A multisociety Delphi consensus statement on new fatty liver disease nomenclature.

The principal limitations of the terms NAFLD and NASH are the reliance on exclusionary confounder terms and the use of potentially stigmatising language. This study set out to determine if content experts and patient advocates were in favor of a change in nomenclature and/or definition. A modified Delphi process was led by three large pan-national liver associations. The consensus was defined a priori as a supermajority (67%) vote. An independent committee of experts external to the nomenclature process made the final recommendation on the acronym and its diagnostic criteria. A total of 236 panelists from 56 countries participated in 4 online surveys and 2 hybrid meetings. Response rates across the 4 survey rounds were 87%, 83%, 83%, and 78%, respectively. Seventy-four percent of respondents felt that the current nomenclature was sufficiently flawed to consider a name change. The terms "nonalcoholic" and "fatty" were felt to be stigmatising by 61% and 66% of respondents, respectively. Steatotic liver disease was chosen as an overarching term to encompass the various aetiologies of steatosis. The term steatohepatitis was felt to be an important pathophysiological concept that should be retained. The name chosen to replace NAFLD was metabolic dysfunction-associated steatotic liver disease. There was consensus to change the definition to include the presence of at least 1 of 5 cardiometabolic risk factors. Those with no metabolic parameters and no known cause were deemed to have cryptogenic steatotic liver disease. A new category, outside pure metabolic dysfunction-associated steatotic liver disease, termed metabolic and alcohol related/associated liver disease (MetALD), was selected to describe those with metabolic dysfunction-associated steatotic liver disease, who consume greater amounts of alcohol per week (140-350 g/wk and 210-420 g/wk for females and males, respectively). The new nomenclature and diagnostic criteria are widely supported and nonstigmatising, and can improve awareness and patient identification.

A multisociety Delphi consensus statement on new fatty liver disease nomenclature.

The principal limitations of the terms NAFLD and NASH are the reliance on exclusionary confounder terms and the use of potentially stigmatising language. This study set out to determine if content experts and patient advocates were in favor of a change in nomenclature and/or definition. A modified Delphi process was led by three large pan-national liver associations. The consensus was defined a priori as a supermajority (67%) vote. An independent committee of experts external to the nomenclature process made the final recommendation on the acronym and its diagnostic criteria. A total of 236 panelists from 56 countries participated in 4 online surveys and 2 hybrid meetings. Response rates across the 4 survey rounds were 87%, 83%, 83%, and 78%, respectively. Seventy-four percent of respondents felt that the current nomenclature was sufficiently flawed to consider a name change. The terms "nonalcoholic" and "fatty" were felt to be stigmatising by 61% and 66% of respondents, respectively. Steatotic liver disease was chosen as an overarching term to encompass the various aetiologies of steatosis. The term steatohepatitis was felt to be an important pathophysiological concept that should be retained. The name chosen to replace NAFLD was metabolic dysfunction-associated steatotic liver disease. There was consensus to change the definition to include the presence of at least 1 of 5 cardiometabolic risk factors. Those with no metabolic parameters and no known cause were deemed to have cryptogenic steatotic liver disease. A new category, outside pure metabolic dysfunction-associated steatotic liver disease, termed metabolic and alcohol related/associated liver disease (MetALD), was selected to describe those with metabolic dysfunction-associated steatotic liver disease, who consume greater amounts of alcohol per week (140-350 g/wk and 210-420 g/wk for females and males, respectively). The new nomenclature and diagnostic criteria are widely supported and nonstigmatising, and can improve awareness and patient identification.

A multisociety Delphi consensus statement on new fatty liver disease nomenclature.

The principal limitations of the terms NAFLD and NASH are the reliance on exclusionary confounder terms and the use of potentially stigmatising language. This study set out to determine if content experts and patient advocates were in favour of a change in nomenclature and/or definition. A modified Delphi process was led by three large pan-national liver associations. The consensus was defined a priori as a supermajority (67%) vote. An independent committee of experts external to the nomenclature process made the final recommendation on the acronym and its diagnostic criteria. A total of 236 panellists from 56 countries participated in 4 online surveys and 2 hybrid meetings. Response rates across the 4 survey rounds were 87%, 83%, 83%, and 78%, respectively. Seventy-four percent of respondents felt that the current nomenclature was sufficiently flawed to consider a name change. The terms "nonalcoholic" and "fatty" were felt to be stigmatising by 61% and 66% of respondents, respectively. Steatotic liver disease was chosen as an overarching term to encompass the various aetiologies of steatosis. The term steatohepatitis was felt to be an important pathophysiological concept that should be retained. The name chosen to replace NAFLD was metabolic dysfunction-associated steatotic liver disease (MASLD). There was consensus to change the definition to include the presence of at least 1 of 5 cardiometabolic risk factors. Those with no metabolic parameters and no known cause were deemed to have cryptogenic steatotic liver disease. A new category, outside pure metabolic dysfunction-associated steatotic liver disease, termed metabolic and alcohol related/associated liver disease (MetALD), was selected to describe those with metabolic dysfunction-associated steatotic liver disease, who consume greater amounts of alcohol per week (140-350 g/wk and 210-420 g/wk for females and males, respectively). The new nomenclature and diagnostic criteria are widely supported and non-stigmatising, and can improve awareness and patient identification.

