Aejaz Habeeb

Comparing Genomes of Helicobacter pylori Strains from the High-Altitude Desert of Ladakh, India

ABSTRACT The genomic diversity of Helicobacter pylori from the vast Indian subcontinent is largely unknown. We compared the genomes of 10 H. pylori strains from Ladakh, North India. Molecular analysis was carried out to identify rearrangements within and outside the cag pathogenicity island (cag PAI) and DNA sequence divergence in candidate genes. Analyses of virulence genes (such as the cag PAI as a whole, cagA, vacA, iceA, oipA, babB, and the plasticity cluster) revealed that H. pylori strains from Ladakh are genetically distinct and possibly less virulent than the isolates from East Asian countries, such as China and Japan. Phylogenetic analyses based on the cagA-glr motifs, enterobacterial repetitive intergenic consensus patterns, repetitive extragenic palindromic signatures, the glmM gene mutations, and several genomic markers representing fluorescent amplified fragment length polymorphisms revealed that Ladakhi strains share features of the Indo-European, as well as the East Asian, gene pools. However, the contribution of genetic features from the Indo-European gene pool was more prominent.

Polymerase chain reaction based analysis of the cytotoxin associated gene pathogenicity island of Helicobacter pylori from saliva: an approach for rapid molecular genotyping in relation to disease status.

Background and Aims: The genetic composition of the intricate cytotoxin associated gene pathogenicity island (cag PAI) of Helicobacter pylori is known to significantly influence the outcome of the disease. Hence, analysis of complete cag PAI of H. pylori isolated from saliva would be of immense importance in standardizing saliva as a reliable non‐invasive diagnostic specimen and also to evaluate the type of H. pylori infection. The aim of the present study was to analyze the genes of cag PAI of H. pylori for their presence and correlating them with the disease status of the patients.

Macrophage migration inhibitory factor, Toll-like receptor 4, and CD14 polymorphisms with altered expression levels in patients with ulcerative colitis

Ulcerative colitis is a multifactorial disease in which genetic factors play a major role. Functional mutations in the genes related to innate immune response exacerbate mucosal damage coupled with persistent inflammation. The cytokine macrophage migration inhibitory factor (MIF), CD14, and Toll-like receptor 4 (TLR4) are the central players with clearly defined roles in inflammation. The aim of this study was to investigate the association between MIF-173G > C, CD14-159C > T, and TLR4-299A > G polymorphisms and mononuclear cell expression in patients with ulcerative colitis (UC). Genotyping of MIF-173G > C, CD14-159C > T, and TLR4-299A > G polymorphisms was performed by amplification refractory mutation system-polymerase chain reaction and allele-specific amplification in 139 and 176 patients with UC and controls, respectively. Simultaneously, the expression levels of intracellular MIF, mCD14, and mTLR4 were determined in mononuclear cells using a flow cytometer. Polymorphisms in CD14-159C > T and TLR4-299A > G significantly affected mCD14 and mTLR4 expression levels and also increased susceptibility to UC. Although intracellular MIF expression levels differed among patient and control groups, the polymorphism in MIF 173G > C was not observed to be associated with a risk of UC.

A simple multiplex PCR assay for diagnosing virulent Helicobacter pylori infection in human gastric biopsy specimens from subjects with gastric carcinoma and other gastro-duodenal diseases

Aim:  To evaluate and develop a multiplex polymerase chain reaction (PCR) assay for diagnosing and specific identification of virulent Helicobacter pylori strains and their main virulence genes cagA, cagE, cagT, vacA and hrgA.

Hepatic stem cells: A viable approach for the treatment of liver cirrhosis

Liver cirrhosis is characterized by distortion of liver architecture, necrosis of hepatocytes and regenerative nodules formation leading to cirrhosis. Various types of cell sources have been used for the management and treatment of decompensated liver cirrhosis. Knowledge of stem cells has offered a new dimension for regenerative therapy and has been considered as one of the potential adjuvant treatment modality in patients with end stage liver diseases (ESLD). Human fetal hepatic progenitor cells are less immunogenic than adult ones. They are highly propagative and challenging to cryopreservation. In our earlier studies we have demonstrated that fetuses at 10-18 wk of gestation age contain a large number of actively dividing hepatic stem and progenitor cells which possess bi-potent nature having potential to differentiate into bile duct cells and mature hepatocytes. Hepatic stem cell therapy for the treatment of ESLD is in their early stage of the translation. The emerging technology of decellularization and recellularization might offer a significant platform for developing bioengineered personalized livers to come over the scarcity of desired number of donor organs for the treatment of ESLD. Despite these significant advancements long-term tracking of stem cells in human is the most important subject nowadays in order to answer several unsettles issues regarding the route of delivery, the choice of stem cell type(s), the cell number and the time-point of cell delivery for the treatment in a chronic setting. Answering to these questions will further contribute to the development of safer, noninvasive, and repeatable imaging modalities that could discover better cell therapeutic approaches from bench to bed-side. Combinatorial approach of decellularization and nanotechnology could pave a way towards the better understanding in determination of cell fate post-transplantation.

