Small intestinal bacterial overgrowth and toll-like receptor signaling in patients with non-alcoholic fatty liver disease.

BACKGROUND AND AIM: The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is multifactorial. There is sparse literature on the role of small intestinal bacterial overgrowth (SIBO) and toll-like receptor (TLR) signaling in NAFLD. The present study evaluated the relationship of SIBO with expression of TLR signaling genes in patients with NAFLD. METHODS: A total of 142 subjects composed of NAFLD (n = 60, mean age 38.7 ± 10.4 years), chronic viral hepatitis (CVH) (n = 32, mean age 39.5 ± 10.6 years), and healthy volunteers (n = 50, mean age 36.56 ± 4.2 years) were enrolled in the study. Duodenal fluid was taken endoscopically in 32 prospective patients with NAFLD for evaluation of SIBO. Hepatic mRNA expression of TLR4, CD14, TLR2, NF-κß, and MD2 and protein expression of TLR4 and TLR2 were studied in 64 patients (NAFLD = 32, CVH = 32) by RT-PCR and immunohistochemistry, respectively. Serum levels of TNF-α, adiponectin, insulin, and endotoxins were also evaluated. RESULTS: Small intestinal bacterial overgrowth was present in 12 (37.5%) out of 32 patients with NAFLD with Escherichia coli as the predominant bacterium. In comparison with those without SIBO, patients with SIBO had significantly higher endotoxin levels and higher CD14 mRNA, nuclear factor kappa beta mRNA, and TLR4 protein expression. Patients with NASH had significantly higher endotoxin levels and higher intensity of TLR4 protein expression in comparison with patients without NASH. Serum levels of TNF-α, endotoxins, and insulin were significantly higher and of adiponectin lower in NAFLD in comparison with CVH and healthy volunteers. CONCLUSIONS: Our study provides the first direct evidence of role of SIBO and endotoxemia and its relation with TLR signaling genes and liver histology in patients with NAFLD.

Levels of hepatitis B virus replicative intermediate in serum samples of chronic hepatitis B patients.

Hepatitis B virus (HBV) cccDNA levels is an absolute marker of HBV replication in the liver of HBV infected patients. This study aimed to quantify the HBV cccDNA levels in sera and liver tissue samples of treatment naïve patients with chronic hepatitis B. Eighty one chronic hepatitis B (CHB) treatment naïve patients were enrolled from January 2009 to June 2011. Total HBV DNA and HBV cccDNA levels were quantified using sensitive real time PCR assay. The mean age of recruited patients was 34 ± 11.5 years. Fifty four (66.7%) patients were HBeAg negative. Liver tissue samples were available from 2 HBeAg positive and 21 HBeAg negative CHB patients. The amount of total intrahepatic HBV DNA ranged from 0.09 to 1508.92 copies/cell. The median intrahepatic HBV cccDNA was 0.31 and 0.20 copies/cell in HBeAg positive and HBeAg negative cases, respectively. Serum HBV cccDNA was detectable in 85.2 % HBeAg positive and 48.1% HBeAg negative CHB patients. Median serum HBV cccDNA was 46,000 and 26,350 copies/mL in HBeAg positive and HBeAg negative subjects, respectively. There was a significant positive correlation between the levels of intrahepatic total HBV DNA and intrahepatic HBV cccDNA (r = 0.533, p = 0.009). A positive correlation was also seen between serum HBV cccDNA levels and serum HBV DNA levels (r = 0.871, p < 0.001). It was concluded that serum HBV cccDNA could be detectable in higher proportion of HBeAg positive patients compared to HBeAg negative patients. Moreover, the median level of serum HBV cccDNA was significantly higher in HBeAg positive patients in contrast to HBeAg negative subjects.

Hepatitis B virus reverse transcriptase mutations in treatment Naïve chronic hepatitis B patients.

