Phylogenetic analysis, based on EPIYA repeats in the cagA gene of Indian Helicobacter pylori, and the implications of sequence variation in tyrosine phosphorylation motifs on determining the clinical outcome.

The population of India harbors one of the world's most highly diverse gene pools, owing to the influx of successive waves of immigrants over regular periods in time. Several phylogenetic studies involving mitochondrial DNA and Y chromosomal variation have demonstrated Europeans to have been the first settlers in India. Nevertheless, certain controversy exists, due to the support given to the thesis that colonization was by the Austro-Asiatic group, prior to the Europeans. Thus, the aim was to investigate pre-historic colonization of India by anatomically modern humans, using conserved stretches of five amino acid (EPIYA) sequences in the cagA gene of Helicobacter pylori. Simultaneously, the existence of a pathogenic relationship of tyrosine phosphorylation motifs (TPMs), in 32 H. pylori strains isolated from subjects with several forms of gastric diseases, was also explored. High resolution sequence analysis of the above described genes was performed. The nucleotide sequences obtained were translated into amino acids using MEGA (version 4.0) software for EPIYA. An MJ-Network was constructed for obtaining TPM haplotypes by using NETWORK (version 4.5) software. The findings of the study suggest that Indian H. pylori strains share a common ancestry with Europeans. No specific association of haplotypes with the outcome of disease was revealed through additional network analysis of TPMs.

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.

Phylogenetic analysis, based on EPIYA repeats in the cagA gene of Indian Helicobacter pylori, and the implications of sequence variation in tyrosine phosphorylation motifs on determining the clinical outcome

The population of India harbors one of the world's most highly diverse gene pools, owing to the influx of successive waves of immigrants over regular periods in time. Several phylogenetic studies involving mitochondrial DNA and Y chromosomal variation have demonstrated Europeans to have been the first settlers in India. Nevertheless, certain controversy exists, due to the support given to the thesis that colonization was by the Austro-Asiatic group, prior to the Europeans. Thus, the aim was to investigate pre-historic colonization of India by anatomically modern humans, using conserved stretches of five amino acid (EPIYA) sequences in the cagA gene of Helicobacter pylori. Simultaneously, the existence of a pathogenic relationship of tyrosine phosphorylation motifs (TPMs), in 32 H. pylori strains isolated from subjects with several forms of gastric diseases, was also explored. High resolution sequence analysis of the above described genes was performed. The nucleotide sequences obtained were translated into amino acids using MEGA (version 4.0) software for EPIYA. An MJ-Network was constructed for obtaining TPM haplotypes by using NETWORK (version 4.5) software. The findings of the study suggest that Indian H. pylori strains share a common ancestry with Europeans. No specific association of haplotypes with the outcome of disease was revealed through additional network analysis of TPMs.

Human fetal liver-derived stem cell transplantation as supportive modality in the management of end-stage decompensated liver cirrhosis.

Liver transplantation is the only existing modality for treating decompensated liver cirrhosis. Several factors, such as nonavailability of donors, combined with operative risks, complications associated with rejection, usage of immunosuppressive agents, and cost intensiveness, make this strategy available to only a few people. With a tremendous upsurge in the mortality rate of patients with liver disorders worldwide, there is a need to search for an alternative therapeutic tool that can combat the above limitations and serve as a supportive therapy in the management of liver diseases. Cell therapy using human fetal liver-derived stem cells can provide great potential to conservatively manage end-stage liver diseases. Therefore, the present investigation aimed to study and prove the safety and efficacy of human fetal liver-derived stem cell transplantation in patients with end-stage liver cirrhosis. Twenty-five patients with liver cirrhosis of different etiologies were infused with human fetal liver-derived stem cells (EpCAM+ve) labeled with Tc-HMPAO through hepatic artery. Our high throughput analysis using flow cytometry, RT-PCR, and cellular characterization exemplifies fetal liver cells with their high proliferation rate could be the best source for rejuvenating the diseased liver. Further, no episodes related to hepatic encephalopathy recurred in any of the subjects following hepatic stem cell transplantation. There was marked clinical improvement observed in terms of all clinical and biochemical parameters. Further, there was decrease in mean MELD score (p < 0.01) observed in 6 months follow-up in all patients. Therapy using human fetal liver stem/progenitor cells offers a potentially supportive modality to organ transplantation in the management of liver diseases.

