin distri- electrophoresis of sucrose density gradient has
bution (NAO fluorescence pattern) [24]. The flow allowed the identification of early-stage melano-
cytometer may be also used for fluorescence reso- some proteins [44]. Two-dimensional differential
nance energy transfer (FRET) analysis. Cells are image-gel electrophoresis (2D-DIGE) and liquid
stained with nonyl acridine orange (NAO) (donor) chromatography tandem-mass spectrometry (LC-
and MitoTracker Orange, a dye with high affinity MS/MS) allow the analysis and identification of
for mitochondrion proteins and voltage sensible the proteic components of the organelles of mela-
(acceptor). The mitochondrial mass and the polar- nocytes with melanosomes at different maturation
ization state of the inner mitochondrial membrane stages [45]. Calreticulin, a soluble Ca2+-binding
can be monitored using FRET index based on chaperone protein, is involved together with cal-
NAO FL1 (green fluorescence) decrease and NAO nexin in the folding of newly synthesized proteins
FL2 (red fluorescence) increase from single to and glycoproteins (including tyrosinase) and for
double-labeled tubes [42]. The limitation associ- quality control pathways in endoplasmic reticu-
ated with flow cytometry is that it is sample con- lum. The LC-MS/MS analysis has highlighted
suming. Several new approaches, based on “fluo that calreticulin expression is dependent on the
world,” are now available. Most of these innova- maturation stage of melanocytes. Even if the pro-
tive technologies have not yet been applied to the teomic assay has been so far carried out on murine
study of vitiligo, but they open promising per- healthy melanocytes, the scenario possibly
spectives. The laser scanning cytometer (LSC) designed by this approach may be crucial for the
permits slide-based cytometry (SBC) and the maturation process leading from nonpigmented
hyperchromatic approach. Its potential applica- melanocytes to pigmented melanocytes in vitil-
tion in vitiligo study arises from its intrinsic igo. Starting from the consideration of vitiligo as
features: nonconsumptive (unlike the flow cytom- metabolic multifactorial disease, an analysis of
eter), iterative restaining, differential photo- the systemic proteomic profile will support its
bleaching (fluorochromes differentiated on the characterization. The proteomic study of the body
basis of their specific photostability), and photo- fluids (plasma, serum, saliva) should start from
activation (for nanoparticles or photo-caged the preliminary remotion of albumin and other
dyes). A single cell can be reanalyzed, whereas major represented proteins, also taking into
the information gained per specimen is only lim- account how and when removing them. The dif-
ited by the number of available antibodies and ference in subcellular derivation (plasma mem-
sterical hindrance [43]. The LSC when combined brane, cytoplasm, mitochondria) has been related
with fine-needle sampling (FNS) may be used to to the nature/severity of the stimulus. Innovative
monitor the cell structural and functional modifi- or traditional disease markers should be as possi-
cations subsequent to in vitro treatment, where ble tissue- and disease- specific. An excellent
ERRNVPHGLFRVRUJ
database reference is provided by the Human metabolites to correlate their changes with patho-
Protein Atlas (HPA), also including data on logical conditions or with drug intake. Accordingly,
mRNA coding for the same proteins. The albumin metabolomics is one of the building blocks of sys-
depletion can be carried out by 2DE with IEF or tems biology. There are two general analytical
sequential precipitation steps. Major plasma/ approaches in metabolomics analysis: targeted and
serum proteins can be removed, in order to untargeted. Whereas the targeted approach considers
improve the sensitivity of proteomic protocols, by a well-defined and known set of metabolites, the sec-
hydrophobic interaction chromatography (HIC) ond one aims to define the quantitative modification
before 2DE. Different companies provide vali- among the analyzed populations (fold change) with-
dated columns, including Agilent, R&D Systems, out standard references and allows the study of
and Sigma. A crucial question should be answered: unidentified metabolites. The untargeted approach is
that related to the cost-effectiveness ratio of the also called metabolite fingerprinting. However, it is
devices. An alternative perspective to depletion of relevant to remember that according to the different
high-abundance proteins is the enrichment of used instrument and method, the final metabolite
low-abundance ones by means of their relative spectra may change. In any case metabolomic study
concentration, according to a process named generates a complex set of data requiring specialized
“protein equalization” and based on saturable and analysis based on cheminformatics, bioinformatics,
specific interaction to a high diversity binding and statistics expertise.
sites present on chromatographic beads. The As regards the specific vitiligo application,
library provides dozen of millions of hexapep- metabolomic research will provide a fingerprint-
tides able to interact with most of proteins in any ing of the metabolic activities related to immune
proteome. The analysis can be performed by deregulation and degenerative process, possibly
SELDI. Some protein modification such as glyco- merging the vitiligo profile with those known and
sylation correlates with specific pathological con- described in other diseases and health population.
ditions, and differently glycosylated proteins can The separation techniques are based on gas chro-
be identified on the basis of the pI and Mr on 2DE matography, high-performance liquid chroma-
assay. During inflammatory process some featur- tography, and capillary electrophoresis; the
ing proteins are released in the plasma/serum detection techniques usually are done by mass
according to acute and chronic phase of the spectrometry or nuclear magnetic resonance.
inflammation itself. The summary of the most rel- Each approach shows different and specific sen-
evant inflammatory markers is provided by sitivity and methodological limitations. Mass
Cytokine & Cells Online Pathfinder Encyclopedia spectrometry is the most widely used approach in
(COPE) website at www.copewithcytokines.org. tandem with chromatography separation method;
An interesting integration to the above atlas is it is sensitive and selective [48].
provided by http://wallace.uab.es/multitask pro- Serum lipidomics is a global profiling of lipid
viding database collecting hundreds of character- molecular species. Lipidomics, defined as the
ized moonlighting multitasking proteins, involved large-scale study of pathways and networks of
in different functions [46]. Recently, proteomic cellular lipids, is an emerging and rapidly expand-
approach was applied to formalin-fixed paraffin- ing research field. It is often necessary to evaluate
embedded tissue [47]. a wide range of molecular species and lipid
classes to gain insights into pathophysiology. In
the future, research in lipidomics will expand to
23.4.3 Metabolomics/Lipidomics include interactions of lipids with lipids, pro-
teins, and other cellular components. Mass spec-
Metabolomics is an emerging field of biological sci- troscopy, nuclear magnetic resonance, and
ence, which considers the highthroughput character- fluorescence spectroscopy have played a crucial
ization of small compounds (>1500 Da) representing role in lipid characterization, identification, and
the final products of cellular metabolism and mirror- quantization [49]. The study of vitiligo pathogen-
ing the chemical fingerprint of an organism at a pre- esis should actually gain a relevant burst from the
cise point. It aims to identify and quantify the metabolomics approach.
ERRNVPHGLFRVRUJ
23.4.4 Transcriptomic 5. Na GY, Paek SH, Park BC, et al. Isolation and
characterization of outer root sheath melano-
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Recently, oligonucleotide-based microarrays have 2006;155:902–9.
been used to explore the pattern of gene expression 6. Vennegor C, Hageman P, Van Nouhuijs H, et al. A
of vitiligo melanocytes. Interestingly, the most monoclonal antibody specific for cells of the melano-
cyte lineage. Am J Pathol. 1988;130:179–92.
upregulated genes are related to the network of 7. Ricard AS, Pain C, Daubos A, et al. Study of CCN3
endosome and lysosome organelles. The next step (NOV) and DDR1 in normal melanocytes and vitiligo
was to analyze the various clusters of the differen- skin. Exp Dermatol. 2012;21:411–6.
tially expressed genes. This approach can represent 8. Wagner RY, Luciani F, Cario-André M, et al. Altered
E-cadherin levels and distribution in melanocytes
the basis for further in-depth analyses to better precede clinical manifestations of vitiligo. J Invest
clarify the complex vitiligo pathomechanisms [50]. Dermatol. 2015;135:1810–9.
9. Caliari SR, Burdick JA. A practical guide to hydro-
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10. Dell’Anna ML, Maresca V, Briganti S, et al.

23.5 Concluding Remarks Mitochondrial impairment in peripheral blood mono-
nuclear cells during the active phase of vitiligo. J
Cell culture models are useful to investigate the 11. Donmez-altuntas H, Sut Z, Ferahbas A, et al. Increased
differences between normal and vitiligo cells and micronucleus frequency in phytohaemagglutinin-
to test potential treatment options in a cell-focusing stimulated blood cells of patients with vitiligo. J Eur
approach. New analytic techniques using a limited Acad Dermatol Venereol. 2008;22:162–7.
12. Giovannelli L, Bellandi S, Pitozzi V, et al. Increased
amount of biological material are very promising. oxidative DNA damage in mononuclear leukocytes in
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therapies using unmodified patient’s cells, the 13. Prunieras M, Regnier M, Schlotterer M. [New proce-
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17. Lee DY, Lee JH, Yang JM, et al. A new dermal

allow the development of vitiligo models using equivalent: the use of dermal fibroblast culture
normal cells bypassing the difficulty to culture vit- alone without exogenous materials. J Dermatol Sci.
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Genetics
24
Richard A. Spritz
Contents
24.1 Genetic Epidemiology 238
24.2 I dentification of Vitiligo Susceptibility Genes 240
24.2.1 The Candidate Gene Approach 240
24.2.2 The Genome-Wide Approach 241
24.3 Opposite Genetic Relationship Between Vitiligo and Melanoma 246
24.4 Current Understanding 246
References 249
Abstract ence has proven that the only analytic

Large-scale epidemiological surveys have approach that produces verified discovery of
shown that most cases of vitiligo occur spo- bona fide disease genes is the genome-wide
radically, though about 15–20% of patients approach. Retrospective analyses of candi-
report one or more affected relatives. The date gene studies have shown that the vast
rationale for genetic studies of vitiligo sus- majority of claimed candidate gene associa-
ceptibility is that underlying genes are tions represent false-positives. Accordingly,
involved in mediating disease causation, the candidate gene approach is no longer con-
either increasing or decreasing risk (protec- sidered valid for de novo disease gene discov-
tive). Three different general approaches have ery and is reserved for confirmatory studies
been used to identify genes that mediate vit- only. Similarly, almost all genes that exhibit
iligo susceptibility: the candidate gene major expression differences between disease
approach, the genome-wide approach, and the and non-disease states turn out to not corre-
gene expression approach. Extensive experi- spond to causal genes, but instead represent
secondary effects, and thus likewise gene
expression studies have generally not led to
R. A. Spritz (*) the discovery of genes that are causal for com-
Human Medical Genetics and Genomics Program,
University of Colorado School of Medicine, plex diseases. Indeed, none of the genes ini-
Aurora, CO 80045, USA tially suggested on the basis of the expression
Department of Pediatrics, University of Colorado approach now appear to be involved in vitiligo
School of Medicine, Aurora, CO 80045, USA causation at all. In contrast, genome-wide
e-mail: Richard.Spritz@ucdenver.edu

ERRNVPHGLFRVRUJ
238 R. A. Spritz
genetic analyses, particularly genome-wide

association studies (GWAS), have proven a iligo might represent a dysregulated
remarkably robust approach to disease gene mechanism of immune surveillance for
discovery, yielding findings that are highly melanoma.
reproducible and which, in aggregate, have • Vitiligo is epidemiologically associ-
provided dramatic advances in understanding ated with a number of other autoim-
the biological basis of many different com- mune diseases, both in vitiligo patients
plex diseases, including vitiligo. Reported themselves and in their close relatives.
candidate gene associations and expression This epidemiologic association has a
difference findings that are not observed in genetic basis, as many vitiligo suscep-
well-powered GWAS of the same population tibility genes have also been shown to
are not now considered to be valid indications contribute to these other autoimmune
of disease-causal genes and thus will not be diseases.
discussed here.
Key Points
24.1 Genetic Epidemiology
• Several different approaches have been
Large-scale epidemiological surveys have
taken to identify vitiligo susceptibility
shown that most cases of vitiligo occur sporadi-
genes. It has become clear that the most
cally, though about 15–20% of patients report
reliable method to identify bona fide
one or more affected relatives [1]. Rarely, large
causal disease genes for “complex dis-
multi-generation families segregate vitiligo in
eases,” such as vitiligo, is the genome-
patterns that suggest autosomal dominant [2] or
wide association study (GWAS), which
autosomal recessive [3] inheritance with incom-
has become the “gold standard” for dis-
plete penetrance. More typically, however,
ease gene identification. In contrast, the
familial aggregation of vitiligo cases occurs in a
great majority of genes suggested on the
non-Mendelian pattern. Hafez [4] and Das [5, 6]
basis of candidate gene studies have
suggested polygenic, multifactorial inheritance
proven to be false-positives.
and estimated vitiligo heritability at 46% [6] to
• To date, approximately 50 vitiligo sus-
72% [4]. Subsequent analyses similarly sug-
ceptibility genes have been identified by
gested polygenic, multifactorial inheritance [1,
GWAS. These genes encode a network
7–13], with approximately 50% heritability
of proteins that regulate aspects of the
[13]. In general, the frequency of vitiligo is
immune system, the cellular apoptosis,
approximately equal in males and females,
the melanocyte, and the interface
when the female sex bias present in most vitil-
between the melanocyte and the immune
igo clinical case series is controlled for appro-
system.
priately [14].
• Vitiligo susceptibility genes that
Strong evidence for genetic factors in the
encode melanocyte components or
pathogenesis of vitiligo comes from studies of
melanocyte regulators have also been
patients’ close relatives. Among Caucasians of
associated with melanoma, involving
European origin (EUR), the risk of vitiligo to a
exactly the same genetic variants but
patient’s siblings is about 6.1% [1], a 16-fold
the opposite alleles. This genetically
increase over the approximately 0.38% preva-
opposite relationship suggests that vit-
lence of vitiligo in at least one EUR population
ERRNVPHGLFRVRUJ
24 Genetics 239
[15]. The overall risk of vitiligo to patients’ autoimmune diseases and that this association
first-degree relatives is 7.1% in EUR, 6.1% in has a genetic basis. Vitiligo is a component of
Indo-Pakistanis, and 4.8% in USA Hispanic/ the APECED (APS1; OMIM #240300) and
Latinos [1], with lower risks to more distant “Schmidt” (APS2; OMIM %269200) multiple
relatives. Generally similar results have come autoimmune disease syndromes (additional
from studies of Han Chinese (CHN) families suggested “autoimmune polyendocrine syn-
[13]. Furthermore, in a study of vitiligo in drome” categories [17] are not widely
EUR monozygotic twins, the concordance for accepted). More importantly, about 10–20% of
vitiligo was 23% [1], more than 60 times the vitiligo patients develop other concomitant
general population risk of 0.38%, and almost autoimmune diseases, as first observed by
four times the 6.1% risk of vitiligo to pro- Thomas Addison in 1855 [18]. Many retro-
bands’ siblings, providing additional strong spective studies have shown that vitiligo is epi-
support for a complex genetic basis of vitiligo demiologically associated with increased
risk. prevalence of autoimmune thyroid disease [1,
Additional evidence for a genetic component 19, 20], adult-onset type 1 diabetes [1], sys-
to vitiligo susceptibility comes from age-of- temic lupus erythematosus [1], pernicious ane-
onset data: among unselected (mostly sporadic) mia [1, 21, 22], Addison’s disease [1, 23], and
EUR vitiligo patients, the mean age of vitiligo perhaps alopecia areata [24, 25]. These same
onset is 24.2 years [16], but among patients in diseases also occur at increased frequency in
families with multiple relatives affected by vit- vitiligo patients’ first-degree relatives, regard-
iligo, the mean age of onset is significantly ear- less of whether or not those relatives have vit-
lier, 21.5 years [14]. Earlier age of disease onset iligo themselves [1]. Families with multiple
in more “familial” cases and diminishing dis- cases of vitiligo have even higher frequencies
ease risk with increasing genetic distance from a of these autoimmune diseases, in both vitiligo
vitiligo case are typical characteristics of a patients and their siblings [14], indicating even
polygenic disorder, and formal genetic segrega- greater genetic susceptibility to autoimmune
tion analyses have indicated that vitiligo is a diseases in such “multiplex” families than in
complex trait, with multiple contributory genetic typical singleton vitiligo patients. Generally
loci [10, 12, 13]. similar results have come from retrospective
While epidemiologic and twin studies thus studies of vitiligo patients in India [26, 27] and
indicate that genes play an important role in vitil- Nigeria [28].
igo pathogenesis, nongenetic, environmental Taken together, these findings suggest that
triggers must also be very important. Identical pathologic variants in specific genes predis-
twins share all of their genes, and incomplete pose to a specific subset of autoimmune dis-
heritability, limited twin concordance and typical eases that includes vitiligo, autoimmune
adult onset underscore the apparent role of envi- thyroid disease, rheumatoid arthritis, adult-
ronmental triggers. While many different environ- onset autoimmune diabetes mellitus, perni-
mental risk factors for vitiligo have been suggested, cious anemia, systemic lupus erythematosus,
epidemiologic data definitively supporting a role Addison’s disease, and perhaps others
any of these in typical vitiligo remain very limited. (Fig. 24.1). As described below, large-scale
Many experts believe that skin damage genetic studies have borne this out, as many
(Koebnerization) perhaps with low-level infection susceptibility genes are shared among these
may be a disease trigger in many cases. autoimmune diseases, in different combina-
Epidemiologic studies have also shown that tions, sometimes involving the same high-risk
vitiligo is strongly associated with other gene variants.
ERRNVPHGLFRVRUJ
240 R. A. Spritz
Autoimmune
thyroid
disease Lupus
Vitiligo Specific
Specific
genes,
genes,
triggers
triggers
Specific
genes,
triggers
Rheumatoid
Type 1 arthritis
Shared
diabetes genes
Specific
genes,
Specific
triggers
genes, Pernicious
Addison’s
triggers anemia
disease
Fig. 24.1 Shared genetic relationships among epidemi- vitiligo patients and their first-degree relatives (even
ologically associated autoimmune diseases. Elevated those without vitiligo) suggests that these autoimmune
prevalence of autoimmune thyroid disease, rheumatoid diseases share some susceptibility genes that increase
arthritis, adult-onset autoimmune diabetes mellitus, per- risk to all or some of these diseases, as well as disease-
nicious anemia, systemic lupus erythematosus, Addison’s specific susceptibility genes and likely environmental
disease, and perhaps other autoimmune diseases in both triggers
24.2 Identification of Vitiligo sion studies have generally not led to the discovery
Susceptibility Genes of genes that are causal for complex diseases.
Indeed, none of the genes initially suggested on
The rationale for genetic studies of vitiligo suscep- the basis of the expression approach now appear to
tibility is that underlying genes are involved in be involved in vitiligo causation at all. In contrast,
mediating disease causation, either increasing or genome- wide genetic analyses, particularly
decreasing risk (protective). Three different gen- genome-wide association studies (GWAS), have
eral approaches have been used to identify genes proven a remarkably robust approach to disease
that mediate vitiligo susceptibility: the candidate gene discovery, yielding findings that are highly
gene approach, the genome-wide approach, and reproducible and which, in aggregate, have pro-
the gene expression approach. Extensive experi- vided dramatic advances in understanding the bio-
ence has proven that the only analytic approach logical basis of many different complex diseases,
that produces verified discovery of bona fide dis- including vitiligo [31, 32]. Reported candidate
ease genes is the genome- wide approach. gene associations and expression difference find-
Retrospective analyses of candidate gene studies ings that are not observed in well-powered GWAS
have shown that the vast majority of claimed can- of the same population are not now considered to
didate gene associations represent false-positives be valid indications of disease-causal genes and
[29, 30]. Accordingly, the candidate gene approach thus will not be discussed here.
is no longer considered valid for de novo disease
gene discovery and is reserved for confirmatory 24.2.1 The Candidate Gene
studies only. Similarly, almost all genes that Approach
exhibit major expression differences between dis-
ease and non-disease states turn out to not corre- Candidate gene studies typically test for nonran-
spond to causal genes, but instead represent dom genetic association of specific DNA
secondary effects, and thus likewise gene expres- sequence variants in specific genes thought to
ERRNVPHGLFRVRUJ
24 Genetics 241
perhaps be involved in susceptibility to vitiligo in multiple different ethnic populations around

on the basis of a priori biological hypotheses. the world. Moreover, genetic associations of vit-
While the variants tested are usually unlikely to iligo with HLA class I and class II alleles appear
be causal for disease, they are assumed to at least to be particularly important in patients from fam-
be in linkage disequilibrium with true pathologi- ilies with various other vitiligo-associated auto-
cal variants. The candidate gene approach obvi- immune diseases, many of which are themselves
ously is limited to testing hypotheses involving associated with genetic variation in the MHC
already known biological candidate genes; it can- class I or class II gene regions.
not discover completely novel genes or pathways. The first genetic association of vitiligo outside
The most common study design is the case- of the MHC was reported by Kemp and cowork-
control analysis, in which allele or genotype fre- ers [42] in the candidate gene CTLA4, which
quencies are assayed and compared between encodes a T-cell co-receptor involved in regula-
ethnically matched unrelated cases and ethnically tion of T-cell activation. CTLA4 is also associated
matched unrelated controls. While the case- with several other autoimmune diseases that are
control study design seems simple, this approach epidemiologically associated with vitiligo, and
is highly subject to false-positive errors due to indeed, CTLA4 association is strongest in vitiligo
imperfect ethnic matching of cases versus con- cases with other concomitant autoimmune dis-
trols, population admixture stratification, inade- eases [43, 44]. While vitiligo association with
quate statistical power and statistical fluctuation, CTLA4 is inconsistent in non-EUR populations,
and inadequate correction for multiple testing. this association has been confirmed by GWAS in
Retrospective analysis of published “genetic EUR [45].
associations” has shown that over 95% represent The only other valid candidate gene associa-
such false-positives, complicated by bias toward tion for vitiligo was also reported by Kemp and
publishing apparently positive results [29, 30]. coworkers [46], with PTPN22, which encodes
Conversely, most published negative genetic LYP protein tyrosine phosphatase. Like CTLA4,
association studies fail to consider the statistical PTPN22 also shows genetic association with
power of the analysis to detect a genetic effect of many different autoimmune diseases. Again,
a given magnitude, which must be considered in genetic association of PTPN22 with vitiligo in
evaluating a negative result. the EUR population has been observed in many
Only three of the very many published candi- other studies [47, 48], and has been confirmed by
date gene associations reported for vitiligo now GWAS, but has not been observed in most studies
appear to be valid: HLA, PTPN22, and CTLA4. of non-EUR populations. Thus, HLA class II,
The earliest genetic studies of vitiligo were case- CTLA4, and PTPN22 are key loci that underlie
control genetic association analyses of genes in both genetic susceptibility to vitiligo and also
the major histocompatibility complex (MHC), epidemiologic association of vitiligo with other
carried out by typing various MHC markers in autoimmune diseases, at least in the EUR popula-
patients with various different vitiligo pheno- tion (Fig. 24.2).
types versus in controls, from many different
populations [11, 33–41]. In general, these studies
have found no consistent association between the 24.2.2 The Genome-Wide Approach
occurrence of vitiligo and specific HLA alleles.
However, Foley and coworkers [36] found asso- Genome-wide genetic approaches scan the entire
ciation between vitiligo and class II HLA-DR4 genome to identify genetic markers that flag
(now known to represent HLA-DRB1*04 alleles), genomic regions that may contain disease sus-
the first true genetic association reported for vit- ceptibility genes. Because genome-wide
iligo. Subsequently, a meta-analysis of multiple approaches are based on genomic location only,
studies found association of vitiligo with class I findings are not limited by known (or surmised)
HLA-A2 [37]. These associations have subse- biology, and these approaches thus offer the pos-
quently been validated by robust GWAS analyses sibility of discovering entirely new disease
ERRNVPHGLFRVRUJ
242 R. A. Spritz
Fig. 24.2 Shared genetic associations of vitiligo with associations based solely on candidate gene association
other autoimmune diseases and with pigmentation and studies are not included. AI hepatitis, autoimmune hepati-
melanoma phenotypes. Blue circles denote genetic asso- tis; AITD, autoimmune thyroid disease (Graves’ disease
ciations shared between vitiligo and other autoimmune and Hashimoto thyroiditis); IBD, inflammatory bowel
diseases. Red circles denote genetic associations shared disease; MG, myasthenia gravis; MS, multiple sclerosis;
between vitiligo and normal pigmentary variation pheno- RA, rheumatoid arthritis; SLE, systemic lupus erythema-
types, as well as malignant melanoma. All associations tosus; T1D, type 1 diabetes mellitus. Reprinted from [32]
shown were identified by GWAS and meet standard crite- with permission
rion for genome-wide significance (P < 5 × 10−8); claimed
susceptibility genes that highlight entirely new genomes of large numbers of such typical single-
pathways to disease. Moreover, genome-wide ton cases, comparing allele or genotype frequen-
approaches allow for measurement of and control cies at hundreds of thousands or millions of
for underlying population stratification and other so-called single-nucleotide polymorphisms
biases and for appropriate correction for multiple (SNPs) across the genome versus in large collec-
testing and thus are far less subject to false- tions of controls. GWAS thus are well-suited to
positive artifacts than are other approaches to dis- analyses of typical cases and predominantly
ease gene discovery. detect relatively common disease susceptibility
There are two quite different genome-wide genes and variants, though approaches to detect
approaches to disease gene discovery, which are uncommon variants are improving rapidly.
applicable in different circumstances and which GWAS have been particularly fruitful in studies
likely highlight different types of genetic loci and of vitiligo, which has proven highly tractable to
underlying causal variants. Genome-wide linkage this powerful approach [31, 32].
studies compare the genomes of affected mem-
bers of “multiplex families” with multiple cases 24.2.2.1 Genome-Wide Linkage
of a given disease, versus relatives without the Studies
disease. Such families are not typical, and indeed The first genome-wide findings relevant to vitil-
they may segregate relatively rare genetic vari- igo came from genetic linkage studies of sys-
ants that have large effects, perhaps different than temic lupus erythematosus that detected a locus
those in more typical singleton cases. Genome- on chromosome 17p13, called SLEV1, in EUR
wide association studies (GWAS) analyze the multiplex lupus families that included at least one
ERRNVPHGLFRVRUJ
24 Genetics 243
relative with vitiligo [49]. SLEV1 was subse- tion in transfected permissive cells by 50% and
quently confirmed by linkage analysis of EUR in vivo might thus interfere with melanoblast/
multiplex vitiligo families with various other melanocyte differentiation or survival, predispos-
autoimmune diseases [50], and fine-mapping in ing to vitiligo [16].
vitiligo families ultimately identified the underly- Genome-wide linkage analyses of smaller
ing gene as NLRP1 (previously termed NALP1) multiplex vitiligo families in both EUR and CHN
[51, 52]. NLRP1 is a key regulator of the innate populations detected a number of additional link-
immune system, particularly Langerhans cells age signals, for most of which specific corre-
and dendritic cells. In response to bacterial sponding genes have not yet been identified. In
“pathogen-associated molecular patterns” EUR families, in addition to NLRP1 on chromo-
(PAMPs), possibly including muramyl dipeptide some 17p, additional vitiligo linkage signals
[53], NALP1 directs assembly of a “NLRP1- were detected on chromosomes 7p13–q21
inflammasome,” which then activates the (termed AIS2), 8p12 (termed AIS3), 9q22, 11p15,
interleukin-1β (IL-1β) inflammatory pathway 13q33, 19p13, and 22q11 [57, 58]. In CHN fami-
[54], which recruits responses by the adaptive lies, genetic linkage studies detected a different
immune system. Next-generation DNA sequenc- set of linkage signals, at chromosomes 1p36,
ing has shown that the most common high-risk 4q13-q21, 6p21-p22, 6q24-q25, 14q12-q13, and
NLRP1 haplotype carries multiple amino acid 22q12 [59, 60]. Association analysis of several
substitution variants, resulting in enhanced acti- candidate genes within the 22q12.1-q12.3 link-
vation of IL-1β in both basal and stimulated age peak suggested that the corresponding gene
states [55]. Together, these findings suggest that may be XBP1 [61], which encodes a transcription
activation of the IL-1β pathway in response to factor important in immune cells. The remaining
bacterial components sensed by skin-resident vitiligo linkage signals have not yet been
innate immune receptors might be one trigger for specifically identified, and it remains uncertain
vitiligo. which represent true vitiligo susceptibility loci
An additional genome-wide linkage study versus false-positives.
investigated a unique, very large, multi-
generation EUR family in which vitiligo and 24.2.2.2 Genome-Wide Association
other autoimmune diseases were inherited as an Studies (GWAS)
apparent autosomal dominant trait with Without question, the most effective approach to
incomplete penetrance. Vitiligo in this family identifying susceptibility genes for vitiligo has

was mapped to a locus termed AIS1, in chromo- been the GWAS approach [31, 32], as has like-
some segment 1p31.3-p32.2, whereas suscepti- wise been the case for almost all other complex
bility to other autoimmune diseases in the context diseases. To date, GWAS have identified approxi-
of a co-inherited AIS1 mutation was mapped to a mately 50 chromosomal loci that appear to be
region of chromosome 6 that included the MHC involved in vitiligo pathogenesis, with high
[2]. Detailed studies of genes in the AIS1 region reproducibility between studies, both within and
of chromosome 1p in this family identified a proin some instances even across major ethnic cate-
moter variant in FOXD3 that increases activity of gories. About two-thirds of these chromosomal
the FOXD3 transcriptional promoter by 50% loci have been resolved to specific genes, and for
[16]; recently, another FOXD3 promoter variant about half of those, the specific causal sequence
associated with vitiligo has also been found that variants and pathobiology have been elucidated,
increases FOXD3 transcription [56]. FOXD3 is providing a working framework of vitiligo patho-
an embryonic transcription factor that regulates genesis for the first time.
differentiation and development of neural crest The first GWAS of vitiligo was carried out in
melanoblasts and some mesodermal elements, a EUR “special population,” an isolated village in
including pancreatic islet cells. The FOXD3 pro- a mountainous region of Romania in which there
moter variant in this family increases transcrip- is a remarkably high prevalence of vitiligo, as
ERRNVPHGLFRVRUJ
244 R. A. Spritz
well as some of the other autoimmune diseases iligo conducted in the EUR population [45,
with which vitiligo is epidemiologically associ- 63–65], as well as another carried out in CHN
ated [3]. This GWAS identified significant asso- [66]. Interestingly, this locus is also in close
ciation with a SNP located on distal chromosome proximity to IDDM8, a linkage and association
6q, which in the context of this relatively low- signal reported in genetic analyses of both type 1
resolution GWAS was interpreted as representing diabetes mellitus and rheumatoid arthritis.
SMOC2 [62]. In fact, however, this SNP is located Indeed, analyzed together in meta-analyses,
immediately adjacent to a robust vitiligo associa- the three GWAS of vitiligo conducted in EUR
tion signal in the RNASET2-FGFR1OP-CCR6 have detected a total of 48 confirmed vitiligo
region detected in three subsequent GWAS of vit- susceptibility loci [45, 63–65] (Table 24.1).
Table 24.1 Vitiligo susceptibility genes identified and confirmed by GWAS

Chromosome Gene or locus Protein Function
1p36.23 RERE Arginine-glutamic acid dipeptide Regulator of apoptosis
repeats
1p13.2 PTPN22 Protein tyrosine phosphatase, Alters responsiveness of T-cell receptors
nonreceptor type 22
1q24.3 FASLG FAS ligand Regulator of immune apoptosis
1q31.3-q32.1 PTPRC Protein tyrosine phosphatase, Regulator of T- and B-cell antigen
receptor type C receptor signaling
2p16.1 PPP4R3B Protein phosphatase 4, regulatory Unknown
subunit 3B
2q13 BCL2L11 BCL2-like 11 Regulator of apoptosis in thymocyte
negative selection
2q24.2 IFIH1 Interferon induced with helicase C Innate immune dsRNA receptor
domain 1
2q33.2 CTLA4 Cytotoxic T-lymphocyte-associated T-lymphocyte checkpoint regulator
protein 4
2q37.3 FARP2-STK25 ? ?
3p24.3 UBE2E2 Ubiquitin-conjugating enzyme E2 Protein ubiquitination pathway; damage
E2 response
3p13 FOXP1 Forkhead box protein P1 Transcriptional regulator of B-cell
development
3q13.33 CD80 T-lymphocyte activation antigen T-cell costimulatory signal
CD80
3q27.3-q28 LPP Lipoma-preferred partner Unknown
3q29 FBXO45-NRROS ? ?
4q24 PPP3CA Serine/threonine protein T-cell calcium-dependent, calmodulin-
phosphatase 2B catalytic subunit stimulated protein phosphatase
alpha isoform
6p25.3 IRF4 Interferon regulatory factor 4 Transcriptional activator in immune cells
and melanocytes
6p25.2 SERPINB9 Serpin B9 Endogenous inhibitor of granzyme B
Association
6p21.3 HLA-A HLA class I histocompatibility Presents peptide antigens to the immune
antigen, A system
6p21.3 HLA-DRB1— HLA histocompatibility antigens, Present peptide antigens to the immune
HLA-DQA1 DRB1 and DQA1 system
6q15 BACH2 BTB domain and CNC homolog 2 Transcriptional activator regulator of
apoptosis
6q27 RNASET2- ? ?
FGFR1OP-CCR6
7p14.3 CPVL Probably serine carboxypeptidase Inflammatory protease; trims antigens for
CPVL presentation
ERRNVPHGLFRVRUJ
24 Genetics 245
Table 24.1 (continued)
Chromosome Gene or locus Protein Function
8q24.22 SLA Src-like adapter Regulator of T-cell antigen receptor
signaling
9q33.3 NEK6 NIMA-related serine/threonine Regulator of apoptosis
protein kinase NEK6
10p15.1 IL2RA Interleukin-2 receptor subunit alpha IL2 receptor regulates regulatory T-cells
10q21.2 ARID5B AT-rich interactive domain- Transcriptional coactivator
containing protein 5B
10q22.3 ZMIZ1 Zinc finger MIZ domain-containing Possible PIAS-family transcriptional or
protein 1 sumoylation regulator
10q25.3 CASP7 Caspase7 Apoptosis executioner protein
11p13 CD44 CD44 antigen Regulator of FOXP3 expression
11q13.1 PPP1R14B- ? ?
PLCB3-BAD-
GPR137-KCNK4-
TEX40-ESRRA-
TRMT112-PRDX5
11q14.3 TYR Tyrosinase Melanocyte melanogenic enzyme; vitiligo
autoantigen
11q21 Gene desert ? ?
12q13.2 PMEL Premelanosome protein PMEL Melanocyte melanosomal type 1
Expression analysis transmembrane glycoprotein
12q13.2 IKZF4 Zinc finger protein Eos Transcriptional repressor; regulates
FOXP3 transcription in regulatory T-cells
12q24.12 SH2B3 SH2B adapter protein 3 Links T-cell receptor activation signal to
phospholipase C-gamma-1, GRB2, and
phosphatidylinositol 3-kinase
13q14.11 TNFSF11 Tumor necrosis factor ligand T-cell cytokine that binds to TNFRSF11A
superfamily member 11 and TNFRSF11B
14q12 GZMB Granzyme B Apoptosis executioner protein of cytotoxic
T-cells
15q12-q13.1 OCA2-HERC2 Oculocutaneous albinism 2 Melanocyte melanogenic protein; vitiligo
autoantigen
16q24.3 MC1R Melanocortin 1 receptor Melanocyte melanogenic protein; vitiligo
autoantigen
17q21.2 KAT2A-HSPB9- ? ?
RAB5C
18q21.33 TNFRSF11A Tumor necrosis factor receptor Regulates interactions between T-cells and
superfamily member 11A dendritic cells
19p13.3 TICAM1 TIR domain-containing adapter TLR3/TLR4 adapter; mediates
molecule 1 NFkappa-B and interferon-regulatory
factor (IRF) activation; induces apoptosis
19q13.33 SCAF1-IRF3- ? ?
BCL2L12
20q11.2 RALY-ASIP Agouti signaling protein Regulator of melanocytes via MC1R
20q13.13 PTPN1 Tyrosine-protein phosphatase, Dephosphorylates JAK2 and TYK2
nonreceptor type 1 kinases cellular response to interferon?
21q22.3 UBASH3A Ubiquitin-associated and SH3 Promotes accumulation of activated T-cell
domain-containing protein A receptors on T-cell surface
22q12.3 C1QTNF6 Complement C1q tumor necrosis Unknown
factor-related protein 6
22q13.2 ZC3H7B-TEF ? ?
Xp21.3-p21.2 IL1RAPL1 Interleukin-1 receptor accessory Unknown
protein-like 1
Xp11.23 CCDC22-FOXP3- ? ?
GAGE
? indicates that specific gene (and corresponding function) has not yet been defined among those listed
ERRNVPHGLFRVRUJ
246 R. A. Spritz
Those loci with the largest individual effects, as 24.3 Opposite Genetic
measured by odds ratios (ORs), include the Relationship Between
MHC class I (HLA-A) and class II (HLA-DRB1- Vitiligo and Melanoma
DQA1) regions, CPVL, ASIP, and TYR.
Altogether, these associations account for An early discovery from GWAS analyses of EUR
approximately 22.5% of vitiligo heritability in subjects [45, 63–65] was that a number of vitiligo
the EUR population [45]. Fine-mapping and susceptibility genes encode melanocyte proteins
functional analyses have identified many of the or regulate melanocyte function and thus affect
specific genes that underlie these associations, as expression of melanocyte proteins. These include
well as many of the specific causal gene variants, TYR, encoding tyrosinase; OCA2, encoding a
providing deep insight into pathobiological path- melanocyte melanogenic protein; MC1R, encod-
ways, mechanisms of disease susceptibility, and ing the melanocortin 1 receptor; IRF4, encoding
even potential targets for new therapies. As has a key melanocytic transcription factor; and ASIP
also been found for most other complex diseases, and PPARGC1B, both of which encode paracrine
the majority of vitiligo causal variants appear to regulators of melanocyte gene expression. TYR,
reside in gene regulatory regions, which may OCA2, and MC1R additionally constitute
prove more tractable as potential drug targets important vitiligo autoantigens. All of these loci
than missense mutations that directly alter pro- have thus far only been associated with vitiligo in
tein structure and function. the EUR population.
Additionally, several GWAS of vitiligo have Unexpectedly, in each case the vitiligo-
been carried out in Asian populations, in general associated SNPs at these loci were protective,
detecting a subset of vitiligo susceptibility loci and in each case the same SNPs are also associ-
identified in EUR. In the CHN Han and Uygur ated with increased susceptibility to melanoma
populations, complex association signals were but with association to the opposite allele. Thus,
detected in the MHC and the RNASET2- for these loci and associated variants, vitiligo and
FGFR1OP-CCR6 regions [66], which were also melanoma risk constitute genetic opposites. This
detected in EUR, as well as PMEL [67] and inverse genetic relationship almost certainly mir-
ZMIZ1 [68], which thus far have only been asso- rors a corresponding inverse biological relation-
ciated with vitiligo in CHN (Table 24.1). A ship. One possibility is that vitiligo may represent
smaller GWAS of vitiligo conducted in Japanese dysregulation of a normal mechanism of immune
detected only an association in the MHC class I surveillance against melanoma [50, 73]. This
(HLA-A) region [69], and another conducted in would be consistent with both the 75% reduction
the Indo-Pakistani population detected only an in melanoma incidence among patients with pre-
association in the MHC class II (HLA-DRB1- existing vitiligo [74, 75] and prolonged survival
DQA1) region [70]. A very small GWAS of of melanoma patients who develop vitiligo dur-
vitiligo in Koreans detected no significant ing immunotherapy [76].
association signals, almost certainly because of
inadequate sample size and thus insufficient
statistical power [71]. 24.4 Current Understanding
An alternative analysis of the EUR GWAS
data considered vitiligo age of onset as a complex The purpose for identifying genes involved in vit-
trait [72]. This analysis detected significant asso- iligo pathogenesis is that such genes are causal.
ciation with the MHC class II (HLA-DRB1- This thus provides a solid basis for the longer-
DQA1) region, indicating that this function may term goals of understanding the pathobiology of
influence occurrence of vitiligo via mediating the the disease, facilitating rational choice of new
effects of environmental triggers that may initiate targets for drugs or other treatment modalities,
or propagate the disease process at various times and enabling genetic-based classification of
during a susceptible individual’s life. patients for specific therapies and eventually pre-
ERRNVPHGLFRVRUJ
24 Genetics 247
symptomatic disease prevention. It is clear that GZMB, TYR, PTPN22, IFIH1, NLRP1, and oth-
genetic studies of vitiligo have gone a long way ers). For vitiligo, as for other complex diseases,
toward enabling all three of these long-term the great majority of causal variants appear to be
goals. regulatory in nature [45], rather than structural,
As shown in Table 24.1, genetic studies have which may prove advantageous for novel phar-
to date identified approximately 50 different macologic interventions.
genetic susceptibility loci for vitiligo, mostly in Almost all identified vitiligo susceptibility
the EUR population. Most of these loci have been genes encode proteins that play roles in immuno-
resolved to specific corresponding genes; this vit- regulation, apoptosis, or melanocyte function,
iligo “parts list” identified by gene discovery pro- consistent with the fact that vitiligo is an autoim-
vides a solid basis for understanding vitiligo mune disease. These proteins furthermore high-
pathobiology. Furthermore, for many of these light a series of pathways and processes that
genes, specific causal gene variants have been comprise a “blueprint” for vitiligo pathobiology
identified (e.g., HLA-A, HLA-DRB1-DQA1, [58] (Fig. 24.3).
Fig. 24.3 Bioinformatic functional interaction network were excluded. Edge colors are as defined by STRING:
of proteins encoded by genes at confirmed vitiligo suscep- teal, interactions from curated databases; purple, experi-
tibility loci. Unsupervised functional interaction network mentally determined interactions; green, gene neighbor-
analysis was carried out using STRING v10.5 (http:// hood; blue, databases; red, gene fusions; dark blue, gene
string-db.org/) [77], using each protein as a node, and per- co-occurrence; pale green, text-mining; black, co-
mitting ≤5 second-order interactions to maximize con- expression; lavender, protein homology. SMEK2 is an
nectivity. Nodes that shared no edges with other nodes alternative name for PPP4R3B
ERRNVPHGLFRVRUJ
248 R. A. Spritz
Thymus Skin
UV Damage
Medullary
epithelial cells Apoptosis
TYR
Cytotoxic
Melanocyte
T lymphocyte
TYR
OCA2 Periphery MC1R
OCA2
Apoptosis
Apoptosis
Immature
T lymphocytes Maturation +
Proliferation
DAMP
MC1R
Apoptosis
Dendritic cell Cytokines
Deltetion of immunocompetent
“self”-reactive immature
T lymphocytes
Fig. 24.4 A general framework of vitiligo pathogenesis. that act as antigens are presented by HLA class II mole-
During embryonic development, a T-cell repertoire is cules on the dendritic cell surface. Immature T-cells that
selected by positive selection of immunocompetent express cognate T-cell receptors bind these self-antigens
immature T-lymphocytes in the cortex of the thymus. and are activated to express costimulatory molecules that
Immunocompetent T-cells that recognize self-antigens result in cell proliferation and differentiation into CD8+
expressed by thymus medullary epithelial cells undergo effector cytotoxic T-cells, with the assistance of CD4+
negative selection and apoptosis. Immunocompetent T-helper cells. The resultant activated cytotoxic T-cells
immature T-cells that do not encounter a cognate self- recognize and bind the cognate self-antigen presented by
antigen then exit the thymus and enter the peripheral cir- HLA class I molecules on the melanocyte surface, assisted
culation. Subsequently, in the skin, many or most cases of by interaction of FAS ligand on the T-cell and FAS on the
vitiligo likely initiate with skin damage, often induced by target melanocyte. The cytotoxic T-cell then elaborates
ultraviolet (UV) exposure or trauma (Koebnerization). granzyme B and perforin, which induce apoptosis of the
Damaged melanocytes apoptose and release molecules target melanocyte. Almost all of these processes involve
that act as damage-associated molecular patterns proteins that are encoded by genes associated with genetic
(DAMPs), which stimulate activation of local dendritic susceptibility to vitiligo. Reprinted from [32] with
cells. Dendritic cells engulf melanocyte proteins, which permission
are degraded in the proteasome, and peptide fragments
While incomplete, and some respects per- (Fig. 24.4). In the thymus, some immunocom-
haps even incorrect, this vitiligo “parts list” and petent immature T-lymphocytes that can recog-
“blueprint” begin to outline what might be con- nize melanocyte self-antigens escape deletion,
sidered a vitiligo “instruction manual” entering the circulation; perhaps this is part of
ERRNVPHGLFRVRUJ
24 Genetics 249
an advantageous system of immune surveillance 3. Birlea SA, Fain PR, Spritz RA. A Romanian popu-
for nascent melanomas. Subsequently, skin lation isolate with high frequency of vitiligo and
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Acknowledgments This work was supported by grants of autoimmune Addison’s disease associated with dif-
AR056292 and AR065951 from the National Institutes of ferent polyglandular autoimmune (PGA) syndromes.
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ERRNVPHGLFRVRUJ
Epigenetics
25
Li Zhou, Henry W. Lim, and Qing-Sheng Mi
Contents
25.1 miRNAs in Melanogenesis and Vitiligo 254
25.1.1 iRNA Biogenesis and Function
m 254
25.1.2 miRNAs in Melanogenesis 256
25.1.3 miRNAs in the Regulation of Immune Tolerance 256
25.1.4 Dysregulated miRNAs in Vitiligo 257
25.2 DNA Methylation Alterations in Vitiligo 258
25.2.1 NA Methylation
D 258
25.2.2 DNA Methylation in Vitiligo Animal Models 259
25.2.3 DNA Methylation in Human Vitiligo 259
25.3 Histone Modifications 260
25.3.1 echanisms of Histone Acetylation and Histone Methylation
M 260
25.3.2 Histone Modification in Autoimmune Diseases 260
25.3.3 Chromatin Modification in Melanocyte Differentiation 261
References 262
Abstract non-Mendelian pattern that suggests a

Even though the pathogenesis of vitiligo is not polygenic, multifactorial inheritance for
completely understood, the most compelling vitiligo. Recent genome-wide association
etiology of vitiligo involves a combination of studies (GWAS) to investigate the genetics of
environmental and genetic factors that con- vitiligo have resulted in the identification of
tribute to autoimmune melanocyte destruc- multiple vitiligo susceptibility genes (Birlea
tion. Large-scale epidemiological studies have et al., J Invest Dermatol 130:798–803, 2010;
shown that about 15–20% of vitiligo patients Quan et al., Nat Genet 42:614–618, 2010),
have one or more affected first- degree many of which are shared by other autoim-
relatives. The familial aggregation takes a mune diseases. These evidences strongly
support the significance of genetic factors
contributing to one’s risk for vitiligo.
L. Zhou (*) · H. W. Lim · Q.-S. Mi Nevertheless, the concordance for generalized
Henry Ford Immunology Program, Department of vitiligo in monozygotic twins is only 23%,
Dermatology, Henry Ford Hospital, Detroit, MI, USA which suggests that environmentally induced
e-mail: lzhou1@hfhs.org; hlim1@hfhs.org; qmi1@
hfhs.org

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254 L. Zhou et al.
epigenetic factors must also play an important

role, and may be even more important than • The mean genomic DNA methylation
genetics. levels were significantly higher in vitil-
Epigenetics is the study of hereditable igo patients.
patterns of gene expression without changes • Histone modifications play critical roles
in the DNA sequence. Epigenetic regulations in the biological processes of the
in response to environmental changes play a immune system as well as the develop-
critical role in determining gene function and ment of autoimmune diseases.
activities. Disorders of epigenetic processes, • Involvement of epigenetic regulations,
which involve DNA methylation, histone especially miRNAs and DNA methyl-
modification, and noncoding RNA expres- ation, in melanocyte development,
sion, were found to be associated with the function, and immune tolerance
pathogenesis of various diseases. Over the regulation.
past few years, great progress has been made
in identifying related epigenetic mechanisms
in autoimmune disease development, includ-
ing vitiligo, to bridge the gap between envi- 25.1 miRNAs in Melanogenesis
ronmental and genetic factors. These and Vitiligo
emerging studies provide new insights into
not only clinical biomarkers for diagnosis 25.1.1 miRNA Biogenesis
and disease progression but also novel targets and Function
for potential epigenetic therapeutic strate-
gies. In this chapter, we summarize the epi- MicroRNAs are 22-nucleotide, single-stranded
genetic regulations in melanocyte biology, RNA that modulates the stability and translation
immune tolerance regulation, as well as of mRNAs posttranscriptionally. To date, more
related epigenetic alterations in autoimmune than 2500 mature miRNAs have been described
vitiligo. in humans [1]. In the past decade, miRNAs have
been recognized as an important component of
epigenetic regulation and a new layer of gene
regulation impacting cellular development, phys-
iology, and disease development [2]. In addition,
Key Points miRNAs represent the best-characterized class of
• Epigenetics is the study of hereditable noncoding RNAs in terms of both expression and
patterns of gene expression without function.
changes in the DNA sequence. As shown in Fig. 25.1, miRNAs are first
• The concordance for generalized vit- transcribed as long, polyadenylated primary pre-
iligo in monozygotic twins is only cursors (pri-miRNA) by RNA polymerase
23%, which suggests that environ- II. They are cleaved into a hairpin-shaped
mentally induced epigenetic factors 70–100- nucleotide precursor (pre-miRNA), by
must also play an important role and Drosha (a nuclear RNase III), which functions in
may be even more important than a complex that contains DGCR8, a dsRNA-
genetics. binding protein. The resulting pre-miRNAs are
• Recent studies identified upregulation then exported to the cytoplasm by exportin-5. In
of miR-224-3p and miR-4712-3p and the cytoplasm, Dicer (another RNase III) in asso-
downregulation of miR-3940-5p in the ciation with a dsRNA-binding protein partner
PBMCs of NSV patients. called TRBP, or PACT in humans, processes pre-
miRNAs into a 19–24-nucleotide miRNA duplex
ERRNVPHGLFRVRUJ
25 Epigenetics 255
microRNA gene or introns
RNA polymerase II
Pri-miRNA
DGCR8
Nucleus
Drosha
Pre-miRNA cytoplasm
Exportin-5
Pre-miRNA
Dicer TRBP
miRNA duplex
RISC
Target mRNA
mRNA degradation or translational repression
Fig. 25.1 MicroRNA biogenesis in animal cells. miRNAs the cytoplasmic RNase III enzyme Dicer in association
are first transcribed as long, polyadenylated primary tran- with a dsRNA-binding protein partner TRBP. The final
scripts (pri-miRNA) by RNA polymerase II from specific step of miRNA maturation is the selective loading of the
miRNA genes. Pri-miRNAs are processed into precursor functional strand of the small RNA duplex onto the RNA-
miRNA (pre-miRNA) stem-loops by the nuclear RNase III induced silencing complex (RISK). Mature miRNAs then
enzyme Drosha and its partner DGCR8. The pre-miRNA guide the RISK to cognate target mRNA and repress target
is then actively transported to the cytoplasm by exportin-5 gene expression by either destabilizing target mRNAs or
and is further processed into ~22-nucleotide duplexes by repressing their translation
by removing the terminal loop. One strand of the specificity [5]. miRNAs control the expression of
resulting duplex is bound by Argonaut to form specific genes, typically by base paring to the 3′
the RNA-induced silencing complex (RISC), untranslated region (3′UTR) of target mRNAs to
which targets mRNA for regulation [3, 4]. mediate repression of that gene either by mRNA
Nucleotides 2–7 of the mature miRNA sequence destabilization, translational inhibition, or both.
create the “seed region” which primarily speci- It is predicted that a single miRNA may target, on
fies the mRNA that the miRNA will bind. It is average, more than a hundred mRNAs, and over
now becoming apparent that base pairing outside 60% of human protein-coding genes contain
the seed region provides a further layer of miRNA-binding sites within their 3′UTRs [2, 4].
ERRNVPHGLFRVRUJ
256 L. Zhou et al.
25.1.2 miRNAs in Melanogenesis determining fish skin color by Yan et al. indi-
cated that miR-429 is a potential regulator of
Microphthalmia-associated transcription factor skin pigmentation. They showed that miR-429
(MITF), a master regulator of melanogenesis and directly regulates expression of Foxd3 by target-
melanocyte function, is a key transcription factor ing its 3′-UTR, which repress the transcription
controlling genes linked to pigmentation. Recent of MITF and its downstream genes, such as
studies have shown the clear interrelationship TYR, TYRP1, or TYRP2 [9].
between miRNAs and MITF, further indicating In addition to the intra-melanocyte localized
the potential involvement of miRNAs in skin miRNA-mediated melanogenesis regulation, a
pigmentation. recent study by Kim and colleagues showed
As a central regulator of microRNA biogene- that miR-675, a long noncoding RNA H19-
sis, Dicer converts pre-miRNAs to fully func- generated miRNA from keratinocytes, could be
tional mature miRNAs. In melanocytes, Dicer released extracellularly and delivered to neigh-
expression was found to be under direct tran- boring melanocytes, in which miR-675 reduces
scriptional regulation by MITF. MITF binds and the expression of MITF through direct binding
activates a conserved regulatory element to its 3′UTR [10]. These results suggest that
upstream of Dicer’s transcriptional start site upon miR-675 derived from keratinocytes could be
melanocyte differentiation. Meanwhile, targeted involved in H19-stimulated melanogenesis
deletion of Dicer is lethal to melanocytes [6]. through targeting.
These observations highlight a central mecha- UVR is the major environmental inducing
nism underlying melanocyte-specific miRNA factor of the melanogenesis process upregulating
regulation and the critical role of miRNAs in a network of genes involved in the pigmentation
melanocyte differentiation and survival. process. To identify miRNAs interfering with the
Nevertheless, multiple recent studies have pigmentary process, Dynoodt and colleagues
shown strong evidence of the regulatory role of performed miRNA profiling on mouse melano-
individual miRNAs on MITF and other related cytes after three consecutive treatments involv-
molecules controlling melanogenesis activities ing forskolin and solar-simulated UV (ssUV)
and skin pigmentation. In a search for miRNAs irradiation [11]. Within the 16 differentially
directly regulating MITF expression, Guo and expressed miRNAs in treated melan-a cells, a
colleagues identified miR-218 as a novel candi- 15-fold downregulation of miR-145 was
date for direct action on MITF expression [7]. detected. The expression of miR-145 is inversely
Ectopic miR-218 expression dramatically correlated with the levels of Sox9, Mitf, Tyr,
reduced MITF expression, suppressed tyrosinase Trp1, Myo5a, Rab27a, and Fscn1. The direct tar-
activity, and induced depigmentation in murine geting of Myo5a by miR-145 was confirmed in
immortalized melan-a melanocytes. mouse and human melanocytes [11]. These
Furthermore, miR-218 also suppressed melano- results suggest a key role for miR-145 in regulat-
genesis in human pigmented skin organotypic ing melanogenesis.
culture (OCT) and human primary skin melano-
cytes through the repression of MITF [7].
Transgenic mice overexpressing miR-137 devel- 25.1.3 miRNAs in the Regulation
oped a range of coat color changes from dark of Immune Tolerance
black to light color. Molecular analyses of the
transgenic mice showed decreased expression of Through extensive use of microarray hybridiza-
the major target gene MITF and its downstream tion and small RNA library sequencing in combi-
molecules, including TYR, TYRP1, and TYRP2, nation with next-generation sequencing
which indicate the regulatory role of miR-137 in approaches, investigators have identified lineage-
melanogenesis and coat color control [8]. A and developmental stage-specific miRNA expres-
recent study on the role of miRNAs in sion profiles in a variety of immune cell types
ERRNVPHGLFRVRUJ
25 Epigenetics 257
[12, 13], which indicate the relevance of miRNAs miRNAs in vitiligo development. To explore
to immune cell development and function. the potential relationship between miRNA dys-
CD4+CD25+Foxp3+ regulatory T (Treg) cells and regulation and vitiligo development, we com-
invariant natural killer T (iNKT) cells are both pared serum miRNA expression profiles in
critical immune regulators and play a pivotal role patients with active NSV and matched healthy
in the maintenance of self-tolerance and immu- controls. Thirty-one miRNAs showed signifi-
nity. Numerical and functional defects of Treg and cantly different expression levels (>2-fold
iNKT cells have been linked to the development of changes, P < 0.05) between the two groups,
a variety of autoimmune diseases, including vitil- while a panel of three miRNAs (miR-16, miR-
igo [14–16]. Our recent clinical study on NSV 19b, and miR-720) [24] appeared to be the best
showed decreased iNKT cell numbers and a trend serum biomarkers to distinguish NSV patients
toward altered iNKT cell phenotype in PBMCs of from healthy controls, with the area under the
active NSV patients compared to age-, gender-, curve (AUC) value of 0.97 [25]. In addition, the
and race-matched healthy controls, suggesting the downregulation of miR-574-3p is correlated
potential involvement of iNKT cells in the patho- with disease severity [25]. Furthermore, we
genesis of vitiligo [17]. Using Treg-specific Dicer also evaluate the serum miRNA expression pro-
or Drosha deletion mouse models, three individual files from an autoimmune vitiligo mouse model,
groups almost simultaneously identified the criti- in which TRP-1-specific CD4+ T cells from
cal role of miRNAs in Treg lineage stability and TRP-1 CD4+ TCR-transgenic mice were adop-
function [18–20]. Tregs with miRNA deletion tively transferred into Rag1KO host [26].
showed impaired peripheral homeostasis, eradi- Twenty miRNAs showed significantly different
cated suppression function, and interrupted Treg expressions between vitiligo and control mice,
cell lineage stability, which ultimately lead to the among which miR-146 [27] and miR-191 [28]
fatal early onset of lymphoproliferative syndrome were significantly upregulated in the serum of
in these mouse models [18–20]. Using bone mar- both vitiligo mouse and NSV patients [25, 26].
row- and thymus-specific Dicer deletion mouse Our studies raise the possibility that miRNAs
models, studies from our laboratory and others may be involved in NSV development and
demonstrate the role of miRNA in iNKT cell could serve as promising biomarkers for differ-
development and function [21–23]. A substantial ential diagnosis, progression, and therapy
reduction of iNKT cell numbers was observed in response of vitiligo. In a recent study, Wang
both thymus and peripheral immune organs from and colleagues evaluated the miRNA expres-
Dicer KO mice. Furthermore, iNKT cells with sion profile in peripheral blood mononuclear
Dicer deletion showed a significantly interrupted cells (PBMCs) of patients with NSV and
capacity of activation and cytokine secretion [22, healthy controls and identified the upregulation
23]. The critical involvement of miRNAs in regu- of miR-224-3p and miR-4712-3p and downreg-
latory immune cell development, homeostasis, and ulation of miR-3940-5p in the PBMCs of NSV
function further emphasize the potential role of patients [29]. Mansuri et al. further analyzed
miRNAs in maintaining immune tolerance and the the miRNA expression profiles of lesional, non-
pathogenesis of autoimmune diseases including lesional skin from NSV patients and healthy
vitiligo. skin from control subjects with the aim to detect
the potential role of skin microenvironmental
miRNAs in the development of vitiligo. In
25.1.4 Dysregulated miRNAs addition to the changed miRNA levels in
in Vitiligo lesional skin compared to healthy skin, nonle-
sional skin from NSV patient showed dysregu-
The critical roles of miRNAs in both melano- lated miRNA expression compared to healthy
genesis and immune tolerance regulation skin, and some miRNAs showed common dys-
strongly suggest the potential involvement of regulations in both lesional and nonlesional
ERRNVPHGLFRVRUJ
258 L. Zhou et al.
Table 25.1 MicroRNAs potentially involved in vitiligo

Changed miRNAs Subjects/tissues Biological function References
miR-16, miR-19b, NSV patients and healthy controls/ Potential serum biomarkers, may be related [24, 25]
miR-720 serum to Th1 response regulation
miR-574-3p NSV patients and healthy controls/ May be associated with NSV lesion [25]
serum severity
miR-146a, miR-191 NSV patients and healthy controls/ Potential serum biomarkers, may contribute [25–28]
serum, Rag1−/− vitiligo mouse and to TNFa over production; proliferation,
control/serum survival of melanocytes
miR-224-3p, NSV patients and healthy controls/ Potential biomarker, potential role in [29]
miR-4712-3p, PBMCs regulating inflammatory cytokine
miR-3940-5p production
miR-135a, NSV patient lesional, nonlesional Potential role of initiation and progression [30]
mijR-183, and helthy control skin of disease
miR-30a-3p,
miR-487a
miR-196a-2 NSV patients and healthy control rs11614913 miR-196a-2 CC-allele [31]
tissues associated with lower risk of vitiligo
miR-25 Human melanocytes and Promote the degeneration of melanocytes [32]
keratinocytes by targeting MITF and inhibiting SCF,
bFGF
skin in NSV patients [30]. These results indi- stress-induced miR-25 overexpression may be
cate the potential role of miRNAs in the initia- potentially involved in vitiligo development [32]
tion as well as progression of vitiligo and that (Table 25.1).
dysregulated miRNAs might be one of the
mechanisms for the increased susceptibility of
vitiligo patients to environmental triggers in 25.2 D
NA Methylation Alterations
disease development. in Vitiligo
To investigate the contribution of genetic vari-
ations in miRNAs to the development of vitiligo, 25.2.1 DNA Methylation
Huang et al. found a significantly lower risk of
vitiligo was associated with the rs11614913 DNA methylation is a dynamic process involving
miR-196a- 2 CC genotype. As the target of methylation and demethylation events in differ-
miR-196a-2, TYRP1 gene expression was down- ent regions of genome, which is crucially impor-
regulated by the rs11614913 C allele in miR- tant for embryogenesis, cellular proliferation,
196a-2, which lowered the levels of intracellular and differentiation [33, 34]. DNA methylation is
reactive oxygen species (ROS) and reduced early catalyzed and maintained by the DNA methyl-
apoptosis in human melanocytes in response to transferase (DNMT) family which is composed
H2O2 treatment [31]. Interestingly, studies from of a group of enzymes, including DNMT1,
the same group showed that oxidative stress DNMT3a, DNMT3b, and DNMT3L. In mam-
induced the overexpression of miR-25 in both mals, DNMTs catalyze the donation of a methyl
melanocytes and keratinocytes, and upregulated group to cytosine (C) and guanine (G) dinucleo-
miR-25 could not only promote the degeneration tides [35]. In the human genome, there are
of melanocytes by targeting MITF but also inhibit regions with enriched CpG dinucleotides, which
the production and secretion of SCF and bFGF are called CpG islands. The human genome con-
from keratinocytes, thus impairing their para- tains about 30,000 CpG islands, 50–60% of
crine protective effect on the survival of melano- which are located in the promoter region of genes
cytes [32]. These results indicate that oxidative [36]. It is believed that methylation of CpG in the
ERRNVPHGLFRVRUJ
25 Epigenetics 259
promoter region leads to the silencing of gene 25.2.3 DNA Methylation in Human
expression by interfering with transcription acti- Vitiligo
vator binding and/or recruitment of chromatin
repressor complexes through the binding of To investigate the potential involvement of epi-
methyl-CpG- binding domain (MBD) proteins, genetic changes in the development of human
which leads to tightly condensed chromatin vitiligo, Lu’s group examined genomic and gene-
around the gene promoter, blocking accessibility specific DNA methylation levels as well as
to transcriptional activators and thereby inhibit-mRNA levels of DNAMTs, MDB proteins in
ing the gene transcription [36, 37]. In addition to
peripheral blood mononuclear cells (PBMCs),
CPG islands, recent genome-wide DNA methyla- from vitiligo patients and controls [43]. Compared
tion studies reveal that non-CpG methylation in to healthy controls, the mean genomic DNA
both the intragenic and intergenic sites is also methylation levels were significantly higher in
important for regulation of differential gene vitiligo patients, which is consistent with some
expression [38]. Demethylation could be either other organ-specific autoimmune diseases such
passive or active. Passive demethylation is as psoriasis and type 1 diabetes mellitus [44, 45].
induced by inhibition of DNMTs, providing the However, some systemic autoimmune diseases,
basis for treatments with the aim of erasing such as systemic lupus erythematosus (SLE) and
abnormal hypermethylation [39], while active systemic sclerosis (SSC), showed global hypo-
demethylation could depend on the activity of methylation in CD4+ T cells compared to controls
cytosine deaminases occurring predominantly in [46, 47]. These results indicate that hypermethyl-
cell differentiation and associated with immune ation may be important in organ-specific immune
cell activation [40]. activation. In addition, DNMT1, the enzyme for
maintaining DNA methylation, was significantly
increased, while some MDB proteins including
25.2.2 DNA Methylation in Vitiligo MBD1, MBD3, MBD4, and MeCP2 were also
Animal Models significantly upregulated in PBMCs from vitiligo
patients, which is consistent with the global
In 1996, Sreekumar et al. reported that DNA hypermethylation identified in vitiligo PBMCs.
methylation inhibitor 5-azacytidine (5-azaC) More interestingly, the expression levels of
induced autoimmune vitiligo in vitiligo-MBD1 and MBD3 were positively correlated
susceptible but normally pigmented chicken with overall methylation levels in PBMCs of vit-
stains, which are parental control strains of the iligo patients, suggesting that increased MBD1
Smyth Line chicken model, due to loss of skin and MBD3 may contribute to global hypermeth-
melanocytes [41]. In this model, both T cells and ylation and the development of vitiligo [43].
B cells are involved in the pathogenesis of the IL-10 is an anti-inflammatory cytokine. It is
disease. Interestingly, chronic low-dose adminis- capable of inhibiting synthesis of pro-
tration of 5-azaC also increased the incidence of inflammatory cytokines and is critically involved
autoimmune thyroiditis in a chicken model for in regulatory T cell-mediated immune regulation.
Hashimoto’s autoimmune thyroiditis [42], which Since IL-10 is particularly sensitive to alterations
supports the interrelationship between vitiligo in methylation status [48, 49] and vitiligo patient
and autoimmune thyroiditis observed in humans. PBMCs have decreased IL-10 transcription [43],
It is possible that DNA hypomethylation caused the DNA methylation status of an IL-10 enhancer
by 5azaC may turn on certain genes that induce region was evaluated. The enhancer region within
autoreactivity in specific T and/or B cells. This intron 4 of IL-10 containing eight CG pairs was
suggests a role for demethylation of genes in the hypermethylated in vitiligo PBMCs, and the
pathogenesis of these autoimmune diseases in overall methylation levels of this region are nega-
genetically susceptible individuals. tively correlated with IL-10 mRNA transcription,
ERRNVPHGLFRVRUJ
260 L. Zhou et al.
which suggests that DNA hypermethylation demethylation occurs as a result of interaction of

within IL-10 gene may contribute to decreased histone demethylase (HDMs). As a major con-
IL-10 expression in vitiligo PBMCs [43]. tributor of epigenetic modification, histone
Nevertheless, future studies in exploring the methylation regulates fundamental processes
gene-specific DNA methylation patterns are such as gene transcription and DNA repair [51].
needed to determine the specific role of DNA Unlike acetylation, the effects of histone meth-
methylation dysregulation in the pathogenesis of ylation on gene regulation depend on the posi-
vitiligo. These information may supply not only tion of residues modified or the number of
novel markers for vitiligo clinical management methyl groups present, resulting in either tran-
but also potential epigenetic therapies for vitiligo scription activation or suppression [52].
in adjusting DNA methylation.
25.3.2 Histone Modification

25.3 Histone Modifications in Autoimmune Diseases
The nucleosome is a repeating subunit that con- Accumulated evidence from recent studies has
sists of 146 base pairs of chromatin in a double indicated that histone modifications play critical
helix wrapped around a protein core composed of roles in the biological processes of the immune
histone octamers H2A, H2B, H3, and H4. As system as well as the development of autoim-
another major source of epigenetic alteration, mune diseases. Dysregulated immune
histone posttranslational modifications, such as homeostasis, activation, and inflammatory cyto-
acetylation, methylation, ubiquitination, and kine production are associated with aberrant his-
phosphorylation, could modulate the chromatin tone and chromatin modification [53, 54]. Type 1
structure, influencing its accessibility to tran- diabetes (T1D) is an organ-specific autoimmune
scription factors at gene promoters and enhanc- disease and shares multiple susceptible genes
ers, therefore modulating related gene expression. with vitiligo. Recent evidence suggests that aber-
Among these processes, acetylation and methyla- rant histone modifications are involved in T1D
tion have been the most extensively studied ones. pathogenesis. The expression of histone deacety-
lase gene, a key regulator of histone modifica-
tion, is reduced in CD4+ T cells from T1D
25.3.1 Mechanisms of Histone patients [55].
Acetylation and Histone By screening histone modification variations
Methylation at known T1D susceptible genes in blood cells,
increased H3K9 acetylation (H3K9Ac) upstream
Histone acetylation and deacetylation are of great regions of HLA-DRB1 and HLA-DQB1 within
importance in gene regulation. Histone acetyla- the IDDM1 locus in T1D monocytes from
tion is catalyzed by histone acetyltransferases T1D. These histone acetylation changes could be
(HATs), adding acetyl group on lysine residues in the cause of the upregulated HLA-DRB1 and
the N-terminal tail and promoting a more open HLA-DQB1 gene expression, which is highly
chromatin structure which links to active genes. associated with T1D [56]. Furthermore, in lym-
In contrast, histone deacetylation catalyzed by phocytes of T1D patients, altered H3K9me2 was
histone deacetylases (HDACs) removes the ace- identified in subset of genes which are involved
tyl groups, causing the DNA to wrap around in autoimmune and inflammation-related path-
nucleosomes more tightly, thereby repressing ways, such as cytotoxic T-lymphocyte-associated
gene expression [50]. protein 4 (CTLA-4), transforming growth factor-
Histone methylation, which adds a methyl beta (TGF-β), nuclear factor-kappaB (NFκB),
group to the histone subunit, is catalyzed by toll-like receptor (TLR), and interleukin-6 (IL-6)
histone methyltransferases (HTMs), while [57]. In patients with rheumatoid arthritis (RA),
ERRNVPHGLFRVRUJ
25 Epigenetics 261
synovial fibroblasts show increased histone 25.4 Concluding Remarks

methyltransferase EZH2 which could silence
secreted frizzled-related protein 1 (SFRP1) gene Growing amount of evidences suggest that gene
expression, thereby abrogating its inhibitory role function depends on not only DNA sequences
on aberrant collagen deposition [58]. These stud- but also the epigenetic modifications, including
ies provide evidence of a novel association DNA methylation, histone modification, non-
between autoimmune diseases and altered his- coding RNA, and their cross talks, which are
tone modifications of key genes that are compo- thought to bridge the gap between environment
nents of disease-related biological pathways. and genetics. Different from genetic factors, epi-
Nevertheless, the association of global or genetic alterations vary among different tissues
gene-specific histone modification patterns with and cell types. Each cell type could be character-
the development vitiligo remains to be explored. ized by a particular epigenome that are associ-
These future studies will not only further eluci- ated with a specific gene expression profile. In
date the epigenetic mechanisms of vitiligo devel- addition, different environmental factors exposed
opment but also provide potential novel could also contribute to their distinction in epi-
therapeutic targets for the treatment of this genetic alteration and disease pathogenesis.
disease. These epigenetic specificities manifested in dif-
ferent diseases, and even individual patients with
the same disease will supply the novel clinical
25.3.3 Chromatin Modification biomarkers for disease diagnosis, prognosis, and
in Melanocyte Differentiation progression monitoring. Furthermore, epigenetic
alterations are potentially reversible, which sup-
Microphthalmia-associated transcription factor ply novel targets for potential epigenetic thera-
(MITF) not only regulates the survival and prolif- peutic treatment and the more personalized
eration of melanocytes but also promotes the application of treatment for autoimmune dis-
transcription of several pigmentation genes. A eases in the future.
comprehensive analysis of the MITF “interac- Similar to other autoimmune diseases, vitiligo
tome” in melanoma cells revealed the nucleo- is a polygenic, multifactorial disease resulting
some remodeling factor (NURF) complex from the complex interactions of genetic and
associates with MITF [59]. More recent studies environmental factors that ultimately contribute
showed that NURF acts downstream of MITF in to melanocyte destruction. As shown in Fig. 25.2,
melanocytes and co-regulate gene expression. In [62–67] genetic polymorphisms, mutations pre-
vivo, mice lacking the NURF subunit BPTF in dispose individuals with dysregulated immuno-
the melanocyte lineage show premature graying surveillance against the melanocytic system. In
due to deficiency in generating mature melano- these individuals, environmental factors induce
cytes from the adult stem cell population [60]. epigenetic alterations including miRNA level,
Therefore, chromatin remodeling by NNURF DNA methylation, and histone modification.
appears to be essential for the transition of the These alterations then affect the expression of
transcriptionally quiescent stem cell to the differ- related genes involved in immune tolerance,
entiated state. Furthermore, NURF subunits, inflammation, and melanocyte homeostasis and
SMARCA5 and BPTF, were also involved in function as summarized in this chapter, which
controlling epidermal stem cell differentiation eventually lead to active immune response and
[61]. Despite extensive characterization of the melanocyte destruction. Even though epigenetic
biochemical properties of the NURF complex studies in vitiligo are still in its infancy, accumu-
and its subunits, the detailed biological functions lated studies have already shown clear evidence
of NURF complex in mammals and its relation- of the critical involvement of epigenetic regula-
ship to the development and maintenance of vit- tions, especially miRNAs and DNA methylation,
iligo remain to be explored. in melanocyte development, function, and
ERRNVPHGLFRVRUJ
262 L. Zhou et al.
Fig. 25.2 Schematic
representation of Genetic factors
environmental factor-
mediated epigenetic
modifications in the
development of Vitiligo
autoimmune vitiligo. In
these genetically
predisposed individuals,
environmental factors,
such as stress,
Disease biomarker
Therapeutic target
chemicals, and
microinfections, induce Immune Melanocytes
epigenetic alterations tolerance
including miRNA level,
DNA methylation, and
histone modification.
These alterations then
affect the expression of DNA Histone
miRNAs modification
related genes involved in methylation
immune tolerance,
inflammation, and Epigenetic alterations
melanocyte homeostasis
and function, which
ultimately lead to
melanocyte destruction Pollution, Environmental
Microinfections Stress
heavy metals chemicals
immune tolerance regulation. Continued studies 7. Guo J, Zhang JF, Wang WM, et al. MicroRNA-218
in epigenetic mechanisms on vitiligo develop- inhibits melanogenesis by directly suppressing
microphthalmia-associated transcription factor
ment will definitely result in more promises in expression. RNA Biol. 2014;11(6):732–41.
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9. Yan B, Liu B, Zhu CD, et al. microRNA regulation of
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and function of EZH2 in synovial fibroblasts: epigen- 67. Spritz RA. Six decades of vitiligo genetics: genome-
etic repression of the Wnt inhibitor SFRP1 in rheuma- wide studies provide insights into autoimmune patho-
toid arthritis. Ann Rheum Dis. 2011;70:1482–8. genesis. J Invest Dermatol. 2012;132:268–73.
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Melanocyte Homeostasis
in Vitiligo
26
Véronique Delmas and Lionel Larue
Contents
26.2 Origin of Skin Melanocytes and Skin Colonization 266
26.2.1 ellular Origin and Migratory Pathway of Melanocyte Progenitors
C 266
26.2.2 Migration of Melanoblasts to the Skin and “the Cadherin Code” 267
26.2.3 Melanoblast Skin Distribution and Differentiation 269
26.3 H omeostasis of Melanocytes 269
26.3.1 The Epidermal Melanin Unit 269
26.3.2 Epidermal Melanocyte Renewal 271
26.4 M elanocyte Stability and Vitiligo 271
26.4.1 The Epidermal Melanin Unit in Vitiligo 271
26.4.2 E-Cadherin and Vitiligo 271
References 274
Abstract the homeostasis of these cells and their renewal

To understand the pathophysiology of during the course of life. We focus on the early
pigmentary disorders, such as vitiligo, it is alterations of melanocyte homeostasis that
essential to understand the development and appeared before the depigmentation of the skin
homeostasis of melanocytes, the cells that proin vitiligo patients and highlight the key role of
duce the melanin pigment. In this chapter, we E-cadherin, the main cell-cell adhesion mole-
will concentrate on classical melanocytes that cule that links melanocyte to the surrounding
give rise to skin colour that can be lost in depig- keratinocytes in the epidermis.
mented region of vitiligo patients. We address
the origins of melanocytes during development,
Key Points
• In vitiligo patients, alteration of melano-
cyte homeostasis leads to the reduction
of basal melanocytes in normal pig-
V. Delmas · L. Larue (*) mented skin.
INSERM U1021, Normal and Pathological • Detached basal melanocytes are
Development of Melanocytes, Institut Curie, PSL
Research University, Orsay, France
replaced until exhaustion of surround-
e-mail: veronique.delmas@curie.fr; ing melanocyte stem cells.
lionel.larue@curie.fr

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266 V. Delmas and L. Larue
also be affected in this disease, but we will not

