Bladder reconstruction, augmentation, substitution, diversion (I)

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233 CYTOTOXIC MONOCLONAL ANTIBODIES AGAINST PROSTATE SPECIFIC MEMBRANE ANTIGEN Yoshihisa

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of the signaling events within actual human biopsies will be critical for patienttailored therapy. Source of Funding: None.

Kinoshita", Katsuyuki Kuratsukuri, Rchard F Jones, Henry D Friedman, Gabriel P Haas, Ching Y Wang, Syracuse, NY INTRODUCTION AND OBJECfIVE: The expression of prostate-specific membrane antigen (PSMA) is relatively specific for prostate epithelial cells and the expression of its gene is upregulated during the carcinogenesis of prostate gland. Since the expression level is especially high in hormone refractory cancer, it is a target for immunotherapy of prostate cancer. Using oligopeptides selected from PSMA as immunogens, a series of monoclonal antibodies (mAb) specific for various epitopes ofPSMA were produced. We investigated the in vitro and in vivo activity of these antibodies against LNCaP cells. METHODS: To test for the specificity of the antibodies, formalin-fixed and paraffin-embedded sections of human tissues were used for immunohistochemical staining. In vitro toxicity against LNCaP cells was determined by MTT and colony formation assays. For in vivo assay, male SCID mice were s.c. injected with 2 million LNCaP cells suspended in 0.1 rnl of 50% Matrigel. When tumors became palpable, they were randomly divided into groups with 6 mice in each group and given weekly i.p, injection of mAb for 4 weeks. HB65, an unrelated monoclonal antibody, served as a negative control. The tumor volume was used as the parameter for the assessment of antitumor activity. RESULTS: Immunohistochemically, these antibodies reacted with the epithelial cells of prostate, renal tubules and intestines. One of the mAb designated as 4E6 was particularly cytotoxic and inhibited c1onogenicity of LNCaP cells in vitro. It also induced apoptosis and caspases 3 and 7 in cultured LNCaP cells. For in vivo antitumor activity, tumor volumes in the mice treated with the mAbs 4E6 and 3D3 were significantly smaller than that in the mice treated with mAb HB65. The induction of apoptosis and Caspases 3 and 7 was observed in the necrotic tumor area of mice treated with PSMA mAbs. CONCLUSIONS: Monoclonal antibodies against various epitopes of the extracellular domain of PSMA were prepared and characterized. Some mAbs specific for PSMA were cytotoxic to LNCaP cells in vitro and in vivo. They activated caspases 3 and 7 and induced apoptosis in LNCaP cells. These mAb may have clinical applications. Source of Funding: VA.

234 SIGNAL PATHWAY PROFILING OF PROSTATE CANCER USING REVERSE PHASE PROTEIN ARRAYS Robert L Grubbs, Bethesda,MD; Valerie S Calvert, Cloud P Paweletz, Bethesda, MD; John L Phillips, W Marston Linehan, John W Gillespie, Michael REmmert-Buck, Lance A Liotta, Bethesda, MD; Emanuel F Petricoin, Bethesda, MD INTRODUCTION AND OBJECTIVE: Reverse phase protein arrays represent a new technology with which to study the fluctuating state of the proteome in minute quantities of cells. The activation status of cell signaling pathways controls cellular fate and deregulation of these pathways underpins carcinogenesis. Changes in pathway activation that occur between early stage prostatic epithelial lesions, prostatic stroma and the extracellular matrix can be analyzed by obtaining pure populations of cell types by laser capture microdissection (LCM). We have applied reverse phase protein array technology to analyze the status of cell signaling involved in pro-survival, mitogenic, apoptotic and growth regulation pathways in the progression from normal prostate epithelium to premalignant lesions (PIN) and then to invasive prostate cancer. METHODS: Whole mount ethanol-fixed/paraffin-embedded or frozen tissue was obtained from radical prostatectomy specimens from men with prostate cancer. Specimens were stained with a modified hematoxylin and eosin protocol. Pure specimens of prostatic ductal epithelium representing normal, premalignant and invasive cancer were obtained by LCM. 3,000·10,000 LCM shots were pooled and lysed in 30 p.Lof lysing buffer containing a I: 1 mixture of 2X SDS electrophoresis buffer and Tissue Protein Extraction Reagent (TPER) for 2 hours at 70°C. After cell lysis, samples were boiled for 3 minutes and 3 nL of the lysate were arrayed with a GMSE 470 microarrayer onto nitrocellulose slides with a glass backing. Staining was performed with an automated stainer (DAKO, Carpintera, CA) using a biotinyl-linked peroxidase catalyzed signal amplification system. Commercially available antibodies against the checkpoints in cell signaling pathways were used in appropriate concentrations. RESULTS: Using multiplexed reverse phase protein arrays coupled with LCM, the states of signaling changes during disease progression from prostate cancer study sets were analyzed. Analysis of dozens of phospho-specific endpoints concomitantly revealed changes in cellular signaling events through disease progression and between patients. CONCLUSIONS: We have used a new protein array technology to study specific molecular pathways believed to be important in cell survival and progression from normal epithelium to invasive carcinoma. With the advent of molecular targeted therapeutics, the identification, characterization and monitoring

Bladder Reconstruction, Augmentation, Substitution, Diversion (I) Moderated Poster Sunday, April 27, 2003

10:00 AM-12:oo PM

235 IS ALL SMALL INTESTINAL SUBMUCOSA (SIS) USED FOR UNSEEDED BLADDER REGENERATION THE SAME? Bradley P Kropp*, Yuanyuan Zhang, Earl Y Cheng, OKC, OK INTRODUCTION AND OBJECTIVE: SIS has been previously shown to promote urinary bladder regeneration utilizing the unseeded technique. Initial results (1994) with hand-made SIS obtained from sows demonstrated consistent and reliable results. Since the original work in the urinary bladder, inconsistent regenerative results have been demonstrated. SIS has been obtained from many different sources and processed in different ways, thereby making it difficult to determine the single most important factor that is required to obtain reliable and consistent bladder regeneration utilizing the unseeded technique. This presentation will review the trials and tribulations of searching for the proper SIS material that is required for urinary bladder regeneration. METHODS: Five different canine bladder augmentation (N=88 dogs) studies have been performed utilizing the following different SIS materials. 1) Market weight SIS vs. Sow weight SIS, 2) Hand-made vs, Machine made, 3) PAA vs. E.-beam vs. Lyophilized (Ethylene Oxide) sterilization, 4) Omentum vs, No omentum and 5) Proximal Jejunal SIS vs, Distal ileum SIS. All animals received a 40% cystectomy with immediate augmentation with one of the above SIS materials. All animals were sacrificed and bladders harvested at 10 weeks postbladder augmentation. All bladders were assessed for amount of bladder regeneraton, SIS graft shrinkage, workability of SIS material, weepiness of SIS materials, and gross and microscopic analysis. RESULTS: Most all materials used demonstrated some evidence of urinary bladder regeneration, however, it appears that only SIS obtained from the distal ileum of sows can produce consistent urinary bladder regeneration without bone formation and severe shrinkage. CONCLUSIONS: After years of research involving urinary bladder regeneration with SIS, it appears that a more reliable material for urinary bladder regeneration has finally been identified. The one major current drawback is that this material has not been tested after terminal sterilization that is required by the FDA prior to human use. Therefore, until this work is completed, studies involving other SIS-like materials should be viewed with great care and caution. From this study, it would appear that different SIS materials do NOT have the same regenerative potential with regards to the urinary bladder. Therefore, SIS is not SIS when it comes to bladder augmentation and hopefully the future will bring a bladder regenerating specific segment of SIS that will be available for clinical use. Source of Funding: Cook Biotech and NIH Grant DK56968.

