>> WELCOME EVERYONE THIS MORNING AND THANK YOU VERY MUCH FOR YOUR PARTICIPATION ON DAY ONE OF THE WORKSHOP. WE'LL GET STARTED AND SO, BASICALLY, I WANTED TO AGAIN EXTEND MY GRATITUDE TO UL AT SPEAKERS FOR ALL OF THEIR EFFORTS IN BRINGING THIS WORKSHOP TOGETHER AS WELL AS THE WORKSHOP STEERING COMMITTEE THAT HELPED TO PUT THIS TOGETHER AND DR. HOOTS AND DR. GIBBONS WHO SPOKE YESTERDAY FOR THEIR SUPPORT OF THIS EFFORT. SO JUST TO RECAP, YESTERDAY WE HAD THREE SESSIONS, BLOOD SCIENCES SESSION 1 AND 2 FOLLOWED BY THE EXOSOME THERAPEUTIC SESSION THAT GRATEFUL A SURVEY OF VARIOUS ASPECTS OF THE BLOOD-BRAIN INTERFACE AND THE ISSUES ASSOCIATED WITH IT AND VARIOUS CONDITIONS FROM NEUROLOGICAL DISEASES TO IMPACT TRAUMA. AND THERE WERE CERTAIN KEY PRIORITIES THAT WERE DISCUSSED AS WELL TOWARDS THE END IN OUR OPEN MIC DISCUSSION WHICH WAS VERY ENTHUSIASTIC AND WE GOT A LOT OF FEEDBACK FROM EVERYONE. THANK YOU VERY MUCH. SO SOME ISSUES ENCOUNTERED WERE REGARDING CLINICAL ENDPOINTS, INTRANASAL DELIVERY, ALSO ISSUES WITH BLOOD-BRAIN BARRIER DISRUPTION AND PERHAPS OMIC APPROACHES TO FOCUS ON SOME OF THESE ISSUES AND AS WELL AS ESTABLISHING COLLABORATIVE GROUPS BEING AWARE OF ACTIVITIES THAT ARE ONGOING ACROSS THE U.S. AND INTERNATIONALLY. SO, WE WILL CONTINUE THIS DISCUSSION AND OF COURSE COME UP WITH RECOMMENDATIONS BASED ON YOUR INPUT FURTHER ON AFTER THE WORKSHOP. TODAY, WE ARE ACTUALLY GOING TO PROGRESS ACROSS THE INTERFACE SOME MORE. SO USING THE FIRST SESSION WILL BE ON BLOOD-BRAIN BARRIER MODELING. SO, USING SOME OF THE ISSUES ENCOUNTERED IN SCIENCE IN BOTH SCIENCES, AND SEEING HOW BEST TO UTILIZE TECHNOLOGIES THAT ARE AVAILABLE AND DEVELOP FURTHER TECHNOLOGIES THAT CAN APPLY TO ADDRESS SOME OF THOSE QUESTIONS. AND THEN WE WILL END WITH A SESSION ON THE BLOOD-BRAIN BARRIER DELIVERY AND TARGETING. THIS IS WHAT YOU'RE THINKING ABOUT. HOW DO WE GET THINGS INTO THE BRAIN, OUT OF THE BRAIN AND WHAT ARE THE SEQUELAE OF BLOOD-BRAIN BARRIER DISRUPTION OR HOW TO GET AROUND IT FOR THERAPEUTIC APPROACHING TO CLINICAL ISSUES AND THINKING ABOUT THE PATIENT IN MIND. AND SO, WITHOUT FURTHER ADIEU, I WANTED TO INTRODUCE THE KEYNOTE SPEAKER FOR TODAY AND IT JUST SO HAPPENED IT WAS VERY FORTUITOUS THAT HIS RECENT PAPER CALLED, FROM BLOOD-BRAIN BARRIER TO BLOOD-BRAIN INTERFACE, NEW TUNES FOR CNT DRUG DISCOVERIES SO IT FELL NICELY INTO THE TOPIC OF THIS WORKSHOP. SO WE DIDN'T COLLABORATE IN ADVANCE BUT IT JUST SO HAPPENED. SO DR. BANKS IS THE ASSOCIATE CHIEF OF STAFF RND AT SEATTLE VA AS WELL AS A PROFESSOR OF MEDICINE AT UNIVERSITY OF WASHINGTON. AND HIS WORK HAS FOCUSED ON BRAIN BODY COMMUNICATION AS MEDIATED BY INTERACTIONS BETWEEN THE BLOOD-BRAIN BARRIER AND PEPTIDES, REGULATORY PROTEINS AND OTHER INFORMATIONAL MOLECULES. AND HE WILL PRESENT OR TALK TODAY THROUGH LUNCH THE SECOND DAY OF THE WORKSHOP ON THE BLOOD-BRAIN INTERFACE. THANK YOU DR. BANKS. >> WILLIAM BANKS: THANK YOU VERY MUCH. THANK YOU TO THE ORGANIZERS FOR INVITING ME. AND I REALLY LEARNED A LOT FROM THE FIRST DAY. I HAVE SEEN THE BLOOD-BRAIN BARRIER DEFINED IN VARIOUS WAYS AND VIEWS FROM A VARIETY OF VIEWPOINTS. BUT I HAVEN'T HEARD ANYONE TALK ABOUT THE PHILOSOPHY OF THE BLOOD-BRAIN BARRIER, WHICH I THINK IS PROBABLY DECIDE EDLY DO YOUEST. I WOULD LIKE TO EXPLORE THIS DICHOTOMY OF THE BLOOD-BRAIN BARRIER. IS IT A BARRIER? IS IT A INTERFACE? IS IT BOTH OR SOMETHING ELSE? SO THIS IS THE OPPOSITE END OF THE SPECTRUM. THIS IS A BLOOD VESSEL GOING THROUGH A KIDNEY -- I'M SORRY, THROUGH A LIVER. THERE IS JUST LOTS AND LOTS OF HOLES GOING THROUGH THE ENDOTHELIAL CELL AND IF WE COULD SEE WHERE IT ATTACHES TO THE NEXT ENDOTHELIAL CELL, YOU WOULD CY SEE A LOT OF GAPS AND JUNCTIONS THERE AS WELL. SO THESE TRANSSECT MARA CELLULAR PATHWAYS PERFORM A VERY IMPORTANT FUNCTION AND THEY PRODUCE THE ULTRAFILTRATE THAT NOURISH THE TISSUES THROUGH WHICH THEY GO. SO, IF WE WERE TO BUILD A BLOOD-BRAIN BARRIER FROM A PERIPHERAL VESSEL, FIRST MODIFICATION WE WOULD HAVE TO DO IS GET RID OF THESE VARIOUS TRANSSIGH TO THETIC PATHWAYS, SEAL THEM UP WITH TIGHT JUNCTIONS. AND NOW WE HAVE THE PHYSICAL BLOOD-BRAIN BARRIER. BUT THE BARRIER HAS OTHER TRICKS BESIDES THAT. THERE IS ALSO ENS MADDIC BARRIER TO MANY SUBSTANCES. MONOAMINES BEING THE CLASSIC EXAMPLE AND EFLUX SYSTEMS PREDOMINATE FOR OTHER SUBSTANCES THAT TRY TO GET INTO THE BRAIN, ARE KEPT OUT BY THE EFLUX SYSTEMS W THESE VARIETY OF MECHANISMS, PHYSICAL, ENZYMATIC, TRANSPORT, THE BLOOD-BRAIN BARRIER BECOMES VERY SUBSTANTIAL. IF WEPT TO GO FURTHER AND BUILD ITS INTERFACE PROPERTIES, WE HAVE TO FIRST SORT OF GIVE BACK SOME OF THE FUNCTIONS THAT WERE TAKEN AWAY BY THE LOSS OF THE TRANSSIGH TO THETIC PROCESS. SO WE CAN GIVE TRANSPORTERS THAT NOW NOURISH THE BRAIN FOR GLUCOSE AND AMINO ACIDS AND ELECTROLYTES, AND ALSO TRANSPORTERS FOR INFORMATIONAL MOLECULES, FOR EXAMPLE THE CYTOKINES. AND INSERT THEM INTO CYTOSIS OR OTHER PROCESSES, DIE PA DEESEIS USES FOR IMMUNE CELLS TO CROSS. SO ALL OF THESE, THE BRAIN IS NOW ABLE TO RECEIVE SUBSTANCES FROM THE BLOOD. THE BLOOD-BRAIN BARRIER IS ALSO IN COMMUNICATION WITH PERIPHERAL TISSUES. BOTH THE CIRCULATING IMMUNE CELLS AND ALSO BY WAY OF THE BLOOD FROM DISTANT ORGANS THAT ARE SECRETED SUBSTANCES INTO THE BLOOD AND ALSO RESPONDS TO SIGNALS FROM THE NEURONS FROM THE BRAIN. AND LISTED JUST THREE HERE ALL OF WHICH ARE NEUROAMINE SUBSTANCES, INTERLEUKINS, NITRIC OXIDE AND PROSTAGLANDINS. BLOOD-BRAIN BARRIER ARE EQUALLY IMPORTANT ALSO RELEASES THESE VARIOUS SUBSTANCES INCLUDING CYTOKINES, THAT DID CREATE A TALK WITH THE PERIPHERAL TISSUES ANNOUNCEMENT WE HAVE EVERYBODY TALKING TO EACH OTHER AND NOW WE HAVE A TRUE INTERFACE. AND INDEED, THERE ARE VERY INTERESTING THINGS THAT HAPPEN, ALL KINDS OF VARIATIONS FOR THIS, FOR EXAMPLE, IN THE BLOOD-BRAIN BARRIER'S ABILITY TO SECRETE CYTOKINES CONSTITUTIVELY, IT CAN ALSO BE INDUCED TO SECRETE THEM. SO ONE THING HERE IS THAT FOR EXAMPLE, THE BLOOD-BRAIN BARRIER CAN RECEIVE IMMUNE SIGNAL ON ONE SIDE OF THE BARRIER AND THEN RELEASE SUBSTANCE ON THE OTHER SIDE. FOR EXAMPLE, IF YOU PUT LPS IT WILL RELEASE IL6 INTO THE LUMINAL SIDE AND IT WILL RELEASE MORE IL6 ON THE LUMINAL SIDE THAN IF YOU PUT THE LPS ON THE LUMINAL SIDE. SO IF WE JUST STOP HERE WE CAN THINK ABOUT, LET'S TAKE THE NEUROIMMUNE AREA FOR EXAMPLE, WHAT HAVE WE HAD HERE? THE BARRIER FUNCTION MAKES THE BRAIN SO-CALLED IMMUNOPRIVILEGED AREA AND YET WE ALREADY DEFINED FOUR DIFFERENT NEUROIMMUNE AXIS THAT ARE THEN REENDOWED BY THE BLOOD-BRAIN BARRIER. THE TRANSPORTER SIGHTY KINES ACROSS THE BLOOD-BRAIN BARRIER THAT,TA LAOS THE BARRIER TO ADHERE SOME OF THE IMMUNE ACTIONS IN THE PERIPHERY. WE HAVE THE BLOOD-BRAIN BARRIER RESPONDING TO CYTOKINES THAT IT MAY RECEIVE FROM EARTH THE BRAIN SIDE OR THE BLOOD SIDE. WE HAVE THE BLOOD-BRAIN BARRIER SECRETING CYTOKINES INTO THE BRAIN OR BLOOD AND WE HAVE THIS KIND OF VERY INTERESTING TRANSDUCTION PROCESS WHERE THE BLOOD-BRAIN BARRIER COULD RECEIVE A SIGNAL ON ONE SIDE AND COMMUNICATE IT OUT OF THE OTHER SIDE. AND FINALLY, IF WE WANT TO BUILD IN NEUROVASCULAR UNIT FURTHER AND FURTHER CROSS TALK AND INTERFACE ASPECTS, WE REALIZE THAT THE BLOOD-BRAIN BARRIER IS TALKING NOT JUST TO NEURONS AND PERIPHERAL TISSUES BUT ALSO THE PERISIGHTS, EXTRACELLULAR MATRIX IS ALSO BEING VIEWED NOW NEUROIMMUNE REGULATIONS AND VARIOUS SIZES. WE ARE TALKING ABOUT MICROGLIA AND ASTROCYTES AND NOW THE MASS CELL IS BACK. PEOPLE HAD FORGOTTEN ABOUT THE MASS CELL FOR ABOUT 20 YEARS. IT'S BACK NOW. SO, THIS MEANS THAT BEYOND THE BARRIER, BESIDES PROTECTING THE BLOOD BRAIN BARRIER, IT IS ALSO PROVIDING NUTRITIONAL ROLE, HOMEOSTATIC SCROLL COMMUNICATION ROLE. AND SO, WITH ALL OF THIS COMPLEX PHYSIOLOGY, THE BLOOD-BRAIN BARRIER IS ABLE TO DO A LOT OF THINGS IN TERMS OF CONNECTING INTERFACE BETWEEN PERIPHERAL TISSUES AND THE CENTRAL NERVOUS SYSTEM AND WITH THIS COMPLEX PHYSIOLOGY, COMES THE OPPORTUNITY FOR DISEASE TO OCCUR. IF SOMETHING WORKS, IT CAN BREAK AND THAT CERTAINLY IS OCCURRING IN DISEASE STATES. AND WHEN THINGS BREAK IT ALSO PROVIDES OPPORTUNITIES FOR DRUG DELIVERY. SO IF THE DISEASE PROBLEMS AT THE BLOOD-BRAIN BARRIER, IT HAS UNIQUE PROPERTY OF PERHAPS BEING ABLE TO TREAT THE CENTRAL NERVOUS SYSTEM DISEASE WITHOUT HAVING THE DRUG CROSS THE BLOOD-BRAIN BARRIER, SORT OF ONE OF THE ASPECTS OF THE BLOOD BRAIN BARRIER. IS IT IN THE BRAIN OR OUT OF THE BRAIN? YES, IT IS BOTH. SO THAT MEANS THAT HALF OF THE BLOOD-BRAIN BARRIER IS IT EXPOSED TO CIRCULATING PERIPHERAL THERAPEUTICS. WHAT I WOULD LIKE TO DO WITH THE REST OF MY TALKING IS EXPLORE THE PHYSIOLOGY THAT ARISES FROM THIS COMPLEXITY. THE DISEASE STATES THAT ARISE AND SOME OF THE DRUG OPPORTUNITIES THAT ALSO ARISE FROM THAT COMPLEXITY. SO, A GREAT EXAMPLE OF HOW THE BLOOD-BRAIN BARRIER IS INVOLVED IN PHYSIOLOGY IS LEPTIN SECRETED BY THE FAT MASS. THE MORE OBESE OR LES OBESE, YOUR LEPTIN LEVELS REFLECT YOUR ADIPOSITY T IS TRANSPORTED ACROSS THE BLOOD-BRAIN BARRIERS 16,000 DALTONS SO -- TOO BIG TO GO BY ANY OTHER WAY. BY ITS OWN TRANSPORT THERE IT INTERACTS WITH BRAIN RECEPTORS IN THE NUCLEUS AND THERE IT TENDS TO PRODUCE ANOREXIA AND TO INCREASE THERMOGENESIS. AND THOSE EVENTS REDUCE FAT MASS SO WHAT WE HAVE HERE IS A NEGATIVE FEEDBACK LOOP MEDIATED IN PART BY THE BLOOD-BRAIN BARRIER. SO WE'LL DEBATE IT BUT LEPTIN IS EXACTLY DOING ITS EVOLUTIONARY ROLE TO INFORM THE BRAIN WHAT YOUR FAT RESERVES ARE. SO THAT YOU CAN THEN USE EXTRA CALORIES INSTEAD OF LOOKING FOR MORE FOOD, YOU CAN GO ON AND THE REPRODUCE OR SUPPORT YOUR IMMUNE SYSTEM OR DO THESE OTHER THINGS THAT ARE VERY EXPENSIVE. THERE ARE A LOT OF OTHER ASPECTS OF THE BLOOD-BRAIN BARRIER THAT WE KNOW ARE RELATED TO VARIOUS PHYSIOLOGICAL FUNCTIONS AND I LISTED JUST A FEW HERE. SOME OF THESE HAVE ALREADY BEEN TALKED ABOUT BY OTHER SPEAKERS. INSULIN IS TRANSPORTED ACROSS THE BLOOD BRAIN BARRIER AND SEEMS TO HAVE COGNITIVE ASPECTS ANDISM THAT'S THE SYMPATHETIC NERVOUS SYSTEM AND CONTROLS 50% OF HEPATIC GLUCOSE PRODUCTION. GHRELIN TRANSPORT STIMULATES APPETITE AND SEEMS TO BE A YING TO THE LEPTIN'S YANG. WE TALKED ABOUT AMYLOID BAIT AT PEPTIDE EFLUX BEING IMPORTANT TO THE REGULATION OF BRAIN LEVELS AND HOW THE BLOOD-BRAIN BARRIER DOES DOES THAT THROUGH TRANSPORT SYSTEMS AND ALSO THROUGH CSF REAB SOUTHERLIES IN THE GLIM FATTICS AND NEUROIMMUNE ADAPTATIONS INCLUDE EVERYTHING FROM SICKNESS BEHAVIORS TO ADAPTATIONS FOR SLEEP. SO, LET'S TALK ABOUT WHEN THE BLOOD-BRAIN BARRIER BREAKS OR WHEN THINGS HAPPEN. THIS IS THE OBOB MOUSE. IT'S A VERY OBESE MOUSE. THIS MOUSE WEIGHS ABOUT 90 GRAMS. LILTER MATES WOULD WEIGH ABOUT 30 GRAMS. THOSE EXTRA 60 GAMS THREE TIMES NORMAL SIZE ARE FAT. AND IT IS BECAUSE IT DOES NOT PRODUCE LEPTIN. SO YOU SEE HOW POWERFUL LEPTIN IS. BUT HUMANS DON'T HAVE LEPTIN DEFICIENCY. WE HAVE LEPTIN RESISTANCE. SO IT'S LIKE TYPE I AND TYPE II DIABETES. WE HAVE LEPTIN RESISTANCE. IT OCCURS AT TWO LEVELS AT THE CNS RECEPTOR AND ALSO PRIOR TO THAT, AND THE BLOOD-BRAIN BARRIER RESISTANCE AND INABILITY OR LOSS OF THE ABILITY TO AS EFFICIENTLY TRANSPORT LEPTIN AS IT SHOULD. AND MODELING SUGGESTS THAT IN EARLY OBESITY AND MODERATE OBESITY, THAT THE BLOOD-BRAIN BARRIER IS THE MAJOR PROBLEM AND THEN AS OBESITY PROGRESSES, EVENTUALLY NOTHING WORK ANYMORE. SICKNESS BEHAVIOR, WHICH IS CONSIDERED AN ADAPTIVE BEHAVIOR S WHAT YOU GET WHEN YOU GET FLU OR COLD AND YOU DON'T FEEL GOOD AND YOU WANT TO REST AND CONSERVE YOUR ENERGY AND THAT IS HOW A MEDIATED BY INTERLEUKIN ONE AND HAS A VARIETY OF SYMPTOMS INCLUDING NOT A GREAT ABILITY TO LEARN. WE ALL HAD THIS. WE JUST WANT TO SLEEP AND DON'T WANT TO TABLING IN NEW INFORMATION AND PROCESS IT. AND WHAT WE ARE SHOWING IS THAT INTERLUKE I BELIEVE 1 CROSSING THE BLOOD-BRAIN BARRIER REGION OF THE BRAIN CALLED THE POSE TEARIER DIVISION OF THE SEP TUM INTERACTS WITH VARIOUS PATHWAYS THAT ULTIMATELY CAUSE COGNITIVE IMPAIRMENT. SO IT'S A DIRECTITANCE PORT OF INTERLEUKIN 1 THAT CROSSES THE BLOOD-BRAIN BARRIER THAT MEDIATES THIS ACTION. INTERLEUKIN 1 ACTS AT THE BLOOD-BRAIN BARRIER TO INDUCE FEVER IN A DIFFERENT WAY AND ACTS TO INDUCE AND PRODUCE PROSTAGLAND THANES MEDIATE THE FEVER. SO AGAIN THE EXAMPLES OF RECEIVING INPUT ON ONE SIDE AND TRANSLATING IT INTO THE OTHER. AND I MENTIONED YESTERDAY THAT THIS MAY BE ONE WAY THAT TRAUMATIC BRAIN INJURY COULD OCCUR. THIS IS VERY SPECULATIVE. THAT COULD OCCUR WHEN THE BODY IS INVOLVED ALSO FOR EXAMPLE THAT SHOCK WAVES GOING THROUGH THE GUT SEEM TO INCREASE LPS LEVELS IN THE BLOOD AND THEN THAT COULD START OFF CYTOKINE CASCADE THAT ALSO IS TRANSDUCED ACROSS INTO THE BRAIN. WE HAVE HEARD TALK ABOUT A BETA DYSREGULATION AND A BETA IS CLEARED FROM THE CENTRAL NERVOUS SYSTEM BY ABSORPTION THROUGH THE LYMPHATICS AND BULK FLOW. AND ALL OF THOSE PROCESSES ARE EITHER DISRUPTED OR IMPAIRED IN NOT ONLY ALZHEIMER'S DISEASE PATIENTS BUT EVEN IN THE ANIMAL MODELS, THE GENETIC MODEL OF SAND PA AS WELL AS THE TRANSGENIC MODELS HAVE ALL THREE OF THESE LESIONS. ANOTHER WAY THE BLOOD BRAIN BARRIER IS INVOLVED IN PRODUCING DISEASES IS DISRUPTION IN DIABETES. WE LEARNED QUITE A BIT ABOUT THIS. SO WHAT HAPPENS IS THE EXCESS GLUCOSE ENTER THE IN BRAIN IS TAKEN YOU UP BY THE VARIOUS TISSUES INCLUDING PAIR I SIGHTS AND REMEMBER THE USE OF GLUCOSE IS NOT INSULIN DEPENDENT IN THE BRAIN. SO THE MORE GLUCOSE YOU HAVE, THE MORE THAT GOES THROUGH THE CREB CYCLE PRODUCING MORE ATP BUT ALSO MORE REACTIVE OXIDATIVE STRESS AND REACTIVE OXIDATIVE STRESS KILLS THE PARASITES AND AFTER 16 WEEKS OF DIABETES IN A RODENT, ENOUGH BERA SITES HAVE DIED THAT BLOOD-BRAIN BARRIER DISRUPTIONS OCCUR LOCALLY. THIS WORK WAS DONE BY JASON HUBER AND THE PARASITE WORK WAS DONE IN OUR LAB WITH COLLABORATORS IN ST. LOUIS. BUT THIS MECHANISM HAS BEEN KNOWN TO UNDERLIE THE RETINOPATHY DISRUPTION OF DIABETES WHICH IS THE MAJOR CAUSE OF BLINDNESS. NOW WE SEE THE SAME PROCESS GOING ON IN THE BLOOD BRAIN BARRIER. SO NOW I'D LIKE TO FLIP OVER NOW THAT WE TALKED ABOUT PHYSIOLOGY AND PATHOPHYSIOLOGY, I'D LIKE TO TALK ABOUT DRUG DEVELOPMENT. WE MIGHT AS WELL START WITH THIS ONE. SO, THIS SATURDAY CREB CYCLE SO THE MORE GLUCOSE YOU HAVE COMING INTO THE SYSTEM THE MORE ATS PRODUCED AND ALSO THE MORE REACTIVE OXYGEN SPECIES. SO IF ONE COULD DISRUPT THIS CYCLE, THEN YOU COULD IN THEORY PREVENT BLOOD-BRAIN BARRIER DISRUPTION IS ONE WAY TO DO IT IS HERE WITH MITOCHONDRIAL CARBONIC HYDRAZINE HIBITOR. TWO ARE ON THE MARKET. THAT WOULD PREVENT PRODUCTION OF BY CARBONATE WHICH WOULD PREVENT PYRUVATE FROM ENTERING THE CREB CYCLE AND THERE SHOULD BE TWO CONSEQUENCES FOR PHYSIOLOGY. ONE, OXYGEN CONSUMPTION WHICH YOU SEE IS INCREASED HERE IN THE HYPERGLYCEMIC STATE, SHOULD GO DOWN. AND THAT IS INDEED WHAT HAPPENS SO WE HAVE KNOCKED OFF THE EXCESS GLUCOSE USE BUT THERE IS THE LACTATE PRODUCTION SHOULD INCREASE BECAUSE THIS WILL SHUT THE PA ROOF 8 SHUTTLED INTO THE LACTATE PATH WAY AND SO, THE HYDROGENS USED WILL GO UP WITH THE HYPOGLYCEMIA AND BE MAINTAINED AND CONTINUE TO BE UP AND THAT IS WHAT WE SEE. SO WE KNOW THIS IS EXACTLY WORKING THE WAY WE SHOULD AND AFTER ABOUT 16 WEEKS OF DIABETES, 10 OF THE FIVE REGIONS OF THE BRAIN THAT WE LOOKED AT HAD STATISTICALLY SIGNIFICANT INCREASES IN BLOOD-BRAIN BARRIER PERMEABILITY TO SUCROSE AND A FEW OTHER AREAS HAD STATISTICAL TRENDS BUT DIDN'T MAKE IT STATISTICALLY AND SURE ENOUGH THREE OF THESE ARE REVERSED WITH BY CARBONATE -- INHIBITORS. AND FOURTH ONE HAD A STATISTICAL TREND. SO IT SEEMS THAT THIS DRUG WOULD WORK PERHAPS FOR RETINOPATHY AS WELL. ANOTHER WAY TO DEVELOP DRUGS IS TO MANUFACTURE A WAY FOR THEM TO CROSS THE BLOOD-BRAIN BARRIER. WE TALKED ABOUT HOW LEPTIN, THE INABILITY OF LEPTIN TO CROSS PREVENTS OR RESULTS IN OBESITY. SO ONE IDEA WOULD BE TO ADAPT A TRANSPORTER TO GET LEPTIN IN. AND WHAT WE HAVE DONE THIS WORK WITH FOLKS AT THE UNIVERSITY OF NORTH CAROLINA, DEVELOP A SERIES OF POPULAR ONTIC LEPTIN THAT IS CAN USE OR AB SOARED PSYCHOTIC MECHANISM TO ENTER THE BRAIN AND ACCESS THE NUCLEUS THAT WAY. SO FAR WE DEVELOPED A DRUG POTENT ENOUGH TO CAUSE WEIGHT LOSS IN OBOB MICE. BUT THAT'S EASY ONE. WE ARE TRYING TO SHOW THAT IT CAN ALSO BE EFFECTIVE IN DIET-INDUCED OBESITY, BUT WE CAN FLIP THIS AROUND AS WELL F WE COULD BLOCK LEPTIN RESISTANCE, WE MIGHT BE ABLE TO TREAT ANOREXIA. MY CLINICAL WORK IS A GERIATRICIAN AND WE SEE A LOT OF OLDER FOLKS WHO JUST MELT AWAY IN THEIR OLD ANAL. THEY JUST START LOSING WEIGHT FOR NO ACTIONER PARENT REASON. SOCIETY, ANOREXIA IS A BIG PROBLEM IN THE ELDERLY AS WELL AS FOR ANOREXIA NERVOSA AND OTHER CONDITIONS. AND INDEED, ONE CAN BLOCK THE TRANSPORTER FOR LEPTIN. WE WORKED WITH COLLEAGUES IN ISRAEL WHO DEVELOPED A PEG LATED LA AND IT PREVENTS THE LEPTIN