ALBANY MEDICAL COLLEGE

RYAN WHITE PART C OUTPATIENT EIS PROGRAM

RYAN WHITE TITLE IV WOMEN, INFANTS, CHILDREN, YOUTH AND AFFECTED FAMILY MEMBERS AIDS HEALTHCARE

RYAN WHITE TITLE IV WOMEN, INFANTS, CHILDREN, YOUTH AND AFFECTED FAMILY MEMBERS AIDS HEALTHCARE

PLK1 REGULATION OF AIRWAY SMOOTH MUSCLE

METABOLIC AND HORMONAL MECHANISMS OF VCID

YERSINIA OUTER-MEMBRANE-VESICLE VACCINES AGAINST PNEUMONIC PLAGUE - SUMMARY YERSINIA PESTIS, THE ETIOLOGIC AGENT OF PLAGUE, HAS BEEN RESPONSIBLE FOR HIGH MORTALITY IN SEVERAL EPIDEMICS THROUGHOUT HUMAN HISTORY. PLAGUE HAS BEEN CLASSIFIED AS A RE-EMERGING DISEASE BY THE WORLD HEALTH ORGANIZATION SINCE THERE ARE SEVERAL THOUSAND REPORTED CASES OF THE DISEASE WORLDWIDE ANNUALLY AND MULTIDRUG-RESISTANT Y. PESTIS STRAINS OCCUR IN RECENT YEARS. THE PLAGUE BACILLUS HAS BEEN USED AS A BIOLOGICAL WEAPON RECORDED IN HUMAN HISTORY AND IS ONE OF THE MORE LIKELY BIOLOGICAL THREATS TO BE USED BY TERRORISTS. CURRENTLY, NO LICENSED PLAGUE VACCINES ARE AVAILABLE IN WESTERN WORLD. ISOLATION OF VIRULENT F1-NEGATIVE Y. PESTIS STRAINS FROM NATURAL SOURCES AND THE EXISTENCE OF LCRV POLYMORPHISMS IN YERSINIA MAY RESULT IN Y. PESTIS VARIANTS THAT ESCAPE PROTECTIVE IMMUNITY INDUCED BY LCRV AND F1 ANTIGENS. THEREFORE, VACCINES SOLELY BASED UPON LCRV AND F1 ANTIGENS IS INSUFFICIENT TO GUARANTEE LONG-TERM DEFENSE AGAINST PLAGUE IN HUMANS. IN ORDER TO OVERCOME THE DRAWBACKS OF SUBUNIT VACCINES COMPOSED OF LCRV AND F1 ANTIGENS, WE PROPOSE TO USE Y. PSEUDOTUBERCULOSIS (YPTB) OMVS AS AN ACELLULAR VACCINE AGAINST PLAGUE: (1) CONSTRUCT YPTB STRAINS WHICH ROBUSTLY PRODUCE HIGHLY IMMUNOGENIC SELF-ADJUVANTING OMVS CARRYING AN ARRAY OF Y. PESTIS PROTECTIVE ANTIGENS; (2) EVALUATE PROTECTIVE IMMUNITY OF OMVS IN RODENTS (MOUSE AND RAT). (3) CAREFULLY DECIPHER MECHANISMS OF IMMUNE PROTECTION INDUCED BY THE YERSINIA OMVS TO PROVIDE FUNDAMENTALS FOR RATIONAL PLAGUE VACCINE DEVELOPMENT. FINALLY, THE SUCCESS OF THIS PROJECT WILL PROVIDE HIGHLY EFFECTIVE AND SAFE PLAGUE VACCINES FOR HUMANS.

INFLAMMATION-RESOLUTION IMPAIRMENTS IN AGING AND ATHEROSCLEROSIS - SUMMARY AGING IS A MAJOR RISK FACTOR FOR ATHEROSCLEROTIC CARDIOVASCULAR DISEASE (CVD) AND YET MECHANISMS AS TO HOW AGING IMPACTS THIS DISEASE REMAINS VASTLY UNDEREXPLORED. AGING IS ASSOCIATED WITH NON-RESOLVING INFLAMMATION (INFLAMMAGING). THE RESOLUTION OF INFLAMMATION REQUIRES THE BALANCE BETWEEN PRO- INFLAMMATORY LEUKOTRIENES (LTS, E.G. LTB4) OR PROSTAGLANDINS (PGS) AND W3-DERIVED SPECIALIZED PRO- RESOLVING MEDIATORS (SPMS), LIKE RESOLVINS. WHILE IT IS NOW KNOWN THAT ATHEROSCLEROSIS IS ASSOCIATED WITH A REDUCED SPM:LT OR PG MEDIATOR RATIO, MAJOR GAPS EXIST IN OUR UNDERSTANDING OF (A) THE MECHANISMS REGULATING IMPAIRED RESOLUTION IN AGE-RELATED ATHEROSCLEROSIS AND (B) HOW SPMS PROMOTE RESOLUTION. OUR OBJECTIVE IS TO INVESTIGATE MECHANISMS OF DEFECTIVE RESOLUTION IN AGING SO THAT WE CAN DEVELOP NOVEL STRATEGIES TO TREAT ATHEROSCLEROSIS IN THE PHYSIOLOGICAL CONTEXT OF AGING. WE FOUND A NEW LINKS IN WHICH AGED ATHEROSCLEROTIC MICE HAD IMPAIRED RESOLUTION OF ATHEROSCLEROSIS, EXHIBITED EXUBERANT GRANULOPOIESIS AND DEFECTIVE SPM SIGNALING IN THE BONE MARROW. WE PROPOSE THREE HIGHLY INTEGRATED, BUT INDEPENDENT, AIMS TO ADDRESS MECHANISMS REGULATING MYELOPOIESIS IN AGING AND ATHEROSCLEROSIS AND TO DETERMINE THE EFFICACY AND MECHANISMS OF RESOLVIN THERAPIES IN AGING AND ATHEROSCLEROSIS. THESE AIMS WILL ADDRESS A FUNDAMENTAL GAP IN OUR UNDERSTANDING OF THE PHYSIOLOGICALLY RELEVANT CONTEXT OF AGING IN ATHEROSCLEROSIS. COMPLETION OF THE PROPOSED STUDIES WILL PROVIDE MECHANISTIC INSIGHT INTO DYSREGULATED RESOLUTION PROCESSES IN AGING, DEFINE CELLS, MEDIATORS AND MECHANISMS RESTRAINING PLAQUE RESOLUTION/REGRESSION IN AGING, AND ESTABLISH RATIONALES FOR NEW THERAPIES. .

ENDOSOME-MITOCHONDRIA INTERACTIONS IN BREAST CANCER CELLS

METABOLIC REGULATION OF HYPERCAPNIC CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD)-DRIVEN SKELETAL MUSCLE DYSFUNCTION - PROJECT SUMMARY: PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD)/PULMONARY EMPHYSEMA OFTEN DEVELOP LOCOMOTOR MUSCLE DYSFUNCTION, WHICH IS ASSOCIATED WITH WORSE CLINICAL OUTCOMES INCLUDING HIGHER MORTALITY. RETENTION OF CO2 IN THE BLOOD, OR HYPERCAPNIA, IS ALSO FREQUENT IN THESE PATIENTS AND SIMILARLY ASSOCIATED WITH HIGHER MORTALITY. THE MECHANISMS THAT REGULATE THESE PROCESSES ARE CURRENTLY UNKNOWN, AND THE AVAILABLE TREATMENTS HAVE NO EFFECTS ON SURVIVAL IN THIS SETTING. THEREFORE, UNDERSTANDING THE MECHANISMS CONTROLLING CO2- RETAINING COPD-DRIVEN MUSCLE DYSFUNCTION COULD HELP DEVELOP STRATEGIES TO PREVENT AND REVERSE THAT, WITH POTENTIALLY SURVIVAL AND QUALITY OF LIFE BENEFITS FOR THESE PATIENTS. MUSCLE DYSFUNCTION IN COPD IS ASSOCIATED WITH ABNORMAL PROTEIN TURNOVER AND METABOLISM. THE PRESENT APPLICATION PROPOSES TO INVESTIGATE THE CONTRIBUTION OF DYSREGULATED CELLULAR METABOLISM TO THE PATHOPHYSIOLOGY OF CO2-RETAINING COPD. THE HYPOTHESIS THAT SUPPORTS THIS APPLICATION IS THAT SUCCINATE DEHYDROGENASE (SDH)/COMPLEX-II SUBUNIT-C DOWNREGULATION REPRESENTS A FUNDAMENTAL EVENT IN COPD-DRIVEN SKELETAL MUSCLE DYSFUNCTION, CAUSING REDUCED ATP-GENERATION AND HIGHER FATIGABILITY; AND THAT HYPERCAPNIA ATTENUATES THIS PROCESS VIA LKB1-AMPK-DRIVEN MITOCHONDRIAL BIOGENESIS. TO INVESTIGATE THAT HYPOTHESIS, THE FIRST AIM IS DEDICATED TO STUDYING THE ROLE OF SDHC DOWNREGULATION IN COPD MYOPATHY USING AN ANIMAL MODEL OF COPD-DRIVEN SKELETAL MUSCLE DYSFUNCTION WE RECENTLY PUBLISHED. GENETIC RESTITUTION OF SDHC WILL ALLOW GAIN-OF-FUNCTION TO ADDRESS THE MECHANISMS LEADING TO METABOLIC DYSFUNCTION IN COPD MUSCLES. THE SECOND AIM OF THE PROPOSAL WILL INVESTIGATE THE SPECIFIC MECHANISMS THAT REGULATE CO2- DRIVEN DYSFUNCTIONAL METABOLISM. AS LKB1/AMPK CONTROLS CO2 SENSING AND PROTEIN TURNOVER IN SKELETAL MUSCLE, HYPERCAPNIA’S EFFECT ON METABOLISM WILL BE INVESTIGATED WITH LKB1 KNOCKOUT CELLS AND ANIMALS EXPOSED TO ELEVATED CO2. WE WILL THEN BLEND COPD AND CO2 ON A SINGLE MODEL AND PERFORM LOSS OF FUNCTION WITH A DOUBLE TRANSGENIC ANIMAL. THIS RESEARCH REPRESENTS A SUBSTANTIVE DEPARTURE FROM THE STATUS QUO BY FOCUSING ON THE CONTRIBUTION OF METABOLISM TO THE LONG-TERM EFFECTS OF COPD-DRIVEN MUSCLE DYSFUNCTION, AND SPECIFICALLY BY IDENTIFYING SDHC AND AMPK AS MAJOR PLAYERS COPD MUSCLE RESPIRATION AND FUNCTION.

HUMAN MECHANISMS OF VIRUS PERSISTENCE IN AN AAV-BASED MOUSE MODEL OF CHRONIC HBV INFECTION

TRANSLATION OF A HIGHLY PROTECTIVE TULAREMIA VACCINE TO THE NHP MODEL - SUMMARY: IDENTIFYING EFFECTIVE VACCINES AND THEIR CORRELATES OF PROTECTION (COPS) FOR HIGHLY LETHAL HUMAN (HU) PATHOGENS IS PROBLEMATIC. TULAREMIA (RABBIT FEVER), A SEVERE ZOONOSES CAUSED BY FRANCISELLA TULARENSIS (FT), IS A PRIME EXAMPLE. STUDIES CONDUCTED IN THE 1960S REVEALED THAT HU VACCINATION WITH THE ATTENUATED LIVE VACCINE STRAIN (LVS) OF FT PROVIDED PARTIAL PROTECTION AGAINST LOW-DOSE AEROSOL CHALLENGE WITH VIRULENT FT SCHUS4 (S4); HOWEVER, PROTECTION AGAINST HIGHER CHALLENGE DOSES (~2,000 CFU) WAS SUBPAR AND NO SURROGATE MARKERS OF HU PROTECTION WERE DEVELOPED. SINCE THE 1960S, NO FURTHER HU VACCINE-EFFICACY TRIALS HAVE BEEN PERFORMED AND THE BULK OF TULAREMIA VACCINE RESEARCH HAS RELIED ON INBRED RODENT MODELS. CURRENTLY, THERE ARE TWO UNMET NEEDS: I) VACCINES THAT PROTECT AGAINST HIGH-DOSE AEROSOL CHALLENGE WITH S4 AND II) HU-RELEVANT COPS TO GUIDE FUTURE CLINICAL TRIALS AS ENVISIONED BY THE FDA ANIMAL RULE. TO ADVANCE VACCINE CANDIDATES AND THEIR COPS, STUDIES ARE NEEDED IN HIGHER OUTBRED MODELS THAT FAITHFULLY REPLICATE HU RESPONSES. WE USED MURINE AND RABBIT (RB) MODELS TO IDENTIFY 2 LEAD VACCINE CANDIDATES (S4ΔAROD AND S4Δ GUABA) THAT WERE EFFECTIVE AGAINST AEROSOLS OF WT S4 (TARGET DOSE ~2,000 CFU). OUR TWO STRAINS ALONG WITH WT LVS AND AN EXTERNALLY DEVELOPED MUTANT (S4CLPB) WERE ALL FOUND TO PROVIDE VARIOUS DEGREES OF VACCINE-MEDIATED PROTECTION. HOWEVER, S4AROD WAS THE ONLY VACCINE THAT PROVIDED SIGNIFICANTLY GREATER PROTECTION THAN LVS. THE S4AROD-MEDIATED PROTECTION WAS ROBUST (75%) UP TO CHALLENGE DOSES OF ~20,000 CFU. DURING THESE EXPERIMENTS, WE ALSO COLLECTED PRE-CHALLENGE SERA FROM INDIVIDUAL RBS. FOLLOWING CHALLENGE OUTCOME, RETROSPECTIVE ANALYSIS OF THESE SERA IDENTIFIED PUTATIVE COPS THAT PREDICT S4 CHALLENGE OUTCOME WITH STATISTICAL SIGNIFICANCE. OUR LONG-TERM GOAL IS TO FOSTER ADVANCEMENT OF THE MOST EFFECTIVE FT VACCINE CANDIDATE TO HU CLINICAL TRIALS. OUR IMMEDIATE OBJECTIVES ARE TO DETERMINE HOW WELL THE S4AROD-MEDIATED PROTECTION AND COPS OBSERVED IN RBS TRANSLATE TO NON-HUMAN PRIMATES (NHP). WE HAVE FORMULATED THE FOLLOWING AIMS: AIM 1. VACCINE EFFICACY IN THE NHP MODEL AND AIM 2. CORRELATE ANALYSIS OF NHP SAMPLES. TO FURTHER HU TRANSLATION, WE HAVE SECURED AN INVALUABLE RESOURCE: ~150 PLASMA FROM LVS-VACCINATED HU BANKED PRIOR TO THE PAUSE IN THE US ARMY’S SPECIAL IMMUNIZATION PROGRAM. THE COMBINED BACTERIOLOGY, VACCINOLOGY, AEROBIOLOGY AND BIOSTATISTICS EXPERTISE WITHIN THIS CONSORTIUM MAKES US EXCLUSIVELY WELL-POSITIONED TO COMPLETE THESE STUDIES; OUR PRODUCTIVITY IN THE PRIOR CYCLE (>20 PUBLICATIONS) DOCUMENTS OUR ABILITY TO ACHIEVE OUR GOALS. BY THE END OF THESE STUDIES, WE WILL HAVE DETERMINED THE EFFICACY OF S4AROD-VACCINATION AGAINST S4 AEROSOL CHALLENGE REGARDING BOTH NHP MORTALITY AND MORBIDITY. WE WILL HAVE PERFORMED A HEAD-TO-HEAD COMPARISON WITH LVS VACCINATION AND DETERMINED IF COPS DEVELOPED IN RBS ARE ALSO PREDICTIVE IN NHPS. WE ENVISION THAT PIVOTAL VACCINE EFFICACY AND CORRELATE DATA DEVELOPED IN THE NHP MODEL WILL PLACE S4ΔAROD ON THE CUSP OF TRANSLATION TO ADVANCED DEVELOPMENT AND HU-CLINICAL TRIALS.

