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Detection of virulence genes and biofilm formation of Enterococci strains isolated from blood samples

Dongdong LI ; Dingxia SHEN ; Ling GUO ; Yanning MA

Chinese Journal of Microbiology and Immunology.2013;(11):865-867. doi:10.3760/cma.j.issn.0254-5101.2013.11.012

Objective To detect the main virulence genes and biofilm formation of Enterococci strains isolated from blood samples .Methods Twenty-eight strains of Enterococcus faecalis ( E.faecalis) and 54 strains of Enterococcus faecium ( E.faecium) were collected from blood samples .Five main virulence genes (asa1, esp, hyl, cylA and gelE) were detected by multiplex PCR.Biofilm formation was investigated by using microtiter dish biofilm formation assay .Results All E.faecalis strains were positive for at least one kind of virulence genes , of which 14 strains were concurrently positive for asa1, esp, cylA and gelE.asa1, cylA and gelE were only detected in E.faecalis strains, while hyl gene only existed in E.faecium strains. Twenty-seven strains of E.faecium were esp positive, of which 12 strains were both hyl and esp positive. None of the 5 virulence genes were identified in 10 strains of E.faecium.85.7% of E.faecalis strains and 63.0%of E.faecium strains could form biofilm.Conclusion Compared with E.faecium strains, more types of virulence genes were detected in E.faecalis strains with higher positive rates .Moreover , E.faecalis strains were more likely to form biofilms than E.faecium strains.

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The role of a novel protein ligand hMSH 2 in Vγ9δ2 T cell-mediated anti-cervix cancer immunity

Yumei DAI ; Chen MO ; Hui CHEN ; Lianxian CUI ; Wei HE

Chinese Journal of Microbiology and Immunology.2013;(11):845-849. doi:10.3760/cma.j.issn.0254-5101.2013.11.009

Objective To explore the role of hMSH2, a novel endogenous tumor-associated pro-tein ligand recognized by Vγ9δ2 T cells, in innate anti-cervix cancer immunity .Methods hMSH2 that ex-pressed on the surface of cervical cancer cell line HeLa cells was blocked by specific antibody .Then the differences in their effects on Vγ9δ2 T cells before and after antibody blockage were evaluated by cytotoxicity of Vγ9δ2 T cells and cytokines secretion .The serum levels of hMSH2 in patients with cervical cancer were detected by ELISA .Distribution of hMSH2 in cervical cancer tissues was analyzed by TMA immunohisto-chemistry .Results Decreased cytotoxicity and IFN-γsecretion were observed in Vγ9δ2 T cells against He-La cells blocked with specific anti-hMSH2 antibody .Serum concentration of hMSH 2 in patients with cervical cancer was slightly higher than that of healthy control .Altered distribution pattern of hMSH 2 was observed in cervical cancer tissues .Conclusion hMSH2 is involved in Vγ9δ2 T cells-mediated innate anti-cervical cancer immunity by enhancing their cytotoxicity and IFN-γsecretion.

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Alteration of T follicular helper cells in children with persistent immune thrombocytopenic purpura

Xin YAO ; Chengrong LI ; Guobing WANG ; Jun YANG ; Changgang LI

Chinese Journal of Microbiology and Immunology.2013;(11):833-838. doi:10.3760/cma.j.issn.0254-5101.2013.11.007

Objective To investigate the role of T follicular helper ( Tfh) cells in the immuno-pathogenesis of persistent immune thrombocytopenic purpura ( pITP) .Methods Twenty children with pITP and twenty healthy controls were enrolled in this study .The proportion of CD4+CXCR5+ICOShigh PD-1high T ( cTfh) cells and the expression of ICOSL on CD 19+B cells in peripheral blood of the patients and healthy subjects were analyzed by flow cytometry .The expressions of Bcl-6, c-Maf, IL-21 and ICOSL at mRNA level were detected by real-time PCR.The plasma concentrations of IL-2, IL-6 and IL-21 were determined by ELISA.Results (1) Compared with the healthy controls , the proportions of cTfh cells increased signifi-cantly in patients with pITP [(17.45±9.04) %vs.(7.57±2.57) %, P<0.05], but decreased with the treatment of dexamethasone (DEX) for 7 days [(5.93±1.64) %vs.(17.45±9.04) %, P<0.05].(2) The expression of Bcl-6, c-Maf and IL-21 at mRNA level in patients with pITP were higher than those in healthy controls (P<0.05).(3) Compared with healthy controls, the expression of ICOSL at mRNA and protein levels in CD19+B cells were significantly up-regulated in patients with pITP (P<0.05), which showed no significant changes after treatment with DEX (P>0.05).(4) The plasma concentration of IL-21 was remarkably elevated in patients with pITP , regardless of DEX treatment (P<0.05).But the plasma concentrations of IL-2 and IL-6 showed no significant changes compared with control group (P>0.05).Con-clusion The immunopathogenesis of persistent immune thrombocytopenic purpura might be associated with the hyper-activation of Tfh cells caused by excessive expression of ICOSL and IL-21.The persistent high expression of ICOSL and IL-21 might be one of the important factors resulting in the recurrence of pITP in children .

