G. Manoj

A simple multiplex PCR assay for diagnosing virulent Helicobacter pylori infection in human gastric biopsy specimens from subjects with gastric carcinoma and other gastro-duodenal diseases

Aim:  To evaluate and develop a multiplex polymerase chain reaction (PCR) assay for diagnosing and specific identification of virulent Helicobacter pylori strains and their main virulence genes cagA, cagE, cagT, vacA and hrgA.

Anti-unlcer and antimicrobial activities of sodium selenite against Helicobacter pylori: in vitro and in vivo evaluation.

Abstract We examined sodium selenite, an inorganic selenium supplement, for its ulcer healing properties and antimicrobial activity against gastric pathogen Helicobacter pylori. Minimum inhibitory concentrations (MIC) were determined using disk diffusion and flow cytometry. The studies were performed over a concentration range of 1 μg/ml to 500 μg/ml sodium selenite. Mild activity was seen at 10 μg/ml and 50 μg/ml, a moderate response at 100 μg/ml and strong response at 500 μg/ml with a MIC value of 10 μg/ml. The compound was found to be active at low pH without any resistance after 10 passages. Flow cytometry data showed a characteristic shift of the viability peak in comparison with the control, thereby confirming the bactericidal effects of sodium selenite. Sodium selenite administered in Wistar rats, pre-ulcerated with naproxen and infected with H. pylori, showed ulcer healing and anti-H. pylori activity at a concentration range of 10–50 μg/rat; however concentrations of 100 μg/rat and 500 μg/rat were found to be toxic in the in vivo studies. In conclusion, sodium selenite shows both ulcer healing and anti-H. pylori activity at a low concentration (10 μg/rat) without toxicity.

Antibacterial and ulcer healing effects of organoselenium compounds in naproxen induced and Helicobacter pylori infected wistar rat model.

Aim of the present study was to evaluate in vitro toxicity and in vivo antibacterial, anti-inflammatory, antiulcer, and antioxidant activities of two organoselenium compounds, selenocystine (SeCys) and ebselen (Ebs). The study was conducted in experimentally induced ulcers in rodent model infected with Helicobacter pylori (H. pylori). In vitro toxicological studies on normal splenic lymphocytes revealed that SeCys and Ebs were non-toxic to the cells even at 100 μM concentration. Antibacterial activity was observed at 500 μg/mL concentration of either of the compounds against H. pylori. In vivo studies after treatment with SeCys and Ebs (500 μg/kg/day) resulted in significant reduction in ROS production and inhibition of lipid peroxidation in gastric tissue. The antioxidant and anti-inflammatory activities of both the compounds were also confirmed by their ability to lower GSH reduction, to induce the expression of antioxidant genes such as GPx-4, and MnSOD and to suppress inflammatory genes namely COX-2, TNF-α and TGF-β. In addition, the immunomodulatory activity of both the compounds was evident by enhance of the CD4 levels and maintenance of the IgG, IL-6 and IL-10 levels. Persistent treatment (500 μg/kg, for 28 days) with both the compounds showed considerable (p<0.05) ulcer healing property supporting its role in gastro protection. In conclusion, the results of our study suggest that both SeCys and Ebs possess broad spectrum of activities without any potential toxicity.

Association of genetic variants of mannan-binding (MBL) lectin-2 gene, MBL levels and function in ulcerative colitis and Crohn’s disease

Ulcerative colitis and Crohn’s disease are the two major forms of inflammatory bowel disease (IBD). A series of reports have hypothesized interplay of genetic and environmental factors in the pathogenesis of IBD. Polymorphism in the mannan-binding lectin-2 (MBL-2) gene is known to affect the structural assembly and function thereby predisposing subjects to various diseases. The present study was designed to evaluate effect of MBL-2 gene polymorphism on MBL levels and function in IBD patients. Genomic DNA was isolated from blood samples collected from 157 ulcerative colitis, 42 Crohn’s disease and 204 control subjects. Genotyping for different polymorphic sites at exon1 of MBL-2 gene was performed by refractory mutation system-PCR and amplification followed by restriction digestion (PCR-RFLP). Serum MBL concentration and C4 deposition levels were estimated using ELISA. Mannan-binding lectin-2 genotypic variants were calculated in IBD and healthy controls. The frequency of single nucleotide polymorphisms at codon 54 was significantly higher in ulcerative colitis patients than controls (P < 0.0001). Ulcerative colitis patients with ‘codon 54’-variation showed low serum MBL concentrations coupled with altered MBL function compared to controls. In conclusion, single nucleotide polymorphism in the MBL-2 gene is an important risk factor significantly affecting MBL levels and function in the development of ulcerative colitis among Indians.

Relevance of Helicobacter pylori genotypes in gastric pathology and its association with plasma malondialdehyde and nitric oxide levels

Persistent infection with Helicobacter pylori confers an increased risk of peptic ulceration and gastric adenocarcinoma. Reactive oxygen and nitrogen species play a crucial role in the progression from normal gastric mucosa to cancer. The aim of the present study was to investigate the plasma malondialdehyde and nitric oxide levels in H. pylori related gastroduodenal diseases and associate their levels with gastric pathology and genotypes of H. pylori. Malondialdehyde and nitric oxide levels in plasma samples of 250 subjects were spectrophotometrically determined. Subsequently, genotypic and histopathological assessment was performed in gastric biopsies obtained during endoscopy. The levels of MDA and NO exceeded in subjects infected with genotype-1 of Hp than those with other genotypes suggesting more precise interaction of highly virulent strains of Hp in eliciting severe tissue damage. In conclusion, the study demonstrates close relationship between the plasma malondialdehyde and nitric oxide levels, gastric histopathology and genotypes of H. pylori.

