Outer Foreskin Research Articles

A pilot study to show that asymptomatic sexually transmitted infections alter the foreskin epithelial proteome.

There is limited data on the role of asymptomatic STIs (aSTIs) on the risk of human immunodeficiency virus (HIV) acquisition in the male genital tract (MGT). The impact of foreskin removal on lowering HIV acquisition is well described, but molecular events leading to HIV acquisition are unclear. Here, in this pilot study, we show that asymptomatic urethral infection with Chlamydia trachomatis (CT) significantly impacts the foreskin proteome composition. We developed and optimized a shotgun liquid chromatography coupled tandem mass spectrometry (MS)-based proteomics approach and utilized this on foreskins collected at medical male circumcision (MMC) from 16 aSTI+ men and 10 age-matched STI- controls. We used a novel bioinformatic metaproteomic pipeline to detect differentially expressed (DE) proteins. Gene enrichment ontology analysis revealed proteins associated with inflammatory and immune activation function in both inner and outer foreskin from men with an aSTI. Neutrophil activation/degranulation and viral-evasion proteins were significantly enriched in foreskins from men with aSTI, whereas homotypic cell-cell adhesion proteins were enriched in foreskin tissue from men without an aSTI. Collectively, our data show that asymptomatic urethral sexually transmitted infections result in profound alterations in epithelial tissue that are associated with depletion of barrier integrity and immune activation.

Pre-Clinical Evaluation of Tenofovir and Tenofovir Alafenamide for HIV-1 Pre-Exposure Prophylaxis in Foreskin Tissue.

Background: HIV-1 pre-exposure prophylaxis (PrEP) has focused predominantly on protective efficacy in receptive sex, with limited research on the dosing requirements for insertive sex. We pre-clinically assessed the ex vivo pharmacokinetic–pharmacodynamic (PK–PD) profile of tenofovir (TFV) and tenofovir alafenamide (TAF) in foreskin tissue. Methods: Inner and outer foreskin explants were exposed to serial dilutions of TFV or TAF prior to addition of HIV-1BaL at a high (HVT) or a low viral titer (LVT). Infection was assessed by measurement of p24 in foreskin culture supernatants. TFV, TAF and TFV–diphosphate (TFV–DP) concentrations were measured in tissues, culture supernatants and dosing and washing solutions. Results: Dose–response curves were obtained for both drugs, with greater potency observed against LVT. Inhibitory equivalency mimicking oral dosing was defined between 1 mg/mL of TFV and 15 µg/mL of TAF against HVT challenge. Concentrations of TFV–DP in foreskin explants were approximately six-fold higher after ex vivo dosing with TAF than with TFV. Statistically significant negative linear correlations were observed between explant levels of TFV or TFV–DP and p24 concentrations following HVT. Conclusions: Pre-clinical evaluation of TAF in foreskin explants revealed greater potency than TFV against penile HIV transmission. Clinical evaluation is underway to support this finding.

Impact of chemokine C\u2013C ligand 27, foreskin anatomy and sexually transmitted infections on HIV-1 target cell availability in adolescent South African males

We compared outer and inner foreskin tissue from adolescent males undergoing medical male circumcision to better understand signals that increase HIV target cell availability in the foreskin. We measured chemokine gene expression and the impact of sexually transmitted infections (STIs) on the density and location of T and Langerhans cells. Chemokine C–C ligand 27 (CCL27) was expressed 6.94-fold higher in the inner foreskin when compared with the outer foreskin. We show that the density of CD4+CCR5+ cells/mm2 was higher in the epithelium of the inner foreskin, regardless of STI status, in parallel with higher CCL27 gene expression. In the presence of STIs, there were higher numbers of CD4+CCR5+ cells/mm2 cells in the sub-stratum of the outer and inner foreskin with concurrently higher number of CD207+ Langerhans cells (LC) in both tissues, with the latter cells being closer to the keratin surface of the outer FS in the presence of an STI. When we tested the ability of exogenous CCL27 to induce T-cell migration in foreskin tissue, CD4 + T cells were able to relocate to the inner foreskin epithelium in response. We provide novel insight into the impact CCL27 and STIs on immune and HIV-1 target cell changes in the foreskin.

