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Early stage mechanism of the specific NF-κB inhibition induced cardiac allograft tolerance

Shuyuan LI ; Chengbiao XUE ; Chao LI ; Yao LI ; Zhishui CHEN ; Ping ZHOU

Chinese Journal of Microbiology and Immunology.2010;30(12):1128-1132. doi:10.3760/cma.j.issn.0254-5101.2010.12.014

Objective To investigate whether allocardiac graft acceptance in the specific NF-κB impaired mice is due to regulatory T cell(Tr) and Th17 cells.Methods Mice abdominal heterotopic cardiac transplantation was performed and then divided in to control group(BALB/c→C57BL/6) and experimental group(BALB/c→IκBα/△N-Tg).Pretransplant and at day 7,30,100 posttransplant,spleens were harvested from the IκBα△ N-Tg mice,and then the Tr were detected by the fluorescence activated cell sorter.At day 5 posttransplant,the CD4 + Th17 cells from the spleens of the two groups were examined by the FACS.Additionally,at day 3 and 5 posttransplant,IL-17 expressed in the cardiac allograft was detected by Western blot.Results In the IκBα/ N-Tg mice group,the cardiac allografts were survived more than day 100,and without obviously lymphocytes infiltration.At the day 7 and 30 posttransplant,the Tr was obviously increased(21.23 ± 3.95,23.17 ± 4.11 vs 11.64 ± 1.96,P ＜ 0.05); however,the Tr decreased at the day 100 posttransplant,and had no difference with before transplant(10.79 ±2.48 vs 11.64 ± 1.96,P ＞0.05).Compared with the control group,at day 5 posttransplant,CD4+ Th17 cells in the IκBα/N-Tg mice and IL-17 expression of the cardiac allograft were both decreased.Conclusion In the early stage after transplantation,specific T cell NF-κB impaired could abrogate the balance of the Tr and Th17 cells,and induce the T cells differentiated into Tr and inhibit the Th17 cells differentiation,and then induce tolerance.

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Analysis of the gene characteristic correspondence on rats with Hantavirus and the human infected Hantavirus in natural epidemic areas of HFRS in Shenzhen

Fan YANG ; Jianfan HE ; Jianjun LIU ; Wushen CHEN ; Huixia XIAN ; Hailong ZHANG ; Hong YANG ; Renli ZHANG ; Yaqing HE

Chinese Journal of Microbiology and Immunology.2010;30(12):1092-1096. doi:10.3760/cma.j.issn.0254-5101.2010.12.007

Objective To compare and contrast the gene characteristic correspondence of hantaviruses(HV) carried by rats in natural epidemic areas of hemorrhagic fever with renal syndrome(HFRS) and infected among HFRS patients in Shenzhen,provide a reasonable scientific basis for controlling of HFRS.Methods We collected the patients serum specimens in acute stage and lung tissues samples of rats.ElISA and direct immunofluorescence were applied to screen the positive samples,respectively.The partial G2 fragments of M segment and S segment were amplified from the representative patients' serum positivesamples and lung tissues positive samples in different areas with reverse transcription-nested-polymerase chain reaction(RT-nested PCR) by the hantaviruses genotype specific primers.The amplified genes were then sequenced,and subjected to genotyping and homology analysis with other known hantaviruses.Results Four hundred and seventy-two rats were trapped in the main epidemic areas,and Hantavirus specific-antigens in lung tissues samples were identified in 47 out of the 472 by direct immunofluorescence.Twelve partial M and S segments were amplified from 60 patients serum specimens positive with specific IgM antibodies against hantavirus with ELISA by RT-nested PCR.The homology of M and S genome segments among 16 strains of Hantaviruses showed more than 95% and 96.5% at nucleotide level,respectively.And the deduced amino acid sequences homology was 98.0% -100% and 98.4%-100%,respectively.The genotype of hantavirus carried by rats and infected among patients were identified to the same subtype-SEO S2.Conclusion The genotype of Hantavirus carried by rats and infected among patients in Shenzhen all belongs to S2 SEOV.The nucleotide homology of SEO type of Hantavirus in the same or nearby areas is higher and the viruses are highly conserved.

