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Morphological Changes in the Oral M
Morphological Changes in the Oral Muc

Viral Hepatitis C Patients: A Cross-Se
Viral Hepatitis C Patients: A Cross-Secti
Vahe Azatyan 1,*, Lazar Yessayan 1, Aelita Sargsyan 2, Anna Khachat
pharmaceutics
Article VaheMelanya
AzatyanShmavonyan
1,*, Lazar Yessayan 1, Aelita Sargsyan 2, Anna
5, Gayane Khachatryan
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Melanya Shmavonyan , Gayane Melik-Andreasyan , Kristina Porkshey
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Morphological Changes in the Oral Mucous

Article Membrane in
Department of Therapeutic Stomatology, Yerev 1
Viral Hepatitis C Patients: AChanges Cross-Sectional Study

Yerevan 0025, Armenia; lazyes@yandex.ru
Morphological in the Oral Mucous
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Department of Therapeutic Stomatology, Yerevan Sta
Article 1
Tuberculosis Research and

2
Response of the Endogenous Antioxidant Defense System

sargsyan.aelita@gmail.com
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Viral Hepatitis C Patients: A Cross-Sectional
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Department of Pathological Anatomy, Yerevan

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Melanya Shmavonyan , Gayane Melik-Andreasyan , Kristina Porksheyan 5 and Mikael Manrikyan 6 7 8

Dextran-Coated Iron Oxide Nanoparticles Laboratory
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Vahe Azatyan *, Lazar Yessayan , Aelita Sargsyan
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1 Department of Biochemistry and Molecular Biology, Faculty
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1. Introduction
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affiliations. www.mdpi.com/
1. Introduction
Int. J. Environ. Res. Public Health 2021, 18, x. https://doi.org/10.3390/xxxxx
Pharmaceutics 2023, 15, 552. https://doi.org/10.3390/pharmaceutics15020552 Hepatitis C virus (HCV) is the main cause of chronic liver dise
https://www.mdpi.com/journal/pharmaceutics
alence of 3% [1]. About 58 million people in the world live with c
Submitted for possible open access
Pharmaceutics 2023, 15, 552 2 of 18
properties [11–13]. It has been reported that the pharmacokinetic and biodistribution be-
havior of the IONPs is strongly correlated with the surface properties and physicochemical
properties of the nanoparticles [14]. In this context, many attempts were made in order to
improve the surface chemistry of IONPs and usually, those attempts involved the coating
and/or functionalization of IONPs with different polymers, such as dextran, chitosan,
starch and dextrin [3,15,16]. Amongst these, some have been successfully transferred to
clinical trials [17], and some have even been approved for use by the U.S. Food and Drug
Administration (FDA) [18] as contrast agents for MRI. One of the most successful polymers
used in the functionalization of IONPs has been dextran, mainly due to its own excellent
biological properties [19]. Dextran is used in the medical field as an antithrombotic because
it has the ability to reduce blood viscosity [20]. Therefore, in this context, dextran-coated
magnetic nanoparticles have been intensively studied over the years for their potential
biomedical applications [21,22]. Even though there have been drawbacks with the iron
oxide nanoparticles developed and approved for use over the years, there are still contin-
uous worldwide efforts made for the optimizations of a synthesis route that will allow
the obtaining of IONPs with reproducible properties and enhanced biological effects for
biomedical applications. Therefore, in the last years, due to the tremendous efforts made
by the scientific community, some iron oxide nanoparticles were approved by the FDA for
Article clinical use, Feraheme® was approved in the treatment of iron deficiency, and Combidex®
Morphological Changes in the Oral Mucous Membrane in

(U.S.) and Sinerem® (Europe) were also approved to be used as magnetic resonance imag-
ing (MRI) agents. In addition, Nanotherm® (MagForce) was approved for being used in

cancer treatment, and Lumirem® was given approval to be used as an imaging agent for the
oral gastrointestinal tract [23–27]. The biological effects of IONPs are dependent on various
factors, including size, surface characteristics, dose, solubility, and cell type [28–30]. The
Vahe Azatyaninteraction
1,*, Lazar Yessayan 1, Aelita Sargsyan 2, Anna Khachatryan 3, Tigran Ghevondyan 4,
of NPs with biological components (e.g., serum proteins) is another important
Melanya Shmavonyan factor that5, modulates
Gayane Melik-Andreasyan
their bioreactivity 6and , Kristina Porksheyan
toxic effects on cells7 and
[31].Mikael Manrikyan 8
Upon intravenous administration, IONPs are opsonized (adsorption of plasma pro-
teins on the NP10 s Department
surface) inofthe bloodstream
Therapeutic beforeYerevan
Stomatology, beingStatephagocytized by macrophages
Medical University (YSMU), 2 Koryun Str.,
and accumulatedYerevan in the 0025,
reticuloendothelial (RES) organs. This process promotes the ac-
Armenia; lazyes@yandex.ru
cumulation of NPs 2 Tuberculosis
in tissues, Research and Prevention
especially in tumorCenter,
ones,6/2 due Adonts
to theStr., 100 Apt., Yerevan
enhanced 0014, Armenia;
permeability
and retention (EPR) sargsyan.aelita@gmail.com
effect [32]. Despite coating with plasma proteins, NPs may be cleared
3 Department of Pathological Anatomy, Yerevan State Medical University (YSMU), 2 Koryun Str., Yer
through opsonin-independent mechanisms by macrophages residing in the liver and spleen.
0025, Armenia; ann_khachatryan@mail.ru
Previously it was shown that either dextran or exposed IONPs could be directly recognized
4 Laboratory of Histochemistry and Electron Microscopy, After Orbeli Institute of Physiology of Natio
by macrophages [33]. Multiple alterations
Academy of Sciences of Republic caused
Armeniaby(NAoS
exposure
RA), 22ofBrothers
macrophages
Orbeli Str.,to IONPs
Yerevan 0028, Armen
were previously reported [34,35]; however,
tigranghevondyan@yahoo.com the specific mechanisms of protective cellular
response induced 5 Department
in the presenceof Infectious Diseases,
of various YerevaninState
IONPs Medical University
macrophages (YSMU),
are not clear.2 Koryun Str., Yereva
Armenia;by
The effects induced sh_melaniya@mail.ru
these NPs on macrophage activation are a concern in terms of
6 National Center of Disease Control and Prevention, Ministry of Health (MoH), 12 Mkhitar Heratsi S
nanomaterial imaging, therapeutic efficacy, or systemic nanotoxicity. Excessive alterations
Yerevan 0025, Armenia; melikandreasyan@mail.ru
drive macrophages to cell death, which has been associated with the development of several
7 Department of Diagnostic Radiology, Yerevan State Medical University (YSMU), 2 Koryun Str., Yere
inflammatory diseases. Macrophages
are equipped with a complex network of protective
mechanisms against oxidative
8 Department of Pediatric Dentistryrelease
bursts (rapid of ROS against
and Orthodontics, Yerevan pathogens) to survive
State Medical University (YSMU), 2 K
during the inflammatoryStr., Yerevan 0025, Armenia; dr.manrikyan@mail.ru
process [36]. How these mechanisms help macrophages to fight
against oxidative * Correspondence:
stress inducedvahe.azatyan@gmail.com;
by IONPs is an intriguing Tel.: +374-91-326773
matter to reveal.
A deeper understanding of the interactions between NPs and macrophages is of great
Abstract: Background:
importance for researchers and clinicians The as
objective was
it can be to revealtoward
directed the most typical changes
generating in oral mucosa
biocompat-
Citation: Azatyan, V.; Yessayan, L.;
ible nanomaterials patients
withand compare
specific themcharacteristics
surface with those in HCV negative
fitting patients. Methods:
the biological environment. The study invo
In this HCVwe
context, patients
aimed and to100 patients without
characterize the HCV whoof
properties applied
newly tosynthesized
a dental clinic. The content of cy
dextran-
coated maghemite
IL-2,nanoparticles
IL-4, IL-10 and(ɤ-INF -Fe2 Oin3 the
NPs) oral fluid
and to was
study determined
the biologicalby ELISA. Buccal mucosa an
implications
of their
K.; Manrikyan, M. Morphological biopsies
interaction with passed histological
macrophages. Our examination.
focus was An
on immunohistochemical
cellular redox study
modulation of mucous me
after
exposure
Changes in the Oral Mucous to biopsies was performed using monoclonal mouse antibodies to CD3+ and CD20+. Results: Th
NPs, including oxidative stress induction and the activity of the endogenous
antioxidant
Membrane in Viral Hepatitis C patients
defense group included 96 (63.5% males), and the non-HCV group included 100 subjects
system.
Patients: A Cross-Sectional Study. males) with lesions of the oral mucous membrane. The lesions of lips and oral mucosa wer
Int. J. Environ. Res. Public Health 2022, frequent in HCV than in the non-HCV group—e.g., erosion (13.5% vs. 1%), cracks in the
18, x. https://doi.org/10.3390/xxxxx corners (42.7% vs. 0%), changes in the oral mucosa surface (89.6% vs. 3.0%), hemorrhages (78
Academic Editor: Paul B. 0%), etc. The pro-inflammatory IL-2 level was higher and anti-inflammatory IL-4 level was l
Tchounwou HCV patients compared with those in the non-HCV group. Conclusions: Morphological c
developed in the microvasculature both worsen the tissue trophism and accelerate the heali
differentiation into coarse-fibrous connective tissue. Immunohistochemical findings indicate
Accepted: 21 July 2022
crease in local humoral immune response.
0025,
Vahe Azatyan 1,*, Lazar Yessayan 1, Armenia; ann_khachatryan@mail.ru
Aelita Sargsyan 2, Anna Khachatryan 3, Center
6 National
Tigran Ghevondyan
of Disease 4, Prevention, Ministry of Health (MoH),
Control2 and
Melanya Shmavonyan , Gayane
5 Melik-Andreasyan 6 , Kristina Porksheyan
Academy of Sciences of Republic Armenia (NAoS
7 Viral Hepatitis
and Viral Hepatitis
Mikael C Patients:CA
Manrikyan Patients:
Cross-Sectional
A Cross-
Tuberculosis Research and Prevention Ce
4 Laboratory of Histochemistry and Electron Microscopy, After Orbeli Institute of Physiology of National
8
RA), 22 Brothers Orbeli Str., Yerevan 0028, Armenia;
7 Department of Diagnostic Radiology, Yerevan State
3 Department of Medical
Pathological University
Anatomy, (YSMUYe
tigranghevondyan@yahoo.com 0025, Armenia; tina129@mail.ru
5 Department of Infectious Vahe Azatyan 1, *, 8Vahe
Lazar Azatyan
Yessayan 1, *,, Lazar
1 Aelita Yessayan
Sargsyan 0025,1 2 Armenia;
, ,Str.,
Aelita
Anna ann_khachatryan@mail.ru
Sargsyan
Khachatryan 2 , Anna 3 , Tigran
Kha
1 Department of Therapeutic Diseases,
Stomatology,Yerevan State
Yerevan Medical
Department
State Medical University
of Pediatric (YSMU),
University Dentistry 2 Koryun
(YSMU), 4and 2Orthodontics,
Koryun Yerevan
Str., 0025, State
Yerevan Medical U
Laboratory of Histochemistry and Electro
Pharmaceutics 2023, 15, 552
Armenia; Melanya
sh_melaniya@mail.ru
Yerevan 0025, Armenia; lazyes@yandex.ru Shmavonyan Melanya
Str., Yerevan Shmavonyan
5, Gayane
0025, Melik-Andreasyan
Armenia; 5, Gayane Melik-Andreasyan
Academy
6, Kristina Porksheyan
of Sciences of
6, Kristina
Republic
3 of 18
7 and
Armeni
6 National Center of Disease Control and Prevention, Ministry of Health (MoH), 12 Mkhitar Heratsi Str.,
2 Tuberculosis Research and Prevention Center, * Correspondence:
6/2 Adonts Str., 100 vahe.azatyan@gmail.com;
Apt., Yerevan 0014, Tel.: +374-91-326773
Armenia;
sargsyan.aelita@gmail.com 5 Department of Infectious Diseases, Yerev
7 Department of Diagnostic Radiology, Yerevan State Medical 1 Department of Therapeutic 1 Department Stomatology,
of Therapeutic
Yerevan Stomatology,
State Medical
3 Department of Pathological Anatomy, Yerevan State Medical University
University (YSMU),
(YSMU), 22Koryun
Koryun Str., Yerevan
Yerevan The
0025, objective
Armenia; was toStr.,
Armenia;
Yerevan
Yerevan
reveal the most
sh_melaniya@mail.ru
lazyes@yandex.ru
0025, Armenia; typical chan
lazyes@yandex.ru
0025, Armenia;
2. Armenia;
0025, Materials tina129@mail.ru
Citation: Azatyan, andV.;Methods
Yessayan, L.;
ann_khachatryan@mail.ru patients Yerevan and 2compare themResearch
with 6 National Center of Disease Control and P
2 those in HCVResearch
8 Department of Pediatric Dentistry and Orthodontics,
4 Laboratory of
Sargsyan, Histochemistry
A.; Khachatryan,and A.; Electron Microscopy, After
Tuberculosis
State Medical
Orbeli Institute University
of
Tuberculosis
and(YSMU),
Physiology
Prevention
of 2negative
Center,and
Koryun
National
patients.
AdontsMetho
6/2Prevention Str.,Ce1
2.1. Sample Preparation HCV patients and 100 patients without HCV who applied to a dental clin
sargsyan.aelita@gmail.com Yerevan 0025,
sargsyan.aelita@gmail.com Armenia; melikandreasyan
Str.,
AcademyYerevan
Ghevondyan,
0025,
of Sciences Armenia;
T.; of
Republic Armenia
Shmavonyan, M.; (NAoS RA), 22 3Brothers Orbeli Str., Yerevan 7 Department 0028, Armenia; of Pathological
Diagnostic Radiology, Yer
IL-4, IL-10Department -Fe2of OinPathological
3 Department Anatomy, of Yerevan StateAnatomy,
Medical Ye
Un
* tigranghevondyan@yahoo.com
The dextran-coated maghemite IL-2,
Tel.: +374-91-326773
nanoparticles and(ɤ-INF 3 the
NPs) oral fluid
suspension was determined
was synthe- by ELISA.
0025, Armenia; 0025,
0025, Armenia;
5 Department
sized byofthe
K.; Manrikyan,Infectious
adapted Diseases,
M. Morphological Yerevan biopsies
coprecipitation State method
Medical passed in histological
University
agreement (YSMU),
4 Laboratory of Histochemistry
examination.
2 Koryun
with 8 anterior Str.,An
Department
4 Laboratory
immunohistochemical
Yerevan
studies
of 0025,
[37–41].
Pediatric Dentistry and stu
Or
and of Electron
Histochemistry
Microscopy, andAfter
Electro O
Abstract:
Armenia; Background:
sh_melaniya@mail.ru
Article Changes in the Oral Mucous The objective was to reveal
biopsies wasthe most
performed typical changes
using in
monoclonal oral
Str., mucosa
mouse
Yerevan 0025,in HCV
antibodies
Armenia; to CD3+
dr.manrikya and
Citation: Azatyan, V.; Yessayan, L.; 6 National
Academy of Sciences ofAcademy
Republic of
Armenia
Sciences (NAoS
of Republic
RA), 22
Armeni
Broth
patients andCenter
compareof Disease Control and Prevention, Ministry of Health (MoH), 12* Mkhitar Heratsi Str.,
in Viralthem with C those inpatients HCV negative patients. Methods: The study involved 96
2.2.Membrane
Physicochemical Characterization Correspondence: vahe.azatyan@gmail.com
Morphological Changes in the Oral Mucous Membrane
Sargsyan, A.; Khachatryan, A.; Hepatitis group tigranghevondyan@yahoo.com
included 96 (63.5% males), and
tigranghevondyan@yahoo.comthe non-HCV group inc
HCV patients and
Information
Patients: A 100 patients
regarding
Cross-Sectional without
Study. the HCV males)who
morphology applied
with lesions
5 to a
Department
and dental
of the
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mucous
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of content
membrane.
Department
Diseases,
dextran-coated
5 of
of cytokines
Yerevan The
Infectious lesions
State
maghemite Diseases,
Medical of lips
Yereva
Unive an
Ghevondyan, T.; Shmavonyan, M.; 7 Department of Diagnostic Radiology, Yerevan State Medical University (YSMU), 2 Koryun Str., Yerevan
IL-2, IL-4, IL-10 and ɤ-INF in the oral fluid was determined Armenia; by ELISA. Abstract:
sh_melaniya@mail.ru
Buccal Armenia;mucosa Background:
sh_melaniya@mail.ru
and gums The objective w
Viral Hepatitis C Patients: A Cross-Sectional Study frequent in HCV than in the non-HCV group—e.g., erosion (13.5% vs.
nanoparticles
Int. J. Environ. (
Res.
-Fe
Public2 O NPs)
Health
3 2022, were determined using transmission electron microscopy. The
Citation: Azatyan, 6 V.; Yessayan, L.;
National Center of Disease
6 National Control Centerand ofPrevention,
Disease Control
Ministry andofP
K.; Manrikyan, M. Morphological 8biopsies
TEM passed
18,
Department studies
x. histological
of Pediatricwere examination.
carried
Dentistry andout using An aimmunohistochemical
corners
Sargsyan, (42.7%
Orthodontics, CM20
Yerevan
A.; vs.
(Philips
State
Khachatryan, 0%), FEI,study
changes
Medical inpatients
Eindhoven,
University of oral
the mucous and
(YSMU),mucosa
The compare
membranesurface
2Netherlands)
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vs. 3.0%
YerevanA.;0025, Armenia; Yerevan
0025, Armenia; melikandreasyan
biopsies was performed
Str.,transmission
Yerevan 0025, Armenia;
electronusing monoclonal0%),
microscope mouse
(TEM) antibodies
etc. The equipped to CD3+
with a and
Lab6 HCV
CD20+. patients
instrument.Results: and
The 100
HCV patients without
Changes in the Oral Mucous Academic Editor: Paul B. Ghevondyan, T.;7pro-inflammatory
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Membrane in Viral Hepatitis C Vahe Azatyan
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group 1,*, vahe.azatyan@gmail.com;
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tina129@mail.ru
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the subjects
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non-HCV
Armenia;
Ghevondyan
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Conclusion
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males) NPs) Shmavonyan
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Int. J. Environ. Res. Public Health 2022, Abstract: Background:

