[Expression of T399I and D299G polymorphisms of TLR4 gene in colorectal cancer cell line by flowcytometery]

Toll-like receptors [TLRs] have been discovered as the most important receptors in innate immunity. One of the most important TLRs is TLR4, the key receptor for the LPS component of gram-negative bacteria. Two polymorphisms, D299G [rs4986790] and T399I [rs4986791], in TLR4 gene are associated with a decreased response to LPS. This study was done to estimate the expression of different polymorphisms of TLR4 gene in colorectal cancer cell line by flowcytometery. In this laboratory study, the HCT116 cells were transfected with plasmids containing different variants of TLR4 gene including; Flag-tagged-TLR4 wild type, flag-tagged D299G and T399I Using TurboFect transfection reagent. Transfection efficiency was evaluated by GFP plasmid. Expression of different variants of TLR4 was assessed in transfected cells by flowcytometery. Data were analyzed using SPSS-11.5 and chi-square test. TLR4 was detected on HT29 and CaCo[2] cell lines at low levels. HCT116 cells did not express detectable amounts of TLR4 by flowcytometery prior to transfection. Gene transfer efficiency for GFP plasmid was about 80% in HCT116 cells by flowcytometery and microscopic analysis. TLR4 expression and LPS responsiveness significantly was higher in HCT116 cells which were transfected with wild type TLR4 gene compared to non-transfected and mutant transfected cells [P<0.05]. Lower expression of TLR4 on cells with mutant TLR4 showed that these polymorphisms affect on expression patterns of TLR4 on colon cancer cells

Human bone marrow-derived mesenchymal stem cells suppress T cell proliferation by inducing cell cycle arrest

Mesenchymal stem cells (MSC) are common residents of bone marrow and are defined by their higher self-renewal ability and multilineage differentiation. MSC play an important role in supporting haematopoiesis and therefore are implicated in influencing immune responses. In line with this, MSC have been utilized to treat graft-versus-host disease (GVHD) in order to suppress unwanted T cell proliferation. In this study, we investigated the immune-suppressive effect of bone marrow derived MSC on T cell proliferation at the cell cycle level. MSC were generated from human bone marrow and confirmed by their immune-phenotyping. Resting or PHA stimulated allogeneic peripheral blood mononuclear cells (PBMC) were co- cultured in the presence or absence of MSC. T cell proliferation was accessed by trypan-blue exclusion assay at day three. Consequently cell cycle analysis was carried out to determine the mechanism of antiproliferation. MSC failed to elicit proliferation at resting T cell. However, proliferation of PHA-stimulated T cells was dramatically inhibited in the presence of MSC in a dose dependent manner (p<0.05). Following the inhibitory activity, MSC prevented activated T cells from entering the S phase of cell cycle by arresting them in the G1 phase. Our findings indicate that MSC escape recognition by T cells and inhibit T cell proliferation by cell cycle arrest at G1 phase. This immune-suppressive effect is most probably mediated by cell-to-cell contact and/or secreted soluble factors.

Biomarkers in cancer: An overview

Traditional tumour markers such as carcinoembryonic antigen (CEA) that have been used for screening gastrointestinal neoplasia for many years are not specific. However, these markers are useful after diagnosis to monitor progress of the disease and recurrence. New biomarkers are constantly being developed to identify individuals with risk of cancer for early detection, to determine prognosis, to detect recurrence, to predict drug responses and to monitor response to treatment. There are several issues involved in the discovery of biomarkers and their development for clinical applications. This article provides a basic overview of the classes of biomarkers, the current status of molecular profiling and discusses the opportunities as well as challenges ahead to improve biomarker development.