Ashish Vyas

Combination of Granulocyte Colony-Stimulating Factor and Erythropoietin Improves Outcomes of Patients With Decompensated Cirrhosis

BACKGROUND & AIMS Patients with decompensated cirrhosis have significantly reduced survival without liver transplantation. Granulocyte colony-stimulating factor (G-CSF) has been shown to increase survival in patients with acute-on-chronic liver failure, and erythropoietin promoted hepatic regeneration in animal studies. We performed a double-blind, randomized, placebo-controlled trial to determine whether co-administration of these growth factors improved outcomes for patients with advanced cirrhosis. METHODS In a prospective study, consecutive patients with decompensated cirrhosis seen at the Institute of Liver and Biliary Sciences, New Delhi (from May 2011 through June 2012) were randomly assigned to groups given subcutaneous G-CSF (5 μg/kg/d) for 5 days and then every third day (12 total doses), along with subcutaneous darbopoietin α(40 mcg/wk) for 4 weeks (GDP group, n = 29), or only placebos (control group, n = 26). All patients also received standard medical therapy and were followed for 12 months. Histology was performed on liver biopsies. The primary end point was survival at 12 months. RESULTS Baseline characteristics of patients were comparable; alcohol intake was the most common etiology of cirrhosis. A higher proportion of patients in the GDP group than controls survived until 12 months (68.6% vs 26.9%; P = .003). At 12 months, Child-Turcotte Pugh scores were reduced by 48.6% in the GDP group and 39.1% in the control group, from baseline (P = .001); Model for End Stage Liver Disease scores were reduced by 40.4% and 33%, respectively (P = .03). The need for large-volume paracentesis was significantly reduced in GDP group, compared with controls (P < .05). A lower proportion of patients in the GDP group developed septic shock (6.9%) during follow-up compared with controls (38.5%; P = .005). No major adverse events were observed in either group. CONCLUSIONS In a single-center randomized trial, a significantly larger proportion of patients with decompensated cirrhosis given a combination of G-CSF and darbopoietin α survived for 12 months more than patients given only placebo. The combination therapy also reduced liver severity scores and sepsis to a greater extent than placebo. Clinicaltrials.gov ID: NCT01384565.

Impaired monocyte-macrophage functions and defective toll-like receptor signaling in hepatitis E virus-infected pregnant women with acute liver failure

Acute viral hepatitis resulting due to hepatitis E viral infection (AVH‐E) is often serious in pregnancy and could result in acute liver failure (ALF). The role of monocytes and macrophages (mono‐macs) in the pathogenesis of AVH‐E and development of ALF‐E in pregnancy is unclear. We investigated the functions of mono‐macs in pregnant (P), AVH‐E (n = 44), ALF‐E (n = 12), healthy controls (HC; n = 20) and compared with nonpregnant (NP) AVH‐E (n = 10), ALF‐E (n = 5), and HC (n = 10). We also recruited non‐hepatitis E virus‐related pregnant (P), ALF‐NE (n = 5) and non‐pregnant (NP), ALF‐NE (n = 12) patients with ALF. Mono‐macs, dendritic cell (DC) phenotypes, and Toll‐like receptor (TLR) expressions were studied by flow cytometry and reverse‐transcriptase polymerase chain reaction. Mono‐macs functionality was determined by analyzing their phagocytic activity and reactive oxygen species (ROS) generation by using flow cytometry. Frequency of mono‐macs and DCs was increased during HEV infection compared to HC (P < 0.001). Macrophages were increased (P < 0.002) in ALF‐E(P) compared to ALF‐NE(P). The macrophage phagocytic activity and Escherichia coli‐induced ROS production was significantly impaired in ALF‐E(P) compared to AVH‐E(P) (P < 0.001), ALF‐E(NP), and ALF‐NE(P) patients (P < 0.02). TLR3 and TLR9 expression and downstream MYD88 signalling molecules IRF3 and IRF7 were significantly down‐regulated in ALF‐E(P) (P < 0.00) compared to AVH‐E(P) and ALF‐NE(P). Conclusion: Functionality of mono‐macs is impaired in pregnant ALF‐E patients compared to AVH‐E(P). Reduced TLR3 and TLR7 expression and TLR downstream‐signaling molecules in pregnant ALF‐E patients suggests inadequate triggers for the innate immune responses contributing to development and severity of ALF‐E. Studies using TLR agonists to activate mono‐macs may be of use and in vitro studies should be undertaken using patient samples.(Hepatology 2015;62:1683–1696)

