Organ-specific matrix self-assembled by mesenchymal cells improves the normal urothelial differentiation in vitro.

PURPOSE: Enterocystoplasty is the gold standard to perform bladder reconstruction. Since this technique has a high morbidity rate, several matrix scaffolds have been proposed to support the urothelial maturation. Unfortunately, epithelial cells failed to fully integrate the cell-matrix interactions and therefore appropriate signalling pathways of normal differentiation. Based on these observations, we proposed to culture bladder urothelial cells (BUC) onto a matrix self-assembled by bladder mesenchymal cells (BMC), to form a vesical model (VM). METHODS: Different serum proportions were assessed to obtain a manipulable matrix deposited by BMC. The BUC were then seeded onto the BMC's matrix to evolve in a three-dimensional culture. Haematoxylin-eosin staining, immunolabeling, scanning electron microscopy, western blot and matrix metalloproteinases analysis were performed for the VM characterization. RESULTS: We were able to obtain an original matrix made of collagen-I and presenting specific organization. Matrix remodelling was observed and led to a cellular compartmentalization. The reconstructed urothelium developed in a pseudostratified arrangement, displaying an adequate cellular polarity and apical membrane remodelling of superficial cells. Like native bladder, cytokeratin 14 immunolabeling was not observed in our VM, which indicate the conformity of the development sequence taken by BUC under the influence of the BMC's matrix. CONCLUSION: Thus, it was possible to elaborate a VM without the use of exogenous matrices. The particular characteristics of the BMC's matrix permitted the development of an urothelium that shared the phenotype of native tissue. The autologous character of our VM, and its appropriate urothelial maturation, could potentially promote a better integration after grafting.

Allantoic cysts and posterior urethral valves: a case report.

Allantoic cysts are a somewhat rare entity. They are identifiable on antenatal ultrasound examination but are not easily distinguishable from pseudocysts, which are often associated with a poor prognosis. Their etiology remains obscure and obstructive uropathies have been proposed to be one of the underlying mechanisms. We report on a case in which both allantoic cysts and a patent urachus were detected antenatally and turned out to be associated with posterior urethral valves (PUV). The cysts were first seen in the early second trimester, with a full fetal bladder and patent urachus. They disappeared by the 29(th) week. During the subsequent sonographic examinations, the bladder was emptying regularly. The kidneys remained normal throughout the pregnancy. It is hypothesized that the increased pressure within the urinary tract kept the urachus patent, led to the formation of allantoic cysts and, later, to their perforation; this allowed the fetus to empty his bladder and kept him from the usual complications of PUV. This case emphasizes the importance of detection and characterization of umbilical cord cysts on antenatal ultasound examination and suggests that obstructive uropathies should be included in the differential diagnosis of umbilical cord cyst communicating with the fetal bladder.

Alternative methods for genetic transformation of Pseudozyma antarctica, a basidiomycetous yeast-like fungus.

Electroporation and Agrobacterium tumefaciens-mediated transformation (ATMT) were adapted and optimized for genetic transformation of the basidiomycetous yeast-like fungus Pseudozyma antarctica as alternatives to the cumbersome PEG/CaCl(2)-mediated transformation of protoplasts. Electroporation yielded 100-200 transformants per mug of DNA per 10(8) cells after 3 days on selective medium. For its part, ATMT yielded 60-160 transformants per 10(6) input cfu after 5-10 days on a selective medium. Transformants obtained from both methods showed stable hygromycin resistance and strong expression of green fluorescent protein. Analysis of integration events revealed a limited number of predominantly tandem insertions in the genome of transformants, an improvement over PEG/CaCl(2)-mediated transformation. Both protocols relied on intact conidia of P. antarctica as starting material and thus eliminated the need for cell wall-degrading or weakening agents such as lytic enzymes or chemicals. Other advantages over protoplast transformation included higher yield of transformants and shorter recovery time of transformed colonies on selective medium.

The potential of Pseudozyma yeastlike epiphytes for the production of heterologous recombinant proteins.

