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Hepatitis B surface antigen (HBsAg) seroconversion is sometimes observed in hepatitis B reactivation (rHBV), probably due to immune resetting and differentiation.To investigate sequential immune differentiation and abrogation of tolerance in patients with rHBV who achieved HBsAg seroconversion.We included 19 patients with chronic hepatitis B (CHBV; HBV DNA log10At 24 weeks, 81% rHBV (n = 67) lost HBV DNA and HBeAg (41%), and 12 (19%) lost HBsAg and made anti-HBs titers10 IU/ml. rHBV patients had higher Th1/17, TNon-seroconverters have increased inhibitory markers on CD4/CD8 T cells. There is a critical play of CD8, Tfh and B cells and subsets in seroclearance, along with checkpoint molecules as a potential therapy for non-seroconverters in HBV infection.

BackgroundPregnancy being immune compromised state, COVID-19 disease poses high risk of premature delivery and threat to fetus. Plasma metabolome regulates immune cellular responses and we aimed to analyze the plasma secretome, metabolome and immune cells in COVID-19 positive pregnant mother and cord blood.MethodsCOVID-19 RT-PCR positive pregnant females (n=112) asymptomatic (n=82), or with mild (n=21) or moderate (n=9) disease and control healthy pregnant (n=10) females were included. Mother’s blood and cord blood (n=80) was analysed for untargeted metabolome profiling and plasma cytokines by high-resolution mass spectrometry (MS) and multiplex cytokine bead array. Immune scan in mothers was done using flow cytometry.ResultsIn asymptomatic SARS-CoV-2 infection, --the amino acid metabolic pathways such as glycine, serine, L-lactate and threonine metabolism was upregulated, riboflavin and tyrosine metabolism, downregulated. In mild to moderate disease, the pyruvate and NAD+metabolism (energy metabolic pathways) were mostly altered. In addition to raised TNF-α, IFN-α, IFN-γ, IL-6 cytokine storm, IL-9 was increased in both mothers and neonates. Pyruvate and NAD+metabolic pathways along with IL-9 and IFN-γ had impact on non-classical monocytes, increased CD4 T cells and B cells but depleted CD8+T cells. Cord blood mimicked the mother’s metabolomic profiles by showing altered valine, leucine, isoleucine, glycine, serine, threonine in asymptomatic and NAD+and riboflavin metabolism in mild and moderate disease subjects.ConclusionsOur results demonstrate a graduated immune-metabolomic interplay in mother and fetus in pregnant females with different degrees of severity of COVID-19 disease. IL-9 and IFN- γ regulated pyruvate, lactate TCA metabolism and riboflavin metabolism with context to disease severity are hall marks of this materno-fetal metabolome.HighlightsSARS-CoV-2 infection alters energy consumption metabolic pathways during pregnancy.Pregnant women with mild to moderate COVID-19 show increased energy demands, and consume stored glucose by upregulating pyruvate and NAD+metabolism.Increased TNF-α and IL-9 in mild COVID-19 disease involve TCA cycle to produce lactate and consume stored glucose by up regulating pyruvate and nicotinamide and nicotinate metabolism.With mild to moderate disease, raised IL-9 and TNF-α, decreased riboflavin pathway, exhaustion of T and B cells cause pathogenesis.Cord blood mimics the metabolic profile of mother’s peripheral blood, SARS- CoV-2 infection reshapes immune-metabolic profiles of mother-infant dyad.Graphical Abstract

Objectives: Visual inspection of the cervix after acetic acid application (VIA) is widely recommended as the method of choice in cervical cancer screening programs in resource-limited settings because of its simplicity and ability to link with immediate treatment. In testing the effectiveness of VIA, human papillomavirus DNA testing, and Pap cytology in a population-based study in a peri-urban area in Andhra Pradesh, India, we found the sensitivity of VIA for detection of cervical intraepithelial neoplasia grade 2 and worse (CIN2+) to be 26.3%, much lower than the 60% to 90% reported in the literature. We therefore investigated the determinants of VIA positivity in our study population.Methods: We evaluated VIA positivity by demographics and reproductive history, results of clinical examination, and results from the other screening methods.Results: Of the 19 women diagnosed with CIN2+, only 5 were positive by VIA (positive predictive value, 3.1%). In multivariate analysis, VIA positivity (12.74%) was associated with older age, positive Pap smear, visually apparent cervical inflammation, and interobserver variation. Cervical inflammation of unknown cause was present in 21.62% of women. In disease-negative women, cervical inflammation was associated with an increase in VIA positivity from 6.1% to 15.5% (P < 0.001). Among the six gynecologists who performed VIA, the positivity rate varied from 4% to 31%.Conclusions: The interpretation of VIA is subjective and its performance cannot be readily evaluated against objective standards.Impact: VIA is not a robust screening test and we caution against its use as the primary screening test in resource-limited regions. Cancer Epidemiol Biomarkers Prev; 19(5); 1373–80. ©2010 AACR.

