A human ex vivo skin model breaking boundaries.

Ex vivo human skin models are valuable tools in skin research due to their physiological relevance. Traditionally, standard cultivation is performed in a cell culture incubator with a defined temperature of 37 °C and a specific atmosphere enriched with CO2 to ensure media stability. Maintaining the model under these specific conditions limits its flexibility in assessing exposures to which the skin is exposed to in daily life, for example changes in atmospheric compositions. In this study we demonstrated that the foreskin-derived skin model can be successfully cultured at room temperature outside a CO2 incubator using a CO2-independent, serum-free media. Over a cultivation period of three days, the integrity of the tissue and the preservation of immune cells is well maintained, indicating the model's stability and resilience under the given conditions. Exposing our Medical University of Graz - human Organotypic Skin Explant Culture (MUG-hOSEC) model to cytotoxic and inflammatory stimuli results in responses analyzable within the supernatant. Besides the common analysis of released proteins upon treatment, such as cytokines and enzymes, we have included extracellular vesicle to obtain a more comprehensive picture of cell communication.

Mono-2-ethylhexyl phthalate-induced downregulation of MMP11 in foreskin fibroblasts contributes to the pathogenesis of hypospadias.

Hypospadias is one of the most common congenital anomalies of the male urogenital system, and di(2-ethylhexyl) phthalate (DEHP), a widely used endocrine-disrupting chemical (EDC), is considered a significant risk factor for this condition. Mono-2-ethylhexyl phthalate (MEHP), the toxic active metabolite of DEHP, has been proven to affect penile development and ultimately result in the hypospadias phenotype. However, while it is acknowledged that hypospadias arises from the aberrant development of multiple penile tissues, the specific impact of MEHP on human foreskin tissue development and its underlying molecular mechanisms of action remain unclear. In this study, we constructed an in vitro toxicity assay for MEHP using human foreskin fibroblasts and employed high-throughput RNA sequencing to investigate the molecular mechanisms subserving the defects in cellular function. We subsequently conducted multi-omics data analysis using public databases to analyze key target genes, and identified MMP11 as a chief downstream gene responsible for the effects of MEHP on HFF-1 cell migration. Through molecular docking analysis and molecular biology experiments, we further demonstrated that the nuclear receptor PPAR-gamma was activated upon binding with MEHP, leading to the suppression of MMP11 expression. Additionally, we found that epigenetic modifications induced by MEHP were also involved in its pathogenic effects on hypospadias. Our research highlights the crucial role of impaired cellular proliferation and migration in MEHP-induced hypospadias. We identified the MEHP/PPAR-gamma/MMP11 pathway as a novel pathogenic mechanism, providing important potential targets for future preventive strategies with respect to hypospadias.

Impact of antimicrobials on penile HIV susceptibility and immunology in uncircumcised men: A randomized phase 1/2 clinical trial.

Within the penile microbiome, bacteria associated with seroconversion, immunology, and cells (BASIC species) enhance HIV susceptibility in heterosexual uncircumcised men by inducing foreskin inflammation and HIV target cell recruitment. This phase 1/2 clinical trial randomizes HIV-uninfected Ugandan men (n = 125) to either oral tinidazole, topical metronidazole, topical clindamycin, or topical hydrogen peroxide to define impact on ex vivo foreskin HIV susceptibility, penile immunology, and BASIC species density. Antimicrobials are well tolerated, and 116 (93%) participants complete the protocol. Topical metronidazole and oral tinidazole reduce the inner foreskin tissue density of HIV-susceptible CD4+ T cells (predefined primary endpoint). Antimicrobials also have varying but substantial effects on reducing prepuce inflammation and BASIC species density, reducing density of foreskin T cell subsets, and increasing foreskin epithelial integrity. Immune alterations correlate strongly with changes in the abundance of BASIC species. Clinical interventions targeting the penile microbiota, particularly topical metronidazole, may reduce HIV susceptibility in uncircumcised men.

Herpes simplex virus spreads rapidly in human foreskin, partly driven by chemokine-induced redistribution of Nectin-1 on keratinocytes.

