>> I'M DELIGHTED TO INTRODUCE JASON BRENCHLEY, OUR DIRECTOR'S LECTURER TODAY. JASON GOT HIS EDUCATION AT IDAHO STATE UNIVERSITY WHERE HE'S ABOUT UNDERGRADUATE MAJOR IN MICROBIOLOGY AND A MASTERS, GOT A MASTERS IN CHEMISTRY. HE DID A POST-DOC AT UT SOUTH WESTERN THEN HE CAME TO THE VACCINE RESEARCH SENT HE WERE WHERE HE WORKED WITH RICK COOP AND DENNY DEWICK AS POST-DOCTORAL FELLOW. TO PROVE THERE'S LIFE AFTER THE VRC. HE WAS RECRUITED AT NIAID AS INVEIGH TOFER AND WAS RECENTLY TENURED IN THE LABORATORY OF MOLECULAR MICROBIOLOGY. HIS INTEREST HAS BEEN MOSTLY ON HIV DEVELOPMENT PATHOGENESIS AND HE'S USED NON-HUMAN PRIMATE MODELS AND OTHER MODELS AS WELL. THE TALK TODAY IS ENTITLED MECHANISMS UNDERLYING MICROBIAL TRANSLOCATION AND THE THERAPEUTIC INTERVENTIONS TO REDUCE MICROBIAL TRANSLOCATION INDUCED IMMUNE ACTVATION. JASON. >> I WOULD LIKE TO THANK DR. GOTTESMAN FOR THE GREAT INTRODUCTION AND THE OPPORTUNITY TO COME PRESENT OUR WORK RELATED TO MICROBIAL TRANSLOCATION IMMUNE ACTIVATION AND DISEASE PROGRESSION. AURELIAN IS INTERESTED IN -- LAB IS INTERESTED IN UNDERSTANDING THE VIROLOGICAL MECHANISMS WHICH UNDERLIE THE DIFFERENCIAL DISEASE OUTCOMES BETWEEN PROGRESSIVELY HIV INFECTED HUMANS AND PROGRESSIVELY HIV AGED MACAQUES COMPARED TO ANIMALS NATURALLY INFECTED WITH HAIR SPECIES SPECIFIC STRAIN OF HIV. HIV # CAME FROM CROSS SPECIES TRANSMISSION OF SIP CPZ WHICH IS VIRUS THAT INFECTS CHIMPANZEES IN AFRICA BUT THERE WAS ANOTHER CROSS SPECIES EVENT IN WESTERN AFRICA WHERESIVE SMM A VIRUS THAT INFECTED -- INFECT HUMANS AND GAVE RISE TO HIV TWO EPIDEMIC AND SIMILAR CROSS SPECIES TRANSMISSION EVENT CAME IN WHEN RHESUS MACAQUE WAS CO-CHANGED WITH ANSIVE PSEUDOMANGABEE AND DEVELOPED PNEUMOSIS AT THIS CASHIA INFECTION. THESE ARE PROGRESSIVE, WHEREAS THE NATURAL SIV INFECTIONS IN AFRICA ARE NON. THEY'RE INSPECTED WITH SPECIES SPECIFIC SIV, THEY DON'T PROGRESS TO AIDS SO WE'RE TRYING TO UNDERSTAND WHAT IT IS THAT'S DIFFERENT ABOUT THE PROGRESSIVE INFECTIONS OF HUMANS AND ASIAN MACAQUES THAT SETS ASIDE SO THESE ANIMALS PROGRESS WHEREAS NATURAL HOSTS DON'T PROGRESS. BASED ON A LOT OF PRE-PUBLISHED DATA IT'S CLEAR ONE OF THE CLEAR DIFFERENCES BETWEEN PROGRESSIVE INFECTION OF HUMANS AND ASIAN ME MACAQUES AND NON-PROGRESSION OF THE AFRICAN GREENS OF THE MULTIFACETED ACTIVATION OF THE IMMUNE SYSTEM WHICH HAPPENS SECONDTIVELY IN PROGRESSIVE HIV INFECTION OF HUMAN AN SEV INFECTION OF ASIAN MACAQUES. EVERY ARM OF THE IMMUNE SYSTEM IS IN A HYPERACTIVATED STATE. IN PROGRESSIVELY INFECTED INDIVIDUAL THERE'S POLYCLONEAL B CELL ACTIVATION. INCREASED TURN OVER OF T-CELLS, HIGH FREQUENCIES OF T-CELLS THAT HAVE AN ACTIVATED PHENOTYPE AND INCREASED HASMATIC LEVELS OF CYTOKINES, CHEMOKINES AND PRO INFLAMMATORY MEDIATORS. SO CLINICALLY THE DEGREE OF IMMUNE ACTIVATION TURN OUT TO BE VERY IMPORTANT TO CHRONICALLY INFECTED INDIVIDUALS THAT THE DEGREE TO WHICH THE IMMUNE SYSTEM IS ACTIVATED IS THE STRONGEST PREDICTOR OF DISEASE PROGRESSION. THE IMMUNE ACTIVATION PREDICTS DISEASE PROGRESSIONko2 THAN VIRAL LOAD OR PERIPHERAL BLOOD CD4 POSITIVE T-CELL COUNT. A QUESTION THAT WE STARTED TRYING TO ASK SEVERAL YEARS AGO IS WHAT'S CAUSING THE IMMUNE ACTIVATION THAT IS OBSERVEED IN CHRONICALLY HIV INFECTED INDIVIDUALS P AND SIV INFECTED MACAQUES. THE VIRUS MUST BE INVOLVED BECAUSE INITIATION WITH HIGHLY ACTIVE ANTIRETROVIRAL THERAPY WHICH REDUCES VIRAL LOADS OF HIV OR SIV TO UNDETECTABLE LEVELS REDUCES IMMUNE ACTIVATION. NOT WITH THE SAME TEMPO AS VIRAL LOAD BUT IN GENERAL IF SOMEONE WHICH IS CHRONICALLY HIV INFECTED INITIATES HIGHLY ANTIRETROVIRAL THERAPY IMMUNE ACTIVATION SLOWLY GOES DOWN. ONE POSSIBILITY OF COURSE IS THAT ALL THIS IMMUNE ACTIVATION IS SIMPLY SPECIFIC FOR THE VIRUS. BUT WE AND OTHERS HAVE LOOKED AT THIS IN QUITE A BIT OF DETAIL AND FOUND ONLY A FRACTION OF THE T-CELLS ACTIVATED OR EFFECTOR TYPES ARE SPECIFIC FOR HIV, MOREOVER TO YOU LOOK AT THE PHENOTYPE OF HIV SPECIFIC CELLS THEY DO NOT UNIFORMLY MANIFEST AN ACTIVATEDDED PHENOTYPE SO IT'S UNLIKELY ALL THE IMMUNE ACTIVATION OBSERVEED IN CHRONICALLY INFECTED INDIVIDUALS IS HIV SPECIFIC. ANOTHER POSSIBILITY THAT PEOPLE TALKED ABOUT IS CYTOKINE INDUCE IMMUNE ACTIVATION WITH ALL THESE PRO-INFLAMMATORY CYTOKINES FLOATING AROUND IT'S POSSIBLE THERE ARE BYSTANDER EFFECTS. BUT IF WE THINK ABOUT THAT CYTOKINES THAT ARE PRO INFLAMMATORY CIRCUMSTANCE INVESTIGATING IN ELEVATED LEVELS ARE THE RESULT OF IMMUNE ACTIVATION. THEY CAN HAVE DOWNSTREAM CONSEQUENCES BUT IF THERE'S ELEVATED TNF SOMETHING MUST HAVE STIMULATED THAT TO BE PRODUCEED IN THE FIRST PLACE SO THE PRO-INFLAMMATORY CYTOKINES ARE THE RESULT OF IMMUNE ACTIVATION. THEN THERE'S QUITE -- THERE WAS A BIG IDEA IN THE FIELD THAT A LOT