Gambierol acts as a functional antagonist of neurotoxin site 5 on voltage-gated sodium channels in cerebellar granule neurons.

The marine toxin gambierol, a polyether ladder toxin derived from the marine dinoflagellate Gambierdiscus toxicus, was evaluated for interaction with voltage-gated sodium channels (VGSCs) in cerebellar granule neuron (CGN) cultures. At concentrations ranging from 10 nM to 10 microM, gambierol alone had no effect on the intracellular Ca2+ concentration [Ca2+]i of exposed CGN cultures. Furthermore, there was no evidence of neurotoxicity in CGN cultures exposed for 2 h to gambierol (1 nM-10 microM). However, gambierol was a potent inhibitor (IC50 = 189 nM) of the elevation of [Ca2+]i that accompanies exposure of CGN cultures to the VGSC activator brevetoxin-2 (PbTx-2). To further explore the potential interaction of gambierol with VGSCs, the influence of gambierol on PbTx-2-induced neurotoxicity was assessed. Gambierol reduced the PbTx-2-induced efflux of lactate dehydrogenase in exposed CGN cultures in a concentration-dependent manner (IC50 = 471 nM). It is noteworthy that the potencies of gambierol as an inhibitor of both PbTx-2-induced Ca2+ influx and cytotoxicity were coincident. Finally, the inhibitory effects of gambierol on PbTx-2-induced elevation of [Ca2+]i were compared with those of brevenal, a natural inhibitor of the toxic effects of brevetoxin isolated from cultures of Karina brevis. Like gambierol, brevenal inhibited PbTx-2-induced elevation of [Ca2+]i in a concentration-dependent manner (IC50 = 108.6 nM). These results provide evidence for gambierol acting as a functional antagonist of neurotoxin site 5 on neuronal VGSCs.

Bladder stretch alters urinary heparin-binding epidermal growth factor and antiproliferative factor in patients with interstitial cystitis.

PURPOSE: The etiology of interstitial cystitis is unknown. Urine from patients with interstitial cystitis has been shown to inhibit urothelial proliferation through a putative antiproliferative factor and to contain decreased levels of heparin-binding epidermal growth factor-like growth factor (HB-EGF) compared to controls. Stretch of detrusor smooth muscle cells is known to stimulate HB-EGF production. Because bladder hydrodistention sometimes alleviates the symptoms of interstitial cystitis, we determined whether the stretch stimulus of hydrodistention alters antiproliferative factor activity and/or HB-EGF in interstitial cystitis urine specimens. MATERIALS AND METHODS: Urine was collected immediately before, and 2 to 4 hours and 2 weeks after hydrodistention from 15 patients with symptoms and cystoscopic findings compatible with interstitial cystitis and 13 controls. Hydrodistention was performed with the subject under general or regional anesthesia and bladders were distended to 80 cm. water 3 times. Urinary HB-EGF was measured by enzyme-linked immunosorbent assay and urinary antiproliferative factor activity was determined by measuring 3H-thymidine uptake by normal human bladder urothelial cells. RESULTS: Hydrodistention significantly increased urinary HB-EGF in patients with interstitial cystitis toward normal control values (before distention p = 0.003, 2 weeks after distention p = 0.67). Urine antiproliferative factor activity decreased significantly after hydrodistention in patients with interstitial cystitis. However, antiproliferative factor activity in interstitial cystitis and control specimens was still statistically different 2 weeks after distention (before distention p = 0.0000004, 2 weeks after distention p = 0.04). CONCLUSIONS: Bladder stretch increased HB-EGF and conversely reduced antiproliferative factor activity in urine from patients with interstitial cystitis but not controls up to 2 weeks after distention. These results provide additional evidence for the possible role of antiproliferative factor and decreased HB-EGF in the pathophysiology of interstitial cystitis. To our knowledge this is also the first human study to show that in vivo bladder stretch can alter urinary factors that regulate cell growth.

Sublingual absorption of leuprolide: comparison between human and animal models.

