Harry S. Nick, PhD - US grants

Reduction of dioxygen, an initial product of which is the superoxide radical 02-, generates free radicals that have been implicated in the genesis of oxygen toxicity reperfusion injury, radiation injury, and in the action of certain xenobiotics. The superoxide dismutases play a critical role in the scavenging of superoxide. Delineation of their molecular biology will provide a basis for the understanding of their role in these pathologies. The primary goal of this proposal is to examine the molecular mechanisms which mediate the expression of both the rat and Mn and Cu/Zn superoxide dismutase (SOD) genes in vivo. We plan to isolate the cDNA clones for both of these genes via the screening of a rat liver gt11 expression library with polyclonal antibodies to each of these enzymes. Oligonucleotide probes derived from conserved regions of published human enzyme amino acid sequence will serve as direct verification of the recombinant clones. The isolated cDNA clones will be utilized as probes in the isolation of genomic clones and in the evaluation of mRNA levels from primary cultures of microvascular rat lung endothelial cells. mRNA levels from these endothelial cells and control cell lines will also be examined in response to stimuli demonstrated to effect SOD activity, specifically, oxygen tension, bacterial endotoxin and paraquat will be tested. In the same context, we plan experiments directed at understanding whether fluctuations in SOD mRNA levels in response to such stimuli are a consequence of transcriptional regulation or the inherent stability of the mRNA. With a basic understanding of transcriptional levels, we then plan to delineate the boundaries of DNA sequences required for cell specific control using studies on the expression of promoter deletion mutants and a detailed analysis of SOD chromatin structure. The extension of these goals of the nucleotide level using genomic sequencing will provide a "fingerprint" unique to each regulatory protein involved in SOD repression and/or induction. This fingerprint will serve as the assay for future isolation and characterization of regulatory proteins, their genes, and their regulatory response to superoxide levels.

The formation of prostaglandins is inhibited by anti-inflammatory steroids in intact cells and isolated organs by specifically hindering release of polyunsaturated fatty acid precursors, namely arachidonic acid. The inhibitory action of steroids is a consequence of RNA and protein synthesis. This acute anti- inflammatory activity has been correlated to steroid- dependent induction and release of phospholipase inhibitory proteins, which directly inhibit arachidonic acid release. Independently isolated proteins have been shown to be closely related and designated lipocortin. Full length cDNAs coding for human lipocortin I and II (also called calpactin) have recently been isolated. The purpose of this research is to undertake a high resolution analysis of the normal and glucocorticoid-mediated mechanisms which control the regulation of lipocortin I gene expression in vivo in the mouse, which offers established genetics, the direct analysis of animal tissues, and a wide variety of appropriate cell lines. The primary strategy of the proposal is as follows: 1) The initial goal is the isolation and total characterization of a complete murine cDNA and genomic clone by restriction and DNA sequence analysis; 2) The evaluation of tissue-specific transcription and the level of glucocorticoid-mediated transcriptional control; 3) The delineation of the promoter boundaries via the generation of deletion mutants of the lipocortin 5' flanking sequence, fusion to a reporter gene coding sequence, and assaying by transient expression and/or in stable transformants; 4) The investigation of lipocortin chromatin structure using DNase I hypersensitive studies; 5) The study of lipocortin gene regulation at nucleotide resolution by directly scanning relevant sequences in vivo to detect protein-DNA interactions using the laboratories expertise in genomic sequencing. These studies are also of medical relevance because lipocortin expression has been correlated to rheumatic diseases such as arthritis and systemic lupus erythematosus and through its steroid-mediated control of prostaglandin synthesis, a vital aspect in the inflammatory response.