OF THE IMMUNE ACTIVATION WAS ATTRIBUTED TO VIRUS INDUCED INNATE IMMUNE ACTIVATION. BUT AGAIN, WE AND OTHERS ROOKED AT IN -- LOOKED AT THIS IN DETAIL AND FOUND POOR CORRELATIONS BETWEEN MARKERS OF THE ACTIVATION AND PLASMA VIRAL LOAD AND CANNOT BE P USED TO DESCRIBE WHAT HAPPENS IN NATURAL SIV INFECTION OF AFRICAN GREEN MONKEYS AND THE PSEUDOMANGABEES WHERE THERE'S HIGH LEVELS OF PLASMA VIRAL LOAD BUT LOW LEVEL CHRONIC IMMUNE ACTIVATION. WHEN I WAS A POST-DOC IN DANNY'S LAB WE THOUGHT ABOUT SPECIFIC PATHOLOGIES THAT HAPPENED WITHIN THE GASTRO INTESTINAL TRACT OF ACUTELY SIV INFECTED ASIAN MACAQUES AND ACUTELY HIV INFECTED INDIVIDUALS. FIRST IT'S CLEAR THAT WITHIN THE GASTRO INTESTINAL TISSUES THERE'S MASSIVE VIRAL REPLICATION DURING ACUTE PHASE. THIS RESULTS IN THE LOSS OF ALMOST ALL GASTROINTESTINAL CD4 POSITIVE T-CELLS. STUDIES HAVE SHOWN INCREASED PERMIBILITY IN INFECTED CATS AND HIV INFECTED HUMANS. THIS IS DUE TO HETEROCYTE APOPTOSIS. SUBSEQUENT STUDIES FOUND SIGNIFICANT FIBROSIS WITHIN MUCOSAL LYMPHOID TISSUE. SO THINKING THESE PATHOLOGIES IN THE GASTROINTESTINAL TRACT OF CHRONICALLY INFECTED INDIVIDUALS WE THOUGHT ABOUT WHAT THIS COULD HAVE AS A CONSEQUENCE TO IMMUNE INFECTED INDIVIDUAL. WE KNOW THE GASTROINTESTINAL TRACT HOUSES TRILLIONS OF BACTERIA WHICH ARE VERY STIMULATORY TO THE IMMUNE SYSTEM. WHICH HYPOTHESIZE ONE CAUSE OF IMMUNE ACTIVATION IS ATTRIBUTED TO MICROBIAL TRANSLOCATION. THIS REFERRED TO TRANSLOCATION OF GUT MICROBES AND MICROBIAL PRODUCTS FROM THE LUMINA OF THE INTESTIN TO PROLIFERATION. YOU CAN GET MICROBIAL TRANSLOCATION IN THINGS LIKE KROHN'S DISEASE AND ALCOHOLISM AND THIS CAN OCCUR WITHOUT OVER BACK TREATMENTIA. SO THEY DON'T HAVE TO BE SEPTIC TO HAVE CONSEQUENT IMMUNE ACTIVATION. SO WE LOOKED P P INTO THE LITERATURE AND FOUND IF WE WANT TO ADDRESS WHETHER OR NOT MICROBIAL TRANSROWCATION OCCURRED THE STANDARD IS LOOKING AT LIPOPOLYSACCHARIDE, GRAM NEGATIVE CELL WALL. SO WE DID THESE STUDIES BACK WHEN I WAS A POST-DOC AND WE FOUND THAT IF WE SAMPLED HIV UNINFECTED INDIVIDUALS OR INDIVIDUALS THAT WERE DIAGNOSED WITH HIV DURING THE ACUTE OR EARLY PHASE WE FOUND NO SIGNIFICANT ELEVATION IN MICROBIAL TRANSLOCATION BUT CERTAINLY IN INDIVIDUALS THAT WERE CHRONICALLY HIV INFECTED OR INDIVIDUALS THAT HAD ACTUALLY PROGRESSED THE FULL BLOWN AIDS BASED UPON CD4 COUNT LESS THAN 200 YOU SEE RAISED LEVELS INDICATING MICROBIAL TRANSLOCATION IS IN FACT OCCURRING. SO TO FOLLOW-UP ON THESE STUDIES WHEN I STARTED MY OWN LAB WE TURNED TO THE NON-HUMAN PRIMATE MODEL USING SIV INFECTED ASIAN MACAQUE TO UNDERSTAND THE MECHANISMS UNDERLYING MICROBIAL TRANSLOCATION TO DEVELOP THERAPEUTIC INTERVENTION. ONE FIRST THING WE WANTED TO DO IS MAKE SURE MICROBIAL TRANSLOCATION WAS OCCURRING AND WAS ACTUALLY BIOACTIVE IN VIVO. IN COLLABORATION WITH JAKE (INDISCERNIBLE) AT NCI WE DEVELOPED IMMUNOHISTOCHEMICAL TECHNIQUES TO SEE MICROBIAL PRODUCTS IN SITU AND SEE IF THEY'RE IN JUXTAPOSITION WITH PRODUCTION OF PROINFLAMMATORY CYTOKINES. THIS IS A SECTION OF A CHRONICALLY SIV INFECTED RHESUS ME TACK THAT BLEW HIS HUMAN TOX LOAD, THE RED IS ANTIBODY AGAINST IL-18 WHICH IS QUINT ESSENTIAL PRO INFLAMMATORY CYTOKINE AND IN THE OLD DAYS USED TO BE CALLED ENDOTOXIN RESPONSIVE SERUM FACTOR. SO IT'S THOUGHT TO BE FAIRLY SPECIFIC FOR LPS STIMULATION. THEN IN BROWN YOU HAVE LPS. YOU CAN SEE THAT IN FACT THERE ARE ELEVATED LEVELS OF LPS WITHIN THE MAZE TEARIC LYMPH NODE OF INFECTED ANIMAL AND THERE TENDS TO BE COLOSSALCATION WITH PRO INFLAMMATORY CYTOKINE IL-18 SUGGESTING THAT THESE MICROBIAL PRODUCTS ARE CAUSING IMMUNE ACTIVATION IN PROGRESSIVELY SIV INFECTED ASIAN MACAQUES. THEN WE TRIED TO FIGURE WHY MICROBIAL TRANSLOCATION WAS OCCURRING. KNOWING SEVERAL GROUPS SHOWED INCREASE IN PERMIBILITY WE HYPOTHESIZED ONE POSSIBILITY WOULD BE FOCAL AREAS OF DAMAGE TO THE STRUCTURAL BARRIER OF THE GI TRACT. SO ADDRESS THIS WE USE CON FOCAL MICROSCOPY. IT'S A COLON SECTION OF CHRONICALLY INFECTED ASIAN MACAQUE. IN RED YOU HAVE ANTIBODY AGAINST E. COLI. GREEN IS THE TIGHT JUNCTION PROTEIN CYTOKERATIN AND BLUE IS DARKSAFFI. THERE ARE FOCAL AREAS OF THE DAMAGE TO THE STRUCTURAL BARRIER OF THE GI TRACT IN THIS INFECTED ANIMAL. WHEN YOU HAVE DAMAGE TO THE BARRIER OF THE GI TRACT YOU HAVE INFILTRATION OF MICROBIAL PRODUCTS INTO THE LAMINA PROPRIA. SO THESE DATA SUGGEST TO US THAT ONE OF THE MECHANISMS UNDERLYING MICROBIAL TRANSLOCATION WAS DAMAGE TO THE STRUCTURAL BARRIER OF THE GI TRACT. SO WE CAN DO QUANTITATIVE ANALYSIS TO DETERMINE THAT THIS IS STATISTICALLY SIGNIFICANT. IF WE LOOK AT DAMAGE IN SIV UNINFECTED ANIMALS AND SACRIFICED DURING ACUTE PHASE WITHIN A WEEK AND A HALF YOU SEE LITTLE DAMAGE TO THE STRUCTURAL BARRIER AND IF WE LOOK