Sanjeev Kumar Sharma

Hepatitis B Virus: Inactive carriers

Inactive carriers forms the largest group in chronic HBV infected patients. Around 300 million people are inactive carriers The inactive HBsAg carrier state is diagnosed by absence of HBeAg and presence of anti-HBe, undetectable or low levels of HBV DNA in PCR-based assays, repeatedly normal ALT levels, and minimal or no necroinflammation, slight fibrosis, or even normal histology on biopsy. Inactive cirrhosis may be present in patients who had active liver disease during the replicative phase of infection. The prognosis of the inactive HBsAg carrier state is usually benign. Long-term follow- up (up to 18 years) of these carriers has indicated that the vast majority show sustained biochemical remission and very low risk of cirrhosis or hepatocellular carcinoma (HCC). Rarely, patients, even noncirrhotics, may develop liver cancer during the inactive HBsAg carrier state. In addition, approximately 20 to 30% of persons in the inactive HBsAg carrier state may undergo spontaneous reactivation of hepatitis B during follow-up. Multiple episodes of reactivation or sustained reactivation can cause progressive hepatic damage and even hepatic decompensation. Introduction

Serotonin transporter promoter variant: Analysis in Indian IBS patients and control population.

Background Studies of serotonin reuptake transporter (SERT-P) polymorphism and irritable bowel syndrome (IBS) have shown diverse results among different populations, which might be due to racial and ethnic difference. Aim This study was to investigate the potential association between the SERT-P polymorphism and clinical subtypes of IBS patients in the Indian population. Method This prospective case-control study included 151 IBS patients. Ninety-two patients were diarrhea-predominant IBS, 44 were constipation-predominant IBS (C-IBS), 15 were alternating diarrhea and constipation IBS, and 100 were healthy controls. SERT gene polymorphism was studied by polymerase chain reaction. Result A genotypic association was observed between SS genotype of SERT-P polymorphism and C-IBS (P<0.05). When the L/S and L/L genotypes were combined into one group, the frequency of the S/S genotype was significantly higher than that of the non-S/S genotype between C-IBS and the control group (P<0.05). There was no significant difference in the SERT-P genotype and allele frequency between c-ibs, alternating diarrhea and constipation IBS, all types of IBS cases, and controls. Conclusions A significant association was observed between the SS genotype of SERT-P polymorphism and C-IBS in the Indian population.

Clinical Significance of Genotypes and Precore/Basal Core Promoter Mutations in HBV Related Chronic Liver Disease Patients in North India

PurposeData on genotypes, basal core promoter (BCP) and precore mutants of hepatitis B virus and their association with different HBV related liver disease have been studied inadequately and are controversial. Thus, the aim of this study was to determine the incidence of BCP and precore HBV mutants and their relationship with HBV genotype and different stages of HBV related liver disease in North Indian patients.MethodsA total 273 patients with different stages of HBV related liver diseases were enrolled. Nested polymerase chain reaction (PCR) was used to amplify the BCP/PC regions. RFLP and direct sequencing were performed to validate the mutations identified in these regions. HBV genotyping was accomplished by multiplex PCR.ResultsGenotype D was the predominant genotype found in each of the various HBV related liver diseases. The BCP mutation was found significantly more often in inactive carriers and compensated cirrhosis as compared to the other groups. The BCP mutation was present in 29.1% of patients with genotype D versus 17.1% with genotype A (Pxa0=xa00.001). The precore mutation was also more frequently observed with genotype D compared with genotype A (36.9 vs. 4.8%, Pxa0=xa00.0007).ConclusionGenotype D is predominant in North Indian patients. The BCP and precore mutations occur in one-third of HBV positive patients in association with the genotype D. We did not find any correlation with severity of liver disease with genotypes and mutations.

Association of alpha 2A adrenergic receptor gene (ADRΑ2A) polymorphism with irritable bowel syndrome, microscopic and ulcerative colitis.

