Platelet-derived major histocompatibility complex class I coating on Treponema pallidum attenuates natural killer cell lethality.

The evasive tactics of Treponema pallidum pose a major challenge in combating and eradicating syphilis. Natural killer (NK) cells mediate important effector functions in the control of pathogenic infection, preferentially eliminating targets with low or no expression of major histocompatibility complex (MHC) class I. To clarify T. pallidum's mechanisms in evading NK-mediated immunosurveillance, experiments were performed to explore the cross-talk relations among T. pallidum, NK cells, and platelets. T. pallidum adhered to, activated, and promoted particle secretion of platelets. After preincubation with T. pallidum, platelets expressed and secreted high levels of MHC class I, subsequently transferring them to the surface of T. pallidum, potentially inducing an immune phenotype characterized by the "pseudo-expression" of MHC class I on the surface of T. pallidum (hereafter referred to a "pseudo-expression" of MHC class I). The polA mRNA assay showed that platelet-preincubated T. pallidum group exhibited a significantly higher copy number of polA transcript than the T. pallidum group. The survival rate of T. pallidum mirrored that of polA mRNA, indicating that preincubation of T. pallidum with platelets attenuated NK cell lethality. Platelets pseudo-expressed the MHC class I ligand on the T. pallidum surface, facilitating binding to killer cell immunoglobulin-like receptors with two immunoglobulin domains and long cytoplasmic tail 3 (KIR2DL3) on NK cells and initiating dephosphorylation of Vav1 and phosphorylation of Crk, ultimately attenuating NK cell lethality. Our findings elucidate the mechanism by which platelets transfer MHC class I to the T. pallidum surface to evade NK cell immune clearance.

Treponema pallidum recombinant protein Tp47 activates NOD-like receptor family protein 3 inflammasomes in macrophages via glycolysis.

Glycolysis is a key pathway in cellular glucose metabolism for energy supply and regulates immune cell activation. Whether glycolysis is involved in the activation of NOD-like receptor family protein 3 (NLRP3) inflammasomes during Treponema pallidum (T. pallidum) infection is unclear. In this study, the effect of T. pallidum membrane protein Tp47 on NLRP3 inflammasome activation in rabbit peritoneal macrophages was analysed and the role of glycolysis in NLRP3 inflammasome activation was explored. The results showed that Tp47 promoted NLRP3, caspase-1, and IL-1ß mRNA expression in macrophages, enhanced glycolysis and glycolytic capacity of macrophage, and promoted the production of macrophage glycolytic metabolites citrate, phosphoenolpyruvate, and lactate. The M2 pyruvate kinase (PKM2) inhibitor shikonin down-regulated the Tp47-promoted NLRP3, caspase-1, and IL-1ß mRNA expression in macrophages, and suppressed the Tp47-enhanced glycolysis and glycolytic capacity. Similarly, si-PKM2 significantly inhibited Tp47-promoted NLRP3, caspase-1, and IL-1ß mRNA expression and the Tp47-enhanced glycolysis and glycolytic capacity in macrophages. In conclusion, Tp47 activated NLRP3 inflammasomes via PKM2-dependent glycolysis and provided a new perspective on the effect of T. pallidum infection on host macrophages, which would contribute to the understanding of the infection mechanism and host immune mechanism of T. pallidum.

Treponema pallidum Promotes the Polarization of M2 Subtype Macrophages to M1 Subtype Mediating the Apoptosis and Inhibiting the Angiogenesis of Human Umbilical Vein Endothelial Cells.

M2 macrophages were related to local immune homeostasis and maternal-fetal tolerance in normal pregnancy; whether M2 macrophages can respond to the stimulation of Treponema pallidum to mediate placental vascular inflammation injury is unclear. In this study, M2 macrophages were constructed to investigate the impact of T. pallidum on macrophage polarization and the underlying signaling pathway involved in this process, and the influence of macrophage polarization triggered by T. pallidum on the apoptosis and angiogenesis of human umbilical vein endothelial cells (HUVEC) was also explored. The results showed that M2 macrophage markers (CD206 and PPARγ) and anti-inflammatory factors (TGFß and CCL18) were decreased, while M1 macrophage marker CD80 and inflammatory cytokines (IL1ß and TNFα) were increased when M2 macrophages were treated with T. pallidum, indicating that T. pallidum promoted the polarization of M2 subtype macrophages to the M1 subtype. Moreover, T. pallidum-induced M1 macrophage polarization was found to be significantly correlated with the activation of Janus kinase 1 (JAK1) and signal transducer and activator of transcription 1 (STAT1). In addition, T. pallidum-induced M1 macrophages were found to promote apoptosis and inhibit the angiogenesis of HUVECs, and JAK1 or STAT1 inhibitors could weaken the apoptosis rate and promote the angiogenesis of HUVECs. These findings revealed that T. pallidum promoted the polarization of M2 macrophages to the M1 subtype through the JAK1-STAT1 signal pathway mediating the apoptosis and inhibiting angiogenesis of HUVECs, which may provide a possible mechanism for T. pallidum-induced adverse pregnancy outcomes.

