Estrogenic regulation of HSP90 kD synthesis in rat urinary bladder.

The role of heat shock protein (HSP90 kD) has been investigated in regard to its association with steroid receptors. HSP90 kD may play a role in steroid receptor stabilization and activation. Oophorectomized Sprague-Dawley rats (n = 25) were placed into five groups and injected subcutaneously with 30 microg beta-estradiol 17-benzoate in sesame oil, with one group injected with carrier oil (control). After estrogen administration, the rats were killed, and their bladders removed for immunostaining, immunoblotting and enzyme-linked immunosorbent assay (ELISA). Immunoblot analysis demonstrated a 90-kD band in bladder homogenates, even in the absence of estrogen. However, the bands were more intense 12 and 24 h after administering estrogen. ELISA showed significant differences in HSP90 kD synthesis as early as 6 h compared to controls (P< 0.05). After 48 h the estrogen-treated rats and controls were identical. The above results were confirmed by immunostaining for HSP90 kD. HSP90 kD synthesis in the rat urinary bladder is under estrogenic regulation. These findings may be relevant in the etiology and pathobiology of interstitial cystitis and menopausal voiding dysfunctions since the bladder is enriched with estrogenic receptors and is under estrogenic influence.

Cyto-injury factors in urine: a possible mechanism for the development of interstitial cystitis.

PURPOSE: In most of our patients with interstitial cystitis (IC), the disease is associated with an increased urothelial permeability whose cause has not been identified. We postulate that both normal urine and the urine of IC patients contains factors capable of injuring the mucosa and causing an increased permeability that would allow urine components to leak into the bladder muscle. To test this hypothesis, we examined fractions of normal urine for toxic effects on bladder smooth muscle and epithelial cells in vitro. In the same in vitro system, we measured the effects of Tamm-Horsfall protein (THP), a normal urinary glycoprotein that may be a scavenger of injurious agents capable of "detoxifying" normal metabolic products. MATERIALS AND METHODS: Human urothelial cells (T24) and rabbit bladder smooth muscle cells were incubated overnight with various fractions prepared from healthy volunteers' urine. The urine fractions of molecular weights >100 Da were incubated overnight with either urothelial or smooth muscle target cells after no treatment or after heating to 56C, preincubation with THP, exposure to heparin, or elution from heparin. Cytotoxicity was determined for each group using a neutral red uptake assay. RESULTS: Urine fractions of molecular weight 500 to 1000 Da were cytotoxic to smooth muscle cells (39%) and urothelial cells (50%). Cytotoxicity levels for THP-treated fractions were significantly lower than those for untreated fractions in both urothelial cells (7% versus 89%, p <0. 001) and smooth muscle cells (8% versus 70%, p <0.01). Fractions exposed to heparin were less cytotoxic to smooth muscle cells (20%) than were untreated fractions (27%). Fractions eluted from heparin were also cytotoxic to urothelial cells (42%). CONCLUSIONS: Normal human urine contains heat labile, cationic components of low molecular weight that bind to heparin. These components, when separated from the bulk of the urinary wastes, are cytotoxic to urothelial cells as well as underlying smooth muscle cells, indicating their potential for causing bladder mucosal injury. The cytotoxic activity can be blocked by the presence of THP. This urinary cytoprotective activity of THP may play an important but unrecognized role in the development of IC.

Urothelial cytoprotective activity of Tamm-Horsfall protein isolated from the urine of healthy subjects and patients with interstitial cystitis.