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.

Relevance of Helicobacter pylori genotypes in gastric pathology and its association with plasma malondialdehyde and nitric oxide levels.

Persistent infection with Helicobacter pylori confers an increased risk of peptic ulceration and gastric adenocarcinoma. Reactive oxygen and nitrogen species play a crucial role in the progression from normal gastric mucosa to cancer. The aim of the present study was to investigate the plasma malondialdehyde and nitric oxide levels in H. pylori related gastroduodenal diseases and associate their levels with gastric pathology and genotypes of H. pylori. Malondialdehyde and nitric oxide levels in plasma samples of 250 subjects were spectrophotometrically determined. Subsequently, genotypic and histopathological assessment was performed in gastric biopsies obtained during endoscopy. The levels of MDA and NO exceeded in subjects infected with genotype-1 of Hp than those with other genotypes suggesting more precise interaction of highly virulent strains of Hp in eliciting severe tissue damage. In conclusion, the study demonstrates close relationship between the plasma malondialdehyde and nitric oxide levels, gastric histopathology and genotypes of H. pylori.

Neural stem cells & supporting cells--the new therapeutic tools for the treatment of spinal cord injury.

Stem cells play important role in the development and in the maintenance of specific tissues. They have been identified in majority of the organs like liver, blood, skin and intestine. Role of stem cells in regenerative medicine have been implicated in many chronic diseases. Stem cell research is a new opportunity to those patients whose organs are damaged or diseased. The discovery of stem cells in central and peripheral nervous system is relatively recent. Spinal cord injury is one of the major neurological disaster affecting mostly young lives. Stem cell transplantation in spinal cord injury patients have shown encouraging results. Different sources of stem cells are being exploited for spinal cord injury as well as other neurological disorders.

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.

Prevalence of hepatitis C virus (HCV) coinfection in HIV infected individuals in south India and characterization of HCV genotypes.

PURPOSE: To determine anti-HCV antibodies and genomic subtype of HCV in 1487 confirmed human immunodeficiency virus (HIV) positive samples. METHODS: A total of 1487 confirmed HIV-positive samples were tested for anti-HCV antibodies by using a third generation ELISA kit (Ortho 3.0) and by RT PCR for HCV. HIV and HCV coinfected samples were selected for HCV genotyping by RFLP and subtyping with NS5-type specific primers. RESULTS: A total of 1487 HIV-infected serum samples were screened for HCV infection, of which, a 1443 (97.04%) were negative and 45 (3.02%) were coinfected. HIV-HCV coinfection was predominant in the age group 41-50 years (51.1%). HCV genotyping and subtyping was done for the 45 HCV RNA-positive specimens of which genotype 1 was observed in 31 (68.8%) and genotype 3 was observed in 14 (31.1%) subjects. Further subtyping analysis showed the genotype 1b in 23 (51.1%), 1a in eight (17.7%), 3a in 10 (22.2%) and 3b in four (8.8%) subjects. CONCLUSION: HIV and HCV seroprevalence is higher in South India, and the most prevalent genotype in coinfection was genotype 1b.

Safety and efficacy of autologous bone marrow stem cell transplantation through hepatic artery for the treatment of chronic liver failure: a preliminary study.

This study was performed to determine the safety and tolerability of injecting autologous bone marrow stem cells (BMC) (CD34+) into four patients with liver insufficiency. The study was based on the hypothesis that the CD34+ cell population in granulocyte colony stimulating factor (G-CSF) mobilized blood and autologous bone marrow contains a subpopulation of cells with the potential for regenerating damaged tissue. We separated the CD34+ stem cell population from the bone marrow. The potential of the BMC to differentiate into hepatocytes and other cell lineages has already been reported. Several reports have also demonstrated the plasticity of hematopoietic stem cells to differentiate into hepatocytes. Recently Sakaida demonstrated reduction in fibrosis in chemically induced liver cirrhosis following BMC transplantation. From a therapeutic point of view, chronic liver cirrhosis is one of the targets for BMC transplantation. In this condition, there is excessive deposition of extracellular matrix and hepatocyte necrosis. Encouraged by this evidence that the CD34+ cell population contains cells with the potential to form hepatocyte-like elements, four patients with liver insufficiency were given G-CSF to mobilize stem cells. CD34+ cells (0.1 x 10(8)) were injected into the hepatic artery. No complications or specific side effects related to the procedure were observed; four patients showed improvements in serum albumin, bilirubin and ALT after one month from the cell infusion.

