CHAPTER ONE

1.0 INTRODUCTION

Persistent infections often lead to chronic inflammation, a well-documented cancer risk factor.

Gastric cancer (GC) generally starts with an inflammatory process mainly associated with

infection by Helicobacter pylori (H. pylori) (IARC, 1994). GC is the fourth most common type of

cancer and the second cause of death by cancer world-wide, affecting particularly Asian and Latin

American countries (Thun et al., 2010). More recently, GC has also been associated with Epstein-

Barr virus (EBV) but the role of the viral infection in early inflammatory gastric responses remains

poorly studied.

The EpsteinBarr virus (EBV), also called human herpesvirus 4 (HHV-4), is one of eight

known viruses in the herpes family, and is one of the most common viruses in humans. It is best

known as the cause of infectious mononucleosis (glandular fever). It is also associated with

particular forms of cancer, such as Hodgkin's lymphoma, Burkitt's lymphoma, gastric

cancer, nasopharyngeal carcinoma, and conditions associated with human immunodeficiency

virus (HIV), such as hairy leukoplakia and central nervous system lymphomas(Maeda et

al.,2009). There is evidence that infection with EBV is associated with a higher risk of

certain autoimmune diseases(Toussirot and Roudier,2008), especially dermatomyositis, systemic

lupus erythematosus, rheumatoid arthritis, Sjgren's syndrome and multiple sclerosis(Dreyfus,

2011; Ascherio and Munger,2010). Some 200,000 cancer cases per year are thought to be

attributable to EBV.Infection with EBV occurs by the oral transfer of saliva and genital secretions

EBV infects B cells of the immune system and epithelial cells. Once EBV's initial lytic infection

is brought under control, EBV latency persists in the individual's B cells for the rest of individual's

1
life (Amon and Farrell, 2004). There are few or no symptoms noticeable when a person contracts

EBV at the adolescence stage of life but when EBV is contracted as an adult it may cause

fatigue, fever, inflamed throat, swollen lymph nodes in the neck, enlarged spleen, swollen liver, or

rash(CDC, 2016). The virus is approximately 122-180 nm in diameter and is composed of a

double helix of DNA which contains about 172,000 base pairs and 85 genes(Amon and Farrell,

2004). The DNA is surrounded by a protein nucleocapsid. This nucleocapsid is surrounded by

a tegument made of protein, which in turn is surrounded by an envelope containing both lipidsand

surface projections of glycoproteins which are essential to infection of the host cell (Odumade and

Hogquist, 2011).

Helicobacter pylori(HP), previously Campylobacter pylori, is a gram-

negative, microaerophilicbacterium found usually in the stomach. It was identified in 1982 by

Australian scientists Barry Marshall and Robin Warren, who found that it was present in a person

with chronic gastritis and gastric ulcers, conditions not previously believed to have

a microbial cause. It is also linked to the development of duodenal ulcers and stomach cancer.

However, over 80% of individuals infected with the bacterium are asymptomatic, and it may play

an important role in the natural stomach ecology(Blaser, 2006).

Up to 85% of people infected with H. pylori never experience symptoms or

complications. Acute infection may appear as an acute gastritis with abdominal pain (stomach

ache) or nausea. Where this develops into chronic gastritis, the symptoms, if present, are often

those of non-ulcer dyspepsia: stomach pains, nausea, bloating, belching, and

sometimes vomiting or black stool. Inflammation of the pyloric antrum is more likely to lead

to duodenal ulcers, while inflammation of the corpus (body of the stomach) is more likely to lead

to gastric ulcers and gastric carcinoma(Butcher and Graham, 2003; Bytzeret al., 2013; Ryan and

2
Kenneth, 2010; Suerbaum and Michetti, 2002). However, H. pylori possibly plays a role only in

the first stage that leads to common chronic inflammation, but not in further stages leading

to carcinogenesis(Brown, 2000).

The pathogenesis of H. pylori depends on its ability to survive in the harsh gastric environment

characterized by acidity, peristalsis, and attack by phagocytes accompanied by release of reactive

oxygen species (Olczaket al., 2002). In particular, H. pylori elicits an oxidative stress response

during host colonization. This oxidative stress response induces potentially lethal and mutagenic

oxidative DNA adducts in the H. pylori genome (O'Rourke et al., 2003).

