Potential diagnostic value of miRNAs in sexually transmitted infections.

MiRNAs are small endogenous non-coding RNAs that have been demonstrated to be involved in post-transcriptional gene silencing, regulating a number of metabolic functions in the human body, including immune response, cellular physiology, organ development, angiogenesis, signaling, and other aspects. As popular molecules that have been studied in previous years, given their extensive regulatory functions, miRNAs hold considerable promise as non-invasive biomarkers. Sexually transmitted infections(STIs) are still widespread and have an adverse effect on individuals, communities, and society worldwide. miRNAs in the regulatory networks are generally involved in their molecular processes of formation and development. In this review, we discuss the value of miRNAs for the diagnosis of STIs.

Structural Changes and Very-Low-Frequency Nonlinear Dielectric Response of XLPE Cable Insulation under Thermal Aging.

The structural changes and very-low-frequency (VLF) nonlinear dielectric responses are measured to evaluate the aging state of cross-linked polyethylene (XLPE) in power cables under various thermal aging conditions. For this purpose, the accelerated thermal aging experiments were performed on XLPE insulation materials at 90 °C, 120 °C and 150 °C with different durations of 240 h, 480 h and 720 h, respectively. The Fourier transform infrared spectrum (FTIR) characterization and differential scanning calorimeter (DSC) were tested to analyze the influence of different aging on physicochemical properties of XLPE insulation. Besides, the VLF dielectric spectra show that the permittivity and dielectric loss change significantly in the VLF range from 1 mHz to 0.2 Hz. A voltage-current (U-I) hysteresis curve referring to a standard sinusoidal voltage and the response current were introduced to characterize the nonlinear dielectric properties of XLPE insulation caused by thermal aging.

Impact of off-hour admission on the MACEs of patients with acute myocardial infarction.

BACKGROUND: In China, on more than 100 weekends or holidays, only on-duty cardiologists are available during admissions. This study aimed to analyze the impact of admission time on major adverse cardiovascular events (MACEs) in patients with acute myocardial infarction (AMI). METHODS: This prospective observational study enrolled patients with AMI between October 2018 and July 2019. The patients were assorted into off-hour (admitted on weekends or national holidays) and on-hour groups. The outcome was MACEs at admission and 1 year after discharge. RESULTS: A total of 485 patients with AMI were enrolled in this study. The occurrence of MACEs was significantly higher in the off-hour group compared with the on-hour group (P < .05). Multivariate regression analysis showed that age (HR = 1.047, 95% CI: 1.021-1.073), blood glucose level (HR = 1.029, 95% CI: 1.009-1.050), multivessel disease (HR = 1.904, 95% CI: 1.074-3.375), and off-hour hospital admission (HR = 1.849, 95% CI: 1.125-3.039) were all independent risk factors for in-hospital MACEs, while percutaneous coronary intervention (HR = 0.210, 95% CI: 0.147-0.300) and on-hour admission (HR = 0.723, 95% CI: 0.532-0.984) were protective factors for MACEs 1 year after discharge. CONCLUSION: The "off-hour effect" still existed in patients with AMI, and the risk of MACEs in the hospital and 1 year after discharge was higher for off-hour admission.

Cold-Sintered ZnO Ceramic Composites Co-Doped with Polytetrafluoroethylene and Oxides.

Grain boundaries play a significant role in determining the performance of ceramic-based materials. The modulation of interfacial structures provides a promising approach to improve the physicochemical and electrical properties of ceramic materials. In this work, the grain boundary structures of ZnO-based ceramics were manipulated by incorporating polytetrafluoroethylene (PTFE) and metal oxides through the cold sintering process (CSP). It was found that the grain size of ZnO-based ceramics can be effectively reduced from 525.93 nm to 338.08 nm with an addition of PTFE and metal oxides of CoO and Mn2O3. Microstructural results show that most of the PTFE phase and metal oxides were distributed along the grain boundaries, which may lead to the increased grain boundary resistance from 1.59 × 106 ohm of pure ZnO to 6.21 × 1010 ohm of ZnO-based ceramics doped with PTFE and metal oxides, and enhanced Schottky barrier height from 0.32 eV to 0.59 eV. As a result, the breakdown field and nonlinear coefficient of the ZnO-based ceramics were improved to 3555.56 V/mm and 13.55, respectively. Therefore, this work indicates that CSP presents a feasible approach to design functional ceramic composites through the integration of polymer and metal oxides.

