Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbv-positive human hepatocelluar carcinoma cell line.

BACKGROUND: Toll like receptors (TLRs) play an important role in innate immunity and various studies suggest that TLRs play a crucial role in pathogenesis of hepatitis B virus (HBV) infection. The present study aims in looking into the status of crucial host and viral gene expression on inciting TLR7. METHODS: The transcription of TLR7 pathway signaling molecules and HBV DNA viral load were quantified by Real Time-PCR after stimulation of TLR7 with its imiquimod based ligand, R837. Cell cycle analysis was performed using flow-cytometry. Expression of TLR7 and chief cell cycle regulator governing G1/S transition, p53 was also seen in liver biopsysss samples of CHB patients. HBV induced alteration in histone modifications in HepG2 cells and its restoration on TLR7 activation was determined using western blot. RESULTS: The TLR7 expression remains downregulated in HepG2.2.15 cells and in liver biopsy samples from CHB patients. Interestingly HBV DNA viral load showed an inverse relationship with the TLR7 expression in the biopsy samples. We also evaluated the anti-viral activity of R837, an agonist of TLR7. It was observed that there was a suppression of HBV replication and viral protein production upon TLR7 stimulation. R837 triggers the anti-viral action probably through the Jun N-terminal Kinase (JNK) pathway. We also observed a downregulation of histone H3K9Me3 repression mark upon R837 treatment in HBV replicating HepG2.2.15 cells, mimicking that of un-infected HepG2 cells. Additionally, the G1/S cell cycle arrest introduced by HBV in HepG2.2.15 cells was released upon ligand treatment. CONCLUSION: The study thus holds a close insight into the changes in hepatocyte micro-environment on TLR7 stimulation in HBV infection.

Transmission dynamics of hepatitis C virus among intra venous drug users in the border state of Manipur, India.

Intra venous drug users (IVDUs) are at high risk for hepatitis C virus (HCV) infection owing to their high rate of drug abuses. The north-eastern part of India has a high prevalence of IVDUs with Manipur being the worst hit state. The aim of the study was to document the molecular epidemiology, the patterns of HCV transmission, genomic variation and recombination events within HCV genome among IVDUs of Manipur, India. 91 anti-HCV sero-reactive blood samples were collected from IVDUs in Manipur. The samples were processed for RNA extraction, nested RT-PCR, sequencing and quantitative viral RNA estimation. Phylogeographic analysis of the sequenced core and NS5B regions of HCV genome was performed to determine the probable transmission route and recombinant HCV strains. 83 out of 91 anti-HCV seropositive samples were RNA positive (91.20%) based on 5'UTR of HCV genome by nested RT-PCR. Of the RNA positive samples, 73 paired partial core and NS5B gene were sequenced. Three major genotype and eight subtypes were detected while no recombinant strains were found. Individuals with genotype 1 had the mean viral load (5.94 ± 0.705 log10IU/ml) followed by genotype 3 (4.91 ± 0.49 log10IU/ml) and 6 (3.96 ± 0.32 log10IU/ml). The viral load was statistically significant among the male individuals at 4.822 ± 1.36 log10IU/ml compared to 4.767 ± 0.49 log10IU/ml for females (t=3.249, p<0.005). The phylogeographic results indicated 3b, 6h originated from Vietnam, 1a had Indian origin, 3a, 6k originated from southern China while 1b originated from Myanmar, respectively. The incidence of eight different subtypes in Manipur reflects the transmission of these strains from the "Golden Triangle" drug trafficking regions. Sequence analysis confirmed the transmission routes of HCV, which is linked to China and Vietnam for the newly emergent genotype 6 in north-eastern India.

Molecular characterization of HBV strains circulating among the treatment-naive HIV/HBV co-infected patients of eastern India.