• Alteration of the level/activity of address this issue. Skin melanocytes are found
E-cadherin in basal melanocytes is an in the dermis and the epidermis. Most are found
early event of vitiligo pathophysiology. in the epidermis where they are located in dif-
• The level/activity of E-cadherin is ferent structures (hair follicles, sweat glands,
reduced according to the genetic status and sebaceous glands) and dispersed throughout
and/or the environment (oxidative and/ the stratum. Skin melanocytes produce different
or mechanical stresses). sets of proteins depending on their location.
Better knowledge of the origins and various
specificities of these melanocyte populations
can lead to a better understanding of the differ-
26.1 Introduction ent types of vitiligo, including segmental
vitiligo.
To understand the pathophysiology of pigmen-
tary disorders, such as vitiligo, it is essential to
understand the features and homeostasis of mela- 26.2 O
rigin of Skin Melanocytes
nocytes, the cells that produce the pigment, mela- and Skin Colonization
nin. Melanocytes are specified at an early stage of
neural crest development during embryonic 26.2.1 Cellular Origin and Migratory
development [1]. Neural crest cells (NCC) are a Pathway of Melanocyte
temporary cell population unique to vertebrates Progenitors
that arise from the embryonic dorsal ectodermal
cell layer and give rise to diverse cell lineages, The embryonic precursor cells of melanocytes,
including melanocytes, craniofacial cartilage and melanoblasts, originate from neural crest cells
bone, smooth muscle, peripheral and enteric neu- (NCC), a transient cell population arising from
rons, and glia. NCC are originally located in the the dorsal part of the neural tube. In humans,
dorsal part of the embryo and subsequently melanoblasts actively proliferate during develop-
migrate to their ultimate destination, acquiring ment, concomitant with specification and migra-
their various properties, including differentiation tion. Melanoblast specification starts 8 weeks
into pigment cells. In the human body, melano- after conception. Dorsolateral migration of mela-
cytes are found in the skin, as well as the iris, noblasts occurs throughout the mesenchymal
inner ear, brain, heart, and fatty tissue [2, 3]. dermis between the 5th and 12th weeks of embry-
Melanocytes in the epidermis synthesize melanin onic development.
and transfer this pigment to the surrounding kera- The mechanisms associated with melanoblast
tinocytes; skin melanocytes are considered to be specification, proliferation, and migration during
“classical melanocytes.” The other melanocytes development have been little studied in humans
found throughout the body do not transfer their for obvious reasons. Most information has been
melanin to surrounding cells and are classified as generated from studies on avian (chicken and
“nonclassical melanocytes.” The function of non- quail), fish (zebrafish), and mammalian (rat and
classical melanocytes and the associated melanin mouse) embryos. Melanoblasts specify, prolifer-
are mostly unknown [2]. However, we do know ate, and first migrate from the vagal region and
the function of some of them, melanocytes then progressively further toward the anterior and
located in the inner ear are essential for hearing, posterior extremities.
and those located in heart valves are involved in In the regions where somites are present,
their stiffness. melanoblasts migrate between the somites and
In this chapter, we will concentrate on classi- the ectoderm from the dorsal region toward the
cal melanocytes that give rise to skin color and belly. This pathway is called the dorsolateral
lost in vitiligo. Pigmentation of the eyes can pathway. In the cephalic region, lacking somites,
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26 Melanocyte Homeostasis in Vitiligo 267
melanoblasts migrate laterally under the ecto- the dorsoventral pathway are poorly understood.
derm. Melanoblasts from different parts of the The SCP signals for melanoblast specification
body thus migrate and interact with various cells, are not fully characterized, but various proteins
extracellular matrices, and/or microenviron- may play a role in regulating the cell fate of
ments. As a consequence, melanoblasts receive SCPs, including NRG/ERBB3 (neuregulin and
information that can vary, differentially influenc- its receptor tyrosine kinase 3), IGFI-IGFII/
ing their potential biological properties or IGF1R (insulin-like growth factor and its recep-
predispositions. tor), and PDGF/PDGFR (platelet-derived growth
Furthermore, although most melanoblasts fol- factor and its receptor) [7].
low the dorsolateral pathway, some emerge from The difference(s), if any, between dorsolateral
the dorsoventral pathway, in between the neural and dorsoventral melanocytes remain(s) unknown.
tube and the somites [4]. It has been observed in For example, it is unclear whether these two cell
several species that the growing nerves project- types have the exact same function and whether
ing through the body serve as a niche for progeni- they colonize the body in a uniform manner.
tor cells, including Schwann cell precursors These questions are of clear importance to better
(SCPs), which give rise to Schwann cells and understanding melanomagenesis and pigmenta-
melanocytes [1]. SCPs are derived from neural tion problems, including vitiligo.
crest cells that delaminate before the dorsolateral
migration of the melanoblasts. These cells ini-
tially follow the dorsoventral pathway, between 26.2.2 Migration of Melanoblasts
the neural tube and somites and not between the to the Skin and “the Cadherin
somites and ectoderm. SCP and Schwann cells, Code”
once differentiated, remain in close contact with
the nerves. In some cases, SCPs lose their contact After specification, dorsolateral melanoblasts
with nerves and may acquire the melanocytic migrate through the dermis and enter the epider-
fate. These melanocytes colonize the dorsal and mis by crossing the basement membrane. Once in
lateral body walls and largely influence limb pig- the epidermis, melanoblasts remain in contact
mentation. Some patients with segmental vitiligo with the basement membrane via the stratum of
have depigmentation that is confined to innerva- the epidermis. In mice, melanoblasts are evenly
tion zones. It is possible that dorsoventral mela- distributed throughout the epidermis of the
nocytes are at the origin of this type of embryo [8]. This suggests that melanoblasts
depigmentation. Many signaling molecules communicate between each other using soluble
(ligands) are required during all stages of mela- factors/receptors and/or keratinocytes to main-
nocyte development [5]. These ligands educate tain a defined and dynamic distance apart from
neural crest cells to specify the melanocytic fate each other. From embryonic day E15.5, melano-
and instruct melanoblasts to proliferate, migrate, blasts migrate toward the matrix of the nascent
survive, and home (final destination), prior to ter- hair follicles, a process that is stimulated by
minal differentiation into pigmented melano- SCF. Just prior birth, melanoblasts of the hair fol-
cytes. The currently best-documented signaling licles are found in the forming hair bulge and hair
pathways implicated in the development of the bulb. The hair bulge is located in the permanent
melanocyte lineage arising from the dorsolateral portion of the hair follicle and contains the niche
pathway are the EDN3/EDNRB (endothelin 3 of the keratinocyte stem cells (KSCs) and mela-
ligand with its endothelin receptor B), WNT/β-- nocyte stem cells (McSCs). The presence of
catenin (Wnt1/3A with one of its mediators, McSCs is required for melanocyte renewal. After
β-catenin), and SCF/KIT (stem cell factor with induction of the McSCs, they proliferate and
its transmembrane tyrosine kinase receptor) sig- migrate toward the forming hair bulb [9]. The
naling pathways [6]. The signaling pathways cells making up this transient cell population are
involved in the development of melanocytes of called TAC (transit amplifying cells). Once the
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cells arrive at the hair bulb, they start to termi- phogenesis, and histogenesis [12]. The signaling
nally differentiate into mature melanocytes and pathways and gene regulation associated with the
express genes encoding enzymes required for morphogenetic and histogenetic functions of cad-
melanin production, including Tyr (tyrosinase) herins are not clear, but β-catenin is known to be
and Tyrp1 (tyrosinase-related protein 1), and dif- involved. Indeed, β-catenin is involved in cell-
ferentiate into mature melanocytes by birth. cell adhesion, signal transduction, and the control
During the entire developmental process, the of gene expression [13]. β-Catenin activity is
environment surrounding the melanoblasts varies tightly regulated by its tyrosine and serine/threo-
dramatically: melanoblasts are first surrounded nine phosphorylation status, depending on spe-
by the mesenchyme of the dermis, then keratino- cific kinases and phosphatases and the
cytes of the epidermis, and finally the various transduction pathways activated by soluble fac-
cells of the hair follicle, including those of the tors, such as Wnt, EGF, and IGF-II.
hair bulge, root sheath, and hair bulb. During this Melanoblasts dynamically express distinct cad-
process, melanoblasts must adapt their behavior herins during development [14]. For example, E-
based on external information (direct cell-cell and P-cadherins are weakly expressed in migrating
interactions, soluble factors, and, potentially, melanoblasts in the dermis, but E-cadherin expres-
exosomes) and their interactions with the extra- sion increases 200-fold when the cells enter the
cellular matrix; their adhesive properties must be epidermis and interact with E-cadherin-expressing
strictly regulated, both temporally and spatially. keratinocytes [15]. The weak expression of
Cadherins constitute a superfamily of cell E-cadherin in the dermis may prevent self-aggre-
adhesion molecules that have been implicated in gation and premature colonization of the epider-
cell-cell recognition and interaction, cell sorting, mis. Melanoblast entry into the hair follicles leads
cell transformation, and histogenesis [10]. to a reduction in the level of E-cadherin and an
Classical cadherins are Ca2+-dependent cell-cell increase in that of P-cadherin (Fig. 26.1).
adhesion molecules. They are transmembrane Melanoblasts in the dermis may differentiate into
proteins comprising an extracellular domain, a melanocytes, producing melanin, but their mela-
membrane-spanning region, and a highly con- nosomes are not transferred to the surrounding
served cytoplasmic portion. The extracellular cells. Dermal melanocytes express N-cadherin as
domain is composed of five subdomains (EC), the surrounding fibroblast, possibly allowing their
each of approximately 110 amino acids. Classical interaction [14]. In conclusion, these dynamic
cadherins are subdivided into two groups (type I changes in cadherin expression appear to aid the
and type II) depending upon their sequence char- melanoblasts in adapting to their local environ-
acteristics [11]. Type I cadherins include ment in the skin. In mice, melanoblast-specific
E-cadherin (E-cad or Cdh1), N-cadherin (N-cad loss of E-cad leads to a reduction in the number of
or Cdh2), P-cadherin (P-cad or Cdh3), and epidermal interfollicular melanoblasts due to a
R-cadherin (R-cad or Cdh4). Type II cadherins proliferative defect of these cells and partial loss of
include cad5 to cad12 (Cdh5 to Cdh12). Cell-cell the pigmentation of the epidermis without any
adhesion is mediated by homophilic interactions effect on coat color, as P-cad is the major cadherin
between the extracellular domains of cadherins in follicular melanocytes [16]. No coat color phe-
on adjacent cells. Cadherins are anchored within notype has been reported in mice lacking
cells by dynamic associations with catenins, P-cadherin. The inactivation of both genes in
which link them to actin filaments. The cytoplas- melanocytes has not been reported yet to evaluate
mic domain of cadherins directly interacts with the importance of E- and P-cadherin for hair folli-
either β-catenin or plakoglobin (γ-catenin). cle melanocyte function. In conclusion, E-cadherin
β-catenin or plakoglobin binds to actin filaments expression in melanoblasts is required specifically
via α-catenin. Cadherins are not only involved in for the establishment of an adequate number of
cell-cell adhesion but also cell signaling, mor- epidermal melanocytes.
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Hair shaft
Epidermis
a b
E-cadherin P-cadherin
c d
McSC
Hair bulb
Mc
Fig. 26.1 Cadherin expression in mouse epidermis and galactosidase antibody. Epidermal and hair bulb
hair follicles. Immunostaining of tail skin of 10-day-old melanocytes express both E- and P-cadherin. However the
Dct::LacZ mice using anti-E- (a, c) or P-cadherin (b, d) relative ratio of these two type 1 cadherins is inversed:
(green) and anti-β-galactosidase (red) antibodies. The epidermal melanocytes express more E-cadherin and less
Dct::LacZ melanocytes expressed β-galactosidase, allow- P-cadherin than hair bulb melanocytes
ing the detection of individual melanocytes using anti-β-
26.2.3 Melanoblast Skin Distribution whereas epidermal melanocytes continue to pro-

and Differentiation liferate and start producing mature melanosomes,
in which melanin synthesis takes place. Epidermal
Once melanoblasts have reached their final desti- melanocytes transfer melanin through their den-
nations, they differentiate into melanocytes. The drites to the surrounding cells, the keratinocytes,
distribution of melanocytes differs depending on by a mechanism, which is not fully understood.
the species. In humans, melanocytes are mostly
located in the basal layer of the epidermis,
whereas they are found mostly in hair follicles in 26.3 Homeostasis of Melanocytes
animals with fur, such as rodents. In humans,
melanocytes have been identified within fetal 26.3.1 The Epidermal Melanin Unit
epidermis as early as 50 days of gestation. The
precise mechanisms that control the organization To understand pigmentation of the skin and the
and number of melanoblasts in the epidermis factors that affect it, it is necessary to focus on
remain unknown, but the initial step is clearly the the intricate cellular and molecular interactions
distribution of migrating melanoblasts in the der- between melanocytes and keratinocytes.
mis and their ability to cross the basement mem- Epidermal interfollicular melanocytes are
brane from the dermis to epidermis [8]. Dermal surrounded by keratinocytes, with which they
melanocytes normally decrease in number during are in contact via their dendrites, at a ratio
gestation and virtually disappear by birth, of approximately 30–40 keratinocytes per
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melanocyte. This association has been called the shed and renewed, but not the melanocytes,
epidermal melanin unit [17]. The cell body of which remain mostly at the basal membrane,
the melanocyte sits on the basal lamina, and its with 5% in the suprabasal layer [16]. Dividing
dendrites come into contact with keratinocytes melanocytes are very infrequently observed in
as far away as the mid-stratum spinosum. The adult epidermis, consistent with the low number
number of keratinocytes between two basal of melanocytes lost during shedding [19]. After
melanocytes varies mostly from 3 to 8, with an 30 years of age, 10–20% of epidermal melano-
average of 6 (Fig. 26.2 and [16]). The first value cytes are lost every decade [20].
(30–40 keratinocytes/melanocytes) must be Melanocytes, keratinocytes, and dermal fibro-
considered in three dimensions, whereas the blasts communicate with each other via secreted
second value (3–8 keratinocytes/melanocytes) factors and cell-cell contact. The cross talk of the
must be considered in two dimensions. Although various signaling pathways between these cells is
the density of epidermal melanocytes varies per a complex network that controls melanocyte
square millimeter in the various regions of the homeostasis. Melanocyte-stimulating hormone
body, the number of epidermal melanocytes in (α-MSH), endothelins (EDN), basic fibroblast
human skin of all types is essentially constant at growth factor (β-FGF), nerve growth factors
an average of 1500 melanocytes per square mil- (NGF), granulocyte-macrophage colony-
limeter of the human epidermis. The major stimulating factor (GM-CSF), steel factor (SCF),
determinant of normal skin color is the activity leukemia inhibitory factor (LIF), hepatocyte
of the melanocytes, i.e., the quantity and quality growth factor (HGF), transforming growth factor
of pigment production, and its transfer to sur- beta (TGFβ), and Jagged1/2 are keratinocyte-
rounding keratinocytes, not their density [18]. derived factors that regulate melanocyte prolifer-
However, this mostly holds true only for nor- ation and differentiation [9, 21]. The production
mal, healthy skin; the number of melanocytes in of some of these factors is stimulated by ultravio-
the epidermis must be sufficient to provide skin let radiation (UVR) exposure and can be influ-
pigmentation, which is clearly affected in vitil- enced by chemical compounds, drugs, and/or
igo. Melanized keratinocytes are constantly stress.
a 12
b
Vitiligo (lesion-free area)
nb of Kc between two basal Mc
Kc
Suparabasal Mc
10 **** Basal Mc
Basal lamina
9 ± 0.4 Kc
8
n=258
Control
Kc
6 Basal Mc
Basal lamina
n=347
6 ± 0.2 Kc
Control Vitiligo
Fig. 26.2 Alteration of melanocyte-keratinocyte distribu- standard errors are indicated in black: mean 6 ± 0.3 for con-
tion in the epidermis of non-lesional skin of vitiligo trols, 9 ± 0.4 for vitiligo. A minimum of 2220 basal kerati-
patients. (a) Means of the number of keratinocytes (Kc) nocytes was analyzed for each group, and a total of 347 and
between two melanocytes (Mc) are represented by color 258 counts were performed for controls and vitiligo skin
horizontal bars (blue for control and red for vitiligo), and sections. (b) Schematic of a control and vitiligo epidermis
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26.3.2 Epidermal Melanocyte Despite their potential for renewal, the McSC
Renewal population is limited, and their number declines
during aging, resulting in graying hair in both
Melanocyte stem cell (McSC) has been identi- humans and mice. In older humans and mice,
fied in the bulge area of hair follicles as a reser- pigmented melanocytes are present in the bulge
voir for the replenishment of melanocytes that region of the outer root sheath and are associated
are lost as adults [9]. McSC in mice appears to with McSC depletion [24]. It will be of great inter-
be similar to the amelanotic population in the est to analyze whether pigmented melanocytes can
lower permanent portion of the human hair fol- be detected in vitiligo patients in the bulge of hair
licle. Histologically, McSC can be identified by follicles as an indicator of McSC depletion. The
their specific cell shape and localization in hair connection between the loss of epidermal mela-
follicles. No specific molecular marker of nocytes and McSC exhaustion in vitiligo requires
McSC has been discovered to date, but these further investigation.
cells are Dct- positive and Ki-67-negative,
retain BrdU, and have very low levels of KIT
and MITF (microphthalmia- associated tran- 26.4 Melanocyte Stability
scription factor). and Vitiligo
Repigmentation in vitiligo patients is often
observed at the hair follicles before spreading 26.4.1 The Epidermal Melanin Unit
out to generate continuous coloring to the skin. in Vitiligo
This observation supports the idea that McSC
migrates from the hair follicles to the basal layer The epidermal melanin unit is totally disturbed
and differentiates into mature epidermal mela- in depigmented areas of vitiligo patients as no
nocytes (for more information see Chaps. 30 melanocytes (or very few) are present and mor-
and 31 please check). Regions of skin in patients phological abnormalities can also be detected in
with vitiligo that lack hair follicles, such as the surrounding keratinocytes. Intriguingly, changes
palms of the hands, can occasionally become in melanocyte distribution in the basement
repopulated by melanocytes. This indicates that membrane are found in normal pigmented skin
stem cell niches are not only present in hair fol- of vitiligo patients located far from the depig-
licles but also in other skin structures such as mentated area. Indeed, the number of keratino-
sweat or sebaceous glands and the dermis. A cytes between two basal melanocytes is higher
subpopulation of dermal stem cells is able to in vitiligo pigmented epidermis than in individ-
migrate to the basement membrane of the epi- uals without vitiligo, average of nine instead of
dermis and differentiate into melanocytes [22]. six keratinocytes (Fig. 26.2 and [16]). Thus, the
The dermal melanocyte stem cells were nega- epidermal melanin unit in normal pigmented
tive for E-cadherin and N-cadherin, whereas skin of vitiligo patients is modified but insuffi-
they acquired E-cadherin expression upon con- ciently to have a visible effect on skin pigmenta-
tact with epidermal keratinocytes. The other tion. Furthermore, the reduction of basal
source of melanocytes is present in sweat glands melanocytes in vitiligo is associated with an
of volar skin both in humans and mice and can increase in the number of suprabasal melano-
provide differentiated melanocytes to the epi- cytes relative to those in a control population:
dermis [23]. Although there is clear evidence 5% versus 25% [16].
that epidermal melanocytes can be replenished
from McSC from the hair bulge, it is unknown
whether melanocyte stem cells from the dermis 26.4.2 E-Cadherin and Vitiligo
and sweat/sebaceous glands could serve as a
source of epidermal melanocytes for vitiligo The reduced number of basal melanocytes
repigmentation. associated with an increase of suprabasal
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melanocytes in vitiligo leads to a number of spe- on melanocytes is associated with oxidative or

cific questions and hypotheses. mechanical stress, melanocyte detachment is
induced in mice and in human epidermal recon-
• What is the mechanism that induces the pres- structed skin [16]. These data are in agreement
ence of suprabasal melanocytes? with the melanocytorrhagy hypothesis (see
• Why are these basal melanocytes not renewed chapter 31, is it still in?) proposing that melano-
by melanocyte stem cells? cytic detachment occurs in response to mechani-
• Are these suprabasal melanocytes alive? cal trauma [25]. Interestingly, a polymorphism
of the E-cadherin gene was recently shown to be
The presence of suprabasal melanocytes associated with vitiligo, providing further evi-
implies a cell adhesion defect in basal melano- dence of a role for E-cadherin in the etiology of
cytes that is corroborated by altered levels of this disease [26]. Another E-cadherin gene poly-
E-cadherin at the membrane of melanocytes of morphism (39 bp away from that identified for
normal pigmented skin of vitiligo patients vitiligo) is associated with Crohn’s disease [27].
(Fig. 26.3 and [16]). An inverse correlation These two SNPs are located in intron 3 of the
between the location of the melanocytes in the E-cadherin gene (Cdh1), between two exons
upper layer of the epidermis and the homoge- encoding the portions of the precursor protein
neous staining of E-cadherin at the membrane of responsible for preventing protein aggregation
melanocytes has been established, indicating before trafficking to the plasma membrane.
that a reduction of E-cadherin in melanocytes Accurate processing of the precursor domain is
correlates with melanocyte detachment. required for E-cadherin targeting to the mem-
Furthermore, when the reduction of E-cadherin brane. The SNPs associated with the Crohn’s
Dct
E-cadherin
E-cadherin
Control Vitiligo
Fig. 26.3 E-cadherin membranous staining is altered in stained with antibodies directed against Dct (red). Images
non-lesional skin of vitiligo patients. Immunofluorescence are merged to visualize E-cadherin staining in melano-
staining of E-cadherin (green) in normal skin or non- cytes more clearly. Note the presence of suprabasal mela-
lesional skin of vitiligo patients. Melanocytes were nocytes in vitiligo epidermis
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disease result in the production of a truncated similar to the alteration of E-cadherin levels seen
mRNA, probably due to an alternative splicing, at the melanocyte plasma membrane in vitiligo.
altering E-cadherin levels at the plasma mem- It is possible that the effect of E-cadherin vari-
brane [27]. Of note, some patients have both vit- ants associated with the unfolded protein
iligo and Crohn’s disease (see Chap. 13, please response pathway, present in the stressed cells of
check). Therefore, the SNP associated with both diseases, results in reduced protein levels at
Crohn’s disease results in reduced E-cadherin the plasma membrane. Importantly, the loss of
levels at the plasma membrane of epithelial cells, E-cadherin initially found only in melanocytes
Normal Vitiligo
a
Non-declared
McSC
b
Non-declared
6 Kc 6 Kc
c
Declared
9 Kc
d
Pre-micro depigmentation
e
Micro-depigmentation
Fig. 26.4 Schematic of vitiligo initiation. Proposed stresses (red arrows). The detachment of melanocytes
mechanisms leading to early loss of pigmentation in vit- increases (due to the loss of E-cadherin), and the renewal
iligo. (a, b) Upper panels show the preclinical vitiligo of the cells at the basal membrane becomes not sufficient
phase (non-declared): melanocytes have primary combi- for normal pigmentation (pre-micro-depigmentation).
natory intrinsic defects leading to reduction of membra- After stresses melanocytes can be seen in the suprabasal
nous E-cadherin in melanocytes and subsequently layer of control skin but are replaced efficiently by the
detachment and transepidermal loss of these cells. (c) The McSC. (e) The lower panel shows the late phase of vitil-
detached melanocytes are not necessarily replaced (aver- igo initiation where the skin is depigmented due to the
age of nine keratinocytes between two melanocytes absence of melanocyte. The intrinsic defects in the mela-
instead of six in the controls—“normal”) indicating a nocytes combined with environmental stresses lead ulti-
defect in melanocyte renewal from the melanocyte stem mately to the total depletion of melanocytes due to
cells (McSCs) located in the niche of the epidermal exhaustion of McSC in the most severe case and create a
appendage (e.g., hair bulge). Skin remains normally pig- micro-depigmentation. The vitiligo progression is cer-
mented even though the vitiligo is declared. (d) This panel tainly followed by an autoimmune response participating
shows the triggering phase after mechanical or chemical and accelerating importantly melanocyte destruction
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in normally pigmented skin is subsequently seen (SFD), INCa, and ITMO Cancer and is under the program
also in melanocytes and keratinocytes in and “Investissements d’Avenir” launched by the French
Government and implemented by ANR LabEx
around the depigmented lesions [28]. Altered CelTisPhyBio (ANR-11-LBX-0038 and ANR-10-
E-cadherin levels in the epidermis (mainly IDEX-0001-02 PSL).
keratinocytes) could impair epithelial integrity
and permeability, consistent with the epidermal
barrier defects observed in vitiligo patients and References
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ERRNVPHGLFRVRUJ
Oxidative Stress and Intrinsic
Defects
27
Mauro Picardo and Maria Lucia Dell’Anna
Contents
27.2 Oxidative Stress 278
27.3 Metabolic Profile in Non-lesional Skin 279
27.4 Mitochondria 280
27.5 The Systemic Oxidative Stress 281
27.6 The Possible Genetic Background 281
References 281
Abstract levels of heme oxygenase 1 in plasma have

Overall, experimental data support intrinsic been linked to the activity phase of the dis-
damage and a close link between oxidative ease and to IL-2 levels. All of these factors
stress and immune responses. For example, are involved both in stress responses and trig-
histological analysis of developing lesions gering of innate immunity. In summary,
shows NALP1 (NACHT, LRR, and PYD genetic, experimental, and clinical studies
domain-containing protein 1), IL-1, and have revealed important pathways in the
catalase expression; moreover, the expression pathogenesis of vitiligo and have identified
targets for the development of new
therapies.
M. Picardo (*) Our unified view considers the intrinsic
Cutaneous Physiopathology and CIRM, San Gallicano defect in melanocytes as the initial event. In
Dermatological Institute, IRCCS, Rome, Italy this model, oxidative stress in the melanocytes
e-mail: mauro.picardo@ifo.gov.it leads to a local inflammatory response and the
M. L. Dell’Anna activation of innate immune processes, which,
Laboratory of Cutaneous Physiopathology, in subjects with a genetic predisposition to
San Gallicano Dermatological Institute IFO, Rome,
Italy develop autoimmunity, generate melanocyte-
e-mail: marialucia.dellanna@ifo.gov.it specific cytotoxic immune responses.

ERRNVPHGLFRVRUJ
278 M. Picardo and M. L. Dell’Anna
27.2 Oxidative Stress

Key Points
• ROS are crucial and essential mediators ROS are physiologically produced during usual
of the intracellular signal transduction. metabolic cellular activities and they play a dou-
On the other hand, when the intracellu- ble role. According to a correct balanced redox
lar ROS level is excessive and uncon- status, at micromolar concentration, ROS are cru-
trolled, an overall cellular impairment cial and essential mediators of the intracellular
takes place, as that we suggest to occur signal transduction. On the other hand, when the
in vitiligo. intracellular ROS level is excessive and uncon-
• ROS also lead to impaired expression or trolled, an overall cellular impairment takes place,
activity of the antioxidant system. as that we suggest to occur in vitiligo [4–14].
• The imbalance of the prooxidants and Melanocytes are intrinsically exposed to high
antioxidant status in vitiligo has been level of toxic compounds because during the
indicated to cause increased sensitivity melanin synthesis, potentially noxious intermedi-
of melanocytes to external prooxidant ates are produced. In addition, melanocytes, due
stimuli and, over time, to induce a pre- to their anatomical localization, are specifically
senescent status. exposed to UV irradiation and physical toxic
• Probably as a consequence of the intra- agents. The early hypotheses regarding the
cellular oxidative stress, they overex- involvement of the oxidative stress, named auto-
press p53 and some of its target genes. cytotoxic and neurogenic, have suggested that
• Mitochondria are key in mediating biochemical alterations leading to the intra- or
melanocyte dysfunction: inhibitors of extracellular generation of free radicals and other
mitochondrial transition pores reduce toxic intermediates can induce melanocyte
ROS levels and cell death, transmem- degeneration [9–16].
brane potential is lost, cardiolipin pat- The attention to the potential pathogenetic
tern and respiratory chain complex role of the catecholamines comes from clinical
expression are altered, and the mito- data, which suggest that psychophysical stress
chondrial mass is increased. (relative death, school tests, work’s problems,
etc.) can be associated with the onset or the wors-
ening of the clinical manifestation of vitiligo and
by the morphological demonstration of the pres-
27.1 Introduction ence of nerve ending close to melanocytes.
Increased plasma level of dopamine and norepi-
Vitiligo is characterized by the loss of functional nephrine, as well as of the urinary metabolites
melanocytes, and multiple mechanisms might con- homovanillic acid and vanillylmandelic acid, in
tribute to this loss, including metabolic abnormali- early and active phase of the disease has been
ties, oxidative stress, generation of inflammatory reported [6, 15–23]. A direct toxic mechanism
mediators, cell detachment, and autoimmune may be due to the toxicity of the quinone and
responses [1–3]. The overall contribution of each of semiquinone moieties and oxyradicals generated
these processes is still under debate. Metabolic alter- by the oxidation of the catecholamines, and an
ations are central to current concepts in pathophysi- indirect mechanism may be related to the vaso-
ology. In our opinion, they induce an increased constriction associated with norepinephrine
generation of reactive oxygen species and suscepti- release and the subsequent production of free
bility to mild exogenous stimuli in the epidermis. radicals. Moreover, an alteration of the epidermal
This produces a biological senescent phenotype of and systemic metabolic synthetic pathway of
skin cells; leads to the release of innate immune mol- biopterins, required for the catecholamine syn-
ecules, which trigger autoimmunity; and ultimately thesis, as well as serotonin and melatonin, possi-
causes dysfunction and death of melanocytes. bly due to a genetic polymorphism, has been
ERRNVPHGLFRVRUJ
27 Oxidative Stress and Intrinsic Defects 279
reported. The alteration could lead to the accu- borders of lesions. This finding possibly explains
mulation of the intermediated product 7-tetrahy- the Koebner phenomenon (i.e., the induction of
drobiopterin, capable of inhibiting melanin new lesions by minor skin trauma) (Chap. 1.5.7)
synthesis and toxic for melanocytes. observed in patients with vitiligo [25, 30].
Besides the occurrence of an initial defect of
the activity or expression of the antioxidant com-
ponents, ROS also lead to impaired expression or 27.3 Metabolic Profile
activity of the antioxidant system. Epidermal in Non-lesional Skin
catalase levels are low, probably as a result of
H2O2-mediated deactivation of the nicotinamide Considering that a general intrinsic defect occurs
adenine dinucleotide (NADH)-binding site of the in vitiligo cells, the biological phenotype and
enzyme, as catalase mRNA expression is metabolic properties of cells derived from
unchanged. Other antioxidant enzymes, includ- normal- appearing skin might help to clarify
ing thioredoxin reductase and thioredoxin, gluta- pathogenesis. Melanocytes from non-lesional
thione peroxidase, glutathione reductase, skin show aberrant signal transduction, including
superoxide dismutases, and the repair enzymes hyperactivation of mitogen-activated protein
methionine sulfoxide reductases A and B, are kinase (MAPK) and cAMP response element-
also altered in vitiligous skin, which indicates binding protein (CREB), and modifications of
ROS generation causes widespread alteration of their membrane lipids. Moreover, probably as a
the antioxidant system [6, 7, 9–11]. consequence of the intracellular oxidative stress,
Oxidative stress compromises the function of they overexpress p53 and some of its target genes.
other cellular proteins and membrane lipids. One This expression induces a senescence-associated
of the affected proteins is tyrosine-related protein secretory phenotype (SASP), which is character-
(TRP)-1, which is important for melanin synthe- ized by the production of interleukin (IL)-6,
sis. Oxidative-driven modification of the TRP-1- matrix metalloproteinase 3, cyclooxygenase-2,
calnexin complex can lead to reduced TRP-1 and insulin-like growth factor-binding proteins 3
stability with subsequent production of toxic and 7 [31, 32]. This pre-senescent profile could
melanin intermediates. Moreover, the dysregula- be melanocyte-specific, which might explain the
tion of some metabolic pathways, even not absence of clinically manifest aging of the entire
strictly related to the melanogenetic activity, has skin. A possible hypothesis could be that
been considered [2]. mitochondrion-driven degenerative damage leads
Modification and inactivation of acetylcholin- to the specific impairment or loss of melanocytes
esterase further promote and maintain skin oxi- alone. p53 might facilitate DNA damage repair,
dative damage. Redox alterations of membrane which maintains genome stability, and can induce
lipids affect lipid rafts, which compromises the the production of POMC (encoding pro-
function of membrane receptors and electron opiomelanocortin) which can modulate, along
transfer and ATP production in mitochondria [2]. SIRT1 (NAD-dependent protein deacetylase sir-
In vitiligo, melanocytes may be thus the the- tuin-1) and FOXO1 (forkhead box protein O1),
ater of the loss of the redox balance because of the energy balance and cell metabolic processes.
the increased generation of free radical and dan- Recently, the ROS-mediated reduced activity
gerous metabolites or because of a defective anti- of MITF, the transcription factor controlling the
oxidant pattern [7, 24]. melanin synthesis pathway, has been proposed.
This imbalance of the prooxidants and anti- MITF is usually phosphorylated and activated
oxidant status in vitiligo has been indicated to by receptor-associated kinases allowing the sub-
cause increased sensitivity of melanocytes to sequent production of the melanogenetic
external prooxidant stimuli and, over time, to enzymes. To bind M-Box and then promote the
induce a pre-senescent status. Stress-dependent transcription of tyrosinase, TRP-1, and DCT,
melanocyte detachment has been observed at the MITF needs the phosphorylation in Ser73 and
ERRNVPHGLFRVRUJ
Ser49. However, the oxidation of MITF can 27.4 Mitochondria

compromise the melanogenetic pathway, as well
as the cell-cycle progression (p16INK4A- Mitochondria have been suggested as a key
dependent) and cell survival (Bcl-2-dependent). source of ROS as they mediate energy production
Finally, the oxidative damage of MITF may and oxidative stress-related aging and control
affect even its ability to antagonize TNF-a activ- apoptosis in healthy cells. How mitochondrial
ity and to bind 6BH4, further contributing to the defects impact metabolism and cell fate in vitil-
oxidative stress. The compromised assembly of igo is under debate. Several lines of evidence
the TRP-1 in a multiproteic complex may affect suggest that mitochondria are key in mediating
the stability of the TRP-1 itself with subsequent melanocyte dysfunction: inhibitors of mitochon-
production of the melanin toxic intermediates. drial transition pores reduce ROS levels and cell
Moreover, the chemical compounds may allow death, transmembrane potential is lost, cardio-
the increased membrane expression of hsp70, lipin pattern and respiratory chain complex
favoring the activation of the dendritic cells and expression are altered, and the mitochondrial
of the immune system. mass is increased.
An altered intracellular membrane-dependent In other degenerative diseases, mitochondrial
signal transduction may take place in vitiligo dysfunction has been connected to cell damage.
melanocytes, possibly affecting the response to To date, a role for the Bcl-2 protein family as
growth factors and dangerous stimuli. A marked modulators of cell survival, metabolism, and
membrane lipoperoxidation, together with loss mitochondrial dynamics is emerging.
and dislocation of the cardiolipin across the mito- Downstream, cytochrome C participates both in
chondrial membrane, has been described in cul- electron transfer and ATP production and acti-
tured vitiligo melanocytes, which could be the vates caspases during apoptosis. Sirtuins are
cause of the increased mitochondrial ROS pro- another example of the cross talk between
duction and of the possible reduced response to metabolism and cell fate. Damaged mitochon-
the specific growth factors. dria modulate sirtuin 1, phosphoinositide
The oxidative-mediated damage has been sug- 3-kinase/Akt, protein kinase A, mTOR (mam-
gested to be involved even in the alteration of malian target of rapamycin), and then nuclear
other epidermal cells, compromising their sur- PGC1 (PPARγ coactivator 1), CREB (cyclic
vival/proliferative potential, and also production AMP-responsive element-binding protein), and
of melanocyte-specific growth factors. Lesional FOXO-1 restoring cellular metabolic normal sta-
keratinocytes, in fact, can present vacuolar degen- tus. As some of the key steps linking mitochon-
eration, which has been associated with the dria to cell fate are defective in vitiligo cells, this
increased exposure to H2O2, and produce an inad- evaluation of cell metabolism can illuminate our
equate amount of the specific melanocyte growth understanding of vitiligo. Altered arrangement
factors, including membrane-bound SCF, which of the ETC proteins affects the ATP level and
could lead to melanocyte apoptosis (Chap. 2.3.8). subsequent mitochondria-nucleus cross talk by
Moreover, keratinocytes themselves, following activating some key mediators, including
the exposure to prooxidant stimuli, can produce PGC1α, resulting in increased compensatory
and release high amount of proinflammatory mitochondrial mass and potentially reverting the
cytokines, such as IL-6, IL-1a, and TNF-a, lead- metabolic impairment [40–50]. Moreover, in
ing to lymphocyte recruitment. However, TNF-a some chronic degenerative processes, the
was reported to directly interfere with some mito- increase in mitochondrial content is an adaptive
chondrial activities through the production of per- mechanism to meet the increased energy demand
oxides (including hydrogen peroxide) leading to a [41]. The inability of vitiligo cells to further
mitochondria-dependent cell death or, at least, to cope with the energetic demand provides the
the activation of the inflammatory genes, through metabolic basis for the pre-senescent phenotype
the nuclear translocation of NF-kB [33–39]. progress [31].
ERRNVPHGLFRVRUJ
27.5 T
he Systemic Oxidative intermediates may be due to an impaired mela-
Stress nosome transport, inner coating of the melano-
some membranes, or defective expression of
In red blood cells and peripheral blood mononu- proteins regulating melanogenic enzyme synthe-
clear cells (PBMC), a defective activity of cata- sis. A catalase polymorphism (T/C SNP), lead-
lase and GPx, associated with an increased ing to an incorrect subunit assembly, was
production of lipid peroxidation by-products, has reported in Caucasian population. A reduced
been described. Melanocytes and PBMC show expression of VIT1 (22q11) could account for an
altered antioxidant pattern, with low activity of altered G/T mismatch repair, calcium homeosta-
catalase and glutathione peroxidase and high sis, and protein degradation. In acrofacial vitil-
activity of SOD and xanthine oxidase, associated igo, COMT polymorphism (G/A, val/met) could
with the presence of index of lipid peroxidation. be responsible for a thermo-sensibility of the
The alteration of the antioxidant apparatus in enzyme with a subsequent high production of
peripheral blood cells has been suggested to be quinones. However, the true value of the differ-
due to the H2O2 produced inside the epidermis ent genetic studies has to be further confirmed,
and systemically distributed by the blood. because they are frequently carried out on a lim-
However, the ROS production could take place ited number of subjects and without validation
inside the nonepidermal cells, independently on by other authors [2, 8, 51, 52].
melanocyte-specific metabolisms. In fact, the
increased ROS production in PBMC has been
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Immunity/Immunopathology
28
Kirsten C. Webb, Steven W. Henning,
and I. Caroline Le Poole
Contents
28.1 Establishment of Vitiligo as an Autoimmune Disease Entity 286
28.2 Cellular Immunity in Vitiligo 287
28.3 Antigen-Presenting Cells in Vitiligo 288
28.4 Heat Shock Proteins in Immune Activation 289
28.5 T Cell Subset Imbalance in Vitiligo 290
28.6 T Cell Trafficking 290
28.7 Cytokines in Vitiligo Skin 291
28.8 Additional Players in Cellular Immunity 291
28.9 Opportunities for Immunotherapy 292
28.10 Vitiligo and Melanoma T Cell Responses 295
References 296
Abstract
For appreciable time, the pathophysiology
leading to ultimate melanocyte destruction
remained uncertain. This is because skin-
infiltrating T cells involved in melanocyte
loss are few in number and are only observed
in actively depigmenting skin; thus, such
K. C. Webb
Division of Dermatology, Department of Medicine, infiltrates are easily overlooked. Moreover,
Loyola University Chicago, Maywood, IL, USA T cells were more difficult to distinguish
S. W. Henning before antibodies became readily available
Lurie Comprehensive Cancer Center, Northwestern for immunohistology. Ample support exists
University, Chicago, IL, USA for autoimmunity as a chief etiopathological
I. C. Le Poole (*) factor in vitiligo: susceptible individuals
Professor of Dermatology, Microbiology and exhibit polymorphisms in immune regulatory
Immunology, Northwestern University at Chicago, genes which promote autoimmunity in the
IL, USA
e-mail: caroline.lepoole@northwestern.edu cutaneous microenvironment; additionally,

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286 K. C. Webb et al.
vitiligo has a well-established association

with other autoimmune diseases. Among the • Promising immunotherapeutic strategies
possibly involved cell populations, are being considered for vitiligo treat-
Langerhans cells might contribute to depigment including modified HSP70 delivery,
mentation on-site (perhaps through contin- chemokine receptor blockade, JAK-STAT
ued melanocyte antigen presentation to inhibitor application, adoptive Treg trans-
cytotoxic T cells), thereby preventing viabil- fer, and anti-cytokine therapies.
ity of any melanocytes attempting to repopu- • Vitiligo development is considered a
late the skin. The HSP- native protein positive prognostic factor in melanoma,
complexes can trigger a local immune as it serves as a marker of generation of
response directed at the cells from which the robust anti-melanocyte tumor responses.
native proteins originate. Upon melanocyte
stress and subsequent HSP70i release, anti-
gen-presenting cells will recruit an initial
cohort of melanocyte-reactive T cells that 28.1 Establishment of Vitiligo
produce IFN-γ upon antigen recognition. as an Autoimmune Disease
This would lead to CXCL10 production and Entity
further recruitment to the epidermis. The
absence of Tregs in vitiligo skin is likewise The disease course of vitiligo is variable, and it
best explained by differential chemokine affects between 0.5% and 1% of the population
expression in lesional skin, mainly involving [1]. The clinical finding of depigmented skin
CCL22. patches correlates with the finding of nearly
complete absence of melanocytes histologically
[2, 3]. This observation prompted studies that
would explain disease etiology in terms of mela-
Key Points nocyte destruction rather than suppression of
• Progressive depigmentation in vitiligo pigmentation. For an appreciable time, the
relies on skin-infiltrating cytotoxic T cells pathophysiology leading to ultimate melanocyte
specific for melanocyte self-antigens. destruction remained uncertain. This is because
• Melanocytes in perilesional skin are also skin-infiltrating T cells involved in melanocyte
sensitive to self-reactive antibodies. loss are few in number and are only observed in
• Regulatory T cells (Tregs) are present in actively depigmenting skin; thus, such infiltrates
decreased numbers in vitiligo-affected are easily overlooked. Moreover, T cells were
skin. more difficult to distinguish before antibodies
• Stress proteins including inducible heat became readily available for immunohistology
shock protein 70 (HSP70i) link initial [4, 5], and, initially, most attention was directed
skin trauma to the adaptive immune toward possible humoral involvement in vitiligo
responses that follow. [6, 7]. Observations including the transmission
• Interferon-gamma (IFN-γ) signaling of disease through adoptive transfer of antibod-
through the JAK-STAT pathway drives ies [8, 9], as well as reduced surface expression
vitiligo pathogenesis by recruiting cyto- of factors such as decay-accelerating factor
toxic T cells to the skin. (DAF) that otherwise protects from complement-
• Stress protein upregulation, activation mediated destruction, also led to antibodies
of antigen-presenting cells, recruitment being considered as the culprits in melanocyte
of melanocyte-reactive T cells, and a destruction [10]. For the morphology of cultured
paucity of regulatory T cells are inter- melanocytes required for such studies, see
twined phenomena that lie at the heart Fig. 28.1. However, as the target antigens
of vitiligo pathogenesis. identified by these studies are not necessarily
expressed in the cell membrane, anti-melanocyte
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28 Immunity/Immunopathology 287
a b
Fig. 28.1 Dendritic morphology of recently plated mela- media. Note that vitiligo melanocytes exhibit multiple,
nocytes in culture. Cells were isolated from (C) neonatal branched dendrites (arrows) in contrast to the more bipo-
foreskin or (V) a scalp biopsy from a vitiligo patient by lar morphology of healthy neonatal melanocytes. K
overnight enzymatic treatment and plated in replete keratinocytes
antibodies were ultimately considered an epi- 28.2 Cellular Immunity in Vitiligo

phenomenon of the disease. Since B cell activa-
tion and T cell activation are intimately related Ample support exists for autoimmunity as a chief
and mutually supportive [11–13], renewed atten- etiopathological factor in vitiligo. As discussed
tion to B cell and antibody involvement is to be above, susceptible individuals exhibit polymor-
expected. phisms in immune regulatory genes which pro-
While many environmental factors can con- mote autoimmunity in the cutaneous
tribute to disease precipitation, the consensus microenvironment. Additionally, vitiligo has a
reached by the Vitiligo European Task Force well-established association with other autoim-
(VETF) is that autoimmunity contributes to all mune diseases [6], the most common of which is
cases of vitiligo [14]. Interestingly, this includes Hashimoto’s thyroiditis [21]. In fact, this associa-
segmental vitiligo [15], which was initially tion is common enough to warrant checking anti-
considered a developmental defect [16]. Vitiligo thyroid antibodies, including antithyroid
likely manifests itself when certain environ- peroxidase antibody and anti-thyroglobulin levels,
mental factors affect individuals with a genetic in addition to baseline thyroid-stimulating hor-
predisposition for the condition. Interestingly, mone (TSH) levels in newly diagnosed vitiligo
many of the proposed vitiligo susceptibility patients [22, 23]. Other associated autoimmune
genes have immune modulatory functions, diseases include alopecia areata, diabetes mellitus
including interleukin- 2 receptor alpha chain type I, pernicious anemia, and Addison’s disease
(IL2RA), ubiquitin- associated and SH3 [21] (see Chap. 13). Some of the more pertinent
domain-containing A protein (UBASH3A), and findings supporting contributory autoimmune
C1q and tumor necrosis factor-related protein mechanisms in vitiligo involve the inflammatory
(C1QTNF6) [17]. Human leukocyte antigens infiltrates consistently identified in newly vitiligo-
(HLA)-A2 [17, 18] and HLA-DR4 [17, 19] afflicted skin. Specifically, lymphocytic infiltrates
have also been linked with vitiligo develop- exhibiting a decreased ratio of CD4+/CD8+ T cells
ment. Indeed, MHC involvement is a hallmark have been identified in perilesional areas of
of autoimmunity [20]. In the following sec- patients with active depigmentation [24, 25]. The
tions, we describe the interplay between the cytotoxic (CD8+) T cells present in perilesional
cutaneous microenvironment and immunologic areas are activated [26] and are located in close
factors that lead to ultimate melanocyte destruc- proximity to melanocytes and melanocyte frag-
tion in vitiligo. ments in the basal layer of the epidermis
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a b
Fig. 28.2 T cells in vitiligo and melanoma. Sections of T cells in vitiligo skin with some cytotoxic T cells
(a) depigmenting vitiligo skin and (b) a melanoma tumor approaching the basal layer of the epidermis. A larger pro-
metastasis were stained with antibodies to CD3 (blue) and portion of the tumor-infiltrating T cells belongs to the
CD8 (red). Note infiltrates consisting of primarily CD8+ CD4+ subset of helper and regulatory T cells (blue)
(Fig. 28.2). Notably, their presence correlates with receptors on resident dendritic cells, resulting in
melanocyte disappearance [25]. These cytotoxic T DC maturation and antigen presentation. This
maturation involves a marked increase in a num-
cell infiltrates are found in perilesional skin of vit-
iligo patients [24], suggesting a role in melanocyte ber of MHC class II and costimulatory molecules
destruction. Indeed, these skin-infiltrating T cells on the DC cell surface [33] and allows DCs to
are reactive with melanocyte self-antigens [27, process antigens and activate T cells efficiently
28]. Circulating melanocyte antigen-specific T [34]. Two important receptor families which com-
cells have been found in increased levels in vitiligomonly stimulate DC maturation are the toll-like
patients as well [29], but, given their location, thereceptors (TLRs) and tumor necrosis factor (TNF)
skin-homing T cells are of greater pathogenic sig- receptors [35–39]. Dendritic cells then endocy-
nificance. Further supporting the role of antigen- tose, process, and present antigenic peptides on
specific T cells in vitiligo pathogenesis is their the cell surface in the context of major histocom-
epidermal tropism [30, 31]. patibility complex (MHC) molecules [40]. After
migrating to skin-draining lymph nodes, they
interact with, activate, and recruit cytotoxic and
28.3 Antigen-Presenting Cells helper T cells specific to the antigens presented
in Vitiligo [41, 42]. Thus, antigen-presenting cells govern
the type of helper T cell response elicited. Through
The development of cutaneous immune responses cross-presentation, melanocyte-specific antigens
begins with antigen-presenting cells (APCs) displayed via MHC class I molecules give rise to
residing in the epidermis and dermis. Indeed, the cytotoxic T cell cohort known to mediate
these APCs ultimately promote T cell activation, melanocyte loss in vitiligo. Interestingly, a viral
which, in turn, begets both further T cell responses trigger may exist in some cases of vitiligo [43].
(cellular immunity), as well as B cell activation Langerhans cells are epidermal antigen-
and antibody formation (humoral immunity). presenting cells that form the first line of defense
Numerous types of professional antigen-against foreign antigens entering via the skin.
presenting cells reside in the skin, including mac- They can stimulate CD4+ T cells [44] and are
rophages, Langerhans cells (LC), and dermal largely responsible for inducing contact hypersen-
dendritic cells [32]. Dendritic cells (DCs) play a sitivity reactions [45]. Sensitization to contact
particularly important role in cutaneous immune allergen was not observed in Langerhans cell-
responses by patrolling for pathogens. Pathogenic deficient skin [46]. The role of Langerhans cells in
signals such as microbial peptides interact with vitiligo is not as clearly established. Interestingly,
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N P
Fig. 28.3 Abundance of CD36+ cells in perilesional cytes, yet the morphology and location of the majority of
vitiligo skin. Overexpression of the thrombospondin cells observed in the dermis of this patient’s non-lesional
receptor CD36 has been associated with monocyte-to- and perilesional skin (detectable as a red AEC precipitate,
macrophage differentiation and activation of the phago- red arrows) support the reported abundance of macro-
cytic process. CD36 can be expressed by other cells phages in depigmenting vitiligo skin (P) as compared to
including thrombocytes, endothelial cells, and melano- non-lesional skin (N)
Langerhans cells reposition themselves, settling Once activated by interferon-gamma (IFN-γ) [54],
into the basal layer of the epidermis in melano- macrophages secrete proinflammatory cytokines.
cyte-depleted vitiliginous skin [47]. Furthermore, Among these, TNF-α can contribute to melano-
repigmentation in response to treatment can be cyte apoptosis [55]. Moreover, phagocytic macro-
associated with Langerhans cell depletion [48]. phages [56] generate proinflammatory cytokines,
Thus, Langerhans cells might contribute to depig- such as IL-8, when engulfing apoptotic cells [57].
mentation on-site (perhaps through continued They may also serve as APCs to promote contin-
melanocyte antigen presentation to cytotoxic T ued activation of autoreactive T cells. Taken
cells), thereby preventing viability of any melano- together, there is ample cause for further studies
cytes attempting to repopulate the skin. This model into the pathogenic role of macrophages in
could be likened to a delayed type hypersensitivity vitiligo.
response, whereby the melanocyte self-antigens
represent the “contact allergen.” Indeed, peptide-
pulsed Langerhans cells can stimulate and expand 28.4 H
eat Shock Proteins
CD8+ MART-I specific T cells [44]. However, as in Immune Activation
Langerhans cells are relatively inefficient at pro-
cessing antigen [49, 50], their actual role in recruit- Heat shock proteins (HSPs) play an integral role
ing melanocyte- reactive T cells and initiating in the development of several autoimmune dis-
disease remains to be firmly established. eases, including vitiligo [58, 59]. Expression of
Macrophages have been implicated in autoim- HSPs is induced by stress [60, 61]. HSPs bind
mune disease development [51], and diabetes will native proteins to prevent misfolding and cellular
not develop in macrophage-deficient mice [52]. apoptosis [62]. In addition to facilitating cellular
Macrophages may likewise be pathogenic in vitil- self-preservation, these HSP-native protein com-
igo as they consistently infiltrate depigmenting plexes can also trigger a local immune response
perilesional vitiligo skin (Fig. 28.3), and their directed at the cells from which the native pro-
appearance correlates with melanocyte loss [24, teins originate [63]. Once the HSP-protein com-
25]. However, the role of macrophages in vitiligo plexes are released into the microenvironment,
has not been clearly established [53]. Macrophages they bind receptors located on antigen-presenting
may phagocytize already apoptotic melanocytes or cells [64, 65]. Surrounding APCs subsequently
may actively contribute to melanocyte destruction. process the bound peptides and cross-present
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them to CD8+ T cells [66], eliciting immune type I cytokines in response to melanocytes, again
responses toward the chaperoned antigens. indicative of cytotoxic T cell involvement [28].
Inducible heat shock protein 70 (HSP70i) can In addition to increased numbers of skin-
also be secreted from live cells under stress as a homing effector T cells [83], decreased skin
“chaperokine” [67]. HSP70 and other stress pro- homing of their inhibitory counterparts, the regu-
teins can activate dendritic cells and subsequent latory T cells (Tregs), is observed [87]. Others
T cell responses, leading to autoimmunity. have reported a decreased abundance of Tregs in
HSP70 mediates development of autoimmune the circulation [88] or mentioned that Treg func-
diabetes in mice [68] and likely plays a role in tion may be reduced among circulating Tregs
other autoimmune diseases as well. [89], but such deficiencies are expected to accom-
Overexpression of inducible HSP70 is sufficient pany more systemic autoimmune consequences.
to precipitate vitiligo disease [69], and vitiligo Decreased numbers or impaired function of Tregs
does not develop in mice that lack expression of have been established in systemic autoimmune
HSP70i [70]. Importantly, the stress protein is disease [90]. This deficiency can well explain
overexpressed in vitiligo skin [71, 72]. These why tolerance to self-antigens is inadequately
findings support a critical role for HSP70i in T maintained [91]. The T cell imbalance in vitiligo
cell-mediated melanocyte destruction. skin thus creates the perfect environment for
Stress exposures that precipitate and worsen depigmentation. Destruction of melanocytes in
vitiligo include trauma to the affected area (koe- the skin not only results in clinical depigmenta-
bnerization) [73], psychological stress [74], UV tion but also leads to a large number of resident
irradiation (oxidative stress) [75], and exposure memory T cells capable of recognizing melano-
to bleaching phenols [76]. HSP70 is upregulated cytic self-antigens [92, 93]. These self-reactive T
in melanocytes after exposure to bleaching phe- cells have been found to persist in the skin long
nols [77], which have a well-established role in after melanocyte destruction [93]. This might
precipitating and worsening depigmentation [76, well explain disease relapse in patients following
78]. Other heat shock proteins are less likely to periods of repigmentation.
induce vitiligo as they are released only after cell
death [79]. HSP70i is unique in that it is secreted
by viable cells under stress, including melano- 28.6 T Cell Trafficking
cytes [80–82], which are ultimately later killed as
the result of HSP70i-mediated cellular immune In order for vitiligo to develop, melanocyte-
activation. specific cytotoxic T cells must migrate toward the
epidermis in response to chemoattractant cyto-
kines. The presence of IFN-γ is crucial for active
28.5 T
Cell Subset Imbalance depigmentation to ensue [94]; it induces the
in Vitiligo expression of the Th1 chemokine, CXCL10 [95],
which is produced by numerous cell types,
Infiltrates with decreased CD4+/CD8+ T cell ratios including keratinocytes, macrophages, fibro-
are found in actively depigmenting vitiligo patient blasts, neutrophils [95, 96], and, importantly,
skin [24, 25, 83]. T cells infiltrating vitiligo skin activated T cells [97, 98]. Upon melanocyte stress
were shown to express the cutaneous lymphocyte and subsequent HSP70i release, antigen-
antigen (CLA), a skin-homing receptor [25]. presenting cells will recruit an initial cohort of
Circulating T cells in vitiligo patients are reactive melanocyte-reactive T cells that produce IFN-γ
with several melanocyte-specific antigens, includ- upon antigen recognition. This would lead to
ing gp-100, tyrosinase, and Melan-A/MART-1 CXCL10 production and further T cell recruit-
[83, 85]. Moreover, a T cell receptor reactive with ment to the epidermis. Indeed, neutralization
the gp100 antigen has been characterized and of CXCL10 prevented depigmentation and
cloned from patient skin [86]. T cells derived promoted repigmentation in mice [99]. Aside
from vitiligo patient skin secrete predominantly from T cells, CXCL10 can recruit monocytes/
ERRNVPHGLFRVRUJ
macrophages and natural killer (NK) cells [96]. inhibits melanocyte proliferation in vivo [110]
Yet, the primary contribution of CXCL10 to vit- and promotes melanocyte apoptosis in vitro [111].
iligo development lies in skin recruitment of self- However, its presence is not required for disease
reactive T cells expressing its receptor CXCR3, development, as vitiligo development ensued in
as found in vitiligo patient skin [99]. TNF-α knockout mice [107]. Cytotoxic T cell-
The absence of Tregs in vitiligo skin is like- mediated apoptosis occurs through either the per-
wise best explained by differential chemokine forin-granzyme pathway [112] or the Fas/Fas
expression in lesional skin. Cells expressing the ligand pathways [112–114]. Because melanocytes
Treg chemoattractant CCL22 are present in proved resistant to Fas ligand-mediated cell death
markedly decreased numbers in vitiligo as com- [115], and most perilesional T cells in vitiligo
pared to control skin [82]. CCL22 is secreted by skin are perforin and granzyme-B immunoreac-
macrophages and dendritic cells [100]. As the tive [25], this has implicated perforin-granzyme-
CCL22 receptor, CCR4, is expressed in similar mediated cell death in vitiligo. However, mice
amounts by circulating Tregs from patients and knockout for perforin can still develop vitiligo
controls, the decreased amounts of CCL22 may [100, 107], as can granzyme knockout mice [115].
be primarily responsible for the significant under- This implies that neither mechanism is exclu-
representation of Tregs in vitiligo skin. sively responsible for depigmentation.
IL-17 is a cytokine with controversial involve-
ment in vitiligo pathogenesis. IL-17 is produced
28.7 Cytokines in Vitiligo Skin by T cells and natural killer cells [116] and is
increased both in vitiligo patient serum and in the
Activated perilesional vitiligo T cells secrete cyto- lesional skin [117]. Furthermore, IL-17A mRNA
kines and chemokines, which either propagate and IL-17A+ T cells were present in increased
immune mechanisms or contribute directly or quantities in perilesional compared to non-
indirectly to melanocyte destruction. Vitiligo lesional vitiligo skin [118]. In mice, adoptive
development is critically dependent on IFN-γ in transfer of Th17 cells induced vitiligo, support-
mouse models of vitiligo [94, 101]. Similarly, T ing a possible role for their cytokines, including
cells derived from vitiligo patient skin secrete IL-17, in vitiligo pathogenesis. However, a role
IFN-γ in response to melanocytes, again indicative for IFN-γ cannot be ruled out [119]. Thus, focus-
of a type 1 cytokine response and cytotoxic T cell ing on the blockade of production or action of the
involvement [28]. IFN-γ-responsive effector che- latter cytokine may be of greater utility.
mokines, including CXCL9, CCL5, and especially
CXCL10, are reportedly elevated in the vitiligo
skin [102] and can serve to facilitate T cell traffick- 28.8 A
dditional Players in Cellular
ing toward resident melanocytes. IFN-γ also Immunity
increases T cell expression of CXCR3, activates
macrophages, and increases ICAM expression on Importantly, melanocytes from vitiligo patients
endothelial cells [54, 103–105]. Each of these fac- are increasingly sensitive to cellular stress, as
tors can facilitate melanocyte destruction. evidenced by dilation of the endoplasmic reticu-
Importantly, IFN-γ also inhibits Treg generation lum [120] and increased levels of oxidative
through STAT1-signaling [106]. However, in mice byproducts [121–123]. When melanocytes
knockout for IFN-γ, spontaneous depigmentation release stress signals into the microenvironment
was restored when Tregs were depleted from the (reactive oxygen species and HSP70i), this acti-
circulation [107], supporting the concept that vates pattern recognition receptors (PRRs), such
IFN-γ involvement is not the sole pathogenic cyto- as Toll-like receptors (TLRs) [124]. In addition to
kine in vitiligo progression. induction of adaptive immune responses via den-
TNF-α contributes to vitiligo pathogenesis by dritic cell activation, there is evidence to support
supporting cytotoxic T cell development [108] that HSP70 also stimulates local innate immune
and enhancing IFN-γ secretion [109]. TNF-α also responses. Indeed, HSP70+ exosomes were found
ERRNVPHGLFRVRUJ
to stimulate activation and migration of natural ability of APCs to activate Th1 cells [134],
killer (NK) cells in vitro [125]. This likely trans- inhibits development of delayed type hyper-
lates to human disease, as patients with vitiligo sensitivity responses [135], and decreases T
were found to have increased amounts of natural cell production of type I cytokines, including
killer cells in both lesional and non-lesional skin IL-12, IL-8, and interferon-γ [136].
compared to their non-vitiligo counterparts [126]. Importantly, UVB was shown to inhibit phos-
This likely renders vitiligo patients capable of phorylation of STAT-1, inhibiting IFN-γ sig-
generating robust innate immune responses. naling [137]. Narrowband UVB (nb-UVB)
Interestingly, NKs taken from lesional skin of also markedly increases lesional and perile-
vitiligo patients expressed high levels of gran- sional abundance of the Treg transcription fac-
zyme B [126], the serine protease that can medi- tor FoxP3 [138], implying that nb-UVB
ate melanocyte destruction. Inflammatory increases the number of Tregs in the cutane-
dendritic cells are also increased in vitiliginous ous environment. This likely contributes to the
skin [59, 72] and are induced by HSP70i in vitro observed treatment success of nb-UVB in the
[127]. Gene transcription evaluation in Smyth clinic [139]. The immunosuppressive proper-
chickens that develop spontaneous vitiligo ties of UVR are evidenced by blunted T cell
revealed increased transcription of innate immune immune- mediated responses as well as
response genes in response to oxidative stress decreased immune surveillance of UV-induced
[128], further supporting the role of innate skin cancers following exposure [140–144].
immune responses in vitiligo. • In addition to its immunosuppressive effects,
The role of humoral immunity in vitiligo UVR has direct effects on melanocyte func-
pathogenesis may be less contributory than cel- tion. Specifically, UVR increases melanosome
lular immune responses, but it likely fuels dis- accumulation and transfer to keratinocytes
ease progression. Vitiligo patients have elevated [145]; treatment with UVR also achieves clin-
anti-melanocyte antibody titers [29, 129]. Such ical repigmentation. Narrowband UVB
antibodies could mediate cytotoxicity toward resulted in increased keratinocyte release of
melanocytes [130]. Moreover, B cells may be bFGF and ET-1, which induce melanocyte
involved in propagating T cell activation [11–13] proliferation; nb-UVB also resulted in
to drive depigmentation. A noteworthy observa- increased melanocyte expression of p125FAK
tion to discriminate between causative and and MMP-2, which enhance melanocyte
bystander roles for autoantibodies is that of rarely migration [146]. Therefore, UVR may induce
reported congenital vitiligo [131, 132], where melanocyte stem cell proliferation, differenti-
depigmentation was postulated to result from ation, and migration from their reservoir site
transplacental transfer of melanocyte- specific upon exposure. The source of repigmenting
IgG antibodies in utero. melanocytes is presumably the hair follicle,
which would clinically explain the appearance
of perifollicular repigmentation as the first
28.9 Opportunities sign of treatment response in UVR-treated vit-
for Immunotherapy iligo patients. Inactive (Dopa-negative, non-
melanin-producing) melanocyte stem cells are
• Phototherapy preserved in the outer root sheath of the hair
• Ultraviolet radiation (UVR) has immunosup- follicles of vitiligo skin [147] and do not yet
pressive properties and is utilized to treat express the target molecules recognized by
many skin conditions including psoriasis, pathogenic T cells. When clinical repigmenta-
atopic dermatitis, cutaneous T cell lymphoma, tion was noted, the first signs microscopically
and vitiligo [133]. Immunosuppression is were an increased number of melanocytes in
mediated by keratinocyte secretion of Th2 the outer root sheath of the hair follicle,
cytokines, primarily IL-10 and IL4 [134]. migrating toward the epidermis and undergo-
Specifically, IL-10 appears to decrease the ing maturation [147]. UVA and nb-UVB
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Fig. 28.4 Immunotherapeutic approaches for vitiligo. (4) Such T cell activation can be inhibited by JAK inhibi-
Stressed melanocytes can secrete inducible HSP70. (1) tors. Within the skin, activated T cells will secrete cyto-
Activation of dendritic cells can be inhibited by overex- kines that can promote ongoing autoimmunity. (5)
pressing a modified version of inducible heat shock pro- Inhibitors of type 1 cytokines can neutralize such immune
tein, namely, HSP70iQ435A. Migratory dendritic cells enhancement. The local environment is conducive to effec-
transporting melanocyte-specific antigens can activate and tor responses in the absence of sufficient regulatory T cells.
recruit T cells from draining lymph nodes. (2) However, (6) By topical application of steroids or calcineurin inhibi-
rapamycin can favor the development of Tregs over inflam- tors, regulatory responses are encouraged, and autoim-
matory IL-17-producing T cells and prevent effector T cell mune responses can be brought to a halt in early stages of
development and recruitment. (3) By introducing inhibi- disease. (7) Adoptive transfer of Tregs can similarly coun-
tors of chemokine receptors such as anti-CXCR3, migra- terbalance ongoing effector responses. In preclinical mod-
tion of effector T cells to the skin can be avoided. When els, most therapeutics not only halt depigmentation but
encountering their target antigens presented by dendritic also allow for subsequent repigmentation of the skin if a
cells or upon arrival in the skin, T cells become activated. reservoir of stem cells is available
(311 nm) and excimer laser (308 nm) have all ient body sites. Therefore, development of
been employed and have each met with vary- additional immunotherapies is prudent. The
ing degrees of success in treating vitiligo principles of UV-based and other immuno-
patients [148, 149]. While phototherapy is an therapeutic strategies are depicted in Fig. 28.4.
efficacious and well-tolerated treatment • Modified HSP70
option for vitiligo [139], it carries some limi- • To be successful, treatment should both halt
tations including limited accessibility to ongoing melanocyte destruction and stimu-
patients and its blunted efficacy in more resil- late repigmentation. HSP70 lends way
ERRNVPHGLFRVRUJ
to melanocyte destruction by activating • JAK inhibitors

dendritic cells, which subsequently leads to • Janus kinases (JAKs) are tyrosine kinases
T cell recruitment and an anti-melanocyte which transmit extracellular cytokine signals
immune response. HSP70iQ435A is a variant to the intracellular space via their association
of inducible HSP70i of potential therapeutic with cytokine receptors [157]. Through their
benefit in vitiligo treatment [71] (=Sci. activation of the STAT transcription pathway
Transl. Med. 2013). Vitiligo-prone mice (JAK-STAT pathway) [158], the JAK kinases
treated with DNA encoding HSP70iQ435A mediate signaling of type I cytokines and
exhibited markedly reduced depigmentation interferons [159–161]. This ultimately affects
[69]. This treatment is currently being lymphocyte activation and proliferation [160,
applied to Sinclair swine with human-like 162, 163]. There are four mammalian JAK
skin and vitiligo lesions that develop in kinases [160, 161]. JAK 1 and 2 are clinically
response to regressing melanomas [148]. In relevant to vitiligo, as they mediate IFN-γ sig-
this application, the gene gun has been nal transduction [164, 165]. Transcription of
replaced by needle-less injectors, which are IFN-γ-inducible genes leads to CXCL10 pro-
more translatable to use in the clinic and duction [166]. A JAK 1/3 inhibitor is currently
which are less traumatic than the gene gun FDA-approved for the treatment of moderate
delivery method [149]. The latter is impor- to severe rheumatoid arthritis. Oral treatment
tant in vitiligo given its tendency to koebner- has also been successfully used for alopecia
ize. Such studies as well as further safety areata [167] and psoriasis [168], and systemi-
testing will be have to be completed before a cally administered treatment can prevent alo-
clinical trial can be considered. By prevent- pecia areata in mice [169]. Randomized
ing dendritic cell activation, autoimmunity controlled trials are required to further define
is prevented, and repigmentation can occur their utility and safety, but given the mecha-
in the absence of infiltrating T cells. nistic relevance of JAK inhibition to vitiligo
• Inhibiting T cell recruitment pathogenesis, their use to treat vitiligo holds
• Effector T cells engage the chemokine recep- promise.
tor, CXCR3, and its ligand, CXCL10, to • An exciting new treatment proposal entails the
migrate to the skin [99, 101, 152]. Recently, use of lipid-lowering statins to treat vitiligo.
serum CXCL10 levels have been found to cor- HMG-CoA reductase inhibitors can target a
relate with progressive disease, whereas levels different aspect of the JAK-STAT signaling
decreased following successful treatment pathway than the JAK inhibitors, by inhibiting
[153]. It follows that one proposed treatment STAT1 activation [170]. Indeed, statins can pre-
strategy includes blocking the chemokine vent progression of vitiligo and promote repig-
receptor, CXCR3, from binding its ligand mentation in mice [171] and likewise promoted
[154]. This intervention has proven efficacious repigmentation in a vitiligo patient [172].
in vitiligo mice in preventing and reversing • Treg-based therapy
disease [99]. CXCL10 is secreted by cytotoxic • The paucity of skin-homing regulatory T cells
T cells in vitiligo skin [97, 98]. Though anti- [87] promotes a local environment whereby
CXCL10 monoclonal antibodies have been cytotoxic T cells may freely attack melano-
tested in treating rheumatoid arthritis and cytes. Cutaneous Treg recruitment fails in vit-
ulcerative colitis [155, 156], their efficacy has iligo patients due to decreased levels of the
not yet been tested in vitiligo patients. It is pos- Treg chemokine CCL22 [87]. In fact, replen-
sible that chemokine depletion will be rela- ishing CCL22 was sufficient to overcome
tively less successful due to its redundancy depigmentation [173]. Vitiligo-prone mice
with, in particular, CXCL11. Overall, however, [174] exhibited reduced depigmentation fol-
interfering with effector T cell homing offers lowing adoptive Treg transfer [107]. Animals
an exciting therapeutic approach in vitiligo. experienced prolonged inhibition of depig-
ERRNVPHGLFRVRUJ
mentation, while transferred Tregs migrated to 28.10 V

itiligo and Melanoma T Cell
the skin [107]. Likewise, vitiligo was success- Responses
fully treated with rapamycin, which promoted
Treg development [107, 175]. When adminis- After decades without marked progress, several
tered with all-trans retinoic acid, rapamycin immune-based treatments are currently emerging
was able to confer both expansion of Tregs for the treatment of malignant melanoma. Their
and biological stability of such Tregs in an efficacy is often associated with depigmentation
inflammatory environment [176]. Rapamycin of the skin. The development of vitiligo is a posi-
as monotherapy also conferred protection tive prognostic factor in melanoma patients
against depigmentation for up to 6 weeks after [183]. This is likely the case because the presence
cessation of therapy in mice, highlighting its of vitiligo denotes efficient anti-melanocyte T
stabilizing effects on expanded Tregs [107]. cell responses against shared tumor and normal
Thus, creating a quantitative increase in Tregs melanocyte antigens [184], including MART-1
in the skin via adoptive Treg transfer or and gp100 [185]. A comparison of T cell infil-
rapamycin administration may serve as a trates in vitiligo and melanoma tissue is shown in
potentially efficacious treatment modality in Fig. 28.2. Meanwhile, a large retrospective cohort
halting progressive vitiligo in humans. study revealed that patients with vitiligo have a
• Cytokine inhibition threefold decreased lifetime risk of developing
• The presence of minute immune infiltrates in melanoma [186]. Adoptive transfer of cytotoxic
depigmenting vitiligo skin warrants the appli- T cells specific for melanoma differentiation
cation of antibodies to neutralize the effects of antigens has been associated with vitiligo devel-
cytokines generated by activated lympho- opment [31, 174, 187], again supporting a patho-
cytes. IFN-γ-neutralizing antibodies were genic role of autoimmune T cells in vitiligo.
shown to markedly reduce self-reactive cyto- Interestingly, the presence of normal or increased
toxic T cell accumulation in the skin and to levels of regulatory T cells in the skin of mela-
prevent depigmentation in a mouse model of noma patients is associated with less effective
vitiligo [94]. In the absence of IFN-γ, T cell clearing of melanoma tumors [188]; depleting
activation is prevented, as is IFN-γ-induced these Tregs is required in order for sufficient
melanocyte apoptosis [177]. Furthermore, cytotoxic T cell-mediated melanocyte destruc-
melanocytes will downregulate the expression tion to ensue [93]. As vitiligo represents autoim-
of MHC, including MHC class II [178]. mune melanocyte destruction in the skin,
TNF-α likely contributes to active depigmen- dermatologists must consider the origin of gen-
tation via its activation of CD8+ T cells. This is eration of these responses. While vitiligo often
supported by the finding that TNF-α inhibition does not have a readily identifiable trigger by his-
has been successful in halting progressive vit- tory, the appearance of this entity beyond adoles-
iligo [179–181], in which CD8+ T cells play cence might be a harbinger of the body generating
an active role [25]. Paradoxically, some indi- immune responses against an underlying mela-
viduals developed de novo vitiligo when noma. This concept is similar to the clinical
treated with anti-TNF-α agents. This may be implications of halo nevi as a potential marker of
explained by the fact that TNF-α can also dysplastic nevi or malignant melanocytic neo-
increase Treg activity; the scale may be tipped plasms [189, 190]. Therefore, new-onset vitiligo
in favor of depigmentation as a result of Treg in this age group warrants a total body skin exam
depletion in these exceptional cases [182]. to screen for melanoma..
ERRNVPHGLFRVRUJ
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ERRNVPHGLFRVRUJ
Cytokines, Growth Factors,
and POMC Peptides
29
Markus Böhm, Katia Boniface, and Silvia Moretti
Contents
29.1 Alterations in Proinflammatory Cytokines 304
29.2 Disturbances in Growth Factor Expression 306
29.3 Dysfunction of the Proopiomelanocortin System 307
References 309
Abstract and melanogenesis, eliciting a dose-dependent

The epidermis and its main constituents, kera- decrease in tyrosinase activity of cultured nor-
tinocytes, produce a vast repertoire of cyto- mal human melanocytes and inhibiting mela-
kines, including interleukins (IL), growth nocyte proliferation, while IFN-γ impedes
factors, colony-stimulating factors, and che- maturation of the key organelle melanosome
mokines. Under normal circumstances most by concerted regulation of pigmentation
of them are not synthesized or not released, genes. Interestingly, a higher expression of
but a number of external stimuli and stressors, these cytokines has been demonstrated by
e.g., infections, chemicals, trauma, or ultravi- various authors in the affected skin of vitiligo
olet radiation, are capable of inducing produc- patients at both protein and transcript levels.
tion and release of such molecules from
keratinocytes. IL-6 and TNF are paracrine
inhibitors of human melanocyte proliferation
Key Points
• IL-6 and TNF are paracrine inhibitors of
human melanocyte proliferation and
M. Böhm (*) melanogenesis.
Department of Dermatology, University of Münster,
• IFN-γ impedes maturation of the key
Münster, Germany
e-mail: bohmm@uni-muenster.de organelle melanosome and induces in
normal human keratinocytes the expres-
K. Boniface
INSERM U1035, BMGIC, Immunodermatology sion of IL-33.
team, ATIP-AVENIR, Université de Bordeaux,
Bordeaux, France
S. Moretti
Division of Clinical Preventive and Oncologic
Dermatology, University of Florence, Florence, Italy

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304 M. Böhm et al.
patients at both protein and transcript levels [6–

