[ association of vitamin D receptor gene BsmI polymorphism with multiple sclerosis in Iranian patients]

1,25-dihydroxyvitamin D 3 [1,25 [OH] 2 D 3], the biologically active form of vitamin D, exerts an immunosuppressive effect through binding to its specific nuclear receptor. The present case-control study was done to examine the possible association of BsmI polymorphism in vitamin D receptor gene [VDR gene] with severity of multiple sclerosis [MS]. 267 Iranian patients with MS and 292 ethnically and sex matched controls were included in this study. BsmI polymorphism in VDR gene was assessed by PCR-RFLP method. No differences in the allelic distribution were observed in the patients as compared to the controls. Also no difference in genotype distribution of VDR-BsmI polymorphism was observed between patients and controls [p=0.43]. Considering the significance of vitamin D 3 as an immune system regulator and its inhibitory effects on MS, investigation on other VDR gene polymorphisms is recommended

[Relation between C.32 A>T Polymorphism in TLR7 and response to treatment in Chronic HCV-infection]

Hepatitis C virus [HCV]-infection leads to development of chronic hepatitis, liver cirrhosis and hepatoma. Both the liver damage and extrahepatic manifestation of HCV are immune-mediated. Since HCV is an RNA virus, a role for toll like receptor 7 [TLR7] in the immune response against HCV is likely. The aim of the present study was to determine the frequency of C.32T allele of TLR-7 in general and chronic HCV hepatitis, and its effect on treatment of HCV. This case -control study was carried out on 154 patients of chronic hepatitis C in 2008-2009. The patients were selected from referrals to Hepatitis clinic at Shahid Motahari Polyclinic affiliated to Shiraz University of Medical Sciences, Shiraz, Iran which had indication of treatment. The patients were randomly selected according to inclusion and exclusion criteria. Control group consisted of 225 healthy subjects. The frequency of C.32T allele of TLR-7 was determined in154 patients with chronic HCV-infection, and in 225 healthy controls. Treatment with interferon-alpha and ribavirin was performed after genotype determination. Sustained virologic response [SVR] and end treatment response [ETR] were determined and effect of C.32T allele of TLR-7 on outcomes of treatment was evaluated. The frequency of C.32T allele of TLR-7 in patients with chronic hepatitis C was 15.33% in male, 14.67% in female and totally 15.2%. The frequency of C.32T allele of TLR-7 in healthy control group was 16.24% in male, 10.3% in female and totally 14.67%. The rate of Sustained Virologic Response [SVR] was 75%, but in patients that had C.32T allele of TLR-7, SVR was 55% [p=0.046]. c.32A>T single nucleotide polymorphism of TLR-7, by impairment of TLR-7 function, can be considered among host factors that had unfavorable effect on response rate to treatment of patients with chronic HCV hepatitis

Isolation, cloning, expression and purification of recombinant RhD antigen from cord blood

Rh [Rhesus] is a highly complex blood group system in man deeply rooted in transfusion medicine. Isolation of RhD from cord blood, cloning and expression of recombinant RhD antigen in bacterial expression system was the aim of this study. Total RNAs were extracted from cord blood [O[+]]. The quality of RNA was determined by electrophoresis. In order to obtain coding sequence of RhD antigen cDNA was synthesized and Rh D gene was amplified by RT-PCR. The isolated RhD gene was cloned to pUCIS vector and transformed to DH5alpha. The confirmed construct was sub cloned into expression vector, pBADgIII/A, and expressed in Top 10 E.coli. The expressed protein was characterized by SDS-PAGE and western blot analysis. Antigenicity of the expressed protein was assessed by ELISA using commercially available human anti-RhD polyclonal antibody with peroxidase conjugated goat anti-human IgG, IgM, IgA as secondary antibody. RhD gene was successfully cloned and expressed. The expected size of recombinant RhD protein was detected in SDS-PAGE, and confirmed by dot and western blot analysis. RhD antibody reacted with recombinant RhD antigen as well as with RhD polypeptide extracted from RBCs membrane. The recombinant RhD may be helpful to further investigate the molecular basis of RhD protein and could be applicable for production anti- D antibody in an animal model

[Isolation and purification of Ag 85 complex from mycobacterium bovis [BCG] and assessment of its cell proliferation response in whole blood]