Characterization of Actinomycetes and Trichoderma spp. for cellulase production utilizing crude substrates by response surface methodology

Laboratory bench scaling was done and an average of 1.85 fold increase by Response Surface Methodology (RSM) optimization was obtained. It was found that the predicted value (4.96 IU/ml) obtained by RSM is in close accordance with observed activity 5.14 IU/ml. Endoglucanases are mainly induced by CMC while Wheat bran (natural substrate) exoglucanase is more active when induced by avicel and cellulose. Addition of substrate beyond a level caused inhibition of cellulase production. The molecular weight of protein as determined by SDS-PAGE is very similar to molecular weight of cellulase of Trichoderma viride (T. viride) cellulase and Trichoderma reesei (T. reesei) endoglucanase. T. reesei β-glucosidase has high enzymatic activity on CMC substrate when compared with T. viride β-glucosidase. Secondary structure analysed by using Circular Dichroism confirmed that composition of celluase system is very similar to other analysed species. The cellulase was found to be active in pH range of 4.8-5.5; while temperature range varied from 50°C to 70°C. Although the enzymatic activity produced by mutants were lesser than the parent, but in one case mutants of Trichoderma reesei’s BGL has shown higher activity on cellulose.

CD4+CD25+CD127low regulatory T cells play predominant anti-tumor suppressive role in hepatitis B virus associated hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide and hepatitis B is one of the commonest causes. T regulatory cells (Tregs) are strong immunomodulators and are likely to play a major role in HCC development. HBV infection is reported to induce expansion of Tregs. We investigated the CD4+CD25+CD127−veFoxP3+ Tregs in HBV-related HCC as compared to non-HBV-HCC. Patients and Methods: Whole blood immunophenotyping was analyzed by multicolor flow cytometry in patients with HBV-related HCC (HBV-HCC, n = 17), non-HBV-HCC (n = 22; NASH = 16, alcohol-related = 6), and chronic hepatitis B infection (CHBV; n = 10). Tregs functionality was checked by in vitro suppression assays using CD4+ CD25+ CD127low Tregs. Levels of serum alpha-fetoprotein (AFP), expression of FoxP3, IL-10, PD1, TGF-β, and Notch in Tregs, and liver explants were analyzed by flow cytometry, immunohistochemistry, and quantitative RT-PCR. Results: CD4+CD25+hi and Foxp3 expression in CD4+CD25+hiCD127low was significantly increased (P = 0.04, P = 0.007) in HBVHCC compared to non-HBVHCC and CHBV patients. HBVHCC also showed high IL-10 and TGF-β secreting CD4 + CD25 + hiTregs. The PD1 expression in CD4 + CD25+hi was significantly decreased in the HBVHCC than non-HBVHCC. In HBVHCC, AFP levels were significantly high (median 941, range 2–727940) than non-HBVHCC (median 13.5, range 2–18,900). In HBVHCC, patients with high AFP (range; 3982–727940 ng/ml) showed positive correlation with Foxp3 expression in CD4+CD25+hi CD127low (r = 0.857, P = 0.014). Reduced PD1 expression in HBVHCC also had negative correlation with FOXP3 in CD4+CD25+hi CD127low (r = −0.78, P = 0.04). However, AFP levels in non-HBVHCC showed negative correlation with (R = −0.67, P = 0.005) with CD4+CD25+hi Tregs. Conclusion: Our results demonstrate that CD4+ CD25+hi Tregs from HBVHCC patients have decreased expression of PD1, resulting in higher IL-10 and TGF-β secretion. Increased suppressive ability of Tregs in HBV-related HCC confers increased anti-tumor suppressive response than in non-HBV-HCC. Modulation of Tregs and PD1 may serve as useful therapeutic targets.

