Presenting Author Mentor Title 1 Andrea Wolf Beilman Safety and Efficacy of Novel Formulations of a Resuscitation Fluid for Hemorrhagic Shock 2 Beth Lusczek

The field of oncolytic adenovirus (OAd) therapy has undergone much growth inrecent years as improvements in vector design have resulted in augmented selectivity andmore potent anti-cancer effects. Before planning a clinical trial, the newly developedOAd must be tested in pre-clinical animal models to access efficacy and toxicity.Currently existing animal models provide very limited information regarding vectorperformance in humans. Mouse models are insufficient because human Ad does notreplicate in murine tissues, thereby barring the possibility to analyze OAd biodistributionand toxicity. Non-human apes have problems of availability and cost. Cotton rats andSyrian hamsters permit OAd replication, but are unsuitable for certain tropism-modifiedvectors since rodent cells lack CD46 and desmoglein2, the primary receptors recentlyidentified for the B-group Ad infection.In this study, we explored a pig as a model to study performance of OAds,including those with Ad3-retargeted tropism. First, we demonstrated the ability of swinecell lines to support replication of Ad5 and Ad5/Ad3. Secondly, we analyzed binding,gene transfer, killing and replication ability of Ads in various non-human cell lines(hamster, murine, rat, bovine, canine and pig) and showed that among tested cell linesonly porcine cells were supportive for binding and active replication of Ad3-retargetedvectors. Of note, Ad5/Ad3-chimeric virus showed stronger replication compared to Ad5in both human and porcine cell lines. Next, we intravenously injected pigs with a highdose (3 x 1012 vp) of each OAd (Ad5 and Ad5/Ad3). Liver biopsies were obtained ondays 1, 2, and 4, and necropsy was performed on day 7. The complete necropsy showedno difference compared to the control. There were no abnormalities in blood parametersand serum chemistry. The blood count including the number of platelets was not affected.AST and LDH, as an indication of both liver and lung damage, showed mild elevation onday 4 and returned to normal at day 7. Liver specific ALT was normal in all pigs and theliver damage marker SDH showed mild elevation at day 7 only in the Ad5 group.Remarkably, viral DNA and replication-dependent luciferase expression were observedin lungs and spleen of Ad-injected pigs, while none was detected in liver samplesthroughout the study. The detailed histology exam of tissue sections revealed signs ofAd-induced interstitial pneumonia in lungs, but no changes in liver. There were nounusual necropsy observations or microscopic lesions in the liver samples. These datacontradict the commonly known observations in mouse models, where subsequent tointravenous injections Ad generally targets the liver. In summary, these studies provide a rationale for the use of pigs to studyperformance of OAds including the vectors with altered tropism. The studies alsoquestion the reliability of a mouse model for analyses of OAd biodistribution and livertoxicity. Therefore, we are currently investigating OAd biodistribution, liver uptake andimmune response in pigs at earlier time points after systemic OAd administration (1, 6,24 hours). We have also started developing a swine model of pancreatic cancer byproducing a genetically-engineered pig with inducible human KRAS and TP53 mutations.We hope this pig model will foster clinical translation of oncolytic viruses. #36Glucose-responsive oxygen consumption rate in islets from chronic pancreatitis patients is size dependent:novel islet quality assessment through bioenergetic phenotyping Joshua J Wilhelm, Zachary A Swanson, Melina D Bellin, Bernhard J Hering Schulze Diabetes Institute, Department of Surgery, University of Minnesota Background: Higher basal oxygen consumption rate (OCR) has been shown to be predictive of outcomes inchronic pancreatitis (CP) patients undergoing total pancreatectomy with islet autotransplantation (TPIAT).However, the assay used required a large islet sample, was not specific to beta cells, and represents only a staticmetabolic state. These islet preparations often contain acinar, which also contributes to OCR and DNA. Thus,we sought to develop a novel assay to study fewer islets, account for impure