236 IS PATIENT SURVIVAL AN APPROPRIATE MEASURE OF THE UTILITY OF RADICAL CYSTECTOMY IN OCTOGENARIANS? Lars Ellison", Mark Schoenberg, Baltimore, MD INTRODUCTION AND OBJECTIVE: Radical cystectomy has been demonstrated to be a safe and effective treatment for muscle invasive bladder cancer in elderly patients. Many centers advocate this treatment even for octogenarians. We asked the question, when compared to radiation therapy, does the survival benefit achieved with this radical cystectomy justify its use in the very elderly? METHODS: We utilized the Surveillance, Epidemiology and End Results data of the National Cancer Institute to examine overall survival after treatment for bladder cancer. We identified all patients diagnosed with bladder cancer between the years 1992 and 1997. Patients were then stratified into age groups: 70-79 and 80 or older. We performed Kaplan-Meier survival analysis and the Lng Rank test to compare treatment strategies across the age groups. In addition we examined actuarial survival with life tables. Differences between groups and strategies were all significantly different. RESULTS: Between 1992 and 1997, 19,697 individuals over the age of 70 were identified with incident localized bladder cancer. Of these, 1082 underwent immediate radical cystectomy and 903 underwent immediate radiation therapy. For patients 70-79, the median survival after surgery was 40 months (versus 19 months

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for radiation therapy). For patients over 80, the median survival after surgery was 19 months (versus 14 months after radiation therapy). Conversely, the percent survival at 1, 3 and 5 years for patients 70-79 after surgery was 76%, 51% and 43% (versus 67% 34% 21% after radiation therapy). For octogenarians, the percent survival at 1,3 and 5 years after surgery was 67%, 35% and 30% (versus 60%, 23% and 16%). CONCLUSIONS: Although octogenarians may derive significant benefit from radical cystectomy (i.e., decrease in local symptoms, bleeding, pelvic pain), these data suggest that there is at best a marginal disease-specific survival benefit following surgery in this particular age group. The clear benefits derived from radical cystectomy by younger patients do not appear to translate in magnitude to patients over the age of 80. For these older patients, the recovery period from surgery may consume the bulk the derived survival benefit. Sourceof Funding: None.

237 BLADDER RECONSTRUCTION WITH CELL·SEEDED AND UNSEEDED SMALL INTESTINE SUBMUCOSA (SIS) GRAFfS IN SUBTOTAL CYSTECTOMY MODEL Yuanyuan Zhang, Earl Y Cheng, Hsueh K Lin, Rick Cowan, Ryan Davis, Bradley P Kropp", OKC, OK INTRODUCTION AND OBJECTIVE: Small intestine submucosa (SIS) is a reasonably good scaffold for histological and functional bladder regeneration in 40% cystectomy model. This study was performed to determine if SIS, seeded or unseeded will promote bladder regeneration in a severely damaged 90% cystectomy canine model. METHODS: Twenty beagles were divided into three groups- Group 1: control (n=8), Group 2: unseeded SIS (n=6) and Group 3: cell-seeded SIS (n"'6). All animals underwent 90% cystectomy. In Group 2, a large piece of SIS (6 x 8 crrr') was used for bladder augmentation one month after 90% cystectomy. In Group 3, to prepare the cell-seeded SIS grafts, dog bladder urothelial cells and smooth muscle cells were harvested separately from the dogs during cystectomy. The bladder cells were then grown on a large piece of SIS for 14 days using layered co-eulture technique in Vitro. The animals at each group were harvested 5 and 9 month follow-ups after the subtotal cystectomy. All animals underwent multiple urodynamic studies, IVP's and blood chemistries. All harvested bladder samples were evaluated grossly and histologically. RESULTS: All 8 animals in Group 1 survived the anticipated period of 5 and 9 months after cystectomy. In Group 2, 3 of 6 dogs were harvested at the intended time points. The other three dogs were sacrificed within 2 months postaugmentation due to bladder or urethra obstruction by stones. When harvested, SIS grafts demonstrated significant shrinkage with moderate adhesions and bone formation in all six dogs. Histologically, the limited bladder regeneration showed all three layers (urothelium, smooth muscle and serosa). In Group 3, 5 of 6 dogs were harvested at the intended time points after 90% cystectomy. The other dog was sacrificed one month after bladder augmentation due to bladder obstruction by stones. Similar to the unseeded-SIS regenerated bladders, the cell-seeded SIS grafts had heavy adhesion, bone formation and shrinkage at all six dogs. CONCLUSIONS: This study demonstrates that the use of seeded or unseeded SIS fails to regenerate normal bladder in this severely damaged 90% cystectomy model. This study provided important insight about the process of regeneration in a severely damaged bladder model and has caused us to re-evaluate the critical elements for complete bladder replacement utilizing tissue engineering techniques. Sourceof Funding: NIH Grant DK56968.

238 RESULTS WITH THE ANTIREFLUXING T·LIMB: DOES IT WORK? John P Stein", Peter Clark, Gus Miranda, Matt Dunn, Eila Skinner, Gary Lieskovsky, Donald G Skinner, Los Angeles, CA INTRODUCTION AND OBJECTIVE: To eliminate the complications associated with the intussuscepted Kock nipple valve an extraserosal flap-valve technique called the "T-limb" was developed. The purpose of this study is to evaluate our intermediate clinical experience with this antirefluxing T-Iimb in patients undergoing an orthotopic bladder substitute (T -pouch). METHODS: From November 1996 through May 2000, 180 patients (142 men [79%D, with a mean age of 67 years (range 33 to 88) underwent an orthotopic T-pouch ileal following cystectomy. All patients had a 4 em T'-lirnb created to prevent urinary reflux. The indication for cytectomy included bladder cancer in 176 patients (97%): 170 for TCC, 3 for squamous cell, and 3 for adenocarcinoma. The median follow-up was 2.8 years (range 1.4 to 5.3). Data were analyzed according to perioperative mortality, early (within 3 months) and late pouch-related complications (with specific attention to the T-limb), radiographic evaluation, and serum creatinine. RESULTS: Four (2%) patients died perioperatively. A total of 9 (5%) early pouch-related complications occurred including: urine leak in 6 patients, mucus retention in 2, and a pouch-cutaneous fistulae in 1. There were no early complications directly related to the T-limb. Eight of the 9 early complications *Presenting author.

