Cytotoxicity and antiangiogenic effects of Rhus coriaria, Pistacia vera and Pistacia khinjuk oleoresin methanol extracts.

Angiogenesis, formation of new blood vessels, play an important role in some diseases such as cancer and its metastasis. Using angiogenesis inhibitors, therefore, is one of the ways for cancer treatment and prevention of metastasis. Medicinal plants have been shown to play a major role in the treatment of a variety of cancers. In this direction, cytotoxic and angiogenic effects of oleo gum resin extracts of Rhus coriaria, Pistacia vera and Pistacia khinjuk from Anacardiaceae family were studied. For IC50 values, cytotoxic effects of the plant extracts were evaluated at different concentrations (1, 10, 20, 40, 80,100 µg/ml) against human umbilical vein endothelial normal cell (HUVEC) and Y79 cell lines using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. In vitro tube formation on matrigel base was used to evaluate angiogenic effects in the presence of increasing concentrations (50, 100, 250 µg/ml) of the extracts. Vascular endothelium growth factor was used as angiogenesis stimulator. Gas chromatography results showed that α-pinene and ß-pinene were the major essential oils constituents of all plant extracts. According to the MTT assay results, the R. coriaria resin extract was more cytotoxic than those of P. vera and P. khinjuk extracts (IC50, 9.1 ± 1.6 vs 9.8 ± 2.1 and 12.0 ± 1.9, respectively; P<0.05). Cytotoxic effects of all extracts against Y79 cell line was significantly higher than those of HUVEC used as a normal cell line (P<0.05). Tube formation assay also showed that extract of R. coriaria resin inhibited angiogenesis more significantly than other tested extracts (P<0.05). It could be concluded that R. coriaria resin extract possess cytotoxic effect and antiangiogenesis against cancer cells and as an anticancer natural product has a good potential for future studies.

Effect of human Wharton's jelly mesenchymal stem cell secretome on proliferation, apoptosis and drug resistance of lung cancer cells.

Multipotent mesenchymal stem cells (MSCs) are recently found to alter the tumor condition. However their exact role in tumor development is not yet fully unraveled. MSCs were established to perform many of their actions through paracrine effect. Thus investigation of MSC secretome interaction with tumor cells may provide important information for scientists who are attempting to apply stem cells in the treatment of the disease. In this study we investigated the effect of human Wharton's jelly derived MSC (WJ-MSCs) secretome on proliferation, apoptotic potential of A549 lung cancer cells, and their response to the chemotherapeutic agent doxorubicin. WJ-MSCs were isolated from human umbilical cord and then characterized according to the International Society for Cellular Therapy criteria and WJ-MSC secretome was collected. BrdU cell proliferation assay and Annexin V-PI staining were used for the evaluation of cytotoxic and proapoptotic effects of WJ-MSC secretome on A549 cells. WJ-MSC secretome neither induced proliferation of lung cancer cells nor affected the apoptotic potential of the tumor cells. We also studied the combinatorial effect of WJ-MSC secretome and the anticancer drug doxorubicinwhich showed no induction of drug resistance when A549 cells was treated with combination of WJ-MSC secretome and doxorubicin. Although MSCs did not show antitumor properties, our in vitro results showed that MSC secretome was not tumorigenic and also did not make lung cancer cells resistant to doxorubicin. Thus MSC secretome could be considered safe for other medical purposes such as cardiovascular, neurodegenerative, and autoimmune diseases which may exist or occur in cancer patients.

Long-term and efficient expression of human ß-globin gene in a hematopoietic cell line using a new site-specific integrating non-viral system.

Targeted integration of a therapeutic gene at specific loci in safe genomic regions by a non-viral vector can restore the function of the damaged gene. This approach also minimizes the potential genotoxic effects of transferred DNA. In this study, we have developed a non-viral vector that functions according to site-specific recombination (SSR). The vector contained a bacterial backbone and puromycin resistance gene (pur(r)), a ß-globin expressing cassette and an attB recombination site. We used phiC31 integrase to insert a copy of the vector into specific genomic locations of a human hematopoietic cell line. Site-specific integration of the vector with one or two copies in the transcriptionally active regions of the genome was confirmed. After genomic integration, we used Cre recombinase to remove the bacterial backbone and pur(r). This removal was verified by negative selection and genomic PCR screening. Following deletion of these sequences, the stable ß-chain expression was continued for several months in the absence of selective pressure. Consequently, this vector may potentially be a powerful tool for ex vivo correction of ß-globinopathies such as ß-thalassemia through successful genomic integration of a functional copy of the globin gene into the patient's target cells.

