Human bone marrow-derived mesenchymal stem cell: a source for cell-based therapy

The ability of mesenchymal stem cells [MSCs] to differentiate into many cell types, and modulate immune responses, makes them an attractive therapeutic tool for cell transplantation and tissue engineering. This project was designed for isolation, culture, and characterization of human marrow-derived MSCs based on the immunophenotypic markers and the differentiation potential. Bone marrow of healthy donors was aspirated from the iliac crest. Mononuclear cells were layered over the Ficoll-Paque density-gradient and plated in tissue cultures dish. The adherent cells expanded rapidly and maintained with periodic passages until a relatively homogeneous population was established. The identification of adherent cells and the immune-surface markers was performed by flow cytometric analysis at the third passage. The in vitro differentiation of MSCs into osteoblast and adipo-cytes was also achieved. The MSCs were CDllb [CR3], CD45, CD34, CD31 [PCAM-1], CD40, CD80 [B7-1], and HLA-class II negative because antigen expression was less than 5%, while they showed a high expression of CD90, and CD73. The differentiation of osteoblasts, is determined by deposition of a mineralized extracellular matrix in the culture plates that can be detected with Alizarin Red. Adipocytes were easily identified by their morphology and staining with Oil Red. MSCs can be isolated and expanded from most healthy donors, providing for a source of cell-based therapy

Endothelial nitric oxide synthase gene t-786c polymorphism in renal transplant recipients

Nitric oxide [NO] is a major mediator in vascular biology, regulating regional blood flow. NO and the enzymes required for its production contribute to ischemia-reperfusion injury. The T-786C functional polymorphism in the promoter region substantially reduces promoter activity of the endothelial nitric oxide synthase [eNOS] gene and compromises endothelial NO synthesis. To examine the association between T-786C [rs 2070744] single nucleotide polymorphism [SNP] in eNOS gene and the development of acute rejection in renal transplant patients. 60 renal transplant recipients [30 with episodes of acute rejection [ARs] and 30 without rejection [non-ARs]], between June 2008 and March 2010, were included in this study. The polymorphism was determined by PCR-restriction fragment-length polymorphism analysis. The distribution of the genotypes were TT/TC/CC 60%, 33.4%, 6.6%, and 43%, 46.7%, 13.3% in ARs and non-ARs, respectively [p=0.28]. The frequency of T-allele was 76.7% and 66.3%; and for C-allele was 66.6% and 33.3% in ARs and non-ARs, respectively [p=0.09]. There were no significant associations between these polymorphisms and acute and chronic kidney allograft rejection. We could not detect any significant association between polymorphism in T-786C of eNOS gene and the development of acute rejection

Evaluation of cytomegalovirus infection after six months of liver transplantation in children in Shiraz, Southern Iran

Liver transplantation [LT] is a life-saving treatment for end-stage liver diseases [ESLD]. Cytomegalovirus [CMV] infection is one of the important causes of morbidity after LT. To evaluate the incidence of late-onset [after 6 months of LT] CMV infection in pediatric recipients. A retrospective analysis was conducted to evaluate 50 pediatric patients who underwent LT for 8 years at the LT Unit of Nemazee Hospital affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. We retrospectively investigated episodes of CMV infection after 6 months of LT proven by CMV antigenemia test. Three recipients [6%] developed late-onset CMV infection. These patients finally responded to ganciclovir. CMV infection is one of the most common post-LT viral infections that usually occurs in the first six months of LT. Our study shows that the incidence of late-onset CMV infection is relatively low, but it still remains a significant problem. Therefore, monitoring and management is crucial for improving the survival of children

[Epidemiological features of rotaviral, bacterial, and parasitic infections among hospitalized children in Jahrom [2006-2007]]

