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).

Incidence, prevention, risk factors, and prediction of venous thromboembolism in Chinese patients after colorectal cancer surgery: a prospective, multicenter cohort study.

BACKGROUND: Venous thromboembolism (VTE) is a common and serious complication after colorectal cancer (CRC) surgery. Few large-sample studies have reported VTE incidence and management status after CRC surgery in China. This study aimed to investigate the incidence and prevention of VTE in Chinese patients after CRC surgery, identify risk factors for developing VTE, and construct a new scoring system for clinical decision-making and care planning. METHODS: Participants were recruited from 46 centers in 17 provinces in China. Patients were followed up for 1 month postoperatively. The study period was from May 2021 to May 2022. The Caprini score risk stratification and VTE prevention and incidence were recorded. The predictors of the occurrence of VTE after surgery were identified by multivariate logistic regression analysis, and a prediction model (CRC-VTE score) was developed. RESULTS: A total of 1836 patients were analyzed. The postoperative Caprini scores ranged from 1 to 16 points, with a median of 6 points. Of these, 10.1% were classified as low risk (0-2 points), 7.4% as moderate risk (3-4 points), and 82.5% as high risk (≥5 points). Among these patients, 1210 (65.9%) received pharmacological prophylaxis, and 1061 (57.8%) received mechanical prophylaxis. The incidence of short-term VTE events after CRC surgery was 11.2% (95% CI 9.8-12.7), including deep venous thrombosis (DVT) (11.0%, 95% CI 9.6-12.5) and pulmonary embolism (PE) (0.2%, 95% CI 0-0.5). Multifactorial analysis showed that age (≥70 years), history of varicose veins in the lower extremities, cardiac insufficiency, female sex, preoperative bowel obstruction, preoperative bloody/tarry stool, and anesthesia time at least 180 min were independent risk factors for postoperative VTE. The CRC-VTE model was developed from these seven factors and had good VTE predictive performance ( C -statistic 0.72, 95% CI 0.68-0.76). CONCLUSIONS: This study provided a national perspective on the incidence and prevention of VTE after CRC surgery in China. The study offers guidance for VTE prevention in patients after CRC surgery. A practical CRC-VTE risk predictive model was proposed.

PHLPP2 is regulated by competing endogenous RNA network in pathogenesis of colon cancer.

Recently, homologous pleckstrin-homology (PH)-domain leucine-rich-repeat protein phosphatases (PHLPP2) has been reported as a tumor suppressor in colon cancer. This study aimed to unravel the possible involvement of long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) regulating PHLPP2 in colon cancer. Expressions of candidate lncRNAs and miRNAs were verified by the RT-qPCR and Western blot analyses in colon cancer. The roles of candidate genes in colon cancer were investigated in HT-29 cells in vitro and in mouse tumor xenograft model in vivo. PHLPP2, a target of miR-141 and miR-424, was downregulated in colon cancer. PHLPP2 upregulation and miR-141 and miR-424 downregulation suppressed the colon cancer cell proliferation, migration, invasion, and epithelial-mesenchymal transition, and promote cell apoptosis, which also resulted in suppression of tumor metastasis and formation. Furthermore, LINC00402, LINC00461, and SFTA1P were identified as the targets of miR-141 and miR-424 and acted as competitive endogenous RNAs (ceRNAs) of PHLPP2. The upregulation of LINC00402, LINC00461, and SFTA1P was verified to enhance the suppressive effects of PHLPP2 in the pathogenesis of colon cancer. Conjointly, our results demonstrated the suppressive effects of PHLPP2 in colon cancer and proved that LINC00402, LINC00461, and SFTA1P acted as ceRNAs of PHLPP2 by competitive binding to miR-141 and miR-424.

Aspirin inhibited the metastasis of colon cancer cells by inhibiting the expression of toll-like receptor 4.

