T32 Training Grant Research Articles

Optimization of a Polycaprolactone (PCL) Scaffold for Islet and Cellular Transplantation

Introduction Islet transplantation is a proven effective therapy for patients with type 1 diabetes; however, donor shortage limits this therapy to very few patients with brittle diabetes. Stem cell therapies for beta cell replacement are quickly moving toward clinical application, but the optimal transplant site and delivery vehicle remains unknown. Previous studies have shown that three dimensional support and structure improves islet survival and function. To this end, we have optimized a polycaprolactone (PCL) polymer scaffold as a biocompatible delivery system for beta cell replacement. In these experiments, we used mouse islets as a proof-of-concept system, prior to application of the device for stem cell delivery. Methods PCL scaffolds of varying thickness were evaluated for maximal loading capacity in vitro with 150 mm PLGA fluorescent microspheres and mouse islets. Insulin secretion was tested in vitro using B6 mouse islets in a perifusion chamber (BioRep, Inc., Miami Lakes, FL). For in vivo testing of islet function and survival within the PCL scaffold, 200 syngeneic islets were transplanted to the epididymal fat pad of STZ-induced diabetic B6 mice, either within a PCL scaffold or directly to the epididymal fat pad. Blood glucose was measured in transplant recipient mice three times weekly for 30 days, at which point islet grafts were explanted and mice were monitored for reversion to hyperglycemia. Normal blood glucose was defined as 250mg/dL for all in vivo experiments. Results/Discussion Thicker PCL scaffolds fabricated to 1.2mm thickness showed higher loading capacity compared to 1mm thick devices (409 versus 305 microspheres, n=6 per device; Figure 1A). slets showed a similar loading pattern, confirming ample carrying capacity of the PCL scaffold. Microspheres distributed within the three-dimensional porosity of the PCL scaffold, as seen by confocal microscopy (Figure 1B). Mouse islets show no limitation or delay in insulin secretion as a result of loading within the PCL scaffold on perifusion testing (data not shown). When PCL scaffolds were loaded with syngeneic islets and transplanted to the epididymal fat pad B6 mice, 8 out of 9 (89%) mice returned to normoglycemia, while 3 out of 9 (33%) control mice receiving free islets to the epididymal fat pad returned to normoglycemia (p=0.041; Figure 2). Upon explant of islet grafts from normoglycemic mice at day 30, all mice subsequently returned to hyperglycemia, confirming function of the transplanted islets. Conclusions Our optimized PCL scaffold supports islet survival and function in a syngeneic B6 mouse model, and may provide a delivery system for future stem cell-based beta cell replacement. Research reported in this publication was supported in part by an NIAID T32 training grant from the National Institutes of Health under an award to the University of California, San Francisco (T32AI125222).

Prevascularization of the Subcutaneous Space Improves Survival of Transplanted Mouse Islets

Introduction Beta cell replacement for the treatment of Type I diabetes has previously been limited by donor availability, invasive surgical techniques, and islet survival post-transplant. Transplantation into the subcutaneous space is ideal for graft accessibility and explant if necessary; however, prior investigations have shown that in comparison to other gold-standard sites (i.e., the kidney capsule), this location lacks the rich blood supply required to meet the high metabolic demands of islets. The aim of this study was to identify and demonstrate the efficacy of a novel prevascularization strategy to improve free islet graft survival in the subcutaneous space. Methods To investigate the ideal prevascularization device, four different materials were implanted into the subcutaneous space of B6 albino mice: polypropylene (PP) mesh, smooth PP, texturized polycaprolactone (PCL), and smooth PCL. Vascularity was assessed using indocyanine green fluorescence measurement at 1-week time points up to 6 weeks post implantation. At selected time points, mice were euthanized and specimens were examined using immunofluorescence histology. B6 mice that had been prevascularized for 2 weeks with textured PCL discs were made diabetic with intraperitoneal streptozosin injection (200 mg/kg). The prevascularization discs were removed, and the mice were then transplanted with 200, 400, 600, and 800 syngeneic mouse islets into the disc capsule. Blood glucose was measured for three weeks to monitor for diabetes cure. Intraperitoneal glucose tolerance test (IPGTT) was performed on transplant recipients to compare their function to diabetic and normal controls. Results When normalized to baseline fluorescence, the peak vascularity as measured by ICG fluorescence was determined for texturized PCL at the 2-week time point (Fig. 1). Histological analysis at 2 weeks (Fig. 2a) and 6 weeks (Fig. 2b) post-implantation demonstrated vascular ingrowth (vWF) and minimal foreign body reaction (macrophages, or F4/80). Diabetes reversal was not observed for 200- or 400-islet transplant recipients. 100% diabetes cure was seen with 800-islet transplants, and partial cure (50%) was seen with 600-islet transplants (Fig. 3). When subjected to IPGTT at 2 weeks post transplant, the 600-islet group showed similar blood glucose kinetics to non-diabetic control in cured mice and impaired glucose tolerance in partially cured and failed mice (Fig. 4).Conclusion Texturized PCL induces improved vascularity in the subcutaneous space of mice while inciting minimal foreign body reaction. Free islets transplanted in the subcutaneous space of mice have improved survival in the prevascularized implant capsule, and allow for comparable function to non-diabetic controls. This strategy may represent a safe and effective translational pathway for the application of subcutaneous islet transplant in human subjects in the near future. Research reported in this publication was supported in part by an NIAID T32 training grant from the National Institutes of Health under an award to the University of California, San Francisco (T32AI125222).

