Subodh Varshney

OCCULT HEPATITIS B VIRUS INFECTION WITH LOW VIREMIA INDUCES DNA DAMAGE, APOPTOSIS AND OXIDATIVE STRESS IN PERIPHERAL BLOOD LYMPHOCYTES

Occult HBV infections (OHBI) are often associated with poor therapeutic response and increased risk of developing hepatocellular carcinoma. Despite a decade of research, OHBI still remains an intricate issue and much is yet to be defined about their possible immune implications. As HBV is known to infect peripheral blood lymphocytes, the present study aimed to explore the molecular mechanisms underlying DNA damage response triggered due to OHBI in host cells. The study was divided into three groups i.e. group A (OHBI patients n=30, viral load <or=100 IU/mL); group B (chronic HBV patients, n=30) and group C (controls, n=30). Peripheral blood lymphocytes were isolated and DNA damage response, apoptosis and oxidative stress were the studied parameters. A significant increase in the phosphorylation of DNA damage response proteins (ATM, ATR, H2AX and p53) in OHBI in comparison to controls suggested that OHBI induces DNA damage in peripheral blood lymphocytes and elicit a PI3 kinase mediated cellular response. In addition, increased DNA fragmentation, circulating nucleosome levels and mitochondrial membrane depolarization observed in OHBI group indicated that this damage might lead to cellular demise and immune hypo-responsiveness. Moreover, OHBI was also observed to be strongly associated with oxidative stress as suggested by the augmented levels of DCF fluorescence and depleted GR activity. Collectively, these results provide the basic knowledge about the genotoxic effects of OHBI in peripheral blood lymphocytes. Such studies may possibly open up new avenues for identifying novel therapeutic targets for viral hepatitis.

Occult hepatitis C virus elicits mitochondrial oxidative stress in lymphocytes and triggers PI3-kinase-mediated DNA damage response

Occult hepatitis C viral infection (OHCI) is a newly reported pathological entity associated with increased risk of developing hepatocellular carcinoma and lymphoproliferative disorders. Although hepatocytes are the primary sites of viral replication, hepatitis C virus is potentially lymphotropic, invading and propagating in cells of the immune system. Lymphocytes, the extrahepatic viral reservoirs, are differentially implicated in the occult and the active forms of the disease. This study aimed to elucidate the implications of mitochondrial oxidative stress on the immune pathophysiological mechanisms of OHCI. We herein report that OHCI induces mitochondrial oxidative stress, leading to DNA double-strand breaks and elicitation of a phosphoinositol 3-kinase-mediated cellular response in peripheral blood lymphocytes. Compared to controls, OHCI subjects showed higher accumulation of pATM, pATR, γH2AX, and p-p53, along with active recruitment of repair proteins (Mre11, Rad50, and Nbs1) and altered mitochondrial DNA content. Increased mitochondrial membrane depolarization and circulating nucleosome levels along with chromatid-type aberrations and decreased T-cell proliferative index observed in the OHCI group further indicated that this damage might lead to Bax-triggered mitochondria-mediated cellular apoptosis. Together our results provide the mechanistic underpinnings of mitochondrial dysfunction in OHCI, a previously unknown paradigm, for explaining the immune pathogenesis in a redox-dependent manner.

Circulating Biomarkers and their Possible Role in Pathogenesis of Chronic Hepatitis B and C Viral Infections

The present study evaluated the plausible role of circulating biomarkers in immune pathogenesis of chronic hepatitis considered a priority in clinical hepatology. Total viral load of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) patients was quantified and correlation studies were performed with circulating levels of Th1/Th2 cytokines; C reactive protein and circulating nucleosomes; glutathione reductase (GR) and superoxide dismutase. To our knowledge, the study is first among its kind that validates strong positive correlation of viral load with IL-4, IL-6, GR in HBV and IL-6, IL-10, GR in HCV infections. Although, multi-centric studies including large cohorts are required for translating our findings to clinical practice, however, role of these biomarkers with potential diagnostic or prognostic significance might be helpful in clinical assessment of high-risk individuals, thereby, designing interventional strategies, towards development of personalized medicare. The results of our study also offer valuable insights of immune signaling mediators engaged in development of hepatocellular carcinoma.

