Association of interleukin-10 with hepatitis B virus (HBV) mediated disease progression in Indian population.

Background & objectives: Interleukin (IL)-10, an anti-inflammatory Th2 cytokine, is one of the key coordinators of the inflammatory responses involved. The present study was designed to evaluate the impact of IL-10 (-819/-592) genotypes, haplotypes, mRNA and the protein levels with risk for hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) development in India. Methods: A total of 390 subjects (145 controls, 62 inactive HBV-carriers, 64 chronic-active HBV patients, 60 HBV related cirrhotics and 59 HBV- HCC subjects) were enrolled in the study. Allele specific (AS)-PCR, ELISA and RT-PCR methods were used for assessing polymorphism, spontaneous blood levels and the mRNA expression, respectively of IL-10. Results: The study revealed that the CC/TA genotype acted as a risk factor for cirrhosis (ORa=2.02; P<0.05) and the subsequent HCC development (ORa=2.20; P<0.05), with controls as reference. However, no significant association was found between the two haplotypes (CC and TA) observed and HCC risk. Moreover, the IL-10 protein and mRNA levels in peripheral blood mono nuclear cells (PBMCs) showed a significant elevation as the disease progressed to cirrhosis. But, no variation was observed in the IL-10 levels in subjects with different IL-10 genotypes. Interpretation & conclusions: These preliminary results suggest a strong association of IL10 (-819/-592) with the HBV infection mediated disease progression, from inactive carrier state to malignancy, in Indian population.

Success of antiviral therapy in chronic hepatitis C infection relates to functional status of myeloid dendritic cells.

Chronic hepatitis C infection poses a major global health predicament and appears to be potent threat to mankind. The treatment in wide use is interferon/ribavirin combination therapy which is generally effective in about 60-70 per cent of patients carrying genotype 3 and causes significant morbidity. The response to therapy is largely guided by limited number of factors such as genotype of virus, rapid virological response, ethnicity, pre-therapy viral load, etc. While involvement of host genetic factors has been a major focus of research in playing an important role in the outcome of disease, the role of immune system cannot be marginalized. Poor cellular trafficking and suboptimal T cell responses in liver, the hall marks of chronic hepatitis C virus infection, might be attributed to defective antigen presentation. Various immunological factors, both innate and adaptive, play role in the pathogenesis of the disease and become dysfunctional in active disease. Recent reports suggest the major impact of functional and numerical status of dendritic cells in deciding the fate of antiviral therapy. In this review we take a look at the involvement of dendritic cells in playing an important role in the response to therapy.

Treatment of chronic hepatitis C in end stage renal disease: experience at a tertiary care centre.

Introduction: Treatment of patients with chronic hepatitis C (CHC) is difficult in the setting of end stage renal disease (ESRD). The present study aimed to analyze the treatment outcome in patients with CHC and ESRD, being evaluated for kidney transplantation. Methods: Data of 65 patients of ESRD with CHC (males: 53, mean age: 39.2±14.4 years) was analysed retrospectively. Patients were treated with either pegylated or conventional interferon (IFN) without ribavirin. Treatment response was assessed for rapid virological response (RVR), early virological response (EVR), end of treatment response (ETR) and sustained virological response (SVR). Results: All patients were receiving hemodialysis (duration 1-60 months). Sixteen patients (25%) (genotype 1: 11, genotype 3: 4, genotype 2: 1) agreed for treatment (13 pegylated IFN and 3 conventional IFN). RVR was achieved in 7 patients (44%) and out of 11 patients (69%) who achieved EVR, ETR was achieved in 7 (44%) patients. Seven patients (44%) dropped out during treatment (2 because of side effects). SVR could be demonstrated in one of 7 patients who achieved ETR (6 patients were lost to follow up after ETR). Conclusions: In our experience, dropouts before, during and after treatment are a major problem in patients with CHC and ESRD. Of those who complete treatment, around half of them are able to achieve the end of treatment response.

Indocyanine green clearance test (using spectrophotometry) and its correlation with Model for End Stage Liver Disease (MELD) score in Indian patients with cirrhosis of liver.

