Treg frequencies in peripheral blood of neurosyphilis patients.

<p>The percentage of CD25^high (<b>A</b>) and Foxp3⁺ (<b>B</b>) among CD4⁺ T cells in peripheral blood of healthy controls, and syphilis patients without (including primary, secondary, latent and serofast syphilis) and with (including both asymptomatic and symptomatic neurosyphilis) neurological involvement are shown. The percentage of CD25^high (<b>C</b>) and Foxp3⁺ (<b>D</b>) among CD4⁺ T cells in peripheral blood of different types of neurosyphilis patients. Individual frequencies for every patient analyzed are shown. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002528#s3" target="_blank">Results</a> represent the mean ± SEM. A and B, Kruskal-Wallis test; C and D, One-way ANOVA. *, P<0.05, ***, P<0.001.</p

Absolute number of leukocytes, percentage and numbers of CD4⁺ T cells, Tregs, and TGF-β levels in CSF from different types of syphilis patients.

a<p>CSF samples from 20 syphilis patients without neurological involvement, 23 asymptomatic, 7 meningeal and 14 parenchymal neurosyphilis patients were analyzed by flow cytometry.</p>b<p>vs. syphilis patients without neurological involvement;</p>c<p>vs. asymptomatic neurosyphilis patients;</p>d<p>vs. meningeal neurosyphilis patients;</p>e<p>percentage (%) among CD45⁺ leukocytes;</p>f<p>percentage (%) among CD4⁺ T cells.</p>g<p>TGF-β levels were examined in 41 syphilis patients without neurological involvement and 63 neurosyphilis patients.</p><p><b>Abbreviations</b>: NS, Not Significant.</p

Association of circulating Treg numbers with serum RPR titers in primary, secondary, latent and serofast syphilis patients.

<p>Serum RPR titers are plotted against circulating CD4⁺ CD25^high Treg frequencies in primary (<b>A</b>, n = 64), secondary (<b>B</b>, n = 96), latent (<b>C</b>, n = 86) and serofast (<b>D</b>, n = 43) syphilis patients. Each dot represents an individual patient. The straight line in each graph is the result of linear regression analysis. Pearson's correlation coefficients (r) and P values are shown.</p

Schematic diagram summarizing the role of Treg in syphilis.

<p>APC, antigen presenting cells; Th1, T helper cell type 1; Treg, regulatory T cells; CSF, cerebrospinal fluid; CNS, central nervous system; DC, dendritic cells.</p

Treg activity in the peripheral blood of early and serofast syphilis patients.

<p>(<b>A</b>) Peripheral blood mononuclear cells (PBMC) were stained for flow cytometric analysis and lymphocytes were gated according to forward and side scatter characteristics. Representative plots show the gates of CD4⁺ CD25^high and CD4⁺ Foxp3⁺ T cells in the lymphocyte gate. CD25 and Foxp3 expression among CD4⁺ T cells are also shown. (<b>B</b>) The percentage of CD25^high and Foxp3⁺ among CD4⁺ T cells in peripheral blood of healthy controls, and in patients with primary, secondary, latent and serofast syphilis. Individual frequencies for every patient analyzed are shown. (<b>C</b>) CD4⁺ CD25⁺ T cells and CD4⁺ CD25⁻ T cells were purified from peripheral blood of healthy donors, and patients with primary, secondary, latent and serofast syphilis. The mean percent inhibition of the proliferative response by CD4⁺ CD25⁺ T cells derived from three to five individuals per group is shown. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002528#s3" target="_blank">Results</a> are representative of three independent experiments. (<b>D</b>) Serum concentrations of TGF-β were measured in healthy controls, and in patients with primary, secondary syphilis, latent, and serofast syphilis. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002528#s3" target="_blank">Results</a> represent the mean or the mean ± SEM. (<b>E</b>) Correlation of serum TGF-β concentration with circulating CD4⁺ CD25^high Treg frequencies in syphilis patients (n = 176). Each dot represents an individual patient. B, Kruskal-Wallis test; C and D, One-way ANOVA; E, Pearson's correlation. *, P<0.05; **, P<0.01; ***, P<0.001.</p

Levels of IL-17 in CSF from patients with different clinical states of syphilis.

<p>Data are given as median (range), or frequencies,</p>a<p>v.s. syphilis patients without neurological involvement.</p>b<p>v.s. asymptomatic neurosyphilis patients.</p><p>IL-17 levels represent the median (range).</p

Increased CSF IL-17 levels in patients with neurosyphilis.

<p>(A) Detection rates of CSF IL-17(≥0.5 pg/ml) in control group (n = 29), syphilis patients without neurological disorders (including primary, secondary, latent and serofast syphilis; n = 69), or syphilis patients with neurological involvement (including asymptomatic and symptomatic neurosyphilis; n = 103). (B) Levels of CSF IL-17 proteins among the samples shown in (A). (C) Detection rates of CSF IL-17 in patients with asymptomatic (n = 40) or symptomatic (including meningovascular, paretic, tabetic and ocular; n = 63) neurosyphilis. (D) Levels of CSF IL-17 proteins among the CSF samples shown in (C). Results represent the median + individual values.</p

Factors that influence normalization of each laboratory measure in the final Cox regression models.

<p>HR, hazard ratio; NS, in the stepwise model and therefore not included in the final model;</p>a<p>Early (secondary and early latent) stage versus late latent stage and unknown duration;</p>b<p>aqueous crystalline pencillin G,4MU intravenous every 4 h for 14 days versus ceftriaxone, 2 g intravenous daily for 10 days;</p>c<p>Greater than the median value versus less than or equal to the median value in subjects with each abnormality. Median values were as follows: CSF WBC count, 8.8 cells/ml; CSF protein concentration, 68 mg/dl; CSF-VDRL titer, 1∶4; serum RPR test titer, 1∶64.</p>d<p>CSF IL-17 positive (≥0.5 pg/ml) versus CSF IL-17 negative (<0.5 pg/ml).</p

Clinical and laboratory characteristics of the control group and the syphilis patients.

<p>Data are given as median (range), or frequencies.</p>a<p>include 13 subjects with primary syphilis, 30 subjects with secondary syphilis, 7 subjects with serofast syphilis and 19 subjects with latent syphilis.</p>b<p>“Duration of primary, secondary syphilis and symptomatic neurosyphilis” means the duration of clinical manifestations; “Duration of latent syphilis” means the period between the patient's high risk behaviors which may be infected with <i>T. pallidum</i> and the confirmed both serum TPPA/RPR positive. “Duration of asymptomatic neurosyphilis” means the period between the patient's high risk behaviors which may be infected with <i>T. pallidum</i> and the confirmed neurosyphilis. “Duration of the serofast” means the period between syphilitic patients who receiving recommended standard treatment, whose nontreponemal test titers remain positive and then was diagnosed as serofast.</p><p><b>Abbreviations:</b> RPR, rapid plasma regain; VDRL, venereal disease research laboratory; TPPA, treponema pallidum particle agglutination assay; WBC, white blood cells; ND, not done.</p><p><b>Healthy donors</b>: peripheral blood mononuclear cells (PBMC) isolation from healthy donors and for measurement of the baseline of the levels of IL-17⁺ cells and the frequency of Th17 cells. <b>Control:</b> patients who undergone orthopaedic or stone surgery and had not experienced syphilis, where CSF was collected prior to spinal anaesthesia. Samples from this group were used for measuring the baseline of the levels of IL-17 in CSF.</p

Comparison of clinical features between patients with CSF IL-17⁺ and IL-17⁻ neurosyphilis.

<p>Data are given as median (range), or frequencies.</p><p>*significant differences between the two groups,*p<0.01.</p><p>**significant differences between the two groups, **p<0.001.</p>a<p>“Duration of asymptomatic neurosyphilis” means the period between the patient's high risk behaviors which may be infected with <i>T. pallidum</i> and the confirmed neurosyphilis. “Duration of symptomatic neurosyphilis” means the duration of clinical manifestations.</p