Association between efficient adhesion to HEp-2 cells and severity of gastritis in Helicobacter pylori strains isolated from children

Volume 1 Number 1 (Spring 2009) 23 30 Association between efficient adhesion to HEp2 cells and severity of gastritis in Helicobacter pylori strains isolated from children Falsafi T* 1, Mahboubi M 1, Zargampour Z 1, Ahmadi M 2 1 Department of Biology, Alzahra University, Vanak, Tehran, Iran. 2 Department of Pathology, Children`s Medical Center of Tehran,Tehran University of Medical Sciences,Tehran, Iran. ABSTRACT Received: September 2008, Accepted: February 2009. Background and objectives: Helicobacter pylori causes chronic gastritis, peptic ulceration, and is a risk factor for gastric cancer. More than half of the world`s population is infected with this pathogen, however the disease outcome varies. Among the virulence factors of H. pylori, specific adhesions to gastric epithelium may be an important step in the induction of active inflammation. H. pylori displays considerable genetic diversity attributed to genetic drift during longterm colonization. Heterogeneity of clinicalisolates has considerably impacted in understanding the role of each factor in infection outcome. However, genetic change may be more limited in the stomach of children in the short period after infection. This work evaluates differences in adherence potential of strains isolated from children with various chronic inflammation status. Materials and Methods: A total of 157 children admitted to Medical Center of Tehran for upper gastrointestinal problems underwent endoscopy for H. pylori infection. Histological examination of their biopsies was performed after H& E and Giemsa staining. Gastritis and inflammation were graded according to the updated Sydney system. 70 culturepositive children, 20 were selected according to their histopathological status. Adherence of their H. pylori isolates to HEp 2 cells was evaluated by viable count of bacteria associated with host cells in an optimized procedure developed in this study. Results: Correlation was seen between inflammation severity score and efficiency of adhesion to HEp2 cells. This correlation was not observed in the KB cell line as an epithelial cellmodel. Conclusion: Specific interaction of H. pylori with host gastric epithelium may be associated with disease outcome Keywords: Helicobacter pylori, Children, Gastr itis, Adherence, HEp2, KB cell line. INTRODUCTION Helicobacter pylori infects the gastric mucosa of more than half of the world`s population (1). The infected individuals usually carry H. pylori for life unless treated. Long term colonization may result in chronic gastritis, peptic ulceration, mucosaassociated lymphoid tissue lymphoma (MALT), and gastric adenocarcinoma (2). The ability of H. pylori to cause these various diseases depends on host, bacterial, and environmental factors (3). Adherence to the host tissue is the first step in pathogenesis of H. pylori infection and bacterial factors essential for colonization of the gastric mucosa include urease, flagella, and adhesive properties * Corresponding author: Dr. Tahereh Falsafi Department of Biology, Alzahra University, Vanak, Tehran, Iran. Phone: ( 9821) 88058 912, Fax: (9821) 8805 8912 E Mail: tfalsafi@yahoo.com factors (3). Vacuolating cytotoxin A (VacA) and the cytotoxin associated antigen (CagA) are the factors that allow H. pylori to persist for decades and induce an intense inflammatory response (46). The interaction of H. pylori with surface mucosa induces release of proinflammatory cytokine interleukin (IL) 8, which leads to influx of neutrophils, mononuclear cells, and Thelper 1 (Th1) cells (23, 7). As H. pylori is not an intracellular pathogen, the Th1 response results in epithelial cell damage rather than in the removal of H. pylori since high levels of tumor necrosis factorα, IL6, IL8, IL10 intensify the inflammation. Thus, longterm infection causes a lifelong inflammatory response that results in cellular damage and initiates the histological damage cascade from chronic active gastritis to cancer (23). Colonization with H. pylori virtually always leads to infiltration of the gastric mucosa at both the antrum and the corpus with neutrophils and mononuclear cells (23). This chronic 23