Screening the B- and T-cell epitope map of TP0136 and exploring their effect in a Treponema pallidum rabbit model.

The systemic immune response, including B- and T-cell reactions, plays a corresponding role in syphilis infections. The TP0136 protein is a target of the immune response in infected hosts and may mediate the immune response. Here, we developed a method that combining reverse vaccine approach with Pepscan/T-cell proliferation to screen and identify three B-cell and two T-cell epitopes of TP0136, and explore the role of the B- and T-cell epitopes in immunized-infected animals. The results showed that immunized with B-cell epitopes not only had no protective effect but also aggravated the syphilitic lesion development. While immunized with T-cell epitopes of TP0136 could induce a strong Th1-cellular immunity response, which could attenuate syphilitic lesion development to a certain extent. The variation in exacerbation or attenuation of skin lesions, induced by distinct B- and T-cell epitopes of Tp0136, within the host's defense against syphilis warrants in-depth exploration.

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.

Treponema pallidum recombinant protein Tp47 enhanced interleukin-6 secretion in human dermal fibroblasts through the toll-like receptor 2 via the p38, PI3K/Akt, and NF-κB signalling pathways.

Interleukin-6 (IL-6) is a multi-effective cytokine involved in multiple immune responses. Whether fibroblasts also turn out to be a cytokine IL-6 factory during interaction with Treponema pallidum is not yet understood. To explore whether fibroblasts participate in inflammation due to syphilis, a series of experiments were performed to explore the role of T. pallidum lipoprotein Tp47 in IL-6 production in human dermal fibroblasts. The Toll-like receptor 2 (TLR2) and participating signalling pathways in this process were also evaluated. The results showed that the expressions of IL-6 and the protein levels of TLR2 in fibroblasts were upregulated after stimulation with Tp47, and this effect was impeded by the TLR2 inhibitor C29. In addition, Tp47 promoted the phosphorylation of p38, PI3K/Akt, and nuclear factor-kappaB (NF-κB), and the translocation of NF-κB in fibroblasts. Moreover, p38, PI3K, and NF-κB inhibitors significantly reduced IL-6 production in fibroblasts stimulated with Tp47. Furthermore, the TLR2 inhibitor C29 inhibited the phosphorylation of p38, Akt, and NF-κB, and the translocation of NF-κB in fibroblasts. In conclusion, our results showed that Tp47 enhanced IL-6 secretion in human dermal fibroblasts through TLR2 via p38, PI3K/Akt, and NF-κB signalling pathways. These findings contribute to our understanding of syphilis inflammation.

Genetic Profiling of the Full-Length tprK Gene in Patients with Primary and Secondary Syphilis.

TprK antigenic variation is acknowledged as an important strategy developed by Treponema pallidum to achieve immune evasion. Previous studies applied short-read sequencing to explore tprK gene sequence diversity in clinical samples; however, due to the limitations of short-read sequencing, it was difficult to determine the linkage between the seven V regions, and crucial information about full-length tprK variants was lost. Although two recent studies explored complete tprK gene profiles in natural human syphilis infection, there are still too few profiled full-length tprK variants among clinical T. pallidum isolates to fully understand the characteristics of TprK coding diversity. Here, Pacific Biosciences (PacBio) long-read sequencing was applied to examine the diversity of full-length tprK variants in 21 clinical T. pallidum isolates from 11 patients with primary syphilis and 10 patients with secondary syphilis. A total of 398 high-confidence full-length sequences, which presented remarkable sequence heterogeneity, were found. However, these full-length tprK variants exhibited limited variation in length and GC content, showing 24 length types and average GC content of 51.5 ± 0.42% and 51.6 ± 0.26% for primary and secondary syphilis samples, respectively. Additionally, the combined patterns of mutated V regions generating new tprK variants were obviously different in primary and secondary syphilis samples. The diversity of tprK gene sequences in primary syphilis samples may represent the underlying variability of the bacterium; conversely, the variability of the tprK gene in secondary syphilis samples may more accurately reflect how T. pallidum escapes host immune clearance. These data highlight the tprK gene as an important coding gene that shows conflicting genetic characteristics but underlies the persistence of spirochete infection. IMPORTANCE The resurgence of syphilis in both low- and high-income countries has attracted attention, and persistent infection by the pathogen has long been a research focus. The tprK gene, encoding the hypervariable outer membrane protein, is thought to be responsible for pathogen immune evasion and persistent infection. Here, PacBio long-read sequencing was applied to examine the diversity of full-length tprK variants in 21 clinical T. pallidum isolates from 11 patients with primary syphilis and 10 patients with secondary syphilis. The results showed that the sequences of the tprK gene were remarkably heterogeneous; however, the sequences presented limited variation in length and GC content. The investigation of the combined patterns of the V regions allowed us to gain insight into the features of the tprK gene generating new variants at different clinical stages. The findings of this study will be helpful for further exploration of the pathogenesis of syphilis.