PEG‐peptide Scavenger Receptor Inhibitors for Non‐viral Gene

The rapid capture and degradation of viral and non‐viral gene delivery nanoparticles by scavenger receptors (SR) on Kupffer cells and fenestrated endothelial cells in the liver results in a decreased half life of all nanoparticles in an in vivo system, as well as a lower percentage of the dose available for delivery to the target organ. Current attempts to achieve SR inhibition involves co‐administering high molecular weight, polyanionic inhibitors. Polyinosinic acid (Poly‐I), the most widely used and potent inhibitor, competes for SR binding and successfully inhibits the uptake and metabolism of viral gene delivery nanoparticles in the liver. However, Poly‐I also activates the immune system, resulting in toxicity in mice, making Poly‐I clinically unacceptable.We have discovered peptide based SR inhibitors that block SR uptake of DNA nanoparticles and improves their metabolic half‐life by forming protein nanoparticles in the blood.Radio‐iodinated PEG‐peptides were used to study the pharmacokinetics and biodistribution to understand the structural properties which influence transfection competency and potency. We hypothesized that the in vivo potency and activity is influenced by the stability of protein nanoparticles formed in the blood. Results show that peptides featuring both hydrophobic and positively charged residues are able to potently inhibit scavenger receptor uptake of non viral gene delivery systems.Support or Funding InformationThis work was supported by the Iowa Biosciences Academy and University of Iowa Pharmacological Sciences T32 training grant. The Iowa Biosciences Academy is supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R25GM058939, the University of Iowa (UI) Office of the Vice President for Research and the UI Chief Diversity Office, the Pharmacological Sciences Training grant T32 GM067795 and NIH grant R01 GM087653.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cardiolipin Remodeling by ALCAT1 Links Oxidative Stress and Mitochondrial Dysfunction to Cellular Senescence

IntroductionCardiolipin is a major mitochondrial phospholipid that is required for ATP production, autophagy/mitophagy, and mitochondrial biogenesis. ALCAT1 promotes cardiolipin peroxidation by catalyzing pathological remodeling of cardiolipin with aberrant fatty acyl chains commonly found in aging, rendering cardiolipin highly sensitive to lipid oxidation by reactive oxygen species (ROS). Oxidized cardiolipin not only causes mitochondrial dysfunction, but also becomes a major source of ROS as lipid peroxides, further exacerbating oxidative stress. Recent development in the field has demonstrated a causative role of cellular senescence in aging and aging related diseases. However, the molecular mechanisms that promote cellular senescence in aging and aging‐related diseases remains elusive.ObjectiveTo determine whether oxidative stress and mitochondrial dysfunction induced by ALCAT1 link aberrant cardiolipin remodeling to cellular senescenceMethodsAdult C57BL/6 wild‐type and ALCAT1 Knockout (KO) mice were used for the studies. Primary vascular smooth muscle cells (VSMC) were isolated and cultured using standard protocols. Young cells were at passage 3 (P3) and old cells were at passage 11 (P11). Senescence associated beta galactosidase (SAβ‐Gal) and aconitase activity were used as biomarkers for cellular senescence and oxidative stress respectively. Cellular bioenergetics were assessed using a seahorse XF24 following treatment with or without xanthine/xanthine oxidase, a combination that generates superoxide. Advanced confocal live cell imaging was used to determine and monitor changes in mitochondrial dynamics, mitophagy, mtDNA, and mitochondrial membrane potential following treatment with hydrogen peroxide. TMRM, mitoTracker Green FM and mitoTracker Red, LysoTracker Green, Hoechst, and PicoGreen were used to label mitochondrial membrane potential, mitochondria, lysosome, nucleus, and mtDNA respectively. Lipid peroxidation products were quantified calorimetrically with a malondialdehyde (MDA) assay kit.ResultsWild‐type VSMC at P11 demonstrated a pronounced SAβ‐Gal staining, significant loss of aconitase activity and mitophagy function, decreased mtDNA, impaired mitochondrial function, and a significant increase in lipid peroxidation products.ConclusionALCAT1 KO protected against onset of cellular senescence by reducing sensitivity to lipid oxidation, which preserved mitochondrial function, mitophagy, and mtDNA. Taken together, our work puts forward a possible potential role of ALCAT1 in promoting cellular senescence.Support or Funding InformationNIH (2R01DK076685‐06A1), American Diabetes Association (1‐14‐BS‐185), the Barth Syndrome Foundation, and support from a NIH T32 training grant through the Barshop Institute's Biology of Aging T32 AG021890 (which has been supporting K.S.M since joining the Shi Lab in May 2016).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Increasing or decreasing the excitability of V2a neurons activates accessory respiratory muscles

Accessory respiratory muscles (ARMs) can be recruited to maintain respiration when the diaphragm is impaired or weakened, such as patients with Amyotrophic Lateral Sclerosis (ALS) or spinal cord injury. We chronically record mouse diaphragm electromyography (EMG), ARM EMG, and whole body plethysmography to assess the effect of activating and silencing V2a neurons with DREADDs on respiratory muscle activity and ventilation. Increasing the excitability of glutamatergic V2a neurons in the brainstem and/or the spinal cord recruits ARMs and enhances ventilation. Interestingly, silencing V2a neurons also activates ARMs. We hypothesize that V2a neurons participate in both excitatory and inhibitory pathways controlling activation of ARMs for breathing.Support or Funding InformationUniversity of Cincinnati T32 Training Grant 2016–2017This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Life and Times of a Carboxysome: Tracking a Single Protein Complex Over Multiple Generations

