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Objective: To generate and to evaluate ex vivo a novel model of bioengineered human bladder mucosa based on fibrin-agarose biomaterials., Methods: We first established primary cultures of stromal and epithelial cells from small biopsies of the human bladder using enzymatic digestion and selective cell culture media. Then, a bioengineered substitute of the bladder lamina propria was generated using cultured stromal cells and fibrin-agarose scaffolds, and the epithelial cells were then subcultured on top to generate a complete bladder mucosa substitute. Evaluation of this substitute was carried out by cell viability and histological analyses, immunohistochemistry for key epithelial markers and transmission electron microscopy., Results: The results show a well-configured stroma substitute with a single-layer epithelium on top. This substitute was equivalent to the control bladder mucosa. After 7 days of ex vivo development, the epithelial layer expressed pancytokeratin, and cytokeratins CK7, CK8 and CK13, as well as filaggrin and ZO-2, with negative expression of CK4 and uroplakin III. A reduction of the expression of CK8, filaggrin and ZO-2 was found at day 14 of development. An immature basement membrane was detected at the transition between the epithelium and the lamina propria, with the presence of epithelial hemidesmosomes, interdigitations and immature desmosomes., Conclusions: The present results suggest that this model of bioengineered human bladder mucosa shared structural and functional similarities with the native bladder mucosa, although the epithelial cells were not fully differentiated ex vivo. We hypothesize that this bladder mucosa substitute could have potential clinical usefulness after in vivo implantation., (© 2015 The Japanese Urological Association.)

Aims: To systematically compare different approaches for evaluating mucosal proliferative activity regarding their diagnostic role for delineating flat lesions of the bladder mucosa., Methods: 32 carcinoma in situ (CIS) and 31 flat non-CIS conditions (low-grade dysplasia and reactive atypia) of the bladder mucosa were assessed by two independent pathologists in two rounds in terms of their proliferative activity assessed by the mitotic counts on H&E-stained sections (mitoses per mm(2)) and immunohistochemically using the MIB-1 antibody and the mitosis marker phosphohistone H3 (PHH3). Two different approaches for immunoscoring (percentage of stained nuclei vs dichotomised height of mucosal staining considering lower half vs full-thickness marker expression) were applied. κ statistics were used to evaluate interobserver and intraobserver reproducibility., Results: Scoring the percentage of Ki67 expressing cell nuclei seems to be superior to dichotomisation of the height of mucosal staining as well as to PHH3 immunostaining and conventional mitotic counts in terms of delineating CIS from flat non-CIS conditions. This approach shows substantial (κ=0.62-0.65; p<0.001) interobserver and substantial to almost perfect (κ=0.67-0.83; p<0.001) intraobserver reproducibility., Conclusions: The MIB-1 antibody is a useful adjunct in the differential diagnosis of conventionally challenging flat lesions of the bladder mucosa. In particular, 16% or more Ki67 positive cell nuclei favours CIS over flat non-CIS conditions, whereas 15% or less Ki67 positive cell nuclei is supportive of non-CIS conditions. However, due to some important limitations of MIB-1 staining, the MIB-1 antibody should be used as a component of a panel.

