Idaho Veterans Research And Education Foundation Inc

IDAHO BIOMEDICAL RESEARCH COLLABORATIVE IN EMERGING/REEMERGING INFECTIOUS DISEASE

METABOLOMIC ANALYSIS AS A TOOL TO UNDERSTANDING THE USE OF NOVEL THERAPEUTICS IN

DIAGNOSIS OF SUB-CLINICAL CRYPTIC GROUP A STREPTOCOCCAL MYONECROSIS

ROLE OF MIR-181A-5P IN ADENOVIRUS 14P1 INDUCED ACUTE LUNG INJURY - PROJECT SUMMARY THERE HAVE BEEN GLOBALLY DISTRIBUTED, REGIONAL OUTBREAKS OF A NOVEL STRAIN OF ADENOVIRUS (AD) 14, AD14P1, THAT INDUCES ACUTE LUNG INJURY SIMILAR TO THAT OBSERVED DURING OUTBREAKS OF INFECTIONS WITH OTHER AD SEROTYPES (E.G., AD 3, 4 AND 7). IT HAS NOT, HOWEVER, PREVIOUSLY BEEN POSSIBLE TO DO COMPARATIVE VIROLOGY AND PATHOGENESIS STUDIES, CONTRASTING A PROTOTYPE, PARENTAL AD STRAIN, SUCH AS AD14, WITH A SINGLE, GLOBALLY DISTRIBUTED GENETIC VARIANT, SUCH AS AD14P1. THE REASONS FOR THE INCREASED SEVERITY OF THESE AD14P1 INFECTIONS ARE POORLY DEFINED. WE HAVE SHOWN THAT CELLS UNDERGOING AD-INDUCED CYTOPATHIC EFFECT (CPE) FROM INFECTION WITH AD14 ELICIT AN IMMUNOREPRESSIVE ACTIVITY OF ACTIVATED HUMAN ALVEOLAR MACROPHAGES BY REPRESSING IL-1B, IL-6, IL-8 AND TNF-ALPHA. IN CONTRAST, AD14P1 CPE CORPSES ENHANCE PRODUCTION OF THESE SAME CYTOKINES, DUE TO DECREASED EXPRESSION OF VIRAL E1B 20K IN AD14P1 CPE CORPSES. CONSISTENT WITH THESE IN VITRO OBSERVATIONS, INFECTION OF SYRIAN HAMSTERS WITH PROTOTYPE AD14 RESULTED IN MINIMAL INFLAMMATION AND LUNG INJURY, WHEREAS AD14P1 INFECTION INDUCED A MARKED ACUTE LUNG INJURY AND ARDS-LIKE PATHOLOGY. IT HAS BEEN SHOWN THAT APOPTOTIC CELLS HAVE ALTERED EXPRESSION OF MICRORNA (MIRNA) THAT CAN REPRESS PRO- INFLAMMATORY RESPONSES AND THAT THESE MIRNA FROM APOPTOTIC CELLS CAN BE DELIVERED TO MACROPHAGES. WE POSTULATED THAT A SIMILAR MECHANISM COULD BE OCCURRING WITH AD14 INFECTED CELLS BUT NOT CELLS INFECTED WITH THE AD14P1 VARIANT. TO TEST THIS, WE INFECTED HUMAN CELLS WITH AD14 OR AD14P1 AND DID SMALL RNA-SEQ ON THE RESPECTIVE CPE CORPSES. THE DATA SHOWED THAT AD14 CPE CORPSES ARE ENRICHED IN FOUR MIRNA (AD14 MIRNA), ALL OF WHICH HAVE BEEN SHOWN TO REPRESS NF-KB DEPENDENT TRANSCRIPTION OF PRO-INFLAMMATORY CYTOKINES, COMPARED TO AD14P1 CPE CORPSES. OUR HYPOTHESIS IS THAT INCREASED EXPRESSION OF THESE SPECIFIC MIRNA IN AD14 CPE CORPSES AND THEIR TRANSFER TO RESPONDER MACROPHAGES ARE THE MECHANISMS THROUGH WHICH AD14 CPE CORPSES REPRESS PRO-INFLAMMATORY RESPONSES. THE STUDIES WE PROPOSE HERE WILL TEST THOSE HYPOTHESES. WE WILL TEST THE ROLE OF AD14 MIRNA IN MODULATING INFLAMMATORY RESPONSES OF MACROPHAGES USING MIRNA MIMICS AND WHETHER E1B 20K MODULATES AD14 MIRNA EXPRESSION DURING INFECTION. WE WILL TEST THE ROLE OF THE MOST ABUNDANTLY EXPRESSED AD14 MIRNA, MIR-181A-5P, AS THE MEDIATOR OF AD14 CPE IMMUNOREPRESSION BY USING 3’ UTR REPORTER CONSTRUCTS AND INHIBITING MIR-181A- 5P WITH MIRZIPS. FINALLY, WE WILL DO TRANSLATIONAL STUDIES TO DETERMINE THE ROLE MIR-181A-5P PLAYS IN THE IMMUNOPATHOGENESIS OF AD14P1 INFECTION IN THE SYRIAN HAMSTER. UPON COMPLETION OF THIS PROJECT, WE EXPECT TO HAVE AN INCREASED UNDERSTANDING OF THE ROLE OF VIRALLY INDUCED CELLULAR MIRNAS IN MODULATION OF MACROPHAGE RESPONSES DURING ACUTE LUNG INJURY IN RESPONSE TO AD14P1. THE POSITIVE IMPACT OF OUR STUDIES WILL BE THE DETERMINATION OF THE POTENTIAL OF MIRNAS AS THERAPEUTIC INTERVENTIONS FOR ARDS.