Received: 28 May 2022 The
frequent in HCV than in the non-HCV group—e.g.,
an FEI Quanta Inspect objective was
microscope. to reveal the
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Armenia; oral
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Yerevan
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were HCV
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Changes in the Oral into
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patients and compare
Accepted:
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2022 with
21 Julyparticle those in Membrane
HCV negative patients.
* nanoparticles.
Correspondence: Methods: The study
vahe.azatyan@gmail.com;
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Correspondence: involved 96 96
vahe.azatyan@gmail.com
Tel.: +374-91-326773
State Medical University (YSMU), 2 ma
group included (63.5%
Sargsyan, A.; Khachatryan, A.; cornersdetermine
(42.7% vs.the changessize distribution
1 inDepartment
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in local humoral
of Therapeutic (89.6%
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immune 3.0%), hemorrhages
response.
Yerevan (78.1% vs. Kor
HCV Published:
patients and date
100 patients without HCV
Ghevondyan, T.; Shmavonyan,
Academic Editor: Paul B. M.; 0%), etc. TheThe mean
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Received: 28 May 2022 ◦Department of Pathological HCVAnatomy,patients Yerevan
and 100HCVState
patientsMedical
patients University
without
and 100
HCV (YSMU),
patients
who 2 Koryu
applied
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Changes in the Oral Mucous biopsies was
effectuated
claims performed
in 3 times
published
differentiation into coarse-fibrous using
maps at 25
and monoclonal
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institu-
Ghevondyan, T.;connectiveShmavonyan,
mouse tissue.
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The The HCV
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0025, mass Academic
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Accepted: 21 July 2022
Membrane in Viral Hepatitis C patients group
crease intional included
Thehumoral
changes
localaffiliations. 96
of (63.5%
immune
residual
Melik-Andreasyan, males),
4 response.
of ann_khachatryan@mail.ru
and
G.; Porksheyan, the non-HCV
dextran,
Melik-Andreasyan,
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Int. J. Environ.Note:
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is the main tional
cause
individuals of
affiliations.
chronic
[3,4]. The most common HCV extrahepatic manifest
Copyright: © 2022 by the authors. The
crease in local humoral macrophage RAW 264.7
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immune response.Tchounwou
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28 May people
2022
* Correspondence:
Medium (DMEM), each
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supplemented with Tel.: +374-91-326773
10% fetal bovine serum
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publication under MDPI stays
terms and neu-
con- Keywords: oral mucosa; viral hepatitis C; cytokines; morphology; differentiation into coarse-fibrous
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live withwww.mdpi.com/journal/ijerph
Copyright: © 2022 by the authors. K.; Manrikyan, M. Morphological
alence of 3% [1].
asAbout 58 million people in the worldmonoclonal
biopsies was performed1.using
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Submitted for possible open access Changes were used
in the Oral Mucous control. Introduction 1. Introduction
with 1.5 million newly infected people each year [1].
publication under the terms and con- Membrane in Viral Hepatitis C patients group included 96 (63.5% males), and the non-HCV group included 100
The extrahepatic
2.3.2. MTT Assay manifestations of viral hepatitisHepatitis C, caused C by
virusHCV (HCV)
Hepatitis
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main
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Health 2021,
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from ativecommons.org/licenses/by/4.0/). individuals
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individuals
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Int. J. Environ. Res. Public Health 2021, 18, x. medium each well
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Received:was28 May 2022
added. After 2 h differentiation
of incubation, the formazan crystals were dissolved in 250 µL finding
into coarse-fibrous connective tissue. Immunohistochemical
Accepted:2-propanol
21 July 2022 solution. The absorbance was measured at 595 nm using a Tecan multi-plate
Published: date
reader (TecanGENios, Grödic, Germany).
Int. J. Environ. Res. Public
Int.Health
J. Environ.
2021,Res.
18, x.
Public Health 2021, 18, x. https://doi.org/10.3390/xxxxx
Publisher’s Note: MDPI stays neu- Keywords: oral mucosa; viral hepatitis C; cytokines; morphology; immunohistoch
2.3.3. Quantification of Intracellular Iron Content
tral with regard to jurisdictional
The
claims in published level
maps of intracellular
and institu- iron was measured as previously described [42]. After
treatment
tional affiliations. with NPs, cells were trypsinized, washed with phosphate-buffered saline (PBS),
centrifuged and counted. The cell pellet was digested in 5 N HCl for 24 h at 37 ◦ C and
1. Introduction
then centrifuged at 2000× g rpm for 10 min. A volume of 50 µL of supernatant was mixed
with 50 µL 1%authors. Hepatitissolution
ammonium persulfate C virus (HCV) is thethe
(to convert main
Fe2+cause
to Feof
3+ chronic liver
ions) and 100disease,
µL with
Copyright: © 2022 by the
offor0.5possible
M potassium alence of 3% [1]. About 58 million people in the world live with chronic H
Submitted open access thiocyanate and shaken for 5 min in order to develop the red colored
with 1.5 million newly infected people each year [1].
publication under the terms and con-
The extrahepatic manifestations of viral hepatitis C, caused by HCV
ken about in the early 1990s [2]. They were shown to develop among 74% o
ativecommons.org/licenses/by/4.0/). individuals [3,4]. The most common HCV extrahepatic manifestations oc
iron-thiocyanate. The formed complexes were spectrophotometrically detected at 450 nm.

A standard curve with FeCl3 •6H2 O ranging from 0–250 µg Fe3+ /mL was used to quantify
the intracellular iron. The total amount of iron was related to the cell number.
2.3.4. Measurement of ROS Production

The level of reactive oxygen species (ROS) generated in RAW 264.7 cells was esti-
mated using the dye 20 ,70 -dichlorodihydrofluorescein diacetate (H2 DCF-DA; D6883, Sigma-
Aldrich, St. Louis, MO, USA). Only two NPs concentrations, selected after cell viability
evaluation, were investigated: 50 and 500 µg/mL. After treatment with NPs, the cells
seeded in a 6-well plate were washed with PBS and incubated at 37 ◦ C in the dark with a
volume of 1.5 mL of 10 µM H2 DCFDA solution for 30 min. Then, the cells were trypsinized,
centrifuged and resuspended in 2 mL of PBS solution. The fluorescence of dichlorofluo-
rescein (DCF) was spectrofluorimetrically recorded at 488 nm ex./515 nm em wavelength.
The RFU values were related to the cell number in each sample.
2.3.5. Preparation of Cell Lysate

RAW 264.7 cells were seeded in 75 cm2 culture flasks at a density of 2 × 106 cells/flask.
After treatment with NPs, the cell suspensions were centrifuged for 5 min at 1500× g rpm,
and the cellular pellets were washed and resuspended in PBS. Cell lysis was obtained through
ultrasonication 3 times for 30 s each, on ice, using a UP50H ultrasonicator (80% amplitude,
1 cycle) from Hielscher Ultrasound Technology (Teltow, Germany). The total extract was
centrifuged at 3000× g rpm for 10 min at 4 ◦ C, and the supernatants were collected. The protein
concentration in each sample was measured using the Bradford method and a calibration
curve of bovine serum albumin (BSA) ranging from 0 to 1.5 mg/mL protein.
2.3.6. Detection of Lipid Peroxidation Products

The level of malondialdehyde (MDA) in RAW 264.7 cells was assessed as an indicator
of lipid peroxidation via the thiobarbituric acid reaction, according to Dinischiotu et al. [43].
Briefly, a volume of 200 µL from cell lysate was mixed with 700 µL 0.1 N HCl and incubated
for 20 min at room temperature. After adding 900 µL of 0.025 M thiobarbituric acid (TBA),
the samples were incubated for another 65 min at 37 ◦ C. Finally, a volume of 400 µL of
PBS was added. The fluorescence of MDA (RFU) was recorded using a 520 nm/549 nm
(excitation/emission) filter. A calibration curve with 1,1,3,3-tetramethoxy propane in the
range of 0.05–5 µM was used to calculate the MDA concentration in each sample. The
results are expressed as nmoles of MDA/mg protein.
2.3.7. Quantification of Reduced Glutathione (GSH) Concentration

The intracellular GSH content was estimated in RAW 264.7 cells using the Glutathione
Assay Kit (CS0260, Sigma-Aldrich, St. Louis, MO, USA). Thus, cell lysates were precipitated
with 5-sulfosalicylic acid (1:1) and centrifugated at 10,000× g rpm for 10 min at 4 ◦ C. A
volume of 10 µL from the supernatant was mixed with 150 µL Assay buffer containing
5,50-dithiobis(2-nitrobenzoic acid) and incubated for 10 min at room temperature. The
5-thio-2-nitrobenozoic acid (TNB) formed was measured spectrophotometrically at 405 nm.
A calibration curve of GSH (3.125–50 µM) was similarly prepared. The GSH levels were
expressed as nmoles of GSH/mg protein.
2.3.8. Measurement of Antioxidant Enzymatic Activity

i. Superoxide dismutase (SOD, EC 1.15.1.1) activity was measured according to the
method of Paoletti et al. [44], which is based on NADPH oxidation by the superoxide
anions. Superoxide anions were generated from oxygen molecules by the addition of tri-
ethanolamine: diethanolamine (100 mM) buffer pH 7.4, containing MnCl2 -EDTA (100 mM
/50 mM) and 2-mercaptoethanol (100 mM) to each sample of cell lysate. After the addition
of 7.5 mM NADPH solution, the decrease of absorbance was followed at 340 nm for 10 min
at 37 ◦ C. A sample-free control was run in parallel. SOD activity was calculated in terms
of U/mg of protein, where one unit (U) of activity was defined as the amount of enzyme
required to inhibit the rate of NADPH oxidation of the control by 50%.
ii. Catalase (CAT, EC 1.11.1.6) activity was assessed by monitoring the disappearance
e of H2 O2 at 240 nm, according to the Aebi method [45]. The cell lysate was mixed with
orphological Changes in the Oral Mucous Membrane in

0.059 M H2 O2 and 0.1 M K2 HPO4 /KH2 PO4 buffer, pH 7.1, and the reaction kinetics was
recorded spectrophotometrically for 1 min. CAT activity was calculated in terms of U/mg
ral Hepatitis C Patients: A Cross-Sectional Study

protein, where 1 U was the amount of enzyme that catalyzed the conversion of one µmole
H2 O2 in 1 min.
iii. Glutathione peroxidase (GPX, EC 1.11.1.9) activity was assessed by the Beutler
Azatyan 1,*, Lazar Yessayan 1, Aelita Sargsyan
method [46] 2, Anna Khachatryan 3, Tigran Ghevondyan 4,
from cell lysate, using 7 mM tert-butyl hydroperoxide and 2 mM NADPH as
nya Shmavonyan , Gayane Melik-Andreasyan
5 substrates.6,The
Kristina Porksheyan
conversion of NADPH 7 andtoMikael
NADPManrikyan
+ was followed
8 by recording the changes
in absorption intensity at 340 nm for 5 min. One unit was defined as the amount of enzyme
1 Department of that catalyzes the conversion of 1 µmole of NADPH per minute. The activity was calculated
Therapeutic Stomatology, Yerevan State Medical University (YSMU), 2 Koryun Str.,
as U/mg
Yerevan 0025, Armenia; protein, using a molar extinction coefficient (εNADPH ) of 6.22 × 103 M−1 cm−1 .
lazyes@yandex.ru
2 Tuberculosis Research and Prevention Center, 6/2 Adonts Str., 100 Apt., Yerevan 0014, Armenia;
Article 2.4. Statistical Analysis
3 Department of Pathological Anatomy, Yerevan State Medical University (YSMU), 2 Koryun Str., Yerevan

Data were obtained from 3 independent experiments and expressed in terms of mean
± standard deviation. The results from each sample were represented on graphs as

percentages
Article tigranghevondyan@yahoo.com
from the control (100%). Statistical analysis was performed through GraphPad
Academy of Sciences of Republic Armenia (NAoS RA), 22 Brothers Orbeli Str., Yerevan 0028, Armenia;
Prism version 8.0.2 (GraphPad Software, Inc., San Diego, CA, USA) using two-way ANOVA

