Advances in Experimental Medicine and Biology 1116

Clinical and Experimental Biomedicine

Mieczyslaw Pokorski Editor

Clinical
Medicine
Research
Advances in Experimental Medicine
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Clinical and Experimental Biomedicine

Volume 1116

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Mieczyslaw Pokorski
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Clinical Medicine
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Mieczyslaw Pokorski
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Clinical and Experimental Biomedicine
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Contents

Bioprogressive Paradigm in Physiotherapeutic and Antiaging

Strategies: A Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Mieczyslaw Pokorski, Giovanni Barassi, Rosa G. Bellomo,
Loris Prosperi, Matteo Crudeli, and Raoul Saggini
Influence of Proprioceptive Neuromuscular Facilitation
on Lung Function in Patients After Coronary Artery Bypass
Graft Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Małgorzata Bujar-Misztal and Andrzej Chciałowski
Remote Ischemic Preconditioning in Renal Protection During
Elective Percutaneous Coronary Intervention . . . . . . . . . . . . . . . . 19
Małgorzata Wojciechowska, Maciej Zarębiński, Piotr Pawluczuk,
Dagmara Gralak-Łachowska, Leszek Pawłowski, Wioletta Loska,
Marzena Goszczyńska, Krzysztof Flis, and Agnieszka
Cudnoch-Jędrzejewska
Prognostic Impact of Extracapsular Lymph Node Invasion
on Survival in Non-small-Cell Lung Cancer: A Systematic
Review and Meta-analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Seyed Vahid Tabatabaei, Christoph Nitche, Maximilian Michel,
Kurt Rasche, and Khosro Hekmat
Influence of Transurethral Resection of Bladder Cancer
on Sexual Function, Anxiety, and Depression . . . . . . . . . . . . . . . . 37
Wojciech Krajewski, Urszula Halska, Sławomir Poletajew,
Radosław Piszczek, Bartosz Bieżyński, Mateusz Matyjasek,
Andrzej Tukiendorf, Fryderyk Menzel, Małgorzata Mazur,
Joanna Rymaszewska, and Romuald Zdrojowy
Cognitive Predictors of Cortical Thickness in Healthy Aging . . . . 51
Patrycja Naumczyk, Angelika K. Sawicka, Beata Brzeska,
Agnieszka Sabisz, Krzysztof Jodzio, Marek Radkowski,
Karolina Czachowska, Paweł J. Winklewski, Karolina Finc,
Edyta Szurowska, Urszula Demkow, and Arkadiusz Szarmach

v
vi Contents

Osteoprotegerin, Receptor Activator of Nuclear Factor

Kappa B Ligand, and Growth Hormone/Insulin-Like Growth
Factor-1 Axis in Children with Growth Hormone Deficiency . . . . . 63
Ewelina Witkowska-Sędek, Małgorzata Rumińska,
Anna Stelmaszczyk-Emmel, Maria Sobol, Urszula Demkow,
and Beata Pyrżak
Inhibition of Cross-Reactive Carbohydrate Determinants
in Allergy Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Maciej Grzywnowicz, Emilia Majsiak, Józef Gaweł,
Karolina Miśkiewicz, Zbigniew Doniec, and Ryszard Kurzawa
Effects of Glutathione on Hydrolytic Enzyme Activity
in the Mouse Hepatocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Iwona Stanisławska, Bożena Witek, Marek Łyp,
Danuta Rochon-Szmejchel, Adam Wróbel, Wojciech Fronczyk,
Agnieszka Kamińska, Adam Kołątaj, and Daniel Załuski
Adaptation to Occupational Exposure to Moderate
Endotoxin Concentrations: A Study in Sewage Treatment
Plants in Germany . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
M. A. Rieger, V. Liebers, M. Nübling, T. Brüning, B. Brendel,
F. Hoffmeyer, and M. Raulf
Effects of Low-Level Laser Therapy in Autism Spectrum
Disorder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Gerry Leisman, Calixto Machado, Yanin Machado,
and Mauricio Chinchilla-Acosta
Epidemiology of Granulomatosis with Polyangiitis in Poland,
2011–2015 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Krzysztof Kanecki, Aneta Nitsch-Osuch, Paweł Gorynski,
Patryk Tarka, Magdalena Bogdan, and Piotr Tyszko
Adv Exp Med Biol - Clinical and Experimental Biomedicine (2018) 3: 1–9
https://doi.org/10.1007/5584_2018_281
# Springer Nature Switzerland AG 2018
Published online: 16 October 2018
Bioprogressive Paradigm
in Physiotherapeutic and Antiaging
Strategies: A Review
Mieczyslaw Pokorski, Giovanni Barassi, Rosa G. Bellomo,
Loris Prosperi, Matteo Crudeli, and Raoul Saggini
Abstract
This review updates the knowledge on the use
of bioprogressive philosophy in current reha-
bilitation paradigms, focusing on age-related
ailments and antiaging strategies. It is a holis-
tic approach that combines aspects of biology
and function into the realm of rehabilitation
therapy. The bioprogressive philosophy, with
assistance of modern technological
developments, such as microgravity-
producing devices and techniques, enables
personalized and targeted therapeutic
approach that seems the most effective in reha-
bilitation and prevention of neuro-myo-sen-
sory disorders that compromise the
homeostatic body harmony, particularly in
old age. The review defines the aging,
discusses the most common physical
dysfunctions, linked to posture, balance, or
gait, and gives cues to modern antiaging reha-
bilitative approaches. The emphasis is put on
M. Pokorski (*)
Opole Medical School, Opole, Poland
e-mail: m_pokorski@hotmail.com
G. Barassi, L. Prosperi, M. Crudeli, and R. Saggini
Department of Medical and Oral Sciences and
Biotechnologies, “G.d’Annunzio” University Chieti,
Chieti, Italy
R. G. Bellomo
Department of Biomolecular Sciences, “Carlo Bo”
University Urbino, Urbino, Italy
the proprio-neuromuscular facilitation, a
combination of stretch technique that steps
into the bioprogressive approach, as currently
the best method in the world of physical
rehabilitation.
Keywords
Aging · Antiaging strategy · Bioprogressive
paradigm · Physical dysfunction ·
Physiotherapy · Proprioceptive neuromuscular
facilitation · Rehabilitation
1 Bioprogressive Rehabilitation
Aging is usually defined as a progressive loss of
function accompanied by increasing morbidity
and mortality. Causes of the aging process are
multifactorial and can be classified into two
groups: intrinsic (notably genetic modifications)
and extrinsic (environmental) factors. Several
studies convincingly demonstrate the fast pace
of aging of the general population worldwide
and how this process is going to advance in the
next few years. As the elderly population grows
in terms of age and size, so do the requests for
assistance for senior persons (Christensen et al.
2009). The main feature of this phenomenon is
the elderly need for care which is required to
maintain their healthy condition, for instance,
performing some kind of exercise aimed to
1
2 M. Pokorski et al.
avoid the risk of falling or to begin a rehabilitation
protocol (Israely et al. 2018; Zur et al. 2018;
Aalen et al. 2014; Arbeev et al. 2009).
A bioprogressive multisensory rehabilitation
approach has proven the most suitable to face a
physiological reduction in stability. The
approach, supported by modern technology,
enables the creation of a changing training envi-
ronment, with the aim to increase proprioceptive
perception and balance reaction (Bellomo et al.
2009). Improvements can be monitored in a mod-
ern gait analysis laboratory to describe variations
in balance reactions, which enables the therapist
to periodically adapt the training program to
changes in the patient condition to reach increas-
ingly important therapeutic goals (Basile et al.
2016). Parkinson’s patients treated with a
bioprogressive rehabilitation paradigm, consisting
of a combination of dynamic antigravity postural
system (SPAD), auditory cue system (metro-
nome), high-intensity vibratory system (VISS),
and active stretching based on virtual reality reha-
bilitation system (VRRS) for several months,
experience a distinct improvement in balance sta-
bility and gait pattern, with a reduction in falls
and overall enhancement of quality of life
(Bellomo et al. 2014). Modern assistance
methods enable the elderly to access the training
facilities at any time and to use them in a safe
environment while continuing to provide
instructions for rehabilitation and monitoring of
progress (Akushevich et al. 2013). Some innova-
tive examples for the preventive and rehabilitative
intervention, which illustrate this increasingly
diversified scope and ever-changing future per-
spective of the intervention program, have been
described by Young (1983).
Aging concerns physical, cognitive, and social
spheres of individuals. The cardiovascular sys-
tem, muscle-skeletal mass, osteoarthritic struc-
ture, immuno-humoral metabolism, and central
nervous system disorders predominate in the
aged, altering the organism’s functionality
(Israely et al. 2018; Carmeli et al. 2016). Diabetes
and cardiovascular diseases, closely linked to
inflammation and oxidative stress, belong to at
least partly modifiable risk factors, with a proper
diet and correct lifestyle. Such pathologic
frameworks often occur in adulthood, and
behaviors are generally modifiable through a
series of rehabilitative interventions (Atkins
et al. 2014; Barzilai et al. 2012; Bergman et al.
2007). Thus, nutrition, frequency of physical
activity, and other crucial factors need to be
examined to develop the appropriated lifestyle,
which would also provide succor to metabolic
activity, cerebral functionality, and postural con-
trol (Si and Liu 2014; Bosma-den Boer et al.
2012; Misirli et al. 2012; Agnoli et al. 2011; Hu
2002).
Studying the inter-system correlations and the
rules that underlie the perceptive-motor pattern
holds a central role in the understanding of
relationships between movement, function, envi-
ronment context, and psycho-behavioral
characteristics of subjects. The human being
forms a unique biosystem whose management
requires a holistic approach, personalized for spe-
cific patient’s characteristics (Saggini et al. 2012).
Posture is identified as the morphological, senso-
rial, and motor expression of the evolved identity.
The somatosensory system, visual and auditory
structures, and vestibular receptors are essential
for maintaining the brain-regulated body motion
and balance, as well as a feel of one’s comfort
(Zimmermann et al. 2013). A potential alteration
of sensory afferentation can be compensated
through the redundancy characteristics of a sys-
tem, which also respond to appropriate
myofascial adaptations (Reghem et al. 2014).
The aging process underlies a progressive deteri-
oration of the delicate mechanisms of neurologi-
cal osteomuscular-fascial optimization,
manifesting as an altered “afferent stimulation”
that determines “postural dysfunction” (Barassi
et al. 2018a, b, c, d).
Already at gestational age week 5, a trilaminar
embryo may be recognized, with the interposition
of mesoderm between ectoderm and endoderm.
The epithelial to mesenchymal cell transition
emerges, during which the structure assumes its
function and form. In this mechanism, muscular
chains and fascial continuity appear to be the
evolutionary expressions of the spinal cord
(Barassi et al. 2018b). Paraxial mesodermal
elements of the skeletal muscular system are
Bioprogressive Paradigm in Physiotherapeutic and Antiaging Strategies: A Review 3

formed as the splanchnic mesodermal derivations,
in particular smooth muscles, derived from the
occipital-to-sacral region somites and head-region
somitomeres (Saggini et al. 1996).
It is known that fascial connective tissue is
able to pick up nociceptive and proprioceptive
information thanks to the presence of mechano-
and nociceptors and its spatial organization (Van
der Wal 2009). Studies also demonstrate that
connective tissue creates a network between the
musculoskeletal, neural, endocrine, circulatory,
respiratory, and immune systems, creating a
milieu for inter-tissue cooperation (Vecchiet
et al. 1999). In particular, fascia is composed of
cells that have the ability to contract and commu-
nicate with one another, ensuring the supervision
of body activity, such as the transmission of ten-
sion created by muscles or management of inter-
stitial fluids (Bordoni and Zanier 2015; Schleip
2003). One of the latest studies performed on
fascia tissue demonstrates, using a confocal laser
endomicroscopy, the presence of “reticular
structures” in visceral submucosa and dermal
periarterial stroma, which are hypothesized to be
an extension of the interstitial space (Benias et al.
2018). There appears a plausibility that such
structures play a central role in the body’s self-
management of edema to recreate homeostatic
harmony (Johnson 1977). Thus, contrarily to the
old physiological tenet of functional neutrality of
connective tissue, purportedly serving the crea-
tion of a parenchymal scaffold for cell arrange-
ment, connective cells appear to host a multitude
of active functions, inter alia, inter-cell communi-
cation, proprioceptive signal transmission, regu-
lation of extracellular space fluid, and others
(Bordoni and Zanier 2015). Nowadays, these
cells appear to underlie the effectiveness of the
bioprogressive rehabilitation paradigms that are
set to affect their function to restore a proper body
neuro-myo-sensory integration, as schematically
depicted in Fig. 1.
2 Stretch Exercise
One of the most common exercise techniques is
muscular-fascial stretch. Stretching technique
modifies the Golgi apparatus activity which, via
the spinal cord, decreases the firing rate of alpha

BODY DYNAMICS

PROPRIOCEPTIVE RECEPTORS SENSORIAL NEURAL STIMULATION

MESODERM
(Connective Tissue)

Microgravity Environment
Tissue Remodelling Rehabilitation Rehabilitation
Action
GOLGI OSTEO-MYO-FASCIAL
APPARATUS SYSTEM

STRETCHING MAV
Mechano-Acoustic Vibration Terrestrial Acquatic

ISOMETRIC PNF ISOTONIC

Proprioceptive
Neuromuscular
Facilitation

BODY DYNAMICS
OPTIMIZATION

Fig. 1 Schematic diagram of the bioprogressive philosophy of physiotherapeutic interventions, pointing to the central
role of connective tissue in maintaining the neuro-myo-sensory integration
4 M. Pokorski et al.
motor neurons, and, in turn, muscle tone, for
instance, as occurs in Yoga posture (Barassi
et al. 2018c; Schleip 2003). There exist various
types of stretching technique. The unresolved
issue with physical stretch is what it should be
like in the setting of health-care practitioners,
particularly those engaged in sports training or
rehabilitative framework in the elderly (Caplan
et al. 2009). There are three types of stretch exer-
cise: static stretching, dynamic stretching, and
proprioceptive neuromuscular facilitation (PNF)
(Fig. 1). A combination of these approaches
guarantees autogenic inhibition, mutual inhibi-
tion, stress relaxation, and a reduction of pain
consequently to the gate control theory, all of
which leads to improvements in the range of
motion of joints (Nelson et al. 2005; Wicke
et al. 2014; Hindle et al. 2012). Static stretching
consists of holding muscles in an elongated posi-
tion for an extended period. Contrarily, dynamic
stretch consists of moving joints through their
range of motion. There are pros and cons of
both types of stretch. Static isometric exercise,
although being physically motionless, is a strong
respiratory stimulant that provides succor to arte-
rial blood and, consequently, tissue oxygenation.
It also increases heart rate and blood pressure
(Pokorski et al. 1990). These effects, thought of
as typical and beneficial from the standpoint of
physical rehabilitation or training, may jeopardize
the frail elderly due to abruptly increased meta-
bolic rate. Static stretch may also lead to muscle
weakness, which hampers subsequent physical
performance. Dynamic stretch, on the other
hand, gives proneness to injuries, which may
actually hamper the range of motion that already
declines with age but is important for daily life
activities (Konrad and Tilp 2014). Dynamic
stretch, however, increases the nerve conduction
velocity, muscle compliance, enzymatic turnover,
and the central control running down from the
brain to muscles (Wicke et al. 2014). The PNF
combines the stretching and contraction of
reciprocally related muscle groups in a cyclical
pattern. “Contract-relax” (CR) and “contract-
relax-antagonist-contract” (CRAC) methods are
the representative techniques of PNF. In the CR
technique, the target muscle is lengthened and
held in that position, while rehabilitant contracts
the muscle isometrically to its maximum for a
certain amount of time, followed by a brief
phase of passive stretching. CRAC is an exten-
sion of the former technique in which after the
maximum isometric contraction of the agonistic
muscle, rehabilitant contracts the corresponding
antagonistic muscle for a certain amount of time.
Ultimately, it seems that a mix of stretch modes,
in particular static stretch followed by dynamic
stretch, optimizes the benefits gained from physi-
cal rehabilitation, simultaneously reducing the
number of exercise-induced muscle impairments
or injuries (Rowlands et al. 2003). To this end,
meditation techniques consisting of a variable
motion ingredient and resembling the CRAC
sequence of stretches, such as is Tai Chi and the
like, advance in popularity of late (Hindle et al.
2012). An additional benefit of meditation is the
improvement in mood, emotional stability, and
cognition (Pokorski and Suchorzynska 2018;
Lam et al. 2014).
3 Aging and Rehabilitation
Old age should not be seen as a disease or pathol-
ogy but as a stage of life that can be fully explored
to pursue personal and social goals. Human func-
tionality has an outstanding plasticity and is capa-
ble of compensating for the lost structural
elements caused by the age-related dampening
of cellular metabolism. That may be exemplified,
for instance, by the adaptive hyperventilatory
responses to strenuous stimuli, such as exercise,
in old age, despite a severe structural deterioration
of the lungs and airways, limiting the alveolar gas
exchange area and thus oxygen diffusion
(Pokorski and Marczak 2003). Likewise, cogni-
tive brain functions are maintained due to a
greater neuronal synchronization and a flexible
use of cognitive reserve or recruitment of alterna-
tive neuronal pathways, normally constituting a
dormant reserve. The antiaging strategy is a pro-
cess that not only involves combating the disease
and its effects but also a large psychological com-
ponent that is closely linked to behavior, person-
ality traits, and other factors (Angel and Hogan
Bioprogressive Paradigm in Physiotherapeutic and Antiaging Strategies: A Review
1992). Exercise is an antiaging strategy linked to
physical and cognitive rehabilitation, develop-
ment of positive stress, and lifestyle changes
(Rebelo-Marques et al. 2018). The mind-to-body
connection is indispensable for a motivated
engagement in antiaging behaviors, such as exer-
cise, and in physical rehabilitation in case of a
disease-induced handicap (Wrycza and Baudisch
2012; Depp and Jeste 2009). Exercise has a pro-
tective effect on cognitive functions and helps
combat the deleterious effects of stress and
aging. The practice of proper physical exercise
can improve physiological and functional
responses, notably cardiovascular fitness and
respiratory function, and thus physical health
and quality of life of the elderly (Bellomo et al.
2017). Exercise also plays a key role in the assess-
ment and prevention of frailty in the elderly
(Singh 2002). In adulthood, aerobic skills and
general physical activity are associated with
lower mortality and morbidity from cardiovascu-
lar diseases. Benefits of physical activity also
include reductions in a risk for stroke, diabetes
mellitus, cancer, and osteoporosis (Ruiz et al.
2008; Lee and Skerrett 2001; Bijnen et al. 1999;
Hakim et al. 1998; Paffenbarger et al. 1993).
Aging is inherently involved with a loss of
muscle mass and function leading to sarcopenia
and, consequently, to postural dysfunction
(Carmeli 2017; Lin and Bhattacharyya 2012).
Sarcopenia is underlain by a reduction in protein
synthesis and an increase in muscle protein deg-
radation (Delli Pizzi et al. 2017). Further, the
ability of muscle regeneration is severely
compromised by aging, which can lead to disabil-
ity, particularly in patients with concomitant
diseases or organ failures. Skeletal muscle dys-
function can be ameliorated by well-suited exer-
cise that improves the function of muscle
mitochondria (Barbieri et al. 2015). Beneficial
effects of focal vibrations on the well-being of
ailing elderly patients are beyond doubt when
analyzing the results of studies on proprioception,
muscle tone, and quality of gait (Toosizadeh et al.
2018; Saggini and Bellomo 2015). Vibrations of
300 Hz effectively increase muscle strength, act-
ing on neuromuscular receptors, Golgi tendon
organs, Pacini corpuscles, as well as through the
5
regulation of genes that are responsible for
expression of sarcomeric proteins and for recruit-
ment of satellite cells. Integrated treatment with
focused mechano-acoustic vibrations has a bene-
ficial effect on bone mineral density (BMD) and
T-score and on muscle strength and quality of life
of osteoporotic subjects (Kwak et al. 2017;
Saggini et al. 2017).
The body’s agility to react to external forces
plays a central role in rehabilitation. Acting on
this mechanism enables the most individual adap-
tation of a therapeutic protocol and thus the use of
the entirety of the rehabilitation power and also
the exposition of compensative reactions and
rebalancing of human body network (Fig. 1). To
this end, microgravity environment is particularly
effective in reversing functional detriments and
limits concerning daily activities in patients with
muscle and balance disorders (Kaneda et al.
2008). The exemplary is the aquatic microgravity
environment, produced by immersion in water,
which improves body stability due to a decrease
in neural system activation caused by a central
redistribution of the blood volume (Barassi et al.
2018a). A decrease in sympathetic activity,
accompanied by increased parasympathetic activ-
ity, promoting vasodilation and blood circulation,
increases blood flow, accelerates cellular metabo-
lism and removal of unwanted materials, and
decreases pain sensitivity (Forestier and Françon
2008). The autonomic nervous system
remodeling positively affects the perception pain
and fatigue associated with musculoskeletal
disorders due mostly to decreasing muscle ten-
sion (Barassi et al. 2018d; Bellomo et al. 2012;
Yasui et al. 2010). The rebalancing of gait pattern
involves the development of relatively stable
changes in spinal and supraspinal networks that
are modeled by proprioceptive and somatosen-
sory sensory information specific for the task.
The development of specialized training
protocols for assisted exercise in the microgravity
environment hinges on the realization of the
importance of locomotor learning and how the
use of specific devices alters the demand stem-
ming from motor tasks (Wolpert et al. 2001). The
literature unambiguously shows that a specific
reeducation protocol in relieving body weight
6 M. Pokorski et al.
can improve the static and dynamic balance, lead-
ing to a better overall motor performance
(Moreno et al. 2013; Threlkeld et al. 2003). In
our daily practice, we implement the single-
photon avalanche detector (SPAD), a photon
detecting system for optical communications.
SPAD is a device able to compose a microgravity
environment during gait motion, which optimizes
the body tridimensional alignment in the body
weight support modality. It has a dual action:
mechanical, which allows for a cortical-
subcortical neuromotor retraining aimed at the
reacquisition of a body scheme in three planes
of the space, and functional, which minimizes the
energy consumption and increases the proprio-
ceptive signal inflow to cortical areas during gait
(Delli Pizzi et al. 2017; Saggini et al. 2004). Thus,
SPAD improves postural stability through an
increase in exteroceptive and proprioceptive sen-
sitivity. The technique is also useful in therapy of
sarcopenic patients for muscle strengthening and
pain resolution, along with a postural and gait
rehabilitation. This innovative therapeutic
approach combines a motor and sensory feedback
task with creation of feed-forward stimulation in
the microgravity environment (Di Pancrazio et al.
2013).
In elderly patients, clinical relevance of
exercise-based rehabilitation and nutritional
interventions, such as increased ingestion of
essential nutrients, results in the long-term
improvements in strength and muscle mass and
in functional capacity and decreases a risk of
chronic metabolic disorders (Snijders et al.
2009). This integrated, personalized therapeutic
and precautionary approach to health and disease,
with characteristics of adaptability and biological
progressiveness, improves quality of life in adult-
hood. This is a holistic strategy, contained within
the biopsychosocial model, developed by Engels
(1977), which emphasizes, but is not limited to,
biological determinants for maintaining health,
and does not overlook the overall importance of
psychological or socioeconomic factors. All of
them may integrate to act as physical traumas to
the body system and compromise the homeostatic
harmony. The neuro-myo-sensory integration of
the bioprogressive philosophy combines aspects
of biology and function into the realm of rehabil-
itation therapy (Fig. 1). It enables the optimiza-
tion of redundant biological circuits and
arrangements through personalized therapeutic
interventions rather than application of rigidly
fixed therapy schemes (Trieschmann 1987). Fur-
ther research should scrutinize the physiological
underpinnings of the bioprogressive approach to
better understand its function and potential
benefits to be gained in physiotherapy.
Ethical Approval This review article has been written in
compliance with the ethical standards of the institutional
and/or national research committees and with the 1964
Helsinki declaration and its later amendments. The article
does not contain any studies with human participants or
animals performed by any of the authors.
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Adv Exp Med Biol - Clinical and Experimental Biomedicine (2018) 3: 11–17
https://doi.org/10.1007/5584_2018_243
# Springer International Publishing AG, part of Springer Nature 2018
Published online: 21 August 2018

Influence of Proprioceptive Neuromuscular

Facilitation on Lung Function in Patients
After Coronary Artery Bypass Graft Surgery

Małgorzata Bujar-Misztal and Andrzej Chciałowski

Abstract surgery of restrictive character, accompanied

Coronary heart disease is the most common
cardiovascular disease. Primary prevention,
medication, and invasive approach, along
with the coronary artery bypass graft
(CABG), are used in cardiovascular prophy-
laxis. Pulmonary rehabilitation, whose main
task is to restore the patient’s optimum effi-
ciency and prevent complications of the respi-
ratory system, particularly resulting from
limited physical activity and immobilization,
plays an important role in the postsurgical
treatment. The aim of this study was to evalu-
ate the influence on lung function of proprio-
ceptive neuromuscular facilitation
(PNF) vs. standard physiotherapy in patients
undergoing CABG surgery. The study
included 42 patients scheduled for surgery,
divided into PNF (n ¼ 22; aged 62.3  8.4)
and standard rehabilitation (n ¼ 20; aged
62.0  7.4) groups. The findings demonstrate
a decrease in post-surgery lung function in
both groups. The increased ratios of FEV1/
FVC and RV%TLC speak for a transient post-
operative disturbance in lung function after
M. Bujar-Misztal (*)
Department of Cardiac Surgery, Military Institute of
Medicine, Warsaw, Poland
e-mail: mbujar@gmail.com
A. Chciałowski
Department of Infectious Diseases and Allergology,
Military Institute of Medicine, Warsaw, Poland
by air trapping. We found a gradual improve-
ment after 2 months of home rehabilitation,
with a significant trend for a better outcome
using PNF. We conclude that PNF holds a
potential for optimizing the outcome of pul-
monary rehabilitation after coronary bypass
surgery.
Keywords
Cardiac rehabilitation · Coronary artery
bypass · Physiotherapy · Proprioceptive
neuromuscular facilitation · Pulmonary
function · Rehabilitation
1 Introduction
Coronary heart disease is the most common car-
diovascular disease (Nichols et al. 2014). An
important treatment element, aside from pharma-
cotherapy, is to counteract risk factors, adjust
physical activity, balance the conditions of work
and rest, and treat comorbid conditions such as
hypertension, diabetes, or obesity (Cassar et al.
2009). In some cases, invasive treatment is
required such as balloon angioplasty, stenting,
or coronary artery bypass graft (CABG) (Bravata
et al. 2007).
CABG surgery is classically performed
through median sternotomy. The violation of the
chest wall and internal structures contributes to

11
12
pain when breathing, chest mobility impairments,
cough reflex weakness, retention of secretions in
the bronchial tree, and sometimes lower lung
lobes collapse, all of which may manifest in
lung dysfunction and arterial blood gas disorders
(El-Kader 2011; Wynne and Botti 2004;
Westerdahl et al. 2001). Pulmonary rehabilita-
tion, whose task is to restore the patient’s opti-
mum breathing efficiency, prevents
complications mostly arising from physical inac-
tivity or immobilization (Pack et al. 2013; Leon
et al. 2005), particularly after surgery. The
existing methods of physiotherapy take into
account the scope of hospital and posthospital
treatment and the possibility of home-based reha-
bilitation (Perk et al. 2012; Dylewicz et al. 2004).
Proprioceptive neuromuscular facilitation
(PNF), known as Kabat-Kaiser rehabilitation, is
currently used in daily physiotherapy practice. In
the method “P” stands for proprioception, i.e.,
sensory receptor-mediated collection of informa-
tion about the executed motion and spatial body
positon, “N” for activation of neuromuscular
structures, and “F” for facilitation. PNF
represents a holistic approach taking into account
the therapeutic influence on the whole person,
including mental and social spheres, rather than
just disease symptoms. The method is
underpinned by a belief that bodily parts are inti-
mately interconnected and the treatment efforts
ought to be addressed to the whole (Adler et al.
2008). Therefore, the aim of this study was to
determine the influence on lung function of
PNF vs. standard physiotherapy in patients after
CABG surgery.
2 Methods
The study was approved by the Ethics Committee
for Scientific Research of the Academy of Physi-
cal Education in Warsaw and by the Ethics Com-
mittee of the Military Institute of Medicine in
Warsaw, Poland, and the patients gave informed
consent for study procedures. Forty-two patients,
aged 48–85, diagnosed with stable coronary
artery disease, scheduled for elective CABG,
were randomly divided into two physiotherapy
M. Bujar-Misztal and A. Chciałowski
groups: PNF (aged 62.3  8.4; n ¼ 22) and
standard rehabilitation (aged 62.0  7.4;
n ¼ 20). The surgery was performed on a beating
heart, without the use of extracorporeal circula-
tion. The principles of the rehabilitation methods
are presented in Table 1.
During the 2-month period after surgery,
patients in both groups continued cardiac rehabil-
itation according to the rules established during
the hospital stay. Spirometry and body
plethysmography examinations were performed
using BodyBox 5500 (Medisoft, Limassol,
Cyprus), according to the guidelines the
European Respiratory Society (ERS) and Ameri-
can Thoracic Society (ATS) (Curtis et al. 1994;
Efthimiou et al. 1991; Berrizbeitia et al. 1989).
Table 2 shows the schedule of pulmonary func-
tion examinations conducted in the study.
Data of respiratory variables are as the percent-
age means SD of predicted values. The Shapiro-
Wilk test was used to assess normality of data
distribution. A paired t-test or Wilcoxon’s test
was used to assess differences in each variable
between the time points of measurement in either
group patients of post-surgery rehabilitation. A
p-value <0.05 defined the statistically significant
differences. Calculations were carried out with R
2.13.0 software for Windows.
3 Results
3.1 Spirometry
We found no signs of obstruction in any of the
patients subjected to either PNF or standard type
of cardiological rehabilitation after CABG sur-
gery. The FEV1%FVC index was around 80%
and 90% in the PNF and standard rehabilitation
groups, respectively, and changed irrelevantly
from the baseline level after 2-month-long post-
surgery rehabilitation (Table 3). In general, all the
variables were modestly reduced after 2-month-
long rehabilitation with either method; the reduc-
tion was more distinct in the PNF group in terms
of statistical significance, albeit the variables
remained in the normal level with the use of
both rehabilitative methods.
Influence of Proprioceptive Neuromuscular Facilitation on Lung Function in. . . 13

Table 1 Cardiological rehabilitation

Proprioceptive neuromuscular facilitation (PNF) Standard rehabilitation
Respiratory exercises Respiratory muscle training
Using proprioceptive stimulation of respiration Learning abdominal breathing
Learning of conscious and controlled breathing Intensified breath
Learning correct posture with the relaxation of shoulder girdle Teaching effective cough with chest stabilization
Learning effective cough with chest stabilization Light exercise tailored to the patient’s condition
Learning intensified breath
Relaxation
Physiotherapy sessions lasting 45 min; 2–3 times a day; 5 days a week
Anticoagulation was an additional pharmacotherapy in both groups

Table 2 Pulmonary function tests

Timeline of examinations
Pre-
surgery Post-surgery
Examination Pulmonary variables Day 3 Day 7 2 months
Spirometry FVC, FEV1, FEV1%FVC, PEF, FEF25, FEF50, FEF75 X – – X
Plethysmography VC, TGV, RV, TLC, RV%TLC X X X X
X marks the time of examination, FVC forced vital capacity, FEV1 forced expiratory volume in 1 s, FEV1/%FVC
Tiffeneau-Pinelli index, FEF25, FEF50, and FEF75 forced expiratory flow at 25%, 50%, and 75% of FVC, VC vital
capacity, TGV thoracic gas volume, RV residual volume, TLC total lung capacity

Table 3 Changes in spirometry variables from the base- standard method in patients with coronary artery bypass
line level after 2-month-long post-surgery rehabilitation graft (CABG)
using proprioceptive neuromuscular facilitation (PNF) or
PNF method Standard method
Variable Pre-surgery baseline 2 months post-surgery Pre-surgery baseline 2 months post-surgery
FVC 99.11  3.99 95.91  3.97 *** 104.98  3.77 96.98  3.65 **
FEV1 96.73  4.01 89.45  3.80 *** 99.54  4.05 90.73  3.66
FEV1%FVC 79.51  8.25 80.81  8.81 90.55  9.33 89.51  7.45
PEF 100.16  4.96 97.26  5.27 *** 104.96  4.02 98.69  4.84
FEF25 89.67  5.68 71.87  6.71* 71.75  8.00 58.33  7.19
FEF50 89.23  7.53 72.74  5.49 *** 90.34  8.73 76.92  6.22 *
FEF75 66.11  5.67 68.56  5.83* 98.45  6.90 93.59  5.74
Data are presented as % predicted value; *p < 0.05; **p < 0.01; ***p < 0.001

3.2 Plethysmography changed to 130.3  23.4% and 134.2  21.3%

In the pre-surgery baseline examination, the RV%
TLC index, i.e., the proportion of residual capac-
ity to total lung capacity, amounted to
113.2  19.1% and 114.0  26.7% predicted in
the patients subjected to PNF and standard reha-
bilitation, respectively. This index increased
significantly to 133.8  26.5% and
133.8  24.5% on Day 3 post-surgery and
on Day 7 post-surgery in the respective groups.
The tests conducted 2 months post-surgery,
after a period of home-based rehabilitation,
showed a decline the RV%TLC index to
109.3  30.1% and 108.2  19.5% in the PNF
and standard rehabilitation groups, respectively,
compared with Day 3 post-surgery as well as with
the baseline pre-surgery examination, albeit the
latter decline failed to reach statistical signifi-
cance in both groups.
14 M. Bujar-Misztal and A. Chciałowski

Table 4 Plethysmography variables in patients treated with proprioceptive neuromuscular facilitation (PNF) and
standard rehabilitation method at baseline pre-surgery and on the third day post-surgery
PNF method Standard method
Variable Pre-surgery baseline Day 3 post-surgery Pre-surgery baseline Day 3 post-surgery
VC 88.38  20.27 49.35  13.53 100.54  20.44 60.75  15.06 *
TGV 112.36  20.69 96.09  20.29 *** 126.82  31.36 101.72  28.34 **
RV 111.39  23.35 90.91  34.44 120.74  40.91 99.47  39.50 *
TLC 93.89  16.79 63.01  16.12 ** 99.89  20.26 69.21  18.76
RV%TLC 113.21  19.09 133.75  26.51 114.04  26.68 133.80  24.49
Data are presented as % predicted value; *p < 0.05; **p < 0.01; ***p < 0.001

Table 5 Plethysmography variables in patients treated with proprioceptive neuromuscular facilitation (PNF) and
standard rehabilitation method on the third and the seventh day post-surgery
PNF method Standard method
Variable Day 3 post-surgery Day 7 post-surgery Day 3 post-surgery Day 3 post-surgery
VC 49.35  13.53 64.31  20.94 * 60.75  15.06 71.68  16.54 ***
TGV 96.09  20.29 101.00  27.70 *** 101.72  28.34 115.56  17.17 **
RV 90.91  34.44 107.12  34.75 *** 99.47  39.50 120.00  35.33 **
TLC 63.01  16.12 77.94  20.77 *** 69.21  18.76 84.57  17.74 ***
RV%TLC 133.75  26.51 130.32  23.41 133.80  24.49 134.20  21.28 **
Data are presented as % predicted value; *p < 0.05; **p < 0.01; ***p < 0.001

Table 6 Plethysmography variables in patients treated with proprioceptive neuromuscular facilitation (PNF) and
standard rehabilitation method on the seventh day and then 2 months post-surgery
PNF method Standard method
Variable Day 3 post-surgery 2 months post-surgery Day 3 post-surgery 2 months post-surgery
VC 64.31  20.94 88.51  21.66 *** 71.68  16.54 97.15  16.61
TGV 101.00  27.70 113.23  29.07 *** 115.56  17.17 118.47  23.49 ***
RV 107.12  34.75 109.02  39.53 *** 120.00  35.33 110.75  29.06
TLC 77.94  20.77 92.33  20.77 *** 84.57  17.74 97.17  16.59 *
RV%TLC 130.32  23.41 109.30  30.12 *** 134.20  21.28 108.17  19.50
Data are presented as % predicted value; *p < 0.05; ***p < 0.001

The results of body plethysmography are post-surgery in both groups. The examination
presented in detail in Tables 4, 5, 6, and 7. conducted 2 months post-surgery showed a sub-
There were a number of significant differences stantial increase in the variables, with an advan-
in specific variables, beyond those in the RV% tage for the PNF treatment.
TLC index above outlined. However, RV%TLC
is a reflection of lung air trapping after CABG
surgery, and thus it is of key importance for lung
4 Discussion
health.
It is worth noticing that the plethysmography
Rehabilitation is an important part of the treat-
variables were generally lower in patients treated
ment for patients after cardiovascular and thoracic
with PNF than those with standard rehabilitation.
surgeries, as it helps improve physical and respi-
The CABG caused a significant decrease in the
ratory capacity. Breathing exercises lead to a
value of individual variables, particularly on Day
reduction or complete elimination of potential
3 post-surgery, with a gradual rebound on Day 7
pulmonary complications resulting from impaired
Influence of Proprioceptive Neuromuscular Facilitation on Lung Function in. . . 15

Table 7 Plethysmography variables in patients treated with proprioceptive neuromuscular facilitation (PNF) and
standard rehabilitation method at baseline pre-surgery and 2 months post-surgery
PNF method Standard method
Variable Pre-surgery baseline 2 months post-surgery Pre-surgery baseline 2 months post-surgery
VC 88.38  20.27 88.51  21.66 *** 100.54  20.44 97.15  16.61
TGV 112.36  20.69 113.23  29.07 *** 126.82  31.36 118.47  23.49 **
RV 111.39  23.35 109.02  39.53 120.74  40.91 110.75  29.06
TLC 93.89  16.79 92.33  20.77 ** 99.89  20.26 97.17  16.59
RV%TLC 113.21  19.09 109.30  30.12 114.04  26.68 108.17  19.50
Data are presented as % predicted value; **p < 0.01; ***p < 0.001

respiratory mechanics, increasing pain, cough,
phlegm retention, or diaphragm impairment
(Efthimiou et al. 1991).
The present study enabled to relate functional
breathing variables in patients after coronary
artery bypass graft surgery to the pre-surgery
baseline and to compare the effects on lung func-
tion of PNF and standard rehabilitation methods
in the extended post-surgery period of up to
2 months. We, generally, noticed reductions in
spirometry indices 2 months after revasculariza-
tion, pointing to impairment of airflow in large
and small bronchi, irrespective of rehabilitation.
The reduction was somehow less in the
PNF-rehabilitated patients. The FEV1/FVC
index did not change significantly at the post-
surgery time marks studied, which was due likely
to the proportional reduction of both components.
Body plethysmography also revealed a decline in
individual variables on the third and the seventh
day post-surgery. The decline was associated with
increased value of RV%TLC that became
normalized 2 months later but only in patients
undergoing PNF rehabilitation.
The PNF method apparently has never before
been used for rehabilitation after CABG surgery,
so there is no comparative reference to the present
findings. However, the general impairment of
lung function after CABG operations is in line
with the results of other studies. A decline, some-
times reaching 40–50%, in FVC and FEV1 has
been observed in the first 3 days post-surgery,
gradually normalizing after a few months
(Wynne and Botti 2004; Dubach et al. 1995;
Berrizbeitia et al. 1989). Rachwalik et al. (2006)
have observed a slight increase in FEV1/FVC,
which points to the trend toward restrictive
changes in the lungs. In a study of Shenkman
et al. (1997), impaired lung function, expressed
as the 25% reductions in FVC, FEV1, and PEF,
was observed 3–4 months after surgery, while
reductions in FEF50 and FEF75 were sustained
for a longer time. A decline in lung function is
due likely to a modified breathing pattern after
chest opening. Damage to ribs limits the chest
wall motion, and respiratory muscles weakness
usually persist for several months. In addition,
atelectatic changes in the basal segments of
lower lung lobes and pleural damage contribute
to respiratory dysfunction (Montes et al. 2004).
The diaphragm dysfunction caused by a transient
phrenic nerve damage as a result of heart hypo-
thermia also is a serious adverse effect that occurs
in about 22–31% of patients, even as late as up to
1 year after surgery (Westerdahl et al. 2003, 2005;
Curtis et al. 1994; Efthimiou et al. 1991). A
weaker cardiac mechanical function with low
ejection fraction may predispose to the develop-
ment of foci of pulmonary edema and an abnor-
mal ventilation/perfusion ratio, followed by
hypoxia (Staton et al. 2005). Vaidya et al.
(1996) have shown that spirometry variables
decrease after mitral valve surgery for up to
3 months, despite improved hemodynamic heart
function.
Staton et al. (2005) have shown significant
reductions in FEV1 and FVC, with unchanged
FEV1/FVC ratio, compared with the pre-surgery
baseline. However, the adoption of FEV1/FVC as
the exponent of a possible restriction has been due
largely to the lack of TLC measurement.
Rachwalik et al. (2006, 2007), after a triple exam-
ination of patients, on the fifth day and 2.5 and
6 months after cardiopulmonary bypass surgery,
16
both off-pump and on-pump, have shown the
presence of restrictive disorders that persisted
for up to 6 months.
It is generally accepted that a decline in FVC,
with normal or elevated to 85–90% of predicted
FEV1/FVC level, may indicate a restrictive lung
disorder due to lung volume reduction (Pinheiro
et al. 2011; Rachwalik et al. 2007; Aaron et al.
1999). However, the criterion for recognition of
restriction is in fact a reduction in TLC, a variable
that cannot be assessed with spirometry. In the
present study, we measured TLC in a body box,
and we found post-CABG-surgery reductions in
both TLC and VC. These observations indicate
that in the early post-surgery period, there is a
propensity for restrictive lung disorders manifest
by reductions in TLC and VC and increases in
FEV1/FVC above 85% predicted, accompanied
by a transient air trapping as indicated by the
increased RV%TLC index.
A limitation of this study was the conductance
of the last lung examination 2 months after
CABG, despite the literature pointing to the
potential persistence of lung function decline up
to 6 months after surgery. A 2-month-long
follow-up was chosen since most patients came
from remote areas of the country, and the symp-
tomless post-surgery period, accompanied by evi-
dent improvement in cognitive functions,
particularly after PNF rehabilitation, was the
most common cause of refusal to submit to pul-
monary testing extended over a longer time
period.
In conclusion, coronary artery bypass graft,
with median sternotomy, triggers short-term,
restrictive disturbances and air trapping in the
early post-surgery period. PNF rehabilitation
improves lung function and respiratory capacity
after surgery. The PNF method has an edge over
the standard rehabilitation in improving lung
function.
Conflicts of Interest The authors declare no conflicts of
interest in relation to this article.
M. Bujar-Misztal and A. Chciałowski
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Adv Exp Med Biol - Clinical and Experimental Biomedicine (2018) 3: 19–25
https://doi.org/10.1007/5584_2018_282
# Springer Nature Switzerland AG 2018
Published online: 29 September 2018