24 FALSAFI ET AL. IRAN. J. ROBIOL. 1 (23 30 ) active gastritis is the primary condition related to H. pylori colonization, and other H. pyloriassociated disorders result from this chronic inflammatory process (23). H. pylori possesses a few adhesion molecules of which three are well characterized (2, 8). The best characterized adhesion molecule is BabA (HopS), a 78kDa BabA protein encoded by the baba gene (89). BabA mediates binding to fucosylated Lewis b (Leb) blood group antigens on the human host cells (89). There are two distinct baba alleles, baba1 and baba2, but only baba2 can encode a fullsized (active) bacterial adhesion protein. BabA is thought to have a role in the virulence of H. pylori, as the baba2 allele is strongly associated with peptic ulcer disease and gastric adenocarcinoma, but this correlation is controversial (2, 89). The second adhesion molecule is OipA (HopH), a 34kDa OipA protein that mediates adhesion to the hostcell (10). The gene encoding the OipA protein is present in all H. pylori strains, but expression is modulated by phase variation. Expression of OipA is strongly associated with increased in vitro and in vivo IL8 expression, but since the OipA and CagA are both required for this expression, further studies should be done to demonstrate contribution of OipA in gastric inflammation (2, 8, 10). The third adhesion molecule is SabA (HopP) that mediates binding to sialic acid containing glycoconjugates (8, 11). H. pyloriinduced gastric inflammation and gastric carcinoma are associated with the replacement of nonsialylated Lewis antigens by sialylated Lex and sialylated Lea and the probable role of SabA is during chronic inflammatory and atrophic gastritis stages (1112). In practice, the pathologists classify chronic gastritis according to the Sydney system that combines topographical, morphological and etiological information into a system that is reproducible and clinically useful. The updated Sydney system (13), is more useful and classifies gastritis into acute, chronic, and special forms and divides the H. pylori associated gastritis into superficial, diffuse antral gastritis (DAG), and chronic antral gastritis (CAG). This system has a scale of 03 for scoring chronic gastritis into mild, moderate, and severe. As H. pylori infection is associated with chronic gastritis, the density of chronic inflammatory cell infiltrate provides a baseline measure in determining and monitoring of the H. pylori infection (13). In addition, neutrophil activity is an almost universal phenomenon in H. pylori gastritis and biopsies contain neutrophils in all cases. In general, the density of intraepithelial neutrphils correlates with the extent of mucosal damage and with the intensity of H. pylori infection (13). H. pylori is genetically heterogeneous, and every H. pyloripositive subject, may either carry one distinct strain or may carry multiple genotypically different strains (14). This genetic heterogeneity may be due to adaptation of H. pylori to the gastric conditions of its host, genetic exchanges during longterm colonization, as well as to the distinct patterns of the hostmediated immune response to H. pylori infection (1517). So, an important factor in understanding the effective role of each virulence factor in outcome of infection is the high level of genetic diversity of the isolates. In other words, the strains isolated in laboratory may not be exactly the same as those involved in the development of disease. Production of genetic diversity could be related to long termcolonization (16) since less heterogeneity exist among the H. pylori isolated from children during early childhood. Therefore, the H. pylori strains isolated from young children may be more helpful in studying the relationship between H. pylori determinants and the development of diverse disease outcomes. The purpose of this study was to observe the relationship between in vivo status of infection based on degree of gastric inflammation and invitro adherence ability of various strains to epithelial hostcells. For this purpose, we