Treponema pallidum membrane protein Tp47 induced autophagy and inhibited cell migration in HMC3 cells via the PI3K/AKT/FOXO1 pathway.

The migratory ability of microglia facilitates their rapid transport to a site of injury to kill and remove pathogens. However, the effect of Treponema pallidum membrane proteins on microglia migration remains unclear. The effect of Tp47 on the migration ability and autophagy and related mechanisms were investigated using the human microglial clone 3 cell line. Tp47 inhibited microglia migration, the expression of autophagy-associated protein P62 decreased, the expression of Beclin-1 and LC3-II/LC3-I increased, and the autophagic flux increased in this process. Furthermore, autophagy was significantly inhibited, and microglial cell migration was significantly increased after neutralisation with an anti-Tp47 antibody. In addition, Tp47 significantly inhibited the expression of p-PI3K, p-AKT, and p-mTOR proteins, and the sequential activation of steps in the PI3K/AKT/mTOR pathways effectively prevented Tp47-induced autophagy. Moreover, Tp47 significantly inhibited the expression of p-FOXO1 protein and promoted FOXO1 nuclear translocation. Inhibition of FOXO1 effectively suppressed Tp47-induced activation of autophagy and inhibition of migration. Treponema pallidum membrane protein Tp47-induced autophagy and inhibited cell migration in HMC3 Cells via the PI3K/AKT/FOXO1 pathway. These data will contribute to understanding the mechanism by which T. pallidum escapes immune killing and clearance after invasion into the central nervous system.

Red Blood Cell Lysis Pretreatment Can Significantly Improve the Yield of Treponema pallidum DNA from Blood.

PCR can be a supplement to Treponema serological testing. However, its sensitivity is not satisfactory for blood sample testing. The aim of this study was to investigate whether pretreatment with red blood cell (RBC) lysis could enhance the yield of Treponema pallidum subsp. pallidum DNA extraction from blood. We developed and verified the efficacy of a quantitative PCR (qPCR) assay that utilizes TaqMan technology to specifically detect T. pallidum DNA by targeting the polA gene. Simulation media with 106 to 100 treponemes/mL were prepared in normal saline (NS), whole blood, plasma, and serum, and RBC lysis pretreatment was performed on a portion of whole blood. Then, blood samples drawn from 50 syphilitic rabbits were divided in parallel into five groups, labeled whole blood, whole blood/lysed RBCs, plasma, serum, and blood cells/lysed RBCs. DNA extraction and qPCR detection were performed. The detection rate and copy number were compared among different groups. The polA assay showed good linearity and an excellent amplification efficiency of 102%. In the simulated blood samples, the detection limit of the polA assay reached 1 × 102 treponemes/mL in whole blood/lysed RBCs, plasma, and serum. However, the detection limit was only 1 × 104 treponemes/mL in NS and whole blood. Among the blood samples from syphilitic rabbits, whole blood/lysed RBCs showed the best detection rate (82.0%), while the detection rate for whole blood was only 6%. The copy number of whole blood/lysed RBCs was higher than that of whole blood. RBC lysis pretreatment can significantly improve the yield of T. pallidum DNA from whole blood, and the yield is better than that from whole blood, plasma, serum, and blood cells/lysed RBCs. IMPORTANCE Syphilis is a sexually transmitted disease caused by T. pallidum that can spread into the blood. T. pallidum DNA can be detected in blood by PCR but with low sensitivity. Few studies have applied RBC lysis pretreatment to blood T. pallidum DNA extraction. This study shows that the detection limit, detection rate, and copy number of whole blood/lysed RBCs were better than those of whole blood, plasma, and serum. After RBC lysis pretreatment, the yield of low concentrations of T. pallidum DNA was improved, and the low sensitivity of blood-based T. pallidum PCR was improved. Therefore, whole blood/lysed RBCs are the ideal sample for acquiring blood T. pallidum DNA.