Changes in the distribution of interstitial cells (IC) are reportedly associated with dysfunctional bladder. This study investigated whether spinal cord injury (SCI) resulted in changes to IC subpopulations (vimentin-positive with the ultrastructural profile of IC), smooth muscle and nerves within the bladder wall and correlated cellular remodelling with functional properties. Bladders from SCI (T8/9 transection) and sham-operated rats 5 weeks post-injury were used for ex vivo pressure-volume experiments or processed for morphological analysis with transmission electron microscopy (TEM) and light/confocal microscopy. Pressure-volume relationships revealed low-pressure, hypercompliance in SCI bladders indicative of decompensation. Extensive networks of vimentin-positive IC were typical in sham lamina propria and detrusor but were markedly reduced post-SCI; semi-quantitative analysis showed significant reduction. Nerves labelled with anti-neurofilament and anti-vAChT were notably decreased post-SCI. TEM revealed lamina propria IC and detrusor IC which formed close synaptic-like contacts with vesicle-containing nerve varicosities in shams. Lamina propria and detrusor IC were ultrastructurally damaged post-SCI with retracted/lost cell processes and were adjacent to areas of cellular debris and neuronal degradation. Smooth muscle hypertrophy was common to SCI tissues. In conclusion, IC populations in bladder wall were decreased 5 weeks post-SCI, accompanied with reduced innervation, smooth muscle hypertrophy and increased compliance. These novel findings indicate that bladder wall remodelling post-SCI affects the integrity of interactions between smooth muscle, nerves and IC, with compromised IC populations. Correlation between IC reduction and a hypercompliant phenotype suggests that disruption to bladder IC contribute to pathophysiological processes underpinning the dysfunctional SCI bladder., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Purpose: Urothelium has 2 main functions. It is a barrier to urine and has a sensory role. In response to stretch urothelium releases various substances that modulate afferent nerve activity. Recent data on the localization of cyclooxygenase type 1, the enzyme responsible for prostaglandin production, suggests that prostaglandin may have complex local action., Materials and Methods: The bladders of 7 guinea pigs were stained for prostaglandin receptors type 1 and 2, and costained for vimentin and cyclooxygenase I., Results: Prostaglandin receptor type 1 staining was seen in urothelial cells and in the suburothelium. Urothelial staining, which was often punctuate and weak, was detected in all urothelial cell layers, including suburothelial cells. In contrast, strong prostaglandin receptor type 2 staining was seen in the urothelium and in suburothelial cells. Cyclooxygenase I was absent in interstitial cells and umbrella cells with the highest concentration in the basal cell layer., Conclusions: Interstitial cells express prostaglandin receptor types 1 and 2, indicating that they can respond to prostaglandin. Umbrella cells do not express cyclooxygenase I. Cyclooxygenase I was present in basal urothelial cells, making them a possible site of prostaglandin synthesis. Thus, prostaglandin produced by urothelium may target prostaglandin receptor types 1 and 2 in the urothelium and suburothelium. Therefore prostaglandin is hypothesized to have a role in signal regulation in the bladder wall., (2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Studies predominantly performed on cystectomy specimens have shown that antibodies against smoothelin can distinguish between muscularis mucosae (MM) (negative or weak stain) and muscularis propria (MP) (strong stain). However, studies on diagnostically difficult (transurethral resection) specimens have not been performed. We studied 34 transurethral resection cases where outside pathologists questioned the presence of MP invasion. Upon expert review of the H&E slides, there was no MP invasion in 18 cases. Smoothelin in MM was negative in 8/18 (44%), weakly positive (1+) in 5/18 (28%), moderately positive (2+) in 4/18 (22%), and moderately/strongly (2-3+) positive in 1/18 (6%). Smoothelin in uninvolved MP present in 8 cases was: 2+ in 2/8 (25%) and 3+in 6/8 (75%). Smoothelin expression in MM was weaker than in MP in 7/8 (88%) cases where both were present. Of 16 tumors with MP invasion, smoothelin in involved MP was: 1+ in 1/16 (6%), 2+ in 3/16 (19%), 2 to 3+ in 9/16 (56%), and 3+ in 3/16 (19%). Smoothelin expression in concurrent uninvolved MP was similar. Our data confirm the relatively distinct staining pattern of smoothelin between MM and MP. However, due to the overlap of intensity between MM and MP, caution should be maintained while using smoothelin immunohistochemistry as a diagnostic tool for MP invasion.

The present article describes the preparation and characterization of anionic collagen gels obtained from porcine intestinal submucosa after 72 h of alkaline treatment and in the form of rhamsan composites to develop injectable biomaterials for plastic reconstruction. All materials were characterized by SDS/polyacrylamide gel electrophoresis, infrared spectroscopy, thermal stability, potentiometric titration, rheological properties, and fluidity tests. Biocompatibility was appraised after the injection of anionic collagen: rhamsan composites at 2.5% in 60 North Folk rabbits. Independently of processing, the collagen's secondary structure was preserved in all cases, and after 72 h of hydrolysis the collagen was characterized by a carboxyl group content of 346+/-9, which, at physiological pH, corresponds to an increase of 106+/-17 negative charges, in comparison to native collagen, due to the selective hydrolysis of asparagine and glutamine carboxyamide side chain. Rheological studies of composites at pH 7.4 in concentrations of 2, 4, and 6% (in proportions of 75:1 and 50:1) showed a viscoelastic behavior dependent on the frequency, which is independent of concentration and proportion. In both, the concentration of the storage modulus always predominated over the loss modulus (G'>G'' and delta<45 degrees ). The results from creep experiments confirmed this behavior and showed that anionic collagen:rhamsan composites at pH 7.4 in the proportion of 50:1 are less elastic and more susceptible to deformation in comparison to gels in the proportion of 75:1, independent of concentration. This was further confirmed by flow experiments, indicating that the necessary force for the extrusion of anionic collagen:rhamsan composites, in comparison to anionic collagen, was significantly smaller and with a smooth flow. Biocompatibility studies showed that the tissue reaction of anionic collagen:rhamsan composites at 2.5% in the proportion of 75:1 was compatible with the application of these gels in plastic reconstruction. These results suggest that the association of collagen with rhamsan may be a good alternative in the replacement of glutaraldehyde to stabilize the microfibril assembly of commercial collagen gel preparations., (Copyright (c) 2005 Wiley Periodicals, Inc.)