Prevalence of hepatitis C virus genotypes and impact of T helper cytokines in achieving sustained virological response during combination therapy: A study from Central India

Characterisation of host immune response to hepatitis C virus (HCV) genotypes may have an important prognostic and therapeutic implication. Genotype-3 was more prevalent in the examined cohort and demonstrated a significantly higher response to combination therapy than genotype-1. Sustained virological response (SVR) was 94.74% in genotype-3 and 45.45% in genotype-1. The patients who achieved SVR reported higher levels of circulating T helper 1 cytokines in comparison to subjects with no SVR in both the studied groups. Besides providing local prevalence, our study might also assist in understanding the host immune mechanisms involved to achieve SVR during combination therapy in chronic HCV patients.

Peri‐operative outcomes for pancreatoduodenectomy in India: a multi‐centric study

BACKGROUND There have been an increasing number of reports world-wide relating improved outcomes after pancreatic resections to high volumes thereby supporting the idea of centralization of pancreatic resectional surgery. To date there has been no collective attempt from India at addressing this issue. This cohort study analysed peri-operative outcomes after pancreatoduodenectomy (PD) at seven major Indian centres. MATERIALS AND METHODS Between January 2005 and December 2007, retrospective data on PDs, including intra-operative and post-operative factors, were obtained from seven major centres for pancreatic surgery in India. RESULTS Between January 2005 and December 2007, a total of 718 PDs were performed in India at the seven centres. The median number of PDs performed per year was 34 (range 9-54). The median number of PDs per surgeon per year was 16 (range 7-38). Ninety-four per cent of surgeries were performed for suspected malignancy in the pancreatic head and periampullary region. The median mortality rate per centre was four (range 2-5%). Wound infections were the commonest complication with a median incidence per centre of 18% (range 9.3-32.2%), and the median post-operative duration of hospital stay was 16 days (range 4-100 days). CONCLUSIONS This is the first multi-centric report of peri-operative outcomes of PD from India. The results from these specialist centers are very acceptable, and appear to support the thrust towards centralization.

Torsion of gall bladder, a rare entity: a case report and review article

IntroductionGallbladder torsion is a rare entity, which is often difficult to diagnose preoperatively. Since its first description in 1898 by Wendel, there have been over 500 documented cases in the literature. It is defined as rotation of the gallbladder on its mesentery along the axis of the cystic duct and cystic artery. Gallbladder torsion is more frequently encountered in the elderly with peak incidence in the 65-75 year old group, and a 3:1 female predominance. Gallbladder torsion typically presents as an acute abdomen requiring emergency surgery, and most cases are found as a surprise at surgery since preoperative diagnosis of gallbladder torsion is difficult. We report a case of acute gallbladder torsion in an elderly male and review the clinical aspect of gallbladder torsion.Case reportA 54-year old male presented to our department with a 5-day history of sudden onset colicky abdominal pain associated with vomiting, progressive abdominal distension and fever. Laparotomy through a chevron incision was performed and findings at operation included a gallbladder, which was necrotic and gangrenous, not attached to the liver by any mesentery. It was hanging by the attachments of cystic duct and cystic artery only, with a 360-degree clockwise torsion.ConclusionGallbladder torsion is rare surgical emergency which requires a high index of suspicion for early preoperative diagnosis and prompt intervention. Treatment consists of cholecystectomy with a prior detorsion to avoid injury to the common duct.

Molecular surveillance of hepatitis and tuberculosis infections in a cohort exposed to methyl isocyanate

ObjectiveThe potential toxic effects on the immune system exerted by occupational and accidental environmental exposures and underlying molecular regulatory mechanisms involved in the etiology and progression of infectious diseases are now being characterized. The Bhopal gas tragedy is undoubtedly one of the worst industrial disasters in the history of mankind. After 25 years of accidental exposure to methyl isocyanate (MIC), severe systemic ailments still continue to preoccupy the lives of the affected population that survived this tragedy. We have performed a molecular surveillance study to characterize hepatitis and tuberculosis infections amongst the first and the second generation of survivors exposed to MIC.Materials and MethodsBoth outdoor and indoor patients referred for molecular diagnosis of hepatitis B virus (HBV), hepatitis C virus (HCV) and Mycobacterium tuberculosis (MTB) were examined. Qualitative analysis for HBsAg, anti-HBc, anti-HCV through ELISA was performed, while BacT/ALERT and Ziehl-Neelson technique were utilized for the assessment of tuberculosis. Detection and quantification of viral and bacterial nucleic acid and characterization of hepatitis genotypes were analyzed using real-time and end-point PCR techniques.ResultsThe results suggest that HBV infections are most common among the MIC-exposed cohort, followed by extra-pulmonary and pulmonary MTB and HCV infections. Genotype 3 is the most prevalent HCV genotype among the survivors. Failure to detect HBsAg, anti-HBc and anti-HCV through ELISA, and tuberculosis by culture and Ziehl-Neelson stain, indicates higher prevalence of occult hepatitis and latent tuberculosis in the affected population.ConclusionsOur study underscores the importance of hospital-based records used as a data source for monitoring possible environmental health hazards. As the risk of progress of infection is often influenced by conditions and periods of environmental chemical exposure, therefore, insights of interconnected molecular pathways will further illuminate the gene-environment association and might offer valuable information for rational drug design.

