Intestine epithelial cell-derived extracellular vesicles alleviate inflammation induced by Clostridioides difficile TcdB through the activity of TGF-ß1.

Background: Clostridioides difficile infection (CDI) has been primarily associated with the toxin B (TcdB), one of the three known protein toxins secreted by C. difficile, which can activate the intestinal immune system and lead to pathological damage. Even though the biological functions of intestine epithelial cell-derived extracellular vesicles (I-Evs) have been well documented, the role of I-Evs in the process of CDI is still unknown. Objectives: The protective effect of I-Evs against C. difficile TcdB was investigated both in cultured murine colon carcinoma MC38 cells and a mouse model used in this study. Results: Mouse I-Evs with mean diameter ranging from 100 to 200 nm and a density of 1.09-1.17 g/mL were obtained and confirmed containing the Ev-associated specific surface markers CD63 and TSG101 as well as high level of TGF-ß1. In MC38 cells, I-Evs were able to decrease the gene expression of IL-6, TNF-α, IL-1ß, and IL-22 induced by C. difficile TcdB, but to increase both the gene expression and protein levels of TGF-ß1. I-Evs treatment via intraperitoneal administration alleviates C. difficile TcdB-induced local colon inflammation in mice and increased their survival rate from 50% up to 80%. Furthermore, I-Evs induced an increase in the proportion of CD4+Foxp3+Tregs in vitro and in vivo through a TGF-ß1-dependent mechanism by activating the TGF-ß1 pathway and prompting phosphorylation of the downstream proteins Smad 2/3. Conclusion: For the first time, our study demonstrated that I-Evs originated from intestine epithelial cells can alleviate inflammation induced by C. difficile TcdB both in vitro and in vivo. Therefore, I-Evs might be potentially a novel endogenous candidate for effective treatment of CDI.

Genomic evolution and virulence association of Clostridioides difficile sequence type 37 (ribotype 017) in China.

Clostridioides difficile sequence type (ST) 37 (ribotype 017) is one of the most prevalent genotypes circulating in China. However, its genomic evolution and virulence determinants were rarely explored. Whole-genome sequencing, phylogeographic and phylogenetic analyses were conducted for C. difficile ST37 isolates. The 325 ST37 genomes from six continents, including North America (n = 66), South America (n = 4), Oceania (n = 7), Africa (n = 9), Europe (n = 138) and Asia (n = 101), were clustered into six major lineages, with region-dependent distributions, harbouring an array of antibiotic-resistance genes. The ST37 strains from China were divided into four distinct sublineages, showing five importation times and international sources. Isolates associated with severe infections exhibited significantly higher toxin productions, tcdB mRNA levels, and sporulation capacities (P < 0.001). Kyoto Encyclopedia of Genes and Genomes analysis showed 10 metabolic pathways were significantly enriched in the mutations among isolates associated with severe CDI (P < 0.05). Gene mutations in glycometabolism, amino acid metabolism and biosynthesis virtually causing instability in protein activity were correlated positively to the transcription of tcdR and negatively to the expression of toxin repressor genes, ccpA and codY. In summary, our study firstly presented genomic insights into genetic characteristics and virulence association of C. difficile ST37 in China. Gene mutations in certain important metabolic pathways are associated with severe symptoms and correlated with higher virulence in C. difficile ST37 isolates.

Exposure to phthalates is associated with grip strength in US adults.

The potential association of exposure to phthalates with muscle strength was reported in previous animal experiments. However, their association was rarely directly investigated in general populations. Thus, we aimed to ascertain the association of exposure to phthalates with grip strength using cross-sectional analysis which included 2436 individuals aged ≥ 20 years from the National Health and Nutrition Examination Survey (NHANES) during 2011-2014. The multivariable linear regression models were performed with the adjustment of related covariates. The results suggested that a one-unit increase in log-transformed phthalate metabolites (µg/g creatinine) was inversely associated with grip strength, including Mono-(2-ethyl)-hexyl phthalate (ß: -2.727 kg, 95% CI: -3.452, -2.002), Mono-(2-ethyl-5-hydroxyhexyl) phthalate (ß: -3.721 kg, 95% CI: -4.836, -2.607), Mono-(2-ethyl-5-oxohexl) phthalate (ß: -4.669 kg, 95% CI: -5.761, -3.577), Mono-2-ethyl-5-carboxypentyl phthalate (ß: -4.756 kg, 95% CI: -5.957, -3.554), Mono-carboxyoctyl phthalate (ß: -1.324 kg, 95% CI: -2.412, -0.235), Mono-carboxynonyl phthalate (ß: -2.036 kg, 95% CI: -3.185, -0.886), Mono-benzyl phthalate (ß: -2.940 kg, 95% CI: -3.853, -2.026), Mono-n-butyl phthalate (ß: -2.100 kg, 95% CI: -3.474, -0.726), Mono-isobutyl phthalate (ß: -2.982 kg, 95% CI: -4.331, -1.633), and Mono-ethyl phthalate (ß: -1.709 kg, 95% CI: -2.368, -1.050). In subgroup analyses, the associations remained largely unchanged when the samples were stratified by gender and age; However they became ambiguous among underweight subjects when the samples were stratified by BMI status. Overall, exposure to phthalates was inversely associated with grip strength among US adults, regardless of their genders and ages. The suggestive potential BMI status-specific effects of phthalates on grip strength were observed.