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Mesenchymal stem cells increase platelet counts in mice with immune-mediated thrombocytopenia and affect T-bet and GATA-3 gene expression

Min XU ; Liang WANG ; Guoqiang LIU ; Guang LU ; Huifang DING ; Jian XING ; Xia ZHAO ; Fang HAN ; Yinghui SHANG

Chinese Journal of Microbiology and Immunology.2013;(11):828-832. doi:10.3760/cma.j.issn.0254-5101.2013.11.006

Objective To explore the effects of mesenchymal stem cells ( MSC ) treatment on platelet counts in mice with immune-mediated thrombocytopenia ( ITP) and the possible mechanism .Meth-ods ITP was induced by daily intraperitoneal injection of anti-platelet membrane CD 41 antibody (MWReg30) into BALB/c mice.The mice were then divided into experiment and control groups with 20 mice in each.Each mouse in experimental group was injected with 2×107 mesenchymal stem cells (MSC) through the tail vein .The numbers of blood platelets in mice from two groups were counted on days 5, 7 and 14 after MSC injection .Reverse transcriptase polymerase chain reaction ( RT-PCR) was performed to meas-ure T-bet and GATA-3 gene expression in peripheral blood mononuclear cells ( PBMCs ) at mRNA level on day 14.The levels of IFN-γ, IL-2, IL-4 and IL-10 in serum were detected by ELISA .Results The platelet counts in experimental group were significantly higher than those in control group on days 7 and 14 after MSC injection [(588.0±81.6)×109/L and (623.0±78.9) ×109/L vs.(317.0±90.1) ×109/L and (288.0± 87.8)×109/L ] (P<0.05).On day 14 after MSC injection, the T-bet expression at mRNA level in PBMCs from mice in experimental group was significantly lower than that in control group [(0.04±0.03) vs.(0.27 ±0.05)] (P<0.05), while the GATA-3 expression at mRNA level was higher than those in control group [ (0.14±0.04) vs.(0.07±0.05)] (P<0.05).Compared with control group, the concentrations of Th1 type cytokines such as IFN-γand IL-2 were remarkably down-regulated in experimental group [(3.1±1.7) pg/ml and (3.2±2.1) pg/ml vs.(10.3±4.8) pg/ml and (16.3±5.7) pg/ml](P<0.05), while the con-centrations of Th2 type cytokines such as IL-4 and IL-10 were up-regulated in experimental group [(88.6± 15.2) pg/ml and (38.3±11.8) pg/ml vs.(32.7±5.7) pg/ml and (22.1±3.4) pg/ml ] (P<0.05). Conclusion MSC treatment can effectively increase platelet counts in mice with immune-mediated thrombo-cytopenia, which may be associated with the suppression of Th 1-dominant response mediated by abnormal ex-pression of T-bet and GATA-3.

