The aim of this study is to evaluat the inhibitory effect of IgY against H. pylori-derived urease on H. pylori in experimental animals.
JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017 STUDY OF THE PREVENTIVE EFFECT OF ANTI-UREASE IGY ON HELICOBACTER PYLORI INFECTION IN WHITE MICE Do Hoang Long*; Do Minh Trung**; Vi Thuat Thang***; Tran Ngoc Dung*** Le Thu Hong***; Le Van Dong**; Nguyen Anh Tuan** SUMMARY Objectives: Urease inhibition with oral specific antibody is a new approach in prevention and treatment of Helicobacter pylori infection This research aimed to investigate the preventive effect of anti-urease IgY on H pylori infection in experimental animal Subjects and method: White mice were prophylactic treated by having food with or without anti-urease IgY a week before H pylori inoculation and continuously till end of the monitoring time Nine weeks after the start of the diet, the mice were examined to determine the reactivity of anti-H pylori IgG in serum and the inflammation and infection status of the stomach biopsy Results: There were significant differences in the anti-H pylori IgG titer, the eradication of H pylori and the reduction of H pylori-associated gastritis between the two groups of infected mice Conclusion: Oral administration of urease-specific IgY prevented H pylori colonization in the inocculated mice * Keywords: Helicobacter pylori; Anti-urease IgY; Oral passive immunization; Preventive effect INTRODUCTION In Vietnam as well as in the world, Helicobacter pylori (H pylori) infection accounts for 75 - 95% of all causes of gastritis and peptic ulcer disease, in which urease secreted by the bacteria is essential for the survival of the bacteria, facilitating the bacteria's invasion into the gastric mucosa [4] In spite of new antibiotics to be used recently, failure in treatment of H pylori induced gastritis and duodenal ulcer is common, due to the antibiotic resistance of the bacteria, especially in the developing and least developed countries [5, 7] Since then, prophylactic treatment with drug or food containing compound that inhibite the bacteria before they get cololization become a new and promissing approach in developed contries such as Korea and Japan [6] Similar to mammals, hens are capable of creating antibodies against pathogen microbes; these antibodies can be transferred from a hen's blood into the egg yolk laid by the hen itself, hence its name IgY (yolk immunoglobulin) Technology and procedures for antigen-specific IgY production have been established elsewhere [3] IgY has been used orally as a passive immunization method in prevention and treatment of some diarrheal diseases of animal and human * Cantho Universiry * Vietnam Military Medical University ** 103 Military Hospital Corresponding author: Le Van Dong (levandong@yahoo.com) Date received: 10/10/2017 Date accepted: 21/11/2017 138 JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017 IgY against urease of H pylori might be effective in preventing H pylori infection, and/or reduce damages caused by the bacteria on gastric mucosa This study aims at: Evaluating the inhibitory effect of IgY against H pylori-derived urease on H pylori in experimental animals SUBJECTS AND METHODS Subjects - Experimental animal: 28 male Swiss mice, weight 18 - 20 gr provided by the Animal House, Department of Equipment and Technical Materials, Vietnam Military Medical University - Other materials: - H pylori (isolates HP3, HP16 and HP18) from patient's gastric mucosa biopsy samples at 103 Military Hospital in our previous study [1] - IgY against urease derived from the isolates of H pylori (HP13, HP16, HP18), purified at Department of Immunology and Applied PharmacoMedical Research Center, Vietnam Military Medical University in our previous study [2] Methods only were feeded with diet containing IgY against urease; group (control group) and group (study group): H pylori infection groups (9 mice per each group), of which mice in group only were feeded with diet containing IgY against urease At day 7th, mice of group were kept overnight without feeding, then infected in the next morning with H pylori by esophageal intubation (108 CFU/mL; 15 mL/kg body weight) After weeks, blood samples of all mice were taken for determination of anti-H pylori antibody titer; mice were all sacrificed and the stomach of the mice were sent out for pathohistological analysis, with focus on severity of gastritis and the presence of H pylori RESULTS AND DISCUSSION Animal activities during experiment Within first weeks of experiment, all mice of the groups were in normal activities, except mice in