IN ANIMALS SACRIFICED DURING THE CHRONIC PHASE INFECTION YOU SEE SIGNIFICANTLY ELEVATED LEVELS OF DAMAGE TO THE STRUCTURAL BARRIER OF THE GI TRACT AND IMPORTANTLY THE DEGREE TO WHICH THERE'S DAMAGE TO THE STRUCTURAL BARRIER OF THE G I TRACT CORRELATES HOW MUCH MICROBIAL TRANSLOCATION THERE IS BASED UPON IMMUNOHISTOCHEMICAL STAINING AND POLYSACCHARIDE. CHRONICALLY SIV ANIMALS HAVE STRUCTURAL BARRIER TO THE GI TRACT THAT IS AN UNDERLYING CAUSE OF MICROBIAL TRANSLOCATION. SO THE NEXT THING WE WANT TO KNOW IS WHETHER OR NOT WE CAN TIE THIS TO SOME IMMUNOLOGICAL ABNORMALITY IN CHRONICALLY INFECTED ANIMALS SO WE STARTED THINKING WHAT TYPES OF IMMUNE RESPONSES ARE THOUGHT IMPORTANT FOR COMBATING EXTRA CELLULAR BACTERIAL ANTIGENS AN MAINTENANCE OF STRUCTURAL BARRIER OF GI TRACT. THE FIRST ARE NEUTRAPHILS BUT IS THERE'S ALSO A RECENTLY DISCOVERED SUBSET OF CD4 + T-CELLS. THESE ARE MEMORY CD4 + T-CELLS A FAMILY OF EFFECTOR CYTOKINES LIKE IL-17 AND 22 BUT NON-INTERFERE GAMMA, IL-4, NOT CLASSICAL CELLS, PRODUCE CYTOKINES IN RESPONSE TO SIMULATION THROUGH THE T-CELL RECEPTOR. IL-17 AND KITEKINES MADE ARE THOUGHT TO BE VERY IMPORTANT FOR ANTI-BACTERIAL IMMUNITY. THIS FAMILY OF CYTOKINES RECRUITS NEUTRAPHILS TO BACTERIAL INFECTION, THIS FAMILY ESPECIALLY IL-22 CAN INDUCE PROLIFERATION OF GAS ROW INTESTINAL -- THESE FAMILY CYTOKINES PRODUCE ANN BACTERIAL DEFENSEINS AND IN MOUSE STUDIES HAS BEEN SHOWN THEY CAN REGULATE PRODUCTION OF DIFFERENT TYPES OF MUSINS. SO WE WENT TO LOOK AT THE FREQUENCY OF CELLS TO PRODUCE IL-17 IN THE COLON OF SIV UNINFECTED ANIMALS AND SIV INFECTED. YOU CAN SEE WE WERE ABLE TO FIND HIGH FREQUENCIES OR ABOUT TEN -- 7 TO 10% CD4 + T-CELLS THAT MADE IL-17 AFTER STIMULATION BUT THERE WERE SUB SETS OF LYMPHOCYTES CAPABLE OF MAKING IL-17 AFTER MITOGENIC STIMULATION. THERE WERE CD8 POSITIVE T-CELLS THAT MAKE IL-17 AND INNATE LYMPHOCYTES THAT PRODUCE IL-17 AFTER MITOGEN HEALTHCARE STIMULATION IN UNINFECTED ANIMALS. HEN WE COMPARE SIV UNINFECTED TO CHRONICALLY INFECTED ANIMALS YOU CAN SEE A SIGNIFICANT REDUCTION IN CELLULAR SOURCES OF IL-17. SO THE IL-17 PATHWAY IS PERTURBED IN THE TRACT OF SIV INFECTED AN L MALLS THAN'S IRRESPECTIVE OF THE CELLULAR SOURCE OF IL-17. THE DEGREE TO WHICH THESE PRODUCING CELLS ARE LOST BE CD4 POSITIVE T-CELLS, CD8 + T-CELLS OR INNATE LYMPHOCYTES, THERE'S A SIGNIFICANT NEGATIVE CORRELATION OF IL-17 AND HOW MANY DAMAGE TO THE STRUCTURAL BARRIER OF THE GI TRACT SUGGESTING THAT LOSS OF THESE IL 17 CELLS IS AN IMPORTANT PHENOMENON FOR DAMAGE OBSERVEED TO THE STRUCTURAL BARRIER OF THE GI TRACT. IMPORTANTLY THE DEGREE WHICH THE IL-17 PRODUCING CELLS ARE LOST IN BOTH THE COLON ON THE LEFT AND THE MESENTERIC LYMPH NODE ON THE RIGHT IS CORRELATING WITH IMMUNE ACTIVATION WHICH WITH WE MEASURE BY EXPRESSION OF KI-67 WHICH IS A NUCLEAR ANTIGENIC CELLS, HIGH LEVELS OF IMMUNE ACTIVATION ARE ASSOCIATED WITH LOSS OF IL-17 PRODUCING CD4, CD8 AND THESE ILCs. SO WE'RE USING IL-17 AS A MARKER OF A PARTICULAR TYPE OF IMMUNOLOGICAL CELL BUT CERTAINLY THERE ARE OTHER EFFECTOR CYTOKINES WHICH CELLS ARE MAKING. SO WE WANTED TO GET AN IDEA OF MECHANISM WHICH IS LOSS OF IL-17 PRODUCTION MIGHT BE LEADING TO DAMAGE TO THE STRUCTURAL BARRIER OF THE GI TRACT. SO WE SORTED AFTER MITOGENIC STIMULATION CELLS THAT MADE IL-17 COMPARED TO CELLS THAT DIDN'T MAKE IT, WE EXTRACTED RNA AND DID GENE CHIP ANALYSIS. 2,000 GENES EXPRESSED BETWEEN CELLS MAKING 17 AND CELLS NOT MAKING 17. AND IF WE KIND OF CATEGORIZE MANY OF THESE GENES THE GENE WHICH WAS MOST DIFFERENCIALLY EXPRESSED BY THE CELLS WE WERE IDENTIFYING MAKING IL-17 WAS IL-17 ITSELF. SO THAT GAVE SOME EVIDENCE TO SUGGEST WE WERE DOING A FAIRLY GOOD JOB OF PULLING CELLS MAKE IL-17. THESE CELL WERE PRODUCING A VARIETY OF CHEMOKINES FOR TRAFFICKING TO THE GASTRO INTESTINAL TRACK. THIS IS INTEGRIN BETA 7 INTO THE GI TRACT AND TESTIMONY GENEpQ„ EXPRESSED FOURTH MOST DIFFERENCIALLY COMPARED TO CELLS THAT DIDN'T MAKE IT, WAS IL-22. SO ABOUT THE TIME WE WERE DOING THE EXPERIMENTS WE'RE IN LUCK COMMERCIALLY AVAILABLE ANTIBODY FOR IL-22 CAME OUT WHICH CROSS REACT WITH ASIAN ME ACTS. SO USING -- MACAQUES. AFTER MITOGENIC STIMULATION HIGH FREQUENCY IL-17 PRODUCE CD4, CD8 AND THESE INNATE LYMPHOCYTE CO-PRODUCED IL-22. SO 40% CD4 THAT MAKE IL-17 IN THE COLON ALSO MAKE IL 22. THIS WAS ALSO TRUE IN THE MESENTERIC LYMPH NODE. CONSISTENT WITH WHAT'S SEEN IN IL-17 PRODUCERS YOU SEE REDUCTION IN FREQUENCY OF CELLULAR SOURCES OF IL-22 LOOK COLON OR MESENTERIC LYMPH NODES OF UNINFECTED COMPARED TO SIV INFECTED ANIMALS. SO NEXT WE WANTED TO TRY TO ANSWER IS WHY THESE IL-17 AND 22 PRODUCERS ARE BEING LOST FROM THE GASTROINTERESTAL TRACT OF CHRONICALLY SIV ANIMALS COMPARED TO