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were treated conservatively. Late reservoir-related complications occurred in 16 (9%) patients including: ureteroileal stenosis in 7, stones in 6, an ischemic reservoir and pouch-vaginal fistulae in 1 each. None of these late complications were attributed to the T-limb. The only late complication directly related to the T-limb was stenosis seen in a patient who received adjuvant radiation to the neobladder. A total of 134 patients underwent a gravity pouchogram study with 11 (8%) demonstrating a radiographic abnormality including: 10 patients with urinary reflux into the T-limb and one with a pouch-vaginal fistulae. Of 142 patients undergoing an IVP, a total of 9 (6%) abnormalities were documented, all demonstrating ureteroileal obstruction. The upper urinary tract otherwise remained unchanged or improved in 94% of patients. There have been no episodes of pyelonephritis or worsening renal function in these patients. CONCLUSIONS: With intermediate follow-up, the antireflux T-mechanism appears to be an effective technique to protect the upper urinary tract and prevent reflux without significant complications. We have not observed complications related to obstruction or stones with the T-limb. Further follow-up is required to assess the long-term results of this technique. Source of Funding: None.

239 SIGMA RECTUM POUCH (MAIMZ POUCH 11): LONG·TERM FOLLOW·UP Markus Woehr*, Sascha Pahernik, Gianluca D'Elia, Sebastian W Melchior, Markus Hohenfellner, Jochen W Thueroff, Mainz.

Germany INTRODUCTION AND OBJECTIVE: The sigma rectum pouch was introduced in 1990 as an alternative to ureterosigmoidostomy. It combines continent urinary diversion with a low pressure, high capacity reservoir. We here report our ten year experience with this type of urinary diversion. METHODS: Between 1990 and 2001 we performed a sigma rectum pouch in 135 patients. Mean age was 42(range 4-73)years. Indications for diversion were transitional cell carcinoma of the bladder in 100 patients, bladder ekstrophy and other malformations of the lower urinary tract in 31 patients and traumatic sphincter damage in 4 patients. The pouch was created out of 30 em detubularized rectosigmoid. The ureters were implanted either in a submucosal tunnel or, in dilated ureters, by serous lined extramural tunnel according to the technique of Abol Enein. Mean follow-up was 57(range 6-136) months. Continence behaviour, early and late complication rate, upper urinary tract function and metabolic disorders were investigated. RESULTS: Complete daytime continence was reported in 97% and complete nocturnal continence in 95 % of the patients. The other patients suffered from mild stress urinary incontinence requiring 1-2 pads for prophylaxis per day. Average frequency was 6.2 times during daytime and 1.3 times during the night. Ureterointestinal stenosis requiring open surgery to protect renal function occured in 7.2 % of 207 evaluated reno-ureteral units. 69% of the patients showed acidosis(base excess < 2,5) and used oral medication. Renal function was stable in all patients and no malignancy at the site of the uretero-intestinal anastomosis was detected until now. CONCLUSIONS: The improvement of the classic ureterosigmoidostomy by creating a low pressure and high capacity reservoir using detubularized sigmorectoid and the use of different types of ureteral implantation techniques show promising results. Continence rate and upper urinary tract complications could be reduced. Thus, the Mainz Pouch II with its simple and reproducable operation technique is a feasable alternative diversion in selected patients. Source of Funding: None.

240 MANAGEMENT OF COMPLEX URINARY FISTULA REFRACTORY TO TRADITIONAL METHODS FOR URINARY DIVERSION: THE ROLE OF PERCUTANEOUS URETERAL OCCLUSION WITH GIANTURCO COILS AND GELATIN SPONGE Steven B Brandes", Hui Zhu, David Hovsepian, St. Louis, MO INTRODUCTION AND OBJECTIVE: Complex urinary fistula due to radiation, malignancy, and/or major pelvic trauma are very difficult to manage and repair. For such patients with poor physiologic reserve, traditional open surgery is usually overly rigorous and unsuccessful. We reviewed our 8-year experience with percutaneous transrenal ureteral occlusion in the management of complex urinary fistula. METHODS: Retrospective chart review of 27 patients (pts.) from 1993-2001. 33 radiation and/or malignancy and 1 pelvic trauma induced urinary fistula were present. All had failed other minimally invasive and endoscopic approaches for urinary diversion. Distal ureteral occlusion was achieved by Gianturco coils alone or in combination with gelatin sponge in total of 48 ureters. Percutaneous nephrostomies were placed for permanent urinary diversion. RESULTS: 27 pts., 20 (74%) female, 7 (26%) male, mean age 57±14 years were evaluated. 26/27 pts. had pelvic malignancies: cervical (14/27), rectal/anal (4/27), prostate (3/27), endometrial ('lJ27), colon ('lJ27), bladder (1/27), and

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urachal (1/27).24/27pts. received prior pelvic irradiation, 10/27 chemotherapy and 15/17 pelvic surgery. PIs. presented with a total of 34 complex urinary fistulae (7 patients with> 2 fistula): 16(47%) vescio-vaginal, 8 (23%)ureteral-abdominall cutaneous, 3 (9%) prostate-rectal, 2(6%) urethral, 2 (6%) entero-vesical, 2 (6%) other bladder, and I (3%) entero-ureteral • 8 associated entero-vaginal and cutaneous fistula were present. Occlusion was successful and sustained in all embolized ureters, with complete or near complete urinary diversion and dryness by 3 days. No repeat embolizations needed. Complications limited to coil migration into the renal pelvis in 2 patients. After urinary diversion, 18/18 pts. reported improved quality of life. 84% (22/26) of the pts, died, with a mean survival of 7.9±ll.6 months after embolization. 16% (4/26) are alive at mean follow-up of 9 months. I patient survived to undergo definitive colo-conduit urinary diversion. CONCLUSIONS: In the management of refractory lower-urinary tract fistula, nephrostomy drainage and ureteral occlusion with Gianturo coils and gelatin sponge are safe and reliable methods for permanent urinary diversion, and improve quality of life (particularly for patients with limited life expectancy). Source or Funding: None.

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gene transfer system. Transferred cells were seeded on SIS disks at 105 cells/em" for 3,7, 14 and 28 days. Cell-SIS constructs were analyzed histologically and with immunohistochemistry. RESULTS: By 7 days in culture, there were multiple layers of cells on the surface of the SIS with evidence of matrix penetration. Histological analysis revealed that BMSC have a similar appearance and growth pattern to that of bladder smooth muscle cells grown on SIS with the exception of greater matrix penetration with BMSC. Immunohistochemical analysis revealed that more than 95% of the cultured BMSC expressed a-smooth muscle actin positivity. Transfected BMSC grew in an identical fashion as the non-transfected BMSC and maintained expression of the EGPP at each of the time points. CONCLUSIONS: SIS supports the 3 dimensional growth of BMSC. These cells may represent an alternative cell source for tissue engineering in bladder regeneration using cell-seeded technology. Use of gene transfer technology for expressing a traceable marker will allow for identification and tracking of these cells in future in vivo studies. Source of Funding: None.

