PVM Research Day

Neuron signaling is important to organism health, sensation, muscle contraction, circadian rhythms, and thought. Drugs or conditions (i.e. injury) can alter how neurons relay signals, but quantifying such changes is sometimes challenging. Better understanding of how neurons signal could improve understanding, treatment, and early detection of diseases like neurodegeneration. Nerve signals involve voltage changes that propagate through and between neurons and other cell types. Measuring voltage changes in an entire cell population provides insight into how the group communicates. However, performing such analysis is often purely qualitative, and/or is very time consuming, limiting the information gained is limited. For example, crosscorrelation is an analysis method that generates a histogram called a correlogram, which provides information about whether the firing of two cells is related, firing patterns of two cells, and whether one cell consistently fires before or after another. Ideally, a correlogram would be created for each pair of neurons in a recorded voltage signal and the three properties (independence, pattern, and relationship) would be quantified. However, correlograms are typically described qualitatively, and only a subset of the relationships in a population are analyzed because it is too time consuming to observe all relationships manually.

To overcome this limitation, we developed an algorithm to analyze voltages from neuronal populations with minimal user effort. The algorithm outputs: (i) statistics on the number of times each cell/signal fired, (ii) statistics on firing intervals (aka. times between firing events), (iv) and information and statistics about relationships between cells. To quantify correlogram information (iv), the algorithm: tests for independent signals by testing for uniform distributions (Kolmogorov-Smirnov test), determines whether one cell consistently fires before/after another by calculating correlogram area left of zero (0.5 for simultaneous firing, < 0.5 for a cell that follows another, and > 0.5 for a cell that leads another), and quantifies firing patterns by counting the number of correlogram peaks, then calculates statistics for each of the three metrics. Algorithm outputs were evaluated by using the algorithm to analyze a previous recording of voltage signals from cells given Bicuculline, a drug that synchronizes firing and simulates seizure in vitro. The algorithm&#8217;s metrics successfully quantified network changes in a population of neurons exposed to bicuculline. The uniformity metric captured synchronization (loss of independent relationships) caused by bicuculline, and showed this synchronization was delayed compared to drug administration. The peak count metric showed that the number of correlogram peaks decreases after bicuculline, suggesting firing patterns are less diverse after the drug. The area left of zero metric showed that bicuculline increases the number of cells that consistently fire before or after others, identifying potential pacemaker cells (red row, blue column in the matrices). In the future, this algorithm could be employed to analyze signals from neuronal populations subjected to other treatments or conditions (such as neurodegeneration or injury) to better understand neuronal communication and gain insight into disease treatment, detection, and management.

The continuous emergence of divergent variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a significant concern for decreased vaccine efficacy, increased virus transmissibility, and decreased accuracy of diagnostic tests. Despite the rapid roll-out of billions of vaccine doses, limitations of the first-generation COVID-19 vaccines were soon realized due to the emergence of several variants of concern (VOCs), including the highly transmissible delta, and omicron variants, as well as omicron subvariants, BA.2, BA.4, & BA.5. The current COVID-19 situation highlights the critical need to design the next generation of vaccines to combat the threat posed by incoming SARS-CoV-2 variants. To develop the next-generation of COVID-19 vaccine, we designed human adenoviral (HAd)-based vectors expressing spike (S), membrane (M), or nucleocapsid (N) proteins of SARS-CoV-2 (Wuhan strain) with or without an autophagy-inducing peptide C5 (AIP-C5) (HAd-S/C5, HAd-S, HAd-M/C5, HAd-M, HAd-N/C5, and HAd-N). Mice were immunized intranasally (i.n.) with HAd vectored vaccines alone or in various combinations leading to the induction of significant levels of antigen-specific humoral (mucosal and systemic) and cell-mediated immune responses. The inclusion of AIP-C5 resulted in significantly higher levels of cell-mediated immune responses. Immunization with vectors expressing S, M & N or S-C5, M-C5 & N-C5 in a vaccine formulation resulted in significantly higher levels of antigen-specific cellular immune responses compared to the groups immunized with the vector expressing a single antigen. Vaccinated animals showed high virus-neutralizing antibody titers against delta and omicron variants. Immunized ACE2 transgenic mice were challenged with SARS-CoV-2 and monitored for morbidity and mortality for two weeks to investigate the protective efficacy. Significant reductions in morbidity, and mortality were observed in the groups immunized with vectors expressing S-C5, M-C5 & N-C5. Our findings suggest that the use of HAd platform in a multi-antigen approach complemented with the addition of C5 peptide is associated with enhanced immune cell-mediated response and improved protection, making it a promising strategy for the development of next-generation SARS-CoV-2 vaccines.

Candida auris is an emerging multi-drug resistant fungal pathogen that colonizes skin and subsequently causes invasive infection in hospitalized patients. Multi-cellular aggregative phenotype is widely reported in the C. auris isolates, but its role in skin colonization and host immune response in not yet known. In this study we generated aggregative phenotype by deleting ACE2 gene in C. auris and determined the fungal colonization, persistence and host immune response of ace2Δ using intradermal mouse model of C. auris skin infection. Our results indicate that ace2Δ infected mice skin had significantly higher fungal load after day 3, but significantly lower fungal load after day 14 compared to the wild-type non-aggregative control. The colonization of ace2Δis associated with increased recruitment of CD11b+ Ly6G+ neutrophils and CD11b+ Ly6 Chi inflammatory monocytes and decreased CD11b+ MHCII+ CD64+ macrophages and CD11c+ MHC II+ dendritic cells. Furthermore, Th17 cells and Type 3 Innate Lymphoid Cells were significantly increased in the skin tissue of ace2Δ infected mice. Our findings suggest that aggregative phenotype of C. auris can colonize in the skin efficiently but cannot persist for a longer period. Further, the colonization is accompanied with increased innate and adaptive antifungal immune responses in the skin.

"Webbed feet" are defined as skin membranes between the digits (connecting the dog's toes). The modern dog has descended from wolves, which likewise have interdigital tissue. People believe this webbing is very specific to certain dog breeds, but no scientific data exists to support or refute this. The present study aims to determine the differences in interdigital tissue length between American Kennel Club (AKC) standard-based "webbed" and "non-webbed" dog breeds and to investigate the effects of sex, paw sidedness, and body size on interdigital tissue length in dogs. A total of 338 dogs, representing 102 breeds, were included in this study. Collected phenotypic data includes height at withers, thoracic and pelvic paw widths when weight-bearing, metacarpal and metatarsal pad widths, and multiple thoracic and pelvic interdigital tissue lengths. The generalized linear model procedures of SAS with fixed effects of webbed/non-webbed groups, sex (male or female), paw sidedness (left or right), and body size (large, medium, and small) were used depending on the trait. There are no differences in interdigital tissue measurements between AKC standard-based webbed and non-webbed dog breeds, and all dogs have interdigital tissue. Sex had no effect on interdigital tissue length. Interestingly, smaller dogs were associated with having more interdigital tissue length compared to larger dogs. Finally, the left paws of dogs had more interdigital tissue, which could be related to their paw preference/dominance; however, further study is needed to prove this concept.

Ichthyosis fetalis (Harlequin ichthyosis) is a rare and fatal autosomal recessive congenital skin disorder characterized by hard skin plaques and deep skin fissures that restrict movement and cause malformation of the eyes, lips, and ears. Individuals affected by this disease are at a high risk of developing a fatal infection(s) due to the compromise of the skin’s protective barrier. Harlequin ichthyosis is the most severe subtype of ichthyosis. Although multiple forms of ichthyosis are described and genetically explained for humans and other mammals, to date there is no scientific literature describing ichthyosis or its genetic cause in domestic cats. Two random-bred Domestic Shorthair kitten littermates were diagnosed pathologically with harlequin ichthyosis, and this study aimed to identify the genetic basis for the disease. We carried out whole genome sequencing of both kittens, and manually examined sequence for known candidate genes. A one base pair deletion leading to a frameshift in the candidate gene ABCA12 was identified, (XM_019838638.1:c.200581732delG), resulting in a premature stop codon. Both affected kittens were homozygous for the frameshift deletion. The mutant allele was not identified in 140 unrelated control cats. Variants in ABCA12 have been previously associated with harlequin ichthyosis in humans, cattle, and mice. Taken together, we can classify the identified ABCA12 frameshift variant as pathogenic and the most likely the causative variant for ichthyosis in these kittens, reporting a genetic cause of ichthyosis in domestic cats for the first time.

Candida auris, an emerging multi-drug resistant fungal pathogen, predominately colonizes the human skin long-term leading to subsequent life-threatening invasive infections. Fungal morphology is believed to play a critical role in modulating mucocutaneous antifungal immunity. In this study, we used an intradermal mouse model of C. auris infection to examine fungal colonization and the associated innate and adaptive immune response to yeast and filamentous C. auris strains. Our results indicate that mice infected with filamentous C. auris had significantly decreased fungal load compared to mice infected with the yeast form. Mice infected with yeast and filamentous forms of C. auris stimulated distinct innate immune responses. Phagocytic cells (CD11b+ Ly6G+ neutrophils, CD11b+Ly6Chi inflammatory monocytes and CD11b+MHCII+CD64+ macrophages) and antigen presenting cells (CD11b+ CD207− DCs, CD11b− CD207+ DCs and CD11b+ CD207+ Langerhans cells) were differentially recruited to mouse skin tissue infected with yeast and filamentous C. auris. Furthermore, filamentous C. auris induced more potent local IL-17 responses eliciting a significantly greater percentage and absolute number of IL-17 producing ILCs, TCRγδ+ and CD4+ T cells in the skin tissue of mice compared to the yeast-locked strain. Collectively, our findings indicate that yeast and filamentous C. auris induce distinct local immune responses in the skin. The decreased fungal load observed in mouse skin infected with filamentous C. auris is associated with potent IL-17 immune response induced by this morphotype.

Vaccine adjuvants are critical components in human and veterinary vaccines as they enhance the immune response and improve vaccine efficacy. Aluminum-based adjuvants are the most widely used vaccine adjuvants and have been added to human vaccines (including hepatitis B and human papillomavirus) for over 90 years. The two types of aluminum adjuvants in licensed vaccines are aluminum hydroxide (AH) and aluminum phosphate (AP) adjuvants with very different physical and chemical differences that affect their interaction with vaccine antigens. The choice of one over the other is based on these differences, but little is known about potential biological and functional differences between these adjuvants. Most experiments aimed at elucidating the mechanism of action of aluminum adjuvants have focused on AH. The goal of the current experiments was to determine if there are biological and immunological differences between AH and AP.

Human monocytic THP-1 cells were differentiated with phorbol ester and then treated with or without LPS prior to incubation with AH or AP. The supernatants were harvested after 48 h and analyzed for IL-1β, IL-1α and IL-18. Pre-treatment with LPS caused increased cytokine production by the aluminum adjuvants, while AP induced significantly more IL-1β, IL-1α and IL-18 compared with AH. The secretion of IL-1β by both AH and AP was inhibited by pretreatment of the cells with cytochalasin D, Ca-074-Me, and MCC950 indicating a requirement for phagocytosis, lysosomal cathepsins, and the NLRP3 inflammasome, respectively. To determine if these differences translate in differences in the inflammation at the injection site, the calf muscle of mice was injected with AH or AP. Muscle tissue collected after 24, 48, and 96 h was digested and analyzed by flow cytometry. There were more neutrophils and monocytes at 96 h following injection of AP compared with AH.

These results suggest that AP is a stronger stimulator of innate immune responses than AH. A better understanding of the biological differences and underlying mechanisms of action of these aluminum adjuvants could help researchers to tailor vaccine formulations to specific applications, and therefore to transition from empirical to rational design of vaccines.

Actively surveillance and androgen deprivation therapy (ADT) are primary treatments for prostate cancer patients. However, recurrence often happens in patients with ADT therapy and tumor spread is the main cause of mortality in prostate cancer, thus effective systematic treatments are needed. Oncolytic adenovirus (oAd) with modification can selectively propagate in tumor cells and significant antitumor effects have been shown in several cancer types. “Naked virus” is easily recognized and neutralized by the host, therefore, mesenchymal stromal cells (MSC), which possess unique characteristics of tumor-tropism and avoid immune rejection, become promising candidates as carriers for oAd. In this study, MSC derived from adipose tissue (ASCs) is used. Our group has extensive expertise in using interleukin 27 (IL27) to regulate both innate and adaptive immunity, showing antitumor effects via effector T cell recruitment to tumors, as well as slowing down bone erosion associated with tumor metastasis. As a result, genetically engineered oAds expressing a reporter gene (green fluorescence) and armed with IL27 will be examined for the ability to exert antitumor effects in vitro and in vivo. Project already reached viral titer determination, optimal non-toxic multiplicity of infection (MOI) in ASCs, mix-culture optimization of ASCs with a prostate cancer cell line (PC3), and cytotoxicity evaluation. A combination of cancer cell spheroids, xenograft mouse models and human tissue slices will be utilized to mimic the complicated microenvironment and enable evaluation of the delivery efficacy of oAds from ASCs, the effectiveness of the oAds, and the synergy with IL27 for achieving optimal treatment effectiveness.

Keywords: Prostate cancer, Oncolytic adenovirus, Interleukin 27

Short spine syndrome is an understudied, rare disease that can occur spontaneously in canids, causing the torso and neck to appear truncated. Recently, a mixed breed dog diagnosed with short spine syndrome presented with shortening of the vertebral column, together with a normal sized head and limbs. Radiographs indicate this dog had multiple misshapen and fused vertebrae, as well as mild scoliosis, mild kyphosis, and malformed ribs. Whole genome sequencing along with variant effect prediction software identified a homozygous 23 base pair deletion in the first exon of the gene MESP2, causing a frameshift mutation. MESP2 encodes for a transcription factor that plays a vital role in the development of vertebrae and somite segmentation. Variants in this gene have been associated with Spondylocostal Dystosis 2 in humans, which is a heritable disease characterized by fused vertebrae, rib malformations, scoliosis, kyphoscoliosis, short stature, and a short neck. This novel variant is likely the cause of short spine syndrome in this dog, and can now be classified as Spondylocostal Dysostosis.

Blast-induced traumatic brain injury (bTBI) is a significant concern for military personnel, yet its effects on sleep patterns and seizure susceptibility remain poorly understood. In this study, we conducted a preliminary investigation using a mouse model to explore chronic sleep characteristics and seizure susceptibility following repetitive moderate-severe bTBI. Six C57BL/6 mice (n.3 bTBI group and n.3 sham group) underwent EEG and EMG electrodes implantation, and video-EEG and EMG recordings were conducted for one week, starting one month post-injury. Sleep phase scoring revealed no significant differences in the distribution of wake, non-rapid eye movement (NREM), and rapid eye movement (REM) stages between the bTBI and sham groups. However, spectral analysis indicated reduced power in the 10-15 Hz range during NREM sleep in the bTBI group. Analysis of sleep spindles (SSPs) revealed significant differences in event duration, amplitude, and frequency between the two groups, suggesting qualitative alterations in NREM sleep post-bTBI. Although no spontaneous seizures were observed, the bTBI group showed a trend towards higher seizure susceptibility, characterized by shorter latency to seizure onset and prolonged post-ictal periods after pentylenetetrazol (PTZ) injection. Further investigation into potential epileptiform events (i.e. hyperactivity events not causing behavioral seizures) and their correlation with sleep alterations post-bTBI is warranted. While this study provides valuable insights, larger sample sizes, behavioral correlation and biochemical correlation studies are necessary to fully elucidate the entity of the sleep disturbances after bTBI and their relationship with PTE development, as well to identify possible therapeutic target.

Candida auris, a globally emerging multi-drug-resistant fungal pathogen, uniquely colonizes the human skin long term, leading to subsequent development of life-threatening invasive infections in humans. The factors regulating skin colonization of C. auris are not well understood. In this study, we established an intradermal mouse model of C. auris infection to define the innate immune response to this emerging pathogen and compare it to Candida albicans. Our results indicate that compared to C. albicans-infected mice, C. auris-infected mouse skin tissue had significantly higher fungal load after 3 days post-infection. Mice infected with C. auris (yeast) had significantly increased number of innate immune cells compared to PBS group. C. auris infection was associated with a significantly decreased accumulation of CD11b+ Ly6G+ neutrophils and increased numbers of CD11b+ Ly6Chi inflammatory monocytes and CD11b+ CD207+ Langerhans cells at the site of infection when compared to C. albicans. Furthermore, when we checked how different clades of C. auris colonize murine skin, we found out that mice infected with different clades does not show any fungal load difference through intradermal route of infection but elicit different levels of accumulation of Langerhans cells. Taken together, our study indicates that C. auris and C. albicans induce differential skin immune responses in mice. We predict clades and species diversity in part regulate the host immune responses in the skin.

Candida auris, an emerging multi-drug-resistant fungal pathogen, uniquely colonizes the human skin long term, leading to subsequent development of life-threatening invasive infections in humans. The factors regulating skin colonization of C. auris are not well understood. In this study, we established an intradermal mouse model of C. auris infection to define the innate and adaptive immune response to this emerging pathogen and compare it to Candida albicans. Our results indicate that compared to C. albicans-infected mice, C. auris-infected mouse skin tissue had significantly higher fungal load after 3- and 14-days post-infection. C. auris infection was associated with a significantly decreased accumulation of CD11b+ Ly6G+ neutrophils and increased numbers of CD11b+ Ly6 Chi inflammatory monocytes and CD11b+ CD207+ Langerhans cells at the site of infection. Furthermore, a significant decrease in the absolute numbers of type 3 innate lymphoid cells and Th17 cells was observed in C. auris-infected skin tissue. Taken together, our findings indicate that the skin immune responses are different between C. auris- and C. albicans-infected mice. The increased fungal load observed in C. auris-infected mouse skin tissue is associated with less potent innate and adaptive immune responses induced by this emerging pathogen relative to C. albicans.

Mechanosensing is a fundamental physiological process, including touch sensation, blood pressure regulation, bladder function, etc. Recently, it has become more evident that mechanosensing plays instructional roles during early embryogenesis. Piezo channels are a group of known ion channels that transduce mechanical stimuli to bioelectric signals. These channels are widely distributed across different cell types and play critical roles in various physiological processes. Dysregulation of the piezo channels has also been linked to various diseases such as hypertension and anemia. However, the detailed function of the piezo genes in early embryonic development remains poorly understood. Gene temporal and spatial expression are good indicators of their functions. In this project, we took advantage of the zebrafish model and examined the piezo gene expression in early staged embryos. We first analyzed vertebrate and invertebrate piezo channel evolution by phylogenetic and syntenic analysis. Our data revealed that piezo2 was duplicated as piezo2a and piezo2b, and we isolated a new piezo gene, piezo3, which is only found in teleosts and very few species in the tetrapod. Then, we performed whole-mount in situ hybridization to comprehensively characterize the expression pattern of the four piezo genes. All four piezo genes have distinct patterns, suggesting each gene might have specialized functions during zebrafish embryogenesis. Moreover, we used Alphafold2 to predict all four zebrafish piezo protein structures, which are relatively conserved in functional domains. In conclusion, our phylogenetic and developmental analyses of the piezo channels shed light on their evolutionary history and potential roles during embryogenesis related to their physiological functions. Our future research will aim to manipulate the piezo genes in vivo in zebrafish to further understand their roles in development and regulation.