Characterization of CD4+ T cell subsets and HIV susceptibility in the inner and outer foreskin of Ugandan men.

Biological mechanisms of foreskin HIV acquisition are poorly defined. The inner foreskin is preferentially infected in explant models, so we hypothesized that this site would be enriched for HIV-susceptible CD4+ T cells and proinflammatory/chemoattractant cytokines. A total of 42 HIV-uninfected Ugandan men without genital symptoms provided foreskin tissues and swabs at the time of elective penile circumcision. The immune phenotype of foreskin-derived CD4+ T cells and entry of a CCR5-tropic HIV pseudovirus was characterized, and specific cytokine levels assayed by multiplexed chemiluminescent ELISA. Unexpectedly, outer foreskin CD4+ T cells more frequently expressed CCR5 (median 29.2% vs 22.9%, P=0.01) and CD69 (median 36.5% vs 15%, P<0.01), and on a per-cell basis, HIV entry was higher. However, overall CD4+ T cell density was approximately twofold higher in the inner foreskin, and several highly susceptible T cell subsets were increased at this site, including Th17 cells (20.0% vs 14.1%, P=0.0021). Specific pro-inflammatory cytokine levels were also higher on the inner foreskin surface (IL-17, IL-8, RANTES and IL-1β; all P<0.05). There was marked heterogeneity in CD4+ T cell populations and immune milieu between inner and outer foreskin tissues. Despite higher per-cell viral entry into CD4+ T cells from the outer foreskin, the higher target cell density and enriched pro-inflammatory cytokines of the inner foreskin suggest that this may be a preferential site for HIV acquisition.

Visualization of HIV-1 interactions with penile and foreskin epithelia: clues for female-to-male HIV transmission.

To gain insight into female-to-male HIV sexual transmission and how male circumcision protects against this mode of transmission, we visualized HIV-1 interactions with foreskin and penile tissues in ex vivo tissue culture and in vivo rhesus macaque models utilizing epifluorescent microscopy. 12 foreskin and 14 cadaveric penile specimens were cultured with R5-tropic photoactivatable (PA)-GFP HIV-1 for 4 or 24 hours. Tissue cryosections were immunofluorescently imaged for epithelial and immune cell markers. Images were analyzed for total virions, proportion of penetrators, depth of virion penetration, as well as immune cell counts and depths in the tissue. We visualized individual PA virions breaching penile epithelial surfaces in the explant and macaque model. Using kernel density estimated probabilities of localizing a virion or immune cell at certain tissue depths revealed that interactions between virions and cells were more likely to occur in the inner foreskin or glans penis (from local or cadaveric donors, respectively). Using statistical models to account for repeated measures and zero-inflated datasets, we found no difference in total virions visualized at 4 hours between inner and outer foreskins from local donors. At 24 hours, there were more virions in inner as compared to outer foreskin (0.0495 +/− 0.0154 and 0.0171 +/− 0.0038 virions/image, p = 0.001). In the cadaveric specimens, we observed more virions in inner foreskin (0.0507 +/− 0.0079 virions/image) than glans tissue (0.0167 +/− 0.0033 virions/image, p<0.001), but a greater proportion was seen penetrating uncircumcised glans tissue (0.0458 +/− 0.0188 vs. 0.0151 +/− 0.0100 virions/image, p = 0.099) and to significantly greater mean depths (29.162 +/− 3.908 vs. 12.466 +/− 2.985 μm). Our in vivo macaque model confirmed that virions can breach penile squamous epithelia in a living model. In summary, these results suggest that the inner foreskin and glans epithelia may be important sites for HIV transmission in uncircumcised men.

The inner foreskin of healthy males at risk of HIV infection harbors epithelial CD4+ CCR5+ cells and has features of an inflamed epidermal barrier.

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.

Male Circumcision and HIV Transmission; What Do We Know?