Testing for H. pylori is recommended if peptic ulcer disease or low-grade gastric MALT

lymphoma is present, after endoscopic resection of early gastric cancer, first-degree relatives h

gastric cancer, and in certain cases of dyspepsia. Several ways of testing exist. One can test

noninvasively for H. pylori infection with a blood antibody test, stool antigen test, or with

the carbon urea breath test (in which the patient drinks Cor C-labelled urea, which the bacterium

metabolizes, producing labelled carbon dioxide that can be detected in the breath (Stenstrmet al.,

2008). Also, a urine ELISA test with a 96% sensitivity and 79% specificity is available. H.

pylori is a major cause of certain diseases of the upper gastrointestinal tract. Rising antibiotic

resistance increases the need to search for new therapeutic strategies; this might include prevention

in form of vaccination (Selgrad and Malfertheiner; 2008). Much work has been done on

developing viable vaccines aimed at providing an alternative strategy to control H. pylori infection

and related diseases, including stomach cancer. Although it is believed that HP infection is the

main risk factor for both types of ulcers, duodenal ulcers have not been associated with GC. EBV

infection has also been associated with GC but a possible viral role in PUD has been poorly studied.

(Blanchard and Nedrud,2010)

3
Coinfected with EBV and HP, an increased risk to develop severe gastric inflammation, gastric

premalignant lesions, and cancer occurs (Cardenas-Mondragon et al., 2015).How EBV

and HP interact at the tissue or cellular level needs to be addressed in subsequent studies.Gastric

cancer (GC) generally starts with an inflammatory process mainly associated with infection

by Helicobacter pylori (IARC, 1994).

The relationship in the of H. pyroli and EBV is majorly associated to gastric carcinoma.Thus, both

H. pylori and EBV infect the human stomach, and therefore may have a synergistic relationship or

some other form of impact on each other (Ryan et al., 2012). Hence, Studies of H. pylori and EBV

have proceeded energetically and revolutionized aspects of the pathogenesis and etiology of gastric

diseases.

4
 CHAPTER 2

2.0 GASTRIC CARCINOMA

Stomach cancer, also known as gastric cancer, is cancer developing from the lining of

the stomach. Early symptoms may include heartburn, upper abdominal pain, nausea and loss of

appetite. Later signs and symptoms may include weight loss, yellowing of the skin and whites of

the eyes, vomiting, difficulty swallowing, and blood in the stool among others. The cancer

may spread from the stomach to other parts of the body, particularly the liver, lungs, bones, lining

of the abdomen and lymph nodes (Ruddon, 2007).

The most common cause is infection by the bacterium Helicobacter pylori, which accounts for

more than 60% of casesCertain types of H. pylori have greater risks than others. Other common

causes include eating pickled vegetables and smoking. About 10% of cases run in families and

between 1% and 3% of cases are due to genetic syndromes inherited from a person's parents such

as hereditary diffuse gastric cancer. Most cases of stomach cancers are gastric carcinomas. This

type can be divided into a number of subtypes. Lymphomas and mesenchymal tumors may also

develop in the stomach. Most of the time, stomach cancer develops in stages over years. Diagnosis

is usually by biopsy done during endoscopy. This is followed by medical imaging to determine if

the disease has spread to other parts of the body (WHO, 2014).

Treatments may include some combination of surgery, chemotherapy, radiation therapy,

and targeted therapy. If treated late, palliative care may be advised.Outcomes are often poor with

a less than 10% 5-year survival rate globally. This is largely because most people with the

condition present with advanced disease.In the United States 5-year survival is 28% while in South

Korea it is over 65% partly due to screening efforts (WHO, 2014).

5
2.1 Signs and symptoms

Stomach cancer is often either asymptomatic (producing no noticeable symptoms) or it may cause

only nonspecific symptoms (symptoms that are specific to stomach cancer and to other related or

unrelated disorders) in its early stages. By the time symptoms occur, the cancer has often reached

an advanced stage (see below) and may have metastasized (spread to other, perhaps distant, parts

of the body), which is one of the main reasons for its relatively poor prognosis.