BACKGROUND: The metastasis of colorectal cancer frequently tends to liver, which is one of the three leading causes of cancer-related deaths worldwide. Growing evidence showed that aspirin could effectively inhibit liver metastasis of colorectal cancer. However, the potential mechanism has not been fully understood. METHODS: Mouse splenic vein metastasis assay was used to examine the metastatic ability of colon cancer cells in vivo. And wound healing and transwell assay were applied to detect the metastasis potential of C26 and HCT116 colon cancer cell lines in vitro. RT-PCR and western blotting were used to explore Toll-like receptor 4 (TLR4) expression in colon cancer cell lines. The functions of TLR4 in the migration of the colon cancer cell line were analyzed by infecting cells with lentivirus containing TLR4 siRNA. RESULTS: We demonstrated that lipopolysaccharides (LPS) could enhance the metastasis potential of C26 and HCT116 colon cancer cell lines. However, aspirin effectively decreased the metastasis capacity of colon cancer cells in vitro and in vivo. We found that the enhancement of LPS on the migration of colon cancer cells by inducing epithelial-mesenchymal transition (EMT) phenotype demonstrated a TLR4-dependent manner. Aspirin treatment lead to the downregulation of TLR4 on C26 cells which resulted in the decrease of C26 cells migration and EMT phenotype that induced by LPS. Additionally, the inhibitory effect from aspirin on the expression of TLR4 on C26 cells leads to the downregulation of NF-κB. CONCLUSION: The results of our study indicate that LPS origin from intestinal flora may promote the metastasis of colon cancer to liver and aspirin may inhibit the metastasis of colon cancer by inhibiting the expression of TLR4.

HIF-1α activates hypoxia-induced BCL-9 expression in human colorectal cancer cells.

B-cell CLL/lymphoma 9 protein (BCL-9), a multi-functional co-factor in Wnt signaling, induced carcinogenesis as well as promoting tumor progression, metastasis and chemo-resistance in colorectal cancer (CRC). However, the mechanisms for increased BCL-9 expression in CRC were not well understood. Here, we report that hypoxia, a hallmark of solid tumors, induced BCL-9 mRNA expression in human CRC cells. Analysis of BCL-9 promoter revealed two functional hypoxia-responsive elements (HRE-B and HRE-C) that can be specifically bound with and be transactivated by hypoxia inducible factors (HIF) -1α but not HIF-2α. Consistently, ectopic expression of HIF-1α but not HIF-2α transcriptionally induced BCL-9 expression levels in cells. Knockdown of endogenous HIF-1α but not HIF-2α by siRNA largely abolished the induction of HIF by hypoxia. Furthermore, there was a strong association of HIF-1α expression with BCL-9 expression in human CRC specimens. In summary, results from this study demonstrated that hypoxia induced BCL-9 expression in human CRC cells mainly through HIF-1α, which could be an important underlying mechanism for increased BCL-9 expression in CRC.

Annexin A4-nuclear factor-κB feedback circuit regulates cell malignant behavior and tumor growth in gallbladder cancer.

Gallbladder cancer (GBC) is the most common malignant tumor of the biliary system. However, the mechanisms underlying its tumor initiation, progression, and metastasis are not yet fully understood. The annexin A4 (ANXA4) gene is highly expressed in GBC tissues and may play an important role in the initiation and progression of this disease. In this study, we examined the up-regulation of ANXA4 in human GBC tissues and cell lines. Elevated ANXA4 correlated well with invasion depth in GBC patients and predicted a poor prognosis. In vitro, GBC-SD and NOZ cells with ANXA4 knockdown demonstrated increased apoptosis and inhibited cell growth, migration, and invasion. Interactions between ANXA4 and nuclear factor-κB (NF-κB) p65 proteins were detected. In vivo, ANXA4 knockdown inhibited tumor growth of GBC cells in nude mice and down-regulated the expression of downstream factors in the NF-κB signaling pathway. Taken together, these data indicate that up-regulation of ANXA4 leads to activation of the NF-κB pathway and its target genes in a feedback regulatory mechanism via the p65 subunit, resulting in tumor growth in GBC.