Treponema pallidum FlaA2 inducing the release of pro-inflammatory cytokines is mediated via TLR2 in keratinocytes.

BACKGROUND: Syphilis, caused by Treponema pallidum (T. pallidum), is a multi-organ, multiple systems, multi-stage sexually transmitted diseases with various clinical manifestations, among of which pathological lesions of skin and mucosa are the typical clinical manifestations of syphilis. However, the immunopathogenesis of this process is poorly understood. T. pallidum flagellin FlaA2, as a part of the important organelle responsible for the causative agent's motility, may contributes to the host skin inflammatory response. OBJECTIVES: To determine the mechanisms of T. pallidum FlaA2 stimulating the expression of pro-inflammatory cytokines in human keratinocytes. METHODS: Recombinant FlaA2 protein was performed to stimulate human keratinocytes. The mRNA transcription levels and protein expression levels of IL-6 and IL-8 were detected by qRT-PCR and ELISA, respectively. Western blot was used to detect the total protein and phosphorylation levels of ERK, p38, JNK and NF-κB, respectively. The intracellular location of NF-κB p65 was detected by immunofluorescence staining. RESULTS: Recombinant FlaA2 could considerably induced the expression of pro-inflammation cytokines IL-6 and IL-8 in HaCaT cells, and FlaA2-induced IL-6 and IL-8 secretion could be decreased by inhibiting TLR2 using pZERO-hTLR2. Further investigation showed that FlaA2 could activate the phosphorylation of ERK, p38 and IκBα and FlaA2-stimulated secretion of IL-6, IL-8 were attenuated by ERK, p38 and NF-κB inhibitors in HaCaT cells. Moreover, FlaA2 activates the ERK, p38 and NF-κB pathways through TLR2 signaling pathway in HaCaT cells. CONCLUSIONS: From the findings above, these results confirm that T. pallidum FlaA2 activates ERK, p38 and NF-κB signaling pathway through TLR2 pathway to induce the production of IL-6 and IL-8, which could contribute to enhance the understanding of the skin inflammatory response induced by the pathogen in syphilis patients.

Aptamers: A prospective tool for infectious diseases diagnosis.

It is well known that people's health is seriously threatened by various pathogens (such as Mycobacterium tuberculosis, Treponema pallidum, Novel coronavirus, HIV, Mucor, etc.), which leads to heavy socioeconomic burdens. Therefore, early and accurate pathogen diagnosis is essential for timely and effective therapies. Up to now, diagnosing human contagious diseases at molecule and nano levels is remarkably difficult owing to insufficient valid probes when it comes to determining the biological markers of pathogens. Aptamers are a set of high-specificity and high-sensitivity plastic oligonucleotides screened in vitro via the selective expansion of ligands by exponential enrichment (SELEX). With the advent of aptamer-based technologies, their merits have aroused mounting academic interest. In recent years, as new detection and treatment tools, nucleic acid aptamers have been extensively utilized in the field of biomedicine, such as pathogen detection, new drug development, clinical diagnosis, nanotechnology, etc. However, the traditional SELEX method is cumbersome and has a long screening cycle, and it takes several months to screen out aptamers with high specificity. With the persistent development of SELEX-based aptamer screening technologies, the application scenarios of aptamers have become more and more extensive. The present research briefly reviews the research progress of nucleic acid aptamers in the field of biomedicine, especially in the diagnosis of contagious diseases.

A Promising Tool in Serological Diagnosis: Current Research Progress of Antigenic Epitopes in Infectious Diseases.