Remote Ischemic Preconditioning

in Renal Protection During Elective
Percutaneous Coronary Intervention

Małgorzata Wojciechowska, Maciej Zarębiński,

Piotr Pawluczuk, Dagmara Gralak-Łachowska,
Leszek Pawłowski, Wioletta Loska, Marzena Goszczyńska,
Krzysztof Flis, and Agnieszka Cudnoch-Jędrzejewska

Abstract criteria of the serum creatinine (SC) and

Remote ischemic preconditioning (RIPC)
exerts protection in remote organs. The pur-
pose of this study was to investigate the poten-
tial of RIPC to prevent contrast induced
nephropathy. One hundred and twenty four
patients were randomized to elective percuta-
neous coronary intervention with or without
RIPC. RIPC was performed using three cycles
of 5-min inflation to 200 mmHg of a standard
upper arm blood pressure cuff. The time
between the last inflation cycle and the coro-
nary intervention was less than 2 h. The pri-
mary endpoint was the incidence of contrast-
induced nephropathy based on the standard
M. Wojciechowska (*)
Department of Experimental and Clinical Physiology,
Center for Preclinical Research, Warsaw Medical
University, Warsaw, Poland
Invasive Cardiology Unit, Western Hospital, Grodzisk
Mazowiecki, Poland
e-mail: gosiawojciech@yahoo.com
M. Zarębiński, P. Pawluczuk, D. Gralak-Łachowska, L.
Pawłowski, W. Loska, M. Goszczyńska, and K. Flis
Invasive Cardiology Unit, Western Hospital, Grodzisk
Mazowiecki, Poland
A. Cudnoch-Jędrzejewska
Department of Experimental and Clinical Physiology,
Center for Preclinical Research, Warsaw Medical
University, Warsaw, Poland
cystatin C (CC) levels. The rates of major
cardiac and cerebral adverse events (MACCE)
during 1 year follow-up were evaluated. We
found that contrast-induced nephropathy
assessed by SC occurred in 4.9% (3/61) patients
with RIPC and in 12.1% (7/58) patients without
it (p ¼ 0.20). Nephropathy assessed by CC
occurred in 1.7% (1/58) patients with RIPC
and 3.5% (2/57) patients without it (p ¼ 0.62).
There was no coincidence between the diagno-
sis of contrast-induced nephropathy based on
SC and CC (McNemar test 0.012, κ ¼ 0.28);
SC was a more sensitive marker of nephropathy
than CC (ten and three cases, respectively). The
MACCE rate during the year of follow-up
tended to be lower with the ischemic
preconditioning than without it, four vs. six
cases, respectively. We conclude that RIPC
prior to percutaneous coronary intervention
has no major influence on the development of
contrast-induced nephropathy and does not
improve the one-year outcome.
Keywords
Coronary intervention · Kidney insufficiency ·
Nephropathy · Percutaneous coronary
intervention · Remote ischemic
preconditioning · Renal protection

19
20
1 Introduction
Contrast-induced nephropathy is defined as an
increase in the serum creatinine
(SC) concentration of >0.5 mg/dL or 25% above
the baseline level within 48–72 h after parenteral
administration of a radiocontrast dye. The inci-
dence of nephropathy, despite the use of low–
osmolar contrast media and appropriate hydra-
tion, remains significant (Bartorelli and Marenzi
2008; Wong and Irwin 2007; McCullough et al.
2006; Rihal et al. 2002). The Mehran risk score is
a simple and well-tested scale that has been devel-
oped to provide the risk of contrast-induced
nephropathy after percutaneous coronary inter-
vention (PCI) (Raingruber et al. 2011; Mehran
et al. 2004).
Serum cystatin C (CC) is an early predictive
biomarker for acute kidney injury. Various stud-
ies demonstrate the superiority of CC compared
to SC, especially to detect a minor reduction in
glomerular filtration rate (GFR). In addition, SC
concentration typically goes up 48–72 h after the
contrast use, whereas CC shows to highest value
after 12–24 h (Herget-Rosenthal et al. 2004;
Dharnidharka et al. 2002; Mussap et al. 2002).
Remote ischemic preconditioning (RIPC) is a
method consisting of multiple brief episodes of
ischemia applied to one organ, which carries pro-
tection against a subsequent prolonged ischemia
applied to remote organs (Zimmerman et al.
2011; Hausenloy and Yellon 2008). Reversed
distribution of blood in the kidneys and ischemia
of the outer kidney medulla are two of the
pathomechanisms for developing contrast-
induced nephropathy (Sendeski 2011;
Goldenberg and Matetzky 2005; Heyman et al.
1994). Suitability of RIPC to prevent kidney
injury after contrast administration has been
evaluated in a few studies. The results are incon-
clusive (Gholoobi et al. 2015; Menting et al.
2015; Yamanaka et al. 2015; Igarashi et al.
2013; Er et al. 2012). Therefore, the purpose of
this trial was to investigate the potential of RIPC
for renal protection during elective PCI.
M. Wojciechowska et al.
2 Methods
2.1 Study Population
This is a prospective, single-center trial that was
conducted from October 2012 to September 2013
in the Invasive Cardiology Unit of Western
Hospital in Grodzisk Mazowiecki, Poland. A
hundred and twenty-four patients aged 18 years
or older, with stable coronary angina and no his-
tory of end-stage renal disease, admitted to the
hospital for elective PCI, were enrolled into the
study. Sixty-two patients were allocated to
receive standard therapy (control group) and
another 62 to receive standard therapy plus
RIPC (RIPC group). One patient from the control
group was excluded due to the acute stent throm-
bosis and the need of urgent angioplasty 2 h after
primary PCI.
2.2 Study Protocol
Patients were numbered depending on the order
of admission to the hospital. They were then
assigned to receive elective PCI (control group)
or PCI with RIPC (RIPC group). All the patients
were given 500 ml of saline and 600 mg of
N-acetylcysteine intravenously twice: before and
after PCI. They were also asked to drink 1.5 L of
still water during the day of the procedure. Con-
comitant drugs, such as diuretics, angiotensin-
converting enzyme inhibitors (ACEI), angioten-
sin II receptor antagonists (AT1 blockers), and
metformin, were permitted.
2.3 Procedures
RIPC was performed by three cycles of 5-min
inflation to 200 mmHg and 5-min deflation of a
standard upper arm blood pressure cuff. The time
between the last inflation cycle and PCI was <2 h.
PCI was performed according to the standard
clinical practice. In all patients, a nonionic
Remote Ischemic Preconditioning in Renal Protection During Elective. . .
iodinated, water-soluble, radiographic contrast
medium (iopromide; iodine concentration
370 mg/ml, osmolality 0.77 Osm/kg H2O at
37  C) was used. Patients from the control
group had blood pressure measured only 2 h
before PCI. The clinicians performing PCI were
not involved in the recruitment of patients into the
study, numbering the patients, or in the
preconditioning procedure.
2.4 Outcomes
The primary outcome was the incidence of
contrast-induced nephropathy defined as SC ele-
vation 25% or  0.5 mg/dL over the baseline
level in a period of 24–72 h after contrast admin-
istration. Additionally, the incidence of contrast-
induced nephropathy, defined as serum CC eleva-
tion 25% 24 h after contrast administration, was
evaluated, and the coincidence between CC and
SC was assessed. Standard assays were used to
measure the serum concentration of creatinine
and cystatin C. The composite endpoint of
major adverse cardiac and cerebrovascular events
(MACCE) consisted of acute coronary syndrome,
transient ischemic attack, or stroke during a
1-year follow-up.
2.5 Statistical Analysis
Continuous variables are presented as arithmetic
means SD for normally distributed variables or
as medians and interquartile ranges in case of a
skewed distribution. Differences between groups
for continuous data were tested by Student’s t-test
or Mann-Whitney rank test, as appropriate. Cate-
gorical variables are reported as counts and
percentages. Differences between categorical
data were assessed by Chi-squared test or Fisher’s
exact test. A two-sided p-value <0.05 was consid-
ered statistically significant. All analyses were
performed with SAS software v9.2 (SAS Insti-
tute; Cary, NC).
21
3 Results
Demographic, clinical, basic biochemical, and
angiographic data of the patients are shown in
Table 1.
3.1 Contrast-Induced Nephropathy
Assessed by Serum Creatinine
(SC)
The assessment of contrast-induced nephropathy
was possible in 119 patients (61 RIPC, 58 control)
as 1 patient from RIPC and 3 patients from the
control group had no complete biochemical data.
The mean increase in SC after 24 and 72 h did not
differ between the two groups (24 h: RIPC
0.01  0.10 vs. control 0.04  0.10; p ¼ 0.087;
72 h: 0.03  0.09 vs. 0.04  0.08; p ¼ 0.623).
Nephropathy measured by SC occurred in 4.9%
patients (3/61) in RIPC group and in 12.1%
patients (7/58) in the control group, p ¼ 0.20
(Table 2).
3.2 Contrast-Induced Nephropathy
Assessed by Serum Cystatin C
(CC)
The assessment of contrast-induced nephropathy
was possible in 115 patients (58 RIPC and 57 con-
trol) as 4 patients from RIPC and 4 patients from
control group had no data on CC after 24 h. The
mean increase in CC after 24 h did not differ
between the RIPC and control groups
(0.01  0.13 vs. 0.01  0.11; p ¼ 0.827).
Nephropathy measured by CC occurred in 1.7%
patients in RIPC and 3.5% patients in the control
group; p ¼ 0.62 (Table 2).
3.3 Coincidence Between Serum
Cystatin C (CC) and Serum
Creatinine (SC)
Data on CC were available in all patients with
contrast-induced nephropathy identified on the
22 M. Wojciechowska et al.

Table 1 Patients’ demographic, clinical, basic biochemical, and angiographic indices

RICP (n ¼ 62) Control (n ¼ 61) p
Dermographics
Age, yr 64.4  10.5 62.9  8.6 0.398
BMI, kg/m2 29.6  4.6 29.3  4.4 0.709
Female, n (%) 20 (32.3%) 19 (31.2%) 0.895
Biochemical indices
Serum creatinine, mg/dL 0.86  0.27 0.86  0.26 0.949
GFR, mL/min 105.6  36.3 107.3  36.5 0.801
Urea, mg/dL 36 (28–45) 37 (30–44) 0.422
Urea-to-creatinine ratio 43.5  9.8 46.3  11.8 0.159
Serum cystatin C, mg/L 0.88  0.23 0.88  0.24 0.855
Troponin Ths, ng/mL 0.01 (0.01–0.02) 0.01 (0.01–0.02) 0.931
Hemoglobin, g/dL 14.2  1.2 13.8  1.3 0.066
Hematocrit, % 41.2  3.0 40.1  3.3 0.052
Clinical data (examination, medical history, and medications)
SBP, mmHg 137.2  19.4 136.4  21.9 0.827
DBP, mmHg 82.3  13.8 80.6  12.6 0.472
LVEF, % 52.5  7.6 53.2  8.6 0.605
CKD, n (%) 3 (4.8%) 2 (3.3%) 1.000
Diabetes, n (%) 13 (21.0%) 16 (26.2%) 0.492
Arterial hypertension, n (%) 45 (72.6%) 47 (77.1%) 0.568
MVD, n (%) 33 (53.2%) 33 (54.1%) 0.923
ACEI/AT1 blockers, n (%) 54 (87.1%) 58 (95.1%) 0.121
Statins, n (%) 56 (90.3%) 55 (90.2%) 0.976
Diuretics, n (%) 29 (46.8%) 31 (50.8%) 0.654
Percutaneous coronary intervention
Contrast volume, mL 155.5  76.7 143.7  55.7 0.331
Duration of PCI, min 45 (30–60) 43 (30–60) 0.978
Mehran risk score 3.2  2.6 2.9  2.3 0.555
Mehran risk score > 2.0 30 (48.4%) 30 (49.2%) 1.000
RIPC remote ischemic preconditioning group, BMI body mass index, GFR glomerular filtration rate, Troponin Ths high-
sensitivity troponin T, SBP systolic blood pressure, DBP diastolic blood pressure, LVEF left ventricular ejection fraction,
CKD chronic kidney disease, MVD multivessel coronary disease, ACEI angiotensin-converting enzyme inhibitors, AT1-
blockers angiotensin II receptor antagonists, TIA transient ischemic attack, PCI percutaneous coronary intervention

Table 2 Remote ischemic preconditioning (RICP) and incidence of contrast-induced nephropathy (CIN)
RICP (n ¼ 62) Control (n ¼ 61) p
Serum creatinine 24-B, mg/dL 0.01  0.09 0.04  0.10 0.087
Serum creatinine 72-B, mg/dL 0.03  0.09 0.04  0.08 0.623
Serum cystatin C 24-B, mg/dL 0.00  0.13 0.01  0.11 0.827
CIN (SC) 3/61 (4.9%) 7/58 (12.1%) 0.197
CIN (CC) 1/58 (1.7%) 2/57 (3.5%) 0.618
CIN (CC) contrast-induced nephropathy assessed by serum cystatin C criteria, CIN (SC) contrast-induced nephropathy
assessed by serum creatinine criteria, Serum creatinine 24-B difference between serum creatinine concentration
measured after 24 h and the baseline (B) level, Serum creatinine 72-B difference between serum creatinine concentration
measured after 72 h and the baseline level, Serum cystatin C 24-B difference between serum cystatin C concentration
measured after 24 h and the baseline level
Remote Ischemic Preconditioning in Renal Protection During Elective. . .
basis of SC. There was no coincidence between
the diagnosis of contrast-induced nephropathy
based on SC and CC (McNemar test: 0.02,
κ ¼ 0.28). Serum creatinine measured twice
(24 and 72 h after PCI) appeared to more accu-
rately identify all patients with contrast-induced
nephropathy than CC measured once (from
10 cases of contrast-induced nephropathy based
on SC, only 3 met criteria to recognize contrast-
induced nephropathy based on CC).
3.4 One-Year Follow-Up
One-year follow-up was possible in case of
95 patients: 9 (90%) with contrast-induced
nephropathy (CIN+) and 86 (79%) without it
(CIN–). The RIPC had no appreciable influence
on the occurrence of major adverse cardiac and
cerebrovascular events during the 1-year follow-
up, nor did it significantly change the MACCE
index (Table 3).
4 Discussion
In this study, general risk for developing contrast-
induced nephropathy was 8.4% (10/119 patients),
and it was compatible with the predicted proba-
bility based on the Mehran risk score (Mehran
et al. 2004). The study demonstrates that RIPC
applied prior to elective PCI failed to provide an
added benefit to hydration and N-acetylcysteine
in decreasing the incidence of contrast-induced
nephropathy. There was a trend toward lower
numerical incidence of contrast-induced nephrop-
athy, assessed on the basis of both SC and CC,
and also lower increase in SC and CC levels after
PCI in the RIPC group; the differences were
however insignificant. As the risk for developing
contrast-induced nephropathy was generally low
in our patients (Mehran risk score about 3 points;
Table 1), these results are in line with similar
studies in the literature, where RIPC failed to
improve kidney function after contrast adminis-
tration in patients with low-to-moderate risk of
contrast-induced nephropathy (Gholoobi et al.
2015; Menting et al. 2015). However, RIPC has
23
been demonstrated to prevented contrast-induced
nephropathy in patients with ST-elevation
myocardial infarction (Yamanaka et al. 2015) or
in coexisting chronic kidney disease (Igarashi
et al. 2013; Er et al. 2012), with a high Mehran
risk score (Mehran et al. 2004). After a thorough
analysis of all the studies, along with the present
results, we conclude that RIPC may be of less
importance in case of elective PCI and low-risk
patients, but it may be particularly useful in unsta-
ble patients with a high to very high risk of
contrast-induced nephropathy.
The mechanisms underlying contrast-induced
nephropathy are multifactorial, and it is possible
that they vary depending on comorbidities and
indications for PCI (elective vs. urgent). In
lower-risk patients, the plausible mechanism of
contrast-induced nephropathy may be a direct
toxicity of contrast on tubular cells, caused or
aggravated by patient’s dehydration (Sendeski
2011). In case of higher-risk patients, the
pathomechanism may be more complicated as
hypoxic injury may be a predominant factor.
RIPC may prevent hypoxic injury and thus may
have a renoprotective effect in higher-risk
patients, but not in the general population.
Urea-to-creatinine (U-to-SC) ratio is used to
determine the patient hydration. An abnormal
value that could indicate hypovolemia and a
reduced renal perfusion is more than 40 (Higgins
2016). In the study, baseline mean U-to-SC ratio
in the patients of both groups was 45.1  1.3, and
it was over 40 in 63.4% of patients. That indicates
that more than 60% of patients could be
hypovolemic on admission. We demonstrate a
positive association between higher baseline U-
to-SC ratio and contrast-induced nephropathy
(55.1  14.8 in CIN+ vs. 43.9  10.0 in CIN–;
p < 0.002), which means that more adequate fluid
therapy should be implemented in selected
patients for preventing nephropathy. In this
study, patients did not interrupt treatment with
potentially nephrotoxic drugs, such as ACEI and
AT1 blockers, so we indirectly demonstrate that
such drugs did not increase the risk of
nephropathy.
CC is considered an early marker of kidney
injury. SC typically goes up between 48–72 h
24
Table 3 Effects on the occurrence of major adverse cardiac and cerebrovascular events of remote ischemic
preconditioning (RIPC) during 1-year follow-up
RICP (n ¼ 62)
n ¼ 47 (75.8%)
TIA/stroke n (%) 1 (2.1%)
ACS (UA/MI) n (%) 4 (8.5%)
MACCE n (%) 4 (8.5%)
TIA Transient ischemic attack, ACS (UA/MI) acute coronary syndrome (unstable angina/myocardial infarct), MACCE
composite endpoint defined as acute coronary syndrome, transient ischemic attack, or stroke
after the use of contrast, whereas CC reaches the
highest value 12–24 h after PCI (Herget-
Rosenthal et al. 2004; Dharnidharka et al. 2002;
Mussap et al. 2002). As patients are usually
discharged from the hospital 24 h after PCI, CC
might be a better option to identify contrast-
induced nephropathy. The present results indi-
cate, however, that SC measured twice, 24 and
72 h after PCI, may be more accurate to detect
nephropathy as we found ten CIN+ cases based
on SC and three on CC.
In previous studies, RIPC performed before
PCI has been shown to improve clinical outcomes
during the following 4 (Yamanaka et al. 2015) or
6 weeks (Er et al. 2012). In this study, RIPC,
performed before elective PCI, failed to influence
the incidence of contrast-induced nephropathy or
the MACCE index during in 1-year follow-up.
Nonetheless, we cannot rule out the appearance
of early post-PCI advantages of RIPC, which
would wane with the time of follow-up. The
main limitation of this study was a small number
of patients investigated. It cannot be ruled out that
in a larger population of patients the differences
between RIPC-treated and control groups would
become significant.
5 Conclusions
We believe we have demonstrated that in patients
referred for elective percutaneous coronary inter-
vention, remote ischemic preconditioning does
not provide statistically significant benefits in
decreasing the incidence of contrast-induced
nephropathy. Cystatin C is considered an early
marker of kidney injury, but measuring it 24 h
M. Wojciechowska et al.
Control (n ¼ 61)
n ¼ 48 (78.7%) p
1 (2.1%) 1.000
5 (10.4%) 1.000
6 (12.5%) 0.740
after percutaneous coronary intervention does not
detect all cases of contrast-induced nephropathy.
Remote ischemic preconditioning before percuta-
neous coronary intervention does not influence
1-year follow-up outcome.
Conflicts of Interest The author declares no conflicts of
interest in relation to this article.
Ethical Approval All procedures in this study were
performed in accordance with the ethical standards of the
institutional research committee and with the 1964
Helsinki Declaration for Human Research and its later
amendments.
Informed Consent Written informed consent was
obtained from all individual participants included in the
study.
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preconditioning: underlying mechanisms and clinical
application. Cardiovasc Res 79:377–386
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Pietruck F, Janssen O, Philipp T, Kribben A (2004)
Early detection of acute renal failure by serum cystatin
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urea: creatinine ratio. www.acutecaretesting.org.
Accessed 24 Aug 2018
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ischemic pre-conditioning alleviates contrast-induced
acute kidney injury in patients with moderate chronic
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cutaneous coronary intervention: development and ini-
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Wever KE, Lemson MS, van der Vliet JA, Wetzels
JF, SchultzeKool LJ, Warlé MC (2015) Ischemic
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Varagnolo M, Nosadini R, Plebani M (2002) Cystatin
C is a more sensitive marker than creatinine for the
estimation of GFR in type 2 diabetic patients. Kidney
Int 61:1453–1461
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M (2011) Using the Mehran risk scoring tool to predict
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PJ, Singh M, Bell MR, Barsness GW, Mathew V,
Garratt KN, Holmes DR Jr (2002) Incidence and prog-
nostic importance of acute renal failure after percuta-
neous coronary intervention. Circulation
105:2259–2264
Sendeski MM (2011) Pathophysiology of renal tissue
damage by iodinated contrast media. Clin Exp
Pharmacol Physiol 38:292–299
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thy. Br J Anaesth 99:474–483
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Tsukuda S, Kazatani Y, Nakamura K, Ito H (2015)
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Adv Exp Med Biol - Clinical and Experimental Biomedicine (2018) 3: 27–36
https://doi.org/10.1007/5584_2018_238
# Springer International Publishing AG, part of Springer Nature 2018
Published online: 29 June 2018

Prognostic Impact of Extracapsular Lymph

Node Invasion on Survival in Non-small-Cell
Lung Cancer: A Systematic Review
and Meta-analysis

Seyed Vahid Tabatabaei, Christoph Nitche, Maximilian Michel,

Kurt Rasche, and Khosro Hekmat

Abstract with publication date since 1990. Prospective

The extracapsular tumor extension (ECE) of
nodal metastasis is an important prognostic
factor in different types of malignancies. How-
ever, there is a lack of recent data in patients
with non-small-cell lung cancer (NSCLC). In
addition, the TNM staging system does not
include ECE status as a prognostic factor.
This systematic review and meta-analysis has
been conducted to summarize and pool
existing data to determine the prognostic role
of ECE in patients with lymph node-positive
NSCLC. Two authors performed an indepen-
dent search in PubMed using a predefined
keyword list, without language restrictions
S. V. Tabatabaei and K. Rasche
Department of Internal Medicine, Division of Pulmonary,
Allergy, and Sleep Medicine, HELIOS Clinic Wuppertal,
Witten/Herdecke University, Wuppertal, Germany
C. Nitche
Department of Internal Medicine, Marien Hospital
Euskirchen, Teaching Hospital of Uniklinik Bonn,
Euskirchen, Germany
M. Michel
Institute of Zoology, University of Cologne, Cologne,
Germany
K. Hekmat (*)
Department of Cardiothoracic Surgery, University Clinic
of Cologne, Cologne, Germany
e-mail: khosro.hekmat@uk-koeln.de
or retrospective studies reporting data on prog-
nostic parameters in subjects with NSCLC
with positive ECE or with only intracapsular
lymph node metastasis were retrieved. Data
were summarized using risk ratios (RR) for
the survival with 95% confidence intervals
(CI). The data was analyzed using Mix 2 (ref:
Bax L: MIX 2.0 – Professional software for
meta-analysis in Excel. Version 2.015.
BiostatXL, 2016. https://www.meta-analysis-
made-easy.com). There 2,105 studies were
reviewed. Five studies covering a total of
828 subjects met the inclusion criteria and
were included in the meta-analysis. Two hun-
dred and ninety-eight (35.9%) patients were
categorized as ECE+, of whom 54 (18.1%)
survived at the end of follow-up. In the
ECE-negative group, 257 patients (48.4%)
survived by the end of follow-up. Thus, ECE
status is associated with a significantly
decreased survival rate: pooled RR 0.45
(95% CI 0.35–0.59), Q(4) ¼ 4.06, Pvalue ¼ 0.39,
and I2 ¼ 68.00% (95 CI 0.00–79.55%). In
conclusion, ECE has a significant impact on
survival in NSCLC patients and should be con-
sidered in diagnostic and therapeutic decisions
in addition to the current TNM staging. Post-
operative radiotherapy may be an option in
ECE-positive pN1 NSCLC patients.

27
28
Keywords
Extracapsular lymph node invasion · Meta-
analysis · Metastases · Non-small-cell lung
cancer · Prognostic factor · Survival rate
1 Introduction
Lung cancer is the most common cancer due to
the increasing number of cigarette smokers and
air pollution exposure (Eckel et al. 2016). Cur-
rently, it is the leading cause of malignancy-
related death in male patients and runs in second
among female cancer patients worldwide (Torre
et al. 2016). Approximately 75–85% of all lung
cancer patients are diagnosed with non-small-cell
lung cancer (NSCLC) (Le Péchoux 2011).
According to the current guidelines, curative
resection by tumor surgery is the first-choice
treatment for stage IA-IIIA NSCLC (Ettinger
et al. 2014; Van Schil et al. 2013). Postoperative
adjuvant chemotherapy and thoracic radiotherapy
have been shown to improve the outcome in
resected stage II and IIIA NSCLC and minimize
the risk of locoregional recurrence (Hui et al.
2015; Gomez and Komaki 2012; Bradley et al.
2005). Bronchovascular reconstructive
procedures are evolving in order to avoid pneu-
monectomy in functionally unsuitable patients
(Maurizi et al. 2016).
Several clinical factors such as tumor differen-
tiation (Kuo et al. 2014), lymphatic (Al-Alao et al.
2014) or blood vessel invasion (Naito et al. 2010),
serum carcinoembryonic antigen elevation, and
tumor size (Igai et al. 2011) are considered to
have a significant influence on survival and
local recurrence rate after a curative tumor
resection. In contrast, tumor histology for
adenocarcinoma vs. squamous cell carcinoma
does not seem to play an important role in prog-
nosis (Kerr and Nicolson 2013). Intriguingly, the
impact of lymph node staging on patient progno-
sis is highly complex. As such, staging of the
mediastinal lymph nodes has been reported to
result in a very heterogeneous picture (Goldstraw
et al. 2007). In NSCLC stage IV, patients with
lymph nodes in the lung (status of N1) have a
S. V. Tabatabaei et al.
better prognosis than patients with N2 status
(hilar lymph nodes on the same side of the
tumor) (Dai et al. 2016). However, even within
the same lymph node stage, prognosis for patients
differs according to the number and the ratio of
the positive mediastinal lymph nodes
(Jonnalagadda et al. 2011; Wisnivesky et al.
2011), single-station or multiple-station involve-
ment (Misthos et al. 2008), and the presence of
skip metastasis or micrometastasis (Zhang et al.
2016; Kerr and Nicolson 2013; Wang et al. 2001).
Importantly, extracapsular lymph node extension
(ECE) has been shown to have a prognostic sig-
nificance in various types of cancer such as
breast (Fujii et al. 2016), head and neck (Kelder
et al. 2012), colorectum (Veronese et al. 2016),
oral cavity (Taghavi and Yazdi 2015), gastric
(Alakus et al. 2010), cervix cancer (Metindir and
Bilir Dilek 2008), melanoma (Khosrotehrani et al.
2014), and vulva cancers (Luchini et al. 2016).
The present study describes the results of a meta-
analysis we conducted to evaluate the prognostic
significance of ECE in NSCLC. The data suggest
that ECE status should be taken into consider-
ation as a prognostic factor in NSCLC.
2 Methods
2.1 Inclusion and Exclusion Criteria
This systematic review adheres to the MOOSE
guidelines (Stroup et al. 2000) and PRISMA
statement (Liberati et al. 2009), using predefined
protocols. The following criteria were employed
for the studies to be eligible for inclusion: (1) pro-
spective or retrospective; (2) use of a curative
surgical approach such as lobectomy or pneumo-
nectomy; (3) presence of a statistical comparison
of survival between ECE+ and ECE– patients;
(4) ECE+ was defined as cancer cells invading
beyond the capsule of a lymph node; (5) non-
small-cell lung cancer needed to be histologically
diagnosed; (6) published in a peer-reviewed jour-
nal; (7) publication year between 1990 and 2017
in any language; (8) patients with nodal status
N1-N2; and (9) NSCLC staging IIa–IIIa. Exclu-
sion criteria were the following: (1) copresence of
Prognostic Impact of Extracapsular Lymph Node Invasion on Survival in. . .
other malignancies; (2) lack of data about prog-
nostic parameters in the title/abstract; (3) insuffi-
cient data to enable a statistical comparison of
survival between ECE+ and ECE– patients;
(4) NSCLC stage IV; (5) palliative therapy of a
subgroup of patients without sufficient data to
exclude them in a secondary analysis;
(6) in vitro or animal studies; (7) neo-adjuvant
chemoradiotherapy; and (8) nodal status N3.
2.2 Data Sources and Literature
Search Strategies
Two investigators independently conducted a
PubMed search in June 2017 with a time limit of
publication date since 1990 using the following
combination of mesh terms: (1) ((“Carcinoma,
Bronchogenic”[Mesh]) AND “Neoplasm
Metastasis”[Mesh]) AND “Lymph Nodes”[Mesh]));
(2) ((“Prognosis”[Mesh]) AND “Carcinoma,
Bronchogenic”[Mesh]) AND “Lymphatic
Metastasis”[Mesh])). References of previously
published related review articles were also consid-
ered. The reviewers screened the titles and abstracts
of all retrieved articles. If extracapsular or
intracapsular tumor extension was mentioned, the
full text was included into the review. The final list
of chosen studies was reached through a consensus.
One investigator extracted the data from the
included articles. Data were validated by another
investigator and inserted into an Excel database.
For each study, the authors’ information, year and
country of publication, number and gender of
patients, age, tumor stage, smoking, vascular
invasion, and duration of follow-up were
extracted.
2.3 Outcomes
The primary outcome was survival after treatment
at the end of the follow-up period. We applied the
Newcastle–Ottawa Scale to evaluate the method-
ological quality of non-randomized trials. The
following three areas of the studies were
evaluated: selection of participants, comparability
of participants, and comparability of outcomes.
29
Scores  5 out of the 9 possible were judged to
indicate a high risk of bias.
The pooled risk ratios (RR) and 95% confi-
dence intervals (CI) of all-cause mortality
between ECE+ and ECE patients were calcu-
lated using DerSimonian–Laird random-effect
models (DerSimonian and Laird 1986). Hetero-
geneity across studies was assessed by the I2
metric and χ2 statistics (Higgins and Thompson
2002).
3 Results
We identified 2105 non-duplicated articles, out of
which 2096, after reviewing the title and abstract,
were judged as inadequate for further elaboration
to meet the goal of this meta-analysis. The
remaining nine full-text articles were retrieved
for more detailed analysis. Out of the nine
articles, four were further excluded for the follow-
ing reasons: one was an editorial (Riquet 1996),
two lacked a comparison of outcomes between
ECE+ and ECE patients (Riquet et al. 1995;
Bollen et al. 1994), and one provided only a
p-value (Borghetti et al. 2016). As a result, five
articles were considered for this meta-analysis
(Liu et al. 2015; Moretti et al. 2009; Lee et al.
2007; Vansteenkiste et al. 1997; Duran-Cantolla
et al. 1994).
3.1 Study and Patient Characteristics
Data from 828 patients were analyzed. The
patients’ clinical and demographic characteristics
are provided in Table 1. Two studies were
conducted in Europe, two in Asia, and one in
the USA. While four of the articles contained
the patients’ characteristics, Duran–Cantolla
et al.’s (1994) did not. There was a greater pro-
portion of men than women patients reported,
with a mean or median age in a range
59–62 years. Two articles investigated a head-
to-head survival of ECE+ and ECE patients
(Liu et al. 2015; Lee et al. 2007). In the remaining
articles, data were extracted from a subgroup
analysis (Moretti et al. 2009; Vansteenkiste et al.
Table 1 Patients’ characteristics

Poor Vascular
Mean/Median differentiation invasion Squamous cell carcinoma Pneumonectomy R0–Resection
age (year) Male (%) T4 (%) N2 (%) (%) (%) (%) Smoking (%) Stage IIIA (%) (%) (%)

Study ECE ECE ECE ECE ECE ECE ECE ECE ECE
Author country NOS + ECE + ECE + ECE + ECE + ECE ECE+ ECE + ECE + ECE + ECE ECE+ ECE + ECE

Lee et al. Taiwan 8 62 (32–-82) 58 42 NR NR NR 67 33 86 14 51 49 57 43 75 25 NR 100

(2007)

Liu et al. China 8 59 (27–79) 21 79 0 24 76 22 78 NR 15 85 21 79 0 28 72 100

(2015)

Duran Spain 7 NR NR NR 33 NR NR NR NR 37 NR NR
Cantolla et al.
(1994)

Vansteenkiste Belgium 7 61 (40–76) 93 9 14 35 21 61 NR NR 80 87

et al. (1997)

Moretti et al. USA NR 61 (35–85) 58 NR 100 NR NR 38 NR 100 NR 85

(2009)

NOS Newcastle–Ottawa Scale score, ECE+ with extracapsular tumor extension, ECE without extracapsular tumor extension, T4 locally advanced lung cancer, N2 cancer is in lymph nodes,
Stage IIIA cancer spread to lymph nodes on the same side of the chest as the tumor, R0-Resection a microscopically margin-negative resection, NR not reported
Prognostic Impact of Extracapsular Lymph Node Invasion on Survival in. . .
1997; Duran-Cantolla et al. 1994). Only Liu
et al. (2015) and Moretti et al. (2009) report a
tumor-free survival as the main outcome. In the
former article, there was neither T4 nor stage IIIa
reported, although these stages were not set as the
exclusion criteria. The median score on the
Newcastle–Ottawa Scale was 7 (range 7–8
points), with no single score pointing to the high
risk of quality bias, i.e.,  5 points.
3.2 Risk Ratio for Overall Survival
Pooling data from the five studies reporting
survival, it turned out that only 18.1% of the
ECE+ patients survived, while the survival rate
amounted to 48.4% in the ECE– patients. This
difference in the survival rate was highly signifi-
cant, showing that ECE-positive status leads to a
distinct decrease in patient survival. The pooled
RR was 0.45 (95% CI 0.35–0.59); Q(4) ¼ 4.06,
Pvalue ¼ 0.39, and I2 ¼ 68.00% (95 CI 0.00–79.55%)
(Fig. 1). The contour-enhanced funnel plot
(Peters et al. 2008) of the studies included in the
meta-analysis indicated no publication bias, as
confirmed by the Begg–Mazumdar test
(Kendall’s τ ¼ 0.00, Pvalue ¼ 1.0) (Fig. 2).
According to classic fail-safe N, 33 nonsignificant
studies are needed to make the pooled Pvalue
nonsignificant.
Fig. 1 Summary of survival risk ratios (RR) in original studies and the pooled estimate; RR risk ratio
31
4 Discussion
The extracapsular tumor extension (ECE) of
nodal metastasis has been known as a prognostic
factor for multiple cancer malignancies, such as
oral cavity, colon, gastric, cervix cancer, and
melanoma. In each case, ECE-positive status is
associated with a higher rate of all-cause mortal-
ity (Chen et al. 2016; Taghavi and Yazdi 2015;
Khosrotehrani et al. 2014; Alakus et al. 2010;
Metzger et al. 2009; Metindir and Bilir Dilek
2008; Wind et al. 2007). The ECE has also been
included in the last versions of staging for squa-
mous cell carcinoma of the vulva (Luchini et al.
2016). Our results emphasize the importance of
ECE status in the prognosis of NSCLC. In this
meta-analysis covering 828 patients, we show
that ECE+ status is associated with poorer out-
come in NSCLC patients. ECE+ status has also
been associated with a higher rate of high-grade
tumors, with vascular and lymph vessel invasion,
and with tumor protein P53 overexpression (Lee
et al. 2007). These could be the physiopathologic
causes behind a worse outcome in ECE+ status.
Postoperative radiotherapy (PORT) has
become the standard therapy in NSCLC patients
with stage II to IIIA in the last decade. The
locoregional relapse of tumor is reported to be
around 40% in these patients, and PORT plays
an important role in patient survival (Jing et al.
2016). In contrast, for patients with pN0 and pN1
nodal disease, PORT is associated with a signifi-
cant decrease in survival. Consequently, PORT is
32
Fig. 2 Publication bias
analysis using a contour-
enhanced funnel plot; RR
risk ratio
reserved only for pN2 and pN3 status. Since the
findings of the present review suggest a decreased
survival in the ECE+ group, we hypothesize that
PORT could benefit the subgroup of pN1 patients
with extra-nodal lymph node invasion. It would
be interesting whether this approach increases
survival compared to no pN1-PORT in NSCLC
(Borghetti et al. 2016; Zaric et al. 2013), similar
to pN1 breast cancer where PORT has been
shown to benefit ECE+ patients (Belkacemi
et al. 2015). Lynch et al. (2014) have shown that
in patients with squamous cell carcinoma of the
tonsil, ECE status is predictive of contralateral
nodal recurrence. Consequently, prophylactic
contralateral radiotherapy has been suggested to
improve overall survival in these patients. The
same could also be true for NSCLC patients
with lymph node status N1 (Borghetti et al.
2016). More data is required to discuss this issue
in NSCLC, particularly with lymph node status
N2 and N3.
Furthermore, Fig. 3 represents a graphical
reconstruction of summarized all-cause mortality
data from studies included in three systematic
reviews published in 2016 and 2007 (Luchini
et al. 2016; Veronese et al. 2016; Wind et al.
2007). As seen, ECE+ is associated with a signif-
icantly higher all-cause mortality in solid tumors.
S. V. Tabatabaei et al.
This result, in conjunction with our finding,
suggests that ECE plays an important role in the
prognosis of solid tumors as well as NSCLC, and
consequently it should be incorporated into the
TNM staging criteria.
It is important to know that the penetration of
the lymph node capsule can be investigated after
surgical lymph node dissection (Gu et al. 2017).
Since ECE is very focal, the surrounding fatty
tissue has to be completely included in surgical
resection of lymph nodes, in order to make a valid
statement about tumor extension (Veronese et al.
2016). In lung cancer patients, endobronchial
ultrasound-guided transbronchial needle aspira-
tion (EBUS-TBNA) has recently been widely
used for mediastinal lymph nodes staging and as
a minimal invasive staging tool has replaced
video-mediastinoscopy. EBUS-TBNA provides
both cytologic and histologic materials, but it
cannot provide reliable information regarding
ECE status.
In the present systematic review and meta-
analysis, we examined three prospective and two
retrospective observational studies involving
828 patients affected by NSCLC with metastatic
lymph nodes. Of these, 298 patients had ECE+ of
lymph node metastasis, while 530 showed only
intra-nodal metastasis. While the results of this
Prognostic Impact of Extracapsular Lymph Node Invasion on Survival in. . .
Fig. 3 Pooled estimate of all-cause mortality in different types of cancer. The data is summarized using a random-effect
model; RR risk ratio
meta-analysis are statistically robust, it is impor-
tant to consider a limitation stemming from the
primary studies. The data on disease-free survival
and cancer-specific mortality were unreported in
three out of the studies included. Consequently, it
was not possible to analyze the NSCLC-related
mortality. Despite this limitation, our meta-
analysis provides evidence that ECE is associated
with a poorer prognosis in NSCLC. Clearly, pro-
spective studies investigating tumor-free survival
and cancer-specific mortality/morbidity are needed
to confirm our findings and specifically to consider
PORT in stage pN1 patients. However, given the
insurmountable evidence for the prognostic role of
ECE+ in diverse cancer types and as shown herein,
and as shown here in NSCLC, we argue that ECE
status should already be included in TNM staging.
Conflicts of Interests The authors declare no conflicts
of interest in relation to this article.
33
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https://doi.org/10.1007/5584_2018_264
# Springer Nature Switzerland AG 2018
Published online: 23 September 2018

Influence of Transurethral Resection

of Bladder Cancer on Sexual Function,
Anxiety, and Depression

Wojciech Krajewski, Urszula Halska, Sławomir Poletajew,

Radosław Piszczek, Bartosz Bieżyński, Mateusz Matyjasek,
Andrzej Tukiendorf, Fryderyk Menzel, Małgorzata Mazur,
Joanna Rymaszewska, and Romuald Zdrojowy

Abstract TURB was prospectively evaluated. The Hos-

The standard of care in non-muscle invasive
bladder cancer consists of transurethral tumor
resection. The aim of this study was to evalu-
ate the influence of transurethral resection of
bladder tumor (TURB) on the patients’ self-
reported depression, anxiety, sexual satisfac-
tion, and erectile dysfunction. Psychological
condition of 252 male patients who underwent
W. Krajewski (*), B. Bieżyński, F. Menzel, M. Mazur,
and R. Zdrojowy
Department of Urology and Oncological Urology,
Wrocław Medical University, Wrocław, Poland
e-mail: wk@softstar.pl
U. Halska
Department of Physiotherapy, Opole Medical School,
Opole, Poland
S. Poletajew and M. Matyjasek
Department of General, Oncological and Functional
Oncology, Medical University of Warsaw, Warsaw,
Poland
R. Piszczek
Department of Urology and Oncologic Urology, Lower
Silesian Specialistic Hospital, Wrocław, Poland
A. Tukiendorf
Department of Social Medicine, Wrocław Medical
University, Wrocław, Poland
J. Rymaszewska
Department of Psychiatry, Wrocław Medical University,
Wrocław, Poland
pital Anxiety and Depression Scale (HADS),
simplified International Index of Erectile
Function (IIEF-5), and Sexual Satisfaction
Questionnaires (SSQ) were administered to
patients before and 10 days after tumor resec-
tion. We found that primary anxiety, depres-
sion, and erectile dysfunction were all worse in
cancer patients than those in the general popu-
lation, and all further worsened after tumor
resection. The post-resection worsening was
influenced by the operation and catheterization
time, complications, and the tumor
characteristics. Taxonomical analysis shows
that the greatest risk of depression aggravation
concerned patients who were younger, had a
higher body mass index, and a medium-sized
tumor. We conclude that transurethral resec-
tion of non-muscle invasive bladder cancer
may adversely affect sexual function, anxiety,
and depression. Patients should be informed
about potential complications to prevent the
abandonment of a follow-up. The findings of
this study stress the role of personalized medi-
cine pursued by a multidisciplinary
medical team.