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BLADDER WALL REGENERATION OF DIFFERENT BLADDER ACELLULAR MATRIX GRAFTS (BAMG) DEPEND ON THEIR COLLAGEN TYPE I AND ill RATIO: A STUDY IN THE CANINE Karl-Dietrich Sievert", Muenster, Germany; J Wefer,

LAPAROCOPIC URINARY DIVERSION AFTER LAPARO· SCOPIC RADICAL CYSTECTOMY AND COMPLEX PELVIC SURGERY Gralf Popken», Berlin, AB, Germany; Tobias Petras, Eckard

Hannover, Germany; Lora Nunes, R Dahja, San Francisco, CA; Lothar Hertle, Muenster, Germany; Emile A Tanagho, San Francisco, CA

INTRODUCTION AND OBJECTIVE: Radical cystectomy or wide ventral pelvic excision and urinary diversion is performed in organ confined, muscle invasive bladder cancer (BC) as well as in advanced gynecological malignancies (GM). In those cases the laparoscopic approach is a challenge for surgeons and urologists. We present ten cases of laparoscopic pelvic intervention with urinary diversion performed in part and completely intracorporally. METHODS: In 7 patients with BC and 3 patients with GM we performed laparoscopic cystectomy or wide pelvic excision and urinary diversions. In I woman additional an ileal and colon stenosis after radiation were resected. In a 5-port transperitoneal technique initially the ureters were mobilized and dissected. In cases of BC a laparoscopic radical cystectomy, in cases of GM a wide ventral pelvic excions was performed with pelvic Iymphnode dissection. Sigma and ileum resection was done with an end-to-end anastomosis for both. In all cases urinary diversion (UD) has been performed extracorporally assisted or complete laparoscopically. Speciments were removed transvaginal or umbilical. RESULTS: We performed 5 conduits, I sigma pouch, 2 ileal neobladders and 2 continent Studer pouches. No peri or postoperative complications appeared. Operation time was about 4.5 to 7 hours, blood loss 200 to 600 ml. Pathohistological examinations revealed pT2/3b,pNO,MO,G2/3 transitional carcinoma in 7 cases and pT4,pNO/I,MO,G3 cervix cancer in 2 cases. In cases of continent urinary diversions there was a mild stress incontinence after removal of transurethral catheters in all cases and 5n dry patients after 3 months. Patients could leave hospital after 6 to 14 days. CONCLUSIONS: Laparoscopic approaches in pelvic alterations and malignancies are possible with all the advantages of minimal invasive surgery as well as performing different continent and incontinent urinary diversions. It is important to perform this procedures with the skill of an interdisciplinary surgical team, especially to improve laparoscopic techniques. Source of Funding: None.

INTRODUCTION AND OBJECTIVE: In urology, the bowel is used primarily for repair, replacement and augmentation of the bladder. However, in follow up, complications might ensue. For years, there have different attempts to use other tissues. In the canine, we used homologous BAMG and found a time-related tissue regeneration of all bladder wall components without any signs of antigenicity. In this trial, we investigated the feasibility of using heterologous BAMG. METHODS: Nine female mongrel dogs underwent a partial cystectomy (greater or equal to 50% loss of bladder wall), followed by grafting of an equal-sized homologous (n= I) or heterologous (n=6) BAMG, prepared from dog (n=I), monkey (n=3) and human (n=3). Two control animals underwent partial cystectomy only. Finally, specimens of the different kinds of normal bladder walls, BAMGs and regenerated bladder tissues (7 months postoperative) were evaluated by H&E, immune histology and saturated picric acid stain. RESULTS: None of the animals demonstrated any pathologic changes of the upper urinary tract. At the seven-month follow-up, all the grafts had smooth muscle bundles in the complete matrix. All components of a normal bladder wall were demonstrated. Additionally, picric acid stain demonstrated no changes in the amount and ratio of collagen Type I and III in the homologous tissue by the BAMG processing as well as in the 7th month follow up. In the heterologous tissue, the amount of collagen Type I increased significantly in the monkey BAMG, but after 7 months, it became equal to the normal monkey tissue. The amount of collagen Type I and III tripled at the time of producing the human BAMG were noticed after month 7. They were still doubled after seven months follow up compared to the normal human bladder, whereas the ratio shifted towards collagen Type III. None of the heterologous specimens demonstrated the collagen Type I and III ratio similar to the host. CONCLUSIONS: Regeneration of the different tissue components are demonstrated up to the center of the augmented matrix without any sign of antigenecity, regardless of the BAMG used. Heterologous BAMG is feasible for bladder augmentation, but the similarity of the texture of collagen, between host and BAMG (homologous BAMG), sustains a faster and more complete regeneration. The amounts of collagen Type I and III and their ratio seem to influence the smooth muscle regeneration. Source of Funding: None.

242 GROWTH OF BONE MARROW STROMAL CELLS ON SMALL INTESTINAL SUBMUCOSA (SIS) : AN ALTERNATIVE CELL SOURCE FOR TISSUE ENGINEERED BLADDER? Yuanyuan Zhang", Earl Y Cheng, Hsueb K Lin, Adam Metwalli, Jarrett Kruska, Robert Epstein, Rick Cowan, Bradley P Kropp, OKC, OK INTRODUCTION AND OBJECTIVE: Bone marrow stromal celIs (BMSC) are multipotent and capable of differentiating into different cell types in the human body. Small intestinal submucosa (SIS) is a biodegradable matrix that has been shown to promote tissue specific regeneration. This study was conducted to see if SIS could support the growth of BMSC that could then potentially be used for bladder reconstruction. METHODS: Bone marrow was obtained from the femur of 5 dogs by direct needle aspiration. BMSC were isolated by density gradient centrifugation utilizing Ficoll-Paque® and cultivated in MI99 with 10% PBS. Primary cultures were transfected with the enhanced green fluorescent protein (EGFP) using a retroviral

Baerlehner, Berlin, Germany

244 FLUORESCENCE IN-SITU HYBRIDISATION ON BIOPSIES FROM CLAM ILEOCYSTOPLASTIES Kenneth D Ivil*, Elizabeth M Parry, James M Parry, Cardiff, UK; Timothy P Stephenson, Chicago, IL INTRODUCTION AND OBJECTIVE: The incidence of carcinoma following clam enterocystoplasty increaseswith time.There is an urgent need to identify patients at risk for developing life-threatening tumors. We have used Comparative Genomic Hybridizationto identifyamplificationson the short-armsof chromosomes9 and 18 in deoxyribonucleic acid (DNA) from biopsies taken from the enterovesicalanastamoses of patients after ileocystoplasty. Amplifications on chromosome 8 were also found in the DNA from the enterovesical anastamoses of two patients who developed adenocarcinomain their ileocystoplasties. The purpose of the current study was to use fluorescent in-situ hybridisation (FISH) to investigate instability at the enterovesical anastamosisin patients who have undergone clam ileocystoplasty. METHODS: Touch sample preparations were made from fresh endoscopic biopsies taken from the enterovesical anastamosis and native bladder remnant (control specimens) in fifteen patients who had undergone clam ileocystoplasty. FISH was performed with fluorescent centromeric probes for chromosomes 8, 9 and 18. Normal values were derived from the third standard deviation from the mean of the control values and was calculated for both monosomy and trisomy for each of the chromosomes studied. RESULTS: Significant aneuploid changes were found at the enterovesical anastamosis in all fifteen patients. Alterations in chromosome 18 copy number

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werethe mostfrequent abnormal findings. Eightpatients had trisomy 18andseven patients had monosomy 18. Nine patients were monozygous for chromosome 9. Monosomy 8 and trisomy 8 wereeachfoundin one patient. No abnormal findings were found in any of the control specimens. CONCLUSIONS: Chromosomal numerical abnormalities occur at the enterovesical anastamosis following clam ileocystoplasty in all patients studied. Chromosome 18 appears to be a good marker of instability. The enterovesical anastamosis is inherently genetically unstable resulting in an increased rate of mutagenesis of the epithelium. Follow-up is required to confirm whether a particular type of chromosomal abnormality is associated with eventual tumor formation. Source of Funding: None.

245 URETEROSIGMOIDOSTOMY AND BONE METABOLISM· IS THERE A RISK OF OSTEOPOROSIS AFTER A FOLLOW·UP OF 12 • 34 YEARS Raimund Stein", Markus Pfiughaupt, Manuela Gesenhoff, Johannes Lotz, Peter Bartenstein, Rudolf Hohenfellner, Joachim W Thiiroff, Mainz; Germany INTRODUCTION ANDOBJECTIVE: Ureterosigmoidostomy hasbeenoneof the oldest techniques for continent urinary diversion. Since 1991 it has been replaced at our institution by the recto-sigmoid pouch. Mild chronic metabolic acidosis is relative common with this type of diversion and could induce osteoporosis. Therefore, we recommend prophylactic alkalisubstitution when the capillary base excess is below -2.5 mmol/l. In this study, we investigated bone density and bone specific blood parameters (cross-laps, bone specific alkine phosphatase, osteocalcin and propeptid of type I collagen). METHODS: Between 1968 and 1991 ureterosigmoidostomy wasperformed in 196patients, 99 diedin the mean time.20 werelost to follow-up. In 37,a complete metabolic follow-up was obtained at our institution, the remaining patients either underwent follow-up examinations elsewhere or did not wantany morefollow-up examinations 20-30yearsafter operation. RESULTS: After median follow-up of 22 years(12-34 years)for 37 patients. the bone specific alkaline phosphatase was elevated in 20% of the patients, osteocalcin in 24%. propeptid of type I collagen in 52% and the cross-laps were elevated in 66%.Bonedensity wasdecreased in 19% and in 13%within the lower limit. At the time of study the base excess was below -2.5 mmoIll 11 patients (35%), only 3 of them took prophylactic alkali medication regularly. CONCLUSIONS: Thereseemsto be a higherrisk of osteoporosis for patients with ureterosigmoidostomy than previously reported. Close follow-up and antiacidotic treatment is strongly recommended. ureterosigmoidostomy or other types of rectal reservoirs should be offered only to those patients with a good long-term compliance. Source of Funding: None.

246 ABDOMINAL SACROCOLPOSUSPENSION USING AUTO· LOGOUS FASCIA LATA Jameel A Brown, Jerilyn M Latini", Karl J Kreder, Iowa City, IA INTRODUCTION AND OBJECTIVE: Numerous techniques and materials have traditionally beenemployed to correctvaginal vault prolapse, including the useof cadaveric fascia, synthetic materials andautologous rectus fascia. Thisstudy evaluates the efficacy, morbidity of. and patient satisfaction with, abdominal sacrocolposuspension usingautologous fascia lata. METHODS: A retrospective database review was performed to identify all women who underwent abdominal sacrocolposuspension using autologous fascia lata for repair of vaginal vault prolapse with at least one-year follow-up. Preoperative and postoperative Pelvic Organ Prolapse Quantification (POP-Q) scores, other relevant clinical and operative parameters along with patient satisfaction wereassessed through clinical chart review and questionnaires. RESULTS: A total of 12 women underwent abdominal sacrocolposuspension usingautologous fascialatabetween 1999and2001 for stages II through IV pelvic organprolapse. Meanagewas63.5years(range35-82years) andfollow-up ranged from 12 to 38 months (average 14 months). One-year follow-up postoperative POP-Q scores improved to andremained at POP-Q stageI or lessin all 12patients. Meanoperative timewas3.47hours(range 1.88-6.25 hours). Meanbloodlosswas 147.7cc (range 50-400cc). Therehasbeenno morbidity or complication associated with fascialata harvest. CONCLUSIONS: The use of autologous fascia lata has advantages over traditional methods. Given the immunologic concerns regarding disease transmission from donoror cadaveric fasciaand thecomplications associated with possible erosion of synthetic materials, the use of autologous tissue has demonstrable advantages. Fascia lata compares favorably in efficacy to other materials and is not associated withsignificant morbidity. Patient satisfaction with the procedure is high. Source of Funding: None.

*Presenting author.