selected H. pylori strains isolated from children with various degrees of inflammation and compared their level of in vitro adherence to epithelial cell models. MATERIALS AND METHODS Patients and H. pylori isolates. Children admitted to the Medical Center of Tehran for upper gastrointestinal problems underwent endoscopy for H. pylori infection. Three similar antral biopsies for culture, histology and rapid urease test (RUT) were taken. RUT was performed using a urea broth according to previously described protocol (18). Clinical symptoms relating to H. pylori infection were recurrent abdominal pain, vomiting, nausea, hematemesis, and melena. The approximate period

H.PYLORI ADHESION TO HEP 2 CELLS 25 of infection was the period that patient suffered from these symptoms. Histological evaluation. Histological examination of the biopsies was performed by standard updated Sydney system as described (8, 19). In brief, routine formalin fixed and paraffin embedded antral biopsies were stained with hematoxylin and eosin (H&E). Based on density of chronic inflammatory cell infiltration, the H. pylori related gastritis was graded into mild, moderate, or severe. The sections were also stained with Giemsa (20) to clearly identify H. pylori organisms inside the biopsies. Bacterial culture. H. pylori was isolated from the biopsy according to previously described method (21). Briefly, antral biopsies were placed within a modified CampyThio medium, composed of thioglycolate base medium (Merck, Germany), 10% sheep blood (SB), and antibiotics (polymyxin B, amphotericin B, vancomycin), transported to laboratory and incubated at 37ºC under a microaerophilic atmosphere. After 3 days, 20 µl of this transportenriched medium was used to streak Modified Campyblood agar plates (CampyBAP) containing brucella agar base (Merck, Germany), 10% SB, and the antibiotics noted above, and incubated until growth. Any gray, translucent gramnegative colonies consistent with morphology of H. pylori were tested for catalase, oxidase, and urease activity, and then stored at 80ºC within a medium composed of skim milk15% glycerol (Merck, Germany), and 1020% fetal bovine serum (Gibco, BRL, Gaithersburg, MD). For laboratory subculture (passage), primary isolates were subcultured and incubated to grow. After ten successive subcultures, the isolates were stored at 80ºC. Epithelial host cells. HEp2 (Laryngeal human epidermal carcinoma) and KB cells (nasopharyngeal human epidermal carcinoma) were supplied by the Pasteur Institute of Iran. Cells were maintained in RPMI containing 50mM Lglutamine, 0.75% sodium bicarbonate, 50 U of penicillin G, 50µg of streptomycin and 2.5µg of amphotericin B per ml (Gibco BRL), complemented with 10% fetal bovine serum (Gibco, BRL, Gaithersburg, MD), and routinely subcultured on plastic tissue culture flasks at 37ºC under 5% CO2 and humidity. Assay to quantitate adherent bacteria. The in vitro adherence assay used in this work was adopted from Wikinson et al. (22) and modified to quantitate viable adherent bacteria. Control experiments. To quantitate adherent bacteria, nonadherent bacteria (unable to associate tightly with host cells) should be eliminated by appropriate number of washings. To determine the appropriate number of washes, adherence of Esherichia coli strain Hb101 to a monolayer of epithelial cells was analyzed. Using various ratios of bacteria to host cells (10 to 1000) and various incubation times (20 min to 360 min), presence of viable bacteria in wash fluid was checked by plating on LuriaBertani agar plates. No viable bacteria were recovered in the wash fluid after four vigorous washes. To analyze the effect of inoculum size and incubation time on adherence efficiency of the H. pylori isolates to host cells, adherence of two randomly selected strains (PP145 and PP23) to HEp 2 cells was analyzed using various ratios of bacteria to host cells (10 to 1000) and various incubation times (20 min to 360 min). To quantitate the adherent bacteria, the hostcells were lysed by a 1530 min incubation at 37 C in the presence of 1 ml of bidistilled sterile water (this step had no inhibitory effect on the viability of H. pylori). The