Value of ctDNA methylation biomarkers in diagnosis of colorectal tumors.

Tweetable abstract DNA methylation alterations have been identified as promising biological markers for early-stage colorectal cancer detection. Here, the authors highlight some recent advances in DNA methylation and its role in the early diagnosis and overall disease course management of colorectal tumors. New insights into DNA methylation biomarkers for colorectal cancer early diagnosis and management are discussed.

The Protective Role of TLR4 in Intestinal Epithelial Cells through the Regulation of the Gut Microbiota in DSS-Induced Colitis in Mice.

BACKGROUND: The cause of ulcerative colitis (UC) is not yet fully understood. Previous research has pointed towards a potential role for mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in promoting the onset and progression of inflammatory bowel disease (IBD) by altering the microbiota of the gut. However, the relationship between toll-like receptor 4 (TLR4) and gut microbiota in IBD is not well understood. To shed light on this, the interaction between TLR4 and gut microbiota was studied using a mouse model of IBD. METHODS: To examine the function of TLR4 signaling in intestinal injury repair, researchers developed Dextran Sulfate Sodium Salt (DSS)-induced colitis and injury models in both wild-type (WT) mice and TLR4 knockout (TLR4-KO) mice. To assess changes in the gut microbiota, 16S rRNA sequencing was conducted on fecal samples from both the TLR4-KO and WT enteritis mouse models. RESULTS: The data obtained depicted a protective function of TLR4 against DSS-induced colitis. The gut microbiota composition was found to vary considerably between the WT and TLR4-KO mice groups as indicated by ß-diversity analysis and operational taxonomic units (OTUs) cluster. Statistical analysis of microbial multivariate variables depicted an elevated abundance of Escherichia coli/Shigella, Gammaproteobacteria, Tenerlcutes, Deferribacteres, Enterobacteria, Rikenellaceae, and Proteobacteria in the gut microbiota of TLR4-KO mice, whereas there was a considerable reduction in Bacteroidetes at five different levels of the phylogenetic hierarchy including phylum, class, order, family, and genus in comparison with the WT control. CONCLUSIONS: TLR4 may protect intestinal epithelial cells from damage in response to DSS-induced injury by controlling the microbiota in the gut.

Discovery of extracellular vesicle-delivered miR-185-5p in the plasma of patients as an indicator for advanced adenoma and colorectal cancer.

BACKGROUND: We aimed to evaluate whether extracellular vesicles (EV)-derived microRNAs (miRNAs) can be used as biomarkers for advanced adenoma (AA) and colorectal cancer (CRC). METHODS: We detected the changes in the plasma EV-delivered miRNA profiles in healthy donor (HD), AA patient, and I-II stage CRC patient groups using miRNA deep sequencing assay. We performed the TaqMan miRNA assay using 173 plasma samples (two independent cohorts) from HDs, AA patients, and CRC patients to identify the candidate miRNA(s). The accuracy of candidate miRNA(s) in diagnosing AA and CRC was determined using the area under the receiver-operating characteristic curve (AUC) values. Logistic regression analysis was performed to evaluate the association of candidate miRNA(s) as an independent factor for the diagnosis of AA and CRC. The role of candidate miRNA(s) in the malignant progression of CRC was explored using functional assays. RESULTS: We screened and identified four prospective EV-delivered miRNAs, including miR-185-5p, which were significantly upregulated or downregulated in AA vs. HD and CRC vs. AA groups. In two independent cohorts, miR-185-5p was the best potential biomarker with the AUCs of 0.737 (Cohort I) and 0.720 (Cohort II) for AA vs. HD diagnosis, 0.887 (Cohort I) and 0.803 (Cohort II) for CRC vs. HD diagnosis, and 0.700 (Cohort I) and 0.631 (Cohort II) for CRC vs. AA diagnosis. Finally, we demonstrated that the upregulated expression of miR-185-5p promoted the malignant progression of CRC. CONCLUSION: EV-delivered miR-185-5p in the plasma of patients is a promising diagnostic biomarker for colorectal AA and CRC. Trial registration The study protocol was approved by the Ethics Committee of Changzheng Hospital, Naval Medical University, China (Ethics No. 2022SL005, Registration No. of China Clinical Trial Registration Center: ChiCTR220061592).

Progress in the diagnosis and treatment of COVID-19 and the role of surgeons in the front line of the pandemic.