Male circumcision (MC) has been shown to be protective against heterosexual HIV transmission and is being explored in some parts of the world as a means of combating the epidemic. The World Health Organization (WHO) recommends that MC be considered as an important component of HIV prevention in high prevalence settings.We review evidence that demonstrates that the inner foreskin is likely to be the main portal of entry for the HIV virus in males. Whether removal of the inner foreskin accounts for all the protection afforded by circumcision is yet to be established.The proposed mechanisms of protection range from inherent immunohistological factors of foreskin such as difference in thickness of keratin layer and density of target cells for HIV between inner and outer foreskin to physiological mechanisms that follow male circumcision such as drying of secretions underneath foreskin after sexual intercourse, loss of microbiome that attract target cells to the genital mucosa and lack of priming the genital mucosa with less abundant sexual transmitted infections among circumcised men.The aim of this review is to give an updated account on the mechanisms proposed so far on the demonstrated 50-70% protection from HIV transmission through heterosexual intercourse, by male circumcision.

Differential Compartmentalization of HIV-Targeting Immune Cells in Inner and Outer Foreskin Tissue

Ex vivo foreskin models have demonstrated that inner foreskin is more susceptible to HIV-1 infection than outer foreskin. In the present study we characterized the compartition of HIV-1 target cells and quantified these cells in the epidermis and dermis of inner and outer foreskins using immunohistochemistry and flow cytometry. Our data showed that the epidermis of the inner foreskin was more enriched with CD4+ T cells and Langerhans cells (LCs), with the co-expression of CCR5 and α4β7 receptors, than the outer foreskin. Interestingly, the vast majority of CD4+ T cells and LCs expressed CCR5, but not CXCR4, indicating that the inner foreskin might capture and transmit R5-tropic HIV strains more efficiently. In addition, lymphoid aggregates, composed of T cells, macrophages and dendritic cells (DCs) in the dermis, were closer to the epithelial surface in the inner foreskin than in the outer foreskin. As dendritic cells are able to capture and pass HIV particles to susceptible target cells, HIV may be able to more efficiently infect the inner foreskin by hijacking the augmented immune communication pathways in this tissue. After the inoculation of HIV-1 particles in a foreskin explant culture model, the level of p24 antigen in the supernatant from the inner foreskin was slightly higher than that from the outer foreskin, although this difference was not significant. The present study is the first to employ both CCR5 and α4β7 to identify HIV target cells in the foreskin. Our data demonstrated that the inner foreskin was more enriched with HIV target immune cells than the outer foreskin, and this tissue was structured for efficient communication among immune cells that may promote HIV transmission and replication. In addition, our data suggests the R5-tropism of HIV sexual transmission is likely shaped through the inherent receptor composition on HIV target cells in the mucosa.

Activating transcription factor 3 is not up-regulated in hypospadias patients in Japan

The aetiology of hypospadias is largely uncharacterized. Some of the researchers have advocated that activating transcription factor 3 (ATF3), an oestrogen-responsive transcription factor, is up-regulated in patients with hypospadias. The purpose is to evaluate the universality of this fact; we studied the expression of ATF3 protein in prepuce tissue obtained from hypospadias and phimosis patients living in metropolitan Tokyo. Prepuce tissue was obtained from outer foreskin at the time of surgery, quickly prepared for paraffin-embedded sectioning and stained immunohistochemically for ATF3. Two researchers blindly evaluated immunoreactivity and scored it semi-quantitatively as nil = 0, weak = 1, or strong = 2, to give a final staining intensity score (SIS). Subjects were 18 hypospadias patients and 17 phimosis patients (as controls) who had surgery between January, 2009 and March, 2010. All subjects lived in metropolitan Tokyo, Japan. Mean ages at surgery were 2.9 ± 1.0 and 3.9 ± 2.4 years, respectively (P > 0.05). SIS was not statistically different between hypospadias patients (1.4 ± 0.5) and controls (1.5 ± 0.5), (P > 0.05). Our data suggest that ATF3 is not highly associated with hypospadias in metropolitan Tokyo. Differences in ethnicity might have influenced our results.

