ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Objective To evaluate inhibitory effect of Chlamydia trachomatis plasmid-encoded protein pORF5 on HeLa cell apoptosis induced by tumor necrosis factor-alpha(TNF-α). Methods The recombinant lentiviral expression vector containing pORF5 gene and helper plasmids were co-transfected into 293T cells to prepare the recombinant lentivirus. Then, the lentivirus particles were collected and concentrated, and used to infect HeLa cells. Flow cytometric screening identified stable pORF5-expressing HeLa (pORF5-HeLa) cells. Meanwhile, the empty plasmid was transfected into HeLa cells to prepare control HeLa cells. The two cell lines were both divided into two subgroups to be treated with 20μg/L TNF-αand fresh culture medium respectively for 6 hours. Then, Hoechst 33258 staining was performed to observe morphological changes of apoptotic cells, flow cytometry to detect cell apoptosis, real-time PCR to measure the mRNA expression of Caspase3, Bax and Bcl-2, and Western blot analysis to determine the protein expression of Bax and Bcl-2. Results After 6-hour treatment with TNF-α, Hoechst 33258 staining showed variable degrees of karyopyknosis and karyorrhexis, and highly-refractive blue apoptotic bodies in the pORF5-HeLa cells and control HeLa cells. The pORF5-HeLa cells and control HeLa cells both showed significantly higher apoptosis rate in the treated subgroup than in the untreated subgroup (pORF5-HeLa cells:35.5%± 4.5%vs. 9.5%± 1.5%, t=13.53, P<0.01;control HeLa cells:63.6%± 5.8%vs. 7.9%± 0.9%, t=32.36, P<0.01). Compared with treated control HeLa cells, treated pORF5-HeLa cells showed significant decreases in mRNA expression of Bax(72.8%)and Caspase 3(84.5%)(t = 35.29, 42.25, respectively, both P < 0.01), as well as in Bax protein expression(t = 17.58,P < 0.01), but significant increases in Bcl-2 mRNA and protein(6.8 times)expression(t = 87.12, 18.93, respectively, both P <0.01). Conclusion pORF5 plasmid protein can inhibit TNF-α-induced HeLa cell apoptosis likely by increasing the expression of anti-apoptotic protein bcl-2 and decreasing the expression of pro-apoptotic proteins Caspase-3 and Bax.
ABSTRACT
Objective To observe the effect of combined administration of Jiang Zhi Tong Luo Soft Capsule(JTSC)and rosu-vastatin on type 2 diabetic with hyperlipemia and its possible mechanisms.Methods Eighty cases of Type 2 diabetic patients with hyperlipemia were divided into combine treatment group (40 cases)and control group (40 cases ).On the basis of routine treat-ment,patients in the combine treatment group were treated by oral 10 mg rosuvastatin (once a day)and oral 100 mg JTSC (three times a day ).Patients in control group were treated by oral 10 mg rosuvastatin (once a day and continuously for 12 weeks).We de-tected the liver function,renal functions,creatine kinase,TC,TG,LDL,HDL and hs-CRP were tested before and after treatments. Results After 12 weeks treatment,the combine treatment group was much better than the control group in reducing the level of TG,TC,LDL,hs-CRP and increasing HDL-C(P <0.05 ).Conclusion Rosuvastatin can effectively treat Type 2 diabetic patients with hyperlipemia.Combined administration of JTSC and rosuvastatin shows better effect than rosuvastatin used alone on the treat-ment of Type 2 diabetic patients with hyperlipemia.
ABSTRACT
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 .
ABSTRACT
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.
ABSTRACT
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 .
ABSTRACT
Objective To study the intracellular localization and temporal expression of CPSIT_0271 in Chlamydia psittaci-infected cells; and to investigate the effects of recombinant GST-CPSIT_0271 protein on the expression of proinflammatory cytokines including IL-6, IL-1βand TNF-αby THP-1 cells.Methods The gene encoding CPSIT_0271 of Chlamydia psittaci was expressed as fusion protein ( GST-CPSIT_0271 ) in E.coli.The polyclonal antibody was prepared by immunizing BALB /c mice with the purified recombinant pro-tein.Antibody titer was determined by ELISA .Indirect immunofluorescence assay ( IFA) was performed to lo-cate the endogenous CPSIT_0271 protein in C.psittaci-infected cells .The expression characteristics of CPSIT_0271 protein were detected by Western blot in C.psittaci-infected HeLa cells at different time points .The levels of IL-6, IL-1βand TNF-αwere analyzed by ELISA after stimulating THP-1 cells with different concentrations of CPSIT_0271 protein.Results CPSIT_0271 protein was found to express in the chlamydia inclusion of C.psittaci-infected HeLa cells .The expression of CPSIT_0271 protein was detected firstly at 36 h and increased at 48 h after C.psittaci infection.The titer of anti-CPSIT_0271 specific antibody in GST-CPSIT_0271 immu-nized mice reached to 1 ∶16 000.GST-CPSIT_0271 protein increased the levels of IL-6, IL-1βand TNF-αin THP-1 cells in a dose-dependent manner in the range of 2 to 5 μg/ml.The levels of TNF-αand IL-1βreached their peaks at 24 h, and IL-6 level peaked at 48 h upon the stimulation by 5 μg/ml of GST-CPSIT_0271 pro-tein.Conclusion CPSIT_0271 expressed in inclusion bodies of Chlamydia psittaci in the infected cells , sug-gesting it might be a late expression gene .GST-CPSIT_0271 protein shows good immunogenicity and enhances the expressions of IL-6, IL-1βand TNF-αin THP-1 cells.