Stomach cancer can cause the following signs and symptoms:

Early cancers may be associated with indigestion or a burning sensation (heartburn). However,

less than 1 in every 50 people referred for endoscopy due to indigestion has cancer. Abdominal

discomfort and loss of appetite, especially for meat, can occur.

Gastric cancers that have enlarged and invaded normal tissue can

cause weakness, fatigue, bloating of the stomach after meals, abdominal pain in the upper

abdomen, nausea and occasional vomiting, diarrhea or constipation. Further enlargement may

cause weight loss or bleeding with vomiting blood or having blood in the stool, the latter apparent

as black discolouration (melena) and sometimes leading to anemia. Dysphagia suggests a tumour

in the cardia or extension of the gastric tumour into the esophagus (Lee and Derakhshan, 2013).

2.3 Causes

Gastric cancer occurs as a result of many factors. It occurs twice as common in males as

females. Estrogen may protect women against the development of this cancer form.

Infections
6
Helicobacter pylori infection is an essential risk factor in 6580% of gastric cancers, but only 2%

of people with Helicobacter infections develop stomach cancer.The mechanism by which H.

pylori induces stomach cancer potentially involves chronic inflammation, or the action of H.

pylori virulence factors such as CagA. It was estimated that EpsteinBarr virus is responsible for

84,000 cases per year (CRU, 2014; Gonzlezet al., 2013;Hatakeyamaand Higashi, 2005)

Smoking

Smoking increases the risk of developing gastric cancer significantly, from 40% increased risk for

current smokers to 82% increase for heavy smokers. Gastric cancers due to smoking mostly occur

in the upper part of the stomach near the esophagus. Some studies show increased risk with alcohol

consumption as well (Thrumurthyet al., 2013).

Dietary factors are not proven causes, but some foods including smoked foods,salt and salt-rich

foods, red meat, processed meat, pickled vegetables, and brackenare associated with a higher risk

of stomach cancer. Nitrates and nitrites in cured meats can be converted by certain bacteria,

including H. pylori, into compounds that have been found to cause stomach cancer in animals.

Obesity is a physical risk factor that has been found to increase the risk of gastric adenocarcinoma

by contributing to the development of gastroesophageal reflux disease (GERD) (Crew and Neugut,

2006).

2.4 Diagnosis

To find the cause of symptoms, the doctor asks about the patient's medical history, does a physical

exam, and may order laboratory studies. The patient may also have one or all of the following

exams:

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 Gastroscopic exam is the diagnostic method of choice. This involves insertion of a fibre

optic camera into the stomach to visualise it.

Upper GI series (may be called barium roentgenogram).

Computed tomography or CT scanning of the abdomen may reveal gastric cancer. It is more

useful to determine invasion into adjacent tissues or the presence of spread to local lymph

nodes. Wall thickening of more than 1 cm that is focal, eccentric and enhancing favours

malignancy (Xu et al., 2013).

CHAPTER 3

3.0 ETIOLOGY OF GASTROINTESTINAL DISEASES BY HELICOBACTER

PYLORI (H. PYLORI) AND EPSTEINBARR VIRUS (EBV).

3.1 Gastric Carcinoma and Helicobacter pylori (H. pylori)

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Gastric cancer (GC) is one of the most common carcinoma and the second leading cause of cancer-

related deaths worldwide. Helicobacter pylori (H. pylori) infection causes a series of precancerous

lesions like gastritis, atrophy, intestinal metaplasia and dysplasia, and is the strongest known risk

factor for GC, as supported by epidemiological, preclinical and clinical studies.H. pylori has been

associated with colorectal polyps and colorectal cancer(Wu et al., 2013). It may also be associated

with eye disease (Saccet al., 2014)

The risk factors for GC consist of H. pylori infection, genetic and environmental factors (Goh et

al., 2014). The outcome of infection is affected by the environmental and genetic factors, the

infection in most individuals does not develop distinct disease or even become beneficial, leading

to the hypothesis that H. pylori might be commensal. However, accumulating evidences support

that H. pylori infection cause a list of diseases, ranging from gastric to extra-gastric diseases, from

chronic gastritis to gastric carcinoma, and thus this bacterium is recognized as the

Classcarcinogenic (Mishra, 2013).