37
38
Keywords
Anxiety · Bladder cancer · Depression ·
Erectile dysfunction · Sexual satisfaction ·
Transurethral tumor resection
1 Introduction
The majority of urinary bladder cancers are
non-muscle invasive cancer at the time of diagno-
sis. This type of cancer in which tumor resides in
the mucosa or submucosal layer of the bladder
contrasts with muscle invasive cancers that infil-
trate or pass through the muscular layer (Babjuk
et al. 2013). The standard of care in non-muscle
invasive bladder cancer consists of transurethral
tumor resection (TURB) with or without addi-
tional intravesical therapy. TURB surgery is a
procedure that allows the visualization of the
urethra and the bladder walls, and the removal
of any suspicious foci. It is commonly performed
under spinal anesthesia with the use of a rigid,
hollow, steel tube (resectoscope) equipped with
an optic system and cutting tools, which are
introduced through the urethra.
Treatment results of a surgical procedure are
evaluated by both clinical outcomes and patient’s
reported outcomes; the latter described by the
patient himself and devoid of any interpretation
by a third party. The vast majority of available
studies concerning the patient’s reported
outcomes in bladder cancer relate to the muscle
invasive cancer and its extensive mutilating
effects (Feuerstein and Goenka 2015). Despite
that TURB is one of the most commonly
performed surgical procedures in urology and
the belief of the operation safety, reports
concerning the self-reported outcomes in those
who survive the malignancy are lacking. It is
noteworthy that patients after TURB surgery
have a significantly higher readmission rate com-
pared with other urological procedures due to a
relatively high occurrence of postoperative lower
urinary tract symptoms, bleeding, and bladder
perforation risk (Rambachan et al. 2014). These
factors raise the possibility that TURB might have
a considerable impact on the patient’s reported
W. Krajewski et al.
outcomes of operation. Therefore, in the present
study, we set out to evaluate the influence on the
self-reported symptoms of anxiety and depres-
sion, and on sexual satisfaction and erectile func-
tion, of TURB surgery in male patients.
2 Methods
The study was approved by the Ethics Committee
of Wroclaw Medical University in Wroclaw,
Poland, and was performed in accordance with
the recommendation of the Declaration of
Helsinki for Human Research. Informed consent
was obtained from all participants enrolled into
the study. Further, the investigating urologist
explained the purpose of the study to each patient,
the protection of participant’s confidentiality, and
the participant’s freedom to drop out of the study
at any time.
2.1 Patients and General Procedures
We conducted a prospective observational analy-
sis of bladder cancer patients who underwent
TURB surgery. The patients were recruited dur-
ing 12 months, from October 2016 to October
2017 in the Department of Urology and Urologic
Oncology of Wroclaw Medical University;
Department of General, Oncological and Func-
tional Oncology of Warsaw Medical University,
Warsaw; and the Department of Urology and
Oncologic Urology of Lower Silesian Specialty
Hospital in Wrocław, Poland. Three hundred
sixty-seven male patients scheduled for TURB
were initially considered for the study. Patients
with a clinical suspicion of a bladder tumor in
whom resection with cutting current was
performed were included into the study. Those
in whom only removal of a tumor with cold
forceps or cold biopsy with fulguration was
performed were excluded. Other exclusion
criteria were the following: age under 18 years
old, inability to understand the information about
the study or to cooperate with psychological
evaluations, and the lack of contact after
Influence of Transurethral Resection of Bladder Cancer on Sexual Function. . .
operation. Patients taking medications that could
affect their mental status were also disqualified.
Psychometric tools used in the study consisted
of the Hospital Anxiety and Depression Scale
(HADS), the Sexual Satisfaction Questionnaire
(SSQ), and the simplified International Index of
Erectile Function (IIEF-5). From the initially con-
sidered population of 367 patients, 68 were
unable to fill out the HADS questionnaire and
were discarded from further evaluation. Forty-
seven patients did not resent the second set of
questionnaires and also were discarded. Those
who stated that they were not sexually active
(56 patients), but filled out the HADS question-
naire, were included into the study. In the end,
252 patients, scheduled for TURB due to con-
firmed bladder cancer, were included in the anal-
ysis (56 with the HADS questionnaire only and
196 with the HADS, SSQ, and IIEF-5
questionnaires). The patients’ mean age was
66
12 years (range 18–98 years). The mean
duration of surgery was 34
21 min (range
10–140 min). The questionnaires were completed
after admission, before surgery. After surgery,
patients were discharged with the addressed enve-
lope containing a second copy of the
questionnaires, which were to be completed in
10–14 days and resent to the departments.
Demographic, clinical, and psychometric data
were collected. Clinical details such as the Amer-
ican Society of Anesthesiologists (ASA) Score –
a score that assesses the physical status of patients
before surgery, body mass index (BMI), nicotine
abuse, history of previous transurethral surgeries,
recurrence/year index, previous intravesical che-
motherapy or Bacillus Calmette-Guérin (BCG)
immunotherapy for bladder cancer were recorded.
Blood and urine biochemical indices were
checked, along with urological data such as
tumor size and focality, surgeon’s experience
(resident/specialist), anesthesia type, obturator
nerve block, and the use of postoperative cyto-
static agent installation. Additionally, catheteriza-
tion and hospitalization times were recorded.
Clinical and pathological stages were evaluated
for all the patients according to the TNM staging
system (Sobin and Gospodarowicz 2010).
39
2.2 Survey Questionnaires
Hospital Anxiety and Depression Scale (HADS)
The HADS, presented by Zigmond and Snaith
(1983), was used in a Polish adaptation by
Watrowski and Rohde (2014). This is a tool suit-
able for different kinds of patients, including
those with both cancerous and noncancerous
diseases. As the items of the questionnaire are
not concentrated on severe symptomatology, the
scale is applicable to the general population
samples (Hinz et al. 2010).
The scale consists of two subscales with seven
items, scored 0–3 each, rating anxiety (HADS-A)
and depression (HADS-D); the higher the score
the worse anxiety and depressive symptoms.
Based on the individual sums of scores, patients
can be assigned to one of the three categories:
non-case/normal (0–7), borderline (8–10), and
definite (11–21) depression and anxiety.
Sexual Satisfaction Questionnaire (SSQ) The
SSQ was designed by Nomejko and Dolińska-
Zygmunt (2014). This tool is dedicated for adults
of all ages and it allows to measure a patient’s
attitude (cognitive and emotional) toward their
sexual activity (Zak–Łykus and Nawrat 2013).
The questionnaire consists of 10 items and the
answers are provided using a 4-point Likert scale:
1 – absolutely not, 2 – rather not, 3 – rather yes,
and 4 – definitely yes. A result of the question-
naire is the sum of points. Questions 1, 3, 5, 8, and
9 are encoded inversely. The theoretical distribu-
tion of the results fits within the 10–40 point
range; the lower the score, the worse perception
of sexual satisfaction. The method does not have
a reference level; the result is interpreted in rela-
tion to the examined population (Nomejko et al.
2012).
Simplified International Index of Erectile Func-
tion (IIEF –5) The IIEF-5 is a condensed version
of the 15-item International Index of Erectile
Function (IIEF) and is composed of five
questions, designed to diagnose the presence
and severity of erectile dysfunction. Each IIEF-5
item is scored on a 5-point scale Likert scale,
40
where the higher the value, the better the erectile
function and intercourse satisfaction. The IIEF-5
score above 21 is considered a normal erectile
function; the lower the score, the worse erectile
function. Erectile dysfunction is classified into
four categories: severe (1–7), moderate (8–11),
moderate (12–16), and mild (17–21) (Rhoden
et al. 2002). The tool has been validated in vari-
ous settings (Tang et al. 2014; Deveci et al. 2008).
2.3 Surgical Procedures
All surgical steps were performed in accordance
with the good quality white-light transurethral
resection of bladder tumors (GQ-WLTURBT)
and European Association of Urology (EAU)
guidelines. Perioperative antibiotics were
administered in all cases. All but two resections
were performed under spinal anesthesia. Obtura-
tor nerve blockade was not performed routinely;
in six cases the blockade was performed due to an
extensive obturator reflex. Rigid resectoscopes
with a monopolar energy source were used.
Each patient had a catheter placed after the resec-
tion, which was removed as soon as reasonably
possible. Immediate administration of cytostatic
drug was given in accordance with EAU
guidelines. Intraoperative and postoperative
complications were categorized according to
Clavien and Dindo (2007) classification.
2.4 Statistical Analysis
Regression and non-regression data analysis was
performed. At first, linear modeling of continuous
outcome variables was conducted. In this
approach, means of intercepts and slopes with
standard errors and p-values were estimated.
Then, univariate and multivariate logistic
regressions were used. The outcomes were
expressed by a classical odds ratio (OR) with a
confidence 95% interval (CI95%) and a p-value.
Moreover, for variables with more than two
response categories, an ordered logistic regres-
sion was implemented which is an extension of
W. Krajewski et al.
logistic regression for binary dependent variables.
Finally, non-regression taxonomy was used for
selected variables to detect the patient types
which were compared to psychological and sex-
ual performance outcomes. The computation was
performed in the R platform (Team 2017).
2.5 Taxonomy
We also constructed dendrograms of taxonomic
distances between the patients, based on selected
variables Marczewski and Steinhaus (1958),
using a complete linkage method of Maechler
(2017). Taxonomy relies on the symmetric differ-
ence between objects (A, B); the distance
(D) between objects is defined as D ¼ (A–B)/
max (A, B). The method enables the defining of
the types of patients grouped on the basis of
shared characteristics.
3 Results
Patients’ clinical characteristics are displayed in
Table 1, and the questionnaire quantitative and
qualitative baseline (before) and post-surgical
results are in Table 2. The mean differences in
the patients’ ailments before and post-surgery
were compared with a t-test for dependent
samples; all of the differences in the
questionnaires were significant.
Linear Regression Linear regression was
conducted to show the associations between clin-
ical variables. Significant (p < 0.05) and border-
line (p < 0.10) relations are displayed in Table 3.
As an example of data interpretation in Table 3,
age was a factor reducing primary sexual satisfac-
tion nearly by 0.2 points per year of advancing
age in the SSQ survey. The other results are to be
interpreted analogously.
Logistic Regression To model the probability of
a worsening in the SSQ, HADS, and IIEF-5
scales after surgery, we used logistic regression
analysis. Significant (p < 0.05) and borderline
results (p < 0.10) are reported in Table 4.
Influence of Transurethral Resection of Bladder Cancer on Sexual Function. . . 41

Table 1 Patients’ characteristics and surgical data

Clinical feature Stratification n (%) Clinical feature Stratification n (%)
Age (year) <65 95 (37.7) Level of training of Specialist 83 (32.9)
65–70 68 (27.0) surgeon Resident 169 (67.1)
70–75 36 (14.3) Number of tumors 1 162 (64.3)
>75 53 (21.0) 2 39 (15.5)
ASA class 1 60 (23.8) 3 51 (20.2)
2 141 (55.9) Tumor size (cm) <1 91 (36.1)
3 51 (20.3) 1–3 108 (42.9)
BMI (kg/m2) 25.0 72 (28.6) >3 53 (21.0)
25.1–30.0 112 (44.4) Hospitalization time 1 202 (80.1)
30.1–35.0 40 (15.9) (days) 2 21 (8.3)
>35.1 17 (6.7) >2 29 (11.5)
Missing 11 (4.4) Catheterization time (h) 24 203 (80.5)
Nicotine abuse Yes 115 (45.6) 24–48 14 (5.6)
No 126 (50.0) >48 35 (14.0)
Missing 11 (4.4) Tumor stage (T) 0 35 (14.0)
Prior TURB 0 101 (40.0) A 111 (44.0)
1 72 (28.6) 1 56 (22.0)
2 37 (14.7) 2 32 (12.1)
3 42 (16.7) Other than 6 (2.4)
UCC
Recurrences/year 0 101 (40.0) Missing 4 (1.6)
<1 81 (32.2) CIS 8 (3.2)
>1 70 (27.8) High grade tumor Yes 87 (34.5)
Preoperative chemotherapy Yes 7 (2.8) No 161 (63.9)
No 242 (96.0) Missing 4 (1.6)
Missing 3 (1.2) Concomitant CIS Yes 35 (13.9)
Preoperative BCG Yes 34 (13.5) No 213 (84.5)
immunotherapy No 216 (85.7) Missing 4 (1.6)
Missing 2 (0.8) Concomitant CD 1 114 (45.0)
Preoperative hematuria Yes 91 (36.1) 2 33 (13.1)
No 150 (59.5) Chemotherapeutic Yes 132 (52.4)
Missing 11 (4.4) instillation No 110 (43.6)
Preoperative pyuria Yes 101 (40.1) Missing 10 (4.0)
No 140 (55.5)
Missing 11 (4.4)
ASA American Society of Anesthesiologists Score, BMI body mass index, TURB transurethral resection of bladder tumor,
BCG Bacillus Calmette-Guérin, CIS carcinoma in situ, CD cancer disease

Taxonomic Analysis Dendrograms of taxonomic
distances grouped patients of a given type into
aggregates, forming hierarchical super-groups
(Figs. 1 and 2). The groups were defined follow-
ing the increment of the joining heights between
objects. After pruning the dendrogram along the
observed cut-off value (red line), six groups of
patients have been formed. These collections
were used for further statistical comparisons.
The assigned taxonomic types of patients were
related to changes in the level IIEF-5 and depres-
sion (one-way ANOVA, Table 5). A rough inter-
pretation of the clinical patterns of patients is as
follows: a population with a relatively average
BMI  30 kg/m2, aged >45, with nonsmoking
status generates the most negative IIEF-5 changes
after surgery, compared to the remaining types of
patients (type 1 in the right-hand part of Table 5).
Further, all the mean differences between the
patient groups are significant.
42 W. Krajewski et al.

Table 2 Questionnaire results at baseline (pre-TURB) and after transurethral resection of bladder (post-TURB) tumor
Psychometric tool/level Pre-TURB Post-TURB
HADS – anxiety (points) 7.9
3.7 (1–19) 8.8
4.4 (1–21)
HADS – anxiety qualitative borderline (n; %) 80 (31.7) 58 (23.0)
HADS – anxiety qualitative definitive (n; %) 56 (22.2) 83 (32.9)
HADS – depression (points) 5.7
4.1 (0–18) 7.3
4.9 (0–20)
HADS – depression qualitative borderline (n; %) 36 (14.3) 45 (17.9)
HADS – depression qualitative definitive (n; %) 35 (13.9) 61 (24.2)
SSQ sexual satisfaction (points) 29.1
5.3 (13–40) 27.3
5.5 (11–40)
IIEF-5 (points) 16.2
5.0 (2–25) 13.4
5.4 (5–25)
IIEF-5 without ED (n; %) 28 (14.3) 13 (6.7)
IIEF-5 mild ED (n; %) 72 (36.8) 44 (22.4)
IIEF-5 mild to moderate ED (n; %) 61 (31.1) 65 (33.2)
IIEF-5 moderate ED (n; %) 24 (12.2) 44 (22.4)
IIEF-5 severe ED (n; %) 11 (5.6) 30 (15.3)
Data are mean point scores
SD (range) or number (%); HADS Hospital Anxiety and Depression Scale, SSQ Sexual
Satisfaction Questionnaire, IIEF-5 simplified International Index of Erectile Function, ED erectile dysfunction. All of the
pre-TURB/post-TURB differences were significant at p < 0.05

interrelationships. We collected a variety of data

4 Discussion
To ensure optimal disease-free survival in
patients suffering from non-muscle-invasive
bladder cancer (NMIBC), lifelong surveillance
with repeated invasive procedures such as
cystoscopies, resections, and in some cases radi-
cal operations are required. That necessitates a
close cooperation between physician and patient
and a good compliance with diagnostic and ther-
apeutic schedules. However, manipulation on
genitourinary tract often causes or aggravates
psychological problems such as anxiety about
losing masculinity, inferior self-esteem and
sense of sexuality, anxiety about the value of
intimate relationships, and a lack of body accep-
tance (Gerharz et al. 2005). Those factors may
lead to the avoidance or total abandonment of the
diagnostic and therapeutic proceedings and lead
to uncontrolled disease development. Thus, it is
crucial to value both clinical and patient-reported
outcomes of therapeutic methods.
In this study, we seek to address changes in
anxiety, depression, erectile function, and sexual
satisfaction in a homogenous group of male
patients who underwent TURB procedure due to
NMIBC. Clinical, peri-, and postoperative data
were also evaluated to find possible
types such as continuous, binary, and categorical
data and adopted both classical regression and
non-regression methods of evaluation, such as
taxonomy statistics, to find the best possible
data fit.
4.1 Hospital Anxiety and Depression
Scale (HADS)
It is known that anxiety and depressive symptoms
are enhanced in oncological patients compared
with healthy individuals (Hinz et al. 2010). Fur-
ther, employment of therapeutic procedures may
additionally increase the patient’s psychological
distress (Palapattu et al. 2004). The present
finding confirmed the presence of elevated anxi-
ety and depressive symptoms in NMIBC patients
on par with other kinds of malignancy (Kulpa
et al. 2016; Kulpa et al. 2014; Hinz and Brahler
2011). We also demonstrated that some cancer
characteristics, such as the number of tumors and
disease aggressiveness, aggravated the
symptoms. It seems that not only the knowledge
of having a tumor but also the awareness of its
advancement reduces the patient’s psychological
wellbeing. We further demonstrated that anxiety
Table 3 Linear regression elaboration
Clinical Clinical Clinical
outcome Regression outcome Regression outcome Regression
Before
tumor After tumor
resection Parameter Mean
SE p Resection Parameter Mean
SE p Δ Difference Parameter Mean
SE p
SSQ Intercept 40.7
2.2 <0.0001 SSQ Intercept 40.8
2.3 <0.0001 SSQ Intercept 0.16
0.41 0.6920
Age 0.2
0.0 <0.0001 Age 0.2
0.0 <0.0001 Surgery time 0.05
0.01 <0.0001
Intercept 33.7
0.8 <0.0001 Intercept 29.1
0.7 <0.0001 Intercept 1.33
0.29 <0.0001
Prim. 0.6
0.1 <0.0001 Surgery 0.1
0.0 0.0046 Cath. Time 0.01
0.01 0.0107
Anxiety time
Intercept 32.0
0.6 <0.0001 Intercept 31.2
0.8 <0.0001 Intercept 3.19
0.69 <0.0001
Prim. 0.6
0.1 <0.0001 Anxiety 0.5
0.1 <0.0001 ASA 0.74
0.34 0.0316
Depression
Intercept 17.7
1.0 <0.0001 Intercept 31.3
0.6 <0.0001 Anxiety Intercept 0.09
0.35 0.7980
Prim. IIEF-5 0.7
0.1 <0.0001 Depression 0.6
0.1 <0.0001 Surgery time 0.03
0.01 0.0016
Anxiety Intercept 8.3
0.3 <0.0001 Intercept 17.6
0.8 <0.0001 Intercept 0.27
0.22 0.2310
Tumor 0.2
0.1 0.0832 IIEF-5 0.7
0.1 <0.0001 Cath. Time 0.01
0.00 <0.0001
focality
Influence of Transurethral Resection of Bladder Cancer on Sexual Function. . .

Intercept 4.9
0.3 <0.0001 Anxiety Intercept 9.2
0.3 <0.0001 Intercept 0.41
0.23 0.0688
Depression 0.5
0.1 <0.0001 TURB 0.3
0.2 0.0426 SSQ change 0.19
0.06 0.0021
Intercept 11.9
0.9 <0.0001 Intercept 8.1
0.4 <0.0001 Depression Intercept 0.65
0.30 0.0307
IIEF-5 0.3
0.1 <0.0001 Tumor size 0.4
0.1 0.0070 SSQ change 0.03
0.01 0.0002
Depression Intercept 9.1
1.0 <0.0001 Intercept 4.5
0.4 <0.0001 Intercept 1.13
0.19 <0.0001
IIEF-5 0.2
0.1 <0.0001 Depression 0.6
0.04 <0.0001 Cath. Time 0.01
0.00 <0.0001
Intercept 1.4
1.4 0.3340 Intercept 12.3
0.8 <0.0001 Intercept 1.22
0.18 <0.0001
Age 0.1
0.0 <0.0001 Sec. IIEF-5 0.3
0.1 <0.0001 SSQ change 0.16
0.05 0.0016
IIEF-5 Intercept 28.9
2.0 <0.0001 Depression Intercept 1.2
1.7 0.4990 Intercept 1.27
0.14 <0.0001
Age 0.2
0.0 <0.0001 Age 0.1
0.0 <0.0001 Anxiety 0.41
0.05 <0.0001
change
43

(continued)
Table 3 (continued)
44

Clinical Clinical Clinical

outcome Regression outcome Regression outcome Regression
Before
tumor After tumor
resection Parameter Mean
SE p Resection Parameter Mean
SE p Δ Difference Parameter Mean
SE p
Intercept 11.9
0.8 <0.0001 IIEF-5 Intercept 0.70
0.48 0.1500
Sec. IIEF-5 0.4
0.1 <0.0001 Surgery time 0.06
0.01 <0.0001
IIEF-5 Intercept 15.1
0.7 <0.0001 Intercept 2.03
0.38 <0.0001
Surgery 0.1
0.0 0.0039 Tumor size 0.40
0.14 0.0061
time
Intercept 2.07
0.34 <0.0001
Cath. Time 0.02
0.01 0.0010
Intercept 1.51
0.25 <0.0001
SSQ change 0.71
0.07 <0.0001
Intercept 2.60
0.28 <0.0001
Anxiety 0.22
0.10 0.0243
change
Intercept 2.03
0.32 <0.0001
Depression 0.49
0.11 <0.0001
change
SSQ Sexual Satisfaction Questionnaire, IIEF-5 simplified International Index of Erectile Function, Cath Catheterization, TURB transurethral resection of the bladder tumor
W. Krajewski et al.
Influence of Transurethral Resection of Bladder Cancer on Sexual Function. . . 45

Table 4 Logistic regression elaboration

Sign of change Risk factor OR CI 95% p-value
SSQ BMI 1.05 (1.00–1.10) 0.072
Age 0.96 (0.93–0.98) <0.001
Weight 1.01 (1.00–1.03) 0.092
Anxiety Catheterization time 0.991 (0.98–1.00) 0.059
Depression Height 1.07 (1.01–1.13) 0.014
Tumor stage (T) 0.79 (0.62–1.01) 0.056
Postoperative chemotherapeutic instillation 2.19 (0.92–5.24) 0.078
IIEF-5 BMI 1.04 (0.99–1.10) 0.088
Age 0.95 (0.93–0.98) <0.001
Preoperative chemotherapy 0.15 (0.02–1.26) 0.081
Operation time 1.02 (1.01–1.03) 0.015
SSQ Sexual Satisfaction Questionnaire, IIEF-5 simplified International Index of Erectile Function, BMI body mass index,
OR odds ratio, CI confidence intervals

Fig. 1 Dendrogram of taxonomic analysis of depressive symptoms. Red line presents the cut-off level for statistically
different patients groups

and depressive symptoms were positively
associated with each other and depressive
symptoms were influenced by the patient’s age
and presurgery urine abnormalities. That lends
support to previous studies that have
demonstrated that urinary infection and lower
urinary tract symptoms (LUTS) might influence
quality of life (Jeong et al. 2015).
We demonstrated that TURB procedure
caused an increase in the level of anxiety and
depressive symptoms. The second post-surgery
of HADS survey, which showed worsened
results, was clearly influenced by surgery, cathe-
terization, and the occurrence of complication.
That may be related to psychological stress stem-
ming from the hospitalization and confirmation of
a neoplasm, bothersome and demeaning catheter-
ization, and physical complaints caused by
LUTS, bleeding, or a post-anesthesia syndrome,
which often occurs after surgery (Ghoneim and
O’Hara 2016). However, in some cases, particu-
larly in TURB naïve patients, we noticed a sub-
stantial decrease in post-surgery anxiety; the
reason could be a “falsely” elevated anxiety
46
Fig. 2 Dendrogram of taxonomic analysis of erectile function based on the International Index of Erectile Function
(IIEF-5). Red line presents the cut-off level for statistically different patients groups
level before unknown circumstances of the first-
time TURB procedure. On the other hand,
patients who experienced multiple TURBs may
accept this situation as a part of their lives and
might not be suffering as they did before
(Yoshimura et al. 2005; Ficarra et al. 2000).
In this study, it was possible to find various
“taxonomical” types of patients that were
associated with changes in depressive symptoms.
Taxonomy enables the extraction of strongly
homogenous subpopulations from the general
patient population, which are characterized by
one or more predefined features and demonstrate
comparable outcomes. Contrarily, the method can
also identify subgroups that are at a significant
distance in terms of the features from other
groups of patients. We demonstrated that the
greatest risk of aggravation of HADS depression
concerned patients who were relatively young,
had greater BMI, and had a medium-sized
tumor. On the opposite side, the youngest age,
the lowest BMI, and the smallest tumor size were
associated with the smallest change in depression
level. We failed to unravel similar statistical
correlates in the taxonomic evaluation of changes
in anxiety.
W. Krajewski et al.
4.2 Sexual Satisfaction
Questionnaires (SSQ)
and International Index
of Erectile Function (IIEF)
Despite the fact that sexual well-being diminishes
with age (Laumann and Waite 2008;
Hatzimouratidis 2007) and that the average age
of a patient diagnosed with bladder cancer is
nearly 70 years (Babjuk et al. 2013), many older
adults still participate in some form of sexual
actions including intercourse, oral sex, and mas-
turbation (Lindau et al. 2007). The patients of this
study displayed erectile dysfunction akin to that
reported in other studies (Tang et al. 2014;
Mazzola et al. 2012; Deveci et al. 2008). We
demonstrated that the basic sexual satisfaction
and erectile function inversely associated with
age and with anxiety and depression. In addition,
patients who stated that they were not sexually
active were older and had a greater score of
depressive symptoms; these findings are in line
with those in other studies (Corona et al. 2010;
Wylie and Kenney 2010). The TURB surgery
adversely affected sexual life. Notably, post-
surgery survey scores decreased in patients with
an extended time of surgery or urethral catheteri-
zation, and with the occurrence of complication
Table 5 Patterns of patients in taxonomic analysis (one-way ANOVA analysis)
Depression (Fig. 1) IIEF-5 (Fig. 2)
Type BMI (kg/m2) Age (year) Tumor size (cm) Depression change Type BMI Age Smoking IIEF-5 change
1 28.3
3.9 60.8
5.1 2.0
0.8 0.6
2.2 1 30.3
3.9 47.7
4.7 No 5.8
6.0
2 27.3
3.2 55.2
9.5 4.5
0.7 2.0
3.6 2 26.2
2.4 63.5
5.1 No 3.2
4.1
3 26.7
2.9 54.8
6.0 0.5
0.4 0.7
1.5 3 24.4
3.2 77.6
7.6 No 1.8
3.3
4 44.9
10.5 61.7
8.1 1.3
1.5 2.3
2.2 4 36.9
9.7 68.5
6.2 No 4.1
3.3
5 26.4
3.5 72.3
7.2 0.7
0.5 1.9
2.5 5 27.8
4.8 67.6
9.7 Yes 2.1
3.4
6 27.1
4.3 73.0
6.3 4.5
1.7 2.0
2.5 p-value <0.0001 <0.0001 – 0.0264
p-value <0.0001 <0.0001 <0.0001 0.0295
Values means
SD
Influence of Transurethral Resection of Bladder Cancer on Sexual Function. . .
47
48 W. Krajewski et al.

or administration of cytostatic agents. A number sexuality, still, true influence of TURB is

of patients reported not engaging in any sexual unknown (Bowen et al. 2015; Placer et al.
activity for a time after surgery (data not shown), 2015). Either type of surgery may lead to erectile
which is consistent with other reports (van der Aa dysfunction due to thermal or chemical tissue
et al. 2009). As in case of depressive symptoms, injury, or due to psychological issues relating to
we identified taxonomical groups related to surgery and hospitalization (Ibrahim et al. 2002;
changes in erectile function. We demonstrate Schmidt et al. 2015; Jaidane et al. 2010). In con-
that patients with a greater BMI ( 30 kg/m2), trast to majority of studies analyzing PROs after
lower age, and nonsmokers were subject to the TURP, we assessed ailments shortly after
most negative IIEF changes after surgery com- procedure. It is possible that during longer fol-
pared to the other patients. low-up the TURB influence on sexual function
The male psychological condition is substan- will blur. However, TURP is often performed
tially influenced by sexuality, including erectile once in a lifetime, and TURB (and similarly
function, sexual desire, ability to have an orgasm, cystoscopies) is in general performed repeatedly.
and the ejaculatory capacity (Gilbert et al. 2013; For that reason, it is important to inform the
Wittmann et al. 2009). On the other side, sexual patient about potential consequences of surgery
function closely associates with the psychological in order to prevent abandonment of follow-up
well-being, also in older age, which helps main- procedures.
tain satisfaction from a relationship and provides
a considerable emotional and physical pleasure
(Thomas et al. 2015; Nomejko and Dolińska-
5 Conclusions
Zygmunt 2014). In turn, quality of sexual life
associates with the health condition (Lindau and
This study was conducted in the high-volume
Gavrilova 2010). Any disorder of the lower abdo-
oncology-orientated medical centers, which
men and pelvis may lead to sexual dysfunction
certifies the quality and repeatability of surgery
due to psychogenic or organic causes. That is
procedures. Nonetheless, TURB, like every sur-
particularly apparent in patients with urological
gical procedure, may be performed a slightly
or gynecological malignancies (Guercio and
different way among urologists. There was a pos-
Mehta 2018). In addition, any medical
sible qualification bias, as we included only
intervention may lead to a worsening of
patients that were able to fill out surveys without
symptoms and to the fear of harming a partner
a help of a third party. We thus lost the
during sexual interaction (Kowalkowski et al.
participants who were unfit to fill out the initial
2014; van der Aa et al. 2009).
questionnaires due to poor health condition or
Literature data concerning sexual function
excessive presurgery stress. Some of the enrolled
refer, almost solely, to patients treated for muscle
patients failed to return the questionnaire or its
invasive blood cancer (Mohamed et al. 2012).
second post-surgery version and were discarded
Radical proceedings in majority of such patients
from the final evaluation. Therefore, this study
cause irreversible erectile dysfunction, signifi-
may not include a complete cross section of
cantly disturbing sexual satisfaction (Asgari
NMIBC patients. We believe, however, we have
et al. 2013; Selvin et al. 2007). Scarce data avail-
conclusively shown that the TURB procedure
able in NMIBC patients demonstrate a high prev-
caused untoward changes in anxiety, depressive
alence of sexual dysfunction compared with an
symptoms, and sexual function. The findings
age- and gender-matched healthy population and
underscore the role of personalized medicine
show that therapy may also adversely influence
based on the effort of a multidisciplinary team
sexual function (van der Aa et al. 2009; Sighinolfi
which includes professionals engaged in the
et al. 2007). When discussing endoscopic
assessment and therapy of psychological health
procedures, only transurethral resection of pros-
apart from somatic ailments.
tate (TURP) was analyzed in terms of changes in
Influence of Transurethral Resection of Bladder Cancer on Sexual Function. . .
Acknowledgments This research did not receive any
specific grants from public, commercial, or nonprofit
agencies.
Conflicts of Interest The authors declare no conflicts of
interest in relation to this article.
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https://doi.org/10.1007/5584_2018_265
# Springer Nature Switzerland AG 2018
Published online: 29 September 2018

Cognitive Predictors of Cortical Thickness

in Healthy Aging

Patrycja Naumczyk, Angelika K. Sawicka, Beata Brzeska,

Agnieszka Sabisz, Krzysztof Jodzio, Marek Radkowski,
Karolina Czachowska, Paweł J. Winklewski, Karolina Finc,
Edyta Szurowska, Urszula Demkow, and Arkadiusz Szarmach

Abstract were examined on 3.0 T MRI scanners. The

This study seeks to define the role of predictive
values of the motor speed, inhibition control,
and fluid and crystallized intelligence in
estimating the cortical thickness in healthy
elderly. Forty-six older healthy subjects
(37 women, 9 men) over 60 years of age
were included in the study. The participants
protocol included standard anatomical
sequences, to exclude brain pathology, and a
high-resolution T1-weighted sequence used to
estimate the cortical thickness. The neuropsy-
chological protocol included fluid intelligence
assessment (Raven Progressive Matrices),
crystalized intelligence assessment

P. Naumczyk (*) and K. Jodzio

Institute of Psychology, University of Gdansk, Gdansk,
Poland
e-mail: psypn@ug.edu.pl
A. K. Sawicka
Department of Bioenergetics and Nutrition, Gdansk
University of Physical Education and Sport, Gdansk,
Poland
B. Brzeska
Department of Biology and Pharmaceutical Botany,
Faculty of Pharmacy, Gdansk Medical University,
Gdansk, Poland
Department of Human Physiology, Faculty of Health
Sciences, Gdansk Medical University, Gdansk, Poland
Second Department of Radiology, Faculty of Health
Sciences, Gdansk Medical University, Gdansk, Poland
A. Sabisz, E. Szurowska, and A. Szarmach
Second Department of Radiology, Faculty of Health
Sciences, Gdansk Medical University, Gdansk, Poland
M. Radkowski
Department of Immunopathology of Infectious and
Parasitic Diseases, Warsaw Medical University, Warsaw,
Poland
K. Czachowska
Department of Human Physiology, Faculty of Health
Sciences, Gdansk Medical University, Gdansk, Poland
P. J. Winklewski
Department of Human Physiology, Faculty of Health
Sciences, Gdansk Medical University, Gdansk, Poland
Second Department of Radiology, Faculty of Health
Sciences, Gdansk Medical University, Gdansk, Poland
Department of Clinical Anatomy and Physiology, Faculty
of Health Sciences, Pomeranian University in Slupsk,
Slupsk, Poland
K. Finc
Center for Modern Interdisciplinary Technologies,
Nicolaus Copernicus University, Torun, Poland
U. Demkow
Department of Laboratory Diagnostics and Clinical
Immunology of Developmental Age, Warsaw Medical
University, Warsaw, Poland

51
52
(information or vocabulary subtest of the
Wechsler Adult Intelligence Scale-Revised
(WAIS-R)), and executive functioning (Color
Traits Test). The findings unraveled several
interdependencies. The higher the intelligence,
the thicker was the grey matter in nine regions
of both hemispheres, but also some paradoxi-
cal reversed associations were found in four
areas; all of them were localized along differ-
ent sections of the cingulate gyrus in both
hemispheres. An inverse association was
found between crystallized intelligence and
the thickness of the pars opecularis of the
right hemisphere. The better the executive
functioning, the thicker was the grey matter
of a given region. The better the motor perfor-
mance, the thicker was the grey matter of the
rostral middle frontal area of the left hemi-
sphere and the lingual gyrus of both
hemispheres. In conclusion, the associations
unraveled demonstrate that the neural
mechanisms underlying healthy aging are
complex and heterogenic across different cog-
nitive domains and neuroanatomical regions.
No brain aging theory seems to provide a
suitable interpretative framework for all the
results. A novel, more integrative approach to
the brain aging should be considered.
Keywords
Aging process · Cortical thickness · Grey
matter · Healthy aging · Inhibition control ·
Intelligence · Motor speed
1 Introduction
There are four major hypotheses describing the
neuronal underpinnings of aging: the frontal
hypothesis, the neurodegenerative hypothesis, the
acceleration in right hemisphere’s aging hypothe-
sis (the right-hemi theory), and the hemispheric
asymmetry reduction in older adults model
(HAROLD). The first two are grounded in struc-
tural studies, whereas the latter two are rooted in
neuropsychological data on healthy aging in which
the subject functions well in everyday life.
P. Naumczyk et al.
The structural brain aging theories seek the
origins of pathological aging in noxious processes
in different areas of the brain. Namely, the frontal
hypothesis focuses on the damage in the anterior
cortical and subcortical regions (West 1996),
whereas the neurodegenerative hypothesis
assumes that aging follows roughly the
Alzheimer’s disease pattern of brain damage,
but at a slower rate (Swerdlow 2007), which
points to the possible brain pathology in the tem-
poral lobes. The expansion of in vivo neuroimag-
ing techniques, such as magnetic resonance
imaging (MRI), sheds some light on this theoreti-
cal debate. In the studies on normal healthy aging,
brain volume shrinkage has been repeatedly
reported (Fjell et al. 2013; Fjell et al. 2009a; Raz
et al. 2010; Raz et al. 2005), with the annual
percentage change of cortical volume up to
0.8% (Fjell et al. 2014). Yet, current data give
more support to the frontal hypothesis. Although
cortical atrophy is widespread and present both in
frontal and temporal areas (Tamnes et al. 2013;
Enzinger et al. 2005), it seems that cortical thin-
ning is more prevalent in the anterior regions
(Fjell et al. 2009b; Resnick et al. 2003). Nonethe-
less, the frontal lobes dysfunction does not
explain the diversity of functional changes
accompanying normal aging. Healthy old people
increasingly seek clinical advice on cognitive
problems. It has been shown that a range of cog-
nitive functions deteriorate with age (Josefsson
et al. 2012; Finkel et al. 2007). The deterioration
primarily concerns working memory (Mattay
et al. 2006; Balota et al. 2000), inhibitory control
(Park and Reuter-Lorenz 2009), the pace of infor-
mation processing (Salthouse 2000), and a long-
term memory – specifically in the episodic mem-
ory retrieval domain (Reuter-Lorenz and Park
2010; Grön et al. 2003).
In the research focused on these four areas,
elderly participants perform worse than the
young ones. Unfortunately, those results are not
easily explained in the framework of the frontal
hypothesis. Although patients with the anterior
brain damage experience deficits in the
information processing, inhibition control, and
working memory, their dysfunction usually far
Cognitive Predictors of Cortical Thickness in Healthy Aging
exceeds what we attribute to the typical pattern of
cognitive aging. To account for the variability in
elderly individuals’ outcomes, the hypothesis of
accelerated aging in the right hemisphere’s, a
right-hemi model has been proposed (Dolcos
et al. 2002). This model suggests an asymmetry
in the age-related vulnerability between the
hemispheres. The right-hemi theory was rooted
in the neuropsychological findings of a corre-
spondence between cognitive performance of
healthy aging individuals and patients suffering
from lateralized brain damage (Taconnat et al.
2007; Uekermann et al. 2006; Royall et al.
2005; Joy et al. 2001). Yet, the studies of struc-
tural brain do not support a notion of asymmetry
in cortical atrophy associated with aging. In the
light of such findings, the HAROLD model was
put forward (Cabeza 2002). It postulates that
aging is reflected mostly in the functional archi-
tecture of the brain in the form of a reduction of
prior specialization of cerebral regions. The
HAROLD model is, to an extent, consistent with
previous structural findings (see Tamnes et al.
2013) suggesting that higher-order cortical
systems are especially prone to atrophy in elderly.
Nonetheless, the current understanding of the
mechanisms underlying age-related brain
changes and healthy cognitive aging remains
inadequate. There are many factors, unaccounted
for, which have a protective or harmful effect on
the brain’s structure and function during aging.
On the other hand, there are no data providing a
direct contrast between the hypotheses mentioned
above, which would require a difficult task of
collecting and matching both anatomical and neu-
ropsychological data. Rather, the two structural or
the two functional models are compared with
each other (Fjell et al. 2009a; Dolcos et al.
2002). Further, some previous studies that try to
link cortical thickness and cognitive functioning
in the elderly have failed to refer to the
hypotheses described (Lee et al. 2016; Persson
et al. 2016; Gautam et al. 2015; Fjell et al. 2006).
The present study seeks to contribute to the
debate on the theories of aging. We focused on
defining neuropsychological correlates of grey
matter thickness in the elderly. Specifically, we
examined the relationships between cortical
53
thickness with motor speed, inhibition control,
and fluid and crystallized intelligence. Whereas
crystallized intelligence is, roughly speaking,
unaffected by normal aging, the other three
factors decline with age. Our goal was to compare
the predictive value of all these factors in the
estimation of the grey matter thickness and its
variations across different brain regions in healthy
elderly. In the study, we remained attentive to the
patterns supporting or undermining the
hypotheses of the brain aging outlined above.
2 Methods
2.1 Participants
The study protocol was approved by the Ethics
Committees of the Universities involved in the
project (permissions NKBBN/296/2017 and
7/2014), and all participants gave written
informed consent for the study procedures. The
participants consisted of 46 pensioners
(37 women and 9 men) over 60 years of age.
Individuals with any psychoneurological
incidents, brain pathology diagnosed in magnetic
resonance scan images, dementia symptoms
based on the Mini Mental State Examination
(MMSE), metal bodily implants, or overt neuro-
degenerative condition excluded from the study.
Detailed demographic data of participants are
depicted in Table 1.
2.2 MRI Acquisition and Processing
The participants were examined in two equivalent
3.0 T MRI scanners (Philips Achieva TX;
Andover, MA and GE 750 Discovery; Chicago,
IL). The protocol included standard anatomical
sequences, as brain pathology reference, and a
high-resolution T1-weighted sequence, later
used for the estimation of cortical thickness. The
T1 sequence parameters for the Philips were:
repetition time (TR) ¼ 8.10 ms, echo time
(TE) ¼ 3.7 ms, voxel size 111 mm, flip
angle 8 ; and for the GE were: TR ¼ 8.19 ms,
TE ¼ 3.18 ms, voxel size 1x1x1 mm, flip
54 P. Naumczyk et al.