Background: Child Turcotte Pugh (CTP) score and Model for End Stage Liver Disease (MELD) are used commonly to assess the prognosis of liver disease but the disadvantage of these static tests is their inability to identify the functional reserve of the liver. Among all quantitative liver function tests indocyanine green (ICG) clearance test is most widely used and has been used to determine operative risk before hepatectomy and to assess prognosis of patients with cirrhosis. Aim: To correlate indocyanine green (ICG) clearance test with MELD score in patients with cirrhosis of liver. Methods: Forty patients with cirrhosis of liver were included and divided into two groups according to their CTP scores. Group A had 20 patients with CTP class A and group B had 20 patients with CTP class B. After ICG injection, ICG retention at 15 minutes (ICGR15) and ICG clearance rate were calculated. Results: In group A, the mean ICGR15 was 32.86% + 6.4% while in group B it was 51.08% + 12.8% (p <0.001). ICG clearance rates were 4.3% + 2.8% and 3.5% + 3.8% per minute in group A and B respectively. MELD score had a strong positive correlation with ICGR15 but a negative correlation with ICG clearance rate. On ROC curve analysis, AUC for MELD was 0.805 vs. 0.88 for ICGR15 in assessing prognosis of patients with cirrhosis. The sensitivity and specificity of MELD score was 60% and 80% respectively while that of ICGR15 was 85% and 90% respectively. Conclusion: ICGR15 has a higher sensitivity and specificity than MELD score in assessing the prognosis of patients with cirrhosis of liver.

HBV specific T-cell responses in hepatitis B.

Background: Cellular immune responses seem to prevail in acute hepatitis, whereas chronically infected patients demonstrate generally suppressed cellular immune responses and significantly greater antibody responses. Aim: To study hepatitis B virus (HBV) specific T cell proliferative responses in HBV related liver diseases. Methods: We analyzed the T lymphocyte proliferative responses to the nonspecific mitogen phytohemagglutinin (PHA) and the HBV specific hepatitis B core antigen (HBcAg) by calculating T cell proliferation index in 10 acute viral hepatitis (AVH) patients, 19 chronic hepatitis B (CHB) patients, 10 HBV cirrhotics, 10 inactive carriers and 10 healthy controls using MTT assay. Results: The mean proliferation index (PI) to PHA was highest in healthy controls (133.2 + 58.1) and lowest in cirrhotics (44.1 + 46.9) with all other groups falling in between. On comparing the mean T cell responses to HBcAg, AVH patients had the highest mean response (186.48 + 116.37) followed by CHB (137.9 + 134.3), inactive carriers (63.2 + 41.2) and cirrhotics (55.5 + 42.7). Conclusions: Patients with AVH had the highest T cell response to HBcAg, which probably explains the clearance of virus in these patients, in contrast to patients with cirrhosis who had the lowest T cell response.

Expansion of peripheral and intratumoral regulatory T-cells in hepatocellular carcinoma: A case-control study.

Background: Hepatocellular carcinoma (HCC) is notorious for poor prognosis with limited therapeutic options. A better understanding of the role of regulatory T-cells (Tregs) in HCC is important for design of immunotherapy based clinical protocol. The objective of the present study was to evaluate the presence of Tregs in tumor microenvironment in patients with HCC compared to chronic hepatitis (CH). Materials and Methods: The frequency of CD4 + CD25 + Treg cells was evaluated from peripheral blood (PB) of 28 patients of HCC and 30 controls including CH cases and healthy donors using flowcytometry. Intratumoral Treg were also analyzed in tissue samples from 17 HCC cases and 15 CH cases. In addition the expression of FOXP3 and CTLA-4 was also studied by RT-PCR. Results: Frequency of CD4 + CD25 + cells in the PBMCs of HCC cases was significantly higher than in HC (10.8 ± 7.64 vs 3.05 ± 1.30, P < 0.005) and CH patients (2.88 ± 1.92, P < 0.005). Also Treg population was significantly higher in HCC tumor microenvironment compared to CH biopsies (15.8 ± 5.32 vs 5.51 ± 3.40, P < 0.05). Expression of FOXP3 and CTLA-4 was also significantly higher in HCC patients ( P < 0.05) compared to CH group. Conclusions: We provide evidence of an increased population of Treg not only in the PB but also in tumor microenvironment of HCC patients, suggesting association of enhanced Treg activity with poor immune responses to tumor antigens. These findings may in future play a significant role in designing immunotherapeutic approaches in HCC.

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.

MDCT venography in the evaluation of inferior vena cava in Budd-Chiari syndrome.