The current struggle to control and contain COVID-19 is critical and surgeons are on the front line in the fight against this virus. Surgeons, and other medical workers in the field of surgery, have a solid foundation and experience in medical treatment and intensive care, and an understanding of the support of respiratory, circulatory, digestive, and other systemic organs. Furthermore, the operative standards of aseptic technique in their daily work enable surgeons to adapt to the working environment in infected areas. As surgeons in the anti-pandemic front line in China, we describe our experience with the diagnosis and treatment of COVID-19 in this country and how the work of surgeons is unfolding during the pandemic.

Toll-like receptor 4 regulates spontaneous intestinal tumorigenesis by up-regulating IL-6 and GM-CSF.

Inflammation is as an important component of intestinal tumorigenesis. The activation of Toll-like receptor 4 (TLR4) signalling promotes inflammation in colitis of mice, but the role of TLR4 in intestinal tumorigenesis is not yet clear. About 80%-90% of colorectal tumours contain inactivating mutations in the adenomatous polyposis coli (Apc) tumour suppressor, and intestinal adenoma carcinogenesis in familial adenomatous polyposis (FAP) is also closely related to the germline mutations in Apc. The ApcMin/+ (multiple intestinal neoplasia) model mouse is a well-utilized model of FAP, an inherited form of intestinal cancer. In this study, ApcMin/+ intestinal adenoma mice were generated on TLR4-sufficient and TLR4-deficient backgrounds to investigate the carcinogenic effect of TLR4 in mouse gut by comparing mice survival, peripheral blood cells, bone marrow haematopoietic precursor cells and numbers of polyps in the guts of ApcMin/+ WT and ApcMin/+ TLR4-/- mice. The results revealed that TLR4 had a critical role in promoting spontaneous intestinal tumorigenesis. Significant differential genes were screened out by the high-throughput RNA-Seq method. After combining these results with KEGG enrichment data, it was determined that TLR4 might promote intestinal tumorigenesis by activating cytokine-cytokine receptor interaction and pathways in cancer signalling pathways. After a series of validation experiments for the concerned genes, it was found that IL6, GM-CSF (CSF2), IL11, CCL3, S100A8 and S100A9 were significantly decreased in gut tumours of ApcMin/+ TLR4-/- mice compared with ApcMin/+ WT mice. In the functional study of core down-regulation factors, it was found that IL6, GM-CSF, IL11, CCL3 and S100A8/9 increased the viability of colon cancer cell lines and decreased the apoptosis rate of colon cancer cells with irradiation and chemical treatment.

Critical role of toll-like receptor 4 (TLR4) in dextran sulfate sodium (DSS)-Induced intestinal injury and repair.

Ulcerative colitis2 (UC) is an inflammatory bowel disease3 (IBD) that causes long-lasting inflammation and ulcers in the human digestive tract. The repair function of TLR4 in the intestinal epithelium is still unknown. Here, wild-type4 (WT) mice, TLR4-knockout mice5 (KO; TLR4-/-) and commensal-depleted mice were used as dextran sulfate sodium6 (DSS)-induced or radiation-induced colitis and injury models to explore the role of TLR4 signaling in intestinal injury. Exogenous lipopolysaccharide7 (LPS) promoted DSS-induced inflammatory cytokines and aggravated intestinal damage. TLR4 deficiency and commensal bacterial depletion inhibited the toxic effects of LPS, but these mice were more susceptible to DSS-induced and radiation-induced intestinal damage. Compared with WT mice, neither DSS nor radiation promoted production of more inflammatory cytokines in the guts of TLR4-KO and commensal-depleted mice. Introducing the cytokine repair factors, PGE2 and GM-CSF, increased the cytokine levels in the guts of DSS-induced colitis mice. We hypothesized that TLR4 and its ligands repaired the epithelium after DSS-induced and radiation-induced intestinal damage by upregulating PGE2 and GM-CSF. Transwell migration assays suggested that LPS, IL6, TNF, PGE2 and GM-CSF promoted intestinal cell migration, and cell viability analysis suggested that these factors protected against radiation-induced intestinal damage. Our data underscore the importance of the balancing role of TLR4 in intestinal injury and repair.

Toll-like receptor 4 (TLR4) deficiency aggravates dextran sulfate sodium (DSS)-induced intestinal injury by down-regulating IL6, CCL2 and CSF3.

