Amniotic fluid stem cell-derived vesicles protect from VEGF-induced endothelial damage.

Injection of amniotic fluid stem cells (AFSC) delays the course of progression of renal fibrosis in animals with Alport Syndrome, enhancing kidney function and improving survival. The mechanisms responsible for these protective outcomes are still largely unknown. Here, we showed that vascular endothelial growth factor (VEGF) signaling within the glomeruli of Alport mice is strongly elevated early on in the disease, causing glomerular endothelial cell damage. Intraventricular injected AFSC that homed within the glomeruli showed strong modulation of the VEGF activity, particularly in glomerular endothelial cells. To investigate this phenomenon we hypothesized that extracellular vesicles (EVs) produced by the AFSC could be responsible for the observed renoprotection. AFSC derived EVs presented exosomal and stem cell markers on their surface membrane, including VEGFR1 and VEGFR2. EVs were able to modulate VEGF in glomerular endothelial cells by effectively trapping the excess VEGF through VEGFR1-binding preventing cellular damage. In contrast, VEGFR1/sVEGFR1 knockout EVs failed to show similar protection, thus indicating that VEGF trapping is a potentially viable mechanism for AFSC-EV mediated renoprotection. Taken together, our findings establish that EVs secreted by AFSC could target a specific signaling pathway within the glomerulus, thus representing a new potential glomerulus-specific targeted intervention.

A novel mutation of anti-Mullerian hormone gene in Persistent Mullerian Duct Syndrome presented with bilateral cryptorchidism: a case report.

BACKGROUND: We report a novel mutation in a case of Persistent Mullerian Duct Syndrome (PMDS). PMDS is characterized by the persistence of female reproductive organs derivatives in a 46,XY subject due to the failure of the Mullerian duct to regress in utero. To date, 53 different mutations of the anti-Mullerian hormone (AMH) gene, including the present one, have been identified. CLINICAL CASE: A 2-week-old male presented with bilateral cryptorchidism and normal male external genitalia. His karyotype was 46,XY. hCG stimulation test was normal. At age 1 year and 4 months, he underwent laparoscopic surgery which revealed a uterus and fallopian tubes. The anti-Mullerian hormone (AMH) level was undetectable (<0.01 ng/mL). Diagnosis of Persistent Mullerian Duct Syndrome, probably due to an AMH mutation, was made. GENETIC STUDIES: A unique homozygous T to G base substitution was found at position 2219, near the middle of the exon 5, changing codon CTG to CGG in anti-Mullerian hormone (AMH) gene. This mutation causes leucine to be converted to arginine at position 426 belonging to a (L)RA(L)LLLKALQ highly conserved sequence in the AMH gene. Both parents are heterozygous for the mutation. CONCLUSION: Persistent Mullerian Duct Syndrome (PMDS) is a rare cause of bilateral cryptorchidism, when in doubt the existence of Mullerian derivatives should be explored by laparoscopy. Assay of serum AMH helps to distinguish between mutations of AMH and AMH receptor. If serum AMH is very low or undetectable, sequencing of the AMH gene usually confirms the presence of a mutation.

Renal differentiation of amniotic fluid stem cells.

OBJECTIVES: The role of stem cells in regenerative medicine is evolving rapidly. Here, we describe the application, for kidney regeneration, of a novel non-genetically modified stem cell, derived from human amniotic fluid. We show that these pluripotent cells can develop and differentiate into de novo kidney structures during organogenesis in vitro. MATERIALS AND METHODS: Human amniotic fluid-derived stem cells (hAFSCs) were isolated from human male amniotic fluid obtained between 12 and 18 weeks gestation. Green fluorescent protein and Lac-Z-transfected hAFSCs were microinjected into murine embryonic kidneys (12.5-18 days gestation) and were maintained in a special co-culture system in vitro for 10 days. Techniques of live microscopy, histology, chromogenic in situ hybridization and reverse transcriptase polymerase chain reaction were used to characterize the hAFSCs during their integration and differentiation in concert with the growing organ. RESULTS: Green fluorescent protein and Lac-Z-transfected hAFSCs demonstrated long-term viability in organ culture. Histological analysis of injected kidneys revealed that hAFSCs were capable of contributing to the development of primordial kidney structures including renal vesicle, C- and S-shaped bodies. Reverse transcriptase polymerase chain reaction confirmed expression of early kidney markers for: zona occludens-1, glial-derived neurotrophic factor and claudin. CONCLUSIONS: Human amniotic fluid-derived stem cells may represent a potentially limitless source of ethically neutral, unmodified pluripotential cells for kidney regeneration.