BLOOD DNA METHYLATION BIOMARKERS OF POST ACUTESEQUELAE OF SARS COV 2 INFECTION (PASC) - PROJECT SUMMARY: DNA 5'-C-PHOSPHATE-G-3' (CPG) METHYLATION IS A COVALENT EPIGENETIC MODIFICATION THAT REGULATES GENE EXPRESSION AND IS HIGHLY SENSITIVE TO AGE AND ENVIRONMENTAL FACTORS. CRITICALLY ILL PATIENTS EXHIBIT ALTERED CIRCULATING BLOOD DNA METHYLATION PROFILES. WE HAVE RECENTLY REPORTED A LARGE SCALE METHYLOME ANALYSIS OF COVID-19 IN ASSOCIATION WITH CLINICAL OUTCOMES, WHICH SUGGESTS AN EPIGENETIC REGULATION OF GENES CONTROLLING DISEASE SEVERITY. RECENT EVIDENCE INDICATES THAT MANY SURVIVING COVID-19 PATIENTS DEVELOP LONG TERM DYSFUNCTIONS AND THE NIH-NHLBI HAS RECENTLY LAUNCHED AN INITIATIVE TO ELUCIDATE THE NATURE OF POST-ACUTE SEQUELAE OF SARS-COV-2 INFECTION (PASC). IT IS CURRENTLY UNKNOWN IF THE RAPID METHYLOME REGULATION EVOKED BY ACUTE ILLNESS PERSIST AFTER SARS-COV-2 RESOLUTION. SUCH PERSISTENCE COULD UNDERPIN LONG TERM SEQUELA ASSOCIATED WITH THIS CONDITION. BECAUSE DNA METHYLATION IS A RELATIVELY STABLE DNA CHEMICAL MODIFICATION THAT COULD INFLUENCE LONG-TERM GENE EXPRESSION PROFILES AND GIVEN THAT WE RECENTLY FOUND THAT MULTIPLE REGIONS DEVELOPED DURING ACUTE ILLNESS PERSIST DIFFERENTIALLY METHYLATED ONE YEAR AFTER HOSPITAL DISCHARGE, WE HYPOTHESIZE THAT COVID-19 INFECTION LEADS TO ENDURING DNA METHYLATION ABNORMALITIES THAT REMAIN AFTER RESOLUTION OF ACUTE ILLNESS IN ASSOCIATION WITH THE POST-COVID-19 CLINICAL PROFILE. IN THIS APPLICATION, WE PLAN TO CONDUCT WHOLE GENOME DNA METHYLATION AND RNA SEQUENCING OF CIRCULATING LEUCOCYTES TO ESTABLISH SUB PHENOTYPES OF PASC, PREDICT FUTURE PASC DEVELOPMENT IN THE ACUTE COVID-19, AND DETERMINE WHICH CIRCULATING LEUCOCYTE LINEAGE CONTRIBUTES TO THAT ENDURING METHYLOME.

TYPE II ALVEOLAR EPITHELIAL CELL-INTRINSIC IL-1 RESPONSE IN PROTECTIVE IMMUNITY AGAINST TUBERCULOSIS - PROJECT SUMMARY/ABSTRACT INTERLEUKIN-1 (IL-1) PLAYS AN IMPORTANT ROLE IN THE HOST DEFENSE AGAINST MYCOBACTERIUM TUBERCULOSIS (MTB), THE BACTERIUM THAT CAUSES HUMAN DISEASE TUBERCULOSIS (TB). IL-1 IS ESSENTIAL FOR ANTI-BACTERIAL IMMUNITY DURING MTB INFECTION IN MICE AND GENETIC POLYMORPHISMS IN IL-1 RECEPTOR (IL-1R1) SIGNALING ARE ASSOCIATED WITH GREATER RISK FROM ACTIVE PULMONARY TB. IL-1R1 IS EXPRESSED BY BOTH THE HEMATOPOIETIC-DERIVED AND STROMAL CELLS IN THE LUNG, BUT THE CELL-TYPE SPECIFIC ROLES OF IL-1R1-SIGNALING IN PROTECTIVE IMMUNITY AGAINST TB REMAINS UNCLEAR. SUPPORTED BY A R56 BRIDGE GRANT FROM NIAID, WE INVESTIGATED THE CONTRIBUTION OF DIFFERENT LUNG CELLS TO THE IL- 1R1 MEDIATED PROTECTIVE IMMUNITY. WE USED BONE MARROW CHIMAERAS AND DISCOVERED THAT IL-1R1 EXPRESSION ON STROMAL CELLS IS CRUCIAL FOR PROTECTING MICE AGAINST SEVERE DISEASE. WE GENERATED A PANEL OF KNOCK-IN AND KNOCK OUT MOUSE STRAINS WHERE IL-1R1 EXPRESSION IS CONDITIONALLY DELETED- OR RESTORED IN SPECIFIC CELLS EITHER IN A WILD TYPE OR IL-1R1-DEFICIENT HOST BACKGROUND. OUR PRELIMINARY RESULTS INDICATED THAT EXPRESSION OF IL-1R1 ON TYPE II ALVEOLAR EPITHELIAL CELLS (AECII) IS BOTH NECESSARY AND SUFFICIENT FOR CONTROLLING BACTERIAL REPLICATION, INFLAMMATION IN THE LUNG AND PREVENTING WASTING DISEASE. AECII-INTRINSIC IL-1R1 SIGNALING IS CRITICAL FOR ANTI- MYCOBACTERIAL IMMUNITY AND PREVENTING TYPE I IFN (IFN-I) DEPENDENT NEUTROPHIL INFLUX. DEPLETING NEUTROPHILS OR IFN-I SIGNIFICANTLY REDUCED BACTERIAL LOAD, IMMUNOPATHOLOGY. MOREOVER, AECII-RESTRICTED OVERPRODUCTION OF GM- CSF IN THE LUNG PROTECTED THE HIGHLY SUSCEPTIBLE GM-CSF DEFICIENT ANIMALS BY REDUCING BACTERIAL LOAD, WEIGHT LOSS AND LUNG PATHOLOGY. IMPORTANTLY, IFN-I BLOCKADE RESCUED ALVEOLAR TYPE I (AEC-I) AND TYPE II (AECII) EPITHELIAL CELLS THAT WERE OTHERWISE DAMAGED DURING PROGRESSIVE DISEASE. BASED ON OUR PRELIMINARY RESULTS, WE HYPOTHESIZE THAT IL-1R1 SIGNALING IN AECII POTENTIATES THE ANTI-MICROBIAL FUNCTION OF MYELOID CELLS BY GM-CSF PRODUCTION AND RESTRAINS THE PATHOLOGICAL LEVEL OF IFN-I PRODUCTION TO MAINTAIN EPITHELIAL BARRIER INTEGRITY. WE HAVE THREE SPECIFIC AIMS. IN AIM1, WE WILL INVESTIGATE THE ROLE OF GM-CSF IN REGULATING ANTIMICROBIAL IMMUNITY BY EXAMINING BACTERIAL FITNESS AND REPLICATION DYNAMICS IN MYELOID CELLS OBTAINED FROM MICE THAT LACK OR EXPRESS IL-1R1 EXPRESSION IN AECII. IN AIM2, WE WILL INVESTIGATE THE ROLE OF AECII-DERIVED PROSTAGLANDIN E2 (PGE2) IN REGULATING IFN-I RESPONSE AND CONSEQUENT PATHOGENIC NEUTROPHIL INFLUX TO PREVENT TISSUE DAMAGE. FINALLY, IN AIM3, WE WILL INVESTIGATE THE IMPACT OF IFN-I ON ALVEOLAR EPITHELIAL CELL REPAIR AND REGENERATION BY PERFORMING LINEAGE TRACING AND SPATIAL ANALYSIS OF LUNG EPITHELIAL CELLS IN THE INFECTED LUNG SAMPLES. OVERALL, THE COMPLETION OF THESE AIMS WILL PROVIDE MECHANISTIC BASIS FOR IL-1R1 MEDIATED PROTECTIVE IMMUNITY AND REVEAL PRINCIPLES TO TARGET AIRWAY EPITHELIAL CELLS FOR BOOSTING ANTIMICROBIAL IMMUNITY AND LIMITING THE INEXORABLE LUNG DAMAGE CAUSED DURING TB THAT CONTRIBUTES TO LUNG FUNCTION IMPAIRMENT IN PATIENTS EVEN AFTER ANTIBIOTIC THERAPY.

TRANSDUCTION OF MECHANICAL STIMULI IN MYELINATION AND PERIPHERAL NERVE REPAIR

IL22 SIGNALING IN EPILEPSY

THE EFFECT OF ADOLESCENT DRUG-INDUCED NEUROIMMUNE SIGNALING IN SEX-SPECIFIC SOCIAL DEVELOPMENT AND REWARD LEARNING. - PROJECT SUMMARY SUBSTANCE USE DISORDERS (SUDS), PARTICULARLY THOSE INVOLVING OPIOIDS, HAVE REACHED EPIDEMIC LEVELS. SUDS ARE HYPOTHESIZED TO BE LEARNING AND MEMORY DISORDERS: THE ASSOCIATION OF DRUG ‘REWARD’ WITH OTHERWISE NEUTRAL STIMULI PROMOTES CONTINUED OR REINSTATED DRUG USE (RELAPSE) WHEN SIMILAR STIMULI ARE RE-ENCOUNTERED. RELAPSE RATES ARE 40-60%, SUGGESTING THAT PREVIOUSLY LEARNED DRUG ASSOCIATIONS ARE A CONSTANT RISK FOR THOSE RECOVERING FROM SUDS. SOCIALLY-MEDIATED LEARNING IS UBIQUITOUS ACROSS SPECIES, AND SOCIAL FACTORS ALSO MODULATE DRUG USE. HOWEVER, HOW SOCIAL FACTORS INFLUENCE DRUG-ASSOCIATED LEARNING, AND ITS EXPRESSION ONCE ESTABLISHED, IS UNCLEAR. THE NUCLEUS ACCUMBENS (NAC) REWARD REGION IS CRITICAL FOR BOTH DRUG ASSOCIATIVE LEARNING AND FOR SOCIAL INFLUENCE OVER BEHAVIOR. WE DETERMINED THAT MICROGLIA, THE RESIDENT IMMUNE CELLS OF THE BRAIN, MEDIATE SOCIAL DEVELOPMENT VIA PHAGOCYTOSIS, OR “PRUNING,” OF SYNAPTIC PROTEINS IN THE NAC CORE DURING EARLY ADOLESCENCE IN MALE RATS, AND PRE-ADOLESCENCE IN FEMALES. ADOLESCENT DRUG USE INCREASES SUD RISK AND SOCIAL DYSFUNCTION LATER IN LIFE; IN FACT, EVEN PRESCRIPTION OPIOID USE IN ADOLESCENCE INCREASES OPIOID MISUSE LATER IN LIFE. THE OPIOID MORPHINE DIRECTLY ACTIVATES A PRO-PHAGOCYTIC RECEPTOR ON MICROGLIA, INCLUDING ON NAC MICROGLIA. THESE DATA RAISE THE POSSIBILITY THAT ADOLESCENT OPIOID USE INCREASES SUD RISK BY CHANGING MICROGLIA-MEDIATED NAC AND SOCIAL DEVELOPMENT, AND IN TURN SOCIAL INFLUENCE OVER REWARD LEARNING. IN THIS PROPOSAL, WE WILL RESTRICT SHORT-TERM MORPHINE EXPOSURE TO EACH SEX-SPECIFIC NAC CORE PRUNING PERIOD IN ADOLESCENCE TO DETERMINE (1) HOW MORPHINE AFFECTS ONGOING DEVELOPMENTAL SYNAPTIC PRUNING ACTIVITY, (2) SOCIAL DEVELOPMENT, AND (3) SOCIALLY-MEDIATED REWARD LEARNING. OUR CORE HYPOTHESIS IS THAT ADOLESCENT MORPHINE EXPOSURE EXACERBATES MICROGLIA-MEDIATED PRUNING IN THE NAC CORE, CAUSING ABNORMAL SOCIAL DEVELOPMENT AND INCREASED SOCIAL INFLUENCE OVER REWARD LEARNING. THIS PROPOSAL WILL BE THE FIRST TO EXAMINE A MECHANISTIC RELATIONSHIP BETWEEN SOCIAL DEVELOPMENT IN ADOLESCENCE AND REWARD LEARNING IN ADULTHOOD, AND MAY IDENTIFY SEX- SPECIFIC ADOLESCENT PERIODS OF VULNERABILITY DURING WHICH LIFETIME SUD RISK AND OTHER MENTAL HEALTH DISORDERS CAN BE INFLUENCED.

THE REGENERATIVE POTENTIAL OF AQP2+ PROGENITOR CELLS - ABSTRACT IDENTIFICATION OF RENAL PROGENITOR CELLS HOLDS PROMISE FOR ELUCIDATING THEIR CONTRIBUTION TO DEVELOPMENTAL DEFECTS AND FOR ISOLATING HUMAN RENAL PROGENITOR CELLS AS A PREREQUISITE TO EVALUATING THEIR THERAPEUTIC POTENTIAL. WHETHER AN ADULT KIDNEY HARBORS PROGENITOR CELLS IS A HOTLY DEBATED ISSUE. BECAUSE MAMMALIAN KIDNEYS CAN REGENERATE NEW CELLS FOLLOWING NORMAL SHEDDING AND INJURY, WE HAVE PUBLISHED A STRICT DEFINITION OF AN ADULT RENAL PROGENITOR CELL REQUIRING IN VIVO DEMONSTRATION OF 1) SELF-RENEWAL, 2) CLONOGENICITY, 3) MULTIPOTENCY, AND PARTICIPATION IN 4) TISSUE MAINTENANCE AND IN 5) INJURY REPAIR. WE HAVE IDENTIFIED A SUBSET OF AQP2+ CELLS THAT WERE ALSO POSITIVELY STAINED WITH AN ANTIBODY RECOGNIZING BOTH V-ATPASE SUBUNITS B1 AND B2 (AQP2+B1B2+) AS THE FIRST POTENTIAL CANDIDATE THAT STRICTLY MEETS THESE 5 REQUIREMENTS. THESE AQP2+ PROGENITOR CELLS (AP) EXHIBITED THE CAPACITY OF SELF-RENEWAL, CLONOGENICITY, AND MULTIPOTENCY, AND GENERATED 5 TYPES OF CELLS INCLUDING PRINCIPAL CELLS (PC) AND INTERCALATED CELLS (IC) TO FORM DCT2, CNT, AND CD DURING DEVELOPMENT. ADULT AP ALSO POSSESSED THESE CAPABILITIES AND REGENERATED ALL CELL TYPES IN DCT2, CNT, AND CD DURING TISSUE MAINTENANCE AND AFTER UNILATERAL URETERAL OBSTRUCTION (UUO). AP EXPRESS IC-SELECTIVE JAG1 AND PC-SELECTIVE NOTCH1, AND MEDIATE REPAIR CORRELATING WITH NOTCH ACTIVATION. OTHERS HAVE REPORTED MARKED SEX BIAS IN THE TRANSCRIPTOME PROFILE OF PC. ALL OF THESE FINDINGS HAVE LAID A SOLID FOUNDATION FOR THIS PROJECT. IN THIS PROPOSAL, WE PROPOSE TO TEST OUR CENTRAL HYPOTHESIS THAT AP POSSESS A UNIQUE MOLECULAR SIGNATURE AND THEIR REGENERATIVE POTENTIAL DIFFERS BETWEEN MALES AND FEMALES AND IS REGULATED BY JAG1. THE SPECIFIC AIMS ARE TO IDENTIFY AND VALIDATE THE AP'S UNIQUE MOLECULAR SIGNATURE (AIM 1), TO INVESTIGATE THE AP'S REGENERATIVE POTENTIAL (AIM 2) AND AP'S REGULATION BY JAG1 (AIM 3) DURING TISSUE MAINTENANCE AND DURING UUO-INDUCED INJURY REPAIR. WE WILL EXPLORE A COMBINATION OF CUTTING EDGE TECHNIQUES/APPROACHES INCLUDING RFP-BASED CELL SORTING TO ENRICH AQP2+ LINEAGE CELLS, SINGLE CELL RNA-SEQ, AQP2ECE/+-BASED LINEAGE TRACING, UNBIASED THYMIDINE ANALOG LABELING, AND A SET OF INNOVATIVE TESTS THAT HAVE BEEN PROVEN TO BE EFFECTIVE FOR VIGOROUSLY VALIDATING B1B2 AS A MARKER OF AP. SUCCESSFUL COMPLETION OF THE PROJECT WILL LIKELY 1) REINFORCE AP AS A NOVEL CONCEPT, WHICH DIFFERS FROM WHAT HAS BEEN REPORTED FOR THE PROXIMAL TUBULES AND COULD SHED NEW LIGHT INTO THE DEVELOPMENTAL, HOMEOSTATIC, AND REGENERATIVE MECHANISMS; 2) YIELD DEEPER INSIGHTS INTO THE DIFFERENTIAL BEHAVIOR OF AP VS. PC AND IC; 3) IDENTIFY AND VALIDATE A UNIQUE MOLECULAR SIGNATURE OF AP FOR THEIR ISOLATION IN THE FUTURE; 4) LINK AP-MEDIATED REPAIR TO NOTCH SIGNALING; 5) ESTABLISH BOTH SEX AND JAG1 AS POTENTIAL REGULATORS OF AP; AND 6) ANSWER MANY FUNDAMENTAL QUESTIONS REGARDING THE ORIGINS OF PC AND IC, HOW THESE CELLS RESPOND TO INJURY THROUGH NOTCH, AND HOW DISRUPTION OF THIS PATHWAY LEADS TO KIDNEY FIBROSIS. IN SHORT, THE FINDINGS ARE SIGNIFICANT FOR HUMAN PATHOLOGY AND STEM CELL BIOLOGY IN GENERAL, AND FOR IMPROVEMENT OF IN VITRO ORGANOID GENERATION.

