Additional file 1 of chngpt: threshold regression model estimation and inference

Supplementary tables and a short tutorial on using chngpt. (PDF 63.5 kb

Additional file 2 of Efficacy of a bivalent killed whole-cell cholera vaccine over five years: a re-analysis of a cluster-randomized trial

Figure S1. (PDF 27.4 kb

Additional file 3 of Efficacy of a bivalent killed whole-cell cholera vaccine over five years: a re-analysis of a cluster-randomized trial

Supplementary Text (Model equations). (PDF 45.5 kb

The Inner Foreskin of Healthy Males at Risk of HIV Infection Harbors Epithelial CD4+ CCR5+ Cells and Has Features of an Inflamed Epidermal Barrier

<div><p>Male circumcision provides partial protection against multiple sexually transmitted infections (STIs), including HIV, but the mechanisms are not fully understood. To examine potential vulnerabilities in foreskin epithelial structure, we used Wilcoxon paired tests adjusted using the false discovery rate method to compare inner and outer foreskin samples from 20 healthy, sexually active Peruvian males who have sex with males or transgender females, ages 21–29, at elevated risk of HIV infection. No evidence of epithelial microtrauma was identified, as assessed by keratinocyte activation, fibronectin deposition, or parakeratosis. However, multiple suprabasal tight junction differences were identified: 1) inner foreskin stratum corneum was thinner than outer (p = 0.035); 2) claudin 1 had extended membrane-bound localization throughout inner epidermis stratum spinosum (p = 0.035); 3) membrane-bound claudin 4 was absent from inner foreskin stratum granulosum (p = 0.035); and 4) occludin had increased membrane deposition in inner foreskin stratum granulosum (p = 0.042) versus outer. Together, this suggests subclinical inflammation and paracellular transport modifications to the inner foreskin. A setting of inflammation was further supported by inner foreskin epithelial explant cultures secreting higher levels of GM-CSF (p = 0.029), IP-10 (p = 0.035) and RANTES (p = 0.022) than outer foreskin, and also containing an increased density of CCR5+ and CD4+ CCR5+ cells (p = 0.022). Inner foreskin dermis also secreted more RANTES than outer (p = 0.036), and had increased density of CCR5+ cells (p = 0.022). In conclusion, subclinical changes to the inner foreskin of sexually active males may support an inflammatory state, with availability of target cells for HIV infection and modifications to epidermal barriers, potentially explaining the benefits of circumcision for STI prevention.</p></div

No differences in keratinocyte turnover and skin homeostasis among inner and outer foreskin mucosa from sexually active men.

<p>A) Fibronectin and D) keratin 6 concentrations in foreskin lysates from all subjects sexually active within the past 2 weeks (n = 17) were measured by MBA. B) Percent of Ki-67+ nuclei among all epithelial nuclei on sections from 9 participants. C) Representative staining for Ki67 and SYTOX orange (nuclei). E) Median epithelial thickness (µm) was computed measuring 3–5 H&E-stained sections of ∼26.5 mm² inner/outer foreskin for each participant (n = 17). F) Representative H&E staining G) Parakeratosis was quantitated from H&E sections from all subjects (n = 17). All p values are from FDR-adjusted Wilcoxon tests.</p

CCR5+ cells accumulate in the inner foreskin in from sexually active MSM.

<p>A) Representative images of foreskin epidermis at 10× magnification stained with CCR5 (pseudocolored green), CD4 (pseudocolored red) and SYTOX Orange (pseudocolored blue) for nuclear identification. All fields from ∼26.5 mm² sections were used for analysis. For isotype controls and larger tissue sections see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108954#pone.0108954.s001" target="_blank">Figure S1</a></b>. White letters mark CD4+ cell aggregates (A), and epidermis (E). B–G) Cell density measured in the B, D, F) whole tissue (dermis and epidermis) or C, E, G) epidermis. Cell types measured are B, C) CD4+, D, E) CCR5+ and F, G) CD4+ CCR5+ cells. All p-values are from FDR-adjusted Wilcoxon tests.</p

Both the inner foreskin epidermis and dermis from sexually active MSM can secrete increased levels of inflammatory cytokines compared to the outer foreskin.

<p>Supernatants from explant cultures from participants who were sexually active within the past 2 weeks (n = 17) were quantitated for GM-CSF, IFN-α, IFN-γ, IL-10, IL-1α, IL-1β, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP1α, MIP1β, RANTES and TNFα using MBA to measure cytokines in dermal and epidermal explants after 48 h culture. All p values are from FDR-adjusted Wilcoxon tests.</p

Sexually active MSM have a slightly reduced SC envelope in the inner foreskin.

<p>A) Median SC thickness (µm) was computed from 3–5 sections of inner/outer foreskin, each taken 5 mm apart. B) Involucrin and C) keratin 1,10 levels were measured by MBA in foreskin lysates. All p values are from FDR-adjusted Wilcoxon comparisons of all subjects sexually active within the past 2 weeks (n = 17).</p