Flow cytometric quantification of apoptotic and proliferating cells applying an improved method for dissociation of spheroids.

Spheroids are a promising tool for many cell culture applications, but their microscopic analysis is limited. Flow cytometry on a single cell basis, which requires a gentle but also efficient dissociation of spheroids, could be an alternative analysis. Mono-culture and coculture spheroids consisting of human fibroblasts and human endothelial cells were generated by the liquid overlay technique and were dissociated using AccuMax as a dissociation agent combined with gentle mechanical forces. This study aimed to quantify the number of apoptotic and proliferative cells. We were able to dissociate spheroids of differing size, age, and cellular composition in a single-step dissociation protocol within 10 min. The number of single cells was higher than 95% and in most cases, the viability of the cells after dissociation was higher than 85%. Coculture spheroids exhibited a higher sensitivity as shown by lower viability, higher amount of cellular debris, and a higher amount of apoptotic cells. Considerable expression of the proliferation marker Ki67 could only be seen in 1-day-old spheroids but was already downregulated on Day 3. In summary, our dissociation protocol enabled a fast and gentle dissociation of spheroids for the subsequent flow cytometric analysis. The chosen cell type had a strong influence on cell viability and apoptosis. Initially high rates of proliferative cells decreased rapidly and reached values of healthy tissue 3 days after generation of the spheroids. In conclusion, the flow cytometry of dissociated spheroids could be a promising analytical tool, which could be ideally combined with microscopic techniques.

Dermal regeneration sheet Integra® in management of recurrent Urethrocutaneous fistula after hypospadias surgery.

BACKGROUND: Urethrocutaneous fistula UCF is the most common complication following surgical repair of hypospadias. Currently, the surgical technique mostly used to prevent recurrence employs preputial dartos or testicular tunica vaginalis flaps as a urethral covering. However, autologous tissues are limited in patients with multiple surgeries, and the use of biomaterials as a urethral coverage may represent a good alternative. OBJECTIVE: The goal of the present study is to assess the results and complications of recurrent UCF correction using a dermal bovine regeneration sheet as a urethral covering. MATERIALS AND METHOD: From May 2016 to January 2019, all patients with recurrent UCF of the authors center were repaired using this technique. The inclusion criteria were patients who had undergone one or more unsuccessful UCF repair surgeries and the absence of preputial tissue. The informed consent has been signed by all the patients. Patients were examined in outpatient consultations where their urinary stream was evaluated and a physical examination of the penis was conducted. RESULTS: A total of 12 patients and 13 UCFs were included in the study. The median follow-up was 18 months, (range: 4-26), and only two patients (15%) developed a recurrence of UCF. No complications were observed in the remaining patients (85%) during their evolution. No patient developed a fibrosis increase or loss of elasticity of the tissues in contact with the dermal matrix. CONCLUSION: The use of an Integra® sheet as a urethral covering during urethral fistula surgery appears to be a safe, effective, and easily reproducible option. However, prospective studies with larger numbers of patients should be performed to corroborate these results.

Anti-proliferative and anti-migratory effects of hyperforin in 2D and 3D artificial constructs of human dermal fibroblasts - A new option for hypertrophic scar treatment?

Hyperforin (HYP), one of the main bioactive compounds in extracts of Hypericum perforatum, is a potential drug candidate for the treatment of skin diseases. Since extracts have proven to support wound healing, in the present study effects of HYP on human dermal fibroblasts (HDF) were evaluated in 2D and 3D in vitro dermal constructs. Viability and cytotoxicity assays as well as a live-dead cell staining were performed to test at which concentration HYP reduces viability and/or shows cytotoxicity. Furthermore a differentiation between cytotoxic, anti-proliferative and anti-migratory effects was done. For the latter purpose a 2D migration assay was performed. HDF-induced contraction of a 3D artificial dermal (AD) construct was determined at given HYP concentration. Induction of apoptosis was examined by determination of caspase 3/7 activities. HYP reduced viability of HDF down to 70% at concentrations of 5-10µM. This decrease was not due to cytotoxicity but to a reduction in proliferation as shown from both the proliferation assay and the cytotoxicity assay as well as from live-dead cell staining. The 2D migration assay showed that HYP reduced migration activity of HDF cells at a concentration of 10µM. At this concentration HYP also reduced the HDF-induced contraction of collagen gels as 3D AD constructs. Apoptotic effects of HYP were excluded performing a caspase 3/7 activity detecting assay. The results show for the first time that HYP may be rather a potential candidate for treatment of hypertrophic scars than promoting effects which are understood as important in wound healing.