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Screening of proteins binding to mouse cytomegalovirus M122 protein from mouse brain cDNA library by yeast two-hybrid system

Hui WANG ; Yufeng ZHOU ; Sainan SHU ; Dan LUO ; Jia TIAN ; Huijuan ZHANG ; Xiaoyi DU ; Feng FANG

Chinese Journal of Microbiology and Immunology.2010;30(10):959-964. doi:10.3760/cma.j.issn.0254-5101.2010.10.020

Objective To investigate the biological function of M122 in pathogenesis of MCMV in developmental brain disorders and brain damage, screening for mouse brain cDNA library interacting with M122 was performed by a yeast two-hybrid system. Methods The reconstructed bait plasmid pGBKT7-M122 was transformed into yeast cells AH109 and screened on the nutrient deficiency medium SD/-Trp. After express of the bait protein in AH109 yeast strains was detected by Western blot analysis, yeast-two hybrid screening was performed by mating AH109 with Y187 containing mouse brain cDNA library plasmid. The diploid yeast cells were plated on the nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade. The second screening was performed with SD/-Trp/-Leu/-His/-Ade containing X-α-gal. The plasmids in positive colonies were extracted and transformed into E. coli JM109 cells. After plasmid DNA in JM109 cells were extracted form positive colonies and sequenced, the results were analyzed by bioinformatic methods. The interactions between M122 protein and the protein obtained from positive colonies were further confirmed by repeating yeast-two hybrid. Then, autoactivations of the proteins obtained from positive colonies were detected.Results The reconstructed bait plasmid was transformed into yeast cells AH109 successfully. The bait protein expressed in the yeast cells AH109 stably. 24 proteins interacting with MCMV M122 were screened, including syntaxin 8 ( Stx8 ), phosphoglucomutase 2 ( Pgm2 ), potassium voltage-gated channel, shaker-related subfamily, beta member 1 ( Kcnab1 ), collagen, type ⅪⅩ, alpha 1 ( Col19a1 ), archain 1 ( Arcn1 ), cytidylate kinase( Cmpk), DnaJ(Hsp40) homolog, subfamily A, member 1 (Dnaja1), ATPase, Na+/K + transporting, beta 3 polypeptide( Atp1b3 ), SH3-domain GRB2-like ( endophilin ) interacting protein 1 ( Sgip1 ),ankyrin repeat domain 17 (Ankrd17), Smg-7 homolog, nonsense mediated mRNA decay factor(Smg7),sperm associated antigen 9 ( Spag9 ), FK506 binding protein 1a ( Fkbp1a), MYST histone acetyltransferase monocytic leukemia 4 ( Myst4), hyaluronan and proteoglycan link protein 1 ( Hapln1), autophagy-related 3 (Atg3), splicing factor, arginine/serine-rich 5 ( Sfrs5 ), zinc finger, C3HC-type containing 1 ( Zc3hc1 ),thioredoxin-related transmembrane protein 1 ( Txndc1 ), adaptor protein complex AP-1, gamma 1 subunit (Ap1g1), Cullin 1 ( Cul1 ), and so on. Three of them were formerly unknown proteins. M122 protein could interact with the proteins obtained from positive colonies in the yeast cells AH109. Ap1g1 and Cul1 were proved to have autoactivation. Conclusion A class of proteins in brain interacting with M122 has been obtained. It is presumed that these proteins are correlated with neuropathogenesis of the brain disorders caused by CMV, but the candidates still need further confirmation for the interaction.