A panel data set on harvest and perfusion decellularization of porcine rectus abdominis.

In this dataset, we particularly depicted the harvest and perfusion decellularization of porcine rectus abdominis (RA), accompanied with displaying of the retained vascular trees within the perfusion-decellularized skeletal muscle matrix (pM-ECM) using vascular corrosion casting. In addition, several important tips for successful pM-ECM preparation were emphasized, which including using anatomically isolated skeletal muscle as tissue source with all main feeding and draining vessels perfused, preserving the internal microcirculation availability, aseptic technique and pyrogen free in all steps, sequential perfusion via artery or vein, and longtime washing after decellularization. The data are supplemental to our original research article describing detailed associations of pM-ECM as a clinically relevant scale, three-dimensional scaffold with a vascular network template for tissue-specific regeneration, "Perfusion-decellularized skeletal muscle as a three-dimensional scaffold with a vascular network template" Zhang et al. (2016) [1].

Perfusion-decellularized skeletal muscle as a three-dimensional scaffold with a vascular network template.

There exists a great need for repair grafts with similar volume to human skeletal muscle that can promote the innate ability of muscle to regenerate following volumetric muscle loss. Perfusion decellularization is an attractive technique for extracellular matrix (ECM) scaffold from intact mammalian organ or tissue which has been successfully used in tissue reconstruction. The perfusion-decellularization of skeletal muscle has been poorly assessed and characterized, but the bioactivity and functional capacity of the obtained perfusion skeletal muscle ECM (pM-ECM) to remodel in vivo is unknown. In the present study, pM-ECM was prepared from porcine rectus abdominis (RA). Perfusion-decellularization of porcine RA effectively removed cellular and nuclear material while retaining the intricate three-dimensional microarchitecture and vasculature networks of the native RA, and many of the bioactive ECM components and mechanical properties. In vivo, partial-thickness abdominal wall defects in rats repaired with pM-ECM showed improved neovascularization, myogenesis and functional recellularization compared to porcine-derived small intestinal submucosa (SIS). These findings show the biologic potential of RA pM-ECM as a scaffold for supporting site appropriate, tissue reconstruction, and provide a better understanding of the importance maintaining the tissue-specific complex three-dimensional architecture of ECM during decellularization and regeneration.

A non-invasive magnetic resonance imaging-based model predicts portal venous pressure.

OBJECTIVE: To establish a non-invasive model for the assessment of portal venous pressure (PVP) based on the magnetic resonance (MR) parameters. METHODS: In this prospective study, contrast-enhanced magnetic resonance imaging (MRI) scan was performed in 109 patients indicated for upper abdominal surgeries after their written consents were obtained, and intraoperative PVP measurements were completed in 92 patients. Altogether 17 patients were excluded for not undergoing surgery or unsuccessful catheterization. A linear model was constructed for estimating PVP levels in 56 patients and further validation was conducted in the other 36 patients. RESULTS: The PVP levels were significantly correlated with MR parameters, including splenic volume (SV), splenic venous diameter (SVD), liver/splenic volume ratio, portal venous diameter, hepatic diameter, portal venous cross-sectional area, ascites, varices and arterial portal shunts. A linear model was established as follows: PVP (mmHg) = 2.529 + 1.572 × SVD (mm) + 0.231 × SV/body mass index (× 10(4) cm(5) /kg) + 3.44 × aspartate aminotransferase-to-platelet ratio index. This model showed excellent accuracy in the detection of portal hypertension, with the area under the receiver operating characteristic curve (AUROC) of 0.945 (95% CI 0.867-1.000), with the sensitivity and specificity of 91.7% and 93.7%, respectively. The agreement analysis revealed that the predictive value using this formula closely reflected the patients' actual PVP level. Moreover, the validation confirmed the accuracy of this model for the assessment of portal hypertension [AUROC 0.935 (95% CI 0.856-1.000)]. CONCLUSIONS: The MRI-based formula has great potential for detecting portal hypertension. As a non-invasive measurement, it may be clinically accepted for the replacement of invasive modalities after further refinement.

