Functional capacity of Th17 cells isolated from the blood of men infected with <i>S</i>. <i>mansoni</i>.

<p>The ability of Th17 cells isolated from the blood to produce a second or third cytokine (IL-22 and/or IFNγ in addition to IL-17A) in response to non-specific stimulation (PMA/iono) was measured using flow cytometry. (A) Proportion of Th17 cells producing additional cytokines was compared between men infected with <i>S</i>. <i>mansoni</i> (shedding eggs), intermediate (presence of worms detected by Urine-CCA, but not shedding eggs), and uninfected men (no detection of worm antigen or eggs). Assessment of polyfunctionality was performed using SPICE v5.35 software with polyfunctionality compared between <i>S</i>. <i>mansoni</i>-infected and uninfected men using a chi-squared distribution (1000 permutations of pies)[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004067#pntd.0004067.ref063" target="_blank">63</a>]. (B) Post-hoc comparison of individual functional subsets between <i>S</i>. <i>mansoni</i>-infected (hatched bars) and uninfected men (related samples Wilcoxon rank test).</p

Differential expression of markers of HIV susceptibility on Th populations isolated from the blood and foreskin.

<p>Expression of (A) CCR5, (B) CD69, and (C) HLA-DR was measured on CD4 T cells isolated from the blood (PBMCs) and foreskin tissue using flow cytometry. “Other” refers to CD4+/CD3+ cells that do not produce IL-17A, IFNγ or IL-22. Expression of surface markers on Th subsets was compared using the Friedman chi-square test; post-hoc pairwise comparisons made using Wilcoxon related samples rank test and Bonferroni adjusted p-values are reported (*p<0.05; **p<0.01; ***p<0.001).</p

Participant demographics.

<p>Participant demographics.</p

Flow cytometry gating strategy for Th subsets isolated from (A) the blood and (B) the foreskin.

<p>Blood cells were isolated by density centrifugation, and foreskin cells by mechanical and enzymatic digestion of tissue. Dead cells and doublets were excluded. Lymphocytes were identified based on characteristic size and granularity in blood samples and gates applied to foreskin samples. CD4 T cells were identified by expression of CD3 and CD4. Th subsets among CD4 T cells were identified by cytokine production in response to non-specific stimuli (SEB shown). Gates for cytokine production were based on unstimulated samples. Th17 cells were identified by production of IL-17A; Th1 cells by production of IFNγ and not IL-17A; Th22 cells by production of IL-22 in the absence of either IFNγ or IL-17A. Representative plots showing expression of CCR5, CD69 and HLA-DR on CD4 T cells are also shown.</p

No Difference in Keratin Thickness between Inner and Outer Foreskins from Elective Male Circumcisions in Rakai, Uganda

<div><p>It has been hypothesized that increased HIV acquisition in uncircumcised men may relate to a more thinly keratinized inner foreskin. However, published data are contradictory and potentially confounded by medical indications for circumcision. We tested the hypothesis that the inner foreskin was more thinly keratinized than the outer foreskin using tissues from 19 healthy, HIV-uninfected men undergoing routine prophylactic circumcision in Rakai, Uganda. Sections from 3 foreskin anatomic sites (inner, outer, and frenar band) were snap-frozen separately. Two independent laboratories each separately stained, imaged, and measured keratin thicknesses in a blinded fashion. There was no significant difference in keratin thickness between the inner (mean = 14.67±7.48 µm) and outer (mean = 13.30±8.49 µm) foreskin, or between the inner foreskin and the frenar band (mean = 16.91±12.42 µm). While the frenar band showed the greatest intra-individual heterogeneity in keratin thickness, there was substantial inter-individual variation seen in all regions. Measurements made by the two laboratories showed high correlation (r = 0.741, 95% CI, 0.533–0.864). We conclude that, despite inter- and intra-individual variability, keratin thickness was similar in the inner and outer foreskin of healthy Ugandan men, and that reduced keratin thickness is not likely to make the inner foreskin more susceptible to HIV acquisition.</p> </div

Frequency of detection of antimicrobial peptides and HIV acquisition.

<p>Frequency of detection of antimicrobial peptides and HIV acquisition.</p

Individual keratin thickness measurements by anatomic foreskin region.

*<p>SD =  standard deviation.</p>**<p><i>p</i>-value based on two-tailed Student’s t-test.</p><p>Bolded <i>p</i>-values indicate significantly thinner inner, outer, or frenar band regions.</p

Demographic and epidemiologic data of male participants.

<p>Demographic and epidemiologic data of male participants.</p

Demographic characteristics of cases and controls at enrollment.

<p>Demographic characteristics of cases and controls at enrollment.</p

Chemokine Levels in the Penile Coronal Sulcus Correlate with HIV-1 Acquisition and Are Reduced by Male Circumcision in Rakai, Uganda

<div><p>Individual susceptibility to HIV is heterogeneous, but the biological mechanisms explaining differences are incompletely understood. We hypothesized that penile inflammation may increase HIV susceptibility in men by recruiting permissive CD4 T cells, and that male circumcision may decrease HIV susceptibility in part by reducing genital inflammation. We used multi-array technology to measure levels of seven cytokines in coronal sulcus (penile) swabs collected longitudinally from initially uncircumcised men enrolled in a randomized trial of circumcision in Rakai, Uganda. Coronal sulcus cytokine levels were compared between men who acquired HIV and controls who remained seronegative. Cytokines were also compared within men before and after circumcision, and correlated with CD4 T cells subsets in foreskin tissue. HIV acquisition was associated with detectable coronal sulcus Interleukin-8 (IL-8 aOR 2.26, 95%CI 1.04–6.40) and Monokine Induced by γ-interferon (MIG aOR 2.72, 95%CI 1.15–8.06) at the visit prior to seroconversion, and the odds of seroconversion increased with detection of multiple cytokines. Coronal sulcus chemokine levels were not correlated with those in the vagina of a man’s female sex partner. The detection of IL-8 in swabs was significantly reduced 6 months after circumcision (PRR 0.59, 95%CI 0.44–0.87), and continued to decline for at least two years (PRR 0.29, 95%CI 0.16–0.54). Finally, prepuce IL-8 correlated with increased HIV target cell density in foreskin tissues, including highly susceptible CD4 T cells subsets, as well as with tissue neutrophil density. Together, these data suggest that penile inflammation increases HIV susceptibility and is reduced by circumcision.</p></div