Previously we reported that the exposure to hepatitis B virus (HBV) infection serves as a major threat among the treatment naive HIV infected population of eastern India. Hence, molecular characterization of these strains is of utmost importance in order to identify clinically significant HBV mutations. A total of 85 treatment naive HIV/HBV co-infected participants were included of whom the complete basal core promoter/precore region, the core and the whole envelope gene could be successfully sequenced for 59, 57 and 39 isolates respectively. Following phylogenetic analysis, it was found that HBV/D was the predominant genotype with HBV/D2 (38.5%) being the most prevalent subgenotype followed by HBV/A1. The major mutations affecting HBeAg expression includes the A1762T/G1764A (13.6%), G1896A (22%) and G1862T mutation (33.9%) which was predominantly associated with HBV/A1. Moreover, the prevalence of G1896A was considerably high among the HBeAg negative HIV/HBV co-infected subjects compared to HBV mono-infection. The main amino acid substitutions within the MHC class II restricted T-cell epitope of HBcAg includes the T12S (15.8%) and T67N (12.3%) mutation and the V27I (10.5%) mutation in the MHC class I restricted T-cell epitope. PreS1/S2 deletion was detected in 3 isolates with all harboring the BCP double mutation. Furthermore, the frequently occurring mutations in the major hydrophilic loop of the S gene include the T125M, A128V and M133I/L. Therefore, this study is the first from India to report useful information on the molecular heterogeneity of the HBV strains circulating among the treatment naive HIV/HBV co-infected population and is thus clinically relevant.

Association of TNF-α Promoter Polymorphism with HBV Associated Disease Outcome Among HBV Infected Patients from Orissa, Southern Part of East India.

BACKGROUND: TNF-α promoter polymorphism has been known to be a potential predictive factor in patients with HBV infection. We therefore tried to investigate whether the TNF-α promoter polymorphism at position -238, -857 and -863 was associated with the outcome of HBV infection in a population from Orissa, southern part of East India. METHODS: A total of 195 patients recruited for the study were classified into 85 controls and 110 HBV infected cases, which included 34 IC, 30 CLD, 32 LC and 14 HCC patients. The polymorphisms at the respective sites were detected by a PCR-RFLP followed by statistical analysis. RESULTS: The frequency of the genotype -238 GG and the allele -238G in the cases (89.0% and 92.7% respectively) was significantly higher than that in the controls (68.2% and 82.2% respectively) (P < 0.001, OR = 3.8 and P = 0.001, OR = 2.73). Whereas the -238 GA genotype was significantly high in the control group (28.2%) when compared to the cases (7.2%) (P < 0.001, OR = 0.2). Similarly, the frequency of -863CC and the allele -863C was significantly higher among the cases (24.5% and 49.5%) compared to controls (1.17% and 34.7%), (P < 0.001, OR = 27.32 and P = 0.003, OR = 1.85), whereas the -863CA genotype was significantly high in the controls (67.0%) when compared to the cases (50.0%) (P = 0.01, OR = 0.49). Haplotype -863C/-857C/-238G in cases was significantly higher than controls (P = 0.002). Multivariate logistic regression analysis indicates that the genotype -863CC bears a negative association with liver disease progression. CONCLUSION: The present study established an association of polymorphisms at site -238 and -863 of the TNF-α promoter with the outcome HBV infection and disease progression.

Evaluation of the wound healing activity of Shorea robusta, an Indian ethnomedicine, and its isolated constituent(s) in topical formulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Different parts of Indian ethnomedicinal plant Shorea robusta is traditionally used for several ailments including wounds and burn by different tribal groups, since ages. Here we have validated, for the first time, the effectiveness and the possible mechanism of action of young leaf extracts of Shorea robusta, used by two distinct tribes of India, and its isolated compounds as a topical formulation in three wound models in rats. MATERIALS AND METHODS: Bioactivity-guided study of the active extract resulted in the isolation of two known compounds. The prepared ointment containing extracts (2.5 and 5%, w/w), fractions (5% w/w) and isolated compounds (0.25% w/w) were evaluated on excision, incision and dead space wound models in rats by the rate of wound closure, period of epithelialisation, tensile strength, granulation tissue weight, hydroxyproline content and histopathology. RESULTS: The animals treated with the extracts and fractions (5%) showed significant reduction in wound area 96.55 and 96.41% with faster epithelialisation (17.50 and 17.86), while the isolated compounds bergenin and ursolic acid heal the wound faster, but complete epithelialisation with 100% wound contraction was evident with 5% povidone-iodine group on 18th post-wounding day. Moreover, the tensile strength of incision wound, granuloma tissue weight, and hydroxyproline content was significantly increased in both the extract and compound(s) treated animals. Furthermore, the tissue histology of animals treated with the isolated compound(s) showed complete epithelialisation with increased collagenation, similar to povidone-iodine group. CONCLUSION: Thus, our results validated the traditional use of Shorea robusta young leaves in wound management.