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247 DOES EXTENDED LYMPHADENECTOMY INCREASE THE MORBIDITY OF RADICAL CYSTECTOMY? Clemens Brossner", Oberwart, Austria; Armin Pycha, Bolzano/Bozen, Italy; Attila Toth, Oberwart, Austria; Christine Mian, Andrea Guddemi, Bolzano/Bozen, Italy; Walter Kuber, Oberwart, Austria INTRODUCTION AND OBJECTIVE: A great number of lymph nodes obtained during radical cystectomy mayimprove stagingandmayincrease patients outcome. Wereportthepost-andperioperative eventsafterradical cystectomy and urinary diversion in bladdercancer in terms of major and minor complications comparing a minimal with an extended lymphadenectomy. METHODS: We analyzed in thisprospective multicenter study92 consecutive patients who underwent radical cystectomy from March 1998 to February 2002. Caseswereselected according tothe American Society of Anesthesiologists (ASA) classification including a score of 2 as well as 3. In 46 patients a minimal lymphadenectomy was performed (group A) compared to 46 patients with an extended lymphadenectomy (group B). Wespecifically evaluated theincidence and type of complications within the rangeof 30 postoperative days. RESULTS: Because of extended lymphadenectomy operation time increased (mean) 71 min (p
248 CONVERSION INTO CONTINENT URINARY DIVERSION Sascha Pahernik", Raimund Stein, Mohsen El-Mekresh, Markus Hohenfellner, Rudolf Hohenfellner, Joachim W Thuroff, Mainz; Germany INTRODUCTION AND OBJECTIVE: In childhood. after ileal or colon conduit diversion some patients, in particular in adolescence. desirea conversion into a continent diversion as elimination of external devices improves quality of life.Wereportour long-term results of conversion fromconduit conversion intoa continent cutaneous diversion. METHODS: Between July 1986to February 2001,32 patients (mean age: 18 years, range 6 to 49 years)underwent conversion of a colon(n= 19)or ilealconduit (n=13) to a ileo-cecal pouchwitha meanfollow-up of 97 months (range 11-185 months). Indications for urinary diversion were mostly neurogenic disease (40%) and bladder exstrophy/epispadias (34%). Conversion was performed using a simplified technique incorporating the preexisting colon/ileal conduit into a i1eocaecal Mainz pouch I. In 37 (61%)renoureteral units(RU)no reimplantation was performed. 16 RU were reimplanted using a serous-lined extramural tunnel (26%). Morphology of theupperurinary tract.renalfunction, continence behaviour and metabolic changes were analyzed. RESULTS: Complications include incontinence (13%). stomastenosis (13%), pouchcalculi (28%) as wellas ureteric stenosis (4/61 RU,7%) requiring surgical intervention. In particular, for dilatedlrefluxive ureters creation of a serous-lined extramuraltunel waseffective in reducing reflux anddilatation of theupperurinary lract.Continence wasachieved in 97%of patients. Faecal frequency wasunchanged in 75% without any treatment. 25% required mediacal therapy (cholestyramine, loperamide), twoof them(6%)suffering fromtherapy refractory diarrhea. During follow-up, theacid-base balance wasmonitored andearlysubstitution of alkalizing agents was performed at a base excess of < -2.5 mmolll for 15 patients (47%). Vitamin B 12 had to be substitued in three patients (9%). None of the patients developed acidosis. Renal function remained stableduring follow-up. All patients are cornletely satisfied. in particular as compared to the previous solution. CONCLUSIONS: The inclusion of the preexisting colon or ileal conduit facilitates conversion and decreases bowel requirements. Accetable complication rate,stablerenalfunction and patients comfort supports theconversion of a conduit into a Mainz PouchI as a safe and viableoptionin the long run. Source of Funding: None.

249 THE USE OF THE CHIMNEY MODIFICATION CAN REDUCE THE RISK FOR URETERAL STRICTURE IN THE ORTHO· TOPIC NEOBLADDER Daniel G Dajusta", Fernando J Bianco, Detroit. MI; David P Wood, Ann Arbor, MI; J Edson Pontes, Detroit, MI INTRODUCTION AND OBJECTIVE: The chimney modification in the neobladder technique maydecrease the incidence of ureteral stricture. Wecompare the results of two groups of patients that underwent neobladder substitution with and without the chimney modification following radical cystectomy for bladder cancer.

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METHODS: From 1996 to 2001. 38 patients underwent neobladder substitution with the construction of a chimney including 15 studer types and 23 w-pouch with the chimney modification. while 45 patients had a w-pouch without the chimney modification. The clinico-pathological data was collected into the Wayne State University multidisciplinary bladder cancer database. RESULTS: The median follow up time was 36 months. 55 months for the non-chimney group and 24 months for the chimney group. Ureteral stenosis was observed in 6 patients of the non-chimney group accounting for a rate of 13% while no ureteral stenosis were recorded on chimney population (p>=0.029). All ureteral stenosis occurred in the non-chimney group within 15 months from surgery. The rate of UTI, urosepsis. ureteral reflux and hydronephrosis were not different in both groups. CONCLUSIONS: The addition of the chimney technique to the neobladder can reduce the incidence of ureteral stenosis following radical cystectomy and neobladder reconstruction. Source or Funding: None.

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from 17.27 preoperatively to 19.61 postoperatively and from 16.95 preop. to 19.37 postop. (Wilcoxon-test p-value 0,05) for the "Functional condition". CONCLUSIONS: Reconstruction of the lower urinary tract using LDDM represents a feasible option in treatment of acontractile bladders. Evaluation of the QoL showed long term satisfactory results for our patients. No patient reported poorer QoL postoperatively according 10 this evaluation. Source of Funding: None.

252 VASCULARIZED BIOLOGICAL SCAFFOLD FOR BLADDER TISSUE ENGINEERING: RESEEDING TECHNIQUE AND SHORT TERM IMPLANTATION IN A PORCINE MODEL Dirk Schultheiss», Alexander I Gabouev, Serghei Cebotari, Heike Mertsching, Stefan Ueckert, Norbert Schlote, Joerg Wefer, Axel Haverich, Udo Jonas, Christian G Stief, Hannover, Germany

INTRODUCTION AND OBJECTIVE: Reconstruction of urinary tissues often employs various types of biomaterials, and adequate material biocompatibility is essential for a successful outcome. In this study primary normal human urothelial cells were used to evaluate the biocompatibility of a number of biomaterials that are either experimentally or clinically used in urinary reconstructive surgery. METHODS: Human urothelial cells were seeded onto 9 naturally derived {intact human bladder submucosa, lyophilized human bladder submucosa. intact pig bladder submucosa, lyophilized pig bladder submucosa, intact small intestinal submucosa, lyophilized small intestinal submucosa, collagen sponge, alginate, alloderm ] and 4 synthetic polymeric biomaterials {poly(glycolic acid). poly-Llactic acid, poly(lactic-co-glycolic acid), Silicone}. Biocompatibility assessment was performed using extraction and direct contact methods, cell viability assay, mitochondrial metabolic activity assay. apoptosis and DNA-synthesis assay. RESULTS: The cytotoxicity and mitochondrial metabolic activity assays showed that all biomaterials, except alginate and latex. did not induce significant changes in cell viability compared to the negative controls (fresh medium with no extracts). The apoptotic activity of the cells with alginate, PGA. and latex was significantly increased. The mitochondrial activity was significantly less with alginate and latex compared to the negative controls. The proliferative activity of cells in direct contact with alginate had a slight but significant reduction in cell proliferation. Direct contact with latex almost completely prevented cell proliferation. CONCLUSIONS: These results indicate that most of the biomaterials tested do not induce significant cytotoxic effects, and exhibit normal metabolic function and cell growth. Normal primary human urothelial cells can be safely seeded and grown on biomaterials for urinary tract tissue reconstruction. Source or Funding: Departmental.

