Helicobacter pylori by Silvia Giono

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Innate immune and proliferative cellular responses to |Helicobacter pylori infection in the gastric mucosa of children.

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Munoz, Leopoldo; Unidad de Investigación en Enfermedades Infecciosas, Hospital de Pediatría, CMN SXXI, Instituto Mexicano del Seguro Social Camorlinga, Margarita; Unidad de Investigación en Enfermedades Infecciosas, Hospital de Pediatría, CMN SXXI, Instituto Mexicano del Seguro Social Hernández, Rogelio; Instituto Nacional de Ciencias Medicas y Nutrición Salvador Subirán, Patología Giono, Silvia; Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Microbiología Ramón, Guillermo; Hospital de Pediatría, Instituto Mexicano del Seguro Social, Patología Muñoz, Onofre; Unidad de Investigación en Enfermedades Infecciosas, Hospital de Pediatría, CMN SXXI, Instituto Mexicano del Seguro Social Torres, Javier; Unidad de Investigación en Enfermedades Infecciosas, Hospital de Pediatría, CMN SXXI, Instituto Mexicano del Seguro Social

Keywords:

apoptosis, childhood gastritis, cell proliferation, immune response, inflammation, neutrophil infiltration

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Innate immune and proliferative cellular responses to Helicobacter pylori infection in the gastric mucosa of children.

Leopoldo Muñoz, Dr.,1 M. Camorlinga, MSc,1, R. Hernández, PhD,2 S. Giono, Dr.,3 G. Ramón, MD,4 O. Muñoz, MD1 and J.Torres, PhD.1

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Unidad de Investigación en Enfermedades Infecciosas, Hospital de Pediatría, CMN SXXI, Instituto Mexicano del Seguro Social (IMSS), México City, México,1 Instituto Nacional de Ciencias Medicas y Nutrición Salvador Subirán, México

City, Mexico,2, Departamento de Microbiología, Escuela Nacional de Ciencias

Biológicas, Instituto Politécnico Nacional, México City, México,3 Servicio de

Patología, Hospital de Pediatría, IMSS, México City, México,4

Reprint request to: Javier Torres, PhD

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Av. Centenario 1707-39; Mexico D.F., C.P. 01580, MEXICO e-mail: jtorresl57@yahoo.com.mx phone: 01152-55-5-627-6940

Running title: Cellular response to H. pylori in children

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Abstract Background. H. pylori infection occurs mostly during childhood; still, few studies in this age group have addressed the innate immune cellular and the proliferative epithelial responses to this infection. Mexico has high prevalence of H. pylori infection in children, but low risk of gastric cancer. The aim of this work was to study the cellular responses of the gastric mucosa to H. pylori infection in Mexican children.

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Methods: Antrum and corpus gastric biopsies were obtained from 44 H. pylori infected (mean age 12Âą3.2) and 44 uninfected (mean age 10Âą3) children. Mucosal cellular responses were studied by immunohistochemistry, using anti-

Ki67 antibodies for proliferation studies, anti-human tryptase for mast cells, and anti-human CD68 for macrophages. T and B lymphocytes were stained with a

commercial integrated system. Estimation of the intensity of cellular responses

was done histologically using the software KS300. Results. In H. pylori infected children proliferative activity of the epithelia, and

infiltration of macrophages, and T and B lymphocytes was significantly higher that that observed in uninfected patients. In H. pylori infected children, a

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balanced response of CD4, CD8 and CD20 lymphocytes was observed; although activated mast cells were decreased and neutrophil and mononuclear cell infiltration was low. Proliferative activity was associated with polymorphonuclear infiltration but not with macrophages or lymphocytes. Conclusions. Mexican children respond to H. pylori infection with a low inflammatory response, a balanced T and B lymphocytes increase and a high

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regenerative activity; this might partially explain the lower predisposition to develop severe gastric lesions.

Introduction. H. pylori infection affects over 50% of the world population and is associated with gastroduodenal ulcer, gastric cancer and MALT lymphomas. Acquisition of the infection occurs predominantly during childhood and once

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acquired, it persists for life in most infected subjects.[1] Early acquisition of H. pylori is a relevant risk factor for the development of gastric cancer,[2] the second leading cause of cancer-related mortality in the world.[3] Gastric cancer

is a multistep and multifactorial process that begins in the first decade of life, with DNA alterations associated with chronic inflammation, and an imbalance of

epithelial cell turnover. [4] In adults H. pylori infection induces cell apoptosis and

stimulates cell proliferation in the gastric epithelium.[5] An inappropriately regulated local inflammatory response to the infection may contribute to the

development of gastric carcinoma.[6] The gastric lamina propria of adults with H. pylori

infection

shows

dense

infiltration

phagocytes,

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neutrophils,

granulocytes, and lymphocytes.[6,7] Although H. pylori infection begins mostly during childhood, there are few reports on the inflammatory and proliferative cell response in this age group. They reported that in children the inflammatory response is poor, with low infiltration of mononuclear and polymorphonuclear cells; [8,9] however, scarce reports exist on the response of mast cells and lymphocytes and on the proliferative response of epithelial cells to the infection.