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.

rs641738C>T near MBOAT7 is associated with liver fat, ALT and fibrosis in NAFLD: A meta-analysis.

BACKGROUND & AIMS: A common genetic variant near MBOAT7 (rs641738C>T) has been previously associated with hepatic fat and advanced histology in NAFLD; however, these findings have not been consistently replicated in the literature. We aimed to establish whether rs641738C>T is a risk factor across the spectrum of NAFLD and to characterise its role in the regulation of related metabolic phenotypes through a meta-analysis. METHODS: We performed a meta-analysis of studies with data on the association between rs641738C>T genotype and liver fat, NAFLD histology, and serum alanine aminotransferase (ALT), lipids or insulin. These included directly genotyped studies and population-level data from genome-wide association studies (GWAS). We performed a random effects meta-analysis using recessive, additive and dominant genetic models. RESULTS: Data from 1,066,175 participants (9,688 with liver biopsies) across 42 studies were included in the meta-analysis. rs641738C>T was associated with higher liver fat on CT/MRI (+0.03 standard deviations [95% CI 0.02-0.05], pz = 4.8×10-5) and diagnosis of NAFLD (odds ratio [OR] 1.17 [95% CI 1.05-1.3], pz = 0.003) in Caucasian adults. The variant was also positively associated with presence of advanced fibrosis (OR 1.22 [95% CI 1.03-1.45], pz = 0.021) in Caucasian adults using a recessive model of inheritance (CC + CT vs. TT). Meta-analysis of data from previous GWAS found the variant to be associated with higher ALT (pz = 0.002) and lower serum triglycerides (pz = 1.5×10-4). rs641738C>T was not associated with fasting insulin and no effect was observed in children with NAFLD. CONCLUSIONS: Our study validates rs641738C>T near MBOAT7 as a risk factor for the presence and severity of NAFLD in individuals of European descent. LAY SUMMARY: Fatty liver disease is a common condition where fat builds up in the liver, which can cause liver inflammation and scarring (including 'cirrhosis'). It is closely linked to obesity and diabetes, but some genes are also thought to be important. We did this study to see whether one specific change ('variant') in one gene ('MBOAT7') was linked to fatty liver disease. We took data from over 40 published studies and found that this variant near MBOAT7 is linked to more severe fatty liver disease. This means that drugs designed to work on MBOAT7 could be useful for treating fatty liver disease.

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.

Hepatic transcriptome signature correlated with HOMA-IR explains early nonalcoholic fatty liver disease pathogenesis.

INTRODUCTION AND OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is multistage with heterogeneous outcomes. We studied the influence of insulin resistance (IR) on the hepatic transcriptome of early NAFLD stages, to understand disease development. MATERIALS AND METHODS: In this cross-sectional study, possible clinicopathological risk factors were compared between mild-NAFL (N = 72) and non-alcoholic steatohepatitis (NASH; N = 51) patients. Liver tissue-transcriptome difference was studied between a subset of 25 mild-NAFL and 20 NASH biopsies and validated on another subset of 12 mild-NAFL and 13 NASH biopsies, using RT-PCR. The relationship between IR driven gene expression changes with fibrosis in NASH was investigated. RESULTS: Significantly higher weight (p = 0.005) and elevated levels of HbA1c (p = 0.009), FBG (p = 0.03) and HOMA-IR (p = 0.009) were found in NASH patients. Five differentially expressed genes (DEGs, fold change > 1.5) were identified in NASH-FABP4, FABP5L2, CD24, PRAP1, and SPP1. The DEGs were positively associated with disease severity and HOMA-IR, and were found to be efficient classifiers of mild-NAFL and NASH. Additional 1218 genes identified related to IR (IrCGs), which can classify NASH-with-fibrosis patients separately from mild-NAFL and NASH patients. IrCGs can promote intra-hepatic fat accumulation, dysregulation of the lipid metabolism, lipotoxicity, and activation of cell survival pathways including activation of cell proliferation and differentiation pathways. CONCLUSIONS: Hepatic expression of genes associated with insulin resistance may drive NAFLD development and progression.

