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Objective To investigate differences in Rab protein expressions in McCoy cells with acute versus persistent Chlamydia trachomatis (Ct) infection.Methods Cultured McCoy cells were infected with different amounts (400,500,550 μl/well) of Ct strain D suspensions,then cultured with the medium containing 100 U/ml penicillin G (persistent Ct infection groups) or that without penicillin G (acute Ct infection groups).Ct-uninfected McCoy cells receiving no penicillin G treatment served as the blank control group,and those receiving penicillin G treatment as the penicillin group.Mter 48-hour culture,McCoy cells were lysed,proteins were collected,and total RNA was extracted from the cells.Enzyme-linked immunosorbent assay (ELISA) was conducted to measure protein levels of Rab4A,Rab6A,Rab10,Rab11A and Rab14,and fluorescence-based quantitative PCR to quantify mRNA expressions of Rab4A and Rab14 (expressed as 2-ΔΔα).Results Protein levels of Rab4A,Rab6A,Rab10,Rab11A and Rab14 were all significantly lower in the acute than in the persistent Ct infection groups (all Z =3.621,P ＜ 0.001),and lower in the persistent and acute Ct infection groups than in the blank control group (all P ＜ 0.008 3),but insignificantly different between the blank control group and penicillin group (all P ＞ 0.05).In addition,the expressions of Rab4A and Rab14 mRNAs were consistent with those of their proteins in these groups.Conclusion The transcriptional and expression levels of Rab proteins are higher in McCoy cells persistently infected with Ct than in those acutely infected with Ct.
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Objective To determine the prevalent herpes simplex virus (HSV) strain in patients with genital herpes (GH),and to evaluate the sensitivity,specificity,positive predictive value (PPV) and negative predictive value (NPV) of anti-herpes simplex virus type 2 (HSV2) IgG and IgM antibodies in the diagnosis of genital herpes (GH) before in vitro fertilization (IVF).Methods Totally,193 HSV2 clinical strains isolated in cell culture from the lesions of patients with GH in the Department of Dermatology,First Affiliated Hospital,Sun Yat-sen University between 2009 and 2011 were typed by using type-specific fluorescein isothiocyanate (FITC)-labelled anti-HSV monoclonal antibodies.Serum samples were obtained from 57 anti-HSV2 IgM/IgG antibody-positive females with suspected GH as well as their husbands (clinical observation group),68 HSV culture-positive patients diagnosed with GH (positive control group),and 120 children aged 8-12 years (negative control group).Enzyme-linked immunosorbent assay (ELISA) was performed to detect anti-HSV1/HSV2 IgG/IgM antibodies in these serum samples.Statistical analysis was carried out using chi-square test.Results There was a significant difference between the positive control group and negative control group in the positivity rate of anti-HSV1 IgG (89.71％ (61/68) vs.40.80％ (49/120),P ＜ 0.01) and anti-HSV2 IgG (91.18％ (62/68) vs.0,P ＜ 0.01),but not in that of anti-HSV1 IgM (20.59％ (14/68) vs.21.70％ (26/120),P ＞ 0.05) or anti-HSV2 lgM (13.24％ (9/68)vs.13.30％ (16/120),P ＞ 0.05).In the clinical observation group,the positivity rate of anti-HSV1 and anti-HSV2 IgM antibodies,anti-HSV1 and anti-HSV2 IgG antibodies was 80.70％ (46/57),91.23％ (52/57),84.21％ (48/57) and 14.04％ (8/57) respectively in the females,19.30％ (11/57),8.77％ (5/57),87.71％ (50/57),12.28％ (7/57)respectively in the males,with significant differences in the positivity rate of anti-HSV1 and-HSV2 IgM antibodies (both P ＜ 0.01),but not in that of anti-HSV 1 or-HSV2 IgG antibodies (both P ＞ 0.05).The sensitivity,specificity,PPV and NPV were 13.24％ (9/68),86.67％ (104/120),36.00％ (9/25) and 63.80％ (104/163) respectively for anti-HSV2 IgM antibody in the diagnosis of GH,91.18％ (62/68),100.00％ (120/120),100.00％ (62/62),and 95.24％ (120/126) respectively for anti-HSV2 IgG antibody.Conclusions HSV2 prevails in the patients with GH in this region,while HSV1 only amounts to 5.18％.The type-specific anti-HSV2 IgG antibody shows a higher specificity,sensitivity,PPV and NPV in the diagnosis of GH than anti-HSV2 IgM antibody,hence,the type-specific anti-HSV2 IgG antibody is superior to anti-HSV2 IgM antibody in diagnosing GH before assisted reproduction.