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Isolation, identification and functional characteristics of murine B10 cells

Yao QIN ; Mei ZHANG ; Ruimei JIANG ; Lulu KONG ; Xinyu XU ; Heng CHEN ; Tao YANG

Chinese Journal of Microbiology and Immunology.2013;(11):819-827. doi:10.3760/cma.j.issn.0254-5101.2013.11.004

Objective To investigate the phenotypes and percentages of B 10 cells in different tis-sues from wild-type mice and to identify their biological functions .Methods The percentages of B10 cells derived from different tissues of mice and their responses to lipopolysaccharide ( LPS) stimulation were ana-lyzed by flow cytometry .Magnetic-activated cell sorting ( MACS ) and fluorescence-activated cell sorting (FACS) were used to purify B10 cells, CD4+CD25-T cells and Treg cells.CD4+CD25-T cells and Treg cells labeled by CFSE were co-cultured with or without B10 cells, and then their proliferation were evaluated after 72 h.Results (1) A subset of CD19+CD5+CD1dhigh regulatory B cells was identified in spleen , pe-ripheral blood and lymph nodes from wild-type mice , of which the highest frequency was detected in spleen (3.95%±0.79%, P<0.05).The isolated B cells from different tissues were stimulated by LPS , PMA, ionomycin and monensin (L+PIM) in vitro to express IL-10.Among them, splenic CD19+IL-10+B cells showed the highest expression of IL-10 (P<0.05).(2) Prolonged LPS stimulation (48 h) to CD5+CD1dhigh B cells enhanced the expressions of IL-10 (P<0.01).(3) CD19+CD5+CD1dhigh B cells inhibited the prolif-eration of CD4+CD25-T cells in vitro in a dose-dependent manner (P<0.01), but increased the secretion of IL-10 by CD4+T cells (P<0.01) and the proliferation of Treg cells in vitro (P<0.01).Conclusion Com-pared with other tissues , the percentage of B10 cell subset in spleen is the highest in wild-type mouse , and B10 cells subset can be activated through Toll-like receptor ( TLR ) signaling pathway .The responses of CD4+CD25-T cells and Treg cells in co-culture with B10 cells are regulated by B 10 cell subset through an increased IL-10 production .B10 cells might be a useful cell population for the treatment of inflammatory au-toimmune diseases.

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Treponema pallidum membrane protein Tpp17 activates vascular endothelial cells in vitro

Ruili ZHANG ; Qianqiu WANG

Chinese Journal of Microbiology and Immunology.2013;(11):807-812. doi:10.3760/cma.j.issn.0254-5101.2013.11.002

Objective To study the effects of Treponema pallidum membrane protein Tpp17 on ac-tivation of human umbilical vein endothelial cells (HUVECs) in vitro and to understand its role in the immu-nopathogenesis of syphilis .Methods HUVECs were co-cultured with recombinant protein Tpp 17.Then the expressions of TNF-α, MCP-1, ICAM-1 and E-selectin at mRNA and protein levels in supernatants were re-spectively detected by enzyme-linked immunosobent assay ( ELISA ) and fluorescent real-time quantitative PCR.The adhesive ability of THP-1 cells was observed by fluorescence microscopy after co-cultured pretrea-ted HUVECs with recombinant protein Tpp 17 with Calcein-AM labeled THP-1.Pretreated HUVECs were cultured in the lower chamber of Transwell with recombinant protein Tpp 17 and monocytic THP-1 cells were cultured in the upper chamber of Transwell .After that, the migration of monocytic THP-1 cells was evalua-ted by using fluorescence microscopy .Results Compared with the blank control group , the expression of TNF-α, ICAM-1, E-selectin, especially the MCP-1, were enhanced by recombinant protein Tpp 17 of Trepo-nema pallidum.Moreover, Tpp17 improved the adhesive ability and migration of monocytic THP-1 cells, es-pecially the latter.Conclusion Treponema pallidum membrane protein Tpp17 might play a certain role in the immunopathogenesis of syphilis by enhancing the expression of TNF-α, MCP-1, ICAM-1 and E-selectin, and by promoting the adherence ability and migration of monocytic THP-1 cells.

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G-test positive serum inhibits ROS-dependent killing of Candida albicans by interfering with internal- ized expression of neutrophil Dectin-1

Bilin DONG ; Ming WANG ; Weihuang LIU ; Dongsheng LI ; Zhongsheng TONG ; Yiqun DUAN ; Wei LIU

Chinese Journal of Microbiology and Immunology.2013;(11):856-864. doi:10.3760/cma.j.issn.0254-5101.2013.11.011

