Effects of Reticuloendotheliosis virus on TLR-3/IFN-Β pathway in specific pathogen-free chickens.

Birds infected by Reticuloendotheliosis virus (REV) are vulnerable to other microorganisms. This immunosuppression is related to the immune organs (thymus, bursa of Fabricius, and spleen) damaged by REV. The regulation of IFN-ß greatly depends on pattern recognition receptor TLR-3 and nuclear factors IRF-7, NF-κB. To address if and how the TLR-3/IFN-ß pathway is disturbed by REV, 60 one-day-old specific-pathogen-free chickens were intraperitoneally injected with RE virus dilution (n = 30) or stroke-physiological saline solution (n = 30). At 1, 3, 7, 21, and 28 days post-infection, after collecting thymuses, bursas, and spleens, we monitor the kinetics of TLR-3, IFN-ß, NF-κB p65, and IRF-7 at transcriptional and translational levels using qPCR, Western blotting, and ELISA separately. As a result, compared with control chickens, the mRNA levels of TLR-3, IRF-7, and NF-κB p65 showed increasingly differences in the early period of REV infection. Synchronal changes occurred at translation levels. In the latter infection period, a decrease of NF-κB p65 was contemporaneous with a fall in IFN-ß at both transcriptional and translational levels in the thymuses and bursas. These data suggest that the changes of IFN-ß content are closely related to NF-κB p65 when REV invades chicken central immune organs. That reveals new insights into the immunosuppression mechanism of REV in avian.

Oxidized low-density lipoprotein stimulates CD206 positive macrophages upregulating CD44 and CD133 expression in colorectal cancer with high-fat diet.

BACKGROUND: Oxidized low-density lipoprotein (ox-LDL), which is abnormally increased in the serum of colorectal cancer (CRC) patients consuming a high-fat diet (HFD), may be one of the risk factors for the development of CRC. Ox-LDL exerts a regulatory effect on macrophages and may influence CRC through the tumor microenvironment. The role of ox-LDL in CRC remains unclear. AIM: To investigate the role of ox-LDL through macrophages in HFD associated CRC. METHODS: The expression of ox-LDL and CD206 was detected in colorectal tissues of CRC patients with hyperlipidemia and HFD-fed mice by immunofluorescence. We stimulated the macrophages with 20 µg/mL ox-LDL and assessed the expression levels of CD206 and the cytokines by cell fluorescence and quantitative polymerase chain reaction. We further knocked down LOX-1, the surface receptor of ox-LDL, to confirm the function of ox-LDL in macrophages. Then, LoVo cells were co-cultured with the stimulated macrophages to analyze the CD44 and CD133 expression by western blot. RESULTS: The expression of ox-LDL and the CD206 was significantly increased in the stroma of colorectal tissues of CRC patients with hyperlipidemia, and also upregulated in the HFD-fed mice. Moreover, an increased level of CD206 and decreased level of inducible nitric oxide synthase were observed in macrophages after ox-LDL continuous stimulation. Such effects were inhibited when the surface receptor LOX-1 was knocked down in macrophages. Ox-LDL could induce CD206+ macrophages, which resulted in high expression of CD44 and CD133 in co-cultured LoVo cells. CONCLUSION: Ox-LDL stimulates CD206+ macrophages to upregulate CD44 and CD133 expression in HFD related CRC.

BARX2 expression is downregulated by CpG island hypermethylation and is associated with suppressed cell proliferation and invasion of gastric cancer cells.

BarH­like homeobox 2 (BARX2), a homeobox gene, is associated with several types of cancers. The present study aimed to determine whether DNA methylation downregulates BARX2 expression and whether BARX2 is associated with suppression of gastric carcinogenesis. BARX2 protein expression in normal and cancerous gastric tissues and various gastric cancer (GC) cell lines was detected using immunohistochemical and western blot assays. BARX2 mRNA levels were detected using both reverse transcription­polymerase chain reaction (RT­PCR) and quantitative PCR (qPCR). Promoter hypermethylation in GC cells was detected using methylation­specific PCR or bisulfite DNA sequencing PCR. Effects of BARX2 expression on GC cell proliferation, clonal formation, and migration were evaluated after lentivirus­BARX2 transfection. The effect of stable BARX2 transfection on tumor formation was assessed in a nude xenograft mouse model. BARX2 was strongly expressed in the normal gastric mucosa, but weakly or not expressed in GC tissues and most GC cell lines. BARX2 expression was negatively correlated with DNMT (a marker for DNA methylation) expression in the gastric tissues. The BARX2 promoter fragment was hypermethylated in the GC cell lines. Overexpression of BARX2 significantly inhibited GC cell proliferation, clonal formation, and migration. Stable BARX2 transfection inhibited tumor formation in xenograft mice, which was correlated with decreased expression of E­cadherin, proliferation markers, and matrix metalloproteinases. In conclusion, BARX2 expression is aberrantly reduced in GC, which is associated with increased DNA methylation of its promoter. BARX2 inhibits GC cell proliferation, migration, and tumor formation, suggesting that BARX2 acts as a tumor suppressor in gastric carcinogenesis.