Immune regulation by T regulatory cells in HBV related Inflammation and cancer

Hepatocellular carcinoma (HCC) is the leading cause of cancer death, and hepatitis B virus (HBV) infection is one of the commonest causes in Asian countries. India has the second largest pool after China for hepatitis B‐infected subjects. HBV clearance is T cell dependent, and one of the reasons for T cells hyporesponsiveness is due to mass production of regulatory T cells (Tregs) through activation of Notch signalling, which suppress CD4/CD8 T cells. Tregs are important to maintain cellular homoeostasis; however, during viral infection increase of Tregs is inversely proportional to HBV DNA titres. Tregs exert their suppressive effect either via cell‐to‐cell contact or through release of interleukin (IL)‐2, IL‐10, TGF‐β and IL‐35. In Chronic hepatitis B virus CHBV infection, PD‐1 pathway also gets activated and is involved in promoting tolerance. However, with Tregs induction, virus‐specific T cell responses also get decreased. Circulatory and intratumoural Tregs promote development of HBV‐specific HCC more by decreasing and impairing the effector functions of CD8 T cells. Antiviral therapies and PD‐1 blockade strategy had shown the inhibition of Tregs and reduction in HBV DNA. However, inhibition of HBV‐specific Tregs is major challenge for future therapies. New cytokine blockade therapies have emerged as potential therapeutic potentials.

Immune Correlates of Hepatitis B surface antigen spontaneous seroconversion in Hepatitis B e Antigen negative chronic hepatitis B patients

Hepatitis B surface antigen (HBsAg) seroconversion in HBeAg −ve chronic hepatitis B (CHB) infection is rare, possibly due to poor antigen processing and impaired humoral response. We investigated the role of dendritic cells (DCs), T follicular helper (TFH) cells and plasma B cells in seroconversion.

Immune balance in Hepatitis B Infection: Present and Future Therapies

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.

Iron-Overload triggers ADAM-17 mediated inflammation in Severe Alcoholic Hepatitis

Severe alcoholic hepatitis (SAH) is associated with iron accumulation in hepatocytes/macrophages. This possibly correlates with inflammation and stress but the exact mechanism still remains obscure. To understand the role of iron and the mechanisms of systemic iron-overload, a transcriptomic study of liver and Peripheral Blood -Mononuclear-Cells (PBMCs) was undertaken in SAH patients, with and without hepatic iron-overload. Our results show that iron-overload in hepatocytes/macrophages is due to an increased expression of iron-loading receptors and CD163 signaling cascade. Increase in labile iron pool induces expression of iron-loading, oxidative-stress and inflammatory genes along with expression of CD163 and ADAM17. Increased liver iron correlated with circulatory iron, TNF-α, macrophage activation (sCD163) and peroxide-stress in CD163+macrophages in patients who were iron-overloaded and died. Circulatory TNF-α and sCD163 levels were associated with poor outcome. Temporal iron/Fenton stress induced in healthy monocyte-derived-macrophage (MDM)/Tohoku-Hospital-Pediatrics-1(THP1) cells showed higher expression of iron-regulatory, inflammatory and oxidative-stress genes. These genes could be suppressed by iron-chelation. These results suggest that iron mediates inflammation through ADAM17 induction, resulting in macrophage activation and increased shedding of TNF-α and sCD163. These events could be inhibited with iron chelation or with ADAM17-blockade, postulating a therapeutic strategy for SAH patients with iron overload.

Incidence and Antibiotic profile of Bacterial Isolates from Neonatal Septicemia in National Medical College and Teaching Hospital, Birgunj, Nepal

Male and female neonatal sepsis was included in this study. Positive blood culture was observed in the study with positive male cases 43.53% than female 41.17%. The gram positive isolates 82.6% were found more than gram negative isolates 16.4%.The most common isolates were Staphylococcus aureus 66 (86.9%), Klebsiella pneumonia 9 (60%) and others. Staphylococcus aureus showed resistant against penicillin G, ceftazidime, cephalexin, cefixime, ampicillin, ofloxacine, ceftriazone, imipenem, azithromycin used antibiotics.

Placental expression of asialoglycoprotein receptor associated with Hepatitis B virus transmission from mother to child

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.