Shelly Vaden

Despite the recent FDA approvals of five checkpoint antibodies for treating advanced or refractory bladder cancer, the majority of advanced bladder cancer patients do not benefit from checkpoint immunotherapy. Furthermore, immune-related adverse events (IRAEs) associated with systemic administration of checkpoint antibodies can be serious to life threatening. Safer and more effective treatments for advanced bladder cancer patients are needed. In more than a decade of preclinical research in mice, intravesical immunotherapy using interleukin-12 (IL-12) formulated with the mucoadhesive biopolymer chitosan (CS), i.e., CS/IL-12, has been shown to eliminate nearly all established orthotopic bladder tumors while inducing robust tumor-specific immunity. In addition to enhanced delivery, the intravesical route also limits systemic exposure and related immunotoxicities. In support of clinical translation, the proposed comparative oncology studies will evaluate the safety and activity of intravesical CS/IL-12 immunotherapy in pet dogs with bladder cancer. There are a number of similarities between canine and human bladder cancers, including tumorigenesis, heterogeneity, molecular subtypes, metastatic behaviors, etc., which make spontaneous canine urothelial carcinoma (UC) an attractive model. These similarities imply that treatments found to be successful in dogs will likely be successful in people. The two objectives of this project are: 1) to demonstrate that intravesical CS/IL-12 immunotherapy can safely induce antitumor immunity against canine invasive UC for the purposes of supporting clinical translation; and 2) to determine if canine bladder cancer is a useful model for the evaluation of novel immunotherapies. The scientific hypothesis is that intravesical CS/IL-12 immunotherapy will minimize systemic exposure to a potent immunotherapy while inducing antitumor immunity via neoantigen-reactive T cells in an advanced bladder cancer setting.

As cats age, they are more likely to develop bladder problems that include bacteria in their urine. Sometimes bladder bacteria can cause signs like urinating more often, straining or pain while urinating, and blood in the urine (i.e., bacterial cystitis). In other cases, cats can have bacteria in their urine without any signs (i.e., subclinical bacteriuria). In both cases, the most common type of bacteria is Escherichia coli. When cats have bacteria in their urine, veterinarians must weigh the risks and benefits of giving antibiotics and consider the cat's other health conditions (e.g., kidney disease). Although in people there is evidence to support not treating women with bacteria in their urine when they have no signs of bladder disease, there is less information for what to do for cats. Giving antibiotics is not without risk (e.g., side effects, disruption to the normal healthy bacteria, developing antibiotic resistance), so knowing more about the effect of bladder bacteria on cats will help veterinarians treat cats presenting for these problems. In people and dogs, E. coli is also the most common bacteria in the bladder, and the gene fimH is the factor that determines how harmful those bacteria are and whether infections will come back. We predict that in cats, E.coli with the fimH gene will lead to worse outcomes and propose following aging female cats with urinary bacteria for up to 6 months to guide veterinarians in making treatment choices for cats with bacteria in their urine.

Title: Early diagnosis of urothelial carcinoma Rationale: Diagnosis of canine urothelial carcinoma, also known as transitional cell carcinoma, occurs commonly late in the disease process, once a mass has developed and causes discomfort noticeable to owners. With 40,000 to 80,000 dogs diagnosed in the United States each year, earlier diagnosis with the promise for improved survival and quality of life would have a significant impact on canine health. When a single mutation in the canine BRAF gene was identified in canine UC/TCC, the CADETSM BRAF Mutation Detection Assay was launched as a tool for early detection of this cancer. With the technical sensitivity of droplet digital PCR, this assay may become a game-changer in diagnosing UC/TCC early and increasing survival in this devastating disease. Hypothesis/Objectives: Our long-term goal is to achieve increased survival of dogs with UC/TCC through early detection and improved treatment. The central hypothesis is that the CADETSM BRAF Mutation Detection Assay is a valid tool for the early diagnosis of urothelial carcinoma (UC) in dogs. To address this hypothesis, we propose to screen free-catch urine samples of population of dogs to identify a cohort with low levels of the BRAF mutation and evaluate the urinary tract of these dogs by ultrasonography and cystoscopy over time. Once a mass is detected we will correlate tumor volume in dogs with BRAF mutation fractional abundance shed in their urine. Study Design: Prospective clinical study ������������������ Owners of ~51,000 dogs from five breeds at an increased risk of developing UC/TCC will be invited to collect urine for the CADETSM BRAF Mutation Detection Assay. An estimated 1,095 samples will be collected, to identify over 40 dogs with BRAF mutation levels of <5% in their urine. Dogs will be invited to attend a detailed clinical assessment through history, physical examination, complete blood count, serum biochemistry, urinalysis urine cytology, and urogenital 2-D ultrasound with tumor volume measurements. We will continue to recruit cases until we have 30 dogs enrolled. This group will be advanced to cystoscopy and biopsy of any visible lesions. For all 30 dogs, the level of BRAF mutation shed into the urine will be monitored monthly. Once the levels reach 10%, dogs will have repeat diagnostics, including ultrasound, urine cytology, and cystoscopy with biopsy. End point will be definitive diagnosis of UC/TCC through urine cytology or histopathology of tumor biopsies. Preliminary Data: Assessment of over 300 pathology-verified specimens of canine UC/TCC with the CADETSM BRAF Mutation Detection Assay showed that 85% are associated with a V595E BRAF mutation. Analysis of free-catch urine samples from over 100 dogs that had biopsy-verified UC/TCC show 100% agreement between the presence of the BRAF mutation detected and UC/TCC. The BRAF mutation has not been detected in hundreds of specimens from dogs without UC/TCC (e.g. polyps, inflammation etc.). Five dogs with low levels of detectable BRAF mutation (i.e. <3%) had no visible mass on ultrasound and no clinical signs of disease. These dogs received no treatment and developed lower urinary signs consistent with UC/TCC within 3-4 months. Three of these dogs were then imaged and all had developed a trigonal mass on ultrasound. One dog was lost to follow up and the fifth dog developed clinical signs six weeks later, at which time ultrasound revealed generalized bladder wall thickening. Monitoring of free-catch urine of all dogs revealed progressive increases in BRAF mutation level, suggesting that as the UC/TCC advances within the urinary tract, the proportion of cells shed into the urine also increases. Expected Results: If our hypothesis is correct, we will have compelling evidence that detection of the BRAF V595E mutation in the urine of dogs is diagnostic for UC/TCC and can be used to initiate early treatment. Budget and Timeline: We are requesting $203,777 to complete this study over 2 years. Potential Impact for Animal Health: With a strong indication that the CADETSM BRAF Mut