TARGETING INFLAMMATION-RESOLUTION PATHWAYS TO LIMIT VCID - ABSTRACT THERE ARE CURRENTLY NO EFFECTIVE THERAPIES TO REDUCE VASCULAR CONTRIBUTIONS TO COGNITIVE IMPAIRMENT AND DEMENTIA (VCID), WHICH CONTRIBUTE TO THE OVERALL BURDEN OF ALZHEIMER’S DISEASE AND RELATED DEMENTIAS (ADRDS). THIS GAP NECESSITATES EXPLORATION OF NEW CELLULAR PROGRAMS AND MECHANISMS ASSOCIATED WITH VCID. DEMENTIA RISK IS INCREASED BY 2.5 FOLD IN PATIENTS WITH ATHEROSCLEROSIS. THUS, ATHEROSCLEROSIS IS A MAJOR CONTRIBUTOR TO ADRDS YET THERAPIES THAT TARGET ADRDS IN THIS CONTEXT IS A MAJOR GAP. CHOLESTEROL LOWERING, WHICH IS THE PRIMARY TREATMENT FOR ATHEROSCLEROSIS, IS NOT SUFFICIENT TO CURTAIL ADRDS, AND WE POSIT THAT NON-TRADITIONAL RISK FACTORS LINKING BOTH ATHEROSCLEROSIS AND VCID MAY REVEAL NEW THERAPEUTIC STRATEGIES TO LIMIT ADRDS. NON-RESOLVING INFLAMMATION IS AN UNDERPINNING FOR BOTH ATHEROSCLEROSIS AND VCID. THE RESOLUTION OF INFLAMMATION IS REGULATED IN PART BY THE BALANCE BETWEEN SPECIALIZED PRO-RESOLVING MEDIATORS (SPMS), LIKE RESOLVINS (E.G RESOLVIN D2, RVD2) AND PRO- INFLAMMATORY MEDIATORS LIKE LEUKOTRIENES AND SOME OF PROSTANOIDS. SPMS RESTRAIN EXCESSIVE NEUTROPHILS (PMN) MIGRATION INTO TISSUES AND ACTIVATE PRO-REPARATIVE MONOCYTES/MACROPHAGES (M), WHEREAS PGS DERANGE BRAIN M FUNCTION WHICH IS ASSOCIATED WITH COGNITIVE AGING. YET, MAJOR GAPS EXIST IN OUR UNDERSTANDING OF: (A) MECHANISMS ASSOCIATED WITH IMPAIRED RESOLUTION IN VCID AND (B) HOW SPMS COULD PROMOTE RESOLUTION IN VCID. THUS, THE OVERALL OBJECTIVE OF THIS R01 IS TO INVESTIGATE MECHANISMS OF DEFECTIVE RESOLUTION IN VCID AND TO HARNESS SPM SIGNALING PATHWAYS TOWARDS A NOVEL TREATMENT STRATEGY. OUR CENTRAL HYPOTHESIS IS THAT ELEVATED AND SUSTAINED MYELOID CELLS PROMOTE VCID IN ATHEROSCLEROSIS, AND THAT TREATMENT WITH SPMS LIMITS MYELOID CELLS AND VCID IN MIDDLE AGE. PROOF-OF-CONCEPT VCID MODELS EXCLUDE PATHOPHYSIOLOGIAL ROLES OF ATHEROSCLEROSIS, AND OUR PROPOSAL OFFERS THIS KEY TRANSLATIONAL FOCUS. THUS, WE AIM TO A) UNCOVER CELL TYPE(S) AND MEDIATORS THAT DRIVE NON-RESOLVING INFLAMMATION (AIM 1), B) DETERMINE CAUSATION FOR ENDOGENOUS RESOLUTION SIGNALING (AIM 2) AND C) TEST SPMS AS A THERAPY (AIM 3) IN VCID. THESE AIMS ADDRESS A FUNDAMENTAL GAP IN OUR UNDERSTANDING OF THE PHYSIOLOGICALLY RELEVANT CONTEXT OF MIDDLE-AGE IN VCID. COMPLETION OF THE PROPOSED STUDIES WILL PROVIDE MECHANISTIC INSIGHT INTO DYSREGULATED RESOLUTION PROCESSES IN MIDDLE-AGE, DEFINE CELLS, MEDIATORS AND MECHANISMS THAT EXACERBATE VCID AND ESTABLISH RATIONALES FOR NOVEL THERAPIES TO REDUCE THE BURDEN OF DEMENTIA.

DECIPHERING MOLECULAR MECHANISMS OF CALCIUM HOMEOSTASIS - PROJECT SUMMARY CALCIUM IS A CRITICAL MEDIATOR OF MANY CELLULAR PROCESSES, INCLUDING MUSCLE CONTRACTION, MITOCHONDRIAL ACTIVITY, TRANSCRIPTION, CELL DIVISION, AND SYNAPTIC VESICLE RELEASE. PARADOXICALLY, CALCIUM CAN ALSO TRIGGER CELL DEATH AND CELLULAR NECROSIS. THEREFORE, DYSREGULATION IN CALCIUM SIGNALING CAN AFFECT CELLS IN DIFFERENT WAYS AND TO VARYING DEGREES. CONSEQUENTLY, CALCIUM LEVELS NEED TO BE TIGHTLY CONTROLLED. INDEED, DEFECTIVE CALCIUM SIGNALING HAS BEEN IMPLICATED IN MANY NEURODEGENERATIVE DISEASES, MUSCULAR DYSTROPHIES AND HEART DISEASE. TO UNDERSTAND THE MECHANISMS REGULATING CALCIUM SIGNALING AND HOW DEFECTS IN THIS PROCESS CAN LEAD TO CELLULAR DYSFUNCTION, WE ARE EXPLOITING THE MODEL SYSTEM CAENORHABDITIS ELEGANS TO IDENTIFY CRITICAL PROCESSES THAT ARE INVOLVED IN THE REGULATION OF CALCIUM HANDLING. WE HAVE RECENTLY DEMONSTRATED THAT SEL-12, THE C. ELEGANS PRESENILIN ORTHOLOG, HAS A ROLE IN MEDIATING ENDOPLASMIC RETICULUM-MITOCHONDRIAL CALCIUM HOMEOSTASIS. DISRUPTION OF PRESENILIN FUNCTION RESULTS IN AN INCREASE IN MITOCHONDRIAL CALCIUM LEVELS AND ALTERS MITOCHONDRIAL METABOLISM PROMOTING PROTEIN HOMEOSTASIS COLLAPSE AND NEURODEGENERATION. PRESENILIN IS A HIGHLY CONSERVED PROTEIN FOUND FROM PLANTS TO HUMANS THAT IS EXTENSIVELY FOUND ON ENDOMEMBRANE STRUCTURES (E.G., ENDOPLASMIC RETICULUM AND LYSOSOME) OF MOST CELL TYPES. HOWEVER, THE ROLE PRESENILIN HAS IN THE ENDOMEMBRANE SYSTEM IS NOT CLEAR. IMPORTANTLY, MUTATIONS IN HUMAN PRESENILIN ARE THE MOST COMMON CAUSE OF EARLY ONSET FAMILIAL ALZHEIMER'S DISEASE. DESPITE THE IDENTIFICATION OF THE INVOLVEMENT OF PRESENILIN IN ALZHEIMER'S DISEASE OVER 20 YEARS AGO, THE FUNCTIONAL CONSEQUENCES OF MUTATIONS IN PRESENILIN CAUSING ALZHEIMER'S DISEASE ARE NOT UNDERSTOOD. TO GAIN FURTHER INSIGHT INTO THE ROLE PRESENILIN HAS IN MITOCHONDRIAL CALCIUM HOMEOSTASIS AND NEURONAL FITNESS, WE HAVE DEVELOPED A NOVEL AND HIGHLY SELECTIVE RNA INTERFERENCE SCREEN TO IDENTIFY GENE PRODUCTS THAT MEDITATE THE ELEVATED ENDOPLASMIC RETICULUM TO MITOCHONDRIAL CALCIUM SIGNALING OBSERVED IN SEL-12 MUTANTS. FROM THIS SCREEN, WE HAVE IDENTIFIED SEVERAL PROTEINS KNOWN TO MEDIATE ENDOPLASMIC RETICULUM CALCIUM RELEASE AND MITOCHONDRIAL CALCIUM UPTAKE BUT WE ALSO IDENTIFIED SEVERAL GENE PRODUCTS WITH AN UNCHARACTERIZED ROLE IN ENDOPLASMIC RETICULUM AND MITOCHONDRIAL CALCIUM SIGNALING. WE PROPOSE TO UTILIZE A MULTIFACETED APPROACH THAT COMBINES GENETIC MANIPULATION, HIGH RESOLUTION LIVE CELL MICROSCOPY TO ANALYZE ENDOPLASMIC RETICULUM AND MITOCHONDRIAL DYNAMICS, MITOCHONDRIAL ACTIVITY ASSAYS, AND BEHAVIORAL ASSAYS TO DETERMINE THE ROLE THESE GENE PRODUCTS AS WELL AS SEL- 12 HAVE IN MITOCHONDRIAL HEALTH AND NEURONAL FITNESS.

INFLAMMATION-RESOLUTION PROGRAMS IN ENDOTHELIAL CELLS IN ATHEROSCLEROSIS - SUMMARY CHOLESTEROL-LOWERING TREATMENTS FOR MANAGING ATHEROSCLEROSIS DO NOT COMPLETELY REDUCE THE INFLAMMATORY RISK AND ASSOCIATED RISK OF CARDIOVASCULAR EVENTS IN MANY INDIVIDUALS. THIS LIMITATION IS LIKELY BECAUSE THE MECHANISMS PROMOTING RESOLUTION OF CARDIOVASCULAR DISEASE (CVD) ARE INCOMPLETELY UNDERSTOOD. INFLAMMATION RESOLUTION REQUIRES A TIGHT BALANCE BETWEEN PRO-INFLAMMATORY CYTOKINES (E.G. INTERLEUKINS 1 AND 6) AND INHIBITORY SIGNALS (E.G. SOCS3) AS WELL AS PROINFLAMMATORY LEUKOTRIENES OR PROSTAGLANDINS (SUCH AS LTB4, PGE2) AND Ω3-DERIVED SPECIALIZED PRO-RESOLVING MEDIATORS (SPMS), LIKE RESOLVINS. ATHEROSCLEROSIS IS ASSOCIATED WITH INCREASED IL-6 SIGNALING AND A REDUCED SPM:LT OR PG MEDIATOR RATIO, AND A “PRO-ATHEROGENIC” (INFLAMMATORY) ENDOTHELIAL PHENOTYPE. HOWEVER, MAJOR GAPS EXIST IN OUR UNDERSTANDING OF (A) THE MECHANISMS REGULATING IMPAIRED RESOLUTION IN CHOLESTEROL-LOWERING SETTINGS, AND (B) HOW SOCS3 AND SPMS PROMOTE RESOLUTION. OUR OBJECTIVE IS TO INVESTIGATE MECHANISMS OF INFLAMMATION RESOLUTION WITHIN ENDOTHELIAL CELLS THAT LIMIT THE PROGRESSION OF ATHEROSCLEROTIC PLAQUES AND PROMOTE REPAIR IN THE CONTEXT OF CHOLESTEROL-LOWERING REGIMENS. WE FOUND NEW REGULATORY SIGNALING LOOPS BETWEEN PRO-INFLAMMATORY CYTOKINES AND PRO-RESOLVING SIGNALS WITHIN ENDOTHELIAL CELLS THAT REDUCE (IL-6) OR INCREASE (SPMS OR SOCS3) ATHEROSCLEROTIC PLAQUE CAP THICKNESS. WE PROPOSE TWO HIGHLY INTEGRATED, BUT INDEPENDENT, AIMS TO ADDRESS MECHANISMS BY WHICH PRO-RESOLVING MEDIATORS ACT ON THE ENDOTHELIUM TO REGULATE ATHEROSCLEROSIS PROGRESSION AND REGRESSION. THESE AIMS WILL ADDRESS A FUNDAMENTAL GAP IN OUR UNDERSTANDING OF HOW ENDOTHELIAL CELLS ORCHESTRATE PRO-RESOLVING SIGNALS TO LIMIT ATHEROSCLEROSIS PROGRESSION. COMPLETION OF THE PROPOSED STUDIES WILL PROVIDE MECHANISTIC INSIGHT INTO DYSREGULATED RESOLUTION PROCESSES IN ATHEROSCLEROSIS, DEFINE THE DIRECT AND INDIRECT ENDOTHELIAL MECHANISMS REGULATING CAP THICKNESS, AND ESTABLISH RATIONALES FOR NEW THERAPIES TO REDUCE THE LINGERING INFLAMMATORY RISK IN SUBJECTS UNDERGOING LIPID-LOWERING TREATMENTS FOR CVD MANAGEMENT. .

MOLECULAR MECHANISMS OF CONFINED CELL MIGRATION - PROJECT SUMMARY/ABSTRACT CELL MIGRATION IS INTEGRAL TO EMBRYONIC DEVELOPMENT, IMMUNE SURVEILLANCE, WOUND HEALING, AND CANCER METASTASIS. IN ORDER TO TRAVERSE THE VARIED PHYSIOCHEMICAL ENVIRONMENTS IN TISSUES, CELLS HAVE BEEN SHOWN TO SWITCH BETWEEN DISTINCT MIGRATION MODES. FOR INSTANCE, WHEN SUBJECTED TO HIGH MECHANICAL CONFINEMENT, CELLS HAVE BEEN SHOWN TO UNDERGO A PHENOTYPIC TRANSITION TO WHAT HAS BEEN TERMED, FAST AMOEBOID (LEADER BLEB- BASED) MIGRATION. FAST AMOEBOID MIGRATION IS CHARACTERIZED BY THE FORMATION OF A LEADER BLEB, WHICH IS A LARGE AND STABLE BLEB. WITH NON-SPECIFIC FRICTION, A RAPID CORTICAL ACTIN FLOW IN LEADER BLEBS PROVIDES THE MOTIVE FORCE FOR FAST AMOEBOID MIGRATION. PREVIOUSLY, WE DEMONSTRATED THAT THE ACTIN CAPPING AND BUNDLING PROTEIN, EPS8, IS REQUIRED FOR LEADER BLEB FORMATION WITHIN A RANGE OF CANCER CELL TYPES. HOWEVER, UNDER CONDITIONS OF HIGH MECHANICAL CONFINEMENT, IMMUNE CELLS HAVE ALSO BEEN SHOWN TO ADOPT FAST AMOEBOID MIGRATION. THEREFORE, CANCER AND IMMUNE CELLS CAN UTILIZE SIMILAR METHODS OF MIGRATION IN CONFINED ENVIRONMENTS. ALTHOUGH IT APPEARS THAT CANCER AND IMMUNE CELLS MAY SHARE SIMILAR MECHANISMS FOR SWITCHING TO FAST AMOEBOID MIGRATION (I.E., CONFINEMENT SENSING), WHETHER CANCER AND IMMUNE CELLS REQUIRE THE SAME SUITE OF FACTOR(S) TO UNDERGO FAST AMOEBOID MIGRATION IS NOT KNOWN. ACCORDINGLY, USING IN VITRO AND IN VIVO APPROACHES, THE PROPOSED WORK WILL DETERMINE THE MOLECULAR MECHANISM(S) REQUIRED BY CANCER AND IMMUNE CELLS FOR MIGRATION IN CONFINED ENVIRONMENTS. THIS IS SIGNIFICANT AS ELUCIDATING THESE MECHANISMS IS A REQUIRED FIRST STEP FOR THE RATIONALE DEVELOPMENT OF SO-CALLED “MIGRASTATICS,” WHICH PREVENT OR ABATE THE MIGRATION OF UNHEALTHY (CANCEROUS) BUT NOT HEALTHY (IMMUNE) CELLS.