FROM CROSSING AND ACTS AS A PERIPHERAL BLOCKER. ITS IS DOES NOT CROSS BUT BY BLOCKING THE TRANSPORT TERPREVENTS LEPTIN FROM GETTING INTO THE RAIN. IT DOES. THIS IS THE FIRST GENERATION. A SECOND GENERATION NOW THAT IS MORE POTENT BUT AFTER THE SECOND DAY OF TREATMENT YOU BEGIN TO SEE INCREASE IN BODY WEIGHT AND WHEN YOU STOP THE DRUG, IT RETURNS TO NORMAL LEVEL. ONE CAN DISCOVER TRANSPORTERS. I THINK THIS AUDIENCE IS VERY AWARE THAT THERE IS MANY TRANSPORTERS THAT ARE KNOWN FOR BLOOD-BRAIN BARRIER BUT WE KNOW THERE ARE MANY THAT AREN'T DISCOVERED YET AND ERIC CHUTE IS DOING A GREAT JOB OF HELPING US CLASS THOSE BUT THERE ARE MANY, MANY MORE THAT ARE UNDISCOVERED. SO WE JUST DISCOVER THEM QUITE OFTEN SERENDIPITOUSLY. THAT WAS THE CASE, FOR EXAMPLE WITH THE PHOSPHOTHIGHOID MOLECULES THAT CAN BE USED AS ANTISENSE. BECAUSE THESE ARE ENZYMATIC ROCKS, THEY HAVE LONG, LONG CIRCULATION TIME IN THE BLOOD. AND HERE IS THE PERCENT OF INJECTED DOSE INTERVENOUSLY INJECTED DOSE OF ANTI-SENT DIRECTED AGAINST APP THAT GETS INTO THE BRAIN. THIS IS ABOUT 50 TIMES MORE THAN THE AMOUNT OF MORPHINE THAT GETS INTO THE BRAIN PLUS SUSTAINED OVER A LONG PERIOD OF TIME AND THIS IS WHAT IT DOES TO A BETA LEVELS IN VARIOUS REGIONS OF THE BRAIN, REDUCES THEM TO ABOUT HALF. AND THIS IMPROVES RETENTION AND ACQUISITION LEARNING AND MEMORY, IN ANIMAL MODEL AD AND IT ALSO RESULTS IN THE RECOVERY OF LRP AND PGP FUNCTION AND ALSO THE RECOVERY OF CSF REABSORPTION PROBLEM WE TALKED ABOUT AND ALSO REDUCES OXIDATIVE STRESS INDUCED BY A BETA. I JUST WANT TO TALK ABOUT OUR EXOSOME WORK. THEY LOOK LIKE THEY DO CROSS THE BLOOD-BRAIN BARRIER. WE HAVE SHOWN THAT IN PARKINSON'S DISEASE WITH COLLEAGUES AT UNIVERSITY OF WASHINGTON, IN DR. JENNINGS LAB, IN THE BLOOD THERE IS INCREASED LEVEL OF ALPHA NUCLIEN EXOSOMES AND WE JUST PUBLISHED THIS WEEK WE THINK THERE IS ALSO INCREASE IN TAU EXOSOMES. WE THINK THESE ARE COMING FROM THE BRAIN. THE REASON WE DO THAT IS BECAUSE IF WE INJECT RADIOACTIVE ALPHA NUCLIEN INTO THE LATERAL VENTRICLE, IT APPEARS IN THOSE EXOSOMES OUT IN THE BLOOD. RADIOACTIVE NUCLIEN. SO WE THINK APPROXIMATE IS GOING THIS WAY COMBINING WITH THE EXOSOMES IN THE BRAIN AND THEN BEING TRANSDUCED OUT ACROSS THE BLOOD-BRAIN BARRIER. ALTHOUGH WE ALSO SHOWN ALPHA SYNUCLEIN TRANSPORTS OUT IN ITS OWN RIGHT. THE COMBINATION COULD BE HAPPENING IN THE PERIPHERY. WE HAVEN'T WORKED THAT OUT YET. THE OTHER WAY IS TO USE FOR DRUG DELIVERY AND AGAIN WITH THE LAB, THEY DEVELOPED SOME MICROPHAGE DERIVED EXOSOMES THAT CROSS THE BLOOD-BRAIN BARRIER AND USE I CAM AND LAF1, ESSENTIALLY IT SEEMS THAT THEY ARE IMITATING WHAT A MACROPHAGE DOES THAT IT IS USING THIS TO ENTER THE BRAIN. AND LIKE A MACROPHAGE IF WE TREAT ANIMALS WITH LPS-INDUCED INFLAMMATION, BDF TRANSPORT INCREASES BY 3 FOLD. SO WE THINK THAT THIS CAN BE USED TO DELIVER DRUGS TO THE BRAIN AND PROBABLY EVEN TO TARGET CERTAIN BRAIN REGIONS. ONE CAN ALSO INHIBIT EFLUX. WE DISCOVERED OR WANTED TO GET PAY CAP INTO THE BRAIN. THERE IS TWO FORMS OF PAY CONDITION. THERE IS PAY CAP 38 WHICH CROSSES QUITE WELL AND IS EFFECTIVE IN STROKE AND AD MODELS AND ANIMAL MODELS BUT THE PROBLEM IS IT IS A VERY YUCKY PEP TIGHT TO WORK WITH. VERY STICKY. WE WOULD LIKE TO USE PACAP27. IT'SITANT PORTED INTO THE BRAIN AS WELL BUT ALSO TRANSPORTED OUT OF THE BRAIN. SO IS IT DOESN'T REALLY ACCUMULATE VERY WELL. SO WE DISCOVERED THAT THE EFLUX TRANSPORTER WAS BETA F ONE ATPAC. WE BUILT THE -- AND SO WE BUILT ANTI-PHOS FADE TO IT AND WE WERE INDEED ABLE TO THUS INCREASE PACAP ACCUMULATION IN THE BRAIN BY FOURFOLD BY JUST INHIBITING E FLUX TRANSPORTER. IT IS VERY SPECIFIC FOR 27PACAP38 WASN'T AFFECTED AND OTHER ANTISENSES WE DEVELOPED DIDN'T AFFECT THE E FLUX EITHER. SO WE HAD QUITE GOOD SPECIFICITY. AND WHEN WE NOW GIVE PACAP27 WE ARE ABLE TO SHOW WE GOT EFFICACY IN STROKE MODELS AND IN ALZHEIMER'S DISEASE MODELS. WE CAN ALSO MODULATE TRANSPORT. LYSOSOMAL STORAGE DISEASES LACK ENZYMES AND THEY DON'T CROSS THE BLOOD-BRAIN BARRIER OF THE ADULT BUT IRONICALLY, THE GUS B, THE ENZYME DISMISSING IN SLY SYNDROME, CROSSES THE BARRIER OF THE NEONATE BUT THE NEONATE LOSES THE ABILITY TO CROSS THE ABOUT. BB WITH MATURATION. IT'S A OCCURRING THEME T JUST GOES AWAY EVERYWHERE. AND WE FOUND THAT THIS IS ACTUALLY THE RECEPTOR THAT IS ALSO ACTING AS A TRANSPORTER FOR THAT. AND WHAT WE CAN DO FOR REASONS I DON'T UNDERSTAND, SERENDIPITOUS FINDING IF WE TREAT THE ANIMAL WITH ALPHA ADD NERGIC, IT REINSTITUTES THE ACTIVITY OF AMMAN 06 PHOSPHATE RECEPTOR AND WE ARE ABLE TO DELIVER MAN 06 PHOSPHATE TO THE BRAIN OF THE MOUSE AT THE SAME RATE AS THAT TRANSPORTER IS IN THE ADULT OR IN THE NEONATE. HERE IS MY SUMMARY. I THINK I'M ALMOST ON TIME. THERE IS A LOT OF NEAR PUTIC TUNES THAT THE BLOOD-BRAIN BARRIER SIMPLY BECAUSE IT IS COMPLEX AND THAT WE TALKED ABOUT VARIOUS WAYS OF DOING THIS, TO USE TRANSPORTERS, TO INHIBIT EFLUX TRANSPORTERS TO GOOSE UP TRANSPORTERS THAT WE ALREADY HAVE THERE, TO INHIBIT TRANSPORT OF SUBSTANCE THAT IS WOULD BE ACTING PHYSIOLOGICALLY. NOTICE SOME OF THESE DON'T REQUIRE THE SUBSTANCE TO ACROSS THE BARRIER. AND TO USE OTHER PATHWAYS. OR THE EXTRACELLULAR PATHWAYS FOR ANTIBODY, LARGE STABLE MOLECULE SYSTEM THAT WE DIDN'T HAVE TIME TO TA -- TALK ABOUT. AND THERE ARE OTHER VARIOUS STRATEGIES AS WELL. THANK YOU VERY MUCH. APPLAUSE. >> THANK YOU. WE MIGHT HAVE TIME FOR JUST ONE QUESTION, OTHERWISE IF YOU'RE AVAILABLE FOR OPEN MIC THIS AFTERNOON. >> YES. >> GREAT. WE'LL HAVE YOU JOIN OPEN MIC DISCUSSION. THANK YOU. SO NOW WE ARE MOVING ON IF THE FIRST SESSION OF THE DAY. THAT IS GOING TO BE CHAIRED BY DR. PARTY SEARSON. DR. PETER SEARSON. >> PETER SEARSON: SO THIS MORNING'S SESSION IS ABOUT BLOOD-BRAIN BARRIER MODELS AND OVER THE LAST 10 YEARS OR SO AS IT SAYS HERE, THERE HAVE BEEN MAJOR DEVELOPMENTS IN A NUMBER OF AREAS STEM CELL, BIOLOGY IN PARTICULAR, TISSUE ENGINEERING, BIOMATERIALS, MICROFABRICATION AND MICROFLUIDICS, THAT PROVIDE NEW OPPORTUNITIES TO ENGAGE ENGINEERS IN PARTICULAR BUT ALSO NEUROBIOLOGIST, NEURO50s PHARMACOLOGISTS AND SO ON TO ADVANCE THE DEVELOPMENT OF IN-VITRO MOTED ELSE OF THE BBB AND THAT WILL BE OUR FOCUS THIS MORNING. SO THE ADVANTAGES OF IN-VITRO MODELS IF YOU CAN MAKE THEM WORK ARE THE POTENTIAL TO USE HUMAN CELL LINES BOTH HEALTHY CELLS AND ALSO MIMIC DISEASE, HAVE WELL-CONTROLLED VARIABLES AND THE ABILITY TO DO HIGH-RESOLUTION IMAGING. AND OF COURSE WHAT WE GIVE UP IS PHYSIOLOGICAL RELEVANCE OF THE LIVING ORGANISM. BUT I THINK YOU'LL SEE AS YOU'LL SEE THIS MORNING FROM THE SPEAKERS, THAT MODELS OVER THE LAST FEW YEARS ARE GETTING MUCH, MUCH CLOSER IN TERMS OF PHYSIOLOGICAL RELEVANCE AND HOPEFULLY WILL ATTRACT MUCH MORE INTEREST IN THE COMMUNITY IN GENERAL. AND THE CHALLENGES I THINK ARE FAIRLY WELL UNDERSTAND IN TERMS OF THE NEED FOR BRAIN-SPECIFIC CELL LINES, CO-CULTURE, UNDERSTANDING CELL-CELL INTERACTIONS AND OR RECAPITULATING THEM AND DEVELOPMENT OF MATRIX MATERIALS AND RIGHT PHYSICAL PARAMETERS AND PROFUSION AND GEOMETRY AND THE BIG ISSUE AT THE MOMENT IS VALIDATION. HOW DO YOU KNOW WHEN YOU HAVE RECAPITULATED WHATEVER ASPECTS OF THE BLOOD BRAIN BARRIER YOU'RE INTERESTED IN. SO THIS MORNING, WE HAVE IN ADDITION TO ME WE HAVE FOUR TALKS FROM JACQUELYN BROWN AT VANDERBILT ON NEUROVASCULAR UNIT ON A CHIP AND IKE SHUSTA ON STARLETTING BLOOD-BRAIN BEAR GER AND HEALTH AND DISEASE. AND LAURA PASQUALUCCI WILL BE TALKING ABOUT NEUROFIERCE, ORGANOGENESIS AND MARTIN WILL BE TALKING ABOUT MOLECULAR DYNAMIC SIMULATIONS, ALL ATOM SIMULATIONS OF TRANSPORT ACROSS THE BBB. SO THIS IS SORT OF MY OVERVIEW IF YOU LIKE OF WHERE WE ARE AT THE MOMENT JUST HIGHLIGHTING ADVANCES IN TISSUE ENGINEERING, STEM CELL TECHNOLOGY, MODELING AND SO ON. SO IN THE TOP BOX IF YOU LIKE, WE HAVE MICROFABRICATION, MICROFLUIDICS AND TISSUE ENGINEERING AND SO ON AND THAT FIELD HAS TWO EXTREMES IF YOU LIKE. THE ORGAN ON A CHIP LEVEL. WE'LL SEE SOME VERY SOPHISTICATED TECHNOLOGY AT THAT END FROM JACQUELYN A LITTLE LATER AT THE OTHER END WE HAVE THE ORGANOGENESIS FIELD WHERE THE IDEA IS BASICALLY TO TAKE STEM CELLS IN THE RIGHT ENVIRONMENT AND HAVE THEM ASSEMBLE BASICALLY ORGANS OR TISSUES. I'M GOING TO TALK ABOUT MY WORK WHICH IS SOMEWHERE IN BETWEEN THE TWO HERE. CELL LINES HAVE LONG BEEN A MAJOR CHALLENGE IN IN-VITRO BLOOD-BRAIN BARRIER MODELS AND I THINK AS MOST OF YOU PROBABLY KNOW, IN PARTICULAR, THE BRAIN MICROVASCULAR ENDOTHELIAL CELLS, THE SOURCE OF A GOOD CELL LINE FOR IN-VITRO MODELS HAS BEEN A MAJOR CHALLENGE FOR MANY YEARS AND LARGELY THANKS TO ERIC SHUSTA, I THINK WE PROBABLY HAVE THAT PROBLEM SOLVED IN THIS POTENTIAL NOW FROM STEM CELL BIOLOGY TO DERIVED ASTROCYTES AND PARASITES AND SO ON. AND I THINK ALSO WITH RESPECT TO MODELING, THAT HAS BEEN LARGELY LIMITED TO ANALYSIS OF PERMEABILITY DATA AND TRYING TO USE THAT WITH INTELLIGENT SYSTEM TO PREDICT PERMEABILITY OF OTHER MODELS BECAUSE WE ARE ALSO GOING TO SEE ADVANCES IN MOLECULAR DYNAMICS SIMULATIONS AND NOW GIVE US THE CAPABILITY TO GO IN THE LAB AND DO AN EXPERIMENT ON THE COMPUTER NOT IN A TRANSWORLD BUT TO GO ON THE COMPUTER AND ACTUALLY CALCULATE PERMEABILITY FOR SMALL MOLECULES FOR PASSIVE DIFFUSION AND THEN EVENTUALLY WE SHOULD BE ABLE TO BUILD IN EFLUX PUMPS AND SO ON. SO I THINK WE HAVE A REALLY INTERESTING RANGE OF TALKS THAT ARE GOING TO HIGHLIGHT THE STATE-OF-THE-ART IN THIS FIELD THIS MORNING. SO, I'M GOING TO TALK A LITTLE BIT NOW FROM MY PERSPECTIVE HERE IN TERMS OF WHAT WE ARE DOING, WHICH AS I SAID, IS SOMEWHERE IN THE MIDDLE FROM THE ORGAN ON THE CHIP LEVEL TO THE ORGAN GENESIS LEVEL IN TRYING TO FIGURE OUT HOW TO BUILD SOME OF THE COMPONENTS OF THE BLOOD-BRAIN BARRIER. SO I THINK I MENTIONED FROM A FABRICATION POINT OF VIEW, WE HAVE RANGE FROM ORGAN ON A CHIP TO ORGAN GENESIS AND THEN THE IMPORTANCE OBVIOUSLY OF CELL LINE STEM CELL TECHNOLOGY IN PARTICULAR. SO I'M GOING TO TALK ABOUT A LITTLE SNIPPETS OF WORK IN 4 AREAS FIRST OF ALL, IN DEVELOPING MICROVESSELS FOR IMAGING DYNAMIC PROCESSES, SECONDLY IN DEVELOPING CAPILLARY NETWORKS, THIRDLY ABOUT CULTURE OF ASTROCYTES, OBVIOUSLY ONE OF THE IMPORTANT COMPONENTS OF THE BLOOD-BRAIN BARRIER AND THEN FOURTH I'M GOING TO TALK A LITTLE BIT ABOUT MORPHOLOGY IN THE ROLE OF CURVATURE ON MORPHOLOGY AND FUNCTION OF BRAIN MICROVASCULAR ENDOTHELIAL CELLS. SO, IN OUR LAB, WE ARE INTERESTED IN FABRICATING MICROVESSEL MODELS, SO THIS MOVIE IS SHOWING A MICROVESSEL ABOUT 150 MICRONS IN DIAMETER ON THE MICROSCOPE PHASE IMAGE, YOU'RE SEEING TOP AND BOTTOM IS THE MATRIX MATERIAL IN THIS CASE COLLAGEN, IT'S A SLIPPED KEL CHANNEL SEEDED WITH HEW VEX IN THIS CASE SO WE ARE VERY FAR FROM A BRAIN MICROVESSEL AT THIS POINT. BUT WE CAN GENERATE THESE FUNCTIONAL VESSELS THAT ARE PRO FUSED AND DOWN ON THE BOTTOM RIGHT IS A CONFOCAL IMAGE IS ONE THAT IS FIXED AND STAINED AND WE STAIN FOR P CAM WITH JUNCTIONAL PROTEIN AND NUCLEUS. SO, THIS IS TYPICAL OF THIS KIND OF WORK THAT WE AND OTHERS ARE DOING IN TERMS OF GENERATING PRO FUSIBLE MICROVESSELS. I'M GOING SHOW YOU A FEW IMAGES OF THE KINDS OF THINGS WE CAN DO AND THE RESOLUTION WE CAN GET CERTAINLY IN COMPARISON IF YOU THINK IN COMPARISON TO MICROSCOPY, WE CAN GET EXQUISITE RESOLUTION. THE NEXT FEW EXAMPLES I'M GOING TO SHOW YOU ARE JUST ILLUSTRATIVE EXAMPLES OF LOOKING AT CANCER. SO WHAT WE ARE LOOKING AT HERE IS A FLUORESCENCE PHASE OVERLAY. THE CELL HERE THAT YOU'RE SEEING INVADING IS A BREAST CANCER CELL, HIGHLY INVASIVE BREAST CANCER CELL LINE THAT IS DUAL LABELED AND THE VESSEL I SHOWED YOU IN A PREVIOUS IMAGE AND WHAT WE ARE LOOKING AT HERE IS BASICALLY THE ENDOTHELIUM SECRETING FACTORS THAT ARE PROMOTING MIGRATION OF THE BREAST CANCER CELL TO THE INTERFACE. SO AGAIN JUST TO GIVE YOU? EXAMPLE OF THE KIND OF THINGS WE CAN DO WITH THESE KIND OF MODELS. SO HERE IS AN EXAMPLE MAYBE ONE OF THE FIRST EXAMPLES YOU HAVE SEEN OF INTRAVERRIZATION. SO IF YOU FOCUS ON THE CELL ON THE BOTTOM HERE, THE VIDEO IS LOOPING. WE ARE SEEING A CELL THAT MIGRATED TO THE INTERFACE AT THE BEGINNING OF THE VIDEO. IT IS MIGRATING ALONG THE INTERFACE BETWEEN THE ECM AND THE ENDOTHELIUM. AND YOU CAN SEE EVENTUALLY IT BALLS UP AND FORCES A DEFECT IN THE ENDOTHELIUM AND FORCES A GAP AND THEN WHEN IT IS EXPOSED TO THE SHEAR FLOW SUFFICIENTLY, THE SHEAR FLOW PULLS IT FROM RIGHT TO LEFT INTO CIRCULATION. I'M GOING TO SAY A FEW WORDS ABOUT MECHANISTICS HERE, ALTHOUGH THE CAVEAT IS OF COURSE WE HAVE NO IDEA WHETHER THIS IS A MECHANISM OPERATING IN-VIVO OR NOT. BUT WHAT YOU PROBABLY NOTICE HERE IS THAT THE CELL IS ROUNDING UP AND SINCE WE HAVE A HISTONE LABELED YELLOW, YOU CAN SEE THE CELL IS BEGINNING TO UNDERGO MITOSIS. SO THE MECHANISM HERE THAT IS HAPPENING IS THAT THE CELL IS INSERTED INTO THE INTERFACE AND UNDERGOES MY TOESIS AND BALLS UP AND THE MATRIX IS STIFFER THAN THE ENDOTHELIUM SO THE CELL PUSHES AGAINST THE ECM INTO THE ENDOTHELIUM AND THAT GENERATES THE FORCE ON THE ENDOTHELIUM TO DISRUPT THE. [ CELL PHONE RINGING ] JUNCTIONS THAT EX-- THE SELL SELL JUNCTIONS THAT EXPOSES SHEAR STRESS. SO WE LOOKED AT MANY HUNDREDS OF VIDEOS AND SEEN 10 EVENTS AND ALL ARE ASSOCIATED WITH MITOSIS. SO THIS IS AN EXAMPLE OF A VIDEO OF THE OTHER EXTREME. THAT WAS SINGLE CELL. THIS IS AN EXAMPLE OF MULTICELL INVASION. SO HERE WHAT WE ARE LOOKING SALT AN ORGANOID THAT IS BEING RESECT FRIDAY A MOUSE MODEL OF BREAST CANCER THAT IS BEING CEDED IN ONE OF OUR MICROVESSELS. SO NOW YOU CAN SEE THE TUMOR IS NOT DISSEMINATING CELLS LIKE IN THE PREVIOUS EXAMPLE. BUT THE ORGINOID IS GROWING AND IT IS EN GOLFING AND COMING AROUND TOP AND BOTTOM OF THE MICROVESSEL AND EVENTUALLY IT PINCHES OFF THE VESSEL AND STARTS SHOOTING OR DISSEMINATING SOME CELLS INTO CIRCULATION THIS IS THE CASE FOR BREAST CANCER CELL FOR EXAMPLE, EXTRA VATATING TO THE BRAIN. THIS THE IS THREE SEPARATE CHANNELS. FLUORESCENCE SHOWING GREEN WHERE WE HAVE DONE A PERMEABILITY EXPERIMENT TO LABEL ENDOTHELIAL CELLS. BREAST CANS ARE CELLS ARE RED AND YELLOW AND THE FAR CHANNEL IS THE CELL BODY STAIN. SO WHAT WE ARE SEEING IS A CELL IN CIRCULATION LIKE A CIRCULATING TUMOR CELL THAT IS ADHERED TO THE ENDOTHELIUM. IT'S UNDER FLOW. YOU CAN SEE AS YOU SEE IN CARTOONS OF EITHER LEUCOCYTE OR TUMOR CELL, IT'S ROUNDED UP AS IT ADHERED TO THE SUFFERS AND A. WHAT YOU CAN SEE CERTAINLY ON THE FAR RIGHT IS THE CELL EVENTUALLY SENDS PROTRUSIONS THROUGH THE ENDOTHELIUM WHICH IS THE FIRST STEP OF EXTRA VASITATION INTO THE SURROUNDING TISSUE. SO THIS IS JUST A FEW EXAMPLES FROM A MICROVESSEL POINTED OF VIEW. WE HAVE A LONG WAY TO GO TO GET BRAIN SPECIFIC BECAUSE THESE ARE NORMAL VASCULAR ENDOTHELIAL CELLS. WE ARE ALSO IN THE PROCESS OF TRYING TO FABRICATE CAPILLARY NETWORKS BY FORMING TWO MICROVESSELS AND USING STIMULATING ANGIOGENESIS TO GROW CAPILLARIES BETWEEN THE TWO VESSELS. SO ONE IS ARTERIAL AND ONE IS A VENNUAL, FOR EXAMPLE. SO YOU CAN SEE IN THE LOWER LEFT HERE WE ARE USING FLUORESCENTLY-LABELED END NEILIAL CELLS. NOT BRAIN SPECIFIC. YOU CAN SEE HERE THIS REGION, 5 CAPILLARIES ARE LABELED THERE AND THEN THE VIDEO ON THE FAR RIGHT IS JUST A VIDEO WHERE WE ARE FLOWING -- BEADS THROUGH THE ARTERIAL AND THE VENNUAL AND YOU CAN SEE IN THE MIDDLE OF THE VIDEO YOU CAN SEE ONE OF THE BEADS CIRCULATING THROUGH THE CAPILLARY, INTERNAL DIAMETER AROUND 8-10 MICRONS, TYPICAL OF A HUMAN CAPILLARY AND THE VELOCITY HERE SERENDIPITOUSLY FOR THE FLOW RATE WE HAVE IS AROUND 2.2 MILLIMETERS PER SECOND -- OR .2 MILLIMETERS PER SECOND WHICH IS A LITTLE LOW BOODLES WHAT YOU WOULD EXPECT IN A BRAIN CAPILLARY. I'M GOING SPEND A COUPLE OF SLIDES TALKING ABOUT DRUG DELIVERY. WE HAVE A NOTION OF IN-VITRO PHARMACOKINETICS. CAN WE UNDERSTAND AND MODEL DRUG DELIVERY IN IN-VITRO MODELS? SO THE VIDEO LOOPING IS AGAIN A VESSEL WHERE WE ARE FLOWING IN LUCIFER YELLOW FOR IN-VITRO PEOPLE DOING EXPERIMENTS IS THE STANDARD MOLECULE USED TO TEST INTEGRITY OF A VESSEL. BY PLOTTING THE INTENSITY IN THE WHOLE FIELD VERSUS TIME, WE SEE AN INITIAL INCREASE DUE TO LOADING LUMEN AND THEN THE STEADY INCREASINGS RELATED TO THE PERMEABILITY. SO WE CAN MEASURE GLOBAL PERMEABILITY. AN EXAMPLE BELOW A SERIES EVER STILLS SHOWS WE CAN MEASURE FOCAL LEAPS. IF WE INDUCE DISRUPTION OF THE ENDOTHELIUM, WE CAN CHARACTERIZE THAT BY MEASURING THE FREQUENCY AND MAGNITUDE OF THESE FOCALLY. SO HERE IS A LITTLE MORE OF AN EXAMPLE OF A FUNCTIONAL EXAMPLE OF IN-VITRO PHARMACOCASEY ANTHONY KINETICS. SHEAR A VESSEL -- PHARMACOKINETICS. WE ARE FLOWING THROUGH A CHEMO THERAPEUTIC THAT IS FLORESCENT. A SINGLE BREAST CANCER CELL YOU CAN SEE EM BREDDED IN THE MATRIX AT THE BOTTOM AND WE CAN CALCULATE PERMEABILITY SO WE ARE SIMULATING SYSTEMIC DELIVERY AND WE CAN FUNCTION ASSAY READ OUTSIDE IN TERMS OF WHAT HAPPENS TO THE CELL. YOU CAN SEE A RED DOT JUST RIGHT TO THE RIGHT OF THE SIX HOUR MARK WHICH IS THE NUCLEUS OF THE CELL WHERE THE DOXORUBICIN IS ACCUMULATING IN THE NUCLEUS. SO I'M GOING GIVE JUST ONE EXAMPLE OF THE QUANTITATIVE ANALYSIS WE CAN DO. SO THE BREAST CANCER CELLS ARE FLOWS ENTLY LABELED. WE CAN COUNT THE NUMBER OF CELL DIVISION EVENTS IN ONE OF THESE VESSELS AS A FUNCTION OF TIME BEFORE AND AFTER INTRODUCING A DRUG. SO, THE PLOT ON THE LOWER LEFT SHOWS THE CELL DIVISION RATE IN PERCENT PER HOUR AS A FUNCTION OF TIME IN FOUR HOUR INCREMENTS BEFORE AND AFTER INTRODUCING DOXORUBICIN. AND YOU CAN SEE WITHIN 16 HOURS THAT PROLIFERATION IS BEEN SWITCHED OFF IN THIS PARTICULAR CASE. WE CAN ALSO TRACK FOR EXAMPLE, THE SPEED OF THE BREAST CANCER CELLS SEE HOW THE DRUG INFLUENCES THAT AND WE CAN ALSO DOT SAME THING WITH THE ENDOTHELIUM. NOW I'M GOING TO SWITCH GEAR AND TALK A LITTLE BIT ABOUT -- I TALKED ABOUT MICROVESSELS AND CAPILLARIES AND I'M GOING TO TALK ABOUT ASTRO SITES. ONE OF THE IMPORTANT COMPONENTS OF THE BBB. THEY ARE USED IN TRANSWELL ASSAYS, CULTURED IN THE DASH OR ASTROCYTE EXTRACT BUT IN TERMS OF BUILDING A MODEL, WE WANT TO BE ABLE TOL CULTURE ASTROS SITES IN 3D AND RECAPITULATE THEM ON THE TOP ARE SLICE TISSUE SECTIONS FROM HUMAN JANE MOUSE BRAIN SHOWING COURT CALL ASTROCYTES. SO IN THE HUMAN BODY, ASTROCYTES EXHIBIT A STAR SHAPED MORPHOLOGY, SMALL CELL BODY, MAIN RADIAL PROCESSES AND 150 MICRON DOMAIN SIZE PRETTY CHARACTERISTIC. IN CELL CULTURE WHEN WE CEDE HUMAN FEELS DERIVED ASTROCYTES ON GLASS FOR EXAMPLE -- FETAL -- THEY LOOK VERY UNHAPPY TO SAY THE LEAST. WHEN WE START TO CULTURE IN 3D, WE STARTED BY CULTURING HIGHLYERONNIC ACID, MAIN COMPONENT OF THE EXTRACELLULAR SPACE IN THE BRAIN. THIS THE IS A MEMBRANE STAIN. YOU CAN SEE THEY BALL UP AND THEY ARE NOT HAPPY AT ALL. SO, THEN WE WENT THROUGH A PROCESS OF SCREENING ABOUT 20 DIFFERENT MATRIX COMPOSITIONS AND WE EVENTUALLY FOUND A MATRIX COMPOSITION ON A FAR RIGHT THERE OF HIGHLYERONNIC ACID COLLAGEN WHERE WE CAN RECAPITULATE THE MORPHOLOGY, SMALL CELL BODY, THE SIZE AND RADIAL PROCESS THAT ARE SEEN IN-VIVO. STIFFNESS IN THE RELAXATION RATE OF THE GEL APPARENTLY PLAY A KEY ROLE IN RECAPITULATING THE MORPHOLOGY. WHAT I DO WANT TO SIGH IS ONE OF THE KEY FEATURES OF ASTROCYTES IS THAT G FAB IS USED AS THE MARKER TO MEASURE ASTROCYTES. IT'S ONLY EXPRESSED WITH ACTIVATED ASTROCYTES AFTER REACTIVE GLIOSIS THEY RESPONDED TO STRESS. SO WE ALSO SPENT SOME TIME FIGURING OUT DERIVING A MATRIX WHERE WE WOULD CULTURE ASTROCYTES IN THE QUIESCENT STATE. SO THE PANEL ON THE TOP LEFT HERE, THED TO COLUMNS ARE AN EXAMPLE FIRST OF ALL OF AN ACTIVE ASTROCYTE SO WE SEE A MEMBRANE STAIN AND THEN A G FAB STAIN. YOU CAN SEE EXPRESSING G FAP THROUGHOUT THE PROCESSES AS WELL AS THE CELL BODY SO THIS WILL BE TYPICAL OF AN ACTIVATED ASTROCYTE. BUT WITH THE RIGHT MATRIX COMPOSITION IF YOU LOOK AT THE COLUMN ON THE RIGHT, THE MEMBRANE STAIN SHOWS CORRECT MORPHOLOGY BUT THE LOWER PANEL D SHOWS VERY LITTLE G FAP EXPRESSION EXCEPT IN THE CELL BODY. WE ALSO DONE A LITTLE BIT OF WORK. WE HAVEN'T CULTURED ASTROCYTES WITH OUR VESSELS BUT WE HAVE DONE PRELIMINARY STUDIES WHERE WE EM EDED ASTROCYTES IN A GEL WITH RODS OF FIBERS OF CAPILLARY DIAMETER THAT ARE FIBRINECT IN COATED. THAT IT IS THE IMAGE IN THE BOTTOM RIGHT THERE AND THE ASTROCYTES AND THIS IS A MEMBRANE STAIN. WILL EXTEND PROCESSES DOWN TO THOSE RODS SO WE HAVE REASONABLE CONFIDENCE WE WILL BE ABLE TO RECAPITULATE THE FORMATION OF PROCESSES TO FORM CAPILLARIES. AND THEN THE FINAL THING I WANT TO SAY JUST A COUPLE OF WORDS ABOUT BRAIN MICROVASCULAR ENDOTHELIAL CELLS. SO, AS I MENTIONED PREVIOUSLY THANKS TO ERIC'S GROUP A FEW YEARS AGO, WHO FIGURED OUT HOW TO DERIVE IPSCs FROM OR DERIVE HUMAN BRAIN MICROVASCULAR ENDOTHELIAL CELLS FROM IPSCs, WE HAVE BEEN ABLE TOL REPRODUCE HIS WORK WITH BC1 AND BC1GFP CELL LINES. THE FLUORESCENCE IMAGE IN THE MIDDLE AT THE BOTTOM SHOWS ONE OF OUR FIRST ATTEMPTS TO FABRICATE VESSELS NOW WITH THESE BRAIN-SPECIFIC STEM CELL DERIVED ENDOTHELIAL CELLS AND THE ELECTRON MICROSCOPY IMAGES ON THE FAR RIGHT BELOW IS IMAGE THAT YOU PROBABLY SEEN MANY OF EM CROSS SECTION OF A RAT BRAIN CAPILLARY SHOWING A TIGHT JUNCTION. THE IMAGE ABOVE IS FROM THE STEM CELL DERIVED ENDOTHELIAL CELLS SHOWING SIMILARLY A CELL/CELL JUNCTION BETWEEN TWO OF THE STEM CELL DERIVED ENDOTHELIAL CELLS THAT OBVIOUSLY LOOKS VERY SIMILAR. SO, THE LAST THING I WANT TO SAY, I THINK AN INTERESTING PHENOMENON WE CAME ACROSS A COUPLE OF YEARS AGO, WE ARE INTERESTED BY THE FACT THAT END NEILIAL CELLS IN BRAIN CAPILLARIES WRAP AROUND AND FORM TIGHT JUNCTIONS WITH THEMSELVES AS WELL AS THEIR UPSTREAM AND DOWNSTREAM NEIGHBORS. SO THIS GOT US TO THINKING ABOUT THE ISSUE OF CURVATURE JUST ONE OVER THE RADIUS OR DIAMETER AND HOW THAT INFLUENCES CELL MOREOVERROLOGY AND FUNCTION. SO WE DEVELOPED -- MORPHOLOGY. SO WE TAKE FIBERS OF DIFFERENT DIAMETERS WE COAT WITH FRY BRING ECK TIN AND CEDE CELLS ON THE SURFACE AND ALLOW THEM TO GROW TO CONFLUENCE AND STAIN THEM AND TAKE CONFOCAL IMAGES AND UNWRAP THE CONFOCAL IMAGES INTO A TWO-D PLANE TO DO QUANTITATIVE ANALYSIS. SO WHAT YOU'RE LOOKING AT OVER ON THE FAR RIGHT AND TOP RIGHT HAPPENED CORN SER A PLOT OF THE INVERSE ASPECT RATIO. A MEASURE OF THE ELONGATION OF THE CELL. IT IS SAY MEASURE OF THE LENGTH OF THE SHORT AXIS TO THE LONG AXIS. AND ON THE BOTTOM SELL A RHYTHMIC SCALE OF THE DIAMETER OF THE FIBER AND THE DATA POINT FOR REFERENCE JUST FROM 2D CULTURE ON THE FAR RIGHT. SO, THE STEM CELL DERIVED EN THIELEIAL CELLS YOU CAN SEE DON'T ELONGATE IN RESPONSE TO CURVATURE. THEY MAINTAIN THE SAME ASPECT RATIO AND FORCOMPARISON HERE WE HAVE RESPONSE OF HUMAN UMBILICAL ENDOTHELIAL CELLS. THE DIAMETER GETS SMALLER AND SMALLER, THEY ELONGATE MORE AND MORE IN RESPONSE TO THE CURVATURE. IF YOU LOOK IN THE BOTTOM RIGHT, THIS IS A PLOT OF FEET ATHE ANGULAR ALIGNMENT. SO THIS IS HOW THE CELLS ALIGN THEMSELVES ALONG THE AXIAL DIMENSION OF THE FIBER OR THE CAPILLARY IF YOU LIKE. SO YOU CAN SEE HEW VEHICLE CELLS AS SOON AS THEY GET EVEN ON SEVERAL HUNDRED MICRON DIAMETER FIBERS -- HUVEC. THEY START ALIGNING WITH THE AXIAL DIRECTION OF THE FIBER. THEY REALLY DON'T LIKE BEING WRAPPED AROUND. THEY DON'T LIKE CURVATURE AND THEY ARE TRYING TO RESIST IT AND THE WAY TO RESCIENTIST IS TO OR I DON'T WANT ALONG THE FIBER DIAMETER. AND IN CONTRAST, THE STEM CELL DERIVED ENDOTHELIAL CELLS SHOW VERY LITTLE ORIENTATION EXCEPT WHEN WE GET DOWN TO SMALL DIAMETERS TOWARDS CAPILLARY DIAMETERS. SO WHAT WE SEE IN THE HUVEC IMAGE, WHEN WE GET DOWN TO FIBERS AROUND CAPILLARY DIMENSIONS, YOU CAN SEE THE CELLS WRAP AROUND AND ELONGATE -- AND WE HAVE TWO OR THREE CELLS AROUND THE PERIMETER. WE CAN NEVER FORCE THEM TO WRAP AROUND AND FORM JUNCTIONS WITH THEMSELVES BECAUSE OF THE WAY THEY RESPOND TO CURVATURE. IN CONTRAST, THE STEM CELL DERIVED END NEILIAL CELLS WILL WRAP AROUND AND FORM A -- [ OFF MIC ] -- AS WELL AS UP STREAM AND DOWNSTREAM NEIGHBORS. SO THIS IMAGE ON THE LOWER LEFT IS WHAT MAYBE GET A HINT FROM EXPERIMENTS. SO, IN SUMMARY, I THINK THERE ARE MANY OPPORTUNITIES AND CHALLENGES IN DEVELOPING THE BUILDING BLOCKS WHICH IS WHAT I'M TALKING ABOUT FOR IN-VITRO MODELS IN THE BLOOD-BRAIN BARRIER AND THERE IS POTENTIAL FOR RECAPITULATING MANY ASPECTS OF BLOOD-BRAIN BARRIER STRUCTURE AND FUNCTION. WE HAVE A LONG WAY TO GO BEFORE WE CAN GET TO THE POINT WHERE HOPEFULLY MANY OF YOU START GETTING INTERESTED IN WHAT WE ARE DOING. SO THANK YOU VERY MUCH FOR YOUR ATTENTION. [ APPLAUSE ] >> THANK YOU VERY MUCH DR. SEARSON. WE WILL HAVE TO HOLD QUESTIONS UNTIL OUR OPEN MIC DISCUSSION. I'D LIKE TO PROCEED WITH DR. JACQUELYN BROWN WHO WILL BE TALKING ABOUT THE NEUROVASCULAR UNIT ON A CHIP. >> CHRISTINE>> IAC LIN. >> JACQUELYN BROWN: I'D LIKE TO START OFF THANGING THE CHAIR FOR THE EXCELLENT INTRODUCTION AND THE ORGANIZER FOR HAVING ME HERE. I'M GOING TO TRY TO COMPRESS THREE YEARS OF RESEARCH IN 15 MINUTES SO WISH ME LUCK AND IF ARE THERE ANY GAPS, WE'LL FILL THEM IN HOPEFULLY DURING THE QUESTION SESSION. AS WAS STATED, WE ARE WORKING ON DOING THESE ORGANS ON A CHIP USING MICROFLUIDIC TECHNOLOGY. I DON'T THINK I HAVE TO WORK TOO HARD TO CONVINCE THIS AUDIENCE THAT FLOW IS INCREDIBLY IMPORTANT FOR BRAIN ENDOTHELIAL CELLS TO REALLY DO THEIR JOB AND BE A BLOOD-BRAIN BARRIER. BUT ONE OF THE OTHER CHALLENGES IS THAT WITH TRADITIONAL CELL CULTURE WE ARE LITERALLY BATHING OURSELVES IN A SEA OF MEDIA. SO ALL OF THOSE SIGNALING MOLECULES THAT WE KNOW ARE SO CRITICAL FOR THE ENDOTHELIAL CELLS TO DO THEIR JOB AND TO SIGNAL TO, ARE ESSENTIALLY DILUTED OUT AND THEY CAN'T REALLY COMMUNICATE WITH EACH OTHER LET ALONE THE OTHER CELL TYPES THAT THEY NEED TO TO PRODUCE THAT NEWER VASCULAR UNIT. SO, REALLY THE GOAL OF MICROFLUIDIC TECHNOLOGY IS TO BRING DOWN THAT MEDIA VOLUME TO THE POINT WHERE THE SIGNALING FACTORS THAT THE CELLS PRODUCE THEMSELVES CAN ACTUALLY HAVE THE INTENDED CONSEQUENCES BECAUSE THEY ARE AT THE CONCENTRATION 35 THEY NEED TO BE. SO IN SOME WAYS, WE ARE LETTING THE MICROFLUIDICS DO SELF ORGANIZATION. WE ARE JUST TRYING TO PROVIDE A SCAFFOLD ON WHICH FOR THEM TO DO IT. SO, AS MANY OF YOU ALREADY SEEN WE HAVE GOT AN ACTUAL BLOOD-BRAIN BARRIER THAT LOOKS LIKE THIS. SO I NEED TO MOD THEY'LL IN SOME WAY SO I CAN INTERROGATE BOTH SIDES OF THE BLOOD-BRAIN BARRIER BECAUSE I DON'T WANT IT TO BE A BLACK BOX. I WANT TO BE ABLE TO GET INFORMATION BACK OUT. SO WHAT WE HAVE DONE IN OUR LAB IS THAT WE HAVE CREATED THIS MICROFLUIDIC DEVICE THAT HAS TWO CHAMBERS, ONE OF WHICH WE ARE CALLING IT THE VASCULAR CHAMBER AND THIS IS THE ONE WE CEDE WITH ENDOTHELIAL CELLS AND THEN WE HAVE A POROUS MEMBRANE TO PROVIDE THAT SCAFFOLD SO THAT ON THE OTHER SIDE WE CAN SEE PARASITES, ASTROCYTES AND NEURONS TO CREATE OUR BRAIN CHAMBER. AND WE CAN GET IN AND OUT OF BOTH SIDES. THIS CHAMBER IS DESIGNED TO HAVE LIER SHEAR STRESS AND THIS IS DESIGNED TO HAVE VERY LOW IF NO SHEAR STRESS. BUT WE CAN STILL MEASURE EFLUENT ON THESE SIDES. AND SO WE ARE LOOKING AT ADDING MAKE ROW GLEIA LATER ON. SO -- MICROGLIA. WE ARE TRYING TO BE ABLE TO MODEL A LOT OF DIFFERENT THINGS NOT JUST TB I AND STROKE, TOXICOLOGY, DRUG TREATMENT, ONE OF THE THINGS THAT WE ARE TRYING TO MAKE SURE THAT WE HAVE IS THAT WE HAVE MULTIPLE CELL TYPES, 3D INSTRUCTION PROFUSION AND DURATION. BECAUSE A LOT OF TIMES THE CELL CULTURE THEY ONLY MAINTAIN THAT CELL MORPHOLOGY WE CARE ABOUT FOR A SHORT TIME BUT THE GOAL OF THESE ORGANS ON A CHIP IS TO HAVE A MONTH, TWO MONTH, 3 MONTHS WHERE THIS ORGAN IS STILL BEHAVING VERY CLOSE TO AN IN-VIVO MODEL. SO, SOME OF THE ENGINEERING FEATS WE HAVE COME UP WITH TO CREATE THIS BLOOD-BRAIN BARRIER IS ONE OF THESE THIS FLIPPABLE BACKPACK PROFUSION SYSTEM. NOW YOU SAW IN THE EARLIER SLIDE THAT I HAD CELLS GROWING IN OPPOSITION ACROSS THE POROUS MEMBRANE. FOR THOSE WHO ARE FAMILIAR WITH TRANSWELLS, YOU KNOW HOW DIFFICULT IT IS TO GET CELLS ON EITHER SIDE OF THE MEMBRANE. SO, I TOLD MY ENGINEERS I WANT TO BE ABLE TO TAKE THOSE AWESOME PUMPS YOU MADE ME TO HELP PRO FUSE MY SYSTEM AND I WANT TO TURN THEM UPSIDE DOWN. THAT'S OKAY, RIGHT? THE OKAY. WE CAN MAKE THAT WORK. SO, TOGETHER WITH SOME OF THE FABULOUS ENGINEERS AT VANDERBILT UNIVERSITY, WE CREATED THIS BACKPACK SYSTEM SO THAT MY FLUID FILES BOTH THE FEED AND THE SYNC FILES GOING ON THE BACK AND REMAIN UPRIGHT AND VENTED WHILE I RUN MY PUMPS EITHER IN THE UPSIDE DOWN MODE OR THE RIGHT SIDE UP MODE SO I CAN GET THE CELLS EASILY WHEN I CEDE THEM TO GROW IN OPPOSITION ACROSS THAT POROUS MEMBRANE. THE OTHER NICE THING ABOUT THIS PROFUSION SYSTEM IS THAT THE ENTIRE FOOTPRINT IS ACTUALLY THE SAME AS A -- SO WHEN YOU TAKE THIS TO TAKE IT OUT AND IMAGE IT, IT FITS ON YOUR STANDARD MICROSCOPE. AND THAT IS ABOUT THE SPACE THAT IS IT TAKES UP IN AN INCUBATOR AS WELL. SO OBVIOUSLY HEIGHT WISE STANDS A LITTLE BIT TALLER. THE OTHER DESIGN FEATURE THAT WE ADDED WAS THIS WATER BACK BECAUSE AS THE PUMPS GENERATE HEAT THEY WERE CAUSING A LOCALIZED AREA OF DEHUMIDIFICATION WHICH OBVIOUSLY FOR MICROFLUIDICS WOULDN'T BE GREAT. AND I WAS BALANCING LITTLE PETRE DASHES OF WATER ON TOP TESTIFY AND THE ENGINEER SAID NO, WE CAN DO BETTER THAN THAT. SO NOW WE ACTUALLY HAVE A WATER BATH THAT NOT ONLY PROVIDES LOCAL HUMIDITY BUT ALSO HELPS TO COOL THE TEMPERATURES THE PUMP TO MAINTAIN THE SAME STABLE TEMPERATURES INSIDE OUR INCUBATOR SO FOR THOSE USING PUMPS INSIDE AN INCUBATOR, YOU PROBABLY ALL DEALT WITH SOME OF THESE CHALLENGES. ONE OF THE OTHER ENGINEERING FEATS WE CREATED WAS OUR OWN CUSTOMIZED TIER DEVICE SINCE ONE OF THE THINGS A LOT OF PEOPLE WANT TO SEE WHEN YOU'RE CREATING A BLOOD-BRAIN BARRIER MODEL IS THE QUESTION OF DO YOU ACTUALLY GET AN INCREASE IN ELECTRICAL RESISTANCE ACROSS THE MEMBRANE? OBVIOUSLY WITH OUR DEVICE, THE TYPICAL CHOPSTICKS SET UP WAS NOT GOING TO WORK. SO WHAT WE HAVE DONE IS THAT IN THOSE TUBES THAT PROVIDE THE PROFUSION, WE SPLICE IN A STAINLESS STEEL BIT OF TUBING AND I CAN CONNECT MY LEADS TO THAT TUBING ON BOTH INLETS AND OUTLET AND THAT'S WHERE I CAN INJECT THE CURRENT AND BREED OUT THE CURRENT. TO SEE CHANGES OVER TIME AND WE CAN DO REALTIME RECORDING SO IF I APPLY A DRUG I CAN LOOK AT HOW TIER CHANGES IN RESPONSE TO APPLICATION OF DRUG. SO, OBVIOUSLY WE HAVE TO KEEP THE CELLS ALIVE AND THE QUESTION IS HOW WELL WE DID. SO FAR, LOOKING AT FOUR WEEKS OUT, WE HAVE GOT PRETTY GOOD CELL SURVIVABILITY. SO THIS IS LIVE DEAD STAIN. THESE TOP PANEL HERE THESE ARE THE SPLITTER NETWORKS THAT ARE GOING INTO THE MAIN BODY OF THE VASCULAR CHAMBER SO THESE ARE BRAIN DERIVED ENDOTHELIAL CELLS. YOU CAN SEE THIS GOING OUT INTO THE MAIN BODY OF THE DEVICE. IF YOU TAKE THAT IMAGE AND GO UP A LITTLE BIT SO NOW WE ARE GOING TO THE OTHER SIDE OF THE MEMBRANE, YOU CAN START TO THE SEE WHERE OUR PARASITES AND ASTROCYTES ARE CEDED AND IF YOU GO STILL FURTHER UP INTO THE DEVICE, YOU GET TO OUR -- WHERE OUR NEURONS AND SOME OF OUR ASTROCYTES ARE EMBEDDED IN A COLLAGEN GEL MATRIX AND THESE ARE THE LITTLE NEURONS PROCESSES BEING SENT OUT. IN TERMS OF ENDOTHELIAL CELL MORPHOLOGY, WE CAN SEE WITH STAINING THAT WE GET NICE EXPRESSION OF ZERO 1 ACROSS THE JUNCTIONS SO WE FEEL LIKE THAT SUGGESTS WE ARE GETTING GOOD TIGHT JUNCTION PROTEIN STAINING. ALSO WHAT WE SEE WITH FLOW IS THAT WE SEE NICE ACTIN BUNDLE STAINING. SO THIS IS NICE BUNDLES AND I THINK THAT YOU CAN SEE FROM THIS THAT A LOT OF THE ACTIN BUNDLES ARE SORT OF ORIENTED IN THIS DIRECTION AND THAT IS IN FACT THE DIRECTION OF FLOW. SO WE HAVE GOT TIGHT JUNCTION PROTEIN STAINING AND WE HAVE NICE CELL POLARITY WITHIN OUR DEVICE. AS FAR AS DIFFUSION, AGAIN THIS IS LOOKING AT BOTH 70KD AND 10KD. WE USED AS OUR CONTROL DEVICE WHERE I PERMALIZED THE CELLS BECAUSE ONE OF THE THINGS WE NOTICE SAID THAT CELLS TEND TO TAKE UP DYES ALL ON THEIR OWN SO YOU MIGHT SAY WELL, DO YOU REALLY SEE REDUCTION IN DIFFUSION BECAUSE THE CELLS ARE DOING THE WORK OF BEING A TIGHT JUNCTIONAL PROTEIN BARRIER OR JUST ABSORBING THE DYE AND THAT IS WHY IT IS NOT GETTING ACROSS? SO WE USE PERMEABLE CELLS SO THEY HAVE HOLES THROUGH THEM NOW SO THEY CAN PASS VERSUS INTACT CELLS AND THERE IS A SIGNIFICANT REDUCTION ALMOST UNDETECTABLE AS FAR AS THE 70KB AND STILL VERY LOW AT THE 10KD IN OUR INTACT DEVICE AT 14 DAYS IN CULTURE. ALSO WE SEE THAT AROUND AGAIN 10 TO 14 DAYS, WE SEE SIGNIFICANT INCREASE IN OUR TEAR READ OUTSIDE. THESE AREN'T USUALLY THE NUMBERS YOU NORMALLY SEE. OUR DEVICE HAS ITS OWN NATURAL IMPEDENCE AS CAN APPRECIATE AS DAY 2. SO WHAT WE REALLY HAVE TO LOOK AT IS THE INCREASE RATHER THAN THE ACTUAL VALUES SINCE THE DEVICE ITSELF HAS ITS OWN RESISTANT PROPERTIES. WE CAN ALSO MODULATE THIS BARRIER SO WE CREATED A BARRIER. WE SHOWN YOU THAT IT HAS TIGHT JUNCTIONS THAT IT HAS RESTRICTION DIFFUSION. CAN WE ALTER IT IN THE WAYS THAT WE BELIEVE IT CAN BE ALTERED IN THE INTACT ANIMAL? NOW, IF YOU SUPPLY GLUTAMATE TO THE BLOOD SIDE IT CAUSES NO CHANGES IN THE BLOOD-BRAIN BARRIER PERMEABILITY. IF YOU SUPPLY GLUTAMATE TO THE BRAIN SIDE BOTH IN ANIMALS AND IN OUR MODEL, YOU CAN SEE THAT THERE IS A SIGNIFICANT DISRUPTION IN BLOOD-BRAIN BARRIER PERMEABILITY. WE ALSO HAVE SHOWN THAT ASCORBIC ACID KNOWN IN HUMANS AT LEAST, TO PRODUCE A TIGHTENING OF THE JUNCTIONAL BARRIER, ALSO CAUSES A TIGHTENING OF OUR JUNCTIONAL BARRIER WITHIN OUR MVU. TRYING TO AGAIN VALIDATE OUR NV. AND ASK THE QUESTION HOW WELL DOES IT RECAPITULATE WHAT WE KNOW OF THE HUMAN BLOOD-BRAIN BARRIER AND ANIMAL MODELS? WE BEGIN ALSO TO LOOK AT CYTOKINE ACTIVATION WHILE THIS IS A MICROFLUIDIC DEVICE AND WE ARE GETTING SMALL VOLUMES OF FLUID OUT, WE ARE STILL ABLE