Roles of P2X7 receptor and NLRP3 inflammasome in inflammatory diseases / 中华微生物学和免疫学杂志

Purinergic 2X7 receptor (P2X7R) is an ionotropic receptor that is involved in various inflammatory diseases through affecting the release of inflammatory cytokines such as IL-1β and IL-18 after inducing the activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3). In recent years, the P2X7R/NLRP3 signaling pathway has become one of the more studied pathways in inflammatory diseases, and some inhibitors of P2X7R and NLRP3 have already been used in early clinical treatment. In this paper, the progress in P2X7R and NLRP3 was summarized, aiming to provide reference for further investigation on the roles of P2X7R/NLRP3 in the pathogenesis of tumors and inflammatory diseases and the potential of P2X7R/NLRP3 as therapeutic targets.

Exploration of training mode for postgraduate education in local universities under the Background of "double first-class" construction / 中华医学教育探索杂志

First-class education for postgraduate is the foundation for the construction of "double first-class" education. Universities play an important role in postgraduate education. This paper explored the measures for the reform and innovation of the construction of "double first-class" education for postgraduate students in our university, which include perfecting the supervisor's responsibility and authority mechanism, deepening the reform on curriculum system, strengthening the construction of sharing platform, and improving the evaluation mechanism of training quality and so on. In conclusion, initial achievement from the reform and innovation of training mode was observed, which provides a useful reference for the construction of "double first-class" education for local universities.

Contribution of NOD2 signal pathway to Chlamydia pneumonia Cpn0423-induced inflammatory response / 中华微生物学和免疫学杂志

Objective:To understand and determine the biological properties of Chlamydia pneumonia (Cpn) hypothetical protein Cpn0423 and the mechanisms of which involved in Cpn0423-induced inflammatory response. Methods:The biological properties of Cpn0423 gene were analyzed using bioinformatic software. The subcellular localization of nucleotide-binding oligomerization domain-like receptor 2 (NOD2) in bone marrow-derived macrophages (BMDMs) was detected by confocal microscope. NOD2-siRNA was used to inhibit the expression of NOD2 at mRNA level. Cpn0423-induced macrophage inflammatory protein 2 (MIP-2) and IL-6 production in BMDMs were detected by ELISA. PCR was performed to detect Cpn0423 DNA in bronchoalveolar lavage fluid (BALF) of Cpn-positive patients.Results:The homology between Cpn0423 and other type Ⅲ secretion system effector proteins of Chlamydia ranged from 85% to 93%. NOD2-siRNA could effectively inhibit the expression of NOD2 at mRNA level in BMDMs ( P<0.001). Moreover, Cpn0423-induced production of MIP-2 [(920.5±99.1) pg/ml vs (130.1±11.5) pg/ml, P<0.001] and IL-6 [(266.2±58.4) pg/ml vs (165.7±21.5) pg/ml, P<0.001] in BMDMs were decreased following NOD2-siRNA pre-treatment. Cpn0423 DNA was detected in the BAlF of 83.3% (10/12) of Cpn-positive cases, but not in Cpn-negative cases. Conclusions:Cpn0423 induced inflammatory response in host cells through NOD2 pathway, which was closely related to the chronic inflammatory injury caused by Cpn.

HO-1 mediates the anti-inflammatory actions of Sulforaphane in monocytes stimulated with a mycoplasmal lipopeptide.