Serum Ubiquitin C-Terminal Hydrolase-L1, Glial Fibrillary Acidic Protein, and Neurofilament Light Chain Are Good Entry Points and Biomarker Candidates for Neurosyphilis Diagnosis Among Patients Without Human Immunodeficiency Virus to Avoid Lumbar Puncture.

BACKGROUND: Laboratory tests to diagnose neurosyphilis using cerebrospinal fluid (CSF) are currently disadvantageous as a lumbar puncture is required, which may result in patients with neurosyphilis missing an opportunity for early diagnosis. Thus, blood biomarker candidates that are more convenient and minimally invasive to collect for diagnosing neurosyphilis is urgently needed. METHODS: This observational study aimed to analyze serum ubiquitin C-terminal hydrolase-L1 (UCH-L1), glial fibrillary acidic protein (GFAP), and neurofilament light chain (NF-L) levels in 153 patients without human immunodeficiency virus (HIV) and to evaluate their diagnostic performance in neurosyphilis compared with CSF. RESULTS: Serum UCH-L1, GFAP, and NF-L levels were significantly higher in patients with neurosyphilis compared with patients with uncomplicated syphilis or non-syphilis. For the diagnosis of neurosyphilis, serum UCH-L1, GFAP, and NF-L revealed sensitivities of 90.20%, 80.40%, and 88.24%, and specificities of 92.16%, 78.43%, and 80.39%, respectively, at cutoff levels of 814.50 pg/mL, 442.70 pg/mL, and 45.19 pg/mL, respectively. In patients with syphilis, serum UCH-L1, GFAP, and NF-L levels correlated strongly or moderately with those in the CSF, with similar or better diagnostic performance than those in the CSF. The testing algorithms' sensitivity and specificity increased to 98.04% and 96.08%, respectively, when subjected to parallel and combination testing, respectively. CONCLUSIONS: To avoid lumbar puncture, each serum UCH-L1, GFAP, and NF-L is a good entry point and biomarker candidate for the diagnosis of neurosyphilis among patients without HIV. These proteins used in concerto can further improve the diagnostic sensitivity and specificity.

Treponema pallidum protein Tp0136 promoting MMPs/TIMPs imbalance via PI3K, MAPK and NF-κB signalling pathways in HDVSMCs.

The invasive capability of Treponema. pallidum is central to its infection process. Matrix metalloproteinases (MMPs), which are specifically inhibited by the tissue inhibitors of metalloproteinases (TIMPs), play a pivotal role in promoting pathogenic invasion by destroying tissue barriers within the body. This study aimed to explore the effect of T. pallidum protein Tp0136 on the balance of MMPs/TIMPs in human dermal vascular smooth muscle cells (HDVSMCs) and the related underlying mechanisms. A number of in vitro studies were conducted to access the impact of recombinant Tp0136 protein on the balance of MMPs/TIMPs in HDVSMCs. The involvement of the PI3K, MAPK, and NF-κB signaling pathways in this process was also investigated. Tp0136 induced the mRNA and protein expressions of MMP1 in HDVSMCs in a concentration-dependent way. In addition, MMP1/TIMP1 and MMP1/TIMP2 ratios were also increased. Furthermore, the study demonstrated that treatment of HDVSMCs with Tp0136 activated the PI3K, MAPK, and NF-κB signaling pathways. Inhibition of PI3K, JNK, P38, and NF-κB, suppressed MMP1 expression and reduced the induction of MMP1/TIMP1 and MMP1/TIMP2 ratios by Tp0136. These findings demonstrate that Tp0136 enhanced the expression of MMP1 involving the PI3K, MAPK, and NF-κB signaling pathways in HDVSMCs, and thus generated the unbalance of MMPs/TIMP, which could contribute to the early spread of T. pallidum and pathogenesis of syphilis.

Evaluation of the efficacy of four anti-SARS-CoV-2 antibodies after vaccination using kits from two manufacturers: A prospective, longitudinal, cohort study at 11 serial time points within 160 days.

