Michael DiSandro

Growth Factors in Bladder Wound Healing

INTRODUCTION Surgical and traumatic injuries to the bladder initiate a complex series of biological processes that result in wound healing. This process involves cellular proliferation, migration and differentiation; removal of damaged tissue; and production of extracellular matrix all of which may be controlled by growth factors. In skin, keratinocyte growth factor (KGF) is induced following incisional injury. We hypothesize that in bladder wound healing KGF and other growth factors are induced to modulate tissue repair. METHODS We have created a model of surgical bladder injury in the rodent. At 12, 24 and 48 hrs and 5 and 7 days after injury, the bladder was bisected and total RNA extracted from the anterior or wounded half and posterior or non-wounded half. Histological analysis of the bladder wound was performed with Masons Trichrome and immunohistochemistry against smooth muscle alpha actin. RNase protection assays were performed to examine the expression of KGF, transforming growth factor (TGF)alpha and TGF beta 2 and 3 as well as the receptors for KGF and epidermal growth factor (EGF). Lastly, the effects of the exogenous administration of KGF on the bladder was tested on neonatal mice by daily injections of 5 micrograms KGF per gram body weight for 5 days. RESULTS At 12 hours after injury KGF mRNA expression in the anterior wounded bladder half and posterior non-wounded bladder half was 8 and 6 times higher respectively, compared to unoperated control bladders. A similar response was seen for TGF alpha, where the 12 hour mRNA expression was 4.5 times higher in the anterior wounded bladder half and 3.5 times higher in the posterior non-wounded bladder half compared to unoperated control bladders. The nadir mRNA expression for both KGF and TGF alpha occurred at 7 days after bladder injury and was the same as in unoperated control bladders. EGFR mRNA expression was approximately 2 times higher in both the anterior wounded and posterior non-wounded bladder halves compared to the nadir levels which occurred at 24 hours after injury. TGF beta 2 and beta 3 mRNA levels did not significantly change in either the anterior wounded or posterior non-wounded bladder halves. Exogenous KGF stimulation resulted in a marked urothelial proliferation when compared to age matched control animals. CONCLUSION During the early phases of bladder wound healing (12-24 hours post injury), mRNA for KGF and TGF alpha increased, whereas TGF beta 2 and beta 3 and the KGFR and EGFR remain unchanged. Additionally, exogenous KGF has a direct effect on urothelial proliferation. KGF and TGF alpha warrant further study as potential mediators of bladder wound healing.

Age at Orchiopexy and Testis Palpability Predict Germ and Leydig Cell Loss: Clinical Predictors of Adverse Histological Features of Cryptorchidism

PURPOSE We determined the relationship between clinical variables and testicular histopathological changes associated with decreased fertility potential in children with cryptorchidism. MATERIALS AND METHODS Testis biopsies of 274 children who underwent orchiopexy and concurrent testicular biopsy between 1991 and 2001 were analyzed for germ and Leydig cell loss, and testicular fibrosis. Multivariable logistic regression was used to determine if age at orchiopexy (analyzed as continuous and ordinal variables), preoperative testis palpability, unilateral vs bilateral disease and/or side of undescended testis was predictive of these pathological outcomes. RESULTS Age at orchiopexy was associated with germ and Leydig cell depletion. Each month of testis undescent was associated with development of moderate/severe germ cell depletion (OR 1.02 for each month of age, p <0.005) and Leydig cell loss (OR 1.01 for each month of age, p <0.02). Nonpalpable testes were associated with severe germ cell depletion. Children with palpable testes had lower odds of germ cell depletion than those with nonpalpable testes (OR 0.46, p <0.005). This finding corresponds to a significant 2% risk per month of severe germ cell loss and 1% risk per month of Leydig cell depletion for each month a testis remains undescended, and a 50% greater risk of germ cell depletion in nonpalpable relative to palpable cryptorchid testes. CONCLUSIONS Testes that remain undescended are associated with progressive loss of germ and Leydig cells, and nonpalpable testes predict severe germ cell loss.

