Variability in the Responses of Hepatitis B Virus D-Subgenotypes to Antiviral Therapy: Designing Pan-D-Subgenotypic Reverse Transcriptase Inhibitors.

Nucleos(t)ide analogues entecavir (ETV) and tenofovir disoproxil fumarate (TDF) are recommended as first-line monotherapies for chronic hepatitis B (CHB). Multiple HBV genotypes/subgenotypes have been described, but their impact on treatment response remains largely elusive. We investigated the effectiveness of ETV/TDF on HBV/D-subgenotypes, D1/D2/D3/D5, studied the structural/functional differences in subgenotype-specific reverse transcriptase (RT) domains of viral polymerase, and identified novel molecules with robust inhibitory activity on various D-subgenotypes. Transfection of Huh7 cells with full-length D1/D2/D3/D5 and in vitro TDF/ETV susceptibility assays demonstrated that D1/D2 had greater susceptibility to TDF/ETV while D3/D5 exhibited poorer response. Additionally, HBV load was substantially reduced in TDF-treated CHB patients carrying D1/D2 but minimally reduced in D3/D5-infected patients. Comparison of RT sequences of D-subgenotypes led to identification of unique subgenotype-specific residues, and molecular modeling/docking/simulation studies depicted differential bindings of TDF/ETV to the active site of their respective RTs. Replacement of signature residues in D3/D5 HBV clones with corresponding amino acids seen in D1/D2 improved their susceptibility to TDF/ETV. Using high throughput virtual screening, we identified N(9)-[3-fluoro-2-(phosphonomethoxy)propyl] (FPMP) derivatives of purine bases, including N6-substituted (S)-FPMP derivative of 2,6-diaminopurine (DAP) (OB-123-VK), as potential binders of RT of different D-subgenotypes. We synthesized (S)-FPMPG prodrugs (FK-381-FEE/FK-381-SEE/FK-382) and tested their effectiveness along with OB-123-VK. Both OB-123-VK and FK-381-FEE exerted similar antiviral activities against all D-subgenotypes, although FK-381-FEE was more potent. Our study highlighted the natural variation in therapeutic response of D1/D2/D3/D5 and emphasized the need for HBV subgenotype determination before treatment. Novel molecules described here could benefit future design/discovery of pan-D-subgenotypic inhibitors. IMPORTANCE Current treatment of chronic hepatitis B relies heavily on nucleotide/nucleoside analogs in particular, tenofovir disoproxil fumarate (TDF) and entecavir (ETV) to keep HBV replication under control and prevent end-stage liver diseases. However, it was unclear whether the therapeutic effects of TDF/ETV differ among patients infected with different HBV genotypes and subgenotypes. HBV genotype D is the most widespread of all HBV genotypes and multiple D-subgenotypes have been described. We here report that different subgenotypes of HBV genotype-D exhibit variable response toward TDF and ETV and this could be attributed to naturally occurring amino acid changes in the reverse transcriptase domain of the subgenotype-specific polymerase. Further, we identified novel molecules and also synthesized prodrugs that are equally effective on different D-subgenotypes and could facilitate management of HBV/D-infected patients irrespective of D-subgenotype.

Diverse facets of MDSC in different phases of chronic HBV infection: Impact on HBV-specific T-cell response and homing.

BACKGROUND AND AIMS: Chronic HBV infection (CHI) is associated with a diverse natural history that includes immune-tolerant (IT), HBeAg-positive chronic hepatitis B (CHB) (EP-CHB), inactive carrier, and HBeAg-negative CHB (EN-CHB) phases. A hallmark of CHI is impairment of HBV-specific T-cell response. Recently, myeloid-derived suppressor cells (MDSCs) have emerged as key regulator of T cells, and their properties are sculpted by their microenvironment. Here, we investigated the distinctive features of MDSCs during CHI, identified factors responsible for their functional discrepancies, and studied their impact on HBV-specific T-cell response and homing. Influence of antiviral therapy on MDSC profile and T-cell response was also assessed. APPROACH AND RESULTS: Flow cytometric analysis indicated that MDSCs in EP-CHB/EN-CHB patients had profound suppressive ability, expressing arginase 1 (Arg1)/inducible nitric oxide synthase (iNOS)/programmed death ligand 1 (PD-L1)/cytotoxic T lymphocyte-associated protein 4 (CTLA-4)/CD40 at significantly greater levels relative to healthy controls (HC). However, in IT, only Arg1+ MDSCs and in inactive carrier, iNOS+ and PD-L1+ MDSCs were higher than HC. In vitro assays demonstrated that high HBsAg titer in IT/CHB induced Arg1+ MDSC. Furthermore, elevated serum TNF-α and IL-4 in CHB potentiated Arg1/PD-L1/CD40/CTLA-4 expression, whereas increased IL-1ß in CHB/IC triggered the expansion of PD-L1+ MDSCs and iNOS+ MDSCs. MDSCs, sorted from CHB/IC, greatly attenuated IL-2/interferon gamma (IFN-γ) production by HBV-specific CD8+ /CD4+ T cells, the effect being more pronounced in CHB. However, MDSCs of IT minimally affected the cytokine production by T cells. Adding Arg1-/iNOS-inhibitor restored only IFN-γ production, while neutralizing PD-L1 recovered both IL-2 and IFN-γ secretion by T cells. Moreover, MDSCs from IT/CHB disrupted virus-specific T-cell trafficking by down-regulating chemokine receptor type 5 on them via TGF-ß signaling. One year of tenofovir therapy failed to normalize MDSC phenotype and HBV-specific T-cell response. CONCLUSIONS: Diversity of MDSCs during CHI affects HBV-specific T-cell response and homing. Hence, therapeutic targeting of MDSCs could boost anti-HBV immunity.