• CXCL10 was shown to be critical for 12], but the exact mechanism explaining how
progression and maintenance of these molecules can affect pigmentation in vitil-
depigmentation. igo is not fully understood. It seems likely that
• GM-CSF and SCF upregulate proteins inflammatory cytokines play a complex role in
required both for proliferation and mela- vitiligo pathogenesis, based both on their inflam-
nogenesis, such as tyrosinase, TRP-1, matory properties and paracrine activity on
and TRP-2. melanocytes.
• Reduced expression of SCF could result Besides their anti-melanogenic properties,
from keratinocyte apoptosis and might IL-1α, IL-6, and TNF can also induce the expres-
be responsible for passive melanocyte sion of adhesion molecules (in particular, ICAM-
death. 1) on melanocyte membrane, thus promoting
further lymphocyte recruitment that can play a
role in melanocyte destruction [13].
TNF can interfere with some mitochondrial
29.1 Alterations activities through the production of peroxides,
in Proinflammatory including hydrogen peroxide, possibly leading to
Cytokines a mitochondria-dependent cell death [14, 15]. In
vitiligo epidermis, besides the high TNF-α levels,
The epidermis and its main constituents, kerati- an impaired nuclear factor (NF)-κBNF activation
nocytes, produce a vast repertoire of cytokines, has been described, and in TNF-treated cultured
including interleukins (IL), growth factors, normal human keratinocytes, the inhibition of
colony-stimulating factors, and chemokines. NF-kB activation resulted in keratinocyte apop-
Under normal circumstances most of them are tosis [9]. These data suggest that NF-κB dysfunc-
not synthesized or not released, but a number of tion may promote TNF-α-dependent apoptosis in
external stimuli and stressors, e.g., infections, keratinocytes from vitiligo lesions and underline
chemicals, trauma, or ultraviolet radiation, are the pivotal role of this cytokine in the epidermal
capable of inducing production and release of cell impairment of vitiligo patients. The close
such molecules from keratinocytes [1]. involvement of this cytokine in vitiligo is also
Human keratinocytes indeed can synthesize a strengthened by its significant reduction in vitil-
number of inflammatory cytokines under proper igo lesions undergoing repigmentation after topi-
stimuli, such as IL-1α, IL-6, tumor necrosis cal tacrolimus treatment [10].
factor (TNF), interferon (IFN)-γ, and more
The proinflammatory properties of TNF stem
recently IL-18, which exert inflammatory and from its activation of the proinflammatory cyto-
noninflammatory effects [2, 3]. kines IL-1 and IL-6 [16] and of numerous nuclear
The abovementioned cytokines deserve spe- transcription factors, most importantly NF-κB,
cial attention in vitiligo since they have been which is involved in maintaining inflammatory
found to be involved in its pathogenesis. IL-6 responses [17]. But TNF may possess also anti-
and TNF are paracrine inhibitors of human mela- inflammatory properties, since it induces activa-
nocyte proliferation and melanogenesis, eliciting tion and proliferation of T regulatory cells (Tregs)
a dose-dependent decrease in tyrosinase activity in vivo [18], so that TNF-α can be considered to
of cultured normal human melanocytes and potentially play both dangerous and protective
inhibiting melanocyte proliferation [4], while roles in vitiligo, by stimulating the cytotoxic T
IFN-γ impedes maturation of the key organelle lymphocytes that are detrimental to melanocytes
melanosome by concerted regulation of pigmen- and activating Tregs [19].
tation genes [5]. Interestingly, a higher expres- It has been reported that increased IFN-γ in
sion of these cytokines has been demonstrated the skin plays a direct role in vitiligo pathogene-
by various authors in the affected skin of vitiligo sis by inducing melanocyte death [20] and that
ERRNVPHGLFRVRUJ
29 Cytokines, Growth Factors, and POMC Peptides 305
persistent IFN-γ treatment induces viability loss, Concerning IL-1, it is known that human kera-
apoptosis, cell cycle arrest, and senescence in tinocytes constitutively express inflammasome
melanocytes [21]. IFNγ also seems important for proteins together with pro-IL-1α and pro-IL-1β
maintaining epidermal pigmentation homeostasis [34, 35]; IL-1b activation in human epidermis
[22]. However, this cytokine can affect pigmenta- can occur via an alternative mechanism involving
tion also through the interaction with other cyto- stratum corneum kallikrein 7, a serine protease
kines. In particular, IL-18, a member of the IL-1 specifically expressed in keratinizing squamous
cytokine family, is released after UVB exposure epithelia [36]. NLR (nucleotide-binding domain
and is capable of increasing melanogenesis [23]; and leucine-rich repeat containing) family, pyrin
such an effect is inhibited by IFN-γ treatment domain-containing protein 1 (NLRP1) is a key
[24]. In addition, IFN-γ inhibits the UVB-induced protein of the inflammasome complex which
expression of protease-activated receptor-2, activates the proinflammatory cytokine IL-1β. In
which is a key mediator of melanosome transfer, perilesional skin of vitiligo patients with active
expressed in keratinocytes [24]; PAR-2 is in turn disease, NLRP1 and IL-1β expression were
reduced in vitiligo epidermis [25]. Furthermore, found to be increased and associated with a
IFN-γ induces in normal human keratinocytes the mostly T-cell inflammatory infiltrate [12], sug-
expression of IL-33, another member of the IL-1 gesting that inflammation represents a crucial
cytokine family [26], which has been demon- moment in vitiligo progression. Furthermore,
strated to be increased in lesional skin and serum IL-β has been reported to inhibit melanocyte
of vitiligo patient; IL-33 was able to reduce activity via microphthalmia transcription factor
expression of both stem cell factor (SCF) and (MITF) regulation [37].
basic fibroblastic growth factor (bFGF) and to IL-6 is a proinflammatory cytokine that has
increase the expression of both IL-6 and TNF in been proposed to link the environmental stressors
primary keratinocytes [27]; these data support and autoimmunity in vitiligo pathogenesis; in
the concept of a vicious network of anti- fact oxidative stress may have a role in vitiligo
melanogenic cytokines in vitiligo. More recently, onset, while autoimmunity contributes to pro-
vitiligo pathogenesis has been attributed also to gression. It was reported that IL-6 and IL-8 are
keratinocyte IFN-γ-induced chemokines which upregulated in melanocytes after the activation of
have been demonstrated to promote T lympho- the unfolded protein response (UPR). This
cyte recruitment to the epidermis where melano- response is activated by the accumulation of mis-
cytes reside [28]. A mouse model of vitiligo with folded peptides derived by the exposure of mela-
focused epidermal depigmentation requires nocytes to damaging phenols, which are able to
IFN-γ for autoreactive CD8+ T-cell accumulation induce oxidative stress, thus resulting in harmful
in the skin [29]. Skin of vitiligo patients and will redox disruptions [38]. In vitiligo, impaired func-
contribute to the recruitment of CXCR3- tion of Tregs that is associated with a widespread
expressing T cells [28, 30]. In addition, CXCL10 activation of cytotoxic T lymphocytes has been
was shown to be critical for progression and described [39], and in psoriasis IL-6 allows cyto-
maintenance of depigmentation in vivo in a toxic T cells to escape from Treg suppression
mouse model of autoimmune vitiligo [28]. IFN- [40]. Thus the upregulation of IL-6 production by
α, produced by plasmacytoid dendritic cells in melanocytes under oxidative stress through the
vitiligo skin, can also induce the release of these UPR activation [38] could reduce Treg modula-
two chemokines by epidermal cells, providing an tion, leading to the activation of the immune
initial signal for CXCR3+ T-cell recruitment dur- response against melanocytes [41]. It was also
ing initiation of the disease [31]. Lastly, the demonstrated that subtoxic levels of hydrogen
release of CXCL16 by keratinocytes and peroxide (H2O2) induce expression of IL-6 in epi-
CXCL12, and CCL5 by melanocytes in vitiligo dermal melanocytes, and both H2O2 and IL-6
under oxidative stress, could be important for the have been reported to be elevated in vitiligo
recruitment of T cells [32, 33]. lesions; H2O2-induced overexpression of IL-6 by
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306 M. Böhm et al.
melanocytes may be another molecular linkage In human nerve growth factors are produced
for the oxidative stress and autoimmune/inflam- and released by keratinocytes and stimulate
matory reactions in vitiligo [42]. melanogenesis and dendritogenesis of melano-
Recently, it was proposed that the proinflam- cytes [49]. This binding activates a cascade of
matory cytokine IL-17 A secreted by the Th17 intracellular signals that lead to transient increase
cells infiltrating vitiligo skin can induce IL-1β, in microphthalmia-associated transcription factor
IL-6, and TNF production in skin resident cells (Mitf) gene expression, which in turn result in
such as keratinocytes and fibroblasts, suggest- upregulation of tyrosinase, tyrosinase-related
ing the presence of mutual cytokine signaling protein (TRP)-1, and TRP-2 (or dopachrome tau-
between skin resident cells and accumulating tomerase, DCT) to allow melanin synthesis [50].
inflammatory cells [43]. Interestingly, in vitro Endothelin (ET)-1 and granulocyte-macrophage
analysis demonstrated that the expression of colony-stimulating factor (GM-CSF) are further
MITF and downstream genes was downregu- keratinocyte-derived factors that regulate the pro-
lated in melanocytes by treatment with the liferation and melanogenesis/dendritogenesis of
abovementioned cytokines [43, 44]. melanocytes in UVB- or UVA-irradiated human
Nonetheless, IL-17 on its own had little effect skin [51–53]. Moreover, SCF is also produced by
on melanogenesis and would rather act indi- keratinocytes in a membrane-bound form. It
rectly on pigmentation through upregulation of stimulates proliferation and dendritogenesis [54]
the inflammatory cytokines cited above. The of epidermal melanocytes in the presence of
presence of a cytokine network induced by dibutyryl adenosine 3′:5′-cyclic monophosphate
IL-17A secreted by Th17 cells infiltrating vitil- (dbcAMP), via the binding to its specific receptor
igo skin may represent another mechanism c-kit [55]. ET-1 (through signaling pathway of
underlying vitiligo pathogenesis but still needs protein kinase C) [56], GM-CSF (via activation
further investigation. of signal transducers and activators of transcrip-
tion STAT-1, STAT-3, and STAT-5 or mitogen-
activated protein (MAP) kinase) [57, 58], and
29.2 Disturbances in Growth SCF (through activation of MAP kinase pathway)
Factor Expression [48] upregulate proteins required both for prolif-
eration and melanogenesis, such as tyrosinase,
In human skin, homeostasis is controlled by TRP-1, and TRP-2. Keratinocyte-derived b-FGF
either endocrine or paracrine communication is well known to stimulate proliferation of epi-
via soluble factors including hormones, growth dermal melanocytes in the presence of dbcAMP
factors, and cytokines or by intercellular [59]. It can also act in synergy with ET-1 but also
communication via cell-cell and cell-matrix MGF to enhance melanogenesis and proliferation
adhesion and gap junctional intercellular com- of epidermal melanocytes [60].
munication [45, 46]. Less is known about the influence of dermal-
At present evidence exists that melanogenic derived cytokines/growth factors on epidermal
paracrine cytokine networks occur between melanocytes. Human fibroblasts seem to induce a
melanocytes and other types of skin cells, includ- decrease of epidermal pigmentation in chimeric
ing keratinocytes and fibroblasts, which regulate human epidermal reconstructs [61], although
melanocytes function [47]. Keratinocytes have they can secrete melanogenic growth factors such
been reported to stimulate the proliferation, as SCF or hepatocyte growth factor (HGF) which
melanogenesis, or dendritogenesis of epidermal are mitogens for human melanocytes in vitro and
melanocytes in normal skin and/or in UVB- in vivo [47, 48]. Some data suggest that upregula-
irradiated skin [48]. In fact, after colonizing in tion of HGF plays a role in skin homeostasis after
the epidermis during development, melanocytes UVA irradiation [62].
may require keratinocyte-derived factors for their Overall, the imbalance of keratinocyte-derived
proliferation and differentiation. cytokines described by some authors in vitiligo
ERRNVPHGLFRVRUJ
epidermis suggests an involvement of epidermal important for skin pigmentation. In explants cul-
cytokines in the pathogenesis of vitiligo. The tures of fibroblasts from vitiligo dermis, a higher
results did not appear univocal, since some gene expression of senescence markers p16, p21,
melanocyte- stimulating cytokines have been and hp1 has been demonstrated at mRNA and
described as decreased [5, 6, 11, 63, 64] or protein level in lesional dermis. Senescence in
increased [59] in lesional epidermis, possibly due vitiligo fibroblasts can decrease the secretion of
to different techniques, diverse choices of con- growth factors and cytokines produced by fibro-
trols, and possibly dissimilar selections of vitil- blasts which may lead to the melanocyte death
igo patients. However, a critical evaluation of the [69, 70].
findings reported in literature suggests that SCF
is actually reduced in depigmented lesions, as
observed in vivo at both protein and transcript 29.3 Dysfunction
levels [5, 6, 11, 63]. In fact keratinocytes in the of the Proopiomelanocortin
depigmented lesions compared to the normally System
pigmented counterpart seem to be more prone to
apoptosis [65, 66] and incapable of producing The proopiomelanocortin (POMC)-derived pep-
adequate amounts of SCF for melanocyte sur- tides include α-, β-, and γ-melanocyte-stimulating
vival [63]. In vitro functional studies have shown hormones (α-, β-, and γ-MSH), adrenocorticotro-
that apoptosis of cultured normal human kerati- pin (ACTH), as well as β-endorphin (β-ED).
nocytes is associated to a concentration- These peptides are players of the classical neuro-
dependent decreased production of SCF mRNA endocrine hypothalamic-pituitary-adrenal (HPA)
and protein and that deprivation of SCF or kerati- stress axis which importantly is also installed in
nocyte feeder in the culture medium induces a the skin as an analogon [71]. Accordingly, epi-
marked decrease in melanocytes as a result of dermal keratinocytes but also several other cuta-
apoptosis [63]. These data strongly suggest that neous cell types express POMC and process this
in vitiliginous keratinocytes a reduced expression precursor into POMC-derived peptides which not
of SCF could result from keratinocyte apoptosis only induce melanogenesis and/or melanocyte
and might be responsible for passive melanocyte proliferation [72–74]. Importantly, melanocortin
death. Further support to the role of keratinocyte- peptides have additional biological actions
derived SCF in regulating melanocyte activity including immunomodulation and cytoprotection
and possible implication in vitiligo is also given against genotoxic stress and oxidative damage
by additional in vitro data, showing that prolif- [75–78] due to expression of functional melano-
eration of cultured melanocytes is enhanced by cortin receptors (MCRs) in skin cells and cells of
tacrolimus-treated keratinocyte supernatant, the immune system. Several studies have thus
whose SCF concentration increases dose- addressed the question as to whether the POMC
dependently with tacrolimus treatment [67]. system in vitiligo is altered.
Concerning other melanogenic cytokines, a Early studies revealed that the peripheral
substantial decrease of GM-CSF and bFGF pro- blood levels of some POMC-derived peptides
tein levels [5, 6, 63] and a reduced expression of and their circadian rhythm are altered.
ET-1 [11] and bFGF [68] transcripts have been Accordingly, plasma levels of β-ED were higher
demonstrated in the depigmented epidermis in in vitiligo patients than in healthy individuals.
vitiligo patients. In addition, a failure in ET-1 Moreover, the circadian rhythm of this POMC
production from vitiligo keratinocytes in peptide was lost in patients with vitiligo [79].
response to ultraviolet-B irradiation has been Increased β-ED plasma levels in vitiligo were
described [68]. subsequently confirmed in another study with
Dermis can probably contribute to vitiligo more patients [80] indicating an increased level
pathogenesis at least in part, since dermal fibro- of stress mediated by the classical neuroendo-
blasts secrete various growth factors which are crine HPA axis or by increased cutaneous
ERRNVPHGLFRVRUJ
308 M. Böhm et al.
production of β-ED in the skin of vitiligo melanocytes and keratinocytes in culture at

patients. However, in a more recent study on 40 RNA and protein level. In support of the former
matched normal individuals, vitiligo patients studies, furin convertase expression was found
had a significantly lower median α-MSH to be significantly lower in both lesional and
plasma level but a higher ACTH plasma level. non-lesional skin of untreated patients with
In contrast, the levels of morning serum cortisol progressive vitiligo compared with healthy
were not altered in vitiligo patients [81]. controls. Antibody-antigen recognition was not
With regard to the cutaneous POMC system, affected by exposure of furin protein to hydro-
it was reported that the immunoreactive gen peroxide. However, treatment with hydro-
amounts of α-MSH in lesional and non-lesional gen peroxide in vitro resulted in a 55% reduction
epidermis of vitiligo are less than in normal in Ca2+ binding [85].
epidermis. Subsequent immunohistochemical Expression of the POMC system was also
studies on skin biopsies (lesional, perilesional, assessed in a more quantitative manner in full-
and non- lesional) from vitiligo patients and thick skin biopsies from vitiligo patients using
control subjects further supported a reduction quantitative real-time RT-PCR [86, 87]. In one
in the level of α-MSH in lesional and perile- study on Estonian vitiligo, patients POMC
sional skin, especially within the melanocytes mRNA levels were somewhat lower in lesional
[82]. Similar to α-MSH a reduction in the num- skin than in control skin. In contrast, the relative
ber of melanocytes staining for PC1 and PC2 in mRNA amounts of both MC-1R and also
vitiligo skin both in non-lesional and perile- MC-4R were found to be slightly elevated in
sional skin compared with control skin was non-lesional skin of vitiligo compared with
seen [82]. Additional studies revealed that epi- lesional skin and control skin [86]. In a more
dermal immunoreactivity for both α-MSH and recent study involving a similar cohort in size
β-ED immunoreactivity is significantly reduced (n = 40), POMC and MC1R mRNA amounts
while that of ACTH is increased in vitiliginous were lower in lesional skin compared with non-
skin [83]. In light of increased amounts of lesional skin and control skin from sex- and
hydrogen peroxide in vitiligo skin, it was pro- age-matched controls [87]. A statistically sig-
posed that oxidation of epidermal POMC pep- nificant positive correlation between lesional
tides leads to structural changes that may alter levels of POMC and MC1R, as well as between
antibody epitope recognition. Based on non-lesional levels of POMC and MC1R in the
FT-Raman spectroscopy and computer model- patients, was found in this study. On the other
ing ACTH, α-MSH and β-ED appear to be hand, a statistically significant negative correla-
redox sensitive resulting in the generation of tion between the lesional and non-lesional lev-
Met sulfoxide when exposed to hydrogen per- els of POMC, as well as between the lesional
oxide. In accordance with this, treatment of and non-lesional levels of MC1R in the patients,
α-MSH and β-ED with hydrogen peroxide was detected [87].
in vitro reduced the melanotropic activity of Therefore, it appears that based on immuno-
this POMC-derived peptide [83]. In support of histochemical studies, quantitative mRNA
this concept, treatment of vitiligo patients with expression analysis and in vitro studies on the
pseudocatalase increased immunoreactivity of action of POMC-derived peptides as well as on
both α-MSH in repigmented skin of patients the function of POMC-processing enzymes the
with vitiligo [83]. Subsequent studies demon- cutaneous POMC system are dysfunctional.
strated that not only POMC-derived peptides Consequences of this deviation could be not only
but also furin convertase, a member of the PC impaired melanogenesis in melanocytes but also
family of POMC-processing enzymes [84], an increased susceptibility to cell death or apop-
could be a target for oxidative stress-induced totic signals including oxidative stress. Moreover,
damage in vitiligo [85]. Furin convertase was impairment of the POMC system may render
found to be expressed in both normal human melanocytes to an immune attack.
ERRNVPHGLFRVRUJ
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KU. The Ca2+−binding capacity of epidermal furin
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Regenerating Melanocytes:
Current Stem Cell Approaches
30
with Focus on Muse Cells
Mari Dezawa, Kenichiro Tsuchiyama,
Kenshi Yamazaki, and Setsuya Aiba
Contents
30.2 Background of Muse Cells 314
30.3 Characteristics of Muse Cells 316
30.4 asic Difference Between Muse Cells and Remainder of Cells,
B
Non-Muse Cells, in Fibroblasts and BM-MSCs 318
30.5 omparison of Differentiation Propensity of Muse Cells
C
from Different Sources 319
30.6 Generation of Melanocytes from Human Dermal-Muse Cells 319
30.7 eaction of Non-Muse Dermal Fibroblasts Against Melanocyte
R
Induction 320
30.8 Generation of Human-Colored 3D-Cultured Skin 322
30.9 Functional Evaluation of Muse Melanocytes In Vivo 324
30.10 Other Stem Cells as Source for Melanocytes 325
30.11 Future Perspectives 325
References 326
Abstract including the dermis and in the bone marrow.

Muse cells are recently found endogenous They are collectable as cells positive for
non-tumorigenic pluripotent stem cells that stage-specific embryonic antigen (SSEA)-3, a
reside in connective tissue of various organs pluripotent surface marker, from tissues, and
are expandable in vitro. Other than SSEA-3,
they express Oct3/4, Nanog and Sox, other
M. Dezawa (*) pluripotent genes. Notably, they are able to
Department of Stem Cell Biology and Histology,
Tohoku University Graduate School of Medicine, differentiate into cells representative of all
Sendai, Japan three germ layers from single cells and are
e-mail: mdezawa@med.tohoku.ac.jp self-renewable, suggesting their pluripotency.
K. Tsuchiyama · K. Yamazaki · S. Aiba Muse cells collected from human dermal
Department of Dermatology, Tohoku University fibroblasts (dermal-Muse cells) were shown
Graduate School of Medicine, Sendai, Japan

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314 M. Dezawa et al.
to efficiently differentiate into melanin-pro- (ES) and induced pluripotent stem (iPS) cells are
ducing functional melanocytes by treating pluripotent cells that have high ability to produce
them with ten factors. Functions of melano- melanocytes [3–8]. However, the ethical
cytes induced from Muse cells (Muse melano- problems of obtaining ES cells [9, 10] and the
cytes) were comparable to that of primary risk of tumorigenesis for both ES and iPS cells
human melanocytes. Melanin-producing abil- are obstacles to clinical use [11–14]. Melanocytes
ity of human Muse melanocytes was retained have also been induced from dental pulp stem
when they were incorporated into human-col- cells (DPSCs), a kind of mesenchymal stem cell
ored three-dimensional (3D) cultured skin and (MSC); however, induction efficiency is not high
even after transplantation of the 3D-cultured because MSCs, including DPSCs, are crude het-
skin into the back of immunodeficient mice. erogeneous population, and subpopulation truly
Since Muse cells are non-tumorigenic and responsible for melanocyte differentiation is not
harvestable from easy accessible sources such identified [15, 16].
as skin biopsy and dermal fibroblasts, Muse This chapter mainly focuses on multilineage-
melanocytes are beneficial for both industrial differentiating stress-enduring (Muse) cells,
and clinical uses. recently found novel type of non-tumorigenic
pluripotent stem cells that are collectable from
human adult skin, adipose tissue, umbilical cord,
Key Points and the bone marrow [17–22]. The technique
• Muse cells are non-tumorigenic endog- newly developed was to generate functional
enous pluripotent stem cells. melanin- producing melanocytes from dermal-
• Muse cells express pluripotency mark- Muse cells by using certain cytokine induction.
ers such as transcription factors Oct3/4, Induced Muse melanocytes were demonstrated
Sox2, and Nanog, as well as pluripotent to function as authentic melanocytes both
surface marker, stage-specific embry- in vitro and in vivo. Of note, three-dimensional
onic antigen (SSEA)-3. (3D) human cultured skin in which Muse mela-
• Muse cells show characters relevant to nocytes were incorporated has been produced
pluripotent stem cells; they are able to by DS Pharma Biomedical Co., Ltd. This tech-
generate cells representative of all three nique is innovative because melanocytes, hard
germ layers from a single cell. to be establish in culture and expanded with
• Muse cells distribute sporadically in the retaining their original properties, can be stably
connective tissue of various organs and supplied with reasonable scale for industrial and
do not look like associated with a struc- clinical use.
tural niche.
30.2 Background of Muse Cells
30.1 Introduction Muse cells, first reported in 2010 by Dezawa

et al., are non-tumorigenic endogenous pluripo-
Since melanocytes are tightly incorporated into tent stem cells [18]. They reside in connective tis-
the basal layer of the epidermis, their isolation is sue of various kinds of organs and in the bone
not easy, and therefore primary human melano- marrow, and thus they are somatic stem cells [23]
cyte culture with high purity is generally hard to (Fig. 30.1).
establish. Somatic stem cells were believed not to
Autologous human melanocytes are potential cross the boundaries between the three germ
cell therapy for vitiligo [1, 2] but are not for gen- layers. For example, neural stem cells generate
eral use because they are difficult to culture and neurons and glial cells but not other lineage
amplify into large scale in vitro. Embryonic stem cells such as cardiomyocytes or hepatocytes
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30 Regenerating Melanocytes: Current Stem Cell Approaches with Focus on Muse Cells 315
Fat Dermis Bone marrow
Trachea Spleen
Umbilical
cord
Fig. 30.1 Muse cells in adult tissues. SSEA-3(+) Muse the bone marrow cavity. Scale bar = 50 μm. (Pictures
cells, detected as HRP+ brown color coded, located in adapted from Dezawa, (2016) Cell Transplant, https://doi.
connective tissue of the human fat tissue, dermis, umbili- org/10.3727/096368916X690881)
cal cord, trachea, and spleen. Muse cells also located in
[24]. Similarly, hematopoietic stem cells are pluripotency [18]. Although in low frequency,
specified stem cells for generating hematopoi- BM-MSCs cultured in general culture media
etic cells and not for other kinds of cells [25]. without any cytokines or reagents (alpha-
However, this dogma was questioned by the minimum essential medium or Dulbecco’s mini-
discovery of mesenchymal stem cells (MSCs) mum essential medium supplied with 10% fetal
[26, 27]. MSCs are harvestable from mesenchy- bovine serum) spontaneously formed cell clus-
mal tissues such as the bone marrow (BM), adi- ters which are very similar to embryoid bodies
pose tissue, umbilical cord, and dental pulp [27, that are formed by embryonic stem (ES) cells in
28]. They were reported to differentiate into suspension culture [18]. Such clusters are occa-
cells representative of all three germ layers by sionally pigmented and have hair-like structures
either gene introduction or cytokine induction (Fig. 30.2a). Interestingly, cells positive for each
into endothelial cells [29], cardiac muscle cells of the ectodermal, endodermal, and mesodermal
[30], skeletal muscle cells [31], hepatocytes markers were detected as mixture in the clusters,
[32], neuronal cells [33], and peripheral glial suggesting that MSCs contain a small subpopu-
cells [34], as pluripotent-like. The differentia- lation of ES-like cells [18, 23].
tion efficiency of MSCs, however, was gener- Generally, tissue stem cells are stress-toler-
ally low, suggesting that a small subpopulation ant, and while normally dormant, they are acti-
was responsible for this phenomenon [28]. In vated by stimuli such as stress [35]. We incubated
fact, MSCs are population that are generally BM-MSCs for over 16 h in trypsin solution con-
harvested simply as adherent cells from mesen- taining no nutrients but only with digestive
chymal tissues, so that they are actually hetero- enzymes in HEPES buffer. This treatment sub-
geneous [27]. stantially increased the ratio of SSEA-3+ cells, a
Muse cells were initially found from cultured surface marker for ES cells, while simultane-
mesenchymal cells, dermal fibroblasts, and ously eliminating cells other than the target,
BM-MSCs, as stress-enduring cells that have allowing us to identify the Muse cells [18]. The
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a b c d
e f g
h i
Fig. 30.2 (a) Human MSCs spontaneously form clusters on gelatin culture contained cells positive for (e) cytokera-
similar to ES cell-derived embryoid bodies. (b) An exam- tin 7 (endodermal marker), (f) alpha-fetoprotein (endoder-
ple of cell sorting of SSEA-3(+) cells from human dermal mal), (g) desmin (mesodermal), (h) smooth muscle actin
fibroblasts. (c) After isolating Muse cells by fluorescence- (mesodermal), and (i) neurofilament (ectodermal). Bars:
activated cell sorting based on SSEA-3, single-Muse cells a, d = 100 μm, c = 20 μm, and e–i = 50 μm. (Pictures
were subjected to single-cell suspension culture to form adapted and modified with permission from Y. Kuroda
characteristic clusters which are very similar to the et al. (2010), 2010 Proceedings of the National Academy
embryoid bodies formed by human ES cells in suspen- of Sciences and with permission from Wakao et al. [22],
sion. (d) Cells expanded out from the adhered adherent and 2011 Proceedings of the National Academy of
cluster, when the cell clusters were transferred onto gela- Sciences)
tin culture. (e–i) The cells that expanded from the cluster
same strategy was also reproduced in fibroblasts 30.3 Characteristics of Muse Cells
(Fig. 30.2b), as well as in adipose-derived MSCs
and umbilical cord-MSCs, showing that Muse • SSEA-3 as a Muse cell marker
cells are not confined to BM-MSCs but are gen- Muse cells express pluripotency markers
erally contained in mesenchymal cultured cells such as transcription factors Oct3/4, Sox2, and
and mesenchymal tissues [17–22] (Fig. 30.1). Nanog, as well as pluripotent surface marker,
These SSEA-3+ cells were further shown to stage-specific embryonic antigen (SSEA)-3.
form cluster which is very similar to ES cell- SSEA-3 is an antibody that recognizes glyco-
derived embryoid body when cultured in single- lipid on cell surface and was first identified in
cell suspension (Fig. 30.2c), suggesting that mouse-fertilized egg, eight-cell stage cells, and
spontaneous formation of embryoid body-like epiblast stem cells and later was found to be
clusters in general culture of BM-MSCs are to be expressed in human ES cells [36, 37]. Thus,
delivered from SSEA-3+ Muse cells. SSEA-3 is relevant to pluripotent stem cells.
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Currently, Muse cells are collected as SSEA- Doubling time of Muse cells is nearly the
3+ cells from various sources [23]. Comparison same as that of fibroblasts, ~1.3 day/cell divi-
of the gene expression between human ES/ sion. Therefore, if we collect 30 ml bone mar-
induced pluripotent stem cells (iPS) and human row aspirate, ~one million Muse cells are
Muse cells reveals that the “expression pat- harvestable within 3 days [18, 23], indicating
tern” of pluripotency genes is very similar that they are practical source for clinical and
between Muse and ES/iPS cells, whereas the industrial uses.
“expression level” is moderate in Muse cells • Pluripotency of Muse cells
compared to ES/iPS cells [22]. Pluripotent stem cells are defined as cells
Muse cells show characters relevant to plu- that can generate cells of all three germ layers
ripotent stem cells; they are able to generate and are self-renewable. As described below,
cells representative of all three germ layers from Muse cells have both abilities at a single-cell
a single cell, and this triploblastic differentiation level.
ability can be reproduced over generations, Differentiation ability of ES cells into trip-
demonstrating that the triploblastic differentia- loblastic lineages is confirmable by transfer-
tion ability is self-renewable [18, 21]. ring embryoid bodies formed from ES cells
• Sources of Muse cells onto gelatin-coated culture plates to let them
MSCs are suggested to locate closely to the spontaneously differentiate. As mentioned
subendothelial region of small blood vessels above, Muse cells form embryoid body-like
[38], while Muse cells distribute sporadically in cluster when they were cultured in single-cell
the connective tissue of various organs and do suspension [18] (Fig. 30.2c). When these
not look like associated with a structural niche single-Muse cell-derived clusters were trans-
[23]. Therefore, Muse cells are collectable ferred to gelatin-coated culture to allow the
from organs; however, such sources are not cells to expand from adhered clusters
practical for both clinical and industrial uses. (Fig. 30.2d), cells positive for markers repre-
Sources that are easy accessible without ethical sentative of all three germ layers, namely, neu-
issues and fully harvestable would be mesen- rofilament (ectodermal), smooth muscle actin
chymal tissues. Fibroblasts can be obtained (mesodermal), and alpha-fetoprotein (endo-
from patients and donors by skin biopsy. In the dermal), are recognized in expanded cells [18,
fibroblasts, both adult dermal and foreskin 21] (Fig. 30.2e–i). Because this differentiation
fibroblasts, 1~5% of SSEA-3+ Muse cells are is generated not by cytokine induction but
reported to be contained [20, 22]. Besides, spontaneously, single-Muse cells were shown
fibroblasts are one of the most accessible cells to have triploblastic differentiation ability. Not
from commercial sources. In fact, 2 commer- only do Muse cells differentiate spontane-
cially obtained fibroblasts contain Muse cell as ously, but they also differentiate at a high rate
several % of total proportion [20, 22]. (~80–95%) into hepatocyte- and neural-
In the bone marrow (BM), Muse cells reside lineage cells as well as into adipocytes when
in the BM cavity at a ratio of nearly 1:3000 the proper cocktail of cytokines is supplied
cells, i.e., comprising ~0.03% of the mononu- [21, 22].
cleated cell fraction [18], and different from Muse cells exhibit self-renewal of pluripo-
those in connective tissue in other organs, tency: single-Muse cell-derived clusters gener-
Muse cells exist as clusters in the BM cavity ated cells positive for endodermal, mesodermal,
[39]. In adipose tissue, a culture of 15 cm3 and ectodermal markers, namely, MAP-2,
(4 cm × 9 cm size) of subcutaneous adipose GATA6, alpha-fetoprotein, and NKX2.5, in
tissue for 3 weeks yielded ~3 × 107 adipose- gelatin-coated culture dish [18, 21]. Other clus-
MSCs which contained 7~8%, namely, ~two ters were individually transferred to adherent
million of SSEA-3+ Muse cells, that were col- culture and allowed to proliferate for a certain
lectable [21]. period, after which they underwent a second
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round of single-cell suspension in culture to • Other characters of Muse cells

generate second-generation single-Muse cell- The two unique characters of Muse cells
derived clusters. The second-generation cluster that are not recognized in other types of plu-
also showed triploblastic differentiation. This ripotent stem cells are their ability to perceive
experimental cycle was repeated several times damage signals and autonomously home into
and clusters from each step exhibited triplo- damaged sites when administered intrave-
blastic differentiation at the single-cell level, nously and to spontaneously differentiate
demonstrating self-renewability of Muse cell in vivo into cells compatible with the homing
triploblastic differentiation ability [18, 21]. tissue after integration. Intravenously injected
Notably, as SSEA-3+ Muse cells directly human Muse cells were demonstrated to home
collected from human bone marrow aspirate into the liver of fulminant hepatitis and muscle
showed expression of Oct3/4, Nanog, and degeneration models of immunodeficient
Sox2 and self-renewability of triploblastic dif- (SCID) mice, respectively, and efficiently dif-
ferentiation, pluripotency of Muse cells is not ferentiated spontaneously into albumin-
the resultant of artificial modification of their producing hepatocytes and dystrophin-positive
original characters in vitro manipulation [18]. skeletal muscle cells after integration, replen-
Since Muse cells are naturally existing ished new cells, and contributed to eventual
somatic stem cells, they generate non-Muse tissue repair [18]. Locally injected Muse cells
cells during proliferation by asymmetric cell also showed remarkable effects. While skin
division [18]. Even if Muse cells are purified ulcers of a diabetes mellitus model are known
100% by cell sorting, they will gradually pro- to be intractable, subcutaneous injected human
duce non-Muse cells by random asymmetrical Muse cells incorporated as dermal and epider-
cell division during proliferation. In this man- mal cells and repaired skin defect in Nod
ner, proportion of Muse cells gradually SCID-diabetes mellitus mice [40]. In a rat
decrease, reaching to one to several percent stroke model, human Muse cells injected into
plateau of total cells, which corresponds to the the cortex spontaneously differentiated into
proportion of Muse cells in general fibroblasts neuronal cells and extended neurites incorpo-
and BM-MSCs [23]. rated into pyramidal tract to recover motor
• Non-tumorigenicity of Muse cells function [41].
Consistent with the fact that Muse cells
reside in adult normal tissue, they are non-
tumorigenic. In fact, the pattern of gene expres- 30.4 B
asic Difference Between
sion of cell cycle-related factors, namely, Muse Cells and Remainder
tumorigenic factors, differs between ES/iPS and of Cells, Non-Muse Cells,
Muse cells [22]. Generally, those factors are in Fibroblasts and BM-MSCs
highly expressed in ES/iPS cells, while very low
expression is observed in Muse cells, and the Cells other than Muse cells in fibroblasts or
level and pattern are similar to that in somatic BM-MSCs, namely, non-Muse cells, sharply
cells, namely, non-Muse cells (Fig. 30.4) [28]. contrast with Muse cells in many points. Non-
Notably, telomerase activity, an indicator Muse cells are SSEA-3-negative, and their
of tumorigenic activity, is very low in Muse expression level of pluripotency genes is gener-
cells compared with iPS and Hela cells and is ally very low or under detection level [22].
rather similar to that in non-Muse cells [21, Methylation level of pluripotency marker is con-
22]. In fact, when human BM- and adipose- sistent; promoter of Oct3/4 and Nanog in non-
derived Muse cells were transplanted into tes- Muse cells is mostly methylated, while those of
tes of immunodeficient mice, no teratoma Muse cells are more demethylated [22].
formation was recognized for up to 6 months While non-Muse cells are able to differentiate
[21, 22]. Therefore, Muse cells are considered into osteocytes, cartilage cells, and adipocytes in
to have a low risk of tumorigenesis. the presence of certain sets of induction
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cytokines, their differentiation rate is substan- While pluripotency is the same among der-
tially lower than that of Muse cells, and the time mal-, BM-, and adipose-Muse cells, their differ-
required for differentiation is longer than that entiation propensity seemed different from each
required for the differentiation of Muse cells [21, other. This suggests that the appropriate tissue
42]. Most importantly, non-Muse cells are unable source should be selected based on the target cell
to cross the lineage boundaries between meso- type and that dermal-Muse cells are the proper
derm, where they originally belong to, to ecto- candidate for generating melanocytes.
derm or endoderm. Even under cytokine
inductions, non-Muse cells do not differentiate
into hepatocytes and neuronal cells although they 30.6 Generation of Melanocytes
show only partial intracellular responses to cyto- from Human Dermal-Muse
kine induction [21, 42]. Cells
Non-Muse cells do not exhibit tissue repair
effect; most importantly, they do not remain in The Wnt3a, endothelin-3 (ET-3), stem cell fac-
the host-damaged tissues after administration and tor (SCF), basic fibroblast growth factor
thus do not differentiate nor replenish lost cells (b-FGF), and cAMP inducers (such as cholera
[18, 41, 43, 44]. toxin and TPA) are known to promote the
For these reasons, utilization of purified Muse expression of transcription factors PAX3,
cell population rather than crude MSCs or fibro- SOX10, CREB, and LEF1 through intracellular
blasts is rational for generating purposive cells signaling [45–47]. These four transcription fac-
and tissue regeneration. tors regulate the promoter of MITF-M, one of
the MITF variants specific for melanocytes, and
have a crucial role in melanocyte differentia-
30.5 Comparison tion. Ascorbic acid stimulates the activity and
of Differentiation Propensity synthesis of tyrosinase [48], which plays an
of Muse Cells from Different important role in functional melanocyte.
Sources Dexamethasone promotes the generation of
melanocytic cells from mouse ES cells [7].
To examine whether Muse cells derived from dif- Based on this knowledge, melanocyte induc-
ferent tissues have different differentiation pro- tion system was set as follows: human dermal-
pensities, gene expression of ectodermal-, Muse cells were seeded at a density of 10,000 cells
endodermal-, and mesodermal-lineages in Muse per 6-well plate and cultured for 1 day in α-MEM
cells derived from the BM, dermal fibroblasts, without serum. The cells were then cultured in dif-
and adipose tissue was compared [21]. ferentiation medium containing 0.05 M dexameth-
Ectodermal-related genes were generally asone, insulin-transferrin-selenium (ITS), 1 mg/ml
higher in both dermal-Muse and BM-Muse cells linoleic acid-bovine serum albumin, 30% low-glu-
than in adipose-Muse cells, and particularly cose DMEM, 20% MCDB-201 medium, 10−4 M
microphthalmia-associated transcription factor l-ascorbic acid, 50% DMEM conditioned by
(MITF) and KIT, genes relevant to melanocytes, L-Wnt3a cells that contain Wnt3a, 50 ng/ml SCF,
were expressed in dermal-Muse cells [42]. On 10 nM ET-3, 20 pM cholera toxin, 50 nM
the other hand, mesodermal-lineage genes were 12-O-tetradecanoyl-phorbol 13-acetate (TPA),
generally higher in adipose-Muse cells than in and 4 ng/ml b-FGF. Cells were maintained in this
dermal- and BM-Muse cells; for example, SP7, differentiation medium for 6 weeks with medium
osteogenic factor, and Pax7, muscle stem cell exchange every 2 days. Cultures were passaged
marker, were only detected in adipose-Muse when cells reached ~80% confluency [42].
cells. In endodermal-lineage genes, BM-Muse The morphology of the Muse cells started to
cells demonstrated the highest levels, particularly change and cells with dendrites appeared within
genes pertinent to hepatocyte- and pancreatic- 3 weeks (Fig. 30.3). Cell size was reduced by
lineages [21]. 5 weeks, and by 6 weeks the cells had morphology
ERRNVPHGLFRVRUJ
Just before
differentiation 3 weeks 5 weeks 6 weeks Melanocytes
Muse cells
NHDFs
Non-Muse cells
Fig. 30.3 Generation of Muse melanocytes. Phase con- lar to original fibroblasts (NHDF). Scale bar = 50 μm.
trast microscopic images of Muse and non-Muse cells (Pictures adapted from Tsuchiyama, K. et al. (2013). J
during melanocyte induction. Muse melanocytes at 6 Invest Dermatol, 133(10), 2425–2435, https://doi.
weeks are similar to human primary melanocytes (mela- org/10.1038/jid.2013.172)
nocytes), while non-Muse cells at 6 weeks are more simi-
very similar to that of human melanocytes cytes, dermal-Muse cells need additional
(Fig. 30.3). Muse cells which originally express differentiation-enhancing factors such as cholera
MITF and KIT newly expressed tyrosinase-related toxin, TPA, and linoleic acid [42].
protein 1 (TRP-1) and gp100 at 3 weeks, dopach-
rome tautomerase (DCT) at 5 weeks, and finally
tyrosinase at 6 weeks, at which point they become 30.7 Reaction of Non-Muse
pigment-producing functional melanocytes that Dermal Fibroblasts Against
are positive for the l-DOPA reaction (Fig. 30.4). Melanocyte Induction
Expressions of tyrosinase, gp100, and MITF were
not only recognized in gene level but were also Muse cells and non-Muse cells have fundamental
confirmed by protein level, immunocytochemis- differences; Muse cells originally express a sub-
try. These results demonstrated that cells with set of pluripotency genes, but those are not
characteristics very close to human primary mela- expressed in non-Muse cells [22]. Non-Muse
nocytes were induced from human dermal-Muse cells show lower ratio of differentiation into
cells [42]. osteocytes and adipocytes by cytokine induction,
Among ten factors used for melanocytes while they did not show ectodermal or endoder-
induction, essential factors were searched. Fang mal differentiation even under the presence of
et al. described a combination of Wnt3a, ET-3, cytokines [21]. Similarly, the reaction of non-
and SCF that is sufficient to induce melanocytes Muse cells to melanocyte induction was different
from pluripotent stem cells [3]. When Muse cells from that of Muse cells.
were cultured in medium lacking any of those Human dermal-non-Muse cells newly
three factors, expression of melanocyte-related expressed TRP-1 at 3 weeks, while its expression
markers was not recognized, and morphology of was not sustained for a longer period and eventu-
cells remained fibroblast-like [42]. Reversely, ally returned to negative by 5 weeks, with the
when Muse cells were cultured only with those cells reverting back to the gene expression pattern
three factors, cells expressed almost all of essen- of fibroblasts, showing a fibroblast-like morphol-
tial melanocyte markers, MITF, KIT, TRP-1, ogy (Figs. 30.3 and 30.4). DCT and gp100 were
DCT, and gp100, but not tyrosinase, one of the never expressed for the entire period of induction.
most important enzymes for melanocyte func- While MITF signal was detected in non-Muse
tion, suggesting that while these three factors cell-derived cells in RT-PCR level at 6 weeks, the
play a central role in differentiation into melano- protein expression level was not high enough to
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a b MITF gp100 Tyrosinase
3 Weeks
Muse cells
MITF
KIT
TRP-1
DCT
gp100
Tyrosinase
Non-Muse cells
β-Actin
es
s
lls
e
lls
DF
at
ce
yt
ce
pl
oc
NH
m
e
e
an
us
us
-te
M
el
M
No
M
n-
No
5 Weeks
Melanocytes
MITF
KIT
TRP-1
DCT
gp100
Tyrosinase
Negative control
β-Actin
es
s
lls
e
lls
DF
at
ce
yt
ce
pl
oc
NH
m
e
e
an
us
us
-te
M
el
M
No
M
n-
No
6 Weeks c Melanocytes Muse cells
MITF
KIT
TRP-1
DCT
gp100
Tyrosinase
β-Actin NHDFs
es
s
lls
e
lls
DF
at
ce
yt
ce
pl
oc
NH
m
e
e
an
us
us
-te
-M
el
M
No
M
n
No
Fig. 30.4 Melanocyte marker expression in Muse mela- differentiation. The positive control was human melano-
nocytes. (a) RT-PCR of melanocyte marker in Muse cells, cytes, and the negative control was naive Muse cells with-
non-Muse cells, human primary melanocytes (melano- out primary antibody. Scale bars = 50 mm. (c) l-DOPA
cytes), human dermal fibroblasts (NHDFs), and non- reaction assay of Muse melanocytes (6 weeks), human
template at 3, 5, and 6 weeks. (b) Immunocytochemistry melanocytes (positive control), and naive NHDFs (nega-
for MITF, gp100, and tyrosinase in immunocytochemical tive control). Scale bars = 100 mm. (Pictures adapted from
analysis of the melanocyte markers MITF, gp100, and Tsuchiyama, K. et al. (2013). J Invest Dermatol, 133(10),
tyrosinase in Muse and non-Muse cells at 6 weeks after 2425–2435, https://doi.org/10.1038/jid.2013.172)
ERRNVPHGLFRVRUJ
be detected by immunocytochemistry (Figs. 30.3 benefit for producing 3D-cultured skin system;

and 30.4). Consequently, none of non-Muse cells animal skins covered with dense hairs show very
were positive for these melanocyte markers, and different histological structure from that of
this suggested that non-Muse cells responded par- human, and thus animal experiment has limitation
tially to melanocyte induction [42]. Therefore, for safety test. Therefore, an in vitro assay system
there is rationality for purified Muse cells to be that mimics human skin is highly demanded.
used for efficient generation of melanocytes. Human skin is comprised of the dermis which
mainly contains fibroblasts and matrix, such as
collagen, and the epidermis which is mainly
30.8 Generation of Human- composed of keratinocytes. Ideally, 3D-cultured
Colored 3D-Cultured Skin skin should show integration of melanocytes at
correct position, the basal to second layer of the
Before evaluation of Muse melanocytes in vivo, epidermal keratinocyte layer, and a dermal
their function was validated in vitro. If they truly papilla-like structure. For this, human fibroblasts,
acquired melanocyte properties, they would suc- human keratinocytes, and collagen type 1 were
cessfully integrate into the place where authentic prepared along with Muse melanocytes.
melanocytes normally reside and express func- A gel layer was created comprising collagen
tional markers in the 3D-cultured skin, which pro- type 1 and human dermal fibroblasts (3.5 × 105
vides visible system to evaluate functional cells/ml), to mimic the dermis, and cultured for
property of Muse melanocytes. There is another ~3 days (Fig. 30.5). After making the dermal
Human dermal Non-Muse cells Induction with 10 factors

fibroblasts 1 Wnt3a 2 Cholera toxin
SSEA-3(-)
3 ET-3 4 Linoleic acid
No pluripotency 5 TPA 6 Dexamethasone
7 SCF 8 ITS supplement
Muse cells 9 b-FGF 10 Ascorbic acid
SSEA-3(+)
Pluripotency (+) DMEM+MCDB 201
Exposure the
Stratum
cultured tissue to
corneum
air-liquid interface
Muse-melanocytes
Epidermis +
Kerotinocytes
Fibroblasts
Dermis +
Type I collagen
Fig. 30.5 Outline of melanocyte generation from dermal- cells did not. 3D-cultured skin was constructed by incorpo-
Muse cells. Human dermal fibroblasts are source of Muse rating Muse melanocytes into basal layer of the stratum
cells. SSEA-3(+) Muse cells and SSEA-3(−) non-Muse corneum. Scale bar = 50 μm. (Pictures adapted and modi-
cells were separated, and each population was subjected to fied from Tsuchiyama, K. et al. (2013). J Invest Dermatol,
induction with ten factors. Muse cells successfully gener- 133(10), 2425–2435, https://doi.org/10.1038/jid.2013.172)
ated melanin-producing melanocytes, while non-Muse
ERRNVPHGLFRVRUJ
equivalent, the epidermal layer was placed onto cells positive for melanocyte-related markers and
the dermal equivalent by mixing Muse melano- Fontana–Masson staining were observed
cytes: keratinocytes at 1:2.5. They were then (Fig. 30.6). These suggest that Muse melanocytes
incubated in Humedia KG2 medium (Kurabo) could integrate into the basal layer of epidermal
for 5 days with a gradually increasing Ca2+ con- layer of 3D human cultured skin with sustaining
centration from 0.15 to 1.5 mM. For the final melanocyte function [42].
step, 3D-cultured skins were cultivated for Even though Muse cells show spontaneous
another 7 days at an air-liquid interface to accel- differentiation into the cells compatible to the
erate keratinization [42] (Fig. 30.5). tissue, they homed in in vivo disease models [18,
As a consequence, 3D skins showed dermal- 41, 43, 44]; naive Muse cells did not spontane-
papilla-like structure, placing pigmented Muse ously differentiate into melanocyte property nor
melanocytes in the basal to second layer of the did they spontaneously become positive for
epidermis (Fig. 30.5). In addition, cells positive melanocyte markers or Fontana–Masson staining
for MITF, tyrosinase, TRP-1, gp100, and S100 when supplied to artificial 3D-cultured skin [42].
were all identified in the basal layer of epidermal This result indicated that naive Muse cells do not
layer, and Fontana–Masson staining revealed the spontaneously differentiate into melanocytes
presence of melanin in the epidermis of both cul- in vitro even if they are integrated into the epider-
tures (Fig. 30.6). When Muse melanocytes were mal layer of 3D-cultured skin, and thus, induc-
replaced with other cell types, such as keratino- tion of Muse cells into melanocytes is required
cytes or non-Muse cells, no pigmented cells or before construction of 3D human cultured skin.
Fig. 30.6 Melanocyte Muse

maker expression in 3D Melanocytes melanocytes Non-Muse cells
skin culture.
Immunohistochemical
analysis of MITF, MITF
tyrosinase, TRP-1,
gp100, and S-100 and
the Fontana–Masson
staining of the
3D-cultured skin. Scale Tyrosinase
bars = 50 μm. (Pictures
adapted from
Tsuchiyama, K. et al.
(2013). J Invest
Dermatol, 133(10),
2425–2435, https://doi. TRP-1
org/10.1038/
jid.2013.172)
gp100
S100
Fontana–
Masson
staining
ERRNVPHGLFRVRUJ
30.9 Functional Evaluation same as grafted human melanocytes (Fig. 30.7).

of Muse Melanocytes In Vivo Furthermore, Muse melanocytes and the neigh-
boring keratinocytes were positive for Fontana–
To investigate whether Muse melanocytes can Masson staining, as seen in the 3D human skin
survive and maintain their melanocyte functions culture with human primary melanocytes
in vivo, 3D-cultured skin, in which human Muse (Fig. 30.7). Following transplantation of
melanocytes were incorporated, was transplanted 3D-cultured skin containing GFP-labeled Muse
onto the back skin of severe combined immune melanocytes, GFP-positive Muse melanocytes
deficiency (SCID) mice. 3D-cultured skin con- were confirmed to be located within the grafted
taining human melanocytes and 3D-cultured skin skin, demonstrating that these GFP-positive cells
containing only keratinocytes were transplanted were transplanted cells and not derived from the
as positive and negative controls, respectively host [42]. Double staining for the melanocyte
(Fig. 30.7). Histologic evaluation revealed that marker S100 and the proliferative marker Ki-67
skin grafts with Muse melanocytes contained revealed the proliferation capacity of Muse mela-
brown color Muse melanocytes located in the nocytes; 9.5% of Muse melanocytes expressed
basal layer, and immunohistochemical analysis both Ki-67 and S100 [42]. In the negative con-
demonstrated that they were positive for MITF, trol, the skin graft did not appear pigmented, and
tyrosinase, TRP-1, gp100, and S100, the no pigmented cells were observed histologically.
a c
Fig. 30.7 Functional characterization of 3D skin culture analysis of MITF, tyrosinase, TRP-1, gp100, S100, and
in combined immunodeficient (SCID) mouse. (a) the Fontana–Masson staining in the skin grafts, showing
Macroscopic observation of skin grafts containing Muse that 3D skin culture maintain melanocyte function of
melanocytes, human melanocytes, and keratinocytes only incorporated Muse melanocytes. Scale bars = 50 μm.
10 days after transplantation. (b) Hematoxylin and eosin (Pictures adapted from Tsuchiyama, K. et al. (2013). J
staining of each skin graft. Scale bars 100 μm (upper pan- Invest Dermatol, 133(10), 2425–2435, https://doi.
els) and 50 mm (lower panels). (c) Immunohistochemical org/10.1038/jid.2013.172)
ERRNVPHGLFRVRUJ
These findings indicated that Muse melano- cells in an undifferentiated state in the absence
cytes homed to the basal layer of the epidermis, of feeder cells. The dermal stem cells, grown as
produced melanin, and delivered it to the neigh- three-dimensional spheres, displayed a capacity
boring keratinocytes in vivo. for self-renewal and expressed NGFRp75, nes-
tin, and OCT4 but not melanocyte markers.
Under conditions favoring melanocyte forma-
30.10 O
ther Stem Cells as Source tion as defined with human ES cells and HFSCs
for Melanocytes [3, 55], some of the attached DSCs develop den-
dritic processes and express the melanocyte
It has been reported previously that other pluripo- markers MITF, DCT, S100, and HMB45. Thus
tent cells, such as ES cells and iPS cells, can be at least a couple of multipotent stem cells reside
differentiated into melanocytes [3, 5–8, 49]. in human skin, and Muse cells and DSCs dif-
However, the potential clinical applications of ferentiate to melanocytes in certain conditions
melanocytes derived from pluripotent ES cells or in vitro.
iPS cells have faced several hurdles. There con-
tinue to be ethical controversies surrounding the
use of ES cells, and for iPS cells, inefficient gen- 30.11 Future Perspectives
eration and the presence of integrated transgenes
are significant limitations, although a lternatives to By full utilization of Muse cells, the technique to
viral vectors or the administration of analogous generate melanin-producing functional melano-
proteins or chemical compounds for cell repro- cytes and the basis for colored 3D human cul-
gramming may ultimately address these problems. tured skin that mimics human skin tissue could
In addition, it is known that the response to differ- be both developed. DS Pharma Biomedical Co.,
entiation cues is variable for some iPS cell lines. Ltd. applied this technique for industrial use and
Multipotent stem cells are reported to reside now producing “POCA® human 3D ‘HADA’
in skin dermis. Toma et al. reported that skin- (High-performance and Advanced Dermal
derived precursors (SKPs) existing in human Assay).” The system is beneficial particularly for
foreskin tissue are multipotent stem cells [50]. safety test for validation of cosmetics and drugs,
SKPs are derived from neural crest cells resid- since animal experiment has limitation for differ-
ing in the skin, and the putative niche of SKPs is ent histological structure of the skin. Since Muse
in human hair papilla [51]. SKPs express a stem melanocytes are incorporated into the 3D skin
cell marker Sox2 and have self-renewal ability culture, validation for adverse effect that may
and express Snail and Slug as characteristic cause vitiligo is expected to be enforceable.
markers. Since Muse cells don’t express Snail Muse cells are non-tumorigenic endogenous
and Slug primarily, SKPs and Muse cells are pluripotent stem cells that show high differentia-
different populations though both of them are tion ability across oligolineage boundaries from
isolated from the dermis. While SKPs are mesodermal to endodermal or ectodermal.
derived from neural crest cells, SKPs do not Melanocyte differentiation is one of their abili-
express melanocyte markers such as DCT nor ties. Notably, the current technique does not
do they differentiate into both neural and meso- require exogenous gene introduction for convert-
dermal progeny [52, 53]. On the other hand, ing dermal-Muse cells into functional melano-
dermis-derived stem cells (DSCs), which are cytes, lowering hurdles for application to clinical
also isolated from human foreskins, differenti- use. For example, patients’ skin biopsy-derived
ate into melanocytes [54]. DSCs are isolated Muse cells could be converted into Muse mela-
from spheres formed from dermis-derived sin- nocytes and transplanted for autologous treat-
gle cells grown in HESCM4 medium, which is ment of vitiligo, a possibility to be validated in a
sufficient to maintain human embryonic stem future study.
ERRNVPHGLFRVRUJ
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51. Fernandes KJ, McKenzie IA, Mill P, Smith KM,
jcs.061598.
Akhavan M, Barnabe-Heider F, et al. A dermal niche 55. Yu H, Fang D, Kumar SM, Li L, Nguyen TK, Acs
for multipotent adult skin-derived precursor cells. G, et al. Isolation of a novel population of multi-
Nat Cell Biol. 2004;6(11):1082–93. https://doi. potent adult stem cells from human hair follicles.
org/10.1038/ncb1181. Am J Pathol. 2006;168(6):1879–88. https://doi.
52. Joannides A, Gaughwin P, Schwiening C, Majed H, org/10.2353/ajpath.2006.051170.
Sterling J, Compston A, et al. Efficient generation
ERRNVPHGLFRVRUJ
Other Defects/Mechanisms
31
Maria Lucia Dell’Anna and Mauro Picardo
Contents
31.1 Local (Dermal) Environment 329
31.2 Detachment 330
31.3 WNT Pathways 331
References 331
Abstract
Key Points
Vitiligo pathogenesis still represents a puzzle
whose pieces are not all at the same level of • Overexpression of senescence-
knowledge and interest among the scientist associated markers has been described
people. The most popular aspects currently in the entire non-lesional epidermis.
considered include genetic, inflammatory, • ECM proteins contribute to melanocyte
autoimmune, oxidative, and metabolic altera- adhesion to the basement membrane.
tions, even if the individual contribution of • Vitiligo fibroblasts appeared flattened
each of these alterations is still unclear. The and enlarged, resembling senescent cells.
role of local environment, detachment process • An increased expression of CCN3 in
and emerging pathways needs renewed atten- keratinocytes in lesional skin was the
tion. These pieces of vitiligo puzzle represent most prominent feature.
a possible link between the most studied • Decreased activation of the WNT path-
mechanisms and all three together are sup- way in vitiligo.
porting each other. • WNT/β-catenin pathway was found to
have a key role in UVB-induced mela-
nocyte stem cell differentiation.
31.1 Local (Dermal) Environment

M. L. Dell’Anna · M. Picardo (*) Increasing evidence suggest the presence of cel-
Cutaneous Physiopathology, San Gallicano
Dermatological Institute, IFO, Rome, Italy
lular and functional abnormalities encompassing
e-mail: mauro.picardo@ifo.gov.it other cutaneous cell populations in both lesional

ERRNVPHGLFRVRUJ
330 M. L. Dell’Anna and M. Picardo
and non-lesional skin [1]. These abnormalities favoring over time the induction of premature
include the overexpression of senescence- senescence. The deregulated dermal-epidermal
associated markers distributed to the entire non- network may also account for the below synthe-
lesional epidermis [2] and redox imbalance and sized melanocytorrhagy.
senescence markers in cultured lesional keratino-
cytes [3, 4]. Dermal components, via extracellu-
lar matrix (ECM) proteins and mesenchymal 31.2 Detachment
cells, exert an important role in the regulation of
melanocyte homeostasis. ECM proteins contrib- The hypothesis of a chronic detachment and tran-
ute to melanocyte adhesion to the basement sepidermal loss of melanocytes is usually named
membrane. Fibroblasts release growth factors melanocytorrhagy. In this theory, the defective
and messengers that are part of a paracrine sig- adhesion of melanocytes is the predisposing fac-
naling network which includes melanocytes. tor, which may or may not lead to further immu-
Most of these mediators, such as hepatocyte nomediated inflammatory steps. Interactions
growth factor (HGF), stem cell factor (SCF), between melanocytes and basement membrane
keratinocyte growth factor (KGF), and neuregu- are mediated by integrins and those between
lin-
1 act as pro-melanogenic factors favoring melanocytes and keratinocytes by cadherins in
melanocyte growth, differentiation, migration, association with β-catenin [13].
and survival. An altered expression of growth Integrin expression seems not affected in NSV,
factors controlling melanocyte homeostasis has and down-modulation of a6 integrin in melano-
been observed in several pigmentary disorders cytes does not alter their localization in skin
including vitiligo [5–7]. However, only few reconstructs. However, an increased expression of
reports point to a specific involvement of the der- CCN3 in keratinocytes in lesional skin was the
mis in vitiligo, and most of the data are focused most prominent feature, whereas melanocytes
on lesional skin. Upregulation of the anti-adhe- remaining in perilesional vitiligo skin did not
sive ECM protein tenascin and of DKK1 has express CCN3. In addition, epidermal expression
been demonstrated in lesional dermis [8, 9]. By of DDR1 and collagen IV were very weak as
contrast, the expression and release of KGF by compared to normal control skin [14].
lesional fibroblasts are reduced [10]. According to the studies on dermal involve-
Most vitiligo fibroblasts appeared flattened ment, these data may also suggest a dermal factor
and enlarged, resembling senescent cells [6, 11]. influencing collagen IV deposition in vitiligo and
Vitiligo fibroblasts also exhibited increased actin a related possibly compensatory effect of dermal-
stress fibers, ROS content, and p53 expression. producing CCN3 cells. CCN2, CCN3, and CCN5
The p53-responsive stress gene was also signifi- are the three most highly expressed transcripts in
cantly induced in vitiligo cells. the dermis, and CCN3 is expressed in the cyto-
High levels of intracellular ROS favor the con- plasm of fibroblast-like cells and endothelial cells
version of fibroblasts into transdifferentiated of the papillary dermis. In skin fibroblasts, CCN3
α-SMA-positive myofibroblasts. works synergistically with TGF-β to upregulate
The ECM glycoprotein fibronectin and the PAI-1 expression to participate in cutaneous
intermediate filament-associated protein vimen- wound healing. CCN2, which is expressed in
tin, both associated with myofibroblast differen- melanocytes, induces ROS production in fibro-
tiation and known to be induced by ROS and in blasts, and CCN3 interacts with CCN2. Altogether,
senescent cells, were also up-modulated in vitil- these data suggest that the CCN proteins in skin
igo, as well as the mesenchymal-derived factor may influence in a complex manner not only
DKK1 [12]. melanocyte adhesion to the basal layer and the
Oxysterols may be involved in the induction composition of the papillary dermis but also
of the myofibroblast phenotype by contributing melanocyte survival and differentiation.
to the unbalanced redox status in the dermis and Accordingly, abnormal deposition of another
ERRNVPHGLFRVRUJ
31 Other Defects/Mechanisms 331
extracellular matrix protein, tenascin, and a fibro- differentiation. Within the skin, the secretion of
blastic influence on epidermal pigmentation have WNT mainly by keratinocytes and melanocytes
been demonstrated. Downregulation of a6 integ- contributes to the differentiation of stem cells in
rin in melanocytes does not alter their localization melanocytes [22].
in reconstructs, whereas CCN3 production by
melanocytes upregulates the DDR1 adhesion
receptor for collagen IV [15–21]. References
1. Picardo M, Dell’Anna ML, Ezzedine K, Hamzavi
I, Harris JE, Parsad D, et al. Vitiligo. Nat Rev Dis
31.3 WNT Pathways Primers. 2015;1:15011.
2. Bellei B, Pitisci A, Ottaviani M, Ludovici M, Cota C,
WNT/β-pathway contributes to the differentia- Luzi F, et al. Vitiligo: a possible model of degenera-
tion of stem cells in melanocytes. Functional tive diseases. PLoS One. 2013;8(3):e59782.
3. Bondanza S, Maurelli R, Paterna P, Migliore E,
analyses support the impact of the decreased Giacomo FD, Primavera G, et al. Keratinocyte cul-
activation of the WNT pathway in vitiligo, as tures from involved skin in vitiligo patients show
decreased expression of LEF1 and decreased an impaired in vitro behaviour. Pigment Cell Res.
activity of the LEF1/TCF promoter were 2007;20(4):288–300.
4. Kostyuk VA, Potapovich AI, Cesareo E, Brescia
observed after oxidative stress, and differentia- S, Guerra L, Valacchi G, et al. Dysfunction
tion of resident stem cells was induced in pre- of glutathione S-transferase leads to excess
melanocytes following ex vivo stimulation with 4-hydroxy-2-nonenal and H(2)O(2) and impaired
WNT activators. The WNT pathway is known to cytokine pattern in cultured keratinocytes and
blood of vitiligo patients. Antioxid Redox Signal.
regulate E-cadherin expression, and interest- 2010;13(5):607–20.
ingly, recent data showed decreased expression 5. Kitamura R, Tsukamoto K, Harada K, Shimizu
of E-cadherin across melanocyte membranes in A, Shimada S, Kobayashi T, et al. Mechanisms
vitiligo patients, leading to decreased adhesive- underlying the dysfunction of melanocytes in vit-
iligo epidermis: role of SCF/KIT protein interactions
ness of these cells to the basal layer under oxi- and the downstream effector, MITF M. J Pathol.
dative and mechanical stress, linking this 2004;202(4):463–75.
pathway to the suggested melanocytorrhagy. 6. Kovacs D, Cardinali G, Aspite N, Cota C, Luzi F,
Oxidative stress decreases WNT pathway activ- Bellei B, et al. Role of fibroblast-derived growth fac-
tors in regulating hyperpigmentation of solar lentigo.
ity in melanocytes and keratinocytes. WNT Br J Dermatol. 2010;163(5):1020–7.
ligands and LEF1 are first decreased in both 7. Lee AY, Kim NH, Choi WI, Youm YH. Less kerati-
melanocytes and keratinocytes; yet, decreased nocyte-derived factors related to more keratinocyte
levels of LEF1 and CDH3 persist in vitiligo skin apoptosis in depigmented than normally pigmented
suction-blistered epidermis may cause passive
ex vivo despite the augmentation of WNT prob- melanocyte death in vitiligo. J Invest Dermatol.
ably owing to compensation of the impaired 2005;124(5):976–83.
pathway. In vitiligo lesions devoid of melano- 8. Le Poole IC, van den Wijngaard RM, Westerhof W,
cytes, the keratinocytes are presumably respon- Das PK. Tenascin is overexpressed in vitiligo lesional
skin and inhibits melanocyte adhesion. Br J Dermatol.
sible for the production of WNT proteins. 1997;137(2):171–8.
Oxidative stress may negatively impact the dif- 9. Oh SH, Kim JY, Kim MR, Do JE, Shin JY, Hann
ferentiation of melanocytes in vitiligo skin. SK. DKK1 is highly expressed in the dermis of vit-
Treatment with WNT agonists or GSK3β inhibi- iligo lesion: is there association between DKK1 and
vitiligo? J Dermatol Sci. 2012;66(2):163–5.
tors induces increased expression of melanocyte 10. Purpura V, Persechino F, Belleudi F, Scrofani C, Raffa
markers, triggering the differentiation of resi- S, Persechino S, et al. Decreased expression of KGF/
dent melanocyte stem cells, not only in the hair FGF7 and its receptor in pathological hypopigmenta-
follicles but also in the dermis, in pre-melano- tion. J Cell Mol Med. 2014;18(12):2553–7.
11. Briganti S, Flori E, Bellei B, Picardo M. Modulation
cytes expressing PAX3 and DCT. Recently, the of PPARγ provides new insights in a stress
WNT/β-catenin pathway was found to have a induced premature senescence model. PLoS One.
key role in UVB-induced melanocyte stem cell 2014;9(8):e104045.
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12. Kuang HB, Miao CL, Guo WX, Peng S, Cao YJ, Duan 18. Nishio K, Inoue A, Qiao S, Kondo H, Mimura

EK. Dickkopf-1 enhances migration of HEK293 cell A. Senescence and cytoskeleton: overproduction of
by beta-catenin/E-cadherin degradation. Front Biosci vimentin induces senescent-like morphology in human
(Landmark Ed). 2009;14:2212–20. fibroblasts. Histochem Cell Biol. 2001;116(4):321–7.
13. Wagner RY, Luciani F, Cario-André M, Rubod A, 19. Powell DW, Mifflin RC, Valentich JD, Crowe SE,
Petit V, Benzekri L, et al. Altered E-cadherin lev- Saada JI, West AB. Myofibroblasts. I. Paracrine
els and distribution in melanocytes precede clini- cells important in health and disease. Am J Phys.
cal manifestations of vitiligo. J Invest Dermatol. 1999;277(1 Pt 1):C1–9.
2015;135(7):1810–9. 20. Waldera Lupa DM, Kalfalah F, Safferling K, Boukamp
14. Ricard AS, Pain C, Daubos A, Ezzedine K, Lamrissi- P, Poschmann G, Volpi E, et al. Characterization of
Garcia I, Bibeyran A, Guyonnet-Dupérat V, Taieb A, skin aging-associated secreted proteins (SAASP)
Cario-André M. Study of CCN3 (NOV) and DDR1 in produced by dermal fibroblasts isolated from
normal melanocytes and vitiligo skin. Exp Dermatol. intrinsically aged human skin. J Invest Dermatol.
2012;21(6):411–6. 2015;135(8):1954–68.
15. Akiba S, Chiba M, Mukaida Y, Sato T. Involvement 21. Yamaguchi Y, Itami S, Watabe H, Yasumoto K,

of reactive oxygen species and SP-1 in fibronectin Abdel-Malek ZA, Kubo T, et al. Mesenchymal-
production by oxidized LDL. Biochem Biophys Res epithelial interactions in the skin: increased expres-
Commun. 2003;310(2):491–7. sion of dickkopf1 by palmoplantar fibroblasts inhibits
16. Desmoulière A, Geinoz A, Gabbiani F, Gabbiani
melanocyte growth and differentiation. J Cell Biol.
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myofibroblasts and in quiescent and growing cultured Reiniche P, Mounier C, Rival Y, Piwnica D, Cavalié
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17. Miyazaki M, Gohda E, Kaji K, Namba M. Increased T. Transcriptional analysis of vitiligo skin reveals
hepatocyte growth factor production by aging human the alteration of WNT pathway: a promising target
fibroblasts mainly due to autocrine stimulation by for repigmenting vitiligo patients. J Invest Dermatol.
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ERRNVPHGLFRVRUJ
Pathophysiology of Segmental
Vitiligo
32
Nanja van Geel, Carole Van Haverbeke,
and Reinhart Speeckaert
Contents
32.1 Neural Mechanisms 334
32.2 Somatic Mosaicism 334
32.3 Microvascular Skin Homing 335
32.4 Conclusion 335
References 336
Abstract Presence of inflammatory infiltrates in progress-

Segmental vitiligo is characterized by its early ing borders of non-segmental vitiligo is a well-
onset, rapid stabilization and unilateral distri- demonstrated finding, and for long autoimmunity
bution (Chap. 6). Prevalences between 3.5% has been considered as a key factor in vitiligo
and 20.5% of all patients with vitiligo have because of the clear association of the disorder
been reported [1]. Many studies across with a personal or familial autoimmune diathesis,
decades and all over the world have attempted which targets in particular the thyroid gland.
to clarify the pathogenesis behind it, but the However, this typical association with other auto-
exact pathophysiology of segmental vitiligo immune diseases is significantly less in segmen-
remains unclear. tal vitiligo [2].
For those reasons, both types were generally
considered to be separate entities because of the
Key Points significant differences in disease presentation,
• Segmental vitiligo, Unilateral, Mosaicism evolution and associated diseases [1, 2]. However,
pathological evidence of inflammation in segmen-
tal vitiligo was described in the first edition of this
book, as well as a case report published in 2012.
N. van Geel, MD, PhD (*) · R. Speeckaert, MD, PhD This case presented an exceptional combination of
Department of Dermatology, Ghent University
Hospital, Ghent, Belgium segmental vitiligo, alopecia areata, psoriasis and
e-mail: Nanja.VanGeel@UGent.be halo naevus. These disorders are supposed to be
C. Van Haverbeke, MD inflammation-driven conditions, strengthening the
Department of Pathology, Ghent University Hospital, likelihood of an immune-mediated pathophysiol-
Ghent, Belgium ogy in segmental vitiligo [3, 4].

ERRNVPHGLFRVRUJ
334 N. van Geel et al.
More recently, more evidence became avail- damage (e.g. in subacute encephalitis, spinal cord
able that segmental and non-segmental vitiligo tumour or following trauma) [6]. Besides these
are not two completely separate entities but findings, physiological abnormalities associated
could represent variants of the same disease with sympathetic nerve function were also
spectrum. In particular, the systematic analysis described in lesional skin of patients with seg-
of segmental vs. non-segmental forms in chil- mental vitiligo (e.g. acetylcholine activity,
dren and the observation of mixed vitiligo (com- increased catecholamine and neuropeptide
bination of segmental and non-segmental release) [7]. In a study of Wu et al., an increased
vitiligo, Chap. 1.5.3) are supporting this new cutaneous blood flow compared to the contralat-
view. eral normal skin was demonstrated along with a
Until today, several theories for the pathogen- significantly increased α- and β-adrenoreceptor
esis of segmental vitiligo have been suggested, response in segmental vitiligo lesions. However,
including neural mechanisms, somatic mosa- this reaction might also be explained as a
icism and microvascular skin homing, whether or bystander effect of inflammation instead of a trig-
not leading to an autoimmune destruction of gering factor. More recently, the exact distribution
melanocytes. of segmental vitiligo was evaluated more in detail.
A study on the clinical profile of patients with
segmental vitiligo demonstrated that the distribu-
32.1 Neural Mechanisms tion of segmental vitiligo lesions was in most
cases not dermatomal, as previously assumed [8].
In 1959, Lerner et al. proposed the ‘neural the-
ory’, a theory based on the suggestion that seg-
mental vitiligo is following the course of 32.2 Somatic Mosaicism
dermatomes [5]. Therefore, neural mechanisms
have been assumed to play a role in the pathogen- Due to the observation that the majority of seg-
esis of this type of vitiligo (Fig. 32.1) [1]. This mental vitiligo lesions did not exactly fit within
theory was also supported by cases of depigmen- the borders of dermatomal lines, a second theory
tation in areas corresponding to local neurological has been suggested. In an observational study,
Microvascular skin
Neural theory Somatic mosacism Three step theory
homing
Increased rate of
Local neurological Immune activation Release of inflammatory
melanocyte apoptosis or
damage factors, neuropeptides and
increased susceptibility
catecholamines
for immune-mediated
destruction
Increased release of
Cytotoxic T cells Increased presentation of
acetylcholine, migrate to the specific melanocyte antigens
catecholamines and segmental vitiligo site (possibly due to somatic
Recruitment of via homing receptors mosacism)
neuropeptides
inflammatory cells
Generation of specific
Melanocyte Melanocyte Melanocyte cytotoxic T cells
destruction destruction destruction
Melanocyte destruction
Fig. 32.1 Neural, somatic mosaicism and microvascular nisms leading to clinical outcome of segmental
skin-homing hypotheses for segmental vitiligo and three- vitiligo (adapted from figure included in Brit J Derm 2012
step theory of several possible pathophysiological mecha- 166:240–6)
ERRNVPHGLFRVRUJ
32 Pathophysiology of Segmental Vitiligo 335
the pattern of segmental vitiligo resembled in in the regional lymph node [1]. A study in 2010
some cases a Blaschkoid distribution, while provides evidence that a melanocyte-specific
other segmental vitiligo lesions corresponded to CD8+ T-lymphocyte-mediated immune response
other patterns of mosaicism (Fig. 32.1) [9]. was involved in the early phases of segmental vit-
Striking similarities were found in this iligo [3]. It seems however unlikely that this the-
study with other mosaic skin disorders, in par- ory of skin-homing receptors can entirely explain
ticular segmental lentiginosis—speckled lentig- the specific disease pattern of segmental vitiligo.
inous nevus—as also noted previously by Hann Furthermore, no other inflammatory skin disease
(Chap. 1.5.2) and verrucous epidermal naevi [9]. has been described showing a similar pattern.
A typical recurring ‘segmental vitiligo pattern’ Melanoma-associated depigmentation after vac-
was observed that can fit within the theory of cination (often delivered by subcutaneous or
mosaicism, but is not yet included in the six sub- intradermal injection) follows a more generalized
types of mosaicism as described by Happle et al. vitiligo-like pattern and not a segmental pattern
[1, 9, 10]. Pigmentary mosaicism has been sug- that could be expected according to the theory of
gested to represent the migration pattern of skin unilateral skin homing [1].
cells during embryogenesis. When visible, pig-
mentary mosaicism (e.g. Blaschkoid lines)
reflects an underlying mosaicism of different 32.4 Conclusion
cell lines [2]. This mosaic distribution has been
observed in several pigmentation disorders such The pathophysiology of segmental vitiligo remains
as Blaschkoid hypopigmentation, epidermal controversial, and several hypotheses lack a clear
naevi and naevus depigmentosus [1, 9]. So far, cascade of events explaining various aspects of
convincing data at the molecular level are still this disease. However, it should be emphasized
missing. A clinical observation, supporting this that the different theories of segmental vitiligo do
theory, is the superior long-term take of epider- not exclude each other. Moreover, the existence of
mal cellular grafting in segmental vitiligo lesions several aetiopathological pathways or different
compared with inferiour results in patients with underlying initial triggers is likely. These different
generalized vitiligo. It indicates that the trans- pathways might all subsequently activate skin
planted cells of autologous donor skin may immune or inflammatory responses leading to
be genetically affected as well in the g eneralized melanocyte destruction. Our group proposed a
type of vitiligo and not in the isolated type of three-step theory, including the different patho-
segmental vitiligo [1]. physiological mechanisms leading to the clinical
presentation of segmental vitiligo, providing
hereby a more integrated view into the current evi-
32.3 Microvascular Skin Homing dence on segmental vitiligo [1]. This three-step
theory includes possible reactions related to neural
Another hypothesis on the pathogenesis of seg- mechanism, somatic mosaicism, microvasular
mental vitiligo suggests that the midline delinea- skin homing and an antimelanocyte-specific
tion in unilateral lesions could represent the immune response. It explains the typical distribu-
migration pattern of cytotoxic T cells from spe- tion pattern and different prognosis of segmental
cific lymph nodes along the efferent microvascu- vitiligo, compared with general vitiligo [11].
lar system via homing receptors (Fig. 32.1). The Although this three-step theory is based on
observation of halo naevi in patients with seg- limited evidence, it summarizes the different pro-
mental vitiligo may support this theory. Patients posed aetiopathogenetic mechanisms for seg-
most often mention that the appearance of halo mental vitiligo into one model. Future research
naevi occurs prior to the development of the seg- needs to shed more light on this hypothesis, and
mental vitiligo lesions, suggesting a clonal the exact pathogenesis of segmental vitiligo is yet
expansion of melanocyte-specific T lymphocytes to figure out.
ERRNVPHGLFRVRUJ
336 N. van Geel et al.
References 5. Mohammed GF, Gomaa AH, Al-Dhubaibi MS.