NFIB functions as an oncogene in estrogen receptor-positive breast cancer and is regulated by miR-205-5p.

Nuclear factor I/B(NFIB) is a prominent transcription factor that plays a critical role in cancer progression. In this study, we found that the protein level of NFIB was significantly upregulated in estrogen receptor (ER) positive breast cancer tissues compared to matched adjacent noncancerous tissues while the NFIB mRNA expression level was not obviously dysregulated. Similarly, ER-positive breast cancer cell line, MCF7 express a high protein level of NFIB, while the mRNA level is not significantly upregulated. The function assays indicated that NFIB promoted MCF-7 cell cycle progression, cell proliferation and suppressed apoptosis in vitro. Furthermore, we explored the molecular mechanisms of NFIB as a target gene of miR-205-5p. Finally, we found that miR-205-5p was significantly downregulated in ER -positive breast cancer, and had the opposite eff ;ects on breast cancer cells compared with NFIB. Taken together, this study highlighted the molecular mechanisms of NFIB as an oncogene in ER-positive breast cancer, which was negatively regulated by miR-205-5p in breast cancer.

A New Method of Using Polyethylene Glycol (PEG) Precipitation of Macroprolactin to Detect Genuine Hyperprolactinemia.

BACKGROUND: The most commonly used method of polyethylene glycol (PEG) precipitation for macroprolactinemia (MP) screening has some significant drawbacks. The aim of this study was to establish a new method using PEG for precipitation of macroprolactin (macroPRL) to detect genuine hyperprolactinemia (genuine HP). METHODS: The optimal PEG concentration for precipitation and the effect of PEG on the precipitation of PRL were analyzed to establish and optimize our PEG precipitation method. The PRL recovery rate and genuine HP detection rate were compared between our method and MP screening method. RESULTS: About 25% PEG6000 was determined to be the optimal PEG concentration for precipitation. Along with an increase in protein concentration in the PRL calibration solution, the PRL recovery rate after precipitation decreased gradually. The PRL recovery rate increased when the precipitation was carried out with diluted PRL calibration solution; the recovery rate reached greater than 90% after a 5-fold dilution of the calibration solution. The genuine HP detection rate and PRL recovery rate using our diluted serum PEG precipitation method were significantly higher than those obtained with the MP screening method. Our method successfully detected 31 cases of genuine HP, which was significantly higher than the detection rate obtained using the MP screening method (25 cases; P < 0.001). CONCLUSION: Precipitation using 5-fold diluted serum with 25% PEG6000 can effectively reduce the macroPRL concentration, increasing the PRL recovery rate and detection rate of genuine HP after precipitation, which is an effective and convenient method for the detection of genuine HP.

Decreased Tim-3 expression is associated with functional abnormalities of monocytes in decompensated cirrhosis without overt bacterial infection.

BACKGROUND & AIMS: Patients with advanced cirrhosis usually exhibit altered monocyte function. However, the molecular mechanisms underlying the functional changes of monocytes are poorly understood. METHODS: We investigated the role of T-cell immunoglobulin domain and mucin domain-containing molecule-3 (Tim-3) in regulating monocyte function in 94 patients with decompensated liver cirrhosis (DC-LC) (decompensation was defined by ascites, hepatic encephalopathy or upper gastrointestinal bleeding), 58 with compensated liver cirrhosis (C-LC) and 52 healthy controls (HC) by characterizing the frequency of Tim-3(+) monocytes, their phagocytosis capacity, HLA-DR expression, cytokine secretion and MAP kinase activation induced by lipopolysaccharide (LPS). RESULTS: Tim-3 expression on CD14(+) monocytes in DC-LC group were significantly lower than that in C-LC and HC and were associated with increased levels of plasma endotoxin, enhanced cytokine production, decreased phagocytic capacity, and reduced HLA-DR expression. Tim-3 expression on monocytes and monocyte function did not differ between C-LC and HC group. Tim-3(+)CD14(+) cells had more potent phagocytic capacity, higher levels of HLA-DR, CD86, CD80, CD163, and CD206 expression, but lower levels of CD1a and CD83, related to that of Tim-3(-)CD14(+) monocytes. In addition, Tim-3(+)CD14(+) cells produced less TNF-α but higher levels of IL-10 in response to LPS. Treatment with anti-Tim-3 antibody significantly reduced phagocytic capacity, but enhanced LPS-stimulated TNF-α, IL-6, and IL-10 secretion. Furthermore, blocking Tim-3 signaling increased p38 MAP kinase phosphorylation in monocytes upon LPS stimulation. CONCLUSIONS: Downregulation of Tim-3 expression was associated with endotoxemia and functional alterations of monocytes in patients with decompensated cirrhosis.