PURPOSE: Tamm-Horsfall protein (THP) is a ubiquitous urinary protein with essentially no known function. We propose that THP is a cytoprotective agent that protects the urothelium from cationic species. To test this hypothesis we isolated THP from normal and interstitial cystitis urine to see if it could protect cultured cells from damage induced by the polyamine, protamine sulfate (PS). METHODS: Tamm-Horsfall protein was extracted from the urine of interstitial cystitis (IC) patients (N=28) and normal volunteers (N=5). Urothelial target cells (T24) were radiolabeled with 51Cr and then exposed to PS (0-1.0 mg/mL) for either 1.5 or 20 h. The resulting cytotoxicity data (dose-response curves) were then compared with the data obtained when PS was preincubated with 0-0.5 mg/mL of THP (IC vs normal), the semisynthetic polysaccharide, pentosan polysulfate (Elmiron), or human serum albumin. RESULTS: Toxicity of PS was significantly reduced by incubation with THP (or Elmiron) prior to evaluation by the chromium release assay, but not reduced by incubating with another protein, albumin. Tamm-Horsfall protein from IC patients' urine was less protective than an equal quantity of THP from normal urine. CONCLUSIONS: These experiments suggest that THP has an important role in bladder mucosal defense mechanisms, protecting the bladder surface from injury. Inability of THP to prevent cytotoxic damage by urinary polyamine or other urinary toxins (cationic species) may be relevant in the etiology of interstitial cystitis, as putative urinary toxic components have been described in the urine of some patients.

Elevated urinary norepinephrine in interstitial cystitis.

OBJECTIVES: To measure urinary catecholamines and determine the extent to which they may be elevated in urine from patients with interstitial cystitis (IC). METHODS: Random urine samples from patients with IC (n = 111) and urine from normal volunteers (n = 92) were acidified on collection (voided and catheterized specimens) and assayed for catecholamine (norepinephrine or normetanephrine) by enzyme-linked immunosorbent assay. Creatinine levels in these urine samples were also measured. RESULTS: Analysis of the data indicated that patients with IC had a higher urinary level of the neurotransmitter norepinephrine compared with the measured levels in the urine of normal volunteers (89.1 +/- 58.3 versus 54.9 +/- 37.1 microg/g creatinine, P <0.05). The metabolite normetanephrine was similar in the urine samples from these two groups. Urine from patients with bladder outlet obstruction (n = 11) did not have elevated amounts of urinary norepinephrine. The norepinephrine levels were not statistically different in the urine samples from patients with symptomatic and asymptomatic IC. The elevated urinary levels in patients with IC did not decrease after treatment with sodium pentosanpolysulfate (Elmiron), heparinoids, dimethyl sulfoxide, or combinations of these during 1 to 15 months of treatment. CONCLUSIONS: Norepinephrine was found to be elevated in the urine from patients with IC compared with urine from normal controls. This would be consistent with increased sympathetic (adrenergic) activity from the bladders of patients with IC or possibly from increased adrenal activity, since stress is associated with symptom increase in some patients with IC. Norepinephrine levels did not decrease with treatment nor did they differ between symptomatic and asymptomatic patients at the time of urine collection.

Elevated stress protein in transitional cells exposed to urine from interstitial cystitis patients.

BACKGROUND: It has been hypothesized that urine from interstitial cystitis (IC) patients may contain one or more toxic factors not present in "normal" urine. Bladder tissues exposed to these toxic factors could have elevated stress proteins. If this assumption is correct, stress protein levels could be a useful marker for identifying patients at risk for developing this syndrome. METHODS: To experimentally investigate this possibility, a sensitive assay (ELISA) was used to measure levels of the 72 kDa stress protein in urothelial target cells after in vitro exposure to urine from IC patients. RESULTS: We observed a modest 12% increase in 72 kDa stress protein in cells treated with urine from IC patients compared to cells exposed to normal urine (1.12 compared to 0.99 ng/microg extracted protein; P < 0.05). In addition, it was possible to demonstrate the 72 kDa stress protein in histologic sections obtained from mucosal biopsies of IC patients. Stress protein was located primarily in the surface urothelial cells of the mucosa. CONCLUSIONS: These results seem to indicate that stress protein could play an important protective role at this particular site. They further suggest that IC urine is more toxic than normal urine and, in contact with underlying urothelial and deeper bladder tissue, may upregulate genes involved in stress protein responses. This may be an important concept in the etiology of IC.

Bladder injury model induced in rats by exposure to protamine sulfate followed by bacterial endotoxin.

