I'M THE DIRECTOR OF THE BLOOD DIVISIONS ANDY RESOURCES AT THE NATIONAL HEART AND LUNG BLOOD INSTITUTE AND IT'S MY PLEASURE TO WELCOME YOU AND WE ARE LOOKING FORWARD TO WHAT WILL BE A VERY EXCITING BLOOD-BRAIN BARRIER CONFERENCE. I HAVE THE AT THE MOMENT OF INTRODUCING MY BOSS TO SAY A FEW WORDS TO YOU AS WELL. BEFORE THAT, I THOUGHT I WOULD GIVE YOU JUST A LITTLE BIT OF PERSPECTIVE ON HOW WE GOT HERE TODAY. IT IS INTERESTING THAT OBVIOUSLY VASCULAR AND NEUROBIOLOGY INTERFACE IN MEDICINE QUITE A LOT. SO IT'S NOT HARD TO THINK ABOUT THE BLOOD-BRAIN BARRIER IN THE TRADITIONAL CONSTRUCTS OF SAY ATRIAL FIBRILLATION GIVING RISE TO EMBOLIC PHENOMENON IN THE BRAIN AND OBVIOUSLY INFECTIOUS ETIOLOGIES, BLOOD BORN BECOMING CENTRAL NERVOUS SYSTEM, ET CETERA. BUT ONE OF THE THINGS THAT REALLY INTRIGUED US AS WE BEGAN TO THINK ABOUT THE INTERFACE BETWEEN BLOOD AND BRAIN WAS FROM KIND OF CULMINATION OF SOME LONG TERM OBSERVATIONS, I THINK, THAT ARE JUST WHERE THE SCIENCE IS STARTING TO CATCH-UP WITH THE CLINICAL SCENARIOS. IN MY EARLY STAGES AND CAREER IN ACADEMIA, I WAS INVOLVED IN THE NATURAL HISTORY STUDY OF TRAUMA BRAIN INJURY IN HOUSTON. IT WAS A LARGE NINDS-FUNDED STUDY WHICH RESULTED IN 3300 BRAIN INJURY PATIENTS BEING RECRUITED BETWEEN BAYOR COLLEGE OF MEDICINE AND UNIVERSITY OF TEXAS OVER A 28-MONTH PERIOD. A PHENOMENON RECRUITAL RATE. AND ONE OF THE OBSERVATIONS WE NOTED BECAUSE MY ROLE IN THAT WAS ON THE COAGULOPATHY SIDE WAS THAT IN PATIENTS WHO SURVIVED THE IMMEDIATE TRAUMATIC BRAIN INJURIES THAT COAGULOPATHY IN LATER SAGES, AFTER 12 HOURS, AFTER THE BLEEDING WAS CONTROLLED, WAS VERY MUCH A COMMON PHENOMENON AND IN FACT, WHEN ONE LOOKED AT THE DATA, PATIENTS WITH POLYTRAUMA PLUS TRAUMATIC BRAIN INJURY HAD PRECISELY THE SAME SORT OF COAGULOPATHY IN SOME CASES ALL THE WAY TO DISSEMINATED INTRAVASCULAR COAGULATION, TO THAT SEEN IN THOSE WITH TRAUMATIC BRAIN INJURY ALONE, COMPARABLY INJURED. SO WE BEGAN IN THAT TIME THINKING ABOUT WELL, OBVIOUSLY SOMETHING FROM BRAIN IS CEDING VASCULAR INJURY STATES ALONG THE ENDOTHELIUM OF THE SYSTEMIC CIRCULATION. WE HYPOTHESIZED THAT BRAIN IN ANIMALS WAS THE CONSTITUENCY FROM THE PLAST INS AND THEREFORE IT MUST BE THAT BRAIN WAS BEING RELEASED INTO THE CIRCULATION AND BEING PRO COREGULAR PATHIC. FAST FORWARD 3 1/2 DECADES, THE MOST RECENT DATA EXISTS EXOSOMES FROM NEURONS AND GLIAL CELLS IN ANIMAL MODELS ARE DOING WHAT WE THOUGHT MIGHT BE HAPPENING DECADES AGO. SIMILARLY, WE ARE STARTING TO SEE, AND THE CHAIR OF OUR FIRST SESSION, WE WERE FORTUNATE ENOUGH TO, JOHN GRIFFIN'S ONE OF OUR GRANTEES, INTERFACE, WE BEGAN DISCUSSING WITH HIM THEIR DATA, THEIR COMBINED DATA, WHICH YOU'LL HEAR ABOUT TODAY, ON REGULATORY ELEMENTS OF VASCULAR INJURY PLAYING A NEWLY-DISCOVERED ROLE IN REGULATORY ELEMENTS IN THE BRAIN ITSELF, SPECIFIC ACTIVATED PROTEIN C, PROTEASE ACTIVATABLE RECEPTORS 1 +. SO, THOSE THINGS STARTED REACHING FOR US INTELLECTUAL CONFLUENT, SAYING THAT THINGS WE FOCUSEDOD MUCH MORE SYSTEMIC SIDE ARE CLEARLY HAVING RELEVANCE MORE BROADLY IN TERMS OF THE BRAIN AND THE INJURY STATES THAT COLLABORATE OR COUNTER OPPOSE EACH OTHER BETWEEN BRAIN AND THE BLOODSTREAM. SO, WE BEGAN DIALOGUES WITH OUR FIRST SESSION CHAIR AND WE REST WITH THE INORDINANT AMOUNT OF WORK AND EFFORT PUT IN BY OCHOCINSKA, IN TODAY'S SESSION AND AGAIN, I WANT TO WELCOME YOU AND WE ARE REALLY EXCITED ABOUT THE DIALOGUE THAT WILL TAKE PLACE OVER THE NEXT TWO DAYS W NO FURTHER ADIEU, IT'S MY PLEASURE TO INTRODUCE MY BOSS, THE DIRECTOR OF THE NATIONAL HEART, LUNG AND BLOOD INSTITUTE, GARY GIBBONS. [ APPLAUSE ] >> A GARY GIBBONS THANK YOU KEITH AND TO YOU. I WANT TO ECHO KEITH'S COMMENTS OF WELCOME. WE APPRECIATE YOUR SHARING THE COLLECTIVE BRAINPOWER THAT IS IN THIS ROOM AS WE CONTEMPLATE WHAT ARE THE SCIENTIFIC OPPORTUNITIES IN THIS VERY INTRIGUING SPACE. PART OF THE ROLE'S OF DIRECTOR OF NHLBI IS THE THAT BECAUSE IT'S THE HEART LUNG, BLOOD, SLEEP RESEARCH PORTFOLIO, I'M CHALLENGED TO ALWAYS BE ACUMENICAL AND EQUIPOISE IN TERMS OF -- I LOVE ALL MY CHILDREN IN THE SCIENTIFIC PORE FOLIO, BUT -- PORTFOLIO -- BUT SOMEONE WHO HAS A CAREER IN VASCULAR BIOLOGY WHEN THERE IS A WORKSHOP THAT WILL PEEKS AND LOOKS AT VASCULATURE, I ADMIT A CERTAIN AMOUNT OF INTRIGUE. BUT THIS IS AN INTRIGUING AREA, ONE THAT WE LIKE TO EXPLORE, IT'S ONE WHERE THERE IS OPPORTUNITIES FOR COLLABORATION AND I DID WANT TO EXPRESS OUR APPRECIATION FOR KEITH AND MARGARET FOR ORGANIZING THIS AND OUR PARTNERS LIKE NCATS AND NCI AND DOD AND OTHERS THAT SHARE INTEREST IN THIS SPACE. I LOVE THAT YOU TALK ABOUT A BLOOD-BRAIN INTERFACE AND THE STRESSING THE INNER COMMUNICATION THAT OCCURS AT A INTRACELLULAR LEVEL, AGAIN SOMETHING THAT I THINK IS A CONCEPT THAT REALLY RESONATES WITH SOMEONE WHO ALWAYS THOUGHT OF A NEUROVASCULAR UNIT IN WHICH OBVIOUSLY A LOT OF FUNCTION IS INFLUENCED BY THAT INTERPLAY AND AS KEITH ALLUDED TO, A LOT OF INNER COMMUNICATION. SO, WE HAVE BEEN ENGAGED IN A STRATEGIC VISIONING PROCESS OVER THE LAST YEAR AND A HALF OR SO, IN WHICH WE HAVE BEEN TRYING TO CHART OUT A FUTURE OF OUR SCIENTIFIC RESEARCH PRIORITIES WITH OUR COMMUNITY AND WE HAVE BEEN VERY INCLUSIVE IN THAT PROCESS, STRATEGIC VISIONING AND THIS WILL PROVIDE US WITH THAT SORT OF ROADMAP IF YOU WILL, AND I THINK THIS SPACE FALLS WITHIN THAT FRAMEWORK. IT HAS FOUR FUNDAMENTAL GOALS OF ADVANCING OUR UNDERSTANDING OF HUMAN BIOLOGY TO APPRECIATE THE NORMAL HUMAN BIOLOGICAL STATE AND APPRECIATE IN THIS CONTEXT, WHAT THE BLOOD-BRAIN INTERFACE IS DOING ON A MOMENT TO MOMENT BASIS TO MAINTAIN NORMAL HOMEOSTASIS. AND WE ARE ENVISIONING A TIME IN THE NEXT 5-10 YEARS WHERE WE COULD APPRECIATE WHAT IS IT ABOUT THE PARTICULAR CHARACTERISTICS OF THAT END THILL YUM AT THAT BLOOD-BRAIN INTERFACE THAT DISTINGUISHES IT FROM ENDOTHELIUM THROUGHOUT THE BODY? WHAT IS THE ADDRESS-OME OF THE ENDOTHELIUM THAT GIVES IT THESE SPECIAL CHARACTERISTICS IN EACH DIFFERENT TISSUE BED AND HOW DOES THAT PLAY A ROLE IN THE COMMUNICATION? ARE THE EXOSOMES INDEED HAVE THAT AS PART OF HOMING AND MOVING PROCESSES AND HOW CAN WE CHARACTERIZE THAT BETTER? OUR GOAL TOO IS AGAIN FOCUSED MORE ON REDUCING THE BURDEN OF HUMAN DISEASE. SO, STARTING TO UNDERSTAND HOW THESE INTERFACES PLAY A ROLE IN DISEASE POTENTIALLY AS MEANS OF INTERVENTIONS. SO AGAIN, WE ARE INTRIGUED BY WHAT YOU'LL BE EXPLORING IN TERMS OF WHAT WE ARE LEARNING ABOUT THE AFFECTS OF BRAIN INJURIES AS KEITH MENTIONED, OR NEURODEGENERATION IN THOSE SIGNALS THAT GO FROM BRAIN ACROSS THAT INTERFACE AND BIDIRECTION ALLEY. THE THIRD GOAL RELATES TO ACCELERATING TRANSLATION AND HOW WE CAN TAKE FUNDAMENTAL DISCOVERY AND TURN IT TO ENHANCEMENTS IN HUMAN HEALTH AND THE NOTION THAT WE MIGHT BE ABLE TO CHARACTERIZE THE ENTIRE IN SITU PROTEOME OF AN END THILL YUM ACROSS AND THROUGHOUT THE BODY, ADVANCES IN NANOTECHNOLOGY THEY MAY ENABLE US TO INDEED MODULATE AND LEVERAGE THESE INTERFACES. AND THEN FINALLY GOING FORWARD RELATING TO OUR JOB AS STEWARDS AND TRAINED IN NEXT GENERATION AND PROMOTING COLLABORATIVE RESEARCH OPPORTUNITIES THAT DRAW INDIVIDUALES FROM VARIOUS DISCIPLINES AND EXPERTISE AND AGAIN I THINK THIS WORKSHOP IS GERMANE IN TRYING TO WORK AT THAT INTERFACE. I MUST ACKNOWLEDGE THAT AS LONG AS WE ARE NOT BEING RECORDED HERE, THAT SOMETIMES IT IS A LITTLE FRUSTRATING THAT CONGRESS IN ITS WISDOM CREATED 27ICs AND CENTERS AND WE ALL HAVE OUR LITTLE SILOED DOMAINS BUT IT IS GREAT TO HAVE WORK AT THESE INTERFACES AND BRING COLLABORATIONS AMONG THE ICs AS WELL AS AMONG YOU ALL BECAUSE WE HAVE COMPLEX PROBLEMS TO SOLVE AND WE CAN ONLY DO IT EFFECTIVELY IF WE LEVERAGE THE COLLECTIVE INTELLIGENCE OF ALL THE FOLKS IN THIS ROOM FROM A VARIETY OF DISCIPLINES. OH, MY GOODNESS! [ LAUGHS ] CLEARLY THERE IS RETRIBUTION HERE. THE SENATE IS SUPPOSED TO MARK UP OUR BUDGET LATER TODAY AND SO, CLEARLIY THERE IS SOME EXPENSES WE NEED TO -- [ LAUGHS ] DEFER MAINTENANCE. BUT SERIOUSLY, AGAIN, JUST TO WRAP UP, WE ARE EXCITED ABOUT THE TOPICS HERE TODAY AND WE SEE THIS AS A GREAT OPPORTUNITY FOR US TO EXPLORE INTRIGUING SPACE TOGETHER. WE LOOK FORWARD TO THE OUTPUT AND HOW IT MAY BE ABLE TO GUIDE HOW WE MAKE INVESTMENTS IN ADVANCING SCIENCE IN THE FUTURE. SO THANK YOU AGAIN FOR YOUR ATTENDANCE AND YOUR CONTRIBUTIONS. [ APPLAUSE ] >> GOOD MORNING, EVERYONE. THANK YOU VERY MUCH. I'D LIKE TO EXTEND MY THANKS TO DR. GIBBONS AND DR. HOOTS FOR THEIR INTRODUCTORY REMARKS AND ALSO FOR THEIR INTEREST IN THE WORKSHOP AND PARTICIPATION IN THESE ACTIVITIES. OF COURSE I LIKE TO THANK ALL THE SPEAKERS FOR THEIR TIME AND EFFORT AND PLANNING THIS WORKSHOP. SO WITH THAT, I WANTED TO JUST VERY BRIEFLY REMIND EVERYONE ABOUT THE PURPOSE AND THE GOALS OF THE WORKSHOP. SO AS DR. GIBBONS MENTIONED, THIS IS AN INTERFACE. THAT'S WHAT WE ARE HOPING FOR. YOU MAY FIND FOR INSTANCE IN VARIOUS MEETINGS CANCER-FOCUSED MEETINGS, BLOOD SCIENCES, HERE WE WANT TO BRING THE VARIOUS GROUPS TOGETHER TO WORK TOGETHER ON THE COMMON GOAL AND THAT GOAL BEING THE PATIENT. SO IT IS REALLY CLINICAL FOCUSED WORKSHOP WITH A TRANSLATION IN MIND TO THINK ABOUT WHAT ARE THE NEXT STEPS, WHAT ARE THE TECHNOLOGIES THAT ARE MOST READY FOR TRANSALATION. SO THAT IS WHY WE TRIED TO GROUP THE VARIOUS SESSIONS ACCORDING TO THEMES. HOWEVER, ALL OF THEM HAVE COMMONALTIES. SO THERE ARE SPEAKERS THAT YOU'LL FIND TALKING ABOUT EXOSOMES IN THE BLOOD SCIENCES SESSION. YOU WILL FIND OTHER SPEAKERS TALKING ABOUT TRAUMATIC BRAIN INJURY IN EXOSOME SESSIONS AND FOR CANCER, TARGETED DELIVERY. OF COURSE THAT IS RELATED TO BOTH BLOOD SCIENCES AS PART OF THE CIRCULATORY SYSTEM BUT ALSO THE BRAIN COMPONENTS. AND PERHAPS ALSO THE BIOMARKER SPACE. SO, TO DETERMINE THE PROCESS THAT IT TAKES FOR NEURODEGENERATION TO OCCUR OR FOLLOWING INJURY TO THE BRAIN. AND SO, AT THE END OF THE DAY TODAY, WE WILL ALSO HAVE AN OPEN MIC DISCUSSION PANEL WHERE EACH OF THE PANELISTS FROM THE BEGINNING OF THE SESSION WILL HAVE AN OPPORTUNITY TO SIT HERE UPFRONT AND ADDRESS ANY OF THE QUESTIONS AND KEY CHALLENGES THAT THEY HAVE IDENTIFIED IN THEIR SPACE. SO, ALONG WITH YOUR PROGRAM BOOK LITTLE, YOU HAVE A HANDOUT FOR THE KEY CHALLENGEOOSE - BOOKLETS. -- SO FEEL FREE TO READ THROUGH THAT AND SORT OF PREPARE SOME QUESTIONS YOU MAY HAVE OR ADDITIONAL RECOMMENDATIONS. BECAUSE I'LL RE-READ THE GOALS OF THE WORKSHOP. TO BRING TOGETHER EXPERTS TO EXAMINE THE KEY CHALLENGES AND PROPOSED RECOMMENDATIONS WITH THE INTERFACE BETWEEN THE CIRCULATORY SYSTEM AND THE BRAIN, AND ADDITIONALLY ATTENDEES WILL HAVE AN OPPORTUNITY TO GENERATE THE SET OF RECOMMENDATIONS. WE ARE LOOKING FOR YOU TO GIVE US SUGGESTIONS HOW BEST TO PROCEED GOING FORWARD. GOING BACK TO THE PATIENT, I WOULD LIKE TO TAKE AN OPPORTUNITY NOW TO INTRODUCE THE KEYNOTE SPEAKER BECAUSE HE ACTUALLY HAD A STUDY IN HUMAN DEMONSTRATING THE DELIVERY THROUGH THE BLOOD-BRAIN BARRIER. SO HIS TALK IS INITIAL EXPERIENCE IN THE PILOT STUDY OF BLOOD-BRAIN BARRIER OPENING FOR CHEMO DRUG DELIVER TOW BRAIN TUMORS BY MRI-GUIDED FOCUS ULTRASOUND, TODD MAINPRIZE HEAD OF THE DIVISION OF SURGERY AT SUNNYBROOK HEALTH SCIENCE CENTER. SO HIS TALK WILL DEMONSTRATE WHY WE SHOULD FOCUS ON BASIC SCIENCE AND DISCOVERY AND ABOUT THE PATIENT AND HOW TO GET THERE TOGETHER AS WE DISCUSS THE CHALLENGES AND OPPORTUNITIES IN THE SPACE. THANK YOU. >> TODD MAINPRIZE: THANK YOU VERY MUCH FOR INVITING ME TO SPEAK HERE. THIS IS A WONDERFUL PROGRAM. LOOKING THROUGH ALL OF THE TALKS THAT ARE GOING TO HAPPEN OVER THE NEXT TWO DAYS, THIS WILL BE ONE OF THE SIMPLEST ONES THAT YOU HEAR FOR SURE. VERY CLINICALLY FOCUSED. SO, I JUST WANTED TO DISCUSS A LITTLE BIT ABOUT HOW THE ULTRASOUND SYSTEM THAT WE USE WORKS. HOW WE CAN DISRUPT THE BLOOD-BRAIN BARRIER AND POTENTIAL APPLICATIONS AND ADDITIONAL TRIALS WE ARE GOING TO BE STARTING SOON UTILIZING OUR ABILITY TO OPEN THE BLOOD-BRAIN BARRIER. SO, IT'S NOT NEWS THAT ULTRASOUND HAS BEEN USED IN MEDICAL APPLICATIONS FOR CENTURY. MOST OF THE DIAGNOSTIC TOOLS THAT WE USE, USE POWER OF LESS THAN ONE WAT PER SQUARE CENTIMETER. SO VERY LOW POWER. LOWER FREQUENCY AND HIGHER INTENSITY ULTRASOUND WAVES CAN BE USED THERAPEUTICALLY. WE CAN DEVELOP HEATING WITHIN TISSUES USING ULTRASOUND FOR OBLATION PURPOSES IF NECESSARY. THERAPEUTIC USES IN THE BRAIN HAVE BEEN USED SINCE THE 50s AND INTERESTINGLY, A NAME WELL-KNOWN TO ANY NEUROSURGEON, LEKSELL, DEVELOPED RADIO SURGERY FIRST WANTED TO USE HIS SYSTEM WITH ULTRASOUND BUT THE SKULL WAS TOO MUCH OF A BARRIER AND HE CHANGED TO IONIZING RADIATION. IN THE 1950s, WE COULD USE ULTRASOUND TO DISRUPT BLOOD-BRAIN BARRIER TO HEAT UP THINGS BUT IT REQUIRED A CRANIECTOMY REMOVAL OF PORTION OF THE SKULL PRIOR TO ULTRASOUND APPLICATION. ESSENTIALLY, THE ULTRASOUND WAVES CAUSED COMPRESSIVE AND RARE WAVES MOVING THROUGH TISSUE AND THERE IS TWO MAIN BIOLOGICAL AFFECTS. THERMAL SO IT CAN BE A DEPOSITION OF HEAT AS THE WAVES ARE ATTENUATED AND THE MECHANICAL DISRUPTION OF THE WAVES THEMSELVES. BOTH CAN BE USED FOR THERAPEUTIC USAGES. WHEN WE HEAT UP THE BRAIN AND CAUSE LESIONS, WE ARE USUALLY TARGETING ABOVE 42 DEGREES, AND FOR EVERY DEGREE THAT WE RAISE IT, WE CAN CAUSE MORE DAMAGE AND HAVE TO USE THE ULTRASOUND LESS TIMES. AND WE HAVE BEEN USING THIS QUITE A BIT IN OUR CENTER FOR TREATMENT OF ESSENTIAL TREMOR AND SOON-TO-BE PARKINSON'S DISEASE BY MAKING SMALL LESIONS IN AREAS OF THE BRAIN THAT CAN HELP ATTENUATE THE TREMOR SITUATION. WE PROBABLY HAVE DONE ABOUT 30 PATIENTS WITH THAT THERMAL AFFECT. NOW, THERE ARE MECHANICAL AFFECTS AS WELL WITH THE ULTRASOUND. AND WHAT CAN YOU SEE IS THE BUBBLES WITH THE ULTRASOUND WAVES WILL OSCILLATE IN SIZE, BUT THERE IS A PROBLEM WITH THAT. ANY TISSUE HAS SMALL MICROBUBBLES IN IT AND THIS NON ASSESSORRA, STABLE OSCILLATION IS THE GOAL FOR WHAT WE ARE USING WITH THE BLOOD-BRAIN BARRIER DISRUPTION, HOWEVER, IT IS UNSTABLE. SO YOU CAN IMAGINE THESE BUBBLES OSCILLATING AND IF THEY DO THAT, WE CAN CAUSE DAMAGE. SO THIS IS WHERE THE PROBLEM COMES IN AND WE HAVE WAYS TO GET AROUND THAT USING HYDROPHONES, AND I'LL SHOW YOU SOME PICTURES OF THAT IN A CLINICAL SETTING. SO, HOW ARE WE GOING TO USE THIS TO TREAT GLIOBLASTOMASSA AND WHY SHOULD WE PICK THAT AS A POTENTIAL TUMOR TO START WITH? GLIOBLASTOMA FIRST OF ALL, IS PROBABLY THE MOST MALIGNANT BRAIN TUMOR THAT THERE IS, EXTREMELY POOR PROGNOSIS. SURGERY, RADIATION, CHEMOTHERAPY, HAS ESSENTIALLY A 14-MONTH MEDIAN SURVIVAL IT'S NOT AN IDEA OF WHEN IT IS -- OR IF IT IS GOING TO OCCUR, IT IS WHEN. AND OVER 90% OF REOCCURRENCES TAKE PLACE WHEREIN TWO CENTIMETERS OF THE ORIGINAL ENHANCING PART OF THE TUMOR. THERAPEUTIC IMPROVEMENTS HAVE VERY MUCH LAGGED BEHIND CELLULAR AND MOLECULAR UNDERSTANDINGS. OUR UNDERSTANDINGS OF THE GENETIC CHANGES, THE CELLULAR DISRUPTIONS OF SIGNALING PATHWAYS, IS ACTUALLY GETTING PRETTY GOOD WITH THIS TUMOR BUT WE HAVE BEEN UNABLE TO REALLY MAKE AN AFFECT IN SURVIVAL. PART OF THAT IS THOUGHT TO BE DUE TO OUR NEED FOR BETTER DRUG DELIVERY MECHANISMS. SO SHEAR A PICTURE OF A GLIOBLASTOMA IN THE LEFT FRONTAL LOBE. THIS IS AN ENHANCING MRI SCAN SO GAD LIN YUM WAS GIVEN. AND THAT WOULD BE REPRESENTED HERE WHERE ALMOST ALL THE CELLS ARE TUMOR CELLS. THE FURTHER YOU GO OUT, SO IF YOU LOOK AT A DIFFERENT TYPE OF MRI SEQUENCE OF FLAIR, YOU CAN SEE CHANGES DISTAL TO THE TUMOR. NOW THIS DOESN'T ENHANCE SO THAT HASN'T ATTACKED THE BLOOD-BRAIN BANIER. 1-10 CELLS ARE TUMOR. FURTHER OUT 100 AND EVEN ON THE CONTRA LATERAL HEMISPHERE WE HAVE KNOWN SINCE THE 80s PEOPLE HAVE DONE BIOPSIES THAT THERE ARE TUMOR CELLS IN THE COULDN'TRA LATERAL HEMISPHERE OF SOMEBODY WITH A GLIOBLASTOMA. SO TRULY A BRAIN-WIDE DISEASE. SO JUST TAKING OUT THIS PART OF THE ENHANCING TUMOR WHERE THE BLOOD-BRAIN BARRIER IS NOT ACTIVE, WON'T HELP SO MUCH AS THERE IS TUMOR CELLS HERE. AND THOSE ARE THE ONES THAT COME BACK AND CAUSE REOCCURRENCE OF THE TUMOR AND THEY ARE PROTECTED BY THE BLOOD-BRAIN BARRIER AS YOU CAN TELL BY THE MRI SCAN. I DON'T HAVE TO TELL THIS GROUP ABOUT THE BLOOD-BRAIN BARRIER. YOU KNOW WAY MORE ABOUT IT THAN I DO I'M SURE. SO THE ENDOTHELIAL CELLS THE CAPILLARIES STUCK TOGETHER MAKE UP VARIOUS PROTEINS, ET CETERA, DOESN'T ALLOW FOR THE PARACELLULAR DIFFUSION. SO THERE HAS BEEN WAYS TO CIRCUMVENT THE BLOOD-BRAIN BARRIER. DIRECT APPLICATION. CHEMOTHERAPY TO THE BRAIN, CONVENTION ENHANCED DELIVERY, PUTTING CATHETERS IN THE INTROSIS YUM OF THE BRAIN AND SLOWLY PUSH THE CHEMOTHERAPY DIRECTLY INTO THE PRANK MA. S ON MOTTIC DISRUPTION. INTERARTERIAL CAROTID DELIVERY AND INTERNASAL APPLICATIONS. SOME OF THESE HAVE BEEN SHOWN TO INCREASE SURVIVAL IN BRAIN TUMOR PATIENTS WHEN THE CHEMOTHERAPY HAS BEEN GIVEN. SO, HOW DO WE DO IT? THERE HERE THIS HAS 1,024 ULTRASONIC TRANSDUCERS THAT ARE MOBILE AND WE CAN MOVE THEM AND AIM THEM. THE PATIENT'S HEAD IS PUT INTO A STEREO TACTIC HEAD FRAME SO SCREWS INTO THE SKULL TO IMMOBILIZE IT. THEY ARE PLACED INTO THE HEAD FRAME AND THERE IS A WATER BATH TO COOL IT DOWN. THE WATER BATH IS NECESSARY IF WE WERE HEATING UP THE BRAIN BUT USING THE BLOOD BRAIN BARRIER DISRUPTION WE USE MUCH LESS POWER, ROUGHLY ABOUT 9 WATS AND WE PROBABLY DON'T EVEN NEED TO COOL THE PATIENT'S HEAD BUT THIS IS THE WAY THE MACHINE IS MADE FOR THE OBLATION PURPOSES. SO, WHAT WE DO IS WE INJECT MICROBUBBLES INTO THE CIRCULATION. THESE ARE THE ROUTINE MICROBUBBLES USED IN RADIOLOGICAL INVESTIGATIONS OF BLOOD VESSELS SO IT'S A CONTRAST AGENT. AND THE ULTRASOUND IS THEN STEERED TO AN AREA THAT WE WANT TO OPEN THE BLOOD-BRAIN BARRIER AND THE ULTRASOUND HITS THESE MICROBUBBLES, THESE MICROBUBBLES OSCILLATE IN SIZE AND THE BLOOD-BRAIN BARRIER IS DISRUPTED. WE THINK IT IS A MECHANICAL DISRUPTION OF THE TIGHT JUNCTIONS ALLOWING FOR IMPROVE PARACELLULAR TRANSPORT, ALTHOUGH IT HAS BEEN SHOWN THAT IT WILL INCREASE THE AMOUNT OF TRANSCELLULAR TRANSPORT AS WELL. OUR COLLABORATORS AT SUNNYBROOK PUBLISHED THIS PAPER AND YOU CAN SEE SOME GOLD STAINING OF THE TIGHT JUNCTION PROTEINS HERE AND AFTER ULTRASOUND, THERE ARE NONE AND YOU CAN SEE THE INCREASED PARACELLULAR TRANSPORT OF FLUID HERE. THEN, THE TIGHT JUNCTIONS START REFORMING AGAIN AFTER ABOUT 12-24 HOURS. AND YOU CAN SEE THE DECREASE IN EXPRESSION OF THE PROTEINS OF THE VARIOUS PROTEINS OF THE TIGHT JUNCTIONS OCCLUDED AND CLOUD EN, AND THEN THEY COME BACK AFTER MORE NORMAL EXPRESSIONS AFTER 6-24 HOURS. AND HERE IS THE MOUSE MODEL SHOWING INCREASED UPTAKE OF GATLIN YUM SHOWING THE OPENING OF THE BLOOD-BRAIN BARRIER IN A MOUSE. THERE HAS BEEN MULTIPLE SAFETY STUDIES DONE UP TO RHESUS MONKEYS AND OTHER THAN THE SMALL A LITTLE BIT OF TREPIDATION OF A FEW RED BLOOD CELLS IN THE AREA OF THE ULTRASONIC APPLICATION, THERE IS NO DAMAGE TO THE EXONS TO THE NEURONS, ET CETERA. IN FACT, REPEATED BLOOD-BRAIN BARRIER DISRUPTIONS IN THE LATERAL BODY IN THE VISUAL CORTEX OF THE MORNINGIES WERE DONE MULTIPLE TIMES AND THERE WERE NO LOSS OF FUNCTION IN THE MONKEYS IN THE VARIOUS VISUAL SYSTEMS. SO WE HAD THIS BLOOD-BRAIN BARRIER PROTOCOL, BEEN TRYING TO GET IT GOING SINCE 2013. THERE ARE INITIAL ISSUES WITH CHEMOTHERAPY AVAILABILITY. IT WAS OPENED IN AUGUST AND WE RECRUITED OUR FIRST PATIENT IN NOVEMBER. AND SO, OUR WORK FLOW FOR THIS STUDY IS A TWO-DAY WORK FLOW. THE PATIENT ON DAY ONE HAS GIVEN A CAELYX, DOCKSY RUBE SIN, WE PICKED IT FOR TWO REASONS. LONG HALF-LIFE IN THE BLOOD AND THE FORM WHICH ALLOWS FOR LESS SYSTEMIC TOXICITY. AND VERY ACTIVE IN GLIOBLASTOMA. SO IT'S A GOOD CHEMOTHERAPY THEY MAY HAVE GOOD ACTIVATION IN PATIENTS. SO THE HEAD IS SHAVED AND THE HEAD FRAME IS PLACED AND THE PATIENT GOES INTO THE HELMET AND AFTER ABOUT AN HOUR AFTER THE IMFUSION WE SON I KATE WITH MICROBUBBLES TWO LESIONS. ONE IN THE TUMOR AND ONE ADJACENT IN THE NORMAL BRAIN BECAUSE WE ARE VERY INTERESTED IN THAT AS WELL. AND THEN ONCE WE VERIFY THAT IT IS OPEN, THE PATIENT IS ADMITTED TO HOSPITAL. ON DAY 2, SHE IS TAKEN TO THE OPERATING ROOM AND WE RESECT THE TUMORS WE NORMALLY WOULD BUT MAKE SURE THE SAMPLES WHERE WE OPENED UP THE BLOOD-BRAIN BARRIER ARE TAKEN FOR BIOCHEMICAL ANALYSIS AND TWO REPRESENTATIVE NON SON INDICATED AREAS. SO A SMALL AREA OF NORMAL TISSUE ON THE TISSUE ON THE WAY TO THE TOMBURE AND A NON SON I INDICATED TISSUE AT THE TUMOR. SO THE ULTRASONIC PARAMETERS THAT WE USED, WE DID A GRADE OF NINE, THREE MILLIMETER DOTS. EACH DOT WOULD BE GIVEN 2.6 MILLISECONDS OF SON INDICATION, 30.4 MILLISECONDS OFF AND THEN THIS WAS REPEATED FOR 300 MILLISECONDS AND THEN IT WOULD STEER TO THE NEXT DOT AND REPEAT THAT FOR 300 MILLISECONDS. AND THEN ONCE IT FINISHED DOT 9 IT WOULD GO TO DOT ONE AND THAT WOULD BE FOR 50 MILLISECONDS. HERE IS THE PATIENT'S TUMOR. IT'S A LARGE GLIOMA WITH A FOCUS OF TRANSFORMATION WITHIN THE TUMOR. SO WE SON I INDICATED IN AN AREA THAT WAS GOING TO BE RESECTED SO THESE LINES ARE SAFETY LINES. WE ARE LOOKING AT ACOUSTIC SCORE. SO IF THESE WAVES GET ABOVE THIS LINE, THAT INDICATES THERE IS A HIGH RISK FOR THAT CAVITATION TO TAKE PLACE. WE WANT TO STAY BELOW THAT. AND THIS MACHINE HAS AN AUTOMATIC SAFETY SHUTOFF IF THERE IS A HIGH RISK OF CAF IITATION. SO, WHAT WE DID WAS INJECTED THE BUBBLES INTO THE ARM AND THEN STARTED SON INDICATING. IT LOOKS LIKE IT TAKES 28 SECONDS FOR THE BUBBLES FROM BLOOD TO BRAIN. SO THE NEXT SON I INDICATION THAT WE REPEATED 9 WATS FOR 50 SECONDS, WE DELAYED TURNING ON THE MACHINE FOR ABOUT 28 SECONDS YOU CAN SEE A GRID OF 9 BLACK DOTS. THIS IS USING THE T2 STAR PROTOCOL WHICH LOOKS AT ANYMORE CHANGES IN BLOOD VESSELS OR ESPECIALLY SMALL AMOUNT OF RED BLOOD CELLS THAT ARE EXTRA VASCULAR NOW. SO WE SEE THAT. WE DIDN'T WANT TO KEEP GIVING HER GAD LID YUM AFTER EVERSON INDICATION. SO THEN WE STEERED TO THE NORMAL BRAIN AND REPEATED THAT AND WE ONLY DID IT ONCE. 9 WATS, 50 SECONDS. AND AGAIN YOU CAN SEE THIS GRID OF 9 DOTS SHE WAS TUMOR RESECTED AND THEN SAMPLES OF NON SON INDICATED AREAS TO MEASURE CHANGES. SO HISTOLOGICAL ANALYSIS DIDN'T SHOW ANY INCREASED HEMORRHAGE T LOOKED LIKE A POSTOPERATIVE SAMPLE OF THE AREA WE DID BIOPSIES ON. WE ARE STILL WAITING QUANTITATIVE ANALYSIS FOR MORE PATIENTS TO BE ACCRUED T LOOKS LIKE A 2-3 FOLD INCREASE IN THE DOCKSY RUBE SIN DELIVERY TO THE AREAS WE OPENED WE ARE AT THE FINAL PHASES OF APPROVAL TO START OPENING UP THE BLOOD-BRAIN BARRIER IN PATIENTS WITH EARLY ALZHEIMER'S DISEASE. THERE IS PRECLINICAL DATA THAT CAME OUT OF OUR HOSPITAL MICE HAD THE BLOOD-BRAIN BARRIER OPENED WITH THE DELIVERY OF AN ANTI--BETA AMYLOID ANTIBODY AND THERE IS SIGNIFICANT DECREASE IN THE PLAQUES. INTERESTINGLY, WHEN THEY LOOKED AT SOME OF THE CONTROL GROUPS, THERE IS ALSO IMPROVEMENT IN THE MICE THAT WERE SOMICATION COMPARED TO THE NON SOMICATION MICE. AND THEN NOT GIVING THEM THE ANTI-AMYLOID ANTIBODIES AND AGAIN RECAPITULATE THE TEST. SO HERE IS WHAT THE SON INDICATION LOOKED LIKE OPENING UP THE BLOOD BRAIN BARRIER THERE IS A SIGNIFICANT IMPROVEMENT IN A COGNITIVE TEST IN THESE TRANSGENIC MICE AND THERE IS ALSO A SIGNIFICANT INCREASE IN NEW NEURONS BEING FORMED IN THE HYPOCAMP I AS WELL AS A SIGNIFICANT IN PROVEMENT IN THE NUMBER OF PLAQUES. SO WE ARE MOVING FORWARD AND PLANNING TO START IN THE NEW YEAR. I WANT TO THANK EVERYBODY INVOLVED IN THE STUDY WE HAVE DONE. DR. HYNYNEN IS THE PHYSICIST AND INSIGHT EC IS THE COMPANY THAT USED HIS RESEARCH AND MADED HELMET AND THEN THE FOCUSED UL TRA SOUND FOUNDATION, THE FUNDERS OF THE STUDY. WE FINISHED THE FIRST PATIENT AND OUR STUDY IS ACTUALLY ON HOLD AGAIN BECAUSE AS YOU CAN IMAGINE WITH NEW DEVELOPMENTS IN THE HELMET, THEY MADE SOME MINOR MODIFICATIONS TO THE HELMET AND NOW WE HAVE TO GO BACK THROUGH APPROVAL AGAIN. SO WE ARE ON HOLD UNTIL WE GET REAPPROVAL. THANK YOU. [ APPLAUSE ] ANY QUESTIONS? [ OFF MIC ] >> SO EITHER MICROGLIA ACTIVATION AND GENERATION OF NEW ANTICIPATE BODIES BECAUSE THERE IS NOW EXPOSURE TO UNSEEN ANTIGENS, OR PRE-EXISTING CIRCULATION OF ANTIBODIES AGAINST THE BETA 42 OR SOMETHING LIKE THAT. I'M NOT SURE. [ OFF MIC ] >> SO, INTERARTERIAL -- BEEN USED BEFORE. TO THE IS TRANSIENT, ET CETERA. DIFFERENCE NON SELECTIVE N A BRAIN TUMOR PATIENT, NON SELECT ACTIVITY MAY BE GOOD. BUT I THINK THAT AS A NEUROSURGEON, I THINK THAT WE COULD MAKE AT LEAST AN IMPACT IF WE COULD CONTROL THAT TWO CENTIMETER RIM AND SO PAINTING THAT WOULD BE BETTER THAN JUST SHOCKING THE WHOLE BRAIN AND DELIVERING FAIRLY TOXIC CHEMOTHERAPY WIDELY THROUGHOUT THE BOTH HEMISPHERES. INTERARTERIAL DISRUPTION OF BLOOD-BRAIN BARRIER IS ALSO HOT TOPIC. I THINK THAT WE HAVE -- WHILE I'M HERE SOMEBODY IS TALKING AT MY INSTITUTION ABOUT THAT FROM McGILL. BUT, THIS IS SELECTIVE. SO ESPECIALLY WHEN IT COMES ALZHEIMER'S DISEASE AND OTHER TYPES OF NEUROSURGICAL PROBLEMS, SELECT ACTIVITY OF OPENING IS INTERESTING. I JUST RECRUITED A PSYCHIATRIST WITH AN INTERESTED IN NEUROSURGERY. WHICH IS A RARE PHENOTYPE. PARAMETICALLY OPPOSED PEOPLE. HE IS GOING TO PUSH FORWARD THE PSYCHOSURGERY OF OPENING UP THE BLOOD-BRAIN BARRIER TO DELIVERING NEW CHEMOTHERAPY 80s, FOR EXAMPLE, DEPRESSION AREA 25 OF THE CINGULATE GYREEROUS AND NONRESPONDER SEEMS TO BE IMPORTANT AND THERE SEEMS TO BE WORK WE CAN DO THERE. [ OFF MIC ] >> SO WHAT I MEANT WAS, USING THE ULTRASOUND TECHNIQUE -- HE JUST ASKED ABOUT BLOOD-BRAIN BARRIER BEING NON SELECTIVE. WHAT I MEANT WAS SELECT THE AREAS OF THE BRAIN WHICH WE CAN OPEN. SO USING ULTRASOUND, WE CAN PICK AN AREA ONE MILLIMETER IN SIZE AND OPEN IT OR WE CAN PAINT LARGE TRACKS. >> HOW LARGE? >> SO RIGHT NOW WE -- IT WOULD TAKE MULTIPLE SHOTS BUT WE CAN OPEN UP TWO CENTIMETER RIM AROUND THE INTERSECTION CAVITY. >> AND YOUR FEELING IS A TWO CENTIMETER RIM IN A MALIGNANT BRAIN TUMOR IS ADEQUATE TO HAVE THERAPEUTIC IMPACT? >> BY NO MEANS OF CARE, BUT I CAN TELL YOU I THINK OUR CHANCES OF CURING THIS WITH CURRENT DRUGS IS LOW BECAUSE IN THE 1940s N JAPAN, THEY WOULD DO HEMISPHERECTOMIES FOR PATIENTS WITH GLYCOMA. IT WOULD COME BACK NINE MONTHS LATER IN THE CONTRA LATERAL SIDE. SO BLOOD-BRAIN BARRIER DISRUPTION IN MY VIEW, IS AN AID TO THE CURRENT THERAPEUTIC REGIMEN. WE NEED TO DO BETTER IN FINDING NEW CHEMOTHERAPIES. >> I THINK THAT IS THE KEY ISSUE. YOU CAN BE AS SELECTIVE WITH NANOTOL AS YOU WANT. JUST DEPENDS ON WHICH VESSEL YOU CAPTURE. >> SURE. >> SO YOU CAN DO VERY SMALL AREAS OR VERY LARGE AREAS. WE HAVE DONE IT 7000 TIMES. THE ONLY TIME WE HAVE COMPLETE AND DURABLE RESPONSES IS WITH MALIGNANT BRAIN TUMORS WHETHER PRIMARY METASTATIC, IS IF WE OPEN THE BLOOD-BRAIN BARRIER IN THE WHOLE BRAIN. EVEN FOUR CENTIMETERS FROM ANY T2 OR FLAIR ABNORMALITY DR. SIBBER GILD HAS SHOWN THAT PATHOLOGIST SAYS WHEN HE BIOPSIES FOUR CENTIMETERS THE TISSUE IS NORMAL. BUT IF HE TAKES THAT TISSUE AND PUTS IT IN THE LABORATORY, THEY ALL GROW OUT TUMORS. >> YES. AND WE HAVE KNOWN THE CONTRA LATERAL HEMISPHERE. WE HAVE KNOWN THAT SINCE THE 40s. >> THE 30s. >> THE PROBABLY BEFORE THAT. >> EXON TOPIC FOR DISCUSSION BECAUSE WE ARE GOING TO HAVE THE OPEN MIC DISCUSSIONS SO ADDITIONAL -- THANK YOU VERY MUCH DR. MAIN PRIZE. THANK YOU. [ APPLAUSE ] AND PLEASE KEEP YOUR QUESTIONS FOR THE DISCUSSION SESSION. THERE WILL BE MORE TIME. NOT TO WORRY. AND I'D LIKE TO PROCEED FORWARD WITH THE BLOOD SCIENCE SESSION WHICH IS OUR FIRST SESSION OF THE DAY PRIOR TO THE BREAK AND THIS ONE IS BEING CHAIRED BY BERISLAV ZLOKOVIC AND I'LL HAVE HIM COME UP AND INTRODUCE THE SESSION. THANK YOU. >> BERISLAV ZLOKOVIC: THANK YOU VERY MUCH MARGARET AND KEITH FOR PUTTING THIS MEETING. WE HAD EXCITING DISCUSSIONS IN SEPTEMBER LAST YEAR AND YOU KNOW, I DIDN'T IMAGINE THAT IT IS GOING TO BE COMING SESSION SUCH AN EXCITING ROSTER OF SPEAKERS IN A MEETING. SO THANK YOU VERY MUCH. AND THANK YOU NHLBI FOR SUPPORT. SO IF WE CAN JUST -- UP LOAD SLIDE. SO LET ME JUST INTRODUCE THE SESSION. I DISCUSSED WITH MARGARET AFTER I INTRODUCE THE SESSION THAT WE CAN MOVE TO MY PRESENTATION SMOOTHLY. SO, I WILL BE THE FIRST TALK HERE AND I WILL TALK ABOUT BLOOD-BRAIN BARRIER IN NEURODEGENERATION TAGGERETS AND TREATMENTS. AND THEN SCOTT DIAMOND WILL TALK ABOUT MICROFLUIDICS FOR BLOOD RESEARCH AND THEN WE GO TO KATERINA TALKING ABOUT FIBRINOGEN NEUROLOGICAL DISEASES. MIKE WILL TALK ABOUT MILEAGE ROW PARTICLES IN TBI AND THERESA WHITESIDE WILL TALK ABOUT EXOSOMES IN GLIOMAS. SO IT IS A VERY INTERESTING. I HAVE BEEN MANY TO BLOOD-BRAIN BARRIER MEETINGS BUT THIS IS A VERY, VERY INTERESTING GROUP, DIVERSE ANGLES ATTACKING THE BLOOD-BRAIN BARRIER PROBLEMS. SO, MY FOCUS HERE WILL BE ON TARGETED POTENTIAL TREATMENTS AND HOW BLOOD BRAIN BARRIER DISRUPTION CAN LEAD TO NEURODEGENERATION AND NEURODEGENERATIVE DISEASES. LET ME JUST GIVE YOU BACKGROUND AND PHYSIOLOGY OF THE BRAIN CIRCULATION. SO THERE IS TIGHT RELATIONSHIP BETWEEN BLOOD VESSELS AND BRAIN CIRCUITS AND THIS IN OUR OPINION, AND STUDIES OVER 25 YEARS, REFLECTS KEY ROLE OF THE VASCULAR SYSTEM IN BRAINS NORMAL FUNCTION, AGING AND DISEASE. PHYSIOLOGICALLY BRAIN IS ONLY 2% OF BODY MASS. BUT RECEIVES 20% OF CARDIAC OUTPUT AND CONSUMES 20% OF OXYGEN IN GLUCOSE AND ALL THESE BLOOD VESSELS ARE 400 MILES LONG. AND THEY HAVE A BLOOD-BRAIN BARRIER THAT YOU HEARD, THAT IS COMPOSED FROM ENDOTHELIAL CELLS AND CAPPED TOGETHER BY GATEKEEPER OF THE BLOOD-BRAIN BARRIER PER I SIGHTS. SO ALL THESE 400 MILES HAVE TO BE COMPLETELY INTACT FOR THE BRAIN TO FUNCTION PERFECTLY. IF YOU HAVE LEAKAGES IN SOME PARTS OF THE BRAIN LIKE HIPPOCAMPUS, YOU CAN GET HEM WE PROBLEMS AND HAVE LEAKAGES DOWN THE SPINAL CORD. YOU CAN GET PROBLEMS RELATED TO THE MOTOR DYSFUNCTION AND ET CETERA. SO, IN AGING AGING AND DEMENTIA AND ALZHEIMER'S DISEASE, THERE IS VASCULAR BLOOD-BRAIN BARRIER DYSFUNCTION AND BRAIN CONNECT ACTIVITY THEY LEADS TO BRAIN DEGENERATION AND DEMENTIA. NOW, BLOOD-BRAIN BARRIER HAS BEEN DEFINED AS A BARRIER THAT PREVENTS BLOOD CELLS AND PATHOGENS INTO THE BRAIN. VERY EXCITING PAPER IN SCIENCE MEDICINE HAS SHOWN THAT MICROBES WHEN THEY COME INTO THE BRAIN CAN SEE THE AMYLOID WHICH LEADS TO PROPAGATION OF THE DISEASE. SO IT'S A VERY IMPORTANT BLOOD-BRAIN BARRIER REMAINS INTACT. WHAT WAS MENTIONED, WE ARE DOING RNA SEQUENCING, THOUSANDS OF TRANSPORTER RECEPTORS IN CELL JUNCTIONS AND I'LL TALK TO YOU ABOUT THEIR ROLE IN HUMAN MONOGENIC DISEASES AND POSSIBLY NEUROLOGICAL DISEASES. WE KNOW FROM HUMAN MONOGENIC RARE DISEASES, THAT BLOOD-BRAIN BARRIER BREAKDOWN IS RELATED TO NEURODEGENERATION AND THIS IS ALSO SHOWN AS WE TALKED ABOUT CHRONIC BREAKDOWN NOT FOR DELIVERY PURPOSES AND THIS IS ALSO SHOWN IN EXPERIMENTAL MODELS. SO HERE IS JUST ONE CORTOON. I WILL JUST FOCUS ON GLUTE 1. TRANSPORTER FOR NUTRIENTS WHICH IS CARRIER TRANSPORTER WHICH CARRIES GLUCOSE IN AND OUT OF THE BRAIN, MOSTLY INTO THE BRAIN ACCORDING TO CONCENTRATION GRADIENTS. WHEN THERE IS DEFICIENCY IN GLUE COTRANSPORTER, EXCLUSIVELY EXPRESSING IN ENDOTHELIUM, WE SEE OVER A PERIOD OF SIX MONTHS, TO ABOUT LONGER A LITTLE BIT, WE SEE COMPLETE NEURONAL DEGENERATION. THIS THE IS MICE THAT IS HAPLOID SOME OF THE GLUTE ONE TRANSPORTEDDER AND HAS A LOSS OF NEURONS IN HIMO CAMPUS AND CORTEX. AND IF YOU CROSS IT WITH AMYLOID BETA MICE WHICH IS ALSO STRONGER. AND THE FIRST NEURONAL INJURIES SEEN HERE BY IMAGING OF PARTICLE MEMBRANE POTENTIAL RESPONSES, ARE SEEN AT ABOUT SIX MONTHS. SO THE BRAIN HAS SOME PLASTICITY TO FIGHT OVER THE TIME BUT IT STARTS LOSING ALSO MASS SO THERE IS A 4% MASS ABOUT SIX MONTHS AND THEN PROGRESSES SO IT'S A MICROCEPHALY. SO THERE IS A PEDIATRIC DISEASE THAT HAS SPECIFIC GENETIC DEFECTS IN ENDOTHELIAL CELLS WHICH IS GLUTE 1 DEFICIENCY SYNDROME. IT SEIZES MICROCEPHALY DEVELOPMENTAL DELAY. THE TRANSPORTER, DISCOVERED A COUPLE OF PAPERS IN NATURE A FEW YEARS AGO, MFSD2A LEADS TO MICROCEPHALY AND INTELLECTUAL SPEECH DISABILITY. MCT8 LEADS TO ALAN MENDON DUDLEY SYNDROME. OCLN IS A TIGHT JUNCTION PROTEINS OF THE BLOOD-BRAIN BARRIER AND LEADS TO SEIZURES AND MICROCEPHALY. SIMILAR WITH PERISITES, A VERY RARE DISEASE CALLED FAR DISEASE -- THAT RESULTS IN CHANGES IN DEFICITS IN -- VERY IMPORTANT ANGIOGENIC SIGNALING SYSTEM DURING DEVELOPMENT. SO THESE ARE CONDITIONS LEADED TO DISRUPTION OF THE BLOOD-BRAIN BARRIER. DISRUPTION OF THE NEUROVASCULAR UNIT AND NEUROLOGICAL DISEASE WITH THE PRIMARY DEFECTS NOT BEING NEURONAL CELLS. SO, WHAT HAPPENS WITHIN THE NEUROVASCULAR UNIT IN CHRONIC NEUROLOGICAL DISEASES SUCH AS ALZHEIMER'S? THIS IS ENDOTHELIAL CELLS. IN ALZHEIMER'S WE KNOW FOR YEARS THERE IS DOWNREGULATION OF NDR1 TRANSPORTER WHICH PREVENTS -- [ INDISCERNIBLE ] THERE IS DOWNREGULATION OF GLUTE 1 TRANSPORTERS THAT ARE ACTUALLY LEADING TO METABOLISM SUPPLY TO THE BRAIN. LWP ONE LEADS TO AMYLOID BETA FROM DOWNREGULATION. THIS TIGHT JUNCTION PROTEINS ARE ALSO DOWN REGULATED. PATHOGENIC RECEPTORS GET EXPRESSED. SO WE DO NOT UNDERSTAND WHAT IT ALL MEANS FOR ALZHEIMER'S DISEASE BECAUSE IT IS SIMPLY NOT THINKING ABOUT IT WE HAVE VERY STRONG EVIDENCE THERE IS BLOOD-BRAIN BARRIER BREAKDOWN AND ALSO POST MORTEM STUDIES HAVE SHOWN THE SAME. SO WHAT WE HAVE DONE RECENTLY, DEVELOPING VERY SENSITIVE DCMRI TECHNIQUE THAT CAN ACTUALLY VISUALIZE IN ALL BRAIN REGIONS AND CALCULATE ALL WHITE MATTER BRAIN REGIONS AND GRAY MATTER BRAIN REGIONS WITH RESOLUTION TO GOING TO HIPPOCAMPAL REGIONS. SO ALL THIS CAN BE DONE. -- IT WASN'T POSSIBLE BEFORE. AND SO WHAT IT FOUND IS AN AGING DEFECT IN HIPPOCAMPUS PARTICULARLY OUR CENTER FOR MEMORY AND LEARNING. WE HAVE PROGRESSIVE BLOOD-BRAIN BARRIER BREAKDOWN IN PEOPLE WITH NO COGNITIVE PROBLEMS. AND IF PEOPLE GET MILD COGNITIVE IMPAIRMENT, THERE IS MUCH MORE PRONOUNCED BLOOD-BRAIN BARRIER BREAKDOWN AND THIS IS HAPPENING OVER HERE AND CORRELATES WITH STRUCTURAL CONNECTIVITY CHANGES IN BRAIN AS SHOWN HERE BY DIFFUSION IMAGING. SO, WHAT IS FIRST HERE? WE STILL DON'T KNOW. WHETHER BREAKDOWN OF DISRUPTIVE CONNECTIVITY. THAT IS THE RESULTS RECENTLY REPORTED LIKE FEW DAYS AGO IN MAY ISSUE BY BELGIUM GROUP ALSO. NOW, THE MAJOR GENETIC INCREASE FACTOR FOR ALZHEIMER'S DISEASE LEADS TO OPENING OF THE BLOOD-BRAIN BARRIER IN HIPPOCAMPUS AND IN COGNITIVELY NORMAL AND ALSO IN PEOPLE WITH MILD COGNITIVE IMPAIRMENT. SO, WE HAVE ACTUALLY SHOWN MY POSTDOCTORAL FELLOW WHO NOW LEADS A PFIZER RESEARCH HAS SHOWN IN OUR GROUP THE PATHWAY. SO WE HAVE A TARGETABLE PATHWAY. THIS PATHWAY IN PSYCHE LOW FILLIAN A, AND ACTIVATION OF MMP9, ENZYME THAT DEGRADES JUNCTION AND BASAL MEMBRANE, LEADS TO BREAKDOWN AND THAT LEADS TO LATER NEURONAL PROBLEMS. THERE ARE ACTUALLY MOLECULES THAT ARE DEVELOPED TO ANTAGONIZE -- THEY ARE IN PHASE II AND PHASE III IN STUDIES OF MS. SO THAT ALSO OPENS UP A THRESHOLD FOR THOSE TO BE USED FOR ALZHEIMER'S DISEASE EVENTUALLY. SO, WE HAVE DEVELOPED A BIOMARKER OF EVERY CELL TYPE IN THE BRAIN OR MOST CELL TYPES AS IT IS SAID. MOST. SO PERRY SIGHTS, END THIELEIA ET CETERA AND HUMAN STUDIES. SO THIS IS UNPUBLISHED DATA BUT IT SHOWS THAT NO COGNITIVE IMPAIRMENT IN MICE IF THEY HAVE CARRIER THEY OPEN OPEN BLOOD-BRAIN BARRIER. AND ALSO THEY HAVE THIS RECEPTORS, SHEDDED RECEPTORS FROM PERISITES. AND THIS CORRELATES MUCH BETTER WITH COGNITIVE IMPAIRMENT FOR EXAMPLE AN EXECUTIVE DYSFUNCTION AND COGNITIVE IN NORMAL PEOPLE AND THEN CLASSICAL BIOMARKERS IN ALZHEIMER'S DISEASE SUCH AS AMYLOID BETA. SO, THE PERISITES IS A CELL THAT HAS BEEN ACTUALLY HAS A VERY, VERY ATTENTION OVER THE LAST 6-7 YEARS AND MODELS A PACE OF DEGENERATION HERE SHOWN BY DISRUPTIVE PDGF-BB SIGNALING SHOWN THAT LEADED TO PROGRESSIVE OPENING OF THE BLOOD-BRAIN BARRIER AND DENSITY THAT HAPPENS VERY EARLY BETWEEN 1-2 MONSAND THE FIRST CHANGES WE SEE IN BRAIN ARE CHANGES IN WHITE MATTER. SO THERE IS DEGENERATION OF WHITE MATTER CHORPUS CALLOSUM SING LUMTHAT PROGRESS AND THIS IS FOLLOWED BY THE DEGENERATION OF GRAY MATTER UPO CAMPO REGIONS AND CORTEX. SO WHAT ARE THE PATHWAYS OF BLOOD-BRAIN DEGENERATION WILL? RECENTLY DESCRIBED IN THE REVIEW BY MY POSTDOCTORAL FELLOWS THEMSELVES? THERE IS LEAKAGE INTO PERIVASCULAR SPACE OF ALL SORTS OF TOXINS. NEAR RED BLOOD CELLS COME IN AND HEMOGLOBIN ARE RELEASING IRON AND IRON GENERATE REACTIVE OXYGEN SPECIES AND THIS IS VERY MUCH NEUROTOXIC. THERE IS FIBRINOGEN TAT REASON WILL TALK MORE ABOUT THAT LEADS TO MICROGLIAL ACTIVATION TOXIC FOR OLIGODENDROCYTE PROGENITOR CELLS AND NEUROTOXIC. PLASMA MATRIX. WE HAVE ANTIBODIES. Y WE HAVE A POSSIBILITY THAT VIRUS AND PATHOGENS COMING TO THE BRAIN SO IT'S A COMPLETE MESS IF WE HAVE A CHRONIC OPENING OF THE BLOOD-BRAIN BARRIER. JUST DEPENDS ON WHICH IT WILL HAPPEN AND WHICH PART OF THE BRAIN. SO THESE ARE PATHWAYS. THERE ARE ALSO SOME TARGETS I WANT TO TALK ABOUT. AND WE HAVE BEEN STUDYING OR DISCOVERED RAGE RECEPTOR IN 2003. AND THERE ARE SEVERAL, ABOUT 50 PAPERS THAT FOLLOW-UP AND CONFIRM THE SAME AND THIS BLOCKING THIS MOLECULE OF THE BLOOD-BRAIN BARRIER IN ALZHEIMER'S DISEASE NOW PROGRESS TO PHASE III STUDIES BY PFIZER ABOUT 800 PEOPLE. AND WE ALSO SHOW LRP1 RECEPTOR THAT CLEARS AMYLOID BETA FROM BRAIN USED IN PHASE II STUDIES AND ANTIBODIES THERAPIES. SO, IN ADDITION TO ACTUALLY THE GENES RECEPTORS, SO FOR AN EXAMPLE, APOE AND PICAL. AND CLUSTERING, VERY MUCH IMPLICATED IN TRANSPORT OF THIS AMYLOID BETA ACROSS THE BLOOD-BRAIN BARRIER. SO THIS IS WHAT IS DESCRIBED RECENTLY IN THE PAPER THAT PICAL. SOME TARGETING AMYLOID BETA. AND ALSO GUIDES FUSION WITH ENDOZOMES WHICH LEADS TO EXOCYTOSIS AND EXPORT OF AMYLOID BETA OF THE BRAIN AND THESE ARE INFLUENCED BY APOE3 AND 4. PARTICULARLY