3.1.1 Mechanisms of Infection

There are multiple ways by which H. pylori manipulates the host to lower the threshold for

carcinogenesis, gastric microbiota, high-salt diet, smoking habit, low iron levels and use of proton

pump inhibitors (PPIs) may enhance risk of H. pylori-associated carcinogenesis (Wroblewskiand

Peek, 2013).

Intercellular apical junctions of epithelial cells are critical in keeping integrity of gastric epithelial

barrier and essential cellular functions. H. pylori disrupts epithelial tight junctions through binding

to specific cellular receptors and stimulating the signaling pathways. As transported into epithelial

cells through T4SS, CagA interacts with junction proteins like E-cadherin and ZO-1, and alters the

tight or adherence junctions. It has been confirmed that E-cadherin, a transmembrane protein,

9
localizes at cell-to-cell junctions and interacts with -catenin to form the E-cadherin/-catenin

complex, which play a key role in interaction of epithelial cells and stabilization of cellular

architecture (Amieva and El-Omar, 2008; Fischer et al., 2009). However, the complex is

destabilized by translocated CagA in a phosphorylation independent manner during H. pylori

infection. As reported, CagA translocation is relevant to mislocalization of ZO-1 in epithelial cells.

Studies revealed that H. pylori altered expression and localisation of claudin-7, a cancer-associated

tight junction protein, in gastroids and human epithelial cells, which was mediated by -catenin

and snail activation. A recent study demonstrated that H. pylori diminished acid-induced

tightening of cell junctions, affected the response of epithelial cells to acid, which took effects in

inflammatory response and alteration of the barrier function (Wroblewskiet al., 2015).

The mechanism underlying H. pylori-related gastric carcinogenesis remains unclear. CagA

interacts with E-cadherin, deregulates -catenin signal transduction and promotes gastric-to-

intestinal trans differentiation. As observed, CagA translocated intracellularly binds to PAR1,

destroys cellular junctions and polarity, and fosters carcinogenesis. Development of gastric and

hematological carcinoma has been observed in the mice that were genetically modified to express

CagA. Studies revealed that cagA+/vacAs1+/vacAm1+H. pylori strains promoted pathogenesis of

intestinal metaplasia and gastric carcinoma. Additionally, H. pylori regulates expression of such

toll-like receptors as toll-like receptor (TLR) 4 and TLR9 in epithelial cells during gastric

carcinogenesis (Wang et al., 2014; Wang et al., 2015).

Also, there environmental factors in H. pylori-related GC There are multiple ways by which H.

pylori manipulates the host to lower the threshold for carcinogenesis, gastric microbiota, high-salt

diet, smoking habit, low iron levels and use of proton pump inhibitors (PPIs) may enhance risk of

H. pylori-associated carcinogenesis. Gastric microbiota Alterations of microbiota inhabiting

10
human digestive tract can favor carcinogenesis. Conventional wisdom espoused the dogma that

pH values < 4 were able to sterilize the stomach, but since the discovery of H. pylori, a complex

community of non-cultivatable inhabitants have been uncovered in the stomach. The interaction

of gastric microbiota with H. pylori likely affects gastric immunobiologyand the outcome of

infection (Brawneret al., 2014). The parietal cell loss caused by H. pylori infection leads to

hypochlorhydria or even achlorhydria, thereby increase the risk of bacterial overgrowth and

detrimental infection (Hagiwaraet al., 2015).

Smoking Studies demonstrated that virulence factors like cagA and smoking might have

synergistic effect in carcinogenesis of GC, and cagA genotype of H. pylori strains was closely

related to active-smoking in population with H. pylori infection. High salt diet as evidenced, CagA

expression is significantly upregulated when certain H. pylori strains are cultured in a medium of

high salt concentrations. Through sequence analysis and site-directed mutagenesis, it was

determined that salt-responsive H. pylori strains were more likely to contain two copies of

TAATGA motif within the cagA gene promoter, while the strains containing only a single copy

of this motif were less likely to possess properties of salt-responsive CagA expression. However,

another study showed that the severity of gastritis in H. pylori infected population might be

unassociated with high-salt diet (Santibez et al., 2015; Lohet al., 2007)

The iron level in the host has also been proved to manipulate the virulence potential of H. pylori.