Supplementary Data from Determinants of VIA (Visual Inspection of the Cervix After Acetic Acid Application) Positivity in Cervical Cancer Screening of Women in a Peri-Urban Area in Andhra Pradesh, India

There is an alarming increase in incidence of fatty liver disease worldwide. The fatty liver disease spectrum disease ranges from simple steatosis (NAFL) to steatohepatitis (NASH) which culminates in cirrhosis and cancer. Altered metabolism is a hallmark feature associated with fatty liver disease and palmitic acid is the most abundant saturated fatty acid, therefore, the aim of this study was to compare metabolic profiles altered in hepatocytes treated with palmitic acid and also the differentially expressed plasma metabolites in spectrum of nonalcoholic fatty liver. The metabolites were analyzed by liquid chromatography-mass spectrometry (LC-MS) platform. Hepatocyte cell lines PH5CH8 and HepG2 cells when treated with 400 μM dose of palmitic acid showed typical features of steatosis. Metabolomic analysis of lipid treated hepatocyte cell lines showed differential changes in phenylalanine and tyrosine pathways, fatty acid metabolism and bile acids. The key metabolites tryptophan, kynurenine and carnitine differed significantly between subjects with NAFL, NASH and those with cirrhosis. As the tryptophan-kynurenine axis is also involved in denovo synthesis of NAD+, we found significant alterations in the NAD+ related metabolites in both palmitic acid treated and also fatty liver disease with cirrhosis. The study underscores the importance of amino acid and NAD+supplementation as promising strategies in fatty liver disorder.

Nonalcoholic or metabolic associated fatty liver disease (NAFLD/MAFLD) is a hepatic reflection of metabolic derangements characterized by excess fat deposition in the hepatocytes. Identifying metabolic regulatory nodes in fatty liver pathology is essential for effective drug targeting. Fatty liver is often associated with circadian rhythm disturbances accompanied with alterations in physical and feeding activities. In this regard, both sirtuins and clock machinery genes have emerged as critical metabolic regulators in maintaining liver homeostasis. Knockouts of either sirtuins or clock genes result in obesity associated with the fatty liver phenotype. Sirtuins (SIRT1-SIRT7) are a highly conserved family of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases, protecting cells from metabolic stress by deacetylating vital proteins associated with lipid metabolism. Circadian rhythm is orchestrated by oscillations in expression of master regulators (BMAL1 and CLOCK), which in turn regulate rhythmic expression of clock-controlled genes involved in lipid metabolism. The circadian metabolite, NAD+ , serves as a crucial link connecting clock genes to sirtuin activity. This is because, NAMPT which is a rate limiting enzyme in NAD+ biosynthesis is transcriptionally regulated by the clock genes and NAD+ in turn is a cofactor regulating the deacetylation activity of sirtuins. Intriguingly, on one hand the core circadian clock regulates the sirtuin activity and on the other hand the activated sirtuins regulate the acetylation status of clock proteins thereby affecting their transcriptional functions. Thus, the Clock-NAD+-Sirtuin connection represents a novel "feedback loop" circuit that regulates the metabolic machinery. The current review underpins the importance of NAD+ on the sirtuin and clock connection in preventing fatty liver disorder.

Presence of dysfunctional senescent hepatocytes is a hallmark feature of liver cirrhosis which finally culminates in liver cancer. We now report the presence of senescent hepatocytes (p21 and p53 positive) in the vicinity of infiltrated immune cells in hepatocellular carcinoma tissue specimens by immunohistochemistry. Hence, we evaluated in vitro, the relevance of senescent hepatoma cells in altering the fate of monocytes and neutrophils by assaying for macrophage polarization and extracellular trap (NETs) formation, respectively. Premature senescence was induced in hepatoma cells (HepG2 and Huh7 cells) by treating cells with doxorubicin. Senescent hepatoma cells showed strong inflammatory phenotype with induced expression of cytokines (IL1β, IL6, IL8 and IL13) as evaluated by flow cytometry. The senescent secretome from hepatoma cells when incubated with healthy monocytes caused it to differentiate predominantly towards M2 fate (CD80

Background Decompensated cirrhosis patients are more prone to bacterial infections. Myeloid derived suppressor cells (MDSCs) expand in sepsis patients and disrupt immune cell functions. GM-CSF therapy helps in restoring immune cell functions and resolve infections. Its role in MDSCs modulation in cirrhotic with sepsis is not well understood. Methods 164 decompensated cirrhotic; 62 without(w/o), 72 with sepsis and 30 with sepsis treated with GM-CSF and 15 healthy were studied. High-dimensional flow cytometry was performed to analyse MDSCs, monocytes, neutrophils, CD4 T-cells and Tregs at admission, day3 and 7. Ex-vivo co-cultured MDSCs with T-cells were assessed for proliferation and apoptosis of T-cells, differentiation to T-regs. Plasma factors and mRNA levels were analysed by cytokine-bead assay and qRT-PCR. Results Frequency of MDSCs and T-regs were significantly increased (p=0.011, and p=0.02) with decreased CD4 T-cells(p=0.01) in sepsis than without sepsis and HC (p=0.000, p=0.07 and p=0.01) at day0, and day7. In sepsis patients, MDSCs had increased IL-10, Arg1 and iNOS mRNA levels (p=0.016, p=0.049 and p=0.06). Ex-vivo co-cultured MDSCs with T-cells drove T-cell apoptosis (p=0.03, p=0.03) with decreased T-cell proliferation and enhanced FOXP3+ expression (p=0.05 and p=0.05) in sepsis compared to no sepsis at day0. Moreover, blocking the MDSCs with inhibitors suppressed FOXP3 expression. GM-CSF treatment in sepsis patients significantly decreased MDSCs and FOXP3+Tregs but increased CD4 T-cell functionality and improved survival. Conclusion MDSCs have immunosuppressive function by expanding FOXP3+ Tregs and inhibiting CD4+ T-cell proliferation in sepsis. GM-CSF treatment suppressed MDSCs, improved T-cell functionality and reduced Tregs in circulation.