PURPOSE: The accuracy of level of anti- severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies is a great concern. We aimed to compare the efficacy of anti-SARS-CoV-2 antibody detection kits from two manufacturers in evaluating the efficacy of SARS-CoV-2 vaccines. METHODS: The immune responses and consistency of four anti-SARS-CoV-2 antibodies were evaluated using two manufacturers' antibody kits (A and B) in 61 subjects within 160 days after vaccination with the CoronaVac vaccine. RESULTS: The total seropositivity rates of neutralizing antibodies and IgM antibodies detected by kit A were higher than those detected by kit B (P = 0.003 and P < 0.001, respectively). Conversely, the total seropositivity rates of total antibodies and IgG antibodies were higher in kit B than kit A (P < 0.001 and P < 0.001, respectively). The consistency rates showed less than 90% agreement between the kits for the detection of the four antibodies, and the κ score showed moderate or substantial consistency. The half-lives of neutralizing antibodies, total antibodies, and IgG antibodies within 160 days after vaccination, detected by kit A were 63.88 days, 80.50 days, and 63.70 days, respectively and by kit B were 97.06 days, 65.41 days, and 77.99 days, respectively. CONCLUSION: The efficacy of antibody detection differed between the two commercial anti-SARS-CoV-2 antibody kits, although there was moderate consistency, which may affect the clinical application and formulation of the vaccine strategy.

Membrane location of cardiolipin antigen in Treponema pallidum: further study on the origin of nontreponemal antibodies.

Aim: The present study examined the membrane location of cardiolipin antigen in treponemes. Materials & methods: The authors used different methods to disrupt the outer membrane of treponemes, detected the location of the cardiolipin antigen and analyzed the immune response in rabbits immunized with various antigens. Results: All organisms were labeled with nontreponemal antibodies on immunoelectron and fluorescence microscopy, except the citrate buffer-treated group, which is a method leading to relatively complete removal. Except for citrate buffer-treated spirochetes, all treponemes produced low-titer, nontreponemal antibodies in immunized rabbits. Conclusion: These findings indicated that the cardiolipin antigen was localized in the outer membrane of spirochetes. This study provided further evidence of the origin of nontreponemal antibodies during Treponema pallidum infection.

A 14+7 day quarantine period and a dual nucleic acid testing reagent strategy detect potentially indiscoverable Coronavirus disease 2019 infections in Xiamen, China.

BACKGROUND: Determining what quarantine period and detection strategy are more effective and sustainable remains a challenge for further prevention and social stability. METHODS: From October 2020 to December 2021, 290,547 inbound overseas travelers were subject to government quarantine in Xiamen, China. The detection rate of COVID-19 during different quarantine periods using dual or single nucleic acid testing reagents. RESULTS: The COVID-19 positive rate was 1.79% (519/290,547). The detection rates during the 7-day, 14-day and 14+7-day quarantine periods using the dual reagents were 78.4%, 91.7%, and 100%, respectively. The detection rate of the 7-day, 14-day and 14+7-day quarantine periods were 73.99%, 86.51%, and 94.22%, respectively, using the Liferiver reagent and 72.25%, 84.59%, and 91.91%, respectively, using the Daan reagent. Based on the 14+7 day strategy, dual nucleic acid testing reagent strategy detected all imported cases, but 30 cases (5.78%) were not detected via Liferiver reagent and 42 (8.09%) cases not detected via Daan reagent. CONCLUSION: A 14+7-day quarantine period and dual nucleic acid testing reagent strategy are effective screening methods for COVID-19 among inbound overseas travelers. The superior detection rate of these strategies reduce the risk of secondary transmission of the SARS-CoV-2 virus.

Anti-Receptor-Binding Domain Immunoglobulin G Antibody as a Predictor of Seropositivity for Anti-SARS-CoV-2 Neutralizing Antibody.

