Summer Scholars Faculty Mentor Summaries

Dr. Benson Akingbemi: Action of xenostrogens in the male gonad and neuroendocrine axis. Our long-term research goal is to understand how developmental exposure to environmental chemicals predispose to adult-onset disease related to male infertility. The increasing incidence of testicular disorders in the population was associated with exposure to chemicals which have the capacity to mimic and/or antagonize hormone action. Of interest, there is increasing public concern that exposures to chemical mixtures may cause unpredictable effects not seen with individual compounds. However, studies on the safety profile of chemicals commonly encountered in the environment are limited. The present study is focused on chemicals which are present in consumer products, including phthalates, bisphenols, 17α-ethinyl estradiol, and soy isoflavones. We hypothesize that exposures to combinations of chemicals intensify disruption of male reproductive function due to their single counterparts. Following exposure of male rats to test chemicals, we will analyze testicular steroid hormone secretion, androgen-stimulated germ cell development, sperm function, and identify mechanisms of chemical disruption of the pituitary-hypothalamus axis. The results will address whether chemical combinations cause additive effects in reproductive tract tissues, describe gene networks mediating chemical action, and inform development of screening protocols for risk assessment of the population. Students will be involved in executing the experimental protocol, sample analyses, data preparation, and presentation of results at scientific meetings.

Dr. Christine Charvet: New tools to translate time across the lifespan of animals. Our goal is to identify corresponding ages across the lifespan of humans and other species. Our work builds on an easily accessible online resource that finds equivalent developmental ages across 19 mammalian species (https://www.translatingtime.org). We previously relied on the timing of abrupt transformations that span prenatal ages of development. Accordingly, our approach was constrained to find corresponding ages at prenatal stages. We are broadening our dataset to find corresponding ages across the lifespan. We have recently collected state of the art neuroimaging scans to visualize brain pathways, RNA sequencing data to detect gene expression, and ATAC sequencing data to capture chromatin accessible regions of the genome. We currently have large datasets capturing transcriptomic and connectomic information from individuals of different ages and from different species (e.g., mice, macaques, marmosets). You will have the opportunity to analyze neuroimaging data and gain programming skills with the programming language R to analyze transcriptional and epigenetic data to translate ages across species. These valuable skill sets in neuroimaging and statistical genetics can be used to address a range of problems in basic and clinical sciences. Our laboratory website houses additional information about our work: www.charvetlab.com.

Dr. Kristine Griffett: Development of Novel Non-Opioid Compounds for Chronic Pain. This project focuses on the development of characterization of novel, non-opioid compounds targeting the nuclear receptor REV-ERB for analgesia. The opioid crisis is a substantial concern worldwide, and new efficacious pain therapies are desperately needed to reduce use and abuse of opioids. We have developed small molecule agonists for the REV-ERB nuclear receptors that appear to have analgesic properties in our preliminary testing. We believe that the inhibitory effects on inflammation at the site of nerve damage is one path that REV-ERB elicits its analgesic properties. The goal is to develop novel therapeutics that will be used for veterinary and human medicine, reducing our dependency on opioids, and reducing potential for abuse among patients, clients, and even providers. Our current model of efficacy testing is in mice, using mechanical and thermal measures to evaluate efficacy of compounds. This work will focus on evaluating animal tolerance to the compounds, efficacy testing, and evaluation of molecular and histological changes in sensory tissues. Techniques including QPCR, behavioral analysis, RNA-seq, IF/IHC or others may be utilized for this work. For more information regarding our work, please check out our website (www.griffettlab.wordpress.com).

Dr. Reid Hanson: A Study of the Visco-elastic and Friction Profiles of Equine Articular Cartilage. Our lab seeks to characterize and compare the material properties of cartilage located within various joints of the equine limb. Specifically, we will investigate the visco-elastic stiffness and friction coefficient of the biphasic cartilage structure. These biphasic properties affect the performance of the joint as it carries different loads and motions. We are investigating to determine if different types of joints with different ranges of motion possess similar or different material properties and which properties are best suited for the joint’s individual conditions. Analyzing the various cartilage surfaces within each joint and between joints will lead to a better understanding of the mechanisms controlling the performance of healthy joints in horses and humans. This data will be used to translate onto the design of better human artificial joints. Articular samples will be extracted from horses and analyzed in the Multiscale Tribology Laboratory, a multidiscipline lab between the Samuel Ginn College of Engineering and the College of Veterinary Medicine. Cartilage surface geometries will be characterized using either nano-scale surface profilometry, scanning and transmission electron microscopy and mathematical/numerical modeling techniques to analyze the structure of the surfaces over many scales. The key is to mesh the geometries at multiple different scales into one complete model.