Non-alcoholic fatty liver disease is one of the most common causes of chronic liver diseases and occurs even in lean individuals having normal or low body mass index (BMI). Crucial issue is understanding the clinical, pathobiologic and metabolic characteristics in comparison to obese patients. Very few studies have compared clinicopathological characteristics between lean and obese. Published literature is generally in a small cohort of patients, rarely included over-weight as separate category, and most often had non-standardized use of BMI criteria with discordant conclusions. There is very sparse published literature on liver biopsy-confirmed cohort and that to from Asia; also, none had explored the role of mediators such as stellate cells, progenitor cells and macrophages.To evaluate the prevalence of NAFLD in lean patients in a large cohort of histology-confirmed NAFLD, and explore clinico-pathological spectrum of lean NAFLD in comparison to over-weight and obese. Also, to investigate role of hepatic stellate cells, macrophage polarization and their relation to hepatic progenitor cells, in particular the relation to fibrosis and to different BMI categories.Prospective analysis of eleven-year retrospective cross-sectional data of all consecutive patients of NAFLD diagnosed in the period between the year 2011 and 2021. All histologically confirmed cases of NAFLD fulfilling inclusion and exclusion criteria were stratified to three groups according to BMI based on Asian criteria. Demographic, lab, metabolic, and histological comparisons between lean and overweight-obese patients were performed. Histological grading and staging of NAFLD components were performed by NAS-CRN score. Immunohistochemical and image analysis-based assessment and quantification of stellate cells, progenitor cells, and macrophage polarization was performed. Appropriate statistical methods were applied, and study was approved by the Institutional ethics committee.Lean patients with biopsy-proven diagnosis constituted 21 % (n = 267) of total NAFLD (n = 1273). Other groups were-over-weight patients (232;18.2 %), and the highest were obese patients (774; 60.8 %). 13.9 % of the lean patients with NAFLD were under-weight with BMI18.5 kg/mPrevalence of NAFLD in lean individuals in a histological-confirmed patient cohort was 21 %. (n = 267/1273). Major strengths of this study are large cohort of lean individuals from a single center, inclusion of only histology-confirmed cases, Asia specific BMI criteria usage, comparative clinical, metabolic, immune-morphologic and image analysis-based characterization, inclusion of over-weight in addition to obese patients, and investigating cross-talk of principal patho-physiologic markers.

Diabetes mellitus is a rising epidemic in most part of the world and is often associated with multiple organ disorders such as kidney, liver, and cardiovascular diseases. Liver is a major metabolic hub, and the metabolic disorders associated with diabetes result in liver dysfunctions culminating in spectrum of liver diseases such as fatty liver disorders, cirrhosis, and hepatocellular carcinoma. The intervention strategies to prevent diabetes-associated liver injury require an overall understanding of the key factors and molecular pathways which can be strategically targeted. The present review focuses on some of the key aspects of fatty acid metabolism, fetuin-A regulation, inflammatory pathways, and genetic factors associated with insulin resistance, dyslipidemia, hyperglycemia, oxidative stress, and so on involved in the nexus between diabetes and liver injury. Further recent interventions, pharmacological target, and newer therapeutic agents are discussed briefly for the better clinical management of diabetes-associated hepatic disorders.

Background Targeting cancer stem cells (CSCs) in hepatocellular carcinoma (HCC) is difficult because of their similarities with normal stem cells (NSCs). EpCAM can identify CSCs from EpCAM+AFP+HCC cases, but is also expressed on NSCs. We aimed to distinguish the two using integrated protein, mRNA and miRNA profiling. Methods iTRAQ based protein profiling and Next Generation Sequencing (NGS) was performed on EpCAM+/EpCAM- cells isolated from HCC (Ep+CSC, Ep- HCC) and EpCAM+ cells from non-cancerous/non-cirrhotic control liver tissues (Ep+NSC). Validations were done using qRT-PCR, flowcytometry and western blotting followed by in vitro and in vivo functional studies. Results 11 proteins were overexpressed (>3 fold) in Ep+CSCs compared to Ep- HCC and Ep+NSC cells. However, RNA-sequencing confirmed the Ep+CSC specific up-regulation of only HSPA8, HNRNPC, MPST and GAPDH mRNAs among these. Database search combined with miRNA profiling revealed Ep+ CSC specific down-regulation of 29 miRNAs targeting these four genes. Of these, only miR-26b-5p was found to target both HSPA8 and EpCAM. Validation of HSPA8 overexpression and miR-26b-5p down-regulation followed by linear regression analysis established a negative correlation between the two. Functional studies demonstrated that reduced miR-26b-5p expression increased the spheroid formation, migration, invasion and tumourigenicity of Ep+ CSCs. Furthermore, anti-miR-26b-5p increased the number of Ep+ CSCs with a concomitant overexpression of stemness genes and reduction of proapoptotic protein BBC3, which is a known substrate of HSPA8. Conclusion miR-26b-5p imparts metastatic properties and helps in maintenance of Ep+ CSCs via HSPA8. Thus, miR-26b-5p and HSPA8 could serve as molecular targets for selectively eliminating the Ep+ CSC population in human HCCs.

