Objective To evaluate the effect of Treponema pallidum membrane protein Tpp47 on vascular endothelial cells.Methods Human umbilical vein endothelial cells (HUVECs) were classified into multiple groups to be cultured with various concentrations (50,100,200,400 and 800 μg/L) of the recombinant protein Tpp47 or lipopolysaccharide (LPS) for different durations (3,6,12,24 and 48 hours).Then,enzyme-linked immunosorbent assay (ELISA) was performed to determine the levels of intercellular cell adhesion molecule-1 (ICAM-1) and E-selectin in the culture supernatant of,fluorescence-based real-time quantitative PCR to quantify the mRNA expressions of ICAM-1 and E-selectin in,HUVECs.The 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay was used to evaluate the proliferation activity of HUVECs treated with Tpp47 (400 μg/L) and LPS (200 μg/L) respectively for 24 hours.To estimate the effect on adhesion ability,some HUVECs were pretreated with Tpp47 (400 μg/L) and LPS (200 μg/L) respectively for 24 hours followed by coculture with THP-1 human monocytic leukaemia cells for 6 hours,then,the adhesion of HUVECs to THP-1 cells was visualized by fluorescence microscopy.The cells receiving no treatment served as the blank control.Results A significant increase was observed in the supernatant level (expressed as the absorbance value at 450 nm) of ICAM-1 for HUVECs treated with Tpp47 of 400 μg/L for 24 hours (1.28 ± 0.03 vs.0.90 ± 0.01,t =18.28,P ＜ 0.05) and that of E-selectin for HUVECs treated with Tpp47 of 400 μg/L for 12 hours (0.51 ± 0.01 vs.0.13 ± 0.03,t =18.19,P＜ 0.05) compared with untreated HUVECs.The adhesion rate to THP-1 cells was significantly higher in HUVECs pretreated with Tpp47 for 24 hours than in untreated HUVECs (56.1％ ± 1.9％ vs.16.3％ ± 2.1％,x2 =12.65,P ＜ 0.05).The cell proliferation rate was 19.5％ ± 1.7％ in HUVECs treated with Tpp47,significantly higher than that in untreated HUVECs (10.0％ ± 3.1％,x2 =3.92,P＜ 0.05),but lower than that in those treated with LPS (41.2％ ± 3.7％,x2 =10.42,P ＜ 0.05).Conclusions The recombinant membrane protein Tpp47 could enhance HUVECs to proliferate and adhere to monocytic THP-1 cells,suggesting a certain role of Tpp47 in the pathogenesis of syphilis.

Objective To investigate the in vitro effects of Treponema pallidum membrane protein Tpp17 on the permeability of endothelial barrier for further investigation on the immunopathogenesis of syphi-lis.Methods A cellular model of in vitro monolayer was established by using human umbilical vein endo-thelial cells ( HUVECs) .Cell-ELISA and a TMB kit were respectively used to measure the expression of VE-cadherin and the flux of horseradish peroxidase ( HRP) by monolayer HUVECs after stimulation with the re-combinant Tpp17 (rTpp17) protein.THP-1 cells stained with Calcein AM were added to the top of HUVEC monolayer in Transwell culture.Then, the numbers of THP-1 cells in the upper wells and beneath the HUVEC monolayer were counted by using a fluorescence microscope.The rTpp17 protein-treated HUVECs were fixed in 4%buffered paraformaldehyde and stained with rhodamine-phalloidin for observing the distri-bution of F-actin under a confocal laser scanning microscope.Results Compared with the control group, the expression of VE-cadherin in HUVECs was decreased, while the permeability of HUVEC monolayer was increased upon the stimulation with rTpp17 protein (P<0.05).Moreover, rTpp17 protein-induced F-actin redistribution and increased transendothelial migration of THP-1 cells were observed in rTpp17 protein-trea-ted HUVECs as compared with those of the control group (P<0.05).Conclusion Treponema pallidum membrane protein Tpp17 could suppress the expression of VE-cadherin and enhance the redistribution of F-actin, resulting in an enhanced transendothelial migration of THP-1 cells and an increased permeability of HUVEC monolayer.The Tpp17 protein might play an important role in the immunopathogenesis of syphilis.