Identification and Evaluation of New Immunoregulatory Genes in Mesenchymal Stromal Cells of Different Origins: Comparison of Normal and Inflammatory Conditions.

BACKGROUND Mesenchymal stromal cells (MSCs) possess potent immunomodulatory properties that increase their value as a cell-based therapeutic tool for managing various immune-based disorders. Over the past years, accumulated results from trials using MSCs-based therapy have shown substantial contradictions. Although the reasons underlying these discrepancies are still not completely understood, it is well known that the immunomodulatory activities mediated by distinct MSCs differ in a manner dependent on their tissue origin and adequate response to inflammation priming. Thus, characterization of new molecular pathway(s) through which distinct MSC populations can exert their immunomodulatory effects, particularly during inflammation, will undoubtedly enhance their therapeutic potential. MATERIAL AND METHODS After confirming their compliance with ISCT criteria, quantitative real time-PCR (qRT-PCR) was used to screen new immunoregulatory genes in MSCs, derived from adipose tissue, foreskin, Wharton's jelly or the bone-marrow, after being cultivated under normal and inflammatory conditions. RESULTS FGL2, GAL, SEMA4D, SEMA7A, and IDO1 genes appeared to be differentially transcribed in the different MSC populations. Moreover, these genes were not similarly modulated following MSCs-exposure to inflammatory signals. CONCLUSIONS Our observations suggest that these identified immunoregulatory genes may be considered as potential candidates to be targeted in order to enhance the immunomodulatory properties of MSCs towards more efficient clinical use.

Foreskin as a source of immunotherapeutic mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) have well-characterized properties and thus represent an attractive cell population for use in several therapeutic applications. Due to the limitations and inconveniences associated with classical sources of MSCs, the identification and characterization of alternative sources are required for safe and efficient cell therapy. The skin tissue is currently referred to as a reservoir of cells with therapeutically relevant functions. Historically considered biological waste, foreskin (FSK) is increasingly used to provide immunotherapeutic MSCs for medicinal products. This review discusses for the first time the nature and profile of MSCs within the foreskin tissue and, in particular, their immunobiology. A better immunological characterization and understanding of foreskin-derived cells will be critical for improving MSC-based cellular strategies for immunotherapeutic applications.

Significant improvement of direct reprogramming efficacy of fibroblasts into progenitor endothelial cells by ETV2 and hypoxia.

BACKGROUND: Endothelial progenitor cell (EPC) transplantation is a promising therapy for ischemic diseases such as ischemic myocardial infarction and hindlimb ischemia. However, limitation of EPC sources remains a major obstacle. Direct reprogramming has become a powerful tool to produce EPCs from fibroblasts. Some recent efforts successfully directly reprogrammed human fibroblasts into functional EPCs; however, the procedure efficacy was low. This study therefore aimed to improve the efficacy of direct reprogramming of human fibroblasts to functional EPCs. METHODS: Human fibroblasts isolated from foreskin were directly reprogrammed into EPCs by viral ETV2 transduction. Reprogramming efficacy was improved by culturing transduced fibroblasts in hypoxia conditions (5 % oxygen). Phenotype analyses confirmed that single-factor ETV2 transduction successfully reprogrammed dermal fibroblasts into functional EPCs. RESULTS: Hypoxia treatment during the reprogramming procedure increased the efficacy of reprogramming from 1.21 ± 0.61 % in normoxia conditions to 7.52 ± 2.31 % in hypoxia conditions. Induced EPCs in hypoxia conditions exhibited functional EPC phenotypes similar to those in normoxia conditions, such as expression of CD31 and VEGFR2, and expressed endothelial gene profiles similar to human umbilical vascular endothelial cells. These cells also formed capillary-like networks in vitro. CONCLUSION: Our study demonstrates a new simple method to increase the reprogramming efficacy of human fibroblasts to EPCs using ETV2 and hypoxia.