ROLE OF A FATTY ACID CHAPERONE IN SCHWANN CELL MYELINATION - CHARCOT-MARIE-TOOTH DISEASE (CMT) IS A GROUP OF DISORDERS AND THE MOST COMMON INHERITED PERIPHERAL NEUROPATHY WITH A PREVALENCE OF 1:2500. APPROXIMATELY 1,000 SMALL MUTATIONS (MISSENSE, NONSENSE, SMALL DELETION OR INSERTION, SPLICE ALTERATIONS) IN MORE THAN 40 GENES ARE RESPONSIBLE FOR CMT. CMT SEVERELY IMPACTS THE IS ORTHOPEDIC QUALITY OF LIFE FOR PATIENTS AND NO EFFECTIVE PREVENTION OR CURE FOR CMT, AND PATIENT CARE IS LIMITED TO PHYSICAL AND OCCUPATIONAL THERAPIES, DEVICES, AND PAIN RELIEF, WHICH IS OFTEN SUBOPTIMAL. ACCOUNTS FOR SIGNIFICANT LIFELONG DISABILITY AND IMPORTANT ECONOMIC LOSS. THERE CMT TYPE 1 IS A DEMYELINATING PERIPHERAL NEUROPATHY CHARACTERIZED BY REDUCED MOTOR NERVE CONDUCTION VELOCITIES (LESS THAN 38 M/S) AND SEGMENTAL DEMYELINATION AND REMYELINATION WITH ONION BULB FORMATIONS ON NERVE BIOPSY. DESPITE THE DIFFERENT SUBTYPES OF CMT ASSOCIATED WITH DISTINCT GENETIC CAUSES AND PATHOGENETIC MECHANISMS, ALL FORMS OF CMT1 EVENTUALLY CONVERGE ON THE LOSS OF MYELINATED NERVE FIBERS. THUS, A COMMON APPROACH BASED ON ENHANCING MYELINATION IN DISEASED NERVES WITHOUT EXACERBATING MYELIN DAMAGES MUST BE IDENTIFIED TO PROVIDE WIDELY EFFECTIVE THERAPY FOR THE ARRAY OF CMT NEUROPATHIES. WE PREVIOUSLY SHOWED THAT ENHANCING AXONAL NRG1TIII SIGNALING INCREASES FATTY ACID LEVELS IN MYELIN, IMPROVES MYELINATION AND NERVE FUNCTION IN MOUSE MODELS FOR INHERITED DEMYELINATING NEUROPATHY, THROUGH AN ALTERNATIVE EGR2-INDEPENDENT PATHWAY. NOTABLY, WE FOUND THAT PMP2, A FATTY ACID-BINDING PROTEIN IS UNIQUELY UP-REGULATED DOWNSTREAM NRG1TIII OVEREXPRESSION IN SCHWANN CELLS. OUR CENTRAL HYPOTHESIS OF THIS PROPOSAL IS THAT OVEREXPRESSION OF THE FATTY ACID-BINDING PROTEIN PMP2 IS A DOWNSTREAM PROMYELINATING MEDIATOR OF NRG1TIII- MEDIATED HYPERMYELINATION AND THAT SUSTAINED OVEREXPRESSION OF PMP2 IS SUFFICIENT TO IMPROVE MYELIN FORMATION, WITHOUT EXACERBATING MYELIN DAMAGES. WE HAVE NOW COMPELLING PRELIMINARY EVIDENCE THAT PMP2 OVEREXPRESSION IN SCHWANN CELLS ENHANCES THE UPTAKE OF FATTY ACID, INCREASES ATP PRODUCTION, AND IS BENEFICIAL TO SCHWANN CELL MYELINATION AND REMYELINATION. THUS, WE PROPOSE TO DETERMINE IF PMP2 OVEREXPRESSION IS A SUITABLE STRATEGY TO IMPROVE MYELINATION DEFECTS IN INHERITED PERIPHERAL DEMYELINATING NEUROPATHY (P0S63DEL), WHICH OF PMP2 FUNCTIONS ARE BENEFICIAL TO IMPROVE MYELIN FORMATION, AND HOW PMP2 EXPRESSION IS BEING REGULATED. THE NEW MECHANISTIC INSIGHTS ON PMP2 OVEREXPRESSION ENHANCING MYELINATION WE WILL GAIN FROM THIS WORK ARE IN LINE WITH NINDS OBJECTIVE SEEKING FUNDAMENTAL KNOWLEDGE ABOUT THE NERVOUS SYSTEM THAT MAY FACILITATE THE DEVELOPMENT OF FUTURE INTERVENTIONS TO REDUCE THE DISEASE BURDEN IN PATIENTS WITH PERIPHERAL NEUROPATHIES.

MOLECULAR MECHANISMS REGULATING PIK3CA-INDUCED VENOUS MALFORMATIONS - SUMMARY PI3’-KINASE (PI3K) SIGNALING IS CRITICAL FOR PROPER ANGIOGENESIS, THE DEVELOPMENT OF NEW BLOOD VESSELS. HOWEVER, ABERRANT PI3K SIGNALING IS ASSOCIATED WITH A WIDE RANGE OF VASCULAR MALFORMATIONS (VMS), INCLUDING VENOUS MALFORMATIONS WHICH CAN ARISE FROM SOMATIC ACTIVATING MUTATIONS IN PIK3CA. VMS ARE FAILURES IN PROPER ENDOTHELIAL CELL DEVELOPMENT AND DIFFERENTIATION GIVING RISE TO DYSFUNCTIONAL BLOOD VESSELS THAT LEAVE AFFECTED PATIENTS WITH SIGNIFICANT MORBIDITY AND OFTEN DISFIGUREMENT. OUR LONG-TERM GOAL IS TO BETTER UNDERSTAND THE MOLECULAR ETIOLOGY OF VASCULAR MALFORMATIONS WITH THE HOPE OF CONTRIBUTING NOVEL APPROACHES TO THE TREATMENT OF THESE CONDITIONS NON-SURGICALLY. WE HAVE DEVELOPED IN VITRO AND IN VIVO MODELS OF PIK3CA-DRIVEN VMS AND FIND THAT THE MTORC1 AXIS MAY BE A CRITICAL COMPONENT OF IMPROPER VASCULAR MORPHOGENESIS. OUR CENTRAL HYPOTHESIS IS THAT THE MTORC1 AXIS IS BEING REGULATED THROUGH A NOVEL MECHANISM; THE INDUCTION OF THE MTORC1 REGULATING PROTEIN RHEB. THIS PROPOSAL SEEKS TO DETERMINE THE ROLE OF RHEB IN DRIVING MALFORMATIONS ARISING FROM PIK3CA MUTATIONS AND REVEAL MOLECULAR INSIGHTS INTO THE MECHANISMS BY WHICH RHEB/MTORC1 AXIS PROMOTES THE DEVELOPMENT OF VASCULAR MALFORMATIONS.

IMPROVING INFLAMMATION RESOLUTION TO MITIGATE ACQUIRED BONE MARROW FAILURE - IMPROVING INFLAMMATION RESOLUTION TO MITIGATE ACQUIRED BONE MARROW FAILURE BONE MARROW FAILURE IS THE DEVASTATING COLLAPSE OF BLOOD PRODUCTION, AND CURRENT TREATMENTS ARE INADEQUATE. FIRST LINE THERAPIES INCLUDE IMMUNOSUPPRESSIVE THERAPIES, AND WHEN FEASIBLE, BONE MARROW TRANSPLANTATION. THESE THERAPIES ARE OFTEN EFFICACIOUS IN THE SHORT-TERM, ESPECIALLY FOR YOUNG PATIENTS, HOWEVER THEY ARE MUCH LESS EFFECTIVE IN OLDER PATIENTS. IMMUNE SUPPRESSION IS NOT WELL TOLERATED, ESPECIALLY IN THE ELDERLY, AND CARRIES INCREASED SUSCEPTIBILITY TO INFECTION. THEREFORE, ALTERNATIVE THERAPIES ARE NEEDED. IT IS WELL ESTABLISHED THAT BONE MARROW FAILURE IS ASSOCIATED WITH NON-RESOLVING INFLAMMATION. THE RESOLUTION OF INFLAMMATION REQUIRES THE BALANCE BETWEEN PRO-INFLAMMATORY LEUKOTRIENES OR PROSTAGLANDINS AND W3-DERIVED SPECIALIZED PRO-RESOLVING MEDIATORS (SPMS), LIKE RESOLVINS. USING A MOUSE MODEL OF ACQUIRED SEVERE APLASTIC ANEMIA (SAA) THAT MIRRORS KEY FEATURES OF HUMAN DISEASE, WE ESTABLISHED A ROLE FOR IMPAIRED INFLAMMATION RESOLUTION PROGRAMS IN DISEASE PATHOGENESIS. OUR PRELIMINARY WORK DEMONSTRATES THE EFFECTIVENESS OF SPMS IN AMELIORATING SAA. THE RATIONALE FOR THE PROPOSED WORK IS THAT RATHER THAN BLUNTING INFLAMMATORY RESPONSES, PROMOTING RESOLUTION AND REPARATIVE PROCESSES MAY BE AN IDEAL THERAPEUTIC STRATEGY FOR BONE MARROW FAILURE. THE PROPOSED STUDIES WILL ADDRESS A FUNDAMENTAL GAP IN OUR UNDERSTANDING OF THE PHYSIOLOGICALLY RELEVANT PROCESS OF INFLAMMATION RESOLUTION IN THE BONE MARROW. WE PROPOSE THREE INTEGRATED, BUT INDEPENDENT, AIMS TO: (1) ADDRESS MECHANISMS OF IMPAIRED RESOLUTION, (2) DETERMINE THE IMPACT OF SPMS IN THE MARROW IN SAA, AND (3) EVALUATE THE EFFECT OF SPMS ON FUNCTION OF HEMATOPOIETIC STEM CELLS. THE PROPOSED WORK WILL SUPPORT A NOVEL APPROACH TO TREAT BMF WITHOUT THE USE OF IMMUNE SUPPRESSION, REPRESENTING AN IMPORTANT DEPARTURE FROM THE CURRENT STANDARD OF CARE. WE BELIEVE THESE STUDIES WILL BE IMPACTFUL THERAPEUTIC OPTIONS FOR BMF PATIENTS, PARTICULARLY OLDER PATIENTS WHERE IST HAS FAILED, AND WHEREIN HOST DEFENSES ARE ALREADY WEAKENED. AT THE SAME TIME, THE PROPOSED STUDIES MAY PROVIDE NEW MECHANISTIC INSIGHT RELEVANT TO OTHER DISEASES CHARACTERIZED BY PROLONGED AND NON-RESOLVED INFLAMMATION, AND THUS WILL HAVE BROAD IMPACT TO HUMAN HEALTH.

EMERGENCY DEPARTMENT ALTERNATIVES TO OPIOIDS AT THE ALBANY MED HEALTH SYSTEM (ED-ALT AMHS) - TO ADDRESS THE URGENT OPIOID EPIDEMIC PUBLIC HEALTH THREAT IN NORTHEASTERN NEW YORK STATE, THE EMERGENCY DEPARTMENT ALTERNATIVES TO OPIOIDS AT THE ALBANY MED HEALTH SYSTEM (ED-ALT AMHS) WILL ADAPT EVIDENCE-BASED PRACTICES TO 1) IMPROVE PERSON-CENTERED CARE (PCC) FOR THOSE WITH PAIN CONDITIONS IN THE ED, 2) IMPLEMENT AN ED-ALT PROGRAM, AND 3) INCREASE CAPACITY AND COORDINATION OF CARE FOR THOSE WITH OPIOID USE DISORDER (OUD) AND PAIN AND OPIOID USE. THE PROJECT WILL BE IMPLEMENTED IN THE AMHS FOUR HOSPITAL EDS WITH A GREATER CATCHMENT AREA OF 2.8 MILLION PEOPLE SPANNING 25 COUNTIES AND A COMBINED TOTAL VOLUME OF OVER 170,000 ANNUAL ED VISITS. AMONG THESE VISITS, THE PROGRAM WILL HAVE AN IMPACT ON AN ESTIMATED 83,000 (49%) PATIENT ENCOUNTERS IN THE ED PER YEAR WITH PERSONS REPORTING PAIN, AMONG WHOM AN ESTIMATED OVER 37,000 (45%) YEARLY WOULD BE EXPECTED TO RECEIVE AN OPIOID EITHER IN THE ED OR UPON DISCHARGE WITHOUT THE PROJECT INTERVENTION (FOR A TOTAL OF OVER 235,000 PATIENT ENCOUNTERS WITH PAIN CONDITIONS IN THE ED AND OVER 106,000 PATIENT ENCOUNTERS IN WHICH OPIOIDS WOULD BE PRESCRIBED OVER THE THREE-YEAR PROJECT PERIOD WITHOUT THE PROJECT INTERVENTION). THE ED-ALT AMHS PROJECT WILL SERVE OUR POPULATION OF FOCUS WHICH INCLUDES PEOPLE RESIDING IN RURAL, URBAN, AND SUBURBAN AREAS WITH AN ESTIMATED SELF-REPORTED 51% FEMALE; 16.1% UNDER 18 YEARS; 23.6% 65 YEARS AND OVER; 80% WHITE; 11.9% BLACK OR AFRICAN AMERICAN; 1.2% ASIAN, NATIVE HAWAIIAN, OR PACIFIC ISLANDER; 0.01% AMERICAN INDIAN/ALASKA NATIVE; AND 3.9% HISPANIC OR LATINO.1 THE PROGRAM AIMS TO IMPROVE THE CULTURE OF PERSON-CENTERED CARE FOR ALL PATIENTS IN THE ED WITH PAIN CONDITIONS, IMPROVE THE USE OF ALTERNATIVES TO OPIOIDS (ALTOS) FOR PAIN, AND STRENGTHEN CAPACITY AND EXISTING RESOURCES FOR SCREENING, MEDICATIONS, AND CARE FOR PATIENTS WITH OUD OR PAIN AND OPIOID USE IN THE ED. THESE AIMS WILL BE ACHIEVED BY IDENTIFYING KEY PERFORMANCE AND OUTCOME GAPS IN THE PROVISION OF PCC FOR PERSONS WITH PAIN CONDITIONS IN THE ED THROUGH PATIENT INTERVIEWS AND ED PROVIDER AND NURSING STAFF FOCUS GROUPS AND DEVELOPING AND IMPLEMENTING LOCALLY-INFORMED PCC STRATEGIES AND TRAINING FOR ED PROVIDERS AND NURSING STAFF. TO IMPROVE THE USE OF ALTOS IN THE AMHS EDS, THE PROJECT TEAM WILL 1) DEVELOP AND IMPLEMENT TRAINING FOR PROVIDERS AND NURSING STAFF IN ALTO BEST PRACTICES AND DIRECTED STRATEGIES TO ADDRESS DIFFERING PAIN CONDITIONS IN THE ED; 2) DEVELOP ELECTRONIC HEALTH RECORD CLINICAL DECISION SUPPORT (ORDER SETS AND BEST PRACTICE ALERTS) AND 3) TRAIN ED PROVIDERS AND NURSES IN TWO INTEGRATIVE NON-PHARMACOLOGIC THERAPIES (MINDFUL BREATHING AND MUSIC THERAPIES). ADDITIONALLY, ED PROVIDER AND NURSE TRAINING WILL BE EXPANDED THROUGH TRAINING AND EDUCATION IN SCREENING, BRIEF INTERVENTION AND REFERRAL TO TREATMENT (SBIRT) AND ED PROVIDER TRAINING IN MEDICATIONS FOR OUD AND BUPRENORPHINE FOR PAIN IN THE ED. FINALLY, THE PROPOSED PROJECT WILL IMPROVE CARE FOR PATIENTS WITH PAIN WHO USE OPIOIDS THROUGH TRAINING AND COORDINATION OF MULTIDISCIPLINARY CARE AND STRENGTHENING PARTNERSHIPS WITH COMMUNITY ORGANIZATIONS.