Although Basidiomycetes represent the most evolved class of fungi, they have been neglected with regard to recombinant gene expression. In this work, basidiomycetous yeasts belonging to Pseudozyma spp. were studied with respect to their amenability to heterologous protein production. Single plasmid or cotransformation experiments routinely afforded 100 to 200 independent transformants for the two tested species of Pseudozyma. Green fluorescent protein (GFP) was expressed in the correctly folded conformation, as demonstrated by fluorescence microscopy, and hen egg white lysozyme (HEWL) was expressed in its active form, as revealed by its lytic activity on Micrococcus lysodeikticus cells. Protease analysis established that Pseudozyma spp. contained equivalent or less extracellular protease activity than yeasts and far less protease activity than ascomycetous filamentous fungi in similar culture conditions. This proteolytic activity was inhibited by over 97% with a combination of PMSF and Pepstatin A. N-glycosylation patterns of native Pseudozyma flocculosa secreted proteins were comprised of one or a few short glycan chains that possess a classic eukaryotic structure typical of higher fungi and animal cells. This is the first report of a Basidiomycete that possesses multiple intrinsic characteristics necessary for use as a heterologous gene expression system.

Laparoscopic dismembered pyeloplasty by a retroperitoneal approach in children.

OBJECTIVES: To report our experience with dismembered laparoscopic pyeloplasty by a retroperitoneal approach in children with pelvi-ureteric junction (PUJ) obstruction. PATIENTS AND METHODS: Between 1999 and 2002, retroperitoneal laparoscopic dismembered pyeloplasty was attempted in 21 children (one bilateral; mean age 8 years, range 1.7-17). In a flank position with four ports (one of 5 or 10 mm and three of 3 mm), the PUJ was resected and the anastomosis made using 6/0 absorbable sutures. Any redundant renal pelvis was reduced when needed. A JJ stent was inserted in all patients. RESULTS: The procedure could not be completed by laparoscopy in four patients, the main reason being difficulty in completing the anastomosis; in the other 18 patients the procedure was successful. An aberrant crossing vessel was found in nine patients and dismembered pyeloplasty enabled ureteric transposition in all, with no conversion. The mean (range) operative duration was 228 (170-300) min and the mean hospital stay 2.5 (2-4) days. In three patients the JJ stents were not in the bladder at the time of removal by cystoscopy, and ureteroscopy was used to retrieve them. All children returned to full activities within 7 days of surgery. The mean (range) follow-up was 12.7 (2-36) months, with six children followed for> 2 years; all were asymptomatic, with imaging confirming improved hydronephrosis. CONCLUSIONS: These mid-term results confirm that retroperitoneal laparoscopic dismembered pyeloplasty is a safe and feasible approach in children. Although the technique is very demanding it has the advantage of duplicating the principles of the open approach. The long operative duration and high conversion rate might be reduced with experience. Before expanding this approach to younger children, refinements in the anastomotic technique are needed.

The predictive value of diagnostic imaging for histological lesions of the upper poles in duplex systems with ureteroceles.

OBJECTIVE: To compare the diagnostic imaging findings with the histological lesions in upper pole nephrectomy (UPN) specimens of duplex system ureteroceles, using renal ultrasonography (US) and nuclear renal scintigraphy. PATIENTS AND METHODS: Between 1992 and 2000, 86 patients with a ureterocele in a duplex system underwent surgery. The results from US were reviewed in 84 patients by a radiologist, for echogenicity, parenchymal thinning and hydronephrosis; 77 nuclear renal scans describing the differential function of the upper poles were also reviewed. Fifty-five patients underwent UPN (25 antenatal, 30 postnatal; 18 intravesical, 37 extravesical) and the specimens were available for independent review by a pathologist, describing five histological categories, i.e. chronic interstitial inflammation, fibrosis, tubular atrophy, glomerulosclerosis and dysplasia. Histological lesions were categorized as severe (> 25%) or minimal (

Retroperitoneal laparoscopic vs open partial nephroureterectomy in children.