Treatment of Crigler-Najjar Syndrome type 1 by hepatic progenitor cell transplantation: a simple procedure for management of hyperbilirubinemia.

Crigler-Najjar Syndrome (CNS) is characterized by mild, chronic unconjugated hyperbilirubinemia resulting from an autosomal-recessive inherited deficiency of hepatic uridine/diphosphoglucuronate-glucuronosyl transferase 1Al since birth. Herein we have reported a confirmed case of CNS type 1 in a 2-year-old girl with an unconjugated hyperbilirubinemia (>30 mg/dL) treated by hepatic progenitor cell infusion through the hepatic artery. No procedure-related complications were encountered. No kernicterus was observed. The total bilirubin started falling at 10 days after cell infusion. Two months after cell infusion the bilirubin fell from 29.0 to 16 mg/dL, with the conjugated bilirubin increasing approximately fivefold, the unconjugated bilirubin decreasing nearly twofold, and the SGPT also decreasing from 210 U/L to 64 U/L. This study demonstrated the efficacy of hepatic progenitor cells to manage hyperbilirubinemia in these patients. As the procedure is simple and the patient has tolerated the cell therapy, infusion can be repeated as required to manage hyperbilirubinemia, which often causes lethal kernicterus. This study was developed to assess the safety, feasibility, and efficacy of hepatic progenitor cell transplantation in a child with CNS type 1.

Management of hyperbilirubinemia in biliary atresia by hepatic progenitor cell transplantation through hepatic artery: a case report.

Cholangiodestruction of bile ducts leads to biliary atresia, a rare disease characterized by intrahepatic and extrahepatic biliary inflammation. If the intrahepatic biliary tree is unaffected, surgical reconstruction by the Kasai procedure of hepatoportoenterostomy of the extra hepatic biliary tract is possible. Untreated, this condition leads to cirrhosis and death within the first year of the life. If the atresia is complete, liver transplantation is the only option. As a result of the shortage of donor livers, hepatocytes have been infused over the past two decades, providing proof of the concept that cell therapy can be effective for the treatment of liver diseases. In the present study, we report a confirmed case of a girl of 1 year of age with increased bilirubin of 28.5 mg/dL and pediatric end-stage liver disease score 20. Biochemical liver function tests showed cholestasis (elevated cholesterol and gamma-GTs) and increased ALT, total bilirubin, conjugated bilirubin, and ALP. The patient was treated with hepatic progenitor cell infusion through the hepatic artery. The total bilirubin and conjugated bilirubin started decreasing during the first month after cell infusion. The level of total bilirubin maintained a threefold decrease after months of cell infusion. The conjugated bilirubin was 16.35 mg/dL before cell infusion, decreasing to eightfold after cell infusion. After 2 months of cell infusion, hepatobiliary scintigraphy showed increased liver cell function. This case demonstrated the efficacy and functionality of hepatic progenitor cells for the management of biliary atresia. Further, as there was a decrease in serum bilirubin, it showed that there was some percentage of the engraftment of the infused cells. As the procedure is simple and the patient has tolerated the infusion therapy, it might be repeated to manage biliary atresia.

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.

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. METHODS AND RESULTS: Genomic DNA from 82 gastric tissues was screened. A master pool of all the ingredients of multiplex reaction was prepared for amplification. Amplicons were sequenced to confirm the amplification of each target genes. Multiplex PCR assay was able to detect all the five target genes in 81.7% and deletions in one or more loci among 18.3%. Genotype cagT +ve/hrgA +ve/cagA +ve/cagE +ve/vacAs1 +ve was more predominant in this study population (67.07%). hrgA, cagT, cagE and cagA genes were present in 100%, 92.7%, 85.4% and 81.7% of the subjects, respectively. The vacAs1 subtype had higher prevalence frequency in patients with overt gastrointestinal disease (78.57%) than with GERD (gastro-esophageal reflux disease) and NUD (non-ulcer dispepsia) (50%). CONCLUSIONS: The multiplex PCR assay developed herein was able to genotype H. pylori isolates based on the main virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to identify H. pylori and the majority of their virulence gene markers by multiplex PCR assay represents a considerable advancement over other PCR-based methods for genotyping H. pylori from large population, and can be explored to gain insights at the genotypic variability exhibited by this pathogen.