TO DO SMALL VOLUME ALIZA ON OUR NVU SO WHEN WE DO IN THIS CASE, STIMULATION WITH LPS, WE CAN LOOK OVER A VARIETY OF TIME COURSES AND WE CAN LOOK AT BOTH THE VASCULAR AND BRAIN SIDE AND ASK HOW OUR CYTOKINES RESPONDING TO THIS NEURON INFLAMMATORY STIMULUS. WE ARE ALSO PARTNERING WITH THE JOHN McCLAIN LAB TO DO SOME REALLY INTERESTING METABOLIC STUDIES ON OUR NVU SO WE ARE USING MASS SPEC TO LOOK AT WHAT ARE THE METABOLIC CONSEQUENCES OF CYTOKINE AND INFLAMMATORY STIMULATION WITHIN THE NVU. YOU CAN SEE FROM HERE THAT STIMULATION OBVIOUSLY CAUSES A RATHER PRONOUNCED CHANGE IN METABOLIC SIGNALING IN OUR NVU AND WE CAN EVEN DO A TIME COURSE RESOLUTION OF THAT METABOLIC SIGNALING. OBVIOUSLY THE BLOOD-BRAIN BARRIER DOESN'T JUST KEEP THINGS OUT. IT NEEDS TO LET THINGS IN. SO THE QUESTION BECAME, HOW GOOD DOES OUR DEVICE LET IN THE THINGS IT NEEDS TO OR TRANSPORT THEM? AS I ALREADY SHOWN YOU THE ASCORBIC ACID TIGHTENS DOWN AS FAR AS PASSIVE DIFFUSITY OVER THE TIME COURSE. IT IS ALSO KNOWN TO BE ACTIVELY TRANSPORTED ACROSS THE BLOOD-BRAIN BARRIER, YOUR BRAIN NEEDS VITAMIN C AND YOU CAN SEE THAT THE LEVELS OF ASCORBIC ACID MEASURED ON OUR BRAIN SIDE OVER THE SAME TIME COURSE THAT THIS DIFFUSION IS BEING MEASURED ARE GOING UP. SUGGESTING THAT THERE IS ACTIVE TRANSPORT OF ASCORBIC ACID ACROSS OUR MODEL OF THE BLOOD-BRAIN BARRIER. THIS WORK HAS BEEN DONE IN COMBINATION WITH PITTSBURGH WASHINGTON AND JOHNS HOPKINS WHO ARE ALSO CREATING THEIR OWN LITTLE ORGANS IN TERMS OF THE GUT, LIVER AND KIDNEYS. SO THERE IS THIS DRUG PAIR IT IS INTERESTING THE LIVER PROCESSES THIS DRUG INTO THE FLOCKSO FEN DINE FORM AND ONE OF THESE PAIRS, THE TERFENADINE CROSSES THE BLOOD-BRAIN BARRIER AND FECES FEN DINE DOES NOT. SO IN MEDIA THAT WERE PROCESSED BY THE LIVER, THEN APPLIED THE LIVER CONDITION MEDIA TO THE VASCULAR SIDE OF MY NVU AND THEN TRIED TO MEASURE THE DRUG CONCENTRATION IN THE BRAIN SIDE OF OUR NVU USING DIRECTED MASS SPECTROMETRY. AND WHAT WE FOUND IS THAT OUR NVU DID IN FACT RECAPITULATE THE HUMAN DATA AND THAT THE TERFENADINE WAS EASILY DETECTED IN THE BRAIN SIDE OF OUR NVU BUT NOT DETECTED, FECES 10A DINE NOT SUPPOSED TO BE TRANSPORTED WAS NOT SEEN ON THE OTHER SIDE. SO I'M GOING TO END THERE AND THANKING ALL OF THE PEOPLE IN MY LAB BECAUSE THIS IS SAY HUGE COLLABORATIVE IN ENGINEERING EFFORT AS WELL AS OUR COLLABORATORS AT CLEVELAND UNIVERSITY WHO ARE ALSO WORKING WITH US AS WELL AS THE SATELLITEINS INSTITUTIONS CREATING THE OTHER ORGAN SYSTEMS AND OUR FUNDING SOURCE WHICH PROVIDES THE MONEY TO MAKE ALL THIS WORK HAPPEN. [ APPLAUSE ] >> THANK YOU VERY MUCH DR. BROWN. SO WE'LL HAVE YOU ANSWER QUESTIONS DURING THE OPEN MIC DISCUSSION. AND WE'LL PROCEED WITH DR. ERIKA SHUSTA WHO WILL BE TALKING ABOUT MODELING AND TARGETING THE BLOOD BRAIN BARRIER AND HEALTH AND DISEASE. >> WHITNEY>> SO YOU NOTICE THE TARGETING IS O I PUT TARGETING IN PRIOR TO REALIZING IT WAS A 15-20 MINUTE TALK. SO I'M GOING TO FOCUS ON MODEL OF THE BLOOD-BRAIN BARRIER AND TELL BUT WORK WE HAVE BEEN DOING OVER THE LAST 4-5 YEARS TO EMPLOY PLURIPOTENT STEM CELL TECHNOLOGY AS A CELL SOURCE FOR BLOOD-BRAIN BARRIER MODELING. AND WE HAVE SEEN A FEW VARIATIONS OF IN-VITRO MODELS ALREADY AND WE SEEN THE NICE THINGS WE CAN DO IN THREE-DIMENSIONS AND WITH FLOW AND WITH MULTICELLULAR CULTURE AND HOW IMPORTANT THOSE CAN BE ON BBB PHENOTYPE, TODAY MY PRIMARY FOCUS WILL BE SOURCING THE BRAIN MICROVASCULAR ENDOTHELIAL CELLS THAT COULD BE USED IN A VARIETY OF CONFIGURATIONS WE HAVE SEEN. SO WHAT I HAVE SHOWN HERE IS THE BASIC TRANSWELMODEL WHERE YOU HAVE A MONOLAYER OF BRAIN ENDOTHELIAL CELLS, FROM IMMORTALIZED OR STEM CELL-BASED SOURCE THAT IS SITS ON A PERMEABLE MEMBRANE AND THE MODELING ITSELF FORMS THE PRINCIPLE DIFFUSION BARRIER BETWEEN TWO LIQUID-FILLED COMPARTMENTS, BLOOD SIDE AND BRAIN SIDE. THEREFORE WE CAN DO A LOT OF DIFFERENTTINES OF STUDIES IN THE PETRE DISH NOT LEAST OF WHICH COULD BE DRUG OR THERAPEUTIC PERMEABILITY SCREENING. WE CAN LOOK AT PARTICULARLY IN THE CASE OF STEM CELLS, WHAT ARE THE DEVELOPMENTAL CUES TO ALLOW FOR SPECIFICATION AND MAINTENANCE OF BARRIER FORMATION. AND CLEARLY CAN STUDY DISEASE PROCESSES AND I'LL SHOW AN EXAMPLE OF THAT WITH PATIENT-DERIVED IPS CELLS. NOW WHEN WE TAKE BRAIN ENDOTHELIAL CELLS OUT OF THE BRAIN AND THAT 3D MICRO-ENVIRONMENT AND PUT THEM UNTIL THIS ARTIFICIAL PETRE DISH AS WE SEEN FROM THE INTRODUCTION OF FLOW AND 3D GEOMETRY AND MULTIPLE CELL TYPES, YOU MAY WANT TO INCLUDE CO-CULTURE CELLS THAT CAN BE IPS SOURCE SUCH AS ASTROCYTES AND NEURONS AND PERICYTES. WHAT I'LL FOCUS ARE A FEW CHARACTERISTICS FOR WHICH WE THINK ARE KEY TO BLOOD-BRAIN BARRIER MODELS AND IT WILL BECOME APPARENT WHEN I SHOW SOME EXAMPLES THE FIRST IS THE PHYSICAL BARRIER THAT DR. BANKS TALKED ABOUT. REALLY CLOSING DOWN PERICELLULAR PERM ABILITY TO HYDROPHILIC SOLUSED. SO WE'LL LOOK AT PROTEINS, KEY TYPE JUNCTION PROTEINS CLOSING DOWN THE PERICELLULAR COURSE BUT THEN ALSO WE AND OTHERS ARE SHOOTING TO TRY TO RECAPITULATE THE IN-VIVO BRAIN BLOOD BRAIN BARRIER AND IN THE IN-VIVO BRAIN MICRO-ENVIRONMENT TO GET GENE EXPRESSION AT A IN-VIVO LEVEL NEITHER OUR GROUP OR OTHER GROUPS HAVE ACHIEVED THIS BUT I THINK THAT SHOULD BE GENERALLY OUR GOAL AND THIS INVOLVES THE CORRECT EXPRESSION OF FUNCTIONAL TRANSPORT SYSTEMS AND IMPORTANTLY THE BLOOD-BRAIN BARRIER IS POLARIZED BECAUSE OF THE PRESENCE OF THE TIGHT JUNCTIONS SO WE SHOULD HAVE THE CORRECT POLARIZATION OF THIS AS WELL. SO WHAT ARE SOME OF THE CHALLENGES OF BLOOD-BRAIN BARRIER ENDOTHELIAL CELL CULTURE? RAZZ WE SEEN IN PREVIOUS TALKS, OFTENTIMES ONE STARTS WITH PRY MARE SOURCING. AND THAT REALLY IS THE FACT THAT THE BBB IS ABOUT .1 PURSE OF THE VOLUME OF THE BRAIN. AT THE END OF THE DAY YOU GET A SMALL PELLET OF BRAIN ENDOTHELIAL CELLS INTRINSICALLY NON PROLIFERATIVE. SO A SMALL AMOUNT OF MATERIAL FROM A RODENT BRAIN YOU MIGHT GET 6-12 OF THESE SET UPS. SO GOOD ENOUGH TO DO SCIENCE MOD TREAT HIGH-THROUGHPUT SCREENING BECOMES VERY DIFFICULT THOUGH WITH THIS LIMITATION ON AMOUNT OF MATERIAL. AND AS AS SOON AS WE TAKE THESE OUT OF THE 3D BRAIN MICRO-ENVIRONMENT AND PUT THEM IN A FLAT CONFIGURATION OF PETRE DISH, A LOT OF THESE CHARACTERISTICS THAT WE SEEN INDUCED BY FLOW AND 3D FROM ANIMAL TO ANIMAL TALKS ARE REALLY LOST. SOME OF THOSE KEY BLOOD-BRAIN BARRIERS SPECIFYING CHARACTERISTICS SUCH AS TIGHTNESS THROUGH THE BARRIER FORMATION AND TIGHT JUNCTION. INFLUX OF NUTRIENTS AND TO THE ACTIVE BARRIER THROUGH A VARIETY OF EFLUX TRANSPORTERS AND THE ENZYMATIC BARRIER. WE WILL BE USING ANIMAL SOURCES. AND OF COURSE THOSE ARE USED BY LOTS OF PEOPLE IN THIS ROOM. THEY ARE AVAILABLE. WE CAN USE THEM WITH TRANSGENIC DISEASE MODELS BUT EVEN FOR THE BEST HANDS, IT'S A BIT OF AN ART FORM. ISOLATIONS COULD BE HETEROGENOUS FROM ANIMAL TO ANIMAL IF WE LOOK AT THE RIGHT-HAND SIDE THIS SATURDAY ABC TRAPS PORTER FAMILY AND ALL YOU NEED TO DO IS LOOK AT THE DIFFERENCE IN THE BAR GRAPHS IN THE SHAPES OF THE BAR GRAPHS TO REALIZE THAT HUMANS HAVE A DIFFERENT SET OF ISONORMIC EXPRESSION COM PARITY TO OTHER SPECIES. SO DEALING WITH THE HUMAN BBB COULD BE ADVANTAGEOUS FOR A VARIETY OF PERSPECTIVES. SO IF WE THINK OF HUMAN MODELS, WIDELY USED IMMORTALIZED LINE, THE D3 LINE, AND THERE ARE OTHER IMMORTALIZED HUMAN LINES AS WELL. BUT ALONG WITH ANIMAL IMMORTALIZED LINES WHAT YOU TYPICALLY FIND IS THEY DON'T REALLY FORM A BARRIER. SO, IF YOU'RE LOOKING AT DOING THIS PERMEABILITY SCREENING, AXEOSOME SCREENING, THINGS LIKE THAT, IT BECOMES DIFFICULT IF YOU CAN'T MEASURE THE THROUGH PUT THROUGH THE CELL OR BETWEEN THE CELL. AND DECONVOLUTING THOSE TWO IS DIFFICULT. THEY CAN BE VERY USEFUL FOR LOOKING AT BLOOD-BRAIN BARRIER BIOLOGY BUT FOR SOME APPLICATIONS THAT WE TALKED ABOUT HERE THAT REQUIRE TIGHTNESS, THIS IS DIFFICULT. OF COURSE WE CAN USE PRIMARY HUMAN BRAIN ENDOTHELIAL CELLS BUT OF COURSE YOU HAVE LIMITATIONS ON BOTH ACCESS BUT THEN ALSO AMOUNT OF MATERIAL COMING FROM SURGICAL RESECTION. SO THIS MOTIVATED US TO TRY TO TAKE ADVANTAGE OF PLURIPOTENT STEM CELL TECHNOLOGY IN ORDER TO SOURCE THE CELLS THAT WE WOULD USE IN BLOOD-BRAIN BARRIER MODELING OF A VARIETY OF CONFIGURATIONS AND I'LL TALK ABOUT THIS HERE AND WE HEARD ABOUT THE OTHER THIS MORNING. AND THE IDEA IS TO TAKE HUMAN PLURIPOTENT STEM CELLS AND DIFFERENTIATE THEM TO BRAIN ENDOTHELIAL CELLS. TASK IS A DIFFICULT PART TRYING TO FIGURE OUT THE RIGHT CONDITIONS TO SPECIFY SOMETHING FROM AN UNDIFFERENTIATED STEM STOLE SOMETHING THAT RESEMBLES A BRAIN ENDOTHELIAL CELL. WE CAN ALSO DOT OTHER SIDE OF THE NEUROVASCULAR UNIT HERE ALSO SOURCING FROM STEM CELLS USING EITHER PRIMARY NEURAL STEM CELLS OR IPS DERIVED ASTROCYTES AND NEURONS FOR MULTICELLULAR CULTURES AND I'LL FOCUS PRIMARILY ON THE BRAIN ENDOTHELIAL CELL COMPONENT. SO WE HAVEN'T TALK TOO MUCH ABOUT STEM CELLS YESTERDAY OR TODAY SO I'LL JUST INTRODUCE THE TWO GENERAL TYPES OF STEM CELLS. SO THERE IS EMBRYONIC STEM CELLS DERIVED FROM THE PLASTO CYSTS AND INDUCED FROM PLURIPOTENT STEM CELLS WHERE SOMATIC CELLS ISOLATED CAN BE REPROGRAMMED THROUGH A FEW TRANSCRIPTION FACTORS TO SOMETHING RESEM EGG WELL A POPULARY POTENT STATE. SO THE KEY HERE IS NOW YOU CAN USE PATIENT DERIVED MATERIALS TO START TO MODEL THE NEURAL VASCULAR UNIT. OF COURSE IF WE GO FROM AN UNDIFFERENTIATED STEM CELL TO ONE THAT RESEMBLES A BRAIN ENDOTHELIAL CELL, WE CAN LOOK AT SIGNALING PATHWAYS INVOLVED IN SPECIFICATION AND MAINTENANCE OF BBB CHARACTERISTICS AND ALSO THE FACT THAT WE HAVE THE HUMAN SOURCING AND SCALE PROVIDED BY THE ESSENTIALLY LIMITLESS EXPANSION OF UNDIFFERENTIATED STEM CELLS THAT ALLOWS US TO GET SCALE ISSUES THAT I TALKED ABOUT EARLIER AS WELL. SO, THIS IS IN CLOSE COLLABORATION WITH DR. SEAN PALAL CHECK, STEM CELL DIFFERENTIATION EXPERT IN THE MY DEPARTMENT IN WISCONSIN AND TWO STUDENTS THAT MOVED ON TO THEIR OWN FACULTY POSITIONS. THEY CAME UP WITH A WAY TO DIFFERENTIATE ENDOTHELIAL CELLS TO GAIN BBB CHARACTERISTICS BY TRYING TO RESEMBLE THE EM BRY ONTIC MICRO-ENVIRONMENT WHICH IS LARGELY CODEVELOPING NEURAL CELL AND ENDOTHELIAL PROGENITORS AS THEY PERVADE THE NEURAL DOWN THEY REPLICATED WITH THE RIGHT TIMING AND MEDIUM AND FACTORS IN THE PETRE DISH AND THEN YOU WANT TO USE THESE AS PURIFIED CELLS DONE BY A ADHESION ON THE COLLAGEN FIBER NECTIN MATRIX. SO BRIEFLY JUST LOOKING AT THESE FILMS AS THEY ARE -- BEING DIFFERENTIATED, YOU GO YOU GET LARGER EXPANDED COLONIES IN ENDOTHELIAL CELLS THAT BEGIN TO GAIN BLOOD-BRAIN BARRIER CHARACTERISTICS. SO PECAM POSITIVE CELLS START TO FORM JUNCTIONS. THE PROTEIN JUNCTIONS ARE INCLUDED IN 4 AND 5. SO WHAT YOU REALLY LIKE TO SEE IMAGES LIKE THIS, THIS CONNIE COMBED IMAGE WHERE THE SEOUL SEOUL CONTACTS ARE THE LOCALIZATION OF YOUR TIGHT JUNCTION PROTEINS. THEY ALSO GAINED A LARGE ABUNDANCE OF GLUTE ONE GLUCOSE TRANSPORTER, THE BBB SELECTIVE GLUCOSE TRANSPORTER AND THEN ALSO BEGIN TO EXPRESS EFLUX TRANSPORTER PROTEINS. THEY RESPOND TO NEURAL CELL CUES IN COCULTURE WITH ASTROCYTES. YOU CAN ROUTINELY GET 7FIRST TOW 1500 OHMS CENTIMETER SQUARED WHICH IS QUITE TIGHT. SO A LOT OF THE FEENEY TYPES WE TALK ABOUT ARE ANALYZED AS PURIFIED CULTURE SO SELECTIVE ADHESION. YOU GET NEARLY PURE POPULATIONS OF BRAIN-LIKENED THIELEIAL CELLS. IF YOU ADD INDUCING AGENT DURING THE DIFFERENTIATION, YOU CAN GENERATE 3000 OHM CENTIMETER SQUARED MONOCULTURED BRAIN ENDOTHELIAL CELLS. AND WHEN YOU ZOOM IN YOU CAN SEE THESE PROTRUSIONS AND MAKE THEM VERY CONTINUOUS JUNCTIONS. SO THEN WE ASKED CAN WE PUT A FEW OF THESE PIECES ITTH AND THINK ABOUT THE ACTUAL DEVELOPMENTAL TIMELINE OF THE BBB DURING EMBRYONIC DEVELOPMENT. AND THIS OCCURS WITH THE ENDOTHELIAL CELLS INVADING THE NEURAL TUBE IN THE PRESENCE OF NEURAL PROGENITORS SO WE REPLICATED SOME OF THAT WITH THE DIFFERENTIATION OF THE STEM CELLS BUT THEN PARASITES COME ON TO INTERGREAT STABILIZE IT AND REDUCE ITS PERMEABILITY PROPERTIES WE PURIFY IN THE PRODUCENCE OF HUMAN BRAIN PERICYTES AND TAKE THIS TRANSWELL, AND DOP IT INTO A WELL THAT HAD DIFFERENTIATESSED MIXTURES PRIMARY NEURAL STEM CELLS OR NOW WE HAVE DONE THIS WITH INDUCED PLURIPOTENT STEM CELLS AS WELL AND I'LL SHOW YOU ONE PIECE OF DATA I THINK IS USEFUL. WE WEREN'T NECESSARILY SHOOTING FOR THIS BUT WHEN YOU PUT ALL THE PIECES TOGETHERS, YOU CAN GET ABOUT 5000 OHM RESISTANCES. AND IT IS INSTRUCT TESTIFY REALIZE THAT IN-VIVO THE HIGHEST MEASURED ELECTRICAL RESISTANCE IS REPORTED TO BE ABOUT 6000 OHM CENTIMETERS SQUARED IN RATS. SO, GETTING TO PHYSIOLOGICALLY RELEVANT TIGHTNESS AND YOU'LL SEE VERY LITTLE IF ANY PASSIVE DIFFUSION BETWEEN CELLS AT THIS LEVEL OF RESISTANCE. NOW THERE IS THAT ACTIVE BARRIER THAT WE HAVE TALKED ABOUT. SO, THESE CELLS EXPRESS P GLYCOPROTEIN MEMBERS OF THE MRP FAMILY AND ALSO BREAST CANCER RESISTANCE PROTEIN AND I WON'T GO THROUGH ALL THE DETAILS BUT YOU CAN EXAMINE USING SPECIFIC SUBSTRATES OF EFLUX TRANSPORTERS AND INHIBITORS OF CERTAIN TRANSPORTERS WHETHER OR NOT THESE CELLS HAVE THE APPROPRIATE ACTIVITY AND JUST AS AN EXAMPLE, THIS IS A RELATIVELY SELECTIVE SUBSTRATE YOU CAN PUT INHIBITOR IN THERE AND SEE A CHANGE IN THE TRANSPORT OF WROTA MEAN AND WE CAN DO THAT WITH A PAN SUBSTRATE LIKE DOXORUBICIN OVER THE MULTIPLE TRANSPORTERS. MEASURE ACCUMULATION AND MEASURE BIDIRECTIONAL TRANSPORT AND LONG STORY SHORT, THESE THREE PROTEINS ARE ALL PRESENT. THEY ARE FUNCTIONAL AND THEY ARE POLARIZED IN THE CORRECT BRAIN TO BLOOD DIRECTION. IF YOU PUT ALL THESE THINGS TOGETHER, THE PHYSICAL BARRIER, ACTIVE BARRIER, CAN YOU PREDICT DRUG UPTAKE. THIS IS A SMALL COHORT OF MOLECULES WE COULD GET OUR HANDS ON AND WHAT WE ARE PLOTTING HERE IS THE PERMEABILITY IN THE IN-VITRO MODEL VERSUS IN-VIVO UPTAKE IN THE BRAIN. ONE END OF THE SPECTRUM YOU HAVE A SMALL LYPOPHILIC MOLECULE READILY TAKEN UP IN-VIVO AND ALSO IN THE MODEL. ON THE OTHER END OF THE SPECTRUM, HAVE YOU SUCROSE WE HEARD ABOUT IT BEING USED AS THE VASCULAR TRACER FOR WHETHER OR NOT THE BLOOD-BRAIN BARRIER OR IS INTACT. THIS SMALL HYDROTHRILLIC MOLECULE SELL EXCLUDED SIGNIFICANTLY IN-VIVO AND ALSO IN THE MODEL. NOTICE GLUCOSE DIFFERENT SIZE THAN SUCROSE BUT A SMALL HYDROPHILIC MOLECULE AND LIKELY DUE TO GLUT1 TRANSPORTER EXPRESSION AND SMALL MOLECULE LYPOPHILIC DRUGS SUBSTRATES ARE EFLUX SO IT'S A COMBINATION BETWEEN INFLUX, EFLUX AND PHYSICAL BARRIER WHICH DETERMINES WHETHER OR NOT YOU CAN BE PREDICTIVE. SO IN THE LAST FEW MINUTES, I WANTED TO GO THROUGH A COUPLE OF VIGNETTES OF USING SOME OF THESE PROPERTIES HOPEFULLY TO OUR BENEFIT. AND HERE WHAT WE HAVE DONE IS WE HAVE TAKEN THESE IPSC DERIVED BRAIN ENDOTHELIAL CELLS TO LOOK AT A NEW CLASS OR IT'S AN OLD CLASS OF DRUGS NEWLY EXAMINEDAL COPHOSPHOCHOLINE DRUGS THAT HAVE THE ABILITY TO TARGET TUMOR AND TUMOR STEM CELL POPULATIONS IN-VIVO IN RODENT MODELS AND IN CLINIC. AND NOT ONLY PERIPHERAL TUMORS BUT THEY HAVE THE ABILITY TO HIGHLIGHT BRAIN TUMORS IN THIS CASE IT'S A GBM PATIENT. YOU CAN SEE FROM THE T2 RELATED MRI, YOU CAN SEE THE TUMOR THAT IS ALSO LIT UP BY THE CLR1404CLR COMPOUND AND THERE IS THE VERSIONS OF THE COMPOUNDS THAT CAN BE USED AS CHEMOTHERAPY AGENTS OR IMAGING AGENTS AND ALSO A DESIRE IN THE NEUROSURGICAL FIELD TOL INDIVIDUAL FLOURO FORES IR OR IN THIS CASE A SAMPLE FLOURO 4 ATTACH FOR IMAGING FOR TUMOR RESECTION. SO THIS IS ALL IN COLLABORATION WITH A NEUROSURGEON AND RIDOLOGIST AT THE UNIVERSITY OF WISCONSIN. SO ONE OF THE THINGS THAT ISN'T KNOWN EACH THOUGH THESE WERE EVALUATED IN RODENT AND HUMAN PATIENTS IS WHAT IS THE ACTUAL BBB PERMEABILITY? WHY DO WE SEE A SIGNAL TO NOISE RATIO FOR BRAIN TUMOR? IS IT BECAUSE THE BARRIER IS DISRUPTED OR IS THERE A SELECTIVE RETENTION BY TUMOR CELLS? AND ARE THERE DIFFERENCES BETWEEN THESE ANALOGUES WE NEED TO CONSIDER WHEN THINKING ABOUT DEPLOYING THEM IN THE CLINIC? SO, WE BASICALLY WENT IN FIRST TO MEASURE THE LYPOPROPERTIES SO DIAZAPAM VERY LYPOPHILIC. SUCROSE NOT. YOU CAN SEE CLRs RANGE IN THE LYPOFELICKITY. THIS IS A LOG BASE SO IT IS PRETTY SUBSTANTIAL IN TERMS OF DIFFERENCES. BUT WHAT YOU'LL NOTICE IS THE PERMEABILITY ACROSS THE BBB MODEL THEY ARE PERMEABLE BUT NOT MUCH MORE PERMEABLE THAN SUCROSE AND MUCH LESS THAN DIAZAPAM DESPITE THE LYPOFLIESITY AND SIZE THAT CAN EXPLAIN WHY THESE ARE TAKEN AT SUCH A LOW-LEVEL. MOREOVER, PHENO1 AND 1502 THEY HAVE THE FLOUR 4S AND HAVE SIMILAR IF NOT LOWERED PERMEABILITY ACROSS THE MODEL. SO I WON'T SHOW YOU ALL THE DATA BUT LONG STORY SHORT THERE IS EVIDENCED THAT THERE IS ACTIVE EFLUX GOING ON THAT IS REALLY LIMITING THE PENETRATION OF THESE COUNTDOWNS ACROSS THE BBB IN THE MODEL. AND HERE I HAVE DONE THAT SAME EXPERIMENT I SHOWED EARLIER LOOKING AT THE VARIOUS SUBSETS OF EFLUX TRANSPORTERS AND SPECIFIC INHIBITORS AND 