Exposure to Mycoplasma pneumoniae leads to lung inflammation through a host defense pathway. Increasing evidence has indicated that the mycoplasma-derived membrane lipoprotein, or its analogue macrophage-activating lipopeptide-2 (MALP-2), excretes LPS as an immune system-stimulating substance and plays a crucial role in pathological injury during M. pneumoniae infection. It has been established that Sulforaphane confers anti-inflammatory properties. However, the underlying mechanism responsible for the inhibitory actions of Sulforaphane in the context of mycoplasmal pneumoniae are poorly understood. Here, we report that Sulforaphane is an inducer of heme oxygenase (HO)-1, a cytoprotective enzyme that catalyzes the degradation of heme through signaling pathways in human monocytes. Sulforaphane stimulated NF-E2-related factor 2 (Nrf2) translocation from the cytosol to the nucleus, and small interfering RNA-mediated knock-down of Nrf2 significantly inhibited Sulforaphane-induced HO-1 expression. Additionally, PI3K/Akt and ROS were also involved in Sulforaphane-induced Nrf2 activation and HO-1 expression, as revealed by the pharmacological inhibitors LY294002 and NAC. Moreover, Sulforaphane treatment inhibited MALP-2-induced pro-inflammatory cytokine secretion and pulmonary inflammation in mice, as well as MALP-2-triggered NF-κB activation. Furthermore, SnPP, a selective inhibitor of HO-1, reversed the inhibitory actions of Sulforaphane, while a carbon monoxide-releasing molecule, CORM-2, caused a significant decrease in MALP-2-induced cytokine secretion. Collectively, these results suggest that Sulforaphane functions as a suppressor of the MALP-2-induced inflammatory response, not only by inhibiting the expression of cytokines and the induction of HO-1 but also by diminishing NF-κB activation in cultured monocytes and the lungs of mice.

IL-10 promotes Chlamydia muridarum infection in mice through inhibiting IFN-γand IL-2 expression / 中华微生物学和免疫学杂志

Objective To study the possible molecular mechanism of IL-10 in promoting Chlamydia muridarum infection in mice. Methods C57BL/6 wild-type and IL-10 gene knockout ( IL-10-/-) mice were infected with Chlamydia muridarum. Indirect immunofluorescence assay was used to detect the growth of Chlamydia muridarum in the intestinal and genital tracts. The severity of genital diseases was assessed by hydrosalpinx scoring. Expression of IFN-γand IL-2 in blood was measured by ELISA. Re-sults Compared with the wild-type group, Chlamydia clearance in the intestinal and genital tracts of IL-10-/- mice was significantly faster, and the expression of IFN-γ and IL-2 increased significantly. In addi-tion, wild-type mice showed more serious hydrosalpinx. Conclusions IL-10 delays Chlamydia trachomatis clearance and promotes Chlamydia infection through inhibiting the expression of IFN-γ and IL-2, which ag-gravates hydrosalpinx.

Identification and drug susceptibility testing of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis / 中华流行病学杂志

Objective To understand the etiological characteristics and drug susceptibility of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis and provide evidence for the prevention and control of infectious mastitis in cows.Methods The milk sample was collected from a cow with mastitis,which was pretreated with 4％ NaOH and inoculated with L-J medium for Mycobacterium isolation.The positive cultures were initially identified by acid-fast staining and multi-loci PCR,then Mycobacterium species was identified by the multiple loci sequence analysis (MLSA) with 16S rRNA,hsp65,ITS and SodA genes.The drug sensitivity of the isolates to 27 antibiotics was tested by alamar blue assay.Results Two anti-acid stain positive strains were isolated from the milk of a cow with mastitis,which were identified as non-tuberculosis mycobacterium by multi-loci PCR,and multi-loci nucleic acid sequence analysis indicated that one strain was Mycobacterium thermoresistibile and another one was Mycobacterium elephantis.The results of the drug susceptibility test showed that the two strains were resistant to most antibiotics,including rifampicin and isoniazid,but they were sensitive to amikacin,moxifloxacin,levofloxacin,ethambutol,streptomycin,tobramycin,ciprofloxacin and linezolid.Conclusions Mycobacterium thermoresistibile and Mycobacterium elephantis were isolated in a cow with mastitis and the drug susceptibility spectrum of the pathogens were unique.The results of the study can be used as reference for the prevention and control the infection in cows.