A Model of Care Optimized for Marginalized Remote Population Unravels Migration Pattern in India.

BACKGROUND AND AIMS: Access to basic health needs remains a challenge for most of world's population. In this study, we developed a care model for preventive and disease-specific health care for an extremely remote and marginalized population in Arunachal Pradesh, the northeasternmost state of India. APPROACH AND RESULTS: We performed patient screenings, performed interviews, and obtained blood samples in remote villages of Arunachal Pradesh through a tablet-based data collection application, which was later synced to a cloud database for storage. Positive cases of hepatitis B virus (HBV) were confirmed and genotyped in our central laboratory. The blood tests performed included liver function tests, HBV serologies, and HBV genotyping. HBV vaccination was provided as appropriate. A total of 11,818 participants were interviewed, 11,572 samples collected, and 5,176 participants vaccinated from the 5 westernmost districts in Arunachal Pradesh. The overall hepatitis B surface antigen (HBsAg) prevalence was found to be 3.6% (n = 419). In total, 34.6% were hepatitis B e antigen positive (n = 145) and 25.5% had HBV DNA levels greater than 20,000 IU/mL (n = 107). Genotypic analysis showed that many patients were infected with HBV C/D recombinants. Certain tribes showed high seroprevalence, with rates of 9.8% and 6.3% in the Miji and Nishi tribes, respectively. The prevalence of HBsAg in individuals who reported medical injections was 3.5%, lower than the overall prevalence of HBV. CONCLUSIONS: Our unique, simplistic model of care was able to link a highly resource-limited population to screening, preventive vaccination, follow-up therapeutic care, and molecular epidemiology to define the migratory nature of the population and disease using an electronic platform. This model of care can be applied to other similar settings globally.

Hepatitis B virus suppresses complement C9 synthesis by limiting the availability of transcription factor USF-1 and inhibits formation of membrane attack complex: implications in disease pathogenesis.

BACKGROUND: The complement system functions primarily as a first-line host defense against invading microbes, including viruses. However, the interaction of Hepatitis B virus (HBV) with the complement-components during chronic HBV infection remains largely unknown. We investigated the mechanism by which HBV inhibits the formation of cytolytic complement membrane-attack complex (MAC) and studied its impact on MAC-mediated microbicidal activity and disease pathogenesis. METHODS: Blood/liver tissues were collected from chronically HBV-infected patients and controls. HepG2hNTCP cells were infected with HBV particles and Huh7 cells were transfected with full-length linear HBV-monomer or plasmids containing different HBV-ORFs and expression of complement components or other host genes were evaluated. Additionally, ELISA, Real-time PCR, Western blot, bioinformatics analysis, gene overexpression/knock-down, mutagenesis, chromatin immunoprecipitation, epigenetic studies, immunofluorescence, and quantification of serum HBV-DNA, bacterial-DNA and endotoxin were performed. RESULTS: Among the MAC components (C5b-C9), significant reduction was noted in the expression of C9, the major constituent of MAC, in HBV-infected HepG2hNTCP cells and in Huh7 cells transfected with full-length HBV as well as HBX. C9 level was also marked low in sera/liver of chronic hepatitis B (CHB) and Immune-tolerant (IT) patients than inactive carriers and healthy controls. HBX strongly repressed C9-promoter activity in Huh7 cells but CpG-island was not detected in C9-promoter. We identified USF-1 as the key transcription factor that drives C9 expression and demonstrated that HBX-induced hypermethylation of USF-1-promoter is the leading cause of USF-1 downregulation that in turn diminished C9 transcription. Reduced MAC formation and impaired lysis of HBV-transfected Huh7 and bacterial cells were observed following incubation of these cells with C9-deficient CHB sera but was reversed upon C9 supplementation. Significant inverse correlation was noted between C9 concentration and HBV-DNA, bacterial-DNA and endotoxin content in HBV-infected patients. One-year Tenofovir therapy resulted in improvement in C9 level and decline in viral/bacterial/endotoxin load in CHB patients. CONCLUSION: Collectively, HBX suppressed C9 transcription by restricting the availability of USF-1 through hypermethylation of USF-1-promoter and consequently hinder the formation and lytic functions of MAC. Early therapy is needed for both CHB and IT to normalize the aberrant complement profile and contain viral and bacterial infection and limit disease progression.

The exosome encapsulated microRNAs as circulating diagnostic marker for hepatocellular carcinoma with low alpha-fetoprotein.