APOE4. CLUSTERING HELPS THE OTHER HAND, TO TRANSPORT AMYLOID BETA FROM BRAIN. SO THESE ARE SOME OF THESE TARGETS. HERE IS THE TRIAL THAT IS BASED ON RAGE INHIBITOR WHICH BLOCKS BASICALLY INFLAMMATORY RESPONSE AND AMYLOID BETA TRANSPORTING DNA BASED ON THIS BIOLOGY OF RAGE FOR DISCOVERY IN 2003. AND WHAT PAGE REALLY DOES BASICALLY BLOCKING RAGE INTERACTION PREVENTS TRANSPORT OF AMYLOID BETA INTO THE BRAIN AND ALSO ACTIVATION OF INFLAMMATORY RESPONSE APOPTOSIS AND CBS SUPPRESSION. SO THIS IS AN ALTERNATIVE APPROACH NOW IN PHASE III STUDIES. WE HAVE DEVELOPED THIRD RAGE BLOCKER, SECOND GENERATION RAGE BLOCKER -- AND IT WAS PUBLISHED IN 2012 AND THERE WAS A NUMBER OF PAPERS THAT FOLLOWED AND FOUND IN CANCER, IN STROKE, IN RED MODEL, THIS IS NOTHING FROM OUR GROUP. THIS IS FROM ALL OTHER PEOPLE THAT ARE USING THIS RAGE BLOCKERS. SO, THE TWO VASCULAR PROCESSES THAT WE PROPOSED SUGGEST THAT BLOOD VESSELS AND BLOOD-BRAIN BARRIER INCLUDING BLOOD-BRAIN BARRIER ARE CONVERGING POINT OF PATHOGENESIS FOR GENETIC FACTORS AND ENVIRONMENT, LIFESTYLE AND VASCULAR FACTORS, HYPERTENSION AND DIABETES AND ET CETERA AND THERE IS THE PATHWAY THAT DOESN'T INVOLVE AMYLOID BETA THAT LEADED TO BLOOD-BRAIN BARRIER DYSFUNCTION AND DAMAGE AND BRAIN HYPERPERFUSION AND A RED PATHWAY THAT ALSO INVOLVES AMYLOID BETA AND INCREASE A BETA FROM AUCTION AND TOGETHER THEY LEAD TO DEVELOPMENT OF PATHOLOGY IN ALZHEIMER'S DISEASE. IN THE A FEW MINUTES I WANT TO TALK ABOUT THE WORK SUPPORTED BY NHLBI ON ACTIVATED PROTEIN C AND THIS IS WHAT KEITH MENTIONED AT THE BEGINNING. PROTEIN C IS AN ENZYME PROTEASE THAT IS ACTIVATEDDED BY -- ON ENDOTHELIAL MEMBRANE INCLUDING BLOOD-BRAIN BARRIER AND REACTS TOL RECEPTORS TO DO CYTOPROTECTIVE SIGNALING AND REDENNATIVE AFFECTS. SO THIS IS THE MOLECULE THAT WE DEVELOPED TO SUPPORT NHLBI FUR A NUMBER OF YEARS THROUGH MIGRANT WITH JOHN GRIFFIN. AND THIS IS MODIFIED AND DOESN'T HAVE ANTICOAGULANT PROPERTIES. SHOW MOLECULE IS NOW IN PHASE II STUDIES SO WENT FROM BENCH TO BEDSIDE AND IS BASICALLY THIS STUDY IS RIGHT NOW ENROLLED 80 PATIENTS AND IT IS CLOSE TO 100 AND IT IS PREPARING FOR PHASE III STUDIES. HAS TREMENDOUS INTERESTING APPLICATION. WE JUST RECENTLY HAVE A PAPER THAT WAS ACCEPTED FOR PUBLICATION AND ALSO FOR COMBINATION WITH STEM CELL THERAPY AND REPAIR AFTER STROKE AND HAS POTENTIAL IN ALZHEIMER'S DISEASE MODELS AND OTHER MODELS. SO, JUST TO BRIEFLY SUMMARIZE, THE KEY POINTS OF THIS PRESENTATION, I TRIED TO PRESENT YOU WITH THE EVIDENCE VERY BRIEFLY PROBABLY FAST, BUT THE GENETIC DEFECTS IN NON-NEURONAL CELL TYPES AND THE EPITHELIUM AND WILL PERISITES, LEADS TO BLOOD-BRAIN BARRIER BREAKDOWN CAUSING RARE MONOGENIC NEUROLOGICAL DISORDERS AND DISEASES. [ READING ] ... AND WE HOPE TO GET TO PHASE III PRETTY SOON. I LIKE TO THANK FOR SUPPORT: I WAS SUPPORTED FOR 10 YEARSOR MORE, 15 YEARS AND ALL TALENTED PEOPLE IN MY GROUP AND LABORATORY AND ALSO JOHN GRIFFIN'S LAB AT SCRIPPS AND IMAGING INSTITUTE. THANK YOU. [ APPLAUSE ] >> MARGARET OCHOCINSKA WE HAVE TIME FOR ONE OR TWO QUESTIONS. IF THERE ARE ANY. >> SINCE THIS IS A WORKSHOP MY NAME IS BILL POLTER, I'M WITH THE NATIONAL INSTITUTE OF MENTAL HEALTH NOW. >> YES, HI. YES, OF COURSE. >> HI. SINCE MANY OF US ARE TRYING TO UNDERSTAND WHAT WE MIGHT LOOK FOR IN TERMS OF EVIDENCE -- THE DEGREE OF ALTERATIONS OF THE BLOOD-BRAIN BARRIER, IN THE PURSUIT OF TRYING TO UNDERSTAND WHEN YOU TRY TO USE THE RAGE INHIBITOR, HAVE YOU DONE ANYTHING OR THOUGHT THROUGH HOW WOULD WE UNDERSTAND BY USING MAYBE SOME PROTEOMIC APPROACHES, SOMETHING LIKE THIS, WHETHER OR NOT THE -- TO WHAT EXCEPT IS THE BLOOD-BRAIN BARRIER ALTER THINGS LIKE ALZHEIMER'SS? FOR INSTANCE, SHOULD THERE BE SOME RELATIONSHIP BETWEEN THE STOKEOMITRY BETWEEN THE RELATIONSHIP BETWEEN THE BLOOD AND THE CEREBRAL SPINAL FLOOD FLUID? I THINK IT IS PRETTY IMPORTANT BECAUSE IF YOU CAN'T STAGE OR STRATIFY PATIENTS ACCORDING TO THE VASCULAR PROBLEMS IN AD, YOU MAY RUN INTO A BIG SIGNAL DETENTION PROBLEM. IN OTHER WORDS, YOU SEE WHAT I'M SAYING? >> ABSOLUTELY. SO BILL, I THINK YOU'RE RIGHT ON THE MONEY AS ALWAYS. THE ONGOING STUDIES RIGHT NOW THAT PEOPLE ARE DOING IN SEVERAL LABS, INCLUDING MINE AND ALSO OTHERS, ARE LETHAL RNA AND PROTEOMIC STUDIES OF BLOOD-BRAIN BARRIER INCLUDING BOTH CELL TYPES AND THAT IS NOT CEREBRAL PROBLEM BECAUSE YOU CAN NOT SEPARATE THESE CELLS EASILY. AND YOU IF DO THEM FROM THE WHOLE BRAIN LIKE A COUPLE OF PAPERS PUBLISHED LAST YEAR, THE SIGNAL THAT COMES FROM -- IS VERY SMALL AND THE NUMBER OF ROADS IN RNA-SEQ STUDIES IS VERY LOW. WE NEED ABOUT 10 OR 60 MILLION READS FOCUSING. SO WHAT WE ARE DOING WITH THE DEPARTMENT AT USC, WE ARE TAKING THE FRESHLY-OPERATED FROM PATIENTS FROM THE CORTICAL VESSELS AND DOING SINGLE CELL RNA ANALYSIS OF HUMAN BLOOD-BRAIN BARRIER. BECAUSE FOR YOUR -- TO ADDRESS YOUR QUESTION, WE DON'T KNOW HOW TO COMPARE THESE. SO THESE ARE ALL IN PROGRESS AND ALL THE DISCOVERIES WITH RAGE AND EVERYTHING ELSE, THEY WERE SPORADIC. IT WAS INTUITEDDIVELY CONNECTED -- SO LET'S SEE WHAT HAPPENS IN BRAIN. RIGHT NOW WE WILL HAVE THAT OPPORTUNITY PRETTY SOON AND BOTH IN ANIMAL MODELS. SO THIS IS ONE OF OUR NEEDS AND CHALLENGE THAT IS NUMBER 1 RNA-SEQ AND PROTEOMICS. YOU WILL SEE THE MICROPHONE SECTION. >> SO A LITTLE BIT RELATED TO THAT. COULD YOU TALK A LITTLE BIT ABOUT SOME OF YOUR WORK THAT INTERACTS WITH APOE3 AND 4 DIFFERENCES AND HOW THAT TRANSLATES TO BLOOD-BRAIN BARRIER? THIS IS CIRCULATING MICROPROTEINS AND ALSO EXPRESSED IN THE BRAIN THAT I THINK ADDRESSES SOME VERY SIGNIFICANT -- >> THAT IS A GREAT QUESTION. WE HAD NHLBI PUT ANOTHER MEETING ON LIPIDS YESTERDAY AND I WAS TALKING JUST SPECIFICALLY ABOUT THAT PART YESTERDAY ABOUT APOE3 AND 4. SO THE THING IS THAT LRP1 RECEPTOR THAT IS IN THE AB LIMINAL SIDE OF THE BLOOD-BRAIN BARRIER HAS AFFINITY TO INTERACT WITH APOE2 AND 3. APOE4 DOES NOT HAVE THAT AFFINITY. AND ACTS MAINLY VLVR IS 1520 TIMES SLOWER IN ENDOSIGH TO THETIC RATE -- SO THAT IS ONE ASPECT -- APOE4 ACTIVATES THIS PRO-INFLAMMATORY PATHWAY IN THE BLOOD-BRAIN BARRIER WHICH LEADS TO DISRUPTION. AND APO3 AND 2 ARE NOT DOING THAT BECAUSE THEY CAN BLOCK PSYCHE LOW THILLIAN A ACTING AGAIN. SO THE KEY IS INTERACTION BETWEEN THIS RECEPTOR. AS YOU KNOW FROM YOUR OWN WORK, CORRECT? AND OUR WORK AND MANY OTHERS, AND NLP1 IS DECREASED IN ALZHEIMER'S WHICH REDUCES CHANCES OF APOE3 CARRIERS. >> THANK YOU VERY MUCH. WE ARE GOING TO PROCEED TO DR. SCOTT DIAMOND AND I'LL JUST SET UP THE PRESENTATION. >> SCOTT DIAMOND: IT'S A PLEASURE TO BE HERE TODAY. WHAT I WANT TO DO IS DISCUSS SOME OF OUR RESEARCH LOOKING AT BLOOD EX-VIVO AND BLOOD OBVIOUSLY HAS THE ADVANTAGE CAN GET YOUR HANDS ON IT AND WORK WITH IT IN THE LABORATORY, WHICH OFFERS TREMENDOUS CAPABILITY TO LEARN ABOUT PATHWAYS WITHIN BLOOD AND ALSO UNIQUE ASPECTS OF AN INDIVIDUAL'S BLOOD, AN INDIVIDUAL PATIENT. WE DO A LOT OF WORK RE-CREATING THE HEMODYNAMIC CONTEXT OF BLOOD, FLOWING BLOOD. SO, BLOOD CLOTTING OBVIOUSLY IS VERY COMPLEX AND BLEEDING DISORDERS ARE VERY COMPLEX. AND WE ARE VERY FOCUSED ON THE SIGNALING PATHWAYS WITHIN PLATELETS AS WELL AS THE COMMUNICATION OF THOSE PLATELETS WITH THE DAMAGED VASCULAR WALL AND ALSO THE PROTEASE CASCADES THAT EXIST WITHIN PLASMA. WE TAKE A VERY INTENSIVE EXPERIMENTAL APPROACH AND ALSO A VERY INTENSIVE COMPUTATIONAL APPROACH TO THESE TYPES OF PROBLEMS BASICALLY TRYING TO DEVELOP IN SILL COREPRESENTATION OF WHAT WE MEASURE IN THE LABORATORY. SOME OF THIS WORK IS DIRECTED TOWARDS THROMBOTIC DISORDERS BUT ALSO CAN BE RELATED VERY DIRECTLY TO BLEEDING DISORDERS. PLATELETS MARGIN 8 TO THE WALL AND WILL THEY CAN, IF THERE IS DAMAGE TO THE WALL THEY CAN INTERACT WITH VARIOUS RECEPTORS AND PULL DOWN UNDER FLOAT CONDITIONS CAN ACCUMULATE AND THOUGHT OF AS A BLOOD CLOT THOUGHT OF AS A TEMPORARY TISSUE IN THAT YOU HAVE CELLS AND ALSO A DEVELOPING MATRIX THAT COAGULATION PATHWAYS GENERATES FIBRIN. ALL OF THIS IS OCCURRING IN SPACE AND TIME UNDER SOMETIMES VERY COMPLEX HEMODYNAMIC CONDITIONS. AND JUST TO EMPHASIZE HOW IMPORTANT THAT TRANSPORT OF THE PLATELETS, MARGINATION OF PLATELETS TO THE SWAIL IF YOU REDUCE THE BLOOD, WILL YOU DRAMATICALLY REDUCE THE ABILITY OF PLATELETS TO GET TO THE WALL. WITH COMPUTER SIMULATIONS AND 3D SIMULATIONS OF PARTICLE TRANSPORT AND PLATELET TRANSPORT TO THE WALL, THIS HAS REALLY IMPORTANT IMPLICATIONS IN NANOPARTICLE DELIVERY TO THE WALL. TRANSPORT OF SMALL PARTICLES IS VERY DIFFERENT BLOOD FLOW THAN IT IS WHEN THINKING OF JUST SMALL MOLECULES THAT ARE DIFFUSIVE. SO PLATELETS ARE SIDES AND SHAPED TO GET TO THE WALL WHERE THEY HAVE TO INTERACT BUT IT REQUIRES A RED BLOOD CELLS TO DO THAT. WE ARE EXTREMELY INTERESTED IN SIGNALING PATHWAYS WITHIN PLATELETS AND THE PLATELET VERY WELL STUDIED AND WE CAN UNDERSTAND THESE PATHWAYS AND THE CONTEXT OF THEIR AVERAGE PROPERTIES BUT ALSO PATIENTS SPECIFIC PROPERTIES. SO, INTRACELLULAR CALCIUM IS PROBABLY THE MOST IMPORTANT ASPECT OF A PLATELET ACTIVATING BUT THERE ARE MANY, MANY WAYS TO ACTIVATE A LITTLE BIT THREAT A CLOTTING OR LACK OF CLOTTING -- PLATELET. IF YOU DON'T HAVE ENOUGH STIMULI AND WE THINK OF A PLATELET AS A COMPUTER SAMPLING THESE DIFFERENT STIMULI IN THE LOCAL ENVIRONMENT TO EITHER STATE RESTING OR TO BECOME ACTIVATED AND WE HAVE A VERY DETAILED COMPUTER SIMULATIONS OF THIS PROCESS TRYING TO UNDERSTAND THE INTERACTION OF DIFFERENT SIGNALING PATHWAYS AS THEY RESULT IN A LITTLE BIT LET ACTIVATION. BUT WE ALSO DEVELOPED A VERY, IN SOME WAYS, SIMPLE LABORATORY TECHNIQUE TO DO HIGH DIMENSIONAL PHENOTYPING OF BLOOD. IN THIS EXPERIMENT, WHAT WE DO IS WE TAKE A SMALL SAMPLE OF BLOOD IN THIS IT AND GIVES US THE PLATELETS OF THE INDIVIDUAL. WE ARE ALLOWED THEN TO PHENOTYPE THAT INDIVIDUAL'S PLATELETS AND WHAT WE DO IS WE MEASURE CALCIUM RESPONSES NOT JUST TO ONE AGONIST BUT TO MANY AGONISTS AND NOT JUST TO VARIOUS INDIVIDUAL AGONISTS BUT TO VARIOUS DOSES OF THOSE AGONISTS AND JUST NOT A SINGLE BUT ALSO TO PAIR OF AGONISTS AT LOW MEDIUM AND HIGH CONCENTRATIONS SO THIS IS A FUNCTIONAL PHENOTYPE THAT JUST TAKES AN AFTERNOON IN THE LABORATORY BUT IT GIVES US A HUGE DATASET TO UNDERSTAND THE FUNCTIONAL RESPONSE OF THAT INDIVIDUAL'S PLATELETS. AND THIS PHENOTYPING METHODS THEN ALLOWS US TO TAKE ALL THIS DATA AND TRAIN NEURAL NETWORKS SO WE HAVE IN SILL COREPRESENTATION OF THAT INDIVIDUAL'S PLATELETS AND WE SEE OR HAVE DOCUMENTED VERY, HOW UNIQUE THE PHENOTYPE IS TO EACH INDIVIDUAL AND THAT IS JUST WITH HEALTHY INDIVIDUALS AND ALL THAT KNOWLEDGE IN THE DATASET CAN BE IMPARTED INTO A MULTI-SCALE MODEL OF CLOTTING UNDER FLOW CONDITIONS. ADDITIONALLY, NOT ONLY DOES THE PLATELET OFFER A UNIQUE PHENOTYPE, THE PLASMA AND THE BLOOD PROTEINS, PROTEASE CASCADES ARE ALSO UNIQUE TO EACH INDIVIDUAL. AND THESE PROTEINS ARE CERTAINLY IMPORTANT TO THIS AUDIENCE WHEN WE THINK ABOUT THROMBINOGENERATION, FIBRINOGENERATION, APC ACTIVATION. WE HAVE A FAIRLY GOOD ABILITY TO SIMULATE THESE TYPES OF REACTIONS AND SPACE AND TIME BUT THEY ARE VERY COMPLEX AND ALSO HAVE THE ABILITY TO INTERROGATE THESE REACTIONS EX-VIVO UNDER MANY, MANY DIFFERENT CONDITIONS. AND THIS IS THE POWER OF WORKING WITH BLOOD EX-VIVO IS THAT WE CAN PHENOTYPE IT NOT JUST UNDER ONE CONDITION BUT UNDERSCORES OF CLOTTING CONDITIONS AND THIS EXPERIMENT I SHOW ON THE RIGHT, WE HAVE ACTIVATED BLOOD UNDER 50 DIFFERENT MODES OF ACTIVATION ALL OF THAT CAN BE SIMULATED BUT THIS GIVES US A VERY DEEP PHENOTYPE OF HOW THE COAGULATION PATHWAY FUNCTIONS IN INDIVIDUALS. SO I SHOWN YOU SOME OF THE PIECES OF BLOOD CLOTTING SUCH AS PLATELET ACTIVATION AND THE ABILITY TO GENERATE THROMBIN IN A CLOTTING EVENT. I WANTED TO DESCRIBE SOME MICROFLUIDIC TECHNOLOGIES THAT WE HAVE DEVELOPED OVER THE LAST 10 YEARS OR SO. AND THESE MICROFLUIDIC TECHNOLOGIES ARE VERY POWERFUL IN THAT THEY CAN ALLOW US TO CONDUCT LITERALLY HUNDREDS AND HUNDREDS OF UNIQUE CLOTTING EVENTS IN AN INDIVIDUAL EXPERIMENTAL STUDY. WE PROBABLY CONDUCTED OVER 5 OR 8000 CLOTTING EVENTS OVER THE LAST 10 YEARS. SO IT GIVES US TREMENDOUS POWER TO RE-CREATE UNIQUE CLOTTING EVENTS OR BIOCHEMICAL EVENTS INVOLVING HUMAN BLOOD. THIS IS ONE DEVICE WHERE WE HAVE BLOOD FLOWING OVER A COLLAGEN FEATURE THAT CONTAINS TISSUES TO MIMIC HEMOSTATIC EVENT WHERE WE HAVE BLOOD FLOWING AT A WALL SHEAR RATE AND ALSO CONTROL THE PRESSURE FROM THE VASCULAR SPACE TO THE EXTRA VASCULAR SPACE. SO THIS IS VERY IMPORTANT IN RE-CREATING HEMOSTAYSIS. NOT ONLY DO WE HAVE BLOOD FLOW IN THIS DIRECTION, OVER THE STIMULATORY SURFACE, BUT WE ALSO HAVE LEAKAGE OF PLASMA CONSTITUENTS ACROSS THIS COLLAGEN WHICH IS HELD ON A POST ARRAY. THIS IS 50 MICRONS AND PLATELETS WILL RAPIDLY ACCUMULATE ON THAT SURFACE BECAUSE WE HAVE TISSUE FACTOR AND WE CAN GENERATE THROMBIN BY THE ENTRIESIC PATHWAY AND MAKE FIBRIN. YOU CAN SEE A HEMOSTATIC BLOOD CLOT IN THIS DEVICE HAS A LAYERED STRUCTURE TO IT. AND THIS ASPECT OF THAT LAYERING IS REFERRED TO AS CORE SHELL ARCHITECTURE OF A HEALTHY HEMOSTATIC EVENT THAT THE PLATELETS IN THE INNER REACH ON ARE ACTIVATED AND DELATING AND PLATELETS ARE LOADED WITH VERY POTENT FACTORS THAT ARE VERY IMPORTANT IN WOUND HEALING EVENTS THIS REACHES A STEADY STATE AND THAT IS HEMOSTAYSIS AND THE OUTER PLATELETS ARE NOT AS ACTIVATED. SO WITH HUMAN BLOOD, WE CAN CREATE THIS ARCHITECTURE OF A HEMOSTATIC EVENT AND THE IT LOOKS VERY SIMILAR TO WHAT ONE WOULD SEE IN LASER INJURY WHERE THE INNER CORE PLATELETS ARE SELECTED POSITIVE -- THE INJURED VESSEL WALL AND THEN THE OUTER LAYER OF PLATELETS ARE ADHERED BUT THEY ARE NOT DELATING THEIR P SELECTED NEGATIVE. WE HAVE A NICE CORRESPONDENCE BETWEEN WHAT ACTIVITIES IN A HEMOSTATIC EVENT IN ANIMAL MODELS AND WE CAN STUDY THAT WITH HUMAN BLOOD. WE HAVE A VARIETY OF DIFFERENT DEVICES TO STUDY CLOTTING PATHWAYS. WE HAVE WORKED WITH BLOOD FROM HEMOFEEL ACKS, OBVIOUSLY EVERYWHERE STRONG DEFECT IN THROMBINOGENERATION AND THAT IS RE-CREATED IN THESE TYPES OF DEVICES. WE ALSO HAVE DONE WORK WITH BLOOD FROM TRAUMA PATIENTS WHERE THERE IS A COAGULOPATHY AND VERY RAPID CHANGE IN BIOLOGY OF THE BLOOD IN THE SYSTEMIC CIRCULATION. SO FROM TRAUMA PATIENCE, WE SEEN IN HALF TRAUMA PATIENTS, THERE IS STRONG DEFECT IN PLATELET FUNCTION AS WE AS A IN THESE MICROFLUIDIC DEVICES. NORMALLY PLATELETS RESPOND INTENSIVELY AND HEALTHY PLATELETS RESPONDENCE TENSELY TO COLLAGEN ON THE SUFFERS AND WILL DEVELOP A VERY RAPID PLATELET AGGREGATE AND IN ABOUT HALF THE TRAUMA PATIENTS WE OBSERVED THEIR PLATELETS ARE HYPOFUNCTIONAL. WE ARE TRYING TO UNDERSTAND THAT CHANGE IN THE SYSTEMIC BLOOD BIOLOGY THAT LEADS TO THAT HYPOFUNCTIONALITY AND CAN LEAD TO BLEEDING PHENOTYPES THAT CAN BE VERY SERIOUS. SO WE HAVE DEVELOPED A LOT OF TECHNIQUES IN THE FIELD OF BLOOD RESEARCH HAS DEVELOPED A NUMBER OF ANTICOAGULANTS THAT ALLOW THE VERY RIGOROUS STUDY OF BLOOD IN THE LABORATORY. AS SOON AS YOU TAKE BLOOD OUT OF THE VASCULATURE, IT IS DIFFERENT. AND WE HAVE TO BE AWARE OF THAT. IF WE ARE JUST STUDYING CELLULAR MECHANICS, EDTA WORKS FINE BUT WE HAVE A VARIETY OF PHARMACOLOGICAL 80s THAT ALLOW US TO STUDY JUST -- AGENTS -- STUDY JUST PLATELET FUNCTION. WE CAN USE IS SOME VERY, VERY SPECIFIC TECHNIQUES. WE ARE ALWAYS WORRIED ABOUT THE CONTACT PATHWAY. NORMALLY WE DON'T THINK OF THIS BUT IT IS BECOMING MORE ARE AND MORE IMPORTANT FACTOR 12A AND A FACTOR 11A AND WILL THROMBOSIS BUT ALSO IN VARIOUS OTHER DISEASES EVEN RELATING TO AMYLOID DEPOSITION THE CONTACT PATHWAY HAS STRONG LYNX TO THE INFLAMMATORY PATHWAYS IN VASCULAR BIOLOGY ALSO. WE CAN REGULATE THE CONTACT PATHWAY WITH TRIPS IN INHIBITOR AND WE HAVE A LOT OF WAYS OF WORKING WITH HUMAN BLOOD EX-VIVO IN GETTING A LOT OF DATA WITHIN 30 MINUTES OR AN HOUR OF COLLECTING THAT BLOOD SAMPLE. SO IN THE CONTEXT OF THIS MEETING, THERE IS A LOT KNOWN ABOUT THE ENGINEERING AND BIOPHYSICS ATTRIBUTES OF THE NEUROVASCULAR UNIT, ISSUES OF SHEAR STRESSES AND PRESSURE, ISSUES OF ELECTRICAL RESISTANCE. AND IN THE MICROFLUIDIC REGIME, THIS WORK IS REALLY AT THE EARLY STAGES. THIS REALLY ONE OF THE FIRST PAPERS TRYING TO RE-CREATE THE NEUROVASCULAR UNITE WHILE MAINTAINING FLOW IN MEASURING THE RESISTANCE OF THAT LAYER. JUST TO SHOW YOU THE IMPORTANCE OF THE PHYSICS OF THIS SYSTEM, AS WELL AS THE BIOLOGY, GOING FROM MONOLAYER, BY ADDING FLUID SHEAR STRESS, YOU CAN INCREASE THE RESISTANCE OF ENDOTHELIAL MODEL LAYER SIGNIFICANTLY AND THEN FOR AN IN-VITRO SYSTEM, THIS IS A REASONABLE LEVEL OF ELECTRICAL RESISTANCE. MOST OF THESE IN-VITRO SYSTEMS NEVER REALLY ACHIEVE THE FULL RESISTANCE OF WHAT IS MEASURED IN-VIVO. ONCE YOU WORK IN A MICROFLUIDIC ENVIRONMENT, YOU HAVE FULL CONTROL OF THE BIOPHYSICS, TRANSPORT, DRUG DELIVERY ISSUES AND YOU CAN FART TO EXPLORE HUNDREDS AND THOUSANDS OF EXPERIMENTAL CONDITIONS AND THIS IS IN TERMS OF THINKING ABOUT TECHNOLOGIES THAT ARE REALLY RIGHT AT THE STAGE OF FURTHER DEVELOPMENT, I THINK THERE IS A LOT OF OPPORTUNITIES USING MICROFLUIDICS. THANK YOU FOR YOUR ATTENTION. [ APPLAUSE ] >> THANK YOU DR. DIAMOND. WE HAVE TIME FOR ONE OR TWO QUESTIONS. >> I SHOULD POINT OUT THAT MOST OF THE WORK I HAVE JUST SHOWN HAS BEEN FOCUSED ON BLOOD FUNCTION. THIS HAS BEEN ABOUT 15 STUDIES OVER THE LAST FIVE YEARS LOOKING AT THE INTERACTION OF BLOOD WITH CULTURED ENDOTHELIUM AND TAKING BLOOD FROM PATIENTS AND THE INTERACTION OF PATIENT BLOOD WITH CULTURED ENDOTHELIUM UNDER FLOW CONDITIONS IN THESE TYPE OF MICROFLUIDIC ENVIRONMENTS. SO WE ARE AT THE EARLY STAGES OF GETTING ENDOTHELIUM IN THESE TYPES OF DEVICES BUT THE NEUROVASCULAR UNIT I THINK STILL AWAITS FURTHER DEVELOPMENT. BUT THERE IS A LOT OF OPPORTUNITY THERE. [ OFF MIC ] >> TALKED ABOUT THE DANGERS THAT RED CELLS COMING IN WITH IRON AND DEPOSITIONS AND OXIDATIVE DRESS THAT KIND OF THING. WONDER BEING PLATELETS? ARE THEY A MIXED BAG ON ONE HAND AND YOU HAVE PROSTAGLANDINS IN OTHER THINGS YOU MAY NOT WANT IN THE BRAIN? ALSO BRINGING FORWARD GROWTH FACTORS. >> A LOT OF GROWTH FACT EXPOSITO KINES AND A VERY INTENSE FOR PRO-COAGULANT ASSEMBLY. SO WHEREVER YOU HAVE THE ACTIVATED PLAT LATE YOU PROBABLY WILL FIND SOME THROMBINGENERATION, SOME FIBRIN DEPOSITION. >> SO WE KNOW WE DON'T WANT RED CELLS IN THE BRAIN IF WHEN WE ARE OPENING THE BLOOD-BRAIN BARRIER. DO WE WANT PLATELETS IN THEM? >> PLATELETS ARE VERY GOOD AT SEALING UP LOCATIONS. SO THEY ARE PROBABLY MORE APT TO RESPOND TO THE SUB ENDOTHELIUM IN TERMS OF ACTIVATIONS SO, WHEREAS RED BLOOD CELLS WE THINK OF AS BEING MAYBE IN THE WRONG PLACE, PLATELETS IF THEY ARE IN THE WRONG PLACE, ARE PROBABLY ACTIVATING TO SOME DEGREE IN DELATING. >> EXCELLENT TALK. YOU SAID SEVERAL TIMES THE SIGNATURES FROM PATIENT SAMPLES WAS VERY UNIQUE. HOW UNIQUE? TALKING AT THE LEVEL OF DNA OR -- >> SO THAT IS THE INTERESTING THING ABOUT PLATELETS THAT IS CHALLENGING IN THE LABORATORY. NO NUCLEUS SO WE CAN'T DO GENETIC STUDIES BUT THEY ARE METABOLICALLY ACTIVE AND THEY HAVE ACTIVE PROTEIN SINT SIS. THEIR SENSITIVITY TOW STIMULI AND MULTIPLE STIMULI CAN VARY SIGNIFICANTLY BETWEEN INDIVIDUALS. AND FOR EXAMPLE, PAR 4 FUNCTION. AND THERE IS GENETIC BASIS TO THIS IN TERMS OF EXPRESSION LEVELS AND FUNCTION OF PAR 4 PROBABLY THE MOST WELL STUDIED IN TERMS OF VARIATION IN HUMANS. >> WITH THE CAPACITY TO CONTROL FLOW AND THE CONSTITUENTS OF THE MEDIA YOU FLOW PAST, HAVE YOU DONE ANYTHING YET IN LOOKING AT CHANGES IN EXOSOMES COMING OUT OF THE END NEILIAL CELL? >> THE ENDOTHELIUM IS CRENNELY SHEAR RESPONSIVE. -- UP INCREDIBLY. THE REASON I SAY THAT IS BECAUSE THERE IS HUNDREDS OF STUDIES GOING BACK SEVERAL DECADES AT THIS POINT. SHOWING ARTERIAL ENDOTHELIUM BEING VERY, VERY SHEAR RESPONSIVE IN TERMS OF THEIR GENE EXPRESSION AND IN IN TERMS OF EXOSOMES. WE KNOW GENE EXPRESSION CHANGES DRAMATICALLY WITH SHEAR N TERMS OF EXOSOME PRODUCTION, I'M NOT AWARE OF STUDIES BUT IT'S AN EXCELLENT POINT. >> THANK YOU VERY MUCH DR. DIAMOND. THAT WAS A GREAT ILLUSTRATION OF THE TRANSFORMATIVE NATURE OF THESE TYPES OF TALKS. SO TALKING ABOUT MICROFLUIDICS, WE WILL HAVE ADDITIONAL SESSION TOMORROW TALKING ABOUT APPROACHES FOR THE NEUROVASCULAR UNITE. NOW I'D LIKE TO MOVE ON TO DR. MICHAEL GOODMAN AND I WILL PUT UP HIS PRESENTATION. >> MICHAEL GOODMAN: GOOD MORNING. I'M REALLY HONORED TO BE HERE AMONG PEOPLE WHO HAVE EXTREME EXPERTISE IN THESE AREAS AND AS A QUICK DISCLAIMER, I'M A SIMPLE TRAUMA SURGEON. AND I THINK I'M PROBABLY THE ONLY ONE IN THE ROOM. AND LOOKING AT THESE PRESENTATIONS, YOU GUYS HAVE DECADES OF DATA AND COMPARED TO THIS, OUR WORK IS VERY NASCENT AND WE ARE STILL WORKING TOWARDS PUTTING THIS TOGETHER. SO, IF THIS SEEMS RAW, PLEASE EXCUSE THAT BUT WE ARE STILL WORKING. SO, INSTEAD OF THE OVERVIEW SLIDE I STARTED WITH OUR GRAPHIC SLIDE HERE TO SHOW THAT AFTER TRAUMATIC BRAIN INJURY, THE THEORY OF PLATE LET ACTIVATION DOES OCCUR, CERTAINLY AS WE MENTIONED THERE IS PLATELET DYSFUNCTION BUT THERE IS A COMPENSATORY PLATELET ACTIVATION FOLLOWING THIS AND WITH THIS WE THINK THERE IS MIKE ROY PARTICLE PRODUCTION AS WELL -- MICROPARTICLE PRODUCTION, POTENTIALLY AS PART OF A COORDINATEED RELEASE FROM PLATELETS AND POTENTIALLY FROM PLATELETS USED IN BREAKDOWN AND THEN OUR NEARY IS THAT THESE MICROPARTICLES CONTRIBUTE TO CLOT INITIATION AND THROMBOSIS IN THE MICROVASCATURE AS WELL AS THE MACRO VASCULATURE. SO THE BACKGROUND FOR WHAT WE DO IS LOOKING AT THE FACT THAT TRAUMATIC BRAIN INJURY CERTAINLY PLAYS A SIGNIFICANT ROLE IN THE MORE BIDDY AND MORTALITY OF THE TRAUMA POPULATION, CERTAINLY AS DR. HOOTS POINTED OUT, TRAUMA PATIENTS HAVE AN EXTREME IMMEDIATE HYPOCOAGABILITY BUT WHAT CONCERNS US MORE WHEN WE TAKE CARE OF PATIENTS THAT SURVIVE TRAUMATIC BRAIN INJURY, THEY HAVE A PERSISTENT AND INCREASING HYPERCOAGABLE STATE WHICH PRESENTS AS DEEPENING THROMBOSIS OR PULMONARY EM BOW LIFIC AND ALSO IN THE MICROVASCULATURE OF THE BRING, LUNG AND KIDNEY WHICH ARE CONTRIBUTING TO MULTI-ORGAN DYSFUNCTION FOLLOWY TBI. SO WE WANTED TO START TO TAKE A LOOK AT THIS, CERTAINLY BRAIN INJURY MODELS IN MICE ARE NOT EASY TO COME BY AND BRAIN INJURY MODELS IN HUMANS ARE EVEN HARDER TO CONSENT FOR. SO, OUR FIRST AIM WAS TO DETERMINE WHETHER TBI CAUSES CHANGES IN COALALATION AND MORE SPECIFICALLY, IF THERE ARE CHANGES IN PLATELET DEPENDENT COAGULATION PARAMETERS AFTER TBI WITH THE HYPOTHESES THAT TBI WILL ALTER COAGULATION IN A TIME-DEPENDENT MANNER AND PLATELET POPULATION HAS CHANGE AFTER TB I AND ALTERATIONS IN THE COAGULATION WILL BE DUE TO THESE CHANGES AND THE PLATELET CONTRIBUTION TO CLOT FORMATION. THE WAY THAT WE DECIDED TO DO THIS WAS WITH A MURINE MOD EF OF TBI. IT'S A CONCUSSIVE TBI BY WEIGHT DROP. 85% OR MORE OF BRAIN INJURIES ARE BLUNT INJURIES. WE TOOK A VERY SIMPLE BLUNT INJURY WITH A 400 GRAHAM WEIGHT DROPPED FROM ONE CENTIMETER AND THE WE VALIDATED THIS BY THE WRITING REFLEX RESPONSE TIME. THE MOUSE AFTER THE CONCUSSIVE INJURY LAYS ON ITS BACK AND THE TIME IT TAKES TO FLIP BACK OVER TO A PRONE STATE IS THE RIGHTING REFLEX RESPONSE TIME. THE COAGULATION ASSESSMENT, BECAUSE I USE SIMPLE TRAUMA APPLICABILITY, SO, WE USE THIS COAGULATION TESTING IN THE LITERATURE NOW IS MOSTLY REPRESENTED AS TEG. THIS IS A COMPETING COMPANY WHICH IS ROTEM. YOU CAN SEE THE DIAGRAM ON THE BOTTOM RIGHT OF THE TEG AND ROTEM AND THIS IS A PICTURE PICK. FOR THOSE UNFAMILIAR WITH THE TESTING, BLOOD IS PUT INTO A CUP. THE CUP IS PLACED INTO A PIN AND EITHER THE CUP OR PIN IS SPUN DEPENDING ON WHETHER IT IS TEG OR ROT EMPLOYMENT AND AS CLOT FORMS, AS THE DYNAMICS OF CLOT OCCUR THE PIN MOVES AND YOU GET THE DISTRACTION OF THE PIN WHICH CAUSES THE CURVE. THE ROTEM FOR THOSE UNFAMILIAR WITH IT, HAS SEVERAL PARAMETERS AND WHAT IS IT EXCITING ABOUT THIS COAGULATION TESTING YOU IS CANNOT ONLY MEASURE HOW CLOTS START BUT HOW QUICKLY CLOTS FORMS AND HOW STRONG THAT CLOT IS THE AND THEN HOW THAT CLOT BREAKS DOWN AS WELL. SO THERE ARE SEVERAL PARAMETERS THAT I'LL GO THROUGH IN OUR RESEARCH. THE CT A CLOTTING TIME, THE INITIATION OF CLOT. THERE IS THE CLOT FORMATION TIME OR THE CFT WHICH IS REALLY THE TIME TO 20 MILLIMETERS OF AMPLITUDE OF DEFLECTION OF THAT PIN WHICH IS USUALLY ASSOCIATED WITH THE FIBROAND POLYMERIZATION AND THE THE MCF, PROBABLY THE MOST IMPORTANT PART FOR US IN TERMS OF OUR RESEARCH IS THE MAXIMAL CLOT FIRMNESS AND THIS IS REALLY MADE UP OF TWO COMPONENTS, PLATELET COMPONENT AND THEN THE PROTEIN COMPONENT MOST OF THE FIBRINOGEN. FOLLOWING THIS 30 MINUTES TO THE SEE HOW MUCH OF THAT CLOT BREAKS DOWN. IN TERMS OF THE TEST THAT YOU'LL SEE OVER THE NEXT FEW SLIDES, THERE IS THE NATEM ON THE ROTEM MACHINE WHICH IS NATIVE WHOLE LOAD SIMPLY RECALLSIFIED BECAUSE THE BLOOD IS CITRATED TO ANTI-COAG LATS AND BLOOD IS NICE YOU CAN TAKE IT OUT BUT YOU HAVE TO MODIFY THE BLOOD SO WE CAN CONTINUE TO WORK WITH IT AND NOT HAVE THE IMMEDIATE CLOTTING. CERTAINLY MICE ARE VERY HARD ESPECIALLY TO WORK WITH FROM A COAGULATION STANDPOINT. THEY TEND TO BE MORE HYPERCOAGULABLE. THE EXTEM MEASURES COAGULATION CATION CADE. THIS IS RECALLSIFIED BLOOD AND WE TAKE THAT FURTHER WITH FIBTEM WHICH IS THE EXTRINSIC COAGULATION CASCADE, RECALLSIFIED TO REVERSE CITRATE AND TISSUE FACTORS ADDED AND ENACTIVATE PLATELETS AND THIS ALLOWS US TO LOOK AT THE FIBRIN-ONLY PORTION OF THE CLOT. SO, IN THE FIRST EXPERIMENT THERE WERE NO CHANGES IN THE NATIVE WHOLE BLOOD AT 24 HOURS AFTER TBI WHICH SEEMING LET SLIGHTLY DISAPPOINTING BUT DISRUPTING THE WHOLE BLOOD COAGULATION OF THE MOUSE CAN BE DIFFICULT. IN ADDITION, THE PLATELET COUNTS WERE UNCHANGED AND THE FIBRINOGEN CONCENTRATION OVER TIME WAS ALSO UNCHANGED. SO WE DECIDED TO LOOK A LITTLE BIT FURTHER AT THE PLATELET CONTRIBUTION TO CLOT WHICH AS I DESCRIBED THESE DON'T LOOK DIRECTLY AT THE PLATELET COMPONENT BUT IF YOU TAKE THIS SIMPLE EQUATION FOR THE PERCENT MCF, A PLATELET, TAKES THE MCF MINUS. [ INDISCERNIBLE ] AND FIND THE PLATELET COUPLEBUTION TO THE CLOT AFTER RUNNING EXTEMAND FIB TEM. THERE IS IN CREASED MCS SEEN AT 24 HOURS AFTER TBI COMPARED TO SHAM MICE. HERE IS THE STANDARD SHAM MOUSE, BLOOD DRAWN, NO INJURY OCCURRED. INTERESTINGLY, 10 MINUTES AFTER THE TBI THERE IS REALLY NO DIFFERENCE IN THE MCF OF THE EXTEMBUT USING EXTRINSIC COAGULATION THERE IS INCREASED MCF AFTER TBI. AND THIS IS CERTAINLY TIME DEPENDENT AT 24 HOURS BUT NOT AT 10 MINUTES. SO WHEN WE BLOCK THE PLATELET CONTRIBUTION OF CLOTS, THERE IS STILL INCREASED MCF AT 24 HOURS AFTER TBI AS YOU CAN SEE IN THE 24-HOUR GROUP COMPARED TO 10 MINUTE GROUP AND SHAM GROUP. SO WHEN WE USE THAT MATHEMATICAL EQUATION TO LOOK AT THE PLATELET CONTRIBUTION TO THE CLOT, INTERESTINGLY, ALTHOUGH TOTAL CLOT IS INCREASED FROM THE EXTEM, THE PLATELET CONTRIBUTION CLOT AT 24 HOURS AND TBI IS DECREASED. SO WE CONCLUDE FRIDAY THIS, WE HAVE INCREASED CLOT STRENGTH BUT THE PLATELET CONTRIBUTION SLOT DECREASED. SO THE PLATE LETCOUNTS DON'T CHANGE SO THEY DON'T EXPLAIN WHY THE CHANGES IN PLATELET CONTRIBUTION IS CLOT FORMATION MAY CHANGE SO WE STARTED TO LOOK AT OTHER FACTORS. PREVIOUS RESEARCH SHOWS PLASMA AND MICROPARTICLES CAN BE INCREASED AFTER TBI AND CEREBRAL HEMORRHAGE AND ALSO BE ASSOCIATED WITH INCREASED MORTALITY AND CAN HAVE CLINICALLY RELEVANT PREDICTIVE VALUE AS YOU CAN LOOK AT DIFFERENT CELL DERIVED MICROPARTICLES AND THEN THEY HAVE BEEN SHOWN TO BE INDEPENDENTLY HYPERCOAGULABLE BUT THAT IS MOSTLY FROM THE SEREIN CONCENTRATION ON THE USED OF THE MICROPARTICLES. SO, HOW MICROPARTICLES ARE ALTERED AFTER TBI HAS NOT BEEN DESCRIBED. SO WE TRIEDED TO DESCRIBE IT. SO MICROPARTICLES EXPRESS DIFFERENT RECEPTORS ON SURFACE MARKERS BASED ON CELL OF ORIGIN SO WE USE CD41 AS A MARKER OF PLATELET-DERIVED MICROPARTICLES CERTAINLY CD41 IS THE SPECIFIC FOR PLATELETS AS WELL AS MEGO CAREIO SITES. THE METHODS OF THIS AGAIN WE USED THE MURINE MODEL OF TBI AND WE USE OUR MICROPARTICLE SITESMENT BY NANOSITE TRACKING ANALYSIS. THIS IS A LITTLE BIT DIFFERENT THAN FLOW. INSTEAD OF FLOW, WE USED LASER-INDUCED MEASUREMENT OF LIGHT SCATTER AND BROWNIAN MOTION TO ALLOW US TO MEASURE SMALLER PARTICLE SIZES AND ABLE TO BE MEASURED ON FLOW. SO WE CAN GET DOWN TAN NANOMETERS RATHER THAN SEVERAL00 WHICH IS THE LOWER LIMIT OF FLOW CYTOMETRY, THE ONLY DISADVANTAGE OF USING THIS IS WE CAN ONLY LOOK AT A SINGLE ANTIBODY TAG AT A TIME. SO, THE NEXT EXPERIMENT THAT WE LOOKED AT WAS CHARACTERIZE TEMPORAL CHANGES OF THE CD41 POSITIVE MICROPARTICLES AFTER TBI. TRYING TO IGNORE THIS. INTERESTINGLY, WHAT WE LOOKED AT THE MICROPARTICLE AFTER TBI, WE FOUND A VERY TIME DEPENDENT CHANGE AND INITIALLY WE THOUGHT THERE IS INJURY MICROPARTICLES WOULD GO UP IMMEDIATELY AND CONTINUE THAT WAY AND WHAT WE FOUND WAS THE OPPOSITE. SO AT THIRT MINUTES AFTER TBI, THERE IS REALLY NO DIFFERENCE IN RIGHT BEFORE TBI BUT AT 3 AND 24 HOURS, THERE IS THIS SIGNIFICANT DECREASE IN THE TOTAL AMOUNT OF MICROPARTICLES. THIS IS A REBOUND BY 72 HOURS WHICH PHASED BACK DOWN 7 DAYS. WHEN WE LOOKED AT THE PLATELET-DERIVED PARTICLE NIKE ROW REENUMERATION, WE SAW THE SAME THING AT 3 AND 24 HOURS, A SIGNIFICANT DECREASE IN THE PLATELET-DERIVED MICROPARTICLES BUT WHEN WE LOOKED AT THE TOTAL PERCENTAGE OF MICROPARTICLES WHICH WERE CD41 POSITIVE, WE FOUND A TREND FROM 30 MINUTES TO 24 HOURS GOING UP TO ALMOST 60% OF THE TOTAL POPULATION OF MICROPALS WHERE CD41 POSITIVE. SO IT CONCLUDED THAT ALTHOUGH THE TOTAL AMOUNT OF MICROPARTICLES ARE DOWN, THE PROPORTION OF THESE WHICH ARE CD41 POSITIVE IS A LOT HIGHER AT THAT 24-HOUR TIME POINT. SO NOW WE WANTED TO LOOK AT WHETHER OR NOT THESE MICROPARTICLES DIRECTLY AFFECT COAGULATION AND WE HYPOTHESIZED THAT HARVESTED FROM SHAM MICE WHICH UNDERGO SHAM INJURY, WELL CHANGE ITS BLOOD COAGULATION TO LOOK MORE LIKE THE SHAM COAGULATION WE SAW WHEREAS THE MICROPARTICLES HARVESTED AFTER TBI WOULD CAUSE DECREASED PLATELET CONTRIBUTION TO CLOT AS SEEN IN THE TRAUMATIC BRAIN INJURY BLOOD I SHOWED IN THE FIRST EXPERIMENT. SO WE ESSENTIALLY DID A CROSSOVER EXPERIMENT WHERE MICROPARTICLES FROM TBI OR SHAM MICE WERE ISOLATED AT 24 HOURS AND THEN GIVEN NOT ONLY TO TBI BLOOD BUT ALSO THE SHAM BLOOD WHICH WAS HARVESTED FOLLOWING THESE INJURIES. AND WE ADDED THESE TO THE ROW TEMTEST. SO YOU SEE HERE, THE TWO GROUPS THIS IS THE SHAM BLOOD AND THE TBI BLOOD GIVEN EITHER SALINE AS A VOLUME CONTROL FOR THE DILUTION. SHAM MICROPARTICLES OR TBI MICROPARTICLES AND INTERESTINGLY, THE EXTEMMCF DID NOT GROSSLY CHANGE. BUT WHEN WE LOOK AT THE PLATELET CONTRIBUTION TO CLOT, WE SEE THAT THE PLATELET CONTRIBUTION TO CLOT IS DECREASED AFTER THE ADDITION OF THE TBI MICROPARTICLES TO THE SHAM BLOOD ON THE LEFT AND THEN INCREASED AFTER THE SHAM MICROPARTICLES ARE ADDED BACK TO THE TBI BLOOD. SO THE MICROPARTICLES REAL DOE ALMOST CHARACTERIZE WHAT WE SEE FROM THESE INJURY GROUPS. AND AGAIN, NO DIFFERENCES IN THE PLATELET COUNTS BETWEEN SHAM AND TBI BLOOD SAMPLES SHOWING THAT THE PLATELETS DON'T ACCOUNT ALONE IN COUNT FOR THE CHANGES THAT WE SEE. SO WHAT WE CONCLUDED FROM THIS IS THAT TBI DERIVED MICROPARTICLES CAUSED A QUALITATIVE ALTERATION IN PLATELET FUNCTION THAT LEADS TO DECRETION OF PLATELETS TO CLOT FORMATION WITHOUT AFFECTING OVERALL COAGULATION. WHICH I THINK IS IMPORTANT BECAUSE WHEN YOU GET CLINICAL TESTS ON THESE PATIENTS, YOU MAY NOT SEE OVERALL COAGULATION CHANGE BUT THE COMPONENTS OF WHAT IS CAUSE TAG COAGULATION CERTAINLY DOES CHANGE. TO CONFIRM THIS, WE WANTED TO LOOK AT WHETHER OR NOT THESE MICROPARTICLES ARE PRO COAGULANT SO WE USED A MICROPARTICLE PRO COAGULANT ASSAY WHICH MEASURES THE PROFIT THOL GOING THROMBIN AND INDEED -- THROMBIN -- THE MICROPALS AFTER 24 HOURS FOLLOWING TRAUMATIC BRAIN INJURY WERE SIGNIFICANTLY MORE PRO COAGULANT IN AND OF THEMSELVES. SO AGAIN BACK TO THE SUMMARY. WE FOUND THAT AFTER INDUCING A TRAUMATIC BRAIN INJURY, I DIDN'T HAVE AS NICE A GRAPHIC OF THE MOUSE BRAIN THERE. WE DO FIND THAT PLATELETS ARE ACTIVATED, RELEASING PLATELET-DERIVED MICROPARTICLES WHICH DO SEEM TO CHANGE EX-VIVO COAGULATION AND POTENTIALLY AFFECT CLOT INITIATION AND THROMBOSIS. SUMMARY POINTS OF THE PRESENTATION CONCUSSIVE TRAUMATIC BRAIN INJURY LEADS TO POST COAGULATION CHANGE IN A MURINE MODEL NOT REPORTED IN THE LITERATURE AS TO BEING ABLE TO USE CLINICAL TESTS TO MEASURE COAGULATION CHANGES IN MICE. THE CHANGES ARE NOT EXPLAINED BY PLATELET COUNT OR FIBRINOGEN CONCENTRATION ALTHOUGH OTHER PRESENTATION HAS PROBABLY ARGUE DESPITE CONCENTRATION BEING THE SAME ACTIVITY MAYBE DIFFERENT AND THAT IS CERTAINLY VALID. POST-TRAUMATIC BRAIN INJURY MICROPARTICLE POPULATION UNDERGO TEMPORAL CHANGES IN NUMBER AND CONTENT AND THESE CAN BE INDEPENDENTLY PRO COAGULANT WHICH MAY CONTRIBUTE TO THE OBSERVED CHANGES IN COAGULATION. [ READING ] SO IF THESE MICROPARTICLES AFFECT PLATELET FUNCTION OR JUST POTENTIALLY SUBSTITUTING FOR PLATELET FUNCTION? DETERMINING INTERACTION OF THESE MICROPARTICLES WITH THE VASCULAR ENDOTHELIUM WHICH I THINK CERTAINLY GUESS TO THIS SYMPOSIUM AND THE THROMBOTIC POTENTIAL AND CEREBRAL AND SYSTEMIC MICROVASCULATURE LOOKING AT THE MODIFIABLE COMPONENTS OF POST TBI MICROPARTICLES AND LOOKING AT WHETHER MICROPARTICLES CAN BE A TARGET FOR POST-TRAUMATIC THROMBOEMBOLIC PROPHYLAXIS. FOR THE NEUROSURGEONS IN THE AUDIENCE, THE IDEA OF GIVING AGES PRIN IMMEDIATELY AFTER TRAUMATIC BRAIN INJURY IS NOT ATTRACTIVE BUT IS THERE ANOTHER THERAPEUTIC WE COULD GIVE WHICH DOESN'T INHIBIT THE PLATELETS BUT MAY ADDRESS THAT POST-TRAUMATIC HYPERCOAGABLE STATE WITHOUT POTENTIALLY LEADING TO INCREASED BLEEDING? THAT'S ALL I HAVE. THANK YOU. [ APPLAUSE ] >> HOW DO MICROPARTICLE -- [ INDISCERNIBLE ] >> SO FOR US IT'S JUST DIFFERENTIAL SENTRI FEWIGATION. >> SO THIS MICROPARTICLE USE -- [ INDISCERNIBLE ] >> ABSOLUTELY. THE EXTRACELLULAR MICROVESICLE POPULATION SUMMERY CAN BE BROAD. WE HAVEN'T BEEN ABLE YET TO CHARACTERIZE THE FULL CELL DERIVED POTENTIAL BUT I CAN TELL YOU THAT LOOKING AT THESE OVER 90% OF THEM ARE IN THE 200-400 NANOMETER RANGE FOR SIZE, ALTHOUGH WE ARE RECOVER LARGER. MOST ARE RIGHT IN THAT SIZE RANGE. >> [ INDISCERNIBLE ] >> SINCE SOME OF THE EARLIER TALKS HAVE ALREADY SHOWN THIS THAT RECOVERY OF THE BLOOD-BRAIN BARRIER CAN BE REALLY RAPID, DID YOU HAPPEN TO LOOK AT EARLIER TIME POINTS RATHER THAN JUST OUT AT 24 HOURS? >> SO, FROM A TEMPORAL PATHWAY, WE LOOKED AT 10 MINUTES, 30 MINUTES, 3 HOURS AND 24 HOURS OUT TO 7 DAYS. WE ALSO NOW COME BACK IN OUR NEXT DATASET TO LOOK AT THE SIX HOUR TIME POINT. INTERESTINGLY, BECAUSE THE 6 HOUR TIME POINT IN THE MURINE TBI MODEL IS REALLY WHERE THE PRO-INFLAMMATORY CYTOKINE CASCADE IS AT ITS PEAK. WE ACTUALLY ARE SEEING AND WE WILL PRESENT THIS WEEKEND AT SHOCK SOCIETY MEETING, THAT THE PLATELET CONTRIBUTION TO CLOT IS INCREASED AT THE 6 HOUR TIME POINT AND MIRRORS THAT SITEO KINE RESPONSE. >> DO YOU HAPPEN TO SEE ANY INDICATION OF POTENTIAL RECOVERY RESPONSE THEN AT THE LATER TIME POINTS? SO FIRST WE HAVE INJURY AND THEN WE RESPOND AND THEN WE DRY GET BACK TO WHERE WE WERE. >> IF WE LOOKED AT OUR MICROPARTICLE NUMBER THERE IS RECOVER TOW BASELINE LEVELS BY 7 DAYS AND WE SUPERINTENDENT GONE OUT THAT FAR WITH OUR RESEARCH YET. -- WE HAVEN'T GONE OUT -- >> FOR THE POST TBI, PROCOAGULANT MICROPARTICLES, HAVE YOU HAD A CHANCE TO DO THE SIMPLE EXPERIMENT TO SEE IT IS BLOCKED TO WHAT EXTENT BY ANTI-TISSUE FACTOR ANTIBODIES? SO OFTEN WITH TRAUMATIC INJURY AND GENERAL TISSUE STIMULATION OR DISRUPTION, TISSUE FACTORS ARE A PRIMARY SUSPECT. >> WE HAVEN'T QUITE DONE THAT. I DIDN'T SHOW WE HAD A SEPARATE TISSUE FACTOR RELATED PROO AGULANT ASSAY FOR MICROPARTICLES AND THERE WERE NO CHANGES IN THAT WHICH WE WERE SURPRISED BY. WE EXPECTED TISSUE FACTOR TO BE A BIG PART OF IT AND AT LEAST FROM THAT ASSAY IT WASN'T. >> THANK YOU VERY MUCH DR. GOODMAN FOR YOUR TALK AND THERE WILL BE MORE TIME FOR DISCUSSION LATER TODAY. I'D LIKE TO MOVE FORWARD WITH KATERINA AKASSOGLOU WHO TALK ABOUT FIBRINOGEN AND NEUROLOGICAL DISEASES AND MECHANISMS AND IMAGING AND THERAPEUTICS. WE ARE DIMMING THE LIGHTS A LITTLE BIT. >> KATERINA AKASSOGLOU: I WOULD LIKE TO START BY THANKING THE ORGANIZERS FOR GIVING ME THE OPPORTUNITY TO PARTICIPATE IN THIS VERY EXCITING WORKSHOP. AS WE STRIVE TO UNDERSTAND THE COMPLEXITY OF NEUROLOGICAL DISEASES IT BECOMES INCREASINGLY CLEAR THAT WE CANNOT STUDY THE BRAIN OR DEVELOP EFFECTIVE TREATMENT FOR DISEASE BY OVERLOOKING GLEIA AND NOT FOCUSING ON EXTRACELLULAR ENVIRONMENT AND HOW IT CHANGES NEUROLOGIC DISEASE. AND THE MAJOR CONSTITUENTS IS THE BRAIN VASCULATURE. YOU CAN SEE HERE THAT THREE-DIMENSIONAL IMMUNOLABELLING OF BRAIN VASCULATURE IN MOUSE BRAIN. YOU CAN SEE THE AREAS HERE OF THE CORTEX LARGE CORTICAL VOLUME AS WELL AS THE HIPPOCAMPUS. AND WITH IMAGING WE ARE NOW ABLE TO LOOK AT OBTAIN MOLECULAR INFORMATION OF THE VASCULATURE IN THIS LARGE VOLUMES. OF COURSE NEUROLOGICALLY SEEING ONE OF THE KEY CHANGES IN THE BRAIN VASCULATURE IN THE DISRUPTION OF THE BLOOD-BRAIN BARRIER. THIS IS IN-VIVO PHOTO IMAGING IN ANIMAL MODEL WITH MST IS VERY DIFFERENT THAN NORMAL VASCULATURE BECAUSE IT IS BLOOD PROTEINS THAT EXTRAP LATE FROM THE LUMEN AND THIS IS DEPICTED HERE WHEN WE USE RED DYE THAT WE INJECT INTO THIS MOUSE, WE CAN SEE OUTSIDE OF THE BLOOD VESSEL. AND YOU CAN SEE THAT THIS IS LARGE AREAS OF THE BLOOD VESSELS ARE ACTUALLY PERMEABLE AND ALLOW THE BLOOD TO GET OUT OF THE BRAIN. AND YOU HEARD FROM PREVIOUS PRESENTATIONS, THIS LEAKAGE OF THE BLOOD AND DISRUPTION IS