To localize and characterize the hypothetical protein CT358 in the chlamydial infected cells. CT358 gene from the Chlamydia trachomatis (C. trachomatis) serovar D genome was amplified and cloned into the pGEX and pDSRedCI vectors. The recombinant plasmid pGEX-CT358 was constructed and expressed as GST fusion proteins. The GST-CT358 fusion protein was used to immunize mice to raise the antibodies, which specifically recognized CT358 without eross-reacting with other unrelated proteins. The antibodies were then used to localize the endogenous CT358 protein and determine the expression pattern in Chlamydial infected cells using an indirect immunofluorescence assay (IFA). Meanwhile, pDSRedC 1-CT358 recombinant plasmid was transfected to HeLa cells to evaluate the effect of CT358 expression on the subsequent chlamydial infection. The hypothetical protein CT358 was identified in the inclusion membrane of C. trachomatis-infected cells for the first time,and it was detected as early as 12 h after C. trachomatis infection and remained in the inclusion membrane throughout the rest of the infection cycle. Cytosolic expression of CT358 via a transgene failed to affect the subsequent ehlamydial infection. These observations together have demonstrated that CT358 is a newly identified chlamydial inclusion membrane protein, giving the potentially importance for further understanding the mechanisms of chlamydial intracellular parasitism.

Chlamydial infection in human urogenital tract induces inflammation and causes tissue damage and scarring. It is thought that cytokine production by the Chlamydia-infected cells plays a key role in chlamydial disease processes. Although many cytokines have been detected during chlamydial infection, little is known about the molecular mechanisms on how Chlamydia triggers and sustains the inflammatory cytokine cascades. In the current study, chlamydial infection of the human cervical epithelial cell line HeLa cells can induce the production of IL-8, IL-1α, IL-1β and IL-6. Using inhibitors for probing intracellular kinase signaling pathways required for the Chlamydia-induced cytokines, it was found that the Chlamydia-activated MAPK / P38 pathway is required for the chlamydial induction of IL-1α and IL-6 while both the Chlamydia-activated MAPK/ERK and MAPK/P38 pathways contribute to the production of IL-8.

To localize and characterize the hypothetical protein CT358 in the chlamydial infected cells.CT358 gene from the Chlamydia trachomatis(C.trachomatis) serovar D genome was amplified and cloned into the pGEX and pDSRedC1 vectors.The recombinant plasmid pGEX-CT358 was constructed and expressed as GST fusion proteins.The GST-CT358 fusion protein was used to immunize mice to raise the antibodies,which specifically recognized CT358 without cross-reacting with other unrelated proteins.The antibodies were then used to localize the endogenous CT358 protein and determine the expression pattern in Chlamydial infected cells using an indirect immunofluorescence assay(IFA).Meanwhile,pDSRedC1-CT358 recombinant plasmid was transfected to HeLa cells to evaluate the effect of CT358 expression on the subsequent chlamydial infection.The hypothetical protein CT358 was identified in the inclusion membrane of C.trachomatis-infected cells for the first time,and it was detected as early as 12 h after C.trachomatis infection and remained in the inclusion membrane throughout the rest of the infection cycle.Cytosolic expression of CT358 via a transgene failed to affect the subsequent chlamydial infection.These observations together have demonstrated that CT358 is a newly identified chlamydial inclusion membrane protein,giving the potentially importance for further understanding the mechanisms of chlamydial intracellular parasitism.

Objective To localize and characterize the plasmid protein pORF5 in the Chlamydia trachomatis(Ct) infected cells. Methods The open reading frame encoding for pORF5 protein from the Ct plasmid was amplified and cloned into the pGEX-6p vector. The recombinant plasmid pGEX-pORF5 was transformed into XL1-blue E. coli to express fusion protein with the glutathione-s-transferase (GST). After purified with Glutathione Sepharose 4B beads, the pORF5 fusion protein was used to immunize mice to make monoclonal and polyclonal antibody. The antibodies were used to localize the endogenous pORF5 protein and detect the expression pattern in Chlamydia-infected cells using an indirect immunofluorescence assay (IFA). At the same time, ELISA was used to determine whether pORF5 plasmid protein was expressed and immunogenic during Ct infection in humans. Results pORF5 was detected a dominant signal in the cytosol of the Chlamydia-infected cells with a pattern similar to that of anti-CPAF. pORF5 also appeared in the RBs and EBs in small quantity. Athough pattern was similarly, pORF5 did not overlap with CPAF. pORF5 protein was strongly recognized antiserum in an ELISA. Conclusion The pORF5 plasmid protein was identified as a secreted protein with good immunogenicity, pORF5 gene was to express the endogenous target protein during human infection.

