Occurrence of virulence genes, 16S rRNA methylases, and plasmid-mediated quinolone resistance genes in CTX-M-producing Escherichia coli from Pakistan.

The aim of the study was to conduct a comprehensive molecular characterization of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli collected from Pakistan. Genetic relatedness among 98 ESBL-producing E. coli was measured by pulsed-field gel electrophoresis (PFGE). The presence of genes encoding ESBLs, virulence factors (VFs), 16S rRNA methylases, plasmid-mediated quinolone resistance (PMQR) encoding elements, plasmid replicon types, phylogenetic groups of E. coli, prevalence of the worldwide disseminated clone E. coli ST131, and phylogrouping of CTX-M enzymes was investigated by polymerase chain reaction (PCR). All isolates carried bla CTX-M genes and, except for one isolate from CTX-M phylogroup 9, they all belonged to CTX-M phylogroup 1. The isolates were genetically diverse with PFGE. Phylogenetic group D (36 %) was most abundant in this collection of E. coli, whereas isolates belonging to B2 (22 %) had the highest content of virulence genes. PMQR genes were found in 84.6 % of the isolates; among them, 93 % isolates were positive for variants of acetyltransferases (aac(6')-lb-cr), whereas qnrB, qepA, and qnrS were present in 11 %, 5 %, and 4 % of the isolates, respectively. Only 3 % of the isolates contained genes encoding 16S rRNA methylases. The most abundant replicon type was IncF (96 %), and 18 % of the isolates belonged to the ST131 clone. Out of 34 investigated VFs, 24 genes encoding different types of adhesins, protectins, toxins, siderophores, and other VFs were found. Although the isolates in this collection were highly resistant to many antimicrobials, susceptibility to amikacin and meropenem was retained.

Pluripotent stem cells - a review of the current status in neural regeneration.

Pharmacological or neurosurgical therapies currently in practice to treat the damage in various neurodegenerative disorders are not efficient in preventing progression or cure of these progressive neurodegenerative processes. Recently, a new approach, cell therapy using stem cell, is being evaluated. However, the use of this therapy in the treatment of these neurological diseases is highly restricted, mainly owing to several technical difficulties and limitations. The strategy of isolation and characterization of neural stem cells from various sources will probably provide a major impetus and open up an interesting, novel therapeutic modality for several neurodegenerative disorders. The high regenerative potential of damaged neural tissues suggests that various embryonic/adult sources serve as a proxy for neural stem cells for cell-based therapy.

An update on hepatic stem cells: bench to bedside.

Liver failure results in impairment of many functions and dependent organs such as brain and kidneys begin to fail, reducing the chance of recovery even further. Orthotopic liver transplantation (OLTx) is the only treatment that improves the survival rate in patients with liver failure. Liver Transplantation (LT), including orthologous liver transplantation (OLT), cadaveric LT, split LT, living donor LT (LDLT) brings hopes to patients with these diseases. Globally, 1.4 million deaths occur annually as a result of chronic liver diseases. The reasons for this high death toll include unavailability of healthy liver donor and highly expensive liver transplantation treatment. Furthermore, some other factors such as operative risks and post-transplant rejection are major limitation of OLT. Isolated hepatocyte transplantation is emerging as alternative bridge support till the healthy donor is arranged. Mature hepatocytes have several drawbacks such as low proliferation both in vitro and in vivo, low viability after cryopreservation, and requirement of large number of cells for infusion. The studies on isolation of hepatic progenitors have shown promising results to overcome these limitations. These cells possess higher proliferative capacity, are less immunogenic and more resistant to cryopreservation, and ischemic injury; properties that could enhance their engraftment within the recipient liver. The hepatic progenitors have been isolated from the intra-hepatic sources and extra-hepatic sources. Fetal cells are one of the ideal sources of hepatic stem/progenitor cells. Autologous bone marrow stem cell transplantation in patients with cirrhosis has shown promising result.

In vitro insulin production and analysis of pancreatic transcription factors in induced human hepatic progenitor cells.

BACKGROUND: beta-Cell destruction and/or insufficient insulin production are the hallmarks of diabetes mellitus (type 1 diabetes). A hepatic progenitor from developing liver is sought to be one of the surrogate sources of insulin production as the pancreas and the liver share a common precursor and signals from the cardiac mesoderm. Production of insulin is possible by transfecting pancreatic transcription factors that play important roles in development of the pancreatic beta-cell. But, there is always the fear of using genetically manipulated cells for therapeutics. Hence, the present study was designed to analyze the feasibility of using primary human fetal hepatic progenitors as a potential source for insulin production. METHODS: Human fetal hepatic progenitors were enriched using CD-326 magnetic cell sorting. The sorted cells were cultured with different concentrations of glucose (5-30 mM) in Dulbecco's modified Eagle's medium. The amount of insulin production was estimated in the cultured cells by the chemiluminescence method. Total RNA isolated from sorted epithelial cell adhesion molecule (EpCAM)-positive cells was reverse-transcribed, and the expression of different beta-cell-producing transcriptions factors was analyzed by polymerase chain reaction (PCR). Immunocytochemical analysis was performed in cultured cells using specific insulin antibodies. RESULTS: The viability of the total liver cells isolated was found to be 95%. The average number of EpCAM-positive cells in the total liver was found to be approximately 15%. An insulin kinetics study using glucose induction with different concentrations showed increased insulin secretion in response to glucose concentrations up to 20 mM. Furthermore, results of immunocytochemical analysis demonstrated intense insulin expression in EpCAM-positive cultured cells. Expression studies of the cultured EpCAM-positive cells using reverse transcription-PCR showed positive expression of the pancreatic transcription factors essential for insulin production. CONCLUSIONS: The present study demonstrates that in vitro differentiation of induced human hepatic progenitors into insulin-producing cells without genetic manipulations may promote strategies for the treatment of type 1 diabetes.