Highlights in pathogenesis of vitiligo. World J Clin
Cases. 2015;3:221–30.
1. Van Geel N, Mollet I, Brochez L, et al. New insights in
6. Nelhaus G. Acquired unilateral vitiligo and poliosis
segmental vitiligo: case report and review of theories.
of the head and subacute encephalitis with partial
Br J Dermatol. 2012;166:240–6.
recovery. Neurology. 1970;20:965–74.
2. Taïeb A, Morice-Picard F, Jouary T, et al. Segmental
7. Wu CS, Yu HS, Chang HR, et al. Cutaneous blood flow
and adrenoceptor response increase in segmental-type
vitiligo lesions. J Dermatol Sci. 2000;23:53–62.
8. Zhou J, Zhong Z, Li J, Fu W. Motor nerve conduc-
2008;21:646–52.
tion velocity is affected in segmental vitiligo lesional
3. van Geel NA, Mollet IG, De Schepper S, et al. First
limbs. Int J Dermatol. 2016;55(6):700–5. https://doi.
histopathological and immunophenotypic analysis
org/10.1111/ijd.13171.
of early dynamic events in a patient with segmen-
9. van Geel N, Speeckaert R, Melsens E, et al. The distri-
tal vitiligo associated with halo nevi. Pigment Cell
bution pattern of segmental vitiligo: clues for somatic
Melanoma Res. 2010;23:375–84.
mosaicism. Br J Dermatol. 2013;186:56–64.
4. Shin J, Kang HY, Kim KH, Park CJ, Oh SH, Lee
10. Happle R. Mosaicism in human skin. Understanding
SC, Lee S, Choi GS, Hann SK. Involvement of T
the patterns and mechanisms. Arch Dermatol.
cells in early evolving segmental vitiligo. Clin Exp
1993;129:1460–70.
Dermatol. 2016;41(6):671–4. https://doi.org/10.1111/
11. van Geel N, De Lille S, Vandenhaute S, et al. Different
ced.12852.
phenotypes of segmental vitiligo based on a clinical
observational study. J EurAcad Dermatol Venereol.
2010;25:673–8.
ERRNVPHGLFRVRUJ
Editor’s Synthesis
33
Mauro Picardo and Alain Taïeb
Contents
33.1 Do All Supposed Mechanisms of Melanocyte Loss Occur In Vivo? 338
33.2 n Enlarged Vision of the Skin Melanogenic Unit May Apply
A
to Vitiligo 339
33.3 Melanocyte Stemness and Vitiligo 339
33.4 The Somatic Mosaicism Hypothesis for SV and Deductions for NSV 340
33.5 Membrane Lipids as Possible Culprits 340
33.6 The Innate Immunity Hypothesis 341
References 341
We have summarized in Table 33.1 the current Concerning clinical, epidemiological, and
status of our understanding of vitiligo pathomech- pathological data reviewed in Sect. 33.1, which
anisms. We have tried to divide it into major should have provided a solid basis for the
fields and levels of evidence. Clearly, in vivo evi- understanding of the disease, we have already
dence in human disease comes first. In vitro noticed intriguing weaknesses in the vitiligo
work, which relies on cultures, can be viewed field. Indeed, the cooperation among research
more cautiously because of artifacts caused by groups has been only recently developing, and
culture conditions, even though appropriate con- variable definitions of type or stage of the dis-
trols are used. Some animal models are probably ease (Chap. 1.2.1) led to difficulties in the inter-
relevant, but definitive evidence is lacking. pretation of some in vivo data, such as the
importance of skin inflammation in the disease.
M. Picardo (*) There has been also a rampant temptation (con-
Cutaneous Physiopathology and CIRM, scious or not) to erase some aspects, which were
San Gallicano Dermatological Institute, IRCCS, not considered as consistent with the pathophys-
Rome, Italy
iological point of view defended by individual
authors.
A. Taïeb
In the following sections, we highlight some
Service de Dermatologie, Hôpital St André, CHU de
Bordeaux, Bordeaux, France gaps in our understanding, underline possible uni-
e-mail: alain.taieb@chu-bordeaux.fr fying scenario, and propose, based on Sect. 33.2

ERRNVPHGLFRVRUJ
338 M. Picardo and A. Taieb
Table 33.1 Summary of some aspects of our current understanding of vitiligo

Level of evidence
Field Good Moderate Limited
Cell biology • Loss of pigment cells in • Loss of melanocyte adhesion • Involvement of non-skin
epidermal/follicular • Melanocyte senescence and stem melanocytes? (not
compartments, melanocyte cell compartment involved completely settled)
structural and functional • Fibroblast functional defects • Apoptosis of melanocytes
anomalies, including • PBMC defects • TNF-induced disease
friction-induced detachment • Mitochondrial impairment • Stratum corneum activation
in vivo • Defective activity of receptors for of inflammasome or other
• Keratinocyte anomalies growth factors melanocyte-targeting
(structural and in culture) • MITF-M deficiency pathways to explain
• Reduced keratinocyte-derived • Propopiomelanocortin system Koebner’s phenomenon
melanocyte growth factors alterations (primary or secondary)
(e.g., SCF, αMSH, ET1)
Genetics • Polygenic background • Causative inflammasome • Several candidate gene
• NALP 1 genetic variants in functional alteration associations (approaches not
NSV associated with • Other causative gene variants or validated/replicated)
autoimmune diseases gene-modifying variants in • Mitochondrial genome
subsets of patients (AIRE, CMH defects (not fully tested)
haplotypes, oxidative stress • Abnormal expression of
regulators, etc.) genes of the FOXD3
pathway (to confirm)
• Protective MC1-R alleles
• Predisposing skin-related
genetic anomaly (applies
mostly to segmental vitiligo)
Biochemistry • Catechols metabolism • Membrane-associated defects, • Melanogenesis defect
anomalies including mitochondria defects exclusively
• Antioxidant systems • Role of environmental toxic
anomalies compounds (phenol, catechols,
• Biopterin pathway anomalies etc.)? (more epidemiologic studies
needed)
Immunology • Humoral (serum) and cellular • Relevant cellular targets of • Contagious disease (but role
(in situ) immune responses to immune responses not clearly of microorganisms not ruled
pigment cell proteins established out; see Role of Herpesvirus
• T-cell lichenoid infiltrates in • Autoimmunity to melanin in the Smyth Chicken Model
progressive NSV, mostly catabolites of Vitiligo (Chap. 2.2.4)
CD8+ • Viral infection (direct) • Injury activation of the skin
• Innate immunity defect to innate immune system
bacterial infections (NALP1?) • Deficient clearance of
apoptotic fragments
review of data, a hierarchy of possible events apoptotic demise of melanocytes, are not yet
occurring upstream of melanocyte loss in the vit- substantiated. In nonsegmental vitiligo, there is
iligo puzzle (Fig. 33.1). now ample evidence that melanocytes of non-
clinically affected areas can be morphologically
or functionally altered in favor of either an
33.1 D
o All Supposed intrinsic abnormality, which could be the basis
Mechanisms of Melanocyte of the Koebner’s phenomenon, or of a systemic
Loss Occur In Vivo? low-key immune disturbance targeting the cuta-
neous melanocytic system, or both. This point is
Table 33.1 indicates a wide possible range of crucial for a better understanding and will need
events, which may provoke melanocyte loss. more in vivo studies using skin biopsies to look
Interestingly, some popular theories, such as the at early changes in the disease. The experimental
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33 Editor’s Synthesis 339
Fig. 33.1 The organized puzzle of the pathogenetic factors
provocation of the Koebner’s phenomenon, as pivotal cells of the innate immune system, being
indicated in Chap. 2.2.2.1, may be a valuable envisioned as maintaining the immune surveil-
tool to detect the chronology of events in vitil- lance of the pigmentary system in case of local
igo and especially a link between trauma and danger. Progresses in the knowledge of the epi-
inflammation. dermal and dermal network governing the release
of specific cytokines and growth factors open
new perspectives in the pathogenesis and therapy
33.2 An Enlarged Vision of vitiligo, if the loss of pigment cells can be first
of the Skin Melanogenic Unit stopped.
May Apply to Vitiligo
If melanocytes are the undisputed target of the 33.3 Melanocyte Stemness

disease, other skin cells affect their function and and Vitiligo
survival. The conventional skin pigmentation
unit was the epidermal melanin unit of Fitzpatrick Until now, the attention has been mostly focused
and Breathnach [1]. A dermal-epidermal melanin on resident epidermal melanocytes. However,
unit was more recently defended [2]. Based on exchanges between hair follicles and interfollicu-
current knowledge of vitiligo pathophysiology, lar compartments have been well demonstrated
the skin melanogenic unit should include, besides for melanocytes (Chaps. 1.3.6 and 2.2.10). The
melanocytes, keratinocytes, fibroblasts, and pos- melanoblasts of the hair follicle niche, mostly
sibly cutaneous nerve endings of the considered studied in the mouse, differentiate and migrate to
unit. Langerhans cells could also play a role as the hair bulb while maintaining a reserve of
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undifferentiated cells inside the bulge [3]. The with the common presentation of NS vitiligo as
niche allows thus the continuous production of an isolated chronic cutaneous disorder in the
new melanocytes avoiding cell exhaustion and majority of patients. Since friction-/pressure-
senescence. Vitiligo may represent a localized prone areas are frequently initially affected, it
senescence process through a mechanism similar would be tempting to speculate that the primary
to the “free radical-mediated graying” [4, 5], skin anomaly affects the upper layers of the epi-
associated with a progressive melanocyte loss dermis to explain the Koebner’s phenomenon; it
from the hair bulb. The presence, or not, of a would explain nicely the subsequent epidermal
functioning follicular reservoir may also be the activation of innate immunity-based mechanisms
critical factor for a successful melanocyte graft via stratum corneum activation of inactive IL1beta
besides loss through the Koebner’s phenomenon. precursors [8]. Pressure may also affect directly
Usually, a minority of melanocyte stem cells basal epidermal layer biology if an abnormal
matures into differentiated and migrating mela- adhesion of melanocytes comes first to explain
nocytes, whereas the bulk of them remains quies- their detachment [9]. Also as envisaged above is
cent. The exhaustion of the quiescent stock will a possible genetic cause limiting survival and
affect the next melanocyte production. The abil- self-renewal of epidermal and follicular melano-
ity to respond to specific growth factors, such as cytes, the latter being hit earlier by the disease in
SCF, may determine the continuous production SV as compared to NSV [6]. The clear associa-
of differentiated and functioning epidermis- tion of NSV to familial hair graying also favors a
committed melanocytes. At this regard, Sect. genetic background affecting melanocyte sur-
33.2 has provided several arguments indicating vival. The expression of several genes influenc-
that the c-Kit/SCF axis appears to be broken in ing such pathways is indeed modified in NSV
vitiligo (Chap. 2.2.8). Vitiligo may be considered [10].
as a possible model for the study of stemness and
senescence processes.
33.5 M
embrane Lipids as Possible
Culprits
33.4 T
he Somatic Mosaicism
Hypothesis for SV The lipidomics approach to cellular signaling indi-
and Deductions for NSV cates that lipids may be considered as “signaling
molecules” between extracellular events and cell
The recently demonstrated association of SV adaptive response [11]. Alterations involving lipid
and NSV has provided new insights in the metabolism may lead to modifications of the struc-
pathogenesis of vitiligo [6]. The main reasoning ture of the membranes, affecting the sensitivity to
behind is that still unknown genetic predispos- prooxidant agents, modulating the intracellular
ing factors may affect first the skin pigmentary redox status (Chap. 2.2.6), and favoring the release
system. The activation of the skin immune/ of haptened lipids, which are, in turn, able to acti-
inflammatory responses would come second vate the immune system. Moreover, the altered
leading to a more severe expression of disease. membrane may participate to the propagation of
This scenario is somewhat derived from that the oxidative damage, responsible for a high level
proposed for another common skin disorder, of intracellular ROS, which are, in turn, also capa-
atopic dermatitis, for which a skin barrier ble to affect the structure and activity of the
genetic dysfunction comes first and may pro- POMC-derived peptides. This phenomenon might
duce direct inflammation [7] and engages in a be the basis for the reduction of αMSH in vitiligo
facultative second step the immune system into epidermis. The responses to the specific growth
an “allergic” Th2-dependent pathway in a subset factors, as well as the processes underlying adhe-
of patients. This two-phase model is consistent sion and survival events, could also be impaired by
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33 Editor’s Synthesis 341
the defective metabolism of lipid messengers. The For unravelling the mysteries of the basement
topical application of sphingolipids-mediated res- level 1, the genetic basis of vitiligo, besides the
toration of Mitf expression and repigmentation in conventional approaches on blood DNA, SV con-
a mouse model of hair graying is in accordance sidered as a common skin genetic mosaic revealed
with this view [4, 5]. postnatally would be a good model. SV is candi-
date disease to explore as a proof of principle for
a new gene discovery strategy in multigenic dis-
33.6 T
he Innate Immunity orders with organ specificity [6, 14]. For a good
Hypothesis angle of attack of the puzzle-like pyramid, we
still lack a definitive argument coming from
Besides the possible exposure of lipid-protein either a clinical or a basic science perspective.
complexes with antigenic properties, apoptosis or The following section will deal with those uncer-
other mechanisms of cellular alteration may acti- tainties for the currently limited available thera-
vate the immune surveillance through the release peutic options.
of membrane fragments. Based on the finding of
NALP variants in immune NSV [12], it has been
hypothesized that such cellular debris or apoptotic References
fragments could activate the NALP1 inflamma-
some complex, activating in turn the IL1 pathway 1. Fitzpatrick TB, Breathnach AS. The epider-
mal melanin unit system. Dermatol Wochenschr.
and alerting the immune system. In addition, 1963;147:481–9.
hsp70 production related to membrane damage 2. Cario-André M, Pain C, Gauthier Y, et al. In vivo and
(Chap. 2.2.7.4) has been widely associated to the in vitro evidence of dermal fibroblasts influence on
activation of antigen-presenting cells. The human epidermal pigmentation. Pigment Cell Res.
2006;19:434–42.
immune-mediated damage, strongly suggested by 3. Yonetani S, Moriyama M, Nishigori C, et al. In vitro
a wide range of clinical and experimental data expansion of immature melanoblasts and their ability
(Chaps. 2.2.3.2, 2.2.4, and 2.2.5), could be thus to repopulate melanocyte stem cells in the hair fol-
the most prominent modality of loss of geneti- licle. J Invest Dermatol. 2008;128:408–20.
4. Picardo M. Lipid-mediated signalling and mela-
cally compromised melanocytes best demon- nocyte function. Pigment Cell Melanoma Res.
strated during flares or acceleration phases of the 2009;22:152–3.
disease. 5. Saha B, Singh SK, Mallik S, et al. Sphingolipid-
mediated restoration of Mitf expression and repig-
mentation in vivo in a mouse model of hair graying.
Pigment Cell Melanoma Res. 2009;22:205–18.
33.7 Concluding Remarks 6. Taïeb A, Morice-Picard F, Jouary T, et al. Segmental
Looking at the most recent experimental somatic mosaicism: implications for common non-
approaches, Le Poole et al. convergence theory 2008;21:646–52.
[13] can tentatively be updated and featured as a 7. Taieb A. Hypothesis: from epidermal barrier dys-
pyramid where melanocyte loss is the tip of an function to atopic disorders. Contact Dermatitis.
enormous puzzle, with a predisposing genetic 1999;41:177–80.
8. Taieb A. NALP1 and the inflammasomes: challenging
background at the basement level (Fig. 33.1). our perception of vitiligo and vitiligo-related autoim-
A variable genetic background (level 1) could, mune disorders. Pigment Cell Res. 2007;20:260–2.
under the influence of also variable and still poorly 9. Gauthier Y. The importance of Koebner’s phenom-
understood environmental triggering factors enon in the induction of vitiligo vulgaris lesions. Eur
J Dermatol. 1995;5:704–8.
(arrows), activate several pathogenic pathways 10. Strömberg S, Björklund MG, Asplund A, et al.

(level 2), occur through different mechanisms Transcriptional profiling of melanocytes from patients
(level 3) and, according to the intensity of the stim- with vitiligo vulgaris. Pigment Cell Melanoma Res.
uli, all may concur to melanocyte loss (level 4). 2008;21:162–71.
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11. Hannun YA, Obeid LM. Principles of bioactive lipid 13. Le Poole IC, Das PK, van den Wijngaard RM, et al.
signalling: lessons from sphingolipids. Nat Rev Mol Review of the etiopathomechanism of vitiligo: a con-
Cell Biol. 2008;9:139–50. vergence theory. Exp Dermatol. 1993;2:145–53.
12. Jin Y, Mailloux CM, Gowan K, et al. NALP1 in
14. Happle R. Superimposed segmental manifestation

vitiligo-associated multiple autoimmune disease. N of polygenic skin disorders. J Am Acad Dermatol.
Engl J Med. 2007;356:1216–25. 2007;57:690–9.
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Part III
Treating the Disease
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Management Overview
34
Contents
34.1 Management-Oriented Evaluation 346
34.2 Treatment Overview 346
34.2.1 V Treatments
U 347
34.2.2 Topical Therapies and Combined Therapies 348
34.2.3 Surgery 348
34.2.4 Camouflage and Depigmentation 348
34.2.5 Other Therapies 349
34.2.6 Counseling 349
34.3 Evidence-Based Guidelines 349
References 349
Abstract to possible aggravating environmental factors

Vitiligo psychosocial impact is essential when and associated autoimmune diseases, in particu-
considering management issues, as well as pre- lar thyroid disease. The first target of therapy is
vious messages given to the patient. Patients in most cases to stop disease progression of the
should be informed that (1) vitiligo is a chronic/ disease, best achieved by combined approaches
relapsing disorder, (2) repigmentation is a slow phototherapy, systemic and local drug interven-
process, and (3) reactivation of the disease in tion. Narrowband UVB is the most useful repig-
different body regions or the reappearance of menting regimen, with the current development
lesions in treated ones may occur, which gives a of home phototherapy devices. Potent topical
rationale for a maintenance treatment. Initial corticosteroid or topical calcineurin inhibitor
assessment should focus on overall severity and therapy may be used first line for localized dis-
course of disease profile, with special attention ease; on the face, calcineurin inhibitors are cur-
rently preferred because of potential side effects
A. Taïeb (*) of prolonged application of steroids and their
Hôpital Saint-André Service de Dermatologie, good safety profile. In addition to psychological
Bordeaux, France
support, offering training to camouflage tech-
niques is particularly helpful in dark-skinned
M. Picardo
patients for facial/hand lesions. Cellular trans-
Dermatological Institute, IRCCS, Rome, Italy plantation or grafting is an option in specialized
e-mail: mauro.picardo@ifo.gov.it centers for patients who have stable and limited

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lesions that are refractory to other therapy. and ability to tan (phototype), disease duration, and
Depigmenting techniques are difficult to handle disease activity are important decision management
and concern a minority of patients. items, as well as the patient psychological profile
and coping with the disease. In some vitiligo/NSV
patients, an “acceleration phase” occurs with a rapid
Key Points disease progression in a few weeks/months which
• Vitiligo psychosocial impact is essential needs a more urgent intervention, such as minipulse
when considering management issues. therapy. Other useful clinical management items
• Patients should be informed that (1) vitil- include previous episodes of repigmentation and
igo is a chronic/relapsing disorder, (2) type, duration, and usefulness of previous treat-
repigmentation is a slow process, and (3) ments. Overall, estimating body surface area (e.g.,
reactivation of the disease in different body rule of 9 using VETF scoring system; see below)
regions or the reappearance of lesions in and dividing by duration of disease give a rough but
treated ones may occur, which gives a useful profile of disease progression. The analysis
rationale for a maintenance treatment. of the Koebner phenomenon and occupations is of
• Initial assessment should focus on overall particular interest for prevention [2]. A simple scor-
severity and course of disease profile, ing system of the Koebner’s phenomenon has been
including aggravating environmental fac- introduced in the clinic [3]. A question about vitil-
tors and associated autoimmune diseases. igo on genitals is included because it causes a strong
• The first target of therapy is to stop dis- embarrassment to patients. A global quality of life
ease progression, best achieved by com- assessment is recommended (“how does vitiligo
bined approaches phototherapy, systemic currently affects your everyday life”, measured over
and local drug intervention. the last week) assessed on a visual analog scale,
• Narrowband UVB is the most useful which is a coarse but useful indicator of coping with
repigmenting regimen. the disease. The patient’s personality and perceived
• Potent topical corticosteroid or topical severity of the disease are predictors of quality of
calcineurin inhibitor therapy may be life impairment [4]. Validated HRQoL scales have
used first line for localized disease. been published and can be filled by patients in the
• In addition to psychological support, waiting room [5].
offering training to camouflage tech- Because of the frequent association of
niques is particularly helpful in dark- vitiligo /NSV with autoimmune thyroid disease,
skinned patients for facial/hand lesions. especially Hashimoto’s thyroiditis, it is recom-
• Cellular transplantation or grafting is an mended to measure on an annual basis the thyro-
option in specialized centers for patients tropin level in patients with antibodies to thyroid
who have stable and limited lesions that peroxidase, which may precede overt thyroiditis
are refractory to other therapy. [6]. Any suggestive manifestations of organ-spe-
• Depigmenting techniques are difficult to cific autoimmune diseases should prompt appro-
handle and concern a minority of patients. priate investigations [7]. The index of suspicion
should be raised when a personal and/or family
history of autoimmune/auto-inflammatory disor-
ders is obtained.
34.1 Management-Oriented
Evaluation
34.2 Treatment Overview
Before discussing management with the patient, a
thorough assessment is needed. An assessment Vitiligo treatments have so far been analyzed
form has been produced by the Vitiligo European using the proportion of treated patients who
Task Force (VETF), which summarizes major his- achieve a specified degree of repigmentation,
tory taking and examination items [1]. Skin color usually starting at >50% for a “good” response.
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34 Management Overview 347
“Good” for this level of skin repigmentation is Table 34.1 General outline of management for vitiligo
indeed debatable in a patient-oriented view in adults
because satisfaction needs, in addition to stop- Type of vitiligo Usual management
ping disease progression, complete restauration Segmental and First line: Avoidance of triggering
limited vitiligo/ factors, local therapies
of pigmentation, especially in visible areas. This
NSV <2–3% (corticosteroids, calcineurin
point has been recently examined in depth by the body surface inhibitors) combined with localized
VGICC [8]. The VETF has proposed a simple involvement NB-UVB therapy, especially
method of assessment which combines analysis excimer monochromatic lamp or
laser, or home NB-UVB
of extent, grading of depigmentation, and pro-
phototherapy
gression [1]. Clinical photographs and if possible Second line: Consider surgical
UV-light photographs are needed for accurate techniques if repigmentation
monitoring of repigmentation. cosmetically is unsatisfactory on
visible areas
For stopping progression, besides UV thera-
Vitiligo/NSV First line: Usual initial combination
pies, systemic steroids have been evaluated mostly >3% BSA total body NB-UVB with systemic/
in open studies and seem to arrest disease progres- topical therapies, including
sion [9–12]. Commonly used repigmentation ther- reinforcement with localized UVB
apies for vitiligo that are supported by data from therapy (see text)
Second line: Consider surgical
randomized trials (RCT) include UV light (whole- techniques in nonresponding areas
body irradiation or UV targeted to lesions) and especially with high cosmetic
topical agents (corticosteroids, calcineurin inhibi- impact
tors, calcipotriol). Camouflaging and depigment- Third line: Consider depigmentation
techniques in nonresponding
ing (in widespread disease) are the other current widespread (>50%) or highly
options. Table 34.1 outlines a stepwise treatment visible recalcitrant facial/hand
approach divided by type of vitiligo and extent, vitiligo
which needs modulation by visibility, age, and (Revised from Taieb A, Gauthier Y. Combining surgical
coping. A zero line is always possible, meaning no and medical therapies. In: Surgical management of
Vitiligo, ed. by Gupta, Olsson and Ortonne. Wiley
treatment if the disease is not bothering the patient. Blackwell 2006. pp. 267–272)
Maintenance therapy is validated for facial lesions A no treatment option (zero line) can be considered in
and intermittent tacrolimus use [13]. patients with a fair complexion after discussion. For chil-
dren, phototherapy is limited by feasibility in the younger
age group and surgical techniques rarely proposed before
prepubertal age. There is no current recommendation vali-
34.2.1 UV Treatments dated to the case of rapidly progressive vitiligo, non-
stabilized by UV or combined therapy. Camouflage,
The currently preferred treatment in adults and psychological support, and maintenance treatment are
indicated in all cases
compliant children with vitiligo/NSV is narrow-
band UVB (NB-UVB), which delivers peak emis-
sion at 311 nm [14]. The color match of Home phototherapy devices are thus more conve-
repigmented skin is excellent, but the response nient and suitable for small extent disease [16].
rate remains low based on patient expectations. The optimal dose may differ at different sites, and
With twice- weekly NB-UVB treatments for an option is shielding the area with the lower
1 year, 48–63% of patients with NSV repigment MED after reaching its optimal dose while con-
75% of the affected areas [15]. At least 3 months tinuing to expose higher MED areas until optimal
and preferably 4 months of treatment is warranted dosing [15]. There is no apparent relationship
before identifying a patient as a nonresponder, between the degree of initial depigmentation and
and approximately 9–12 months of treatment is the response to NB-UVB treatment [14], but the
usually required to achieve the maximal repig- duration of disease is inversely correlated with the
mentation. Unfortunately, access to this treatment degree of treatment-induced repigmentation [15].
is frequently limited in some areas due to low The best results are achieved on the face, followed
availability or long distances to treatment centers. by the trunk and limbs. The poorest outcomes
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have been noted for hands and feet lesions that at Calcineurin inhibitors can promote repigmentation
best show a moderate response. Relapses are without immunosuppression [25]. The combined
common at all sites; around 60–70% of patients use of UV and calcineurin inhibitors is not yet fully
resume depigmentation in areas repigmented by approved but recommended by the VETF/EDF/
treatment within 1 year whether the regimen is EADV/UEMS guidelines [40]. Alternative sources
PUVA or NB-UVB [17]. of light such as the helium-neon laser (red light)
Responses of segmental vitiligo to narrow- need more development [26]. Topical corticoste-
band UVB are at best limited when the same roids can also increase the efficacy of UVB [41].
NB-UVB therapy is applied for 6 months [15]. Topical calcipotriene RCT studies indicate limited
Earlier and more aggressive treatment schemes or no effect in isolation and a possible minor
are thus advisable. The use of targeted high-flu- enhancer effect on repigmentation in combination
ency UVB (excimer laser or monochromatic with UV or topical corticosteroids [19].
excimer lamp, both at 308 nm) which may reach
deeper targets such as amelanotic melanocytes of
the hair follicle, and also avoid irradiation of 34.2.3 Surgery
uninvolved skin, may improve outcomes, as well
as combined approaches. Results are also prom- Surgical methods such as minigrafting, which
ising in cases of vitiligo/NSV involving limited consists in transplanting punch grafts from an
areas [4, 18–35]. Red light helium-neon laser autologous donor site [21]—currently less used
phototherapy has also been reported as promot- when other options are possible—or cellular trans-
ing repigmentation in SV [36]. plantation using autologous epidermal cell sus-
pensions containing melanocytes [23] or ultrathin
epidermal grafts [31] or a combination of cellular
34.2.2 Topical Therapies transplantation and ultrathin grafting, are used in
and Combined Therapies cases of focal/segmental vitiligo if medical
approaches fail. Several variants have been
Topical therapies are not suitable for widespread recently developed. UV irradiation is frequently
NSV but may be effective in cases with more local- associated [42]. Patients with vitiligo/NSV are
ized disease (including SV). Combined treatments considered good candidates for surgical tech-
are frequently considered when phototherapy alone niques depending on availability and cost, if their
does not show efficacy after 3–4 months or in an disease is stable (over the preceding year) [3] and
attempt to accelerate response and reduce cumula- has a limited extent (2–3% of body surface area).
tive UV exposure [37]. As compared with PUVA Contrary to SV, in which the grafted cells come from
which determines a predominant perifollicular pat- apparently disease-free areas, the survival of living
tern of repigmentation, topical corticosteroids (and but potentially abnormal transplanted melanocytes
topical calcineurin inhibitors—TCI—such as tacro- is less predictable in vitiligo/NSV. Koebnerization
limus and pimecrolimus) exhibit a diffuse type, limits efficacy (hands in particular). Based on a
which is faster but less stable [33]. Based on a meta- RCT, after a strict preoperative selection for dis-
analysis, class 3 (potent) topical corticosteroids ease stability in vitiligo/NSV, cellular transplanta-
achieve more than 75% repigmentation in 56% of tion plus UV results in repigmentation of at least
patients [22]. TCI are preferred for face and neck 70% of the treated area [42].
lesions, because they do not cause skin atrophy. The
efficacy of TCI is enhanced by exposure to UV
radiation delivered by high-fluency UVB devices 34.2.4 Camouflage and
[35] but not clearly by conventional NB-UVB [28]. Depigmentation
The concerns about the risk of cutaneous or even
extracutaneous cancer provoked by topical calcineu- Topical camouflaging products mask esthetic skin
rin inhibitors have been disproportionate [38, 39]. disfigurement on a transient, semipermanent, or
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permanent basis (tattoos). Benefits can be 34.3 Evidence-Based Guidelines

obtained by the skilled use of corrective cos-
metics. For dihydroxyacetone (DHA), the most General guidelines for adults and children have
used self tanner, the higher the concentration, been elaborated by the British Association of
the better the response observed particularly in Dermatologists [24] and guidelines for surgery
darker phototypes [10]. Chemical or laser by the IADVL Dermatosurgery Task Force [32].
depigmentation can definitely be a choice in a A treatment algorithm has been also proposed
small subset of carefully selected patients, but based on evidence-based medicine principles
results are variable both in efficacy and dura- [27]. In addition to the Indian and Japanese
tion [30]. guidelines, the European VETF/EDF guidelines
have been published in 2011 [40] and are cur-
rently in revision. Beyond guidelines, personal-
34.2.5 Other Therapies ized/stratified strategies have been proposed [45].
The use of topically applied antioxidants has not

yet been supported by long-term follow-up stud-
ies [40]. Yet, the topical application of the syn- References
thetic analog of the catalase (PC-KUS) has been
1. Taïeb A, Picardo M, VETF Members. The definition
proposed as consistent therapy for the NSV both and assessment of vitiligo: a consensus report of the
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efficacy of natural health products (Chinese 2007;20:27–35.
herbs, plant extracts, vitamins) for vitiligo exist 2. Diallo A, Boniface K, Jouary T, Seneschal J, Morice-
Picard F, Prey S, Cario-André M, Mazereeuw-Hautier
but are of poor methodological quality and con- J, Taieb A, Ezzedine K. Development and validation
tain significant reporting flaws [43]. An RCT of the K-VSCOR for scoring Koebner’s phenom-
supports a moderate adjunctive effect of enon in vitiligo/non-segmental vitiligo. Pigment Cell
Polypodium leucotomos to NB-UVB [29] and Melanoma Res. 2013;26:402–7.
3. Ezzedine K, Lim HW, Suzuki T, Katayama I, Hamzavi
another that of systemic antioxidants in addition I, Lan CC, Goh BK, Anbar T, Silva de Castro C, Lee
to NB-UVB [20]. Ginkgo biloba as a monother- AY, Parsad D, van Geel N, Le Poole IC, Oiso N,
apy [34] is not recommended. Benzekri L, Spritz R, Gauthier Y, Hann SK, Picardo M,
Taieb A, Vitiligo Global Issue Consensus Conference
Panelists. Revised classification/nomenclature of vit-
iligo and related issues: the Vitiligo Global Issues
34.2.6 Counseling Consensus Conference. Pigment Cell Melanoma Res.
2012;25:E1–13.
The involvement of a psychologist or psychiatrist 4 . Kostopoulou P, Jouary T, Quintard B, Marques S,
Boutchnei S, et al. Objective vs subjective factors
may be helpful in patients who experience diffi- in the psychological impact of vitiligo: the experi-
culty coping with the diagnosis. Sunscreens are ence from a French referral center. Br J Dermatol.
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JM, Jouary T, Ballanger F, Boralevi F, Taieb A, Taieb C,
because moderate sun exposure (heliotherapy) is Ezzedine K, The Vitiligo Impact Patient Scale (VIPs).
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Repeated frictions for applying sunblockers 6. Gey A, Diallo A, Seneschal J, Léauté-Labrèze
C, Boralevi F, Jouary T, Taieb A, Ezzedine
without real sunburn risk may be more detrimen- K. Autoimmune thyroid disease in vitiligo: multivari-
tal than beneficial. It is important to make time ate analysis indicates intricate pathomechanisms. Br J
available to discuss photoprotection issues vs. Dermatol. 2013;168:756–61.
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significance of organ-specific autoimmune disorders
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Passeron T, Böhm M, Anbar T, Goh BK, Lan Clin Exp Dermatol. 2007;32:631–6.
C-CE, Lui H, Ramam M, Raboobee N, Katayama 21.
Falabella R. Treatment of localized vitiligo
I, Suzuki T, Parsad D, Seth V, Lim H, van Geel N, by autologous minigrafting. Arch Dermatol.
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ERRNVPHGLFRVRUJ
Medical Therapies
35
Alain Taïeb
Contents
35.1 Topical Therapies 354
35.1.1 Topical Corticosteroids 354
35.1.2 Topical Calcineurin Inhibitors 356
35.1.3 Other Topical Therapies 362
35.1.4 Topical 365
35.2 Immunosuppressive Systemic Therapies 366
35.2.1 Corticosteroids 366
35.2.2 Immunosuppressants/Biologics 368
35.3 Other Systemic Therapies 372
35.3.1 Vitamin Supplementation 372
35.3.2 l-Phenylalanine 372
35.3.3 Systemic Antioxidants 372
35.3.4 Afamelanotide 374
References 374
Abstract only topical anti-inflammatory drugs for lim-

Medical therapies of vitiligo have a twofold ited vitiligo. Maintenance treatment for long
aim: stopping inflammation and regenerating term control of microinflammation remains
pigment cells. So far, the first aim has been more problematic since this issue has not been
generally fulfilled for short periods with addressed convincingly in large studies. The
courses of systemic corticosteroids or with second aim relies mostly on phototherapies,
which can also participate to the control of
microinflammation. The most effective medi-
A. Taïeb (*)
Service de Dermatologie, Hôpital St André,
cal approaches combine anti- inflammatory
Bordeaux, France and regenerative strategies. After psoriasis
e-mail: alain.taieb@chu-bordeaux.fr and atopic dermatitis, new targeted and semi

ERRNVPHGLFRVRUJ
354 A. Taïeb
targeted therapies (such as JAK inhibitors) are 35.1 Topical Therapies

currently in development for vitiligo and alo-
pecia areata. 35.1.1 Topical Corticosteroids
Wietze Van der Veen, Bas S. Wind,

and Alain Taïeb
Key Points
• Topical corticosteroids (TCS) and topi- 35.1.1.1 General Background
cal calcineurin inhibitors (TCI) are Currently, topical glucocorticosteroids (TCS) or
commonly used as a first line treatment topical calcineurin inhibitors (TCI) are the usual
in limited vitiligo. first line treatments of vitiligo. TCS have been
• TCS and TCI stabilize active progress- used widely as topical and sometimes intrale-
ing lesions and can promote repigmen- sional therapy in vitiligo since their introduction
tation in combination with UV. in dermatology in the 1950s. However, based on
• Topical calcineurin inhibitors are pre- current criteria for clinical trials, the studies are
ferred on the face. of poor quality.
• In extrafacial locations, potent TCS are The mechanism of glucocorticoid (GC) action
first choice and should be given not at the gene level can be divided into transrepres-
more than once daily with a discontinu- sion, which is responsible for a large number of
ous chronic application regimen to desirable anti-inflammatory and immunomodu-
avoid skin atrophy. lating effects; and transactivation, which is asso-
• TCS could be a useful adjunctive treat- ciated with frequently occurring side effects as
ment after skin grafts, and can increase well as with some immunosuppressive activities
UV repigmentation efficacy. [1].
• Controversy still exists about the effi- TCS have the well-known anti-inflammatory
cacy of TCI alone to induce repigmenta- effects of GC, useful to control the inflammatory
tion on strictly UV protected areas. phase of vitiligo which is well demonstrated at
• The therapeutic effect of vitamins and the margin of lesions. TCS, when used in combi-
antioxidants is at best limited and seems nation with tretinoin and hydroquinone in the
to be obtained only in combinatory treatment of the hypermelanoses, suppress the
treatment with phototherapy or topical biosynthetic and secretory functions of melano-
steroids. cytes, and thus melanin production. Used alone,
• The safety of prolonged administration they are also depigmenting agents, and the loss of
of topical and systemic antioxidants pigment or delay in pigmentation induced may
requires further evaluation. help the migration of pigment cells from the fol-
• Week end oral minipulses of corticoste- licular compartment towards the epidermis dur-
roids (OMP) is a well tolerated regimen ing repigmentation.
proposed for stabilizing progressive
vitiligo. 35.1.1.2 Studies
• Optimal duration of OMP therapy to Most patients included in published studies had
stop vitiligo progression is situated vitiligo/NSV and the duration of the intervention
between 3 and 6 months. was limited. The use of a highly potent (clobeta-
• Systemic immunosuppressants and sol) or potent (betamethasone) topical steroid can
immunomodulators including biologics repigment NSV but only in a limited proportion
and JAK inhibitors have been tested on a of cases in the small published series [2, 3].
too limited scale in vitiligo to make firm Clayton [2] found 15–25% repigmentation in
conclusions. 10/23 subjects and >75% in 2/23 (the other 11
showed no response); Kandil [3] found 90–100%
ERRNVPHGLFRVRUJ
35 Medical Therapies 355
repigmentation in 6/23 subjects and 25–90% in 3

(with 6 showing ‘beginning’ repigmentation).
Clayton [2] found that all steroid users had skin
atrophy with clobetasol, a highly potent topical
steroid (used for 8 weeks), whilst Kandil [3]
noted hypertrichosis in two subjects and acne in
three subjects, related to 4 months use of the
potent topical steroid, betamethasone.
Topical fluticasone (a potent TCS) alone or
combined with UV-A has been studied in 135
adults [4]. Fluticasone used alone for 9 months
induced a mean repigmentation of only 9% (com-
pared to UV-A alone of 8%) whereas the combi-
nation of fluticasone and UV-A induced a mean
repigmentation of 31%; no steroid atrophy was
noted in steroid users.
In a meta-analysis on non-surgical therapies in
vitiligo [5] of data extracted of three RCT on
potent [3, 6, 7] and two RCT’s on very potent
corticosteroids [2, 6], 75% repigmentation was
found in one third of the patients treated with Fig. 35.1 Patient treated with betamethasone: before
potent corticosteroids, while no significant differ- (up) and after (down) the therapy
ences were found between very potent corticoste-
roids and their placebo. In larger non-randomized on clinical observations [13]. The 308 excimer
patient series however, potent and very potent laser was also shown to be synergic with hydro-
TCS appeared to be equally effective (more than cortisone 17-butyrate [9]. Carbon Dioxide laser
75% repigmentation) in about 55% of the dermabrasion or fractional CO2 laser plus TCS
patients. As in all topical therapies, corticoste- and narrowband ultraviolet B has been proposed
roids achieve the best results on sun-exposed for refractory vitiligo [14, 15].
areas like face and neck [4, 5]. Other indications
for successful treatment are dark skin [8] and 35.1.1.3 Application Scheme
lesions of short duration [9] (Fig. 35.1). There is a general agreement to use TCS, what-
In children, the topical use of the highly potent ever their indication, on the basis of once daily
steroid clobetasol dipropionate was noted to application, to limit side effects. Optimal dura-
induce better repigmentation than PUVAsol tion for vitiligo is not established, as well as the
alone, >50% repigmentation in 15/22 (vs. 4/23 use of a continuous vs. discontinuous regimen
for PUVAsol) used for 6 months, but 6 steroid [16]. The amount used should be monitored
users developed skin atrophy [10]. Another study (number of tubes/month is the most manageable
in 20 children over an 8-week period compared like in atopic dermatitis). Potent TCS should be
topical clobetasol and tacrolimus and found 41% used first. When no results are seen after 3 months
repigmentation for clobetasol vs. 49% repigmen- of sincere trial we suggest either to try very
tation for tacrolimus [11, 12]. potent TCS if no side effects have been noted or
Westerhof et al. [4] demonstrated a three times to stop the medication.
higher repigmentation rate in vitiligo patients
treated with fluticasone propionate (FP) com- 35.1.1.4 Side Effects
bined with UVA as compared to patients treated Side effects like atrophy have been described in
with either UVA or FP alone. A synergic effect of 14% of cases with very potent TCS as compared
UVB or calcipotriol plus TCS is possible based to 2% with potent TCS [1]. Systemic absorption
ERRNVPHGLFRVRUJ
356 A. Taïeb
is a concern on thin skin. Children with head and/ and genital area [25, 26]. Furthermore, because
or neck affected areas are eight times more likely of the limited percutaneous penetration, signifi-
to have an abnormal cortisol levels compared cant systemic absorption has not been reported
with children affected in other body areas [17]. following normal use [27]. However, they are
For local therapy of vitiligo potent TCS should more expensive than topical steroids, and the
therefore be preferred over very potent TCS that black box warning issued by the FDA in 2006
have more side effects. The lower classes of local (see below) because of fear of increased skin can-
corticosteroids have not demonstrated benefit. cer risk in case of combination with UV treat-
A major improvement in the therapeutic index ments has limited their use in atopic dermatitis
of GCs has been made with the development of and vitiligo.
compounds that display only local activity. This Tacrolimus and pimecrolimus are topical
limited activity is due to the instability of the ascomycin immunomodulating macrolactams,
compounds that markedly reduces undesired sys- and act as calcineurin inhibitors, affecting the
temic effects. For example, mometasone furoate, activation/maturation of T-cells, and subse-
methylprednisolone aceponate and budesonide quently inhibit the production of various Th1
locally inhibit pro-inflammatory cytokines and and Th2 type of cytokines (IL-2, IL-3, IL-4,
chemokines very effectively with negligible sys- IL-5, IL-10, GM-CSF, TNFα and IFNγ). This
temic effects. has led to speculate that this mechanism may
Selective modulation of GR action is a prom- interfere with the autoimmune/inflammatory
ising concept for separation of anti-inflammatory mediated loss of melanocytes in vitiligo lesions.
effects from side effects, which has led to the The inhibition of TNFα production was sus-
design of qualitatively new drugs, such as selec- pected by some authors to be especially impor-
tive glucocorticoid receptor agonists (SEGRAs). tant in vitiligo, as TNFα can inhibit melanocyte
These innovative steroidal or non-steroidal mol- proliferation and melanogenesis, and that it can
ecules induce transrepression, while transactiva- induce ICAM-1 expression on melanocytes,
tion processes are less affected [1]. Limitation of through which T-lymphocyte induced destruc-
atrophic side effects by combination with reti- tion of melanocytes may occur [28]. In addition,
noids (etretinate) seems also promising for long in vitro evidence of a direct interaction between
term treatments [18]. tacrolimus and keratinocytes has been obtained,
creating a favourable milieu for melanocytic
growth and migration by inducing the release of
35.1.2 Topical Calcineurin Inhibitors stem cell factor (SCF) and enhancing matrix
metalloproteinase (MMP)-9 activity [29].
N. van Geel, B. Boone, I. Mollet and J. Lambert Furthermore, Kang and Choi [30] demonstrated
that tacrolimus could even directly stimulate
35.1.2.1 General Background melanogenesis and migration of cultured human
The topical calcineurin inhibitors (TCI)—tacroli- melanocytes.
mus ointment 0.1% and 0.03% and pimecrolimus
cream 1% —have been specifically developed for 35.1.2.2 Studies
the treatment of inflammatory skin diseases, and Since 2002 the beneficial effect of TCI has been
approved for the short term and intermittent long reported for patients with vitiligo. The first clini-
term treatment of atopic dermatitis in several cal trial regarding the efficacy of TIMs was car-
countries [19–24]. In contrast to TCS, they do not ried out in six patients with generalized vitiligo.
have the risk of local side effects, such as skin Five out of six patients achieved >50% repigmen-
atrophy, telangiectasia, and glaucoma after pro- tation of their treated areas using topical tacroli-
longed use. Therefore they are preferentially mus for 1–5 months [31].
used in areas more susceptible to these side Many reports have followed this initial trial,
effects, such as head and neck region, flexures investigating mainly tacrolimus, and since 2003
ERRNVPHGLFRVRUJ
Table 35.1 Case reports and clinical studies on tacrolimus in the treatment of vitiligo
Reference Study design N Study duration
Treatment regimen Results
[32] Open pilot study 6 4–6 months 0.1% Tacrolimus Repigmentation in 5/6 patients.
More than 25% repigmentation in
3/6 in UV protected areas
[33] Open pilot study 12 8 months 0.1% tacrolimus Good to excellent repigmentation
twice daily in 50% of patients
[34] Open pilot study 25 children 12 weeks 0.03% tacrolimus Complete repigmentation in
twice daily 57.9% of patients, best results in
face and hear bearing sites
[21] Retrospective 57 children 3 months 0.03% or 0.1% At least partial repigmentation in
review tacrolimus once or 84% of patients, best results in
twice daily head and neck region
[35] Open prospective 23 24 weeks 0.1% tacrolimus Varying levels in 89% of patients,
study twice daily best result head and neck region
[20] Randomized, 20 children 2 months 0.1% tacrolimus Tacrolimus almost as effective as
double-blind, versus 0.05% clobetasol propionate on several
comparative trial clobetasol body locations
propionate
[22] Case report 3 2–4 months 0.1% tacrolimus Complete repigmentation in
twice daily 100% of patients
[36] Open pilot study 15 1.5–9.5 months 0.1% tacrolimus At least partial repigmentation in
twice daily 87% of patients on several body
(+ sunlight locations
exposition in some
patients)
[28] Open pilot study 6 1–5 months 0.03% or 0.1% Moderate to excellent
tacrolimus twice repigmentation in 83% of patients
daily
[37] Case report 1 18 months 0.1% tacrolimus 90% repigmentation in face and
twice daily scalp region
also pimecrolimus (Tables 35.1 and 35.2). treated lesions were localized on the extremi-
Unfortunately, only few randomized trials have ties with 9 out of 20 (45%) on the back of the
been published, some of them in combination hands [43].
with UVB (Table 35.3) and only one study com-
pared pimecrolimus and tacrolimus in vitiligo 35.1.2.4 Combination Therapy
(Table 35.4). Two studies demonstrated that addition of topi-
cal tacrolimus to excimer laser therapy can
35.1.2.3 Tacrolimus and Pimecrolimus improve laser efficacy [42, 49]. In another study
Monotherapy narrow band UVB phototherapy was combined
One randomized, double blind, left-right com- with tacrolimus ointment in 110 vitiligo patients
parative trial has been conducted and showed that [40]. The authors observed more than 50% of
tacrolimus is effective similarly to clobetasol repigmentation in 42% of the lesions. The
propionate 0.05% [12]. This study has been con- repigmentation rate appeared strictly related to
firmed in children [34]. the site: an improvement of >50% was obtained
A double-blind vehicle controlled study, for lesions located on the face (83%), followed
evaluating the efficacy and safety of pimecroli- by the limbs (68%) and trunk (53.5%) com-
mus cream 1% predominantly on extremities of pared to lesions on both extremities and genital
20 vitiligo patients did not show efficacy on the areas, where responses were more disappoint-
actively treated site, possibly because all the ing. In a randomized, placebo-controlled, dou-
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358 A. Taïeb
Table 35.2 Case reports and clinical studies on pimecrolimus in the treatment of vitiligo
Study
Reference Study design N duration
Treatment regimen Results
[38] Case report 1 5 months
1% pimecrolimus Significant improvement with
versus calcipotriol both, but mainly with
cream pimecrolimus of facial lesions
[39] Case report 2 children 3–4 months 1% pimecrolimus Almost complete repigmentation
twice daily of eyelid and genital lesions
[12] Retrospective study 8 11 months 1% pimecrolimus Mean percentage repigmentation
twice daily in face 72.5%
[40] Open prospective 26 6 1% pimecrolimus Repigmentation in 57.7% of
study twice daily lesions. Mean repigmentation of
62% head and neck region
[41] Open prospective 30 12 weeks 1% pimecrolimus Repigmentation in 57.7% of
study twice daily lesions. Best results face and
truck (mean repigmentation 31%
and 36% respectively)
[42] Open prospective 19 6 months 1% pimecrolimus >25% repigmentation in 68% of
study once daily patients
[43] Randomized, 20 6 months 1% pimecrolimus No significant difference and
placebo-controlled, twice daily versus effect on extremities/hands
double-blind trial Placebo
[11] Comparartive 10 2 months 1% pimecrolimus Comparable rate of
prospective, non versus 0.05% repigmentation in non facial
blind trial clobetasol propionate areas
[29] Case report 1 5 months 1% pimecrolimus Percentage of repigmentation:
>90%
Table 35.3 Topical calcineurin inhibitors in combination with UVB

Study
Reference Study design N duration Treatment regimen Results
[44] Open prospective 110 16 weeks 0.03–0.1% tacrolimus once Repigmentation of >50% in 42%
study daily + UVB of lesions, best results in face
(83%)
[45] Randomized, 8 12 weeks 0.1% tacrolimus + UVB No statistically significant
placebo-controlled, versus placebo + UVB difference in non facial areas
double-blind trial
[46] Comparative 9 12 weeks 0.1% tacrolimus + UVB No repigmentation with
prospective, non versus 0.1% tacrolimus tacrolimus monotherapy on UV
blind, pilot study protected areas
[25] Comparative, 14 12 weeks 0.1% tacrolimus + excimer Combination therapy is superior to
prospective, laser versus excimer laser excimer laser monotherapy in
randomized, UV-resistant areas
intra-individual
study
[30] Prospective, 8 10 weeks 0.1% tacrolimus + excimer Significantly greater degree of
double-blind, laser versus repigmentation with combination
placebo-controlled placebo + excimer laser therapy on elbows and knees
study
[47] Case report 1 4 months 0.1% tacrolimus + UV-B Repigmentation 95% on face
Majid Left-right 8 6 months 0.1% tacrolimus + UVB vs. Earlier and better repigmentation
controlled study 0.1% tacrolimus with combination therapy
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Table 35.4 Comparative studies between tacrolimus and pimecrolimus

Study Study
Reference design N duration Treatment regimen Results
[48] Case 1 18 months 1% pimecrolimus Tacrolimus site 88% and pimecrolimus
report versus 0.1% tacrolimus under 73% site repigmentation
overnight occlusion
ble-blind study of Mehrabi and Pandya [50], potential therapeutic alternative with a good
narrow-band-UVB therapy with and without benefit:risk ratio.
tacrolimus ointment was compared in eight
patients. They did not demonstrate an additional 35.1.2.6 Application Scheme
effect of tacrolimus. However, it has to be men- Data about the most effective treatment scheme
tioned that this study was underpowered (n = 8) using TCI in vitiligo are still missing. In the
to detect significant differences. Besides, the available vitiligo studies the application fre-
evaluated lesions were all located on non-facial quency is varying between once and twice daily.
areas. Twice daily gives better results [37]. Duration of
treatment mentioned in the studies ranged from
35.1.2.5 Interpretation 10 weeks to 18 months. Good results are usual
TCI seem to affect repigmentation differently for facial vitiligo, in vitiligo/NSV and SV
according to the anatomical location of the
lesions. Most studies show beneficial results
mainly in the head and neck region. This may be
explained by the greatest density of hair follicles
in these areas and thus of melanocyte reservoir.
Besides, the influence of a reduced epidermal
thickness might be of importance as this facili-
tates penetration of large molecules into the
skin. Moreover, the head and neck region is a
sun-exposed areas and UV therapy is a well-
known treatment option of vitiligo. Probably,
UV light exposure during TCI treatment seems
to play an important or even synergistic role
[41]. Sardana et al. [31] reported that there is
sufficient evidence that tacrolimus monotherapy
is useful to produce repigmentation, as illus-
trated by their two cases. However, it is still not
known whether monotherapy with tacrolimus
ointment requires longer periods to induce
repigmentation as compared to combined treat-
ment with UVB. A correlation between the
repigmentation rate and patients’ age or duration
of the disease has not been consistently demon-
strated. According to the literature, the results in
children using TCI seem to be comparable to
those obtained in adult patients [12, 22, 26, 51].
Generally, therapeutic options in childhood vit- Fig. 35.2 Before (up) and after (down) tacrolimus
iligo patients are more limited, and TCI are a tratment
ERRNVPHGLFRVRUJ
360 A. Taïeb
Fig. 35.3 Patients treated with tacrolimus. Segmental vitiligo patient treated with topical tacrolimus for 6 months
(courtesy Dr A. Salhi)
ERRNVPHGLFRVRUJ
Fig. 35.4 Before and after tacrolimus under occlusion (hydrocolloid dressing) for 7 weeks
(Figs. 35.2 and 35.3). Information about the because of good efficacy and limited side effects
minimal or ideal treatment period in vitiligo is as compared with potent TCS, and use potent or
not available. However, intermittent use after very potent TCS intermittently on other body
repigmentation, on the model of proactive treat- sites, optimally as combination therapies with
ment of atopic dermatitis, has proven to be help- natural or UV light.
ful to limit relapse in a controlled study [47].
Repeated application may limit Koebner’s phe- 35.1.2.7 Side Effects
nomenon [52]. Furthermore, long-term results The most common reported side effects for TCI
about the course of disease after treatment inter- within the first days of treatment are local appli-
ruption are missing or incomplete. Interestingly, cation reactions such as burning sensation, pruri-
the a dditional overnight occlusion of 0.1% tacro- tus, and erythema. However, the incidence of this
limus with polyurethane and hydrocolloid foils side effect is lower in vitiligo patients than in the
can lead to a good repigmentation on the extremi- published atopic dermatitis studies. This is prob-
ties after a previous response failure when used ably due to the presence of an intact epidermis in
without occlusion [53]. It was mentioned that the vitiligo patients versus the altered skin barrier of
hydrocolloid dressings might be more suitable the atopic dermatitis patient. Treatment with TCI
for improving trans-cutaneous penetration com- has not been associated with a statistically sig-
pared to polyurethane dressings, as they produce nificant increase of skin infections or systemic
a much stronger water-binding capacity in the infections in patients with atopic dermatitis
stratum corneum (Fig. 35.4). In general most vit- [45, 54, 55].
iligo specialists prefer for long term control of A tacrolimus induced hyperpigmentation in a
the disease to use TCI on the face and neck vitiligo lesion of the infraorbital area has been
ERRNVPHGLFRVRUJ
362 A. Taïeb
related to sun exposure. The hyperpigmentation up-regulation of c-Kit. Morphological modifi-

was temporary, with reappearance of depigmen- cations are detected in number and length of the
tation within 1 month of discontinuing topical dendrites. For keratinocytes, vitamin D induces
tacrolimus application. Temporary tacrolimus differentiation, decreased proliferation and
induced perilabial lentiginose has also been release of the pro-inflammatory cytokines IL-8
observed [35]. Less frequently reported side and IL-6, as well as increased production of
effects in the face are acne, hypertrichosis and IL-10. In vitiligo skin, both melanocytes and
rosaceiform eruptions [54]. keratinocytes have been shown to have an alter-
Since January 2006 a black box warning for ation of Ca2+ uptake and a decreased intracellu-
tacrolimus and pimecrolimus was announced by lar concentration of Ca2+, which could
the FDA, because of concerns of potential safety compromise melanogenesis by increasing the
risks including skin cancer and lymphoma. level of reduced thioredoxin, which inhibits
However there is up till now no evidence of a tyrosinase [56].
causal relationship between the sporadically Vitamin D3 analogues have been reported to
reported lymphomas and the use of a topical cal- interfere with intracellular Ca2+ fluxes, cause G1
cineurin inhibitor. It should be noted that most block, induce cKit upregulation, and inhibit
studies with TIM’s are conducted in patients with TNFα, IL8, and RANTES production. The func-
atopic dermatitis, in which the barrier function of tional consequences of these activities affect cell
the skin is disturbed. In vitiligo, the barrier func- survival, proliferation and differentiation pro-
tion of the skin is normal which results in a lesser cesses, including melanogenesis. Moreover, vita-
degree of penetration of TIMs. So far the use of min D analogues are believed to target the
TIMs has not been reported to be associated with immune response interfering with activated T
significant systemic immunosuppression or cells and inhibit the expression of several
increased risk for skin cancer and other malig- cytokine genes, such as those encoding TNFα
nancies in clinical vitiligo trials [45]. and IFNγ.
Vitamin D3 analogues have been mainly pro-
posed as a supporting therapy during PUVA,
35.1.3 Other Topical Therapies NB-UVB, or excimer (308 nm) phototherapy
[61–63].
35.1.3.1 Vitamin D Analogues Initially, open studies reported repigmentation
Mauro Picardo of vitiligo lesions using calcipotriol, a synthetic
The idea that vitamin D analogues may be con- analogue of calcitriol [1,25(OH)2D3] with
sidered an option for the treatment of the vitil- PUVAsol or solar exposure. Subsequent studies
igo arose from the casual observation of the reported contradictory results. The combination
occurrence of cutaneous perilesional hyperpig- of calcipotriol and PUVA was reported as more
mentation after local application on psoriatic effective than PUVA alone (70% versus 52% of
plaques in combination with phototherapy. the patients obtained mild-moderate repigmenta-
Starting from this clinical evidence, several tion), in particular in initiating repigmentation
in vitro and in vivo data supported the therapeu- [64–66], also suggesting the up-regulation of
tic role of the calcipotriol and tacalcitol for vit- c-kit expression as possible melanocyte-mediated
iligo [56–60]. Skin cells, including keratinocytes, mechanism of action. However, a right/left com-
melanocytes, and dermal fibroblasts possess parative open study did not shown that the addi-
vitamin D receptors (VDR), the activation of tion of calcipotriol increased the response rate to
which induces the expression of several genes PUVA [67].
associated with proliferation, differentiation Some studies have suggested the enhancement
and immune responses. Exposure of human of NB-UVB phototherapy when calcipotriol or
melanocytes to vitamin D promotes tyrosinase tacalcitol was locally applied (Fig. 35.5) before
activity and melanogenesis, together with the or after UVB exposure [68–70], providing earlier
ERRNVPHGLFRVRUJ
Fig. 35.5 Two patients, with different localization of the lesions, treated with tacalcitol plus NB-UVB
pigmentation with lower total UVB dosage, thus performed on 80 patients reported that the
reducing the cost and the duration of the treat- combination of tacalcitol with heliotherapy has
ments. However two single blinded left-right no additional advantage when compared to
clinical studies and one RCT did not show any heliotherapy alone [75].
improvement in treatment outcome when adding However, the published trials are mainly based
topical calcipotriol [71]. on small numbers of enrolled patients and differ-
Another synthetic analogue of vitamin D, top- ent phototherapy protocols were implemented,
ical tacalcitol [1-24(OH)2D3], in combination characterized by different temporal sequences
with bi-weekly NB-UVB phototherapy, improved and modality of vitamin D3 analogue applica-
the extent of pigmentation and increased the rate tion, and variable/different period of treatment-
of response as compared to phototherapy alone washout before starting the tested therapy, so that
in a left-right RCT. Another RCT using tacalcitol a comparison of the results is not easy. The most
in combination with monochromatic excimer recent studies of vitamin D analogues-UVB com-
light (308 nm MEL) confirmed that tacalcitol bined treatment conducted in India gave also
improved the efficacy of phototherapy, achieving opposite results [76, 77]. In any case, most of the
earlier pigmentation with lower dosage [72]. studies reported best results when lesions were
However the true effectiveness of the combina- localized on the face or on the trunk.
tory treatment, it is still considered controversial Small studies and isolated case-reports
because other studies failed to find improvement described initially the effectiveness of calcipot-
when various sources of UVB phototherapies riol or tacalcitol in children [78, 79]. Calcipotriol
were associated with vitamin D3 analogues has been evaluated in combination with
[73, 74]. Moreover, a vehicle-controlled RCT corticosteroids (betamethasone dipropionate) in
ERRNVPHGLFRVRUJ
364 A. Taïeb
Table 35.5 Summary of the studies

Vitamin D
analogue UV Study Administration Duration Effectiveness Reference
Calcipotriol No Children (18) 2/day 4–6 months Yes [79]
Tacalcitol Sun Case-report 1/day 1 month Yes [78]
children
Calcipotriol PUVA Adults (26) 2/day 3–9 months Yes [83]
Tacalcitol NB-UVB Randomized 2/week UV ± 1/day cream 6 months Yes [82]
assessor-blind
adults (32)
Tacalcitol 308 nm Single-blind 1/week MEL ± 2/day cream 12 sessions Yes [62]
MEL adults (38)
Tacalcitol Sun Double-blind 1/day 16 weeks No [63]
randomized
placebo controlled
(80 adults)
Calcipotriol NB-UVB Comparative 3/week UV ± 2/day cream 30 sessions No [84]
prospective, 40
adults
Calcipotriol No Left-right open 1/day 3–6 months No [85]
(24 adults)
randomized trials involving also some pediatric lipid peroxidation are the major metabolic
subjects. Good results (80% of success) in terms alterations reported in the literature [86–88]. The
of onset, degree, and stability of repigmentation occurrence of a redox unbalance, both at epider-
were obtained with the combinatory therapy mal and systemic level, has been described in vit-
[80, 81]. A possible benefit of the association was iligo patients (Chap. 31). Usually, the redox
to limit atrophogenic effect of TCS. The most unbalance is caused by a hyperproduction of
frequent side effects in all studies was mild to ROS not associated with the increased activity of
moderate erythema or itching, a typical symptom the adequate detoxifying apparatus. In vitro and
associated with the application of vitamin D ana- clinical data suggests that several different cellu-
logues [82] (Table 35.5). lar metabolisms may lead to the unwanted pro-
duction of radical species, possibly associated
35.1.3.2 Antioxidants with the lipoperoxidative processes. The high
Mauro Picardo and Maria Lucia Dell’Anna lipid content of the cellular and mitochondrial
membranes seems to play a relevant role in the
General Background for Topical propagation of the peroxidative events, mainly
and Systemic Antioxidant Therapy the mitochondrial ones due to their strictly physi-
in Vitiligo cal association with the main cellular source of
The therapeutic approach with antioxidants origi- free radicals. Moreover, some studies indicate
nated from the description of the occurrence of that the reactive species, released as side-products
the oxidative stress and possible vitamin defi- of specific epidermal metabolisms, can diffuse
ciency in vitiligo patients, involving melanocytes systemically, and can target any cell type [89].
and other epidermal as well as non-epidermal Antioxidants form an integrated network inside
cells [86, 87]. Increased production of H2O2, the cells, and the activity of each compound
biopterins and catecholamines, defective expres- depends on the presence and function of the rest
sion and/or activity of the antioxidant enzymes of the antioxidant molecules. The participating
catalase and glutathione peroxidase in addition to enzymes and non enzymatic molecules act as
ERRNVPHGLFRVRUJ
4
placebo before
3,5 lipoic acid
3
2,5
2
1,5
1
0,5
0
no repigm medium good excellent
Cat lipoacid combi after

150
140 Cat placebo
130 H2O2 lipoacid combi
120
110 H2O2 placebo
100
% of T0
90
80
70
60
50
40
30
20
T2 T6
Fig. 35.6 Modification of oxidative stress in patients treated with NB-UVB alone (placebo) or in combination with
lipoacid. Clinical response of a patients treated with a pool of antioxidants
scavengers of the free radicals and are able to 35.1.4 Topical

reduce the oxidised compounds inside the
network, allowing the restoration of a correct 35.1.4.1 Pseudocatalase
redox balance. The system glutathione/glutathi- This antioxidant treatment containing pseudo-
one peroxidase, catalase, superoxide dismutases, catalase and calcium chloride was initially sug-
α-lipoic acid, vitamin E, and vitamin C are the gested by Schallreuter and colleagues [88].
main components of this antioxidant network. Pseudocatalase is a low molecular weight coordi-
α-Lipoic acid is a lipophilic and hydrophilic com- nation complex [bis-manganese III-EDTA-
pound acting as a fatty acids peroxyl and hydroxyl (HCO3-)2] able to produce O2 and H2O from H2O2
radical scavenger, lipoxygenase inhibitor and glu- at a rate higher than catalase. Solar or UVB expo-
tathione synthesis promoter. Moreover, α-lipoic sure is required for the chemical activation of
acid is involved in recycling vitamins C and pseudocatalase [90] but not for the therapeutic
E. Vitamin C is a hydrophilic antioxidant; whereas effects, since the amount of UVB administered
vitamin E is a lipophilic free-radical scavenger (0.15–0.3 J/cm2 per session, two to three times a
and inhibits lipid peroxidation helping to maintain week) is lower than that used during conventional
membrane integrity. According to the physiologi- phototherapic protocols in association with low
cal redox-based system, the most effective thera- UVB doses [88]. A group of 33 vitiligo patients
peutic approach should provide a balanced pool of were treated for 15 months and repigmentation
antioxidant molecules, with the aim of restoring was found excellent in 90% of the subjects,
the correct intracellular network (Fig. 35.6). mainly on the face and dorsum. The clinical
ERRNVPHGLFRVRUJ
366 A. Taïeb
improvement has been related to the reduction of compared to pseudocatalase cream [98]. Another
epidermal H2O2 level. Subsequently, the negative small trial has been reported [99].
combination pseudocatalase plus Dead Sea cli-
matotherapy—dead sea water contains metals
able to activate pseudocatalase—has been shown 35.2 Immunosuppressive
to shorten the time required for the onset of repig- Systemic Therapies
mentation (10 days versus some weeks) in nearly
all the patients treated [91], as compared to UVB- Infammation and dysimmunity definitely play a
activated pseudocatalase. The recovery in the role in vitiligo, which has suggested to try sys-
epidermal enzymatic activities of catalase, tetra- temically various immunomodulators including
hydrobiopterin dehydratase, acetylcholinester- corticosteroids, immunosuppressants, and more
ase, and dihydropteridine reductase (Chap. 2.3.4) recently biologics and targeted small molecules
which can be inactivated by the oxidation of con- such as JAK inhibitors.
stitutive aminoacids such as methionine, cyste-
ine, tryptophan [11, 92], has been reported after
the treatment. The clinical validity of the pseudo- 35.2.1 Corticosteroids
catalase regimen has been debated, because large
scale double-blinded versus placebo studies have Davinder Parsad and Dipankar De
not been published and since data have not been
confirmed by others authors [90, 93, 94]. The 35.2.1.1 General Background
effectiveness of pseudocatalase, however, seems Though topical steroids are used extensively in
to be dependent on the formulation of the cream, the management of vitiligo, studies on systemic
and the PC-KUS® brand is reported to be suc- steroids have been sparingly reported in the lit-
cessful whereas different pseudocatalase prepa- erature. The cause of this discrepancy is not
rations have been used by other groups. PC-KUS® known. Systemic steroids can arrest the activity
has been also successfully tested in pediatric of the disease, if they are used in sufficient doses
population and a retrospective study carried out for a sufficient period of time [100, 101]. They
on 71 children, 61 with diffuse and 10 with seg- are in general not effective in repigmenting stable
mental disease. The progression of the disease vitiligo. Moreover side effects associated with
was stopped, without side effects and more than long-term use of daily systemic corticosteroids
75% of repigmentation was observed in 93% of may act as deterrent against their common use.
the patients, associated with the H2O2 removal by Pulse therapy refers to the administration of
epidermis. Only feet and hands lesions appeared large (supra-pharmacologic) doses of drugs in an
to be poorly responsive [95]. The effectiveness of intermittent manner to enhance the therapeutic
the treatment was found independent of disease effect and reduce the side effects of a particular
duration and skin phototype. drug [102]. The credit of first use of corticosteroids
Vitix®. A formulation containing an extract of in pulse form goes to Kountz and Cohn [103] who
Cucumis melo vehiculated by beads of palm oil used it to prevent renal graft rejection. Subsequently,
(Vitix®) has been proposed for the treatment of pulse corticosteroids have been used in various
vitiligo. Indeed, the antioxidant properties of the dermatological as well as non-dermatological indi-
extract evaluated in an in vitro model, was attrib- cations. Oral minipulse (OMP), i.e. intermittent
uted mainly to superoxide dismutase, glutathione administration of betamethasone/dexamethasone
reductase and catalase activities [96]. The applica- was pioneered in India by Pasricha et al. [101].
tion of the gel, twice daily, with a simultaneous They first used OMP in vitiligo. Subsequently,
sun exposure for 30 min or NB-UVB photothera- OMP has been used successfully in various other
pyhas been reported to induce repigmentation dermatoses like extensive alopecia areata [104],
after 1–3 months [97]. Yet, others failed to confirm cicatricial alopecia, extensive/bullous lichen pla-
both the ability of the gel to remove H2O2 from the nus [105], trachyonychia [106], infantile periocu-
epidermis and the clinical effectiveness, when lar haemangioma [107] etc. As the indications of
ERRNVPHGLFRVRUJ
use of OMP suggest, it is understandable that in majority of patients within 15 weeks of starting
dermatoses where steroid therapy is effective, treatment. No side effects were reported [109]. In
OMP may be used, maintaining efficacy and cut- the study by Radakovic-Fijan et al. [110], 29
ting down on side effects. patients, 25 with progressive disease and 4 with
stable disease were included. The daily dose of
35.2.1.2 OMP Studies dexamethasone was significantly increased to
In the first reported study on OMP in vitiligo by 10 mg/day and the treatment was continued for a
Pasricha et al. [101], betamethasone or dexa- maximum period of 24 weeks. In addition,
methasone, both corticosteroids with a long half- plasma cortisol and corticotrophin levels were
life (36–54 h), were given as a single oral dose of measured to detect hypothalamo-pituitary-
5 mg on 2 consecutive days per week. This dose adrenal axis suppression before and up to 6 days
of steroids was decided upon arbitrarily. after the dexamethasone pulse in the first and
Progression of the disease was arrested in 91% of fourth weeks of treatment in 14 patients. Disease
the patients. A degree of repigmentation was activity was arrested in 88% of patients with pro-
observed in a proportion of patients and the side gressive disease after an average treatment period
effects were either not significant or altogether of 18.2 weeks. Marked repigmentation was
absent. observed in 6.9% and moderate or slight repig-
In the subsequent trial of 40 patients—36 with mentation in 10.3%, while 72.4% had no response
progressive and 4 with static disease—the same in repigmentation. A tendency towards better
OMP regimen was used [108]. In children, the treatment results was observed with an increas-
dose was proportionately reduced. In adults who ing number of pulses. Side effects were observed
did not respond to the standard dose of cortico- in 69% patients, which included weight gain,
steroids, the dose was increased to 7.5 mg/day insomnia, agitation, acne, menstrual distur-
then reduced to 5 mg/day when disease progres- bances, and hypertrichosis. Plasma cortisol and
sion was arrested. Within 1–3 months of starting corticotrophin levels, though markedly decreased
treatment, 89% patients with progressive disease after one pulse, returned to normal before starting
stabilized, while within 2–4 months, repigmenta- the next pulse. The authors observed that ethnic
tion was observed in 80% of total patient cohort. background may have an impact on therapeutic
The area of repigmentation continued to progress response [110].
as treatment continued, though none of the
patient achieved complete repigmentation. 35.2.1.3 Interpretation
Seventeen of 40 (42%) had at least one side and Recommendations
effect, though they were not significant. The side OMP with either betamethasone or dexametha-
effects profile included weight gain, dysgeusia, sone can arrest progression of spreading disease.
headache, transient mild weakness, acne, mild However, it is not usually suitable alone for
puffiness of the face alone, perioral dermatitis, repigmentation of vitiligo lesions. In addition, in
herpes zoster, glaucoma and amenorrhoea. The patients with fast spreading vitiligo, disease pro-
authors presumed that the repigmentation gression is not stopped immediately. There are no
observed was spontaneous and thus varied from RCT confirming that either speed or magnitude
patient to patient and lesion to lesion [108]. of response to phototherapy and photochemo-
Subsequently Kanwar et al. [109] assessed the therapy in patients with generalized fast spread-
efficacy of OMP in vitiligo. They used dexameth- ing vitiligo might be potentiated by concomitant
asone in a dose of 5 mg/day on 2 consecutive administration of OMP. As noted before, the dos-
days per week and the dose was halved in chil- age of dexamethasone used in OMP has been
dren 16 years of age or younger. Of 37 patients arbitrarily chosen. In the majority of the studies,
included with actively spreading disease, 32 were a dose of 5 mg every day for 2 consecutive days
evaluable at the end of the study. 43.8% had mild per week has been used. For those who not
to moderate repigmentation without appearance respond, 7.5 mg/day may be used, then reduced
of new lesions. The pigmentation appeared in to 5 mg/day if disease progression is arrested.
ERRNVPHGLFRVRUJ
368 A. Taïeb
The lower OMP regimen 2.5 mg for 2 consecu- supported by the identification of circulating
tive days is however sufficient in the majority of melanocyte-specific antibodies in the serum of
cases [111]. The drug can be preferably given on affected patients. A good response was seen at
weekends to increase the compliance as short the dosage of 50 mg twice a day in about the 27%
therm side effects may be troublesome in some. of the cases, while no response was observed in
If the 5 mg/day dose is used, the drug can be 33% of the treated patients. Haematological tox-
gradually tapered off over 6 months, after desired icity, hair loss and nausea have been reported as
response is achieved. However, when a lower common side effects during the treatment, limit-
dose is used (2.5 mg/day), the treatment can be ing its use [115, 116].
stopped abruptly without any noticeable manifes- Low dose azathioprine (at the maximal dos-
tation of hypothalamo-pituitary-adrenal axis sup- age of 50 mg/day) in association with PUVA has
pression. Minocycline has been tested as an been also proposed. Azathioprine is able to
alternative with less side effects to OMP in pro- inhibit the cellular immune response and is
gressive vitiligo, and shown to have similar stabi- widely used in inflammatory bowel disorders
lizing effects [112]. Concerning stable vitiligo, and in rheumatology, and in dermatology for
NB-U.V.B plus OMP and Nb-U.V.B alone were autoimmune dermatoses, including pemphigus,
found as expected to be clinically superior over at the dosage of 50–100 mg/day. A study per-
OMP alone [113]. formed on 60 patients randomized to receive
either azathioprine (0.6–0.75 mg/kg/day) in
association with PUVA or PUVA therapy alone,
35.2.2 Immunosuppressants/Biologics has demonstrated a potential synergic effect of
the two approaches, but true positive results
Markus Böhm have been demonstrated only in a minority of
the subjects, with the limitation of lack of vali-
35.2.2.1 Rationale for the Use dated, standardized measures for vitiligo assess-
of Systemic ment [117].
Immunomodulators Starting from the pathogenetic concept of a
As described in Chap. 2.3.5 there is a large systemic over-activation of cellular immunity in
body of data indicating that vitiligo is an vitiligo, systemic cyclosporine has been anec-
immune-mediated inflammatory disease. This dotally used in patients with diffuse disease, at
concept is further corroborated by the beneficial the dosage of 5 mg/kg/daily with difficult to
effects from anti-inflammatory and/or immuno- interpret responses. Vogt-Koyanagi-Harada
modulatory treatment, e.g. therapy with corti- patients have benefited cyclosporine for eye
costeroids or topical calcineurin inhibitors. In involvement but vitiligoid changes have not been
this view, different systemic imunomodulators assessed precisely. Considering the kidney and
have been proposed as an alternative to cortico- liver toxicity of cyclosporine, experience remains
steroids for the treatment of progressive limited [118].
vitiligo. Methotrexate is now widely used in chronic
inflammatory skin diseases but has not been
35.2.2.2 Systemic much investigated in vitiligo. A study in unstable
Immunosuppressants vitiligo has shown comparable stabilizing effects
Low dose cyclophosphamide has been proposed of 10 mg methothrexate/week to low dose OMP
in the early 1980s. Cyclophosphamide inhibits over 6 months [119].
the production of antibodies by B lymphocytes
[114]; the rationale for its use in vitiligo derived 35.2.2.3 Biologics
from the experience in other autoimmune The clinical experience in vitiligo of biologics
skin diseases, such as pemphigus, and was introduced in dermatology for the treatment of
ERRNVPHGLFRVRUJ
psoriasis is limited. They include anti-TNF-α injections of the IFN-γ antibody were also per-
targeted therapies (etanercept, infliximab, formed. A gradual diminishment of the border
adalimumab) and efalizumab, the latter being an between the depigmented area and normal skin
antibody that binds to the CD11a subunit of was seen [122, 127]. In addition, the authors
LFA-1. reported on the clearance of vitiligo in another
patient with autoimmune polyglandular syn-
Anti-IFN-γ Strategy drome (APS)-4, i.e. vitiligo and alopecia areata
The pioneering concepts and preclinical observa- [129]. It is unclear for how long systemic anti-
tions of Skurkovich et al. deserve to be summa- IFN-γ therapy was given in the latter individual.
rized [120, 121]. These scientists were amongst Moreover, the above preclinical study lacks
the first who proposed not only to remove certain detailed description of patient’s characteristics as
types of IFNs but also TNF-α to treat various well as follow-up data.
autoimmune diseases. Based on their longstand- The group of John Harris using a transgenic
ing research on the pathogenetic role of proin- mouse model reported that the IFN-γ-induced
flammatory cytokines in immune-mediated chemokine CXCL10 is expressed in lesional skin
inflammatory diseases, Skurkovich et al. initially from vitiligo patients, and that it is critical for the
proposed that IFN-γ should be removed in auto- progression and maintenance of depigmentation
immune diseases such as rheumatoid arthritis, in this model [130]. Following Skurkovich, they
multiple sclerosis, type I diabetes, psoriasis vul- hypothesized that targeting IFN-γ signaling
garis, alopecia areata or vitiligo [122]. In this might be an effective new treatment strategy.
context it is worth mentioning that IFN-γ mRNA Activation of signal transducer and activator of
levels have been shown increased in lesional and transcription 1 (STAT1) is required for IFN-γ sig-
in adjacent uninvolved skin of patients with vit- naling and other studies revealed that simvas-
iligo [28]. It is also well known that systemic tatin, an FDA-approved cholesterol-lowering
administration of IFN-α can induce or aggravate medication, inhibited STAT1 activation in vitro.
vitiligo [123–125] although improvement of pre- They found that simvastatin both prevented and
existing vitiligo under pegylated IFN-α-2A was reversed depigmentation in their mouse model of
reported in a patient with hepatitis C [126]. In a vitiligo, and reduced the number of infiltrating
small preclinical case series, Skurkovich et al. autoreactive CD8(+) T cells in the skin [131].
injected polyclonal IFN-γ antibodies (both IgG Unfortunately a subsequent trial of simvastatin in
and/or F(ab′)2 antibody fragments) into patients human vitiligo failed [132]. The recent develop-
with various Th-1-mediated autoimmune dis- ment of JAK inhibitors (below) is an alternative
eases [122]. The protein concentration of these to block the JAK-STAT signalling pathway in
antibodies was about 33 mg/ml with an IFN-γ vitiligo.
neutralizing capacity of more 66 μg/ml [127].
Four patients with vitiligo (12–14 years old) AntiTNF-α Approach
received intradermal injections of F(ab′)2 frag- Since TNF-α protein expression and immuno-
ments (titer: 24 × 103 IU/ml) generated from goat reactivity is elevated in lesional skin of patients
antibodies to human IFN-γ [128]. Aliquots of with vitiligo [133, 134] it is not surprising that
0.1 ml of the antibody were given perilesionally anti-TNF-α drugs had been investigated in this
for 10 days. All treated patients experienced sus- indication. However, anti-TNF-α therapy, in
tained erythema after 3 days of therapy followed accordance with its potential to trigger other
by development of small, slightly infiltrated autoimmune phenomena (such as alopecia
pinkish papules in the depigmented areas. On day areata and lupus erythematosus-like syn-
10, the authors observed loss of the well-defined dromes) can induce de novo vitiligo.
borders between the normal and depigmented Accordingly, a 61-year-old Caucasian suffering
skin in all treated patients [128]. Intramuscular from rheumatoid arthritis was reported to
ERRNVPHGLFRVRUJ
370 A. Taïeb
develop vitiligo lesions on the dorsa of the and generalized vitiligo for 11 years received
hands 6 months after intravenous therapy with 350 mg infliximab intravenously at weeks 0, 2
infliximab at 3 mg/kg [129]. Infliximab was not and 6, and then every other week for 10 months.
discontinued and the patient’s vitiligo was sub- With this treatment the patient’s disease activ-
sequently treated with Polypodium leucotomos ity score and functional indices for arthritis
extracts and topical pseudocatalase to regain improved as well as his vitiligo: 6 months after
50% of his pigmentation. In another case infliximab spreading of two vitiligo spots at
report, a 66-year-old white man receiving both axilla and pretibial areas was halted. In
adalimumab for the treatment of his psoriasis is addition, several other vitiligo spots (located
described [135]. Within 4 months of 40 mg on the trunk, finger joints, face and pretibial
adalimumab administered subcutaneously areas) revealed partial repigmentation or even
every other week his psoriasis had cleared. disappeared. In accordance with the established
However, depigmented skin was noticed in role of TNF-α as an inhibitor of melanocyte
those areas previously affected by psoriasis but proliferation [137] and melanogenesis [138]
not in unaffected areas. A skin biopsy specimen these data would suggest some potential of
from the depigmented skin confirmed the pres- antiTNF-α agents in the treatment of vitiligo,
ence of vitiligo. Concomitant psoriasis and vit- as shown in other reports suggesting a possible
iligo is already described, with the common stabilizing effect [139, 140].
occurrence of koebnerization (Chap. 1.5.7).
Regarding the therapeutic efficacy of TNF-α Cell Skin Recruitment Blocking Strategy
T
antibodies in patients with pre-existing vitiligo Two reports have described a beneficial effect
the data are limited and controversial. of efalizumab in vitiligo as a T cell targeted
Rigopoulos et al. [136] assessed the therapeutic recombinant antibody binding to the CD11a
potential of etanercept in a small open-label subunit of LFA-1 [141, 142]. As a consequence
pilot study consisting of four male patients of such an immunomodulatory approach by an
with vitiligo vulgaris (mean age: 29.3; mean anti-LFA-1 antibody, the interaction between
duration of vitiligo: 7.5 months). All patients LFA-1 and intercellular adhesion molecule
had progressive disease with development of (ICAM)-1 expressed by numerous activated
new lesions within the previous 3 months. Most resident skin cells is blocked. Expression of
of the patients had an involvement of extremi- ICAM-1 had previously been detected in per-
ties including hands and feet. All of the patients ilesional melanocytes around active vitiligo
had not received any treatment for vitiligo for patches [143]. Regarding expression of
the last 2 months. The treatment protocol con- LFA-1 in vitiligo patients, it was shown that
sisted of weekly injections of etanercept (50 mg LFA-1 immunoreactivity in leukocytes is
subcutaneously) for 12 weeks followed by higher in minigrafts of non-responders than in
25 mg of etanercept weekly for another responders [144].
4 weeks. Treatment success was evaluated pho- In the first report, a 52-year-old male
tographically and photometrically. Although Surinamese Hindustani man with plaque psoria-
the overall tolerability was good none of the sis and universal vitiligo received efalizumab
patients had any repigmentation. However, no [142]. He had been suffering from vitiligo since
aggravation of their vitiligo was noticed. The more than 10 years. His previous anti-psoriatic
authors suggested that monotherapy with therapies had included ultraviolet (UV) light and
TNF-α antibodies should not be considered as a psoralen, methotrexate, cyclosporine and
treatment option for vitiligo. In another case fumaric acid. Six weeks after starting subcutane-
report, vitiligo improvement was observed in a ous efalizumab at 1 mg/kg once per week the
patient with ankylosing spondylitis under inf- patient developed spotty repigmentation in viti-
liximab treatment [135]. A 24-year-old male liginous areas of his face. Facial repigmentation
with ankylosing spondylitis since the age of 18 continued in a perifollicular pattern under efali-
ERRNVPHGLFRVRUJ
zumab treatment for 6 further weeks but treat- JAK2, whereas the major TH17 cytokine IL-23
ment had to be terminated due to the deterioration activates JAK2 and Tyk2, and the major TH2
of his psoriasis. Subsequently, facial vitiligo cytokines IL4 and IL13 activate JAK1 and 3.
became worse. In the second report, Fernandez- Selective JAK-inhibitors have anti-
Obregon [141] reported on a 43-year-old male inflammatory properties and have been approved
Hispanic with a history for vitiligo vulgaris for in several countries for the treatment of rheuma-
more than 10-years and plaque psoriasis for toid arthritis (tofacitinib, baricitinib) and myelo-
5 years. Before starting efalizumab he had fibrosis or polycythemia vera (ruxolitinib).
received topical high-potency steroids, anthra- Currently both topical [145] and systemic admin-
lin, and UVB phototherapy. However, UVB istration of JAK inhibitors is under investigation
therapy was not tolerated and his vitiligo deteri- following very promising preliminary observa-
orated. Upon systemic acitretin he developed tions [146, 147].
pruritus and therapy had to be stopped.
Efalizumab was started at 0.7 mg/kg and there- 35.2.2.4 Concluding Remarks
after continued at 1 mg/kg/week. At the time Studies with classic immunosuppressants, such
when the patient’s psoriasis improved as methotrexate and azathioprine, are limited. No
(6–8 weeks after beginning with efalizumab) his study has been reported with mycophenolate
vitiligo also showed some improvement, espe- mofetil. As in many reported vitiligo interven-
cially on his trunk and lower extremities. Later, tion, a better differentiation of interest on stop-
the patient received systemic methypredniso- ping disease progression vs. promoting
lone followed by methotrexate upon which his repigmentation would be useful. Combination
vitiligo continued to improve. therapies with UV need to be studied for the sec-
These two reports suggested some therapeutic ond type of outcome.
effect of efalizumab even in long standing vitil- For biologics, neutralization of IFN-γ, TNF-α
igo vulgaris, but the product has been withdawn and LFA-1, limited data regarding efficacy have
because of the risk of progressive multifocal been reported. Antibodies against IFN-γ are not
encephalopathy by reactivation of JC virus yet routinely available whereas off-label use of
infection. TNF-α antibodies in vitiligo is possible.
However, TNF-α antibodies should be consid-
Multicytokine Blockade: JAK Inhibitors ered with great caution. First, they may trigger
The Janus kinase (JAK) and signal transducer de novo development of vitiligo. The data on the
and activator of transcription (STAT) pathway is use of anti-TNF-α antibodies in pre-existing vit-
a ubiquitous intracellular signaling network. iligo are controversial regarding the efficacy of
Janus kinases (named after the two-faced Roman these agents with no or only a limited repigmen-
god) are a particular group of tyrosine kinases tary response at best, but combined treatment
that associate with cytokine receptors. When with UV needs further investigation. Recent evi-
cytokines bind to their receptor, JAKs (denoted dence that vitiligo infiltrating memory T cells
as JAK1, JAK2, Tyk2 etc.) associated with differ- produce both IFN-γ and TNF-α would suggest a
ent receptor units cross-phosphorylate each other dual blockade to be helpful [148]. The promising
on tyrosine residues. The receptors are then phos- current development of JAK inhibitors may fill
phorylated by JAKs, creating phosphotyrosine- the gap. Among other therapeutic possibilities,
docking sites for SH2-containing proteins, in this the apparent beneficial use of efalizumab, no
case a specific group of proteins called STATs. longer available, suggests that blocking T cell
Activation of each STAT depends on the type of entry in the skin or derived strategies may help.
cytokine. In most cases, cytokine receptors are Local administration of biologics or targeted
composed of multiple subunits, each of which drugs such as JAK inhibitors could become an
binds to one of the four JAK family members. alternative option in selected vitiligo patients in
The major TH1 cytokine IFNγ uses JAK1 and future.
ERRNVPHGLFRVRUJ
372 A. Taïeb
35.3 Other Systemic Therapies 1992 in the USA in an open study in association
with vitamin C [151]. Another trial was con-
Several alternative treatments using supplemen- ducted in Sweden, involving 100 patients treated
tation with antioxidants and vitamins have been with folic acid (5 mg) and vitamin B12 (1 mg) for
proposed [149]. 3 months and advised to expose their skin to sun.
Repigmentation was observed in nearly half 52
the treated patients [153].
35.3.1 Vitamin Supplementation Low vitamin D levels have been associated
with several autoimmune diseases. Although no
Vitamin B12 and folic acid have been found to convincing evidence has been published of a vita-
be decreased in sera of vitiligo patients, even in min D deficiency in vitiligo, the topical rhodo-
absence of specific clinical manifestations dendrol induced vitiligo observed epidemically
[150, 151], but the initial mechanism accounting in Japan responded to oral vitamin D in associa-
for the reduction has been poorly evaluated. tion with phototherapy [154].
Folic acid and vitamin B12 derivatives interact It is known that para-aminobenzoic acid
in the one-carbon cycle and the folate form N-N- (PABA) induces hair and skin darkening, not spe-
methylene tetrahydrofolate gives the methyl cifically in vitiligo but in patients treated for dif-
group to homocysteine producing, in a vitamin ferent diseases. The trial conducted in vitiligo
B12-dependent manner, methionine. This enzy- patients indicated that 1000 mg/day, together
matic reaction determines the level of homocys- with vitamin C and B12 and folic acid, enhanced
teine, and the depigmentation may be due to the the pigmentation in 12 out of 20 enrolled patients
deficient methionine synthesis and homocyste- [151. However, this study was limited by the low
ine build-up. Taking into account that homocys- number of enrolled subjects as well as by the
tinuria can cause pigmentary dilution, inconsistent outcome measures.
characterized by fair skin and hair [152], and
that the alteration of the homocysteine metabo-
lism may be related to the catalase polymor- 35.3.2 l-Phenylalanine
phism, is interesting to point out that in vitiligo
patients the serum level of homocysteine has Four trials evaluated the effectiveness of l-
been found increased and positively correlated phenylalanine (50–100 mg/kg up to 18 months)
with the activity of the disease [152]. The pteri- even as supporting therapy of UVA or UVB pho-
dine in folic acid might refund the pteridine defi- totherapy, or of other approaches. All studies
ciency, which inhibits the melanogenesis by reported beneficial effects (30–90% of repigmen-
lowering tyrosine; indeed, the pteridine from tation in 25–60% of the patients) even if they
folic acid may stop the altered recycling of the show high patients dropout or inconsistent out-
reduced pterines (6BH4 and 7BH4) proved in come measures [153, 155, 156].
vitiligo epidermis. Finally, the addition of vita-
min B12 and folic acid may support the
UV-induced stimulation of melanocyte stem 35.3.3 Systemic Antioxidants
cells, through a mechanism independent on the
serum level of folic acid [150]. An open study Vitamin E. A clinical trial evaluated the effects
indicated that vitamin- induced repigmentation of vitamin E on the recovery of skin lipid per-
occurs mainly in patients with vitiligo more oxidation induced by PUVA treatment in a
recent than 10 years, independently on the activ- group of 30 patients [86]. The study reported
ity of the disease [150]. 75% of repigmentation in 60% of the patients
The combination folic acid and vitamin B12 treated with PUVA and vitamin E whereas the
was investigated first with promising results in same percentage of repigmentation was
ERRNVPHGLFRVRUJ
Fig. 35.7 A semplified

schematic representation
of the enzymatic and
non-enzymatic
antioxidant network
obtained in 40% out of 15 subjects exposed to Polypodium leucotomos. A further approach