Infectious diseases, caused by various pathogens in the clinic, threaten the safety of human life, are harmful to physical and mental health, and also increase economic burdens on society. Infections are a complex mechanism of interaction between pathogenic microorganisms and their host. Identification of the causative agent of the infection is vital for the diagnosis and treatment of diseases. Etiological laboratory diagnostic tests are therefore essential to identify pathogens. However, due to its rapidity and automation, the serological diagnostic test is among the methods of great significance for the diagnosis of infections with the basis of detecting antigens or antibodies in body fluids clinically. Epitopes, as a special chemical group that determines the specificity of antigens and the basic unit of inducing immune responses, play an important role in the study of immune responses. Identifying the epitopes of a pathogen may contribute to the development of a vaccine to prevent disease, the diagnosis of the corresponding disease, and the determination of different stages of the disease. Moreover, both the preparation of neutralizing antibodies based on useful epitopes and the assembly of several associated epitopes can be used in the treatment of disease. Epitopes can be divided into B cell epitopes and T cell epitopes; B cell epitopes stimulate the body to produce antibodies and are therefore commonly used as targets for the design of serological diagnostic experiments. Meanwhile, epitopes can fall into two possible categories: linear and conformational. This article reviews the role of B cell epitopes in the clinical diagnosis of infectious diseases.

Identification of key genes and pathways in syphilis combined with diabetes: a bioinformatics study.

We noticed that syphilis patients seem to be more susceptible to diabetes and the lesions often involve the kidneys, but the pathogenesis is not yet completely understood. In this study, microarray analysis was performed to investigate the dysregulated expressed genes (DEGs) in rabbit model of syphilis combined with diabetes. A total of 1045 genes were identified to be significantly differentially expressed, among which 571 were up-regulated and 474 were down-regulated (≥ 2.0fold, p < 0.05). Using the database visualization and integration discovery for the Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analysis. The downregulated DEGs were significantly enriched for biosynthesis of antibiotics, carbon metabolism and protein digestion, while the upregulated DEGs were mainly enriched for cancer and PI3K-Akt signaling pathway. Molecular Complex Detection (MCODE) plugins were used to visualize protein-protein interaction (PPI) network of DEGs and Screening for hub genes and gene modules. ALB, FN1, CASP3, MMP9, IL8, CTGF, STAT3, IGF1, VCAM-1 and HGF were filtrated as the hub genes according to the degree of connectivity from the PPI network. To the best of our knowledge, this study is the first to comprehensively identify the expression patterns of dysregulated genes in syphilis combined with diabetes, providing a basis for revealing the underlying pathogenesis of syphilis combined with diabetes and exploring the goals of therapeutic intervention.

Laboratory Diagnostic Tools for Syphilis: Current Status and Future Prospects.

With the increasing number of patients infected with syphilis in the past 20 years, early diagnosis and early treatment are essential to decline syphilis prevalence. Owing to its diverse manifestations, which may occur in other infections, the disease often makes clinicians confused. Therefore, a sensitive method for detecting T. pallidum is fundamental for the prompt diagnosis of syphilis. Morphological observation, immunohistochemical assay, rabbit infectivity test, serologic tests, and nucleic acid amplification assays have been applied to the diagnosis of syphilis. Morphological observation, including dark-field microscopy, silver-staining, and direct fluorescent antibody staining for T. pallidum, can be used as a direct detection method for chancre specimens in primary syphilis. Immunohistochemistry is a highly sensitive and specific assay, especially in the lesion biopsies from secondary syphilis. Rabbit infectivity test is considered as a sensitive and reliable method for detecting T. pallidum in clinical samples and used as a historical standard for the diagnosis of syphilis. Serologic tests for syphilis are widely adopted using non-treponemal or treponemal tests by either the traditional or reverse algorithm and remain the gold standard in the diagnosis of syphilis patients. In addition, nucleic acid amplification assay is capable of detecting T. pallidum DNA in the samples from patients with syphilis. Notably, PCR is probably a promising method but remains to be further improved. All of the methods mentioned above play important roles in various stages of syphilis. This review aims to provide a summary of the performance characteristics of detection methods for syphilis.

A comparison of genotyping tool in Treponema pallidum: Review and meta-analysis.