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An early childhood infection has been associated with higher risk for gastric cancer. Mexico is a country with high prevalence of H. pylori infection early during childhood; still, the rate of gastric cancer is low.[10] Studies of the characteristic of the cellular response of the gastric mucosa to H. pylori infection in children might help explain the outcome of the infection in adults. The aim of this study was to add knowledge on the innate immune cellular response and on the proliferative epithelial response of the gastric mucosa to H. pylori infection in

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Mexican children.

Materials and methods.

Biopsies. Gastric biopsies from antrum and corpus were obtained from 44

H. pylori infected children, 2 to 16 years of age (mean age 12Âą3.2) and 44 H.

pylori uninfected children, 4 to 16 years of age (mean age 10Âą3). These children attended the Pediatric Hospital at Centro Medico Nacional Siglo XXI, Instituto Mexicano del Seguro Social in Mexico City, because of chronic abdominal pain.

They underwent diagnostic upper gastrointestinal tract endoscopy between 1995

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and 2002. The study was approved by the ethical committee of the Pediatric Hospital.

H.pylori infection. H. pylori infection was diagnosed by urea breath test, serology, culture and histology, as described. [9] Infection was considered positive when at least three of the four diagnostic tests were positive. Absence of infection was defined when all four test resulted negative. Infection with cagA(+) H. pylori was determined by serology. [9]

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Histological assessment of inflammation. Paraffin sections of both, antrum and corpus were stained with hematoxylin-eosin. Neutrophil and mononuclear cell infiltration was graded according to the updated Sydney sytem. [11] Immunohistochemistry for proliferation, lymphocytes, macrophages and mast cells. Immunohistochemistry was performed in antral and corpus biopsy specimens. For proliferation studies, sections were incubated with anti-Ki67 monoclonal antibodies (NeoMarkers, Fremont, CA) followed by incubation with

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polyvalent biotinylated link (anti-mouse and anti-rabbit) and with horseradish peroxidase-streptavidin label (Cell Marque, Hot Springs, AR). Sections were stained in 0.05% diaminobenzidine hydrochrolide, and counterstained with

hematocxylin. Proliferative activity was quantitated using the KS300 software

(Carl Zeiss, Jena, Germany). At least five fields (20X magnification) along the

proliferation zone were measured per biopsy. The ratio of the stained area (Âľ2) to the total area (mm2) was calculated and expressed as percentage. For T and B lymphocytes studies, biopsies were stained with the Ventana

Medical System's integrated instrument-reagent systems (Tucson, AZ), as

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described by the manufacturer. For T lymphocytes, monoclonal antibodies against-CD3 and anti-CD8 were used, and for B lymphocytes monoclonal antiCD20 was used. For mast cells, monoclonal anti-human mast cell tryptase (Dako, Carpinteria, CA) was used; and for macrophages, monoclonal anti-human CD68 (Dako, Carpinteria, CA) was used.

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Lymphocytes, mast cells and macrophages were measured as follows: stained cells were counted and the total tissue area studied was recorded (mm2). The average number of labeled cells was calculated and results were expressed as number of cell/mm2. Statistical analysis. The association between infiltration of inflammatory cells with H. pylori infection was assessed using the chi2 test. Correlation between intensity of inflammation and cell proliferation was analyzed with the

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Spearman correlation test. Man-Whitney U-test was used to evaluate differences between H. pylori infected and uninfected biopsies regarding cell proliferation, lymphocytes, macrophages and mast cells. Results.

Intensity of inflammation. In H. pylori infected children, mononuclear cell

infiltration was mild in most children <10 years (64%) and moderate to severe in most children >10 years of age (61%) (p<0.05); whereas a mild degree of infiltration was observed in all non-infected patients. Concerning infiltration of

polymorphonuclear cells in H. pylori infected children, it was absent in about

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25%, and mild to moderate in over 50%, regardless of the age of the children; however, severe infiltration was observed only in children older than 10 years (p<0.05).

