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The inhibiting effect on rat C Ⅱ TA and MHC Ⅱ gene expression by plasmid vector-based RNAi technulogy

Gang LIU ; Xianghui HE ; Na ZHAO ; Liwei ZHU

Chinese Journal of Microbiology and Immunology.2009;29(4):375－379-. doi:10.3760/cma.j.issn.0254-5101.2009.04.021

Objective To investigate the inhibitory effect on rat MHC Ⅱ transactivator (C Ⅱ TA) and MHC Ⅱ gene expression by plasmid vector-based RNAi technology. Methods According to C Ⅱ TA ge-netic information, three short hairpin RNA (shRNA) sequences were designed and the corresponding plas- mid vectors were constructed. Rat dendritic cell (DC) was transfected in vitro and rat spleen was transfected in vivo. Real time RT-PCR and flow cytometry were used to detect the expression of C Ⅱ TA and MHC Ⅱ in DC and spleen after transfection. Results C Ⅱ TA-shRNA plasmids were successfully constructed. Com-pared with control groups, the mRNA transcription levels of C Ⅱ TA and MHC Ⅱ and the expression level of MHC H were significantly inhibited in all three pC Ⅱ TA-shRNA experimental groups ( P < 0.01 ). There was positive correlation between the expression of C Ⅱ TA and MHC Ⅱ. Among the three shRNA groups, the first one showed the strongest inhibitory effect. Conclusion The expression of rat C Ⅱ TA and MHC Ⅱ can be obviously inhibited by plasmid vector of shRNA targeting C Ⅱ TA gene, which can be used for further investi-gation of gene therapy.

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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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NOD2 plays an important role in the inflammatory responses of macrophages to Staphylococcus aureus

Xuhua XIE ; Lili WANG ; Fengyun GONG ; Chao XIA ; Ying SONG ; Jianxin SONG

Chinese Journal of Microbiology and Immunology.2011;(3):193-200. doi:10.3760/cma.j.issn.0254-5101.2011.03.001

Objective To investigate the effects of NOD2 on inflammatory responses of macrophages to Staphylococcus aureus. Methods Real-time RT-PCR detected NOD2 gene expression of macrophages infected by S. aureus. Synthesis of siRNA against NOD2 and interfere with macrophages, observed the effects of NOD2 gene silencing to phagocytosis of 5. aureus, cytokine secretion, activation of nuclear transcription factors, cell apoptosis of the macrophages infected by S. aureus using F.I .IS A, flow cytometry etc. Results S. aureus infection of macrophages can cause increased expression of intracellular NOD2. NOD2 gene silencing of macrophage lead to the decreased ability of phagocytosis with S. aureus, the lower levels of cytokines secretion, deficiencies of NF-κB activation. S. aureus can cause macrophage apoptosis, with the apoptosis rate increased with time. Conclusion The intracellular pattern recognition receptor NOD2 play a key role in pathogen recognition, signal transduction, activation of nuclear transcription factors in the process of macrophages infected by S. aureus.

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CheA/CheY signaling system responsible for chemotaxis in vitro and colonization in vivo of Campylobacter jejuni

Yuan WANG ; Hongqiang LOU ; Huan WANG ; Weilin HU ; Jie YAN

Chinese Journal of Microbiology and Immunology.2011;(3):201-207. doi:10.3760/cma.j.issn.0254-5101.2011.03.003

Objective To determine the effects of Che A and CheY proteins of Campylobacter jejuni regulating the bacterial chemotaxis in vitro and colonization in vivo. Methods By using pET42a plasmid and E. coli BL21DE3 as expression vector and expression strain, respectively, prokaryotic expression systems of cheA and cheY genes of C. jejuni strain NCTC11168 was constructed. Rabbits were immunized with the target recombinant expression proteins, rCheA and rCheY, to prepare the antisera. rCheA-IgG and rCheY-IgG in the antisera were separated using DEAE-52 ion exchange column. pBluescript- II -SK was applied to construct suicide plasmid which used to generate cheA gene knock-out mutant (cheA-). A chemotaxis model in vitro of C. jejuni based on DOC-HAP, the chemotactic ability of cheA' mutant as well as the effect of rCheA-IgG and closantel inhibiting the bacterial chemotaxis were demonstrated. The phosphorylation levels of CheA and CheY after DOC treatment were examined by using either rCheA-IgG or CheY-IgG capture method. The difference of colonization ability between cheA- mutant and wild-type of C. jejuni in mice were checked and then compared. Results The constructed prokaryotic expression systems could efficiently express rCheA and rCheY. The data from PCR and sequencing confirmed the cheA gene knock out from cheA- mutant chromosome. cheA- mutant lost its chemotactic ability towards DOC. Both the rCheA-IgG and closantel could inhibit the chemotaxis of wild-type of C. jejuni (P < 0.05 ). When treatment of DOC, the phosphorylation levels of CheA and CheY in wild-type of C. jejuni rapidly decreased (P < 0. 05 ). The colonization ability in murine jejunum of cheA- mutant was also lower than that of the wild-type ( P<0.05 ) . Conclusion Chemotaxis-associated two-component signaling system (Che-TCSS) of C. jejuni are composed of CheA and CheY, and the two proteins are activated by dephosphorylation. CheA in the Che-TCSS play a critical role in chemotaxis in vitro and colonization in vivo of C. jejuni, and this protein can be used as a target for developing novel anti- C. jejuni drugs.