Synthesis and characterization of folate-targeted dextran/retinoic acid micelles for doxorubicin delivery in acute leukemia.

Folate and retinoic acid grafted/dextran (FA-RA/DEX) copolymers with different molecular weight of DEX were synthesized using carbonyldiimidazole and dimethylaminopyridine for targeted delivery of doxorubicin (DOX) in acute myelogenous leukemia (AML). The copolymers structure was confirmed by (1)H NMR and FTIR. Critical micelle concentration (CMC) of each copolymer was determined using pyrene as a fluorescent probe. DOX was loaded in micelles by the direct dissolution method. Physical properties of micelles, including particle size, zeta potential, drug loading efficiency, and drug release profiles, were examined. The orientation of the folate ligand on the surface of the micelles was studied by X-ray photoelectron spectroscopy (XPS) technique. The cytotoxicity of micelles loaded with DOX at different concentrations was studied in KG1 cells using MTT assay and their cellular uptake by flow cytometry technique. FTIR and (1)H NMR spectra confirmed successful production of the targeted micelles and XPS spectra showed the surface orientation of folate. R15D10F7 copolymer produced micelles with particle size of 82.86 nm, polydispersity index of 0.3, zeta potential of -4.68 mV, drug loading efficiency of 96%, and release efficiency of 63%. DOX loaded in folate-targeted micelles of RA/DEX was more toxic than that in nontargeted micelles and free drug and seems promising in reducing drug resistance in AML.

In vitro cytotoxic evaluation of some synthesized COX-2 inhibitor derivatives against a panel of human cancer cell lines.

Celecoxib is a non-steroidal anti-inflammatory drug (NSAID) developed as a selective inhibitor of cyclooxygenase-2 (COX-2) for the treatment of rheumatoid arthritis disease. Recently some other mechanisms have been identified for anti cancer activity of these agents including induction of apoptosis, inhibition of tumor vascularization, stimulation of antitumor immune responses and inhibition of cellular protein synthesis. The cytotoxic effects of four synthesisized analogues of celecoxib (coded as D, E, F and G) were evaluated against Hela, MDA-MB-231, A-2780-s and HT-29 cancer cells, using MTT assay; Also their induction of apoptosis using DNA fragmentation analysis were studied. MTT assay showed that cell survival percent of COX-2 positive cell lines (HT-29, MDA-MB-231 and Hela; p≤0.05) were decreased significantly after exposure to the tested COX-2 inhibitors while little effect was observed on the COX-2 negative cell line (A-2780-s). Results also showed that A-2780-s and Hela were the most resistant and the most sensitive cell lines to these compounds, respectively. Moreover, in DNA fragmentation assay, induction of apoptosis was confirmed by electrophoretic pattern of separated DNA fragments in Hela cell line. Compounds E and G in comparison with D and F exerted more cytotoxic effect on COX-2 positive cell lines (Hela, HT-29 and MDA-MB-231). This could be due to the hydrophobic substituent (Cl, CH3) located at the para position of phenyl ring leading to more lipophilicity and cell uptake. In addition, these COX-2 inhibitors induced apoptosis on Hela cell-line, which could be considered as one of the cytotoxic mechanisms of these compounds as potential anti cancer agents.

Polymeric delivery of siRNA for dual silencing of Mcl-1 and P-glycoprotein and apoptosis induction in drug-resistant breast cancer cells.