UNINFECTED. PEOPLE ATTRIBUTE IT TO PREFERENCIAL TARGETING BY SIV ITSELF. IF WE THINK ABOUT IT CD8 CELLS AND INNATE LYMPHOSITES DON'T EXPRESS THE RECEPTOR FORSIVE CD4 SO UNLIKELY THAT THEY CAN BE INFECTED BY SV IN VIVO. WE CAN USE QUANTITATIVE REAL TIME PCR TO DETERMINE INFECTION FREQUENCIES AND WE HAVE FOUND NO PREFERENCIAL INFECTION OF CD4 POSITIVE THE T-CELLS MAKING IL-17 COMPARED TO CELLS THAT DON'T MAKE IT. SO WE DON'T THINK THAT THIS LOSS OF IL-17 AND 22 PRODUCERS IS ATTRIBUTED TO PREFERENCIAL HIV OR SIV INFECTION IN VIVO. THE MECHANISM UNDERLYING LOSS OF CELLS IS, SEEMS TO BE SHARED AMONG THE DIFFERENT CELLS BECAUSE WE FIND FAIRLY GOOD CORRELATIONS BETWEEN LOTS OF CD8 CELLS MAKING IL-17 VERSUS CD4, AND LOOK AT LOSS OF ILC MAKING 17 CD8 CELLS MAKING IL-17. SO WHATEVER PHENOMENON LEADING TO LOSS OF THESE PRODUCERS SEEMED TO BE SHARED AMONG ALL IL-17, 22 POSITIVE CELLS. SO IF WE THINK ABOUT WHAT TYPES OF CELLS ARE IMPORTANT FOR ORCHESTRATING PRODUCTION OF IL-17 OR 22, BY LYMPHOCYTE OF COURSE WE START TO THINK ABOUT ANTIGEN PRESENTING CELLS. AND IN MICE STUDIES, THERE ARE SEVERAL THAT SUGGESTS THERE'S A PARTICULAR SUBSET OF ANTI-GENERAL PRESENTING CELLS THAT EXPRESS CD 3 CRITICALLY IMPORTANT FOR INDUCTION AND MAINTENANCE OF IL-17 PRODUCING CELLS. USING RECENT STUDY IN MICE SUGGESTED THAT THE CD 3 POSITIVE CELLS EXPRESS VITAMIN A METABOLIZING ENZYME CALLED ROTENOIC ACID DEHYDROGENASE. THE MAJORITY OF ACTIVITY IS WITHIN THE CD 103 POSITIVE SUBSET. SO WE KNOW VITAMIN A METABOLISM IS VERY IMPORTANT FOR MAINTENANCE AND INDUCTION OF IL-17 PRODUCERS. SO WE STARTED THINKING ABOUT 103 POSITIVE APCs IN THE GASTROINTESTINAL TRACT OF CHRONICALLY INFECTED AND UNINFECTED ANIMALS SO WE MEASURE FREQUENCY OF CD 103 POSITIVE APC IN COLON AN MESENTERIC LYMPH NODE OF UNINFECTED AND SIV INFECTED ANIMALS AND YOU SEE SIGNIFICANT REDUCTION IN FREQUENCY OF 103 POSITIVE DC IN BOTH ANATOMICAL SITES. DEGREE TO WHICH THE 103 POSITIVE DCs ARE LOST ARE CORRELATED POSITIVELY HOW MANY IL-17 PRODUCERS ARE LOST. SO LOSS OF THE IL-17 PRODUCING C. CXFC 4, CD8 AND THESE INNATE LYMPHOCYTE IS CORRELATED DIRECTLY WITH LOSS OF CD 103 POSITIVE DENDRITIC CELLS. SO WE WOULD ARGUE THAT ONE POTENTIAL MECHANISM UNDERLYING LOSS OF THESE IL-17 PRODUCING CELLS WHICH ARE CRITICALLY IMPORTANT FOR MAINTENANCE OF THE STRUCTURAL BARRIER OF THE GI TRACK IS ATTRIBUTED TO SOME ABNORMALITY WITHIN THE ANTIGEN PRESENTING CELL SUB SETS. SO WE STARTED THINKING ABOUT WHY IS IT THAT THE CD 103 POSITIVE ANTIGEN PRESENTING CELLS IMPORTANT FOR MAINTENANCE OF IL-17 PRODUCERS SO WE CYTOMETRICLY SORTED DENDRITIC CELLS EXPRESSING CD 103 VERSUS NOT EXPRESSING FROM GASTROINTESTINAL TRACTS APPROXIMATE THERE WERE 2,000 GENES DIFFERENCIALLY EXPRESSED. IF WE DO PATHWAY ANALYSIS ONE KEY PATHWAY THAT LIGHTS UP AMONG THE 103 POSITIVE ANTIGEN PRESENTING CELLS IS HIGHLIGHTED TO BE IMPORTANT MAINTENANCE AND PROMOTION OF IL-17 PRODUCERS. TO ANSWER THIS DIRECTLY WE DID COCULTURING EXPERIMENTS TO SORT CD 103 POSITIVE ANTIGEN PRESENTING CELLS OR CD 103 NEGATIVE AND CO-CULTURED THEM WITH NAIVE CD4 POSITIVE T-CELLS FROM NON-HUMAN PRIMATES AND CULTURED THEM IN TH-17 CONDITIONS IN THE PRESENCE OR ABSENCE OF IL-6. WE DID THIS SPECIFICALLY BECAUSE IT LOOKEDEh LIKE 103 POSITIVE WERE DOINGING A GOOD JOB OF MAKING IL-6 THEMSELVES AND THEN WE LOOKED HOW WELL THESE NAIVE CD4 POSITIVE T-CELLS ARE DIFFERENTIATED INTO TH-17 CELLS BASED UPON PRODUCTION OF IL-7 MESSAGE OR GAMMA C WHICH IS A TRANSCRIPTION FACTOR EXPRESSED BY TH-17 CELLS. YOU CAN SEE EVEN IN ABSENCE OF IL-6, THE CD 103 POSITIVE DENDRITIC CELLS DO BETTER JOB OF INDUCING TH-17 CELLS COMPARED TO THE CD 103 NEGATIVE DCs. THE QUESTION NOW IS WHY IS IT WE'RE LOSING THE CD 103 POSITIVE ANTIGEN PRESENTING CELLS. LOSS OF APC THAT EXPRESS 103 OR INFILTRATION THAT NON-ANTIGEN PRESENTING CELLS. SO TO START TO GET THIS WE USED IMMUNOHISTOCHEMISTRY AND WE'RE USING IMMUNOHISTOCHEMICAL STAINING DIE OXGENASE SOME THIS IS AN ENZYME EXPRESSED BY A CERTAIN SUBSET OF ANTIGEN PRESENTING CELLS THOUGHT SOMEWHAT PROINFLAMMATORY IN NATURE. LEFT IS MESENTERIC LYMPH NODE OF UNINFECTED ANIMAL, AND YOU SEE VERY LITTLE EXPRESSION. BUT IN THE CHRONICALLY INFECTED ANIMAL YOU SEE SIGNIFICANT LEVELS OF STAINING AND WHEN WE DO QUANTITATIVE IMAGE ANALYSIS YOU CAN SEE THAT'S THE AREA FRACTION OF THE COLON AND MESENTERIC LYMPH NODE STAINING POSITIVE FOR THIS IDO ENZYME SIGNIFICANTLY HIGHER COMPARED TO SIV UNINFECTED ANIMALS. THE DEGREE TO WHICH THE COLON IS STAINING POSITIVE FOR IDO, CORRELATES NEGATIVELY WITH LOSS OF 103 POSITIVE DENDRITIC CELLS IN COLON AND -- WITHIN BOTH COLON AND MESENTERIC LYMPH NODE. YOU HAVE SEEN THAT MUCOSAL IL-17 AND 22 PRODUCERS ARE DECREASED AFTER SIV