Leuprolide is a potent luteinizing hormone releasing hormone agonist used for the treatment of hormone-dependent diseases. It is a decapeptide drug currently administered by subcutaneous and intramuscular injection because it is not orally bioavailable. In the present study, sublingual gel formulations of leuprolide were administered to dogs, monkeys and humans. Plasma samples were analyzed by radioimmunoassay. Absorption and pharmacokinetics of leuprolide following sublingual administration were compared and evaluated. It was found that the extent and rate of absorption were similar between humans and monkeys following sublingual dosing of leuprolide formulations. A prolonged absorption of up to approximately 6 h after dosing was observed in both humans and monkeys. The rate and extent of absorption were significantly higher in dogs than in humans. The estimate of absolute bioavailability of leuprolide was 46.7% in dogs compared with 2.7% in monkeys at an equivalent dose of 0.45 mg/kg. Absolute bioavailabilities in humans were 2.0, 2.3 and 2.4% at doses of 1.125, 2.25 and 4.5 mg, respectively. Based on these results, the dog is not an appropriate animal model for evaluating sublingual absorption of leuprolide. This work suggests that monkey is a preferred model for the development and assessment of sublingual formulations of leuprolide.

Treatment of urinary incontinence in children by endoscopically directed bladder neck injection of collagen.

PURPOSE: We assessed optimal selection criteria for and expected outcome of bladder neck injection of collagen in children with intrinsic sphincteric deficiency. MATERIALS AND METHODS: We retrospectively reviewed the charts of children with intrinsic sphincteric deficiency treated with bladder neck injection of collagen at 3 pediatric urology centers. RESULTS: We reviewed the records of 12 boys and 6 girls 6 to 18 years old (median age 10.5). The underlying etiology of incontinence was neurogenic bladder in 10 patients, the epispadias-exstrophy complex in 6, ureterocele in 1 and trauma in 1. Previous surgery included bladder augmentation in 4 cases, bladder neck plasty in 7 and epispadias repair in 4, and concurrent medical management comprised anticholinergics in 8, alpha-agonists in 1 and intermittent catheterization in 9. Skin tests in all patients were negative for collagen. Seven patients had 1 injection, 9 had 2 and 2 had 3. The volume of collagen injected ranged from 2.4 to 13 cc (median 5) per treatment and 3 to 28 cc (median 7) per patient. Followup ranged from 5 to 21 months (median 15). Of the 14 patients evaluated 5 are dry, 4 are improved, 1 is unchanged and 4 underwent further continence surgery. No treatment related morbidity was seen. CONCLUSIONS: Endoscopically directed injection of collagen around the bladder neck is successful in 64% of children with intrinsic sphincteric deficiency. It is an outpatient treatment with no significant morbidity. Further continence surgery is not precluded when necessary. Patients with adequate capacity and compliant neurogenic bladders, and those with the exstrophy-epispadias complex and failed bladder neck plasty seem to be optimal candidates for collagen injection.

Endoscopic treatment of vesicoureteral reflux with collagen: preliminary report and cost analysis.

PURPOSE: We determined therapeutic and operative cost efficacy of endoscopic collagen injection for vesicoureteral reflux. MATERIALS AND METHODS: We performed a multicenter study of 10 male and 23 female sequential patients (45 renal units) with vesicoureteral reflux and a surgical indication. Patients were skin tested with collagen and when negative, they underwent endoscopic injection. Renal ultrasound/cystography was done 3 months after treatment. Cost analysis was performed at 1 institution. RESULTS: Reflux was grade I in 2 renal units, II in 18, III in 15 and IV in 10. The majority of cases were single systems with primary reflux. We performed 1 treatment in 32 renal units, 2 in 6, 3 in 6 and 4 in 1. Cure was evident in 31 of 38 renal units (81.6%) and 21 of 27 patients (78%) 3 months after the last injection. Patient morbidity was minimal. Operative cost per renal unit was $1,599.68 for collagen injection and $9,144.47 for reimplantation. CONCLUSIONS: Endoscopic injection of collagen is effective treatment for vesicoureteral reflux. Furthermore, it causes minimal morbidity and may effect cost savings in health care management.

The C-terminus of IFN gamma is sufficient for intracellular function.

We have previously shown that murine interferon gamma (IFN gamma) and its C-terminal peptide, muIFN gamma (95-133), bind to a region on the cytoplasmic domain of the IFN gamma receptor contained in the synthetic peptide, MIR(253-287). This region of the murine receptor bears considerable homology (approximately 80%) to its human counterpart. Here we report that not only do human IFN gamma and the human IFN gamma C-terminal peptide, huIFN gamma(95-134), bind to the cytoplasmic domain of the human IFN gamma receptor, but also that this interaction is species non-specific. MuIFN gamma(95-133) binds to human IFN gamma receptor cytoplasmic peptide HIR(252-291), and huIFN gamma(95-133) binds to MIR(253-287). Furthermore, treatment of murine macrophage cell lines with C-terminal peptides of either murine or human IFN gamma results in 10-fold upregulation of MHC class II molecule expression and increased resistance to infection with vesicular stomatitis virus (VSV) (10(6)-10(9)-fold reduction in yield). These data suggest a direct role for the C-terminus of IFN gamma in the initiation of intracellular signalling processes and may be indicative of a more general mechanism of action for extracellular signalling molecules.