Diagnosis of hepatocellular carcinoma (HCC) remains challenging to clinicians, particularly in a patient with low alpha-fetoprotein. Here, in silico, ex vivo and in vitro data were combined to identify liver-specific exosomal miRNAs as an early diagnostic marker for HCC. Transcriptome profiling for mRNA and small RNA in same HCV-HCC and normal liver tissues followed by cross-validation of 41 deregulated miRNAs (log2 FoldChange > 1.5, Padj < .1) with GEO/TCGA datasets of HCV/HBV related HCC vs normal/adjacent tissue revealed three miRNAs were commonly deregulated (miR-10b/miR-21/miR-182) among all HCC irrespective of viral etiology. Targets of top deregulated miRNAs were identified by TargetScan/miRwalk and validated in mRNA transcriptome data followed by Panther/Gene Ontology enrichment/Cytoscape analysis suggested that targets were mostly from carcinogenesis pathways. Hence, those miRNAs were validated in normal and HCV-HCC tissues by qRT-PCR and subsequently in plasma-derived-exosomes of both HBV/HCV infected non-HCC (chronic hepatitis [CH]/liver cirrhosis [LC]) and HCC samples, and in liver-specific Anti-Asgr2 immuno-enriched exosomes. Exosomes were verified using Nanosight/TEM/immune-blotting with anti-Alix/anti-GRP78/anti-Asgr2. Along with miR-21-5p, miR-10b-5p/miR-221-3p/miR-223-3p was found significantly upregulated in the exosome of HCC patients than CH/non-HCC. The comparable expression pattern was seen in anti-Asgr2 immuno-precipitated exosomes. Interestingly, the AFP level was found below 250 ng/mL in about 94% of HCV-HCC and 62% of HBV-HCC patients. ROC analysis showed that miR-10b-5p + miR-221-3p + miR-223-3p + miR-21-5p could differentiate CH/non-HCC(CH + LC) from HCC with AUROC: 0.86 (97.5% CI: 0.77-0.94)/0.80 (97.5% CI: 0.70-0.89), sensitivity: 74%/58% and specificity: 86%/95% while miR-10b-5p + miR-221-3p + miR-223-3p showed AUROC: 0.84 (97.5% CI: 0.74-0.94)/0.74 (97.5% CI: 0.63-0.84), sensitivity: 86%/86% and specificity:66%/53% for low AFP-HCC vs CH/non-HCC, respectively, having better sensitivity than the combination of four miRNAs. Multivariate analysis further revealed low Albumin and high miR-21-5p as probable independent risk factor for HCC.

A novel microRNA boosts hyper-ß-oxidation of fatty acids in liver by impeding CEP350-mediated sequestration of PPARα and thus restricts chronic hepatitis C.

Imbalance in lipid metabolism induces steatosis in liver during Chronic hepatitis C (CHC). Contribution of microRNAs in regulating lipid homoeostasis and liver disease progression is well established using small RNA-transcriptome data. Owing to the complexity in the development of liver diseases, the existence and functional importance of yet undiscovered regulatory miRNAs in disease pathogenesis was explored in this study using the unmapped sequences of the transcriptome data of HCV-HCC liver tissues following miRDeep2.pl pipeline. MicroRNA-c12 derived from the first intron of LGR5 of chromosome 12 was identified as one of the miRNA like sequences retrieved in this analysis that showed human specific origin. Northern blot hybridization has proved its existence in the hepatic cell line. Enrichment of premiR-c12 in dicer-deficient cells and miR-c12 in Ago2-RISC complex clearly suggested that it followed canonical miRNA biogenesis pathway and accomplished its regulatory function. Expression of this miRNA was quite low in CHC tissues than normal liver implying HCV-proteins might be regulating its biogenesis. Promoter scanning and ChIP analysis further revealed that under expression of p53 and hyper-methylation of STAT3 binding site upon HCV infection restricted its expression in CHC tissues. Centrosomal protein 350 (CEP350), which sequestered PPARα, was identified as one of the targets of miR-c12 using Miranda and validated by luciferase assay/western blot analysis. Furthermore, reduced triglyceride accumulation and enhanced PPARα mediated transcription of ß-oxidation genes upon restoration of miR-c12 in liver cells suggested its role in lipid catabolism. Thus this study is reporting miR-c12 for the first time and showed its' protective role during chronic HCV infection.

CD8+CD28- T cells: key cytotoxic players impacting disease pathogenesis in chronic HBV infection.