BACKGROUND: To decipher the molecular epidemiology of the Treponema pallidum subspecies, pallidum, researchers have developed different molecular typing schemes which identify strains type from clinical specimens. However, the results of these studies show remarkable diversity. METHODS: We searched for literature in PubMed, MEDLINE, Web of Sciences, and OVID from January 1998 to January 2019, in order to compare the efficiency of typing schemes using published evidence for systematic reviews and meta-analyses. RESULTS: From the 43 studies included, the overall typing efficiency of Treponema pallidum was 71.4% (95% CI: 63.2-78.9%). Subgroup analyses indicated that the typing efficiency of CDC-typing (CDCT, 68.2%, 95% CI: 53.6-81.2%) was worse than those of enhanced CDC-typing (ECDCT, 72.3%, 95% CI: 60-83.1%), CDC-rspA (81.6%, 95% CI: 76.1-86.6%), multi-locus sequence typing (MLST, 67.1%, 95% CI: 61.1-72.7), and sequencing-based molecular typing (SBMT, 71.6%, 95% CI: 50-89.2%). A limitation of this review is that the studies included employed different criteria to collect and investigate samples of Treponema pallidum, which could contribute to heterogeneity. CONCLUSIONS: This analysis suggests that CDCT is an inferior scheme in molecular typing, the discriminatory power was very similar for ECDCT and SBMT. Other factors contributing to the heterogeneity between typing studies warrants further study.

The N-terminal D1 domain of Treponema pallidum flagellin binding to TLR5 is required but not sufficient in activation of TLR5.

Syphilis is a chronic bacterial infection caused by Treponema pallidum (T pallidum) and the pathogenesis that T pallidum infection induces immunopathological damages in skin and other tissues remains unclear. We have previously reported that recombinant flagellins of T pallidum can elicit IL-6 and IL-8 transcriptions via TLR5 pathway. To identify the domains which induced the pro-inflammatory activity and the importance of the interactions between TLR5 and domains, homology-based modelling and comparative structural analyses revealed that Tpflagellins can combine with TLR5 directly. Deletion mutations showed that the ND1 domain binding to TLR5 is required but not sufficient in TLR5 activation. Moreover, site-directed mutagenesis analysis indicated that the arginine residue (Tpflagellins R89) of the ND1 domain and its adjacent residues (Tpflagellins L93 and E113) constitute a hot spot that elicits IL-6, IL-8 transcriptions and TLR5 activation, and affects the binding of Tpflagellins to TLR5. Taken together, these results give insight into the pathogenesis of T pallidum and may contribute to the future design of Tpflagellins-based therapeutics and syphilis vaccine.

Performance of novel infection phase-dependent antigens in syphilis serodiagnosis and treatment efficacy determination.

OBJECTIVE: The objective of this study was to screen new antigens for syphilis serodiagnosis. METHODS: First, we determined whether the Treponema pallidum proteins Tp0971, Tp0768 and Tp0462 were infection phase-dependent antigens by observing serum reactivity differences in New Zealand rabbits infected with activated or inactivated T. pallidum. A non-infection phase-dependent antigen, the Tp92 membrane protein, was used as the negative control. Next, Tp0971-, Tp0768- and Tp0462-based ELISA was performed on 2138 human serum samples and compared with the T. pallidum passive particle agglutination assay (TPPA) and LiZhu™ Tp-ELISA. In addition, another 60 paired serum samples from patients at follow-up were analysed to evaluate the relationships between titre changes and differences in the A450 nm values of the Tp0971, Tp0768, Tp0462 and Tp92 antibodies measured by ELISA. RESULTS: Compared with Tp92 (negative control), Tp0971, Tp0768 and Tp0462 were determined to be infection phase-dependent antigens. Compared with those of the TPPA, the sensitivities of Tp0971-, Tp0768- and Tp0462-based ELISA were 96.4%, 96.9% and 93.0%, respectively, and the specificities were 97.7%, 95.4% and 98.9%, respectively, resulting in consistencies of 97.1%, 96.2% and 95.9%, respectively. Compared with those of the LiZhu™ Tp-ELISA, the consistencies of Tp0971-, Tp0768- and Tp0462-based ELISA were 95.1%, 94.2% and 94.0%, respectively, with kappa values of 0.902, 0.884 and 0.880, respectively. Tp0971, Tp0768 and Tp0462 demonstrated high sensitivities and specificities, as well as high conformity to the TPPA and LiZhu™ Tp-ELISA. Moreover, a significantly positive Spearman rank correlation coefficient (0.82,*P < 0.05) was found between the difference in the A450 nm values of the Tp0971 antibody and the RPR titre change. CONCLUSION: The infection phase-dependent antigens Tp0971, Tp0768 and Tp0462 are promising for syphilis diagnosis, and Tp0971 may be utilized to monitor curative effects during syphilis treatment.