HIV-1 gp120 protein downregulates Nef induced IL-6 release in immature dentritic cells through interplay of DC-SIGN.

HIV-1 replication is a tightly controlled mechanism which demands the interplay of host as well as viral factors. Both gp120 (envelope glycoprotein) and Nef (regulatory protein) have been correlated with the development of AIDS disease in independent studies. In this context, the ability of HIV-1 to utilize immature dentritic cells for transfer of virus is pivotal for early pathogenesis. The presence of C-type lectins on dendritic cells (DCs) like DC-SIGN, are crucial in inducing antiviral immunity to HIV-1. Both gp120 and Nef induce the release of cytokines leading to multiple effects of viral pathogenesis. Our study elucidated for the first time the cross-talk of the signaling mechanism of these two viral proteins in immature monocyte derived dentritic cells (immDCs). Further, gp120 was found to downregulate the IL-6 release by Nef, depending on the interaction with DC-SIGN. A cascade of signaling followed thereafter, including the activation of SOCS-3, to mediate the diminishing effect of gp120. Our results also revealed that the anti-apoptotic signals emanated from Nef was put to halt by gp120 through inhibition of Nef induced STAT3. Thus our results implicate that the signaling generated by gp120 and Nef, undergoes a switch-over mechanism that significantly contributes to the pathogenesis of HIV-1 and widens our view towards the approach on battling the viral infection.

Association of Interleukin-1ß and Gene Polymorphisms with Liver Pathogenesis in Hepatitis B Virus Infection among Eastern Indian Population.

BACKGROUND: Interleukin-1ß (IL-1ß) is an important member of the family of the proinflammatory cytokines that modulate outcome of hepatitis B virus (HBV) infection. OBJECTIVES: This study was designed to investigate the relationship between the polymorphic genotypes of the interleukin-1ß (IL-1ß) promoter region and the interleukin-1 receptor antagonist gene (IL-1RN) and disease outcome in HBV-infected individuals. METHODS: DNA was extracted from 395 study subjects including HBV carriers with varying clinical presentations, as well as healthy controls and spontaneously recovered cases (SRC). Polymorphisms in IL-1ß (at position -511) and IL-1RN (variable nucleotide tandem repeats, VNTR) were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR based assay respectively. RESULTS: Among the study subjects, different IL-1ß (at position -511) (CC, CT and TT) and IL-1RN (1/1, 1/2, 2/2 and 1/3) polymorphic genotypes were found at variable proportions. Logistic regression analysis revealed, no notable difference at the level of IL-1ß promoter (P = 0.244; OR = 0.78; 95% CI = 0.52-1.18) or IL-1RN genotype polymorphism (P = 0.840; OR = 1.03; 95% CI = 0.78-1.36) among the HBV carriers and controls or SRC cases. Pairwise proportion testing showed, IL-1ß -511 genotype CC was significantly higher among asymptomatic carriers (ASC) in comparison with liver cirrhosis (LC) patients (P value = 0.028) and healthy control group (P-value = 0.036). IL-1RN genotype 2/2 was considerably higher in LC group than SRC as well as control group. Combinations of IL-1ß (-511) and IL-1RN polymorphisms were associated with disease progression, such as CC-1/2 with ASC and TT-2/2 with LC. CONCLUSION: IL-1ß polymorphisms are found to be associated with disease severity. Different polymorphic combinations are associated with degree of disease severity. Overall this is the first report from Eastern India, which shows association of IL-1ß polymorphisms with HBV-related hepatic complications.