Range of Normal Liver Stiffness and Factors Associated With Increased Stiffness Measurements in Apparently Healthy Individuals.

BACKGROUND & AIMS: Transient elastography (TE) is a noninvasive technique used to measure liver stiffness to estimate the severity of fibrosis. The range of liver stiffness measurements (LSMs) in healthy individuals is unclear. We performed a systematic review to determine the range of LSMs, examined by TE, in healthy individuals and individuals who are susceptible to fibrosis. METHODS: We collected data from 16,082 individuals, in 26 cohorts, identified from systematic searches of Embase, Ovid MEDLINE, Cochrane Central Register of Controlled Trials, and Cochrane Database of Systematic Reviews for studies of liver stiffness measurements. Studies analyzed included apparently healthy adults (normal levels of liver enzymes, low-risk alcohol use patterns, and negative for markers of viral hepatitis). The presence of diabetes, hypertension, dyslipidemia, or steatosis, based on ultrasound examination, was known for most participants. We performed a meta-analysis of data from individual participants. The cohort was divided into 4 groups; participants with a body mass index <30 kg/m2 were examined with the medium probe and those with a body mass index ≥30 kg/m2 were examined with the extra-large probe. Linear regression models were conducted after adjusting for potential confounding factors of LSMs. We performed several sensitivity analyses. RESULTS: We established LSM ranges for healthy individuals measured with both probes-these did not change significantly in sensitivity analyses of individuals with platelets ≥150,000/mm3 and levels of alanine aminotransferase ≤33 IU/L in men or ≤25 IU/L in women. In multivariate analysis, factors that modified LSMs with statistical significance included diabetes, dyslipidemia, waist circumference, level of aspartate aminotransferase, and systolic blood pressure at examination time. Significant increases in LSMs were associated with the metabolic syndrome in individuals examined by either probe. Diabetes in obese individuals increased the risk of LSMs in the range associated with advanced fibrosis. CONCLUSIONS: In a systematic review and meta-analysis of data from individual participants, we established a comprehensive set of LSM ranges, measured by TE in large cohorts of healthy individuals and persons susceptible to hepatic fibrosis. Regression analyses identified factors associated with increased LSMs obtained by TE with the medium and extra-large probes.

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.

Sociodemographic Determinants of Preterm Birth and Small for Gestational Age in Rural West Bengal, India.

Preterm birth and small for gestational age (SGA) are major contributors to neonatal mortality in India. However, social determinates of preterm births and SGA are poorly characterized. We use population-based data from a health and demographic surveillance system (HDSS) in rural West Bengal, India, to examine risk factors for preterm birth and SGA. HDSS pregnancy cohort data for 2430 pregnant women were used in this analysis. Of 2430 pregnancies, 16% were preterm births and 38.2% were SGA. Results from logistic regression reveal that higher maternal education (≥11 years) was associated with reduced risk of preterm births and SGA. Greater wealth quintile was also associated with decreased risk of preterm births and SGA (p-value for trend: <0.05). In light of the findings, ensuring effective coverage of preterm and SGA interventions among women of low socioeconomic status will be essential to mitigate the large burden of preterm births and SGA.

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.

Antimicrobial activity of ellagic acid against Helicobacter pylori isolates from India and during infections in mice.

Objectives: Because of the rise in antimicrobial resistance, an inexpensive, diet-based treatment against Helicobacter pylori infection would be of great interest. The present study was performed to assess the in vitro effects of ellagic acid against clinical H. pylori strains that were resistant to antibiotics used for therapy and also to observe the morphological structure following treatment with ellagic acid. The effectiveness of ellagic acid in eradicating H. pylori infection in a murine (C57BL/6) infection model, one of the standard inbred mouse lines often used for experimental infection, was also assessed. Methods: A total of 55 strains were screened. The agar dilution method was used to determine the susceptibility of isolates to test compounds. Transmission electron microscopy was used to observe the morphology following treatment with ellagic acid. The antibacterial activity of ellagic acid in an H. pylori SS1-infected mouse model and its effect on gastric mucosal injury were determined by histology and PCR. Results: Ellagic acid inhibited the growth of all 55 of the H. pylori strains tested. The MIC of ellagic acid ranged from 5 to 30 mg/L, showing its bactericidal properties in vitro. Ellagic acid also demonstrated anti-H. pylori efficacy in eradication of this organism in an in vivo model, as well as restitution and repair of H. pylori-induced gastric mucosal damage. Conclusions: The present study paves the way for the preventive and therapeutic use of ellagic acid against H. pylori infection and, thus, ellagic acid can be considered a promising antibacterial agent against H. pylori-associated gastroduodenal diseases in humans.