Response and duration of serum anti-SARS-CoV-2 antibodies induced by the third dose of an inactivated vaccine: A prospective longitudinal cohort study at 21 serial time points over 641 days.

Given the prevalence of low-pathogenic but highly infectious Omicron variants, a cohort study was conducted to assess the response and duration of novel coronavirus-inactivated vaccine-induced antibodies 1 year after the third dose (Day 641). Blood samples were collected and anti-spike neutralizing antibodies (neutralizing antibody), total antibodies against the receptor-binding domain of the spike protein (total antibody), and immunoglobulin G antibodies against the spike protein (IgG antibody) were determined. Antibody kinetics and attenuation were evaluated. The results showed that the levels of neutralizing, total, and IgG antibodies on Day 641 were 98.05 IU/mL, 152.8 AU/mL, and 7.68 S/CO, respectively. Levels of anti-SARS-CoV-2 antibodies were higher in the younger subgroup than in the older subgroup at several time points after the second and third doses. The seropositive rate of neutralizing antibodies providing protection from infection or severe infection was 46.87% and 87.5%, and the seropositive rates of total antibody and IgG antibody were maintained at 100% and 90.63%, respectively. The half-lives of neutralizing, total, and IgG antibodies were 186.89, 363.04, and 417.50 days, respectively. Collectively, anti-SARS-CoV-2 antibodies may provide a certain degree of protection from infection 1 year after the third dose and high protection from severe infection.

Development and Evaluation of a Novel One-Step RT-qPCR Targeting the Vero Gene for the Identification of False-Positive Results Caused by Inactivated Virus Vaccine Contamination.

To identify false-positive SARS-CoV-2 test results caused by novel coronavirus inactivated vaccine contamination, a novel RT-qPCR targeting the ORF1ab and N genes of SARS-CoV-2 and Vero gene was developed. The amplification efficiency, precision, and lower limit of detection (LLOD) of the RT-qPCR assay were determined. A total of 346 clinical samples and 132 environmental samples were assessed, and the diagnostic performance was evaluated. The results showed that the amplification efficiency of the ORF1ab, N, and Vero genes was 95%, 97%, and 93%, respectively. The coefficients of variation of Ct values at a concentration of 3 × 104 copies/mL were lower than 5%. The LLOD for the ORF1ab, N, and Vero genes reached 8.0, 3.3, and 8.2 copies/reaction, respectively. For the 346 clinical samples, our RT-qPCR assay identified SARS-CoV-2-positive and SARS-CoV-2-negative samples with a sensitivity of 100.00% and a specificity of 99.30% and novel coronavirus inactivated vaccine-contaminated samples with a sensitivity of 100% and a specificity of 100%. For the environmental samples, our RT-qPCR assay identified novel coronavirus inactivated vaccine-contaminated samples with a sensitivity of 88.06% and a specificity of 95.38%. In conclusion, the RT-qPCR assay we established can be used to diagnose COVID-19 and, to a certain extent, false-positive results due to vaccine contamination.

Anti-SARS-CoV-2 IgM Secondary Response Was Suppressed by Preexisting Immunity in Vaccinees: A Prospective, Longitudinal Cohort Study over 456 Days.