Laboratory variables of bladder autoaugmentation in an animal model.

OBJECTIVES: Evaluations of the functional, radiologic, and pathologic outcomes of autoaugmentation by two surgical techniques (vesicomyectomy versus vesicomyotomy) were compared. Autoaugmentation or vesicomyotomy is being increasingly considered as a simplified method of bladder augmentation in the hypertonic decreased-capacity bladder. METHODS: In a series of 35 laboratory sessions, creation of an animal model approximating the small-capacity hypertonic bladder was achieved. Sixteen vesicomyotomies and 16 vesicomyectomies were performed on the 32 stabilized one-third reduced bladders. Three stabilized one-third reduced bladders were used as controls. RESULTS: Radiologic studies show a large diverticulum. A 17.2% net increase in surface area was achieved, compared with the reduced bladder, at the time of pathologic examination. Functional capacity was increased by 43.5% on urodynamic studies, and leak point pressure was decreased by 48.1%. Histologic and morphometric examinations of the autoaugmentation area showed a few muscle fibers with serosal deposition of collagen. There was less muscle ingrowth at the periphery of the autoaugmentation site utilizing vesicomyectomy. CONCLUSIONS: There was no statistical difference between vesicomyotomy and vesicomyectomy with respect to radiologic, pathologic, or urodynamic outcome.

A short-term study of nitrofurantoin prophylaxis in children managed with clean intermittent catheterization.

OBJECTIVE: Because there is no evidence for the effectiveness of antibiotic prophylaxis in children with neurogenic bladder, the value of once-daily nitrofurantoin macrocrystals was assessed in a selected population with neurogenic bladder due to meningomyelocele. METHODS AND TRIAL POPULATION: Children with significant urinary tract abnormalities other than neurogenic bladder were excluded. A urinary tract "infection" was defined as > or = 10(8) colony forming units of bacteria/L of urine together with pyuria of > or = 50 x 10(6) leukocytes/L, and/or symptoms consistent with an urinary tract infection. Fifty-six children participated in a 24-week double-blinded, placebo-controlled cross-over study. The infection status was assessed at two weekly intervals or if relevant clinical manifestations occurred. RESULTS: For the whole trial the average percentage of "infections" per urine sample for each patient was reduced from 39% on placebo to 19% on single daily dose prophylaxis (P < .0003). For the first 12 weeks of the trial corresponding figures were 45% on placebo and 22% on prophylaxis (P < .0018). There was evidence for a marked carryover protective effect of nitrofurantoin into the placebo arm of the trial. CONCLUSION: Nitrofurantoin is an effective prophylactic agent during a 3-month period. Long-term studies are needed to confirm the reasonable expectation of a beneficial effect on urinary tract damage.

Gonadal tumors in disorders of sexual differentiation.

OBJECTIVE: To study the prevalence and distribution of gonadal tumors in patients with disorders of sexual differentiation. METHODS: Retrospective review of pathologic materials and clinical data on all patients diagnosed with mixed gonadal dysgenesis, pure gonadal dysgenesis, androgen insensitivity, and true hermaphroditism between 1982 and 1990. RESULTS: Twenty-one patients were identified and all underwent bilateral gonadectomy at the time of diagnosis. Nine of 21 patients had a gonadal tumor for a prevalence of 44 percent. Those at greatest risk for tumor were patients with mixed gonadal dysgenesis (6 of 11 patients) and pure gonadal dysgenesis (2 of 3 patients). There were four gonadoblastomas, two dysgerminomas, and one each of teratocarcinoma, seminoma, cystadenofibroma, and juvenile granulosa cell tumor. CONCLUSIONS: The high prevalence of gonadal tumors in children with mixed and pure gonadal dysgenesis warrants consideration of early, bilateral, prophylactic gonadectomy once the diagnosis is established with certainty.

Cryopreservation of rat Leydig cells for in vitro and in vivo studies.