BACKGROUNDnAlpha 2 adrenergic receptors (alpha2 ARs) play a central role in the regulation of systemic sympathetic activity. Prejunctional alpha 2A adrenoceptor regulates through negative feedback at presynaptic nerve ending. A-1291 C>G polymorphism located in alpha2-adrenergic receptor gene (ADRAlpha2A) has been identified. We investigated the possible association between 1291 C>G polymorphism in the promoter region of ADRAlpha2A in clinical subtypes of IBS, ulcerative and microscopic colitis patients.nnnMETHODSnThis prospective case control study included 92 patients with diarrhea predominant IBS (D-IBS), 44 with constipation predominant IBS (C-IBS), 15 with alternating diarrhea and constipation IBS (M-IBS), 75 ulcerative colitis (UC), 41 microscopic colitis (MC) and 100 healthy controls. The subjects were genotyped by using PCR amplification of the promoter region of ADRAlpha2A gene followed by digestion with the restriction enzyme MspI. The study was approved by the institute ethical committee.nnnRESULTSnA strong genotypic association was observed between alpha2A-1291 C>G polymorphism and D-IBS (chi2=6.38, df=2, p<0.05). There was no significant difference in alpha2A-1291 C>G genotype and allele frequency between C-IBS, M-IBS, UC, MC cases and control subjects.nnnCONCLUSIONSnA significant association was observed between alpha2A-1291C>G polymorphism and D-IBS. Thus, alpha2 AR gene may be a potential candidate involved in the pathophysiology of D-IBS.

Cytosolic phospholipase A2 (cPLA2) IVA as a potential signature molecule in cigarette smoke condensate induced pathologies in alveolar epithelial lineages

BackgroundSmoking is one of the leading causes of millions of deaths worldwide. During cigarette smoking, most affected and highly exposed cells are the alveolar epithelium and generated oxidative stress in these cells leads to death and damage. Several studies suggested that oxidative stress causes membrane remodeling via Phospholipase A2s but in the case of cigarette smokers, mechanistically study is not yet fully defined. In view of present perspective, we evaluated the involvement of cytosolic phospholipase A2 (cPLA2) IVA as therapeutic target in cigarette smoke induced pathologies in transformed type I and type II alveolar epithelial cells.MethodsTransformed type I (WI26) and type II (A549) alveolar epithelial cells were used for the present study. Cigarette smoke condensate (CSC) was prepared from most commonly used cigarette (Gold Flake with filter) by the Indian population. CSC-induced molecular changes were evaluated through cell viability using MTT assay, reactive oxygen species (ROS) measurement using 2,7 dichlorodihydrofluorescin diacetate (DCFH-DA), cell membrane integrity using fluorescein diacetate (FDA) and ethidium bromide (EtBr) staining, super oxide dismutase (SOD) levels, cPLA2 activity and molecular involvement of specific cPLA2s at selected 24xa0h time period.ResultsCSC-induced response on both type of epithelial cells shown significantly reduction in cell viability, declined membrane integrity, with differential escalation of ROS levels in the range of 1.5–15 folds and pointedly increased cPLA2 activity (pu2009<u20090.05). Likewise, we observed distinction antioxidant potential in these two types of lineages as type I cells had considerably higher SOD levels when compared to type II cells (pu2009<u20090.05). Further molecular expression of all cPLA2s increased significantly in a dose dependent manner, specifically cytosolic phospholipase A2 IVA with maximum manifestation of 3.8 folds. Interestingly, CSC-induced ROS levels and cPLA2s expression were relatively higher in A549 cells as compared to WI26 cells.ConclusionsThe present study indicates that among all cPLA2s, specific cPLA2 IVA are the main enzymes involved in cigarette smoke induced anomalies in type I and type II lung epithelial cells and targeting them holds tremendous possibilities in cigarette smoke induced lung pathologies.

Therapeutic potential of arachidonyl trifluromethyl ketone, a cytosolic phospholipaseA2 IVA specific inhibitor, in cigarette smoke condensate-induced pathological conditions in alveolar type I & II epithelial cells