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Study on fast diagnostics of activated infection of human herpes virus type 6 in infants with fever and rash

He SUN ; Yosikawa TETSUSHI

Chinese Journal of Microbiology and Immunology.2010;30(10):953-955. doi:10.3760/cma.j.issn.0254-5101.2010.10.018

Objective To explore the fast diagnostics of activated infection of human herpes virus type 6 (HHV-6) with IEA/ex3( an antibody specific for an immediate early protein encoded by variant B HHV-6 U90 gene)in infants with fever and rash . Methods To collect the 55 samples of peripheral blood mononuclear cells (PBMCs) from clinical infants whose having a fever and rash, age are below 2 years old.The immediate early antigen (IEA) of HHV-6 were analyzed with IEA/ex3 in PBMCs by indirect immunofluorescence then using confocal microscope to observe, and using fluoromicroscope to observe after the PBMCs of 44 samples were incubated with cord blood cell at 37℃ in a humidified 5% CO2 atmosphere for 48 hours. The virus was isolated from the PBMCs. Results The conformation rate of the HHV-6 IEA using confocal microscope to observe and virus isolation was 92.73%, sensitivity was 90.32%, specificity was 95.83% and it was respectively 87.50%, 77.27%, 100% using fluoromicroscope to observe after the PBMCs were incubated for 48 hours. Conclusion The study suggested that detection of HHV-6B IEA in PBMCs using confocal microscope to observe after the PBMCs were incubated for 48 hours can fast diagnose HHV-6 activated infection of infant with fever and rash than technique of virus isolation.

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The cellular immune response produced in BALB/c mice immunized with HPV6b L1/Ct MOMP multi-epitope chimeric DNA

Zhaohui SHI ; Shanli ZHU ; Wen XU ; Lijun LU ; Lingling LI ; Lifang ZHANG

Chinese Journal of Microbiology and Immunology.2010;30(10):942-948. doi:10.3760/cma.j.issn.0254-5101.2010.10.015

Objective To study on the specific cellular immune response produced in BALB/c mice immunized with human papillomavirus (HPV) type 6b capsid protein L1 and Chlamydia trachomatis (Ct) major outer membrane protein(MOMP) multi-epitope chimeric DNA (HPV6b L1/Ct MOMP multiepitope) , and the enhancement of the specific cellular immune response to Ct MOMP multi-epitope by HPV6b L1. Methods The Ct MOMP multi-epitope gene was connected to the C terminal of HPV6b L1,the gene of HPV6b L1 had been optimized according to the codon usage of eukaryotic system, and then the HPV6b L1/Ct MOMP multi-epitope chimeric gene was cloned to pcDNA3.1 ( + ) vector. After identification by restriction enzyme digestion and DNA sequencing, the recombinant plasmid was transfected into COS-7 cells, Indirect Immunofluorescence (IIF) was used to confirm the expression of proteins. Then, BALB/c mice were randomly assigned to receive (intramuscular injection) either pcDNA3.1 ( + )/HPV6b L1/Ct MOMP or pcDNA3.1 ( + )/Ct MOMP or pcDNA3.1 ( + ) or PBS ( n = 12, 150 μg/time), and the same immunization schedule was repeated third times at 2 week intervals. The level of cytokine( IFN-γ, IL-4, IL-10) -producing CD3+ T cells in spleen, the cytotoxicity of Ct MOMP-specific and HPV6b L1-specific cytotoxic T lymphocyte (CTL) in spleen were detected by intracellular cytokine staining-fluorescence activated cell sorter (ICS-FACS) and LDH release assays, respectively. Results After immunization, when the efCTL (44.56%±4.02%, 35.35% ±2.89% ) and HPV6b L1 specific cytotoxicity of CTL (27.08% ±2.04%, 21.68% ±4.06% ) in pcDNA3.1 ( + )/HPV6b L1/Ct MOMP multi-epitope chimeric DNA immunized mice, were significantly higher than that in pcDNA3.1 ( + )/Ct MOMP multi-epitope DNA (35.50%±2.68%, 30.24% ±1.75%; 12.27% ±3.36%, 9.32% ±3.07%) and other control groups(F=72.87, F=114.55, P＜0.05; F=30.04, F=10.47, P＜0.05), and Ct MOMP multi-epitope specific cytotoxicity of CTL in pcDNA3.1 ( + )/Ct MOMP multi-epitope DNA immunized mice were significantly higher than that in control groups( F = 58.85, F = 120.21; P<0.05). The level of intracellular cytokine IFN-γ in pcDNA3.1 ( + )/HPV6b L1/Ct MOMP multi-epitope DNA immunized mice(4.34% ±0.06%)was higher significantly than that in pcDNA3.1 ( + )/Ct MOMP multi-epitope DNA immunized mice(3.14% ± 0.18%, P<0.05 ) and other control groups ( F = 473.83, P<0.05 ), while, the levels of IL-4 ( F =0.97, P ＞ 0.05 ) and IL-10 ( F = 2.25, P ＞ 0.05 ) had no significant difference between groups. Conclusion Both Ct MOMP and HPV6b L1 protein specific cellular immune response could be induced in BALB/c mice immunized with HPV6b L1/Ct MOMP multi-epitope chimeric plasmid, and the HPV6b L1 gene optimized by eukaryotic codon could significantly enhance the cellular immune response induced by Ct MOMP multi-epitope gene in BALB/c mice.