Identification and drug susceptibility testing of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis / 中华流行病学杂志

Objective To understand the etiological characteristics and drug susceptibility of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis and provide evidence for the prevention and control of infectious mastitis in cows.Methods The milk sample was collected from a cow with mastitis,which was pretreated with 4％ NaOH and inoculated with L-J medium for Mycobacterium isolation.The positive cultures were initially identified by acid-fast staining and multi-loci PCR,then Mycobacterium species was identified by the multiple loci sequence analysis (MLSA) with 16S rRNA,hsp65,ITS and SodA genes.The drug sensitivity of the isolates to 27 antibiotics was tested by alamar blue assay.Results Two anti-acid stain positive strains were isolated from the milk of a cow with mastitis,which were identified as non-tuberculosis mycobacterium by multi-loci PCR,and multi-loci nucleic acid sequence analysis indicated that one strain was Mycobacterium thermoresistibile and another one was Mycobacterium elephantis.The results of the drug susceptibility test showed that the two strains were resistant to most antibiotics,including rifampicin and isoniazid,but they were sensitive to amikacin,moxifloxacin,levofloxacin,ethambutol,streptomycin,tobramycin,ciprofloxacin and linezolid.Conclusions Mycobacterium thermoresistibile and Mycobacterium elephantis were isolated in a cow with mastitis and the drug susceptibility spectrum of the pathogens were unique.The results of the study can be used as reference for the prevention and control the infection in cows.

Recombinant protein CPSIT_0846 induces protective immunity against Chlamydia psittaci infection in BALB/c mice.

Chlamydia psittaci is an obligate intracellular bacteria that causes respiratory disease in poultry and humans. Currently, there are no licensed vaccines against chlamydial infection in humans. The transmembrane head protein CPSIT_0846 of C. psittaci is a putative member of the larger Inc protein family. In this study, we investigated immunogenicity and protective efficacy of the recombinant CPSIT_0846 protein in BALB/c mice. Mice immunized with CPSIT_0846 developed strong T-lymphocyte responses that were recalled by the immunogen CPSIT_0846 in an in vitro restimulation assay. These T cells displayed a strong Th1-biased cytokine profile with high levels of IFN-γ. At the same time, a strong humoral immune response was also detected in the immunized mice with high titers of Chlamydia psittaci-specific serum IgG antibodies. More importantly, the robust immune responses correlated well with significantly reduced chlamydial burden and inflammatory pathology in the mouse lungs upon an airway challenge infection. The above results together suggest that the CPSIT_0846 protein may be a potential vaccine candidate antigen for inducing protection against C. psittaci infection and disease in the airway.

Treponema pallidum membrane protein Tp0971 induce macrophages to produce cytokines via TLR2/NF-κB pathway / 中国免疫学杂志

Objective:To investigate the production of cytokines from macrophages induced by Treponema pallidum membrane proteins Tp0971.Methods: The Tp0971 was amplified by PCR from a preparation of T.pallidum genomic DNA and then sub-cloned into the prokaryotic expression vector pET28a(+)to construct the recombinant plasmid pET28a(+)/Tp0971.The recombinant plasmids were transfected into E.coli Rosseta strain to express recombinant protein Tp0971 by IPTG induction.The expression products were purified by Ni-NTA affinity chromatography,and the concentration was determinated by BCA method.Detoxi-Gel was used to remove endotoxin contamination in during the protein preparation.After induced by PMA,cells were incubated with various concentrations of recombinant proteins Tp0971.The expression levels of IL-8,IL-1β and IL-6 were detected by ELISA.Cells were pretreated with anti-TLR2 antibody or TLR2 siRNA,or pyrrolidine dithiocarbamate,an inhibitor of NF-κB,for evaluation of the role of TLR2 and NF-κB in the production of cytokines by ELISA.Results: Tp0971 gene were amplified successfully by PCR,and the recombinant plasmids were confirmed by enzyme digestion and sequencing.SDS-PAGE results showed three recombinant proteins were expressed as the soluble with a relative molecular weight of 29 kD.0.5-10 μg/ml of Tp0971 could stimulate macrophages to produce IL-8,IL-1β and IL-6 dose-dependently.After cells were pretreated with siRNA or neutralizing antibody targeting TLR2 or the PDTC,the Tp0971 induced protein expression levels of proinflammatory cytokines were significantly reduced in macrophages.Conclusion: Tp0971 induces macrophages to produce proinflamatory cytokines via TLR2/NF-κB pathway.