A COMEDY NOMINATOR IN A VARIETY OF DISEASES INCLUDING MS, STROKE, BRAIN TRAUMA, AND CLASSIC NEURODEGENERATION LIKE ALZHEIMER'S DISEASE. HOWEVER, THERE IS ALSO THE CHICKEN AND EGG QUESTION. IS THE LEAKAGE OF BLOOD A CONSEQUENCE OF PATHOLOGY OR IS IT POSSIBLE THAT THIS LEAKAGE OF BLOOD PROTEINS CAN BE A DRIVER OF THIS PATHOLOGIC DISEASES? AND OF COURSE THE ONLY WAY WE CAN ANSWER IN BIOLOGY CHICKEN AND EGG QUESTIONS IS WHEN WE START TO UNDERSTAND PERIPHERAL TRIGGERSES AND MOLECULAR DETERMINANTS WITH SPECIFIC GENETIC MODELS AND INHIBITORS AND CAUSALITY NECERTAIN PATHWAYS. AND IN MY LAB, I CHARACTERIZED BLOOD CLOTTING FACTORS AND FIBRINOGEN AND SHOWN THAT IT PLAYS IMPORTANT ROLE IN BOTH DRIVING NEURODEGENERATION AND INFLAMMATION OF THE BRAIN. COORDINATING COMMUNICATION BETWEEN PERIPHERAL AND NEW SYSTEM AND THE BRAIN INTERFACE, AND ALSO AT THE SAME TIME THIS IS A REALLY GOOD NICHE TO DEVELOP NOVEL IMAGING TECHNIQUES TO IMAGE NEUROVASCULAR INTERFACE AS WELL AS ANOTHER NICHE FOR THERAPEUTICS. THE CONCEPT OF THE LEAKAGE OF BLOOD IS A KEY COMPONENT OF NEUROPATHOLOGY AND DEFINITELY NOT NEW. THIS IS A HUMAN BRAIN IN MS PATIENT AND YOU CAN IDENTIFY VERY LARGE LEGION HERE WHERE DESCRIBED FROM ONE OF THE FIRST DEFINITIONS OF THE EARLY 1800's. LESIONS WERE DESCRIBED AS SMALL LUMENS OF SMALL VESSELS IN COONS WITH BLOOD WITH ALTERITIONS OF INDIVIDUAL -- CHRONIC INFLAMMATION. SO, THIS HYPOTHESES WE HAVE IS THAT IT IS UNEXPLORED WHETHER BLOOD PROTEINS ARE CONTRIBUTORS TO DISEASE. OF COURSE THE MAIN CHALLENGE IS WHICH BLOOD PROTEIN TO STUDY? THERE ARE IS SO MANY. SO WHICH ONE TO FOCUS? AND OUR RESEARCH WAS ENTIRELY HYPOTHESES DRIVEN AND PRESENTED THREE KEY COMPONENTS WHAT MAKES THIS SO ATTRACTIVE TO STUDY IN THE BRAIN. FIRST ABANDONED EVIDENCE FROM IMMUNOPATHOLOGY ABOUT THE PRESENCE IN NEUROLOGIC DISEASE. THIS IS FROM DIFFERENT PAPERS TO THE LITERATURE OF IMMUNOHISTOLOGY FOR FIBRINOGEN IN FUME BRAIN AND YOU CAN SEE MS, TBI AND ALZHEIMER'S DISEASE WHERE THERE IS ABANDONED AND SUSTAINED DEPOSITION OF FIBRIN OVER THE COURSE OF THE DISEASE. THIS IS ALSO THE CASE IN ANIMAL MODELS. THIS IS LABELING IN A CLEAR SPINAL CORD AND YOU CAN SEE HERE FIBRINOGEN LABELED IN RED, SPANNING OVER SEVERAL MILLIMETERS INTO THE LESIONS OF THESE MICE SUGGESTING THAT INDEED, THIS IS PRESENT IN THE BRAIN. THE FIBRINOGEN HAS A VERY PROFOUND PRO-INFLAMMATORY FUNCTION WHEN IT IS CONVERTED TO FIBRIN. IT'S SOLUBLE IN THE BLOOD AND THROMBIN IS CONVERTED TO FIBRIN AND PLAYS A ROLE IN BLOOD CLOTS AND INFLAMMATION. AND THE ABILITY FOR FIBRINOGEN TO PLAY THIS DUAL ROLE IS UNIQUE MOLECULAR STRUCTURE. IT CONTAINS A BINDING GAMMA TRAIN, A BINDING SITE THAT IS. [ INDISCERNIBLE ] WE KNOW THAT THE DRUGABLE INTERACTION, THE DRUGABLE DEVELOPED BY BARRY KOEHLER IS ACTUALLY A THROMBOLYTIC INTERACTION OF FIBRIN WITH THE PLATELET BUT THIS DRUG DOES NOT DEVELOP ANTI-INFLAMMATORY FUNCTIONS BECAUSE IN FLORIDA -- [ INDISCERNIBLE ] WE COULDN'T HELP BUT ASK THE QUESTION WOULD IT BE POSSIBLE TO SELECTIVELY TARGET THE PRO-INFLAMMATORY FIBRIN AND IN THIS WAY, SUPPRESSING INFLAMMATION WITHOUT ASSESSING THE BLOOD CLOTTING. THE FIRST ORDER OF BUSINESS IS TO SHOW NEUROLOGIC DISEASE. IN MY LAB TOGETHER WITH MANY OTHERS IN THE FIELD WHAT WE SHOWED IS WHEN WE DETECT FIBRIN IN MICE, GREAT RANGE OF NEUROLOGIC DISEASES, MS, BRAIN TRAUMA, ALZHEIMER'S DISEASE, THE MICE RECOVER BETTER, HAVE LESS NEUROLOGIC SIGNS AND LESS INFLAMMATION OF THE BRAIN. SO JUST GETTING RID OF FIBRIN IS REALLY PROTECTIVE IN A WIDE RANGE OF NEUROLOGIC DISEASES. OF COURSE HOW DOES THIS HAPPEN? AND WHEN WE STARTED THIS WORK, IT WAS NOT KNOWN. SO WE IDENTIFIED THE MICROGLIA CELL, THE FIRST CELLULAR TARGET IN THE BRAIN. WE SHOWED MICROGLIA BINDS AND MEDIATES ACTIVATION. THESE CELLS IDEALLY POSITIONED TO RECEIVE SIGNALS FROM THE VASCULATURE. THIS IS ONE OF THE FIRST SCHEMATICS SHOWING VASCULAR MICROGLIA IN POSITION AROUND BLOOD VESSELS AND WILL PERFORM LIVE IMAGING OF THE BRAIN WHERE WE CAN USE THE PHOTO MICROSCOPY TO IDENTIFY THE DYNAMICS OF THIS CELL IN THE MOUSE BRAIN AND CAN SUBSTANTIALLY EXTEND THE PROCESSES ALLOW CAN BLOOD VESSEL WALLS. IT ALSO CAN BE VERY RESPONDED TO VASCULAR INJURY. WE INJURE BLOOD VESSEL IN THE BRAIN AND SEE HOSTELS EXTEND INTO THE SITE OF THE VASCULAR DAMAGE. ON THE LEFT HERE YOU YOU CAN SEE INJECTION OF THE CONTROL VEHICLE WHILE IMAGE AT THE SAME TIME IN THE LIVING MOUSE AND YOU CAN SEE THE BASELINE MICROGLIA. HOWEVER, WHEN WE INJECT FIBRINOGEN, THERE IS VERY RAPID HEMOTACTIC RESPONSE OVER TO THE END OF THE NEEDLE. AND THE SIMILAR CORRELATION OF THIS BLOOD-BRAIN BARRIER IN MICROGLIA ACTIVATION HAPPENS IN MS AND HERE ON THE LEFT YOU CAN SEE LIVE IMAGING BIG RED CLOUD IS LEAKING AND CORRELATES WITH THE AREA WHERE THESE CELLS HAVE -- WHAT WE DID IS USED FIBRINOGEN MOUSE MUTATED WITHIN THE BINDING ITEM A AND THIS MOUSE DOESN'T HAVE ANY DEFECTS IN COAGULATION. [ INDISCERNIBLE ] ALSO DEGREES EXONAL DAMAGE AND DECREASE OF CHEMICAL SIGNS. SUGGESTING THAT SIGNALING OF THIS SIGNALING REQUIRED FOR MICROGLIA IN EXONAL DAMAGE. SO THIS IS A SUMMARY FOR WORKING MODEL WHERE WE CONSIDERED FIBRINOGEN TO BE REALLY ONE OF THOSE TRIGGERS IN THE EXTRACELLULAR ENVIRONMENT AND ACTIVATE MICROGLIA TO LEAK BOTH INTO IMMUNE PROCESSES LIKE FOR EXAMPLE, NEURODEGENERATIVE PROCESSES AND THE CONCEPT OF TRIGGER, I THINK IT'S A VERY IMPORTANT NEUROLOGICAL DISEASE. THIS IS WITH THE COVER OF NATURE WHEN THE WHOLE GENOME SEQUENCING WAS PERFORMED. TWO IDENTICAL TWIN SISTERS, ONE WITH MS AND ONE WITHOUT. NO GEETIC OR EPIGENETIC CHANGES WERE FOUND AND THE CONCLUSION WAS THERE HAS TO BE A TRIG THEY'RE CAUSES TO DEVELOP AND THE OTHER TO NOT. SO BLOOD-BRAIN BARRIER DISRUPTION AND THE DIFFERENT TRIGGERS IN NEUROLOGIC DISEASE COULD BE SEEN AS ONE OF THOSE SIGNALS AS MICE BRING PATHOLOGY TO ONE INDIVIDUAL VERSUS ANOTHER EVEN THOUGH THEY HAVE A SIMILAR GENETIC BURDEN FOR A SPECIFIC DISEASE. SO, GEETIC MODEL SHOWED TARGETING RECEPTOR COULD BE IMPORTANT FOR ELIMINATING FUNCTIONS OF FIBRIN. SO HOW COULD WE DO THIS? SO, WHAT WE FOUND IS THAT THIS IMPORTANT FEATURE. [ INDISCERNIBLE ] NORMALLY EXPOSED AFTER FIBRINOGEN IS CONVERTED TO FIBRIN. SO WE DEVELOPED CLINICAL ANTIBODY TO TARGET THIS AND MAYBE THIS ANTIBODY WILL BE FIBRIN-SPECIFIC AND NOT RECOGNIZE FIBRINOGEN. AND BECAUSE IT IS NOT OVERLAPPING WITH PLATELET BINDING SITE, WE HOPE IT WOULD NOT BE ANTICOAGULANT. THOSE SELECTED FOR FIBRIN USE -- [ INDISCERNIBLE ] SO WE DID NOT THINK NOT CLEARLY DESIRABLE S SO WE DEVELOPED A SERIES OF ANTIBODIES AND INDEED CRITERIA WE SET FOR THIS ANTIBODY. IT'S A HIGH BINDER AGAINST THE FIBRIN EPITOPE WHICH IS WHAT WE DESIRED BUT THIS ANTIBODY FOR HIGH PREFERENCE TO BIND TO FIBRIN COMPARED TO FIBRINOGEN. RECOGNIZES PATHOGENIC FORM. THIS ANTIBODY WAS IN-VITRO AND ACTIVATION ASSAY MICROIVATING MICROGLIAL CELLS -- [ INDISCERNIBLE ] THE ANTIBODY BLOOD GENE EXPRESSION CHANGES OF PATTERNS INDUCED IN MICROGLIA AND ALSO MACROPHAGE CELLS. YOU CAN SEE SEVERAL GENES UPREGULATEOD MACROPHAGES 5B8. THIS IS ONE OF THOSE GENES. SUBFRAGMENTS CAN ALSO BLOCK GENE EXPRESSION BUT THE ANTIBODY SPECIFIC FOR FIBRIN-INDUCED ACTIVATION WHEN WE USE THIS ACTIVATOR -- INFECTION WILL USE THIS MECHANISM, THIS PATHWAY REMAIN INTACT IN THE BRAIN IN THE PERIPHERY. WE HAVE DONE SEVERAL CLINICAL STUDIES NOW -- [ INDISCERNIBLE ] THIS IS A MODEL FOR MS. THE MICE PARALYZE AND TREATMENT AFTER PERILLIZATION OF THESE MICE AND ADMINISTRATION OF THE ANTIBODY IS REALLY USING RELAPSE AND THE PARALYSIS OF THESE MICE AFTER ADMINISTRATION. ANTIBODY BLOCKS MICROGLIAL ACTIVATION AND INFLAMMATION AND WHAT THEY FIND PRO DOWNED ABILITY TO BLOCK EXONAL DAMAGE AND ALSO GENERATION OF SPECIES IN THESE MODELS. ANOTHER FEATURE THAT IS ALSO PROFOUND FOR THE ANTIBODY IS THE DOE CREASE OF PRO-INFLAMMATORY CELLS WE OBSERVED IN THE BRAIN SUGGESTING THAT RECRUITMENT IS ALSO REDUCED BECAUSE PERIPHERAL INFLAMMATORY CELLS IS IMPORTANT FOR NEUROLOGIC DISEASES. IMPORTANTLY, THE ANTIBODY DOES NOT NEED AFFECT FIBRIN POLARIZATION IN-VITRO OR BLOOD CLOTTING. SO YOU CAN SEE FIBRINOGEN DISCERN YOU CAN SEE HERE ANTIBODY THAT IS NOT AFFECTING THE POLYMERIZATION IN-VITRO. THIS IS THE CLOTTING TIME FROM MICE IN-VIVO AFTER DOSE WITH ANTIBODY. WE HAVE DONE PROLONGED STUDIES AND STUDIES WITH 3 AND 4 TIMES THE DOSE OF THERAPEUTIC DOSE AND THE ANTIBODIES NOT ANTICOAGULANT. SO THIS SUGGESTS THAT PROOF OF PRINCIPAL THAT PHARMACOLOGICALLY WE CAN DISSECT THE DAMAGING FUNCTIONS TARGETED -- WITHOUT AFFECTING BENEFICIAL -- [ INDISCERNIBLE ] IMPLICATIONS AS A TREATMENT TO TARGET INNATE IMMUNE ACTIVATION BY DISRUPTION IN BRAIN AND IN THE PERIPHERY. THIS COULD ALSO TIE IN WELL WITH CURRENT -- IN MS, MOST OF THE ANTI-INFLAMMATORY TREATMENT, MODIFYING THERAPIES, TARGET DOWNSTREAM. THEY ARE ALL IMMUNE TREATMENTS TARGETING FIBRIN INTERPASSION WITH THE MICROGLIA IS MORE ABSENT TREATMENT TARGETING THESE PATHWAYS AND ALSO WOULD HAVE THE ADDIVE AFFECT THAT IT MIGHT NOT ONLY SUPPRESS IMMUNE FUNCTIONS BUT ALSO NEURODEGENERATION. AND ALSO WITH THE VALIDATED MS, AND WE ARE WORKING ON MODELS OF TRAUMATIC BRAIN INJURY AND ALZHEIMER'S DISEASE TO SEE WHETHER THESE PATHWAYS WOULD BE ALSO APPLICABLE IN THESE AREAS. I WOULD LIKE TO THANK MY LAB AND VERY TALENTED TEAM THAT DOES THIS WORK. [ READING ] *6 ALL MY WORK HAS BEEN FUNDED BY NIND IS AND SUPPORT TO MY PROGRAM AND THE NATIONAL MULTIPLE SCLEROSIS SOCIETY AND DIFFERENT FOUNDATIONS AND A GRANT FROM THE HILTON FOUNDATION TO DO BIOMARKER STUDY IN PATIENTS TO SEE IF THE POPULATION IS CHANGING AND DIFFERENT FOUNDATIONS AND POSTDOC FELLOWSHIPS AND GRANTS IN MY TEAM. THANK YOU VERY MUCH FOR YOUR ATTENTION. [ APPLAUSE ] >> WE'D LIKE TO THANK DR. KATERINA AKASSOGLOU. IF THERE ARE ANY QUESTIONS WE HAVE TIME FOR ONE OR TWO QUESTIONS. [ OFF MIC ] >> YES, EXCELLENT QUESTION. OUR STUDIES WITH THE FIBRINOGEN AND SOMETIMES WE USE FLUORESCENCE, YOU CAN SEE IT CLOTTING RIGHT IN FRONT OF YOUR EYES STOW CLOTS IN SECONDS. SO THERE SEEMS TO BE SOME VERY HIGH PROCOAGULANT ACTIVITY THAT MAKES IT CLOT REALLY QUICKLY. SO EVEN THOUGH THE ANTIBODIES ARE USED FOR HISTOLOGY TO NOT REALLY DISCRIMINATE, I THINK A MAJORITY OF WHAT IS STAYING IS FIBRINOGEN AND NOW WE STARTED USING THERAPEUTIC ANTIBODY FOR HISTOLOGY BECAUSE IT IS FIBRIN SPECIFIC SO IT IS NICE TO BE ABLE POLICY WHERE YOU HAVE FIBRIN AND FIBRINOGEN. WE DIRECTLY IMAGED COAGULATION OF THE BRAIN AND DEVELOPING MOLECULAR PROBE FOR THROMBIN ACTIVATION AND THIS IS STUDY WE PUBLISHED TWO YEARS AGO IN ANNULS OF NEUROLOGY AND WORKING IT WITH ROGER TO BEG YOUR PARDON A NOVEL PROP TO RECOGNIZE -- TO DEVELOP A NOVEL PROP -- WHEN WE INJECT ANIMAL MODS ELSE FOR MS, THERE IS HIGH COACTIVATION ACTIVITY OF MODELS AND THIS IS NOW VALIDATED MODELS OF STROKE AS WELL IN ARTHROSCLEROSIS. WITH MOLECULAR IMAGING OR WITH STATUS OF IMAGE FIBRINOGEN IN THE BRAIN, I THINK THE ANSWER IS YES, I THINK IT CLOTS ALMOST IMMEDIATELY. >> YES, GO AHEAD, FEEL FREE TO COME TO THE MICROPHONE SO IT IS EASIER TO HEAR. THANK YOU. >> VERY ELEGANT. DO YOU HAVE ANY SENSE OF THE HISTORICOMITRY? IS IT ORDERS EVER MAGNITUDE? THE REASON I ASK THE QUESTION, FIBRINOGEN CONCENTRATION AND FIBRIN MONOMER CROSSLINKING. IS THAT OBVIOUSLY, WE KNOW THAT IN BRAIN INJURY AND OBVIOUSLY NEINFECTION, ACUTE PHASE REACTANT AND OVER A TIME OF LIKE A LIFETIME, IT JUST, I WONDER IF THE AVAILABILITY OF THIS PARTICULAR SEGMENT OF THE GAMMA CHAIN IS CONCENTRATION DEPENDENT ENOUGH SO THAT IF YOU HAD A CHRONIC INFLAMMATORY DISEASE SYSTEMICALLY THAT IT MATED HAVE A -- AND HU ANY MINOR REACHES OF THE BLOOD-BRAIN BARRIER, THAT YOU MIGHT ACTUALLY ENHANCE CHI NECESSITY OR CHRONIC UPTAKE OF THE NEURONAL ACTIVATION OR GLIAL CELL ACTIVATION AND NEURONAL INJURY? >> IT IS AN EXCELLENT QUESTION AND A DIFFICULT ONE. SO, IF I UNDERSTAND CORRECTLY, HOW TO EXPLORE THE EPITOPE AND SO THE CONVERSION TO FIBRIN DEFINITELY BY THROMBIN IS GIVING THE BEST EXPOSURE. FOR US TO BE ABLE TO DEVELOP THE ASSAY STRUCTURAL AND SCREEN ANTIBODIES, WE FOR TUS IS DEVELOPMENT AND WE COMPARED NINE DIFFERENT PREPARATIONS OF FIBRIN WITH ANY IMAGINABLE WAY INCLUDING DEGRADATION PRODUCTS OF FIBRIN TO FIND WHICH ONE WOULD BE THE ACTUAL IN-VITRO SUB STATE THAT WOULD BE BIOACT AND I HAVE MIMIC THE FUNCTIONS IN-VIVO. THAT WAS THE BIGGEST CHALLENGES FOR THIS PROJECT AND FOR DRUG DISCOVER DISCOVERY IN GENERAL. SO WHAT WE CAN SAY WITH CENTEREDY IS -- THE BEST EXPOSURE OF THE EPITOPE. HAVING SAID THAT, IMMOBILIZATION OF -- ALSO EXPOSES EPITOPE. SO THE GENE EXPRESSION CHANGES ARE MAYBE IN SOME GENES, 100 FOLD DIFFERENCE SO YOU CAN GET PERHAPS JUST MOBILIZED FIBRINOGEN 5-FOLD INCREASE IN PRO-INFLAMMATORY GENE THAT WOULD BE 80-FOLD IF YOU HAVE IS THE EXPOSED EPITOPE. SO IT IS ALSO STUDY INFLAMMATION NEEDS TO BE CAUTIOUS IN THE CONTEXT OF HOW DO WE STUDY FIBRINOGEN IN-VITRO? BECAUSE IT'S NOT THE SAME LIKE STUDYING IT IN THE BRAIN. >> JUST WONDERING. SO IT'S CLEAR IN A VERY INTERESTING PATHWAY TO TARGET. DO YOU THINK IT IS POSSIBLE TO MODEL THE OTHER SIDE OF THE PATHWAY OF A CERTAIN GENES IN MICROGLIA CONSTANTLY WHICH INTERACTING WITH THE FIBRINASE A THERAPEUTIC APPROACH FOR MS? >> TO MODEL SOME GENES AND TO MOD EEL -- >> MOG LATE EXPRESSION, I DON'T CARE. WITH OTHER TECHNOLOGY CONSTANT. >> OF COURSE. WE HAVE A STUDIES UNDERGOING IN THE LAB TO BE ABLE TO DEFINE THE PATHWAYS BOTH AT THE RNA LEVEL BUT ALSO AT PROTEOMIC LEVEL OF THE LIGAND ACTIVITY OF THE INNATE IMMUNE ACTIVATION. SO WE HAVE DESIGNED AND WE ARE HALFWAY THERE IS TO COMPARE THE ACTIVATION PATHWAYS FROM LPS, FIBRIN AND COMPLIMENT. BECAUSE COMPLIMENT AND FIBRIN SHARE THE SAME RECEPTOR. HOWEVER, THE GENE EXPRESSION IS DIFFERENT AND THE GENE EXPRESSION IS DIFFERENT. SO WHAT GIVES THE SPECIFICITY AND HOW DOES THE LIGAND INTERACTION MAKE THAT SPECIFIC? SO WE ARE EXTREMELY INTERESTED IN THE LIGAND SELECT ACTIVITY AND DOING THIS BOTH WITH RNA-SEQ APPROACHES AND ALSO PROTEOMICS. >> AS SOON AS YOU DEFINE IT, THEN IT IS POSSIBLE TO TARGET -- >> EXACTLY. WE DON'T KNOW OTHER THAN THE RECEPTOR, WE HAVE IDENTIFIED THE PATHWAY. KINASE IS ACTIVATED AND ROLE IS ACTIVATED AND BUT TO HOW MANY SELECTIVE AND TO WHAT EXTENT SELECT FOR SPECIFIC DISEASE, I THINK THAT IS -- >> WHAT DO YOU THINK ABOUT THE STRATEGY OF JUST FULLY MODULATING OR SILENCING THE LEVEL OF RECEPTOR EXPRESSIONING IN MICROGLIAL? >> SO THE RECEPTOR OF COURSE IT IS ALWAYS A QUESTION YOU GO AFTER THE LIGAND OR RECEPTOR? SO, THE RECEPTOR IS A LOT OF PROTECTIVE FUNCTIONS IN MICROGLIA AND IN GENERAL. IN MANY CASES IN PROTECTION FROM MODELS DEPENDS ON THIS RECEPTOR AND THE ABILITY TO BE ACTIVATED BY COMPLIMENT AND NOT BY FIBRIN. IN AS? PARTICULAR, THIS RECEPTOR HAS BEEN LINKED WITH SPINE ELIMINATION AND THE RECEPTOR IS ASSOCIATED WITH AUTISM AND ALSO VERY IMPORTANT TO DEVELOPMENTAL FUNCTION. SO WE LIKE TO AVOID -- IT WAS TO AVOID TARGETING RECEPTOR AND THE LIGAND I THOUGHT WOULD GIVE US MORE SELECTIVITY. >> THANK YOU. >> THANK YOU VERY MUCH DR. KATERINA AKASSOGLOU FOR YOUR PRESENTATION. WE LIKE TO MOVE FORWARD WITH DR. THERESA WHITESIDE WHO WILL BE PRESENTING EXOSOMES IN YOMA THEIR POTENTIAL AS CARRIERS OF INFLAMMATION BETWEEN THE TUMOR AND IMMUNE CELLS. >> THERESA WHITESIDE: GOOD MORNING, EVERYBODY. I'M GOING TO TALK ABOUT EXOSOMES, THAT WE CALL TECHS AND WE ARE INTERESTED IN WHETHER THEY SERVE AS BIOMARKERS OF PROGNOSIS OR CARRIERS OF INFORMATION BETWEEN THE TUMOR CELLS AND NORMAL CELLS. SO, FIRST OF ALL, A BRIEF LESSON ABOUT TUMOR DERIVED EXOSOMES. THEY ONLY, ONE TYPE OF MANY DIFFERENT EXTRACELLULAR VESICLES THAT CELLS PRODUCE. THEY ARE CHARACTERIZED BY SIZE, BY SPECIFIC DENSITY AND A UNIQUE PROTEIN PROFILE AND BY FUNCTION THE EXOSOMES MEDIATE. THEY TRAVEL FREELY, DISTRIBUTED THROUGHOUT ALL BODY FLUIDS OF ALL INDIVIDUALS BUT THEY ARE PARTICULARLY ABUNDANT IN DISEASES. THEY ARE ABUNDANT IN CNS OF PATIENTS WITH BRAIN CANCERS BECAUSE IN TUMOR CELLS THEY PRODUCE MORE EXOSOMES THAN NORMAL CELLS AND THEREFORE THE FRACTION EXOSOMAL FRACTION IN PLASMA OF THESE PATIENTS ARE ENLARGED AND SO THE RATIO OF TEX TO NON-TUMOR DERIVED EXOSOMES IS LARGE. SO HERE IS WHAT THEY LOOK LIKE IN TRANSMISSION ELECTROMICROSCOPY UP ON TOP. THEY ARE -- YOU CAN SEE A VARIETY OF SIZES BUT THEY RANGE IN SIZE FROM 30 TO 150 IN THEM SO VIRUS SIZE VESICLES SURROUNDED BY DOUBLE MEMBRANE AND WHEN YOU LOOK AT THEM BY SCANNING ELECTROMICROSCOPY AT THE BOTTOM, THEY LOOK LIKE BALLS. NOW, WHAT DO THEY CARRY? ON THE SURFACE, THEY CARRY VARIETY OF LIPIDS, A VARIETY OF PROTEINS INCLUDING RECEPTOR AND MOLECULES, AND TUMOR ASSOCIATED ANTIGENS, ET CETERA. IN THE LUMEN, THEY CARRY NUCLEIC ACIDS, DNA, mRNA AND RNA AND VARIOUS PROTEINS, INCLUDING ENZYMES, PROTEINS AND AN COPROTEINS. NOW, EXOSOMES DIFFER FROM OTHER MICROVESICLES BY UNIQUE BIOGENESIS. THEY COME FROM THE ENDOCYTIC COMPARTMENT AND YOU CAN SEE AS THE EARLY EXOSOME IS FORMED, A PART OF THE SURFACE MEMBRANE END UP IN THE EARLY ENDOSOME AND THEN THERE THEIR IS THIS REVERSE IN VAGINATION WHERE THE INTRALUMINAL VESICLES ARE FORMED IN THE EARLY OR LAYTEXOSOME AND THEN THIS BECOMES MULTIBODY FULL OF THIS INTRALUMINAL VESICLES AND WHEN THE MULLEY VESSICULAR BODY COMES TO THE SURFACE, THERE IS FUSION AS EXOSOMES GET RELEASED. BUT THE IMPORTANT THING TO SEE HERE IS THAT THE TO POOING FEE OF THE EXOSOMES THAT ARE BEING RELEASED IS SORT OF AT LEAST IN PART, REMINDS US OF THE SURFACE OF THE MOTHER CELL. SO, BECAUSE OF THAT, TEX ARE THOUGHT OF AS TUMOR SURROGATE IF YOU WISH. THEY CARRY UNIQUE LOAD OF APRIL COOLS. THAT AT LEAST IN PART -- LOAD OF MOLECULES -- THAT REFLECTS THE MOLECULAR AND GENETIC COMPONENTS OF THE PARENT CELL AND WILL THEY MANAGE TO DELIVER THIS TO RECIPIENT CELL AND BY DOING SO, CHANGE TO ALTER THE FUNCTIONS AND ALSO IN TERMS OF IMMUNE CELLS AND I'M AN IMMUNOLOGIST SO I'M PARTICULARLY INTERESTED IN AFFECTS ON IMMUNE CELLS, THEY DOWN REGULATE ANTI-TUMOR IMMUNE RESPONSES IN THE IMMUNE EFFECTOR CELL. BUT, BECAUSE OF THIS SIMILARITY IF YOU WITH ISSUE TO THIS SURFACE OF MOTHER TUMOR CELL, THEY ARE EXPECTED TO SERVE AS LIQUID TUMOR BIOPSY AND THERE IS A GREAT INTEREST IN THIS TO SEE WHETHER INDEED THIS WILL COME TO BE. SO, TEX ALTERED FUNCTIONS OF NORMAL TISSUE CELLS IN THE BRAIN TUMOR MACRO ENVIRONMENT AND THIS HAS BEEN ALREADY SHOWN IN YEARS AGO BY SCOTT IN THIS NATURE CELL BIOLOGY PAPER. DR. SKOG TOOKBLYO BLASTOMA CELLS IN CULTURE RADIO - GLIOBLASTOMA CELLS AND HAVE THEM PRODUCE EXOSOMES. AND THESE EXOSOMES WERE LABELED WITH PKH DYE, GREEN, AND WERE FED TO HUMAN BRAIN MICROVASCULAR ENDOTHELIAL CELLS YOU SEE HERE UNDER A. AND YOU CAN SEE INSIDE THE CELLS GREEN LABELED EXOSOMES. THEY WERE TAKEN IN BY THESE MICROVASCULAR CELLS AND AT THE SAME TIME, YOU CAN ALSO SEE UNDER D, THAT THEY EXPRESS GLUTE G. NOT ONLY IN -- SO THIS VASCULAR ENDOTHELIAL CELLS CO-INCUBATED WITH EXOSOMES AND ACQUIRED GLUTE ACTIVITY. NOT ONLY THAT, THESE GBM EXOSOMES ALSO STIMULATED ANGIOGENESIS IN-VITRO IN THIS CASE. YOU CAN SEE THAT THERE WAS INCREASE FORMATION OF THE LENSE WHEN THE CELLS WERE INCUBATED WITH MICROVESICLES AS COMPARED IN THE BLACK ON THE RIGHT WHERE THEY WERE INCUBATED WITH FACTORS THAT INDUCED ANDROGEN SIS. IN ADDITION, THE GBM EXOSOMES CARRIED ANGIOGENIC PROTEINS. ON THE RIGHT THERE IS A MICROVESICLE CONTENT. ON THE LEFT ACTUAL CELLS THEMSELVES AND YOU CAN SEE IN THE BLOOD THAT SOME OF THE PROTEINS SUCH AS ANGIOGENESIS, IL8 AND OTHERS, ARE CARRIED BY MICROVESICLES AND BY EXOSOMES PRODUCED BY GLIAL BLASTOMA CELLS. SO THIS SEVERS AS A PROOF-OF-CONCEPT THAT TUMOR DERIVED EXOSOMES CARRY A GENETIC MESSAGE AND DELIVER IT TO IN THIS CASE, VASCULAR ENDOTHELIAL CELLS. SO, WITH THIS IN MIND, WE WANTED TO LOOK WHETHER THE EXOSOMES IN PATIENTS WITH GLIAL BLASTOMA WHO WERE TREATED WITH A VACCINE, WHETHER THESE EXOSOMES TAKEN FROM PATIENTS COULD TELL US ANYTHING ABOUT PATIENT RESPONSE TO THE VACCINE. SO WE HAD A TRIAL WITH DENDRITIC CELLS ACTIVATED DENDRITIC CELLS PULSED WITH MANY EPITOPES WHICH WERE GLIOMA-ASSOCIATED PEPTIDES, AND THIS WAS THE TRIAL FOR PATIENTS WITH RECURRENT MALIGNANT GLIOMA. THE TRIAL WAS FINISHED, PUBLISHED IN A JOURNAL OF CLINICAL ONCOLOGY AND WE ASKED, COULD A CHANGE IN EXOSOMA PROTEIN, EXOSOMES TAKEN HERE AND TAKEN FROM mRNA EXPRESSION LEVELS COULD SERVE A SURROGATE MARKER OF IMMUNOLOGICAL AND CLINICAL RESPONSE IN THE PATIENTS RECEIVING THIS VACCINATION THERAPY? SO, WE OBTAINED PLASMA SENTIMENT PRIOR TO VACCINATION THERAPY IN EIGHT WEEKS. THIS IS AFTER THEY RECEIVED ALREADY THE VACCINE AT WEEK 8, AND WE HAD PLASMA AVAILABLE FROM 20 OF 22 PATIENTS THAT WERE ENROLLED. THIS WAS A SORT OF MIXED GROUP OF PATIENTS, 12 OF THEM HAD GLIOBLASTOMA AND 5 HAD ANAPLASTIC ASTROCYTOMA AND THEY WERE SAMPLED FOR TUMORS AS WELL. 8 OF 12 ACTUALLY HAD MEASURABLE IMMUNE RESPONSE TO THE VACCINES. THEY ARE VERY CAREFUL IN MONITORS BY BOTH TETRAMERS AND BY -- SO WE WERE LUCKY IN THIS GROUP, WOO HE SOME PATIENTS WHO HAD IMMUNOLOGICAL RESPONSES AND OTHERS DID NOT. IN ADDITION, WE OBTAINED PLASMA SPECIMENS FROM OTHER GLIOBLASTOMA PATIENTS LARGELY TO COMPARE PROTEIN, EXOSOMAL PROTEIN LEVELS OUR PATIENTS VERSUS ADDITIONAL PATIENTS. WE ISOLATED EXOSOMES BY DIFFER SHALL -- AND THEN CHARACTERIZED THEM BY PROTEIN CONTENT AND THEN ALSO BY PARTICLE SIZE. AND THEN WE EXTRACT mRNA FROM THE EXOSOME AND WE TEST THEM IN QUANTITATIVE RT-PCR FOR EXPRESSION OF 24 IMMUNE RESPONSE OR GLIOPROGRESSION-RELATED GENES. HERE THE GENES THE 24 WE MORE OR LESS SELECTED ARE QUANTITATIVE RTPCR ASSAYS WERE DONE BY OUR COLLABERATOR. AND MEASURED mRNA EXPRESSION LEVELS IN THE LEVELS ABOVE THESE GENES. SO, FIRST OF ALL, LET'S TALK ABOUT PROTEIN LEVELS IN THE EXOSOMAL TRACTION. WHAT I'M SHOWING HERE IS THAT IN THE GLIOMA PATIENT PLASMA THERE WAS SIGNIFICANT INCREASE IN THE PROTEIN CONTENT OF THE EXOSOME COMPARED TO NORMAL CONTROL. CLEARLY THERE WAS VERY WIDE DISTRIBUTION BUT IS NOT SURPRISING. THIS IS A TOTAL EXOSOMAL FRACTION. THESE ARE NOT JUST TEX DERIVED. SO PRESUMABLY THE RATIO OF TEX VERSUS EXOSOMES FROM NORMAL CELLS MAY VARY. NEVERTHELESS, WE ALSO SAW AN INTERESTING FACT THAT PATIENTS WHOSE TUMOR GRADE WAS HIGHER SO GRADE 4 AS COMPARED TO GRADE III, HAD MORE PROTEIN IN LARGER PROTEIN FRACTIONS OF THE EXOSOMES. BUT, WE ALSO LOOKED AT PROTEIN CONCENTRATION FROM EXOSOME ISOLATED PRIOR TO VACCINATION THERAPY AND AT 8 WEEKS AFTER THE VACCINATION THERAPY. AND YOU CAN SEE THAT THERE WAS A SIGNIFICANT DIFFERENCE SHOWING A DECREASE IN THE PLASMA LEVEL OF EXOSOMES. IN SOME CASES, THE DECREASE WAS QUITE STRIKING AND IT APPEARED THAT THE HIGHER THE LEVEL PRE-VACCINATION, THE GREATER THE DECREASE IN THE CONTENT, PROTEIN CONTENT OF THE EXOSOMES. SO WE THEN WONDERED DO THESE CHANGES IN PROTEINS HAVE ANYTHING TO DO WITH THE TUMOR SIZE AND TUMOR VOLUME? AND SO, MY COLLEAGUES OF COURSE HAVE DONE MRI AND WE KNEW EXACTLY WHETHER THESE TUMORS DECREASE IN SIZE OR INCREASE IN SIZE AND YOU CAN SEE IN THIS WATER PLOT, A PLOT HERE, THAT THE CHANGES IN THE TUMOR SIZE FROM BASELINE TO A, AND IN SOME CASES, THERE WAS FAIRLY GOOD CORRELATION THAT WHEN THE TUMOR GOT BIGGER, THE PROTEIN FRACTION AND EXOSOMES WENT UP AND VICE VERSA AT THE OTHER END OF THE WATER PLOT. THEN, WE LOOKED AT THE CHANGES IN mRNA EXPRESSION LEVEL OF THE SELECTED GENES THAT WE ISOLATED AT THE BASELINE AND AFTER THE VACCINE. AND YOU CAN SEE THAT ACTIN B SERVING AS A SORT OF CONTROL GENE, YOU CAN SEE THAT WE SAW SIGNIFICANT CHANGES IN THE MRNA LEVELS FOR 4 DIFFERENT GENES ALL OF WHICH ACTUALLY ARE RELATED IN ONE WAY OR ANOTHER DESCRIBED TO BE RELATED TO GLIOBLASTOMA. AND THE DATA HERE YOU CAN SEE THAT CT VALUES INCREASED IN ALL FOUR OF THE GENES. IN ACTIN B THEY DID NOT SO WE DID NORMALIZE ACTIN B. AND YOU HAVE TO REMEMBER THAT ACTUALLY HIGHER CT VALUES MEAN DECREASED mRNA LEVELS. YOU CAN SEE THE SAME THING WHEN WE DID HEAT MAPS AT BASELINE ON TOP AND 8 WEEKS POST VACCINATION AT THE BOTTOM. CLEARLY A SIGNIFICANT DECREASE IF YOU WISH, IN mRNA LEVELS OF THESE GENES. SO WE THEN ASKED, WE WONDERED WHETHER THESE CHANGES IN mRNA IN ANY WAY, CORRELATED TO DISEASE PROGRESSION? SO HERE IS KAPLAN MIRE PLOT FOR THIS GROUP OF PATIENTS AND THIS WAS FOLLOWED FROM ANYWHERE FROM 25 WEEKS TO 64 WEEKS. THERE WERE THREE PATIENTS THAT WERE ALIVED. THIS IS PATIENT 4 AND PATIENT 15 AND PATIENT 16. I DON'T KNOW WHY I DON'T -- THERE IT IS. ANY A ALL THREE PATIENTS WERE STILL ALIVE AT THE TIME THIS WILL PLOT WAS MADE AND PATIENT 15 IS PARTICULARLY IMPORTANT BECAUSE HE WAS DISEASE-FREE AT 52 WEEKS OR 54 WEEKS, AFTER THERAPY. AND ALL THREE OF THESE PATIENTS ACTUALLY HAD IMMUNOLOGIC RESPONSE AT MEASURED BY IMMUNOLOGIC ASSAYS. AND ALSO THEY HAD CHANGES IN MRNA THAT I SHOWED YOU IN A PREVIOUS SLIDE. SO THEN WE NOTED TO SEE WHETHER THERE WAS ANY CORRELATION BETWEEN DELTA CT VALUES FOR THE EXPRESSION OF THE FOUR GENES THAT I SHOWED YOU IN EXOSOMES AND OVERALL SURVIVAL OR PROGRESSION-FREE SURVIVAL. AND SO WE DID RATIOS THAT IS CALCULATED THEM AND IT TURNED OUT THIS WAS NOT A VERY SIGNIFICANT EXERCISE BECAUSE ONLY DELTA CT FOR IL8 EXPRESSION SHOWED A TREND AT THE HAZARD RATIO OF 1.56 WITH CLOSE SIGNIFICANT. SO, THEN WE WONDERED, OKAY, DO THESE CHANGES IN mRNA LEVELS THAT WE OBSERVED 44 GENES, CORRELATE AT ALL WITH THE IMMUNOLOGIC PARAMETERS? SO THE SPEAR MAN CORRELATION WAS DONE AND BASICALLY, THE ONLY THING THAT YOU CAN SEE HERE AND I HAVE THE CIRCLE OVER THERE S THAT TGF BAIT AT IN IL8 WERE THE ONLY CHANGES IN GENE EXPRESSION THAT CORRELATE TO GP100 RESPONSE AND TO RESPONSE TO IL13 RECEPTOR ALPHA TWO. SO THERE WAS SOME POSITIVE AND ONE NEGATIVE CORRELATION WHERE THESE GENE EXPRESSION CORRELATED TO THE EXPRESSION. SO, I WANT TO MAKE A COMMENT ON PATIENT 15 BECAUSE THAT WAS QUITE INFORMATIVE. THIS WAS A MAN WITH PRIMARY RECURRENT AA. AT 54 WEEKS AFTER VACCINE HE SHOWED MARSHAL RESPONSE AND PROGRESSION FREE. AT TWO YEARS LATER, THE LESIONS DECREASED STILL FURTHER AND WHEN WE LOOK AT EXOSOMES OF THIS PATIENT EIGHT WEEKS POST VACCINE, THE TOTAL PROTEIN LEVELS INCREASED QUITE SIGNIFICANTLY IN THIS PATIENT AND THERE WAS VERY STRONG UPREGULATION OF mRNA FOR PD-1. SO PD-1 IS IMPORTANT RECEPTOR, SUPPRESSION IMMUNOSUPPRESSIVE RECEPTOR, IF YOU WITH WISH, AND WE THOUGHT THEREFORE WE THOUGHT WE HAD A LOT AVAILABLE FROM THIS PATIENT AND MEASURED THE PERCENTAGES OF A CD4 POSITIVE PD-1 POSITIVE CELLS SO CELLS IMMUNE CELLS WHICH EXPRESS PD-1, IMMUNOREPRESSIVE RECEPTOR CHECKPOINT INHIBITOR AND WE SHOWED THAT THE PROPORTIONS OF THE CELLS THAT ARE PD-1 POSITIVE ACTUALLY DECREASED OVER TIME AFTER THE VACCINE. AND THIS DECLINE IN INHIBITORY RECEPTORS OF LYMPHOCYTES, SUGGESTS THAT THEY HAD IMMUNOLOGIC RECOVERY AND IT WAS REFLECTED IN EXOSOMES. SO THEY WERE NOT ONLY TELLING US ABOUT WHAT WAS HAPPENING TO THE TUMOR BUT ALSO AT LEAST IN THIS PATIENT, THEY WERE TELLING US HOW THE IMMUNE CELLS IN THIS PATIENT BEHAVED AFTER THE VACCINE. SO, IN CONCLUSION, TOTAL EXOSOME PROTEIN LEVEL AND mRNA EXPRESSION LEVELS FOR GENES THAT WERE INVOLVED IN IMMUNE REGULATION OR GLIOMA PROGRESSION APPEARED TO REFLECT VACCINE-INDUCED CHANGES IN THE TUMOR CELLS AND IMMUNE CELLS. THIS WAS A SMALL STUDY. DATA LIMITED BY DATA OBTAINED AT SHORT-VACCINATION TIME PERIODS. THERE WAS A SMALL NUMBER OF GENES AND WE DIDN'T USE TEX PURIFIED BUT WE USED TOTAL EXOSOMAL FRACTION. ALL THESE ARE LIMITING FACTORS. NEVERTHELESS, WE WERE ABLE TO SHOW THAT EXOSOME ISOLATED FROM PLASMA OF GLIOMA PATIENTS MAY HAVE A PROMISE AS PREDICTORS OF RESPONSE TO VACCINATION THERAPY AND I BELIEVE THIS WAS THE FIRST STUDY THAT REALLY SHOWED THE POTENTIAL CORRELATION EXISTED AND PROBABLY WE SHOULD LOOK AT IT FURTHER. AND I WANT TO ACKNOWLEDGE THE CONTRIBUTION OF LAUREN MILLER, A SURGEON NOW IN SWITZERLAND WHO WAS A POSTDOCTORAL FELLOW AND MY COLLEAGUES AND OUR STATISTICIAN AND THE PI OF THE CLINICAL TRIAL THAT I DESCRIBED TO YOU. THANK YOU. [ APPLAUSE ] >> A OKLAHOMA: THANK YOU VERY MUCH DR. WHILE MARGARET OCHOCINSKA -- WE MAY HAVE TIME FOR ONE QUESTION. >> DO YOU THINK THAT EXOSOMES CAN BE DETECTED FROM SMALLER VOLUME DISEASE, FOR INSTANCE BRAIN THAT TAFT CEASE? >> WELL -- METASTASES -- >> WE USED APPROXIMATELY ONE ML OF PLASMA AND THAT IS ENOUGH TO ISOLATE THE SUFFICIENT AMOUNT OF EXOSOMES TO BE ABLE TO DO PROTEINS LIKE mRNA STUDIES -- [ OFF MIC ] >> THANK YOU VERY MUCH. I ALSO WANT TO THANK THE SPEAKERS FROM THIS SESSION AND THERE WILL BE AN OPEN MICROPHONE DISCUSSION WITH THESE SPEAKERS UP HERE AT THE PANEL TABLE AT 3:00 THIS AFTERNOON. RIGHT NOW WE HAVE TIME FOR 10 MINUTE BREAK. SO EVERYONE CAN GET SOME COFFEE AND REFRESHMENTS AND BE BACK AT 10:40. THANK YOU. FOR THE ONLINE AUDIENCE, ITS PROGRAM BOOKLET IS THE AVAILABLE ON THE REGISTRATION WEBSITE. SO IF YOU WOULD LIKE TO LOOK AT THE PROGRAM BOOKLET IF YOU'RE NOT HERE, PLEASE DO SO. THE REGISTRATION WEBSITE. WITH THAT, I'D LIKE TO OPEN THE SECOND SESSION, BLOOD SCIENCE SESSION AND THE SESSION CHAIR IS DR. TAMARA CROWDER FROM THE CASUALTY CARE RESEARCH PROGRAM. THANK YOU. >> TAMARA CROWDER: SO, GOOD MORNING. MY NAME IS TAMARA CROWDER, THE DOD NEUROTRAUMA RESEARCH PORTFOLIO MOTHER-IN-LAW AND I FALL UNDER THE U.S. ARMY MEDICAL RESEARCH FOR MATERIAL COMMAND COMBAT CASUALTY CARE RESEARCH PROGRAM. AND REALLY FORTUNATE TO BE THE CHAIR OF THIS SESSION. WE HAVE FOUR DISTINGUISHED SPEAKERS REPRESENTING INDUSTRY, ACADEMIA AND THE DOD. WE'LL HER DR. KENDALL JENSEN FOR NON-INVASIVE DIAGNOSTICS SPEAK ON MONITORING THE CENTRAL NERVOUS SYSTEM THROUGH PERIPHERAL BIOFLUIDS. DR. AISLED SLADE FROM BCU WILL SPEAK ON BRAIN INJURE NEBLOOD FOLLOWED BY DON MARION FROM THE DOD SPEAKING TO THE ROLE OF THE BLOOD-BRAIN BRARRIER AND POST-TRAUMATIC CEREBRAL BLOOD FLOW AND THE NEUROVASCULAR UNIT AND DR. ROBERT CLARK FROM THE UNIVERSITY OF PITTSBURGH, TRANSPORTERS AT BLOOD-BRAIN INTERFACES TO REGULATE THE METABOLOMIC AND PHARMACOLOGIC MICRO-ENVIRONMENT. SO, BEFORE I TURN THIS OVER TO OUR SPEAKERS, I WANT TO TALK ABOUT URGENCY FROM A COMBAT CASUALTY CARE PERSPECTIVE. I REALIZE THE STUDY IS MUCH BIGGER THAN TRAUMA BUT OUR GOAL IS THE SAME. WE MEASURE OUR SUCCESS THE SAME WAY. DELIVERING A CLINICAL TOOL OR A PRODUCT THAT CHANGES CLINICAL PRACTICE. THIS REQUIRES A SOLID STRATEGIC PLAN THAT COVERS SPECTRUM OF RESEARCH FROM BASIC PROOF-OF-CONCEPT THROUGH ADVANCED DEVELOPMENT AND FIELDING. IT REQUIRES A COLLABORATIVE EFFORT OF INVESTIGATORS ACROSS MULTIPLE CENTERS, DISCIPLINES AND COUNTRIES. YOUR RESEARCH IS PART OF AN OVERARCHING STRATEGY, STRATEGIC PLAN TO DELIVER MATERIAL AND KNOWLEDGE PRODUCTS THAT IMPROVE LIVES. IN COMBAT CASUALTY CARE, OUR PROGRAM STATEMENT STARTS WITH THIS. STAY AHEAD OF THE CURVE. THAT IS THE DIRECTIVE. THAT IS THE GOAL. THAT'S THE PLAN. IT'S REALLY EASY TO SAY THAT BUT THE EXECUTION OF THE PLAN IS REALLY DIFFICULT BECAUSE PLANS ARE ALWAYS TWISTING AND DEVIATING FROM THE NORM AND THE NORM CHANGES FROM DAY-TO-DAY. THIS IS THE TACTICAL COMBAT CASUALTY CORRIDOR. IT IS FILLED OUT FOR EVERY CASUALTY. YOU CAN SEE THE OPPORTUNITIES FOR BLOOD-BRAIN BARRIER DISRUPTION. LOOK AT MECHANISMS OF INJURY. LITERALLY EVERY MECHANISM OF ENTRY IS AN OPPORTUNITY FOR DISRUPTION. SO COMPOUND THIS WITH A TYPICAL COMBAT CASUALTY THAT SUSTAINED A TBI AND SIGNIFICANT BLOOD LOSS, HEMORRHAGE, AMPUTATION AND/OR BURN. AFTER HEMORRHAGE, THE GOAL IS TO INCREASE BLOOD PRESSURE. AFTER NEUROTRAUMA THE GOAL IS TO SUSTAIN BLOOD PRESSURE. AND BURNS HAVE IMPACT ON THE INTEGRITY OF THE BLOOD-BRAIN BARRIER. FOR US, IT'S THE DELICATE BALANCING ACT TO PROVIDE LIFE-SAVING MEASURES UNDER THESE CONDITIONS TO GIVE THE CASUALTY THE BEST CHANCE OF SURVIVAL. WE ALL KNOW THAT BLOOD-BRAIN BARRIER INTEGRITY IS CRITICAL TO PRESERVING BRAIN FUNCTION, CIVILIAN OR MILITARY CASUALTIES, THE OVERARCHING GOAL IS TO DECREASE MORBIDITY AND MITIGATE INJURY AND PUSH CAPABILITIES FORWARD WITH THE GOAL OF IMPROVING OUTCOMES ENSURING THE BEST POSSIBLE RECOVERY AND WHERE POSSIBLE, CHANGING WHAT IS CURRENTLY NON SURVIVABLE TO A SURVIVALLABLE INJURY. SO EARLIER, I MENTIONED THE IMPORTANCE OF A STRATEGIC PLAN IN THIS STATEMENT FROM THE DIRECTOR OF THE DEFENSE BUDGET ANALYSIS AND CENTER FOR STRATEGIC AND INTERNATIONAL STUDIES SAYS IT VERY WELL. [ READING ] FOR US, THE DRAWDOWN IN FORCES OF IRAQ AND AFGHANISTAN POSES DECREASED FUNDING FOR TRAUMA-RELATED RESEARCH. I REALIZE THE RESEARCH FUNDING COMES FROM MANY AREAS NOT JUST TRAUMA ALLOCATION BUT THE MESSAGE IS THE SAME. IN ADDITION TO A PLAN, SUCCESS WILL REQUIRE A SUSTAINED COMMITMENT TO FUNDING NOT JUST DRIBS AND DRABS TO FUND A FEW BASIC PROOF-OF-CONCEPT STUDIES BUT FUNDING THAT ALLOWS FOR TRANSLATION OF MATERIAL AND KNOWLEDGE PRODUCTS TO CLINICAL PRACTICE. I ASK US ALL TO ALWAYS BE THINKING OF PARTNERSHIPS AND FDA REGULATIONS AND CLINICAL DEVELOPMENT. I ASK US TO ENSURE THAT ALL OF OUR RESEARCH GOALS ADDRESS BASIC MECHANISMS, BASIC QUESTIONS, ARE MAPPED TO FUTURE STUDIES DESIGNED TO CHANGE CLINICAL PRACTICE. WHEN WE GET INTO DISCUSSION LATER, WE WANT TO HEAR YOUR THOUGHTS, IDEAS, FOR HOW TO BEST DEVELOP PLAN TO ADVANCE THE STATE OF THE SCIENCE OF FUNCTION AND PRESERVATION AND REPAIR IN TRAUMA AND DISEASE. STAY AHEAD OF THE CURVE. IT'S A PHRASE THAT REQUIRES ACTION, INHERENTLY ASSUMES TIRELESS EFFORT FROM FRONT-END RESEARCHERS WHILE ALSO REQUIRING IMMENSE SACRIFICE FROM BACK-END USERS. HOW DO WE GET THERE? WE START HERE. WE START HERE TODAY. WITH A COMMITMENT TO THE HIGHEST LEVEL OF RESEARCH AND DEVELOPMENT AND IMPROVEMENT. AGAIN, I'M REALLY HONORED TO BE ABLE TO CHAIR THIS SESSION. THANK YOU FOR YOUR TIME AND I'M GOING TO TURN IT OVER NOW TO OUR SPEAKERS BEGINNING WITH KENDALL JENSEN FOR NON-INVASIVE DIAGNOSTICS. [ APPLAUSE ] >> KENDALL JENSEN: THANK YOU. SO I'M GOING TO TALK ABOUT WORK THAT WE HAVE BEEN DOING IN OUR LABORATORY FOR A FEW YEARS NOW AND I'M GOING TO TRY NOT TO BE TOO WILDLY INTO SPECULATION BUT THAT IS EASY TO DO AT THE BEGINNING PART OF THIS TALK. SO, I JUST FROM OUR PERSPECTIVE, WE HAVE BEEN WANTING TO MONITOR THE CENTRAL NERVOUS SYSTEM SO LOOK FOR EXTRACELLULAR RNA INFORMATION THAT COMES FROM THE BRAIN OR SPINAL CORD THAT INDICATES INJURY OR DISEASE SO I'M LOOKING AT THINGS THAT GET OUT. SO, THIS IS PROBABLY NOT A SURPRISING SLIDE. THERE IS NOTHING TOO SHOCKING ON HERE. WE WANT TO HAVE THE ABILITY TO MONITOR THE CENTRAL NERVOUS SYSTEM. IT WOULD BE INVALUABLE TO BE ABLE TO ASSESS THE THERAPEUTIC EFFICACY OF THINGS. FOR INSTANCE WE HAVE SEVERAL PROJECTS LOOKING AT PATIENTS WITH ALS. IF YOU COULD -- THEY HAVE A SHORT WINDOW OF TIME BETWEEN DIAGNOSIS AND THE TIME OF DEATH AND IF YOU COULD PUT A THERAPEUTIC IN AND SEE IF IT WAS DOING SOMETHING, DO SOMETHING IN REALTIME OR MORE QUICKLY THAN WE CAN NOW, THAT WOULD BE EXTREMELY BENEFICIAL TO THAT PATIENT POPULATION. WE ALSO WANT TO KNOW HOW DISEASE PROGRESSES. IF WE COULD MONITOR THINGS LONGITUDINALLY, MORE FREQUENTLY, THAT WOULD BE EXCEPTIONALLY USEFUL. AND ALSO TRAUMATIC INJURY. IF WE COULD EVALUATE OR EVEN PREDICT SECONDARY INJURY THAT WOULD INCREASE OUR DIAGNOSTIC ACCURACY AND THERAPEUTIC POTENTIAL. SO, ONE THING THAT WE WANT TO DO IS WE WANT TO LOOK AT EXTRACELLULAR RNA. THE THING OUR LAB HAS BEEN LOOKING AT FOR QUITE A WHILE. HOW DO THEY GET OUT OF TISSUES AND THE CENTRAL NERVOUS SYSTEM? I DON'T KNOW THE ANSWERS TO ANY QUESTIONS. I'M JUST ASKING THEM. IS IT AN ACTIVE OR PASSIVE PROCESS? WHAT IS GOING TO BE THE REPRESENTATION? THE MOST ABUNDANT INTERRUPT? WILL IT TELLING SOMETHING ABOUT THE DISEASE? WHAT ARE WE GOING TO LOOK AT? DOES DISEASE DECREASER INCREASE THE PERMEABILITY OF THE BLOOD-BRAIN BARRIER? NONE OF THIS I CAN ANSWER BUT I CAN SHOW YOU SOME TRANSCRIPTS AND SORT OF WHAT IT ACCIDENT LOO LIKE IN DIFFERENT BIOFLUIDS. -- WHAT IT LOOKS LIKE -- ARE THERE GOING TO BE SOME BIOFLUIDS THAT ARE MORE REPRESENTATIVE OF SOME TISSUES FOR CENTRAL NERVOUS SYSTEM? YOU'RE GOING TO SEE MORE INFORMATION IN THE CEREBRAL SPINAL FLUID. OR OTHER BIOFLUIDS THAT ARE NON-INVASIVE AND EASY TO COLLECT? WHAT DO DETECTED EXTRACELLULAR RNA REPRESENT? INFORMATION ESPECIALLY IN THE LARGEST ORGANS? THE MOST ACTIVE ORGANS OR THE ONES THAT ARE MORE VASCULAR? SO, I'M GOING TO TALK ABOUT EXTRACELLULAR RNA. I KNOW THERE ARE A LOT OF PEOPLE GOING TO TALK ABOUT EXOSOMES TODAY AND IN THIS CASE, I DIDN'T FRACTIONATE ANYTHING INTO PARTICULAR VESICLE TYPES OR INTO RNA BINDING PROTEIN CARRIERS. EVERYTHING IS AS LOOKED AT AT ONCE. SO ALL EXTRACELLULAR RNA. JUST FREE CELLS. AND ORIGINALLY, WHEN WE STARTED DOING THIS 5 OR 6 YEARS AGO, THE GOAL WAS TO IDENTIFY RNAs THAT COULD TRACK PATHOLOGY AND WHAT WHAT WERE OUR CHOICES? CEREBRAL SPINAL FLOOD, BLOOD, URINE, SALIVA, OR SOMETHING ELSE? FOR SOME PROJECTS, CEREBRAL SPINAL FLUID WASN'T HARD TO COLLECT FOR OUR PROJECTS LOOKING AT ALZHEIMER'S AND PARKINSON'S, IT WAS SOMETHING THAT WAS ACHIEVABLE. IF YOU'RE LOOKING AT POTENTIAL CONCUSSION OR MILD TRAUMATIC BRAIN INJURY, CEREBRAL SPINAL FLUID IS PROBABLY NOT SOMETHING PEOPLE ARE TYPICALLY GOING TO DO. SO IN CASES OF THAT WE LOOKED INTO OTHER BIOFLUIDS TO BE SUCCESSFUL. SO THERE IS CURRENTLY NO WAY, AND I'M NOT GOING TO TELL YOU OF ANY WAY TODAY, THAT YOU CAN TELL WHERE EXTRACELLULAR RNAs EXFROM. ALL YOU CAN DO IS SAY THEY ARE EXTRACELLULAR RNAs. YOU CAN TRY TO GUESS WHERE THEY MIGHT HAVE COME FROM. AND THERE ARE STRATEGIES CURRENTLY UNDERWAY BY MANY DIFFERENT LABS AND EVEN WE ARE TRYING TO DO SOME OF THAT AS WELL. WE ARE TRYING TO ENRICH FOR SOME INFORMATION THAT COMES FROM THE BRAIN. SO I'M JUST GOING TO DESCRIBE A COUPLE OF EXPERIMENTS OR JUST IDEAS. I'M REALLY JUST GOING TO SHOW YOU PROFILES OF BIOFLUIDS. SO WE TOOK THE RNA-SEQ ATLAS FROM EMBL HAS CODING INFORMATION FROM 32 DIFFERENT TISSUES CURRENTLY. ONE OF THE BETTER REPOSITORIES FOR A BODY ATLAS OF RNA-SEQ AND IT IS FROM 122 DIFFERENT INDIVIDUALS. USUALLY THERE IS 3-4 DIFFERENT TISSUES REPRESENTED FOR EACH TISSUE TYPE. ALL OF THAT IS RNA SEQUENCED. WE HAVE SAMPLES FROM ADULTS, NOT THE PERFECT SAMPLE SETS BUT THIS IS SORT OF AN IDEA OF WHAT WE HAVE. AND WHAT WE ARE COMPARING TO. SO WE HAVE A LOT OF SAMPLES FROM ADULTS WITH HEMORRHAGE. WE HAVE PEDIATRIC ABOUT 100 SAMPLES, WE HAVE ABOUT 150 SAMPLES HERE FROM INTRAVENTRICULAR HEMORRHAGE OR NEURAL TUBE DEFECT. WE HAVE PLASMA SAMPLES. 