806 URINARY CHEMOKINES AS NON-INVASIVE PREDICTORS OF ULCERATIVE INTERSTITIAL CYSTITIS

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804 IRRITATION-INDUCED BLADDER OVERACTIVITY IS SUPPRESSED BY TIBIAL NERVE STIMULATION IN CATS Mang Chen*, Changfeng Tai, Bing Shen, Jicheng Wang, James Roppolo, William deGroat, Pittsburgh, PA INTRODUCTION AND OBJECTIVES: Tibial neuromodulation is becoming more widely used for management of overactive bladder symptoms. We therefore sought to investigate the effects of tibial nerve stimulation on bladder overactivity induced by acetic acid (AA) irritation. METHODS: Cystometry was performed in 10 ␣-chloralose anesthetized female cats by infusing saline or AA through a urethral catheter that was secured by a ligature around the urethra. Intravesical infusion of 0.25% AA was used to irritate the bladder and induce bladder overactivity. Multiple cystometrograms (CMGs) were performed before, during, and after tibial nerve stimulation to determine the inhibitory effect on the micturition reflex. RESULTS: Infusion of 0.25% AA irritated the bladder, induced bladder overactivity, and significantly reduced the bladder capacity to about 20% of the control capacity. Tibial nerve stimulation at either low (5 Hz) or high (30 Hz) frequency significantly increased bladder capacity to about 40% of the saline control capacity when it was applied during the AA infusion CMG. The amplitude of bladder contractions was smaller during AA irritation than during saline distention due to a significantly smaller bladder capacity. However, tibial nerve stimulation at 5 Hz increased both the bladder capacity and the amplitude of bladder contractions. CONCLUSIONS: Stimulation of the tibial nerve of cats can partially reverse bladder overactivity induced by intravesical administration of a chemical irritant that activates C-fiber afferent nerves. This data provides pre-clinical evidence for the use of tibial neuromodulation in treating overactive bladder symptoms. Source of Funding: This study is supported by NIH under grants DK-068566 and DK-077783, and by the Christopher and Dana Reeve Foundation.

Infections/Inflammation of the Genitourinary Tract: Interstitial Cystitis Podium 20 Monday, May 16, 2011

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805 PREDICTIVE FACTORS OF PAIN SEVERITY IN PATIENTS WITH INTERSTITIAL CYSTITIS/BLADDER PAIN SYNDROME Jennifer Rothschild*, Marguerite Thomer, Susan Messing, Robert Mayer, Rochester, NY INTRODUCTION AND OBJECTIVES: Patients with the visceral hypersensitivity syndrome Bladder Pain Syndrome/Interstitial Cystitis (IC) present with a wide spectrum of severity of pain as well as urinary frequency. The causes of such diversity is complex but the severity of pain does impact the distress experienced as well as the modalities of therapy needed to control suffering. Finding correlates of pain severity may allow better insight into etiology and therapy. METHODS: Institutional approval was obtained for data review of 186 patients with a clinical diagnosis of BPS/IC who completed an intake form including characteristics of their IC/BPS as well as comorbid illnesses and report of current pain on a 0-to-10 scale. Statistics were performed using SAS Statistical software with P⬍0.05 as possibly clinically significant. RESULTS: Pain severity did not appear to correlate by age or gender, duration of symptoms, rapidity of onset, report of dietary food sensitivities or exacerbation of symptoms during menstrual cycle.

Increased pain scores were associated with reported dysmenorrhea, IC onset with a precipitating event of pelvic surgery, any history of pelvic surgery, more than 5 vaginal infections a year, abuse, asthma, and migraine. A logistic regression model looked at the association of severe (7–10) vs mild (0 –3) pain with history of abuse, dysmenorrhea and history of onset of IC symptoms following pelvic surgery. The patients with history of abuse, dysmenorrhea, and pelvic surgery have an 82% (95%CI, 49 –96%) predicted probability of a severe pain score (⬎ 7) while those having none of these factors have a predicted probability of 5% (95%CI, 1–20%). CONCLUSIONS: Although the causes of BPS/IC are unknown and likely multifactorial, the findings suggest that once the bladder does develop a hypersensitive state, those with a history of prior pelvic noxious stimuli will be at risk for experiencing substantial pain possibly related to sacral sensitization and in contrast, actual duration of IC itself does not appear to be significant. Risk factors for dysfunctional coping (social stress and history of abuse) also predict more severe pain though likely by different mechanisms. Finally, those patients with migraines and/or asthma also have increased pain profiles and possible mechanisms for this are worthy of additional investigation although these do not appear to be strong independent indicators of severe pain. Source of Funding: None