Clostridium difficile (C. difficile) is a significant anaerobic Gram-positive bacterium known for causing severe and sometimes fatal diarrhea. To understand the C. difficile pathogenesis and identify potential treatment targets, we conducted a mouse study involving symptomatic, asymptomatic, and uninfected mice. After antibiotic pre-treatment and subsequent infection, mice were monitored for signs of disease and euthanized to collect sections from the intestine. These sections were then utilized for spatial transcriptomics technology to analyze differential gene expression in these mice's intestinal mucosa and submucosa. Spatial transcriptomics allows for the dimensional assessment of gene transcription, providing insight into how individual cells utilize mRNA and proteins in the intestine. We aim to use spatial transcriptomics technology to analyze differential gene expression in these mice's intestinal mucosa and submucosa. This study found that specific pathways, primarily related to innate immunity, significantly differed between symptomatic and asymptomatic mice. Specifically, genes related to neutrophil degranulation, an important pathway in C. diff infection, exhibited significant dysregulation. Two genes, S100a8 and S100a9, were notably upregulated in symptomatic mice compared to asymptomatic mice, suggesting their potential role in exacerbating mucosal inflammation and contributing to disease severity.

During inflammation, immune cells produce pro-inflammatory cytokines in order to mount a defense response. Under normal conditions, a pro-inflammatory response is followed by an anti-inflammatory response to repair damaged tissue. Macrophages exhibit plasticity, allowing them to regulate both pro and anti-inflammatory responses. Chronic inflammation interrupts the body’s ability to mount an anti-inflammatory response, leading to tissue damage near the inflammation site and in some cases the development of cancer. The goal of this project is to investigate the ability of nanoparticle delivery to macrophages to act as a long-term regulator of inflammation through the Stimulator of Interferon Genes (STING) pathway. Employing Flash NanoPrecipiation (FNP) we seek to nanoencapsulate novel STING antagonist small molecules that have shown evidence of being able to polarize macrophages into their anti-inflammatory phenotype. FNP allows the encapsulation of strongly hydrophobic small molecules or small molecules with ionizable functional groups. Through the use of an amphiphilic stabilizer such as PEG-PCL, we can achieve slow-release formulations for STING antagonists. Furthermore, adding hydrophobic glucocorticoids, such as cholecalciferol, to the nanoparticles we can enhance anti-inflammatory properties as well as potentially improve the stability to achieve a slower release rate. Following an LPS + IFNg challenge, to induce a pro-inflammatory phenotype, we will then treat the cells with the nanoparticles and measure their conversion from the pro-inflammatory to anti-inflammatory phenotypes via flow cytometry. Studying the complex interactions between STING pathway and inflammatory response will better inform us on how nanomedicine can be used for long-term solutions to chronic disease symptoms.

Sarcocystis neurona, an obligate intracellular apicomplexan and the causative agent of the neurological disease equine protozoal myeloencephalitis (EPM), results in substantial losses to the equine industry. In apicomplexans, the dense granules, a specialized secretory organelle, releases dense granule proteins (GRAs) to aid in intracellular development, survival, and host manipulation. Dense granules are abundant in S. neurona, but their repertoire remains unidentified. Transcriptomic analysis of S. neurona developmental stages (extracellular merozoites and intracellular schizonts) has identified a dense granule protein, SnGRA9. We have performed in silico analysis of SnGRA9, epitope-tagged the native locus to express SnGRA9-HA [GRA9-hemagglutinin (HA) fusion protein], and performed immunolocalization to begin understanding its role in S. neurona biology. In silico analysis of SnGRA9 revealed an intrinsically disordered domain and a conserved motif for cleavage by an aspartyl protease, ASP5, indicating SnGRA9’s possible maturation and translocation into the host cell. Additionally, Western blot assays of the schizont stages show a ~25kD SnGRA9 peptide consistent with the location of the ASP5 cleavage site. The absence of this GRA9 peptide from merozoites raised speculation about differential roles for SnGRA9. Immunofluorescence assays further confirm the differential expression of this protein between the two stages, and during schizont development (day 1 through day 5). SnGRA9-HA seemed to be localized in the host cell cytoplasm. Further experiments are underway to confirm the secretion of SnGRA9 into the host cell, its processing by SnASP5, and the identification of interaction partners. This study marks an essential step in understanding the role of dense granules in S. neurona

Alzheimer’s Disease (AD) is a progressive neurodegenera=ve disease afflic=ng 50 million individuals worldwide. It is characterized by mul=ple pathological hallmarks, including synap=c and neuronal loss, and brain atrophy, leading to substan=al cogni=ve decline. Current animal models mainly focus on familial AD (fAD), which is caused by gene=c muta=ons. However, models for studying sporadic AD (sAD), which represents over 95% of AD cases without a specific gene=c cause, are s=ll limited. Notably, the significant species differences between humans and animals might be the leading cause of clinical failures for AD therapeu=cs that have shown success in animal models, highligh=ng the importance of developing more transla=onal human-centric models for studying AD, par=cularly sAD. In this study, we developed a novel human-induced pluripotent stem cell (hiPSC)-based vascularized neuroimmune organoid model. These organoids contain mul=ple cell types in the human brain, including human neurons, microglia, astrocytes, and blood vessels. We further induced AD pathologies by challenging organoids with AD pa=ent-derived brain homogenate. Our results demonstrated that AD brain homogenate-treated organoids successfully recapitulated mul=ple AD pathological hallmarks, including amyloid plaques, tau tangles, neuroinflamma=on, elevated microglial synap=c pruning, and synap=c and neuronal loss, as compared to the control group. Remarkably, aYer 2 weeks of treatment with Lacanemab, an FDA-approved drug targe=ng amyloid beta, we observed a significant reduc=on of amyloid burden. In summary, the neuroimmune organoid model provides a unique opportunity to study sAD in a human cell se]ng. This innova=ve model also holds promise to facilitate AD drug development, par=cularly for an=body-based AD treatment.

The placenta is the least understood and only disposable organ of the human body. Nonetheless, it plays a crucial role in providing the fetus with oxygen and nutrients. Immune responses at the maternal-fetal interface are essential in facilitating implantation, maternal tolerance to the fetus, and parturition. Various immune cells, cytokines, and chemokines have shown to play an important role in the development of maternal immune tolerance and the formation and growth of the placenta itself. The fractalkine receptor CX3CR1 is highly expressed on monocytes and macrophages within the placenta and accumulating evidence has indicated a contribution of CX3CR1 in the pathogenesis of several obstetric disorders. Utilizing C57BL/6 wildtype and CX3CR1-knockout mice in combination with flow cytometry and Immunohistochemistry we demonstrate a crucial involvement of CX3CR1 in the placental immune microenvironment and placental development. Indeed, our results show that the majority of CX3CR1 expressing cells within the placenta are monocytes and macrophages, from both maternal and fetal origin. Comparing placentas from wildtype and CX3CR1-knockout mice revealed a significant reduction of cell numbers in immune cell populations in CX3CR1-knockout placentas, including populations that express little to no CX3CR1. Additionally, a decrease in overall placental size is seen in the CX3CR1-knockout mice. Finally, we show that the adoptive transfer of wildtype bone marrow monocytes early (E4.5) into CX3CR1-knockout mice pregnancy could restore the immune cell populations in the placenta. These results point towards monocytes/monocyte-derived cells needing functional CX3CR1 to influence the placental immune environment.

The nematode, Baylisascaris procyonis, in the intestines of raccoons sheds its eggs in the raccoon feces, contaminating the environment. Accidental ingestion of these eggs by humans and 160 other hosts results in neural larva migrans, wherein 5-7% of larvae from ingested eggs migrate to the brain, leading to permanent neurological impairments or fatalities. Differential gene expression analysis between the larvae and adults of B. procyonis was performed to reveal transcripts abundant in the larvae. For this, Illumina sequencing was performed on B. procyonis mRNA isolated from aseptically hatched, in vitro-cultured larvae, and adults obtained from roadkill raccoons. The transcriptome data from larvae and adults were aligned to the B. procyonis reference genome to investigate differential gene expression. Genes upregulated in the larvae were annotated and classified using various bioinformatic techniques. A set of 7387 Baylisasaris genes were differentially expressed, with 5409 genes upregulated in the larvae. Venom allergen proteins, C-type lectins, and cuticular collagens with putative roles in host invasion, tissue migration, feeding, larval development, and immunomodulation were among the top 20 larval-abundant transcripts. Repeat antigen 1, currently used for serodiagnosis, and other Baylisascaris-specific transcripts were also among these abundant transcripts. Gene ontology and KEGG pathway analysis have identified enriched biological and metabolic processes associated with B. procyonis larvae, presumably contributing to larval adaptation and survival strategies. This data will promote future studies investigating the roles of specific genes in the pathogenesis of neural larva migrans, thus advancing understanding of the host-parasite interactions of this emerging zoonosis.

Traumatic brain injury (TBI) is a global public health crisis affecting up to 74 million people annually. Individuals in war zones are acutely susceptible to TBI due to explosive blast shockwaves or Blast Induced Neurotrauma (BINT). In mild-BINT, the initial physical, or primary, injury may not result in immediate cognitive changes. However, pathological biochemical cascades or secondary injury begin immediately following blast wave exposure. These cascades can extend for weeks or even years beyond the initial physical injury, resulting in chronic physical and/or psychiatric degeneration. Analyzing and measuring neuronal communication changes in the brain following injury is vital for understanding the contribution of secondary injury to long-term neurodegeneration. However, neuronal networks are difficult to characterize and study in whole animals. Therefore, an in vitro blast model is critical to better understand the immediate and long-term neuronal functional alterations due to mBINT. This study aimed to investigate changes in neuronal network dynamics following blast injury using primary murine cortical cultures grown on microelectrode arrays (MEAs). Networks exposed to a mild blast overpressure injury exhibited immediate changes in activity parameters: The average spike activity of the networks decreased by 33.73 ± 8.06% (n=5), along with alterations in spiking patterns and dynamics. Cross-correlation analysis of pre- and post-blast signals revealed changes in firing hierarchy and network oscillations. This model offers a unique opportunity to study the immediate functional consequences of blast shockwaves on neuronal signaling patterns, providing insights that could inform the development of therapeutic interventions for mild blast-induced neurotrauma.

Aging is a major risk factor for neurodegenerative diseases. It is estimated that 20% of Americans above the age of 55 live with some form of dementia. Furthermore, as the world’s aging population increases, so does the number of patients suffering from cognitive decline. To date, there are no treatments to mitigate, let alone cure, neurodegenerative diseases. One of the main reasons behind the lack of strategies to combat dementia is the complexity of the molecular signatures that contribute to aging-associated neurodegeneration. Therefore, it is paramount that we understand the mechanisms governing neurodegeneration in a holistic way. In this study, we focus on unveiling the changes in the proteome, kinome and phosphoproteome of aging mice brains to elucidate the molecular signatures of age-related neurodegenerative processes. Our analysis showed differential regulation of 446 proteins in an age-dependent fashion. Gene set analyses revealed a marked increase in proteins associated with neuroinflammation, synaptic function, and protein folding among proteins upregulated in aged mice. We also found that several proteins involved in glutamatergic signaling, neuroinflammation, and cytokine signaling had an increase in their phosphorylation status in old mice. These findings are corroborated with our observations suggesting a reorganization of the mouse kinome. Kinase enrichment analysis using upregulated phosphosites indicated the activation of PI3K-AKT-mTOR signaling pathway, that, taken together with the hyperphosphorylation of several proteins, including key proteins in the onset of Alzheimer’s and Parkinson’s diseases, such as Mapt and Dpysl2, sheds light on novel (phospho)proteins and pathways that play a role in age-dependent neurodegeneration.

Traumatic brain injury (TBI) is a leading cause of death and long-term disability worldwide. Further, seizure development is one of the most serious sequalae post-TBI. Unfortunately, the underlying mechanisms linking TBI and seizures are not well understood. Identifying cell-scale pathophysiological changes in the brain post-injury will be paramount for identifying therapeutic targets. Therefore, we utilized our novel TBI-on-a-chip model to investigate seizure-like activity (SLA) in neuronal networks post-TBI. This system simulates the pathophysiology of concussive TBI by applying clinically relevant, rapid acceleration injuries to murine cortical networks on microelectrode arrays, while providing real-time, cell-scale monitoring of electrophysiological and morphological changes. Utilizing extracellular recordings of network spike activity, we reveal the spontaneous appearance of SLA in networks exposed to 10 rapidly (4-6 sec) administered 30 g impacts. Additionally, analysis of cross-correlation profiles revealed significant changes in network dynamics post-impact injury. Furthermore, networks exposed to single impacts of 30, 100, and 200 g showed that fluorescence intensities of the astrocyte reactivity marker, GFAP, and the lipid peroxidation product and oxidative stress marker, acrolein, progressively increase with larger g forces. Astrocytes can play a significant role in modulating neuronal function, and acrolein can exacerbate neuroinflammation and oxidative stress, which are hallmarks of TBI. Further experiments are ongoing to investigate the role of astrocyte reactivity and acrolein in seizure development post-TBI. In summary, our TBI-on-a-chip model could provide vital insights into functional and morphological changes post-TBI, while enabling the investigation of underlying SLA mechanisms, which could lead to the identification of potential therapeutic targets.

Acute Respiratory Distress Syndrome (ARDS) is an acute inflammatory lung injury associated with pulmonary edema and characterized by the presence of alveolar damage and lung stiffness. Globally, about 40% of ICU patients suffer from ARDS, half of which are categorized as severe ARDS with 90% mortality rate. Current therapy is limited to supportive care in the form of mechanical ventilator, which highlights the dire need of targeted therapies. In ARDS, lung injury triggers activation of immune cells, which release pro-inflammatory cytokines aimed at eliminating the cause of injury. Prolonged upregulation of IL-1β, IL-8, IL-6, and TNF-α contribute to macrophage and neutrophil recruitment and accumulation, promoting further release of toxic mediators that perpetuate chronic inflammation and alveolar damage. Therefore, therapies capable of modulating cytokine production while stimulating tissue repair may improve ARDS patients’ prognosis. Interleukin-27 (IL-27), a multifunctional cytokine involved in innate and adaptive immunity and in promoting tissue repair, has been hypothesized as a candidate to rebalance an ARDS inflammatory microenvironment. To test this hypothesis, we first established an LPS-induced in vitro model and characterized gene expression responses in lung epithelial cells (BEAS-2B) and macrophages (THP-1). Cytokines typically have short half-lives; thus we utilized a gene therapy approach to deliver IL-27 by transfecting an IL-27-expressing plasmid into human adipose mesenchymal/stromal cells (hASC). Our results showed that IL-27-hASC conditioned media reduces pro-inflammatory cytokine gene expression in LPS-induced BEAS-2B and THP-1, both in monoculture and co-culture settings. We are currently testing our hypothesis in an in vivo model of LPS-induced ARDS, further determining the potential of IL-27 therapy in reducing inflammation in an ARDS model.

Background: Tuberculosis (TB) remains a significant public health concern. This infectious disease is still among the ten leading causes of death by a single pathogen. It is the primary cause of death among acquired immunodeficiency syndrome (AIDS)/human immunodeficiency virus (HIV-1) patients. There is a synergistic relationship between these two pathogens, in which TB enhances viral infection and HIV-1 predisposes the development of active TB. Nevertheless, the mechanisms behind it are still not fully understood, and there are few studies investigating TB/HIV-1 coinfection. MicroRNAs are small, non-coding RNAs that regulate gene expression post-transcriptionally. Therefore, they have been largely studied in both diseases for diagnostic and prognostic purposes. Yet, no study has focused on the expression of microRNAs within the granuloma level.

Methods: We compared the expression of microRNAs from lung granulomas of human autopsied TB+/HIV-1- patients (n = 14) and TB+/HIV-1+ (n = 16), after laser capture microdissection (LCM) by small RNA sequencing.

Results: There were 267 microRNAs commonly expressed between TB+/HIV-1- and TB+/HIV-1+ patients, 91 expressed only in TB+/HIV-1- patients, and 14 expressed only in TB+/HIV-1+ patients. From those, miR-137-3p and miR-650 were upregulated in TB+/HIV-1+ compared to TB+/HIV-1-, while miR-205-5p, miR-378c, and miR-150-5p were downregulated.

Conclusion: As expected, microRNAs were also differentially expressed at the granuloma level between TB+/HIV-1- and TB+/HIV-1+ human patients, but functional studies are still needed to understand their role in TB/HIV-1 coinfection at the granuloma level more specifically.

The placenta is a vital organ during pregnancy, serving as a connec9on between mother and fetus, by supplying oxygen, nutrients and removing waste. Nonetheless, it is suscep9ble to infec9ons that can result in miscarriage or developmental complica9ons that affect the fetus. Indeed, infec9ons are accounted for up to 15% of early miscarriages and up to 66% of late miscarriages. A major pathogen that is responsible for complica9ons during pregnancy is Toxoplasma gondii. In the United States alone, nearly 6,000 cases of congenital toxoplasmosis are reported every year. Moreover, up to 85% of the in-utero infec9ons result in severe birth defects including developmental complica9ons such as choriore9ni9s, blindness, hydrocephaly, deafness, and fetal demise. Major gaps remain in understanding the precise mechanisms of how T. gondii crosses the placental barrier and this has been highlighted as one of the main ques9ons in the toxoplasmosis field. Moreover, the molecular mechanisms that regulate immune responses during T. gondii infec9on are yet to be explored, underscoring the need for further research. This study focuses on revealing how T. gondii crosses the placenta and infects the fetus and how the placental immune cell microenvironment responds to the infec9on. My preliminary data indicate that T. gondii infects both hematopoie9c and non-hematopoie9c cells, leading to the hypothesis that the parasite u9lizes them to traverse through the placental barrier. Moreover, fluorescence analyses showed that there is a significant decrease in T and B cell popula9ons in the placenta, implica9ng that they are part of the T. gondii pathogenesis.

Purpose: Increase in intraocular pressure (IOP) causes changes to the essential components of the sclera, the scleral fibroblasts, and their extracellular matrix. This also causes damage to the optic nerve head (ONH), and its supporting cells including the ONH astrocytes and Lamina Cribrosa (LC) cells. This affects the biomechanical response of the optic nerve head to elevated intraocular pressure which translates to glaucomatous optic neuropathy. Dogs have been proven to be useful models in the study of glaucoma as they have similar eye anatomy as humans and can also naturally develop glaucoma as in humans. There are also similarities in the pathogenesis of canine and human glaucoma, and human glaucoma treatments have been found to be effective in dogs. The purpose of our study was to isolate, culture and characterize with immunocytochemistry the sclera fibroblasts, ONH astrocytes and LC cells of normal and glaucomatous canine eyes.

Methods: Freshly enucleated normal (n=10) and glaucomatous (n=8) canine eyes were obtained and used for primary cell culture. At 37°C and 5% CO2 humidified incubator, sclera fibroblasts were grown from peripapillary sclera (PPS) explant, while ONH astrocytes and LC cells were separated as two cell populations from LC explant. Sclera fibroblasts were fixed and stained with vimentin, laminin, CD45 and cytokeratin antibodies. ONH astrocytes stained with GFAP, Vimentin, and α-SMA. LC cells stained with α- SMA and GFAP. Immunofluorescence microscopy for visualization of the proteins was done with Nikon Eclipse Ni Microscope using the NIS Elements imaging system. Statistical analysis was done with a twosample t-test using R studio.

Results: We were able to successfully extract and culture primary scleral fibroblasts from the PPS tissues, ONH astrocytes and LC cells from LC explants of canine eyes with and without glaucoma. Appreciable differences were noted in the morphology of these cells between the samples with and without glaucoma. Consistent with the immunostaining results in humans, the canine scleral fibroblasts were vimentin and laminin positive, and negative for cytokeratin and CD45. Whereas ONH astrocytes were GFAP positive but negative for vimentin and α-SMA, LC cells were α-SMA positive and GFAP negative. Vimentin and laminin intensity stain were noted to be significantly lower in the scleral fibroblasts of the glaucoma group (P<0.05).