The Effect of Testosterone on Androgen Receptors and Human Penile Growth

PURPOSE Recent rat studies suggest that early exposure to exogenous testosterone accelerates the loss of androgen receptors and compromises eventual penile length. In humans we hypothesize that down regulation of the androgen receptor is not the mechanism that stops penile growth. To test this hypothesis we investigated the effects of androgen deprivation and supplementation on the developing human penis. MATERIALS AND METHODS A total of 15 normal human fetal penises at 7 to 19 weeks of gestation (mean plus or minus standard deviation 12 +/- 4.5) was divided in half sagittally. Specimens were grafted beneath the renal capsule of male athymic nude mice or nude rats. Three groups of host animals were prepared, including 10 with no testosterone that were castrated at grafting, 15 with testosterone and 5 with super testosterone in which 50 mg. testosterone propionate pellets were implanted subcutaneously at grafting. Each fetal penile specimen was its own control, since half was implanted into an intact animal and the other into a castrated or super testosterone host. Six weeks after grafting the specimens were analyzed for gross size (length), histology and expression of androgen receptors. RESULTS All human fetal penile specimens grew from the nadir size and appeared as white exophytic growths on the surface of the host kidneys. Normal grafts were larger than castrate specimens (mean 6.9 +/- 2.1 versus 3.9 +/- 2.1 mm., p = 0.014). Mean length of the super testosterone specimens (7.3 +/- 2.3 mm.) was not significantly greater than that of normal specimens (p = 0.797). Histological analysis revealed that all specimens were composed of viable penile tissue. Cellular density of the castrate penises was approximately 2 times greater than that of the normal and super testosterone specimens (40.6 +/- 5.9 versus 25.1 +/- 2.8 cells per cm.2, p > 0.001), as calculated on enlarged micrographs. Supraphysiological doses of testosterone did not change the histology compared to controls. Immunohistochemical localization revealed androgen receptors expressed throughout the corporeal bodies, surrounding stroma and penile skin with intracellular localization to nucleus. The mean proportion of cells expressing androgen receptors was higher in the castrate (29.4 +/- 5.2 cells per cm.2) than in the normal (24.0 +/- 3.7) and super testosterone (24.7 +/- 4.5) grafts (p = 0.005). However, in regard to growth there was no change in the proportion of androgen receptor positive cells among the groups. CONCLUSIONS Testosterone influences penile growth, possibly as a result of extracellular stromal expansion. The number of androgen receptor positive cells in the human fetal penis did not change among the castrate, normal and super testosterone hosts. These experiments support the hypothesis that penile growth cessation is mediated by mechanisms other than down regulation of the androgen receptor. Furthermore, these data support the hypothesis that early administration of androgen to prepubertal male individuals does not result in a shorter phallus in adulthood.

Mesenchymal-epithelial interactions in bladder smooth muscle development: epithelial specificity.

PURPOSE We previously showed that mesenchymal-epithelial interactions are necessary for the development of bladder smooth muscle. Specifically without bladder epithelium embryonic bladder mesenchyme does not differentiate into smooth muscle. We determine whether this process is specific to bladder epithelium or whether epithelial cells from other organ systems induce bladder mesenchyme to differentiate into smooth muscle, as well as whether epithelial age is an important variable. MATERIALS AND METHODS We recombined 14-day bladder mesenchyme before smooth muscle differentiation with rat epithelium from 14-day, 19-day, newborn and adult bladder, ureter, colon, ileum, stomach, cornea and epidermis. In addition, bladder epithelium was recombined with 14-day embryonic small intestinal, 14-day embryonic gastric and newborn seminal vesicle mesenchyme. All tissue recombinants were grafted under the renal capsule of an adult rat syngeneic host for 3 weeks. RESULTS Immunohistochemical analysis with antibodies directed against smooth muscle alpha-actin revealed that all epithelial types studied induced bladder mesenchyme to differentiate into smooth muscle, although to different degrees. Induction of smooth muscle was independent of urothelial age. In addition, bladder epithelium induced intestinal, gastric and seminal vesicle mesenchyme to differentiate into smooth muscle and express an overall morphological pattern indicative of the bladder fibromuscular wall. CONCLUSIONS The mechanism whereby urothelium induces bladder mesenchyme to differentiate into smooth muscle is not specific to embryonic urothelium. Older urothelium and heterotypic epithelium also induce smooth muscle differentiation. With the common use of bowel, stomach and ureteral segments for bladder augmentation it is important to understand the interaction of different types of epithelium with the native bladder.

