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Isolation and preliminary identification of 74×103 molecule as putative Japanese encephalitis virus receptor

Junping REN ; Yingfeng LEI ; Wei ZHANG ; Jing YANG ; Wenyu MA

Chinese Journal of Microbiology and Immunology.2009;29(4):307－311-. doi:10.3760/cma.j.issn.0254-5101.2009.04.005

Objective To isolate and identify the putative Japanese encephalitis virus (JEV) re-ceptors from C6/36 and Vero cells. Methods Molecules binding with JEV were isolated from C6/36 and Vero cells by co-immunoprecipitation (Co-IP) approach, identified by mass spectrometry, and detected by Western blot. The location of putative JEV receptor on cells membrane and the binding with JEV were ob-served by laser scanning confocal microscopy (LCM). Results Several molecules binding with JEV were isolated from C6/36 and Vero cells by Co-IP, and only one molecule was identified as heat shock cognate 70 (HSC70) by mass spectrometry. Antibody against HSC'70 was able to detect a 74 ×103 protein isolated by Co-IP from C6/36 and Vero cells membrane in Western blot assays. It was observed by LSCM that when JEV attached on the surface of C6/36 cells, JEV and HSCT0 protein were co-localization. Conclusion 74 x 103 molecular identified as HSC70 protein from C6/36 cells may be JEV receptor.

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Study on adhesion of Lactobacillus acidophilus to mice Peyer's patches and inhibition of pathogen invasion

Jin SUN ; Tingting ZHOU ; Guifang CHANG ; Guowei LE ; Yonghui SHI

Chinese Journal of Microbiology and Immunology.2009;29(4):361－365-. doi:10.3760/cma.j.issn.0254-5101.2009.04.018

Objective To investigate the adhesion mechanism of Lactobacillus acidophilus FN001外to Peyer's patches. Methods Adhesion of L. acidophilus FN001 to mice Peyer's patches was studied in vitro using a fluorescent quantization method. The nature of adhesion mediator was studied by the effects of physical, chemical and enzymatic pre-treatments of the bacteria on their adhesion and effect of sugars on in- hibition of adhesion. The presence of lectin-like proteins in the cell surface was determined by hemagglutina- tion. Effect of L. acidophilus FN001 on inhibition of adhesion of pathogens to Peyer's patches was also stud- ied. Results The adhesion of L. acidophilus FN001 was strongly inhibited in the presence of D-mannose and methyl-ct-D-mannoside. Pretreatment of L. acidophilus FN001 with pepsin and trypsin decreased the ad- hesive capacity indicating that cell surface proteins are involved in adhesion to Peyer's patches. L. acidophi- lus FN001 could agglutinate rabbit red cell in mannose specific manner and protease pretreatment could de-crease hemagglutinin, suggesting that L. acidophilus FN001 has mannose specific lectin (s). In adherence inhibition assay, L. acidophilus NF001 could significantly inhibit adhesion of E. coli ATCC25922 to Peyer's patches when L. acidophilus NF001 were applied to Peyer's patches first or at the same time with pathogen. Conclusion It was concluded that a mannose-specific protein mediated adhesion of L. acidophilus FN001 to the Peyer's patches, and L. acidophilus FN001 could inhibit adhesion of pathogen with similar lectins speci- ficity to Peyer's patches.

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The variety of the mRNA expression of perforin, granzyme B by expanded NK cells with different cytokines from human peripheral blood

Jian MA ; Xiaohong LI ; Chunji GAO ; Dandan ZHAO

Chinese Journal of Microbiology and Immunology.2009;29(4):336－339-. doi:10.3760/cma.j.issn.0254-5101.2009.04.012

Objective To study the relationship between the expression of perforin, granzyme B and the cytotoxicity of NK cells, after NK cells were purified and expanded with different cytokines. Meth-otis It was detected that the mRNA expressions of perforin, ganzyme B from 8 donors, and the cytotoxcity of NK cells to target I(562 cells by competitive qualitative RT-PCR. Results The mRNA expressions of perforin, granzyme B by purified and expanded NK cells with different cytokines were markedly enhanced, and IL-2 +IL-15 group, IL-2 + IL-12 + IL-15 group were significantly higher than others. It is consistent that the relationship between the mRNA expressions of NK cells and the cytotoxicity of NK cells to I(562 cells. Conclusion The mRNA expressions of perforin, granzyme B of NK cells with different cytokines were obviously.

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Mechanisms of loss of immune tolerance in the production of antiplatelet autoantibodies in primary immune thrombocytopenia

Juping ZHAI ; Yang HE ; Binghua YANG

Chinese Journal of Microbiology and Immunology.2016;36(10):790-794. doi:10.3760/cma.j.issn.0254-5101.2016.10.014

Primary immune thrombocytopenia ( ITP) is an organ-specific autoimmune hemorrhagic disease. The etiology of ITP is still unclear, but loss of immune tolerance to platelet surface antigens is con-sidered as a fundamental cause of ITP. Therapeutic strategies that prevent the activation and proliferation of autoreactive cells have been suggested, which includes clearance of autoreactive cells ( apoptosis) , receptor editing, induction of anergy and extrinsic cellular suppression. Failure at any of these steps may lead to the production of autoantibodies against platelet and megakaryocyte glycoproteins. An improved understanding of the mechanisms for autoantibody production will provide theoretical basis for optimal diagnosis and treatment of ITP.