806 URINARY CHEMOKINES AS NON-INVASIVE PREDICTORS OF ULCERATIVE INTERSTITIAL CYSTITIS Pradeep Tyagi, PhD*, Kim Killinger RN, MSN, Vikas Tyagi, MD, Michael Chancellor, Kenneth Peters, MD, Royal Oak, MI INTRODUCTION AND OBJECTIVES: Bladder biopsy studies done on ulcerative interstitial cystitis/painful bladder syndrome (IC/ PBS) patients have noted increased mRNA expression of CXCR3 receptor binding chemokines such as CXCL-9,10,11. We hypothesized that increased transcript of these chemokines in bladder tissue will be reflected by the increased levels of respective proteins in urine. In the present study, we investigated whether a multi-marker panel of 2 or 3 chemokines in urine can non-invasively differentiate ulcerative IC/PBS from non-ulcerative IC/PBS and potentially replace the invasive and expensive cystoscopy based diagnosis. METHODS: Urine samples were collected from 10 ulcerative IC/PBS patients with ulcer, 10 non-ulcerative IC/PBS patients, and 10 age, race, and sex-matched asymptomatic control subjects. Presence of ulcer in ulcerative group and absence in non-ulcerative group of IC/PBS patients was confirmed by cystoscopy at time of urine collection. Eight different chemokines/cytokines in urine were measured by MILLIPLEX™ MAP Human Cytokine/Chemokine multiplex immunoassay. Kruskal-wallis ANOVA followed by Dunn’s test was used to check for significance among various groups and values are expressed as mean ⫾ SEM. RESULTS: Urine levels of most chemokines/cytokines were 10 –100 fold lower in asymptomatic controls relative to IC/PBS patients. Univariate comparison of urine levels of eight tested proteins between ulcerative and non-ulcerative group of patients revealed a significant 10 fold elevation of CXCL1 (170⫾60.55 vs 12.55⫾3.38 pg/ml) and 5 fold elevation of CXCL-10 (IP-10) (684.7⫾68.10 vs 123.3⫾45.35 pg/ml) in the urine of ulcerative patients (ANOVA, p⬍0.001). Urine levels of IL-6 (16.06⫾6.99 vs 2.42⫾1.94 pg/ml) were also significantly elevated in ulcerative IC/PBS group, but quantitatively the levels compared to CXCL-1 and CXCL-10 were several fold lower and closer to the detection limit of the assay. CONCLUSIONS: The chemokines and cytokines (CXCL-1, IL-6 and IP-10) identified here are key players responsible for inflammatory cell adhesion and infiltration into inflamed tissue. The elevated urinary levels in ulcerative IC/PBS patients reflects the ongoing bladder inflammation noted at the site of bladder ulcer. Lower urinary levels of IL-6 noted here in IC/PBS patients agree with reports from other groups.

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Based on our findings, we conclude that a multi-marker urinary chemokine panel can non-invasively discriminate ulcerative IC/PBS from non-ulcerative IC/PBS in an inexpensive manner. Source of Funding: None

807 BLADDER EPITHELIAL CELL EXPLANTS FROM INTERSTITIAL CYSTITIS/PAINFUL BLADDER SYNDROME PATIENTS EXHIBIT DECREASED AKT/WNT SIGNALING AS COMPARED TO MATCHED CONTROLS Susan Keay*, Chen-Ou Zhang, Baltimore, MD INTRODUCTION AND OBJECTIVES: Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic disorder with bladder epithelial thinning or ulceration, pain, urinary frequency and urgency. Bladder epithelial cells from IC/PBS patients make a glycopeptide antiproliferative factor or “APF” (Neu5Ac␣2-3Gal␤1-3GalNAc␣-O-TVPAAVVVA) that binds to CKAP4 and inhibits cell growth. Because the peptide portion of APF is 100% homologous to a segment of frizzled 8, a Wnt receptor, we determined whether IC/PBS explants have evidence for decreased Akt/Wnt signaling, and whether abnormalities in Akt/Wnt signaling could be induced by APF treatment of normal cells. METHODS: Primary bladder epithelial explants from 6 IC/PBS patients and 6 age- and gender-matched asymptomatic controls were plated in Minimal Essential Medium with supplements, then cultured in completely defined serum-free medium; additional normal cells were similarly plated but treated with active synthetic APF or inactive unglycosylated control peptide (both from PolyPeptide Laboratories). Gene expression was measured by quantitative real-time polymerase chain reaction. Specific protein expression was determined by Western blot and/or enzyme-linked immunosorbent assay. Autoradiogram bands were quantified by scanning densitometry and normalized to ␤-actin. Significance of a difference in mean values between groups was determined by two-tailed Student’s t-test for each assay, with p⬍.05 considered significant. RESULTS: IC/PBS bladder epithelial cell explants had significant decreases in Akt ser473 and GSK3␤ ser9 phosphorylation; nuclear ␤-catenin and JunB protein levels; and cellular MMP2 expression, as compared to matched controls. Similarly, treatment of normal bladder epithelial cells with synthetic APF induced significant changes in phosphorylation or expression of the same cell proteins, as compared to treatment with control peptide. CONCLUSIONS: Explanted IC/PBS bladder epithelial cells have decreased Akt/Wnt signaling, resulting in significantly decreased nuclear ␤-catenin and JunB, plus decreased downstream MMP2 expression. Each of these abnormalities could also be induced in normal cells by APF. As Akt/Wnt signaling and MMP2 expression are important for cell proliferation and epithelial to mesenchymal transition, these findings suggest that inhibition of Akt/Wnt signaling by APF may play a role in the decreased proliferation and altered differentiation of IC/PBS bladder epithelial cells previously noted in several laboratories. Source of Funding: NIH/NIDDK R01DK52596