5 Department of variance
Infectious Diseases,
with theYerevan State Medical University
Geisser-Greenhouse (YSMU),
correction 2 Koryun
followed byStr., Yerevan
Sidak’s 0025,
multiple comparison
Vahe Azatyan
Armenia; 1,*, Lazar Yessayan 1, Aelita Sargsyan 2, Anna Khachatryan 3, Tigran Ghevondyan 4,
sh_melaniya@mail.ru
test. Values of * p < 0.05, ** p < 0.01, and *** p < 0.001 were considered statistically significant
6 National Center of Disease Control
Melanya Shmavonyan vs. and
5, Gayane Prevention, Ministry of Health
Melik-Andreasyan (MoH), Porksheyan
6, Kristina 12 Mkhitar Heratsi Str.,Mikael Manrikyan 8
7 and
ViralYerevan
Hepatitis
0025, Armenia;C Patients: A Cross-Sectional Study
(each sample control).
Department of 3.
7 Diagnostic
ResultsRadiology,
and DiscussionYerevan State Medical University (YSMU), 2 Koryun Str., Yerevan
0025, Armenia; tina129@mail.ru 1 Department of Therapeutic Stomatology, Yerevan State Medical University (YSMU), 2 Koryun Str.,
Vahe Azatyan
8 Department
1,*, Lazar Yessayan 1, Aelita Sargsyan 2, Anna Khachatryan 3, Tigran Ghevondyan 4,
Nanoparticles
of Pediatric Dentistry and in general,
Yerevan and maghemite
0025, Armenia;
Orthodontics, Yerevan State Medicalnanoparticles
lazyes@yandex.ru University (YSMU), in 2particular,
Koryun due to their
Melanya Str.,
Shmavonyan 5, Gayane Melik-Andreasyan
Tuberculosis
Yerevan 0025, Armenia; dr.manrikyan@mail.ru
adaptability, represent
2 a promising 6, Kristina Porksheyan 7 and Mikael Manrikyan 8
Research and Prevention
platform that Center,
could 6/2 Adonts
improve Str., 100
therapeutic Apt., Yerevan
delivery 0014,
andArmenia;
* Correspondence: vahe.azatyan@gmail.com; sargsyan.aelita@gmail.com
Tel.: +374-91-326773
controlled release in the area of different affected organs. The size of the particles and their
3 Department of Pathological Anatomy, Yerevan State Medical University (YSMU), 2 Koryun Str., Ye
stability 1are two very
Department ofimportant
Therapeutic parameters
Stomatology, that
Yerevancharacterize
State Medical the behavior
University of nanoparticles.
(YSMU), 2 Koryun Str.,
Abstract: Background: TheYerevan
objective 0025,
was Armenia;
to reveal ann_khachatryan@mail.ru
the most typical changes in oral mucosa in HCV
Thus, studies that contribute
0025, Armenia; to the understanding
lazyes@yandex.ru of these nanometric suspensions
4 Laboratory of Histochemistry and Electron Microscopy, After Orbeli Institute of Physiology of Nati
are
: Azatyan, V.; Yessayan, L.;
patients and compare very them with those Research
2 Tuberculosis
important. in HCV and negative patients.
Prevention Methods:
Center, 6/2 AdontsThe study
Str., 100 involved
Apt., Yerevan96 0014, Armenia;
n, A.; Khachatryan, A.; Academy of Sciences of Republic Armenia (NAoS RA), 22 Brothers Orbeli Str., Yerevan 0028, Arme
HCV patients and 100The patients without HCV
and who
morphology applied
structure
tigranghevondyan@yahoo.com ofto a dental
the clinic.
prepared The content of cytokines
dextran-coated maghemite nanoparti-
dyan, T.; Shmavonyan, M.; 3 Department of Pathological Anatomy, Yerevan State Medical University (YSMU), 2 Koryun Str., Yerevan
ndreasyan, G.; Porksheyan,
IL-2, IL-4, IL-10 and
cles ( ɤ-INF
-Fe O
2 3in the 5 oral
NPs) werefluid
Department was of determined
Infectious
investigated by by
TEM ELISA.
Diseases, Yerevan
(Figure Buccal
State mucosa
Medical
1a–d). The and gums
University
large (YSMU),
bright 2 Koryun
field TEMStr., Yerev
0025, Armenia;
Armenia; ann_khachatryan@mail.ru
sh_melaniya@mail.ru
rikyan, M. Morphological biopsies passed histological
image (Figure examination.
1a) revealed An thatimmunohistochemical
the dextran-coated study
iron of
oxidemucous membrane
nanoparticles
have a uniform
6 National Center of Disease Control and Prevention, Ministry of Health (MoH), 12 Mkhitar Heratsi S
s in the Oral Mucous biopsies was performed
particle using monoclonal
distribution
Academy of of mouse
nanometric
Sciences antibodies
of Armenia;
Republic sizes to
and
ArmeniaCD3+ andRA),
spherical
(NAoS CD20+. Results:
shapes.
22 Brothers TheStr.,
Moreover,
Orbeli HCV the particle
Yerevan size
0028, Armenia;
Yerevan 0025, melikandreasyan@mail.ru
ne in Viral Hepatitis C patients group distribution
included 96tigranghevondyan@yahoo.com
(63.5%
was7 alsomales), and the non-HCV
determined group included
by measuring the mean100 subjects D,
diameter, (62.0%
of approximately
Department of Diagnostic Radiology, Yerevan State Medical University (YSMU), 2 Koryun Str., Yer
5 Department of Infectious Diseases, Yerevan State Medical University (YSMU), 2 Koryun Str., Yerevan 0025,
: A Cross-Sectional Study. males) with lesions of the
500 particles. oral mucous 0025,membrane. The lesions of lips and oral mucosa were more
viron. Res. Public Health 2022, frequent in HCV than The Armenia;
in average
the non-HCV sh_melaniya@mail.ru
graingroup—e.g.,
8 Department erosion
sizeofdistribution
Pediatric (13.5%
andvs.
obtained
Dentistry 1%),
fromcracks in theimage
theYerevan
Orthodontics, TEM mouth
State is depicted
Medical Universityin(YSMU), 2 K
ps://doi.org/10.3390/xxxxx Figure 1b, and it Str.,
was Yerevan
emphasized 0025, Armenia;
that dr.manrikyan@mail.ru
the particle
corners (42.7% vs. 0%), changes in the oral mucosa surface (89.6% vs. 3.0%), hemorrhages (78.1% vs. size distribution was approximately
Yerevan * 0025, Armenia; melikandreasyan@mail.ru
12.4 nm.7 Furthermore,
0%), etc. The pro-inflammatory IL-2 level the
washigh-resolution
higher TEM image,Tel.:
and anti-inflammatory IL-4+374-91-326773
presented
level in Figure
was lower 1c, revealed the
in
ic Editor: Paul B. Department of Diagnostic Radiology, Yerevan State Medical University (YSMU), 2 Koryun Str., Yerevan
existence
HCV patients compared with of regular
0025,those
fringes
in the
Armenia; non-HCV group. Conclusions: Morphological changes which is char-
in the
tina129@mail.ru
nanoparticle having a distance of 2.51 Å,
wou
acteristic8 of Abstract:
the (311) Background:
ofinterplanar The
distance objective
of theand was Yerevan
cubic to revealState
maghemite. the Medical
most
Thetypical
clear changes
lattice in oral
fringe mucosa
developed in the
Citation: Azatyan, Yessayan, L.;Department
V.;microvasculature both Pediatric
worsen Dentistry
the tissue and Orthodontics,
trophism accelerate the healing University
with (YSMU), 2 Koryun
d: 28 May 2022 observed inStr., patients
theYerevan
HRTEM and
0025, compare
Armenia;
image them with those
highlighted the obtainingin HCV negative
of nanoparticles patients. Methods:
with a crystalline The study invo
differentiation into coarse-fibrous
Sargsyan, A.; Khachatryan, A.; connective tissue. Immunohistochemical findings indicated a de-
d: 21 July 2022 nature. The HCV
* Correspondence:
SAED patients
patterns and 100 patients
for the without
obtained HCV whoiron
Tel.: +374-91-326773
dextran-coated applied
oxide to ananoparticles
dental clinic. Thewerecontent of cy
crease
Ghevondyan,in local humoral immune
T.; Shmavonyan, M.; response.
ed: date indexed to be ofIL-2, cubic IL-4, IL-10 and ɤ-INF
maghemite, -Fe2 Oin3 ,the
andoralthefluid was determined
obtained rings werebyattributedELISA. Buccalto themucosa an
(220), Abstract:
(311), (400), Background:
biopsies
(422), passed
(511) The
and objective
histological
(440) was to reveal
examination.
planes, the
An
respectively most typical changes
immunohistochemical
in agreement in
with oral
study mucosa in HCV
of mucous
existing me
er’s Note: MDPI stays Citation:
neu- Keywords:
K.; Manrikyan,
Azatyan, oral mucosa;
M. Morphological
V.; Yessayan, L.; viral hepatitis C; cytokines; morphology; immunohistochemistry
th regard to jurisdictional Changes in the Oral reported patients
data and
[47,48]. compare
biopsies was them
performed with those
using in HCV
monoclonal negative
mouse patients.
antibodies Methods:
to CD3+ The
and study
CD20+.involved
Results: 96T
Sargsyan, A.; Khachatryan, A.; Mucous
n published maps and institu- Membrane HCV
Furthermore,
in Viral Hepatitis C patients and 100 patients without HCV who applied to a dental clinic.
patients group included 96 (63.5% males), and the non-HCV group included 100 subject
additional information about the morphology of the The content
dextran-coated of cytokines
filiations. Patients: G.;
Melik-Andreasyan,
maghemite
A Cross-Sectional
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IL-2,nanoparticles
IL-4, IL-10with
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-Fe2 Oinof
ɤ-INF 3 the
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wasmucous wasgathered
also determined
membrane. by lesions
The
using ELISA.
scanning Buccal
of lips mucosa
and
electron oral and gums
mucosa
mi- we
Int. J.M.
Environ. Healthbiopsies
croscopy.
Res. Public passed
frequent
The results
2022, histological
in
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in
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SEM An immunohistochemical
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studies group—e.g.,
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in Figure study
(13.5% ofvs.
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The1%),
SEM membrane
cracks in the
1. Introduction
K.; Manrikyan, Morphological
Changes in18, thex.Oral Mucous micrograph
https://doi.org/10.3390/xxxxxbiopsies was performed
presented
corners in Figure
(42.7% using
vs. 2a,monoclonal
0%), as well in
changes asmouse
theoral
the antibodies
inset of the
mucosa tohigh
CD3+
surface and CD20+.
magnification
(89.6% Results:
vs. 3.0%), SEMThe HCV
hemorrhages (7
Hepatitisimage C virus(Figure
(HCV)
patients isgroup
2b),the main
confirmed cause
included 96
the of(63.5%
chronic
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obtained disease,
fromthe with
non-HCV
the TEM a global
group
studies prev-
included
and 100
revealed subjects
the (62.0%
Membrane
ht: © 2022 by the authors. in Viral Hepatitis C
Academic Editor: Paul B. 0%), etc. The pro-inflammatory IL-2 level was higher and anti-inflammatory IL-4 level was
alence of 3% [1]. About 58particles
males)
million
with
people
lesions
in
of the
the worldsizes
oral mucous
live with
membrane.
chroniclesions
HCV infection,
Patients: A Cross-Sectional Study.
ed for possible open access Tchounwou
obtaining of HCV having
patients nanometric
compared with those and the The
inspherical
non-HCV shapes. of lips
group. and oral mucosa
In Conclusions:
addition, were more
theMorphological
SEM
Int. J.
with
Environ. Res.
1.5
Public
million
Health
newlyfrequent
micrograph
2022,
infected
also people
in HCV than
depicted each
the year [1].
in microvasculature
the non-HCV group—e.g.,
uniformity a homogeneity erosion
of the (13.5%
particles. vs. 1%),and
More cracks
than in thethe
that, mouth
ion under the terms and con- developed in the both worsen the tissue trophism accelerate heal
18,At- Received:The 28 extrahepatic
May 2022
x. https://doi.org/10.3390/xxxxx manifestations
corners (42.7%
of viral hepatitis
vs. 0%), changes
C, caused bysurface
in the oral mucosa
HCV were(89.6%
first spo-
vs. 3.0%), hemorrhages (78.1% vs.
of the Creative Commons differentiation into coarse-fibrous connective tissue. Immunohistochemical findings indicat
ken about
Accepted: in the
21 July 2022 early 1990s [2]. They were shown to develop among 74% of HCV infected
n (CC BY) license (https://cre-
Academic Editor: Paul B. 0%), etc. The pro-inflammatory IL-2 level
crease in local humoral immune response. was higher and anti-inflammatory IL-4 level was lower in
mmons.org/licenses/by/4.0/). individuals
Published: date [3,4]. The most common HCV extrahepatic manifestations occur in the oral
Tchounwou HCV patients compared with those in the non-HCV group. Conclusions: Morphological changes
Publisher’s Note: MDPI staysdeveloped neu- in the microvasculature
Keywords: oral mucosa; viral bothhepatitis
worsen C; thecytokines;
tissue trophism and accelerate
morphology; the healing with
tral with regard to jurisdictional differentiation into coarse-fibrous connective tissue. Immunohistochemical findings indicated a de-
Pharmaceutics 2023, 15, x FOR PEER the particle

REVIEW size distribution obtained using the SEM micrograph determined the grain6 size
of 18
distribution at 13.1 nm, which is consistent with the one obtained from the TEM image.
Article

Vahe Azatyan 1,*, Lazar Yessayan 1, Aelita Sargsyan 2, Anna Khachatryan 3, Tigran Ghevondyan 4,
Melanya Shmavonyan 5, Gayane Melik-Andreasyan 6, Kristina Porksheyan 7 and Mikael Manrikyan 8
1 Department of Therapeutic Stomatology, Yerevan State Medical University (YSMU), 2 Koryun Str.,

Article
Armenia; sh_melaniya@mail.ru
Morphological Changes in the Oral Mucou


7 Department of Diagnostic Radiology, Yerevan State Medical University (YSMU), 2 Koryun Str., Yerevan
Viral Hepatitis C Patients: A Cross-Section

8 Department of Pediatric Dentistry and Orthodontics, Yerevan State Medical University (YSMU), 2 Koryun
Str., Yerevan 0025, Armenia; dr.manrikyan@mail.ru

* Correspondence:Vahe Azatyan 1,*, LazarTel.:
vahe.azatyan@gmail.com; Yessayan 1, Aelita Sargsyan 2, Anna Khachatryan
+374-91-326773 , Ti 3
Melanya Shmavonyan , Gayane Melik-Andreasyan , Kristina Porksheyan 7 a

5 6
Abstract: Background: The objective was to reveal the most typical changes in oral mucosa in HCV
Citation: Azatyan, V.; Yessayan, L.; Figure
Figure 1.1.TEM
TEM
patients and image
image (a),particle
compare
(a), particle
them with sizedistributions
size distributions
those obtained
in HCVobtained
negative
1 Department
fromlarge
patients.
from large areaTEM
Methods:
of Therapeutic
area TEM Theimage
study
Stomatology,
image (b),
(b), high- 96
involved
Yerevan
high- State Me
Sargsyan, A.; Khachatryan, A.; resolution TEM (HRTEM) image (c) and selected area electron diffraction (SAED) (d) of dextran-
Pharmaceutics 2023, HCV
resolution patients
TEM and
(HRTEM) 100 patients
image (c) without
and HCV
selected who
area applied
Yerevan
electron to a dental
0025,
diffraction Armenia;clinic.
(SAED) The content
lazyes@yandex.ru
(d) of of
dextran-coatedcytokines
Ghevondyan, T.;15, x FOR PEER
Shmavonyan, M.; REVIEW
coated maghemite nanoparticles (ɤ-Fe2O3 NPs). 7 of 18
IL-2, IL-4,
maghemite IL-10 and(ɤ-INF
nanoparticles -Fe2 Oin Tuberculosis by
the oral fluid was2 determined Research
ELISA. and Prevention
Buccal mucosaCenter,
and6/2 Adonts S
gums
Melik-Andreasyan, G.; Porksheyan, 3 NPs).
K.; Manrikyan, M. Morphological biopsies passed histological examination. An immunohistochemical study of mucous membrane
The average grain size distribution obtained from the TEM
3 Department image is
of Pathological depicted
Anatomy, in Fig-
Yerevan State Medic
Changes in the Oral Mucous biopsies was performed using monoclonal mouse antibodies to CD3+ and CD20+. Results: The HCV
0025,distribution
ure 1b, and it was emphasized that the particle size was approximately 12.4
Membrane in Viral Hepatitis C
nm.patients group included 96 (63.5% males), and 4the
Furthermore, the high-resolution TEM image, non-HCV
Laboratory
presentedof group included
Histochemistry
in Figure and
1c,100 subjects
Electron
revealed (62.0% Aft
Microscopy,
the
Patients: A Cross-Sectional Study. males) with lesions of the oral mucous membrane. The lesions of lipsofand
existence of regular fringes in the nanoparticle having a distance of 2.51 Å, which is char- RA), 22 B
Academy of Sciences oral
Republic mucosa
Armeniawere(NAoSmore
frequent in HCV tigranghevondyan@yahoo.com
(311)than in the non-HCV
distancegroup—e.g., erosion (13.5% vs. The1%), cracks in fringe
the mouth
Int. J. Environ. Res. Public Health 2022,
acteristic of the interplanar of the5 cubic maghemite. clear lattice
Department of Infectious Diseases, Yerevan State Medical U
18, x. https://doi.org/10.3390/xxxxx cornersin(42.7%
observed the HRTEMvs. 0%), changes in the oral mucosa
image highlighted surface (89.6%
the obtaining vs. 3.0%), hemorrhages
of nanoparticles (78.1% vs.
with a crystal-
Academic Editor: Paul B. 0%), etc. The pro-inflammatory IL-2 level was
line nature. The SAED patterns obtained for the dextran-coated higher
6 and anti-inflammatory
National Center of Disease
IL-4
Control
iron oxide level was lower in
and Prevention, Ministr
nanoparticles
Tchounwou HCV patients compared with those
were indexed to be of cubic maghemite, ɤ-Fe2O3,Yerevan in the non-HCV group.
and the Conclusions:
0025, Armenia;rings
obtained Morphological
were attributed changes
developed in the microvasculature both worsen 7the tissue
Department trophism
of and
Diagnostic
to the (220), (311), (400), (422), (511) and (440) planes, respectively in agreement with ex- accelerate
Radiology, the healing
Yerevan with
State Medica
Received: 28 May 2022 0025, Armenia; tina129@mail.ru
differentiation
isting reported data into[47,48].
coarse-fibrous connective tissue. Immunohistochemical findings indicated a de-
Accepted: 21 July 2022 8 Department of Pediatric Dentistry and Orthodontics, Yerev
crease in local humoral
Furthermore, additional immune response. about the morphology of the dextran-coated
information
Published: date Str., Yerevan 0025, Armenia; dr.manrikyan@mail.ru
maghemite nanoparticles (ɤ-Fe2O3 NPs) was also * gathered
Correspondence:
usingvahe.azatyan@gmail.com;
scanning electron micros- Tel.: +374-91-32
Publisher’s Note: MDPI stays neu- Keywords:
copy. oral obtained
The results mucosa; viral from hepatitis
the SEM C; cytokines;
studies are morphology;
depicted immunohistochemistry
in Figure 2a–c. The SEM
micrograph presented in Figure 2a, as well asAbstract: the insetBackground:
of the highThe objective was SEM
magnification to reveal the m
claims in published maps and institu- Citation: Azatyan, V.; Yessayan, L.;
image (Figure 2b), confirmed the data obtained patients
from and TEM
the compare them and
studies with revealed
those in HCV the negativ
tional affiliations. Sargsyan, A.; Khachatryan, A.;
Figure 2.
obtaining
Figure SEM
2. SEM image
of image
particles(a), inset
(a), having of high
nanometric
inset of high magnification HCV
sizes and
magnification SEM patients
image
SEMspherical
image (b)and
(b) 100
and
shapes.
and patients
particle
particle without
size
In addition,
size HCV
distributions
the SEM
distributions who applie
1. Introduction
obtained
micrograph
obtained fromSEM
from SEMimage
also image(c)
depicted (c)ofofdextran-coated
the dextran-coated
uniformity a maghemite
IL-2,
homogeneity
maghemite nanoparticles
IL-4, IL-10
of the
nanoparticles and ( (ɤ-Fe
ɤ-INF
particles.
-Fe 2O3 NPs).
O in the
More
NPs).oral fluid
than was determ
that,
Melik-Andreasyan, G.; Porksheyan, 2 3
Hepatitis
the particle C virus
size distribution (HCV) is
obtained
K.; Manrikyan, M. Morphological
the main
using cause of
biopsies
the SEM chronic
passed liver
micrograph disease,
histological with a global
examination.
determined the An
grainprev-
immuno
Copyright: © 2022 by the authors. The stabilization of nm,
maghemite nanoparticles by world
the adsorption of a layer ofTEM
dextran
sizealence of 3% at
distribution [1].13.1 About 58
which
Changes in the Oral Mucousmillion
is people
consistent in the
biopsies
with was
the one live with
performed
obtained chronic
using
from HCV
monoclonal
the infection,
mouse
im- antibo
Submitted for possible open accessaround the particles is very important in biological and medical applications. The for-
age.with 1.5 million newly infected
Membrane in Viral Hepatitis C people each year [1].
patients group included 96 (63.5% males), and the non
publication under the terms and con-mation of the dispersant layer (adlayer, inofour case, dextran)
The extrahepatic
Patients: A manifestations
Cross-Sectional Study. viral hepatitis
males) C, was
with lesions caused very important
of thebyoral HCV werefor
mucous
thespo-
first
membrane. The
stabilization
ken aboutofinmaghemite
the early suspensions.
1990s [2]. They Itwere
is known
shown that
to the thinamong
develop layers 74% on the of surface
HCV of
infected
tribution (CC BY) license (https://cre- Int. J. Environ. Res. Public Health 2022, frequent in HCV than in the non-HCV group—e.g., e
the particles
individualslead[3,4].
to the agglomeration
The most common of the
HCV particles. For thismanifestations
extrahepatic purpose, DLS occur studies inwere
the oral
ativecommons.org/licenses/by/4.0/). 18, x. https://doi.org/10.3390/xxxxx corners (42.7% vs. 0%), changes in the oral mucosa surfa
The stabilization of maghemite nanoparticles by the adsorption of a layer of dextran