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Preliminary screening of different permeation enhancers in transcutaneous immunization with inactivated human highly pathogenic avian influenza vaccine

Yanli SUN ; Honggang ZHANG ; Yanhua SUN ; Wenbao LU ; Xiliang WANG

Chinese Journal of Microbiology and Immunology.2010;30(10):925-930. doi:10.3760/cma.j.issn.0254-5101.2010.10.012

Objective To screen the potent permeation enhancers used in transcutaneous immunization with inactivated highly pathogenic avian influenza vaccine. Methods Five different permeation enhancers, ethanol, propylene glycol, dimethyl sulfoxide, ratinoic acid, oleic acid, were used to treat the skin of BALB/c mice before transcutaneous immunization. Sera were collected before the flist transcutaneous immunization and every two weeks post immunization. The titers of influenza virus-specific humoral IgG and IgA were assayed in serum, lung and nasal lavages by ELISA. The titers of hemagglutination inhibition ( HAI), IFN-γand IL-4 produced by splenic lymphocytes were also detected. Except that, clinical symptom of the skin in different time points and skin pathological changes were observed. Results The serum IgG titers, HAI titers and the influenza virus-specific lgA and IgG in lung and nasal lavages in the groups of HA +CT + DMSO, HA + CT + RA and HA + CT + OA were significantly higher than those of HA and HA + CT groups( P ＜0.05). Moreover, the numbers of splenic lymphocytes producing IFN-γ and IL-4 were increased in the above three groups than those in control groups. In addition, no evident clinical symptoms were observed, but stratum corneum of the skin in different groups showed different changes. Conclusion DMSO,RA and OA are potent permeation enhancers in mouse model inoculated with inactivated high pathogenic avian influenza vaccine transcutaneously.

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Subtype and sequence analysis of the gag and env genes for HIV-1 strains isolated in Hebei Province

Cuiying ZHAO ; Qiaomin LI ; Hongru ZHAO ; Xinli LU ; Baojun LI ; Yuqi ZHANG ; Xiangfen MIAO ; Hui XING ; Xiang HE

Chinese Journal of Microbiology and Immunology.2010;30(10):914-918. doi:10.3760/cma.j.issn.0254-5101.2010.10.009