During chronic hepatitis B (CHB), CD8+ T cells down-regulate CD28, the primary co-stimulation molecule for T-cell activation. Diverse functional attributes of CD8+CD28- T cells are suggested in various disease contexts. The present study aimed to characterize CD8+CD28- T cells in different phases of chronic Hepatitis B virus (HBV) infection (CHI)- Immune-tolerance (IT), Hepatitis B e-antigen-positive CHB (EP-CHB), Inactive carriers (IC) and Hepatitis B e-antigen-negative CHB (EN-CHB), to appraise their contribution in HBV-related disease pathophysiology. Flow cytometry analysis of T cells in peripheral blood of study subjects revealed enhanced CD8+CD28- T-cell accumulation in EP-/EN-CHB, compared with IT/IC and they expanded equivalently in HBV-specific and non-specific CD8+ T-cell compartments. Profound increase in CD8+CD28- T cells expressing perforin/granzyme-B/CD57/IFN-γ/TNF-α and markers of terminal differentiation were observed exclusively in EP-/EN-CHB. Further, activation with anti-NKG2D resulted in heightened IFN-γ/TNF-α production selectively from CD8+CD28- T cells, suggesting NKG2D-mediated alternative co-stimulation. CD8+CD28- T cells sorted from CHB patients induced enhanced apoptosis of peripheral blood mononuclear cells (PBMC), including CD4+ T cells. However, NKG2D-ligand (major histocompatibility complex class I chain-related molecule A/B (MICA/B)) was preferentially expressed by HBV-specific CD4+ T cells of CHB patients, making these cells a potential target to NKG2D-dependent CD8+CD28- T-cell killing. Both CD28+ and CD28- T cells in CHB expressed CXCR3 at similar levels and thus capable of homing to the liver. A positive correlation was seen between CD8+CD28- T-cell frequency and serum-alanine transaminase (ALT) levels and CHB-derived CD8+CD28- T cells caused pronounced cell death in HBV-transfected Huh7 cells. Immunofluorescence staining identified greater intrahepatic incidence of CD8+CD28- T cells but decline in CD4+ T cells in CHB than IC. Collectively, CD8+CD28- T cells demonstrated differential distribution and phenotypic/functional skewing in different CHI phases and contribute to disease progression by Perforin-Granzyme- or IFN-γ-TNF-α-mediated cytotoxicity while restraining antiviral immunity through NKG2D-dependent HBV-specific CD4+ T-cell depletion.

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

Despite widespread distribution of hepatitis B virus (HBV)-genotype D, the clinical implications of its ten subgenotypes (D1-D10) have not been well documented. Here, we have investigated the impact of two major circulating HBV/D subgenotypes, D1 and D3 in Eastern India towards pathogenesis of liver disease progression to hepatocellular carcinoma (HCC). HBV subgenotypes were determined using full-length genome sequences of HBV isolates from patients with chronic hepatitis B (CHB), liver cirrhosis (LC) and HCC. Impact of D1 and D3 on viral lifecycle and disease progression was assessed by several in vitro assays. Phylogenetic tree analysis revealed that HBV/D1 and HBV/D3 were the two predominating HBV subgenotypes circulating in Eastern India. Interestingly, the frequency of patients infected with HBV/D1 was noticed progressively rising from CHB to HCC through LC while the increasing frequency of HBV/D3 declined suddenly in HCC implicating HBV/D1 might have greater oncogenic potential than HBV/D3. Similar to higher viral load noted in HCC patients infected with HBV/D1 than HBV/D3, the larger amount of intracellular/extracellular viral DNA and secreted HBsAg levels in transfected cell lines also implicated that HBV/D1 might replicate faster than HBV/D3. Again, higher expression of marker genes related to endoplasmic reticulum stress, epithelial-mesenchymal transition, DNA double strand breaks, angiogenesis etc. and faster rate of cellular migration and anchorage independent growth cumulatively suggested that compared to HBV/D3, HBV/D1 generates more liver injuries which eventually culminates into HCC. Therefore, our results highlight the importance of determination of subgenotypes of HBV in CHB patients, so that high-risk individual can be monitor periodically that may help to detect HCC at early stages.

Hepatic miR-126 is a potential plasma biomarker for detection of hepatitis B virus infected hepatocellular carcinoma.

Controversies about the origin of circulating miRNAs have encouraged us to identify organ specific circulating miRNAs as disease biomarkers. To identify liver-specific miRNAs for hepatocellular carcinoma (HCC), global expression profiling of miRNAs in liver tissue of HBV-HCC and HBV-control with no or mild fibrosis was evaluated. A total of 40 differentially expressed miRNAs were identified in HCC. Among ten highly altered miRNAs, six miRNAs were successfully validated in tissues, whereas only two miRNAs, miR-126 and miR-142-3p showed increased expression in plasma of HBV-HCC compared to HBV-non-HCC patients. Subsequently, ROC curve analysis revealed that neither miR-126 nor miR-142-3p performed better than AFP in discriminating HCC from non-HCC while combination of each with AFP showed significantly higher efficiency rather than AFP alone (AUC: 0.922, 0.908 vs. 0.88; sensitivity: 0.84, 0.86 vs. 0.82 and specificity: 0.92, 0.94 vs. 0.86 respectively). Interestingly, triple combination of markers (miR-126 + miR-142-3p + AFP) showed no additive effect on efficiency (AUC: 0.925) over the dual combination. Again, the expression of only miR-126 was noticed significantly higher in HBV-HCC patients with low-AFP [<250 ng/ml] compared to either non-HCC or liver cirrhosis (AUC: 0.77, 0.64, respectively). Furthermore, no alteration in expression of mir-126 in HCV-HCC or non-viral-HCC revealed that miR-126 + AFP might be specific to HBV-HCC. To understand the physiological role of these two miRNAs in hepato-carcinogenesis, target genes related to cancer pathways (APAF1, APC2, CDKN2A, IRS1, CRKL, LIFR, EGR2) were verified. Thus, combination of circulating miR-126 + AFP is a promising noninvasive diagnostic biomarker for HBV-HCC and may be useful in the management of HCC patients.