Objectives: To investigate tumour tissue and non-malignant tumour-surrounding bladder mucosa (NTSBM) for expression of human telomerase RNA (hTR) as a possible marker for premalignant transformation of urothelial cells., Material and Methods: From 67 patients with superficial transitional cell carcinoma (TCC), sections with representative tumour tissue and NTSBM were selected for evaluation. Sections with moderate or severe dysplasia were omitted from evaluation. hTR expression was detected with in situ hybridization using a 35S-UTP-labelled riboprobe, and analysed semiquantitatively by counting the hybridization signals., Results: In 45 of the 67 patients hTR expression was moderate or strong in tumour tissue, and in 20 hTR expression was moderate or strong in NTSBM. Moderate or strong hTR expression was detected in the NTSBM from 19 of 60 patients with pTa/pT1 tumours. Of the 56 patients who were treated conservatively, eight had tumour recurrence, of whom five had moderate or strong hTR expression in the TSBM, compared with only 14 of 48 patients without tumour recurrence., Conclusion: Detecting hTR expression and the morphological distribution of hTR hybridization signals in NTSBM by in situ hybridization might indicate premalignant alterations involved in tumour recurrence.

Seven chemicals, six of which are known to induce epithelial neoplasms of the urinary bladder in rats, were assayed for their ability to induce DNA damage in primary cultures of rat and human cells from urinary bladder mucosa, and in urinary bladder, liver and kidney of intact rats. Significant dose-dependent increases of DNA fragmentation, as measured by the Comet assay, were obtained in cells from both rats and humans with the following concentrations of five test compounds: 2-naphthylamine and N-nitrosodi-n-butylamine 0.5 and 1 mM, phenacetin 2 and 4 mM, cyclophosphamide from 2 to 8 mM, and o-toluidine 16 and 32 mM. Nitrilotriacetic acid (1-4 mM), a rat bladder carcinogen, and 4-aminobiphenyl (0.125-0.5 mM), a bladder carcinogen in humans but not in rats, gave a weak positive response in rats cells and a more marked response in humans cells. In terms of DNA-damaging potency, 4-aminobiphenyl, cyclophosphamide, phenacetin and 4 nitrilotriacetic acid were more active in human than in rat cells, whereas the converse occurred with 2-naphthylamine. Consistently with the results observed in vitro statistically significant dose-dependent increases in the average frequency of DNA breaks were detected in the urinary bladder mucosa of rats given p.o. single doses corresponding to 14 and 12 LD50 of six of the seven test compounds; the only one which gave a substantially negative response was 4-aminobiphenyl. With the exception of N-nitrosodi-n-butylamine which caused DNA damage in liver and of phenacetin and nitrilotriacetic acid which caused damage in kidney in agreement with their tumorigenic activity, any substantial evidence of DNA lesions in these two organs was absent in rats treated with 12 LD50 of the other 4 test compounds. These findings give evidence that urinary bladder genotoxic carcinogens may be identified by the DNA damage/Comet assay using as targets cells of urinary bladder mucosa, and show that the effect may be quantitatively different in cells from rats and from human donors.