Cyanobacteria are abundant primary producers in the biosphere that require only CO2 and light as carbon and energy sources to grow. Cyanobacteria contain an icosahedral proteinaceous nanostructure known as the carboxysome, which functions by increasing the concentration of CO2 around the carbon‐fixing enzyme Rubisco, thereby maximizing carbon fixation rates. While this core functionality of these macromolecular protein complexes is understood, dynamic activities of individual carboxysomes are largely unknown due to a lack of insight at the single molecule level. Since the average cyanobacterium contains 3–4 carboxysomes per cell, it is easy to obtain an average carbon fixation rate per cell, but no way to determine the precise activities of each individual carboxysome through population‐level experiments. As carboxysomes last for multiple generations, it is likely that their carbon‐fixing ability decreases with time, which would have a substantial impact on carbon fixation, and subsequently, overall cell fitness and physiology.To understand precisely how a single carboxysome affects cell growth and fitness, we have created a strain of cyanobacteria where carboxysome expression is controlled via an inducible system, thus enabling us to reduce the number of carboxysomes per cell to one. Since only a single carboxysome is present in each cell, all of carbon fixation and growth can be accredited to that individual carboxysome. By tracking single cells with single carboxysomes, it is possible to estimate carboxysome functionality over multiple generations by measuring cell growth rates. The results from this experiment can be used to quantify the in vivo functional lifetime and stability of individual carboxysomes. In addition to understanding how carboxysome age affects function, real‐time visualization of carboxysomes in single cells will allow for analysis of their spatiotemporal dynamics and potentially the identification of a novel carboxysome degradation pathway.Understanding these dynamic properties of carboxysomes is critical for an accurate model of cyanobacterial growth and physiology. It is also valuable information for industrial and biotechnological applications that involve optimizing growth and carbon‐fixation rates, and is critically important for engineering more stable and active carboxysomes. This is the first example of tracking the enzymatic activity of a single protein complex in vivo over its entire lifetime and across multiple generations. Basic properties discovered here are likely to apply to diverse bacterial microcompartments, and possibly to entirely unrelated large macromolecular complexes.Support or Funding InformationFunding for Nicholas Hill is provided by the NIH T32 Training Grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rotimi Mesubi: Time Well Spent.

Olurotimi (Rotimi) Mesubi is currently a Clinical Cardiac Electrophysiology Fellow at Johns Hopkins University School of Medicine where he also completed his postdoctoral fellowship in Cardiovascular Disease. Originally from Nigeria, where he earned both a Bachelor of Medicine and Bachelor of Surgery at the University of Ilorin, Rotimi continued gaining an impressive amount of experience in the United States. He earned his Master of Public Health at Harvard University and residency and internships at MetroHealth Medical Center (Case Western Reserve University). It is his research as a Cardiovascular Disease Fellow at the University of Iowa—initiated under his mentor, Dr. Mark Anderson, before they moved to Johns Hopkins University—that earned Rotimi the American Heart Association BCVS (Basic Cardiovascular Sciences) Melvin L. Marcus Young Investigator Award. Rotimi Mesubi As the first of 4 children, I was fortunate to grow up in a nurturing environment. My dad, a college chemistry professor, and mom, a high school chemistry teacher, encouraged my siblings and me to be curious about the world and pursue our dreams. The seeds of my interest in science were sown in elementary school, when I would spend after school hours in my dad’s chemistry lab and interact with his graduate students. Later, I was attracted to medical school, which I attended in Nigeria. One of the reasons I moved to the United States was to pursue postgraduate training; I always dreamed of being at the cutting edge of scientific discovery at the best institutions in the world. I felt then, and still do today, that the US is a unique place that offers such opportunities to someone like me from a foreign country. I began my internship and residency training in internal medicine and pediatrics at MetroHealth Medical Center (Case Western Reserve University) with a plan to become an infectious disease—HIV—specialist. …

Abstract 2502: Liquid diet introduction of tracers into mice for stable isotope-resolved metabolomics (SIRM) investigations

Abstract Tracer-based mapping of metabolic networks in vivo is a powerful approach for revealing metabolic reprogramming in human cancer. However, current in vivo labeling techniques for model animals face important challenges including insufficient depth of pathway coverage (e.g. inability to detect labeled nucleotides, proteins, and lipids) and stress-related artifacts. Here, we report stress-free administration of 13C6-glucose via liquid diet into mice. 13C enrichment was observed in metabolites of glycolysis, the Krebs cycle, the pentose phosphate pathway, nucleobases, UDP-sugars, as well as macromolecules glycogen, lipids, and proteins from major organs in NSG mice. We have applied the liquid diet method to map the glucose metabolic networks in NSCLC tumors in a patient-derived xenograft (PDX) model. We observed a high enrichment in the metabolites of glycolysis, Krebs cycle, and PPP as well as de novo synthesized nucleotides and amino acids by IC-UHR-FTMS analysis. Lung PDX displayed unexpected metabolic complexity, such as the use of pyruvate to fuel anaplerosis as well as gluconeogenesis. We also found high 13C enrichment in both tumor and plasma glutamine, which implies that glutamine in the PDX tumors largely came from other organs via the blood rather than being synthesized in situ. Our data showed that liquid diet is an effective and noninvasive means for comprehensive analysis of glucose-associated metabolic networks in human tumor xenografts, which can also be extended to SIRM studies with other fuel sources. Acknowledgements: This work was supported in part by grants: 1R01ES022191-01 (to TWMF and RMH), 1P01CA163223-01A1 (to ANL and TWMF), and 1U24DK097215-01A1 (to RMH, TWMF, and ANL) R.C. Sun was supported by a T32 training grant to M. Vore (5T32ES007266-25) Citation Format: Ramon C. Sun, Marc O. Warmoes, Ye Yang, Pan Deng, Qiushi Sun, Andrew N. Lane, Richard M. Higashi, Teresa W-M Fan. Liquid diet introduction of tracers into mice for stable isotope-resolved metabolomics (SIRM) investigations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2502. doi:10.1158/1538-7445.AM2017-2502

Human Osteopontin Isoforms Differentially Promote Post‐Ischemic Neo‐vascularization via Divergent Effects on Macrophages in vivo and in vitro