The "free radical theory of oxygen toxicity" links the deleterious pulmonary effects of hyperoxia, cellular oxygen metabolism and the respiratory burst of activated inflammatory cells to highly reactive metabolic products of oxygen. These reactive oxygen species can inactivate cellular enzymes, damage DNA and destroy lipid bilayers. To protect cells from these cytotoxic oxygen metabolites, a system of cooperative antioxidants have evolved with the primary defense being the superoxide dismutases (SODs). It has been shown repeatedly that elevated levels of the manganese SOD (MnSOD) provide an effective antioxidant defense which is strongly associated with the cell's tolerance to superoxide induced injury and survival. Therefore, understanding the lung's normal mechanisms for stimulating endogenous antioxidant defenses may lead to logical steps in the development of therapeutiC regimens that are.effective in preventing or ameliorating free radical mediated pulmonary toxicity. To this end, the goals of this proposal are to understand the molecular mechanisms which control stimulus-dependent gene expression and mRNA stability of the MnSOD gene in pulmonary epithelial cells. We plan to evaluate the interaction of promoter and enhancer elements, relative to their role in mediating the MnSOD gene's response to the inflammatory mediators: LPS, IL-I, and TNF. Using in vivo footprinting, we plan to delineate, at single nucleotide resolution, the position of cis-acting regulatory sequences in the enhancer. The nucleotide contacts defined by in vivo footprinting will be utilized to identify and clone the appropriate stimulus-linked transacting factors and their coding sequences. Antisense and dominant negative strategies will be used to demonstrate functional relevance of each trans-acting factor to MnSOD gene expression. Finally, we will identify mRNA binding proteins involved in controlling MnSOD mRNA half-life using RLPCR and UV cross-linking.

DESCRIPTION (Applicant's Abstract): Of the known biologically active mediators generated by cells, both normally and during the inflammatory response, few have received more attention than those generated in the arachidonic acid cascade. Cytokines are established regulators of the arachidonic acid cascade in lung cells. The levels of various arachidonic metabolites distinguish between the normal and pathogenic states of the human lung. The arachidonyl-selective, cytosolic phospholipase A2 (cPLA2) is ubiquitously present in human lung and is most likely the rate-limiting step in eicosanoid generation. We therefore propose to study the molecular regulation of this pivotal gene in human lung fibroblasts and epithelial cells in response to glucocorticoids and pro-inflammatory cytokines. We will detect basal and stimulus-specific DNase I hypersensitive (HS) sites located in distant regions of the cPLA2 locus by utilizing a novel strategy involving field inversion gel electrophoresis (FIGE) to analyze altered regions of chromatin structure. A regulatory element trapping protocol will be employed to functionally identify regulatory sequences and complement our chromatin analysis. Deletion analysis will be used to precisely delineate cis-acting regulatory elements functionally by transient transfection in control, stimulated and repressed pulmonary cells. To identify, at single nucleotide resolution, the location of transcriptionally relevant protein-DNA contacts in living cells, we will employ in vivo footprinting using ligation mediated PCR (LMPCR) in conjunction with site-directed mutagenesis, EMSA and Transcription Factor Decoy (TFD) analysis. The knowledge gained by studying the molecular mechanisms controlling arachidonate levels will provide the basis for an understanding of the pathophysiology of acute lung injury that occurs in pulmonary diseases, such as asthma, acute respiratory distress syndrome, pulmonary fibrosis, and cystic fibrosis.