PUVA only. The clinical result was associated has been performed with Polypodium leucotomos
with a decreased level of lipoperoxidation extract, a fern native to the tropical and
products. subtropical regions of the Americas, with history
Balanced pool. Taking into account the mech- of use as a folk remedy in Honduras. The pres-
anisms underlying the correct synthesis and ence of phenolic derivatives, accounts for the
recycling of the antioxidant network, the admin- antioxidant and immunomodulatory activities
istration of a balanced pool of antioxidants and [150, 158]. Polypodium leucotomos, adminis-
molecules involved in their recycling has been trated both locally and systemically, quenches
proposed [157]. According to that, a pool of free radicals and counteracts the lipoperoxida-
α-lipoic acid, vitamin E and C, has been evalu- tion. In addition, a shift of the lymphocytes with
ated in a double-blind placebo controlled trial, the Th1 phenotype versus the Th2 one, and lower
enrolling 35 patients, in order to test its role in serum levels of TNFα and IFNγ were observed.
reducing the UV dosage and in improving the Two double- blinded placebo controlled trials
rate of repigmentation [157]. The oral intake of have suggested its effectiveness. The association
the balanced pool promoted the effectiveness of with PUVA increases the percentage of patients
the NB-UVB phototherapy, increasing the extent with more than 50% of repigmentation [152]. A
of repigmentation and lowering the UV dosage. clear trend of repigmentation of neck and head in
At the same time the activity of the catalase was patients treated with Polypodium leucotomos
partially restored and the intracellular level of during NB-UVB phototherapy (44% versus 27%
ROS decreased. Excellent and good repigmenta- out of the 50 enrolled patients) has been described
tion was reported in 40% and 20% of antioxidant- in the second study [159], supported by digital
treated patients respectively (versus 22% and photography performed at the beginning and at
10% respectively in patients exposed to NB-UV two different subsequent time points of the study.
alone). Clinical data have been also confirmed by The oral intake of antioxidants do not inter-
digital photography performed at the beginning fere with other drugs or other diseases (such as
and at two different subsequent time points of the thyroiditis, diabetes, hypertension possibly
study (Fig. 35.7). occurring in vitiligo patients). Even if long-term
ERRNVPHGLFRVRUJ
374 A. Taïeb
follow-up studies have not been still published, significant difference was noted in patients with
the oral intake of antioxidants is not associated SPT III [161]. Despite these encouraging
with the occurrence of side effects, allowing results, the drug has not been developed further
thus their repeated and prolonged administra- for vitiligo.
tion. However, recent data have raised some
concerns for cancer prevention, following very
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Surgical Therapies
36
Boon Kee Goh
Contents
36.1 Cellular Grafting for Vitiligo 382
36.2 C ultured Cellular Grafting 383
36.2.1 Cultured Melanocyte Transplantation 383
36.2.2 Cultured Epidermal Sheet Transplantation 384
36.3 N on-cultured Cellular Grafting 385
36.3.1 Non-cultured Epidermal Cell Suspension Transplantation (NCES) 385
36.4 Factors Influencing Outcome in NCES 389
36.5 Simplified Non-cultured Cellular Grafting 390
36.6 on-cultured Outer Root Sheath Hair Follicle Cell Suspension
N
(NCORSHFS) Transplantation 392
36.7 nnex A: Surgical Protocol for NCES (Adopted and Modified
A
from [27]) 395
References 396
Abstract split skin graft. Under proper training, harvest-

Cellular grafting for vitiligo has evolved since ing an ultrathin split skin graft (not beyond the
its inception. Non-cultured cellular grafting has papillary dermis) is fast and results in no or
superseded cultured techniques as the preferred minimal scarring. Innovative methods to sim-
modus operandi in clinical practice. The rea- plify non-cultured cellular grafting have also
sons are straightforward: it can be completed in been successfully achieved.
a few hours, does not require the use of xenobi- Melanocytes can now be extracted not only
otics (except trypsin) or culture media and is of from the epidermis for transplantation but
significantly lower costs. Although it does not from outer root sheath of hair follicles.
offer the possibility of cell expansion and cryo- Non-cultured cellular grafting has proven to be
preservation for future transplant, this can be a safe and effective surgical treatment for stable
supplanted by harvesting a new piece ultrathin vitiligo, substantiated by various long-term studies
over the last two decades. The imposition of strict
legislative regulation on this technique is unwar-
B. K. Goh (*) ranted and will only drive up cost and deprive
Skin Physicians Pte Ltd, Mount Elizabeth Medical patients’ access to this innovative procedure.
Centre, Singapore, Singapore

ERRNVPHGLFRVRUJ
382 B. K. Goh
36.1 Cellular Grafting for Vitiligo

Key Points
• Non-cultured cellular grafting has Over the last two decades, cellular grafting for
superseded cultured techniques as the vitiligo has achieved significant progress. This
preferred modus operandi in clinical surgical approach is indicated for stable vitiligo
practice for stable vitiligo. refractory to medical therapy. The procedure
• It is not time-consuming and does not involves harvesting epidermal cells from autolo-
require the use of xenobiotics (except gous donor skin and transplanting them (with or
trypsin) or culture media and is of sig- without prior selective cultivation) onto vitiligi-
nificantly lower costs. nous recipient sites (Fig. 36.1). Unlike tissue
• Under proper training, harvesting an grafting, where the treatment area is limited by
ultrathin split skin graft (not beyond the the size or number of tissue grafts, cellular graft-
papillary dermis) is fast and results in no ing allows treatment of achromic skin manifold
or minimal scarring. larger than the donor area. It also has the capabil-
• Innovative methods (the “6-well” and ity of repigmenting leukotrichia in vitiligo, pos-
“4-well” plate techniques) to simplify sibly due to retrograde migration of transplanted
non-cultured cellular grafting have also melanocytes into the hair follicles [1, 2].
been successfully achieved, including Cellular grafting can be broadly categorized
using the body as the natural incubator. into (a) cultured melanocyte or epidermal cell
• Melanocytes can now be extracted from transplantation (cultured cellular grafting) and
the outer root sheath of the hair follicles. (b) non-cultured transplantation of epidermal
• The imposition of strict legislative regu- cells or outer root sheath hair follicle cells (non-
lation on this technique is unwarranted. cultured cellular grafting) (Fig. 36.2). The key
advantage of in vitro culture is that the cells can
Extract
Harvest
Cultured Non-cultured
Dermabrade and Transplant
Fig. 36.1 Cellular grafting involves harvesting epidermal cells from autologous donor skin and transplanting them
(with or without prior selective cultivation) onto vitiliginous recipient sites
ERRNVPHGLFRVRUJ
36 Surgical Therapies 383
Fig. 36.2 Types of Cellular grafting

cellular grafting for
vitiligo
Cultured Non-cultured
Epidermal cells (NCES)

Melanocytes
Outer root sheath hair follicle cells

Epidermal cells (NCORSHFS)
be expanded and cryopreserved for future use. In 1988, it was found that basic fibroblast
However, the legislative restriction on cultivating growth factor (bFGF) is an important
cells for clinical use has limited the widespread keratinocyte-derived factor influencing melano-
application of cultured cellular grafting. cyte survival and proliferation, a natural mitogen
for melanocytes [6]. By using a defined medium
that includes bFGF, it became feasible to culti-
36.2 Cultured Cellular Grafting vate human melanocytes in vitro without TPA (a
tumour promoter) and fetal calf serum (FCS). In
36.2.1 Cultured Melanocyte 1993, Olsson and Juhlin successfully expanded
Transplantation melanocytes in culture with a medium free of
phorbol esters, pituitary extract and serum. The
Until the 1980s, it had not been feasible to culti- medium they used was PC-1 (Ventrex, USA),
vate melanocytes in vitro in large quantities. An supplemented with bFGF, dibutyryl cyclic ade-
important reason was the preferential over- nosine monophosphate and antibiotics. They
growth of keratinocytes over melanocytes in transplanted the cultured cells (1000–2000 mela-
culture. It was not until 1982 that a breakthrough nocytes/mm2) onto superficially dermabraded
occurred with the introduction of melanocyte achromic skin of ten patients with vitiligo, out of
mitogens. Eisinger and Marko selectively which nine had good cosmetic outcome [7].
cultivated human melanocytes from neonatal Further evaluation of this technique was reported
foreskin and adult skin by introducing a year later. By excluding geneticin in the culture
12-O-tetradecanoylphorbol-13-acetate (TPA) medium (which suppresses proliferation of kera-
and cholera toxin (CT) into the culture medium tinocytes and fibroblasts), Lontz et al. trans-
[3]. Subsequent enhancement was achieved by planted 16 patients with stable or active vitiligo
the addition of isobutylmethylxanthine (IBMX), with cultured melanocytes. Of them, 8 had 50%
a phosphodiesterase inhibitor, which works syn- to beyond 90% repigmentation in their grafted
ergistically with TPA [4]. sites, while the remaining 8 had less than 50%.
By supplementing culture medium with these The major factor that influenced outcome was
mitogens, cultivation of large quantities of mela- neither the age of the patient nor the disease
nocytes became a reality. These advances opened activity but anatomic location of the recipient
up the possibility of culturing human melano- sites. The face and trunk achieved the best repig-
cytes for clinical use. The first translational mentation outcome, while the fingers and elbows
experiment was undertaken by Lerner and col- were most refractory. Ultrastructural examina-
leagues, who cultivated melanocytes from a tion of the transplanted site revealed that it
patient with piebaldism using a medium contain- resembled that of an uninvolved healthy skin,
ing TPA, IBMX, CT, bovine serum and pituitary although the melanocytes were positioned
extract. By injecting these cells into the achromic slightly higher among basal keratinocytes. The
skin (raised by suction blisters) of the patient, investigators concluded that this procedure was a
successful melanocyte engraftment and repig- viable therapeutic option for vitiligo when con-
mentation of piebaldism were achieved [5]. ventional treatments had failed [8].
ERRNVPHGLFRVRUJ
384 B. K. Goh
Besides having the advantage of expanding segmental or focal) (n = 80), stable generalized
melanocytes from a small skin biopsy, cultured vitiligo (n = 26) and active generalized vitiligo
cellular grafting offers the attractive option of (n = 14). Patients’ melanocytes were cultivated in
cryopreserving excess cells for future use. The Hu16 medium, which consisted of Ham’s F12
successful reimplantation of cryostored melano- mixture (Gibco, USA), supplemented with
cytes was reported in four patients with vitiligo in recombinant human bFGF, CT, IBMX, 20% FCS
1994 [9]. Cultured melanocytes were preserved and gentamicin. The bFGF concentration was
in a cryoprotectant, consisting of 8% dimethyl much higher than that used by Olsson and Juhlin
sulphoxide (DMSO) and newborn calf serum, (20 ng/ml vs. 5 ng/ml), based on the premise that
and were frozen for 6–12 months in −70 °C. The lipoproteins in the serum bind bFGF and lower
melanocyte survival after thaw was around 70%. the growth factor’s attachment to its receptors.
The value of cryopreservation is that it allows After ablating the recipient sites with CO2 laser,
regrafting of incompletely repigmented vitiligo the melanocyte suspension was applied to the
after the initial transplant or regrafting of large denuded areas at a density of 600–1000 cells/
achromic areas after successful test grafts. mm2, followed by a silicone gauze dressing. The
Over time, further progress was made in cul- patients were followed up for 6–66 months. The
tured melanocyte transplantation. outcome of transplantation was impressive, and
De-epithelization of the recipient site had tradi- disease stability was the main outcome determi-
tionally been achieved by cryotherapy, suction nant: 90–100% repigmentation was recorded in
blister or dermabrasion. These interventions 84% of cases in the stable localized vitiligo
work well for vitiligo areas that are accessible. group, 54% in the stable generalized group and
But for delicate anatomic sites (such as eyelids 0% in the active generalized group (p < 0.01).
and nasal folds) and irregular lesions, they may Age, gender and size of lesion did not influence
be impractical [10]. Although costs may be a the outcome of transplantation, while the effect
concern, laser ablation using Erbium-YAG or of anatomical location could not be evaluated sta-
carbon dioxide (CO2) lasers offers a superior tistically because most of the treated areas were
alternative, because of excellent precision over in the head and neck.
margin and depth [11].
Cumulative data on the outcome of cultured
melanocyte transplantation in the treatment of 36.2.2 Cultured Epidermal Sheet
vitiligo also began to emerge. Olsson and Juhlin Transplantation
reported their series on 100 vitiligo patients.
Using the surgical approach they previously Instead of selectively cultivating melanocytes
described, 72 patients had good to excellent through special media, epidermal cells (keratino-
repigmentation (40 patients had repigmentation cytes and melanocytes) can be cultured into lay-
rates exceeding 94%; and 32 achieved repigmen- ered sheets and used in repigmenting stable
tation rates between 65% and 94%).The best vitiligo. The basis of using cultured epidermal
results were seen in patients with stable vitiligo grafts stems from its long-standing application in
for at least 1–2 years. Fingers, elbows and knees the treatment of burns and chronic non-healing
were particularly refractory to repigmentation. wounds. When cultured epidermal grafts are
Colour mismatch was initially common but transplanted into full-thickness burns that have
improved over 6–8 months. No significant scar- been excised to the fascia (hence devoid of local
ring was reported, and repigmentation remained melanocyte reservoir), repigmentation can be
unchanged in patients followed up for 1 and observed after re-epithelization, indicating that
2 years [12]. Chen and colleagues reported their melanocytes in the graft have repopulated the
series consisting of 120 patients followed up over recipient site [14, 15].
a 5-year period [13]. They divided their cohort In 1989, Brysk and colleagues reported their
into three groups: stable localized vitiligo (i.e. experience using culturing epidermal cells for the
ERRNVPHGLFRVRUJ
treatment of stable vitiligo [16]. Autologous epi- attributed to loss of initial vitiligo stability and
dermal cells were harvested from shave biopsies difficulty in securing the grafts over joints and
of normally pigmented skin and were cultured in acral sites.
MCDB-153 medium (Clonetics, USA). This Cultured epidermis is delicate to handle, and
TPA-free medium supports clonal proliferation its manipulation can affect cell viability and graft
of keratinocytes and melanocytes, but not fibro- survival. This problem can be mitigated by grow-
blasts. The cells were seeded onto collagen- ing epidermal cells on a chemically defined sur-
coated membranes, and after 50–70% confluence, face, forming a composite sheet that is easy to
the composite was transplanted as an autologous handle. Examples of such biomaterial supports
graft onto dermabraded vitiliginous sites. include collagen-coated membranes and acrylic
Re-epithelization was complete in 2 weeks, and acid-coated silicone sheets. In 1998, Andreassi
repigmentation was evident after 4 weeks. The and colleagues assessed the utility of a flexible
investigators extended this technique in treating membrane made up of a biopolymer of hyal-
four vitiligo patients, of whom three had 40–90% uronic acid esterified by benzyl alcohol [20].
repigmentation of grafted areas at 1-year follow- This membrane is perforated with a regular array
up, and the colour match with the surrounding of laser-drilled micropores for the seeding of
skin was good [17]. cells and larger pores for drainage. After cultur-
Cultivation of melanocyte-bearing epidermal ing keratinocytes on 3T3 fibroblast feeder layer,
grafts was also successfully carried out by they were seeded onto the membrane. Autologous
Falabella and colleagues [18]. Autologous mela- transplants using this composite epidermal sheet
nocytes and keratinocytes were cocultured in were carried out in 11 patients with facial and
Eagles’ minimal essential medium with Hanks’ truncal vitiligo. No side effects were reported,
balanced salt solution, supplemented by fetal and 5 of the patients had almost complete repig-
bovine serum, hydrocortisone, glutamine and mentation. Epidermal cells can also be grown on
antimicrobials. Instead of using a feeder layer to silicone sheets coated with acrylic acid, prepared
promote epidermal differentiation, adjustment of by plasma polymerization. Successful transfer of
pH and calcium concentration was utilized. The melanocyte-bearing epidermal sheets was suc-
pH of the culture medium was adjusted to 7.2, cessfully carried out in human wound bed equiv-
and after achieving keratinocyte confluency, the alents [21–23].
calcium concentration was switched from 1.2 to
3.0 mmol/l. Epidermal sheets were obtained after
21 days of plating and were released from the 36.3 Non-cultured Cellular
substratum by dispase. They were then attached Grafting
to a supporting layer of petroleum gauze and
transplanted to the recipient sites (denuded by 36.3.1 Non-cultured Epidermal Cell
cryotherapy) of three patients with segmental or Suspension Transplantation
focal vitiligo. Satisfactory repigmentation was (NCES)
clinically evident, and recolonization of melano-
cytes in the grafted sites was demonstrated by Although cultured cellular grafting offers the
electron microscopy. Following this pilot suc- possibility of cryopreserving excess cells for
cess, the investigators extended the procedure in future transplantation, it is not without draw-
treating eight patients with generalized vitiligo backs. Cell cultures are expensive to maintain
and one patient with segmental vitiligo [19]. In and time-consuming, as it takes several weeks for
this trial, the calcium concentration used in cultured cells to reach confluence. The procedure
promoting keratinocyte differentiation was
also requires highly qualified personnel and
1.8 mmol/l. Five out of the nine patients achieved involves the use of culture medium and xenobiot-
at least 60% repigmentation at 1–2 years of fol- ics, which invokes the imposition of good manu-
low-up. Failure of or poor repigmentation was facturing practice (GMP) standards. This
ERRNVPHGLFRVRUJ
386 B. K. Goh
essentially restricts the treatment to highly spe- engraftment of the cells; all four treated patients
cialized centres or industries. An alternative is to with stable vitiligo achieved more than 80%
transplant autologous epidermal cells in the same repigmentation. Instead of using cryotherapy to
surgical setting without first expanding the cells prepare the recipient bed, carbon dioxide laser
in culture. Large vitiliginous areas, five- to ten- was utilized. The advantage of using laser abla-
fold larger than the size of the donor skin, can tion was mentioned in the earlier section; notably
still be treated in an outpatient setting and the it confers better speed, precision and depth con-
entire procedure completed just over a few hours. trol. Finally, trypsin is not accepted by the regula-
This form of transplantation, non-cultured cel- tory agency of some EU members as it is an
lular grafting, was pioneered by Gauthier and enzyme classified together with restriction
Surleve-Bazeille in 1992 [24]. Donor skin was enzymes for DNA. Consequently, the mechanical
harvested from the occipital scalp and incubated dissociation should be currently preferred.
in cold trypsin for 18 h, after which the epidermis Because wound healing on its own can theo-
was separated from the dermis using a pair of fine retically induce repigmentation by activation of
forceps. Epidermal cells were extracted and sus- stem cell reservoirs and associated pathways, it is
pended in PBS. The cellular suspension was sub- important to clarify if the repigmentation of leu-
sequently injected into blisters raised by liquid koderma by cellular grafting is contributed
nitrogen at the recipient areas of segmental and mainly by the engraftment of the transplanted
focal vitiligo as well as naevus depigmentosus. In cells. The seminal paper that addressed this was
8 out of 12 patients treated, a repigmentation rate the elegant work done by Van Geel et al. [27]. In
of more than 70% was achieved. Six years later, this double-blind placebo-controlled trial, 33
Olsson and Juhlin published a comparable tech- paired vitiligo lesions of 28 patients were treated
nique using a basal cell layer enriched suspen- either with epidermal cell suspension enriched
sion [25]. The donor skin, however, was harvested with hyaluronic acid or the vehicle fluid alone.
from the gluteal region, and the time for The transplantation of epidermal cells resulted in
trypsinization was reduced to 50 min, which
repigmentation of at least 70% in 77% of the
made it possible to complete the entire surgical treated areas at 12 months, whereas none of the
procedure within the same day. The cell suspen- placebo group achieved this (and at any time
sion was directly applied onto dermabraded vit- point). The investigators concluded that repig-
iligo lesions, before securing it with a thin mentation is caused by engrafted melanocytes,
collagen film, moistened gauze and Tegaderm. rather than postinflammatory reaction induced by
Out of 17 patients treated, 12 with vitiligo vul- epidermal injury or a response to previously
garis had a repigmentation rate of at least 80%, failed phototherapy.
while the remaining 5 patients with segmental Long-term follow-up studies on autologous
vitiligo had complete repigmentation. NCES transplantation concur that it is a safe pro-
These techniques, however, have several prac- cedure and is effective in repigmenting vitiligo
tical problems. Blisters are particularly difficult that is stable. Many reports have been published
to raise by liquid nitrogen on bony eminences since 2002. In general, segmental vitiligo shows
and can be associated with leakage of cell sus- the best repigmentation outcome and retention of
pension. Significant hypopigmentation can also colour, followed by focal vitiligo. For vitiligo
result from cryo-damage of melanocytes. The vulgaris, the average repigmentation does not
fluidity of the cell suspension can also lead to sig- exceed 50% in long-term evaluations. The key
nificant “run-off” over contoured surfaces and reason is attributed to the resurgence of vitiligo
result in loss of viable cells. Van Geel et al. activity or that the condition has not been com-
mitigated this problem of “run-off” by increasing pletely stable over time. The role of postsurgical
the viscosity of the cell suspension using hyal- phototherapy or sun exposure has also been eval-
uronic acid (Alcon, France) [26]. Turning the uated; it is reasonable to conclude that the ultra-
suspension into a gel-like paste did not impair violet light exposure enhances the speed of the
ERRNVPHGLFRVRUJ
repigmentation but does not affect the final repig- (n = 5) vitiligo fared less well (mean repigmenta-
mentation outcome [28]. tion of 70% and 37%, respectively). Final repig-
Olsson and Juhlin were the first group who mentation was achieved within a mean of
reported their transplantation experience using 10 months post-grafting; however, a perfect
basal cell layer suspension in patients treated in colour match (between treated areas and sur-
the preceding 1–7 years [29]. For segmental vit- rounding normal skin) was seldom achieved.
iligo and piebaldism, all patients achieved and Although 80% of the patients had some colour
retained 95–100% repigmentation; for focal vit- mismatch, the outcome was not disturbing to a
iligo, the repigmentation was 100%. However, majority of them.
for generalized vitiligo, the intervention achieved Our team evaluated the 12- and 60-month
only an average repigmentation of 49%. Mulekar outcome of a large multi-racial cohort of vitiligo
reported his results in the treatment of segmental patients (n = 177) following NCES transplanta-
(n = 49) and focal (n = 15) vitiligo for the preced- tion [28]. Several practical issues, additional to
ing 1–5 years, using the surgical technique simi- previous reports, were addressed: the role of
lar to that of Olsson and Juhlin. Segmental post-grafting phototherapy, the comparison of
vitiligo achieved better repigmentation outcomes collagen dressing versus hyaluronic acid in
compared to focal vitiligo; 84% of patients with reducing “run-off” and the time interval to repig-
segmental vitiligo achieved at least 95% repigmentation among different ethnicities. We
mentation, compared to 73% of those with focal adopted the transplant technique described by
vitiligo. In both types of vitiligo, the repigmenta- Van Geel et al. but replaced hyaluronic acid with
tion was retained until the end of the respective collagen dressing as the biomaterial to reduce
follow-up period, and no adverse effects were “run-off” in a proportion of patients. At 12-month
reported [30]. These two reports, however, did follow-up, good to excellent repigmentation (i.e.
not address several important outcome measure- >50% repigmentation) was seen in 88% of
ments and other factors that influence final patients with segmental vitiligo, significantly
results, such as time interval to achieve final higher than 71% of patients with non-segmental
repigmentation, colour matching and the role of vitiligo. For patients who were followed up to
phototherapy or sun exposure post-grafting. 60 months, 83% of them retained >75% repig-
Van Geel et al. addressed these concerns in mentation. This underscores again that NCES is
their long-term follow-up study: percentage of effective in repigmenting vitiligo, and segmental
repigmentation was assessed in 82 patients, and vitiligo fares the best. Significantly more patients
long-term outcomes were evaluated retrospec- treated with collagen dressing (Neuskin-F®,
tively by 54 patients using a questionnaire up to Eucare Pharmaceuticals, India) (82%) achieved
7 years post-grafting. Their surgical technique good to excellent repigmentation compared to
was largely similar to that of Olsson and Juhlin, those who received hyaluronic acid (63%). The
with the exception that hyaluronic acid was advantage of using collagen dressing is that the
added to increase the viscosity of the epidermal biomaterial is highly pliable and adheres well to
cell suspension [31]. In their study, NCES the wound bed after applying a small aliquot of
achieved a high percentage of repigmentation, cell suspension. It prevents “run-off” on con-
with more than 75% repigmentation achieved in toured surfaces better than hyaluronic acid.
71% of patients, which was maintained during Interestingly, post-grafting targeted photother-
the follow-up in the majority of patients. Those apy did not significantly affect repigmentation
who lost the repigmentation were patients (7%) outcome at 12 months. We believe that the final
with generalized vitiligo. Corresponding to repigmentation rests upon the successful engraft-
previous reports, best results (mean repigmenta- ment of transplanted melanocytes (and other epi-
tion of 85–91%) were obtained in segmental vit- dermal cells) and is unaffected by UVB
iligo (n = 30), piebaldism (n = 3) and halo naevi stimulation. In other words, UVB stimulation
(n = 5), whereas generalized (n = 33) and mixed may speed up repigmentation but does not alter
ERRNVPHGLFRVRUJ
388 B. K. Goh
the final outcome. Although unpublished, we 6–8 weeks) and Caucasian (around 8–12 weeks)
also observed that the first signs of post-grafting patients. The technique of NCES adopted by our
repigmentation are faster in Indian patients group (including the surgical pearls) is described
(within 4 weeks) than our Chinese (around in Annex A and Figs. 36.3a–g and 36.4.
a b
Fig. 36.3 (a) Harvesting an ultrathin epidermal graft centrifugation. (e) Application of cell suspension onto
using a silver dermatome. (b) An ultrathin epidermal graft laser-ablated vitiliginous recipient site. (f) Placing colla-
in a petri dish containing 0.25% trypsin-EDTA. (c) The gen sheets to hold cell suspension in place and prevent
graft is cut into smaller pieces and incubated in 0.25% “run-off”. (g) Securing suspension and collagen sheets
trypsin-EDTA at 37 °C. (d) Completion of cellular extrac- using Hypafix dressings
tion: a dense cell pellet at the base of the Falcon tube after
ERRNVPHGLFRVRUJ
f g
Fig. 36. 3 (continued)
a b
Fig. 36.4 (a and b) Successful repigmentation of segmental vitiligo after cellular grafting
36.4 F
actors Influencing Outcome failed medical treatments and phototherapy. The
in NCES vitiligo has to be stable (preferably more than a
year) before surgical transplant, and the patient
From the above discussion, it is clear that for does not display Koebner phenomenon. The abil-
NCES to be successful, the selection criteria of ity to meet patients’ expectation also has to be
patients have to be strict. A surgical intervention carefully assessed before surgery. In addition, the
is indicated only if a patient with vitiligo has concentration of melanocytes transplanted and
ERRNVPHGLFRVRUJ
390 B. K. Goh
the presence of inflammation in the recipient (231 ± 27/mm2). In other words, there was a sig-
sites also influence surgical outcomes. nificant attrition of cells during the harvesting
Rao et al. studied the clinical, biochemical and process. It is reasonable to conclude instead that
immunological factors determining stability of a higher donor-recipient area ratio offers a better
disease in patients with generalized vitiligo repigmentation (1:3 fares better than 1:5) and a
undergoing surgical transplantation [32]. The transplant density of 210–250 melanocytes/mm2
success rates were highest among patients whose provides >75% repigmentation in half of the
vitiligo is clinically stable for 2 years or more. treated cases.
Poor repigmentation after transplantation was
associated with shorter disease stability
(13.2 ± 14.3 vs. 56.6 ± 52.4 months) and higher 36.5 Simplified Non-cultured
percentages of CD8+ (3.0 ± 2.1 vs. 1.0 ± 1.4) and Cellular Grafting
CD45RO+ (1.7 ± 2.4 vs. 0) cells in the lesional
biopsy. These findings suggest that the presence Although NCES transplantation is effective in
of an inflammatory milieu at the recipient site repigmenting stable vitiligo, the technique
portends disease instability and is associated with requires extraction of epidermal cells that
poor surgical success. involves multiple stages and reliance on a labora-
There is a dearth of studies determining the tory space and equipment (such as a laminar flow
optimal concentration of melanocytes required chamber and centrifuge machine). This largely
for successful NCES. Olsson and Juhlin limits the use of this technique to academic insti-
postulated that a melanocyte count of 190/mm2 tutions. Over the last decade, ways to simplify
was sufficient for adequate repigmentation [25]. this procedure have been explored. ReCell® kit
In a randomized prospective study, Tegta et al. (Avita Medical, Australia) is a portable battery-
compared the efficacy of two different dilutions operated cell-harvesting device that was initially
of melanocytes in achieving early and acceptable developed for the treatment of superficial burns
repigmentation [33]. In one group, the epidermal through epidermal cell transplantation. Sodium
cell suspension was prepared from a donor graft lactate is used as the delivery fluid. During the
one-third the size of the recipient area, while in treatment of burns, it was observed that the resul-
the other group, the graft was one-fifth that of the tant healing was accompanied by repigmenta-
recipient area. The former group received a tion, which led to the use of this device for
higher density of melanocytes (231 ± 27 cells/ treatment of stable vitiligo. In a small pilot study
mm2) and had significantly greater extent involving ten lesions in five patients with stable
repigmentation (50% patients achieving >75% vitiligo (one segmental and four generalized vit-
repigmentation) than the latter group with lower iligo), Mulekar et al. evaluated the effectiveness
melanocyte density (154 ± 27 cells/mm2) (none of the ReCell® kit in repigmentation compared to
of the patients achieved >75% repigmentation). conventional NCES transplantation [35]. The
The investigators concluded that the minimum results were comparable: with ReCell®, two
number of cells to produce acceptable repigmen- lesions showed 100%, one 65%, one 40% and
tation is in the range of 210–250/mm2. What one 0% repigmentation, while with NCES, three
defines as an “acceptable” repigmentation rate is showed 100%, one 30% and one 0% repigmenta-
subject to debate, and practitioners should reflect tion. Another conclusion that can be drawn from
if 50% of subjects failing to achieve >75% repig- this study, which is useful and important in prac-
mentation is an acceptable outcome. It is impor- tice, is that it is unnecessary to use a melanocyte
tant to note that melanocyte density harvested medium as a carrier fluid for the cell suspension.
from the donor graft in this study was low (694– Sodium lactate or PBS suffices, as the recipient
771/mm2) compared to published numbers wound bed provides serum and adequate nutri-
(1700 ± 139/mm2; thigh area) [34], and the num- ents to neutralize the effects of trypsin and sup-
bers were even lower in the final cell suspension port the survival and proliferation of the
ERRNVPHGLFRVRUJ
transplanted epidermal cells. However commer- times with a pipetter. This suspension is then
cial kits like ReCell® have a certain drawback, withdrawn from the fifth well and dispensed
and that is cost. These devices are expensive, and into the sixth well through the microfilter to
affordability becomes an important limiting fac- remove residual tissue debris. The filtrate, con-
tor for their routine use. taining the final cell suspension, is then applied
To circumvent this issue, our team developed to laser-ablated recipient sites. Using this cell
an inexpensive, simplified grafting technique extraction process, we treated two patients with
that obviates the need of a laboratory set-up segmental vitiligo, two with focal vitiligo and
[36]. The term “6-well plate technique” was one with piebaldism. All treated lesions repig-
coined. It makes use of a 6-well tissue culture mented, and the percentage of repigmentation at
plate in the preparation of the epidermal cell 6 months ranged between 65% and 92%. This
suspension. Moving in a clockwise manner, “6-well plate” set-up differs from commercially
soybean trypsin inhibitor (Sigma) is added into available devices in a few ways. It does not have
the first well, while PBS is added into the sec- a built-in automated heater; this can easily be
ond to fourth wells. The fifth well is left empty, circumvented by warming the harvested skin
and a 40-μm cell strainer is placed in the sixth tissue in a petri dish containing trypsin using a
well (Fig. 36.5). After harvesting the autologous small benchtop portable incubator. Soybean
donor tissue, the ultrathin split skin graft is cut trypsin inhibitor is used to neutralize trypsin,
into smaller pieces in a petri dish and incubated but it can be replaced by Ringer’s lactate solu-
in trypsin-EDTA 0.25% for 40 min at 37 °C. The tion. The cost of this set-up is only a fraction of
“trypsinized” tissues are subsequently placed in that in commercial devices, making it an appeal-
the first well, where the effects of trypsin are ing and inexpensive alternative.
neutralized by soybean trypsin inhibitor Kumar et al. simplified this technique further
(Fig. 36.5a). The tissues are then rinsed in PBS by using a four-compartment petri dish [37]. The
in the next three wells and placed in the empty basic principles, however, remain the same.
fifth well. Using a pair of fine forceps, the epi- Instead of using a separate petri dish, the har-
dermis is separated from the dermis of each tis- vested donor tissue is placed in the first compart-
sue piece in this well, and the tissue surfaces are ment of the petri dish containing trypsin-EDTA
scraped to mechanically dislodge the epidermal 0.25%. This is then incubated for an hour at
cells (Fig. 36.5b). PBS is then added, and sus- 37 °C. The tissue is subsequently rinsed in PBS
pension is mixed evenly by aspirating several in the next two compartments to remove residual
a b
Fig. 36.5 (a) Using a 6-well culture plate to extract epi- to fourth wells). (b) Separation of the epidermis and der-
dermal cells. Trypsinized ultrathin skin graft is washed in mis, followed by mechanical dislodgement of epidermal
soybean trypsin inhibitor, before rinsing in PBS (second cells (by scraping), takes place in the fifth well
ERRNVPHGLFRVRUJ
392 B. K. Goh
trypsin. In the fourth compartment, the epidermis dressing. The investigators treated five patients
of the tissue is separated from the dermis using a with stable vitiligo (three NV and two SV), all of
pair of fine forceps and the epidermal cells dis- whom had excellent repigmentation (Fig. 36.6f).
lodged. Tissue debris are removed using forceps, A key concern with this procedure is the safety
and PBS is added and the suspension mixed by of trypsin, incubating within the suction blister.
aspirating several times using a sterile syringe. Recombinant trypsin is noted to be safe by the
The final suspension is then applied to derm- investigators, and its minute quantity, even if
abraded vitiliginous recipient sites. Six patients absorbed, is neutralized promptly by serum.
(four with segmental and two with focal vitiligo) Because trypsin breaks intercellular epidermal
were treated with this simplified NCES trans- bonds, it has no effects on dermal tissue, and the
plantation. The repigmentation was 90–100% in healing of the donor site was observed to be
four of the patients and more than 75% in two of unaffected.
them at 16 weeks. Although the effects of trypsin These techniques simplify NCES transplanta-
may not be fully removed by PBS rinses (thereby tion, obviating the need of a laboratory set-up and
impairing cell viability), this is overcome by reagents such as soybean trypsin inhibitor. It
serum present on the recipient wound bed after makes the procedure more cost-effective and
dermabrasion. accessible in a clinic setting. Although the total
In the absence of an incubator, cold trypsin- number of viable cells extracted may arguably
ization can take place instead [24]. This can be not be as high as conventional methods per unit
done by placing the petri dish containing the tissue, the repigmentation rates have been shown
donor tissue in trypsin 0.25% in a refrigerator at to be acceptable. Future studies, comparing the
4 °C overnight. Gupta et al. [38], however, two methods (i.e. simplified vs. conventional),
devised an ingenious way of harnessing the will be helpful in determining if there are signifi-
human body as a natural incubator and as a cant differences in cell densities and viability and
“mini-laboratory.” Cell separation and harvest- in clinical outcomes.
ing are performed inside suction blisters induced
on a patient’s thigh (Fig. 36.6). After a suction
blister has been raised using standard procedures
(Fig. 36.6a), the blister fluid is aspirated using a 36.6 N
on-cultured Outer Root
24G hypodermic needle attached to a 2-ml Sheath Hair Follicle Cell
syringe. This needle is introduced into the blister Suspension (NCORSHFS)
cavity through the adjacent skin via a subcutane- Transplantation
ous route (Fig. 36.6b,c). With the needle still in
place, the syringe is then replaced with another Besides the epidermis, melanocytes can be
syringe containing 1 ml of 0.25% trypsin, which extracted from human hair follicles. The hair fol-
is instilled into the blister cavity (Fig. 36.6d). licle contains a rich reservoir of melanocytes and
The needle is removed, and the solution is left in their precursors. Melanocyte-lineage antigens
the blister cavity for 45 min, during which epi- and c-Kit-positive cells are localized in the outer
dermal cells (melanocytes and keratinocytes) root sheath (ORS) of the infundibulum and mid-
will begin to dislodge to form a cell suspension. follicle, as well as in the hair bulb matrix [39].
This suspension is then aspirated and transferred The perifollicular connective tissue sheath and
to a petri dish (Fig. 36.6e). The roof of the blister papilla are also potential sources of mesenchy-
is subsequently excised, placed in the same petri mal stem cells. Unlike the epidermal-melanin
dish, and its dermal surface scraped to mechani- unit which has 1 melanocyte to every 36 kerati-
cally dislodge residual basal cells. The final sus- nocytes, the follicular-melanin unit has a signifi-
pension is then applied to dermabraded patches cantly higher ratio of 1:5 [40]. In addition,
of recipient vitiligo. The excised blister roof is melanocytes in anagen hair bulb are functionally
returned to the donor site to serve as a biological more active and have remarkable synthetic
ERRNVPHGLFRVRUJ
a b c d e
f Before surgery 2 Weeks
4 Weeks 16 Weeks
Fig. 36.6 In-vivo technique of harvesting epidermal cells for cellular grafting (Courtesy of Dr. Somesh Gupta)
capacity in comparison to epidermal “plucked” hair follicles for grafting stable vitil-
melanocytes [41]. These physiological features igo. Three out of five patients they treated had
make hair follicle a more attractive source of almost complete (>90%) repigmentation.
melanocytes for cell-based therapies in vitiligo. Although this technique is simple, non-invasive
The use of hair follicle cells for repigmenting and highly accessible, the cellular yield from
stable vitiligo was first explored by Vanscheidt plucked hairs can be low and inadequate for
and Hunziker [42]. In small case series, the inves- transplantation. Kumar et al. evaluated the yield
tigators derived single-cell suspension from of CD200+ cells (a marker for hair follicle bulge
ERRNVPHGLFRVRUJ
394 B. K. Goh
stem cells) derived from plucked hairs compared by rotating a 1-mm biopsy punch in the direction
with follicular unit extraction (FUE); the latter of each hair follicle to the level of the mid-dermis.
has significantly higher yield. They concluded The follicular unit was pulled out using a follicle-
that the stem cell niche is partially lost in follicu- holding forceps; transected hairs were discarded.
lar plucking [43]. Fifteen to 25 follicular units were extracted and
Using FUE of anagen hairs, Mohanty et al. collected in a medium containing Dulbecco’s
extracted cells from the outer root sheaths and Modified Eagle’s Medium (DMEM; Sigma-
grafted vitiligo lesions with the suspension [44]. Aldrich, USA) supplemented by antibiotics. In
The technique they adopted is as follows the laboratory, these units were washed in PBS
(Fig. 36.7). Anagen hairs were selected from the and then incubated in 0.25% trypsin–0.05%
occipital scalp and trimmed to 2 mm in length. EDTA (Gibco, USA) at 37 °C for 90 min to sepa-
Field block anaesthesia was administered using rate ORS cells. After every 30 min, the extracted
2% lignocaine. The follicular units were extracted hair follicles were placed in a new tube of
a b
c d
Fig. 36.7 Extraction of outer root sheath cells for cellular grafting (Courtesy of Dr. Somesh Gupta)
ERRNVPHGLFRVRUJ
trypsin-EDTA and the reaction in the previous transplantation in practice. Furthermore, FUE
tube aborted with soybean trypsin inhibitor (followed by ORS melanocyte extraction) is tech-
(Sigma-Aldrich, USA). Post-trypsinization only nically more laborious and time-consuming com-
keratinous hair shafts remained, which were dis- pared to harvesting a single piece of split skin
carded. The cell suspension of all three tubes epidermal graft. In a randomized study, Singh
were combined in a single tube and filtered et al. compared the treatment outcome in patients
through a 70-μm cell strainer. The filtrate was with stable vitiligo treated with NCORSHFS and
centrifuged for 5 min at 1000 rpm to obtain a cell NCES methods [46]. Although not statistically
pellet, which was subsequently resuspended in a significant, excellent (90–100%) and good
small amount of DMEM, before application onto (>75%) repigmentation were achieved in more of
dermabraded vitiligo patches, and covered with the treated areas using NCES (83% and 92%,
collagen dressings (Neuskin-F®, Eucare respectively) than those with NCORSHFS (65%
Pharmaceuticals, India). Fourteen patients (3 SV and 78%). Furthermore, patients in the NCES
and 11 NSV), stable for at least 3 months, were group were significantly more satisfied with the
treated; out of them, 9 patients achieved >75% outcome than those in the NCORSHFS group.
repigmentation. The mean percentage of repig-
mentation was 65.7 ± 36.7%. Similar to previous
studies, segmental vitiligo fares better than non- nnex A: Surgical Protocol for NCES
A
segmental vitiligo; and patients with at least a (Adopted and Modified from [27])
year of clinical stability have significantly better
repigmentation than those with less than a year 1. The first step is always the most important. It is
(78.6 ± 29.1% vs. 18.3 ± 16.1%, p = 0.02). crucial to harvest an ultrathin split skin graft.
These results were reproducible by other We usually choose the left lateral hip as the
investigators. Vinay et al. evaluated the repig- donor site, and the size is one-third to one-fifth
mentation rates of NCORSHFS transplantation the vitiligo area to be transplanted. The donor
in 30 patients with 60 lesions of stable vitiligo. site is cleansed with chlorhexidine 4% and out-
They also assessed the factors that determine the lined with skin marking ink, before performing
treatment outcome [45]. Twenty-one out of 60 an anaesthetic field block using 2% lignocaine.
(35%) lesions achieved a repigmentation rate of A silver dermatome is used to harvest a split
>75%, and out of them, 10 had >90% (or “excel- skin graft not beyond the papillary dermis,
lent”) repigmentation. Concurring with previous which can be gauged by the translucency of the
reports, segmental vitiligo fared the best in that graft (Fig. 36.3a). Too thick a graft will impede
33% of segmental vitiligo lesions achieved excel- trypsinization and can lead to scarring of the
lent repigmentation, compared to 25% of focal donor site. This is a skill to be acquired and a
vitiligo and only 4% of generalized vitiligo. The learning curve. It requires the help from an
investigators found that optimal (>75%) repig- assistant in stretching the skin taut (using, e.g. a
mentation was associated with a higher number sterilized wooden spatula), so that the surface is
of melanocytes (mean = 1187 vs. 796 cells/cm2) as flat as possible. Sterile liquid paraffin pro-
as well as a higher number of hair follicle stem vides good lubrication for the dermatome to
cells (mean = 756 vs. 494 cells/cm2) transplanted. glide on the donor skin surface.
The presence of dermal inflammation in the viti- 2. The split skin graft is placed in a sterile tray
liginous recipient site was associated with poor and washed in normal saline, before placing it
repigmentation, due to its deleterious effects on in a petri dish containing 10 ml of trypsin-
transplanted cells. EDTA 0.25%, pre-warmed to 37 °C
Although there are physiological advantages (Fig. 36.3b). The graft is then cut into smaller
of using ORS melanocytes over epidermal mela- pieces, and the petri dish is covered and placed
nocytes for vitiligo transplant, the repigmentation in an incubator, set at 37 °C, for 30 min
outcome is not superior to that in NCES (Fig. 36.3c).
ERRNVPHGLFRVRUJ
396 B. K. Goh
3. After incubation, the petri dish containing the not required in our experience. Patients can
donor tissue is placed in a laminar flow cham- expose the recipient sites to sunlight daily for
ber, wherein the extraction of epidermal cells 5–10 min.
takes place. The lid of the petri dish is lifted, 9. If the grafting is successful, signs of repig-
inverted and placed on the benchtop of the mentation are usually evident by 4 weeks in
chamber. The tissue pieces are removed from Indian, 8 weeks in Chinese (Fig. 36.4a, b) and
trypsin and placed on the inverted lid. Here, 12 weeks in Caucasian patients.
using fine forceps, the epidermis is peeled
away from the dermis and their surfaces
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Depigmenting Therapies
37
Abdulrahman Aljamal, Mohammed Aljamal,
Sanjeev Mulekar, and Aleissa Ahmed
Contents
37.2 Ideal Depigmenting Agent 400
37.3 Patient Selection 400
37.4 Agents for Depigmentation 401
37.4.1 Monobenzyl Ether of Hydroquinone (MBEH) 401
37.4.2 Monomethyl Ether of Hydroquinone/4-0 Methoxyphenol 404
37.4.3 88% Phenol Solution 405
37.4.4 Laser Therapy 405
37.4.5 Q-Switched Ruby (QSR) Laser (694 nm) 406
37.4.6 Q-Switched Alexandrite Laser (755 nm) 406
37.4.7 Cryotherapy 407
37.4.8 Imatinib 408
37.4.9 Imiquimod 408
37.4.10 Diphencyprone (DPCP) 408
References 409
Abstract
Key Points
Depigmentation may be suggested when other
possible treatment failed and when the lesions • Only patients with extensive vitiligo
are perceived as disfiguring. Chemical or should be treated, when other possible
physical approaches are available, depending therapies failed.
on the site and the extent of the lesions. • Incomplete or trichrome repigmentation
Hydroquinone and phenol derivatives are the may cause more disfigurement.
most appreciated chemical depigmenting • MBEH is the most potent and mainstay
agents, whereas lasers and cryotherapy may depigmenting agent, and it can induce
be relevant alternatives even if the last ones depigmetation at distant sites.
must be applied in hospital. • Repigmentation occurs in MBEH
because it often does not destroy follicu-
lar melanocytes.
A. Aljamal · M. Aljamal · S. Mulekar · A. Ahmed (*) • 4MP is as effective as MBEH, but the
The National Center of Vitiligo and Psoriasis, side effects like skin irritation are less
Riyadh, Kingdom of Saudi Arabia

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400 A. Aljamal et al.
Functional studies have demonstrated that HQ

common and less severe. It required lon- and other phenolic compounds, such as tert-
ger treatment. buthyl-phenol, may act even through different
• Lasers are fast, effective, and safe with mechanisms, including the oxidation of TRP1,
short follow-up durations. and by interfering with RNA and DNA synthe-
• Patches with positive Koebner respond sis. HQ has been identified as the main depig-
to cryosurgery. menting agent, whereas among the several
phenolic derivatives, the monobenzyl ether of
hydroquinone (MBEH) appeared as the more
handfuls one. In this chapter, we will review and
37.1 Introduction compared various established and potential
depigmentation agents and emerging therapies
The depigmenting approach is quite recent, that can be used in extensive and universal vit-
deriving from the observation of unwanted iligo (Figs. 37.1 and 37.2).
depigmenting action of the phenol derivatives
[1]. On the basis of this clinical observation, the
researchers aimed to define the possible mecha- 37.2 Ideal Depigmenting Agent
nisms of action of this class of compounds. The
first suggested target was the enzyme tyrosi- An ideal depigmenting agent should have a
nase, and the capability of different phenol potent, rapid, and selective effect on melano-
derivatives to act as alternative substrate of the cytes; it should lead to permanent depigmenta-
enzyme or as competitive inhibitor was evalu- tion and should be nontoxic with least side
ated. Consequently, it was hypothesized that effects [3]. Presently, there is no ideal agent for
this class of substances, or some of them, may depigmentation.
be used for the treatment of the skin disorders
due to hyperpigmentation or melanocyte hyper-
proliferation. Structural studies have indicated 37.3 Patient Selection
the role of the position and of the type of substi-
tutes in the phenolic ring to allow the compound The first step in depigmentation is to choose an
to be hydroxylated or oxidated by tyrosinase appropriate patient. Only patients with extensive
[2]. Hydroquinone (HQ) belongs to the phenol/ vitiligo should be treated, and this should be done
catechol class of chemical agents. HQ inhibits only after trying other possible therapies. The
tyrosinase through the interaction with the cop- patient should be informed that these therapies
per at the active site, as well as decreases the are a potent depigmenting agent and not used a
amount of intracellular glutathione and induces cosmetic purpose [1, 2]. The subjects with high
the production of oxygen-reactive species. HQ skin phototypes (V and VI), have a disfiguring -
acts as alternative substrate, according to most especially when exposed areas are involved (face
part of phenol/catechol compounds, because it or the hands), may be candidate to depigmenta-
is similar to tyrosine. The enzyme can thus oxi- tion. Moreover, incomplete or trichrome repig-
dize HQ without generating the pigment. In mentation (e.g., when using UV light) may cause
addition, the produced quinones are able to react more disfigurement. The patients should be
with the sulfhydryl residues of the proteins gen- informed that the repigmentation might occur in
erating oxidative damage and affecting the cell vitiligo lesions, causing further depigmenting
growth. The oxidative damage, involving both cycles. Patients must be informed that these treat-
lipids and proteins of the cellular membranes, ments lead to a definitive irreversible
may thus account for the depigmenting action. depigmentation.
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37 Depigmenting Therapies 401
a b
Fig. 37.1 Residual patches of pigmentation (a) Results after monobenzone therapy (courtesy prof. Falabella)
Table 37.1 Patients in whom depigmenting therapy

HO OH Hydroquinone should be considered
1. Vitiligo involving >50% of BSA
2. Patients with progressive disease and fail to
regimentation therapy
O OH Monobenzone 3. Vitiligo involving face and hands which affect
severally patient quality of life
4. Vitiligo on area resistant to treatments, e.g., hands,
malar area
OH 4-tert-Buthyl-phenol
37.4 Agents for Depigmentation
Fig. 37.2 Chemical structure of some hydroquinone-
related compounds with depigmenting activity 37.4.1 Monobenzyl Ether
of Hydroquinone (MBEH)
Depigmentation therapy should be avoided in
children less than 12 years of age [4]. Younger MBEH is a derivate of hydroquinone, also known
patients should be given the option of repigmenta- as monobenzone or p-(benzyloxy) phenol. It is
tion [5]. Table 37.1 shows the patients in whom the most potent and mainstay depigmenting
depigmenting therapy should be considered, and agent. It is the only drug USFDA approved for
Table 37.2 depicts the possible current approaches. depigmentation in vitiligo [3].
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402
Table 37.2 Summary of depigmenting agents

Mechanism of action Dose/concentration Outcome Side effect Remarks
MBEH After converting to quinone, it 10%, 20%, 30%, 40% 1 month Dermatitis, pruritus, xerosis, USFDA approved
induces melanocyte death Once to twice daily conjunctival melanosis, and distal Patient use it at home
depigmentation
Tretinoin with Inhibition of enzyme glutathione 0.025–0.1% 10 days Erythema, desquamation, dermatitis, With combination you can
MBEH S-transferase in melanocytes and distressing hyperpigmentation avoid high concentration of
MBEH
4-Methoxyphenol Like MBEH 20% cream 4–12 months Skin irritation but less than MBEH Take longer time than
MBEH
88% phenol Compromise melanocytic activity 88% Two sessions, Scar, dyschromia, high-dose is toxic Cheap, no complications in
solution 45 days apart experienced hands
Laser Selectively targeting melanocytes for Q-switched lasers like Might require Painful; requires local anesthesia Rapid result
destruction by photothermolysis 1064, 755, 694 and multiple sessions Costly
532 nm Only in clinic
Cryotherapy Intracellular ice formation leads to Liquid nitrogen for 4 weeks Permanent scarring, if performed Only small area treated
irreversible melanocyte damage 10–20 s aggressively Cheap
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Pain, edema, bullae On clinic
Imatinib Tyrosine kinase inhibitor 800 mg/day for 4 weeks Periorbitaledema, fluid retention,
6 months by the nausea, emesis, diarrhea and
systemic route myelosuppression
Imiquimod Stimulation of the innate immune 5% cream 3 months Burning, itching and pain at the Depigmentation not involve
response and cell-mediated adaptive target site untreated area
immunity
Diphencyprone Immunomodulatory action 0.0001% 10.5 months Local eczema with blistering,
regional lymphadenopathy and
contact urticaria
A. Aljamal et al.
37.4.1.1 Mechanism of Action ple, 5% on the neck, 10% on the face, and 20% on
There are many theories on how MBEH causes the arms and legs. There are some patients who
depigmentation. These include the following [6]: fail to lighten with 20% MBEH over a course of
3–4 months; in these patients, the concentration
1. Reacting with tyrosinase as the key enzyme of MBEH can be increased to 30% and then, if
during melanin synthesis forming quinone. there is no response over a similar time period, to
The quinone formed fastens with cysteine 40%. Concentrations of 30% and 40% MBEH
found in tyrosinase proteins (sulfhydryl (-SH) have been used primarily on the extremities, espe-
group) to form hapten-carrier compounds. cially the elbows and knees. Concentrations
This means that formation of neo-antigens in greater than this are not recommended [5].
the tyrosinase proteins enables a systemic, When the vitiligo has been stable for years, a
melanocyte destruction and an inflammatory longer duration of therapy and higher concentra-
reaction. tions of MBEH may be required [5].
2. MBEH produces reactive oxygen species
Gradually, depigmentation occurs over a
(ROS), for example, peroxide, by inducing period of 4–12 months [5]. Depigmentation is
cellular oxidative stress in the unprotected mostly irreversible and histologically associated
pigmented cells. This in turn leads to lyso- with loss of melanosomes and melanocytes [3].
somal degradation of melanosomes by
autophagy. Additionally, there is interference 37.4.1.3 Precautions
of the melanosome structure and membranes. Patients should always be informed about some
Henceforth, the major histocompatibility precautions while using MBEH:
complex (MHC) class I and II routes and ini-
tiation of melanocyte Ag-specific T-cell 1. Application of MBEH at one site can lead to
responses cause an increase in surface expres- loss of pigment at distant body sites, i.e.,
sion of melanosomal antigens. application of MBEH to the arm may result in
3. Generation of ROS contributes to immune loss of pigment on the face [4].
response resulting from the release of exo- 2. The pigment loss is permanent.
somes containing tyrosinase and MART-1 3. Avoid application over areas close to the
antigen. eye [5].
4. MBEH-exposed skin presents with rapid and 4. Avoid skin-to-skin contact with another per-
persistent innate immune activation. Moreover, son because it can cause a decrease in pig-
MBEH is a contact-sensitizer inducer of a type mentation at the site of contact in the other
IV delayed-type hypersensitivity response person.
against the quinone-hapten. However, this 5. Daily use of sunscreens with a sun protection
only occurs if there is production of pro- factor (SPF) of 30 or more throughout the year
inflammatory cytokines such as interleukin is essential to prevent repigmentation as well
(IL)-1b and IL-18 by the Langerhans cells or as sunburn reactions [4].
keratinocytes. 6. Follicular repigmentation can occur spontane-
ously or after sun exposure over the exposed
37.4.1.2 Treatment Procedure areas of face and forearms. It may vary from
The application of MBEH is usually done by the as soon as therapy is discontinued [7] to
patient at home. First, test spot is advised over a months and years [5]. Repigmentation occurs
normal pigmented skin on the forearm to assess in MBEH because it often does not destroy
the development of contact dermatitis. If there is follicular melanocytes. One theory is that
no contact dermatitis, the cream can be used on there is a lack of penetration of the MBEH to
the face or areas of top priority and then move in the level of the hair matrices, while a second
stages for low-priority areas. Different concentra- theory is that two populations of melanocytes
tions of MBEH can be used at one time, for exam- exist in the skin, the population more resistant
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to vitiligo as well as to depigmentation ther- 37.4.2.1 Mechanism of Action

apy residing in the hair follicle. Hair, eye- This is similar to that of MBEH. Through a dose-
brows, and eyelashes may be resistant to dependent response manner, melanocytes in the
depigmentation for the same reason [5]. hair follicles may also be affected by
4-
methoxyphenol (MP), but because of their
37.4.1.4 Side Effects deeper localization, these melanocytes are less
Irritant contact dermatitis, more than allergic, can susceptible to the compound compared with the
develop mainly in areas of pigmented rather than epidermal melanocytes [3].
vitiliginous skin [8]. If this happens, MBEH is
withheld, and open wet dressings are applied to 37.4.2.2 Treatment Procedure
the affected area along with topical steroids. When The compound can be used in 20% concentration
the dermatitis subsides, MBEH can be reconsti- in an oil/water cream base. Patients are instructed
tuted at a lower concentration of 5%. Frequency of to first apply the cream on a normal pigmented
application is also reduced and gradually advanced test spot (as big as 5 cm2) to observe whether an
as tolerated. Mixing MBEH with emollients at the allergic reaction would occur in the next 48 h.
time of application may also decrease irritant reac- Patients with a negative allergic reaction are
tions [5]. Other side effects include exogenous allowed to apply the cream on the remaining pig-
ochronosis [9], unmasking of telangiectasias and mented skin areas twice daily until complete
phlebectasias on the lower extremities [5], pruri- depigmentation is observed [12]. The effective-
tus, xerosis, erythema, rash, edema, conjunctival ness of 4MP has been correlated with the dura-
melanosis, and distant depigmentation [4]. Risk of tion of use of the cream; the longer the cream was
carcinogenesis cannot be ruled out, and hence it is used, the better the results [3].
banned from European Union since 2001 in cos- Combination product of 2% 4-hydroxyani-
metics [10]. sole (mequinol) and 0.01% tretinoin was tested
in a double-blind multicenter study and was
37.4.1.5 H ow to Increase the Efficacy found to significantly improve solar lentigenes
of Monobenzyl Ether and related hyperpigmented lesions of the face
of Hydroquinone? and hands after a twice-daily application of up
MBEH alone may need to be used occasionally to 24 weeks [3].
even beyond 1 year to maintain depigmentation.
MBEH therapy can be combined with all-trans 37.4.2.3 Side Effects
retinoic acid (ATRA) to enhance its depigment- Like MBEH, 4MP also produces side effects like
ing and melanocytotoxic effects via inhibition mild burning or itching, irregular leukoderma,
of enzyme glutathione S-transferase in melano- contact dermatitis, ochronosis, and risk of carci-
cytes. This way, contact dermatitis can be nogenesis cannot be ruled out [10]. Protection
avoided by using high concentrations of 40% from sunlight is necessary or repigmentation risk
MBEH. However, combination of ATRA- is high [3, 10].
MBEH did not affect hair pigmentation in ani-
mal studies [11]. 37.4.2.4 4-Methoxyphenol vs.
Monobenzyl Ether
of Hydroquinone
37.4.2 Monomethyl Ether 4MP is as effective as MBEH, but the side effects
of Hydroquinone/4-0 like skin irritation are less common and less
Methoxyphenol severe. Melanocytotoxic properties of 4MP are
comparable with those of MBEH. However,
This compound is a phenol derivative and is compared with MBEH cream, a disadvantage of
also known as p-hydroxyanisole (HA) or 4MP is the longer time required prior to the onset
mequinol [3]. of visible depigmentation (between 4 and
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12 months), whereas it was previously reported 37.4.3.3 Post-procedure Care

that depigmentation with MBEH may already be Delicate cleaning with saline and use of antibi-
evident after 1 month [3]. otic ointment with steroids of mild to moderate
potency and sun blocks should be done. Antiviral
use is indicated for patients with a history of her-
37.4.3 88% Phenol Solution pes simplex.
Phenol is inexpensive and has been used topi- 37.4.3.4 Side Effects
cally for chemical peelings. A non-occluded In experienced hands, 88% phenol solution does
88% phenol application in a small area (area not produce any complications. However, some-
<20% of face or neck area) does not demand times 88% phenol solution produces complica-
necessary care as with Baker-Gordon’s phenol tions such as non-esthetic scar formation,
formula (40–50% phenol). This is because 88% dyschromia, and development of herpetic eczema.
phenol, being more concentrated, rapidly coagu- High-dose phenol usage is toxic, so it should not
lates epidermis thus itself stopping its own fur- be applied over large areas. Its cellular uptake is
ther percutaneous penetration; on the other hand, both rapid and passive because of its lipophilic
Baker-Gordon’s formula, being more dilute, character and signs of systemic toxicity develop
does not coagulate epidermis, hence showing soon after exposure. Phenol’s main target organs
enhanced penetration and consequently systemic are the liver, kidney, respiratory, and cardiovascu-
absorption [13]. lar systems. Cardiovascular shock, cardiac arhyth-
mias, and bradycardia, as well as metabolic
37.4.3.1 Mechanism of Action acidosis, have been reported within 6 h of skin
All phenol compounds are toxic to melanocytes. peeling procedures with phenol. Repigmentation
Transient or definite hypopigmentation is a fea- may occur if patients do not protect themselves
ture of phenol cauterizing, and this is due to the properly from ultraviolet radiation [13].
development of a melanocytic incapacity to nor-
mally synthesize melanin, i.e., phenol does not 37.4.3.5 8 8% Phenol vs. Monobenzyl
cause melanocyte destruction; rather, it compro- Ether of Hydroquinone
mises its activity [13]. On the other hand, other With phenol, the side effects are lesser. It can be
depigmenting agents, such as hydroquinone and used in areas where MBEH is not available. It is
MBEH, destroy melanocytes. a cheap, practical product, with no complications
Protein coagulation is observed in the epider- in experienced hands [13].
mis immediately after application of 88% phenol
solution. If phenol is re-applied, depth will be
greater, with the capacity of reaching reticular 37.4.4 Laser Therapy
dermis [3].
Lately, laser therapies have been recommended
37.4.3.2 Treatment Procedure for vitiligo depigmentation due to their fast,
First the skin is cleaned with gauzes soaked with effective, and safe nature with short follow-up
alcohol. The use of a swab moistened with phe- durations.
nol is used to treat small areas, till cutaneous They have been reported to be more effective
frosting occurs. The patient feels a burning sensa- for positive Koebner phenomenon vitiligo
tion for approximately 60 s, which gradually patients [14].
decreases in intensity, and it can last from min- Additionally, lasers are advocated for MBEH
utes to hours. After two sessions, 45 days apart, and other bleaching agent failure cases more so
total elimination of residual pigmented areas is on areas such as the face where short-term rapid
noticed. No signs of repigmentation have been depigmentation is required. Moreover, to over-
seen till after 1½ year of therapy [13]. come side effects of topical therapies, e.g., itch-
ERRNVPHGLFRVRUJ
ing, redness, burning, high failure rates with only gauze and patients advised to avoid sunlight
partial depigmentation/possible repigmentation, exposure for a period of 6 weeks. For larger
and long treatment follow-up, laser has been doc- affected areas, multiple lasers are performed at
umented to overcome these disadvantages. 2–4-week intervals to achieve total depigmenta-
Additionally risks of scar formation are mini- tion [12].
mized with laser therapies [12].
37.4.5.3 T anning of Skin Prior
to Using Q-Switched Ruby
37.4.5 Q-Switched Ruby (QSR) Laser Laser
(694 nm) Selective photothermolysis targets activated
melanocytes that are triggered by tanning of the
Njoo et al. reported a successful vitiligo depig- skin. Henceforth, performing QSR laser after
mentation QSR laser [12]. They noted that for tanning can permanently destroy activated mela-
extremities big confluent pigment areas, topical nocytes. Kim et al. [7] reported no repigmenta-
therapy can be used first with combined therapy tion after 1 year follow-up where QSR was
giving better results but with risks of rapid depig- performed after tanning.
mentation as early as 1 or 2 weeks after therapy.
37.4.5.1 Mechanism of Action 37.4.6 Q-Switched Alexandrite Laser

Laser therapies have been demonstrated to be (755 nm)
very effective in identifying the melanocytes that
cause destruction and therefore resulting to In a case study by Rao and Fitzpatrick [15], a
depigmentation. QSR lasers in particular have 68-year-old woman had 18 sessions of QSR laser
wavelengths between 600 and 800 nm, which are and 20% MBEH application in 5 years with a
known to prompt selective photothermolysis for residual pigmentation clearance failure. It was
pigmented lesions due to their faster absorption reported that there was noted repigmentation
by melanin. Moreover, QSR laser duration of within 3 months of QSR laser session. Henceforth,
pulse energy is known to be shorter than melano- ten sessions (mean per session, 3.4–6.0 J/cm2,
somes relaxation time thereby releasing no 3–4 mm, 484–1636 pulses) of Q-switched alex-
energy to the surrounding tissues [3]. andrite (QSA) laser (755 nm, 50–100 ns) were
performed to the resistant pigmented patches
37.4.5.2 Treatment Procedure with discontinuation of topical MBEH therapy. A
QSR laser releases energy pulses of 694 nm in notable clearance of all treated sites was noted
wavelength, within 25–28 ns in 1–1.2 Hz fre- within 22 months and minimal repigmentation at
quency [12]. Depending on the skin type, a 5 mm 12 months follow-up.
to the pigmented spot can be administered pulse QSA 755 nm has more advantages compared
energy of 0–10 J/cm2, which can be varied to to the 694 nm QSR due to its faster frequency
10–40 J/cm2 accordingly [14]. Initially, a 5 cm2 that helps in rapid therapy and improved results
test spot is treated and clinical depigmentation from its higher tissue penetration [15].
evaluated after 8 weeks. Where depigmentation However, there are other potential Q-switched
is noted, more laser treatment is done until total lasers that can selectively destruct melanocytes
depigmentation is achieved. In cases of no depig- which include neodymium-yttrium aluminum
mentation, laser treatment is stopped. For pain garnet (Nd:YAG) laser (1064 nm) and the
management, laser procedure is done under frequency-doubled Nd:YAG laser (532 nm) [3].
eutectic mixture of 25 mg/g lidocaine (EMLA)
and prilocaine each. 37.4.6.1 Side Effects
However, a maximum of 80 cm2 is allowed per The main disadvantage is that the procedure is
session with the area treated covered with sterile painful requiring local anesthesia. Additionally,
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this treatment is expensive as it can only be done in uneven areas of the nose, cyoprobes with smaller
a clinic setting. There is also possible failure in diameters may be required. No more than one
removing pigmented patches even after several freeze-thaw cycle is advised. However, another
treatment months because follicular repigmenta- study has used two freeze-thaw cycles also [16].
tion due to movement of perifollicular melanocytes After a week, a depigmented, unscarred,
to the epidermis showing that they were not com- slightly atrophic, and erythematous smooth area
pletely destroyed by laser treatment. This condition appears. The best cosmetic result is obtained
is known as Koebner phenomenon. Patients with 4 weeks after cryotherapy. The depigmentation is
active vitiligo respond better to laser treatments permanent, although more than one session may
compared to those with stable vitiligo. Henceforth, be required for partially depigmented lesions,
patients who are Koebner negative relapse [12]. with 4–6 weeks intervals, before complete depig-
mentation occurs [10].
In those with more extensive pigmented
37.4.7 Cryotherapy patches, cryotherapy can be performed in several
sessions 1–3 weeks apart in order to prevent dis-
When rapid depigmentation is desirable, physical comfort to the patient. Spot cryotherapy can be
agents like cryotherapy and lasers work faster used for areas which repigment.
than bleaching agents.
37.4.7.3 Advantage
37.4.7.1 Mechanism of Action Cryotherapy has been suggested to depigment
Vitiligo patches with positive Koebner responds MBEH-resistant skin. The procedure requires no
well to cryosurgery. Intracellular ice formation anesthesia and can be performed in an outpatient
leads to irreversible tissue damage. In this aspect, department. No dressing, sedatives, or antibiotics
melanocytes are more sensitive to cryotherapy are required. Preparation time is short and inex-
damage in comparison with other epidermal pensive. The risk of infection is low and wound
cells. The degree of damage depends on the rate care is minimal. This method is simple, easy to
of cooling and minimum temperature achieved. perform, safe, efficacious, and cost effective.
Inflammation develops within 24 h of treatment, Depigmentation developed by cryotherapy is per-
further contributing to destruction of lesion manent and without scarring if performed by
through immunologically mediated mechanisms. experienced dermatologists. Many patients pre-
Mild freezing leads to a dermoepidermal separa- fer a single short-term procedure than applying
tion, which is useful in treating epidermal lesions. an expensive compound for 10 months or more
with unpredictable effects and a considerable
37.4.7.2 Treatment Procedure failure rate.
Spot testing by a single freeze-thaw cycle is done,
and when the edema and erythema subside, the 37.4.7.4 Disadvantage
patches are treated with cryotherapy 3–6 weeks A qualified experienced person is required for
later. Both CO2 and liquid N2 can be used. A 2-cm this, and hence treatment is hospital based. Also,
flat-topped and round cryoprobe is held approx cryotherapy is suitable for small lesions, and a
40 mm from the skin surface. The whole patch single sitting cannot be utilized for depigmenting
can be frozen with a single freeze-thaw cycle extensive areas unlike lasers.
from the periphery and then by forming succes-
sive rows inward. Procedure should be termi- 37.4.7.5 Side Effects
nated when a narrow (<1 mm) frost rim forms Immediate side effects include edema, pain, and
around the periphery of the cryoprobe. The rim bulla formation. If cryotherapy is performed
can develop within 10–20 s by a cryogun con- aggressively, it can lead to permanent scarring.
nected to a container with barometric pressure Cryotherapy should be used by experienced
above 80 kg/cm2. For lesions around the orbits or person.
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37.4.8 Imatinib prominent in the pathogenesis of vitiligo [3].

