Clostridium Difficile Toxin B Research Articles

Clostridium difficile toxin B-induced apoptosis of colon cancer cells and related mechanisms

Objective To analyze the effects of Clostridium difficile toxin B (TcdB) on the proliferation and apoptosis of colon cancer cell line SW480 and the possible mechanisms related to cell apoptosis. Methods SW480 cells were treated with different concentrations of TcdB. Cell proliferation was detected by MTT assay. Cell apoptosis and mitochondrial membrane potential were measured with flow cytometry. Results TcdB significantly inhibited the proliferation of SW480 cells in a time-concentration dependent manner and the inhibition rate reached 46.36% at 48 h. Flow cytometry results showed that TcdB could induce the apoptosis of SW480 cells in a time-concentration dependent manner and a 20.83% apoptosis rate was induced by 800 ng/ml of TcdB at 48 h. Conclusions TcdB could inhibit the proliferation and induce the apoptosis of colon cancer SW480 cells, and the possible mechanisms might be relate to the initiation of mitochondrial apoptosis pathway. Key words: Clostridium difficile toxin B; Colon cancer SW480 cells; Proliferation; Apoptosis

Diarrhea pathogens in rotavirus-negative infants in Hangzhou

Objective To investigate the distribution of diarrhea pathogens in infants without rotavirus-detection in Hangzhou. Methods 605 stool samples of children with rotavirus-negative diarrhea were collected from Hangzhou First People's Hospital of Zhejiang University, Children's Hospital of Zhejiang University and Hangzhou Children's Hospital from March 2017 to June 2018. The routine test results were analyzed retrospectively and Bristol score was used for the characteristics of stool samples. DNA and/or RNA were extracted from fecal samples with DNA and RNA extraction kit. The extracted DNA and RNA-reversed cDNA were used as templates. 7 common pathogens DNA and/or RNA were amplified by polymerase chain reaction (PCR). The amplified products were detected by agarose gel electrophoresis. The positive rates of pathogens were analyzed by chi-square test. Results Among 605 children, 375 were male(28±11) months and 230 were female (29±10) months. Bristol score of stool samples was mainly in type 6 (496, 82%), followed by type 7 (85, 14%) and type 5 (24, 4%). Among 605 results 97 cases were occult blood positive (positive rate 16%) and 170 cases were white blood cell positive (positive rate 28%).452 of 605 stool samples were positive for pathogen target genes. The positive rate was 74.7%. 319 cases detected single pathogen gene fragments. 127 cases detected two pathogen genes and 6 cases detected three pathogen gene fragments. The positive rate of Clostridium difficile toxin B (48.9%, 296/605)was the highest than the others, followed by Salmonella (20.0%, 121/605) and Norovirus (10.9%, 66/605). The positive rate of Clostridium difficile toxin A was 1.0% (6/605). The positive rates of pathogens in male and female children were 86.7% (325/375) and 86.5% (199/230) respectively, with (χ2=0.002, P=0.959). Conclusions Salmonella and Norovirus were the main pathogens in children with diarrhea who were negative for rotavirus detection in Hangzhou. The high positive rate of Clostridium difficile toxin B may be related to the colonization of Clostridium difficile in the gastrointestinal tract of infants rather than the pathogen of diarrhea because of the low positive rate of Clostridium difficile toxin A. There was no gender difference in the detection rate of diarrhea pathogens. Key words: Diarrhea; Salmonella; Norovirus; Bacterial toxins; Polymerase chain reaction; Epidemiologic studies

Evaluation of a real-time PCR assay for the qualitative detection of Clostridium difficile toxin gene and its clinical application