Coronary and peripheral artery diseases result in vessel occlusion and ischemia, initiating neovascularization to restore blood flow and preserve function. We previously demonstrated that osteopontin (OPN), a secreted cytokine, is critical to ischemia‐induced neovascularization. Unlike rodents, humans express 3 OPN isoforms (a, b, and c); however, the roles of these isoforms in neovascularization and cell migration remain undefined. To investigate if human OPN isoforms have differential effects on functional perfusion recovery in vivo, OPN−/− mice underwent hind limb ischemia (HLI) and received 1.5×106 lentivirus particles expressing OPNa, OPNb or OPNc at the time of surgery by intramuscular (IM) injection of the adductor muscle using a microsyringe. By utilizing this experimental approach, the only OPN in the system is what we add back, thus allowing us to determine how each human OPN isoform functions to influence neovascularization in vivo. OPNa and OPNc significantly improved limb perfusion, as measured by laser Doppler perfusion imaging, by 30.4%±0.8 and 70.9%±6.3, respectively (d14; p<0.001 vs. LVGFP, p<0.05 vs. hOPNb), and we confirmed by ELISA that these effects were not due to different protein expression levels (n=6, p=ns). OPNa and OPNc rescued limb function compared to control and OPNb‐treated animals (61.1%±8.2; 76.2%±9.7; p<0.05), as assessed by voluntary running wheel use. Additionally, OPNa and OPNc‐treated animals exhibit significant increases in arteriogenesis and macrophage infiltration in vivo (n=4, p<0.05 vs. GFP, OPNb). Therefore, we further hypothesized that divergent effects of OPN isoforms on arteriogenesis are due to differential effects on macrophage migration, survival, and/or polarization. Although OPN isoforms had no differential effects on macrophage polarization in vitro, OPN isoforms did differentially promote microphage migration in vitro, assessed using a modified Boyden chamber assay. While OPNa and OPNc both increased macrophage migration in vitro, OPNc was a more potent migratory stimulus than OPNa and OPNb in vitro (n=4, p<0.001 vs. no trx, OPNa, OPNb, and MCP‐1), consistent with what was observed in vivo. To determine if OPN isoforms have divergent effects on macrophage survival in vitro, OPN−/− macrophages were serum‐deprived to induce apoptosis and treated with recombinant OPNa, OPNb or OPNc proteins in vitro. Survival was assessed by FACS using FITC‐annexin V and propidium iodide staining. All isoforms significantly increased macrophage survival compared to no treatment, with OPNc increasing survival significantly greater than OPNa and OPNb (n=4, p<0.05 vs. OPNb, p<0.001 vs. no trx, and OPNa). In conclusion, human OPN isoforms exhert divergent effects on neovascularization through differential effects on arteriogenesis and macrophage infiltration in vivo and differential direct effects on Macrophage migration and survival, but not polarization, in vitro. Taken together, these data support that human OPN isoforms may represent novel therapeutic targets to improve neovascualrization and preserve tissue function in patients with obstructive artery diseases.Support or Funding Information1. T32 Training Grant #T32HL007745, NIH/NHLBI, W. Robert Taylor (PI), 01/03/2016 to present: Research Training in Academic Cardiology (Role: Post‐doctoral trainee) The goal of this training grant is to support for the highly successful postdoctoral training program in cardiovascular research in the Division of Cardiology at Emory University School of Medicine. The training program is structured around a mentor‐based environment supplemented with an extensive array of didactic educational opportunities for multidisciplinary training in basic and clinical vascular biology. Successful trainees will be well‐equipped to initiate a research career in academic cardiology. 2. K99/R00 HL119567, NIH/NHLBI, Alicia N.Lyle (PI), 04/2014–06/2019: The Role of Human Osteopontin Isoforms in Collateral Formation This project uses a novel translational therapeutic strategy to determine how the different human osteopontin (OPN) isoforms influence collateral formation.

Loss of Myosin Vb in Intestinal Enterocytes Results in Aberrant Expression of Key Apical Transporters in vitro

BackgroundInactivating mutations in myosin Vb (MYO5B) result in Microvillus Inclusion Disease (MVID). MVID is a rare form of congenital diarrhea characterized by morphological intestinal enterocyte abnormalities and aberrations in the localization of brush border proteins and enzymes. Despite recent attention on MVID, the origin of the diarrheal disorder remains unclear. Our lab has generated a mouse model of MVID with a germline deletion of MYO5B (MYO5B KO). These mice rapidly develop diarrhea, have failure to thrive and demonstrate numerous microvillus inclusions in the intestinal enterocytes.HypothesisWe hypothesize that loss of MYO5B results in the improper localization of key apical membrane components away from the brush border resulting in inadequate absorption of nutrients and water.MethodsThe 3D culture of intestinal epithelial organoids, known as enteroids, allows for the in vitro propagation of all of the intestinal cell types. Enteroids were used as a simplified model to study epithelial alterations that result from loss of MYO5B. Enteroids were generated from the duodenum of neonatal mice with a germline deletion of MYO5B (MYO5B KO) and heterozygous or wildtype littermates (control). Enteroids were differentiated for 3–5 days by withdrawal of Wnt3A and analyzed for the localization of apical transporters by immunostaining. Quantitative real time PCR was used to determine the relative gene expression of transporters in MYO5B KO and control derived enteroids.ResultsF‐actin staining using phalloidin conjugates showed a mature brush border in enteroids derived from neonatal control mice. MYO5B KO derived enteroids also showed apical F‐actin staining, but F‐actin and ezrin positive inclusions were observed within enterocytes. These inclusions closely resembled inclusions seen in the intestinal tissue of MYO5B KO mice. SGLT1 and DPPIV were expressed on the apical membrane of control enteroids. In contrast, MYO5B KO derived enteroids had diffuse, subapical SGLT1 and DPPIV expression. NHE3 immunofluorescence showed apical expression of NHE3 in control enteroids and decreased expression in MYO5B KO enteroids. qRT‐PCR data showed a significant increase in SGLT1 and NHE3 mRNA expression in MYO5B KO enteroids compared to control enteroids. This could indicate that SGLT1 and NHE3 mRNA may be up‐regulated in an attempt to compensate for degradation or improper localization. Enteroids generated from MYO5B KO mice also showed a significant increase in proliferation compared to control derived enteroids, consistent with an increase in proliferation observed in the intestinal crypts of MYO5B KO mice.ConclusionsCollectively these data suggest that enteroids from MYO5B KO mice maintain the aberrant enterocyte characteristics seen in vivo and thus will be a good model for elucidating the mechanism by which loss of MYO5B results in untreatable diarrhea. The enteroid model of loss of MYO5B demonstrates intestinal epithelial changes that suggest that the improper localization of apical transporters is responsible for the diarrheal pathology seen in patients with MVID.Support or Funding InformationNIDDK Grant RO1 DK048370 and T32 Training Grant 2T32CA106183‐11.