Influence of HIV-associated degree of immune suppression on molecular heterogeneity of hepatitis B virus among HIV co-infected patients.

We have investigated the molecular diversity of Hepatitis B virus (HBV) among the HIV co-infected patients from eastern-India. HBsAg/HBV-DNA positive subjects (n=73) from 874 HIV-infected patients were analyzed by sequencing followed by genetic diversity quantification. HBV/genotype-D and HBV/sugenotype-D2 were predominant. HBV/D2 isolates from patients with low CD4 count manifested significantly lower non-synonymous substitutions (p<0.0001) and Shannon entropy (p=0.0006) in their surface and polymerase gene in comparison to those from moderately increased CD4 count. ART-induced immune-reconstitution therefore might raise non-synonymous immune/therapy escape substitutions among these HBV/D2 isolates. Decreased genetic diversity and increased viral load in the HBV/D2 isolates might facilitate the maintenance of their wild type characteristics in the low CD4 count, leading to its increased prevalence in this group. Interestingly, genetic diversity in HBV/A1, the next common subgenotype, was modified in the opposite manner. Together our results underscore the need for proper HBV molecular monitoring in HIV co-infection.

Subgenotype D5, BCP and MHR mutations in hepatic complications among hepatitis B virus infected patients from Orissa, India.

The study was undertaken to investigate the clinical implications of hepatitis B virus (HBV) genotypes, basal core promoter (BCP), precore (PC) and surface gene mutations in HBV infected patients from Orissa, southeastern India. HBV infections were identified by serology testing and HBV DNA amplification by polymerase chain reaction among the 152 patients. After sequencing, surface gene mutation were studied by sequence analysis as well as by using BLOSUM scores and BCP mutations were studied only by sequence analysis. A high proportion of HBV/D5 (66.0%) was found among the study samples having significant relation with liver cirrhosis (LC) and hepatocellular carcinoma (HCC) patients (p<0.05). The BCP mutation, TA (81.4%) and C1753/TA (75.0%) was found in significant proportion (p<0.05) among HCC cases and in fact a gradual increase in these mutations were noted between inactive carriers (IC) to HCC group and also showed higher viral load. An increasing trend of major hydrophilic region (MHR) mutations in S gene was also observed from IC (56.0%) to chronic liver disease (CLD) (60.4%) to LC (72.4%) to HCC (95.0%) patients. In conclusion, our study suggests that the predominant HBV subgenotype HBV/D5 with high viral load and BCP mutations (double and triple) and high mutations in MHR region was significantly associated with advanced liver disease (LC and HCC) and might act as predictor of severe hepatic complications.

Anti-hepatitis B core antigen testing with detection and characterization of occult hepatitis B virus by an in-house nucleic acid testing among blood donors in Behrampur, Ganjam, Orissa in southeastern India: implications for transfusion.

BACKGROUND: Occult hepatitis B virus (HBV) infection might transmit viremic units into the public blood supply if only hepatitis B surface antigen (HBsAg) testing is used for donor screening. Our aim was to evaluate the prevalence of occult HBV infection among the HBsAg negative/antiHBc positive donations from a highly HIV prevalent region of India. METHODS: A total of 729 HBsAg negative donor units were included in this study. Surface gene and precore region were amplified by in house nucleic acid test (NAT) for detection of occult HBV infection and surface gene was analyzed after direct sequencing. RESULTS: A total of 220 (30.1%) HBsAg negative donors were antiHBc positive, of them 66 (30%) were HBV DNA positive by NAT. HBV DNA positivity among 164 antiHBc only group, was 27.1% and among 40 antiHBs positive group was 30.0%. HBV/D (93.3%) was predominant and prevalence of both HBV/C and HBV/A was 3.3%. Single or multiple amino acids substitutions were found in 95% samples. CONCLUSION: Thus, a considerable number of HBV infected donors remain undiagnosed, if only HBsAg is used for screening. Addition of antiHBc testing for donor screening, although will lead to rejection of a large number of donor units, will definitely eliminate HBV infected donations and help in reducing HBV transmission with its potential consequences, especially among the immunocompromised population. The HBV genetic diversity found in this donor population are in accordance with other parts of India.