Objective In comparison of the performances for the detection of Clostridium difficile toxin B genes from stool between BD MAX Cdiff assay and a laboratory-developed (LD) assay.The LD assay was evaluated in clinical application. Methods This study was a clinical application research. A total of 147 stool specimens from patients with diarrhea in Hangzhou First Hospital affiliated with Zhejiang Chinese Medical University were detected by the two assays from 1 July to 30 September 2014. DNA extraction and amplification of the tcdB gene were performed automatically on the BD MAX platform. Meanwhile, the tcdA and tcdB gene were detected by the LD real-time PCR assay after DNA extraction. Then, the results were analyzed by use of SPSS 10.0. Results A total of 147 stool samples were collected. There were 33 C. difficile positive cases and 114 negative cases detected by both of two assays. However, there were four stool samples had incongruent results. In comparison with BD MAX, the LD assay had a sensitivity of 93.94% (31/33), a specificity of 98.25% (112/114), a positive predictive value of 93.94% (31/33), and negative predictive value 98.25% (112/114). Furthermore, the results of the LD assay were statistically coherent with that of the BD assay (Kappa=0.922, P<0.01). Conclusions The LD assay was highly sensitive and accurate as BD MAX Cdiff assay in the detection of toxigenic Clostridium difficile. Furthermore, this LD assay could be also applied to detection of clinical stool samples directly with low cost. The assay will be more promising in diagnosis of toxigenic C. difficile in clinical application in China due to no additional instrument needed.(Chin J Lab Med, 2017, 40: 511-514) Key words: Clostridium difficile; Bacterial toxins; Molecular diagnostic techniques

The expression of receptor-binding region of Clostridium difficile toxin B

Objective To express receptor-binding region of Clostridium difficile toxin B by gene cloning,providing foundation forestablishmentof a rapid method to detect Clostridium difficile. Methods Recover Clostridium difficile,extract genomic DNA and amplify the targeted gene by PCR.The recovered PCR product was linked to p GEM-T by TA cloning.pGEM-T-CDB3 was identified by PCR,restriction enzyme cleavage and gene sequencing.The plasmids p GEM-T-CDB3 and pGEX-4T-1 were digested.The target gene CDB3 and pGEX-4T-1 were recovered,ligated and transformed into E.coli BL21 (DE3) to construct the recombinant plasmid.The expression of target protein was induced by IPTG,while the molecular weight and specificity of the protein were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Results The PCR product of Clostridium difficile toxins B receptor-binding domain (CDB3) was about 1851 bp.Plasmid pGEMT-CDB3 was about 4800 bp,obtained by recombinatiom the cloned vector pGEM-T with CDB3.The recombinant expression plasmid p GEX-4T-1-CDB3 was constructed and the target protein was expressed after induction. Conclusion The prokaryotic expression vector p GEX-4T-1-CDB3 is constructed successfully and the target protein is expressed greatly,which will lay a good foundation for rapid detection of Clostridium difficile toxin B and development of a vaccine.(Chin J Lab Med,2013,36:324-328) Key words: Clostridium difficile; Bacterial proteins; Bacterial toxins; Polymerase chain reaction

Cell expression of a four extra octarepeat mutated PrP C modifies cell structure and cell cycle regulation

RK13 cell lines generated to express bovine PrP C with a four extra octarepeat insertional mutation (Bo-10ORPrP C) show partially insoluble PrP C and lower rates of cell growth when compared to either the same cells expressing wild type Bo-6ORPrP C or the original RK13 cell line. The expression of Bo-10ORPrP C in cell cultures was also associated with changes in cell size and reorganization of the actin cytoskeleton. This last process was reversed by Clostridium difficile toxin-B, a specific inhibitor of small GTPase proteins. Further, in clones expressing Bo-10ORPrP C, increased proportions of cells at cell cycle stage G2/M were observed. Proteasome inhibitors caused a further expansion of G2/M-stage cells that was more marked in cell lines expressing Bo-10ORPrP C than those expressing Bo-6ORPrP C, while this effect was minimal or null in the original RK13 cell line. Hence, the presence of Bo-10ORPrP C in RK13 cells promotes cell cycle arrest at G2/M, and the effect is amplified by proteasome inhibition. These findings suggest a role for PrP C in cell morphology and cell cycle regulation, and open new avenues for understanding the mechanisms underlying PrP mutation-associated diseases.