PURPOSE: A bladder injury model was developed using protamine sulfate (PS) and endotoxin lipopolysaccharide (LPS) administered intravesically to female Sprague-Dawley rats. MATERIALS AND METHODS: Experimental and control animals were catheterized and intravesically exposed to PS-LPS, PS, LPS, or phosphate buffered saline. After 4, 24 or 72 hours, rats were sacrificed. Urines and bladder tissues were then obtained. Bladder mucosal permeability was evaluated by measuring 14C-urea uptake 24 hours after injury. Repeated instillations of PS/LPS were also made in another group of rats over a period of 5 weeks to attempt to establish a more serious mucosal injury, possibly reflected by altered staining of the collagen IV component of the urinary basement membrane (UBM). RESULTS: Histological examination of the tissues indicated a maximal inflammatory response in the mucosa 4 hours after instillation of PS/LPS. Neutrophils and macrophages in close proximity to the UBM and intraepithelially could be demonstrated. Bladder permeability was significantly altered (26.9% 14C-urea uptake) in rats assayed 24 hours after the PS/LPS treatment, but not after exposure to PS or LPS alone (11.9 and 17.5%, respectively). Protease activity detected in urines from experimental, but not control, animals coincided with the appearance of inflammatory cells in the lamina propria. Inflammatory injury did not appear to alter the collagen IV staining of the UBM. CONCLUSIONS: This rat bladder injury model is useful for examining controversial issues regarding bladder wall structure-function alterations induced by inflammation and possibly important in the pathobiological mechanisms involved in some patients with interstitial cystitis.

Selective type IV collagen defects in the urothelial basement membrane in interstitial cystitis.

PURPOSE: The study sought to identify changes in the urothelial basement membrane (UBM) associated with interstitial cystitis (IC). MATERIALS AND METHODS: Immunohistochemical assessment of bladder biopsies from IC patients and controls was compared with clinical and histologic findings. RESULTS: Selective decreases or loss of type IV collagen staining, but not laminin, were found in the UBM of 5 of 11 IC patients with no change in type IV collagen staining of other bladder wall sites. CONCLUSIONS: The loss of type IV collagen may represent a primary or secondary event and could alter the UBM's role in permeability, thereby contributing to the pathogenesis of IC in the subset of IC patients exhibiting this change.

Intestinal de-epithelialization and augmentation cystoplasty: an animal model.

OBJECTIVES: An animal model of augmentation cystoplasty was developed in New Zealand rabbits to study the effects of intestinal de-epithelialization on subsequent re-epithelialization by bladder urothelium. METHODS: Twenty-four rabbits underwent augmentation cystoplasty using intestinal segments that were either treated with protamine sulfate and urea solution or else anastomosed with an intact epithelium. Half of the rabbits receiving the de-epithelialized intestinal segments were subjected to glycosaminoglycan replacement therapy by administration of intravesical heparin. Experimental and control rabbits were sacrificed at 1-, 2-, and 3-month intervals. RESULTS: Histologic examination of the augmented sections showed small areas of urothelium growing over the intestinal epithelium (approximately 15%). The heparin-treated group demonstrated the greatest amount of re-epithelialization. There was no obvious histologic difference in the amount of collagen present in the augmented tissues in any of the experimental groups. CONCLUSIONS: In a preliminary study, New Zealand rabbits appear to be satisfactory as an experimental animal for studying the augmentation cystoplasty procedure and for the development of therapeutic interventions for enhancing epithelial growth. Protamine and urea will de-epithelialize the bowel and heparin may promote epithelialization of augmented intestinal segment by transitional epithelium.

Characterization of microbial presence at the surface of silicone mammary implants.