Table 1 Demographic and neuropsychological characteristics of the study group

Demographic variable Mean  SD Min–Max
Age (year) 69.0  4.6 63–86
Education (years) 14.4  3.4 7–26
Retirement (years) 10.3  7.6 0–31
Gender n (%) –
Women 37 (80) –
Men 9 (20) –
Cognitive function tests
Fluid intelligence 75.3  25.5 3–99
(RPM-S percentile score)
Fluid intelligence 7.4  1.8 2–10
(RPM-S sten score)
Crystallized intelligence 13.1  2.7 8–18
(WAIS-R calculated normative score)
Motor speed and visual attention 55.7  15.6 32–97
(time of CTT part 1 completion (s))
Executive control and visual attention 112.0  33.0 57–194
(time of CTT part 2 completion (s))
Inhibition control 1.1  0.6 0.02–2.70
(CTT interference score)
RPM-S Raven Progressive Matrices-standard version, WAIS-R Wechsler Adult Intelligence Scale-Revised, CTT Color
Traits Test

angle ¼ 12 . All anatomical scans were processed thickness of each region was measured by aver-
with FreeSurfer v.6.0 software (Athinoula aging all individual metrics of vertices for a given
A. Martinos Center for Biomedical Imaging at region. The evaluation resulted in 68 cortical
Massachusetts General Hospital, Boston, MA) thickness values for every subject (66 regions
following the recommended processing stream and 2 mean cortical thickness scores).
(http://surfer.nmc.mgh.harvard.edu/), previously
described in detail works (Dale et al. 1999; Fischl
et al. 2004a, b; Fischl et al. 2002; Fischl et al.
2.3 Neuropsychological Assessment
1999). In short, the processing includes transla-
tion into Talairach space and removal of
All of the participants underwent a thorough neu-
non-brain tissue from the images, followed by a
ropsychological assessment that took place about
conversion of the voxel-based images into the
10 days after the MRI session. The neuropsycho-
surface-based 3D representations of triangles
logical protocol included fluid intelligence assess-
(vertices), basing on white and gray matter
ment with the Raven Progressive Matrices
boundaries. The procedure allows for a precise
(RPM-S) standard version, crystallized intelli-
subject-to-subject and subject-to-template
gence assessment with the Wechsler Adult Intel-
registrations upon individual gyral and sulcal
ligence Scale-Revised (WAIS-R) or vocabulary
topology. Anatomical landmarks derived from
subtest of the WAIS-R, and the executive func-
the Desikan-Killiany brain atlas (Desikan et al.
tioning assessment with the Color Traits Test
2006) were fitted into the data resulting in
(CTT).
66 regions of interest, 33 per hemisphere, for
As normative fluid intelligence scores, both
each participant. The atlas is presented in Fig. 1.
percentile and sten scores, were non-normally
The closest distance from the grey matter bound-
distributed (Shapiro-Wilk (46) ¼ 0.787,
ary to the pial boundary at each vertex represents
p < 0.001 and Shapiro-Wilk (46) ¼ 0.901,
cortical thickness at a given vertex. The cortical
Cognitive Predictors of Cortical Thickness in Healthy Aging 55

Fig. 1 The Desikan-Killiany brain atlas used for automated cortical parcellations. (Reproduced from Klein and
Tourville (2012) under the terms of the Creative Commons Attribution License)

p ¼ 0.001, respectively), the normally distributed
raw scores of the RPM-S were used for further
evaluation. For crystallized intelligence, a calcu-
lated normative score of a given subtest was cho-
sen to account for differences in the subtests used.
As for the CTT, three metrics were derived from
the test: the time taken to complete part 1 –
marker of visual attention and motor speed, the
time taken to complete part 2 – marker of execu-
tive control and visual attention, and the interfer-
ence index – marker of the flexibility of voluntary
attention, especially over the inhibition processes.
The last marker was computed as a difference
between the time needed for part 2 and part
1 completion, divided by the time needed for
part 1 completion. The interference score is 0, if
there is no difference in the performance between
the test parts. The higher the interference score,
the worse the executive functioning, as more time
is needed to account for the increased
inhibition load.
2.4 Statistical Evaluation
The evaluation focused on the relationship
between the neuropsychological metrics
obtained, i.e., fluid IQ raw score, crystallized IQ
56
calculated normative score, time for part 1 of the
CTT, time for part 2 of the CTT, and the interfer-
ence index of CTT, age, and the cortical thickness
score derived from the MRI examination. The
evaluation was conducted in three steps. First,
the normality of variable distribution was
verified, based on the Shapiro-Wilk test and
Z-skewness/Z-kurtosis estimations. For
non-normally distributed variables, a log transfor-
mation was performed and the normality test was
repeated. For eight variables (time of CTT – both
part 1 and part 2 – and grey matter thickness of
lateral occipital area of left hemisphere (LH), LH
posterior cingulate cortex, LH insula, caudal ante-
rior cingulate of right hemisphere (RH), and RH
superior frontal area), the transformation resulted
in normal distribution, so that the log-version of
scores was further compared. For seven variables
(participants’ age, and grey matter thickness of
LH isthmus cingulate area, LH lateral
orbitofrontal area, LH pars opecularis, RH pars
triangularis, RH posterior cingulate cortex, and
RH precentral gyrus) the log-transformation was
not sufficient, so that they were further evaluated
with nonparametric tests. The second step
included paired correlations between all of the
variables with Pearson’s and Spearman’s tests,
depending on whether a given variable was
distributed normally. The third step, for all of
the anatomical regions of interest (ROIs), for
which more than one of the neuropsychological/
demographical variables correlated with cortical
thickness, a hierarchical stepwise regression (for
normally distributed variables) or a partial
Spearman’s correlation (for non-normally
distributed variables) was performed to determine
which of the factors examined explained most of
the variance of grey matter thickness.
Additional measures of the effect size were
computed for all significant associations to deter-
mine the percentage of variance in the cortical
thickness scores explained by the variables of
interest. Those measures were the adjusted r2
metric for regression models and Pearson’s/
Spearman’s r2 coefficient for correlation models.
Statistical analyses were performed using a com-
mercial statistical package for social sciences –
SPSS v24 (IBM; New York, NY).
P. Naumczyk et al.
3 Results
3.1 Neuropsychological Data
The study participants’ both fluid intelligence
(mean percentile score on RPM-S of
75.3  25.5; range 3–99) and crystallized intelli-
gence (normative score on WAIS-R of
13.1  2.7, range 8–18) are grossly in line with
the average level present in the Polish elderly
population at large. Likewise, participants
showed normal executive functioning. The mean
interference index of 1, achieved in our sample,
indicates that it took the subjects twice as long to
perform part 2 of the test, compared with part
1. The neuropsychological results are
summarized in Table 1.
3.2 Cognitive Predictors of Cortical
Thickness
To account for multiple comparisons, a Bonferroni
correction was applied, so that p < 0.05 was
corrected to p < 0.007. As this is a highly conser-
vative approach, the uncorrected results also are
reported to verify the trends in data. After account-
ing for the shared variance (see Methods for
details), cortical thickness of 30 anatomical regions
in both hemispheres and the global metric of grey
matter thickness of the right hemisphere were, to
different extents, explained by neuropsychological
or demographical data. Out of those, five measures
of thickness remained significant after the rigorous
multiple comparisons correction.
Only six of the uncorrected relationships were
explained by participants’ age. The age-related
effects were found only in the right hemisphere,
mostly in the temporal lobe, with a fraction of
variance explained varying from 9% (middle tem-
poral area, RH) to 26% (inferior temporal area,
RH; this link remained significant after
Bonferroni correction). All of them were a
reversed linkage, meaning the higher the
participants’ age, the thinner the grey matter.
The neuropsychological variables explained
the rest of the associations found (24 uncorrected
Cognitive Predictors of Cortical Thickness in Healthy Aging
significant linkages in both hemispheres, 4 signif-
icant corrected linkages). The factor that
accounted for most of them (13 uncorrected and
2 Bonferroni corrected) was fluid intelligence.
Higher fluid intelligence was associated with
thicker grey matter in nine regions of both
hemispheres, but also some paradoxical reversed
associations were found in four areas; all of them
were localized along different sections of the
cingulate gyrus in both hemispheres. The same
reversed direction was significant for crystallized
intelligence and the thickness of the pars
opecularis of the right hemisphere.
There was an inverse relationship between the
interference index and cortical thickness; the
higher the interference index, the worse the exec-
utive performance. This relationship indicates
that the better participants’ executive functioning,
i.e., the smaller the interference index, the thicker
the grey matter of a given region. Thus, executive
functioning explained underlying differences in
variance in five regions of both hemispheres
(one region after Bonferroni correction). Also,
the better was the participants’ motor perfor-
mance, the thicker the grey matter of the rostral
middle frontal area of the left hemisphere and the
lingual gyrus of both hemispheres (the left
hemisphere’s association was significant after
Bonferroni correction). The results, with the
corresponding measures of the effect size, are
provided in Table 2.
4 Discussion
This study defined the predictive value of age and
cognitive variables for cortical thickness in differ-
ent anatomical regions. Specifically, fluid and
crystallized intelligence, motor speed, and inhibi-
tion control were chosen as factors with declining
versus roughly invariable developmental
trajectories. After correcting for multiple
comparisons, significant “predictor-cortical thick-
ness” pairs were found in five regions. It is worth
noting that the uncorrected results were in line
with the ones that passed the rigorous threshold;
therefore, those trends will be briefly discussed
as well.
57
Age-related effects were notable only in the
right hemisphere, mostly in the temporal lobe.
This result, particularly the mean thickness score
prediction, is in line with the right-hemi model
described in the introduction. Despite the tempo-
ral location, it does not support the neurodegener-
ative hypothesis, as the Alzheimer’s pathologies
affect primarily parahippocampal and entorhinal
gyri (Braak and Braak 1991). Still, this is a
finding contradictory to previous studies, where
no profound asymmetry of age-related effects has
been found (Lee et al. 2016; Zhou et al. 2013;
Fjell et al. 2009b; Raz et al. 2005). There are a
few explanations possible. The first refers to the
gender inequality present in this study. Our group
had 37 women and only 9 men, and there is
research suggesting that age-related brain atrophy
is less pronounced or even statistically insignifi-
cant in females, when compared to peer males
(Thambisetty et al. 2010; Hutton et al. 2009).
Another distinguishing feature is the demo-
graphic structure. Our participants were all
elderly, with age ranging from 62 to 86 years,
which was a rather low age disparity compared to
other studies in which the spread was as wide as
18–93 years (Fjell et al. 2009a), 20–79 years (Lee
et al. 2016), 22–60 years (Hutton et al. 2009), or
5–59 years (Zhou et al. 2013). It might be that in
our group the difference in brain atrophy was not
so much apparent within a 24-year span between
the eldest and youngest participants. In the main,
our group also was older than some of the others,
which could affect regional variability in the cor-
tical damage observed. An interesting possibility,
however, cannot be ruled out that the method we
employed in this study enabled a direct compari-
son of the predictive value of age with variables
associated with cognitive functioning; the latter is
evincibly a better predictor of brain atrophy in
multiple regions.
The relationship between fluid and crystallized
intelligence, and grey matter thickness, on the
other side, we unraveled was twofold. For some
associative regions throughout the frontal, parie-
tal, and temporal lobes of both hemispheres, the
linkage was positive, meaning that higher intelli-
gence accompanied thicker grey matter, whereas
for others (localized in different sections of
58 P. Naumczyk et al.

Table 2 Cognitive and demographic predictors of the grey matter thickness

Variable (indicator)
Brain region % variance explained Beta/r p (uncorr) p (corr)
Age
Mean thickness, RH 9 0.292 0.049 ns.
Middle temporal, RH 9 0.296 0.046 ns.
Supramarginal, RH 10 0.318 0.031 ns.
Precentral RH 11 0.325 0.040 ns.
Superior temporal, RH 12 0.352 0.016 ns.
Inferior temporal, RH 26 0.521 <0.001 0.01
Intelligence (fluid – RPM-S)
Caudal anterior cingulate, RH 22 0.466 0.001 0.05
Isthmus cingulate, RH 19 0.459 0.001 0.05
Rostral anterior cingulate, LH 13 0.387 0.008 ns.
Isthmus cingulate, LH 10 0.316 0.032 ns.
Temporal pole, RH 10 0.311 0.036 ns.
Inferior parietal, LH 10 0.312 0.035 ns.
Fusiform, LH 11 0.326 0.027 ns.
Superior parietal, LH 9 0.332 0.024 ns.
Inferior temporal, LH 12 0.352 0.016 ns.
Banks STS, RH 11 0.354 0.016 ns.
Temporal pole, LH 13 0.36 0.014 ns.
Lateral orbitofrontal, RH 13 0.363 0.013 ns.
Pars orbitalis, LH 15 0.389 0.008 ns.
Intelligence (crystallized – WAIS-R)
Pars opecularis, RH 10 0.314 0.034 ns.
Motor speed and visual attention (time of CTT – part 1 completion)
Lingual, RH 12 0.346 0.018 ns.
Rostral middle frontal, LH 12 0.369 0.012 ns.
Lingual, LH 16 0.402 0.006 0.05
Inhibition control (interference index of CTT)
Lateral occipital, LH 9 0.297 0.045 ns.
Pars triangularis, RH 10 0.311 0.036 ns.
Inferior parietal, RH 9 0.326 0.027 ns.
Superior parietal, RH 11 0.326 0.027 ns.
Precuneus, LH 21 0.453 0.002 0.05
Significance of results after Bonferroni correction for multiple comparisons is marked in the last right-hand column; %
variance explained, the percentage of variance in cortical thickness score of a given region explained by a variable; Beta
standardized regression coefficient, r Pearson’s/Spearman’s correlation coefficient, p(uncorr) significance threshold
unaccounted for by multiple comparisons, p(corr) significance threshold accounted for by multiple comparisons, ns
not significant
RPM-S Raven Progressive Matrices-standard version, WAIS-R Wechsler Adult Intelligence Scale-Revised, CTT Color
Traits Test, LH left hemisphere, RH right hemisphere, uncorr significance threshold unaccounted for by multiple
comparisons, corr significance threshold accounted for by multiple comparisons, ns not significant

cingulate cortex for the fluid intelligence and in
the pars opecularis of the right hemisphere for
crystallized intelligence), it was negative, mean-
ing that better intelligence related to thinner grey
matter. As the positive linkages are in line with
previous studies (Lee et al. 2016; Menary et al.
2013), the negative ones raise more interest. It is
intriguing that such paradoxical associations
between the global and/or crystallized intelli-
gence and grey matter thickness, although previ-
ously reported, have been left out of discussions
by the authors (Fjell et al. 2006). Although some
Cognitive Predictors of Cortical Thickness in Healthy Aging
of our results should be taken with caution, as
they were not corrected in the multiple compari-
son analysis, those previous reports of the para-
doxical associations lend support for interpreting
them as non-incidental findings.
There are a variety of possible mechanisms
underlying this reversed (paradoxical) relation-
ship (higher intelligence – thinner cortical grey
matter) found in this and previous studies. One
possible explanation comes from research on the
developing brain. During childhood and adoles-
cence, cortical thickness metrics follow a reversed
U-shape trajectory. Starting at some age (different
between regions and individuals), the thickness of
grey matter continuously decreases
(Khundrakpam et al. 2016). That is interpreted
as a healthy process of synaptic pruning, which
plays a key role in regional functional specializa-
tion of the brain (Haartsen et al. 2016). Menary
et al. (2013) has provided support to this hypoth-
esis, demonstrating a reversed relationship
between lexicon scores and grey matter thickness
of frontal areas, meaning that the youth with
extensive vocabulary had thinner cortices. Thus,
it may be that thinner grey matter of the cingulate
cortex and pars opecularis we found, rather than
atrophy, represents a greater higher degree of
specialization of those regions with increased
participants’ intelligence.
The mechanism above outlined lends support
for the HAROLD model. This theory puts empha-
sis on the dedifferentiation of previously
specialized brain regions in triggering the cogni-
tive performance changes observed in the elderly.
As fluid and crystallized intelligence are known
preservative cognitive factors, their predictive
value for a greater specialization of certain brain
regions would suggest that these variables play a
key role in restraining the asymmetry reduction
with aging. From this perspective, it is interesting
that it was fluid, not crystallized, intelligence that
accounted for most of the associations unraveled
in this study. There are noteworthy differences
between the two constructs that may contribute
to this result. Fluid intelligence, often synony-
mous with abstract reasoning, is based on the
ability to solve novel problems creatively,
59
whereas crystallized intelligence is based on
declarative knowledge and the ability to utilize
facts (Reber and Reber 2001). Therefore, the for-
mer has a strong hereditary component (Gray and
Thompson 2004), while the latter is acquired
through learning and then shaped by experience
(Cattell 1963). Our results suggest that a stronger
prognostic is congenital traits.
The present results also identified five regions
in which the core predictor of grey matter thick-
ness was the performance of inhibitory control in
the elderly. Those were localized mostly in asso-
ciative regions of the frontal, parietal, and occipi-
tal lobes. Worse inhibitory control was related to
greater cerebral atrophy in all the linkages found.
This is a novel finding, as in previous studies on
neuroanatomy-cognition correspondence in the
elderly, the executive domain has either been
left out of investigation (Raz and Rodrigue
2006; Persson et al. 2016) or few significant
results have been found (Fjell et al. 2006). Also,
an additional finding in the present study was the
predictive value of the motor speed and visual
attention outcome for thickness of three areas of
the cerebral cortex. This factor also was not of
primary interest in previous studies. Lee et al.
(2016) have identified a principal component
they attributed to motor performance, which is
associated with cortical thickness of multiple
regions, such as inferior temporal, olfactory,
parahippocampal, and inferior frontal orbital
areas. In contrast, our findings were few, which
may be a result of using univariate scores of only
one test as an indicator in the study.
Nonetheless, the predictive power of the
elderly’s executive and motor performance is
hardly interpretable under any of the theoretical
contexts provided. The results do not support the
frontal hypothesis (as only two linkages in ante-
rior regions were found), nor do they confirm a
higher effect in the temporal areas (as the neuro-
degenerative model postulates). Also, no obvious
lateralization was present or absent in the data;
thus, it cannot be interpreted as enhanced deterio-
ration of the right hemisphere, nor as a reduction
in asymmetry between the hemispheres
(as HAROLD model assumes). It seems rather
60
that the structure-function relation is
multidirectional, both when higher order (such
as inhibition control or visual attention) and
lower-order (such as motor speed) cognitive
processes are considered.
5 Conclusions
Multiple factors influence grey matter thickness
over the course of life. Our study, focused on the
elderly group, was aimed at identifying the sig-
nificance of the prevailing ones from among the
following: age, cognitive performance in the
executive domain (specifically inhibition con-
trol), motor speed, and visual attention, as well
as individual fluid and crystallized intelligence.
We directly compared the predictive value of the
aforementioned variables to rule out any possible
shared variance.
The associations unraveled indicate that the
neural mechanisms underlying healthy aging are
complex and heterogeneous across different cog-
nitive domains and neuroanatomical regions.
None of the main brain aging theories seem to
provide a suitable interpretation framework for all
the results. Thus, a novel, more integrative
approach should be reconsidered. It should be
emphasized that cognitive factors investigated in
this study were associated with cortical thickness
in multiple cerebral regions. It further confirms
the role of proper neuropsychological testing in
the understanding of neuronal changes occurring
with aging. As all of the participants were healthy
at the study time, an opportunity arises to track
the possible ongoing prodromal deterioration or
dementia in this group in the future.
Acknowledgments The work was funded by the
National Science Center grants: Preludium UMO-2013/
09/HS6/N/02634 and Miniatura UMO-2017/01/X/NZ4/
00779. The FreeSurfer’s calculations were carried out at
the Academic Computer Center in Gdańsk.
Conflicts of Interest The authors declare no conflicts of
interest in relation to this article.
P. Naumczyk et al.
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https://doi.org/10.1007/5584_2018_274
# Springer Nature Switzerland AG 2018
Published online: 5 October 2018

Osteoprotegerin, Receptor Activator

of Nuclear Factor Kappa B Ligand,
and Growth Hormone/Insulin-Like
Growth Factor-1 Axis in Children
with Growth Hormone Deficiency

Ewelina Witkowska-Sędek, Małgorzata Rumińska,

Anna Stelmaszczyk-Emmel, Maria Sobol, Urszula Demkow,
and Beata Pyrżak

Abstract the GH-deficient and GH-sufficient children.

The growth hormone/insulin-like growth
factor-1 (GH/IGF-1) axis is involved in the
regulation of the receptor activator of nuclear
factor kappa B ligand (RANKL)/RANK/
osteoprotegerin (OPG) system, but the exact
mechanism of the associations is not fully
explained. In this study we investigated the
serum OPG and total sRANKL concentrations
in short children who had differences in GH
secretory status. We also investigated the
associations between the GH/IGF-1 and
OPG/RANKL systems in GH-deficient
children during GH treatment. There were no
significant differences in any anthropometric
or biochemical parameters evaluated between
E. Witkowska-Sędek (*), M. Rumińska, and B. Pyrżak
Department of Paediatrics and Endocrinology, Medical
University of Warsaw, Warsaw, Poland
e-mail: ewelina.witkowska-sedek@wum.edu.pl
A. Stelmaszczyk-Emmel and U. Demkow
Department of Laboratory Diagnostics and Clinical
Immunology of Developmental Age, Medical University
of Warsaw, Warsaw, Poland
M. Sobol
Department of Biophysics and Human Physiology,
Medical University of Warsaw, Warsaw, Poland
The OPG content and total alkaline phospha-
tase (ALP) activity increased significantly
after the initiation of GH treatment, while
total sRANKL remained unchanged. The
variables baseline BMI SDS for height-age
(β ¼ 0.42; p < 0.05), baseline ALP activity
(β ¼ 0.36; p < 0.05), weight SDS for height-
age at 6 months of GH treatment (β ¼ 1.86;
p < 0.01), and total ALP activity at 6 months of
GH treatment (β ¼ 0.48, p < 0.01) were
identified as independent predictors of
ΔOPG6-month-baseline. We conclude that OPG
and total sRANKL concentrations are inde-
pendent from GH secretory status in short
children. OPG elevation during GH treatment
is independently associated with total ALP
activity and nutritional status in GH-deficient
children.
Keywords
Bone turnover · Children · Growth hormone ·
Hormone replacement therapy ·
Osteoprotegerin · RANKL/RANK/OPG
pathway

63
64
1 Introduction
The receptor activator of nuclear factor kappa B
ligand (RANKL)/receptor activator of nuclear
factor kappa B (RANK)/osteoprotegerin (OPG)
system is the main trimolecular complex which
mediates osteoblast-controlled osteoclastogenesis
and bone degradation and maintains physiologic
bone remodeling (Kohli and Kohli 2011; Yates
and Górecki 2006; Theoleyre et al. 2004; Khosla
2001; Yasuda et al. 1998; Simonet et al. 1997).
The balance between OPG and RANKL/RANK
is crucial for normal osteoclastic bone resorption.
RANKL is expressed as a surface-bound mole-
cule on cells such as osteoblasts, bone marrow
stromal cells, chondrocytes, activated T
lymphocytes and CD4+/CD8+ thymocytes or as
a soluble molecule released by tumor necrosis
factor-alpha convertase. RANK receptors are
expressed on preosteoclasts and on other cells of
this lineage. OPG is produced and secreted by
osteoblasts and by many other cell types, such
as the heart, kidney, liver, spleen, and B cells
(Boyce and Xing 2008; Kearns et al. 2008; Li
et al. 2007; Takayanagi 2007; Vega et al. 2007;
Wada et al. 2006; Ikeda et al. 2001). The interac-
tion between RANKL and RANK stimulates
osteoclast formation, differentiation, and survival
in bone by activation of a number of transcription
factors and signaling pathways, whereas OPG
acts as a soluble decoy receptor by binding
RANKL and consequently inhibits RANKL-
induced osteoclastic bone resorption (Rubin
et al. 2002; Mundy et al. 2001). Both local and
systemic factors such as various cytokines and
calciotropic hormones, which are crucial in the
regulation of osteoclastogenesis and bone
remodeling, are associated with changes in the
stromal RANKL/OPG ratio. The discovery of
those interactions has helped to understand the
pathophysiology of several disorders which affect
bone metabolism, such as glucocorticoid-induced
osteoporosis, postmenopausal osteoporosis, rheu-
matoid arthritis, multiple myeloma, vascular cal-
cification, chronic periodontitis, and rare genetic
disorders of mineral metabolism (Friedmann et al.
2014; Vega et al. 2007; Kruk et al. 2002). Factors
E. Witkowska-Sędek et al.
increasing the RANKL/OPG ratio and thereby
favoring osteoclast recruitment, which result in
stimulation of bone resorption, include parathor-
mone (PTH), 1,25-dihydroxyvitamin D, and
glucocorticoids (Kohli and Kohli 2011; Ma
et al. 2001; Hofbauer and Schoppet 2004). On
the other hand, factors that decrease the RANKL/
OPG ratio, such as estrogens, androgens, and
leptin, inhibit bone resorption and consequently
decelerate bone turnover (Rubin et al. 2002;
Burguera et al. 2001; Szulc et al. 2001; Lee and
Lorenzo 1999).
In healthy children and adolescents, bone mass
depends on the balance between bone formation
and bone resorption, which are regulated in grow-
ing bone by several systemic and local factors.
The growth hormone/insulin-like growth factor-1
(GH/IGF-1) and RANKL/RANK/OPG pathways
seem to be the major determinants of those pro-
cesses (Guerra–Menéndez et al. 2013; Kohli and
Kohli 2011; Boyce and Xing 2008; Hofbauer and
Schoppet 2004), but the clinical application of the
RANKL/RANK/OPG pathway may be difficult
due to a lack of well-established reference data.
Growth hormone deficiency and IGF-1 defi-
ciency are some of the main risk factors for mark-
edly reduced bone mineral density, increased
fracture risk, and decreased bone remodeling
(Giustina et al. 2008). GH replacement therapy
increases bone turnover as evaluated by changes
in biochemical markers of bone formation and
bone resorption, which peak at 3–6 months after
the start of GH treatment, suggesting the activa-
tion of both osteoblasts and osteoclasts
(Witkowska–Sędek et al. 2016, 2017, 2018;
Andersson et al. 2015; Giustina et al. 2008;
Nielsen et al. 1991). Experimental studies have
shown that the GH/IGF-1 axis is involved in the
regulation of the RANKL/RANK/OPG pathway
and subsequently modulates bone formation and
bone resorption, but the exact mechanism of those
associations is not fully explained (Guerra–
Menéndez et al. 2013; Mrak et al. 2007; Wang
et al. 2006; Rubin et al. 2002; Ueland et al. 2002).
Moreover, it is emphasized that OPG and
RANKL serum levels do not fully reflect their
concentrations in bone microenvironment, which
Osteoprotegerin, Receptor Activator of Nuclear Factor Kappa B Ligand, and. . .
is a further difficulty in the exact evaluation of the
relationships between the GH/IGF-1 axis and the
RANKL/RANK/OPG pathway.
This study seeks to define serum OPG and
total sRANKL concentrations in short children,
with different GH secretory status, and to evalu-
ate the associations between the GH/IGF-1 axis
and the OPG/RANKL pathway in GH-deficient
children during GH replacement therapy.
2 Methods
2.1 Subjects and General Protocol
This study was conducted at the Department of
Pediatrics and Endocrinology of the Medical Uni-
versity of Warsaw, Poland, and included 41
GH-deficient and 20 GH-sufficient short children
and adolescents recruited prospectively. All the
children underwent GH testing in order to evalu-
ate the GH secretory status. GH deficiency was
diagnosed according to the following criteria:
height below the 3rd percentile for sex and age
according to Polish growth charts, height velocity
less than –1 SD below mean for sex- and
age-matched Polish population, bone age delay,
exclusion of causes of short stature other than GH
deficiency, and the GH level below 10 ng/mL in a
test of spontaneous nocturnal growth hormone
secretion (five measurements of GH
concentrations made every 30 min during the
first 2 h after falling asleep) and in two pharma-
cological tests with different stimuli such as clo-
nidine, insulin, glucagon, or arginine. Children
with GH release equalling or exceeding 10 ng/
mL were considered as GH-sufficient. The maxi-
mum release of GH (GHmax) in a particular
patient was defined as the highest GH concentra-
tion in any measurement in three tests in
GH-deficient children and in any measurement
in the test or tests performed in GH-sufficient
children.
The protocol included baseline anthropometric
and biochemical evaluation of both GH-deficient
and GH-sufficient children and anthropometric
and biochemical evaluation at 6 and 12 months
of GH replacement therapy in GH-deficient
65
children. In the 41 strong group of GH-deficient
children, 18 of them were treated with GH for
12 months, 14 were treated for 6 months, and in
9 children only baseline data were available. All
GH-deficient children received recombinant
human GH subcutaneously once daily at bedtime.
Mean GH doses are reported in Table 2. Height,
weight, and body mass index (BMI) were
expressed as standard deviation scores (SDS)
according to standards of the Institute for Mother
and Child in Warsaw, Poland. BMI was calcu-
lated as weight in kilograms divided by height in
square meters (kg/m2). Height was normalized for
chronological age, while weight and BMI were
normalized for height-age. Height-age was
defined as the age that corresponds to the patient’s
height when plotted at the 50th percentile on a
growth chart. Baseline height velocity (HV) was
calculated in all children using data from the
period of 6–18 months before the initiation of
GH treatment or taking baseline anthropometric
and biochemical measurements in GH-sufficient
children. Bone age was evaluated using the
standards of Greulich and Pyle (1969). Pubertal
status was assessed according to staging system
of Tanner (1962).
2.2 Biochemical Analysis
Serum OPG, total sRANKL, IGF-1, PTH,
25-hydroxyvitamin D (25(OH)D), calcium and
phosphorus content, total alkaline phosphatase
(ALP) activity, and 24-h urine calcium and phos-
phorus excretion were measured in all children at
baseline and at 6 and 12 months of GH replace-
ment therapy in the GH-deficient group. Serum
content of OPG and total sRANKL were
measured with ELISA (DRG Instruments
GmbH, Marburg, Germany) and those of GH,
IGF-1, and PTH with an immunoassay using the
IMMULITE 2000 Xpi analyzer (Siemens,
Erlangen, Germany). The serum content of 25
(OH)D was measured by immunoassay using
the ARCHITECT analyzer (Abbott Diagnostics,
Abbott Park, IL). The serum ALP activity, cal-
cium and phosphorus content, and urine calcium
and phosphorus excretion in 24-h urine samples
66 E. Witkowska-Sędek et al.

were measured by dry chemistry system using the
VITROS 5600 chemistry analyzer (Ortho Clinical
Diagnostics, Raritan, NJ). IGF-1 concentrations
were normalized for sex and bone age and were
expressed as SDS according to the normative data
provided by the manufacturer (Siemens
Healthcare Diagnostics Inc., Deerfield, IL).
2.3 Statistical Evaluation
the Spearman correlation analysis for nonpara-
metric data. The independent predictors of OPG
elevation during the first 6 months of GH treat-
ment were analyzed using backward stepwise
regression beginning with the three variable
regression models. A p-value <0.05 defined sta-
tistically significant differences. The analysis was
performed using a commercial Statistica v13.1
package (StatSoft, Tulsa, OK).

Data were reported as means SD or medians 3 Results

with interquartile ranges. Data normality was
checked with the Shapiro-Wilk test. Comparisons 3.1 Anthropometric and Biochemical
between the GH-deficient and GH-sufficient chil- Parameters at Baseline
dren were conducted using a t-test or Mann and During Growth Hormone
Whitney U test. Comparisons between baseline Therapy
and treatment values for the corresponding
parameter were conducted using the repeated At baseline there were no significant differences
measures ANOVA with Bonferroni post hoc test in any evaluated anthropometric or biochemical
for parametric data and the Friedman test with parameters between the GH-deficient and
post hoc comparisons for nonparametric data. GH-sufficient children (Table 1).
Correlation analyses were performed using the After the initiation of GH treatment, as
Pearson correlation test for parametric data and expected, HV increased significantly

Table 1 Anthropometric and biochemical parameters in GH-deficient and GH-sufficient children at baseline
Parameter GH-deficient children GH-sufficient children p
Number of patients 41 20 –
Prepubertal/pubertal status) 31/10 13/7 –
Age (years) 10.3  3.5 10.8  3.4 ns
Bone age (years) 8.2  3.5 9.7  3.7 ns
Height SDS 2.59  0.58 2.60  0.57 ns
Weight SDS for height-age 0.09  0.72 0.36  0.57 ns
BMI SDS for height-age 0.08  1.00 0.45  0.75 ns
HV (cm/year) 5.3  1.4 5.2  1.4 ns
IGF-1 SDS 0.37  1.04 0.20  1.41 ns
OPG (pmol/L) 5.6 (3.6–10.1) 9.4 (4.8–11.5) ns
Total sRANKL (pmol/L) 226.3 (164.5–317.3) 243.8 (179.9–337.2) ns
Total ALP (U/L) 202.0 (171.0–231.0) 193.5 (160.0–236.5) ns
PTH (pg/mL) 20.7 (12.2–24.1) 17.6 (11.1–22.9) ns
25(OH)D (ng/mL) 25.8 (22.2–34.0) 27.6 (24.3–34.6) ns
Serum Ca (mg/dL) 10.0 (9.8–10.3) 10.1 (10.0–10.2) ns
Serum P (mg/dL) 5.0  0.46 5.1  0.53 ns
Urine Ca excretion (mg/kg/24 h) 2.9 (1.5–4.0) 2.5 (1.0–4.2) ns
Urine P excretion (mg/kg/24 h) 16.8 (14.0–20.1) 19.5 (16.1–23.1) ns
Data are presented as means SD or medians with interquartile ranges (IR) as appropriate; ns not significant, GH growth
hormone, SDS standard deviation score, BMI body mass index, HV height velocity, IGF-1 insulin-like growth factor-1,
OPG osteoprotegerin, sRANKL receptor activator of nuclear factor kappa B ligand, ALP alkaline phosphatase, PTH
parathormone, 25(OH)D 25-hydroxyvitamin D, Ca calcium, P phosphorus
Osteoprotegerin, Receptor Activator of Nuclear Factor Kappa B Ligand, and. . . 67

(p < 0.001), which led to an increase in height significantly higher than that at baseline after
SDS observed after the first 6 months of treatment 12 months of GH treatment (p < 0.05), but its
(p < 0.001). Weight SDS and BMI SDS for 24-h urine excretion did not change significantly
height-age did not change significantly during during therapy. Nor did serum phosphorus con-
the first 12 months of GH treatment. The mean tent change during GH treatment. The anthropo-
IGF-1 SDS increased significantly after 6 months metric and biochemical parameters at baseline
of GH treatment compared to the baseline value and at 6 and 12 months of GH replacement ther-
(p < 0.001) but then declined back toward the apy are presented in Table 2.
baseline value after another 6 months of treat-
ment, remaining insignificantly higher than that
at baseline. The OPG content and total ALP
3.2 Associations Between OPG or
activity increased significantly after the first
Total sRANKL
6 months of GH treatment (p < 0.01 and
and Anthropometric
p < 0.05, respectively) and then reached a plateau,
and Biochemical Parameters
remaining significantly higher than that at base-
line after 12 months of treatment (p < 0.001 and
Baseline associations between OPG or total
p < 0.05, respectively). Total sRANKL, PTH, and
sRANKL and the anthropometric and biochemi-
25(OH)D did not change significantly during GH
cal parameters investigated, taken as independent
treatment. Serum calcium content was
variables, in GH-deficient and GH-sufficient

Table 2 Anthropometric and biochemical parameters at baseline and during GH treatment in GH-deficient children
Parameter Baseline 6 months 12 months pa pb pc
Number of patients 41 32 18 – – –
Age (years) 10.3  3.5 11.3  3.4 11.7  3.6 – – –
Bone age (years) 8.2  3.5 – 9.9  3.5 – <0.001 –
Height SDS 2.59  0.58 2.23  0.59 2.01  0.64 <0.001 <0.001 <0.01
Weight SDS for height-age 0.09  0.72 0.17  0.72 0.06  0.72 ns ns ns
BMI SDS for height-age 0.08  1.00 0.18  0.98 0.03  1.01 ns ns ns
HV (cm/year) 5.3  1.4 – 9.8  1.03 – <0.001 –
GH dose (mg/kg/week) – 0.19  0.01 0.19  0.01 – – –
IGF-1 SDS 0.37  1.04 2.02  2.51 0.94  1.06 <0.001 ns <0.05
OPG (pmol/L) 5.55 7.57 9.70 <0.01 <0.001 ns
(3.56–10.05) (4.31–12.16) (6.36–12.42)
Total sRANKL (pmol/L) 226.3 260.9 316.4 ns ns ns
(164.5–317.3) (155.4–354.0) (129.3–511.4)
Total ALP (U/L) 202.0 244.0 264.0 <0.05 <0.05 ns
(171.0–231.0) (212.0–291.0) (225.0–282.0)
PTH (pg/mL) 20.7 (12.2–24.1) 19.2 (11.2–30.3) 16.6 (12.6–22.5) ns ns ns
25(OH)D (ng/mL) 25.8 (22.2–34.0) 27.3 (23.1–31.2) 28.0 (23.1–31.8) ns ns ns
Serum Ca (mg/dL) 10.0 (9.8–10.3) 10.2 (9.9–10.3) 10.2 (10.0–10.5) ns <0.05 ns
Serum P (mg/dL) 5.00  0.46 5.30  0.36 5.40  0.80 ns ns ns
Urine Ca excretion (mg/kg/ 2.9 (1.5–4.0) 2.3 (2.0–3.2) 2.0 (1.2–4.4) ns ns ns
24 h)
Urine P excretion (mg/kg/ 16.8 (14.0–20.1) 20.1 (14.5–26.1) 17.8 (14.3–22.3) ns ns ns
24 h)
Data are presented as means SD or medians with interquartile ranges (IR) as appropriate; pa p-value baseline vs. 6 months,
pb p-value baseline vs. 12 months, pc p-value 6 vs. 12 months, ns not significant, GH growth hormone, SDS standard
deviation score, BMI body mass index, HV height velocity, IGF-1 insulin-like growth factor-1, OPG osteoprotegerin,
sRANKL receptor activator of nuclear factor kappa B ligand, ALP alkaline phosphatase, PTH parathormone, 25(OH)D
25-hydroxyvitamin D, Ca calcium, P phosphorus
68 E. Witkowska-Sędek et al.