Mesodermal differentiation of human pluripotent stem cells increases Mitofusin‐2 mediated Mitophagy

Pluripotent stem cells rely on glycolysis for their energy needs and shift their metabolism towards mitochondrial oxidative phosphorylation during differentiation in order to adapt to the metabolic needs of mature differentiated cells. Oxidative phosphorylation generates reactive oxygen species and can thus lead to collateral protein oxidation and damage. Excessive mitochondrial damage leads to mitochondrial autophagy or mitophagy during which mitochondria are marked for degradation. A key regulator of mitophagy is the outer mitochondrial membrane protein Mitofusin‐2 (Mfn2) which either directs mitochondria towards fusion with other mitochondria to optimize mitochondrial function or towards mitophagy. We hypothesized that differentiation of pluripotent stem cells would necessitate increased mitophagy to contain the oxidative damage due to enhanced oxidative phosphorylation.Human induced pluripotent stem cells (iPSCs) of human embryonic stem cells (hESCs) were treated with Bone Morphogenetic Protein 4 (BMP4) to induce differentiation to the mesodermal lineage. These cells exhibited an expected increase in the mesodermal transcription factor Brachyury. Cells were stained with MitoTracker to visualize mitochondria and assess mitochondrial network formation. We observed an increase in mitochondrial fusion during mesodermal differentiation. There was also a two‐fold increase in Mitofusin 2 protein levels with BMP4 treatment. The ratiometric fluorescent sensor mitoKeima which detects the pH of mitochondria and assesses mitophagy indicated significantly higher basal mitophagy after three days of BMP4 induced differentiation with an increase in the relative mitophagy index from 2.7 to 9.7 (p<0.01) within three days.These findings suggest that mitophagy and mitochondrial networking are critical Mitofusin‐2 mediated adaptive processes required for the metabolic reprogramming of differentiating stem cells.Support or Funding InformationNIH T32 Training Grant

The Mechanisms of Gut‐dependent Protection against Cardiac Injury

The gut is the main location for immunological regulation and is influenced by the flora that reside there. This flora, also known as the gut microbiome, is necessary for maintaining immune health and disruptions in the microbiome result in systemic, metabolic and inflammatory disorders including obesity, diabetes, and cardiovascular disease. Therefore a possible approach to treat these disorders involves modulating the gut microbiome through the administration of probiotics. Probiotics are defined by the World Health Organization as, “live microorganisms that confer a health benefit to the host when consumed in adequate amounts.” Data from our laboratory indicate that a specific strain of probiotic, Bifidobacterium animalis sub‐species lactis 420 (B420), reduces the extent of cardiac injury following ischemia/reperfusion in a mouse model of myocardial infarction. This cardioprotection is attributed to a reduction in cardiac inflammation, however, it is unclear precisely how B420 mediates this effect. Here, we hypothesize that the mechanism of gut‐dependent cardioprotection observed in vivo is through epigenetic and post‐translational regulation of intestinal, epithelial and immune cell function. We further hypothesize that the initiation of this mechanism is not uniform throughout the GI tract, and may be regionally localized. To this end, we developed a system to track the probiotic in the gut to better understand where and how they interact with the gut microbiome. Indocyanine Green (ICG) is a near infrared(NIR) stable aqueous, sterile, fluorescent, and clinically effective agent. In this study we aim to develop an inexpensive and effective method to track B420 bacteria in the gut‐microbiome. ICG was cultured with B420 and analyzed with LagoX imaging software at NIR wavelengths. ICG did not inhibit growth of B420 culture, and was a suitable reagent for labeling B420 bacteria. The ICG‐B420 co‐culture was gavaged in a treatment group of C57BL/6 mice at 2mg/kg, while control mice received B420 only. The GI tract was imaged in vivo and ex vivo bioluminescence. Bacterial genomic DNA from C57BL/6 treated and untreated mice gut epithelia was quantified using B420 primers and quantitative PCR(qPCR), and verified localization of bacteria colonization observed during fluorescent imaging.Support or Funding InformationCardiovascular Training Grant in Biomedical Engineering NIH T32 training grant

Next‐generation nutritional screening

The biochemical identification of essential vitamins and minerals during the early 20th century led to major public health advances through treatment and prevention of deficiency diseases. Today, we face an epidemic of overnutrition. Chronic metabolic conditions such as obesity and diabetes are responsible for millions of deaths each year and constitute an enormous global health burden. Despite the major influence of diet on this epidemic, nutrition knowledge has not been applied as successfully as for vitamin and mineral deficiency diseases. In addition to vitamins and minerals, the foods we eat contain thousands of biologically active nutritional compounds that are managed at the cellular level by molecular networks which sense the cellular nutritional state and respond to maintain homeostasis. A key hub in these networks, and the focus of this project, is the endoplasmic reticulum (ER), a cellular organelle that is a broad sensor of nutritional and metabolic status. Impaired ER function is implicated in the development of obesity and diabetes, and improvement of ER function is a common therapeutic target that can potentially improve health in many tissues and disease states. Among its many functions, the ER is a major site for metabolism of lipids, a class of nutritional compounds that includes fatty acids and bile acids. Enzymes located on the ER membrane metabolize lipids from the diet, and also synthesize lipids de novo. While lipid synthesis at the ER has been widely studied, little is known about how the ER handles the many lipids from the diet, and how the response of the ER to dietary lipids impacts health. Without new tools to detect how cells sense and respond to nutritional compounds, we are unable to make continued progress in nutrition to reduce chronic metabolic conditions.The aim of this project is to create a novel cellular screening system to detect the effects of nutritional compounds. This project will focus on lipids, as they are rich in diversity and biology, and our lab is well‐equipped to study them. I will begin by curating a small collection of the predominant lipids in the human diet. These lipids are included in an existing collection of 688 lipids that our lab has established in collaboration with the Broad Institute for screening. In order to detect the cellular effects of lipids, I will focus on indicators of ER function. We have developed luminescent cellular reporters for real‐time monitoring of ER function in live cells. I will introduce these reporters into adipocytes, cells that manage systemic nutrient levels through storage and release of lipids. These reporter cells will be integrated into a microfluidic chip the size of a microscope slide. It will contain chambers, where we will seed cells expressing the ER reporters, and channels to flow culture medium and nutritional compounds to the cells. This system will enable high‐throughput screening of dietary lipids, continuous sampling of the culture medium for evaluation of the dynamic response of the ER, and co‐culture of related cell types, such as adipocytes and macrophages, to model cellular interactions.Support or Funding InformationWe acknowledge the generous support of the Sabri Ülker Food Research Foundation, Sabri Ülker Center, and Harvard Training Program in Nutritional Metabolism T32 training grant (4T32HD052961‐10).