The purpose of this project was to examine the incidence of microbial presence on the surface of mammary implants and its correlation with clinical presentation. The significance of microbial presence without signs of overt infection is questioned. Several issues are raised, including whether the presence of micro-organisms may immunize the host, trigger autoimmune reactions, or locally change the course of healing (resulting in capsular contracture). A total of 150 explanted silicone mammary implants from 87 patients were cultured. Cultures of 81 devices were positive (54%); the predominant isolate was Staphylococcus epidermidis (found on 68 implants, or 84%). Bacteria were detected on 76% (62 of 82) of implants surrounded by contracted capsules and on 28% (19 of 68) of those without capsular contracture (p < 0.05). Among 40 patients (46%) who had no general health problems, 11 (28%) had positive cultures of explanted devices (15 of 62 explants, or 24%). In the remaining 47 patients (54%) who complained of myalgia (77%), arthralgia (68%), chronic fatigue (38%), skin rashes (21%), cognitive problems (19%), dry mucosal membranes (19%), episodes of low-grade fever (17%), and hair loss (13%), 38 (81%) had positive cultures (66 of 88 explants, or 75%) (p < 0.05). The hypothesis that capsular contracture or problems that might be related to chronic infection and immunization are associated with subclinical infection is supported by this study.

A new method for cytodestruction of bladder epithelium using protamine sulfate and urea.

Bladder epithelium relies primarily on the presence of a surface glycosaminoglycan (GAG) layer and the structural integrity of cell-cell contact to maintain impermeability to toxic urinary wastes. Previous clinical studies evaluating bladder permeability characteristics in interstitial cystitis patients had indicated that epithelial desquamation occurs after treatment with protamine sulfate (PS) followed by hypertonic urea. The following study was performed using rabbits to further investigate this finding. The urinary bladder was evaluated for optimal treatment conditions for epithelial removal. Protamine sulfate (1 to 10 mg./ml.) and urea (100 to 200 gm./ml.) were instilled into the bladder at volumes ranging from 5 to 60 ml. to that required for near maximum distention. After incubation at room temperature for 15 minutes, the bladders were fixed and evaluated histologically for epithelial removal. The maximum epithelial removal occurred when the bladders were distended, and when PS concentration was 5 to 10 mg./ml. and urea at 200 gm./l. There was greater epithelium removal after repeated treatments. Epithelial cells that were removed were not viable based on Trypan blue staining. There was no significant increase of C14 labeled urea in the plasma after 15 minutes. Rabbits that were followed for 6 weeks after treatment did not show any histological evidence of increased collagen deposition and/or fibrosis. This procedure may have important clinical value since it may remove sufficient bladder epithelium in patients with transitional cell carcinoma to have therapeutic benefit. This offers a realistic option for selective, nontoxic destruction of bladder epithelium.

Abnormal sensitivity to intravesical potassium in interstitial cystitis and radiation cystitis.

The urgency-frequency syndrome (UFS) (non-bacterial cystitis, interstitial cystitis) may well represent a heterogenous group with several etiologies. This study was based on the hypothesis that one subset of UFS patients has a leaky (to solutes) epithelium and cations such as potassium could thereby diffuse subepithelially and provoke symptoms. It was also hypothesized that normal impermeable transitional epithelium would not allow cations to diffuse across the cells during the K+ provocation test and no symptoms would be experienced. If the epithelium was permeable ("leaky"), diffusion would occur and provoke symptoms. Water or 0.4 M KCl was placed intravesically into normal volunteers and interstitial cystitis (IC) patients. Water did not provoke symptoms in either group but KCl provoked 4.5% of normals and 70% of IC patients. Differences were significant (P < 0.0001). This test provides a valuable diagnostic tool for UFS and a valuable research tool to separate epithelial permeability problems from other subsets of patients. A third group, consisting of 11 IC patients in remission on heparinoid therapy, was also tested and only 18% were provoked by KCl. Four patients with radiation cystitis were also examined and all four (100%) were provoked by the potassium.

Proteoglycan core protein syndecan in bladder biopsies.

The etiology of interstitial cystitis (IC) may be related to a dysfunctional epithelium caused by an abnormal permeability barrier. The presence of deleterious urinary substances (quaternary amines) that alter an otherwise normal epithelium may also be contributory. IC disease could reflect an inability of the bladder to repair its protective surface-coat material (glycosaminoglycans and proteoglycans), which is constantly exposed to a toxic urine environment. Bladder biopsy tissue from IC patients and derived explant cells were investigated to determine if mRNA for a proteoglycan core protein could be extracted and evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Syndecan was chosen for this investigation because the available sequence information permitted PCR primers to be synthesized. The results indicated that biopsy tissue and explant cells could be utilized for the isolation of syndecan core protein mRNA. This proteoglycan was also demonstrated in mouse bladders by immunostaining and immunoblotting (but not in human tissues) using a syndecan-specific monoclonal antibody (281-2). Quantitative differences in IC tissues versus normal bladder tissue with respect to gene expression for this proteoglycan core protein can now be determined.

