Tolerance induction by CD40 silenced dendritic cells through antisense

One of the valuable tools for inhibiting the specific gene expression is antisense technique. To determine T cell responses, co-stimulatory molecule expression on the antigen presenting cells is important. In the present study, the effects of high affinity antisense against CD40 mRNA on the function and phenotype of DCs [dendritic cells] were investigated. The DCs were separated from the mice spleens and then cultured in vitro. By means of lipofectamine 2000, the antisense was delivered into the cells and the efficacy of transfection was estimated by flow cytometry. Also, the mRNA expression and protein synthesis were assessed by real time PCR and flow cytometry, respectively. The DCs were transfected with 6 M antisense and 2 l lipofectamine 2000. The percentage of CD40 expression in DCs was 38%. The results showed that CD40 expression is reduced in DCs to 22% and 24%. By annexine V and propidium iodine staining, we could evaluate the viability of the transfected cells. The inhibition of CD40 gene expression was associated with the increase in IL-4 secretion. This shifted the DCs to stimulate Th2 cytokine production from the allogenic T cells. In addition, in the MLR, the DCs without CD40 expression showed poor allostimulatory effects. This finding is valuable in the study of the costimulatory molecules of DCs. These data demonstrate that direct interference of the cell surface expression of CD40 at transcriptional level by antisense confers tolerogenecity potential of DCs. This approach is a useful tool through which DCs become tolerogenic and can be studied as a potential therapeutic option for the autoimmune diseases and allograft rejection

Effect of pregnant mouse serum on induction of indolamine 2, 3-dioxygenase in dendritic cells

Several studies have shown that many factors are involved in the maternal tolerance to the fetus. Indolamine 2, 3- dioxygenase [IDO] enzyme which catabolizes tryptophan is one of the factors that have been reported to play an important role leading to a successful pregnancy. The objective of this study was to evaluate the effects of pregnant mouse serum on the induction of indolamine 2, 3 dioxygenase in dendritic cells [DCs] which may be used as a basis for practical studies on the immunological bases of recurrent abortions. Allogenic pregnant mice sera were collected in mid-pregnancy. DCs were isolated from Balb/c mouse spleen through a three-step method, including: Collagenase digestion of spleen tissue, low density cells separation via the Nycodenz density gradient centri-fugation and plastic adherence. T cells were isolated from C57BL/6 mouse lymph nodes through nylon wool method. As stimulator cells, pregnant and non-pregnant mice sera treated DCs were irradiated and co-cultured with purified T cells [allogenic MLR]. 1-methyl-tryptophan [1-MT], as the specific inhibitor of IDO, was added to some wells of MLR assay in different concentrations and T cells proliferation response was measured by [3]H-Thymidine incorporation. The MLR supernatant was also analyzed by HPLC for its tryptophan and kynurenin [Trp metabolite] content. All tests were repeated for 5 times. Man-Whitney's non- parametric test was used to evaluate the differences among groups. Confidence interval was 95% and p-values <0.05 were regarded as significant. The results showed the ability of pregnant mice sera to reduce the dendritic cells ability in T cell proliferation induction compared to non-pregnant mice sera but addition of 1-MT did not have any significant effect on this inhibition. Additionally, IDO metabolites concentration assessment in the presence or absence of 1-MT, through HPLC method, did not show any significant difference. There are many factors in pregnant mice sera such as progesterone, IL-10, Vit D3, etc. Which might cause inhibition of T lymphocyte proliferation response in allogenic MLR through affecting DCs' efficiency. Although it seems that IDO expression by DCs is not responsible for decrease in T cell proliferation after treatment of DCs by pregnant mice sera, thus some other mechanisms might be responsible for this phenomenon which their identification needs more investigation

[Preparation of fetal calf serum alternatives and their effects on growth and secretion of hybridoma cell lines]

