LEVELS OF HBD2 AND HBD3 IN PATIENTS INFECTED AND NON-INFECTED BY HELICOBACTER PYLORI

Introduction: Infection with Helicobacter pylori (H. pylori) evokes inflammatory and immune responses of the host, which most likely determine the clinical outcome of H. pylori infection. In the gastric epithelium, expression of human alpha-defensins, beta-defensins (hBD)-1, -2 and -3 has been detected in vivo. In particularly hBD3 as well hBD2 is known to be induced in gastric epithelial cells infection by the H. pylori and may be involved in the pathogenesis of H pylori-associated gastritis, possibly through its function as immune and inflammatory mediator. However, during prolonged infection, hBD3 was subsequently downregulated by the H. pylori virulence determinant CagA. Materials and Methods: Patients. In this study, serum and biopsies of 33 healthy individuals stratified according to the H. pylori infection (17 positive and 11 negative) was analyzed. Elisa Assay and Real Time PCR. Sera of patients were assayed by enzyme-linked immunosorbent (ELISA) assay for detection of both hBD- 2 (Phoenix Pharmaceuticals, Inc.) and hBD-3 (Alpha Diagnostic International) levels, according to the manufacture’s protocol. Protein concentration was expressed as pg/ml of serum volume. Real time PCR was carried out with the LC Fast Start DNA Master SYBR Green kit (LightCycler 2.0 Instrument, Roche; Milano, Italy) and PCR products were examined on 1.4% agarose gel. 192 Posters P 136 IMPACT OF HBV GENOTYPES A AND D GENETIC VARIABILITY ON INFECTION EVOLUTION Noemi Urone, Donatella Ferraro Sezione di Microbiologia “A. Chiarini”, Dipartimento di Scienze per la Promozione della Salute e Materno Infantile “G. D’Alessandro”, Università degli Studi di Palermo - Italy Introduction: HBV is characterized by a high genetic variability and several studies have demonstrated the correlation between viral variants and severe forms of acute and chronic liver disease. Few data are available about the impact of genetic variability on the evolution of acute infection. Recently, 5 SNPs (C504, C801, G1171, T1785 and A1786) into HBV genome were reported as potential novel markers linked to a lower rate of chronicity. The aim of this study was to analyze the genetic variability of HBV genotypes A and D isolates from cases of self-limiting acute (AHB) and chronic hepatitis (CHB) in order to identify HBV variants associated with chronicity or resolution of infection. Patients and Methods: The preS-S, preC-C and the overlapped P and X genes, were sequenced from sera of 33 AHB and 46 CHB Sicilian patients and analyzed with 92 HBV-A and D sequences from Gene Bank in order to identify HBV geno/subtype by phylogenetic analysis and to study the genetic variability. Results: Eighteen strains of HBV-D and 15 of HBV-A from AHB, 42 of HBV-D and 4 of HBVA2 from CHB patients were obtained. Two in frame deletions, between preS1 and preS2 regions (Δ173-321, Δ1-29) and in the S gene (Δ489-516) were detected in an AHB-D3 strain while 2 CHB-D1 strains showed the deletions Δ37-52 and Δ36-57 into preS2 region. The mutation M01I at preS2 start codon was detected in 2 AHB-D1 and a CHB-D1strains. Mutations of immune-escape were not found but >30% of strains showed many amino acid polymorphisms in “a determinant” domain. Secondary drug resistance mutations on RT-domains were identifiedGenotyping of H.pylori. The DNA of each biopsy, estracted using a High Pure Template Preparation Kit (Roche), was used to amplify following genes: ureA, cagA. Results: Protein levels of both studied defensins, hBD-2 and hBD-3, are lower or unchanged in serum of H. pylori- positive compared to those H. pylori- negative subjects. However, mRNA levels of hBD-2 and hBD-3 are up-regulated in most gastric biopsies of H. pylori-positive subjects, except in patients belonging to the genotype cagA+ vacA s1m1 EPIYA ABC. Conclusion: Although it has been demonstrated that H.pylori infection was associated with significant upregulation of hBD-2 and hBD-3 transcript levels in gastric mucosa, our data suggest that no similar association was found on protein level in serum of patients. In addition to, the upregulation of hBD-2 and hBD-3 transcript levels is absent in gastric biopsies of some H. pylori- positive subjects. These results suggest that, most likely, H. pylori strains belonging to a particular genotype not only inhibit pathogen-derived hBD-2 and hBD- 3 induction, but also block their constitutive expression to escape from the strong antimicrobial effects.