Vertical transmission of hepatitis B virus (HBV) from the mother to the newborn often results in viral persistence. To understand mechanisms of maternofetal HBV transmission, we studied maternal immunity and peripheral blood mononuclear cell (PBMC) transcriptome in mothers and newborns. We included 50 mothers and babies who were hepatitis B surface antigen (HBsAg) positive: 22 HBV transmitting mothers (group [Gr.] I) and 28 HBV nontransmitting mothers (Gr. II) to newborns and 10 healthy mother–baby pairs (Gr. III). PBMCs were analyzed for HBV‐specific dendritic cells (DCs), T cells, T follicular helper (TFh) cells, B cells, functional immune responses, and cytokine levels as well as transcriptome signatures to identify immune gene expression correlates for protective immunity. Group II mothers had lower HBsAg levels (3.82 × 103 versus 1.493 × 104; P

Background and Aims: Non-dividing hepatocytes in end-stage liver disease indicates permanent growth arrest similar to senescence. Identifying senescence in vivo is often challenging and mechanisms inhibiting senescence are poorly understood. In lower organisms mitochondrial unfolded protein response (UPRMT) helps in increasing longevity; however, its role in senescence and liver disease is poorly understood. Aim of this study was to identify hepatocyte senescence and the role of UPRMT in cryptogenic cirrhosis. Methods: Doxorubicin was used to induce senescence in non-neoplastic hepatocytes (PH5CH8) and hepatoma cells (HepG2 and Huh7). Senescence-associated markers and unfolded protein response was evaluated by fluorescence microscopy, immunoblotting and gene expression. Explants/biopsies from normal, fibrosis, compensated and decompensated cirrhosis without any known etiology were examined for presence of senescence and UPRMT by immunohistochemistry and gene expression. Results: Accumulation of senescent hepatocytes in cryptogenic cirrhosis was associated with reduced proliferation, increased expression of γH2AX and p21, together with loss of LaminB1. Dysfunctional mitochondria and compromised UPRMT were key features of senescent hepatocytes both in vitro and also in decompensated cirrhosis. Intriguingly, compensated cirrhotic liver mounted strong UPRMT, with high levels of mitochondrial protease, CLPP. Overexpression of CLPP inhibited senescence in vitro, by reducing mitochondrial ROS and altering oxygen consumption. Conclusions: Our results implicate a role of hepatocyte senescence in cryptogenic cirrhosis together with a crucial role of UPRMT in preventing hepatocyte senescence. A compromised UPRMT may shift the fate of cirrhotic liver toward decompensation by exaggerating hepatocyte senescence. Restoring CLPP levels at least in cell culture appears as a promising strategy in mitohormesis, thereby, preventing senescence and possibly improving hepatocyte function. Keywords: Cryptogenic Liver Cirrhosis, Mitochondrial Respiration, Mitochondrial Unfolded Protein Response, Oxidative Stress

Two breakthrough techniques that have totally revolutionized biology in last 1 decade are the discovery of genome editing tools and growing the stem cells/primary tissue explants in defined 3D culture. In this regard the discovery of CRISPR-Cas9 as a specific gene editing tool and organoid culture from adult stem cell has provided easy handy tools to uncover the process of organ development and also modeling cancer. Genetically modified organoids have been developed by sequential knockout and knockin of driver mutations by genome editing followed by niche-based selection. The modified organoids when xenotransplanted in animal models faithfully recapitulate the neoplastic events of human tumors. The present review focuses on the merging of these two powerful technologies in understanding the complexities of colon and liver cancer.