Polymorphonuclear infiltration was absent in 100% of uninfected

patients. Mononuclear and polymorphonuclear infiltrate was significantly higher in biopsies of H. pylori infected children. Among H. pylori infected children mononuclear infiltrate was higher in the antrum than in the corpus; however, the difference was not statistically significant

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(p=0.06); whereas polymorphonuclear infiltrate was not different between antrum and corpus. Most children infected with cagA (-) H. pylori strains had a mild inflammatory infiltrate (86%, p<0.0001); whereas most cases infected with cagA (+) strains showed moderate to severe infiltrate (73%, p<0.005). Proliferative activity. Immunoreactivity for Ki-67 in antrum and corpus biopsies of uninfected H. pylori children was observed mainly in epithelial cells at the neck region of the crypts. Whereas in biopsies from H. pylori infected

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children, the proliferative activity extended toward both, the tip and bottom of the gastric glands. Proliferative activity in antrum and corpus of H. pylori infected biopsies was significantly higher that that observed in uninfected patients (table1).

Proliferation and cell responses. Antrum and corpus biopsies from 20 H.

pylori infected and 20 uninfected children were evaluated for proliferative activity in the different compartments of the gastric foveola. In H. pylori infected children, a significant increase of proliferative activity was observed in the lower border

and isthmus zone when compared with uninfected patients (Fig 1) (p<0.0001). A

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significant correlation between the mononuclear and polymorphonuclear infiltration with proliferative activity was observed in both, antrum and corpus of H. pylori-infected children (p=0.045 and p= 0.02, respectively) (Figs 1C and 2). Macrophage infiltration. Macrophages infiltration was significantly higher in H. pylori infected than in uninfected biopsies, in antrum and corpus (fig 3). Macrophages localized mostly in lamina propria.

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Mast cell infiltration. Infiltration of active mast cells was significantly lower in the antrum and corpus of H. pylori infected that in uninfected biopsies (fig 4). Activated mast cells were mainly in lamina propria. Detection of T and B lymphocytes. Antrum and corpus biopsies from 20 H. pylori infected and 30 uninfected children were studied. Infiltration of both, T lymphocytes (CD3 and CD8) and B lymphocytes (CD20) was significantly increased in H. pylori-infected biopsies, in antrum and corpus (Table 2). T

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lymphocytes (CD3) were found in lamina propria and in the intraepithelial region. (Fig 5A); CD8 T lymphocytes represented most of the intraepithelial lymphocytes (Fig 5B). B lymphocytes were observed exclusively in the glands zone (Fig 5C).

No correlation was found between the degree of proliferative activity and

infiltration of T lymphocytes, B lymphocytes, mast cells and macrophages.

Discussion.

H pylori infection is invariably associated with an inflammatory response in the gastric mucosa.[12]

In adults, H. pylori-associated chronic gastritis is

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characterized by surface epithelial degeneration, infiltration of the mucosa by chronic inflammatory cells, and a characteristic neutrophils active component. [13] In spite of the fact that H. pylori infection occurs mostly during childhood

[14]

few studies have addressed the innate immune cellular response and the proliferative epithelial response to the infection in this age group. In this work we aimed to study these issues in children of a country with high prevalence of H. pylori infection, but low risk of gastric cancer.

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We found that in children with H. pylori infection the infiltration of mononuclear and polymorphonuclear cells was mild to moderate in most cases. Of note, in almost 30% of infected children no polymorphonuclear infiltration was observed; this low inflammatory response in children has also been observed by others. [8,9,15] Our results showed that mononuclear but not polymorphonuclear infiltrate is higher in antrum than in corpus; previous studies in children have also reported a higher inflammatory response in antrum.[15] Our study also

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demonstrate that inflammatory infiltrate is higher in older children and in children infected with cagA (+) H. pylori strains. In contrast to the low inflammatory response, a significant proliferative

activity was observed in epithelial cells of H. pylori infected children, with no

significant difference between antrum and corpus. In contrast, other studies in H.

pylori infected children have reported higher proliferative activity in antrum than in corpus. [12,15] This discrepancy may be due to differences in the number of children studied, our groups were larger; or the age of the children, in our study

they were younger. Of interest, in previous studies.[15,16] some children

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presented gastroduodenal ulcer or gastric atrophy, whereas none of our cases presented these lesions; probably the increased epithelial proliferation observed in our population function as an efficient regenerative activity, preventing mucosal damage and development of severe lesions. It should be noted than in adults with H.pylori infection, increased proliferation of epithelial cells is link to mucosal damage and higher risk of gastric cancer; we suggest this is not the case in children, where proliferation might play an important regenerative activity.

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We found a significant correlation between the degree of inflammation (mononuclear and polymorphonuclear infiltrate) and the intensity of proliferation. H. pylori infection may cause epithelial cell death by direct injury (production of ammonia or toxins) or by reactive oxygen species produced by neutrophils.