Objective To investigate mechanisms underlying the regulation of the permeability of vascular endothelial cells by the Treponema pallidum membrane protein Tpp47.Methods Human umbilical vascular endothelial cell (HUVEC) monolayers were established as a model,and were directly cultured with the presence of recombinant Tpp47 protein (rTpp47-treated group),or boiled and inactivated rTpp47 (negative control group).Some HUVEC monolayers,which were pretreated with the RhoA/ROCK signal pathway inhibitor Y-27632 for 30 minutes and then cultured with the presence of rTpp47,served as the pretreatment group.After 1-and 4-hour additional culture,enzymelinked immunosorbent assay (ELISA) was performed to estimate the permeability of these cell monolayers to horseradish peroxidase (HRP).After 12 hours of culture,rhodamine-phalloidin was used to stain cytoskeletal proteins,and confocal laser scanning microscopy was performed to observe the arrangement of the cytoskeletal protein F-actin.Western-blot analysis was conducted to measure the expressions of RhoA in HUVECs treated with rTpp47 or inactivated rTpp47.Results The supernatant level of HRP (expressed as the absorbance value at 450 nm) was significantly higher in the rTpp47-treated group than in the negative control group (0.81 ± 0.10 vs.0.39 ± 0.09,P ＜ 0.05),but no significant difference was observed between the pretreatment group (0.51 ± 0.10) and rTpp47-treated group or negative control group (both P ＞ 0.05) after 1-hour culture.Similarly,the rTpp47-treated group showed significantly increased levels of HRP compared with the pretreatment group and negative control group (2.31-± 0.14 vs.1.21 ± 0.12 and 0.73 ± 0.12,both P ＜ 0.05),while there was no significant difference between the pretreatment group and negative control group after 4-hour culture.The expression of RhoA in HUVECs treated with rTpp47 was significantly higher than that in those treated with inactivated rTpp47.Confocal laser scanning microscopy showed that rTpp47 treatment led to the rearrangement of F-actin in HUVECs followed by the formation of stress fibers in cytoplasm,while Y-27632 could partly inhibit the rearrangement of F-actin.Conclusion The recombinant Treponema pallidum membrane protein Tpp47 can regulate the permeability of vascular endothelial cells through the RhoA/ROCK signal pathway.

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.

Objective To investigate the effects of broadband ultraviolet B (BB-UVB) on the proliferation of, tyrosinase activity and melanogenesis in melanocytes.Methods Melanocytes isolated from human foreskin were subjected to primary culture.Some cultured primary melanocytes were irradiated with BB-UVB at 10, 20, 30, 40, 50, 100, 200 and 300 mJ/cm2.Then, CCK-8 assay was performed to evaluate the proliferative activity of melanocytes, dopa oxidation assay to estimate the activity of tyrosinase, and sodium hydroxide (NaOH)-lysis method was used to determine melanin content.Real-time fluorescence-based quantitative PCR was conducted to measure the mRNA expressions of genes involved in non-canonical Wnt pathways in melanocytes after irradiation with BB-UVB at 30, 50 and 100 mJ/cm2.Western blot was carried out to determine the expressions of proteins involved in non-canonical Wnt pathways in melanocytes before and after irradiation with BB-UVB of 100 mJ/cm2.The melanocytes receiving no treatment served as the control group.Statistical analysis was carried out by one-way analysis of variance followed by least significant difference (LSD)-t test for multiple group comparisons and by the independent sample t test for two-group comparisons.Results After irradiation with BB-UVB at 10-300 mJ/cm2, the proliferative activity of melanocytes was gradually reduced compared with the control group (all P ＜ 0.05), and the survival rate of melanocytes was less than 50％ when the irradiation dose of BB-UVB was higher than 100 mJ/cm2.Furthermore, tyrosinase activity gradually increased in melanocytes after irradiation with BB-UVB at 10-100 mJ/cm2 compared with the control group, and the increase was statistically significant at the radiation dose of 100 mJ/cm2 (P ＜ 0.05).Compared with the control group, the WIF-1 mRNA expression level decreased, while c-Jun N-terminal kinase (JNK), microphthalmia-associated transcription factor (MITF), Ras-related C3 botulinum toxin substrate 1 (RAC 1) and tyrosinase (TYR) mRNA expression levels increased in melanocytes after irradiation with BB-UVB at 30, 50 and 100 mJ/cm2 (all P ＜ 0.05);the WNT5A mRNA expression significantly decreased in melanocytes irradiated with 30 and 50 mJ/cm2 BB-UVB, but increased in those irradiated with 100 mJ/cm2 BB-UVB (all P ＜ 0.05).The radiation with 100 mJ/cm2 BB-UVB significantly decreased the expression of WIF-1 protein, but enhanced the expressions of WNT5A, JNK, MITF, RAC1 and TYR proteins in melanocytes compared with the control group (all P ＜ 0.05).Conclusions BB-UVB can decelerate the proliferation of, elevate tyrosinase activity and melanin level in, melanocytes.The WIF-1 gene may inhibit melanogenesis, and the decrease in its expression may promote melanogenesis by activating the JNK/MITF/TYR pathway through the combined effects of proteins involved in non-canonical Wnt pathways.