Conclusion: This is the first study describing the primary culture of canine scleral fibroblasts, ONH astrocytes and LC cells. We believe the techniques will be useful for future investigations to understand the cellular and molecular changes with glaucoma development in dogs as a large animal model for naturally occurring glaucoma. Future studies planned in our lab include proteomic analysis and biomechanical tests of established cells from the eyes with different types and stages of glaucoma.

Aging is a major risk factor associated with cancer and neurological diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). However, the mechanisms that underpin the process of aging are not well understood. Like genes and proteins, lipids play key structural, regulatory, and signaling roles. Hence, profiling lipid changes in different organs due to aging process provides useful information for understanding the molecular mechanisms underlying this process and for developing treatments or cures for age-related diseases. Despite their significance, still, very little is known about the composition and age-dependent changes of lipids in the brain and liver, the two most lipid-rich organs after adipose tissues. In this study, we performed exploratory lipidomic analysis of mice brain and liver at different ages via Multiple Reaction Monitoring Mass Spectrometry (MRM-MS) to determine changes in different classes of lipids and correlate the differences in lipid profiles with the age groups. The MRM-MS analysis was performed using ion transitions based on precursor (Prec) and neutral loss (NL) scans obtained from LIPID MAPS database for screening each sample independently for 24 different classes of lipids including different phospholipid classes such as phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), ceramides, di- and tri-acylglycerols, and acylcarnitine. We identified age-dependent changes in different lipids including tri- and di-acylglycerols, phospholipids and ceramides. In our presentation, we will discuss the importance of these lipids in the context of aging and age-related disorders will be discussed.

Understanding how organ size and shape are determined is crucial for developmental biology, and vertebrate limb/fins have been widely studied as a model system for this purpose. We have characterized two long-fin zebrafish mutants (Dhi862 and Dhi4458) generated by retroviral insertional forward genetic screening, which were found to have changes in the inwardly rectifying potassium channel (kir) gene, kcnj10a/kir4.1. Despite this identification, the specific mechanism of how the kcnj10a mutation induces long fins remains elusive. The following study characterize the mutant’s morphology through fin and body size measurements in adult and larval zebrafish, coupled with in depth analysis of kcnj10a gene expression using whole mount in situ hybridization and qPCR techniques. Expression patterns of developmental genes in fin morphogenesis were compared between mutants and wildtype siblings, and the kcnj10a coding region was mutated using the CRISPR-Cas9 system. Our results showed that one and two copy transgenic Dhi862 and Dhi4458 mutants had elongated fins with altered fin ray segments compared to wildtype siblings. We identified transient ectopic expression of kcnj10a in the entire somite and notochord of mutant fish embryos, with subsequent removal of ectopic expression using the CRISPR-Cas9 system rescued the Dhi862 long-finned phenotype. These findings suggest that the ectopic expression domains of kcnj10a in the somite and notochord are responsible for the long-fin development in these mutants.

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary myocardial disorder characterized by the pathological replacement of normal myocardial tissue with adipose and fibrous tissue. While prevalent in Boxer dogs, ARVC also affects humans, cats, and other dog breeds, resulting in severe clinical consequences such as malignant ventricular arrhythmias, cardiac dilation, syncope, and sudden death. However, diagnosing ARVC often requires invasive histopathological examinations, primarily postmortem. Given the limitations of current diagnostic criteria, particularly in Boxer dogs, identifying non-invasive biomarkers for early diagnosis and prognosis is imperative. MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression, are appealing biomarkers due to their stability in tissues and bodily fluids.

Objective: This study aims to discover miRNA-based biomarkers for the early detection and prognosis of ARVC in Boxer dogs.

Material and Methods: We obtained total RNA from Formalin-Fixed Paraffin-Embedded (FFPE) cardiac samples of Boxer dogs affected by ARVC, including those with sudden cardiac death (SCD group; n=4), non-cardiac related deaths (NCD group; n=4), and healthy mixed-breed controls that were euthanized as part of a prior study (CTRL group; n=4). These samples underwent small RNA next-generation sequencing (sRNA NGS).

Results: Our analysis identified fifty-seven miRNAs (FDR<0.01) displaying differential expression between the SCD and CTRL groups, with only one showing a distinction between SCD and NCRD (FDR=0.009). Conclusion: These unique miRNA expression patterns are promising as diagnostic and prognostic markers for ARVC in Boxer dogs. Their validation and testing in larger cohorts and liquid biopsy samples are particularly interesting.

The risk of many chronic diseases increases with age. With the rapidly growing elderly population, better understanding the biology of aging is critical. Advances in proteomics have allowed for the assessment of thousands of proteins in biological matrices, which can be translated into clinical biomarkers. This project aimed to investigate the biology of aging and identify potential biomarkers of aging utilizing proteomics techniques. We performed both untargeted and targeted proteomic analyses of liver tissues collected from young and old mice to identify specific proteins that are differentially regulated with age. First, we generated a protein library using comparative proteomics to identify differentially regulated liver proteins. Then used this library to develop Multiple-Reaction-Monitoring-Mass Spectrometry (MRM-MS) assays which facilitate the accurate quantification of proteins within different samples. For MRM assays, we selected several previously reported senescence-related proteins as well as several newly identified proteins from our study to quantify and assess differences in the specific peptides corresponding to the proteins of interest. MRM-quantification of target peptides allowed for the precise determination of which proteins are up- or down-regulated in old mice relative to young counterparts. These proteins include an apoptosis-inducing factor and eukaryotic translation initiation factor. Using these proteins that were differentially regulated during biological aging let us develop a method that identifies age-related biomarkers in a tissue-specific manner by studying the pathways they are involved in using a core protein database of known protein-protein interactions. This information is necessary for the future development of therapies to prevent age-associated diseases and promote proper tissue repair in older individuals.

Acanthamoeba is amphizoic amoeba majorly responsible for causing Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). The whole genome assembly along with proteomic profiling can help us better understand pathogenic and non-pathogenic Acanthamoeba isolates. Illumina and Nanopore sequencing were performed for keratitis, encephalitis, and non-pathogenic environmental isolates. A hybrid assembly was prepared for the AK and GAE isolates, while only the Illumina reads were utilized for a non-pathogenic environmental isolate. Based on the sequencing data analysis, genes including lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein (MBP) were selected as probable pathogenic targets that were evaluated for their relative gene expression in the keratitis and amoebic encephalitis animal model. In addition, liquid chromatography-mass spectrometry (LC-MS/MS) was performed for keratitis, encephalitis, and non-pathogenic environmental isolate. The expression analysis revealed phospholipase, lysophospholipase, and MBP genes to be significantly upregulated during AK while phospholipase, lysophospholipase, S8/S53 peptidase, and carboxylesterase genes were significantly upregulated during GAE in the animal model. The proteomic data revealed differential protein expression in pathogenic versus non-pathogenic isolates. The gene expression data suggests that the selected probable markers could play a role in the contactdependent and independent mechanisms of Acanthamoeba pathogenesis. In addition, the proteomic profiling of the 3 isolates revealed differential protein expression crucial for parasite growth, survival, and virulence. Our results provide baseline data for selecting possible pathogenic targets that could be utilized for designing knockout experiments in the future.

Spinal cord injury (SCI) is a devastating condition that causes variable and often significant sensory, motor, and autonomic dysfunction. Despite extensive research, the limited efficacy of clinical treatments highlights the need for a better understanding of the complex pathology underlying SCI in order to identify new treatment strategies for functional restoration. SCI pathology consists of an initial mechanical injury (primary injury) followed by biochemical cascades (secondary injury) which exacerbates the extent of damage and worsens functional outcomes. Acrolein, a highly-reactive α,β-unsaturated aldehyde, has been demonstrated to play a pivotal role in secondary injury events including oxidative stress, mitochondrial dysfunction, and lipid peroxidation. Excitotoxicity is another known secondary injury mechanism in SCI that results from an increase in extracellular glutamate levels. However, the interaction between acrolein pathology and excitotoxicity after SCI has not been investigated in detail. Under normal conditions, synaptic glutamate levels are tightly regulated primarily by astrocytic glutamate transporter-1 (GLT-1) receptors. Evidence showing that GLT-1 could be compromised by other reactive aldehydes suggests its potential as a key target for acrolein-mediated dysfunction that could contribute to excitotoxicity and neurodegeneration following SCI. Thus, we sought to first characterize GLT-1 expression and function following SCI, and then determine if acrolein mediates these findings by interacting with GLT-1. Using a clinically relevant T10 contusion model of SCI, we show that: 1) GLT-1 and acrolein levels display an inverse expression pattern as revealed by immunoblotting and immunohistochemical labeling, 2) acrolein interacts with GLT-1 as revealed by co-immunoprecipitation, and 3) the injection of exogenous acrolein to intact spinal cord tissue significantly reduced GLT-1 expression. Together, these findings suggest the causal role of acrolein in glutamate excitotoxicity following SCI by affecting GLT-1 protein expression. Furthermore, the implications of this study reveal potential targets for neuroprotective therapeutic intervention following acute SCI.

MPNST (Malignant peripheral nerve sheath tumor) is a highly aggressive sarcoma with a poor prognosis and a high rate of relapse. Surgical removal is the mainstay of clinical treatment, but it is always limited due to the tumor size and the location. Currently, no effective targeted therapy is available to this type of malignancy. Thus, a novel and efficient therapeutic drug target is desired. PRMT5, a type II protein arginine methyltransferase, has recently been reported as a promising therapeutic target for various types of human cancers with the MTAP gene deleted. Because the loss of MTAP causes cancer cells heavily dependent on the PRMT5 activity. MTAP deletion in MPNSTs led us to hypothesize that PRMT5 is an effective therapeutic target for MPNST. We first conducted IHC and discovered a decreased ratio of MTAP/PRMT5 in the MPNSTs compared to the benign tumor neurofibromas, indicating MTAP loss and consequential increased PRMT5 expression in cancer cells. Then, we inhibited PRMT5 activity by using shRNAs and the potent chemical inhibitors in the 2 pairs of MPNST cell lines with high or low MTAP/PRMT5 ratios. We found that cell growth of MTAP/PRMT5-low MPNST cell lines was suppressed, while limited effects on cell growth of MTAP/PRMT5-high MPNST cell lines. Mechanistically, we found compromised double-stranded DNA break repair in the PRMT5-inhibited MTAP/PRMT5-low MPNST cell lines. Overall, our results suggested that PRMT5 is a therapeutic target for MTAP-deficient MPNST.

Influenza viruses result in nearly a billion cases globally every year and pose a significant threat to the public health. Several potential pandemic influenza viruses have emerged in the last 2-3 decades, and some of these viruses have resulted in high mortality in humans. Currently available seasonal influenza vaccines provide strain-specific protection and thus are less effective against antigenically distinct strains. Therefore, a universal influenza vaccine is needed to provide comprehensive protection against a wide range of influenza viruses. This study targeted the influenza virus's conserved internal protein, nucleoprotein (NP), to develop a universal influenza vaccine. The NP of influenza A virus linked to the autophagy-inducing protein C5 (AIP-C5) to enhance NP-specific cell-mediated immunity (CMI) and were expressed in the replicative defective bovine adenoviral (Ad) [BAd] or human Ad [HAd] vector platform. A single intranasal immunization of mice with the BAd or HAd vector showed significant increases in number of interferon-gamma (IFN-γ) or interleukin 2 (IL-2) expressing cells in the spleen, mediastinal lymph node, and lungs, indicating the development of robust NP-specific CMI. Besides, there were significant increases in NP-specific IgG, IgG1, and IgG2a, antibody titers in the serum, signifying the induction of humoral immunity. Subsequently, groups of BAd vaccine vector-immunized animals were challenged with several heterosubtypic influenza A viruses, and protection efficacies were monitored by morbidity and mortality for two weeks post-challenge or lung virus titers at day three post-challenge. Significant protection from morbidity and mortality or reductions in lung virus titers were observed, suggesting the value of NP, AIP-C5, and BAd vector in designing an effective universal influenza vaccine.

The present abstract discusses the challenges associated with traditional checkpoint blockade immunotherapy for prostate cancer which is characterized as an immuno-excluded tumor with limited CD8+ T cell infiltration in the tumor microenvironment (TME). Cytotoxic CD8+ T cells have a crucial role in antitumor immune response associated with conventional immunotherapy, as they dominate the TME of inflamed tumors. The low presence of CD8+ T cells in the TME has been attributed to the abundance of immunosuppressive cells, such as Myeloid Derived Suppressive Cells (MDSCs) and Tumor-Associated Macrophages (TAMs), which are found in tumor parenchyma. Previous studies in our laboratory identified FRβ as a marker for immunosuppressive MDSCs and TAMs in the TME of human and murine prostate tumors. Those studies also reported that FRβ expression is restricted to the MDSCs and TAMs in the tumor. In collaboration with Dr. Phillip Low's group, we have targeted immunosuppressive cells in the TME, MDSCs, and TAMs, using FRβ-targeted Toll-Like Receptor 7 agonist (FA-TLR7a), an immunostimulatory drug conjugated with folate. FA-TLR7a binds to FRβ, is internalized, and activates TLR7 in immunosuppressive MDSCs and TAMs. The objective is to reprogram MDSCs and TAMs from protumorigenic to antitumorigenic myeloid cells. In this study, we found that reprogramming leads to lower immunosuppressive activity, increased CD8+ T cell infiltration in the TME of prostate adenocarcinoma, and significant antitumor activity. The antitumor response is mediated by CD8+ T cells. These findings demonstrate that targeting FRβ with a strong immunostimulatory ligand modifies the tumor microenvironment and induces antitumor activity.

Unregulated, systemic inflammation can result in significant production losses and negative health outcomes in dairy cows. Soluble mediators and non-immune cells (e.g., platelets) have been studied for their expansive role in mediating inflammation. However, little research is presently available evaluating these mediators in dairy cows. Our objective was to compare the plasma oxylipin and endocannabinoid profiles and the platelet and plasma proteomic profiles of healthy cows against cows suffering from elevated systemic inflammation as indicated by plasma haptoglobin (Hp) concentrations. Postpartum cows at 3 DIM with plasma Hp concentrations ≥ 0.50 g/L and no clinical disease were enrolled into the high-inflammation group (n=8). Cows with plasma Hp concentrations ≤ 0.1 g/L and no clinical disease were enrolled into the lowinflammation group (n=8). Blood samples were collected for plasma lipidomics, proteomics, and platelet isolation. Lipidomics data was assessed using a mixed liner regression model where parity was included as a fixed effect. Targeted lipidomics were completed and revealed differences in the plasma oxylipin and endocannabinoid profiles in high- and low-inflammation cows. Cows in the high-inflammation group had increased plasma concentrations of the oxylipins 9(S)-HpOTrE, 9(S)-HOTrE, 13(S)-HpOTrE, and 9,10-EpOME, and an increased concentration of the endocannabinoid anandamide. Platelet proteomics identified 2,074 unique proteins at a 1% false discovery rate (FDR). 155 platelet proteins were identified as differentially abundant between the high- and low-inflammation groups, and included proteins involved in platelet granule release and cellular iron uptake. Plasma proteomics identified 331 unique proteins at a 1% FDR. 24 differentially abundant proteins were identified, including proteins involved in autophagy and immune mediation. These results suggest that cows suffering from systemic inflammation may have impaired disease resistance and that platelets could contribute to their inflammatory state. More research is needed to elucidate the biological function of many of the proteins and lipid mediators identified in this study.

Cervical cancer is the fourth most common cancer in women. There is a significant disparity in cervical cancer burden and death, and 90% of diagnosed cases occur in low/middle-income-countries (LMICs), where there is limited access to screening tests due to their unavailability and affordability. Thus, there is a vital need to develop an affordable, easy-to-use point-of-care (POC) screening test. Therefore, this work aims to create a lateral flow immunoassay (LFIA) for screening Cyclin-dependent kinase inhibitor 2A (CDKN2A or p16INK4a) protein overexpressed in neoplastic epithelial cells of the cervix.

The test is based on a biochemical interaction of antigen-antibody. It consists of four parts: a sample pad, a conjugate pad, a reaction membrane, and an absorbance pad, which will be housed in a backing card. A sample from cervical swabs will be added will be placed on the pretreated sample pad. Then, the dried conjugate will be applied to the conjugate pad, interact with the analyte in the sample, and migrate into the reaction zone. At this point, a nanoparticle-conjugated antibody is added to the membrane.

We have studied the binding kinetics of three antibodies to our target protein and tested their conjugation ability and stability to gold nanoparticles. We chose the range of nanomolar to picomolar to indicate a strong binding affinity of our target antibody. The highest intensity for our antibody-to-gold nanoparticle was achieved at the 100:1 molar ratio.

Our developed POC test is designed to be simple, cost-effective, and will significantly impact cervical cancer treatment, particularly in LMICs.

Objectives: To determine if an ultrasonic Doppler-guided technique (UDGT) leads to improved placement efficacy (time, success) of feline dorsal pedal arterial catheters versus the traditional palpation-guided technique (TPT).

Materials and methods: A total of 26 adult, client-owned cats requiring sedation or general anesthesia for any reason, greater than 12 months of age and 3.0 kg, and with Doppler blood pressures of at least 80 mmHg were enrolled. Each rear limb was randomly assigned for dorsal pedal arterial catheterization using either the UDGT or TPT. With the UDGT, the location of the artery was identified by an audible sound using the Doppler. Successful catheter placement was confirmed by visualization of an arterial pressure waveform using a transducer and monitor system attached to the catheter. The Kaplan-Meier method and log rank test were used to compare the two techniques.

Results: The overall proportion of successful arterial catheterization was 17% (9/52); 19% (5/26) via UDGT and 15% (4/26) via TPT. Among successful arterial catheterizations (n=9), the average time to catheterization was 339  198 seconds: 328  237 seconds (n=5) with UDGT and 353  171 seconds (n=4) with TPT. The log rank test showed the two techniques were not significantly different in likelihood of successful arterial catheter placement or time to successful catheterization (p=0.698). An arterial flash occurred in 62% (32/52) of the limbs, 58% (15/26) with the UDGT and 65% (17/26) with the TPT. Complications (self-limiting bruising, hematoma formation) were observed equally between UDGT (3/26 limbs) and TPT (3/26 limbs) in 6 cats.

Conclusions and relevance: The UDGT did not improve the efficacy of catheter placement compared to the TPT. Few complications were associated with arterial catheterization.

Antimicrobial resistance is a growing concern globally. While studies have atempted to quantify the use of antimicrobials in humans and livestock, there has been litle data available to estimate the frequency of use in pets. In the US, most antimicrobials used in pets are prescribed by veterinarians during visits or surgeries. Recently, the Morris Animal Foundation (MAF) has made available data from a longitudinal study of >3000 Golden Retrievers called the Golden Retriever Lifetime Study (GRLS). This dataset provides information on the dogs’ medical histories including their dates of entry into the study, age, sex, neuter status, diagnoses, and medications prescribed. For the purposes of this analysis, the nine most commonly prescribed antimicrobials were selected. Prescriptions were summarized across time through a cohort analysis. These antimicrobials represent several classes of antibiotics that have implications for both human and animal health. The total time observed for this cohort was 17,055 dog-years. Preliminary analysis of the data has shown that the most prescribed drug, Metronidazole, has seen a decrease in the rate of prescriptions, from once in every 4 dog-years in 2013 – to once in every 200 dog-years in 2021, although the average age of the dog cohort also increased during this time-period. Entire female dogs were prescribed Metronidazole at a significantly lower rate than all other dogs in the study. This is the first time to our knowledge that this type of study has been conducted using a large sample of pets that is representative of the US population. Furthermore, this data represents the sole freely available source for the long-term tracking of antimicrobial usage in pets. After completing univariable analysis, we plan on conducting multivariable analysis. There is potential for future studies that can provide further insight into this area.

