Analysis of human papillomavirus infection in 1 902 patients and drug treatment efficacy in 266 patients in Tianjin / 中华微生物学和免疫学杂志

Objective:To analyze the incidence of human papillomavirus (HPV) infection in 1 902 patients and to evaluate the efficacy of drug treatment in 266 patients, aiming to provide reference for the treatment of HPV infection.Methods:The subtypes of HPV isolated from 1 902 patients aged 15-86 years visiting the venereology outpatient clinic of Tianjin Medical University General Hospital from October 2019 to May 2021 were identified by polymerase chain reaction-reverse dot blot hybridization. Drug treatment efficacy in 266 patients of them was retrospectively analyzed.Results:The overall incidence of HPV infection in the 1 902 patients was as high as 53.84% (1 024/1 902). It was 52.60% (689/1 310) in males and 56.59% (335/592) in females. There was no significant difference in the incidence between males and females ( P>0.05). The most common HPV genotype in males and females was HPV6 [15.27% (200/1 310) and 21.96% (130/592)], followed by HPV16 [10.61% (139/1 310) and 9.46% (56/592)], HPV11 [9.31% (122/1 310) and 8.61% (51/592)], HPV52 [6.79% (89/1 310) and 8.95% (53/592)] and HPV43 [5.64% (87/1 310) and 8.45% (50/592)]. The majority of HPV-positive patients were aged between 20 and 39 years. There were 476 cases (25.03%, 476/1 902) of single-type infection and 548 cases (28.81%, 548/1 902) of multiple infection. The incidence of multiple infection was higher than that of single-type infection ( P<0.05). The incidence of multiple infection in females was higher than that in males ( P<0.05). Among the 266 patients, 106 were treated with Paiteling, a traditional Chinese medicine (TCM) preparation, and 68 of them tested negative (64.15%) after treatment. Fifty-eight patients were treated with recombinant human interferon α2b and 22 of them (37.93%) tested negative after treatment. Twenty out of the 56 subjects treated with imiquimod tested negative after treatment. Eight out of the 46 patients without treatment also turned negative. Conclusions:The incidence of HPV infection in the 1 902 patients visiting the venereology outpatient clinic was very high, and most of them were young adults. Multiple infection was more common than single-type infection. Topical application of drugs such as Paiteling, recombinant human interferon α2b and imiquimod was effective in treating HPV infection.

Effects of five proteins secreted by Chlamydia trachomatis on phagocytosis of mouse macrophages and dendritic cells / 中华微生物学和免疫学杂志

Objective To analyze the effects of five proteins secreted by Chlamydia trachomatis on the phagocytosis of macrophages and dendritic cells derived from bone marrow cells of C3H/HeJ mice. Methods Glutathione S-transferase ( GST)-CT311, GST-GIgA, GST-cHtrA, GST-OmcBc and GST-Pgp3 proteins were prepared through an Escherichia coli prokaryotic expression system and purified by GST Mag-Beads. Chlamydia membrane protein GST-IncA was also prepared as a control. Proteins of interest were ob-tained by cleaving off GST-tag with PreScission protease. Macrophages (MΦ) and dendritic cells (DC) were prepared from bone marrow cells of C3H/HeJ mice and pretreated with either 100 μg/ml or 500 μg/ml of the above proteins. LPS was used as a control to testify the specificity of the proteins' functions. Four hours after pretreatment,fluorescent beads were added to culture media to evaluate the changes in phagocytosis with direct immunofluorescence assay. Results LPS and low concentration (100 μg/ml) of these proteins had no significant influence on the phagocytosis of DC and MΦ,while high concentration (500 μg/ml) of Pgp3, cHtrA and CT311 could significantly promote the phagocytosis of DC and MΦ. Conclusion Pgp3, cHtrA and CT311 can promote the in vitro phagocytosis of DC and MΦ,which may facilitate the in vivo dissemina-tion of Chlamyida trachomatis.