1404 ITSELF DID NOT SHOW ANY STATISTALLY SIGNIFICANT ALTHOUGH THERE SEEMS TO BE A LITTLE BIT OF EFLUX CONTRIBUTION. HOWEVER THE FLOR FORD MODIFIED VERSIONS SHOWED EFLUX WHICH CAN HELP EXPLAIN WHY THESE LYPOPHILIC MOLECULES ARE EXCLUDED IN THE BBB MODEL AND THE IDEA NOW IS THAT THIS SIGNAL NO NOISE RACEIO IN THE HUMAN PATIENT MAY BE LARGELY DUE TO PASSING A RUPTURED BBB BECAUSE THE INTACT BBB SEEMS TO BE QUITE IMPERMEABLE. MOREOVER, THE FLUOROPHORES AREN'T ONLY EXPRESSED AT THE BLOOD BRAIN BARRIER BUT ALSO IN MANY DIFFERENT CANCERS SO IT WOULD BE IMPORTANT FOR INTEROPERATIVE IMAGING IN THE BRAIN AND ELSEWHERE. SO THE LAST COUPLE OF MINUTES I'LL TALK ABOUT TAKING ADVANTAGE OF THE FACT THAT THESE CAN BE PATIENT DERIVED STEM CELL LINES THAT WE THEN DIFFERENTIATE TO BRAIN ENDOTHELIAL CELLS AND OTHER NEURAL CELL TYPES. AND THERE ARE MONOGENIC DISEASES THAT BLOOD-BRAIN BARRIER ASSOCIATED AND ONE OF THESE IS A RARE DISEASE CALLED ALAN HERN ON DUDLEY SYNDROME. THIS IS USED AS OUR EXAMPLE DISEASE MODEL WELL BBB BECAUSE IT IS DUE TO A MUTATION IN TRUNCATION IN THE MCTA THYROID HORMONE TRANSPORTER. AND IT IS LINKED TO SEVERE MENTAL RETARDATION IN YOUNG BOYS AS AN X-LINKED PHENOMENON. AND THIS IS IN COLLABORATION WITH CEDAR SINAI. THE IDEA IS TO TAKE THE BBB MODEL AND EXAMINE IF NCT8 DEFICIENCY IS KEY IN REPLICATING REPLICATI NG THIS FE NON NON A HIGH PLASMID THYROID HORMONE LEVEL BUT A VERY LOW CORTICAL OR CSF LEVEL IN HUMANS. SO FIRST QUESTION IS CAN WE TAKE IPS LINES DERIVED FROM PATIENTS AND MAKE BRAIN-LIKE ENDOTHELIAL CELLS? AND INDEED WE CAN HERE JUST A FEW OF THE MARKERS. THESE ARE PARENTAL CONTROLS AND THESE ARE ACTUALLY THE BOY'S. IS THESE HAVE TWO DIFFERENT TYPES OF MCT8 MUTATIONS. THESE CAN FORM TIGHT MONOLAYERS WITH HIGH ELECTRICAL RESISTANCE AND HAVE UNIFORM EFLUX TRANSPORTER EXPRESSION EXCEPT FOR MCT8 THAT YOU EXPECT TO BE DEFICIENT IN THE MUTATED LINES. NOW, A LONG STORY SHORT, THIS PICTURE OR THIS FIGURE GIVES US MOST OF THE INFORMATION. IF WE LOOKED AT THE PARENTAL LINES, THESE FIRST TWO BARS HERE, YOU CAN SEE T3 TRANSPORTER ACROSS THE IPS DERIVED MODEL. IF YOU LOOK AT THE MUTATIONS YOU SEE IMPAIRED T3 TRANSPORT VIA MUTATION OF THE MCT8. WHAT IS NICE ABOUT USING STEM CELLTECHINOLOGIY IS WITH GENETTING TOOLS YOU CAN IN THAT SAME GENETIC BACKGROUND RESTORE MCT8 TRANSPORTED OR TAKE THE PARENTAL CONTROL AND INDUCE THE DEFECT AND SO THAT MEANS THE ENTIRE GENETIC BACKGROUND YOU'RE SHOWING THAT MCT8 IS KEY FOR THIS T3 DERIVED TRANSPORT PHENOTYPE. SO I THINK THE LAST DATE AT SLIDE HERE THEN IS JUST SHOWING THE DIRECTIONALITY WHICH WAS QUITE INTERESTING. WHAT WE FIND IS FOR ALL OF THOSE LINES THAT HAVE INTACT MCT8 THERE IS A BLOOD TO BRAIN PREFERENCE FOR T3 TRANSPORT THAT WHEN YOU ELIMINATE MCT8 YOU SEE DIFFERENCE BIDIRECTIONALLY SO SUGGESTIONING A UNIDIRECTIONAL OR ENHANCED IMPORT OF T3 AS A FUNCTION OF MCT8. SO, LONG STORY SHORT AGAIN, WE LOOKED AT THE NEURAL CELLS AS WELL. THEY DIDN'T SHOW ANY DEVELOPMENTAL DEFICITS IN THE PETRE DISH BUT CERTAINLY THIS BLOOD-BRAIN BARRIER DEFICIT WAS PRONOUNCED AND MAY EXPLAIN INPATIENTS WHY THIS IS HAPPENING. SO IF YOU THINK ABOUT THE LAST BULLET POINT HERE THEN IN RED BEING ABLE TO USE THE SAME MODEL WHERE YOU HAVE THE READ OUT OF IMPAIRED T3 TRANSPORT AND YOU CAN LOOK FOR SMALL MOLECULES FOR INSTANCE THAT COULD ENHANCE T3 UPTAKE FOR PATIENTS SUFFERING FROM THESE DISEASES. SO IN SUMMARY, HOPEFULLY I CONVINCED YOU ALONG WITH THE OTHER TALKS THIS MORNING THAT STEM CELLS CAN BE USED TO FORM BLOOD-BRAIN BARRIER ENDOTHELIAL CELLS AND DO MULTICELLULAR CULTURES IT'S VERY SCALABLE. ONE OF THESE PAPERS WE WENT THROUGH 10,000 FILTERSOR SOMETHING LIKE THAT WHICH WOULD HAVE BEEN MANY, MANY ANIMALS. AND HOPEFULLY I HAVE CONVINCED YOU THERE ARE NICHES IN TERMS OF EXPLORING THE INTERACTIONS BETWEEN BLOOD BORN COMPONENTS OF THE BBB AND THEN ULTIMATELY WHAT GETS INTO OUR OUT OF THE BRAIN. SO I'D LIKE TO THANK ALL OF MY COLLABORATORS I MENTIONED DURING THE TALK AND FUNDING SOURCES. THANK YOU VERY MUCH. [ APPLAUSE ] >> THANK YOU VERY MUCH. WE'LL HAVE TO HOLD QUESTIONS FOR THE OPEN MIC DISCUSSION SO WE'LL MOVE ON TO THE NEXT TALK BY DR. LAURA SERGIU PASCA. AND HE WILL BE TALKING ABOUT DEVELOPING TRIDIMENSIONAL MODELS OF HUMAN CEREBRAL CORTEX IN-VITRO. MY LAB IS NOT DIRECTLY WORKING ON THE BBB OR INTERFACE. WE ARE INTERESTED IN OVERALL MODELING BRAIN DISORDERS AND IN PARTICULAR NEURODEVELOPMENTAL DISORDERS BUT I THINK THAT SOME OF THE WORK THAT WE HAVE BEEN DOING IS PROBABLY RELEVANT TO SOME OF THE QUESTIONS THAT WE HAVE TRIED TO ADDRESS. THE BEST WAY TO ILLUSTRATE MY LAB IS DOING AND THE PROGRAMMING APPROACH HAS ALREADY BEEN ILLUSTRATED IS THROUGH A DRAWING THAT ONE OF MY FIRST PATIENT WITH AUTISM DID WHEN I OPENED MY LAB. SO, HE DREW THIS AND HE THOUGHT THIS IS HOW WE ARE STUDYING NEURODEVELOPMENTAL DISORDERS. HE THOUGHT WHAT WE ARE DOING IS CLIMBING UP A LADDER AND POKING HOLES IN PEOPLE'S BRAINS AND LOOKING OF COURSE DESTROYING THE BBB BUT LOOKING AT BOTH NEURONS AND ASTROCYTES IN ADDITION. OF COURSE, THIS OUTPRICESLY WHAT WE ARE DOING SO ACTUALLY AFTER TALKING TO HIM AND HE IS HIGH FUNCTIONING, THE NEXT DAY HE SENT ME ANOTHER DRAWING THAT I THINK IS PROBABLY A LITTLE BIT MORE ACCURATE FOR PRESENTATION OF OVERALL THE APPROACH OF MODELING DISORDERS IN A DISH. AND SO, THIS IS THE ILLUSTRATION OF THE PARADIGM SO WHAT WE ARE DOING IS STARTING WITH SKIN CELLS FROM THIS PATIENT AND DOING SOME MUMBO-JUMBO IN HIS OWN WORDS TO TURN THEM BACK INTO CELLS THAT LOOK LIKE STEM CELLS AND BEHAVE LIKE STEM CELLS, INDUCED POPULARY POTENT STEM CELLS AND THEN DRIVING THEM INTO BECOMING NEURONS. AND WHILE MAYBE THE FIRST TWO STEPS OF THE HARVESTING OF THE SKIN CELLS AND REPROGRAMMING ARE NOW STANDARDIZED ACROSS LABS, ONE OF THE CHALLENGES THAT WE ARE FACING THAT THE MOMENT IN THE FIELD IS REALLY IN DEVELOPING THE BEST METHODS FOR DERIVING NEURONS IN A DISH. AND SO WE ALREADY USE THIS PROCESS BEFORE FOR MODELING A MONOGENIC FORM OF AUTISM, WHAT I WANT TO SHOW YOU HERE. BUT I DO WANT TO POINT OUT IS THAT MOST OF THE PHENOTYPES THAT WE FOCUS ARE ACTUALLY IF I COULD CALL SIMPLE PHENOTYPES, AND THE SENSE THAT FOR INSTANCE THIS IS -- SO THIS MONOGENIC FORM OF AUTISM IS CAUSED BY A CALCIUM CHANNEL T IS HIGHLY PENETRANT AS ONE OF THE MOST PENETRANT FORMS OF AUTISM WE KNOW OF. AND THIS PATIENTS HAVE AUTISM IN ABOUT 80% OF THE CASES AND THE SYNDROME. SO WHAT WE WERE ABLE TO SHOW IS THAT IF YOU DO DERIVE NEURONS FROM THESE PATIENTS YOU CAN USE CALCIUM IMAGING AND SORT OF SHOW THAT IN THE PATIENT NEURONS DO ALLOW MORE CALCIUM TO GET IN THE CELLS ONCE THEY ARE DEPOLAR AUDIOS AND THIS CAN BE BLOCKED WITH A BLOCKER OF THIS CHANNEL. WE ALSO FOUND LIKE NEW THINGS FOR INSTANCE ONE OF THE PHENOTYPES WE IDENTIFIED HAD TO DO WITH CAT COLA MEAN. AND WE FOUND THE PATIENT CELLS WERE PRODUCING EXCESSIVE ECK TOPICALLY TOURISM HYDROXYLASE AND PRODUCING HIGH LEVELS OF DOPAMINE IN THE DISH. PHENOTYPE THAT WE USED TO IDENTIFY SMALL MOLECULE THAT WORTH RESCUING THE PHENOTYPE AS WELL AS THE PHENOTYPES WE IDENTIFIED IN THIS PATIENT INCLUDING THE DENDRITE PHENOTYPE. SO, WHAT I WANTED TO POINT OUT HERE IS THAT THIS PLATFORM CAN BE USED FOR IDENTIFYING PHENOTYPES BUT WE ARE LIMITED IN CERTAIN ASPECTS DUE TO THE PLATFORM. SO, THIS IS HOW NEURONS IN A DISH LOOK LIKE AND THERE ARE VARIOUS LIMITATIONS TO THIS CULTURE. ONE OF THEM THEY ARE QUITE LABORIOUS AND THEY TAKE A LONG TIME TO CULTURE. THEY ARE FUNCTIONALLY MATURE SO ONLY HALF OF THE NEURONS FIRE IN MOST PROTOCOLS AND MAYBE MORE IMPORTANTLY BECAUSE THESE ARE CORTICAL NEURONS THERE IS NO SITE FOR ARCHITECTURE SO YOU DON'T SEE LAYERING AND MAYBE MORE IMPORTANTLY FOR ME AS A NEUROSCIENTIST, THERE ARE NO SYNAPSES. AND THIS IS DUE TO THE FACT THAT ASTRO GENESIS DOESN'T HAPPEN IN THE SYSTEM SO YOU HAVE TO BRING ASTROCYTES FROM EITHER RODENTS OR YOU HAVE TO DERIVE THEM SEPARATELY AND THEN COCULTURE THEM WHICH MAKES IT VERY COMPLICATED. HOW DO WE GET FROM THIS A LITTLE BIT CLOSER TO THE ARCHITECTURE AND THE FUNCTIONALITY OF THE HUMAN CORTEX? AND SO, WHAT MY LAB HAS DONE AND THIS IS WORK PUBLISHED A YEAR OR SO AGO, IS DEVELOP A METHOD FOR DERIVING IN 3D CULTURES THROUGH A SIMPLE MUCH MORE SIMPLER METHOD THAN WHAT WE HAD BEFORE, WHAT WE CALL CORTICAL STEROIDS. AND THESE ARE ESSENTIALLY DERIVED BY LIFTING THE IPS COLONIES AND PROVIDING A FEW GUIDING CUES VERY EARLY ON. AND THERE ARE ALL THE TIME NOT EMBEDDED IN EXTRA SELL LAYER MATRIXES AND THIS SORT OF LIKE DIFFERENTIATED FROM ORGANOID APPROACHES WHICH ARE LESS DIRECTED IN DIFFERENTIATION. SO THE STEROIDS LOOK LIKE THIS. THEY ARE ABOUT A COUPLE OF HUNDRED OF MICROMETERS AFTER A COUPLE OF WEEKS IN DIAMETER. THEY VERY EARLY SHOW NEURONAL DIFFERENTIATION. IF YOU WAIT LONG ENOUGH THEY DEVELOP INTO A LARGER THAT YOU HAVE 4 MILLIMETERS IN SIZE WHICH IS VERY SIMILAR TO THE SIZE OF A MOUSE BRAIN AND E12.5. IF YOU CONTINUE TO GROW THEM FOR LONGER, THEY REACH ABOUT 5 MILLIMETERS IN SIZE AND THEY STOP. THE GREAT VALUE OF THE SYSTEM IS THAT IT IS VERY SCALABLE SO YOU CAN GET HUNDREDS. WE HAVE THOUSANDS OF THE STEROIDS FROM ANY PARTICULAR PATIENT AND SORT OF LIKE DRIVE THEM IN PARALLEL. SO THIS IS HOW THEY LOOK OVER TIME. AND WE DID A LOT OF WORK IN CHARACTERIZING THEM FIRST A TRANSCRIPTIONAL LEVEL SO WE SHOWN BY MAPPING INTO ALL THE DATA THAT WE NOW HAVE ON THE TRANSCRIPTOME OF THE HUMAN CORTEX AND SHOW THEY DO MATURE FASTER THAN MONOLAYER CULTURES. MOST CULTURES MAP TO 8-10 POST CONCEPTION WEEKS AND WE WERE GETTING UP TO 50 DAYS TO THE SECOND TRIMESTER AND BEYOND. ANOTHER GREAT ADVANTAGE OF THE SYSTEM IS THE SITE OF ARCHITECTURE BECAUSE THEY ARE SO BIG YOU CAN FIX THEM AND EMBED THEM AND TREAT THEM LIKE BRAINS LIKE RODENT BRAINS. SO IF YOU LOOK INSIDE YOU CAN SEE MOST OF THEM HAVE VENTRICLE WHICH IS USUALLY ON THE SIDE AND YOU SEE THE ARRANGEMENT OF THE CELLS IN A VERY PECULIAR WAY. WE CAN SJOGREN THE VARIOUS SUBPOPULATION OF PROGENITORS, THE PAC 6 WHICH ARE REGULAR GLEIA. SUB VENTRICULAR PROGENITORS ARE RANGE ON THE OUTER SIDE AND YOU CAN SEE EVEN THE RATE OF PROCESSES OF THE RADIAL GLEIA THAT ARE PERPENDICULAR TO THE VENTRICAL-LIKE STRUCTURE INSIDE. EVEN THE CELL DIVISIONS HAPPENING IN A VERY PECULIAR WAY AND THIS IS ONE OF THE GREATER ADVANTAGES OF THE SYSTEM YOU CAN IMAGE THEM LIVE. SO, THIS RADIAL GLEIA DIVIDE IN A VERY PECULIAR WAY HAS BEEN SHOWN FOR A LONG TIME. THEY ACTUALLY MOVE THEIR SOMA CLOSE TO THE VENTRICLE WHEN THEY ARE DIVIDING AND THEN THE DAUGHTER CELL WHICH IS VERY OFTEN A YOUNG NEURON, IS MOVING ON UPON TO OF THE MOTHER CELL. AND SO YOU CAN IMAGE THIS LIVE. YOU WILL SEE HOW THE CELLS LABELED WITH THE CELL SPECIFIC REPORTER, ARE GOING TO DIVIDE IN THIS PECULIAR WAY MOVING CELL SOMA CLOSER TO LOOM IN AND THEN THE DAUGHTER CELL MOVING ON TOP. SO IF YOU WAIT LONG ENOUGH YOU START SEEING THE VARIOUS LAYERS OF THE CORTEX FORMING IN A DISH SO FOR INSTANCE YOU CAN SEE UPPER LAYER OF NEURONS ON THE SURFACE AND POSITIVE CELLS AND THEN IF YOU WAIT 130 DAYS AND THIS IS WHERE WE ESTIMATED THAT CORTICOGENESIS WAS COMPLETE SO THIS IS STILL ALMOST 20 WEEKS OF CULTURE. YOU CAN SEE DEEP LAYER NEURONS ARRANGE AROUND THE VENTRICLE IN SUPERFICIAL LAYERS ON THE OUTSIDE AND VARIOUS MARKERS THAT WE HAVE QUANTIFIED AS WELL AS THEIR TIMING SO YOU CAN SEE DEEP LAYERS FORMING FIRST AND UPPER LAYERS FORMING LAST EXACTLY LIKE IT IS HAPPENING IN THE HUMAN CORTEX. YOU CAN ALSO SEE OTHER FEATURES FOR INSTANCE DOUBLE WHICH IS EXPRESSED VERY HIGHLY DURING GESTATION AND IT GOES DOWN TOWARDS THE END. IF YOU WAIT FOR ABOUT A YEAR, DOUBLE DOES GO DOWN IN THE CELLS AND THE CELLS ARE STILL HEALTHY. THERE ARE OTHER WAYS OF LOOKING AT THE CORTICAL LAYER IDENTITIES. SO FOR INSTANCE HERE AT THE TOP GENES IDENTIFIED BY THE ALLEN BRAIN INSTITUTE AS BEING LAYER SPECIFIC IN HUMANS AND SHOWING YOU GET THE CORTICAL LAYERS WITH THE CELLS. SO, WE SPEND A LOT OF TIME AND I'M NOT GOING TO GO INTO THIS EXPERIMENTAL RELIABILITY BECAUSE WE WANT TO THE USE THEM FOR MODELING DISEASE. SO FOR INSTANCE WE ESTIMATED LIKE WHAT IS THE VARIESABILITY OF CROSS MULTIPLE STEROIDS WITHIN ONE CULTURE? WHAT IS THE VARIABILITY ACROSS DIFFERENT DIFFERENTIATIONS OF THE SAME LONG OR DIFFERENT DIFFERENTIATIONS OF SISTER CLONES FROM THE SAME INDIVIDUAL? AND SO, AS I WAS MENTIONING ONE OF THE LIMITATIONS IS THE MATURATION OF MONOLAYER CULTURES AND THE FACT THAT YOU DON'T HAVE ASTROCYTES. SO, WE ACTUALLY TREATED A CONDITION IN TRYING TO OBTAIN ASTROCYTES. SO IF YOU WEIGHT FOR ABOUT 10 WEEKS, YOU START TO SEE THAT OUTSIDE OF THOSE ZONES THERE ARE GFAP POSITIVE CELLS AND ACTUALLY IF YOU USE THE RIGHT TOMOGRAPHY AND RECONSTRUCT THIS, YOU CAN SEE THIS VERY THIN PROCESS REALLY ARE ALL ACROSS THE NEUROPHIL AND THE NUMBER OF GFAP POSITIVE CELLS INCREASES WITH TIME AND GETS STABILIZED AT AROUND 20% OF ALL THE CELLS. NOW, THIS WORK IS DONE IN COLLABORATION WITH BEN WHO IS A PIONEER OF ASTROCYTE BIOLOGY SO WE USE THE TECHNIQUES THAT HIS LAB DEVELOPED OVER THE LAST 20 YEARS OR SO. ONE OF THE THINGS YOU CAN DO IS ISOLATE THE ASTROCYTE AND SEE THEM IMMUNOPURIFIED SO YOU CAN SEE HOW THEY ARE RECOVERING AFTER THIS PROCESS OF BEING ASSOCIATED FROM THE 3D CULTURES. IF YOU LOOK CLOSELY YOU CAN SEE THAT THEY LOOK STAR-LIKE AND THEY HAVE A THIN PROCESS EVEN AFTER SELF DAYS OF MAINTAINING IN-VITRO. IF YOU PUT SERUM THEY BECOME FLAT AND UPREGULATE GFAP AS A NUMBER OF INFLAMMATORY CYTOKINES AND THEY BECOME REACTIVE. AND ANOTHER ADVANTAGE OF THE SYSTEM IS YOU CAN DO LONG TERM CULTURES. AND SO, WE HAVE BEEN TRYING TO SEE HOW LONG CAN WE ACTUALLY GO WITH THIS CULTURES AND HOW MUCH MATURATION THERE IS FOR SOME OF THE CELLS. SO, THIS IS HOW ASTROCYTES LOOK IF THEY ARE MAINTAINED UP TO TWO YEARS. NOW THEY ARE BEYOND TWO YEARS OF BEING MAINTAINED IN A DASH. THIS IS HOW THEY ARE RIGHT AFTER THE ASSOCIATION SO YOU CAN ALREADY SEE THAT THE PROCESSES ARE MUCH MORE PROMINENT IN CELLS IN ASTROCYTES THAT HAVE BEEN MAINTAINED FOR A LONG PERIOD OF TIME. THIS IS HOW THEY LOOK AFTER THEY RECOVER AND YOU CAN CLEARLY SEE THE NUMBER OF BRANCHING OF THE CELL, THE BRANCHING COMPLEXITY INCREASES OVER TIME. THIS IS HOW AN ASTROCYTES LOOKS AFTER OVER A YEAR OF CULTURE IN THE 3D CULTURE SO VERY COMPLEX PROCESSES. SO, WE SPEND A LOT OF TIME LATELY CHARACTERIZING THEM OVER TIME AT THE MOLECULAR TRANSCRIPTIONAL LEVEL AS WELL AS FUNCTIONAL. I WON'T SHOW ALL THE FUNCTIONAL DATA TODAY. FOR INSTANCE, AND THIS IS WORK DONE BY A WONDERFUL STUDENT STEVEN SLONE WHO ISOLATED ASTROCYTES OVER TIME UP TO 600 DAYS IN CULTURE FROM THIS 3D CORTICAL STEROIDS. SO THE FIRST THING THAT HE ACTUALLY DID IS HE LOOKED AT THE TOP GENES THAT THE LAB ESSENTIALLY IDENTIFIED IN HUMAN ASTROCYTES TO BE UPREGULATING DURING THE MATURATION OF THE CELL. SO THESE ARE THE TOP 100 GENES EXPRESSING IN FETAL ASTROCYTES VERSUS FOR INSTANCE POSTNATAL ASTROCYTES. SO IF YOU LOOK AT THE EXPRESSION OF THESE GENES IN ASTROCYTES THAT HAVE BEEN ISOLATED FROM THIS CULTURE OVER TIME, YOU CAN CLEARLY SEE THAT EARLY ON THEY REALLY EXPRESS GENES EXPRESSING IN PRIMARY FETAL ASTROCYTES BUT IF YOU WAIT LONG ENOUGH, YOU START TO SEE SOME OF THE MATURE GENES BEING EXPRESSED ANOTHER WAY OF SHOWING. SO ONE OF THE GENES FOR INSTANCE THAT IS EXPRESSED EARLY ON ARE GENES THAT HAVE TO DO WITH CELL CYCLE AND LATER ON YOU CAN SEE THOUSANDS OF FOLDS OF UPREGULATION OF AQUAPORE IN VERY EARLY ON IS VERY EXPRESSES VERY LOW LEVELS. SO, WE THINK THIS OFFERS A OPPORTUNITY TO STUDY THE MATURATION OF SOME OF THE CELLS WHICH HAS NOT BEG YOUR PARDON ATTAINED BEFORE. AND ANOTHER THING WE SPEND A LOT OF TIME WITH IS DEVELOPING TOOLS FOR IMAGING OF THE CELLS SO THIS IS WORK WE DID WITH OUR LAB AT CALL TECH, A PAPER THAT WAS PUBLISHED IDENTIFYING VIRAL VECTORS EFFICIENTLY TRANSDUCE THE BRAIN AND THE ORTCALL STEROIDS. SO THIS IS KNOW WHO OF THE VIRUSES THAT AFFECTS THE STEROIDS AND ONE OF THE THINGS YOU CAN DO AND ONE OF THE MOST EXCITING EXPERIMENTS THAT WE HAVE DONE. I STILL DON'T KNOW EXACTLY WHAT IT MEANS BUT I WANT TO SHARE IT WITH YOU. SO THIS IS AN EXPERIMENT WHERE WE INFECTED THE SPHERES WITH CELL SPECIFIC REPORTERS SO YOU CAN SEE ALL THE NEURONS IN RED AND ALL THE RADIO GLEIAS AND ASTROCYTES IN GREEN. SO, ONE OF THE STEROIDS THAT HAS BEEN CULTURED FOR MANY WEEKS HAS ACTUALLY BEEN CUT ACUTELY AND PUT OFF THE TOP OF THE COVER SLEEVE. AND ALTHOUGH WE USE GERMAN GLASS WHICH IS THE BEST GLASS YOU CAN GET, THE GLASS IS RIGHT ON THE SIDE. YOU WILL SEE THAT VERY SOON AFTER BEING PLATED, ASTROCYTES IMMEDIATELY RECOGNIZE THAT SURFACE. AND THEY SEND THIS LONG PROCESS SORT OF LIKE TRYING TO EXPLORE AND SEE WHAT HAPPENS. AND THEY AGAIN NO MATTER HOW MUCH WE LOOK IN 3D CULTURES, WE DON'T SEE