TLR4, IgA and EpCAM Expression in Colorectal Cancer and Their Possible Association with Microbiota as a Pathogenic Factor; An Immunohistochemical and Genetic Study.

BACKGROUND: The pathogenesis of inflammatory bowel disease (IBD) and colorectal cancer (CRC) is thought to be related to immune response against gut microbiota. TLR4, IgA, and EpCAM have a role in intestinal local immune response and their altered expression related to both IBD and CRC. Lipopolysaccharide (LPS) is the main activator of TLR4. The objective of this study is to evaluate the possible role of intestinal microbiota in the pathogenesis of IBD and CRC through expression of TLR4, IgA and EpCAM. METHODS: One hundred five cases were divided into (Group 1/ Control: 10 sections of normal colonic mucosa, Group 2/CRC: 51 cases, Group 3/IBD: 44 cases). Immunohistochemistry for TLR4, IgA, and EpCAM was done. LPS was assessed in all groups. TLR4 gene and protein expression were assessed in colorectal cancer cell line by RT-PCR and immunocytochemistry. RESULTS: There was a significant correlation between TLR4 and tumor grade (P value 0.003 and 0.01 respectively). A significant correlation was found between IgA expression and T stage (P value 0.02) and between EpCAM expression and histologic type (P value 0.02). In comparison of CRC patients to controls; there was a statistically significant different expression of TLR4 positivity, IgA positivity and EpCAM (P value <0.001, 0.004, <0.001 respectively). Patients with CRC were compared to colitis patients and there was a statistically significant different expression of IgA positivity and EpCAM expression (P value <0.001). There was significant higher expression of TLR4 in CRC cell line than the fibroblast by both PCR and immunocytochemistry (P-value: 0.003 and 0.024 respectively). LPS level in CRC patients was significantly higher than the control and IBD groups (P values <0.001 and <0.001 respectively). CONCLUSION: TLR4, IgA, EpCAM expression in both CRC and IBD might be related to the pathogenic role of microbiota and could represent potential prevention modalities and therapeutic targets.

Determination of a CD4+CD25+ Foxp3+T cells subset in Egyptian Colorectal Cancer Patients.

Human tumors including colorectal cancers (CRC) are often infiltrated by immune cells predominantly T lymphocytes especially regulatory T (Treg) cells expressing the forkhead box protein 3 (Foxp3). It has been suggested that CD25+CD4+Foxp3+ regulatory T cells (Tregs) might hamper effective immunosurveillance of emerging cancer cell. The aim of this study was to measure the frequency of total CD4+CD25+ Tregs & CD4+CD25+Foxp3+ subset of Treg cells in peripheral blood of Egyptian CRC patients and their correlation with the tumor stage, histopathology of the tumor and lymph node affection. A total of 31 CRC patients were enrolled in the study. The tumor was categorized using a TNM staging system. Peripheral blood samples were collected within the first 24 h of surgery. The frequency of total CD4+CD25+ Tregs & CD4+CD25+ Foxp3+ subset of Treg cells in peripheral blood mononuclear cells (PBMCs) were measured by flow cytometry and absolute count was determined. High frequency of Tregs was detected in cancer patients with distal margin involvement (44-48 cells/µL) compared with those with free distal margin (5-32 cells/µL). Similarly, higher frequency of Tregs were detected (16-44 cells/µL) in cancers with lymph node involvement compared with cancers without lymph node involvement (5-32 cells/µL). Higher frequency of CD4+CD25+Foxp3+ Tregs were found in mucinous adenocarcinomas than in other histopathological types, although both observations were statistically insignificant. The median value for total absolute lymphocyte count/ µL was 639, out of which CD4+CD25+ subset constituted 35 cells, and about half of this subset were Foxp3+Tregs. In conclusion, CD4+CD25+Foxp3+ Tregs may be a useful marker for predicting invasion, metastasis, and prognosis of colorectal cancer in Egyptian patients.

Gene polymorphisms of Interleukin 1? and Metalloproteinase 3 in Hepatitis C Infected Patients and Hepatocellular Carcinoma Patients.

Cytokines as IL 1? and matrix metalloproteinases (MMPs) are involved in tumor growth, invasion, and remote metastasis in various cancers. IL-1? ?31T/C polymorphism in the promoter region has been linked to an elevated risk of Hepatocellular carcinoma (HCC). MMP3 polymorphism at 1171 has also been linked to an elevated risk and a highly invasive type of HCC. Studying the association between IL-1? 31C/T gene polymorphism and MMP3 11715A/6A gene polymorphism in cases of Hepatitis C virus (HCV) and HCC. The study was done on 20 HCV infected patients, 20 HCC patients and 15 healthy controls. IL-1? 31 C/T and MMP3 1171 5A/6A gene polymorphisms was investigated using restriction fragment length polymorphism discrimination assay. Data revealed that the frequency of IL-1? C/T and MMP3 5A/6A polymorphisms was higher in patient groups (HCC and HCV) compared with healthy controls. A significant association was found between IL-1? C/T, MMP3 5A/6A polymorphism and HCC susceptibility. In conclusion, IL-1? C/T and MMP3 5A/6A polymorphisms are associated with an increased risk of developing HCC in Egyptian patients.

MicroRNA 26a Expression in Peripheral Blood Mononuclear Cells and Correlation with Serum Interleukin-17 in Relapsing-Remitting Multiple Sclerosis Patients.

Multiple sclerosis (MS) is a chronic multifocal inflammatory demyelinating disease. One of the main cells that play a crucial role in pathogenesis of MS is T helper 17 (Th 17). There are growing interests in nominating microRNAs in Th17 cell differentiation and suggesting new therapeutic modalities. The aim of the study was to assess microRNA 26a (miR26a) expression in peripheral blood mononuclear cells of relapsing - remitting MS patients as compared to healthy control subjects and examine association of these levels with serum IL17. Forty (40) relapsing - remitting MS patients were enrolled based on the MacDonald criteria (20 in relapsing phase and 20 in remitting phase). In addition, twenty (20) healthy control subjects were included. Blood samples were subjected to quantitative polymerase chain reaction (qPCR) for miR26a and ELISA for serum IL 17 levels. A significant upregulation of miR26a relative expression level (∆∆ Ct) and serum IL17 level (pg/ml) was found in total MS patients and remitting MS patients when compared with controls (P < 0.001). Among the relapsing group, a significant increase in miR26a expression levels (P= 0.004) but not serum IL17 level was demonstrated. Insignificant correlation between miR26a expression and serum IL17 in MS patients was detected (r= 0.08, P= 0.62). In conclusion, a significant increase of these two biomarkers (miR26a & IL17) occurs in relapsing - remitting MS patients, and this reflects their important role in pathogenesis and disease development.