Providing fetal calf serum [FCS] alternatives as cell culture supplements is an important field of research to compensate for the FCS supply shortage.This study focused on preparation of fetal calf serum alternatives and their effects on growth and secretion of hybridoma cell lines.Outdated human platelet units undergo extraction for its growth factors to be obtained. Human AB blood group plasma was also converted to serum and its growth effect was compared to FCS, hypoxanthine-thymidine [HT] and RPMI1640 as cell culture media and supplement. Cell growth indices were preliminary counting of cells, confluency as surface area of plates filled with cells, and titration of monoclonal anti-A and anti-B blood group antibodies collected from cultured mouse hybridoma cells. Statistical analysis including one sample t-test, logarithmic multiple regression curve fit, and factor analysis was done by SPSS v12 software. The four nutritional supplements of [1] human serum AB [AB], [2] human platelet extract [PLT], [3] equal mixture of AB and PLT [ABP], and [4] fetal calf serum as cell culture were examined on mouse hybridoma anti-A and anti-B monoclonal antibody producer cell lines for cell growth indices and compared with the same indices on RPMI1640 media. The growth-stimulating effects in descending order of values were [1] ABP5%, [2] FCS10%, [3] ABP10%, [4] AB5%, [5] AB10%, [6] PLT5%, [7] ABP20%, [8] PLT10%, [9] PLT20%, and [10]HT; but AB20% inhibited growth of mentioned hybridoma cell lines. The titer of anti-A and anti-B monoclonal antibodies produced by cultured hybridoma on 5 and 10 percent concentration of AB, PLT and ABP compared to FCS5-10% at descending order were [1] PLT5%, [2] PLT10%, ABP5%, ABP10%, AB10%, and [3] AB5%. In general FCS had the following effects on curves of cell growth: [1] the highest increase on slope of multiplication [ascending] phase, [2] the highest increase on slope of death [descending] phase, and [3] the lowest duration of stationary phase. Then, FCS can be appropriate for growth of cells at initial low cell count. Human serum AB, human platelet extract, and equal mixture of both at optimum concentrations [these supplements at high concentrations killed cells] compared to FCS showed [1] decreased slope of multiplication phase, [2] decreased slope of death phase, and [3] increased duration of stationary phase. Thus, AB and PLT may be suitable for continuous cell culture systems in which cell survival during longer times is required. Factor analysis was introduced as a model to evaluate kinetics of cell growth at different supplements

[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

[Allostimulatory efficiency of splenic dendritic cells from pregnant and non pregnant mice]

Mammalian reproduction looks like an immunological paradox, because fetal alloantigens encoded by father genes should induce cell mediated immune responses leading to fetal loss. Maternal immune system, in addition to local modulation, undergoes systemic modulations during pregnancy. Dendritic cells [DCs], as professional antigen presenting cells, play a key role in initiation and control of immune response and it seems that functional changes in these cells during pregnancy may contribute to the systemic immune tolerance. To address this issue, in this study we isolated and purified DCs from pregnant mice and evaluated their stimulatory potential to induce proliferative response of allogenic T cells in unidirectional mixed leukocyte reaction [MLR]. Following collagenase digestion of splenic tissue, using density gradient centrifugation [13% Nycodenz] and adherence properties of DCs to the bottom of tissue culture dish, 7x10[5] DCs were isolated from each spleen with more than 95 percent purity. Allogenic T cells were isolated by nylon wool column, using their non-adhesive character to nylon wool. After radiation, isolated dendritic cells from pregnant and non-pregnant Balb/c mice were used in mixed leukocyte culture with C57BL/6 mice T lymphocytes. T lymphocyte proliferation was measured after 72 hours by [3]H- thymidine incorporation. 7x 10[5] dendritic cells with the purity of >95% were isolated from each spleen. Also the yield of T- lymphocyte form Inguinal and Brachial lymph nodes was about 3-5x10[5] with the purity of%85-90. The results showed that there is no statistical difference between stimulatory potential of DCs form pregnant [cpm=33000] and non- pregnant [cpm=35000] mice in induction of allogenic T-Cell proliferation. These findings can result from low concentration of immune suppressor factors in circulatory system of pregnant mice or due to separation of dendritic cells from pregnancy microenvironment and their maturity in vitro in the absence of the immune suppressor factors

Nitric oxide production following vaccination with killed mycobacterium tuberculosis [H37Rv] and BCG

Protective immune response induced by viable BCG has been suggested by several investigators. Both killed BCG and Mycobacterium tuberculosis are able to induce response. Nitric oxide [NO] is one of the non- specific responses produced against these agents. To survey the effect of alive, killed BCG and also killed Mycobacterium tuberculosis [H37Rv strain] on NO Production. 6-8- week-old female BALB/c mice were used. Three groups were vaccinated with viable, killed BCG and killed Mtb, respectively. One group received PBS as a control. After 5-8 weeks of vaccination, peritoneal cells of all groups were collected in usual manner and plated out in 96-well plates. Cells were treated with killed H37Rv, killed BCG and viable BCG alone or with rIFN gamma and NO inhibitors [aminoguanidine and NGMA]. Supernatant of each well was collected after 24h. NO level was estimated by Griess method by ELISA reader at 540nm absorbance. Results indicated that NO induction level in vaccinated groups were higher than control [P