Table 3 Spearman’s correlation coefficients (r) between baseline serum OPG (pmol/L) or total sRANKL (pmol/L)
concentrations and baseline anthropometric and biochemical parameters in GH-deficient and GH-sufficient children
GH-deficient children GH-sufficient children
Parameter OPG Total sRANKL OPG Total sRANKL
Age (years) ns ns ns 0.45 (<0.05)
Bone age (years) ns ns ns 0.46 (<0.05)
Height SDS ns ns ns ns
Weight SDS for height-age 0.33 (<0.05) 0.46 (<0.01) ns ns
BMI SDS for height-age ns 0.41 (<0.01) ns 0.47 (<0.05)
HV (cm/year) ns ns ns ns
GHmax (ng/mL) ns ns ns ns
IGF-1 SDS ns ns 0.55 (< 0.05) ns
Total ALP (U/L) ns ns ns ns
PTH (pg/mL) ns ns ns ns
25(OH)D (ng/mL) ns 0.34 (<0.05) ns ns
Serum Ca (mg/dL) ns ns 0.46 (<0.05) ns
Serum P (mg/dL) ns ns ns ns
Urine Ca excretion (mg/kg/24 h) ns ns ns ns
Urine P excretion (mg/kg/24 h) ns ns ns ns
GHmax maximum growth hormone release, SDS standard deviation score, BMI body mass index, HV height velocity,
IGF-1 insulin-like growth factor-1, OPG osteoprotegerin, sRANKL receptor activator of nuclear factor kappa B ligand,
ALP total alkaline phosphatase, PTH parathormone, 25(OH)D 25-hydroxyvitamin D, Ca calcium, P phosphorus, ns not
significant

children, are presented in Table 3. There were no
significant associations between either maximum
GH release, baseline height SDS or HV and either
baseline OPG or total sRANKL levels in GH-
deficient and GH-sufficient children. In
GH-deficient children, the strongest associations
were found between the total sRANKL content
and the nutritional parameters such as weight
SDS for height-age and BMI SDS for height-age
(r ¼ 0.46; p < 0.01 and r ¼ 0.41; p < 0.01,
respectively). In GH-sufficient children, OPG
content was adversely associated with IGF-1
SDS (r ¼ 0.55, p < 0.05), while total sRANKL
was adversely associated with age (r ¼ 0.45;
p < 0.05), bone age (r ¼ 0.46; p < 0.05), and
BMI SDS for height-age (r ¼ 0.47; p < 0.05).
There were no statistically significant
associations between OPG or total sRANKL
levels at 6 months of GH treatment and baseline
anthropometric and biochemical parametres. Fur-
ther analysis revealed that the increment in OPG
concentration in the first 6 months of treatment
(ΔOPG6-month-baseline) was adversely associated
with GHmax (r ¼ 0.41; p < 0.05) and positively
with baseline height SDS (r ¼ 0.49; p < 0.01),
weight SDS and BMI SDS for height-age
(r ¼ 0.54; p < 0.01 and r ¼ 0.58; p < 0.001,
respectively), and with baseline IGF-1 SDS
(r ¼ 0.46; p < 0.01). Total sRANKL content did
not change significantly during GH treatment.
Likewise, we did not find any associations of
significance between OPG concentrations after
the first 6 months of GH treatment and any of
the anthropometric or biochemical parameters
measured after the first 6 months of GH treatment.
The increment in IGF-1 during the first 6 months
of therapy (ΔIGF-16-month-baseline) and GH doses
administered in that period were not associated
with OPG concentrations after 6 months of ther-
apy either. ΔOPG6-month-baseline was associated
with height SDS (r ¼ 0.46; p < 0.01), weight
SDS, and BMI SDS for height-age (r ¼ 0.61;
p < 0.001 and r ¼ 0.50; p < 0.01, respectively)
and total ALP activity (r ¼ 0.39; p < 0.05) and
was adversely associated with 25(OH)D
concentrations (r ¼ 0.45; p < 0.05) evaluated
after the first 6 months of GH treatment. Total
sRANKL concentration after the first 6 months of
Osteoprotegerin, Receptor Activator of Nuclear Factor Kappa B Ligand, and. . .
GH treatment was associated only with total ALP
activity at 6 months of therapy (r ¼ 0.42;
p < 0.05).
To evaluate the relationship between the
ΔOPG6-month-baseline, considered as a dependent
variable, and the independent study variables,
measured at baseline (models 1, 2, and 3) and at
6 months of GH treatment (models 4, 5, and 6),
backward stepwise regression analysis was used.
Regression models included the following inde-
pendent variables: model 1, baseline height SDS,
weight SDS, and BMI SDS for height-age; model
2, GH max, baseline bone age, and IGF-1 SDS for
bone age; model 3, baseline total ALP, PTH, and
25(OH)D; model 4, height SDS, weight SDS, and
BMI SDS for height-age, all at 6 months of GH
treatment; model 5, IGF-1 SDS for bone age,
ΔIGF-16-month-baseline, and GH dose administered
in the first 6 months of treatment; model 6, total
ALP, PTH, and 25(OH)D, all at 6 months of GH
treatment. The variables, baseline BMI SDS for
height-age (β ¼0.42; p < 0.05), baseline ALP
activity (β ¼0.36; p < 0.05), weight SDS for
height-age at 6 months of GH treatment (β
¼1.86; p < 0.01), and total ALP activity at
6 months of GH treatment (β ¼ 0.48; p < 0.01),
were identified as independent predictors of
ΔOPG6-month-baseline in models 1, 3, 4, and
6, respectively. Neither GH max, nor IGF-1
SDS or increase in IGF-1 SDS after the initiation
of GH treatment, nor GH dose, nor bone age was
found to be significant independent predictors of
OPG elevation after the initiation of therapy.
After the first 12 months of GH treatment, we
failed to find any significant associations between
the OPG content or ΔOPG12-month-baseline and any
of the anthropometric and biochemical
parameters. GH doses administered in the first
12 months of replacement therapy were adversely
associated with OPG at 12 months (r ¼ 0.60;
p < 0.05). We also noticed that there was a ten-
dency for greater elevation in OPG in the first
12 months of therapy in children treated with
lower GH doses, but that result was statistically
insignificant (r ¼ 0.49; p ¼ 0.06). Total
sRANKL content at 12 months of GH treatment
was not associated with any of the anthropometric
and biochemical parameters evaluated at 12
69
months of therapy. Backward stepwise regression
analysis was not performed at 12 months of GH
treatment because there were too few patients
who could be included in such analysis.
4 Discussion
Several authors have investigated the relationships
between the GH/IGF-1 axis and the RANKL/
RANK/OPG pathway in experimental and clinical
studies (Xia et al. 2015; Guerra–Menéndez et al.
2013; Flint et al. 2009; Wang et al. 2006; Lanzi
et al. 2003; Ueland et al. 2003), but such studies in
GH-deficient patients, especially in children and
adolescents, are scarce. IGF-1 influences OPG and
RANKL concentrations in a dose- and time-
dependent manner in vitro and in vivo, which
confirms its role as a coupling agent in direct
activation of bone formation and indirect, through
OPG/RANKL, activation of bone resorption
(Rubin et al. 2002). It is hypothesized that GH
induces OPG production and is able to modulate
bone remodeling by directly influencing
osteoblast-osteoclast cross talk (Mrak et al.
2007). The results of studies evaluating OPG and
RANKL concentrations in children and
adolescents are divergent. Further multicenter
studies, including large cohorts of children and
adolescents, are needed to establish valuable refer-
ence data for pediatric population and to foster the
clinical use of the RANKL/RANK/OPG pathway.
Buzi et al. (2004) have found that the OPG level in
healthy children is gender independent but changes
with age and is significantly higher in the first
4 years of life than at 4–14 years of age, while
RANKL content slightly declines with age. Those
authors have also reported that serum OPG content
in children affected by Turner’s syndrome
(TS) and early or precocious puberty (PP) is com-
parable to those in healthy peers, while that in
children with rheumatoid arthritis (RA) is signifi-
cantly higher than in controls. Simultaneously,
they have also found that serum RANKL does
not differ significantly between TS-, PP-, and
RA-afflicted children and age-matched healthy
population. On the other hand, Wasilewska et al.
(2009) have suggested that in healthy children and
70
adolescents, OPG concentration is independent of
gender, age, and pubertal status, while RANKL is
higher in boys than girls and increases with age
and weight. Meazza et al. (2014), in turn, have
failed to find any significant differences in the
OPG concentration between healthy controls and
GH-deficient children before the initiation of GH
treatment. Studies, also those in the adult popula-
tion, show a positive association between serum
OPG and age, which is considered a compensatory
response aimed at counteracting bone loss in the
aging skeleton, while serum RANKL seems to
decrease with age (Abrahamsen et al. 2005;
Kudlacek et al. 2003). In different stages of patho-
logical bone metabolism with elevated bone
resorption, OPG content usually increases, which
could be interpreted as a compensatory bone
response to increased osteoclastic activity (Siomou
et al. 2011; Kearns et al. 2008; Buzi et al. 2004;
Ueland et al. 2001). Both untreated GH deficiency
and IGF-1 deficiency, which are characterized by
decreased bone turnover and increased fracture
risk, and GH replacement therapy, which leads to
an increase in bone turnover, could be reflected in
changes in serum RANKL and OPG
concentrations (Ueland and Bollerslev 2005;
Lanzi et al. 2003).
In the present study, we set out to evaluate the
differences in serum OPG and total sRANKL
content between GH-deficient and GH-sufficient
short children. We hypothesized that the OPG
and total sRANKL content could be associated
with the GH secretory status and IGF-1 concen-
tration. We found that although GH-deficient
children showed a tendency for higher BMI and
lower IGF-1 SDS for bone age, accompanied by a
greater bone age delay, their OPG and sRANKL
were not different than those detected in
GH-sufficient children. Similar results have been
previously reported by Flint et al. (2009), who
further conclude that that neither OPG nor
RANKL correlates with BMD z-score in the
short children. In addition, those authors have
revealed that IGFBP-3, which is closely related
to GH secretory status, is the only significant
negative predictor of serum OPG concentration.
The authors put forward a notion that increased
OPG content might represent a compensatory
E. Witkowska-Sędek et al.
response to the missing anabolic actions of GH
and IGF-1 in GH-deficient children, but the lack
of significant associations between OPG, IGF-1,
and the maximum GH release in diagnostic tests
would weigh against such relationships. The pres-
ent study revealed that in both GH-deficient and
GH-sufficient children, neither OPG nor total
sRANKL content was associated with maximum
GH release, but there was an inverse association
between OPG concentration and IGF-1 SDS for
bone age in GH-sufficient children. We found that
OPG concentration was inversely associated with
weight SDS for height-age, while total sRANKL
concentration was positively associated with
nutritional status in GH-deficient children. In con-
trast, in GH-sufficient children, there was an
inverse association of sRANKL content with
age and bone age. In our opinion, the main limi-
tation of this study, and that of Flint et al. (2009),
is a small number of short children investigated.
A larger sample size of short children and
adolescents should be evaluated to better explore
the relationships between the RANKL/RANL/
OPG system and GH secretory status.
In the present study, we also set out to evaluate
the effects of GH replacement therapy on OPG
and total sRANKL content and the relationship
between OPG or RANKL and other biochemical
parameters, including total ALP activity that is
considered a bone formation marker, and
calcium-phosphorus balance in GH-deficient
children. We found that after the initiation of
GH treatment, both OPG content and ALP activ-
ity significantly increased, while total sRANKL,
PTH, and 25(OH)D remained unchanged. The
content of serum calcium also increased during
GH treatment, but its urine excretion did not
change significantly. Nor were there changes in
the content of serum phosphorus and its urine
excretion. We also noticed that the increase in
OPG in the first 6 months of GH treatment was
adversely associated with the maximum GH
release and positively associated with height
SDS and nutritional status at baseline and at
6 months of therapy, with baseline IGF-1 SDS
for bone age, and with total ALP activity at
6 months of therapy. The backward stepwise
regression models revealed that only ALP activity
Osteoprotegerin, Receptor Activator of Nuclear Factor Kappa B Ligand, and. . .
and nutritional status independently predicted the
elevation of OPG after the initiation of GH treat-
ment. We also found that GH doses were
adversely associated with the increase in OPG in
the first year of replacement therapy. The plausi-
ble association between GH administration and
OPG content ought to be confirmed in a larger
group of patients. Meazza et al. (2014) have also
investigated the effects of 12-month GH treat-
ment on OPG content and on various metabolic
parameters, including adipokine profile, in
GH-deficient children, and report, in opposition
to the present study, a lack of significant changes
after the initiation of GH treatment. Those authors
have found that height SDS, both at baseline and
after 12 months of GH treatment, was adversely
associated with the OPG content in GH-deficient
children. They also found an adverse association
between the baseline OPG and IGF-1 content
before treatment. The discrepancy between the
present findings and those above outlined points
to the need of further exploration of OPG content
changes due to GH treatment.
The results of studies in adult populations par-
tially correspond to those reported in children.
Ueland et al. (2001) have reported that serum
OPG content does not depend on GH secretory
status and does not differ between GH-deficient
and healthy adults. Lanzi et al. (2003), who
investigated the effects of 6-month-long GH treat-
ment in both childhood and adult-onset GH defi-
ciency, have reported a significant increase in
plasma OPG content after the first 6 months of
GH treatment, which is accompanied by signifi-
cant increases in total serum calcium and urinary
calcium excretion with unchanged PTH and 25
(OH)D levels. In the same study, the authors also
evaluate the associations between the OPG level
during GH treatment and two bone turnover
markers: osteocalcin, considered a bone formation
marker, and urine deoxypyridinoline excretion,
considered a bone resorption marker. They have
reported an inverse association of OPG increase
during GH treatment with the increase in
osteocalcin and with deoxypyridinoline excretion.
Xia et al. (2015) have revealed that OPG, despite
its associations with bone metabolism, might also
play a role in the development of cardiovascular
71
disorders in GH-deficient adults. They demon-
strate that plasma OPG content is higher in
GH-deficient adults than in healthy controls and
is associated with age, BMI, waist and hip circum-
ference, waist-hip ratio, and the content of fasting
insulin, total cholesterol, triglycerides, high sensi-
tivity C-reactive protein, and interleukin-6. The
authors suggest that OPG could be a potential
therapeutic target in cardiovascular disorders.
Those results suggest that the RANKL/RANK/
OPG pathway is associated not only with bone
turnover but also with metabolic status and could
be used as a marker of impaired metabolic status
leading to increased cardiovascular risk. Although
those observations have been made in
GH-deficient adults, it is possible that the same
associations could also apply for GH-deficient
children and adolescents. Erol et al. (2016) have
found that OPG content is lower in obese than
nonobese children, but they do not find any
associations between OPG and insulin resistance.
In conclusion, we found that serum OPG or
sRANKL was independent from GH secretory
status and IGF-1 content in short children. After
the initiation of GH replacement treatment, serum
OPG content increased significantly, while total
sRANKL remained unchanged. The increase in
OPG during GH treatment was positively
associated with total ALP activity and nutritional
status in GH-deficient children. The exploration of
mutual relationships between the RANKL/RANK/
OPG pathway and both bone turnover and
glucolipid metabolism could lead to new avenues
of clinically applicable research concerning GH
deficiency and GH replacement treatment.
Conflicts of Interest The authors declare no conflicts of
interest in relation to this article.
Ethical Approval All procedures performed in this study
were in accordance with the ethical standards of the insti-
tutional research committee and with the 1964 Helsinki
Declaration and its later amendments. The study protocol
was approved by the Bioethics Committee of the Medical
University of Warsaw (permit: KB/93/2016).
Informed Consent Informed written consent was
obtained from all individual participants included in the
study or from their parents and legal guardians.
72 E. Witkowska-Sędek et al.

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Adv Exp Med Biol - Clinical and Experimental Biomedicine (2018) 3: 75–79
https://doi.org/10.1007/5584_2018_266
# Springer Nature Switzerland AG 2018
Published online: 23 September 2018
Inhibition of Cross-Reactive
Carbohydrate Determinants in Allergy
Diagnostics
Maciej Grzywnowicz, Emilia Majsiak, Józef Gaweł,
Karolina Miśkiewicz, Zbigniew Doniec, and Ryszard Kurzawa
Abstract
Despite being clinically largely irrelevant,
antibodies against cross-reactive carbohydrate
determinants (CCD) are an important issue in
the in vitro diagnostics, as they may produce
false positive or falsely elevated results of the
immunoglobulin E class (asIgE) in relation to
the actually present level of asIgE. The present
chapter demonstrates an effective resolution of
this diagnostic issue by the use of a CCD
inhibitor in in vitro tests. A synthetic CCD
inhibitor, Polycheck® CCD inhibitor, was
used in the laboratory diagnostics of 24 chil-
dren diagnosed with allergic diseases. The
anti-CCD antibody content was measured in
the serum using a Polycheck® Atopic 30-I
panel (Biocheck GmbH; Münster, Germany),
a screening assay for the quantitative determi-
nation of multiple allergen-specific IgE. We
found that the baseline anti-CCD antibody
content, without the CCD inhibitor, ranged
from 0.7 to 3.5 kU/L in the sera of the majority
of 16 out of 24 children. When the CCD
M. Grzywnowicz and E. Majsiak
Polish-Ukrainian Foundation of Medicine Development,
Lublin, Poland
J. Gaweł, K. Miśkiewicz, Z. Doniec, and R. Kurzawa (*)
Allergology and Pulmonology Clinic, Institute of
Tuberculosis and Lung Diseases, Regional Branch in
Rabka-Zdrój, Rabka-Zdrój, Poland
e-mail: rkurzawa@igrabka.edu.pl
inhibitor was applied, the anti-CCD antibody
content decreased in 16, remained unchanged
in 3, and increased in 5 samples. In samples
positive for plant allergens, the asIgE content
dropped by an average of 72% when the CCD
inhibitor was used in the assay, except the
antibodies to tree and grass pollen allergens,
for which the asIgE content remained above
100 kU/L. We conclude that the use of a CCD
inhibitor in in vitro assays is a viable option to
mitigate the influence of anti-CCD antibodies
on the measured level of asIgE immunoglobu-
lin, which increase the reliability of testing
particularly in cases displaying multiple
allergies.
Keywords
Allergy · Anti-CCD antibodies · CCD
inhibitor · Cross-reactive carbohydrate
determinants (CCD) · IgE immunoglobulin
1 Introduction
Cross-reactive carbohydrate determinants (CCD)
are structurally simple carbohydrate residues
which become attached to amino acid protein
chains during glycosylation as part of post-
translational processing in eukaryotic cells. This
process, which leads to glycoprotein production,
is common in both plants and animals. What
75
76 M. Grzywnowicz et al.

makes CCD different from other carbohydrate positive levels of anti-CCD antibodies. To this
residues is their antigenicity and ability to stimu- end, we compared the asIgE content in relation
late the production of allergen specific immuno- to 30 allergens using multiparametric
globulin E (asIgE). The term “cross-reactive” Polycheck® tests with and without a CCD
determinants was first used more than 30 years inhibitor.
ago (Aalberse et al. 1981). Subsequent research
has demonstrated that anti-CCD antibodies are of
no clinical relevance, since their presence is not
2 Methods
associated with any symptoms (Mertens et al.
2010; Muller et al. 2009; Mari et al. 2008; Ebo
The study was approved by the institutional
et al. 2004; Mari 2002). While basophil activation
Ethics Committee and was performed according
tests have demonstrated bioactivity of anti-CCD
to the guidelines of the Helsinki Declaration for
asIgE, no connection has been substantiated to
Human Research. There were 24 children of
clinical symptoms in patients (Mertens et al.
either genders (12 boys and 12 girls) aged 3–10
2010; Foetisch et al. 2003).
(average 5 years) enrolled into the study. They
Despite the seeming clinical irrelevance, anti-
were hospitalized with chronic upper and lower
CCD antibodies pose a serious problem in case of
respiratory tract symptoms and skin symptoms in
the in vitro asIgE diagnostics with respect to the
the Regional Branch of Warsaw Institute of
allergens in which they appear, making it harder to
Tuberculosis and Lung Diseases in the mountain-
correctly diagnose the allergy source. That is
ous city of Rabka-Zdrój from July to August
caused by the structural heterogeneity of CDD
2017. The goal of hospitalization was to conduct
and their resemblance in various allergen sources.
the diagnostics procedures and to determine ther-
The most important type of CCD, having a
apy. Seventeen children were diagnosed with
sequence of N-glycan with alpha(1,3)-fructose
asthma, associated with allergic rhinitis in 8 of
attached to the polypeptide chain through nitrogen
them, and 7 children were diagnosed with moder-
atom in the asparagine radical, is present in both
ately severe atopic dermatitis. Anti-CCD
plant allergens, including inhalable allergens and
antibodies were found in the sera of all children
various food allergens, hymenoptera venom, and
in a range of 0.43–25 kU/L (median 1.5 kU/L).
latex (Altmann 2016). Therefore, when an exam-
The Polycheck® Atopic 30-I (Biocheck
ined person produces anti-CCD antibodies, they
GmbH, Münster, Germany), an allergy test rou-
can bind with allergens which contain CDDs dur-
tinely used in allergy screening, was the diagnos-
ing the test, giving a false positive or falsely ele-
tic method applied. This test is a multiparametric,
vated result in relation to the actual asIgE content.
quantitative test with a lower limit of detection of
Taking into account that the prevalence of anti-
0.15 kU/L. As recommended by the producer, it
CCD antibodies in people subjected to the in vitro
was assumed that positive results start from the
asIgE testing can reach 25%, they pose a serious
level of 0.35 kU/L. The allergens included in the
diagnostic problem in the allergy testing (Muller
assay were the following: cow’s milk, alpha-
et al. 2009; Ebo et al. 2004; Mari et al. 2008). The
lactalbumin, beta-lactoglobulin, casein, egg
prevalence of anti-CCD antibodies is even higher
white, egg yolk, codfish, peanut, cacao, soybean,
among young people, amounting to 35%.
apple, carrot, tomato, flour mix, chicken meat,
One way to circumvent or eliminate the prob-
citrus mix, rice, grass mix, rye, dog epithelia, cat
lem posed by anti-CCD antibodies in the in vitro
epithelia, Cladosporium herbarum, Alternaria
asIgE diagnostics is to perform the component-
alternata, Aspergillus fumigatus,
resolved diagnostics, based on the antigens free
Dermatophagoides pteronyssinus, D. farinae,
from CCD or utilizing a CCD inhibitor. The
hazel pollen, birch pollen, and mugwort pollen.
premise behind the present study was to evaluate
The test also includes a CCD marker, pineapple
the influence of a CCD inhibitor on the asIgE
bromelain.
content in individuals previously diagnosed with
Inhibition of Cross-Reactive Carbohydrate Determinants in Allergy Diagnostics 77

A Polycheck® CCD inhibitor was used Table 1 Anti-CCD antibody content assayed without and
according to the producer’s recommendations, with the CCD inhibitor (kU/L)
added at a ratio of 1:50 to each tested serum. CCD inhibitor Difference
This synthetic inhibitor is composed of polyva- Sample Without With %
lent CCD structures of bromelain, with up to four 1 25.00 6.30 75
amino acids left after the proteolysis and purifica- 2 0.47 0.22 53
tion processes, which guarantees the lack of pep- 3 0.78 0.41 47
tide epitopes. The glycopeptide obtained in such a 4 1.00 0.56 44
way is attached to a protein carrier, which is 5 2.40 1.40 42
6 1.90 1.20 37
highly purified human serum albumin.
7 8.90 6.10 31
Data were presented in the tabular form as two
8 0.89 0.62 30
nominal variables (sample number and the 9 0.80 0.59 26
absence or presence of CDD inhibitor) and one 10 0.82 0.63 23
measurement variable (content of anti-CCD anti- 11 0.76 0.60 21
body). Differences in the content of anti-CCD 12 2.70 2.30 15
antibody assayed without and with the addition 13 0.56 0.49 13
of a CCD inhibitor, expressed as the percentage 14 2.80 2.50 11
of the former, were statistically analyzed using 15 0.87 0.80 8
the Wilcoxon signed-rank test. A p-value <0.05 16 3.40 3.30 3
defined a statistically significant difference. Sta- 17 1.50 1.50 0
tistical analysis was performed using a commer- 18 2.00 2.00 0
19 2.20 2.20 0
cial GraphPad Prism v7 package (San Diego,
20 1.50 1.60 +7
CA).
21 1.00 1.10 +10
22 0.43 0.48 +12
23 2.40 2.90 +21
3 Results 24 6.30 7.90 +25

We found that the anti-CCD antibody content was

significantly lower when the samples were Table 2 Anti-CCD antibody content assayed without and
incubated in the serum containing the CCD inhib- with the CCD inhibitor in a patient positive for plant-based
allergens (kU/L)
itor than in those without the inhibitor (p < 0.05).
The results for individual samples are presented Polycheck®
in Table 1. The anti-CCD content decreased in Atopic 30-I CCD inhibitor %
16, remained unchanged in 3, and increased in Without With
5 samples. In most of the samples (16 out of the Peanut 3.50 2.00 43
24), the initial anti-CCD antibody content, with- Cacao 0.34 0.25 26
Soybean 0.78 0.38 51
out the inhibitor, was in a range 0.7–3.5 kU/L,
Apple 6.20 0.42 93
i.e., in radioallergosorbent test (RAST) class
Carrot 56.00 16.00 71
2, which is in line with the studies presented in
Tomato 4.20 0.15 96
the literature (Holzweber et al. 2013). Flour mix 4.00 0.58 86
From among the samples analyzed, special Citrus mix 9.20 0.72 92
attention should be paid to those displaying a Rice 9.00 0.65 93
positive result in relation to plant allergens. Grass mix >100 >100 –
Table 2 presents an example of the presence of a Rye >100 >100 –
multitude of plant allergens in a patient’s serum Hazel pollen >100 >100 –
and their changes after the addition of the CDD Birch pollen >100 >100 –
inhibitor. After application of the inhibitor, there Mugwort pollen >100 >100 –
was an across-the-board decrease in the anti-CCD CCD 0.82 0.63 23
78 M. Grzywnowicz et al.

antibodies to plant allergens, except for the the limit of detection, completely mitigating the
antibodies related to tree and grass pollens false positive. On average, the level of the entire
which remained at a level exceeding 100 kU/L. anti-CCD antibody content was lowered by 72%,
despite a 23% decrease in the CCD marker. That
clearly illustrates an advantage of the use of a
CCD inhibitor, instead of testing different levels
4 Discussion
of a CCD marker. Unfortunately, in the patients
investigated in this study, despite positive values
A CCD inhibitor is used in the asIgE detection
for CCD, only one sample displayed numerous
test to limit false positive reactions, by competi-
positive results for asIgE. It is worth emphasizing
tively binding anti-CCD antibodies in the sample
that the use of multiparametric tests makes it
assayed before they become bound with allergens
much easier for a physician to identify the pres-
containing a CCD residue. In general, CCD inhib-
ence of anti-CCD IgE antibodies than when
itor, as opposed to allergen-extract molecules, is
interpreting individual results (Altmann 2016;
composed of a mix of molecules that are unasso-
Holzweber et al. 2013).
ciated with the solid phase of the test, which is
The CCD inhibitor is a tool for in vitro asIgE
why the CCD inhibitor-anti-CCD IgE complexes
testing, which helps reduce the effect of anti-CCD
can be washed out along with the other unbound
antibodies on the test results, and thereby
elements during the procedure, and thus do not
increases the specificity of the method. The appli-
influence the test result. The CCD inhibitor used
cation of this reagent can contribute to the better
in this study has been tested before in a group of
and faster diagnostics in cases of multiple
more than 1,300 CCD-positive patients, effec-
allergies tested in vitro. The present findings
tively reducing the number of false positives
lead to a conclusion that the use of CCD inhibi-
(Holzweber et al. 2013).
tion is a viable option in a clinical setting.
The present study shows that the use of a CCD
Multiparametric tests in the diagnostics of
inhibitor lowers the content of anti-CCD
IgE-dependent allergies facilitate the identifica-
antibodies directly analyzed in the test, which
tion of an influence of anti-CCD antibodies on
complies with the previous observations
the results for other allergens. However, due to
(Altmann 2016). The absence of an inhibitory
additional costs stemming from the need to retest
effect in some samples we analyzed remains
with CCD inhibitor, the use of a CCD inhibitor
unexplained. That could stem from the lack of
should be considered on a case-by-case basis.
duplicate measurements. Also, a small quantity
of the material tested or unique patient’s antibody
Conflicts of Interest The authors declare no conflicts of
characteristics may constitute some limitation. A interest in relation to this article.
low content of asIgE found in the present sample
of patients (median 1.5 kU/L, with no inhibitor)
can be yet another factor making the statistical
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Adv Exp Med Biol - Clinical and Experimental Biomedicine (2018) 3: 81–87
https://doi.org/10.1007/5584_2018_280
# Springer Nature Switzerland AG 2018
Published online: 29 September 2018

Effects of Glutathione on Hydrolytic

Enzyme Activity in the Mouse
Hepatocytes

Iwona Stanisławska, Bożena Witek, Marek Łyp,

Danuta Rochon-Szmejchel, Adam Wróbel, Wojciech Fronczyk,
Agnieszka Kamińska, Adam Kołątaj, and Daniel Załuski

Abstract weight, whereas the latter received 12 μL/g of

In this study, the effect of glutathione (GSH) on
the activity of hydrolytic enzymes of lysosomal,
microsomal, and cytosolic fractions was studied
in the mouse hepatocytes. The experiments
involved 30 Swiss male mice, divided into the
experimental and control groups of 15 mice each.
The former group received injections of 12 μL/g
of GSH solution at a dose of 100 μg/g body
I. Stanisławska, M. Łyp (*), and A. Kołątaj
Department of Dietetics, College of Rehabilitation,
Warsaw, Poland
e-mail: marek.lyp@wsr.edu.pl
B. Witek
Department of Animal Physiology, The Jan Kochanowski
University, Kielce, Poland
D. Rochon-Szmejchel
“Dandiete” Diet Outpatient Clinic, Nowe Miasto
Lubawskie, Poland
A. Wróbel
“Expertdent”, Kielce, Poland
W. Fronczyk
Institute of Genetics and Animal Breeding, Polish
Academy of Sciences, Magdalenka, Poland
A. Kamińska
The Cardinal Stefan Wyszyński University, Warsaw,
Poland
D. Załuski
Department of Pharmacognosy, The Ludwik Rydygier
Collegium Medicum, Nicolaus Copernicus University,
Bydgoszcz, Poland
physiological saline, all given intraperitoneally
daily for 7 days. Then, fragments of liver tissue
were collected from the euthanized animals and
processed to obtain lysosomal, microsomal, and
cytosolic fractions of hepatocytes. The activity of
the following enzymes was investigated in vitro:
β-glucuronidase, β-galactosidase, β-glucosidase,
N-acetyl-hexosaminidase, lysosomal esterase and
lipase, acid phosphatase, cathepsin D and L, leu-
cine aminopeptidase, and alanine aminopepti-
dase. We found that GSH, administered in vivo
in the mouse, in the main (73% cases), increased
the in vitro activity of the majority enzymes
abovementioned, although the effect was some-
how variable, depending on the fraction of
hepatocytes and the type of enzyme. The findings
imply that GSH supplementation may intensify
the rate of cellular hydrolytic degradation, i.e., the
rate of disposal by the cell of unwanted materials.
Keywords
Cellular fraction · Glutathione · Hepatocytes ·
Hydrolytic enzymes · Mouse liver
1 Introduction
The liver plays a key role in the metabolism of
glutathione (GSH), a peptide involved in the

81
82
regulation of cellular redox state (Kalinina et al.
2015; Hultberg and Hultberg 2006). GSH also
plays a protective role against oxidative stress
and damage in the hepatocytes, scavenging a
variety of free radicals (Wang et al. 2015). GSH
is notably present in the hepatocyte cytosol
(approx. 90%), where it is synthesized in a
two-step reaction, and in the mitochondrial matrix
(10%) to which it passes from the cytosol
(Koehler et al. 2006).
Lysosomes are the membrane-bound cellular
entities that contain hydrolytic enzymes whose
main role, although not explicitly limited to, is
to degrade and dispose of unwanted by the cell
material and its debris (Settembre et al. 2013).
Oxidative stress contributes to the destabilization
of lysosomal membranes causing a release of
hydrolases into the cytosol, with a subsequent
induction of cellular apoptosis or necrosis (Bidere
et al. 2003; Jóźwik et al. 2003).
Recently, GSH supplementation is a heady
trend in the general public. The peptide is consid-
ered a food additive with the presumed enhancing
role in the cellular clearance and energy pro-
cesses, which also makes it of use in the sports
field to increase physical efficacy. However, the
influence of GSH on the activity of specific lyso-
somal processes is largely unknown. The present
study seeks to examine the effect of exogenous
GSH, applied in vivo, on the in vitro activity of
hydrolytic enzymes in the lysosomal, micro-
somal, and cytosolic fractions of the mouse liver.
2 Methods
2.1 Animals and Study Protocol
The experiment was performed in 30 Swiss male
mice, aged 8 weeks, weighing 25.0  1.2 g. The
animals were kept in standard cages at a room
temperature of 20–22  C, relative humidity of
50–60%, with water ad libitum, and a 12 h light/
dark cycle (lights on at 8:00 a.m. and off at 8:00 p.
m.). All animals were maintained in a good phys-
ical condition and were under a professional
veterinary care.
I. Stanisławska et al.
After weaning at 4–5 weeks, animals obtained
feed consisting of 16% protein for another 7 days.
The composition and nutritional value of the feed
are presented in Table 1. Then, the animals were
divided into the glutathione- and 0.9% NaCl-
treated (control) groups, consisting of 15 animals
each. Over the next 7 days, the mice received
reduced glutathione (Sigma-Aldrich; St. Louis,
MS) in a dose of 100 μg/g dissolved in 12 μL/g
body mass of 0.9% NaCl or saline alone. All
injections were made i.p., twice daily, between
8.15–8.45 a.m. and 4.15–4.45 p.m. 6 h after the
last injection, under brief diethyl ether anesthesia,
the mice were euthanized by cervical dislocation
and were decapitated. Fragments of liver tissue
were immediately isolated, perfused with 0.9%
NaCl solution at 5  C, and placed in a 0.25 M
saccharose solution containing 2 mM EDTA, at a
concentration of 1 g tissue per 6 ml solution. The
fragments were homogenized at 200 rpm in a
Potter-Elvehjem tissue grinder with a Teflon
pestle (Omni International; Kennesaw, GA),
according to Beaufay’s (1972) method.
Homogenates were subjected to sequentially
increasing centrifugations: 8 min at 5,500 rpm in
a K26D centrifuge, 20 min at 14,000 rpm (Sorvall
RC-5C), and 60 min at 10,000 rpm (Beckman
L7–66; GMI, Ramsey, MN), according to the
method of Marzella and Glaumann (1980).
The lysosomal and microsomal sediments
were suspended in 100 mM phosphate-buffered
saline (PBS), pH 6.0, with the addition of 0.1%
Table 1 Compound and energy content of the mouse
feed used in the experiment
Ingredient Content (%)
Premix LSM 1.0
Salt 0.39
Chalk 1.6
Phosphates 1.0
Milk powder 5.8
Soybean meal 10.1
Corn meal 64.7
Cereal germs 15.4
Total energy (MJ/kg) 14.0
Leucine (% in protein) 8.9
Lysine (% in protein) 4.7
Effects of Glutathione on Hydrolytic Enzyme Activity in the Mouse Hepatocytes
Triton X100. They were then frozen and thawed
twice and centrifuged for 10 min at 700 rpm in a
K-26D centrifuge. After another double freezing
and thawing, the cytosolic fractions were then
assayed.
2.2 Cellular Enzymes
Activity of cellular enzymes was determined in
each of the subcellular fraction obtained during
the preparation procedures described in the sub-
section above, i.e., lysosomal, microsomal, and
cytosolic fractions, using the appropriate
substrates and methods depicted in Table 2. A
Nicolet Evolution 300 BB spectrophotometer
setup (Thermo Electron Corporation, Rugby,
Warwickshire, UK) was used for the enzyme
activity determination.
Activities of β-GlcUr, β-Gal, β-Glu, Hex, AcP,
EL, and LL were determined using 4-nitrophenyl
substrates at 420 nm wavelength according to
Barrett and Heath’s (1977) micromethod. Cath
D and Cath L were determined using 6%
azocasein as a substrate at 366 nm wavelength
according to Langner et al.’s (1973) method.
LeuAP and AlaAP were determined using Fast
Blue BB salt (4-benzoylamino-2,5-diethoxyben-
zenediazonium chloride) substrates at 540 nm
wavelength according to McDonald and Barrett’s
(1986) method. The enzyme activity was
expressed in nmol/mg protein/h. The protein
Table 2 Lysosomal enzymes analyzed in the study
Enzyme
β-glucuronidase (β-GlcUr)
β-galactosidase (β-Gal)
β-glucosidase (β-Glu)
N-acetyl-hexosaminidase (Hex)
Acid phosphatase (AcP)
Esterase (E)
Lipase (L)
Cathepsin D (Cath D)
Cathepsin L (Cath L)
Leucine aminopeptidase (LeuAP)
Alanine aminopeptidase (AlaAP)
(”) – same as above
83
was determined using a modified method of
Kirschke et al. (1972). All substrates were pur-
chased from SERVA Feinbiochemica GmbH &
Co (Heidelberg, Germany).
Data were presented as means SD.
Differences between group means of the glutathi-
one and control groups were compared using
analysis of variance ANOVA. A p-value <0.05
defined statistically significant differences. A
commercial statistical package of SAS/STAT
software was used (SAS Institute Inc., Cary, NC).
3 Results
In the glutathione group, activity of β-GlcUr sig-
nificantly decreased, on average, in the lysosomal
fraction of hepatocytes (to 64.4% of that in the
control group) and increased in microsomal
(to 174.9% of control) and cytosolic (to 119.9%
of control) fractions. The activity of β-Gal and
β-Glu increased in all the fractions studied, while
the activity of Hex markedly increased in lyso-
somal and cytosolic fractions only (to 130.5%
and 136.2% of control, respectively) (Table 3).
GSH-fortified animal feed distinctly enhanced the
activity of all remaining hydrolytic enzymes in
the subcellular fractions of mouse liver, with the
exception of esterase, leucine and alanine
aminopeptidases, and cathepsins in the cytosol
and lipase in the microsomal fraction.
Enzyme code Assay method
EC 3.2.1.31 Barrett and Heath (1977)
EC 3.2.1.23 ”
EC 3.2.1.21 ”
EC 3.2.1.30 ”
EC 3.1.3.2 ”
EC 3.1.1.2 ”
EC 3.1.1.13 ”
EC 3.4.23.5 Langner et al. (1973)
EC 3.4.22.15 ”
EC 3.4.11.1 McDonald and Barrett (1986)
EC 3.4.11.2 ”
84 I. Stanisławska et al.

Table 3 Activity of glycosidases in lysosomal (L), microsomal (M), and cytosolic (C) fractions of liver tissue in the
in vivo glutathione (GSH)-treated and control untreated mice
Enzyme Fraction Control GSH % of Control
L 303.1  30.0 195.1  17.0** 64.4
β-GlcUr M 94.0  9.2 164.4  16.0** 174.9
C 62.4  5.0 74.8  6.1* 119.9
L 347.8  35.0 450.5  40.0** 129.5
β-Gal M 59.5  5.8 112.9  11.0** 189.7
C 325.5  27.0 394.6  32.0** 121.2
L 117.1  12.0 215.3  19.0** 183.9
β-Glu M 23.9  2.2 34.2  3.3** 143.1
C 134.0  11.0 157.2  12.0* 117.3
L 842.1  84.0 1098.7  99.0** 130.5
Hex M 287.1  29.0 245.5  24.0 85.5
C 225.0  19.0 306.5  25.0** 136.2
L 1979.4  198.0 2571.4  233.0*** 129.9
AcP M 795.7  79.0 1226.9  122.0*** 154.2
C 1520.0  127.0 2444.5  203.0*** 160.8
L 1434.1  143.0 1932.0  175.0*** 134.7
E M 864.9  86.0 1017.7  101.0* 117.78
C 320.5  27.0 302.2  25.0 94.3
L 574.8  58.0 728.8  66.0*** 126.8
L M 592.6  59.0 576.4  57.0 97.3
C 134.2  11.0 157.6  12.0 117.4
L 95.6  9.7 143.7  12.0*** 150.3
AlaAP M 208.2  21.0 328.7  32.0*** 157.9
C 134.1  11.0 132.3  10.0 98.7
L 146.8  15.0 236.3  21.0*** 161.0
LeuAP M 232.5  23.0 359.8  35.0*** 154.8
C 75.1  6.1 80.7  6.6 107.5
L 22.7  2.4 33.7  2.9*** 148.5
Cath. D & L M 3.0  0.1 3.9  0.2*** 130.0
C 0.89  0.2 0.55  0.1*** 61.8
β-GlcUr β-glucuronidase, β-Gal β-galactosidase, β-Glu β-glucosidase, Hex N-acetyl-hexosaminidase, AcP acid phos-
phatase, E esterase, L lipase, AlaAP alanine aminopeptidase, LeuAP leucine aminopeptidase, Cath A & L cathepsins D
and L. Enzyme activity was expressed in nmol/mg protein/h; *p  0.05; **p  0.01, and ***p  0.001 for the intergroup
difference

rate, so that they are exposed to large amounts of

4 Discussion
reactive oxygen species (ROS) and permanent
oxidative stress. It is known that oxidative stress
The mechanisms responsible for maintenance of
contributes to cellular injury through a reduction
cellular glutathione homeostasis are not fully
in the content of cellular GSH, shifting the bal-
well-understood. GSH is synthesized in hepatic
ance toward the oxidative side of glutathione
cytosol, where it is transported into the
disulfide (GSSG). Oxidation of the sulfhydryl
mitochondria (Fernandez-Checa and Kaplowitz
groups in cysteine residues of proteins forms
2005), with the resulting high content of GSH in
disulfides (Mosharov et al. 2000). Cysteine is
liver cells. The rate of GSH synthesis in
the rate-limiting element in cellular glutathione
hepatocytes is balanced by its secretion into the
biosynthesis (Radtke et al. 2012) and this
blood and bile (Zhu et al. 2014). Hepatocytes are
proteinogenic amino acid is hardly replenishable
rich in mitochondria and have a high respiratory
Effects of Glutathione on Hydrolytic Enzyme Activity in the Mouse Hepatocytes 85

Table 4 Synopsis of in vivo GSH supplementation on the in vitro activity of all hydrolases investigated in the cellular
fractions of mouse hepatocytes
Activity of hydrolases
Hepatocyte fraction Increase (n) No change (n) Decrease (n)
Lysosomal 9 0 1
Microsomal 8 2 0
Cytosolic 5 4 1
Total; n (%) 22 (73.4) 6 (20.0) 2 (6.6)

with foods. We reasoned that exogenously in effect intensifying hydrolytic degradation by

supplemented glutathione could protect
hepatocytes from oxidative stress by inhibiting
mitochondrial lipid and protein peroxidation or
by chelating nonheme iron that easily
accumulates in liver mitochondria, the processes
that lead to mitochondrial and lysosomal dysfunc-
tion and damage (Seo et al. 2008). Oxidative
stress-induced injury also is mediated through
lysosomal damage (Ollinger and Brunk 1995),
thereby decreasing the ability of the cell to dis-
pose of unwanted materials. Thus, we deemed it
warranted to examine whether and how the
administration of exogenous glutathione would
influence the process of hydrolytic degradation
mediated by hydrolases in the in vitro
preparations of lysosomal, microsomal, and cyto-
solic fractions of mouse hepatocytes. This kind of
study design finds support in the knowledge that
GSH-fortified diet can appreciably influence GSH
cellular content (Valencia et al. 2001). The
findings of this study, in the main, are in line
with the rationale above outlined. We found that
in vitro activity of all hydrolases investigated,
with a single exception of β-GlcUr, increased in
the lysosomal fraction. The enzymes activity,
with a few exceptions, also increased in the
other cellular fractions, the least so in the cytosol
(Table 4). The predominant effect on the activity
of lysosomal hydrolases of exogenous GSH has
also been described in renal tissue by Śliwa-
Jóźwik et al. (2004), which is suggestive of a
high demand of liver and renal cells for the anti-
oxidant. There is a biological plausibility that
improvement in structural functionality of
lysosomes, due to the antioxidative action of
GSH, leads to enhanced activity of hydrolases,
the cell of unwanted materials.
The content of GSH in the liver increases
along with protein intake (Hum et al. 1992;
Morini et al. 1991), providing succor to protective
antioxidative membrane processes. McCarty and
DiNicolantonio (2015) have shown that protein
intake increases the pool of amino acids available
to glutathione synthesis. In this study we used a
high-protein diet along with GSH supplementa-
tion, which may contribute to enhanced activity
of endogenous peptidases noticed. The exception
was cathepsins whose content decreased in the
cytosolic fraction of hepatocytes. Cathepsins are
released from lysosomes into the cytosol in
response to apoptogenic factors, including oxida-
tive stress. Their release induces changes in the
outer mitochondrial membranes (Bidere et al.
2003). Thus, decrease in cathepsins content
speaks for the possibility of less oxidative stress
and less propensity for apoptosis, as an effect of
GSH supplementation noticed in this study.
The enzyme family of lipases and esterases
shows a great degree of complex functional diver-
sity (Anthonsen et al. 1995). These proteins are
usually activated on contact with the aggregated
substrate. Thus, increase in their activity noticed
in this study also is in line with the augmentation
of cellular degradative processes, induced by
GSH given in vivo.
In conclusion, the findings of this study sug-
gest that GSH-supplemented diet activates degra-
dation of unwanted cellular material in
hepatocytes, particularly in the most hydrolyti-
cally active lysosomal fraction. That points to
the potential role of exogenous GSH as an agent
modulating the reactivity of the degradative lyso-
somal enzymes in the liver. GSH-enriched diet
86
might thus play a protective role in liver dysfunc-
tional states by developing adaptive responses
aimed at rebalancing of a distorted “redox state”
through the enhancement of cellular antioxidant
capacity. The findings of this study imply a poten-
tial benefit of GSH supplementation for decreas-
ing oxidative damage, particularly in tissues of
high metabolic demand, at the cellular level.
There is an urgent need to explore the safety of
GSH-fortified diet, which requires alternative
study designs.
Conflicts of Interest The authors declare no conflict of
interest in relation to this article.
Ethical Approval All procedures performed in this study
were in accordance with the ethical standards of the insti-
tution at which the studies were conducted and with the
1964 Helsinki Declaration and its later amendments. All
applicable international, national, and/or institutional
guidelines for the care and use of animals were followed.
The experiments were carried out with the approval of the
Bioethics Committee of the Świętokrzyska Chamber of
Physicians in the city of Kielce in Poland (permit
no. 49/2016).
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Adv Exp Med Biol - Clinical and Experimental Biomedicine (2018) 3: 89–109
https://doi.org/10.1007/5584_2018_261
# Springer Nature Switzerland AG 2018
Published online: 5 October 2018