bacteria were dispersed by vigorous pipetting and serial dilutions (10 1 to 10 7 ) were made in 0.9% NaCl before plating on Modified campy BAP. No significant increase in the number of adherent bacteria was observed after 1 h incubation. Also, no significant increase in the number of adherent bacteria was observed when inoculum was more than 100 bacteria to host cell. Adherence was assessed using bacteria/host ratio of 100:1, incubation time of 1h, and four washes. Adherence assay. Confluent stock culture was trypsinized (Gibco, BRL, Gaithersburg, MD) and cells were adjusted to 8 10 4 cells/ml, then transferred into 24well tissue culture plates (Gibco, BRL, Gaithersburg, MD), and incubated until the cells reached 45 10 5 /well. H. pylori strains, submitted to no more than two subculturing, were grown on ModifiedCampy BAP for 23 days, harvested into Hanks balanced salt solution (HBSS) and adjusted to 5.108 bacteria/ ml. E. coli Hb101 (negative control) was grown on

26 FALSAFI ET AL. IRAN. J. ROBIOL. 1 (23 30 ) standard LB agar plate overnight. The bacterial count in the inoculums was estimated by plating dilutions of H. pylori (103, 105, and 107) on modifiedcampy BAP. The cellmonylayers were washed with HBSS to remove traces of antibiotics and were incubated in 0.9 ml RPMI medium complemented with 2% fetal bovine serum. The monolayer was then inoculated with 100 µl of the bacterial suspension (with a ratio of 100 bacteria to each cell) and incubated for 1 h. At the end of the incubation period, the monolayers were vigorously washed four times with 1 ml HBSS, the host cells were lysed and adherent bacteria were enumerated as described above. For determination of the adhesion index, the number of CFU for each well was counted separately and the mean result was reported to the mean number of host cell. For this purpose, 6 wells were infected with each strain, 3 wells out of which, were trypsinized for enumeration of host cells (by hemacytometer) and 3 wells were used for enumeration of bacteria by CFU. were all isolated from children with mild chronic active gastritis () except PP49 that was isolated from a moderate active chronic gastritis. The most adherent strains to Hep2 cells were PP23, PP53, PP44, PP157, PP153, PP87, PP58, PP62, and PP68, respectively, that were all isolated from children with severe or moderate chronic active gastritis except PP68 that was isolated from mild chronic gastritis (Table.2). A significant difference (p value < 0.005) was observed between the adhesion index of strains from mild chronic gastritis and that of the strains from severe /or moderate chronic gastritis. Adherence to KB cells. Adherence tests with 5 out Statistics. Standard statistical analysis was used to evaluate reproducibility of adherence in various wells (standard deviations) and to observe significant difference (p value) between the adhesion index of the strains from mild chronic gastritis and that of the strains from severe or moderate chronic gastritis. RESULTS Histological observation. Classification of chronic gastritis showed mild active chronic gastritis in 11 children (7 girls, 4 boys), moderate active chronic gastritis in 3 children (2 girls, 1 boy) and severe active chronic gastritis in 6 children (4 boys, 2 girls). Staining of the biopsies with Giemsa showed clear presence of H. pylori in 6 of the samples. No significant difference was observed between the two sexes concerning severity of disease. Table 1 contains all information related to these children concerning histology, age, gender, and the approximate period of infection. Figures (1 2) show results from a moderate active chronic gastritis case stained with H & E and Giemsa, respectively. Adherence to HEp2 cells. Comparison of adhesion indices showed that the least adherent strain to Hep2 cells was PP156, followed by PP70, PP85, PP10, and PP21, PP01, PP13, PP74, PP145, PP49, and PP20, respectively. These strains Fig. 1. Antral mucosa with moderate infiltration of lymphoplasma cells in lamina propria (moderate chronic gastritis), Hematoxylin Eosin stain, X 400 Fig. 2. Helicobacter pylori in superficial mucus and crypts of antral mucosa, Giemsa stain, X 1000.