The bacteria harvested from gerbils with low iron levels were found to assemble more T4SS pili

per bacterium, translocate increased amounts of CagA, and augment more IL-8 secretion compared

to those isolated from gerbils with normal iron levels. Furthermore, the H. pylori strains isolated

from patients with low ferritin levels induce significantly higher levels of IL-8 compared to the

strains from patients with the highest ferritin levels, suggesting that iron deficiency in the host

11
might enhance the bacterial virulence and the risk for carcinogenesis of gastric tissues. PPIs It has

been evidenced that long-term use of proton PPIs might aggravate corpus atrophic gastritis in H.

pyloriinfected patients (Lohet al., 2012; Noto et al., 2013).

3.1.2 Signs and Symptoms

Acute infection may appear as an acute gastritis with abdominal pain (stomach ache)

or nausea(Butcher and Graham, 2003). Where this develops into chronic gastritis, the symptoms,

if present, are often those of non-ulcer dyspepsia: stomach pains, nausea, bloating, belching, and

sometimes vomiting or black stool (Ryan and Kenneth, 2010).

Individuals infected with H. pylori have a 10 to 20% lifetime risk of developing peptic ulcers and

a 1 to 2% risk of acquiring stomach cancer(Chang and Parsonnet, 2010). Inflammation of

the pyloric antrum is more likely to lead to duodenal ulcers, while inflammation of

the corpus (body of the stomach) is more likely to lead to gastric ulcers and

gastric carcinoma(Suerbaum and Michetti, 2002). However, H. pylori possibly plays a role only

in the first stage that leads to common chronic inflammation, but not in further stages leading

to carcinogenesis(Brown, 2000). A meta-analysis conducted in 2009 concluded the eradication

of H. pylori reduces gastric cancer risk in previously infected individuals, suggesting the continued

presence of H. pylori constitutes a relative risk factor of 65% for gastric cancers; in terms

of absolute risk, the increase was from 1.1% to 1.7% (Fuccioet al., 2009).

3.1.3 Diagnoses of H. pylori

Colonization with H. pylori is not a disease in and of itself, but a condition associated with a

number of disorders of the upper gastrointestinal tract (Kusterset al. 2006). Testing for H. pylori is

recommended if peptic ulcer disease or low-grade gastric MALT lymphoma is present,

12
after endoscopic resection of early gastric cancer, first-degree relatives h gastric cancer, and in

certain cases of dyspepsia(Stenstrm et al., 2008), not routinely(Kusterset al. 2006). Several ways

of testing exist. One can test noninvasively for H. pylori infection with a blood antibody test,

stool antigen test, or with the carbon urea breath test (in which the patient drinks Cor C-

labelled urea, which the bacterium metabolizes, producing labelled carbon dioxide that can be

detected in the breath) (Stenstrmet al., 2008). Also, a urine ELISA test with a 96% sensitivity

and 79% specificity is available. None of the test methods is completely failsafe. Even biopsy is

dependent on the location of the biopsy. Blood antibody tests, for example, range from 76% to

84% sensitivity. Some drugs can affect H. pylori urease activity and give false negatives with the

urea-based tests. The most accurate method for detecting H. pylori infection is with

a histological examination from two sites after endoscopic biopsy, combined with either a rapid

urease test or microbial culture(Logan and Walker, 2001) .

3.2 Epstein-Barr virus (EBV)

3.2.1 History of EBV

The EpsteinBarr virus was named after Michael Anthony Epstein (born 18 May 1921), now a

professor emeritus at the University of Bristol, and Yvonne Barr (19322016), a

1966 Ph.D graduate from the University of London, who together discovered(McGrath

andPaula,2014) and, in 1964, published on the existence of the virus. In 1961, Epstein,

a pathologist and expert electron microscopy, attended a lecture on "The Commonest Children's

Cancer in Tropical AfricaA Hitherto Unrecognised Syndrome." This lecture, by Denis Parsons

Burkitt, a surgeon practicing in Uganda, was the description of the "endemic variant" (pediatric

form) of the disease that bears his name. In 1963, a specimen was sent from Uganda to Middlesex

Hospital to be cultured. Virus particles were identified in the cultured cells, and the results were

13
published in The Lancet in 1964 by Epstein, Bert Achong, and Barr. Cell lines were sent to Werner

and Gertrude Henle at the Children's Hospital of Philadelphia who developed serological markers.