Adaptation to Occupational Exposure

to Moderate Endotoxin Concentrations:
A Study in Sewage Treatment Plants
in Germany

M. A. Rieger, V. Liebers, M. Nübling, T. Brüning, B. Brendel,

F. Hoffmeyer, and M. Raulf

Abstract plants. Subjects were matched to pairs

Acute or chronic inhalation of endotoxin may
lead to changes of lung function and inflam-
matory markers in the airways. Adaptation to
workplace exposure may be possible. In this
study, we investigated the possible difference
in inflammatory markers assessed in nasal
lavage fluid (NALF) in chronical exposure
compared to voluntary subjects exposed
acutely to endotoxin. We sought to define the
variability of inflammatory markers in NALF
and the dose-related changes after moderate
exposure in naïve subjects. Endotoxin expo-
sure (4–1039 EU/m3) resulted from routine
work during one shift in sewage treatment
M. A. Rieger (*)
Institute of Occupational and Social Medicine and Health
Services Research, University Hospital of Tübingen,
Tübingen, Germany
e-mail: monika.rieger@med.uni-tuebingen.de
V. Liebers, T. Brüning, F. Hoffmeyer, and M. Raulf
Institute for Prevention and Occupational Medicine of the
German Social Accident Insurance (IPA), Ruhr University
Bochum, Bochum, Germany
M. Nübling
Research Center for Occupational and Social Medicine
(FFAS), Freiburg, Germany
B. Brendel
Institute for Clinical Epidemiology and Applied Biometry,
Tübingen University Hospital, Tübingen, Germany
(8 workers escorted by 10 students).
Inflammatory markers were investigated
before, directly after, and 16 h after the shift
end. Additional NALF samples were collected
in students without any specific exposure after
3 days. In NALF, total cell count, and interleu-
kin (IL)-8 and IL-1β concentrations were sig-
nificantly higher in workers than in students at
all times pointing to workplace-related long-
lasting exposure resulting in adaptation. How-
ever, concentration of inflammatory markers
without specific exposure in students showed
a great variability, covering the whole range of
values recorded in the workers. The findings of
this study make us to recommend a repeated
assessment of inflammatory markers in
healthy volunteers before the investigation of
exposure-related changes and a sample size
adequate for statistical analysis.
Keywords
Cumulative effect · Dose-response
relationship · Endotoxin exposure ·
Inflammation · Nasal lavage fluid ·
Occupational exposure · Sewage

89
90
1 Introduction
Occupational exposure to endotoxins is typical
for working conditions in the waste or wastewater
industries and agriculture. Exposure to endotoxin
may induce three different kinds of reactions:
acute effects due to a single exposure or chronic
effects due to chronic or repeated exposure during
a longer period (Rylander 1995). Depending on
the dose, location, route, and time of release in the
blood, the response to endotoxin varies. Several
studies have shown adverse health effects
induced by acute or chronic inhalation of endo-
toxin. Changes in lung function and inflammatory
markers such as ECP, interleukin (IL)-8, IL-1β,
tumor necrosis factor (TNF)-α, and C-reactive
protein (CRP) in the airways or blood are well
recognized (Liebers et al. 2008; Liebers et al.
2006; Rylander 2002; Thorn 2001). As it is
known from different studies, no-effect levels of
endotoxin range from 9 to 170 ng/m3. Castellan
et al. (1987) have described a threshold of
90 endotoxin units (EU)/m3 for acute mucous
membrane irritation and pulmonary change
among cotton workers. Against this backdrop,
the Dutch Expert Committee on Occupational
Standards of the National Health Council has
proposed a health-based recommended occupa-
tional exposure limit (HBROEL) for endotoxin
in the workplace of 90 EU/m3 in 2010 (8-hour
time-weighted average) (Health Council of the
Netherlands 2010).
Several studies have been undertaken to
describe a dose-response relationship concerning
the clinical symptoms (Castellan et al. 1987) or
inflammatory markers (Danuser et al. 2000;
Michel et al. 1997), but data still seem to be
insufficient in work-related (Thorn 2001) or envi-
ronmental exposure (Walser et al. 2015). Clinical
and epidemiological findings have linked the
endotoxin exposure at the work place to fever,
cough, shortness of breath, wheezing, headache,
nose and throat irritation, chest tightness, acute
airway flow restriction, and inflammation
(Liebers et al. 2006). The effects of acute expo-
sure to higher levels of endotoxin have been
summarized in the so-called organic dust toxic
M. A. Rieger et al.
syndrome (ODTS). It has been shown in farmers
that ODTS is a major predictor of chronic bron-
chitis (Radon et al. 2003). Yet, especially when
exposed to moderate endotoxin concentrations,
many workers seem to develop no symptoms,
but they adapt to exposure (von Essen and
Romberger 2003) or develop endotoxin tolerance
(Bakirci et al. 2007). Therefore, there might be a
difference in the reaction pattern to chronic expo-
sure to endotoxin compared with voluntary
subjects exposed acutely.
In this study, we focused on inflammatory
markers in the nasal lavage fluid (NALF) such
as the total cell count, proportion of neutrophils,
total protein, IL-8, IL-6, IL-1β, TNF-α, and
sCD14, and we wanted to answer the following
two questions:
1. Is there a difference in the content of inflam-
matory markers in NALF between chronically
exposed (workers of sewage treatment plants)
and just acutely exposed subjects (students)?
2. Are there any differences in the patterns of
inflammatory markers in NALF of healthy
volunteers after exposure to a moderate endo-
toxin concentration (work in a sewage treat-
ment plant) compared with nonspecific
exposure? Are there any dose-related or
cumulative changes?
2 Methods
2.1 Study Design
Study protocol was created in accordance with
the Declaration of Helsinki and was approved
by the Ethics Committee of the University of
Wuppertal. All participants gave written informed
consent to the study protocol. As the exposure
scenario we chose the areas of the chamber filter
press or the sand trap in sewage treatment plants.
Our previous measurements in these areas have
shown rather high concentrations of endotoxin
(up to 942 EU/m3 during flushing of the chamber
filter press after treatment of sewage sludge with
Adaptation to Occupational Exposure to Moderate Endotoxin Concentrations: A. . .
iron (III) chloride and lime) combined with low
concentrations of other biological hazards (e.g.,
total bacteria: 105–106 CFU/m3 and total molds:
<103 CFU/m3), and no relevant exposures to
ammonia (Rieger and Diefenbach 2007;
Steinberg 2001). Based on those results and fur-
ther exploratory measurements, we selected four
sewage treatment plants of a regional association
for sewage treatment in North Rhine-Westphalia
(Germany) for the present study. Data were
assessed in 2002–2003.
To get insight into the adaptation to endotoxin
exposure, we compared chronically exposed
workers on the sewage treatment plants to just
acutely exposed voluntary subjects (students)
regarding the possible changes in various
outcomes during one working shift. The workers
and students were matched in pairs, i.e., the
workers were escorted by students during their
ordinary work on the plant. The students
documented the workers’ activities in a log. To
investigate a possible dose-response relationship,
students were asked to participate in the study
several times with intervals of several months
between the repeated exposures. Four students
were purposefully exposed to high concentrations
of endotoxin on two following days to assess a
possible cumulative effect on inflammatory
markers. Lastly, specimens were obtained from
five students not only during their stay on sewage
treatment plants but also after 3 days free of any
specific exposure in offices and lecture rooms at
the university. Thus, variability of parameters
within 1 day could be compared with changes
evoked by specific exposure.
2.2 Study Sample
As described above, a major goal of the study was
a comparison of chronically exposed workers in
sewage treatment plants (n ¼ 8) to just acutely
exposed voluntary subjects, represented by
students (n ¼ 10). In addition, a possible dose-
response relationship of the immunological
reactions and a cumulative effect of exposure to
endotoxin on two following days were
investigated.
91
2.2.1 Inclusion Criteria
and Sociodemographic Data
Students The following inclusion criteria were
applied for students: healthy male non-smokers,
without any clinically manifested allergy and not
living or working in a nearby farm, sewage treat-
ment plant, or waste industry. The students were
aged 24.7 (range 22–29) years and had a body
mass index (BMI) of 23.7 (range 20.7–26.6) kg/m2
(Table 1). After inclusion in the study, a screening
test was performed at least 1 month prior to expo-
sure. The test consisted of a skin prick test with
common aero-allergens. In addition, specific
immunoglobulin E (sIgE) in response to a variety
of ubiquitous aero-allergens was assessed in the
serum (atopy screen sx1 Phadiatop®, CAP-FEIA;
ThermoFisher Scientific, Waltham, MA). Allergen
sIgE 0.35 kU/L was considered positive. Based
on the screening results and medical history, 4 out
of the 10 students were classified as having atopy.
On the screening day, students also were trained by
a laboratory’s specialist to gather NALF.
On the day of examination, sociodemographic
data, general symptoms, and the judgment of the
stay on the sewage treatment plant were assessed
by means of a standardized questionnaire. None of
the students indicated any concurrent possible
exposure to biological hazards due to occupation
or leisure time activities. Concerning the occupa-
tional history, only two students indicated former
occupational exposure to airborne biological
hazards (agricultural work years before the study)
and none had any other previous inhalation expo-
sure. Concerning the judgment of stress due to the
working environment of the sewage treatment
plant, most of the inquired aspects were rated as
little-to-moderate (heat, dust, chemicals, humidity,
cold, and time pressure) and four aspects as
moderate-to-high (noise, humidity, infectious
agents, and odor), which was assessed on a
3-point Likert scale: 1 ¼ little stress and
3 ¼ high stress. The self-reported students’ health
status was rated to be very good (mean 1.4), based
on the following scale: 1 ¼ very good, 2 ¼ good,
and 6 ¼ insufficient. The evaluation of general
complaints and medical history showed no specific
92 M. A. Rieger et al.

Table 1 Sociodemographic data of students

Agea BMIa Atopyb SHSc (1 ¼ very good Subjective evaluation of work environment on
Students (years) (kg/m2) (yes/no) 6 ¼ insufficient) sewage plantd (range 9–27 points)
s1 29 25.3 Yes ni 14
s2 25 22.7 Yes 2 16
s3 29 26.3 No 2 14
s4 23 20.7 No 1 18
s5 24 22.8 No 1 17
s6 23 26.6 No 1 17
s7 23 22.8 Yes 1 16
s8 22 22.7 Yes 2 15
s9 25 22.8 No 2 15
s10 24 24.2 No 1 ni
Mean 24.7 23.7 – 1.4 15.8
a
In case of a repeated participation, the age, body mass index (BMI), and indications in the questionnaire of the first day
of examination are given
b
Assessed by skin prick test using common aero-allergens, confirmed by specific immunoglobulin E in response to a
variety of ubiquitous aero-allergens (atopy screen sx1 Phadiatop) and medical history
c
SHS Subjective health status
d
Working environment was judged by the students regarding nine aspects: heat, noise, dust, chemicals, infectious agents,
odor, humidity, cold, and time pressure (1 ¼ little stress, 2 ¼ moderate stress, 3 ¼ high stress)
ni no indication

findings. Spirometry revealed no abnormalities in
the morning of the examination day.
Workers The workers were recruited among the
staff of selected sewage treatment plants working
at the chamber filter press or sand trap. Only
workers fit for work on the day of examination
were asked to participate in the study. All workers
were men, 3/8 workers were current and 2/8 were
former smokers. The mean age was 40.9 (range
24–56) years. The BMI was 28.4 (range
23.6–37.6) kg/m2, with one worker being obese
according to the definitions of WHO. The mean
working duration on the sewage treatment plant
was 13.4 (range 4.0–33.8) years and the workers
indicated to work >6 h/day in the chamber filter
press hall (Table 2). Beyond the work on sewage
treatment plants, only two workers reported a
previous occupational exposure to airborne
biological hazards (agricultural work or sewage
sludge treatment of about 7 years’ duration) and
one worker reported a former inhalation exposure
from mining of 6 years’ duration. In a
standardized questionnaire, the workers judged
the stress as little-to-moderate by such aspects of
the work environment as heat, noise, dust,
chemicals, humidity, cold, and time pressure and
as moderate-to-high due to infectious agents and
odor. The majority of workers (7/8) indicated a
high work satisfaction. The evaluation of general
complaints and medical history showed no spe-
cific findings. The subjective ability to work was
rated slightly better (mean 2.3, range 1–4) than
the own health status (mean 2.8, range 2–4)
(1 ¼ very good, 2 ¼ good, and 6 ¼ insufficient).
In the morning of the examination day, spirome-
try resulted in regular findings in all but one
worker; the latter presenting with expiratory
flow limitation. Based on sIgE (in all but one
worker) and the medical history, one worker
was described as being atopic.
As the recruitment of sewage treatment plants
turned out difficult, the time interval between the
measurements in single plants was between 1 and
9 months. That made it possible that four students
(s4, s7, s8, and s9) could be examined on two
different plants and one student (s5) on three
plants. According to the usual size of the sewage
treatment plant, only two or three workers per
plant could be recruited for participation in the
study.
Table 2 Sociodemographic data of workers
Daily work in Work on Subjective evaluation of Work satisfaction Subjective ability to
SHSb (1 ¼ very chamber filter the sewage work environment on (single item; 1 ¼ little, work (school grades,
Age BMI Atopya good press hall plant sewage plantc (range 9–27 2 ¼ medium, 1 ¼ very good
Workers (years) (kg/2) (yes/no) 6 ¼ insufficient) (hours) (years) points) 3 ¼ high) 6 ¼ insufficient)
w1 38 28.1 No 4 >6 8.3 15 3 3
w2 43 27.8 No 4 >6 6.0 17 3 2
w3 38 26.2 No 2 >6 8.3 12 3 2
w4 24 23.6 No 2 >6 4.0 17 3 2
w5 49 29.1 No 3 >6 19.8 15 3 4
w6 44 26.3 Yes 3 >6 17.7 15 2 2
w7 56 37.6 No 2 <3 33.8 10 3 1
w8 35 28.7 No 2 Highly variable 9.5 16 3 2
All Mean Mean Yes: 2.8 > 6 h: 6/8 Mean 13.4 Mean 14.6 Mean 3 Mean 2.3
workers 40.9 28.4 1/8
a
Assessed by antibodies against inhalative allergens (IgE, sx1-test) and medical history
b
SHS Subjective health status
Adaptation to Occupational Exposure to Moderate Endotoxin Concentrations: A. . .

c
Working environment was judged by the workers regarding nine aspects: heat, noise, dust, chemicals, infectious agents, odor, humidity, cold, and time pressure (1 ¼ little stress,
2 ¼ moderate stress, 3 ¼ high stress)
93
94
2.3 Assessment of Exposure
Exposure to endotoxin was assessed by personal
sampling of aerosols from the inhalable dust frac-
tion using the PGP-GSP system (GSA
Messgerätebau GmbH; Ratingen, Germany).
Usually the sampling equipment was carried by
workers during their routine work at the sewage
treatment plant. Endotoxin was assessed using a
commercially available chromogen-kinetic Limu-
lus amoebocyte lysate (LAL) assay (COAMATIC
Chromo-LAL®; Chromogenix, Hemochrom) as
described elsewhere (Liebers et al. 2006; Rieger
et al. 2005). Endotoxin exposure was measured
twice per day for a period of 112–265 min (mean
166 min, median 178 min) and reached
concentrations between 4 and 121 EU/m3
(median 24.5 EU/m3) during routine work and
between 162 and 1039 EU/m3 (median 589.5
EU/m3) in areas with high exposure (Table 3). A
great variability of exposure on single working
days and some rather low endotoxin
concentrations during the day resulted from the
different tasks the workers performed during their
daily work, including periods without or with
only low specific exposure, such as administra-
tive or monitoring work in an office near the
chamber filter press hall (mean 131 min/day,
range 70–215 min/working day) or legal breaks.
There was no relevant exposure to airborne bac-
teria (mean 2.4  104 CFU/m3) or molds (mean
3.8  102 CFU/m3) during the working day. All
analyses were performed by the same laboratory
(Mikrobiologisches Labor Dr. Balfanz –
Dr. Lohmeyer GbR, Münster, Germany).
2.4 Assessment of Effects
To assess a broad spectrum of possible effects due
to the exposure to endotoxin, subjective
(symptoms – standardized questionnaire) and
objective (cellular and soluble inflammatory
markers in blood and NALF; lung function)
parameters were repeatedly gathered. The present
evaluation focuses on NALF with regard to
inflammatory markers.
M. A. Rieger et al.
2.4.1 Time Points for Data Assessment
During Exposure
According to the cross-shift approach, the
parameters were assessed at three time points
and intervals were chosen according to similar
studies (e.g., Doreswamy et al. 2011; Danuser
et al. 2000; Sigsgaard et al. 2000; Wang et al.
1997):
Time point A: morning, before exposure (i.e.,
6:30–9:30 am, depending on the single plant)
Time point B: afternoon, 8 h after start of expo-
sure (i.e., 2:45–4:00 pm, depending on the
single plant)
Time point C: next morning (usually about 16 h
after time point B, i.e., 6:45–10:00 am)
2.4.2 Nasal Lavage Fluid (NALF): Cellular
and Soluble Inflammatory Markers
Before the collection of NALF, the subjects were
asked to blow their nose. Then a syringe with a
suitable adapter containing 7 mL of sterile physi-
ological saline solution was introduced into a
nostril, followed by its aspiration; the procedure
was repeated five times (Raulf et al. 2015; Raulf-
Heimsoth et al. 2011; Raulf-Heimsoth et al.
2000). The residual volume of NALF was
recorded (3 mL being considered as good result),
sent overnight under standardized conditions to
the laboratory and centrifuged (5 min at
1300 rpm). The cell-free supernatant was divided
into aliquots and frozen at 80  C until further
analysis of soluble inflammatory markers. The
cell pellets were resuspended, and the total cell
number was determined using the Neubauer
counting chamber. For differential cell counts,
slides were prepared by cytospin (Cytospin 2;
Shandon Corp., Pittsburgh, PA) and stained with
May-Grünwald-Giemsa. Two independent
observers counted 200 cells on each slide under
light microscopy. The results were expressed as
the percentage and calculated as absolute cell
count based on the total cell number.
The inflammatory markers were determined in
the thawed cell-free supernatants. All samples
underwent only one freeze-thaw cycle. The
Adaptation to Occupational Exposure to Moderate Endotoxin Concentrations: A. . . 95

Table 3 Endotoxin exposure on sewage treatment plants during the days of examination assigned to study participants
EU/m3
Plant no. (date) Students Workers Two measurements/daya; average
1 s1 w1 90/46; 68.0
(23/4/2002) s2 w2 17/16; 16.5
s3 w3 12/7; 9.5
2 s4 w4 32/41; 36.5
(22/5/2002) s5 w4 32/41; 36.5
s6 w5, (w6)b 99/91; 95.0
s7 – 121/47; 84.0
3c s7 w7 10/7; 8.5
(12/2/2003) s8 w7 10/7; 8.5
s9 w8 4/5; 4.5
s5 w8 4/5; 4.5
(13/2/2003) s10 w7 58/13; 35.5
s5 w8 12/33; 22.5
s9 – 51/8; 29.5
4c,d s4 318/234; 276.0
(3/11/2003) s5 798/885; 841.5
s8 – (No personal sampling); 465.0e
s9 394/162; 278
(4/11/2003) s4 536/643; 589.5
s5 231/1039; 635.0
s8 – (No personal sampling); 659.7e
s9 866/643; 754.5
a
Exposure was assessed by personal sampling during routine work on sewage treatment plants (especially in chamber
filter press hall and sand trap) before and after lunch break, sampling equipment usually carried by the worker(s), duration
of measurements: 112–265 min (mean 166 min, median 178 min)
b
W6 no full exposure due to part-time work on the day of examination
c
Plant 3 and plant 4: measurements on two following days
s student, w worker, EU endotoxin unit
d
Plant 4: students only, no routine work but high exposure near the sand trap
e
Student s8 no personal sampling but exposure assigned as mean value of exposure of students s4, s5, and s9 (each
personal sampling)

following soluble markers were measured: inter-
leukin (IL)-1β, IL-5, IL-6, IL-8, soluble (s)CD14,
tumor necrosis factor (TNF)-α, eosinophilic cat-
ionic protein (ECP), NO, total protein content,
and albumin. The present evaluation of cellular
and soluble inflammatory markers in NALF
focuses on total cell count, proportion of
neutrophils, and the concentrations of total pro-
tein, IL-8, IL-6, TNF-α, IL-1β, and sCD14.
Therefore the methods of these selected soluble
markers are described in more detail.
Interleukins, sCD14, and TNF-α were
measured using purchased monoclonal “sand-
wich” enzyme immunoassay (EIA) kits. IL-1β
and TNF-α were measured with the respective
PeliKine™-Tool sets (CLB, Amsterdam,
Netherlands) in a standard range of 0.4–300 pg/
mL for IL-1β and a standard range of
1.4–1000 pg/mL for TNF-α. IL-8 was measured
with the OptEIA™ ELISAs (BDBiosciences
Pharmingen, Heidelberg, Germany) in a standard
range of 3–200 pg/mL and most of the samples
must be diluted to reach the standard range. IL-6
and sCD14 were determined with the DuoSet™
ELISA (R&D Systems, Wiesbaden, Germany) in
a standard range of 3–600 pg/mL for IL-6 and of
250–16,000 pg/mL for sCD14. Total protein con-
tent was determined according to the method of
Bradford (1976) with bovine serum albumin as a
standard solution (range 10–100 μg/mL). For all
96
soluble markers, concentrations below the limit of
quantification (LOQ) were set at 2/3 of LOQ.
2.4.3 Spirometry
Vital capacity (VC), forced vital capacity (FVC),
and forced expiratory volume in 1 s (FEV1) as
well as peak expiratory flow (PEF), peak inspira-
tory flow (PIF), and maximal instantaneous
forced expiratory flow when x% of the FVC
remains to be expired (MEFx%; 25%, 50%, and
75%) were measured pre- and post-shift, as well
as in the next morning with a portable spirometer
(Jaeger SpiroPro®; Viasys Würzburg, Germany)
using the software V1.0. Measurements were
performed according to the guidelines of the
American Thoracic Society (ATS 1995), but
repeated measurements were not possible at all
time points due to organizational constraints on
the sewage treatment plants. Reference values
were chosen according to Quanjer et al. (1993).
2.4.4 Questionnaires
The occupational and medical history (including
smoking), the current occupation, potential other
sources of exposure (e.g., leisure time activities,
or place of living), and the subjective stress
caused by the working conditions on the sewage
treatment plant were assessed by means of a
standardized questionnaire and by the subjective
evaluation of health and workability. In addition,
the study participants were asked to indicate the
extent of symptoms in line with a mucous mem-
brane irritation syndrome (e.g., symptoms from
the eyes, nose, and throat), general effects of
exposure to endotoxin conforming to an organic
dust toxic syndrome (e.g., limb pain, fever, and
feeling like getting a flu), or other symptoms (e.g.,
nausea and diarrhea) at time point A, B, and C,
using a Likert scale (0 ¼ not at all, 4 ¼ very
strong).
2.5 Sample Size for Single Research
Issues
1. Comparison of chronically and only acute
exposed subjects:
M. A. Rieger et al.
For this major goal of the study, 12 pairs of
workers and students were combined and
investigated on three sewage treatment plants
(see Table 4)
2. Variability of inflammatory markers without
specific exposure, possible dose-response rela-
tionship, and cumulative effects of the endo-
toxin exposure:
Five students (s4, s5, s7, s8, and s9) were
investigated without any specific exposure with
a time interval of at least 3 weeks after the last
exposure to endotoxin (s4, s5, s8, and s9:
3 weeks, s7: 9 months). This examination was
repeated twice each with a 1-week interval.
Dose-related changes of inflammatory markers
concentrations were investigated based on
20 values as some students participated repeat-
edly in the study. The students’ values were
grouped according to three categories of endo-
toxin concentration: <50 EU/m3 – eight students;
50–200 EU/m3 – three students; >200 EU/m3 –
four students. Possible cumulative effects could
be investigated in four students (s4, s5, s8, and s9)
who were exposed to high endotoxin
concentrations on plant 4 on two following days
(see Table 5).
2.6 Statistical Analysis
Data were expressed as median with min–max
range (tables) or stem and leaf plots (box plots)
illustrating the interquartile range (75th percentile
– 25th percentile) and minimum and maximum
(figures). As the concentration of inflammatory
markers in NALF showed no normal distribution,
logarithmic (total cells, total protein, IL-8, IL-1β,
and sCD14) or dichotomous values (neutrophils
categorized below/equal or above 40%, IL-6 and
TNF-α categorized as below/equal or above the
respective limit of quantification) were used for
statistical analysis comparing workers and
students (research issue 1). In this context, after
univariate analysis (logarithmic values: regres-
sion analysis and analysis of variance, with a
UNIANOVA procedure; dichotomous values:
Table 4 Cellular and soluble inflammatory markers in nasal lavage fluid (NALF) before exposure (time point A) in chronically exposed (workers) and so far not exposed
subjects (students). These data were assessed on plant 1–3 (only Day 1)
Students (n ¼ 10; n ¼ 12 values) Workers (n ¼ 8; n ¼ 8 values) Students vs. workersa
Number of values >LOQ (%; Number of values >LOQ (%; Univariate
NALF 100% ¼ 12) Median (range) 100% ¼ 8) Median (range) analysis GEE
Total cell count 12 (100%) 6.0 (4.0–12.0) 8 (100%) 10.0 (4.0–24.0) p ¼ 0.090 p ¼ 0.079
(104)
Neutrophils (%) 12 (100%) 78.8 (27.5–93.3) 8 (100%) 35.8 (0.0–94.0) ns/nt ns/nt
Total protein 12 (100%) 58.5 (18.1–160.3) 8 (100%) 61.3 (10.0–370.3) ns/nt ns/nt
IL-8 (pg/mL) 12 (100%) 147.1 (38.3–802.3) 8 (100%) 231.9 (59.1–1162.1) p ¼ 0.024 p ¼ 0.128
IL-6 (pg/mL) 1 (8%) 2.0 (2.0–43.2) 2 (25%) 2.0 (2.0–18.4) ns/nt ns/nt
TNF-α (pg/mL) 8 (67%) 1.6 (0.9–90.9) 5 (63%) 1.6 (0.9–20.2) ns/nt ns/nt
IL-1β (pg/mL) 10 (83%) 1.5 (0.3–6.3) 8 (100%) 2.1 (0.8–36.6) p ¼ 0.019 p ¼ 0.085
sCD14 (pg/mL) 12 (100%) 3461.0 (1657.8–12083.8) 8 (100%) 3907.6 (1266.3–12876.2) ns/nt ns/nt
a
This statistical analysis is based on the values of all three time points; at time point A, a marked difference was found for the total cell count only
ns no statistically significant difference between students and workers (i.e., p > 0.05)
Adaptation to Occupational Exposure to Moderate Endotoxin Concentrations: A. . .

nt no tendency regarding significant differences between students and workers (i.e., p > 0.100)
LOQ limit of quantification, GEE general estimating equation
97
 98

Table 5 Cellular and soluble inflammatory markers in nasal lavage fluid (NALF) in students at time point A (morning) and B (afternoon) according to endotoxin exposure during
the day
Time point A
(morning) Time point B (afternoon after endotoxin exposure)b
(n ¼ 10 students/ No specific exposurea < 50 EU/m3 (n ¼ 8 50–200 EU/m3 (n ¼ 3 > 200 EU/m3 (n ¼ 4/n ¼ 8 values); median (range)
n ¼ 31 values); (n ¼ 5 students/n ¼ 14 students/n ¼ 9 values)c; students/n ¼ 3 values)c;
NALF median (range) valuesd); median (range) median (range) median (range) Day 1c Day 2c
Total cellsa 8.0 (4.0–16.0) 4.0 (4.0–12.0) 8.0 (4.0–12.0) 8.0 (8.0–8.0) 4.0 (4.0–8.0) 4.0 (4.0–4.0)
104
Neutrophils 67.7 (0.0–93.3) 38.7 (0.0–84.7) 56.1 (9.0–94.0) 27.0 (16.0–81.3) 12.2 (1.0–55.0) 60.3 (7.0–66.0)
(%)
Total 73.6 (6.7–359.1) 74.7 (21.6–372.7) 37.5 (21.3–150.9) 42.9 (21.5–43.3) 49.6 (44.2–164.7) 47.9 (33.6–133.5)
protein (μg/
mL)
IL-8 246.7 373.6 (96.2–2982.0) 137.1 (38.8–572.2) 105.5 (89.0–414.2) 379.9 (169.2–636.9) 558.2 (51.7–1324.2)
(pg/mL) (38.3–1590.9)
IL-6 2.0 (2.0–43.2) 2.0 (2.0–33.6) 2.0 (2.0–26.2) 2.0 (2.0–2.0) 2.0 (2.0–2.0) 2.0 (2.0–5.7)
(pg mL)
TNF-α 0.9 (0.9–90.9) 1.7 (0.9–3.2) 1.5 (0.9–9.5) 6.6 (0.9–7.6) 0.9 (0.9–7.7) 0.9 (0.9–1.6)
(pg/mL)
IL-1β 2.8 (0.3–17.7) 1.2 (0.3–15.7) 0.9 (0.3–4.2) 0.6 (0.5–1.7) 1.8 (1.4–5.7) 1.4 (0.9–3.4)
(pg/mL)
sCD14 3548.8 3337.4 (1563.8–18374.0) 2968.0 (943.2–7415.5) 2495.9 (2046.5–3473.3) 3108.2 (1937.3–6620.7) 2426.8 (1943.2–7741.1)
(pg/mL) (275.3–13574.1
The number of values is based on repeated participation of students in the study (see Table 3)
a
No specific exposure: offices and lecture rooms of the university
b
Endotoxin exposure derived from the students’ stay on a sewage treatment plant (plant 1–3: escorting workers during their routine working day)
c
Concentrations per category: <50 EU/m3: median 17 EU/m3 (4–36 EU/m3), 50–200 EU/m3: median 84 EU/m3 (70–95 EU/m3); >200 EU/m3: Day 1 median 356 EU/m3
(162–885 EU/m3; mean: 465 EU/m3), Day 2 median 643 EU/m3 (231–1039 EU/m3; mean 659.7 EU/m3)
d
Student 8: did not participate on the third day without specific exposure
M. A. Rieger et al.
Adaptation to Occupational Exposure to Moderate Endotoxin Concentrations: A. . .
logistic regression) regarding the possible effects
either due to group (chronic or only acute expo-
sure), time points (A, B, and C), or the
interactions of group and time points, generalized
linear models (GLM) with generalized estimating
equations (GEE) were calculated, as some
students participated several times in the study.
In addition, the possible effect of group and plant
on the values gathered at time point A was
analyzed by univariate analysis (logarithmic
values) or logistic regression (dichotomous
values). Due to a limited number of subjects, we
additionally reported results ignoring the cluster-
ing caused by repeated measurements of the same
subjects. A two-sided significance level of 0.05
was chosen for all tests. According to small num-
bers, no statistical analysis was possible for
research issue 2. Data were analyzed using a
commercial SPSS software v22 and v23 (IBM
Co, Armonk, NY).
3 Results
3.1 General Findings
None of the healthy volunteers cancelled the
exposure on the sewage treatment plant. Only
after the exposure to a high endotoxin concentra-
tion on plant 4, some students indicated
symptoms in line with a mucous membrane irrita-
tion in the standardized questionnaire (increase
from “no symptoms” to “little” or “clear” irrita-
tion especially of the nose) which were no longer
prevalent on the next morning. One student
reported stomach ache, diarrhea, and nausea on
the morning of the second day of exposure on
sewage plant 4, but the symptoms eased during
the day. The sewage workers did not report any
changes of symptoms during the working day in
the standardized questionnaire.
3.1.1 Lung Function
All but one worker (w8) had normal values in the
spirometric examination before the exposure and
showed no clinically relevant changes after the
work shift. Worker w8 had a peripheral flow
limitation which seemed to increase during a
99
work shift. In addition, workers w3 and w4
showed a tendency of exposure-induced changes
in lung function, whose significance remains
unclear due to methodological reasons i.e. no
possibility of repeated measurements due to orga-
nizational constraints on the sewage treatment
plants. All students had normal spirometry results
at all time points and showed no signs of
exposure-induced changes in lung function.
3.2 Research Issue 1: Effects
of Chronic Endotoxin Exposure
The emphasis of the present study was on the
possible effects of the endotoxin exposure on
cellular and soluble inflammatory markers in
NALF. For the comparison of chronically
exposed workers with acutely exposed students,
12 pairs of workers and students based on data of
8 workers and 10 students could be examined on
3 sewage treatment plants. There were significant
differences between both groups in IL-8 (univari-
ate analysis: p ¼ 0.024, GEE: p > 0.128) and
IL-1β (univariate analysis: p ¼ 0.019, GEE:
p ¼ 0.085), each with a lower concentration in
students than workers. Similarly, total cell count
tended to be lower in students than workers (uni-
variate analysis: p ¼ 0.090, GEE: p ¼ 0.079).
TNF-α was the only inflammatory marker with a
significant concentration difference at the three
time points (univariate analysis: p ¼ 0.079,
GEE: p ¼ 0.016) with fewer values below LOQ
at time point A, compared to time point C. For
TNF-α, no significant effect or interaction with
the variable “group” could be detected. For the
other inflammatory markers, no effect or interac-
tion regarding “group” or “time point” could be
seen in the statistical analysis.
Concerning the possible effect of
chronic vs. acute exposure (variable “group”) or
the effect of plant on the concentration resulting
at time point A, a tendency toward a significant
difference was found for the variable “group”
only in the total cell count in NALF (p ¼ 0.064)
(lower concentration in students) and the variable
“plant” turned out to be significantly associated
with a higher concentration of this marker on
100
plant 3 than on plant 1 (p ¼ 0.034). Likewise,
logistic regression showed a significant effect of
the variable “plant” on TNF-α (p ¼ 0.027), with
more results below the limit of detection on plant
1 (five samples) than on plant 2 or 3 (only one
sample each). Concerning both markers, there
was no significant interaction between the
variables “group” and “plant”. For the other
inflammatory markers gathered at time point A,
no such effects of the two variables could be seen
in this univariate analysis. The concentrations of
inflammatory markers in students and workers at
time point A are given in Table 4.
3.3 Research Issue 2: Different
Patterns of Inflammatory
Markers in NALF After Exposure
to a Moderate Endotoxin
Concentration as Compared
to Nonspecific Exposure. Dose-
Response Relationship of Acute
Exposure, Including
a Cumulative Exposure
The inflammatory markers in NALF at time point
B for all students participating in the study were
analyzed according to the respective endotoxin
exposure during the day (plant 3: values of the
first day of exposure only) and compared to time
point B values without specific exposure and to
all values gathered before the exposure (i.e., time
point A) (Table 5). The values gathered from five
students at time point B, on 3 days without any
specific exposure (see Table 5 – column “no
specific exposure”), and the respective time
point A- and C-values (data not shown) showed
a great variability and there was no marked dif-
ference between A-, B-, and C-values. Due to this
great variability, comparison of NALF markers
gathered on sewage plant 4 with those gathered in
the same four students during 3 days without
specific exposure revealed no significant differ-
ence between both conditions (see Table 5). Only
the percentage of neutrophils at time point B1
tended to be lower after exposure to a high
M. A. Rieger et al.
endotoxin concentration than that without spe-
cific exposure (median: 12.2% (exposure >200
EU/m3) vs. 53.3% (no specific exposure)). Gen-
erally, concentrations of inflammatory markers
without specific exposure included most other
values gathered after endotoxin exposure in the
samples investigated in the present study (see
Table 5).
Concerning the pattern of inflammatory
marker concentrations, a higher endotoxin con-
centration seemed associated with an increased
concentration of total protein, IL-8, IL-1β, and
sCD14, and with a decrease in the percentage of
neutrophils and in the concentration of IL-6 in
descriptive analysis. Differently, comparing the
inflammatory marker concentrations in the after-
noon of the first and second days with high endo-
toxin exposure on plant 4 (mean endotoxin
concentration 465 EU/m3 (Day 1) and 659.7
EU/m3 (Day 2) (for details regarding the exposure
see Table 3 and legend to Table 5)), marked
differences between the values gathered at time
point B on the first and second days seemed
detectable regarding the total cells (lower on
Day 2), neutrophils (higher on Day 2), IL-8
(higher), IL-6 (higher), and sCD14 (lower) (see
last columns in Table 5 and Fig. 1). Compared to
the initial findings on the morning of Day 1 (time
point A1), the concentrations found on the morn-
ing of Day 2 (C1/A2) and Day 3 (C2) during the
cross-shift examination showed almost complete
recovery regarding the neutrophils (C1 and C2)
but slight changes regarding IL-6 and especially
IL-1β (C1 and C2), and an increase in variability
especially at time point C1 regarding the total
cells, total protein, TNF-α, and sCD14 (Fig. 1).
Due to a small sample size, combined with a
great variability in the concentration of all
markers investigated, no statistical analysis was
possible to compare the values at time point B,
according to different kinds of exposure or to
compare between all of the time points (A1 to
C2), or between the three time points of each day
(A1 to C1 and A2 to C2), with or without specific
exposure. The study’s main finding was the great
variability of inflammatory marker concentrations
in naïve subjects without specific exposure.
 12 100
NALF total cells (10e4)

NALF neutrophils (%)

80
10
60
8
40
6
20

4 0

A1 B1 C1/A2 B2 C2 A1 B1 C1/A2 B2 C2

15 14

NALF TNF-a (pg/mL)

12
NALF IL-6 (mg/mL)

10
10
8
6
4
5
2
0
A1 B1 C1/A2 B2 C2 A1 B1 C1/A2 B2 C2
Time of collection (Day 1 & Day 2) Time of collection (Day 1 & Day 2)

350
NALF total protein (mg/mL)

1000
300
NALF IL-8 (pg/mL)

250
200
100
150
100
50
0 10

A1 B1 C1/A2 B2 C2 A1 B1 C1/A2 B2 C2

6 12000
10000
NALF sCD14 (pg/mL)
NALF IL-1b (pg/mL)

5 8000
6000
4
4000
3
2
2000
1
0

A1 B1 C1/A2 B2 C2 A1 B1 C1/A2 B2 C2
Time of collection (Day 1 & Day 2) Time of collection (Day 1 & Day 2)