Objective To investigate the mechanism of soluble β-1, 3-D-glucan in G-test positive serum in inhibiting ROS-dependent killing of Candida albicans ( C.albicans ) mediated by neutrophil Dectin-1.Methods The expression and distribution of internalized Dectin-1 and triggered ROS in human neutrophils were detected by using confocal/two-photon laser scanning microscopy upon stimulation with C.albicans (MOI=10) which was pretreated with β-1, 3-D-glucanase (10 U/ml) or not.Abrogation test was used to analyze whether intracellular Dectin-1 was involved in C.albicans-triggered ROS production in human neutrophils.Furthermore, flow cytometry analysis was performed to detect the expression of intracel-lular Dectin-1 and ROS in neutrophils which were pretreated respectively with G-test positive serum at differ-ent dilutions for 60 min and then stimulated with C.albicans for another 60 min at 37℃.Results After stimulated with C.albicans (MOI=10) for 60 min, the expression of Dectin-1 in neutrophils was recruited to the spores of opsonophagocytized C.albicans, and partly co-localized with the triggered ROS production . However, the expression of intracellular Dectin-1 was not observed in neutrophils when stimulated with β-1, 3-D-glucanase pretreated C.albicans for 60 min at 37℃.Abrogation test further showed that C.albicans-trig-gered ROS production in neutrophils was partly and irreversibly inhibited by adding Dectin -1 blocking mAb of 5 μg/ml.In addition , both the triggered expression of intracellular Dectin-1 and ROS production in neu-trophils stimulated with C.albicans ( MOI=10 ) in the presence of G-test positive serum were significantly lower than those of neutrophils stimulated only with C.albicans (LSD-t test, P<0.01).Linear regression a-nalysis suggested that the triggered intracellular Dectin-1 and ROS production in neutrophils upon stimulation with C.albicans were both inhibited by soluble β-1, 3-D-glucan in a dose-dependent manner (Dectin-1,R2=0.702,P<0.01;ROS,R2=0.588,P<0.01 ).Conclusion Taken together, these results demonstrated that the soluble β-1, 3-D-glucan in G-test positive serum may play a role in inhibiting the ROS-dependent killing of C.albicans by interfering with internalized expression of neutrophil Dectin-1.

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Establishment and application of a flow cytometry-based rapid drug susceptibility test for Candida species

Yongze ZHU ; Qingfeng HU ; Yonglie ZHOU ; Jinfang ZHAO ; Jie YAN

Chinese Journal of Microbiology and Immunology.2013;(11):850-855. doi:10.3760/cma.j.issn.0254-5101.2013.11.010

Objective To establish a flow cytometry-based drug susceptibility test for the rapid de-tection of antifungal susceptibility or resistance of Candida isolates.Methods The gate selection and opti-mal experimental conditions of flow cytometry-based drug susceptibility test were determined by using Candi-da albicans strain ATCC90029 as the test strain and propidium iodide ( PI) as the fluorescent dye .The es-tablished flow cytometry-based drug susceptibility test was used to detect the susceptibility or resistance to fluconazole or voriconazole of 110 isolates belonging to Candida species, and the obtained results were com-pared with those by using typical M 27-A3 constant dilution method .Results The killed and viable Candida albicans ATCC90029 strains were clearly divided into two groups on the figure of SS /log (FL3) by regulating voltages.There was a high correlation between the results of susceptibility test and the proportions of killed and viable fungi in mixture (r=0.999).The flow cytometry-based drug susceptibility test could provide the results within 30 min and its optimal concentration of fungal suspension , time of drug-fungus incubation , dyeing method and time were 1.0×106/ml, 3 h incubation and sodium deoxycholate-pretreated plus PI dye-ing for 5 min, respectively .The total coincident rates between the established test and the constant dilution method were 98.2%and 87.3%in the detection of drug susceptibility of 110 fungal isolates to fluconazole and voriconazole .Conclusion The flow cytometry-based drug susceptibility test shows advantages of rapidi-ty, accuracy and high sensitivity compared with the constant dilution method .It has a great potential for clin-ical application .