The objectives of this study were to identify complication and short-term mortality rates for partial and full thickness cecal and colonic surgery, as well as to explore risk factors with a specific emphasis on lack of supervision by an experienced board-certified surgeon. We hypothesized that dogs undergoing full thickness cecal and colonic surgery would have both increased dehiscence rate and short-term mortality rate when compared to partial thickness colonic surgery. We also hypothesized that board-certified surgeon assistance would significantly decrease complications and increase survival to discharge compared to surgery performed by residents without supervision. Dogs were included if they had undergone surgery that involved the cecum or the colon. Medical records from three tertiary hospitals were reviewed for patient demographics and clinical data. The complication and mortality rates for full thickness cecal and colonic surgeries was higher than partial thickness surgeries. There was no indication of influence of board-certified surgeon presence in surgery on complications or mortality (p=0.99 and p=0.99 respectively). The findings of this study are consistent with previous literature in veterinary medicine and human medicine where an experienced surgeon presence had no significant benefit in terms of reducing complication rates or mortality. The main limitations of this study include lack of standardized follow up, diagnostics, treatment plans and the relatively low sample size despite the multi-institutional sample population. Partial thickness cecal and colonic surgery has potentially lower complication rate and mortality compared to full thickness surgery. Presence of board-certified surgeon has no influence on complication rate or mortality.

Background: Oxidative stress is well-documented in people with hemolytic diseases and is reported in canine immune-mediated hemolytic anemia (IMHA). Oxidative stress induces erythrocyte membrane phosphatidylserine externalization in people, causing eryptosis and thrombus formation. This has not been evaluated in dogs. Acute thromboembolism is the leading cause of natural death in canine IMHA despite thromboprophylaxis, highlighting the need for novel therapeutic targets.

Objectives: To determine if oxida,ve stress induces PS externaliza,on in canine erythrocytes (objective 1), and if exposure to antioxidants prevents such changes (objective 2).

Methods: In vitro, experimental study using blood from five healthy adult purpose-bred research Beagles. Blood was collected from each dog and erythrocytes harvested. For objective 1, erythrocytes were exposed to pro-oxidant agents tert-butyl hydroperoxide (TBHP) at 2, 3, or 4 mM, or 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) at 30, 40, or 50 mM. For objec,ve 2, erythrocytes were exposed to 3 mM TBHP and N-acetylcysteine-amide (NACA) at various concentrations (0, 1, or 3 mM). PS externalization was assessed using flow cytometry (Annexin V assay) with median fluorescence intensity (MFI) recorded.

Results: Tert-butyl hydroperoxide at 3 and 4 mM caused increased PS expression in erythrocytes (P < 0.05). AAPH at all concentrations caused increased PS expression (P < 0.01). NACA at all concentrations prevented significant PS expression from erythrocytes exposed to TBHP (P < 0.01).

Conclusions: Oxidative stress causes PS externalization in canine erythrocytes and NACA ameliorates this effect. Future studies are needed to determine if this occurs in IMHA dogs and its role in promoting thromboembolism.

Background: Currently, there is growing interest in using tumor immune signatures to guide more effective therapeutic strategies for dogs with invasive urothelial carcinoma (InvUC). A non-invasive, urine-based assay to characterize tumor-associated immune responses would be valuable in this setting. The objective of this study was to use a urine-based method to characterize gene expression signatures in dogs with InvUC, focusing on immune-related genes.

Methods: RNA was isolated from archived paired tumor and urine specimens from dogs with biopsy-confirmed InvUC (n= 20), and from normal bladder mucosa (n= 4) and urine sediments (n = 9) from normal dogs. Tissue and urine RNA were queried using the nCounter Canine IO Panel (NanoString Technologies, Seattle, WA). Differential gene expression profiles were compared using Rosalind. nCounter expression profiles from tumor samples were then compared to tumor bulk RNA-seq data.

Results: nCounter analysis identified 65 differentially expressed genes in InvUC tumor samples and 288 differentially expressed genes in InvUC urine sediment samples. Differentially expressed genes identified in InvUC urine sediment included tumor subtype markers, immune response markers, and genes associated with the p53 and MAPK pathways. Correlation of NanoString results from tumor and urine samples from the same individual was high, while correlation of NanoString and RNA-seq results was moderate, as expected with two different technologies. Gene signatures associated with major leukocyte populations were detected in both tumor and urine samples.

Conclusions: Counter analyses can identify signatures from immune cell populations, markers of immune function, and cancer signaling pathways in tumor and urine specimens from dogs with InvUC. Further investigations are underway to classify tumors based on urine immune signatures and monitor changes over time.

Balamuthia mandrillaris is a free-living amoeba that can cause severe central nervous system (CNS) infection and skin lesions, leading to a high mortality rate (~90%). The current diagnostic methods for this amoeba have many limitations and are not very sensitive and specific. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the 18S rRNA gene for B. mandrillaris. LAMP assays detected 5 different B. mandrillaris strains, no cross-reactivity with the DNA of other free-living amoeba, selected protozoa or bacteria. The lower limit of detection for a positive signal was 10fg/μL of extracted DNA, 10 original trophozoites, or 1 heated trophozoite/100 μL of media, which showed 10~100 fold higher sensitivity than PCR. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing B. mandrillaris, particularly in resource-poor areas.

Background: Balloon valvuloplasty (BVP) is standard treatment for severe valvular pulmonic stenosis (PS) in dogs. Efficacy and safety of low-pressure balloon catheters (LP) and high-pressure balloon catheters (HP) for BVP has not been compared.

Hypothesis/Objectives: There will be no difference in pressure gradient reduction between HP and LP. Cardiac troponin I (cTnI) will be higher with HP compared to LP immediately following BVP.
Animals: Twenty-three (23) client owned dogs with severe PS.

Methods: Prospective, randomized study. Patients matched based on pulmonic annulus size. Echocardiographic measures of PS severity included transvalvular pressure gradient (PGmax) and aorta-to-pulmonary artery velocity time integral ratio (Ao/PAVTI) at baseline, 18-24 hours following BVP and at 3-month recheck. Serum cTnI was measured at each time point.

Results: There were 11 dogs in the LP group and 12 in the HP group with no difference in sex, weight or age between groups. There was no significant difference in PS severity between groups at baseline [(Ao/PAVTI; p=0.376), (PGmax; p=0.406)], 18-24 hours post-BVP [(Ao/PAVTI; p=0.270), (PGmax; p=0.263)], or at 3-month recheck [(Ao/PAVTI; p=0.418), (PGmax; p=0.184)]. There was no significant difference in serum cTnI between groups at baseline (p=0.069), 18-24 hours post-BVP (p=0.378), or at 3-month recheck (p=0.705). Major complications were rare with no difference between HP and LP.

Conclusions and Clinical Importance: There was no difference in reduction of PS severity between HP and LP. The use of HP is not expected to provide superior reduction in PS severity compared to LP or be associated with increased myocardial injury.

This research aimed to evaluate the outcomes of a radiotherapy protocol, consisting of five fractions of 4Gy each, resulting in a total dose of 20Gy for apocrine gland anal sac tumors and local lymph nodes in canines. This protocol was assessed as a palliative treatment for macroscopic tumors alone, or in combination with additional therapies under different scenarios. Medical records from fifty canine patients met the inclusion criteria and were divided into different treatment groups: radiotherapy alone (n=22, 44%), radiotherapy with chemotherapy or targeted therapy with toceranib (n=18, 36%), surgery with radiotherapy (n=5, 10%), and surgery with radiotherapy and chemotherapy or targeted therapy with toceranib (n=5, 10%). Patients who received radiotherapy alone had a median survival time of 384 days (95% CI 198–569), and 628 days (95% CI 579–676) for RT + additional therapies. The median time to progression for patients with radiotherapy alone was 337 days (95% CI 282–391 days), and 402 days (95% CI 286–517 days) for radiotherapy plus additional treatments. Acute side effects were mild, with the majority having diarrhea (66%), and only one patient developed grade III late effects VRTOG v1 classification. Results demonstrated that radiotherapy alone under this protocol provided a comparable median time to progression vs radiotherapy plus additional treatments while maintaining acceptable side effects. The combination of this protocol with other treatment modalities offers attractive results for local disease control and survival while maintaining acceptable toxicities. Overall, these findings contribute to the growing evidence supporting the role of radiotherapy in managing apocrine gland anal sac adenocarcinoma in dogs.

Clinical outcome, details of vascular aberrance, treatment complications, and prognosis have been well-documented for the typical small breed dogs with congenital extrahepatic portosystemic shunts (EHPSS). Less frequently, certain larger breed dogs have EHPSS. There is a lack of understanding of the clinical presentation and general outcomes associated with these dogs. The aim of this study was to describe demographics, clinical presentation, shunt anatomy, clinical progression, and complications in large dogs ≥15 kg with EHPSS treated with or without surgery. Medical records from 11 tertiary institutions of dogs ≥15 kg diagnosed with EHPSS between January 01, 2005 and December 31, 2020 were reviewed, with minimum follow-up of 90 days. Clinical signs, diagnostics, vascular anatomy, treatments, and perioperative complications were assessed. Sixty-three dogs were included. Median age was 21.9 months (IQR: 9-36.8). Of the surgically treated dogs, 14/45 (35.6%) had short-term complications, and 3/45 (6.7%) had shunt-related deaths. Medical management was discontinued in 15/40 and reduced in 9/40 of surviving dogs who had surgical attenuation. All medically managed, nonattenuated dogs (18/18) were maintained on their original shunt-related medication regimens.

Clinical presentation of dogs ≥15 kg with extrahepatic portosystemic shunts was similar to the more commonly reported small breed dogs. Surgical management of single EHPSS in large dogs ≥15 kg had similar clinical short-term outcomes as small breed dogs. Clinicians should be aware that large breed dogs with EHPSS share similar characteristics and clinical outcomes to small breed dogs. Surgical treatment is a viable option for large breed dogs with EHPSS.

The periparturient period is a challenging time for dairy cows, with high incidences of metabolic and infectious diseases. Excessive systemic inflammation in the postpartum is associated with increased risk of diseases. Haptoglobin (Hp), a major acute-phase protein, is widely used as an inflammatory marker in cattle. The objective of this study was to establish thresholds for plasma Hp at 1, 3, 5, and 7 days in milk (DIM) associated with disease and culling within 60 DIM. Blood samples were collected from 513 cows from 2 commercial herds in IN at 1, 3, 5, and 7 DIM, and plasma Hp concentrations analyzed via ELISA. Multivariable Poisson regression models were built to evaluate the associations of Hp at each DIM with the risks of metritis, clinical mastitis, clinical ketosis (CK) and(or) displaced abomasum (DA), and culling. Critical thresholds were derived from receiver operating characteristic curve analysis for significant DIM. Plasma Hp at 1 and 3 DIM were associated with the risk of metritis (thresholds: 0.20 and 0.44 g/L; risk ratios: 3.0 and 6.8, respectively). Plasma Hp at 3 DIM was associated with the risk of CK and(or) DA (threshold: 0.52 g/L; risk ratio: 9.4). Plasma Hp at 1, 3, 5, and 7 DIM were associated with the risk of culling (thresholds: 0.09, 0.50, 0.30, and 0.13 g/L; risk ratios: 5.5, 7.0, 5.2, and 5.6, respectively). Haptoglobin in the first week postpartum is associated with negative health events, and DIM of measurement must be considered when evaluating these associations.

There is an absence of studies evaluating the lipidomic profile of Gram-positive (GP) bacteria using Multiple Reaction Monitoring Profiling (MRM-Profiling), as well as comparing the effects of new antibiotics with similar targets and close structures. The significance of this work lies in how antibiotics' impact on bacterial surface lipids. Understanding antibiotic-induced lipid changes offers insights into novel drug targets and strategies against resistance. In this study, we have performed a comparison between Vancomycin, HSGN238 and HSGN94 and their impact on the lipidomic profiles of the strain USA300. In this in vitro challenge (3h, 37°C, 200 rpm, 5mL of TSB), half-MIC of the compounds were used, with samples prepared for analysis at the LC-MS/MS guided by MRM methods. From the classes of lipids that can be discovered by MRM, the majority were TG (101), DG (62), PG (45) and PC (19). Univariate analysis showcased distinct lipidomic signatures among the antibiotic treatments, demonstrating a clear separation, highlighting that is possible to track specificities from the groups analyzed, even in different culture medias (MH and TSB). The sublethal doses were able to challenge the strain USA300, causing this modification on the lipid standards, leading to the concept that this method can sensitively detect lipidic changes playing a role at the early stages of AMR. This innovative S. aureus lipidomics approach paves the way for understanding antibiotic effects on bacterial lipid profiles and their relation to antimicrobial resistance. By revealing membrane alterations, it can lead to an early resistance detection and targeted therapy development.

Liquid biopsy technology has become an emerging, non-invasive method for identifying tumor-associated mutations that may play a role as a biomarker in human and veterinary oncology. This study investigates the use of cell-free DNA (cfDNA) isolation and next-generation sequencing technology in order to identify and quantify mutated genes from circulating tumor DNA (ctDNA) in dogs with invasive urothelial carcinoma (InvUC). A total of 8 dogs with InvUC were included. cfDNA was isolated using a Nonacus BeadXtract cfDNA kit (Nonacus, Quinton, UK) and quantified using cfDNA ScreenTape analysis. Mutations were identified following sequencing and processing through a variant calling pipeline of 68 genes developed in the CanCan Diagnostic Lab. Mutations were observed in all 8 samples of dogs with iUC, with at least 20 mutations identified in >75% of samples. Some of the most frequently mutated genes included: TTN, NF-1, FBXW7, TET2, MAP3K14, TP53, MTAP, and BRCA2; with a number of those genes having known roles in cell signaling and regulatory pathways. Mutations in BRAF were found in 4/8 (50%) of dogs with InvUC. The results of our study demonstrates that mutations in cfDNA can be isolated and quantified from the plasma of dogs with iUC. This cfDNA mutational analysis and quantification may be a potentially useful tool for cancer detection and monitoring changes in the tumor during therapy and over time.

Wildlife overpopulation has detrimental consequences for the sustainability of ecosystems. Contraceptive vaccination using native porcine zona pellucida (nPZP) proteins isolated from ovaries is among the most humane, safe, and least disruptive options to mitigate this. However, improvements in the longevity, safety, and preparation efficiency of current vaccines are needed. This study compared the humoral response and fertility outcomes in female mice immunized with different vaccine formulations. Antigens included nPZP, recombinant PZP2 and PZP3 and recombinant equine IZUMO1 derived from Chinese hamster ovary (CHO) cells, and PZP3 derived from GnTI deleted HEK293 cells. Antigens were formulated with an AS03-like emulsion adjuvant, AddaS03, or with a combination adjuvant comprised of a plant-derived nanoparticle, Nano-11, and a stimulator of interferon genes (STING) agonist, ADU-S100. Serum antibody responses to nPZP and IZUMO1 were determined by ELISA. The IgG, IgG1 and IgG2b levels were significantly increased after the third dose with the highest titer seen in mice immunized with nPZP with AddaS03. Although least abundant, IgG2a levels were highest in Nano-11/ADUS100 groups, indicating a more balanced Th1/Th2 response. Fertility was assessed by fetal count, and only the nPZP with AddaS03 group had a significant decrease in fertility. To conclude, the nPZP with AddaS03 formulation appears to be a promising alternative contraceptive vaccine, although trials with wildlife species are necessary for further formulation refinement.

The gold standard for determining disease progression of glaucoma in research settings involves optic nerve axon counting, which is performed ex vivo. With the advancement of noninvasive imaging techniques, it is possible to image details of the retina and optic nerve head (ONH) in vivo. This study sought to determine the relationship between the number of axons at the ONH and various parameters using optical coherence tomography (OCT) and confocal scanning laser ophthalmoscopy (cSLO) in dogs with various stages of primary open angle glaucoma. Beagles (n=6 eyes) with open angle glaucoma and age matched non-glaucoma dogs (n=2 eyes) were included in the study. OCT and cSLO images were taken of each eye, capturing the ONH, and a built-in software was used to measure neuroretinal rim area, ONH area and diameter, and optic cup diameter. Total retinal thickness, ganglion cell complex (GCC), and outer retinal thickness were also measured. Slides with the ONH samples were scanned and axons were manually counted using Image J software. A strong positive correlation existed when comparing the following parameters to the number of axons: neuroretinal rim area (r=0.91, p<0.01), ONH area (r=0.73, p<0.04), total retinal thickness (r=0.75, p=0.02), and GCC (r=0.82, p<0.01).There was a strong negative correlation (r=-0.75, p=0.03) between the number of axons and optic cup area and a moderate negative correlation (r=-0.70, p=0.12) between number of axons and age. The strong positive and negative correlations between the number of optic nerve axons and the various parameters measured support the utility of OCT and cSLO as useful noninvasive imaging techniques to assess the progression of glaucoma in vivo in dogs with open angle glaucoma.

Clostridioides difficile, a Gram-positive, spore-forming, anaerobic bacterium, is an opportunistic pathogen that causes severe colitis and death in humans and animals. Antibiotic treatmentinduced disturbances in the gut microbiota frequently exacerbate infections caused by this bacterium. There is substantial evidence in the literature demonstrating that host stress can lead to changes in the gut microbiota. The gut is the epicenter of hormonal exuberance during stress. Stress modifies the gut physiology while modulating the gut microbiome. Previous studies have shown that norepinephrine, an abundant hormone in the gut experiencing stress, significantly affects the growth and virulence of many Gram positive and Gram negative bacteria. This research project aims to investigate the extent to which norepinephrine influences the virulence of C. difficile. The RNA seq and RT-PCR were performed to study the changes in expression and abundance on virulence genes of C. difficile. The effect of increasing concentrations of norepinephrine on the growth and virulence of C. difficile was evaluated. Our findings provide valuable insights that can inform treatment modalities and guide patient management decisionmaking processes. Results from this study could help us develop a treatment strategy that could include adrenergic blockers in treating C. difficile colitis.

Early detection of pathogens is imperative for the health of laboratory zebrafish and to ensure reproducible scientific results. While most pathogens are present as subclinical or chronic infections, their presence can be a confounding factor in data collection, and some infections can affect zebrafish health and reproduction. Current methods to test for pathogens sample a myriad of sources, including cage swabs, detritus, water collection or filtration, and whole sentinel fish PCR or histopathology. Sentinel mice have been used in the past for mouse health monitoring, but there has been a recent shift to replace sentinel animals with filters in rack exhausts. We wished to see if such methods could be translated from mouse racks to zebrafish systems. We placed filters in the sumps of zebrafish racks to be collected and tested for pathogens at monthly intervals using PCR, and results were compared to those detected on filters in which water was actively vacuum pumped through, swabs of sump biofilm, and whole fish PCR. Results suggest that the efficacy of filters may wan with prolonged use, with sensitivity being greatest at 60 days and then decreasing at 90. Results also showed the limitations of current testing methods for zebrafish health monitoring, with efficacy of detecting pathogens varying widely based on the method of collection.

It is well-known that animals in laboratory facilities require environmental enrichment to allow them to display their natural behaviors. Examples of environmental enrichment include, but are not limited to, toys, nesting materials, gnawing materials, food and treats, and additional shelters. However, when given enrichment, it is unknown whether mice actually benefit from a specific enrichment, or if they have a preference as to the specific type of enrichment they receive. To see whether mice do indeed have a preference in their enrichment, several types of commercially available enrichment were placed in cages with singly housed C57BL/6 mice. After a few days of acclimation, acute behavioral trials and physiological analyses were conducted to see how the mice reacted to their enrichment. Later, chronic behavioral trials and physiological analyses were conducted to measure long-term effects of whether the provided environmental enrichment benefited the mice. Using the results from the behavioral trials and blood samples, we will observe the behavioral markers of evident stress along with analyzing the white blood cell counts for evidence of stress. This will allow visualization of any benefits from certain types of enrichment, allowing researchers to purchase that enrichment over others in the future.

