[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE AND DESIGN: This study evaluated the effect of an antioxidant, Vitamin E, on cultured chondrocytes against H2O2-induced damage in vitro. MATERIAL: Rat chondrocytes isolated from articular cartilage. TREATMENT: Chondrocytes were pretreated with either 50 or 100 μM Vitamin E or serum-free medium for 24 h followed by their exposure to 200 μM H2O2 for 3 h. Chondrocytes without exposure to H2O2 served as control group. METHODS: The effect of Vitamin E pretreatment was evaluated by examining proteoglycan contents, nitrite levels, viability, apoptosis, and senescence of cultured chondrocytes. RESULTS: Proteoglycan contents increased in groups treated with Vitamin E. Semi-quantitative real-time PCR data also correlated with these results and demonstrated that Vitamin E up-regulated expression of Agc1, Col2a1, and PCNA genes along with down-regulation in the expression of Col1a1 and Casp3 genes. The differentiation index improved after Vitamin E pretreatment. Nitrite levels were reduced with a corresponding increase in cell viability. Reduction in apoptosis and senescence was also observed after Vitamin E pretreatment. Moreover, a dose-dependent effect of Vitamin E was seen. In contrast to 50 μM Vitamin E, 100 μM was more potent in inducing protection of chondrocytes from H2O2-induced oxidative damage. CONCLUSION: Vitamin E reversed the oxidant-induced alterations in chondrocytes and may be a good option to pretreat chondrocytes before transplantation.
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION: The clinical correlation between hepatitis C virus (HCV) NS5A gene mutations and response to antiviral treatment in HCV-3a-infected patients is not as well known as that in HCV-1a-infected and HCV-1b-infected patients. OBJECTIVES: This study attempted to determine mutations in the NS5A gene in isolates of HCV-3a obtained from infected Pakistani patients treated with interferon-α standard therapy and to determine its association with several host factors. METHODS: A total of 71 consecutive chronically infected HCV patients [43 men and 28 women, mean age 38 years (range, 16-70 years)] without previous antiviral therapy, who fulfilled other criteria of the current study were enrolled for antiviral therapy. RESULTS: Of the 71 patients enrolled, 53 completed the course of antiviral therapy. Among them, 23 (43.3%) patients showed an end-of-treatment response, 20 (41.4%) showed a sustained virological response, and three (5.6%) patients were HCV-RNA negative at the end of treatment but developed a relapse thereafter. Only seven (13.2%) patients were virologic nonresponders (NR). Probable transmission risk factors were previous major/minor surgery (20%), transfusion of blood or blood products (2%), dental surgery (10%), or sporadic (unknown; 60%). The estimated duration of infection varied from 6 months to 20 years. The correlation between the number of mutations and several factors was also studied. The first factor that was associated with the number of mutations in the NS5A gene was age older than 40 years, where the difference was statistically significant between viral sequences of patients showing an end-of-treatment response and NR (P<0.001). Further, the difference in the average number of mutations was statistically significant between patients showing sustained virological response and NR (P<0.005). The second and third factors that were found to be associated with number of mutations in the NS5A gene were sex and viral load, respectively. CONCLUSION: Along with host factors such as age older than 40 years, female sex, and low baseline viral load, the less number of mutations in the NS5A gene of HCV-3a is associated with a positive outcome of treatment response in HCV patients receiving interferon plus ribavirin therapy.
European journal of gastroenterology & hepatology 04/2013;
[Show abstract][Hide abstract] ABSTRACT: To identify pathogenic mutations responsible for retinal dystrophy in three consanguineous Pakistani families.
A thorough ophthalmic examination including fundus examination and electroretinography was performed, and blood samples were collected from all participating members. Genomic DNA was extracted, and genome-wide linkage and/or exclusion analyses were completed with fluorescently labeled short tandem repeat microsatellite markers. Two-point Lod scores were calculated, and coding exons along with exon-intron boundaries of RPE65 gene were sequenced, bidirectionally.
Ophthalmic examinations of the patients affected in all three families suggested retinal dystrophy with an early, most probably congenital, onset. Genome-wide linkage and/or exclusion analyses localized the critical interval in all three families to chromosome 1p31 harboring RPE65. Bidirectional sequencing of RPE65 identified a splice acceptor site variation in intron 2: c.95-1G>A, a single base substitution in exon 3: c.179T>C, and a single base deletion in exon 5: c.361delT in the three families, respectively. All three variations segregated with the disease phenotype in their respective families and were absent from ethnically matched control chromosomes.
These results strongly suggest that causal mutations in RPE65 are responsible for retinal dystrophy in the affected individuals of these consanguineous Pakistani families.