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The study on epidemic characteristics of an outbreak of panresistance Klebsiella pneumoniae

Yanyan WANG ; Hong LIU ; Xin DU ; Gang LI ; Quhao WEI ; Xiaoyun CHEN ; Xiaofei JIANG

Chinese Journal of Microbiology and Immunology.2011;(3):208-212. doi:10.3760/cma.j.issn.0254-5101.2011.03.004

Objective To understand the epidemic characteristics of an outbreak of panresistance Klebsiella pneumoniae occurred between 2006 and 2009 in a university hospital of Shanghai, China. Methods A total of 57 panresistance K. pneumoniae isolates were collected from August 2006 to December 2009.Antibiotic susceptibility of the isolates were determined by Kirby-Bauer disc diffusion method and microbroth dilution (MBD). ESBLs-producing initial screen test and phenotypic confirmatory test and carbapenemase-producing modified Hodge test ( MHT) were performed to detect the resistance phenotype of the isolates. Be-ta-lactamases were studied by IEF, PCR and the product sequencing. While conjugation assay were conducted to understand the transferability of these genes. The genetic relationship between isolates was established by ERIC-PCR and multilocus sequence typing (MLST). Except for the antibiotics recommended by CLSI guideline in the routine test, the other antibiotics were added to find out the effective drugs to treat the infection. Results All 57 isolates were highly resistant to all examined antibiotics. All isolates produced ESBLs and carbapenemase. IEF revealed that each isolate produced four beta-lactamases. All isolates carried blaKPC-2,blaCTX-M-14,blaSHV12,blaTEM-1,qnrB and aac(6') - I b-cr. Forty-four of the 57 (77.2% ) isolates were successful to transfer their resistance genes to E. coli recipient J53 by conjugation assay. By RAPD, all 57 isolates were grouped into two genotypes that were further identified as members of MUST types 423 and 11.Sequence types 423(ST423) only occurred before May 2008 and ST11 occurred (52 isolates) after May 2008. Most of isolates of the outbreak were ST11 (91. 2% ). A part of isolates were susceptive to added antibiotics. Conclusion The outbreak of panresistance K.pneumoniae was caused by those isolates which carried multiple resistant genes. There is a different ability of dissemination between different ST types K. pneumoniae isolate. It was necessary to add the antibiotics to find out the effective drugs to treat the infection.

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Activation of TLRs/NF-κB signal pathway and occurrence of different functional cytokines during invasive pulmonary aspergillosis

Xiaomei XIE ; Xiang LI ; Hongdan LUO ; Qing SHI ; Haihong ZHANG ; Zhenhua RAO

Chinese Journal of Microbiology and Immunology.2011;(3):213-219. doi:10.3760/cma.j.issn.0254-5101.2011.03.005

Objective To study the activation of TLRs/NF-κB signal pathway and production of different functional cytokines during invasive pulmonary aspergillosis( IPA) , in order to probe the pathogene-sis of IPA. Methods Mouse were randomly divided into normal, normal + inoculated with Aspergillus fumigatus( normal inoculation group), and immune suppression + inoculation with Aspergillus fumigatus (IPAmodel group) , the mouse were killed at different time points after inhaling Aspergillus fumigatus spores by nose. Removing the lung tissue in a sterile manner and making pathological section respectively, counting Aspergillus fumigatus colony, dynamiclly detecting the expression of TLR2, TLR4 mRNA, variation of NF-κB p65 protein, pro-inflammatory cytokines TNF-α, IL-1β and anti-inflammatory cytokines IL-10 levels in the lung tissue by RT-PCR and Western blot method during Aspergillus fumigatus infection in mouse. Results (1) When it's 72 h after inhaling Aspergillus fumigatus by nose, IPA model emerged severe lung tissue inflammation, and generated a large number of hyphae, meanwhile, burthen of Aspergillus fumigatus was higher than normal inoculation group at each time point. (2)Compared with the normal inoculation group, IPA group whose TLR2 mRNA was low expression at early stage of infection (24 h), and emerged high expression at late stage of infection (120 h, 144 h); and TLR4 mRNA has been at a state of low expression in the infection process; NF-κB p65 suddenly increased at early stage of infection(24 h) and then continued to decline. (3) After infected by Aspergillus fumigatus in normal mouse, proinflammatory cytokine TNF-α, IL-1β in lung exhibited high expression at the early stages of infection, and the highest expression levels appeared at 48 h or 72 h, then decreased and recovered to normal level. And the expression level of anti-inflammatory cytokine IL-10 rised at late stage of infection; The IP A mouse released a lot of anti-inflammatory cytokine IL-10 at early stage of infection, which significantly reduced at late stage, and released pro-inflammatory cyto-kines TNF-α, IL-1β at slow and low level. Conclusion The abnormal activation of TLRs/NF-β signaling pathway caused the loss of dynamic balance between pro-inflammatory cytokines and anti-inflammatory cytokines, leading to the occurrence and development of IPA.