Mesenchymal-epithelial interactions in the bladder

SummaryDuring bladder development, undifferentiated mesenchymal and epithelial cells undergo an orderly sequence of differentiation defined by the expression of smooth-muscle (α-actin, myosin, vinculin, desmin, vimentin, and laminin) and epithelial (cytokeratins 5, 7, 8, 14, 18 and 19) protein markers. This process requires mesenchymal-epithelial interactions with bladder epithelium (urothelium) necessary for the differentiation of bladder smooth muscle. Peptide growth factors such as keratinocyte growth factor (KGF) and transforming growth factors (TGF) α and β are likely candidates as mediators of these mesenchymal-epithelial interactions. Transcripts for KGF, TGF α, and TGF β are regulated during bladder development and during smooth-muscle hypertrophy secondary to bladder-outlet obstruction. Finally, two experimental bladder models — (1) partial outlet obstruction and (2) regeneration of bladder smooth muscle into an acellular tissue matrix — are described in the context of mesenchymal-epithelial interactions in the bladder.

Outcomes of continent catheterizable stomas for urinary and fecal incontinence: comparison among different tissue options.

To retrospectively review the outcome of appendix, transverse tubularized intestine segments, caecal flap, gastric tube and others tissue options used as a continent stoma for urinary and fecal incontinence.

MESENCHYMAL-EPITHELIAL INTERACTIONS IN BLADDER SMOOTH MUSCLE DEVELOPMENT: EFFECTS OF THE LOCAL TISSUE ENVIRONMENT

PURPOSE We have previously shown that mesenchymal-epithelial interactions are necessary for the development of bladder smooth muscle. Specifically without fetal or adult urothelium embryonic rat bladder mesenchyma does not differentiate into smooth muscle. The mechanism responsible for this interaction is not known, although it is postulated that diffusable growth factors have a role. Our hypothesis is that diffusable factors within adult rat bladders influence smooth muscle differentiation. MATERIALS AND METHODS Chimeric bladders were created by surgically implanting 14-day embryonic rat bladder mesenchyma before smooth muscle differentiation into the detrusor space of adult syngeneic hosts to test whether the host urothelium would induce smooth muscle differentiation without being in direct contact with fetal bladder mesenchymal tissue. Sub-detrusor pockets were created between the serosa and smooth muscle layer, between the smooth muscle layer and lamina propria, and between the lamina propria and urothelium in direct contact with urothelium. Controls consisted of intact 14-day embryonic rat bladders with the urothelium not removed, and 14-day embryonic bladder mesenchyma recombined with urothelium (direct contact) placed within the sub-detrusor space of the bladder and under the renal capsule. RESULTS Immunohistochemical staining with antibodies directed against smooth muscle alpha-actin and urothelium (cytokeratin 7) revealed smooth muscle differentiation in intact embryonic bladders and bladder mesenchyma plus urothelium recombinants in contrast to bladder mesenchyma alone, which had no alpha-actin staining (morphometric smooth muscle analysis p = 0). There was no alpha-actin staining in chimeric bladders even when bladder mesenchymal grafts were placed directly in contact with host urothelium. In addition, bladder mesenchyma plus urothelial recombinants within the host bladder had less alpha-actin staining than their counterparts placed under the renal capsule (p = 0.001). CONCLUSIONS A diffusable factor most likely exists within adult rat bladders that inhibits smooth muscle differentiation.