In 1967, a technician in their laboratory developed mononucleosis and they were able to compare

a stored serum sample, showing that antibodies to the virus developed(Epstein and

Anthony 2005). In 1968, they discovered that EBV can directly immortalize B cells after infection,

mimicking some forms of EBV-related infections (Henle andHenle,1980), and confirmed the link

between the virus and infectious mononucleosis(Young,2009).

3.2.2 Subtypes of EBV

EBV can be divided into two major types, EBV type 1 and EBV type 2. These two subtypes have

different EBNA-3 genes. As a result, the two subtypes differ in their transforming capabilities and

reactivation ability. Type 1 is dominant throughout most of the world, but the two types are equally

prevalent in Africa. One can distinguish EBV type 1 from EBV type 2 by cutting the viral genome

with a restriction enzyme and comparing the resulting digestion patterns by gel

electrophoresis(Odumadeand Hoguist, 2011).

3.2.3 Mechanism of Epstein-Barr virus (EBV)

The Epstein-Barr virus (EBV) is detected in about 10% of gastric carcinoma cases throughout the

world. In EBV-associated gastric carcinoma, all tumor cells harbor the clonal EBV genome.

Gastric carcinoma associated with EBV has distinct clinico-pathological features, occurs

predominately in men and in younger-aged individuals, and presents a generally diffuse

histological type. Most cases of EBV-associated gastric carcinoma exhibit some histology rich in

lymphocyte infiltration. The immunological reactiveness in the host may represent a relatively

14
preferable prognosis in EBV-positive cases. This fact highlights the important role of EBV in the

development of EBV-associated gastric carcinoma (Tokunaga et al., 1993).

The Epstein-Barr virus (EBV) is associated with a variety of tumors derived from B cells, T cells,

natural killer (NK) cells, and epithelial cells. Burkitt lymphoma, post-transplant

lymphoproliferative disease, and Hodgkins disease are B-cell tumors. Peripheral T-cell

lymphomas and NK/T-cell lymphomas are T-cell tumors and NK-cell tumors, respectively.

Nasopharyngeal carcinoma and gastric carcinoma (GC) are epithelial tumors (Rickinson and Kieff,

2007; Young et al., 1989; Zuret al., 1970)

EBV-associated GC has definite histological relevance to GC with lymphoid stroma (GCLS),

which was originally described by Watanabe et al. as a subtype of the carcinoma. GCLS is a poorly

differentiated adenocarcinoma with diffuse and intense lymphocyte infiltration similar to EBV-

associated nasopharyngeal lymphoepithelioma. Further infiltration of the carcinoma (tumor cells)

into the submucosa is occasionally accompanied by EBV-associated GC generally exhibiting a

characteristic histology referred to as GCLS. EBV-associated GC has a null or gastric phenotype

as determined by the expression pattern of the mucin molecules MUC5AC and MUC6 and is

characterized by a relative lack of intestinal phenotypic expression, including Cdx2. According to

these findings, the targets of EBV infection and their subsequent transformation are seemingly the

precursor cells possessing intrinsic differentiation potential toward the gastric cell type (Baruaet

al., 2006; Fukayama, 2010; Nakamura et al., 2005)

Entry in the cells

EBV can infect both B cells and epithelial cells. The mechanisms for entering these two cells are

different. To enter B cells, viral glycoprotein gp350 binds to cellular receptor CD21 (also known

as CR2). Then, viral glycoprotein gp42 interacts with cellular MHC class II molecules. This
15
triggers fusion of the viral envelope with the cell membrane, allowing EBV to enter the B cell

(Odumade et al., 2011). Human CD35, also known as complement receptor 1 (CR1), is an

additional attachment factor for gp350/220, and can provide a route for entry of EBV into CD21-

negative cells, including immature B-cells. EBV infection downregulates expression of

CD35(Ogembo et al., 2013).

