The Interaction Of Treponema Pallidum With Host Immunity And Evaluation Of Ceftriaxone In Treatment Of Early Syphilis

Section 1 A preliminary study on signal pathways for TP0126 induces pro-inflammatory cytokines production in human THP-1 macrophage-like cellsObjective:To explore potential pro-inflammatory activity of recombinant TP0126 protein and the signal pathways of TP0126.Methods:Recombinant TP0126 was expressed in E.coli expression system.Varied concentrations of TP0126 were used to stimulate human THP-1 macrophage-like cells at different time.ELISA was used to detect the production of TNF-α,IL-1β,and IL-6 for identifying the pro-inflammatory activity of TP0126.TP0126 was used to stimulate the target cells pre-treated with PDTC and/or SB202190,which are specific inhibitors of NF-κB and p38MAPK pathways respectively,then ELISA was employed to detect the expression of TNF-α,IL-1β,and IL-6.Furthermore,western blot was used to detect the expression of phosphorylated IκBα and phosphorylated p38 in the NF-κB and p38MAPK pathways respectively before and after inhibition for identifying its signal pathways.Results:Recombinant TP0126 protein was successfully expressed and purified.TP0126 was able to induce human THP-1 macrophage-like cells to produce TNF-α,IL-1β and IL-6.The optimal stimulatory concentration of TP0126 was 5 μg/ml.The optimal stimulatory time for TNF-a and IL-6 production was 12 h,and for IL-1β was 24 h.PDTC and SB202190 could significantly inhibit the expression of TNF-α,IL-1β,and IL-6 induced by TP0126 in target cells.The expression of phosphorylated IKBa and phosphorylated p38 protein reached a peak at 60 min after stimulation with TP0126 in human THP-1 macrophage-like cells.After pretreatment with PDTC and/or SB202190,the expression of phosphorylated IKBα and phosphorylated p38 protein were significantly reduced respectively.Conclusion:TP0126 has pro-inflammatory activity and could be able to induce human THP-1 macrophage-like cells to produce TNF-α,IL-1β and IL-6 in vitro,which is regulated by NF-κB and p38MAPK signal pathways.Section 2 Dynamic observ ation on Treponema pallidum load and host immune response in peripheral blood of experimental syphilitic rabbitsObjective:To observe the occurrence of bacteremia in rabbits infected with Treponema pallidum(Tp)and to investigate whether Thl/Th2 and Th17/Treg in peripheral blood are involved in the immune response of early syphilis.Methods:Virulent Tp or heat-killed Tp(HKTP)was inoculated into the testes and veins to construct animal models.The inoculated rabbits were divided into TP testicular test group,HKTP testicular test group,HKTP venous test group and normal control group,with 6 rabbits in each group.Real-time fluorescence quantitative PCR was used to detect the Tp DNA load in peripheral blood from pre-inoculation to post-inoculation 1,3,7,14,28,and 42 days in all rabbits;Using RPR and TPPA to detect the development of humoral immunity during infection;on the 42nd day after innoculation(when orchitis was completely subsided),pathological changes of different organs of rabbits in each group were examined by histopathology.In the observation period,the expression of IL-6,IL-10,IL-17,IFN-γ,RORγt,TGF-γ and Foxp3 mRNA in PBMC were detected by RT-qPCR from pre-infection to post-infection 14,28,and 42 days in TP testicular test group.Furthermore,the expression of IL-6,IL-10,IL-17,IFN-y and TGF-P protein were detected by ELISA.Results:In the TP testicular test group,orchitis and high titer serological reaction occurred at 7 days after inoculation,orchitis peaked at 14 days.When orchitis completely subsided at 42 days,mild pathological changes can be found in heart,liver,kidney,teste and even brain.In HKTP testicular test group and HKTP venous test group,rabbits did not show any clinical presentations,but seroconversion appeared.The titer of TPPA and RPR were significantly lower than those in TP testicular test group(p<0.01),and the RPR titer kept short duration.In the TP testicular test group,Tp DNA was detected in peripheral blood at 1,3,7 and 14 days after inoculation.The Tp DNA load peaked at 14 days,hereafter Tp DNA were not detected again;In HKTP testicular test group,we failed to found the Tp DNA at all times;whereas in the HKTP venous test group,Tp DNA were detected in all.6 rabbits at 1 day after inoculation(2903-6748 copies/ml).The expression of IFN-γ,IL-6,IL-10,TGF-β,Foxp3,IL-17.RORyt mRNA in PBMC of rabbits in the TP testicular test group at 14 days,28 days and 42 days after inoculation were not statistically different from that at pre-infection.Furthermore,the expression of IFN-y,IL-6,IL-10,TGF-P,IL-17 protein in plasma of rabbits in the TP testicular test