INFECTION. THIS INCLUDES PRACTICALLY EVERY CELLULAR SOURCE THAT WE CAN THINK ABOUT OF THESE EFFECTOR CYTOKINES INCLUDING CD4s, CD8 AND INNATE LYMPHOCYTE. LOTS OF CELLS AFTER IMMUNODEFICIENCY VIRUS INFECTION ASSOCIATED WITH DAMAGE TO THE CHRONIC EPITHELIUM. LOSS OF CELLS EXPRESS GENES KNOWN IMPORTANT FOR MUCOSAL HOMEOSTASIS. ONE MECHANISM UNDERLYING LOSS OF IL-17 PRODUCERS IS AN ALTERATION IN THE LANDS ESCAP OF THE LOCAL ANTIGEN PRESENTING CELLS AND THAT WE SEE LOWER LEVELS OF CD 103 POSITIVE ANTIGEN PRESENTING CELLS WHICH EXPRESS GENES ASSOCIATED WITH PROMOTION OF IL-17 AND IL-# 2 POSITIVE CELLS AND HAVE SHOWN THESE CELLS DIRECTLY PROMOTE ROR GAMMA C AND IL-17 BY STIMULATED NAIVE CELLS. THESE DATA ARE ONE OF THE FIRST POSSIBLE MECHANISMS DESCRIBING DAMAGE TO THE STRUCTURAL BARRIER OF THE GASTRO INTESTINAL TRACT, MICROBIAL TRANSLOCATION, IMMUNE ACTIVATION DURING PROGRESSIVE SIV INFECTION AND SIMILAR STUDIES ARE ONGOING IN CHRONICALLY INFECTED INDIVIDUALS. IF ALL THESE PHENOMENON ARE REALLY IMPORTANT FOR PROGRESSIVE NATURE OF HIV INFECTION IN HUMANS AN SIV INFECTION IN NON-HUMAN PRIMATES OF ASIAN MACAQUES, THE PREDICTION WOULD BE THAT THESE PHENOMENON WOULD NOT OCCUR IN NATURALLY SIV INFECTED AFRICAN GREENS. SO IF WE DID A SUMMARY TABLE HERE AND LOOK AT NATURAL HOST ON THE LEFT, THESE ARE ANIMALS LIKE AFRICAN GREEN GREEN MONKEYS AND PSEUDOMANGABEES WHO DON'T PROGRESS TO AIDS. VERSUS NON-NATURAL HOST, THESE WOULD BE THE ASIAN MACAQUES AND HUMAN, HIGH VIRAL LOADS YES ON BOTH CASES. DEPLETION OF GASTROINTESTINAL CD4 + T-CELLS, NEARLY COMPLETE AND PROGRESSIVELY INFECTED INDIVIDUALS, IT'S FAIRLY MINOR IN NON-PROGRESSIVELY INFECTED INDIVIDUALS, DEPLETION OF PERIPHERAL BLOOD CD4 POSITIVE T-CELLS OCCURS IN NATURAL HOSTS. ALMOST ALWAYS HAPPENS IN PROGRESSIVELY INFECTED HOSTS, PREFERENCIAL DEPLETION OF TH-17 CELLS, WE HAVE SEEN THAT HAPPENS IN PROGRESSIVE INFECTION. OTHER STUDIES HAVE SHOWN THIS DOESN'T HAPPEN IN NON-PROGRESSIVE INFECTION. NO IMMUNE ACTIVATION IN NON-PROGRESSIVE INFECTION, NO MICROBIAL TRANSLOCATION AN OF COURSE NO PROGRESSION TOIZE. SO USING PSEUDOMANGABEES WE DID IMMUNOHISTOCHEMISTRY LOOKED FOR INFILTRATION IN THE LAMINA PROPRIA OF FOUR CHRONICALLY INFECTED MANGABEES AND YOU SEE VERY LITTLE IN THE LAMINA PROPRIA. THESE ARE RECTAL BIOPSIES SO NOT ABLE TO THOROUGHLY INVESTIGATE THE INTEGRITY OF THE STRUCTURAL BARRIER OF GI TRACT BUT WHEN WE HAVE EPITHELIUM IT'S INTACT. SO IT DOESN'T LOOK LIKE THE GI TRACT IS PERTURB AND THERE'S LITTLE TRANSLOCATION. WE'RE ABLE TO LOOK AT THE FREQUENCY OF IL-17 PRODUCING CD4s, CD8 P AND THESE INNATE LYMPHOCYTES FROM RECTAL BIOPSY OF SIV UNINFECTED AND CHRONICALLY INFECTED MANGABEES AND NO REDUCTION IN FREQUENCY OF ANY OF THE IL-17 PRODUCING CELLS SUBz3oL?– SETS. IMPORTANTLY, THESE ALSO MAINTAIN HEALTHY FREQUENCIES OF ANTIGEN PRESENTING CELLS THAT EXPRESS CD 103. SO THE QUESTION AFTER PRESENTING DATA ARE WHETHER OR NOT IT'S CLINICALLY IMPORTANT TO CHRONICALLY HIV INFECTED INDIVIDUALS BECAUSE WE HAVE NOW FOR 30 YEARS HAD A BROAD CLASS OF ANTIRETROVIRAL MEDICINES WHICH CAN REDUCE VIRAL REPLICATION TO UNDETECTABLE LEVELS FOR THE LIFE SPAN OF CHRONICALLY HIV INFECTED LIFE SPANS. SO THE ANSWER SEEMS TO BE MAYBE, MAYBE NOT. BUT RECENTLY WE HAVE BEEN ABLE TO LOOK AT MORTALITY OF HIV INFECTED INDIVIDUALS ON HIGHLY ACTIVE ANTIRETROVIRAL THERAPY. THERE'S CURRENT HIGHLY ANTIRETROVIRAL DRUG REGIMENS AROUND FOR YEARS AND WHAT PEOPLE HAVE SHOWN IS THAT COMPARED TO HEALTHY POPULATION CONTROL, INDIVIDUALS WHO ARE HIV INFECTED AND VIROLOGICALLY SUPPRESSED THERAPEUTICALLY WITH DIFFERENT TYPES OF ARV REGIMENS ARE DYING PREMATURELY COMPARED TO POP POPULATION CONTROLS SO STUDY SHOUGHS INCREASE MORTALITY AMONG ARV TREATED INDIVIDUALS IS ATTRIBUTED TO INFLAMMATION. THEY'RE DYING OF CARDIOVASCULAR DISEASE AND DIFFERENT TYPES OF CANCERS AN DIFFERENT TYPES OF KIDNEY FAILURES. SO THE QUESTION IS WHAT'S CAUSING RESIDUAL INFLAMMATION IN CHRONICALLY INFECTED INDIVIDUALS VIRAL LOGICALLY SUPPRESSED WITH ARVs? SEVERAL STUDIES SUGGEST INCREASED INFLAMMATION NCI AMONG ARV INDIVIDUALS TO MICROBIAL TRANSLOCATION. THAT THERE'S BIOLOGICAL REASONS CAN BE ONGOING IN INDIVIDUALS THAT ARE VIROLOGICALLY SUPPRESSED WITH ARCRVs. THERE'S MASSIVE FIBROSIS WITHIN NOT ONLY THE LAMINA PROPRIA BUT LYMPHOID FOLLICLES WITHIN THE GASTROINTESTINAL TRACT. THERE'S LOW RECONSTITUTION OF CD4 + T-CELLS SO YEARS AFTER ADMINISTRATION OF ARV, CHRONICALLY INFECTED INDIVIDUALS ON ARV DO NOT RECONSTITUTE GASTRO INTESTINAL CD4 T-CELLS TO HEALTHY LEVELS. THERE'S DECREASED FREQUENCY OF TH-17 CELLS. THOUGHT DECREASED HOMING OF T-CELLS TO THE LAMINA PROPRIA. THERE'S DECREASED EXPRESSION OF GENES IMPORTANT FOR EPITHELIAL REPAIR AND THERE'S