Evaluation of urothelial Tamm-Horsfall protein and serum antibody as a potential diagnostic marker for interstitial cystitis.

Demonstration of adherence of Tamm-Horsfall protein to bladder epithelium has been suggested as a potential diagnostic test for interstitial cystitis. Bladder specimens from 18 interstitial cystitis patients were evaluated by indirect immunoperoxidase techniques using a Tamm-Horsfall protein specific monoclonal antibody to determine the diagnostic value of the staining results. The study population consisted of 7 severely diseased patients who required cystectomy with urinary diversion and 11 other patients meeting National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases criteria for interstitial cystitis. We were unable to detect intraepithelial or surface-bound Tamm-Horsfall protein in any of the biopsy tissues. Human kidney tissue, similarly fixed and processed, consistently demonstrated Tamm-Horsfall protein staining of the kidney tubules. The monoclonal antibody also reacted on Western blots against urinary Tamm-Horsfall protein. Although antibody (alpha-Tamm-Horsfall protein) reactivity was measured by enzyme-linked immunosorbent assay in sera from interstitial cystitis patients, the titers did not differ statistically from those measured in sera from those without interstitial cystitis. Together, these results make it unlikely that immunohistochemical detection of Tamm-Horsfall protein will have diagnostic value in interstitial cystitis. Whether Tamm-Horsfall protein has a role in the pathogenic processes involved in this disease is not yet known. These findings do not eliminate the possibility that some interstitial cystitis patients will have abnormalities associated with the biochemical and physiological functions of Tamm-Horsfall protein.

Diagnosis and therapy of subclinically infected prostheses.

We believe 5 to 7 percent of prosthetic devices are "subclinically" infected by Staphylococcus epidermidis. These infections are manifested by chronic pain, migration and late extrusion of the devices. To examine this problem, we cultured penile and mammary prostheses. For the experimental arm, we cultured painful penile and mammary prostheses that were being removed because of symptoms (pain). For patients in a control group, we cultured penile prostheses being replaced because of mechanical failure (no pain) and mammary tissue expanders that were temporarily installed. Actual parts of the device were cultured in Trypticase Soy Broth. There were 14 and 12 painful penile and mammary prostheses, 13 and ten, respectively, were cultured positive, for an infection rate of 88 percent. The primary organism identified was S. epidermidis. The nonpainful penile prostheses (zero of five and three of 22 mammary prostheses) grew S. epidermidis. The differences were highly significant (p < 0.001), suggesting that the painful prosthesis is infected. In an attempt to resolve the problem of the painful prosthesis, ten prosthesis were removed and exchanged for new devices. Patients received preoperative and postoperative antibiotics. All ten had positive cultures and nine of ten were successfully exchanged (no pain).

Prevention of acrolein-induced bladder injury by pentosanpolysulfate.

The active metabolite of cyclophosphamide, acrolein, which is capable of damaging the transitional epithelium of the bladder, was evaluated in both in vivo and in vitro models to determine if its damaging effect could be reduced by the presence of a sulfated polysaccharide pentosampolysulfate. It was discovered that in all models pentosanpolysulfate was capable of reducing transitional cell injury due to acrolein.

Intraocular granuloma: a Schistosoma mansoni model of ocular inflammation.