Significance: PuO2 measurement, a robust surrogate marker for medullary hypoxia, could be an excellent real-time biomarker for diagnosing AKI and its progression. This proposal is significant and innovative because PuO2 will be measured continuously cage-side, representing a substantial advancement in current AKI diagnosis standards. Hypothesis/Objectives: Medullary hypoxia is associated with the development of AKI in dogs, and PuO2 can be used as a real-time marker for the development and progression of AKI. Objective 1: To determine the incidence of medullary hypoxia in dogs with AKI, we will measure PuO2 over 48 hours in dogs with AKI and without AKI. Objective 2: To establish PuO2 as a predictor of AKI progression, we will monitor PuO2 and serum creatinine over 72 hours in dogs with AKI. Study Design: We will recruit 30 dogs diagnosed with AKI and 15 dogs without any signs of AKI. A urinary oxygen tension monitoring device will be placed in line with the urinary collection system. The device will continuously record PuO2 and urine flow. They will be recorded and monitored for 72 hours in dogs with AKI, and for 48 hours in the control dogs. Serum creatinine and plasma lactate concentrations will be measured every 24 hours. Expected Results: We predict that the PuO2 will be significantly lower in dogs with AKI than the control dogs. We also anticipate finding an inverse correlation of increasing PuO2 with decreasing serum creatinine and a decreasing or persistently low PuO2 associated with ongoing or progressing AKI in the dogs with AKI group. Anticipated Outcomes: The finding of this study will support our hypothesis that medullary hypoxia is associated with pathophysiological factors involved in canine AKI. Demonstrating a low PuO2 in dogs with AKI will further support PuO2 measurement as a real-time novel kidney biomarker for AKI diagnosis and ongoing kidney injury. Measuring PuO2 will enable clinicians to develop a new therapeutic strategy for AKI in dogs and help us with the real-time diagnosis of AKI.

Rationale: Urinary tract infections (UTI) are one of the most commonly encountered infections requiring antimicrobial therapy in dogs [1, 2]. Extra-intestinal Pathogenic E. coli (ExPEC) are the most frequent cause of UTI and are often co-isolated with Enterococcus spp. (ENC) [1, 2, 3-7]. Female dogs are predisposed to UTI and can develop persistent infections requiring multiple rounds of antimicrobials [1-7]. With the rise of antimicrobial resistance (AMR) in both ExPEC and ENC, there is a clear need to develop new therapies [2, 3, 8-10]. To date, UTI therapy and research have targeted ExPEC while ENC has largely been ignored. We recently discovered a novel in vitro growth synergy between ExPEC and ENC. When grown in iron-limited conditions that mimic the urinary tract, co-culture with ENC provides marked growth advantages to ExPEC. Therefore, the overall goal of this proposal is to investigate the genetic drivers of this phenomenon and define the clinical relevance of this synergy. Hypothesis/Objectives: We hypothesize ExPEC harboring genetic elements that allow for a benefit from ENC co-infection will cause canine UTI with worse clinical outcomes and increased AMR. We will test this hypothesis in two objectives. Objective 1: Identify genetic drivers of ExPEC growth advantage in co-culture with ENC. Objective 2: Compare clinical outcomes of dogs with ExPEC:ENC co-infection to dogs with single agent infections.