Cell free HIV-1 virus can infect inner and outer foreskin polarized explants

Methods First, we have developed cryopreservation methods that allow infection of foreskin tissue explants after thawing (654 +/178.3 pg of p24/gr of tissue) and provide comparable infection rates to fresh samples( 716.5 +/446pg of p24/gr of tissue). Second, we have developed an ex vivo assay that uses human foreskin explants to replicate the polarized viral entry. After 12h of polarized exposure, HIV entering the explant is amplified for 6 days of culture with activated PBMCs and measured using p24 ELISA.

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

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.

A novel operative technique for concealed penis secondary to penoscrotal web: a case report

Contemporary surgical techniques for concealed penis defined in the literature are sophisticated and, because of this, we decided to perform a simple technique. A child with penoscrotal web was admitted to our Pediatric Surgery Clinic. The patient had recurrent urinary tract infections without urinary tract disease and, therefore, web reconstruction was thought to be useful. A modified unfurling technique and penoscrotal angle reconstruction was used. The main characteristic of our procedure can basically be described as an oblique incision of the outer foreskin leaf which guides the next steps. We report the operative and clinical course of the patient. We demonstrated that the penoscrotal web was treated simply and effectively as an outpatient procedure. In addition, in the follow-up period, we found that the patient had acceptable and cosmetic penile skin.

Enhanced cellular responses and environmental sampling within inner foreskin explants: implications for the foreskin's role in HIV transmission

The decrease in HIV acquisition after circumcision suggests a role for the foreskin in HIV transmission. However, the mechanism leading to protection remains undefined. Using tissue explant cultures we found that Langerhans cells (LCs) in foreskin alter their cellular protein expression in response to external stimuli. Furthermore, we observe that upon treatment with TNF-α, tissue-resident LCs became activated and that stimulatory cytokines can specifically cause an influx of CD4+ T-cells into the epithelial layer. Importantly, both of these changes are significant in the inner, but not outer, foreskin. In addition, we find that LCs in the inner foreskin have increased ability to sample environmental proteins. These results suggest differences in permeability between the inner and outer foreskin and indicate that HIV target cells in the inner foreskin have increased interaction with external factors. This increased responsiveness and sampling provides novel insights into the underlying mechanism of how circumcision can decrease HIV transmission.

Keratinization of the adult male foreskin and implications for male circumcision

The theory that a more thinly keratinized inner foreskin leads to increased HIV-1 susceptibility has been based on relatively little published data. We sought to quantify the keratin thicknesses of the inner and outer foreskin to determine the plausibility of this hypothesis. We took repeated measurements of the keratin layer of 16 adult male foreskins to determine whether differences existed between the inner and outer foreskin. Adult foreskins were collected from consenting donors undergoing elective male circumcision for unknown medical indications in Chicago, Illinois, USA. Specimens were processed, sectioned and stained for keratin using antifilaggrin fluorescent antibodies. Slides stained with hematoxylin and eosin were used as controls and compared with results from previously published studies using this method. Keratin layers were measured in a standardized fashion for each specimen. Comparing our fluorescence-based analysis with previously published immunohistochemical methods revealed that our method was highly accurate for measuring foreskin keratin thickness. There was significant heterogeneity in the keratin thickness of the inner and outer aspects of the male foreskin within and between the different donors. There was no significant difference between the inner and outer foreskin keratin thickness (25.37 +/- 12.51 and 20.54 +/- 12.51 microm, respectively; P = 0.451). We found no difference between the keratinization of the inner and outer aspects of the adult male foreskin. Keratin layers alone are unlikely to explain why uncircumcised men are at higher risk for HIV infection.

OA011-01. Early events of human immunodeficiency virus type 1 (HIV-1) ex vivo penetration in the foreskin mimicking HIV-1 sexual transmission

Methods Two novel models of the adult human foreskin mucosa were established: 1) ex vivo polarized inner and outer foreskin explants; 2) in vitro immuno-competent reconstructed foreskins, which include fibrous sheets produced by foreskin fibroblasts that are topped by differentiating foreskin keratinocytes (from either inner or outer foreskin) and Langerhans cells (LCs). These models permit to inoculate comparatively in a polarized manner at the mucosal pole either HIV-1-infected cells (that are present in all secretions vectorizing HIV-1) or cell-free HIV-1.