[17]

Cell death is compensated by an increase in cell proliferation;[12] in fact, in our study and that of Lynch et al.[12] increased proliferative activity was observed in regions with higher infiltration of polymorphonuclear cells. This suggests that

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proliferative activity is the epithelial regenerative response to cell damage caused by inflammatory mediators.

In this study, infiltration of macrophages was higher in H. pylori infected

children. Macrophages may contribute to the establishment of a chronic irritating

inflammatory response which persistently disturbs the gastric mucosa, increasing

the risk to develop gastric carcinoma. In fact, in children of countries with high risk of gastric cancer, infiltration of macrophages was higher than in children from communities with low gastric cancer risk.[18]

Mast cells are a main pro-inflammatory cells; they release a wide range of

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potent mediators, such as histamine, a strong stimulant of gastric acid.[19] Previous reports suggested that mast cells might be involved in the pathogenesis of gastroduodenal disease caused by H. pylori.[20] Contrary to what we expected, we found that infiltration of active mast cells was significantly lower in H. pylori infected, as compared with uninfected children. A previous study in children, [21] found that mast cell density was significantly greater in children with chronic gastritis, regardless of H. pylori infection. Studies in adults have also

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shown that mast cells are increased in the antrum of patients with functional dyspepsia; regardless of the presence of H. pylori infection.[22,23] These results suggest that mast cells are associated with gastritis and clinical symptoms and not necessarily with H. pylori infection. Other reports have also shown that mast cells are degranulated by exposition to H. pylori-derived products.[24] In our study, we measured active tryptase and probably at this point of the infection most mast cells were already activated and the enzyme exhausted.

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The mucosal inflammation induced by H. pylori seems to be T-cell dependent, as H. pylori infection does not induce gastritis in T-cell-deficient mice.[25] There are few reports on the lymphocyte subsets present in the gastric

mucosa of H. pylori infected children. Our results showed that infiltration of both,

T and B lymphocytes was increased in H. pylori infected children. This

recruitment of T and B lymphocytes early in during infection suggests a role for them during the initial establishment of the innate and inflammatory mucosal response to the infection. In a recent report.[18] infiltration of T lymphocytes was

found increased during H. pylori infection and was more prominent in children of

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a population with high-risk for gastric cancer as compared with children from a low- risk population; the predominance of cytotoxic T lymphocytes suggested that they may predispose to more severe damage in high risk populations. It is interesting to note that we found a rather balanced increased of both, T and B lymphocytes, with similar numbers of each recruited to the mucosa, which would in this case suggests lower predisposition to severe damage.

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In our study, T lymphocytes were mainly in lamina propria, with CD8 more abundant in the intraepithelial zone. In contrast, B lymphocytes were below the lamina propria in the glands zone. Recent reports in infected volunteers found CD4 cells significantly higher than CD8 cells [25] which are similar to that observed during natural infection in adults. [26] In our study, CD8 T lymphocytes represented about 45% of the total T lymphocytes, suggesting that in our children there is equilibrium in the response of helper and cytotoxic lymphocytes; which,

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again, differs from findings in children of a population with high-risk for gastric cancer.[18]

In contrast to the observed correlation of polymorphonuclear infiltration

with proliferative activity, we found no correlation of proliferation with the number

of macrophages, mast cells, T and B lymphocytes. These results suggest that

mediators produced by these cells are not responsible for the death of epithelial cells which leads to increased regenerative activity. In conclusion, Children of Mexico, a country with high prevalence of early

H. pylori infection, but low risk for gastric cancer, respond to H. pylori infection

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with a low inflammatory response, a balanced increased in T and B lymphocytes and a high regenerative activity; this might partially explain the lower predisposition to develop severe gastric mucosal lesions.

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Acknowledgements:

This work was supported by CONACYT (grant 34565-M), MEXICO; and by the Coordinaci贸n de Investigaci贸n en Salud, IMSS, MEXICO.

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from Helicobacter pylori activates monocytes to induce lymphocyte dysfunction and apoptosis. J Clin Invest 2001; 108:1221–28. 7

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Lynch DA, Mapstone NP, Clarke AM, et al. Correlation between

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Bodger K, Crabtree JE. Helicobacter pylori and gastric inflammation. Br Med Bull 1998; 54:139-150.

Webb PM, Knight T, Greaves S, et al. Relation between infection with Helicobacter pylori and living conditions in childhood: evidence for person to person transmission in early life. BMJ 1994; 308:750-53.