Dr. Katie Horzmann: Developmental toxicity of trichloroethylene metabolites in the zebrafish model. I work with emerging and legacy environmental toxicants and study the effects of developmental exposure to these chemicals using the zebrafish (Danio rerio) biomedical model. One legacy contaminant is trichloroethylene (TCE), an industrial solvent and degreaser that contaminates over half of all Superfund sites, is a known carcinogen, and is linked to other adverse health outcomes including congenital cardiac defects and neurotoxicity. TCE is rapidly metabolized and the metabolites are thought to contribute to the overall toxicity. Our laboratory’s hypothesis is that the metabolites of TCE mediate aspects of developmental TCE toxicity. Student scholars would be able to join in a project investigating the developmental toxicity of TCE metabolites in embryonic and larval zebrafish. In addition to learning zebrafish husbandry and handling skills, scholars would evaluate embryonic mortality and hatching, embryonic behavior, larval behavior, and larval morphology in zebrafish with developmental exposure to TCE metabolites.

Dr. Jeff Huang: Lineage trace cells of gonadal origin in the adrenal gland. The adrenal cortex can be divided into different zones based on its histological features and function. The current model of the adrenal cortex development is based on two lineage tracing mouse lines. Data from these two genetic models suggested that the adrenal cortex is composed of two distinct populations: the fetal zone and the adult zone. The fetal zone originates from the adrenaogonadal primordium, whereas the adult zone is developed from the stem/progenitor cells reside in the adrenal capsule. During development, cells in the fetal zone undergo regression and are replaced by the continuously renewing adult cortical cells from the capsule. AMHR2 (Anti-Müllerian Hormone Receptor Type 2) is mainly found in Müllerian duct, the primordial anlage of the female reproductive tract. It is also expressed in fetal ovary and testis. Surprisingly, by using the lineage tracing mouse model that labels Amhr2-positive cells, we found that the Amhr2-positive cell population contributes to both fetal zone and the adult zone. This is the first lineage tracing model that labels cells in both fetal zone and adult zone. The student will use double immunostaining to characterize the cell population originates from the Amhr2-positive cell population to further confirm this groundbreaking finding.

Dr. Dana Levine: Evaluation of the regulation of Neutrophil Extracellular Trap (NET) formation by immune-mediated hemolytic anemia (IMHA) therapies. Immune-mediated hemolytic anemia (IMHA) is a common cause of severe anemia in dogs. IMHA is an autoimmune disease in which autoantibodies develop that target normal red cells for destruction. Unfortunately, IMHA is associated with a tragically high mortality rate (up to 80%) and mortality is mostly attributed to fatal thromboembolic events. The cause of thromboembolism is poorly understood. Treatment of IMHA involves generalized immunosuppression with various immunotherapies and clot prevention with various antithrombotic medications. Despite these therapies, fatal thrombi still occur. Furthermore, we lack useful data comparing the efficacy of different immunosuppressive or antithrombotic therapies in dogs with IMHA, leaving clinicians to select drugs without any evidence. When activated, neutrophils can extrude neutrophil extracellular traps (NETs), webs of DNA, nucleosomes, histones, and granular proteases. NETs are designed to trap and kill invading microorganisms. However, NETs can also activate clot formation and induce thrombosis. We believe that NETs are important in the pathogenesis of thrombosis in IMHA and we have shown that markers of NETs are increased in the blood of dogs with IMHA and that increased NET markers are predictive of death in dogs with IMHA. As such, NETs are a logical new therapeutic target in IMHA. We hypothesize that some current IMHA therapies will prevent canine neutrophils’ ability to generate NETs, i.e. NETosis. We will assess current immunosuppressive and antithrombotic therapies for their ability to prevent canine neutrophils from undergoing NETosis ex vivo. In so doing we will determine which therapies can prevent NETosis and thus which might be better frontline IMHA treatments. Through this project you will learn to isolate canine neutrophils and stimulate NETosis and will image NETs with immunofluorescence microscopy and live cell imaging. You will also have the opportunity to practice venipuncture skills and you will become an expert on IMHA.