Retrospective analysis of penciclovir combined with foscarnet sodium in the treatment of herpes zoster / 中华实验和临床病毒学杂志

Objective@#To evaluate the therapeutic efficacy of penciclovir combined with foscarnet sodium in the treatment of herpes zoster.@*Methods@#The clinical datas of 135 herpes zoster patients from the ward of Department of Dermatology, Tianjin Medical University General Hospital were collected. Among them 64 patients received penciclovir and foscarnet sodium, and the remaining 71 patients only received penciclovir alone.Their general information, the time for vesicle stopped emerging, rash began to scab, pain to relief obviously, the adverse reaction and if they got the postherpetic neuralgia were recorded and included into statistical analysis.@*Results@#The general information showed no significant differences between the 2 groups(all P>0.05). The time for vesicle stopped emerging, rash began to scab, pain to relief obviously in combination group was shorter than the penciclovir group (all P<0.001). The number of patients who developed postherpetic neuralgia of combination group was fewer than that of penciclovir group(P=0.013). There was no statistical significance between the 2 groups the adverse reaction(P=0.928).@*Conclusions@#The penciclovir and foscarnet sodium combination therapy showed rapid therapeutic effects on herpes zoster patients, the incidence of postherpetic neuralgia was low, and there was no more side effects than penciclovir alone therapy. The combined therapy may be a reliable way to treat herpes zoster.

In vitro activity of rifampin against and rpoB mutations in Chlamydia trachomatis clinical isolates / 中华皮肤科杂志

Objective To evaluate the susceptibility of Chlamydia trachomatis clinical isolates to rifampin, and assess the relationship between rpoB mutations and antibiotic resistance in them.Methods A microculture method was used to determine the minimal inhibitory concentration (MIC) of rifampin in 52 Chlamydia trachomatis clinical isolates.The rpoB gene was amplified from all the clinical isolates and a standard strain of Chlamydia trachomatis followed by single-strand conformation polymorphism (SSCP)analysis.Sequencing of PCR products was carried out for two clinical isolates.Results No rifampin-resistant strain was found among these clinical isolates.The MIC of rifampin varied from 0.004 to 0.030 mg/L Neither SSCP analysis nor sequencing showed rpoB mutations.Conclusions No rpoB mutations were found in Chlamydia trachomatis isolates from patients unresponsive to rifampin.The unresponsiveness to rifampin may be attributed to multiple factors.

Identification of cell invasion-related virulence genes in chlamydial plasmids in vitro / 中华皮肤科杂志

Objective To compare the infectivity of several transformed Chlamydia trachomatis (Ct) mouse pneumonitis (Mopn) strains to host cells, and to identify cell invasion-related virulence genes in Chlamydial plasmids. Methods Several Ct strains, including wild-type Ct Mopn strain(WT strain), plasmid-free Ct strain(CMUT3 strain), Ct Mopn strain transformed with the shuttle vector carrying pGFP and the complete C. muridarum (CM) plasmid (pGFP::CM strain)and Ct Mopn strains transformed with shuttle vectors carrying pGFP and mutant CM plasmids with in-frame deletions of Pgp3, 4, 5 or 7 (pGFP::CM△Pgp3, 4, 5, 7 strains), were cultured, amplified and collected. After the concentrations of Ct were determined, each of these strains was divided into four groups to be inoculated at a same amount(1.5 × 104 inclusion forming units(IFU)) followed by four different treatments respectively: centrifugalization +DEAE group treated with centrifugalization followed by ion-exchange chromatography on diethylaminoethyl(DEAE)-cellulose columns, centrifugalization group treated with centrifugalization only, DEAE group treated with chromatography on DEAE-cellulose columns only, control group receiving no treatment. After additional culture for 20 - 24 hours, indirect immunofluorescence assay was performed to count the number of chlamydial inclusions. At 20, 40 and 60 hours after infection, the growth rate and area of chlamydial plaques were assessed after three continuous passages. Lugol′s iodine staining was conducted to observe glycogen synthesis in bacterial inclusions. Results The inclusion number in the centrifugalization + DEAE group, centrifugalization group, DEAE group and control group was 10.20 ± 1.30, 6.80 ± 0.44, 3.00 ± 1.22 and 0.80 ± 0.45 respectively for the pGFP::CM△Pgp4 strain, 6.40 ± 0.89, 3.80 ± 0.83, 1.60 ± 0.89 and 0.60 ± 0.54 respectively for the CMUT3 strain. Under same experiment conditions, the pGFP::CM△Pgp4 strain and CMUT3 strain showed similar infectivity, and formed less inclusions compared with the other Ct strains (all P < 0.01). The number of inclusions formed by the same Ct strains were significantly different among the 4 groups(F = 845.310, P <0.01), and were highest in the centrifugalization + DEAE group for all the strains. The pGFP::CM△Pgp4 strain showed significantly lower growth rate and area of plaques with an abnormality in glycogen accumulation compared with the other strains at 20, 40 and 60 hours after infection. Conclusion The plasmid-encoding gene Pgp4 may be a cell invasion-associated virulence gene in chlamydial plasmids.