around the particles Article is very important in biological and medical applications. The formation
Morphological Changes in the Oral Mucous

of the dispersant layer (adlayer, in our case, dextran) was very important for the stabilization
of maghemite suspensions. It is known that the thin layers on the surface of the particles
Viral Hepatitis C Patients: A Cross-Sectional

lead to the agglomeration of the particles. For this purpose, DLS studies were carried out to
determine the hydrodynamic diameter of maghemite nanoparticles covered with dextran
in suspension. The zeta potential was also determined for the studied suspensions.
Figure 3 shows Vahe Azatyan the distribution 1,*, Lazar Yessayan 1, Aelita Sargsyan 2, Anna Khachatryan 3, Tigra
based on volume (a) and the distribution based on
numbers (b). Both Melanya Shmavonyan
the distribution based 5, Gayane Melik-Andreasyan 6, Kristina Porksheyan 7 and
on volume and the one based on numbers clearly
show that the suspension of maghemite nanoparticles covered with dextran does not
present Article
aggregates. As can be seen from the volume 1 Department size distribution,
of Therapeutic the analyzed
Stomatology, sample
Yerevan State Medica
Article
Morphological Changes in the Oral Mucous Mem
is composed of approximately 45.4 nm particles (Figure 3a). The
Yerevan 0025, Armenia; lazyes@yandex.ru number distribution

presented in Figure 3b was monomodal, with2anTuberculosis average peak Research
at 38and nm. Prevention
Figure Center, 3c revealed 6/2 Adonts Str., 1
Viral Hepatitis C Patients: A Cross-Sectional Stud

the intensity particle size distribution obtained for maghemite nanoparticles coated with sargsyan.aelita@gmail.com
3 Department of Pathological Anatomy, Yerevan State Medical U

dextran in suspension. The graph shows a monomodal
at 45.7 nm without indicating the presence of4 aggregates in the studied sample. The ratio
distribution with a distinct peak
Laboratory of Histochemistry and Electron3 Microscopy, After O
Article DH /D Vahe
TEM Azatyan
representing 1,*, Lazar Yessayan 1, Aelita
the hydrodynamic diameter SargsyanH )2, calculatedAnna Khachatryan DLS,(NAoS Tigran Ghevon
Academy(D of Sciences of Republic from Armenia studies RA), 22 Broth
Vahe Azatyanand 1,*, Lazar Yessayan 1, Aelita 5Sargsyan 2, Anna Khachatryan
Melanya Shmavonyan diameter, Gayane Melik-Andreasyan 3, Tigran Ghevondyan
6, Kristina Porksheyan 4,
7 and Mikael M
the nanoparticle calculated by TEM (D ) was
TEM 3.7 nm. On the other hand,
Melanya Shmavonyan the ratio D5,HGayane Melik-Andreasyan
/DSEM representing 6, Kristina
the hydrodynamic 5 Department Porksheyanof Infectious
diameter (D 7 and Mikael Manrikyan 8
H )Diseases,
calculated Yerevan from StateDLS Medical Univ
Article
studies and the nanoparticle diameter calculated Armenia; by
1 Department of Therapeutic Stomatology,
sh_melaniya@mail.ru
SEM (D ) was 3.5 nm. Both the
SEM Yerevan State Medical University
6 National Center of Disease Control and Prevention, Ministry of
Morphological Changes in the Oral M

