The Collaborative Cross: A Systems Genetics Resource for Studying Host-Pathogen Interactions

Host genetic variation plays an important role in shaping infectious disease susceptibility. Noll et al. review the application of a genetically diverse mouse reference population, the Collaborative Cross, to study variation in disease response across multiple pathogens, highlighting advances in model development and genetic mapping. © 2019 Elsevier Inc.Host genetic variation has a major impact on infectious disease susceptibility. The study of pathogen resistance genes, largely aided by mouse models, has significantly advanced our understanding of infectious disease pathogenesis. The Collaborative Cross (CC), a newly developed multi-parental mouse genetic reference population, serves as a tractable model system to study how pathogens interact with genetically diverse populations. In this review, we summarize progress utilizing the CC as a platform to develop improved models of pathogen-induced disease and to map polymorphic host response loci associated with variation in susceptibility to pathogens

Preclinical Animal Modeling in Medicine

The results of preclinical animal research have been successfully implemented in various medical and biological practices. The use of animals in medicine is based on significant anatomical, physiological, and molecular similarities between humans and animals. Particularly, mammals that have vast biological commonalities with humans represent not only a valuable model to explore the mechanisms of varied human diseases, but also to define new diagnostic and treatment strategies. This book covers broad but important aspects of animal modeling for scientific medicine as well as for translational systems and biological sciences. Alternative methods such as cell culture and in vitro experiments that do not require the sacrifice of an animal are encouraged for scientific and medical studies

Discoveries of Targets and Novel Agents for the Treatment of Ischemic Retinopathy and Neovascular Disease

Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are among the most common causes of blindness in adults. Vision loss can occur during the advanced stages of DR and AMD as a consequence of unregulated and dysfunctional growth of new blood vessels, or neovascularization (NV) in the retina or choroid. NV can also be triggered by numerous other ocular insults and diseases including radiation retinopathy (RR) and retinal vein occlusion. These latter cases are generally less common but, like DR and AMD, they are characterized by an initial injury, chronic inflammation, and ischemia which perpetuates episodes of retinal neovascularization (RNV). Current targets for RNV include vascular endothelial growth factor, VEGF which is achieved through anti-VEGF protein therapeutics aimed at sequestering the growth factor and preventing the activation of its receptor. However, prospective studies show that anti-VEGF resistance has become a major clinical concern in patients receiving long-term therapy. Thus, targeting downstream signaling proteins linked to pathological RNV represents an alternative or adjunctive approach to approved anti-VEGF treatments, which may provide better patient outcomes through enhanced efficacy of antiangiogenic therapy. Our first goal was to understand how RNV progresses from early stage injury to proliferative ischemic retinopathy, in order to justify protein targets for drug discovery. We first began with an investigation into the causality of radiation injury itself to identify mechanisms of radiation sensitivity and/or resistance in the genetically diverse, murine BXD strains using a total-body irradiation (TBI) model. Our studies suggested mean survival time (MST) over 30 days may in fact be related to genetic variation in genes associated with endothelial progenitor cells (EPC) localization, wound healing, and focal adhesion (FA) dynamics involving both the hematopoietic and gastrointestinal systems. We targeted these mechanisms of tissue repair by blocking the homing of hematopoietic-derived cells to sites of irradiation (IR) injury which proved fatal to mice treated with an integrin-paxillin inhibitor, 6-B345TTQ. In a physiological flow-based assay, we inhibited circulating leukocytes from interacting with an inflamed endothelium, in vitro. These results suggested that the reparative/inflammatory angiogenic response triggered by radiation could be blocked by targeting FA signaling, a central process of RNV progression in ischemic retinopathies. Findings in BXD studies linked tissue reparative processes involving ischemia- induced angiogenesis with mortality. We hypothesized that by targeting early injury in retinal endothelial cells (REC), we could prevent late-stage RNV. Thus, we first explored how RECs respond to radiation injury at levels high enough to cause significant vision impairments in RR. Previously identified radioprotectant, KZ-41, was used in these studies to ameliorate IR-induced injury to RECs through decreased inflammatory stress kinase activation, cell death, and subsequent IR-induced proliferation, in vitro. FA activation through paxillin was found to be a crucial mechanism by which KZ-41 inhibited ischemia-induced RNV in the murine oxygen-induced retinopathy (OIR) model. Targeting stress kinase activation of FA signaling post-IR injury served as a way to prevent the pathological progression of RNV, in vivo. However, it is difficult to predict when or how to treat the inflammation early in ischemic retinopathy, especially in chronic conditions such as diabetes, when the injury has already occurred. Therefore, we sought to target the common focal point of ischemic disease by focusing on drivers of late stage RNV, the focal adhesion signaling complex. Using VEGF as the driver of in vitro angiogenesis, we explored growth factor-induced FA signaling in RECs to validate target proteins Src, focal adhesion kinase (FAK), and paxillin as crucial to RNV progression. Our work helped to identify a novel paxillin modulator, JP-153 which afforded excellent antiangiogenic activity, in vitro. JP-153 achieved potent inhibition of RNV in the OIR model through topical application by disrupting paxillin activation. Together, these data suggested paxillin is a key driver of RNV and may serve as a viable target for the treatment of neovascular eye disease. In Chapter 6, we characterized the pharmacokinetic profile of JP-153 with regard to its absorption, distribution, metabolism, and elimination (ADME) after both oral and intravenous administration. We found that JP-153 exhibited rapid metabolism in rats with an oral bioavailability of approximately 30%. During these studies, we successfully developed a sensitive and selective analytical method using mass spectrometry in order to detect JP-153 concentrations in rat plasma. JP-153 possessed a relatively rapid clearance profile, which is an ideal characteristic for ocular therapeutics. Lower systemic exposures decrease the risk of cardiovascular side effects, a common concern with antiangiogenic therapies. Though, further work to characterize its ocular pharmacokinetic profile is needed to identify the proper dosing regimen in future studies. Thus, these data herein have served as a basis for further development of JP-153 series analogs, used either as a topical or systemic therapeutic for in vivo efficacy studies and pre-clinical work. In conclusion, our work has successfully provided rationales for new drug targets and clinically relevant pharmacological agents to halt RNV. The following chapters describe and discuss novel ways in which we target inflammatory signaling and protein-protein interactions related to FA protein paxillin to effectively stop angiogenesis in the retina. Importantly, targeting paxillin has much broader implications in treating angiogenesis in general, and work studying paxillin modulation in cancer cells represents interesting hypotheses for future work in our laboratory