Objective: To investigate whether progesterone receptors are present in the mucosa of the urinary bladder of continent premenopausal women compared with continent postmenopausal women., Materials and Methods: Fifty-seven biopsies from the mucosa of the trigone and lateral wall of the urinary bladder were examined by the avidin-biotin-peroxidase immunohistochemical technique for the presence of estrogen and progesterone receptors. The specimens were obtained at cystoscopy performed to investigate hematuria in 42 patients and neoplasia in 15. The study group (n = 29) comprised non-pregnant premenopausal women in the luteal phase of their menstrual cycle and the control group (n = 28) comprised postmenopausal women. None of the subjects had urinary incontinence or was taking medication with hormones. In no case did the primary lesion involve the specimen used for laboratory analysis., Results: There was positive immunostaining with estrogen in 28 patients of the study group (96.5%) and 4 (14.4%) in the control group (p<0.0001). The 28 samples of the study group also showed positive immunostaining for progesterone receptors. There was positive immunostaining with progesterone in 18 samples (64.3%) of the control group (p<0.01). Fourteen samples (50%) of the control group thus showed positive immunostaining for progesterone but no evidence of positive immunostaining with estrogen. Immunostaining for estrogen and progesterone receptors was similar in trigonal and lateral wall samples., Conclusion: In continent pre- and post-menopausal women, a direct progestogenic effect on the mucosa of the urinary bladder seems likely in addition to estrogen.

The urinary bladder is lined by transitional epithelium, the glycocalyx on the luminal surface has interesting properties and is implicated in protective functions. Glycoconjugates are major components of the glycocalyx, but their biochemical nature is not well understood. Previous studies on rabbit bladder indicated the presence of significant levels of sialoglycoproteins compared to glycosaminoglycans in the epithelium. In this study, rabbit explant cultures were radiolabeled by precursor sugars or amino acids and a major lectin-reactive glycoprotein of rabbit bladder mucosa was isolated by affinity chromatography on jacalin-agarose. The radiolabeled glycoprotein was purified to homogeneity by a second cycle on the lectin column, followed by gel filtration and density gradient centrifugation. The average molecular mass of the glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDS-PAGE, respectively. Its buoyant density was 1.40 g/ml, suggesting a carbohydrate content of approximately 50%. The percent distribution of glucosamine-derived tritium label in sialic acid, galactosamine, and glucosamine was 30, 52, and 18, respectively. The glycoprotein consisted entirely of small sialylated and neutral oligosaccharides O-glycosidically linked to serine and threonine residues. The same glycoprotein could be immunoprecipitated with an antibody against the carboxy terminal 17 amino acid peptide of human MUC1 mucin glycoprotein. This suggests that this mucin glycoprotein is the rabbit homolog of MUC1 glycoprotein, which has been previously established to be a component of human bladder urothelium and has been purified from human urine and biochemically characterized.

The glycocalyx of the mucosal surface of urinary bladder acts as an effective barrier against invasion by pathogenic microorganisms and injury from toxic substances in the urine. Defects in these bladder mucosal components could thus be important factors in the development of diseases such as interstitial cystitis and lower urinary tract infections. However, information on the nature of glycoconjugates of mammalian bladder mucosa is very limited. In this study, the glycoconjugates of rabbit bladder were examined histochemically using biotinylated lectins with specificities for a variety of carbohydrate moieties. Three [Artocarpus integrifolia (Jacalin), Datura stramonium (DSL), and Maackia amurensis II (MAL-II)] of the lectins bound predominantly to the luminal cell layer, with decreased binding to the basal layers of the epithelium. In contrast, Ricinus communis I and Sambucus nigra lectins did not bind to the cells in the epithelium but strongly interacted with the subepithelial layers, especially the lamina propria. The intensity of the staining by Jacalin and MAL-II was significantly reduced by prior treatment of the bladder sections with O-sialoglycoprotein endopeptidase, indicating that the ligands of these lectins are primarily mucin glycoproteins. In parallel biochemical studies, a high-molecular-weight glycoprotein with characteristics typical of epithelial mucins was purified from the mucosa of rabbit bladder explant cultures metabolically labeled with [(3)H]glucosamine. Quantitative analysis of the sialic acid, uronic acid, and hexosamine contents of delipidated rabbit bladder mucosa revealed a larger proportion of sialoglycoproteins compared with glycosaminoglycans. Taken together, the results of histochemical and biochemical analyses indicate that glycoproteins rather than glycosaminoglycans are the major components of the bladder epithelium, and that the former include a mucin., (Copyright 2000 Academic Press.)