ICU PHYSICIAN SERVICES - SSA

CALCIUM/CALMODULIN ACTIVATED KINASES IN SMOOTH MUSCLE

ENDOTHELIAL MECHANISMS OF MULTIORGAN DYSFUNCTION - PROJECT SUMMARY THREE DIFFERENT FATES AWAIT THE MILLIONS OF CRITICALLY ILL PATIENTS ADMITTED TO INTENSIVE CARE UNITS EVERY YEAR. CLOSE TO 30% WILL RECOVER WITHOUT OBVIOUS SEQUELAE, 15% SUCCUMB TO THE ACUTE ILLNESS, AND THE REMAINDER 55% WILL DEVELOP VARIOUS DEGREES OF LONG-TERM IMPAIRMENTS IN COGNITIVE, IMMUNE, CARDIOVASCULAR, OR RENAL FUNCTIONS, LEADING TO INCREASED OVERALL MORTALITY. THESE SEQUELAE ARE DIAGNOSED UNDER THE UMBRELLA TERM POST-INTENSIVE CARE SYNDROME (PICS). WE LACK THE KNOWLEDGE TO IMPROVE ACUTE SURVIVAL, AND TO PREDICT AND TREAT PICS. LARGELY, THERAPIES FOR SEPTIC SHOCK AND OTHER CRITIALLY ILL PATIENTS ARE LIMITED TO INFECTIOUS SOURCE CONTROL AND HEMODYNAMIC SUPPORT. SEVERE SYSTEMIC INFLAMMATORY REACTIONS, INCLUDING SEPSIS, OFTEN LEAD TO SHOCK, ORGAN FAILURE AND DEATH, IN PART THROUGH AN ACUTE RELEASE OF CYTOKINES THAT PROMOTE VASCULAR DYSFUNCTION. THE CURRENT BODY OF WORK, INCLUDING OUR OWN RESEARCH, STRONGLY ARGUES FOR A CRITICAL ROLE FOR THE ENDOTHELIUM IN DETERMINING THE OUTCOMES OF CRITICAL ILLNESS THROUGH EXPRESSION OF MULTIPLE PROTEINS TO PROMOTE DISSEMINATED INTRAVASCULAR COAGULOPATHY, LEUKOSTASIS AND EDEMA. HOWEVER, SIMPLY BLOCKING CYTOKINE ACTIVITY DOES NOT IMPROVE SURVIVAL, IN LARGE PART DUE TO THE IMMUNOSUPPRESIVE ACTIONS OF THESE TREATMENTS. IT IS IMPERATIVE TO RETHINK THE PROBLEM. WE POSIT THAT A BETTER UNDERSTANDING OF THE ENDOTHELIAL MECHANISMS DOWNSTREAM OF CYTOKINE SIGNALING WILL LEAD TO IMPROVED THERAPIES TO PREVENT ORGAN DAMAGE AND MORTALITY WITHOUT INTERFERING WITH THE REQUIRED PATHOGEN CLEARANCE. LITTLE IS KNOWN ABOUT THE ENDOTHELIAL SIGNALING PATHWAYS REGULATING THE TRANSCRIPTIONAL PROFILE IN FAILING ORGANS. THIS PROPOSAL IS DESIGNED TO TAKE FULL ADVANTAGE OF THE INNOVATIVE TOOLS AND KNOWLEDGE WE DEVELOPED DURING THE LAST SEVERAL YEARS TO ASK FUNDAMENTAL MECHANISTIC QUESTIONS ON THE ROLE OF ENDOTHELIAL SIGNALING AND TRANSCRIPTIONAL RESPONSES DURING SEVERE INFLAMMATION. SOURCING OF HUMAN PRIMARY ENDOTHELIAL CELLS IN-HOUSE ALLOWS US TO PERFORM MECHANISTIC STUDIES IN A COST-EFFECTIVE MANNER, A PANEL OF ENDOTHELIAL-SPECIFIC TRANSGENIC MICE ENABLES US TO STUDY KEY REGULATORS OF TRANSCRIPTION IN THE CONTEXT OF MULTIORGAN DYSFUNCTION, AND CLINICAL COLLABORATORS PROVIDE US WITH UNIQUE HUMAN SPECIMENS TO ENSURE THE TRANSLATABILITY OF OUR RESEARCH. OUR PRIOR FINDINGS OF A CRITICAL ROLE FOR THE IL6-STAT3-SOCS3 SIGNALING AXIS IN THE ENDOTHELIUM PROVIDES A STRONG SCIENTIFIC BASIS FOR THE PROPOSED WORKING MODEL, AND OUR NEW UNPUBLISHED BIOINFORMATICS ANALYSIS OF ENDOTHELIAL TRANSLATOME OF FAILING ORGANS SUGGEST SEVERAL NOVEL IL6 EFFECTORS OF ENDOTHELIOPATHY, PROVIDING INITIAL TARGETS FOR FURTHER RESEARCH. WE AIM AT DETERMINING WHICH CHANGES DICTATE THE SEVERITY OF ACUTE SHOCK (AND THUS SHORT-TERM SURVIVAL), AND WHICH LEAD TO LONG-TERM CONSEQUENCES WELL BEYOND THE RESOLUTION OF THE INITIAL SHOCK. KEY QUESTIONS DRIVING OUR RESEARCH ARE: 1) WHAT ARE THE EFFECTORS DOWNSTREAM OF A CYTOKINE STORM THAT WE CAN TARGET TO LIMIT ORGAN DYSFUNCTION WITHOUT LIMITING THE IMMUNE RESPONSE? 2) WHAT ARE THE MAIN DRIVERS OF LONG-TERM CONSEQUENCES AND CHRONIC INFLAMMATION AFTER SHOCK RECOVERY? 3) HOW CAN WE TAKE ADVANTAGE OF THE COMPLEXITY OF THE ENDOTHELIAL RESPONSE TO TAILOR IT TOWARDS A PRO-IMMUNE RESPONSE WHILE LIMITING THE COLLATERAL DAMAGE? THE OUTCOME OF OUR EFFORTS IN ANSWERING THESE CRITICAL QUESTIONS IS THE DISCOVERY OF KEY DETERMINANTS OF ORGAN FAILURE. THE KNOWLEDGE GAINED MAY LEAD TO INNOVATIVE NON-IMMUNOSUPPRESSIVE THERAPEUTIC STRATEGIES TO LIMIT ORGAN DYSFUNCTION.

MICROGLIAL IGF1 IN EPILEPTOGENESIS - EPILEPTOGENESIS IS A PATHOLOGICAL PROCESS THAT TRANSFORMS A NORMAL BRAIN INTO AN EPILEPTIC BRAIN, TYPICALLY INITIATED BY GENETIC MUTATIONS AND NEUROLOGICAL INSULTS SUCH AS STATUS EPILEPTICUS (SE). EXCESSIVE ACTIVATION OF MTOR SIGNALING IS RECOGNIZED AS A MECHANISM UNDERLYING BOTH GENETIC AND ACQUIRED FORMS OF EPILEPSY. MODELING HYPERACTIVATION OF MTOR SIGNALING IN NEURONS AND ASTROCYTES RECAPITULATES THE EPILEPTOGENIC ACTIVITY. HOWEVER, THE ROLE OF MICROGLIA IN EPILEPTOGENESIS IS UNDEREXPLORED. WHILE THE INFLAMMATORY RESPONSE OF MICROGLIA HAS LONG BEEN POSTULATED TO BE EPILEPTOGENIC, WE RECENTLY DEMONSTRATED THAT MICE WITH ELEVATED MTOR ACTIVITY IN MICROGLIA DEVELOP SEVERE SPONTANEOUS RECURRENT SEIZURES (SRS) WITHOUT SIGNIFICANT INDUCTION OF PRO-INFLAMMATORY CYTOKINES. THIS NOVEL FINDING INDICATES THERE MUST BE AN ALTERNATIVE EPILEPTOGENIC ROUTE INDEPENDENT OF THE INFLAMMATORY RESPONSE. MOREOVER, OUR PRELIMINARY DATA REVEALED THAT MTOR ACTIVATION INDUCES ROBUST EXPRESSION OF INSULIN-LIKE GROWTH FACTOR 1 (IGF1), WHEREAS INHIBITION OF MTOR SUPPRESSES EXPRESSION OF IGF1 IN MICROGLIA. WHILE IGF1 HAS BEEN KNOWN AS A POTENT ACTIVATOR OF MTOR, OUR DISCOVERY THAT MTOR ACTIVATION IN TURN UP-REGULATES THE EXPRESSION OF IGF WAS ENTIRELY UNEXPECTED. THESE DATA SUGGEST THAT THE MICROGLIAL MTOR/IGF1 AXIS PROPAGATES MTOR SIGNALING INTO OTHER CELLS IN THE CNS. OUR OVERARCHING HYPOTHESIS IS THAT ELEVATED MTOR SIGNALING IN MICROGLIA UP-REGULATES IGF1 EXPRESSION, WHICH IN TURN PROPAGATES MTOR SIGNALING TO THE SURROUNDING CELLS THROUGH A PARACRINE ROUTE INVOLVING IGF1, CONSEQUENTLY DISRUPTING BRAIN HOMEOSTASIS AND RESULTING IN SRS. THREE SPECIFIC AIMS ARE PROPOSED TO TEST OUR HYPOTHESIS. AIM 1 WILL CHARACTERIZE THE PROPAGATION OF MICROGLIAL IGF1 SIGNALING TO NON-MICROGLIAL CELLS. AIM 2 WILL EVALUATE THE ROLE OF MICROGLIAL IGF1 IN CNS HOMEOSTASIS. AIM 3 WILL DETERMINE IF IGF1 PLAYS A ROLE IN EPILEPTOGENESIS. OUR STUDY WILL NOT ONLY SIGNIFICANTLY ADVANCE OUR UNDERSTANDING OF HOW THE MTOR SIGNALING MEDIATES EPILEPTOGENESIS, BUT COULD ALSO LEAD TO NEW STRATEGIES TO PREVENT EPILEPTOGENESIS.

ROLE OF TUMOR BIOMECHANICS ON DRUG-RECEPTOR ENGAGEMENT - PROJECT SUMMARY / ABSTRACT MONOCLONAL ANTIBODY (MAB) THERAPIES TARGETING THE HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR 2 (HER2) HAVE TRANSFORMED THE TREATMENT OF HER2-POSITIVE BREAST CANCER. HOWEVER, MANY PATIENTS DEVELOP RESISTANCE OR FAIL TO ACHIEVE DURABLE REMISSION, UNDERSCORING THE LIMITATIONS OF RECEPTOR ABUNDANCE AS A PREDICTOR OF THERAPEUTIC RESPONSE. INCREASING EVIDENCE INDICATES THAT THE TUMOR MICROENVIRONMENT (TME), PARTICULARLY EXTRACELLULAR MATRIX (ECM) STIFFNESS, COLLAGEN DENSITY, AND VASCULAR DYSFUNCTION, CRITICALLY INFLUENCE DRUG DELIVERY, RECEPTOR ACCESSIBILITY, AND BINDING KINETICS. STIFF AND POORLY PERFUSED REGIONS CREATE PHYSICAL BARRIERS THAT HINDER THE DIFFUSION OF LARGE THERAPEUTIC ANTIBODIES SUCH AS TRASTUZUMAB (TZM), REDUCING EFFECTIVE DELIVERY AND HER2 ENGAGEMENT. THE GOAL OF THIS PROJECT IS TO INVESTIGATE THE MECHANISTIC BASIS OF ANTIBODY ACCESSIBILITY, RECEPTOR BINDING, AND THERAPEUTIC EFFICACY IN INTACT TUMOR TISSUES. ANIMAL TUMOR MODELS ARE REQUIRED BECAUSE ANTIBODY DELIVERY, RECEPTOR-DRUG ENGAGEMENT, VASCULAR PERFUSION, ECM STIFFNESS, COLLAGEN ORGANIZATION, AND CELLULAR DENSITY ARE COUPLED PROPERTIES OF THE INTACT TUMOR MICROENVIRONMENT THAT CANNOT BE ADEQUATELY REPRODUCED IN VITRO. XENOGRAFT AND PATIENT-DERIVED XENOGRAFT MODELS WERE SELECTED TO PROVIDE BIOLOGICALLY RELEVANT VARIATION IN RECEPTOR EXPRESSION, COLLAGEN CONTENT, CELLULAR DENSITY/DISTRIBUTION, AND VASCULAR ORGANIZATION. APPLYING THE MESOSCOPIC IMAGING PLATFORM TO THESE EXCISED TUMOR TISSUES WILL GENERATE HETEROGENEOUS, SPATIALLY RESOLVED DATASETS TO TEST HOW TUMOR BIOMECHANICS AND VASCULATURE REGULATE DRUG–TARGET ENGAGEMENT AND SUPPORT FUTURE TRANSLATION TO CLINICAL TUMOR SPECIMENS. OUR CENTRAL HYPOTHESIS IS THAT TUMOR BIOMECHANICAL AND VASCULAR HETEROGENEITY REGULATES ANTIBODY ACCESSIBILITY, HER2 BINDING, AND THERAPEUTIC EFFICACY, AND THAT PHARMACOLOGIC MODULATION OF ECM STIFFNESS WILL INFLUENCE ANTIBODY PENETRATION AND RESPONSE. THIS HYPOTHESIS IS SUPPORTED BY OUR PRELIMINARY FINDINGS SHOWING THAT COLLAGEN-RICH, POORLY VASCULARIZED TUMORS EXHIBIT MARKEDLY REDUCED TZM–HER2 ENGAGEMENT COMPARED WITH MORE COMPLIANT, WELL-PERFUSED TUMORS, EVEN WHEN HER2 EXPRESSION IS EQUIVALENT, DEMONSTRATING THAT TME BIOMECHANICS, NOT RECEPTOR LEVELS ALONE, DETERMINE EFFECTIVE DRUG BINDING. DESPITE THE CLEAR IMPACT OF TME, NO EXISTING IMAGING PLATFORM CAN SIMULTANEOUSLY QUANTIFY DRUG–TARGET BINDING, BIOMECHANICAL STIFFNESS, AND VASCULAR FEATURES IN INTACT, WHOLE TUMORS. TO ADDRESS THIS CRITICAL GAP, WE WILL DEVELOP AND APPLY MFLIO², A FIRST-IN-CLASS MULTIMODAL IMAGING SYSTEM THAT INTEGRATES MESOSCOPIC FLUORESCENCE LIFETIME IMAGING FÖRSTER RESONANCE ENERGY TRANSFER (MESOFLI-FRET), OPTICAL COHERENCE TOMOGRAPHY (OCT), AND OPTICAL COHERENCE ELASTOGRAPHY (OCE). THIS CO-REGISTERED PLATFORM WILL PROVIDE QUANTITATIVE MAPS OF TUMOR MOLECULAR INTERACTIONS, STRUCTURAL ORGANIZATION, AND BIOMECHANICAL PROPERTIES WITHIN THE SAME SPECIMEN. ARTIFICIAL-INTELLIGENCE-DRIVEN REGISTRATION AND RECONSTRUCTION PIPELINES WILL SPATIALLY ALIGN THESE DATASETS WITH HISTOLOGICAL GROUND TRUTH, ENABLING VOXEL-LEVEL CORRELATION BETWEEN ANTIBODY BINDING, ECM STIFFNESS, AND VASCULAR ARCHITECTURE. THIS INTEGRATED FRAMEWORK DIRECTLY LINKS MOLECULAR DRUG ENGAGEMENT TO THE PHYSICAL AND VASCULAR STATE OF THE TME AT CLINICALLY RELEVANT MESOSCALE RESOLUTIONS. AIM 1 WILL DEVELOP AND VALIDATE THE INTEGRATED MFLIO² PLATFORM. WE WILL IMPLEMENT STRUCTURED-LIGHT MESOSCOPIC FLI-FRET FOR QUANTITATIVE WHOLE-TUMOR MAPPING OF ANTIBODY–RECEPTOR INTERACTIONS, INCORPORATE OCT AND OCE MODULES FOR CONCURRENT ASSESSMENT OF VASCULAR AND MECHANICAL FEATURES, AND DEPLOY AI-BASED MULTIMODAL REGISTRATION TO ALIGN OPTICAL DATA WITH HISTOPATHOLOGY. AIM 2 WILL APPLY MFLIO² TO HER2. XENOGRAFT AND PATIENT-DERIVED XENOGRAFTS TUMOR MODELS TO ELUCIDATE HOW TME STIFFNESS AND ECM COMPOSITION OR VASCULAR ORGANIZATION MODULATE TZM–HER2 BINDING, DRUG RESPONSE AND TREATMENT THERAPY. PHARMACOLOGIC AGENTS THAT REMODEL ECM COMPOSITION