CONTEXT.­: Neutralizing antibody detection can assess the incidence of COVID-19 and the effectiveness of vaccines. However, commercial reagents for neutralizing antibodies were developed after the anti-SARS-CoV-2 immunoglobulin (Ig) G and IgM antibodies. Therefore, some laboratories did not perform neutralizing antibody testing services because of multiple factors. OBJECTIVE.­: To find a fast, accurate, and economic alternative for the detection of neutralizing antibodies for the development of COVID-19 screening programs. DESIGN.­: The response and correlation of 3 antibodies (anti-spike protein neutralizing antibody, total anti-receptor-binding domain [RBD] antibody, and anti-RBD IgG) were determined by observing the dynamics in 61 participants for 160 days after vaccination. RESULTS.­: The levels of neutralizing and anti-RBD IgG antibodies reached their peak values on day 42 after vaccination (120.75 IU/mL and 14.38 signal-to-cutoff ratio [S/CO], respectively). The total antibody levels peaked at 138.47 S/CO on day 35 after vaccination. The strongest correlation was found between neutralizing and anti-RBD IgG antibody levels (r = 0.894, P < .001). The area under the receiver operating characteristic curve for total antibody levels for the prediction of seropositivity for neutralizing antibodies was 0.881 (P < .001), and that for anti-RBD IgG antibody levels was 0.937 (P < .001). CONCLUSIONS.­: Neutralizing and anti-RBD IgG antibody levels were strongly correlated, and thus anti-RBD IgG antibody levels can be used for the accurate assessment of immunity following SARS-CoV-2 infection or vaccination.

Genome-wide long noncoding RNA and mRNA expression profiles demonstrate associations between exposure to inorganic elements and the risk of developing hepatocellular carcinoma.

BACKGROUND: Long noncoding RNAs (lncRNAs) are closely associated with the development of hepatocellular carcinoma (HCC). The present study conducted a genome-wide microarray analysis and qPCR validation to obtain comprehensive insights into this issue. METHODS: Thirty male HCC patients with chronic HBV infection were included in the present study. Primary HCC tissue and normal tissue were collected. Double-stranded complementary DNA synthesized from 10 pairs of samples was labeled and hybridized to a microarray chip. Further analyses, such as hierarchical clustering, gene ontology (GO) and pathway analyses, were performed. In addition, qPCR validation was performed on tissue samples and additional serum samples. RESULTS: The microarray analysis identified 946 upregulated and 571 downregulated lncRNAs and 1720 upregulated and 1106 downregulated mRNAs. Among these RNAs, ENST00000583827.1 (fold change: 21) and uc010isf.1 (fold change: 18) were the most over- and underexpressed lncRNAs in the HCC tissues, respectively. For the mRNAs, KIF20A (fold change: 26) and HEPACAM (fold change: 50) were the most over- and underexpressed in the HCC tissues, respectively. The GO analysis demonstrated that the most differentially expressed mRNAs were related to the response of metal ions. The pathway analysis also suggested that the most enriched pathway was mineral absorption. CONCLUSIONS: The subsequent qPCR validation exhibited high consistency with the microarray analysis, except for three lncRNAs. The qPCR analysis also demonstrated that TCONS_00008984 had a 767-fold overexpression level in HCC tissues when compared with normal tissues, and this finding was confirmed in the serum samples; therefore, TCONS_00008984 has the potential to serve as a diagnostic marker or prognostic indicator. The GO and pathway analyses indicated that exposure to inorganic elements may be involved in HCC risk.

Meta-omics characteristics of intestinal microbiota associated to HBeAg seroconversion induced by oral antiviral therapy.

Tenofovir and entecavir are currently designated as the preferred oral antiviral drugs for chronic hepatitis B. However, only less than 40% of patients can achieve HBeAg seroconversion. We aim at investigating the role of intestinal microbiome in HBeAg seroconversion induced by oral antiviral therapy and describe multi-omics characteristics of HBeAg seroconversion associated intestinal flora. In this study, we prospectively collected fecal samples at baseline from the patients with HBeAg positive chronic hepatitis B who would have oral antiviral therapy. 16S rDNA sequencing and metabolomics were performed. We identified HBeAg seroconversion-related microbial signature and constructed prediction model for HBeAg seroconversion. Thirty-seven of these subjects achieved HBeAg seroconversion within 156 weeks after the initiation of oral antiviral therapy, while 41 subjects remained HBeAg positive even after over 156 weeks of therapy. A computational statistical and machine learning approach allowed us to identify a microbial signature for HBeAg seroconversion. Using random forest method, we further constructed a classifier based on the microbial signature, with area under curve being 0.749 for the test set. Patients who achieved HBeAg seroconversion tended to have lower abundance of certain fecal metabolites such as essential amino acids, and several dipeptides. By analyzing the fecal microbiota from the patients with and without HBeAg seroconversion, we showed intestinal microbiome play a critical role in HBeAg seroconversion induced by oral antiviral therapy. We also identified intestinal microbial signature that is associated with HBeAg seroconversion after oral antiviral therapy.