Enhanced survival mechanisms of malignant cells in combination with elevated levels of drug transporters can sustain an undesirable resistance against drug therapy. Short interfering RNA (siRNA) delivery against targets involved in aberrant mechanisms is a promising approach and we hypothesize that simultaneous silencing of multiple targets could prove more advantageous than common approach to silence individual targets. To explore this approach, we targeted anti-apoptotic proteins myeloid cell leukemia 1 (Mcl-1) and survivin along with the efflux pump P-glycoprotein (P-gp) in drug-resistant breast cancer cells. Polymeric siRNA delivery was employed for this purpose by using small polyethylenimine (PEI) substituted with lipids. While silencing Mcl-1 caused ∼90% cell death in wild-type cells, this effect was less significant in P-gp over-expressing cells. An additive effect for Mcl-1 and P-gp silencing was evident in the latter cells, where simultaneous silencing of these targets created a significantly higher effect compared with silencing each individual target. Prolonged exposure of wild-type cells to doxorubicin (DOX) resulted in upregulation of P-gp, breast cancer resistance protein, survivin and Mcl-1. Dual silencing of P-gp and Mcl-1 again resulted in an additive effect in resistance-induced cells, which displayed an increased dependency on Mcl-1 for survival. Cytotoxic effect of DOX was also enhanced in resistance-induced cells after silencing Mcl-1. We conclude that polymer-mediated siRNA delivery can silence multiple targets simultaneously and reverse drug resistance.

Use of magnetic folate-dextran-retinoic acid micelles for dual targeting of doxorubicin in breast cancer.

Amphiphilic copolymer of folate-conjugated dextran/retinoic acid (FA/DEX-RA) was self-assembled into micelles by direct dissolution method. Magnetic iron oxide nanoparticles (MNPs) coated with oleic acid (OA) were prepared by hydrothermal method and encapsulated within the micelles. Doxorubicin HCl was loaded in the magnetic micelles. The characteristics of the magnetic micelles were determined by Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), transmission electron microscopy (TEM), and vibrating sample magnetometer (VSM). The crystalline state of OA-coated MNPs and their heat capacity were analyzed by X-ray diffraction (XRD) and differential scanning calorimetry (DSC) methods, respectively. The iron content of magnetic micelles was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). Bovine serum albumin (BSA) was used to test the protein binding of magnetic micelles. The cytotoxicity of doxorubicin loaded magnetic micelles was studied on MCF-7 and MDA-MB-468 cells using MTT assay and their quantitative cellular uptake by fluorimetry method. TEM results showed the MNPs in the hydrophobic core of the micelles. TGA results confirmed the presence of OA and FA/DEX-RA copolymer on the surface of MNPs and micelles, respectively. The magnetic micelles showed no significant protein bonding and reduced the IC50 of the drug to about 10 times lower than the free drug.

Synthesis and biological evaluation of bidentate 3-hydroxypyridin-4-ones iron chelating agents.

A series of 3-hydroxypyridin-4-one derivatives (HPOs) were synthesized and their partition coefficient values (K(part)) were determined. The cytotoxic effects of these iron chelators against Hela cancer cells were also evaluated. The IC(50) of HPOs was determined using MTT assay. Among these ligands, compound 4e (K(part)=5.02) with an IC(50) of 30 µM and 4f (K(part)=0.1) with an IC(50) of 700 µM showed the lowest and highest IC(50)s, respectively. In conclusion, the introduction of a more hydrophobic functional group (such as butyl in compound 4e) on the nitrogen of pyridinone ring resulted in higher cytotoxic activity of ligands.

Effect of cerium lanthanide on Hela and MCF-7 cancer cell growth in the presence of transferring.

The anti-cancer activity of metal ions in the lanthanide group is being considered recently. It has been reported that cerium salts might stimulate the metabolism and therefore, produce anti-cancer effects. However, little is known about the effects of protein-cerium complex in controlling cancer cell growth. The aim of the present study was to elucidate the possible pathways for the cytotoxic effect of cerium in the presence of apo-transferrin on two cancer cell lines (Hela and MCF-7), that express transferrin receptors 3-4 fold higher than normal cells. The effect of different concentrations of cerium (0.1, 1, 10, 100 µM) in the presence and absence of transferrin for 48 h and 72 h incubation periods (37°C, 5% CO2 and 95% humidity) was studied using the MTT assay. The results showed that cerium has a cell-proliferation inhibitory activity which is significantly increased by transferrin protein. Compared with the direct treatment of cancer cells with cerium, the presence of transferrin assisted inhibition of cell proliferation by 20% and 40% in Hela and MCF-7 cells, respectively. Though apo-transferrin could lightly induce cell growth particularly in MCF-7 cells by itself, this phenomenon could not overcome the cerium-protein cell-proliferation inhibition activity. In conclusion, our results indicate that at a certain concentration, the cerium compounds could be possibly involved in the control of cell proliferation and inhibiting the growth of cancer cells.