Protective effect of interferon-γ on Chlamydia psittaci acute infection / 中国人兽共患病学报

We investigated the effects of IFN-γ on Chlamydia psittaci (Cps) infection.HeLa cells were treated with different concentrations of recombinant human IFN-γ (5 ng/mL,25 ng/mL,50 ng/mL) after infecting with C.psittaci 6BC,then the number and morphology of C.psittaci inclusion bodies were examined after 48 hours.C57BL/6J mice were intranasally infected with 2 × 106 IFUs C.psittaci 6BC,and intraperitoneally administrated with 10 μg recombinant murine interferon-γ 24 hours prior or post infection,then body weight,activity and survival rate were recorded.The histopathology of mice livers and lungs was analyzed by HE staining on day 5 or day10 post infection.And the chlamydial inclusion bodies were titrated in the lung homogenates of mice sacrificed on day 5 after infection.The inclusion body numbers of recombinant human IFN-γ treated groups (by 5ng/mL,25ng/mL,50ng/mL) were significantly less than that in the control group (23.8±5.1)× 106,(10± 3.58) × 106,(8.0±2.22) × 106,(43.3±11.05)× 106,respectively).And the morphology of inclusion bodies in IFN-γ treated HeLa cells was irregular and much smaller.We also found that IFN-γ could significantly improve the survival rate,reduce acute clinical manifestations and pathological injurery of lung and liver in C.psittaci respiratory tract infected mice model.So we summarized that IFN-γ can mediate strong immunological protection during acute C.psittaci early infection.

Analysis on human T cell epitopes polymorphisms of five specific antigens of Mycobacterium tuberculosis in 13 areas of China / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the polymorphisms of the coding gene and the human T cell epitopes of antigen GlnA1, Mpt70, LppX, GroES and LpqH on Mycobacterium tuberculosis complex (MTBC) strains in thirteen provinces of China.</p><p><b>METHODS</b>A total of 173 clinical MTBC isolates from thirteen provinces were selected to test the gene sequences of the five antigens, using PCR and DNA sequencing methods. Sequences were compared and sliced by BioEdit, and the variations of the human and nonhuman T cell epitopes were analyzed. The rates on synonymous mutation (dS), non-synonymous mutation (dN) and dN/dS values were calculated by Mega 6.0 software.</p><p><b>RESULTS</b>Among the 173 strains, there were two non-synonymous mutations in the non-epitope region of glnA1, one non-synonymous mutations in epitope domain of mpt70, one non-synonymous mutation and one synonymous mutation in the epitope domain of lpqH; while groES showed no mutation. lppX had five non-synonymous mutations and one synonymous mutation in the epitope domain. Nine strains presented higher polymorphism at the same gene locus of position 152 in lppX. And seven of the fifteen epitopes contained in lppX were altered and the dN/dS value of this gene was 0.19.</p><p><b>CONCLUSIONS</b>Data from the human T cell epitope domains of MTBC antigens Mpt70, LppX and LpqH contained epitope diversity, indicated that these antigens may have involved in diversifying the selection to evade the host immunity. GlnA1 had the polymorphism in epitope domain, which might have little influence on the immuno-response. While GroES seemed relatively conservative, it could play an important role on identification, diagnosis and the development of potential Mycobacterium tuberculosis vaccine.</p>

Prediction and identification of B-cell epitopes of Treponema pallidum repeat protein F / 中国人兽共患病学报