DH /DTEM ratio1 and the DH /D
Department SEM ratio
of Therapeutic Yerevan show 0025,
Stomatology, that
Armenia;the dextran
Yerevan lazyes@yandex.rupresent
State Medical on the (YSMU),
University surface2 of Koryun Str.,
the maghemite nanoparticles Yerevan 0025, Armenia; resultsTuberculosis
2 in an 7increase
lazyes@yandex.ru Research inandthePrevention
size of the Center, 6/2 Adonts
obtained Str., 100 Apt., Yer
sample.
Vahe Azatyan 1,*, Lazar Yessayan , Aelita Sargsyan 2, Anna Khachatryan 3,Department
Tigran
of Diagnostic Radiology,
Ghevondyan 4, Yerevan State Medical Un
Viral Hepatitis C Patients: A Cross-Se
This 1result is in Tuberculosis
2full agreement Research
with and sargsyan.aelita@gmail.com
Prevention
previous Center,
studies 6/2 Adonts
[49–52]. The Str.,dispersion
100 Apt., Yerevan stability 0014,of Armenia;
Melanya Shmavonyan 5, Gayane Melik-Andreasyan
dextran-coated 6, Kristina3Porksheyan
maghemite suspension Department
was measured of7 Pathological
and Mikael
8 Department through Anatomy,
Manrikyan Yerevan8 State
theDentistry
absolute value Medicalof zeta University (YS
of Pediatric and Orthodontics, Yerevan S
3 Department of Pathological 0025,Anatomy,
Armenia;Yerevan ann_khachatryan@mail.ru
State Medical Universityhas (YSMU), 2 Koryun Str., Ye
potential. According to previous studies [53], it Str.,
can Yerevan
be said 0025,
that Armenia;
a nanofluid dr.manrikyan@mail.ru
4 Laboratory of Histochemistry and Electron Microscopy, After Orbeli Institu
a colloidal
0025, Armenia; Vahe ann_khachatryan@mail.ru
Azatyan 1,*,
1dispersion stability if the Stomatology,
absolute zeta potential * Lazar
is greaterYessayan
Correspondence: 301, Aelita Sargsyan 2,Tel.:
thanvahe.azatyan@gmail.com;
mV. The colloidal Anna Khachat
+374-91-326773
Department of Therapeutic
4 Laboratory of Histochemistry Yerevan
Academy State
and of Medical
Sciences
Electron University
of Republic
Microscopy, (YSMU),
Armenia
After 2 (NAoS
Orbeli Koryun
Institute Str.,ofstability
RA), 22Physiology
Brothers Orbeliof Nati S
ofYerevan
dextran-coated
0025, Armenia; maghemite
Academy
Melanya
lazyes@yandex.ru suspension Shmavonyan
using
of Sciences of tigranghevondyan@yahoo.com zeta 5, Gayane Melik-Andreasyan 6, Kristina Pork
potential was presented
Republic Armenia (NAoS RA), 22 Brothers Orbeli Str., Yerevan 0028, Arme in Figure 3d.
2TheTuberculosis
zeta potential Research and Prevention
measured
tigranghevondyan@yahoo.comfor Center, 6/2 Adonts
5 Department
dextran-coated Abstract:
of Str.,
InfectiousBackground:
100
maghemite Apt., Yerevan
Diseases, The
suspension 0014,
Yerevan objective
Armenia;
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was was
Medical
−38.4 toUniversity
reveal
mV. the(YSMU mos
Since the measured 5 Department
value isof higher Infectiousthan Armenia;
Diseases, patients
30 mV, Yerevan and
sh_melaniya@mail.ru
it can compare
Statebe1 Medical
them
said University
that of with
the those
(YSMU),
dextran-coatedin HCV
2Stomatology,negative
Koryun Str., Yerev pa
3 Department of Pathological
Department Therapeutic Yerev
maghemite suspension Armenia; Anatomy, Yerevan
sh_melaniya@mail.ru
isT.;atShmavonyan,
good colloidal
6 National State Medical
Center
HCV patients
stability of University
Disease
condition.and (YSMU),
Control and
100 patients 2 Koryun
Prevention, Str., Yerevan
Ministry
withoutlazyes@yandex.ru of Health
HCV who applied to (MoH
Ghevondyan, M.; Yerevan 0025, Armenia;
6 National Center of Disease Yerevan
Control 0025,and Armenia;
Prevention, melikandreasyan@mail.ru
Ministry ofO Health (MoH), 12 Mkhitar Heratsi 6S
The numerical value
Melik-Andreasyan,
of the polydispersity IL-2,
index IL-4, IL-10
(PDI) and
the ɤ-INF
2 ofTuberculosis
-Fe 2 in3 the
Research
NPs oral
wasand fluid
0.165. was
Prevention Indetermin
Center,
Yerevan 0025, andG.; Porksheyan,
Electron
Armenia; Microscopy,
7 Department After
of
melikandreasyan@mail.ru Orbeli Institute
Diagnostic Radiology, of Physiology
Yerevan State of National
Medical University (YS
agreement biopsies a passed sargsyan.aelita@gmail.com
histological examination. Ansize immunohist
Academy ofwith Sciences
K.;previous
of Republic
7 Manrikyan,
Department
studies
M. of Armenia [54,55],
Morphological
Diagnostic (NAoS
0025,
theRA),PDI
Armenia;
Radiology,
istina129@mail.ru
22Yerevan
Brothers representation
Orbeli
State Str., Yerevan
Medical
3 Department
of the
University
population
0028, Armenia;
(YSMU), 2 KoryunYerevanStr., Yer
of Pathological Anatomy,
distribution in a
tigranghevondyan@yahoo.com sample.
Changes
0025,inArmenia;
Moreover,
the Oral Mucous 8 previous biopsies
studies was
[56] performed
have using
demonstrated
Department of Pediatric Dentistry and Orthodontics, Yerevan State
tina129@mail.ru monoclonal that mouse
values antibodie
Medical
Department
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materials
University the non-H
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Moreover, 0025,PDI
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Armenia;
Study. values males) vahe.azatyan@gmail.com;
between with
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andAcademy theoforal Tel.:
the+374-91-326773
mucous fact membrane.
that the The(NA les
Sciences of Republic Armenia
analyzed sample * is
Int. J.Correspondence:
uniform
Environ. Res. in
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relation
Health 2022, to the frequent
particle size Tel.:
in HCV +374-91-326773
[57]. than
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tigranghevondyan@yahoo.com non-HCV group—e.g., erosi
zeta potential
Yerevan 0025, Armenia; melikandreasyan@mail.ru Abstract: Background: The objective was toinreveal themucosa
most
studies
7 Department
showed 18,that
x. the dextran layer
https://doi.org/10.3390/xxxxx on the surface
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(42.7% Department
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vs. 0%), changes of Infectious
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oral atypical
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Citation: Azatyan, Radiology,
V.; Yessayan, L.; Yerevan State Medical University (YSMU), 2 Koryun Str., Yerevan
fairly significant Abstract:
thickness, Background:
which led The
patients
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and good
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Armenia;
as could
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sh_melaniya@mail.ru
IL-2fromchanges
negative
level the in
waszeta oral mucosa
patients.
higher and Met an
0025,
Citation: Azatyan, V.; Yessayan, Armenia;
L.; A.; tina129@mail.ru
Sargsyan, Khachatryan,
Academic Editor: A.; Paul B. 6 National Center of Disease Control and Prevent
8potential analysis. patients and compare HCVthem with
patients those
and in
100 HCV
patientsnegative
without patients.
HCV Methods:
who applied The to study
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Sargsyan, A.; Khachatryan, Department
Ghevondyan,
A.; of Pediatric
T.; Shmavonyan,
Tchounwou Dentistry M.; and Orthodontics, HCV Yerevan patients compared
State Medical Universitywith those
(YSMU), in
Yerevan 0025, Armenia; melikandreasyan@mail
2the non-HCV
Koryun group
Str., Yerevan
Furthermore, 0025, HCVArmenia;
the patients andof 100
stability the patients
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and who
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in the
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Ghevondyan, T.; Shmavonyan, Melik-Andreasyan,
M.; G.; Porksheyan, developed in the microvasculature
7 2 Department
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of Diagnostic both Radiology,
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Yerevan
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Correspondence:
Images
K.; Manrikyan,
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IL-4,
28 May
ofMorphological IL-10
dextran-coated 2022and biopsies ɤ-INF
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Accepted:
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biopsies was examination.
performed An immunohistochemical study of mucous me
in localusing monoclonal mouse antibodies toOrthodo
CD3+ a
glass
K.; Manrikyan, M. Morphological vials over different
Changes in the Oral Mucous periods of time are depicted
crease
in Figure
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humoral
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immune
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response.
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Abstract:
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Background:
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monoclonal
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mouse
the 96 changes
antibodies
(63.5%
Str.,
images Yerevan in0025,
males), oral
to
presented CD3+ mucosa
and and
Armenia;the
in in HCV
CD20+.
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dr.manrikyan@ma
Figure groupT
patients and Acompare patients them with
group those
included in HCV
males) 96 negative
(63.5%
with males), patients.
and Methods:
* oral
Correspondence:
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group involved
included 96
100 Tel.
subjects
Keywords: oral mucosa; viral hepatitis C; cytokines; of
lesions of the mucous membrane. The lesions lips
Membrane
Sargsyan, A.; Khachatryan, A.; in Viral the suspensions
HepatitisPatients:
C were
Cross-Sectional
Publisher’s stableStudy.
Note: over
MDPI time,
stays and
neu- there was no sign of precipitation or deposition morph
Patients: M.; HCV
of thepatients
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nanoparticles
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the bottom
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regard 2022, HCV
offrequent
of
to jurisdictionalthe who
theoral in
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mucous
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membrane.
than in the clinic.
Figure The4a
non-HCV The
lesions content
depicts of lips
group—e.g.,the ofandcytokines
image oral of
erosion mucosa
(13.5% we
Ghevondyan, T.; Shmavonyan,
Int. J. Environ. Res. IL-2,
the
Public IL-4, IL-10
dextran-coated
18,
Health x. and ɤ-INF
frequent -Fe
2022, claims in in
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published
2 3 the
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than fluid
suspensions
and in was
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cornersV.;(42.7%
institu- determined
non-HCV
obtainedvs.L.; by
from Abstract:
ELISA.
the Background:
Buccal
synthesis mucosa
0%), changes in the oral mucosa surface (89.6% vs.the
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Pharmaceutics 2023,
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2023, 15, FOR PEER REVIEW 8 8of
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Pharmaceutics 2023,
Pharmaceutics 2023,15,
15,x552
FOR PEER REVIEW 9 of 18 9
Article Article
Article
Morphological Changes
Morphological
in the Oral
Changes
Mucous
in the
Membr
Ora
Morphological Changes in the Oral Mucous Membran
Viral Hepatitis CViral
Patients:
Hepatitis
A Cross-Sectional
C Patients: AStudy
Cross
Vahe Azatyan 1,*, Lazar Yessayan Vahe
1, Aelita
AzatyanSargsyan
1,*, Lazar2, Anna
Yessayan
Khachatryan
1, Aelita Sargsyan
3, Tigran Ghevondy
2, Anna Kh
VaheMelanya
AzatyanShmavonyan
1, *, Lazar Yessayan 1 ,Melanya
5, GayaneAelita Sargsyan
Shmavonyan
Melik-Andreasyan 2 , Anna Khachatryan
6,5,Kristina , Tigran
Gayane Porksheyan
Melik-Andreasyan
3 Ghevondyan
7 and Mikael 4,
6, Kristina
Man
Melanya Shmavonyan 5, Gayane Melik-Andreasyan 6, Kristina Porksheyan 7 and Mikael Manriky
1 Department of Therapeutic Stomatology, 1 Department
Yerevan State
of Therapeutic
Medical University
Stomatology(YSM
1 Yerevan 0025, Armenia; lazyes@yandex.ru
Department of Therapeutic Stomatology, Yerevan Yerevan 0025,
State Armenia;
Medical lazyes@yandex.r
University (YSMU), 2
2 Tuberculosis Research and Prevention
0025, Armenia; lazyes@yandex.ru Center,
2 Tuberculosis
6/2 Adonts
Research
Str.,and
100 Prevention
Apt., Yerevan C
Yerevan
2 sargsyan.aelita@gmail.com
Tuberculosis Research and Prevention Center, sargsyan.aelita@gmail.com
6/2 Adonts Str., 100 Apt., Yerevan 0014
3 Department of Pathological Anatomy, 3 Yerevan
DepartmentStateofMedical
Pathological
University
Anatomy,
(YSMU Ye
3 0025, Armenia; ann_khachatryan@mail.ru 0025, Armenia; ann_khachatryan@mail.r
Department of Pathological Anatomy, Yerevan State Medical University (YSMU), 2 K
0025, Laboratory of Histochemistry and Electron
4 4 Laboratory
Microscopy,
of Histochemistry
After Orbeli Institute
and Electr of
4 Academy of Sciences of Republic Armenia
Academy
Laboratory of Histochemistry and Electron Microscopy, (NAoSofRA),
Sciences
After 22 Brothers
of Republic
Orbeli Orbeli
Institute Armen
of Str.,
PhysY
Academy tigranghevondyan@yahoo.com
of Sciences of Republic Armenia (NAoStigranghevondyan@yahoo.com
RA), 22 Brothers Orbeli Str., Yereva
5 Department of Infectious Diseases, Yerevan 5 Department
State Medical
of Infectious
University
Diseases,
(YSMU),
Yerev 2
5 Armenia;
Department sh_melaniya@mail.ru
of Infectious Armenia;
Diseases, Yerevan State sh_melaniya@mail.ru
Medical University (YSMU), 2 Kory
6 National Center of Disease Control and 6 National
Prevention,
Center
Ministry
of Disease
of Health
Control
(MoH),
and P 1
6 NationalYerevan
Center 0025, Armenia;
of Disease melikandreasyan@mail.ru
Control YerevanMinistry
and Prevention, 0025, Armenia;
of Health melikandreasya
(MoH), 12 Mk
7 Department of Diagnostic Radiology, 7 Yerevan
Department
StateofMedical
Diagnostic
University
Radiology,
(YSMU Ye
7 0025, Armenia; tina129@mail.ru
Department of Diagnostic Radiology, Yerevan 0025, Armenia;
State Medicaltina129@mail.ru
University (YSMU), 2 Ko
0025, Department of Pediatric Dentistry and
8 8 Department
Orthodontics, ofYerevan
PediatricState
Dentistry
MedicalandUn O
8 Str., Yerevan
Department 0025, Armenia;
of Pediatric Dentistry dr.manrikyan@mail.ru
Str., Yerevan
and Orthodontics, 0025,State
Yerevan Armenia;
Medicaldr.manriky
Universit
Str.,* Yerevan
0025, Armenia; * Correspondence:
dr.manrikyan@mail.ru Tel.: +374-91-326773
vahe.azatyan@gmail.co
* Correspondence: vahe.azatyan@gmail.com; Tel.: +374-91-326773
Abstract: Background: The objective Abstract:
was to reveal
Background:
the most Thetypical
objective
chang w
Citation: Azatyan, V.; Yessayan, L.; Citation: Azatyan, V.; Yessayan, L.;
patients and compare The them
objective
withwas
thoseto reveal
patients
in HCV
andthe most typical
negative
compare themchanges
patients. with Method
thosein
Sargsyan,
Citation: Azatyan, A.;V.;
Khachatryan,
Yessayan, L.;A.; Sargsyan, A.; Khachatryan, A.;
patients
HCV and compare
patients andthem with those
100 patients in
without
HCVHCVHCV negative
patients
whoand patients.
applied to aMethods:
100 patients dentalwithout
clinicTh
Ghevondyan,
Sargsyan,
Figure T.;
A.; Khachatryan,
4. Shmavonyan,
Visual A.;appearance of Ghevondyan,
M.; prepared T.; Shmavonyan, M.;
dextran-coated ɤ-Fe 2O3 NPs (a–c), dextran-coated ɤ-
Figure 4. Visual
Melik-Andreasyan,
appearance
G.; Porksheyan,
ofHCV patients
IL-2,
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and ɤ-INF
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without
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and a
determined dental
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in ELISA.
the The
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Ghevondyan, T.; Shmavonyan, M.;3+
NPs with 500 μg Fe /mL (d–f)
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histological
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mouse
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antibodies using
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Changes in the Oral
K.; Manrikyan, M. Morphological Mucous Changes in the Oral Mucous
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Changes in the Oral MucousViral Hepatitis C biopsies was
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of hydroxyl groups on the surface. This behavior HCV patients
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in the microvasculature bothnon-HCV
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Received: 28 May[58,59].
2022 As can beReceived:
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developed in the microvasculature
differentiation into coarse-fibrous bothconnective
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single July 2022
step in the temperature Accepted:range
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21 July 2022
of 180–750
into coarse-fibrous
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°C.
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response. local
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Published:
Accepted: 21 July 2022date Published: date
Fe2O3 NPs takes place crease in two stages.
in local humoral Theimmune
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Published: date
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with a mass Note: loss
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of 150–400 oral mucosa;
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1 Department of Therapeutic
1 Department
Stomatology,
of Therapeutic
Yerevan State
Stomatology,
Medical University
Yerevan State
(YSMU),
Med
Yerevan 0025, Armenia;Yerevan
lazyes@yandex.ru
0025, Armenia; lazyes@yandex.ru
Tuberculosis Research 2 and
2Tuberculosis
Prevention Research
Center, and
6/2 Adonts
Prevention
Str., Center,
100 Apt.,
6/2Yerevan
Adonts001 Str
Pharmaceutics
Pharmaceutics2023,
2023,15,
15,x552
FOR PEER REVIEWof Therapeutic Stomatology,sargsyan.aelita@gmail.com
1 Department Yerevan State Medical sargsyan.aelita@gmail.com
University (YSMU), 2 Koryun Str., 10 of 18 10 of 1
3 Department
Anatomy, of Yerevan
Pathological
StateAnatomy,
Medical University
Yerevan State
(YSMU),
Medica 2K
0025, Armenia;
2 Tuberculosis Research and Prevention Center, ann_khachatryan@mail.ru
6/2 Adonts Str., 100 Apt., Yerevan 0014, Armenia;
4 Laboratory and Electron
of Histochemistry
Microscopy, and After
Electron
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3 Department of Pathological Anatomy, Yerevan of Medical
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0025, Armenia; ann_khachatryan@mail.ru tigranghevondyan@yahoo.com tigranghevondyan@yahoo.com
5 Department of Infectious5 Department
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Academy of Sciences of Republic Armenia Armenia; sh_melaniya@mail.ru
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Orbeli sh_melaniya@mail.ru
Str., Yerevan 0028, Armenia;
6 National Center of Disease
6 National
ControlCenter
and Prevention,
of Disease Control
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8 Department of Pediatric 8 Department
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Yerevan
ing to0025, Armenia;
previous melikandreasyan@mail.ru
studies [63], the percentage of dextran molecules attached to the surface
Str., Yerevan
7 Department of Diagnostic Radiology, Yerevan 0025, Armenia;
State Medical Str., Yerevan
University dr.manrikyan@mail.ru
(YSMU),0025, Armenia;
2 Koryun dr.manrikyan@mail.ru
Str., Yerevan
of ɤ-Fe2O3 NPs can be simply* calculated by measuring
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* Correspondence: in residual weight
Tel.: +374-91-326773 Tel.: +374-91-326
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Department of Pediatric Dentistry and Orthodontics, Yerevan State Medical University (YSMU), 2 Koryun
the
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in
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* Correspondence:
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Yessayan,
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+374-91-326773
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patients and comparepatients
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andthose
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Sargsyan, A.; Khachatryan,Sargsyan,
A.; A.; Khachatryan, A.;
obtained from TGA analysis HCV are in agreement
patients and 100 with
HCV previous
patientspatients
without andstudies
HCV [63] applied
100 patients
who that obtained
without to aHCV
dentala clinic.
who applied
Th
Article Ghevondyan, T.; Shmavonyan, Ghevondyan,
M.; T.; Shmavonyan, M.;
dextran-coating The objective
efficiency of was to
magnetite
IL-2, reveal
IL-4, IL-10 theandmost
nanoparticles
IL-2,
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in
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of
the
IL-10oral and in
fluid oral
approximately was
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in in
50.12%
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the
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oral and
fluid
by ELISA.
was determ
Bucc
Figure 5. TGA
Figure 5. TGA curves curves
of dextran (a), dextran-coated
of dextran (a), dextran-coated -Fe2 O3 NPs (b) and -Fe2 O3 NPs (c).
2O 3 NPs (b) and 2 O 3 NPs (c).
patients
53.89%,and compare
respectively. them with those in HCV negative patients. Methods: TheAn study involved 96 An immunoh
Sargsyan, A.; Khachatryan, A.; K.; Manrikyan, M. Morphological K.; Manrikyan, biopsies passed
M. Morphological histological
biopsiesexamination.
passed histological immunohistochemical
examination. study o
HCV patients The
The and
cell
cell100 patientsof
viability
viability without
of RAW
RAW HCV
264.7
264.7 who
murineapplied
murine cellsto
cells a dental
exposed
exposed clinic.
to
to The content
dextran-coated
dextran-coated of cytokines
maghemite
maghemite
Ghevondyan, T.; Shmavonyan, M.; The obtained
Changes in the Oral Mucous results biopsies
were
Changes in the Oral Mucous was
in performed
good biopsies
agreement using was
monoclonal
with performed
previous mouseusing antibodies
monoclonal
studies to CD3+
mouse
[60–62]. andantibod
AccordCD2
IL-2,nanoparticles
IL-4, IL-10 and(ɤ-Fe
nanoparticles ( ɤ-INF
-Fe 22OO in the oral
33NPs)
NPs) in
in afluid
a was
concentration
concentration
patients group determined range
range
includedpatientsby
96of
of ELISA.
5–500
5–500
(63.5%
group Buccal
μg
µg
males), Fe
included
3+mucosa
3+ /mL
/mL
and 96 the and males),
varied
varied
(63.5%
non-HCV gums
in ina adose
dose
group
and the
include
non
ing toinprevious
Membrane Viral Hepatitis studies
Membrane
C in [63], the percentage
Viral Hepatitis C of dextran molecules attached to the surfac
K.; Manrikyan, M. Morphological biopsies
and
and
Patients:
passed histological
time-dependent
time-dependent
A Cross-Sectional
examination.
manner
manner (Figure
(Figure
males) An
with immunohistochemical
6).
6). For
For
lesions doses
doses
ofmales)
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oralwith study
250
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simply calculated Study. by measuring the difference in residual weigh
Vahe
ChangesAzatyan 1,*, Lazar Yessayan
in the Oral Mucous biopsies was
1, Aelita
decrease
decrease performed
Sargsyan
of
of viability,
Int. J. Environ. Res. Public Healthusing
viability, 2, Annamonoclonal
compared
compared
Int. J.2022, Khachatryan
to
to
frequent
Environ. Res. mouse
control,
control,
in HCV
Public Health antibodies
3, was
Tigran
was
2022, than to
noticed
frequent CD3+
Ghevondyan
noticed and
after
after
in the non-HCV
in HCV CD20+.
2424 ,
4and
and
than Results:
4848
group—e.g., hh from
from
in the non-HCV The HCV
treatment
treatment
erosion group—e.g.,
(13.5% vs. 1%), er
patients
between
except group
uncoated
forincluded
the dose 96 of ɤ-Fe
6,(63.5%
100μg 2O3 NPs
µg 3+3+
males),
Fe /mLand
and atdextran-coated
at the non-HCV
24 h. For group
the same ɤ-Fe
included
2O3 NPs. This result reveals tha
exposure 100 8 subjects
intervals, (62.0%
the dose
Melanya
Membrane in Shmavonyan
Viral Hepatitis C 5, Gayane 18,Melik-Andreasyan
except for the dose 18,
x. https://doi.org/10.3390/xxxxx ofx. Kristina
100 Fe Porksheyan
corners/mL (42.7%
https://doi.org/10.3390/xxxxx 24 h.
vs. and
7 For
0%), Mikael
the
corners same
changes Manrikyan
(42.7%
inexposure
the vs.oral
0%), intervals,
mucosa
changes surface
in the
the dose
(89.6%
oral of
mucosa
vs. 3.0%),
surfac
he
Patients: A Cross-Sectional Study. males) the
of
500 with
500coating
μg lesions
µg3+Fe
Fe /mL efficiency
3+
of/mL
the oral
induced inducedaofsignificant
mucousthe ɤ-Fe2O
membrane.
a significant 3 NPs
Thewas
reduction
reduction of about
lesions of
cellular 52.13
of cellular
lips and %. The
oral
viability mucosa
viabilitybyhigherTGA
35%bywere35%
andresults
more
and
38%,wasare
38%, in agree
re-higher
Academic Editor: Paul B. results
Academicobtained 0%), etc. The pro-inflammatory
0%), etc. TheIL-2pro-inflammatory
level
Editor: Paul B.from DLS and zeta potential studies. Moreover, our result was IL-2
and level
anti-inflammatory and
I
Int. J. Environ. Res. Public Health 2022, frequentment in
respectively,
spectively, withcompared
HCV the
than in
compared the non-HCV
to to control
control group—e.g.,
cells.
cells. After
After erosion
72 72h (13.5%
hof vs.
oftreatment,
treatment, 1%), cracks
allalldoses
doses in the mouth
above
above 2525μgµg
1 Department of Therapeutic HCV patients compared HCV with
patients
those compared
in the non-HCV
with those
group. in the
Conclusions:
non-HCV gro
M
Tchounwou
cornersFe 3+3+
obtained
/mL
(42.7% from
caused a Stomatology,
Tchounwou
TGA analysis
significant Yerevan State Medical University (YSMU), 2 Koryun Str.,
are
decrease in agreement
in this with
parameter. previous
Two ofstudies
doses of NPs [63]ofandthat
50 500 obtained
and
0025, vs. 0%),achanges in the oral mucosa surface (89.6% vs.Two
3.0%), hemorrhages (78.1% vs.
18, x. https://doi.org/10.3390/xxxxx Fe /mL caused significant decrease in this parameter. doses NPs of 50both
Yerevan Armenia;
3+ lazyes@yandex.ru developed in the microvasculature
developed in the
bothmicrovasculature
worsen the tissue trophism
worsenand theaccel
tissu
20%),
500
etc.
Received:
μg µg
The28 Fe
May have
dextran-coating
Fe
Tuberculosis
3+ /mL /mL
pro-inflammatory
2022 been
Research
have
andReceived: been
IL-2
28 May
efficiency
chosen
Prevention
chosen
level
2022
for was
ofthe
Center,
for the
higher
magnetite
6/2following
Adonts
following
and determinations.
anti-inflammatory
nanoparticles
determinations.
Str., 100 Apt., Yerevan IL-4
of
0014, level was
approximately
Armenia; lower in 50.12% and
Academic Editor: Paul B. differentiation into coarse-fibrous
differentiation connective
into coarse-fibrous
tissue. Immunohistochemical
connective tissue. Immfin
HCV Accepted:
patients 21 July
53.89%, 2022
compared
respectively. Accepted:
with 21 July
those in2022
the non-HCV group. Conclusions: Morphological changes
Tchounwou crease in local humoral crease
immune in local
response.
humoral immune response.
Published: date
3 Department of Published: date Yerevan State Medical University (YSMU), 2 Koryun Str., Yerevan
developed in
The thePathological
microvasculature
cell viabilityAnatomy,
ofbothRAW worsen264.7 themurine
tissue trophism and accelerate
cells exposed the healing with maghemit
to dextran-coated
Received: 28 May 2022 0025, Armenia; ann_khachatryan@mail.ru
differentiation
Publisher’s
nanoparticles
4 Laboratory
intoMDPI
Note: coarse-fibrous
stays
Publisher’s
(ɤ-Fe neu-
2O3 NPs)connective
Keywords:
Note: MDPI
in tissue.
a staysoral
neu-Immunohistochemical
concentrationmucosa;Keywords:
viral
range hepatitis
oral findings
mucosa;
ofof5–500 C; cytokines;
μg indicated
viral 3+/mLa varied
hepatitis
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FeNational de-
C; cytokines;
immunohi
in a dosmo
Accepted: 21 July 2022 of Histochemistry and Electron Microscopy, After Orbeli Institute Physiology of
crease in
traland
Academy of local
with humoral
regard to immune
jurisdictional
tral with
Sciences of Republic manner
time-dependent response.
regard
Armenia (NAoSto jurisdictional
(Figure RA), 22 6).Brothers
For doses Orbeli Str.,
below Yerevan 2500028, μgArmenia;
Fe mL, no significan
3+/
Published: date
claims in published maps and
tigranghevondyan@yahoo.com claims
institu-
in published maps and institu-
decrease of viability, compared to control, was noticed after 24 and 48 h from treatmen
Publisher’s Note: MDPI stays neu- Keywords:
5 oral mucosa;tional
Department of Infectious viralaffiliations.
hepatitis
Diseases, YerevanC;State
cytokines;
Medicalmorphology; immunohistochemistry
University (YSMU), 2 Koryun Str., Yerevan 0025,
tral with regard to jurisdictional except for the dose of 100 μg Fe /mL at 24 h. For the same exposure intervals, the dose o
3+
500 Center
μg Feof3+Disease
/mL induced 1. significant
Introductionreduction 1. Introduction
claims in published maps and institu- 6 National Control anda Prevention, Ministry of Healthof cellular
(MoH), viability
12 Mkhitar Heratsiby Str.,35% and 38%, re
tional affiliations. Yerevan 0025, Armenia;
spectively, compared melikandreasyan@mail.ruHepatitis C virus (HCV)
Hepatitis
is theC main
virus
to control cells. After 72 h of treatment, all doses cause
(HCV) of is
chronic
the main
liver
cause
above disease,
25ofμg
ch
Copyright: © 2022 by theCopyright:
authors. © 2022 by the authors.
7 Department of Diagnostic Radiology,
3+/mL caused a significant
FeArmenia; alence
Yerevan of 3% [1]. About
State
decrease
Medical alence58ofmillion
University 3% [1].people
(YSMU), 2 Aboutin
Koryun 58the
Str., million
worldpeople
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in the
chro
w
1. 0025,
Introduction
Submitted for possible open
Submitted
access for possible
tina129@mail.ru open accessin this parameter. Two doses of NPs of 50 and 50
with 1.5 million newly
with infected
1.5 million
people
newlyeach
infected
year [1].
people each year [1
μg Feunder
publication
8 Department
Hepatitis of/mL
3+
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Pediatric
virus been
termsDentistry
(HCV) ischosen
publication
and con- and
under
the forcause
the terms theYerevan
and
Orthodontics,
main following
con- determinations.
State Medical
of chronic liver University
disease, (YSMU),
with 2 Koryun
a global prev-
Copyright: © 2022 by the authors. Str., Yerevan
ditions 0025, Armenia;
The extrahepatic manifestations
The extrahepatic of viral
manifestations
hepatitis C,ofcaused
viral hepa
by H
alence ofof 3%
the Creative Commons
[1]. About ditions
58 At-
of the Creative Commons At-
million ken people
about in
in the
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early
ken live with
1990s
about[2].
in chronic
They
the early
were HCV
1990s
showninfection,
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toThey
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were among
shown 7t
Submitted for possible open access * Correspondence: vahe.azatyan@gmail.com;
tribution (CC BY) license (https://cre- Tel.:(https://cre-
tribution (CC BY) license +374-91-326773
with 1.5 million newly infected people each year
individuals [3,4].[1].
The
individuals
most common
[3,4]. The
HCV most
extrahepatic
common HCV
manifestation
extrahe
publication under the terms and con- ativecommons.org/licenses/by/4.0/).
The extrahepatic manifestations of viral hepatitis C, caused by HCV were first spo-
ditions of the Creative Commons At- Abstract: Background: The objective was to reveal the most typical changes in oral mucosa in HCV
Citation: Azatyan, V.; Yessayan, L.; ken about in the early 1990s [2]. They were shown to develop among 74% of HCV infected
tribution (CC BY) license (https://cre- patients and compare them with those in HCV negative patients. Methods: The study involved 96
ativecommons.org/licenses/by/4.0/). individuals [3,4]. The most common HCV extrahepatic manifestations occur in the oral
HCV Int. patients
Figure 6.and
J. Environ. 100
Cell
Res. patients
viability
PublicInt.
Health without
of RAW
J. Environ. HCV
264.7
18,
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cellsapplied
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ax.dental
and 72 clinic. The content
h exposure of cytokines
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Ghevondyan, T.; Shmavonyan, M.; Figure 6. Cell viability of RAW2021, 264.7 cells Health
after 24, 48,18,
2021, and https://doi.org/10.3390/xxxxx
72 h exposure to different doses of dex- www.m
IL-2, IL-4, IL-10
dextran-coated and ɤ-INF
-Fe Oin the
NPs oral fluid was
(corresponding determined
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Data
tran-coated ɤ-Fe2O3 NPs (corresponding to 5–500 μg Fe mL). Data (n = 3) are expressed as relative
2 3 3+/ mucosa
(n = 3) and
are gums
expressed as
K.; Manrikyan, M. Morphological biopsies passed histological
values (%) examination.
related to control An
± immunohistochemical
SD. The results were study
considered of mucous
values (%) related to control ± SD. The results were considered statistically significant at * p < 0.05,at
relative membrane
statistically significant
Int. J. Environ. Res. Public
Changes in the Oral Mucous Health 2021,biopsies
18, x. p <was performed p <using
***https://doi.org/10.3390/xxxxx
0.05,
0.01 ** p ***
and < 0.01 monoclonal
and ***
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www.mdpi.com/journal/ijerph
Membrane in Viral Hepatitis C patients group included 96 (63.5% males), and the non-HCV group included 100 subjects (62.0%
Patients: A Cross-Sectional Study. males) with The internalization
lesions of NPs membrane.
of the oral mucous was quantified by measuring
The lesions of lips andtheoral
level of iron
mucosa in the
were treated
more
RAW 264.7 cells with NPs corresponding to doses of 50 µg Fe 3+ /mL and 500 µg Fe3+ /mL,
Int. J. Environ. Res. Public Health 2022, frequent in HCV than in the non-HCV group—e.g., erosion (13.5% vs. 1%), cracks in the mouth
18, x. https://doi.org/10.3390/xxxxx respectively (Figure 7a). The intracellular concentration of Fe3+ increased in time, de-
corners (42.7% vs. 0%), changes in the oral mucosa surface (89.6% vs. 3.0%), hemorrhages (78.1% vs.
pending on the applied dose. The lower dose determined a significant increase of Fe3+
Academic Editor: Paul B. 0%), etc. The pro-inflammatory IL-2 level was higher and anti-inflammatory IL-4 level was lower in
concentration by about 42%, 101%, and 132% after 24 h, 48 h, and 72 h, respectively, whereas
the higher one generated a significant raise of the intracellular Fe3+ level by about 383%,
Received: 28 May 2022 721%, and 997% respectively, compared to control cells. According to Figure 7b, the pro-
differentiation into coarse-fibrous connective tissue. Immunohistochemical findings indicated a de-
Accepted: 21 July 2022 duction of ROS in RAW 264.7 murine cells exposed to NPs increased in a concentration and
Published: date time-dependent manner up to 48 h of exposure, followed by a slight decrease after 72 h.
Figure 6. Cell viability of RAW 264.7 cells after 24, 48, and 72 h exposure to different doses of dex
Publisher’s Note: MDPI stays neu- Keywords: oral mucosa;
tran-coated ɤ-Fe2Oviral hepatitis
3 NPs C; cytokines;
(corresponding to morphology;
5–500 μg Feimmunohistochemistry
3+/mL). Data (n = 3) are expressed as relativ
tral with regard to jurisdictional values (%) related to control ± SD. The results were considered statistically significant at * p < 0.05
claims in published maps and institu- ** p < 0.01 and *** p < 0.001 (sample vs. control).
Fe3+/mL, respectively (Figure 7a). The intracellular concentration of Fe3+ increased in time,
depending on the applied dose. The lower dose determined a significant increase of Fe3+
concentration by about 42 %, 101%, and 132% after 24 h, 48 h, and 72 h, respectively,
whereas the higher one generated a significant raise of the intracellular Fe3+ level by about
Pharmaceutics 2023, 15, 552 383%, 721%, and 997% respectively, compared to control cells. According to Figure11 7b,
of the
18
production of ROS in RAW 264.7 murine cells exposed to NPs increased in a concentration
and time-dependent manner up to 48 h of exposure, followed by a slight decrease after 72
h. The
The dosedose
of 50ofµg/mL
50 μg/mL determined
determined a significant
a significant raiseraise of ROS
of ROS levelslevels by about
by about 17%, 17%, 75%,
75%, and
and 57% after 24 h, 48 h, and 72 h, respectively, whereas the higher dose
57% after 24 h, 48 h, and 72 h, respectively, whereas the higher dose generated a significant generated a sig-
nificant increase of these by 104%, 185% and 130% after the same
increase of these by 104%, 185% and 130% after the same intervals of exposure, comparedintervals of exposure,
tocompared to control
control cells. cells. Malondialdehyde
Malondialdehyde (MDA) is anofend-product
(MDA) is an end-product the oxidativeof the oxidative
degradation of
degradation of fatty
polyunsaturated polyunsaturated
acids (PUFA) fatty
and isacids (PUFA)ofand
an indicator is an
cellular indicator
oxidative of cellular
stress. In the
oxidative
RAW 264.7stress.
murine Incells
the RAW 264.7
exposed formurine
24 h tocells
bothexposed
doses offorNPs,24 hthe
to MDA
both doses of NPs, the
concentrations
MDA concentrations increased significantly by 56% (lower dose)
increased significantly by 56% (lower dose) and 212% (higher dose) in comparison withand 212% (higher dose)
control cells (Figure 7c). The treatment of RAW 264.7 cells with a dose of 50 µg Fe /mLdose
in comparison with control cells (Figure 7c). The treatment of RAW 264.7 cells with
3+ a for
48ofh50 μg72
and Feh3+/mL for 48 insignificant
generated h and 72 h generated
increases insignificant
in MDA levels increases
by 7% and in MDA levels by 7%
25%, respectively,
and 25%, respectively,
compared to the control. compared
Instead,toexposure
the control.
to NPsInstead, exposure totoNPs
corresponding µg Fe3+ /mL
500corresponding
Article 3+
to 500 μg Fe
determined /mL determined
significant raises of significant raises of MDA
MDA concentrations by 82%concentrations
and 48% after by4882% and7248%
h and h,
after 48 h and
respectively, 72 h, respectively,
relative to the control.relative to the control.

