A Rare HBV Subgenotype D4 with Unique Genomic Signatures Identified in North-Eastern India –An Emerging Clinical Challenge?

BACKGROUND/AIMS: HBV has been classified into ten genotypes (A-J) and multiple subgenotypes, some of which strongly influence disease outcome and their distribution also correlate with human migration. HBV infection is highly prevalent in India and its diverse population provides an excellent opportunity to study the distinctiveness of HBV, its evolution and disease biology in variegated ethnic groups. The North-East India, having international frontiers on three sides, is one of the most ethnically and linguistically diverse region of the country. Given the paucity of information on molecular epidemiology of HBV in this region, the study aimed to carry out an in-depth genetic characterization of HBV prevailing in North-East state of Tripura. METHODS: From sera of chronically HBV infected patients biochemical/serological tests, HBV DNA quantification, PCR-amplification, sequencing of PreS/S or full-length HBV genomes were done. HBV genotype/subgenotype determination and sequence variability were assessed by MEGA5-software. The evolutionary divergence times of different HBV subgenotypes were estimated by DNAMLK/PHYLIP program while jpHMM method was used to detect any recombination event in HBV genomes. RESULTS: HBV genotypes D (89.5%), C (6.6%) and A (3.9%) were detected among chronic carriers. While all HBV/A and HBV/C isolates belonged to subgenotype-A1 and C1 respectively, five subgenotypes of HBV/D (D1-D5) were identified including the first detection of rare D4. These non-recombinant Indian D4 (IndD4) formed a distinct phylogenetic clade, had 2.7% nucleotide divergence and recent evolutionary radiation than other global D4. Ten unique amino acids and 9 novel nucleotide substitutions were identified as IndD4 signatures. All IndD4 carried T120 and R129 in ORF-S that may cause immune/vaccine/diagnostic escape and N128 in ORF-P, implicated as compensatory Lamivudine resistance mutation. CONCLUSIONS: IndD4 has potential to undermine vaccination programs or anti-viral therapy and its introduction to North-East India is believed to be linked with the settlement of ancient Tibeto-Burman migrants from East-Asia

Influence of polymorphisms in TNF-α and IL1β on susceptibility to alcohol induced liver diseases and therapeutic potential of miR-124-3p impeding TNF-α/IL1β mediated multi-cellular signaling in liver microenvironment

Background and aimsAlcoholic liver disease (ALD) is the leading cause of the liver cirrhosis related death worldwide. Excessive alcohol consumption resulting enhanced gut permeability which trigger sensitization of inflammatory cells to bacterial endotoxins and induces secretion of cytokines, chemokines leading to activation of stellate cells, neutrophil infiltration and hepatocyte injury followed by steatohepatitis, fibrosis and cirrhosis. But all chronic alcoholics are not susceptible to ALD. This study investigated the causes of differential immune responses among ALD patients and alcoholic controls (ALC) to identify genetic risk factors and assessed the therapeutic potential of a microRNA, miR-124-3p.Materials and methodsBio-Plex Pro™ Human Chemokine analysis/qRT-PCR array was used for identification of deregulated immune genes. Sequencing/luciferase assay/ELISA detected and confirmed the polymorphisms. THP1 co-cultured with HepG2/LX2/HUVEC and apoptosis assay/qRT-PCR/neutrophil migration assay were employed as required.ResultsThe combined data analysis of the GSE143318/Bio-Plex Pro™ Human Chemokine array and qRT-PCR array revealed that six genes (TNFα/IL1β/IL8/MCP1/IL6/TGFβ) were commonly overexpressed in both serum/liver tissue of ALD-patients compared to ALC. The promoter sequence analysis of these 6 genes among ALD (n=322)/ALC (n=168) samples revealed that only two SNPs, rs361525(G/A) at -238 in TNF-α/rs1143627(C/T) at -31 in IL1β were independently associated with ALD respectively. To evaluate the functional implication of these SNPs on ALD development, the serum level of TNF-α/IL1β was verified and observed significantly higher in ALD patients with risk genotypes TNF-α-238GA/IL1β-31CT+TT than TNF-α-238GG/IL1β-31CC. The TNF-α/IL1β promoter Luciferase-reporter assays showed significantly elevated level of luciferase activities with risk genotypes -238AA/-31TT than -238GG/-31CC respectively. Furthermore, treatment of conditioned medium of TNF-α/IL1β over-expressed THP1 cells to HepG2/LX2/HUVEC cells independently showed enhanced level of ER stress and apoptosis in HepG2/increased TGFβ and collagen-I production by LX2/huge neutrophil infiltration through endothelial layer. However, restoration of miR-124-3p in THP1 attenuated such inter-cellular communications and hepatocyte damage/collagen production/neutrophil infiltration were prohibited. Target analysis/luciferase-reporter assays revealed that both TNF-α/IL1β were inhibited by miR-124-3p along with multiple genes from TLR4 signaling/apoptosis/fibrogenesis pathways including MYD88, TRAF3/TRADD, Caspase8/PDGFRA, TGFβR2/MCP1, and ICAM1 respectively.ConclusionThus, rs361525(G/A) in TNF-α and rs1143627(C/T) in IL1β gene may be used as early predictors of ALD susceptibility among East Indian population. Impeding overexpressed TNF-α/IL1β and various genes from associated immune response pathways, miR-124-3p exhibits robust therapeutic potential for ALD patients

Coinfection of Leptomonas seymouri and Leishmania donovani in Indian Leishmaniasis

Leishmania donovani is considered the causative organism of visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). Testing of 4/29 DNA samples from VL and PKDL patients as well as 2/7 field isolates showed an aberrant internal transcribed spacer 1 (ITS1) restriction fragment length polymorphism (RFLP) pattern, which upon sequencing strongly matched Leptomonas seymouri, thus confirming its presence in Indian leishmaniasis

Mitogen-activated protein kinases and nuclear factor-kappaB regulate Helicobacter pylori-mediated interleukin-8 release from macrophages.