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Rapid identification and susceptibility testing of positive blood culture caused by gram negative bacteria

Yueru TIAN ; Feiyi RUAN ; Hong LIU ; Fuqi AI ; Yimin MA ; Ye JIANG ; Xiaofei JIANG

Chinese Journal of Microbiology and Immunology.2011;(3):220-224. doi:10.3760/cma.j.issn.0254-5101.2011.03.006

Objective To reduce the turnaround time for laboratory diagnosis of bacteremia, the feasibility of rapid identification and susceptibility testing using samples taken directly from positive blood culture bottles was evaluated. Methods The growth of microorganisms in blood culture bottles was screened by the BACTEC 9000 blood culture system. 65 positive blood culture bottles containing gram-negative bacteria were adopted to test. Culture fluid was injected into BD SST vacutainer and centrifuged to pellet blood cells. After collecting required McFarland units, they were cultured on Phoenix 100 NMIC/ID-4(identification-gram-negative bacteria and susceptibility testing) cards using 0.25 McF and 0.5 McF methods respectively. They were also evaluated by the standard method, involving subculture tests from positive blood culture bottles. Results 63 of 65 gram-negative bacteria (96. 9% ) were correctly identified with 0. 25 McF method. 59 of 65 gram-negative bacteria(90.8% ) were correctly identified with 0.5 McF method. For antimicrobial susceptibility testing, the 0.25 McF direct method had an agreement rate more than 94% , the 0.5 McF method was more than 85.7% and direct blood sample KB method was more than 93.8% compared to the standard method. But the overall minor error rate in susceptibility testing of direct blood sample KB method is higher than other methods. Conclusion Applying 0. 25 McF and 0. 5 McF rapid identification and susceptibility test was practical. During to possessing more prominent advantages, laboratory put the 0. 25 McF direct method into practice had a timely, remarkable significance.

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Immunogenicity comparison of DNA vaccines encoding HIY-1 AE2f Tat, Rev and Nef in different gene fusion formulations

Guocai Lü ; Lin ZHENG ; Yida YANG

Chinese Journal of Microbiology and Immunology.2011;(3):225-228. doi:10.3760/cma.j.issn.0254-5101.2011.03.007

Objective To construct DNA vaccine expressing HIV-1 AE2f gp145-tat-rev-nef fusion gene( AE-Gp145TRN) and to compare the immunogenicities of DNA vaccines expressing Tat, Rev and Nef in gene fusion formulations of tat-rev-integrase(c-half)-vif-nef( AE-TRIVN) and AE-Gpl45TRN. Methods DNA vaccine was constructed by inserting the codon optimized HIV-1 AE2( gp145-tat-rev-nef fusion gene into mammalian expression DNA vector. In vitro expression efficiency of the constructed DNA vaccine was determined by Western blot and the immunogenicities of AE-Gpl45TRN and AE-TRIVN were compared by immunizing female BALB/c mice. IFN-r ELISPOT assay was used to read out the specific T cell immunity. Results Western blot assay showed the constructed DNA vaccine could be expressed efficiently in vitro. After vaccination, AE-TRIVN mounted significantly higher T cell responses against Tat, Rev and Nef[(148±91)SFCs/106 splenocytes]than Gpl45TRN[(55±28) SFCs/106 splenocytes]. Specific T cell responses elicited by AE-TRIVN predominantly targeting Rev, whereas Gpl45TRN could significantly enhance T cell responses against Nef. Conclusion AE-TRIVN and Gpl45TRN induced distinct T cell response modalities, which implied different gene fusion formulations may affect the immunogenicity of specific DNA vaccines.

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