Simplified and versatile method for bisulfite-based DNA methylation analysis of small amounts of DNA.

Epigenetic alterations of gene function play a central role in the pathogenesis of many tumors and in the process of aging. Abnormal methylation at transcriptional sites of genes results in epigenetic silencing of the genes that protect against tumor formation or that repair DNA. To date, several studies have analyzed methylation status by oligonucleotide arrays, restriction analysis (COBRA), methylation-specific amplification, and sequence analysis. This requires high concentration of bisulfite-treated DNA, which mandates use of commercially available expensive kits, and is an often laborious and time-consuming task. In this article, we report a simplified high-throughput method, which can serve as a surrogate for screening methylation profiles of various genes and has high sensitivity compared with the other methods described previously.

Prognostic significance of genotyping Helicobacter pylori infection in patients in younger age groups with gastric cancer.

BACKGROUND: Malignant tumours of the stomach are common, but the incidence of stomach cancer varies from country to country, probably a result of genetic, epigenetic and environmental factors. Stomach cancer often occurs in older people whose stomachs produce only small quantities of acid. Although infection with Helicobacter pylori has been proven beyond doubt in the aetiopathogenesis of various gastric disorders, not much is known about the genotypes of H pylori infection in early-onset gastric cancer. AIM: To ascertain the genotypes of H pylori in gastric cancer. METHODS: Ninety-two patients were separated into three groups on the basis of their endoscopic findings: group 1, gastric cancer; group 2, gastric ulcer; group 3, non-ulcer dyspepsia. Gastric biopsy specimens were obtained for culture and DNA isolation; additional specimens were taken from subjects with gastric cancer for histopathological analysis. Amplification was performed using specific oligonucleotide primers to obtain genotypic data. Four samples from each group were randomly selected for sequence analysis. RESULTS: Genotypic analysis showed cagT+ve/hrgA+ve/cagA+ve/cagE+ve/vacAs1+ve to be highly prevalent in 79% of cases of H pylori infection. This genotype was found in 88% of subjects in group 1 and 78% in group 2. Intestinal-type adenocarcinoma was found in 35 subjects (83%), 32 (9%) of which harboured this genotype. Sequence analysis showed no significant strain-specific variations. CONCLUSIONS: Certain genotypes of H pylori have higher predictive value for the development of intestinal-type carcinoma at an early age. Genotyping of H pylori may well be a useful tool for screening people at increased risk of developing malignancy.

Journey from hepatocyte transplantation to hepatic stem cells: a novel treatment strategy for liver diseases.

Acute liver failure (ALF) carries high morbidity and mortality (>80%) even in the best centres. Orthotopic liver transplantation (OLTx) is the only viable approach to the treatment of ALF. This has significantly improved the survival in these patients. The major limitations of OLTx are non availability of the donor liver, requirement of a major surgical procedure, high cost and longterm immunosuppression. Isolated hepatocyte transplantation is emerging as an appealing method for the treatment of ALF because of its technical simplicity and easy availability of cells. Transplantation of allogenic/xenogenic hepatocytes transplantation in experimentally induced ALF has shown an increased survival rate. Clinical studies in acute, chronic liver failure and metabolic disorders have also been undertaken in a few centres and have shown encouraging results. To maintain the continuous supply of cells, xenogenic source of hepatocytes (porcine, rabbit, canine) have offered a hope. A major concern regarding the use of xenogenic donors is the risk of transmission of zoonosis and immunogenicity. Recently, Porcine endogenous retrovirus (PERV) has been shown to infect human tissue in vitro. The problem of immunogenicity of xenogenic hepatocytes can be overcome to some extent by immunoisolation, encapsulation technique, which may also provide protection to the hepatocytes during cryopreservation. The knowledge of adult hepatic stem from tissue offered a new hope for the treatment of various chronic and metabolic diseases. Further, the transdifferentiation potentiality of haematopoietic stem cells to hepatic lineage has strengthened cell therapy.

Prevalence study to elucidate the transmission pathways of Helicobacter pylori at oral and gastroduodenal sites of a South Indian population.

INTRODUCTION: Since the discovery of Helicobacter pylori (H. pylori), much progress has been made worldwide in the field of its epidemiology. In spite of these advancements, many aspects of epidemiology still remain unclear, particularly among populations with low socio-economic status. The present study was designed to elucidate the different routes of transmission of H. pylori in the Hyderabad (South India) population and to investigate the impact of certain factors, such as age, gender, and lifestyle. METHODS: Samples used for the study included saliva and biopsy samples of 400 symptomatic subjects from Hyderabad, India. The patients were retrospectively grouped, based on histopathology of the biopsy and 16S rRNA amplification of both saliva and biopsy as H. pylori positive and negative. RESULTS: This study showed that the prevalence of H. pylori in both saliva and biopsy samples increased with age. In addition, the H. pylori infection was found more commonly in the saliva and biopsy samples among males (64 percent and 60 percent, respectively) than females (53.3 percent and 64 percent, respectively). Similarly, 71.6 percent and 73.5 percent of those who consumed municipal water acquired H. pylori (which were respectively found in their saliva and biopsy samples) compared to a lesser proportion (12.6 percent and 12.6 percent, respectively) of those who consumed boiled or filtered water. The study also found that subjects who preferred home-cooked food (57.1 percent and 57.7 percent) showed a lower prevalence of H. pylori in saliva and biopsy samples, respectively, compared to those (80 percent and 88 percent) who frequently ate out. CONCLUSION: The results of the present study suggest that besides the oral-oral route, the transmission of H. pylori also takes place through the consumption of food prepared under unhygienic conditions. Consumption of municipal tap water also has a high impact in the transmission of H. pylori.