To obtain more insight into IgM in anti-SARS-CoV-2 immunity a prospective cohort study was carried out in 32 volunteers to longitudinally profile the kinetics of the anti-SARS-CoV-2 IgM response induced by administration of a three-dose inactivated SARS-CoV-2 vaccine regimen at 19 serial time points over 456 days. The first and second doses were considered primary immunization, while the third dose was considered secondary immunization. IgM antibodies showed a low secondary response that was different from the other three antibodies (neutralizing, total, and IgG antibodies). There were 31.25% (10/32) (95% CI, 14.30-48.20%) of participants who never achieved a positive IgM antibody conversion over 456 days after vaccination. The seropositivity rate of IgM antibodies was 68.75% (22/32) (95% CI, 51.80-85.70%) after primary immunization. Unexpectedly, after secondary immunization the seropositivity response rate was only 9.38% (3/32) (95% CI, 1.30-20.10%), which was much lower than that after primary immunization (p = 0.000). Spearman's correlation analysis indicated a poor correlation of IgM antibodies with the other three antibodies. IgM response in vaccinees was completely different from the response patterns of neutralizing, total, and IgG antibodies following both the primary immunization and the secondary immunization and was suppressed by pre-existing immunity induced by primary immunization.

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.

[Roles of PET/CT in Predicting the Prognosis of Diffuse Large B Cell Lymphoma Patients Treated with Chimeric Antigen Receptor T Cell Therapy].

OBJECTIVE: To investigate the prognosis prediction value of PET/CT in DLBCL patients treated with CAR-T therapy. METHODS: The effects of PET/CT were retrospectively explored on 13 R/R DLBCL patients who were treated with CAR-T therapy. Parameters reflecting tumor metabolic burden, such as metabolic tumor volume (MTV) and total lesion glycolysis (TLG) were measured before and after CAR-T treatment. RESULTS: Patients with larger baseline MTV or longer sum of longest diameters showed shorter overall survival (OS) time than those with low tumor burden. Patients achieved complete remission (CR), partial remission (PR) and minor remission (MR) determined by response evaluation criteria in lymphoma (RECIL) in 12 weeks showed progression-free survival and OS time superior to those of patients with no remission. In addition, it was found that 2 patients with residual masses classified as PR by contrast-enhanced CT of patients were evaluated as complete metabolic response by PET/CT imaging. CONCLUSION: PET/CT shows a great value in the evaluation of prognosis and response in CAR-T-treated R/R DLBCL patients.

Giant barocaloric effect in hexagonal Ni2In-type Mn-Co-Ge-In compounds around room temperature.

The most widespread cooling techniques based on gas compression/expansion encounter environmental problems. Thus, tremendous effort has been dedicated to develop alternative cooling technique and search for solid state materials that show large caloric effects. An application of pressure to a material can cause a change in temperature, which is called the barocaloric effect. Here we report the giant barocaloric effect in a hexagonal Ni2In-type MnCoGe0.99In0.01 compound involving magnetostructural transformation, Tmstr, which is accompanied with a big difference in the internal energy due to a great negative lattice expansion(ΔV/V ~ 3.9%). High resolution neutron diffraction experiments reveal that the hydrostatic pressure can push the Tmstr to a lower temperature at a rate of 7.7 K/kbar, resulting in a giant barocaloric effect. The entropy change under a moderate pressure of 3 kbar reaches 52 J kg(-1) K(-1), which exceeds that of most materials, including the reported giant magnetocaloric effect driven by 5 T magnetic field that is available only by superconducting magnets.

Giant negative thermal expansion in bonded MnCoGe-based compounds with Ni2In-type hexagonal structure.

MnCoGe-based compounds undergo a giant negative thermal expansion (NTE) during the martensitic structural transition from Ni2In-type hexagonal to TiNiSi-type orthorhombic structure. High-resolution neutron diffraction experiments revealed that the expansion of unit cell volume can be as large as ΔV/V ∼ 3.9%. The optimized compositions with concurrent magnetic and structural transitions have been studied for magnetocaloric effect. However, these materials have not been considered as NTE materials partially due to the limited temperature window of phase transition. The as-prepared MnCoGe-based compounds are quite brittle and naturally collapse into powders. By using a few percents (3-4%) of epoxy to bond the powders, we introduced residual stress in the bonded samples and thus realized the broadening of structural transition by utilizing the specific characteristics of lattice softening enforced by the stress. As a result, giant NTE (not only the linear NTE coefficient α but also the operation-temperature window) has been achieved. For example, the average α̅ as much as -51.5 × 10(-6)/K with an operating temperature window as wide as 210 K from 122 to 332 K has been observed in a bonded MnCo0.98Cr0.02Ge compound. Moreover, in the region between 250 and 305 K near room temperature, the α value (-119 × 10(-6)/K) remains nearly independent of temperature. Such an excellent performance exceeds that of most other materials reported previously, suggesting it can potentially be used as a NTE material, particularly for compensating the materials with large positive thermal expansions.