The Diagnostic Performance of lncRNAs from Blood Specimens in Patients with Hepatocellular Carcinoma: A Meta-Analysis.

OBJECTIVE: Long noncoding RNAs (lncRNAs) are widely involved in the carcinogenesis and development of cancers. We conducted a meta-analysis to evaluate the diagnostic performance of lncRNAs in hepatocellular carcinoma (HCC). METHODS: After the inclusion and exclusion process, relevant information was extracted. Heterogeneity between studies was evaluated, and data synthesis was conducted by employing a bivariate random-effects model. RESULTS: In total, 20 eligible studies were enrolled. The pooled sensitivity and specificity were 0.86 (95% confidence interval [CI], 0.80-0.90) and 0.88 (95% CI, 0.82-0.92), respectively. The pooled positive likelihood ratio, pooled negative likelihood ratio, and pooled diagnostic odds ratio were 7.1 (95% CI, 4.9-10.2), 0.16 (95% CI, 0.11-0.23), and 44 (95% CI, 25-79), respectively. The results of the linear regression method and visual inspection of the Deeks funnel plot did not indicate significant publication bias. CONCLUSION: Our meta-analysis suggested that lncRNAs have high diagnostic performance for HCC and have the potential for clinical application.

Molecular Characterization Based on MLST and ECDC Typing Schemes and Antibiotic Resistance Analyses of Treponema pallidum subsp. pallidum in Xiamen, China.

In total, 49 clinical samples were analyzed using two typing schemes, Enhanced Centers for Disease Control and Prevention (ECDC) and multilocus sequence typing (MLST), to describe the molecular characteristics of circulating Treponema pallidum isolates in Xiamen between 2016 and 2017. In addition, genetic mutations potentially related to antibiotic resistance of T. pallidum were also analyzed. Forty five samples were fully typed by ECDC, and 14 different subtypes were detected. The most common subtype was 16d/f (24.4%), followed by 14d/f (20.0%). All forty nine samples were successfully typed by MLST, while only four allelic profiles were identified, including three SS14-like profiles and one Nichols-like profile. Among them, the major allelic profile was 1.1.8 (85.7%). Interestingly, the allelic profile 1.3.1 widespread in Europe and North America was not detected in this region. Additionally, A2058G mutation in 23S rRNA was found in all detectable samples (38/38), and no mutation in 16S rRNA was observed (36/36). Four non-synonymous single-nucleotide polymorphisms in penicillin-binding protein genes were found in the 35 samples eligible for Sanger sequencing. Among them, the variant in tp0500 (P564I) can only be found in the SS14-like isolates. Homoplastic changes in tp0760 (I415F/I415M) and tp0705 (A506V/A506T) were found. Moreover, the variant tp0705 A506V and the variant tp0705 A506T separately appeared in the SS14-like isolates and Nichols-like isolates, respectively. This study showed that the genotypes of T. pallidum isolates in Xiamen between 2016 and 2017 were different from those in other geographic areas. The resistance-related variants of T. pallidum isolates identified in this study could provide awareness for clinicians in the treatment of syphilis.

Suppressed Expression of CXCL14 in Hepatocellular Carcinoma Tissues and Its Reduction in the Advanced Stage of Chronic HBV Infection.