STILL ONGOING INCREASE APOPTOSIS. SO THE CONCLUSION IS INCREASED MICROBIAL TRANSLOCATION MAYBE A CAUSE OF INFLAMMATION IN ARV TREATED INDIVIDUALS AND THIS MIGHT BE WT'S DRIVING THEIR INCREASED MORTALITY COMPARED TO CHRONICALLY -- UNINFECTED INDIVIDUALS. WE STARTED THINKING ABOUT THERAPEUTIC INTERVENTIONS USING TO DECREASE MYCONTROLIAL TRANSLOCATION IN IMMUNE ACTIVATION. NO MONOHUMAN PRIMATES ASIAN MACAQUE WITH SIV. SO PROBIOTICS ARE BACTERIA THOUGHT BENEFICIAL TO THE HOST. THOUGHT THE INHIBIT PRODUCTION OF PRO-INFLAMMATORY CYTOKINES, THOUGHT THE DECREASE PERMIBILITY IN PART BTHEIR PRODUCTION OF SHORT CHAIN FA SAY ASIT -- FATTY ACIDS AND DECREASE GROWTH OF POTENTIAL PATHOGENS BY COMPETIN FOR NUTRIENTS WITHIN THE GASTROINTESTINAL TRACT. IMPORTANTLY, PROBIOTIC ORGANISMS HAVE BEEN SHOWN IN CLINICAL PLACEBO CONTROL TRIALS TO BE BENEFICIAL IN MANY DISEASES WHERE MICROBIAL TRANSLOCATION IS IMPLICATEDINCLUDING FATTY LIVER DISEASE, INFLAMMATORY BOWEL DISEASE, BACTERIAL OVERGROWTH, ALCOHOL INDUCED HEPATITIS AND VIRAL DIARRHEAL DISEASE. SO WE DECIDED TO TRY TO INTRODUCE THERAPEUTICS OF PROBIOTICS IN CHRONICALLY INFECTED ASIAN MACAQUES. DESIGN WAS TO TREAT AFTER SIV INFECTION TO CHRONIC INFECTION TO SEE IF PROBIS WOULD DO ANYTHING. SO WE FOLLOW ANIMALS LONGITUDINALLY AND IT DIDN'T LOOK LIKE TH PRO BIOTICS BY THEMSELV WERE IMPARTING A BENEFIT TO CHRONICALLY INFECTED MACAQUES. VIRAL LOADS WERE HIGH, LOSING CD4 POSITIVE T-CELLS, IMMUNE ACTIVATION WAS HIGH. SO WE BOOSTED THESE ANIMALS WITH AN ARV REGIM SO THESE ARE ARV THAT CROSS REACT WITH SIV AND FOLLOWED FOR FIVE MONTHS AND SACRIFICE THE ANIMALS AND DID A FAIRLY INVASIVE STUDY WHETHER OR NOT PROBIOTICSAD AN EFFECT. AND IN WITH THE SAME VIRUS SIV 39, DAY 160 THEY STARTED THE SAME REGIMEN, THEY WENT FOR FIVE MONTHS AND WE SACRIFICED THE ANIMALS. THE PRO BIOTIC REGIMENS, ONE IS CALLED VSL-3 A COMBINATION OF 8 DIFFERENT PR BOOTIOTIC ORGANISMS ANDCTOBACILLUS, THE TRADENAME IS CULTUREELL SO WE CHOSE THESE TWO PROBIOTIC ORGANISMS VSL-3 AND CULTUREELL AS THE PROBIOTIC ORGANISMS WITH THE MOST HISTORY OF USE IN OTHER TIMES OF DISEASE WHERE MICROBIAL TRANSLATION IS IMPLICATED. D THE ARV REGIMEN, THERE WERE REVERSE TRANSCRIPTASE INHIBIT HUBTORS AND TWO INTEGRATION INHICKTORS -- INHIB DONATED FROM M GID. SO THE ANIMALS THAWE CHEESE THOSE USE FORPEUTIC INTERVENTION TRIAL WERE PIG TAIL MAES AND WE SPECIFICALLY CHOSE PIG TAIL MACAQUES BECAUSE WE PREVIOUSLY SHN THAT THESE ASIAN MACAQUES ARE DYING PREMATURELY AFTER SIV MACK 239 ON COMPARED TO CONONCAL US FOR THESE TYPES OF STUDIES RHESUS MACAQUES SO THEASE MORTALITY AMONG PIG TAILS IS ATTRIBUTED TO PIG TAILS HAVING HIGH LEVELS MICROBIAL TRSLOCATION AND IMMUNE ACTIVATION BUT FOR THEY'RE INFE SOD WE CHOSE PIGILS BECAUSE WE THOUGHT INCREASE MORTALITY WAS ATTRIBUTED TO MIIAL TRANSLOCATION. THE FIRST THING WE WANTED TO LOOK AT IS WHETHER OR NOT THE ARV REGIMEN IS DECREASING VIRAL LOAD. SO IN RED WE LOOK AT VIRAL LOADS IN ANIMALS THAT GOT ARV AND PROBIOTICS. THE BLACK LINEARE ANIMALS WITH ARV ALONE YOU N SEE AFTER ADMTRATION OF THIS ARV REGIMEN WE DID A GOOD JOB OF SHUTTING DOWN VIRAL REPLATION WITH THE EXCEPTION OF ONENIMAL WHO ENDED UP BEIN ARVAND PROBIOTIC. HIS LOAD DIDN'T COME TO UNDETECTABLE LEVELS UNTIL TIM OF NECROPSY. HE WAS NIGH REAMIC THE MAJORITY THEME SO HIGHLIGHTED BY A DIFFERENT SYMBOL IN SUEQUENT SLIDES. WE FOUND NOGNIFICANT DIFFERENS IN THE PERIPHERAL OOD POSITIVE T-CELLS COUNTS O ANIMALS COARED TO P ALS WITH ARV ALONE. IN BOTH GROUPS C T COUNTS TENDED TO INCREASE AFTER ADSTRATION OF ARV. s. THNEXT THING WE WANTED TO LOOK AT WAS COMPOSITION OF GASTROININAL MICROBIOME. SO WE DIDONGITUDINAL ANALYSIS OF THE FECAL MICROBIOME INEE ANIMALS THAT WERE GOING TO B -- IN THE ARV ONLY GROUP AND FOUR ANIMALS THAT WERE IN THE ARV AND PRIOTIC GROUP. WE DID THESE STUDIES IN COLLABORATIO WITH JULIE -- AND CLASSIFIEDE 454 SEQUENCING OF GUT MICROBIE AND CLASSIFD THE BUGS USING THE RIBOSOMAL DATABASE PROJECT CLASSIFICA SO BARS ON THE TOP ARE BACTERIAL IDEDES A TTOM ARE TERM CUTINGS. LONGITUDINAL ACROS-- TERM CUTES. THERE'S NO STRIKING DIFRENCE IN COMPOSITION OF THE GASTROINTESTINAL MICROBIOME, AT LEAST THE FECAL MICROBIOME. THERE'S NO DIFFERENCE AND WE CAN LOOKT PROTEOBTER WHICH HOUSES MORE OF THE PATHOGENIC BUGS AND WE'RE NOT SEEING SIGNIFICANTNCREASE IN THE FECAL MICRO BE TO THIS PROTEOBACTER PHYLUM COMPARED TO ANIMALS UNINFECTED. IF WE LOOK MOR CAREFULLY AND LOOK Ať<1 COMPOSITION THAT ARE LOST STRIDIALES INCLUDE A GROUP OF BACTERIA CD SESSION MEANTEIMEMTIS BACTERIUM GOOD AS IL-17 PRODU. SO THEULL GE IN THE ANALS PRE-SIVCOMPARED TO ALL POST SIV BEFORE THERAPEUT INTERVENTION, NOFICANT DIFFERENCE. W IF WE LOOK TONIMALS THAT GOTARV AND PROBIOTICS