MODERNIZATION OF SMALL ANIMAL CAGING FOR INFECTIOUS DISEASE STUDIES AT THE BOISE VAMC - PROJECT SUMMARY WHILE STATIC CAGING FOR HUSBANDRY OF SMALL ANIMALS IS ACCEPTED BY THE GUIDE, VENTILATED CAGING HAS BECOME THE GOLD STANDARD FOR SMALL ANIMAL HUSBANDRY. STATIC CAGING RESULTS IN INCREASED TEMPERATURE AND HUMIDITY IN THE CAGES. THIS RESULTS IN WET BEDDING MATERIALS THAT CAN FOSTER GROWTH OF MICRO-ORGANISMS. AS A RESULT, INCREASED CHANGING OF ANIMAL BEDDING IS REQUIRED TO MAINTAIN SANITARY LIVING CONDITIONS. INCREASED FREQUENCY IN CAGE CHANGING HAS MULTIPLE DETRIMENTAL EFFECTS. FIRST, MORE BEDDING IS REQUIRED TO HOUSE THE ANIMALS AND GENERATES MORE BEDDING WASTE. SECOND, MULTIPLE CAGE CHANGES PER WEEK INCREASES THE WATER CONSUMPTION AND DIRTY WASTEWATER GENERATION AS CAGES REQUIRE SANITIZATION IN HOT WATER CAGE WASHERS. THIRD, INCREASED HANDLING OF ANIMALS CAN HAVE NEGATIVE EFFECTS ON RESEARCH STUDIES. FOURTH, FREQUENT CAGE CHANGING INCREASES THE EXPOSURES OF VMU PERSONNEL TO BOTH ANIMAL DANDER AND INFECTIOUS AGENTS. THE GOAL OF THIS PROJECT IS TO OBTAIN FUNDS TO UPGRADE THE CAGING AT THE BOISE VETERANS AFFAIRS MEDICAL CENTER VETERINARY MEDICAL UNIT (VMU) FROM STATIC CAGING TO VENTILATED CAGING. BY MOVING FROM STATIC CAGING TO VENTILATED CAGING THE ANIMALS WILL BE IN HOUSING THAT PROVIDES BETTER AIR FLOW ALLOWING FOR BETTER CONTROL OF TEMPERATURE AND HUMIDITY IN THEIR CAGES, IMPROVING NOT ONLY THE LIVING CONDITIONS FOR THE SMALL ANIMALS BUT INCREASING THE RIGOR AND REPRODUCIBILITY OF THE EXPERIMENTS CONDUCTED IN THE VMU AS WELL AS SAFETY OF VMU PERSONNEL.