Actomyosin Cortical Mechanical Properties in Nonadherent Cells Determined by Atomic Force Microscopy.

The organization of filamentous actin and myosin II molecular motor contractility is known to modify the mechanical properties of the cell cortical actomyosin cytoskeleton. Here we describe a novel method, to our knowledge, for using force spectroscopy approach curves with tipless cantilevers to determine the actomyosin cortical tension, elastic modulus, and intracellular pressure of nonadherent cells. We validated the method by measuring the surface tension of water in oil microdrops deposited on a glass surface. We extracted an average tension of T ∼ 20.25 nN/µm, which agrees with macroscopic experimental methods. We then measured cortical mechanical properties in nonadherent human foreskin fibroblasts and THP-1 human monocytes before and after pharmacological perturbations of actomyosin activity. Our results show that myosin II activity and actin polymerization increase cortex tension and intracellular pressure, whereas branched actin networks decreased them. Interestingly, myosin II activity stiffens the cortex and branched actin networks soften it, but actin polymerization has no effect on cortex stiffness. Our method is capable of detecting changes in cell mechanical properties in response to perturbations of the cytoskeleton, allowing characterization with physically relevant parameters. Altogether, this simple method should be of broad application for deciphering the molecular regulation of cell cortical mechanical properties.

Activity of the external urethral sphincter evoked by genital stimulation in male rats.

AIMS: To determine whether the external urethral sphincter (EUS) fasciculi of male rats respond to the mechanical stimulation of genital structures and to characterize the pattern of the electromyographic (EMG) activity of the three regions of the EUS: the cranial (CrEUS), the medial (MeEUS) and the caudal (CaEUS). METHODS: Electromyographic signals were recorded from the CrEUS, MeEUS and CaEUS regions of the male rat's EUS, before, during and after the mechanical stimulation of the urogenital structures. RESULTS: The CrEUS, MeEUS and CaEUS regions responded when brushing and squeezing the foreskin and glans as well as to penile and prostatic urethral distension. The CaEUS EMG amplitude (P < 0.01) and frequency (P < 0.05) were lower in comparison to the CrEUS and MeEUS responses to the mechanical stimulation. In addition, the CaEUS was characterized by a short or no afterdischarge. In contrast, the CrEUS and MeEUS responded by presenting a long discharge after the penile or prostatic urethral distension. CONCLUSIONS: The activity of the EUS is modulated by both, cutaneous and visceral genitourinary stimuli, with motor units being activated by mechanoreceptors located in the foreskin, glans, bladder, and urethra. The CrEUS, MeEUS and CaEUS have differential EMG patterns, indicating that the EUS consists of three anatomically and functionally different regions. Precise coordination in the muscular activity of these regions may be crucial for the control of male expulsive urethral functions, i.e., during voiding and ejaculation. Neurourol. Urodynam. 35:914-919, 2016. © 2015 Wiley Periodicals, Inc.

Risks, benefits, complications and harms: neglected factors in the current debate on non-therapeutic circumcision.

UNLABELLED: Much of the contemporary debate about the propriety of non-therapeutic circumcision of male infants and boys revolves around the question of risks vs. BENEFITS: With its headline conclusion that the benefits outweigh the risks, the current circumcision policy of the American Academy of Pediatrics [AAP] (released 2012) is a typical instance of this line of thought. Since the AAP states that it cannot assess the true incidence of complications, however, critics have pointed out that this conclusion is unwarranted. In this paper it is argued that the AAP's conclusion is untenable not only for empirical reasons related to lack of data, but also for logical and conceptual reasons: the concept of risk employed-risk of surgical complications-is too narrow to be useful in the circumcision debate. Complications are not the only harms of circumcision: the AAP and other parties debating the pros and cons of circumcision should conceptualize their analysis more broadly as risk of harm vs. prospect of benefit, thereby factoring in the value of the foreskin to the individual and the physical and ethical harms of removing it from a non-consenting child.

Age-related changes in the composition of the cornified envelope in human skin.