COMPARED TOLS THAT GOT AB ARV ALONE YOU CAE IN THE ANIMALS THAT GOT ARV AN PROBIOTIC THE CH IN CLOSTRIDIUM INCRSED IN ALLMALS. ONE WAS THE SAME T OTHER TWO DECREASED. THIS CHANGE IF WERE ARCRV AND PRO BIOTICLSARED ONE, APPROACHED STATISTICAL SIGNANCE. SO IT LOOKED LIKE WE WERESEEING R CHANGES IN THE COMPOSITION OF GUT MICROBIOME ATTRIBUTED TO THE TREATMENT WITH PROBIOTICS. WE THEN NTED TO SEE THE THERE WAS CHANGE IN E IMMUNE SYSTEM OASTROINTESTI TRACT SO WE CYTOMETRICLY ASSORTED LEUKOS AND THERE'S 400 G DIFFERENCIA EXPRESSED IN ANIMALS THAT G ARV AND PROBIOTICS COM ANIMWITH ARV ALONE. MANY G KIND OF CLUSTERED GENES WE THINK TO BE EXPRESSED BYIGEN PRESENTINGLLS SO A LOT OF DIFFERENT TY HLA GENE, IL-1 BETA. SO WE WONDERED HER OR NOT THERE WAS INCRTHE FREQUENCY OFUKOSITES WITHIN THE GASTRO STROW INTESTINAL TRACT. SO WE MEASUREDHE FREQUENCY OF APC IN THE TRACT OF ANI THAT GOTARV A PROBI COMPARED TO ANIMALS WITH ARVNE AND YOU SEE A HIGHER FREQUENCY OF APITHIN THE GASTROINTESTIN TRACT O ANIMALS WITH ARV AND PRO BIO THIS WAS NOT ATTRIBUTED TO CREASED EXPRESSIONOF H BY ANY GIVEN ANTIGEN PRESENTING CELLS, MEDIAN FLUORESCENT INTENSY IN ANIMALS WITH ARV D PROBIOTIC COMPARE TO ANIMALS ALONE. NO SIGNIFICANT DIFFERENCE. WIIN THE ANTIGENRESENTING CELLS AN SEE AN INCREASED FREQUENC THE APCs TT KETHIS PRO-INFLAMMATORY -- THIS EFFECTOR CYTOKINE IL-23 WHIS VERYMPORTANT FOR INCTION OF I INTRODUCERS IN T ANIMALS THAT GOT ARV AND PROBIOTICS COMPARED TO ANIMAL WITH ARV ALONE. SO PROBIOTICS WERE CHANGING THE LAE OF INSTRUCTION STROW INTESTINAL ANTIGEN PRESENTING CELLS. WE WONDER WHETHER OR NOT WITH THIS INCR APC FREQUENCY WOD HAVE AN FECT ON FREQUENC T-CELLS THAT MADE 7. SO WE USED MITOGENICMULATION AND AT FREQUENCY OF IL-17 PRODUCG CELLS IN GASTROINTESTINAL TRACT OF SIV UNFECTED, CHRONICALLYFECTED ANIMALS YOU SHEVERY STEREO TYPICAL REDUCTION IN IL-17 FREQUENCIES. AND ANIMALS ARV AND PROBIOTICS COMPARED TO ARCRV A. WE DID NOT SEECONSTITUTIOF THE-17 PRG CD4 POSITIVE T-CELLS WITHIN THE STROINTESTIN TRACT OF ANIMALS WITH A AND PROBIOTS. CAN LOOKT THE ACTUAL FUNCTIONALITY OF THE CELLS THAT ARE MAKING IL-. N MANY OTHER STUDIES THE F POLYFUNCTIONALTY IS THOUGHT TOE A VERY GOOD G FORIALLY NON-PROGRESSIVE NATURE OF HIV INFECTION IN INDIDUALS THOL VIRAL REPLICION WITH IMMUNE SM. SO WE CAN LOOK AT POLYFUNCTIONALTY OF IL-17 ING CELLS. CO-PRODUCTIOSIMULTANEOUSLY OF IL-17, IL-2 SO THESE ARE MAKING THREE FUNCTIONS OR TNFIN PINK AND CELLS MAKING ONLY IL-17 AREURPLE AND YOU CAN SEE INHE ANIMALS WITH ARV ANDIOTICS THERE'S A SIT INCR IN FUNCTIONALITY OF TH- CELL SO WE DIDN'T E INCREASE IN IL-17 INTRODUCING CELLS BUT THE IL-1CELLS THAT WERE THERE WERE LYFUNCTIONAL. TH-1 CELLALSO TRUE AMONG THE SO EVENHOUGH WEIDN'T S SIGNIFICANT INCREASE IN FREQUENCY OF CD4 CELLS M CD4,F WE LOOKL FREQUENCY CD4 POSITIVE T-CELLS WITHIN THE GASTROINTESTINAL TRACT CAN SEE FIVE MONTHS AFTER ADSTRATION OF IRAND PROBIOTICS IN ANIMALS, THESE WEREE TO RECONSTITUTE GASTROINTESTINAL CD4 POSITIVE T-CELL FREQUENCY NEAR HEA LEVELS. SO AND THE ANIMALED THAT THAT HAD RESIDUALVIREMIA IS H THE SQUARE SO THIS ANIMAL WAS VIREMIC. AND PRO BIOTIC HADOWEST OF CD4 POSITIVE T-CELLS IN THE GASTROINTESTINAL T BUT ANY OF THE ANIMALS THAT GOT A ALO AND ALL THESE ANIMALS WE VIRAL LO SUPPRESSED SO FIVE MONTHS AFTER ADMINISTRATION OF ARV AND PROBIOTICS WAS SUFFICIENT IN THESEANIMALS TO RECONSTITUTE C POSITIVE T-CELLS IN TSUE TOEAR HEALTHY LEVEL. THE DEGREE TO WHICH THOSE CD4 + T-CES W RECONSTITUTED NDED TO CORRELATE TH INCREASED FCIES OF ANTIGEN PRESENTING CELLS WITHIN GASTROINTESTINAL TRACT OF ANIMALS WHO HAD ARV AND PROB. IS INCREASE RECONSTITUTION OF CD4 POSITIVECELLSTHIN THE GAINTESTINAL TRACT OF ANIMALS WITH A AN PROBIOTICS TREATMENAS NOT ASSOCIATED WIHESE ANIMALS HAVI HIGHER FREQUENCY POSITIV T-CELLS PRIOATMENT. SORE ABLE TO DO BUY I D'T THINK I DOT KNOWSIS AND YOU CAN SEE BOT ANIMALS GOING TO GET IOTIC COMPARED TOANIMALS THAT WERE GOI NG TO GET ARV ONLY YEE AN EQU AND MASSIVE DEPLN OF CD4 POSITIVE T-CELLS. T ONLY WERE THERE INCREAS LEVELS OF RITUTION OF4 POSITIVE T-CELLS THERE WAS A REDUCTION IMNE ACTIVATI WIROINTESTINALES AND WE MEASURE BYLOOKING AT PRESSION OF KI-67 BY POSITIVE T-CELLS IN T GASTROINTESTINAL TIS AND THE ANIMAL WHO WAS STILL VIREMIC ARV REGIMENIDN'T SUPPRESS VIRAL REPLICATION WAS ONE OF THE HIGHEST STILL LOWER THAN PRACTICALLY ANIMALS GOT ALONE. SO TRYING TO THINK WHYGHT THEY ENHANC RECONSTION OF GUEST STROW INTESTINAL CD4 POSITIVE T-CEL WE THOUGHT ABOUT FIBROSIS. MEARED IT WN ISOLATED LYMPHOIDFOLLICLES BY NOHISTOCHEMISTRY, THESE ANIMALS GOT ARV PROBIOTIC AND IMMUSTOCHEMICAL STAIN FOR FIBRIL NECK TIN WHICH MARKS MYOFIBROSIS. THE BLUE IS HEMOTOXULIN. YOU CAN SEE