Sarah Bartolone

Addressing challenges in underactive bladder: recommendations and insights from the Congress on Underactive Bladder (CURE-UAB)

Underactive bladder (UAB) is an expanding troublesome health issue, exerting a major influence on the health and independence of older people with a disproportionally low level of attention received. The 2nd International Congress on Underactive Bladder (CURE-UAB 2) convened in Denver, CO on December 3 and 4, 2015, and comprised of top clinicians, scientists, and other stakeholders to address the challenges in UAB. A series of workshops aimed to define UAB and its phenotype, define detrusor underactivity (DU) and create a subtyping of DU, evaluate existing animal models for DU, and lastly to establish research priorities for UAB.

Modeling of chronic radiation-induced cystitis in mice

Purpose Radiation cystitis (RC), a severe inflammatory bladder condition, develops as a side effect of pelvic radiation therapy in cancer patients. There are currently no effective therapies to treat RC, in part from the lack of preclinical model systems. In this study, we developed a mouse model for RC and used a Small Animal Radiation Research Platform to simulate the targeted delivery of radiation as used with human patients. Methods and materials To induce RC, C3H mice received a single radiation dose of 20 Gy delivered through 2 beams. Mice were subjected to weekly micturition measurements to assess changes in urinary frequency. At the end of the study, bladder tissues were processed for histology. Results Radiation was well-tolerated; no change in weight was observed in the weeks after treatment, and there was no hair loss at the irradiation sites. Starting at 17 weeks after treatment, micturition frequency was significantly higher in irradiated mice versus control animals. Pathological changes include fibrosis, inflammation, urothelial thinning, and necrosis. At a site of severe insult, we observed telangiectasia, absence of uroplakin-3 and E-cadherin relocalization. Conclusions We developed an RC model that mimics the human pathology and functional changes. Furthermore, radiation exposure attenuates the urothelial integrity long-term, allowing for potential continuous irritability of the bladder wall from exposure to urine. Future studies will focus on the underlying molecular changes associated with this condition and investigate novel treatment strategies.

Development of an interstitial cystitis risk score for bladder permeability

Background Interstitial cystitis/bladder pain syndrome (IC) is a multifactorial syndrome of severe pelvic and genitalia pain and compromised urinary function; a subset of IC patients present with Hunner’s lesions or ulcers on their bladder walls (UIC). UIC is diagnosed by cystoscopy, which may be quite painful. The objective of this study was to determine if a calculated Bladder Permeability Defect Risk Score (BP-RS) based on non-invasive urinary cytokines could discriminate UIC patients from controls and IC patients without Hunner’s ulcers. Methods A national crowdsourcing effort targeted IC patients and age-matched controls to provide urine samples. Urinary cytokine levels for GRO, IL-6, and IL-8 were determined using a Luminex assay. Results We collected 448 urine samples from 46 states consisting of 153 IC patients (147 female, 6 male), of which 54 UIC patients (50 females, 4 male), 159 female controls, and 136 male controls. A defined BP-RS was calculated to classify UIC, or a bladder permeability defect etiology, with 89% validity. Conclusions The BP-RS Score quantifies UIC risk, indicative of a bladder permeability defect etiology in a subset of IC patients. The Bladder Permeability Defect Risk Score is the first validated urine biomarker assay for interstitial cystitis/bladder pain syndrome.

Rapid Detection of Zika Virus in Urine Samples and Infected Mosquitos by Reverse Transcription-Loop-Mediated Isothermal Amplification

Infection with Zika virus (ZIKV) is of growing concern since infection is associated with the development of congenital neurological disease. Quantitative reverse transcription PCR (qRT-PCR) has been the standard for ZIKV detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper testing. Studies have suggested that ZIKV detection in urine is more sensitive and has a longer window of detection compared to serum and saliva. The objective of this study was to develop a urine diagnostic test that could be completed in under 30 minutes. Urine samples spiked with ZIKV or dengue virus were tested using RT-LAMP as well as by conventional quantitative qRT-PCR. These techniques were then validated using crude lysates made from ZIKV infected mosquitoes in addition to urine and serum samples from ZIKV infected patients. RT-LAMP specifically detected ZIKV in urine and serum for ZIKV infected patients and crude mosquito lysates. This test was performed in under 30 minutes and did not require RNA extraction from urine nor mosquitos. This approach could be used for monitoring of exposed individuals, especially pregnant women, couples wanting to conceive, or individuals with suspicious symptoms as well as surveillance of mosquito populations.

Crowdsourcing Disease Biomarker Discovery Research: The IP4IC Study

Purpose: Biomarker discovery is limited by readily assessable, cost efficient human samples available in large numbers that represent the entire heterogeneity of the disease. We developed a novel, active participation crowdsourcing method to determine BP‐RS (Bladder Permeability Defect Risk Score). It is based on noninvasive urinary cytokines to discriminate patients with interstitial cystitis/bladder pain syndrome who had Hunner lesions from controls and patients with interstitial cystitis/bladder pain syndrome but without Hunner lesions. Materials and Methods: We performed a national crowdsourcing study in cooperation with the Interstitial Cystitis Association. Patients answered demographic, symptom severity and urinary frequency questionnaires on a HIPAA (Health Insurance Portability and Accountability Act) compliant website. Urine samples were collected at home, stabilized with a preservative and sent to Beaumont Hospital for analysis. The expression of 3 urinary cytokines was used in a machine learning algorithm to develop BP‐RS. Results: The IP4IC study collected a total of 448 urine samples, representing 153 patients (147 females and 6 males) with interstitial cystitis/bladder pain syndrome, of whom 54 (50 females and 4 males) had Hunner lesions. A total of 159 female and 136 male controls also participated, who were age matched. A defined BP‐RS was calculated to predict interstitial cystitis/bladder pain syndrome with Hunner lesions or a bladder permeability defect etiology with 89% validity. Conclusions: In this novel participation crowdsourcing study we obtained a large number of urine samples from 46 states, which were collected at home, shipped and stored at room temperature. Using a machine learning algorithm we developed BP‐RS to quantify the risk of interstitial cystitis/bladder pain syndrome with Hunner lesions, which is indicative of a bladder permeability defect etiology. To our knowledge BP‐RS is the first validated urine biomarker assay for interstitial cystitis/bladder pain syndrome and one of the first biomarker assays to be developed using crowdsourcing.

Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth defects. The goal of this protocol is to quickly detect ZIKV in both human and mosquito samples. The current gold standard for ZIKV detection is quantitative reverse transcription PCR (qRT-PCR); reverse transcription loop-mediated isothermal amplification (RT-LAMP) may allow for a more efficient and low-cost testing without the need for expensive equipment. In this study, RT-LAMP is used for ZIKV detection in various biological samples within 30 min, without first isolating the RNA from the sample. This technique is demonstrated using ZIKV infected patient urine and serum, and infected mosquito samples. 18S ribosomal ribonucleic acid and actin are used as controls in human and mosquito samples, respectively.

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.