Detection of Chlamydia trachomatis phage Vp1 gene in clinical swab specimens as well as anti-Vp1 antibodies in serum specimens / 中华皮肤科杂志

Objective To detect Chlamydia trachomatis phage Vp1 gene in clinical swab specimens and anti-Vp1 antibodies in serum specimens.MethodsCervical and urethral swab as well as serum specimens were collected from attendees to the sexually transmitted disease(STD) clinic in the Tianjin Institute of STD,during March 2008 to March 2011.PCR was conducted to detect chlamydial phage Vp1 gene in swab samples,enzyme linked immunosorbent assay(ELISA) and Western blot to detect anti-Vp1 antibody in sera.The swab specimens positive for Vp1 gene were subjected to cell culture followed by the detection of Vp1 protein with an immunofluorescence-based method.ResultsTotally,36 out of 1542 swab specimens turned out to be positive for Vp1 gene,and 23 out of 453 serum specimens for anti-Vp1 antibody.No positive results were obtained in the Vp1 gene-positive swab specimens by cell culture and immunofluorescence-based assay.ConclusionThe Vp1 gene of Chlamydial trachomatis phage and anti-Vp1 antibody are successfully detected from clinical swab and serum specimens respectively.

Preparation and preliminary application of monoclonal antibody against Vp1 protein of chlamydiaphage ΦCPG1 / 中华皮肤科杂志

Objective To express recombinant Vp1 protein of chlamydiaphage φCPG1, prepare monoclonal antibody against Vp1 protein and utilize it to screen clinical isolates of Chlamydia trachomatis. Methods The Vp1 protein was obtained by prokaryotic expression, and monoclonal antibody against this protein was prepared by hybridoma technique. ELISA and Western blot were used to identify monoclonal antibodies. Then,the monoclonal antibody was prepared in quantity by injecting hybridoma cells into the abdominal cavity of BALB/C mice, and purified by using protein G affinity chromatography. Clinical isolates of Chlamydia trachomatis were screened for the chlamydiaphage by immumofluorescence assay using the prepared monoclonal antibody.Results Purified Vp1 protein was obtained. The monoclonal antibody against Vp1 protein was gained after 3times of sub-clone and consistently identified as IgG1. Three hybridoma cell strains that stably secreted monoclonal antibody were generated. Chromosome analysis of hybridoma cells showed that the mean number of chromosome was 96, most of them were telocentric and a few were submetacentric. The titer of purified monoclonal antibody was more than 1: 12 800. Twenty clinical isolates were screened by using the monoclonal antibody and no positive results were obtained. Conclusions The monoclonal antibody against Vp1 protein of chlamydiaphage φCPG1 is successfully prepared, while no chlamydiaphage is detected by immumofluorescence assay using the prepared antibody in 20 clinical isolates of Ct.