Immunogenicity and protective efficacy against Treponema pallidum in New Zealand rabbits immunized with plasmid DNA encoding flagellin.

Plasmid DNA encoding flagellin FlaB3 was used as a vaccination candidate for the evaluation of immunogenicity and protection against Treponema pallidum subsp. pallidum dissemination. First, intramuscular injection of the flagellin encoded by the plasmid DNA into New Zealand rabbits elicited both humoral and cellular immune responses. Total IgG production increased in response to flagellin. In addition, serum IFN-γ secretion and CD8+ cells were substantially greater in the rabbits immunized with the plasmid encoding flagellin FlaB3 than those in the rabbits immunized with recombinant flagellin. The flagellin encoded by the plasmid DNA induced significant upregulation of serum IL-6 and IL-8 compared to that of the control rabbits. Subsequently, intradermal challenge of the vaccinated New Zealand rabbits with 1 × 107T. pallidum resulted in a significant reduction of the bacterial organ burden in the blood, liver, spleen, and testicles in the flagellin plasmid DNA-vaccinated rabbits. Furthermore, the histopathological analysis demonstrated that the rabbits immunized with the plasmid DNA-encoded flagellin (FlaB3) showed better immune protection. These findings provide evidence that plasmid DNA-encoded flagellin (FlaB3) may be useful as a potential immunization route for future development of a vaccine to inhibit T. pallidum dissemination in related animals.

Intramuscular primary immunization by nucleic acid vaccine pcDNA/Gpd-IL-2 and enhanced immunization with mucosal adjuvant CpG-ODN and Gpd-IL-2 recombinant protein effectively induced strong mucosal immune responses and immune protective effects against Treponema pallidum skin infection.

The present study aimed to evaluate the immune effect of intramuscular primary immunization by the nucleic acid vaccine pcDNA/glycerophosphodiester phosphodiesterase-interleukin-2 (pcDNA/Gpd-IL-2) and enhanced immunization 2 weeks later with the combination of mucosal adjuvant CpG-oligodeoxynucleotides (ODN) and Gpd-IL-2 recombinant protein on skin infection caused by Treponema pallidum (Tp) in New Zealand rabbits. At week 8 following immunization, MTT assay was used to detect spleen cell proliferation, while enzyme-linked immunosorbent assay was performed to detect the cytokine and secretory immunoglobulin A (SIgA) levels. At week 10 after primary immunization, rabbits were inoculated with 105 Tp (Nichols strain). Alterations in the skin redness, swelling and ulceration were recorded for 0-60 days. In addition, positive rate of Tp in skin lesions and ulcer formation rate were examined using dark field and silver staining. The results indicated that intramuscular primary immunization by nucleic acid vaccine pcDNA/Gpd-IL-2 followed by enhanced immunization via nasal feeding with mucosal adjuvant CpG-ODN and Gpd-IL-2 recombinant protein induced the higher levels of Tp Gpd specific antibodies, increased the secretion of IL-2 and interferon-γ, and promoted the proliferation of T cells in the first 8 weeks after immunization. Furthermore, this immunization strategy stimulated the production of mucosa specific SIgA antibody. Thus, this strategy led to the lowest Tp positive and ulcer formation rates at the Tp infection sites, as well as healing of skin lesions on the earliest time point (day 42). In conclusion, immunization by nucleic acid vaccine pcDNA/Gpd-IL-2 followed by enhanced immunization with a combination of mucosal adjuvant CpG-ODN and Gpd-IL-2 recombinant protein is an effective immune strategy to induce strong mucosal immune responses and immune protective effects.

The outer membrane protein Tp92 of Treponema pallidum induces human mononuclear cell death and IL-8 secretion.