150 OF THOSE. 150 URINE SAMPLES AND SALIVA SAMPLES AND WE ISOLATED THE TOTAL CELL-FREE RNA CONTENT AND IN THIS PARTICULAR CASE, WE LOOKED AT ALL THE LONG RNAs. THE WHOLE TRANSCRIPTOME. TYPICALLY PEOPLE LOOKING AT BIOMARKERS AND LOOKING AT EXTRACELLULAR RNAs LOOKED AT SMALL RNA SO I'M GOING TO TELL YOU ABOUT THE WHOLE TRANSCRIPTOME BECAUSE I THINK IT WORKS NICELY WITH TALKING ABOUT SOME OF THIS INFORMATION THAT IS KNOWN FROM THE RNA BODY ATLAS. SO WE TOOK SOME GENE THAT IS HIGHLY EXPRESSED IN BRAIN COMPARED TO ALL THE OTHER TISSUES. THIS IS JUST EXEMPLIFIED GENES THAT ARE HIGHLY EXPRESSED IN THE BRAIN COMPARED TO ALL OTHER TISSUES LISTED. SO SOME OF THEM AREN'T SURPRISING GFAP WAS HIGHLY EXPRESSED AND LOWLY EXPRESSED IN THE OTHER TISSUES JUST IN THE SINGLE-DIGITS. PLP1 WHICH IS A COMPONENT OF MYELIN IS ALSO EXPRESSED VERY HIGHLY IN BRAIN. BUT IT IS ALSO EXPRESSED IN SOME OTHER TISSUES AS WELL BUT NOT SUPER HIGH. POTENTIALLY IF YOU WERE GOING TO LOOK IN PLASMA SAMPLE WOULD YOU KNOW THAT THIS TRANSCRIPT CAME FROM THE BRAIN OR WOULD YOU THINK THAT MAYBE IT CAME FROM TESTIS IN MALES OR FROM SOMEWHERE ELSE? SO WE ALSO LOOKED AT A COUPLE OF OTHER ONES. SLC1A2 IS HIGHLY EXPRESSED IN THE BRAIN AND ALSO EXPRESSED IN A FEW OTHER TISSUES AS WELL. SO THIS IS JUST AN EXAMPLE OF WHAT I'M TALKING ABOUT. SO THESE ARE THE FIVE GENES WE LOOKED AT. AND YOU CAN SEE WE TRIED TO NORMALIZE EVERYTHING SO WE COULD COMPARE THEM ACROSS. SO THESE ARE REALLY JUST GFAP IS REALLY HIGH IN THE BRAIN AND IF YOU SUMMED UP ALL THE OTHER TISSUES, WHAT WOULD YOU BE LOOKING SNAT SO THIS IS VERY BRAINCENTRIC. SO EVERYTHING YOU WOULD SUM UP FROM THE OTHER TISSUES COMES 32 COUNTS. SO MOST THINGS YOU SEE GFAP SOMEWHERE ELSE YOU BELIEVE MADE IT AT DOES COME FROM BRAIN. IT'S PROBABLY ONE OF THE BEST MARKERS THAT WE LOOKED AT. PLP1 HIGH EXPRESSION IN THE BRAIN IF YOU ADD UP ALL THE OTHER TISSUES, YOU SEE ABOUT 600 COUNTS. SO THERE IS A CONTRIBUTION FROM OTHER TISSUES. AND THEN SAME FOR THE OTHER ONES COMING DOWN IN SIZE AND THESE ARE SAME RELATIVELY THE SAME. SO, WHAT DO WE SEE IN BIOFLUIDS? SO IF WE LOOK IN CEREBRAL SPINAL FLUID YOU CAN SEE THAT GFAP IS HIGHLY EXPRESSED EVEN HIGHER IN THE PEDIATRIC CASES. IT COULD BE ASSOCIATED WITH THEIR DISEASE BUT YOU CAN SEE THAT THERE IS A LOT OF GFAP PRESENT IN CSF. YOU WILL SEE THERE ARE DIFFERENCES. THIS IS EXPRESSIONED ALMOST THE SAME IN THE BRAIN BUT EXPRESSED DIFFERENTLY IN THE CSF. THERE WILL BE TRANSCRIPTS THAT DON'T MAKE IT OUT INTO THE PERIPHERY EVEN THOUGH YOU THINK THEY ARE HIGHLY EXPRESSED IN THE TISSUE ORGAN YOU'RE LOOKING AT. AGAIN, THIS IS EXPRESSED FAIRLY HIGHLY BUT NOT IN A SIMILAR FASHION. IT'S NOT VERY HIGH OUT IN THE CEREBRAL SPINAL FLUID AND THEN THE NEXT GENE. THIS IS HARDLY EXPRESSED IN CSF. SO PLASMA SAMPLES. SO YOU LOOK AT THE SAMPLES AND SEE THERE IS A DRAMATIC DECREASE NOW OF THE AMOUNT OF GFAP YOU CAN DETECT IN PLASMA SAMPLES. AND THEN EACH THING IS DROPPING SIGNIFICANTLY. BUT THERE IS STILL POTENTIAL INFORMATION THERE. IT'S JUST GETTING HARDER AND HARDER TO SAY THAT THAT IS ACTUALLY FROM THE BRAIN. AND IF YOU LOOK AT URINE, OR SALIVA, URINE IS WIERD IN THAT YOU JUMP UP IN SOME OF THESE OTHER RNAs OR SOME OF THESE BRAIN-RICH PROTEINS SO, THAT IS INTERESTING. BUT MAYBE I CAN SHOW SOME OF WHY THAT MIGHT BE HAPPENING TOO. SO IF YOU LOOK AT PLASMA CSF SAMPLES, PLASMA SAMPLES IN URINE AND SALIVA, THE NEXT TISSUE WITH THE MOST INDIVIDUALS 7 IN THE HEART. SO MOST OF THIS GFAP IS PROBABLY COMING FROM THE BRAIN. IT'S ALSO NOT VERY DETECTABLE IN BIOFLUIDS OUTSIDE OF THE CSF. IF YOU LOOK AT PLP1, IT'S EXPRESSED AT 74 READS PER MILLION IN SKIN. SO THESE OTHER THINGS ARE THEY REALLY COMING FROM THE BRAIN OR ARE THEY COMING FROM OTHER AREAS AND AGAIN, IT'S NOT KNOWN BUT THERE IS NOT HIGHLY EXPRESSED BUT BETTER IN THE CSF. AND THEN FINALLY SLC1A2 YOU CAN SEE THAT IT IS HIGHLY EXPRESSED IN ALL THE BIOFLUIDS T IS EXPRESSED HIGHLY IN THE LIVER AND IN THE ADRENAL GLAND WHICH MAY BE CONTRIBUTING TO WHY YOU SEE A SIGNIFICANT PEAK IN URINE. SO MAYBE IT IS PROXIMITY TO THE ORGANS THAT YOU'RE LOOKING AT AND THE INFORMATION YOU'RE GOING TO GET. SO PEOPLE ARE -- AND OUR LAB TOO HAS BEEN TRYING TO ENRICH FOR BRAIN SPECIFIC INFORMATION LOOKING AT L1 CAM OR LOOKING FOR OUTPUTS FROM TO YOU AND IT IS HIGHLY EXPRESSED IN BRAIN BUT IN THE RNA-SEQ ATLAS THESE GENES ARE ALSO EXPRESSED IN OTHER AREAS PRETTY SIGNIFICANTLY. AND SO, WHEN YOU DO THIS YOU LOOK AT THE BRAIN'S INFORMATION AND THEN ADDING UP ALL THE OTHER TISSUES TOGETHER YOU SEE L1 CAM COMING FROM THIS TISSUES AND SO IT WOULD BE HARD TO SAY IN A PLASMA SAMPLE IF THIS IS REALLY COMING FROM THE BRAIN OR FROM SOME OTHER GROUP OF ORGANS OR SOME ADDITION OF ORGANS. SO, ALL IS NOT LOST. I STILL THINK YOU CAN LOOK FOR BIOMARKERS ASSOCIATED WITH EXTRACELLULAR RNAs AND I'M SURE THERE WILL BE LOTS OF TECHNIQUES AND WAYS WE WILL ENRICH FOR THINGS AND I'M JUST GOING TO GIVE TWO EXAMPLES WHERE I THINK WE SAW SOME RELEVANT INFORMATION WHEN WE WERE MONITORING THE CENTRAL NERVOUS SYSTEM. SO THIS IS ONE OF THE FIRST STUDIES WE DID. IT WAS APROOF OF PRINCIPLE STUDY. SO, ALL THE PATIENTS WERE POST MORTEM. SO WE HAD ALL THE PATHOLOGY REPORTS AND ALL THE TANGLE PLAQUE INFORMATION. WE ALSO HAD A CSF AND A SERUM SAMPLE FROM EACH ONE. SO WE COULD COMPARE THE TWO AND WE COULD SAY WHAT SORT OF PATHOLOGY THEY HAD IN THE BRAIN RELATED TO THE INFORMATION WE HAD FROM SERUM. SO, REALLY THIS IS JUST AN EXAMPLE AND I DON'T EXPECT ANYBODY TO REMEMBER WHAT ANY microRNAs DO. WHAT WE HAD WAS MORE DIFFERENTIALLY EXPRESSED GENE. SO IT HAS MORE microRNAs DIFFERENTIALLY EXPRESSED THAN THE SERUM THAT I'M GOING TO SHOW ON THE NEXT PAGE. 73% OF THE microRNAs HAD BEEN FOUND TO BE DIFFERENTIALLY EXPRESSED IN THE BRAIN OF PATIENTS WITH ALZHEIMER'S DISEASE. SO FOR US TO FIND THEM OUT IN THE PERIPHERY, WE WERE PRETTY EXCITED. WHEN WE LOOKED AT SERUM SAMPLES THAT GOES DOWN A LITTLE BIT. 55% OF THE microRNAs WE FOUND DIFFERENTIALLY EXPRESSED WERE DETECTABLE IN SERUM AND THERE WERE FEWER OVERALL MICRORNAs THAT COULD BE DETECTED. THIS WAS A FULL LIST AND THE LIST FOR CSF WAS PROBABLY TWICE AS LONG. SO ONE OF THE THINGS THAT WE WERE TESTED TRYING TO DO, WE WERE DOING PROJECTS FOR THE MICHAEL J. FOX FOUNDATION AND THEY WANTED TO SEE IF WE COULD DETERMINE ANY INFORMATION ABOUT THE DEMENTIA FROM SOME OF THE PATIENTS THEY HAD BIOMARKERS LOOKING FOR BIOMARKERS OF DEMENTIA. ONE OF THE THINGS WE DID WAS COMPARED PATIENTS WITH PARKINSON'S DISEASE AND WITH DEMENTIA AND CONTROL SUBJECTS VERSUS ALL I'M SORRIERS AND LOOKED AT THE INTERSECTION OF THE microRNAs THAT WERE SICKLY DIFFERENT. AND ONE OF THE TOP ON BOTH LIST WAS MIR34C5P AND THAT WAS INTERESTING BECAUSE WE WENT BACK AND LOOKED AT THE LITERATURE AND IT IS HIGHLY EXPRESSED IN THE BRAIN ESPECIALLY IN THE HIPPOCAMPUS. AND WE WERE DETECTING IT IN SERUM SAMPLES. AND AGAIN, IF YOU LOOK AT THE PREVIOUS DATA YOU WOULDN'T NECESSARILY BELIEVE THAT IT HAD TO HAVE COME FROM THE BRAIN. PROBABLY OTHER TISSUES AND THE microRNA DATABASE IS NOT AS GOOD AS THE WHOLE TRANSCRIPTOME DATABASE SAYING WHAT MICRORNAs ARE PRESENT ANYWHERE. SO IT WAS HIGHLY EXPRESSED IN THE BRAIN ESPECIALLY IN THE ALZHEIMER'S PATIENTS AND THESE PATIENTS WERE AGE MATCHED SO THERE WAS NO DIFFERENCE IN AGE. IT WAS ALSO INTERESTING BECAUSE IT'S KNOWN THAT MIR34C CAN INTERACT WITH MEMORY GENES AND THAT WHEN IT ALSO INTERACTS AND DECREASES CERTAIN 1 EXPRESSION AND IS -- CERTIFICATE 1 CAN AFFECT MEMORY GENES AND THIS IS TESTED IN TRANSGENIC MICE AND PEOPLE USED IT SORT OF OR LOOKED AT THIS INFORMATION SEPARATELY. AND THEN ONE MONTH AFTER OUR PAPER WAS PUBLISHED THERE WAS ANOTHER GROUP THAT FOUND THE EXACT SAME microRNA TO BE DIFFERENTIALLY EXPRESSED IN PATIENTS WITH ALZHEIMER'S AND THIS WAS MORE REASSURING. THIS WAS FOUND IN PLASMA SAMPLES AND LIVING PATIENTS IN SERUM AND POST MORTEM AND FOUND THAT THERE WAS INCREASE, SO THIS IS HIGHER LEVELS OF MIR34C AND LOWER LEVELS OF CT SCALE. THEY GO UP WITH ALZHEIMER'S DISEASE. AND THEN JUST ONE LAST SORT OF EXAMPLE THAT I'M GOING TO SHOW AS WE HAVE THIS PROJECT WHERE WE ARE LOOKING AT THE ARIZONA STATE UNIVERSITY FOOTBALL PLAYERS AND WE HAVE THEM ENCODERS IN THEIR HELMETS SO THEY HAVE EVERY SINGLE IMPACT FROM THE BEGINNING OF THE SEASON TO THE END OF THE SEASON TRACKED. WE HAVE BEEN DOING THAT FOR FOUR YEARS NOW. AND WE HAVE SOME GUYS WHO HAVE BEEN IN THE STUDY THE WHOLE FOUR YEARS AND TRACKED EVERY SINGLE HIT FROM EVERY SINGLE PRACTICE AND EVERY SINGLE GAME. AND WE CAN LOOK AT THE INFORMATION YOU GET DIFFERENT INFORMATION ABOUT THE IMPACT, LOCATION, AND EACH POSITION THEY HAVE SORT OF DIFFERENT INFORMATION AS WELL. AND WHAT WE FOUND IS THAT IF WE TOOK SOME OF OUR EARLY URINE SAMPLES AND WE LOOKED AT THE WHOLE TRANSCRIPTOME, WE COULD LOOK AT SUBJECTS WHO HAVE BEEN HIT WITH LOW FREQUENCY THE WEEK BEFORE THIS URINE SAMPLE WAS TAKEN AND SUBJECTS HIT WITH HIGH FREQUENCY THE WEEK BEFORE THE URINE SAMPLE WAS TAKEN AND THEY BROKE INTO TWO CLUSTERS BUT YOU CAN REALLY SEE WITH YOUR EYES THEY BREAK INTO A THIRD CLUSTER AS WELL AND THAT THIS GROUP HAD MUCH HIGHER HITS THAN ANY OF THE OTHER GROUPS. I WILL SAY THAT THERE WAS ONE THAT DIDN'T CLUSTER QUITE RIGHT WITH THIS GROUP AND WAS THIS INDIVIDUAL HERE AND THAT PERSON WE WENT BACK TO SEE WHY THIS PERSON WHO HAD 114 HITS MIGHT HAVE ENDED UP IN THIS CLUSTER AND TWO DAYS AFTER THIS URINE SAMPLE WAS TAKEN HE WAS DIAGNOSED WITH A CONCUSSION. SO MAYBE THERE IS SOME SORT OF INCREASE INJURY MARKERS ASSOCIATED WITH THIS CLUSTER AS WELL. AND SO I JUST LIKE TO THANK A LOT OF PEOPLE WHO ARE INVOLVED IN THAT ASU FOOTBALL STUDY. IT TAKES A TON OF PEOPLE TO RUN IT. ALSO DAN WHO IS ONE OF THE REASONS WHY I WAS ASKED TO GIVE A TALK HERE AND ALSO DR. KALANI WHO WORKS WITH ME ON LOOKING AT THESE EXTRACELLULAR RNAs. [ APPLAUSE ] >> THANK YOU VERY MUCH DR. JENSEN. WE HAVE TIME FOR ONE OR TWO QUESTIONS. [ OFF MIC ] >> COULD YOU COME TO THE MICROPHONE? >> WITH THE ASU FOOTBALL STUDY ARE YOU MAPPING THE ACCELEROMETER DATE - TO CONCUSSIONS? IS THERE ANYTHING YOU CAN SHARE ABOUT THAT? >> WE DO. SO ORIGINALLY FOUR YEARS AGO WHEN I WAS YOUNG AND NAIVE, WE REALLY THOUGHT THAT WHAT WE WERE GOING TO LOOK AT ARE ALL THESE CONCUSSIONS AND LOOKED AT THOSE -- AND I THOUGHT EVERYBODY WOULD HAVE A CONCUSSION. BUT THERE ARE THREE A YEAR MAYBE. SO WE MAP THE INFORMATION TO THE CONCUSSIONS BUT WE HAVEN'T HAD A LOT OF CONCUSSIONS. WHAT WE HAVE BEEN DOING IS LOOKING AT REPETITIVE HEAD INJURY. WHAT IT MEANS AND AS WE CAN SEE MARKERS OF REPETITIVE HEAD INJURY AND IF MAGNITUDE OR FREQUENCY PLAYS A ROLE IN THAT. >> I NOTICED IN THE SCATTER GRAM OF THE OTHER PAPER THAT THE microRNA SORT OF APPARENTLY INFORMATIVE microRNA WAS WELL ABOVE THE NORMAL RANGE IN A SUBSET OF THE SUBJECTS. SO AGAIN, GETTING TO THE THING I ASKED EARLIER, DO YOU BELIEVE THAT THE PEOPLE WHO SHOW THE REALLY HIGH INCREASES, IS THAT A REFLECTION OF THE BLOOD-BRAIN BARRIER AND ARE YOU WORKING WITH ANYBODY TO EXPLORE THAT? I KNOW YOU SAID YOU DON'T HAVE THE ANSWERS BUT AGAIN, THIS GETS TO HOW CAN WE DEFINE AND USE SOME OF THESE MARKERS TO STRATIFY INDIVIDUALS WHO WE THINK THAT IT IS INFORMING US ABOUT SOME OTHER PROCESS OTHER THAN JUST THE BRAIN INJURY ITSELF BUT MAYBE THE BLOOD-BRAIN BARRIER ENTRY AS WELL. >> THAT'S PERFECT. EXACTLY WHY I'M HERE. I'M REALLY HOPING WE CAN GET INFORMATION ABOUT THAT AND WORK ON PROJECTS OR FIND COLLABORATIONS BECAUSE I DON'T KNOW THE ANSWER TO THAT AND THAT WORRIES ME AS A PERSON LOOKING FOR A BIOMARKER. WHY IN ONE PERSON WOULD YOU GET HIT AND SEE SOMETHING AND ANOTHER PERSON I WOULD NOT. IS THAT LOCATION ON THE HEAD? IS THAT IMPACT? THERE IS A MILLION DIFFERENT THINGS I REALLY WOULD LIKE TO KNOW ABOUT THAT. >> REGARDING YOUR microRNA DATA, CAN YOU SEE A DIFFERENCE WHETHER THESE ARE SOLUBLE IN PLASMA OR CONTAINED WITHIN EXTRACELLULAR VEHICLES OR SOME OF BOTH? >> SO WE ARE ABOUT TO FIND OUT. SO WE JUST DID A 250 SUBJECT STUDY LOOKING AT JUST IN VESICLES OR ASSOCIATED WITH RNA PROTEIN BARRIERS SO WE WILL BE ABLE TO LOOK AT THE PROFILES OF THOSE SPECIFICALLY. [ OFF MIC ] >> BUT THE DATA I PRESENTED SO WE ARE GOING BACK NOW TO LOOK AND SEE IF THOSE VESICLES OR RNAs RELATED IN THE SAME THING. THE HARD THING THERE IS THAT EVERY PROFILE IS REALLY DIFFERENT. EXTRACELLULAR RNA PROFILE, TOTAL SELL-FREE LOOKS DIFFERENT THAN THE VESICLES AND LOOKS DIFFERENT THAN RNA BINDING PROTEINS. >> ARE YOU IMAGING THESE KIDS? >> NO. WE ARE NOT. THAT'S SOMETHING THAT WE HAVE ALWAYS WANTED TO ADD TO THE STUDY. WE JUST HAVEN'T HAD FUNDING FOR IT. >> BECAUSE STANDARD IMAGING IS NOT SENSITIVE ENOUGH. I'D BE INTERESTED TO TALK TO YOU. >> OKAY. THANK YOU. >> THANK YOU VERY MUCH DR. JENSEN. NOW WE WOULD LIKE TO PROCEED WITH DR. AISLED SLADE AND HIS PREALEX VALADKA. >> THANK YOU. IT IS A PLEASURE TO BE HERE SO JUST BY WAY OF INTRODUCTION. I'M A NEUROSURGEON WHO JUST LAST FALL RETURNED TO VIRGINIA COMMONWEALTH UNIVERSITY WHERE I DONE MY RESIDENCY AND I WENT THERE SPECIFICALLY BECAUSE I WAS TOLD IT WAS THE BEST PLACE IN THE WORLD FROM BRAIN INJURY SEARCH AND I'M HOPING TO BRING THAT DEPARTMENT BACK TO THAT LEVEL. BUT, IN MY WORLD, PEOPLE TEASE ME AND TELL ME THAT PREOPTATIVE AND POSTOPERATIVE COMATOSE PATIENTS DIFFER ONLY BECAUSE THEY ARE COMATOSE. I'LL CRY TOO KEEP YOUR ATTENTION. THREE MAIN THINGS I'M GOING TO DO -- IF YOU USE THE -- CAN YOU SEE THE ARROW IN THE OTHER ROOM? ON THE OTHER SCREEN? I'LL TRY TO REMEMBER TO DO THAT SO YOU CAN SEE WHAT I'M REFERRING TO. THREE MAIN THINGS I'LL TRY TO COVER TODAY AND IN A WAY, THIS TALK -- KENDEL TALKED ABOUT RNA AND I'M GOING TO FOCUS ON PROTEIN-BASED MARKERS. THE FIELD IS VERY RAPIDLY EVOLVING BUT IT IS PRETTY CLEAR NOW THAT YOU CAN USE BLOOD BASED MARKERRERS OF TBI FOR DIAGNOSIS. PROGNOSIS PERHAPS AND MANAGEMENT NOT AS MUCH. ALTHOUGH I THINK THAT IS WHERE WE NEED TO GO ESPECIALLY IN THE SCENARIOS THAT TAMMY LAID OUT IN THE INTRODUCTION IF YOU HAVE THE SERVICE MEMBER WHO IS PINNED DOWN AND CAN'T GET TO A AIRCRAFT CARRIER FOR SEVERAL DAYS. IT IS POSSIBLE TO MANAGE THE EVOLVEMENT OF A BRAIN INJURY? AND KENDEL MENTIONED THERE IS ALSO A SECONDARY INJURIES AND CLOSE INTERACTION OF WHAT HAPPENS WITH THE BODY IN TERMS OF HYPERTENSION AND HYPOXIA AND THINGS LIKE THAT THAT CAN AFFECT THE BRAIN INJURY. BUT I'LL MENTION THERE HAS BEEN SOME QUESTIONS RAISED, SOME UNCENTER THESE FORCED US TO RETHINK HOW WE ARE ARE GOING TO DO THESE AND WE'LL EXPLORE THE OPPORTUNITIES IN THE FUTURE. SO I'M GOING HAVE THREE MAIN AREAS FOR THIS TALK AND THE FIRST IS SHOWING SOME OF THE UTILITY OF THIS. A FRIEND OF MINE DESCRIBED THIS FIELD OF BLOOD BASED MARKERS OF BRAIN INJURY IN TBI DIAGNOSTICS A FIELD THAT IS A MILE WIDE AND AN INCH DEEP. THAT MAY HAVE BEEN TRUE A FEW YEARS AGO WHEN THIS WAS BRAND NEW BOTTLINGS ARE STARTING TO -- BUT THINGS ARE STARTING TO DECLARE THEMSELVES A LITTLE BIT. THIS SLIDE IS PERHAPS SOMEWHAT ASPIRATIONAL BUT IT IS MAYBE POSSIBLE TO IDENTIFY CERTAIN PARTS OF THE NEURON AND TIE CERTAIN MARK TOURS CERTAIN AREAS. SO YOU WILL HEAR A LOT ABOUT UCHL1. UBIQUITIN CAR C TERMINAL HYDROLACE L1 THAT IS ONE OF THE MOST POPULAR FREQUENTLY STUDIED ONES. AND PERHAPS YOU CAN LOOK AT DIFFERENT PARTS OF THE CELL BODY, LOOK AT THE NECROSIS VERSUS APOPTOSIS. WE ALREADY HEARD A LITTLE BIT ABOUT THE NEUROVASCULAR UNIT. I THINK DON WILL COVER THAT NEXT AND WE'LL HEAR MORE ABOUT IT LATER. MIGHT BE MARKERS PARTICULAR TO THAT. ESPECIALLY CHRONICALLY PERHAPS IN CYTOKINES. WE'LL GET INTO THAT A LITTLE BIT LATER. SO THE POINT IS THAT MAYBE THIS IS A LITTLE BIT IDEALIZED BUT IT WOULDN'T BE INTERESTING TO BE ABLE TO FIGURE OUT WHETHER THE EXON IS INJURED MORE THAN A DEN DRIED OR ASSOCIATED VAC LAATURE OR MYELIN SHEET. THIS IS PRETTY MUCH A STATE-OF-THE-ART RIGHT NOW. MOST OF THESE PAPERS ARE A LOT OF THEM AT LEAST, COME FROM THE COLLEAGUES IN EMERGENCY DEPARTMENTS. THEY ARE TRYING TO FIGURE OUT DO I NEED A CT SCAN ON ALL THESE PATIENTS? ESPECIALLY AS COSTS ARE GOING UP AND INCREASING AWARENESS OF THE DANGERS OF EXCESSIVE RADIATION EXPOSERS ESPECIALLY IN PEDIATRICS. SO IF YOU MEASURE LEVELS OF CERTAIN THINGS ABOVE A CERTAIN THRESHOLD, MAYBE YOU DON'T NEED TO SCAN THAT PERSON AND MAYBE YOU DON'T NEED A FOLLOW-UP CT SCAN. THAT IS INTERESTING. I THINK THAT IS ONLY ONE VERY PRELIMINARY TYPE OF APPLICATION OF THIS. NOW THIS SLIDE IS A LITTLE BIT BUSY. SO LET ME EXPLAIN WHAT IS GOING TO HERE. THE LEVEL OR TWO PANELS ON TOP OF CSF. THE TWO ON THE BOTTOM ARE SERUM AND THESE ARE LEVELS OF UCHL1 AND ON THE LEFT-HAND SIDE THESE ARE THE PATIENTS WHO DIED. THESE ARE NON SURVIVORS. AND RIGHT-HAND SIDE YOU HAVE PATIENTED WHO SURVIVED. AND THIS IS ABOUT A WEEK OR SO. SAMPLES OBTAINED EVERY SIX HOURS. SO YOU CAN SEE WHAT HAPPENS IN PATIENTS WHO DIDN'T DO WELL. THEIR LEVELS ARE PRETTY HIGH AND THE Y AXIS ARE THE SAME UNITS HERE. 100, 200, 300. LOWER LEVELS IN CSF ARE THE PATIENTS WHO SURVIVED. IF YOU LOOK AT SERUM, VERY HIGH LEVELS ESPECIALLY EARLY ON AND UCHL1 AND THE PATIENTS WHO DID NOT MAKE IT YOU ABOUT THE PATIENTS WHO SURVIVED TENDED TO DO BETTER. THERE IS A LOT OF STUFF ON THIS SLIDE. PERHAPS MOST IMPORTANT THING IS THIS PAPER THAT LINDA PUBLISHED RECENTLY IN JAMA NEUROLOGY. EMERGENCY MEDICINE PHYSICIAN LOOKED AT PATIENTS FOR AN ENTIRE WEEK. THE BLUE LINES HERE ARE GFAP. THE ORANGE IS UCHL1. AND THIS IS THE WHOLE SERIES OF PATIENTS. IF YOU LOOK OVER HERE, THESE ARE PATIENTS WHO HAD NO CLINICAL EVIDENCE OF MILD OR MODERATE TBI BUT THESE ARE THE ONES WHO DID. THE TAKE HOME MESSAGE YOU SEE A BUMP IN U WHY. HAD. L1 IN ABOUT 20 HOURS -- UCHL1 -- ROUGHLY A DAY AFTER INJURY AND THEN IT CAME DOWN AS OOH POSED TO UC HAD. L1 HIGH INITIALLY AND THEN CAME DOWN. THIS PAIR OF SLIDES OVER HERE, THESE ARE PATIENTS WHO HAD CT SCAN EVIDENCE OF BRAIN INJURY. SO BLOOD SOMEWHERE INSIDE THE SKULL. AND AGAIN, VERY SIMILAR PATTERN. UCHL1 ABOUT A DAY LATER AND THEN COMES DOWN -- GFAP PEAKED WHEREAS UCHL1 WAS HIGH AND THEN CAME DOWN. AND THESE PATIENTS UNDERWENT SOME SORT OF NEUROSURGICAL PROCEDURE AND THE SAME PATTERN. A PEAK IN GFAP A DAY LATER AND UCHL1 COMES DOWN AND THEN PATIENTS WITH NO INTERVENTIONS STAYED LOW THE WHOLE TIME. AND ANOTHER SLIDE THIS IS SAY PAPER PUBLISHED BY RAMONE DIAZ WHO IS THERE IN THE AUDIENCE BACK THERE. THIS CAME OUT A COUPLE OF YEARS AGO. AND THE DATA WERE SPREAD OUT BY DOING TRANSFORMS. WHAT IS INTERESTING HERE, YOU CAN READ IN THE LEGEND IF YOU SEE A PATIENT -- LOOK AT THE ONES WHO HAD ELEVATED UCHL1s SO EVERYTHING TO THE RIGHT OF THIS GRAPH, MOST ARE ASSOCIATED WITH HIGH GFAPs BUT THE FLIP SIDES ISN'T TRUE. PATIENTS HIGH GFAPs, EVERYTHING ABOVE THIS, YOU HAD A LOT OF LOW UCHL1 LEVELS TOO. MAYBE THERE IS DIFFERENT KINETICS OF HOW THESE THINGS GET IN YOUR BLOOD, MAYBE DIFFERENT INJURY PATTERNS WE ARE NOT AWARE OF HERE. AND THIS IS A LITTLE BIT IDEALIZED BUT THE POINT HERE IS THAT THESE ARE PATIENTS WITH THE DIFUSE BRAIN INJURY. SO THEY HAVE MAYBE SOMETHING LIKE A COMPLICATED CONCUSSION OR A COMPLICATED MILD BRAIN INJURY WHERE THERE IS BLOOD IN THE HEAD OR MAYBE SOMETHING MORE SEVERE. THESE ARE PATIENTS WHO HAD A MASS LESION IN THEIR BRAIN EITHER CONTUSION IN THE BRAIN OR EP DO YOU RECALL HEP TOME A SO THE RATIO OF GFA36789 TO UCHL1 WAS HIGHER IN PATIENTS WHO HAD STRUCTURAL DAMAGE EVIDENCE BY BLOOD SOMEWHERE IN THERE. AND AGAIN, PERHAPS IT IS POSSIBLE THAT IF YOU LOOK AT SOME OF YOUR ACK SEMERATION AND DECELLERATIONS WITH THE FOOTBALL PLAYERS, DIFFUSE INJURY SEEMS TO LEAD TO A HIGHER LEVELS OF UCHL1 AS COMPARED TO GFPA MAYBE BECAUSE YOUR PROMINENCE INJURY MECHANISMS THERE IS NEURONAL CELL DEATH. AS OPPOSED TO A BIG LESION WHERE YOU SEE HIGHER LEVELS OF GFAP COMPARED TO UCHL1 MAYBE BECAUSE OF PRIMARY PATHOPHYSIOLOGY GOING ON THERE IS GLIOSIS. THIS IS A VERY BUSY SLIDE BUT IT IS INTERESTING IF WE UNPACK THIS, EACH ONE OF THESE LINES ON THE GRAPH, THIS IS WHAT HAPPENS ACUTELY AND THIS GOES INTO THE CHRONIC PHASE HERE. HERE WE HAVE MINUTES GOING HOURS AND DAYS AND NOW MONTHS AND YEARS. SO ACUTELY, THE THING THAT HAPPENS WHEN YOU HAVE A BRAIN INJURY IS IMMEDIATE INJURY TO THE NEURONS, AXONS, DENDRITES. AND IT SEEMS AS IF YOU CAN MEASURE THAT BY VERY EARLY SPLICING THINGS LIKE UCHL1, SPECTRUM BREAKDOWN PRODUCTS. MAP 2. A FEW MINUTES LATER YOU START TO GET DAMAGE TO THE VASCULATURE AND THE BLOOD-BRAIN BARRIER. AND AGAIN SOME MARKERS AVAILABLE FOR THAT. A SHORT WHILE LATER, GPAP SEEMS TO PEAK AND THEN YOU START SEEING APOPTOSIS. THIS IS EVIDENCE EARLY ON AND SEEMS TO PEAK LATER. THERE ARE MARKERS FOR THAT. AND FOUNDING DOWN THE LINE YOU START TO GET NEURODEGENERATION. INTERESTING HERE, LOOK AT MONTHS AND YEARS. A LOT OF THESE THINGS SEEM TO COME BACK UP. I'M GOING TO COVER THIS A LITTLE BIT AT THE END. ONE OF THE OTHER THINGS YOU SEEN IS SOME OF THE PREVIOUS SLIDES IS WE HAVE BEEN FOCUSING A LOT ON THE BLOOD-BRAIN BARRIER. EVERYONE IS TALKING ABOUT STUFF THAT GETS IN THE BLOOD INTO THE BRAIN. WE REALLY HAVEN'T BEEN COVERING AS MUCH ABOUT THE BRAIN BLOOD BARRIER. AND THOSE TWO ARE NOT NECESSARILY THE SAME THING AS WHEN DR. GIBBONS GAVE INTRODUCTION, HE TALKED ABOUT BLOOD-BRAIN INTERFACE. MAYBE THAT SAY PHRASE WE SHOULD BE USING. THIS IS HOW THEY THINK OF THE BLOOD-BRAIN BEARIER, THESE TIGHT JUNCTIONS IN THE ENDOTHELIAL CELLS. THERE IS ALSO A BARRIER OUT HERE. IT IS STUFF THAT IN THE BRAIN MAY NOT HAVE SUCH AN EASY TIME GETTING INTO THE BLOOD ONCE WE BREAK THIS BARRIER DOWN HERE. SO WE NEED TO APPRECIATE SOME OF THE BIDIRECTIONALITY HERE. SO LET'S GO OVER A FEW SLIDES. THE SECOND OF THE THREE PARTS OF MY TALK HOW WE NEED TO GET A BETTER UNDERSTANDING HOW TO INTERPRET THIS DATA. AND THE WAY TO THINK ABOUT THIS IS THIS IS SUPPOSED TO BE SORT OF SEQUENCE OF EVENTS MOVING FROM LEFT TO RIGHT. SO IF THERE IS A BRAIN INJURY HERE, THE CELLS GET INJURED. THINGS GET FROM THE INTEREST CELLULAR TO EXTRACELLULAR COMPARTMENT IN THE BRAIN AND WE HEARD ABOUT EXOSOMES AND WE'LL HEAR MORE ABOUT THEM AND THE MARKERS THEMSELVES MAY GET OUT. THERE IS BREAK OUT AT THE BRAIN BLOOD BARRIER AND THAT IS HOW WE GET MARKERS IN THE CSF AND FROM THERE WE CAN GET INTO THE BLOOD. AS KENDEL MENTIONED, URINE AND START LOOKING AT SALIVA AND SWEAT AND EVEN A FEW OTHER WAYSES TO TRY TO SAMPLE THIS AND SOMETHING THAT MAY BE NON-INVASIVE, SOMETHING THAT YOU COULD POTENTIALLY DO SERIALLY. YOU COULD IMAGINE IF YOU GET BEDSIDE DIAGNOSTICS TO DO SOME OF THIS STUFF WITH THE FINGER STICK OR A SALIVA SWAB OR SOMETHING ALONG THOSE LINES. IF YOU WANT TO THINK ABOUT THIS IS FOR YOUR BIOMARKERS COMING FROM, THIS IS HOW WE DETECT THEM SOMEWHERE IN THE BALANCE OF THAT WE START TO GET INTO THE BIOKINETICS OF THESE THINGS PERHAPS WHY SOME OF THEM PEAK AT CERTAIN HOURS. SOME MUCH EARLIER AND SOME MUCH LATER. LET'S GO OVER A COUPLE OF ARTICLES HERE. SO HERE IS ONE LOOKING AT THE AMERICAN FOOTBALL PLAYERS AFTER A GAME TRANSIENT BLOOD-BRAIN BARRIER -- [ READING ] ... SOUNDS GREAT BUT THEY ASSUMED THAT ELEVATED F100 BETA COMES FROM BBB OPENING EVEN THOUGH YOU CAN FIND IT IN PERIPHERAL TISSUE. IF YOU'RE ON ORTHOPAEDIC SERVICE, THEY WILL HAVE ELEVATED S100 BETA WITH BONE FRACTURES. THEY DIDN'T DISTINGUISH BETWEEN BLOOD GETTING GETTING INTO BRAIN OR BRAIN INTO BLOOD. AND FINALLY IN ANY ONE OF THESE STUDIES, WE NEED TO HAVE PROPER CONTROLS. SO EXERCISE CAN OPEN THE BLOOD-BRAIN BARRIER AND IN TRAUMA STUDY, YOU NIDE TRAUMA PATIENTS WHO HAVEN'T HAD CENTRAL NERVOUS SYSTEM INJURY. SO BREAK EN ANKLES OR ABDOMINAL INJURY OR SOMETHING LIKE THAT. HERE IS ANOTHER PAPER. [ READING ] ... WELL, AGAIN, THEY ASSUME THAT THE ALBUMIN QUOTIENT REFLECTS BLOOD BRAIN BARRIER STATUS. AGAIN DIDN'T DISTINGUISH BETWEEN THE BBB. WE TALKED ABOUT THE CONTROLS ALREADY. VERY SMALL NUMBER OF PATIENTS. THEY LOOKED AT TIME POINTS 12, 24 AND 40 YOU HOURS. ONLY THE FIRST WAS SIGNIFICANT. NOW SOME OF THIS IN TERM OF MAKING SURE THAT ARTICLES DON'T GET PUBLISHED IS OUR RESPONSIBILITY AS PEER REVIEWERS AND EDITORS OF JOURNALS, BUT NOTICE THIS WAS 2011. IN THIS FIELD IT IS EVOLVING VERY RAPIDLY. BACK THEN THAT MAY HAVE BEEN GOOD ENOUGH BUT NOW AS WE LEARNED A LOT MORE ABOUT HOW THIS WORKS, THIS ARTICLE MAY NOT BE ACCEPTABLE TODAY EVEN THOUGH IT WAS STATE-OF-THE-ART JUST A FEW YEARS AGO. ONE MORE INTERESTING PAPER HERE THAT AGAIN LOGARITHMIC TRANSFORMATION. SO NOTICE IF WE LOOK AT CSF VERSUS SERUM LEVELS OF GFAP, THERE IS A PRETTY GOOD CORRELATION HERE. NOT SO FOR UCHL1. THE CORRELATION IS ONLY .19 AND THEY ARE LUMPED OVER THERE. THESE BIOMARKERS ARE VERY DIFFERENT AND THEY MAY REFLECT NOT ONLY DIFFERENT PARTS OF THE NERVE CELL AND THE AREAS THAT ARE INJURED BUT EVEN DIFFERENTTINES OF PATHOPHYSIOLOGY. A CLOSED DIFFUSE INJURY VERSUS A MASS LEGION BEING THERE. SO FINALLY LET'S TALK ABOUT SOMETHING THAT WE MAY BE ABLE TO MAKE MORE USE OF IN THE FUTURE JUST TO GIVE ONE EXAMPLE ABOUT REALLY UNDERSTANDING THE PATHOLOGY OF THIS. NOT JUST ACUTELY IN A SCENARIO THAT TAMMY OUTLINED BUT EVEN MONTHS DOWN THE LINE. BECAUSE WE ARE ACTUALLY PRETTY GOOD AT TAKING CARE OF PATIENTS ACUTELY BUT THEN THEY HAVE TO GO TO REHAB FACILITY AND I ALWAYS SAY THAT THE SURGEONS HAVE THEM FOR A COUPLE OF WEEKS BUT THEN THEY HAVE TO DEAL WITH THEIR PROBLEM AND DISABILITIES FOR THE REST OF THEIR LIVES. SO IT'S POSSIBLE THAT THERE IS A SEQUENCE OF EVENTS GOING ON WE SEE ACUTE EXONAL DAMAGE, FOCAL INFLAMMATION AND APOPTOSIS WHERE THE BLOOD-BRAIN BARRIER GETS OPENED OR THE BLOOD-BRAIN BARRIER. WE GET MICROBLEEDS AND LOCAL INFLAMMATION AND DEGENERATION OF BRAIN TISSUE THAT TURNS INTO A SELF PERPETUATING CYCLE. HERE IS A PAPER FROM ALENA WHO IS MADE HER AN ADJUNCT FACULTY IN HER DEPARTMENT. THERE IS A LOT ON THIS SLIDE. BUT THE UNILATERAL CONTROL CORTICAL IMPACT, SO THIS IS A RODENT STUDY. THE TYPE OF INJURY WE CAN USE LOOKING AT ONE DAY A WEEK, A MONTH, TWO MONTHS AND 3 MONTHS. YOU SEE THAT INITIALLY THERE IS A SORT OF DIFFUSE INCREASE IN STAINING FROM THE IGG WHICH IS GOING AWAY BUT YOU SEE PROGRESSIVELY INCREASING OF PUNKITATED AREAS OF IGG STAINING AND THAT IS ILLUSTRATED HERE. THIS IS THE DIFFUSE BACKGROUND THAT GOES AWAY AND HERE ARE NUMBERS OF THINGS THAT GO UP AFTER THREE MONTHS WHICH CORRELATES WELL WITH AREAS OF MICROBLEEDING. YOU DON'T SEE MUCH INITIALLY BUT WITH TIME THERE IS MORE AND MORE AREAS OF FOCAL HEMORRHAGE. AND IF YOU LOOK AT THE CHORPUS CALLOSUM, YOU SEE AS WE GO FROM SHAM CONTROLS TO 24 HOURS A MONTH, TWO MONTHS, 3 MONTHS, YOU SEE PROGRESSIVE DECREASE IN THE CHORPUS CALLOSUM, WHITE MATTER TRACKS THERE, AND AGAIN IF YOU LOOK AT THE CD68, A MARKER INDICATING MICROGLIAL AND MONOCYTE ACTIVATION, YOU SEE THAT TENDS TO INCREASE OVER TIME AS WELL GOING UP MORE AND MORE. SO, IT'S POSSIBLE THAT OVER TIME, IF YOU COMPARE SHAM INJURIES TO INJURIES THREE MONTHS LATER, WE MAY HAVE ONGOING BREAKDOWN OF THE BLOOD-BRAIN BARRIER, PERHAPS THE BRAIN BLOOD BARRIER, NOT JUST ACUTE BUT SOMETHING THAT IS BAD AND GETTING WORSE ALL THE TIME. SO AGAIN, TRYING TO WRAP ALL OF THIS UP, I THINK THIS IS SOMETHING WE NEED TO GET INVESTIGATORS TO THINK ABOUT MORE AND MORE. THE BLOOD-BRAIN BARRIER IS BIDIRECTIONAL. HAVE PEOPLE SPECIFY WHETHER THEY ARE LOOKING AT BLOOD-BRAIN BIRRIER OR BRAIN BLOOD BARRIER AND STANDARDIZE YOUR CONTROL FOR WHAT YOU'RE ASSUMING AND THE OTHER BIG PROBLEM IS THAT THE OPENING MAY NOT BE A PATHOLOGICAL EVENT. IF YOU ARE ALLOWED TO RUN A FEW MILES OR LIFT WEIGHTS, YOU MAY BE OPENING YOUR BLOOD-BRAIN BARRIER OR YOUR BRAIN BLOOD BARRIER. SO AGAIN, NEED TO EXCLUDE NON-SPECIFIC OPENING EVENTS LIKE EXPRESS OR OTHER PATHOLOGIES. AND FINALLY WE NEED TO DEVELOP STANDARDIZED PROCEDURES AND METHODS SO WE ARE ALL SPEAKING THE SAME LANGUAGE HERE. THIS IS CONFUSING ENOUGH JUST IN THE TRAUMA WORLD AND WE HAVE PEOPLE HERE FROM TUMOR AND TRAUMA AND DEGENERATIVE DISEASE AND OTHER AREAS. SO WE HAVE A LOT OF WORK TO DO IN THIS FIELD. SO I NEED TO THANK RON HAZE, ALENA AND A LOT OF OTHER PEOPLE WHO -- THINK ABOUT THIS, IT WAS FUN TO THINK ABOUT AND SPECULATE PUTTING THIS TOGETHER. WE HAVE TIME FOR QUESTIONS? ONE OR TWO QUESTIONS. SORRY. I RAN A LITTLE LONG. IF YOU COME TO THE MICROPHONE -- [ APPLAUSE ] FOR PEOPLE WHO MAY BE LISTENING PLEASE STATE WHO YOU ARE AND WHERE YOU'RE FROM SO WE CAN GET TO KNOW EACH OTHER A LITTLE BIT BETTER ALSO. CLEAR AS MUD? THANK YOU. >> ALL RIGHT. SO IF THERE ARE NO QUESTIONS, AT THIS THE CURRENT TIME WE LIKE TO PROCEED WITH THE NEXT PRESENTATION, DR. MARION, POST-TRAUMATIC CEREBRAL BLOOD FLOW AUTO REGULATION OF THE NEUROVASCULAR UNIT. >> DON MARION: SO THANK YOU TAMMY AND ORGANIZERS OF THIS WORKSHOP FOR INVITING ME. I FEEL A LITTLE BIT LIKE A FISH OUT OF WATER HERE. BUT I CERTAINLY DON'T HAVE THE INSIGHT AND EXPERIENCE WITH BLOOD-BRAIN BARRIER THAT MOST OF THE PEOPLE IN THIS ROOM DO BUT I WOULD LIKE TO TALK TO YOU A LITTLE BIT ABOUT SEIZ RECALL BLOOD FLOW. I RECALL -- CEREBRAL BLOOD FLOW. FIRST OF ALL, MY DOD FLAG. I'M SPEAKING FOR MYSELF AND DO NOT SPEAK IN AN OFFICIAL CAPACITY FOR THE DEPARTMENT OF DEFENSE. I JUST WANT TO START OUT BY SAYING THAT I RECALL AS A RESIDENT AND ESPECIALLY JUNIOR RESIDENT, JUST TWO OR THREE YEARS -- NO MORE THAN THAT AGO -- [ LAUGHS ] BUT THE STANDARD OF CARE FOR MANAGEMENT OF TRAUMA PATIENTS, ESPECIALLY SERIOUSLY HEAD INJURED PATIENTS WAS AGGRESSIVE HYPERVENTILATION AND THAT IS WHAT WAS DONE BY THE PRE-HOSPITAL PERSONNEL AND ALL THESE PATIENTS THAT THE CAME IN AND THE RATIONAL FOR THAT WAS THAT IF YOU HYPERVENTILATED THE PATIENT THE ASSUMPTION WAS YOU PRODUCE CEREBRAL BLOOD FLOW AND THEREFORE REDUCE CRANIAL PRESSURE AND ASSUMED THE COMATOSE HEAD INJURED PATIENT HAD PROBLEMS WITH ELEVATED GRANULAR PRESSURE AND THAT IS WHAT WAS GOING TO KILL THEM IF THEY WERE GOING TO HAVE A POOR OUTCOME. OF COURSE WE KNEW FROM PHYSIOLOGISTS DECADES BEFORE THAT THAT WAS A FLAWED WAY OF THINKING SINCE MOST OF THE CEREBRAL BLOOD VOLUME IS -- AND HYPERVENTILATION ONLY EXPECTED TO AFFECT THE ARTERIAL SIDE OF THE CIRCUIT OR THE MUSCULAR ARTERIES OR ARTERIALS. SO WE AGAIN LOOKING AT AN ISSUE WITH SEIZ RECALL BLOOD FLOW STUDIES AND THAT IS GOING TO BE -- CEREBRAL BLOOD FLOW -- AND THAT IS THE CORE OF THE TALK AND I'LL TRY TO RELATE SOME POSSIBLE MECHANISMS VIS-A-VIS THE BLOOD-BRAIN BARRIER AND THEN FINISH WITH SOME OF THE CLINICAL STUDIES THAT WE DID. SOME OF THE THINGS WE KNOW IS THAT THERE IS A SIGNIFICANT DECREASE IN CBF IMMEDIATELY AFTER TRAUMA PROBABLY IN THE ORDER OF 30-40% AND EVEN MORE IN FOCAL REGIONS ESPECIALLY AROUND CONTUSIONS. AND THERE IS A DISRUPTION OF PRESSURE AUTOREGULATION AND ALSO DECREASE OR INCREASE IN CHEMICAL AUTOREGULATION OF CO2 AUTOREGULATION. THERE IS A SIGNIFICANT NUMBER OF -- IN THE MILITARY, A SIGNIFICANT NUMBER OF SEVERE BLAST TBI VICTIMS THAT DEVELOPED VASCULAR PROBLEMS THROUGH ANEURYSMS AND VASOSPASM. 80% OF PATIENTS WITH VASEO SPASM WERE SHOWN SUSTAINED A BLAST TRAUMA SEVERE VASOSPASMS SOMETIMES LASTED AS MUCH AS 14 DAYS. THE BLAST VASCULAR PATHOLOGIES ALTERATIONS OF THE VASCULAR EXTRACELLULAR MATRIX AND SUSTAINED MICROGLIAL AND ASTROGLIAL REACTIONS AND PERIPHERAL INFLAMMATORY RESPONSE THAT COULD LAST FOR QUITE SOMETIMES. IN ADDITION, THERE IS THIS PHYSIOLOGIC RESPONSE, NEUROSTIMULATION THAT EVOKES INCREASE IN CEREBRAL BLOOD FLOW AND THIS IS THE BASIS FOR FUNCTIONAL MRI. AND ALSO IONIC FLUXES THROUGH ARRAY OF ION CHANNELS WITH ALLOW THE NEUROVASCULAR UNIT TO ENGAGE IN MULTICELLULAR SIGNALING AND THIS LIKELY PLAYS A ROLE IN LOCAL HEMODYNAMICS. IT'S UNDERSTOOD FROM A NUMBER OF INVESTIGATORS INCLUDING THE STUDY THAT ION CHANNELS MODULATE THE ARTERIAL SMOOTH MUSCLE MEMBRANE POTENTIAL. THERE IS NO DIRECT CONNECTION AND WE'LL TALK ABOUT THAT IN A MINUTE. THERE IS NO DIRECT CONNECTION BETWEEN THE FOOT PLATE OF THE ASTROCYTES AND THE VASCULAR SMOOTH MUSSEL SO NO DIRECT CELL TO CELL CONTACT BETWEEN ASTROCYTE AND END. SO IT IS MOST LIKELY MOLECULAR MAYBE INVOLVING EXOSOMES AND SUCH. IN MURINE STUDIES, THERE IS A REDUCTION IN CEREBRAL BLOOD FLOW DURING HYPOXIA THAT RELATES TO AREAS WITH COMPROMISED BLOOD-BRAIN BARRIER AND SUGGESTS THAT THERE IS A LINK BETWEEN THAT HYPOXIA AND THIS IS KIND OF BEEN, AT LAST ONE MECHANISM THAT IS ALWAYS ATTRACTIVE TO ME SINCE I WAS CONVINCED THAT AT THE TIME OF SEVERE INJURY IN THE MOTOR VEHICLE CRASH FOR EXAMPLE, THAT THERE WAS PROBABLY A BRIEF% OF HYPOXIA OR PERHAPS -- A BRIEF PERIOD OF HYPOXIA IN A FEW MINUTES. IN TERMS OF MOLECULAR MECHANISMS AND THE NEUROCHEMICALS THAT MAY BE INVOLVED, MY COLLEAGUE DR. CLARK, WHO IS WITH US HERE TODAY, AND WILL FOLLOW ME, LOOKED AT SEVERAL YEARS AGO ALMOST A DECADE OR SO AGO NOW, AND SUGGESTED THAT ADEN SIN MIGHT PLAY A ROLE IN THESE VASCULAR CHANGES AND SO WHAT HE DID IS HE SHOWED THAT IF YOU LOOKED AT THE ARTERIAL JUGULAR VENUES DIFFERENCE IN TBI PATIENTS, HE DEFINED CORRELATION BETWEEN ADEN SIN LEVELS AND THE AMOUNT OF OXYGEN EXTRACTED FROM THE BLOOD -- BY THE BRAIN SO THE LARGER THE HIGH -- OR THE HIGHER THE NUMBER, THE MORE OXYGEN WAS EXTRACTED AND THE LOWER THE ACTIONER DEN SIN. SO IT APPEARED TO HAVE AN FACT -- ADEN SIN. CBF IS TYPICALLY 30-40% LOWER THAN NORMAL. IF YOU DO THESE DIRECT BLOOD FLOW STUDIES AND DO IT IN VERY EARLY AFTER THE INJURY, AND THEN FOLLOW THEM ALONG WITH STUDIES, WHAT YOU TYPICALLY FIND IS THE LOWEST OF THE LOWS ARE WITHIN THE FIRST FEW HOURS AFTER INJURY. AND THAT PATIENTS WITHOUT SURGICAL MASS LEGIONS WHO DIE HAVE LOWEST LOWS AND THOSE WITH THE BEST OUTCOMES HAVE THE HIGHEST FLOWS EVEN THOUGH THEY MAY COME IN WITH SIMILAR OR SAME GCF ON ADMISSION. 