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\begin{document}

\title{Determination of Adenovirus 5 and 37 seropositivity in obese patients}



\author[1]{Nur Gamze Bostan}%
\author[1]{Özge Altınok}%
\author[2]{Süleyman Büyükaşık}%
\author[2]{Halil Alış}%
\author[1]{Yaşar Ali Öner}%
\affil[1]{Istanbul Aydin University}%
\affil[2]{Istanbul Aydin University Faculty of Medicine VM Medical Park Hospital}%


\vspace{-1em}



  
  \date{May 28, 2024}


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\selectlanguage{english}
\begin{abstract}
Obesity is seen as one of the most important health problems of our age
due to its accompanying diseases such as diabetes, high blood pressure
and cholesterol. It is an epidemic that started in the 1980s and
continues worldwide, regardless of the level of development of the
countries, and is associated with many factors. In the face of the
rapidly increasing prevalence of obesity, it has been thought whether
microorganisms can play a role in the formation of obesity. Based on
this idea, the concept of `infectobesity' was introduced and studies on
this subject have shown that some microorganisms cause obesity in
experimental animal models such as mice, chickens and non-human
primates. One of the microorganisms thought to cause obesity is
Adenoviruses. Especially Adenovirus-36 (Ad36), Adenovirus-5 (Ad5) and
Adenovirus-37 (Ad37) serotypes are thought to have effects on fat
regulation in the body. There are very few studies investigating the
relationship between Ad5 and Ad37 serotypes and obesity in humans. The
aim of our study is to increase the research on this subject, to develop
a new perspective on the infectious etiology of obesity, and to
contribute to the development of new treatment approaches for obesity
and obesity-related diseases.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Obesity is considered one of the most significant health problems of our
time due to its accompanying diseases such as diabetes, hypertension,
and high cholesterol (1). It started in the 1980s and has continued
worldwide regardless of the countries' levels of development, being
associated with various factors. Among these factors are environmental,
genetic, endocrine factors, nutrition, and lifestyle (2). Despite the
emphasis on nutrition, changes in nutrition and lifestyle have not been
seen to completely solve the problem.

In the face of the rapidly increasing prevalence of obesity, the role of
microorganisms in the formation of obesity has been considered (3).
Based on this idea, the concept of ''infectobesity'' has been put
forward, and studies on this subject have shown that some microorganisms
can cause obesity in animal models such as mice, chickens, and non-human
primates. Since 1982, viruses associated with obesity have been
identified, including Canine distemper virus (CDV), Rous-associated
virus-7 (RAV-7), Borna disease virus (BDV), Scrapie agent prion, and
SMAM-1 from animal viruses; and from human viruses, only Adenovirus 36
(Ad-36), Adenovirus 37 (Ad-37), and Adenovirus 5 (Ad-5) from the
Adenoviridae family (4).

Adenoviruses are non-enveloped, icosahedral symmetric, and medium-sized
viruses. All adenoviruses that infect humans share the same common
genome; they contain a single linear and double-stranded DNA molecule.
There are more than 50 serotypes infecting humans (5).

Adenoviruses associated with obesity are believed to cause triglyceride
accumulation in fat cells (adipocytes) and differentiation of immature
adipocytes (pre-adipocytes) into mature adipocytes (3). It has been
determined that Ad-5 and Ad-37 increase the formation of fat cells in
animals and contribute to the development of obesity. Ad-36 is the most
studied human adenovirus to date, and its association with human obesity
has been proven (6).

Leptin hormone is secreted by adipocytes of adipose tissue, reduces
appetite, increases energy expenditure, stimulates the breakdown of fat
cells, and inhibits lipid synthesis. It is known that insufficient
synthesis of leptin leads to more fat storage. To investigate the
mechanism of adipose tissue formation associated with adenovirus, the
secretion of leptin in human pre-adipose cells infected with
Adenoviruses was examined. It was found that in cells infected with
Ad-37, leptin mRNA synthesis was suppressed, and lipid accumulation
increased simultaneously. Additionally, changes in enzymes such as
intracellular glycerol 3-phosphate dehydrogenase (GPDH) induced the
transformation of pre-adipocytes into mature adipocytes (3).

The aim of our study is to investigate the seropositivity of Adenovirus
5 and 37 in blood samples taken from 48 obese patients and 42 normal
weight individuals without underlying chronic diseases, not using any
medication routinely, and without immune system problems, and whether
there is a significant difference between obese and normal weight
individuals. Increasing the number of studies on this subject and
developing a new perspective on the infectious etiology of obesity will
contribute to developing new treatment approaches for obesity and
obesity-related diseases.