Hypothesis: Up-titration of enalapril dose to maximally reduce urine protein excretion in dogs with glomerular disease will improve survival time, slow rate of decline in kidney function, and delay onset of azotemia as compared to the standard enalapril dose. Objective 1: Determine and compare 12, 24, 36, and 52-week survival rates of dogs with non-azotemic glomerular disease treated with standard vs. up-titrated doses of enalapril. Objective 2: Determine and compare rate of decline in kidney function and time until diagnosis of azotemia in dogs with glomerular disease treated with standard vs. up-titrated doses of enalapril. Objective 3: Quantify and compare renin-angiotensin-aldosterone system suppression in dogs with non- azotemic glomerular disease treated with standard vs. up-titrated doses of enalapril, and determine whether patient survival or rate of decline in kidney function is associated with amount of suppression. Objective 4: Determine whether the following differ between dogs with glomerular disease treated with standard vs. up-titrated doses of enalapril: need for additional anti-hypertensive therapy; prevalence of glomerular disease complications; association between glomerular disease histologic type and patient survival or rate of GFR decline.

On this project, we will oversee the clinical examinations and testing required of the approximately 6 cases (cats), and the submission of these results to the Ohio State University for use in their research study. Cats will be screened with three view abdominal radiographs, CBC, biochemistry, urinalysis, and urine culture. Cats enrolled into this clinical trial will return to NCSU every 2 weeks for recheck exams including body weight, urinalysis and abdominal radiographs until radiographic dissolution. A CBC, biochemisty, urinalysis, and urine culture will be performed at the time of dissolution or day 56 in cats without dissolution. For cats with no evidence of dissolution at day 56, a cystotomy will be performed to remove the uroliths. These will be submitted to the UC-Davis Urolith Laboratory for quantitative analysis.

Urinary incontinence affects more than 20% of spayed female dogs, with medium and large breeds more commonly affected. In the majority of the cases urinary incontinence is caused by dysfunction of the muscles controlling the urethral sphincter. This results in uncontrolled loss of urine and can lead to serious bladder and kidney infections, in addition to irritation and/or ulceration of the skin in contact with the urine. Treatment can include hormone therapy, drugs designed to strengthen the muscle tone of the urethral sphincter, collagen injections, or surgery. Recently, we have reported that injection of muscle progenitor cells into damaged urethral sphincters can restore normal function in dogs. The purpose of this project is to extend those observations and examine the usefulness of cultured muscle cells for the restoration of function of the urethral sphincter in dogs with naturally occurring urinary incontinence. Briefly, a small muscle biopsy will be obtained from the affected dog, and muscle progenitor cells isolated and grown in culture. After expansion, the cells will be injected into the urethral muscle layer and the dogs allowed to recover. The effects of the procedure will be determined by owner reported continence scoring and by direct measurements of sphincter function. The animals will be followed for a period of 24 months to determine the long term effects of the procedure.

Congestive heart failure (CHF) causes difficulty breathing because of fluid accumulation in the lungs. It is an important clinical problem because it is common and has potential to cause suffering when breathing comfortably becomes impossible. Mitral valve regurgitation and dilated cardiomyopathy are common causes of CHF in dogs, which can develop as these conditions progress in severity. Because there currently is no cure for these heart diseases, the treatment focus has been on prolongation of the time to CHF development, relieving signs of fluid retention when CHF occurs, and supporting the function of a failing heart. Diuretic medications (����������������water pills���������������) cause increased urination after removal of fluid from the lungs and are the most effective treatment for CHF. Most dogs respond to diuretics initially, but over time, progression of heart disease and maladaptive mechanisms result in less urine production and return of CHF signs with potential for suffering, death, or euthanasia. Loss of responsiveness to diuretic medications may be multifactorial, and so identification of the underlying reason could guide medical interventions to restore urine production in response to diuretics and improve quality of life for these dogs. We have identified several candidate blood and urine variables that correlate with urine production in dogs and therefore may be useful to indicate responsiveness to diuretic medications. The purpose of this study is to evaluate these variables in dogs with progressive stages of naturally occurring heart disease, with intention to identify dogs that are poorly responsive to diuretic medications and determine underlying causes.

There is a critical need for safe and effective analgesics to treat the pain associated with the most common disease of cats - degenerative joint disease (DJD). There is a particular need in populations where non-steroidal anti-inflammatory drugs are contraindicated, such as cats with chronic kidney disease (CKD). One of the biggest hurdles in the development of analgesics for long-term (chronic) pain in cats has been the difficulty in measuring this pain. Several avenues of evidence suggest that DJD in cats is due to an altered state of immune function, leading to increased inflammatory mediators. We believe cytokine/chemokine profiles can be used as a measure of degenerative joint disease (DJD)-associated pain, and further, that cytokine/chemokine profiles will inform of novel targets for development of effective, safe analgesics in cats. In this study, we propose to evaluate cytokine/chemokine profiles in cohorts of cats from which we have detailed clinical information, allowing us to determine the relationship between cytokines/chemokines and DJD-associated pain, and the differences in inflammatory burden when CKD is also present. We will evaluate normal cats, cats with DJD and cats with DJD and concurrent CKD. Our pilot data indicates we will successfully identify differences, and results of this study may lead to a biomarker (or biomarker profile) measure of chronic pain associated with DJD and the combination of DJD and CKD. Additionally, evaluation of biomarker profile differences between groups might lead to discovery of the mechanisms of pain in these cats, or even the mechanisms underlying these chronic diseases.