Leydig cells lose their ability to secrete testosterone following short-term in vitro culture. A procedure for effective storage of these cells would be useful. In this study, rat Leydig cells were cryopreserved in the presence of 10, 15 or 20% dimethylsulfoxide (DMSO) at approximately 1 degree C/min to -70 degrees C and then stored in LN2. After thawing, the cells cryopreserved in the presence of 15% DMSO showed the highest viability of over 75%. These cells secreted basal levels of testosterone in vitro as well as responded to hCG stimulation by secreting over 9-fold increase in testosterone. The viability of these cells was further confirmed by the demonstration of 3 beta-HSD positive cells under the kidney capsule of rats isografted with cryopreserved Leydig cells. This study demonstrated that purified rat Leydig cells can be cryopreserved and the cryopreserved cells retained normal function and were responsive to hCG stimulation. Cryopreservation is a simple procedure for long-term storage of functional Leydig cells.

Appraisal of the stability of leukocytes in refrigerated urine.

There is limited evidence that urinary leukocytes are rapidly destroyed in alkaline hypotonic urine. We assessed the stability of leukocytes in urine specimens provided by 90 children with neurogenic bladder who attended a meningomyelocele clinic. No significant correlation was found between urine specific gravity and leukocyte survival after an interval of 4 h in a sample of 30 specimens from these patients. The survival of leukocytes was determined at 2 h and at 4 h in aliquots of these 30 specimens directly, and after adjustment to pH values of 5.0, 7.0, 8.0, 8.5, and 9.0. Statistically significant leukocyte destruction only occurred at pH 9.0 at 2 h (16%), at pH 8.5 at 4 h (19%), and at pH 9.0 at 4 h (57%). Only one of a further sample of 180 routine specimens had both a pH of > or = 8.5 and an interval to laboratory examination of > 2 h. No specimen had a specific gravity of < 1.002, and 93.9% had values of > or = 1.005. Urine pH and tonicity were not therefore important determinants of leukocyte stability in refrigerated samples examined within 4 h from this clinic population.

Recurrent urinary tract infections in children.

Urinary tract infections are common in children and present in various ways. Diagnosis is based on findings of pyuria and bacteriuria. Management includes adequate and timely investigation, appropriate antibiotics, treatment of underlying contributing factors, and follow-up advice.

Application of a leukocyte and nitrite urine test strip to the management of children with neurogenic bladder.

A urine leukocyte count of > or = 50/mm3 together with a bacterial count of > or = 10(5) colony-forming units (CFUs) per milliliter was used to define significant infection in 160 children with neurogenic bladder and evaluate the leukocyte and nitrite components of the Chemstrip 9 test. A Chemstrip 9 leukocyte reading of < or = 25 together with a negative nitrite reaction occurred in 99 children and had a sensitivity of 83.5% and a negative predictive value for infection of 97.0%. A Chemstrip 9 reading of > or = 500 leukocytes together with a positive nitrite reaction occurred in 18 children and had a sensitivity of 40% with a 100% positive predictive value for infection. Other combinations of Chemstrip 9 leukocyte and nitrite reactions were unhelpful or of uncertain value. Selection of up to three specimens from each patient increased the number of samples to 360 and provided general confirmation of the above conclusions. Nitrofurantoin may reduce the sensitivity of the nitrite strip reaction.

Cutaneous vesicostomy in the young child: indications and results.

With newer methods of managing lower urinary tract pathology in the young child, the role of cutaneous vesicostomy may be changing. This prompted a review of 50 consecutive patients treated with initial vesicostomy at our center over ten-year period. These children underwent vesicostomy diversion at a median age of 5.8 months and, of the 34 vesicostomies which have been subsequently closed, for a median duration of twenty-five months. Our indications agree with series reported previously and include patients with meningomyelocele, posterior urethral valves, or other forms of congenital or acquired lower urinary tract anomaly or dysfunction, along with complicating factors such as vesicoureteral reflux, recurrent infections, and/or renal deterioration. However, we also have identified a major group--those with primary gross vesicoureteral reflux--not previously included in detail. Follow-up averaged thirty-eight months. Improvement or stabilization of upper urinary tracts was achieved in over 90 percent of cases, and this trend continued after vesicostomy closure. As well, cutaneous vesicostomy allowed ureteral dilation to normalize, decreasing the degree of reflux and need for subsequent ureteral tapering and reimplantation at closure. Finally, though our stomal revision rate of 20 percent is high, modified techniques are being pursued.

Creation of a pedicle valve unit (PVU) for establishment of enteric continence. Experimental observations.