OBJECTIVES: To compare the results of retroperitoneal laparoscopic with open partial nephroureterectomy. PATIENTS AND METHODS: Laparoscopic retroperitoneal partial nephroureterectomy was undertaken in 15 children (13 upper and two lower poles; median age at the time of surgery 61 months, range 5-212). A three-trocar retroperitoneal approach was used. The polar vessels were identified and either coagulated or clipped before transecting the parenchyma using a harmonic scalpel. An additional 13 consecutive children underwent similar procedures (11 upper and two lower poles) by conventional open surgery (median age at surgery 16 months, range 1.5-72). RESULTS: One patient in the laparoscopy group required conversion to open surgery because of a peritoneal tear and was excluded from the analysis. The mean (range) operative duration was 146 (50-180) and 152 (75-240) min for the open surgery and laparoscopy groups, respectively. The blood loss was minimal (< 20 mL) in both groups. The mean (range) hospital stay was 1.4 (1-3) and 3.9 (3-5) days for the laparoscopy and open groups, respectively (P < 0.001). Eight patients were discharged on the day after the laparoscopic procedure. In addition, of those patients in the laparoscopy group who had a lower-pole partial nephrectomy, one had a urinoma after surgery. CONCLUSION: Laparoscopic retroperitoneal partial nephrectomy is a safe and feasible procedure in children. It requires a similar operative duration to that of an open procedure. The main advantage to the laparoscopic approach is that it significantly decreases the hospital stay compared with that after an open procedure.

The use of tolterodine in children after oxybutynin failure.

OBJECTIVE: To assess the safety and efficacy of tolterodine tartrate prescribed to children who previously failed to tolerate oxybutynin chloride. PATIENTS AND METHODS: We reviewed 34 children, followed for>1 year, who were prospectively crossed-over from oxybutynin to tolterodine because of side-effects. The initial diagnosis was dysfunctional voiding in 31 patients. All patients were placed on a behavioural modification protocol. When their symptoms did not improve after 6 months, treatment with an anticholinergic agent was considered. Urodynamic studies were conducted in 20 patients, confirming uninhibited contractions in 19. The remaining 14 patients were empirically started on antimuscarinic or anticholinergic agents. The 34 patients were treated with oxybutynin for a median (range) of 6 (2-84) months. When significant side-effects were reported, they were crossed over to tolterodine. The efficacy of tolterodine was assessed as defined by the International Children's Continence Society, with tolerability assessed and side-effects documented using a questionnaire. RESULTS: The mean age at the first dose of tolterodine was 8.9 years; the dose was 1 mg twice daily for 12 patients and 2 mg twice daily for 22. The median treatment with tolterodine was 11.5 months, with 20 (59%) patients reporting no side-effects; six described the same but tolerable side-effects as with oxybutynin. Eight patients discontinued tolterodine because of side-effects after a median (range) of 5 (1-11) months. The efficacy of tolterodine was comparable with that of oxybutynin, as reported by the questionnaire and voiding diaries. The reduction in wetting episodes at 1 year was> 90% in 23 (68%), more than half in five and less than half (or failure) in six patients. CONCLUSION: Tolterodine is tolerated well in children. In this subgroup of patients who could not tolerate oxybutynin, 77% were able to continue tolterodine treatment with no significant side-effects.

Organized sports for children and preadolescents.

Participation in organized sports provides an opportunity for young people to increase their physical activity and develop physical and social skills. However, when the demands and expectations of organized sports exceed the maturation and readiness of the participant, the positive aspects of participation can be negated. The nature of parental or adult involvement can also influence the degree to which participation in organized sports is a positive experience for preadolescents. This updates a previous policy statement on athletics for preadolescents and incorporates guidelines for sports participation for preschool children. Recommendations are offered on how pediatricians can help determine a child's readiness to participate, how risks can be minimized, and how child-oriented goals can be maximized.

Microtubule disruption induced in vivo by alkylation of beta-tubulin by 1-aryl-3-(2-chloroethyl)ureas, a novel class of soft alkylating agents.