The main function of the epidermis is to protect us against a multitude of hostile attacks from the environment. Its main cell type, the keratinocytes have a sophisticated system of different proteins and lipids available to form the cornified envelope, which is responsible for the barrier function of the skin. During ageing, dramatic changes are taking place. Some proteins of the SPRR-, S100- and LCE3-family are massively up-regulated, whereas others like loricrin, filaggrin and the LCE1&2 protein families are significantly down-regulated. The latter ones are known to be under control of calcium and/or 'calcium response elements'. We were able to show that the calcium peak specific for the stratum granulosum, which is the site where loricrin and the LCE1&2 families are synthesized, is reduced during ageing. The resulting cornified envelope in old skin has an extensively changed composition on the molecular level compared to young skin. This knowledge is of critical importance to understand chronic wound formation and ulcers in old age.

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.

The 2010 Royal Australasian College of Physicians' policy statement 'Circumcision of infant males' is not evidence based.

Infant male circumcision (MC) is an important issue guided by Royal Australasian College of Physicians (RACP) policy. Here we analytically review the RACP's 2010 policy statement 'Circumcision of infant males'. Comprehensive evaluation in the context of published research was used. We find that the Statement is not a fair and balanced representation of the literature on MC. It ignores, downplays, obfuscates or misrepresents the considerable evidence attesting to the strong protection MC affords against childhood urinary tract infections, sexually transmitted infections (human immunodeficiency virus, human papilloma virus, herpes simplex virus type 2, trichomonas and genital ulcer disease), thrush, inferior penile hygiene, phimosis, balanoposthitis and penile cancer, and in women protection against human papilloma virus, herpes simplex virus type 2, bacterial vaginosis and cervical cancer. The Statement exaggerates the complication rate. Assertions that 'the foreskin has a functional role' and 'is a primary sensory part of the penis' are not supported by research, including randomised controlled trials. Instead of citing these and meta-analyses, the Statement selectively cites poor quality studies. Its claim, without support from a literature-based risk-benefit analysis, that the currently available evidence does 'not warrant routine infant circumcision in Australia and New Zealand' is misleading. The Statement fails to explain that performing MC in the neonatal period using local anaesthesia maximises benefits, safety, convenience and cost savings. Because the RACP's policy statement is not a fair and balanced representation of the current literature, it should not be used to guide policy. In the interests of public health and individual well-being, an extensive, comprehensive, balanced review of the scientific literature and a risk-benefit analysis should be conducted to formulate policy.

ZNF750 is a p63 target gene that induces KLF4 to drive terminal epidermal differentiation.

Disrupted epidermal differentiation characterizes numerous diseases that impact >25% of the population. In a search for dominant mediators of differentiation, we defined a requirement for ZNF750 in terminal epidermal differentiation. ZNF750 controlled genes mutated in numerous human skin diseases, including FLG, LOR, LCE3B, ALOXE3, and SPINK5. ZNF750 induced progenitor differentiation via an evolutionarily conserved C2H2 zinc finger motif. The epidermal master regulator, p63, bound the ZNF750 promoter and was necessary for its induction. ZNF750 restored differentiation to p63-deficient tissue, suggesting that it acts downstream of p63. A search for functionally important ZNF750 targets via analysis of ZNF750-regulated genes identified KLF4, a transcription factor that activates late epidermal differentiation. ZNF750 binds to KLF4 at multiple sites flanking the transcriptional start site and controls its expression. ZNF750 thus directly links a tissue-specifying factor, p63, to an effector of terminal differentiation, KLF4, and represents a potential future target for disorders of this process.

Possible function of the frenulum of prepuce in penile erection.

Fremulum of prepuce was the ruffle of foreskin while there was little about the function of fremulum. This study discusses the possible function of the frenulum of prepuce in penile erection. Twelve patients had premature ejaculation (PE) whose frenula were short. Two patients suffered unsatisfied intercourse whose frenula were damaged and departed 12 or 6 months earlier. We prolonged the short frenulum and reconstructed the ruptured frenulum. All patients reported satisfied sexual intercourse after 3-6 months. It is concluded that the frenulum is important in penile erection. PE might be treated by lengthening the frenulum.

Keratinocyte-specific stat3 heterozygosity impairs development of skin tumors in human papillomavirus 8 transgenic mice.