Underweight among rural Indian adults: burden, and predictors of incidence and recovery.

OBJECTIVE: To study the magnitude and predictors of underweight, incident underweight and recovery from underweight among rural Indian adults. DESIGN: Prospective cohort study. Each participant's BMI was measured in 2008 and 2012 and categorized as underweight (BMI<18·5 kg/m2), normal (BMI=18·5-22·9 kg/m2) or overweight/obese (BMI ≥23·0 kg/m2). Incident underweight was defined as a transition from normal weight or overweight/obese in 2008 to underweight in 2012, and recovery from underweight as a transition from underweight in 2008 to normal weight in 2012. Bivariate and multivariable logistic regression analyses were employed. SETTING: The Birbhum Health and Demographic Surveillance System, West Bengal, India. SUBJECTS: Predominantly rural individuals (n 6732) aged ≥18 years enrolled in 2008 were followed up in 2012. RESULTS: In 2008, the prevalence of underweight was 46·5 %. From 2008 to 2012, 25·8 % of underweight persons transitioned to normal BMI, 12·9 % of normal-weight persons became underweight and 0·1 % of overweight/obese persons became underweight. Multivariable models reveal that people aged 25-49 years, educated and wealthier people, and non-smokers had lower odds of underweight in 2008 and lower odds of incident underweight. Odds of recovery from underweight were lower among people aged ≥36 years and higher among educated (Grade 6 or higher) individuals. CONCLUSIONS: The current study highlights a high incidence of underweight and important risk factors and modifiable predictors of underweight in rural India, which may inform the design of local nutrition interventions.

Probable psychiatric disorder in a rural community of West Bengal, India.

PURPOSE: India faces multiple challenges to mitigate a high burden of psychiatric disorders. The risk of developing psychiatric disorder among the rural Indian population is poorly investigated. This study aims to understand the factors associated with probable psychiatric disorder (PPD) among a select rural Indian population. METHODS: Data from the Birbhum population project of the society for health and demographic surveillance, West Bengal, India, were utilized. Cross-sectional data covering a sample of 31,135 respondents (male 15,384 and female 15,751) aged ≥ 16 years were used. The General Health Questionnaire-28 was administered and the responses were computed into three categories: psychological case, psychological caseness, and normal. Bivariate and multivariate ordered logit regression analyses were applied to attain the study objective. RESULTS: Of the total population, 26% of respondents were identified with PPD. People aged ≥ 60 years, females, divorced/separated/widowed individuals, the unemployed and people with no formal education, individuals from the poorest economic group, and people with a history of selling or mortgaging assets towards their healthcare expenditure had a higher prevalence of psychiatric case within their respective group. CONCLUSIONS: A high burden of PPD was estimated in the select rural community. While designing an intervention for measuring and addressing psychiatric disorders, the socioeconomic gradient of PPD could be helpful.

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.

Alcohol Use-Related Problems Among a Rural Indian Population of West Bengal: An Application of the Alcohol Use Disorders Identification Test (AUDIT).

AIMS: To examine alcohol use and related problems among a rural subset of the Indian population. METHODS: The Alcohol Use Disorders Identification Test (AUDIT) was used as part of Health and Demographic Surveillance of 36,611 individuals aged ≥18 years. From this survey data on 3671 current alcohol users were analysed using bivariate and multivariate ordered logit regression. RESULTS: Over 19% of males and 2.4% of females were current alcohol users. Mean ethanol consumption on a typical drinking day among males was estimated to be higher (96.3 gm) than females (56.5 gm). Mean AUDIT score was 11 among current alcohol users. AUDIT showed in the ordered logit regression estimated alcohol use-related problems to be low among women, Scheduled Tribes and unmarried people, whereas alcohol use-related problems registered high among Muslims. CONCLUSION: This rural population appears to be in need of an effective intervention program, perhaps targeting men and the household, aimed at reducing the level of alcohol use and related problems.