INTRODUCTION AND OBJECTIVE: A vascularized scaffold for tissue engineering ensures efficient nutrition of all cellular components both during in vitro repopulating with functional cells and immediately after in vivo implantation and anastomosis to the recipients blood circulation. For this purpose we used modified acellular small intestinal submucosa segments with preserved arterial and venous vascular network reseeded with autologous endothelial cells. METHODS: Porcine small bowel segments with preserved mesenteric vascular pedicles were decellularized mechanically and chemically. The acellular scaffold was repopulated with porcine primary bladder smooth muscle cells and urothelial cells. isolated from bladder biopsies, and cultured in a perfusion bioreactcr for 21 days. Cell characterization was controlled by immuno-staining for alpha-actin and keratin 7. Cellular metabolic activity was evaluated by sequential MTS-formazane assays and contractility tested by Carbachol exposure in the organ bath system. In a parallel setting endothelial progenitor cells were used for reendothelisation of the vascular scaffoldnetwork, First implantation experiments with anastomosis of the arterial and venous pedicles to the iliac vessels of the recipient were performed in pig model (n>=7). Patency of vessels was controlled intraoperatively by Doppler ultrasound. Finally. grafts were harvested for histological examination after one hour. RESULTS: The scaffolds were reseeded with multilayers of cultured bladder smooth muscle cells and a urothelial cell layer as documented by respective immunostaining. At the end point of in vitro cultivation a high metabolic activity of the vital cells was measured, whereas no contractile response was registered in the organ bath. Endothelial recellularization of all vascular structures resulted in confluent monolayers with the cells expressing endothelial specific proteins (vonWiIlebrand factor. CD62E) . First implantation experiments with these endothelial reseeded scaffolds showed intact intraoperative perfusion in both pedicles and lack of thrombosis in macroscopic or microscopic vascular system after one hour. Respective implantation of acellular matrix resuited in early thrombosis of the vascular network. CONCLUSIONS: We present an acellular matrix with functional vascular system reseeded with endothelial progenitor cells. After additional repopulation with primary bladder cells this graft seems highly feasible for bladder substitution. Source of Funding: DFG SCHU 1457/1-1,2-1.

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253

DO PATIENTS WITH BLADDER ACONTRACTILITY PROFIT FROM LDDM SURGERY? Germar- M Pinggera", Amulf Stenzl,

THE EFFECTS OF DESMOPRESSIN ON NOCTURNAL URINARY VOLUME IN PATIENTS WITH ORTHOTOPIC NEOBLADDER Salvatore Siracusano*, Valentina Pecorari, Trieste,

250 EVALUATION OF NATURALLY DERIVED AND SYNTHETIC BIOMATERIALS FOR BLADDER TISSUE RECONSTRUCTION Jean -Louis Parientes, Byungsoo Kim, James J Yoo, Anthony Atala, Boston, MA

Milomir Ninkovic, Roland Prosser, Johann Kinzl, Georg Bartsch, Hannes Strasser, Innsbruck, Austria INTRODUCTION AND OBJECTIVE: As shown previously, reconstruction of the lower urinary tract using autologous latissimus dorsi detrusor myoplasty (LDDM) represents a feasible and effective option in treatment of bladder acontractility. It was the aim of this study to evaluate quality of life (QoL) after this invasive surgical treatment METHODS: Between 9/95 and 12/00 21 patients (14 males, 7 females, mean age: 39,5 years in males and 41.1 years in females) with bladder acontractility underwent transfer of an innervated autologous latissimus dorsi muscle flap as previously published by Stenzl et al (Lancet, 1998 May). Three validated questionnaires were disllibuted before as well as after operation to measure QoL (mean follow up 42 months): the FLZ questionnaire (to compare the patients with a representative standard population), the Symptom Check List (SLC-90R, to investigate the psychological and physical complaints) and the FACT-G (Functional Assessment of Cancer Therapy, to allow for self estimation of QoL). RESULTS: 1. FLZ: the average scores for all but 2 individuals increased and showed an improvement of individual QoL life postoperatively. Mean FLZ-sum increased from 452.07 to 497,85 (p-value 0.40). 2. SLC-90: Evaluation showed a reduction of all scores, which also indicated that the patients did not suffer from particular psychological stress. 3. FACT-G: Evaluation of this questionnaire showed an improvement of QoL. All four subscales demonstrated higher values postoperatively. The average value of the subscale "Emotional condition" rosed

Italy; Paolo Di Benedetto, Udine, Italy; Gianluca D'Aloia, Trieste, Italy; Paolo Tomasi, Sassari, Italy; Anna Tiberio, Silv io Stener, Emanuele Belgrano, Trieste, Italy INTRODUCTION AND OBJECTIVE: The orthotopic neobladder (ON) causes the loss of bladder sensitivity obliging the patients to void in predetermined times. with discomfort in the night and worsening of the quality of life. It is not clear whether these patients are affected by polyuria and if the administration of desmopressin(DDVP)can reduce the need to wake up during the night. Aim of this study was to verify the nocturnal urinary volume in patients with ON in comparison with controls and to evaluate whether DDVP produces a significant reduction of the nocturnal urinary volume. METHODS: We studied 13 patients with ON and 8 normal volunteers without disorders of the lower urinary tract. In the first phase the evaluation of the total nocturnal urinary volume (sum of the urinary volume produced during each wake-up) without administration of DDVP was performed in the group of normal volunteers and in the pathological group. In the II phase we administered to the pathological group 0,2 rng of oral DDVP at 11 p.m. with an evaluation of the total nocturnal urinary volume. The Mann-Whitney test was performed to define the statistical differences concerning the total nocturnal urinary volume. between the two groups, and any differences in the pathological group in relation to the total nocturnal urinary volume before and after administration of DDVP (p<0.05 was considered significant).

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Vol. 169, No.4, Supplement. Sunday, April 27, ZOO3

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RESULTS: The statistical analysis revealed no difference concerning total nocturnal urinary volume production between normal volunteers and the pathological group without administration of DDVP (p=0.238) and a significant difference conceming total nocturnal urinary volume production between the pathological group without administration of DDVP and the pathological group with administration of nDVP (p=O.OO4). CONCLUSIONS: Our results showed that the patients who underwent ON do not modify the total nocturnal urinary volume production in comparison with normal volunteers. The DDVP determined a significant reduction of total nocturnal urinary volume production in the pathological group compared to the same group without DDVP administration. totsl nocturnal urinary vClIume (mt)

normal voluntelll1l patient, without admlnlslration of DDVP patientswith admlnistndion of DOVP

meanSE 67.52 80.99 4.97

mean 661.25 793.08 484.23

so 190.97 292.02 163.55

Source of Funding: None.