Treponema pallidum is the pathogen that causes syphilis, a sexually transmitted disease; however, the pathogenic mechanism of this organism remains unclear. Tp92 is the only T. pallidum outer membrane protein that has structural features similar to the outer membrane proteins of other Gram-negative bacteria, but the exact functions of this protein remain unknown. In the present study, we demonstrated that the recombinant Tp92 protein can induce human mononuclear cell death. Tp92 mediated the human monocytic cell line derived from an acute monicytic leukemia patient (THP-1) cell death by recognizing CD14 and/or TLR2 on cell surfaces. After the stimulation of THP-1 cells by the Tp92 protein, Tp92 may induce atypical pyroptosis of THP-1 cells via the pro-caspase-1 pathway. Meanwhile, this protein caused the apoptosis of THP-1 cells via the receptor-interacting protein kinase 1/caspase-8/aspase-3 pathway. Tp92 reduced the number of monocytes among peripheral blood mononuclear cells. Interestingly, further research showed that Tp92 failed to increase the tumour necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-10, IL-18 and monocyte chemotactic protein 1 (MCP)-1 levels but slightly elevated the IL-8 levels via the Nuclear Factor (NF)-κB pathway in THP-1 cells. The data suggest that Tp92 recognizes CD14 and TLR2, transfers the signal to a downstream pathway, and activates NF-κB to mediate the production of IL-8. This mechanism may help T. pallidum escape recognition and elimination by the host innate immune system.

Screening and identification of immunoactive FlaB protein fragments of Treponema pallidum for the serodiagnosis of syphilis.

Flagellin is a classical pathogen-associated molecular pattern that can evoke a robust immune response. We have demonstrated previously that three full-length flagellins of Treponema pallidum, namely FlaB1, FlaB2 and FlaB3, did have diagnostic value in the serodiagnosis of syphilis. Here, we selected and constructed three recombinant fragments of each complete FlaB, both the conserved N-terminal and the C-terminal region, and the middle variable part, with the goal of exploring fragments unique to Treponema pallidum for use as antigen targets in a fragment-based serological test. The diagnostic performance of fragments was evaluated using different panels of serum specimens (= 332) by indirect IgG enzyme-linked immunosorbent assay. The data showed that all the conserved fragments exhibited excellent sensitivities (91.1-95.0%) but poor specificities (64.1-78.4%), while the three middle regions demonstrated higher sensitivities and specificities for detecting IgG antibody, with 92.7% and 96.1% for FlaB1M ('B1M'), 91.6% and 94.8% for B2M, and 95.0% and 100% for B3M, respectively. In comparison, the sensitivity and specificity of Architect Syphilis TP was found to be 95.5% and 94.8%, respectively. These findings revealed that the middle portion of each FlaB had epitopes specific for Treponema pallidum and identified B3M as a promising candidate antigen for the serodiagnosis of syphilis.

Treponema pallidum flagellins stimulate MMP-9 and MMP-13 expression via TLR5 and MAPK/NF-κB signaling pathways in human epidermal keratinocytes.

Syphilis is a chronic disease caused by Treponema pallidum and the pathogenesis is still unclear. T. pallidum infection induced inflammatory responses are involved in the immunopathological damage in skin and other tissues. Flagellin, the monomeric subunit of bacterial flagella, is a classic pathogen associated molecular patterns (PAMPs) that interacts to TLR5 and induces inflammatory responses. Keratinocytes, as immune sentinels recognize the PAMPs via TLRs, play an important role in skin innate immune response. Matrix metalloproteinases (MMPs) expressed by keratinocytes are involved in skin inflammatory responses and promoting pathogens invasion. In this study, we demonstrate that FlaB1, FlaB2 and FlaB3, the flagellins of T. pallidum, induced MMP-9 and MMP-13 production in human immortalized keratinocytes cell line HaCaT. Silencing of TLR5, but not TLR2 and TLR4 attenuated MMP-9 and MMP-13 expressions induced by T. pallidum flagellins. MMP-9 and MMP-13 expressions were also be abrogated by transfection with a dominant negative (DN) plasmid of MyD88. We also found that treatment of HaCaT cells with FlaB1, FlaB2 and FlaB3 activate the MAPK and NF-κB signaling pathways. Inhibited of ERK, JNK, p38 and NF-κB suppressed MMP-9 expression induced by the FlaB1. MMP-13 expression was found to be suppressed by pretreatment with inhibitors of ERK, JNK and NF-κB, but not p38. These findings demonstrate that T. pallidum flagellins (FlaB1, FlaB2 or FlaB3) can stimulate MMP-9 and MMP-13 expression through TLR5 and MAPK/NF-κB signaling pathways in human epidermal keratinocytes, which could contribute to the pathogenesis of T. pallidum infection.

Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for the Detection of Treponema pallidum DNA in the Peripheral Blood of Secondary Syphilis Patients.

Secondary syphilis (SS) has always been puzzling for the clinicians because of the similarity of the appearance of skin rashes with other dermatoses. Serological assays are useful, but less sensitive at an early stage of SS or when patients are immunodeficient. Therefore, there is an urgent need to develop a rapid and effective tool for the diagnosis of SS, which may play an important role in the control of epidemic syphilis outbreaks. In this study, we evaluated a loop-mediated isothermal amplification (LAMP) assay, targeting gene encoding the basic membrane protein of Treponema pallidum, to detect the presence of circulating T. pallidum DNA in the blood of SS patients. The specificity of LAMP was validated using three strains of Spirochaetales and six common clinical bacteria. The clinical applicability of LAMP assay was assessed using 642 blood samples from clinically suspected SS patients and 80 samples from healthy blood donors, showing a sensitivity of 82.1% and a specificity of 100.0% in the diagnosis of SS. Thus, our results indicate that the LAMP can be used as a supplementary method for the diagnosis of SS.

Treponema pallidum flagellins elicit proinflammatory cytokines from human monocytes via TLR5 signaling pathway.

The tissue damage caused by syphilis infection may be associated with inflammation. However, the virulence factors of Treponema pallidum are still unclear, nor are the molecular mechanisms for leading to the productions of proinflammatory cytokines. Flagellin, a classic pathogen-associated molecular pattern (PAMP), is a potent immunogen that induces inflammation. In the present study, we have demonstrated that stimulations of human monocytes with Treponema pallidum FlaB1, FlaB2, and FlaB3 result in the up regulation of interleukin (IL)-6 and IL-8. Moreover, silencing of the Toll-like receptor 5 (TLR5) gene by using small interfering RNA was found to abrogate the T. pallidum flagellins-induced IL-6 and IL-8 expressions. Similarly, transfection with the dominant negative plasmid encoding MyD88 (pDeNy-hMyD88) was also giving rise to the down regulation of IL-6 and IL-8. We further investigated the relative contributions of mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling to transcriptions and translations of IL-6 and IL-8. Western Blot and immuno fluorescence experiments revealed that flagellins-mediated IL-6 and IL-8 expressions are heavily dependent on ERK, p38, and NF-κB. In addition, inhibitions of p38 kinase, ERK, and NF-κB were found to attenuate the productions of IL-6 and IL-8. Taken together, our results indicate that T. pallidum flagellins can upregulate IL-6 and IL-8 generations via TLR5 and MAPK/NF-κB signaling pathways in THP-1 cells, which will improve our understanding of the pathogenesis of T. pallidum.

Treponema pallidum flagellin FlaA2 induces IL-6 secretion in THP-1 cells via the Toll-like receptor 2 signaling pathway.

Treponema pallidum subsp. pallidum membrane proteins are considered as potent inducers in the initiation and development of inflammation. In the present study, the mechanism that leads to the production of interleukin 6 (IL-6), one of the key proinflammatory cytokines, by human monocytic THP-1 cells when these cells are treated with T. pallidum flagellin FlaA2 was investigated. Stimulation with flagellin FlaA2 can induce IL-6 expression in human monocytes and augment the phosphorylation of ERK, p38, and NF-κB, but has no effect on the phosphorylation of JNK. Likewise, FlaA2-induced IL-6 production was found to be attenuated by inhibitors for ERK, p38, and NF-κB, but not by JNK inhibitor. Immunofluorescence analysis showed that flagellin FlaA2 could stimulate the translocation of IκBα from the cytosol to the nucleus, and this phenomenon could be inhibited by the specific inhibitor BAY11-7082. FlaA2-induced IL-6 expression was also proved to be abrogated by transfection with dominant negative (DN) plasmid of MyD88. We further demonstrated that transfection with DN-TLR2 was sufficient to attenuate IL-6 expression and the phosphorylation of ERK, p38, and IκBα. These results suggest that flagellin FlaA2 induces IL-6 production via signaling pathways involving TLR2, MyD88, ERK, p38, and NF-κB in monocytes, which could contribute to the pathogenesis of T. pallidum.