Characteristics and Early Outcomes of Older Adults Undergoing Kidney Transplantation

BackgroundKidney transplantation (KT) has increased over the last decades due to longer life expectancy and a higher incidence of end stage renal disease (ESRD). Knowledge gaps remain regarding outcomes and infectious complications in older adults. Our primary aim was to describe the infectious complications, rejection episodes, graft and patient survival among older KT recipients (KTr) during the first 12-months post-transplant.MethodsRetrospective cohort study of all KT performed at Duke on older adults aged ≥65 years between 2011 and 2015. Demographic, clinical and outcome data were collected from the medical chart. Descriptive analyses were performed.ResultsNinety-one kidney-only transplants had been performed between 2011–2015. 60.4% were males, predominantly Caucasian (61.5%) and had a median age of 68 (range 65–75). 73.6% were on chronic dialysis prior to transplant. Frequent comorbidities included hypertension (93.4%), diabetes mellitus (47.3%), cardiovascular disease (49.5%) and obesity (36.3%). 92.3% of this KTr population encountered at least one infectious complication in the 12 months after transplant, while only 7.7% had a rejection episode. Delayed graft function occurred in 26.4%. 71.4% were admitted at least once in the 12 months after KT. The most frequent reason for admission were infections (58% of the admissions), followed by lab abnormalities (25.5%) and surgical complications (20.4%), while only 1.3% were due to a cardiovascular event. Mortality at one year was 9.9%, graft loss 3.3%.ConclusionInfectious complications occur at very high frequency in older KTr which in turn results in frequent hospital readmissions in the year following transplants. Despite these complications, the majority of older KTr were alive with a functioning graft one-year post-transplant. Cardiovascular events in this population are not as frequent as described in the literature. We postulate that a better understanding of immunosuppressive regimens and antimicrobial prophylaxis might be able to mitigate infectious complications.Disclosures M. Hemmersbach Miller, T32 Research Grant (NIH): I’m currently on a T32 Transplant ID Training Grant, Grant recipient and T32 Training Grant recipient - 4T32AI100851-04. K. Schmader, Merck: Investigator, Research grant. GlaxoSmithKline: Investigator, Research grant

A Novel CD34/ETO2/Ifngr gene Regulatory Axis Is Implicated in Poor-Prognosis Cases of t(8;21) AML

Abstract Background:Roughly 15% of AML cases are derived from the t(8;21) translocation, a molecular event that results in the production of the AML1-ETO (AE) fusion transcription factor. This aberrant protein induces a broad dysregulation of the transcriptome and causes expansion of leukemia stem cells (LSCs), which predisposes clones to an increased risk of second-hit mutations. In isolation, without the presence of type I mutations, the t(8;21) mark is considered to be one of favorable prognosis. However, a number of LSC characteristics have been shown to influence patient survival, including expression levels of the cell surface glycoprotein, CD34, and of the truncated isoform of AE, AML1-ETO9a (AE9a). Despite these findings, a unified model of how these factors interact to further the progression of t(8;21) AML has not been determined. Here, we use a combination of in vitro assays and bioinformatic analyses of patient datasets with the hopes of identifying an underlying connection between independent, prognostic factors. Results: We first performed gene knockdown studies with shRNA constructs (against AE) in a pKLO.1 backbone, which was transduced into the AML-model cell line Kasumi-1. Subsequent RT-PCR showed a large decrease in CD34 expression and, serving as a control, an increase in RASSF2, a cytoplasmic signaling protein normally repressed by AE. This led us to perform gene overexpression of cDNA encoding AE or AE9a into CD34-enriched cord blood HSCs. Interestingly, we were able to show that both AE and AE9a increased the expression of CD34, and AE9a was able to do so to a greater extent. This prompted us to analyze previous ChIP-seq data from Kasumi-1 cells, and we discovered high levels of AE binding to an upstream enhancer of CD34. While this site was free of any E-box binding motifs, we nonetheless observed binding peaks of the E-protein HEB, and further gene knock-down studies showed that HEB recruitment to this site depended on AE and, in turn, CD34 expression depended on HEB recruitment. Next, we used differential gene expression analyses to determine the CD34-coexpressed genes from a publically available patient microarray dataset (GSE14468, n=516). CD34-high patient samples showed an accompanying enrichment of JUP, KIT, CD133, HEB, E2A and ETO2, and a decrease in immune signaling molecules such as IFNGR1 and IFNGR2. ETO2 is the only ETO family member significantly expressed in HSCs and is an important corepressor of E-proteins HEB and E2A; it is thought to help repress the pro-differentiation and anti-proliferation effects of E-proteins. ETO2, being an ETO family member, is also able to oligomerize with other ETO family members, such as ETO, and thus may play a role in modifying the functions of the AE-transcriptional complex. Intriguingly, we have evidence that AE and AE9a repress ETO2, although AE9a does so to a lessened extent. We then performed Kaplan-Meier analyses on subgroups of non-CBF AML and t(8;21) AML cases. Unsurprisingly, we found that both groups experienced lower event-free survival when CD34 and ETO2 were high (p=.028 and p=.040), and the t(8;21) group when IFNGR was low (p=.014). Additionally, although CD34 expression was higher in t(8;21) patients compared to non-CBF AML (p=.0027), ETO2 expression was lower (p=.025). If both subgroups were further divided into CD34-high and CD34-low groups, ETO2 expression was, in both cases, higher in the CD34-high patients and lower in the CD34-low patients, suggesting a possible regulatory link between these two genes. Finally, we performed ETO2 gene knockdown experiments in CD34+ HSCs and observed an increase in IFNGR1 and IFNGR2 expression. Conclusions: Our findings suggest a clinically relevant association between CD34, ETO2, and IFNGR in both t(8;21) and non-CBF AML. We believe this regulatory axis is especially relevant in relapse likelihood and may be hijacked by LSCs to preserve their "stemness" and resistance to therapy. Our findings may suggest that AE simultaneously activates CD34 and represses ETO2, providing explanation for why full-length AE is slow to induce leukemia in mouse models and a relatively good prognostic mark in humans. Additionally, our findings suggest a loss of ETO2repression as a potential mechanism by which the AE9a isoform, and perhaps other second-hit mutations, converts LSCs to a more dangerous phenotype. Supported by NIH grants R01HL093195-01A1 to JZ and T32 training grant T32GM008306-26A1 Disclosures No relevant conflicts of interest to declare.