SIGFICANT RUCED LEVELSF FIBROSIS I THE ISOLAT LYMPHOID FOLLICLE. WHEN YOU DO IMAGESIS YOU SEE THAT WAS STATISTICALLY GNIFICANT WITHIN THE ISOLATE LYMPHOID FOLLICLESF THE AN WHOSE GOTRV PROBIOTS. NEGATI CORRELATED WITH RECONSTITUTION OF CD4 POSITIVE T- SO ARVATED --AND PROOTIC TREAD ANIMALS WITH ER FREQUENCIES OF POSITIVE T-CELLS ALSO REDUCED FIBROSIS WITHIN THE LYMD FOLLICLES. TO SUMMAHIS PART YOU HAVE EN PROBIOTICS ENHANCE IMMUNITY INCREASING ANTIGEN PRESEG CELL FREQUENCY ORNALITY. INSED EXPRESON OF APC ASSOCIATED GENES. INCREASED IL-23 PRODUCTION APC AND INCEDMS TO ENHANCKEY CELLNITY. THERE'S INCREED CD4 POSITIVE LL FUNCTIONALITY INCREASE RETITUTION OF CD4 + TLS, DECREA IMMUNE ACTIVATION WITHIN THE TRACT AND DECREASED OSIS. SO WE'RE FOLLOWING UP ON THESEc UDIES, TRYING TO ESSIVELY LOO AT THE EPITHELIAL BARRIER INTEGRITY, TRYING TO CHARACTERIZE BACT WHICH ADHERE DIRTLY TO THEEPITHELIUM, TRYING TOCHARACTERIZE TH E APC AR INCREASED IN FREQUENCY AND NOW TRYING T THINK ABOUT ADDITIONAL THAPIES THAT MIGHT INCE IL-1 PO CELLREQUENCIES. IRRESPECTIVE DATARONGLY SUGGEST BIOTIC MENTATION OF HEART IN HIV INFECTEDIVIDUALS MAYMPROVE THEIR PROGNOSIS. SO IL CLOSE THERE AND THANK PEOPLED IN THIS WORK. TANTED POSTOC WHO JUST LE WEDNESDAO HAS GONE TO BIGGER AND BETTER THINGS AT ERSITY OF WASHINGTON,KI CHAT. THE STUDIES INVOLVING SAUDI MANGABEES, DONE A GRADUATE STUDENT IN EMORY TY, STUD INVOLVING AFRICAN GREEN MONKEY, YASMEEN (INDISCERNIBLE NG ING103 +APC. IMMUNOHISTOCHEMISTRY DONE IN COLLABORATION WITH JAKE ESTES.THE SEQUENCI NG O T MICROBIOM LABORATION WITJUDY (INDISRNIBLE) AT NHGRI. ALL THE SAMPLES COME FROM (INDIBLE) AT EMORY. R GENE CHIP ANALYSIS DONE INBORATION WITHELLIOT ADAD AT (INDISCERNIE) FLORIDA AND THEY WERE DONATED MICHAEL MILL WHORE US RT INHIBI AND DARIA (INDRNIBLE) WHO GAVEHE INTEGRAT INHIBITORS. NO STUDY WOULD BE POSWITHOUT DEDICATERINA RIAN STAFF ATHER. AND WE HAVE FUNDING FROM T OFFICE OF AIDS RESEARCH AND FINALLY YOU FOR YOUR TIME AND ATTENTI I'LLENTERTAIN ANTIONS. [AUSE] >>OULD I A TO GO TO THE MICROPNE SINCE WE AR TRANTING. >> CAN YOU SAY ANYTHIN ABOUT THE PROBIOTYOU USED IN YOUR STUDIES WITH MACAQUES WHICH THINK ARE OPTIMIZEDOR HUMANS ANDOW MIGHT THEY IMPROVED? >>UTELY. THINK THA, WHEN WE THOUGHT ABOUWHAT TYPES OF PROBIOTICS T USE, THERE ARE HUNDREND HUNDREDS OF STRAINS PROBIOTI AND EVERYBODY THINKS THEIRS IS BEST. SO WE CHOSE ONE U PLACEBO CONTROL TRIALSO SHOW A POSITIT IN S OTHER DISEASES MICROBIAL ANSLOCATION IS IMPLICATED. IN HUMANS ABSOLULY. SO THIS IS R I'M SURE THE FI S IN A ME CONTACT THERE'S ALLOF REASONS E CAN IMPROVE BASED ON THE COMPOSITION OF MICROBIOME WITHINS MAQUES. >> DID YOU LOO AT TGFELSIN BROSIS? >>THE IDEA'S IMPORTANT FORFIBROSIS. THERE ARE STUDIES THAT SIT'S INCR LEV OF TH CEPTOR WHICH IS ABO TO SOP UPGF BUT WE'RE DOI THAT, TRYING TO SEE IF P DECREASE EXPRESSION BIOACTIVITY OF TGF BETA. >> THERE WER BACK TO BACK PAPERS IN I COMPARING ISGs OF THE PATHOGENESIS RESISTANT AND SUSCEPTIBLE NOMAN PRIMATE SPEC THERE APPEARED TO BE A CRITICAL INT AROUND FOUR B TO DISTINGUIS ACUT PHASE CHRONI HAVE YOU HAD OPPORTY TO LOOK BETWEEN TWO WEEKS AND SIX WEEKS AT THE OFPRESSION OR CELLS? -17, 22 AND CD 103 CELLS? >> GREATUESTION. IS WE HAVE NOT BUT 'RE TRYING TOOSE STUDIES NOW. THIS IS ONE OFHE GREAT ADVAES OF THE NON-HUMAN PRIMATES IS WE KNOW THEY'RE FECTED SO WE CAN SCE THEM AT KNOWN T PERIODS AFTER INFECT SO IN A VER PRELIMINARY STUDY OF TWO ANIMALS, ITKS LIKE LOSS103 APC PERCEE LOS OF IL-17 PRODUCERS. THATNS AROUND 2 TO 3 WEEKS POST INFON. >> DO YOU TRON SE GENES PLA A ROLE? ABSOLUTELY. ONE THE I THINKTHAT IS UNDERLYING LOTS O3 POSITIVEPC DFERENT OF PRO INFLAM APC SO BY NUMBERS THE 103 POSITIVE CELLS NOT AFFECTE BY FR OUTNUMBERED BY 1 NEGATIVE C THAT EXPRESS LIKE (INDISIBLE). THERE ANY CORRELA SES IN I PRODUCTION? >> WE T LOOKED E'RE LOOKING AT THAT NOW. >>YOU LOOKEDT THE ROLE OF VITAMIN A DEFICIE E THOUGHT ABTHIS A THERE'S STUDIES IN MICE AT SUGGEST YOU HE A VITAMIN A DECIENT MOUSE HAS AART TIME MAKINGIL-17 PRODUCING CELLS. THE STUDIESARE DO IS YOU GIVING MOM VIN A DEFICT a TH PUP VITAMIN A DICIENT. THE LIVER IS A GREAT STORAGE FA FOR SITE MINUTE A. SO KEEPING ANIMA ON VITAMIN DIET LONG ENOUGHIME IT BECOMES DEFICIENT IS VERY,ERYDIFFICT. SO WE'RE STARTING TOHINK A ING THIREGNANT RHES MACAQUES TO LOOK AT LEVEL OF -17 PN VITAMIN A FICIENT ANIMAL >> ANYO DO CLINICAL TR? THERE ARE MANY PEOPLE DOING CLINICAL TRI PROBIOTICS. >> HERE? >> I DON'TKNOW IF ANYBODY I G ITTHE CLINICALENTER HERE BUT VERAL GROUPS ARE THINKING ABOUT THIS W.>> ANY OTHERION? IF NO OTHE QUESTION JASON, THANK YOU FOR AERY STIMULATING WE'LL LOOK FORWARDO HEARING MORE. >>SHOULDWE ALL GO AND GET PRTIC >> I DON'T KW IF EVERYBODY >> T.