24 HOURS AFTER INJURY FLOWS TYPICALLY INCREASE IN GROUPS THAT HAVE A GOOD OUTCOME. THEY MAY STAY LOW FOR A DAY OR EVEN WEEKS THAN THOSE WHO DON'T. AND THIS IS A CLASSIC STUDY I THINK FROM SNIDER AND HIS GROUP AT UCLA, PUBLISHED IN 1997 BUT I THINK IT IS ONE OF THE FIRST AND ONE OF THE MOST IMPORTANT THAT SHOWED A MISMATCH BETWEEN METABOLISM AND CEREBRAL BLOOD FLOW. SO THIS IS A PERSON WITH A SUBDUAL HEMOTHOMA AND PROBABLY A CONTUSION. A CORRESPONDING PET SCAN WAS DONE SHOWING HYPOGLYCEMIA OR INCREASED GLUCOSE METABOLISM AND THE BLOOD TISSUE UNDERLYING THAT BUT THAT IS ASSOCIATED WITH AN AREA OF LOW FLOW, DARK AREAS BEING LOW FLOW AND THE RED BEING THE HIGHER OR NORMAL FLOWS. IN ADDITION, HYPERVENTILATION CAN CAUSE FLOW ISCHEMIA AND IN OUR STUDIES WE FOUND THAT AT 24-36 HOURS THERE WAS A 10% OR MORE INCREASE IN THE CONCENTRATIONS OF GLUTAMATE IN THESE PATIENTS AND 10% OR MORE INCREASED IN LACTATE AND 10% WERE MORE INCREASED IN LACTATE HIGH ROWIVATE RATIO. ALL MARKERS FOR CEREBRAL ISCHEMIA. THE EFFECTIVE POST-TRAUMATIC LESIONS ON MULTIPLE CO2 PATIENTS WITHIN CONTUSIONS SURROUNDING HYPERACTIVITY WAS SUGGESTING A HYPERSENSITIVITY TOW HYPERVENTILATION THERAPY. WE FOUND CONTUSIONS WERE PARTICULARLY VULNERABLE TO SECONDARY INJURY AND THIS MAY BE CAUSED BY HYPOTENSION OR AGGRESSIVE HYPERVENTILATION AND IN ONLY TO THE OF THE PATIENTS WE STUDIED THE CONTUSION WAS BAYSO REACTIVITY LESS THAN 1%. TYPICALLY IT WAS HYPERREACTIVE AND ESPECIALLY IN THIS RIM OF BRAIN TISSUE SURROUNDING THE CONTUSION QUITED VARIABLE ACTUALLY AND THIS IS AN EXAMPLE OF THAT. THIS IS A PATIENT WE HAVE WITH CONTUSIONS OF THE FRONTAL AND TEMPORAL LOBES. AT NORMAL VENTILATION, BLOOD FLOWS WEREN'T BAD. STILL SOME ISCHEMIA OR SOME LOW FLOW AROUND THE CONTUSION BUT INTERESTINGLY, WHEN WE HYPERVENTILATED THE PATIENT AND THEN REPEATED THE STUDY 15 MINUTES LATER, YOU SAW A VERY LOW SLOW SURROUNDING THE CONTUSION AND HIGH FLOWS WITHIN THE CENTER OF IT. SO WE INTERPRETED THIS AS SUGGESTING THAT THERE REACTIVITY OR THE ABILITY OF THE VESSELS TO CONTROL FLOW WAS GONE IN THE DEAD TISSUE, MAXIMALLY BASAL DILATED SO YOU HAD POOLING OF BLOOD AND THAT DEAD TISSUE BUT IN THE SURROUNDING TISSUE THAT COULD HAVE SURVERIFIED, THERE WAS WHERE WE WERE DRIVING TISSUE INTO VERY LOW FLOW OR PERHAPS ISCHEMIA. SO, SAME FINDINGS WERE OBSERVED BY NYE COLLEAGUE AT PITTSBURGH CHILDREN HOSPITAL IN A PEDIATRIC SUDY AND ALSO FINDING ABNORMALLY BLOW FLOWS ON AVERAGE AND COMATOSE KIDS ON ADMISSION THAT TYPICALLY INCREASED BY A WEEK OR SO AFTER ADMISSION IN THOSE WHO DID WELL. SO, IN SUMMARY, OUR CONCLUSIONS FROM THIS IS THAT CBF CHANGES FOLLOWING TRAUMA ARE COMPLEX. AND CAN RESULT FROM THE MECHANICAL DISRUPTION OF THE BRAIN TISSUE OR PERHAPS FROM MOLECULES NITRIC OXIDE OR ADENOSINE. IMPORTANTLY THERE APPEARS TO BE DISRUPTION OF AUTOREGULATORY MECHANISMS. I'M PROUD, I GUESS, TO THINK THAT I PLAYED A SOME SMALL PART IN AN IMPORTANT CHANGE IN PRACTICE. AS I SAID IN 1980 THERE WAS AGGRESSIVE HYPERVENTILATION IN THE PRE-HOSPITAL SETTING AND IN IN 2016 WE JUST REVISED THE GUIDELINES FOR THE MANAGEMENT OF TBI IN THEATER IN THE MILITARY AND THE RECOMMENDATION NOW IS TO MAINTAIN A PCO2 OF 35-40 FOR THOSE INDIVIDUALS. SO, WITH THAT, ARE THERE ANY QUESTIONS? [ APPLAUSE ] >> JUST ARM EDUCATIONAL QUESTION. SO -- A TISSUE IN THE BRAIN RESPONSIBLE FOR CSF PRODUCTION AND FOR -- THIS IS ONLY TISSUE IN THE BRAIN WHICH IS EXTREMELY EASY TO TARGET AND MODULATE GENE EXPRESSION. JUST FOR MANY REASONS BECAUSE IF YOU INJECT IN CSF, THIS IS WHERE EVERYTHING GOES RIGHT AWAY. SO, THERE ARE WELL STUDIED MECHANISMS WHICH REGULATE CSF FLOW PRODUCTION WHICH SEEMS TO BE SOME KIND OF ENTER PLAY BETWEEN SOLUBLE FORM OF VGF RECEPTOR ONE AND FLEET 1 AND MEMBRANE BOUND FRACTION. DO YOU THINK THAT MODULATING THE WEIGHT OF CSF PRODUCTION WHICH SHOULD INCREASE WEIGHT OF CSF FLOW MIGHT BE CONSIDERED AS INTERPRETIVE INTERVENTION STRATEGY FOR TRAUMATIC BRAIN INJURY? OR HAVE ANYBODY LOOKED AT IT? I JUST DON'T KNOW THIS FIELD AT ALL. >> IT'S BEEN LOOKED AT. AND PEOPLE HAVE LOOKED AT DRUGS THAT DO MODULATE CCSF REDUCTION AND MY AGING PREALZHEIMER'S BRAIN CAN'T REMEMBER THE NAME OF THE DRUG YET. BUT THAT IS -- I THINK THE PROBLEM WITH THAT IS THAT THE ORDER OF MAGNITUDE OF THE SWELLING AND INTERCRANIAL HYPERTENSION IS MUCH GREATER THAN EXTRA EXPECT TO SEE WITH MEDICAL REDUCTION OF CSF REDUCTION FROM THE CORE PLEX US. IN OTHER WORDS, IT'S A MUCH GREATER PROBLEM THAN WILL BE RESOLVED. >> SO PRACTICALLY INCREASING THE PRODUCTION OF CSF WOULD NOT COMPENSATE FOR THE LOCAL SWELLING WHICH INTERFERES -- >> VERY OFTEN IT'S NOT JUST LOCAL SWELLING. IT'S MORE GLOBAL SWELLING TOO. >> THANK YOU. QUESTIONS REGARDING SOME OF THE DATA. SO PREVIOUS VIEW OF ALL OF THIS IS LOOKING AT MACRO CIRCULATION AND THE ROLE AND WE SEE REPEATEDLY THAT AFFECTING THE MACRO CIRCULATION HAS VERY LITTLE AFFECT IN ENDPOINT. AND THAT IS REALLY THE MICROCIRCULATORY FAILURE THAT IN A LOT OF THESE CASINGS IS THE COMMON MECHANISM OF SECONDARY INJURY. ARE YOU FAMILIAR WITH ANY TOOLS AND DEVELOPMENT FOR STUDYING THIS MICROCIRCULATORY COMPONENT A LITTLE BIT BETTER OR ANYBODY IN THE ROOM? >> YES, I MEAN, I THINK THAT MRI TECHNIQUES ARE MUCH BETTER THAN WHAT WE USED TO HAVE AS CBS FOR EXAMPLE FOR STUDYING THAT. >> ONE POSSIBILITY IS USING THESE NANOPARTICLES. YOU CAN SEE BLOOD VESSELS DOWN TO ABOUT 50 MICRONS JUST ON THE 3T MAGNET. YOU CAN SEE IT SPECIFICALLY EARLY TIME POINTS IN THE BLOOD POOL AGENT. YOU CAN SEE THE INTACT LYMPHATICS IN AN INTACT ANIMAL. YOU CAN SEE AT A LATER TIME POINT, THEY ARE SPECIFICALLY TARGETED CD11BC AND CD68 CELLS. YOU CAN SEE AS FEW AS EIGHT LABELED CELLS IN THE BRAIN. SO THE SUPER PARAMAGNETIC MOLECULES ARE LOOKING AT MICROCIRCULATION AND LOOKING AT INFLAMMATION ARE VERY SENSITIVE. >> THANK YOU. AND AN EXPERT IN THIS AREA THANK YOU. >> THANK YOU VERY MUCH DOCTOR MARION. WITH THAT WE ARE READY TO MOVE ON TO THE NEXT TALK, DR. ROBERT CLARK PRESENTING EMPLOYING TRANSPORTERS AT BLOOD-BRAIN INTERNATIONS TO REGULATE THE BRAIN'S METABOLOMIC AND PHARMACOLOGIC MICRO-ENVIRONMENT. >> ROBERT CLOCKER: >> ROBERT CLARK: I APPRECIATE THE INVITATION FROM DR. CROWDER TO SPEAK THIS MORNING. SINCE OTHERS HAD DISCLAIMERS, AND THERE WAS A MENTION OF VARIOUS PHENOTYPES EARLIER TODAY, HAVING FOLLOWED TWO NEUROSURGEONS, I WILL SAY I AM A PEDIATRICIAN BY TRAINING, WHICH I BELIEVE TO BE THE OPPOSITE PHENOTYPE OF YOUR TYPICAL NEUROSURGEON. I REALLY HAVE NO DISCLOSURES. A COUPLE NIH GRANTS AND IMPORTANTLY I HAVE NO SPECIFIC TIES TO PHARMA. I WILL DISCUSS A COUPLE OF DRUGS HERE. THE OBJECTIVES OF MY TALK ARE REALLY TO DISCUSS MEMBRANE TRANSPORTERS AT BLOOD-BRAIN INTERFACES WHICH I THINK ARE REALLY UNDER APPRECIATED BRARRIER TO EFFECTIVE DRUG DELIVERY AND WE HEAR A LITTLE BIT ABOUT THIS FROM ALEC'S TALK THIS MORNING. A LOT OF PEOPLE FOCUSED WHEN THE BLOOD-BRAIN BARRIER IS BREACHED EITHER VIA DISEASE PROCESS OR USING SOME SORT OF ULTRASOUND OR OTHER INTERVENTION. WE ARE PAYING A LOT OF TAKES THO TO HOW THINGS GET NIN BUT NOT AS MUCH TO HOW THINGS GET OUT AFTERWARDS. THE MIDDLE PORTION OF THE TALK WILL BE TO DESCRIBE. >>> GEES TO CAPITALIZE ON SOME OF THESE XENOBIOTIC TRANSPORTERS OF THE BLOOD-BRAIN INTERFACE TO OPTIMIZE BRAIN EXPOSURE OF POTENTIALLY NEUROPROTECTIVE COMPOUNDS AND THEN FINALLY I'LL REVIEW THE ROADMAP FOR CLINICAL DEVELOPMENT OF A PROTOTYPE COMBINATION STRATEGY USING N ASEATIL CYSTEIN AS A SUBSTRATE AND PRO BEN SID AS THE INHIBITOR. SO, THERE ARE A BE IN OF TRANSPORTERS AND WE HAVE SEEN A LITTLE BIT AND SNIPPETS OF OTHER PEOPLE'S SLIDES REGARDING THESE TRANSPORTERS OF BLOOD-BRAIN AND BRAIN CSF BARRIERS. THERE IS THE SOLITUDE CARRIERS AND I THINK WE SAW SLC 20-SOMETHING -- A8. INCLUDING ORGANIC ION TRANSPORTERS AND TRANSPORTER PEPTIDES. THERE IS ALSO ATP BINDING CASSETTE TRANSPORTERS AND P GLYCOPROTEIN AND ONE OF THE MOST WELL DESCRIBED MEMBRANE TRANSPORTER OF THE BLOOD BRAIN BARRIER AND ALSO MULTIDRUG RESISTANCE ASSOCIATED PROTEINS WHICH WE HEARD EARLIER MAY BE IN PART ASSOCIATED WITH TRANSPORTERRING OTHER XENOBIOTICS AND IMPORTANT TO THE BRAIN. AND THEN I'LL TALK A LITTLE BIT ABOUT TRANSPORTER SUBSTRATES INCLUDING ENDOGENOUS MOLECULES WHICH ARE VERY, VERY IMPORTANT BUT ALSO XENOBIOTICS. YOU THINK ABOUT THE TRANSPORTERS ARE MEANT TO GET RID OR KEEP BALANCE THE BIOCHEMICAL MILL YOU IN THE BRAIN AFTER INJURY. THERE IS A LOT OF ENDOGENOUS SUBSTRATES BEING TRANSPORTED BACK AND FORTH AND BUT THIS WAS ALL REALLY BEFORE THE AGE OF DRUGS AND PHARMA AND TRYING TO GET STUFF IN THE BRAIN AND KEEP IT IN THE BRAIN IF THAT IS WHERE YOUR THERAPEUTIC TARGETS ARE. SO THESE ARE JUST SOME OF THE TRANSPORTERS. THIS IS RELATIVELY OLD DESCRIPTION. 2012 IS WHEN THIS WAS PULLED OFF THE WEB ACCEPT I ADDED A COUPLE MORE BUT YOU CAN SEE -- THE WEB SITE -- THERE ARE NUMEROUS TRANSPORTERS ON NUMEROUS CELL TYPES IN NUMEROUS PLACES IN THE BLOOD-BRAIN BARRIER AND THE BRAIN CSF BLOOD BARRIERS AS WELL. SO, WHAT I HAVE BEEN FOCUSING ON RECENTLY AS PART OF THIS EFFORT IS AGAIN NOT REALLY LOOKING BOTH AT HOW DRUGS GET INTO THE BRAIN. FOR THAT I'M A PRETTY SMALL FISH IN A PRETTY BIG POND. ACTUALLY IT IS ONE OF THE FOCUSES OF THIS MEETING PROBABLY IS HOW TO GET DRUGS INTO THE BRAIN. AND WHERE I'M FOCUSING A LITTLE BIT MORE ON IS AGAIN HOW THESE DRUGS ONCE THEY GET INTO THE BRAIN, HOW TO KEEP THEM INTO THE BRAIN. AND AGAIN ALEX SORT OF ALLUDED TO THIS EARLIER BUT I THINK IT IS IMPORTANT TO NOT THINK OF THE BRAIN AS THE HOTEL CALIFORNIA. I MEAN ONCE STUFF GETS IN, IT DOESN'T DOUBLE NEGATIVE BUT DOESN'T NOT NEVER LEAVE T DOES LEAVE THE BRAIN AND EVEN LARGER MACRO MOLECULES SUCH AS PROTEINS, WE HEARD A LITTLE BIT ABOUT THE LYMPHATIC SYSTEM, CAN BE CLEARED VIA THAT PATHWAY AS WELL. SO THERE ARE CERTAINLY AT THIS CONFERENCE WHERE THERE IS A VARIETY OF PEOPLE STUDYING VARIOUS DISEASES, IT IS IMPORTANT TO SORT OF LOOK AT WHEN THE BLOOD-BRAIN BARRIER IS INTACT AND ALSO WHEN THE BLOOD-BRAIN BARRIER IS DISRUPTED, AND AT LEAST AS IT RELATES TO THIS TALK, THE IMPACT OF DISEASE ON SOME OF THESE MEMBRANE TRANSPORTERS. IN OTHER WORDS, MORE THAN JUST -- LOOKING AT MORE THAN JUST MECHANICAL DISRUPTION OF THE BLOOD-BRAIN BARRIER BUT ALSO THE FUNCTIONAL DISRUPTION OR FUNCTIONALLY AFFECTING THE BBB DISEASE. AND INTERESTINGLY, I WAS FOR ANOTHER SUBJECTIVES TRYING TO LOOK AT WHAT WAS KNOWN ABOUT THE WINDOW OF BLOOD-BRAIN BARRIER DISRUPTION IN HUMANS AFTER TRAUMA AND IT IS VERY, VERY DIFFICULT TO FIND THOSE DATA. AND THE DOGMA WHEN I WAS TRAINING A COUPLE OF YEARS AFTER DON WAS A RESIDENT, THE THOUGHT THAT WAS BLOOD-BRAIN BARRIER IS DAMAGED AFTER TRAUMA AND SEALS UP RELATIVELY QUICKLY, FOR THE MOST PART WITHIN HOURS WITH THE EXCEPTION OF THE PERICONTUSIONAL AREA. BUT THE COUPLE OF STUDIES I COULD FIND, ONE WAS KAUFFMAN'S DATA LOOKING AT ALBUMIN PERMEABILITY AND THIS IS THE MOST RECENT THING, WHICH IS STILL IMPRESSED KIND OF PAPER BUT IT SUGGESTS THAT THE BLOOD-BRAIN BARRIER MAY BELIEVE DISRUPTED AFTER TRAUMA FOR QUITE A WHILE IN HUMANS AND THEY DID A STUDY USING THAT LOW MOLECULAR WEIGHT TRACER AND FOUND THAT EVEN PATIENTS THAT WERE EVALUATED TWO DAYS, 5 DAYS LATER, THERE STILL APPEARED TO BE A DEGREE OF DISRUPTION. SO MECHANICALLY IT STILL MAY BE DISRUPTED BUT AGAIN, I'M KIND OF INTERESTED IN WHAT HAPPENS AFTER THE SUBSTANCE GETS INTO THE BRAIN. SO THESE WERE JUST SOME OF THE STUDIES IN TERMS OF THE IMPACT OF DISEASE ON MEMBRANE TRANSPORTERS. THIS IS ANTHONY WILLYARD, A CLINICAL FELLOW WHO NOW IS IN PHOENIX BUT HE WAS LOOKING AT WHAT HAPPENS TO ABC, C1 AND ABC B1, WHICH ARE P GLYCOPROTEIN AND MRP1 IN THE BRAIN. AND INTERESTINGLY, IT LOOKS LIKE THESE ARE HUMAN TISSUES OBTAINED IN PITTSBURGH FROM A TRAUMATIC BRAIN INJURY. THERE APPEARS TO BE AN UPREGULATION OF MRP1 AFTER TRAUMA. BOTH VIA WESTERN BLOT AND IMMUNOHISTOCHEMICALLY AND YOU CAN SEE IT ON THE BLOOD VESSELS BUT YOU CAN ALSO SEE IN WHAT APPEARED TO BE NON VASCULAR RELATED CELLS AS WELL. AND WE DO KNOW THERE ARE REPORTS OF MRP1 BEING OR BCC1 BEING IN NEURONS FOR EXAMPLE. P GLYCOPROTEIN, NO CHANGE IN AMOUNT OR RELATIVE AMOUNT BUT THERE DID APPEAR TOO BE REDISTRIBUTION MORE TOWARDS THE OR AWAY FROM THE VASCULAR SPACE INTO THE EXTRA VASCULAR SPACE BUT THERE WAS NO DIFFERENCE. WE ALSO HAVE DONE STUDIES LOOKING AT THE IMPACT OF MEMBRANE TRANSPORTERS - TO THE DISEASE AND THESE ARE SOME POLYMORPHISM STUDIES WE DID -- A MEDICAL STUDENT WORKING WITH ME IN PITTSBURGH AND NOW SHE EMERGENCY MEDICINE PHYSICIAN IN CINCINNATI. HE IS THE ONE ON YOUR LEFT. CERTAIN POLYMORPHISMS ASSOCIATED WITH -- WE WEREN'T SURE -- APPEARED TO BE ASSOCIATED WITH GAIN OF FUNCTION AND APPEARED TO BE ASSOCIATED WITH CLINICAL OUTCOMES IN THESE PATIENTS AFTER TRAUMATIC BRAIN INJURY AND THIS WAS THE CASE FOR ABC B1 AND ABC C1. SO, THAT SAID ABETTED OF A INTRODUCTION, HOW ARE WE GOING TO CAPITALIZE ON THE FACT THAT MEMBRANE TRANSPORTERS ARE SITTING THERE AND THERE IS A LONG-STANDING PHARMACEUTICAL RECORD OF DEVELOPING COMPOUNDS AND THINGS THAT ARE BOTH SUBSTRATE AND INHIBITORS OF THESE TRANSPORTERS. SO, THIS IS BECOMING A LITTLE BIT MORE OF A POPULAR FIELD OF STUDY. THERE WAS A CURRENT PHARMACEUTICAL DESIGN THAT INDICATED A ISSUE TO THIS ESSENTIALLY WHETHER TRANSPORTERS THE CNS BARRIER SITES REPRESENT OBSTACLES OR OPPORTUNITIES FOR DRUG DELIVERY. SO, THIS IS JUST MEANT TO -- THIS IS JUST FOUR OF THEBA ZILLION TRANSPORTERS OUT THERE NOTING SOME OF THE MORE COMMON INHIBITORS AND ALSO THE SUBSTRATES AND YOU'LL SEE IN THIS LIST, YOU CAN CHERRY PICK OUT THINGS THAT HAVE BEEN REPORTED TO BE NEUROPROTECTIVE. AND IN TERMS OF TRAUMATIC BRAIN INJURY, BUT ALSO ISCHEMIC BRAIN INJURY AND THEN THERE IS ALSO A NUMBER OF THINGS LIKE CHEMO THERAPEUTIC AGENTS AND CERTAINLY SOME OF THESE INHIBITORS HAS BEEN USED AS A CHEMO SENSITIZING AGENT. AND CERTAINLY USED IN THE ANTIBIOTIC WORLD PARTICULARLY FOR MDR RESISTANT BACTERIA FOR ANTIBIOTIC USAGE. AND THEN THERE IS AGAIN A NUMBER OF POTENTIAL CANDIDATES UP THERE. SO, WHAT WE DID AFTER WE THOUGHT OF THIS IDEA WAS THOUGHT THAT WE COULD BE BETTER AT THIS THAN GOOGLE AND SORT OF HANDPICKING OUT STUFF WHICH IS HOW WE CAME UP WITH A INCOME FOR BENEFITS. SO THE CHAIR OF SYSTEM SYSTEM AND COMPUTATIONAL BIOLOGY AT PITTSBURGH, SHE HAS DONE A LOT OF THIS SORT OF IN SELL COWORK AND AGAIN, THIS IS JUST MEANT TO SHOW YOU IF YOU TYPE IN PRO BENEFIT TO THE THIS WEBSITE, YOU GET A NUMBER OF POTENTIAL BOTH RECEPTOR OR BOTH TRANSPORTERS BUT ALSO POTENTIAL CANDIDATES THAT MAY OR MAY NOT WANT TO NEWS COMBINATION WITH INHIBITORS TO TRY TO KEEP STUFF IN. SO THE IS THAT THE GEE WE THOUGHT OF WAS TO IDENTIFY A POTENTIALLY NEUROPROTECTIVE SUBSTRATE OF A VERY PROMISCUOUS INHIBITOR. AND PRO BEN SID IS PROMISCUOUS. NAC IS A PRECURSOR FOR GLUTATHIONE. AND THERE WAS SOME EVIDENCE THAT IT COULD BE TRANSPORTED VIA PRO BEN SID. PENICILLIN WAS IN SHORT SUPPLY SO THEY COLLECT THE URINE FROM THE SOLDIERS AND SIPHONED OUT THE PENCIL ON AND GAVE IT BACK TO THEM. SO IT WAS DEVELOPED DURING WORLD WAR II TO PREVENT EXCRETION OF PENICILLIN FROM THE KIDNEYS. AND IT HAS BEEN SHOWN TO BE NEUROPROTECTIVE IN PRECLINICAL MODELS INCLUDING SOME OF THE STUDIES WE HAVE DONE IN TISSUE CULTURE. ACETAMINOPHEN IS FDA APPROVED ANTIDOTE FOR ORANT SEATAL CYSTEIN IS APPROVED ANTIDOTE FOR TYLENOL OVERDOSES, HEPATIC NECROSIS AND NEUROPRO TICKETTIVE IN CLINICAL MODELS AND RELATIVE TO MEMBERS OF THIS GROUP IT CERTAINLY HAS BEEN REPORTED IN +1 TO BE AN AFFECT OF COUNTER MEASURE FOR BLAST INDUCED MILD TRAUMATIC BRAIN INJURY. THE ONE THING ABOUTANT SEATAL CYSTEIN IT HAS VERY LIMITED AT LEAST IF YOU READ THE LITERATURE, LIMITED BLOOD-BRAIN BANIER PENETRATION AND BRAIN EXPOSURE. SO, I THINK I'M TRYING TO FIGURE OUT HOW MUCH -- I DON'T HAVE TO TALK QUITE THIS FAST. SO AGAIN REVIEWING THE ROADMAP FOR ONE OF THESE STRATEGIES. SO, WE PICKED THIS AND THE FIRST QUESTION WAS DOES COADMINISTRATION OF PROBENECID INCREASE SERUM IN BRAIN NAC EXPOSURE? AND THESE ARE NAIVE RATS AND IF YOU GIVE AN SEATAL CYSTEIN TO NAIVE RATS YOU CAN MEASURE IN THE SERUM. IF YOU COADMINISTER PROBENECID, IT INCREASES LEVELS IN THE SERUM BY ABOUT 60%. IT INCREASES THE AMOUNT IN THE BRAIN BY MUCH HIGHER THAN THAT WHICH SUGGESTS THERE IS BOTH SOME OF AFFECT IN TERMS OF SERUM ELIMINATION OR EXCRETION IN THE KIDNEY AND ALSO SUGGEST THERE MAY BE TRANSPORTER AFFECTS WHICH KEEPS IT INSIDE THE BRAIN AND THESE WERE DONE IN THE SCHOOL OF PHARMACY AT UNIVERSITY OF PITTSBURGH BY PHIL -- AND WE LOOKED CLOSER AND THESE DATA ARE IMPRESSED NOW IN XENOBY ON THE CA. THE REASON IT TOOK SO LONG IS PHARMACISTS ARE -- I DON'T KNOW IF THERE ARE ANY PHARMACISTS IN THE AUDIENCE BUT PHARMACISTS ARE DIFFERENT, THINK DIFFERENTLY THAN AT LEAST CLINICIANS. SO FOR CLINICIANS, WE WANT 95% CERTAINTY. PHARMACISTS THEY WANT UP TO 7 DIGITS OF PRECISION IN TERMS OF SOME OF THESE THINGS. SO IT TOOK US A WHILE BUT WE HAD TO GO THROUGH A DOZEN TRANSPORTERSES TO FIND OUT WHICH TRANSPORTERS TRANSPORT AND CAME UP WITH OAT 3 AND OAT 1,. BUT PRETTY CLEAR THAT AN SEATAL CYSTEIN DID APPEAR TO BE A SUBSTRATE FOR BOTH OF THESE INHIBITORS AND BOTH ARE INHIBITABLE BY PROBENECID. SO, THIS AND A NUMBER OF PRECLINICAL STUDIES BOTH IN RODENTS AND IN CELL CULTURE LED US TO THIS PROPOSED BIOLOGICAL MODEL FOR HOW THIS MAY BE WORKING AND THE THOUGHT IS YOU GIVE AN SEATAL CYSTEIN, IT CAN GO IN TO A LESSER DEGREE ACROSS THE INTACT BLOOD-BRAIN BARRIER BUT TO A GREATER DEGREE ACROSS THE DAMAGED BLOOD-BRAIN BARRIER. ONCE IT IS INSIDE, IF YOU HAVE OAT 3 AT THE BLOOD-BRAIN BARRIER, IT WILL PUMP IT BACK OUT AGAIN. IF YOU HAVE A FUNCTIONAL OAT 3. IF IT STAYS AROUND FOR A WHILE, IT CAN BE A CYSTEIN TO CYSTEIN INTO A NEURON WHERE VIA -- OR SERVE A A CYSTEIN DONOR FOR THE FORMATION OF GLUTATHIONE. IT CAN THEN BE CONGREGATED GLUTATHIONE CAN BE CONGREGATED TO VARIOUS SUBSTANCES IN XENOBIOTICS AND TRANSPORTED BACK OUT OF THE ION F IT WANDERS INTO THE CSF, THEN IT CAN BE PUMPED OUT BACK INTO THE BLOOD BY OAT 1 AND OUT 3 TRANSPORTERS AT THE CSF BLOOD INTERFACE. SO, THIS WAS A PROPOSED MODEL. AGAIN, WE WERE JUST AS MUCH INTERESTED IN HOW IT GETS IN AS HOW TO KEEP IT INSIDE THE BRAIN. SO THIS - AUTO OTHER THING WE DID WAS IN PART RESPONSE TO AN RFA THAT MONA HICKS PUT OUT SEVERAL YEARS AGO LOOKING AT COMNATIONAL THERAPIES AND WE PROPOSED TO DO SORT OF A TRIPLE ATTACK IN-VITRO, IN-VIVO AND CLINICAL STUDY SIMULTANEOUSLY BECAUSE OF THE FACT THAT THESE DRUGS WERE HAVING VERY FAVORABLE SAFETY PROFILE AND THESE ARE 14 CHILDREN, 7 GOT PLACEBO AND 7 GOT COMBINATION OF DRUGS AND SO THIS IS LOOKING AT THE CSF LEVELS OF AN SEATAL CYSTEIN AND YOU CAN SEE THEY ARE READILY DETECTABLE LEVELS IN THE CSF AND I GUESS MOST IMPORTANTLY SINCE THIS WAS A PHASE I STUDY, THERE WERE NO ADVERSE EVENTS OR PHYSIOLOGICAL DEARRANGEMENTS ATTRIBUTED TO TREATMENT. I THINK WE HEARD A COMMENT ASKING ABOUT CSF PRODUCTION AND THINGS LIKE THAT. WE WERE A LITTLE BIT WORRIED BECAUSE PROBENECID IS SO PROMISCUOUS AND DOES TRANSPORT A LOT OF SUBSTANCES OUT OF THE BRAIN THAT IT MAY AFFECT THE,YOU AND MAY SEE A SIGNAL IN INTRACRANIAL PRESSURE AND THAT DOESN'T APPEAR TO BE THE CASE. SO WE DIDN'T EXPECT THERE TO BE A DIFFERENCE BUT WE WERE RELIEVED THAT THERE DIDN'T APPEAR TO BE A TREND TOWARDS HIGHER INTERCRANIAL PRECIOUS IN THE PATIENTS AFTER TBI. AND THEN AS A BIOLOGICAL -- WE LOOKED AT CSF GLUTE AT THIGH OWN CONCENTRATIONS AND IT DOES APPEAR TO BE STATISTICALLY HIGHER ACTOR TREATMENT WITH AN SEATAL CYSTEIN AND THAT GROUP BUT AS YOU CAN SEE, A LOT OF IT MIGHT BE CAPTURED INSIDE THE CELL SO IT WAS REALLY MUCH, MUCH MORE COMPLICATED. SO, THE CAVEAT AS I ALLUDED TO IS THAT CERTAINLY IT WILL ALTER XENOBIOTICS IF COMMONLY USED IN TRAUMATIC BRAIN INJURY PATIENTS LIKE ANTIBIOTICS. BUT ALSO THE ENDOGENOUS ORGANIC ACID STUB STRAITS INCLUDING THOSE THAT MIGHT BE BENEFICIAL OR DETRIMENTAL. AND OTHER THINGS THAT MAY HAVE COMBINATIONS OF BOTH IN BALANCE ROOST GLAND IN SPECIES AND HETEEs. SO, THIS IS SO TO ADDRESS THIS, WE BASICALLY ARE DOING A METABOLOMICS STUDY IN REMAINING CSF FROM THESE PATIENTS. SO, WE HAVE ESSENTIALLY LOOKING AT THE ORGANIC TRANSPORTER PROBENECID METABOLOME IF YOU WANT TO THINK OF IT THAT WAY. AND WE HAVE A NUMBER OF COMPOUNDS THAT ARE HITS. WE HAVEN'T GOT AROUND TO SORT OF DOING PRINCIPLE CLONING ANALYSIS OR TRYING TO EVALUATE THESE CLEARLY BUT FORTUNATELY PROBENECID WAS HIGHER IN THE PROBENECID TREATED PATIENTS. BUT THEN THERE ARE A NUMBER OF SUBSTANCES YOU THINK WOULD BE PROBENECID SUBSTRATES THAT APPEAR TO BE NOT ONLY HIGHER IN THE -- APPEAR TO BE INJURY AFFECT WITH TRAUMA, SO THAT IS THE MIDDLE ORANGE COMPONENT THERE VERSUS HEALTHY CONTROLS BUT APPEAR TO BE FURTHER HIGHER WITH PROBENECID AND THESE ARE JUST SOME ACID WHICH IN THE 60s OR 70s PEOPLE WERE USING PROBENECID TO DECIDE IF THINGS GOT PUMPED OUT OF THE BRAIN OR NOT IN HUMAN VOLUNTEERS. SO THIS WAS ALL SORT OF INTERNAL VALIDITY THAT WHAT WE WERE LOOKING AT WAS GOOD. WE SEEN A LOT OF POTENTIALLY EXCITING OTHER SUBSTRATES INCLUDING PROSTAGLANDIN SPECIES AND HEATS AND EATS AND SOME ENVIRONMENTAL TOXINS THAT APPEAR TO BE SITTING IN THE CSF BUT THAT IS FOR FUTURE AND SO, JUST TO SORT OF FINISH UP HERE. ONE OF THE EARLIER SPEAKERS ALREADY SHOWED HIS PAPER ON THE PANEL ON THE RIGHT THERE BUT THE -- TO WRAP UP, THERE IS ALSO INDUCERS OF SOME OF THESE MEMBRANE TRANSPORTERS AS WELL. SO IN SOME DECEASES, YOU MAY WANT TO -- DISEASE ES AUGMENT CAPACITY FOR TRANSPORESSERS AND INCREASE THEIR FUNCTION OR GAIN OF FUNCTION, AND YOU KNOW THAT THERE HAS BEEN SOME STUFF THAT IN ALZHEIMER'S DISEASE WHERE A LOT OF THESE MEMBRANE TRANSPORTERS INCLUDING THE P VALUE PROTEIN AND ABC C1 AS WELL AS ABC G2 WHICH USED TO BE KNOWN AS BREAST CANCER RECEPTOR -- SOMETHING OR OTHER -- APPEAR TO BE ALTERED IN ALZHEIMER'S DISEASE AND OTHER NEURODEGENERATIVE DISEASES. AND JUST AS AN EXAMPLE HERE IS SOME LISTED INDUCERS OF P GLYCOPROTEIN. SO, I THINK JUST TO CONCLUDE, I TRY TO TAKE THE WRAP UP USING THE FRAMEWORK PROVIDED BY MARGARET AND SO, WHAT TECHNOLOGIES IN TERMS OF TECHNOLOGIES CLOSER TO TRANSLATION? I THINK THAT APPLICATION OF OLD TECHNOLOGY OR REPURPOSING SOME OF THESE DRUG COMBINATIONS IS ALREADY READY FOR PRIME-TIME AND AGAIN WE HAVE ALREADY DONE A PHASE I TRIAL WITH THESE. I THINK THE STRATEGY IS BROADLY APPLICABLE AS IT -- AS IT AFFECTS ITS FUNCTION OF THE BLOOD BRAIN INTERFACE VERSUS JUST REQUIRING STRUCTURAL BREAKDOWN. POTENTIAL HARM AND I THINK WE ARE DOING THE METABOLOMIC STUDIES TO PARTIALLY ADDRESS THIS QUESTION. COLLABORATIONS ARE ESSENTIAL FOR THIS PROJECT AS YOU'LL SEE IN A SECONDARY NUMBER OF COLLABORATORS. THE PRO DABBING ONE STUDY WAS A POSSIBLE ROADMAP FOR HOW THIS COULD BE DONE -- PRO NAC -- AND FOR THIS TYPE OF PROJECT, THE INCELLICO PREDICTIVE MODELING IS A REALLY, REALLY VALUABLE TOOL FOR SORT OF SELECTING THINGS AND THEN TAKING THOSE TO THE LABORATORY AND DOING PRECLINICAL STUDIES THAT ESCALATE TO MORE HIGH FIDELITY CLINICAL MODELS AND PICK A COMBINATION THAT MIGHT BE USEFUL CLINICALLY. AND JUST WANT TO ACKNOWLEDGE SOME OF THE PEOPLE THAT HELPED IN THIS PROJECT. THANKS. [ APPLAUSE ] >> THANK YOU VERY MUCH DOCTOR CLARK. WE HAVE TIME FOR PERHAPS ONE QUESTION. >> COULD I ASK A QUESTION? OF COURSE P GLYCOPROTEINS ARE DRIVEN BY ATP. HOW MANY OF THESE TRANSPORTERS ARE POWERED BY PROTON OR OTHER IONIC RADIANTS. >> CERTAINLY ALL THE ATP, ABC TRANSPORTERS ARE ATP DEPENDENT. THE SOLITUDE CARRIERS, SLCs THAT WERE FOR THAT WE USED TARGETING THE BLOOD-BRAIN INTERFACE FOR USING PROBENECID ARE BOTH ION DRIVEN. THAT'S HOW THEY WORK IN THE KIDNEY. >> WHAT ION? >> I DON'T KNOW. SORRY. >> THANK YOU. LAST QUESTION. >> SO I REALLY LIKE YOUR TALK. BUT WITH REGARDS TO THE NAC, WE SHOWN TWICE IT CROSSES THE BLOOD-BRAIN BARRIER QUITE WELL. FIRST TIME WAS 15 YEARS AGO. WE KEPT READING IT DIDN'T DOSS AND GREAT AFFECTS IN BRAIN SO WE BOUGHT SOME RADIO ACTIVITY AND SHOWED THAT IT CROSSED VERY WELL. AND THAT WAS PUBLISHED IN J OF NEUROCHEM BUT THE LITERATURE SAID IT WASN'T CROSS. SO HOW COULD ALL THESE PEOPLE BE WRONG? WE COULDN'T FIND ANY EVIDENCE IT DIDN'T CROSS SO WE REPEATED THE EXPERIMENT AGAIN. FOUND IT CROSSED AND IT WAS ENHANCED IN NEUROINFLAMMATORY CONDITIONS. SO, I THINK THIS REALLY FITS WELL WITH YOUR DATA THAT SHOWS THAT -- >> I THINK YES, I CAN RECONCILE THAT AND THAT IS KIND OF WHAT WE HEARD WHEN WE WERE THINKING ABOUT USING AN SEATAL CYSTEIN AND EVERYONE SAID IT DOESN'T CROSS THE BLOOD-BRAIN BARRIER CRUDING VERY PROMINENT PEOPLE ON STUDY SECTIONS. >> WHAT DOSE DID YOU GET? >> WE GAVE THE SAME DOSES THAT YOU GIVE FOR TYLENOL OVERDOSES WHICH IS -- >> 150 MIGS PER KEG? >> BUT SO THAT IS HOW TO RECONCILE THAT IS I THINK IT DOES CROSS THE BLOOD-BRAIN BARRIER T DEPENDS ON WHEN YOU MEASURE IT TO SEE -- [ OFF MIC ] >> THAT WOULD BE GREAT BUT SO WE HAVE SEEN THE ONLY OTHER PAPER I SAW IN CSF IN HUMANS WAS RECENT PAPER IN FOR PARKINSON'S DISEASE AND THEY SAW LEVELS IN CSF THAT WERE A BIT HIGHER THAN OURS BUT THEY WERE LOOKING AT PEAK LEVELS VERSUS OUR MORE NOT QUITE STEADY STATE BUT BUT I THINK THAT TO YOUR POINT, I THINK THAT IT GETS IN. I THINK THAT PEOPLE MAY HAVE BEEN DEPENDING ON THEIR METHODS, MAY HAVE BEEN MISLED BY THE FACT THAT IT ALSO GETS PUMPED OUT AND SO SOARED OF SLOWING THE TRANSITION OUT OF THE BRAIN USING SOMETHING LIKE PROBENECID IS KIND OF OUR RATIONAL FOR DOING IT. AND I GUESS WHERE THIS LEADS TO IS THE -- WE ARE PLANNING TO DO A SUBSEQUENT STUDY OBVIOUSLY PROBABLY MULTICENTER BUT WE WILL NEED TO INCLUDE A NAC-ONLY GROUP IN ADDITION TO THE PRO-NA R AND THAT MAY BE AN ADAPTIVE DESIGN OR NOT DEPENDING ON WHAT OUR IMPORTS ARE. >> BUT YOUR EXCEPT ABOUT RETOOLING. NAZ A GREAT EXAMPLE. BOTH AN SEATAL CYSTEIN AND SODIUM SULPHATE, IF YOU SEPARATE THEM WITH THE BLOOD-BRAIN BARRIER, CAN -- 80% OF CHILDREN GET CISPLATIN FOR SOLID TUMORS AND 80% OF THOSE GET HIGH FREQUENCY HEARING LOSS WHICH IS DEVASTATING AND IF YOU GIVE EITHER OF THOSE TWO DRUGS, YOU CAN PROTECT THE OUTER HAIR CELLS OF THE COCHLEA WITHOUT PROTECTING THE TUMOR. AND THERE IS TWO PHASE III TRIALS THAT WERE JUST REPORTED. RETOOLING OLD DRUGS. >> THANK YOU. >> THANK YOU VERY MUCH TO DR. CLARK AND EVERYONE ELSE WHO HAS PARTICIPATED IN THIS SESSION. I WANT TO REMIND EVERYONE THAT THE SAME PANEL OF SPEAKERS WILL BE AVAILABLE FOR THE OPEN MIC DISCUSSIONS STARTING AT 3:40 THIS AFTERNOON. RIGHT NOW WE HAVE OUR LUNCH BREAK SO FOR EVERYONE, ANYONE WHO ORDERED, BOXED LUNCHES THEY ARE AVAILABLE NEXT TO THE REGISTRATION DESK. ALL THE LITTLER, THERE IS FOOD AVAILABLE JUST NEXT DOOR IN THE CENTER. THERE IS A MAP SO YOU CAN FIND OUT EXACTLY HOW TO GET THERE. AND FOR ANYONE WHO HAS ORDERED BOXED LUNCHES FEEL FREE TO GIVE MONEY TO THE FRONT DESK STAFF THERE. THANK YOU. AND PLEASE COME BACK AT 1:10 THIS AFTERNOON. ACTUALLY I SEE THE CHAIR FOR THE NEXT SESSION SO WE CAN HAVE HIM START AND I WOULD LIKE TO INTRODUCE DR. RICHARD KRAIG, UNIVERSITY OF CHICAGO MEDICAL CENTER AND I WILL HAVE HIM START OUR AFTERNOON SESSION. THANK YOU. >> OKAY. AGAIN, THANK YOU TO EVERYBODY FOR HAVING THE OPPORTUNITY TO SHARE AND INVITE -- BE INVITED TO SPEAK THIS AFTERNOON. I'M GOING TO TRY THIS WITH THE MOUSE SO BOTH SIDES CAN SEE. THIS AFTERNOON WE'RE GOING TO TALK IN MORE DETAIL ABOUT EXOSOME THERAPEUTIC. BUT ONLY FOUR OF US WILL TALK THERAPEUTICS. I WILL ANASTASIA WILL, DR. ZHANG WILL AND AC WILL AND DEPEOPLEIUS WILL REVERSE COURSE AND TELL US HOW EXOSOMES LEAD BRAIN AND BE A BIOMARKER. I WON'T BELABOR BUT TO MAKE A POINT ABOUT THAT. FROM THOSE TRYING TO DELIVER EXOSOMES TO THE BRAIN ARE HAVING A DIFFICULT TIME. YOU WILL HEAR US TALK ABOUT HOW WE'RE USING THE INTRANASAL GROUP. THE STUFF GETS OUT. IT'S GOING TO GET IN. PART OF WHAT YOU'RE HEAR IN MY TALK IS HOW SPECIFIC CELLS AND SPECIFIC STIMULI DETERMINE THE SURFACE PROPERTIES OF THE EXOSOMES SO I THINK THAT WILL BE A KEY ASPECT OF GETTING THEM TO WHERE YOU WANT TO GO. SO WE HAD A NICE INTRODUCTION THIS MORNING. ABOUT EXOSOMES. WHICH COVERED MOST OF WHAT I WANTED TO SAY BUT I DO WANT TO MAKE TWO MAJOR POINTS ABOUT THIS. YES, THEY COME FROM A PARENT CELL AND THEY'RE REELED BY BLEBBING OR ENDOCYTOSIS FROM THE MICROHAVESIS LARBO DI. THE TWO POINTS I WANT TO MAKE ARE THE FOLLOWING. THEY HAVE EFFECTS IN THE NERVOUS SYSTEM BY VIRTUE OF SURFACE PROTEINS, BY VIRTUE OF SURFACE PROTEINS AS THEY INTERACT WITH RECIPIENT CELL MEMBRANE OR AS THEY FUSE WITH THE MEMBRANE AND THEY'RE CONSTITUENTS ROLLED OUT INTO THE CELL, THAT'S THE FIRST POINT. SECOND POINT, THE TYPE OF EXOSOME THAT IS RELEASED AND THE CORE GO THAT IS CONTAINED IS NOT ONLY SPECIFIC CELL DEPENDENT BUT STIMULUS DEPENDENT. I WILL BARE THAT HOME IN MY TALK. I WILL TALK TO YOU TODAY, I WAS GOING TO FOCUS ON HOW DENDRITIC CELL EXOSOMES CAN BE TRAFFICKED TO THE BRAIN. BUT I WANT TO TAKE IN THE 15 MINUTES THROUGH THE WHOLE CASCADE OF THE WORK THAT WE HAVE HAD THE PRIVILEGE OF BEING PART OF THE U GRANT COMMON FUND. I HAVE TO SAY THANKS TO THESE GUY BECAUSE I TALK A LITTLE FAST AND HAVE A STREAM OF CONSCIOUSNESS APPROACH, THIS IS THE FIRST GRANT IN OVER 30 YEARS OF DOING NIH STUFF, THAT'S KEPT ME ON TRACK. TO DO THE TASKS AT HAND, THAT'S IMPORTANT BECAUSE WE DEVELOPED A PI ON THIS, WE ARE A THERAPEUTICS THAT WILL REMYELIN DAMAGED BRAIN, NOT ONLY APPLICABLE TO TRAUMATIC BRAIN INJURY BUT MIGRAINE, MULTIPLE SCLEROSIS AND STOPS OXIDATIVE STRESS. UNIVERSITY OF CHICAGO INNOVATION FUND IS HELPING COMMERCIALIZE THAT AND THIS RO1 I HAVE HAD FOR QUITE A WHILE DEALS WITH MICROGLIAL EXOSOMES AND PREVENTATIVE HEALTH. THE FOCUS OUR LABORATORY IS TO SEARCH OUT AND FIND ENVIRONMENTAL ENRICHMENT BASED NEAR THERAPEUTICS. INCREASED INTELLECTUAL AND SOCIAL ACTIVITY ENRICHES BRAIN, IT'S SHOWN IN ANIMALS AND HUMANS FROM TRAUMATIC BRAIN INJURY TO EPILEPSY STROKE, PARKINSON'S DISEASE BEHAVIORAL DISORDERS TO MIGRAINE TO IMPROVE THESE DISORDERS ON THE ORDER OF 50%. IF RETIRED PERSON WALKS THREE QUARTERS OF A MILE A DAY, FIVE MILES A WEEK, THEY HAVE A 50% LESS DECLINE IN ALZHEIMER'S THAN THEIR SEDENTARY COLLEAGUES. COGNITION IS PROPORTIONAL TO MYELIN AND ENVIRONMENTAL ENRICHMENT INCREASES MYELINATION OF THE BRAIN. IN ADDITION IT REDUCES OXIDATIVE STRESS, WHICH ALSO PROMOTES MYELINATION. WHAT I'M GOING TO SHOW YOU IS FIRST SERIES OF EXPERIMENTS IN THE RAT, WE HAVE THE IDEA TO LOOK AT EXOSOMES BECAUSE I HAD A SMART STUDENT. NOT COMING BACK ON NEW PROGRAM WITH ME. WE DID MICRORNA SCREENING OF THE SERUM EXOSOMES. AND WE COUNT THEY CONTAIN AMONG OTHER THINGS BUT ESPECIALLY A HIGH CONTENT OF MICRORNA 219. 219 AS TURNS OUT IS NECESSARY AND SUFFICIENT TO PROMOTE OLIGODENDROCYTE PRECURSORS INTO DIFFERENTIATED MYELINATING CELLS. SO IF WE CAN MAKE THIS IN A DISH PACKAGE AND GIVE TO PEOPLE WE HAD THE FIRST THERAPEUTIC THAT REMYELINATES DAMAGED BRAIN. NOT JUST STOP DEGREE OF DEMYELINATION, ALL CURRENT THERAPEUTICS DO, LESS THAN DEGREE OF INFLAMMATION BUT PROMOTE ACTUAL REMYELINATION. I'M GOING TO SHOW PA SERIES OF EXPERIMENTS THAT CONFIRM THAT AND MOVE ON. WHAT WE DO IS USE THE SLICE CULTURE AS SCREEN PROOF OF PRINCIPAL PREPARATION FOR OURSELVES. THESE ARE P-10 ABOUT MALLS SLICED LIKE LUNCH MEAT BRAIN DOWN ON PLATFORM, GROW IT FOR A MONTH AND THEN USE IT. SO THESE ACTUALLY MATURE TO A 21 DAY OLD WEANED RAT IN TERMS OF CELLULAR MORPHOLOGY, CHEMISTRY AND MOLECULAR BIOLOGY AND SYNAPTIC FUNCTION. THIS IS TOP VIEW WHEREVER MY MOUSE WENT. TOP VIEW IS NEUROAL ARCHITECTURE. YOU CAN SEE VIA EM THERE'S PLENTY CONSISTENT WITH THAT SEEN IN VIVO. I WON'T SHOW YOU THE SLIDES THAT SAID WE CREATED A SIGNIFICANT INCREASE IN MYELINATION. WE DID. AND WE FOUND THIS 2 IS IT SO WE WENT AFTER THE TARGETS OF 219. AND REMEMBER, 219 IS A MICRORNA THAT INHIBITS THINGS SO INHIBITING AND INHIBITOR CAUSES THE PROGENITOR CELLS TO GO ALL THE WAY THROUGH AND MYELINATE. SO ALL THREE OF THESE WERE SIGNIFICANTLY REDUCED, SUGGESTING THAT THE TARGET WE WERE LOOKING FOR WAS CORRECT, 219, EFFECTIVE WITH THREE OF THE TARGETS THAT HIT IN THE MYELINATION PROCESS. IN A ADDITION WE CAME HERE AND TRANSFECTED TESTS WITH AN TAG NEAR SO WE THOUGHT THAT'S GOOD. THERE WERE SERUM EXOSOMES. I WILL TELL YOU SOMEONE STARTED A TRIAL USING HUMAN PLASMA TRANSFUSED TO DEMEANTING ELDERLY PEOPLE TO SEE IF IT WILL LESSEN THE DEMENTIA, WE THOUGHT WE WOULD BE SLICKER THAN THAT. AND WORK JUST A LITTLE BIT HARDER. WE BEGAN WITH THE FOLLOWK DATA. ENVIRONMENTAL ENRICHMENT OR EXERCISE, THIS IS STRAIGHT EXERCISE. THERE'S ACTUALLY A DROP IN INTERFERON GAMMA WITH ACUTE EXERCISE, MORE SEVERE EXERCISE, A BIGGER DROP BUT EPIGENETIC SIGNALING SUCH AS WOULD BE NECESSARY TO IMPROVE BRAIN FUNCTION IS PHASIC EXERCISE WITH REST IN BETWEEN TO KEEP YOUR BRAIN FRESH, SIMILAR TO PHASIC EXERCISE NOW BEING -- EXERCISE BEING ADDED TO GATED FOR IMPROVING MUSCLE TONE. PHASIC EXERCISE CAUSE A SIGNIFICANT INCREASE IN INTERFERON GAMMA AFTER A MONTH. THIS IS TALK A WALK, REST, TAKE A WALK, RIDE A BIKE A FEW MINUTES STOP THE EYE BICYCLE, START THE BICYCLE ON A 30 SECOND INTERVAL. INTERFERON GAMMA SIGNIFICANTLY INCREASED. THAT GAVE A CLUE TO THE DISH WHERE WE CHOSE DENDRITIC CELLS AND THE DISH TO GENERATE OUR EXOSOMES. WE CHOSE DENDRITIC CELLS BECAUSE THEY'RE A PROFOUND SOURCE OF EXOSOMES. WE HAVE AN IMPORTANT CAVEAT HERE. WE KEEP THE IMMUNE CELLS IMMATURE AND STIMULATE ONLY WITH INTERFERON GAMMA. WE DON'T WITH A CO-STIMULUS SUCH AS INTERFERON GAMMA AND PATHOGEN BECAUSE THAT MAKE IT IS CELLS MATURE AND THEY SPIT OUT A COMPLETELY DIFFERENT CADRE OF EXEXOSOMES THAT IN FACT MAY DEMYELINATE. SO IMMATURE EXOSOMES STIMULATED WITH INTERFERON GAMMA, WE PRODUCE EXOSOMES IN A DISH. THAT EXACTLY EMULATED THE EFFECTS OF ENVIRONMENTAL EPIRICHMENT, THE INCREASE -- ENRICHMENT, INCREASE IN MYLEN BASIC PROTEIN, LESSEN DEGREE OF DEMYELINATION AND CHEMICAL MODEL OF MULTIPLE SCLEROSIS, (INDISCERNIBLE) A SOAP THAT DEMYELINATES BRAIN, YOU U PUT IT ON, YOU GET DEMYELINATION HERE YOU PUT ON AND GIVE EXOSOMES AND WE HAVE A 75% RETURN WITHOUT FOOLING WITH DOSE RESPONSES YET. WE HAD NO EFFECT ON STEM CELLS OR TOXICITY TO CELLS IN CULTURE. THE NEXT THICK WE DISCOVERED IS THESE ARE MICROGLIA STAINED IN RED, A LITTLE SMALL BUT MAKE MY POINT HERE, THAT'S GREEN STAINING IS GLUTATHIONE. SO WE'RE EXOSOMES NOT ONLY INCREASE REMYELINATION BUT MATURING OLIGODENDRIA SITE PRECURSORS MAY PROMPT INCREASE IN ANTIOXIDANT POTENTIAL IN BRAIN. THE RIGHT SHOWS HISTOGRAM REDUCING OXIDATIVE STRESS WITH MITOCHONDRIAL INHIBITOR. THEN WE PRETREAT WITH EXOSOMINGS THAT INDUCE MITOCHONDRIAL INHIBITOR AND HAVE SIGNIFICANT REDUCTION IN OXIDATIVE STRESS SEEN. THIS AGAIN IS NEXT COLUMN IS A SCRAMBLE, THE INHIBITOR, WHICH NOW INHIBIT 219, AND RESTORE OXIDATIVE STRESS BACK TO THE LEVEL THAT WAS PREVIOUSLY SEEN. FINALLY WE DID ANOTHER EXPERIMENT ADDING SCRAMBLE OF 219 INEFFECTIVE WHILE 219 INHABIT FUNCTION TO REDUCE OXIDATIVE STRESS AND SURE ENOUGH IT DID. NOW COLLECTING SEVERAL PIECE OF EVIDENCE THAT SAYS OUR EXOSOMES NOT ONLY REMYELINATE BUT REDUCE OXIDATIVE STRESS SO THEY MAYBE VALUABLE ON THE FRONT ENMS WHICH IS AN INFLAMMATORY DISEASE. WE NEXT TAG THE EXOSOMES TO SEE WITH WHERE THEY'RE GOING. WITH QUANTUM DOTS. THIS IS OLIGODENDRIA SITE, MICROGLIA, ASTROCYTE, NEURON, THESE ARE QUANTUM DOTS IN RED, IMMUNOHISTOCHEMISTRY ON BOTTOM AND YOU CAN SEE THAT MOST OF THE QUANTUM DOTS WHEN STILL LATED WITH INTERFERON GAMMA GO TO 78% GO TO OLIGODENDRIA SITES. THE REST OF THEM GO TO MICROGLIA. BUT NOTICE THE UNSTIMULATED CELLS, I MENTIONED TWICE ALREADY THE SURFACE PROTEINS OF THE EXOSOMES CAN BE CHANGED BY THE WAY YOU STIMULATE A CELL. THE GRAY -- GREEN BARS ARE UNSTIMULATED DC EXOSOMES ADDED TO CULTURE. THEY HAVE A COMPLETELY DIFFERENT PROFILE. THEY GO TO MICROGLIA, MOSTLY ASTROCYTES WITH NONE OF THESE, BY THE WAY, REALLY EVER GOING TO NEURONS. REQUEST THAT DONE WE STARTED TURNING OUR ATTENTION, I WILL SPEND REST OF MY TIME SHOWING FUNCTION IN VIVO AND THIS DISTRIBUTION STRUCTURALLY OF THE EXOSOMES. WE HAVE THREE ANIMAL MODELS OF SHOWING THAT WE CAN TAKE OUR DENDRITIC CELL EXOSOMES AND CAUSE A SIGNIFICANT IMPACT ON WHOLE ANIMAL BRAIN FUNCTION. THIS IS FLORAL MYELIN, A FLUORESCENT MARKER OF MYLEN. THE TREATED SIDE, UNTREATED SIDE, THREE DAYS AFTER TREATMENT, SIGNIFICANTLY DIFFERENT. IMMUNOHISTOCHEMICAL STAINING FOR MYLEN-BASED PROTEIN, THE VELCRO THAT HOLDS MYLEN TOGETHER SO -- MYELIN TOGETHER SO A GOOD MARKER FOR WRAPPING DEGREE OF WRAPPING MYELIN ITSELF AND FINALLY FOR COMPLETENESS THE LAST HISTOGRAM HERE IS MYELIN BASED PROTEIN. AGAIN, BACK TO THAT SOAP MODEL WE USED, LIE SELLETH SIN INJECT TOAD THE CORPUS CALLOSUM THAT CAUSE AS LESION BUT IF YOU PRE-TREAT WITH OUR EXOSOMES AND MONITOR THREE DAYS LATER, THIS IS SIGNIFICANT REDUCTION IN DEGREE OF DEMYELINATION. TIME PREVENTS ME FROM TALKING ABOUT MY FAVORITE BUT MULTIPLE SCLEROSIS PATIENT VERSUS A TWO TO THREE FOLD HIGHER INCIDENCE OF MIGRAINE HEADACHE. WE HAVE SHOWN IN LABORATORY USING SPREADING COMPRESSION, I STUDIED OVER 30 YEARS, PROPAGATING WAVE OF ELECTRICAL SILENCE THOUGHT TO BE MIGRAINE AURA AND WE BELIEVE IT IS ALSO PART OF THE MIGRAINE PAIN, IT CAUSES DEMYELINATION JUST LIKE MULTIPLE SCLEROSIS BUT IT'S TRANSIENT. THESE ARE THE UVOs ON PEOPLE'S MRI SCANS. THAT DEMYELINATION RECOVERS WITHIN SEVEN DAYS, ITS T-CELL DEPENDENT AND INTERFERON GAMMA DRIVEN AND IS ALSO AMPLIFIED BY OXIDATIVE STRESS. OXIDATIVE STRESS MAKES THE BRAIN MORE EXCITABLE. IT DOESN'T BRAIN TRAUMA, DOES IT IN EPILEPSY AND MIGRAINE. OUR TREATMENT IS AN ANTIOXIDANT. SO WHEN YOU PUT IT TO THE SLICE CULTURE, IT STOPS SPREADING THE PRESSURE THRESHOLD IN SLICE CULTURES AND RAISE SPREADING COMPRESSION THRESHOLD 57 FOLD WITHOUT DOSE RESPONSE AGAIN, IN THE WHOLE ANIMAL. AFTER TRYING TO INDUCE SPREADING COMPRESSION THERE. THE LAST WHAT I'M GOING TO SHOW YOU IS SOMETHING NEW WE'RE TRYING TO DO, WE HAVE SHOWN YOU FUNCTION IN SEVERAL MECHANISMS BUT EVERYBODY WANTS TO SEE WHERE DID IT GO. WELL, NASALLY APPLYING EXOSOMES FIRST WITH SALINE THEN WITH REAL -- WHEN ANIMAL SIX HOURS LATER USING A PRETTY SMART DYE, THIS IS IN CLING, A DYE TO STUDY TRANSPORT OF VESICALES. IT'S CAPABLE OF ALLOWING SUPERRESOLUTION MICROSCOPY WHICH ALLOWS IMAGING DOWNING TO 20 NANOMETERSMETERS SO YOU CAN DO LIVE IMAGING OF EXOSOME MOVEMENT OR REAL RESOLUTION OF EXOSOMES. PLAIN OLD CON FOCAL MICROSCOPY ONLY HAS RESOLUTION OF 257 NANOMETERS. SO YOU WILL NEVER SEE INDIVIDUAL EXOSOMES. THESE PICTURES I'M SHOWING YOU HERE ARE CON FOCAL 248 BY 248 TILED TOGETHER AND YOU CAN SEE THAT IT'S DARK, ALONG THE PEELED SURFACE. I WILL SHOW YOU MORE EMPHATICALLY HERE, THAT IT'S DARKER AT THE PEEL SURFACE, CERTAINLY COMPARED TO THE SHAM BUT NOTICE THE OLFACTORY FIBERS. LOOK VERY MUCH AS IF THEY'RE FOLLOWING ALONG THE FIBERS. THEY'RE NOT THE FIBERS. THAT'S THE INTERSTITIAL SPACE ALONG THE FIBERS. WE PROVIDE SIMILAR EVIDENCE HERE OF HOW THEY'RE GETTING IN AND THAT IS THERE'S A GRADIANT FROM THE PEEL SURFACE INWARD. THESE ARE THE PICTURES ONE, TWO, THREE, THAT ARE ONE, TWO, THREE AND HERE IS THE SHAM. SO THE DARKEST BRIGHTEST FLUORESCENCE IS AT THE PEEL SURFACE AND DROPS OFF AS YOU GO INTO THE BRAIN. FINALLY, WE HAVE VERY PRELIMINARY EVIDENCE BUT WE HAVE EVIDENCE THAT SAYS DYE IS MOVING ALONG THE PERIVASCULAR SPACE. THESE AREN'T EXOSOMES YOU'RE LOOKING AT, THOSE ARE THREE TO SIX MICRON THINGS. I THINK THOSE THREE TO SIX MICRON THINGS ARE PERIVASCULAR MACROPHAGES. THEY ENDOCYTOSE LIKE CRAZY AND TAKE -- TURN OVER 100% CELL MEMBRANE SURFACE EVERY HOUR. SO THEY WOULD BE -- YOU WOULD SEE THINGS THAT ARE CONCENTRATION DEPENDENT. IF THE CONCENTRATION OF THE EXOSOMES IS HIGHEST IN THE PERIVASCULAR SPACE YOU WILL SEE CELLS HIGHEST, YOU DID SEE IN TISSUE TOO. LET ME SHOW YOU WHERE THAT KIND OF THINKING COMES FROM. IT'S A REAL SMART GUY, GOT TRAINED WITH MY THESIS ADVISOR, THIS IS CHARLES NICKELSON STUDIED MOLECULES DIFFUSED IN EXTRA CELLULAR SPACE FOR 30 YEARS. A FOLLOW NAMED BARNES AT UNIVERSITY OF MADISON, THIS IS HIS WORK PUBLISHED IN JOURNAL OF CEREBRAL BLOOD FLOW METABOLISM 2015. H E SHOWS WITHIN TWO MINUTES OF GIVING 3-D -- 3K DECKS TON, HIT ENTERS THE TRIGERMINAL NUCLEUS, IT PASSES THROUGH THE OLFACTORY EPITHELIUM, THE PERINEURONNAL SPACE OF SENSORRY FIBERS OF THE TRIGERMINAL NERVE AND DIFFUSES TO THE BRAIN BUT IT DIFFUSES IN THE BRAIN VIA BULK FLOW SO IT'S GOING FAST AS A ROCKET. 