Cytotoxic evaluation of doxorubicin in combination with simvastatin against human cancer cells.

Doxorubicin is a broad spectrum antibiotic used in the treatment of cancers. Its dose dependent cardiotoxicity is the most serious side effect causing withdrawal of drug from hard chemotherapeutic regimen. Statins are shown to be cytotoxic in concentrations higher than the effective doses for the treatment of hypercholesterolemia (40 mg/day). Co-administration of statins and chemotherapeutic agents suppose to be synergic although there are some controversies in the literature. In this study, cytotoxic effects of doxorubicin alone and in combination with simvastatin on Hela tumor cell line were evaluated. Different concentration of doxorubicin and simvastatin were added to the cultured cells and incubated for 72 h. Cell survival was evaluated using MTT and trypan blue exclusion assays. The results indicated that simvastatin in low concentration (0.25 µM) seems to be growth stimulator although cell viability was reduced in concentrations of ≥2 µM. Doxorubicin alone at all tested concentrations (0.1, 1 and 2 µM) was a cell growth inhibitor. It was also shown that percent cell viability was reduced in a decreasing manner with the following protocols: 1) co-administration of doxorubicin and simvastatin in different concentrations; 2) addition of simvastatin after incubation of cells with doxorubicin and 3) addition of doxorubicin after incubation of cells with simvastatin. It could be concluded that between 3 tested protocols combination of doxorubicin and simvastatin after 72 h incubation, showed the highest cytotoxicity against Hela cells.

Spontaneous intrauterine "ping-pong" fracture: review and case illustration.

We report a case of a closed outer-table parietal "ping-pong" skull fracture occurring in a 4 190-gram female infant born at 39 weeks and 5 days gestation after an uneventful Cesarean section (Apgar scores of 9 and 9 at one and five minutes). There was no maternal history of abdominal trauma during pregnancy and there were no complications or difficulties with Cesarean section delivery. Neurological examination was normal. Computed tomography with three-dimensional reconstruction images showed a 4 x 5 cm depression in the right parietal bone with a medial lucency consistent with a fracture of the superior margin of the skull and leftward deviation of the sagittal suture and sinus. Spontaneous resolution did not occur by one month of age and the skull fracture was repaired with excellent cosmetic results. Rarely has a case of spontaneous intrauterine skull fracture been reported in an atraumatic Cesarean delivery. We believe this fracture resulted from a chronic in utero process without associated trauma as evidenced by deviation of the sagittal suture and sinus.

Failure to obtain durable results with collagen implantation in children with urinary incontinence.

PURPOSE: We studied the efficacy and durability of injection of glutaraldehyde cross-linked bovine collagen for treating urinary incontinence in children. MATERIALS AND METHODS: From April 1994 to July 1995, 12 boys and 8 girls 4 to 18 years old (mean age 9.5) underwent endoscopically directed collagen injections into the bladder neck. Followup ranged from 9 to 23 months (mean 15.2). The etiology of incontinence included myelodysplasia in 12 patients, exstrophy/epispadias in 4, and epidural abscess, sacral agenesis, imperforate anus and posterior urethral valves in 1 each. RESULTS: Collagen was injected once in 9 patients, twice in 10 and 3 times in 1. Injected volume ranged from 3 to 18 cc (mean 7.3). Followup urodynamic studies were available for 10 patients. Leak point pressure increased from 28.7 to 34.9 cm. water. One patient (5%) was dry, 5 (25%) had improvement, 10 (50%) had transient improvement for 2 to 90 days (mean 52) and 4 (20%) remained incontinent. Five children underwent bladder neck sling procedures. In 1 patient transient sciatic nerve irritation developed due to gluteal hematoma. CONCLUSIONS: The previously reported high success rate of collagen injection for urinary incontinence in children is unsupported by this study. Improvement in continence was temporary or inadequate in the majority of patients. Collagen therapy may only delay the need for surgery for managing organic urinary incontinence in children.

Ureteral bladder augmentation.