Hepatocyte nuclear factor 4α suppresses the aggravation of colon carcinoma.

Hepatocyte nuclear factor 4-α (HNF4α), a nuclear receptor, is expressed at lower levels in colon carcinoma tissues than in adjacent normal tissues. However, the relation between HNF4α and colon cancer progression and the underlying molecular mechanisms remain unclear. Here, we investigated the role of HNF4α in the progression of colon carcinoma. We showed that HNF4α mRNA and protein were downregulated in colon carcinoma specimens. HNF4α expression was related to pT classification (P < 0.001), lymph node metastasis (P = 0.002), distant metastasis (P < 0.001) and clinical stage (P < 0.001) in colon carcinoma patients. Patients with low or negative HNF4α expression had worse 3-year progression-free survival (PFS, P = 0.006) and overall survival (OS, P = 0.005) than patients with high HNF4α expression. Low HNF4α expression was an independent prognostic factor for 3-year PFS (hazard ratio 2.94; 95% confidence interval 1.047-8.250; P = 0.041). Ectopic expression of HNF4α inhibited colon carcinoma cell (HT29, LoVo, and SW480) proliferation, migration, and invasion, induced G2/M phase arrest and promoted apoptosis. Ectopic expression of HNF4α upregulated E-cadherin and downregulated vimentin in vitro, and suppressed SW480 xenograft tumor growth and liver metastasis in vivo. Furthermore, HNF4α overexpression downregulated the expression of snail, slug and twist. HNF4α inhibited EMT through its effect on the Wnt/ß-catenin signaling pathway, and HNF4α downregulation may be mediated by promoter methylation in cancer tissues. Our results suggest that downregulation of HNF4α plays a critical role in the aggravation of colon carcinoma possibly by promoting EMT via the Wnt/ß-catenin signaling pathway and by affecting apoptosis and cell cycle progression.

Risk factors for the development of metachronous bone metastasis in colorectal cancer patients after curative resection.

OBJECTIVE: Metachronous bone metastasis (MBM) occurs in 6-10% of colorectal cancer (CRC) patients following surgical treatment. The aim of this study is to determine the risk factors affecting the development of MBM in CRC patients following curative resection. METHOD: Clinical and pathological records of 516 CRC patients who underwent curative resection were retrospectively studied. The association between clinicopathological variables and development of MBM was investigated using univariate and multivariate analyses. RESULT: The incidence of MBM was 6.0% and the median time of developing MBM was 15 (range, 1-89) months. Univariate analysis identified that lymph node involvement (p = 0.001), tumor stage (p = 0.020) and tumor location (p = 0.015) were significantly correlated with development of MBM. Multivariate analyses showed tumor location (p = 0.039) and lymph node involvement (p = 0.003) were independent risk factors contributing to the occurrence of MBM. CONCLUSION: This study indicated that tumor location and lymph node involvement were independent risk factors for development of MBM in CRC patients after curative resection.

Allogeneic blood transfusion and the prognosis of gastric cancer patients: systematic review and meta-analysis.