Objective To give an empirical study on the interests claims of employees in public hospitals. Methods By questionnaire investigation, to sum up data using factor analysis and pairedsamples T test, and to compare difference between different kinds of patients using ANOVA. Results The interests claims of employees can be summed up to seven factors and there is some significant difference between different kinds of employees. Conclusion At current stage, public hospitals need pay more attention to material interests claims of employees.

Objective To study the white matter fiber integrity in patients with posttraumatic stress disorder by using MR diffusion tensor imaging (DTI).Methods Twelve patients with posttraumatic stress disorder and twelve healthy volunteers were examined with MR T1WI、T2WI and DTI.Fractional anisotropy maps、directionally encoded color maps were created with the software of DTV-II.Fractional anisotropy (FA) were measured in the genu (anterior),body and splenium (posterior) of corpus callosum,horizontal part and posterior part of the bilateral cingulste fibers.Results In normal volunteers group,FA measurements in white matter regions were normal and reconstructed FA images and directionally encoded color(DEC) maps could display main white fibers in normal controls.The FA value of the splenium (posterior) of corpus callosum and horizontal part of the bilateral cingulate fibers was significant lower in PTSD patients than in normal controls (P<0.05).Conclusions The fiber bundle of the limbic system in patients with PTSD may have structural abnornalities.

ObjectiveTo study the potential of using temporal clustering analysis(TCA)technique in localizing an epileptogenic zone.MethodsTwelve patients with epilepsy were examined using resting functional MRI(fMRI). The patients had detectable focal lesions on cranial MRI.TCA was performed to analyze resting fMRI data in order to identify the timing of interictal epileptiform discharges (IEDs).Standard event-related fMRI analysis in SPM99 was used to generate maps of the activation induced by epileptic brain activities.Comparisons were made between TCA Resultsand SPM motion trochoid.ResultsEight of the twelve subiects showed activations in the brain regions that were consistent with those lesions determined on anatomic MRI.The remaining four subiects showed no clear activation in the areas of detectable lesions. In addition, correlation was found between TCA Resultsand motion trochoids.ConclusionsTemporal cluster analysis,an exploratory data-driven technique,may provide the timing information about interictal epileptiforill discharges.However,the Resultsfrom this novel fMRI analytical technique need to be interpreted with caution as it is vulnerable to motion artifact.

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.

ObjectiveTo purify and characterize the monoclonal antibody (McAb) against Chlamydia trachomatis pORF5 plasmid protein.Methods The hybridoma cells stably secreting specific McAb against pORF5 were cultured in a large scale,and protein G purification by affinity chromatography was used to purify 2H4 McAb.ELISA was used to determine the antibody titer,and identify McAb isotype.Immunofluorescence assay (IFA) and Western blot were performed to detect McAb specificity.Results The purity of 2H4 antibody was 93％,the titer reached 1:1024,and 2H4 McAb was identified to belong to IgG2a isotype,2H4 McAb reacted strongly with the GST-pORF5 fusion protein and endogenous pORF5 protein expressed by Chlamydia trachomatis serovar A,D,L2,Chlamydia muridarum ( MoPn ),Chlamydia psittaci 6BC,but not other chlamydial plasmid proteins and Chlamydia pneumoniae(Cpn) AR39 strain.Conclusion2H4 McAb against pORF5 protein was successfully purified with a high titer and specificity which lay a foundation for further study on pORF5 protein structure and function.

ObjectiveTo construct a mouse model for studying pathophysiology and mechanism of human Chlamydia trachomatis genital infection.MethodsInnate immunity-deficient C3H/HeJ female mice were infected intravaginally with human C.trachomatis serovar D urogenital isolates for screening the highest violent clinical strain.The clinical strain UT0603 as well as standard strain D/UW-3/CX were then used to reinfect na(i)ve mice,the lower genital tract shedding were monitored by swabbing every 3-7 day over the entire infection period by culture.Some mice were sacrificed at early infection stage to detection of in site Chlamydia growth by immunofluorescence assay,then all the mice were sacrificed at later infection stage to evaluate upper genital tract gross pathology and histopathological characterization.ResuIts In the lower genital tract,Chlamydia shedding time course were significantly prolonged in clinical strain infected mice.Chlamydia not only growth in the lower genital tract,the live organism also ascending and growth in the upper genital tissue.The gross appearance under naked eyes and dilation and inflammation scores under microscope all showed that the genital tract pathology from the clinical strain infected mice were much more severe than standard strain infected control mice.Conclusion Together,all these results demonstrated that a mouse model for Chlamydia genital infection was constructed.