Objective To assess the role of multidetector computed tomography (MDCT) venography in the evaluation of the inferior vena cava (IVC) in Budd-Chiari syndrome (BCS), its accuracy as compared to digital subtraction venography (DSV) and the potential of this technique to replace venography for the defi nitive diagnosis of BCS. Methods Twenty-fi ve suspected cases of BCS were prospectively enrolled in this study and underwent both MDCT venography and DSV. Two observers independently evaluated and graded both the axial and reformatted MDCT images for the presence, site, degree and length of IVC narrowing. The collateral pathways and the hepatic veins were also assessed in all cases. The degree of correlation between MDCT venography and DSV was expressed using Spearman’s rank correlation coeffi cient (Rs). Results There was excellent correlation between MDCT venography and DSV in predicting the presence of stenosis and in grading the degree and length of IVC stenosis (Rs=0.58, p<0.05). Four patients had presence of a web within the IVC and the reconstructed MDCT venography images detected the fl ap of the membrane in all of them. In three cases of complete obstruction the cranial extent of the obstruction could be determined on the reconstructed MDCT venography images, while double catheter access through the femoral and jugular routes was needed to determine the same on DSV. MDCT venography was signifi cantly more informative in depicting the presence and site of both intrahepatic and extrahepatic collaterals as compared to DSV. Conclusion MDCT venography, in the present study, accurately provided information of both conventional CT and IVCgraphy, in the evaluation of the IVC in a non-invasive way. It helped overcome the shortcomings of CT in the evaluation of IVC and was better than DSV for the evaluation of collaterals, calcifi cation and complete IVC obstruction. We suggest that CT venography can be used as a frontline investigation for the diagnosis of IVC obstruction and for planning surgery or percutaneous endovascular intervention.

Minimal hepatic encephalopathy: time to recognise and treat.

Minimal hepatic encephalopathy represents a part of the spectrum of hepatic encephalopathy and is the mildest form. While patients with hepatic encephalopathy have impaired intellectual functioning, personality changes, altered levels of consciousness, and neuromuscular dysfunction, patients with minimal hepatic encephalopathy have no recognisable clinical symptoms of hepatic encephalopathy but have mild cognitive and psychomotor deficits. The prevalence of minimal hepatic encephalopathy has been reported to vary between 30% and 84% in patients with liver cirrhosis and is higher in patients with poor liver function. The diagnosis is usually made by neuropsychological and/or neurophysiological testing in cirrhotic patients who are otherwise normal on neurological examination. Minimal hepatic encephalopathy is a clinically significant disorder that impairs the health-related quality of life, predicts the development of overt encephalopathy and is probably associated with a poor prognosis. Thus screening all patients with cirrhosis for minimal hepatic encephalopathy using psychometric testing is recommended. Pharmacologic therapy is recommended for patients diagnosed with minimal hepatic encephalopathy. The pathogenesis of minimal hepatic encephalopathy is considered similar to that of overt hepatic encephalopathy and ammonia plays a key role. Thus ammonia lowering agents such as lactulose, L-ornithine and L-aspartate that have good safety profiles are recommended. Future studies will better define the role of probiotics, levocarnitine and sodium benzoate.

Multidrug resistance in amoebiasis patients.

BACKGROUND & OBJECTIVES: Amoebiasis, caused by Entamoeba sp. a protozoan parasite, is a major public health problem in tropical and subtropical countries. The symptomatic patients are treated by specific chemotherapy. However, there are reports of treatment failure in some cases suggesting the possibility of drug resistance. The present study was therefore planned to assess the presence and expression of mRNA of multidrug resistance (MDR) gene in clinical isolates of Entamoeba histolytica and E. dispar. METHODS: Forty five clinical isolates of Entamoeba sp. [E. histolytica (15) and E. dispar (30)] were maintained in polyxenic followed by monoxenic medium. DNA and total RNA were extracted from clinical isolates of Entamoeba sp. and from sensitive strain of E. histolytica (HM1: IMSS) and subjected to polymerase chain reaction (PCR) and multiplex reverse transcription (RT)-PCR techniques. RESULTS: The 344 bp segment of E. histolytica DNA was seen by PCR using primers specific to EhPgp1 in all clinical isolates and sensitive strain of E. histolytica. Over expression of EhPgp1 was observed only in resistant mutant of E. histolytica; however, transcription of EhPgp1 was not seen in any clinical isolates and sensitive strain of E. histolytica. INTERPRETATION & CONCLUSION: The findings of the present study indicate that, so far, drug resistance in clinical isolates of E. histolytica does not seem to be a major problem in this country. However, susceptibility of clinical isolates of E. histolytica against various antiamoebic drugs needs to be investigated for better management.

Minimal hepatic encephalopathy.