INTRODUCTION: CXCL14 was a significantly under-expressed mRNA in hepatocellular carcinoma tissues according to our microarray analysis, as well as head and neck squamous cell carcinoma and cervical squamous cell carcinoma. CXCL14 was considered a tumor suppressor in some studies; however, its role in HBV infection has not been identified. METHODS: CXCL14 mRNA expression was quantified from 20 male HCC patients, and the fold change in cancer tissues was calculated by comparisons with normal adjacent tissues. Overall, 212 patients with chronic HBV infection and 180 HBV-free controls were recruited to investigate the association between CXCL14 polymorphisms and HBV progression as well as liver function parameters. Serum CXCL14 levels were determined by enzyme-linked immunosorbent assay (ELISA), and comparisons were made between different HBV status and different CXCL14 genotypes. RESULTS: The mRNA expression of CXCL14 was 0.33-fold in HCC tissues when compared with adjacent tissues. The frequencies of rs2237062 and rs2547, but not rs2237061, were significantly different between patients with mild hepatitis and moderate-to-severe hepatitis. Moreover, rs2237062 and rs2547 polymorphisms correlated with impaired liver function parameters. ELISA results suggested that HBV-free controls had the highest level of CXCL14, while mild hepatitis patients had low levels, and patients with moderate-to-severe hepatitis had the lowest level. GA+AA genotypes of rs2547 were associated with reduced levels of serum CXCL14 because it introduced a stop codon at residue 109. CONCLUSION: CXCL14 was significantly suppressed in HBV-related HCC tissues, and its polymorphisms were linked with advanced stage chronic HBV infection and impaired liver function.

Evaluation of Real-Time PCR Coupled With Multiplex Probe Melting Curve Analysis for Pathogen Detection in Patients With Suspected Bloodstream Infections.

Background: This study aimed to evaluate real-time polymerase chain reaction coupled with multiplex probe melting curve analysis (PCR-MCA) for pathogen detection in patients with suspected bloodstream infections (BSIs). Methods: A PCR-MCA assay was developed for simultaneous identification of 28 kinds of the most common pathogens and two resistance genes within a few hours. The diagnostic performance of the PCR-MCA assay was determined and compared to the results of blood culture. Results: A total of 2,844 consecutive new episodes of suspected BSIs in 2,763 patients were included in this study. There were 269 episodes of pathogens identified by blood culture. For all the pathogens tested, the PCR-MCA assay exhibited a sensitivity of 88.8% (239/269), specificity of 100% (2,575/2,575), and agreement of 98.9% (2,814/2,844). For the pathogens on the PCR-MCA list, the PCR-MCA results had a sensitivity of 99.2% (239/241), specificity of 100% (2,575/2,575), and agreement of 99.9% (2,814/2,816) compared with the results of blood culture. For seven samples with multiple pathogens identified simultaneously during one blood culture investigation, the PCR-MCA assay verified the results of the blood culture, with an agreement rate of 100% for each. Conclusion: The PCR-MCA assay could discover 88.8% of the pathogens in clinical practice, showing excellent diagnostic performance vs. that of blood culture for pathogen detection in patients with suspected BSIs, and would contribute to rapid diagnosis and correct antibiotic administration.

Metabolic Disorders in Patients with Central Nervous System Infections: Associations with Neurosyphilis.