Objectives To test a simple method for genotyping of C.trachomatis isolates and to investigate the clinical significance of the genotypes. Methods A part of the chlamydial genome encoding the major outer membrane protein(omp1) was amplified by polymerase chain reaction (PCR). The products were digested by endonucleases to see the characteristic patterns, after silver staining on 10% polyacrylamide gels. Results The omp1 genes of 15 serovars of C. trachomatis were amplified by PCR,which generated an 871 base pair gene fragment. AluⅠ digestion of the product gave characteristic patterns for the 15 serovars,but group C presented closely similar patterns. A triple digestion with HpaⅡ, followed by HinfⅠ and EcoRⅠ, would allow the differentiation of serovars in group C. Analysis of 74 clinical isolates revealed serovars E, F, D, G as the most prevalent genital serovars in the studied populations. Serovars B, H, J were occasionally identified. A mixed infection with serovars F and D was seen in a clinical sample. No significant relationship was observed between clinical manifestations of urogenital chlamydial infections and serovars,however,serovar D was more often associated with high titer of anti chlamydial antibody than other serovars. Conclusion The omp1 genotyping technique seems to be promising for epidemiological studies.

Objective To explore the association of tumor necrosis factor(TNF)-α gene-308G/A single nucleotide polymorphism with susceptibility to syphilis.Methods The study includes 56 patients with early symptomatic syphilis,38 with eady latent syphilis,and 102 normal human controls.The-308G/A single nucleotide polymorphism of TNF-α gene was detected in all subjects by real-time fluorescent quantitative PCR.Results The frequency of TNF-α-308 G and A allele was 0.926 and 0.074 in patients with early syphilis,0.941 and 0.059 in the controls,0.929 and 0.071 in patients with early symptomatic syphihs,0.921 and 0.079 in patients with early latent syphilis.No significant difference was found between patients with early syphilis and the controls or between patients with early symptomatic syphilis and those with early latent syphilis in the frequency of either allele (MG) at-308 position of TNF-α gene(all P>0.05).Conclusion There seems to be no evidence for association between TNF-α gene-308G/A single nucleotide polymorphism and susceptibility to syphilis.

Neurosyphilis is a serious clinical stage of syphilis caused by Treponema pallidum invading the nervous system, and risk and predictive factors of neurosyphilis are different between syphilis patients with and without HIV infection. The risk factors for neurosyphilis in HIV-negative patients with syphilis mainly include gender, age, clinical stage of syphilis, treatment, etc.; the predictive factors include serological titers, changes in some indicators of cerebrospinal fluid, neurological or ophthalmic symptoms. HIV viral load, CD4 + T cell counts and antiretroviral treatment are the main predictors and risk factors for neurosyphilis in HIV-positive patients with syphilis.

Objective To study the association of U. urealyticum biovar and genotype with nongonococcal, nonchlamydial mucopurulent cervicitis. Methods The study population consisted of two groups: patient group (226 female patients with nongonococcal, nonchlamydial mucopurulent cervicitis) and control group (118 healthy women). The biovar and genotype of U. urealyticum were identified in specimens positive for U. urealyticum culture by using PCR-single-strand conformation polymorphism (PCR-SSCP) analysis. Results The most common genotype in both groups was mba 3/14 in biovar 1 with the detection rate being 30.98%(57/184) in patients with mucopurulent cervicitis and 43.42% (33/76) in the controls. A significant difference was observed in the prevalence of genotype 2B in biovar 2 between the patients and controls [16.30% (30/184) vs 6.58% (5/76), χ2 = 4.367, P= 0.037). The genotype 1, 3/14 and 6 in biovar 1 predominated in the controls with their total prevalence being 81.58%. Conclusion The genotype 2B in biovar 2 of U. urealyticum may be associated with nongonococcal, nonchlamydial mucopurulent cervicitis among female patients attending an STD clinic.

Objective To observe the attachment of Treponema pallidum to human brain microvascular endothelial cells (HBMECs) in vitro.Methods Some primary cultured HBMECs were inoculated into in 24-well plates to be cocultured with the suspension of T.pallidum at a concentration of 1.6 × 107 treponemes/ml.After 0.5,2 and 4 hours of co-culture,scanning electron microscopy was conducted to observe the attachment of T.pallidum to HBMECs.Some HBMECs were cocultured with the presence of T.pallidum suspensions at different concentrations (4 × 106,8 × 106,1.6 × 107 treponemes/ml) for 2,4,6 and 16 hours,then,dark-field microscopy was performed to count the number of treponemes that attached to single HBMECs.Statistical analysis was carried out by using repeated-measures analysis of variance.Results As scanning electron microscopy showed,treponemes gathered at some regions on the surface of HBMECs when they attached to HBMECs.In addition,T.pallidum partly merged with the membrane of HBMECs at the site of attachment.After co-culture with T.pallidum suspensions,the number of treponemes that attached to single HBMECs was significantly different among different time points (F =387.72,P ＜ 0.001) and among different concentrations of T.pallidum suspensions (F =593.23,P ＜ 0.001),with an interaction effect between the concentration of T.pallidum suspensions and incubation period (F =98.74,P ＜ 0.001).Concretely speaking,the number of treponemes that attached to single HBMECs increased over time until 6 hours after the start of coculture,then showed a decreasing trend,and reached the nadir value at 16 hours.Conclusion T.pallidum can adhere to cultured HBMECs in vitro,likely by the merger of its end with the membrane of HBMECs at some regions.