THIS FLAT PROCESS. THEN YOU'LL SEE AT ONE POINT THE RADIO GLEIA HERE THAT DIVIDES IN THAT VERY PECOOLIER WAY I SHOWED YOU BEFORE WHEREAS YOU WILL SEE SOME NEURONAL PROCESSES OF THE NEURONS DON'T EXTEND TOWARDS THE -- THEY DON'T REALLY CARE ABOUT THAT. YOU WILL SEE ONE HERE THAT IS LIKE ISOLATED AND TRYING TO FIND PARTNERS. SO THIS IS SOME OF THE THINGS THAT YOU CAN DO WITH THIS SYSTEM SO BECAUSE WE DO HAVE ASTROCYTES, WE FIND ABUNDANCE ISN'TOGENESIS. SO THIS IS RATE TOMOGRAPHY SHOWING NEURONS AND ON TOP OF THESE SNAP TICK PROTEINS, YOU CAN SEE ABUNDANT SYNAPSE FORMATION IN THE CULTURES. THIS IS ALSO RECONSTRUCTION OF ONE SINGLE SYNAPSE. YOU CAN SEE THREE MARKERS IN A RATE TOMOGRAPHY. SO THIN 70 NANOMETER SECTIONS THROUGH A SYNAPSE AND STOW YOU CAN SEE THE OB CISION OF SYNAPSE IN ONE SIDE AND PSD95 ON THE OTHER SIDE. STRUCTURAL SYNAPSE MEANS NOTHING TO AN ELECTROPHYSIOLOGIST. AND YOU CAN HAVE A LOT OF STRUCTURAL SYNAPSES WITHOUT NECESSARILY BEING FUNCTIONAL SO WE SPENT A LOT OF TIME CHARACTERIZING AT THE ELECTRICAL LEVEL THIS STARED SO WE USED EITHER PATCH-CLAMPING AND WE FOUND 90% OF THE CELLS DO FIRE ACTION POTENTIALS AND WE USED CALCIUM IMAGING TO CUT OPEN SOME OF THE STEROIDS SO EVEN NOW IMAGE THEM ENTIRELY AND LOOK AT THE ACTIVITY OF INDIVIDUAL NEURONS AND HOW THEY ARE LIKE PRESUMABLY TALKING TO EACH OTHER IF I'M NOT INTERPRETING TOO MUCH. AND SO, AGAIN THE OTHER THING THAT WE HAVE DONE BEYOND CALCIUM IMAGING IS TRY TO LEVERAGE THIS SYSTEM AND MAINTAIN THE CYTOARCHITECTURE. SO, IF YOU DO LOOK AT SYNAPTIC ACTIVITY, 90% OF THEM ARE CONNECTED. THEY ARE ALL EXCITATORY CELLS AS YOU EXPECT. ONE OF THE THINGS YOU CAN DO AND THIS IS WORK WE DID WITH A LAB AT STANFORD IS YOU CAN APPLY SLIDES RECORDING TECHNOLOGIES SO LITERALLY TAKE ONE OF THE STEROIDS AND SECONDS THEM AND THEN -- SECTION THEM AND THEN REPORT FROM ONE SIDE AND STIMULATE ON THE OTHER SIDE ELECTRICALLY OR USING GENETIC METHODS AND RECORD THE ELECTRICAL ACTIVITY YOU TRIGGER BY STIMULATION. AND SO WE THINK THAT THIS OFFERS ANOTHER LEVEL OF RESOLUTION IN TERMS OF PROBING SOME OF THE COMPLICATED NEURONETWORKS. I'LL STOP HERE BECAUSE I THINK I PROBABLY RAN OUT OF TIME. I WANT TO SUMMARIZE. THIS IS THE LOGO OF MY LAB. I WANT TO SUMMARIZE SOME OF THE ADVANTAGES OF THIS CULTURE WHICH AGAIN I'M A BIG FAN OF 3D CULTURES. IT'S JUST THAT THEY ARE HALF TO BE USED IN COMBINATION WITH OTHER METHODS AS WELL AND ALTHOUGH THERE ARE SIMPLER WAYS THERE ARE MORE COMPLEX IN OTHER WAYS. SOME OF THE ADVANTAGES OF 3D CULTURES IS THE FACT THAT YOU DO MAINTAIN SOME OF THE CYTO-ARCHITECTURE THAT THE MATURITY FUNCTIONALLY AND TRANSCRIPTIONALLY DOESN'T PUSH FORWARD AND THE FACT YOU'RE GETTING ASTROCYTES IN THESE CULTURES WHICH PROMOTES SIN ANTIGEN SIS AND THE FACT THAT YOU CAN APPLY MORE COMPLEX ELECTROPHYSIOLOGICAL METHODS TO THIS SYSTEM AND WITH THIS, I WOULD LIKE TO ACTUALLY ACKNOWLEDGE THE PEOPLE IN MY LAB AND IN PARTICULAR AN CAAND AND IN PARTICULAR AN CAAND STEVEN WHO HAVE DONE MOST OF THE WORK AND OUR KEY COLLABORATORS IN PARTICULAR BEN AND JOHN AND MY FUNDING SOURCES AND I GUESS WE'LL TAKE QUESTIONS AT THE END. THANK YOU. [ APPLAUSE ] >> THANK YOU VERY MUCH DR. PASCA. YOU'RE CORRECT. WE WILL TAKE QUESTIONS AT THE OPEN MIC DISCUSSION. AND WE'LL MOVE FORWARD WITH PRESENTATION BY DR. MARTIN ULMSCHNEIDER. REVEALING THE TRANSPORT MECHANISMS KINETICS AND ENERGETICS OF DRUGS DIFFUSING THROUGH MEMBRANES OF THE BLOOD-BRAIN BARRIER. MARTIN ULMSCHNEIDER: THANK YOU FOR INVITING ME AND FOR PUTTING TOGETHER THIS VERY NICE MEETING. IT'S BEEN REALLY EXCITING. SO I'M SORT OF A BIT OF THE ODD ONE OUT HERE I THINK BECAUSE I'M GOING TO TALK ABOUT COMPUTATIONAL METHODS AND YOU HAVE TO BEAR WITH ME. THIS IS REALLY WORK THAT IS IN INFANCY. THESE TECHNOLOGIES ONLY BECOME AVAILABLE TO THESE KIND OF PROX IN THE LAST THREE-FOUR YEARS MAYBE. SO THE KEY QUESTIONS I WANT TO ADDRESS HERE IS, CAN MOLEC LARSENALATIONS DETERMINE MECHANISMS OF DIFFUSION ACROSS A MEMBRANE? CAN WE PROVIDE QUANT AT A PREDICTIONS FOR SOLUTE TRANSPORT CREASES MEMBRANE? AND I'M GOING TO INTRODUCE THE TECHNIQUE. SO WHAT SAY MOLECULAR DYNAMIC SIMULATION? AND I'M GOING TO TRY TO SHOW WHAT KIND OF INSIGHTS CAN BE GAINED. AND I WANT TO STRESS HERE THAT I'M TALKING ABOUT EQUILIBRIUM, ARK TOMMIC DETAIL SIMULATION. SO A LOT OF MODELS AND IMPLICIT MODELS AND A LOT OF DEDICATED MODELS. THESE ALL HAVE VERY SERIOUS ASSUMPTIONS. THIS LITERALLY IS THE EQUIVALENT OF DOING EXPERIMENT IN A CITY. BUILDING A SYSTEM OR ATOM. NOT PULLING. YOU'RE HITTING THE ENTER BUTTON AND WAIT A LONG TIME AND SEE WHAT HAPPENS. IF THE YOU BUILD IT CLOSE ENOUGH TO THE SYSTEM IN NATURE YOU CAN GET VERY ACCURATE INFORMATION. SO, FIRST OF ALL, WHAT IS MOLECULAR DYNAMICS? IT'S BASICALLY A CLASSIFICATION OF ATOMIC WORLD IF YOU LIKE. SO IT'S MECHANICS. YOU HAVE BONDS DONE WITH HARMONIC POTENTIAL, ANGLES DONE WITH ANOTHER HARMONIC POTENTIAL, AND ANOTHER COSIGN EXPANSION AND YOU GENERALLY NEED 3-4 TERMS AND THIS IS VERY ACCURATE SO IF YOU DO A QUANTUM MECHANIC CALCULATION YOU CAN MAP THERE PRECISELY. WHAT YOU DO IS YOU PUT A PARTIAL CHARGE ON EVERY ATOM AND THEN THE NON-BONDED OR THE ACTIONS USUALLY SHOW POTENTIALS THAT ARE PRETTY PRECISE. THEN YOU CALLCULATE THE FORCE ON ANY ATOM IN THE SYSTEM DUE TO ALL OF THAT IN THE SYSTEM AND JUST ENTER GRACE THE SECOND LAW OF MOTION AND YOU GET A TRAJECTORY OF CONSECUTIVE STATES. THIS IS OBVIOUSLY A FEW MORE ALGORITHM THAT ARE INVOLVED. YOU NEED HEAT BATH AND PRESSURE COUPLINGS. BUT IN PRINCIPLE THIS IS HOW IT, WHO. THE TRICKY PART COMES IN WITH THESE PARAMETERS THAT I HIGHLIGHTED. SO THE EQUILIBRIUM DISTANCE OF A BOND AND THE ANGLE AND ALSO THE FORCE CONSTANT. IF THESIS ARE WRONG THEN YOUR WATER MOLECULE HAS THE WRONG ANGLE OR THE WRONG VI OPERATIONAL MODES AND YOU WILL ULTIMATELY GET WRONG CONFIRMATIONS AND WRONG STRUCTURESES AND WRONG KINETIC DATA. THIS IS FANS FEEL CALLED A FORCE FIELD. I DON'T KNOW WHY THEY NAMED IT LIKE THAT BUT THIS IS WHERE THE KEY OF THE PROBLEMS ARE. THERE IS A HANDFUL OF FORCE FIELDS AROUND THAT ESTIMATE EACH ONE IS ABOUT 10,000 POSTDOC YEARS IN WORK. SO THESE ARE REALLY HARD THINGS TO DO AND DIFFICULT TO FIX IF THEY ARE BROKEN. SO I SHOW HERE ONCE YOU MAKE THE EFFORT AND YOU MAKE A FORCE FIELD, THIS IS A LIPID FORCE FIELD BEING DEVELOPED. THIS IS THE KIND OF INFORMATION YOU GET SO YOU SEE THIS IS JUST A LITTLE LOOP IN NANOSECONDS. YOU CAN SEE THE LIPIDS FUSING HERE. THIS IS WATER UP HERE AND HYDROPHOBIC TAILS AND THIS IS THE KIND OF STUFF YOU USUALLY GET FROM EXPERIMENTS. SO YOU HAVE SCATTERING DATA, YOU CAN RESOLVE THE PRINCIPLE GROUPS, PHOSPHATES AND CHOLINE AND HERE IS THE OL CANES AND THE METALS, THE TIP OF THE LIPID TAILS. AND YOU CAN SEE IF YOU JUST -- YOU GET THAT KIND OF DENSITY PROFILE. HERE IT IS JUST TO CONVINCE YOU THAT IF YOU BOTHER YOU CAN GET THIS CORRECTLY. THIS IS THE EXPERIMENTAL DATE AT FROM NEUTRON SCATTERING AND SIMULATION DATA FROM LIPIDS. YOU CAN GET KINETIC DATA AND FUSION COOFFICIALS AND DOWN HERE GET THINGS LIKE PHASE TRANSITIONS RIGHT. SO YOU SEE THE BILAYER FROM THE CRYSTALLINE PHASE AND THE EXPERIMENT IS THE BLUE SOLID LINE AND HERE YOU ISING GOING TO THE PHASE TRANSITION AROUND 20 DEGREES. AND YOU GET ALSO FLUCTUATION. NOW THIS IS MANY, MANY ITERATIONS TO GET THESE DOTS ON THE CURVE BUT ONCE YOU HAVE DONE IT, THEY WORK BEAUTIFULLY. THE MICROPHONE. SORRY. SO, IN A NUTSHELL YOU CAN GET THIS VERY PRICE. HOW CAN WE USE THIS KIND OF SIMULATION SYSTEM FOR THE BLOOD-BRAIN BARRIER? TYPICALLY, THE PROBLEM HERE REALLY IS WE DON'T HAVE ATOMIC DETAIL STRUCTURE OF A BLOOD-BRAIN BARIER. WHAT WE HAVE IS VARIOUS CARTOONS AND MICROSCOPY DATA. SO STARTING WITH THIS CARTOON PICTURE, YOU SEE BLOOD AND VARIOUS TRANSPORT PATHWAYS AND SOME GO THROUGH THE CELLS AND SOME MAYBE GO PARACELLULAR AND SOME INVOLVE PROTEINS AND SOME GO DIRECTLY THROUGH. SO, WHAT THE SIMULATIONS CAN OFFER IS A MOLECULAR PICTURE BUT NOT OF THE WHOLE THING. THAT IS TOO DIFFICULT AT THE MOMENT BUT WE CAN ZOOM INTO A CERTAIN MODE OR CERTAIN PIECE HERE AND WE CAN TRY AND DO ATOMIC DETAIL SIMULATIONS. THEY WILL GIVE YOU DYNAMICS AND ACTIONS, PHYSICAL PHENOMENA. I JUST ZOOMED INTO A PATCH OF THE BASAL LATERAL PROTEIN OR TO A ION SIMULATION AND THEN I CAN GET VERY DETAILED INFORMATION. SO, THIS IS JUST TO SHOW THIS WAS A PAPER I SAW YESTERDAY SOMEBODY SHOWED NEARLY 2000 LIPIDS. SO, WE TOOK SIX. THIS IS FROM A PAPER HERE AND THESIS ARE THE VARIOUS LIPIDS AND COULD BUILD THE BAYS AT LATERAL MEMBRANE. THIS IS ATOMIC RESOLUTION AGAIN AND I'M GOING TO APPLY TO DIFFUSION PROPERTIES. SO LIKE I SAID, TWO TYPES OF TRANSPORT. YOU CAN INDIVIDUAL DIFFUSION THROUGH A BILAYER OR HAVE TRANSPORT THAT CAN BE EITHER PASSIVE THROUGH A PORE, MEMBRANE PORE, A PROTEIN PORE, OR IT CAN BE ACTIVE MEANING TRANSPORTED OR PUMPED ACROSS. I'M GOING TO TALK ABOUT THAT A LITTLE BIT LATER BUT MAINLY FOCUS ON DIFFUSION PART BECAUSE IT'S THE EASIER BIT. SO I BUILT MY SYSTEM HERE AND THIS SHOULD PLAY NOW. AND THEN, I PRESENTER. SO YOU SEE, I HIGHLIGHTED THE ONE MOLECULE THAT WILL GO THROUGH. THESE ARE ALL THE OTHER ETHANOLS I REMOVED THE BORDER FOR CLARITIY AND IT IS BOBBING AROUND HERE AND THIS KIND OF SIMULATION TAKES ABOUT A MONTH. SO YOU HAVE TO BE PATIENT. NOW IT'S GOING THROUGH AND HERE IT IS ON THE OTHER SIDE. SO YOU GET A LOT OF DETAILS INFORMATION HERE IN THIS MOVIE IT SHOWS YOU HOW THIS MOLECULE GOES THROUGH. I'M GOING TO ZOOM IN SO YOU CAN MAKE SNAPSHOTS AND SHOW THE VARIOUS POSITIONS. I HIGHLIGHTED A MOLECULE GOING THROUGH AND NOW YOU CAN ZOOM IN TO YOUR HEART'S CONTENT AND SEE HERE, AT THIS POSITION, IT IS BONDING WITH ECHOLINE AND THERE IS ANOTHER OR HEREAFTER AND THE BLUE THING IS THE WATER. THE YELLOW IS PHOSPHATES AND THIS IS NITROGEN. AND DOWN HERE YOU CAN SEE IT STICKS TO THE METHYL GROUP TOWARDS THE TAILS WHILE MAKING A HYDROGEN BOND WITH WATER MOLECULES. SO YOU GET NICE INFORMATION ON HOW IN ATOMIC DETAIL THIS GO THROUGH THROUGH THIS BILAYER. WHAT YOU CAN ALSO GET IS TRACK EACH INDIVIDUAL MOLECULE SO UNLIKE THE REAL WORLD, IN A SIMULATION YOU HAVE EVERY ATOM A NUMBER AND YOU CAN TRACK IT AND SEE WHAT IT IS DOING IN THREE DIMENSIONS AND THIS IS THE MEMBRANE NORMAL AND THIS SYSTEM IS DRAWN OVER HERE TO SCALE. AND EACH COLOR IS ONE ETHANOL MOLECULE MOVING UP AND DOWN THROUGH THE SYSTEM. SO HERE THEY ARE IN WATER. HERE THEY ARE ON THE INTERFACE. YOU CAN SEE THIS GAP HERE. SO THEY DON'T LIKE TO BE AT THIS POSITION. AND THESE ONES HERE ARE WHEN THEY GO THROUGH THE BILAYER. SO GET A DENSITY PROFILE. AND THIS IS THE POSITION WHERE THIS ETHANOL MOLECULES LIKE TO BE. IT'S BLOWN UP BY TIMES 5 OTHERWISE TOO SMALL. I CAN ALSO HISTOGRAM THE STRUCTURAL ROUTES AND I CAN SEE THE INDIVIDUAL TRANSPORTER EVENTS FOR BILAYER. SO GET A LOT OF INFORMATION FROM THESE SYSTEMS AND OBVIOUSLY HERE WE HAVE TRANSPORT EVENTS SO WE CAN CALCULATE RATES. SO FOR THIS KIND OF SYSTEM HERE YOU GET A RATE OF ABOUT 1.6 PER MICROSECOND PER NANOMETER SQUARED AND MOST OF THE MOLECULES WE HAVE DONE SO FAR WE HAVE HAD A ORDER OF MAGNITUDE OF EXPERIMENTAL RATES. SO IT IS SEEMING LIKE IT IS RELIABLE AND CAN BE USED. WHAT CAN YOU ALSO DO IS YOU CAN DO A TRANS-BILAYER ENERGY PROFILE AND GETS FROM THE DENSITY HERE BY JUST TAKING A LOGARITHM AND MULTIPLYING AND THAT GIVES YOU FREE ENERGY PROFILE. THIS IS JUST TO SHOW VARIOUS METHOD, UMBRELLA SAMPLING METHOD SO IT'S NOT THE EQUILIBRIUM METHOD. THIS IS. THEY ALL CONVERGING TO THE CORRECT ANSWERS SO YOU HAVE ASSURANCE THIS KIND OF DATA IS RELIABLE. THIS SHOWS YOU THE ORDER OF MAGNITUDE OF WHAT YOU NEED. YOU NEED ABOUT 2-4 MICROSECONDS FOR CONVERGENCE AND JUST I'LL TALK ABOUT IT AT THE END BUT THESE ARE LONG SIMULATIONS BY TODAY'S TIME STAMP. SO A MICROSECOND IN COMPUTER STIMULATION A LONG TIME. AND WILL EXPERIMENT IS PROBABLY EXTREMELY SHORT TIME. YOU CAN DO DIFFERENT MOLECULES AND JUST TO SHOW YOU HOW THE BARRIERS VARY WHEN YOU CHANGE THE MOLECULES GOING FROM SMALL TO LARGE HERE, OR VAGUELY YOU CAN HAVE DIFFERENT BARRIERS. YOU CAN HAVE LIKE A GLYCEROL OR A SINGLE STRAIGHT BROAD BARRIER IN THE MEMBRANE. SO I DID THE GRAY IS THE MEMBRANE HERE. AND THIS IS THE FREE ENERGY HERE. SO THIS JUST PLOTS THE BARRIER, THE MOLECULE WILL FEEL WHEN YOU DRAG IT ACROSS THE MEMBRANE. AND YOU CAN HAVE SOMETHING LIKE AMMONIA WHICH HAS A LITTLE WELIN THE MILT T STILL IS A SINGLE BARRIER IN WATER BUT THEN YOU HAVE MOLECULES LIKE CAFFEINE AND ETHANOL THAT LIKE TO SIT AT THE INTERFACES SO YOU SEE THEY HAVE LITTLE WELLS HERE AND THEY LIKE TO BE HERE AND ICE PROPANOL AS WELL AND SOME HAVE SINGLE BARRIER IN THE MIDDLE OR A LITTLE WELL IN THE MIDDLE AND THEN SOMETHING LIKE CARBON DIOXIDE HAS BARRIERS BUT REALLY LIKES TO BE IN THE MEMBRANE. SO YOU CAN SEE THERE IS A LOT OF COMPLEXITY HERE THAT WHEN YOU COMPARE THIS ALL OF THESE MOLECULES SATISFY THE RULE OF 5 MEANING THEY ARE SMALL, HAVE A SMALL MOMENT AND DON'T HAVE TOO MANY HYDROGEN BONDS AND THEY HAVE A SMALL PETITION COEFFICIENT SO ALL SATISFY THIS DRAGGABILITY RULE OF LIPINSKI BUT FROM THIS TABLE IT IS HARD TO GET THIS KIND OF INFORMATION. YOU GET DETAILED INFORMED TO HELP YOU SELECT DRUGS AND MAYBE DESIGN DRUGS AS WELL. YOU CAN DO TEMP DEPENDENT. SO THIS IS THE BARRIER PROFILE FOR ETHANOL WHEN YOU VARY THE TEMP AND THE SIMULATION AND YOU CAN PROBABLY SPOT HERE WAY ABOVE BOILING. YOU CAN DO THIS IN SIMULATIONS BECAUSE YOU CAN BLOCK -- SO ULTIMATELY THESE ARE SUPER HEATED LIQUIDS AND NOTHING MUCH CHANGES. THE BILAYER STAYS STABLE UP TO 530 CENTER GRADE. SO YOU CAN DO THESE KIND OF EXPERIMENTS IN THE COMPUTER IF YOU LIKE. SIMULATIONS NOT EXPERIMENTS. AND YOU CAN SEE HOW THE BARRIER PROFILE CHANGES BUT THE INTERESTING THING HERE IS THAT THE HEIGHT OF THE KEY BARRIER VARIES LINEARLY WITH TEMP. SO THEN THAT TELLS IF YOU HAVE A MOLECULE THAT HAS TOO HIGH A BARRIER TO GO ACROSS, I CAN GO TO HIGHER TEMPERATURES LOWER THESE BARRIERS AND STILL PREDICT WHAT THE BARRIER WILL BE AT A LOWER TEMP. ALL THE MOLECULES WE LOOKED AT PEPTIDES AS WELL, HAVE THIS LINEAR BEHAVIOR. WE DON'T KNOW WHY. BUT IT IS PRETTY SOLID ACROSS ALL THE SIMULATIONS WE HAVE DONE SO FAR. NOW, IN TERMS OF KINETICS, I HAVE SHOWN THIS BEFORE. THESE ARE INDIVIDUAL TRANSPORT EVENTS AND THESE ARE THREE DIFFERENT TEMP REGIMES AND ALL TO THE SAME SCALE SO YOU CAN SEE BY LOOKING AT IT THAT THE MOMENT YOU HEAT IT UP, YOU REALLY HAVE VASTLY IMPROVED KINETICS AND IF I PLOT IT DOWN HERE, THE TRANSITION RATE GROWS AND IF I DO A PLOT SO YOU'RE PLOTTING THE LOGARITHM OF RATE AGAINST INVERSE TEMP, AT WHICH IS BASICALLY REPLOT OF THIS DATA HERE AND YOU SEE PERFECTLY LINEAR BEHAVIOR AND AGAIN I CAN EXTRAPOLATE DOWN TO ROOM TEMP AND PREDICT THE RATE THAT IT WILL VAULT ROOM TEMP EVEN IF THE MOLECULE IS TOO SLOW TO MOVE IN THE SIMULATION. SO, WITH THAT, THIS IS THE SIMPLE DIFFUSION TO A BILAYER AND OF COURSE THE BLOOD-BRAIN BARRIER INVOLVED MEMBRANES INVOLVE MUCH MORE COMPLICATED SYSTEM SO WHAT I HAVE HERE IS A PCP -- PGP TRANSPORTER AND A BILAYER. THESE ARE HUGE SYSTEMS. THE ONES I SHOWED YOU BEFORE ARE 50,000 ATOMS RUNNING PRETTY EFFICIENTLY. THIS IS ABOUT 200,000 ATOMS. SO THESE ARE SLOW AND VERY DIFFICULT AND ALSO THE TIME SCALES OF OPERATION OF THESE MOLECULES ARE MILLISECONDS PLUS. THAT'S VERY HARD TO DO THAT AT THE MOMENT. THERE IS ONLY ONE MACHINE IN THE WHOLE WORLD ALCOHOL SITS IN TIMES SQUARE IN NEW YORK BUILT BY A BILLIONAIRE AND HE CAN SIMULATE THESE SYSTEMS AND I THINK THE NIH IS GOING TO GET ONE. SO, WE ARE APPLYING FOR TIME ON THAT. BUT THESE ARE REALLY DIFFICULT SYSTEMS ALSO BECAUSE IF YOU HAVE A SLIGHT FORCE FIELD PROBLEM ON THIS SYSTEM IT IS REALLY GOING TO SHOW. GETTING AN ETHANOL MOLECULE PARAMETERIDES CORRECTLY IS NOT THAT HARD BUT GETTING A BIG PROTEIN LIKE THIS CORRECT IS HARD. SO, WHAT ARE THE CURRENT LIMITATIONS OF THIS METHOD? YOU HAVE TIME SCALES. SO, THE TIME SCALES TYPICALLY THE LIMIT REALLY FOR DIRECT DIFFUSION LIKE A 50,000 ATOM SYSTEM IS REALISTICALLY 100 MICROSECONDS. ANYTHING ELSE IS NOT FEASIBLE. IT WOULD TAKE MORE THAN 5 YEARS TO SIMULATE. AND I'M TALKING BIG SUPER COMPUTERS HERE. SO, THAT MEANS YOUR DETECTABLE RATE OF DIFFUSION HAS TO BE A MILLION PER SECOND WHICH IS OKAY. DO ION CHANNELS, EQUIPORE INS AND FAST DIFFUSING MOLECULES. IF YOU SIMULATE TOO SHORT AND THIS IS WHY THIS IS IMPORTANT, YOU CAN SEE THIS IS JUST A DIFFERENT TECHNIQUE AGAIN BUT IT SHOWS YOU IF YOU SIMULATE TOO SHORT YOUR BARRIER IS WRONG. SO YOU NEED TO BE IN THIS REGIME HERE 8 MICROSECONDS DOWN HERE TO GET THE CONVERGED BARRIER OTHERWISE IT IS NOT GOING TO BE ACCURATE. THE SECOND PROBLEM AND THIS IS A TECHNICAL PROBLEM REALLY. SIZE. YOU CAN -- I HAVE SHOWN THIS IS THE BIGGEST SIMULATION I HAVE RUN SO FAR ABOUT 