An experimental uveitis model was developed in New Zealand rabbits by an intraocular injection of Schistosoma mansoni eggs. An inflammatory response was clinically apparent after 5 days and histologically was characterized by an eosinophilic infiltrate into the vitreous and choroid. The chorioretinitis that developed resulted in the disruption of the photoreceptor layer. After 30 days, eggs were enveloped by a granulomatous host response similar to that observed in animals infected systemically with Schistosoma mansoni. Reduction (immunomodulation) of granuloma size and cellularity compared with controls was observed in paraffin sections of eyes challenged (100 eggs) 4 weeks after a priming injection (500 eggs) in the contralateral eye. The granulomatous response was not evident when heat-killed eggs were injected intraocularly. Extracts made from viable eggs also induced an intense vitreous infiltrate 12 hr after injection. Serum collected from rabbits injected with 500 or more eggs showed antibody (7s) reactivity for 125I-labeled bovine S antigen, as demonstrated by immunoprecipitation with Staphylococcus aureus (Pansorbin). This model is useful for analyzing immunologic parameters involved in ocular granulomatous and parasitic diseases, humoral and cellular responses mediating autosensitization to retinal or other ocular antigens, and possible for screening chemotherapeutic agents for immunomodulation of potentially injurious host inflammatory responses.

Binding of retinoblastoma and normal sera to retinoblastoma-derived cultured cells.

We have observed increased binding of retinoblastoma patients' sera to a retinoblastoma-derived cultured cell line (Y-79). This reactivity was mediated by the serum IgG fraction and was directed toward different tumor or target cell (Y-79, Molt, Raji, and fibroblasts) cultured in media containing fetal calf serum. Normal pooled serum IgG fractions did not demonstrate any similar binding. When target cells were cultured in media containing human serum instead of fetal calf serum, a considerable reduction in retinoblastoma sera binding activity was observed. Reactivity against target retinoblastoma cells could be reduced but not entirely eliminated by quantitative absorption with nonretinoblastoma (Molt) cells grown in media with fetal calf serum. Retinoblastoma and normal sera binding to autologous fibroblasts, nonautologous fibroblasts, and cultured melanoma cells was also minimal. These findings suggest that residual binding activity in the sera tested may be directed against retinoblastoma tumor antigens. The fetal calf serum component responsible for reactivity with certain retinoblastoma sera was shown by immunoprecipitation, competitive inhibition, and gel electrophoresis to be bovine serum albumin.

Fc and C3b receptors on cultured retinoblastoma cells.

Fc and C3b receptors were identified on cultured retinoblastoma cells. Labeled receptor protein bound to affinity gels prepared with IgG, Aggregated IgG, and Fc but not to control gels prepared from Fab'2 or Sepharose-4B alone. Eluted Fc receptors was partially characterized by polyacrylamide gel electrophoresis. Molecular weight of the isolated receptor or its subunit was approximately 4.3 X 10(4) daltons. Cultured retinoblastoma cells were found to rosette with human indicator erythrocytes specific for C3b and Fc receptors. This study indicates that considerable reactivity between retinoblastoma patients' sera and cultured tumor cells may be mediated by these receptors as well as by reactivity toward retinoblastoma antigens.

Characterization of retinoblastoma immune complexes.

Immune complexes from retinoblastoma sera were characterized with molecular sieve chromatography, affinity chromatography, and polyacrylamide gel electrophoresis (PAGE). Retinoblastoma patients' sera had two well-defined peaks of immune complex activity after molecular sieve chromatography. These protein fractions had a molecular weight of approximately 1.6 x 10(5) and 2.0 x 10(6) daltons. Affinity chromatography with Sepharose 4B-protein A and analytical PAGE demonstrated that IgG was the predominant immunoglobulin in these immune compelxes. Immune complexes also had affinity for Sepharose-concanavalin A, indicating the glycoprotein nature of the antigen component.

Bge snail cell-line antigens: ineffectiveness as antischistosomal vaccine in mice.

Mice and rabbits were immunized with antigens derived from Bge cells, Biomphalaria glabrata hemolymph, or Schistosoma mansoni. Antisera from mice given molluscan antigens did not form immunoprecipitates with soluble antigen from adult worms, but their binding to surfaces of sporocysts, cercariae, and schistosomules suggests the presence of cross-reacting determinants. In vitro, cell-mediated immune responses to Bge antigens were not demonstrable in infected nor in immunized mice. Mice immunized with Bge cell-line antigens and challenged with S. mansoni cercariae showed no reduction in worm burden when compared with control mice.