Impact of household hygiene and water source on the prevalence and transmission of Helicobacter pylori: a South Indian perspective.

INTRODUCTION: In developing countries, the Helicobacter pylori (H. pylori) infection rate is high, especially in lower socioeconomic groups. The populace in developing countries lives in conditions that are highly conducive to the acquisition of microorganisms. Poor hygiene, crowded household conditions and deficient sanitation mark their day-to-day life. We aimed to find out the roles of household hygiene and water source in the prevalence and transmission of H. pylori infection among the South Indian population using polymerase chain reaction (PCR) assay. METHODS: The selected population consisted of 500 adults of varying ages ranging from 30 to 79 years, with upper gastrointestinal tract symptoms. Each participant in the study was given a questionnaire to complete. Samples to assess H. pylori infection included three gastric biopsies (two from the antrum and one from the corpus region). Infection was detected by PCR amplification of the 16S rRNA gene of H. pylori. The data was then examined statistically by univariate and multivariate analyses. RESULTS: The overall prevalence of H. pylori was detected to be 80 percent. Prevalence increased with an increase in age and it was found to be 90 percent in the 70-79 year age group (p-value is less than 0.01). The prevalence of infection among people who drank water from wells was 92 percent compared with 74.8 percent of those who drank tap water (p-value is less than 0.001). H. pylori infection prevalence was found to be higher in people with low clean water index (CWI) (88.2 percent) than in those with higher CWI (33.3 percent) (p-value is less than 0.001). While the prevalence of H. pylori in the subjects with lower socioeconomic status was 86.1 percent, in higher groups, it was 70 percent (p-value is less than 0.001). The prevalence of H. pylori was also found to be higher in subjects who lived in overcrowded houses. It was 83.7 percent with high crowding index, 76.6 percent with medium crowding index, and 71.3 percent with low crowding index (p-value is less than 0.05). CONCLUSION: The results of the present study suggest that the risk of acquisition and transmission of H. pylori can be prevented to a large extent by following improved household hygienic practices, proper waste disposal measures as well as the regular use of boiling water for drinking purposes.

Characterization of hepatic progenitors from human fetal liver using CD34 as a hepatic progenitor marker.

AIM: To enrich putative hepatic progenitors from the developing human fetal liver using CD34 as a marker. METHODS: Aborted fetuses of 13-20 wk were used for the isolation of liver cells. The cells were labeled with anti CD34; a marker used for isolating progenitor population and the cells were sorted using magnetic cell sorting. The positive fractions of cells were assessed for specific hepatic markers. Further, these cells were cultured in vitro for long term investigation. RESULTS: Flow cytometric and immunocytochemical analysis for alphafetoprotein (AFP) showed that the majority of the enriched CD34 positive cells were positive for AFP. Furthermore, these enriched cells proliferated in the long term and maintained hepatic characteristics in in vitro culture. CONCLUSION: The study shows that aborted human fetal liver is a potential source for isolation of hepatic progenitors for clinical applications. The study also demonstrates that CD34 can be a good marker for the enrichment of progenitor populations.

Stem cell therapy-present status.

Stem cell research is a new field that is advancing at an incredible pace with new discoveries being reported from all over the world. Scientists have for years looked for ways to use stem cells to replace cells and tissues that are damaged or diseased. Stem cells are the foundation cells for every organ, tissue, and cell in the body. Stem cells are undifferentiated, "blank" cells that do not yet have a specific function. Under proper conditions, stem cells begin to develop into specialized tissues and organs. They are self-sustaining and can replicate themselves for long periods of time. Embryonic stem cells are pluripotent cells, isolated from the inner cell mass of the blastocyst-stage mammalian embryo. They have the ability to differentiate into several somatic or somatic-like functional cells such as neurons, hepatocytes, cardiomyocytes, and others. Adult stem cells are specialized cells found within many tissues of the body where they function in tissue homeostasis and repair. They are precursor cells capable of differentiation into several different cells. The knowledge of stem cells from various sources offered a new hope for the treatment of various diseases.