Dr. Aime Johnson: Viral vectored contraceptive GnRH vaccine for cat population control. Euthanasia to control overpopulation kills more cats than infectious diseases combined. While surgical sterilization is effective and permanent, it remains costly and stressful. Challenges in pet cats include co-morbidity making anesthesia dangerous or owner desired non-surgical alternatives to spay or neuter. A non-surgical, easily administered method of controlling fertility is needed. An effective vaccine could be used in feral and pet cats, both male and female. Gonadotropin Releasing Hormone (GnRH) is the master regulator of reproduction, and antibodies that inhibit this key hormone would suppress all reproductive function, including fertility. Anti-GnRH antibodies have been shown to block GnRH function, however, traditional GnRH protein vaccines have fallen short in both efficacy and duration. This project will assess an established non-replicating adenovirus vectored GnRH vaccine (Ad-GnRH) already proven to be successful in mice and horses by our lab. Pilot data utilizing this Ad-GnRH vaccine in the domestic cat has also demonstrated an induction of antibody response to GnRH. Our Ad-GnRH vaccine successfully enters the cells and allows for continuous antibody production. We will treat eight prepubertal male cats with a single injection of Ad-GnRH. Our goal is to retard gonadal development, block fertility, and suppress breeding behavior. Unimmunized age matched male cats will control for normal reproductive system development. Anti-GnRH vaccines are not new, but there are only a few labs working with viral vectored GnRH vaccines. Our viral vectored vaccine should improve upon antibody response and longevity of response as compared to traditional GnRH protein vaccine. The student would be in charge of all aspects of the study for the summer including the animal work (blood collection, hormone assay, semen collection and analysis, etc).

Dr. Shankumar Mooyottu Clostridium difficile – a One Health pathogen Clostridioides difficile (CD) is a spore-forming, strictly anaerobic bacterium that causes a toxin-mediated enteric disease in humans and animals. CD is a nosocomial pathogen that has been associated with the use of antibiotics that results in disruption in normal enteric microflora (gut-dysbiosis), subsequent pathogen colonization and severe toxin-mediated colitis in hospital settings. However, the number of C. difficile infection (CDI) in human patients who do not have any history of hospitalization or antibiotic treatment has been increasing in the community settings. In such cases, household pets (dogs) are suspected to be the source of community-associated CDI. Interestingly, dogs never contract clinical CDI even though 20% of dogs carry CD in their gut, and many of them are closely related to human pathogenic strains. Our lab (Gut Pathogen-Microbiome Laboratory) has been working on an NIH-funded project on the pet-borne transmission of CDI (https://www.fic.nih.gov/Grants/Search/Pages/eeid-r01ai174289.aspx). On the contrary, we have identified a bacterium in dog gut that confers the CDI resistance, based on which we developed a microbiota-based therapeutic agent to control CDI in other animals and humans. For testing such therapeutics, we have developed a novel mouse model and a surgical swine ileal loop model of gut-microbiome-pathogen interface.

Maria C. Naskou: Stem Cell derived Extracellular Vesicles for modulation of Neuroinflammation. Neuroinflammation complicates the clinical progression of many neurodegenerative diseases and is mainly characterized by reactive astrogliosis and microgliosis, which further exacerbate the severity of neurological disorders via the release of pro-inflammatory mediators and neurotoxic factors. Stem cell derived Extracellular Vesicles (SC-EVs) are heterogenous populations of plasma membrane-shed microvesicles that derive from the endocytic pathway and have been shown to alleviate neuroinflammation by exhibiting an anti-inflammatory effect via suppression of astrogliosis and microgliosis and release of inflammatory mediators. Our aim is to first isolate and characterize the phenotype of feline derived astrocytes and microglia via flow cytometry. Subsequently we will evaluate the anti-inflammatory effect of SC-EVs in vitro on feline derived astrocytes and microglia that derive via gene and cytokine expression. Conclusions will provide insights for the potential use of SC-EVs as anti-inflammatory agents for a variety of diseases in which inflammation is part of the pathology and enable us to generate innovative therapeutic strategies for various neurodegenerative diseases.