Vahe Azatyan 1,*, Lazar Yessayan 1, Aelita Sargsyan 2, Anna Khachatryan 3, Tigran Ghevon
Melanya Shmavonyan 5, Gayane Melik-Andreasyan 6, Kristina Porksheyan 7 and Mikael M
1 Department of Therapeutic Stomatology, Yerevan State Medical University

2 Tuberculosis Research and Prevention Center, 6/2 Adonts Str., 100 Apt., Yer
3 Department of Pathological Anatomy, Yerevan State Medical University (YS

4 Laboratory of Histochemistry and Electron Microscopy, After Orbeli Institu
Academy of Sciences of Republic Armenia (NAoS RA), 22 Brothers Orbeli S

5 Department of Infectious Diseases, Yerevan State Medical University (YSMU
6 National Center of Disease Control and Prevention, Ministry of Health (MoH

7 Department of Diagnostic Radiology, Yerevan State Medical University (YS

8 Department of Pediatric Dentistry and Orthodontics, Yerevan State Medical

Abstract: Background: The objective was to reveal the most typical ch

patients and compare them with those in HCV negative patients. Met
Figure
Figure 7. Cellular
7. Cellular iron
iron uptake and HCV
levels
levels of ofpatients
oxidative
oxidative and 100 patients
stress
stress without
parameters
parameters ininRAWHCV
RAW who
264.7
264.7 applied
cells
cells to24,
after
after a dental cl
24,
48,48,
andand 72 72
h h exposure
exposure to to
two
two
doses IL-2,
doses
of of IL-4, IL-10
dextran-coated
dextran-coated and
ɤ-Fe 2ɤ-INF
O-Fe O
3 NPsin
2 3 the
NPs oral fluid
corresponding
corresponding was
to 50 determined
to
and 50 and
500 μg by ELIS
Fe 3+/mL. 3+
500 µgK.;Fe (a)
/mL. M.
Manrikyan, intracellular iron biopsies
intracellular iron
(a)Morphological content quantified passed histological
by
content quantified iron-thiocyanate examination.
reaction;
by iron-thiocyanate An immunohistochemical
(b) level
reaction; of reactive
(b) level of
oxygenoxygen
reactive species detecteddetected
Changes in thespecies
Oral Mucous
by H2DCF-DAby Hbiopsies
assay; was
2 DCF-DA performed
(c)assay;
level of using monoclonal
(c)malondialdehyde
level mouse
estimated
of malondialdehyde byantibodies
TBA reac-
estimated byto CD3+ a
tion. Data
TBA reaction. (n = 3) are expressed as relative
patientsvalues
group(%) related
included to control
96 (63.5% ± SD. The
males),±and results
SD.the were
non-HCVcon- group
Membrane Data in Viral(n = 3) are
Hepatitis C expressed as relative values (%) related to control The results
sidered statistically significant at * p < 0.05, ** p < 0.01 and *** p < 0.001 (sample vs. control).
Patients: A Cross-Sectional
were considered significantmales)
statisticallyStudy. at * p <with
0.05,lesions of the
** p < 0.01 andoral
*** pmucous
< 0.001membrane.
(sample vs. The lesions of lips
control).
Int. J. Environ. Res. Public Health 2022, frequent in HCV than in the non-HCV group—e.g., erosion (13.5%
The cellular antioxidant defense
18, x. https://doi.org/10.3390/xxxxx system
corners is formed
(42.7% vs. 0%), by non-enzymatic
changes and enzymatic
in the oral mucosa surface (89.6% vs. 3
molecules. Reduced glutathione (γ-glutamyl-cysteinyl-glycine)
0%), etc. The pro-inflammatory
is
IL-2
a tripeptide
level was
thatand
higher
repre-
anti-inflamm
sentsAcademic
the main Editor: Paul B.
non-enzymatic molecule. The exposure of RAW 264.7 cells to NPs corre-
Tchounwou HCV patients compared with those in the non-HCV group. Conclus
sponding to 50 µg Fe3+ /mL determined a significant increase of GSH level by 148% after
developed in the microvasculature both worsen the tissue trophism an
Received: 28
24 h related toMay
the2022
control (Figure 8a). At longer periods of treatment, this increase was
differentiation into coarse-fibrous connective tissue. Immunohistochem
Accepted:
by about 81% 21 and
July 2022
63% after 48 h and 72 h, respectively, compared to the control. Even if
Published: date
this increase is diminished in a time-dependent manner, the level of this non-enzymatic
antioxidant remains higher in comparison with the control. In the case of treatment with the
Publisher’s Note: MDPI stays neu- Keywords: oral mucosa; viral hepatitis C; cytokines; morphology; imm
higher dose, the level of GSH decreased by 66% and 42% after 24 h and 48 h, respectively,
1. Introduction
Hepatitis C virus (HCV) is the main cause of chronic liver d
alence of 3% [1]. About 58 million people in the world live wi
molecules. Reduced glutathione ( 𝛾 -glutamyl-cysteinyl-glycine) is a tripeptide that
represents the main non-enzymatic molecule. The exposure of RAW 264.7 cells to NPs
corresponding to 50 μg Fe3+/mL determined a significant increase of GSH level by 148%
after 24 h related to the control (Figure 8a). At longer periods of treatment, this increase
was by about 81% and 63% after 48 h and 72 h, respectively, compared to the control. Even
Pharmaceutics 2023, 15, 552 if this increase is diminished in a time-dependent manner, the level of this non-enzymatic 12 of 18
Article antioxidant remains higher in comparison with the control. In the case of treatment with

the higher dose, the level of GSH decreased by 66% and 42% after 24 h and 48 h,
respectively,
and increasedand increased
by 44% byh,44%
after 72 after 72
although at h, although
this at this
time point, thetime
resultpoint, the statistically
was not result was
Viral Hepatitis C Patients: A Cross-Sectional Studynot statistically

significant. significant.
1 Department of Therapeutic Stomatology, Yerevan State Medical University (YSMU), 2 Koryun Str.,