Fig. 1 Cellular and soluble inflammatory markers in nasal lavage fluid (NALF) in four students at time point A (before
exposure), time point B (afternoon of Day 1 and Day 2), and time point C (the morning after exposure during Day 1 and
Day 2); mean endotoxin exposure of 465 EU/m3 (Day 1) and 659.7 EU/m3 (Day 2)
102
4 Discussion
4.1 Study Design and Study Sample
In the present study, we chose a quasi-
experimental design to investigate the effects of
an endotoxin exposure lasting several hours on
inflammatory markers in NALF and to compare
chronically and acutely exposed subjects. We
chose not to expose the study participants in a
laboratory setting, but rather to assess the expo-
sure during routine work in selected areas of
sewage treatment plants in Germany (plant 1–3:
chamber filter press, plant 4: sand trap) where our
previous studies have shown high endotoxin
concentrations (>900 EU/m3) and at the same
time a rather low exposure to bacteria, molds,
and chemical hazards (Rieger and Diefenbach
2007; Steinberg 2001). Likewise, high
concentrations of endotoxin in certain areas of
sewage treatment plants (e.g., 729–887 EU/m3
during cleaning of the filter press) have been
reported in a Dutch study (Visser et al. 2006).
This particular exposure to endotoxin, combined
with a much lower concentration of other
biological hazards and without dust, is different
from the situation, e.g., in swine confinement
buildings used for similar investigations by
other authors (Wang et al. 1997).
The exposure to a “natural mixture” of endo-
toxin was different from laboratory settings where
usually lipopolysaccharide derived from one spe-
cific bacterium have been used for standardized
exposure (Danuser et al. 2000; Sigsgaard et al.
2000; Rylander et al. 1989). Generally, the use of
a working environment for an investigation of
possible effects of endotoxin exposure has
facilitated the recruitment of workers and a simul-
taneous examination of several study participants,
such as pairs of chronically and acutely exposed
subjects. Yet it lacks the possibility of a
standardized exposure, a feature of a laboratory
setting.
The advantage of the present quasi-
experimental design was a very realistic exposure
scenario and a rather good possibility to address
chronically exposed subjects. Yet, as the
M. A. Rieger et al.
recruitment of sewage treatment plants and
workers proved to be difficult, the time gap
between the single study days was rather long.
In addition, the endotoxin exposure on a single
sewage plant was very heterogeneous and, nota-
bly, it was much lower than expected as the
workers performed a variety of tasks during the
working day. Therefore, a fourth sewage treat-
ment plant was chosen, where only students
were exposed to high endotoxin concentrations
in a specific plant area on two following days.
Exposure Assessment We used personal sam-
pling to assess the amount of endotoxin inhaled
by the workers and the escorting students. The
assessment of endotoxin concentrations followed
the German specifications for standardized
measurements (Rieger et al. 2005; BGIA-
Arbeitsmappe 1989) using a commercially avail-
able chromogen-kinetic Limulus amoebocyte
lysate (LAL) test as described elsewhere (Rieger
et al. 2005). The LAL test has turned out suitable
for the comparison of exposure in different sew-
age treatment plants (Reynolds et al. 2005) and
the assessment of exposure during working
activities (Liebers et al. 2008).
Due to a surprisingly high proportion of
administrative and monitoring tasks and duties
related to the work in the chamber filter press
hall, the exposure due to routine work on plants
1–3 was lower than expected based on previous
studies (Rieger and Diefenbach 2007; Visser et al.
2006; Steinberg 2001), but it reflected a well-
known variability of exposure typical for
biological hazards (Liebers et al. 2008). The
concentrations we found clearly exceeded the
HBROEL recommended in 2010 in the
Netherlands (Health Council of the Netherlands
2010) only on plant 4 (median 356 EU/m3 (Day
1) and 643 EU/m3 (Day 2)) and ranged from
4 EU/m3 up to 95 EU/m3 (i.e., 0.4 ng/m3 up to
9.5 ng/m3) for the routine work on plants 1–3,
where pairs of workers and students were
investigated. Thus, in only 6/22 measurements
on plant 1–3, the endotoxin concentrations
exceeded the HBROEL recommendations of
50 EU/m3 at the time of the study (DECOS 1998).
Adaptation to Occupational Exposure to Moderate Endotoxin Concentrations: A. . .
Concerning condition “no specific exposure”,
we decided not to perform any exposure assess-
ment, as all office and lecture rooms, where the
students stayed during the 3 days of the investi-
gation, were in a good condition and had a good
indoor climate with no apparent risk of elevated
concentrations of endotoxin, as reported to occur
due to dampness in schools (Jacobs et al. 2014;
Jacobs et al. 2013).
Inflammatory Markers in NALF The noninva-
sive collection of NALF turned out well applica-
ble for this field study, and it has been applied in
many studies addressing the reaction to endotoxin
exposure in both field and laboratory settings
(Raulf et al. 2017; Sigsgaard et al. 2015; Wouters
et al. 2002; Douwes et al. 2000). According to the
concept of “united airway disease” in the occupa-
tional setting, inflammatory markers assessed in
NALF may not only point to the inflammatory
processes in the upper but also lower airways
(Quirce et al. 2010). As the students were trained
in collecting their NALF at the study onset, the
recovery rate and quality of specimens were gen-
erally high. The time points for NALF collection
(B about 8 h and C about 24 h after time point A)
were chosen according to other studies, where
NALF was gathered at 1 h (Danuser et al.
2000), 3 h (Sigsgaard et al. 2000), 6/7 h
(Doreswamy et al. 2011; Wang et al. 1997), or
23/24 h or later after endotoxin exposure
(Danuser et al. 2000). In most of those studies,
the exposure-related effect in inflammatory
markers has occurred 3 or 6 h after exposure,
with a recovery approximately 24 h after a single
exposure. Thus, time points B and C seemed to be
chosen properly in the present study.
Study Sample The constitution of pairs of
workers (i.e., chronically exposed subjects) and
acutely exposed healthy volunteers (students),
escorting the workers, was designed to compare:
(a) the baseline concentration of inflammatory
markers in NALF in the morning and (b) the
patterns of inflammatory markers in NALF after
exposure to endotoxin in the afternoon and on the
next morning in both groups. To our knowledge,
103
the latter question has not yet been addressed in
the occupational settings, where endotoxin is the
leading biological hazard, as opposed to studies
examining workers and naïve controls in occupa-
tional settings with more mixed biological
exposures, e.g., waste collection (Wouters et al.
2002) or pig farming (Sahlander et al. 2012; von
Essen and Romberger 2003). As the workers on
the sewage treatment plants were only men, we
recruited no female students. Thus, we could not
examine any gender effect despite this might have
been interesting according to the findings of a
Taiwanese study addressing the recovery in lung
function after cessation of exposure to organic
dust (Lai et al. 2015).
There were some differences in the
sociodemographic data of workers and students.
The workers were older, some were smokers, and
some showed signs of peripheral flow limitation
before (one worker) or after (three workers) the
work shift. This is in line with cross-sectional
studies in wastewater industries (Steinberg
2001), waste collectors (Raulf et al. 2017;
Schantora et al. 2015), compost workers
(Hoffmeyer et al. 2014), or agricultural workers
(Spierenburg et al. 2015) presenting with respira-
tory symptoms during their employment. How-
ever, with respect to the ATS recommendations,
methodological limitations of our spirometry
assessment have to be quoted. In general, there
was no possibility of repeated measurements due
to organizational constraints on the sewage treat-
ment plants. On the other hand, students were
only non-smokers and had normal spirometry
findings.
Atopy was determined in 1/8 workers and 4/10
students by objective parameters. Students rated
their general health better than workers
(1.4 vs. 2.8; 1 ¼ very good, 6 ¼ insufficient)
and indicated a higher stress than workers due to
some aspects of the working environment on the
sewage treatment plants, e.g., noise 2.7
(students) vs. 1.8 (workers) or humidity 2.4
(students) vs. 1.4 (workers) (1 ¼ little, 3 ¼ high
stress), whereas both groups indicated a similarly
high stress due to odor and infectious hazards.
104
The fact that the workers, despite partly
longstanding work on sewage treatment plants,
indicated a rather good health status and even
better ability to work in conjunction with a high
work satisfaction might point to a healthy worker
survival effect. Yet, one out of eight workers
showed pathologic findings in spirometry before
exposure, and he and two additional workers
showed a tendency of exposure-induced changes
in lung function at time point B. According to the
questionnaire, some students experienced slight
symptoms of mucous membrane irritation, but no
severe complaints. To contrast the findings
derived from the high exposure, some students
were also asked to participate in the assessment
during 3 days without specific exposure. Yet, no
data of chronically exposed subjects were avail-
able in either condition.
4.2 Research Issue 1: Effects
of Chronic Endotoxin Exposure
We aimed to answer the question of whether there
was a difference between chronically exposed
(workers of sewage treatment plants) and acute
exposed subjects (students) regarding the markers
in NALF before and after exposure to endotoxin
during a routine work on a plant. The results of
univariate analysis and GEE pointed to a certain
difference between students and workers, which
can be interpreted as the effect of chronic expo-
sure: workers showed higher concentrations of
some inflammatory markers in NALF (i.e., total
cell count, IL-8, and IL-1β) across all three time
points but without significant clinical symptoms
or findings due to the exposure. Focusing only on
the values gathered at time point A, this differ-
ence occurred in the total cell count. To summa-
rize, the effect of chronic exposure may result in
higher concentrations of inflammatory markers in
NALF. Yet, these findings are based on only a
small sample size (n ¼ 10 students, n ¼ 8
workers). In addition, regarding the comparison
of concentrations at time point A, the uneven
distribution due to repeated participation of
some students has to be accounted for, as this
M. A. Rieger et al.
was not reflected in statistical analysis (n ¼ 12
values from students, n ¼ 8 values from workers).
The difference between workers and only
acutely exposed students is similar to the findings
comparing current and former compost workers
to white collar workers. There, current workers
show a higher concentration of inflammatory
markers in induced sputum than former compost
workers or nonexposed subjects (Raulf et al.
2015). Similarly, before exposure, workers from
biofuel plants have a higher IL-8 concentration in
NALF compared to controls from conventional
fuel plants (Zheng et al. 2014). Pig farmers com-
pared to naïve controls show signs of local and
systemic inflammation associated with altered
innate immunity, which has been ascribed to
adaptation to chronic endotoxin exposure
(Sahlander et al. 2012; von Essen and Romberger
2003). So far, this effect has not been described
for workers in wastewater industries.
Other than in studies comparing farm workers
to naïve subjects in swine confinement (Sahlander
et al. 2012; von Essen and Romberger 2003), in
the present study there was no definable higher
concentration of inflammatory markers in
students than in workers at time point B or C, as
a result of exposure to endotoxin on plant 1, 2, or
3. This might be due to a rather low endotoxin
concentration associated with routine work on the
days of examination and the fact that the sample
size was too limited to detect differences between
workers and students.
4.3 Research Issue 2: Different
Patterns of Inflammatory
Markers in NALF After Exposure
to a Moderate Endotoxin
Concentration as Compared
to Nonspecific Exposure. Dose-
Response Relationship of Acute
Exposure, Including
a Cumulative Exposure
We found a great intra- and interindividual
variability concerning the concentrations of
inflammatory markers in NALF gathered in five
healthy students in the morning and afternoon of
Adaptation to Occupational Exposure to Moderate Endotoxin Concentrations: A. . .
3 days without any specific exposure. Due to a
small sample size we could not perform any sta-
tistical analysis, but descriptive analysis suggests
that there were no circadian differences in the
inflammatory markers investigated.
In the literature, we could not find any study
focusing on the variability of inflammatory
markers in NALF (or other specimens) in naïve
subjects without a specific exposure. Yet, hints
for a great variability can be driven from studies
where the results of (sometimes repeated)
measurements before exposure are reported. A
broad concentration range of inflammatory
markers in NALF has been reported, e.g., in for-
mer waste collectors, especially in never smokers
(Raulf et al. 2017), in former compost or white
collar workers in induced sputum (Raulf et al.
2015), or in NALF and exhaled breath condensate
in workers from conventional fuel plants (Zheng
et al. 2014). A repeated collection of NALF with-
out exposure (0 min, 30 min, and 2, 6, and 24 h)
in six healthy volunteers or pre-exposure in
16 healthy volunteers (Raulf-Heimsoth et al.
2010) has shown a broad range of inflammatory
markers comparable to the present findings.
Unfortunately, other authors have not reported
the range of repeated measurements of inflamma-
tory markers in healthy subjects, but just the
means or medians, e.g., in 20 healthy volunteers
with repeated measurements (Kitz et al. 2008) or
in 9 healthy volunteers with rather “similar”
concentrations (Michel et al. 1997). Therefore,
no comparison is possible between the present
results and those reported previously. Interest-
ingly, the median concentration of inflammatory
markers at different time points after saline instil-
lation reported by Danuser et al. (2000) do not
show such high variability as found in the present
study. Thus, our findings seem rather new as a
great variability of inflammatory markers has so
far been described explicitly only in response to
organic dusts (Sigsgaard et al. 2005).
In the present study, the concentration of
inflammatory markers without a specific endo-
toxin exposure in the students entailed the major-
ity of values collected after the exposure (see
Table 5). When possible dose-related changes in
the marker concentration were investigated by
105
comparing time point B values of the students
exposed to different endotoxin concentrations,
higher endotoxin concentrations seemed
associated with increased concentration of total
protein, IL-8, IL-1β, and sCD14, and with
decreased percentage of neutrophils and IL-6
concentration. A cumulative exposure to the
endotoxin concentration > 200 EU/m3 (Day 1:
median 356 EU/m3 (162–885 EU/m3; mean:
465 EU/m3), Day 2: median 643 EU/m3
(231–1039 EU/m3; mean 660 EU/m3)), for
about 8 h each, seemed associated with a differ-
ence in the concentration of time point B values
collected on the first and second day. The differ-
ence, particularly, were present in total cell count
(lower on Day 2), percentage of neutrophils
(higher on Day 2), and concentrations of IL-8
(higher), IL-6 (higher), and sCD14 (lower). Yet
these effects should be considered cautiously
against the background of a great intra- and inter-
individual variability of inflammatory markers in
NALF specimens collected at time point B in five
students on 3 days without any specific exposure,
as well as compared to a broad range of values
collected at time point A in the students.
Beside a small sample size combined with
great variability of concentrations, a reason for
the lack of a distinguished dose-dependent reac-
tion in NALF inflammatory markers in the pres-
ent study could be that the endotoxin exposure
during routine work (plant 1–3) or at specific
places known to result in a high endotoxin expo-
sure (plant 4) was too small to induce a detectable
response. So far, there have been several studies
where the dose-dependent changes of inflamma-
tory markers are investigated, mainly with the
goal to describe the effect of the lowest possible
level of endotoxin exposure in healthy volunteers
or chronically exposed workers (Danuser et al.
2000; Michel et al. 1997).
Soluble inflammatory markers in NALF are
the main outcomes in an experimental study of
Danuser et al. (2000), where seven subjects were
exposed to 0, 10, and 40 μg lipopolysaccharide
(LPS) by nasal instillation for 10 s. The instilla-
tion of 10 μg LPS increased IL-6 over 2-fold and
that of 40 μg caused an increase in nasal
symptoms and a 13-fold increase in IL-6
106
compared to saline treatment, with a peak at 6 h.
As there were no repeated measurements in single
individuals, the variability of the effects is
unknown. Yet, comparing the present findings
to those of that study suggests that the exposure
on plant 4 was high enough to produce a distinct
reaction in the NALF inflammatory markers.
Michel et al. (1997) published the results of a
study on the dose-response relationship to inhaled
endotoxin, focusing on clinical symptoms, blood
and induced sputum indices, and lung function in
nine healthy volunteers after a weekly bronchial
challenge with increasing doses of 0.5, 5, and
50 μg LPS. They noticed significant increases in
the number of neutrophils and monocytes in the
sputum after inhalation of 5 μg LPS, which was
significantly greater after 50 μg LPS, compared to
saline. No repeated measurements were reported.
Thus, variability of the effects in the nine
volunteers is unknown. The authors have set a
threshold value for no response in sputum inflam-
matory cells at 0.5 μg inhaled LPS, which
corresponds to the exposition for 10 h a day and
lung ventilation of 1 m3 per hour of air containing
50 ng LPS. This concentration corresponds to
about 500 EU/m3, which was exceeded by the
mean concentration the majority of students
were exposed to for about 8 h on plant 4 in the
present study. Yet, in the cited study, significant
effects in sputum inflammatory cells were
observed only at a concentration of 5 μg LPS
and higher, corresponding to a rather high expo-
sure of about 5000 EU/m3. Thus, exposure even
on sewage treatment plant 4 possibly was too low
to evoke distinct effects.
Concerning the effect of repeated exposure
(Day 1 and Day 2 on plant 4) at time point B on
the total cell count (lower on Day 2), neutrophils
(higher on Day 2), IL-8 (higher), IL-6 (higher),
and sCD14 (lower on Day 2), our present results
can be compared with a study of Doreswamy
et al. (2011). Those authors have reported a sig-
nificant increase in neutrophils and a decrease in
sCD14 and IL-8-concentration in NALF after a
6-hour experimental exposure (instillation of
2 μg LPS in the nostril) on the third day com-
pared to pre-exposure or 6 h after just a single
exposure in 15 healthy volunteers. Those results
M. A. Rieger et al.
are akin to the present findings concerning
neutrophils and sCD14, at variance with IL-8.
To wrap it up, there still seems to be a lack of
replicated inhalation studies in healthy volunteers
describing a consistent dose-response relation-
ship or cumulative effects due to endotoxin expo-
sure. That holds true for both work-related (Thorn
2001) and environmental exposures (Walser et al.
2015). Concerning the present study, it remains
unclear whether exposure to a far higher endo-
toxin concentration than found on plant 4 would
have induced changes in inflammatory markers
distinct from the greatly variable concentrations
found without any specific exposure in the
students. Inversely, it could be that the apparent
changes described in the experimental studies
cited would not come to sight if those studies
accounted for the variability of findings without
specific exposure.
5 Conclusions
We replicated previous findings of a great
variability of the concentration of inflammatory
markers in NALF in healthy subjects without
specific exposure at different time points, i.e.,
within 1 day and between several days. The con-
centration range was even broader than the appar-
ent reaction after exposure to a rather high
endotoxin concentration (mean: 465 EU/m3
(Day 1)/660 EU/m3 (Day 2)). Therefore, in our
study we could not replicate findings on a dose-
response relationship or cumulative effects
obtained by other authors.
Based on our findings, it seems necessary to
carefully assess individual ranges of concentra-
tion for single inflammatory markers without any
specific exposure before investigating a reaction
to a specific exposure such as endotoxin or other
hazardous substances. In addition, a great
variability of findings in single individuals
underscores the importance of a sample size,
which should be large enough for the use of
non-parametric tests (i.e., 20 subjects) or corre-
spond to the sample size indicated in the test-
specific table accounting for the predictable
great variability of findings (Wilcoxon 1945).
Adaptation to Occupational Exposure to Moderate Endotoxin Concentrations: A. . .
Concerning the adaptation to chronic endo-
toxin exposure, our results suggest a higher con-
centration of inflammatory markers in chronically
versus acutely exposed subjects. Yet, as in our
study the exposure during routine work was not
high enough to induce specific effects, further
research is needed to compare this pattern of
concentration changes of inflammatory markers.
Acknowledgments The study was financed by a grant of
the Lieselotte und Dr. Karl Otto Winkler Stiftung für
Arbeitsmedizin and own research funds of the institutions
involved (Department of Applied Physiology, Occupa-
tional Medicine and Infectiology, University of
Wuppertal, Germany, and Department of Allergology/
Immunology, Institute for Prevention and Occupational
Medicine, Institute of the German Social Accident Insur-
ance, Ruhr University Bochum, Germany). The work of
the Institute of Occupational and Social Medicine and
Health Services Research (IASV) is supported by an unre-
stricted grant of the employers’ association of the metal
and electric industry Baden-Württemberg
(Südwestmetall).
The authors thank the Emscher-Genossenschaft/
Lippeverband (EGLV) for giving access to their sewage
treatment plants, all volunteers for study participation, and
the involved staff of IPA, IASV, and University
Wuppertal, especially Monika Lahr, Heiko Diefenbach,
and Nicole Blomberg for technical assistance in collection
of specimens and on the sewage treatment plants
and Gerda Borowitzki, Susanne Freundt, and Sabine
Bernard for technical assistance in analysis of the NALF
samples.
Conflicts of Interest The authors declare no conflicts of
interest in relation to this article.
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Adv Exp Med Biol - Clinical and Experimental Biomedicine (2018) 3: 111–130
https://doi.org/10.1007/5584_2018_234
# Springer International Publishing AG, part of Springer Nature 2018
Published online: 29 June 2018
Effects of Low-Level Laser Therapy
in Autism Spectrum Disorder
Gerry Leisman, Calixto Machado, Yanin Machado,
and Mauricio Chinchilla-Acosta
Abstract
The study examined the efficacy of low-level
laser therapy, a form of photobiomodulation,
for the treatment of irritability associated with
autistic spectrum disorder in children and
adolescents aged 5–17 years. Twenty-one of
the 40 participants received eight 5-min
procedures administered to the base of the
skull and temporal areas across a 4-week
period (test, i.e., active treatment participants).
All the participants were evaluated with the
Aberrant Behavior Checklist (ABC), with the
global scale and five subscales (irritability/agi-
tation, lethargy/social withdrawal, stereotypic
behavior, hyperactivity/noncompliance, and
inappropriate speech), and the Clinical Global
Impressions (CGI) Scale including a severity-
of-illness scale (CGI-S) and a global improve-
ment/change scale (CGI-C). The evaluation
took place at baseline, week 2 (interim),
week 4 (endpoint), and week 8 (post-
procedure) of the study. The adjusted mean
G. Leisman (*)
Faculty of Social Welfare and Health Sciences, University
of Haifa, Haifa, Israel
National Institute for Brain and Rehabilitation Sciences,
Nazareth, Israel
Faculty ‘Manuel Fajrdo’, University of Medical Sciences,
Havana, Cuba
e-mail: g.leisman@edu.haifa.ac.il
C. Machado, Y. Machado, and M. Chinchilla-Acosta
Institute for Neurology and Neurosurgery, Havana, Cuba
difference in the baseline to study endpoint
change in the ABC irritability subscale score
between test and placebo participants was

15.17 in favor of the test procedure group.
ANCOVA analysis found this difference to be
statistically significant (F ¼ 99.34, p < 0.0001)
compared to the baseline ABC irritability sub-
scale score. The study found that low-level
laser therapy could be an effective tool for
reducing irritability and other symptoms and
behaviors associated with the autistic spectrum
disorder in children and adolescents, with pos-
itive changes maintained and augmented
over time.
Keywords
Autism spectrum disorder · Brain · Clinical
trial · Low-level laser therapy · Neuronal
networks · Photobiomodulation
1 Introduction
Autism spectrum disorder (ASD) is a complicated
syndrome characterized clinically by language
impairment, dysfunction in social engagement,
stereotypical movements and behaviors, and var-
ious cognitive deficits (McPartland and Volkmar
2012; South et al. 2012; Zappella 2012; Melillo
and Leisman 2010). In earlier work, we have also
noted an excess of high-frequency EEG,
suggesting an imbalance in the excitation-
111
112
inhibition homeostasis in the neocortex
(Machado et al. 2015).
Recent theories have proposed that deficits in
the integration of transient activity patterns in
diverse brain regions suggest a possible temporal
binding deficit in ASD (Leisman et al. 2012;
Testa–Silva et al. 2012; Vissers et al. 2012;
Melillo and Leisman 2009). Binding denotes the
effective incorporation of dissimilar neural infor-
mation in shaping an individual’s unified experi-
ence of the world and self, reportedly impaired in
ASD (Machado et al. 2015; McPartland and
Volkmar 2012; South et al. 2012; Zappella
2012; Melillo and Leisman 2010). We have
suggested that synchronous activity in the neo-
cortex may be critical for sensory integration and
is associated with integrated perceptual
experiences (Ding et al. 2017), selective attention
(Salo et al. 2017), and working memory (Griffiths
and Kumar 2017), all of which are part of the
myriad deficits reported in ASD. The evidence
from both animal and human studies
demonstrates that changes in neuronal synchrony
occur during all of these processes.
Augmented coherence values, reflecting
inefficiencies in brain networking noted in ASD,
likely reflect rigid neuronal networks that could
explain the typical manifestation of repetitive
behaviors, deficiencies in social interaction, and
communication and imagination, characteristics
of ASD (Machado et al. 2015; Gadow and
Drabick 2012; Zappella 2012).
Brain specialization does not fully explain
most aspects of brain function. Confirmatory
studies indicate that integrative functions over
multiple distributed systems and areas support
diverse cognitive processes such as visual recog-
nition (Behrmann and Plaut 2013), language
(Friederici and Gierhan 2013), cognitive control
(Power and Petersen 2013), emotion (Pessoa
2012), and social cognition (Barrett and Satpute
2013). The neural substrate enabling integration
of distributed neural information and the emer-
gence of a coherent cognitive state depends partly
on neural communication among specialized
brain regions existing within a network of interre-
gional projections (Fuster 1997; Goldman-Rakic
1988). The result is associated with large-scale
G. Leisman et al.
patterns of synchronization and information flow
(Brovelli et al. 2004) between connected
elements.
A significant literature exists on the ability of
low-level light therapy (LLLT) to penetrate the
skull. Low-energy light passes the skull and a
therapeutic effect likely exists. LLLT systems
employ the so-called quantum optically induced
transparency effect (Scherman et al. 2012; Weis
et al. 2010). This effect controls optical properties
of dense media enhancing transparency contrast
by a factor of five. Therefore, the skull, spine, or
joints can be penetrated even with moderate
intensity light reaching deep layers in muscles,
connective tissue, and even bone, enabling
transcranial effects of LLLT (Hamblin 2018;
Hiwaki and Miyaguchi 2018; Grover Jr et al.
2017; deTaboada et al. 2006).
LLLT achieves a therapeutic effect by
employing non-ionizing light, including lasers,
light-emitting diodes, or broadband light in the
visible red (600–700 nm) and near-infrared
(780–1100 nm) spectra (Shanks and Leisman
2018; deFreitas and Hamblin 2016). LLLT is a
nonthermal process occurring when a chromo-
phore is exposed to a suitable wavelength of
light. Chromophores are responsible for the
color associated with biological compounds
such as hemoglobin and cytochromes (Cotler
et al. 2015). With chromophore absorption of a
photon of light, an electron transits to an excited
state, with a physiologic effect occurring when
photons dissociate the inhibitory signaling mole-
cule nitric oxide (NO) from cytochrome-C-oxi-
dase, increasing the electron transport,
mitochondrial membrane potentials, and produc-
tion of mitochondrial products such as ATP and
NADH (deFreitas and Hamblin 2016; Wang et al.
2016, 2015). Other effects include the production
of reactive oxygen species (ROS) which activate
transcription factors, leading to the cellular pro-
liferation and migration (Farivar et al. 2014).
Based on these complex characteristics, LLLT
possesses physiologically modifying properties
associated with light characteristics such as wave-
length and irradiance, varied by exposure
parameters, such as energy density, irradiation
duration, and treatment frequency. On the basis
Effects of Low-Level Laser Therapy in Autism Spectrum Disorder 113

of the above, we investigated behavioral and cog- their parents seeking treatment for the symptoms
nitive changes in ASD as a consequence of the of ASD and from among the individuals who
delivery of red LLLT. responded to recruitment flyers and print ads.
Participants did not receive any financial compen-
sation for participation.
2 Methods
2.2 Inclusion and Exclusion Criteria
2.1 Participants
Each eligible participant satisfied each of the fol-
The study received an approval from the Helsinki
lowing inclusion criteria and none of the exclu-
Committee of the Institute for Neurology and
sion criteria. Males or females aged 5–17 years
Neurosurgery in Havana, Cuba, and was
meeting the criteria of the Diagnostic and Statis-
registered with the FDA (Identifier:
tical Manual of Mental Disorders, 4th Edition, for
NCT03379662). Informed written consent was
ASD, are diagnosed by a qualified medical pro-
obtained from the parent or guardian of each
fessional. Diagnosis was confirmed by the
participant after a full explanation of the
Autism Diagnostic Interview (ADI-R)
procedures to be undertaken. The informed con-
(Constantino et al. 2003). Each participant
sent forms, research protocol, and approvals are
demonstrated “irritable” behaviors such as
available for inspection in the Office of Research
tantrums, aggression, self-injurious behavior, or
Integrity at the Institute of Neurology and Neuro-
a combination of thereof. The participant’s Aber-
surgery in Havana, Cuba.
rant Behavior Checklist (ABC) irritability sub-
The participants consisted of 40 individuals
scale (Rojahn et al. 2003) score was >18; the
distributed at baseline in the fashion described in
Clinical Global Impressions Severity Scale
Tables 1, 2, and 3. Participants in both groups
(CGI-S) (Berk et al. 2008) score was 4
spanned 5–16 years, with the mean participant
(4 ¼ moderately ill). The participants’ therapeu-
age of approx. 8 years. A t-test for independent
tic/intervention plan had been consistent/stable
samples revealed no statistically significant dif-
for 3 months. They abstained from undertaking
ference in age between test and placebo group
new treatments during the study time.
participants (μa-μb ¼
0.13; t ¼
0.13;
Exclusion criteria were the following: history
df ¼ 38; p(two-tailed) ¼ 0.90 ( p > 0.05)).
of a primary or concurrent diagnosis of another
All study participants were recruited from
disorder, including neurological, use of a psycho-
among the investigator’s normal pool of new
tropic drug, or any participation in a research
and existing patients who voluntarily came with
study within 30 days prior to the commencement
of the current study.
Table 1 Age of study participants by the procedure group
Age (years) Testa (n ¼ 21) Placebo (n ¼ 19)
Mean 8.2 8.4 2.3 Baseline Concomitant
Standard deviation 3.0 3.2 Medications and Low-Level Light
Range 5–16 5–16 Therapy (LLLT)
a
Test group was the active treatment group
Table 4 presents the OTC and prescription
Table 2 Participant gender breakdown for the test and medications reported by participants at the base-
placebo group participants line evaluation used in the past by both test and
Gender Testa (n ¼ 21) Placebo (n ¼ 19) placebo group participants. No participant took
Male 16 14 any OTC medication during the study time.
Female 5 5 Table 5 lists the non-drug therapies routinely
a
Test group was the active treatment group used by both test and placebo group participants
114 G. Leisman et al.

Table 3 Participant ethnicity by the procedure group

Ethnicity Testa (n ¼ 21) Placebo (n ¼ 19)
Caucasian 3 4
Hispanic 9 1
African 8 13
Hispanic and African 1 0
Asian 0 1
a
Test group was the active treatment group

Table 4 Prior medication used to treat the symptoms of per week, 3–4 days apart at the investigator’s test
autistic disorder by the procedure group site. A pulsed laser of 635 nm with a power output
Medicationa Testb (n ¼ 21) Placebo (n ¼ 19) of 15 mW and a red 635 nm LED were used as
Carbamazepine 10 7 treatment and placebo, respectively.
Risperdal 9 8 Participants were required to maintain their
Valproate 2 3 regular medication schedule and treatment
Methylphenidate 3 1 regimens, as reported at the baseline evaluation,
Haloperidol 1 3 to treat symptoms related to autistic disorder
Chlorpromazine 1 2 throughout the study time. All of them complied
Risperidone 1 1 with this requirement.
Conductosa 1 1
Thioridazine 0 2
Levomepromazine 0 1
a 2.5 Outcome Measures
Some participants previously used several medications;
therefore, medication use by participant group adds up to
greater than the participant sample size per group; btest Pre-post treatment outcome measures included
group was the active treatment group the Aberrant Behavior Checklist (ABC). The
global score and the five subscale scores
to treat the symptoms of ASD at the time of entry consisted of (a) irritability and agitation,
to the study. Therapy used to treat symptoms of (b) lethargy and social withdrawal,
ASD was minimal and equal between test and (c) stereotypic behavior, (d) hyperactivity and
placebo group participants. noncompliance, and (e) inappropriate speech.
Medications routinely used by both test and The ABC is an informant rating instrument
placebo group participants at the time of study empirically derived by principal component anal-
enrollment for indications other than to treat the ysis. The global score for the ABC has not been
symptoms of autistic disorder are listed in psychometrically derived and is not statistically
Table 6. valid (Farmer and Aman 2012).
The second outcome measure consisted of the
Clinical Global Impressions (CGI) scale includ-
2.4 Procedure ing a severity-of-illness scale (CGI-S) and a
global improvement/change scale (CGI-C).
All the 40 participants completed the course Leucht and Engel (2006) have noted that both
according to the protocol. Twenty-one of them the CGI and the Brief Psychiatric Rating Scale
were randomized to the test (active treatment) (BPRS) are often employed in drug trials. Those
procedure group, and 19 were randomized to the authors have found that by calculating the effect
placebo procedure group. size and its 95% confidence interval for both
Participants received eight 5-min laser light continuous (standardized mean differences) and
applications to the base of the skull and temporal dichotomous (odds ratio) outcomes, there was no
areas with the Erchonia® EAL Laser (active or significant differences between tests, which
sham) across a 4-week period: two applications indicates a good inter-test reliability.
Effects of Low-Level Laser Therapy in Autism Spectrum Disorder
Table 5 Therapies used to treat the symptoms of autistic disorder by the procedure group
Therapy
Logopedic therapy for language
Hydrotherapy for hyperactivity
a
Test group was the active treatment group
Table 6 Baseline non-autism-related concomitant medication use by the procedure group
Medication (Indication)
Ketotifen (allergies)
Benadryl (allergies)
Meclizine (allergies)
Fluticasone (Asthma)
Vitamins A, B6, and C
a
Test group was the active treatment group
The ABC and CGI-S and CGI-C were
administered prior to treatment and then 2 (mid-
point) and 4 weeks (endpoint) of treatment and
finally 8 weeks after treatment (post-procedure
evaluation).
3 Results
3.1 Pre-procedure Measures
Aberrant Behavior Checklist (ABC) Mean and
standard deviation baseline (pre-procedure) of
the ABC global and subscale scores for test and
placebo group participants are provided in
Table 7.
T-tests for independent samples revealed no sta-
tistically significant differences in the baseline
ABC global score or in any of the five ABC
subscale scores between test and placebo group
participants ( p > 0.05) as shown in Table 8.
Clinical Global Impressions Severity (CGI-S)
Score Table 9 shows the CGI-S baseline score
for the test and placebo group participants. The
majority of participants in both groups had a
baseline CGI-S score of 6, corresponding to
“severely ill”.
115
Testa (n ¼ 21) Placebo (n ¼ 19)
2 2
1 0
Testa (n ¼ 21) Placebo (n ¼ 19)
3 1
0 1
0 1
1 0
0 1
3.2 Primary Efficacy Outcome
Analysis
The evaluation time point at which study success
was analyzed (study endpoint) was predetermined
as 4 weeks following baseline (pretreatment)
evaluation. The study was predetermined to be
considered successful if, using the intent-to-treat
(ITT) last observation carried forward (LOCF)
analysis, the primary endpoint was statistically
significant at the 0.05 level. The primary efficacy
outcome measure was predefined as the mean
change from baseline to endpoint (end of the
4-week treatment period) in the ABC irritability
and agitation subscale score, with a minimum
mean difference of
8.5 points between the test
and placebo groups.
The primary outcome measure was evaluated
for the following two populations:
1. Intent-to-Treat (ITT). ITT analysis included all
participants with valid measurements at base-
line, randomized to a procedure group.
Dropouts and terminated participants were
handled by carrying forward the last observa-
tion for all time points following the dropout
(LOCF). If a participant was not a dropout but
had no data in a relative day range, the last
observation prior to the time point being
analyzed was employed.
2. Per-Protocol Population. Per-protocol analy-
sis of results intended to corroborate
116 G. Leisman et al.

Table 7 ABC global and subscale scores by the procedure group

Testa (n ¼ 21) Placebo (n ¼ 19)
Mean  SD Mean  SD
Global score 30.5  6.7 29.6  6.8
Irritability and agitation 23.1  9.3 24.7  5.1
Lethargy and social withdrawal 13.7  4.1 12.3  5.6
Stereotypic behavior 32.8  7.8 36.9  7.9
Hyperactivity and noncompliance 7.2  3.1 6.4  4.0
Inappropriate speech 107.3  20.3 104.7  28.7
a
Test group was the active treatment group

Table 8 T-test results for differences in ABC global and subscale scores between the two procedure group
(test vs. placebo)
μa-μb t df p
Global score 0.95 +0.44 38 0.66
Irritability and agitation
1.64
0.68 38 0.50
Lethargy and social withdrawal 1.40 +0.91 38 0.37
Stereotypic behavior
4.09
1.64 38 0.11
Hyperactivity and noncompliance 0.77 +0.69 38 0.49
Inappropriate speech 2.60 +0.33 38 0.74

Table 9 CGI-S baseline scores by the procedure group

CGI-S Score Testa (n ¼ 21) Placebo (n ¼ 19)
7: Among the most extremely ill patients 3 0
6: Severely ill 14 15
5: Markedly ill 4 4
a
Test group was the active treatment group

conclusions drawn from the ITT analysis. This
analysis excluded the participants with major
protocol deviations and incompletes
(dropouts, non-compliant participants,
disqualified participants, etc.). All participants
in this study completed all study visits and
procedures and had all study measurements
recorded through to the study endpoint evalu-
ation. Therefore LOCF was not applied.
3.3 General Statistical Evaluation
For the ABC irritability subscale score, changes
were evaluated by analysis of covariance
(ANCOVA), with baseline measures as a covari-
ate and a procedure group (active or placebo) as
the main effect. Table 10 shows the observed and
adjusted means for the absolute change in ABC
irritability subscale score from baseline to end-
point, adjusting for the covariate of baseline ABC
irritability subscale scores.
The adjusted mean difference in the baseline to
endpoint change in the ABC between the test and
placebo groups was
15.2, almost double the
pre-established study success criteria of
8.5
points between the procedure groups, in favor of
the test group (F ¼ 99.3; p < 0.0001). Table 11
shows the observed mean and standard deviation
at baseline and endpoint for the ABC score and
change between the evaluations by the procedure
group.
The ABC score decreased 14.8 points for
participants in the test group, while it increased
by 0.32 points for participants in the placebo
group, resulting in an observed difference
between the two groups in the ABC irritability
Effects of Low-Level Laser Therapy in Autism Spectrum Disorder
Table 10 Change in ABC irritability subscale score from baseline to endpoint, adjusting for baseline ABC irritability
subscale score
ABC irritability subscale score
Observed mean
Adjusted mean
a
Test group was the active treatment group
Table 11 ABC irritability subscale score from baseline to endpoint by the procedure group
ABC irritability subscale score
Baseline
Endpoint
Change
a
Test group was the active treatment group
subscale score from baseline to endpoint evalua-
tion of
15.1.
T-Test for Two Correlated Samples A correlated
sample t-test was used to compare the difference
in the mean change in ABC score from study
baseline to endpoint. The evaluation for partici-
pant groups separately found the mean change to
be significant for the test group (t ¼ +10.6;
df ¼ 20; p < 0.001), but not for the placebo
group (t ¼
1.0; df ¼ 18, p ¼ 0.33).
T-Test for Independent Samples A t-test for two
independent samples, used to compare the mean
ABC score change from baseline to endpoint
between the test and placebo groups, found that
the mean difference of
15.1 was significant in
favor of the participants in the test group
(t ¼
10.1; df ¼ 38; p < 0.0001).
3.4 Secondary Efficacy Outcome
Analysis
3.4.1 Positive Responder Rate (PRR)
The evaluation of the difference in PRR between
the procedure groups was performed as a
predetermined secondary efficacy outcome mea-
sure to provide support for the primary efficacy
analysis. The PRR was defined as satisfaction
with both of the following: (a)  25% reduction
from baseline to endpoint in the ABC score based
117
Testa (n ¼ 21) Placebo (n ¼ 19)

14.81 0.32

14.83 0.34
Testa (n ¼ 21) Placebo (n ¼ 19)
Mean  SD Mean  SD
30.5  6.7 29.6  6.8
15.7  9.9 29.9  6.6

14.8  6.4 0.3  1.4
on the participant’s primary caregiver’s rating and
(b) CGI-I rating of 1 (very much improved) or
2 (much improved) at study endpoint as deter-
mined by the clinician’s evaluation.
Participants attaining a  25% reduction
from baseline to endpoint in the ABC irritability
subscale score. Eighteen (86%) of the 21 test
group participants attained a minimum of 25%
reduction in the ABC score from baseline to end-
point, while none of the 19 placebo participants
did. Table 12 shows the mean and standard devi-
ation of the percent (%) change in ABC scores
from baseline to endpoint for the test and placebo
groups.
ABC Irritability Subscale A t-test for two inde-
pendent samples was performed to compare the
mean difference in percent change in ABC scores
from baseline to endpoint between the test group
and placebo groups. The mean difference of
-52.5% was significant in favor of the test group
participants (t ¼- 9.79; df ¼ 38; p < 0.0001).
CGI-I Ratings Seventeen (81%) of the 21 test
group participants received a CGI-I rating of
1 or 2 at the study endpoint evaluation, while
none of the 19 placebo participants did.
Positive Responder Rate Successes Table 13
shows the number and percentage of test and
placebo group participants meeting the dual
criteria for PRR. There was a difference of 81%
118
Table 12 Percentage change in ABC irritability subscale score by the procedure group
% change in ABC irritability score
Mean  SD
a
Test group was the active treatment group
Table 13 Positive responder rate by the procedure group
Participants
Meeting success criteria (n, %)
a
Test group was the active treatment group
Table 14 Comparison of the proportion of positive responder rates between the procedure groups
2  2 table Positive responder rate (PRR)
Testa group 17
Placebo group 0 19
p < 0.00001 17
a
Test group was the active treatment group
in the proportion of participants who met the PRR
between procedure groups: 81% of test group
participants met the PRR criteria compared with
none in the placebo group. Fisher’s exact test for
two independent proportions was conducted to
compare the proportion of PRR between the
groups (Table 14). The difference in this pro-
portion was significant ( p < 0.00001). The
greater PRR from baseline to endpoint for the
test group relative to the placebo group also was
significant.
3.4.2 Aberrant Behavior Checklist (ABC)
Global and Subscale Scores
The evaluation of the mean change from baseline
to endpoint for each of the ABC global score and
the remaining four ABC subscale scores (leth-
argy/social withdrawal, stereotypic behavior,
hyperactivity/noncompliance, and inappropriate
speech) between the two procedure groups was
performed as a predetermined secondary efficacy
outcome measure to provide support for the pri-
mary efficacy analysis outcome. Table 15 shows
the mean and standard deviation of baseline and
endpoint ABC global and subscale scores and the
changes for the test and placebo participants
(Aman et al. 1985a, 1985b). The ABC global
and subscale scores were evaluated with
ANCOVA, with the baseline measure for each
G. Leisman et al.
Testa (n ¼ 21) Placebo (n ¼ 19)

51.0  22.4 1.5  6.6
Testa (n ¼ 21) Placebo (n ¼ 19)
17 (81) 0 (0)
Negative responder rate (NRR)
4
23
score as a covariate and the group as a main effect
(Tables 16 and 17). The F values were statistically
greater for the mean change for each of the ABC
scores between groups ( p < 0.0001). With
removal of individual differences in baseline
scores, the two adjusted means significantly dif-
fered ( p < 0.0001), and the means were signifi-
cant for the test but not for placebo group
participants (Tables 18 and 19).
A t-test analysis for two independent samples
comparing differences in the mean change in the
ABC scores from baseline to endpoint between
the two groups is provided in Table 20; the dif-
ference was significantly greater for the test than
placebo group participants.
The evaluation of the mean change in each of
the ABC global score and the five subscale sores
(irritability/agitation, lethargy/social withdrawal,
stereotypic behavior, hyperactivity/noncompli-
ance, and inappropriate speech) across the study
duration between the procedure groups was
performed as a predetermined supportive measure
providing support for the primary efficacy analy-
sis. The ABC scores were recorded at the follow-
ing four evaluation points: baseline
(pre-procedure), week 2 (interim procedure
administration), week 4 (study endpoint), and
week 8 (i.e., 4 weeks post-procedure). Table 21
shows these data for the test and placebo groups.
Effects of Low-Level Laser Therapy in Autism Spectrum Disorder 119

Table 15 ABC global and subscale scores from baseline to endpoint by the procedure group
Testa (n ¼ 21) Placebo (n ¼ 19)
Mean  SD Mean  SD
Global score Baseline 107.3  20.3 104.7  28.7
Endpoint 63.8  30.5 105.4  28.4
Change
43.5  19.1 0.7  2.6
Lethargy and social withdrawal Baseline 23.1  9.3 24.7  5.1
Endpoint 13.8  8.8 24.7  5.1
Change
9.3  5.8 0.1  0.2
Stereotypic behavior Baseline 13.7  4.1 12.3  5.6
Endpoint 8.2  5.1 12.3  5.6
Change
5.5  4.0 0.0  0.0
Hyperactivity and noncompliance Baseline 32.8  7.8 36.9  7.9
Endpoint 21.1  9.5 37.3  7.4
Change
11.7  7.5 0.4  1.1
Inappropriate speech Baseline 7.2  3.1 6.4  4.0
Endpoint 4.9  2.4 6.4  3.9
Change
2.3  2.3 0.0  0.3
a
Test group was the active treatment group

Table 16 Change in ABC scores from baseline to endpoint, adjusted for baseline score
ABC global and subscales Mean scores Testa (n ¼ 21) Placebo (n ¼ 19)
Global score Observed
43.53 0.74
Adjusted
43.60 0.82
Lethargy and social withdrawal Observed
9.29 0.05
Adjusted
9.44 0.22
Stereotypic behavior Observed
5.47 0
Adjusted
5.43
0.05
Hyperactivity and noncompliance Observed
11.67 0.37
Adjusted
11.95 0.68
Inappropriate speech Observed
2.29 0
Adjusted
2.21
0.08
a
Test group was the active treatment group

Table 17 Significance of the mean baseline to endpoint change in the ABC global and subscale scores between the test
and placebo groups
Observed difference Adjusted difference F p
Global score 44.3 44.4 99.0 <0.0001
Lethargy and social withdrawal 9.7 9.3 58.1 <0.001
Stereotypic behavior 5.5 5.4 33.8 0.000001
<0.00005
Hyperactivity and noncompliance 12.0 12.6 50.3 <0.0001
Inappropriate speech 2.3 2.1 19.3 0.000091
<0.0001

Figures 1, 2, 3, 4, 5, and 6 show the mean indicate that each ABC score in the test (active
changes in each of the ABC global and five sub- treatment group) decreased significantly from the
scale scores across the study duration by the pro- baseline level across the three evaluation points;
cedure group. The figures as well as Table 21 the decrease was progressively augmented over
120 G. Leisman et al.

