Bernadette Zwaans

Challenges and Opportunities in Radiation-induced Hemorrhagic Cystitis.

As diagnosis and treatment of cancer is improving, medical and social issues related to cancer survivorship are becoming more prevalent. Hemorrhagic cystitis (HC), a rare but serious disease that may affect patients after pelvic radiation or systemic chemotherapy, has significant unmet medical needs. Although no definitive treatment is currently available, various interventions are employed for HC. Effects of nonsurgical treatments for HC are of modest success and studies aiming to control radiation-induced bladder symptoms are lacking. In this review, we present current and advanced therapeutic strategies for HC to help cancer survivors deal with long-term urologic health issues.

Void spot assay procedural optimization and software for rapid and objective quantification of rodent voiding function, including overlapping urine spots

Mouse urinary behavior is quantifiable and is used to pinpoint mechanisms of voiding dysfunction and evaluate potential human therapies. Approaches to evaluate mouse urinary function vary widely among laboratories, however, complicating cross-study comparisons. Here, we describe development and multi-institutional validation of a new tool for objective, consistent, and rapid analysis of mouse void spot assay (VSA) data. Void Whizzard is a freely available software plugin for FIJI (a distribution of ImageJ) that facilitates VSA image batch processing and data extraction. We describe its features, demonstrate them by evaluating how specific VSA method parameters influence voiding behavior, and establish Void Whizzard as an expedited method for VSA analysis. This study includes control and obese diabetic mice as models of urinary dysfunction to increase rigor and ensure relevance across distinct voiding patterns. In particular, we show that Void Whizzard is an effective tool for quantifying nonconcentric overlapping void spots, which commonly confound analyses. We also show that mouse genetics are consistently more influential than assay design parameters when it comes to VSA outcomes. None of the following procedural modifications to reduce overlapping spots masked these genetic-related differences: reduction of VSA testing duration, water access during the assay period, placement of a wire mesh cage bottom on top of or elevated over the filter paper, treatment of mesh with a hydrophobic spray, and size of wire mesh opening. The Void Whizzard software and rigorous validation of VSA methodological parameters described here advance the goal of standardizing mouse urinary phenotyping for comprehensive urinary phenome analyses.

MP31-04 THE POWER OF CROWDSOURCING: NOVEL METHOD FOR DISCOVERY OF URINE BIOMARKERS

quantitative sensory testing, and urine sample collection. Temporal summation to evoked, thermal cutaneous pain was performed with a Medoc Thermal Sensory Analyzer at .4 Hz, a frequency known to elicit C-fiber mediated wind-up in the dorsal horn of the spinal cord. Subjects were asked to rate their pain (0 e 100 VAS) during each of a sequence of 10 brief (.5 second) heat pulses to 49 C. Temporal summation was defined as the difference in pain rating between the maximum and first pain ratings. An individual with a difference in pain ratings or a first pain rating greater than 1 SD above controls after normalization was designated as demonstrating CS. Mid-stream urine samples collected from each patient were subjected to metagenomic sequencing targeting the V3-V4 region of the 16S-rRNA gene. Relative bacterial abundances were compared using the QIIME and the Wald test statistic in the MGLM package among women with and without OAB and CS. RESULTS: 23 patients comprised the study cohort. 6/10 (60%) subjects with OAB demonstrated CS, 2/8 (25%) subjects demonstrating CS did not have OAB. Bacterial abundance differed significantly between patients with and without OAB (Wald test statistic 316, p<0.01) and CS (Wald test statistic 80, p<0.01). Relative bacterial abundances were similar in patients with OAB and CS (Table). Relative to those without OAB and CS, Enterobacteriaceae, Chitinophagaceae and Burkholderiaceae were more abundant in subjects with OAB and CS and Lactobacillaceae and Prevotellaceae less abundant. CONCLUSIONS: C-fiber activation related to elevated CS and alterations in the urinary microbiome may represent a combined mechanism of action for the development of refractory LUTS in some patients that warrants further study.

MP29-07 DEVELOPMENT OF THE ULCERATIVE INTERSTITIAL CYSTITIS RISK SCORE (ICUS): A URINE-BASED MULTIPLE PROTEIN ASSAY TO PREDICT ULCERATIVE INTERSTITIAL CYSTITIS

INTRODUCTION AND OBJECTIVES: Ifosfamide-induced hemorrhagic cystitis and bladder hypersensitivity can be difficult to manage when mesna fails to prevent them. Bladder hypersensitivity associated with various forms of cystitis may be refractory to multiple treatment modalities. Prior work suggests interleukin-4 (IL4) alleviates ifosfamide-induced hemorrhagic cystitis and resiniferatoxin (capsaicin receptor agonist)-induced bladder pain. IL4-inducing principle of Schistosoma mansoni eggs (IPSE) is a host modulatory protein that binds immunoglobulins on leukocytes thereby inducing IL4 production and translocates into host nuclei to alter gene transcription. We sought to determine if the S. haematobium homolog of IPSE (H-IPSE) would reduce ifosfamideand resiniferatoxin-induced bladder pathology. METHODS: We cloned and expressed H-IPSE and a nuclear localization sequence (NLS)-deficient mutant H-IPSE (H-IPSE[NLS]). H-IPSE IgE binding was measured by ELISA. H-IPSE activation of IgEbearing basophils was assayed using RSATL8 basophilic reporter cells. Cellular uptake and NLS-dependent nuclear translocation of H-IPSE and H-IPSE(NLS) were confirmed using HTB9 urothelial cells and fluorescence microscopy. We administered IL4, H-IPSE, H-IPSE+antiIL4 antibody, H-IPSE(NLS), or H-IPSE(NLS)+anti-IL4 antibody to mice prior to ifosfamide (with and without mesna) or resiniferatoxin. Negative controls were administered saline only. Positive controls were administered ifosfamide only. Previously published metrics for pain and urinary frequency were interpreted in blinded fashion. Bladder histology was interpreted in blinded fashion. Bladder hemoglobin was quantified using Drabkin0s assay. Bladder gene expression was assessed via realtime PCR. RESULTS: H-IPSE bound IgE in vitro and activated IgE-bearing RSATL8 cells. Nuclear translocation of H-IPSE but not H-IPSE(NLS) was confirmed. H-IPSE was superior to mesna and IL4 in suppressing ifosfamide-induced bladder hemorrhage (IL4-dependent). H-IPSE was comparable to mesna in dampening ifosfamide-triggered pain behaviors (NLS-dependent) and urinary frequency (NLS-dependent). H-IPSE reduced resiniferatoxin-mediated freezing behaviors (IL4and NLSdependent). H-IPSE reduced mRNA expression of proinflammatory mediators and increased expression of uroplakin mRNA. CONCLUSIONS: Our work suggests a uropathogen-derived host modulatory protein has therapeutic effects in bladder disease models.