250,000 ATOMS. IT'S A HEXAMERIC CHANNEL AND A CHANNEL FOR HELICOBACTER PIE HORRY NEEDS THIS TO SURVIVE IN THE STOPPLE ACK. -- STOMACH. IF YOU BLOCK THIS, WILL YOU KILL THE BUG. THIS IS 6 CHANNELS HERE AND THE PROBLEMS IS NOT JUST THAT IT IS HUGE, YOU NEED A BIG COMPUTER FOR IT BUT THE MOLECULES MOVE REALLY FAST SO IF YOU LOOK AT THE CARBON DIOXIDE HERE AND HERE AND HERE AND AMMONIA AND SO YOU NEED TO WRITE OUT EVERY 10 PICO SECONDS. EACH FRAME IS 10 MEGABYTES SO YOU CAN MANAGE FINN YOU'RE RUNNING A FEW MICROSECOND SIMULATION YOU CAN BUY A NEW HARD DRIVE AND THEN THE PROBLEM AS WELL IF YOU'RE TRYING TO ANALYZE THIS, YOUR ANALYSIS CODES WILL JUST CAN'T READ IN A TERABYTE SIZE FILE. SO WE SPENT A LOT OF OUR ANALYSIS CODE NOW ON DATA LOADING JUST TO GET AROUND THESE KIND OF PROBLEMS. THAT SAID, I WANTED TO END UP WITH A POSITIVE NOTE. SO WHEN I STARTED MY PH.D. IN 1998, WE DID ONE NANOSECOND SIMULATIONS SO MY BOSS USED TO CALL THIS EXTENDED MD. AND BASICALLY YOU PUT A PEPTIDE IN A MEMBRANE AND IT WOBBLES A LITTLE BIT AND YOU WRITE A PAPER AND EVERYBODY IS EXCITED. BUT NOTHING HAPPENS. SO, WE MOVED ON A LOT FROM THAT AND IN THE MID TO LATE 90s YOU COULD DO ION CHANNEL CONDUCTIONS AND YOU SEE IONS DROPPING IN THE MIDDLE A CHANNEL AND WE HAVE DONE THIS KIND OF WORK AS WELL RECENTLY AND WE CAN ACCURATELY REPRODUCE CONDUCTION DATA AND OTHER THINGS. AND THEN SORT OF THIS BILLIONAIRE CAME ALONG AROUND 2010 AND BUILT HIS NEW COMPUTER. THIS IS CLASSICAL CPU, THINGS YOU CAN BUY OFF THE SHELF AND THEN THIS GAY CAME ALONG AND BUILT A SUPER COMPUTER 100 TIMES FASTER THAN A LITTLE CPU BUT IT JUST DOES MOLECULAR DYNAMICS. HE REALLY SHIFTED THE GAME NOW HE CAN DO -- HE HAS DONE A PAPER ON ION CHANNEL GATING A MILLISECOND PROCESS AND I THINK IN THE NEXT FIVE TO 10 YEARS YOU REALLY WILL HAVE MOST OF THE PROBLEMS OF BIOLOGY ACCESSIBLE TO THESE KIND OF TECHNIQUES BECAUSE THERE IS NO REASON WHY THIS SHOULDN'T KEEP ON GROWING LIKE THIS. MICRODYNAMICS IS A PARALLEL PROBLEM. THE SYSTEM CAN BE SPLIT INTO LITTLE BITS. YOU CAN FARM THE BITS TOUT DIFFERENT COMPUTERS. SO IF YOU HAVE A BIGGER COMPUTER, YOU CAN RUN BIGGER SIMULATIONS AND THERE IS NO REASON WHY THIS SHOULD ABATE IN THIS PLACE. SO, LIKE I SAID, 10,000 FOLD INCREASE JUST FOR STANDARD CPUs IN 15 YEARS I THINK THAT IS PRETTY PHENOMENAL. I WANT TO FINISH AND THANK PETER WHO IS THE COLLABORATOR IN DOING ALL THE EXPERIMENTS ON THE DIFFUSION RATES AND YUKON, A POSTDOC WHO HAS DONE MOST OF THE SIMULATIONS HERE AND DPRA FOR FUNDING AND THANK YOU. [ APPLAUSE ] >> THANK YOU VERY MUCH DR. MARTIN ULMSCHNEIDER. SO WE WILL DEFER QUESTIONS TO THE OPEN MICROPHONE DISCUSSION FOR THIS SESSION I WANTED TO THANK ALL THE SPEAKERS AND THEIR OPEN MIC DECISION WILL BE AT 12:15 FOLLOWING THE NEXT SESSION. RIGHT NOW WE HAVE TIME FOR A 15 MINUTE BREAK FOR EVERYONE TO REGAIN SOME OF THEIR ENERGY AND FEEL FREE TO HAVE SOME COFFEE AND WATER AND SNACKS OVER HERE. THANK YOU WE ARE ABOUT TO START. WE ARE GOING TO START WITH JULIA LJUBIMOVA. THANK YOU VERY MUCH. >> OKAY. LET'S START OUR LAST SESSION FOR THIS VERY WELL ORGANIZED MEETING AND OUR SESSION IS DEDICATED FOR DEVELOPMENT OF NEWEST TECHNOLOGIES FOR BLOOD-BRAIN BARIER DELIVERY TARGETING AND TREATMENT OF BRAIN TUMORS. WE HAVE EXPERTS IN THE FIELD AND IT IS JUST IN THIS DIRECTION TO FOLLOW ME WE HAVE FIVE SPEAKERS INCLUDING ME, DR. EDWARD NEUWELT CARTOON SUMMARY AND HE WILL SPEAK ABOUT INFUSION OF SOLUTION AND REVERSIBLE OPENING OF BLOOD-BRAIN BARRIER. HE HAS THREE PARTS OF BIG ACHIEVEMENTS IN TREATMENT, CLINIC, HE WILL SPEAK ABOUT IT. NEXT SPEAKER WILL BE DR. HAINES AND HE WILL SPEAK ABOUT BRAIN PENETRATING NANOPARTICLES AND GUIDED ACOUSTIC -- NO. NOT ACOUSTIC. HELP ME. ULTRASOUND. HE DELIVERS THEM INTO THE BRAIN VERY INTERESTING. IT'S NANOPARTICLES RECENTLY PUBLISHED BY DR. . [ INDISCERNIBLE ] AND DR. STEGH WILL SPEAK ABOUT SPHERE CALL NUCLEIC ACIDS AND LATEST DEVELOPMENT AND LATE GENERATION OF SPHERICAL NUCLEAR ACIDS ENTERING CLINICAL TRIAL WHERE VERY INTRIGUED TO HEAR HOW IT WORKS AND WHAT THEY CAN DO. AND I WILL SPEAK ABOUT ACTIVE TRANSPORT OF BIOCOG GATES TO IMAGE AND TREAT BRAIN TUMORS. SO NOW LET'S SWITCH TO MY PRESENTATION. SO, RECENTLY WE PUBLISHED SEVERAL PAPERS IN 2013 AND 2016 WHICH SUMMARIZED MOLECULAR PROFILING OF GLIOMA WHICH REVEALS MULTIPLE TARGETS. OF COURSE PATIENTS WITH GLIAL TUMORS, BRAIN TUMORS, THEY WOULD LIKE US TO SERVE THEM TODAY NOT IN A DECADE. AND CRITICAL QUESTION FOR CLINIC IS, HOW TO TREAT GLIOMAS BY OPERATING WITH MULTIPLE CANCER BIOMARKERS? HOW TO TREAT PATIENTS WHEN GENE PROFILES FROM MULTIPLE BIOPSIES OF ONE TUMOR SHOW DIFFERENT PATTERNS. SO WHICH DRUG, WHICH BIOMARKER IS MOST IMPORTANT AND WHICH DRUG WE CAN USE FOR TREATMENT, OR COMBINATION OR TREATMENT REGIMEN, HOW SHOULD BE CREATED AND WHAT TECHNOLOGY WE CAN USE IMMEDIATELY MODIFY AND REPLY FOR TUMOR GENETIC CHANGES AND PROFILES. AND ALSO TUMOR PROGRESSES AND WE KNOW THAT GENETIC PROFILE OF REOCCURRENCES OR METASTASES ARE TOTALLY DIFFERENT FROM PRIMARY TUMOR. SO, WE DEVELOPED THE TECHNOLOGY AND WE HAVE NOW FAMILY OF CONJUGATES WHICH WE CAN USE FOR TUMOR DIAGNOSTIC IMAGING AND TREATMENT. SO, OUR FAMILY OF NANODRUGS WE CALL THEM BIONANOPOLYMERS BECAUSE THEY BASED ON NATURAL POL MERE. IT'S A ACID WHICH WE DERIVED FROM ONE CELL ORGANISM AND IT IS A BOND STRUCTURE WHICH WORKS IN THE RIGHT PLACE IN THE RIGHT TIME HOW WE DESIGN IT. WE SYSTEMICALLY INJECT OUR NANODRUG WHICH HAS TO PASS BLOOD-BRAIN BARRIER. WE USE MONOCLONAL ANTIBODIES AND PEPTIDES AND THEN SECOND TARGETING ANTIBODY PEPTIDES THEY HAVE TO RECOGNIZE CANCER CELL MEMBRANE, SPECIFIC CANCER CELL. BY BARRIER ON THE LOW PH WE HAVE SPECIAL PEPTIDES. IT IS IN THE SOMAL UNIT ESCAPE AND WE BREAK END SOMAL MEMBRANE AND THEN mRNA, SRNA DIFFERENT ONE. THEY CAN BLOCK mRNA AND PROTEIN SYNTHESIS OF SPECIFIC CANCER PROTEINS. VERY IMPORTANT FOR TREATMENT IN PARTICULAR BRAIN TREATMENT THAT OUR NON CONJUGATES OUR DRUGS SHOULD BE FULLY BIODEGRADABLE AND END PRODUCTS ARE WATER AND CO2 AND ANOTHER VERY IMPORTANT THINGS THAT THEY METABOLIZE TO PRODUCTS WHICH ARE NON TOXIC AND NON IMMUNOGENIC. SO, THIS IS CARTOON I WOULD LIKE TO DEMONSTRATE BECAUSE WHEN WE STAIN MICROVESSELS OF THE BRAIN WE SEE THAT WE DO NOT DELIVER OUR NANODRUGS INTO THE NON TUMOR CELLS. WE SPECK ABOUT BBB AND SPECIFIC CANCER CELL DELIVERY. SO WE'LL LABEL OUR CONJUGATE AND HERE WE NOT ONLY SEE THE TRAPPING OF OUR NANODRUGS IN THE BRAIN BECAUSE IT MIGHT BE IN THE LUMINA, DRUG REALLY PASSED BBB AND WE CAN SEE RED COLOR LABELED THE DRUG IN A SIGHT AIRPLAYSM OF TUMOR SELLS -- CYTOMASM. SO FIRST EXAMPLE I WOULD LIKE TO SHOW HOW WE CAN RECOGNIZE BY SPECIFIC MARKERS USING MRI VIRTUAL BIOPSY, WE CALL THIS TECHNOLOGY BRAIN LESIONS. SO THIS IS MRI WHICH RADIOLOGISTS CAN SEE AND YOU SEE SEVERAL ENHANCEMENTS. AND THIS ENHANCEMENTS MIGHT BE METASTATIC TUMORS FROM DIFFERENT LOCATIONS, IT MIGHT BE PRIMARY BRAIN TUMOR, IT MIGHT BE INFECTION, BECAUSE THIS IS IMAGE OF A WOMAN WHO WAS TREATED WITH CHEMOTHERAPY FOR HER PRIMARY POSITIVE BREAST CANCER IMMUNE SYSTEM GOES DOWN. IT MIGHT BE INFECTION FOR EXAMPLE ENHANCEMENT NUMBER 3. AND NEUROLOGICAL SYMPTOMS ON INITIAL PRESENTATION ARE OFTEN THE SAME FOR REOCCURRENCE OR BRAIN INFECTION. THIS IS A BRAIN. I KNOW THAT A LOT OF NEUROSURGEONS IN THIS AUDITORIUM BUT YOU KNOW THAT YOU CAN NOT BIOPSY FROM ALL THESE FIELDS TO UNDERSTAND HOW TO TREAT AS A BRAIN WITH ANTIBIOTICS OR DIFFERENT DRUGS, DEPENDS WHAT WILL BE. OR, BIOPSIES TUMORS DURING THE TUMOR REOCCURRENCE. AND IT IS VERY INVASIVE AND SOMETIMES IMPOSSIBLE. SO WE DEVELOPED MOUSE MODEL WHERE WE IN OC LATED TWO DIFFERENT CELLS, CANCER CELLS, FROM RAT IT IS POSITIVE BREAST CANCER CELLS AND FROM LEFT EGFR PRIMARY BRAIN TUMOR. IT IS U87 CELL LINE. IN 21 DAYS WHEN TUMOR FORMED WE INJECTED NONIMAGING AGENT WHICH RECOGNIZED HERT 2 POSITIVE TUMORS AND YOU CAN SEE FROM ONE HOUR AFTER THE INJECTION, WE SEE IT IS THE SAME MICE, YOU CAN -- SAME MOUSE, YOU CAN SEE HERT 2 POSITIVE SIGNAL AND WE DO NOT DETECT EGFR POSITIVE TUMOR IN THE BRAIN. SO THIS IS ALSO INJECTION FOR EGF -- HERT 2 POSITIVE PART OF THE BRAIN AND THIS IS OUR NON CONJUGATE. IT'S A ACID BLOOD FORM AND ATTACH TWO DIFFERENT MONOCLONAL ANTIBODIES OR PEPTIDE AND IT IS. [ INDISCERNIBLE ] DAUGHTER FOR MRI DETECTION SO IN 60 MINUTES IN 20 MINUTES, WE CAN SEE TWO TUMORS. FROM ONE HOUR TO 103 MINUTES, FROM 1-3 HOURS, WE CAN DETECT HERT 2 POSITIVE BRAIN TUMOR AND WE HAVE COVERAGEITATIVE ALGORITHM FOR RADIOLOGIST SO PRACTICALLY PATIENT CAN LEAVE DEPARTMENT OF RADIOLOGY WITH PRECISE DIAGNOSIS WHAT KIND OF LESION IT MIGHT BE. WE CAN DETECT INFLAMMATION IF WE CAN USE GNF ALPHA MONOCLONAL ANTIBODY OR MANY OTHER LESIONS ONLY WE HAVE TO SELECT RIGHT BIOMARKER. THIS IS OPPOSITE SITUATION WHERE WE WANTED TO DETECT EGFR POSITIVE TUMOR IN THE MICE AND SAME STORY IN 20 MINUTES WE HAVE PEAK FOR TWO TUMORS HERT 2 IS SMALLER AND NOT SUCH A GOOD DETECTABLE IN 20 MINUTES. IT'S A TIME WHEN IN THE CLINIC USUALLY MRI DETECTION OBTAINED AND FROM ONE HOUR TO 3 HOURS WE HAVE VERY WELL DEFINED EGFR POSITIVE TUMOR VISIBLE. SO, AFTER WE DEMONSTRATE PROOF PRINCIPAL FOR MOLECULAR DIAGNOSIS MADE BY MRI WE KNOW HOW TO TREAT TUMORS. AND THIS IS A LITTLE BIT DIFFERENT PART. THIS IS EXAMPLE OF OUR TREATMENT OF HERT 2 POSITIVE METASTASES. AND SYSTEMIC TREATMENT OF LUNG METASTASES IN THE BRAIN AND TRIPLE NEGATIVE BREAST CANCER. SO WE SIGNIFICANTLY IMPROVED SURVIVAL OF THESE ANIMALS AND I WOULD LIKE TO STRESS ONE MORE TIME THAT THERAPEUTIC MONOCLONAL ANTIBODY, IGG1, WHICH SUCCESSFULLY WE USE IN THE CLINIC TO TREAT FOR EXAMPLE HERT2 POSITIVE BREAST CANCER, THEY DO NOT CROSS BLOOD-BRAIN BARRIER. AND THIS IS WHY IT IS IMPORTANT TO DELIVER THEM AS A PART OF OUR TECHNOLOGICAL DRUG FOR THE TREATMENT. DIAGNOSTIC AND TREATMENT BASED ON MOLECULAR MARKERS. SECOND PART OF MY PRESENTATION DEDICATES TO NANOTECHNOLOGY WHICH ALLOWS US TO BLOCK VERY COMPLICATED PROTEINS. IT'S EXTRACELLULAR MATRIX PROTEINS IN OUR CASE IT IS LAMININ 4 ONE INN 1 AND WE CAN BLOCK LAMININ 411 AND REGULATE NOTCH SIGNALING PATHWAY KEY PLAYERS DURING BRAIN CANCER PROGRESSION. SO, BRIEFLY, LAMINNINE IS A MAJOR STRUCTURAL PROTEIN OF BLOOD VESSELS AND NORMAL BLOOD VESSELS IN THE BRAIN THEY OVER EXPRESS ALPHA 4 BETA 1 GAMMA 1 CHAIN OF LAMININ. NOT OVER EXPRESS. JUST EXPRESS. YES. MALIGNANT LAMININ, ALPHA 4 BETA 1 GAMMA 1. IT'S A SWITCH FROM ONE BETA CHAIN TO ANOTHER BETA CHAIN AND LOOK HOW DRAMATICALLY SITUATION CHANGE WITH THIS SWITCH. SO, WE HAVE A DIFFERENT LAMININ WHICH IS MUCH MORE PERMEABLE, LAMININ 411, MALIGNANT, AND WHICH FACILITATE THE CELL MIGRATION AND GLIOMA CELLS WITH THIS BAD LAMININ WHICH THEY PRODUCED BY THE WAY. YES. THEY MUCH EASIER TO FORM REOCCURRENCE AND MIGRATE. THIS IS A PAPER WE PUBLISHED MANY YEARS AGO WHEN WE JUST FOUND LAMININ 411 IN TABLE OHM AS. YOU CAN SEE NORMAL BRAIN AND -- GLIOMAS -- AND RED COLOR OVEREXPRESSED NOW IN GBM, BETA 1 IS A KEY CHAIN OVEREXPRESSED IN GBM AND GOOD LAMIN IN, BETA 2, BECAUSE GOOD LAMIN IN ALPHA 4 BETA 2 GAMMA 1 IS ALMOST ABSENT IN GBM. HOWEVER, PRESENT IN NORMAL BRAIN GREEN COLOR AND GLIOMA GRADE II. SO, IT IS CLINICAL DATA OBTAINED FROM PATIENTS 123GBF PATIENTS AND WITH BETA 1 OVEREXPRESSION MEDIUM SURVIVAL TIME IS 10 MONTHS VERSUS MEDIAN SURVIVAL TIME, SOMEONE WHO HAS BETA 2 IS FROM GOOD TIME NONMALIGNANT BRAIN TIME, AND REOCCURRENCE ALSO DEVELOPS MUCH SOONER THAN IN THE GROUP WITH BETA 2 OVEREXPRESSION. SO, THIS IS A CORRELATION BETWEEN LAMIN IN BETA 1 AND STEM CELLS MARKER NOTCH 1. WE DO NOT DETECT LAMIN IN BETA 1 IN NORMAL BRAIN PATIENTS. WE SEE NOTCH 3 AND BETA 1 OVEREXPRESSION IN GRADE III, GRADE 4 GLIOMAS SAME WITH CD133 AND BETA 1 AND BETA 1 AND CMYC. THEN WE WERE VERY HAPPY TO SEE THIS PAPER PUBLISHED IN PARALLEL WITH A GERMAN GROUP FIVE YEARS AGO. THEY FORMULATED THE ENDOTHELIAL CONCEPT WHICH ALLOWS THEM TO UNDERSTAND THE INITIAL STEPS OF SPROUTING ANGIOGENESIS. SO LAMIN IN 411 THROUGH ITS RECEPTOR IN THE GREENS AND LIGAND DLL4 REGULATE NOTCH PATHWAY. AND IT PLAYS INITIAL ROLE FOR DIVIDING OF T-CELLS, ENDOTHELIAL CELLS AND FORM NEW TUMOR CELLS. BUT THIS IS IN-VITRO STUDY. WE WERE VERY HAPPY TO READ THE PAPER AND WE TRIED TO SEE WHAT WE CAN DO. SO WE DEVELOPED NON BIOCONJUGATE, ABLE TO BLOCK TWO CHAINS, ALPHA 4 AND BETA 1. IF WE BLOCK ONLY ONE CHAIN OF THIS PROTEIN BY REACTIONS THAT LAMIN IN 411 WILL ASSEMBLE FROM SOME OTHER CHAINS WHICH ARE ALWAYS PRESENT IN EXTRACELLULAR MATRIX. SO, THIS IS OUR NANODRUG. WE ARE ABLE TO BLOCK ALPHA 4 AND BAIT AT 1 OF LAMIN IN 411 AND WE HAVE DIFFERENT MONOCLONAL ANTIBODIES AND PEPTIDES I DO NOT HAVE PEPTIDES TO SPEAK ABOUT IT BUT OUR NANODRUG WAS VERY EFFECTIVE TO BLOCK GLIOMA PROLIFERATION AND THIS IS SURVIVAL EXPERIMENT AND THIS IS TUMOR SIZE INDEPENDENT EXPERIMENT WHERE WE SACRIFICED ALL ANIMALS IN AGE GROUP IN ONE DAY AND WE MEASURED THE TUMOR SIZE. IT'S UNTREATED ANIMALS AND THIS IS TREATED ANIMALS. THEN, WE CHECK EXPRESSION OF STEM CELL MARKERS AND RECEPTORS AND LIGANDS OF THIS LAMIN IN NOTCH PATHWAY AND THIS IS EXPRESSION OF NOTCH 1 IN TREATED AND UNTREATED ANIMALS AND AFTER TREATMENT, WE SUPPRESSED NOTCH 1. WE SUPPRESSED BETA 1 INTO GREEN WHICH IS THE RECEPTOR FOR LAMIN IN 411, DLL4 LIGAND FORM NOTCH, CD133 ALSO AND SUPPRESSED AND NEST IN AND C-MYC WHICH REFLECTS TUMOR PROLIFERATION TODAY IT CALLS STEM CELL MARKER. SO, WE CONFIRM THAT USING OUR NANOBIOCONJUGATE ABLE TO BLOCK BIOMARKERS AT THE SAME TIME THROUGH IN THE GREENERY CEPTOR, WE CAN AND DLL4, WE CAN BLOCK NOTCH PATHWAY AND THROUGH CD133 C-MYC WE CAN SUPPRESS CELL PROLIFERATION, DIFFERENTIATION AND MIGRATION OF GLIOMA CELL. SO IN CONCLUSION, WE HAVE TO DEVELOP TECHNOLOGY BASED ON POLIMATIC ACID THROUGH BBB FOR DIAGNOSTIC MRI IMAGING AND PRICE TUMOR TREATMENT AND WE HAVE SHOWN THAT CONJUGATES ARE POWERFUL REGULATORS OF THE INTERACTIONS BETWEEN TUMOR MICRO-ENVIRONMENT LAM NIN 411 INTRACELLULAR MATRIX PROTEIN AND BETA ONE NOTCH SIGNALING PATHWAY DURING BRAIN CANCER PROGRESSION. THANK YOU VERY MUCH. I'M VERY HAPPY TO WORK WITH MY TEAM. HE FORMS A CONCEPT OF OUR IMAGING AND DRUG TREATMENT AND WE HAVE EXCELLENT SYNTHETIC CHEMISTS AND WE HAVE NEUROSURGEONS AND WE HAVE VERY GOOD BIOLOGISTS AND 400 MEMBERS PARTICIPATED. A LOT FOR OUR DATA. THANK YOU VERY MUCH. [ APPLAUSE ] >> THANK YOU VERY MUCH DR. AND WE'LL HOLD QUESTIONS DURING THE OPEN MICROPHONE DISCUSSION FOR YOUR SESSION WHICH WILL START AT 12:45. THE NEXT SPEAKER WILL BE DR. EFSTATHIOS KARATHANASIS AND HE WILL PRESENT NANOTECHNOLOGY TAKES AIM AT THE BLOOD-BRAIN BARIER. >> THANK YOU FOR THE INTRODUCTION. THIS IS MUCH BETTER THAN MY WIFE. IT TOOK HER FIVE YEARS TO SAY WHAT SHE JUST SAID. SO, WITH THAT, I HAVE A CONFESSION TO MAKE IF THAT WILL WORK. SO, MY BACKGROUND IS IN CHEMICAL ENGINEERING SO I CANNOT HELP OF THINKING OF ALL OF YOU AS A COLLECTION OF PIPES AND REACTORS AND THINGS HAVE TO SWIM AND THEY HAVE TO GET UP TO A SPECIFIC LOCATION AT A SPECIFIC COMPOSITION, REACT, KEEP WHAT YOU WANT, REMOVE UNDESIRED BIOPRODUCTS. SO WHENEVER I THINK OF SMALL OR CONVENTIONAL THERAPEUTICS WITH SMALL MOLECULAR WEIGHT,IC OF THEM LITTLE LUNATICS. I THROUGH SOME NUMBERS OF FLOW AND DIFFUSION AND SO, IF WE ARE TO MAKE A SIMPLE ASSUMPTION THAT THE HUMAN BODY IS A BODY OR A BOTTLE OF WATER. ONCE YOU INJECT SMALL MOLECULE IN ESSENCE IT IS TRYING TO DIFFUSE THROUGHOUT THE ENTIRE THING. THAT IS A SIMPLE ASSUMPTION OF AN ENGINEER BUT NOT THAT FAR AWAY FROM REALITY. SO IF YOU WANT TO INCREASE YOUR LEVELS OF YOUR DRUG AT A SPECIFIC LOCATION, MANY TIMES YOU HAVE TO RAISE THE CONCENTRATION THROUGHOUT THE ENTIRE BOTTLE. SO, DON'T HAVE MANY DEGREES OF FREEDOM AND THE ONLY DEGREE YOU HAVE IS PRETTY MUCH BIOCHEMICAL INTERACTIONS TARGETED BETTER. NOW ONCE YOU RAISE YOUR SCALE OF YOUR THERAPEUTIC AND YOU GO TO THE NANOSCALE, NOW YOU ADD MANY MORE DEGREES OF FREEDOM. YOU HAVE MORE DESIGNS AND ENDLESS DESIGNS. YOU CAN ASK A CHEMIST. HE CAN MAKE ANYTHING, NANO, ANY SHAPE OUT OF ANY MATERIAL. AND WE HAVE DONE THAT. YOU HAVE TO STOP THEM. SO, AT THE SAME TIME, YOU ARE ASKING NOW THE QUESTIONS THAT YOU CAN START FILLING THE FLOW. SO WHERE WOULD YOU GO AND THEN AT THE SAME TIME YOU SAY WELL, I HAVE SEEN A PIECE OF WOOD FOR EXAMPLE IN A RIVER AND HAS NO REASON TO TURN 90 DEGREES AND START SWIMMING TOWARDS THE BANKS OF THE BLOOD VESSEL WALL IN OUR CASE BUT WE HEARD YESTERDAY THAT RED BLOOD CELLS PLAY A ROLE IN NANOPARTICLES PROBABLY MANAGERINATE THEMSELVES IN THE CELL FREE LAYER AND BOUNCE BACK AND FORTH BETWEEN THE BLOOD VESSEL WALL AND THE EPITHELIAL CELLS SO HAVE YOU VERY INTERESTING TRANSPORT PHENOMENON, ENDLESS DESIGNS TO ACTUALLY REASONABLY DESIGN THERAPEUTICS TO GO AFTER A