Virtually all segments of the gastrointestinal tract have been used successfully in augmentation cystoplasty. The complications inherent in enterocystoplasty are well described. Megaureters subtending effete kidneys (poorly or nonfunctioning) provide a novel and excellent source of augmentation material with urothelium and muscular backing, free of the electrolyte and acid base disturbances, and mucus production that plague enterocystoplasty. Augmentation cystoplasty using detubularized, reconfigured, otherwise disposable megaureter, with or without ipsilateral total or partial nephrectomy, was performed in 16 patients (mean age 8.8 years, range 1 to 25) with inadequate and dysfunctional bladders. Postoperative followup varied between 8 and 38 months (mean 22). The overall renal function and radiographic appearance of the remaining upper tracts have remained stable or improved in all patients. Of the 16 patients 15 require intermittent catheterization and 1 voids spontaneously. Ten patients are continent day and night, 5 have improved continence (4 damp at night and 1 stress incontinence) and 1 has failed to gain continence despite good capacity and compliance. Complete postoperative urodynamic evaluations in 12 of 13 patients show good capacity, low pressure bladders with no instability. Complications occurred in 5 patients, including transient urine extravasation in 2, contralateral ureterovesical obstruction in 2 and Mitrofanoff stomal stenosis in 1. Augmentation ureterocystoplasty combines the benefits common to all enterocystoplasties without adding any of the untoward complications or risks associated with nonurothelial augmentations.

Bladder augmentation in patients with neurogenic bladder and vesicoureteral reflux.

Current treatment of noncompliant neurogenic bladder associated with significant vesicoureteral reflux that is refractory to intermittent self-catheterization and anticholinergic therapy includes bladder augmentation coupled with a procedure to eliminate reflux. Antireflux surgery is often difficult in such a clinical setting. The diseased and thickened detrusor makes reimplantation into the bladder difficult, and successful reimplantation into the intestinal component is tricky and time-consuming. Augmentation alone was done in 14 patients with significant vesicoureteral reflux in the face of a noncompliant, high pressure neurogenic bladder. No effort was made to correct reflux surgically because, in theory, reflux is secondary to abnormal bladder pressure. Of the 13 patients who have had adequate evaluation with postoperative cystograms 12 no longer have reflux. The reflux in the remaining patient has improved from grade IV to grade II. Postoperative cystometric examination in 12 patients demonstrated low pressure and adequate volume. Correction of bladder dynamics alone reversed the reflux. Conversely, persistence of reflux postoperatively is an indication that augmentation has not successfully returned the bladder to a low pressure reservoir. Our experience indicates that antireflux procedures are not routinely needed in this group of patients.

Percutaneous balloon dilation of ureteral strictures after failed surgical repair in children.

We attempted treatment of long-standing strictures of ureterovesical or ureterosigmoid anastomosis in 6 children by percutaneous balloon dilation. The 4 girls and 2 boys were between 2 and 19 years old (mean age 9 years) at the time of dilation. Obstruction occurred at the ureterosigmoid anastomosis in 3 patients and at the ureterovesical junction in 3. Obstruction was confirmed by diuresis renography, antegrade pyelography and pressure perfusion studies. An attempt at dilation was unsuccessful for 1 patient. For the remaining 5 patients the ureter has remained patent for a followup period of 7 to 18 months (mean 12 months). Percutaneous balloon dilation of established ureterointestinal and ureterovesical obstruction after failed surgical repair can be successful. The hospital stay is reduced and major surgery with its accompanying morbidity is avoided. If balloon dilation is unsuccessful, surgical repair remains an option. Balloon dilation should be seriously considered as an initial form of treatment in this group of patients.

Success of the artificial urinary sphincter after failed surgery for incontinence.