The lymphatic system serves five primary roles: 1) removal of excess body fluids; 2) absorption and transport of fatty acids/chyle to the circulatory system; 3) blood filtration; 4) mounting the primary defense against infections and cancer through immune cell production and activation; and 5) generation and activation of regulatory immune cells that protect against autoimmune/autoinflammatory disease. Given these key physiological functions, we deem it important to develop a three-dimensional (3D) tissue map for three major lymphoid organs of the human immune system: spleen, thymus and lymph nodes (Organ Specific Projects 1- 3, respectively). As one of our primary strengths, we have over 10 years of experience in the procurement of transplant-quality organs for research, including those of the lymphatic system. As part of this 24/7/365 effort, we have well-established relationships with the U.S. Organ Procurement Organizations (OPO), having referrals from 56/58 over the last decade. Our standard operating procedures (SOPs), both published and web-based, include assessment of normality, quality control assays, and systematic anatomical dissection/storage. We also participate in National QA/QC programs to evaluate tissue processing/banking procedures. Our approach to developing assay pipelines towards the common goal of a 3D tissue map will initially involve acquiring a macro image of the intact tissue and addressing tissue morphology, using our strengths in magnetic resonance imaging (MRI). The proposed optical microscopy pipeline will address microanatomical features using formalin fixed paraffin embedded (FFPE) and optimal cutting temperature (OCT) compound embedded sections as well as tissue optical clearing and expansion. These specimens will be studied from nm to mm resolution using stochastic optical reconstruction microscopy (STORM), confocal, multiphoton and light sheet fluorescence microscopies (LSFM), with all pipelines sharing a common file format for simplified 3D reconstruction. Based on the unique role for lymphatic organs in production and trafficking of immune cells, fluorescence activated cell sorting (FACS) of cells from blood and each lymphatic organ will provide a comparison of the patient- specific immune cell repertoire and serve in subsequent single cell RNA-seq analyses. To co-register biomolecules to their cognate cells, we will employ imaging mass cytometry (IMC), multiplex single molecule fluorescence in situ hybridization (smFISH) and multiplexed error-robust FISH (MERFISH) to map cellular protein and mRNA expression, ultimately on each 3D tissue atlas. Our experience in organ procurement, the availability of the National High Magnetic Field Laboratory (NHML) Advanced Magnetic Resonance Imaging and Spectroscopy (AMRIS) Facility at UF, multiphoton/confocal microscopy, LSFM, state of the art FACS, 10X GENOMICS Chromium controller, and high throughput cDNA library sequencing for RNA-Seq at the University of Florida, as well as world leading IMC and 3D data analysis facility at the University of Zurich, ideally positions us to provide the highest quality 3D Human BioMolecular Atlas (HuBMAP) of the lymphatic organs.

Extensive clinical research studies are urgently needed to inform patient management strategies and develop pharmaceutical countermeasures to combat the 2019 novel coronavirus disease (COVID-19). Currently, COVID-19 infections and hospitalizations are surging across the state of Florida; hence, we propose to establish the University of Florida (UF) as a subject-enrollment and sample collection center for the Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) study at three Florida health science centers. In this nationwide prospective observational study, peripheral blood and nasal swabs will be frequently collected from consented hospitalized patients with confirmed COVID-19, with endotracheal aspirates also collected if the patient requires intubation. After participants are discharged, blood and nasal swabs will be collected during outpatient visits held at three-month intervals over the course of one year, allowing for longitudinal analysis of the virus?s effects on the immune system both during active infection and following recovery. Through the OneFlorida Clinical Research Consortium (CRC), we already have established infrastructure including trained staff to support participant consenting/enrollment and sample collection, as well as on site laboratory facilities for sample processing. In Aim 1, we propose to integrate three OneFlorida CRC collection sites (Tampa General Hospital/University of South Florida, UF Health Jacksonville Medical Center, and UF Health Shands Hospital in Gainesville) into the IMPACC study. These sites have sufficient COVID-19 caseload to support enrollment of 100 participants over the course of the four-month recruitment period. In Aim 2, samples will be processed immediately upon collection and shipped to six core laboratories for precision medicine genotyping, viral sequencing, proteomics/metabolomics, antibody measurement and isotyping, as well as immunophenotyping by CyTOF. From these collective data, IMPACC seeks to link viral burden with immune signatures as biomarkers of acute disease severity, mortality, the development of durable immunity, and long-term outcomes in survivors. Moreover, extensive immunophenotyping data has the potential to uncover new therapeutic targets to mitigate the disease severity. Initially, COVID-19 mortality was attributed to a cytokine storm and enhanced thrombosis, supporting treatment with immunosuppressive drugs in patients with severe disease. However, in an effort to support the power of immmuophenotyping to provide key information, we provide preliminary data suggesting that immunosuppression is a primary concordant feature of the disease, potentially arguing against the routine use of immunosuppressant medications. These data also demonstrate our ability to perform single cell RNA sequencing (scRNAseq), scATACseq, spectral flow cytometry, and ELISpot to evaluate gene expression, chromatin accessibility, protein expression, and immune cell function, respectively. With the consortium?s approval to use residual IMPACC samples, these site-specific assays could be funded through outside mechanisms and the data shared across the IMPACC consortium.