HSV infects keratinocytes in the epidermis of skin via nectin-1. We established a human foreskin explant infection model to investigate HSV entry and spread. HSV1 entry could only be achieved by the topical application of virus via high density microarray projections (HD-MAPs) to the epidermis, which penetrated beyond one third of its thickness, simulating in vivo microtrauma. Rapid lateral spread of HSV1 to a mean of 13 keratinocytes wide occurred after 24 hours and free virus particles were observed between keratinocytes, consistent with an intercellular route of spread. Nectin-1 staining was markedly decreased in foci of infection in the epidermis and in the human keratinocyte HaCaT cell line. Nectin-1 was redistributed, at the protein level, in adjacent uninfected cells surrounding infection, inducible by CCL3, IL-8 (or CXCL8), and possibly CXCL10 and IL-6, thus facilitating spread. These findings provide the first insights into HSV1 entry and spread in human inner foreskin in situ.

Antimicrobial susceptibility rates in gram-positive catalase-negative cocci from sheep and goat genital microbiota.

Gram-positive catalase-negative cocci (GPCNCs) are significant components of the genital microbiota in sheep and goats. However, characterizing them can be difficult due to overlapping culture features and the limited information on their susceptibility to antibiotics. In this study, 97 foreskin and 13 vaginal swabs were investigated using a culturomic approach. Of 110 animals, 76 (69.09 %) hosted GPCNCs, including strains from Streptococcaceae (37, 33.64 %), Aerococcaceae (30, 27.27 %), Enterococcaceae (6, 5.45 %) and other minor species. With increasing antimicrobial resistance rates in livestock, surveillance programs are globally required, so we conducted a pilot study on GPCNCs isolated from the genital mucosa surfaces of sheep and goats using the minimal inhibitory concentration assay (MIC). Due to gaps in interpretative standard breakpoints, normalized resistance interpretation was used for setting epidemiological susceptibility cut-off values (COWTs). Of 57 suitable strains, the majority (80.71 %) showed high COWTs with decrease susceptibility to at least one antimicrobial class, with 22.81 % displaying multiresistant profiles. Of interest, combined resistances to beta-lactams, macrolides, lincosamides, and tetracyclines were detected in strains of Streptococcus plurianimalium. Further combinations, including resistance to beta-lactams, pleuromutilins, aminoglycosides, and lincosamides, were also recorded in both Streptococcus uberis and Enterococcus spp. strains. Being beta-lactams, macrolides, and tetracyclines the most used antibiotics in livestock worldwide, our results highlight the need for their prudent use. Collectively, our findings highlight that small ruminant genital microbiota can serve as reservoirs for opportunistic severe pathogens, often zoonotic, carrying multidrug resistances, thus standing for high risks for both animals and humans.

Tratamiento de las pápulas perladas peneanas mediante uso de láser de dióxido de carbono fraccionado / Treatment of pearly penile papules using fractionated carbon dioxide laser

Las pápulas perladas del pene son angiofibromas acrales, lesiones asintomáticas benignas, no infecciosas, ubicadas alrededor del glande masculino. Debido su aspecto suelen causar angustia, disminución de autoestima y dificultades en la intimidad sexual. Todo esto lleva a un deterioro de la calidad de vida sexual. Métodos. Se incluyeron pacientes varones con pápulas perladas peneanas que recibieron tratamiento con láser de dióxido de carbono fraccionado entre diciembre de 2023 y abril de 2014. Se trataron en total 75 pacientes. Previo al tratamiento se les realizó una encuesta para saber si había realizado consulta previa con especialista y los motivos para realizar el tratamiento. Se les realizó el primer control postratamiento entre 10 a 15 días post tratamiento. En caso de presentar pápulas perladas peneanas residuales se procedió a un segundo o tercer tratamiento. Se les realizó segundo y tercer control postratamiento entre 10 a 15 días postratamiento. En dicho segundo o tercer control se les realizó encuesta de satisfacción. Resultados. Todos los pacientes incluidos en nuestro estudio informaron que se lograron eliminar sus pápulas perladas peneanas: 13% (10 pacientes) en primera sesión, 82% (62 pacientes) en segunda sesión y solo 4% (3 pacientes) requirieron una tercera sesión. Conclusiones. Las pápulas perladas peneanas, aun siendo de etiología benigna y no contagiosa, reducen significativamente la calidad de vida sexual de los varones, especialmente los jóvenes, y merecen de un tratamiento médico. El tratamiento de las pápulas perladas peneanas mediante uso de láser de dióxido de carbono fraccionado demostró ser un tratamiento rápido, seguro y eficaz

Pearly penile papules are acral angiofi bromas, benign, non-infectious, asymptomatic lesions around the male glans. Due to their appearance, they often cause distress, decreased self-esteem, and difficulties in sexual intimacy. All this leads to a deterioration in the quality of sexual life. Methods. Male patients with pearly penile papules who received fractionated carbon dioxide laser treatment between December 2023 and April 2014 were included. A total of 75 patients were treated. Before the treatment, a survey was conducted to fi nd out if they had previously consulted with a specialist and what motivated them to undergo the treatment. They underwent their first post-treatment control between 10 to 15 days after treatment. If residual pearly penile papules were present, a second or third treatment was performed. A second and third post-treatment control was carried out between 10 to 15 days after treatment. In said second or third control, a satisfaction survey was carried out. Results. All patients included in our study reported that their pearly penile papules were eliminated: 13% (10 patients) in the fi rst session, 82% (62 patients) in the second session, and only 4% (3 patients) required a third session. Conclusions. Pearly penile papules, even though they are of benign and non-contagious etiology. They signifi cantly reduce the quality of sexual life of men, especially young men, and deserve medical treatment. The treatment of pearly penile papules using fractionated carbon dioxide laser proved to be fast, safe, and effective.

Decellularized skin pretreatment by monophosphoryl lipid A and lactobacillus casei supernatant accelerate skin recellularization.

BACKGROUND: Bioscaffolds and cells are two main components in the regeneration of damaged tissues via cell therapy. Umbilical cord stem cells are among the most well-known cell types for this purpose. The main objective of the present study was to evaluate the effect of the pretreatment of the foreskin acellular matrix (FAM) by monophosphoryl lipid A (MPLA) and Lactobacillus casei supernatant (LCS) on the attraction of human umbilical cord mesenchymal stem cells (hucMSC). METHODS AND RESULTS: The expression of certain cell migration genes was studied using qRT-PCR. In addition to cell migration, transdifferentiation of these cells to the epidermal-like cells was evaluated via immunohistochemistry (IHC) and immunocytochemistry (ICC) of cytokeratin 19 (CK19). The hucMSC showed more tissue tropism in the presence of MPLA and LCS pretreated FAM compared to the untreated control group. We confirmed this result by scanning electron microscopy (SEM) analysis, glycosaminoglycan (GAG), collagen, and DNA content. Furthermore, IHC and ICC data demonstrated that both treatments increase the protein expression level of CK19. CONCLUSION: Pretreatment of acellular bioscaffolds by MPLA or LCS can increase the migration rate of cells and also transdifferentiation of hucMSC to epidermal-like cells without growth factors. This strategy suggests a new approach in regenerative medicine.

Preputial pedicle flap ICG blood flow assessment during proximal hypospadias repair: Development of a standardized protocol.

INTRODUCTION: Blood supply to preputial flap drives outcomes of hypospadias repair. Unfortunately, we only have surgeon's subjective assessment to evaluate flap perfusion which may not be accurate. Indocyanine green (ICG) has been used in a multitude of surgeries for perfusion assessment, however, no standardized protocol has been described for use of ICG in hypospadias repairs. The aim of this study is to develop a standardized protocol of ICG use in proximal hypospadias and establish perfusion patterns of preputial flaps. STUDY DESIGN: A pilot study was conducted using ICG in patients with proximal hypospadias undergoing first stage repair with a preputial flap. The Stryker SPY PHI system and novel quantification software, SPY-QP, were used for ICG imaging. An adaptive approach was taken to develop and implement a standardized protocol (Summary Figure). Per the protocol, ICG was administered at 3 time points which were felt to be critical for assessment of flap perfusion. Of the study patients who have undergone second stage repair, ICG was also used to reassess the flap prior to tubularization of the urethra. RESULTS: A total of 14 patients underwent first stage hypospadias repair with preputial flaps and intraoperative use of ICG. Median ICG uptake of the prepuce after degloving (dose 1) was 58.5% (IQR 43-76). ICG uptake decreased after flap harvest and mobilization (dose 2) with a median ICG uptake of 34% (IQR 26-46). ICG uptake remained stable after securing the flap in place and closing the skin (dose 3) with a median ICG uptake of 34% (IQR 25-48). ICG was able to delineate subtle findings in the preputial flaps not visible to the naked eye and in one case impacted intraoperative decision making. To date, 5 patients have undergone second stage repair. Flap assessment prior to tubularization of the urethra showed hypervascularity with a median ICG uptake of 159%. CONCLUSIONS: A standardized protocol for ICG use in proximal hypospadias was successfully developed and implemented. ICG uptake in the preputial flap decreased with increasing manipulation and mobilization of the flap. ICG was able to detect changes to flap perfusion which were not able to be seen with the naked eye. Reliance on surgeon's subjective assessment of flap perfusion may be inadequate and ICG could provide a useful tool for surgeons to improve preputial flap outcomes. ICG may also enhance the learning experience for trainees and early career urologists in these complex surgeries.