Introduction: Tuberculosis [TB] caused by Mycobacterium tuberculosis remains a major worldwide health problem. The only TB vaccine currently available is an attenuated strain of mycobacterium bovis termed Bacillus Calmette -Guerin [BCG]. The efficacy of BCG remains controversial. Mycobacterium secretory proteins are generally considered important antigens for protection against TB. A major protein component of mycobacterial culture filtrate is Antigen 85 complex which is a promising potential vaccine candidate

Material and Methods: Antigen 85 complex was purified from Mycobacterium bovis [BCG] culture filtrate by sequential chromatography on phenyl Sepharose 4B, DEAE-Sephacel and Superdex G75. Purification was confirmed by SDS-PAGE and immunoblotting with a specific monoclonal antibody. The in vitro ability of Ag 85 complex to stimulate cell proliferation was compared with that of Purified Protein Derivative [PPD] and the polyclonal T cell mitogen PHA in a whole blood assay in which the target cells were derived from 25 healthy PPD-positive and 25 healthy PPD-negative subjects

Results: Antigen 85 complex was found to have a molecular weight of 30-32 KDa by SDS-polyacrylamide gel electrophoresis and reacted strongly by immunoblotting with the monoclonal antibody specified against Ag 85. The responses to Ag 85 and PPD were significantly higher in cells of PPD- positive than in cells of PPD-negative donors. Eighty eight percent [22/25] of the PPD- positive cells responded to Ag 85 whereas only 16% [4/25] of the PPD-negative cells responded

Conclusion: The cell proliferation response to Ag 85 complex is significantly different between cells of skin-test positive and skin- test negative subjects and may suggest an immuno-protective role for Ag 85 complex against M. tuberculosis infection

Correlation of corneal allograft rejection with tumor necrosis factors-alpha gene polymorphism

Correlations between bone marrow, heart, kidney, liver, skin and lung transplant rejection or survival with human cytokine gene polymorphisms have been described. There are also reports about the role of cytokines and Tumor Necrosis Factors-Alpha [TNF-alpha] on corneal transplant in animal models. Further studies are needed to clarify the role of cytokines in corneal allograft rejection in humans. To study whether corneal allograft rejection is associated with TNF-alpha gene polymorphism. A total of 105 cases of corneal transplant were followed for a mean period of 25.9 months and the episodes of rejections recorded. We determined allele-specific PCR [ASPCR] TNF-alpha gene polymorphism of the patients and evaluated their association with rejection. Results; The overall incidence of corneal graft rejection and its subsequent recovery were 21% and 63.6% respectively. Rejection was more common in the vascularized corneas [5.4 folds; P<0.001], and in eyes with anterior synechia [3.9 fold; P<0.05]. There was no correlation between TNF-alpha gene polymorphism and the chance of allograft rejection. No association was found between human TNF-alpha 308 G/A promoter gene polymorphism and corneal allograft rejection in our cases of uncomplicated penetrating keratoplasty

Production of prostate-specific antigen [PSA] by a breast cancer cell line, SK-Br-3

PSA is a 33-KDa serine protease that is produced predominantly by prostate epithelium. However, it has been shown that about 30-40% of female breast tumors produce PSA and its production is associated with the presence of estrogen and progesterone receptors. We have now developed a new tissue culture system to study PSA production in breast cancer and its association with prognostic factors such as progesterone receptor and c-erbB- 2. For this purpose we investigated the ability of PSA production in five different cell lines, including two breast cancer cell lines, SK-Br-3 and MDA-MB-453. The PSA in tissue culture supernatant and cytoplasm of the Sk-Br-3 cell line was detected by western blotting and immunoperoxidase, respectively. Furthermore, we found lower expression of c-erbB-2 in Sk-Br-3 than non-PSA producer breast cancer cell line, MDA-MB453. Progesterone receptor was expressed by both PSA-positive and -negative cell lines and only the intensity of staining and the number of positive cells in SkBr-3 population was higher than MDA-MB-453. According to our findings PSA can be considered as a good prognostic factor in breast cancer and we suggest that these two cell lines are a good in vitro model to study the relationship of different breast cancer prognostic factors and their regulations