To enter epithelial cells, viral protein BMRF-2 interacts with cellular 1 integrins. Then, viral

protein gH/gL interacts with cellular v6/v8 integrins. This triggers fusion of the viral

envelope with the epithelial cell membrane, allowing EBV to enter the epithelial cell (Odumadeet

al., 2011). Unlike B cell entry, epithelial cell entry is actually impeded by viral glycoprotein gp42.

Once EBV enters the cell, the viral capsid dissolves and the viral genome is transported to the cell

nucleus.

16
Figure 1: Endoscopic image of an Epstein-Barr virus-associated gastric carcinoma in the upper

gastric body. The tumor shows a protruded shape probably because of the abundant lymphocyte

infiltration (Nishikawaet al., 2014).

3.2.4 Signs and Symptoms

EBV is contracted as an adult it may cause fatigue, fever, inflamed throat, swollen lymph nodes in

the neck, enlarged spleen, swollen liver, or rash(CDC, 2016).

Diagnosis of Epstein-Barr includes; heterophile antibody agglutination (monospot) test, specific

EBV antibody serologies by testing for EBV nuclear antigen (EBNA) and EBV viral capsid

antigen (VCA).

Treatment of EBV is mostly supportive with analgesics, increased, fluid intake, and rest. However,

in cases of extreme tonsillar hypertrophy, observation for intravenous fluid therapy may be

required (Rogers, 2012).

CHAPTER 4

4.0 RELATIONSHIP BETWEEN H. PYLORI AND EPSTEIN BARR VIRUS

INFECTIONS

H. pylori infection is acquired early in life during childhood, and the presence of the bacteria

induces an inflammatory reaction in the gastric mucosa which in most cases causes no disease.

However, in some individuals the chronic long lasting inflammation triggers serious damage to

the gastric epithelium increasing the risk to develop precancerous lesions, which in turn increase

the risk to end up with a life threatening GC (Hisamatsuet al., 2010).

17
Some of the co-factors that promote a more severe inflammatory reaction may present early on

during childhood. The inflammatory nature of GC denotes a critical role for an immunogenic agent

and in accordance with this, GC is a cancer primarily of infectious etiology. While the association

of H. pylori with GC and early inflammatory lesions is well documented, only a few studies have

analyzed the participation of EBV infection in patients with gastritis, three of them in adults over

40 years old (Kitayama, Honda and Sugimura, 2000) and two case reports in young women of 18

and 17 years old(Hisamatsuet al., 2010). The latter is the only case of EBV and gastritis reported

to date in a pediatric patient in which high serum levels of anti-VCA antibodies (IgG and IgM)

were found. Other studies have examined the presence of EBV sequences in gastritis samples. A

recent report found EBV sequences by quantitative PCR in 15/50 and 5/6 of pediatric and adult

gastritis, respectively (Ryan et al., 2012). Recent study in adults found that about 90% (12/13) of

chronic atrophic gastritis present EBV sequences (Hirano et al., 2003). Taken together these data

support an important role for EBV in early inflammatory reactions of the gastric mucosa.

Studies in adults with NPC have found that disease progression correlates with increased

antibodies against EBV reactivation antigens (Li et al., 2010). We hypothesized that a similar

phenomenon may occur in GC and that the study of children with EBV infection may help identify

patients with severe inflammation in the gastric mucosa, potentially at higher risk to develop

precancerous lesions. Antibodies against proteins of the EBV lytic cycle could reflect higher levels

of infection of the upper digestive tract epithelia and thus serve as a marker for the lesion

progression. However, when we analyzed EBV infection in children, we did not observe the

expected correlation with severe gastritis, and higher levels of anti-EBV antibodies either IgG or

IgM, reflecting acute or chronic infections, were found preferentially in cases with mild gastritis.

Thus, EBV single infection does not explain the cases of severe gastritis.

18
Studies have been limited to H. pylori infection and have shown that in children it is usually

associated with a mild to moderate inflammation (MN cell infiltration) and a mild to absent activity

(PMN cell infiltration), although a severe inflammation and activity is observed in a few cases.