groups and showing less activities, refusal of eating, and ruffled hair Mice in group showed weight loss after infection and during the following weeks until experiment ended * Preparing mice diet containing IgY against H pylori's urease: Effect of diet containing IgY against urease on body weight of the mice The food was prepared by mixing the yolk protein suspension rich in IgY against urease with conventional nutrition-enriched mice food (4 mg IgY per gr of food, according to concentration estimation method described by Nomura) In non-H pylori infected mice, average body weight (BW) of the mice in group and increased during the experiment, being 16.8 and 16.4 grs at the beginning of the experiment, respectively, and 18.2 grs at the end of the experiment There was no significant difference between BW of the mice in the groups In addition, diet containing IgY against urease had no significant effect on mice’s body weight * Grouping of mice and experiments: Mice were divided into groups: group and (5 mice per group): non-H pylori infection groups, of which mice in group 139 JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017 Conversely, in H pylori infected mice, average BW of the mice in group decreased, being 17.5 grs at the beginning of the experiment, and 17.4 grs at the end of experiment (with net weight loss of 0.1 grs) Meanwhile, average BW of mice in group (having IgY against H pyloriderived urease) were 17.3 grs and 19.1 grs at the beginning and the end of experiment, respectively (with net weight gain of 1.7 grs) No significant diffenrence in BW of mice in groups and was observed; however, the weight gain of mice in group revealed a positive effect of IgY against H pyloriderived urease (used as a preventive measure) on development of H pyloriinfected mice It was showed by our observation in this study that, those mice feeded with (1) food containing IgY against H pyloriderived urease (both in control group and in H pylori-infected group) all had better weight gain compared to the others This weight gain was probably due to the fact that IgY, by having protein’s nature, had provided more nutrition which helped mice developed better Interestingly, IgY is a protein originated from hen’s egg, and in this study, it showed no immunogenicity when orally administered to mice Taking all into account, these findings partly proved the non-toxic property of the IgY preparation, and thus, the safety of the preparation for oral administration Additionally, the increase in body weight of H pylori-infected mice feeded with the IgY preparation revealed the inhibitory effect of IgY against H pylori-derived urease on the bacteria (2) Graph & 2: Average body weight of mice: (1) non-H pylori infection, and (2) H pylori infection Determination of anti-H pylori antibody in H pylori-infected mice All blood samples from mice in group (no prevention by anti H pylori-derived urease IgY) showed reactive with anti-H pylori antibody; meanwhile, only 66.6% of samples from mice in group (IgY prevention group) were reactive with the antibody Additionally, activity of anti-H pylori antibody in H pylori-infected mice with IgY prevention was lower than that of mice without IgY prevention (p = 0.003) 140 JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017 Severity of gastric mucosa inflammation in mice All H pylori-infected mice without anti-urease IgY prevention developed gastritis; meanwhile, only 77.7% of mice infected with H pylori but had IgY prevention presented with gastritis Besides, the gastritis situation in mice without IgY prevention was more severe than that in mice with IgY prevention (p = 0.004) Gastric submucosa’s bacterial density analysis No presence of H pylori was recorded in gastric submucosa’s tissue samples of mice in groups and 2; meanwhile, the bacteria was present in all samples from H pylori-infected mice without IgY prevention (group 3) Bacterial density recorded in gastric submucosa’s tissue samples from mice in group (IgY prevention group) was lower (44%) Similarly, H pylori bacterial density on gastric mucosa of mice without IgY prevention was higher than that of mice with IgY prevention (p 0.0001) (3) (4) Graph & 4: Gastritis in H pylori-infected mice: (3) severity of gastritis, and (4) H pylori bacterial density (1) (2) Figure & 2: Histological gastric injuries in mice N3-C1: (1) H&E staining, and (2) Giemsa staining 141 JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017 Regarding the preventive effect of IgY against H pylori-derived urease, our findings were in accordance with results published by Nomura et al Firstly, all mice