We implanted the artificial urinary sphincter in 15 incontinent patients for whom multiple urethral and bladder neck operations, including sphincter placement, had been unsuccessful. The 5 male and 10 female patients ranged from 3 to 26 years old (mean age 11 years). The etiology of incontinence was neurogenic bladder in 10 patients, epispadias in 2, exstrophy in 1, ectopic ureters in 1 and traumatic urethral disruption in 1. Of the 15 patients 13 required augmentation enterocystoplasty and clean intermittent catheterization. The initial anti-incontinence procedures were Young-Dees-Leadbetter bladder neck reconstruction in 10 patients, artificial urinary sphincter placement in 4 and bladder neck suspension in 1. Causes of failure of the primary treatment were erosion (artificial urinary sphincter), and incontinence and/or difficult catheterization (Young-Dees-Leadbetter). Followup from the last salvage operation averaged 21 months (range 6 to 37 months). A total of 58 operations was performed. Among the 4 patients in whom the artificial urinary sphincter eroded the bladder neck repeated attempts to place the cuff at the same site were unsuccessful and erosion occurred in all 4 within 1 year. Sphincter placement was more successful among the 11 patients who initially underwent Young-Dees-Leadbetter bladder neck reconstruction or bladder neck suspension; acceptable continence was attained in 8 patients (73%). We conclude that placement of the sphincter cuff around a previously eroded bladder neck probably will result in erosion. Sphincter implantation should be attempted in patients in whom bladder neck reconstruction has failed. Persistence in the treatment of these patients is essential because multiple operations often are necessary to achieve continence.

The influence of HLA matching on renal allograft survival at a single pediatric center.

Between January 1, 1969, and February 2, 1988, 331 pediatric renal transplants were performed at a single pediatric center. Of these 225 were first cadaveric allografts. Graft allocation was independent of the outcome of human lymphocyte antigen (HLA) matching. The results of class I antigen matching were analyzed in 224 transplants, and actuarial graft survivals were calculated. Class II antigen matching was analyzed in 80 patients from January 1, 1982, and submitted to the same analysis. HLA matching could not be demonstrated to be beneficial. It is proposed that any matching benefit is small and obscured in our series by the dominance of other factors.

Urinary tract disorders in patients with chronic granulomatous disease.

Twelve of the 25 patients with chronic granulomatous disease treated at our institution between 1957 and 1987 were found to have urinary tract disorders. All 12 patients were male and 22 years of age or younger when chronic granulomatous disease was diagnosed. Six patients had hydroureteronephrosis in association with recurrent episodes of pyelonephritis, retroperitoneal lymphadenitis, and granuloma formation. The other six patients had genital lesions or dysuria. Among the six patients with hydroureteronephrosis, a nephrectomy was performed in two, ureterolysis was used to relieve obstruction in one, and hydroureteronephrosis resolved after antibiotic therapy alone in three. We conclude that complications involving the genitourinary system occur frequently in patients with chronic granulomatous disease. Periodic imaging of the urinary tract may detect asymptomatic hydroureteronephrosis or other treatable genitourinary abnormalities in these patients.

Nonintubated Anderson-Hynes repair of ureteropelvic junction obstruction in 60 patients.

We reviewed our experience with 68 consecutive Anderson-Hynes ureteropyeloplasties. The 64 infants, children and young adults ranged from 2 days to 28 years old (median age 2 years), and 28 were less than 1 year old. Intubation was used in only 4 patients: 2 who also underwent ureteral reimplantation for vesicoureteral reflux, 1 with stones in the renal pelvis and 1 with pyonephrosis. We successfully repaired 60 of 64 nonintubated renal units (93.4 per cent). Temporary postoperative ureteral stenting was required for extravasation from 8 renal units (12.5 per cent). Two patients later underwent repeat ureteropyeloplasty for recurrent obstruction. Nephrectomy was performed for pyonephrosis in 1 patient and for a nonfunctioning kidney that had exhibited poor function preoperatively in 1. Prolonged ileus necessitated extended hospitalization in 3 patients (4.6 per cent). We conclude that nonintubated dismembered ureteropyeloplasty for uncomplicated, primary ureteropelvic junction obstruction can be performed safely and successfully, and should be considered the standard treatment. Positioning of the Penrose drain is critical to avoid urinoma formation. When persistent urinary leakage occurs temporary diversion is easy and well tolerated. Hospital stay averaged 12.1 days for patients with extravasation compared to 4.3 days when no extravasation occurred. Immediate preoperative retrograde pyelography did not seem to contribute to postoperative urinary extravasation by causing edema of the ureteral orifice.

Congenital ureteral valves in children: case report and review of the literature.

Congenital ureteral valves are a rare cause of ureteral obstruction. While only 17 pediatric cases have been reported in the literature, this condition should be considered in the differential diagnosis of the child with upper urinary tract obstruction. The pediatric cases are reviewed, and we present an additional case in which unilateral hydronephrosis caused by a ureteral valve was first detected by ultrasound in utero.