New surgical techniques in pediatric urology.

More sophisticated endoscopic instruments, combined with a better understanding of bladder and urethral pathology, have significantly improved the therapeutic approaches for both posterior urethral valves and ureteroceles. New generation lithotripters have allowed for a safe and efficient method of treating urinary calculi in children, which was once thought too injurious a process with first-generation machines. The rapidly advancing field of laparoscopy, aided by the development of more optically refined and diminutive instruments, has allowed for its application in a wide variety of surgical interventions in pediatric urology. The tubularized incised plate urethroplasty has challenged more traditional approaches to hypospadias repair and is now considered by many pediatric urologists to be the best approach for midshaft and distal hypospadias. The one-stage approach to exstrophy repair may hold the answer to improved continence without a formal bladder neck reconstruction. Finally, the field of tissue engineering leads the way to new advances in autologous biological substitutes in the surgically-challenged patient where there is a shortage of local tissues at the surgeon's disposal.

Urethral atresia: long-term outcome in 6 children who survived the neonatal period.

PURPOSE: Urethral atresia is incompatible with life unless an alternative communication between the bladder and amniotic sac exists. Although antenatal intervention may improve perinatal mortality, clinical consequences remain. We outline the outcome after treatment of 6 patients born with urethral atresia. MATERIALS AND METHODS: We reviewed the charts of 6 patients with urethral atresia treated at the Children's Hospital of Michigan between 1982 and 1999. Patient age ranged from 3 to 17 years (mean 9). All patients were males (46 XY) and presented at birth or in early infancy. RESULTS: Oligohydramnios was demonstrated in 4 of the 6 patients (67%). A vesico-amniotic shunt had been placed in 2 patients (at more than 30 weeks of gestation and in 1 at 17 weeks of gestation). The remaining 3 patients presented at birth with either a vesicocutaneous fistula or patent urachus. Bilateral or unilateral hydronephrosis was identified in 4 patients while 2 had severe renal dysplasia. Cystography identified moderate to high grade vesicoureteral reflux in all patients, and 5 (83%) had the prune belly syndrome. Mean serum creatinine at age 1 year was 1.3 mg/dl (range 0.5 to 2.1). Renal failure occurred in 5 patients (83%) before age 10 years and 4 of them have received a renal transplant. An average of 7.8 (range 9 to 14) urological procedures were performed on each patient. Progressive urethral dilation was not successful in the majority of our cases and ultimately 67% required some form of supravesical diversion. CONCLUSIONS: Our study demonstrates that urethral atresia is not necessarily fatal. Prenatal decompression allows survival and in some cases may even lead to normal bladder and renal function. A complicated clinical course requiring extensive reconstruction is to be expected.

Stop-flow in mediastinum and thorax for resistant lymphoma.