Sponging of five tumour suppressor miRNAs by lncRNA-KCNQ1OT1 activates BMPR1A/BMPR1B-ACVR2A/ACVR2B signalling and promotes chemoresistance in hepatocellular carcinoma.

Diverse mechanisms have been established to understand the chemoresistance of hepatocellular carcinoma (HCC), but the contribution of non-coding RNAs is not surveyed well. Here, we aimed to explore the lncRNA-miRNA axis in Hepatitis C and B virus (HCV and HBV) infected HCC to investigate the molecular mechanism of chemoresistance and to identify a potential therapeutic target for HCC. The small RNA transcriptome analysis followed by qRT-PCR validation with the liver tissues of both HCV and HBV infected HCC patients revealed that miR-424-5p, miR-136-3p, miR-139-5p, miR-223-3p, and miR-375-3p were the most downregulated miRNAs in HCC compared to normal (log2 fold change ≤-1.5, Padj ≤ 0.05). In silico pathway analysis with the validated targets of each miRNA revealed that the signalling pathway regulating pluripotency of stem cells is commonly targeted by these five miRNAs. Subsequent validation by 3'UTR-luciferase assay and western blot analysis unveiled that these five miRNAs impeded either same or diverse genes, but all linked to BMP signalling pathway such as BMPR1A/BMPR1B by miR-139-5p, miR-136-3p, and miR-375-3p, and ACVR2A/ACVR2B by miR-424-5p and miR-223-3p. Furthermore, restoration of each miRNA in Huh7/SNU449 cells inhibited phosphorylation of downstream SMAD1/5 and ERK1/2, and attenuated Epithelial-mesenchymal transition, stemness, spheroid formation, chemoresistance, invasion and migration of cells. To investigate the mechanism of suppression of these miRNAs, "DIANA" tool was employed and lncRNA-KCNQ1OT1 was retrieved as interacting partner of all the five miRNAs. In vitro RNA pull-down assay revealed that lncRNA-KCNQ1OT1 physically interacted and sequestered these five miRNAs in the cytoplasm. Hence, KCNQ1OT1 was suppressed in Huh7/SNU449 cells using CRISPR technology and observed regression of oncogenic properties with enhanced chemosensitivity and reduced metastasis in cancer cells. Shrinkage of tumour size and volume in NOD-SCID mice injected with KCNQ1OT1-sgRNA cells further strengthened our observations. Thus, lncRNA-KCNQ1OT1 is the main regulator, which reduces the level of beneficiary miRNAs in the tumour milieu and modulates BMP signalling pathway to promote chemoresistance in HCC, suggesting lncRNA-KCNQ1OT1 might have robust potential to be a therapeutic target in HCC.

Influence of polymorphisms in TNF-α and IL1ß on susceptibility to alcohol induced liver diseases and therapeutic potential of miR-124-3p impeding TNF-α/IL1ß mediated multi-cellular signaling in liver microenvironment.

Background and aims: Alcoholic 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 methods: Bio-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. Results: The combined data analysis of the GSE143318/Bio-Plex Pro™ Human Chemokine array and qRT-PCR array revealed that six genes (TNFα/IL1ß/IL8/MCP1/IL6/TGFß) 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 IL1ß were independently associated with ALD respectively. To evaluate the functional implication of these SNPs on ALD development, the serum level of TNF-α/IL1ß was verified and observed significantly higher in ALD patients with risk genotypes TNF-α-238GA/IL1ß-31CT+TT than TNF-α-238GG/IL1ß-31CC. The TNF-α/IL1ß 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/IL1ß over-expressed THP1 cells to HepG2/LX2/HUVEC cells independently showed enhanced level of ER stress and apoptosis in HepG2/increased TGFß 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/IL1ß were inhibited by miR-124-3p along with multiple genes from TLR4 signaling/apoptosis/fibrogenesis pathways including MYD88, TRAF3/TRADD, Caspase8/PDGFRA, TGFßR2/MCP1, and ICAM1 respectively. Conclusion: Thus, rs361525(G/A) in TNF-α and rs1143627(C/T) in IL1ß gene may be used as early predictors of ALD susceptibility among East Indian population. Impeding overexpressed TNF-α/IL1ß 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.

Multifaceted Defects in Monocytes in Different Phases of Chronic Hepatitis B Virus Infection: Lack of Restoration after Antiviral Therapy.