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CTLA-4 and PD-1:the potential immunotherapeutic targets for malignant lymphoma

Xiaohui JIA ; Xianhuo WANG ; Huilai ZHANG

Chinese Journal of Microbiology and Immunology.2016;36(10):783-789. doi:10.3760/cma.j.issn.0254-5101.2016.10.013

The human immune system has the regulatory functions of eradicating pathogens and limiting excessive inflammation, which protect the surrounding tissue from being damaged. The immune bal-ance of self-limiting is mainly controlled by complex interactions between antigen-presenting cells ( APCs ) and T cells. However, the immune balance is destroyed in cancer, which results in immune evasion and tumor metastasis or promotes the development of drug resistance. Immune checkpoints play critical roles in the immune system. Therefore, blocking tumor immune evasion by targeting the immune checkpoints has be-come a research focus in the treatment of relapsed or refractory malignant tumors. Currently, in the studies of malignant lymphomas, some phaseⅠ/Ⅱclinical studies of immune checkpoint inhibitors have achieved sur-prising results. This review will discuss the regulation and immunotherapy of immune checkpoints in malig-nant lymphomas.

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Establishment of a double-antibody sandwich ELISA for the detection of shiga toxin typeⅡin shiga toxin-producing Escherichia coli infection

Fengjuan SHI ; Xiaoyan ZENG ; Lu SONG ; Zhiyang SHI ; Xiling GUO ; Yongjun JIAO

Chinese Journal of Microbiology and Immunology.2016;36(10):771-774. doi:10.3760/cma.j.issn.0254-5101.2016.10.010

Objective To establish a double-antibody sandwich ELISA for the rapid detection of shiga toxin typeⅡ ( StxⅡ) in shiga toxin-producing Escherichia coli ( STEC) infection. Methods A pool of murine hybridomas was used to screen out the optimal antibody pair for the establishment of double-anti-body sandwich ELISA. The established ELISA system was used to detect StxⅡin the culture supernatants of 16 clinical strains of STEC. Specificity and sensitivity of the established ELISA system were also evaluated. Results Two antibodies, S2D8 and S2C6, were successfully screened out, based on which the double-anti-body sandwich ELISA was set up. StxⅡand its variants rather than StxⅠwas detected in the culture super-natants of STEC with a lowest detection limit of 4 ng/ml. Its performance was consistent with that of commer-cial colloidal gold test kit, indicating the characteristics of good specificity and sensitivity. Conclusion The S2D8/S2C6-based ELISA laid a foundation for researches which designates the shiga toxin as a potential can-didate on the diagnosis and therapy of STEC infection.

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Effectiveness of varicella vaccine:a case-control study

Yaping REN ; Yi FEI ; Shaotan XIAO ; Yi PENG

Chinese Journal of Microbiology and Immunology.2016;36(10):758-761. doi:10.3760/cma.j.issn.0254-5101.2016.10.007

Objective To evaluate the vaccine effectiveness ( VE) in children who received one dose of live attenuated varicella vaccine. Methods A questionnaire-based 1 ∶ 2 matching case-control study was conducted during May, 2015 to April, 2016 to evaluate the VE based upon the demographic data, vacci-nation status and clinical symptoms of 127 children with varicella and 254 healthy subjects under 12 years old. Results Compared with the healthy subjects, 83. 46% of the patients with varicella were immunized with one dose of varicella vaccine, which was lower than that of the control group (90. 55%) (χ2=4. 08, P<0. 05). The overall protective rate of one dose of varicella vaccine reached 64. 29% (95% CI:42. 00%-78. 00%). Of the 127 cases of varicella, 83. 46% were breakthrough cases immunized with one dose of var-icella vaccine. Primary cases presented more severe clinical symptoms than breakthrough cases. The longer the interval between vaccination and varicella-zoster virus infection was, the more severe the breakthrough cases would be. Conclusion Varicella vaccine is effective for preventing varicella, but the VE of one dose of varicella vaccine is limited. A two-dose varicella vaccination strategy should be proposed in our country as soon as possible.