Injectable hydrogel harnessing foreskin mesenchymal stem cell-derived extracellular vesicles for treatment of chronic diabetic skin wounds.

Chronic skin wounds are a serious complication of diabetes with a high incidence rate, which can lead to disability or even death. Previous studies have shown that mesenchymal stem cells derived extracellular vesicles (EVs) have beneficial effects on wound healing. However, the human foreskin mesenchymal stem cell (FSMSCs)-derived extracellular vesicle (FM-EV) has not yet been isolated and characterized. Furthermore, the limited supply and short lifespan of EVs also hinder their practical use. In this study, we developed an injectable dual-physical cross-linking hydrogel (PSiW) with self-healing, adhesive, and antibacterial properties, using polyvinylpyrrolidone and silicotungstic acid to load FM-EV. The EVs were evenly distributed in the hydrogel and continuously released. In vivo and vitro tests demonstrated that the synergistic effect of EVs and hydrogel could significantly promote the repair of diabetic wounds by regulating macrophage polarization, promoting angiogenesis, and improving the microenvironment. Overall, the obtained EVs-loaded hydrogels developed in this work exhibited promising applicability for the repair of chronic skin wounds in diabetes patients.

Foreskin neurovascular structure: A histological analysis comparing 0-3 years and 6-11 years children.

BACKGROUND: Circumcision is a surgical operation that is frequently performed throughout the world due to religious, cultural, and medical reasons. The best age for circumcision is still debatable, with different procedures depending on geography, culture, and surgeon preference. OBJECTIVE: This study aims to immunohistochemical examination using S100 staining and histologically evaluate the neurovascular structures in foreskin samples obtained from children aged 0-3 years and 6-11 years. The goal is to provide guidance in determining an appropriate age for circumcision based on these data. STUDY DESIGN: Concerns regarding potential effects on glans sensitivity and sexual function led to the investigation and comparison of sensory innervation in the foreskin of children aged 0-3 and 6-11 years, a total 54 samples, divided into pre-phallic (0-3 years) and post-phallic (6-11 years) groups, were examined. The mean number of Meissner and Pacinian corpuscles, Ruffini endings, free nerve endings and the diameters of arteries were investigated. RESULTS: Our findings show that compared to the 6-11 age group, the 0-3 age group had considerably lower sensory innervation in terms of, Meissner's corpuscles, Pacinian corpuscles, Ruffini endings and free nerve endings. Additionally, the diameter of arteries was noticeably smaller in the 0-3 age group. CONCLUSIONS: In conclusion, this study supports the idea that circumcision performed in the early years of life may be associated with less adverse effects on neurovascular structures.

Histological and morphological development of the prepuce from birth to prepubertal age.

PURPOSE: To study the histological changes of the preputial tissue from birth to prepubertal age in order to define unnoticed morphological changes. MATERIALS AND METHODS: Prepuce samples were obtained from 79 healthy boys who underwent routine ritual circumcision. Specimens were divided into six groups according to the boys' age: newborn, 0-1 year of age, 2-3 years of age, 4-5 years of age, 6-7 years of age, and 8-9 years of age. Histologic analysis of the specimens was performed by H&E, Masson's trichrome, Verhoeff-Von Gieson, immunohistochemical staining. RESULTS: Microscopic examinations showed that average epithelial thickness increased after the neonatal period (p=0.001). When collagen fiber density was evaluated, no significant differences between groups were found (p=0.083). When the elastic fibers in the dermis were evaluated, it was determined that the number and thickness of elastic fibers increased with age. Immunohistochemical examinations showed that the number of peripheral nerves marked with S100 was lower in the neonatal period than at other ages (p=0.048). When the vessels marked with CD105 antibody were counted, there was no significant difference between the groups (p=0.078). CONCLUSIONS: This is the first study to examine the age-related structure of connective tissue elements in the foreskin. Our results showed that the prepuce's prepubertal maturation process is continuous, and the first 2 years of life are appropriate not only in relation to the physiological effects of age but also the optimum structural changes for wound healing, such as vessel diameter, epithelium thickness, peripheral nerve count.