Abstract A057: CX3CR1 distinguishes three distinct migratory memory CD8 T cell subsets

Abstract CD8 T cells are crucial for the protection against tumors and intracellular pathogens via their ability to generate long-term memory. Memory T cells (TMem) are however not a homogenous population. Traditionally, blood-borne TMem have been sub-divided into central memory (TCM) and effector memory (TEM) cells based on different migration behaviors and cytotoxic potential. We recently discovered the existence of a novel subset of CD8 TMem that has unique migratory properties and a superior homeostatic self-renewal capacity. The hitherto unrecognized memory CD8 T cell subset is characterized by intermediate expression levels of CX3CR1, while classical TCM were CX3CR1−, and TEM were CX3CR1high. The fraction of CX3CR1int cells among antigen-experienced TMem was remarkably stable over the course of a year, while TEM gradually declined and TCM increased to be the largest subset by 10 months. Interestingly, CX3CR1int TMem had the highest proliferation rate of the three TMem subsets. While TCM and TEM were phenotypically stable over >10 weeks at steady state, CX3CR1int TMem not only self-renewed, but also gave rise to bonafide TCM. Consequently, CX3CR1int TMem contributed to the gradual increase in TCM. The newly identified CX3CR1int TMem displayed unique migratory properties. Unlike CX3CR1high TEM, CX3CR1int gradually acquired lymph node homing capability. Moreover, parabiosis experiments and analysis of recirculating memory cells in thoracic duct lymph revealed that CX3CR1int TMem, not TEM, are the predominant T cell subset surveying non-lymphoid peripheral tissues. Supported by a postdoctoral fellowship of the Cancer Research Institute Irvington Fellowship Program and a Rubicon fellowship (Netherlands Organization for Scientific Research, NWO) to CG, NIH T32 Training Grant in Hematology 5T32-HL07623-20 to EAM, NIH F31 grant CA171339 to SML, NIH T32 grant HL066987 to DA, the Ragon Institute of MGH, MIT and Harvard and NIH/NIAID RO1 AI069259, PO1 AI078897 and PO1 AI112521 to UHvA. Citation Format: Carmen Gerlach, E. Ashley Moseman, Scott M. Loughhead, David Alvarez, Anthonie J. Zwijnenburg, Lisette Waanders, Rohit Garg, Juan C. de la Torre, Ulrich H. von Andrian. CX3CR1 distinguishes three distinct migratory memory CD8 T cell subsets [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A057.

Could targeted, antibiotic-loaded gold nanoconstructs be a new magic bullet to fight infection?

Could targeted, antibiotic-loaded gold nanoconstructs be a new magic bullet to fight infection?

SU-G-201-10: Experimental Determination of Modified TG-43 Dosimetry Parameters for the Xoft Axxent® Electronic Brachytherapy Source

Purpose: The establishment of an air kerma rate standard at NIST for the Xoft Axxent® electronic brachytherapy source (Axxent® source) motivated the establishment of a modified TG-43 dosimetry formalism. This work measures the modified dosimetry parameters for the Axxent® source in the absence of a treatment applicator for implementation in Xoft's treatment planning system. Methods: The dose-rate conversion coefficient (DRCC), radial dose function (RDF) values, and polar anisotropy (PA) were measured using TLD-100 microcubes with NIST-calibrated sources. The DRCC and RDF measurements were performed in liquid water using an annulus of Virtual Water™ designed to align the TLDs at the height of the anode at fixed radii from the source. The PA was measured at several distances from the source in a PMMA phantom. MCNP-determined absorbed dose energy dependence correction factors were used to convert from dose to TLD to dose to liquid water for the DRCC, RDF, and PA measurements. The intrinsic energy dependence correction factor from the work of Pike was used. The AKR was determined using a NIST-calibrated HDR1000 Plus well-type ionization chamber. Results: The DRCC was determined to be 8.6 (cGy/hr)/(µGy/min). The radial dose values were determined to be 1.00 (1cm), 0.60 (2cm), 0.42 (3cm), and 0.32 (4cm), with agreement ranging from (5.7% to 10.9%) from the work of Hiatt et al. 2015 and agreement from (2.8% to 6.8%) with internal MCNP simulations. Conclusion: This work presents a complete dataset of modified TG-43 dosimetry parameters for the Axxent® source in the absence of an applicator. Prior to this study a DRCC had not been measured for the Axxent® source. This data will be used for calculating dose distributions for patients receiving treatment with the Axxent® source in Xoft's breast balloon and vaginal applicators, and for intraoperative radiotherapy. Sources and partial funding for this work were provided by Xoft Inc. (a subsidiary of iCAD). This work was also supported by the Radiological Sciences T32 Training Grant through the University of Wisconsin-Madison Medical Physics department (5T32CA009206-37).