Monocytes play an important role in the control of microbial infection, but monocyte biology during chronic hepatitis B virus (HBV) infection (CHI) remains inadequately studied. We investigated the frequency, phenotype, and functions of monocyte subsets in different phases of CHI, namely, immune tolerance (IT), hepatitis B early antigen (HBeAg)-positive/HBeAg-negative chronic hepatitis B (EP-/EN-CHB, respectively), and inactive carrier (IC), identified factors responsible for their functional alterations, and determined the impact of antiviral therapy on these cells. Flow cytometric analysis indicated that HLA-DR+ CD14++ CD16- classical monocytes were significantly reduced while HLA-DR+ CD14++ CD16+ intermediate and HLA-DR+ CD14+ CD16++ nonclassical monocytes were expanded in IT and EP-/EN-CHB compared with those in IC and healthy controls (HC). In comparison to IC/HC, monocytes in IT and CHB exhibited diminished expression of Toll-like receptor 2 (TLR-2)/TLR-4/TLR-9 and cytokines interleukin-12 (IL-12)/tumor necrosis factor alpha (TNF-α)/IL-6 but produced higher levels of IL-10/transforming growth factor ß (TGF-ß). Further, monocytes in CHB/IT showed impaired phagocytosis and oxidative response relative to those in IC/HC. In vitro assays indicated that high titers of hepatitis B surface antigen (HBsAg) present in IT/CHB and of IL-4 in CHB triggered the functional defects in monocytes via induction of ß-catenin. Additionally, monocyte-derived M1 macrophages of CHB/IT produced fewer proinflammatory and more anti-inflammatory cytokines than those of IC/HC, while in CHB/IT, the monocytes skewed the differentiation of CD4+ T cells more toward regulatory T cells and a Th2 phenotype. Moreover, monocytes in CHB and IT overexpressed chemokine receptor CCR2, which coincided with increased intrahepatic accumulation of ß-catenin+ CD14+ cells. One year of tenofovir therapy failed to normalize monocyte functions or reduce serum HBsAg/IL-4 levels. Taken together, monocytes are functionally perturbed mostly in IT and EP-/EN-CHB phases. Targeting intramonocytic ß-catenin or reducing HBsAg/IL-4 levels might restore monocyte function and facilitate viral clearance. IMPORTANCE Chronic HBV infection (CHI) is a major cause of end-stage liver disease for which pharmacological treatments currently available are inadequate. Chronically HBV-infected patients fail to mount an efficient immune response to the virus, impeding viral clearance and recovery from hepatitis. Monocytes represent a central part of innate immunity, but a comprehensive understanding on monocyte involvement in CHI is still lacking. We here report a multitude of defects in monocytes in chronically HBV-infected patients that include alteration in subset distribution, Toll-like receptor expression, cytokine production, phagocytic activity, oxidative response, migratory ability, polarization of monocyte-derived macrophages, and monocyte-T-cell interaction. We demonstrated that high levels of hepatitis B virus surface antigen and IL-4 potentiate these defects in monocytes via ß-catenin induction while therapy with the nucleotide analog tenofovir fails to restore monocyte function. Our findings add to the continuing effort to devise new immunotherapeutic strategies that could reverse the immune defects in CHI.

Intact cag pathogenicity island of Helicobacter pylori without disease association in Kolkata, India.

Several genes including the cagA in the cag pathogenicity island (cag PAI) of Helicobacter pylori are thought to be associated with the gastroduodenal diseases and hence variation in the genetic structure of the cag PAI might be responsible for different clinical outcomes. Our study was undertaken to characterize the cag PAI of H. pylori strains from duodenal ulcer (DU) patients and asymptomatic or non-ulcer dyspepsia (NUD/AV) subjects from Kolkata, India. Strains isolated from 52 individuals (30DU and 22NUD/AV) were analyzed by PCR using 83 different primers for the entire cag PAI and also by dot-blot hybridization. Unlike H. pylori strains isolated from other parts of India, 82.6% of the strains used in this study had intact cag PAI, 9.6% had partially deleted cag PAI, and 7.7% of the strains lacked the entire cag PAI. Dot-blot hybridization yielded positive signals in 100% and 93.8% of PCR-negative strains for HP0522-523 and HP0532-HP0534 genes, respectively. An intact cagA promoter region was also detected in all cagA-positive strains. Furthermore, the expression of cagA mRNA was confirmed by RT-PCR for the representative strains from both DU and NUD/AV subjects indicating the active cagA promoter regions of these strains. A total of 66.7% of Kolkata strains produced a ∼390-bp shorter amplicon than the standard strain 26695 for the HP0527 gene, homologue of virB10. However, sequence analyses confirmed that the deletion did not alter the reading frame of the gene, and mRNA transcripts were detected by RT-PCR analysis. The strains isolated from DU and NUD/AV express CagA protein and possess a functional type IV secretion system, as revealed by Western blot analyses. Interestingly, no significant differences in cag PAI genetic structure were found between DU and NUD/AV individuals suggesting that other bacterial virulence factors, host susceptibility, and environmental determinants also influence the disease outcome at least in certain geographical locations.

Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages.

We report the identification and functional analysis of nine genes from Gram-positive and Gram-negative bacteria and their phages that are similar to lambda (lambda) bet or Escherichia coli recT. Beta and RecT are single-strand DNA annealing proteins, referred to here as recombinases. Each of the nine other genes when expressed in E. coli carries out oligonucleotide-mediated recombination. To our knowledge, this is the first study showing single-strand recombinase activity from diverse bacteria. Similar to bet and recT, most of these other recombinases were found to be associated with putative exonuclease genes. Beta and RecT in conjunction with their cognate exonucleases carry out recombination of linear double-strand DNA. Among four of these foreign recombinase/exonuclease pairs tested for recombination with double-strand DNA, three had activity, albeit barely detectable. Thus, although these recombinases can function in E. coli to catalyze oligonucleotide recombination, the double-strand DNA recombination activities with their exonuclease partners were inefficient. This study also demonstrated that Gam, by inhibiting host RecBCD nuclease activity, helps to improve the efficiency of lambda Red-mediated recombination with linear double-strand DNA, but Gam is not absolutely essential. Thus, in other bacterial species where Gam analogs have not been identified, double-strand DNA recombination may still work in the absence of a Gam-like function. We anticipate that at least some of the recombineering systems studied here will potentiate oligonucleotide and double-strand DNA-mediated recombineering in their native or related bacteria.