BACKGROUND: Perioperative allogeneic blood transfusion (ABT) may be a deleterious predictor on the prognosis of gastric cancer (GC) for subjects who had undergone curative surgeries. In this article we proposed to figure out the effect of ABT with a systematic review and meta-analysis. METHODS: Relevant articles were identified by searching Pubmed and Embase to March 2014. A random-effects model or fixed-effects model was used to calculate pooled odds ratios (ORs). Sensitivity analysis, meta-regression, stratified analysis, dose-response meta-analysis were conducted, and publication bias tested. RESULTS: Eighteen studies (9120 GC patients) were included, of which 36.3% received transfusions. ABT was associated with increased all-cause mortality (OR, 2.17; 95% confidence interval [CI], 1.72-2.74; p<0.001; I2=75%). Sensitivity analysis showed significant changes in ORs while meta-regression had little influence on ORs. Galbraith plot revealed the OR reduced to 2.10 (95% CI, 1.86-2.37; p<0.001) with tau2 reduced to 0.00 and I2 reduced to 0%. RESULTS of stratified analysis were robust and consistent. Dose-response meta-analysis revealed that all-cause mortality was significantly lower in patients transfused with ≤800 mL of blood than those transfused with >800 mL (OR, 0.58; 95% CI, 0.37-0.92; p=0.02; I2=54%). ABT was also associated with increased cancer-related mortality (OR, 2.57, p=0.011) and recurrence (OR, 1.52, p=0.017). CONCLUSIONS: In GC patients undergoing curative surgeries, ABTs are associated with a worse prognosis, including all-cause mortality, cancer-related mortality and recurrence. Patient blood management should be investigated further to minimize use of ABT.

Proteomic identification of tumor biomarkers associated with primary gallbladder cancer.

AIM: To identify potential biomarkers of primary gallbladder cancer (PGC). METHODS: Fresh PGC, cholecystitis and normal gallbladder tissue specimens collected from 10 patients, respectively, were subjected to comparative proteomic analysis. The proteomic patterns of PGC were compared with those of cholecystitis and normal gallbladder tissues using two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were then identified using a MALDI-TOF mass spectrometer (MS) and database searches. To further validate these proteins, 20 samples of PGC tissues and normal tumor-adjacent tissues were collected for Western blot, quantitative real-time PCR, and immunohistochemical staining assay. RESULTS: Seven differentially expressed protein spots were detected by 2-ED analysis by comparing the average maps of PGC, cholecystitis and normal gallbladder tissues. Six of the seven differentially expressed proteins were identified using MALDI-TOF MS, with three overexpressed and three underexpressed in PGC tissue. Protein levels of annexin A4 (ANXA4) were significantly elevated, and heat shock protein 90-beta (Hsp90ß) and dynein cytoplasmic 1 heavy chain 1 (Dync1h1) were decreased in PGC tissues relative to the normal tumor-adjacent tissues as shown by Western blot analysis. However, levels of actin, aortic smooth muscle and gamma-actin were unchanged. In addition, the mRNA levels of all 5 proteins showed similar changes to those of the protein levels (P < 0.01). Further validation by immunohistochemical analysis showed the upregulated expression of ANXA4 and decreased expression of Hsp90ß and Dync1h1 in the cytoplasm of PGC tissues relative to the normal tumor-adjacent tissues. CONCLUSION: Three proteins are identified as potential biomarkers of PGC using proteomic analysis. The functions of these proteins in the carcinogenesis of PGC remain to be studied.

Retrograde vs Conventional Dissection Technique in Pancreaticoduodenectomy: A Pilot Study.

IMPORTANCE: A retrograde dissection technique of pancreaticoduodenectomy in a caudocranial direction has been described recently. OBSERVATIONS: Fifteen consecutive patients who underwent retrograde pancreaticoduodenectomy were compared with 15 consecutive patients operated on through a conventional approach. The mean (SD) intraoperative blood loss was 407 (202) mL in the retrograde group compared with 423 (253) mL in the conventional group (P = .84). The mean (SD) operative duration was 255 (57) minutes in the retrograde group compared with 264 (54) minutes in the conventional group (P = .66). The overall morbidity was 7 of 15 patients (47%) in the retrograde group and 6 of 15 (40%) in the conventional group (P > .99). Neither group had a positive resection margin or a perioperative death. CONCLUSIONS AND RELEVANCE: The retrograde dissection technique had no significant difference in perioperative outcomes compared with the conventional dissection technique and could serve as an alternative dissection approach in pancreaticoduodenectomy.