Equine protozoal myeloencephalitis (EPM) is a rare, but economically devastating, degenerative neurological disease caused by Sarcocystis neurona, an intracellular protozoan. Despite high seroprevalence of S. neurona, very few horses develop EPM or present with broad neurological signs of weakness, ataxia, and neurogenic muscle atrophy. Akin to sister genera, S. neurona relies on discharge of excretory-secretory proteins (ESPs) from its apical organelles to invade the host cell and survive intracellularly. Investigation into S. neurona ESPs may reveal important virulence factors associated with EPM progression. Therefore, the aim of this study was to evaluate the antigenicity and generate a proteomic profile of S. neurona ESPs for future studies and development of additional diagnostic tests. Cell-culture derived live S. neurona underwent induced secretion and the ESPs were collected for analysis. Sera and cerebrospinal fluid from five horses of known EPM status were tested for antibodies to S. neurona ESPs by Western blot. Reactivity at two distinct molecular weight ranges was observed and amino acid sequencing is needed to establish the identity of these unknown proteins. Bottom-up proteomics of in-gel digested ESPs was performed via Mass Spectrometry and 92 S. neurona proteins were identified. 21 proteins were found to be from secretory organelles, 22 from other cellular locations, and the remaining are unstudied with unknown localization. Further optimization of sample preparation and data analysis is required for deeper characterization. Overall, this study has provided a glimpse into S. neurona ESPs and establishes a foundation for their use in future research aimed at developing new diagnostic tools for EPM.

Understanding the vascular anatomy of commonly used laboratory animals is necessary to improve research outcomes of studies focusing on image analysis (e.g. magnetic resonance angiography) and surgical approaches of comparative models. The rat is a popular model to use in experimental studies. However, due to the small size of the rat it can be difficult to visualize some of the anatomical details we wish to study. Attempts to perfuse the vascular system of the rat often omit the smallest of vessels. This method development study aims to determine the best procedure to generate casting of the rat laryngeal vascular system. Various combinations of saline, latex, and formalin were used for casting of the vascular system. Perfusion was attempted with both a perfusion pump and the rat heart pumping. Freeze thaw specimens fixed with formalin and latex produced the best cast, with the superior thyroid artery visible. Rats that were formalin fixed and then casted with latex produced the best perfusion results. In the future, a dehydration study is planned that will study how dehydration changes the effect of estrogen on ultrasonic vocalizations, blood vessel geometry, and the vocal fold tissue of the rat larynx. These casting method results will be used as a model for that dehydration study.

It has been shown that changes in sympathetic innervation to the heart are correlated with arrhythmogenesis, as it can lead to heterogeneous changes in cardiac electrophysiology. The cervicothoracic (stellate) ganglia are one of the final common pathways for extrinsic cardiac sympathetic fibers, and thus changes in its activity have been linked with arrhythmia development. Traditionally, stellate ganglia nerve activity (SGNA) has been measured invasively by surgically implanting electrodes directly into the ganglia or the subcutaneous space above them. More recently, studies have shown that sympathetic nerve activity can be measured on the skin’s surface, and that this method is accurate in estimating SGNA. Currently, the canine research models have relied on artificially induced arrhythmia. However, this method does not capture the change in nerve activity that occurs during progressive heart disease. The present study examined skin sympathetic nerve activity in dogs with naturally occurring arrhythmias via a non-invasive recording technique. Conventional ECG electrodes were placed over the approximate area of the stellate ganglion, on either side of the body. Simultaneously, we recorded traditional ECG activity using standard clips and procedures. Ganglion activity in dogs with good cardiac health was also obtained to evaluate sympathetic tone in diseased versus healthy patients. Examining sympathetic activity in the skin of dogs with naturally occurring cardiac disease can provide more clinically applicable insight. Understanding how the sympathetic nervous system affects cardiac electrophysiology, can lead to the design of more efficacious treatments for malignant arrhythmias in the future.

Three-dimensional (3D) modeling using computed tomography (CT) scans is becoming increasingly popular in veterinary medicine. CT scans create a series of images that can be used to generate 3D models. Error during 3D modeling has been reported. In human medicine, femurs are positioned perpendicular to the CT scan, but anatomical differences in veterinary medicine do not allow for this positioning. Standard procedures for CT scans used in the generation of 3D models have not yet been developed in veterinary medicine. The goal of this research is to examine the effect of femur angle during CT scan on 3D modeling. Soft tissue was dissected from three pairs of femurs from beagles. All six femurs were placed on a custom jig in a CT scanner and one scan was obtained at each 0, 20, 40, 60, and 80 degrees relative to the table. From these scans, 3D models were generated using open-source 3D modeling software. This yielded five models of each femur, one at each listed angle. Surface area and volume of each segmentation were calculated. The five models of each femur were overlayed and an iterative process was used to minimize error. Hausdorff distances were calculated and heat maps generated comparing models from each angle to the model from the 0 degree angle scan. Repeated measures ANOVA will be run to analyze the effect of femur angle during CT on surface area, volume, and maximum and mean Hausdorff distances. We expect a decrease in surface area and volume and an increase in maximum and mean Hausdorff distances as femur angle increases. We expect errors in the model to be localized to the proximal and distal ends of the femur where bone geometry is more complex.

Marwa Alhashimi ¹, Ahmed Elkashif ¹, Shubhada K Chothe ^2,3, Ekramy E Sayedahmed ¹, Wen-Chien Wang ¹, Padmaja Jakka ^2,3, Abhinay Gontu ^2,3, Santhamani Ramasamy ^2,3, Lindsey Labella ^2,3, Meera Surendran Nair ^2,3, Ruth Nissly ^2,3, Suresh V. Kuchipudi ^2,3, and Suresh K. Mittal ¹

¹Department of Comparative Pathobiology, Purdue Institute of Inflammation, Immunology and Infectious Disease, and Purdue University Center for Cancer Research, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA.

²Department of Veterinary and Biomedical Sciences, Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, USA.

³Animal Diagnostic Laboratory, Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, PA, USA.

The continuous emergence of variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease-2019 (COVID-19), remains a major global health threat. The efficacy of the first-generation COVID-19 vaccines has been seriously undermined by the emergence of immune escape and highly transmissible variants, including the recent Omicron subvariants BA.2, BA.4, & BA.5. To develop the next-generation of the COVID-19 vaccine, we designed human adenoviral (HAd)-based vectors expressing spike (S), membrane (M), or nucleocapsid (N) proteins of SARS-CoV-2 with or without an autophagy-inducing peptide C5 (AIP-C5) (HAd-S/C5, HAd-S, HAd-M/C5, HAd-M, HAd-N/C5, and HAd-N). Mice were immunized intranasally (i.n.) with HAd vectored vaccines alone or in various combinations leading to the induction of significant levels of antigen-specific humoral (mucosal and systemic) and cell-mediated immune responses. The inclusion of AIP-C5 resulted in significantly higher levels of cell-mediated immune responses. Immunization with vectors expressing S-C5, M-C5 & N-C5 in a vaccine formulation resulted in considerably higher levels of antigen-specific cellular immune responses compared to the groups immunized with the vector expressing a single antigen. Vaccinated animals showed high virus-neutralizing antibody titers against delta and omicron variants. Immunized ACE2 transgenic mice were challenged with SARS-CoV-2 and monitored for morbidity and mortality for two weeks to investigate the protective efficacy. Significant reductions in morbidity and mortality were observed in the groups immunized with vectors expressing S-C5, M-C5 & N-C5. Our results suggest that the inclusion of M and N antigens with S correlated with improved protection and, therefore, can serve as a promising strategy for the next-generation SARS-CoV-2 vaccines.

Nanoparticulate silver (AgNP) is a versatile product with established bactericidal activity against a range of bacteria. The study had two objectives: 1) establish the  in vitro bactericidal activity of AgNP against Methicillin-resistant  Staphylococcus pseudintermedius (MRSP) and  Escherichia coli using commercially available AgNP and 2) compare the in vitro bactericidal activity of free AgNP and AgNP incorporated into carriers for sustained release (SR-AgNP). It was hypothesized 1) antibacterial activity would be demonstrated against both MRSP and  E. coli by free AgNP and 2) antibacterial activity would not differ between free AgNP and SR-AgNP. Meuller-Hinton broth was inoculated with bacteria at a variety of concentrations and incubated with 10 µg/ml AgNP in a microdilution assay plate, sub-cultured to blood agar, and incubated for determination of colony forming units (CFU). Results showed reduced bacterial growth up to a bacterial concentration of 10 ¹ CFU/ml for MRSP and 10 ³ CFU/ml for  E. coli. Then, Meuller-Hinton broth was inoculated with bacteria at concentrations determined in objective one and incubated with 10 ug/ml AgNP incorporated into gelatin sponge, calcium sulfate hemihydrate (CSH) beads, or poloxamer 407 gel, sub-cultured to blood agar plates, and incubated for CFU determination. Free AgNP had greater antibacterial activity compared to SR-AgNP gelatin sponge and SR-AgNP CSH beads, but not SR-AgNP poloxamer 407 gel. This study did not assess the effect of wound bed characteristics. AgNP exhibits antimicrobial activity against MRSP and  E. coli and has the potential for use in veterinary wound care.

N. Bhowmik, K. J. Ekenstedt Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN

Canine short spine syndrome is a collection of rare malformations affecting vertebral development, causing shortening and compression of various spinal segments; it has occurred sporadically since at least the 17th century. Two Coonhounds and one mixed breed dog were diagnosed with “short spine” via gross and radiographic examinations. Whole-genome sequencing (WGS) was performed in three related Coonhounds (two affected and one unaffected) and one affected mixed breed dog. The private variants of all four dogs were identified via comparison to 728 WGS control dogs representing 64 breeds. These variants were next filtered based on their predicted impacts (high, moderate, low) and how they altered protein sequences. VarElect software was then used to rank and prioritize disease-causing candidate genes. Both affected Coonhounds had a homozygous A to G missense variant in exon 7 of the DLL3 (Delta like Canonical Notch Ligand 3) gene. The related, unaffected Coonhound was heterozygous for this variant. Thirty-four unrelated and unaffected Coonhounds were also all homozygous wild type. The mixed breed dog had a splice acceptor variant (T>G) at the beginning of exon 5, also in the DLL3 gene. An additional 722 dogs representing 158 breeds, including mixed breeds, wild canids, and village dogs, were homozygous wild-type for both discovered DLL3 variants. The DLL3 gene has been previously associated with spondylocostal dysostosis, a heterogeneous group of axial skeletal disorders in humans [1]. The DLL3 protein is involved in the embryonic segmentation clock (part of the Notch signaling pathway); dysfunction affects segmentation of the vertebral column [2].

References

1) Bulman MP, Kusumi K, Frayling TM, McKeown C, Garrett C, Lander ES, Krumlauf R, Hattersley AT, Ellard S, Turnpenny PD (2000) Mutations in the human delta homologue, DLL3, cause axial skeletal defects in Spondylocostal dysostosis. Nature Genet 24: 438-441.
2) Online Mendelian Inheritance in Man (OMIM), https://www.omim.org. Entry #602768. Accessed March 21, 2023.

Jeanna M Blake1, Andrew D. Miller2, Kari J. Ekenstedt1

1Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA

2Department of Population Medicine and Diagnostics Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA

Glycogen storage diseases (GSDs) are rare, typically inherited, disorders caused by various defects in glycogen metabolism enzymes, generally resulting in the accumulation of glycogen in various tissues. Recently, postmortem histopathology of two adult Basset Hound (BH) littermates resulted in a diagnosis of GSD, manifesting in cardiac and smooth muscle. Using whole genome sequencing, a splice site donor variant was identified in a homozygous form in both littermates and in a heterozygous form in their unaffected sire; this splice donor variant is in exon 8 of RBCK1, a gene which encodes for an E3 ubiquitin ligase. The presumptive loss of the splice site donor introduces a stop codon within 22 codons following the inclusion of the intron. Screening for the variant in related (n = 6) and unrelated (n > 40) BHs identified one additional affected littermate and five additional familial heterozygous carriers. No variant alleles were present in the unrelated BH population, establishing the novelty of the identified mutation. Variants in RBCK1 have been associated with polyglucosan body myopathy 1, a type of GSD characterized by skeletal muscle myopathy and cardiomyopathy, as well as GSD due to glycogen branching enzyme deficiency in humans. To date, no reported variants in RBCK1 have been identified in dogs associated with GSD. The findings of this study add to the mutational spectrum of GSDs in dogs, permitting broader genetic testing for early diagnosis and for disease prevention through targeted breeding strategies.

Candida auris, an emerging multi-drug resistant fungal pathogen poses a serious threat and causes invasive infection in humans. The majority of C. auris isolates exhibit resistance to currently FDA-approved antifungal drugs. Thus, there is an urgent need to understand its pathogenesis to develop novel therapeutics to combat this pathogenic yeast. C. auris primarily colonizes in the skin leading to systemic invasive infections. Therefore, understanding the factors regulating C. auris colonization in the skin is critical to gain insights into the pathogenesis of this fungal pathogen. In this project, human beta defensin-3 (hBD-3) which is one of the major antimicrobial peptides expressed in the human skin was tested against C. auris. Antifungal activity of hBD-3 against C. auris isolates were tested using physiologically relevant conditions via in vitro assays. Future research efforts will focus on understanding the antifungal activity of hBD-3 using additional in vitro assays as well as in vivo murine models. These studies may potentially open the door to host modulation and using antimicrobial peptides to prevent and treat this deadly fungal pathogen in humans.

Ying Cheng Chen1, Lucas D. Johnson 1, Charles S. Umbaugh 1, Herman O. Sintim 2, and Marxa L. Figueiredo 1

1Department of Basic Medical Sciences, Purdue University, West Lafayette, IN, USA

2Department of Chemistry, Purdue University, West Lafayette, IN, USA

Laminin receptor, 67 LR, and its precursor, 37 LR (collectively LR), are upregulated in various tumor types, including in prostate cancer, where LR is thought to aid in proliferation, metastasis, and reduced survival. LR has been shown to interact with the glycoprotein pigment epithelium-derived factor (PEDF), an anti-angiogenic factor, and this interaction is promising in reducing tumor growth. However, current delivery of PEDF in recombinant form involve peptide formulation or gene delivery which are expensive, unscalable, and complicated. Thus, a small molecule that could mimic the function of PEDF and interacts with LR to promote anti-tumorigenic and anti-angiogenic phenotypes could be of high interest and potential for cancer therapy. In our previous in silico work, a novel small-molecule, C3, showed promise as a candidate PEDF mimic. In vitro studies were conducted to analyze the efficacy of C3 through the criteria of anti-viability, anti-wound healing, anti-cancer signaling properties, and activation of PEDF signaling-related genes. In the present project, we validated the efficacy of C3 in vivo against prostate tumor growth using a syngeneic model of immune competent C57BL/6 male mice. Mice were implanted with TRAMP-C2Ras (TC2R), murine prostate cancer cells, subcutaneously and C3 efficacy was assessed by reduction in tumor size relative to the vehicle control group (2.5% DMSO) delivered intraperitoneally at two doses, 5 mg/kg (Low) and 10 mg/kg (High). The overall health of mice was continuously monitored through qualitative observations as well as quantitative measurements such as weight as a function of time.

Zhixia Chi1,2, Xuehong Deng1, Guangjun Zhang2, Michael K. Wendt1.

1. Purdue University Department of Medicinal Chemistry and Molecular Pharmacology, West Lafayette, IN.

2. Purdue University Department of Comparative Pathobiology, West Lafayette, IN.

Background: Prostate cancer (PCa) is the second most common cancer-related death in men

worldwide. Androgen receptor (AR) is expressed in nearly all primary prostate cancer. Inhibiting

AR function via androgen-deprivation treatment (ADT) or androgen receptor antagonists is the

first-line treatment for clinically localized and metastatic PCa. However, those methods

inevitably lead to castration-resistant prostate cancer (CRPC). Protein arginine methyltransferase

5 (PRMT5) is a type II methyltransferase. Increased expression of PRMT5 is positively

correlated to the progression of prostate cancer tissues. In recent decades, targeting the

enzymatic active site or the S-adenosyl methionine (SAM) binding pocket of PRMT5 has been

utilized to develop compounds and circulated them in clinical trials. Nevertheless, these

compounds could potentially impose irreversible damage to normal tissues, given the fact that

the catalytic and substrate-binding function of PRMT5 is critical for normal cell survival.

Cumulative evidence suggests developing inhibitors for PRMT5 protein-protein interaction (PPI)

could be attractive therapies for cancer treatment. We recently demonstrated that PRMT5

collaborated with pICln to promote prostate cancer growth via epigenetic activation of AR

expression. Utilizing the near-atomic cryo-EM structure of PMRT5:pICln and the structuralbased

virtual screen, our collaborators identified a small molecular therapeutic inhibitor (HJL-1)

of PRMT5:pICln protein-protein interaction. However, whether HJL-1 could provide a

promising treatment for prostate cancer therapy remains to be determined.

Methods: Utilizing prostate cancer cell lines, xenograft mouse model, Co-immunoprecipitation

(Co-IP) and bimolecular fluorescence complementation (BiFC), we investigated if HJL-1 could

decrease AR expression and imped prostate cell proliferation by disrupting PRMT5:pICln PPI.

Results and conclusion: Our CO-IP and BiFC results indicated HJL-1 impeded PRMT5:pICln

PPI in prostate cancer cells. Moreover, the proliferation and target gene expression of prostate

cancer cells that express the wild type of AR and drug-resistant AR-V7 splice variant was

decreased by HJL-1 in a time- and dosage-sensitive manner. Thus, our data suggest HJL-1 could

be a novel inhibitor of PRMT5:pICln PPI for prostate cancer therapy.

Diprasom Das1, Harm HogenEsch1,2 and Shankar Thangamani1,2*

1Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47906

2Purdue Institute for Immunology, Inflammation and Infectious Diseases (PI4D), West Lafayette, IN 47906

Candida auris, an emerging multi-drug resistant fungal pathogen, causes invasive infections in humans. The factors regulating the colonization of C. auris in host niches are not well understood. In this study, we examined the effect of antibiotic-induced gut dysbiosis on C. auris intestinal colonization, dissemination, microbiome composition and the mucosal immune response. Our results indicate that mice treated with cefoperazone alone had a significant increase in C. auris intestinal colonization compared to untreated control groups. A significant increase in the dissemination of C. auris from the intestine to internal organs was observed in antibiotic-treated immunosuppressed mice. Intestinal colonization of C. auris alters the microbiome composition of antibiotic-treated mice. Relative abundance of firmicutes members mainly Clostridiales and Paenibacillus were considerably increased in the cefoperazone-treated mice infected with C. auris compared to cefoperazone-treated uninfected mice. Next, we examined the mucosal immune response of C. auris infected mice and compared the results with C. albicans infection. The number of CD11b+ CX3CR1+ macrophages was significantly decreased in the intestine of C. auris infected mice when compared to C. albicans infection. On the other hand, both C. auris and C. albicans infected mice had a comparable increase of the number of Th17 and Th22 cells in the intestine. Significant increase in the C. auris-specific IgA was observed in the serum of C. auris infected mice compared to uninfected groups. However, no significant increase in the C. albicans-specific IgA was observed in the C. albicans infected mice compared to uninfected groups. Taken together, treatment with broad-spectrum antibiotic increased the colonization and dissemination of C. auris from the intestine. Furthermore, findings from this study for the first time revealed the microbiome composition, innate and adaptive cellular immune response to intestinal infection with C. auris.