Expansion of Regulatory T Cells Promotes the Clearance of Apoptotic Cells by Macrophages in Murine Atherosclerotic Plaques

A key process in inflammation resolution is efferocytosis, the clearance of apoptotic cells (AC) by phagocytes. Defective efferocytosis has been implicated in diseases characterized by chronic, nonresolving inflammation, including atherosclerosis. In atherosclerotic plaques, the accumulation of uncleared apoptotic cells leads to necrotic core formation, thereby increasing plaque instability, likelihood of rupture, and risk for acute cardiovascular events such as heart attack and stroke. Expansion of regulatory T cells (Tregs) in murine atherosclerosis decreases aortic root lesion area through mechanisms that are not fully understood. Given that Treg coordination of macrophage function has been reported, we hypothesized that expansion of the Treg compartment during advanced atherosclerosis promotes lesional efferocytosis. First, we used zymosan induced peritonitis as a proof‐of‐concept model to interrogate how Tregs may affect the efferocytic capacity of macrophages. Using mice in which the human diphtheria toxin (DT) receptor sequence is knocked into the 3′ UTR of the Treg lineage‐specific gene, Foxp3, we used DT to deplete Tregs in the days following injection with 0.1 mg Zymosan. Twelve days after injury, mice were ip injected with fluorescently labelled apoptotic neutrophils. After 45 minutes, the peritoneal cavity was lavaged and efferocytosis was measured by flow cytometry. Interestingly, uptake of labelled AC by macrophages in Treg‐depleted mice was significantly reduced compared to PBS‐injected control mice (30% vs 52%, p<0.05). Similarly, when assayed ex vivo, efferocytosis was again found to be defective in macrophages harvested from Treg‐depleted mice (p<0.05). Next, to determine whether Tregs could promote efferocytosis in atherosclerotic lesions, low density lipoprotein receptor deficient (Ldlr−/−) mice were placed on a western diet for a total of 15 weeks. Starting at week 12, mice were treated with with an IL‐2/anti‐IL2 antibody complex (IL2C) that is known to specifically expand Foxp3+ regulatory T cells. After three weeks of treatment, Tregs were significantly increased in the spleen and blood of treated, but not control, mice. We investigated efferocytosis in aortic root lesions by immunofluorescence staining of tissue sections to identify the association of macrophages (F4/80+) and apoptotic cells (TUNEL+). Quantification of the ratio of macrophage‐associated to “free” AC revealed that efferocytosis was significantly increased in aortic root lesions of IL2C treated mice (p<0.05). Interleukin‐10 has been reported to be elevated during Treg expansion in athero‐prone mice and is known to be a Treg effector molecule in other settings. To identify whether IL‐10 is an important player in the promotion of efferocytosis during Treg expansion, we treated mice with an IL‐10 neutralizing antibody or IgG during the expansion period. Strikingly, IL‐10 neutralization abrogated the improvement in lesional efferocytosis without affecting Treg expansion. Cumulatively, these data demonstrate a positive relationship between Foxp3+ Tregs and efferocytosis. Future investigations aim to elucidate the signaling mechanism through which this occurs.Support or Funding InformationThis work was supported in part by a T32 training grant from the NIH (HL007343)

Paks integrate polarity and vesicle transport signaling in ovarian epithelial cancers

P21 activated kinases (Paks) are effectors for small RhoGTPases and have been widely implicated in regulation of cytoskeleton arrangements and cell architecture. Paks gained a lot of momentum when several members of the family have been shown to be over‐expressed in a variety of cancers, including ovarian, breast, prostate and colon. These discoveries led to augmented efforts to discover new and improved Pak inhibitors to be used in clinic. Recent data shows however that the absence or complete inhibition of Pak2 leads to lethality in adult animals. It is thus imperative that we have a better understanding of Pak regulated pathways and their direct substrates.In an effort to unveil new molecular efforts in Pak signaling, we employed a modified kinase assay in conjuncture with peptide identification by LC/MS. A high percentage of the newly discovered Pak substrates fell in the category of molecules that control cell communication. Cell communication relates to all modes of inter and intra cell messaging, including cytoskeleton arrangement and vesicle transport. Importantly, these are also key elements that when deregulated lead to epithelial cancer initiation and progression.Among the newly identified Pak direct targets that regulate cell communication CDK16 and NSF have been previously shown to regulate each other and together to control polarity and vesicle transport respectively. We found that Pak1 and Pak2 phosphorylate CDK16 at S119 and NSF at S207. Our data has shown that unphosphorylatable CDK16 mutants are unable to bind Pak1 and 2 and that inactive Paks do not bind CDK16. This suggests that functional protein complexes are formed between CDK16 and Pak1 and 2. Furthermore, we found that in an ovarian carcinoma cell line overexpression of an unphosphorylatable CDK16 mutants can induce stress fiber and focal adhesion disassembly (link to deregulated polarity), while a CDK16 mutant that mimics a constitutively phosphorylated state can partially rescue stress fiber disassembly induced by Pak inhibition.These data strengthen our hypothesis that Paks regulate epithelial cancer progression by phosphorylating CDK16 and NSF, proteins that integrate polarity and vesicle trafficking pathways. To confirm this hypothesis we will first investigate the temporal and spatial regulatory events that take place between Pak, CDK16 and NSF and how these events influence polarity and vesicle transport. Secondly, we will investigate how the dis regulation of Pak/CDK16/NSF pathway leads to epithelial cancer initiation and spread. Thirdly, we will consider the possibility of combining Pak and CDK16 inhibition to arrest the growth various epithelial carcinomas.Our results address the possibility to target Pak signaling in Pak overexpressing cancers by inhibiting CDK16, a downstream kinase effector and to override some of the critical effects of current Pak inhibitors.Support or Funding InformationNIH T32 Training grant; NCI CA06927 to Fox Chase Cancer Center