When we analyzed co-infection with both H. pylori and EBV we found that children infected with

both pathogens had the strongest association with severe gastritis, as measured by severe

infiltration of MN (inflammation) and PMN cells (activity) in the gastric mucosa. In contrast,

patients HP/EBV+ preferentially presented mild inflammation (MN cell infiltration) and no

activity (absence of PMN cells); while those HP+/EBV presented moderate inflammation and

mild activity. Our results suggest that co-infection with EBV and H. pyori is necessary to cause

severe gastritis, supporting an important role for EBV, at least in pediatric patients. This increased

effect was also true for patients infected with H. pylori CagA+ nstrains, already known to be

associated with greater virulence and increased risk for GC. Children co-infected with

CagA+/EBV+ showed a significantly stronger association with severe gastritis than infected with

CagA+/EBV, further supporting the need of EBV to cause severe gastritis even in the presence

of highly pathogenic H. pylori CagA+ strains. Interestingly, co-infection by EBV and H.

pylori CagA negative strains did not trigger severe stages of gastritis, arguing against EBV

complementing the pathogenesis of less virulent CagA negative H. pylori strains and thus still

supporting the importance of CagA(Hisamatsuet al., 2010).

It is postulated that in order to infect epithelial cells, EBV must exit first from B cell latency, a

step triggered by expression of transcriptional factor Zta, which is the main orchestrator of the

lytic cycle. However, the extracellular signals in the upper digestive tract that trigger Zta

expression are unknown. CagA is an oncoprotein that among many documented functions induces

loss of polarity in epithelial cells, allowing the transport of basolateral proteins towards the apical

19
face (Saadatet al., 2007). It might be possible that CagA signaling could function as trigger of the

EBV lytic cycle, and CagA induced loss of cell polarity could also favor EBV tropism for epithelial

cells. Arguing against the latter, we observed more pediatric patients infected with EBV than

with H. pylori, which might indicate that infection with the bacteria does not necessarily occur

first.

It is important to address how EBV and H. pylori interact in the gastric mucosa in future studies.

Two possible mechanisms are envisioned: one is simply through additive inflammatory responses

causing increased damage to the tissue. A second mechanism is through more intimate interactions

between EBV and H. pylori genes, e.g. the above-mentioned putative CagA and Zta

cooperation. H. pylori infection has also been positively linked to MALT gastric

lymphoma(Sagaertet al., 2010) supporting increased activation/signaling of B cells transiting

through the gastric mucosa. In this scenario, one of the main inducers of Zta expression and the

EBV lytic cycle in B cell cultures is protein kinase C (PKC)(Baumann et al., 1998) and CagA is a

known activator of this kinase(Brandt et al., 2009). Ectopic expression of CagA in transgenic mice

supports that this protein is the first described bacterial oncoprotein and as such it behaves similarly

to viral oncoproteins(Ohnishi et al., 2008). Interestingly, CagA and EBV oncogenes LMP1 and

LMP2A trigger activation of NFB and MAP Kinases, important signaling pathways for increased

cell survival and proliferation during oncogenic transformation (Dawsonet al.,,2012). The

common activation of signaling pathways by both pathogens suggests a common mechanism of

infection/transformation of the gastric epithelium.

Although data supports that co-infection of EBV with H. pylori is necessary to cause severe gastric

inflammation and activity, EBV requirement seems to be greater for induction of severe activity

20
in the gastric mucosa. Severe activity is given by a large infiltrate of polymorphonuclear cells

permeating the lamina propria and the gastric glands, which in turn activate and secrete more pro-

inflammatory mediators, thus enhancing and perpetuating the local inflammatory reaction and

increasing the risk for severe and permanent damage to the gastric mucosa. In H. pylori infection,

IL-8 seems to be the main neutrophil attracting cytokine(Camorlinga-Ponce et al., 2003; Kabir,

2011). IL-1 has been found overexpressed in NPC tumor samples, an important cytokine for PMN

recruiting. It is possible that the combined role of IL-8 and IL-1 results in the increased activity

observed in EBV and H. pylori co-infected patients.

In conclusion, EBV is involved in the pathophysiology of severe gastric lesions in children infected

with H. pylori. Also in the absence of EBV infection, H. pylori is not sufficient to trigger severe

gastritis in children. This study points out the need to study both pathogens jointly to understand

the mechanism behind severe damage of the gastric mucosa early in life, which could have

implications for the correct disease diagnosis and treatment; also, it could help to identify children

with increased risk to present more serious lesions later in life.

21
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