TARGETING LIPOLYTIC STEROL SIGNALING TO FOSTER FAVORABLE FOAM CELL MODULATION AND ATHEROPROTECTION. - PROJECT SUMMARY A LARGE PROPORTION OF LIPIDS IN ATHEROSCLEROTIC LESIONS ARE STORED IN CYTOPLASMIC LIPID DROPLETS (LDS) OF FOAMY MACROPHAGES. ALTHOUGH CERTAIN PROCESSES DRIVING FOAM CELL FORMATION CAN TRIGGER PRO-INFLAMMATORY SIGNALS, RECENT ANALYSES OF LESIONAL CELLS LINK FOAM CELLS TO ALTERNATIVELY ACTIVATED METABOLIC PHENOTYPES. THIS SUGGESTS PROTECTIVE MODULATORY MECHANISMS THAT, IF IDENTIFIED, COULD LEAD TO NOVEL ATHEROPROTECTIVE STRATEGIES. THE LIVER X RECEPTOR (LXR) PATHWAY IS A CENTRAL REGULATOR OF FOAM CELL BIOLOGY THAT FACILITATES ADAPTATION TO CHOLESTEROL OVERLOAD AND REPRESSES INFLAMMATION. THE NATURAL LXR LIGANDS ARE SIDE-CHAIN OXYSTEROLS AND CERTAIN CHOLESTEROL SYNTHESIS INTERMEDIATES. LIKE CHOLESTEROL, THESE STEROLS ARE ABUNDANTLY STORED AS ESTERS THAT ARE NOT READILY AVAILABLE TO ACCESS THE RECEPTORS. LIPID DROPLET-ASSOCIATED HYDROLASE (LDAH) IS A LIPASE/ESTERASE WITH HIGH AFFINITY FOR LDS. IN ATHEROMA LDAH IS ASSOCIATED WITH METABOLIC MACROPHAGE CLUSTERS POOR IN INFLAMMATORY MARKERS, AND GAIN- AND LOSS-OF-FUNCTION STUDIES IDENTIFIED LDAH AS AN ATHEROPROTECTIVE PLAYER THAT ALSO PROMOTES STABLE LESION ARCHITECTURES. VARIOUS LIPIDOMIC ANALYSES OF MACROPHAGES AND ATHEROMA SHOW THAT LDAH MOBILIZES STEROL STORES AND HAS A ROBUST IMPACT ON REGULATORY SPECIES, WHILE TRANSCRIPTOMIC PROFILES SHOW THAT LDAH INDUCES PROTOTYPICAL LXR TARGETS AND PROMOTES A REPARATIVE PHENOTYPE. OUR CENTRAL HYPOTHESIS IS THAT LDAH FACILITATES ADAPTIVE RESPONSES TO LIPID OVERLOAD THROUGH LIPOLYTIC RELEASE OF STEROIDAL LXR LIGANDS FROM LDS, WHILE CONNECTING LIPID METABOLISM WITH FAVORABLE MODULATION OF LESIONAL CELLS. IMPORTANTLY, LDAH’S INDUCTION OF LXR TARGETS IS NOT PARALLELED BY INDUCTION OF LIPOGENIC GENES, SUGGESTING THAT THIS MECHANISM OF ENDOGENOUS LXR ACTIVATION IS DEVOID OF LIPOGENIC SIDE EFFECTS THAT HINDER THE USE OF SYNTHETIC AGONISTS. OUR OVERARCHING GOAL IS TO ESTABLISH AN LDAH-LXR AXIS AS A DRIVER OF FAVORABLE MODULATION OF VASCULAR CELLS AND ATHEROPROTECTION AND IDENTIFY STRATEGIES TO ENHANCE THIS MECHANISM. AIM 1 WILL TEST THE HYPOTHESIS THAT LDAH PROTECTS THROUGH LXR- DEPENDENT MECHANISMS AND PLAYS A MAIN ROLE IN ENDOGENOUS LXR ACTIVATION. IN AIM 2 NEWLY GENERATED REPORTER AND LDAH-FLOXED MODELS WILL BE USED TO TEST THE HYPOTHESIS THAT SMOOTH MUSCLE CELLS ARE INDIRECTLY MODULATED BY LDAH-DRIVEN CHANGES IN MYELOID CELL ACTIVATION AND/OR DIRECTLY MODULATED BY LDAH’S ACTION ON THEIR OWN LDS, AND SIGNIFICANTLY CONTRIBUTE TO THE DEVELOPMENT OF FIBROPROTECTIVE ARCHITECTURES. AIM 3 WILL ASSESS WHETHER PROTECTIVE LXR ACTIVATION CAN BE ENHANCED THROUGH COORDINATED STEROL MOBILIZING INTERVENTIONS AND WILL LEVERAGE A STABLISHED DRUG SCREENING PLATFORM TO IDENTIFY COMPOUNDS THAT ACTIVATE THIS MECHANISM. THE INNOVATIVE ASPECT OF THIS PROJECT LIES IN LEVERAGING THE METABOLISM OF STEROLS THAT ACCUMULATE ABUNDANTLY IN ARTERIAL CELLS TO ENHANCE LXR ACTIVATION. IF SUCCESSFUL, THESE STUDIES MAY LEAD TO NOVEL TRANSLATIONAL OPPORTUNITIES.

MODULATING THE HIPPO PATHWAY IN CHARCOT-MARIE-TOOTH DISEASE - MODULATING THE HIPPO PATHWAY IN CHARCOT-MARIE-TOOTH DISEASE CHARCOT-MARIE-TOOTH DISEASE TYPE 1A (CMT1A) IS THE MOST COMMON INHERITED PERIPHERAL NEUROPATHY, AFFECTING MILLIONS WORLDWIDE AND PROFOUNDLY IMPAIRING QUALITY OF LIFE. CHARACTERIZED BY A TOXIC OVEREXPRESSION OF PMP22, CMT1A LEADS TO PROGRESSIVE DEMYELINATION AND NERVE DEGENERATION. WHILE ADVANCEMENTS IN GENE THERAPY HAVE SOUGHT TO MITIGATE THE DISEASE, CLINICAL OUTCOMES REMAIN MODEST, HIGHLIGHTING THE NEED FOR INNOVATIVE THERAPEUTIC STRATEGIES. THIS PROPOSAL INVESTIGATES THE MODULATION OF THE HIPPO PATHWAY AS A NOVEL APPROACH TO TREATING CMT1A. SPECIFICALLY, WE FOCUS ON TARGETING THE YAP/TAZ-TEAD1 TRANSCRIPTIONAL COMPLEX, WHICH REGULATES PMP22 EXPRESSION IN SCHWANN CELLS. USING ADVANCED GENETIC MODELS AND REPURPOSED TEAD INHIBITORS, WE AIM TO EVALUATE THE POTENTIAL OF REDUCING PMP22 LEVELS AND ALLEVIATING DISEASE PATHOLOGY. FIRST, WE WILL EXAMINE THE MOLECULAR AND CELLULAR EFFECTS OF YAP/TAZ/TEAD1 INHIBITION IN SCHWANN CELLS, LEVERAGING TEAD INHIBITORS TO REDUCE PMP22 EXPRESSION AND MITIGATE CMT1A-ASSOCIATED IMPAIRMENTS. SECOND, WE WILL EXPLORE THE THERAPEUTIC POTENTIAL OF TAZ ABLATION AND TEAD INHIBITORS IN VIVO, ASSESSING THEIR ABILITY TO PREVENT OR REVERSE MYELIN DEFECTS AND RESTORE NERVE FUNCTION IN A PRECLINICAL MOUSE MODEL. FINALLY, COMPREHENSIVE ANALYSES, INCLUDING SPATIAL TRANSCRIPTOMICS AND LONG-TERM SAFETY EVALUATIONS, WILL DEEPEN OUR UNDERSTANDING OF THE MECHANISMS UNDERPINNING THESE INTERVENTIONS. BY INTEGRATING PHARMACOLOGICAL AND GENETIC APPROACHES, THIS TOOL COMPOUND STUDY AIMS TO PROVIDE A TRANSFORMATIVE STRATEGY FOR CMT1A, REPURPOSING EXISTING DRUGS TO ACCELERATE THERAPEUTIC DEVELOPMENT. THE FINDINGS COULD HAVE BROAD IMPLICATIONS, OFFERING A FOUNDATION FOR ADDRESSING OTHER PERIPHERAL NEUROPATHIES AND EXPANDING THE THERAPEUTIC APPLICATIONS OF HIPPO PATHWAY MODULATION.

ARTIFICIAL INTELLIGENCE ENHANCED CANCER CELL CLASSIFICATION BASED ORGANELLE MORPHOLOGY AND TOPOLOGY - ABSTRACT BREAST CANCER IS A HIGHLY HETEROGENOUS DISEASE, BOTH PHENOTYPICALLY AND GENETICALLY. THE QUANTITY AND SUBCELLULAR LOCATION OF CANCER PROTEIN BIOMARKERS ARE USED TO CLASSIFY BREAST CANCER TYPES. TRANSCRIPTOMICS, MULTIPLEXED IMAGING, OR MASS CYTOMETRY HAVE BEEN USED TO CLASSIFY BREAST TUMOR CELL HETEROGENEITY WITH VARYING SUCCESS. ALTHOUGH GENOMICS AND PROTEOMICS HAVE BEEN SUCCESSFUL IN THE IDENTIFICATION OF TUMOR CELL POPULATIONS INVOLVED IN METASTATIC PROGRESSION, THE ABILITY TO DETERMINE WHETHER PATIENT TUMORS CONTAIN METASTATIC SUBPOPULATIONS IS STILL LACKING. RECENTLY, ORGANELLE MORPHOLOGY AND FUNCTION HAS BEEN USED AS A DIRECT READOUT OF THE FUNCTIONAL PHENOTYPIC STATE OF AN INDIVIDUAL CANCER CELL. WE PROPOSE TO USE THE SPATIAL CONTEXT OF ORGANELLES, SPECIFICALLY THEIR SUBCELLULAR LOCATION AND INTER-ORGANELLE RELATIONSHIPS (TOPOLOGY), TO CLASSIFY NOVEL AND DISTINCT METASTATIC CANCER CELL SUBPOPULATIONS. WE DEVELOPED AN ORGANELLE TOPOLOGY-BASED CELL CLASSIFICATION PIPELINE (OTCCP) TO QUANTIFY, FOR THE FIRST TIME, THE TOPOLOGICAL FEATURES OF SUBCELLULAR ORGANELLES, DEFINED AS THE DISTANCE BETWEEN EACH ORGANELLE OBJECT AND ALL ITS NEIGHBORS WITHIN A CELL. UNDER RFA-CA-21-013 (DEVELOPMENT OF INNOVATIVE INFORMATICS METHODS AND ALGORITHMS FOR CANCER RESEARCH AND MANAGEMENT), WE WILL ADAPT OR DEVELOP MACHINE LEARNING AND DEEP LEARNING METHODOLOGIES TO ACCELERATE AND AUTOMATE OTCCP-BASED ORGANELLE- BASED TOPOLOGY CANCER CELL CLASSIFICATION TO IDENTIFY SUBPOPULATIONS OF METASTATIC CELLS WITHIN HETEROGENEOUS PRIMARY TUMORS WITH POTENTIAL DIAGNOSTIC AND PROGNOSTIC VALUE. THIS APPROACH WILL ALSO HAVE MAJOR IMPACT AS A DISCOVERY TOOL TO ADVANCE OUR UNDERSTANDING OF CANCER CELL BIOLOGY ON A SUBCELLULAR LEVEL.

DEVELOPING NEW MOUSE MODELS OF ANDROPAUSE FOR ADRD RESEARCH - PROJECT SUMMARY/ABSTRACT AGING AND AD ARE ASSOCIATED WITH A DECLINE IN SEX HORMONES. APPROXIMATELY 40% OF MEN OVER 45 YEARS OLD SUFFER FROM LOW ANDROGEN LEVELS. ENDOCRINE AGING IN MEN, TERMED ANDROPAUSE, IS CHARACTERIZED BY AGE-RELATED DECLINES IN ANDROGENS (SUCH AS TESTOSTERONE). THESE DECLINES IN ANDROGENS ARE ASSOCIATED WITH INCREASED AD RISK AND COGNITIVE DECLINE. DESPITE ADVANCES IN MENOPAUSE MODELS, MODELS OF ENDOCRINE AGING IN MALES (ANDROPAUSE) HAVE BEEN STALLED FOR OVER A CENTURY AND ARE LIMITED TO GONADECTOMY. GONADECTOMY CAUSES ALMOST A COMPLETE LOSS OF ANDROGENS, AND THUS DOES NOT ACCURATELY MIMIC REDUCED ANDROGEN LEVELS OBSERVED IN ANDROPAUSE IN MEN. LACK OF MORE ACCURATE MODELS OF ANDROPAUSE THAT MORE CLOSELY MIMIC THE HUMAN HORMONAL PROFILE IS A CRITICAL TECHNICAL GAP THAT IS SEVERELY LIMITING THE FIELDS OF AGING AND DEMENTIA. OUR LONG-TERM GOAL IS TO DEVELOP MOUSE MODELS OF DEMENTIA THAT MORE ACCURATELY REFLECT THE HORMONAL ENVIRONMENT IN HUMANS AND THUS INCREASE THE TRANSLATIONAL POTENTIAL OF THERAPIES THAT ARE TESTED IN THESE MODELS. THE OBJECTIVE OF THIS PROPOSAL IS TO DEVELOP TWO NEW MOUSE MODELS OF ANDROPAUSE. FIRST, WE WILL DETERMINE THE TIME COURSE OF NATURAL ENDOCRINE AGING IN WT MICE AND SEVERAL MOUSE MODELS OF ADRD (AIM 1). NEXT, WE WILL MODEL ENDOCRINE AGING IN MALES VIA GONADECTOMY AND HORMONE REPLACEMENT WITH GRADUAL DECREASING OF ANDROGEN REPLACEMENT OVER TIME (AIM 2). FOR A NON-SURGICAL MODEL OF ANDROPAUSE, WE WILL DETERMINE IF A REPRODUCTIVE TOXIN CAN BE USED TO INDUCE GRADUAL ANDROGEN LOSS IN MALE MICE (AIM 3). FOR EACH MODEL, WE WILL ASSESS AGING PHENOTYPE (DECREASED STRENGTH, REDUCED ACTIVITY, INCREASED ANXIETY, AND IMPAIRED MEMORY) AND AD NEUROPATHOLOGY. WE PREDICT THAT MICE WITH LOW ANDROGEN LEVELS WILL EXHIBIT A MORE ACCELERATED AGING PHENOTYPE AND INCREASED AD NEUROPATHOLOGY. THIS WOULD REPRESENT THE FIRST EVER MODELS OF ANDROPAUSE WITH CAREFULLY CONTROLLED, GRADUAL HORMONE LOSS. WITHOUT THESE NEW, IMPROVED MODELS OF ANDROPAUSE, THE FIELD WILL CONTINUE TO TEST THERAPEUTICS FOR AD IN MALE MICE WITH AN ENDOCRINE STATUS DOES NOT MATCH THAT OF AGING MEN. THIS HINDERS THE TRANSLATIONAL POTENTIAL OF THESE THERAPIES. OUR NEW MODELS HAVE THE POTENTIAL TO TRANSFORM THE AGING FIELD.