Purpose: The aim of this study was to examine the histopathology of neobladder mucosa biopsy specimens obtained routinely as part of postsigmoidocolocystoplasty (SCP) follow-up., Methods: One hundred cases of SCP (mean age at surgery, 10.6 years) performed by the authors were examined for the presence of dysplasia or malignant changes in the mucosa of the neobladder using H&E and proliferating cell nuclear antigen (PCNA) staining., Results: No dysplastic or malignant changes were identified in any case. Metaplasia was found in 5 cases and hyperplasia in 2. There were no major differences found on H&E and PCNA staining of specimens obtained after different periods of follow-up post-SCP; follow-up was short term (up to 5 years) in 44 cases, medium term (from 5 to 10 years) in 48 cases, and long term (over 10 years) in 8 cases. PCNA staining was significantly more intense in subjects who stopped regular bladder irrigations (BI) post-SCP and in subjects in whom bladder stones developed (P < .05; Welch's t test), compared with subjects who continued BI and subjects in whom bladder stones did not develop., Conclusions: After SCP, patients are advised to continue BI. Regular biopsies should be part of routine follow-up, especially in subjects with bladder stones.

3,3'-Dimethoxybenzidine (DMB), a congener of benzidine used in the dye industry and previously found to be carcinogenic in rats, was evaluated for its genotoxic activity in primary cultures of rat and human hepatocytes and of cells from human urinary bladder mucosa, as well as in liver and bladder mucosa of intact rats. A similar modest dose-dependent frequency of DNA fragmentation was revealed by the alkaline elution technique in metabolically competent primary cultures of both rat and human hepatocytes exposed for 20 h to subtoxic DMB concentrations ranging from 56 to 180 microM. Replicating rat hepatocytes displayed a modest increase in the frequency of micronucleated cells after a 48-h exposure to 100 and 180 microM concentrations. In primary cultures of human urinary bladder mucosa cells exposed for 20 h to 100 and 180 microM DMB, the Comet assay revealed a clear-cut increase of DNA fragmentation. In rats given one-half LD50 of DMB as a single oral dose, the GSH level was reduced in both the liver and urinary bladder mucosa, whereas DNA fragmentation was detected only in the bladder mucosa. Taken as a whole, these results suggest that DMB should be considered a potentially genotoxic chemical in both rats and humans; the selective effect on the rat urinary bladder might be the consequence of pharmacokinetic behavior.

The acid-soluble extract of the bladder mucosal surface was obtained by washing out the bladder with dilute acetic acid in the presence of protease inhibitors. The wash-out materials from rats, rabbits, pigs, and humans manifested strong bactericidal activity against E. coli in vitro. The ultrafiltrate of the human material, which contained two major peptides with apparent molecular masses of 6.7 kD and 8.5 kD, respectively, showed potent bactericidal activity against E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus sanguis. Three antibacterial polypeptides (PiBPs) were purified from the porcine material. The molecular masses of PiBP-5, PiBP-11 and PiBP-25 were 5773.3 Da, 11127.8 Da and 25073 Da, respectively. PiBP-5 was unusually rich in glycine, serine and threonine residues (20.0, 16.3 and 10.4 mol%, respectively), and N-terminal amino acid sequencing revealed that PiBP-5 was homologous (83.3% identity in an 18 residue overlay) to the "tail" of human cytokeratin-7. Although the amino acid compositions of PiBP-11 and PiBP-25 were established, both had blocked N-termini and primary sequence data were not obtained. These results provided evidence indicating that the presence of peptides in the bladder mucosa could enable it to kill adherent bacteria.

Multistep alterations of chromosome 17 in the progression of human urinary bladder neoplasia were studied by superimposed histologic and genetic mapping. The p53 gene was included in the analysis as a model tumor suppressor gene that is frequently involved in urothelial carcinogenesis. The strategy provided a systematic approach to the study of multistep genomic alterations that occur as neoplasia progresses from precursor intraurothelial conditions to invasive cancer. This was accomplished by sampling the entire mucosa of the organ and displaying microscopically identified invasive cancer and precursor conditions in the form of a histologic map. Subsequent isolation of DNA provided a set of samples in which the search for genetic alterations was performed and superimposed on the histologic map. This approach disclosed multifocal allelic losses of chromosome 17 in the early preinvasive phases of urothelial neoplasia. The alterations were predominantly confined to the p12-13, q22-11 and q24-25 regions. Mutations and allelic losses of the p53 gene were mapped to early preinvasive phases of urothelial neoplasia. The data provide detailed analysis of chromosome 17 allelic losses that occur in the development and progression of urothelial neoplasia and represent the first step for genome-wide modeling of multistep human urothelial carcinogenesis.