Effect of cryopreservation on CD4+ T cell subsets in foreskin tissue.

Voluntary medical male circumcision (VMMC) reduces HIV acquisition by at least 60%, but the determinants of HIV susceptibility in foreskin tissues are incompletely understood. Flow cytometry is a powerful tool that helps us understand tissue immune defenses in mucosal tissue like the inner foreskin, but foreskin flow cytometry has only been validated using fresh tissue samples. This restricts immune analyses to timepoints immediately after surgical acquisition and hinders research in this area. We compared fresh analysis with whole tissue cryopreservation and later thawing and digestion to analyze CD4+ T cell populations relevant to HIV susceptibility (CCR5, CD25, CD127, CCR4, CXCR3, CCR6, CCR10, HLA-DR, and CD38). Eight foreskin samples from HIV-negative males aged >18 years were collected after VMMC. For each sample, half the foreskin was immediately cryopreserved for later digestion and flow cytometry analysis, while the remaining tissues were analyzed fresh. We demonstrate no significant impact of cryopreservation on CD4+ T cell expression of CD25, CCR4, CCR6, HLA-DR, CCR10, or CD127. Although expression levels of CCR5, CD38, and CXCR3 were increased after cryopreservation, the relative ranking of participants was retained. In conclusion, cryopreserved foreskin tissues may be suitable for subsequent digestion and flow cytometry phenotyping of HIV-susceptible T cell populations.

Extracellular vesicles derived from human foreskin cells (hFS-Exo) accelerate cell migration and angiogenesis through MAPK pathway: an in vitro study.

BACKGROUND: Wound healing is one of the important processes in the body. Attempts to create new drugs are of interest due to the side effects of natural and chemical wound healing compounds. To overcome this obstacle, stem cells have been used as healing agents. However, both difficulties in collection and risks such as rejection and teratoma in the recipient body have limited the use of stem cells, directly. Since the potential content of the stem cells can be transferred to the recipient cells by vesicles, small extracellular vesicles have recently become prominent agents. METHODS AND RESULTS: The wound-healing effect of extracellular vesicles derived from foreskin cells was investigated in both keratinocyte and endothelial cells. Migration assay, RT-PCR, Col1a1 ELISA and Western Blot experiments were utilized to reveal healing effect of EVs and its possible molecular pathways. EV-treated groups exhibited more proliferative, invasive, and migrative characteristics. When comparing to the control group, new vessel formation was induced in EV groups. An increase in gene levels of growth factors related to wound healing and change in the mitogen-activated protein kinase (MAPK) signaling pathway proteins in EV-treated groups were determined. Possible molecular mechanisms underlying cell movements were associated with the MAPK pathway. It was found that human foreskin cell EVs (hFS-Exo) may have a potential to heal wounds in a short period of time by triggering the MAPK pathway. CONCLUSIONS: hFS-Exo could be a new promising wound healing agent in the future.

Comparison between a deep-learning and a pixel-based approach for the automated quantification of HIV target cells in foreskin tissue.

The availability of target cells expressing the HIV receptors CD4 and CCR5 in genital tissue is a critical determinant of HIV susceptibility during sexual transmission. Quantification of immune cells in genital tissue is therefore an important outcome for studies on HIV susceptibility and prevention. Immunofluorescence microscopy allows for precise visualization of immune cells in mucosal tissues; however, this technique is limited in clinical studies by the lack of an accurate, unbiased, high-throughput image analysis method. Current pixel-based thresholding methods for cell counting struggle in tissue regions with high cell density and autofluorescence, both of which are common features in genital tissue. We describe a deep-learning approach using the publicly available StarDist method to count cells in immunofluorescence microscopy images of foreskin stained for nuclei, CD3, CD4, and CCR5. The accuracy of the model was comparable to manual counting (gold standard) and surpassed the capability of a previously described pixel-based cell counting method. We show that the performance of our deep-learning model is robust in tissue regions with high cell density and high autofluorescence. Moreover, we show that this deep-learning analysis method is both easy to implement and to adapt for the identification of other cell types in genital mucosal tissue.