group at 14 days,28 days and 42 days after inoculation has no significant difference from that at pre-infection.Conclusion:Before the local immune response occurs,low load of Tp bacteremia can be found,causing infection at distant sites and causing mild pathological changes.After local immune and humoral immune are established,bacteremia can still be detected.But we neither detected significant changes in expression of IFN-γ,IL-6,IL-10,TGF-β,Foxp3,IL-17,RORyt mRNA in PBMC,nor in expression of IFN-γ,IL-6,IL-10,TGF-β,IL-17 protein in plasma,suggesting that Thl,Th2,Treg,Thl7 are not involved in the immune response of early syphilis.Section 3 Comparison of efficacy of treatments for early syphilis:a systematic review and meta-analysisObjective:To assess the efficacy of ceftriaxone and doxycycline/tetracycline in treating early syphilis relative to that of penicillin,and thereby to determine which antibiotic is a better replacement for penicillin.Methods:By searching literatures from PubMed,Cochrane Central Register of Controlled Trials,Embase,the Web of Science,and ClinicalTrials.gov and systematically screening relevant studies,eligible randomized controlled trials(RCTs)and observational studies on treatments with penicillin,doxycycline/tetracycline,and ceftriaxone for early syphilis were identified and combined in this systematic review.Estimated risk ratios(RRs)and 95%confidence intervals(CIs)were utilized to compare their serological response and treatment failure rates.At 12-month follow up,serological response rates were compared by a direct meta-analysis and network meta-analysis(NMA),while treatment failure rates were compared with a direct meta-analysis.Results:Three RCTs and seven cohort studies were included in this research.The results of NMA demonstrated that no significant differences existed in serological response rate at 12-month follow-up between any two of the three treatments(doxycycline/tetracycline vs.penicillin RR=1.01,95%CI 0.89-1.14;ceftriaxone vs.penicillin RR=1.00,95%CI 0.89-1.13;ceftriaxone vs.doxycycline/tetracycline RR=0.99,95%CI 0.96-1.03),which was consistent with the outcomes of the direct meta-analysis.In addition,the direct meta-analysis indicated that,at 12-month follow-up,penicillin and ceftriaxone treatment groups had similar treatment failure rates(RR=0.92,95%CI 0.12-6.93),while treatment failure rate was significantly lower among penicillin recipients than among doxycycline/tetracycline recipients(RR=0.58,95%CI 0.38-0.89).Conclusion:Ceftriaxone is as effective as penicillin in treating early syphilis with regard to serological response and treatment failure rate.Compared with doxycycline/tetracycline,ceftriaxone appears to be a better choice as the substitution of penicillin.A careful follow-up should be conducted for patients receiving doxycycline/tetracycline so as to identify treatment failure early.Section 4 Evaluation of the efficacy of different doses of ceftriaxone in treatment of experimental syphilis in rabbitsObjective:To assess the efficacy of different doses of ceftriaxone for experimental syphilis.Methods:Experimental spirochete infections were established by intratesticular inoculation in rabbits.Treatment was initiated at 2 weeks after inoculation.There were five experimental groups each comprising three animals;four groups were inoculated,which received intramuscular injection of 100,000 u/kg of benzathine penicillin G weekly for 2 weeks,intramuscular injection of 42.0 or 4.2 mg/kg of ceftriaxone daily for 10 days,or no treatment.The other group was not inoculated,constituting a normal control.The efficacy was evaluated using the mean time of orchitis healing,seronegative conversion,and disappearance of spirochete DNA from tissues.Results:Strikingly different outcomes were noted between the untreated and three treated groups,while no discernible differences existed among the three treated groups.In all treated groups,orchitis healed shortly after the initiation of therapy(5 day),nontreponemal antibody titers decreased rapidly and converted negative,and no spirochete DNA were detected in brain,heart,liver,spleen,kidney and testis.On the other hand,in the untreated control group,the regression of orchitis occurred from 21 to 28 days after the initiation of therapy;nontreponemal antibody titers remained positive,standing at a higher level(>1:8)at 98 days after the end of treatment;and spirochete DNA were found in testes of all rabbits with the mean value of 3710 copies/μg.Conclusion:The efficacy of ceftriaxone is remarkable for early experimental syphilis.The low dose of ceftriaxone is shown to be as effective as its high dose and benzathine penicillin G,which could cure the early syphilis.