ROLE OF WNT/Β-CATENIN PATHWAY IN ALVEOLAR EPITHELIAL REPAIR DURING TUBERCULOSIS AND ITS REGULATION BY CHRONIC TYPE I INTERFERON SIGNALING - PROJECT SUMMARY TUBERCULOSIS (TB) REMAINS A LEADING CAUSE OF MORTALITY WORLDWIDE, WITH LUNG DAMAGE BEING A KEY DRIVER OF DISEASE SEVERITY AND POOR OUTCOMES. ALVEOLAR EPITHELIAL CELLS ARE CRITICAL FOR MAINTAINING LUNG HOMEOSTASIS AND PROMOTING REPAIR, PROCESSES THAT ARE TIGHTLY REGULATED BY WNT/Β-CATENIN SIGNALING. OUR PRELIMINARY DATA INDICATE THAT MYCOBACTERIUM TUBERCULOSIS (MTB)-INDUCED INFLAMMATION DISRUPTS ALVEOLAR EPITHELIAL INTEGRITY IN TB-SUSCEPTIBLE MICE, LEADING TO IMPAIRED SURFACTANT PRODUCTION AND DEFECTIVE LUNG REGENERATION. STRIKINGLY, THIS IS ASSOCIATED WITH A SIGNIFICANT REDUCTION IN Β-CATENIN LEVELS. NOTABLY, INHIBITION OF TYPE I INTERFERON (IFN-I) SIGNALING RESTORES Β-CATENIN EXPRESSION, SUGGESTING A PREVIOUSLY UNRECOGNIZED ROLE OF IFN-I IN SUPPRESSING WNT/Β-CATENIN ACTIVITY AND ALVEOLAR REPAIR. WE AIM TO INVESTIGATE HOW CHRONIC IFN-I SIGNALING IMPAIRS WNT/Β-CATENIN FUNCTION, LEADING TO DEFECTIVE EPITHELIAL REPAIR AND EXACERBATED LUNG PATHOLOGY IN TB. SPECIFICALLY, IN AIM1, WE WILL DEFINE THE ROLE OF WNT/Β-CATENIN IN ALVEOLAR EPITHELIAL REPAIR FOLLOWING MTB INFECTION. WE WILL ASSESS HOW WNT/Β-CATENIN ACTIVATION OR INHIBITION INFLUENCES ALVEOLAR TYPE 2 (AT2) CELL PROLIFERATION, DIFFERENTIATION, AND STEMNESS USING MURINE AND HUMAN PRIMARY ALVEOLAR CELLS. ADDITIONALLY, WE WILL EVALUATE LUNG HISTOPATHOLOGY, EPITHELIAL MARKER EXPRESSION, AND AT2 CELL DIFFERENTIATION IN TB-RESISTANT AND SUSCEPTIBLE MICE. IN AIM2, WE WILL DETERMINE HOW IFN-I SIGNALING SUPPRESSES WNT/Β-CATENIN ACTIVITY DURING TB-INDUCED LUNG DAMAGE. USING GENETIC AND PHARMACOLOGICAL APPROACHES, WE WILL INVESTIGATE THE MOLECULAR MECHANISMS BY WHICH IFN-I SIGNALING MODULATES WNT/Β-CATENIN FUNCTION AND IDENTIFY KEY MEDIATORS OF IFN-I–WNT/Β-CATENIN CROSSTALK AS POTENTIAL THERAPEUTIC TARGETS. FINALLY, IN THE AIM3 WE WILL EVALUATE THE THERAPEUTIC POTENTIAL OF TARGETING WNT/Β-CATENIN AND IFN-I PATHWAYS TO ENHANCE ALVEOLAR REPAIR. WE WILL TEST WNT/Β-CATENIN ACTIVATORS, SUCH AS GSK3Β AND PORCUPINE INHIBITORS, AS WELL AS IFN-I BLOCKADE USING ANTI-IFNAR ANTIBODIES IN MURINE TB MODELS. THERAPEUTIC EFFICACY WILL BE ASSESSED THROUGH HISTOPATHOLOGICAL ANALYSIS, EPITHELIAL BARRIER INTEGRITY, INFLAMMATORY RESPONSES, AND BACTERIAL BURDEN. THIS STUDY WILL PROVIDE NOVEL INSIGHTS INTO THE INTERPLAY BETWEEN IFN-I SIGNALING AND WNT/Β-CATENIN IN TB PATHOGENESIS, UNCOVERING MECHANISMS THAT IMPAIR ALVEOLAR REPAIR. BY IDENTIFYING HOST-DIRECTED THERAPEUTIC STRATEGIES, WE AIM TO ENHANCE LUNG RECOVERY AND IMPROVE OUTCOMES FOR TB PATIENTS.

ROLE OF LRRC8 ANION CHANNELS IN BRAIN TAURINE SIGNALING AND BODY FLUID HOMEOSTASIS - SUMMARY: VOLUME-REGULATED ANION CHANNELS (VRACS) ARE UBIQUITOUS CHLORIDE/ANION CHANNELS FORMED BY FIVE PROTEINS FROM THE LEUCINE-RICH REPEAT-CONTAINING FAMILY 8 (LRRC8A-E), AND FOUND IN ESSENTIALLY ALL TYPES OF VERTEBRATE CELLS. THESE CHANNELS ARE ACTIVATED BY CELL SWELLING AND ENABLE CELL VOLUME CONTROL THROUGH THE RELEASE OF CYTOSOLIC CHLORIDE AND A VARIETY OF SMALL ORGANIC MOLECULES (OSMOLYTES). THE BRAIN HAS BEEN VIEWED AS THE PLACE WITH THE HIGHEST PHYSIOLOGICAL AND PATHOLOGICAL RELEVANCE FOR VRACS. EVEN MINOR CHANGES IN NEURAL CELL SIZE HAVE A PROFOUND IMPACT ON THE GEOMETRY OF EXTRACELLULAR SPACE, IONIC BALANCES, AND NEUROTRANSMITTER SIGNALING, THUS EMPHASIZING THE IMPORTANCE OF VRACS AND CELL VOLUME CONTROL. HOWEVER, SUCH CONTROL COMES AT A STEEP PRICE: OPENING OF VRACS RELEASES THE EXCITATORY NEUROTRANSMITTERS GLUTAMATE AND ASPARTATE, WHICH DRIVE EXCITOTOXICITY IN STROKE, TRAUMATIC BRAIN INJURY, AND EPILEPSY. FOR THIS REASON, INHIBITION OF VRAC-INDUCED EXCITA- BILITY IS PURSUED AS A NEUROPROTECTIVE STRATEGY. YET, SUCH THINKING MAY BE OVERLY SIMPLISTIC BECAUSE IT DOES NOT CONSIDER VRACS’ CONTRIBUTIONS TO THE RELEASE OF INHIBITORY MOLECULES. MICE WITH CONDITIONAL BRAIN DELETION OF THE ESSENTIAL VRAC SUBUNIT LRRC8A (NESTIN-CRE DRIVEN BLRRC8A KO) DIE FROM SPONTANEOUS SEIZURES DURING ADOLESCENCE AND EARLY ADULTHOOD. THE MOLECULAR MECHANISMS RESPONSIBLE FOR THIS LETHAL PHENOTYPE REMAIN AN ENIGMA. THE CURRENT PROJECT WILL ADDRESS THE CRITICAL, BUT PREVIOUSLY UNKNOWN, ROLE OF VRACS IN CONTROL OF BRAIN EXCITABILITY. OUR OVERARCHING HYPOTHESIS IS THAT LRRC8/VRAC CHANNEL FUNCTION IN GLIAL AND NEURONAL CELLS IS RESPONSIBLE FOR TONIC AND STIMULATED RELEASE OF INHIBITORY NEUROTRANSMITTERS AND NEUROMODULATORS. SUCH RELEASE OF INHIBITORY MOLECULES PROVIDES CONTROL OF BRAIN EXCITATION, AND ITS ABROGATION MAY BE RESPONSIBLE FOR LETHAL SEIZURES IN BLRRC8A KO MICE. THE CURRENT EXPLORATORY PROJECT WILL ADDRESS OUR MAIN HYPOTHESIS USING TWO WORKING MODELS. WORK IN SPECIFIC AIM 1 WILL CONCLUSIVELY PROVE THE CONTRIBUTION OF LRRC8A-CONTAINING VRACS TO INHIBITORY CONTROL THROUGH THEIR REGULATION OF EXTRACELLULAR TAURINE AND GABA LEVELS IN MAMMALIAN BRAIN. THIS WILL BE DONE USING A MICRODIALYSIS APPROACH IN THE ALREADY VALIDATED BLRRC8A KO MICE. STUDIES IN AIM 2 WILL ASCERTAIN THE FUNCTIONAL ROLE FOR ASTROCYTIC VRAC-MEDIATED TAURINE RELEASE IN HYPOTHALAMIC REGULATION OF BODY FLUID-ELECTROLYTE HOMEOSTASIS. THIS WILL BE DONE BY MEASURING CHANGES IN BLOOD OSMOLARITY AND ACTIVITY/C-FOS IMMUNOREACTIVITY OF HYPOTHALAMIC MAGNOCELLULAR NEURONS IN MICE CARRYING ASTROCYTIC DELETION OF LRRC8A (ALDH1L1-CREERT2-DRIVEN ALRRC8A KO). OVERALL, THIS EXPLORATORY STUDY WILL ADDRESS A SUBSTANTIAL GAP IN KNOWLEDGE: THE ROLE OF BRAIN LRRC8/VRAC CHANNELS IN THE NEGATIVE CONTROL OF NEURONAL EXCITABILITY. THIS NEW INFORMATION IS VERY IMPORTANT FOR THE RATIONAL DESIGN OF NEUROPROTECTIVE AGENTS THAT TARGET VRAC ACTIVITY IN STROKE AND OTHER BRAIN PATHOLOGIES.

A NOVEL C-DI-AMP-BASED RECOMBINANT BCG VACCINE - SUMMARY TUBERCULOSIS (TB) REMAINS A GLOBAL EPIDEMIC, WITH ONE-FOURTH OF THE CURRENT WORLD POPULATION INFECTED AND APPROXIMATELY 10 MILLION NEW ACTIVE CASES ANNUALLY. HOWEVER, OUR KNOWLEDGE ABOUT THE CAUSATIVE AGENT, MYCOBACTERIUM TUBERCULOSIS (MTB), IS STILL LIMITED, AND THE ONLY LICENSED TB VACCINE (BCG) IS INADEQUATE TO CONTROL THE EPIDEMIC. IT IS CRITICAL TO BETTER UNDERSTAND THE BIOLOGICAL DIFFERENCE BETWEEN MTB AND BCG IN ORDER TO DEVELOP A MORE EFFECTIVE TB VACCINE. OUR LONG-TERM GOAL OF THIS PROJECT IS TO DEVELOP A NOVEL RECOMBINANT BCG VACCINE TO BETTER CONTROL TB. RECENTLY, CYCLIC DI-AMP (C-DI-AMP) HAS BEEN RECOGNIZED AS A NEW BACTERIAL SIGNALING MOLECULE AND A POTENT VACCINE ADJUVANT. WE HAVE DEMONSTRATED THAT MTB RV3586 (DISA) ENCODES A DIADENYLATE CYCLASE AND RV2837C (CNPB) ENCODES A C-DI-AMP PHOSPHODIESTERASE. COMPARED TO THE MTB WILD-TYPE (WT), CNPB SECRETES A SIGNIFICANTLY LARGER AMOUNT OF C-DI-AMP AND STIMULATES A STRONGER TYPE I INTERFERON (IFN-I) RESPONSE IN THE INFECTED MACROPHAGES. WE HAVE ALSO REVEALED THAT BCG CNPB PRODUCES BUT DOES NOT SECRETE C-DI-AMP; BOTH C-DI-AMP SECRETION AND MTB REGION OF DIFFERENCE 1 (RD1) ARE REQUIRED FOR THE C-DI-AMP-INDUCED IFN-I RESPONSE. INTERESTINGLY, IT IS WELL KNOWN THAT BCG IS DEFECTIVE IN INDUCING IFN-I, AND ADDITION OF IFN-I ENHANCES BCG'S IMMUNOGENICITY. MOREOVER, C-DI-AMP HAS BEEN UTILIZED AS A POTENTIAL VACCINE ADJUVANT THAT ELICITS STRONG HUMORAL AND CELLULAR IMMUNE RESPONSE. OVERPRODUCTION OF C- DI-AMP IN BCG BY EXPRESSING DISA ALSO RESULTS IN BETTER PROTECTION IN INFECTED ANIMALS. HOWEVER, THE IMPROVEMENT IS MODERATE LIKELY BECAUSE THAT THE RECOMBINANT BCG IS STILL UNABLE TO SECRETE C-DI-AMP AND INDUCE SUBSTANTIAL IFN-I. THEREFORE, WE HYPOTHESIZE THAT MANIPULATION OF C-DI-AMP HOMEOSTASIS AND SECRETION IN BCG WILL ENABLE BCG TO PROVIDE A BETTER PROTECTION AGAINST TB. THE OBJECTIVE OF THIS APPLICATION IS TO CONSTRUCT A RECOMBINANT BCG THAT SECRETES C-DI-AMP AND INDUCES OPTIMAL LEVELS OF IFN-I RESPONSE DURING VACCINATION. WE PROPOSE TWO SPECIFIC AIMS: (1) TO CONSTRUCT C-DI-AMP-SECRETING RECOMBINANT BCG STRAINS, AND (2) TO EVALUATE THE RECOMBINANT BCG STRAINS IN INDUCTION OF IFN-I AND PROTECTION AGAINST MTB INFECTION. THESE RECOMBINANT BCG STRAINS WILL BE VERY LIKELY SUPERIOR TO THE CURRENT BCG IN VACCINE EFFICACY. FURTHERMORE, OUR FINDINGS WILL ALSO PROVIDE FUNDAMENTAL INSIGHTS INTO VACCINE STRATEGIES FOR OTHER BACTERIAL PATHOGENS. THUS, THIS PROPOSAL HAS A BROAD IMPACT ON PUBLIC HEALTH.