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ObjectiveTo observe the ultrastructural changes of Chlamydia trachomatis after treatment with azithromycin.Methods The Chlamydia trachomatis laboratory strain (D/UW-3/Cx) was cultured in McCoy cells with or without the presence of azithromycin of 0.0667,0.1340,0.1900,0.2680 and 0.3330 mg/L for 48 hours.The ultrastructural changes of host cells andChlamydia trachomatis were observed by transmission electron microscopy.ResultsAfter 48-hour culture,vesicles increased in number both inside and outside of the inclusion bodies with the rise in azithromycin concentration; there were abnormally large reticulate bodies,some of which experienced abnormal division and even necrosis or breakdown; the number of elementary bodies was decreased,while their size was enlarged,with a more wrinkled outer membrane.No inclusionbodieswereseenwhentheconcentrationofazithromycinwas0.333mg/L. Conclusions Azithromycin can induce an increment in the outer membrane of Chlamydia trachomatis,formation of vesicles,abnormal enlargement or breakdown of reticulate bodies,and a decrease in elementary bodies.
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Objective To investigate the role of cHSP60 in the pathogenesis of murine cervicitis.Methods Fifty female C3H/HeN mice were randomly and equally classified into 5 groups, including the control group receiving no treatment and 4 groups receiving intravaginal inoculation of cHSP60 (cHSP60 group),live elementary bodies of Chlamydia trachomatis mouse pneumonitis (MoPn group), inactive elementary bodies of MoPn (inactive MoPn group) and growth medium (medium group), respectively. Five days after the inoculation,cervical tissue was resected from these mice and subjected to pathological examination. Results There were varying degrees of inflammatory reaction characterized by neutrophil infiltration, necrosis and shedding of mucosal cells in the cervices of mice in cGSP60 and MoPn groups. No statistical difference was observed in the incidence of cervicitis (90% vs. 80%, P ＞ 0.05), neutrophile count [76.00 (25.0 - 80.0) vs. 25.00 (8.75 -32.5), P＞ 0.05] or inflammation score [12.5 (11.5 - 14.25) vs. 9.00 (8.00 - 11.5), P ＞ 0.05] between the cHSP60 and MoPn group. The inflammatory reaction was weak with decreased incidence of cervicitis (40%),inflammation score [0.00 (0.00- 12.50)] and neutrophile count [0.00 (0.00- 15.50)] in inactive MoPn group compared with the cHSP60 and MoPn groups (all P ＜ 0.05). A small number of neutrophils migrated into the superficial layer of cervical mucosa in only 2 mice in the medium group. Conclusion cHSP60 may be a primary pathogenic factor in chlamydial genital tract infection.
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Objective To clone and express Chlamydia trachomatis (Ct) heat shock protein 60 (hsp60) gene. Methods The hsp60 gene fragment was amplified from Ct chromosomal DNA by PCR. After purification and digestion with enzymes Sal I and Not I , the hsp60 gene fragment was inserted into the compatible site of prokaryotic expression vector pET-28a. The constructed recombinant plasmid was identified by PCR, restriction enzyme cleavage and sequencing, then, it was transfected into an expression strain Escherichia coli BL21 (DE3). The expression of fusion protein was induced by isopropy-β-D- thiogalactoside (IPTG) in the host bacteria, and the expressed product was identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western-blot. Results PCR and restriction enzymes cleavage analysis confirmed that the hsp60 gene was successfully cloned into the recombinant plasmid. DNA sequencing showed that the sequence of cloned gene was fully consistent with the published sequence in Genebank. As revealed by SDS-PAGE, the size of expressed fusion protein approximated 60 kilodaltons, and Western-blot confirmed the expressed product to be the expected protein. The final concentration of fusion protein was 17.85 mg/L with a purity of more than 90%. Conclusions A recombinant expression plasmid pET-28a-hsp60 is successfully constructed in this study, and soluble hsp60 protein is expressed by the recombinant plasmid-transfected E. coli.
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20%ALA cream were applied topically to condylomata acuminata.The cream was kept in place for 4 h.He-Ne laser light at 630nm was used,and the dose of light was 100 J/cm2 for all of the patients.After three treatment,the complete removal rate(CRR)of urethral and other genital mucossa were 93.4%and 88.9%,significantly higher than genital skin 39.1%(P＜0.05).The adverse reactions of ALA-PDT are mainly local minor erosion,short-term pain,but no scar.It showed that ALA-PDT is an effective,minimally invasive treatment for condylomata acuminata,especially for the lesions on urethral and genital mucosa.