Candida albicans (CA) , a commensal gut fungus (mycobiome) and opportunistic eukaryotic organism, frequently inhabits the gastrointestinal (GI) tract. Analysis of the gut microbiota so far has predominantly focused attention on the microbiome, while failing to underscore the critical role of the mycobiome in health and disease. Importantly, the factors regulating the colonization of mycobiome components in the GI tract and the role of gut fungi in the health and disease remain poorly understood. To address this gap in knowledge, our lab utilizes a combination of targeted metabolomics, 16S ribosomal RNA amplicon gene sequencing, and in-vivo mouse models to divulge the complex interaction between mycobiome, the metabolome and the microbiome. Our findings indicate that taurocholic acid (TCA), a major bile acid present in both humans and mice control the balance between commensalism and invasive CA infection originating from the gut. Oral administration of TCA through drinking water is sufficient to induce colonization and dissemination of CA in mouse models. Recent findings indicate that TCA regulates CA by controlling immune cells in the intestine. Here, we report that oral administration of TCA to CA-infected mice significantly decreased the number of mononuclear phagocytes and CD4+ IL17A+ T helper 17 cells that play a critical role in controlling CA in the intestine. Collectively, our results indicate that TCA modulates mucosal innate and adaptive immune responses to promote CA colonization in the intestine. Future studies to elucidate the mechanism(s) by which TCA regulates host defense will provide in-depth understanding of bile-mediated regulation of CA colonization in the intestine. Taken together, the gut mycobiome is gaining recognition as a fundamental part of our gut microbiota. Mycobiome have been increasingly found to contribute and play an active role in inflammatory bowel disease, microbiota-gut-brain axis, colonization and pathogenesis of enteric pathogens. Our findings will have broad implications for our understanding of the gut mycobiome in health and disease which is an underappreciated aspect of microbiome research.

Authors: Ziyu Dong, GuangJun Zhang Affiliations: Department of Pathobiology, PVM, Purdue University

Mechanosensing is a fundamental physiological process, including touch sensation, blood pressure regulation, bladder function, etc. Recently, it is more evident that mechanosensing play instructional roles during early embryogenesis. Piezo are a group of known ion channels that transduce mechanic stimuli to bioelectric signals. Dysregulation of piezo channels has also been linked to various diseases such as hypertension. However, the detailed function of piezo genes remains poorly understood. Gene temporal and spatial expression are good indicators of their functions. In this project, we took advantage of the zebrafish model, and examined piezo gene expression in early staged embryos. We cloned 4 piezo genes from zebrafish and our phylogenetic analysis revealed that piezo2 was duplicated as piezo2a and piezo2b, and we isolated new piezo gene, piezo3, which is only found in the teleost, but not the tetrapod. Moreover, we performed whole mount in situ hybridization to comprehensively characterize the expression pattern of the four piezo genes. All the four piezo genes have distinct patterns, suggesting each gene might specialized functions during zebrafish embryogenesis. Future research will aim to manipulate piezo genes in vivo in zebrafish to further understand their roles in development and regulation.

Prostate cancer (PCa) is considered one of the highly frequent diagnosed malignancies among males global wide. PCa is considered a cold tumor with only few T and NK immune cells infiltrating tumor microenvironment (TME). PCa cells eventually evade CD8 T cell cytotoxicity by losing MHC I antigen presentation. Chimeric antigen receptor T (CART) cells (engineered T cells) could target an antigen on cancer cell surface independently of MHC-I engagement but their efficiency on solid tumors, i.e. PCa, is still not clear.

In this collaborative study with Dr. Philip Low, we used universal mouse Anti-FITC CART cells to target mouse PCa cells expressing human PSMA (MyC Cap PSMA). We performed LDH cytotoxicity assay, cytokine array analysis and RT-PCR to study the effect of CART cells on PCa cells in vitro. To assess the efficacy of CART cells on prostate tumor growth, MyC Cap and MyC Cap PSMA cells were injected subcutaneously in FVB immunocompetent mice. The mice were treated with Dupa-FITC (adaptor) for 1 month, and the body weight of mice, tumor volume and bioluminescence were measured. Dupa-FITC activated CART cells induced cytotoxicity of MyC Cap PSMA in a time and dose dependent manner compared to control MyC Cap cells. There was an elevation in the mRNA level of IL2 and protein level of IFNγ, IL13 and TNFα with downregulation of GM-CSF, VEGFA and CXCL1 upon incubation of Dupa activated CART cells with MyC Cap PSMA cells. Importantly, CART cells decreased MyC Cap PSMA bioluminescence and tumor volume and weight in FVB mice compared to control MyC Cap tumor. In addition, CART induced remarkable apoptosis of MyC Cap PSMA cancer cells. Blood and spleen of tumor implanted mice showed CART cells expansion compared to control mice. We concluded that targeting PSMA using universal CART cells could be a promising avenue in treatment of local PCa.

E.A Hartman, DVM ¹
D. Pena-Hernandez, DVM ²
M. Risselada, DVM, PhD, DECVS, DACVS-SA ¹
H.Y Weng, BVM, MPH, PhD ²
M.G. Papich, DVM, MS ³
S.Y. Kim, DVM, MS, DACVS-SA ¹

^{1Department of Veterinary Clinical Sciences, Purdue University College of Veterinary Medicine, West Lafayette, Indiana, USA}

^{2Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, West Lafayette, Indiana, USA}

^{3Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, USA}  

The objective of this project was to compare antimicrobial activity as demonstrated by zone of inhibition (ZOI) produced by antibiotic-impregnated calcium sulfate (CaSO ₄) beads after storage for 0, 3, 6, 9 and 12 months. Three millimeter diameter CaSO ₄ beads impregnated with vancomycin (50mg/mL) or amikacin (250mg/mL) or without antibiotic (control) were created at the onset of the study. Beads were separated for storage (light, temperature and humidity protected) for 3,6,9, or 12 months until testing. The ZOI against methicillin-resistant Staphylococcus pseudintermedius , methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa was recorded with serial re-plating on a fresh lawn of bacteria every 24 hours until beads dissolved, fractured or failed to produce a ZOI. Elution samples were collected at 24 hours for objective measurement of antibiotic concentration via high-performance liquid chromatography (HPLC) The ZOIs and their changes were compared with mixed-effect linear models. Descriptive statistics of eluted concentrations of vancomycin measured via HPLC were reported. Zones of inhibition were consistent at 24 hours regardless of time since formulation, except vancomycin against P. aeruginosa which did not generate a ZOI at any point. The daily changes of ZOI and duration of activity of antibiotics did not vary between ages ( p > 0.05). There was no consistent change in eluted concentration of vancomycin between different ages of beads. In conclusion, storage for up to 12 months did not impair the in-vitro activity of antibiotic-impregnated CaSO ₄ beads as demonstrated through ZOIs.

Juan F Hernandez-Franco ¹ and Harm HogenEsch ^1,2

¹ Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University; ² Purdue Institute of Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN 47907.

Cancer vaccines are being developed for preventive and therapeutic applications to target and eliminate tumors. Cancer vaccines activate dendritic cells (DCs) and promote their migration to lymph nodes, where they stimulate CD8 ⁺ T cells to generate tumor-specific immunity. New adjuvants for cancer vaccines are needed as clinical trials have demonstrated only modest success thus far. This study investigated whether a combination adjuvant (NanoS100) containing cationic plant-derived adjuvant nanoparticles (Nano-11) and the synthetic STING agonist ADU-S100 (MIW815) enhances cross-presentation and antitumor immunity. NanoS100 increased CD80 and CD86 expression on DCs and stimulated cross-presentation of ovalbumin (OVA) as indicated by activation of SIINFEKL- specific B3Z CD8 ⁺ T cells in vitro. To demonstrate the induction of cytotoxic T lymphocyte (CTL) activity by NanoS100, we injected CFSE-stained donor splenocytes pulsed with SIINFEKL into vaccinated mice. A reduction of CFSE-positive cells confirmed the cross-presentation of tumor antigen by NanoS100-mediated CTL lysis of SIINFEKL-bearing target cells. Intradermal vaccination with NanoS100+OVA induced prophylactic immunotherapy by preventing the development of subcutaneously inoculated B16-OVA (melanoma) and E.G7-OVA (lymphoma) tumor cells in mice. The antitumor immunity of NanoS100 was confirmed by depletion of CD8 ⁺ T cells. To mimic therapeutic immunotherapy, subcutaneous inoculation of B16-OVA and E.G7-OVA tumor models were established in mice, followed by intradermal vaccination with NanoS100+OVA. The NanoS00+OVA immunotherapy significantly reduced tumor size and increased the survival rate of mice. The antitumor immunity produced by NanoS100 was validated by the establishment of potent humoral and cell-mediated responses, as shown by the induction of germinal center B cells, follicular helper T cells (Tfh), antigen-specific plasma cells, IgG, IgG1, and IgG2c production, and the differentiation of antigen-specific Th1 and CD8 ⁺ IFN-γ ⁺ T cells. This study supports the further development of NanoS100 as a novel adjuvant platform for the design and delivery of STING-target immunotherapies, as it protected mice against tumorigenesis both prophylactically and therapeutically.

Annapoorani Jegetheesan1, Daniel K. Howe2 and Sriveny Dangoudoubiyam1

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West

Lafayette, IN. 2Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY.

Sarcocystis neurona, the causative agent of equine protozoal myeloencephalitis is a unique

apicomplexan lacking rhoptries in both merozoite (extracellular) and schizont (intracellular)

development stages. In apicomplexans, secretions from organelles viz., micronemes,

rhoptries and dense granules are crucial for successful intracellular parasitism. Rhoptry

proteins (ROPs) released via rhoptries facilitate invasion and establish a niche inside the host

cell. Surprisingly, despite lacking rhoptries, several ROP homologs were identified in S.

neurona proteome, of which SnROP9 was abundant. In this study, we have performed invitro

assays to determine protein characteristics of SnROP9 and its expression in S. neurona

developmental stages. For this purpose, CRISPR-Cas9 aided SnROP9-HA expressing S.

neurona line was successfully generated. The native (SnROP9) and fusion (SnROP9-HA)

forms were used as required. Analyses of SnROP9 protein demonstrated that is soluble and

secreted by the merozoites. In the intracellular schizont stages, SnROP9 showed consistent

expression throughout the schizont development. Immunolocalization revealed that SnROP9

localizes to either poles of the merozoite while it is scattered throughout the schizont body

during intracellular development. Also, SnROP9 did not localize to the micronemes and

therefore unlikely to be trafficked this organelle. Lack of dense granule markers precludes colocalization

assays to determine if SnROP9 homes to dense granules. Interestingly, both

SnROP9 and SnROP9-HA proteins were observed as multiple bands across all stages of

development. Presence of intact HA in all these forms is indicative of potential N-terminus

processing of SnROP9, not uncommon in apicomplexans. Additional experiments are

required to assess its biological role in S. neurona development.

Shreya Kumar and Marxa L. Figueiredo

Department of Basic Medical Sciences, Purdue University, West Lafayette, IN, USA

Prostate cancer is the second leading cause of cancer-related death among American men. It exhibits significant tropism for the bone and once metastasis occurs, survival rates fall significantly. Current treatment options are not curative and focus on symptom management. Immunotherapies are rapidly emerging as a possible therapeutic option for a variety of cancers including prostate cancer, however, patient sensitivity remains a concern. Chemotherapies, like cabozantinib, can have immune-priming effects which sensitize tumors to immunotherapies. Additionally, lower doses of chemotherapy can be used in this context which can reduce patient side effects. We hypothesized that a combination of chemotherapy (cabozantinib) and immunotherapy (Interleukin-27) can treat bone-metastatic prostate cancer and can exert pro-osteogenic effects. IL-27 is a multi-functional cytokine, which recruits immune cells to the tumor, and promotes bone repair. To test this hypothesis, we performed in vivo experiments where syngeneic C57BL/6J mice were implanted with intratibial (cortical bone) TRAMP-C2ras tumors (TRAMP-C2 cells were infected with Lv-HrasG12V to make them suitable for growth in bone). Immunotherapy was administered in the form of gene therapy. Plasmid expressing IL-27, or an empty plasmid control, was injected intramuscularly. Ultrasound was used to aid gene delivery. Various gene-delivery methods were tested and optimized through in vivo studies with microbubbles in combination with ultrasound emerging as the best method. Following immunotherapy, the animals received either cabozantinib or a vehicle control by oral gavage. Bioluminescence imaging (BLI) was used to monitor tumor size. Based on a pilot study, we anticipate that this combinatorial therapy will inhibit tumor growth. Further, RNA sequencing and cytokine arrays will be used to investigate the mechanisms involved. We hope that such chemo-immunotherapy will emerge as a novel therapeutic strategy for treating bone-metastatic prostate cancer while reducing chemotherapy-associated toxicity, improving sensitivity to immunotherapy, and promoting healthy musculoskeletal tissue repair.

Source of research support: R01CA196947

Vishnu Manikantan1, Uma Aryal1,2 and Sriveny Dangoudoubiyam1

1Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, Purdue University, West Lafayette, IN. 2Purdue Proteomics Facility, Bindley Bioscience Center, Purdue University, West Lafayette, IN

Baylisascaris procyonis is one of the three important parasitic causes of eosinophilic meningitis in humans that either ends fatally or causes life-long neurological deficits. Infection occurs upon ingestion of infective B. procyonis eggs from environmental sources contaminated with excreta of raccoons harboring adult worms in their intestine. In humans, ingested eggs hatch in the intestine, releasing larvae that undergo extra-intestinal migration, and 5-7% of larvae reach the brain. Migrating larvae release a repertoire of proteins, excretory-secretory products (ESPs) that facilitate penetration, destruction and inflammation of the host-tissue. In this study, aseptically hatched B. procyonis larvae were maintained in standard tissue culture medium to enable release of ESPs by metabolically active larvae. This larval ESP rich culture medium was collected, dialyzed, concentrated and analyzed by mass spectrometry. The Baylisascaris larval proteomics data was processed using MaxQuant and various bioinformatics tools. In the absence of B. procyonis “-Omics” data, putative identities of 269 larval ESPs were established by matching with proteome data of related nematodes. Proteins belonging to transthyretin-like family and those with calcium binding domains were predominant of the secreted proteins. Potential immunomodulatory proteins like macrophage migration inhibitory factor, glutathione S-transferase, galectins and peroxiredoxins were identified. Homologs of nematode-specific proteins viz., polyprotein allergens (immunodominant antigens), calreticulin (vaccine candidate) and calumenin (drug target) were found in Baylisascaris larval ESPs. Using B. procyonis larval transcriptome assembly currently under generation, we envision to further analyze B. procyonis larval ESPs that will be fundamental for development of strategies to mitigate harmful effects of eosinophilic meningitis.

Rodrigo Mohallem1,2 & Uma K Aryal1,2

1Department of Comparative Pathobiology, Purdue University, USA; 2Bindley Bioscience Center, Purdue University, USA.

Somatic cells accumulate several mutations during an organism’s life span. Cells, however, have developed intrinsic mechanisms to prevent tumorigenesis upon deleterious mutations, most notably, oncogene-induced senescence (OIS). OIS is a process in which cells enter a state of permanent cell cycle arrest in response to the activation of proto-oncogenes, such as RasG12V. In this study, we explore the mechanisms of OIS using an integrated proteomics and molecular genetics approaches. As a model of OIS, we use IMR90 human diploid fibroblasts that were transduced with the inducible protein ER:RasG12V lentivirus. OIS was induced by treating IMR90-ER:RasG12V cells with 100nM (Z)-4-Hydroxytamoxifen (4-OHT) for 0, 2, 4 or 6 days. Purified nuclear proteins and phosphoproteins were analyzed by LC-MS/MS. Our results demonstrate that protein phosphorylation is significantly increased after Ras activation, and a majority of differentially regulated phosphosites during OIS were ERK1/2 target sites. Interestingly, phosphorylation at these motifs is recognized by the Prolyl Isomerase Pin1 protein, which isomerizes phosphorylated proteins and regulates their localization, function and interactions. Our results show that Pin1 is a key regulator PML-NB dynamics, and it is required for the senescence-dependent increase in PML-NB foci numbers. We have found that the constitutive PML-NB protein SP100 protein levels increase only in the control cells but not when Pin1 is depleted, with an observed increased cell proliferation and loss of senescence phenotype. Our data provides new evidence that Pin1 acts as a tumor suppressor in fibroblast cells during OIS.

Since 2020, COVID-19 pandemic has forever changed millions of lives worldwide, with over 600 million positive cases and 6 million deaths globally. Preventative measures, such as vaccines have since been developed and distributed, and yet breakthrough infections are seen even in vaccinated individuals. Moreover, current therapies are mostly treating symptoms, thus there is still a dire need of targeted therapies that can prevent lung damage and mortality. COVID-19 patients are shown to have an imbalance in their cytokine expression, mainly upregulation in pro-inflammatory cytokines such as IL-6, IL-8, IL-1 and TNF-α leading to cytokine storm, organ damage and even death. Therefore, perhaps a therapeutic that can both reduce SARS-CoV-2 infection and rebalance the cytokine profiles through immunomodulation is crucial to prevent and treat SARS-CoV-2 infection. Interleukin-27 (IL-27) is a multifunctional cytokine involved in multiple inflammatory signaling pathways and has shown promise in its ability to rebalance lung and other tissue microenvironments. We hypothesize that the multifunctional IL-27 biologic agent can rebalance the COVID-19 inflammatory microenvironment tissue culture models. Our previous in silico data analysis showed the potential of IL-27 in inducing antiviral genes and rebalancing cytokine imbalance. We now test our hypothesis in an in vitro setting by examining the potential of our ACE2-targeting IL-27 in preventing the entry of GFP-expressing SARS-CoV-2 Spike pseudotyped lentivirus in epithelial cells with overexpression of ACE2 receptor (HEK293-ACE2). We subjected HEK293-ACE2 in various ACE2 blocking conditions prior to the administration of lentivirus, followed by analysis of GFP expression using IncuCyte. Our results shows promise of ACE2 targeting IL-27 in human adipose stromal cell conditioned media to reduce lentivirus entry compared to control and muscle cell conditioned media. However, no significant difference is yet to be seen between non-targeting and ACE2 targeting IL-27 conditioned media. Thus, we will continue to improve our current assay, particularly using alternative means to increase baseline GFP expression and ACE2 targeting peptides used in our therapy. We hope to continue determining the potential of ACE2-targeting IL-27 therapy in reducing SARS-CoV-2 infection and restoring cytokine imbalance, which could lead to novel approaches to reduce the progression and severity of COVID-19.

Authors: Sung Jun Park, GuangJun Zhang

Affiliations: Department of Comparative Pathobiology, Purdue University

Background: Vertebrate limb/fin is a great model for us to understand how organ size and shape are determined. Despite the significant impact of patterning mechanisms in vertebrate zebrafish fins, the detailed patterning mechanisms remained largely unexplored. A retroviral insertional forward genetic screening generated two long-fin zebrafish mutants, Dhi862 and Dhi4458. Genetic analysis revealed that changes in the kcnj10a gene are responsible for their long fins in both mutants.

Methods: First, we performed morphological characterization of the two mutants by measuring adult and larva fin, body size and whole mount in situ hybridization on zebrafish mutant embryos to access the kcnj10a gene expression. To further validate the function of kcnj10a transient and ectopic expression, we utilized the CRISPR-Cas9 system to mutate the kcnj10a coding region.