Mutations in the reverse transcriptase (RT) region of the hepatitis B virus (HBV) genome lead to decreased susceptibility to nucleos(t)ide analogs approved for treatment of HBV infection. The aim of this study was to detect and analyze pre-existing HBV RT mutations in treatment naïve patients with chronic hepatitis B. Seventy one chronic HBV treatment naïve patients were enrolled from January 2009 to June 2011. HBV RT sequence analysis was done by using direct bidirectional sequencing of semi-nested PCR products. HBV genotypes were determined by multiplex PCR. Genotype D was found in 64 patients (90.1%) followed by genotype C and A which were present in 5 (7.0%) and 2 (2.8%) patients respectively. The results of the RT sequence analysis showed mutations in 34 (47.9%) patients. The rtH248N mutation was the most common mutation, accounting for 47.1% patients. Other common mutations included rtD263E/S, rtM129L, rtF122L/V/I, rtS135Y/H, rtQ149K, rtL91I, rtH126R, rtC256S/G, rtY257W, rtS259T and rtE271D, which were present in 26.5% (9/34), 29.4% (10/34), 20.6% (7/34), 20.6% (7/34), 20.6% (7/34), 17.6% (6/34), 14.7% (5/34), 14.7% (5/34), 11.8% (4/34), 11.8% (4/34) and 11.8% (4/34) patients respectively. The known primary drug resistance mutations were found in 3 (8.8%) patients. The present study shows the presence of RT amino acid substitutions in treatment-naïve patients with chronic hepatitis B, which may decrease susceptibility to available oral antiviral drugs. On the basis of the finding of this study, genotypic testing is recommended before the start of therapy in naïve patients, so that suitable antiviral drugs can be prescribed.

Serum levels of angiogenic and anti-angiogenic factors: their prognostic relevance in locally advanced hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a prototype tumor wherein angiogenesis plays a vital role in its progression. The role of VEGF, a major angiogenic factor in HCC is known; however, the role of anti-angiogenic factors simultaneously with the angiogenic factors has not been studied before. Hence, in this study, the serum levels of major angiogenic [Vascular Endothelial Growth Factor (VEGF), angiopoietin-2 (Ang-2)] and anti-angiogenic (endostatin, angiostatin) factors were analyzed and correlated with clinico-radiological features and with outcome. A total of 150 patients (50 HCC, 50 cirrhosis and 50 chronic hepatitis) and 50 healthy controls were enrolled in this study. Serum levels of VEGF, Ang-2, endostatin, and angiostatin were estimated by enzyme-linked immunosorbent assay. HCC shows significantly elevated serum levels of angiogenic factors VEGF and Ang-2 and of anti-angiogenic factors endostatin and angiostatin. ROC curve analysis for serum VEGF yielded an optimal cut-off value of 225.14 pg/ml, with a sensitivity of 78 % and specificity of 84.7 % for a diagnosis of HCC and its distinction from other group. Using this value, the univariate and multivariate analysis revealed significantly poor outcome in patients with higher levels of serum VEGF (p = 0.009). Combinatorial analysis revealed that patients with higher levels of both angiogenic and anti-angiogenic factors showed poor outcome. Serum VEGF correlates with poor survival of HCC patients and, therefore, serves as a non-invasive biomarker of poor prognosis. Moreover, elevated levels of anti-angiogenic factors occur endogenously in HCC patients.

Angiogenic and anti-angiogenic factor gene transcript level quantitation by quantitative real time PCR in patients with hepatocellular carcinoma.

Tumor angiogenesis, a major requirement for tumor growth and metastasis, is regulated by pro- and anti-angiogenic factors. The aim of this study was to quantify the expression of angiogenic (VEGF, HIF-1α, Angiopiotein-2) and anti-angiogenic (endostatin, angiostatin and Thrombospondin-1) factors and to discern their clinical relevance. A total 90 patients (67 HCC, 9 cirrhosis and 14 chronic hepatitis) were enrolled in the study. Tissue transcript levels of angiogenic (VEGF, HIF-1α, Ang-2) and anti-angiogenic (endostatin, angiostatin and TSP-1) factors were analyzed by quantitative real time-polymerase chain reaction (qRT-PCR) in the tissue samples. The tissue transcript levels of VEGF, HIF-1α and endostatin were found to be significantly higher in HCC in comparison to cirrhosis and chronic hepatitis. Although Ang-2, angiostatin and TSP-1 tissue transcript levels were higher in HCC group than the others groups but the difference was not statistically significant. In univariate analysis both VEGF and HIF-1α were found to be associated with poor survival of HCC patients. Multivariate analysis by the cox proportional hazard model revealed only VEGF as an independent factor predicting poor survival of the HCC patients. Angiogenic and anti-angiogenic factors are all highly expressed in HCC patients. Upregulation of tissue anti-angiogenic factors indicates the urgency for the alternative of anti-angiogenic therapies.

Reactive Oxygen Species in Regulating Lymphangiogenesis and Lymphatic Function.