[Show abstract][Hide abstract] ABSTRACT: Our aim was to improve the survival and reduce the apoptosis of chondrocytes derived from mesenchymal stem cells from Wharton's jelly of human umbilical cord (WJMSCs) by Lovastatin supplementation under hydrogen-peroxide-induced injury conditions to simulate the osteoarthritic micro-environment. Chondrocytes were differentiated in vitro from WJMSCs. The cultured WJMSCs expressed CD90 (84.07%), CD105 (80.84%), OCT4 (26.90%), CD45 (0.42%) and CD34 (0.48%) as determined by flow cytometry. Increased aggregation of proteoglycans observed by Safranin-O staining accompanied by increased expression of COL2A1, ACAN, SOX9 and BGN shown by immunocytochemistry and reverse transcription with the polymerase chain reaction (PCR) confirmed the chondrogenic differentiation of the WJMSCs. The in vitro differentiated chondrocytes were subjected to oxidative stress by exposure to 200 μM hydrogen peroxide, either in the presence or absence of Lovastatin (2 μM) for 5 h. Lovastatin treatment resulted in decreased apoptosis, senescence and LDH release and in increased viability and proliferation of WJMSC-derived chondrocytes. Real time PCR analysis showed markedly up-regulated expression of prosurvival, proliferation and chondrogenic genes (BCL2L1, BCL2, AKT, PCNA, COL2A1, ACAN, SOX9 and BGN) and significantly down-regulated expression of pro-apoptotic genes (BAX, FADD) in the Lovastatin-treated group in comparison with injured cells. The reduced expression of VEGF and p53 as determined by enzyme-linked immunosorbent assay and PCR suggests the suitability of the use of Lovastatin in adjunct to WJMSC-derived chondrocytes for the treatment of osteoarthritis. We conclude that Lovastatin protects WJMSC-derived chondrocytes from hydrogen-peroxide-induced in vitro injury.
[Show abstract][Hide abstract] ABSTRACT: Human hereditary deafness at the DFNB29 locus on chromosome 21q22.1 is caused by recessive mutations of CLDN14, encoding claudin 14. This tight junction protein is tetramembrane spanning that localizes to the apical tight junctions of organ of Corti hair cells and in many other tissues. Typically, the DFNB29 phenotype is characterized by prelingual, bilateral, sensorineural hearing loss. The goal of this study was to define the identity and frequency of CLDN14 mutations and associated inner ear phenotypes in a cohort of 800 Pakistani families segregating deafness. Hearing loss in 15 multi-generational families was found to co-segregate with CLDN14-linked STR markers. The sequence of the six exons and regions flanking the introns of CLDN14 in these 15 families revealed five likely pathogenic alleles. Two are novel missense substitutions (p.Ser87Ile and p.Ala94Val), whereas p.Arg81His, p.Val85Asp and p.Met133ArgfsX23 have been reported previously. Haplotype analyses indicate that p.Val85Asp and p.Met133ArgfsX23 are founder mutations. The p.Val85Asp accounts for ∼67% of the mutant alleles of CLDN14 in our cohort. Combined with the previously reported data, CLDN14 mutations were identified in 18 of 800 Pakistani families (2.25; 95% CI, 1.4-3.5). Hearing loss in the affected individuals homozygous for CLDN14 mutations varied from moderate to profound. This phenotypic variability may be due to environmental factors (for example drug and noise exposure) and/or genetic modifiers.Journal of Human Genetics advance online publication, 13 December 2012; doi:10.1038/jhg.2012.143.
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C is the major health problem over the globe affecting approximately 200 million people worldwide and about 10 million Pakistani populations. Developing countries are especially facing the problems of HCV infection. Hence the goal of the study was to find out the antigenic epitopes that could be effective vaccine targets of HCV genotype 1 of Asian origin against HLA alleles frequently distributed in Asian countries. A total of 85 complete genome sequences of HCV 1 of Asian origin were retrieved from the HCV sequence database. Using in silico tools, T cell epitopes were predicted from conserved regions of all the available HCV 1 subtypes against Asian HLA alleles. Using 10 MHC I supertypes 51 epitopes was predicted as promiscuous binders. MHC class I supertypes A2 and B7 were found to be good promiscuous binders for a large number of predicted epitopes. Other alleles of MHC I supertypes (B57, B27, BX, B44) either were not respondent as promiscuous binders or responded only to a limited number of epitopes. Against 8 predominantly found Asian alleles of DRB1 supertype, 42 epitopes was predicted as promiscuous binders. MHC class II alleles DRB1-0101, DRB1-0701 and DRB1-1501 were the highest binders to promiscuous predicted epitopes while DRB1-0301 was the least binder for the predicted promiscuous epitopes of HCV 1 genotype of Asian origin. Literature review survey of predicted epitopes via IEDB also confirmed that great numbers of predicted epitopes are true positive. Hence, sophisticated selection of viral proteins and MHCs provided conserved promiscuous epitopes that can be used as effective vaccine candidates for all Asian counties. ABBREVIATIONS: HCV - hepatitis C virus, MHC - major histocompatability complex, HLA - human leukocyte antigen, CTL - cytotoxic T lymphocytes.