50 IN DIFFUSION GOES MILLISECONDS MICRONS AND MILLISECONDS INCHES AND HOURS. NEVER GO BY 50 IN DIFFUSION. THE WAY IT GETS INTO THE BRAIN IT'S IN THE CENTER OF THE BRAIN IN 30 MINUTES H. THORN HAS SHOWN THAT RADIO ACTIVE IGF 1 OR INTERFERON GAMMA REACHES THE CENTER OF A MONKEY BRAIN WITHIN 30 TO 60 MINUTES. SO BULK FLOW VIA THE PERINEURONNAL SPACES IS HOW THESE THINGS GET IN AND THERE'S A CONFIRMATION OF IT, THE ARTERIES ARE I CAN TAKING IT LIKE THAT. ARE TAKING IT LIKE THAT. LET ME CONCLUDE BY SAYING WE THINK WE HAVE FOUND OURSELVES AT LEAST CERTAIN IN TERMS OF OUR PASSION, AND MY CLINICAL DESIRES, TO LOOK AT ENVIRONMENTAL ENRICHMENT AS A MEANS TO SERGE NEW THERAPEUTICS. THIS HAS THE POWER OF PROVIDING SOLID SCIENCE AND NOT HOMOOPATHY WHERE PATIENTS SAY THIS IS THE SCIENCE HOW YOU CAN MAKE YOURSELF BETTER. SINCE IT'S MOTHER NATURE'S OWN SIGNALING IT WILL HAVE A HIGH BENEFIT TO RISK RATIO, FOR THOSE PEOPLE THAT CAN'T GET OUT AND WALK, EXERCISE OR USE THEIR MINDS BY DRIFTING AROUND IN A PARK, WE'LL DO IT FOR THEM. WE'LL DO IT FOR THEM WITH A KNEW METIC THAT CONSISTS OF INVENTORY DENDRITIC CELLS THAT EMULATE WHAT YOU CAN DO FOR YOURSELF BY WALKING AROUND. AND WHAT WE HOPE IS REMARKABLE IS THAT FOR THE FIRST TIME WE'RE REMYELINATING, NOT PROVIDING A STEM CELL, THAT GENERATES AN EMBRYONIC TISSUE, WE'RE TAKING A STEM CELL AND DIFFERENTIATING IT INTO MATURE FUNCTIONING TISSUES. THAT IS, MYELIN. BY THE SAME TIME WE'RE REDUCING OXIDATIVE STRESS. SO THE GOALS OF WHERE WE'RE HEADED WITH OUR COMMON FUND ACTIVITIES, OF COURSE ARE TO MOVE THESE TO HUMAN CELLS, CAN WE NOW START PRODUCING THIS VIA GMP QUALITY AND SUFFICIENT QUANTITIES FOR HUMAN STUDIES. I ALREADY HAVE SOME EVIDENCE THAT I CAN BEGIN TO TELL YOU THAT THERE IS NO IMMUNOGENIC REACTION. THEY ARE INVISIBLE. IMMUNOGENICALLY. THIS IS DONE BY MOLECULAR BIOLOGY AND IMMUNOSTAINING. FOR OUR CONVERSATION TODAY, IT'S THE OPTIMAL ROUTE. I SHOW YOU WHAT I HOPE IS GOOD RESULTS ABOUT NASAL DELIVERY BUT WE'RE ANXIOUS TO GO THIS SUMMER ON INTRAVENOUS DRIVERRY, AS I STARTED -- DELIVERY, AS I START ENDOMETRYIUS TO SAY MORE MORE OF, IF IT GETS OUT IT CAN GET IN, IT DEPENDS ON SPECIFIC CELL TIME AND WAY YOU STIMULATE CELLS. UNSTIMULATED DENDRITIC CELLS DON'T DO A THING. STIMULATED DENDRITIC CELLS NOT WAY INFECTION OCCUR, WITH INTERFERON GAMMA AND CO-STIMULUS BUT INTERFERON GAMMA ALONE, DOSED CORRECT LIZ CREATES PRECISELY THE EXOSOMES MOTHER NATURE CREATES WHEN DOING ENVIRONMENTAL ENRICHMENT, I WILL STOP THERE, MAYBE TIME FOR A COUPLE OF QUESTIONS. [APPLAUSE] >> THANK YOU VERY MUCH, DR. KRAIG. WE HAVE TIME FOR ONE OR TWO QUESTIONS. >> HAVE YOU EVER CONSIDER TRYING TO INCREASE AMOUNT OF FORMULATED MERE 219 AND THIS IS ANY FUNCTIONAL IMPACT? >> I THINK I DROPPED IT BUT I READ YOUR APP TO TRY AND INTRODUCE OR TALKS WHICH I GUESS I DIDN'T DO WELL. IT LOOKS MORE PLAUSIBLE. WE TRIED THAT. IT WASN'T SO POSSIBLE. SO WE QUIT. BUT I WILL TELL YOU, THERE ARE DIFFERENCES IN SPECIES THAT GIVE LOTS MORE 219 SO YOU MAY NOT HAVE TO LOAD IT BUT PLAN WAS TO LOADS IT. WHAT I'M AFRAID OF IN LOADING IS, I FOCUS A BIT ON -- WHEN YOU LOOK AT OUR MI RNA SCREENING, THERE'S A WHOLE CADRE OF MYELIN STIMULATING mRNAs. I DON'T KNOW THAT IT'S -- I KNOW ONE IS DOING 99% BUT MAY NEED A LITTLE PEPPER AN SALT FROM THE OTHER TWO OR THREE. SO THAT'S THE PLAN EVENTUALLY TO TRY TO LOWER THE COMBINATION. IF WE'RE CAPABLE OF DOING THAT B WE SUCH OUT OF FDA BASED SIDE OF THERAPY CONVERSATION AND INTO THE BIOLOGIC CONVERSATION BUT I'M PRETTY GOOD ON THIS SIDE RIGHT NOW. >> SO THESE CHANNELS THAT THE BULK FLOW PERINEURAL OR VASCULAR CHANNELS WHAT'S THE RELATIONSHIP BETWEEN THEM AND THE GLYMPHATIC CHANNELS? >> LIKE A SALIVA, BACK FROM OLD DAYS FROM '70s, THOSE FOLKS DOING BLOOD BRAIN BARRIER STUFF, 75% OF CEREBRAL SPINAL FLUID IS MADE BY THE CHORIO PLEXIS. 25% IS BY ASTROCYTES. THERE'S A RIVER THROUGH THE NERVOUS SYSTEM IN THE PERIVENTRICULAR SPACE AND FROM THE BRAIN OUT TO THE PIA AND THROUGH AND DOWN STRAIGHT TO THE SPINAL CORD. WHERE YOU HAVE THE DATA THIS SUMMER TO SAY DO THESE THING ACTUALLY MAKE IT TO THE SPINAL CORD. IF IT'S BULK FLOW THEY'RE GOING TO MAKE IT TO THE SPINAL CORD IN HALF AN HOUR. PARDON ME? >> THIS IS GOOD VECTOR FLOW. THE FLOW IS PRETTY FAST. >> I HAVE A QUESTION ABOUT THE IMMUNOGENICITY AND THE REASON I'M ASKING IS DID WE ACTUALLY MADE EXOSOMES FROM HUMAN DENDRITIC CELLS THAT WERE ACTIVATED WITH INTERFERON GAMMA PLUS A COUPLE OF OTHER CYTOKINES. AND ACTUALLY THESE EXOSOMES WERE INCUBATED WITH IMMUNE CELLS INDUCED AT MASSIVE UPREGULATION OF RNA. EXPRESSION IN THESE -- IN IMMUNE CELLS. SO THE QUESTION OF IMMUNOGENICITY IS AN IMPORTANT ONE I THINK. >> YES. WITHOUT BEING TOO VAGUE, MY ANSWER IS SENTENCER ON APPLICATION DIRECTLY TO THE NERVOUS SYSTEM. ANOTHER STATEMENT ABOUT THE CELLS PASSING THROUGH THE NIGHT. 20 YEARS AGO T-CELLS B CELLS AN DENDRITIC CELLS DIDN'T PASS THROUGH THE NORMAL NERVOUS SYSTEM. NOW IT'S CLEAR THAT T-CELLS ARE NECESSARY FOR MEMORY FORMATION FROM THE DENTATE GYRUS AND THERE'S A CADRE OF B CELLS AND T-CELLS TRAVELING THROUGH SO THEY DO MOVE THROUGH. BUT OUR MARKERS SO FAR HAVE BEEN IN SLICE CULTURE WHICH HAS MICROGLIA WHICH ARE IMMUNE CELLS AND IN FACT WE FIND SIGNALING THAT IMPROVES NOT WORSENS. SO WHETHER MY HE CANSOMES ARE DIFFERENT THAN YOURS, THE AGING OF THE EXOSOME, THAT'S THE KIND OF THING THAT THE GMP HAS TO SORT OUT. (OFF MIC) >> YES. (OFF MIC) >> NOT BY US. THE LITERATURE IS ACCUMULATING. LET ME FINISH BY SAYING OUR CULTURE WE HAVE CAPACITY TO WATCH THESE CULTURES OVER A MONTH, 60 DAYS AND WE WATCHED THE ANIMALS FOR A MONTH CARRIED OUT. THIS HAD ABSOLUTELY NO GENESIS, THAT'S WHAT I EXPECT FROM MESENCHYMAL CELLS, NOT THESE CELLS. >> WE CAN WATCH PARTICLES GO DOWN FROM CSF WITHIN 15 MINUTES AND IN THE DEEP CERVICAL LYMPH NODES, THAT EAR NOT TO THE SUPERFICIAL OR ANYWHERE ELSE. AND IT'S VERY FAST. IT'S AMAZING. >> THANK YOU VERY MUCH, DR. KRAIG. WE'RE READY TO PROCEEDS TO THE NEXT TALK BY DR. ANASTASIA KHVOROVA. HER TALK IS HIGH CONTENT PROTEOMICS LIPIDOMICS ANALYSIS ON A PATH TOWARD UNDERSTANDING THE MECHANISM OF EXOSOME MEDIATED CELLULAR UPTAKE AND BLOOD BRAIN BARRIER CROSSING. WE'LL DIM LIGHTS A LITTLE BIT FOR HER PRESENTATION. THANK YOU. >> THANK YOU FOR GIVEK ME AN OPPORTUNITY TO COME AND TALK HERE TODAY. AND ESSENTIALLY IT WAS VERY INTERESTING TO LISTEN TO THE TALKS THROUGH THE MORNING WHICH LOOK AT DISRUPTION OF THE BLOOD BRAIN BARRIER AS A PROBLEM BECAUSE IN OLIGONUCLEOTIDE THERAPEUTIC WORLD THERE'S A LONG STANDING QUESTION OF HOW WE LEARN OR TEACH OLIGONUCLEOTIDES TO CROSS THE BLOOD BRAIN BARRIER. AND WE HAVE GOT ME INVOLVED ME EXOSOME CONSORTIUM, IS ACTUALLY A CONCEPT THAT EXOSOMES POTENTIALLY CAN BE USE AS A VESICAL FOR DELIVERING THERAPEUTIC OLIGONUCLEOTIDES TO THE CNS TISSUES. SO IF YOU LOOK AT OLIGONUCLEOTIDE FIELD AS A WHOLE, THERE'S TWO MAJOR -- ONE CAMP IS SAYING WHICH MOST OF MY LAB BELONG TO, THAT MODULATING THE CHEMICAL COMPOSITION OF THE OLIGO ITSELF IS A WAY TO MAKE DRUG OUT OF IT AND CLEARLY FROM CLINICAL UTILITY PERSPECTIVE AND TO SCIENCE WHERE ACTIVITY OR DELIVERY IS DEFINED BY PHOSPHATIDE BACKBONE MODIFICATIONS OR CONJUGATE ARE CLINICAL AND MOST ADVANCED WITH SOME DRUGS DELIVERED EXPECTED TO GET REGULATORY APPROVAL NEXT YEAR. THE SECOND CAMP SAID NO, YOU HAVE TO FORMULATE MICRORNAS AND THERE HAVE BEEN A LOT OF WORK ON DIFFERENT FORMULATIONS, AND THE CONCEPT OF EXOSOMES WAS APPEALING BECAUSE CLEARLY THOSE VESICLES WHICH ARE NATURALLY RESPONSIBLE FOR TRANSPORT OF SMALL RNA FROM CELL TO CELL, AND WHILE THERE'S DISCREPANCY IN THE FIELD, OVERALL THERE MORE LABS AND RIGHT NOW THERE'S A LOT OF PUBLICATION, INDICATION THAT INDICATING THAT THIS CROSS OF INFORMATION TRANSFER OF INFORMATION DOES HAPPEN. SO HOW DO WE EXPLORE USING EXOSOMES AS A HOG GO NUCLEOTIDE DELIVERY -- OLIGOKNEW THREE OWE TIDE DELIVERY VEHICLE? FIRST THING THAT CAME TO MIND IS IT WOULD BE NICE TO CLEMICALLY DEFINE THE CARGO RATHER THAN REWIND WHAT IS IN THERE BIOLOGICALLY AND HOW WOULD ONE LOAD ARTIFICIAL CARGO AS A TRADITIONAL METHOD EXPIRATION IT'S EFFECTIVELY NO GO FROM A STARTING POINT. SO WITH MY LAB WORKS ON WAS WORKING ON AT THE TIME, IT WAS A SMALL CHEMICALLY STILLIZED AUDIO METRIC HYBRID WHICH ARE HYPOMODIFIED. THOSE ARE MOLECULES WHERE THE DUPLEX IS 11 TO 15 BASE PAIRS, SUFFICIENT TO ENABLE ABILITY OF THOSE COMPOUNDS TO ENTER INTO RICH COMPLEX, THEY HAVE THE LONGER GUIDE STRAIN WHICH IS FULLY TOES TO FATIZED AND IT'S MODIFIED WITH A HYDROPHOBIC MEDICATIONS AND IN THE CYCLICAL CONFIGURATIONS. MAJOR FEATURE OF THOSE COMPARTMENTS AS THEY BINDS TO CELL MEMBRANE WITHIN VERY QUICKLY WITHIN MINUTES OF EXPOSURE THEN GET INTERNALIZED AND I'M NOT GOING TO GO BY -- TALK ABOUT MECHANISM BUT THEY INDUCE VERY POTENT RNAi BASED GENE SILENCING SO LOW THE BETTER THE CELL EFFICACY. SO WHAT IT SHOWS THE NEXT SLIDE IS FIRST TEN MINUTES OF CELL LINE INTERNALIZATION EVENTS AND WHAT IS IMPORTANT FOR TODAY'S DISCUSSION IS OBSERVATION THAT IMMEDIATELY WITHIN SECONDS OF EXPOSURE, THERE'S A LOT OF COMPOUNDS WOUND TO CELL MEMBRANE FOLLOWED UP THROUGH THE ENDOCYTOSIS, SOME TYPE OF ENDOCYTOSIS PROCESS AND INTERNALIZATION. SO WHAT WE WHAT IS IMPORTANT TO UNDERSTAND ABOUT THIS CHEMISTRY, IT BINDS TO CELLS AND CELL NEURONS AND WHEN YOU INJECT IN THE BRAIN, THEY STAY AROUND SITE OF INJECTION AND AROUND SITE OF INJECTION INDUCE POTENT SILENCING AND THIS HAS BEEN PUBLISHED. SO WHEN WE DECIDED TO EXPLORE IS WHETHER WE CAN USE ABILITY OF THOSE HYDROPHOBIC MICRORNAs TO BOND CELLULAR MEMBRANE AS A WAY TO LOAD OLIGONUCLEOTIDE WITH EXOSOMES. AND INDEEDS IF YOU JUST TAKE EXOSOME PREP AND WHAT WE DO IN THE LAB RIGHT NOW WE USE STANDARD DIFFERENTIAL PROTOCOL, YOU TAKE THE HE CANSOME AND MIC THEM TOGETHER IN A TUBE, WITH HYDROPHOBICLY MODIFIED SI RNAs, INCUBATE FOR TEN MINUTES TO HALF AN HOUR, YOU CAN SEE THIS VAST MAJORITY OF THE OLIGO IS ASSOCIATED WITH THE EXOSOME. IF YOU TAKE THE PELLET AND THEN YOU REST AND REPEAT THE POE PROCEDURE YOU CAN SEE THE MAJORITY OF OLIGOSTILL WITH THE EXOSOME. WHAT WE KNOW RIGHT NOW, THIS IS A SATURATABLE PROCESS. YOU CAN LOAD AROUND 2000 OLIGOS PER EXOSOME AND YOU CAN CONTINUE TO ADD MORE OLIGONUCLEOTIDE STAIN SOLUTION AND BY THAT POINT WE'RE EFFECTIVELY SATURATING THE EXOSOMAL MEMBRANE. SO LOOK AT THE PROPERTIES OF THE EXOSOMES WHICH ARE NATIVE OR EXOSOMES WHICH HAVE BEEN LOADED WITH HYDROPHOBIC THEIR SIZE SEEMS TO BE THE SAME QUANTITY SEEMS TO BE THE SAME, MICROGRAM SEEMS THE SAME, ONLY THING WE SEE IS SLIDE DECREASE IN MEMBRANE POTENTIAL WHICH IS ALL INDICATIVE THAT SIGNIFICANT FRACTION OF THE OLIGOS ARE STILL ASSOCIATED WITH THE MEMBRANE. BUT IT'S VERY INTERESTING FOR US TO SEE THAT WHILE HYDROPHOBIC IS MODIFIED SI RNAs TRAFFIC INSIDE THE CELLS VERY EFFECTIVELY BY ITSELF, IT IS A QUICK EVENT THAT HAPPENS MOMENTARILY, YOU SEE MEMBRANE ASSOCIATION AND SOME CELLS INSIDE, WHEN YOU LOAD THEM IN EXOSOMES THE TRAFFIC AND PATTERNS CHANGE DRAMATICALLY AND THEY HAVE BEEN REALLY DOMINATED BY THE TRAFFIC AND PATHWAY USED BY EXOSOMES FOR CELL INTERNALIZATION. SO IF YOU LOAD SH RNAs AND EXOSOMES WHAT YOU CAN SEE, YOU CAN SEE THAT THE PROCESS OF UPTAKE IS MUCH SLOWER, YOU SEE NO MEMBRANE BINDING, YOU SEE AUTOMETRIC BARRIER NUCLEAR LOCALIZATION. THIS SHOWS YOU THE KINETIC EVENT IN HELO CELLS WHICH SHOWS THE SAME THING IN A MORE QUANTITATIVE MANNER AND WHEN WE LOOK AT NEURONS WE SEE EXACTLY THE SAME THING, EXOSOMES TAKE LONGER TO GET INTERNALIZED BY THE CELLS. BUT THEY DO GET IN AND INDUCE FUNCTIONALLY REALLY SHOWS VERY NICE DOSE RESPONSE AND DOSE DEPENDENT SILENTING OF THE GENES AND I'M SHOWING THE DATA FOR THE GENE ONE OF THE TARGETS WE USED IN THE LAB BUT WE HAVE DEMONSTRATED FOR SEVERAL OR TARGETS AS WELL. SO WHERE WE STARTED THIS WHOLE PROJECT THE IDEA WAS THAT MAYBE IF WE FORMULATE OLIGONUCLEOTIDES TO THE EXOSOMES THIS MIGHT BE A WAY TO ENHANCE DISTRIBUTION FOR THE BRAIN TISSUE. WHAT WE DID NEXT, WE TOOK THE FORMULATED HE CANSOMES AND INJECT THEM IN THE BRAIN, INFUSED FOR PERIOD OF TIME AND THEN TEN DAYS LATER LOOK AT THE OVERALL DISTRIBUTION THROUGHOUT THE BRAIN. WHAT WE HAVE SEEN IS THAT VERY DIFFERENT PICTURE WHEN WE JUST INJECT A NON-FORMULATED OLIGO. SO YES THERE IS SOME BLUSH AROUND THE SITE OF INJECTION AROUND THIS DAMAGE WHICH IS SIGNIFICANT BUT YOU CLEARLY SEE DIFFUSION OF THE FLUORESCENT OLIGONUCLEOTIDE TO THE NON-INJECTED SIDE OF THE BRAIN. SO LOOKING AT THE FLUORESCENCE PICTURES ARE GREAT BUT THEY IN GENERAL PROMPT ARTIFACTS SO WE WANTED TO CONFIRM THAT WHAT WE OBSERVE BY FLUORESCENCE IS ACTUALLY HAPPENING REALLY HAPPENS. AND TO DO THAT WE USE ASSAY CALLED PNA HYBRIDIZATIONS ASSAY. WE DIDN'T INVENT IT, WE TOOK IT FROM A COMPANY CALLED AXIO LABS. THIS IS A STANDARD CLINICAL STAGE ASSAY WHICH HAS BEEN USED FOR DETECTION OF MICRORNAs AND S RNAs IN A CLINICAL SAMPLES. THE CONCEPT OF THE ASSAY IS EXTREMELY SIMPLE. SYNTHESIZE PEPTIDE NUCLEIC ACIDS WHICH IS NON-CHARGED VERSION OF OLIGONUCLEOTIDE WHICH HAS A HIGH AFFINITY AND PERFECT COMPLIMENTARITY TO THE GUIDE STRAND OF THE THERAPEUTIC OLIGO. AND THEN YOU SPIKE LABEL PNA IN BIOPSY OF THE TISSUES AND RUN WERE YOU SEPARATE OUT FLUORESCENCE THIS HYBRID DUPLEX VERY EASILY, IT'S A GREAT ASSAY WITH A SENSITIVITY OF TEN PICA MOLE PER GRAM OF TISSUE AND SEVERAL LOG OF MAGNITUDE LINEARITY RANGE. IT HAS BEEN APPROVED BY FDA AS CLINICALLY ACCEPTABLE ASSAY TO LOOK AT MICRORNA AND S RNA DISTRIBUTION AND CLINICAL SAMPLE. SO WHEN WE LOOK AT WHAT WE HAVE SEEN IS SIMILAR TO WHAT WE HAVE SEEN BY FLUORESCENCE, IF WE INJECT OLIGO ITSELF WE SEE INJECTED SITE BUT DON'T SEE THE NON-INJECTED SITE AND WHEN WE FORMULATE THE EXOSOMES IT'S TWO SEQUENCES TWO DIFFERENT EXPERIMENTS WHERE WE STILL HAVE SLIGHTLY MORE EJECTED SIDE WE ABSOLUTELY CLEARLY CAN DETECT IT ON NON-INJECTED SIDE OF THE BRAIN, INDICATING THAT FORMULATION OF OLIGONUCLEOTIDE EXOSOMES WAS RESPONSIBLE FOR THE TRANSFER THROUGH THE BRAIN MATRIX. IT'S NOT JUST GETTING THERE, IT'S INDUCING SILENCING AND HIGHLY STATISTICAL SIGNIFICANCE SILENCING THROUGH THE LEVEL, MAGNITUDE OF THE FACTORS, 35, 40%, WHICH MY COLLABORATOR, CLINICAL COLLABORATOR SAYING IT'S SUFFICIENT, IN MY EXPERIENCE YES IT'S REPRODUCIBLE BUT WE PROBABLY WOULD LIKE TO SEE HIGH LEVEL OF MODULATION TO TRANSLATE IT TOWARDS INDICATIONS. SO WHAT I TALK ABOUT SO FAR IS THE FACT THE SIMPLE STIMULATION AND HYDROPHOBIC HYBRIDIZATION, IT CAN BE USED AS AN EFFICIENT WAY TO LOAD ARTIFICIAL CARGO IN THE EXOSOMES. THE EXOSOMES DO HAVE A DIFFERENT KINETIC PATHWAY AND THEY DO ENABLE BILATERAL BRAIN DISTRIBUTION. EVERYTHING WHICH I HAVE PRESENTED SO FAR WAS DONE WITH EXOSOMES DERIVED FROM UA TO 7 GLIOBLASTOMA TUMOR CELL LINE. THE REASON WE USE THIS CELL LINE ORIGINALLY BECAUSE OF PUBLICATION SAYING THERE'S EXOSOME TRAFFIC WELL AND WE HEARD ABOUT THIS TODAY FROM PREVIOUS SPEAKERS AND THE SECOND IS IT PRODUCED TONS OF EXOSOMES TO RELATIVELY EASY CELL LINE TO WORK WITH. WHEN WE CONSIDER CLINICAL INDICATION MOVING TUMOR DERIVED EXOSOMES INTO CLINIC, GENERATED QUITE A BIT OF CONCERNS AND QUESTIONING SO WE DECIDED IT MIGHT BE BETTER IF SHIFT MORE TOWARDS MORE CLINICAL ACCEPTABLE SOURCE OF CELLS AND WHEN WE DECIDED TO DO IS LOOK AT MESENCHYMAL STEM CELLS BECAUSE THERE'S SO MANY CLINICAL TRIALS GOING ON WITH MESENCHYMAL STEM CELLS AND IF FDA ALLOWS TO INJECT CELLS PROBABLY CONDITION MEDIA IS GOING TO BE FINE. WHEN WE START WORKING WITH IT, WHEN WE REALIZE HOW WE TREAT STRESS WHAT SIGNALING PROVIDE TO THE MEDIA, DRAMATICALLY IMPACTED ABILITY OF EXOSOMES TO TRANSFER OLIGONUCLEOTIDE CARGO INTO THE NEURONS. WHAT DO YOU SEE OVER HERE IS JUST AN EXAMPLE OF THE CURVE IN PRIMARY NEURON SHOWING YOU THAT WHEN WE SERUM STARVE MESENCHYMAL STEM CELLS THEY PRODUCE EXOSOMES WHICH ARE MUCH MORE EFFECTIVE IN THEIR ABILITY TO TRANSFER OLIGONUCLEOTIDES INTO PRIMARY NEURONS. THIS IS JUST REPEAT OF SAME EXPERIMENT WITH EXOSOME DERIVED FROM DIFFERENT TYPE OF MESENCHYMAL STEM CELLS, SHOWING TIME OVER TIME WHENEVER YOU STRESS A CELL, IN OUR CASE IT'S SERUM DEPRIVATION, YOU -- THE EXOSOMES DERIVED FROM THIS PROCEDURE ARE MORE EFFECTIVE IN ABILITY TO TRANSPLANT NUCLEOTIDE CARGO INSIDE CELLS. IN SOME CASES THE DIFFERENCE IS DRAMATIC. IN SOME CASES, THEY'RE MINOR. BUT THEY ALWAYS GO IN THE DIRECTION. THIS IS VERY COMMON TEAM AND YOU HAVE HEARD FROM PREVIOUS TALK THAT WHAT VESICALES POPULATION WE WORK WITH IS REALLY AFFECTED WHAT'S HAPPENED TO THE CELL OR TO THE ANIMAL OR THE TISSUE IN HOURS BEFORE THOSE VESICLES ARE PRODUCED WHICH OVERALL LOGICALLY MAKES A LOT OF SENSE BUT MAKE AN EXPERIMENT WORK VERY COMPLICATED AND REQUIRING A LOT OF CONTROLS. WE ALSO HAVE TO MENTION WE DO NOT OBSERVE THIS WITH MICROVESICLES, WHICH IS A LARGER FRACTION OF THE EXOSOMES. AND WE DON'T HAVE TO OBSERVE FROM THE MICROVESICLES DERIVED FROM THE SAME CELL CULTURE WAS COMFORTING TO ME BECAUSE THIS IS ANOTHER INDICATION THE EFFECT IS ACTUALLY SPECIFIC. WHICH BRINGS ME TO THE LAST QUICK PART OF THE TALK, OUR PAST VENTURE TO TRYING TO UNDERSTAND, WHAT IS THE DIFFERENTIAL FEATURES ON THE SURFACE OF EXOSOMES WHICH HAS BEEN CHANGED WITH CONDITIONAL TREATMENT OF THE CELLS, WHICH MIGHT BE RESPONSIBLE FOR FUNCTIONALLY TRANSFER. IF YOU LOOK AT STRUCTURE WE HAVE TWO CANDIDATES. PROTEINS WHICH IS COVERING THE EXOSOME OR IT'S LIPIDS WHICH CONSTITUTE MAJORITY OF THE MEMBRANE. SO WHAT WE DECIDED TO DO, WE DECIDED TO DO PARALLEL HIGH THROUGH PUT PROTEOMICS OF LIPIDOMICS ANALYSIS OF EXOSOMES AND MICROVESICLES DERIVED FROM A DIFFERENT CELL TYPE, CELL SOURCES AND WE INTENTIONALLY USED VERY DIFFERENT CELL SOURCES WHICH WAS MINIMUM STEM CELLS, GLIAL BLASTOMA CELLS AND CARCINOMA CELLS. WE GENERATED THREE COMPLETELY INDEPENDENT BIOLOGICAL REPLICAS SO DIFFERENT WEEKS, DIFFERENT PREPS, INDEPENDENTLY. AND THEN WE TOOK CELLS, WE TOOK 10G PALATE, MICROVESICALES AND WE TOOK 100G PALATE, WHICH IS EXOSOMES AND WE PERFORM HIGH THROUGH PUT PROTEOMICS, SO WE DETECTED 3,531 PROTEINS AND WE ALSO PERFORMED HIGH THROUGH PUT LIPIDOMICS IN COLLABORATION WITH THE COMPANY CALLED BURKE AND WE WERE ABLE TO DETECTION AROUND 2000 DIFFERENT LIPID PATIENTS. THIS IS JUST ON QUALITY CONTROL MEASURES, SHOW YOU YES, EXOSOMES LOOK LIKE EXOSOMES. AND MICROVESICALES HAVE SIZES, MAKES CLEARLY NOT PRESENT CERTAIN KNOWN MARKETERS OF EXTRA CELLULAR VESICALES ARE PRESENT IN THOSE. THEN DATA STARTED TO BE VERY INTERESTING AND WE ARE PUBLISHING IT, IT WILL BE OUT VERY SOON AND HOPEFULLY THERE ARE PEOPLE WHO ARE BETTER ABLE TO ANALYZE THIS TYPE OF INFORMATION. FIRST I WOULD LIKE TO MENTION THAT EXOSOME PROTEIN COMPOSITION IS DRAMATICALLY DIFFERING FROM THE PROTEIN COMPOSITION OF THE CELL. WHICH IS INDICATIVE WHEN LINE OF PROOF LINE OF ACTIVE PROTEIN SOURCING. VERY INTERESTING OBSERVE INVESTIGATION WAS EXOSOMES FROM TWO DIFFERENT CANCER CELL LINES, THE PROTEIN COMPOSITION IS SIMILAR BETWEEN EACH OTHER. WE ARE TALKING ABOUT THIS IS PRINCIPLE COMPONENT ANALYSIS, THERE'S TWO DOTS ESSENTIALLY OVERLAP AND THIS IS DEGREE OF CORRELATION AT 0.8. SO COMPLETELY DIFFERENT CELL SOURCES, THE ONLY COMMONALITY THIS IS CANCER AND WHILE CELL COMPOSITION OF PROTEIN IS VERY DIFFERENT, ACTUALLY WOULD GET SORTED IN EXOSOMES IS EXTREMELY SIMILAR. NOW THE VERY INTERESTING OBSERVATION YES PERFORM THE SAME ANALYSIS FOR LIPIDOMICSK WE REALIZE THAT PROTEIN AND LIPID SOURCING DO NOT GO TOGETHER. WHICH WAS SURPRISING. AND WHAT WE HAVE SEEN HERE IS THIS ACTUALLY WHAT LOOKS MORE THE SAME, EXOSOMES DERIVED FROM MESENCHYMAL STEM CELLS AND HD 7 CELLS. LATER ON WE WERE ABLE TO LINK -- LOOK AT THE CORRELATION, 0.93. THIS IS PRINCIPLE COMPONENT ANALYSIS, EFFECT TVLY OVERLAP, BUT ACTUALLY DEFINES IT IS THAT WEIGHT OF EXOSOMEAL PRODUCTION AND I HAVE NO TIME TO TALK ABOUT IT RIGHT NOW. I WILL KEEP GENERAL ANALYSIS SLIDES BECAUSE OF TIME AND WANT TO MENTION ONE THING, THAT IT WAS VERY INTERESTING FOR US TO SEE AND OVERALL APPRECIATED THAT LIPID COMPOSITION OF EXOSOMAL MEMBRANE IS VERY, VERY DIFFERENT. AND CAN BE SIGNIFICANT CONTRIBUTOR TO EXOSOME TRAFFICKING PROPERTIES AS WELL AS POTENTIAL CONTRIBUTOR TO HOUSE PROTEINS ON EXOSOMAL MEMBRANE OVERREPRESENTED. ONE -- THE DATA SET FOR PUBLIC SO EVERYBODY WOULD BE WELCOME TO DIG IN BUT ONE THING I WOULD LIKE TO MENTION IS WE HAVE SEEN IN EXOSOMES WHICH ARE VERY GOOD IN NEURONAL TRAFFICKING IS ARTIFICIALLY HIGH PERCENTAGE OF CARDIO LIPIDS WHICH IS ONE OF THE LIPIDS RESPONSIBLE FOR CURVATURE, ENHANCEMENT OF THE CURVATURE, THERE'S ALMOST NO PHOSPHO-- THERE'S MUCH HIGHER CONCENTRATION OF FATTY ACIDS AND OTHER INTERESTING FINDINGS. AND WITH THAT, I WOULD LIKE TO CONCLUDE AND THANK THE PEOPLE WHO DID THE WORK SPECIFICALLY (INDISCERNIBLE), SHE DID ALL THE PROTEOMICS AND LIPIDOMICS AND MARIE (INDISCERNIBLE) IS SENIOR POST-DOC AND REST OF THE TEAM FROM THE NIH COMMON FUND. THANK YOU FOR YOUR ATTENTION. [APPLAUSE] >> THANK YOU VERY MUCH, PLEASE HOLD YOUR QUESTIONS FOR DURING THE OPEN MICROPHONE DISCUSSION, THERE WILL BE THE SESSION EXOSOME THERAPEUTIC SESSION WILL START AT 3:40 THIS AFTERNOON. SO WE WOULD LIKE TO PROCEED WITH THE NEXT TALK, DR. ZHANG WILL BE PRESENTING, EXOSOME LIKE NANOPARTICLES DELIVERING THERAPEUTIC AGENTS THROUGH AN INTRANASAL ROUTE INHIBIT BRAIN TUMOR PROGRESSION. >> SO (INDISCERNIBLE) EXOSOME THERAPEUTIC AND BECAUSE THIS WORKSHOP FOCUS ON DENDRITIC SO I'M GOING TO TALK ABOUT BIOLOGIC FACT, FOCUS ON -- YOU CAN SEE HERE IT DRIVERS THERAPEUTIC AGENTS THROUGH THE VIRUS OR LIPSOME OR NON-PARTICLE HAS TREMENDOUS BOOST IN DELIVERING ADVANCE BUT STILL HAVE MANY OBSTACLE TO BE OVERCOME. I HAVE (INAUDIBLE) TO THE MAJOR CHALLENGE FOR YOU TO FIRST TO DELIVER THERAPEUTIC. SO WE HAVE INTRODUCED THE FIRST ONE WHICH IS EDIBLE PLANT WITH EXOSOME LIKE NON-PARTICLE. (INDISCERNIBLE) FIRST OF ALL IN THE PAST COUPLE OF YEARS WE HAVE STUDIED MAMMALIAN LYSOSOMAL THERAPEUTIC EFFECT. HOWEVER, MANY (INAUDIBLE) ONE MAJOR OBSTACLE IS LARGE QUANTITY GMP SPECIFIC PRODUCT TO TREAT PATIENTS. AND IN CONTRAST, THE PLANT DERIVE EXOSOMAL REALLY INDUCE -- AS MUCH AS YOU WANT, I GAVE AN EXAMPLE, THE EXOSOME LIKE NON-PARTICLE TREAT THOSE FOR EACH PATIENT. CON SEPTEMBER DERIVED EXOSOMAL DOSE. SO (INDISCERNIBLE). HOW MUCH YOU CAN MAKE TWO TECHNICIAN FULL TIME TO WORK ON THAT TYPE EXOSOME. THEREFORE I WANT TO FOCUS ON PLANT TO STUDY TWO STUDIES IN THE LAB. MAMMALIAN EXOSOMAL BIOLOGICAL FACT. THE EDIBLE PLANT EXOSOMAL -- BODIES HOMEOSTASIS OF THE GUT. I WON'T GIVE THE TALK FOR TIME LIMITATIONS TODAY BUT WE HAVE TRIALS GOING ON, YOU ARE EITHER GREAT DERIVED EXOSOMEAL -- CHEMOSTRUCTURE (INDISCERNIBLE) IN CANCER PATIENTS. SECOND CLINICAL TRIAL IN COLON CANCER TREATED WITH GRAPEFRUIT (INAUDIBLE). ALMOST -- EXOSOMAL TO TREAT OBESITY PATIENTS. SO ON TO CLINICAL TRIALS, BUY LOGICAL FACT (INDISCERNIBLE) PARTICULARLY FOOD FRUIT OR VEGETABLE DERIVE EXOSOMAL LIKE PARTICLE, ONE BIOLOGICAL FACT, SO I WON'T HAVE TAKE TO TALK ABOUT THAT TODAY BUT VERY EXCITING, I'M REALLY IN COLLABORATION WITH INVESTIGATOR WHO CLINICIAN DIFFERENT KIND EXOSOMAL TREAT DIFFERENT TYPE DISEASE. IN PARTICULAR (INAUDIBLE) HOW THE GUT COMMUNICATE WITH BRAIN THROUGH EXOSOMAL THROUGH THE THINGS WE ARE EATING. IN COLLABORATION WITHICALLY IN ADDITION INTERESTED IN (INAUDIBLE) DISEASE EDIBLE PLANT EXOSOMAL TO TREAT BRAIN ASSOCIATED DISEASE. (INDISCERNIBLE) WHAT AM I GOING TO TALK ABOUT TODAY IS EDIBLE PLANT DERIVED IN PARTICULAR EACH CASE IS GREEN FOOD DERIVE EXOSOMAL LIKE PARTICLE TO CARRY THERAPEUTIC PATIENT TO TREAT DISEASE IN PARTICULAR BRAIN CANCER. EDIBLE PLANT CELL DERIVED EXOSOMAL NON-PARTICLE FOR THERAPEUTIC DELIVERY. SO THEREFORE I'M GOING TO FOCUS ON MICRORNA. FOR TREAT BRAIN DISEASE. THIS SLIDE SHOWS THE PLANT EDIBLE PLANT DOES RELEASE -- EXOSOMAL LIKE NANOPARTICLE. THERE'S FOUR DIFFERENT TYPE PLANT, ELECTROMICROPHOTOGRAPH, LEFT SIDE HERE IS PURIFIED BAND, IN THIS CASE BAND 2, GRAPEFRUIT OR GINGER. WE HAVE MANY, MANY MORE EXAMPLES HERE, WE HAVE LIBRARY ALL DIFFERENT KINDS OF PLANTS. SO WE CAN TREAT ALL DIFFERENT KINDS OF DISEASE AS CARRIER, DEPENDINGEN WHAT KIND OF DISEASE, WHAT TYPE CELLS. SO THE PAST NUMBER OF YEARS WE HAVE PUBLISHED A NUMBER THAT SHOWS ABLE PLANTS IN THIS CASE GRAPEFRUIT PLANT EXOSOMAL CAN CARRY THERAPEUTIC AGENT. CARRY BY -- CARRIED BY -- I DON'T HAVE TIME TO TALK ABOUT THIS (INDISCERNIBLE) PUBLISH. ALSO PUBLISH HAVE HUGE GRAPEFRUIT DERIVED EXOSOMAL PARTICLE LIKE TARGETING SPECIFIC CELLS. IN THIS CASE TUMOR CELLS. SO THIS MANUSCRIPT WE TALK ABOUT USE THE HIGH GLUE DA SITE MEMBRANE PROTEIN, HYBRID WITH GRAPEFRUIT NON-PARTICLE, THEREFORE IT CAN ACHIEVE TWO DOSE, NUMBER ONE, IT CAN CARRY THERAPEUTIC AGENT, SECOND, BECAUSE SURFACE EXOSOMAL DERIVED NON-PARTICLE, IN THIS CASE NON-PARTICLE SURFACE CODE WITH MEMBRANE, DERIVED FROM GLUE DA SITE. THEREFORE ABLE TO PEN TRAIT TISSUE WHERE IT HAS THE INFORMATION. THIS CASE WE USE FOUR DIFFERENT TYPE, MY INFORMATION MODEL TO DEMONSTRATE THE WORK. ABLE TO TARGET DELIVER THERAPEUTIC AGENT TO THE TISSUE WHERE ITED THAT INFORMATION. PRE-CLINICAL TRIAL, FOUR DIFFERENT MOUSE MODEL. SO WHAT AM I GOING TO FOCUS ON TODAY IS CAN WE USE GRAPEFRUIT DERIVED EXOSOME TO CARRY THERAPEUTIC AGENT THROUGH INTRANASAL TO DELIVER THERAPEUTIC AGENT INTO THE BRAIN? HERE I SHOW YOU THE LIVE MOUSE IMAGING, MICE CERTIFIED DELIVERED WITH EITHER LABELED EXOSOME WHICH FOR US BECAUSE HERE IS WHEN GRAPEFRUIT VESICAL LABELED SEROTYPE -- IN CONTRAST WITH THE STANDARD LIPOSOME CANNOT GET INTO THE BRAIN THROUGH INTRANASAL ADMINISTRATION. AND (INDISCERNIBLE) DOMINATE SIGNAL IN THE BRAIN AND SEVERAL QUESTIONS WERE. SO (INAUDIBLE) AND THEN THE NEXT QUESTION WAS INTRANASAL DELIVER CAN BE A THERAPEUTIC AGENT. USE THIS HIGHLY EFFICIENCY ABLE TO BIND TO THE RNA, THEREFORE WE WANT TO FURTHER GRAPEFRUIT DERIVED NON-VESICAL COGNITION WITH HIGH EFFICIENCY TO DELIVER TO MAKE THEM BETTER THERAPEUTIC DELIVERY VEHICLE. WE'RE HAPPY WITH THIS LIPID DYE AND EFFICIENCY FIRST WAS MAJOR SIZE, MAJOR CHANGE, WHERE DIFFERENT LIPID CHANGE COMPARED WITH GRAPEFRUIT NON-VESICLE WITH THOSE IPSI, AND HYBRID HAVE SHIFT FROM CHARGE TO POST CHARGE, DEFICIENCY, CLEARLY IN THE SLIDE SHOWS YOU THE EFFICIENCY OF MAP MAKE HYBRID WITH PI LIPID DYE WITH THE EXOSOME EXTRACTING FROM THE GRAPEFRUIT NON-VESICAL. SO THIS HYBRID VESICAL INTO THE BRAIN, INTRANASAL, HERE IS 90 MINUTES AFTER INTRANASAL GIVEN. AND FURTHER THE LARGE SECTION HERE YOU CAN SEE THE COLOR IS LABELED WHICH ENCAPSULATION TO GRAPEFRUIT NON-VESICAL AND LABELED VAS CAN YOU A LAR LABELED GRAPEFRUIT LIPID, YOU CAN SEE HUGE AMOUNT OF RNA GET INSIDE THE BRAIN. AND WITHIN VESICLES. CON FOCAL MICROSCOPIC YOU CAN SEE IS SIGNAL IN THE BRAIN AFTER DELIVER, COMPARED WITH LOW DYE AND THOSE ARE INDICATED IN THE BRAIN AS WELL YOU CAN SEE HERE. AFTER DELIVERY OF THERAPEUTIC AGENT, ONE MAJOR CHALLENGE IS IN THE PAST IF YOU CARRY -- DELIVER VEHICLE ONE MAJOR CHALLENGE IS TOXICITY TO GI. VERY TOXICITY WHEN INDUCE INFLAMMATION BECAUSE HERE IN THE BLAIN THEREFORE A MARK FOR THE MACROPHAGE, HUGE AMOUNT OF MACROPHAGE INFILTRATION IN THE BRAIN. ALSO THE MICROGLIA CELL WHICH ARE MARKER BY ANTIBODY WHICH IS PARTICULAR STAINING MICROGLIAL CELLS, BOTH INCREASE, IF YOU -- INTRANASAL GIVEN GI CARRY -- IN CONTRAST, TOXICITY BY EXOSOMAL LIKE PARTICLE DERIVED ISOLATED GRAPEFRUIT NON-PARTICLE YOU CAN SEE BASICALLY NO (INAUDIBLE) MACROPHAGE AND BRAIN. SO NEXT QUESTION WAS TO -- CAN WE CARRY THERAPEUTIC AGENT TO THE BRAIN DISEASE. IN THIS CASE WE KICK OFF (INDISCERNIBLE) MACROPHAGE OR THE MICROGLIAL CELLS TUMOR CELLS. SO WHEN GRAPEFRUIT NON-PARTICLE -- UPTICK BY MICROGLIAL TUMOR CELLS, THEY SHUT DOWN ONE MOLECULE, THEREFORE IT CAN ACTIVATE NATURAL KILLER CELLS WHICH IS (INDISCERNIBLE) BECOME IMMUNE RESPONSE. SHOW A COUPLE OF SLIDES FOR VERIFICATION. YOU CAN SEE HERE IS (INDISCERNIBLE) CARRY BY THE EXOSOMAL GRAPEFRUIT NON-PARTICLE CAN'T GET INTO BRAIN. WE FIRST LABEL WITH REDS DYE, FROZEN DYE AND ALSO WE HAVE HERE THIS RECEPTOR NO TUMOR DELIVER CELLS AND LABEL CO-STIMULATION CON FOCAL SCOPE MAJORITY HERE. ALSO FOAT, IF YOU FURTHER ADD ONE MORE LAYER TARGETED TO THE MICROGLIAL TUMOR CELLS, MICROGLIAL TUMOR CELLS HAVE HIGHER LEVEL THEREFORE IN THIS VECTOR FOOD DERIVED NON-PARTICLES CARRY MACROPHAGE, WITH FOLIC ACID TARGETING TO MICROGLIAL TUMOR CELLS. YOU SEE CON FOCAL SCOPE ALSO HERE. IF YOU CARRY FOLIC ACID, THE AIRER, OR THE GRAPEFRUIT NON-PARTICLE YOU CAN SEE LIGAND ON THE VECTORS. -- ALSO HAVE A THERAPEUTIC EFFECT SO ALSO DELIVERED IS A BIOLOGICAL FACT, (INDISCERNIBLE) AND SHUT DOWN MHC 1 THEREFORE SUBJECT TO NATURAL KILLER CELLS. SO HERE WE SHOW -- FIRST ONE WE HAVE DONE WITH FOLIC ACID LIGAND BINDING TO THE VECTORS, SECOND MIDDLE COLUMN WITH FOLIC ACID CODE WITH VECTORS THIRD LINE ONLY TUMOR MICE IN TREATMENT. NO TUMOR (INAUDIBLE). YOU CAN SEE HERE THE LABELED VECTORS YOU CAN SEE GREEN ONE, THE TUMOR IS TUMOR TISSUE. SO THIS HERE, IS A TUMOR TISSUE. RESULT. YOU CAN SEE PARTICULARLY IN THROUGH THE BRAIN, IN THE TUMOR TISSUE, SIGNAL AND (INDISCERNIBLE) IF VECTOR CODE WITH FOLIC ACID LIGAND HAVE HIGH SUFFICIENCY TUMOR CELLS WHICH IS GI 26 TUMOR CELL LINE, IF THAT'S NOT ENOUGH, THERAPEUTIC AGENT, IN THIS CASE TUMOR TISSUE, YOU CAN SEE HERE LABELED GREEN FLUORESCENCE HERE, AND ALSO HERE, EFFICIENCY MUCH (INAUDIBLE) THIS ONE COMPAREDDED TO THIS ONE. SUMMARY, SO FAR WHAT I SHOW IS INTRANASAL GRAPEFRUIT DERIVED NANOPARTICLE CARRIES THERAPEUTIC AGENT ABLE TO GET INTO BRAIN, IF THIS VECTOR FURTHER COATED WITH TARGETED LIGAND WITH FOLIC ACID MICROABLE TO HAVE EFFICIENCY TO DELIVER THERAPEUTIC AGENT INTO THE MICROGLIAL TUMOR CELLS WHICH IS TR 26 TUMOR CELLS. SO THIS SLIDE HERE IS LIVE IMAGING BRAIN TUMOR SO CLEARLY TUMOR INJECTION AND THEY OFFER INJECTION YOU CAN SEE HERE, BUT DAY 28 AFTER TUMOR INJECTION GR 26 TUMOR WHICH IS TR 26 TUMOR HAVE STABLE (INDISCERNIBLE) GENES ASSAY, TUMOR GROWS IN THE BRAIN. YOU CAN SEE THIS GROUP MICE WHICH IS TREATED WITH FOLIC ACID COATED WITH VECTOR, CARRIES THERAPEUTIC AGENT, IN THIS CASE HAVE MUCH LESS TUMOR GROWTH COMPARED WITH CONTROL ONE, OR WITH (INAUDIBLE). THAT'S NOT ENOUGHND YOU CAN SEE FURTHER QUOTATION HERE ALSO SHOW MORE IMPORTANTLY VIABILITY AS -- FOR MICE TREATED WITH VECTOR, GRAPEFRUIT NON-VECTOR CARRIES THERAPEUTIC AGENT (INAUDIBLE) FOLIC ACID. VIABILITY (INAUDIBLE) LAST, INCREASE ACTIVATION -- HERE YOU HAVE TWO MORE SLIDES, YOU CAN SEE HERE GENES YOU SHOW HERE, MHC 1 HERE, WHICH THIS FOLIC ACID, YOU CAN SEE AFTER STAINING. FURTHERMORE THIS CONTROL VECTOR COMPARE WITH OTHER CARRIER (INAUDIBLE). ALSO LITERATURE NK CELL ACTIVATION. IT'S A MARKER FOR NK ACTIVATION. YOU CAN SEE HERE IS ONLY THIS GROUP HERE DRAMATICALLY NK CELLS AS WELL AS MHC 1 EACH OTHER. HIGH LEVEL ACTIVATION, LOWER LEVEL MHC 1, THEREFORE TUMOR MASS GENE EXPRESSION LESS TUMOR GOES IN THE BRAIN. COMPARED WITH OTHER CONTROL GROUPS. SO THIS IS INTRANASAL DELIVER MAYBE NOT ENOUGH, ONE SELECTION HERE, INTRAVENOUS, ALSO ABLE TO (INAUDIBLE) IN THE BRAIN AS WELL. IN THIS CASE GRT MOTIF, GRAPEFRUIT NANOPARTICLE. THIS IS THE WORK DONE IN THE LAB REMEMBER ALSO HAVE NUMEROUS COLLABORATOR AS WELL (INAUDIBLE). THANK YOU. [APPLAUSE] >> THANK YOU VERY MUCH, DR. ZHANG. IF YOU CAN ALSO HOLD YOUR QUESTIONS FOR DR. ZHANG FOR THE OPEN MIC DISCUSSION. WEE HAVE TO MOVE FORWARD WITH THE PRESENTATION OF DR. KAPOGIANNIS. THAT WILL BE PLASMA EXOSOMES ENRICHED FOR NEURONAL ORIGIN, SOURCE FOR BIOMARKERS FOR NEUROINFLAMMATORY DISEASES. >> THANK YOU. I WOULD LIKE TO THANK THE ORGANIZERS FOR THIS KINDS INVITATION. I'M GOING TO BE ONE OUT OF FOUR IN THIS WORKSHOP THAT TALKS DIAGNOSTIC. SO THIS IS ONE SLIDE, THE ONLY SLIDE ACTUALLY THAT DOES NOT DESCRIBE OUR DATA OR WORK. BUT IT COMES FROM MY COLLABORATOR NORMAN HOWIE AT JOHNS HOPKINS, IT'S A MODEL OF BLOOD BRAIN BARRIER AS GOOD AS ANY MODEL CAN BE WHERE IT SHOWS UP FORMATION EXOSOMES IN ASTROCYTES, THEIR DELIVERY BY TWO ENDOTHELIAL CELLS, UPTAKE BY ENDOTHELIAL CELLS AND THEIR RELEASE IN THE LUMINAL IN THE LUMINAL SURFACE. SO IT IS AT LEAST ONE OF THE WAYS THAT EXOSOMES CAN FIND THEIR WAY FROM BRAIN TO PERIPHERY BY CROSSING THROUGH ENDOTHELIAL CELLS. I GOT EDUCATED MYSELF IN THE VARIOUS OTHER ROUTES BY WHICH THIS IS POSSIBLE. PERINEURAL SPACES, THE GLYMPHATIC SYSTEMS. SO BOTTOM LINE FOR ME IS THAT THERE IS A WAY OUT FOR BRAIN EXOSOMES TO MAKE IT THROUGH THE PERIPHERY. AND THAT'S WHERE ACTUALLY MY WORK STARTS FROM HERE ON. SO I WILL PRESENT THE WORK WE'RE DOING ON ISOLATION AND MOLECULAR CHARACTERIZATIONS OF EXOSOMES ENRICHED OR EB ENRICHED FROM ROMAN ORIGIN AND USE OF THEM AS A SOURCE FOR BIOMARKERS FOR VARIOUS DISEASES. I NEED TO REMIND THE AUDIENCE ABOUT CONTENT AND THE DIFFERENT MOLECULES THAT ARE PRESENT IN AND ON EXOSOMES AND OTHER EVs, IN PARTICULAR I WILL POINT YOUR ATTENTION THAT IT HAD BEEN KNOWN SOME TIME TARGETING ADHESION MOLL EXCUSE THAT HAVE IN GENERAL RELATIVE TISSUE SPECIFICITY, HAVE BEEN KNOWN TO BE ON THE SURFACE OF EXOSOMES. IN PARTICULAR WE WERE GOT INTERESTED IN N CON NEURAL CELL ADHESION MOLECULES WHICH HAVE RELATIVE THOUGH NOT ABSOLUTE TISSUE SPECIFICITY CELL SPECIFIC FOR NEURONS AND TOOK ADVANTAGE FROM THAT TO ISOLATE SUBSET OF VESICLES THAT EXPRESS THESE MARKERS. SO OUR APPROACH IS TO START IN GENERAL FROM PLASMA OR SERUM SAMPLES FROM PATIENTS, USE PRECIPITATION TECHNIQUE WHICH IS HIGH THROUGH PUTTY NEARBY, NOT THE PURE WAY NOT THE BEST TO ISOLATING HIGHLY PURE VESICLES BUT YOU CAN DO IT IN LARGE NUMBERS FAIRLY EFFICIENT, WE GET ISOLATION OF TOPO EV OUT OF THIS, THESE EVs ARE A MIXTURE OF DIFFERENT VESICLES AND THEY HAVE DIFFERENT ORIGINS. IN THE SECOND STEP WHAT WE DO IS USING ANTIBODIES AGAINST ONE CHARACTERISTIC SURFACE MARKER, WE PRECIPITATE ONE SUBSET OF THEM AND NOW WE HAVE AN ENRICHED POPULATION OF EVs THAT HIGHLY EXPRESS THIS MARKER END BY EXTANT THEY ARE HIGHLY ENRICHED FOR THE TISSUE OF ORIGIN OF THIS MARKER. SO HOW DO THESE LOOK? TAKE THROUGH PRECIPITATE IN GENERAL THE WHOLE NATURE OF THIS PEG MESH THAT CREATE, IT'S DIRTY LOOKING, AFTER IMMUNOPRECIPITATION STEP, WE HAVE CLEANER PREPARATION. THIS IS NOT THE BEST THING THAT YOU WILL SEE AND WE'RE ACTUALLY WORKING TO IMPROVE IT BUT YOU CAN APPRECIATE IF WE REMAIN FOR L 1 WE SEE NUMBER OF VESICLES THAT EXPRESS, MANY EXPRESSION I WOULD SAY THE VAST MAJORITY EXPRESS L 1 AND SOME EXPRESS MULTIPLE MOLECULES ON THE SURFACE. SO WE DO GET ENRICHED PREPARATIONS THAT THEY HAVE L 1 ON THEIR SURFACE. THEN WE FURTHER CHARACTERIZE THEM AND THIS IS ON NANOSITE, THIS IS FOR THOSE FAMILIAR WITH -- THIS IS A LASER BASED TECHNIQUE AT THE BROWNIAN MOTION WHICH CAN LOOK AT VERY SITE DISTRIBUTION AND CONCENTRATION OF THESE VESICLES IN BUY LOGICAL FLUID. THEY WILL GET VL-1 EXOSOMES, WE HAVE A PEAK AROUND A HUNDRED, SOME SLIGHTLY LARGER SO WE DEPENDING HOW PURE RESPONSE WANTS TO BE IN TERMINOLOGY, WE CAN REFER TO THEM AS EV, SUBSET ARE EXOSOMES MAYBE SOME ARE NOT. IF WE TALK NUMBERS, THE L-1 EXOSOMES COMPARED TO NEGATIVE. CD 1 EXOSOMES IN TOTAL 5 TO 10% TOPS OF TOTAL EXOSOMES IN A NUMBER OF TIMES WE HAVE DONE THAT CHARACTERISTIC EXAMPLE. 