Result: We found that one or two copy transgenic alleles in Dhi862 and Dhi4458 mutants developed elongated fins compared to wildtype siblings. The fin ray segments of Dhi862 and Dhi4458 were altered in segment number and length. In addition, we identified transient ectopic expression of kcnj10a in the whole somite and notochord in mutant fish embryos. Removal of the ectopic expression using the CRISPR-Cas9 system, Dhi862 long-finned phenotype can be rescued.

Conclusion: Here, we characterized the fin morphological changes of the two mutants and discovered a transient activation and ectopic expression of kcnj10a gene in somite and notochord, suggesting these ectopic expression domains cause the long-fin development.

Worapat Sawatwong1,2, and Russell P. Main1,2

1Musculoskeletal Biology and Mechanics Lab, Department of Basic Medical Sciences, Purdue

University, West Lafayette, IN, USA

2Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA

Osteocytes (Ots) sense and transduce mechanical signals, and mediate bone (re)modeling in response to mechanical loading. Previous studies showed that loadinduced strain distribution influences the osteogenic response of the skeleton. Among various in vivo loading models, bone appears to be more sensitive to non-physiological strain distribution compared to strains elicited during normal locomotion. However, the effect of tissue-level strain distribution on the activity of Ots remains unclear. This study aims to reveal the effects of differences in strain distribution on the gene expression-level of the (re)modeling response. Four female C57BL/6 mice with strain gauges attached to the tibiae were subjected to the tibial axial compressive (AC, physiological) and medial-lateral cantilever-type (ML, non-physiological) loading modes. Finite element analysis was performed to calibrate the strain at the posteriorlateral (PL) region where Ots are exposed to opposite strain directions in AC and ML models. The AC model requires a relatively higher load (3.5N) than the ML model (2N) to generate the absolute strain magnitude of +1000 μe in the PL region. Our next step is to acquire 16wk old C57Bl/6J mice subjected to either tibial AC or ML loading for three days (n=4/group). The tibial cross-sectional cortical bone will be collected and subjected to spatial transcriptomics analysis to reveal the strain distribution effects on different Ot gene expressions and potential causes for bone (re)modeling. The outcome of this study is essential for understanding mechanosensation and mechanotransduction at the Ot, which could contribute to better osteoporosis clinical intervention through mechanical stimulation or drug development.

Dingxun Wang1, Chang-Deng Hu2, GuangJun Zhang1

1 Department of Comparative Pathobiology, Purdue University 2 Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University

Malignant peripheral nerve sheath tumor (MPNST) is a highly aggressive sarcoma with a poor prognosis and high relapse rate. Currently, surgical removal is still the mainstay of the treatment, which is always limited due to tumor size and location. No effective targeted therapy is available for this type of malignancy. Thus, a novel and efficient therapeutic drug target is urgently desired. PRMT5, a type II protein arginine methyltransferase, has recently been reported as a promising therapeutic target for various types of human cancers with methylthioadenosine phosphorylase (MTAP) deleted. The loss of MTAP caused cancer cells to become heavily dependent on the PRMT5 arginine methyltransferase. The high reported rate of MTAP deletion led us to hypothesize that PRMT5 is an effective therapeutic target for MPNST. We performed immunohistochemistry on 19 human MPNST patient samples and found that PRMT5 expression was significantly increased compared to the adjacent normal tissue. Consistently, we observed relatively high expression of PMRT5 in most human MPNST cell lines compared to the immortalized Schwann cells. Furthermore, we inhibited PRMT5 using shRNA and potent chemical inhibitors (JNJ-64619178 and MRTX1719) in the MPNST cell lines with high or low MTAP levels. We discovered that cell growth of MTAP-low MPNST cell lines was significantly suppressed while limited effects on cell growth of MTAP-proficient MPNST cell lines were observed. Our results suggested that PRMT5 potentially be a therapeutic target for MTAP-deficient MPNST patients.

Wen-Chien Wang1, Ekramy E. Sayedahmed1, Marwa Alhashimi1, Ahmed Elkashif1, Suryaprakash Sambhara2, Suresh K. Mittal1

1Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN; 2Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA

Influenza viruses are responsible for millions of cases globally and significantly threaten public health. Since pandemic and zoonotic influenza viruses have emerged in the last 20 years and some of the viruses have resulted in high mortality in humans, a universal influenza vaccine is needed to provide comprehensive protection against a wide range of influenza viruses. Currently available seasonal influenza vaccines provide strain-specific protection and thus are less effective against mismatched strains. In this study, we target the conserved influenza domains, including the stem region of hemagglutinin (HA2) and the ectodomain of matrix protein 2 (M2e), to evaluate their roles in developing a broadly protective influenza vaccine. The secretory or anchored form of HA2 stem and tandem M2e linked with the autophagy-inducing protein (AIP) C5 (AIP-C5) were expressed in bovine or human adenoviral (Ad) vectors. The prime-boost immunization of mice with bovine and human Ad showed a comparable increase in the expression of interferon-gamma (IFN-γ) and interleukin 2 (IL-2) in splenocytes, mediastinal lymph node cells, and lung lymphocytes, indicating the development of HA2-specific cellular immunity. Besides, the comparable antibody level against HA was shown in all the candidate vaccines, signifying the induction of humoral immunity. Immunization and challenge study with an H5N1 reassortant influenza virus demonstrated a comparable decrease in lung viral titers in all candidate vaccine groups. Further challenge studies with other influenza subtypes will determine the broadly protective efficacy of HA2- and M2e-based Ad vaccines.

Free-living amoeba (FLA) can act as vectors for maintaining and transmitting pathogens in the environment. This study uses mathematical modeling to investigate the population dynamics of FLA and Mycobacterium avium subsp. paratuberculosis in drinking water troughs used for cattle. A novel compartmental model was developed, and numerical simulations were run in R, to explore the impacts of different parameter values.

Preliminary results show that carrying capacity and water flow rates are important drivers of amoebal growth in drinking water troughs and other factors. These findings suggest that this new mathematical model canbe used to explore the complex relationships between microorganisms and their environments. In conclusion, this study provides a foundation for future research efforts to investigate the growth and dynamics of amoebae-bacterial systems using mathematical modeling techniques.

In Malawi, poultry are the most commonly owned livestock among households nationally; in Lilongwe, the capital city, around 50% of households own poultry. Small-scale poultry farming plays an important role in meeting household nutrition requirements and mitigating food insecurity while also reducing gender inequality and poverty. However, contact between backyard poultry and their owners presents an avenue for the transmission of infectious diseases including foodborne, zoonotic, and drug-resistant pathogens. We hypothesize that small-scale poultry owners may have relatively high rates of direct contact with domestic poultry which in turn have high rates of direct contact with wild birds, increasing the risk of infectious diseases transmission between wild birds, domestic poultry, and humans posing potential societal risks including zoonotic transmission to human owners, transmission to commercial farms which may result in regional economic impacts and food supply challenges, and the emergence of pathogens with outbreak and pandemic potential. Biosafety and biosecurity, antimicrobial use, and animal husbandry practices in small-scale backyard poultry systems in urban and peri-urban settings in Lilongwe have not been well described; our aim was to fill this gap and to qualitatively describe backyard poultry owners’ perceptions of poultry health and disease. We utilized purposive sampling to interview 13 poultry farmers across 3 communities in Lilongwe; study recruitment continued until thematic saturation was reached. We analyzed data using thematic analysis and summarize findings across 7 thematic areas.

Nelly O. Elshafie1, Ekramy. E. Sayedahmed1, Marwa Ali1, Michael O. Childress2, Andrea P. dos Santos1

1Purdue University, Department of Comparative Pathobiology , 2Purdue University, Department of Veterinary Clinical Sciences

Lymphoma is one of the most communal malignancies in dogs and occurs in various forms, including multicentric, thymic, gastrointestinal, cutaneous, and solitary forms. Canine diffuse large B-cell lymphoma (DLBCL) is the highest reported and aggressive Non-Hodgkin lymphoma (NHL) subtype in dogs, with much-shared similarities with its equivalence in humans. The disease progression is hasty, and the treatment can be overwhelming. Investigators are studying using microRNAs as a prognostic indicator to enhance outcomes. In this research, we examined the correlation between progression-free survival (PFS) in dogs with DLBCL and the level of miRNA expression. MicroRNAs (miRNAs), small non-coding RNA molecules, are crucial for downstream gene transcription regulation. They participate in several pathophysiologic processes, including the growth and progression of cancer. Based on the analysis of small RNA sequencing (sRNA-seq) data using PFS, we validated and quantified the expression of the microRNAs using quantitative PCR from 44 archived lymph nodes retrieved from dogs with DLBCL with known outcomes. We then correlated these expression values with PFS. According to our findings, a particular miRNA subset is strongly linked to lower PFS in dogs with DLBCL. MiR-16-5p, miR-125a, miR192-5p, and miR-187-3p, in particular, were markedly upregulated in dogs with lower PFS. This result suggests that miRNA profiling offers promise for foreseeing PFS in DLBCL-affected dogs. Creating individualized treatment plans for canine DLBCL patients may eventually result from identifying particular miRNAs that correlate with the course of the disease.

Hamideh Esmaeilzadeh1*, Camila Benaduce Emanuelli Mello1, Nelly O Elshafie1, Sarah Malek2, Andrea Pires dos Santos1 Purdue University College of Veterinary Medicine, 1 Department of Comparative Pathobiology, 2 Department of Veterinary Clinical Sciences

Osteoarthritis (OA) is a progressive and destructive joint disease. The activated phenotypes of macrophages with folate receptor-ß (FRβ) are involved in OA-related inflammation in humans and are potential targets for early detection and therapeutic interventions. The presence of FRβ in activated synovial macrophages in dogs with OA has not been previously demonstrated. Our objectives were to evaluate the expression of FRβ in synovial macrophages and determine the difference in polarization of macrophages into inflammatory phenotype (M1) and antiinflammatory phenotype (M2) between OA and control dogs. Synovial fluid from the knee was collected from ten dogs with OA due to cranial cruciate ligament rupture and ten controls.

Straight preparations were made to estimate total nucleated cell count, and cytocentrifuge preparations fixed with acetone. Immunocytochemical staining for FRβ, M1 marker inducible nitric oxide synthase (iNOS), and M2 marker arginase 1 (ARG1) were performed. Positive large mononuclear cells were counted. The number and percentage of FRβ-positive cells in the OA samples were increased compared to controls (p < .05). The number of iNOS-positive cells was increased in the OA compared to control samples (p < .05). The percentage of iNOS-positive cells was higher than ARG1-positive cells in the OA samples. In conclusion, activated macrophages with FRβ are present and detectable in synovial fluid of dogs. Higher expression of FRβ in activated macrophages in canine OA may be further explored as potential therapeutic targets. A higher percentage of iNOS-positive cells in canine OA indicates a polarization of synovial macrophages toward a proinflammatory phenotype.

Authors: Stefanie H. Hansen ¹ and Timothy B. Lescun ¹

Affiliations: ¹Department of Veterinary Clinical Sciences, Purdue University College of Veterinary Medicine

Abstract: The biomechanical strength of the tension band created by screw fixation of proximal sesamoid bones (PSB) as used in arthrodesis of equine metacarpophalangeal joints is unknown. The aim of this study was to compare the biomechanical properties of two screw sizes (4.5-mm and 5.5-mm cortex screws) inserted into the PSB. Ten pairs of skeletally normal forelimbs were used in the study. Two cortex screws were placed using lag technique through the third metacarpal condyles into the center of each PSB. Following screw insertion, 5 limb pairs were loaded in axial compression from 250N – 1.5kN for 3600 cycles in non-destructive cyclic testing. Subsequently 10 pairs underwent single cycle to failure under controlled axial loading. Mean stiffness (kN/mm) and failure load (kN) were compared between groups. Cyclic testing showed no difference in construct stiffness between the first and last 5% of cycles. All constructs failed by transverse fracture of the PSBs. There was no difference in construct stiffness between the 4.5-mm (0.448 ± 0.278kN/mm) and 5.5-mm (0.437 ± 0.197kN/mm) screw groups (P = 0.907). Mean failure load was similar between groups ((2.932 ± 0.485kN; 4.5-mm vs 3.385 ± 0.761kN;5.5-mm, P= 0.103). Limitations of this study include small sample size and lack of cyclic testing to failure. Our results show that in this mode of loading, screw size is not a critical determinant of tension band strength. The suspensory apparatus and PSBs are the weakest part of the construct when lag technique of the PSB is used in metacarpophalangeal joint arthrodesis. Results supports reported successful use of both screw sizes in clinical cases.

Jobin Kattoor1, Andrea dos Santos1, Rebecca Wilkes1

1Department of Comparative Pathobiology, Purdue University.

Pathogen surveillance has been performed with next-generation sequencing (NGS) metagenomics because of its potential to detect any pathogen in a sample; however, it lacks the sensitivity needed for effective diagnostic use. Targeted NGS is the selective capture or amplification of nucleic acids of interest in a sample prior to sequencing, and if targeting pathogens, it can be used for clinical diagnostics because of the sensitivity and specificity of this method rival that of real-time PCR. Given the ability to include thousands of primers in a targeted method, it can be expanded for detecting pathogens for multiple syndromes or pathogens from multiple animal species, making it also ideal for surveillance.

We developed a targeted NGS panel for detecting 75 canine and feline pathogens using AmpliSeq custom-designed primers, automated library prep, and an Ion GeneStudio S5. This panel detected vector-borne pathogens from nucleic acids extracted from whole blood samples from 149 dogs from nine Indigenous communities in southeast and southern Brazil.

This test method detected nine different vector-borne disease pathogens in these dogs. Co-infection with multiple vector-borne agents was standard in this population. Though vector-borne diseases were of specific interest, we were able to detect five additional unrelated pathogens of clinical significance (including canine parvovirus and canine distemper virus) because of the comprehensiveness of the panel, and sequences obtained from the samples with this method confirmed the results.

Targeted NGS is an effective method for surveillance use for canine populations with unknown infectious disease history.

Authors: Eric Kontowicz1, Daryl Ragland2, Max Moreno-Madrinan3, Wendy Beauvais1

Affiliations: 1. Department of Comparative Pathobiology College of Veterinary Medicine, Purdue University, 2. Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, 3. Global Health Program, DePauw University

This work funded by the National Pork Board Grant NFB #21-100

The purpose of our study was to assess risks of interspecies influenza transmission (pig-to-worker or worker-to-pig) within a typical indoor pig grower unit in the midwestern United States.

We adopted the World Organization for Animal Health (WOAH) risk assessment framework and conducted separate risk assessments for H1N1, H1N1pdm09, H3N2, H3N2v, H1N2, and H1N2v. Semi-quantitative probability assessments (ranging from negligible to highly likely) were based on literature review, influenza surveillance data, and author agreement where data was lacking. We assumed a single production cycle (~26 weeks) on an indoor pig grower farm with 4,000 pigs, two workers, and no influenza vaccination. Uncertainty categories (ranging from low to high) were based on data availability and agreement between sources.

We assessed the following conditional risk steps: What is the likelihood that…1a) at least one piglet brought onto the farm is infected with influenza?; 1b) at least one worker is infected with influenza from an infected pig on the farm;1c) an infected worker develops influenza symptoms?; 2a) at least one swine worker is infected with influenza outside of swine farm?; 2b) at least one pig is infected by a worker; 2c) the newly infected pig will develop symptoms of influenza?

The probability of interspecies transmission events during a production cycle was found to be high for most influenza subtypes and associated with high uncertainty levels due to an overall lack of empirical data. Further studies are needed to improve the precision in estimates for frequency of interspecies transmission in an indoor growing pig setting.

Oppenheimer Lúgaro V, Weng HY, Hui TY, Guptill L

Background – Recent evidence suggests the proportion of multidrug-resistant (MDR) urinary tract infections (UTIs) in dogs is increasing.

Objective – To characterize antimicrobial resistance trends in canine UTIs and determine risk factors associated with development of MDR UTIs.  

Animals – 1,106 bacterial isolates from 759 dogs treated at a veterinary teaching hospital

Methods – Isolate susceptibility data from urine cultures were retrospectively evaluated over 2 time periods (2009–2013; 2017–2021). Information extracted from medical records included signalment, urinalysis findings, classification of sporadic or recurring UTI, presence of lower urinary tract signs, urolithiasis, neoplasia, chronic kidney disease, endocrinopathies, urinary tract anatomical disorders, micturition disorders, previous antimicrobial or immunosuppressive drug use, and history of indwelling catheterization. Chi-square tests and random-effect logistic regression were used to assess temporal changes and risk factors.

Results – There were increases in MDR isolates between the 2 time periods ( P < .001) and extensively-drug resistant (XDR) isolates from 2017–2021 ( P < .001). Common isolates included Escherichia coli (40.1%), Staphylococcus spp. (14.3%), and Enterococcus spp. (12.9%), all with an increasing trend in antimicrobial resistance from 2009–2021 ( P < .001). Significant risk factors for MDR UTIs included previous antimicrobial and immunosuppressive therapy ( P < .001 and P = .044 respectively), micturition disorders ( P = .005), and previous catheterization ( P < .001).

Conclusions and Clinical Importance – The increase in MDR/XDR UTIs emphasizes the urgent need for antimicrobial stewardship and the importance of urine culture and susceptibility, especially in dogs with previous antimicrobial or immunosuppressive drug use, micturition disorders, or previous catheterization.

Arielle Ostrager VMD1; George E. Moore DVM, PhD2; R. Timothy Bentley BVSC (Dist), MRCVS, DACVIM (Neurology)3 1, 3: Department of Veterinary Clinical Sciences, Purdue University College of Veterinary Medicine 2: Department of Veterinary Administration, Purdue University College of Veterinary Medicine

Background: The prognosis of dogs with meningoencephalomyelitis of unknown etiology (MUE) is difficult to predict. The purpose of this study was to determine the frequency of normal MRI patients with MUE, and to determine whether the absence of MRI lesions affects prognosis in patients with MUE.

Methods: 73 client-owned dogs with a clinical diagnosis of MUE presenting to Purdue University Veterinary Hospital from 2010-2020 had their medical records reviewed. Patient clinical data (presenting complaint, MRI findings, CSF results, MRI findings, presence of mass effect, administration of prednisone and immunosuppressive medications, survival in months, and cause of death were documented. Survival due to disease was compared between normal- MRI and abnormal-MRI dogs.

Results: Death due to MUE occurred in 5% of normal-MRI dogs and 33% in abnormal-MRI dogs. The incidence of death due to MUE significantly differed between normal-MRI and abnormal-MRI dogs (p= 0.016)

Conclusions: Survival due to disease exceeded the 10-year study period in >50% of patients in both groups, suggesting improved survival time than has been previously reported in patients with MUE. Normal-MRI patients with MUE have improved survival compared to abnormal- MRI patients with MUE. .

Daniela Peña1,2,3, Joneson Jessica2, G. Kenitra Hendrix1,2

Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine1, Indiana Animal Disease Diagnostic Laboratory2 correspondence: dpeahern@purdue.edu

Nosocomial salmonellosis in hospitalized animals is a recognized hazard, especially in large animal clinics. Mitigation of outbreaks, as well as active surveillance efforts, require an effective and time-sensitive diagnosis. A standardized culture protocol (SCP) for detecting Salmonella spp. in environmental samples using a 48-hour enrichment step results in a 5-day turnaround time for negative results. The RapidChek® SELECT™ Salmonella (RCSS) test system offers detection of organisms in 22-44 hours through a double enrichment and a lateral-flow immunoassay (LFIA). Negative results are reported within 48 hours. At the Indiana Animal Disease Diagnostic Laboratory (IN-ADDL), we compared the performance of RCSS to a SCP on recovering Salmonella spp. from environmental samples. The study included ten environmental samples collected at the large animal Purdue Veterinary Hospital (LA-PVH) using electrostatic (Swiffer®) wipes and 20 wipes artificially spiked with two different concentrations of ATTC25923 S. Typhimurium. After completion of comparison experiments, a clear time advantage and higher sensitivity led to the adoption of RCSS as the primary method for testing environmental samples submitted by the LA-PVH. RCSS was used according to manufacturer instructions, including confirmation of positive LFIA results by culture in a selective and differential agar. For morphologically compatible isolates, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was chosen as the confirmation method. Eight-hundred and seventy-two environmental samples were tested over 12 months. Here, we report the performance data of RCSS in term of sensitivity, specificity, and positive predictive value. We also provide guidelines on reporting results obtained using this system.