To predict and identify the dominant B‐cell epitopes of conserved region of Treponema pallidum repeat protein F (TprFN ) and provide the basis for development of polyvalent epitope‐based syphilis vaccine ,the amino acid sequence of TprFN was obtained from GenBank and analyzed with comprehensive meta‐analysis Mobyle ,ABCpred and IEDB online software .The peptides containing predicted epitopes were artificially synthesized . To obtain and measure the titers of antibodies against TprFN ,New Zealand rabbits were immunized with recombinant protein TprFN expressed in E .coli and identified by Western blot (WB) .Sera from TprFN‐immunized rabbits ,syphilis patients ,and normal human and normal rabbits were used to deter‐mine the immunoreactivity and specificity of 7 predicted peptides of TpFN by indirect ELISA .Comprehensive meta‐analysis of online software showed that P1 (43‐62AA) ,P2(57‐71AA) ,P3(81‐88AA) ,P4(89‐103AA) ,P5(125‐138AA) ,P6(231‐251AA) and P7(268‐279AA) might be the B‐cell epitopes .A protein was expressed in a soluble form and identified as TpFN by WB .The ELISA indicated that P1 and P3 were active with TprFN‐immunized rabbit sera and syphilis patient sera but not with negative control sera .These results indicate that P1 and P3 are the potential dominant B‐cell epitopes .

Macrophage-activating lipopeptide-2 induces the expression of hemeoxygenase-1 in THP-1 cells and its possible mechanism / 中华微生物学和免疫学杂志

Objective To investigate whether macrophage-activating lipopeptide-2 ( MALP-2) in-duces the expression of hemoxygenase-1 ( HO-1 ) in THP-1 cells and to further elucidate its possible regulatory mechanism for a better understanding of protective response upon mycoplasma infection .Methods THP-1 cells were cultured in vitro and stimulated by MALP-2 at different concentrations for 12 h.THP-1 cells were incubated with TLR 2 or TLR6 neutralizing antibodies , or transfected with their dominant negative plasmids to evaluate the effects of TLR 2 and TLR6 on HO-1 expression .Phosphorylation of Akt was detected by Western blot.PI3K inhibitor LY294002 was used to investigate the role of PI3K in HO-1 expression.Im-munofluorescence and electrophoretic mobility shift assay ( EMSA ) were performed to observe the nuclear translocation and DNA-binding activity of nuclear factor Nrf 2.Small interfering RNA ( siRNA) was used to silence the genes encoding Nrf2 and HO-1.Cobalt protoporphyrin (CoPP), an inducer of HO-1, was used to treat THP-1 cells.The expression of HO-1 was detected by Western blot .The secretion of TNF-αand IL-1βby THP-1 cells were measured by ELISA .Results MALP-2 induced the expression of HO-1 in THP-1 cells.However, the expression of HO-1 was inhibited by TLR2 and TLR6 neutralizing antibodies and expres-sion of their dominant negative plasmids .Moreover, PI3K pathway was activated by MALP-2, and with the use of PI3K inhibitor, the expression of HO-1 was decreased.The translocation of Nrf2 to the nucleus and itsDNA-binding activity were enhanced by MALP-2, but were inhibited by the treatment of PI3K inhibitor.Theexpression of HO-1 was significantly down-regulated upon the interference of Nrf2 gene expression withsiRNA.Silenced expression of HO-1 increased the level of TNF-αand IL-1β, while CoPP treatment decreasedthe secretion of MALP-2-induced cytokines.Conclusion MALP-2 might induce the expression ofHO-1 in THP-1 cells through TLR2,6/PI3K/Nrf2 pathways.The expression of HO-1 could negatively regulatethe hyper-secretion of cytokines.