5 TO 8% RZ RICH, WE MAYBE LEAVING SOME BEHIND BUT IN ANY CASE, WHAT WE RECOVER IS 5 TO 8% FOR SUBSET OF PLASMA EV EXPRESSED AT ONE TIME. IF WE CHARACTERIZE FOR SOME ARRAY WE SEE THEY EXPRESS POSITIVE AND NEGATIVE CD8 1 POSITIVE, THEY EXPRESS COMMON NEURONAL MARKERS BUT THEY HAVE SOME DIFFERENCES IN THE NUMBERS THEY EXPRESS. IMPORTANTLY, THE ENRICHED POPULATION HERE EXPRESS MUCH LESS MARKERS THAT SHOULDN'T BE THERE, EPITHELIAL MARKER TELLING US THAT WE'RE GETTING A DIFFERENT PREPARATION THAT AT LEAST DOES NOT CONTAIN MUCH EPITHELIAL ORIGIN DISEASE. ALSO ONE WE PRECIPITATE BY L-1 WE SEE THAT TO A LARGE EXTENT THESE VESICLES EXPRESS NCOM WHICH IS A DIFFERENT, NEURONAL MARKER. WE HAVE ENRICHED PREPARATION THE QUESTION IS WHAT IS IN IT. THIS IS SOME FRESH DATA THAT I HAVE. WE HAVE A ANYMORE OF ARRAYS AS A QUICK EASY STEP TO GO THROUGH A NUMBER OF MOLECULES SOME WHICH MAYBE ALSO POTENTIAL BIOMARKERS FOR A DIFFERENT DISEASE. THERE ARE A NUMBER OF THINGS THAT POP OUT AND MAKE US VERY INTRIGUED. FIRST THE ARE TO EXPLAIN HOW THESE WORK, THESE ARE COLOR CODED FIGURES BUT THE REAL DATA ARE JUST DOT BLOTS. WE DID THEM BY LOADING THE SAME NUMBER OF DISEASE FOR PREPARATION. L-1 AND ALTERNATIVE IMMUNOPRECIPITATION FOR EPITHELIAL WE HAVE THE SAME AMOUNT SO THE DIFFERENCES ARE NOT BECAUSE OF TOTALS ARE MORE AND L # COMs ARE MET, SO FORTH. THEY SEEM TO HAVE A LOT MORE BIOMARKERS IN THEM AND MAYBE THIS IS A SUBSET HETEROGENEOUS POPULATION IF YOU LOOK AT THEM ANY KIND OF SIGNAL IS KIND OF WASHED OUT BUT ANOTHER POINT IS WE SEE MANY RECEPTORS BEING PRESENT. I WILL POINT YOUR ATTENTION WITH LEPTON AN LEPTON RECEPTOR. LEPTON ITSELF IS NOT AND SAME HOLDS THROUGH FOR MANY OTHER MOLECULES THESE CHANGE THE WAY THAT YOU WOULD THINK ABOUT BIOMARKER PURSUED INTO THINKING MORE ABOUT RECEPTOR MOLECULES, THAT NORMALLY YOU THINK ABOUT THEM AS PRESENT ON CELL SURFACE, RATHER THAN SOLUBLE MOLECULES. ANOTHER THAT HIGHLIGHTS MORE DRAMATICALLY, HERE IS A -- HERE IS ARRAY OF KINASES AND PHOSPHOKINASES AND DIFFERENT INTRACELLULAR MOLECULES. YOU SEE L-1 ARE HIGHLY ENRICHED FOR MOLECULES SUCH AS IRK, AKT, MTOR, KREB, MOLECULES THAT YOU THINK OF INTRACELLULAR SIGNALING ONES, WHEREAS TOTALLY THESE ARE -- THEY BECOME MUCH LESS OF THIS. SO WHETHER THAT REFLECTS SUBSET OF MOLECULES COMING FROM VERY HIGHLY ACTIVE METABOLICALLY ACTIVE CELLS, WHICH (INAUDIBLE) BY THE WAY T. WHAT DO WE DO WITH THEM? PURSUIT IS TO FINDS BIOMARKERS FOR ALZHEIMER'S DISEASE AND DIFFERENT DISEASES. SO AFTER WE ISOLATE OUR ENRICHED PREPARATION, WE TYPICALLY LIGHT THEM THEN WE PURSUE SOME TARGETS. MOST OF THE TIME WE RUN SO WE ARE ELISAS. WE PUBLISHED A NUMBER OF CASE CONTROL STUDIES SO FAR, THESE ARE SMALLER DATA SETS OF PATIENTS WITH ALZHEIMER'S DISEASE AGAINST CONTROLS, TYPICALLY WE SELECT VERY CLEAN CLEAR WELL DEFINED ALZHEIMER'S PATIENTS AND VERY CLEAN CONTROLS. FOR PROOF OF CONCEPT, SO FIRST STUDY WE PURSUED THE PATHOGENIC MOLECULES OF ALWAYS SHIMMERS DISEASE. I USE THE EXOSOMINGS IN THESE INTERCHANGEABLY IN MY SPEECH, I HOPE THAT DOESN'T OFFEND THE PUREED BUT IN ANY CASE I CAN'T HELP IT SOMETIMES. SO WE HAVE SEEN L 1 POSITIVE HAVE HIGHER E BETA THOUGH DIFFERENCE NOT DRAMATIC THE OTHER RESULTS HAVE A BETA BUT WHERE WE SEE A DRAMATIC DIFFERENCE IS PTAU, INTRACELLULAR, NORMALLY UNDETECTABLE IN PLASMA AND L 1 COM HAVE TONS OF BACK LOADS OF PHOSPHOTAU WHERE THE REST DO NOT. IT'S ONE OF THE HIGH -- THAT'S THE TYPE OF MOLECULES TO PURSUE POTENTIAL BIOMARKERS IN THESE. IN OUR INITIAL STUDY PUBLISHED A COUPLE OF YEARS AGO, WE SAW DRAMATIC DIFFERENCES IN 2 TOES TOE TAU SPACES BETWEEN PATIENTS AND CONTROLS, WITH NO OVERLAP. ALSO IN THAT PARTICULAR CASE WE SAW A SIMILAR DIFFERENCE IN A BETA. WE FOUND VERY SIGNIFICANT SEPARATION BETWEEN PATIENTS AND CONTROLS WITH 96% ACCURACY. WE'RE REALLY KATEING THE RESULTS IN MUCH LARGER DATA SETS, STILL BLINDED. WE HOPE IT WILL HOLD THOUGH THIS WAS A TIGHT CASE CONTROL STUDY SO THEY MAY BE LESS IMPRESSED, IN ANY CASE WE'LL SEE SOON. ANOTHER TARGET WE PURR SOD IN ALZHEIMER'S DISEASE IS RELATED TO INSULIN RESISTANCE. IT'S KNOWN INSULIN RESISTANCE OCCURS IN ALZHEIMER'S BRAIN. MOLECULAR BASIS FOR INSULIN RESISTANCE OR AT LEAST THE CANDIDATE IS THIS MOLECULE INSULIN RECEPTOR SUBSTRATE ONE WHICH EXISTS IN A SERUM PHOSPHORYLATED FORM WHICH MARKS AN INSULIN RESISTANT FORM AND TYROSINE PHOSPHORYLATED FORMS. SO THIS HAS BEEN KNOWN TO BE TRIGGERED BY BETA AMYLOID, AN ACTIVE PROCESS FOR INDUCING INLYNN RESISTANCE IN ALZHEIMER'S DISEASE AND PRESENT IN PATHOLOGICAL PROGRESSION INTHE ALZHEIMER'S DISEASE. SERINE PHOSPHORYLATED SPECIES. SO ERS 1 IS AN INTRACELLULAR MOLECULE, NORMALLY NON-FOUND IN PLASMA BUT WE DO FIND IN OUR EXOSOMES. WE DID A STUDY WE MEASURED IRS 1 L 1 EXOSOMES IN CONTROLS PATIENTS WITH ALZHEIMER'S DISEASE AND SUBJECTS COGNITIVELY NORMAL WITH DIABETES AS WELL AS STD AND WHAT WE FOUND IS WHEREAS TOTAL IRS 1 IS THE SAME IN ALL GROUPS, PATIENTS WITH ALZHEIMER'S DISEASE HAVE A LOT OF THE SERINE PHOSPHORYLATED IRS 1, IF YOU WISH, MUCH LESS GOOD EFFICIENT IRS 1 AND RATIO OF THE TWO WAS HIGH LIE DIAGNOSTIC. SO THIS IS ALSO A MARKER WE'RE PURSUING IN LARGER ALZHEIMER'S DATA SETS. NOW WE'RE BESIDES ALZHEIMER'S DISEASE, VERY INTERESTING FOR THIS GROUP IN PARTICULAR VERY BRIEFLY IN ADDITIONAL STUDIES TWO DISEASE STATES. VERY BRIEFLY, IN MS. MS THROUGH COLLABORATORS AT JOHNS HOPKINS WE GOT OUR HANDS ON A DEATH SET AND WHAT WE FOUND THERE, IT'S ACTUALLY A SIGNIFICANT DECREASE IN NUMBER OF L-1 EXOSOMES. WE HAVEN'T PURSUED ANY MARKERS WITHIN THESE EXOSOMES YET BUT IT'S FIRST DISEASE STATE WHERE WE SEE A DIFFERENCE BETWEEN PATIENTS AND CONTROLS IN THE NUMBER OF OF L-1 AND INTERESTINGLY THIS NUMBER SEEMS TO RELATE TO DISEASE DURATION. THE OTHER THE TBI THROUGH GOOD COLLABORATOR, NURSING RESEARCH INSTITUTE, JESSICA IS GREAT SUPERB RESEARCHER IN TBICS, WE HAVE ALREADY COMPLETED A STUDY AND NOW ANALYZING THE DATA OF PATIENTS BUT MILITARY SERVICEMEN THAT HAVE TBI AND SOME ARE RESIDUAL NEUROLOGICAL IMPAIRMENT AND SOME DID NOT, WE LOOK AT TAU AND A BETA IN THESE VESICLES WE FIND GOOD SEPARATION IN PARTICULAR FOR THOSE THAT HAVE IMPAIRMENT. STUDIES THAT WE'RE CONDUCTING, WE'RE CONTINUING TO EXPAND. SO THESE ARE SOURCE OF BIOMARKERS ONE DOE FOR STUFF STUDYING DIFFERENT CELLULAR PROCESSES AND PERHAPS THE GOAL IS TO HAVE MARKERS TO FOLLOW RESPONSE TO TREATMENT. THIS IS THE TEAM THAT CONDUCTS IT AND SPECIAL THANKS IN PARTICULAR TO MY MENTOR, DR. (INDISCERNIBLE) WHO DEVELOPED THESE MOLECULAR TECHNIQUES. THANK YOU. [APPLAUSE] >> THANK YOU VERY MUCH, FROM KAPOGIANNIS. KEEP YOUR QUESTIONS TO THE OPEN MIC DISCUSSION. THE NEXT PRESENTER WILL BE DR. MARTIN PRESENTING HER-2 TARGETED EXTRA CELLULAR VESICAL DELIVERY OF THERAPEUTIC mRNA FOR PRO DRUG THERAPY. THANK YOU. >> FIRST I WOULD LIKE TO THANK THE ORGANIZERS FOR INVITING ME TO THIS MEETING. AND OF COURSE TO MY SUPPORTERS, COMMON FUND EERC FUND, THAT'S GIVEN ME THE OPPORTUNITY TO CONDUCT THIS RESEARCH. THESE THE TOPIC OF MY TALK, USE OF THE NEW PRODRUG REGIMEN FOR SPECIFIC TARGETING OR HER-2 POSITIVE BREAST CANCER CELLS. USING DIRECT FIT EXTRA CELLULAR VESICLES ISSUE WITH WHAT ARE EVs ANDS WHAT ARE EXOSOMES. EV IS MUCH MORE INCLUSIVE TERM. EXOSOMES ARE OFTEN USED AS A SUBSTITUTE FOR THEM. I WON'T GO INTO DETAILS OF IT BUT BECAUSE EXOSOMES IS A MORE COMMONLY USED TERM,THAT IS THE TERM I WOULD STICK TO. I WILL ALSO TALK ABOUT SOME STUDIES WE HAVE DONE ON EV AS THE BLOOD BRAIN BARRIER. THESE ARE PEOPLE WHO COLLABORATED WITH US. WE'RE ALSO VERY ABLE STANFORD COLLEAGUES NUMBER OF VERY ABLE COWORKERS BOTH IN MY LAB AS WELL AS IN THE LAB OF MY COLLABORATORS THAT -- NOW THE TIME IS SHORT BUT THEY RECOGNIZE IDENTIFY AND PUBLICATION AN CERTAINLY WILL BE YET TO COME. FIRST NEW PRODRUG REGIMEN, BECAUSE IT CONSISTS OF A HARMLESS COMPOUND CALLED PHENOL. THAT'S ACTIVATED BY ENZYME CALLED CHRR THEN IT IS CONVERTED INTO FCHB. WHERE (INAUDIBLE) IS HARMLESS NCHB IS HIGHLY TIE TOE TOXIC. AND THIS IS REDUCKTIVE ACTIVATION. AND AS I SAID IT IS HIGHLY CYTOTOXIC. IT ALSO HAS THE INTERESTING PROPERTY THAT IT IS FLUORESCENT AND THAT ENABLES US TO EXAMINE ASPECTS OF IT RELATIVELY PAINLESSLY, FOR INSTANCE THE FACT THAT IT HAS A VERY EFFECTIVE BYSTANDER EFFECT, WHICH MEANS THAT ONLY A FEW CELL MUTE GENERAL FOR THE NEIGHBORING CELLS TO BE KILL. NOT ONLY THAT, YOU CAN/IZE IT NON-INVASIVELY IN LIVING MICE. AND THAT MEANS THAT YOU CAN EASILY TELL WHETHER WHETHER THE -- YOUR ATTEMPT AT TRYING TO CONFINE THE ACTIVATION OF -- ONLY TO THE TUMOR IS SUCCEEDING OR NOT. HERE IS A CASE WHERE IF IT IS SUCCEEDING THIS IS TUMOR, HERE IS THE CASE WHERE IT IS NOT SUCCEEDING, YOU CAN SEE THAT FCHB IS MAGENTA COLOR IS FORMED AS WELL. THIS IS CONVENIENT, IT GIVES YOU A RAPID WAY OF SCREENING A DIFFERENT NUMBER OF DIFFERENT APPROACHES TO DECIDE WHICH TO FOCUS ON FOR MORE INVOLVED STUDIES. THIS IS THE ENZYME WE AT THIS COVERED AS WELL. WE HAVE A PATENT ON IT, IMPROVED IT BY MANY METHODS AND X-RAY CRYSTALLOGRAPHY SHOWED THE -- IMPROVING -- SECRET FOR IMPROVING IT, IT IS A TETRAMER AND IF YOU CAN INCREASE THE DIMER DIMER INTERACTION, MAKE IT STRONGER, THE ENZYME ACTIVITY INCREASES AND SECONDLY, IF YOU ENHANCE THE CAVITY SIZE OF ACTIVE SITE WHICH IS -- HARBOR FMN THEN TWO YOU INCREASE ACTIVITY OF THIS ENZYME AND WE HAVE DONE IT SEVERAL FOLD AND IT IS IMPROVED NOT ONLY FOR -- OUR PRO DRUG BUT MANY OTHER PRODRUGS AS WELL. IT IS QUITE AN EFFECTIVE REGIME, YOU CAN SEE THAT THESE ARE MICE IMPLANTED WITH ORTHO TOPIC 41 BREAST TUMORS HIGHLY AGGRESSIVE. IN THE TREATED CASE YOU HAVE 40% SURVIVAL AT 150 DAYS WHEREAS ALL THE UNTREATED MICE ARE DEAD BY DAY 25. MORE RECENTLY WE HAVE DONE PKPD STUDIES TO PREDICT MORE EFFECTIVE SCHEDULE OF ADMINISTRATION OF THE REGIMEN. FROM AND SLIDE HERE PURR POTS TO SHOW WE HAVE SUCCEEDED IN INCREASING EFFECTIVENESS BY THIS NEW ADMINISTRATION REGIMEN. AND IN ORDER TO PREVENT METASTASIS OF CANCER. TARGETING EXOSOMES TO HER-2 LIGANDS. TO DO THIS, WE MADE THE CHIMERIC PROTEIN WHICH CONSISTS OF C 1 C 2 DOMAINS OF (INDISCERNIBLE), C 1 C # 2 DOMAINS OF -- BINDS TO SERINE WHICH IS PRESENT ON THE SURFACE OF EXOSOME. SO ANYTHING CONTAINING C 1, C 2 WOULD BIND TO THE SURFACE OF THE EXOSOMES. TO THIS WE COMBINE SINGLE CHAIN VARIABLE FRAGMENT ANTIBODY WHICH HAS A HIGH AFFINITY FOR THE HER-2 LIGAND. WE HAVE THE SEQUENCE TO ENSURE THAT PROTEIN WITH WOULD MIGRATE TO SURFACE OF THE EXOSOME AND WE HAVE A TAG TO PURIFY THE PROTEIN. WE PUT THIS DNA CONSTRUCT INTO PLASMID. AND WHEN YOU INJECT -- TRANSFECT CELLS WITH 293 CELLS, TRANSFECT WITH PLASMID THEY MAKE THE EXOSOMES AN THESE EXOSOMES CONTAIN THIS CHIMERIC PROTEIN WHICH HAS IT ON THE SURFACE. THE SURFACE EXHIBITS LIGAND AS WELL. THIS SHOWS THAT WE ISOLATED THIS PROTEIN AND SHOWED THAT IT HAS 70 KD MOLECULAR WEIGHT WHICH WAS EXPECTED. AND IT IS THE 3-D STRUCTURE OF IT SHOWING THAT THE SC IS EXPRESSED ON SURFACE, THESE ARE THE C 1 C 2 DOMAINS. SO YOU SEE HECK 293 EXOSOMES, YOU HAVE BEEN BEEN HEARING, ARE FINE FOR PROOF OF CONCEPT STUDIES BUT THESE ARE CANCER CELLS. WE CAN USE FOR CLINICAL PURPOSES. SO WE ISOLATED DENDRITIC CELLS FROM HUMAN BLOOD, THEN DIFFICULT PROCESS, EXPENSIVE AND ALL THAT BUT ONE BIG ADVANTAGE OF EXOSOMES OF DENDRITIC CELLS IS THAT BECAUSE THEY DON'T HAVE (INAUDIBLE) THE HE CANSOMES THEY MAKE ARE BOLD. SO THEY HAVE A GREATER SURFACE AREA TO BIND TO CHIMERIC PROTEIN WHEN YOU ADD FROM THE OUTSIDE. SO I MENTIONED ALREADY PURIFIED LARGE QUANTITY OF THIS CHIMERIC PROTEIN. THEN MIXED WITH DENDRITIC CELLS ESOMES AND YOU CAN SEE MUCH OF THE SURFACE IS COVERED PRIMARILY WITH THIS HER-2 DIRECTED C 1 C 2 CHIMERIC PROTEIN. THIS IS THE NANOSITE SIZING. YOU SAW THE NANOSITE VIEW MOW IT LOOKS LIKE AND THE ARCH IS ABOUT SAME HUNDRED AVERAGE TIME, HUNDRED NANOMETERS. SO WHAT DO THESE EXOSOMES DO? CAN THEY REALLY BIND? WHAT WE DID FIRST IS USE ELISA AND THE PLATE HAS HER-2 LIGAND AT ITS BOTTOM, WE HIT IT WITH THE EV MADE FROM HER-2, 93 CELLS OR EVs, RECONSTITUTED EVs FROM THE DENDRITIC CELLS. AND YOU CAN SEE THAT THE CONTROLS HAVE NO BINDING. HER-2, 93 CELLS DO BIND. AND ONES FROM DENDRITIC CELLS BIND TO MUCH GREATER DEGREE FOR OBVIOUS REASONS. BECAUSE MUCH GREATER SURFACE OF THE EXOSOMES DISCOVERED -- IS COVEREDDED WITH CHIMERIC TARGETING PROTEIN. THIS IS THE ELISA, HOW ABOUT CELLS THEMSELVES? WHAT WE DID HERE IS TO ADD EXOSOMES TO HER-2 QUOTE UNQUOTE NEGATIVE CELLS T HAVE LOW CONTENT OF HER-2 LIGAND. BT 474 CELLS ARE VERY HIGH IN HER-2 CONTENT. THERE'S ALMOST NONE. VERY DIFFICULT TO SEE ANY GREEN THING IN HERE, THE EXOSOMES ARE LABELED GREEN. AND YOU CAN SEE THAT THERE IS A VERY CONSIDERABLE BINDING OF THE CELLS BY THESE EXOSOMES OF THE HER-2 POSITIVE CELLS. YOU CAN SEE NOT ALL THE CELLS ARE BOUND. THE REASONS ARE SEVERAL. I CAN DISCUSS LATER ON BUT ONE THING I WOULD LIKE TO POINT OUT, WHICH IS THAT BECAUSE THE BYSTANDER EFFECT OF MCHV IS VERY STRONG, IT IS NOT NESS THAT ALL THE CELLS IN THE TUMOR BE CONVERTED OR BOUND TO THE AGENT THAT CONFERS ON CAPABILITY TO CON VET (INDISCERNIBLE) CHB. NOW SO WE HAVE NOW HER-2 TARGETED EXOSOME BUT THEY HAVE TO HAVE THE GENE THAT CAN ACTIVATE CONVERT C LOG TO CHMB. WE DECIDED THAT BETTER GENE DELIVERY IS mRNA. THE REASON IS OBVIOUS. THAT IF YOU PUT DNA THE FIRST TO CYTOSOL AND THEN TRANSFER TO NUCLEUS, TRANSCRIBED THERE THE mRNA HAS TO COME OUT. IN FACT, THE EFFICIENCY IS VERY, VERY LOW. mRNA ON THE OTHER HAND GOES DIRECTLY SO THAT WOULD BE A BETTER WAY OF DOING THINGS. SO THE GROWING WAVE OF PUTTING NUCLEOTIDES TO EXOSOMES HAVE BEEN THROUGH ELECTROPORATION. AND THAT WORKS FINE WITH MICRORNAs. AND WE HOPE IT WOULD WORK ALSO WITH MESSENGER RNA BUT DID NOT. WE TRIED MANY THINGS BUT IT DIDN'T. IN COLLABORATION WITH (INDISCERNIBLE) OF SBI WE MADE A NEW PLASMID, IN WHICH THE CHRR GENE, THE GENE THAT ENCODES THE ACTIVATING ENZYME, WAS COMBINED WITH THIS ZIP CODE. YOU JUST HEARD THAT THE EXOSOME CONTENT WITH RESPECT TO PROTEIN mRNA OR WHATEVER IS QUITE DIFFERENT FROM THAT OF THE PARENT CELL. BUT OF COURSE WHICH MEANS THERE'S A SELECTIVE MECHANISM AND SELECTIVE MECHANISM FOR MRNA INTRODUCTION INTO EXOSOMES IS THE ZIP CODE SPECIFIC SEQUENCES THAT ARE PRESENT ON mRNAs THAT ARE DESTINED TO GO INTO THE EXOSOME. SO WE USED ONE OF THESE ZIP CODES AND WE DID QRTPCR AND SHOWED INDEED mRNA GOT INTO IT. SO IS IT ACTUALLY FUNCTIONAL? SO HERE WHAT WE DID, WAS TO TAKE NOW BT 47 # CELLS, WE ADDED TO THEM DIRECTED LOADED EXOSOMES, ADDED DIRECTED AND LOADED WITH THIS M RNA THAT ENCODES CHRR. AND THEN WE HIT THEM WITH CMO. THE POINT IS THAT IF THIS M RNA IS FUNCTIONAL, THE ENZYME WOULD BE FORMED PANNED MCHB WOULD BE FORMED. AND MCHB IS FLUORESCENT, IT'S A REPORTER AND YOU CAN DETERMINE WHETHER A DELIVERY OF THE GENE BY mRNA HAS SUCCEEDED. INDEED IT HAS. THROUGH CONTROL THEY ONLY SHOW BACKGROUND FLUORESCENCE, WHEREAS THE BT 474 CELLS HIT WITH RODENT EVs SHOW A MUCH HIGHER DEGREE OF FLUORESCENCE, WHICH MEANS MUCH HIGHER DEGREE OF ACTIVATION OF C LOG AN GENERATION OF MCHB. NOW, IF FBAB IS GENERATED THE CELLS OUGHT TO BE KILLED. INDEED THEY ARE. THIS IS WHERE YOU SEE THESE OTHER CONTROLS AND THERE'S A 50% KILL. THIS IS JUST THE BEGINNING AND WE HAVE WAYS TO ENHANCE STABILITY OF mRNA TRANSLATION OF IT AND THINGS LIKE THAT SO THAT IS WHAT WE ARE DOING NOW. WE ALSO SHOWED THAT THE EV OR EXOSOMES HAVE THE CAPABILITY OF DONATING DNA INTO IN VIVO. SO WE USE HERE CRE LOGS MICE, CONTAINING STOP CO-DON. IF YOU CAN BRING IN THE CRE RECOMBINASE THE STOP CO-DON IS REMOVED AND YOU CAN SHOW THIS ACTUALLY WORKED. SO WHAT WE SHOWED WAS THE EVs ARE CAPABLE OF DELIVERING DAN THAT ENCODES CRE RECOMBINASE AND LIMB TOE SITE IS FORMED IN LIVING MICE. NOW, EXOSOMES AND BBB, THE BLOOD BRAIN BARRIER. THE NORMAL BRAIN. WE DID IT WITH THIS WAS USED TO VISUALIZE THE VASCULATURE, BACKGROUND IMAGING AND THEN DID THE INJECTED GFP EVs VIA RETRO ORBITAL RANGE. IN THE VENUE THAT I'M GOING TO SHOW YOU, YOU CAN SEE EV AS WAY SPOTS OR WIDE STREAKS. FEW THINGS TO NOTICE. EV, AFTER INJECTION EV LAST ONLY A SHORT TIME, LAST ONLY FOR A VERY SHORT TIME IN THE SITUATION. TWO TO FOUR MINUTES. THIS IS CONSISTENT WITH WHAT OTHER PEOPLE HAVE FOUND AS WELL. SECONDLY, THE EXOSOMES DO NOT COMBINE WITH BLOOD BRAIN BARRIER, WITH THE VASCULATURE OF THE BRAIN. NONE OF THEM WOULD YOU SEE TO BE THE VASCULATURE HERE. THAT'S THE NORMAL ONE. BUT IT IS DIFFERENT WITH THE CARCINOGEN, WITH THE BRAIN WITH CANCER. AND IMMEDIATELY UPON INJECTION, OF EXOSOMES, YOU CAN SEE THAT FEW ARE BINDING TO THE VASCULATURE. THIS INCREASES WITH TIME. NOT ONLY THAT, YOU CAN FIND THEM IN THE -- EXTRAVASCULAR SPACE. SO ABLE TO CROSS THE BARRIER HERE. AT SIX HOURS, YOU HAVE A GREATER DEGREE OF THIS. SO I GUESS -- OKAY, NOW FINALLY, C 1 C 2 METHOD OF MAKING DIRECTED THIS OR THAT IS USEFUL AND WHAT WE'RE PLANNING TO DO NOW IS HAVE A MULTI-FUNCTIONAL EXOSOME. EXOSOME LABELED WITH DECK CRATED WITH HER-2 TARGETING LIGAND AS WELL AS THOSE THAT TARGET THE NERVES OR NEURAL ELEMENTS. SO WE JUST HEARD OF NCCAM FROM THE PREVIOUS SPEAKER, (INAUDIBLE) IS ONE OF THEM SO WE HOPE THIS WAY, WE MAY BE ABLE TO ENHANCE THE CAPABILITY OF THESE EXOSOMES TO CROSS THE BLOOD BRAIN BARRIER AND THEN TO ATTACK SPECIFICALLY THE METASTASIS OF HER-2 POSITIVE CANCER TO THE BRAIN. SO WITH THAT, I STOP. THANK YOU. [APPLAUSE] >> THANK YOU VERY MUCH, DR. MATIN. WE'LL HOLD QUESTIONS UNTIL AFTER BREAK. WE'RE GOING TO SWITCH GEARS, FIRST WE E HAVE COFFEE AND RECOUP RATE, GET ENERGY AND THEN SWITCH GEARS TO AN OPEN MICROPHONE DISCUSSION WHICH WILL BE EXCITING WITH ALL THOSE SPEAKERS FROM EACH SESSION UP HERE FOR DISCUSSION OF KEY PRIORITIES AND KEY RECOMBINATIONS FROM THIS MEETING. THANK YOU. AND SO PLEASE BE BACK AT 3:10. WE HAVE HALF HOUR FOR EACH PANEL, SESSION 1 HALF HOUR UNTIL 3:40, SESSION 2 WILL START AFTER THAT WITH DR. CRADER AND WE'LL FINISH WITH DR. KRAIG, EXOSOME THERAPEUTIC. THANK YOU. >> PLEASE TAKE YOUR SEATS, IT'S A SESSION WITH THE OPEN MICS. CAREFUL AND POLITE AT THE MICROPHONES. I THINK IT WAS A GOOD IDEA WHEN YOU ASK A QUESTION TO ANNOUNCE YOURSELF AND SAY YOU YOU'RE FROM OR STATEMENT ABOUT WHO YOU ARE. THAT'S I THINK EVERYONE WILL BELIEVE THAT THAT WILL HELP TO AS MODERATOR MY GOAL IS TO WATCH THAT YOU DO NOT GO TO THE TIME OF THE NEXT SESSION SO I WILL BE STRICT AND LIMIT EACH SESSION TO 30 MINUTES, AFTER THAT. >> FIRST, IT ET GREAT TO BE BACK WITH NHLBE BECAUSE ANDREA AND I GO LONG WAY BACK WITH THE GRANT WITH JOHN GRIFFITH ON PROTEIN C AND THIS ONE CAME AS I SAID IN MY TALK TO FROM BENCH STUDIES AN BASIC DISCOVERIES TO PHASE 2 FOR STROKE. THAT WAS THE FIRST TALK WE HAD. DIFFERENT TARGETS AN TREATMENT AND GENETIC CAUSE CONSIST OF HUMAN MONOGENIC DISEASES AS YOU CAN SEE ON THE SIDE, SO I WOULD LIKE TO GUIDE YOU THROW OUR OPPORTUNITIES AND CHALLENGES IF I CAN AND WE CAN OPEN DISCUSSION AND WITH ALL SPEAKERS AS YOU CAN SEE IT IS REALLY THAT WE DON'T HAVE RIGHT NOW COMPREHENSIVE PROTEOMICS MORE CHALLENGING THE RNA SEQ BUT BOTH CHALLENGES -- WHEN I SAY B TO B ENDOTHELIAL CELLS, PARASITES IN HUMANS AND ANIMAL MODELS. SO WE DON'T HAVE IT AND THAT WOULD BE GREAT RESOURCE IMAGING MOLECULAR BIOMARKERS IN MOLECULAR FUNCTION AN BIOFLUIDS WE HEARD IN DIFFERENT SESSION BUT WE HAVE NOW IMMANAGING BIOMARKERS FOR THE BLOOD BRAIN BARRIER FOR SUBTLE CHANGES, WE HAD FOR TUMORS AS YOU HEARD IN THE FIRST TALK TODAY. WE DO HAVE THEM AND PEOPLE ARE USING IT SO THAT'S NEW. BENCHMARKER BEST PRACTICE FOR IN VITRO MODELS BUT THE BARRIERS TO PROGRESS THAT Z I WANT TO SYSTEM RISE FOR YOU, IN T ROLE OF BLOOD BRAIN BARRIER IN BLOOD BLOOD BRAIN BARRIER IN CATRY THAT'S TALK AND CIRCULATING EXOSOME, WHAT IS ROLE OF PATHOGENESIS ALZHEIMER'S AND OTHER BRAIN DISEASES SUCH AS TRAUMA, STROKE, LS, MS, THE UNDERLYING MECHANISM REMAIN TODAY LARGELY UNDERRESEARCHED REPRESENTATIVE BARRIER IN THE FIELD. WHAT ARE THE OPPORTUNITIES THAT CAN COME FROM HERE? WE HAVE PROOF IN ANIMAL MODEL BUT WHAT IN THE HUMAN BRAIN THAT HAVE DISTURBANCES, ALZHEIMER'S OR, ET CETERA, WHAT IS THE NEUROVASCULAR CHANGES BLOOD DERIVED FACTORS, WHAT IS THAT BREAK DOWN OF THE BLOOD BRAIN INTERFACE. CAN THIS DRIVE THE PATHOGENIC EVENT IN PREVENT TOXIC MATERIAL FROM PATHOGENS, BLOOD, MICROBES, BLOOD COMPILATION FACTORS THAT LEAD THE DISRUPTIVE BRAIN COGNITION IMPAIRMENT AND NEUROLOGICAL SYMPTOMS. AS WE FOUND DIFFERENT DISORDERS, WHAT IS THE ROLE. IF WE -- DISSECT MOLECULAR MECHANISMS THROUGH BIOMARKERS. WHETHER THEY REPRESENT DRUGGABLE TARGETS CAN WE USE THEM TO PREVENT AND TREAT NEUROLOGICAL DISORDER, SOME CURRENTLY USED, I MENTION EXAMPLE OF -- IN MULTIPLE SCLEROSIS THERE'S ALSO EFFORT TO BLOCK SIGH CROW FILL INS THAT LEAD TO DISRUPTION OF BARRIER, ET CETERA, SO THESE ARE PHASE 2 STUDIES, PHASE 3 STUDIES. NOW WHETHER IMAGING BLOOD BRAIN BARRIER INTERFACE, SERVE AS RELIABLE TOOLS. WHAT IT MEANS IF YOU FIND REDUCED PERMEABILITY IN THE HIPPOCAMPUS OR SOME OTHER BRAIN REGIONS DOES THIS SUGGEST THERE WILL BE NEURODURATION COMING NEUROLOGICAL CHANGES INJURY AND ALSO -- THESE ARE ALL GOOD QUESTIONS, I JUST SUMMARIZE FOR YOU WE HAVE A GREAT GROUP OF SPEAKERS THAT COVER GROUND HERE. SO PLEASE LET'S ANSWER THE QUESTIONS. >> FEEL FREE TO COME UP TO THE MICROPHONES. HELLO. >> I'M JACKIE BROWN VANDERBILT UNIVERSITY. ONE OF THE THINGS THAT I WOULD LIKE TO HEAR FROM THE PANEL ABOUT IS I THINK A LOT OF TIMES WE THINK THIS IS A BARRIER AND THEREFORE ANY TIME IT'S OPEN, IT'S BAD. OBVIOUSLY THAT'S NOT THE CASE WHAT NEEDS TO HAPPEN TO BETTER UNDERSTAND WHEN THE GATE NEEDS TO BE OPENED VERSUS WHEN IT NEEDS TO BE CLOSED? >> MAY I SAY ONE THING, THEN I WILL HAVE MY COLLEAGUES ALSO TO RESPOND. WHEN WE TALK ABOUT THE (INDISCERNIBLE) ON DEMAND, THIS IS THERAPEUTIC OPENING, WE TALK ABOUT CHRONIC OPENING THE BARRIER, RIGHT? BUT OPEN BARRIER NEUROLOGICAL DISEASE ARE TUMOR DOESN'T MEAN YOU CAN DELIVER EVERYTHING. YOU HAVE P GLYCO PROTEIN RECEPTOR MDR 1 GENE, YOU START TRYING DRUGS IN AND THE NDR IS TRYING TO PULL IT BACK IN CIRCULATION. SO THIS IS JUST TO LITTLE BIT OF BE CAREFUL ABOUT TERMINOLOGY HOW WE'RE GOING TO CALL IT. THE OPENING ON DEMAND IMPLY HOW TO REGULATE AND CONTROL IT WHICH ALSO HAS IMPLICATIONS IN CLOSING ON DEMAND. PANNED I THINK IN A LOT OF THERAPEUTIC SETTINGS NOT ONLY DO YOU HAVE ACTIVE AGENT BUT YOU HAVE CONCEPT OF A REVERSAL AGENT. THE THERAPY HAS AGAINST TIME OF OPERATION AND IF YOU' GOING TO OPEN THE BLOOD BRAIN BARRIER BY SOME SPECIFIC INTERVENTION YOU MAY WANT TO HAVE ENOUGH CONTROL ALSO WOULD BE SIMILAR AMONG DIFFERENT INDIVIDUALS. SO IT IS POSSIBLE THAT SOME PEOPLE MIGHT BE MORE SUSCEPTIBLE TO INFLUX OF BLOOD PROTEINS THAN OTHERS. FOR EXAMPLE, WHEN THERE'S BRAIN TRAUMA, MANY TIMES YOU HAVE -- YOU SEEK A BRAIN TRAUMA, SPORTS ACCIDENTS THAT LEAD TO DIMINUTION OF THE BRAIN AND DOESN'T DEVELOP TO BECOME A MESS. THESE REPAIRS PERFECTLY AND THE PATIENTS NEVER HAVE ANY OTHER INCIDENTS, RIGHT? SO OBVIOUSLY THERE IS A COMBINATION OF OPENING THE BARRIER WITH GENETIC SUSCEPTIBILITY WHETHER THEY BE AUTOIMMUNE DEGENERATIVE DISEASE OR AMYLOID DRIVEN DEGENERATIVE DISEASE. SO I THINK SOME UNDERSTANDING OF DRIVERS OF SUSCEPTIBILITY THAT WE DON'T FULLY KNOW RIGHT NOW,TY STRATIFY PATIENTS TO OPEN BARRIER VERSUS OTHERS THAT COULD BE A DETRIMENTAL EFFECT. >> JUST BRIEFLY, PROBABLY THINK TWO TERM. VASCULAR PLASTICITY AND NEURALPLASTICITY, THERE A PERIOD OF TIME WHICH VASCULAR SYSTEM CAN REGENERATE AND GET BACK AND ALSO NEURONS. BUT THIS IS NOT FOREVER. SO THIS WINDOW IS STILL DON'T KNOW WHAT IT IS, WE KNOW IN ANIMAL STUDIES. WHEN THE BARRIER IS OPENED, ONE HAS TO BE CAREFUL THAT EVEN DURING THAT DURATION, SOME HARM DOESN'T TAKE PLACE. WE USE IN THE LABS AND MANY OTHERS FOR DIFFERENT PURPOSE TO INJURE THE BRAIN. SO THE -- DEPENDS ON SIGNAL,TY PENDS ON AMPLITUDE SO THAT'S ALWAYS KIND OF STILL IN A NAYSANT STAGE BECAUSE WE DELIVERED TWO, THREE, FOUR SESSIONS AND WE GET BEAUTIFUL LOTS OF NEURONS DEGENERATION BUT WE USE HIGH AMPLITUDE. SO THAT'S ALL OF COURSE WHEN YOU TRANSPLANT LAIT TO HUMANS BECOMES EVEN MORE PROBLEMATIC. IS >> SO THE QUESTION I WOULD LIKE TO A ADDRESS, MAYBE IT'S BROAD BUT WE HAVE A PROBLEM WITH CHRONIC DISEASES LIKE ALZHEIMER'S DISEASE. SO NOW THAT WE BELIEVE THE BLOOD BRAIN BARRIER IS OPENED WE CAN SET UP THE NEUROPATHOLOGICAL EVENTS THAT TAKE TIME TO DEVELOP OR EVEN FUNCTION IN THE NDU EVOKE THOSE PATHWAY BUS THE WE IS WE -- PATHWAYS BUT WE STILL LACK THE INITIATING EVEN. BUT SUBMIT NOW THAT WE'RE USED TO THE IDEA OF A BLOOD BRAIN INTERFACE, WE CAN BEGIN TO GET AROUND THAT PROBLEM. AS AN EXAMPLE WE CAN THINK OF TRAUMATIC BRAIN INJURY. MOST ARE TBBI. THEY'RE TRAUMATIC BRAIN AND BODY INJURY. ONE CAN REMEMBER THAT IN THE GULF WAR, THE TBIs CAUSE OF DEATH WAS USUALLY LUNG HEMORRHAGE BECAUSE THAT WAS THE MOST SENSITIVE TISSUE. NOW THAT WE HAVE THE VESTS TO PROTECT FOLKS NOW WE'RE BEGINNING TO SEE MORE TBI. BUT THE POINT IS, THAT PERIPHERAL TISSUES ARE BEING ENTERED INCLUDING THE GAP WHICH COULD BE SOURCE OF LPS TO BLOODSTREAM. WE HEARD SPEAKERS TALK ABOUT THAT HAPPENING EVEN WITH JOGGING TODAY. SO NOW WE HAVE INFLAMMATORY EVENTS GOING ON IN THE PERIPHERY. WE KNOW INFLAMMATORY EVENTS PRODUCE MANY CHANGES IN ALZHEIMER'S DISEASE SO I WOULD SUBMIT THE NEXT STEP TO BEGIN THINKING ABOUT PERIPHERAL INFLUENCE ON WHAT ALTERS NDU IN BLOOD BRAIN BARRIER PERMMENT. >> THE FIRST THING I WAS GOING TO SAY, WE HAVE GREAT COMMENTS A FEW MINUTES AGO ABOUT THE BLOOD BRAIN BARRIER OPENING CLOSING WHAT MAYBE DIRECTING IN TERMS OF GENETIC INFLUENCES. WE NEED TO ALSO LOOK AT THERAPEUTIC INFLUENCES. WE HAVE SIMPLE WAYS TO OPEN UP BUT CAN WE DO BETTER? CAN WE AIM FOCALLY, CAN WE FLIP A SWITCH, TUSH IT ON AND OFF ON DEMAND AND HOW QUICK WE CAN DO THAT, THAT APPLIES TO EVERYTHING, TRAUMA, STROKE, TUMORS, DEGENERATIVE DISEASE AND THE OTHER COMMENT ABOUT THE RELATIONSHIP BETWEEN BODY AND BRAIN IS ON TARGET. MORE AND MORE I THINK WE'RE REALIZING THAT NEUROCRITICAL CARE, THERE WAS A GREAT EDITORIAL A FEW MONTHS AGO JOURNAL OF AMA BY DAVID MENON AND RON DISASTER WHO POINTED OUT THE SAME THING, THEY NEED TO TAKE A MUCH MORE INTEGRATIVE VIEW, STROKE PATIENTS HAVE BEEN TREATED SEPARATELY FROM THE REST OF THE BODY. TRAUMATIC BRAIN INJURY, DIFFERENT OTHER TRAUMA PATIENTS SO WE NEED TO GET AWAY FROM THAT, THE BLOOD BRAIN BARRIER IS AN IDEA INTERFACE TO LOOK AT THE RELATIONSHIP BETWEEN THE BRAIN AND REST OF THE BODY. >> I'M INTRIGUED BY THE DEGREE AS WELL AS THE KIND OF BLOOD BRAIN BARRIER COMPROMISE. IN SYSTEMIC CIRCULATION OBVIOUSLY CELLS CAN EGRESS OUT OF THE VASCULAR SPACE BY DIE YAPDESIS LIKE INFLAMMATORY CELLS WHERE YOU HAVE JUST ONE ENDOTHELIAL BARRIER WITH REGULATABLE TYPE JUNCTIONS. WE HEARD THE BAD THINGS THAT HAPPEN WHEN RED CELLS GET IN BRAIN, IRON BEING STRESS AND STUFF. WE TALKED SMALL MOLECULES, WE TALKED LARGER MOLECULES, NIGH WRITTEN GENERAL, NANOPARTICLES. SO IS THERE A GOOD UNDERSTANDING, WE KNOW PARASITES ARE AN ADDITIONAL LAYER, BEYOND ENDOTHELIUM THAT YOU DON'T HAVE IN SYSTEMIC CIRCULATION. BUT IS THERE ANY SENSE OF SERIES OF GATES THAT COULD BE IN A NON-INJURY STATE REGULATED TO ALLOW THE THINGS TO PASS WHETHER EXOSOMES, WHETHER SMALL MOLECULES NANOPARTICLES, WHATEVER, LIKE TRAUMA AND BRAIN INJURY YOU HAVE COMPLETE OBLITERATION OF ENDOTHELIAL SURFACE IN THE INJURY AREA, PLUS THE PARASITES PRESUMABLY DAMAGE AS WELL, SO THERE'S ESSENTIALLY EGRESSION OF CELLS, PROTEINS, AND CYTOKINES ALL AT ONE FELL SWOOP. >> I'M SUPPOSED TO ANSWER QUESTIONS BUT I HAVE A QUESTION AND THAT RELATES TO WHAT YOU JUST SAID. WHAT ABOUT EXOSOMES. ALL CELLS PRODUCE THEM, NOT JUST DISEASE CELLS SO PRESUMABLY THEY PLAY A PHYSIOLOGICAL ROLE. THE QUESTION I'M ALWAYS THOUGHT THAT EXOSOME IS SORT OF FREE THROUGH THE BLOOD BRAIN BARRIER OR SHUFFLED THROUGH TAKEN OUT BY ENDOTHELIAL CELLS OR PARASITES AND THEN WHAT IS THE STORY? I DON'T KNOW. >> I CAN ONLY ANSWER FROM SYSTEMIC CIRCULATION, I'M NO EXPERT ON BARRIER, I'M TALKING TODAY AND TOMORROW. BUT CERTAINLY THERE, THE GATING CAN HAPPEN PRETTY -- PROBABLY IN PHYSIOLOGIC AND PATHOLOGIC STATES BOTH. AND IS PROBABLY RELATED TO OTHER TYPE CELL ADHESION MOLECULES THAT LINK ENDOTHELIUM TOGETHER AND THEY CAN BE REGULATED FROM DNA OF THE ENDOTHELIUM ITSELF TO SIGNAL TO ALLOW JUNCTIONS TO COME AND GO BUT I DON'T KNOW IF THAT'S ANALOGOUS COMPLETELY TO WHAT HAPPENS IN BLOOD BRAIN BARRIER BUT I'M SPECULATING. >> I THINK GREET POINT ABOUT SELECTIVE OPENING OF THE BLOOD BRAIN BARRIER. I THINK THAT GENETIC MODELS OF DIFFERENT TYPE JUNCTION OF MICE HAVE SHOWN THIS IS POSSIBLE. SO FOR EXAMPLE, KNOCK OUT FOR CLONING, HAVE A DIFFERENT PERMEABILITY SO WHEN DECKS TONS USED FROM TEN KILODALTON TO 100 KILODALTON, WE RECALL THE CODING, THE 40 KILODALTON, SO IT'S POSSIBLE THAT BY MANIPULATING SPECIFIC MOLECULES TIGHT JUNCTION INTERFACE YOU MAY CREATE THOSE. SO THERE MIGHT BE SOME APPLICATION THERE BUT KNOWLEDGE FROM A PHARMACOLOGIC TOOL THAT MIMIC THE GENETIC MODELS. >> OTHER THING TO REMEMBER, I'M NOT SURE HOW CREATIVE THE INTERACTION OF MICROVESICALES AS A WHOLE WITH ENDOTHELIUM ITSELF, SO WE ASSUME THE BLOOD BRAIN BARRIERS OPEN OR CLOSED WHEREAS MICROVESICLES AND WE STARTED TO LOOK AT HOW THEY ARE ACTUALLY TAKEN IN TO THE ENDOTHELIUM AND DEPENDINGEN WHAT'S ON THE MICROVESICAL CAN AFFECT THAT ENDOTHELIAL FUNCTION DIRECTLY ALLOWING FOR THE OTHER MICROVESICALES TO EGRESS ONE WAY OR THE OTHER. >> COULD I MAKE A COMMENT? I DON'T THINK WE SHOULD TALK ABOUT EXOSOMES AS IF THEY WERE A SINGLE ENTITY. BECAUSE I THINK THAT SOME OF THIS HAS COME OUT TODAY AND I WILL SHOW YOU DATA TOMORROW WITH MACROPHAGE DERIVED EXOSOME THEY ECHO OUT LIKE MACROPHAGES SO THEY'RE PROBABLY ALL GOING TO BE IF THERE'S A THEME AND TRANSSIGH TOTTIC LIKE MECHANISM, IT'S NOR DIE DEBTIC THE CLOSER TO INMINE CELL PROBABLY USING OTHER MECHANISMS IF WE PUT OTHER THINGS, WHICH MEANS HOPEFULLY WE CAN BEGIN TO TARGET THEM AT LEAST IN THE VASCULATURE VARIOUS BRAIN REGIONS. (OFF MIC) (LOST AUDIO)) LOST AUDIO) (LOST AUDIO) WITHIN THE SAME BRAIN HAVING DIFFERENT PERMEABILITY TO DIFFERENT SIZE DEXTRANS SO IT IS A HETEROGENEOUS PHENOMENON AN SITE SELECTIVE PHENOMENON. WHEN TALKING MICROVESICLES I ECHO THE COMMENTS THAT WE HEARD. 'S INTERESTING TO THINK ABOUT NOT ONLY IN THE DISEASE BRAIN BUT IN A FUNDAMENTAL SENSE IN NORMAL BRAIN WHAT IS THE ROLE OF MICROVESICAL SIGNALING VEESIA THE NEUROVASCULAR UNIT AND SINCE IT'S NOT WIDE OPEN, HOW DO THESE THINGS TRAFFIC AND HOW THEY SIGNAL TO THE PERIPHERY OR VICE VERSA. THAT MIGHT LEAD TO INTERESTING INSIGHTS WHAT CONSTITUTE DISEASE. >> (INDISCERNIBLE) I HAVE A QUESTION ABOUT ANY STANDARD TECHNOLOGY OR PROCEDURE ABLE TO QUANTIFY CLOSE AN OPEN THE DB BARRIER GATE? >> YES. >> GREAT. SO THE OTHER ONE -- >> DCMRI, THE LAST ONE, HUMAN BEING IN ANIMAL MODELS, EVEN HUMAN BRAIN. >> I NEED TO TALK TO YOU AT DINNER. (INDISCERNIBLE) HUMAN OBESITY, MUCOSAL BARRIER IS -- WE CALL PERMEABILITY, WITH HIGH FAT DIET, (INAUDIBLE) OBESITY. AND YOU -- THAT LEADS TO MASS EFFECT. THAT ALSO HIGH FAT EJECTION FUNCTION PROTEIN AS WELL. SO WE CAN MEASURE THAT IN LIKE MICE TO QUANTIFY THE OPEN AND CLOSE CAN CHANGE OVER TIME COURSE, THAT WOULD BE GREAT. >> THANK YOU VERY MUCH. >> I THINK ON THAT, IT SORT OF GOES I GUESS TO THE NEXT SESSION ABOUT BIOMARKERS BUT I THINK IN IMAGING MODALITY IS NICE BUT CLINICALLY YOU WON'T RUN A PATIENT DOWN TO SAME IMAGING MODALITY EVERY FEW HOURS TO SEE SO THE OTHER CHALLENGE, IS THERE A SERUM BIOMARKER IDENTIFIABLE OF OPEN CLOSE VARIABLE STATE OF ENDOTHELIAL DYSFUNCTION SPECIFIC TO THE BRAIN. YOU CAN LOOK AT GENERALIZED THINGS LIKE THE DEGRADING OF GLYCO KALIX BUT I DON'T THINK ESPECIALLY IN THE POLYTRAUMA PATIENT THAT WAS NEXT MENTIONED, THAT'S NOT SPECIFIC TO THE BRAIN SO HOW DO YOU TELL IF THE POLYTRAUMA PATIENT BRAIN BARRIER DYSFUNCTION OR NOT. >> QUESTION WAS BROUGHT UP SELECTIVE AND TIMED OPENING OF BLOOD BRAIN BARRIER IS IMPORTANT AND LEAD US ON A STUDY AND UNDERSTAND A LITTLE BIT ABOUT WHAT THAT DEFECT IN THE BLOOD BRAIN BARRIER DOES. WHAT ABOUT AREAS WHERE THERE IS NO BLOOD BRAIN BARRIER? CAN THOSE BE POTENTIAL AREAS WE CAN STUDY FOCUS MORE ATTENTION ON EXTERNAL STIMULI FOR EXAMPLE AND WHETHER YOU GUYS ARE EXPERIENCED IN THAT. >> THESE TRADITIONALLY CALLED VINTRICULAR ORGANS. SUPER OPTIC NUCLEUS OR EVEN CHOROID PLEXIS. SO THEY'RE -- THEY'RE ABOUT SIX OR SEVEN (INDISCERNIBLE) THESE LEAD THE MOLECULES TO GRADUALLY GOING -- NOT EXPLORED RECENTLY BUT WORK BY JERRY COSLOSKY WHO SHOW THAT IGG SLOWLY PENETRATE AND HAVE EXCHANGES WITH THE BRAIN. BUT WE HAVE TO KEEP IN MIND THAT THERE ARE RECEPTORS LEAK S -- FCRS AND OTHERS THAT PULL THAT OUT. H COMES IN SO MUCH WHEN YOU GET DISTRIBUTED -- IT IS ALL ABOUT THE MAXIMUM CAPACITY WE PULLED OUT. SO I DON'T THINK THEY REALLY CONTRIBUTE AS A POSSIBILITY FOR TARGETED DELIVERY. A LOT OF IT IS CLEANED OUT. AND IF IT -- IF THAT WOULD BE A WAY, WE WILL SHORT OUT THE WAY FOR DECISIONS. >> SORRY, (INAUDIBLE) BANKS FROM SEATTLE. THAT'S HOW YOU REMEMBER AND RODRIGUEZ FINALLY DEFINED WHAT THAT BARRIER WAS, THAT INTERFACES BETWEEN THE CVO AND THE NEURAL -- SO IT DOESN'T SEEM THAT THAT IS A SOURCE FOR LEAKAGE THOUGH THERE'S SOME OLDER DATA SUGGESTING THAT SUBSTANCES CAN GET THERE AND THIS WE NOW KNOW ENDOTHELIAL CELL IS CAN RELEASE OTHER SUBSTANCES LIKE PROSTAGLANDINS, THIS MAYBE A MECHANISM, TO TRANSDUCE FEVER FOR EXAMPLE IN CERTAIN CIRCUMSTANCES. BUT I THINK IS DRUG DELIVERY. SO PEOPLE ARE VERY CORDIAL SO I WILL BE NASTY AND SAY I HAVE PROBLEM WITH THE IDEA OF DISRUPTING THE BLOOD BRAIN BARRIER IN THE CLASSIC SENSE THAT WE'RE TALKING ABOUT EVEN MINUTELY BECAUSE NO MATTER HOW SMALL YOUR DRUG IS, IT'S PROBABLY GOING TO BE NOT THAT MUCH SMALLER EVEN BIGGER THAN A MONOAMINE. WE DON'T WANT MONOAMINES IN THE BRAIN. I DON'T SEE HOW WE CAN DO CLASSIC BLOOD BRAIN BARRIER DISRUPTION AND NOT RESULT IN TOXICITY INCLUDING INTRODUCTION INFLAMMATION. SO I GET NERVOUS ABOUT THIS. >> THANK YOU, BILL. >> WE SHOULDN'T DISRUPT THE BLOOD BRAIN BARRIER TO SYSTEM UK CIRCULATION. NOT TALKING THE WAY YOU DO IT, YOU DO IT RIGHT BUT THE IDEA OF DISRUPTING MOST OF THE TIME. DISRUPTING BLOOD BRAIN BARRIER WITH DRUG OR CHEMICALS OR DISSOLVING AN ALLOWING THINGS TO LEAK IN BECAUSE A LOT OF THINGS THAT THE BLOOD BRAIN BARRIER WAS MEANT THE KEEP OUT ARE GOING TO LEAK IN AS WELL. >> I AGREE WITH YOU BUT REMEMBER WAY THIS CONVERSATION IS EVOLVED GET BACK TO COMMENTS WE HEARD, TRADITIONAL WAY TO QUOTE DISRUPTING UNQUOTE THE BLOOD BRAIN INTERFACE IS A BLUNT INSTRUMENT AND I THINK WE'RE TALKING THE POSSIBILITY TO BE MORE REFINED AND EVEN SOME OF THE SHOWING ON MY TALK WHY ARE SOME MARKERS OF INJURY HIGH RIGHT AWAY AND THEN DOWN AND OTHERS DON'T APPEAR UNTIL 24 HOURS LATER? IF I THINK THERE'S A LOT THAT WE OBVIOUSLY DON'T KNOW ABOUT DIFFERENTIAL TRANSPORT MECHANISMS DIFFERENT TYPE SIZES OF MOLECULES, TO PRODUCE THOSE. SO THAT'S KIND OF WHERE WE'RE GOING IS REALIZE THAT OUR BLUNT INSTRUMENT MAYBE FINE IN OCCASIONAL CASES BUT WE CAN DO BETTER THAN THAT. >> WE HAVE TIME ONLY FOR ONE MORE QUESTION. OR COMMENT. >> MAKE I CAN PROVOKE THE DEFINE IN BLOOD BRAIN BARRIER WHEN YOU WANT THINGS TO GET IN, IT DO DOESN'T LIKE YOU WHEN YOU DON'T WANT THEM IN. RIGHT? (OFF MIC) >> OKAY. THANK YOU VERY MUCH. WE HAVE NOW SPACE FOR THE MEMBERS OF PRESENTERS OF THE BLOOD SESSION NUMBER 2. SO ARE WE READY TO QUESTIONS, COMMENCE TO THE MEMBERS OF SESSION NUMBER 2? DR. CROUDER YOUR MICROPHONE. >> THANKS. SO I'M GOING TO TURN THIS OVER TO THE SPEAKERS. I'M JUST A NEUROSCIENTIST PROGRAM MANAGER AND M RNC. THIS PANEL WANTS TO START WITH THE END IN MIND WHICH IS IMPROVING OUTCOMES. WHAT ARE OUR TOP NEEDS, WHAT ARE OUR TOP OR MOST CHALLENGING BARRIERS AND WHAT ARE THE LEAST CHALLENGING BARRIERS OR LOW HANGING FRUIT, IS THAT SOMETHING WE SHOULD GO AFTER FIRST? WHAT OPPORTUNITIES ARE OUT THERE. I LOVE THE SECOND OPPORTUNITY WHICH IS NOT ON THIS PAGE BUT IT WILL COME UP WHICH IS ESTABLISHMENT OF A DOD INTERAGENCY BLOOD BRAIN BARRIER SPECIAL INTEREST GROUP. TASKED WITH SYSTEMIC RESPONSES TO BARRIERS IDENTIFIED BY THE CONFERENCE IN ASSOCIATED WITH THE JOINTLY AUTHORED PEER REVIEW PUBLICATION. WE JUST HEARD A PERFECT EXAMPLE OF THIS, WE CAN'T POSSIBLY DO JUSTICE TO THE FEEL OF OR STATE OF THE SCIENCE OF BLOOD BRAIN BARRIER TODAY. A PERFECT EXAMPLE SHOULD WE DISRUPT THE BLOOD BRAIN BARRIER OR NOT. IT'S SOMETHING THAT COULD BE INCLUDED IN A POSITION PAPER AND WE GOT TRULY THE GREATEST MINDS IN BLOOD BRAIN BARRIER RESEARCH IN THIS ROOM, IF Y'ALL FORMED AN INTERAGENCY SPECIAL INTEREST WORKING GROUP I CAN SEE DOING GREAT THINGS. I'M GOING TO TURN IT OVER TO Y'ALL AND THE REST OF THE PANELISTS. >> I WANT TO ADD THE COMMENTS DR. CROUDER JUST REFERRED TO ARE IN YOUR HAND OUTS, YOU HAVE A HAND OUT WITH YOUR PROGRAM BOOKLET SO REFER TO THE THIRD PAGE BECAUSE WE WEREN'T ABLE TO FIT THE POINTS ON THE SLIDE DECK. >> THEY HAVE A LOT OF IDEAS. >> WELL I WILL SAY THIS, WHAT DO YOU THINK ABOUT THE IDEA OF FORMING A SPECIAL INTEREST WORKING GROUP? FOLKS IN THE ROOM THAT WOULD BE INTERESTED IN THAT? GO ALEX. >> IT IS A GREAT IDEA. TRY FOG GET MY ARMS AROUND THAT OR MY BRAIN AROUND THAT. WE HAVE DIVERSITY OF INTEREST IN THIS ROOM, WE HAVE HEARD VERY ELEGANT TALKS ABOUT VERY SPECIFIC TYPES OF EXOSOMES OR VESICALES, THEN WE HEARD CLINICALLY ORIENTED TALKS ABOUT NEEDS OF INJURED SERVICE MEMBERS IN THE FIELD. HOW DO YOU DESIGN SOMETHING WHERE YOU CONTINUE TO KEEP THE HATFIELDS AND MCCOYS TALKING TO EACH OTHER WITHOUT THIS DEGENERATING TO A BUNCH OF LITTLE CAMPS. THAT'S GOING TO BE A MAJOR NEED. FROM (OFF MIC) >> START -- START WITH E IN MIND THAT WORKS BEST. (OFF MIC) # >> CAN WE USE THE MIC. >> UNIVERSITY OF CHICAGO. YOU ALREADY HAVE -- HOW DO YOU ADVANCE THIS THIS FIELD AND GET THE BRAINS TOGETHER? AND MAKE A CLINICAL END POINT. WHAT ARE THE NECESSARY STEPS TO MAKE EXOSOMES OR EXTRA CELLULAR VESICALES A CLINICAL TREATMENT PHENOMENA, WHAT DO I NEED TO DO. I TELL YOU THE FIRST ANSWER YOU ALREADY RECOGNIZE, I DON'T WANT TO MAKE IT SELF-SERVING BUT IT WILL SOUND THAT WAY, SIX OR SEVEN TALKS SHOWED THAT INTRANASAL DELIVERY OF EXOSOME CLEARLY MAKE IT TO THE BRAIN AND HAVE FUNCTIONAL SIGNIFICANCE. YOU DON'T NEED TO FIGHT ABOUT IT. BUT FIGHT ABOUT IT WHEN THE SCIENCE COMES TO BE. BUT THE OPTIMAL ROUTE INTO THE NERVOUS SYSTEM FOR NANOPARTICLES IS INTRANASAL. NEXT IS HOW BLOOD CELLS YOU'RE GOING THE MAKE, WHAT ARE YOU GOING TO TREAT AND WHEN YOU BEGIN TO TALK TO THE FDA THEY ASK YOU QUESTIONS LIKE PICK THE BIGGEST TARGET OR MOST LETHAL DISEASE YOU CAN HAVE THE BIGGEST IMPACT. WOULDN'T THAT BE TRAUMATIC BRAIN INJURY? THOSE ARE THE -- >> I WAS GOING TO SAY THOSE ARE GREAT COMMENTS BUT IN A WAY YOU MADE MY POINT, YOU'RE GETTING STUFF INTO THE BRAIN, I WANT TO MEASURE THE STUFF COMING OUT OF THE BRAIN. >> THERE'S TWO WAYS OUT OF THE BRAIN. ONE -- 50%, THE OTHER -- ONE IS THE -- INTACT CELLS GET OUT EASILY WITHIN TWO OR THREE DAYS YOU CAN MAKE THE CSF INTO CRE WITH LYMPHOCYTES. AND THE ANIMALS WILL BE FINE, THE CELLS WILL GET OUT GRANULATION WITHIN 24 TO 48 HOURS. SO THE BLOOD BRAIN BARRIER IS REALLY FROM THE OUTSIDE IN. THE OTHER EXOSOME FLUX PATHWAY, YOU CAN IMAGE THE WHOLE THING. YOU CAN IMAGE THE GLYCO LYMPHATICS, WHICH GO RAPIDLY AS AS WAS SHOWN DOWN PARALYMPHATIC TO THE DEEP CERVICAL NODE. NOT THE SUPERFICIAL NODES OR ANYWHERE ELSE. YOU CAN IMAGE THAT. WITH REGARD TO TRAUMA, ANATOMIC IMAGING DOESN'T HAVE IT. AS WAS POINTED OUT BY NEUROSURGICAL COLLEAGUE. IT IS THE MICROCIRCULATION THAT CAUSES NEURONIC DEFICITS. YOU CAN MEASURE THOSE USING NEW TECHNOLOGY CALLED STEADY STATE. IT IS A WHOLE ORDER OF