A combination of circulating microRNA-375-3p and chemokines CCL11, CXCL12, and G-CSF differentiate Crohn's disease and intestinal tuberculosis.

Differentiation of Crohn's disease (CD) from intestinal tuberculosis (ITB) is a big challenge to gastroenterologists because of their indistinguishable features and insensitive diagnostic tools. A non-invasive biomarker is urgently required to distinguish ITB/CD patients particularly in India, a TB endemic region, where CD frequency is increasing rapidly due to urbanization. Among the three differentially expressed miRNAs obtained from small RNA transcriptomic profiling of ileocaecal/terminal ileal tissue of ITB/CD patients (n = 3), only two down-regulated miRNAs, miR-31-5p, and miR-215-5p showed comparable data in qRT-PCR. Out of which, only miR-215-5p was detectable in the patient's plasma, but there was no significant difference in expression between ITB/CD. On the other hand, miR-375-3p, the pulmonary TB specific marker was found in higher amount in the plasma of ITB patients than CD while reverse expression was observed in the ileocaecal/terminal ileal tissues of the same patients. Next, using Bioplex pro-human cytokine 48-plex screening panel, only three chemokines, Eotaxin-1/CCL11, SDF-1α/CXCL12, and G-CSF have noted significantly different levels in the serum of ITB/CD patients. ROC analysis has revealed that compared to a single molecule, a combination of miR-375-3p + Eotaxin-1/CCL11 + SDF-1α /CXCL12 + G-CSF showed a better AUC of 0.83, 95% CI (0.69-0.96) with 100% specificity and positive predictive value while sensitivity, negative predictive value, and accuracy were 56%, 69%, and 78% respectively in distinguishing ITB from CD. This study suggests that a combination of plasma markers shows better potential in differentiating ITB from CD than a single marker and this panel of markers may be used for clinical management of ITB/CD patients.

Unique hepatitis B virus subgenotype in a primitive tribal community in eastern India.

Hepatitis B virus (HBV) strains isolated from members of the primitive Paharia ethnic community of Eastern India were studied to gain insight into the genetic diversity and evolution of the virus. The Paharia tribe has remained quite separate from the rest of the Indians and differs culturally, genetically, and linguistically from the mainstream East Indian population, whose HBV strains were previously characterized. Full-length HBV DNA was PCR amplified, cloned, and sequenced. Phylogenetic relationships between the tribal sequences and reference sequences from the mainstream population were assessed, and divergence times of subgenotypes of HBV genotype D were estimated. HBV was found in 2% of the Paharias participating in the study. A predominance of hepatitis B e antigen-negative infection (73%) was observed among the Paharias, and the genome sequences of the HBV strains exhibited relative homogeneity, with a very low prevalence of mutations. The novel feature of Paharia HBV was the exclusive presence of the D5 subgenotype, which was recently identified in Eastern India. Analysis of the four open reading frames (ORFs) of these tribal HBV D5 sequences and comparison with previously reported D1 to D7 sequences enabled the identification of 27 specific amino acid residues, including 6 unique ones, that could be considered D5 signatures. The estimated divergence times among subgenotypes D1 to D5 suggest that D5 was the first to diverge and hence is the most ancient of the D subgenotypes. The presence of a specific, ancient subgenotype of HBV within an ethnically primitive, endogamous population highlights the importance of studies of HBV genetics in well-separated human populations to understand viral transmission between communities and genome evolution.

Course of disease and survival after onset of decompensation in hepatitis B virus-related cirrhosis.

BACKGROUND: Data regarding the outcome of hepatitis B virus (HBV)-related cirrhosis after the onset of decompensation is scanty. METHOD: From January 1998 to December 2008, a retrospective-prospective inception cohort study involving HBV-related decompensated cirrhotics was performed. Predictors of death and clinical events after the onset of decompensation were evaluated. Patients with co-infection with hepatitis C virus and/or human immunodeficiency virus, alcohol consumption to any degree and diabetes diagnosed before the detection of liver disease were excluded. RESULT AND ANALYSIS: Two hundred and fifty-three patients (231 males, 139 e-negative), including 102 untreated patients, were analysed. The mean (+/-SD) age was 43.0 (+/-12.0) years. The mean (+/-SD) follow-up period was 47 (+/-47) months. Decompensation was the first presentation of liver disease in 210 (83%) patients. Ascites (70%) and variceal bleed (28%) were predominant modes of decompensation. Forty-three (17%) patients died (22 vs 14% in untreated and treated cohort, respectively; P=0.002). Type 2 hepato-renal syndrome was the commonest cause of death (32%). Survival was independent of e-antigen status. In the total cohorts, predictors of death were occurrence of sepsis with systemic inflammatory response (SIRS), ascites as the initial mode of decompensation, absence of antiviral therapy and events of high-grade hepatic encephalopathy [hazards ratios (HR) of 4.4, 3.6, 2.2 and 1.7 respectively]. In the untreated cohort, initial decompensation with ascites and development of sepsis with SIRS were independent predictors of death (HR 8.5 and 2.3 respectively), while 5-year survival was higher in patients having initial decompensation with variceal bleed vs ascites (29 vs 16%, respectively, P=0.002). CONCLUSION: Decompensation with ascites and sepsis with SIRS predict reduced survival. Antiviral therapy beyond 6 months improves outcome.