808 THE EFFECTS OF CAFFEINE ON THE SYMPTOMS OF INTERSTITIAL CYSTITIS/PAINFUL BLADDER SYNDROME: A RANDOMIZED, PLACEBO-CONTROLLED DOUBLE BLIND STUDY Amin Herati*, Barbara Shorter, Mostafa Sadek, Helen Levey, Zhamshid Okhunov, Robert Moldwin, Lake Success, NY INTRODUCTION AND OBJECTIVES: Caffeine is a widely consumed stimulant among the adult US population and well known to exacerbate symptoms of interstitial cystitis/painful bladder syndrome (IC/PBS). The purpose of this study is to define the relationship between caffeine and IC symptoms by assessing changes in irritative voiding symptoms and pelvic pain.

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METHODS: Consecutive IC/PBS patients were randomized to receive either a pill containing 100mg of caffeine or placebo using a four block randomization scheme. All subjects were asked to abstain from caffeine for one to two days prior to the start of the study and to complete a questionnaire consisting of a modified O’Leary Sant symptoms and problem index (0SSPI), global response assessment (GRA), and a visual analog pain score (VAS). Patients were also instructed to maintain a voiding diary starting two days prior to commencing the study and continuing until two days after taking his or her assigned pill. Symptoms were followed by asking the patients to record their pain levels using the GRA and VAS pain scores every 4 hours for the first 16 hours and then once every eight hours for the next 32 hours, for a total of 48 hours. Continuous variables were compared using a two sample t-test, and Pearson’s correlation was used to determine if an association was present between the OSSPI scores and the observed pain response and voided volumes. RESULTS: Thirty patients with IC/PBS were enrolled in this study, fourteen of which were randomized to the caffeine arm and sixteen were randomized to the placebo arm. Four patients (28.6%) in the caffeine arm and one patient (6.2%) in the placebo arm dropped out before completing the study. Reasons cited for withdrawal included fear of the discomfort associated with the caffeine pill and caffeine withdrawal headaches. Both arms were comparable in baseline VAS scores, IC Symptom and Problem index scores (p ⫽ 0.73, p⫽0.08, and p⫽ 0.1, respectively). No significant differences were found in the VAS and GRA at each of the time intervals assessed between the two groups after administration of caffeine or placebo pill. No correlation was also identified between the baseline symptom scores, GRA and VAS pain scores, and the volume voided after administration of the assigned pill. CONCLUSIONS: Although caffeine has been anecdotally associated with symptom exacerbation in IC/PBS, our randomized, placebo-controlled, double-blind study did not demonstrate a difference in irritative voiding symptoms or a difference in voiding volume. Source of Funding: The Fishbein Family IC Research Foundation Grant

809 ESTROGEN RECEPTOR EXPRESSION AND POTENTIAL FUNCTION IN INTERSTITIAL CYSTITIS Wu-Jiang Liu*, Jie Jin, Yun-Xiang Xiao, Li-Li Liang, Hai-Feng Wang, Qun He, Ying Wang, Ying-Lu Guo, Beijing, China, China, People’s Republic of INTRODUCTION AND OBJECTIVES: Interstitial cystitis (IC) is a disease seen mostly in women, and estrogen has been implicated in the etiology of this disease. This study aimed to elucidate estrogen receptor (ER) expression and its relationship with mast cell in interstitial cystitis. METHODS: Bladder biopsy samples were obtained from IC patients with informed consent. Estrogen receptor expression was investigated by real-time PCR and Western-blot. Estrogen receptor location was indentified by immunohistochemical stain. The bladder epithelium and connective tissue beneath the epithelium were collected separately by microdissection, and then investigated by real-time PCR. Bone marrow-derived murine mast cell was cultured in vitro, stimulated with estradiol. The histamine in supernatant was measured by ELISA. Mast cell related cytokines were assayed by RT-PCR and Westernblot. Cyclophosphamide-induced cystitis model were used for isolating mast cell from bladder by high power flow cytometry cell sorting, the ER expression in mast cell was investigated by real-time PCR and immunofluorescence. RESULTS: Both ER␣ and ER␤ were expressed in bladder tissue from patients. ER␤ is mainly expressed in the basal cell layer of the urothelium, while ER␣, although very little, was expressed in the connective tissue beneath the epithelium. Estradiol can activate the mast cell evidenced by releasing histamine and expressing proinflam-