7 Department of Diagnostic Radiology, Yerevan State Medical University (YSMU), 2 Koryun Str., Yerevan

8 Department of Pediatric Dentistry and Orthodontics, Yerevan State Medical University (YSMU), 2 Koryun

Abstract: Background: The objective was to reveal the most typical changes in oral mucosa in HCV
patients and compare them with those in HCV negative patients. Methods: The study involved 96
HCV Figure
patients
Figure 8.8.and 100 patients
Antioxidant
Antioxidant withoutof
response
response HCV
of RAW
RAW who applied
264.7
264.7 to a dental
cells after
cells after 48,clinic.
24, 48,
24, and 72
and The
72 hh content
exposure
exposure of to
cytokines
to two
two doses
doses of
of
IL-2, IL-4, IL-10 and ɤ-Fe
dextran-coated
dextran-coated ɤ-INF Oin
-Fe22O the corresponding
33 NPs
NPs oral fluid was to
corresponding determined
to 50 and 500by μgELISA.
µg 3+
Fe /mL.
Fe 3+
Buccal
/mL. (a) mucosa
(a)level
level of and gums GSH
ofintracellular
intracellular GSH
K.; Manrikyan, M. Morphological biopsies passed histological
concentration;
concentration; (b) examination.
(b) enzymatic
enzymatic activityAn
activity of immunohistochemical
ofSOD;
SOD; (c)
(c)enzymatic
enzymaticactivity study
activity of of
ofCAT;mucous
CAT; (d) membrane
(d) enzymatic
enzymatic activity
activity
of
biopsies GPX. Data
was performed
of GPX. (n = 3)
Data (n = using are expressed
3) are monoclonal as
expressed asmouserelative values
antibodies
relative values (%)(%) related
to CD3+ and
related to control
toCD20+. ±
controlResults:SD.
± SD. TheThe results
HCV were
Theresults were
Changes in the Oral Mucous
considered statistically significant at * p < 0.05, ** p < 0.01 and *** p < 0.001 (sample vs. control).
Membrane in Viral Hepatitis C patients group included
considered 96 (63.5%
statistically males),
significant at * and the non-HCV
p < 0.05, ** p < 0.01 group
and ***included
p < 0.001100 subjects
(sample (62.0%
vs. control).
Patients: A Cross-Sectional Study. males) with lesions of the oral mucous membrane. The lesions of lips and oral mucosa were more
Antioxidant enzymesenzymes such such asassuperoxide
superoxidedismutase
dismutase (SOD), catalase (CAT), and and
Int. J. Environ. Res. Public Health 2022, frequent inAntioxidant
HCV than in the non-HCV group—e.g., erosion (13.5% vs.(SOD),
1%), catalase
cracks in (CAT),
the mouth glu-
glutathione
tathione peroxidase
peroxidase (GPX)(GPX) workwork together
together to to counteract
counteract the the toxic
toxic ROS ROS produced
produced inin cells.
cells.
18, x. https://doi.org/10.3390/xxxxx corners (42.7% vs. 0%), changes in the oral mucosa surface (89.6% vs. 3.0%), hemorrhages (78.1% vs.
The specific activity
The specific activity of of SOD
SOD(Figure
(Figure8b) 8b)increased
increased in in
thethecellscells exposed
exposed to NPsto NPscor-
Academic Editor: Paul B. 0%), etc. The pro-inflammatory IL-2 level was higher and anti-inflammatory IL-4 level was lower in
corresponding
responding to to 50 50µgμg Fe3+
Fe/mL
3+ /mL by 58% after 24 h and returned
by 58% after 24 h and returned to the control level level after
after
48
48 hh and 72 h. However, post-treatment
post-treatment with with NPsNPs corresponding
correspondingto to500500µg μgFe Fe3+3+/mL,
/mL, this
this
Received: 28 May 2022 enzymatic
enzymatic activity decreased by 15% after 24 h, increased significantly by 57% after 48
activity decreased by 15% after 24 h, increased significantly by 57% after 48 hh
differentiation into coarse-fibrous connective tissue. Immunohistochemical findings indicated a de-
Accepted: 21 July 2022 and
and returned
returned to to the
the control level after 72 h.
Published: date The
The specific
specific activity of CAT
CAT (Figure
(Figure8c)
8c)increased
increasedsignificantly
significantlyby byabout
about40%40%after
after2424h
hand
and4848h hofofexposure
exposuretotoNPsNPscorresponding
correspondingtoto5050µg 3+3+/mL.
μgFeFe /mL. After 72 h, this
this enzymatic
enzymatic
Publisher’s Note: MDPI stays neu- Keywords: oral mucosa; viral hepatitis C; cytokines; morphology; immunohistochemistry
activity
activityraised
raisedsignificantly
significantlybyby 193%
193% compared
compared to the control.
to the The treatment
control. of RAW
The treatment of 264.7
RAW
claims in published maps and institu- 264.7with
cells cellsNPs
withcorresponding
NPs corresponding to 500toμg500
Feµg Fe3+determined
3+ /mL /mL determined no significant
no significant changechange
in the
tional affiliations. in the specific
specific activityactivity
of CAT of CAT
after after
24 h24andh and a significant
a significant increasebyby171%
increase 171%and and 510%,
510%,
respectively, after 48 h and 72 h.
respectively,
1. Introduction
The specific activity of GPX (Figure 8d) in the RAW 264.7 cells exposed to NPs cor-
Hepatitis
responding C virus
to 50(HCV)
µg Fe3+ is/mL
the main cause of
decreased chronic liveronly
significantly disease,
afterwith
72 h,aby
global
20%prev-
related to
alencecontrol.
of 3% [1].
High About
dose58 million people
administration in the world
significantly live with
inhibited chronic
the specificHCV infection,
activity of GPX by
with 1.5
25%million newly
only after infected
24 h, people
then after each
48 h, onlyyear [1]. activation by 18% was noticed, and after
a slight
publication under the terms and con-
The
72 h,extrahepatic manifestations
the level returned to normal. of viral hepatitis C, caused by HCV were first spo-
ken about The
in theRAWearly 1990s
246.7 [2]. They
murine were shown
macrophage cellto develop
line is usedamong 74%natural
to screen of HCV andinfected
engineered
ativecommons.org/licenses/by/4.0/). individuals
products[3,4]. The most
to predict common
their potentialHCV extrahepatic
effect in vivo [64]. manifestations
Macrophage occur in the
responses tooral
different
NPs are influenced by dose, size, composition, and surface properties and uptake these by
endocytosis or phagocytosis, undergoing polarization [65].
Int. J. Environ. Res. Public Health 2021, 18, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/ijerph
In the presence of serum-containing culture media, NPs’ surfaces form protein coronae
that confer properties involved in their interaction with cells. Previously, it was shown
that the uptake of NPs by macrophages was directly related to the amounts of proteins
adsorbed on the NPs’ surface [66]. Also, studies in vivo revealed that dextran-coated
supermagnetic iron (SPIO) NPs presented on their surface, mannan-binding lectins bound
to the dextran coating, and histidine-rich glycoprotein and kininogen bound to the iron
oxide part. Also, secondary binders such as complement lectin and contact clotting factors
were identified [33].
In our study, the uptake of NPs containing protein corona is highlighted by the increase
of Fe3+ ions concentration in the treated RAW 246.7 cells, these ions being released from
the maghemite core. There is a significant probability that Fe3+ ions to be complexed by
low molecular weight compounds such as citrate, nucleotides, and glycosaminoglycans.
These Fe3+ -chelates could be reduced to Fe2+ by NAD(P)H-dependent flavoenzymes, lipoyl
dehydrogenase being probably the dominant catalyst within cells [67]. As a result, the level
of Fe2+ probably increased, and the Fenton reaction occurred, generating hydroxyl radicals
(HO•) and hydroxide ions (OH−).
On the other hand, due to the interaction of NPs with RAW 246.7 cells, NADPH
oxidase could be activated, producing superoxide anions (O2 •− ), as was proved in the
case of other types of NPs [68]. Metabolism of iron occurs predominately in mitochondria,
and its overload can also affect mitochondrial function because this transition metal is
involved in ROS production, which can diminish the expression of mitochondrial-encoded
respiratory chain subunits [69]. Superoxide passage by simple diffusion inside cells is
highly unfavorable [70], but this anion can use anion channels of membranes to enter
cells [71]. Superoxide can also react with H2 O2 in the Haber-Weiss reaction, catalyzed by
iron [72], also generating hydroxyl radicals (HO•).
ROS can attack PUFA and generate MDA, which is an end-product of lipid peroxi-
dation. These considerations are supported by the same pattern of cellular Fe3+ and ROS
levels up to 48 h of exposure. The highest level of MDA was registered after 24 h, being
correlated with ROS concentration. Later on, MDA concentrations decreased in a time-
dependent manner, probably due to the activation of the non-enzymatic and enzymatic
antioxidant systems.
Alternatively, superoxide can be dismutated in the reaction catalyzed by SOD, gen-
erating H2 O2 and molecular oxygen [73]. In mammalian cells, there are 2 types of SOD:
Cu/Zn-SOD, present mainly in the cytosol, and mitochondrial MnSOD. The lower dose
(50 µg Fe3+ /mL) of NPs determined an increase in the total SOD activity after 24 h of
exposure, followed by a decrease to the control level after 48 h and 72 h, respectively.
Probably, this decrease occurred due to the significant level of H2 O2 generated, which
could inhibit Cu/Zn-SOD [74]. Also, the exposure to the higher dose (500 µg Fe3+ /mL) of
NPs slightly diminished the total SOD after 24 h, probably due to the same reason. Instead,
the increase of total SOD after 24 h in cells exposed to the lower dose we assumed it might
be a consequence of the Mn-SOD activation. As shown in a previous study [75], Mn-SOD
activity could be induced in macrophages as a mechanism for the acquirement of oxidative
stress resistance through the participation of PARP1 in the regulation of Mn-SOD gene
expression. Mn-SOD is possible to respond to an endogenous redox imbalance that involves
superoxide leakage as a result of disruption of mitochondria functions in iron-overloaded
cells. However, how much Mn-SOD contributes to the strongly elevated level of total SOD
after 24 h is unclear and needs to be further investigated. Possibly, after 48 h, the incorpora-
tion of iron instead of Mn into Mn-SOD switched its activity to a prooxidant peroxidase
that utilizes H2 O2 for the oxidation of other molecules [76]. Previously, it was also proved
that the accumulation of iron decreased the expression and activity of Mn-SOD [77]. In
our opinion, after 48 h, the high concentration of Fe2+ generated an increase in Mn-SOD
activity due to the two-ion switch. After 72 h, possibly the accumulation of Fe2+ decreased
the expression and activity of Mn-SOD.
Article
CAT catalyzes the reaction of decomposition of H2 O2 , a product of the reaction Morphological Changes in the Oral
catalyzed by SOD, in water and molecular oxygen. CAT is a porphyrin-containing tetramer
that is located mainly in peroxisomes [78]. The variation of CAT-specific activity in time Viral Hepatitis C Patients: A Cross-
can be correlated with that of total SOD in the case of treatment with the lower dose. In the
case of the higher dose, the significant Vahe increase
Azatyanof 1,*, Lazar Yessayan 1, Aelita Sargsyan 2, Anna Kha
CAT-specific activity after 48 h could be
correlated with the high total SOD Melanya
activity.Shmavonyan
Possibly, the high 5, Gayane Melik-Andreasyan 6, Kristina
quantity of H2 O2 generated
Article after the switch of Mn withArticle iron ions was involved in the up-regulation of Mn-SOD and
Morphological Changes in the OralMorphological

Mucous Membrane
Changesinin the Oral M
CAT gene transcription [79]. 1 Department of Therapeutic Stomatology,
Article The level of H2 O2 produced in cells can be controlled not onlyYerevan by CAT activity
0025, Armenia; butlazyes@yandex.r
also
Viral Hepatitis C Patients:Changes

Morphological A Cross-Sectional
Viral Hepatitis
in the OralStudy
C Patients:
Mucous A Cross-Se
Membrane in
by GPX. This latter enzyme catalyzes H2 O2 decomposition in the 2 Tuberculosis
presence ofResearch GSH toand formPrevention Ce
water and oxidized glutathione (GSSG). The Michaelis constant forsargsyan.aelita@gmail.com H2 O2 is 50 times higher
for CAT compared to that of GPX [80]. GPX is the first enzymeDepartment of Pathological Anatomy, Ye
3

Vahe Azatyan *, Lazar Yessayanhigh
1, 1 , Aelita
levelsSargsyan
of ROS, ,playing
2 Anna Vahe Khachatryan
Azatyan , role
an important 3 Tigran
1, *, Lazar
as aGhevondyan
Yessayan
first line of4, defense
1 0025,
Aelita
4 ,
that is activated
Armenia;
Sargsyan 2
against oxidative
under
, Anna Khachatr
Laboratory of Histochemistry and Electro
Melanya Shmavonyan 5, Gayane stressMelik-Andreasyan 6 , KristinaMelanya
Porksheyan Shmavonyan 7
[81]. However, taking into account the high level of CAT activity, and Mikael
5 , Gayane Manrikyan
Melik-Andreasyan
Academy
8 we of could
6, Kristina Porks
Sciencesconsider
of Republic Armeni
Vahe Azatyan 1,that
*, Lazar Yessayan
theArticle 1, Aelita Sargsyan 2, Anna Khachatryan 3, Tigran Ghevondyan 4,
quantity of H2 O2 generated was removed by CAT action.tigranghevondyan@yahoo.com
Melanya Shmavonyan 5, Gayane Melik-Andreasyan 6, Kristina GSH Porksheyan 57 and Mikael ofManrikyan
1 DepartmentGPX of also catalyzes
Therapeutic the reaction
Stomatology, Yerevanbetween
State Medical and lipid
University peroxides,
1 (YSMU),
Department Department playing
2 Koryun
of Infectious
Therapeutic
Str., a role the8
inDiseases,
Stomatology, Yerev
Yereva
detoxification
Yerevan 0025, Armenia; of these molecules. After exposure to the lower
lazyes@yandex.ru Yerevan Armenia;
dose, 0025, sh_melaniya@mail.ru
GPX-specific
Armenia; activity
lazyes@yandex.ru
6 National Center of Disease Control and P