BACKGROUND: Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) that causes long-lasting inflammation and ulcers in the human digestive tract. The repair role of TLR4 in the intestinal epithelium is still unknown. METHODS: By comparing to wild-type (WT) mice, Toll-like receptor 4 (TLR4)-knockout mice (TLR4-KO) were used as dextran sulfate sodium (DSS)-induced colitis models to explore the role of TLR4 signaling in intestinal injury. High-throughput RNA-Seq, RT-qPCR and ELISA were performed to screen and verify key differences in gut genes between WT and TLR4-KO mice. Functional study of core dysregulated factors was performed in intestinal cell lines. RESULTS: We found that DSS-induced intestinal injury was aggravated by LPS (TLR4 agonist) and TLR4-KO. When compared to WT mice, IL6, CCL2, CSF3, IL11, Ccnb1, Ccnd1 and TNF-α significantly decreased and Fas and FasL have increased in the gut of TLR4-KO mice. IL6, CCL2, CSF3, Fas and FasL have all increased in CT-26 cells treated with LPS. Combined with the above data and KEGG enrichment, it can be assumed that TLR4-KO might aggravate DSS-induced intestinal damage by attenuating cell cycle, cytokine-cytokine receptor interaction, and Toll-like receptor signaling pathway, and enhancing the apoptosis pathway. In the functional study of core dysregulated factors, it was found that LPS, IL6, IL11, CSF3, CCL2, S100A8, S100A9 and Mmp3 have improved viability of colon cancer cell lines and decreased apoptosis rate of mouse colon cancer cells when these were treated with DSS. However, Jo-2 (Fas agonistic monoclonal antibody) played the opposite role in colon cancer cells treated with DSS. CONCLUSIONS: TLR4 had a repairing effect on DSS-induced intestinal damage and it up-regulate IL6, CCL2 and CSF3. Fas and FasL enhanced DSS-induced colon injury in mice, but might have little to do with TLR4 signaling.

Apoptosis of multiple myeloma cells induced by agonist monoclonal antibody against human CD28.

CD28 is expressed abnormally on human multiple myeloma (MM) cells but the significance had not been identified until now. In this paper, we are suggesting that abnormal expression of CD28 might be a marker of tumour progression. We therefore took the approach of generating a hybridoma cell line capable of secreting agonist monoclonal antibody directed against human CD28 (agonist anti-CD28 mAb) and then determined the expression of CD28 molecules on the MM cell lines U266 and XG1. The biological effects of agonist anti-CD28 mAb on cell growth and proliferation of U266 and XG1 cell lines were then analysed. Our results showed that the expression of CD28 on U266 and XG1 was significantly higher than that of PBTC or Jurkat cells. We found that by adding the agonist anti-CD28 mAb to cultures of U266 and XG1 cells their rate of growth and proliferation was obviously inhibited. Further morphological and molecular analyses found that U266 and XG1 incubated with agonist anti-CD28 mAb showed signs of nuclear condensation, chromatin marginal changes, cells membrane breaking, and cytoplasmic shrinkage. Vacuoles and apoptotic bodies were also observed using a transmission electron microscope and the development of typical DNA laddering patterns were found by the use of electrophoresis assays, suggesting that U266 and XG1 cells were undergoing apoptosis induced by agonist anti-CD28 mAb in vitro.

Time courses of B7 family molecules expressed on activated T-cells and their biological significance.

B7 family molecules are mainly expressed on the outer membrane of antigen-presenting cells. Here, our results demonstrate that CD80, CD86, and PD-L1 molecules are also expressed on T-cells that have been activated by simultaneous exposure to anti-CD3 and anti-CD28 mAbs, but PD-L2 and GL50 molecules were not detectable during the first six days of culture that follow such stimulation. We have analysed the time course of B7 family molecule expression on activated T-cells. CD28 and its ligands, CD80/CD86, have a high degree of co-localization and exhibit compartmental distribution on the membrane of activated T-cells, which is visualized by confocal microscopy. Interestingly, the co-localization of PD-1 and its ligand also exhibit similar phenomenon. Additionally, we provide evidence indicating that the CD80, CD86, and PD-L1 molecules are functional, since T-cells expressing B7 family molecules are able to stimulate the proliferation of highly purified allogeneic or autologous T-cells. Anti-CD80, anti-CD86, and soluble CD28-Ig protein could significantly attenuate the proliferation of T-cells, whereas anti-PD-L1 mAb may lead to the expansion of activated T-cells. We can conclude that activated T-cells expressing B7 family molecules could act as "APC" to trigger purified T-cells, and B7 family molecules play important roles during the activation of T-cells. These results indicate a need for further work, exploring the regulatory roles these molecules may play in immune responses.