Patterns of de novo allo B cells and antibody formation in chronic cardiac allograft rejection after alemtuzumab treatment.

Even though the etiology of chronic rejection (CR) is multifactorial, donor specific antibody (DSA) is considered to have a causal effect on CR development. Currently the antibody-mediated mechanisms during CR are poorly understood due to lack of proper animal models and tools. In a clinical setting, we previously demonstrated that induction therapy by lymphocyte depletion, using alemtuzumab (anti-human CD52), is associated with an increased incidence of serum alloantibody, C4d deposition and antibody-mediated rejection in human patients. In this study, the effects of T cell depletion in the development of antibody-mediated rejection were examined using human CD52 transgenic (CD52Tg) mice treated with alemtuzumab. Fully mismatched cardiac allografts were transplanted into alemtuzumab treated CD52Tg mice and showed no acute rejection while untreated recipients acutely rejected their grafts. However, approximately half of long-term recipients showed increased degree of vasculopathy, fibrosis and perivascular C3d depositions at posttransplant day 100. The development of CR correlated with DSA and C3d deposition in the graft. Using novel tracking tools to monitor donor-specific B cells, alloreactive B cells were shown to increase in accordance with DSA detection. The current animal model could provide a means of testing strategies to understand mechanisms and developing therapeutic approaches to prevent chronic rejection.

Identification of differentially-expressed proteins between early submucosal non-invasive and invasive colorectal cancer using 2D-DIGE and mass spectrometry.

Early detection and diagnosis of colorectal cancer (CRC) are closely related to a better therapeutic outcome, and the five-year survival rate of early CRC is over 90 percent. Though endoscopic minimally invasive treatment has become a quick and effective therapy for early CRC, endoscopic biopsies are usually not deep enough to obtain tissues from the submucosal layer and it is difficult to determine whether early CRC has infiltrated into the submucosa. Therefore, in the present study, we constructed tumor models of early submucosal non-invasive CRC (SNICRC) and submucosal invasive CRC (SICRC) in Fischer-344 rats induced by N-methyl-N-nitrosourea (MNU). The differentially-expressed proteins were analyzed and identified in SNICRC, SICRC and normal control (NC) tissues using highly sensitive two dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS). Proteomic data revealed 132 protein spots between SNICRC and SICRC, 162 protein spots between SICRC and NC and 154 protein spots between SNICRC and NC which were found differentially expressed. These differential spots were picked, in-gel digested and peptide mass fingerprints were obtained by MALDITOF-MS/MS. Finally, five differentially-expressed proteins in SNICRC, SICRC and NC were identified, and increases in Transgelin, peptidylprolyl isomerase A (PPIA) and tropomyosin alpha isoform d were observed, while decreases in carbonic anhydrase 2 (CAII) and an unnamed protein were detected in SICRC compared with SNICRC and NC. Furthermore, Fluorescence-based quantitative polymerase chain reaction (FQ-PCR), Western blotting and immunohistochemistry assays also revealed significant upregulation of Transgelin expression and down-regulation of CAII expression in SICRC tissues. In conclusion, 2D-DIGE is confirmed to be an efficient strategy that enables us to identify differentially expressed proteins between early SNICRC and SICRC. The potential biomarkers such as Transgelin and CAII may be used for the detection of early SICRC.

Effects of miR-223 on colorectal cancer cell proliferation and apoptosis through regulating FoxO3a/BIM.

OBJECTIVE: Colorectal cancer is a common malignant tumor of the digestive tract. It frequently occurs at the junction of the rectum and sigmoid colon. It is characterized by high mortality and poor prognosis. Bcl-2 interacting mediator of cell death (BIM) plays a role in the regulation of cell proliferation and apoptosis, and involves in the pathogenesis of colorectal cancer. The transcription factor forkhead, transcription factor O subfamily 3a (FoxO3a) plays a role in the regulation of BIM expression and is associated to the pathogenesis of colorectal cancer. Bioinformatics analysis suggests that there is a targeted relationship between FoxO3a and microRNA-223 (miR-223). This study aims to investigate effects of miR-223 on the regulation of FoxO3a/BIM signaling pathway and colorectal cancer cell proliferation and apoptosis. MATERIALS AND METHODS: Colorectal cancer cell line SW620 and normal colorectal epithelial cell line NCM460 were cultured in vitro. Dual luciferase reporter assay was used to validate the relationship between miR-223 and FoxO3a. Flow cytometry was adopted to detect apoptosis. EdU staining was applied to test cell proliferation. Western blot was selected to determine FoxO3a and BIM protein expressions. RESULTS: There was targeted regulatory relationship between miR-223 and FoxO3a. MiRa-223 up-regulated, FoxO3a and BIM expressions reduced, and cell proliferation was enhanced in SW620 cells compared with NCM460 cells. MiR-223 inhibitor or pIRES2-FoxO3a transfection significantly increased FoxO3a and BIM expressions, attenuated cell proliferation, and enhanced cell apoptosis. CONCLUSIONS: MiR-223 targeted inhibited expression of FoxO3. Down-regulating the expression of miR-223, it increased the expressions of FoxO3a and BIM, weakened SW620 cells proliferation and induced apoptosis.

MicroRNA-455 suppresses the oncogenic function of HDAC2 in human colorectal cancer.

Colorectal cancer (CRC) is the fourth leading cause of cancer-induced mortality. Histone deacetylase 2 (HDAC2) is involved in prognosis and therapy of CRC. This study aimed to explore novel therapeutic targets for CRC. The alteration of HDAC2 expression in CRC tissues was estimated by qRT-PCR. After lentivirus transfection, HDAC2 knockdown was confirmed by western blot analysis. The effect of HDAC2 knockdown on cell proliferation was then assessed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Screened by TargetScan, microRNA (miR)-455 was predicted to bind to 3'UTR of HDAC2 and the prediction was verified by luciferase assay. Finally, cells were transfected, respectively, with miR-455 mimics or miR-455 negative control (miR-NC) and the expression of HDAC2, cell proliferation and apoptosis of transfected cells were respectively evaluated by western blot analysis, MTT assay and flow cytometry. Results showed that the HDAC2 expression was up-regulated in CRC tissues (P<0.05). HDAC2 knockdown significantly decreased cell viability at day 3 (P<0.05), day 4 (P<0.01), and day 5 (P<0.001) after infection. Then, miR-455 was verified to directly target HDAC2, resulting in a significant difference in luciferase activity (P<0.01). Moreover, miR-455 decreased the expression of HDAC2 (P<0.01). miR-455 remarkably decreased cell viability at day 3 (P<0.05), day 4 (P<0.01), and day 5 (P<0.001) after transfection while inducing cell apoptosis (P<0.001). In conclusion, miR-455 inhibited cell proliferation while inducing cell apoptosis by targeting HDAC2 in CRC cells.