Cigarette smoke is responsible for multiple disorders and causes almost 10 million annual deaths globally but underlying mechanisms are still underexplored. Continuous exposure of Cigarette smoke condensate (CSC) leads to cytosolic phospholipase A2 (cPLA2) mediated high free radicals where cPLA2s seems to play crucial role in generated various patho-physiological conditions such as chronic inflammation, oxidative stress and cancer. In this view, we assessed the therapeutic potential of arachidonyl trifluromethyl ketone (ATK), a cPLA2 inhibitor, via pharmacological inhibition of most expressible CSC-induced cPLA2 group IVA in type-I and type-II alveolar epithelial cells. The In Vitro inhibitory effect of ATK on CSC-induced PLA2 activity and its cellular role were assessed in terms of cell viability, fluorescein diacetate (FDA) dye uptake assay for membrane integrity, reactive oxygen species (ROS)/reactive nitrogen species (RNS) levels and pro apoptotic as well as anti apoptosis markers via flow cytometry, along with extracellular signal-regulated kinases (ERK) levels using enzyme-linked immunosorbent assay (ELISA). The experimental findings demonstrated that ATK acts as potent inhibitor of cPLA2 activity and shown its effectiveness as therapeutic agent by significantly mimicking CSC-induced levels of free radicals, primary apoptosis, ratio of pro-apoptotic/apoptotic proteins and levels of ERK whereas protected cells from loss of cell viability and membrane integrity. Thus, this study is an important step towards the opening up of avenues for the applicability of the cPLA2 isoform specific inhibitors such as ATK for pre-clinical and clinical studies and could be beneficial during smoking-induced lung pathological conditions.

Abstract 3347: Establishment of four novel HPV-16 positive cell lines of invasive cervical carcinoma of Indian origin and characterization of CD133 positive cancer stem cells

Background and Aim: Cancer of the cervix is the 2nd most common cancer in Indian women. Cell lines are essential in vitro models in cancer research. Till date, available cell lines in cervical cancer were highly passaged, developed from Caucasian, Black or Asian origin; hence, establishment of a low-passage cervical cancer derived cell line was the first aim of this study. Cancer stem cells (CSCs) are proposed to be responsible for metastasis and therapy resistance. Previously, we reported CD133 to be a putative marker for CSC, and they were further characterized. Materials and Methods: 25 women were recruited after informed consent and approval from the Institute Ethics committee. A small portion of the biopsy was collected in DMEM/F12 media and subjected to primary culture. Successful propagation was possible in 4 cases. Their morphology, epithelial nature and HPV status were evaluated. Variable Number Tandem Repeat (VNTR) assay, cell cycle and kinetic studies were performed. Tissue expression of CD133 and CD49f stem cell markers was evaluated by direct immunofluorescence on frozen tumor samples. Adherent cells were sorted into CD133+ vs CD133- tumor bulk from all 4 cell lines by FACS. These two populations were evaluated for differences in tumorosphere formation, chemosensitivity to cisplatinum, expression of stemness (SOX2, OCT4, NANOG) and EMT (SNAIL, SLUG, TWIST, VIMENTIN and E-CADHERIN) markers at the transcript level. Observations: Four cell lines designated RSBS-9, RSBS-14, RSBS-23 derived from non-keratinizing squamous cell carcinoma and RSBS-43 derived from adenocarcinoma cervix were established and passaged up to 50 times. The epithelial nature of cell lines was confirmed by cytokeratin and epithelial membrane antigen positivity. VNTR assay confirmed derivation of cell lines from respective parental tissue sample. All 4 cell lines were HPV-16positive. The cell doubling time was approximately 48 hours. Immunofluorescence on tissue samples revealed diffuse positivity for CD49f indicating that it may not represent a CSC marker as previously reported whereas CD133 staining showed scattered positive cells. No particular location of CD133+ CSC was observed. Comparison of CD133+ with CD133- bulk population cells revealed increased tumorosphere formation; however, no significant difference in the chemosensitivity to cisplatinum was seen. Real time quantitation of transcripts of stemness and EMT markers revealed CD133+ cells showing upregulation of EMT markers VIMENTIN, SNAIL, SLUG in the RSBS-23 cell line and of VIMENTIN and TWIST in RSBS-9 cell line whereas there was no significant difference in the RSBS-14 and RSBS-43 cell lines for any of the markers studied. Conclusion: Low passage cell lines are useful model systems for understanding the biology of cervical cancer. CD133+ Cancer Stem cells exhibit some EMT markers in 2 of the 4 cell lines established. Citation Format: Shifa Javed, Bal Krishan Sharma, Sanjeev Kumar Sharma, Swati Sood, Rashmi Bagga, Shalmoli Bhattacharyya, Radhika Srinivasan. Establishment of four novel HPV-16 positive cell lines of invasive cervical carcinoma of Indian origin and characterization of CD133 positive cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3347.