Background: Myeloid-derived suppressor cells (MDSCs) are immunosuppressive in nature, originate in the bone marrow, and are mainly found in the blood, spleen, and liver. In fact, liver acts as an important organ for induction and accumulation of MDSCs, especially during infection, inflammation, and cancer. In humans and rodents, models of liver diseases revealed that MDSCs promote regeneration and drive the inflammatory processes, leading to hepatitis, fibrogenesis, and cirrhosis, ultimately resulting in hepatocellular carcinoma. Summary: This brief review is focused on the in-depth understanding of the key molecules involved in the expansion and regulation of MDSCs and their underlying immunosuppressive mechanisms in liver diseases. Key Message: Modulated MDSCs can be used for therapeutic purposes in inflammation, cancer, and sepsis.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein involved in redox signalling and programmed cell death. The role of AIF has been well recognized in diabetes and obesity. However, the aspect of AIF deficiency in the development of hepatic steatosis and liver injury is unknown. Therefore, in the current study, Harlequin (Hq mutant) mouse with markedly reduced content of AIF was investigated to explore the role of AIF on the initiation of liver injury. The wild type (WT) developed physiological and pathological features of non-alcoholic fatty liver disease (NAFLD) that were not seen in the Hq mice with AIF deficiency, when fed on high fat high fructose (HFHF) diet. Following bile duct ligation (BDL), the liver associated pathological changes were less conspicuous in Hq mice as compared to WT mice. The expression of AIF protein and apoptosis was markedly lesser as compared to their respective control in Hq mice on HFHF diet. Furthermore, the genes involved in fatty acid metabolism were also altered in the group of treated Hq mice. In conclusion, Hq mice failed to develop diet induced hepatic steatosis, suggestive of a role of AIF mediated pathway in the initiation and progression of liver inflammation. Thus, partial loss of AIF appears to be hepatoprotective. SIGNIFICANCE OF THE STUDY: AIF deficiency has multiple roles in altered pathology processes and cellular metabolism, thereby compromising the cellular homeostasis. Considering the molecular functions of AIF in other organ pathology little is known about its role in diet induced liver injury. Hence, the aim of the current study was to investigate the role of AIF deficiency in liver injury and diseases with focus on NAFLD. The study will help to deliniate the mechanisms of NAFLD using Harliquin Mice.

In multicellular organisms majority of the cells remain in a non-dividing states of either quiescence (reversible) or senescence (irreversible). In the present study, gene expression signatures unique to quiescence and senescence were identified using microarray in osteosarcoma cell line, U2OS. It was noted that certain genes and pathways like NOD pathway was shared by both the growth arrest conditions. A major highlight of the present study was increased expression of number of chemokines and cytokines in both quiescence and senescence. While senescence-associated secretory phenotype (SASP) is well known, the quiescence-associated secretory phenotype (QASP) is relatively unknown and appeared novel in this study. ARID5A, a subunit of SWI/SNF complex was identified as a quiescence associated gene. The endogenous expression of ARID5A increased during serum starved condition of quiescence. Overexpression of ARID5A resulted in more number of cells in G0/G1 phase of cell cycle. Further ARID5A overexpressing cells when subjected to serum starvation showed a pronounced secretory phenotype. Overall, the present work has identified gene expression signatures which can distinguish quiescence from senescence.

Chronic hepatitis B virus (HBV) infection affects millions of people worldwide and about half a million people die every year. India represents the second largest pool of chronic HBV infections with an estimated 40 million chronically infected patients. Persistence or clearance of HBV infection mainly depends upon host immune responses. Chronically infected individuals remain in immune tolerant phase unless HBV flares and leads to the development of chronic active hepatitis or acute-on-chronic liver failure. Strategies based on inhibition of viral replication (nucleoside analogues) or immune modulation (interferons) as monotherapy, or in combination in sequential therapies, are currently being used globally for reducing HBV viral load and mediating HBsAg clearance. However, the immune status and current therapies for promoting sustained virological responses in HBV-infected patients remain suboptimal. Elimination of cccDNA is major challenge for future therapies, and new molecules such as NTCP, Toll-like receptor (TLR)7 agonist (GS9620) and cyclophilin have emerged as potential targets for preventing HBV entry and replication. Other than these, HBV cccDNA elimination is the major target for future therapies.

In metabolic disorders like obesity, NAFLD and T2DM, adipocytes are dysfunctional. Hence, pharmacological interventions have importance in preventing differentiation of adipocytes and stimulating lipid uptake. We, therefore, investigated the effects of arbutin (ARB), purpurin (PUR), quercetin (QR), and pterostilbene (PTS) on adipocyte differentiation and lipid uptake using 3T3-L1 adipocytes. Further, in silico docking studies were achieved to investigate interactions of ARB, PUR, QR, and PTS with beta-ketoacyl reductase (KR) and thioesterase (TE) domains of fatty acid synthase (FAS) enzyme. Mature 3T3-L1 adipocytes were used to investigate the anti-adipogenic effect of selected pharmacological agents by Oil Red O staining and in vitro fatty acid uptake analysis. Molecular docking studies were performed to predict the binding interactions of selected compounds with KR and TE domains of FAS enzyme. All these agents significantly decrease the adipocyte differentiation and showed the stimulatory effect on fatty acid uptake in 3T3-L1 adipocytes. However, PTS and PUR proved to be anti-adipogenic, whereas ARB and QR showed significant effect on fatty acid uptake, compared to others. Similarly, all the compounds displayed significant binding interactions with KR and TE domains of FAS enzyme, supporting the results of in vitro studies. Graphical abstract.