Imiquimod also stimulates CD8 cells to become
Skin depigmentation (whitening/generalized cytotoxic and enhances antigen presentation [19].
hypopigmentation) from imatinib first reported in Recently, it was reported that human melanocytes
patients of chronic myeloid leukemia [17]. Skin express toll-like receptor 7 (TLR7). When applied
became darker during the discontinuation period topically, imiquimod binds to TLR7 followed by
and began lightening again once imatinib mesyl- stimulation of various cytokines, which induce the
ate treatment was resumed. abovementioned T lymphocytic response [20].
Imiquimod also has a direct action on melanocytes
37.4.8.1 Mechanism of Action via apoptosis of melanocytes. This action is related
It is postulated that imatinib mesylate, being a to reduction of expression of Bcl-2 and/or an
tyrosine kinase inhibitor, may interfere with the increase in the proapoptotic stimulus (cytotoxic T
production of melanin, resulting in decreased lymphocytes, natural cytotoxic T cells/killer cells,
pigmentation of the skin. After patients begin granzymes B, Fas, TNF, Bax, etc.) [21].
receiving imatinib mesylate, hypopigmentation Therefore, it is possible that imiquimod may
can be observed within 12 weeks. It is difficult to cause elimination of melanocytes by direct influ-
define the onset exactly, because the change is ence on cells as well as inducing acquired immu-
gradual. An ethnic and/or genetic basis has also nity indirectly, thus inducing vitiligo-like
been considered [17]. hypopigmented lesions.
37.4.8.2 Side Effects 37.4.9.2 Method of Application

The side effects of imatinib mesylate are perior- Five percent imiquimod use may be followed by
bital edema, fluid retention, weight gain, muscu- erythema which gradually turns to depigmented
loskeletal pain, headache, nausea, diarrhea, and patches over a period of 3 months. No repig-
myelosuppression. In addition, a number of der- mentation has been seen till 6 months after the
matological side effects have been documented, depigmentation. Also, depigmentation did not
such as follicular mucinosis, erythroderma, and extend to areas that had not been treated with
lichenoid eruption. Imatinib mesylate can also imiquimod [22]. The depigmenting effects of
induce local or generalized hyperpigmentation. imatinib and imiquimod have only been reported
Severe congestive cardiac failure is an uncom- in a few studies, and randomized control trials
mon but recognized side effect of imatinib. are lacking. Hence, further studies are required
Imatinib in children can delay normal growth, on these agents and other similar molecules
although a proportion will experience catch-up before they can be used as mainstream depig-
growth during puberty [3]. menting agents.
37.4.9.3 Side Effects

37.4.9 Imiquimod The most common side effects of imiquimod
are burning, itching, pain, erythema, erosions,
Imiquimod is a novel imidazoquinoline immune and scabbing/crusting at the target site which
response modifier, frequently used for topical occur more frequently with twice-daily appli-
treatment of anogenital warts and basal cell carci- cation [3].
nomas [18].
37.4.9.1 Mechanism of Action 37.4.10 Diphencyprone (DPCP)

Imiquimod increases production of pro-
inflammatory cytokines, mainly interferon (IFN)-α, Topical application of DPCP when used for the
tumor necrosis factor (TNF)-α, and IL-6, IL-8, treatment of alopecia areata was found to pro-
IL-10, and IL-12, all of which augment the type 1 duce depigmentation as part of its side effects.
helper T-cell (TH1) response which is found to be Duhra and Foulds [23] reported a case of
ERRNVPHGLFRVRUJ
alopecia totalis in whom sensitization therapy 9. Charlin R, Barcaui CB, Kac BK, Soares DB, Rabello
Fonseca R, Azulay-Abulafia L. Hydroquinone
with topical DPCP was commenced. There was induced exogenous ochronosis: a report of four
marked reaction with erythema and edema on cases and usefulness of dermoscopy. Int J Dermatol.
the forearm after 3 days, but the scalp mani- 2008;47:19–23.
fested only slight macular erythema. The fore- 10. Radmanesh M. Depigmentation of the normally

pigmented patches in universal vitiligo patients
arm reaction subsided after 2 weeks and was by cryotherapy. J Eur Acad Dermatol Venereol.
replaced 6 weeks later by a depigmented patch. 2000;14:149–52.
Similar depigmented areas appeared on the nape 11. Kasraee B, Fallahi MR, Ardekani GS, Doroudchi G,
of the neck and the midline of the back. These Omrani GR, Handjani S, et al. Retinoic acid synergis-
tically enhances the melanocytotoxic and depigment-
remained unchanged for 2 years after discon- ing effects of MBEH in black guinea pigs skin. Exp
tinuing DPCP therapy. Electron microscopy and Dermatol. 2006;15:509–14.
incubation with dopa in affected skin revealed 12. Njoo MD, Vodegel RM, Westerhof W. Depigmentation
an absence of melanosomes and melanocytes. therapy in vitiligo universalis with topical 4methoxy-
phenol and the Q-switched ruby laser. J Am Acad
DPCP-induced vitiligo is rare and may repre- Dermatol. 2000;42:760–9.
sent a Koebner phenomenon in predisposed indi- 13. Zanini M. Depigmentation therapy for generalized
viduals. Vitiligo can develop even with DPCP vitiligo with topical 88% phenol solution. An Bras
concentrations as low as 0.0001% [23]. Dermatol. 2005;80:415–6.
14. Thissen M, Westerhof W. Laser treatment for fur-
ther depigmentation in vitiligo. Int J Dermatol.
37.4.10.1 Side Effects 1997;36:3868.
Adverse effects include local eczema with blis- 15. Rao J, Fitzpatrick RE. Use of the Q-switched 755 nm
tering, regional lymphadenopathy, hyperpigmen- alexandrite laser to treat recalcitrant pigment after
depigmentation therapy for vitiligo. Dermatol Surg.
tation, hypopigmentation, and vitiligo [18]. 2004;30:1043–5.
16. Di Nuzzo S, Masotti A. Depigmentation ther-

apy in vitiligo universalis with cryotherapy
References and 4- hydroxyanisole. Clin Exp Dermatol.
2010;35:215–6.
1. Nordlund JJ. Depigmentation for the treatment of 17. Leong KW, Lee TC, Goh AS. Imatinib mesyl-

extensive vitiligo. In: Hann SK, Nordlund JJ, editors. ate causes hypopigmentation in the skin. Cancer.
Vitiligo. Lucon: Blackwell Science; 2000. p. 207–13. 2004;100:2486–7.
2. Solano F, Briganti S, Picardo M, et al. Hypopigmenting 18. Halder RM, Taliaferro SJ. Vitiligo. In: Wolff K,

agents: an updated review on biological, chemical and Goldsmith LA, Katz SI, Gilchrest BA, Paller AS,
clinical aspects. Pigment Cell Res. 2006;19:550–71. Lefell DJ, editors. Fitzpatrick’s dermatology in gen-
3. Alghamdi KM, Kumar A. Depigmentation therapies eral medicine, vol. 1. 7th ed. New York: McGraw Hill;
for normal skin in vitiligo universalis. J Eur Acad 2008. p. 61622.
Dermatol Venereol. 2011;25:749–57. 19. Zirvi TB, Costarelis G, Gelfand JM. Vitiligo-like

4. Drake LA, Dinehart SM, Farmer ER, Goltz RW, hypopigmentation associated with imiquimod
Graham GF, Hordinsky MK, et al. Guidelines of care treatment of genital warts. J Am Acad Dermatol.
for vitiligo. J Am Acad Dermatol. 1996;35:620–6. 2005;52:715–6.
5. Bolognia JL, Lapia BK, Somma S. Depigmentation 20. Kang HY, Park TJ, Jin SH. Imiquimod, a toll-like
therapy. Dermatol Ther. 2001;14:29–34. receptor 7 agonist, inhibits melanogenesis and pro-
6. Van doon Boorn JG, Melief CJ, Luiten liferation of human melanocytes. J Invest Dermatol.
RM. Monobenzone induced depigmentation: from 2009;129:243–6.
enzymatic blockade to autoimmunity. Pigment Cell 21. Kim CH, Ahn JH, Kang SU, Hwang HS, Lee MH,
Melanoma Res. 2011;24:673–9. Pyun JH, et al. Imiquimod induces apoptosis of human
7. Kim YJ, Chung BS, Choi KC. Depigmentation ther- melanocytes. Arch Dermatol Res. 2010;302:301–6.
apy with Q-switched ruby laser after tanning in vit- 22. Senel E, Seckin D. Imiquimod-induced vitiligo-like
iligo universalis. Dermatol Surg. 2001;27:969–70. depigmentation. Indian J Dermatol Venereol Leprol.
8. Lyon CC, Beck MH. Contact hypersensitivity to 2007;73:422–3.
monobenzyl ether of hydroquinone used to treat vit- 23. Duhra P, Foulds IS. Persistent vitiligo induced by
iligo. Contact Dermatitis. 1998;39:132–56. diphencyprone. Br J Dermatol. 1990;123:415–6.
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Combined/Sequential/Integrated
Therapies for Vitiligo
38
Thierry Passeron
Contents
38.1 Combination of Surgical Therapies and Phototherapy 412
38.2 Combination of Surgical Therapies and Corticosteroids 412
38.3 Combination of Phototherapy and Topical Steroids 412
38.4 Combination of Phototherapy and Topical Calcineurin
Inhibitors (TCI) 413
38.5 Combination of Phototherapy and Topical Vitamin D 414
38.6 Combination of Phototherapy and Antioxidants 414
38.7 ombination Therapies Using Dermabrasion or Fractional
C
Ablative Lasers 415
38.8 Maintenance Therapy 416
38.9 Conclusions 417
References 417
Abstract we have many strategies to repigment vitiligo

Vitiligo is a challenging disorder to treat, and patches. Association of surgical procedures
to date no treatment provides truly satisfactory with phototherapy was the first to demonstrate
results. However, from molecules that target that combination approaches could further
the immune system to therapies that stimulate increase repigmentation rates. The interest of
the proliferation and differentiation of mela- combining medical approaches is now also
nocytes and/or melanocyte progenitors and well demonstrated in several prospective ran-
others that fight against radical species or domized studies. This is mainly true for the
bring new melanocytes to the affected areas, association of topical treatments, such as cal-
cineurin inhibitor or topical corticosteroids,
T. Passeron (*) with phototherapy. Other interesting associa-
University Côte d’Azur, Department of Dermatology, tions are those between antioxidants and
University Hospital of Nice, lasers, but the data remain limited. New strate-
Nice Cedex 3, France
gies have been also reported aiming to reduce
University Côte d’Azur, INSERM U1065, the risk of relapse after achieving repigmenta-
team 12, C3M, Nice, France
e-mail: passeron@unice.fr tion. The development in the near future of

ERRNVPHGLFRVRUJ
412 T. Passeron
targeted immune treatments but also new topi- narrowband UVB (NB-UVB) or PUVA were
cal drugs aiming to enhance the differentiation clearly superior to phototherapy alone for repig-
of melanocyte stem cells should lead to new menting vitiligo [11].
and hopefully more effective combination or Combination of NB-UVB with surgical thera-
sequential protocols adapted to the severity pies has been less studied but also enhances
and the activity of each vitiligo. repigmentation [11–13]. One prospective ran-
domized intra-individual study performed on a
limited number of patients (n = 14) found no dif-
Key Points ference between NB-UVB and excimer laser
• Combination approaches were first after punch grafting [14]. To the best of our
reported associating surgical procedures knowledge, there is no direct comparison between
and phototherapy. NB-UVB and PUVA as a synergic agent for sur-
• Increasing data have emphasized the gical therapies available in the literature. A series
interest of combining also medical of five patients who failed to repigment after the
approaches for enhancing repigmenta- combination of grafting plus PUVA has been
tion rates. reported to finally repigment when the surgical
• Recent data also reported effective strat- procedure was combined with NB-UVB [15].
egy for decreasing the risk of relapses Although interesting, this report is not sufficient
after achieving effective repigmentation. to prove the superiority of NB-UVB in this indi-
cation, and further studies are required.
38.1 Combination of Surgical 38.2 Combination of Surgical

Therapies and Phototherapy Therapies
and Corticosteroids
Surgical therapies of vitiligo such as punch or
blister or split thickness grafting or more recently Although most combined approaches using sur-
transplanted autologous melanocyte or epider- gical therapies have been performed with photo-
mal cell suspensions have been the first thera- therapy, topical and systemic steroids have also
peutic options to be associated with phototherapy. been evaluated. In a prospective study performed
In 1983, Bonafé et al., first reported the com- on 50 vitiligo patients, the combination of punch
bined use of skin grafts followed with psoralen grafting with topical steroids (fluocinolone ace-
plus ultraviolet A (PUVA) therapy [1]. The tonide 0.1%) has been shown to be as effective as
combination of several surgical therapies with punch grafting followed by PUVA [2].
PUVA has been then further studied and remains An open series and a case report have sug-
today the best studied combination in vitiligo gested that low doses of oral steroids might be
treatment. Indeed, several studies have shown beneficial in addition to surgical procedures [16,
that adjunction of PUVA following surgical 17]. This has to be confirmed in a controlled trial.
treatment enhances the repigmentation rate of
vitiligo patches [2–10]. Most of authors add
phototherapy several weeks after the surgical
38.3 Combination
procedures (usually 3 or 4 weeks after) and use of Phototherapy and Topical
standard phototherapy protocols for vitiligo Steroids
treatment. The interest of combining photother-
apy and surgical procedures has been proven in The combined use of UVA and topical steroids
a prospective, randomized, double-blind has been studied by the Westerhof group. In a pro-
study that clearly showed that autologous trans- spective, randomized, controlled, left–right com-
planted epidermal cell suspensions followed by parison study, it was shown that the combination
ERRNVPHGLFRVRUJ
38 Combined/Sequential/Integrated Therapies for Vitiligo 413
a b
Fig. 38.1 (a) Vitiligo patches of the face. (b) Clinical aspect after 12 weeks of twice-weekly 308 nm excimer laser
combined with daily application of desonide 0.05% cream
of UVA and fluticasone propionate was much was applied twice a day. The results were similar
more effective than UVA or topical steroid alone and showed a greater efficiency with the com-
[18]. Lately, topical hydrocortisone 17-butyrate bined treatment as compared to laser monother-
cream combined to 308 nm excimer laser was apy. Tolerance was good, and side effects were
found in a prospective randomized trial to be limited to constant erythema, sticking due to
more effective in the treatment of vitiligo of the ointment and rare bullous lesions. These results
face and neck compared to excimer laser alone have been confirmed by many others, showing
[19]. However, studies having assessed the asso- that tacrolimus and pimecrolimus used topically
ciation of topical steroids to NB-UVB or achieve greater results when combined to excimer
excimer laser/light remain limited preventing to laser/light but also to NB-UVB [30–34]. Studies
draw definitive conclusions as emphasized performed in the pediatric population also under-
recently by meta-analyses of the literature line the efficacy of the combination of topical
(Fig. 38.1) [20, 21]. tacrolimus or pimecrolimus with NB-UVB or
excimer laser in children suffering from vitiligo
[35, 36]. Recent meta-analysis of the literature on
38.4 Combination the combination of NB-UVB or excimer laser/
of Phototherapy and Topical light confirmed the superiority of combining TCI
Calcineurin Inhibitors (TCI) to these phototherapies compared to photother-
apy alone [20, 21, 37].
TCI such as tacrolimus and pimecrolimus have The potential increased risk of skin cancers
demonstrated their effectiveness for treating vit- promoted by the association of two immunosup-
iligo, but the best results are achieved in sun- pressive treatments remains to be taken in con-
exposed areas and especially on the face [22–26]. sideration. However, reassuring data on the use
Treatment with such drugs in monotherapy for of TCI have been reported [38, 39]. Moreover,
other areas provides disappointing results [27]. penetration of high quantities of calcineurin
Two studies first evaluated if the combination of inhibitors can mostly be observed when used
308 nm excimer laser and topical tacrolimus over large surfaces in atopic dermatitis patients
could be synergistic. They have compared the where the skin barrier is altered, which is not the
efficiency of 308 nm excimer combined with case for vitiligo skin. TCI have been used for vit-
tacrolimus ointment to 308 nm excimer laser iligo alone or combined with phototherapy for
monotherapy [28] or associated with placebo more than 10 years without any indication of risk.
ointment [29]. In both cases, a total of 24 ses- Taken together these data are very reassuring
sions were evaluated, and tacrolimus ointment concerning the use of TCI combined with UV
ERRNVPHGLFRVRUJ
414 T. Passeron
a b
Fig. 38.2 (a) Vitiligo of the leg and knee before treatment. (b) Clinical aspect after 12 weeks of twice-weekly 308 nm
excimer laser combined with twice-daily application of 0.1% tacrolimus ointment
exposures in vitiligo patients; however, a total meta-analysis of the literature couldn’t find suf-
follow-up of 20–25 years may be required to be ficient evidence to support the interest of combin-
completely reassured concerning a potential ing phototherapy to vitamin D analogues [20,
increased risk of skin cancers. Thus, the risk– 21]. Thus, accordingly to the existing data, com-
benefit ratio needs to be discussed with patients bination approaches with phototherapy should
when TCI are proposed in combination to photo- clearly prefer the use of topical steroids or TCI.
therapy or sun exposures (Fig. 38.2).
38.6 Combination
38.5 Combination of Phototherapy
of Phototherapy and Topical and Antioxidants
Vitamin D
Oxidative stress has been shown to be involved in
The occurrence of repigmentation of vitiligo in the pathogenesis of vitiligo. Pseudocatalase has
patients treated with calcipotriol (a vitamin D3 the ability to remove hydrogen peroxide and so
analogue) for psoriasis has suggested that it could be interesting in the treatment of vitiligo.
might be efficacious in treating vitiligo. It appears The combination of topical pseudocatalase with
now quite clear that calcipotriol in monotherapy UVB has shown very promising results in a pilot
is useless for treating vitiligo [40]. The use of non-RCT study (complete repigmentation on the
topical vitamin D with sun, PUVA, or NB-UVB face and the dorsum of the hands in 90% of
provided controversial data [41–53]. Recent patients) [54]. Unfortunately, these results were
ERRNVPHGLFRVRUJ
not confirmed in another study [55]. Finally, a response to therapy was better using the com-
prospective randomized placebo-controlled study bination therapy with 50 lesions (78%) achiev-
showed that pseudocatalase cream does not add ing a moderately or marked repigmentation
any incremental benefit to NB-UVB alone [56]. compared to 14 lesions (22%) of similar effec-
Oral antioxidant supplementation was also tiveness in the monotherapy group (p < 0.0001).
reported to increase the effectiveness of UVB Quite surprisingly, only moderate pain during
phototherapy in a prospective double-blind ablation or at sites of 5-FU application was
placebo- controlled study [57]. Although this reported in all cases. As 5-FU, dermabrasion
study concerned a relative small number of and UVB were combined in this study, the
patients with only 80% having completed the respective role of the 5-FU and the dermabra-
entire treatment, the results are certainly encour- sion in potentiating the UVB is difficult to
aging. This potential interest of oral antioxidants determine. The impact of the laser-assisted
was also supported by a prospective double- dermabrasion on the repigmentation of vitiligo
blind trial that studied the combination of was assessed in a prospective randomized
Polypodium leucotomos extract with NB-UVB study performed on symmetrical patches of
[58]. Better repigmentation was observed with vitiligo located only in difficult-to-treat areas
combination of Polypodium leucotomos extract (such as bony prominences and extremities)
and UV, but the difference was slight and only [60]. Twenty patients were treated with a com-
observed in a subgroup of patients. Of note, no bination of topical steroids and NB-UVB for
study has been published since 2007 using 3 months. On the side that was randomly
Polypodium leucotomos extract in combination assigned, a pretreatment with an erbium laser-
to phototherapy. Thus, despite the demonstrated assisted dermabrasion was performed. The cri-
role of the oxidative stress in the pathophysiol- teria of success were defined as a repigmentation
ogy of vitiligo, further studies are mandatory to of at least 50% 1 month after the end of the
confirm the interest of oral antioxidants com- treatment. Two patients were lost to follow-up,
bined with phototherapy. and a total of 24 symmetrical lesions were
treated. The combination with a preceding
dermabrasion provided significant better
38.7 Combination Therapies results with a rate of success of 46% compared
Using Dermabrasion or to 8% in the group with only topical steroids
Fractional Ablative Lasers and UVB (p < 0.0001). However, side effects
were important with high pain, delayed healing
Skin ablation with mechanical superficial and two hypertrophic scars in the group using
dermabrasion combined with topical applica- dermabrasion. These side effects strongly limit
tion of 5% 5-fluorouracil (5-FU) was intro- the usefulness of this combination approach in
duced in 1983. The supposed mechanism of daily practice, but the results clearly show the
action is mainly based on the combination of beneficial role of a preceding dermabrasion.
epidermal removal and induction of irritation The mechanisms involved in this enhancing
mediated by 5-FU, which through the produc- effect remain to be elucidated.
tion of inflammatory cytokines and prostaglan- A similar approach but using fractional abla-
dins stimulates melanocyte migration and tive CO2 laser was then conducted in a prospec-
proliferation. In a prospective trial, 50 adult tive randomized trial [61]. Ten patients with
subjects with 64 symmetrical lesions of non- symmetrical vitiligo patches were treated with
segmental vitiligo were enrolled [59]. One side NB-UVB with one side also receiving two ses-
was treated with ER:YAG laser ablation, fol- sions of CO2 fractional ablative laser at
lowed by 5-FU application before simultane- 2 months interval. Two months after the end of
ous NB-UVB therapy of both sides for a the treatment, the combination approach was
maximum period of 4 months. The overall more effective with three patches achieving
ERRNVPHGLFRVRUJ
416 T. Passeron
moderated or marked improvement compared

to none with UVB alone (p = 0.034). Although
the enhancing repigmentation rate appears to be
much lower with fractional CO2 laser compared
to laser-assisted dermabrasion, the tolerance
was much better without significant side effects
using the fractional ablative laser. These results
were corroborated in a small prospective trial
showing that fractional CO2 laser and sun pro-
vide better results compared to sun exposure
alone [62]. More recently a prospective ran-
domized intra-individual study was conducted Fig. 38.3 Vitiligo lesion of the back under Wood’s lamp
on 27 patients with 27 pair-lesions of non-seg- examination after 12 weeks of NB-UVB and daily appli-
mental vitiligo located on hands [63]. All cation of topical steroids. Left side has received a pretreat-
lesions received NB-UVB phototherapy and ment with an erbium laser-assisted dermabrasion
compared to right side that did not received any
0.05% clobetasol propionate cream, and one pretreatment
side was randomly assigned to receive also
fractional CO2 laser (ten sessions at 1-week
interval). One patient was lost to follow-up, and
six vitiliginous lesions (23.1%) in combination 38.8 Maintenance Therapy
group achieved good to excellent repigmenta-
tion compared with one lesion (3.9%) in group After successful repigmentation, the rate of
with topical steroid and NB-UVB (p = 0.065). relapse in vitiligo patches is about 40% [67]. In
Interestingly, a prospective intra- individual atopic dermatitis proactive treatment with topical
study performed on extremities and/or bony steroids or calcineurin inhibitors has demon-
prominences of 25 vitiligo patients showed that strated their efficacy to decrease flares of the dis-
topical steroids associated to fractional CO2 ease [68]. In a two-centre, prospective randomized
laser and NB-UVB is more effective than frac- study, the use of bi-weekly application of 0.1%
tional CO2 and phototherapy without steroids tacrolimus ointment was compared to placebo
[64]. These studies support the interest of com- [69]. Thirty-five patients with 72 non-segmental
bining the three approaches (laser, photother- vitiligo lesions who achieved at least 75% of
apy and topical treatment) to achieve optimal repigmentation after phototherapy, topical treat-
results. However, studies performed on larger ment or a combination approach were included.
population are still required to confirm the After 6 months 40% of lesions showed depig-
effectiveness of fractional ablative lasers in mentation in the placebo group compared to
potentiating repigmentation in vitiligo and to 9.7% with the tacrolimus (p = 0.0075). The toler-
determine the optimal modalities of the ance was good, and the side effects were limited
sessions. to transient erythema and stinging or burning
Interestingly, the holes made by these frac- sensations. This study shows that twice-weekly
tional ablative lasers but also by microneedles applications of 0.1% of tacrolimus are effective
have been shown, using an ex vivo model, to suc- for decreasing vitiligo relapses.
cessfully deliver melanocytes into the skin [65]. Accordingly to the data available in atopic
A pilot study recently suggested that ablative dermatitis and the comparable efficacy of topical
fractional lasers could be used to prepare the bed steroids and TCI for treating vitiligo, it may be
grafting before using epidermal suspension sug- hypothesized that topical steroids could also be
gesting that such laser procedure could be also of effective for preventing vitiligo relapse. Many
interest for surgical treatments of vitiligo questions remain. How long should this preven-
(Fig. 38.3) [66]. tive treatment be continued? Are applications
ERRNVPHGLFRVRUJ
three times a week more effective than only two to choose the more suitable treatments that could
and thus could further reduce the risk of relapse? be used in combination or sequentially depend-
We actually proposed this maintenance treatment ing on each individual case.
only in patients with active vitiligo or patients
who already had relapses after having achieved Take-Home Pearls
repigmentation, and we continue this proactive • Combination approaches have now clearly
approach for at least 6 months without any sign showed their interest in treating vitiligo. The
of disease activity. However, further studies are usefulness of associating treatments is dem-
clearly required to answer to these questions. onstrated for surgical and medical approaches.
• Best evidences are reported with phototherapy
(PUVA, NB-UVB, excimer laser/light) asso-
38.9 Conclusions ciated with surgical procedures or with topical
calcineurin inhibitors.
There are three aims that need to be reached for • The use of 0.1% tacrolimus ointment twice
the optimal care of vitiligo patients. First halting weekly is the first approach demonstrated to
the disease progression, then allowing complete reduce the risk of relapse after achieving
repigmentation of lesional areas and finally pre- repigmentation of vitiligo lesions.
venting relapses. Combination approaches have • Recent advances in the understanding of vit-
now clearly showed their interest in treating vitiligo pathophysiology lead to great hope using
iligo. The usefulness of associating treatments is new therapeutic strategies for halting disease
demonstrated for surgical and medical progression or repigmenting vitiligo lesions
approaches. Due to the difficulties for achieving that could be used in association or sequen-
full repigmentation and the length of treatments tially depending on each patient and the cur-
that often requires 6–24 months to provide opti- rent course of his disease.
mal results, such combination approaches appear
of great interest in most vitiligo patients.
However, the current approaches mainly aim to
repigment vitiligo lesions. Strategies to prevent References
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tive right-left comparative clinical trial. J Eur Acad mental vitiligo: a prospective, randomized half-body
Dermatol Venereol. 2015;29:925–32. comparative study. Br J Dermatol. 2012;166:658–61.
49. Kullavanijaya P, Lim HW. Topical calcipotriene
62. Helou J, Maatouk I, Obeid G, et al. Fractional laser
and narrowband ultraviolet B in the treatment of for vitiligo treated by 10,600 nm ablative fractional
vitiligo. Photodermatol Photoimmunol Photomed. carbon dioxide laser followed by sun exposure. Lasers
2004;20:248–51. Surg Med. 2014;46:443–8.
ERRNVPHGLFRVRUJ
420 T. Passeron
63. Vachiramon V, Chaiyabutr C, Rattanaumpawan P,

68. Schmitt J, von Kobyletzki L, Svensson A, et al.

et al. Effects of a preceding fractional carbon diox- Efficacy and tolerability of proactive treatment with
ide laser on the outcome of combined local narrow- topical corticosteroids and calcineurin inhibitors for
band ultraviolet B and topical steroids in patients with atopic eczema: systematic review and meta-analysis
vitiligo in difficult-to-treat areas. Lasers Surg Med. of randomized controlled trials. Br J Dermatol.
2016;48:197–202. 2011;164:415–28.
64. Li L, Wu Y, Sun Y, et al. Triple combination treat- 69. Cavalie M, Ezzedine K, Fontas E, et al. Maintenance
ment with fractional CO2 laser plus topical beta- therapy of adult vitiligo with 0.1% tacrolimus oint-
methasone solution and narrowband ultraviolet B for ment: a randomized, double blind, placebo-controlled
refractory vitiligo: a prospective, randomized half- study. J Invest Dermatol. 2015;135:970–4.
body, comparative study. Dermatol Ther. 2015;28: 70. Rashighi M, Agarwal P, Richmond JM, et al. CXCL10
131–4. is critical for the progression and maintenance of
65. Regazzetti C, Alcor D, Chignon-Sicard B, et al.
depigmentation in a mouse model of vitiligo. Sci
Micro holes for delivering melanocytes into the skin: Transl Med. 2014;6:223ra23.
an ex vivo approach. Pigment Cell Melanoma Res. 71. Regazzetti C, Joly F, Marty C, et al. Transcriptional
2016;29:481–3. analysis of vitiligo skin reveals the alteration of WNT
66. Silpa-Archa N, Griffith JL, Williams MS, et al.
pathway: a promising target for repigmenting vitiligo
Prospective comparison of recipient-site preparation patients. J Invest Dermatol. 2015;135:3105–14.
with fractional carbon dioxide laser vs. dermabrasion 72. Richmond JM, Bangari DS, Essien KI, et al.

and recipient-site dressing composition in melanocyte- Keratinocyte-derived chemokines orchestrate T cell
keratinocyte transplantation procedure in vitiligo: a positioning in the epidermis during vitiligo and may
preliminary study. Br J Dermatol. 2016;174:895–7. serve as biomarkers of disease. J Invest Dermatol.
67. Nicolaidou E, Antoniou C, Stratigos AJ, et al. Efficacy, 2016;137(2):350–8.
predictors of response, and long-term follow-up 73. Wang XX, Wang QQ, Wu JQ, et al. Increased expres-
in patients with vitiligo treated with narrowband sion of CXCR3 and its ligands in patients with vit-
UVB phototherapy. J Am Acad Dermatol. 2007;56: iligo and CXCL10 as a potential clinical marker for
274–8. vitiligo. Br J Dermatol. 2016;174:1318–26.
ERRNVPHGLFRVRUJ
Camouflage
39
Alida DePase and Thomas Jouary
Contents
39.2 Why Camouflage and Cosmetic Rehabilitation Are Needed 422
39.3 Camouflage as a Medical Intervention? 422
39.4 A Brief History of Camouflage 423
39.5 Camouflage Controlled Studies 424
39.6 Self-Tanning Creams, Lotions and Sprays 424
39.7 Cover Creams, Foundations and Sticks 425
39.8 Vitiligo of the Lips 426
39.9 Leukotrichia 426
39.10 Permanent and Semi-permanent Camouflage 426
39.11 Precautions of Use 426
39.12 Conclusion: Camouflage as a Balm for “Bruised” Souls 427
References 427
Abstract
It is only recently that camouflage has been
recognised as being a medical intervention,
when there are no other satisfactory options
to really help the patient. Non permanent
techniques such as self tanners should be usu-
A. DePase ally preferred. The Koebner’s phenomenon
Associazione Ricerca Informazione per la Vitiligine
(ARIV), Cernusco Lombardone, LC, Italy
can be observed in areas where an adherent
camouflage needs an intense frictional wash-
T. Jouary (*)
Service de Dermatologie, Hôpital Saint André,
ing to pull it off. Cosmetic tattoo may be suit-
CHU de Bordeaux, Bordeaux, France able for depigmented lips, especially in black
e-mail: thomas.jouary@ch-pau.fr people, and for depigmented nipples.

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422 A. DePase and T. Jouary
The expressed fear of many vitiligo patients is

Key Points that that the disease may spread to visible areas.
• It is only recently that camouflage has Indeed, patients with visible vitiligo are usually
been recognized as being a medical inter- seeking for a treatment more actively than others.
vention, when there are no other satisfac- They are logically searching help first for patches
tory options to really help the patient. in the visible areas and only secondly for areas of
• It is important to discuss with the patient lesser aesthetic importance.
his or her lifestyle to choose products Standard and experimental vitiligo treatment
(self-tanners, cover creams, etc.) suit- options are demanding from the psychological
able for his/her case. point of view and may be disappointing for
• Camouflage can improve the quality of both patient and dermatologist. Moreover
life of vitiligo patients. repigmentation is often partial and relapses may
occur after stopping treatment. The treatment of
hand and face lesions is particularly difficult,
39.1 Introduction because of Koebnerization (Chap. 2.2.2.1).
Segmental vitiligo of the face starts frequently in
The World Health Organization defines “health” childhood. At this age, melanocyte cell grafts are
as a physical and psychological well-being. rarely considered for ethical reasons and because
Considering all physical and psychological some repigmentation may appear spontaneously
aspects of vitiligo, dermatologists should suggest in the future. In all these situations, when classi-
a therapy when feasible but should also recom- cal medical treatments fail or cannot be consid-
mend psychological approaches and the use of ered, efficient camouflage techniques should be
camouflage [1–7]. Benefits can be obtained by proposed to the patient.
the skilled use of corrective cosmetics which are
presented in the following chapter.
39.3 Camouflage as a Medical
Intervention?
39.2 Why Camouflage
and Cosmetic Rehabilitation Dermatologists as a rule don’t know much about
Are Needed camouflage; in the past, they have been dismis-
sive of what they considered merely make-up.
Camouflage refers to a range of special products, They are uninformed about the wide range of
specially developed to disguise aesthetic skin dis- available camouflage products and the different
figurement of any kind, requiring special applica- techniques of application. They are equally
tion techniques. Cosmetic rehabilitation is about unaware of the benefits that can be obtained by
encouraging patients to achieve a positive image the skilled use of corrective cosmetics, such as
of the self. self-tanners, stains, dyes, whitening lotions,
Vitiligo is a disfiguring disease, pervasive to tinted cover creams, compact, liquid and stick
the patient. In an attempt to cope, patients may foundations, fixing powders, fixing sprays,
drastically change their way of life. They choose cleansers, semi-permanent and permanent tattoos
clothes with the sole aim of covering the patches. and dyes for facial and head white hair.
They feel obliged of wearing long sleeves and It is only recently that camouflage has been
long trousers even in the hottest summer. recognized as being equally worthy of consid-
Patients presenting with face and hand lesions eration as a medical intervention, when there
demonstrate usually a higher impairment of are no other satisfactory options to really help
their quality of life and self-body image than the patient and when the disease is recalcitrant
others. to all standard and alternative therapies.
ERRNVPHGLFRVRUJ
39 Camouflage 423
Table 39.1 Self-tanner and cover creams: application Candidates for vitiligo camouflage are
method patients of both genders and of all ethnic origins
Preparative and ages. As with all patients, it is important for
Facilities phase Cleaning the camouflage practitioner to learn about their
Room or Cotton Soap
prior history, current medical situation, emo-
bathroom at washcloth to
medium-low exfoliate the tional state and attitude towards camouflage. The
temperature skin patient’s ability and desire to perform the vari-
(sweating is not ous camouflage techniques should be discussed,
good)
and the patient should be asked whether he/she
Band or tie to Skin lotion (feet,
keep hair off the ankles, knees, has experienced allergic reactions to cosmetics
face elbows, hands, in general in the past. It is important to discuss
wrists) for areas with the patient his or her lifestyle to choose
with fine lines products (self-tanners, cover creams, etc.) suit-
Fingernail scrub
brush
able for his/her case.
Baby wipes to
remove the
unwanted trace 39.4 A
Brief History
Latex gloves of Camouflage
Sponge paintbrush
(for self-tanner on
the back) Since ancient times, an unblemished face has
Dark-coloured universally been considered a symbol of beauty
bathing suits (for and therefore sought after at all costs. In ancient
whole body)
Roman times, camouflage was used by the
Loose outfit
(during the slaves who had gained their freedom, became
product drying) rich, and were determined to leave their past
Dark T-shirt behind. This consisted of an ochre-coloured
Moisturizer (for paste made of clay and helped to cover the mark
the subsequent made on their forehead with a hot iron, the igno-
period)
minious stigma of the slave. Quinto Sereno
Sammonico, doctor of the second century AC,
Camouflage consultations have been developed in the Chapter Cutis et faciei vitiis propellendis
in some dermatology departments. The aim of of his Liber Medicinalis, proposes a remedy to
these consultations is to educate patients about eliminate freckles: “Invida si maculat faciem
camouflage (Table 39.1 details the application lentigo decoram nec prodesse valent naturae
method of commonly used products). These dona benignae, erucam atque acidum laticem
multidisciplinary consultations, composed of simul inline mali Saepiolae cineres ex ossibus
camouflage trained nurses, dermatologists and omnia levant…” (If the horrible ephelides spoil
camouflage specialized persons, have had a the skin, spoiling its natural beauty, spread a
favourable impact on patients. Camouflage lotion made of vinegar and rocket on the skin).
therapy or education means also to enable Until the early twentieth century, make-up in
patients to apply the cosmetics themselves. It general was used only by the rich, theatre actors
is worthwhile when the patient can master the or prostitutes. It was only in the twentieth cen-
correct procedure with the products chosen and tury, with the birth of the film industry, that
the various techniques of application. Patients camouflage and make-up in general became a
should also be informed on where and how to must for film stars. Products resistant to the
obtain camouflage products that suit their indi- effect of stage lighting were requested for actors
vidual needs. with imperfections to hide. After the First World
ERRNVPHGLFRVRUJ
War, many soldiers came back from the front stratum corneum and gives a tan resembling the
severely burnt or disfigured, and camouflage solar UV-induced tan. This is due to the so-called
was a necessary blessing for them. The make-up “Maillard’s” reaction, after the author who stud-
and camouflage era was born, and mass produc- ied the chemical reaction that goldens the crust of
tion for the general public became a reality. bread in the oven. Recent studies have demon-
strated that DHA reacts totally and only with the
first cells with which it comes into contact, and
39.5 Camouflage Controlled therefore it remains on the surface of the stratum
Studies corneum, until it is eliminated with the normal
turnover of the epidermis. Preparations contain-
Clinical research on camouflage in vitiligo is ing DHA are stable between pH 4 and pH 6. At
very limited given the practical importance of neutral pH, brown grumes are formed inactivating
this field. A Cochrane database review in 2006 pigmentation. It has been noticed that the pres-
did not find published trial in this field [8]. Some ence on the skin or in the product of little organic
authors report the efficacy and safety of dihy- or inorganic molecules may alter the DHA colour-
droxyacetone (DHA) in healthy volunteers and ing capability. Traces of metals such as iron, tita-
vitiligo patients. These open and/or retrospective nium, zinc or alpha-hydroxy acids such as lactic
studies have compared different DHA concentra- acid can inactivate the product. So to avoid the
tions in patients of various phototypes. The frequent risk of lack of uniformity, a good rule is
higher the concentration, the better the response not to utilize self-tanners after using creams with
observed particularly in darker phototypes [8]. zinc or titanium or after washing the skin with
Only one study demonstrated the positive impact alkaline or lactic acid-based soaps largely present
of self-tanning interventions on the quality of life in products being defined by manufacturers as “at
in a cohort of vitiligo patients [9]. These studies physiologic pH” [1]. They can be used throughout
were principally conducted with DHA-derived the year, they are waterproof, and the fake tan
products which are described in detail thereafter. developed does not stain clothes or sheets.
However, sea water makes them fade away
quickly, while swimming pool water does not.
39.6 S
elf-Tanning Creams, Lotions No sunless tanner currently available contains
and Sprays adequate sunscreen, so sun shielding products
may be applied during the day, and moisturizers
Self-tanners in gel, cream, lotion or spray give as well, but only after the desired colour intensity
the skin a brown colour that resembles a natural has been obtained. Before applying self-tanners,
tan and normally lasts 3–5 days. The tanning of it is advisable to gently rub the skin with a very
the skin develops in about 3–24 h after the appli- soft brush to eliminate dead cells, especially on
cation. Instant colour self-tanners, available from elbows, knees and knuckles, in order to obtain an
some manufacturers, thanks to a colour guide, even skin colour. The skin should be perfectly
give a tanned colour instantly. These products are dry. It is advisable not to apply these products
popular among those who cannot or do not like during the hot hours of the day in summer,
sun exposure and can be considered camouflage because excessive sweat can result in uneven
products as they disguise depigmentation suc- application and may prevent the active substance
cessfully. Marketed for about 40 years, at first to develop colour properly. Moreover, the appli-
self-tanners were not successful because they cation on eyebrows or near the forehead hairline
provided a yellowish and uneven colour, while should be avoided. Only a small quantity of the
today the latest formulas give excellent aesthetic product should be applied first, and if the desired
results. Unluckily they are not suitable for colour is not achieved, it is possible to intensify
coloured people, and the best results are in with additional daily applications. If too much of
Caucasians of phototypes I to III. the product is used, the result will be unnatural.
The active ingredient is dihydroxyacetone Particular spare amount should be applied on the
(DHA), a sugar that reacts with the proteins of the face and neck, because this part of the skin takes
ERRNVPHGLFRVRUJ
39 Camouflage 425
a b
Fig. 39.1 (a and b) Camouflaging of hands and forearms
a b
Fig. 39.2 (a–b) Camouflaging of facial vitiligo in one dark-skinned patient
to self-tanners quite well. If the hair is short, the shoes and bras for 1 h, if the products have been
product should be applied behind the ears. used on the body.
For small-sized vitiligo lesions, it is advisable
to use a self-tanner in lotion (not in cream) with a
q-tip. The lotion is spread from the centre of the 39.7 C
over Creams, Foundations
lesion towards the outside, up to 1–2 mm from and Sticks
the lesion edge. A different technique is to spread
the product over the entire area, for instance, Highly pigmented creams come in compact, liq-
hands and arms, including the normal pigmented uid or stick formulations and are available from
areas and subsequently repeat over only the white several manufacturers in a wide range of natural
areas using a q-tip dipped in the product skin colours. They are lightweight and easy to
(Figs. 39.1 and 39.2). apply, usually free of contact allergens, but der-
Most instructions for sunless tanners men- matitis and allergic reactions may occur, due to
tion rub it well until it is absorbed, but this takes fragrances and preservatives present in some
too much time and the hands will become brands. The texture is denser than traditional
orange. Instead, the product should be applied foundation creams used by the general public, as
quickly but thoroughly, spreading in a circular their aim is to provide effective cover. They may
motion to avoid streaking. Finally, after apply- contain up to 50% mineral oils and wax. The tex-
ing the product, washing for 3 h should be ture, different from normal foundation products,
avoided. Any sweat-inducing physical activity is also due to titanium dioxide, used as a thicken-
sould be avoied for 1 h: no tight jeans, belts, ing and shielding agent, offering sun protection,
ERRNVPHGLFRVRUJ
Table 39.2 Cover cream application lips, suitable also for male patients. They are an
Cover Applied with the ball of the middle finger and alternative to lip tattoos. These very popular cos-
cream smoothed over the discoloured area with a metics are easily available in almost all depart-
light-pressing motion. A flat brush can be ment stores and pharmacies.
used to feather out the outer edges of the
thick, opaque cover cream solution until it is
so well-blended to become undetectable
Fixing Colourless powder to stabilize the 39.9 Leukotrichia
powder foundation, applied with a small cotton pad.
This procedure makes the application
waterproof and resistant to smudges and
Leukotrichia can affect visible areas on the beard,
friction. A few minutes for the talc to be moustaches, eyebrows and eyelashes. In these
absorbed, then the excess is dusted off cases it is advisable to dye the white hair in a
Fixing It maintains the corrective make-up for a hairdressing salon the first time, and subsequently
spray whole day. Vaporized at a distance of 40 cm,
the dyeing can be practiced at home with prod-
it creates, thanks to its silicon and polymers
formula, an elastic film that guarantees ucts formulated for these delicate areas.
coverage
39.10 Permanent and Semi-

while the colours are provided by iron oxides. permanent Camouflage
Blended to complement the individual’s particu-
lar skin colour, they can conceal disfiguring vit- Also referred to as micro-pigmentation, cosmetic
iligo patches of exposed areas. Suitable for men, tattooing and dermal pigmentation are techniques
women and children, their waterproof character- that require an experienced technician, qualified
istics allows shower and swimming. When they to offer these services. Results vary according to
are applied on the face, they should be removed the skill of the practitioner and his/her experience
every day. It is crucial to choose one’s basic with colours. Cosmetic tattoo may be suitable for
colour and then the various shades that can vary depigmented lips, especially in black people, and
indefinitely, according to the different parts of for depigmented nipples. As regards other vitil-
the body, to the seasons or simply from one day igo areas, results may be very disappointing.
to another. Sometimes it is necessary to mix Pigments especially formulated for cosmetic
more colours of different brands. application are implanted beneath the epidermis
For a correct application, the skin should be into the dermal layer, by microinsertion. Topical
cleaned. Any previous make-up has to be gently anaesthetics are used to minimize discomfort,
removed. The patient has to learn proper make-up allergic reactions are rare, and sterile needles and
removal techniques, that is, gentle movements to surgical gloves are used for each procedure.
avoid the Koebner phenomenon. Using a sample Semi-permanent camouflage, or cosmetic tattoo-
palette of cover creams, two or three shades can ing, differs from traditional tattooing, where per-
be mixed, to achieve the desired colour match. manent skin inks and dyes are inserted into the
More than three colours will make the procedure skin. Camouflage can be called permanent if
too complicated and expensive. An optimal blend compared to normal make-up, but as it will fade
of colours should be created, and when applied, with time, which usually lasts from 2–5 years, it
this should match the colour of the surrounding is actually “semi-permanent”.
skin as closely as possible (Table 39.2).
39.11 Precautions of Use

39.8 Vitiligo of the Lips
Some precautions should be considered when a
Transfer-resistant lip colour lasting up to 12 h of camouflage technique is used or proposed to the
wear, in matte formulas, is available in many patients. First, the patient should be informed
shades that duplicate the natural colour of the that camouflage has to be removed smoothly to
ERRNVPHGLFRVRUJ
39 Camouflage 427
avoid intensive friction by using washcloths. This therapy” can do to improve the quality of life of
is of importance as the Koebner phenomenon is vitiligo patients.
observed in areas where an adherent camouflage
needs an intense frictional washing to pull it off Take-Home Messages
(Chap. 2.2.2.1). Similarly, fixing powder should • Non-permanent techniques such as self-
be used cautiously and avoided whenever possi- tanners should be usually preferred.
ble. Smooth, liquid and light instant colour self- • The Koebner phenomenon can be observed in
tanner and stains should be preferred for these areas where an adherent camouflage needs an
reasons. intense frictional washing to pull it off.
Another point of importance is the risk with • Cosmetic tattoo may be suitable for depig-
permanent camouflage. Indeed, vitiligo course is mented lips, especially in black people, and
highly unpredictable. Even after many years, for depigmented nipples.
stable large patches can resolve spontaneously.
Giving these considerations, permanent camou-
flage and tattoos should be considered with par- References
ticular caution. The development of a
depigmented patch around an area previously 1. DePase A. Importanza del Camouflage in Pazienti
con Vitiligine. In: Lotti T, editor. La Vitiligine, nuovi
treated with these techniques can lead to inaes- Concetti e Nuove Terapie. Milano: Utet; 2000.
thetic results. Thus, if performed in vitiligo, the 2. DePase A. La Voce dei Pazienti. In: EBD evidence
colour of the tattoo has to be very close to the based dermatology. Milano: Masson; 2003.
natural pigmentation of the patient, which is very 3. DePase A, Naldi L. Vitiligo, some reflections on clini-
cal research in a rather elusive disorder Centro Studi
difficult. Since the colour of the tattoo is defini- Gised. Bergamo: Ospedali Riuniti; 2004.
tive and does not follow the UV-induced tanning 4. DePase A. Vitiligo, au-delà de la maladie I. In:
changes, the contrast between tattooed skin and Ortonne JP, editor. Cutis and psyche. France; 2004.
natural pigmented areas may cause problems. 5. DePase A. The view of the cosmetologist. In: Lotti
T, Hercegova L, editors. Vitiligo: problems and solu-
tions. New York: Marcel Dekker Inc.; 2004.
6. DePase A. Il Camouflage, Chapter 69. In: Naldi L,
39.12 Conclusion: Camouflage Rebora A, editors. Dermatologia Basata sulle prove di
as a Balm for “Bruised” Souls Efficacia. Milano: Masson; 2006.
7. Ongenae K, Dierckxsens L, Brochez L, et al. Quality
of life and stigmatization profile in a cohort of
Camouflage increases the patient’s confidence vitiligo patients and effect of the use of camouflage.
and improves his/her quality of life. It is readily Dermatology. 2005;210:279–85.
accepted by women, who, unlike children and 8. Whitton ME, Ashcroft DM, Barrett CW, González
U. Interventions for vitiligo. Cochrane Database Syst
men, may be already accustomed to use make-up Rev. 2006;(1):CD003263.
products. But both men and adolescents can eas- 9. Rajatanavin N, Suwanachote S, Kulkollakarn
ily learn how to apply the products. S. Dihydroxyacetone: a safe camouflaging option in
There are many ways to conceal small or large vitiligo. Int J Dermatol. 2008;47:402–6.
areas of vitiligo and the natural result increases
the patient’s confidence. No more embarrassing
questions or intrusive staring, wearing a short- Further Reading
sleeved shirt or shorts in the summer, no hands
http://www.skin-camouflage.net
hidden in pockets and greeting with a handshake http://www.redcross.org.uk/standard.asp?id=49354
without fear, these are some of what “camouflage
ERRNVPHGLFRVRUJ
Photoprotection Issues
40
Alessia Pacifico, Giovanni Leone,
and Mauro Picardo
Contents
40.1 Normal and Vitiligo Skin UV Sensitivity 430
40.2 Photoadaptation of Vitiliginous Skin to UV Irradiation 431
40.3 Vitiligo and Skin Cancer 432
40.4 Practical Photoprotection 433
References 434
Key Points • Hyperkeratosis and epidermal hyperpla-

• Using Fitzpatrick’s system, the physi- sia would develop in vitiligo exposed to
cian can estimate the relative risk for UV to compensate for the lack of
each phototype of developing acute and pigment.
chronic changes related to UV • Antioxidant status may account for the
exposure. SPT-related differences which is the
• Stratum corneum protects against UV antioxidant status of the skin.
mainly because of reflection of radia- • Low incidence of actinic damage, basal
tion, absorption of the radiant energy and squamous cell carcinomas in vitil-
and internal scattering. igo could be due to a protective function
• Melanin is a potent UV absorber and a of upregulated wild-type p53.
protective factor against UV radiation. • For photoprotection in sunny and tropi-
cal climates, avoidance of peak sunny
hours and clothing is the major step.
A. Pacifico · G. Leone (*) • In patients with skin types >III usually
Phototherapy Unit, San Gallicano Dermatological two different sunscreens (the first one
Institute, IFO, Rome, Italy
e-mail: gleone@ifo.it
on the vitiliginous lesions with SPF
around 15 and another one with SPF
M. Picardo
50+ on surrounding healthy skin) are
Dermatological Institute, IRCCS, Rome, Italy recommended.

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430 A. Pacifico et al.
40.1 Normal and Vitiligo by Cario-André et al. assessed the photoprotective

Skin UV Sensitivity role of melanocytes in the epidermis and studied
the effects of UV B on epidermis reconstructed
A number of mechanisms have been developed with and without melanocytes. To address more
during evolution to protect human skin from exces- specifically the role of melanin in fair-skinned
sive UV radiation. To classify the susceptibility of individuals, experiments were done with cells
human subjects to develop erythema following UV obtained from human skin of low skin types (II–
exposure, Fitzpatrick’s system differentiates six III). In order to study the effect of constitutive
sun-reactive skin phototypes (SPT) [1] (Table 40.1). melanin and possibly that of newly synthetized
In this system, the capacity to tan is equally impor- melanin precursors, a single dose of UVB (4–5
tant to help to categorize individuals of any colour MED) was administered to reconstruct, and the
or ethnic background. Using this method, the phy- effects were then monitored over the first 24 h. The
sician can estimate the relative risk of developing results clearly showed that low phototype melano-
acute and chronic changes related to UV exposure. cytes protect epidermal basal cells against UVB-
A study by Carrettero-Mangolis and Lim confirms induced apoptosis as well as necrosis and may thus
that there is a correlation between minimal ery- preserve epidermis integrity after UVB irradiation.
thema dose (MED) and SPT, i.e. the higher the On the other hand, such melanocytes do not seem
SPT, the higher the MED [2]. to have a protective role against DNA damage and
There is evidence that the stratum corneum (SC) consequently may not prevent skin cancer [7].
protects against UV mainly because of reflection of In a recent study by Yamaguchi et al. DNA
radiation, absorption of the radiant energy and damage and apoptosis in different skin types
internal scattering. In the palms where the SC is before and after UV exposure have been reviewed.
very thick, the minimal erythema dose is 16 times Their results, accordingly with other published
higher than on the back. It was observed that in reports, indicate that UV-induced DNA damage
areas stripped from SC, sunburn develops more is more effectively prevented in darker skin types.
easily than in the unstripped areas of the skin [3]. This paper also demonstrated that rates of repair
Melanin is a potent UV absorber, and thus skin of DNA damage may differ greatly among differ-
pigmentation is another natural protective factor ent individuals and that UV-induced apoptosis is
against UV radiation. It is already well known that significantly greater in darker skin. These results
pigmented skin is more resistant to sunburn than suggest that UV damaged cells are more effi-
poorly pigmented skin [4, 5]. Delayed effects of ciently removed in darker skin [8].
chronic UV exposure, such as development of skin In patients with vitiligo, it is generally
cancer and photoageing are also more pronounced assumed that lesional skin of SPT I–VI would be
in individuals with fair skin colour [6]. Skin very sensitive to UV radiation owing to the fact
responds to repeated exposure by tanning, espe- that pigment is absent in such lesions, but it has
cially in skin types III and IV who tan easily and been recently established that there are several
burn rarely, which is the result of melanogenesis mechanisms that contribute to modulate UV sen-
and associated SC thickening. A study performed sitivity in vitiliginous skin. Depigmented area of
vitiligo patients does not contain pigment and
Table 40.1 Fitzpatrick SPT classification thus protection against UV is afforded by the SC
SPT I Always burns, never tans and the rest of epidermis. It might thus be sus-
SPT II Burns easily, tans minimally pected that hyperkeratosis and epidermal
SPT Burns moderate, tans gradually to light brown hyperplasia would develop in vitiligo exposed to
III
SPT Burns minimally, alias tans well to moderate
UV to compensate for the lack of pigment. The
IV brown importance of hyperplasia in photoprotection
SPT V Rarely burns, tans profuse to dark was validated in studies looking at vitiligo skin
SPF Never burns, deeply pigmented which is characterized by a lack of melanocytes.
VI Gniadecka et al. assessed solar simulated radia-
ERRNVPHGLFRVRUJ
40 Photoprotection Issues 431
tion (SSR) MED on amelanotic and adjacent nor- phototoxicity in their skin despite increasing doses
mally pigmented skin of 14 vitiligo patients. of UV radiation [12]. Thus, these patients devel-
Erythema and melanin content were quantified oped photoadaptation, a frequently observed phe-
using a reflectance device and SC thickness was nomenon. Photoadaptation has been described in
determined from frozen skin samples. These different SPT, and it is probably due to both pig-
authors reported that the predominant protective mentary and non-pigmentary influences. Several
factor in both vitiligo and pigmented skin is the studies have indicated that factors involved in pho-
SC where it accounted for 57% of the total photoadaptation include hyperkeratosis, acanthosis,
toprotection in pigmented skin. Repeated expo- melanogenesis as well as an unknown factor that
sures to this skin was shown to elicit a protection may be due to DNA repair or an immune related
factor of 15 in the absence of melanocytes. In process. Photoadaptation was described by Oh
contrast, selective activation of melanocytes by et al. as a “number of changes occurred that are
UVA that does not induce SC thickening only adaptive, in the sense that they result in a dimin-
gave a protection factor of 2–3. Gniadecka et al. ished future response to equivalent doses of radia-
suggested that SC accounted for over two-thirds tion” [13]. One of the methods to measure
of photoprotection observed in normal skin and photoadaptation is MED phototesting.
hence was far more significant in this role than In phototherapy protocols, vitiliginous skin
induced tanning [9]. The significance of SC in has been always classified as Fitzpatrick’s SPT I
photoprotection may be greater in fair-skinned or II because of the lack of pigment. Caron-
individuals than in pigmented individuals espe- Schreinmachers et al. have recently showed that
cially in vitiligo skin where the SC may represent by elicitation of the MED on areas of vitiliginous
the only source of protection. However, studies skin, UVR sensitivity varied with total body skin
performed by Sheehan et al. did not support a sig- type even in skin without pigment. Unlike previ-
nificant photoprotective role for stratum corneum ous studies where we only had information about
thickening [10]. The different results obtained by the relation between MED and SPT from normal
Gniadecka and Sheehan may be due to several skin of different SPT, in this study it has been
variables. It is known in fact that UVR sensitivity proved that there is a linear relationship between
vary between body sites on the same person. the SPT of non-affected skin of vitiligo patient
Kaidbey et al. investigating epidermal UVR and the sensitivity to NB UVB irradiation of
transmission in black skin (skin type VI), as well lesional skin (Fig. 40.1) [14]. Since melanocytes
as Caucasian skin (skin types I–III), found that in are absent or scarcely present in lesional vitiligo
skin types I, II and III, the SC is the main site of skin, this difference in photosensitivity cannot be
UVR screening, absorbing over 50% of the inci- due to melanin, but this protection must be based
dent radiation. However, samples were taken from on other mechanisms. We know that epidermis
previously sun-exposed sites (abdominal skin) [4]. thickness increases after UV irradiation. In the
Kaidbey and Kligman also reported in an earlier study performed by Caron-Schreinmachers et al.,
study that melanogenesis (without appreciable however, patients’ skin had not been exposed to
thickening of the SC) induced by repeated UVA UV for at least 3 months, and tests were carried
exposure afforded a protection factor of 2–3 [11]. out in regions that had not been exposed to sun so
skin thickening could not be responsible for the
observed differences. One mechanism that pos-
40.2 Photoadaptation sibly accounts for the SPT-related differences is
of Vitiliginous Skin the antioxidant status of the skin. It has been
to UV Irradiation already well established that antioxidants provide
photoprotection. Bessou-Touya et al. have shown
A recent retrospective study showed that most that melanocytes of Caucasian subjects, which
patients with vitiligo treated with narrow band have a higher content of unsaturated fatty acids
UVB (NB UVB) phototherapy did not develop in their cell membrane, are more prone to the
ERRNVPHGLFRVRUJ
Fig. 40.1 MED on

lesional and nonlesional
skin
p eroxidative effects of UV light and that kerati- is a major causal factor in the development of
nocytes participate in photoprotection via photo- skin cancer. Non-melanoma skin cancers
type-dependent antioxidant enzyme activities (NMSC) are initiated for the most part by
[15]. Picardo et al. demonstrated that there are chronic sunlight exposure and can readily be
significant differences in antioxidant status in produced by experimental exposure to ultravi-
normal skin between people with high (III–V) olet radiation in animal models [17, 18].
and low (I–II) SPT [16]. Possibly the same differ- Ultraviolet B (UVB) is the major active wave-
ences in antioxidant status between the different band region that causes direct photochemical
SPT exist also in vitiligo skin. damage to DNA, from which gene mutations
Overall the studies performed indicate that arise. Unlike UVB, ultraviolet A (UVA) could
photoadaptation occurs following UVR exposure have more indirect effects on DNA via the gen-
also in depigmented areas of vitiligo subjects and eration of reactive oxygen species. In contrast
that the normally pigmented skin phototype with NMSC, cutaneous melanoma is more
should be taken in account when a photoprotec- commonly associated with sporadic burning
tion strategy (or phototherapy) is recommended. exposure to sunlight, especially early in life,
but the wavelengths responsible have not been
clearly identified. There are several indications
40.3 Vitiligo and Skin Cancer that UVA might have an important role in the
pathogenesis of melanoma [19]. However, this
Sun exposure is the main cause of photocar- involvement has recently been questioned,
cinogenesis, photoageing and photosensitivity. since only UVB could induce melanoma in a
Unprotected exposure to ultraviolet radiation transgenic mouse model [20].
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The action spectrum for UV-induced tanning 40.4 Practical Photoprotection

and erythema are almost identical. Indirect evi-
dence suggests that UVA has a greater role in All patients with vitiligo, but particularly those
long-term sun damage than it does in acute effects with fair skin, should have a photoprotective
such as sunburn or vitamin D synthesis, which counselling adapted to their phototype, environ-
are overwhelmingly attributable to UVB [21, 22]. mental exposure risk and therapy plan. Indeed,
Considering that patients with vitiligo have repigmentation needs UV stimulation, and an
patches lacking pigment and that a group of exceedingly high photoprotection could be coun-
patients develop the disease during childhood, terproductive. However, for photoprotection in
it would be expected that these subjects with sunny and more risky areas such as tropical cli-
the involvement of the head, neck and hands, mates, avoidance of peak sunny hours and cloth-
which are sites at constant risk of sun damage, ing are the major steps. It is thus most important
should develop an increased risk for NMSC to explain to the patient the importance of wear-
[23]. A review of the literature indicates an ing sun-protective clothing (such as wide
even lower risk of NMSC. The rarity of NMSC brimmed hats) to help prevent tanning and sun-
in vitiligo was noted earlier in a study by burns. Sunblockers come as second line.
Calanchini-Postizzi and Frank on 23 patients Sunscreen that provides protection from both
who had a mean duration of vitiligo of UVA and UVB should be used. Besides helping
15.1 years. These investigators found only three to protect the skin from sunburn and resulting
actinic keratoses in light-exposed vitiligo koebnerization and long-term damage of suscep-
patches in those 23 patients. In addition, the tible depigmented areas, sunscreens also mini-
number of “sunburn cells”, commonly consid- mize tanning, which makes the contrast between
ered as the morphological sign of UV damage normal and depigmented skin less noticeable
to keratinocytes, was found significant lower [28]. A waterproof, broad-spectrum sunscreen
compared to healthy controls [13]. Schallreuter with a sun protection factor of at least 15 should
et al. more recently hypothesized that the low be used on all exposed skin, pigmented and
incidence for actinic damage and basal and depigmented. Particular attention should be given
squamous cell carcinomas as documented in to the formulation of the sunscreen to allow easy
vitiligo could be due to a protective function of spreading (and removal if needed) without risk of
upregulated wild-type p53 induced by the con- skin trauma (Koebner’s phenomenon).
stant H2O2 stress existing in all the epidermis of Sunscreen act by one of two mechanisms,
vitiligo patients [24]. either by absorbing UV rays or by blocking or/
The association between vitiligo and mela- and scattering these rays. Chemical sunscreens
noma is interesting because both diseases affect function by absorbing UVB and/or UVA.
melanocytes and that immunological mecha- Nowadays, protection against both UVA and
nisms play a part in both conditions. Different UVB is very common, and as a result, sunscreens
clinical studies report the connection between often contain a mixture of light-absorbing chem-
malignant melanoma and vitiligo, and also sev- icals. Sunscreen’s efficacy in absorbing UVB is
eral authors suggest that the appearance of depig- measured by the sun-protective factor (SPF).
mentation during the course of malignant UVB absorbers have been commonly used
melanoma or its treatment with interferon can be worldwide for decades, whereas most UVA and
considered a good prognostic sign [25–27]. broadband absorbers have been developed in
Thus a series of arguments indicate that either recent years. Since a sunscreen has to protect
the chronic adaptation to UV stress via increased against the entire UV spectrum, different filters
antioxidant responses or reinforced disease- have to be combined in the same product. The
driven immunosurveillance against melanocyte cinnamates (2-ethyl p-methoxycinnamate) are
antigens may naturally protect vitiligo patients. by far the most popular UVB absorbers in both
However, large-scale epidemiological studies are the USA and Europe, and they are used in com-
needed to clarify this important issue. bination with other UVB absorbers to achieve a
ERRNVPHGLFRVRUJ
high SPF. The second most popular filters during and vitiliginous skin and nevertheless allowing
the recent past are camphor derivatives. part of the UV spectrum to stimulate pigmenta-
Salicylates and para-aminobenzoic acid (PABA) tion on affected skin [30].
and its derivatives are among the oldest commer-
cially available UVB filters, and they are still
used worldwide. The increasing need for broad- References
band agents and improved photostability has led
to the introduction of a new generation of filters, 1. Fitzpatrick TB. The validity and practicality of sun-
including methylene bis-benzotriazolyl tetra- reactive skin types I through VI. Arch Dermatol.
1988;124:869–71.
methylbutylphenol (Tinosorb M) and bis- 2. Carretero-Magolis C, Lim HW. Correlation between
ethylhexyloxyphenol methoxyphenyl triazine skin types and minimal erythema dose in narrow
(Tinosorb S), both manufactured by CIBA band UVB (TL-01) phototherapy. Photodermatol
Specialty (Basel, Switzerland), as well as tere- Photoimmunol Photomed. 2001;17:244–6.
3. Pathak MA, Fitzpatrick TB. The role of natural pho-
phthalylidene dicamphor sulfonic acid (Mexoryl toprotective agents in human skin. In: Pathak MA,
SX) and drometrizole trisiloxane (Mexoryl XL), Harber LC, Seljl M, Kukita A, editors. Sunlight
produced by L’Oreal (Clichy, France). The mex- and man, normal and abnormal photobiologic
oryls and tinosorbs are not licensed in the USA responses. Tokyo: University of Tokyo Press;
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and Japan [29]. 4. Kaidbay KH, Agin PP, Sayre RM, Kligman
Physical sunscreens act by blocking or scatter- AM. Photoprotection by melanin-a comparison of
ing UV rays. Micronized formulations of zinc black and Caucasian skin. J Am Acad Dermatol.
oxide and titanium dioxide are gaining in popu- 1979;1:249–60.
5. McFadden AW. Skin disease in the Cuna Indians.
larity. Mixtures of these minerals, along with Arch Dermatol. 1961;84:1013–23.
chemical sunscreens, have led to a marked reduc- 6. Taylor CR, Stern RS, Leyden JJ, Gilchrest
tion in the transmission of both UVB and UVA. In BA. Photoaging/photodamage and photoprotection. J
particular, the use of broad-spectrum sunscreens Am Acad Dermatol. 1990;22:1–15.
7. Cario-André M, Pain C, Gall Y, et al. Studies on epi-
alone limiting tan contrast may be an effective dermis reconstructed with and without melanocytes:
therapy in vitiligo patients with skin type I or melanocytes prevent sunburn cell formation but not
II. Many of these agents are also used in cosmetic appearance of DNA damaged cells in fair skinned
products such as eye shadow, foundation and Caucasians. J Invest Dermatol. 2000;115:193–9.
8. Yamaguchi Y, Beer JZ, Hearing VJ. Melanin mediated
powders. Zinc oxide, titanium dioxide, talc, apoptosis of epidermal cells damaged by ultraviolet
kaolin and calamine are examples of physical radiation: factors influencing the incidence of skin
agents. In the past, formulations containing these cancer. Arch Dermatol Res. 2008;300:s43–50.
agents were often opaque, and as a result, patients 9. Gniadecka M, Wulf HC, Mortensen NN, Poulsen
T. Photoprotection in vitiligo and normal skin. A
found them cosmetically unacceptable. More quantitative assessment of the role of stratum cor-
recently, brown colours, such as iron oxide, were neum, viable epidermis and pigmentation. Acta Derm
added as ingredients; not only does this make the Venereol. 1996;76:429–32.
physical sunscreen more appealing cosmetically, 10. Sheehan JM, Potten CS, Young AR. Tanning in
human skin types II and III offers modest photo-
it also helps to scatter the UV rays making the protection against erythema. Photochem Photobiol.
formulation more effective. 1998;68:588–92.
It is recommended that the sunscreen be reap- 11. Kaidbay KH, Kligman AM. Sunburn protection by
plied approximately every 90 min. In patients longwave ultraviolet radiation induced pigmentation.
Arch Dermatol. 1978;114:46–8.
with darker skin types (>III), we usually recom- 12. Hamzavi I, Deleon S, Yue K, Murakawa
mend the use of two different sunscreens: the one G. Repigmentation does not affect tolerance to NB
on the vitiliginous lesions with SPF around 15 UVB light in patients with vitiligo. Photodermatol
and another one with SPF 50+ on surrounding Photoimmunol Photomed. 2004;20:117.
13. Oh C, Hennessy A, Ha T, et al. The time course of
healthy skin, if possible with a high UVA protec- photoadaptation and pigmentation studies using a
tion factor. We find this strategy particularly use- novel method to distinguish pigmentation from ery-
ful in minimizing the contrast between healthy thema. J Invest Dermatol. 2004;123:965–72.
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Caron-Schreinemachers ALDB, Kingswijk MM, UVA protection of sunscreens: summary and recom-
Bos JD, Westerhof W. UVB 311 nm tolerance of vit- mendations. J Am Acad Dermatol. 2001;44:505–8.
iligo skin increases with skin photo type. Acta Derm 22. Morison WL. Clinical practice. Photosensitivity. N
Venereol. 2005;85:24–6. Engl J Med. 2004;350:1111–7.
15. Bessou-Touya S, Picardo M, Maresca V, et al.
23. Calanchini-Postizzi E, Frank E. Long term actinic
Chimeric human epidermal reconstructs to study damage in sun exposed vitiligo and normally pig-
the role of melanocytes and keratinocytes in pigmented skin. Dermatologica. 1987;174:266–71.
mentation and photoprotection. J Invest Dermatol. 24. Schallreuter KU, Behrens-Williams S, Khaliq TP,

1998;111:1103–8. et al. Increased epidermal functioning wild-type p53
16. Picardo M, Maresca V, Eibenschutz L, et al.
expression in vitiligo. Exp Dermatol. 2003;12:268–77.
Correlation between antioxidants and phototypes 25. Buljan M, Situm M, Lugovic L, Vucic M. Metastatic
in melanocytes cultures. A possible link of physi- melanoma and vitiligo: a case report. Acta
ologic and pathologic relevance. J Invest Dermatol. Dermatovenerol Croat. 2006;14:100–3.
1999;113:424–5. 26. Gogas H, Ioannovich J, Dafni U, et al. Prognostic sig-
17. Dumaz N, van Kranen HJ, de Vries A, et al. The role nificance of autoimmunity during treatment of mela-
of UVB light in skin carcinogenesis through the anal- noma with interferon. N Engl J Med. 2006;354:709–18.
ysis of p53 mutations in squamous cell carcinomas of 27. Michail M, Wolchock J, Goldberg SM, et al. Rapid
hairless mice. Carcinogenesis. 1997;18:897–904. enlargement of a malignant melanoma in a child with
18. Madan V, Hoban P, Strange RC, et al. Genetics and vitiligo vulgaris after application of topical tacroli-
risk factors for basal cell carcinoma. Br J Dermatol. mus. Arch Dermatol. 2008;144:560–1.
2006;154(Suppl 1):5–7. 28. Halder RM, Brooks HL. Medical therapies for vit-
19. Wang SQ, Setlow R, Berwick M, et al. Ultraviolet iligo. Dermatol Ther. 2001;14:1–6.
A and melanoma: a review. J Am Acad Dermatol. 29. Palm MD, O’Donoghue MN. Update on photoprotec-
2001;44:837–46. tion. Dermatol Ther. 2007;20:360–76.
20.
De Fabo EC, Noonan FP, Fears T, Merlino 30. Paro Vidolin A, Barbieri, L, Aurizi C, Marcassoli

G. Ultraviolet B but not Ultraviolet A radiation initi- LG, Leone G. Photoprotection in vitiligo patients: a
ates melanoma. Cancer Res. 2004;64:6372–6. new approach. Poster n. 13 Abstracts of the Vitiligo
21. Lim HW, Naylor M, Honigsman H, et al. American International Symposium (VIS), Detroit, 2018;9–10.
Academy of Dermatology consensus conference on
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Treating the Disease: Age, Gender,
Ethnic Skin, and Specific Locations
41
Savita Yadav and M. Ramam
Contents
41.2 A ge 438
41.2.1 Treatment of Vitiligo in Childhood 439
41.2.2 Vitiligo in Older Adults 442
41.3 Gender 442
41.4 High Phototypes 442
41.4.1 edical Treatment
M 443
41.4.2 Surgery 444
41.4.3 Vitiligo Phobia 445
41.5 Specific Locations 445
References 447
Abstract in children. In spite of all these limitations,

Vitiligo treatment has to be tailored to every response to medical as well as surgical treat-
individual after considering several individual ments is generally stated to be better in chil-
features, including age, gender, and ethnic dren compared to adults. Vitiligo in older
skin type as well as the specific location of patients does not usually produce as much dis-
patches. Vitiligo affects people of all age tress as in young people since the psychoso-
groups though the highest incidence is seen in cial burden is considerably less at this stage of
the second and third decades of life. Treatment life when getting married and securing
options are limited in childhood vitiligo employment are not of concern. The social
because some systemic therapies are contrain- stigma and cosmetic disfigurement of vitiligo
dicated, phototherapy is difficult to adminis- affect both men and women, but the burden is
ter, there are age-specific adverse effects of significantly heavier in women, particularly in
different medicines, and, finally, there are communities where marriages are arranged by
difficulties in undertaking surgical treatment families. Treatment options and response are
similar in men and women. Vitiligo in subjects
S. Yadav · M. Ramam (*) with darker skin poses particular problems
Department of Dermatology & Venereology, All India because of the greater visibility and disfigure-
Institute of Medical Sciences, New Delhi, India