INTRODUCTION: Recently, neurosyphilis was found to be associated with diabetes mellitus (DM). Whether the association was specific to neurosyphilis among central nervous system (CNS) infections, and whether neurosyphilis is associated with other prevalent metabolic disorders deserves further study. METHODS: An in-depth cross-sectional study was conducted with 74 neurosyphilis patients and 74 sex- and age-matched patients with other CNS infections. DM-, hypertension-, and dyslipidemia-related factors were compared between patients with neurosyphilis and those with other CNS infections. RESULTS: The prevalence rates of hypertension and hyperlipidemia in neurosyphilis patients were 45.9 and 21.4%, respectively, which were higher than those in patients with other CNS infections (45.9 vs. 28.4%, p = 0.027; 21.4 vs. 8.3%, p = 0.028). In addition, neurosyphilis patients had significantly higher systolic blood pressure (BP; median 139 mm Hg; interquartile range [IQR] 121-151 mm Hg), -diastolic BP (median 83 mm Hg; IQR 76-89 mm Hg), total cholesterol (median 4.86 mmol/L; IQR 3.80-5.51 mmol/L), low-density lipoprotein (median 3.39 mmol/L; IQR 2.52-3.95 mmol/L), and apolipoprotein A1 (apoA1; median 1.31 g/L; IQR 1.06-1.52 g/L) levels and lower apoB/A1 ratios (median 0.67; IQR 0.49-0.99) than patients with other CNS infections (p< 0.05). There were no differences in the DM-related factors between patients with neurosyphilis and those with other CNS infections (p> 0.05). CONCLUSION: Potential association between neurosyphilis and metabolic disorders was found among CNS infections. The results could have important implications for clinical practice, alerting more clinicians to this issue.

Recombinant Treponema pallidum protein Tp47 promotes the migration and adherence of THP-1 cells to human dermal vascular smooth muscle cells by inducing MCP-1 and ICAM-1 expression.

Vascular inflammation is a complex and multifactorial pathophysiological process that plays a crucial role in all stages of syphilis and is responsible for tissue damage. Little is known about the interactions of infiltrating immunocytes with human dermal vascular smooth muscle cells (HDVSMCs) in arterioles during the immunopathogenesis of syphilis. The Treponema pallidum subsp. pallidum membrane protein Tp47 is considered a major inducer of inflammation initiation and development. In this study, we demonstrated that Tp47 promoted the migration and adhesion of THP-1 cells to HDVSMCs. Furthermore, Tp47 increased monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) mRNA and protein expression levels in a dose- and time-dependent manner. The migration and adhesion of THP-1 cells to HDVSMCs were significantly suppressed by anti-MCP-1 and anti-ICAM-1 neutralizing antibodies, respectively. Further studies revealed that treatment of HDVSMCs with Tp47 activated the PI3K/Akt, p38 MAPK and NF-κB signalling pathways. Inhibition of PI3K/Akt, p38 MAPK and NF-κB suppressed the MCP-1 and ICAM-1 expression induced by Tp47. In addition, the migration and adhesion of THP-1 cells to Tp47-treated HDVSMCs were significantly decreased by pretreatment with PI3K/Akt, p38 MAPK and NF-κB inhibitors. These findings demonstrate that Tp47 promotes the migration and adherence of THP-1 cells to HDVSMCs by inducing MCP-1 and ICAM-1 expression, which is mediated by activation of the PI3K/Akt, p38 MAPK and NF-κB pathways. This study provides a novel potential therapeutic strategy for controlling the vascular inflammatory response in syphilis patients.

Metabolite Profiles of the Cerebrospinal Fluid in Neurosyphilis Patients Determined by Untargeted Metabolomics Analysis.

The mechanism underlying the stealth property of neurosyphilis is still unclear. Global metabolomics analysis can provide substantial information on energy metabolism, physiology and possible diagnostic biomarkers and intervention strategies for pathogens. To gain better understanding of the metabolic mechanism of neurosyphilis, we conducted an untargeted metabolomics analysis of cerebrospinal fluid (CSF) from 18 neurosyphilis patients and an identical number of syphilis/non-neurosyphilis patients and syphilis-free patients using the Agilent, 1290 Infinity LC system. The raw data were normalized and subjected to subsequent statistical analysis by MetaboAnalyst 4.0. Metabolites with a variable importance in projection (VIP) greater than one were validated by Student's T-test. A total of 1,808 molecular features were extracted from each sample using XCMS software, and the peak intensity of each feature was obtained. Partial-least squares discrimination analysis provided satisfactory separation by comparing neurosyphilis, syphilis/non-neurosyphilis and syphilis-free patients. A similar trend was obtained in the hierarchical clustering analysis. Furthermore, several metabolites were identified as significantly different by Student's T-test, including L-gulono-gamma-lactone, D-mannose, N-acetyl-L-tyrosine, hypoxanthine, and S-methyl-5'-thioadenosine. Notably, 87.369-fold and 7.492-fold changes of N-acetyl-L-tyrosine were observed in neurosyphilis patients compared with syphilis/non-neurosyphilis patients and syphilis-free patients. These differential metabolites are involved in overlapping pathways, including fructose and mannose metabolism, lysosomes, ABC transporters, and galactose metabolism. Several significantly expressed metabolites were identified in CSF from neurosyphilis patients, including L-gulono-gamma-lactone, D-mannose, N-acetyl-L-tyrosine, and hypoxanthine. These differential metabolites could potentially improve neurosyphilis diagnostics in the future. The role of these differential metabolites in the development of neurosyphilis deserves further exploration.