BACKGROUND Endothelial progenitor cells (EPCs) help in neovascularization and endothelial repair during injury. Patients with cirrhosis show increased number and function of EPCs in circulation. METHODS Since natural killer (NK) cells regulate EPCs, we investigated the relationship between the 2 in alcoholic cirrhosis (AC, n = 50) and severe alcoholic hepatitis (SAH, n = 18) patients and compared with nonalcoholic cirrhosis (n = 15) and healthy controls (HC, n = 30). Levels of systemic inflammatory cytokines were measured, and coculture assays were performed between EPCs and NK cells in contact-dependent and contact-independent manner. NK cell-mediated killing of EPCs was evaluated, and expression of receptors including fractalkine (FKN) on EPCs and its cognate receptor CX3CR1 on NK cells was studied by RT-PCR assays. RESULTS Patients with SAH had higher regulated on activation, normal T cell expressed and secreted (RANTES) (p = 0.01), vascular endothelial growth factor (VEGF) (p = 0.04), IL-1β (p = 0.04), and IL-6 (p = 0.00) growth factors and proinflammatory cytokines as compared to AC and HC. Distinct populations of CD31+ CD34+ EPCs with low and high expression of CD45 were significantly lower in SAH than HC (CD45low , p = 0.03; CD45hi , p = 0.04) and AC (CD45low , p = 0.05; CD45hi , p = 0.02). SAH patients, however, showed increased functional capacity of EPCs including colony formation and LDL uptake. NK cells were reduced in SAH compared with AC (p = 0.002), however with higher granzyme ability (p

Background Hepatocellular carcinoma (HCC) is characterized by marked phenotypic and molecular heterogeneity. Clinico-morphologic phenotypes and associations are important surrogate markers of molecular aberrations; therefore have immense relevance for targeted therapy. There is paucity of published literature on critical analysis of HCC heterogeneity and morphological alliance. Aims: To assess the heterogeneity and dominance of histomorphological features, and to explore clinicopathological associations in HCC. Methods Retrospective cross-sectional study of 217 HCC tissue specimens was performed for the assessment of prevalence of major histological patterns, cytological features, and clinicopathological correlation. Results Homogeneous architecture with a single dominant histological pattern was a rarity. Single pattern constituting ≥50 % of the tumour was found in less than 1/5th of the cases. Macrotrabecular HCC represented 9.2 % of cases. The simultaneous presence of 2–3 patterns or atypical variants and/ or cytological characteristics was recorded in 25 % and 30 % respectively. Significant clinicopathological associations: Pseudoglandular with microtrabecular pattern-cholestasis, showed better differentiation and early-stage; macrotrabecular pattern frequently occurred with pleomorphic giant cells, higher tumour stage, higher AFP levels; solid pattern often showed clear cells. Noticeable mutual exclusions were MD bodies with microtrabecular and pseudoglandular patterns; Compact pattern with neutrophilic clusters and cholestasis. Larger tumours were significantly more heterogeneous; however, heterogeneity did not correlate with outcome Conclusions HCC displays immense heterogeneity with an amalgamation of different histomorphological patterns and features; nevertheless, there are certain reproducible associations and omissions. Tumor biopsies agree fairly well with large specimens. Characterization of phenotypic heterogeneity, dominance, associations, and exclusions in individual patients provides vital information.

Background and Aims Non-dividing hepatocytes in end-stage liver disease indicates permanent growth arrest similar to senescence. Identifying senescence in vivo is often challenging and mechanisms inhibiting senescence are poorly understood. In lower organisms mitochondrial unfolded protein response (UPRMT) helps in increasing longevity; however, its role in senescence and liver disease is poorly understood. Aim of this study was to identify hepatocyte senescence and the role of UPRMT in cryptogenic cirrhosis. Methods Doxorubicin was used to induce senescence in non-neoplastic hepatocytes (PH5CH8) and hepatoma cells (HepG2 and Huh7). Senescence-associated markers and unfolded protein response was evaluated by fluorescence microscopy, immunoblotting and gene expression. Explants/biopsies from normal, fibrosis, compensated and decompensated cirrhosis without any known etiology were examined for presence of senescence and UPRMT by immunohistochemistry and gene expression. Results Accumulation of senescent hepatocytes in cryptogenic cirrhosis was associated with reduced proliferation, increased expression of γH2AX and p21, together with loss of LaminB1. Dysfunctional mitochondria and compromised UPRMT were key features of senescent hepatocytes both in vitro and also in decompensated cirrhosis. Intriguingly, compensated cirrhotic liver mounted strong UPRMT, with high levels of mitochondrial protease, CLPP. Overexpression of CLPP inhibited senescence in vitro, by reducing mitochondrial ROS and altering oxygen consumption. Conclusions Our results implicate a role of hepatocyte senescence in cryptogenic cirrhosis together with a crucial role of UPRMT in preventing hepatocyte senescence. A compromised UPRMT may shift the fate of cirrhotic liver toward decompensation by exaggerating hepatocyte senescence. Restoring CLPP levels at least in cell culture appears as a promising strategy in mitohormesis, thereby, preventing senescence and possibly improving hepatocyte function., Graphical abstract