The aim of this study was to develop a natural tissue valve that could be anastomosed into any area of the gastrointestinal (GI) tract to act as a fecal "brake" and so establish enteric continence at that site. A 4-cm-long valve created from an intussuscepted small bowel pedicle was anastomosed into the cecum and brought out through the abdominal wall as a stoma in 11 rabbits. The animals were re-explored five weeks later for assessment of valve viability and continence and microscopic appearance. In all cases, the valve was fully continent in vivo. All valves were viable, and there was no anastomotic leakage. Pressure testing of the valve at reoperation revealed that 7 of 10 valves tested withstood pressures of 30 mmHg before and after catheterization and 6 of 10 were fully continent to cecal "blanching" pressure (50 mmHg). Valve failure was due to deintussusception in three cases. In four cases, valves were continent over 50 mmHg and showed no tendency to incontinence to bursting pressure of the cecum. We conclude that a continent pedicle valve unit (PVU) for placement in a variety of locations in the GI tract is feasible. The PVU has implications in the management of short-gut syndrome, incontinent ileostomy, continent cecostomy, and as a continent valve placed in the perineum for restoration of perineal defecation following proctectomy.

Intestinal obstruction complicating pregnancy.

Intestinal obstruction is a rare but serious complication of pregnancy with significant maternal and fetal mortality. The reported incidence of intestinal obstruction complicating pregnancy varies widely, from 1 in 66,431 to 1 in 1,500 deliveries. A retrospective review of 66 cases of intestinal obstruction complicating pregnancy and the puerperium, including 2 cases from our institution, revealed that the most common causes of mechanical obstruction were adhesions (58%), volvulus (24%), and intussusception (5%). Seventy-seven percent of the patients with obstruction due to adhesions had undergone previous abdominal or pelvic surgery. Presenting symptoms and signs were similar to those of the nonpregnant patient; abdominal pain was present in 98% of patients, vomiting in 82%, and tenderness to palpation in 71%. In 82% of patients, obstruction was evident on radiographic evaluation. Prompt management of obstruction is essential; the median length of time from admission to laparotomy in the 66 patients was 48 hours. Bowel strangulation requiring resection was present in 23% of patients. Thirty-eight percent of patients completed term pregnancies after operative resolution of obstruction; total maternal mortality was 6%, and total fetal mortality 26%. Thus, both mother and fetus are at risk when intestinal obstruction complicates pregnancy. Clinical suspicion of the presence of obstruction and aggressive intervention are required to decrease the morbidity and mortality of this rare complication of pregnancy.

A comparison of two continent catheterizable vesicocutaneous techniques.

In a rabbit model we describe and compare two continent catheterizable diversion techniques for which the bladder is intact and the stoma is situated in the lower abdomen. Both mechanisms are formed from a short segment of ileum; one utilizes the principles of the encircling loop technique as described by Koff and the second, the Kock principle (intravesical nipple). Both models were evaluated for the following parameters: ease of catheterization (87%, 79% respectively), continence (91%, 86% respectively), and ease of construction. We present evidence that both techniques are applicable to the clinical setting.

Tisseel in hypospadias repair.

Tisseel (Immuno AG, Vienna, Austria), a fibrin sealant, adhesive and hemostatic agent made up of sterilized human and bovine components, was used in hypospadias repairs. The incidence of fistula and significant edema and the duration of hospitalization were all reduced in 30 patients who underwent modified Mustardé repairs in which Tisseel was used when compared with a matched group of 30 repairs without Tisseel. In addition, the rate of complications in fistula repairs and complex revisions was reduced when Tisseel was used on urethral suture lines and under all skin flaps.

Augmentation ileocystoplasty in children with myelodysplasia.

Over the past 8 years 20 children suffering from myelodysplasia were selected to undergo augmentation ileocystoplasty for urinary incontinence refractory to treatment by clean, intermittent catheterization and anticholinergic agents, or as part of urinary undiversion. The children ranged in age from 5 to 17 years, and the follow-up ranged from 6 months to 7 years. Three children underwent undiversion, two had vesicostomy closure and six had bladder outlet repair at the time of augmentation. In one patient, surgery was technically impossible. Postoperatively 16 children were fully continent on clean, intermittent catheterization and anticholinergic medication. Three boys failed to achieve satisfactory continence. Delayed perforation, a potentially serious complication, occurred in two patients several months postoperatively.

Wilms tumorlet, nodular renal blastema and multicystic renal dysplasia.

We reviewed 60 cases of multicystic renal dysplasia collected during a 10-year period. Differentiated nonproliferative nodular renal blastema occurred in 6.7 per cent of the cases, which is similar to the incidence of nodular renal blastema in kidneys of other infants with congenital obstructive uropathy. A unique case of cystic dysplasia containing nodular renal blastema and Wilms tumorlet indicates the possibility that a persistently proliferative nephroblastomatous lesion may rarely occur.