254 A CONTINENT RIGHT COLON RESERVOIR UTILIZING A CATHETERIZABLE SUBMUCOSSALY EMBEDDED APPENDICOSTOMY John P Steins, Maurice M Garcina, Siamk Daneshmand, Gus Miranda. Eila Skinner, Gary Lieskovsky, Donald G Skinner. Los Angeles, CA INTRODUCTION AND OBJECTIVE: To report the surgical technique and clinical outcomes of patients undergoing lower urinary tract reconstruction by means of a continent cutaneous right colon reservoir utilizing a catheterizable submucosally embedded appendicostomy. METHODS: From February 1993 to January 2002. a total of 27 patients underwent a continent right colon urinary reservoir by means of a bilateral uretero-ileal coloappendicostomy. The ascending colon is detubularized and folded and forms the reservoir component of the diversion, the terminal ileum as the afferent limb, and the ileocecal valve as the anti-reflux mechanism. The continence mechanism incorporates the submucosally tunneled appendiX with preservation of the mesentery in a flap-valve technique. A total of 15 males (56%) and 12 females underwent this form of continent urinary diversion, with an average age of 65 years (range 39-82 years) and were evaluated. Clinical outcomes were analyzed including: peri-operative mortality, early complications «3 months). late complications (>3 months), upper urinary tract status and reservoir evaluation, frequency of catheterization, and continence status. RESULTS: All 2S patients underwent the continent right colon reservoir without intra-operative complications. One perioperatively death occurred secondary to a myocardial infarction. Five patients died within the first 4 months of surgery (all related to the primary disease). These 6 patients were considered unevaluable. Mean follow-up of the Zl evaluable patients was 27 months (range 3-75 months). There were no early pouch related complications. Four of21 patients (19%) developed a late complication of stomal stenosis, and all were successfully repaired with open surgical stomal revision. One patient (5%) developed right ureteral hydronephrosis secondary to a uretero-enteric stricture which was successfully treated with ureteral dilation. Of the 21 patients whose continence status was known, all (100%) reported complete continence day and night with ease of catheterization and a mean frequency of catheterization of 5.4 hours (range 4-7 hours). CONCLUSIONS: This continent cutaneous right colon reservoir with bilateral uretero-ileal coloappendicostomy has demonstrated good functional results with excellent continence. This technique is a reasonable alternative for cutaneous urinary diversion when an intact appendix is present. Source of Funding: None.

Interstitial Cystitis Moderated Poster Sunday, April 27, 2003

10:00 AM-12:00 PM

255 GLOBAL CHANGES IN GENE EXPRESSION IN IC BLADDERS Jordan D Dimitrakov", Georgi Staikov, Dimiter Staikov, Plovdiv, Bulgaria; Dorian Y Dikov, Lagny-sur-Marne Cedex, France INTRODUCTION AND OBJECTIVE: Interstitial cystitis (IC) is an enigmatic clinical condition which represents a subset of neuropathic pain syndromes. IC pain is persistent and bladder nerve injury produces sensory/motor deficits and other paradoxical sensations of a qualitative nature leading to a reorganization of sensory *Presenting author.

transmission pathways. A global understanding of the molecular mechanisms involved in IC pain is needed for a better understanding of its pathophysiology and treatment. METHODS: We studied 30 serial bladder biopsies and blood samples from patients diagnosed with interstitial cystitis (IC) on the basis of the NlliINIDDK criteria and 30 bladder biopsies from age-matched controls. Global gene expression changes were examined as well as the pathobiology at the cellular level using DNA microarray, quantitative real-time PCR and immunohistochemistry. Total RNA from each sample was prepared using Trizol(TM) (Life Technologies, Galthersburg, MD, USA), followed by RNeasy(TM) (Qiagen, Hilden, Germany). RNA samples were analyzed by denatured gel electrophoresis. In addition, total RNA quality was assessed by capillary electrophoresis (Bioanalyzer 2100 Agilent, Palo Alto, CA, USA) to ensure that the 28S:18S rRNA ratio was >1.0 for each sample. Hybridization probes were prepared and analyzed according to Affymetrix: instruction. RESULTS: We found that the persistent pain state characteristic of the IC bladder is accompanied by changes in gene expression which can be categorized as follows: (1) immediate early genes (ATF 3, NGFI-A); (2) genes such as ion channels (Nav1.8 (SNS), Nav1.9 (NAN), NAvl.l) and signaling molecules that contribute to the excitability of neurons; and (3) genes that are indicative of secondary events such as neuroinflammation (OX44, ll.-lbeta. ll.-6, TNF, ll.-18, MIP-l). In addition. we studied gene regulation in both IC and non-IC bladders by quantitative PCR, and observed differential gene regulation in these two populations of bladders. Furthermore, we demonstrated unexpected co-regulation of many genes, especially the activation of neuroinflammation markers. CONCLUSIONS: The results of our study provide a new picture of the molecular mechanisms that underlie the complexity of IC pain and suggest that it shares common pathobiology with progressive neurodegenerative disease. Source of Funding: None.

256 GENE THERAPY OF BLADDER PAIN WITH GENE GUN PARTICLE ENCODING PRO·OPIOMELANOCORTIN (POMC) CDNA fao-Chi Chuang", A-K Chou, L-C Yang. T-R Yu, Kaohsiung Hsien, Taiwan; Michael B Chancellor, Pittsburgh, PA INTRODUCTION AND OBJECTIVE: Interstitial cystitis is a bladder supersensitivity disease associated with bladder pain, which has been a major challenge to understand and treat. We hypothesize that targeted and localized expression of endogenous opioid peptide in the bladder could be useful for the treatment of bladder pain. Pro-opiomelanocortin (POMC) is one of such precursor molecules. In this study, we developed a gene-gun method for the transfer of POMC cDNA in vivo, and investigated its therapeutic effect on acetic acid-induced bladder hyperactivity in the rats. METHODS: Human POMC eDNA was cloned into a modified pCMV plasmid and delivered into the bladder of adult female rats by gene-gun. Three days after gene therapy, continuous cystometrograms (CMGs) were performed under urethane-anesthetized by filling the bladder (0.08 mllmin) with solution of saline, followed by 0.3% acetic acid. Bladder immunohistochemical testing was used to detect endorphin after POMC cDNA transfer. RESULTS: Intercontraction interval (ICI) was decreased after exposure to acetic acid (72.7% decrease), however, rats which received POMC eDNA via gene-gun showed a significantly reduced response (ICI 32.5% decrease). This effect was antagonized by intramuscular naloxone, an opioid antagonist (lmglkg). Increased endorphin immunoreactivity with anti-endorphin antibody was detected in the bladder in gene-gun treated animals at day 3 after injection. CONCLUSIONS: The POMC gene can be transferred in the bladder using gene-gun and that increased expression of endorphin in bladder can suppress nociceptive responses induced by bladder irritation. Thus POMC gene-gun delivery may be useful for the treatment of IC and other types of visceral pain. Source of Funding: Chang Gung Memorial Hospital Kaohsiung and National Science Council, Taiwan.

257 GENE THERAPY OF URINARY BLADDER PAIN USING REPLICATlON·DEFECTIVE HERPES SIMPLEX VIRUS (HSV) VECTORS EXPRESSING PREPROENKEPHALIN (PPE) Jun Nishiguchi", Michael B Chancellor, Katsumi Sasaki, Jang H Kim, Michael E Franks. James R Goss, William F Goins, David J Fink. Joseph C Glorioso. Naoki Yoshimura, Pittsburgh, PA INTRODUCTION AND OBJECTIVE: Painful bladder symptoms in patients with interstitial cystitis are often difficult to treat. The present study investigated the effects of enkephalin gene therapy using replication-deficient herpes simplex virus (HSV) encoding preproenkephalin (PPE), a precursor of enkephalins, on bladder pain responses induced by intravesical irritation. METHODS: Viral suspension (5XI08 plaque forming unit: pfu/mi) of replication-deficient HSV vectors encoding PPE, which contains an HCMV IEp-