tissue, and be specific to beta-cellfunction. We have adapted the Seahorse extracellular flux (XF) technology to dynamically measure bioenergeticchanges in both OCR and ECAR (extracellular acidification rate) during glucose stimulation, as well as during aseries of drug injections to assess mitochondrial (mito) quality and energy utilization. This “mito stress test” haspreviously been used to assess the other tissues like hepatocytes, adipocytes, and immune cells wherebioenergetic phenotype (use of oxidative vs glycolytic pathways for ATP production) is closely related to viability,maturation, and/or differentiation state. Methods: Our assay has evolved over time, using a 500-1000 IEQ aliquot of unpurified tissue taken just prior toinfusion into TPIAT patients, plated in 24-well plates with assay media (5.6mM glucose). Most recently, isletsfrom 14 consecutive TPIAT patients were handpicked, and large and small pure islets were plated separately, aswere embedded islets (if present), and acinar tissue. After equilibration in assay media, OCR & ECAR aredetermined every 8 minutes, with an initial baseline determined prior to sequential injection of glucose(final=20.6mM), oligomycin (50uM), FCCP (5uM), and rotenone/antimycin A (10uM each) to determine glucoseresponsiveness and mito quality. After the assay, wells were stained with dithizone and each was digitallyimaged for islet size measurements. Islet size index (ISI) was calculated for each well as a ratio of IEQ:IPN. Wellscontaining islets with similar average sizes were grouped and glucose responsiveness and mito qualityparameters analyzed. Results: With glucose stimulation, wells with the smallest islets (average ISI 0-1) showed an increase in OCR of61.32 +/62.09%, compared to wells with ISI >5 (7.93 +/16.59%; p<0.001), as did wells with ISI 1-2 (47.65 +/-55.56; p<0.001). Any islet wells with an average ISI 3-5 never reached greater than a 14.5% OCR increase(P>0.05). Acinar tissue and embedded islets exhibited a slight decrease in OCR (Table 1). Small islets (ISI 0-1 andISI 1-2) also exhibited a higher spare capacity (98.84 +/84.67% and 82.03 +/45.46%, respectively) compared tolarge islets (ISI >5; 16.11 +/17.02%). Interestingly, differences in various metabolic parameters were notedbetween patients with known insulin resistance and diabetes, suggesting a functional change in the metabolismof islets between normal and diabetic or perhaps even pre-diabetic patients. Conclusions: The ability of tissue to increase OCR in response to glucose is islet-specific and may be animportant functional parameter corresponding to ability to reverse diabetes. Our results have distinguisheddifferent bioenergetic profiles of acinar, pure islet and embedded islet tissue. Smaller islets were especiallydistinct, including ability to increase OCR and ECAR in response to glucose, suggesting a possible advantage ofsmaller islets as previously reported in this patient population. We hypothesize that mito quality andbioenergetic phenotype hold potential to detect novel and more subtle differences in islet quality that will helpfurther our interpretation of differences in islet quality that result from progression of disease and onset of type3c diabetesand may also help better refine our predictions of clinical metabolic outcomes. Figure 1. Changes in oxygen consumption rate (OCR) for one representative non-diabetic TPIAT patient islets (33 yo,M, Pancreas divisum, 15 yr disease, HgA1c 5.6%, Fasting/stimulated c-peptide 1.2/3.2) Table 1. Bioenergetic profile of pure islets grouped by size index, embedded islets, and acinar tissue from 14 TPIATpatients 0-1 1-2 2-3 3-4 4-5 5+ Glucose Stimulation(ΔOCR %)61.32 +/-62.09* t47.65 +/-55.56* t22.18 +/-32.89*9.04 +/-23.9614.51 +/-18.337.93 +/-16.59 -7.38 +/8.16 -4.91 +/-12.41 Spare Capacity (%)98.84 +/-84.67t82.03 +/-45.46t55.24 +/-54.8424.89 +/-16.1134.86 +/-30.3416.11 +/-17.02*21.13 +/-27.31*58.40 +/-69.86 Basal Operating % ofMax Respiration55.42 +/-16.16t53.90 +/-13.79t62.91 +/-25.15t78.01 +/-11.3973.65 +/-16.3084.98 +/-15.51*83.59 +/-17.37*64.98 +/-25.76 ECAR Increase (%) 131.56 +/-193.52*102.92 +/-177.34*49.20 +/-55.0285.80 +/-95.1060.44 +/-71.4759.86 +/-82.4742.33 +/-66.14-3.21 +/-100.38EmbeddedIslets AcinarPure Islets *p<0.05 compared to Acinar categoryt p<0.05 compared to 5+ category