To establish a method for reasonable clinical use of adriamycin (ADM) and pirarubicin (THP) in the intravesical chemotherapy for superficial bladder cancer, intracellular concentrations of these drugs were examined in culture cell lines (T-24, T-24/ADM and FHS736b1) with variable durations of contamination. The intracellular concentration of THP showed a plateau at 15-30 min. contamination in T-24, and in T-24/ADM, and showed the time dependence of contamination in FHS736b1, human normal bladder mucosa cell line. The intracellular concentration of ADM showed the time dependence of contamination in T-24, T-24/ADM and FHS736b1. And these concentrations of THP were 20 times higher than those of ADM. In conclusion, it seems better that THP was retained for 5-15 min. in the bladder in the intravesical chemotherapy, from the point of view of drug efficacy and preventing side effects. And it seems good that ADM was retained for more than 30 min. in the case with drug sensitive tumors.

An impairment of the mucosal glycoconjugates could be an important factor in the development of bladder disorders such as interstitial cystitis. However, very little definitive biochemical information is available on the glycoco-jugate components of the mammalian bladder mucosa. In this-study, the mucosa from metabolically radiolabeled rabbit bladder was separated, delipidated, and digested with protease, and the released glycosaminoglycans and glycopeptides were fractionated. About 80 and 36% of the nondialyzable tritium and 35S activities, respectively, was associated with the sialoglycopeptide fractions. The balance of the total tritium activity in the protease digest was in glycosaminoglycans identified as hyaluronan, chondroitin sulfates, and heparan sulfate. Immunohistochemical examination using anti-heparan sulfate antibodies, including one against mouse syndecan-1, indicated the presence of heparan sulfate proteoglycan in the epithelium. In contrast, there was no significant staining of the bladder epithelium with anti-chondroitin-4- and 6-sulfate antibodies or hyaluronan-binding protein. The lamina propria and muscle layers showed strong staining with anti-chondroitin-4-sulfate antibody and hyaluronan-binding protein and weak staining with anti-chondroitin-6-sulfate antibody. The insignificant levels of glycosaminoglycans in the glycocalyx of bladder mucosa epithelium suggest that glycosaminoglycans may be less important than other glycoconjugates in maintaining normal epithelial function and in bladder disorders such as interstitial cystitis.

Intravesical administration of drugs has been used commonly in spinal cord injury patients to suppress detrusor hyperreflexia (eg oxybutynin, verapamil, terodiline) or, to initiate a micturition reflex (eg 15S 15-methyl prostaglandin F2 alpha, protaglandin E2); however, the response has been variable and sometimes, unpredictable. This prompted us to study the presence of nerve fibres in the vesical urothelium and submucosa in mucosal biopsies taken from the dome and trigone (obtained prior to performing a therapeutic procedure eg, vesical lithotripsy, or a diagnostic cystoscopy) in 47 consecutive, unselected paraplegic/tetraplegic patients with a neuropathic urinary bladder. Nerve fibres were demonstrated by routine immunohistochemical technique using commercially available monoclonal and polyvalent antibodies against S-100 (DAKO A/S, Glostrup, Denmark) and Neurofilament (MILAB, Malmo, Sweden). Biopsy specimens were graded for the presence of nerve fibres on a 0-3 scale for urothelium, and superficial/deep submucosa separately in a blind and randomised manner. Virtually no fibre presence was found in one paraplegic patient and no superficial or single fibres were noted in a tetraplegic patient. Absence of C-fibre hyperplasia (Grade 0) was found in nine cases (paraplegic: 4; tetraplegic: 5); Grade 1 hyperplasia was observed in 17 cases (paraplegic: 4; tetraplegic: 13); Grade 2 hyperplasia was seen in 11 cases (paraplegic: 7; tetraplegic 4); and Grade 3 hyperplasia was noticed in eight cases (paraplegic 3: tetraplegic: 5). The magnitude of C-fibre hyperplasia was not significantly different between paraplegic and tetraplegic patients (chi(2) = 4.64; P = 0.3262). The relationship, if any, between the degree of C-fibre hyperplasia and duration of paralysis was studied by categorising patients as < 5 years, and > 5 years of paralysis. No evidence of single fibre or fibre bundle hyperplasia (Grade 0) was seen in five and six cases, grade 1 hyperplasia in six and 11 cases, grade 2 hyperplasia in two and nine cases, and grade 3 hyperplasia in three and five cases respectively in these two categories of patients. (chi(2) = 1.92; P = 0.58). The possible relationship between C-fibre hyperplasia in the vesical mucosa/submucosa and (i) the vesical response to intravesical drug administration; (ii) the vesical urothelial proliferation arrest; (iii) the electrical stimulation of urinary bladder by implanted electrodes (sacral anterior root stimulator); and (iv) long-term indwelling urethral Foley catheter drainage, are discussed with illustrative case reports. In conclusion, mucosal biopsy and study of nerve fibres in urothelium and submucosa of neuropathic bladder has helped to generate hypotheses on the association between C-fibre hyperplasia and response to intravesical pharmacotherapy and the predictive value of such a study in identifying those patients who are likely to respond to intravesical pharmacotherapy.