Simplified and versatile method for bisulfite‐based DNA methylation analysis of small amounts of DNA

Epigenetic alterations of gene function play a central role in the pathogenesis of many tumors and in the process of aging. Abnormal methylation at transcriptional sites of genes results in epigenetic silencing of the genes that protect against tumor formation or that repair DNA. To date, several studies have analyzed methylation status by oligonucleotide arrays, restriction analysis (COBRA), methylation‐specific amplification, and sequence analysis. Thisrequires high concentration of bisulfite‐treated DNA, which mandates use of commercially available expensive kits, and is an often laborious and time‐consuming task. In this article, we report a simplified high‐throughput method, which can serve as a surrogate for screening methylation profiles of various genes and has high sensitivity compared with the other methods described previously. J. Clin. Lab. Anal. 23:172–174, 2009.

Association of Helicobacter pylori restriction endonuclease-replacing gene, hrgA with overt gastrointestinal diseases

BACKGROUND AND AIM Helicobacter pylori has been proven to be responsible for causing various gastrointestinal disorders including gastric adenocarcinoma. Several genes of pathogen (the genes of the cag-PAI, vacA, iceA, and babA) either in combination or independently have been reported to significantly increase the risk of ulceration/gastric carcinoma, with the cagA gene having the strongest predictive value. Pursuit to identify new genes which could serve as a marker of overt disease progression, lead to the discovery of hrgA gene. METHODS Fifty-six indigenous strains of H. pylori from subjects with various gastric disorder were screened to assess the status of hrgA gene along with the cagA gene using simple polymerase chain reaction using specific oligonucleotide primers. Post-amplification, amplicons were subjected for sequencing to identify any strain specific variations in sequences from the H. pylori isolated from different disease manifestations. Histopathological analysis was done to ascertain any significant change in the histological scores of subjects infected with cagA+/hrgA+ and cagA-/hrg+ strains. RESULTS All the 56 (100%) subjects amplified with the oligonucleotide primers specific to hrgA gene, whereas 81.71% subjects showed the presence of cagA gene. Sequencing of the amplimers showed 99% homology. Histology of the cagA+/hrgA+ and cagA-/hrg+ subjects did not show any significant difference. CONCLUSION hrgA gene of Helicobacter pylori is not a ideal surrogate marker for identifying individuals with higher risk of developing overt gastro-duodenal diseases such as neoplasia of the stomach.

PP-030 Risk of glandular atrophy, intestinal metaplasia and dysplasia in subjects with vacA positive and complete or disrupted cagE, cagT Helicobacter pylori infection

PP-029 Management of oesophageal varices by reloading inexpensive hemorrhoidal O-rings for band ligation at Evangel Hospital, Nigeria N.G. Ladep1 *, J. Sule2, P. Ushie2, R. Ugiagbe1, M. Topazian3, W. Ardill2. 1Department of Medicine, University of Jos and Jos University Teaching Hospital, PMB 2076, Jos, Plateau state, Nigeria, 2ECWA Evangel Hospital, Zaria Bye-pass, Jos, Plateau state, Nigeria, 3Mayo Clinic, Rochester MN, USA

OL-030 Synergistic interplay between Helicobacter pylori virulence genes and host COX-2 and iNOS enhances the risk of premalignant and malignant lesions

Background and Aim: H. pylori (Hp) strains vary in their carcinogenic potential. Both host factors and bacterial factors have been postulated to contribute to the variable outcome. Over expression of host COX-2 and iNOS has been implicated in the development of gastric carcinoma. Furthermore, the link between genotypes in relation to COX-2 and iNOS expression and disease status needs to be determined. Therefore the present study addressed to identify Hp-bearing hosts who are at greatest risk of developing precancerous lesions. Methods: A total of 240 subjects with various gastric disorders were screened. Genotyping based on cagA, cagE, cagT, vacA signal region and hrgA genes of Hp was performed using DNA from gastric biopsies. Expression of COX-2 and iNOS was assessed by RT-PCR and immunoblotting. Histological scoring of antral and corpus biopsies for the presence precancerous lesions was done. Results: The genotype cagA+/cagE+/cagT+/hrgA+/vacAs1 showed high prevalence 177 (73.7%). Among which 81.1% had overt gastric disorders whereas 46% subjects had less severe gastric disease. Histology revealed presence of atrophy in 52% vs 18%, IM in 32% vs 9% and dysplasia in 20% vs 4% respectively (Statistically significant at p < 0.01). RT-PCR and immunoblotting data showed high expression patterns of COX-2 and iNOS in overt gastric disorders than with less severe gastrointestinal disorders. Conclusion: Genotype cagT+ve/hrgA+ve/cagA+ve/cagE+ve/ vacAs1+ve and heightened expression levels of COX-2 and iNOS have higher differentiating and predictive value for the development of severe disease manifestations. This suggests that Hp induced gastric inflammatory reaction to be influenced by multiple factors, and probably results from the synergistic effect of bacterial virulence and host factors, which work together in a complex way causing various diseases in the host.