Gender Identity in Disorders of Sex Development: Review Article

OBJECTIVES Many concerns have been raised regarding the treatment and long-term outcome of infants born with complex genital anomalies. Debate among clinicians, psychologists, ethicists, and patient advocate groups regarding the optimal management of these individuals is ongoing. Although determining the most appropriate gender is a difficult task, this review will help clarify some of the issues at hand. METHODS A literature review which addresses the challenges of advising families about gender identity in infants and children with disorders of sex development. RESULTS The evidence for endocrine effects on neurobiological development with regard to sexual behavior is compelling, although the existing outcome studies are largely anecdotal and somewhat contradictory. CONCLUSIONS Gender assignment in infants born with a disorder of sex development remains only one of the many difficult decisions faced by both the treatment team and the family. Improved long-term follow-up of these patients will provide much needed feedback on previous and contemporary management.

Do adult men with untreated hypospadias have adverse outcomes? A pilot study using a social media advertised survey

OBJECTIVE Hypospadias is usually treated in childhood. Therefore, the natural history of untreated mild hypospadias is unknown. We hypothesized that men with untreated hypospadias, especially mild, do not have adverse outcomes. MATERIALS Facebook was used to advertise an electronic survey to men older than 18 years. Men with untreated hypospadias identified themselves and indicated the severity of hypospadias with a series of questions. Outcomes included: Sexual Health Inventory for Men (SHIM), penile curvature and difficulty with intercourse, International Prostate Symptom Score (IPSS), Penile Perception Score (PPS), psychosexual milestones, paternity, infertility, sitting to urinate, and the CDC HRQOL-4 module. RESULTS 736 men completed self-anatomy questions and 52 (7.1%) self-identified with untreated hypospadias. Untreated hypospadias participants reported worse SHIM (p < 0.001) and IPSS scores (p = 0.05), more ventral penile curvature (p = 0.003) and resulting difficulty with intercourse (p < 0.001), worse satisfaction with meatus (p = 0.011) and penile curvature (p = 0.048), and more sitting to urinate (p = 0.07). When stratified by mild and severe hypospadias, severe hypospadias was associated with more adverse outcomes than mild hypospadias. CONCLUSION Men with untreated hypospadias reported worse outcomes compared with non-hypospadiac men. Mild untreated hypospadias had fewer adverse outcomes than severe hypospadias. Research is needed to determine if treatment of childhood hypospadias improves outcomes in adults, especially for mild hypospadias.

Obesity Does Not Decrease the Accuracy of Testicular Examination in Anesthetized Boys With Cryptorchidism

PURPOSE Given that the prevalence of childhood obesity is increasing in the United States, we tested the timely hypothesis that obesity hinders physical examination based localization of the cryptorchid testis. MATERIALS AND METHODS Body mass index and percentiles of weight for height and body mass index for age were calculated for boys undergoing surgery for cryptorchidism at the University of California San Francisco Childrens Hospital and Childrens Hospital of Oakland. Two definitions of obesity were examined, ie greater than 85% or greater than 95% for either percentile. Patients were examined in the office and under general anesthesia before the skin incision. Intraoperative testicular location was recorded for each patient. The numbers of correct and incorrect preoperative determinations of testicular location were stratified by weight classification. Results were analyzed using contingency tables and Fishers exact test. RESULTS A total of 161 boys were recruited, accounting for 171 testes. The predictive value of palpating a suspected testis preoperatively with patients under anesthesia was greater than 95% for all weight classifications (p <0.0001). The predictive value of not palpating a testis preoperatively under anesthesia was greater than 56% for obese boys and greater than 42% for nonobese boys (p <0.0001). The concordance rates between examinations in the office and those performed under anesthesia were 90.9% and 82.7% for obese and nonobese boys, respectively (p = 0.51). The predictive value of not palpating a suspected cryptorchid testis in the office was higher in nonobese boys than in obese boys (81% vs 22%, p <0.0001). CONCLUSIONS In our series childhood obesity did not make preoperative testicular examinations under anesthesia less accurate. However, office examinations may be more accurate in nonobese boys.