The distribution of neurons expressing NADPH diaphorase activity was examined histochemically in whole mount preparations of the neonatal guinea pig urinary bladder. NADPH diaphorase positive neurons were abundant in the intramural ganglia in both the detrusor and trigone regions of the bladder. Labelled nerve fibres were found in the ganglion interconnectives and in smooth muscle bundles. Mucosal epithelial cells and endothelial cells lining the blood vessels supplying the bladder were also found to express NADPH diaphorase activity. In order to verify that NADPH diaphorase activity represented the presence of nitric oxide synthase in bladder neurons, a well characterised tissue culture preparation was employed. This also provided an opportunity to estimate the proportion of the total population of bladder neurons which expressed NADPH diaphorase activity. Using a combination of histochemical and immunohistochemical techniques, NADPH diaphorase positive neurons were found to constitute approximately 90% of the total neuronal population, which was identified by labelling with an antiserum to the general neuronal marker protein gene product 9.5. Almost all neurons (99%) which expressed NADPH diaphorase activity in culture were also found to be immunoreactive for nitric oxide synthase. These findings indicate that nitric oxide may play a role in the neural control of bladder function, and this possibility is discussed.

Acid-soluble extract of human bladder mucosal surface was obtained by washing out the bladder of accidentally dead male human with 1% acetic acid in the presence of proteinase inhibitors. Acid urea polyacrylamide gel electrophoresis (AU-PAGE) indicated that the acid-soluble extract had more than 10 main protein bands, but the currently known antibiotic peptides, e. g., Lysozyme and defensins, were not found in this extract. The quantitation assay of lysozyme showed that lysozyme only amounted to 2.2 microgram per milligram protein. When tested for antibacterial activity by using ultrasensitive radial diffusion assay, the acid-soluble extract effectively killed E. coli ML-35P. The antibacterial activity of the acid-soluble extract was further analysed by gel overlay technique and the result showed that one main protein band, which was referred to as human bladder protein (HBP), was potently antibacterial against E. coli ML-35P. The HBP accounted for 4% of the total acid-soluble extract protein. These results suggest that the presence of antibacterial protein in the bladder mucosa may be the molecular base of the bladder antibacterial defence mechanisms.

Partial obstruction of the rabbit bladder outlet induces a rapid hypertrophy characterized by increased bladder mass, increased smooth muscle content, and increased collagen deposition. In addition, partial outlet obstruction induces decreased contractile responses to both field stimulation and postsynaptic receptor stimulation. Although the morphological and contractile responses to partial outlet obstruction have been well characterized, there is little information on the cellular and molecular mechanisms of these changes. In a previous study, we demonstrated that one of the earliest genes to be expressed following partial outlet obstruction in rabbits was the gene expressing stress protein-70 (HSP-70). In order to further define the genetic and molecular basis of these responses, the expression of stress gene products HSP-70 and HSP-90 in rabbit urinary bladder subjected to partial outlet obstruction has been quantitatively evaluated by Western blot coupled with laser densitometry using anti-HSP-70 and -90 monoclonal antibodies. The data show that stress gene products HSP-70 and HSP-90 are constitutively expressed in control rabbit bladder tissue and transiently increased following partial outlet obstruction. Increased content of HSP-70 was detected at 6 hr after obstruction and reached a maximum (2.7-fold over the control level) at 24 hr. Increased HSP-90 was also detected at 6 hr but reached a maximum (4.5-fold over the control level) at 12 hr. By 7 day post-obstruction, the content of these two proteins returned to the control levels.(ABSTRACT TRUNCATED AT 250 WORDS)