Treatment of Chronic Hepatitis due to Hepatitis C Virus (CH-C) in India: A Randomized Controlled Trial Comparing Daily Interferon-alfa-2b and Ribavirin with Daily Interferon-alfa-2b and Glycyrrhizin—A Multicenter Study

BACKGROUND AND AIM Pegylated-interferon-alfa (PEG-IFN-α) with ribavirin is an established treatment in chronic hepatitis due to hepatitis C virus (HCV) (CH-C). Such treatment is expensive and in resource-poor countries such as India, alternative less expensive therapy is needed. METHODS Multicenter randomized controlled trial comparing two treatment regimens (interferon-alfa-2b [IFN-α-2b] 3 million unit/day [MU/day] and ribavirin 1000 mg/day [I+R] vs IFN-α-2b 3 MU/day and glycyrrhizin 250 mg [I+G]) in CH-C. Viral, host characteristics and therapeutic responses were assessed (ICMR-6 months trial for chronic hepatitis-CTRI/2008/091/000105). RESULTS One hundred and thirty-one patients meeting the inclusion criteria were randomized to I + G (n=64) or I+R (n=67) during the period February 2002 to May 2005. About 85% (I+G=53, I+R=58) completed 6 months of treatment and 89% of them (I+G=46, I+R=53) completed 6 months of follow-up after completion of treatment. Hepatitis C virus genotype 3 was the major type detected (71% patients). The mean log10 viral load (copies/mL), histological activity index, and fibrosis stage for all patients were 5.1 ± 0.98, 5 ± 2, and 2± 1.5, respectively. Sustained viral response (SVR) was significantly higher in I + R group than in I + G group (65.7% vs 46.9%, OR=2.2, P = 0.03). Treatment with I + G was associated with significantly lower frequencies of leukopenia (2% vs 17%, P <0.01) and anemia (8% vs 40%, P <0.001) as compared to treatment with I + R. CONCLUSION Genotype 3 HCV infection with low viral load is prevalent in India. Daily IFN with ribavirin showed significantly better responses. Leukopenia and anemia were significantly more in ribavirin group. Responses observed with IFN + ribavirin were similar to the reported response rates with PEG-IFN suggesting that this modality may be considered as a cheaper alternative of treatment for chronic hepatitis C.

Identification of rare hepatitis C virus genotype 5a among Indian population

In Indian population, hepatitis C virus (HCV) genotypes 1 and 3 are prevalent and predominant with the highest frequency. However, other genotypes are seldom reported, and among them the HCV genotype 5a is exceptionally rare. The presented case had no history for either blood transfusion or using any type of IV drugs and never traveled to any other country. He was serologically positive with HCV antibodies and HCV RNA. 5′UTR-specific amplification and sequencing of infected viral genome confirmed that he had been infected with HCV genotype 5a which is not closely related to other common prevalent genotypes like 1a, 1b, 3a, and 3b in India. Patient’s wife and children tested negative for anti-HCV and HCV-RNA. This unique case report could be attributed to circulation of HCV genotype 5a from other geographic area at very low frequency in India as determined by phylogenetic analysis and nucleic acid-sequencing methods.

Bioengineered humanized livers as better three-dimensional drug testing model system

AIM To develop appropriate humanized three-dimensional ex-vivo model system for drug testing. METHODS Bioengineered humanized livers were developed in this study using human hepatic stem cells repopulation within the acellularized liver scaffolds which mimics with the natural organ anatomy and physiology. Six cytochrome P-450 probes were used to enable efficient identification of drug metabolism in bioengineered humanized livers. The drug metabolism study in bioengineered livers was evaluated to identify the absorption, distribution, metabolism, excretion and toxicity responses. RESULTS The bioengineered humanized livers showed cellular and molecular characteristics of human livers. The bioengineered liver showed three-dimensional natural architecture with intact vasculature and extra-cellular matrix. Human hepatic cells were engrafted similar to the human liver. Drug metabolism studies provided a suitable platform alternative to available ex-vivo and in vivo models for identifying cellular and molecular dynamics of pharmacological drugs. CONCLUSION The present study paves a way towards the development of suitable humanized preclinical model systems for pharmacological testing. This approach may reduce the cost and time duration of preclinical drug testing and further overcomes on the anatomical and physiological variations in xenogeneic systems.

Transplantation of Epcam+Ve Human Hepatic Stem Cells in Liver Cirrhosis Patient and Cellular Immune Response Running Title: Transplantation and Cellular Immune Response

Liver transplant is considered is the only treatment for liver cirrhosis. Despite the success of organ transplantation, the treatment requires lifelong immunosuppression and major limitation is the availability of donor liver. Hepatic progenitor cell transplantation (HSCT) offers novel but challenging alternatives therapy to liver transplantation for management of liver cirrhosis. In this study, we investigated the cellular immune response by monitoring T-cell, NK-cell and cytokines which play major role in cellular rejection. A total of 5 patients with decompensated liver cirrhosis were enrolled in the study. T-cell (CD3, CD4 and CD8), NK-cells (CD16) was monitored before (Day-1) and after transplantation (Day 1, 7, 15, 30) of human fetal liver-derived EPCAM+Ve cell by flowcytometry. Before and after transplantation, Cytokinelevels (IL2, TNFβ, IFNα, IFNγ and INFβ) were also measured by ELISA. Study has demonstrated that after HSCT patient showed marked clinical recovery and decline in the MELD score and there was no significant increase found in cell mediated response and cytokine levels between pre and post transplantation. Hence this preliminary study demonstrated human fetal liver- derived EPCAM+Ve stem cell transplantation is safety for end stage liver cirrhosis.