A mycoplasma lipopeptide induces hemeoxygenase-1 expression via TLR2, 6/c-Src/PI3 K pathway in monocytes / 中国免疫学杂志

Objective:To observe the molecular mechanism involved in expression of hemeoxygenase -1 (HO-1) induced by a macrophage-activating lipopeptide-2 (MALP-2).Methods:THP-1 cells were cultured in vitro and stimulated by MALP-2 for 12 h, expression of HO-1 was detected by Western blot .TLR2 and TLR6 neutralizing antibodies incubation , dominant negative plasmids transfection were used to assess the functional of TLR 2,6 in mediating HO-1 expression.Phosphorylation of c-Src and Akt were detec-ted by Western blot, and c-Src siRNA and PI3K inhibitor LY294002 were used to investigate the role of c-Src and PI3K in HO-1 ex-pression.Results:MALP-2 induced c-Src phosphorylation , and TLR2 and TLR6 neutralizing antibodies , or their dominant negatively plasmids could abrogate this effect .In addition, siRNA of c-Src could decrease the phosphorylation level of Akt , and the PI3K inhibi-tor could inhibit HO-1 expression.Conclusion: MALP-2 can induce THP-1 cells expression of HO-1 through TLR2,6/c-Src/PI3K pathways .

Mycoplasma MALP-2 induces the expression of heme oxygenase-1 via MAPKs and Nrf2 pathways / 中华微生物学和免疫学杂志

Objective To investigate whether macrophage-activating lipopeptide-2 ( MALP-2) in-duces the expression of heme oxygenase-1 ( HO-1 ) in THP-1 cells and its possible mechanism .Methods Human monocyte cells THP-1 were cultured in vitro and then were incubated with various concentrations (0, 0.01, 0.1, 1.0 or 5.0 ng/ml) of MALP-2 for 16 h, or were stimulated by 5.0 ng/ml MALP-2 for different length of time (0 h, 4 h, 8 h, 12 h, 16 h or 24 h).The expression of HO-1 at mRNA and protein levels were detected by real-time PCR analysis and Western blot assay .The enzyme activity of HO-1 was detected by colorimetric analysis.THP-1 cells were pre-incubated with 30 μmol/L of SB203580, PD98059 and SP600125 for 30 min and then were cultured with 5.0 ng/ml MALP-2 for 16 h to investigate the role of mito-gen-activated protein kinases (MAPKs) signaling pathway in HO-1 production.After incubating THP-1 cells with 5.0 ng/ml MALP-2 for different periods of time, NF-E2-related factor 2 (Nrf2) protein was detected by Western blot assay to study the effects of Nrf2 pathway on MALP-2-induced HO-1 expression.Nrf2 and HO-1 proteins were measured by Western blot assay after transfecting THP-1 cells (1×106/well) with Nrf2 siRNA at a final concentration of 100 nmol/L.Results MALP-2 enhanced the expression of HO-1 at mRNA and protein levels as well as the enzyme activity of HO-1 in THP-1 cells in a concentration-dependent manner.The expression of HO-1 protein induced by MALP-2 was significantly inhibited by 30 μmol/L MAPKs specific inhibitors ( SB203580 , PD98059 and SP600125 ) .MALP-2 induced Nrf2 translocation at a concentration of 5.0 ng/ml.The expression of Nrf2 and HO-1 proteins were significantly decreased in Nrf 2 siRNA-transfected THP-1 cells.Conclusion MAPKs and Nrf2 signaling pathways were involved in the MALP-2 induced HO-1 expression .