Assessment of tumor antigen-loaded solid lipid nanoparticles as an efficient delivery system for dendritic cell engineering

AIM The work attempts to overcome tumor-associated immune tolerance using a surface-modified solid lipid nanoparticle (SLNP) delivery system for dendritic cell (DC) immunotherapy. MATERIALS & METHODS Different formulations of SLNPs (SLNPs-alone, cationic SLNPs and mannosylated SLNPs) were prepared using tumor cell lysates. Prepared nanoparticles were characterized and their ability to activate DCs to induce a tumor cell-specific response was assessed. RESULTS SLNPs induced a strong phagocytic signal to DCs without any significant toxicity. Comparatively, mannosylated SLNPs evoked an optimum and effective cell-mediated immune response with no significant toxicity. CONCLUSION Surface-modified SLNPs may play a pivotal role in designing a clinically translatable DC-based immunotherapy for gastrointestinal malignancies. This novel approach may also facilitate the treatment of residual disease, following standard therapy.

Cefoperazone-Sulbactam for Treatment of Intra-Abdominal Infections: Results from a Randomized, Parallel Group Study in India

BACKGROUND Combinations of a third-generation cephalosporin and metronidazole, with or without an aminoglycoside, often are used for the treatment of intra-abdominal infections in surgical settings. Simpler regimens that preserve an adequate spectrum of coverage, but allow easier administration and have fewer side effects, may be a more desirable option. METHODS This randomized, open-label, active comparator study evaluated the effectiveness (non-inferiority hypothesis) of the beta-lactam/beta-lactamase inhibitor combination cefoperazone-sulbactam (2-8 g/day), compared with ceftazidime (2-6 g/day)-amikacin (15 mg/kg/day)-metronidazole (500 mg three times daily) in 154 and 152 subjects, respectively, having intra-abdominal infections. The study was conducted at 17 centers in India. RESULTS Non-inferiority of cefoperazone-sulbactam (91.9%) compared with ceftazidime-amikacin-metronidazole (81.8%) was demonstrated for continued resolution of clinical signs and symptoms at the 30-day follow-up (primary endpoint) with a treatment difference of 10.1% (95% confidence interval 2.1%, 18.1%; pre-defined non-inferiority limit > -12.5%). Superiority of cefoperazone-sulbactam also was demonstrated for this endpoint, with significantly more subjects achieving continued resolution at the 30-day follow-up than in the comparator group (p = 0.015). On microbiologic outcomes, cefoperazone-sulbactam had higher success rates than ceftazidime-amikacin-metronidazole (92.9% vs. 80.0%). The pathogens (202 isolated) isolated most commonly were Escherichia coli (38.6%) and Klebsiella spp. (12.9%). The incidence of treatment-related adverse events was 6.5% and 16.4% in the cefoperazone-sulbactam and ceftazidime-amikacin-metronidazole groups, respectively, with more discontinuations due to treatment-related adverse events in the comparator arm (3.2% vs. 9.9%). CONCLUSION Empirical cefoperazone-sulbactam monotherapy could be a useful adjunct to surgical intervention for intra-abdominal infections.

Diagnosis of gastrointestinal tuberculosis: Using cytomorphological, microbiological, immunological and molecular techniques — A study from Central India

The present study included three groups: (A) age and gender matched control (n=24) with no previous signs of M. tuberculosis complex (MTBC) infection, (B) patients (n=28) diagnosed with gastro-intestinal TB (GITB), (C) patients (n=50) with clinical and histo-pathological signs of GITB, but were culture and AFB negative. Real time assay performed using fluorescence resonance energy transfer hybridization probes showed a positivity index of 36 % in group C, i.e. 18 were found reactive from the total 50 cases studied. In addition, immune characterization of these 18 cases showed depleted CD4+ count and increased levels of IFN-γ and TNF-α cytokines. No positive case was found in group A, while in group B, out of total 28 cases studied 27 were found positive. A combinatorial diagnostic approach for rapid detection and characterization of GITB might provide specific therapeutic strategies for prevention and treatment of the infection in future.