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium ulcerans, the causative agent of Buruli ulcer, is the third most common mycobacterial disease after tuberculosis and leprosy. The present treatment options are limited and emergence of treatment resistant isolates represents a serious concern and a need for better therapeutics. Conventional drug discovery methods are time consuming and labor-intensive. Unfortunately, the slow growing nature of M. ulcerans in experimental conditions is also a barrier for drug discovery and development. In contrast, recent advancements in complete genome sequencing, in combination with cheminformatics and computational biology, represent an attractive alternative approach for the identification of therapeutic candidates worthy of experimental research. A computational, comparative genomics workflow was defined for the identification of novel therapeutic candidates against M. ulcerans, with the aim that a selected target should be essential to the pathogen, and have no homology in the human host. Initially, a total of 424 genes were predicted as essential from the M. ulcerans genome, via homology searching of essential genome content from 20 different bacteria. Metabolic pathway analysis showed that the most essential genes are associated with carbohydrate and amino acid metabolism. Among these, 236 proteins were identified as non-host and essential, and could serve as potential drug and vaccine candidates. Several drug target prioritization parameters including druggability were also calculated. Enzymes from several pathways are discussed as potential drug targets, including those from cell wall synthesis, thiamine biosynthesis, protein biosynthesis, and histidine biosynthesis. It is expected that our data will facilitate selection of M. ulcerans proteins for successful entry into drug design pipelines.
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) has been considered to be a significant risk factor in developing liver associated diseases including hepatocellular carcinoma all over the world. HCV is an enveloped positive strand virus comprising a complex between genomic RNA and viral envelope glycoproteins (E1 and E2), which are anchored within host derived double-layered lipid membrane surrounding the nucleocapsid composed of several copies of core protein. HCV cell entry is the first step in infection and viral replication into host cells mainly hepatocytes. HCV cell entry is a complex process involving both the viral (envelope glycoproteins E1/E2) and host factors (cellular receptors and associated factors i.e. CD81, SR-BI, LDL-R, CLDN1, Occludin, DC-SIGN, L-SIGN and Glycosaminoglycans). Besides these the expression of certain other conditions such as polarization and EWI-2 expression inhibits the viral cell entry. Exploring the mechanism of HCV entry will help to better understand the viral life cycle and possible therapeutic targets against HCV infection including viral and host factors involved in this process. New strategies such as RNAi represents a new option for targeting the host or viral factors for prevention and therapeutic against HCV infection. In the current review we try to summarize the current knowledge about mechanism and interaction of cellular and viral factors involved in HCV cell entry and its implication as therapeutic target to inhibit HCV infection.
Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 08/2012; 12(8):1699-1709.
[Show abstract][Hide abstract] ABSTRACT: Liver fibrosis is a major health problem worldwide and poses a serious obstacle for cell based therapies. Mesenchymal stem cells (MSCs) are multipotent and important candidate cells for future clinical applications however success of MSC therapy depends upon their homing and survival in recipient organs. This study was designed to improve the repair potential of MSCs by transplanting them in sodium nitroprusside (SNP) pretreated mice with CCl(4) induced liver fibrosis.
SNP 100 mM, a nitric oxide (NO) donor, was administered twice a week for 4 weeks to CCl(4)-injured mice. MSCs were isolated from C57BL/6 wild type mice and transplanted in the left lateral lobe of the liver in experimental animals. After 4 weeks, animals were sacrificed and liver improvement was analyzed. Analysis of fibrosis by qRT-PCR and sirius red staining, homing, bilirubin and alkaline phosphatase (ALP) serum levels between different treatment groups were compared to control.
Liver histology demonstrated enhanced MSCs homing in SNP-MSCs group compared to MSCs group. The gene expression of fibrotic markers; αSMA, collagen 1α1, TIMP, NFκB and iNOS was down regulated while cytokeratin 18, albumin and eNOS was up-regulated in SNP-MSCs group. Combine treatment sequentially reduced fibrosis in SNP-MSCs treated liver compared to the other treatment groups. These results were also comparable with reduced serum levels of bilirubin and ALP observed in SNP-MSCs treated group.
This study demonstrated that NO effectively augments MSC ability to repair liver fibrosis induced by CCl(4) in mice and therefore is a better treatment regimen to reduce liver fibrosis.
Journal of Translational Medicine 04/2012; 10:75.
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