MAGNITUDE MORE SENSITIVE THAN DSE WHICH IS -- YOU HAVE TO DO ON A SINGLE PATH WHERE STEADY STATE WITH BLOOD POOL FERMOXITOL THE DIFFERENCE IS NIGHT AND DAY. YOU CAN LOOK AT THE INFLAMMATION OF CRANIAL CEREBRAL TRAUMA, ACUTELY AND CHRONICALLY OR OF OTHER DISEASES INCLUDING ALZHEIMER'S DISEASE BECAUSE THESE NANOPARTICLES ARE VERY SPECIFICALLY FOR CD 11 BC AND CD 68 CELLS. WE HAVEN'T HEARD ABOUT M RWITH REGARD TO ALL THESE THINGS. AND THERE'S SO MUCH TECHNOLOGY, BENEFIT GOT STUFF WITH DCE ALSO WHICH IS PERMEABILITY BUT THE PROBLEM WITH PERMEABILITY IS YOU HAVE TO HAVE NORMALIZED VASCULATURE. THE NEURORADIOLOGISTS CALL PERMEABILITY K TRANS WHEREAS STANLEY RAPAPORT TALK ABOUT PS PRODUCT BUT PS PRODUCT DOESN'T CORRELATE WITH IT UNLESS YOU YOU HAVE A NORMALIZED VASCULATURE, IF YOU HAVE AN NORMAL K TRANSDOESN'T WORK. >> SO BACK TO KRAIG'S COMMENT ABOUT BEST WAY IN IS THROUGH NASAL ADMINISTRATION. FROM A COMBAT CASUALTY CARE PERSPECTIVE ESPECIALLY ATTRACTIVE, FOR ME THAT'S WITH THE END IN MIND. WE KNOW WHAT THE GOAL NOW IS AND NEXT AREA OF OPERATION IS UNLIKELY TO BE RELATIVELY CLOSE TO COMBAT SUPPORT HOSPITALS AND GOAL MIGHT BE 72 HOURS. FIRST RESPONDERS CAN'T START -- THEY CAN, THEY'RE CAPABLE OF STARTING IVs BUT NOT IN THE FIELD BECAUSE OF RISK OF INFECTION, SO NOW LET'S GO TO PENNED DOWN FOR UP TO 72 HOURS INTRANASAL ADMINISTRATION OF THERAPEUTIC TO HELP TO PREVENT OR MITIGATE SECONDARY BRAIN INJURY FOR US IS SUPERRER ATTRACTIVE. YOU GO TO THE FDA WITH THAT, APPROACH COMBAT CASUALTY CARE WITH THAT PROPOSAL, WE'RE ALL OVER IT. >> THAT'S OOH ALL DONE IN SMALL ANIMALS. ONE OF THE REASONINGS ALL THESE BRILLIANT STROKE THERAPIES HASN'T WORKED IS YOU'RE DEALING WITH TINY BRAIN. AND WHEN YOU GET BIG ANIMALS LIKE WOMEN BRAIN 100-GRAMS BIGGER THAN MEN, IT'S A DIFFERENCE PROBLEM. I DON'T KNOW ANYONE -- WE CAN'T GET THE KINDS OF DISTRIBUTION THAT I HAVE SEEN TODAY WITH EXOSOMES WITH NANOPARTICLES THROUGH THE NOSE. HAS ANYBODY DONE IT IN LARGE ANIMAL? >> THEY HAVEN'T DONE IT YET. BUT I DON'T WANT TO LIMIT Y'ALL'S INGENUITY OR THE INDUSTRY PARTNERS OUT THERE. >> IS IT'S A NEW WORLD. >> JUST TO FOLLOW-UP, MRI IS GREAT, AGAIN STARTEDDED WITH THE END IN IN MIND AS TAMMY SAID IT MAY NOT BE REAL iIC FOR THE IMMEDIATELY INJURED PERSON ON A BATTLE FEEL BUT CONCEIVABLY GET THEM TO TERMINATE THE NEXT CONFLICT IS GOING TO BE MIDDLE EAST SOMEWHERE, THAT CAN MAKE MORE HELP ON THE CIVILIAN SETTING IF YOU CAN GET A MULTIPLY INJURED UNSTABLE PATIENT TO A MAGNET, THAT'S HARD ENOUGH. BUT THAT WOULD BE THE END IN MIND. SO IF WE WANT TO TRY SOMETHING LIKE THAT, I THINK LARGE ANIMAL MODELING IS GOING TO HAVE TO GO AND WORKING BACKWARDS, WHAT NEEDS TO BE DONE. BECAUSE A LOT OF THINGS IN THE LAB HAVE NOT PANNED OUT AT ALL, CLINICALLY TAKING THE TRAUMA PATIENTS. >> I LIKE THIS NOTION OF LEAVE ON A POSITIVE NOTE. THERE ARE COMMERCIAL AND ACADEMIC UNITS IN THE COUNTRY NOW THAT WILL MODEL ENTRY OF LARGE MOLECULES ALL THE WAY THE NANOPARTICLES INTO THE BRAIN. I CITED FOR YOU WORK THAT WAS DONE IN THE MONKEY BRAIN, IT SAYS WITHIN 30 MINUTES RADIO ACTIVE INTERFERON GAMMA 150 KILODALTON MOLECULE IN THE CENTER OF THE BRAIN WITHIN 30 MINUTES. THE WORK HASN'T BEEN DONE, IT HASN'T BEEN DONE WITH IMMUNE CELLS, IT NEEDS TO BE DONE AND THE EXCITEMENT AT BEING ABLE TO EFFECT BRAIN FUNCTION IS TRAUMATIZED. WITH A NASAL AGENT, I THINK IS SOMETHING THAT SHOULD GET PEOPLE EXCITED. >> I THINK WE HAVE A NUMBER OF PEOPLE WAITING BY THE MICROPHONE AND TO SUMMARIZE THE PREVIOUS DISCUSSION OF NASAL DELIVERY, I CAN SAY ON A QUESTION TO BETTER GO THROUGH THE BLOOD BRAIN BARRIER, THEY HONESTLY GO AROUND IT. PLEASE. >> MIKE FROM CINCINNATI, A SIMPLE TRAUMA SURGEON SO LET ME START WITH THE SIMPLE PRESENTATIONS UP THERE. I'M NOT EVEN A NEUROSURGEON. SO ESPECIALLY DR. JENSON, I THINK THE IDEA OF FAR FOLD AND GIVING A THERAPEUTIC INTRANASALLY, WHAT HAVE YOU IS GREAT. YOU HAVE TO MAKE THE DIAGNOSIS FIRST. ARE WE THERE YET WITH THE SERUM BIOMARKERS AND IF NOT, FROM YOUR PERSPECTIVE, WHAT IS IT GOING TO TAKE TO HAVE THAT BEDSIDE URINE PREGNANCY TEST TO SAY NEUROLOGIC INJURY, YES, NO. IBILITY HOW MUCH? DOES IT MATTER? AND CAN WE FOLLOW ITSELF SERIALLY WITH THE SAME BIOMARKERS? >> VERY BRIEFLY, COUPLE OF MARKERS UCHL # AND GFAP ARE SOON HOPEFULLY SOON TO BE FDA APPROVED FOR TESTING TBI AND THE COMPANY BEHIND THAT IS LARGER COMPANIES AT BEDSIDE FINGER STICK TYPE TESTING AVAILABLE. SO NOT PERFECT, IT MAY HAPPEN SOON, I KNOW NUMBERS HAVE BEEN INVOLVED WITH HOE DIANE AND RAMONE. >> LET ME COMMENT QUICKLY, THAT TALKING ABOUT MODERATE AND SEVERE INJURY ARE NOT SO MUCH TALKING ABOUT CONCUSSION WHICH IS 80% OF TBI IN MILITARY. AND THAT'S THE SERVICE MEMBER YOU WANT TO IDENTIFY, THE GUY THAT MIGHT HAVE HAD A CONCUSSION DON'T WANT TO SEND HIM BACK TO BATTLE TOO SOON. DAVE TELLS US MULTIPLE CONCUSSIONS CAUSE A REAL PROBLEM FOR THIS GUY. THAT'S MY CONCERN ABOUT THE BIOMARKERS UCHL 1 AND GFAP. ANY OF THOSE, THEY'RE NOT GOOD FOR IDENTIFYING CONCUSSION. >> U THINK THOSE THINGS DO HAVE POTENTIAL. BUT I THINK CERTAIN THINGS ARE MISSING. I DON'T KNOW IF SOMETIME WHEN I SEE SOMETHING FROM SOME OF THE FOOTBALL PLAYERS OR SOME OF THE OTHER STUDY WE CAN DO, IT'S WHERE THEY WERE HIT AND HOW THEY WERE HIT THAT WE SEE SOMETHING. IT STILL NEEDS TO BE WORKED OUT HOW INFORMATION GETS THERE AND MAKE IT LESS VARIABLE. SO MY BIGGEST CONCERN, I SEE VARIABILITY. (OFF MIC) >> PLEASE TAKE THE MIC. >> SORRY. THOUGHT I WAS LOUD ENOUGH. A QUICK FOLLOW-UP ON THAT. SO GREAT WORK WITH ASU FOOTBALL PLAYERS THEY TAKE HE INJURY AND A LOT OF BODY INJURY. SO DO YOU HAVE ANY WORK ON ISOLATED HEAD INJURY BOXERS THAT ARE ONLY TAKING REPEATED HEAD INJURIES? >> YOU CAN CONTROL IT IN A BOUGHT IF YOU PUT THEM THERE AND DIDN'T HAVE ANY -- NO BODY SHOTS. >> WE DON'T HAVE ANYTHING LIKE THAT YET. WE DO HAVE A LOT OF TRACK AND FIELD CONTROLS WHO EXERCISE AND DO OTHER THINGS THAT THEY DON'T GET THEIR HEADS IN BUT NOTHING TO REALLY CONTROL FOR THE OTHER HITS THEY'RE GETTING, TO ME AND OTHER INJURIES, I'M HOPING THAT'S A BACKGROUND THING TO THEM GETTING THEIR HEADS HIT AND US MONITOR THAT AND SAY THIS IS HOW MANY TIME THEY HIT THEIR HEAD. I DON'T KNOW HOW MANY TIMES THEY HIT THEIR KNEE OR THEIR SHOULDER. IT'S A PROBLEM WITH THOSE STUDIES TOO. >> SO WHAT I WOULD LIKE TO BRING UP IS SOMETHING THAT HASN'T BEEN DISCUSSED. IT'S POTENTIALLY USING MEASURES OF BLOOD BRAIN BARRIER INTEGRITY AS A PHARMACODYNAMIC BIOMARKER WHEN BLOOD BRAIN BARRIER IS DISRUPTED ALMOST ALWAYS THERE'S ALSO SOME NEURODEGENERATION GOING ON, NEUROINFLAMMATION GOING ON SOME ACTIVE PROCESS, IT'S PART OF AN ONGOING PATHOLOGY RATHER THAN -- MOST CASES NOT A GENETIC DEFECT CERTAIN TBI AND STROKE AND MS AND OTHER CONDITIONS. AND I THINK IT'S LIKELY AND CERTAINLY THERE'S ANIMAL DATA FOR THIS, THAT ANY MOST NEUROPROTECTIVE AGENTS IN ADDITION TO DOING GENERAL NEUROPROTECTION THEY'RE ALSO PROTECT THING THE NEUROVASCULAR UNIT. I WONDER WHAT ANYONE'S THOUGHTS ARE ABOUT USING EITHER DCMRI OR SOME OTHER MEASURE OF BLOOD BRAIN BARRIER INTEGRITY AS A PHARMACODYNAMIC BIOMARKER TO MAYBE ASSESS WHETHER A PARTICULAR DRUG IN ANIMALS OR ULTIMATELY IN HUMANS HAVING AN EFFECT ON THE INTEGRITY. >> IF WE CAN TRACK EFFECTIVENESS OF THERAPIES THAT WOULD BE USED, THAT'S ONE OF THE HOLY GRAILS THAT WE HAVE IN TBI AS YOU KNOW >> I AGREE. TO GET EVEN MORE GRANULAR, RAMONE YOU KNOW SOME PATIENTS RESPOND TO ONE TREATMENT NOT ANOTHER. SORT OF A FAIL FAST, YOU HAVE A PATIENT AND YOU THINK THIS DRUG WILL WORK MAYBE YOU CAN IDENTIFY THAT QUICKLY AND IF IT'S NOT GOING TO WORK MOVE TO SOMETHING ELSE. >> I KNOW THERE'S A COUPLE OF COMPONENTS IN THE AUDIENCE BUT THERE IS A MRICS METHOD FOR LOOKING AT GLUTATHIONE. FOR EXAMPLE, YOU HAVE TO ADMINISTER SOME IV TRACER, BUT THAT WOULD BE SOME WAY TO TRACK EFFECTIVENESS OF SISTINE AND TERM HOW MUCH IS WORKING AT THE BRAIN LEVEL NECESSARILY VERSUS THE VASCULAR LEVEL. BUT I JUST HAVE ONE POINT MOST EFFECTIVE WAY TO GET ACROSS THE BLOOD BRAIN BARRIER, YOUR FREAKING NEUROSURGEON, DO A CRANIOTOMY AND DUMP IT IN. >> I THINK I HAVE DONE THAT. ULTIMATELY WE'RE HUE HERE REPRESENTING THE MILITARY DOD -- THAT MAY NOT BE SOMETHING DIRECTLY APPLICABLE FORWARD, CERTAINLY SEVERAL YEARS LATER YOU COULD BUT MAYBE TOO LATE. >> PARTICULARLY A LOT OF HE CANSOME THINGS THAT ARE TARGETING INFLAMMATION, AND MAYBE AUTOIMMUNE YOU SHOW THE SLIDES THINGS ARE HAPPENING MONTHS DOWNSTREAM, THAT MAYBE SOMETHING THAT'S SET UP EARLY AND YOU CAN ADDRESS IT MUCH, MUCH EARLIER DOWN THE ROAD IN TERMS OF DEVELOPING SORT OF AN AUTOIMMUNE RESPONSE TO BRAIN INJURY THAT WHEN YOU GET YOUR SECOND BLOOD BRAIN BARRIER PERMEABILITY ISSUE LIKE GETTING OLD OR WHATEVER, AND THAT LYMPHOCYTES ARE ABLE TO LEAK IN THERE AND THEY'RE EXPOSED TO THE ANTIBODIES THAT HAVE BEEN AUTOANTIBODIES GENERATED RELATED TO ANTIGENS FLOATING INTO THE CERVICAL LYMPH NODES, THE MEMORY B CELLS T-CELLS WHATEVER, YOU HAVE THE AUTOANTIBODY FORMATION RATHER THAN LYMPHOLYTICS LATER DOWN THE COURSE, YOU USE SOME TARGETED EXOSOME THAT PREVENTS AUTOANTIBODY FORMATION AGAINST BRAIN ANTIGEN LIKE YOU SEE WHATEVER YOUR FAVORITE ONE IS. >> THAT MIGHT BE SOMETHING TO DO IN 24 HOURS ONCE YOU'RE IN GERMANY. >> JOHN GRIFFIN SCRIPTS RESEARCH INSTITUTE LA JOLLA. I WOULD LIKE THE PANEL TO CONSIDER ANOTHER DIRECTION OR OPPORTUNITY THAT'S NOT TOUCHED UPON THAT FOLLOWS FROM DR. CLARK'S DISCUSSIONS ABOUT THE MANY TRANSPORTER AND OLD METABOLITE, BECAUSE YOU STUDIED OR WE DID, IN IN BIOCHEMISTRY AS STUDENTS. IN SISTINE. BUT TODAY I THINK THAT ONE OF THE LATEST OMICS OF THIS CENTURY OPPOSED TO GENE OMICS AND PROTEOMICS IS METABALOMIC. YOU MEASURE THOUSANDS OF METABOLITES WITH MASS SPECK. SOME OF THE MASS SPEC WE UNWHAT THEY ARE, ABOUT 2000 OF THEM, ABOUT 6,000 WE STILL DON'T KNOW WHAT THEY ARE. THERE'S TREMENDOUS OPPORTUNITY IN TRAUMA FOR EXAMPLE IF THERE'S WHOLE BODY TRAUMA OR BRAIN TRAUMA, TO HAVE IDENTIFY WHAT ARE THE METABOLITES THIS, THOSE THAT ARE PROHE CAN THETIVE, WITH INFLUENCE THOSE ARE PATHOLOGIC. WHAT ARE BALANCES OF THE VARIOUS THOUSANDS OF METABOLITES THAT ARISE IN CHRONIC OR ACUTE OR WHETHER INJURY IS RELATED TO THE BRAIN OR WHOLE BODY. AND LEARN FROM BASIC KNOWLEDGE. FROM THAT ONE CAN IMAGINE TRANSLATING THAT KNOWLEDGE INTO DOING SOMETHING AS DR. CLARK POINTED OUT ABOUT POTENTIAL VALUE OF INACETYL AS A SIMPLE NEOTAB LIGHT. THERE'S CLASSES OF CATEGORIES WHOSE INFLUENCES WE DON'T EVEN KNOW ABOUT BUT IF WE LEARNED AND STUDIED AS A FUNCTION OF TIME, PATIENTS THAT HAD BRAIN TRAUMA, OTHERNDst THAT HAD WHOLE BODY TRAUMA, WHAT ARE THE BALANCES OF METABOLITES THAT CAN INFLUENCE THE OUTCOME? SO I THINK I WILL LIKE TO POINT THIS OUT AS POTENTIAL OPPORTUNITY FOR THE LONG RANGE FUTURE FOR FIRST DISCOVERY AND THEN TRANSLATION. >> I THINK ON A SMALL EARLY STUDY THAT WAS JUST PUBLISHED IN JAMA SURGERY, I DON'T KNOW IF YOU REMEMBER THE DETAIL, IT'S A SMALL NUMBER OF TRAUMA PATIENTS THAT RECEIVE BRAIN INJURY, LOOKING AT THAT, AS YOU MIGHT EXPECT THEY CORRELATE SEVERITY OF INJURY WITH INCREASE IN IN CYTOKINE, THINGS LIKE THAT. AIN'T MUCH BUT AT LEAST IT'S FIRST STEP IN TO OURLY THINKING ALONG THE SAME LINES. THOSE ARE PROBABLY TARGETED. >> YOU CAN DO IT BUT NOW YOU'RE TALKING -- UNTARTED -- >> I AGREE. >> WE HAVE APPLIED THAT TO THROMBOSIS TO VENUS THROMBOSIS AN DISCOVERED A CLASS OF LIPIDS, ACYL CARNITINESES THAT HAVE REMARKABLE COAGULANT ACTIVITIES, NO ONE KNOWS THIS. THERE REALLY IS A TREMENDOUS OPPORTUNITY FOR MINING DISCOVERY WITH THE NEWESTOMICS. FOR TRAUMA SITUATIONS, FOR TRAUMA ESPECIALLY IT'S THERE, MORE CHALLENGING FOR CHRONIC BUT WITH TRAUMA YOU CAN HAVE THE TIME SAMPLES FROM THE SAME PATIENT. SO YOU DON'T HAVE ALL THE PROBLEMS OF STUDY METABOLITES FROM 20 INDIVIDUALS OR 200 INDIVIDUALS, YOU HAVE GOT TIME COURSE FOR THE SAME WHERE YOU HAVE A LOT OF CONTROL. >> CERTAINLY I COMPLETELY AGREE. WE HAVE CSF AND WE HAVE THE OPPORTUNITY TO LOOK NOT ONLY WITH TRAUMA VERSUS CONTROL BUT WITH DRUG INTERVENTION. WE ALSO HAVE A STUDY THAT IS ENROLLED THE PATIENT WHERE IT'S A HIGH FIBER DIET MICROBIOME RELATED STUDY. THE ADVANTAGE WITH TBI PATIENTS AT LEAST VICESBERG AND VIRGINIA YOU PUT A VENTRICULAR DRAINAGE INTO MONITOR INTRACRANIAL PRESSURE SO YOU HAVE A ACCESS TO CSF SO YOU CAN SEE THE CSF VERSUS SYSTEMIC MICROBIOME, WE HAVE SERUM FROM THESE PATIENTS TOO. AS YOU ARE AWARE, METABALOMICS THINGS ARE EXHAUSTIVE FOR THE PERSON RUNNING THE ASSAY SO NOT ME, I HAVEN'T DONE THE CLINICAL STUFF, THAT'S HARD PART BUT ACTUALLY AT LEAST WITH THE METABALOMIC PEOPLE TELL ME IS THAT I'M ASKING FOR A HUGE, HUGE EFFORT ON THEIR PART TO RUN THESE THINGS. BUT I CERTAINLY THINK THAT IT'S CERTAINLY WORTH EXPLORING BOTH IN THE SYSTEMIC AND BRAIN LEVEL AND CSF USING INTERVENTION IN INJURY EFFECT AND INTERVENTIONAL EFFECT WILL JUST HELP ENHANCE WHAT WE LEARN FROM THESE STUDIES EVEN MORE. >> SO WE'RE GETTING TIGHT ON TIME. I WANT TO ASK -- WE ALL WANT HEAR FROM EVERYONE BUT I WANT YOUR QUESTION TO BE SUCCINCT PLEASE. >> I LIKE TO COMMENT, IT'S NOT A MAJOR QUESTION, BUT I WOULD LIKE TO ADDRESS THE QUESTION ABOUT THE ESTABLISHMENT OF A STUDY GROUP AND I WOULD WANT TO POINT OUT THAT IN TWO WEEKS FROM NOW THERE'S GOING TO BE A DOOREDDEN CONFERENCE IN NEW HAMPSHIRE CALLED BARRIERS IN THE CNF. THERE'S 172 PEOPLE ATTENDING THAT ARE WORLD EXPERTS ON BAIN BARRIER AND BARRIERS IN THE CNF. I HEARD A LOT TODAY ABOUT NEW ISSUES DEALING WITH BRAIN BARRIERS. A LOT OF DISEASE MODEL, A LOT OF SCIENTIFIC INTEREST ESPECIALLY IN THE CLINICAL COMMUNITY, FOR BRAIN BARRIERS SO I WAS ENCOURAGED RATHER THIS THAN HAVING ANOTHER INDEPENDENT GROUP OR STUDY GROUP THAT THERE'S IMPACT OF NUMBER. THERE'S POWER IN NUMBERS. AND HAVING SOME COLLABORATIVE EFFORTS OF LEARNING FROM BARRIERS IN CNS TYPE GROUP, THE GROUP HERE IS VERY VALUABLE. I THINK EVERYONE THERE WOULD BENEFIT FROM HEARING FROM YOU, BUT I THINK ALL OF US HERE WOULD BENEFIT A LOT FROM LEARNING AND HEARING FROM THEM. SO I WOULD ENDOOR RAJ RATHER THIS THAN HA -- >> ABSOLUTELY. ABSOLUTELY THANK YOU FOR SHARING THAT. >> WE HAVE TIME FOR THREE SHORT QUESTIONS. >> WE HAVE HEARD A LOT ABOUT THE ADVANTAGE OF THE NASAL ROUTE TO GET TO THE BLOOD BRAIN BARRIER. THE QUESTION IS WHY IS THIS EFFECTIVE. THE REASON I'M ASKING IS THAT NOT KNOWING -- I DON'T KNOW WHY THIS IS SO EFFECTIVE. THE QUESTION, ARE THERE SIDE EFFECTS WHICH ARE NOT BENEFICIAL IF YOU WERE TO USE NASAL ROUTE FOR APPROACHING THE BLOOD BRAIN BARRIER? >> ONE COMMENT. CLINICALLY, OBVIOUSLY YOU WANT TO MAKE SURE THERE'S NO SKULL BASE FRACTURE, YOU HAVE TO MAKE SURE IF YOU'RE PUTTING STUFF INTO NOSE IT IS GETTING TO THE RIGHT PLACE, AND YOU GET SOME PATIENTS WITH EXTENSIVE FACIAL INJURIES, IT MAY NOT BE AN OPTION. THOSE THINGS ALL COME UP. ALWAYS COME UP. (OFF MIC) >> NASAL UNFOE IS GREAT BUT MIND SET IS SOMEBODY IN THE FIELD, IDEALLY PRE-HOSPITAL AS SOON AS THEY GET THERE, SO IT'S A SLIGHTLY DIFFERENT ENVIRONMENT. >> LET'S JUMP TO THE NEXT QUESTION. >> MY NAME IS (INDISCERNIBLE) NEUROSURGEON AT THE NEUROLOGICAL INSTITUTE. I WORK WITH KENDALL JOHNSON WHO IS THERE. ONE ISSUE WE ENCOUNT IRED AND I URGE PEOPLE TO THINK ABOUT IN THE BIOMARKER DEVELOPMENT FOR DISEASES THAT ARE QUOTE UNQUOTE DIRTY LIKE TRAUMA INVOLVED IN MULTIPLE SYSTEMS, STROKE, SECURE FACT TIN, HEMORRHAGE, WITH SMALL INTERVENTIONS THAT HAPPEN IN THE ICU FOR OTHER CAUSES ARE GOING TO DRASTICALLY AFFECT THE BIOMARKER PROFILE. SO IF YOU'RE STUDYING SPASM IN A SUBARACNAL HEMORRHAGE PATIENT AND THAT PATIENT DEVELOPS PNEUMONIA OR DEVELOPS INVENTORY TRICKLITIS AND SOMEBODY STICKS A BUNCH OF ANTIBIOTIC THAT WILL CHANGE YOUR PROFILING AND YOU SHALL BE COGNIZANT AND CAUTIOUS ABOUT THOSE ALTERATIONS THAT MAY NOT -- MAY NOT COME UP FIRST GLANCE REVIEWING CLINICAL SAMPLES. >> >> YOU'RE RIGHT. THAT'S GOING TO BE THE BUG A BOO, I REALIZE, BRAIN JUST DOESN'T LIVE IN A BOX IN ISOLATION. EVERYTHING HAPPENS TO THE BODY AFFECTS THE BRAIN, VICE VERSA. SO (INDISCERNIBLE) IS GOING TO BE DIFFICULT. >> THE ONE QUESTION AND COMMENT. THE QUESTION IS, I THINK THE BLOOD BRAIN BARRIER CONFERS BALANCE WITH BRAIN BLOOD BARRIER (INDISCERNIBLE) MY QUESTION IS FIZZ OTHER -- ANY FACTORS (INDISCERNIBLE) OPEN CLOSE BETWEEN BLOOD BRAIN BARRIER AND THERE'S A LOT OF PHYSIOLOGIC THINGS THAT HELP GET STUFF FROM THE BRAIN BACK INTO THE BLOOD SLEEP ALLEGEDLY IF YOU'RE RODENT INCREASES FLUX. INTRACRANIAL PRESSURE, BLOOD PRESSURE THINGS LIKE THAT CAN HELP ENHANCE WASTE DISPOSAL, WHATEVER FLEX OUT OF THE BRAIN. SO I THINK -- AND ALL THE MEMBRANE TRANSPORTERS THERE'S A LOT OF THINGS THAT HELP REGULATE THINGS GOING ON. SOMETHING AS SIMPLE AS CSF FLUX HELPS PUSH THINGS OUT OF THE BRAIN. SO GETTING OUT THERE CAN BE SORT OF VERY HOLISTIC OR SIMPLE MECHANISMS FOR THAT. BUT IN TERMS OF GETTING IN, I THINK IS WHERE THE HARD PART IS. >> MY COMMENT IS FOR THE INTRANASAL DELIVER EXOME OR PLAN HOE TREAT AGENT TO THE PARENT TO REGULATE THESE PHYSIOLOGICAL, HIGH EFFICIENCY. REASON FOR THAT IS INTRANASAL DELIVERS VESICAL LIKE VASCULAR BY MACROPHAGE. SO THE MACRO PHAGO(INAUDIBLE) INDUCTIONS BUT DELIVERED, THE LONGITUDINAL INFECTED THEREFORE SEVERAL PROFILE CHANGE PRIMARY (INDISCERNIBLE) NOW BECOME PRIMARY TARGET OF THE DISEASE AND CYTOKINE SO THEREFORE YOU CAN HAVE SYSTEMIC EFFECT. THEREFORE DEFICIENCY IS NOT A MAJOR ISSUE FOR INTRANASAL DELIVERY. >> THANK YOU VERY MUCH. I THINK THAT THIS IS TIME TO LET OTHER SESSION ANSWER THE QUESTIONS AND PLEASE EXOSOME THERAPEUTICS SESSION MEMBERS PLEASE COME IN. THE MICROPHONE GOES TO DR. RICHARD KRAIG. >> THOSE ARE ISSUES WE PROMISE TO SETTLE IN 20 MINUTES AN RECEIVE YOUR COMMENTS IN TEN. >> YOU CAN HAVE 30 MINUTES. >> DOES ANYBODY HAVE ANY QUESTIONS FOR THE PANEL OR COMMENTS ON WHAT WE THINK ARE KEY QUESTIONS TO GO FORWARD? >> MAY I START WITH THE FIRST QUESTION? AND ACTUALLY THIS IS THE QUESTION FOR RECEIVED BY THE EMAIL. YOU'LL TRY TO REPEAT ALMOST EXACTLY. THE QUESTIONS FOUND LIKE THAT, THERE ARE A NUMBER OF COMPANIES THAT CLAIM THEY CAN HAVE BLOOD BASED TEST FOR ALZHEIMER'S DISEASE. WITH SENSITIVITY OF 90%. HOWEVER, THERE IS NO BLOOD BASED TEST FOR ALZHEIMER'S DISEASE CURRENTLY APPROVED. DO YOU KNOW WHAT IS THE STATUS AND WHAT IS THE FINE LINE? QUESTION IS NOT MINE, THE QUESTION IS SOMETHING RECEIVED ONLINE. >> I AM AWARE OF MANY EFFORTS, ACADEMIC EFFORTS TO DO IT. THEN THERE'S A PARALLEL WORLD, STEN COMPANIES AN CLAIMS WE NEED ANALYSIS OF VERY LARGE COHORTS, VERY LARGE DATA SETS SO THAT THESE EARLY SUCCESSES CAN BE CONFIRMED AND VALIDATED BEFORE WE CONSIDER GOING TO THE FDA OR SOMETHING THAT WILL MAKE IT A TEST CLINICALLY AVAILABLE. SO I THINK THAT SCIENCE IS EVOLVEING. I WOULDN'T PUT IT A CLOSER TO IT. >> ERIC SHUSTA, WISCONSIN. I WAS TRYING TO PUT DATA I SAW FROM THE SPEAKERS REGARDING EXOSOMES AND THE CIRCULATION, AND EXOSOMES IN THE BRAIN. AND CONCENTRATION OF NANOPARTICLE AND LYSOSOME DESIGN GOING ON A COUPLE OF DECADES. I WANT COMPARE AND CONTRAST. ONE SET OF DATA I SAW WAS THAT THE HE CANSOMES ARE CLEARED VERY QUICKLY. MUCH LIKE NAKED PARTICLE IN THE BLOOD. THAT MAKES SENSE. ANOTHER SET OF DATA WE SAW WAS FOCAL INJECTION THAT DIFFUSED BOTH BRAIN HEMISPHERES. THINK CLASSIC LITERATURE THAT SHOWS INTRODUCTION OF PROTEINS BECAUSE METABOLISM AND BINDING TO CELLS AND SO ON, THE PENETRATION IS VERY LOW. IN THAT CASE IN PARTICLE DESIGN, WE WILL PROBABLY HEAR IT TOMORROW, BUT PEGLATION OR HYPERPEGLATION SEEMS TO HELP PENETRATION THROUGH TISSUES BUT THAT ALSO WOULD HELP WITH RETENTION IN THE BLOODSTREAM. SO I GUESS WHAT IS IT ABOUT HE CANSOMES, THAT ALLOW THEM TO PENETRATE READILY WITHIN TISSUE WHOSE EXTRA CELLULAR SPACE PROBABLY HAS DIAMETER ON THE ORDER OF SIZE OF EXOSOMES. THANK YOU. >> FANTASTIC QUESTION. WE DO NOT KNOW. WE HAVE A HYPOTHESIS IN THE CASE OF BRAIN WHAT IS CONTRIBUTING FACTOR TO CROSSING THE HEMISPHERE IS EXONAL TRAFFICKING. EXONAL TRAFFICKING IS REALLY EFFICIENT IN TRANSFERRING THE PARTICLES AND I THINK IN PART IT'S A CONTRIBUTE FACTOR. THIS HAS NOTHING TO DO WITH THE CROSSING OF THE BLOOD BRAIN BARRIER, IT'S JUST A QUESTION OF MECHANISM OF THE DISTRIBUTION OF THE VESICALES OR PARTICLES WITHIN A BRAIN. WE ALSO DO KNOW THAT IF YOU JUST MAKE A NEUTRAL LIPSOME AND -- LIPOSOME AND DO NOT PLAY WITH ANYTHING YOU DON'T GET THE SAME DEGREE OF THE OTHER HEMISPHERE, SO THERE'S SOMETHING IN EXOSOMAL MEMBRANE LIPID COMPOSITION, PROTEIN COMPOSITION RESPONSIBLE FOR THOSE PROPERTIES AND WE ARE IN THE MOSES OF TRYING TO FIGURE IT -- PROCESS OF TRYING TO FIGURE IT OUT. IN SYSTEMIC DELIVERY, I WANT TO GET IT OFF MY CHEST, THAT IF YOU INJECT EXOSOMES SYSTEMATICALLY, TALKING IV, THEIR PK BEHAVIOR IS VERY SIMILAR TO PK BEHAVIOR OF GENERALLY SMALL LIPOSOMES OR NEUTRAL LIPOSOMES WHICH VAST MAJORITY CLEARED THROUGH THE LIVER AND WHEN I SAY VAST MAJORITY, MORE THAN 90%. THIS HALF LIFE IN A BLOODSTREAM IS LESS THAN HALF HOUR. SYSTEMIC INJECTION. THERE MIGHT BE SOME DELIVERY AND CROSS THROUGH THE BLOOD BRAIN BARRIER IN SEDATION WHERE EXOSOMES ARE USED IF SAY I DELIVERED SYSTEMICALLY VAST MAJORITY DELIVERED ESSENTIALLY WILL BE CLEARED WITHIN VERY SHORT PERIODS OF TIME AND IT'S CLEARANCE IS NOT THROUGH THE BRAIN. INTRANASAL IS A DIFFERENT QUESTION. >> LET ME ADD TO THAT, COMES TO ME FROM WHERE FOR ART THOUSAND, EXOSOMES ARE BONA FIDE. THEY HAVE A NATURAL NATURAL SURFACE STRUCTURE AND PARTICIPATE IN VESICULAR TRAFFICKING WHICH IN NERVOUS SYSTEM IN ONE ORGAN STRUCTURE IS EXCELLENT. THERE'S VESICALES EVERYWHERE. SO THAT'S INSIDE A CELL, NOT SO SURPRISING TAKE THEM OUTSIDE A CELL AND MOVE ALONG THE EXTRA CELLULAR SPACE THAT UNDELAYED IN SIZE AND GOES ANYWHERE FROM 15 TO 25% WITH NEURAL ACTIVITY. WHAT I WANT TO SAY ABOUT EXOSOME PARTICLES IN THE PERIPHERY, PARTICLES IN THE PERIPHERY, WHEN YOU DUMP A BUCKET IN ARE GO TO BE SUCKED UP BE I THE LIVER, IT'S WHAT ITS ORGAN IS SUPPOSED TO DO. THE LUNG DOES THE SAME. I FOOLED AROUND EARLY DAYS WITH SODIUM ELECTRODES. WE WENT TO TO PASS YUM ELECTRODES PH ELECTRODES, AN FOUND TEN TO THE MINUS NINE, I DISCOVERED CYTOKINES WHICH ARE SIGNALING. STILLNALINGS ARE SIGNALING EXOSOMES WHICH MAYBE AT A MOLAR SIGNALING. IF I GET A EXOSOME ACROSS THE BRAIN AND MYELINATES IT 150% HIGHER THAN USED TO, I HAVE TO CONSIDER NOT JUST NUMBER OF PARTICLES I'M TRANSFERRING BUT THE EFFICACY THAT WE'RE TRANSFERRING. SOMETHING WE DIDN'T SPEAK TO BUT I WILL LIKE TO SPEAK TO IN TERMS OF THOSE INTERESTED IN MICROGLIA. WE STILL DON'T REALLY KNOW IF I PLANT AN EXOSOME IN THIS MICROGLIAL CELL DO THEY SELF-PROPAGATE LIKE JOE APPLE SEED OR DO I PUT STIMULUS IN INTERFERON GAMMA HERE AND PUT IT HERE AMEND PUT IT HERE. IF HAY THAN TO CELL PROPAGATE GAME OVER. YOU GET ONE, TWO, THREE, EXOSOMES ACROSS THE BLOOD BRAIN BARRIER, YOU'RE CHAIN CHANGING FUNCTION, IN ADDITION I WOULD LIKE TO SAY FROM OUR PAST SESSION, THEN I WILL BE QUIET, THE NOTION OF OPENING AND CLOSING THE BLOOD BRAIN BARRIER. I WOULD PREFER TO ALMOST TAKE THE COMPOSITE OF PEEP'S THOUGHTS WHERE YOU BUILT IT TO BE INTACT TO KEEP PERIPHERY AWAY FROM THE CENTRAL. YOU USE IT DIAGNOSTICALLY OFF CSF TO SEE THAT YOU HAVE BROKEN BARRIER AN DISEASE. SO LEAVE IT INTACT. FULL WITH FILTERING PROCESSES THAT ARE ABLE TO GET THINGS THROUGH. WHEN YOU TICK A R ASHS WHISKER BARREL, YOU EXERCISE AN INCREASE IGF 1, PART OF THE PSYCHKINE BURST, WHEN YOU HAVE SENRY SIMULATION OF JUST TICKING A WHISKER BARREL IGF 1 PREFERENCIALLY TRANSFERS INTO THE BRAIN ON THE SENSORY PATHWAYS THAT WERE ACTIVATED. SO I SUSPECT WHEN YOU THINK IGF 1 GOES INTO YOUR HIPPOCAMPUS, YOU'RE GOING TO SELECTIVELY CHANGE BLOOD BRAIN BARRIER FUNCTION BASED ON NEURAL ACTIVITY, THAT'S SUGGESTING TRANSMAGNETIC STIMULATION, MAYBE ALTERED BLOOD BRAIN BARRIER FUNCTION IN A PHYSIOLOGIC FUNCTION BUT THE NOTION PERMEABILITY ACROSS THE BLOOD BRAIN BARRIER IS I WOULD SUGGEST WE SHOULD LEAVE IT ALONE, GET INTACT SO IT KEEPS THOSE BAD THINGS OUT. AND THEN ALLOW WHAT WE WANT AS THERAPISTS IN. >> KEITH WHIT KER U.S. ARMY RESEARCH CATEGORY. UP LIKE PLEAINGS IN THE NON-MEDICAL SIDE OF THE HOUSE, MY QUESTION IS A LITTLE BIT OF A CURVE BALL, I'M INTERESTED TO GET YOUR THOUGHTS ON POTENTIAL USE OF EXOSOMES IN THE FUTURE IN HEALTHY POPULATIONS IN ORDER TO ENHANCE SOME OF THE PERFORMANCE PARAMETERS PHYSICALLY AND COGNITIVELY, SPECIFICALLY BECAUSE IT SEEMS LIKE IT WILL BE TEMPORARY AN REVERSIBLE. THANKS. OKAY I GOT THAT ONE. I'M A MIGRAINE DOC 20% OF THE TIME, I'M A REAL GOOD GUY AS CHAIRMAN OF PSYCHOLOGY AT THE UNIVERSITY OF ILLINOIS CHICAGO. WE DIDN'T TREAT THEM WELL AT THE UNIVERSITY OF CHICAGO. I ASKED THEM MANY PATIENTS ARE ON -- TAKING SEDATIVE HYPNOTICS, WE'RE STARTING TO USE PROVIGILON AS AWAKENING DRUG. HE CAME THROUGH AND SAID ARMY HAS DONE A STUDY. THEY GAVE THE KIDS KIDS BECAUSE THEY ARE 15 TO 25, YOUR MOTOR SKILLS ARE SHARPEST WHILE THE BOOKS ON SHELF AREN'T SO FULL. MOTOR SKILLS ARE SHARPEST. HE SAID THE KID SHOT BETTER WITH COFFEE THAN THEY DID WITH -- BECAUSE THERE'S NOTHING WRONG WITH THE KID. THE TROUBLE WITH COFFEE IS, YOU CAN GET PSYCHOTIC WHEN YOU TAKE TOO MUCH OF A DOSE. BUT REFLECTSES GO FASTER. THE QUESTION I HOPE THIS LEADS TO IS I'M NOT TRYING TO BE SELF-SERVING BUT THAT GUIDED US WITH THE ENVIRONMENTAL ENRICHMENT, IS COGNITIVE DECLINE FROM AGING. IF YOU DON'T THINK YOU DEMYELINATE YOUR BRAIN. IT'S CLEARLY BEEN ESTABLISHED THE OLDER YOU GET DECADE BY DECADE, 60, 70, 80, THE MORE CREATIVE OR EXPERIENTIAL YOUR THINKING IS, THE MORE YOU WILL MYELINATE YOUR BRAIN. SO IT MAY NOT BE MYELINATE ABOVE NORMAL, IT MAY MEAN MYELINATE TO KEEP NORMAL AS YOU HAVE A PROGRESSIVE DECLINE FROM AGING. BUT I DON'T THINK WE'RE GOING TO USE IT LIKE THINGS LIKE THIS LIKE A SUPER DRUG. THEY'RE USED TO MAINTAIN HEALTH, AND RESTORE HEALTH AND THEN YOU'RE TOLD TO DO IT YOURSELF. >> IF I MAY I WILL GO BACK TO EXOSOMES. I THINK WE HAVE A MAJOR BARRIER IN TERMS OF DEALING WITH EXOSOMES. THAT'S BECAUSE -- MOST OF US WHO WORK WITH PLASMA DERIVED NOT TUMOR -- NOT CELL LINE DERIVED BUT PLASMA DERIVED, BODY FLUID EXOSOMES, WORK WITH TOTAL FRACTIONS. AND I LIKE YOUR TALK VERY MUCH BECAUSE WHAT YOU SHOWED US IS THAT YOU ACTUALLY IMMUNOCAPTURED THE EXOSOME THAT CARRY RELEVANT MARKERS. FOR ALZHEIMER'S DISEASE. AND THEY ONLY REPRESENT 10 TO WHATEVER 15% OF THE TOTAL POPULATION. BUT AFTER YOU PULL THEM OUT THEN THAT CORRELATED VERY NICELY AND HAD A PRETICK GIVE VALUE AND DIAGNOSTIC VALUE IN ALZHEIMER'S DISEASE. I THINK THIS IS TRUE FOR ANY OTHER WORK WITH EXOSOMES. WE NEED TO LEARN HOW TO SEPARATE TUMOR DERIVED OR NOT TUMOR DERIVED ONES. NEURAL HE CANSOMES FROM OTHER EXOSOMES. I THINK THIS IS A MAJOR BARRIER. BUT I CAN TELL YOU YOU USE PRECIPITATION METHOD FOR ISOLATION OF YOUR EXOSOME AND TO ME IT LOOKED LIKE DIRT. SO I SPENT TWO YEARS OF MY LIFE LEARNING HOW TO ISOLATE MORPHO LOGICALLY INTACT BEAUTIFUL EXOSOMES THAT ARE FUNCTIONALLY ACTIVE BIOLOGICALLY ACTIVE FROM HUMAN PLASMA, WE DO IT WITH NORMAL DONORS WE DO IT WITH CANCER PATIENTS. I THINK IN PARTICULAR IF WE WANT TO USE IMMUNOCAPTURE METHOD AFTER ISOLATION OF TOTAL FRACTION, THE INTEGRITY OF THE EXOSOME AND WHAT THEY CARRY IS VERY, VERY IMPORTANT. SO I LIKE THE METHOD FOR EXAMPLE WHERE SITE EXCLUSION CHROMATOGRAPHY ALLOW ME TO DEPLETE UNWANTED PROTEINS. MOST NOT ALL, MOST IMMUNOGLOBULIN ALBUMINS AND THEN WITH IMMUNOCAPTURE METHOD. SO HOW WE ISOLATE EXOSOME TO DO WHATEVER WE WANT TO DO WITH THEM I THINK IS CRITICAL. >> MAY I? FIRST OF ALL, I LOVE YOUR COMMENTS AND I WOULD LOVE TO GET YOUR INPUT TO DO THIS BETTER. ACTUALLY WE TRIED THEM TO START WITH THIS -- WE'RE NOT COMMITTED TO PRECIPITATION METHODS, THE GENERAL IDEA IS TO GET TOTAL EXOSOMES AND POPULATION, AS YOU KNOW, IF WE'RE STARTING WITH SOLICITATION AND ANTIBODIES YOU, WE DO GET SOME NON-SPECIFIC BINDING OF SOLUBLE MATERIAL WHICH WE ACTUALLY WOULDN'T LIKE. WE TRIED NOT ONLY BY A LONG TIME BUT BY DIFFERENT MOLECULES AND FOR DIFFERENT CELL TYPE,, SOME OF THIS WORK IS STILL UNPUBLISHED SO I CAN'T SAY TOO MUCH, BUT WE DO GET DESPITE THE DIRT WE GET VERY DIFFERENT POPULATIONS, THEY HAVE VERY DIFFERENT MOLECULES INSEED THEM AND NO DOUBT WE GET SOME DIRT ON TOP OF THAT, SO IT'S AN ONGOING PROCESS TO TRY TO INCREASE THE SIGNAL AND DECREASE THE NOISE. SEC IS GOOD BUT IT GIVES YOU DILUTE IT'S GOING TO BE A BALANCE BETWEEN PURITY GETTING RID OF SOLUBLE MATERIAL AND GETTING RID OF DECENT ENOUGH NUMBER OF VESICLES FROM A GIVEN BIOLOGICAL SAMPLE AND IF WE HAD LIKE TONS OF MATERIAL, ONE IS VERY IMPURE. (OFF MIC) >> IN THE MATERIAL TO GIVE YOU ENOUGH PROTEIN AND WOULD NOT TO -- SO I WOULD LIKE TO TALK TO YOU. >> IT'S NOT TURNING ON. RECENTLY WE DID SOME WORK ON THE HETEROGENEITY OF EV WE'RE TALKING ABOUT AND WE WERE ABLE TO DIFFERENTIATE WHAT WE CALL MICROVESICALES AND WHAT WE CALL EXOSOMES. THE FUNNY THING IS THE SIZE WAS THE SAME. THE DENSITY WAS DIFFERENT AND THEIR ABILITY TO TRANSFER BIOMOLECULES IS QUITE DIFFERENT. THE MICROVESICLES COULD TRANSFER DNA BUT NOT THOUGHT TO BE THE THOUGHT TO BE EXOSOMES. CD 63 WAS ABSENT FROM THE MICROVESICLES PRESENT ON EXOSOMES. SO IT IS REALLY A COMPLICATED FIELD. AND ONE HAS TO BE CAREFUL, EVENTUALLY I THINK WHEN -- IF THIS GETS TRANSFERRED TO THE CLINIC. THIS HAS TO BE SERIOUSLY CONSIDERED. (OFF MIC) >> (INDISCERNIBLE) TO EXOSOME BUT (INDISCERNIBLE) EXOSOMAL HOW CLOSE TO SO THEREFORE I THINK FOR HERE ANTIVIRAL DERIVED MICROVAS SCHOOL IS A GENERAL TERMINOLOGY INCLUDE EXOME VESICAL. THE QUESTION IS OUR BODY BIOFLUID, THE (INDISCERNIBLE) MICROVASCULAR POPULATION, SUBPOPULATION. AND BIOFLUID, IS REALLY PREDOMINANT CIRCULATION. POPULATION, YES. HE CANOMAL IS NOT PREDOMINANT POPULATION OR BIOFLUID. WE PUBLISHED LAST WEEK. AND ONCOGENE TARGETING. THAT IS NOT POPULATION. VERY SIMPLE. EXOME, CLICK THROUGH IT, APPLY TO IT, YOU UNDERSTAND (INDISCERNIBLE) LOW SPEED HIGH SPEED, IN THE PROCESS COULD MUCH EASIER TO GET IT CLOSE ABROGATION. YOUR SAMPLE. THEREFORE -- HE CANSOMAL NOT TRULY HE CANSOMAL IN POWER FLUID STANDPOINT YOU COLLECT FROM (INDISCERNIBLE). SO THE QUESTION IS, IF YOU HAVE TECHNOLOGY ABLE TO DEVELOP INDIVIDUAL (INAUDIBLE) (INDISCERNIBLE) DATA GENERATED FROM THOSE MIGHT BE MORE CLOSE THAN WHAT HAPPENING IN OUR BODIES. >> AS REGARDS >> THEY CAN DAMAGE -- >> YES, I HAVE A QUESTION, COMMENTS BECAUSE THEY NEVER WORK IN THE FIELD OF ENDOSOMES. I WORK IN THE FIELD OF MOLECULAR THERAPY. DRUG DELIVERY. THIS IS A NEW FIELD FOR ME, I WAS VERY HAPPY TO HEAR GREAT TALK AND I LOVE YOUR TALK AS WELL. BECAUSE IT'S A PLUS SIZE DEVELOPMENT FOR TISSUE CELL RECOGNITION. SO BIOLOGISTS SHOULD BE -- YES, HOWEVER, I UNDERSTOOD TODAY THAT ENDOSOMES THEY ARE REALLY HETERO GENIUS. AND TO TREAT SYSTEMIC DISEASE LIKE BRAIN TUMOR OR ALZHEIMER'S HOW WE CAN DELIVER THEM, WHAT SIZE THEY HAVE, OLFACTORY DELIVERY, ONE OR TWO ENDOSOMES AS A TRIGGER OR TO TREAT PATHOLOGICAL CONDITION? WHO CAN EXPLAIN THIS, HOW DO IT WORK? SIZE, SIZE OF ENDOSOMES. HOW THEY ARE -- WILL BE DELIVERED TRANSCYTOSIS MECHANISM. MAYBE MEDIATED ENDOCYTOSIS BECAUSE YOU HAVE SOME PROTEINS. FROM MS.ON THE SURFACE. >> LARGER SCALE OR FUTURE PRODUCTION OF EXOSOMES AS THEY PREACH THERAPEUTICS ARE GOING TO HAVE TO ENTAIL ASPECTS OF WHAT YOU SAY. WE NEED STANDARDIZED PROTOCOLS FOR PRODUCTION, THAT HAS STANDARDIZED ASSAYS TO SAY THIS GOOD, THAT BAD, SIZING WILL BE IMPORTANT. AND SIZING OF SOME CELL TYPES ARE VERY UNIFORM. (OFF MIC) >> MICROPHONE PLEASE. >> 80 TO 19 NANOMETERS. THE 80 TO 100. >> 120 NANOMETERS. >> YOU THINK IT'S A OPTIMAL SIZE FOR B BB DELIVERY? 80, 100 NANOMETERS? >> I THINK IT'S IMPORTANT TO REVERSE THE THINKING FOR A MOMENT AND THAT IS, IF YOU TAKE AN EXSOME AND THROW IT AT A NERVOUS SYSTEM NASALLY OR HOWEVER YOU GOT IT IN AND YOU HAD A SIGNIFICANT EFFECT AND YOU MEASURE FOR TOX LOGICAL SIDE EFFECTS, AND YOU DO NOT HAVE ANY, YOU HAVE THE PHYSIOLOGY ESTABLISHED THAT SAYS THIS IS WORTHER PURSUING FOR ME TO CHARACTERIZE THE BIODISTRIBUTION OF MY PRODUCT AND THE STANDARDIZATION OF THE PRODUCT. IF IT DOESN'T WORK WHO CARES? IF IT WORKS YOU BEGIN TO DO THAT CHARACTERIZATIONS. >> THIS IS DRAWBRIDGE PHARMACEUTICAL COMPANY HAS TO DO FOR -- BUT FOR US SCIENTIST WHOSE HAS TO DEMONSTRATE PROOF PRINCIPAL FOR TREATMENT -- >> MANY UNIVERSITIES ARE BEGINNING TO BECOME ENTREPRENEURIAL IN THEIR ACTIVITIES AND ARE PROMOTING THAT KIND OF WORK TO BE DONE IN A LABORATORY. PUT YOURSELF ON MUCH MORE SOLID GROUND. (OFF MIC) >> MICROPHONE PLEASE. >> WITH STANDARDIZATION OF VIRUSES WE HAVE LONG HAD COLONY FORMING UNITS AND VECTOR GENOME COPIES IS THE TWO GOLD STANDARDS. COUNTING PARTICLES ARE FIRST STEP BUT KNOWING ACTIVE BIOLOGICAL DOSE WAS CRITICAL FOR VIRUS DEVELOPMENT WHAT DO YOU SEE AS REALLY THE GOLD STANDARD FOR IN VITRO ASSAY FOR ACTIVE BIOLOGICAL DOSE? >> THERE IS NO GOLD STANDARD. THIS IS A PROBLEM. I WANTED TO COMMENT ON SCALING UP MANUFACTURING OF EXOSOMES AND DEFINING THE STAN CARD IN MATRIXES AND THERE ARE CURRENTLY NOT A LOT IN PLACE. THE ISSUE WITH EXCEPTION OF PLAN DERIVED EXOSOMES IS QUANTITATIVE. JUST TO GIVE YOU AND RICHARD RUN INTO THESE PROBLEMS WE RUN INTO THESE PROBLEMS A LOT AND MANY OTHER PEOPLE WHO ARE CONSIDERING EXOSOMINGS AS THERAPEUTIC IS SCALE UP. TODAY IF YOU ARE USING TISSUE CULTURE CONDITION MEDIA TO MAKE ENOUGH EXOSOMES TO RUN ONE WELL POWERED ANIMAL STUDY AND WHEN I'M SAYING WELL POWERED ANIMAL STUDY, IT'S AT LEAST N OF 1012, WITH ALL CONTROLS INCLUDED IN ONE STUDY. WE'RE TALKING ABOUT GROWING CELLS IN A DOUBLE STOCK TISSUE INCOW BAYTOR FEELED WITH FLUSK FOR SIGNIFICANT PERIODS OF TIME SO IF IT TAKES THAT MUCH EFFORT TO DO A SINGLE ANIMAL RODENT STUDY YOU UNDERSTAND THAT THE QUANTITIES REQUIRED FOR NON-HUMAN PRIMATE IN HUMANS ARE SIGNIFICANTLY HIGHER WITH UNDERSTANDING THAT MAYBE THERE'S SOME SMALL SUBFRACTION OF POPULATION WHICH IS SUPER POTENT, THIS IS FINE. THEN THE NEXT QUESTION, WE HAVE BEEN HAVING A LOT OF DISCUSSIONS AS A CONSORTIUM IN TERMS OF HOW TO SCALE UP AND DEFINE THE BEST METHODOLOGY FOR PURIFICATION. SO FAR RIGHT NOW, THE WINNER IS, BUT THERE'S NOT A LOT OF PEOPLE DOING IT, IT'S DIFFERENTIAL SITUATION. AND THE BEAUTY IS THAT THERE'S SO MUCH EFFORT AND MONEY WHICH HAVE BEEN SPENT IN MONOCLONAL ANTIBODY FIELD AND VIRAL FIELD AND DEFINING AND OPTIMIZING THOSE CONTINUOUS FLOW INCUBATORS, DIFFERENTIAL SITUATION UNITS SO MOST LIKELY THIS CAN BE ADOPTED FOR PRODUCTION. >> SEEN FOR VIRUS PRODUCTION WHERE THERE'S BEEN PROBABLY A THOUSAND FOLD GAIN IN YIELD VIRUS PER CELL. >> PRECISELY. THIS IS WHERE THE WHOLE FIELD IS GOING. WHEN WE HAD PRELIMINARY DISCUSSION WITH FDA WHAT IS THEIR REQUIREMENT FOR QUALITY ASSURANCE, ACTUALLY AS EXPECTED THE RESPONSE WAS VERY AWAKE. THE RESPONSE WAS TELL US THERE'S CERTAIN THINGS THAT MOVE TO CLINIC WITH ESSENTIALLY MINIMAL QUALITY ASSURANCE CRITERIA COUNTING HOW MANY CONFIRMING CERTAIN BIOMARKERS ARE THERE. THE PROBLEM LIKE FUNDAMENTAL SCIENTIFIC PROBLEMS, IF WE ASSUME THERE IS ONE PERCENT, 0, 1 PERCENT SUPER DUPER EXOSOMES WHICH ARE EXTREMELY IMPORTANT WE HAVE TO DEFINE WHAT BIOMARKERS HOW TO QUALIFY THE PREP TO ASSURE 0.1 OR 1% IS ACTUALLY THERE. >> TO ANSWER YOUR QUESTION. WAS IT GRAPE OR GRAPEFRUIT THE BIOLOGICAL UNIT ASSUMES STANDARDIZED BY INDUCTIONS OF (INDISCERNIBLE) EXOSOME (INDISCERNIBLE) PARTICLE IN VITRO IN MACROPHAGE (INDISCERNIBLE) PER EXOMAL PARTICLE, >> I LIKE ONE CAVEAT AS A CHAIR TO FINISH. THE ANSWER IS EXOSOMES ARE NEW. THOSE CONCERNS ARE PROBLEMS ARE BEING WORKED ON BY MANY OF US I SUSPECT THEY WILL BE SOLVED AT LEAST TO SOME EXTENT TO MAKE THIS PLAUSIBLE IN THE NEAR FUTURE. THEY ARE BUT THE VIRUS CONVERSATION IS VALUABLE. BEFORE IT GOT FIXED, THIS IS NEW, THERE'S A LOT OF POTENTIAL -- DON'T GET FIXED. THE SCALE UP, THE STANDARDIZATION, THE REPRODUCIBILITY IS ALL PART OF MAKING THIS GO FORWARD. THE CLEANER, YOUR VARIANCE IS ON EXPERIMENTAL MEASURES, THE BETTER THAT MEANS STANDARD ERROR IS ON YOUR EXPERIMENTAL VALUES. THAT HOLDS TRUE TO THE BIOLOGY OF THE EXOSOMES YOU'RE APPLYING. SO WE'RE ALL WORKING ON IT. WE HAVE TO STOP THERE. IT'S BEEN A LOT OF FUN. [APPLAUSE] TBRZ >> THANK YOU VERY MUCH. I WANT TO BRIEFLY DO A FEW CLOSING REMARKS AS EVERYONE GATHERS THEIR THINGS. I WANT TO THANK EVERYONE FOR THEIR TIME AND EFFORTS AND PARTICIPATION IN TODAY'S SESSION. AND WE HAVE HEARD A LOT ABOUT SOME POTENTIAL RECOMMENDATIONS WHICH WE WILL ALL TAKE NOTE OF, WE HAVE THE -- WILL BE GENERATING A WORKSHOP REPORT THAT'S ONE OF THE DELIVERABLES FROM THIS MEETING. ESPECIALLY ANYTHING THAT INVOLVES COLLABORATIVE APPROACHES FOR INSTANCE TOUCHING BASE WITH THE BARRIERS IN CNS UPCOMING SYMPOSIUM TAKING INTO ACCOUNT AND SO AGAIN WITH THAT I WANT TO ALSO MENTION THERE'S INFORMAL DINNER FOR ANYONE WHO IS AVAILABLE THIS EVENING AT 6:30, IT'S WILL BE AT THE HOTEL VENUE, EXIST AT THE BACK YOUR BOOKLET, JUST LOOK AT THE ADDRESS THERE. THE SHUTTLE BUS FOR ALL THE SPEAKERS I WILL TAKE THEM BACK TO THE HOTEL, WILL BE OUTSIDE HERE BUT AT 5:15. IN THE MEANTIME IF YOU WANT TO HAVE MORE COFFEE OR WATER FEEL FREE. ONE LAST THING SECURITY WAS THOSE RETURNING TOMORROW WITH YOUR BADGES, TO KEEP THOSE AND YOU CAN WALK WITH THOSE ID TAGS SO WHEN YOU CHECKED IN INITIALLY IF WE STILL HAVE IT PLEASE KEEP IT WITH YOU. I DO HAVE A FEW PARKING STICKERS FOR ANYONE WHO HAS DRIVEN IN WHO IS A PARTICIPANT IN THE WORKSHOP. THANK YOU. LOOKING FORWARD TO SEEING YOU BRIGHT AND EARLY TOMORROW MORNING. AND AT DINNER TONIGHT. THANK YOU.