We have previously reported that 4-tert-butyl-[3-(2-chloroethyl)ureido] benzene (4-tBCEU), a potent cytotoxic agent, modulates the synthesis of tubulins, suggesting that its cytotoxicity may be mediated through an antimicrotubule mechanism. Indeed, 4-tBCEU and its 4-iso-propyl (4-isopropyl [3-(2-chloroethyl)ureido] benzene) and 4-sec-butyl (4-sec-butyl [3-(2-chloroethyl)ureido] benzene) homologues induced disruption of the cytoskeleton and arrest of the cell cycle in G2 transition and mitosis. To better understand the mechanisms responsible for microtubule disruption by 1-aryl-3-(2-chloroethyl)ureas (CEU), we first examined their cytotoxicity on Chinese hamster ovary cells resistant to vinblastine and colchicine due to the expression of mutated tubulins (CHO-VV 3-2). These cells showed resistance to CEU, e.g., 4-tBCEU having an IC50 of 21.3+/-1.1 microM as compared with an IC50 of 11.6+/-0.7 microM for wild-type cells, suggesting a direct effect of the drugs on tubulins. Western blot analysis confirmed the disruption of microtubules and evidenced the formation of an additional immunoreactive beta-tubulin with an apparent lower molecular weight on SDS polyacrylamide gel. Incubation of MDA-MB-231 cells with [urea-14C]-4-tBCEU revealed the presence of a radioactive protein that coincided with the additional beta-tubulin band, indicating that CEU could covalently bind to the beta-tubulin. The 4-tBCEU-binding site on beta-tubulin was identified by competition of the CEU with colchicine, vinblastine, and iodoacetamide, a specific alkylating agent of sulfhydryl groups of cysteine residues. Colchicine, but not vinblastine, prevented the formation of the additional beta-tubulin band, suggesting that 4-tBCEU alkylates either Cys239 or Cys354 residues near the colchicine-binding site. To determine the cysteine residue alkylated by 4-tBCEU, we incubated the radiolabeled drug with human neuroblastoma cells (SK-N-SH) that overexpress the betaIII-tubulin, an isoform where Cys239 is replaced by a serine residue. The results clearly showed that betaIII-tubulin is not alkylated by [urea-14C]-4-tBCEU, suggesting that cysteine 239 residue is essential for the reactivity of 4-tBCEU with beta-tubulin. Taken together, these findings indicate that the mechanism of cytotoxicity of CEU involves microtubule depolymerization through alkylation of beta-tubulin.

Differences in neuropsychological and academic achievement between adolescent delinquents and status offenders.

OBJECTIVE: This study sought to determine whether neurocognitive factors could discriminate delinquents brought before a juvenile court in a large urban area from nondelinquent status offenders brought before the same court. METHOD: Psychological tests were administered to 216 adolescents, aged 13-15 years, presenting to a large urban juvenile court. One hundred ten delinquents (65 male and 45 female) were compared to 106 high-risk nondelinquents (65 male and 41 female) on the WISC-R subtests, the Wide Range Achievement Test, and the Memory for Designs Test. RESULTS: Discriminant analysis revealed that the male delinquents could not be discriminated from the comparison group of male status offenders on the basis of scores on any of the measures. Among the female subjects, scores on reading, arithmetic, digit span, and picture completion subtests and the Memory for Designs Test differed significantly between groups, with some of the findings favoring status offenders and others favoring delinquents. CONCLUSIONS: Overall, the findings did not support the hypothesis that inferior intelligence is an independent risk factor for delinquent behavior.

Child maltreatment histories among runaway and delinquent children.

The records of 378 children presenting to a juvenile court were reviewed for histories of child maltreatment, based on evidence from a formal investigative process by the Department of Social Services. The children came from two groups: delinquents, who had engaged in criminal activity, and status offenders, whose legal involvement was due to non-criminal behavior, such as running away and truancy. Fifty-five percent of the status offenders and 45% of the delinquents had substantiated histories of maltreatment. The percent of status offenders who had been sexually abused was seven times higher among runaways (35%) than among the other members of the group (5%). The percent of delinquents who had been physically maltreated was significantly greater among those convicted of committing violent crimes (27%) than among the non-violent delinquents (14%). Thus, maltreatment may contribute significantly to delinquent behavior.

Clinical evaluation of juvenile delinquents: who gets court referred?

This study examines which alleged delinquents in a large urban juvenile court are selected for referral to the court's psychiatric clinic. A number of demographic factors, probation officer impressions, index charges, and past delinquency record variables were examined for all minor delinquency cases referred in a six-month period and for a random selection of nonreferred cases. In general, referral was associated with lower socioeconomic status (SES), and with a variety of probation impressions of child and family dysfunction. Little relation to index charge or past record was found. The authors interpret these results as showing that probation referrals appear to be made more on the basis of high risk than on the basis of favorable prognosis for treatment.