\selectlanguage{polish}\textbf{MATERİALS AND METHODS}\selectlanguage{english}

This study was conducted with the approval of the Istanbul Aydin
University Non-Interventional Clinical Research Ethics Committee with
decision number 2023/16. Study groups were formed based on volunteerism,
and individuals aged between 18-60 years without any chronic diseases
and immune deficiencies were selected for obese and control groups. For
obese patients, individuals with BMI [?]30 and for healthy groups,
individuals with BMI greater than 18.5 and less than 25 were selected.
10 ml of fasting blood was taken from 48 patients and 42 healthy
individuals who met these criteria. The collected blood samples were
centrifuged to separate the sera and stored at -20\selectlanguage{ngerman}°C until the study was
conducted.

The seropositivity of Ad-37 and Ad-5 in the collected study samples was
investigated using ELISA test kits, which can demonstrate
antigen-antibody reactions through enzymes. The serum samples of obese
and healthy individuals were tested according to the instructions of the
test kits. Optical density measurements at a wavelength of 450 nm were
made using the Biotek-Epoch brand ELISA microplate reader.

The obtained results were interpreted as positive or negative by taking
the average of the standard samples included in the ELISA kits and
accepting it as the cutoff value. Normality analyzes were examined to
analyze the data, and it was observed that the data were not normally
distributed. Independent two-group Mann-Whitney U test, a non-parametric
analysis, was used to determine the statistical significance of the
results and the relationship between them, and a significance level of p
\textless{}0.05 was accepted.

\textbf{RESULTS}

The average age of the patient group from which the samples were
collected was found to be 39.69 ± 12.17, and the average age of the
control group was 24.71 ± 7.79. In the patient group, there were 20
females with an average age of 42.35 ± 10.47 and 28 males with an
average age of 37.65 ± 13.16. In the control group, there were 26
females with an average age of 24.84 ± 6.19 and 16 males with an average
age of 24.5 ± 10.10. The physical characteristics of the patient and
control groups are shown in Table I.

\textbf{Table I. Mean and standard deviation values of physical
characteristics of patient and control groups}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
& Patient & Control\tabularnewline
\midrule
\endhead
Height & 170,08 \selectlanguage{ngerman}± 13,2 & 169,09 \selectlanguage{ngerman}± 11,1\tabularnewline
Weight & 119 \selectlanguage{ngerman}± 21,11 & 63,15 \selectlanguage{ngerman}± 10,9\tabularnewline
Body Mass Index & 40,72 \selectlanguage{ngerman}± 6,8 & 22,16 \selectlanguage{ngerman}± 1,97\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

In the patient group (n=48), Ad-37 antibody positivity was detected in
39 samples, while in the control group (n=42), it was detected in 24
samples. According to the Mann Whitney U test result, a significant
difference (p =0.011) was found in favor of the patient group.

For Adenovirus 5, antibody positivity was detected in 26 individuals in
the patient group and 26 individuals in the control group. According to
the Mann Whitney U test result, no statistically significant difference
(p=0.461) was found.

More positivity for Adenovirus 37 was detected in the patient group
compared to the control group. However, for Adenovirus 5, the number of
positive individuals was equal in both patient and control groups. IgG
positivity for both Ad-5 and Ad-37 was detected in 28 individuals in the
patient group and 22 individuals in the control group. In 9 patient
individuals who tested negative for Ad-37, Ad-5 was also negative. Ad-37
and Ad-5 were found to be more positive in male individuals in the
patient group and female individuals in the control group. Comparison of
Adenovirus 5 and 37 seropositivity by gender distribution in patient and
control groups is shown in Table II.

\textbf{Table II. Distribution of Ad-37 and Ad-5 antibody presence in
patient and control groups by gender.}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& Adenovirus 37 IgG & Adenovirus 37 IgG & Adenovirus 5 IgG & Adenovirus
5 IgG\tabularnewline
\midrule
\endhead
& \textbf{Positive} & \textbf{Negative} & \textbf{Positive} &
\textbf{Negative}\tabularnewline
Patient (n=48) \emph{Female (n=20)} \emph{Male (n=28)} & 39 \emph{14}
\emph{25} & 9 \emph{6} \emph{3} & 26 \emph{10} \emph{16} & 22 \emph{10}
\emph{12}\tabularnewline
Control (n=42) \emph{Female (n=26)} \emph{Male (n=16)} & 24 \emph{16}
\emph{8} & 18 \emph{10} \emph{8} & 26 \emph{15} \emph{11} & 16 \emph{11}
\emph{5}\tabularnewline
\bottomrule
\end{longtable}

There was no significant relationship found between the gender of
individuals in patient and control groups and the positivity of Ad-5 and
Ad-37 antibodies. The results of this relationship according to the Mann
Whitney U test are shown in Table III.