Sirtuins belong to the family of class III histone deacetylases; its role in neoplasia is controversial as both tumor-suppressive and promoting functions have been reported. There are very few reports available, where expressions of sirtuin isoforms are comprehensively analyzed during neoplasia. Therefore, in the present study, the expression of SIRT1, SIRT2, and SIRT7 during different stages of cervical cancer progression was analyzed. The normal cervical epithelium showed feeble expression of sirtuin isoforms, SIRT1, SIRT2, and SIRT7. A significant increase in SIRT1 expression was noted in the cytoplasm as well as in the nucleus of proliferative layers of cervical epithelium in squamous intraepithelial lesions (SIL); however, in the squamous cell carcinomas (SCC), a heterogeneous pattern of SIRT1 expression varying from low to high was noted. A progressive increase in the expression of both SIRT2 and SIRT7 was noted during cancer progression in the following order: normal

BACKGROUND Asialoglycoprotein receptor expression on hepatocytes has been associated with endocytosis, binding and uptake of hepatitis B virus. The role of asialoglycoprotein receptor in hepatitis B virus vertical transmission and its expression on placenta has not yet been studied. PATIENTS AND METHODS Thirty-four HBsAg+ve and 13 healthy pregnant mothers along with their newborns were enrolled. The former were categorized into transmitting and non-transmitting mothers based on their newborns being hepatitis B surface antigen and hepatitis B virus DNA positive. Expression of asialoglycoprotein receptor and hepatitis B surface antigen in placenta and isoform of asialoglycoprotein receptor on dendritic cell in peripheral and cord blood dendritic cells were analysed using flowcytometry, immune histochemistry, immune florescence and qRT-PCR. RESULTS Twelve HBsAg+ve mothers transmitted hepatitis B virus to their newborns whereas the rest (n = 22) did not. Hepatitis B virus-transmitting mothers showed increased expression of asialoglycoprotein receptor in trophoblasts of placenta. Immunofluorescence microscopy revealed colocalization of hepatitis B surface antigen and asialoglycoprotein receptor in placenta as well as in DCs of transmitting mothers. There was no significant difference in the expression of asialoglycoprotein receptor on peripheral blood mononuclear cells or chord blood mononuclear cells between the 2 groups. However, hepatitis B virus-transmitting mothers and their HBsAg+ve newborns showed increased mRNA levels of isoform of asialoglycoprotein receptor on dendritic cell in peripheral blood mononuclear cells. Hepatitis B virus-transmitting mothers and their HBsAg+ve newborns showed an increased expression of isoform of asialoglycoprotein receptor on dendritic cell on circulating dendritic cells compared to hepatitis B virus non-transmitting mothers and their negative newborns. CONCLUSIONS This study revealed that increased expression of asialoglycoprotein receptor in placenta and colocalization with hepatitis B surface antigen strongly indicates its role in intrauterine transmission of hepatitis B virus. Asialoglycoprotein receptor-blocking strategy can be used for therapeutic intervention of vertical transmission.

The calcineurin-NFAT signaling pathway regulates cell proliferation, differentiation, and development in diverse cell types and organ systems. Deregulation of calcineurin-NFAT signaling has been reported in leukaemias and few solid tumors such as breast and colon. In the present study, we found elevated calcineurin protein levels and phosphatase activity in cervical cancer cell lines and depletion of the same attenuated cell proliferation. Additionally, nuclear levels of NFAT2, a downstream target of calcineurin, viz, was found elevated in human papillomavirus (HPV) infected cells, HeLa and SiHa, compared to the HPV negative cells, HaCaT and C33A, indicative of its higher DNA binding activity. The nuclear levels of both NFAT1 and NFAT3 remain unaltered implicating they have little role in cervical carcinogenesis. Similar to the in vitro studies, the HPV infected human squamous cell carcinoma specimens showed higher NFAT2 levels compared to the normal cervical epithelium. Depletion of NFAT2 by RNAi attenuated growth of SiHa cells. Overexpression of HPV16 oncoproteins viz, E6 and E7 increased NFAT2 expression levels and DNA binding activity, while knockdown of E6 by RNAi decreased the same. Briefly, we now report an activation of calcineurin-NFAT2 axis in cervical cancer and a novel role of HPV oncoprotein in facilitating NFAT2 dependent cell proliferation.

Background Acute viral hepatitis E (AVH-E) can often result in acute liver failure (ALF) during pregnancy. microRNAs serve as mediators in drug induced liver failure. We investigated their role as a biomarker in predicting ALF due to HEV (ALF-E). Methods We performed next generation sequencing and subsequent validation studies in PBMCs of pregnant (P) self limiting AVH-E, ALF due to HEV (ALF-E) and compared with AVH-E in non-pregnant (NP) females and healthy controls. Findings Eleven microRNAs were significantly expressed in response to HEV infection; importantly, miR- 431, 654, 1468 and 4435, were distinctly expressed in pregnant self-limiting AVH-E and healthy females (p = 0.0005), but not in ALF-E. Sixteen exclusive microRNAs differentiated ALF-E from self limiting AVH-E in pregnant females. miR-450b which affects cellular proliferation and metabolic processes through RNF20 and SECB was predominanlty upregulated and correlated with poor outcome (ROC 0.958, p = 0.001). Interpretation Our results reveal that a specific microRNA profile can predict fatality in ALF-E in pregnancy. These microRNAs could be exploited as prognostic biomarkers and help in the development of new therapeutic interventions.