The lymphatic system is pivotal for immunosurveillance and the maintenance of tissue homeostasis. Lymphangiogenesis, the formation of new lymphatic vessels from pre-existing vessels, has both physiological and pathological roles. Recent advances in the molecular mechanisms regulating lymphangiogenesis have opened a new area of research on reparative lymphangiogenesis for the treatment of various pathological disorders comprising neurological disorders, cardiac repair, autoimmune disease, obesity, atherosclerosis, etc. Reactive oxygen species (ROS) produced by the various cell types serve as signaling molecules in several cellular mechanisms and regulate various aspects of growth-factor-mediated responses, including lymphangiogenesis. The ROS, including superoxide anion, hydrogen peroxide, and nitric oxide, play both beneficial and detrimental roles depending upon their levels and cellular microenvironment. Low ROS levels are essential for lymphangiogenesis. On the contrary, oxidative stress due to enhanced ROS generation and/or reduced levels of antioxidants suppresses lymphangiogenesis via promoting lymphatic endothelial cell apoptosis and death. In this review article, we provide an overview of types and sources of ROS, discuss the role of ROS in governing lymphangiogenesis and lymphatic function, and summarize the role of lymphatics in various diseases.

Comparison of 7 staging systems in north Indian cohort of hepatocellular carcinoma.

BACKGROUND & AIM: Many liver staging systems have been proposed for patients with hepatocellular carcinoma (HCC); however it is still controversial which staging system is best. The aim of this study was to compare the ability of 7 different staging systems in predicting survival in an Indian cohort of patients with HCC. METHODS: In this prospective study, 101 HCC patients were diagnosed and stratified according to 7 different staging systems; along with analysis of independent predictors of survival and their correlation with it (Kaplan-Meier analysis). RESULTS: CLIP, Tokyo score and BCLC staging system showed a significant difference in the probability of survival. All other staging systems failed to show a significant difference in survival. Age, portal vein thrombosis, serum bilirubin, MELD score showed a significant difference with survival in univariate analysis. However, serum bilirubin was the independent predictor of survival with a hazard ratio of 1.609 (95% CI 1.015-2.553, p = 0.043). CONCLUSION: The CLIP, Tokyo score and BCLC are the most useful staging systems in an Indian cohort.

Id1 Promotes Obesity by Suppressing Brown Adipose Thermogenesis and White Adipose Browning.

Obesity results from increased energy intake or defects in energy expenditure. Brown adipose tissue (BAT) is specialized for energy expenditure, a process called adaptive thermogenesis. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) controls BAT-mediated thermogenesis by regulating the expression of Ucp1 Inhibitor of differentiation 1 (Id1) is a helix-loop-helix transcription factor that plays an important role in cell proliferation and differentiation. We demonstrate a novel function of Id1 in BAT thermogenesis and programming of beige adipocytes in white adipose tissue (WAT). We found that adipose tissue-specific overexpression of Id1 causes age-associated and high-fat diet-induced obesity in mice. Id1 suppresses BAT thermogenesis by binding to and suppressing PGC1α transcriptional activity. In WAT, Id1 is mainly localized in the stromal vascular fraction, where the adipose progenitor/precursors reside. Lack of Id1 increases beige gene and Ucp1 expression in the WAT in response to cold exposure. Furthermore, brown-like differentiation is increased in Id1-deficient mouse embryonic fibroblasts. At the molecular level, Id1 directly interacts with and suppresses Ebf2 transcriptional activity, leading to reduced expression of Prdm16, which determines beige/brown adipocyte cell fate. Overall, the study highlights the existence of novel regulatory mechanisms between Id1/PGC1α and Id1/Ebf2 in controlling brown fat metabolism, which has significant implications in the treatment of obesity and its associated diseases, such as diabetes.

The cell polarity protein Scrib functions as a tumor suppressor in liver cancer.

Scrib is a membrane protein that is involved in the maintenance of apical-basal cell polarity of the epithelial tissues. However, Scrib has also been shown to be mislocalized to the cytoplasm in breast and prostate cancer. Here, for the first time, we report that Scrib not only translocates to the cytoplasm but also to the nucleus in hepatocellular carcinoma (HCC) cells, and in mouse and human liver tumor samples. We demonstrate that Scrib overexpression suppresses the growth of HCC cells in vitro, and Scrib deficiency enhances liver tumor growth in vivo. At the molecular level, we have identified the existence of a positive feed-back loop between Yap1 and c-Myc in HCC cells, which Scrib disrupts by simultaneously regulating the MAPK/ERK and Hippo signaling pathways. Overall, Scrib inhibits liver cancer cell proliferation by suppressing the expression of three oncogenes, Yap1, c-Myc and cyclin D1, thereby functioning as a tumor suppressor in liver cancer.

Genetic polymorphism in CD14 gene, a co-receptor of TLR4 associated with non-alcoholic fatty liver disease.