Objective To study the epidemic situation and characteristic of subtypes of HIV-1 strains prevalent in Hebei Province. Methods Viral RNA was extracted from plasma samples, HIV-1 genes (env and gag ) were amplified by RT and nested-PCR using specific primer pairs and sequenced directly.The acquired sequences were compared with international subtypes references and their phylogenetic-trees were analyzed to determine the subtype. Results Among 154 HIV-1 antibody positive cases , 148 HIV-1 gene fragments were amplified and analyzed. There were 6 kinds of HIV-1 subtypes and recombinants, moreover unidentified 2 cases in 148 samples, of which 61 (41.2%) cases of B', 59(39.9% ) cases of CRF01_AE, 16( 10.8% ) cases of CRF07_BC, 6(4.1% ) cases of CRF08_BC, and 2( 1.4% ) cases of C and B01 each. HIV-1 B01 was detected firstly in Hebei Province. Conclusion Six HIV-1 subtypes were identified in Hebei Province. B' and CRF01_AE are the primary subtypes and recombinants of HIV-1 existed in Hebei Province. The surveillance of HIV-1 gene variation should be paid more attention to.

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Effects of deoxyribozyme against respiratory syncytial virus infection in mice with different immune functions

Juan ZHOU ; Xiaodong ZHAO ; Liping JIANG ; Xiqiang YANG

Chinese Journal of Microbiology and Immunology.2010;30(10):902-908. doi:10.3760/cma.j.issn.0254-5101.2010.10.006

Objective To investigate the effects of deoxyribozyme(DZ) against respiratory syncytial virus(RSV) infection in BALB/c mice and nude mice. Methods RSV infected BALB/c mice and nude mice were nasally dripped with DZ. Pulmonary viral titers were detected by plaque forming experiment,and viral mRNA expression was assayed by RT-PCR. Leukocytes and the subgroup cells in bronchoalveolar lavage fluid (BALF) were counted, cytokines of TNF-α, IL-12, IFN-γand IL-10 in BALF were assayed by ELISA. Pulmonary histopathology was examined to realize the inflammation of airway. Results Pulmonary titers of 0.2 mg, 0.4 mg and 0.8 mg DZ treated BALB/c mice were lg(3.65 ±0.12) PFU/g lung,lg( 3.25 ± 0.10) PFU/g lung and lg( 3.03 ±0.08 ) PFU/g lung, decreased as compared with that of infected control BALB/c mice lg(4.35 ± 0.11 ) PFU/g lung ( P<0.05 ). Meanwhile viral titers of 0.2 mg,0.4 mg and 0.8 mg DZ treated nude mice were lg(4.82 ±0.15) PFU/g lung, lg(4.47 ±0.12) PFU/g lung and lg(4.21 ±0.11 ) PFU/g lung, declined dramatically as compared with that of infected control nude mice lg(6.23 ± 0.15) PFU/g lung( P<0.01 ). 0.2 mg, 0.4 mg and 0.8 mg DZ reduced BALB/c mice pulmonary viral mRNA expression by 30.51% ,47.38% ( P<0.05 ) and 53.97% ( P<0.01 ) and nude mice by 36.59% (P ＜0.05 ), 48.72%, 59.78% ( P<0.01 ) respectively as compared with their infected control groups. In 0.4 mg DZ treated BALB/c mice and nude mice, total numbers of leukocytes in BALF were decreased dramatically and pulmonary histology was significantly improved compared with their infected controls( P<0.05 ). And the treatment of 0.4 mg DZ reduced productions of TNF-α, IL-12 and IFN-γin BALF of RSV infected nude mice ( P<0.05 ). Conclusion DZ effectively inhibits viral replication and reduces airway inflammation in RSV infected BALB/c mice and nude mice, and the effects in nude mice are more significant. DZ is a potential therapeutic agent against RSV infection in vivo.