H.PYLORI ADHESION TO HEP 2 CELLS 27 Table 1. Patients information relating to the various isolates Isolate Histopathological H.p a Gender Age (year) Infection period b PP157 4 PP153 13 PP53 07 4 months PP44 11 2years PP23 09 PP49 11 PP58 12 >1 years PP87 04 1 year PP62 12 PP68 03 <1 year PP145 10 PP01 08 PP156 11 PP70 10 1 week PP13 10 PP20 09 PP85 12 >1 year PP74 04 PP21 07 PP10 05 <1 year a: presence of H. pylori in sections after Giemsa staining. *: Not available; b: Approximate period during which the patients complains from the symptoms of disease. of 20 selected strains isolated from mild and severe active chronic gastritis showed a lower adherence rate (Table.3). No difference was observed between the adherence efficiency of strains obtained from mild, moderate/or severe chronic gastritis. Effect of serial passage on adherent and nonadherent H. pylori strains. In order to examine the effect of multiple laboratory passages on the adherence of diverse H. pylori strains, five strains isolated from mild, moderate and severe chronic active gastritis, were submitted to

28 FALSAFI ET AL. IRAN. J. ROBIOL. 1 (23 30 ) Table 2. Adhesion of twenty strains to HEp2 Table 3. Adhesion of five selected strains to KB cells Isolate Pathologic status a Adhesion b Isolate Pathologic status a Adhesion b PP157 31.5±1 PP 62 8.5±1.48 PP153 31±2.16 PP1450 10±98 PP53 42.5±6.29 PP58 12±0.83 PP44 36±3.9 PP23 11.4±1.55 PP49 12±0.62 PP53 10.3±0.84 PP23 PP58 PP87 58±8.6 26.3±0.48 28.6±0.94 a:, and : Sever and Moderate active chronic gastritis; : Mild active chronic gastritis. b: Mean of adhesion index from three Independent wells with standard deviation. PP62 PP68 26±0.5 21±2.6 Table 4. Adhesion of five selected strains (subcultured 10 times) to HEp 2 cells PP85 3 ±0.49 Isolate Pathologic status a Adhesion b PP145 11 ±0.64 PP53 3.2 PP01 7.44±.53 PP23 10 PP74 9±1.5 PP58 5.3 PP156 2±0.17 PP62 5.4 PP70 2.3±0.28 PP145 5.6 PP13 PP20 PP21 PP10 7.5±2.7 12±0.47 4.8±1.12 3.8±0.71 a:, and : Severe and Moderate active chronic gastritis; : Mild active chronic gastritis. b: Mean of adhesion index from three Independent wells with standard deviation. ten successive laboratory passages. Adhesion rates of these strains to Hep2 cells showed a considerable diminution in comparison to those of initial strains (Table 4). DISCUSSION We assessed the relation between in vitro adherence ability of freshly isolated H. pylori strains from children with various degrees of inflammation to HEp2. Available information on the approximate time of infection (the a:, and : Sever and Moderate active chronic gastritis; : Mild active chronic gastritis. b: Mean of adhesion index from three Independent wells with standard deviation. period during which a patient complains from symptoms) was also taken into consideration (Table 1). This factor may be important, since H. pylori strains in the stomach of infected patients may be less heterogeneous in the short period after infection, especially in very young children. In this work a significant difference (p value < 0.005) was observed between the adhesion index of strains from mild chronic gastritis and that of the strains from severe /or moderate chronic gastritis (Tables 12). Case IP68 did not demonstrate a correlation between severity of gastritis and efficient adherence to HEp2. This may be due either to a possible handling error during histopathology analysis or to the specific age of the patient (~3 years old). A direct relation between more efficient adherence and observation of H. pylori in histological sections was also observed (Table 1, columns 3). This may be related to more efficient