AIM: To evaluate the pathogenic role of toll-like receptor (TLR) gene polymorphisms in patients with non-alcoholic fatty liver disease (NAFLD). METHODS: Two hundred and fifty subjects (NAFLD = 200, healthy volunteers = 50) underwent polymerase chain reaction and restriction fragment length polymorphism to assess one polymorphism in the toll-like receptor 2 (TLR2) gene (A753G), two polymorphisms in the TLR4 gene (TLR4 Asp299Gly and Thr399Ile allele), and two polymorphisms in the cluster of differentiation 14 (CD14) (C-159T and C-550T) gene, a co-receptor of TLR4. Association of TLR gene polymorphisms with NAFLD and its severity was evaluated by genetic models of association. RESULTS: On both multiplicative and recessive models of gene polymorphism association, there was significant association of CD14 C (-159) T polymorphism with NAFLD; patients with TT genotype had a 2.6 fold increased risk of developing NAFLD in comparison to CC genotype. There was no association of TLR2 Arg753Gln, TLR4 Asp299Gly, Thr399Ile, and CD14 C (-550) T polymorphisms with NAFLD. None of the TLR gene polymorphisms had an association with histological severity of NAFLD. CONCLUSION: Patients with CD14 C (-159) T gene polymorphism, a co-receptor of TLR4, have an increased risk of NAFLD development.

Response to potent anti-HBV agents in chronic hepatitis B and combined effect of HBV reverse transcriptase mutations.

BACKGROUND AND AIM: Response to nucleos(t)ide analogue therapy against HBV infection depends on a number of factors. One of them is appearance of drug resistance mutations. The present study aimed to investigate the efficacy of ETV and TDF as anti-HBV agents and to analyze the role of HBV-RT mutations in reducing the efficacy of mentioned drugs. MATERIAL AND METHODS: Sixty nine treatment naïve CHB patients (mean age 33.8 ± 11.9 years) were enrolled and treated with ETV or TDF for one year. Complete virological response (CVR) was defined as undetectable serum HBV DNA after 12 months of therapy. Amino acid and nucleotide sequence analyses of HBV-RT region were performed using Geno2pheno HBV drug resistance tool. The 3D model of HBV-RT protein was built by I-TASSER server and RMSD was calculated between wild type and mutated HBV-RT protein. RESULTS: After 12 months of treatment, four CHB patients did not achieve CVR and all of them were with HBV genotype D. HBeAg seroconversion was achieved in 56% HBeAg positive patients after 12 months of antiviral therapy. The HBV-RT amino acid sequences from these four patients were used for in-silico analysis. It was found that the presence of many mutations in HBV-RT region of HBV isolated from these patients led to a high degree of variation in configuration of atoms of HBV-RT protein and also caused displacement of active site of this protein. CONCLUSION: The efficacy of antiviral drugs in inhibiting HBV replication may be reduced by combined effect of many HBV-RT mutations; however, an in vitro study is needed to validate the findings.

Clinical utility of prothrombin induced by vitamin K absence in the detection of hepatocellular carcinoma in Indian population.

BACKGROUND: Alpha-fetoprotein (AFP) is a well known widely used biomarker for the detection of hepatocellular carcinoma (HCC); however, it suffers from a low sensitivity and specificity. Protein or prothrombin induced by vitamin K absence or antagonist II (PIVKA-II) is another tumor marker elevated in HCC but not extensively used. AIM: Evaluation of PIVKA-II and AFP in diagnosing HCC in India. PATIENTS AND METHODS: The study group consisted of 70 consecutive HCC patients, 38 patients with cirrhosis, 30 patients with chronic hepatitis, and 30 normal healthy subjects. All patients were evaluated for PIVKA-II and AFP levels by ELISA. RESULT: The mean plasma concentration of PIVKA-II in HCC, cirrhotic, chronic hepatitis patients and healthy controls was 101.07 ± 78.30 ng/ml, 2.45 ± 4.25 ng/ml, 1.50 ± 0.98 ng/ml and 0.79 ± 0.75 ng/ml, respectively. Receiver operating characteristic (ROC) curve was plotted for PIVKA-II and AFP. At a cutoff level of 9.2 ng/ml for PIVKA-II a sensitivity of 80% and a specificity of 92.1% was found, whereas AFP at a cutoff level of 13.02 ng/ml showed 72.9% sensitivity and 65.8% specificity. No significant relationship of plasma levels of PIVKA-II was observed in HCC with HBsAg/antiHCV positivity and associated portal vein thrombosis, but a positive correlation was seen with the tumor size (P = 0.001). However, no such significant association was found with AFP. CONCLUSION: PIVKA-II was more sensitive and specific than AFP for diagnosing HCC in the Indian population.