Phylogenetic analysis of the p24-p7 region of the human immunodeficiency virus type 1 gag gene to determine subtype distribution among female sex workers in Calcutta, India.

Human immunodeficiency virus type 1 (HIV-1) subtype C, based on the envelope region, has been reported to be predominant in India. We sequenced the p24-p7 gag region from 51 HIV-1 seropositive female sex workers in Calcutta, India, for more-detailed molecular characterization. Subtype C was found to be prevalent, although no strong monophyletic cluster was observed.

Determination of gag subtypes of HIV type 1 detected among female sex workers in Calcutta, India.

HIV-1 subtyping is important to study the changing scenario of genetic variation. The gag-based heteroduplex mobility assay (gag-HMA) was developed and evaluated as a powerful and reliable technique for identifying the HIV-1 group M subtypes A to H and the circulating recombinant forms (CRFs). To study the subtype distribution of HIV-1 strains from the eastern part of India, we used the gag-based HMA, followed by the sequencing and phylogenetic analysis. Blood samples from HIV-1-seropositive female sex workers in Calcutta were subjected to gag-HMA. The most prevalent subtype was found to be the C type, among which the C4 subsubtype was prevalent. However, a number of nontypable C strains were found in gag-HMA. Phylogenetic analysis revealed the discrete nature of the C strains and no monophyletic cluster was noticed. This result might indicate a growing tendency of variations among the HIV-1 type C strains circulating in eastern India.

Determination of gag and env subtypes of HIV-1 detected among injecting drug users (IDUs) in Manipur, India: evidence for intersubtype recombination.

The majority of HIV-1 transmission in Manipur, one of the northeastern states of India, is through the sharing of needles and syringes among the injecting drug users (IDUs). A total of 28 HIV seropositive samples were used to determine the HIV-1 subtypes with respect to both gag and envelope genes. The specific regions within gag and envelope genes were amplified from PBMC DNA by nested PCR using appropriate primers. These amplicons were used in heteroduplex mobility assay followed by DNA sequencing. Phylogenetic analysis of the nucleotide sequences of respective regions showed that 89% of samples (25/28) were infected with subtype C with respect to both gag and envelope genes; however, 11% of the samples (3/28) showed subtype discordance with respect to the envelope (C2-V3) and gag (p24-p7) genomic regions. Simplot analysis of the discordant samples showed the presence of intersubtype recombination between subtype C and Thai B; two samples were found to be subtype C in envelope but Thai B in gag, whereas, one sample was found to be subtype Thai B in envelope and 'C' in gag region.

HIV type 1 subtypes circulating in eastern and northeastern regions of India.

Genetic subtyping has been a powerful tool in tracking the global spread of HIV. To determine the HIV-1 subtypes circulating in eastern and northeastern regions of India blood samples were collected from female sex workers in Calcutta and intravenous drug users (IDUs) in Manipur. Fifty-four samples from Calcutta and 25 samples from Manipur were analyzed for HIV-1 subtyping by heteroduplex mobility assay (HMA). Twenty-six samples from these regions were sequenced. HMA and sequencing of the samples from these regions revealed subtype C as the major subtype, circulating within both eastern and northeastern regions. In Manipur, subtype ThaiB was also detected as the second major subtype. Some of the IDUs from Manipur were found to be dual infected with subtype C and ThaiB.

A molecular approach (multiplex polymerase chain reaction) for diagnosis of rhinosporidiosis.

A New multiplex PCR have been developed in our laboratory using primer sets, aiming amplification of both D.N.A target fragments obtained in 18S RNA of commonly encountered fungi in human being and in Rhinosporidium seeberi using F1-fw/F2-rev (500 bp target) and Rhino-fw/ Rhino-rev (.177 bp target). This multiplex PCR has been found to be able to delect R. seeberi from clinical samples and differentiate it from other fungi. Furthermore, by this multiplex PCR, R. seeberi, phylogenitically appears to belong to a member of so called DRIPs clade of fish parasite not a cyanobacterium as claimed previously, by some workers.