Resveratrol reduces the proinflammatory effects and lipopolysaccharide- induced expression of HMGB1 and TLR4 in RAW264.7 cells.

BACKGROUND: Resveratrol (Res) is a polyphenol anti-inflammatory agent. We have studied the link between the anti-inflammatory effects of Res and the high mobility group box 1(HMGB1) signaling pathway. METHODS: Murine macrophage-like RAW264.7 cells (RAW264.7 cells) were either untreated (control) or treated with Res, LPS, or LPS + Res. Levels of IL-6, NO, and TNF-α were measured by ELISA and colorimetric assays. Expression of HMGB1 was detected by qRT-PCR, western blot, and immunofluorescence assays. Protein and mRNA expression levels of TLR4 were also examined. RESULTS: Res significantly reduced the levels of IL-6, NO, and TNF-α in RAW264.7 cells exposed to LPS. Expression levels of HMGB1 (mRNA and protein) and of TLR4 in the LPS + Res-treated cells were lower than in cells treated with LPS alone. CONCLUSIONS: Res can block the inflammatory effects induced by LPS in RAW264.7 cells. Down-regulation of HMGB expression may be one of the mechanisms of action of Res. Res may also influence TLR4 expression in the HMGB1-TLR4 signaling pathway.

Association between Mannose-binding lectin gene polymorphisms and hepatitis B virus infection: a meta-analysis.

OBJECTIVE: The results of studies on the relation between Mannose-binding lectin gene (mbl2) polymorphism and HBV infection were contradictory and inconclusive. In order to shed a light on these inconsistent findings and to clarify the role of mbl2 polymorphisms in susceptibility or progression of chronic hepatitis B (CHB), a meta-analysis was performed. METHODS: PubMed and Embase were searched for available articles. A meta-analysis was performed to examine the association between mbl2 polymorphisms and chronicity or progression of hepatitis B infection. Odds ratio (OR) and its 95% confidence interval (CI) served as indexes. RESULTS: A total of 17 eligible studies were involved, including 2151 healthy controls (HC), 1293 spontaneous recovered (SR) patients with acute infection, 2337 cases with chronic hepatitis B (CHB) and 554 cases with progressive hepatitis B. There was no evidence of significant association between mbl2 exon1 polymorphisms and CHB risk in any genetic model or pairwise comparisons when compared with HC group or SR group. In the stratified analysis of ethnic groups, also no obvious relation between mbl2 polymorphism and CHB risk was identified. There was still no significant association between the complete mbl2 genotypic profile (including both the exon1 and the promoter gene) polymorphisms and CHB risk, as compared with SR group. However, it was found that there was an association between the mbl2 AO/OO genotype and severe hepatitis B (SHB) or liver cirrhosis (LC) (LC vs. HC:OR=3.66, 95%CI, 2.38-5.63; SHB vs. HC, OR=3.88, 95%CI, 2.26-6.64), but there was no relationship between the mbl2 AO/OO genotype and hepatocellular carcinoma (HCC) (OR=1.26, 95%CI, 0.82-1.94). CONCLUSION: The present meta-analysis indicated that mbl2 exon1 polymorphisms might not significantly associate with chronicity of HBV infection, but might be significantly related to the progressive HBV such as SHB and LC.

[Alteration of mi-RNA expression profile after interferon treatment in HCV-infected Huh7.5.1 cells].

OBJECTIVE: To screen the mi-RNA expression profile after interferon treatment in cells infected with hepatitis C virus (HCV). METHODS: Huh-7.5.1 cells was infected with HCV by in vitro transcription and cultured with interferon. The mi-RNA microarray was used to measure the mi-RNA expression in the control group, HCV transcription group and interference group. Intra-group differences were analyzed by the 2 ((-delt delt CT)) method. RESULTS: With mi-RNA expressed in normal Huh-7.5.1 cells as a benchmark, expressions of 13 kinds of mi-RNAs were up-regulated after HCV infection and then down-regulated following interferon treatment; 7 were down-regulated after HCV infection and then up-regulated following interferon treatment. CONCLUSION: mi-RNA10a, mi-RNA21, mi-RNA149, mi-RNA152 and mi-RNA210 may be related to hepatitis C virus replication and transcription.