UNDERSTANDING AND INTEGRATING THE METABOLOME, MICROBIOME, AND INNATE IMMUNITY IN DIABETIC WOUNDS

DEVELOPMENT OF ANTIBODIES TO SPECIFIC CELL SURFACE MARKERS TO ASSESS MACROPHAGE POLARIZATION DURING ADENOVIRUS 14 AND 14P1 INFECTION IN THE SYRIAN HAMSTER - PROJECT SUMMARY ADENOVIRUS (AD) NORMALLY INDUCES MILD, SELF-LIMITED INFECTIONS IN IMMUNOCOMPETENT HUMAN HOSTS. A MORE VIRULENT STRAIN OF AD14, AD14P1, FIRST EMERGED IN THE U.S. MILITARY AND HAS SINCE SPREAD GLOBALLY TO CIVILIAN POPULATIONS RESULTING IN SEVERE INFECTIONS, SOMETIMES LEADING TO ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS). THE INCIDENCE OF ARDS IN THE U.S. IS ~200,000 CASES ANNUALLY, WITH A HOSPITAL MORTALITY RATE OF ~40%. MOST ARDS TREATMENT MEASURES TARGET THE INFLAMMATORY RESPONSE; HOWEVER, ALL HAVE FAILED TO SHOW A MORTALITY BENEFIT. WE HAVE SHOWN THAT THE AD E1B 20K (20K) GENE PRODUCT CONTROLS MODULATION OF THE MACROPHAGE INFLAMMATORY RESPONSE TO CELLS DYING FROM AD INFECTION (AD CPE CORPSES). ABSENT OR LOW LEVEL 20K GENE EXPRESSION GENERATES AD CPE CELLS THAT ARE PRO-INFLAMMATORY, WHEREAS NORMAL (WILD TYPE VIRUS) 20K GENE EXPRESSION GENERATES ANTI-INFLAMMATORY AD CPE CELLS. AD14P1 EXPRESSES ONLY 20% OF THE 20K EXPRESSED BY WILD TYPE AD14. THIS REDUCED 20K EXPRESSION INDUCES PRO-INFLAMMATORY AD14P1 CPE CORPSES, WHEREAS WILD TYPE AD14 CPE CORPSES ARE ANTI- INFLAMMATORY. THE CENTRAL HYPOTHESIS OF THIS APPLICATION IS THAT THIS VIRAL GENETIC CHANGE IN THE IMMUNOMODULATORY EFFECT OF INFECTION WITH EMERGENT AD14P1 IS THE KEY BIOLOGICAL DIFFERENCE THROUGH WHICH THIS EMERGENT VIRUS INCREASES THE INCIDENCE AND SEVERITY OF ACUTE LUNG INJURY (ALI) THAT CAN RESULT IN ARDS AND DEATH. THE SYRIAN HAMSTER, MESOCRICETUS AURATUS, IS PERMISSIVE FOR INFECTION WITH HUMAN ADENOVIRUSES. SYRIAN HAMSTERS ARE ALSO SUSCEPTIBLE TO MANY OTHER HUMAN VIRUSES SUCH AS INFLUENZA, HANTAVIRUS, SARS-COV, SARS-COV-2, MARBURG AND EBOLA. WE HAVE SHOWN THAT INFECTION OF HAMSTERS WITH AD14P1 REPLICATES MANY OF THE KEY FEATURES OF HUMAN ALI/ARDS INCLUDING PATCHY BRONCHOPNEUMONIA, INCREASED PRO-INFLAMMATORY CYTOKINE EXPRESSION, EDEMA AND NEUTROPHIL INFILTRATION INTO THE LUNG AND AIRWAYS. A PROBLEM WITH THE SYRIAN HAMSTER MODEL SYSTEM HAS BEEN THAT THERE ARE NO TRANSGENIC HAMSTERS AND THAT THERE IS A LACK OF IMMUNOLOGICAL REAGENTS AVAILABLE, SUCH AS THOSE FOR THE MOUSE AND HUMAN. DEVELOPMENT OF THE CRISPR/CAS9 SYRIAN HAMSTER HAS REMOVED ONE OF THOSE OBSTACLES. THIS PROJECT ADDRESSES THE OTHER PROBLEM BY CREATING ANTIBODIES THAT ARE SPECIFIC FOR HAMSTER CELL SURFACE MARKERS EXPRESSED ON MACROPHAGES. THESE ANTIBODIES WILL ALLOW IDENTIFICATION AND ISOLATION OF MACROPHAGE SUB- POPULATIONS AND COMPARATIVE CHARACTERIZATION OF MACROPHAGE ACTIVATION IN RESPONSE TO AD14 AND AD14P1 INFECTIONS. THE ANTIBODIES WILL COMPLEMENT THE SMALL NUMBER OF EXISTING HAMSTER-SPECIFIC ANTIBODIES AVAILABLE FOR FLOW CYTOMETRY STUDIES. THESE ANTIBODIES WILL ALLOW US TO BEGIN TO UNDERSTAND THE EFFECTS OF AD14 AND AD14P1 INFECTION ON THE INNATE IMMUNE RESPONSE AND TO DEVELOP MECHANISTIC STUDIES OF HOW THESE TWO VIRUSES GENERATE SUCH DIFFERENT IMMUNE RESPONSES. UNDERSTANDING THOSE KEY FACTORS WILL ALLOW DEVELOPMENT OF TARGETED THERAPEUTICS TO TREAT NOT ONLY AD-INDUCED ALI/ARDS BUT POTENTIALLY ARDS TRIGGERED BY OTHER CAUSES OF ALI. IN ADDITION, THESE ANTIBODIES CAN BE USED TO STUDY THE EFFECTS OF OTHER HUMAN PATHOGENS ON MACROPHAGES AND NEUTROPHILS IN THE SYRIAN HAMSTER.