Abby J. Reising1, Christopher M. Fulkerson1, Cleveland G. Shields2, Michael O. Childress1

Affiliations: 1Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University 2Department of Human Development and Family Science, College of Health and Human Sciences, Purdue University

Chemotherapy is widely used in veterinary oncology, but carries real and perceived risks of adverse events (AEs). Human cancer patients perceive AEs from chemotherapy as more severe than do their attending physicians. It is currently unknown if this discrepancy also exists in veterinary oncology. This survey study’s aim was to assess differences in the ways that dog owners and veterinary oncologists perceive the acceptability of chemotherapy-related AEs. We hypothesized that oncologists would accept higher grade AEs and a greater risk of AEs of any grade than owners. Two surveys were generated for owners and veterinarians. Respondents were asked to define maximally acceptable AE scores and risks of AEs given three hypothetical outcomes of treatment: 1) cure; 2) extension of life in the absence of cure; and 3) improved quality of life in the absence of cure or extension of life. T-tests were used to compare mean response scores between groups. Responses from 56 dog owners and 73 veterinarians were analyzed. Owners accepted higher grade AEs if chemotherapy would improve quality of life (p = 0.004). Owners accepted greater risk of moderate (p = 0.0001) or severe (p = 0.0001) AEs if chemotherapy was expected to cure their pet’s cancer. This is the first study to assess how owners and veterinary oncologists differ in their perception and acceptance of chemotherapy-related AEs. These preliminary results may help to frame discussions with dog owners on the goals and expectations of chemotherapy.

Laura Machado Ribas1, Jasmine Aggarwal2, Kerstin Muner2, Nelly Elshafie2, Andrea Pires dos Santos2, Suzanne Cunningham3, Luis Felipe Neves dos Santos1

1 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA
2 Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA 3 Department of Clinical Sciences, Cummings Veterinary Medical Center, Tufts University, North Grafton, MA, USA

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease reported in dogs, cats, and humans. It manifests as malignant ventricular arrhythmias, cardiac dilation, and sudden death. Although multiple mechanisms have been implicated in the pathogenesis of the disease, there is no reference standard ante mortem diagnostic test in dogs or people and, since limited diagnostic criteria are available to characterize ARVC in Boxers, additional biomarkers for this disease are needed. MicroRNAs (miRNAs) are known to regulate the expression of target genes and have been implicated in cardiac diseases, attracting particular interest as potential non-invasive biomarkers. Their presence has been recently reported in the myocardium and serum of end-stage ARVC patients. This study aimed to investigate miRNAs expression in the cardiac tissue of Boxer dogs. We hypothesized that miRNAs would correlate with the clinical outcomes of the disease. Cardiac samples (right and left ventricles) from twelve dogs were analyzed. Four Boxer dogs presented with sudden cardiac death (SCD group) and a definite histopathological diagnosis of ARVC, and another four Boxers presented for non-cardiac related death (NCRD group); cardiac tissues from four non-Boxer dogs were also evaluated (control group). Four miRNAs (miR-133b, miR-145-5p, miR-185-5p, and miR-494-3p) were chosen among those known to be expressed in humans with ARVC, but their expression in cardiac tissues was not significant among groups and did not correlate with clinical outcomes between Boxer dogs’ groups. The absence of consistent connections could be related to the analytical approaches and possible different associated pathways from humans. Further investigation is needed into the potential use of miRNAs as clinical biomarkers and their involvement in the pathogenesis of ARVC in Boxers.

Jannatul Shabnam and Sriveny Dangoudoubiyam

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47906

Baylisascaris procyonis, a zoonotic nematode responsible for larva migrans in at least 70 avian species. Both free-range and captive birds may risk contracting infections by ingesting infective eggs from their surroundings contaminated with feces of raccoons harboring adult B. procyonis. Infected birds display clinical signs such as difficulty perching or flying, torticollis, wing paralysis, disorientation, and may eventually die. Antemortem diagnostic tests are currently unavailable to perform timely diagnosis or determine prevalence of this infection in birds. In our study we have optimized recombinant B. procyonis RAG1 protein (rBpRAG1)–based ELISA and Western blot assay for detection of Baylisascaris infection in birds. Sera was collected at multiple time points from cockatiels experimentally exposed to B. procyonis larval antigens either by hyperimmunization with B. procyonis larval excretory-secretory proteins (group I, n=5, HI) or by oral infection with infective eggs (group IIa, n=9, 1400 eggs and group IIb, n=9, 2200 eggs). Both groups had their own control cockatiels. On day 28, all five HI cockatiels showed high serum antibody levels with mean OD values ranging from 1.027 to 1.930. These values were 9-16 fold higher than the pre-exposure mean ODs (0.094 to 0.126). Cockatiels in groupII showed variable antibody response and seroconversion was evident in only 56% (5 of 9) of groupIIa and 67% (6 of 9) of groupIIb cockatiels. Western blot results were congruent with ELISA results. The results suggested that rBpRAG1-based serological assays will be useful to assess antibody response in birds exposed to B. procyonis.

Authors: Go Togawa, DVM, PhD1; Melissa Lewis, VMD, PhD, DACVIM (Neurology)1; Dillon Devathasan DVM, MS, DACVIM (Neurology)2

Affiliations: 1. Purdue University College of Veterinary Medicine, 2. Auburn University College of Veterinary Medicine

Background: Fibrocartilaginous embolic myelopathy (FCEM) and acute non-compressive nucleus pulposus extrusion (ANNPE) are common causes of canine spinal cord injury with clinical similarities. Limited information is available regarding the prognosis of paraplegic dogs with FCEM and ANNPE.

Hypothesis/Objectives: To describe the clinical features and outcome of paraplegic deep pain positive (DPP) and deep pain negative (DPN) dogs with FCEM and ANNPE.

Animals: Thirty-one client-owned paraplegic dogs with thoracolumbar FCEM or ANNPE presenting to university hospitals between 2012-2022.

Methods: Multi-center retrospective study. Paraplegic dogs with a clinical and magnetic resonance imaging diagnosis of FCEM or ANNPE were included. Logistic regression analysis was performed to investigate associations between diagnosis, clinical and imaging variables, and outcome (recovery of independent ambulation or not).

Results: On initial presentation, 14 dogs were paraplegic DPP (8 FCEM, 6 ANNPE), 17 dogs were paraplegic DPN (11 FCEM, 6 ANNPE). Outcome was available for 26 dogs (14 DPP, 12 DPN) with a median follow-up time of 182 days (range, 0 – 2311). Excluding 2 DPN dogs euthanized at diagnosis, 1/10 DPN dogs (10%) regained independent ambulation, whereas 9/14 DPP dogs (64%) regained independent ambulation. DPN dogs had a significantly higher risk of not regaining independent ambulation compared to DPP dogs (OR: 29.3, CI: 2.1-419.2 P = 0.013).

No other variables were associated with outcome (P > 0.05).

Conclusions and clinical importance: While the recovery of ambulation is possible, the absence

of pain perception appears to be a useful negative prognostic indicator in dogs with severe

Glucose monitoring is crucial in a critical patient, especially in young animals, as those have less capacity to maintain blood glucose levels. However, repeated venipunctures in a juvenile veterinary patient can be challenging due to patient size, stress, and the potential iatrogenic anemia.

Interstitial glucose monitors are routinely used in diabetes care. It provides continuous real time glucose readings of the interstitial fluid - which correlates with the blood glucose - without the need of any needle stick. These monitors have not been evaluated in juvenile dogs in a clinical setting.

The aim of this study is to compare an interstitial glucose monitor (IGM) against a portable blood glucose monitor (PBGM) in sick juvenile dogs in a veterinary intensive care unit.

Paired interstitial and blood glucose measurements (n=159) were collected from 16 client owned dogs under one year of age with systemic illness. Pearson’s correlation, mean absolute relative difference (MARD), bias, 95% limits of agreement and Parkes Consensus Error Grid (CEG) analysis was performed.

Between the interstitial and blood glucose correlation was r = 0.65, MARD 15.4%, bias -2.6%, with the 95% limits of agreement ranging from -42.5% to 37.4%. 100% of the pairs fell into clinically acceptable zones (A+B) on CEG analysis.

Our results indicate good clinical accuracy with no or minimal differences in clinical outcome between IGM and PBGM in the target population. Further clinical studies with larger sample size, particularly in the hypoglycemic range are needed to assess the IGM performance in the lower glucose range.

Allyson Jones, Jessica Craig, Eric Kontowicz, Wendy Beauvais

Department of Comparative Pathobiology (Craig, Kontowicz, Beauvais), College of Veterinary Medicine (Jones), Purdue University, West Lafayette, IN

Avian Cholera, caused by the bacteria Pasteurella multocida, is a respiratory and septicemic disease of domestic and wild avian species. Disease control is challenged by a lack of effective vaccines, identifying reservoirs of the disease agent, and predicting drivers of outbreaks. We analyzed publicly available outbreak reports to explore the spatio-temporal distribution of avian cholera outbreaks in the US.1 Reports of sightings of 24 species of birds from each county in the US between January 2002 and May 2022 were extracted from eBird. Daily precipitation, temperature, and humidity data for each county were also extracted. Exploratory mapping and logistic regression were conducted to determine the association between temperature, precipitation, and the distribution of avian cholera within the US. Over the study period, there were 263 reported cases of suspected or confirmed avian cholera events with a median number of 241 birds affected per event. Snow geese (Anser caerulescens) and American Coots (Fulica americana) were the most common avian species present during outbreaks and appeared in 144 and 131 events, respectively. We hypothesize that avian cholera is associated with higher temperatures. The visualization of these spatial and temporal trends in avian cholera could be used to target surveillance in regions and during times where vulnerable species are most at risk.

Research Grant: Morris Animal Foundation

Student Support: College of Veterinary Medicine, Purdue University

Amyloid deposits have been detected in the majority of feline diabetic patients. These deposits originate from islet amyloid polypeptide (IAPP or amylin). IAPP is a normally a satiety hormone that is produced and co-secreted with insulin by beta-cells, which are the most common cell type in the islets of Langerhans in the pancreas. However, IAPP misfolding leads to the development of amyloid deposits, which have been associated with beta-cell death during the progression of diabetes. IAPP aggregation can be inhibited by several molecular entities such as silibinin and resveratrol. However, these agents have poor bioavailability and cause a variety of pharmacological effects. Currently, there is no commercially available effective means to stop or prevent pancreatic amyloidosis in diabetes mellitus using small drug-like molecules. The goal of this project is to identify selective and general inhibitors of feline IAPP fibril formation, and to demonstrate that the aggregation of feline IAPP (fIAPP) can be modulated by IAPP-interactive compounds in vitro. A series of urea-based compounds were developed for this purpose, and their potency[?right word] to reduce the formation of fibrils and toxic oligomers from IAPP was assessed in vitro using biophysical methods such as Thioflavin T (ThT) fluorescence assays, dynamic light scattering, [TERM FOR ZETASIZER], and transmission electron microscopy (TEM). Both selective and non-selective inhibitors of IAPP fibril formation were identified. This study has the potential to point toward new therapeutic strategies for type 2 diabetes.

Mary Nowak ¹, Nelly Elshafie ¹, Kerstin Muner ¹, Thiago Macedo Lopes Correia ^1,2, Jasmine Aggarwal ¹, Andrea Pires dos Santos ¹

¹Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana.

²Universidade Federal da Bahia, Vitória da Conquista, Bahia, Brazil

Background: Canine hemangiosarcoma (HSA) commonly manifests as a visceral tumor that constitutes approximately 5% of cancers in dogs. The prognosis for visceral HSA is poor due to the aggressive nature of the tumor and lack of specific clinical signs until significant infiltration has occurred. Hence, most dogs present with metastatic disease that responds poorly to standard surgical and chemotherapeutic intervention. Grading systems for HSA have poor prognostic significance. Thus, improved markers are imperative to guide a patient's course of treatment. Non-coding microRNAs regulate gene expression and may serve as predictive biomarkers for HSA. Objective: To investigate the potential of microRNAs in the prognostic assessment of canine splenic HSA. Methods: Retrospective study using archived splenic biopsies from 18 cases of canine splenic HSA divided into three groups based on survival times (G1: <90 days, G2: 90-180 days, and G3: >180 days). Expression of four microRNAs (miR-126, miR-150, miR-214, miR-456) with documented roles in canine hemangiosarcoma was assessed by quantitative PCR. Quantification cycle (Cq) values were normalized using the exogenous control UniSp6 and results expressed as fold change. Differences in expression were determined via one-way ANOVA, followed by Tukey's test. Results: From the four microRNAs analyzed, miR-214 was significantly upregulated in G1 and G2 compared to G3 (p<0.05). Conclusions: Our findings suggest miR-214 may serve as a prognostic marker for canine HSA as upregulation was associated with decreased survival times. miR-214 has been associated with regulating pro-tumorigenic processes in canine HSA and other cancers. Confirmatory studies are needed to evaluate this marker.

Marissa Ramón, Siyuan Sun, and Riyi Shi

Center for Paralysis Research, Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana

Spinal cord injury (SCI) is marked by primary injury (physical impact) and a secondary injury (chemical injury) that amplifies the damage and functional deficits triggered by the primary trauma. An important hallmark of secondary injury is oxidative stress. Acrolein, a key player in oxidative stress, is a toxic aldehyde that is elevated significantly following SCI. Acrolein increases reactive oxygen species (ROS) and lipid peroxidation, thus furthering the damage. Acrolein is of special importance because it has a longer half-life than known ROS and inhibits important endogenous antioxidative stress enzymes. Previous research has identified aldehyde dehydrogenase 2 (ALDH2) as an important antioxidative enzyme. ALDH2 metabolizes acrolein to suppress oxidative stress, but can also be inhibited by acrolein, especially during acrolein overload. Over 600 million people worldwide exhibit an inactive form of isoenzyme ALDH2 (ALDH2*2) that is linked to several diseases, such as Alzheimer's, Parkinson's Disease, and alcohol flushing response. The overall objective of this study was to assess the role and the potential therapeutic value of ALDH2 and the neuroprotective effect of Alda-1, an ALDH2- selective agonist, in SCI, using a transgenic mouse model with ALDH2*2. There were two central hypotheses for this study: 1) transgenic mice would exhibit a higher concentration of acrolein compared to wild-type following SCI, and 2) treatment with Alda-1 would amplify ALDH2 function in both wild-type and transgenic mice, reducing acrolein concentration in the spinal cord. Findings from this study further illustrated ALDH2 as a target for attenuating secondary injury of SCI and introduced Alda-1 as a potential treatment for SCI.

Student Support: Purdue University College of Veterinary Medicine Summer Research Scholarship

Background: Estradiol has a role in the body’s response to systemic dehydration. Vocal folds are negatively affected by systemic dehydration. Additionally, they are a known target organ for estradiol. This research sets the groundwork for future studies investigating the role of sex hormones in influencing the adverse effects of dehydration and the voice.

Objective: This study investigates the histopathology of the larynx in gonadectomized rats undergoing systemic dehydration.

Methods: Twelve female and 12 male Sprague Dawley rats were divided into dehydrated (n=12) and euhydrated groups (n=12). Each hydration group had intact (n=6) and gonadectomized (n=6) rats. Blood was collected at beginning and end of experiment to measure and compare serum estradiol levels (plus androgen for males), packed cell volume, and total protein. Body weight and water intake were measured daily. The dehydrated rat groups received 4 ml water/100 g of baseline body weight (approximately 35% less than baseline average intake) compared with ad lib water in the euhydrated group. Sections of larynx were stained with hematoxylin and eosin and immunohistochemically for β-estrogen receptors.

Results: On HE staining, no difference in the vocal fold morphology was identified between euhydrated and dehydrated groups, intact or gonadectomized. β-estrogen receptor labeling was variable between individuals; however, no significant differences between groups were identified.

Kathryn A. Wolfert, Deepika Dhawan, Seung-Oe Lim, Kimaya Bakhle, Alexander W. Enstrom, and Deborah W. Knapp

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN (Wolfert, Dhawan, Bakhle, Enstrom & Knapp)

Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN (Lim)

Immune checkpoint inhibition has become a promising treatment option in a number of canine and human cancers, such as invasive urothelial carcinoma (InvUC). As one of these checkpoint molecules, the programmed cell death 1 receptor (PD-1) is primarily expressed on mature cytotoxic T lymphocytes (CTLs), with ligands PD-L1 and PD-L2 expressed on tumor cells and antigen-presenting cells. The interaction between PD-1 and its ligands on tumor cells leads to CTL inactivation and immune tolerance of the tumor, making the PD-1/PD-L1 axis a key immunotherapeutic target for both veterinary and human oncology.  PD-1 has also been documented to be present on the surface of a number of tumor cell types, though its function in this context is unclear. To investigate the influence of this tumor cell-intrinsic PD-1 and PD-L1 interaction on cancer cell growth, a canine InvUC cell line that overexpresses canine PD-1 (K9TCC-PU-Nk-cPD1) was developed via lentiviral transduction. Surface expression of canine PD-1 (cPD-1) in this line was confirmed via flow cytometry. Using CellTiter-Glo, soft agar, and Western blot assays, we assessed the proliferation, colony formation, and downstream signaling in the MAPK/ERK and PI3K-PKB/Akt pathways of this engineered cell line in the presence of varying amounts of cPD-L1-Fc protein. We further characterized the influence of cPD-1 on immune evasion via T-cell killing assay using activated canine PBMCs. This work in understanding the role of tumor-cell intrinsic PD-1 has significant implications for prognosis and treatment recommendations for PD-1 expressing cancers of all species.

Research Grant: Bladder Cancer Research fund

Student Support: Morris Animal Foundation

Ex vivo biomechanical and microscopic comparison of two cortical screw sizes in fetlock joint arthrodesis.

Authors: Maria X. Yañez, Timothy B. Lescun, and Stefanie Hansen.

College of Veterinary Medicine, Purdue University, West Lafayette, IN

Abstract: In horses, osteoarthritis (OA) is especially prevalent in the metacarpophalangeal (MCP) joint. The current treatment for refractory OA of the MCP joint is surgical arthrodesis using locking compression plates in combination with a palmar tension band. One method of palmar tension band application is lag screw fixation of the proximal sesamoid bones (PSBs) to the third metacarpal bone condyles. The aim of this study was to compare the biomechanical properties and microscopic damage of two cortex screws (4.5-mm and 5.5-mm diameter) inserted into the PSB after loading. We hypothesize that increased screw diameter decreases construct failure load and that microscopic damage will be greater in medial lag screws due to horses bearing greater weight in the medial side of the limb. Screws were inserted in five pairs of cadaver forelimbs. After single cycle to failure axial loading, screws were removed and evaluated by low power stereomicroscopy and high resolution scanning electron microscopy. All constructs failed by transverse fracture of the PSB through the screw holes. There was no significant difference in construct stiffness or mean failure load between the 4.5-mm and 5.5-mm screw groups. These results reveal how screw size is not a critical determinant for tension band strength. Surgeons can be confident that either screw size will provide proper strength to the construct. This allows them to prioritize other factors for screw selection such as patient size, screw availability, or price.