2 Tuberculosis
was not Research
changed 1 and Prevention
after 24 hofandCenter, 6/2 Adonts
decreased Str., 100
after the Apt., Yerevan
other Tuberculosis
0014, Armenia;
Research and Prevention Center,
Str., 6
2 time intervals, suggesting
Department Therapeutic Stomatology, Yerevan State MedicalYerevan University (YSMU),
0025, Armenia; 2that
Koryun
melikandreasyan
this enzyme wasYerevan not involved in the removal
0025, Armenia; lazyes@yandex.ru of MDA. sargsyan.aelita@gmail.com
Taking into account the pattern
7 Department of Diagnostic Radiology, Yer
3 Department of Pathological Anatomy, Yerevan State Medical University (YSMU), 3 Department 2 Koryun of Pathological
Str.,Yerevan
of GSH variation 2 Tuberculosis
corresponding Research toand
this Prevention
dose, probably Center, 6/2this Adonts Str.,
tripeptide 0025, 100Armenia;
Apt.,
was aYerevan
substrate Anatomy,
0014,
tina129@mail.ru
for Yerevan S
Armenia;
0025, Armenia;
Vahe ann_khachatryan@mail.ru
Azatyan 1,*, Lazar Yessayan 1, Aelita Sargsyan0025,
sargsyan.aelita@gmail.com 2, Anna Armenia; ann_khachatryan@mail.ru
Khachatryan 3, Tigran Ghevo
glutathione-S-transferase (GST), which could
4 Laboratory of Histochemistry and Electron Microscopy, After Orbeli Institute
be implicated in MDA removal [82]. At
8 Department of Pediatric Dentistry and Or
4 Laboratory
the
3 Department of Pathological Anatomy, Yerevan State of6 Physiology
Medical of Histochemistry
University of National
(YSMU), 2 and
Koryun Electron Yerev
7 and Str.,
Micr
same Melanya
time, in the
Academy of Sciences of0025,
Shmavonyan
case
Republic
of Armenia
5, Gayane
treatment (NAoS
of RAW
RA),
Melik-Andreasyan
22
246.7
Brothers
cells with
Orbeli
the
Str.,
,
Academy
Kristina
Str.,
higher
Yerevan Yerevan
of
0028,
Porksheyan
dose
Sciences 0025, Armenia;
ofRepublic
Armenia;
of
NPs, GPXArmenia
Mikael
dr.manrikya
(NAo
Armenia; ann_khachatryan@mail.ru * Correspondence: vahe.azatyan@gmail.com
was not efficient
tigranghevondyan@yahoo.com in MDA detoxification.
4 Laboratory of Histochemistry and Electron Microscopy, tigranghevondyan@yahoo.com
After Orbeli Institute of Physiology of Nation
cle 5 Department of Infectious
In the present Diseases,
study,ofat
Academy Yerevan
Sciences State
a lower 1 Medical
dose
of Republic
(50
Department University
µg Fe
Armenia
3+
of (NAoS (YSMU),
/mL),
Therapeutic
5 Department
RA),
the2Stomatology,
22 Koryun
GSH level
Brothers Str.,
ofOrbeli
Infectious
Yerevan
decreased
Yerevan Diseases,
0025,Medical
Str., State
Yerevan
after Yerevan
0028, Stat
Universi
Armenia
Abstract: Background: The objective w
orphological Changes in the Oral Mucous Membrane in
Armenia;
24 h and sh_melaniya@mail.ru
increased in a time-dependent manner up to 72 h Armenia;
when it sh_melaniya@mail.ru
became higher than the
6 National Center of Disease Control and Prevention, Citation:Yerevan
Azatyan, 0025, Armenia;
V.; Yessayan, L.;6 lazyes@yandex.ru
control level. 5The Department
higher dose of Infectious
(500 Ministry
2Diseases,
µg Fe 3+ /mL)
Tuberculosis of Health
Yerevan State(MoH),
Research
increased National
and
Medical 12patients
the Mkhitar
Prevention Center
University
GSH Heratsi
and of compare
Center, Disease
(YSMU),
level by Str.,
6/2 Control
Adontsthem
2 Koryun
150% and
with
Str.,
after Preventi
thoseY
100Yerevan
Str., Apt.,
Yerevan 0025, Armenia;Armenia; Sargsyan, A.; Khachatryan, A.;
24 h exposure andmelikandreasyan@mail.ru Yerevan 0025, Armenia;and melikandreasyan@mail.
ral Hepatitis C Patients: A Cross-Sectional Study decreased later on, sargsyan.aelita@gmail.com
sh_melaniya@mail.ru remaining higher than HCV
the patients
control level 100
at allpatients
time without
7 Department of Diagnostic Radiology, Yerevan Ghevondyan,
State T.; Shmavonyan,
Medical University M.;
(YSMU),
7 Department 2 Koryun of Str.,
Diagnostic
Yerevan Radiology, Yerevan
intervals. 6 National Center of Disease
Previous studies revealed that Department
3 Control
dextran-coated of Pathological
and Prevention, maghemite Anatomy,
Ministry IL-2, Yerevan
ofnanoparticles
Health
IL-4, (MoH),
IL-10 State
and12(Medical
Mkhitar
ɤ-INF
-Fe OinUniversity
Heratsi
the oral StrS(
0025, Armenia; tina129@mail.ru Melik-Andreasyan, G.; Porksheyan, 0025, Armenia; tina129@mail.ru 2 3
NPs) modulate
8 Department of Pediatricthe GSH level
Dentistry and in aK.;cell-type-dependent
Orthodontics,
Manrikyan, Yerevan State
M. Morphological manner
Medical8 Departmentbiopsies
University[47,83]. ofpassed
(YSMU),
Pediatric histological
2 Koryun
Dentistry andexaminati
Orthodon
7 Department of Diagnostic Laboratory
4 Radiology, of Histochemistry
Yerevan State
4, Medical and Electron
University Microscopy,
(YSMU), After
2 Koryun Orbeli
Str., Insti
Yerev
e Azatyan 1,*, Lazar Yessayan 1, Aelita
Str.,Sargsyan
Yerevan
Our0025, 2, Anna
Armenia;
results Khachatryan
highlight the need3, Tigran
dr.manrikyan@mail.ru to
Changes evaluate
in the
Ghevondyan
Oral in-depth
Mucous theStr., biopsies
Yerevan
interactions was
0025, performed
Armenia;
between using monoclo
dr.manrikyan@mai
IONPs
0025, Armenia; tina129@mail.ru Academy of Sciences of Republic Armenia (NAoS RA), 22 Brothers Orbel
anya Shmavonyan 5, Gayane Melik-Andreasyan
* Correspondence:
and cells to take , Kristina Porksheyan
6vahe.azatyan@gmail.com;
full advantage
8 Department Tel.:
ofMembrane
of Pediatric and
the7Dentistry Mikael
+374-91-326773
intrinsic
in Manrikyan
properties
and
Viral C of
Orthodontics,
Hepatitis * these
Correspondence:
8
Yerevan patients
NPs ingroup
State vahe.azatyan@gmail.com;
biological
Medical includedsystems.
University (63.5%2Tel.:
96(YSMU), ma
Ko
Str., Yerevan 0025, Armenia; 5 Department
Patients: Adr.manrikyan@mail.ru
Cross-Sectional of Infectious
Study. males)
Diseases, with lesions
Yerevan State Medicalof theUniversity
oral mucous (YSM
Abstract:
4. Background:
Conclusions * The objective
Correspondence: was to reveal the
Armenia;
vahe.azatyan@gmail.com; most typical
sh_melaniya@mail.ru
Tel.: changes
Abstract:
+374-91-326773 in
frequentBackground:
oral mucosa in
The HCVobjective was to r
Int.
1 Department of Therapeutic Stomatology, Yerevan State Medical University (YSMU), 2 Koryun
J.
Citation: Azatyan,
V.; Yessayan, L.;
6 National
Health 2022, Str., in HCV than in the non-HCV
patients and compare them with those in HCV negative Center
patients.of the
Disease
Methods: Control
patients The
and and
study Prevention,
compare involved
them Ministry
with
96 of Health
those in HC (M
The
Sargsyan, A.; Khachatryan, A.; Yerevan 0025, Armenia; lazyes@yandex.ru
present study was focused 18, on
x. the response
Sargsyan, A.; Khachatryan,
of corners (42.7% vs. 0%), changes in the
endogenous
A.; Armenia; melikandreasyan@mail.ru
antioxidant defense o
Abstract: Background: The Yerevan
objective 0025,
was to reveal the most typical changes in oral mucosa inw
2 Tuberculosis HCVsystempatients and 100 patients without
Research and Prevention Center, 6/2 Adonts Str.,
induced in RAW 264.7 HCV
macrophages who applied to a dental
100 Apt., Yerevan 0014, Armenia;
upon exposure HCV
clinic.
to patients
The content
dextran-coated
0%), etc. and
The 100
of cytokines
patients
maghemite
pro-inflammatory without HCV
IL-2 lev
Ghevondyan, T.; Shmavonyan, M.; Azatyan, V.; Yessayan, L.;
Citation: Ghevondyan, Academic7 T.;Department
Shmavonyan,
Editor: Paul of M.;
B. Diagnostic Radiology, Yerevan State Medical University (Y
IL-4, IL-10 and(patients
IL-2, nanoparticles -Fe2 Oin3and
ɤ-INF the
NPs). compare
oral Thefluid them with
was0025,
dextran-coated
Melik-Andreasyan,
those maghemite
determined
G.;Armenia;
Porksheyan,
inby HCV negative
ELISA.
IL-2,
tina129@mail.ru
Buccal
IL-4, patients.
IL-10
nanoparticles
HCV mucosa
patients andMethods:
and-Fegums
ɤ-INF
(compared 2O inThe
3 the
NPs)study
with oral involv
fluid
those inw
3 Department Tchounwou
of
biopsies Pathological
were passed
obtained Anatomy,
HCV
histological
using Yerevan
patients State
and
examination.
an adapted 100Medical
patients
An University
without
8 immunohistochemical
coprecipitation (YSMU),
HCV
method. 2
who Koryun
applied
biopsies
study
The Str.,
of toYerevan
a
passed
morphology dental
mucous clinic.
histological
membrane
of the The content
examination.
dextran- of cyto
An
K.; Manrikyan, M. Morphological
Ghevondyan, T.; Shmavonyan, M.; Department
K.; Manrikyan, M. Morphological of Pediatric Dentistry and Orthodontics,
developed in the microvasculature both Yerevan State Medic
biopsies
coated was performed
using IL-10
monoclonal
nanoparticles and ( ɤ-INF
Received:
mouse
-Fe Oin28
Str., the
May
antibodiesoral
2022
Yerevan
NPs) was fluid
to
0025, CD3+was determined
biopsies
investigatedand
Armenia; CD20+. was by
Results:
performed
by TEM ELISA.
and The
SEM Buccal
using
HCV mucosa
monoclonal
studies. andmo
Changes in the Oral Mucous Melik-Andreasyan,
4 LaboratoryG.; of Porksheyan,
Histochemistry and Electron Microscopy, Changes inAfter the Oral
2 3 Mucous
Orbeli Institute of Physiologydifferentiation
of National into coarse-fibrous conn
biopsies passed Accepted:
histological *Viral 21 July 2022
Correspondence:
examination. vahe.azatyan@gmail.com;
An immunohistochemical Tel.:study
+374-91-326773
of mucous mem
Membrane in Viral Hepatitis K.; C Academy
Manrikyan, patients
M. of Thegroup
Sciences SEM
Morphological included
and
of Republic TEM 96micrographs
Armenia (63.5%
(NAoS males),
Membrane 22and
RA),highlighted the
inBrothers non-HCV
Hepatitis
Orbeli that the
C Str., group
Yerevan patients
included
particles 0028, group
were
crease 100
in
Armenia; subjects
included
well-dispersed,
local humoral(62.0%
96 (63.5%pre-
immune males),
respon an
Published: datespherical shape. The particle size distributions
males) senting
Study.tigranghevondyan@yahoo.com
Patients: A Cross-SectionalChanges in the Oral Mucous a biopsies
with lesions of the oral mucous
nanometric was
size performed membrane.
distribution
Patients: usingand monoclonal
A Cross-Sectional The lesions mouse
Study. of lips antibodies
males)
and oral to
withmucosa CD3+
lesions were and
of themoreCD20+. Results:
oral mucous memb The
Int. J. Environ. Res. Public Health5 Department of
Membrane frequent
2022, in Viral Infectious
inC HCV
determined
Hepatitis patients
Diseases,
than
from in the
TEM group
Yerevan State
non-HCV
and included
SEM
Int. Medical Abstract:
96
group—e.g.,Res.(63.5%
University
micrographs
J. Environ. Public Background:
males),
(YSMU),
erosion
were
Health 2andKoryun
(13.5%
2022,12.4 The
the
frequent
nmvs. objective
non-HCV
Str.,
1%),
and Yerevan
incracks
13.1HCV was
group
nm.0025,
in toMoreover,
than reveal
the included
inmouth
thethe most
100
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Citation: Azatyan, V.; Yessayan, L.; Publisher’s Note: MDPI stays neu- Keywords: oral mucosa; viral hepatitis
18, x. https://doi.org/10.3390/xxxxx Armenia; sh_melaniya@mail.ru
HRTEM
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vs. 0%), males)
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Melik-Andreasyan, G.; Porksheyan, 2 3
Tchounwou Department Diagnostic Yerevan State
TchounwouMedical University (YSMU), 2 Koryun Str., Yerevan
studies,
developed the
inK.;the dextran
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Academic 0025, Armenia;
Editor: Paul B.tina129@mail.ruManrikyan, Morphological 1. Introduction
Received: 28 May 2022 Received: 28 May 2022
biopsies wasinperformed using monoclonal mouse
8 Department of
Tchounwou
ensure
Pediatric
differentiation the very
Dentistry
into
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Yerevan with nanoparticles.
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Conclusions:
2 Koryun
findings into
indicated aantibodies
nanoparti-
coarse-fibrousMorphological
de- to CD3+
connective ch
Accepted: 21 July 2022 Str., Yerevan cles0025, functionalized
Armenia; dr.manrikyan@mail.ru
with Accepted:
dextran 21 patients
could Julybe 2022 group
successfully includedapplied 96 (63.5%
in Hepatitis
males),
different C virus
and
biologicalthe
(HCV)
non-HCV
and isgrou
the
crease in local Membrane
humoral developed
in Viral
immune inresponse.
Hepatitis the
C microvasculature
Copyright: © 2022both by the worsen
authors. the tissue
crease trophism
in local humoral andimmuneaccelerate the healin
response.
Published: date * Correspondence:
Received: 28 May 2022medical vahe.azatyan@gmail.com; Tel.:Published: date
+374-91-326773 males)
alence of 3% [1]. About 58 million
fields.
Patients: Adifferentiation
Cross-Sectional Study.
into coarse-fibrous
Submitted for with lesions
connective
possible of the oral
tissue.
open access mucous membrane.findings
Immunohistochemical The lesions of li
indicated
Accepted: 21 July 2022 with 1.5 million newly infected peo
Publisher’s Note: MDPI stays neu- RAWInt.
Keywords: oral mucosa; J. 246.7
Environ. cells
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MDPI in
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immunohistochemistry group—e.g.,
by the
Keywords: oral mucosa; viral hepatitis C; cyt
exposureerosion (13.5%
Abstract:
date Background: The objective was to reveal theregard
most typical thechanges in oral mucosa The extrahepatic manifestation
in thein HCV
Published: to these18,NPs. This statement
tral with
on: Azatyan, regard toL.; jurisdictional
V.; Yessayan,
x. https://doi.org/10.3390/xxxxx tralis with
supported
ditions of thetoby
corners (42.7%
Creative increased
vs. 0%),
Commons
jurisdictional At- GSH
changes level andoral the
mucosa diminished
surface (89.6% vs
patients and compare
MDA them with those in HCV negative patients. Methods: The ken
study about
involved in the
96 early 1990s [2]. The
claims
yan, A.; in published
Khachatryan, A.; maps and institu- Note: MDPI
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Keywords:
246.7
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emphasized dental clinic.
affiliations. The content
that dextran-coated individuals
of cytokines
maghemite [3,4]. The most
nanopar- commo
ndyan, T.; Shmavonyan, jurisdictional
Tchounwou HCV compared with those in the non-HCV group. Conclu
IL-2, IL-4, IL-10
Andreasyan, G.; Porksheyan, claims in published maps and
ticles -Fe2 Oin3 the
(ɤ-INF
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in the microvasculature method andcould
both gums be used
worsen in thetrophism
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1. Introduction 1. Introduction
nrikyan, M. Morphological tionalbiopsies passed
affiliations. histological
medical Received:
and examination.
28 May 2022
pharmaceutical An immunohistochemical
fields due to their study
physicochemical of mucous and membrane
biological
differentiation into coarse-fibrous connective tissue. Immunohistoch properties.
biopsies was performedHepatitis C virus
Accepted:
using 21 July(HCV)
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Copyright: © 2022 by the authors. 1. Introduction Copyright: Int. J.
©crease
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Submitted C
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Submitted for possiblegroup open access included subjects (62.0%
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[1]. main cause
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ts: A Cross-Sectional
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https://doi.org/10.3390/xxxxx corners
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Author Contributions: Conceptualization, D.P. and A.D.; methodology, D.P. and A.D.; software,
M.B., D.P. and S.L.I.; validation, D.P., A.D., M.B., S.L.I. and N.B.; formal analysis, D.P.; investigation,
M.B. and S.L.I. resources, D.P., A.D. and N.B.; data curation, D.P., A.D., M.B., S.L.I. and N.B.;
writing—original draft preparation, D.P., A.D., M.B. and S.L.I.; writing—review and editing, D.P.,
A.D., M.B. and S.L.I.; visualization, D.P., A.D., M.B., S.L.I. and N.B.; supervision, D.P., A.D., M.B.,
S.L.I. and N.B.; project administration, D.P.; funding acquisition, D.P. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is available on demand from the corresponding authors.
Conflicts of Interest: The authors declare no conflict of interest.
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