Rotavirus is one of the most common cause of diarrhea and one of the major causes of severe gastroenteritis in very young children. To follow up and genotype the agents of rotavirus infection as well as assessing the bacterial and parasitic organisms among hospitalized children with gastroenteritis in the city of Jahrom, Iran. This cross-sectional descriptive study was carried out during October 2006 to October 2007. A total of 163 stool samples from hospitalized children less than 5 years old with severe diarrhea were collected from two hospitals in Jahrom. Culture, microscopy, EIA, and RT-PCR were used for detection of bacterial, parasitic and rotaviral agents. Data were analyzed using SPSS 14 and descriptive statistics including chi-square test, ANOVA, and Fisher exact test. A p value less than 0.05 was considered to be statistically significant. Of total samples, 46.02% were positive for group A rotavirus by EIA. The predominant genotypes were G[1] [17.33%], G[4] [30.66%], and nontypable [30.66%]. Also, E.coli, Shigella spp., Shigella spp. + E.coli, E.coli + rotavirus, Salmonella spp., E. histolytica/E.Dispar, and other infectious agents were identified in 7.97%, 17.18%, 1.83%, 15.20%, 3.66%, 10.84%, and 6.28% of cases, respectively. According, to the data obtained from the present study, rotavirus infections in Jahrom mostly occur within the cold months of the year, epidemiologically

Association of the co-stimulatory molecules polymorphisms with CMV infection in liver transplant recipients

Co-stimulatory molecules play a critical role in regulating T-cell function during CMV infection after liver transplantation. To investigate the relationship between the polymorphisms of the co-stimulatory genes and the susceptibility to CMV infection after liver transplantation. Single nucleotide polymorphisms [SNPs] in PD-1 gene [PD1.1 A/G, PD1.3 A/G, PD1.9 C/T] ICOS [-693 A/G, 1720 C/T], CTLA-4 gene [-318 C/T, 1722 T/C, 1661 A/G, 49 A/G] and CD28 [+17 C/T] were analyzed by PCR-RFLP in 70 liver transplant patients. CMV infection was determined in these patients by antigenemia test. CTLA-4 49G showed significant association with CMV infection [p=0.03, OR=3.82, 95% CI: 0-3.5; p=0.01, OR=004, 95% CI: 0-1.3]. G and T alleles in CTLA-4 gene [-318 C/T and 1661 A/G] [p=0.03, OR=0, 95% CI: 0-3.5; p=0.01, OR=0.04, 95% CI: 0-1.3] were significantly higher in CMV-infected rejector group. CTLA-4 have significant role in CMV pathogenesis and rejection among CMV-positive liver transplant patients

molecular and antigenic tissue impact of viral infections on liver transplant patients with neonatal hepatitis

Pathogenesis of neonatal hepatitis relates to various underlying causes including viral infections. Both hepatotropic and non-hepatotropic viruses may induce liver failures in infants before birth, during delivery, or shortly after birth. The tissue impact of HCMV, HSV, HBV, HCV, and rotavirus and adenovirus infections was evaluated in studied infants with neonatal hepatitis. The history of viral infections was analyzed in paraffin-embedded biopsy and autopsy tissues of 22 infants with neonatal hepatitis between years 1996 and 2007, retrospectively. The tissue molecular presentation of HBV, HCV, HCMV, HSV, adenovirus, and rotavirus was evaluated by different qualitative simple and nested PCR and RT-PCR protocols. Immunohistochemistry [IHC] method was used for studying the antigenic prevalence of HSV-1, 2; HBV, HCMV and adenovirus infections. Also the laboratory liver indices of all patients with neonatal hepatitis were analyzed. The HBV and HSV genomes were detected in 3 [14%] of 22 infants. The rotavirus and HCV-RNA and also the HCMV-DNA were detected separately in 1 [4%] of 26 paraffin-embedded autopsy and biopsy tissues. The HBV and HSV-1 specific antigens were separately diagnosed in 1 [4%] of 26 neonatal samples by IHC protocols. Also the HSV-2 antigen was seen in 5 [23%] of 22 liver autopsy and biopsy specimens. Co-infections with HCMV, HSV, HBV, HCV, and rotavirus were detected in these infants with hepatitis. Diagnosis of single and mixed molecular and antigenic traces of HCMV, HSV, HBV, HCV and rotavirus underlines the etiologic role of these viruses in clinical pathogenesis of neonatal hepatitis

[Molecular and serological characterization of group A rotavirus isolates obtained from hospitalized children in Jahrom]