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438 S. Yadav and M. Ramam
ment along with the widespread prejudices both molecular and clinical level, has led
and taboos associated with the disease in these advances in the arena of medical and surgical
communities. The location of patches is an treatment. Though we are some way from a cure
important determinant of response with vitil- for this disease, it has become possible to provide
igo on the hands and feet, knees and elbows, efficacious treatment to most patients with the
and the mucosae responding poorly to all cur- presently available range of options.
rent treatment modalities. Choice of surgical Vitiligo treatment has to be tailored to every
technique, dressing, and postoperative care individual after considering several demo-
and instructions are guided by the site affected. graphic and disease factors. In this chapter, we
will address the effect of age, gender, and ethnic
skin type as well as the specific location of
patches on choice of intervention and the treat-
ment response.
Key Points
• Treatment should be defined for the spe-
cific patient based on features related to 41.2 Age
the disease and the individual.
• Treatment options are somewhat limited Vitiligo affects people of all age groups though
in childhood vitiligo, but response to the highest incidence is seen in the second and
medical as well as surgical treatments is third decade of life. The disease commonly starts
generally stated to be better in children in childhood or young adulthood [7, 8]. There are
compared to adults. rare reports of congenital vitiligo as well [9].
• Vitiligo is usually less distressing in Expectedly, the psychological impact of the dis-
older subjects as the psychosocial bur- ease is marked in adolescence, a stage at which
den is low. concern about personal appearance is high lead-
• The social stigma and cosmetic disfig- ing to difficulty in coping with the stigma of dis-
urement of vitiligo affect both men and ease [10].
women, but the burden is significantly Vitiligo starts in the first decade of life in
heavier in women. about a fourth of all patients [11]. There is a
• Greater visibility and societal responses slight female preponderance in children com-
pose serious problems in subjects with pared to adults. The commonest morphological
dark skin, particularly in communities type is vitiligo vulgaris (generalized vitiligo) fol-
where vitiligo is stigmatized. lowed by focal, segmental, and acrofacial vitiligo
• Acral, periorificial, and mucosal vitiligo [11, 12]. Vitiligo in children has certain features
respond poorly to treatment. that are different from adult vitiligo. The propor-
• Choice of surgical technique is guided tion of focal and segmental vitiligo is higher in
by the site and total area affected. children compared to adults. It is usually more
extensive and associated with a higher proportion
of familial disease (12–35% compared to 6.25–
30% in general) [13]. Koebnor phenomenon,
41.1 Introduction halo nevi, and leukotrichia also appear to be more
frequent in children [11, 12, 14, 15]. Trauma-
Vitiligo is a common cutaneous disorder of prone sites like knee, elbow, and hands are fre-
depigmentation affecting around 1% of world quently involved. Childhood vitiligo can result in
population [1–4]. It carries a significant social significant psychological trauma in the form of
stigma which has a great impact on personal and teasing by others, loss of self-esteem, avoidance
social life [5, 6]. Research over the last decade, at of social gatherings, and anxiety.
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41 Treating the Disease: Age, Gender, Ethnic Skin, and Specific Locations 439
41.2.1 Treatment of Vitiligo

in Childhood
Before initiating treatment, a detailed discussion

regarding the nature of disease, its course, treat-
ment options, and expectations is necessary.
Treatment options are limited in childhood vitil-
igo because some systemic therapies are contra-
indicated, phototherapy is difficult to administer,
there are age-specific adverse effects of different
medicines, and, finally, there are difficulties in
undertaking surgical treatment in children. In Fig. 41.1 Hypopigmentation around macules of vitiligo
spite of all these limitations, response to medical following the use of a potent topical corticosteroid
as well as surgical treatments is generally stated
to be better in children compared to adults [16].
after using potent topical corticosteroids for
41.2.1.1 Medical Treatment 4–6 weeks [19]. Perilesional hypopigmentation
Topical steroids form the first-line therapy for (Fig. 41.1) is another side effect of potent topical
both adults and children with vitiligo by virtue of corticosteroids irrespective of indication but is of
good efficacy and low cost with side effects that particular significance in vitiligo as this adverse
are mainly local. Potent topical corticosteroids effect may be misinterpreted to be a sign of dis-
can be safely used for short durations in limited ease activity or failure of treatment. We have
disease involving less than 20% body surface found that stopping the potent topical corticoste-
area [17–19]. Two studies of the efficacy of roid leads to a reversal of this side effect in a few
potent topical corticosteroids conducted primar- weeks. Resumption of therapy with a less potent
ily in children showed that more than 50% repig- topical corticosteroid keeps it from happening
mentation could be achieved in 34% and 68% of again. In fact, we now initiate therapy with less
patients [20, 21]. potent preparations to forestall the development
Clobetasol propionate was found to be more of this distressing adverse effect.
effective than calcipotriol [22]. Kose et al. noted When topical corticosteroids are used for pro-
marked to complete improvement (75–100%) in longed periods over large surfaces, especially on
8 out of 20 patients treated with clobetasol and in the head and neck, there may be suppression of
none of the patients treated with calcipotriol. the hypothalamo-pituitary axis. Kwinter et al.
There was no to minimal improvement (0–50%) [23] found abnormal cortisol levels in 29% of
in 18 out of 22 patients receiving calcipotriol and tested children and recommended assays of early
in 7 out of 22 patients receiving clobetasol. Potent morning plasma cortisol levels at baseline and
topical corticosteroids showed better results than every 4 weeks in children treated with moderate-
with tacrolimus, though this difference was not to high-potency topical corticosteroids. However,
statistically significant [17]. this testing schedule may be burdensome to
The main limitations of topical corticosteroid undertake in clinical practice, and a more practi-
therapy are adverse effects which can be both cal approach would be to use topical corticoste-
local and systemic. Local side effects are com- roids cautiously and for short durations when
moner and include thinning of skin, telangiecta- treating children with extensive disease.
sia, and striae. These can be minimized by regular Topical tacrolimus has been found effective in
examination of skin and by alternating potent the treatment of vitiligo in children, both in the
with less potent corticosteroids every 6–8 weeks strengths of 0.03% and 0.1% [17, 24–26]. In our
or by providing steroid free intervals of 4–6 weeks practice, we almost exclusively use the higher
ERRNVPHGLFRVRUJ
strength, applied once daily. Silverberg et al. efficacy with clobetasol found calcipotriol had a
treated vitiligo in 57 children with tacrolimus weak effect. Calcipotriol and calcitriol are
(0.03% and 0.1%) with good response. Vitiligo therefore not recommended as monotherapy [22].
on the head and neck was more likely to respond Travis and Silverberg treated 12 children with
and also showed more complete repigmentation, topical corticosteroids in the morning and calci-
and twice daily dosing was found to be more effi- potriene in the evening. Ten (83.3%) patients
cacious. A randomized trial comparing tacroli- repigmented and no adverse effects were seen.
mus 0.1% ointment with clobetasol 0.05% This may suggest a role for calcipotriol in combi-
ointment found the mean percentage repigmenta- nation with topical corticosteroids, but most
tion with these agents was 41.3% and 49.3%, workers now do not use vitamin D derivatives in
respectively [17]. Ho et al. found similar results the treatment of vitiligo [30].
with an intermittent regimen of clobetasol and a
continuous regimen of tacrolimus administration 41.2.1.2 Phototherapy
for 6 months for both facial and non-facial vitil- Phototherapy can be considered for the treat-
igo [27]. On the face, successful repigmentation ment of extensive vitiligo involving more than
(>50% improvement) was noted in 58% of 20% of the skin surface and for less extensive
patients in both the clobetasol and tacrolimus but refractory disease. Narrow-band ultraviolet
groups. On non-facial sites, successful repigmen- B (NB-UVB) is preferred for phototherapy in
tation was noted in 39% and 23% of patients in children as it is well tolerated and effective.
the clobetasol and tacrolimus groups, respec- Phototherapy is best administered in a UV
tively. Even though it is significantly more expen- chamber when there is extensive involvement.
sive than topical corticosteroids, topical Data on the maximum dose of NB-UVB that
tacrolimus can be considered as the primary can be administered safely is lacking, but ordi-
treatment option for pediatric vitiligo at sites narily it is given for up to 1 year. If further treat-
such as the face and flexures where the skin is ment is necessary, a phototherapy-free period is
prone to topical corticosteroid-induced atrophy. recommended followed by limited exposure
In addition, it could be used as an alternative to [31]. Njoo et al. treated 51 children with a mean
topical corticosteroids at other sites. The repig- body surface area involvement of 15% with
mentation induced by tacrolimus is often darker twice weekly NB-UVB therapy for a maximum
than the surrounding normal skin and may take period of 1 year. Following treatment, 80% of
several weeks to normalize. Other side effects are children had stabilization of disease, and 53%
uncommon. of children had greater than 75% repigmenta-
Pimecrolimus was used in 60 children in com- tion. Vitiligo on the hands, feet, fingers, toes,
bination with microdermabrasion to enhance and bony prominences did not repigment well.
penetration. Three vitiliginous patches in the Minor side effects observed with NB-UVB ther-
same anatomical location were chosen for treat- apy included itching, erythema, and xerosis. No
ment in each patient [28]. The patches were photoallergic or phototoxic reactions have been
treated with pimecrolimus 1% cream alone for reported with NB-UVB, and there does not
10 days or pimecrolimus 1% cream application appear to be an increased risk of malignancy.
for 1 day after microdermabrasion or placebo However, there are some practical problems
application for 10 days. Clinical response (>50% with NB-UVB therapy in children. First, the
repigmentation) was seen in 60%, 32%, and 1.7% child has to be accompanied by an adult to the
patients in the microdermabrasion, pimecroli- clinic every time thus increasing the cost of
mus, and placebo group, respectively. treatment. Second, some children may not be
Calcipotriol showed some efficacy in an open comfortable standing alone in the phototherapy
label trial with 11 (77.8%) of 14 patients who chamber. Also, long-term safety data in children
completed the study showing some improvement exposed to NB-UVB during the early years of
[29]. However, a randomized study comparing its life is not available.
ERRNVPHGLFRVRUJ
Systemic photochemotherapy either as pso- Fourth, exposure to natural sunlight should be

ralen and ultraviolet A (PUVA) therapy adminis- avoided as far as possible, and sunscreen should
tered in a phototherapy chamber or with sunlight be used on photoexposed parts.
(PUVASOL) is contraindicated in children below
12 years of age because of the unknown long- 41.2.1.3 Surgical Treatment
term effects of oral psoralen, increased risk of The last two decades have seen a lot of work in
carcinogenesis, and premature aging of skin [32, the surgical management of stable vitiligo.
33]. However, this restriction may not be appli- Several surgical techniques have been described
cable in children with brown skin who have a (see Chap. 3.2.3), but our focus here is on the role
70-time lower risk of sunlight-induced cutaneous of surgery in stable childhood vitiligo. There is a
malignancies. dearth of literature on this subject, and studies
Children with localized disease refractory specifically addressing children are scarce. Most
to medical treatment can be offered locally studies on surgical treatment include either adults
directed photochemotherapy or phototherapy. alone or have a mixed study population of chil-
Topical PUVA or PUVASOL therapy is an dren as well as adults, but some conclusions can
option for localized disease [21]. A response be drawn.
rate of 53% was observed with paint PUVA There are several practical problems when
photochemotherapy in children [34]. Topical operating on children. First, the appropriate type
PUVA is generally less preferred over NB-UVB of anesthesia should be administered; otherwise,
because of the risk of unpredictable phototoxic the child may not cooperate during the proce-
reactions, perilesional hyperpigmentation, dure. The type of anesthesia required will depend
increased contrast between lesional and pig- upon the size and site of area to be treated, age,
mented skin, and the need for longer treatment and maturity of the child and type of surgery. In
though these can be minimized by careful use general, it is preferable to give some sedative
of the modality and detailed counseling of prior to surgery, use topical anesthetic creams
caregivers [31]. Recently, handheld NB-UVB under occlusion as far as possible, and avoid
units have become available and can be used to injectable anesthetic agents. Also, parents need to
treat localized vitiligo without whole-body be carefully taught what to do and what to avoid
exposure. Excimer laser has also been used following surgery to ensure good results.
similarly and found to be effective in children, Mulekar et al. [40] reported satisfactory
more so when combined with topical calcineu- results (more than 65% repigmentation) with the
rin inhibitors or corticosteroids which help to non-cultured epidermal cell suspension (NCES)
reduce the cumulative UV dose [35–38]. Cho technique in 80% of treated children and adoles-
et al. found that children respond better than cents and have suggested that this technique can
adults to excimer light and did not show any be considered the first choice to treat clinically
significant adverse effects [39]. stable segmental vitiligo in children [41]. As in
In a retrospective review of different modes of adults, focal and segmental vitiligo in children
phototherapy in children, Koh et al. observed that respond better to surgery than generalized vitil-
patients not responding to one mode may be igo in the amount, spread, and homogeneity of
responsive to other modes [34]. repigmentation.
Some guiding principles should be kept in As in adults, small stable patches can be man-
mind when using phototherapy in children, irre- aged with either mini-punch grafting (MPG) or
spective of the type of treatment [31]. First, parts suction blister epidermal grafting (SBEG) [42].
of the body which are not affected should be cov- In children, mini-punch grafting has some advan-
ered during treatment. Second, areas which have tages over suction blister epidermal grafting as
repigmented should be protected in subsequent the former procedure is quicker and does not
sessions. Third, genitals should be shielded as need require lying in a fixed position for long and
they invariably do not respond to phototherapy. the chances of post-procedure graft displacement
ERRNVPHGLFRVRUJ
are less than with suction blister epidermal graft- psychosocial burden is considerably less at this
ing. Studies suggest that repigmentation is faster, stage of life when getting married and securing
and there is greater peri-graft spread of pigment employment are not of concern. The stigma of
in young patients compared to old, with both vitiligo may affect the marriageability of their
techniques [16, 43]. Some have postulated that offspring, and they may worry about spreading
this may be due to a greater release of cytokines the disease to young grandchildren. Education
in young patients following the injury of grafting, and counseling may suffice for some patients.
which then stimulates melanocytes to proliferate Camouflage, even as the only intervention, is
and migrate briskly [44]. more acceptable at this stage of life. It is under-
Melanocytes from younger persons prolifer- taken in order to overcome the extended effects
ate better in in vitro cultures than those from of social stigma on children and other members
older people [45, 46]. These in vitro data sug- of the family. Localized disease can be addressed
gest that autologous melanocyte culture and with topical corticosteroids or calcineurin inhibi-
transplantation should give good results in chil- tors. For those who seek more aggressive therapy
dren. In a clinical study, Hong et al. treated vit- for extensive disease, photo(chemo)therapy is a
iligo in 12 children, 20 adolescents, and 70 good option as it avoids the adverse effects of
adults with cultured pure melanocyte transplan- immunosuppressive systemic agents in patients
tation with a mean extent of repigmentation of who may have other comorbidities such as diabe-
80.7%, 78.9%, and 76.6%, respectively, though tes mellitus and hypertension.
this difference was not statistically significant.
These results indicate that the technique is a
useful option in children with refractory stable 41.3 Gender
disease covering an extensive area [47].
Acceptability of this technique is higher among Vitiligo occurs with almost equal frequency in
parents as a large area of vitiligo is treated from men and women though some studies suggest a
a small donor area. mild female preponderance [48–50]. The social
To conclude, surgical techniques lead to good stigma and cosmetic disfigurement of vitiligo
results in children with stable vitiligo but require affect both men and women, but the burden is
detailed planning of both the procedure and anes- significantly heavier in women, particularly in
thesia along with good postoperative care by par- communities where marriages are arranged by
ents to achieve the best outcome. families. This may lead to pressure on the derma-
tologist to treat girls and women more aggres-
41.2.1.4 Treatment of Psychosocial sively. Most studies indicate that the response to
Aspects of Disease medical and surgical treatment is similar in men
Besides medical and surgical treatment, coun- and women. Women and their families may
seling of the child and parents is important as require greater psychosocial support.
the disease course is chronic and repigmentation
takes time. Sometimes, the help of professional
counselors/psychiatrists may be necessary in 41.4 High Phototypes
case preliminary assessment reveals significant
psychological distress in the child and/or The prevalence of vitiligo varies from 0.1% to
parents. 4% worldwide [51–55]. There is some data to
suggest that the disease is more frequent in peo-
ple with darker skin and begins earlier in life [3,
41.2.2 Vitiligo in Older Adults 48, 56]. Vitiligo vulgaris is the most common
type in Southeast Asia, and most studies from
Vitiligo in older patients may produce as much this region indicate that the disease occurs with
distress as in younger people, but more often the equal frequency in men and women [48, 57–61].
ERRNVPHGLFRVRUJ
Vitiligo in darker skin types poses particular

problems because of the greater visibility and
disfigurement along with the widespread preju-
dices and taboos associated with the disease in
these communities, including the confusion of
vitiligo and leprosy in some [62, 63]. Patients and
their families report facing social ostracism, dif-
ficulties in getting a job, and formidable barriers
to getting married [64].
Because of the social stigma, patients want
every small patch treated and resist suggestions
to forgo treatment for small patches on covered Fig. 41.2 Follicular repigmentation in vitiligo
areas, for example. Leukotrichia in the absence
of adjacent skin involvement is more visible in
darker skin, and often patients demand treatment
for this. Even when people have immigrated to
countries where the social stigma of vitiligo is
not as burdensome, they continue to respond to it
in the manner of their communities of origin.
The various medical, surgical, and photother-
apy treatment options are similar for people with
darker skin as for other skin types. There are minor
variations in protocols and differences in the
response rates that we will discuss in this section.
Fig. 41.3 Hyperpigmentation following

41.4.1 Medical Treatment photochemotherapy
Topical corticosteroids steroids and tacrolimus can dose-related to a great extent. While inadequate
be safely used, and the response is similar to that in doses will result in a poor response, overdosing
other skin types [65]. Vitiligo on the head and neck of oral psoralen or using a higher strength of
responds best, while there is a relatively poorer topical psoralen solution leads to repigmentation
response on the trunk and limbs. The pattern of that is excessively dark (Fig. 41.3). When sun-
repigmentation is usually diffuse on the head and light is used for photochemotherapy, the duration
neck, while vitiligo on the trunk and extremities of light exposure should be increased gradually,
shows both diffuse and follicular patterns (Fig. 41.2) and patients should be encouraged to take treat-
of repigmentation with roughly equal frequency. ment at the same time of day as the intensity of
Tacrolimus is safe for long-term use, but caution sunlight varies at different times of day. Patches
needs to be exercised while using topical steroids. that are completely repigmented can be covered
In darker skin types, corticosteroid-induced telangi- when exposing to light to prevent further darken-
ectasias and atrophy are less obvious, so these ing. As the risk of cutaneous malignancy is rela-
adverse effects must be looked for carefully and tively low in dark skin, photochemotherapy can
treatment modified when they are noted. be given for longer periods. Response to
Photo(chemo)therapy is effective in produc- photo(chemo) therapy tends to be better than in
ing repigmentation in vitiligo [66]. When used in those with lighter skin [67]. Conversely, as pig-
darker skin types, there are special considerations mented skin tans easily, this may heighten the
to keep in mind. Both oral and topical psoralen contrast between unaffected skin and vitiligo
should be dosed appropriately as the response is leading to an apparent worsening of disease.
ERRNVPHGLFRVRUJ
Lastly, depigmented patches on exposed skin et al. [69] reported excellent repigmentation (90–
should be protected from sunlight to avoid 100%) in 71% (20 out of 28 patches) of the lesions
phototoxicity. in the NCES group compared to 27% (7 out of 26
Some studies have compared the different patches) of the lesions in the SBEG group at the
photo(chemo)therapeutic techniques in pig- end of the study period of 16 weeks [69]. Color
mented skin. A study comparing PUVA therapy match and pattern of repigmentation were similar
administered using light chambers with PUVAsol in the two groups. Initial repigmentation was
reported quicker onset and greater total repig- faster in vitiligo treated with suction blister grafts,
mentation and greater improvement in quality- but pigment spread and homogeneity of repig-
of-life with PUVA at the end of 9 months of mentation were better with non-cultured epider-
treatment [66]. Another study comparing mal suspension. Hyperpigmentation is a common
NB-UVB therapy with systemic PUVA therapy adverse event in darker skin types, both at the
found that the median repigmentation achieved at donor and the recipient site, and this was the most
the end of 6 months was similar, 45% with common adverse event when treating with suction
NB-UVB and 40% with PUVA. However, the blister grafting (SBG) [43].
color match was better in the NB-UVB group, Leukotrichia responds poorly to medical treat-
while side effects were more frequent with PUVA ment. It is amenable to surgical treatment to some
(57%) than with NB-UVB (7.4%) [68]. extent provided the disease is stable. Holla et al.
retrospectively analyzed the improvement in leu-
kotrichia in patients who had undergone NCES
41.4.2 Surgery for vitiligo [70]. They found improvement in leu-
kotrichia in 88% of 42 treated patches.
Several different surgical techniques including Improvement was faster in body hair than pubic
mini-punch grafting, ultrathin split-thickness skin hair and hair on the face and scalp. Initial repig-
grafting, suction blister grafting, and non-cultured mentation was observed as early as 2 months on
epidermal cell suspension (Fig. 41.4a, b) were the trunk and extremities with almost complete
first developed and are regularly used in darker improvement in body hair at 6 months.
skin patients. We would like to present the differ- Some workers have used tattooing with dark
ential response to treatment in this particular skin colored dyes to camouflage refractory vitiligo in
type. For somewhat larger patches, non-cultured individuals with dark skin. This technique is
epidermal suspension (NCES) is a good surgical reported to work well for patches over the nipple,
approach which showed better results than suc- areola, lip, genitalia, and scalp where a good
tion blister grafting in an Indian study. Budania color match can be achieved with proper s election
a b
Fig. 41.4 Vitiligo (a) before and (b) after grafting with non-cultured epidermal cell suspension
ERRNVPHGLFRVRUJ
of dye color and appropriate technique. The pro- topical steroids, patients should be closely moni-
cedure can be done under local anesthesia using tored for cutaneous atrophy. Ophthalmological
medical grade tattooing dye and an electrical tat- adverse effects such as glaucoma and cataract are
tooing machine [71]. The right shade is made by uncommon but must be kept in mind. Patients
mixing the basic pigment and matching with the with eyelid vitiligo undergoing phototherapy in a
color of the surrounding skin. Results are imme- UV chamber for more extensive vitiligo can just
diate and permanent and the procedure is inex- keep the eyes closed to protect them and avoid
pensive. However there are problems with using goggles which would prevent the beneficial
leaching of dye, color mismatch, change in shade effects of UV light on eyelid skin.
over time, and the risk of infection. For these rea- Sometimes, in refractory lesions, surgical
sons, tattooing is no longer a recommended treat- management is warranted. Non-cultured epider-
ment technique. mal suspension (NCES) is a good technique at
this site as it does not require immobilization and
dressing of eyelids and also provides the best
41.4.3 Vitiligo Phobia color match. However, since the eyelids are a
curved surface, when pouring the suspension
The fear of developing vitiligo prompts consulta- over the recipient site, the head should be posi-
tions for a variety of hypopigmented macules in tioned such that the runoff of the suspension is
communities where vitiligo is stigmatized. minimal. Suction blister epidermal grafting
Dermatologists should remember that the treat- (SBEG) also provides a reasonably good color
ment of such patients is incomplete unless it is match, and graft uptake tends to be very good
clearly stated that the disease is not vitiligo, even because of the rich vascularity of this area. A dis-
if the patient has not explicitly asked the ques- advantage is the need for postoperative dressing
tion. Similarly, patients who have vitiligo should and immobilization of the eyelid for 1 week.
be reassured that every hypopigmented macule Tissue glue can be used to prevent the displace-
they notice on their skin does not represent an ment of grafts. Mini-punch grafting (MPG) is
extension of the disease. preferred less as the skin is very thin over the eye-
lid making it technically challenging and cobble
stoning, and polka dot appearance can compro-
41.5 Specific Locations mise the results.
Recipient-site preparation is difficult in eyelid
Apart from type of vitiligo and extent of disease, vitiligo. When administering anesthesia, the skin
response to treatment depends to a great extent should be infiltrated enough to make it tumes-
on the site of lesions. Vitiligo on the head and cent, and injection into the eyelid margin should
neck and hair-bearing areas responds more rap- be avoided to prevent damage to eyelash roots.
idly and completely to treatment [17, 18, 48] On Laser dermabrasion is preferred as it allows more
the other hand, vitiligo on the hands and feet, precise control, while mechanical or motorized
bony prominences (knees, elbows, ankles), and dermabrasion is difficult at this site as there is no
mucosa responds poorly to both surgical and underlying bony support. Care must be exercised
medical treatment. to avoid injury to the eye during vitiligo surgery.
We describe below the issues related to treat- Perioral vitiligo is relatively resistant to treat-
ment of vitiligo at particular body sites. ment. Medical management is similar to that of
Periocular vitiligo can be easily managed with eyelid vitiligo but is usually less effective. In
medical treatment alone in most patients. addition, the role of recurrent herpes labialis in
Repigmentation tends to be brisk and complete producing and perpetuating depigmentation
at this site. Topical steroids (low to mid-potency) needs to be considered. If recurrences are fre-
or calcineurin inhibitors (tacrolimus and quent, suppressive antiviral therapy may help to
pimecrolimus) are effective options. When using control both herpes and vitiligo.
ERRNVPHGLFRVRUJ
Localized phototherapy with a handheld vitiligo in 20 patients, before and after PUVA
NB-UVB therapy device may be tried but is therapy. They found that factors such as inher-
unlikely to be helpful. Surgical treatment may be ent low melanocyte density, lower melanocyte
considered if the disease is stable. Like the eye- stem cell reservoirs, and lower baseline epider-
lid, the lip is a curved surface, and runoff of the mal stem cell factor may possibly be playing a
suspension has to be avoided during the non- role. Most patients get <25% or 25–50%
cultured epidermal suspension (NCES) technique repigmentation with medical treatment and/or
by proper positioning of the head. Suction blister phototherapy, and complete repigmentation is
epidermal grafting (SBEG) on the lip runs a risk hardly ever achieved. Many authors have tried
of graft displacement. This can be avoided by combination therapies in order to get better
immobilizing grafts with fibrin glue [72] and sur- results (Table 41.1).
gical tapes [73], restricting oral intake to liquids In the study by Bayoumi et al. [81], laser
during the first few postoperative days, and dermabrasion enhanced the treatment response of
restricting the movement of perioral muscles. A vitiligo over resistant sites to combination ther-
study comparing mini-punch grafting (MPG) and apy with steroids and phototherapy. Complete
SBEG concluded that both techniques were repigmentation was seen in 16.7% of lesions on
effective, but MPG gave a better color match. the dermabraded side but in none of the lesions
Cobble stoning was the most common adverse on the control side. Unfortunately, side effects
event with MPG and hyperpigmentation with were common in the dermabrasion group and
SBEG [74]. made this a less preferred option in spite of the
Postoperative care is imperative irrespective good results. Dermabrasion possibly enhances
of the technique in order to achieve good results the biological UV impact by increasing the pen-
as the operated area is affected by brushing, eat- etration of UV rays as well as topical steroids,
ing, talking, and drinking. The patient should be enhancing production of inflammatory cytokines
instructed to take only liquids through a straw or with a pro-pigmenting action and removing
using a vessel with a spout for the first 2 days and affected keratinocytes.
a semisolid diet for the next 5 days. Every effort Surgery (NCECS and SBEG) can be tried
should be made to keep the area clean and dry but repigmentation is not as good as at other
and movement of the perioral muscles (as in talk- sites [84]. Postoperative immobilization is
ing) avoided, as far as possible. difficult in these areas, and complications
Genital vitiligo can have profound psycho- such as delayed healing and secondary infec-
sexual effects on the patient and/or the partner. tion are more common. For small patches,
Topical calcineurin inhibitors are the preferred punch grafting is preferred and gives good
treatment at this site [75]. Topical steroids (low to results even on acral areas. Gupta et al.
mid-potency) can also be used for short dura- reported good results with suction blister epi-
tions. However, the treatment response tends to dermal grafting (SBEG) on acral areas [43].
be poor [76]. The response to phototherapy is Results may be enhanced by using PUVA-
also not good. Surgery is relatively difficult com- exposed epidermal grafts [85].
pared to other body sites but has been attempted Scalp vitiligo is more obvious when the over-
[77, 78]. lying hair is white. It can be managed with a
Elbows, knees, hands, and feet respond topical corticosteroid lotion that may be easier to
poorly to all treatment modalities: medical, sur- use than a cream or ointment. Localized photo-
gical, and phototherapy. In an attempt to find an therapy with NB-UVB combs may be helpful. If
explanation for the poor response, Esmat et al. leukotrichia is not extensive, plucking out the
[79] took biopsies from vitiliginous and perile- white hair is a simple and rapidly effective solu-
sional skin on the trunk and proximal limbs and tion. White hair may regrow but can be plucked
compared them with similar biopsies from acral out again.
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Table 41.1 Different combination treatments tried to treat vitiligo at resistant sites
Author and
S No year Study design Patient no. Results Comments
1 Garg et al. Treated all patches with 40 vitiligo 50% patches showed Only minor adverse
[80] combination of patches in marked repigmentation effects
microdermabrasion 22 patients after 1 or max 4 sessions
(2 weekly, 1–5 sittings) of microdermabrasion
and topical 5% 5-FU
twice daily
2 Bayoumi Randomized left and right 18 At least 50% Laser dermabrasion
et al. [81] comparative study done to repigmentation achieved improved the
find the effect of ErYAG in 50% and 4.2% treatment response
laser dermabrasion on patches in active and significantly but had
response to treatment with control group, side effects like
combination of topical respectively delayed healing,
steroid and phototherapy hypertrophic scar,
and pain
3 Li et al. [82] Active side—CO2 laser 25 patients 44% patches in active Combination
dermabrasion along with with group achieved more therapy works better
topical steroid and symmetrical than 50% in resistant patches
phototherapy. stable resistant repigmentation which
Control side—CO2 laser vitiligo was higher than the
dermabrasion and patches control group
phototherapy
4 Mohamed Group I—5-FU 68 adult 50% patches in Group Only minor adverse
et al. [83] Group II—CO2 laser patients with III achieved Grade 4 effects
Group III—combination symmetric repigmentation
acral vitiligo
9. Kedward AL, Gawkrodger DJ. Congenital stable

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Psychological Interventions
42
Panagiota Kostopoulou and Alain Taïeb
Contents
42.1 Why Psychological Support Is Important 452
42.2 Screening Patients in Need of Psychological Support 452
42.3 Psychological Interventions 453
References 453
Abstract
A low self-esteem and high levels of per-
Key Points
ceived stigma seem to be important factors
• A low self-esteem and high levels of
for quality of life impairment in vitiligo
perceived stigma seem to be important
patients. A simple visual analog scale from 0
factors for quality of life impairment in
to 10 with the notice ‘How much does your
vitiligo patients.
skin disease bother you currently?’ helps to
• A simple visual analog scale from 0 to
measure coarsely how patients feel about
10 with the notice ‘How much does
their disease independently of medical find-
your skin disease bother you currently?’
ings. Patients with high perceived severity are
helps to measure coarsely how patients
the best targets of psychological interven-
feel about their disease independently of
tions. Cognitive-behavioural therapy might
medical findings.
help improve the quality of life, self-esteem
• Patients with high perceived severity
and perceived body image.
are the best targets of psychological
interventions.
• Cognitive-behavioural therapy might
help improving the quality of life, self-
esteem and perceived body image.
P. Kostopoulou · A. Taïeb (*)

Service de Dermatologie, Hôspital St André,
Bordeaux, France

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452 P. Kostopoulou and A. Taieb
42.1 W
hy Psychological Support study, we found that perceived severity of the dis-
Is Important ease and patient’s personality are important fac-
tors to consider when assessing the psychological
Although vitiligo does not lead to severe physical impact of vitiligo. If self-body image is more
illness, patients experience a variable degree of influenced by gender, perceived severity is more
psychosocial impairment. The psychological influenced by patient’s personality than by objec-
impact of vitiligo has been shown in different tive criteria of the disease (Fig. 42.1) [17].
studies all over the world. Patients with vitiligo
suffer from poor body image, low self-esteem
and social isolation, caused by feelings of embar- 42.2 S
creening Patients in Need
rassment, and they experience a considerable of Psychological Support
level of disability [1–7]. The prevalence of psy-
chiatric morbidity associated with vitiligo ranges Objective criteria like percentage of the body
from 25% to 30% in Western Europe [2, 8] and area affected and staging score [15] are important
from 56% to 75% in India [3, 9, 10]. A low self- but not enough for patient’s assessment and fol-
esteem and high levels of perceived stigma seem low-up. A psychologically oriented interview is
to be important factors for quality of life impair- essential in order to evaluate the perceived impact
ment in vitiligo patients [9, 11–16]. The majority of the skin disorder. This perceived impact is
of patients with vitiligo found their disfigurement important to tailor the management to the
moderately or severely intolerable [14], and most patients’ needs and difficulties. A simple visual
of them said that vitiligo had affected their lives analog scale from 0 to 10 with the notice ‘How
recently [9]. Psychological interviews with much does your skin disease bother you cur-
patients at our department c onfirm that vitiligo rently?’ helps to measure coarsely how patients
has an important impact on their daily lives. feel about their disease independently of medical
Visible or not directly visible lesions influence findings. A simple questionnaire of quality of life
not only their personal relationships and their such as the DLQI [18, 19] which is very easy and
professional career but also their social life. rapid to use can be also be used to measure
Patients said they avoided daily activities and dif- patients’ daily difficulties associated to their skin
ferent social events in order to protect themselves disorder.
from embarrassing comments [17]. In the same
Fig. 42.1 Relative 25
importance of factors
affecting the variance of
perceived severity 20
23
15
%
16
10
5
1
0
1 2 3
Factors associated 1=demographic factors 2=objective
severity 3=personality
ERRNVPHGLFRVRUJ
42 Psychological Interventions 453
42.3 Psychological Interventions 42.4 Concluding Remarks
Different types of psychological support and help Psychological support has primarily the intention
can be proposed. Most published articles concen- to help the patients express their psychological
trate on the type of psychological difficulties and suffering and the negative feelings associated
the factors that influence them, but do not men- with the disease. It is also aimed at helping the
tion specific interventions. The only psychologi- patients to accept themselves as they are, with or
cal intervention published in vitiligo is that of without the disease. In case of beneficial impact
Papadopoulos et al. [12], who have used a of the intervention, patients may be able not only
cognitive-behavioural therapy. They indicated it to resume their activities and to participate in
can help improve the quality of life, self-esteem their social life without feeling handicapped by
and perceived body image. They also suggested their skin disorder but also to develop their own
that cognitive-behavioural therapy may influence personality without being restricted by disease
the progression of the condition itself. Even if considerations [16, 20–26].
their findings are based to a very small sample of
patients and if their conclusions are difficult to
acknowledge without reservation, this work References
offers new perspectives. Dermatologists might
consider adding psychosocial interventions to 1. Firooz A, Bouzari N, Fallah N, et al. What patients
standard medical treatment [13]. with vitiligo believed about their condition. Int J
In addition, based on our experience, a sim- Dermatol. 2004;43:811–4.
2. Kent G, Al Abadie M. Factors affecting responses on
ple psychological interview and a supportive dermatology life quality index items among vitiligo
attitude can also be beneficial. Patients are sufferers. Clin Exp Dermatol. 1996;21:330–3.
happy to have the opportunity to express diffi- 3. Matoo SK, Handa S, Kaur I, et al. Psychiatric morbid-
culties resulting from their disease. Being ity in vitiligo: prevalence and correlates in India. J Eur
understood and listened to is a part of the global 4. Porter JR, Beuf AH, Lerner A, Nordlund
management of vitiligo. Sometimes, a simple J. Psychosocial effect of vitiligo: a comparison of
discussion with a professional can help the vitiligo patients with ‘normal’ control subjects, with
patient becoming free from the complexes psoriasis patients and patients with other pigmentary
disorders. J Am Acad Dermatol. 1986;15:220–4.
caused by the disease and embarrassing com- 5. Porter JR, Beuf AH, Lerner A, Nordlund
ments he has endured. Throughout this process, J. Psychological reaction to chronic skin disorders: a
patients may move from a state of discomfort to study of patients with vitiligo. Gen Hosp Psychiatry.
a greater acceptance of their image. During the 1979;1:73–7.
6. Porter JR, Beuf AH, Lerner A, Nordlund J. The
interview, we can also evaluate the patient effect of vitiligo in sexual relationships. J Am Acad
socio-professional context and the stigmatisa- Dermatol. 1990;22:221–2.
tion caused by the disorder either at work or 7. Sampogna F, Raskovic D, Guerra L, et al.
during daily activities. Different strategies to be Identification of categories at risk for high qual-
ity of life impairment in patients with vitiligo. Br J
accepted and to better enjoy social life can be Dermatol. 2008;159:351–9.
proposed. Accordingly, a multidisciplinary team 8. Picardi A, Abeni D, Melchi CF, et al. Psychiatric mor-
should manage, mainly at first visit, the patient. bidity in dermatological outpatients: an issue to be
Patients who can benefit a psychological follow- recognised. Br J Dermatol. 2000;143:983–91.
9. Kent G, Al’Abadie M. Psychologic effects of vitiligo:
up can receive more attention and may just feel a critical incident analysis. J Am Acad Dermatol.
better because of that. An objective is that 1996;35:895–8.
patients’ negative thoughts related to the disease 10. Matoo SK, Handa S, Kaur I, et al. Psychiatric morbid-
should be gradually replaced by other more pos- ity in vitiligo and psoriasis: a comparative study from
India. J Dermatol. 2002;28:424–32.
itive based on their personality and qualities. By 11. Harlow B, Poyner T, Finlay AY, Dykes PJ. Impaired
doing so, patients should improve their self- quality of life of adults with skin disease in primary
esteem and socialisation. care. Br J Dermatol. 2000;143:979–82.
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12. Papadopoulos L, Bor R, Legg C. Coping with the dis- vitiligo: using clinical significance to measure the
figuring effects of Vitiligo: a preliminary investigation potential effectiveness of enhanced psychological
into the effects of Cognitive-Behavioural Therapy. Br self-help. Br J Dermatol. 2014;171(2):332–7.
J Med Psychol. 1999;72:385–96. 21. Chan MF, Chua TL. The effectiveness of thera-

13. Picardi A, Abeni D. Can Cognitive-Behavioural
peutic interventions on quality of life for vitiligo
Therapy help patients with vitiligo? Arch Dermatol. patients: a systematic review. Int J Nurs Pract.
2001;137:786–8. 2012;18(4):396–405.
14. Salzer B, Schallreuter KU. Investigation of the per- 22. Eleftheriadou V, Whitton ME, Gawkrodger DJ,

sonality structure in patients with vitiligo and a pos- et al. Vitiligo priority setting partnership. Future
sible association with catecholamine metabolism. research into the treatment of vitiligo: where
Dermatology. 1995;190:109–15. should our priorities lie? Results of the vit-
15. Taieb A, Picardo M. The definition and assessment of iligo priority setting partnership. Br J Dermatol.
vitiligo: a consensus report of the Vitiligo European 2011;164(3):530–6.
Task Force. Pigment Cell Res. 2007;20:27–35. 23. Wang KY, Wang KH, Zhang ZP. Health-related quality
16. Bonotis K, Pantelis K, Karaoulanis S, et al.
of life and marital quality of vitiligo patients in China.
Investigation of factors associated with health-related J Eur Acad Dermatol Venereol. 2011;25(4):429–35.
quality of life and psychological distress in vitiligo. J 24. Thompson AR, Clarke SA, Newell RJ, Gawkrodger
Dtsch Dermatol Ges. 2016;14(1):45–9. DJ, Appearance Research Collaboration (ARC).
17. Kostopoulou P, Jouary T, Quintard B, et al. Objective Vitiligo linked to stigmatization in British South Asian
vs subjective factors in the psychological impact of women: a qualitative study of the experiences of liv-
vitiligo: the experience from a French referral centre. ing with vitiligo. Br J Dermatol. 2010;63(3):481–6.
Br J Dermatol. 2009;161(1):128–33. 25. Ongenae K, Van Geel N, De Schepper S, Naeyaert
18. Finlay AY, Khan GK. Dermatology Life Quality
JM. Effect of vitiligo on self-reported health-related
Index (DLQI). A simple practical measure for routine quality of life. Br J Dermatol. 2005;152:1165–72.
clinical use. Clin Exp Dermatol. 1994;19:210–6. 26. Whitton M, Pinart M, Batchelor JM, Leonardi-Bee
19.
Finlay AY. Quality of Life Measurement in J, Gonzalez U, Jiyad Z, Eleftheriadou V, Ezzedine
Dermatology: a practical guide. Br J Dermatol. K. Evidence-based management of vitiligo: sum-
1997;136:305–14. mary of a Cochrane systematic review. Br J Dermatol.
20. Shah R, Hunt J, Webb TL, Thompson AR. Starting to 2016;174(5):962–9. https://doi.org/10.1111/bjd.14356.
develop self-help for social anxiety associated with Epub 2016 Mar 25. Review.
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Patients’ Perspectives
43
Jean-Marie Meurant
Contents
43.2 The Contrasting Perceptions of Doctors and Patients 457
43.3 The Case for Providing Holistic Care for Sufferers 457
43.4 The Skin and the Sufferer: Two Responses to Two Distinct Issues 458
43.5 Trust, the Doctor-Patient Relationship, and Phototherapy 458
43.6 The Case for Care Schemes 459
43.7 Existing Transitional Care 459
43.7.1 sychological Care
P 460
43.7.2 Corrective Makeup 460
43.7.3 Sunscreen Cover 461
43.8 Patient/Sufferers’ Associations 461
43.9 Conclusion 462
Reference 462
Abstract
isolation, depression, and self-harm, as well as
It is important to take into account the major weakening the doctor-patient relationship and
psychological effects of vitiligo in direct suf- the ability to educate the patient to follow their
ferers and their immediate family (parents of treatment against vitiligo and diagnosed
children, siblings, etc.) towards treatment. comorbidities. Announcing a diagnosis of vit-
Above all, the seriousness of the disease iligo may entail psychological support being
should not be minimised by doctors and/or arranged for sufferers, and their families, soon
sufferers’ friends and family because doing so after the announcement, by clinical psycholo-
entails the risk of increasing the risk of gists specialising in skin diseases. In addition,
implementing individual or group psychologi-
cal support such as support groups and/or cor-
rective makeup workshops in individual or
J.-M. Meurant (*)
Association Française du Vitiligo, Paris, France group sessions, assisted by professionals and
e-mail: jean-marie.meurant@afvitiligo.com clinical psychologists, is recommended in

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456 J.-M. Meurant
order to help sufferers cope with the difficult

situations which can often arise. The use of clinical psychologists specialising in
phototherapy devices at home is recommend- skin diseases.
able for better treatment monitoring and long- • Implement, or direct patients towards,
term efficacy, in view of work and transport transitional care schemes pending ther-
considerations. There is a need to educate apy solutions, in the form of individual
patients about favourable sun exposure condi- or group psychological support such as
tions, recommending sunscreen creams and support groups and/or corrective
solar-protective clothing to avoid being “shut makeup workshops in individual or
away”, as witnessed by a large number of vit- group sessions, assisted by profession-
iligo sufferers. The awareness of the risks of als and clinical psychologists, in order
depigmentation caused by repeated rubbing to help sufferers cope with the difficult
should be raised. A global care perspective situations which can often arise.
enables better compliance, allowing sufferers • Recommend the use of phototherapy
to become less withdrawn and thus avoiding devices at home for better treatment mon-
loss of self-esteem, resulting in better inclu- itoring and long-term efficacy, in view of
sion in family, school, and professional life. work and transport considerations.
Providing information about any local vit- • Educate patients about favourable sun
iligo sufferers’ associations is important: these exposure conditions, recommending
are useful points of contact for the various sunscreen creams and solar protective
players in the healthcare system, overcoming clothing to avoid being “shut away”, the
the isolation of sufferers, speaking on their experience of a large number of vitiligo
behalf, and moderating their statements by sufferers. Raise awareness of the risks
providing objective information and serving of depigmentation caused by repeated
as credible references. rubbing.
• Arrange global care to enable treatment
to be followed better, enabling sufferers
to become less withdrawn and thus
Key Points avoiding loss of self-esteem, resulting in
• Take into account the major psychologi- better inclusion in family, school, and
cal effects of vitiligo; direct sufferers and professional life.
their immediate family (parents of chil- • Provide information about any local vitil-
dren, siblings, etc.) towards treatment. igo sufferers’ associations: these are use-
• Above all, the seriousness of the disease ful points of contact for the various players
should not be minimised by doctors and/ in the healthcare system, overcoming the
or sufferers’ friends and family; doing isolation of sufferers, speaking on their
so entails the risk of increasing the risk behalf, and moderating their statements
of isolation, depression, and self-harm, by providing objective information and
as well as weakening the doctor-patient serving as credible references.
relationship or “therapeutic alliance”
and the ability to educate the patient to
follow their treatment against vitiligo
and diagnosed comorbidities. 43.1 Introduction
• Announcing a diagnosis of vitiligo may
entail psychological support being Vitiligo sufferers very frequently say that their
arranged for sufferers, and their fami- future as a human being is at stake, that their life
lies, soon after the announcement, by is at risk of collapse, and that their relationships
with others have radically changed. Sufferers are
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43 Patients’ Perspectives 457
ashamed of having “patches” and become with- Sufferers vent their despair on social media,
drawn and afraid of others’ gaze; they suffer from weep in doctors’ surgeries, break down in sup-
inner feelings of guilt that undermine and may port groups, lock themselves up at home, cut
even seriously damage their self-esteem. themselves off from the world, and gradually slip
The suffering entailed by this state of affairs into depression: this can lead to self-harm and
may be exacerbated by a number of factors: attempted suicide.
Vitiligo is not simply a disease that affects the
• The sufferer’s environment being steeped in appearance. It is a full-blown auto-immune dis-
the exaggerated cults of beauty and health. ease with its related comorbidities and harmful
• Beliefs and rites entailing the risk of not being effects on the psyche; it therefore has serious
able to marry because of depigmentation, psychological repercussions.
being forced to divorce, being rejected, etc. As it loses its pigment, the skin—the protective
envelope for the inner self—creates a sensation of
The following are aggravating factors: having “flaws” and “holes” in this protection, weakening
a dark skin type, being in frequent contact with the person as a whole. Vitiligo also has comorbidi-
the public, and fragile personal circumstances. ties, and thus is not simply a weakness, but rather
Any major life changes (house move, change of an overall deficiency of the whole person, causing
school, job, etc.) can further exacerbate these isolation, depression, and risks of suicide.
situations. A large number of parents, pregnant mothers,
and women who want a baby ask questions about
whether they are to blame for having passed on
43.2 T
he Contrasting Perceptions vitiligo or indeed whether they might pass on
of Doctors and Patients their vitiligo or other related diseases. The seri-
ous thing about vitiligo is that contrary to a non-
The appearance of depigmented areas is almost visible disease, depigmentation is terribly and
systematically a traumatic experience for suffer- constantly visible on the face and hands, making
ers, as well as for their friends and family in the sufferer prey to stigma at all times.
many cases. This trauma may be assessed or
deemed to be unwarranted and exaggerated by
the examining physician or the sufferer’s partner. 43.3 T
he Case for Providing
This in no way changes the way in which suffer- Holistic Care for Sufferers
ers see themselves, their suffering, or the distress
that consumes and isolates them. Sufferers who attend a medical consultation for
Worse still, playing down or ignoring the their vitiligo have previously been through a lot,
painful feelings of sufferers increases their dis- both personally and in private, experiencing
tress and their feelings of guilt and shame. anxiety about the disease and its development
The increasing, deliberate visibility of “differ- combined with an overwhelming fear of the
ent” models who suffer from vitiligo (such as diagnosis. Almost systematically, sufferers will
Winnie Harlow in Canada, Tysca in France, and have already carried out Internet searches which
April Star in the USA) and the use of their ser- reinforce their anxiety about a potential lack of
vices by PR agencies, particularly in fashion treatment.
shows, should not obscure the reality experienced Hesitations about the diagnosis and fear
by the vast majority of sufferers, afflicted with caused by the announcement of the disease lead
depigmentation, who cannot or can no longer put patients into web searches that end up making
up with their skin, have to endure the stigma of them more vulnerable still.
other people looking at them, and feel excluded— Sufferers are constantly searching on the
all of which is reinforced by a lack of care and Internet. They go back and forth between medi-
satisfactory medical solutions. cal sites and sufferers’ associations, as well as
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458 J.-M. Meurant
websites where certain solutions are presented as There needs to be a radical shift from analy-
providing definitive treatment (from the Dead sis conducted solely in terms of the medical
Sea, Cuba, Tunisia, plant-based, aloe vera, etc.). issue of skin depigmentation and the reactions it
Each time, the sufferer will attempt to find a com- causes.
forting solution to bring an end to their tireless
quest for healing. This never-ending search leads
them to web surfing with all the related financial 43.4 T
he Skin and the Sufferer:
costs and risks (purchasing products with Two Responses to Two
untested effects, exotic treatments, travel, etc.). Distinct Issues
Vitiligo sufferers arrive in doctors’ surgeries
with high levels of anxiety. Sufferers have very The diseased organ should not obscure the patient
high expectations, which are vital in terms of themselves. Depigmented skin must not conceal
their conscious and subconscious self-projection the desperate sufferer, who is in dire need of
into a decidedly “patchy” future, one that will assistance, if other more serious damage is to be
revolutionise their lives. avoided. The latter is far costlier for the commu-
The physician that sees the sufferer is often nity around the sufferer and their family (with-
unable to provide a direct response in terms of drawal, academic difficulties, the consequences
treatment; this makes it difficult for them to of stigma and bullying, isolation, unemployment,
address the sufferer’s expectations. episodes of depression, suicide, etc.). This is also
A brief, swift consultation (involving a short the case for the comorbidities associated with vit-
time spent looking at the affected skin, so short it iligo: these require long-term treatment in a cli-
may even be perceived as being non-existent) mate of trust between the patient and their
will be experienced as a failure; it is as if the dis- physician (the main comorbidities currently
ease, what the patient has to say, and what they known are thyroid disorders, autoimmune diabe-
feel are not being taken into account. tes, pernicious anaemia, Addison’s disease, pso-
The patient may well understand that there riasis, rheumatoid arthritis, lichen planus, coeliac
are legitimate limits to the extent of possible disease, and alopecia).
treatment, but this will once again bring them
face to face with the fact that they must suffer
alone and confront them once more with the 43.5 Trust, the Doctor-Patient
rejection they feel and the stigma they regularly Relationship,
experience. and Phototherapy
Sufferers are therefore often tense and some-
times aggressive. As suffering human beings, The treatments which may be proposed to vitil-
they may be subject to outbursts of tears and/or igo sufferers are long term, lasting several
various types of decompensation: these are diffi- months: these are often felt to be a bind in busy
cult for physicians to cope with, given the techni- schedules, due to their frequency during the
cal and economic considerations of surgeries, be week, and often require non-negligible or even
they private or public, even in countries with long journey times.
well-developed healthcare systems. The demands of phototherapy sessions on suf-
In this context, many physicians report that ferers are clear: leaving their work or classes,
they find relations with vitiligo sufferers difficult. having to make the journey to the doctor’s sur-
They are only too aware of how the patient is gery or hospital, etc. The impact of these consid-
affected, the limited treatment possibilities, and erations may cause them to give up on their
the need to refer them to systems which are treatment for a time or even permanently.
beyond their own dermatology specialisation, Some sufferers may have already bought pho-
and so they prefer to minimise the extent to which totherapy devices for the face and hands on the
they are confronted with this pathology, given the Internet, to make it easier for them to follow their
lack of clear and/or alternative solutions. treatment.
ERRNVPHGLFRVRUJ
The possibility of prescribing home use of friends and families, and provide communication
phototherapy systems rented from recognised resources and solutions to mitigate the with-
professionals should be examined during the drawal and the stigma to which sufferers are very
course of the doctor-patient relationship. often subjected.
Procedures need to be identified; they include Psychological care, preferably immediately or
educating the patient by means of guidelines and alternatively at a later date, during consultations,
clear instructions for use, with regular monitor- clearly appears to be called for, in order to meet
ing of the treatment by the prescribing physician. the following three needs:
Checking the proper use of appliances and expo-
sure duration, together with the ease of use of • A medical need, with consultations focus-
home devices, clearly favours treatment being ing on being attentive to the appearance and
followed and increases its chances of being development of the disease, the diagnosis of
successful. the form of vitiligo present, potential solu-
In 2006, the Journal of Cutaneous Medicine tions, the treatment choices to be made, pre-
and Surgery (the official publication of the cautions to be taken (moisturising,
Canadian Dermatology Association) published a sunscreen, monitoring for related diseases),
study [1] in this respect which concludes as fol- and referral to global and individual psy-
lows: “Narrow-band ultraviolet B home photo- chological care.
therapy was found to be an effective form of • A psychological need, with the swift provi-
therapy for photoresponsive diseases, when com- sion of psychological care (this may even be
pared to the therapies delivered in hospitals. It is immediate in certain hospitals), giving an
safe and presents few side effects when patients opportunity for the sufferer’s private thoughts
receive appropriate guidelines, teaching, and to be expressed, as well as their experience of
follow-ups. Not only is it convenient; it also pro- the disease and its effects, its impact on their
vides effective savings for patients who are life and those of their friends and family, the
unable to attend the hospital owing to time, travel, professional and/or academic consequences,
and interference with work schedule. All patients its effect on self-esteem, etc. This care must
on home therapy were satisfied with their evaluate whether additional referrals should
treatment, plan to continue it, and recommend it be made in coordination with the GP and/or
to others in similar situations”. the physician who made the diagnosis and is
Some may be concerned that patients may not treating the vitiligo.
use the appliances correctly or that family mem- • A need to control healthcare costs by provid-
bers may misuse them; however, the latter con- ing overall care for the patient, in order to
cern also applies for any drugs kept in family avoid the all-too-frequent pitfall that consists
medicine cabinets, despite which it is not an in going from doctor to doctor to get as many
overriding issue in such cases. Vitiligo sufferers opinions as possible, leading to Internet surf-
are no different from other responsible patients ing and the risk of falling into the various traps
and should be trusted accordingly. of miracle solutions, unverified doses that may
pose risks to the patient’s health, and costly
travel and/or treatment.
43.6 The Case for Care Schemes
The progress of vitiligo research (aetiology, 43.7 Existing Transitional Care

potential avenues for treatment, clinical trials,
etc.) and the time required to develop therapeutic Transitional care should not be restricted simply
and drug-based solutions mean that the organisa- to individual psychological care, even though this
tion of transitional care schemes is called for. is one of the fundamental aspects for recovering
These can reduce the psychological impacts, as self-esteem and reducing the risks of depression.
well as their consequences for sufferers and their Drug-based treatments suited to the most difficult
ERRNVPHGLFRVRUJ
460 J.-M. Meurant
situations must be prescribed by specialists or acknowledged and are no different from others.
GPs. Support groups should be in addition to indi-
Individual psychological care tends to main- vidual therapy, rather than a replacement; indi-
tain the sufferer in isolation, as do individual cor- vidual therapy may open the way for this
rective makeup sessions. additional support.
Dedicated support groups for vitiligo sufferers Support groups overseen by clinical psychol-
and group corrective makeup workshops allow ogists allow sufferers to escape from their isola-
individuals to be less isolated and to discuss their tion by allowing them to discuss the disease and
experiences and feelings. They can see that other their coping practices and begin the process of
men and women are suffering too and are able to resilience. Sympathetic and benevolent listen-
talk about best practices and tips together. ing by other sufferers can help restore
self-confidence.
Social events and other meetings organised
43.7.1 Psychological Care by associations, in particular by people suffer-
ing from the same disease (after-work eve-
Vitiligo sufferers often need to be listened to and nings, themed days, holidays, etc.), are all
expect advice over and above specific treatment ways of combating isolation, particularly in
advice for the areas of depigmented skin. view of the discussions initiated by such
In hospitals, consultation with a psychologist events. They also facilitate recognition of the
may sometimes be tied in with dermatology extent to which the disease is burdensome on
departments; this can make patients attending sufferers. The presence of clinical psycholo-
consultations aware of the need to benefit from gists is also advisable, to facilitate and encour-
this kind of care and follow-up. This is however age benevolent expression.
rare in hospitals and in private practices.
Patients are disoriented by the diagnosis and
are greatly sometimes even deeply distressed. 43.7.2 Corrective Makeup
Sufferers often interpret oxidative stress in the
melanocytes as meaning “you are too stressed” The way others see sufferers of the disease, in
and thus feel they are being blamed for their particular when the depigmented zones affect the
condition. face and hands, is experienced as a stigma, even
Guilt contributes to a loss of self-esteem and if people are only looking out of compassion or
leads to withdrawal; this may even become seri- curiosity (“Is it contagious?”, “Does it hurt?”,
ous depression. etc.). The gaze of others is perceived as appor-
A personal interview with a clinical psycholo- tioning blame for being unlike other people. This
gist is one form of care. This allows the patient to is more particularly the case in countries in which
work on expressing their fears and anxiety and skin phototypes are dark and/or in jobs in contact
relate their personal journey with the disease and with the public, as well as in situations in which
its impact. individuals are especially vulnerable in terms of
However, faced with the many questions sur- self-image and how other people look at them
rounding the disease, other people’s opinions, (teenagers, young adults, etc.).
and so on, the possibility of offering additional It is therefore appropriate to arrange psycho-
care in the form of a support group overseen by logical care (or provide additional care) in the
clinical psychologists is a desirable one. form of actions aimed at lessening the difference
Support groups (or focus groups, e.g. work in colour between pigmented and depigmented
and vitiligo, children and vitiligo, etc.) allow zones.
discussion that relieves the patient from the Individual corrective makeup advice from a
weight of their experience as they share feelings professional makeup artist, or from tutorials to be
and emotions. At last, they feel that they are found on social media, can improve patients’
ERRNVPHGLFRVRUJ
quality of life by providing immediate solutions To meet differing expectations, specialised

which they can implement. However, this per- workshops can be arranged for children, teen-
sonal advice or Internet viewing has the draw- agers, and dark skin phototypes; advanced
back of maintaining sufferers seeking solutions workshops can also be organised. Workshops
in difficult, isolated situations. now tend to be mixed, with increasing num-
Corrective makeup workshops have four bers of men registering.
goals: Each participant can come with a friend or
family member; the latter’s presence will pro-
• Making patients less isolated, by giving them vide reassurance for them, encourage them to
the opportunity to meet other individuals who attend, and allow them to hear what the other
have the same skin disease, enabling them to participants have to say about the difficulty of
discuss their experience and the coping strate- living with a visible disease.
gies they use, share information about Participants are almost 100% satisfied. At
research, etc., as well as hear from other suf- one of the French Vitiligo Association makeup
ferers whose kind words will reassure other workshops, the following comment was made
participants. at the end of the session: “Thanks to you, I’ve
• Giving reasoned advice and answering ques- got my wife back”. This comment embodies
tions about skin care, sunscreens, etc. Such the extent of the changes that can take place—
workshops offer an opportunity for a direct and which are perceived by both participants
presentation and explanation of preconceived and their families.
ideas about sun exposure, the Koebner phe-
nomenon, and rubbing.
• Experimenting with a wide range of both spe- 43.7.3 Sunscreen Cover
cific and non-specific products for correcting
depigmented zones in order to find the “best” Although vitiligo sufferers do not need to avoid
shade, in the participant’s appraisal, for the the sun altogether, they do need to protect them-
correction they wish to benefit from. The sole selves regularly all year round, not simply during
arbiter of the effectiveness of the skin correc- holidays in the sun.
tion is the way individual patients see Sunscreen creams and UV protective clothing
themselves, even though the opinions of other are vital aspects to be included in advice, as well
patients present clearly play a moderating as in the financial cover provided by social secu-
role, in that they are deemed to be more impar- rity systems.
tial than individuals who do not suffer from
vitiligo.
• Providing participants with a greater degree of 43.8 Patient/Sufferers’
autonomy by giving them their very own Associations
opportunity to experiment with techniques
during the makeup workshop, benefit from Over the past few years, vitiligo patients’ associ-
personal advice dispensed by professional ations have become more organised, creating
corrective makeup artists and beauticians, and informational websites and a presence on social
choose suitable products from among those media, as well as taking part in public health
presented (the actual availability of the latter commissions and other similar bodies.
in stores or on the Internet should be checked Associations managed by sufferers for suffer-
beforehand). ers are becoming helpful points of contact for the
These workshops should be run according to a various players in the healthcare system
clearly defined protocol and be supervised by (researchers, organisations of healthcare profes-
one or two clinical psychologists and two cor- sionals, pharmaceutical firms, ministries and
rective makeup artists. government bodies, etc.), speaking on behalf of
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462 J.-M. Meurant
sufferers about their concerns and priorities, with 43.9 Conclusion

a view to promoting and contributing to more
efficient action and research. Sufferers are in the position of anxiously await-
Associations’ familiarity with the disease, the ing a therapeutic solution.
ability to mobilise sufferers, and credibility in the Minimisation of the disease (“it’s not seri-
eyes of sufferers are all assets for researchers ous!”), the dismissal of vitiligo as being solely a
when constructive collaboration is put into place question of appearance (“it’s only skin deep!”),
for the purposes of research. the paucity of the therapeutic solutions offered,
Sufferers’ associations also act as moderators and the perceived lack of compassion all increase
of their message, offering a detached way of the risk of withdrawal and major depressive epi-
informing sufferers via online communications, sodes. It is therefore appropriate to provide assis-
hotlines, answers to emails, and information tance through transitional solutions, throughout
meetings organised on their initiative. the journey of vitiligo patients, not simply from a
Patients’ and sufferers’ associations are genu- dermatological point of view but holistically.
ine sources of resilience. The skin must be treated—but the human who
They allow sufferers to escape from isolation has it on and who lives inside it must not be
and make them active participants in their own forgotten.
treatment—and potentially, their healing.
Sufferers are no longer helpless victims; instead,
they become informed and can help others thanks Reference
to their own experience as victims, thereby
becoming resource individuals. Such an achieve- 1. Haykal K-A, DesGroseilliers J-P. Are narrow-band
ultraviolet B home units a viable option for continu-
ment is an outstanding way of overcoming their ous or maintenance therapy of photoresponsive dis-
suffering. eases? J Cutan Med Surg. 2006;10(5):234–40.
ERRNVPHGLFRVRUJ
Discussion on Empirical,
Traditional, and Alternative
44
Treatments
Mauro Picardo
Contents
44.2 Chinese Traditional Products 464
44.3 Plant-Derived Extracts 465
44.4 Melagenin 465
44.5 Aspirin 465
44.6 Statins 466
44.7 Dermabrasion Combined with 5-Fluorouracil 466
44.8 Others 467
References 467
Abstract The pro-melanogenic effect may be related

Alternative treatments, such as traditional to antioxidant or anti-inflammatory proper-
Chinese products, plant-derived photosensi- ties; however, the main limitation of the pub-
tizing agents, and herbal and vitamin supple- lished studies concerns the non-complete list
ments, can certainly improve therapeutic of the used products as well as their chemical
outcomes in vitiligo. composition. Similarly, the effectiveness of
Traditional Chinese medicine is currently melagenin is still lacking the knowledge of the
attracting interest in dermatological research underlying mechanisms of repigmentation.
looking at the possible development of new On the basis of the in vitro study on immuno-
drugs. This approach is supported by the polit- modulatory action, aspirin and statins may be
ical strategies of Western medicine commu- considered for the treatment.
nity toward China. More controlled studies should be per-
formed before considering most of the new
emerging options based on unconventional
M. Picardo (*) drug or combinatory approaches.

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464 M. Picardo
and Singapore, many non-Asian countries have,

Key Points only in the last decades, recognized the usefulness
• The pro-melanogenic effect of prod- of this traditional practice in improving health and
ucts by traditional Chinese medicine providing supplementary options for patients
may be related to antioxidant or anti- looking for an alternative approach to their care.
inflammatory properties. Traditional Chinese medicine is currently
• The main limitation of the published attracting interest in dermatological research
studies concerns the non-complete list looking at the possible development of new
of the used products as well as their drugs. This approach is supported by the political
chemical composition. strategies of Western medicine community
• Melagenin, aspirin, or statins may act toward China.
through an immunomodulatory action. The use of herbal products and vitamin sup-
• Dermabrasion associated with 5FU may plements as enhancers or modulators of melano-
be applied in stable vitiligo. genesis is becoming particularly popular. This
• More controlled studies should be pro- pro-melanogenic effect may be related to antioxi-
vided, and increased knowledge of the dant or anti-inflammatory properties; however,
underlying mechanism of action should the main limitation of the published studies con-
support the use of these new approaches. cerns the non-complete list of the used products
as well as their chemical composition.
An early randomized controlled study per-
formed by Jin et al. in more than 200 subjects
44.1 Introduction described a complete or more than 60% repig-
mentation in 13% and 17% of patients treated
Clinical evidence has shown that alternative with undefined mixture of Chinese herbs [2]. The
treatments, such as traditional Chinese prod- results were carried out by comparing herbal
ucts, plant-derived photosensitizing agents, and treatment versus oral corticosteroids (15 mg/day,
herbal and vitamin supplements, can certainly decreased to 5 mg every 2–4 weeks), psoralen
improve therapeutic outcomes in vitiligo. Some (topical, 30%), or corticosteroids combined with
prospective controlled, double-blind, and ran- herbal products. After 2 months of treatment,
domized studies have been performed to iden- corticosteroids were more effective than herbal
tify mechanisms of action and potential adverse mixture since 31% and 14% of patients, respec-
effects supporting the efficacy and safety of tively, showed an excellent and a good (more
these products. A recent review article identified than 60%) repigmentation. However, the combi-
27 studies involving alternative treatments for nation of corticosteroids and herbal drugs resulted
vitiligo including prospective controlled trials, in the best treatment since it produced a complete
prospective uncontrolled trials, and retrospec- repigmentation in 31% and more than 60% in
tive studies [1]. 14% of treated patients.
A further study described the occurrence of
complete or good (more than 60%) repigmenta-
44.2 Chinese Traditional Products tion in 95% of patients treated with Xiaobai mix-
ture, which is an aqueous extract of walnut, red
Traditional Chinese medicine refers to a system flower, black sesame, black beans, zhi bei fu
of medical practice originated in ancient China ping, lu lu tong, and plums (1 ml contains 0.1 g of
more than 2000 years ago and based on empirical raw medication). The mixture has been adminis-
evidence and theories. Although traditional tered (160 ml) every day for 3 months [3].
Chinese medicine was widely practiced in Asian Many issues surrounding the efficacy of
populations such as China, Hong Kong, Taiwan, traditional Chinese medicine remain unresolved.
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44 Discussion on Empirical, Traditional, and Alternative Treatments 465
The main criticism is the absence of high-quality Some studies evaluated the effect of oral
clinical trials performed with sufficient number administration of Ginkgo biloba and Polypodium
of patients and well-characterized products [4]. leucotomos in combination with photo- or photo-
Moreover, none of the published treatments have chemotherapy chapter 44) to improve antioxi-
been unequivocally confirmed. dant activities of the skin.
Therefore, the use of such alternative treat-
ments may be considered as a supplement of
established therapies since biochemical and 44.4 Melagenin
molecular mechanisms underlying the effect on
vitiligo are clearly unknown. A recent review and The extract of the human placenta named “mela-
meta-analysis examined 48 studies that assessed genin” has been tested, and the first use was in
phototherapy in combination with oral Chinese 1991 [10]. However, the underlying mechanisms
herbal medicine in the treatment of vitiligo, and of repigmentation induced by melagenin have not
they reported that combination therapy resulted been thoroughly clarified. A study evaluated the
in a higher repigmentation rate than phototherapy efficacy of topical melagenin for repigmentation
alone [5]. Nevertheless, there is limited available in 22 child patients with vitiligo treated twice
evidence of long-term follow-up and poor meth- daily with 1.2 mg/ml aqueous melagenin in com-
odological of the trials. bination with 20 min of infrared exposure [11].
The general effective rate of melagenin-infrared
combination treatment was 45.5%, and the treat-
44.3 Plant-Derived Extracts ment has shown minimal side effects. Some
in vitro studies suggested that melagenin is
Some plant-derived substances like khellin, extremely rich of bioactive molecules since
Polypodium leucotomos, and Ginkgo biloba have human placental extracts contain keratinocyte
been proposed in the management of vitiligo. growth factor [12], endothelin-1 [13], and sphin-
Khellin is an extract of the Mediterranean fruit golipids [14]. Melagenin may act on pigmenta-
Ammi visnaga, and it is structurally similar to tion by modulating melanocyte proliferation and
psoralens used in PUVA. Khellin has been shown melanogenesis. The melanoblast cell line
to provide benefit alone and in combination with NCCmelb4M5 proliferates and differentiates
UVA or UVB phototherapy, but its systemic use after melagenin treatment as demonstrated by
may be limited by potential side effects such as increased expression of c-KIT, tyrosinase, and
liver toxicity, nausea, and orthostatic hypotension MITF [15].
[6, 7]. Therefore, research interest focused on
topical application of khellin within liposomes in
combination with phototherapy [8]. Hofer et al. 44.5 Aspirin
reported the results obtained from 28 patients
with generalized vitiligo treated at least for Acetylsalicylic acid (aspirin) has been reported
3 months with 100 mg of khellin and UVA. Forty- to have free radical scavenging property [16] and
one percent of patients showed 70% repigmenta- to be a potent antioxidant in many oxidative
tion, and nausea was experienced by 29% of stress-related diseases [17]. Previously, Zailaie
patients. Moreover, long-term follow-up demon- reported that acetylsalicylic acid was beneficial
strated no instances of actinic damage or skin to vitiligo patients by inhibiting melanocyte lipid
cancer. peroxidation and increasing cell proliferation
A study investigated the topical application [18]. The mechanism of action has been attrib-
effectiveness of Cucumis melo extract [9] in com- uted to the inhibition of leukotriene synthesis and
bination with UVB, but no differences toward the to the induction of catalase/glutathione peroxi-
placebo cream have been detected. dase activities [19]. Moreover, the author reported
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466 M. Picardo
a reduction of soluble IL2 receptor (sIL2R) con- 44.7 Dermabrasion Combined

centration of the serum IL1β, IL-6, IL-8, and with 5-Fluorouracil
TNFα levels as well as of anti-melanocyte
antibodies [20, 21]. Recently, Jian and co-work- Application of 5-fluorouracil (5-FU) after
ers demonstrated that pretreatment with acetyl- mechanical dermabrasion, as a treatment for vit-
salicylic acid protects human melanocytes against iligo, was introduced by Tsuji and Hamada in
H2O2-induced oxidative stress via Nrf2-driven 1983 [27], and its efficacy was successively con-
transcriptional activation of HO-1 [22] and inhi- firmed by other authors [28]. Only patients with
bition of HO-1 expression abrogates acetylsali- stable disease and limited extension of the mani-
cylic acid protective effects. Therefore, although festations can be treated because of the risk of
more preclinical and clinical research is needed, Koebner phenomenon induced by the treatment.
aspirin might represent a promising new thera- Successful repigmentation of areas devoid of
peutic agent for vitiligo. hair follicles was first reported by Anbar after
ER:YAG laser skin ablation followed by topical
application of 5-FU and Nb-UVB phototherapy
44.6 Statins [29], but it was difficult to understand why a
topical drug, such as 5-FU, well known for its
Statins are inhibitors of the 3-hydroxy-3- antimitotic activity, might improve the prolifera-
methylglutaryl-coenzyme A reductase (HMG- tion and migration of melanocytes. The mecha-
CoA reductase), the rate-limiting enzyme of the nism of action is mainly based on the combination
cholesterol synthesis pathway. In 2004, Noel of epidermal removal and induction of irritation
et al. described an unusual case of regression of mediated by 5FU, which through the production
vitiligo in a patient treated with statins [23]. of inflammatory cytokines and prostaglandins
The clinical effectiveness has been related to stimulates melanocyte migration and prolifera-
immunomodulatory and anti-inflammatory tion. Moreover, based on histological findings
activities of this family of compounds through obtained by Gauthier et al. on guinea pig skin, it
the inhibition of TNFα, IL-6, and IL-8 release was possible to propose a possible “scenario”
and of the LFA1 and ICAM1 expression. including several successive events, allowing us
Moreover, statins are also able to in vitro inhibit to give an explanation for successful repigmen-
the expression of MHC class II molecules con- tation after skin ablation combined with 5-FU
tributing thus to the immune suppression [24]. [30]. As was demonstrated in vitro, 5-FU can
A different possible mechanism of action has exert a selective cytotoxic effect on different
been related to the interference with the phos- types of epidermal cells. Melanocytes seem to be
phatidylinositol-3-kinase/Akt signal transduc- less vulnerable than keratinocytes to 5-FU, and
tion pathway [25]. their ability to proliferate in cultures appears to
A recent study reported the ability of simvas- be preserved [31]. After dermabrasion followed
tatin, an HMG-CoA reductase inhibitor approved immediately by application of 5-FU ointment for
by the Food and Drug Administration for the 2 days, many keratinocytes implicated in the
treatment of hypercholesterolemia, to both pre- new epithelialization are chemically damaged.
vent and reverse established depigmentation in a Consequently, a strong inflammatory reaction is
mouse model of vitiligo by reducing the number seen, and the healing is considerably delayed.
of infiltrating autoreactive CD8+ T cells in the On day 15, some degenerative keratinocytes
skin and IFN-γ production [26]. Based on these located in the lower layers still persist, but due to
in vitro and in vivo studies, a clinical trial for the persistence of a local edema, the intercellular
patients with vitiligo treated with high-dose sim- spaces of the basal layer remain very enlarged
vastatin would be useful to determine whether for a long time. Active melanocytes migrate
simvastatin may be a safe, targeted treatment through these enlarged intercellular spaces from
option for patients with vitiligo. the pigmented to the achromic epidermis.
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44 Discussion on Empirical, Traditional, and Alternative Treatments 467
During this period, inflammatory mediators better results has been also assessed. After
such as leukotrienes C4 and D4 (LTC4 and 3 months of treatment, repigmentation degree of
LTD4) are locally released, which could stimu- treated lesions has been measured. Latanoprost
late melanocyte proliferation and migration [32]. was found to be better than placebo and compa-
Moreover, during epidermis remodeling, kerati- rable with NB-UVB in inducing skin pigmenta-
nocytes synthesize and secrete metalloprotein- tion. This effect was enhanced by the addition of
ases, which are involved in the degradation of NB-UVB exposure.
the extracellular matrix. These favorable condi-
tions, which persist for a long time, could explain
the successful migration of melanocytes from References
the pigmented to the adjacent achromic area
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5. Chen YJ, Chen YY, Wu CY, Chi CC. Oral Chinese
the secreted phospholipase A2, which is a com- herbal medicine in combination with phototherapy
ponent of the bee venom, has been considered for vitiligo: a systematic review and meta-analysis of
[33]. When tested in vitro, bee venom was able randomized controlled trials. Complement Ther Med.
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6. Hofer A, Kerl H, Wolf P. Long-term results in the
ity, migration, and tyrosinase activity probably treatment of vitiligo with oral khellin plus UVA. Eur J
by modulating intracellular signal transduction Dermatol. 2001;11:225–9.
pathways related to PKA, Erk, and PI3K/Akt. 7. Ortel B, Tanew A, Honigsmann H. Treatment of
The water extract (0.1 mg/ml) of Piper nigrum, vitiligo with khellin and ultraviolet A. J Am Acad
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and its main alkaloid piperine, has been demon- 8. De Leeuw J, Assen YJ, van der Beek N, et al.
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9. Khemis A, Ortonne JP. Comparative study of vege-
Several beneficial effects in terms of repig- table extracts possessing active superoxide dismutase
mentation response have been reported following and catalase (Vitix) plus selective UVB phototherapy
the use of prostaglandins in the treatment of vitil- versus an excipient plus selective UVB photother-
igo. Parsad et al. [35] and Kapoor et al. demon- apy in the treatment of common vitiligo. Nouvelles
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strated efficacy of topical PGE2 in the treatment of 10. Suite M, Quamina DB. Treatment of vitiligo with
localized stable vitiligo. The application of a gel topical melagenine - a human placental extract. J Am
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tion in twenty-two child patients with vitiligo on the
On the other hand, Anbar et al. evaluated the scalp. Chin Med J. 2004;117:199–201.
effects of latanoprost, a PGF2α analogue used in 12. Chiu ML, O’Keefe EJ. Placental keratinocyte growth
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mentation in 22 patients with vitiligo [37], and epidermal growth factor. Arch Biochem Biophys.
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they compared the results with those obtained by 13. Wilkes BM, Susin M, Mento PF. Localization of

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Moreover, the combined effect for achieving centa. J Histochem Cytochem. 1993;41:535–41.
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placental extracts: skin pigmenting activity and gross cil in vitiligo. Arch Dermatol. 1983;119:722–7.
chemical composition. Int J Dermatol. 1995;34: 28. Sethi S, Mahajan BB, Gupta RR, Ohri A. Comparative
61–6. evaluation of therapeutic efficacy of dermabra-
15. Zhao D, Li Y, Wang P, et al. Melagenin modulates sion, dermabrasion combined with topical 5%
proliferation and differentiation of melanoblasts. Int 5- fluorouracil cream, and dermabrasion combined
J Mol Med. 2008;22:193–7. with topical placentrex gel in localized stable vitiligo.
16. Maniglia FP, Costa JA. Effects of Acetylsalicylic acid Int J Dermatol. 2007;46:875–9.
usage on inflammatory and oxidative stress markers 29. Anbar TS, Westerhof W, Abdel-Rahman AT, et al.
in hemodialysis patients. Inflammation. 2015. https:// Effect of one session of ERG:YAG laser abla-
doi.org/10.1007/s10753-015-0244-8. tion plus topical 5Fluorouracil on the outcome of
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acid treatment until surgery reduces oxidative stress of non-segmental vitiligo: a left-right comparative
and inflammation in patients undergoing coronary study. Photodermatol Photoimmunol Photomed.
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vitiligo. Saudi Med J. 2004;25:1656–63. Dermatol. 2013;2013:852497. https://doi.
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tured melanocytes of active vitiligo. Saudi Med J. 5-fluorouracil on human skin melanocytes and malig-
2004;25:1439–44. nant melanoma cells in vitro. Acta Derm Venereol.
20. Zailaie MZ. Decreased proinflammatory cytokine
1986;66:474–8.
production by peripheral blood mononuclear cells 32. Morelli JG, Yohn JJ, Lyons MB, et al. Leukotrienes
from vitiligo patients following aspirin treatment. C4 and D4 as potent mitogens for cultured human
Saudi Med J. 2005;26:799–805. neonatal melanocytes. J Investig Dermatol.
21. Zailaie MZ. Aspirin reduces serum anti-melanocyte 1989;93:719–22.
antibodies and soluble interleukin-2 receptors in vit- 33. Jeon S, Kim NH, Koo BS, et al. Bee venom stimu-
iligo patients. Saudi Med J. 2005;26:1085–92. lates human melanocyte proliferation, melanogen-
22. Jian Z, Tang L, Yi X, et al. Aspirin induces Nrf2- esis, dendriticity and migration. Exp Mol Med.
mediated transcriptional activation of haem oxy- 2007;39:603–13.
genase-1 in protection of human melanocytes from 34. Lin Z, Liao Y, Venkatasamy R, et al. Amides from
H2O2-induced oxidative stress. J Cell Mol Med. Piper nigrum L. with dissimilar effects on mela-
2016;20:1307–18. nocytes proliferation in vitro. Pharm Pharmacol.
23. Noel M, Gagné C, Bergeron J, et al. Positive pleio- 2007;59:529–36.
tropic effects of HMG-CoA reductase inhibitor on 35. Parsad D, Pandhi R, Dogra S, et al. Topical prosta-
vitiligo. Lipids Health Dis. 2004;3:7–11. glandin analog (PGE2) in vitiligo-a preliminary study.
24. Namazi MR. Statins: novel additions to the dermato- Int J Dermatol. 2002;41:942–5.
logic arsenal? Exp Dermatol. 2004;13:337–9. 36. Kapoor R, Phiske MM, Jerajani HR. Evaluation of
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β1b in MS. Neurology. 2002;59:990–7. 37. Anbar TS, El-Ammawi TS, Abdel-Rahman AT,

26. Agarwal P, Rashighi M, Essien KI, et al. Simvastatin Hanna MR. The effect of Latanoprost on vitiligo:
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ERRNVPHGLFRVRUJ
Beyond Guidelines
45
Tag S. Anbar, Rehab A. Hegazy, and Amira A. Eid
Contents
45.2 Repigmentation 470
45.3 Depigmentation 474
45.4 Camouflage 474
45.5 ombination Between Repigmentation, Depigmentation, and/or
C
Camouflage 474
45.6 tabilization Throughout the Process of Repigmentation/
S
Depigmentation 475
45.7 Follow-Up 476
45.8 Conclusion 476
References 478
Abstract
The therapeutic plan must start from the
T. S. Anbar (*) shared decision of patient and medical doctor,
(in memory) Department of Dermatology and taking into account that repigmentation,
Andrology, Faculty of Medicine, Al Minya depigmentation, camouflage, or a combina-
University, Al Minya, Egypt
tory approach should depend on localization,
R. A. Hegazy extent, and duration of the lesions. In repig-
Department of Dermatology, Faculty of Medicine,
mentation option, the available melanocytes
Cairo University, Cairo, Egypt
useful for migration have to be evaluated, as
A. A. Eid
well as in depigmentation or camouflage ones.
Department of Dermatology, Venereology
and Andrology, Faculty of Medicine, Alexandria An algorithm will support the physician in the
University, Alexandria, Egypt modulation of the therapeutic plan.