Association of perioperative allogeneic blood transfusions and prognosis of patients with gastric cancer after curative gastrectomy.

BACKGROUND: The relationship between perioperative allogeneic blood transfusions (PABTs) and prognosis in patients with gastric cancer remains controversial. METHODS: Six hundred five consecutive patients with gastric cancer who underwent curative gastrectomy from a single center were enrolled in this retrospective study. Clinical and pathologic variables were prospectively collected. The effect of PABT on the long-term survival of patients with gastric cancer after curative gastrectomy was evaluated by univariate and multivariate analyses. RESULTS: The overall 5-year survival rate was 65.0%. On univariate analyses, PABT had a statistically significant negative impact on 3-year and 5-year survival rates (66.3% vs 80.5% [P = .005] and 38.7% vs 76.4% [P < .001], respectively). However, multivariate analyses revealed that duration of operation (P = .009), tumor size (P = .001), and tumor stage (P < .001), instead of PABT, were independent prognostic factors. CONCLUSIONS: Our study indicates that PABT is not an independent prognostic factor for long-term survival in patients with gastric cancer after curative gastrectomy.

Therapeutic effects of p75 tumor necrosis factor receptor monoclonal antibody on a rat model of traumatic arthritis.

BACKGROUND: The aim of the present study was to investigate the therapeutic effects of p75 tumor necrosis factor receptor monoclonal antibody D8F2 on a traumatic arthritis model in rats, and to explore the underlying mechanism. METHODS: Forty male Sprague Dawley rats were randomly divided into five groups: (A) sham operation control group, (B) traumatic arthritis model group, (C) low-dose D8F2 group (1 mg/kg), (D) medium-dose D8F2 group (3 mg/kg), and (E) high-dose D8F2 group (10 mg/kg). Joint fluid samples were collected at 72 h after surgery, and enzyme-linked immunosorbent assay was performed to measure the following inflammatory factors: tumor necrosis factor α (TNF-α) and interleukin 1ß. One week after the surgery, rats were killed, and immunohistochemical staining was applied to detect the matrix metalloproteinase (MMP1 and MMP3) expression in the synovium. In cultured synovial fibroblast experiments, the D8F2-induced ubiquitination of TNF receptor-associated factor 2 (TRAF2) was examined by immunoprecipitation, and nuclear translocation of p65 nuclear factor-κB (p65NF-κB) mediated by TNF-α and D8F2 was analyzed by western blotting. RESULTS: In the traumatic arthritis model group, the inflammatory factors and MMPs were significantly increased relative to the sham operation control group (P < 0.05), whereas D8F2 could downregulate these factors in a dose-dependent manner (P < 0.05). The results from in vitro studies indicated that D8F2 can induce TRAF2 ubiquitination and inhibit the nuclear translocation of p65NF-κB mediated by TNF-α. CONCLUSIONS: p75 Tumor necrosis factor receptor monoclonal antibody has a therapeutic effect on traumatic arthritis, which may occur via the downregulation of inflammatory factors and MMPs at the transcription level because of TRAF2 degradation and inhibited activation of NF-κB.

A study on inhibition of inflammation via p75TNFR signaling pathway activation in mice with traumatic brain injury.