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Day AS, Jones NL, Lynett JT, et al. cagE is a virulence factor associated with Helicobacter pylori-induced duodenal ulceration in children. J Infect Dis. 2000; 181:1370-5.

Matthews GM, Butler RN. Cellular mucosal defense during Helicobacter

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Bedoya A, Garay J, Sanzon F, et al. Histopathology of gastritis in

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Otaka M, Watanabe S. Role of mucosal mast cells in Helicobacter pylori infection. J Gastroenterol 2002; 37:70-2

Bamba N, Nakajima S, Andoh A, et al, Stem Cell Factor Expressed in

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Nakajima S, Krishnan B, Ota H, et al. Mast cells involvement in gastritis with or with Helicobacter pylori infection. Gastroenterol 1997; 113:746754.

Nakajima S, Bamba N, Hattori T. Histological aspects and role of mast cells in Helicobacter pylori-infected gastritis. Aliment Pharmacol Ther 2004; 20 Suppl 1:165-70.

Nurgalieva ZZ, Conner ME, Opekun AR, et al. B-cell and T-cell immune

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responses to experimental Helicobacter pylori infection in humans. Infect Immun 2005; 73:2999-3006. 26

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Table 1. Epithelial cell proliferation in antrum and corpus (Âľ2/mm2) of children according to H. pylori infection. Region

No.

H. pylori +

H. pylori -

p value

Antrum

16,4 (4,7-36,8)

8,6 (2,3-17,990)

<0.0001

Corpus

15,0 (4,0-33,4)

6,6 (2,7-14,836)

<0.0001

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Values are median (range).

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Table 2. Comparison of the intensity of T and B lymphocytes infiltration in antrum and corpus of the gastric mucosa of H.pylori Infected and non-infecred.

H. pylori+

H. pylori-

p value

CD3

121 (10-357)

14 (0-50)

<0.0001

CD8

57 (6-163)

4 (0-37)

<0.0001

5 (0-23)

<0.0001

Marker

CD20

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125 (25-515)

Values are median (range).

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Text to figures.

Figure 1. Immunohistochemical staining for Ki-67 antigen in biopsies from the gastric antral mucosa. (A) biopsy from a children without H. pylori infectrion (200X). (B) biopsy from a H.pylori Infected children, showing increased epithelial cell proliferation in the lower border and in the isthmus

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zone of the gastric foveola (200X). (C) biopsy from a H.pylori Infected children, showing a correlation between increased cell proliferation and increased inflammatory cells (100X).

Figure 2. Correlation between infiltration of inflammatory cells and

epithelial cell proliferation. (A) Correlation between mononuclear cell

infiltration and `proliferation in antrum and corpus biopsies. (Spearmanâ&#x20AC;&#x2122;s correlation analysis, r=0.217, p=0.045; n=84). (B) Correlation between

polymorphonuclear cell infiltration and proliferation in antrum and corpus biopsies. (Spearmanâ&#x20AC;&#x2122;s correlation analysis, r= 0.249, p=0.022; n=84).

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Figure 3. Intensity of macrophage infiltration in antrum and corpus biopsies of H..pylori Infected and non-infected children. (Man-Whitney Utest, p= 0.013).

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Figure 4. Intensity of mast cell infiltration in antrum and corpus biopsies of H.pylori Infected and non-Infected children. (Man-Whitney U-test, p<0.0001). Figure 5. Immunohistochemical staining for CD3 (A), CD8 (B) and CD20 (C) lymphocytes. (A) Biopsy specimen showing severe infiltration of CD3+ T lymphocytes, localized in lamina propria and in the intraepithelial region.

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(200X). (B) Biopsy specimen showing an increased infiltration of CD8 T lymphocytes, localized mainly in the intraepithelial region (200X).

(C)

Biopsy specimens showing marked stromal infiltration by B lymphocytes

(original magnification 200X).

vi ly

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Helicobacter

r Fo A

Figure 1

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Helicobacter

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35000

Ki-67(Area/mm2)

30000 25000 20000 15000 10000 5000

r Fo 1 2 3 Mononuclear cell infiltration

35000

25000 20000

Ki-67 (Area/mm2)

30000

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Helicobacter

15000 10000 5000 0

Polymorphonuclear cell infiltration Figure 2

Helicobacter

p= 0.013

400

CD68/mm2

300

200

100

r Fo Hp-

Hp+

Figure 3

vi ly

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Helicobacter

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p<0.0001

700 600

Triptasa/mm2

500 400 300 200 100

r Fo Hp -

Hp+

Figure 4

vi ly

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Helicobacter

Figure 5

r Fo ew

Re ly

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Helicobacter