Profile of the tprK gene in primary syphilis patients based on next-generation sequencing.

BACKGROUND: The highly variable tprK gene of Treponema pallidum has been acknowledged to be one of the mechanisms that causes persistent infection. Previous studies have mainly focused on the heterogeneity in tprK in propagated strains using a clone-based Sanger approach. Few studies have investigated tprK directly from clinical samples using deep sequencing. METHODS/PRINCIPAL FINDINGS: We conducted a comprehensive analysis of 14 primary syphilis clinical isolates of T. pallidum via next-generation sequencing to gain better insight into the profile of tprK in primary syphilis patients. Our results showed that there was a mixture of distinct sequences within each V region of tprK. Except for the predominant sequence for each V region as previously reported using the clone-based Sanger approach, there were many minor variants of all strains that were mainly observed at a frequency of 1-5%. Interestingly, the identified distinct sequences within the regions were variable in length and differed by only 3 bp or multiples of 3 bp. In addition, amino acid sequence consistency within each V region was found among the 14 strains. Among the regions, the sequence IASDGGAIKH in V1 and the sequence DVGHKKENAANVNGTVGA in V4 showed a high stability of inter-strain redundancy. CONCLUSIONS: The seven V regions of the tprK gene in primary syphilis infection demonstrated high diversity; they generally contained a high proportion sequence and numerous low-frequency minor variants, most of which are far below the detection limit of Sanger sequencing. The rampant variation in each V region was regulated by a strict gene conversion mechanism that maintained the length difference to 3 bp or multiples of 3 bp. The highly stable sequence of inter-strain redundancy may indicate that the sequences play a critical role in T. pallidum virulence. These highly stable peptides are also likely to be potential targets for vaccine development.

Treponema pallidum promotes macrophage polarization and activates the NLRP3 inflammasome pathway to induce interleukin-1ß production.

BACKGROUND: The involvement of inflammasome activation and macrophage polarization during the process of syphilis infection remains unknown. In this study, A series of experiments were performed using human macrophages to research the role of NLRP3 inflammasome regulation in interleukin (IL)-1ß production and its influence on macrophage polarization triggered by T. pallidum. RESULTS: The results showed that in M0 macrophages treated with T. pallidum, the M1-associated markers inducible nitric oxide synthase (iNOS), IL-1ß and TNF-α were upregulated, and the M2-associated markers CD206 and IL-10 were downregulated. In addition, we observed NLRP3 inflammasome activation and IL-1ß secretion in T. pallidum-treated macrophages, and the observed production of IL-1ß occurred in a dose- and time-dependent manner. Moreover, the secretion of IL-1ß by macrophages after T. pallidum treatment was notably reduced by anti-NLRP3 siRNA and caspase-1 inhibitor treatment. NAC, KCl, and CA074-ME treatment also suppressed IL-1ß release from T. pallidum-treated macrophages. CONCLUSIONS: These findings showed that T. pallidum induces M0 macrophages to undergo M1 macrophage polarization and elevate IL-1ß secretion through NLRP3. Moreover, the process of NLRP3 inflammasome activation and IL-1ß production in macrophages in response to T. pallidum infection involves K+ efflux, mitochondrial ROS production and cathepsin release. This study provides a new insight into the innate immune response to T. pallidum infection.