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

Mechanism of CEA release from the malignant cells of colorectal carcinoma

Carcinoembryonic antigen [CEA] is one of the most frequently used tumor markers which is over-expressed in a variety of human cancers with epithelial origin such as colorectal, gastric, pancreatic, lung and breast cancers. The precise mechanism by which CEA is released from the cell surface into serum in cancer patients has not been clarified so far. CEA is attached to the plasma membrane by a glycophosphatidyl inositol [GPI] anchor. There have been some evidences indicating a possible role for involvement of an enzyme in CEA release from the cell surface. The GPI-anchor is a substrate for specific phospholipases. According to these information, we investigated the possible role of GPI-specific PI-PLD [GPI-PLD] in hydrolysis and CEA release. We have, therefore, used reverse transcription-polymerase chain reaction [RT-PCR] to verify GPI-PLD expression in some cell lines of colorectal adenocarcinoma. The amount of CEA released from a high CEA producing cell line in the presence or absence of specific activators/inhibitors of this enzyme was measured. Using RT-PCR, expression of GPI-PLD gene in these cell lines can be demonstrated. The amount of CEA released from high producing cell lines are increased or decreased in the presence or absence of specific activators/inhibitors. According to this study there are evidences implicating the role of GPI-PLD enzyme in releasing CEA from the surface of the cells of colorectal carcinoma