In vitro susceptibilities of urogenital Chlamydia trachomatis to five antimicrobial agents alone or in combination / 中华皮肤科杂志

Objective To test the in vitro acitivity of azithromycin, minocyline, moxifloxacin, doxycycline and rifampicin alone or in combination against three standard strains of urogenital Chlamydia trachomatis,i.e. D-UW-5/Cx, E-UW-5/Cx and G-UW-5/Cx. Methods Three standard strains of C. trachomatis were inoculated into McCoy cells, and used to susceptibility testing after more than 90% McCoy cells were infected.Microdilution method was applied to determine the activity of the 5 antimicrobial agents alone, and checkerboard array to determine that in combination. Results The in vitro minimal inhibitory concentrations (MICs)were 0.25 mg/L for azithromycin, 0.06 mg/L for moxifloxacin, 0.063 - 0.25 mg/L for doxycycline, 0.032 -0.064 mg/L for minocyline, 0.008 mg/L for rifampicin. And, the fractional inhibitory concentration index was constantly 0.75 for the combination of azithromycin with moxifloxacin, doxycycline and rifampicin, 2.5, 2.83and 4 for the combination of minocyline with azithromycin, moxilloxacin and rifampicin, respectively. Conclusions As far as the activity against C. trachomatis is concerned, there is a synergism for the combination of azithromycin with moxifloxacin, doxycycline and rifampicin, but antagonism for the combination of minocyline with azithromycin, moxifloxacin and rifampicin.

Cloning, expression and identification of phage's capsid Vp3 protein of guinea pig inclusion conjunctivitis chlamydia / 中华传染病杂志

Objective To get phagc's capsid Vp3 gene and protein of guinea pig inclusion conjunctivitis (GPIC) chlamydia. Methods The genome DNA was extracted from the φCPG1 phage.The full sequence of Vp3 gene was amplified by polymerase chain reaction (PCR) from the above genome DNA. The Vp3 gene was digested by restriction endonuclease and then inserted into prokaryotic plasmid vector pET30a (+). The recombinant plasmid was transformed into E. coil BL21, and was identified by restriction endonuclease, PCR and sequencing. The E. coil BL21 with expected recombinant plasmid was induced and the expressed recombinant Vp3 protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, then purified by agarose gel. Results The recombinant gene was sequenced and proved to be 447 bp which was consistent with the φCPG1 Vp3 gene sequence in GenBank. A 25 000 capsid protein was expressed and confirmed by SDS-PAGE and Western blot. The purified protein was obtained. Conclusion The capsid Vp3 protein of φCPG1 is successfully expressed and purified, which is helpful for the further study on its mechanism and clinical applications.

In vitro susceptibilities of urogenital Chlamydia trachomatis clinical isolates to azithromycin alone and in combination with other antimicrobial agents / 中华微生物学和免疫学杂志

Objective To test the in vitro activity of azithromycin against recent clinical isolates of urogenital Chlamydia trachomatis, and combined activity of azithromycin with moxifloxacin, rifampicin, doxycycline and minocyline. Methods When more than 90% McCoy cells were infected, the 41 strains tested were collected to investigate minimal inhibitory concentrations (MICs) of 5 antimicrobials alone. Checkerboard array was used to calculate the fractional inhibitory concentrations(FICs) and then detected the interactions among the various combinations. Results In vitro, synergism or additivity effect of 51.22%,53.66% and 58.54% strains was found in azithromycin-moxifloxacin, azithromycin-doxycycline and asithromycin-rifampicin combinations, respectively. No difference was observed in all of the combinations ( P ＞0.05). However, antagonism effect of 90.24% strains was observed in azithromycin-minocyline combination. Conclusion This study indicates that the combination of azithromycin with moxifloxacin, doxycycline or rifampicin is more effective against Chlamydia trachomatis than individual antimicrobials. Therefore, these antimicrobials combinations might be recommended against Chlamydia trachomatis recurrent or persisitent infection. However, the combination of azithromycin with minocyline exhibited a markedly antagonism activity against Chlamydia trachomatis. Combined sensitivity test to a certain extent can compensate for some shortcomings of individual susceptibility test.