Immunogenicity analysis of a recombinant protein CPSIT_p7 and its expression in HeLa cells during persistent Chlamydophila psittaci infection / 中华微生物学和免疫学杂志

Objective To construct a prokaryotic expression plasmid for CPSIT_p7 gene from Chlamydophila psittaci ( Cps) 6BC strain and to evaluate immunogenicity of the recombinant protein His-CPSIT_p7 and detect its dynamic expression at mRNA and protein levels in HeLa cells during persistent Cps infection.Methods The fusion protein His-CPSIT_p7 was expressed in E.coli BL21 and purified by Ni-NTA affinity chromatography .BALB/c mice were immunized with the recombinant protein to prepare polyclonal antibody for evaluation of the immunogenicity of His-CPSIT_p7 by ELISA.Penicillin sodium was used to establish a model of Cps persistence infection .RT-PCR and Western blot assay were performed to de-tect the expression of CPSIT_p7 at mRNA and protein levels during Cps persistent infection .Results The fusion protein His-CPSIT_p7 was successfully expressed with the use of constructed recombinant expression plasmid pET30 a-CPSIT_p7 and purified .ELISA result showed that the specific antibody titer against CPSIT_p7 reached 1 ∶1 000 000 on the 40th days after immunization .The expression of CPSIT_p7 at mRNA and protein levels were increased in a time-dependent manner in Cps-infected HeLa cells .The peak of mRNA level was reached at the time point of 36 hours after infection , followed by a time-dependent decrease during Cps acute infection .However , the expression of CPSIT_p7 at mRNA and protein levels were not decreased until 60 hours after infection during Cps persistent infection .Conclusion His-CPSIT_p7 protein was suc-cessfully expressed in the prokaryotic expression system and purified , showing an advantage of good immuno-genicity.Highly expressed CPSIT_p7 at mRNA and protein levels were detected during Cps persistent infection.

Effect of CD14 on lipid-associated membrane proteins of Mycoplasma genitalium activate NF-kappaB / 中国人兽共患病学报

To investigate the effects of CD14 on nuclear transcription factorκB (NF-κB) was activated by lipid-associated membrane proteins (LAMPs) of Mycoplasma genitalium (Mg) ,THP-1 cells were pretreated with serum human or CD14 neu-tralizing antibody ,and then were stimulated by LAMPs .The activation of NF-κBp65 was detected by ELISA .After LAMPs was pretreated with sCD14 stimulated Hela cells with the co-transfection ,the activity of NF-κB luciferase were detected by the dual-luciferase reporter gene to analyze the role of CD14-mediated NF-κB activation by LAMPs .The activation of NF-κBp65 was significantly up-regulated in LAMPs activated THP-1 cells by human serum .It’s suggested that CD14 neutralizing anti-body could inhibit the activation of NF-κBp65 in LAMPs stimulated THP-1 .The activation of NF-κB was significantly up-regu-lated in LAMPs activated Hela cells by mCD14 or sCD14 .CD14 could augment the activation of NF-κB by Mg LAMPs .

Preliminary study on genital tract immune injury in mice induced by pORF5 plasmid protein of Chlamydia trachomatis / 中华微生物学和免疫学杂志

Objective To investigate the immune injury in genital tract of BALB/c mice induced by plasmid protein pORF5 of Chlamydia trachomatis and its possible mechanism.Methods GST(glutathione-S-transferases)-pORF5 fusion protein was expressed and digested with PreScission Protease to obtain the target protein without GST tag.After further purification and endotoxin removal,pORF5 protein was injected into the posterior fornix of BALB/c mice on day 1,3 and 6,while the control groups were injected with PBS or GST protein respectively,and then all the mice were sacrificed on day 7 to evaluate genital tract gross pathology and histopathological characterization.The levels of TNF-α,IL-1β and IL-6 in serum,splenocytes culture supernatant and vaginal douche were detected by ELISA.Results Mice in pORF5 group developed different degrees of swelling in isthmic portion and ampulla of uterine tube,connective tissue adhesion and hydrosalpinx in the genital tract tissues,while the PBS group and the GST group did not show any obvious change.The inflammatory score showed that the genital tract pathology in pORF5 group was much more severe than PBS and GST control groups (P＜0.O1).The levels of TNF-α,IL-1β and IL-6 in vaginal douche and splenocytes culture supernatants in pORF5 group were obviously higher than those of PBS and GST groups (P＜0.05).The levels of TNF-α and IL-1β in serum were also higher than those of GST and PBS groups (P＜0.01).Conclusion pORF5 plasmid protein could induce pathological immune response in the genital tract of BALB/c mice,which may be associated with the increase of the production of the inflammatory cytokines TNF-α,IL-1β and IL-6 in BALB/c mice.