\textbf{Table III. Ad-37 and Ad-5 antibody positivity according to
gender distribution in patient and control groups.}

Control Group\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& \textbf{Gender} & \textbf{n} & \textbf{Rank Average} & \textbf{Rank
Sum} & \textbf{U} & \textbf{P}\tabularnewline
Ad-5 & Female & 26 & 22,38 & 582,00 & 185,000 & 0,479\tabularnewline
& Male & 16 & 20,06 & 321,00 & &\tabularnewline
Ad-37 & Female & 26 & 20,58 & 535,00 & 184,22 & 0,468\tabularnewline
& Male & 16 & 23,00 & 368,00 & &\tabularnewline
Patient Group & Patient Group & Patient Group & Patient Group & Patient
Group & Patient Group & Patient Group\tabularnewline
Ad-5 & Female & 20 & 23,50 & 470,00 & 260,00 & 0,628\tabularnewline
& Male & 28 & 25,21 & 706,00 & &\tabularnewline
Ad-37 & Female & 20 & 21,80 & 436,00 & 226,000 & 0,095\tabularnewline
& Male & 28 & 26,43 & 740,00 & &\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{polish}\textbf{DİSCUSSİON AND CONCLUSİON}\selectlanguage{english}

Obesity is defined as excessive weight gain resulting from the
accumulation of fat in the body, and it is a rapidly spreading health
problem worldwide. Factors such as increased consumption of processed
foods, sedentary lifestyle, and processed food habits brought about by
modern life are effective in the formation of obesity. Additionally,
various factors such as nutritional and psychological disorders,
endocrine system disorders, medication use, and genetic factors can
contribute to obesity (2). In recent years, studies have been conducted
on the concept of ''infectobesity,'' suggesting that some microorganisms
may also contribute to obesity. According to this theory, certain
infections can alter fat regulation in the body through various
mechanisms and increase body mass index (4). This theory is supported by
numerous pieces of evidence. Many reports investigating this issue have
explained how infections caused by pathogens alter adipose tissue
structure and support the role of infected adipocytes in obesity by
disrupting components such as fat tissue signaling pathways (7).

Understanding these microorganisms as etiological factors is essential
for effective management of obesity. Viral agents that are likely to
play a role in fat regulation in the body have been found in human and
animal studies conducted since 1982, including Canine Distemper Virus
(CDV), Borna Disease Virus, Scrapie Agent, Rous-associated virus-7,
SMAM-1, and Adenoviruses (8). Adenoviruses generally cause various
infections in humans, primarily respiratory tract infections. Studies
have shown that Ad-5, Ad-36, and Ad-37 may also cause obesity in humans.
It is believed that these adenovirus serotypes have a negative effect on
leptin production in infected adipocytes, leading to increased fat
storage (9, 10).

The metabolic effects of adenoviruses have attracted more attention
since it was shown in the 1990s that SMAM1 increases fat cell counts in
chickens and that antibodies against SMAM-1 are associated with human
obesity. Since then, a total of nine adenoviruses, including Ad-2, Ad-5,
Ad-8, Ad-9, Ad-36, and Ad-37, have been investigated for their metabolic
effects in cell cultures, animal studies, and human epidemiological
studies. Among these, Ad-36 is the most studied serotype and has been
most strongly associated with human obesity. While the association
between Ad-2, Ad-8, and Ad-9 serotypes and fat accumulation in cells was
not found in most studies, Ad-5 and Ad-37 have been identified as
potentially related to obesity in different studies (11).

In a study investigating the effects of different adenovirus serotypes
(Ad-36, Ad-2, Ad-9, and Ad-37) on leptin secretion and fat accumulation
in 3T3-L1 preadipocyte cells, it was observed that leptin secretion in
cells infected with Ad-36 was 50\% lower than in uninfected cells, while
lipid accumulation was significantly higher in cells infected with Ad-9,
Ad-36, and Ad-37, with lower leptin secretion. Ad-2 was found not to
affect lipid accumulation or leptin secretion (12). The decrease in
leptin, also known as the appetite-suppressing hormone, is thought to be
associated with weight gain at this level.