ABSTRACT
ObjectiveTo screen a 12-mer phage display peptide library by the polyclonal antibody (pAb) against the recombinant adhesion protein of Mycoplasma genitalium (rMgPa) in order to obtain the antigenic mimic epitopes of MgPa.MethodsThe purified pAb was used to screen the immunodominant mimic epitopes of MgPa by a random 12-peptide phage display library.Seventy-four recombinant phage clones were randomly selected,and then DNA sequence analysis and computer-based bioinformatics analysis were performed to define the consensus amino acid residues of the mimotopes by MIMOX.The binding specificities of the selected phage-displayed peptides to the purified pAb were confirmed by ELISA,competitive ELISA and Western blot analysis.Results After four rounds of biopanning,a significant enrichment of phages was achieved,the inserts from 74 phage clones distinguished 45 peptides based on the different amino acids sequences.Amongst 45 peptides,36 peptides were ELISA positive and 23 peptides that absorbance values were higher than 1.5 showed high reactivities with pAb and effectively inhibited the binding of pAb to rMgPa.Immunoscreening via phage display peptide library revealed three different mimptopes of adhesion protein of M.genitalium,P-S-A-A/V-X-R-F/W-E/S-L-S-P,A-K-I/L-T/Q-X-T-L-X-L and K-S-L-S-R-X-D-X-I.Results of bioinformatics analysis by MIMOX demonstrated that S,A,F for cluster 1,A,K,I,T and L for cluster 2,K,S,L,R,D and I for cluster 3,may be the key consensus amino acid residues in the aligned mimotopes,respectively.ConclusionAntigenic mimics on MgPa were successfully identified and the motif P-S-A-A/V-X-R-F/W-E/S-L-S-P,A-K-I/L-T/Q-X-T-L-X-L and K-S-L-S-R-X-D-X-I may represent the immunodominant mimic epitopes of MgPa.And S,A,F K,I,T,L,R and D may be the key amino acid residues for the epitopes of MgPa.
ABSTRACT
Objective To clone and express the immunodominant domain of the chlamydial proteaselike activity factor(CPAF) from Chlamydophila psittaci(Cps) and evaluated the diagnosing value of the recombinant protein in Cps infection.Methods The immunodominant region epitope of CPAF (CPAFm,A196-A450)from Cps was chosen according to bioinformatics analysis and references.The specific primer was designed and the gene was amplified by PCR and then ligated into a pGEX6p-2 vector.Recombinant protein was induced to express by IPTG and analyzed by SDS-PAGE and Western blot.Indirect EL1SA method of serological diagnosis was established with the reorganization protein as coating antigen.One hundred and eighty sera samples from ducks with respiratory tract infection symptoms were detected with the established indirect ELISA and a commercial ELISA-kit to assess the value of the recombinant protein in serodiagnosis.The results were further identified with Western blot.Results Prokaryotic expression vector pGEX6p-2/CPAFm was constructed and a 54x103 fusion protein was attained.The indirect ELISA method was established with reorganization protein for envelope antigen.Using the indirect ELISA to detect Cps lgG positive and negative reference sera,the sensitivity and specificity were both 100％ (20/20).And the recombinant protein has no cross reaction with either Chlamydophila pneumoniae or Chlamydophila trachomatis.The concordance rate between the indirect ELISA and Western blot to 180 ducks sera samples was 100％,while the concordance rate of the commercial ELISA kit was 77.5％-95.0％.Conclusion The prepared recombinant protein of the CPAF immunodominant region epitope gene from Gps can highly benefit on developing new indirect ELISA as methods to detect specific anti-Cps antibodies.