Immune responses induced by recombinant major outer membrane protein of Chlamydia trachomatis serovar E / 中华皮肤科杂志

Objective To investigate specific immune responses in mice induced by recombinant major outer membrane protein(rMOMP)of C.trachomatis serovaf E.Methods Thirty-six female BALB/cmice aged 3 to 4 weeks Were divided into three groups.i.e.,adjuvant group vaccinated、with purified rMOMP and Freund's adjutant,solitary group vaccinated with rMOMP only and control group vaccinated with phosphate buffered saline(PBS).All the mice were intramuscularly vaccinated on week 0,2 and 4.Blood samples and vaginal washes were obtained from these mice on week 6,then,mice were challenged with elementary body(EB)of C.trachomatis serovar E at the footpad followed by the observation of delayed hypersensitivity.On week 7.mice were genitally infected with C.trachomatis EB;one week later,blood samples and vaginal washes were obtained again;six weeks later,spleen lymphocytes were isolated from the mice and stimulated bv C.trachomatis or ConA followed by the detection of cell proliferation with MTT assay.In vitro neutralization assay was also performed.ELISA was used to determine the titers of Chlamydia-specific IgO antibody in sera and IgA antibody in vaginal washes,as well as the level of IFN-γ in culture supernatant of lymphocytes and sefa of mice.Vaginal swabs were collected after genital challenge and subjected to C.trachomatis culture.Results The absorbance at 405 ms of Chlamydia-specific IgG antibody and proliferation index of lymphocytes were 0.641±0.059 and 5.085±1.291.respectively,in mice immunized with rMOMP and Frennd's adjuvant.significantly higher than those in mice immunized with rMOMP only(0.424±0.015 and 3.123 ±0.840.both P<0.05).The thickness of right hind footpad increased by 0.324±0.054 mm and 0.272±0.064 mm,respectively,in solitary group and adjuvant group,respectively,with significant difference between the two groups(P<0.05).A significant increase was also observed in the adjuvant group compared with the control group in the above three parameters(all P<0.01).Conclusion The rMOMP of C.trachomatis could efficiently induce Chlamydia-specific humoml and cellular immune responses in mice.

Regulation of melanin expression in both cell and gene by retinoic acid / 中华医学美学美容杂志

Objective To investigate the effects and mechanism of retinoie acid on B16 marina mel-anoma cells and human melanocytes in vitro. Methods B16F10 murine melanoma cells and human mela-noeytes were cultured in culture medium which contains different concentration of components, including retinoic acid. Using reverse transcription-polymerase chain reaction (RT-PCR) mRNA expression of the tyrosinase was detected. Tyrosinase activity, melanin content and cell proliferation rate were also deter-mined. Results Retinoieacid exhibited an inhibitory effect on the expression of tyrosinase mRNA. As the concentration of retinoic acid was 100 μmol/L, treating for 72 h, the expression of tyrosinase mRNA de-creased 30.13 %, retinoic acid exhibited an inhibitory effect on tyrosinase activity and melanin production at high concentration (>500 μmol/L), and it could promote the cell proliferation. Retinoic acid and hy-droqninone could be cooperative at high concentration (1 000 μmol/L), and enhanced the down regulation of tyrosinase activity and melanin content. Retinoic acid could also mitigate the inhibitory effect of hydro-quinone on cell proliferation, so as to protect the cells from injury. Hydroquinone had no effect on tyrosi-nase gene expression at mRNA level. Conclusion Retinoic acid inhibits the synthesis of melanin by the genetic regulation at mRNA level.