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Analysis of correlation between resistance of carbapenem agents in Pseudomonas aeruginosa and mutation and altered expression of oprD2

Xiaoyan LI ; Chao ZHUO ; Guangyao JIN ; Xiaoqiang LI ; Shunian XIAO ; Nanshan ZHONG

Chinese Journal of Microbiology and Immunology.2010;30(11):1048-1052. doi:10.3760/cma.j.issn.0254-5101.2010.11.016

Objective To investigate the impact on the resistance of carbapenem with the expression of OprD2 or OprD2 mutation in Pseudomonas aeruginosa. Methods One hundred and one clinical strains of Pseudomonas aeruginosa with MIC for imipenem ≥8 μg/ml were studied. MIC were determined by the broth microdilution method, and the antibiotics tested were imipenem(IPM ), biapenem( BPM), meropenem(MEM) and panipenem(PEM). The expression of the oprD2 gene in Pseudomonas aeruginosa were analyzed by real-time reverse transcriptase PCR(RT-PCR). For the Pseudomonas aeruginosa with normal expression of OprD2 and resistance to imipenem, full-length oprD2 gene was amplified by PCR and the products were sequenced. Results According to the result of the expression of oprD2 gene, 101 strains of Pseudomonas aeruginosa were divided into two groups: group1 with diminished expression of OprD2, and group2 with normal expression of OprD2. Comparing isolates with MIC of 4 kinds of carbepenem agents ≥ 16 μg/ml in two groups. Data showed the amount of OprD2 expression were different between two groups(P ＜0.01 or P ＜ 0.05). In group1, there are 28 isolates with MIC ≥ 16 μg/ml of all the 4 kinds of carbapenems, among which 25 isolates have obviously diminished expression of OprD2 ( ＜ 0.4). Negative correlations tendency appeared between the level of OprD2 transcription and MICs of 4 kinds of carbepenem agents in Pseudomonas aeruginosa. In group2, 16 strains with OprD2 mutation divided into 4 types according to the pattern of alteration. Compared with PAO1, these strains have increased MIC with different degree to IPM,BPM, MEM and PEM. Conclusion The deletion or diminished expression of OprD2 resulted in resistance to imipenem in Pseudomonas aeruginosa. The level of OprD2 transcription and antimicrobial activities for carbapenem agents proved to be highly correlated in Pseudomonas aeruginosa. The mutation of OprD2 in Pseudomonas aeruginosa probably decreased the sensitivity of carbapenem agents against Pseudomonas aeruginosa.

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The relationship between marOR mutations and the antibiotic resistance in Shigella spp

Jingchao REN ; Guangcai DUAN ; Haiyan YANG ; Ruili Lü ; Weidong ZHANG ; Yuanlin. XI

Chinese Journal of Microbiology and Immunology.2010;30(11):1044-1047. doi:10.3760/cma.j.issn.0254-5101.2010.11.015

Objective To detect the influence of marOR mutations on antibiotic resistance in Shigella spp. Methods The marOR gene with four-base deletion was amplified by overlap PCR, then inserted in a T-vector and transformed into DH5α. The clone of marOR gene with four-base deletion and three point mutations was prepared from the strain having these mutations. Electrophoresis and sequencing were preformed to certify the correction of the cloned genes. Drug susceptibility tests were preformed for the strains harbouring the different clones [DH5α, DH5α (T), DH5α (marOR), DH5α (marOR-CATT), DH5α(marOR-CATT + 3m)]. Results Compared with the control strain (DH5α-T), the antibiotic resistances of marOR with four-base deletion [DH5α (marOR-CATT)] were higher to streptomycin, tobramycin, cefazolin and cefalexin, and the antibiotic resistances of marOR with four-base deletion and three point mutations [DH5α (marOR-CATT + 3m)] were higher to streptomycin and to tetracycline. The antibiotic resistances of DH5α (marOR-CATT) and DH5α (marOR-CATT +3m) to streptomycin, tetracycline, chloramphenicol, cefazolin, levofloxacin, ciprofloxacin and norfloxacin were higher than DH5α (marOR). The diameters of the antibiotics except the trimethoprim between DH5α (marOR-CATT) and DH5α (marORCATT +3m) had not significant disparity. Conclusion The four-base deletion in 1376-1379 sites of the marOR gene increased the resistance of Shigella spp to some antibiotics. The point mutations in 1411, 1417,1435 sites of the marOR gene have little influence on the antibiotic resistance of Shigella spp.

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