OBJECTIVE: To investigate the effects of and mechanisms underlying the activation of the p75 tumor necrosis factor receptor (p75TNFR) signaling pathway in inflammatory responses in mice with traumatic brain injury. METHODS: We first generated hybridomas that produced antibodies specific for p75TNFR, by inoculating BALB/c mice with antigenic peptides derived from mouse p75TNFR, which is critical to the binding of tumor necrosis factor-alpha (TNF-α) and p75TNFR. The isotype, epitope, titer, specificity, and affinity constant of monoclonal antibodies (mAbs) were determined using commercial kits and enzyme-linked immunosorbent assay. We then screened the agonist antibody via L929 cytotoxicity assay. The levels of inflammatory factors were detected in C57BL/6 mice with traumatic brain injury and then the mice were injected with either saline (control) or p75TNFR agonist mAb. Furthermore, we investigated the effects of p75TNFR agonist mAb on p38MAPK and nuclear factor-κB signals. RESULTS: Seven mAbs against p75TNFR were generated. Among them, the mAb D8F2 could markedly enhance the cytotoxicity of TNF-α on L929 cells. In a traumatic brain injury model, D8F2 could inhibit the levels of inflammatory factors and downregulate RNA transcription of these factors by suppressing the activation of p38 mitogen-activated protein kinase and nuclear factor-κB. CONCLUSION: The mAb D8F2 could inhibit posttraumatic inflammatory responses effectively. In this study, we developed an agonist anti-mouse p75TNFR mAb, which may be used in the future to devise new strategies for the clinical treatment of inflammation after trauma.

Identification and assessment of new biomarkers for colorectal cancer with serum N-glycan profiling.

BACKGROUND: The objectives of this study were to identify and validate the diagnostic value of N-glycan markers in colorectal cancer (CRC) and to uncover their underlying molecular mechanism. METHODS: In total, 347 individuals, including patients with CRC, patients with colorectal adenoma, and healthy controls, were divided randomly into a training group (n = 287) and retrospective validation groups (n = 60). Serum N-glycan profiling was analyzed by DNA sequencer-assisted/flurophore-assisted carbohydrate electrophoresis (DSA-FACE). Two diagnostic models were constructed based on N-glycan profiling with logistic stepwise regression. The diagnostic performance of each model was assessed further in retrospective, prospective (n = 43), and follow-up (n = 46) cohorts. Lectin blot and reverse transcriptase-polymerase chain reaction were used to analyze the total core-fucosylated residues and molecular expression involved in core-fucosylation modifications in CRC. RESULTS: Two diagnostic models designated CRCglycoA and CRCglycoB were constructed to differentiate CRC from normal and adenoma, respectively. The areas under the receiver operating characteristic curves (AUC) of both CRCglycoA and CRCglycoB were higher than the AUC of carcinoembryonic antigen (CEA) (CRCglycoA, 0.92 vs 0.81; CRCglycoB, 0.81 vs 0.73). The sensitivity and accuracy of CRCglycoA improved from 21.7% to 25% and from 11.63% to 18% in the training cohort, the retrospective cohort, and the prospective cohorts compared with the sensitivity and accuracy of CEA. The sensitivity of CRCglycoB improved from 20% to 28.23%. Both altered N-glycans, and results from the diagnostic models were reversed after curative surgery. The level of total core fucose residues and fucosyltransferase were decreased significantly in CRC. CONCLUSIONS: The current results indicated that the N-glycan markers based diagnostic models are new, valuable, noninvasive alternatives for identifying CRC. The authors concluded that decreased fucosyltransferase may be responsible for decreased levels of total core-fucosylated modification in both tissues and serum from patients with CRC.

Impact of intra-subunit interactions on the dimeric arginine kinase activity and structural stability.

Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by ATP, yielding the phosphoarginine. In this research, six conserved residues located on the intra-subunit domain-domain interfaces were mutated to explore their roles in the activity and structural stability of dimer AK. The mutations D69A, E70A, E71A and F80A led to pronounced loss of AK activity and structural stability. Although the mutations V75A and F76A had little effect on AK activity and structure, they caused gradually decreased the stability and reactivation of dimer AK. Our results suggested that the mutations might affect the correct positioning of the N-loop and C-loop thus disrupted the efficient recognition and interactions between the N-terminal domain and C-terminal domain which may influence the compact dimer structure, and result in decreased activity and structural stability.