H.PYLORI ADHESION TO HEP 2 CELLS 29 colonization of stomach; a potential which is manifested by more efficient adhesion to Hep2 cells. We observed that H. pylori adherence to cultured epithelial cells differed with celllineage, since no association was observed between severity of gastritis and adherence to KB cells. This observation suggests a possible role of specific interaction between H. pylori and host cell receptors on Hep2 cells but no on KB cells. Furthermore, we observed that adherence of clinical isolates to cultured cells was reduced after repeated subcultures that may reflect the controversial results obtained from in vitro experiments, using standard or clinical strains undergone repeated subcultures. Similar observation was made by Kim et al. (23). In vitro adhesion of H. pylori to the epithelium of mucosal surface has been studied by several investigators. Ismail et al. (24) have observed that H. pylori outer membrane vesicles modulate gastric epithelial cells proliferation and IL8 production. Guruge et al. and Peek et al. (25, 26), have observed that epithelial attachments alter the outcome of H. pylori infection. Recently, Linden et al (27) have found that four modes of adhesion used during H. pylori binding to human mucins in the oral and gastric niches, and various factors including mucin type. They have also demonstrated H. pylori strain may play a role during long term infection. The results of this study provide a baseline for better understanding the role of strain specific adherence, that is attributed to the specific adherence factors diversely present in H. pylori strains. These adherence factors that are responsible for specific interaction of H. pylori with host gastric epithelium may be associated with disease outcome. ACKNOWLEDGMENTS We particularly thank Dr. Mehri Najafi and Dr. Fatemeh Mahjoub (Children Medical Center of Tehran) for their collaboration. We also thank Mrs. Madadi and Mrs.Tafreshi for their technical assistance. This project was supported by a grant from Vice Chancellor for Research, Alzahra University, Vanak, Iran. REFERENCES 1. Kikuchi S, and Dore P. 2005. Epidemiology of Helicobacter pylori Infection. Helicobacter. 10 (Suppl 1):14. 2. Kusters JG, Van Vliet AHM, and Kuipers EJ. 2006. Pathogenesis of Helicobacter pylori infection. Clin Microbiol Rev. 19(3):449490. 3. Penta R, De Falco M, Iaquinto G, De Luca A. Helicobacter pylori and gastric epithelial cells : from gastritis to cancer. J Exp Clin Cancer Res 2005; 24(3):337 45. 4. Warburton, S. E., N. P. Mapstone, A. T. R. Axon, P. Hawkey, M. F. Dixon. 1998. Clinical and Histological association of caga and vaca in Helicobacter pylori gastritis. J Clin Pathol. 51:5561. 5. Backert S, Schwarz T, Miehlke S, Kirsch C, Sommer C, Kwok T, et al. Functional analysis of the cag pathogenecity island in Helicobacter pylori isolates from Patients with gastritis, peptic ulcer, and gastric cancer. Infect Immun 2004; 72(2):10431056. 6. Nilsson C, Sillen A, Ericksson L, Strand ML, Enroth H, Normark S, Falk P, and Engstrand L. Correlation between cag pathogenecity island composition and Helicobacter pylori Associated Gastroduodenal Disease. Infect Immun 2003; 71(11):65736581. 7. Beckhed F, Torstensson E, Seguin D, RichterDahlfor A, and Rokbi B. Helicobacter pylori Infection induce Interleukin8 receptor expression in the human gastric epithelium. Infect Immun 2003; 71(6):33573360. 8. Aspholm M, Kalia A, Ruhl S, Arnqvist A, et al. Helicobacter pylori Adhesion to carbohydrates. Methods Enzymol 2006; 417:293339. 9. AspholmHurtig M, Dailide G, Lahmann M, Kaalia A, Ilver D, Roche N, et al. Functional adaptation of BabA, the H. pylori ABO blood group antigen binding adhesin. Science 2004; 305(5683):51922. 10. Yamaoka, Y., S. Kikuchi, H. M. elzimaity, O. Gutierrez, M. S. Osato, and D. Y. Graham. Importance of Helicobacter pylori oipa in clinical presentation, gastric inflammation, and mucosal interleukin 8 production. Gastroenterology 2002; 123:414 424. 11. Asphom M, Olfat FO, Norden J, Sonden B, et al. SabA is the H. pylori Hemagglutinin and is Polymorphic in Binding to Sialylated Glycans. PLoS Pathogens 2006; 2(10):9891001. 12. Mahdavi J, Sonden B, Hurtig M, Olfat FO, Forsberg L, Roche N, et al. Helicobacter pylori SabA adhesion in persistant infection and chronic inflammation. Science 2002; 297(5581):5738. 13. Dixon MF, Genta RM, Yardley JH, and Gorrea P. Classification and grading of gastritis: the updated Sydney System. International workshop on the histopathology of gastritis, Houston, Texas. Am J Surg Pathol 1994; 20:1161 1181. 14. Israel DA, Salama N, Krishna KU, Rieger UM, Atherton JC, Falkow S, and Peek Jr RM. Helicobacter pylori genetic diversity within the gastric niche of a single

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