Human papillomaviruses (HPV) of the genus ß are thought to play a role in human skin cancers, but this has been difficult to establish using epidemiologic approaches. To gain insight into the transforming activities of ß-HPV, transgenic mouse models have been generated that develop skin tumors. Recent evidence suggests a central role of signal transducer and activator of transcription 3 (Stat3) as a transcriptional node for cancer cell-autonomous initiation of a tumor-promoting gene signature associated with cell proliferation, cell survival, and angiogenesis. Moreover, high levels of phospho-Stat3 have been detected in tumors arising in HPV8-CER transgenic mice. In this study, we investigate the in vivo role of Stat3 in HPV8-induced skin carcinogenesis by combining our established experimental model of HPV8-induced skin cancer with epidermis-restricted Stat3 ablation. Stat3 heterozygous epidermis was less prone to tumorigenesis than wild-type epidermis. Three of the 23 (13%) Stat3(+/-):HPV8 animals developed tumors within 12 weeks of life, whereas 54.3% of Stat3(+/+):HPV8 mice already exhibited tumors in the same observation period (median age for tumor appearance, 10 weeks). The few tumors that arose in the Stat3(+/-):HPV8 mice were benign and never progressed to a more malignant phenotype. Collectively, these results offer direct evidence of a critical role for Stat3 in HPV8-driven epithelial carcinogenesis. Our findings imply that targeting Stat3 activity in keratinocytes may be a viable strategy to prevent and treat HPV-induced skin cancer.

Proteomic profiling reveals a catalogue of new candidate proteins for human skin aging.

Studies of skin aging are usually performed at the genomic level by investigating differentially regulated genes identified through subtractive hybridization or microarray analyses. In contrast, relatively few studies have investigated changes in protein expression of aged skin using proteomic profiling by two-dimensional (2-D) gel electrophoresis and mass spectrometry, although this approach at the protein level is suggested to reflect more accurately the aging phenotype. We undertook such a proteomic analysis of intrinsic human skin aging by quantifying proteins extracted and fluorescently labeled from sun-protected human foreskin samples pooled from 'young' and 'old' men. In addition, we analyzed these candidate gene products by 1-D and 2-D western blotting to obtain corroborative protein expression data, and by both real-time PCR (RT-PCR) and microarray analyses to confirm expression at the mRNA level. We discovered 30 putative proteins for skin aging, including previously unrecognized, post-translationally regulated candidates such as phosphatidyl-ethanolamine binding protein (PEBP) and carbonic anhydrase 1 (CA1).

Cigarette smoke-induced Egr-1 represses T beta R-II expression in human skin dermal fibroblasts.

Tobacco smoking is one of the many factors that contribute to premature skin aging, but the exact mechanism by which smoking induces facial wrinkling is still poorly understood. To investigate the regulatory potential of early growth response-1 (Egr-1) on the premature skin aging by smoking, this study examined the hypothesis that cigarette smoke-induced Egr-1 represses T beta R-II expression in human skin dermal fibroblasts (HSDFs). The protein and mRNA expressions of Egr-1 and T beta R-II were detected using Western blot and real-time RTPCR in HSDFs after exposure to cigarette smoke extract (CSE). Egr-1 and T beta R-II promoter activities were analyzed in CSE-exposed fibroblasts using luciferase assay. T beta R-II promoter activity was also evaluated in HSDFs to be transfected with Egr-1 overexpression vector. To investigative Egr-1-specific effects, we utilized Egr-1 small interfering RNA (siRNA) to inhibit Egr-1 expression. The expressions of Egr-1 protein and mRNA were increased in a time and dose-dependent manner. CSE also induced Egr-1 at the transcription level. Egr-1 was induced though phosphorylation of Erk1/2 after CSE exposure in HSDFs. We also observed the immunostained Egr-1 proteins were mainly localized from the cytoplasm to the nucleus after CSE treatment by immunocytochemical analyzes. Furthermore, T beta R-II protein and mRNA levels were decreased in a dose-dependent manner by CSE and T beta R-II promoter activity was significantly repressed by CSE. HSDFs transfected with Egr-1 overexpression vector showed significantly reduced T beta R-II promoter activity. In addition, T beta R-II mRNA levels were upregulated in HSDFs transfected with Egr-1 siRNA, suggesting that T beta R-II expressional downregulation by CSE is induced via an Egr-1-dependent mechanism. This study suggests that the downregulation of T beta R-II expression by cigarette smoke-induced Egr-1 may contribute to smoking-induced premature skin aging.