ERRNVPHGLFRVRUJ
Regardless of the pattern, repigmentation depends

Key Points on the presence of a new generation of functional
• Discuss the current clinical profile and melanocytes recruited to the affected area from
the expected outcome, as well as the one or more of the following sources [6]:
possible therapeutic options (repigmen-
tation, depigmentation, camouflage, or 1. Follicular melanocytes
combined), at the first visit. Melanocyte stem cells were detected in the
• Stabilization is a continuous process bulge area residing in the niche, from where
going hand in hand with repigmentation they usually migrate to differentiate into mela-
and depigmentation. nocytes. However, under certain circum-
• Flexibility in changing the plan of ther- stances they can migrate upwards and
apy according to the patient’s response differentiate into melanocytes in the basal
is a must. layer of the epidermis [7–9]. This vertical
• Follow-up does not only give an option migration of melanocytes is thought to be
of treating vitiligo in its early stages, but responsible for the perifollicular pattern of
it also can help through Wood’s light repigmentation. In addition to the bulge, a
examination in controlling the disease secondary possible melanocyte germ was
in its subclinical state. described in the infundibulum following the
treatment with narrowband ultraviolet B [10].
2. Marginal melanocytes
They provide the source for the marginal
45.1 Introduction repigmentation pattern, where active perile-
sional melanocytes may gradually migrate
The therapeutic plan must start from the shared toward the lesion and produce melanin, the
decision of patient and treating physician. so-called horizontal melanocyte migration.
Accordingly, based on the clinical data and the Unfortunately, they are only responsible for
expected sources of melanocytes for repigmenta- repigmentation of the peripheral 2–3 mm of
tion in each individual lesion, the decision of repig- the lesion, which alone is clearly not enough
mentation, depigmentation, and/or camouflage is to induce repigmentation in large lesions [11].
taken after a thorough explanation to the patient; 3. Other possible sources
keeping in mind that this plan will be a joint doctor- In vitiligo, amelanotic, DOPA-negative melano-
patient decision. We propose the use of a simple cytes are spared [12]. Small amelanotic melano-
drawing that will not only facilitate in the treatment cytes containing abnormal melanosomes [13],
description to the patient, but also it will help the as well as Melan-A+ cells [14], have been
doctor in subsequent follow-ups at a glance. detected in epidermis of patients with long-
standing vitiligo. Reactivation of those amela-
notic melanocytes could possibly be the reason
45.2 Repigmentation behind the diffuse type of pigmentation seen in
vitiligo. Furthermore, repigmentation that occa-
Over the years, a number of treatment guidelines sionally occurs in areas devoid of hair follicles
for vitiligo have been published, aiming to pro- such as the palms and soles raised the possibil-
vide an approach to the available therapeutic ity of the presence of melanocyte precursors or
modalities for vitiligo management [1–3]. epidermal stem cells related to melanocytes in
Whether spontaneous or medically induced, five glabrous skin [15, 16]. However, this deduction
main repigmentation patterns have been reported awaits further research.
in vitiligo lesions, namely, marginal, perifollicu- To achieve repigmentation, any treatment
lar, diffuse, combined [4], and medium spotted, plan should target the available melanocyte
which has been recently described in children in sources. Owing to the speculative and contradic-
areas with scanty or no hair follicles [5]. tory data concerning interfollicular epidermal
ERRNVPHGLFRVRUJ
45 Beyond Guidelines 471
a b
Fig. 45.1 The effect of narrowband stimulation on vitiligo skin. (a) Perifollicular pigmentation in lesions with black
hair. (b) Erythema in lesions with white hair
stem cells and melanocyte precursors, only (b) Pathological factors

follicular and marginal melanocytes will be
Since the hair follicle is considered the main
addressed in our repigmentation approach. reservoir of melanocytes, cases with leuko-
Several factors can influence the decision of trichia are probably associated with melano-
whether to target the follicular, or marginal mela- cyte reservoir exhaustion resulting in the
nocytes, or both in the treatment plan, including: absence of amelanotic melanocytes in the
(a) Anatomical factors bulge area [18]. As a result, narrowband
Repigmentation is dependent on function- stimulation which usually results in perifol-
ing melanocytes migrating to the lesion licular pigmentation in lesions with black
and producing melanin. Given the fact that hair will only cause erythema in lesions
follicular melanocytes represent the main with leukotrichia without any pigmentary
source for repigmentation, areas devoid of changes related to these white hairs
hair, such as mucous membranes, palms, (Fig. 45.1a, b). On the other hand, the pres-
and soles, will depend mainly on the hori- ence of pigmented hairs in the lesion carries
zontal migration of marginal melanocytes a good prognostic sign, but is not a 100%
toward the lesion resulting in marginal guarantee for repigmentation. The duration
repigmentation, which is of clinical value of the lesion is inversely proportional to the
in small lesions only. Similarly, areas with presence of these cells in black hair, thus the
scanty hair distribution, for example, the early intervention is mandatory [18, 19].
flexor aspect of the wrist, the dorsum of the
foot, and periungual skin, will exhibit mar- Moreover, the size of the lesion, the hair den-
ginal repigmentation in addition to mini- sity in the lesion, as well as the duration of vitil-
mal perifollicular repigmentation, which igo will affect the choice of therapy.
will result in scattered small pigmented Larger numbers of pigmented hairs as well as
macules of negligible clinical value. shorter disease durations are generally associated
A third clinical scenario is encountered in with better results [18]. The size of the lesion influ-
areas with numerous pigmented hairs such ences the choice of the technique used to induce
as the face and trunk. Since the outcome of repigmentation. Logically, targeted phototherapy,
the vertical migration outweighs the out- excimer laser, or topical medications can be of
come of horizontal one, it can be deduced value if small areas are affected; meanwhile if
that the degree of repigmentation will be large or multiple small areas are affected, the
largely dependent on the density of pig- choice of whole-body phototherapy with or with-
mented hairs in such lesions [17]. out topical medications will be more feasible [20].
ERRNVPHGLFRVRUJ
In case of absence or scarcity of pigmented in this condition represents a major challenging

hairs in vitiligo lesions, marginal melanocytes factor. The resultant improvement in repigmenta-
become the primary melanin provider in the repig- tion will be considered marked in small lesions
mentation process. Owing to their limited and poor in large affected areas (Fig. 45.2a–e).
(2–3 mm) migratory capacity, the size of the lesion Consequently, horizontal m igration is not enough
Fig. 45.2 (a)
a
Diagrammatic
illustration of the
different degrees of
improvement in relation
to the size of the lesion.
(b and c) Complete
repigmentation in small
lesions (white arrows)
and moderate
improvement of slightly
larger lesions (black
arrows). (d and e) Poor
response in a large
lesion. N.B. The
difference in response in
the three given examples
occurred despite the
same size of resultant
marginal repigmentation
(courtesy of Dr.
Mohamed T. Anbar) b c
d e
Before After
ERRNVPHGLFRVRUJ
to repigment lesions more than 1 cm2 [20, 21]. degeneration and local releases of inflammatory
Enhancing migration of marginal melanocytes and mediators. This in turn will be responsible for mela-
accordingly pigment spread can be achieved by dif- nocyte stimulation, proliferation, and migration
ferent modalities such as phototherapy [22], topical through the enlarged intracellular spaces from the
tacrolimus [23], and 5-FU applied on skin ablated normally pigmented to the achromic epidermis,
by Erbium-YAG laser [24, 25]. The clinical success eventually leading to r epigmentation [11].
of the latter technique in the treatment of small areas In lesions with an expected minimum thera-
with scanty hair such as periungual areas peutic impact for either source of melanocytes,
(Fig. 45.3a–f) is believed to be due to k eratinocytes the need for melanocytes from remote sources,
a b
c d
e f
Before After
Fig. 45.3 Erbium-YAG laser followed by 5-FU in vitiligo
ERRNVPHGLFRVRUJ
i.e., surgery, arises. Such decision is taken in can be achieved using Q-switched ruby laser
most cases during the first visit after careful anal- (QSR 694 nm) [28], Q-switched alexandrite laser
ysis of the repigmentation influential factors. In (QSA 755 nm) [29], Q-switched Nd-YAG laser
some patients surgery would be their only thera- [30], and cryotherapy [31].
peutic line, for example, a single stable lesion. In
other instances, similar lesions can be present
accompanying medically treatable lesions, in 45.4 Camouflage
whom it is more advisable to start with the medi-
cal treatment and then continue with surgery. Camouflage as a therapeutic option can be consid-
This would ultimately aid in gaining the patient’s ered if cosmetically acceptable repigmentation or
confidence and improving stabilization chances. depigmentation cannot be achieved. It can also be
To complete the picture, one must state that not of value during the course of treatment to conceal
uncommonly does the surgical decision impose the lesion until the final outcome of therapy is
itself during the course of therapy, for example, reached. Camouflage in vitiligo can be temporary
in case of an un-expected poor response in one or or permanent. Temporary options include self-tan-
more lesions receiving phototherapy together ning agents, dyes, tinted cover creams, liquid and
with topical medications. stick foundation, and fixing powders and creams,
The timing of surgery is an important factor whereas the permanent options include micro-pig-
that affects the success of the operation, as the mentation or tattooing. Temporary methods are
stability of the lesion is the mainstay of success. generally preferred over the permanent ones, which
Furthermore, taking into account the size of the should be used with caution given the unpredict-
lesion is crucial for the decision of surgery, con- able nature of the disease and the metallic nature of
sidering the need for an adequate donor area. the injected pigments [32, 33].
Apart from psychological effect, the delay in
surgery does not affect the outcome of the opera-
tion, as opposed to the delay in the medical inter- 45.5 Combination Between
vention that decreases the opportunity of success Repigmentation,
as described above. Depigmentation, and/or
Though different from vitiligo/NSV in various Camouflage
aspects, segmental vitiligo can also be assessed
using the same approach, keeping in mind the We can face situations necessitating combina-
short time for leukotrichia development hence tions between the abovementioned therapeutic
the importance of very early medical interven- lines, for example, a patient with a history of
tion, otherwise surgery or camouflage will be the long-standing vitiligo affecting the trunk and
only hope. hands, and with concern about the newly devel-
oping facial lesions, as well as the unsightly
remaining colored islands on the dorsum of the
45.3 Depigmentation hands. In such a situation, we can use camouflage
or extend the scope of depigmentation to entail a
Although when this decision could be adopted is localized approach to the hands; meanwhile, the
not clearly agreed upon, depigmentation by recent facial lesions can be repigmented. The
chemical or physical means may be considered as choice between depigmentation and camouflage
a therapeutic option when vitiligo involves >50% in the management of his hands will depend on
of the body surface area of patients with refrac- the original skin color of the patient and the
tory vitiligo [1]. Monobenzyl ether of hydroqui- patient’s desire. In patients with dark skin photo-
none 20% (MBEH) [1, 26], 4-methoxyphenol, types (V and VI), depigmentation of the remain-
and 88% phenol are capable of inducing chemical ing pigmented island on the hands will yield
depigmentation [27]. Physical depigmentation marked contrast between the hands and face that
ERRNVPHGLFRVRUJ
will be clearly apparent revealing the nature of by lymphocytes [37]; hence, no more melanin is
the patient’s condition. In such a situation, the transferred to the keratinocytes. Despite the fact
patient’s preference plays a vital role in the deci- that this step exhibits histopathological activity in
sion; if having a uniform skin color is more the form of the inflammatory infiltrate, there is no
important to him than the resultant contrast, then clinical evidence of what is occurring; thus, a
the decision of depigmentation will be possible. biased decision of stability can be taken. The sec-
On the other hand, if this contrast will be unac- ond stage soon follows, in which the amount of
ceptable to the patient, then a decision of camou- melanin in the epidermis will gradually decrease
flage will be made. An important advantage of due to lack of melanosome production, as well as
depigmentation over camouflage is that it is done the continuous turnover of keratinocytes. The
once, and it does not require daily or every other decrease in melanin might not be clinically evi-
day care like camouflage, but again the patients’ dent even by Wood’s light examination, but fur-
preference is what matters at the end, and the ther progress of the process will lead to the final
doctor’s role is to explain and clarify. Needless to stage where clinically visible hypo- and eventu-
say, ensuring disease stability is essential regard- ally depigmented lesions are present [6, 38].
less the patient’s decision. Interestingly enough, by the time the lesion
becomes clinically evident in the third stage, the
underlying inflammatory process might have
45.6 Stabilization Throughout already subsided; hence, our decision of disease
the Process activity might be inaccurate. In other words, clini-
of Repigmentation/ cally stable lesions or even normal skin may be
Depigmentation the site of activity in the first stage; meanwhile the
increase in lesion size or even the appearance of
Vitiligo is considered stable if there are no new new lesions in the third stage may be associated
lesions, no increase in size of pre-existing lesions, with cessation of the activity process.
and no Koebner phenomenon over a certain It was recently reported that amelanotic
period. However, there is no consensus even on lesions with well-defined borders are clinical
the duration to define a stable lesion [34]. markers of stability, while disease activity should
Determining the stability of a lesion might be considered on finding hypomelanotic lesions
appear to be a simple task, but this is far from true with ill-defined borders [39, 40]. Meanwhile the
because all the abovementioned criteria of stability increased expression of CXCL10 [41] and the
depend mainly on the patient history, which was deficiency of E-cadherin [42] were found to be
recently proved to be of questionable value com- preclinical markers of vitiligo activity. Despite
pared to objective methods [35]. In this context, the these valuable findings, the distinction between
question of the reliability of our clinical evaluation stability and activity is still not crystal clear; and
is raised, in other words, does what we see clini- until more accurate biochemical and clinical
cally reflect what is going on beneath the surface? markers of disease activity are well established,
In the absence of a disease process, constitu- the combined subjective data supplied by the
tive skin color is determined by melanosomes patient history together with the objective evalu-
synthesized by epidermal melanocytes that are ation through careful lesion examination, photos,
then transferred to neighboring keratinocytes, and planimetry [43] will still be the only practical
[36], aiding us to expect the sequence of events methods of evaluation in our hands.
occurring during vitiligo activity. It could be Stabilization during repigmentation and depig-
assumed that the affected areas will pass through mentation is quite different. In cases requiring
three stages, each of which has its histopathologi- depigmentation, stabilization is achieved
cal and clinical characteristics. In the first stage, primarily through photo-protective measures.

regardless of the underlying pathogenic mecha- However in cases in which repigmentation is the
nism, the melanocytes are lost after being attacked plan, stabilization is more complicated and can be
ERRNVPHGLFRVRUJ
Fig. 45.4 Value of

Wood’s light in a
follow-up
achieved by general, local, or systemic measures. 45.7 Follow-Up

General measures include avoiding Koebner phe-
nomenon [44], photoprotection, and controlling Our role is not restricted to reaching the most
psychological stress. Patient education on how to acceptable color to the patient, but it extends to
avoid trauma, whether at home or at work, after include follow-up visits. These follow-up visits
the discussion of his daily activities, together with can be regular every 6 months or situational as
avoiding the use of rough or sharp objects in his in case of the patient’s suspicion about the
clothes [45], is integral to this process. Topical appearance of new lesions or after vacations in
measures were assumed to be of benefit in the sta- sunny climates that can pose a threat to the
bilization process if applied on extensions of patients. The use of Wood’s light in the follow-
lesions existing in naturally aggressive sites such up visits is mandatory because it can help detect
as the periungual areas or after cure of a lesion if early lesions not yet discernible by the naked
relapse is highly expected on discontinuation of eye especially in children where dealing with
treatment [20]. This assumption was recently subclinical vitiligo (Fig. 45.4a, b) is easier and
proved when maintenance therapy with tacroli- less stressful.
mus 0.1% twice weekly was reported to be effec-
tive in preventing depigmentation of vitiligo
lesions [46]. However, topical measures are not 45.8 Conclusion
applicable for stabilization of newly occurring
lesions, since their site cannot be predicted. This personalized and stratified approach is sum-
Systemic measures include systemic corticoste- marized in an algorithm (Fig. 45.5) [20], which
roids, phototherapy (NB-UVB or excimer laser), offers a step-by-step guide from the patient’s first
systemic antioxidants, immunosuppressants, and visit onwards. Its individualized outcome-
biologics [47–49]. They can control the progres- oriented plan is flexible in dealing with the man-
sion of existing lesions together with the preven- datory modifications that imposes itself due to
tion of the development of new lesions. different patients’ responses.
ERRNVPHGLFRVRUJ
Q1: Is your patient stable?
Yes No
Advise about general measures of stabilization

Q2: What is the therapeutic plan Stabilize
topical, and/or systemic
Vitiligo < 75% BSA Vitiligo > 75% BSA

Camouflage*
Regiment Depigment
Q3: What is the hair content in vitiligo lesion?
Profuse black hair Scanty and/or white hair

(A) Rely on vertical Q4: What is the size of lesion?
melanocyte migration
Small Large or < 1 cm2 > 1 cm2

multiple small
• Targeted phototherapy (B) Rely on horizontal
(C) Rely on remote
• Excimer laser • Phototherapy ± melanocyte migration:
melanocyte migration:
• Topical medication • Systemic medication -Targeted phototheraphy
i.e. surgery.
-Excimer laser
-Topical medication
Success Failure+ Success Failure+
Success Failure+ Success

End of Go to (C) End of
treatment treatment
Failure++ End of
End of Go to (C) treatment
treatment
< 20% BSA > 20% BSA
Go to (C) Camouflage Successful graft Failure of graft

taking with taking
depigmentation
Repeat up
Stabilize to 3 trials
Success Failure
End of Camouflage
treatment
• It is done when repigmentation and depigmentation are not affordable and/or not accepted by the patient. It might be
applied hand in hand with both options.
•+; Failure is considered when the treatment modality yields no response in a duration of 3 monts, or shows a non-
satisfactoy improvement for 6 months, keeping in mind the individual side effects of each modality (similar to the
parameters of treatment discontinuation applied after NBUVB treatment).8
•++; Failure of surgery occurs either due to improper attachment of donor tissue to the recipient area, or due to loss of
pigment from the donor area due to disease activity.
•BSA; body surface area, ±; with or without
Fig. 45.5 An algorithm depicting the personalized stratified approach for management of vitiligo [20]
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2003;16:322–32. ing stability in vitiligo, assessed by vitiligo disease
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39. Benzekri L, Gauthier Y, Hamada S, et al. Clinical iligo. J Am Acad Dermatol. 2001;44:814–7.
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ERRNVPHGLFRVRUJ
Editor’s Synthesis and Perspectives
46
Contents
46.1 From EBM Guidelines to Clinical Practice 482
46.2 Perspectives 482
References 483
Abstract
EBM guidelines have many limitations in a
Key Points
field like vitiligo but can help to fight charlatan-
• EBM guidelines have many limitations
ism and harmful interventions. Inflammation/
in a field like vitiligo but can help to fight
autoimmunity are the major targets to stop dis-
charlatanism and harmful interventions.
ease progression. Combination therapies using
• Inflammation/autoimmunity are the
systemic/topical antiinflammatory-immuno-
major targets to stop disease progression.
suppressant drugs with UVB therapy are the
current mainstay approach for vitiligo. More • Combination therapies using systemic/
targeted immunosuppressants such as Jak topical antiinflammatory-immunosup-
inhibitors are currently in clinical development pressant drugs with UVB therapy are the
Novel approaches should promote melanocyte current mainstay approach for vitiligo.
survival and stability within the basal mem- • More targeted immunosuppressants
brane zone of the epidermis. such as Jak inhibitors are currently in
Regenerative medicine is the next important clinical development.
development because of the exhaustion of • Novel approaches should promote mela-
spontaneous regeneration especially in acral nocyte survival and stability within the
locations. basal membrane zone of the epidermis.
• Regenerative medicine is the next
important development because of the
A. Taïeb (*) exhaustion of spontaneous regeneration
Hôpital Saint-André Service de Dermatologie, especially in acral locations.
Bordeaux, France
M. Picardo

ERRNVPHGLFRVRUJ
Table 46.1 General outline of management for vitiligo

Type of vitiligo Usual management
Segmental and limited First line: Avoidance of triggering factors, local therapies (corticosteroids,
nonsegmental <2–3% body calcineurin inhibitors) combined with NB-UVB therapy, Excimer monochromatic
surface involvement lamp if available or home phototherapy. Treatment to monitor closely over 6 to 12
months before going to second line.
Second line: Consider systemic intervention (see below) if disease flare during first
line treatment, and total body NB-UVB treatment.
Third line: Consider surgical techniques if repigmentation is cosmetically
unsatisfactory on visible areas
Nonsegmental First line: Avoidance of triggering/aggravating factors. Stabilization with NB-UVB
therapy, at least 3 months. Optimal duration at least 9 months if response.
Combination with systemic/topical therapies, including reinforcement with
localized UVB therapy, possible
Second line: Consider systemic steroids (e.g. 3–4 month minipulse therapy) or
immunosuppressants if rapidly progressing disease or absence of stabilization under
NB-UVB. New molecules especially Jak inhibitors in development.
Third line: Consider surgical techniques in non-responding areas especially with
high cosmetic impact. However, Koebner phenomenon limits the persistence of
grafts. Relative contraindication in areas such as dorsum of hands
Fourth line: Consider depigmentation techniques (hydroquinone monobenzyl ether
or 4-methoxyphenol alone or associated with Q-switch ruby laser) in non-
responding widespread (>50%) or highly visible recalcitrant facial/hands vitiligo
A no treatment option (zero line) can be considered in patients with a fair complexion after discussion. For children,
phototherapy is limited by feasibility in the younger age group and surgical techniques rarely proposed before prepu-
bertal age. There is no current recommendation applicable to the case of rapidly progressive vitiligo, not stabilized by
UV therapy, but classic immunosuppressants and Jak inhibitors can be started. For all subtypes of disease or lines of
treatment, psychological support and counselling including access to camouflage instructors are needed
46.1 F
rom EBM Guidelines awareness on limitation of the Koebner phenom-
to Clinical Practice enon which is rarely taken into account.
Nevertheless, a stepwise treatment approach
The major interest of EBM reviews and meta- divided by type of vitiligo and extent, which
analyses is to point to potentially harmful inter- needs modulation by visibility, age and coping, is
ventions or those with limited interest. Given the outlined in Table 46.1 [2]. A zero line is always
importance of charlatanism in the vitiligo field, possible, meaning no treatment if the disease is
counselling patients to avoid some therapies of not bothering the patient. The environmental fac-
dubious efficacy is indeed a major step. tors (occupation, Koebner’s phenomenon, sus-
As partly stated in the Cochrane review [1], tained stress or anxiety) should be always
there are many limitations to derive a valuable discussed in the management plan. This stepwise
algorithm of treatment for all vitiligo patients approach should be considered as a proposal
based on RCTs. First, RCTs are rare and often based mostly on EBM data. However, there is
lacking important methodological steps or much room for modulation and innovation based
details. Second, studies have often been con- on this scheme.
ducted in heterogeneous groups in terms of vitil-
igo duration or progression, if not mixing
localized, segmental and nonsegmental forms. 46.2 Perspectives
Third, confounding factors are many, e.g. light
exposure in long-term interventions for which Part II of this book has put the emphasis on numer-
light sources may influence outcome, nutritional ous potential therapeutic resources, based on a bet-
intake if antioxidant status is considered or ter understanding of the crucial steps of melanocyte
ERRNVPHGLFRVRUJ
46 Editor’s Synthesis and Perspectives 483
mechanisms of disappearance and repigmentation derived from progenitors or reprogrammed skin

schemes. For a common disorder like vitiligo, there cells [8] will probably further increase our surgi-
are probably subtypes in terms of mechanisms of cal possibilities of intervention.
melanocyte loss, but the best documented is
of inflammatory/immune origin, with the activation
of memory T cells during flares [3]. Currently,
based on these principles, a more aggressive anti- References
inflammatory therapy is becoming more widely
1. Whitton M, Pinart M, Batchelor JM, Leonardi-Bee
accepted for stopping disease progression and pro- J, Gonzalez U, Jiyad Z, Eleftheriadou V, Ezzedine
mote repigmentation in combination with UV light. K. Evidence-based management of vitiligo: sum-
If the initial step preceding inflammation comes mary of a Cochrane systematic review. Br J Dermatol.
2016;174(5):962–9. https://doi.org/10.1111/bjd.14356.
from a local predisposition of melanocytes to attach
Epub 2016 Mar 25. Review. PubMed PMID: 26686510.
poorly to the basement membrane, a more precise 2. Taieb A, Alomar A, Böhm M, Dell’anna ML, De
description of the basic impairment is obviously Pase A, Eleftheriadou V, Ezzedine K, Gauthier
needed to improve melanocyte stability. Another Y, Gawkrodger DJ, Jouary T, Leone G, Moretti
S, Nieuweboer-Krobotova L, Olsson MJ, Parsad
possible therapeutic target is an underlying impair-
D, Passeron T, Tanew A, van der Veen W, van
ment of melanocyte survival mechanisms, Geel N, Whitton M, Wolkerstorfer A, Picardo M,
and enhancing the Wnt/betacatenin pathway is a Vitiligo European Task Force (VETF); European
possibility to explore in addition to the MSH ana- Academy of Dermatology and Venereology (EADV);
Union Europe´enne des Me´decins Spe´cialistes
logs [4]. The issue of self-renewal (stemness) apti-
(UEMS). Guidelines for the management of vit-
tude of melanocytes has been raised especially for iligo: the European Dermatology Forum consen-
SV, which clearly benefits from autologous grafting sus. Br J Dermatol. 2013;168(1):5–19. https://doi.
[3]. It is yet unclear if this is a real issue in vitiligo/ org/10.1111/j.1365-2133.2012.11197.x. Epub 2012
Nov 2. PubMed PMID: 22860621.
NSV and if this is related to the cellular environ-
3. Boniface K, Seneschal J. Vitiligo as a skin memory
ment rather than directly to melanocyte themselves, disease: The need for early intervention with immuno-
as suggested by Guerra [5]. modulating agents and a maintenance therapy to target
When melanocyte loss has been stopped, resident memory T cells. Exp Dermatol. 2019; https://
doi.org/10.1111/exd.13879. [Epub ahead of print]
therapy tries to address repigmentation. New
Review. PubMed PMID: 30636075.
repigmenting therapies are emerging such as 4. Lim HW, Grimes PE, Agbai O, Hamzavi I,
He-Ne lasers [6] and prostaglandin E2 [7]. The Henderson M, Haddican M, Linkner RV, Lebwohl
field of regenerative medicine is booming and M. Afamelanotide and narrowband UV-B photother-
apy for the treatment of vitiligo: a randomized mul-
applications to vitiligo are expected. The use of
ticenter trial. JAMA Dermatol. 2015;151(1):42–50.
melanocyte precursors in the hair follicle is https://doi.org/10.1001/jamadermatol.2014.1875.
promising, as well as the clinical development of PubMed PMID: 25230094.
mesenchymal stem cells. If we can better stimu- 5. Bondanza S, Maurelli R, Paterna P, et al. Keratinocyte
cultures from involved skin in vitiligo patients show
late the emigration of those stem cells towards
an impaired in vitro behaviour. Pigment Cell Res.
the epidermis and understand why they usually 2007;20:288–300.
stop migrating when becoming pigmented, a 6. Lan CC, Wu CS, Chiou MH, et al. Low-energy
major step should be achieved. There is also the helium-neon laser induces locomotion of the imma-
ture melanoblasts and promotes melanogenesis of
issue of dormant residual (dedifferentiated)
the more differentiated melanoblasts: recapitulation
melanocytes which could be resuscitated in of vitiligo repigmentation in vitro. J Invest Dermatol.
interfollicular zones and opening some new ther- 2006;126:2119–26.
apeutic avenues. A better appreciation of this 7. Kapoor R, Phiske MM, Jerajani HR. Evaluation of
safety and efficacy of topical prostaglandin E2 in
issue is clearly needed to consider newer possi-
treatment of vitiligo. Br J Dermatol. 2009;160:861–3.
bilities for medical treatments. Surgical treat- 8. Takahashi K, Tanabe K, Ohnuki M. Induction of plu-
ments for limited and disfiguring disease have ripotent stem cells from adult human fibroblasts by
dramatically improved. Newer technologies defined factors. Cell. 2007;131:861–72.
ERRNVPHGLFRVRUJ
Index
A inherent melanocyte alterations, 211

Acceleration phase, 346 spontaneous autoimmune vitiligo, 207–209
Acetylcholinesterase, 279 uveitis, 212
Acetylsalicylic acid, 465–466 water buffalo, 213
Acrofacial vitiligo (AFV), 13, 45 Annular lichenoid dermatitis of youth (ALDY), 84, 85
Afamelanotide, 9, 374 Antigen-presenting cells (APCs), 288–289
Aging Anti-IFN-α approach, 369–370
in childhood Anti-IFN-γ strategy, 369
medical treatment, 439–440 Antioxidants, 279, 364–365
phototherapy, 440–441 combined therapies, 348
psychosocial aspects, 442 intracellular network, 340
surgical treatment, 441–442 occurrence, 414–415
in older adults, 442 Polypodium leucotomos, 415
psychological impact, 438 Anti-PD-1 therapies, 164–166
Alabras, 5 Anti-TNF-α strategy, 295
Alexithymia, 175 APECED, see Autoimmune polyendocrinopathy-
Alezzandrini syndrome, 109 candidiasis-ectodermal dystrophy
Allergic contact dermatitis, 122 APS, see Autoimmune polyendocrine syndrome
All-trans retinoic acid (ATRA), 384 Aspirin, 465–466
Alopecia areata, 126, 127 Assessment
Alopecia areata-like feathering defect, 213 chronic autoimmune/inflammatory diseases, 172
Ammi majus Linnaeus (AML), 7–9 interobserver variability, 174–175
Animal models scoring system, 172–174
mouse models skin biopsy, 172
FH mouse, 218 variables, 175
h3T-A2 mouse, 218–219 Ataxia-Telangiectasia (AT) syndrome, 136–137
Krt14-Kitl∗ mouse-pmel-1 T cell adoptive Atharva Veda, 4
transfer model, 217–218 Autoantibodies against aromatic l-amino acid
melanocyte-specific immune responses, 214–215 decarboxylase (AADC), 134
Pmel-1 mouse, 217 AutoCAD 2000, 197
TCR transgenic hosts, 215–219 Autoimmune/inflammatory diseases, 172
TrpHEL mouse, 218 Autoimmune polyendocrine syndrome (APS), 134–136,
TRP1 transgenic mouse, 218 172, 239
vitesse mouse, 219 Autoimmune polyendocrinopathy-candidiasis-
naturally occurring vitiligo, 207 ectodermal dystrophy (APECED), 134, 135,
Smyth line (SL) chicken 183
alopecia areata-like feathering defect, 213 Autoimmune regulator (AIRE), 134
autoimmune hypothyroiditis, 212 Autoinflammatory/autoimmune diseases
avian model, 213 alopecia areata, 126, 127
blindness, 212 atopic diseases, 128
characteristics, 207 autoimmune thyroiditis, 126, 128
environmental factor, 211–212 combinatorial pathogenic effects, 126
genetics basis, 208–209 epidemiological studies, 126, 128
immune system, 209–211 follicular vitiligo, 128
impaired vision, 212 Hashimoto’s thyroiditis, 127

M. Picardo, A. Taïeb (eds.), Vitiligo, https://doi.org/10.1007/978-3-319-62960-5
ERRNVPHGLFRVRUJ
486 Index
Autoinflammatory/autoimmune diseases (cont.) transfer-resistant lip colour, 426

immunodeficiencies treatments, 346–349
B-cell deficiencies, 132 WHO, 422
combined/T-cell immunodeficiencies, 128 cAMP response element-binding protein (CREB), 279
CVID, 128, 131 Candidate gene approach, 240–241
heterozygous C4 deficiency, 132 Canities, 95, 97
HIV infection, 126, 128–131 Cardiac melanocytes, 109
ICTL, 131 Catecholamines, 278
IRIS, 131 CD4+CD25+Foxp3+ regulatory T (Treg) cells, 257
oxidative stress dysregulation, 129 CD8+ T-cells, 122, 128, 165, 305
monogenic disorders, 132, 133 Cell isolation and culture
AADC, 134 Eagle’s minimal essential medium, 227
AIRE, 134 fibroblasts, 227
APECED/APS1, 134, 135 hair follicle keratinocytes, 227–228
autoimmune polyendocrine syndromes, 134–136 hair follicle melanocyte, 227–228
autosomal dominant vitiligo, 132 hydrogels, 228
breakage disorders, 136–138 keratinocytes, 226–227
mitochondrial disorders, 136 melanocytes, 226–227
organ-specific autoimmunity, 134 PBMC, 228
piebaldism, 132 transgenesis, 228
Val620Ala mutation, 132 CellnTechn-07 (Chemicon), 227
vitiligoid patches, 132 Cellular immunity, 291–292
vitiligo-related phenotypes, 132 Cephalic melanocytes, 108–109
prevalence, 127 Charak, 4
psoriasis, 128 Chemically induced vitiligo, 122
5-Azacytidine (5-azaC), 259 Childhood vitiligo
and autoimmune thyroid disorders, 145
clinical features, 144
B differential diagnosis, 145
Baker-Gordon’s formula, 385 early-onset group, 144
Baras, 5 epidemiology of, 142–143
Basal membrane, 34 family history, 144–145
Basic fibroblast growth factor (bFGF), 383, 384 later-onset group, 144
Bcl-2 protein family, 280 NSV vitiligo, 143
β-catenin, 268 post-pubertal onset, 144
Bilateral mosaicism, 78–79 prepubertal onset, 144
Blaschkoid hypopigmentation, 335 prevalence of, 142
Bloom syndrome (BS), 138 psychological morbidity, 145
Blue vitiligo, 45 quality of life, 145
Bone marrow (BM), 315, 317 segmental vitiligo, 143
Bordeaux VGICC classification, 12, 14 therapeutic considerations, 146
Breakage disorders, 136–138 treatment, 144
on trunk and groin, 143
Cholera toxin (CT), 383
C Chroma Meter CR 200/CR 300, 198, 199
Cadherin (cell adhesion), 228, 268, 269 CHS, see Contact hypersensitivity
Calcineurin inhibitors, 348, 351–354 CIV, see Clinically inflammatory vitiligo
Calcipotriol, 363, 440 Classical melanocytes, 266
Calcitriol, 440 Classic oculocutaneous albinism, 17
Calreticulin, 233 Clinical aspects, 39
Camouflage Clinical evaluation
cosmetic rehabilitation, 422 autoimmune/inflammatory disorders, 170, 172
cover creams, foundations and sticks, 425–426 checklist for NSV, 171
DHA, 424 clinical data, 184
guidelines, 474 hair graying, 170, 171
history, 423–424 importance, 182
intervention, 422–423 predictive assessment, 183
leukotrichia, 426 systematic pathology assessment, 183
permanent and semi-permanent camouflage, 426 Wood’s lamp examination, 170, 171
precautions, 426–427 Clinically inflammatory vitiligo (CIV)
self-tanning creams, lotions and sprays, 424–425 clinical features and presentation, 82, 83
ERRNVPHGLFRVRUJ
Index 487
confetti-like leukoderma, 89 Deep phenotyping, 182, 183

differential diagnosis, 82, 85 Delayedamelanotic (DAM) chicken, 208
follicular vitiligo, 85, 88 Dental pulp stem cells (DPSCs), 314
histopathological examination, 84–85 Depigmenting therapy, 379–389
hypochromic vitiligo/vitiligo minor, 88–89 cryotherapy, 387
leukoderma punctata, 89 DPCP, 388–389
marginal hyperpigmentation, 83 extensive and universal vitiligo, 380
nummular lichenoid lesions, 83 guidelines482
other infectious/inflammatory diseases, 83 hydroquinone, 380
patient history, 83–84 imatinib, 388
and psoriasis, 82 imiquimod, 388
PUVASOL therapy, 89 laser therapy, 385–386
with raised borders, 82 MBEH, 386
symptoms and signs, 82 ATRA, 384
vitiligo guttata/punctata, 89 definition, 381
Clobetasol propionate, 439 mechanism of action, 383
Collagen-glycosaminoglycan-chitosan, 229 precautions, 383–384
Combined therapy, 347, 386 side effects, 384
dermabrasion/fractional ablative lasers, 415–416 treatment procedure, 384
maintenance therapy, 416–417 4-0 methoxyphenol, 384–385
phototherapy patient selection, 380–381
and antioxidants, 414–415 88% phenol solution, 385
and TCI, 413–414 QSA laser, 386–387
and topical steroids, 412–413 QSR laser, 386
and vitamin D, 414 stabilization, 475–476
surgical therapies and corticosteroids, 412 Depression, 175, 178
surgical therapies and phototherapy, 412 Dermabrasion/fractional ablative lasers, 415–416
Common variable immunodeficiency (CVID), 128, 131 Dermal components, 330
Computerized digital image analysis system, 197 DermaSpectrometer, 199
Contact hypersensitivity (CHS), 122–123 Dermatology Life Quality Index (DLQI),
Contact vitiligo, 122 154, 179
Corel Draw 9.0, 197 Dermis, 34–35
Corrective makeup workshops, 455–456 Dermis-derived stem cells (DSCs), 325
Corticosteroids, 347–348, 412 Dermoscopy, 199, 203
See also Topical corticosteroids Detachment process, 330–331
Counseling, 349 Dicer, 256, 257
CpG islands, 258–259 Differential diagnosis
C1q and tumor necrosis factor-related protein hypopigmented mycosis fungoides, 35
(C1QTNF6), 287 inherited/genetically induced hypomelanoses
Crohn’s disease, 272, 273 hypomelanosis of Ito, 14, 18
Cryotherapy, 387 monogenic hypomelanoses, 14, 15
CTLA4 gene, 241 piebaldism, 14–15, 17
Cultured epidermal sheet transplantation, 384–385 tuberous sclerosis, 15, 17
Cultured melanocyte transplantation, 383–384 Waardenburg’s syndrome, 15, 18
Cutaneous disorder, 458 melanoma-associated depigmentation, 19, 20
Cutaneous mosaicism, 22, 58, 191 melasma, 21, 22
CVID, see Common variable immunodeficiency microscopical features, 35
CXCR3 chemokine receptor, 165 morphologic abnormalities, 35
Cyclophosphamide, 48 nevus depigmentosus, 22, 23
Cytokine & Cells Online Pathfinder Encyclopedia occupational and drug-induced depigmentation, 22
(COPE) website, 234 para-infectious hypopigmentation, 20–21
Cytokines, see Proinflammatory cytokines para-malignant hypomelanoses, 19, 20
Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), pityriasis alba, 35
164, 260 post-inflammatory hypomelanoses, 18, 19
posttraumatic leukoderma, 21
Digital epiluminescence dermoscopy, 199
D Digital image analysis, 197–198
Damage-associated molecular patterns (DAMPs), 248, Digital photographs, 197
249 Dihydroxyacetone (DHA), 349, 424
Dead de-epidermized dermis (DDD), 229 Diphencyprone (DPCP), 388–389
Decay-accelerating factor (DAF), 286 DLQI, see Dermatology Life Quality Index
ERRNVPHGLFRVRUJ
488 Index
DNA methylation, epigenetics otic melanocytes, 107–108

in animal models, 259 types, differences, and changes, 110
CpG islands, 258–259 VKH syndrome, 109
in human vitiligo, 259–260 Waardenburg syndrome, 109
DNA methyltransferase (DNMT), 258, 259
DOPA-stained skin sheets, 9
Dorsolateral pathway, 266, 267 F
Dulbecco’s Modified Eagle’s Medium (DMEM), 394 Fetal calf serum (FCS), 383
FH TCR transgenic mouse vitiligo model, 218
Fibroblasts, 190, 191
E Fine-needle sampling (FNS), 233
Eagle’s minimal essential medium, 227 Fitzpatrick’s system, 430
Ebers Papyrus, 3, 4, 7 Fluorescence resonance energy transfer (FRET) analysis,
EBM guidelines, 482 233
E-cadherin, 268, 269, 271–274 Fluorochrome-conjugated monoclonal antibodies, 232
Electron microscopy, 32 5-Fluorouracil (5-FU), 415, 466–467
Embryonic stem (ES), 314 Fluticasone, 352
EMU, see Epidermal melanin unit Fluticasone propionate (FP), 352
Endothelin-3 (ET-3), 319, 320 Focal vitiligo, 14
Environmental factors, 121, 122 Follicular melanogenesis, 96
Epidemiological data, 182 Follicular unit extraction (FUE), 394
Epidermal melanin unit (EMU), 229, 269–271 Follicular vitiligo, 14, 85, 88, 128
Epidermal melanocytic target, 184 Fontana–Masson staining, 26, 323
Epidermal naevi, 335 Forkhead box protein O1 (FOXO1), 279
Epidermal pigmentation, 65, 194, 219, 305, 306, 331 FOXD3 transcriptional promoter, 243
Epigenetics Free radical-mediated graying, 340
definition, 254
disorders of, 254
DNA methylation G
in animal models, 259 Gender, 462
CpG islands, 258–259 Generalized vitiligo
in human vitiligo, 259–260 blue vitiligo, 45
environmental factors, 261, 262 depigmentation, 42, 43
histone modifications distribution of, 43–44
in autoimmune diseases, 260–261 hyperpigmented lesional borders, 42
chromatin modification in melanocyte multichrome vitiligo, 44–45
differentiation, 261 natural course, 44
histone acetylation, 260–261 symmetrical lesions, 42, 43
histone methylation, 260–261 symptoms, 42
miRNAs in melanogenesis Wood’s lamp examination, 43, 44
biogenesis and function, 254–255 Genetically modified cells in monolayer, 230
immune tolerance regulation, 256–257 Genetic factors, 122
miR-145, 256 Genetics
miRNA dysregulation, 257–258 bioinformatic functional interaction network of
MITF, 256 proteins, 247
solar-simulated UV, 256 candidate gene approach, 240–241
UVR, 256 DAMPs, 249
Epigenome editing, 230, 231 epidemiology
Erythema index (EI), 198 autoimmune diseases, 239, 240
Evidence-based guidelines, 349 autoimmune polyendocrine syndrome, 239
Extracellular matrix (ECM) proteins, 330 EUR population, 238, 241
Extra-cutaneous melanocytes Han Chinese families, 239
Alezzandrini syndrome, 109 males and females, 238
cardiac melanocytes, 109 twin studies, 239
cephalic melanocytes, 108–109 genome-wide approach, 241–242
distribution of melanocytes in human tissues, 104 genome-wide association studies, 243–246
in hereditary pigmentary disorders, 109 genome-wide linkage studies, 242–243
lymphangioleiomyomatosis, 109 purpose of gene identification, 246–247
melanin biosynthesis, 109 vitiligo and melanoma relationship, 246
ocular melanocytes, 105–106 vitiligo pathogenesis framework, 248
ERRNVPHGLFRVRUJ
Index 489
Genital vitiligo, 446 histone methylation, 260

Genome-wide association studies (GWAS), 243–246 Histopathology
Genome-wide genetic approach, 241–242 amelanotic lesions, 30
Genome-wide linkage studies, 242–243 basal layer vacuolization, 30
Ginkgo biloba, 465 biopsy site selection, 26
Global assessment scale (GAS), 196, 197 epidermal and dermal changes, 26, 30
Graft versus host disease (GVHD), 84 epidermal-clustered lymphocytes, 28
Graves’ disease, 126, 127, 135, 172, 242 focal spongiosis, 28
Growth factor follicular melanocytes, 28
bFGF protein levels, 307 Fontana-Masson silver staining, 26
granulocyte-macrophage colony-stimulating factor, functional melanin-producing melanocytes, 26, 27
270, 306, 307 marginal melanocytes, 28
hepatocyte growth factor, 306 melanophages, 30
keratinocytes, 306 of normal-appearing skin, 30–31
microphthalmia-associated transcription factor, 306 phototherapy, 30
Guillet-Westerhof syndrome, 21 pro-melanogenic mediators, 30
reparative/protective phenomenon, 29, 30
residual melanocytes, 28
H spongiosis, 30
Hair follicle keratinocytes (HFK), 227 of stable lesional vitiligo, 26, 27
Hair follicle melanocyte (HFM) culture, 227–228 vacuolar degeneration, 29
Hair follicles, 339 HIV infection-associated inflammatory vitiligo, 84
Hair graying, 170 H3K9 acetylation (H3K9Ac), 260
Halo nevus HLA alleles, 241
clinical features, 94–95 HLA-DQB1 gene expression, 260
diagnosis, 100 H3T-A2 mouse model, 218–219
histopathology, 97–98 Human leukocyte antigens (HLA)-A2, 287
hydrogen peroxide concentration, 99 Human Protein Atlas (HPA), 234
with non-segmental vitiligo, 99 Hydrogels, 228
pathogenesis, 96 Hydrophobic interaction chromatography (HIC), 234
treatment, 100 Hydroquinone (HQ), 381
with Vogt-Koyanagi-Harada syndrome, 99 p-Hydroxyanisole (HA), 384
Hank’s balanced salt solution, 229 3-Hydroxy-3-methylglutaryl-coenzyme A reductase
Harvesting epidermal cells, 412 (HMG-CoA reductase), 466
Hashimoto’s autoimmune thyroiditis, 259 Hypochromic vitiligo/vitiligo minor, 88–89
Hashimoto’s thyroiditis, 94, 126, 127, 131, 145, 172, Hypothalamic-pituitary-adrenal, 307
212, 259, 287, 346
Heat shock proteins (HSPs), 289–290
Hemicorporeal hypomelanosis of Ito, 18, 22 I
Hepatocyte growth factor (HGF), 330 IADVL Dermatosurgery Task Force, 349
Hereditary pigmentary disorders, 109 “Ideal observer” method, 174, 175
Herodotus, 7 Idiopathic CD4+ T-cell lymphocytopenia (ICTL), 131
Highly active antiretroviral treatment (HAART), 130 IL-10 gene, 259
High phototypes Image-Pro Plus 4.5, 197
leukotrichia, 443 Imatinib, 388
medical treatment, 443–444 Imiquimod, 388
prevalence, 442 Immune reconstitution inflammatory syndrome
surgery, 444–445 (IRIS), 131
vitiligo phobia, 445 Immunity/immunopathology
Histone acetylation, 260 antigen-presenting cells, 288–289
Histone acetyltransferases (HATs), 260 autoimmune disease, 286–287
Histone deacetylases (HDACs), 260 cellular immunity, 287–288
Histone demethylase (HDMs), 260 cytokine inhibition, 295
Histone methylation, 260 environmental factors, 287
Histone methyltransferases (HTMs), 260 heat shock proteins, 289–290
Histone modifications HSP70+ exosomes, 291–292
in autoimmune diseases, 260–261 inhibiting T cell recruitment, 294
chromatin modification in melanocyte JAK inhibitors, 294
differentiation, 261 melanoma T cell responses, 295
histone acetylation, 260 modified HSP70, 293–294
ERRNVPHGLFRVRUJ
490 Index
Immunity/immunopathology (cont.) epidermis reconstruction

morphology of cultured melanocytes, 286 dead de-epidermized dermis, 229
phototherapy, 292–293 fibroblasts and collagen-glycosaminoglycan-
T cell subset imbalance, 290 chitosan, 229
T cell trafficking, 290–291 Langerhans cells, 229
toll-like receptors, 291 melanocytes and keratinocytes, 229
Treg-based therapy, 294–295 functional studies
Immunohistochemistry, 31–32 chromatin modifications, 231
Immunosuppressants, 476, 482 epigenome editing, 230, 231
Immunosuppressive systemic therapies genetically modified cells in monolayer, 230
anti-IFN-α approach, 369–370 keratinocyte cultures, 229
anti-IFN-γ strategy, 369 melanocyte cultures, 229
classic immunosuppressants, 371 reconstructed epidermis, 231–232
corticosteroids single-cell epigenomics approach, 231
oral minipulse, 366–368 In vivo confocal microscopy, 183
pulse corticosteroids, 366 In vivo reflectance confocal microscopy (RCM)
side effects, 366 concept, 199
immunomodulators, 368 half rings/scalloped border-like rings, 201
JAK Inhibitors, 371 limitations, 194, 202
systemic immunosuppressants, 368 papillary rings, 201, 202
T cell skin recruitment blocking strategy, 370–371 stability of vitiligo, 202
TNF-α antibodies, 371 UVB therapy, 201, 202
Independent component analysis, 197 Isobutylmethylxanthine (IBMX), 383
Indeterminate leprosy, 20
Induced pluripotent stem (iPS) cells, 314
Inflammatory infiltrate, 32 J
Inherited/genetically induced hypomelanoses Janus kinases (JAKs), 294
classic oculocutaneous albinism, 17
hypomelanosis of Ito, 14, 18
monogenic hypomelanoses, 14, 15 K
piebaldism, 14–17 Keratinocyte growth factor (KGF), 330
tuberous sclerosis, 14, 17 Keratinocytes, 32–34, 190, 191
Waardenburg’s syndrome, 14, 18 Keratinocyte stem cells (KSCs), 267
Innate immunity hypothesis, 341 Khellin, 465
Integra®, 229 Kilas, 4, 178
Integrin expression, 330 Koebner phenomenon (KP), 13, 19, 98, 99, 165, 166,
Intercellular adhesion molecule (ICAM)-1, 352 184, 346
Interleukin-1β (IL-1β) inflammatory pathway, 243 classification, 115, 116
Interleukin-2 receptor alpha chain (IL2RA), 287 assessment by patient history, 115
International Consensus, 13 by clinical examination, 115
Interobserver variability, 174–175 by lesions, 115
Invariant natural killer T (iNKT) cells, 257 clinical differentiation, 115
In vitro study clinical presentation, 117–119
analytic techniques depigmentation, 116
fluorescence-based assays, 232–233 etiopathogenesis, 117
lipidomics, 234 on friction areas (Type 2A), 117
metabolomics, 234 incidence of, 115, 117
proteomic assay, 233–234 melanocyte loss, 338–339
transcriptomics, 235 mini-grafting test, 117
cell isolation and culture post-inflammatory hypopigmentation, 118
Eagle’s minimal essential medium, 227 Wood’s light examination, 118
fibroblasts, 227 Krt14-Kitl∗ mouse-pmel-1 T cell adoptive transfer
hair follicle keratinocytes, 227–228 model, 217–218
hair follicle melanocyte, 227–228
hydrogels, 228
keratinocytes, 226–227 L
melanocytes, 226–227 L∗, a∗, b∗ color parameters, 198
PBMC, 228 Langerhans cells, 34, 229
transgenesis, 228 Laser scanning cytometer (LSC), 233
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Index 491
Late-onset vitiligo Melanocytic targets, 182

clinical features, 148 Melanoma-associated depigmentation, 19, 20
definition, 147 Melasma, 21, 22
and diabetes mellitus, 148–149 Membrane lipids, 340–341
epidemiology, 147–148 Mesenchymal stem cell (MSC), 314, 315
therapeutic considerations, 149 Metabolomics, 234
Lesional keratinocytes, 32, 230, 280, 330 Metastatic melanoma, 164–166
Leukoderma, 6 4-Methoxyphenol (MP), 384–385
Leukoderma punctata, 89 Methyl-CpG-binding domain (MBD) proteins, 259
Leukotrichia, 426, 443, 444 Mexameter, 199
clinical features, 95–96 MGM4 BulletKit (Lonza), 227
diagnosis, 100 Microinflammation, 183
pathogenesis, 96–97 Microphthalmia-associated transcription factor (MITF),
treatment, 100–101 165, 256, 261, 279, 280, 319, 320
Lipidomics, 234 Microvascular skin homing, 335
Liquid chromatography tandem-mass spectrometry Minimal erythema dose (MED), 347, 426
(LC-MS/MS), 233 Mini-punch grafting (MPG), 441, 445
Low self-esteem, 97, 178, 452 Minolta CR-200 Chroma Meter, 199
Lymphangioleiomyomatosis (LAM), 109 miRNAs in melanogenesis
biogenesis and function, 254–255
immune tolerance regulation, 256–257
M miR-145, 256
M154 (Gibco), 227 miRNA dysregulation, 257–258
M254 (Gibco), 227 MITF, 256
“Maillard’s” reaction, 424 solar-simulated UV, 256
Maintenance therapy, 416–417 UVR, 256
Major histocompatibility complex (MHC), 241 MITF, see Microphthalmia-associated transcription
Management-oriented evaluation, 346 factor
MatriDerm™, 229 Mitochondria, 136, 280
MBEH, see Monobenzyl ether of hydroquinone Mitogen-activated protein kinase (MAPK), 279
MCDB153 (Sigma), 227 Mixed vitiligo (MV), 13, 59, 60
McSCs, see Melanocyte stem cells bilateral mosaicism, 78–79
MED, see Minimal erythema dose clear segmental blaschkolinear pattern, 74
Medical therapies definition, 74
antioxidants, 411–413 diagnosis, 76–77
calcineurin inhibitors, 351–354 genetic link SV-vitiligo/NSV, 77–78
combination therapy, 352–353 halo nevi, 78
Melagenin, 465 leukotrichia, 78
Melanin, 430 nonsegmental involvement, 74–76
Melanin index, 198 risk factors, 77
Melanoblasts segmental involvement, 74, 76
cadherins, 268, 269 therapeutic intervention, 74
dorsolateral pathway, 266, 267 univariate analysis, 77
dorsoventral pathway, 267 UVB phototherapy, 76
keratinocyte stem cells, 267 Wood’s lamp examination, 77
melanocyte progenitors migratory pathway, 266–267 M2 medium (PromoCell), 227
melanocyte stem cells, 267 Modified HSP70, 293–294
skin distribution and differentiation, 269 Monobenzyl ether of hydroquinone (MBEH),
Melanocyte homeostasis 48, 387
epidermal melanin unit, 269–270 ATRA, 384
epidermal melanocyte renewal, 271 definition, 421
Melanocyte loss, 338–339 mechanism of action, 384
Melanocytes, 32, 34, 319–320, 325 vs. 4-methoxyphenol, 384–385
Melanocyte-specific T lymphocytes, 335 vs. 88% phenol solution, 385
Melanocyte stability precautions, 383–384
E-cadherin and vitiligo, 271–274 side effects, 384
epidermal melanin unit, 271 treatment procedure, 383
Melanocyte stem cells (McSCs), 267, 271, 273, 274 Monogenic hypomelanoses, 14, 15
Melanocyte stemness, 339–340 Mono-segmental vitiligo, 14
ERRNVPHGLFRVRUJ
492 Index
Mouse models Nijmegen breakage syndrome (NBS), 137–138

FH mouse, 218 NLRP1 gene, 243
h3T-A2 mouse, 218–219 Nonclassical melanocytes, 266
Krt14-Kitl∗ mouse-pmel-1 T cell adoptive transfer Non-cultured epidermal cell suspension (NCES)
model, 217–218 transplantation, 385–389
melanocyte-specific immune responses, 214–215 age, childhood in, 441
Pmel-1 mouse, 217 basal cell layer suspension, 387
TCR transgenic hosts, 215–219 culture medium and xenobiotics, 385–386
TrpHEL mouse, 218 donor skin, 386
TRP1 transgenic mouse, 218 factors influencing outcome, 389–390
vitesse mouse, 219 hyaluronic acid, 387
MPG, see Mini-punch grafting hypopigmentation, 386
MSC, see Mesenchymal stem cell laser ablation, 386
Mucosal pigmentary system, 182 long-term follow-up, 386–387
Multichrome vitiligo, 44–45 protocol, 395–396
Multifactorial nature of autoimmune diseases, 206 simplification, 390–392
Multilineage-differentiating stress-enduring (Muse) cells ultrathin epidermal graft, 388–389
in adult tissues, 314, 315 UVB stimulation, 387–388
BM-MSCs, 315, 316, 318–319 wound healing, 386
characteristics Non-cultured outer root sheath hair follicle cell
non-tumorigenicity, 318 suspension (NCORSHFS), 392–395
pluripotency, 317–318 Non-lesional skin, metabolic profile, 279–280
SCID mice, 318 Non-melanoma skin cancers (NMSC), 432
sources, 317 Non-Muse cells, 318–319
SSEA-3, 316–317 Non-segmental vitiligo (NSV), 13, 45, 333, 340
fibroblasts, 318–319 Normally pigmented skin, 35
functional evaluation, 324–325 Nucleosome remodeling factor (NURF), 261
future perspectives, 325 Nummular lichenoid lesions, 83
hematopoietic stem cells, 315
human-colored 3D-cultured skin, 322–323
melanocytes, 319–320, 325 O
non-Muse cells, 320–322 Occupational and drug-induced depigmentation, 22
propensity, 319 Occupational vitiligo, 122
somatic stem cells, 314–315 Ocular melanocytes
Multisegmental vitiligo, 13 eye anatomy, 105
MV, see Mixed vitiligo negative ocular electrophysiologic findings, 106
Mycosis fungoides, 19, 20 ocular pigmentary changes, 106
Myoclonic epilepsy associated with ragged-red fibers in retinal pigment epithelium, 105, 106
(MERRF) syndrome, 136 in uveal tract, 105–106
Myopathy, encephalopathy, lactic acidosis, and Oligonucleotide-based microarrays, 235
stroke-like episodes (MELAS) syndrome, 136 Oral minipulse (OMP), 366–368
Otic melanocytes, 107–108
Outer root sheath (ORS), 392, 394
N Oxidative stress, 189, 190, 278–279
Naevus cells, 183, 184 Oxysterols, 330
Naevus depigmentosus, 335
Narrowband UVB (NB-UVB), 146, 147, 157–159, 292,
347–348, 353, 362, 363, 412 P
Natural psoralens, 8 Para-aminobenzoic acid (PABA), 372, 434
NBS, see Nijmegen breakage syndrome Para-infectious hypopigmentation, 20–21
N-cadherin, 268, 271 Para-malignant hypomelanoses, 19, 20
NCES, see Non-cultured epidermal cell suspension Partial depigmentation, 46, 174, 386
transplantation Pathogen-associated molecular patterns (PAMPs), 243
Neural crest cells (NCC), 266 Pathology, 183
Neuronal theory, 191 Pathophysiology
Neuropeptides, 179 animal models, 191
Nevus depigmentosus, 22, 23 environmental factors, 189
Nicotinamide adenine dinucleotide (NADH)-binding genetic factors, 189
site, 279 in vitro approach, 191
ERRNVPHGLFRVRUJ
Index 493
melanocyte loss, 189 Principal component analysis, 197

mitochondrial dysfunction, 190 Programmed cell death-1 (PD-1), 164, 165
non-segmental vitiligo, 190, 191 Proopiomelanocortin (POMC), 279
oxidative stress, 189, 190 agouti signaling protein, 309
prooxidant/antioxidant status, 190 α-MSH, 308
redox alteration of lipids, 190 β−endorphin plasma levels, 307, 308
segmental vitiligo, 190, 191 Furin convertase, 308
stressed melanocytes, 190 hypothalamic-pituitary-adrenal, 307
Patients’ perspectives MC1R, 308, 309
associations, 461–462 mRNA levels, 308
care schemes, 459 Prooxidants, 278–281
doctor-patient relationship, 458–459 Propionibacterium acnes, 21
doctors perceptions, 457 Prostaglandins, 467
factors, 457 Protein equalization, 234
holistic care for sufferers, 457–458 Proteomics, 233–234
issues, 458 Pseudocatalase (PC), 349, 365–366, 387
phototherapy, 458–459 Psoralea corylifolia, 7, 8
transitional care Psoralen and ultraviolet A (PUVA) phototherapy
corrective makeup, 460–461 GVHD, 96–97
drug-based treatments, 459–460 mycosis fungoides, 96
psychological care, 460 Psoriasis, 128
sunscreen cover, 461 Psychiatric morbidity, 154, 178, 452
Pattern recognition receptors (PRRs), 291 Psychological factors, 175
PBMCs, see Peripheral blood mononuclear cells Psychological interventions
P-cadherin, 268, 269 cognitive-behavioural therapy, 453
Perceived severity, 175, 176, 346, 452 perceived severity, 452
Perifollicular repigmentation, 174 prevalence, 452
Perilesional hypopigmentation, 439 screening patients, 452
Periocular vitiligo, 445 Psychosocial effects, 178
Perioral vitiligo, 445–446 PTPN22 gene, 241
Peripheral blood mononuclear cells (PBMCs), 228, 257, PUVA therapy, 9
281
Personalized stratified approach, 476, 477
Phenolic/catecholic derivatives, 122 Q
88% Phenol solution, 385 Q-switched alexandrite (QSA) laser,
l-Phenylalanine, 134, 372 386–387
Photoprotection issues Q-switched ruby (QSR) laser, 386
normal and vitiligo skin UV sensitivity, 430–431 Quality of life (QoL)
practical photoprotection, 433–434 DLQI, 179
skin cancer, 432–433 historical perspective, 177–178
UV irradiation, 431–432 psychological stress, 179
Phototherapy, 292–293 psychosocial effects, 178
Phototherapy chamber or with sunlight (PUVASOL), 441 socio-economic and educational consequences,
Phytophotodermatitis, 8 178–179
Piebaldism, 14–17, 132 vitiligo impact scale, 179
Pigmentary mosaicism, 335
Pimecrolimus, 348, 356, 358, 359, 440
Plant-derived extracts, 465 R
Pmel-1 mouse model, 217 Reactive oxygen species (ROS), 189–190, 278–281
Point-counting method, 197 ReCell® kit, 390, 391
Poliosis, 95, 97 Reflectance spectroscopy
Polyclonal antibodies, 232 colorimetric examination, 198
Polypodium leucotomos, 349, 370, 373, 415, 465 DermaSpectrometer, 199
POMC, see Proopiomelanocortin erythema index, 198
Post-inflammatory hypomelanoses, 18, 19 melanin index, 198
Posttraumatic leukoderma, 21 Mexameter, 199
Predictive assessment, 183 tristimulus colorimeter, 198
Premature canities, 97 Regenerative medicine, 483
Premature hair graying, 94, 98, 99, 170, 183 Relative melanin index (RMI), 199
ERRNVPHGLFRVRUJ
494 Index
Repigmentation sympathetic denervation, 55

anatomical factors, 471 tacrolimus ointment, 67–68
Erbium-YAG laser, 473 treatment, 68
follicular melanocytes, 470 of trunk, 62, 63
lesion size, 472 twin-spotting phenomenon, 57
marginal melanocytes, 470 white macule, 63, 64
pathological factors, 471 Senescence-associated secretory phenotype (SASP), 279
sources, 470–471 Serum lipidomics, 234
stabilization, 475–476 Sexual relationship, 178
timing of surgery, 474 Shirabito, 4
RNA-induced silencing complex (RISC), 255 Shweta kustha, 178
ROS, see Reactive oxygen species Single-nucleotide polymorphisms (SNPs), 242, 246
“Rule of nines,” 196 Sirtuins, 280
6-well plate technique, 391
Sjögren’s syndrome, 84
S Skin biopsy, 172
Salicylates, 434 Skin-derived precursors (SKPs), 325
SBEG, see Suction blister epidermal grafting Skin melanocytes
Scalp vitiligo, 446 cellular origin, 266–267
Schmidt (APS2) syndrome, 135 melanoblasts
Schwann cell precursors (SCPs), 267 cadherins, 268, 269
SCORAD index, 170 dorsolateral pathway, 266, 267
Scoring/grading methods dorsoventral pathway, 266
global ssessment scale, 196 keratinocyte stem cells, 267
point-counting method, 197 melanocyte progenitors migratory pathway, 266
“rule of nines,” 196 melanocyte stem cells, 267
VASI, 196, 197 skin distribution and differentiation, 269
VIDA score, 196, 197 Skin melanogenic unit, 339
Scoring system, 172–174 Skin of color
Secreted frizzled-related protein 1 (SFRP1) gene, 261 clinical subtypes of vitiligo, 156
Segmental vitiligo (SV) depigmentation therapy, 158–159
alpha-and beta-adrenoreceptor response, 56 etiology and pathogenesis, 155–156
bilateral involvement, 63–65 quality of life, 154–155
blaschkolinear/developmental hypothesis, 56 treatment, 157–158
blaschkolinear distribution patterns, 56–58 Slide-based cytometry (SBC), 233
body leukotrichia, 60 Smyth line (SL) chicken, 259
causes, 54 alopecia areata-like feathering defect, 213
cephalic SV, 61–62 autoimmune hypothyroiditis, 212
classification, 61 avian model, 213
clinical and pathological features, 58–60 blindness, 212
depigmentation of hairs, 61 characteristics, 207
dermatomal/neural hypothesis, 54 environmental factor, 211–212
eyebrow and eyelashes, 61 genetics basis, 208–209
helium-neon (He-Ne) laser, 67 immune system, 209–211
immunologic mechanisms, 54 impaired vision, 212
ischemic depigmentation, 55 inherent melanocyte alterations, 211
Koga’s experiments, 54, 56 spontaneous autoimmune vitiligo, 207–209
leukotrichia-associated SV, 61 uveitis, 212
NBUVB, 65–67 Social isolation, 178, 452
neural mechanisms, 334 Social status of vitiligo patients, 6–7
and nevus spilus, 57, 59 Socio-economic and educational consequences, 178–179
vs. non-segmental forms, 334 Solar simulated radiation (SSR), 430–431
microvascular skin homing, 335 Soybean trypsin inhibitor, 391
physostigmine-induced sweating response, 56 Spreading, 173, 174
prevalences, 333 SSR, see Solar simulated radiation
poliosis, 61 Staging, of vitiligo, 172–174
PUVA treatment, 65–66 Statins, 466
recurrence of vitiliginous lesions, 62, 63 Stem cell factor (SCF), 330
repigmentation, 65–66 Stratum corneum (SC), 430, 431
similar segmental distribution, 54, 55 Suction blister epidermal grafting (SBEG), 441, 444, 445
somatic mosaicism, 56, 58, 334–335, 340 Sun-protective factor (SPF), 433–434
sympathetic anomalies, 56 Sun-reactive skin phototypes (SPT), 430
ERRNVPHGLFRVRUJ
Index 495
Sunscreen cover, 461 therapeutic options, 359

Suprabasal melanocytes, 272 TNFα production, 356
Surgical therapies, 348 topical corticosteroids, 354
cellular grafting Topical corticosteroids (TCS)
cultured epidermal sheet transplantation, 384–385 application scheme, 353, 355
cultured melanocyte transplantation, 383–384 betamethasone, 355
types, 382, 383 fluticasone, 352, 355
vitiliginous recipient sites, 382 fluticasone propionate, 352
history, 9 hypermelanoses treatment, 354
NCES intralesional therapy, 354
basal cell layer suspension, 387 mechanism of glucocorticoid action, 354
culture medium and xenobiotics, 385–386 NSV, 349
donor skin, 386 side effects, 355–356
factors influencing outcome, 389–390 skin atrophy, 351
hyaluronic acid, 387 steroid clobetasol dipropionate, 355
hypopigmentation, 386 TCS, 351
laser ablation, 386 with tretinoin and hydroquinone, 354
long-term follow-up, 386–387 Topical glucocorticosteroids (TCS), 353–354
protocol, 395–396 Topical therapies, 348
simplification, 390–392 antioxidants, 349
ultrathin epidermal graft, 388–389 calcineurin inhibitors, 351
UVB stimulation, 387–388 combination therapy, 352–353
wound healing, 386 pseudocatalase, 414
NCORSHFS transplantation, 392–395 tacrolimus and pimecrolimus, 352–353
SV, see Segmental vitiligo topical corticosteroids
SvetaKhista, 4 fluticasone, 352
Sweat secretion stimulation test, 54 fluticasone propionate, 352
Symptomatic inflammation, 184 NSV, 349
Syndromic vitiligo, 183 side effects, 353
Systematic pathology assessment, 183 skin atrophy, 351
Systemic antioxidants, 372–374 TCS, 351
Systemic lupus erythematosus (SLE), 259 vitamin D analogues, 414
Systemic oxidative stress, 281 Traditional Chinese medicine, 464–465
Systemic sclerosis (SSC), 259 Transcriptomics, 235
Transgenesis, 228
Transit amplifying cells (TAC), 267
T T regulatory cells (Tregs), 190, 304
Tacrolimus, 356, 357, 359, 413, 439–440 Tristimulus colorimeter, 198
T cell skin recruitment blocking strategy, 370–371 TRP1 transgenic mouse model, 218
T cell subset imbalance, 290 Tuberous sclerosis, 14, 17
T cell trafficking, 290–291 Two-dimensional differential image-gel electrophoresis
TCI, see Topical calcineurin inhibitors (2D-DIGE), 233
TCS, see Topical glucocorticosteroids Type 1 diabetes (T1D), 260
T1D susceptible genes, 260 Tyrosine-related protein (TRP)-1, 279, 280
12-O-Tetradecanoylphorbol-13-acetate (TPA), 383
Th1/Th2 predominant diseases, 182
3D-cultured skin system, 322–323 U
Thyroglobulin, 172 Ubiquitin-associated and SH3 domain-containing A
Thyroid peroxidase (TPO), 172 protein (UBASH3A), 287
Toll-like receptors (TLRs), 291 Ultraviolet (UV) treatments, 347–348
Topical calcineurin inhibitors (TCI), 348 3' Untranslated region (3' UTR), 255, 256
application scheme, 359–361
for atopic dermatitis, 356
beneficial effect, 356 V
case reports and clinical studies, 357 VASI, see Vitiligo area scoring index
combined therapies, 348, 357, 359 VDR, see Vitamin D receptors
in combination with UVB, 357, 358 VETF, see Vitiligo European Task Force
for inflammatory skin diseases, 356 Vinay Pitak, 4
interpretation, 353–354 Vitamin B12, 97
pimecrolimus, 356, 358, 359 Vitamin C, 386
side effects, 361–362 Vitamin D, 414
tacrolimus monotherapy, 357, 359 Vitamin D analogues, 362–364, 414
ERRNVPHGLFRVRUJ
496 Index
Vitamin D receptors (VDR), 155 diagnosis, 48

Vitamin E, 372–373 management, 49
Vitamin supplementation, 157, 372 MBEHQ, 48
Vitesse mouse model, 219 non-segmental vitiligo, 45
Vitiligo area scoring index (VASI), 194, 196, 197 oral beta-carotene, 48
Vitiligo disease activity (VIDA) score, precipitating factors, 46–48
194, 196, 197, 202 prevalence, 45, 46
Vitiligo during pregnancy, 146–147 true segmental vitiligo (SV), 45
Vitiligo European Task Force (VETF), Vitiligo vulgaris, 13
12, 170, 172, 195, 287, 346 Vitix®, 366
Vitiligo fibroblasts, 330 Vogt-Koyanagi-Harada (VKH) syndrome,
Vitiligo Global Issues Consensus Conference 48, 82, 83, 109, 182, 183, 213, 218
(VGICC), 12 VU, see Vitiligo universalis
Vitiligo impact scale (VIS), 179
Vitiligo-like depigmentation,
94, 96, 164–166, 214, 215 W
Vitiligo locations Waardenburg’s syndrome (WS), 14, 18, 109
elbows, knees, hands, and feet, 446, 447 Water buffalo, 213
genital vitiligo, 446 White leprosy, 5
periocular vitiligo, 445 Wnt/betacatenin pathway, 483
perioral vitiligo, 445–446 WNT pathways, 331
scalp vitiligo, 446 “Wood’s filter,” 194
Vitiligo minor/hypochromic vitiligo, 14 Wood’s lamp, 170, 171, 174, 194, 195
Vitiligo punctata, 14 Wood’s light
Vitiligo specific quality of life (vitiQoL), 154, 179 examination, 194–195
Vitiligo-specific quality of life measurement follow-up, 476
(VIS-22), 154 World vitiligo day, 179
Vitiligo universalis (VU), 13
autoimmune/autoinflammatory disorders, 48
clinical features, 46, 47 Z
clinical manifestation, 45 Zoorat, 4, 5
ERRNVPHGLFRVRUJ

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Vitiligo

ISBN 978-3-319-62958-2 ISBN 978-3-319-62960-5 (eBook)

© Springer-Verlag Berlin, Heidelberg 2010 First Edition

clearly needed. They should be confronted by genetics and other investigative

Bordeaux, France Alain Taïeb

and surgical armamentarium for vitiligo. Fortunately, things should change

Bordeaux, France Alain Taïeb

Part I Defining the Disease

1 Historical Aspects of Vitiligo ���������������������������������������������������������� 3

12 Environmental Triggers and Occupational/Contact

Part II Understanding the Disease

20 Pathophysiology Overview�������������������������������������������������������������� 189

Part III Treating the Disease

34 Management Overview�������������������������������������������������������������������� 345

43 Patients’ Perspectives���������������������������������������������������������������������� 455

Setsuya Aiba Department of Dermatology, Tohoku University Graduate

Maria Lucia Dell’Anna Cutaneous Physiopathology, San Gallicano

Cheng-Che Eric Lan Department of Dermatology, Kaohsiung Medical

Richard A. Spritz Human Medical Genetics and Genomics Program,

Abstract word “Zoorat” in the bible corresponds to a

© Springer Nature Switzerland AG 2019 3

Fig. 1.1 Papyrus

Fig. 1.2 From the

Fig. 1.3 Ammi majus

Born by night art thou, O plant

According to Indian medical books, the two most

exposed to sunlight [13]. Extensive research 1.6  odern History of Surgical

References 16. Lim HW, Grimes PE, Agbai O, et al. Afamelanotide

Abstract (VGICC). Vitiligo/nonsegmental vitiligo

© Springer Nature Switzerland AG 2019 11

meetings and workshops a set of consensus state-

Table 2.1 Bordeaux VGICC classification of vitiligo (2011)

2.2 International Consensus “vulgaris,” as synonymous with “common,” con-

2.3.2.2 Segmental Vitiligo p­ igmentation. The relation to true vitiligo comes

Table 2.2 Monogenic hypomelanoses

Fig. 2.3 Hypomelanosis of Ito (courtesy of Dr O Fig. 2.4 Waardenburg’s syndrome

2.5.1.3 Para-malignant 2.5.1.4 Para-malignant Hypomelanoses/

Fig. 2.6 Mycosis fungoides can present as hypopig-

Fig. 2.8 Indeterminate leprosy (a elbow, b forehead)

Fig. 2.12 Vitiligoid depigmentation of dorsum of hands

via c-Kit signaling downregulation [20], as well

2.5.2 Conditions to Exclude

Conference Panelists. Revised c­lassification/nomen-

Abstract ered. However, the increasing interest in

© Springer Nature Switzerland AG 2019 25

increasing interest in predicting the stability of

Table 3.1 Special stains and antibodies to detect melanocytes

Fig. 3.3 Skin biopsy of

3.6 Melanocytes 3.7 Keratinocytes

with these observations, Prignano et al. described

Abstract In addition, specific clinical aspects are

© Springer Nature Switzerland AG 2019 39

© Springer Nature Switzerland AG 2019 41

d­ efinition limited to the face, head, hands, and 5.1 Introduction

but total d­ epigmentation of the scalp hair may 5.2.1 Distribution

The evolution of generalized vitiligo is unpre-

AFV. There are no series looking specifically at

5.2.3.2 Blue Vitiligo 5.4 Vitiligo Universalis

childhood. The population prevalence of VU is

5.4.1 Clinical Features

In VU, nearly all the skin becomes completely

Fig. 5.7 An Indian woman with vitiligo universalis and

Occasionally before complete depigmentation,

Bordeaux, France Alain Taïeb

Bordeaux, France Alain Taïeb

Part I Defining the Disease

1 Historical Aspects of Vitiligo �� 3

12 Environmental Triggers and Occupational/Contact

Part II Understanding the Disease

20 Pathophysiology Overview�� 189

Part III Treating the Disease

34 Management Overview�� 345

43 Patients’ Perspectives�� 455

exposed to sunlight [13]. Extensive research 1.6 odern History of Surgical

2.2 International Consensus “vulgaris,” as synonymous with “common,” con-

2.3.2.2 Segmental Vitiligo p igmentation. The relation to true vitiligo comes

2.5.1.3 Para-malignant 2.5.1.4 Para-malignant Hypomelanoses/

2.5.2 Conditions to Exclude

Conference Panelists. Revised classification/nomen-

3.6 Melanocytes 3.7 Keratinocytes

d efinition limited to the face, head, hands, and 5.1 Introduction

but total d epigmentation of the scalp hair may 5.2.1 Distribution

5.2.3.2 Blue Vitiligo 5.4 Vitiligo Universalis

5.4.1 Clinical Features

5.4.6 Management Q-switched alexandrite lasers have proven to be

5.5 Conclusions 9. Fargnoli MC, Bolognia JL. Pentachrome vitiligo. J

8.4 Differential Diagnosis

8.6 Rare Variants

8.6.1 Follicular Vitiligo

8.6.3 Vitiligo Guttata/Punctata,

8.7 Summary and Concluding References

9.5 Diagnosis ing vellus hair using dermatoscopy, before initi-

10.7 Conclusion functions, autoimmunity, or malignancy war-

Abstract 11.1 Introduction