Table 18 t-test analysis for the mean change in the ABC global and subscale scores for the test group participants
t df p
Global score +10.45 20 <0.0001
Lethargy and social withdrawal +7.34 20 <0.0001
Stereotypic behavior +6.34 20 <0.0001
Hyperactivity and noncompliance +7.14 20 <0.0005
Inappropriate speech +4.59 20 <0.0001

Table 19 t-test analysis for the mean change in the ABC placebo groups was performed as a
global and subscale scores for the placebo group predetermined secondary efficacy outcome mea-
participants
sure providing support for the primary efficacy
t df p analysis outcome. Table 24 shows the number of
Global score
1.25 18 0.23 participants by the CGI-S rating at baseline and
Lethargy and social withdrawal
1.00 18 0.33 endpoint for both groups of participants. All
Stereotypic behavior –0.0 18 –1.00
21 test (active treatment) participants showed a
Hyperactivity and noncompliance
1.44 18 0.17
one-category or greater improvement in CGI-S
Inappropriate speech 0.0 18 1.00
ratings from baseline to endpoint. In contradis-
tinction, the majority of placebo participants
Table 20 t-test analysis for the mean change in the ABC (17) showed no change in CGI-S rating, one
scores between the test and placebo groups demonstrated a worsening of one category, and
t df p another demonstrated an improvement of
Global score
10.01 38 <0.0001 one-category rating from baseline to endpoint. A
Lethargy and social
7.01 38 <0.0001 2  3 Fisher’s exact test for matched categorical
withdrawal data was conducted to compare the proportion of
Stereotypic behavior
6.02 38 <0.0001 participants whose CGI-S rating improved,
Hyperactivity and
6.92 38 <0.0005 showed no change, or worsened from baseline
noncompliance
to endpoint in the test and placebo groups
Inappropriate speech
4.32 38 <0.0001
(Table 25).
A difference in the proportion of participants
time, including a 4-week follow-up period during whose CGI-S rating changed one or more
which no further treatment occurred. Conversely, categories from baseline to endpoint between the
there was no such change in the placebo group. test and placebo groups was significant
One-way ANOVA and Tukey’s HSD post hoc ( p < 0.00001), with a greater proportion of test
analyses were performed to evaluate the mean group participants who had improved CGI-S
change in each ABC global and subscale scores ratings from baseline to endpoint relative to the
across the four study evaluation points in the test placebo group (Table 26). In both test and pla-
and placebo groups. Table 22 shows that the score cebo groups, all participants (100%) were rated at
improvement in each of the ABC subscales was baseline in the top three severity-of-condition
significant across almost all the comparative ratings, i.e., marked, severe, and most extreme.
evaluations in the test group. Conversely, score All placebo group participants (100%) retained
changes in the placebo participants were not sta- these top CGI-S ratings (categories 5–7 in
tistically significant (Table 23). Table 26) by week 8, while only 3 of the 21 of
active test treatment (14%) received the same top
3.4.3 Clinical Global Impressions ratings. This continuously progressive and sub-
Severity-of-Illness (CGI-S) Ratings stantial improvement for the test over placebo
The evaluation of the change in CGI-S ratings group participants was illustrated in Fig. 7 which
from baseline to endpoint between the test and indicates the percentage of participants who
Effects of Low-Level Laser Therapy in Autism Spectrum Disorder 121

Table 21 ABC global and subscale scores across the study duration by the test and placebo groups
Testa (n ¼ 21) Placebo (n ¼ 19)
Mean  SD Mean  SD
Global score Baseline 107.3  20.3 104.7  28.7
Week 2 89.3  24.1 110.0  19.4
Week 4 63.8  30.5 105.4  28.4
Week 8 48.8  25.1 105.3  31.6
Irritability Baseline 30.5  6.7 29.6  6.8
Week 2 23.4  7.4 29.9  7.5
Week 4 15.7  9.9 29.9  6.5
Week 8 11.8  10.0 29.9  6.6
Lethargy and social withdrawal Baseline 23.1  9.3 24.7  5.1
Week 2 18.2  9.3 24.7  5.1
Week 4 13.8  8.8 24.7  5.1
Week 8 10.7  9.8 24.7  5.1
Stereotypic behavior Baseline 13.7  4.1 12.3  5.6
Week 2 11.6  4.4 12.3  5.6
Week 4 8.2  5.1 12.3  5.6
Week 8 6.9  5.1 12.6  5.7
Hyperactivity and noncompliance Baseline 32.8  7.8 36.9  7.9
Week 2 30.3  8.2 36.7  7.8
Week 4 21.1  9.5 37.3  7.4
Week 8 16.0  10.5 37.3  7.4
Inappropriate speech Baseline 7.2  3.1 6.4  4.0
Week 2 5.8  2.6 6.4  4.0
Week 4 4.9  2.4 6.4  3.9
Week 8 3.5  2.2 6.5  3.9
a
Test group was the active treatment group

received the 5–7 categories of CGI-S ranking
across the study duration by the procedure group.
3.4.4 Clinical Global Impressions
Improvement/Change (CGI-C)
Ratings
The evaluation of CGI-C ratings at study end-
point between the procedure groups was
performed as a predetermined secondary efficacy
outcome measure to provide support for the pri-
mary efficacy analysis outcome. The number of
participants by the CGI-C rating at endpoint for
the test and placebo groups is shown in Table 27.
Twenty out of the 21 test group participants
showed some degree of improvement in the
autism-related symptoms at study endpoint
relative to baseline, with the majority (13) receiv-
ing a rating of “much improved”. Conversely, no
placebo group participant was rated as
demonstrating symptom improvement at end-
point relative to baseline, with the majority
(17) rated as “no change” and the remaining
2 as “minimally worse”. A proportion of
participants in both test and placebo groups
whose CGI-C rating at study endpoint, relative
to baseline, showed “improvement” (CGS-C
rating of 1, 2, and 3), “no change” (CGI-C rating
of 4), or “worsening” (CGI-C rating of 5, 6, and
7) in symptoms, evaluated with 2 x 3 Fisher’s
exact test, is shown in Table 28. A difference in
this proportion between the two groups was sig-
nificant ( p < 0.00001). There was a greater
122 G. Leisman et al.

Fig. 1 Aberrant Behavior 120

Checklist (ABC) global
score in the test (active 100
treatment) and placebo

Points
group participants 80

60

40
Baseline Week 2 Week 4 Week 8

Test Placebo

Fig. 2 Aberrant Behavior 35

Checklist (ABC) irritability 30
subscale score in the test
Points
(active treatment) and 25
placebo group participants 20
15
10
Baseline Week 2 Week 4 Week 8

Test Placebo

Fig. 3 Aberrant Behavior 30

Checklist (ABC) lethargy/
social withdrawal subscale 25
Points

score in the test (active

treatment) and placebo 20
group participants
15

10
Baseline Week 2 Week 4 Week 8

Test Placebo

Fig. 4 Aberrant Behavior 14

Checklist (ABC)
stereotypic behavior
12
Points

subscale score in the test 10

(active treatment) and
placebo group participants 8

6
Baseline Week 2 Week 4 Week 8
Test Placebo
Effects of Low-Level Laser Therapy in Autism Spectrum Disorder 123

Fig. 5 Aberrant Behavior 40

Checklist (ABC) 35
hyperactivity/

Points
noncompliance subscale 30
score in the test (active
25
treatment) and placebo
group participants 20
15
Baseline Week 2 Week 4 Week 8

Test Placebo

Fig. 6 Aberrant Behavior 8

Checklist (ABC) 7
inappropriate speech Points
subscale score in the test 6
(active treatment) and
5
placebo group participants
4
3
Baseline Week 2 Week 4 Week 8

Test Placebo

Table 22 One-way ANOVA and Tukey’s HSD post hoc follow-up results for the change in the Aberrant Behavior
Checklist (ABC) score across the study duration in the test (active treatment) group participants
ABC scale/subscale F p Tukey’s HSD
Global score 101.6 <0.0001 Changes between each possible evaluation
Combination was significant at p < 0.01
Irritability 114.5 <0.0001 Changes between each possible evaluation
Combination was significant at p < 0.01
Lethargy and social 49.6 <0.0001 Changes between each possible evaluation
withdrawal Combination was significant at p < 0.01, except week 4–8 which was
significant at p < 0.05
Stereotypic behavior 38.2 <0.0001 Baseline to week 2 significant at p < 0.05
All others significant at p < 0.01, except week 4–8 which was not
significant
Hyperactivity and 59.4 <0.0001 All significant at p < 0.01, except baseline to week 2 which was not
noncompliance significant
Inappropriate speech 26.9 <0.0001 All significant at p < 0.01, except week 2–4 which was not significant
124 G. Leisman et al.

Table 23 One-way ANOVA results for the change in Aberrant Behavior Checklist (ABC) score across the study
duration in the placebo group participants
ABC scale/subscale F p
Global score 0.4 0.789
Irritability 0.4 0.739
Lethargy and social withdrawal 1.0 0.399
Stereotypic behavior 1.0 0.399
Hyperactivity and noncompliance 2.4 0.081
Inappropriate speech 0.4 0.739

Table 24 Clinical Global Impressions severity-of-illness (CGI-S) ratings at baseline and endpoint by the procedure
group
Testa (n ¼ 21) Placebo (n ¼ 19)
CGI-S category Baseline Endpoint Baseline Endpoint
7: Most extreme of all autism patients 3 – – –
6: Severe 14 2 15 16
5: Marked 4 1 4 3
4: Moderate – 13 – –
3: Mild – 4 – –
2: Borderline – 1 – –
1: Normal – – – –
a
Test group was the active treatment group

Table 25 Fisher’s exact test for matched categorical data to compare the proportion of participants whose Clinical
Global Impressions severity-of-illness (CGI-S) ratings improved
2  3 table Improved Unchanged Worsened n
Testa group 21 0 0 21
Placebo group 1 17 1 19
Total 22 17 1 40
a
Test group was the active treatment group; p < 0.00001

Table 26 Clinical Global Impressions severity-of-illness (CGI-S) ratings across the study duration by the procedure
groups
Testa (n ¼ 21) Placebo (n ¼ 19)
CGI-S Baseline Week 2 Week 4 Week 8 Baseline Week 2 Week 4 Week 8
7: Most extreme 3 2 – 1 – – – –
6: Severe 14 1 2 2 15 15 16 15
5: Marked 4 15 1 – 4 4 3 4
4: Moderate – 3 13 9 – – – –
3: Mild – – 4 6 – – – –
2: Borderline – – 1 3 – – – –
1: Normal – – – – – – – –
a
Test group was the active treatment group; p < 0.00001
Effects of Low-Level Laser Therapy in Autism Spectrum Disorder 125

%
100

80

60

40

20

0
Test Placebo

Baseline Week 2 Week 4 Week 8

Fig. 7 Percentage of participants receiving a 5–7 rating in the severity-of-illness subscale of the Clinical Global
Impressions Scale (CGI-S) across the study duration

Table 27 Clinical Global Impressions improvement/change subscale (CGI-C) ratings at the study endpoint by the
procedure group
CGI-C category Testa (n ¼ 21) Placebo (n ¼ 19)
1: Very much improved 4 –
2: Much improved 13 –
3: Minimally improved 3 –
4: No change 1 17
5: Minimally worse – 2
6: Much worse – –
7: Very much worse – –
a
Test group was the active treatment group; p < 0.00001

Table 28 Fisher’s exact test comparing the proportion of participants with Clinical Global Impressions improvement/
change subscale (CGI-C) ratings of ASD symptoms at the study endpoint
2  3 table Improved Unchanged Worsened n
Test groupa 20 1 0 21
Placebo group 0 17 2 19
Total 20 18 2 40
a
Test group was the active treatment group; p < 0.00001

proportion of participants demonstrating primary efficacy analysis outcome. CGI-C ratings

“improvement” at study endpoint in the test than
placebo group, which is viably attributed to the
application of LLLT.
The evaluation of change in the CGI-C ratings
across the study duration by the procedure group
was performed as a predetermined supportive
analysis to provide additional support for the
were recorded across the following evaluation
points: after 2 weeks (interim procedure adminis-
tration), 4 weeks (study endpoint), and 8 weeks
(4 weeks post-procedure). Table 29 shows the
number of participants by CGI-C rating category
for the test and placebo groups. The CGI-C
ratings for the test (active treatment) participants
126 G. Leisman et al.

Table 29 Clinical Global Impressions improvement/change subscale (CGI-C) ratings across the study duration by the
procedure group
Testa (n ¼ 21) Placebo (n ¼ 19)
CGI-C rating Week 2 Week 4 Week 8 Week 2 Week 4 Week 8
1: Very much improved – 5 10 – – –
2: Much improved 9 12 8 – – –
3: Minimally improved 11 3 3 – – –
4: No change 1 1 – 18 17 17
5: Minimally worse – – – 1 2 2
6: Much worse – – – – – –
7: Very much worse – – – – – –
a
Test group was the active treatment group; p < 0.00001

indicated a continuous progressive improvement
in symptoms. As early as 2 weeks into the 4-week
procedure, 20 out of the 21 participants received
CGI-C ratings of improvement, relative to base-
line, with 11 showing “minimal improvement”
and 9 “much improvement”. By week 8, the pre-
sentation of autistic symptoms and behaviors was
reported as “very much improved” for almost half
(48%) of active treatment participants. Con-
versely, there was essentially no change in the
CGI-C ratings across the study duration for the
placebo participants, indicating no observable
symptomatic improvement.
4 Discussion
Baseline ABC irritability subscale scores
indicated that LLLT was significantly more effec-
tive than placebo in treating symptoms of ASD in
children and adolescents independent of baseline
ABC scores, which accounted for 6% of the vari-
ance in the ABC score change from baseline to
endpoint. The difference in the proportion of PPR
between the LLLT and placebo groups was sta-
tistically significant ( p < 0.00001).
Considering the baseline score as a covariate,
F values were statistically significant
( p < 0.0001) for the mean change from baseline
to endpoint between the LLLT and placebo
groups and within participant groups for both
ABC global and subscale scores. Removing indi-
vidual differences in the baseline score, the
adjusted means significantly differed
( p < 0.0001), supporting the conclusion that
LLLT is more effective than placebo in effecting
a positive change in the ASD symptoms.
All participants of the active treatment showed
a one-category or greater improvement in CGI-S
from baseline to endpoint, while 17 of the placebo
participants showed no CGI-S rating change. The
difference in the proportion of participants whose
CGI-S rating changed one or more categories
from baseline to endpoint between the two groups
was significant ( p < 0.00001). This difference is
attributable to the efficacy of LLLT compared
with placebo. All 21 active treatment participants
showed a one-category or greater improvement in
CGI-S from baseline to endpoint.
Twenty out of the 21 active treatment
participants showed some degree of improvement
in autism-related symptoms at endpoint relative to
baseline. The majority (13) received ratings of
“much improved”. No placebo group participant
demonstrated improvement in symptoms at end-
point relative to baseline. The majority
(17) demonstrated “no change”, and the
remaining 2 placebo participants rated “mini-
mally worse”. We found that the ABC global
and five subscale scores decreased progressively
and significantly from baseline across each of the
three successive evaluation points; the decrease
progressed over time, including a 4-week follow-
up during which no further LLLT occurred. Con-
versely, the placebo group demonstrated no sig-
nificant change across the study duration,
demonstrating the effectiveness of LLLT in
reducing ASD-associated symptoms.
For both test and placebo group, all
participants were rated at baseline in the top
Effects of Low-Level Laser Therapy in Autism Spectrum Disorder
three severity conditions. By week 8, all placebo
participants retained a 5–7 CGI-S score, while
only 3 of the 21 active treatment (14%) received
this rating, which speaks for a progressive
improvement in the symptom presentation across
the study time. As early as 2 weeks into the
4-week active treatment, 20 out of the 21 test
group participants received improved CGI-C
ratings, with 11 participants showing “minimal”
and 9 demonstrating “much improvement”. By
week 8, symptom presentation of ASD was
reported as being “very much improved” for
almost half (48%) of the test group participants,
whereas no significant change in the CGI-C rating
was noted for the placebo group participants.
There is a well-established literature on
photobiomodulation, supporting improvement in
dysfunctional neuronal activity with the use of
low intensity red and near-infrared (NIR) light
(Hiwaki and Miyaguchi 2018; Naeser et al.
2014). Ten-year experience in animal studies
has indicated that transcranial photobiomo-
dulation has a positive effect in animal models
of traumatic brain injury (Wu et al. 2012a; Oron
et al. 2007), Alzheimer’s (Wu et al. 2012b),
depression (Purushothuman et al. 2015), and
stroke (Hiwaki and Miyaguchi 2018; Lapchak
and deTaboada 2010; Oron et al. 2006), while
human studies have included traumatic brain
injury (Naeser et al. 2011), depression (Schiffer
et al. 2009), and stroke (Zivin et al. 2009). Fur-
ther, low-level light energy has been found safe
for humans in the stroke studies, providing a high
benefit-to-risk ratio, with no reported side effects
of LLLT.
Almost all aspects of cognitive function
require integration of widely distributed neural
activity. Network analysis of human brain con-
nectivity has reliably classified sets of neocortical
areas essential for supporting optimized neuronal
signaling and communication. Optimal “brain
hubs” exist in the networks of effective connec-
tivity, supporting the notion of the hub’s function
in a wide range of cognitive tasks and a dynamic
coupling in and across effective functional
networks. A high level of brain hub centrality
renders these networks vulnerable to dysfunction
and disconnection (van den Heuvel and Sporns
127
2013). Abnormal anatomical connectivity and
performance of hub regions have been
hypothesized to be associated with cognitive and
behavioral dysfunction in numerous neurological
and psychiatric disorders (Bullmore and Sporns
2012; Seeley et al. 2009). For example, analyses
of structural and functional connectivity in
schizophrenia have shown reduced frontal hub
connectivity (Fornito et al. 2012).
We have recently noted that altered functional
connectivity, i.e., synchronous brain activity,
might be associated with the deficits characteris-
tically found in ASD (Machado et al. 2015). Of
specific importance is the integrity of functional
connectivity in the default mode network (DMN),
a network active during inactive resting states,
and in cognitive functions linked to the
ASD-related social dysfunction. Assaf et al.
(2010) have found a decreased functional connec-
tivity between the precuneus/medial/prefrontal/
anterior/cingulate cortices and default mode net-
work core areas, with the degree of functional
connectivity in these regions inversely correlated
with the ASD communication and social deficits.
These results support the hypothesis that default
mode networks’ under-connectivity contributes
to the core deficits seen in ASD.
Of particular interest is the association of
default mode networks with ASD (Buckner
et al. 2008). Numerous studies have found vari-
ous “hubs” with the effectiveness of the entire
network highly dependent on the hub status. The
most common ASD-related hubs are those
reported by Raichle et al. (2001) and Greicius
et al. (2003) being associated with the ventrome-
dial and dorsomedial prefrontal, posterior cingu-
late, precuneus, lateral parietal, and entorhinal
cortices (Raichle 2015). Assaf et al. (2010) have
examined three default mode sub-networks
obtained from the resting fMRI scans of
16 ASD individuals and 16 matched controls,
using independent component analysis. The
ASD individuals demonstrate a reduced func-
tional connectivity between the medial-prefron-
tal/anterior-cingulate/precuneus cortices and
other default mode sub-networks areas. The
degree of functional connectivity in these regions
128
is inversely associated with the severity of social
and communication deficits.
LLLT promotes cell and neuronal repair
(Dawood and Salman 2013) and brain network
rearrangement (Erlicher et al. 2002) in many neu-
rologic disorders identified with lesions in the
hubs of default mode networks (Buckner et al.
2008). LLLT facilitates a fast-track wound-
healing (Dawood and Salman 2013) as
mitochondria respond to light in the red and
near-infrared spectrum (Quirk and Whelan
2011). On the other hand, Erlicher et al. (2002)
have demonstrated that weak light directs the
leading edge of growth cones of a nerve. There-
fore, when a light beam is positioned in front of a
nerve’s leading edge, the neuron will move in the
direction of the light and grow in length (Black
et al. 2013; Quirk and Whelan 2011). Nerve cells
appear to thrive and grow in the presence of
low-energy light, and we think that the effect
seen here is associated with the rearrangement
of connectivity.
Reports are now emerging that LLLT and
photobiomodulation significantly upregulate
brain-derived neurotrophic factor (BDNF), a fac-
tor highly associated with dendritic sprouting,
neuroplasticity, and brain reconnectivity
(Hamblin 2018). In particular, photobiomo-
dulation influences the ubiquitous transcription
factor CREB (cAMP-response element-binding
protein) to increase BDNF and consequently den-
drite growth (length, spine density, and branching
in the hippocampal neurons). Functional mag-
netic resonance studies demonstrate the activity
modulation in intrinsic brain networks, including
default mode networks, likely to be dysfunctional
in a variety of conditions. All of that supports the
notion that LLLT can modify functional and
effective connectivity in the neocortex. Research
on photobiomodulation reveals the beneficial
effects of LLLT for a rapidly expanding list of
conditions, making this method increasingly
accepted by the mainstream medicine, even
though its mechanisms of action remains by far
an area of limited knowledge.
Acknowledgments The project was funded by Erchonia
Corp., Melbourne, FL, the producers of the equipment
G. Leisman et al.
employed in this study. The authors would like to thank
Ms. Elvira Walls for her assistance with the statistical
analysis and research methodology.
Conflicts of Interest None of the authors have any fidu-
ciary interests in the research reported herein or with the
source of funding.
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# Springer International Publishing AG, part of Springer Nature 2018
Published online: 4 July 2018

Epidemiology of Granulomatosis
with Polyangiitis in Poland, 2011–2015

Krzysztof Kanecki, Aneta Nitsch-Osuch, Paweł Gorynski,

Patryk Tarka, Magdalena Bogdan, and Piotr Tyszko

Abstract rate for GPA was 94%. In conclusion, the

Granulomatosis with polyangiitis (GPA) is a
form of vasculitis that affects small- and
medium-sized vessels in many organs. The
aim of the study was to describe the epidemi-
ology of GPA in Poland in 2011–2015, includ-
ing the incidence and prevalence rates. The
authors conducted a retrospective,
population-based study, using hospital dis-
charge records with GPA diagnosis. GPA inci-
dence was estimated on the basis of the data
from the Polish hospital morbidity study car-
ried out by the National Institute of Public
Health. The final study group consisted of
1491 patients (749 females, 742 males) who
were first time hospitalized with the diagnosis
of GPA. The average annual incidence of GPA
in Poland was 7.7/1,000,000 (95% CI,
4.1–11.4), and the point prevalence at the end
of 2015 was 36/1,000,000. A statistically
significant decrease in the GPA incidence
was noticed in this study. A 1-year survival
K. Kanecki (*), A. Nitsch-Osuch, P. Tarka,
and M. Bogdan
Department of Social Medicine and Public Health,
Medical University of Warsaw, Warsaw, Poland
e-mail: kanecki@mp.pl
P. Gorynski
National Institute of Public Health–National Institute of
Hygiene, Warsaw, Poland
P. Tyszko
Institute of Rural Health in Lublin, Lublin, Poland
incidence and prevalence rates of GPA in
Poland are similar to that reported in other
European countries. The study provides recent
epidemiological data on GPA in Poland,
which may be useful for comparisons with
other geographical regions.
Keywords
Autoimmune disease · Disease prevalence ·
Epidemiological data · Granulomatosis with
polyangiitis · Systemic vasculitis
1 Introduction
Granulomatosis with polyangiitis (GPA), previ-
ously known as Wegener’s granulomatosis, is a
systemic necrotizing vasculitis which affects
small- to medium-sized vessels and is related to
anti-neutrophil cytoplasmic antibodies.
Clinically, it can involve multiple systems,
commonly pulmonary system in 78%, upper
respiratory tract in 86%, and kidneys in 61% of
cases (Langford 2012). Elucidation of the
mechanisms underlying this type of vasculitis,
frequently accompanying lung diseases, is com-
plicated because of its rarity, which hampers the
undertaking of large-scale studies, difficulties in
the classification, and multifaceted clinical
presentations, which suggest the existence of sev-
eral etiologic pathways (Gibelin et al. 2011).

131
132
GPA can occur at all ages, and it has been
reported from infancy to old age.
Results from the population-based surveys
support the existence of differential international
variations in the occurrence of GPA. It has
become a well-accepted notion that the geo-
graphic distribution of GPA decreases with the
decreasing latitude, both north and south of the
equator. In the Nordic countries, the incidence or
prevalence of GPA is roughly two- to sevenfold
higher than that in the southern Europe. A similar
discovery was made in the southern hemisphere,
with a threefold higher incidence of GPA, con-
trolled for age, sex, and ethnicity, in the southern-
most part of New Zealand compared with the
northernmost part (O’Donnell et al. 2007). Ethnic
origin is also thought to play a part in the devel-
opment of GPA. In a multiethnic population of
France, GPA is twice as prevalent among popula-
tion of the European versus non-European
descent; and non-European cases of GPA are
rather rare (Mahr et al. 2004). Relatives of
patients with GPA are at increased risk of being
diagnosed with other autoimmune and inflamma-
tory diseases, indicating a shared susceptibility to
those diseases (Knight et al. 2010a).
HLA-DPB1*0401 is a GPA susceptibility allele,
and the population frequency of the allele may
help explain variations in the GPA incidence
described in the literature (Watts et al. 2015).
1.1 Incidence and Prevalence
of Granulomatosis
with Polyangiitis (GPA)
GPA is a rare disease. Therefore, collection of a
large population of patients to determine the inci-
dence and prevalence takes time, which makes
studies rather scarce. In a study conducted in
1990–2005, using of the General Practice
Research Database in the UK, 295 patients with
newly diagnosed GPA have been identified.
The overall annual incidence of GPA was 8.4
(95% confidence interval (95% CI), 7.5–9.4)
per million. In that study, the annual rate was
8.1 (95% CI, 6.8–9.6) per million and 8.8 (95%
CI, 7.4–10.3) per million in women and men,
K. Kanecki et al.
respectively. The incidence was stable throughout
the study period. There was an increase in the
annual prevalence from 28.8 per million in 1990
to 64.8 per million in 2005 (Watts et al. 2009).
Another British study has reported 462 GPA
cases diagnosed in 1997–2013 (Pearce et al.
2017). In that study, the overall estimate of GPA
incidence was 11.8 (95% CI, 10.7–12.9) per mil-
lion person-years. The incidence was 0.88 (95%
CI, 0.40–1.96) in children (aged <16 years) and
14.0 (95% CI, 12.8–15.4) in adults. The preva-
lence in 2013 was 134.9 (95% CI, 121.3–149.6)
per million population.
In a study from northern Germany in
1998–2005, GPA incidence ranged between
6 and 12 new cases per million per year (median
8.5) (Herlyn et al. 2008). In another German
study in 1994–2006, the prevalence of GPA rose
from 58 to 98 per million population (Herlyn
et al. 2014). In a Swedish study conducted until
30 June 2008, using hospital records and serology
database, the annual incidence for GPA was
estimated at 9.8 (95% CI, 7.4–12.2) per million
(Mohammad et al. 2009). In a retrospective Nor-
wegian cohort study, using hospital discharge
records, the annual incidence increased from 5.2
(95% CI, 2.7–9.0) during 1984–1988 to 12.0
(95% CI, 8.0–17.3) during 1994–1998 per mil-
lion. The point prevalence increased from 30.4
(95% CI, 16.6–51.0) to 95.1 (95% CI,
69.1–129.0) per million population (Koldingsnes
and Nossent 2000). A Finish study has shown an
annual increase in the incidence from 1.9 (95%
CI, 1.4–2.6) during 1981–1985 to 9.3 (95% CI,
8.1–10.6) during 1996–2000 per million (Takala
et al. 2008). In the region of Lugo of northwest
Spain, a study based on the data from 1988–2001,
coming from a single reference hospital and
regarding all patients aged 15 or older, has
reported the annual GPA incidence of 2.95
(95% CI, 1.44–6.05) per million population
(Gonzalez-Gay et al. 2003). In the Costa del Sol
region of southern Spain in 2010, the annual
incidence and prevalence were 2.1 and 15.8 per
million, respectively (Romero-Gómez et al.
2015). In northern Italy, the mean annual GPA
incidence increased from 1.7 during 1995–1999
to 3.4 per million per year during 2005–2009.
Epidemiology of Granulomatosis with Polyangiitis in Poland, 2011–2015
The point prevalence increased from 17.8 (95%
CI, 7.7–35.1) in 1999 to 34.3 (95% CI,
20.3–54.2) per million in 2009 (Catanoso et al.
2014). In a French urban multiethnic population,
GPA prevalence was estimated at 23.7 (95% CI,
16–31) per million adults (Mahr et al. 2004). In
Lithuania, the annual GPA incidence is reported
at 2.1 (95% CI, 1.1–4.1) (Dadoniene et al. 2005),
whereas in Turkey at 4.8 per million population
(Pamuk et al. 2016). Between 2000 and 2004,
GPA prevalence in the Australian Capital Terri-
tory and the surrounding rural region was
148.0 (95% CI, 125.1–173.9) per million, and
the disease-specific incidence was 8.4 per million
per year (Ormerod and Cook 2008). A 5-year inci-
dence of GPA in South Australia was 56.0 (95%
CI, 44.1–68.4) per million over the period
2001–2005 (Hissaria et al. 2008). A 5-year preva-
lence of GPA varies in New Zealand between
29–75 cases per million population (de Zoysa
2013). Using the National Inpatient Sample, the
largest all-payer inpatient database in the USA,
the annual hospitalization rate for patients with
the principal diagnosis of GPA increased by 24%,
from 5.1 to 6.3 per 1 million US population, in the
period 1993–2011 (p < 0.0001 for trend) (Wallace
et al. 2017). The annual incidence of GPA in
Japan is 2.1 per million adult population (95%
CI, 0.6–3.7 per million) (Fujimoto et al. 2011).
Another study performed in a southern hemisphere
region shows a 5-year GPA prevalence of
152 (95% CI, 117–186) per million population
(Gibson et al. 2006).
In Poland, the average annual GPA incidence
has been estimated at 4.9 per million in the gen-
eral population, 5.8 per million in the adult popu-
lation, and 1 per million in the population below
18 years of age (Kanecki et al. 2014). In view of
the paucity of epidemiological data, the present
study seeks to define a current burden of GPA in
Poland. The updated information would also help
compare recent trends in GPA morbidity in
Poland with those in other geographical regions.
There is a growing interest in the development
of surveillance ways for GPA, using the existing
resources such as hospital morbidity databases. In
a New Zealand study, a 5-year incidence of GPA
in South Australia over the period 2001–2005
133
was determined on the basis of the International
Classification of Diseases (M31.3) for the dis-
charge diagnosis using the Integrated South
Australian Activity Collection (Hissaria et al.
2008). A different study also shows that discharge
records might be a useful element in the epidemi-
ological evaluation of eosinophilic
granulomatosis with polyangiitis, a necrotizing
vasculitis (Kanecki et al. 2017).
2 Methods
This study was approved by the institutional
board for clinical studies. It is a retrospective,
population-based study based on a review of hos-
pital discharge files reporting the diagnosis of
GPA in Poland in 2011–2015. The incidence of
GPA was estimated on the basis of data obtained
from the Polish hospital morbidity study carried
out by the National Institute of Public Health.
Data on all inpatients, discharged and those who
passed away, excluding psychiatric and military
hospitals, were sent to the National Institute of
Public Health, on a monthly basis. The
anonymized data included the information on
hospitalization with ICD10-coded diagnoses,
date of admittance and discharge, birth date, gen-
der, and the place of residence. We evaluated the
patients with the first-time hospitalization for
GPA. The incidence rate was calculated using
the number of GPA patients and the
corresponding census data. In addition, demo-
graphic data for the general Polish population
were obtained from the Central Statistical Office
in Poland (CSO 2016).
2.1 Statistical Analysis
Categorical data were expressed as percentages.
Assuming the Poisson distribution of the cases
analyzed, 95% confidence intervals (CI) were cal-
culated. Nonparametric tests were used when the
normality assumptions were unmet. A p-value of
<0.05 defined statistically significant differences.
The incidence rate was calculated using the num-
ber of disease cases divided by the population
134 K. Kanecki et al.

figure. Linear regression was used for trend anal-
ysis. All analyses were performed using Statistica
software v2013 and WINPEPI software
(Abramson 2011).
3 Results
There were 6995 cases of hospitalization with
GPA diagnosis in 2011–2015, out of which 1491
cases represented patients with the first-time GPA
diagnosis. The majority consisted of adult patients,
1455 (97.5%;
18 years of age). The whole
patient group comprised 742 (49.8%) males and
749 (50.2%) females. The number of GPA patients
by gender per year is presented in Fig. 1. The
number of GPA patients in rural and urban regions
per year is presented in Fig. 2. There were signifi-
cant differences in the incidence rate between peo-
ple in urban (995 patients) and rural areas
(496 patients); 67% vs. 33%, p < 0.001.
The age distribution of patients at their first
hospitalization with GP diagnosis is presented in
Fig. 3. The median and mean age of all patients
were 55 and 52 years, respectively. The average
annual incidence of GPA in Poland was
estimated at 7.7 per million population (95%
CI, 4.1–11.4), and the point prevalence was
38.4 per million at the end of 2015. GPA inci-
dence decreased significantly over time in
Poland between 2011 and 2015 (12.9 vs. 6.7
per million, respectively; p < 0.001). Of note,
there was a significant seasonal decreasing trend
in the first-time GPA incidence from January to
December, presented in Fig. 4 (p < 0.01). There
were no significant differences in age according
to gender at the time of GPA diagnosis. In all
6,995 hospitalized cases of GPA, there were
108 deaths during the study period (54 females
and 54 males; mean age 60 years, range
19–86 years). Fifty-one out of the 108 deaths
were observed in patients hospitalized with
newly diagnosed GPA (27 females and

Fig. 1 Incidence of 300 Female

granulomatosis with
Number of Patients

250 Male
polyangiitis (GPA) by
gender in 2011–2015 200
150
100
50
0
2011 2012 2013 2014 2015
Calendar Year

Fig. 2 Incidence of 350 Urban

Number of Patients

granulomatosis with 300 Rural

polyangiitis (GPA) by place 250
of living in 2011–2015
200
150
100
50
0
2011 2012 2013 2014 2015
Calendar Year
Epidemiology of Granulomatosis with Polyangiitis in Poland, 2011–2015 135

Fig. 3 Age distribution of patients diagnosed with granulomatosis with polyangiitis (GPA) in 2011–2015

Fig. 4 Seasonal monthly incidence of granulomatosis with polyangiitis (GPA) in 2011–2015

24 males). During the study period, A 1-year
survival rate for GPA was 94%.
4 Discussion
On the basis of the inpatient discharge files, we
estimated that the average annual incidence of
GPA in Poland was 7.7 per million population.
This finding is comparable with data from other
European countries (Pearce et al. 2017; Romero-
Gómez et al. 2015; Catanoso et al. 2014;
Mohammad et al. 2009; Watts et al. 2009; Herlyn
et al. 2008; Takala et al. 2008; Dadoniene et al.
2005; Gonzalez-Gay et al. 2003; Koldingsnes and
Nossent 2000). Moreover, the average annual
incidence in 2011–2015 was higher than that
reported in a previous epidemiological study
from Poland (Kanecki et al. 2014). The GPA
incidence was also higher than that previously
reported in a recent British study (Pearce et al.
2017). The GPA prevalence in northern Germany
rose from 58 to 98 per million population in
1994–2006 (Herlyn et al. 2014). However in
136
another study, it amounted to 6–12 new cases per
million per year in the period of 1998–2005
(Herlyn et al. 2008). A high GPA incidence in
2011 in Poland and a significant drop in succes-
sive years may be related to environmental,
infectious, or other unknown factors.
Additionally, we noticed a significantly
decreasing trend in GPA incidence month-to-
month, with the highest rate of newly diagnosed
GPA in January. It is a long-held belief that the
etiology of systemic vasculitides could be infec-
tious. For vasculitides with a frequent pulmonary
involvement, this hypothesis is all the more rele-
vant because lung diseases are notably initiated
by airborne infections. The most compelling
observation to support the infectious cause
comes from the circumstantial evidence showing
that the onset of these diseases is preceded by
infectious events and from the studies showing
that GPA first becomes evident during the fall and
winter months, the seasons with frequent
respiratory infection epidemics. However, there
is a controversy regarding the concept of season-
ality concerning the onset of disease, with other
studies suggesting either no GPA seasonality
(Mahr et al. 2006a) or its increased occurrence
in the summer (Mahr et al. 2006b).
We found that there were significantly more
patients who lived in urban rather than rural
areas. Likewise, a greater GPA prevalence was
observed in urban areas in northern Germany
(Herlyn et al. 2014). An Australian study has
also reported a trend toward a higher incidence
of GPA in rural areas (Ormerod and Cook 2008).
In a multicultural population of New Zealand,
GPA predominates in Europeans (de Zoysa
2013). A long-standing exposure to silica may
be associated with GPA onset (Hogan et al.
2007). Other positive relationships were
suggested for GPA and farming, particularly
due to a contact with livestock and organic
solvents (Lane et al. 2003). The role of occupa-
tional exposures as determinants of GPA risk is
at variance with a recent study that demonstrates
no such relationship between (Knight et al.
2010b).
GPA occurs primarily during adulthood, but
its age-specific incidence rates are not uniformly
K. Kanecki et al.
distributed across age groups. Population-based
studies indicate that GPA may occur in a wide
range of age (Lane et al. 2003; Koldingsnes and
Nossent 2000). A wide range of age at the first-
time GPA hospitalization was also noticed in the
present study, the median and mean age of 55 and
52 years, respectively. The wide age ranges may
imply that stochastic events are at work in the
disease pathogenesis (Gibelin et al. 2011). In the
UK, median age of patients with newly diagnosed
GPA was 59 years (interquartile range
47–70 years), with a mean of 52 years (Watts
et al. 2009). In other studies, mean age at the
diagnosis was 64 years (Herlyn et al. 2014) and
55–65 years (Herlyn et al. 2008). In Finland,
mean age at the diagnosis was 53 years (Takala
et al. 2008), and in Norway it was 50 (range
10–84 years). The highest incidence rate occurred
in Norwegian men aged 65–74 (Koldingsnes and
Nossent 2000).
In the present study, male to female ratio was
0.99. Likewise, 51–52% of GPA patients in the
British studies were male (Wallace et al. 2016;
Watts et al. 2009). Yet another British study has
also reported that GPA incidence is lower in
females (adjusted incidence rate ratio ¼ 0.68;
95% CI, 0.56–0.81), with the greatest incidence
in those aged 55–69 years (adjusted incidence
rate ratio ¼ 6.8, 95% CI, 4.9–9.6; reference
group 16–39 years of age) (Pearce et al. 2017).
In Norway, 62% of GPA patients are male
(Koldingsnes and Nossent 2000), whereas only
49% of male patients have been reported in
Finland (Takala et al. 2008).
During the present study period, 108 deaths in
all GPA patients were observed, with a 1-year
survival rate for GPA was 94%. All the deaths
were observed in adults. Among the dead, there
were 51 patients with newly diagnosed GPA. In a
Swedish study, 5-year survival rate for GPA was
83% (Mohammad et al. 2009). In the UK, mortal-
ity was reported at 13.6% for the 1-year rate
(Pearce et al. 2017). In New Zealand, 1- and
10-year probability of survival in GPA patients
were 91% and 62%, respectively (Khan et al.
2012). A meta-analysis of GPA cases indicates a
similar mortality risk, with a standardized mortal-
ity ratio of 2.63 (95% CI 2.02–3.43) (Tan et al.
Epidemiology of Granulomatosis with Polyangiitis in Poland, 2011–2015
2017). A population-based British study suggests
that the survival rate of GPA patients has
improved considerably over the past two decades,
affirming the benefits of recent trends in the man-
agement of GPA and its complications (Wallace
et al. 2016). Using the National Inpatient Sample,
the largest all-payer inpatient database in the
USA, it has been reported that inhospital deaths
in patients with the principal diagnosis of GPA
declined by 73%, from 9.1% to 2.5% (p < 0.0001
for trend) from 1993 to 2011 (Wallace et al.
2017). Patients with GPA have a ninefold
increased risk of death in the first year of the
disease, attributed to infection, active vasculitis,
and renal failure. The risk is at its lowest between
the second and the eighth year of the disease,
although higher than that in the control popula-
tion. There is an increased mortality from the
ninth year onward which remains unexplained
(Luqmani et al. 2011).
This study has several limitations. The data-
base which was used provides general informa-
tion regarding hospitalization of GPA patients,
thereby allowing the authors to present an epide-
miological report. However, it did not allow
examining risk factors or other correlates of the
disease in individual patients. Another limitation
is that the evaluation relied on discharge files
from inpatient hospitalizations, which excludes
patients who were seen at the outpatient basis.
This inaccuracy may result in the underestimation
of the incidence rate. On the other hand, this type
of vasculitis is a systemic necrotizing disease with
multi-organ involvement and usually requires
treatment or advanced diagnostic procedures to
be performed at short-notice in the inpatient
setting. In addition, the first-time GPA diagnosis
in the inpatient discharge file may not necessarily
be the very time of the disease occurrence. A
longer duration of the observation may improve
the estimation.
5 Conclusions
The average annual incidence of GPA in Poland
was estimated at 7.7 per million population (95%
confidence interval, 4.1–11.4) and the point
137
prevalence at 36 per million at the end of 2015.
The incidence rate of GPA in Poland is similar to
the data reported by other European countries. A
higher incidence of GPA was reported in urban
than rural regions of Poland. During the study
period, a 1-year survival rate for GPA was 94%.
Trends in GPA incidence observed in this study
may be related to the environmental, infectious,
or other unknown factors.
Conflicts of Interest Authors declare no conflict of inter-
est in relation to this article.
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