Expression, purification and immunocompetence analysis of a Treponema pallidum recombinant protein TP0993 / 中华皮肤科杂志

Objective To evaluate the value of a Treponema pallidum (TP) recombinant protein TP0993 in the serodiagnosis of syphilis.Methods A bioinformatics method was used to obtain the sequence of TP0993 gene.The open reading frame (ORF) without upstream non-coding region of TP0993 gene was ligated into the expression vector PET-28a (+),which was then transformed into Escherichia coli Rosetta.Isopropyl-β-d-thiogalactoside (IPTG) was used to induce the expression of TP0993 protein.The expressed protein was purified with nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography.Western blot was performed to evaluate the immunoantigenicity of the protein.New Zealand rabbits were immunized with the recombinant protein for immunogenicity evaluation.Indirect enzyme linked immunosorbent assay (ELISA) was developed by using the purified recombinant protein to coat microwell plates.Anti-TP antibodies were detected by the established ELISA and TP particle agglutination assay (TPPA) in 480 clinical serum samples.Results The prokaryotic expression vector PET-28a (+)-0993 was successfully built,and a fusion protein with a relative molecular weight of about 34 000 Da was attained after IPTG-induced expression and purification.Western blot proved that the recombinant protein could specifically react with clinical sera positive for anti-TP IgG antibodies.Specific humoral response was elicited in New Zealand rabbits by the recombinant protein.Compared with TPPA,the established indirect ELISA showed a sensitivity of 88.3％ and a specificity of 85.8％.There was a consistency of 86.5％ between the indirect ELISA and TPPA.Conclusion The expressed recombinant protein showed favorable immunocompetence,and may serve as a candidate antigen for serodiagnosis of syphilis.

Observation on the immunological competence of multiple antigen peptides based on the mimic epitope of the adhesion protein of Mycoplasma genitalium / 中华微生物学和免疫学杂志

Objective To provide experimental evidence for the development of multi-epitope-baseded marker vaccines through investigating the humoral and cellular immune responses in BALB/c mice induced by the multiple antigen peptides (MAPs) with the mimic epitope.Methods Three types of MAPs in eight branched forms containing the mimic epitope of Mycoplasma genitalium adhesion protein (MgPa) were prepared using poly-lysine as the core matrix.The purity of MAPs was analyzed by reverse phase high performance liquid chromatography (RP-HPLC).The molecular weights of MAPs were characterized by Mass Spectrometry.The BALB/c mice were immunized intramuscularly for four times with single or mixed MAPs.The specific IgG antibody and the subtype of IgG antibody in serum of the immunized mice were detected by indirect ELISA.The proliferative responses of the spleen lymphocytes were detected using MTT assay.The ELISA were used to detect IFN-γ and IL-4 levels in the cultured supematant of spleen lymphocytes.Results The three types of MAPs containing the mimic epitopes were successfully prepared with high purity.They,could stimulate mice to produce specific IgG antibodies,of which,the major antibody isotype was Th1 immune response-associated IgG2a.Compared with the single MAP immunization group,the mixed-MAPs immunized mice produced more IgG,IgG1 and IgG2a antibody (P＜0.05).Furthermore,these MAPs could enhance the specific proliferation of spleen lymphocytes in immunized mice and induce the production of IFN-γ and IL-4.The levels of IFN-γand IL-4 in mixed-MAPs group were significantly higher than those of the single MAPs group (P＜0.01).Conclusion The three types of MAPs could induce strong specific cellular and humoral immune responses.The immunological competence of the mixed-MAPs was stronger than those of the single MAP.