Gastric infection, as well as inflammation, caused by Helicobacter pylori, activates the production of cytokines and chemokines by mononuclear cells; interleukin-8 (IL-8) is one of the major inflammatory chemokines. Since H. pylori does not invade mucosal tissue, we observed the effect of the water extract of H. pylori (HPE), containing shed factors, on the production of IL-8 by human peripheral blood monocytes and the human monocyte cell line THP-1. HPE-treatment induced activation of the mitogen-activated protein kinases (MAPKs) ERK (extracellular signal-regulated kinase), p38 and JNK (c-Jun N-terminal kinase), an effect which was not dependent on the presence of the cag pathogenicity island. p38 MAPK activation was sustained. The specific inhibitors, U0126 (for ERK1/2 signalling) and SB203580 (for p38 MAPK signalling), both abrogated IL-8 secretion from HPE-treated THP-1. Dominant-negative mutants of the upstream kinases MEK1 (MAPK/ERK kinase 1), MKK (MAPK kinase) 6 and MKK7 also inhibited IL-8 secretion, pointing to a role of all three MAPKs in HPE-mediated IL-8 release. The inhibitory effects of polymyxin B and anti-CD14 antibody suggested that the effect of HPE on MAPKs was mediated by H. pylori lipopolysaccharide (LPS). By analysis of IL-8-promoter-driven luciferase gene expression, we observed that the effects of HPE-induced nuclear factor-kappaB (NF-kappaB) activation and MAPK signalling were mediated at the level of the IL-8 promoter. While ERK1/2 activation could be linked to enhanced DNA binding of activator protein-1 (AP-1), p38 MAPK signalling did not affect AP-1 DNA binding. Taken together, these results provide the first evidence that LPS from H. pylori stimulates IL-8 release from cells of the monocytic lineage through activation of NF-kappaB and signalling along MAPK cascades. The stimulation of MAPK signalling in macrophages by LPS of H. pylori amplifies the inflammatory response associated with gastric H. pylori infection and needs to be taken into consideration when developing therapeutics based on these signalling pathways

Oncogenic potential of Hepatitis B virus subgenotype D1 surpasses D3: significance in the development of hepatocellular carcinoma

This article does not have an abstract

microRNA-124 ameliorates alcohol induced liver injury by restricting multiple components involved in intracellular crosstalk

This article does not have an abstract

Environmental Isolates of Aeromonas spp. Harboring the cagA-Like Gene of Helicobacter pylori

We investigated the presence of cagA-like gene of Helicobacter pylori in environmental isolates of Aeromonas spp. from different water samples of Calcutta, India, by colony hybridization using a cagA-specific DNA probe and by PCR with cagA-specific primers. Nucleotide sequencing of five PCR products revealed 97 to 98% homology to canonical cagA of H. pylori 26695 as well as to four clinical H. pylori strains from Calcutta. The cagA-like gene of the environmental isolates was unstable in laboratory conditions and tended to be lost upon subculturing

Indistinguishable cellular changes in gastric mucosa between Helicobacter pylori infected asymptomatic tribal and duodenal ulcer patients

AIM: To investigate the changing pattern of different histological parameters occurring in the stomach tissue of Helicobacter pylori (H pylori) infected tribal populations and duodenal ulcer patients among ethnic Bengalis and correlation of the genotypes of H pylori with different histological parameters. METHODS: One hundred and twelve adult individuals were enrolled into this study between 2002 and 2004. Among them, 72 had clinical features of duodenal ulcer (DU) from ethnic Bengali population and 40 were asymptomatic ethnic tribals. Endoscopic gastric biopsy samples were processed for histology, genotyping and rapid urease test. Histologically, haematoxylin and eosin staining was applied to assess the pathomorphological changes and a modified Giemsa staining was used for better detection of H pylori. For intestinal metaplasia, special stainings, i.e. Alcian blue periodic acid-Schiff and high iron diamine-Alcian blue staining, were performed. PCR was performed on bacterial DNA to characterize the presence or absence of virulence-associated genes, like cagA, and distribution of different alleles of vacA and iceA. RESULTS: Intraglandular neutrophil infiltration, a hallmark of activity of gastritis, was present in 34 (94%) of tribals (TRs) and 42 (84%) of DU individuals infected with H pylori. Lymphoid follicles and aggregates, which are important landmarks in H pylori infection, were positive amongst 15 (41%) of TRs and 20 (40%) of DU subjects. Atrophic changes were observed in 60% and 27.7%, respectively, among DU cases and tribals (P > 0.003). Metaplastic changes were detected in low numbers in both groups. Moderate to severe density distribution of H pylori in the gastric mucosa was 63% among TRs, whereas it was 62% in DU subjects. There were no significant differences in the distribution of virulence-associated genes like cagA, vacA and iceA of H pylori strains carried by these two populations. CONCLUSION: Our study showed almost similar distribution of inflammatory cells among asymptomatic tribals and DU Bengali patients. Interestingly, the tribal population are free from any clinical symptoms despite evidence of active histologic gastritis and infection with H pylori strains carrying similar virulence markers as of strains isolated from patients with DU. There was an increased cellular response, especially in terms of neutrophil infiltration, but much lower risk of developing atrophy and metaplastic changes among the tribal population