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Study on the expression of TLR2 and TLR4 in peripheral blood mononuclear cells and their relation- ship with Th1/Th2 immune response in patients with Henoch-Schonlein purpura

Hong CHANG ; Qiuye ZHANG ; Na CHENG ; Shouqing ZHANG ; Yi LIN

Chinese Journal of Microbiology and Immunology.2013;(11):839-844. doi:10.3760/cma.j.issn.0254-5101.2013.11.008

Objective To explore the role of TLR2 and TLR4 in the pathogenesis of Henoch-Schonlein purpura ( HSP) by investigating their expression at mRNA and protein levels in peripheral blood mononuclear cells ( PBMCs ) and their influences on Th 1/Th2 immune response in children with HSP . Methods 64 hospitalized children with HSP in the Affiliated Hospital of Qingdao University Medical Col -lege from October 2011 to November 2012 were enrolled in the study .They were further divided into non-He-noch-Schonlein purpura nephritis ( NHSPN ) group ( n =36 ) and Henoch-Schonlein purpura nephritis (HSPN) group (n=28).30 age-matched healthy children from Child Health Division of the same hospital were selected as controls .The expression of TLR2 and TLR4 at mRNA level in PBMCs were detected by re-al-time fluorescent polymerase chain reaction .The expression of TLR2 and TLR4 at protein level and T cells subset were detected by flow cytometry .The levels of IFN-γ, IL-4 and IL-6 in plasma were determined by enzyme-linked immunosorbent assay (ELISA).Results (1)Compared with the control group , the expres-sion of TLR2 and TLR4 at mRNA and protein levels were remarkably increased in children with HSP , espe-cially in HSPN group.(2)Compared with the control group, the percentage of CD3+T cells and CD3+CD4+T cells were down-regulated in HSP group , but the percentage of CD 3+CD8+T cells and CD3+HLADR+T cells were up-regulated.(3)The level of IFN-γand the ratio of IFN-γ/IL-4 in plasma from children with HSP were significantly lower than those of the controls , while the level of IL-4 and IL-6 were remarkably higher than those of the controls .(4)The expression of TLR2 and TLR4 at protein level in PBMCs from chil-dren with HSP showed significant positive correlations with the expression of TLR 2 and TLR4 at mRNA level and plasma concentration of IL-4 and IL-6, but a negative correlation with the ratio of IFN-γ/IL-4.Conclu-sions The aberrant activation of TLR 2 and TLR4 might be correlated with the immunological pathogenesis of HSP by enhancing Th2 immune response.The hyper-activation of TLR2 and TLR4 might result in renal injury in patients with HSP .

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Evaluation of immunogenicity of an immune complexed hepatitis B vaccine

Jing XU ; Juan WANG ; Gang WU ; Jiaqi ZHOU ; Pu SHAN ; Jilai LI ; Lifeng XU

Chinese Journal of Microbiology and Immunology.2013;(11):813-818. doi:10.3760/cma.j.issn.0254-5101.2013.11.003

Objective To evaluate the immunogenicity of an immune complexed hepatitis B vac-cine ( HBsAg-HBIG immune complexes , IC) in mouse and cynomolgus monkeys by using recombinant hepa-titis B vaccine ( Saccharomyces cerevisiae, HBsAg) as the control .Methods BALB/c mice were vaccinated with single dose of IC and single dose of HBsAg respectively and then serum samples were collected at differ -ent time points for the detection of dynamic anti-HBs by using ELISA .The serum anti-HBs titers in BALB/c mice vaccinated with different immunization strategies were also analyzed .ELISPOT assay was performed to detect the numbers of IFN-γSFC and IFN-γpositive rate in splenocytes of BALB/c mice intramuscularly im-munized with IC, HBsAg or standard hepatitis B vaccine at 5μg/mouse.ED50 was measured to evaluate the stability of IC.Twelve cynomolgus monkeys were equally divided into two groups and immunized with high dose (100 μg) and low dose (20 μg) of IC respectively and then , serum anti-HBs levels at different time points were detected .Results The serum anti-HBs titers in IC immunized group at different time points were higher than those immunized with HBsAg .Moreover, the anti-HBs titer induced by two doses of IC reached a level comparable to that elicited by three doses of HBsAg .ELISPOT assay showed that both the numbers of IFN-γSFC and IFN-γpositive rate were the highest in IC immunized group as compared with those immunized with HBsAg and standard hepatitis B vaccine .IC had a lower ED50 than HBsAg, indicating a good long term stability .Cynomolgus monkeys immunized with high or low dose of IC produced high levels of anti-HBs titer during a long time period .Conclusion IC has a higher immunogenicity inducing both hu-moral immunity and cellular immunity as compared with HBsAg or standard hepatitis B vaccine .

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