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Hepatitis B virus X protein induces CRT transcription by activating C/EBPα

Xin YUE ; Chunhui WAN ; Peiwei ZHAO ; Xiaonan CAI ; Xuelian HE

Chinese Journal of Microbiology and Immunology.2016;36(10):740-745. doi:10.3760/cma.j.issn.0254-5101.2016.10.004

Objective To investigate the molecular mechanism of calreticulin ( CRT) transcription induced by HBV and its viral proteins. Methods The human hepatocellular cell line, HepG2, was trans-fected with pHBV1. 3 and eukaryotic expression plasmids of HBV viral proteins, respectively. The expres-sion of CRT was measured after transfection. A reporter plasmid of CRT promoter was constructed to analyze the induction of CRT promoter by pHBV1. 3 and HBV viral proteins. Furthermore, two truncated and one C/EBPα site deficient mutants were constructed to evaluate the regulatory effects of HBx on CRT promoter. Fi-nally, HepG2 cells were transfected with HBx expression plasmids and the cellular localization of C/EBPαwas analyzed. Results In this study, pHBV1. 3 could significantly up-regulate the expression of CRT at mRNA and protein levels as well as enhancing the activity of CRT promoter. Among the seven HBV viral proteins, HBx could enhance the activity of CRT promoter and the expression of CRT at mRNA and protein levels. HBx could not induce the transcription of CRT when the C/EBPα binding site was deleted from the CRT promoter. The expression of HBx could promote the nuclear translocation of C/EBPα. Conclusion HBV and its viral protein HBx could up-regulate the CRT expression at transcriptional level. The transcrip-tional factor C/EBPα played a critical role in HBx-induced transcriptional activation of CRT.

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Mechanisms and molecular typing of carbapenem-resistant Proteus mirabilis strains deficient in swarming motility

Long SUN ; Xi LI ; Xiaoting HUA ; Keren SHI ; Yanfei WANG ; Ranfeng CHEN ; Yunsong YU

Chinese Journal of Microbiology and Immunology.2016;36(10):734-739. doi:10.3760/cma.j.issn.0254-5101.2016.10.003

Objective To investigate the mechanism and epidemiological characteristics of carbap-enem-resistant Proteus mirabilis ( PM) strains deficient in swarming motility. Methods PM strains were isolated from Hangzhou General Hospital of CAPF ( Chinese People′s Armed Police Forces) during January 2013 to December 2014. Bacterial motility and flagella of the PM strains were observed through semi-solid agar culture and flagella staining. Pulsed-field gel electrophoresis ( PFGE) was performed for homology anal-ysis. Antimicrobial susceptibility test and phenotypic confirmatory test were also carried out. PCR analysis and DNA sequencing were performed to confirm the genotype of resistant genes. Plasmid electroporation and S1-PFGE in combination with Southern blot hybridization were used to determine the location of the carbap-enem-resistant genes. Genetic structure of the blaKPC-2 gene was obtained by PCR mapping. Results A total of 42 PM isolates deficient in swarming motility were screened out and the resistance rates to imipenem and meropenem were 57. 1% and 52. 4%, respectively. PCR analysis and DNA sequencing confirmed that 24 carbapenem-resistant PM isolates deficient in swarming motility carried blaKPC-2 gene and belonged to three clones as indicated by the results of PFGE. Southern blot hybridization indicated that the blaKPC-2 gene was located on plasmids varying in size (26 kb, 55 kb and 139 kb). In addition, some of the strains harbored several resistant genes, such as blaTEM-1 , blaCTX-M-65 and rmtB. The genetic structures of strains carrying blaKPC-2 gene were ISKpn8, blaKPC-2 and ISKpn6-like from upstream to downstream. Conclusion Compared with the PM strains with swarming motility, the carbapenem-resistance rate was significantly higher in these PM strains deficient in swarming motility. Carbapenemases KPC-2 played an important role in the carbapen-em-resistant PM strains deficient in swarming motility. There was a cloning spread trend for carbapenem-re-sistant PM strains in our hospital. Clinicians should pay more attention to the risk of spreading.

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Laboratory test for 18 imported Zika cases in China

De WU ; Huan ZHANG ; Qiqi TAN ; Jiufeng SUN ; Huiqiong ZHOU ; Dan NING ; Dawei GUAN

Chinese Journal of Microbiology and Immunology.2016;36(10):721-726. doi:10.3760/cma.j.issn.0254-5101.2016.10.001

Objective To provide scientific evidences for Zika virus detection by clarifying the means by which Zika virus was discharged and the duration of corresponding processes. Methods Various samples of Zika cases were collected at different times and detected by using real-time RT-PCR. The positive samples were inoculated into cells and suckling mice through intracranial injection. The whole genome se-quences of those isolated Zika virus strain were sequenced and the results were further analyzed by comparing with the sequences of Zika virus from GenBank. Results The positive rates of Zika virus in urine, saliva and serum samples were 82. 4% (14/17), 82. 4% (14/17) and 52. 9% (9/17) respectively. The longest period of detected presence of Zika virus was found in urine samples amongst the three types of samples, fol-lowed by saliva and serum samples. Six Zika virus strains were isolated from 9 positive serum samples. Phy-logenetic analysis showed that the six genomes of Zika virus all belonged to Asia lineage, but located in two branches by Samoa and Venezuela strains. Conclusion This study indicated that urine, saliva and serum all could be used as the samples for routine detection of Zika virus. Urine and saliva samples showed higher detection rates of Zika virus RNA in comparison to serum samples, while Zika virus could be easily isolated from positive serum samples. Suckling mice were better for Zika virus isolation than cell lines.

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