Myeloid-derived suppressor cells induce regulatory T cells in chronically HBV infected patients with high levels of hepatitis B surface antigen and persist after antiviral therapy.

BACKGROUND: CD4+ regulatory T-cells (Tregs) expand during chronic hepatitis B virus (HBV) infection and inhibit antiviral immunity, although the underlying mechanism remains largely elusive. Myeloid-derived suppressor cells (MDSC) have been linked with T-cell dysfunction but questions remain regarding their persistence/profile/function in chronically HBV infected patients. AIM: To characterise MDSC in different phases of chronic HBV infection namely, immune-tolerant (IT), hepatitis B e-antigen-positive chronic hepatitis B (EP-CHB), inactive carriers (IC) and hepatitis B e-antigen-negative chronic hepatitis B (EN-CHB), to investigate their role in Treg induction and evaluate the effect of anti-viral therapy on these cells. METHODS: Multiparametric flow cytometry, cell-sorting and co-culture assays were performed along with longitudinal immune monitoring of CHB patients receiving tenofovir. RESULTS: HLA-DR- CD11b+ CD33hi -Monocytic-MDSC (M-MDSC) were enhanced in IT, EP-CHB and EN-CHB compared with IC, and this was related to increasing hepatitis B surface antigen (HBsAg) concentration. IT and EP-/EN-CHB displayed elevated frequency of CD4+ CD25+ FOXP3+ Treg that positively correlated with that of M-MDSC. However, both M-MDSC and HLA-DR- CD11b+ CD33low -granulocytic-MDSC from IT and EP-/EN-CHB expressed high transforming growth factor beta (TGF-ß) and interleukin-10 (IL-10). Co-culture of sorted HLA-DR- CD33+ -MDSC with autologous MDSC depleted-PBMC from IT and CHB but not from IC, increased CD4+ CD25+ FOXP3+ -iTreg and CD4+ FOXP3- IL-10+ -Tr1-cells through a cell-contact independent mechanism. While MDSC-derived TGF-ß and IL-10 promoted development of iTreg, only IL-10 appeared to be crucial for Tr1 induction. One year of tenofovir treatment failed to normalise MDSC frequency/function or reduce Treg percentage and serum HBsAg levels, despite reduction in viral load. CONCLUSIONS: We established a previously unrecognised role of MDSC in Treg development in IT and EP-/EN-CHB via TGF-ß/IL-10-dependent pathways and both cell-types persisted after anti-viral therapy. Hence, therapeutic targeting of MDSC or reducing circulating HBsAg level together with tenofovir-therapy might be more effective in restricting HBV persistence and disease progression.

Distinctiveness in virological features and pathogenic potentials of subgenotypes D1, D2, D3 and D5 of Hepatitis B virus.

Distinct clinical features of HBV infection have been associated with different viral genotype/subgenotype. HBV Genotype-D comprised of 10 subgenotypes, D1-D10, whose clinical implications still remain elusive. We investigated for the first-time, the virologic characteristics and cytopathic effects of four non-recombinant D-subgenotypes, D1/D2/D3/D5. Expressions of viral/host genes were evaluated in Huh7 cells transfected with full-length, linear-monomers of HBV/D-subgenotypes or pGL3-Basic vector carrying subgenotype-specific HBx. Intracellular HBV-DNA and pregenomic-RNA levels were high in D1/D2 than D3/D5. Expressions of PreC-mRNA and HBx were highest for D2 and D1 respectively, whereas PreS2/S-transcript was significantly reduced in D5. Increased apoptotic cell death and marked upregulation in caspase-3/Bax/TNF-R1/FasR/TRAIL-R1/ROS/MCP-1/IP-10/MIP-1ß expression were noticed specifically in D2- and also in D3-transfected cells, while D5 resulted in over-expression of ER-stress-markers. D-subgenotype-transfected Huh7 cells were co-cultured with PBMC of healthy-donors or LX-2 cells and significant increase in pro-inflammatory cytokines in PBMC and fibrogenic-markers in LX-2 were noticed in presence of D2/D3. Further, Huh7 cells transfected with D1, in particular and also D5, displayed remarkable induction of EMT-markers and high proliferative/migratory abilities. Collectively, our results demonstrated that D2/D3 were more associated with hepatic apoptosis/inflammation/fibrosis and D1/D5 with increased risk of hepatocarcinogenesis and emphasize the need for determining HBV-subgenotype in clinical practice.