The biochemical composition of physiologically moist mucosa and serosa of rabbit bladder before and after bladder outlet obstruction was determined by means of FT-IR spectroscopy with the ATR method and second-derivative analysis. A predominantly beta-sheet structure was found in the amide I band for mucosa and serosa before and after obstruction, but the random coil structure increased in both obstructed bladder samples. However, the major beta-sheet structure associated with some alpha-helical structure in the amide II band of mucosa and serosa for non-obstructed bladder changed into a predominantly alpha-helical structure after bladder obstruction. The obstructed bladder serosa was more pronounced. The amount of glycoproteins doubled in the obstructed bladder serosa, but did not change in the bladder mucosa.

Water and electrolyte transport in turtle urinary bladder closely resembles that present in the mammalian collecting tubule. Although cAMP is known to participate in the control of mucosal transport processes, the GTP-binding inhibitory Gi and stimulatory Gs proteins which link receptors on the cell surface to the adenylate cyclase system remain to be identified in this urinary epithelium. To this end, individual cells harvested from the mucosal surface of the turtle bladder were isolated using a discontinuous density Ficoll gradient. Examination by electron microscopy of the material from the different layers of the Ficoll gradient confirmed that bands II and III contained carbonic anhydrase-rich cells and granular cells, respectively. Identification of Gi and Gs in carbonic anhydrase-rich and granular cells was accomplished using pertussis (PT) and cholera toxins to promote [32P] ADP ribosylation of the proteins. Separation of Gi and Gs from other cell proteins was accomplished using polyacrylamide gel electrophoresis and autoradiography. Pretreatment of cells with 0.2% triton X-100 substantially magnified the ADP-ribosylation of Gi by PT. A doublet form of Gi was present in the 40-kD region and indicated heterogeneity of the PT substrate in granular and carbonic anhydrase-rich cells. Gs was observed as a single polypeptide at the 42-kD region in both cell types. A distinct 45-kD peptide not present in mammalian collecting tubule was identified by both toxins in granular cells and by cholera toxin in carbonic anhydrase-rich cells. In summary, this investigation identified and characterized Gi and Gs proteins in carbonic anhydrase-rich and granular cells from the mucosa of turtle urinary bladder.(ABSTRACT TRUNCATED AT 250 WORDS)

This study deals with the effect of mucosal UO2(2+) on the Ca(2+)-blockable, poorly selective cation channels in the apical membrane of frog skin and toad urinary bladder. Our data show that UO2(2+) inhibits the Na+ currents through the amiloride-insensitive cation pathway and confirm a previously described stimulatory effect on the amiloride-blockade Na+ transport. Noise analysis of the Ca(2+)-blockable current demonstrates that the divalent also depresses the low-frequency Lorentzian (fc = 11.7 Hz) in the power density spectrum (PDS) and reveals the presence of high-frequency relaxation noise (fc = 58.5 Hz). The action of UO2(2+) is not reversed upon washout and is not accompanied by noise, typically induced by reversible blockers. The divalent merely depresses the plateau of the low-frequency Lorentzian, demonstrating a decrease in the number of conductive cation channels. Similarly, with mucosal K+ and Rb+, UO2(2+) also unmasks the high-frequency Lorentzian by depressing the noise from the slowly fluctuating cation channels (type S). In all experiments with mucosal Cs+, the PDS contains high-frequency relaxation noise (fc = 75.1 Hz in Rana temporaria, and 65.4 Hz in Rana ridibunda). An effect of UO2(2+) on the Cs+ currents and Lorentzian plateaus could not be demonstrated, suggesting that this monovalent cation does not pass through type S channels. Experiments with the urinary bladder revealed only a UO2(2+)-insensitive pathway permeable for Na+, K+, Rb+, and Cs+. We submit that in frog skin two cation-selective channels occur, distinguished by their spontaneous gating kinetics, their sensitivity to UO2(2+), and their permeability for Cs+. In toad urinary bladder, only one kind of cation-selective channel is observed, which resembles the UO2(2+)-insensitive channel in frog skin, with fast open-closed kinetics (type F).