Langerhans’ cell density and degree of keratinization in foreskins of Chinese preschool boys and adults

Recent epidemiological studies have proposed that male circumcision reduces the relative risk of acquiring HIV-1. Here, we evaluated the density of Langerhans' cell and degree of keratinization in the foreskins of Chinese preschool boys and adults. Sixty preschool boys and 20 healthy men without infectious history following male circumcisions were included. The keratin thickness and Langerhans' cells were quantified by using keratin staining, immunohistochemistry, and image analysis. The extent of keratinization was much greater in the inner foreskin than in the outer foreskin in adults and boys with infectious history. It was likely to be less keratinized in boys' foreskins compared with those of adults. The density of Langerhans' cells was higher in the outer foreskin than in the inner foreskin of adults and healthy boys. Furthermore, an increased density of Langerhans' cells of inner foreskin was also found in boys with infectious history compared with healthy boys. There was much higher Langerhans' cell density in boys' foreskin compared with those of adults. These findings suggest that Chinese men may have a different feature of keratin in the foreskin, and a higher Langerhans' cells density in boys' foreskin may be due to it being less keratinized.

Potential HIV-1 target cells in the human penis

To study the distribution of HIV-1 receptors and degree of keratinization in the human penis. Formalin-fixed penises were obtained from nine uncircumcised cadavers. Foreskins were obtained from 21 healthy adult men undergoing elective circumcision for social reasons. Uncircumcised penises were obtained within 24 h of death from eight men. All tissues were stained for keratin and HIV-1 receptors. Penises from nine formalin fixed cadavers aged 64-80 years were obtained from the Department of Anatomy, University of Melbourne. Foreskins were obtained from 21 men aged 18-64 years following circumcision performed at either the Freemason's or Mercy Private Hospitals, Melbourne, Australia. Fresh penile necropsy specimens from eight uncircumcised men aged 23-63 years were obtained from the Victorian Institute of Forensic Medicine, Melbourne. The degree of keratinization was scored, and the distribution of HIV-1 susceptible cells was mapped in the glans penis, penile urethra, urethral meatus, frenulum and foreskin. Cells with HIV-1 receptors were present in all penile epithelia, but Langerhans' cells were most superficial in the inner foreskin and frenulum. The inner foreskin had a significantly thinner keratin layer (1.8 +/- 0.1 units), than the outer foreskin (3.3 +/- 0.1), or glans penis (3.3 +/- 0.2), P < 0.05. Superficial Langerhans' cells on the inner aspect of the foreskin and frenulum are poorly protected by keratin and thus could play an important role in primary male infection. These findings provide a possible anatomical explanation for the epidemiologically observed protective effect of male circumcision.

The transverse outer preputial (TOP) island flap: an easy method to cover urethroplasties and skin defects in hypospadias repair

Objective The transverse outer preputial (TOP) island flap, a simple procedure inspired by the double-faced island flap, is proposed as a substitute for the Byars repair to provide skin coverage for most of the current reconstructive techniques for hypospadias. Patients and methods From 1996 to 2003, 108 consecutive children (aged 1–8 years) had a primary repair of various forms of hypospadias with different degrees of chordee; the TOP island flap was used in 66. The surgical technique is based on the use of a dorsal transverse skin flap mobilized with its own vascular pedicle from the outer foreskin, then ventrally rotated longitudinally after removing the inner part of the prepuce. Results Partial necrosis of the lateral border of the flap was infrequent (<2%) and did not induce urethral complications. There was no total necrosis in the series. The rate of fistula was comparable with children operated using other techniques (8%). The cosmesis was good, with no ventral bulkiness or hypertrophic scarring. Conclusions The TOP island flap is a safe method for covering a new urethra and penile skin defects, with good cosmetic results.