BACKGROUND/AIMS: Management of patients with heavily pretreated malignant lymphoma failing frontline treatment and salvage high-dose chemotherapy and autologous peripheral stem cell rescue, is problematic. A pilot study was conducted to evaluate isolated thoracic perfusion of drugs by means of stopflow technique. METHODOLOGY: Six patients were enrolled in the study; diagnoses included 4 advanced Hodgkin's disease, 1 primary mediastinal B-cell lymphoma, and 1 anaplastic large cell lymphoma. Patients were aged 18-37 years; 4 presented with bulky mediastinum. They had never achieved a complete response since all had progressed from front-line treatment, and 3 had even failed salvage high-dose chemotherapy with autologous peripheral stem cell rescue. Cisplatin (100 mg/m2) and melphalan (35 mg/m2) were used. Carmustine (100 mg/m2) were added to these 2 drugs and cytarabine (2000 mg/m2) in patients not previously treated by carmustine, etoposide, cytarabine, and melphalan. Epidoxorubicin (70 mg/m2) was added in patients who previously received a suboptimal dosage of antracycline. Drugs were delivered monthly via aortic perfusion performed by means of Aigner's stop-flow technique. RESULTS: Overall 13 cycles of perfusional chemotherapy were administered with a median number of 2 cycles. During the procedures there were no technical, hemodynamic, or vascular complications, and no deaths occurred during surgery. After 1 month, 6 (100%) objective responses after isolated thoracic perfusion were recorded, 3 (50%) of which were complete. Tolerance to therapy was excellent. Hematological toxicity was mild and transfusional support was needed only in one course. At the last follow-up, 2 patients are alive (1 complete response and 1 very good partial response, maintained). CONCLUSIONS: This new therapeutical approach seems very active in recurrent/refractory malignant lymphoma and may play an important role in this setting.

Neurogenic bladder in infants born with anorectal malformations: comparison with spinal and urologic status.

BACKGROUND/PURPOSE: Spinal dysraphism and neurovesical dysfunction (NVD) frequently are associated in children with anorectal malformations (ARM). This study compares the urodynamic data from a selected group of patients with the results of their spinal and urologic imaging studies. METHODS: Twenty-six children (20 with isolated imperforate anus and six with persistent cloacal malformations) were investigated. All patients were evaluated with leak point pressures (LPP), renal ultrasound scan, and voiding cystourethrography. Eight children had urodynamics performed before and after posterior sagittal anorectoplasty (PSARP). The spinal cord was assessed using ultrasonography or magnetic resonance imaging. Current urologic status was obtained to provide long-term follow-up. RESULTS: Twenty-one of 26 children demonstrated elevated LPPs above the established normal value of 40 cm H2O, and 15 of these children had normal spinal imaging study findings. Uroradiographic studies findings showed that 12 of 21 children with elevated LPPs had hydronephrosis or vesicoureteral reflux with seven of these patients having normal spinal cords. LPPs in the eight patients with pre- and postoperative studies were 74 +/- 14.7 cm H2O and 68 +/- 31.8 cm H2O (mean +/- SD), respectively. CONCLUSIONS: These urodynamic and radiographic data confirm that NVD (elevated LPP) is common in patients with anorectal malformations despite normal spinal cords. Bladder dysfunction does not appear to be a sequelae of a properly performed PSARP. Patients with ARM and any uroradiographic or clinical urologic abnormality should undergo urodynamic testing even though the spinal studies are normal.

An alternative ureteroileal reimplantation used in augmentation cystoplasty for neurogenic bladder with bilateral vesicoureteral reflux.

PURPOSE: We describe the use of a serous lined extramural tunnel for ureteral reimplantation during augmentation of a neurogenic bladder to prevent reflux. MATERIALS AND METHODS: A 46-year-old male C6 spinal cord injury patient presented with a high pressure bladder, detrusor-sphincter dyssynergia and bilateral grade II/III vesicoureteral reflux. Despite maximal anticholinergic therapy and intermittent catheterization, detrussor pressures were between 80 and 100 cm. water at volumes of 100 to 150 cc with consistent leakage between catheterizations. Preoperative ultrasound and voiding cystourethrogram demonstrated moderate bilateral hydronephrosis and a heavily trabeculated bladder. Augmentation cystoplasty with the formation of 3 cm. extramural ureteral tunnels as described by Ghoneim was performed. The serosa of the adjacent limbs of the ileal segment were opposed to form the back wall of a serosal lined tunnel. RESULTS: At 3 weeks postoperatively a cystogram demonstrated no extravasation or reflux. At 8 weeks an excretory urogram showed prompt function and excretion bilaterally with marked improvement of preoperative hydronephrosis. CONCLUSIONS: Subserosal ureteral tunnels can be used as an alternative antireflux technique during augmentation cystoplasty when ureteral reimplantation is required. Two advantages of this technique include the elimination of staples and avoidance of ischemic problems associated with an afferent intussuscepted nipple valve.