Rotaviruses are the most common factors causing gastroenteritis in children. Considering the high rate of mortality in developing countries, WHO proposed hospital based surveillance in order to identify the prevalent genotype profile. This study was conducted to determine the prevalent genotypes of Rotavirus by RT-PCR method in hospitalized children in Jahrom city. In this study, 163 stool samples were collected from children [<5 years old] with sever diarrhea who were hospitalized in two hospitals in Jahrom city during 2005-2006. At first, group A rotavirus were distinguished by Enzyme Immunoassay [EIA] test and then the genotypes of positive samples were determined using 9 specific primers by Nested RT-PCR method. From the total of 163 collected samples, 75 samples were positive by ELISA. The frequency of G1,G2,G3,G4,G9 and the mix genotypes were 17.33%, 13.34%, 2.67%, 30.66%, 2.67% and 2.67%, respectively. The most common prevalence of positive rotavirus was observed in winter with 22.69% compared to the least prevalence in summer with 4.29% [P<0.05]. Considering the high prevalence of positive rotavirus in studied population and also prevalence of rare genotypes, we suggest a broader research in other parts of the country

role of clinical, therapeutic and laboratory findings in monitoring of HCMV infection in bone marrow transplant recipients

Human cytomegalovirus [HCMV] has been an enormous threat for bone marrow transplant [BMT] recipients. For active and/or latent HCMV infection, diagnosis of the risk factors which increase the risk of posttransplant morbidity and mortality seems necessary. In this research, some of the HCMV risk factors were monitored and compared with HCMV molecular diagnostic methods for better detection of HCMV infection in BMT patients. HCMV risk factors including clinical, biological, biochemical, haematological indexes, and also anti-HCMV and transplant prophylactic and therapeutic conditioning regimens were monitored from March 2002 to March 2006, in 104 BMT patients referred to BMT Unit of Nemazee Hospital in Shiraz University of Medical Sciences and was compared with HCMV molecular methods for BMT donors and recipients' pre- and posttransplantation. Anti-HCMV-lgM was detected in 9.6% and 6.7% of BMT recipients and donors, respectively. Anti-HCMV-lgG was also detected in 8.7% and 9.1% of recipients and donors, pre-transplant, respectively. HCMVPCR results were positive in 20% of recipients and 33.3% of donors. Significant correlations were observed between HCMV positive results and the use of a therapeutic dose, but not the prophylactic dose of glucocorticoids and cyclosporine, pre and post-transplantation. Fasting blood sugar, creatinine, globulin, and liver enzymes levels such as alkaline phosphates and asparagine transpherase significantly correlated with detection of HCMVDNA in transplant patients. Also, negative results of HCMV-PCR significantly correlated with the use of prophylactic dose of acyclovir in BMT patients. Significant correlations of positive and negative HCMV-PCR results with HCMV disease risk factors suggest the possible role of these factors on prognosis and monitoring of HCMV disease in BMT recipients preand post-transplantation

[Genomic evaluation of 5'-UTR sequence of fusion gene in vaccine strain of measles virus [AIK-C] following passage in MRC-5 cell line]

Multiple point mutations and deletions have been detected in the 5'-UTR non-coding region of F gene of measles virus vaccine strains that may affect the function of fusion protein and viral penetration. To study the possible alterations in 5'-UTR non-coding sequence of F gene following a change in measles virus cell culture from chicken fibroblast to MRC-5 cell line. Concerning the importance of F gene in immunogenicity and the cellular tropism of this protein, the present study was performed at the Biotechnology Department of Razi Vaccine and Serum Research Institute, Karaj, Iran. After cloning of 5'-UTR non-coding sequence of F gene and amplification using a two-step RT-PCR method, the recombinant plasmid was sequenced. Comparing this F gene 5'-UTR sequence with standard vaccine strains by DNAMAN program, the possible changes which might have been occurred in F gene non-coding sequence, was determined. Comparing the F gene 5'-UTR sequence of AIK-C vaccine strain with two sequences of Parks and Mori vaccine strains was indicative of two nucleotide variations in bases 156 and 288 after shifting the measles virus vaccine strain cell lines from chicken fibroblast to MRC-5. Regarding the changes in two nucleotides of F gene 5'-UTR non-coding sequence after cell culture shift from fibroblast to MRC-5 cell line for multiplication of measles virus vaccine strain, these alterations may affect the selection of AUG initiation codon, promote the translational level of fusion protein, and therefore leading to possible reduction in immunogenicity of this newly propagated virus vaccine strain