In our study, Ad-5 and Ad-37 serotypes were investigated in serum
samples collected from obese patients and normal-weight control groups
using ELISA kits. Ad-37 was found to be positive in 39 out of 48 obese
patients and in 24 out of 42 individuals in the control group, while
Ad-5 was positive in 26 individuals in both the patient and control
groups. According to the statistical analysis results, a significant
difference was found between the patient and control groups in terms of
Ad-37 positivity, supporting our hypothesis. The positivity of Ad-37
serotype was interpreted as a possible contribution to the development
of obesity. However, no significant difference was found for Ad-5,
contrary to our hypothesis.

In a study by Whigham et al., chickens were inoculated with Adenovirus
2, 31, and 37, and food intake and weight were monitored for three and a
half weeks. It was observed that chickens infected with Ad-37 had over
three times the visceral fat and over twice the total body fat compared
to the uninfected control group, despite consuming the same amount of
feed (13).

In a study conducted on 268 patients with non-alcoholic fatty liver
disease, the seropositivity of Ad-36 and Ad-37 was investigated. Ad-37
was positive in 65 patients, while both Ad-37 and Ad-36 were negative in
82 patients. The prevalence of obesity defined as a body mass index [?]30
did not differ significantly between patients positive for Ad-37 (11/65;
16.9\%) and those negative for Ad-37 (15/82; 18.2\%) (14).

In a two-stage study, antibodies for Ad-2, Ad-31, Ad-36, and Ad-37 were
searched for in the serum of 502 obese and non-obese individuals, and no
difference in antibody status was found between obese and non-obese
individuals. In the second stage, the body mass index and body fat of
twins discordant for Ad-36 antibody were compared between obese and
normal-weight individuals, and it was found that the twin positive for
Ad-36 antibody was heavier than their sibling. However, this
relationship could not be established for Ad-2, Ad-31, and Ad-37
serotypes (15).

In the molecular-level investigation of epithelial cells infected with
Ad-5, it was observed that Ad-5 has a metabolic effect similar to Ad-36
in triggering adiposity-related intracellular signaling mechanisms (11).

In a study by Cakmakl\selectlanguage{polish}ı\selectlanguage{english}o\selectlanguage{polish}ğ\selectlanguage{english}ullar\selectlanguage{polish}ı \selectlanguage{english}in 2011 investigating the seropositivity
of Ad-5, Ad-8, and Ad-36 in obese and non-obese children, the positivity
of Ad-36 and Ad-5 antibodies in the obese group was found to be
statistically significant compared to the non-obese group. The study
concluded that there was an association between Ad-5 and Ad-36 serotypes
and obesity in children (8).

In a study by So et al., mice injected with Ad-5 showed significantly
greater weight gain than the control group after 21 weeks. Additionally,
there was no significant difference in food intake between the study and
control groups, indicating that the weight difference was due to
adenovirus infection (16).

In a study investigating the effect of Ad-5 on hamsters fed a balanced
diet and a high-calorie diet, the experimental group was inoculated with
Ad-5, and body weight was measured weekly for short-term (22 weeks) and
long-term (44 weeks) periods. Adenovirus infection was shown to induce
hyperglycemia and hyperlipidemia in animals fed a balanced diet. After
44 weeks, a 30\% increase in body weight was reported compared to
unvaccinated animals, along with morphological changes associated with
non-alcoholic fatty liver disease. Similar but more severe changes were
observed in animals fed a high-calorie diet, with the animals with the
most morphological and functional changes having the lowest body weight
(17).

Studies investigating the relationship between Ad-5 and Ad-37 and human
obesity are limited worldwide. A definitive conclusion about whether
these serotypes can cause obesity has not been reached. Our study found
significant Ad-37 IgG positivity in obese individuals compared to the
control group. This result will contribute to the literature by
demonstrating the relationship between Ad-37 and obesity. However, Ad-5
IgG yielded insignificant results in both the obese and control groups.
The presence of twenty-four Ad-37 positive individuals in the control
group suggests previous exposure to this virus due to consumption under
unhygienic conditions.

Further research on this topic in different geographical areas could
provide clearer epidemiological information on viral obesity and
infectobesity in general. As our knowledge of the relationship between
microorganisms and obesity increases, the inclusion of antimicrobials in
the treatment options for obesity seems likely in the near future.

\textbf{REFERENCES}

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