Minimal hepatic encephalopathy (MHE) is the mildest form of spectrum of hepatic encephalopathy (HE). Patients with MHE have no recognizable clinical symptoms of HE but have mild cognitive and psychomotor defi cits. The prevalence of MHE is high in patients with cirrhosis of liver and varies between 30% and 84%; it is higher in patients with poor liver function. The diagnostic criteria for MHE have not been standardized but rest on careful patient history and physical examination, normal mental status examination, demonstration of abnormalities in cognition and/or neurophysiological function, and exclusion of concomitant neurological disorders. MHE is associated with impaired health-related quality of life, predicts the development of overt HE and is associated with poor survival. Hence, screening all patients with cirrhosis for MHE using psychometric tests, and treatment of those patients diagnosed to have MHE has been recommended. Ammonia plays a key role in the pathogenesis of MHE, which is thought to be similar to that of overt HE. Thus, ammonia-lowering agents such as lactulose and probiotics have been tried. These agents have been shown to improve cognitive and psychometric defi cits, and have good safety profile. Future studies will better defi ne the role of other drugs, such as rifaximin, acetyl L-carnitine and L-ornithine L-aspartate.

Poor responses to recombinant HBV vaccination in patients with HIV infection.

The study was conducted with an aim to assess the efficacy of recombinant HBV vaccination in untreated HBV seronegative HIV/AIDS subjects as compared to normal controls. The second objective was to identify differences in CD4 and CD8 T cell numbers/kinetics/functions and levels of TH2 cytokines (IL4 and IL10) in different groups during the three-dose vaccination regimen. 40 HIV/AIDS patients were subdivided into groups 1A where patients had a high CD4 (> 200/mm3) count and IB where patients had a low CD4 (< 200/mm3) count. Twenty normal healthy control subjects were also recruited in the study (group II). Patients received 40 micro and controls received 20 micro of recombinant HBV vaccine in each dose. All subjects received 3 doses of the vaccine. Detection of CD4 and CD8 cells was done by flowcytometry. TH2 type of cytokines IL4 and IL10 were estimated in the culture supernatant of PHA stimulated leukocyte rich plasma by sandwich ELISA. Anti-HBs levels were estimated in the serum by ELISA. Anti-HBs response was severely compromised in patients as compared to controls. Groups II, 1A and 1B showed titers of 16906 +/- 21303, 8834 +/- 14136 and 462 +/- 814 m/U/m/ respectively. Both CD4 and CD8 cells increased significantly after vaccination in all the groups irrespective of the disease status. On the other hand, IL4/IL10 responses to PHA stimulation in the HIV-positive groups were much lower than in controls (P< 0.1). Despite a double dose of vaccine in patients, the antibody response was significantly lower which correlated with a lower CD4 count. Cytokines IL4 and IL10 which regulate antibody response, were also lower in-patients and this together with a low CD4 count possibly accounted for the low anti-HBs levels. All patients with high CD4 lymphocyte count were responders while only 47% of patients with low CD4 lymphocyte count responded to immunization. Patients with a CD4 count of less than 50 failed to respond. Thus early immunization is advocated in all HIV patients at a stage when they are still capable of mounting an adequate immune response.

Unconjugated hyperbilirubinemia in nonalcoholic steatohepatitis--is it Gilbert's syndrome?

BACKGROUND: Patients with nonalcoholic steatohepatitis (NASH) have normal liver function tests except for raised transaminases until they have progressed to cirrhosis of liver. The objective of this study was to evaluate patients of NASH for the presence of hyperbilirubinemia at presentation. METHOD: Sixty-seven patients of NASH were studied for the presence of hyperbilirubinemia at presentation. All patients were worked up for the presence of cirrhosis and hemolytic work up and fasting test were done in those found with unconjugated hyperbilirubinemia. RESULTS: Five out of 67 patients (7.5%) of NASH were found to have unconjugated hyperbilirubinemia. Though the fasting test was not positive, they all had a negative hemolytic workup and none of them had underlying cirrhosis. Clinical characteristics of patients with unconjugated hyperbilirubinemia were similar to those with normal serum bilirubin levels. CONCLUSION: Unconjugated hyperbilirubinemia in patients with NASH may suggest an associated Gilbert's syndrome.

Comparative evaluation of hexokinase isoenzyme typing and antigen detection for differentiation of Entamoeba histolytica and Entamoeba dispar.

Entamoeba histolytica, the causative organism of invasive amebiasis is a potential pathogen, while asymptomatic infection is caused by E. dispar. Differentiation of the species is not possible on the basis of morphological characters by microscopic examination. In the present study an attempt has been made to differentiate E. histolytica from E. dispar in 45 isolates obtained from culture and direct stool samples respectively on the basis of hexokinase isoenzyme analysis and Tech Lab ELISA. A 100% correlation was found between these two techniques. However, Tech Lab E. histolytica antigen detection test was found to be both rapid and technically simple. Its use in diagnosis and epidemiological studies is recommended.