Dr. Maninder Sandey: Immune checkpoint inhibitors (ICIs) have shown unprecedented clinical activity in a wide range of malignancies. However, their efficacy remains limited in many malignancies due to primary or acquired resistance. Targeted therapies that activate tumor necrosis factor receptor superfamily (TNFRSF) members, like OX40 & 4-1BB, are currently explored to augment the clinical efficacy of ICIs. In this study, we have constructed a novel-nanobody (Nb) based Agonist-Redirected Checkpoint (ARC) platform that consolidates immune checkpoint blockade (ICB) and TNFRSF agonism in a single biologic. Our long-term goal is to utilize this Nb-based ARC platform to develop novel immunotherapeutics to treat canine and human cancer patients. In this project, we will: (Aim 1) assess the safety and pharmacokinetic (PK) profile of aPD1-Fc-OX40L; (Aim 2) conduct a phase II/III clinical trial of aPD1-Fc-OX40L in canine patients with oral melanoma (OM); and (Aim 3) assess the functional activities of aPD1-Fc- OX40L over monotherapies. We will perform clinical correlative studies (using flow cytometry and immunohistochemistry (IHC)) on tumor and peripheral blood samples collected before and after administration of aPD1-Fc-OX40L to understand the cellular and molecular mechanisms that determine the antitumor immune response.

Dr. Melissa Singletary and Dr. Sarah Krichbaum: The detection canine is the most capable tool for the detection of many hazardous and other targeted substances. The Canine Performance Sciences (CPS) Program conducts research and development to enhance the technology of using dogs for detection of such substances. This effort includes research and development focused on health and welfare management of working dogs throughout all life stages from production to field operations and retirement. CPS summer scholar participants usually perform research that is at aimed toward direct application of working dog performance in a supportive partnership with the department of clinical sciences and services such as theriogenology which provide our summer scholars with an opportunity to become familiar with the production, clinical assessments, and health care of performance/working dogs. Throughout exposure and experience across the program, Summer Scholars at CPS will learn about the development, training, and application of detection dogs in addition to performing a chosen project examining metrics for working dog health, welfare and performance in behavioral/cognitive, physical fitness and olfactory-based tasks.

Dr. Scarlett Sumner and Dr. Maria C. Naskou: Characterization of platelet derived products against wound healing. Platelet alpha granules contain numerous cytokines and growth factors that can affect tissue regeneration, alleviation of inflammation and influence anabolic processes, while releasing numerous antimicrobial, anti-inflammatory and analgesic factors. Specifically, platelet derived products such as platelet rich plasma and platelet lysate from various species have been shown to aid in wound healing by promoting granulation tissue formation, epithelialization, angiogenesis, and collagen deposition. However, the manufacturing process of such products can affect growth factor and cytokine concentration as well as their antimicrobial properties. The aim of this study is to a) characterize the concentration of growth factors and cytokines related to wound healing from various preparations of platelet derived products and b) assess their anti-microbial properties against bacteria that are commonly present in skin wounds. The completion of this project will allow us to standardize the preparation method of platelet derived products while retaining its antimicrobial properties and evaluate in vivo its role in wound healing and the management of wound related microbial contamination.

Dr. Kamoltip Thungrat: Genetic Characterization and Comparison of Virulence and Antimicrobial Resistance in Uropathogenic Canine Escherichia coli. Escherichia coli (E. coli) is commonly associated with canine urinary tract infections (UTIs). Dogs with UTIs express varying severities of clinical signs, ranging from absent (asymptomatic bacteriuria; ABU) to severe (pyelonephritis). Severity is based, in part, on the presence of virulence factors, which facilitate E. coli isolates survival in the urinary tract environment. In ABU, bacteria colonize the urinary tract without provoking symptoms. It is not indicated for antimicrobial therapy; the treatment may increase antimicrobial resistance, and removal may allow infections with more virulent uropathogens. The aim of this study is to characterize E. coli from canine UTIs associated with differing severities of clinical signs, with the specific goal of describing genetic profiles of virulence factors and antimicrobial resistance in ABU. Ultimately, the data from this project will be used to develop the rapid diagnostics of virulence and antimicrobial resistance. The Summer Scholar will join the Clinical Pharmacology Laboratory and learn microbiological and molecular techniques (e.g., bacterial culture, antimicrobial susceptibility testing, PCR). The Scholar will be involved in the experimental design, data analyses and interpretation, and preparing a research presentation at regional or national scientific meetings.