ROLE OF PKC EPSILON IN ATHEROPROTECTION - PROJECT SUMMARY CARDIOVASCULAR DISEASE AFFLICTS APPROXIMATELY 50% OF OLDER AMERICANS AND IS THE MAJOR CAUSE OF DEATH WORLDWIDE. IT IS A CHRONIC INFLAMMATORY DISEASE IN WHICH MACROPHAGES (MØ) PLAY A CENTRAL ROLE; THEIR DELETION REDUCES PLAQUE SIZE AND COMPOSITION. USUALLY, INFLAMMATION IS SELF-LIMITING, WITH RESOLUTION FOLLOWING INITIATION. IN ATHEROSCLEROSIS THIS YIN AND YANG IS OUT OF BALANCE, WITH MØ FAILING TO SHIFT TOWARDS RESOLUTION. WHILE MANY OF THE PLAYERS IN THE INFLAMMATION (CYTOKINES, CHEMOKINES) AND RESOLUTION (SPECIALIZED PRO-RESOLVING LIPIDS INCLUDING RVD1) PHASES HAVE BEEN IDENTIFIED, THE INTRACELLULAR SIGNALING NETWORK THAT LINKS UPTAKE OF OXLDL TO RESOLUTION, AND THE ROADBLOCKS TO THAT RESOLUTION IN ATHEROSCLEROSIS, REMAIN TO BE DEFINED. PRELIMINARY DATA IMPLICATE PKCE IN BOTH INFLAMMATION AND RESOLUTION BY RESTRICTING EXCESSIVE INFLAMMATION WHILE PROMOTING RELEASE OF SPECIALIZED PRO-RESOLVING LIPID MEDIATORS (SPM). HOWEVER, HOW PKCE ACHIEVES THIS IS A FUNDAMENTAL UNANSWERED QUESTION. WE GENERATED A PKCELOXP/LOXP MOUSE THAT WAS CROSSED WITH LYSM CRE TO GENERATE A NOVEL STRAIN IN WHICH PKCE IS SELECTIVELY DELETED IN THE MYELOID LINEAGE (MEKO). WHEN MADE HYPERCHOLESTEROLEMIC, MEKO MICE HAVE SIGNIFICANTLY MORE PLAQUE THAN WT ANIMALS. THIS RESULT IMPLICATES MYELOID PKCE AS A NOVEL ATHEROPROTECTIVE GENE. USING OUR MEKO MICE WE WILL TEST THE HYPOTHESIS THAT MYELOID PKCE CONTRIBUTES TO INFLAMMATION BUT ALSO SIGNALS IN THE PRODUCTION OF SPM, THUS PROMOTING RESOLUTION; ITS ABSENCE SUSTAINS INFLAMMATION AND REDUCES RESOLUTION THEREBY EXACERBATING ATHEROSCLEROSIS. THE HYPOTHESIS WILL BE TESTED IN VITRO AND IN VIVO. IN VIVO EXPERIMENTS INCLUDE ZYMOSAN PERITONITIS, A SELF-RESOLVING INFLAMMATION THAT WILL ENABLE US TO DETERMINE WHERE PKCE ACTS ALONG THE INFLAMMATION RESOLUTION AXIS. CELLS AND PERITONEAL LAVAGE FLUID WILL BE RECOVERED OVER TIME (4-72 H). CELL METRICS INCLUDE NUMBER AND RATIO OF NEUTROPHILS TO MACROPHAGES (MØ) AND THE POLARIZATION STATE OF THE MØ. LAVAGE FLUID WILL BE ASSAYED FOR CYTOKINES, CHEMOKINES, AND SPMS. SECONDLY, ARCHIVED AORTIC ROOTS FROM HYPERCHOLESTEROLEMIC WT AND MEKO MICE WILL BE ANALYZED FOR HISTOLOGICAL DIFFERENCES (EG, LOCALIZATION AND POLARIZATION STATE OF MØ, AMOUNT AND LOCATION OF COLLAGEN, AMOUNT OF NECROSIS AND APOPTOSIS.) FINALLY, IN VITRO STUDIES WILL DEFINE THE DIFFERENCES IN THE RESPONSES OF WT AND MEKO MØ TO OXIDIZED LDL AND DURING EFFEROCYTOSIS. UNDERSTANDING HOW PKCE ACTS IN INFLAMMATION AND RESOLUTION MAY REVEAL NOVEL NODES OF REGULATION THAT CAN BE THERAPEUTICALLY TARGETED TO PROMOTE RESOLUTION IN THE CONTEXT OF HYPERLIPIDEMIA. IN CONCLUSION, OUR FINDINGS WILL PROVIDE THE FOUNDATION UPON WHICH TO BUILD A MORE MECHANISTIC GRANT TO UNDERSTAND HOW PKCE SIGNALS DURING THE SHIFT FROM INFLAMMATION TO RESOLUTION. TRANSLATIONALLY, THE RESULTS MAY BE APPLICABLE TO THOSE DISEASES IN WHICH PKCE IS DYSREGULATED AND MØ PLAY A PROMINENT ROLE (EG, CANCER, SCLERODERMA, ALZHEIMER’S, ATHEROSCLEROSIS, ETC.).

IGF::CL::IGF THORACIC SURGEON SERVICES

POP2 AS A NOVEL THERAPEUTIC IN RHEUMATOID ARTHRITIS - PROJECT SUMMARY: INFLAMMATION UNDERLIES THE DISABLING MANIFESTATIONS AND CO-MORBIDITIES OF RHEUMATOID ARTHRITIS (RA). TREATMENT REGIMENS FOR RA PATIENTS AIM TO CONTROL INFLAMMATION AND PROGRESSIVE JOINT DAMAGE MEDIATED BY INFLAMMATORY CYTOKINES INCLUDING TNFA, IL-1SS, AND IL-6. RECENT STUDIES DEMONSTRATE THAT HUMANS POSSESS FOUR GENES CODING PYRIN-ONLY PROTEINS (POPS) THAT LIMIT NF-B SIGNALING AND INFLAMMASOME ACTIVATION PATHWAYS, EVENTS CRITICAL FOR ELABORATION OF THE CYTOKINES MEDIATING INFLAMMATION. TNF, IL-1SS, AND IL-6 ARE CRITICALLY REGULATED BY NF-B TRANSCRIPTION FACTORS. WE HAVE PREVIOUSLY DESCRIBED THE ANTI-INFLAMMATORY PROPERTIES OF POP2 IN-VITRO AND IN-VIVO. HERE WE PROPOSE PROOF-OF-CONCEPT STUDIES TO EVALUATE HOW POP2 AND POP2-DERIVED PEPTIDES IMPACT EXPERIMENTALLY INDUCED RA WHEN ADMINISTERED EXOGENOUSLY, USING A MURINE PRECLINICAL MODEL. POP2 PEPTIDE THERAPY REPRESENTS A NOVEL THERAPEUTIC APPROACH TO AMELIORATING EXCESSIVE INFLAMMATION THROUGH DUAL INHIBITION OF BOTH NF-B SIGNALING AND INFLAMMASOME PATHWAYS. THE IMMUNOLOGICAL AND MOLECULAR BASIS FOR OUR HYPOTHESES STEMS FROM OUR PUBLISHED AND PRELIMINARY DATA SHOWING THE NATURALLY EXPRESSED, NON-TOXIC POP2 PEPTIDE DAMPENS CYTOKINE PRODUCTION IN INFLAMMATORY RESPONSES WITHOUT COMPROMISING HOST IMMUNITY. OUR STUDIES WILL PROVIDE INSIGHT INTO HOW TREATMENT REGIMENS BASED ON POP2 PEPTIDE THERAPY MAY BE UTILIZED OR IMPROVED UPON. IN VITRO CHARACTERIZATION, DOSE-ESCALATION STUDIES AND IN-VIVO ASSESSMENT OF DISEASE PARAMETERS WILL INFORM APPROACHES TO DEVELOP POP2 PEPTIDES AS A NOVEL THERAPEUTIC.

ROLES OF EPIDERMAL INTEGRIN Α3Β1 IN AGE-DEPENDENT CHANGES DURING CUTANEOUS WOUND HEALING - PROJECT SUMMARY EVEN WITHOUT COMORBIDITIES ASSOCIATED WITH AGING, WOUND HEALING IN HEALTHY ELDERLY PEOPLE (>65) IS DELAYED, POSING A HIGH RISK OF INFECTION. HOWEVER, MECHANISMS FOR THE AGE-RELATED DECLINE IN WOUND HEALING ARE LARGELY UNKNOWN. INTEGRIN ADHESION RECEPTORS ON EPIDERMAL KERATINOCYTES BIND TO EXTRACELLULAR MATRIX (ECM) AND HAVE CRITICAL ROLES IN CONTROLLING THE WOUND MICROENVIRONMENT AND CRITICAL PARAMETERS OF WOUND HEALING. AGE-DEPENDENT CHANGES IN ADHESION RECEPTORS MAY CONTRIBUTE TO REDUCED WOUND HEALING IN AGED SKIN, BUT THESE HAVE NOT BEEN EXPLORED. THIS IS AN IMPORTANT KNOWLEDGE GAP, AS INTEGRINS ARE CELL SURFACE RECEPTORS THAT CAN BE READILY TARGETABLE THERAPEUTICALLY. INTEGRIN Α3Β1 IS AN EXCELLENT CANDIDATE FOR MEDIATING EPIDERMAL WOUND FUNCTIONS THAT DIMINISH WITH AGE. INDEED, OUR PRELIMINARY DATA SHOW THAT Α3Β1 EXPRESSION DECREASES IN AGING MURINE SKIN. FURTHERMORE, OUR NEW GENETIC MODEL OF INDUCIBLE, EPIDERMIS-SPECIFIC Α3KO (IΑ3EKO) HAS BEGUN TO ELUCIDATE Α3Β1-DEPENDENT WOUND FUNCTIONS IN ADULT SKIN, FREE FROM COMPENSATION AND/OR BASEMENT MEMBRANE DEFECTS CAUSED BY EMBRYONIC AND SUSTAINED DELETION OF EPIDERMAL Α3Β1 IN THE WIDELY USED CONSTITUTIVE Α3KO MICE. OUR PRELIMINARY DATA INDICATE THAT ACUTE LOSS OF Α3Β1 IN EPIDERMIS OF YOUNG ADULT MICE (1) SLOWS WOUND REEPITHELIALIZATION AND (2) IMPAIRS EPIDERMAL FACTORS THAT MEDIATE IMMUNE CELL INFILTRATION. INDEED, REEPITHELIALIZATION AND IMMUNE CELL INFILTRATION ARE COMPROMISED IN AGING SKIN OF HUMANS AND RODENTS, AND CONTRIBUTE TO IMPAIRED WOUND HEALING IN THE ELDERLY. THE PROPOSED WORK WILL USE AN AGING MODEL IN COMBINATION WITH OUR IΑ3EKO MODEL TO TEST OUR HYPOTHESIS THAT Α3Β1 IN EPIDERMIS PROMOTES WOUND HEALING BY REGULATING TWO DISTINCT PROCESSES: (1) EPIDERMAL MIGRATION OVER THE PROVISIONAL WOUND ECM, AND (2) INDUCTION OF EPIDERMAL FACTORS THAT MEDIATE IMMUNE CELL INFILTRATION, AND THAT THESE Α3Β1-DEPENDENT FUNCTIONS ARE DIMINISHED DURING INTRINSIC (CHRONOLOGICAL) AGING, IMPEDING THE ABILITY TO HEAL WOUNDS. WE PROPOSE TWO SPECIFIC AIMS TO TEST OUR HYPOTHESIS AND ANSWER THE QUESTION: DOES WANING EPIDERMAL INTEGRIN Α3Β1 FUNCTION IN AGING SKIN CONTRIBUTE TO WOUND HEALING DEFICIENCIES IN THE ELDERLY? WE WILL DETERMINE, IN VIVO AND IN VITRO, THE ABILITY OF YOUNG, MIDDLE, AND AGED KERATINOCYTES WHICH EXPRESS OR LACK Α3Β1 TO (AIM 1) PROLIFERATE AND MIGRATE DURING RE-EPITHELIALIZATION, AND TO (AIM 2) SECRETE SOLUBLE IMMUNE CELL-STIMULATING FACTORS THAT PROMOTE IMMUNE CELL RECRUITMENT DURING WOUND HEALING. AIM 2 IS A PARTICULARLY INNOVATIVE ASPECT, AS IT EXTENDS OUR STUDIES BEYOND TRADITIONAL ROLES FOR INTEGRINS IN ADHESION/MOTILITY TO FURTHER EXPLORE KERATINOCYTE-DRIVEN PARACRINE CROSSTALK. THIS WORK WILL ENHANCE UNDERSTANDING OF HOW AGE-RELATED CHANGES IN THE FUNCTIONS OF EPIDERMAL INTEGRINS CONTRIBUTE TO REDUCED WOUND HEALING.

CAMPUS BASED-FWS

IGF::OT::IGF TUITION AND FEES

IGF::OT::IGF TUITION AND FEES

IGF::CL::IGF ENT SERVICES

IGF::OT::IGF TUITION AND FEES

THORACIS SURGERY SERVICES FOR BENEFICIARIES OF THE VA MEDICAL CENTER ALBANY, NEW YORK.

DECIPHERING THE ROLE OF HUMAN-FC RI IN CROSS-PRESENTATION FOR OPTIMAL CD8 T-CELL RESPONSE IN THE RESPIRATORY MUCOSA - PROJECT SUMMARY/ABSTRACT AS CD8⁺ T-CELL RESPONSES ARE CRITICAL TO IMMUNITY AGAINST CANCER AND CYTOSOLIC PATHOGENS, INNOVATIVE STRATEGIES TO ENHANCE CROSS-PRESENTATION (XP) AND IMPROVE CD8⁺ T-CELL RESPONSES HOLD GREAT POTENTIAL FOR ADVANCING IMMUNOTHERAPIES AND VACCINES FOR THESE DISEASES OF MAJOR PUBLIC HEALTH CONCERN. NEVERTHELESS, CURRENT VACCINE TECHNOLOGIES PREDOMINANTLY FOCUS ON INDUCING ANTIBODY (AB) RESPONSES RATHER THAN BOOSTING T- CELL-MEDIATED IMMUNITY. WE HAVE DEMONSTRATED THAT TARGETING PROTEIN ANTIGENS (AGS) TO HUMAN-FCΓRI (HFCΓRI) CAN EVOKE CD8⁺ T-CELL RESPONSES. HOWEVER, A CRITICAL GAP REMAINS IN FULLY UNDERSTANDING THE PRECISE ROLE OF HFCΓRI IN PROMOTING XP AND CD8⁺ T-CELL RESPONSES. IN THIS STUDY, WE AIM TO INVESTIGATE THE INVOLVEMENT OF HFCΓRI IN XP AND ITS IMPACT ON THE ENSUING CD8⁺ T-CELL RESPONSES. PREVIOUSLY, WE DEMONSTRATED THAT PROTEIN AGS FUSED TO Α-HFCΓRI ELICIT CD8 T-CELL RESPONSES. THIS PROMPTED OUR HYPOTHESIS THAT TARGETING AGS TO HFCΓRI WILL PROMOTE XP OF THE TARGETED AGS LEADING TO ENHANCED CD8 T-CELL MEDIATED IMMUNITY. HERE WE PROPOSE TO TEST THIS HYPOTHESIS USING A MODEL THAT INTEGRATES AN AB RAISED AGAINST THE HFCΓRI – NAMED Α-HFCΓRI, AND A MOUSE STRAIN THAT EXPRESSES HFCΓRI ON PROFESSION ANTIGEN PRESENTING CELLS (APCS). SINCE WE ONLY USE THE VARIABLE FRACTION (AB-FV) OF THIS AB (WITHOUT THE FC FRACTION), THIS APPROACH ALLOWS SELECTIVE-TARGETING OF HFCΓRI WITHOUT ENGAGING OTHER FC RECEPTORS (FCRS). THIS IS UNLIKE THE APPROACHES UTILIZING IMMUNE COMPLEXES (ICS) THAT CANNOT ISOLATE THE ROLE OF INDIVIDUAL FCRS IN XP, DUE TO BROAD SPECIFICITY OF MOST AB ISOTYPES. ADDITIONALLY, TARGETING TO THE ACTIVATING RECEPTOR HFCΓRI WILL INDUCE A MORE POTENT RESPONSE THAN THE ICS - WHICH POTENTIALLY ENGAGE BOTH ACTIVATING AND INHIBITORY RECEPTORS. MOREOVER, SINCE THE EXPRESSION OF HFCΓRI IS RESTRICTED TO THE PROFESSIONAL APCS I.E. MACROPHAGES (MΦS) AND DENDRITIC CELLS (DCS), IT IS LIKELY TO REDUCE UNWANTED ACTIVATION OF OTHER CELL TYPES EXPRESSING A VARIETY OF FCRS. THIS PROPOSAL HAS TWO MAJOR AIMS: 1) TO INVESTIGATE THE ROLE OF HFCΓRI-TARGETING IN XP; AND 2), TO EXAMINE THE ROLE OF VARIOUS DC SUBSETS IN THIS PROCESS, IN PARTICULAR FOLLOWING INTRANASAL DELIVERY OF THE MODEL VACCINE. OVERALL, THIS STUDY AIMS TO FILL A SIGNIFICANT KNOWLEDGE GAP IN UNDERSTANDING THE ROLE OF HFCΓRI IN XP, WHICH IS CRUCIAL FOR ENHANCING CD8 T-CELL RESPONSES OF PROTEIN SUBUNIT VACCINES. THE FINDINGS WILL IMPACT INNOVATIVE VACCINE STRATEGIES THAT EFFECTIVELY PROMOTE CELL-MEDIATED IMMUNITY AGAINST CANCER AND INTRACELLULAR PATHOGENS, WHILE MINIMIZING UNWANTED IMMUNE ACTIVATION AND INFLAMMATION.

IGF::CL::IGF THORACIC SURGEONS

IGF::OT::IGF TUITION AND FEES