in group in our study showed a reduced activity of anti-H pylori antibody, yet a complete eradication of the bacteria from gastric mucosa in the H pylori-infected mice was not fully observed Secondly, 55.6% of mice in group got a complete bacterial eradication (HP testing negative), which was lower compared to that in Nomura’s study (100%); nonetheless, among HP (-) mice in our study, 40% of mice was free of gastritis, partly revealing that IgY against H pylori-derived urease helped eradicate the bacteria It was much likely that the preventive effect of the IgY preparation helped protecting the mice’s gastric mucosa from the infection of H pylori; in other words, the IgY worked by eradicating the cause of gastritis The preventive effect of IgY against H pylori-derived urease was revealed by significant reduction of H pylori bacterial density on the gastric mucosa (p < 0.0001) Furthermore, the severity of gastritis in mice of group was proportionally decreased along with the decrease of H pylori on gastric mucosa (p = 0.004) In general, the H pylori infection rate of mice in group was the highest compared to the others; in this group, the activity of anti-H pylori antibody and the severity of gastritis was higher than those in the others as well This observation suggested that IgY against H pylori-derived 142 urease had probably contributed to improvement of H pylori-induced gastritis, as well as decrease of urease activity at the gastric mucosa by inhibiting H.pylori’s urease, and thus, eradicating the survival factor for H pylori, leading to a reduction of gastric mucosa’s injuries and making the bacteria itself easier to be eradicated IgY against H pylori-derived urease had been administered to the experimental mice before H pylori infection, with the aims of evaluating the preventive effect of the IgY preparation It was revealed by the results that in mice with IgY prevention, the infection rate and the gastritis severity were lower compared to those without IgY prevention The IgY preparation was proved to be non-toxic and safe by oral administration In other words, IgY against H pylori-derived urease exerted a preventive effect on H pylori infection CONCLUSIONS We have successfully established an animal model of H pylori infection on Swiss mice by injecting H pylori suspension, 108 CFU/mL/mouse through esophageal intubation The infection rate was 100% by anti-H pylori antibody testing on blood samples and the presence of the bacteria at the gastric submucosal tissue of mice being infected A continuous diet containing IgY against urease (4 mg IgY/gram of food) for week before infection and thereafter showed a preventive effect of H pylori infection on experimental mice JOURNAL OF MILITARY PHARMACO-MEDICINE N09-2017 REFERENCES Đỗ Hoàng Long, Đỗ Minh Trung, Lê Thu Hồng CS Nghiên cứu phân lập vi khuẩn tách chiết enzym urease từ mơi trường ni cấy Helicobacter pylori Tạp chí Y - Dược học quân 2014, 39 (1), tr.12-16 Đỗ Hoàng Long, Đỗ Minh Trung, Lê Thu Hồng CS Nghiên cứu chế tạo kháng thể từ lòng đỏ trứng gà kháng urease Helicobacter pylori Tạp chí Y - Dược học quân 2014, 39, tr.35-39 Ko K.Y, Ahn D.U Preparation of immunoglobulin Y from egg yolk using ammonium sulfate precipitation and ion exchange chromatography Poultry Science 2007, 86 (2), pp.400-407 Kusters J.G, van Vliet A.H.M, Kuipers E.J Pathogenesis of Helicobacter pylori infection Clinical Microbiology Reviews 2006, 19 (3), pp.449-490 Miwa H, Ghoshal U.C, Gonlachanvit S et al Asian Consensus report on functional dyspepsia Journal of Neurogastroenterology and Motility 2012, 18 (2), pp.150-168 Nomura S, Suzuki H, Masaoka T et al Effect of dietary anti-urease immunoglobulin Y on Helicobacter pylori infection in Mongolian gerbils Helicobacter 2005, 10 (1), pp.43-52 WHO Geneva Proposal for the inclusion of a proton pump inhibitor for the treatment of dyspepsia in the WHO model list of essential th medicines 17 Expert Committee on the Selection and Use of Essential Medicines 2009, pp.1-31 143 ... after infection and during the following weeks until experiment ended * Preparing mice diet containing IgY against H pylori' s urease: Effect of diet containing IgY against urease on body weight of. .. helped protecting the mice s gastric mucosa from the infection of H pylori; in other words, the IgY worked by eradicating the cause of gastritis The preventive effect of IgY against H pylori- derived... account, these findings partly proved the non-toxic property of the IgY preparation, and thus, the safety of the preparation for oral administration Additionally, the increase in body weight of H pylori- infected