[Expression of recombinant protein B subunit pili from vibrio Cholera]

Vibrio cholerae is a gram-negative bacterial pathogen that causes cholera disease. Following ingestion by a host and entry into the upper intestine, V. cholera colonizes and begins to emit enterotoxin. One of the most pathogenic factors of Vibrio cholera is toxin-coregulated pili [TCP]. Toxin-Coregulated pili is as the primary factor requiered for the colonization and insistence of bacteria in the small intestine. The toxin-coregulated pili are bundle-forming pili that are coordinately regulated with cholerae toxin [CT]. The CT operon is part of the genome of the cholera toxin bacteriophage [CTXQ] which utilizes TCP as its receptor. The aim of this study is to produce a recombinant vaccine for V. cholerae in the future. The tcpB gene was amplified by Polymerase chain reaction [PCR] method and subcloned into pET32a expression vector. Escherichia coli BL21 [DE3] plysS competent cells were transformed by pET32a - tcpB recombinant plasmid. In different media with changing the parameters of nutrient content like glucose as carbon source and yeast extract as nitrogen source, protein expression was induced by using IPTG. Recombinant protein were purified by affinity chromatography [Ni-NTA]. The concentration of Recombinant proteins measured according to Bradford assay. The sequencing results by Sanger method showed a similar sequence as tcpB gene. Escherichia coli BL21 plysS was transformed with TCPB-pET32a and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography and Ni-NTA kit. Recombinant protein tcpB was produced in the cytoplasm of Escherichia coli BL21 plysS, by pET32a expression vector. Therefore, utilization of this protein in Escherichia coli BL21 plysS by expression vectors such as pET32a is possible

[Anti-human herpes virus-6 antibodies titer in patients with multiple sclerosis in Markazy Province]

Multiple sclerosis [MS] is a auto-immune disease of central nervous system. The etiology of MS is unknown, but environmental factors such as viruses are involved in the development of MS. In this study, MS patients were assessed for antibodies titers against Human Herpes virus-6 [HHV-6] in Markazi Province. In this case-control study, 31 new cases of MS patients and 60 healthy subjects were selected with similar demographic criteria such as sex, age and location. Antibodies titer [IgM and IgG] against HHV-6 were examined by ELISA and Immunofluorescence methods. Data were analyzed using Logistic regression and Odds ratio. Data indicates that 74.2% of case group and 34.2% of control group were identified as positive for IgM against HHV-6. The difference between the two groups in terms of IgM against HHv-6 was statistically significant [p=0.001]. Incidence of IgM positivity against HHV-6 was increased more than five times in MS patients compared to control group. Also there was a statistically significant difference between case and control groups in IgG titer [p=0.019]. Acute infection of HHV-6 is a risk factor for MS

[Effect of vitamin D3 on IFN-T and IL-10 levels in mice with experimental a utoimmune encephalomylitis]

Multiple sclerosis [MS] is a chronic autoimmune disease with unknown etiology affecting the central nervous system. The pre9alence of MS is highest where environmental supplies of vitamin Dare lowest. Some studies have shown a strong protective effect of vitamin D3 in experimental autoimmune encephalomyelitis [EAE]; a model of MS. However, it is not known whether vitamin D3 has a protective effect in EAE. Vitamin D3 may be inhibit EAE by having an effect on TH1 and TH2 immune responses. To address this question, the effect of vitamin D[3] on cellular immune responses in C57BL/6 mice with experimental autoimmune encephalomyelitis was investigated. Male C57HL/6 mice matched in age and weight were placed in two therapeutic groups [n10 per group] as follows: Vitamin D3-treated EAE mice [5 micro g/kg/every two days of vitamin D3 given i.p from day -3 until day +19 after disease induction]. Non-treated EAE mice [EAE control] received vehicle alone with same schedule. 20 days after immunization, the mononuclear cells [MNCs] of the spleen were isolated from mice and cultured in the presence and absence of MOG35-55 for 96 hours. The supematant of cultured cells was collected and produced cytokines [IL-10 and IFN-gamma] were assayed by ELISA. The results showed that vitamin D3-treated mice had less severe clinical signs and synptoms of EAE [3.2 +/- 0.8] than non-treated EAE induced mice [5.3 +/- 0.44]. [p=0.001]. Also, there was a significant difference regarding the day of onset of disease in the vitamin D3-treated and non treated EAE-induced mice [day 15 +/- 1 and day 11 +/- 1, respectively]. There was no significant difference in IFN-y production between treated and non-treated mice, but the amount of 11-10 production in the D3-treated mice was higher than the non-treated group [p=0.001]. Considering the role of TH1 in the pathogenesis of EAE and MS. it is suggested that vitamin D3 can reduce or delay the onset of EAE by shifting immune responses to TH2 and IL-10 production. Thus, vitamin D3 as an immune modulatory agent is potentially important for treatment of MS