Health Disparities 2018: Closing the Gap

With the theme of “Health Disparities: Closing the Gap,” this symposium aims to showcase the work done by researchers here in the Valley and beyond toward improving the health and well-being of the communities they serve and society as a whole

Investigation of the mechanisms underlying the regulation of macrophage activation and glomerular inflammation by JunD

Macrophages are the predominant immune effector cell found in one of the most severe causes of renal failure in humans, crescentic glomerulonephritis (Crgn). The Wistar Kyoto (WLY) rat is uniquely susceptible among rat strains to Crgn and susceptibility genes have previously been mapped to seven quantitative trait loci (Crgn1-7). The AP-1 transcription factor JunD is markedly overexpressed in WKY macrophages and was identified as a major determinant of macrophage activation associated with Crgn susceptibility at Crgn2. The work presented in this thesis aimed to investigate mechanisms underlying the regulation of macrophage activation and glomerular inflammation by JunD in the NTN-susceptible WKY rat strain and in patients with Crgn. Genomic-based approaches, including microarray analysis of bone marrow-derived macrophage transcriptomes, RNA interference and cistrome analysis using ChIP-Seq, along with histological techniques and genotyping studies, were used to identify key genes and pathways underlying JunD-mediated activation of macrophages. Transcriptome analyses showed that the Crgn2 locus regulates expression of over 800 genes in WKY macrophage responses to lipopolysaccharide (LPS). Acute changes in Jund expression by RNA interference also modulated gene expression in WKY macrophages, including effector genes for the macrophage LPS response. Cistrome analysis revealed that genetically determined differences in Jund expression alter its genome binding pattern and the increased numbers of JunD-bound genes in WKY macrophages were functionally linked with signalling pathways and cell activation. Integration of the data identified 47 genes, that were associated with JunD binding peaks and whose expression was both regulated by Crgn2 and altered by siRNA knockdown of Jund, as the key candidates through which JunD determines macrophage activation. Overall, this work provides the basis for understanding genes and pathways through which JunD regulates macrophage activation and has identified novel gene targets for modulation of macrophage phenotypes in WKY rat and human macrophages

Autoimmune Disorders

The present edition entitled "Autoimmune disorders - Pathogenetic aspects" aims to present the current available evidence of etiopathogenetic insights of both systemic and organ specific autoimmune disorders, the crossover interactions among autoimmunity, cardiovascular morbidity and malignancy as well as novel findings in the exciting fields of osteoimmunology and immunology of pregnancy

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin