Epidemiological Study On Helicobacter Heilmannii And The Evaluation Of Pathogenicity In Vivo

In addition to Helicobacter pylori(Hp).Helicobacter heilmannii is the only helicobacter species that can colonize in human stomach.H.heilmannii is related with a variety of gastric diseases.including acute and chronic gastritis,peptic ulcer.gastric cancer,MALT and esophageal disease.which should pay attention to.In 1990s,the infection rate of H.heilmannii in Guangdong province is higher than abroad(Western0.25%-0.95%.domestic 1.78%-7.32%).As the Nobel Prize for the discovery of H.pylori,people's approval for the relationship between H.pylori and chronic gastritis result in the routine antibiotic treatment.It is unknown, however,whether the epidemic status of H.heilmannii have changed.In view of the unculturable H.heilmannii in vitro,rapid diagnosis is depend mainly on the morphology of the organism.A great deal of attention has focused on the epidemiological investigation,case reports and molecular identification in foreign research,while study is still in vacancy in our country after 90s.The exact mechanism of H.heilmannii causing chronic gastritis is still unkown and there is no strain in vitro. So it's essential to build a animal model with persistent colonization before obtaining the H.heilmannii stain.It appears to be the necessary means and inevitable paths for further study of H.heilmannii.Therefore,the purpose of the study is to learn the epidemiological features at the Present Stage,to culture H.heilmannii in vitro.to establish the long-term model of H.heilmannii infected mice and to clear the pathogenic mechanism preliminarily.Materials and Methods:1.The clinical data,endoscopic reports and pathologic specimens of 2339 patients during 2005-2006 and Jan.-Apr.1994 which collected from Gastroscopy Centre of Nanfang Hospital,were analysed.2.Screening H.heilmannii infected patients:in the first half year of 2007.1 of 703 cases with upper digestive tract endoscopic examination was detected by gastric mucosal biopsy smear.H.heilmannii passaged in mice preservation.3.Isolation and culture of H.heilmannii in different media.temperature and gas conditions.4.Eestablishment of long-term animal model infected with H.heilmannii: 4-6-week-old male BALB/c mice(n=36),female C57BL/6 mice(n=36) and nude mice were randomly assigned to experimental group(n=30) and control group(n =6) for each respective strain.Experimental group was inoculated with H.heilmannii slurry 0.2ml.and euthanatized at 4,8,12.24 and 36 weeks postinoculation(PI).while Control group was inoculated with sterile broth 0.2ml.2 were randomly euthanatized at 4.12. and 36 weeks(nude mice euthanatized at 4,8 and 12 weeks).served as control.Then we observed the bacterial colonization and histopathology.5.To clear the pathogenesis and immunity of H.heilmannii.levels of mRNAs of mucosal cytookines(IFN-γ,,IL-1β,IL-4,IL-10) were assessed using real-time reverse transcription-PCR.Results:1.Of 2339 cases,7 cases of H.heilmannii infected specimens were detected.The detection rate was 0.30 percent.and it was within the framework of foreign reports. but lower than the domestic literature(P= 0.033).2.1 of 703 cases with H.heilmannii was detected by smear.and H.heilmannii was passaged in mice.We try to culture in different conditions.but has not achieved success by now.3.A long-term model of H.heilmannii colonization was successfully established in infected BALB/c mice,C57BL/6 mice and nude mice.H,heilmannii colonization increased with time in three strains,there was significant difference(Mann-Whitney U rank test,P＜0.05) in infected BALB/c mice.Chronic inflammation score reached the peak at 12th weeks and subsequently decreased and increased again in BALB/c mice (P=0.042),while C57BL/6 mice maintained at a higher level without significant difference(P=0.877).There was no markable Lymphoplasmacytic gastritis in infected nude mice.but it significantly appears neutrophil infiltration throughout the whole process(Mann-Whitney U rank test.P＞0.05 ).Acute inflammation score reached the peak at 12^th weeks and showed the lowest point at 36^th weeks(Mann-Whitney U rank test.P＜0.05) in BALB/c mice.The activity of inflammation showed the lowest scores in C57BL/6 mice,but became steady after 8^th weeks(Mann-Whitney U rank test,P＞0.05 ).Two strains of eu-thymic mice began to appear mild atrophy,intestinal metaplasia and lymphoid follicle.Control group had no obvious histological changes.4.The expression of IFN-γ,.IL-1β,IL-4,IL-10 all decreased,compared with control group at different time,One-Way ANOVA analysis showed significant difference(P＜0.05).IL-1βreached its maximum levels at 4 weeks PI,while IFN-γ.IL 4 and IL-10 at 8 or 12 weeks.Two strains of eu-thymic mice showed similar trend of cytokine expression.IFN-γhad not changed distinctly after 12 weeks in C57BL/6 mice,but in BALB/c mice decreased obviously.At 36 weeks IL-4 was lower than the control group in C57BL/6 mice(P＜0.05).Both IL-1βand IL-4 mRNA showed significant difference in nude mice(P＜0.05).while IFN-γ,and IL-10 mRNA showed no significant difference.The correlation for IL-1βand IFN-γ,.IL-10,1L-4 was detected (Spearman correlation analysis test,P＜0.05).IFN-γ.IL-1βand IL-4 correlate with chronic inflammation(Spearman correlation analysis test,P＜0.05).espatially IFN-γ,.Conclusion:1.Infection with H.heilmannii has a lower incidenc in the digestive tract disease now than in 90s.Atrophic gastritis were mostly seen in the infected patients,which suggested that H.heilmannii is an important pathogenic bacteria in the Atrophic gastritis.2.Both BALB/c mice and C57BL/6 mice can establish a long-term colonization model infected with H.heilmannii.3.As represented by T Lymphocyte,Cellular immunity plays a major role in the H. heilmannii infection.while humoral immunity also plays a certain role.4.Th1-type immune responses play important roles in C57BL/6 mice infected with H.heilmannii.And BALB/c mice show both Th1-type and Th2-type immune responses.