Effect of Sesame oil on leukocyte infiltration into the brain of C57/BL6 mice with experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis [EAE] is an animal model of multiple sclerosis distinguished by infiltration of leukocytes into the central nervous system. Changes in composition and levels of unsaturated fatty acids, affect the integrity of blood-brain barrier. In this study, we evaluated the effect of Sesame oil on the leukocyte infiltration into the brain of MOG[35-55] induced EAE male C57BL/6 mice. In this experimental study, male C57BL/6 mice were placed in two therapeutic groups [n=10 per group] with age and weight-matched as follow: 1.Sesame oil-treated EAE mice received 4ml/kg/day of Sesame oil given i.p. from day -3 until day +19 after disease induction, 2.Non-treated EAE mice [EAE control] received Phosphate buffer alone with same schedule. EAE was induced by immunization of mice with MOG[35-55] peptide and complete Freund's adjuvant. Leukocytes infiltration into the brain was investigated 20 days after immunization. Data was analyzed using Mann-Whitney U test. The results show that Sesame oil-treated mice had significantly less clinical score of EAE [2.6 +/- 0.4] than non-treated EAE induced mice [4.2 +/- 0.6], [p<0.001]. Also, there was a significant difference at number of the infiltrating cells in brain between Sesame oil-treated [80 +/- 20] and non treated EAE-induced mice [150 +/- 30], [p<0.01]. These results indicate that Sesame oil reduces infiltration of leukocytes into the brain of EAE mice, therefore lessening the histological changes and clinical signs and thus ameliorating the disease

[Effect of vitamin E on the inhibition of experimental autoimmune encephalomyelitis in C57BL/6 mouse]

Free radical-mediated per-oxidation of biological molecules, such as lipids, is implicated in the pathogenesis of multiple sclerosis and it's animal model experimental allergic encephalomyelitis [EAE]. Low concentration of antioxidant vitamin E has been observed in serum of multiple sclerosis. However, it is not known whether vitamin E has protective effect in EAE. Vitamin E may inhibit EAE by effect on the level of uric acid and Nitric Oxide [NO] production. In this experimental study some male C57BL/6 mice were placed in two therapeutic groups [n=8 per group] with age and weight-matched as follow: 1] Vitamin E-treated EAE mice [10mg/kg/every two days of vitamin E given i.P from day-3 until day + 19 after disease induction, 2] Non-treated EAE mice [EAE control] received vehicle alone with same schedule. In addition, 5 age and weight-matched male C57BL/6 mice served as normal [non-EAE] controls. Clinical score of disease, uric acid and NO levels of the groups were analysed. Results showed that vitamin E-treated mice had significantly less clinical score of EAE [4 +/- 0.8] than non-treated EAE induced mice [5.3 +/- 0.44], [p< 0.01]. Also, there was difference at the onset day of the disease between vitamin E-treated and non-treated EAE-induced mice [day 13 +/- 1 and day 11 +/- 1, respectively], although was not significant. Concentration of uric acid in vitamin E treated mice were significantly lower than EAE control [p< 0.001]. There was no difference at the level of NO between the groups. Vitamin E had no effect on NO level, but decreased serum uric acid level. It suggests that vitamin E can reduce or delay the onset of EAE by increasing uric acid consumption

[Effect of sesame oil on the development of experimental autoimmune encephalomyelitis in C57BL/6 mice]

Experimental autoimmune encephalomyelitis [EAE] is a inflammatory demyelinating autoimmune disease of the CNS that servers as an animal model for multiple sclerosis [MS]. Seame oil effect was evaluated in the treatment of EAE in C57BL/6 mice. Seame oil shows profound anti-inflammatory activity and has been traditionally used to treat inflammatory disorders. EAE was induced by immunization of 8 week old mice with MOG[35-55] with complete freunds adjuvant. Therapy with sesame oil was started on day 2 before the immunization. total antioixdant capacity [TAC] was assessed by ferric reducing-antioxidant power [FRAP] method. nitric oxide [NO] producation was also estimated by Griess reaction. After daily intraperitoneal dosage the seame oil significantly reduced the clinical symptoms in c57BL/6 mice with EAE [p< 0.01]. Also, treated mice displayed a significantly delayed disease onset compared with control mice. seame oil significantly increased TAC [p< 0.05], but had no effect on serum nitrie production. Our results suggest for the first time that seame oil therapy may be effective in the prevention of symptomatic EAE. this resistance to encephalomyelities may be associated with inhibition of oxidative stress

[Designing of a mixed ELISA method for detection of IgM and IgA rheumatoid factors]

Different isotypes of antibody can be produced by immune system after antigen contact. Detection and measurement of different classes of antibody against the antigen is very important in some cases. The aim of this study is designing of an ELISA method on the basis of inhibition of enzyme activity by using a non-competitive inhibitor. Therefore in this study rheumatoid factor is used as a model for the detection of different other classes of antibodies against the antigen.In this cross sectional analytical study, we measured IgM and IgA rheumatoid factors in sera of 10 patients with rheumatoid arthritis and positive latex test, by mixed and routine ELISA. In mixed ELISA the activity of the first conjugated enzyme was blocked by a non-competitive inhibitor after adding the substrate, then the next conjugated antibody, which was specific for another isotype, was added. By optical density, results was comparisoned with routine ELISA.The obtained results showed that the average optical density is lower when compared with routine ELISA, but the difference is not statistically significant. However these two methods did not show any significant difference in quantifying antibody isotypes. Also there is a positive association between mixed and routine ELISA [r =0.9, p=0.001].Lower optical density in mixed ELISA is probably because of stick hindrance by the first conjugate. So, because there is no significant difference between the results of these two types of ELISA, and also no need to repeat the test for each isotype in this method, it is recommended to use the new method instead of the routine one to save time and reagents