Calcineurin (CN) is a major calmodulin binding serine/threonine phosphatase which plays a crucial role in numerous mammalian signal transduction pathways. Calcineurin inhibitors represent a valuable tool for elucidating CN dependent cellular processes. The present work deals with the synthesis and comprehensive characterization of a new polymer anchored peroxo niobium complex, Nb-2(O-2)(6)(carboxylate)(2)]-PA (Nb-2) PA=poly(sodium acrylate)], and identification of a set comprising of neat homoleptic as well as polymer immobilized peroxo complexes of vanadium(V) and niobium(V) as potent CN inhibitors. The in-vitro effect of the complexes on calmodulin mediated dephosphorylation activity of CN was investigated using a physiological substrate of calcineurin, RII-phosphopeptide as well as a non-protein substrate p-nitrophenyl phosphate (p-NPP). Enzyme kinetic analysis data revealed that the compounds inhibit function of CN via uncompetitive pathway with K-i values ranging between 1-3M, suggesting the formation of an enzyme-inhibitor-substrate complex during the course of inhibition.

Sirtuins belong to a class of NAD-dependent deacetylases, and include seven distinct isoforms, of which SIRT7 is the least studied member. In the present study, the subcellular expression of SIRT7 in primary fibroblasts undergoing senescence was evaluated by immunocytochemistry and immunoblot assay. Expression of nucleolar SIRT7 in young fibroblast was very prominent, decreased in pre-senescent cells, and became undetectable in the senescent cells. Interestingly, we observed previously unreported staining for cytoplasmic SIRT7 in fibroblasts, and report the existence of a steady-state level of SIRT7 in cytoplasm. Selective localization of the high-molecular-mass (47.5 kDa) SIRT7 in the cytoplasmic fraction and the low-molecular-mass (45 kDa) SIRT7 in the nuclear fraction was observed in the immunoblot analysis of various cell types. The specificity of the N-terminal antibodies for detection of cytoplasmic SIRT7 was confirmed by RNAi and peptide competition assays. The two forms of SIRT7 showed reciprocal expression following serum starvation, nocodazole and okadaic acid treatments, and also during senescence. Using a combination of deletion constructs and site-directed mutagenesis, we defined the role of two distinct SIRT7 sequences in the N-terminal region (amino acids 61-76, LQGRSRRREGLKRRQE) and the C-terminal region (amino acids 392-400, KRTKRKKVT) for nuclear and nucleolar import, respectively. In conclusion, we report for the first time the existence of a cytoplasmic pool of SIRT7 in addition to its well-known nucleolar form, identify distinct localization signals for its nuclear/nucleolar targeting, and describe an association between loss of nucleolar SIRT7 and replicative senescence.

Sirtuins (SIRT) belonging to the NAD+ dependent histone deacetylase III class of enzymes have emerged as master regulators of metabolism and longevity. However, their role in prevention of organismal aging and cellular senescence still remains controversial. In the present study, we now report upregulation of SIRT2 as a specific feature associated with stress induced premature senescence but not with either quiescence or cell death. Additionally, increase in SIRT2 expression was noted in different types of senescent conditions such as replicative and oncogene induced senescence using multiple cell lines. Induction of SIRT2 expression during senescence was dependent on p53 status as depletion of p53 by shRNA prevented its accumulation. Chromatin immunoprecipitation revealed the presence of p53 binding sites on the SIRT2 promoter suggesting its regulation by p53, which was also corroborated by the SEAP reporter assay. Overexpression or knockdown of SIRT2 had no effect on stress induced premature senescence, thereby indicating that SIRT2 increase is not a cause of senescence; rather it is an effect linked to senescence-associated changes. Overall, our results suggest SIRT2 as a promising marker of cellular senescence at least in cells with wild type p53 status.

Hydrogen peroxide is often required in sublethal, millimolar concentrations to show its oxidant effects on cells in culture as it is easily destroyed by cellular catalase. Previously, we had shown that diperoxovanadate, a physiologically stable peroxovanadium compound, can substitute H2O2 effectively in peroxidation reactions. We report here that peroxovanadate when anchored to polyacrylic acid (PAPV) becomes a highly potent inhibitor of growth of lung carcinoma cells (A549). The early events associated with PAPV treatment included cytoskeletal modifications, increase in GTPase activity of Rac1, accumulation of the reactive oxygen species, and also increase in phosphorylation of H2AX (gamma H2AX), a marker of DNA damage. These effects persisted even at 24 h after removal of the compound and culminated in increased levels of p53 and p21 together with growth arrest. The PAPV-mediated growth arrest was significantly abrogated in cells pre-treated with the N-acetylcysteine, Rac1 knocked down by siRNA and DPI an inhibitor of NADPH oxidase. In conclusion, our results show that polyacrylate derivative of peroxovanadate efficiently arrests growth of A549 cancerous cells by activating the axis of Rac1-NADPH oxidase leading to oxidative stress and DNA damage.