SPECIFIC MICRO-ENVIRONMENT. SO YOU HAVE SEEN THIS OR A VARIATION OF THAT. THIS IS HOW THE FIELD OF NANOMEDICINE WAS BUILT ON. ENHANCED PERMATION OF RETENTION AFFECT. IT WAS PROPOSED AS THE ULTIMATE DRUG SMUGGLER. IT EXPLOITS THE VASCULATURE OF ANGIOGENIC BLOOD VESSELS AND MOST SOLID TUMORS WILL HAVE THIS AMPLEIOGENIC ACTIVITY. BY NOW WE KNOW THIS DOESN'T WORK. MOST TUMOR -- MOST OF THE TIME THIS IS NOT THE CLINICAL SCENARIO. THE PRIMARY SIDE WILL BE REMOVED AND YOU WILL BE GIVEN SYSTEMIC THERAPIES TO GO AFTER INVISIBLE RESIDUAL DISEASE OR MICROMETASTATIC DISEASE. THERE ARE CASES THAT THIS IS THE CLINICAL SCENARIO BRAIN TUMORS IS ONE OF THEM. BUT THEN HIGH PRESSURES IN HOMOGENEOUS ACROSS DIFFERENT BLOOD VESSELS NUT WHAT WE SEEN MULTIPLE TIMES IN CLINICAL SPECIMENS OR ANIMAL STUDIES. THIS RESULTS IN A NEAR PERIVASTULAR DISTRIBUTION OF NANOPARTICLES. YOU WILL INTERACT WITH SOME CANCER CELLS. YOU WILL MISS MOST OF THEM. MOSTLY IN THE DEEP TISSUE. SO, I HAVE CATEGORIZED THE THREE MAJOR CHALLENGES AS I SEE THEM TO DELIVER DRUGS INTO GLIOMAS. THIS IS MY FIELD OF STUDY. ONE IS THE BLOOD-BRAIN BARRIER AND ALL OF YOU KNOW WAY MORE THAN I DO. THE WAY I SEE IT IS THAT YOU HAVE TO DELIVER LIPPHILIC DRUGS MOST OF THE TIME AND ESCAPE THE PUMPS AND THEN AN ADDITIONAL PROBLEM IS THAT GLIOMAS HAVE A VERY DIFFUSED PHENOTYPE. SO THEY WILL ACTUALLY IMVADE AND IT WAS THE FIRST TALK YESTERDAY YOU WILL HAVE THE PRIMARY SIDE WITH THE MAJORITY OF THE CELLS ARE GLIOMAS BUT THEY SHOULD GO UPWARDS. YOU STILL HAVE CELLS GLIOMA CELLS THAT ARE BURIED IN AN OCEAN OF CELLS OF NORMAL BRAIN CELLS. SO YOU END UP HAVING ALMOST A NEARLY IMPOSSIBLE SURGICAL CURE AND AT THE SAME TIME PHARMACOLOGICAL INTERVENTION THE NEED TO BE EXTRAORDINARILY PRECISE AND TARGET CELLS EXPLICITLY. I THE THURSDAY ONE TURNS OUT GLIOMAS IN BRAIN TUMORS ARE SMARTER THAN THE OTHER TYPES OF CANCER. THEY HAVE MORE STEM-LIKE BEHAVIORS AND THAT ADDS AN EXTRA LAYER OF CELL PLASTICITY AND THE RESULTS IN A MUCH HIGHER DRUG RESISTANCE TO CONVENTIONAL THERAPIES. SO, I TALKED ABOUT THE TRANSPORT. SO YOU HAVE A LITTLE MOLECULE THERE IS A MISMATCH EITHER WILL SWIM WELL IN THE WATER IN BLOOD OR IT WILL PENETRATE VERY WELL THE BLOOD-BRAIN BARRIER AND THEN DEPENDS ON WHETHER IT WILL OVERCOME THE EFLEX PUMPS BUT AT THE SAME TIME, THIS IS A MISMATCH IN THE DESIGN OF THE MOLECULE IN ITS PHYSICAL CHEMICAL PROPERTIES. NOW THE OTHER HAND, WE MAKE NANOPARTICLES AND WE THINK THAT THERE IS ONE DESIGN THAT MATCHES EVERYTHING SO WE ASK IT TO EVADE THE IMMUNE SYSTEM, CIRCULATE FOR LONG TIME, GIVE IT ENOUGH TIME TO INTERACT MEANINGFULLY WITH A VASCULAR BED, CLOSE IT AND HAVE A MEANINGFUL AND DEEP TRANSPORT. FIND THE CELL MEMBRANE YOU AND WANT CROSS IT AND DELIVER MOST OF THE TIMES THE GOODS INTO THE NUCLEUS. SO, TYPICALLY FROM ALL THAT STORY YOU CAN APPRECIATE THAT ONE DESIGN PROBABLY CANNOT DO IT. WE ARE ABUSING THE TERM AND WHAT I WILL TRY TO PERSUADE YOU IS NANOPARTICLES ARE IDEAL FIT TO TARGETING THE VASCULAR BED WHAT I CALL VASCULAR TARGETING. THEY ARE NOT THAT GOOD FOR GOOD TARGETING AND CANNOT GO THAT DEEP INSIDE THE TISSUE. SO WE USE LOTS OF IMAGING AND WE ARE VERY FORTUNATE NOWADAYS WE HAVE VERY EXPENSIVE TOYS AND ALWAYS TELL MY KIDS THAT I HAVE THE TOYS THAT THEY WILL NEVER HAVE AT HOME. SO WE HAVE ALL THOSE COOL IMAGING TOYS THAT IN A QUANTITATIVE MANNER AND DIME DEPENDENT MANNER, WE CAN TRACK PARTICLES AND MEASURE PARAMETERS AND UNDERSTAND WHICH DERIVED PRINCIPLES AND RULES THAT DESCRIBE HOW PARTICLES TRANSPETER AND THE WHERE DO THEY GO. I HAVE A PLOT HERE, I WILL NO THE GO THROUGH DATA BUT IT SHOWS ON THE X AXIS WE HAVE A PARAMETER IN BLOOD FLOW AND ON THE Y AXE SIS DEPOSITION OF THE REGION OF NANOPARTICLES IN DIFFERENT REGIONS OF CANCERS. AND I WANT TO EMPHASIZE THE Y AXIS IS ACTUALLY A SCALE SO WHAT YOU SEE IS THAT PARTICLES OF DIFFERENT SIZE SO THESE ARE PHYSICAL CHEMICAL PROPERTIES AND HOW THESE ARE AFFECTED BY FLOW. BIOCHEMICALLY INTERACTIONS. SOME PARTICLES ARE DECORATED WITH LIGANDS THAT TARGET OVEREXPRESS THE SURFACE ON CANCER CELLS AND YOU CAN SEE THAT IT IS ALL OVER THE MAP. ONE DESIGN WOULD HAVE MISSED MASSIVELY REGIONS. SO THE MICRO-ENVIRONMENT IS VERY IMPORTANT AND KNOWLEDGE OF THAT IS HIGHLY IMPORTANT. SO ALL OF THOSE THINGS CAME INTO PLAY AND WE CAME TO CONCLUSION THAT WHEN YOU WANT TO DO MICROVASCULAR TARGETING OF MICROCAPILLARRIES IN THE BRAIN, IT ACTUALLY IT WILL BENEFIT FROM A DESIGN THAT HAS OB LONG ISOMETRIC SHAPE AND FLEXIBILITY. SO WE CAME UP WEAPON AN ELEGANT WAY OF TAKING ANY NANOPARTICLE AND CONVERTING ONE SIDE OF IT TO EXHIBIT A DIFFERENT CHEMICAL FUNCTIONALITY. TYPICALLY NANOPARTICLES WILL HAVE A POL I MER. MOST OF THE TIMES PEG AND MOST OF THE TIMES THEY WILL HAVE AN AMINE FUNCTIONAL GROUP SO YOU CAN CHANGE THAT. SO WHAT YOU HAVE IS A NANOPARTICLE THAT HAS TWO PHASES WHICH FIT OR SERVE AS FITTINGS SO YOU CAN START PUTTING ONE AFTER THE OTHER. THIS IS MY SON. HE TOOK AFTER HIS MOTHER, NOT ME. SO WE CAN TAKE DIFFERENT NANOPARTICLES AND NOW THAT THEY HAVE THOSE FITTINGS, WE CAN PUT ONE AFTER THE OTHER WITH HIGH PRECISION AND WE CAN ACTUALLY SCALE IT UP. AND I HAVE PICTURE FOR YOU TO BELIEVE ME. WE CAN ADD DIFFERENT LAYERS OF DIFFERENT PARTICLES. SO YOU WILL ENHANCE YOUR SHAPE AND START ADDING FUNCTIONS USEFUL FOR APPLICATION. SO ONE OF THE DESIGNS IS A FEW NANOPARTICLES. THEY ARE HIGHLY STABLE. AND WE HAVE A LIPOSOME. THIS IS ALMOST THE SMALLEST SIZE THE HIGHEST CURVATURE OF THE LIPID MEMBRANE BEFORE IT WILL BE UNSTABLE AT THESE CONDITIONS. AND THAT COMES INTO PLAY. SO WE PICKED THE LIPOSOME SO LET ME MOVE ON. SO ONE OF THE FEATURES IT HAS IS IT INTERACTS IN A VERY, VERY UNIQUE WAY WITH RADIO FREQUENCY FIELDS. TYPICALLY YOU WOULD TRIGGER THE RELEASE FROM A NANOPARTICLE OR A IMPLANT USING HEAT MEDIATED PHENOMENON. THERMOSENSITIVE POLYMER LIPID THAT WOULD REQUIRE A LOT OF PARTICLES IN THERE TO ACCUMULATE TO CONVERT ENERGY OF THE WAVE INTO HEAT. THIS HAPPENS AT THE SINGLE PARTICLE SCAPE. SO WE USE RADIO FREQUENCIES AT THE FM RADIO RANGE, VERY LOW RANGE, HIGH PENETRATION, VERY SAFE, GO TO BEST BUY AND 1000 DOLLARS LATER AND AN AMPLIFIER, WE CAN BUILD SYSTEMS. AND VERY LOW COST AND QUICK. AND YOU ALSO HAVE A SUPER POWER MAGNETIC TAIL THAT ONCE IT IS STARTING TO WRAP THE MAGNETIC FIELD, IT IS TRYING TO ALIGN MAGNETIC MOMENTS BUT BECAUSE THEY ARE LINKED WITH THIS OTHER, IT VIBRATES. YOU HAVE A LIPID BUBBLE THAT DOESN'T TAKE MUCH SO ONCE YOU STOP SHAKING IT IN YOU BURST LIPID BUBBLE IN YOUR COMMAND AND TRIGGER RELEASE OF THE DRUG. SO, LET ME SUMMARIZE THE WHOLE -- I'M JUST SHOWING YOU TWO VERY TYPICAL DRUGS THAT HAVE DIFFERENT MECHANISMS OF ACTION AND A LOT OF THEM IN THOSE SYSTEMS HAVE PRETTY MUCH THE SAME RELEASE RATE AS EXPECTED. SO, THE CONCEPT IS THAT YOU PICK DRUGS THAT THEIR PHYSICAL CHEMICAL PROPERTIES AND IN PARTICULAR, THE COEFFICIENT WOULD ALLOW IT TO CROSS THE BLOOD-BRAIN BARRIER. BUT WE LOADED ON TO THE BUS AND TO TELL THE BUS TO GO TO THE GLIOMA SIDE AND THEN ONCE THAT HAPPENS, VASCULAR TARGETING OF GLIOMAS ARE OTHER TUMORS IT IS HIGHLY EFFICIENT AND EXTRAORDINARILY QUICK. SO WITHIN HALF HOUR WE ACHIEVE MAXIMUM DEPOSITION AT THE GLIOMA SIDE. THEN WE APPLY RADIO FREQUENCY FIELD AND THE PARTICLES VIBRATE AND THE DRUG, NOW YOU LET THE LUNATICS TO DO THEIR JOB WHICH ARE BEST FOR. LET THEM DISBURSE INSIDE THE TISSUE. OF COURSE YOU WILL HAVE SOME IN THE BLOOD SITE BUT YOU HAVE ENORMOUS AMOUNT OF DRUG THAT DISBURSES INTO THE DEEP TUMORS AND I'LL SHOW YOU SOME DATA. SO, FIRST WE TRIED PRETTY MUCH EVERYTHING. I DEPENDENT WANT TO HAVE A LONG PLOT. SO WE HAVE THE FREE DRUG, DOXORUBICIN WITH HIGH PARTITION COEFFICIENT. THIS IS MATCHING VERY WELL CLINICAL DATA. THIS IS THE MOD CHEL IS GLIOMA MODEL. THE CLINICAL PRODUCT VARIATION OF IT, LIPOSOMAL DOXORUBICIN HAVE BEEN CLINICAL TRIALS 15 YEARS AGO. THEY HAVE ABOUT LESS THAN 1% OF THE INJECTED DOSE INTO THE GLIOMA SIDE NOT MUCH IN THE NORMAL BRAIN WHICH IS GOOD NEWS BUT NOT THAT MUCH ACTUALLY INSIDE THE GLIOMA ALSO. AND OUR ANIMAL STUDIES MATCH THOSE NUMBERS ALSO. WHAT IS VERY INTERESTING IS THAT WE GET THE 15-20 FOLD ACCUMULATION OF THE DRUG AT THE DRUG SITE, AT THE GLIOMA SIDE BY UTILIZING DESIGNED NANOPARTICLES IN CONJUNCTION WITH VASCULAR TARGETING. WHAT WE ARE TARGETING? EVERYTHING THAT IS OVEREXPRESSED ON THE VASCULAR BED OF CANCERS. IN MOST CASES, THE DISEASE WILL, ANY DISEASE MOST OF THE TIME WILL MANIFEST ITSELF ON A VASCULAR BED. SO THERE WILL BE QUITE A FEW VASCULAR MARKERS YOU CAN GO AFTER. SO THIS FOR EXAMPLE, WE HAVE TRIED VEGF RECEPTORS AND WE VIED MANY DIFFERENT MOLECULES THAT SHOW UP WHEN YOU HAVE COMPROMISED BRAIN TUMOR INTERFACE. AND THE RESULTS COME TO BE THE SAME IN NUMBERS BUT WE TARGET DIFFERENT LOCATIONS OF THE TUMOR. HERE IS JUST A NORMAL BIODISTRIBUTION STUDY. OUR PARTICLE ENDS UP WHERE MOST NANOPARTICLES END UP IN THE ORGAN, LIVER AND SPLEEN. WHAT IS VERY INTERESTING IS THAT USING TWO DIFFERENT MODELS OF GLIOMA, WE TARGET REALLY VERY WELL AND WITH SIMILAR NUMBERS BUT NORMAL BRAIN THAT BLUE LINE OVER THERE, IS JUST NOISE BUT WE DON'T MEASURE MUCH IN THE NORMAL BRAIN. SO, THIS IS A HISTOLOGICAL IMAGE OF THE CNS1 MODEL. HIGHLY INVASIVE. YOU CAN SEE EVEN THE EDGES ARE VERY INFILTRATING AND YOU CAN SEE THAT MIGRATE AND YOU HAVE A NEW COLUMN THAT GROWS. SO I WILL ZOOM IN THAT INVASIVE SITE AND YOU CAN SEE IN RED THE BLOOD VESSELS AND YOU CAN SEE -- [ OFF MIC ] ARE ASSOCIATED WITH A DISEASE. SO, IF I WAS NOT TO SEE RADIO FREQUENCY FIELD, YOU WILL NOT SEE THE DRUG. IN THIS APPLICATION, WE USED THE DOXORUBICIN NOT BECAUSE IT IS THE BEST CYTOTOXIC DRUG FOR GLIOMAS BUT THE MILD FLORESCENT SO WE CAN SEE DISTRIBUTION. SO WHILE IT IS STILL IN THE LIPOSOME, IT IS CLEANSED BUT ONCE WE RETHESE YOU CAN SEE IT WITH YOUR SCOPE. SO THE DRUG IS RELEASED AFTER RADIO FREQUENCY AND WILL DISSURES AND CROSS CELL MEMBRANES AND NUKE TIED WITH KEIM CHEMOTHERAPY. SO WE USE MANY DIFFERENT MODELS FROM STEM CELL DERIVED MODELS AND TO PEDIATRIC MODELS. WE DO BELIEVE THAT KIDS ARE NOT SMALL ADULTS. SO WE TRY TO SEE IF THERE ARE DIFFERENT TARGETS WE SHOULD GO. IS THE BBB SIMILAR IN TERMS OF DRUG DELIVERY? SO, HERE I'M SHOWING YOU HOW INVASIVE THIS IS AND HOW MIGRATORY THOSE CELLS ARE AND HOW THEY ARE IN GREEN AND YOU CAN SEE I'M ONLY SHOWING THE DRUG HERE IN RED AND HOW WELL WE TARGET PRETTY MUCH THOSE LOCATIONS AND WE DISBURSE OUR DRUGS. SO WE ARE NOT NAIVE. EVEN THOUGH WE ARE ACHIEVING VERY GOOD SURVIVAL RATES, FOR EXAMPLE, IN THE CNS1 MODEL, WE SIGNIFICANTLY PROLONGED SURVIVAL AND UNLESS INVASIVE MODEL WE INCLUDE SOME ANIMALS AND HAVE MANY MORE DATA AND ANIMAL MODELS AND SO, BUT WE DO UNDERSTAND THAT SITE OTOXIC DRUGS ALONE WILL NOT BE THE ULTIMATE THERAPY. SO, WE ARE WORKING CLOSELY WITH GLIOMA STEM CELL BIOLOGISTS AND BRAIN TUMOR BIOLOGISTS TO IDENTIFY THOSE ADDITIONAL PHARMACOLOGICAL INHIBITORS THAT WILL GROW SPECIFICALLY FOR THE DIFFERENT SUBPOPULATIONS OF STEM CELLS. I'M ALSO SHOWING HERE MARK GRIZZ WHO WOULD WHO BUILDS OUR RADIO FREQUENCY FIELDS. AND OF COURSE THIS WORK IS PRIMARILY SUPPORTED BY THE NCI ALLIANCE FOR NANOTECHNOLOGY AND THE PEDIATRIC SIDE OF IT IS FROM A FOUNDATION. AND OF COURSE IT TAKES AVILLAGE TO RAISE A SUCCESSFUL NANOTECHNOLOGY AND I'M VERY FORTUNATE TO HAVE A LOT OF SMART PEOPLE THAT WORK IN MY LAB AND A LOT OF OTHER SMART PEOPLE THAT WANT TO COLLABORATE WITH US WITHIN THE COMPREHENSIVE CANCER CENTER WITH THAT, I WILL TAKE YOUR QUESTIONS PROBABLY IN THE END. [ APPLAUSE ] >> THANK YOU VERY MUCH. AND JUDGES, WE WILL TAKE MORE QUESTIONS DURING THE OPEN MIC DISCUSSION. AND WE'LL PROCEED NOW TO DR. ALEXANDER STEGH WHO WILL SPEAK ABOUT SPHERE CALL NUCLEIC ACIDS FOR THE PRECISION TREATMENT OF MALIGNANT GLIOMA. >> THANK YOU VERY MUCH FOR THE INVITATION. IT'S BEEN A GREAT MEETING. SO IN MY PRESENTATION I'D LIKE TO GIVE OVERVIEW OF SOME OF THE WORK WE HAVE BEEN DOING ON USING RNA IN THE NANOMATERIALS TO DELIVER RNAI STABLY AND ROBUSTLY TO INTERCRANIAL TUMOR SITES AND THIS IS A TRUE COLLABORATION BETWEEN MY LABORATORY AND THE LABORATORY OF DR. MURPHY AT NORTHWESTERN UNIVERSITY AND THE WORK THAT I'M SHOWING YOU TODAY IS MAINLY FOCUSED ON THE CONSTRUCT THAT WE REFER TO AS SPHERICAL NUCLEIC ACIDS. SO IN MY LABORATORY MAINLY FOCUS ON GBM -- LETHAL IN CURABLE CANCER. A VERY CONCISE SUMMARY OF THE DISEASE AS WELL AS CHALLENGES WE ARE FACING AND I LISTED MY VERSION OF IT ASY WOO KNOW AND THAT IS WHY WE ARE HEREMENT WE VERY TO CONSIDER DELIVERY ASPECTS OF THEY WERE SPEC HAVE TO NEGOTIATE PASSAGE TO BLOOD-BRAIN AND BLOOD TUMOR BARRIERS AND THESE BARRIERS CAN PREVENT ACCUMULATION OF THERAPEUTIC DRUG CONCENTRATION WITHIN THE DILUTIONS AND ONE QUESTION WE ARE WORKING ON IS WHETHER WE CAN DEVELOP NOVEL DELIVERY PLATFORMS MORE EFFECTIVELY AND MORE SELECTIVELY DELIVER PAYLOADS TO THE TUMOR. AND IT'S NOT JUST DELIVERY. THIS IS ALSO THE ABILITY TO TRACK THESE PARTICLES BECAUSE OFTENTIMES IN CLINICAL TRIALS IT IS NOT REALLY BEING EVALUATED WHY A DRUG IS IN ACTIVE. IT COULD BE SIMPLY BECAUSE IT IS INACTIVE DRUG AND DOESN'T HIT TARGET OR BECAUSE IT DOESN'T GO WHERE IT NEEDS TO BE. WE ALSO HAVE TO ACKNOWLEDGE THE EXISTENCE OF STEM CELLS WHICH MANY BELIEVE ARE CRITICAL DRIVERS OF GLIOMA REOCCURRENCE AND THERAPY RESISTANCE AND HAVE TO UNDERSTAND ON A FUNDAMENTAL BIOLOGICAL LEVEL THE COMPLEXITIES OF THE DISEASE AND NEED TO GET INTO A POSITION WHERE WE ARE ABLE TO NOT ONLY TARGET ISOLATED CANCER GENES AND ONCOGENES BUT WHERE WE ARE ABLE TO TARGET ENTIRE ONCOGENIC SIGNATURES COLLABORATE TO DRIVE CANCER PROGRESSION. SO THIS IS BASICALLY A SUMMARY OF THE FIRST GENERATION THAT WE ARE BEGINNING TO CHARACTERIZE PRECLINICALLY. SO WE ARE LOOKING AT A GOLD NANOPARTICLE THAT HAS A DIAMETER OF 30 NANOMETERS AND WE CAN FUNCTIONALIZE THESE PARTICLES WITH SMALL RNA OL GO NUCLEOTIDES AND microRNAS AND ONCE WE ARRANGE THEAL GO NUCLEOTIDES IN THIS VERY SPECIFIC 3 DIMENTIONAL ARCHITECTURE THESE PARTICLES HAVE INTERESTING ACTIVITY PROFILES. SO LOOKING AT SINGLE ENTITY AGENT CAPABLE. GENE TRANSFECTION AND TREATMENT REGULATION. PARTICLES ARE VERY STABLE IN THE ENVIRONMENT AND RESISTENT AND MANY DIFFERENT CELL TYPES PENETRATE TISSUE EFFECTIVELY AND I'M GOING TO ISSUE SOME DATA WE HAVE ON GLIAL TISSUE AND VERY LITTLE TOXICITY DUE TO VERY LITTLE TARGET AFFECTS AND VERY MODULAR ACTIVATION OF IMMUNE RESPONSE. TO SHOW YOU SOME DATA ON CELLULAR UPTAKES, YOU CAN SEE A PATIENT DERIVED GLIOMA CELL CULTURE ENRICH FOR GLIOMA STEM CELLS AND WE CAN MODIFY WITH AND TRACK INTRACELLULAR DISTRIBUTION OF PARTICLES VERY EASILY AND WITHIN 30-60 MINUTES WE -- THE SPHERE CULTURE IS NICELY PENETRATED. THIS IS AN ACTIVE PROCESS SO IT IS RECEPTOR MEDIATED ENDOCYTOTIC PROSSO WHEN WE BLOCK JAF GER -- OR KNOCK DOWN INDIVIDUAL MEMBERS OF THE FAMILY WE SEE A VERY SIGNIFICANT REDUCTION OVER ALL UPTAKE OF PARTICLES. AND THIS IS A NONCONTACT CULTURE MODEL AND I LIKE TO ILLUSTRATE IN IN-VITRO BLOOD-BRAIN BARRIER THESE MODELS HAVE THE CAPACITY TO SEE THEM VERY PENETRATING THE BRAIN ENDOTHELIAL COMPARTMENT AND THE SKIN AND ALSO BE DETECTED WITHIN THE COMPARTMENT BUT MORE IMPORTANTLY IN-VIVO EXPERIMENTS WE ASK CAN WE INJECT PARTICLE SYSTEMICALLY THROUGH TAILOR INJECTIONS AND CAN WE SEE BRAIN UPTAKE AND SEE GLIOMA SPECIFIC UPTAKE AND THE ANSWER IS YES, SO WE USE A BUNCH OF DIFFERENT MODEL SYSTEM. ON THE LEFT SIDE YOU CAN SEE EXPERIMENTS WITH MODELS AND WE TOOK A NON TUMOR BEARING MOUSE AND INJECTED PARTICLES TO THE TAIL AND THEN IMAGING THE BRAIN CAN UPTAKE THESE PARTICLES. WE CAN ALSO TAKE DOWN THESE BRAIN EXTRACTS AND MEASURE VERY PRECISELY CONTENT PROGRAM AND TISSUE AND SEE 10 TO THE 10 PARTICLES OF TISSUE IN NON TUMOR BEARING MOUSE. IF WE THEN GO AHEAD TO PDX BEARING MOUSE, SO THESE ANIMALS HAVE A VERY DIFUSELY INVADING TUMOR GROWING IN THEIR BRAIN UPTAKE OF THESE INJECTED PARTICLES AND SIGNIFICANTLY HIGHER THREE ORDERS OF MAGNITUDE WE SEE APPROXIMATELY 10-13th PARTICLES PROGRAM OF TISSUE AND WE CAN ALSO VISUALIZE PARTICLE UPTAKE THROUGH MANY METHODS AND DO THIS ON BRAIN SECTIONS USING SILVER STAINING WHERE WE PRECIPITATE THE GOLD AND YOU CAN SEE ONCE IN THE BRAIN WE CAN SEE A TUMOR SELECTIVE UPTAKE MOST LIKELY THROUGH ENHANCED PERMEABILITY AND RETENTION OF THE BLOOD-BRAIN BARRIER AND YOU CAN SEE THAT THE PARTICLES PREDEPARTMENTLY LOCALIZE IN TUMOR ELEMENTS OVER NON TUMOR ELEMENTS AND WE CAN ALSO GO AHEAD AND COFUNCTIONALIZE PARTICLES NOT JUST WITH RNAs OR microRNAs AND ALSO FUNCTIONALIZE THEM WITH GADOLINIUM AND ATTACH PARTICLES TO SHORT DNA LINKER SO WE HAVE A DIAGNOSTIC AGENT ATTEMPT AND IF WE DO THIS AND THIS IS THE THREE-DIMENSIONAL RECONSTRUCTION OF IMAGES, WE CAN SEE EXTENT OF TISSUE DISTRIBUTION AND LASTLY WE CAN ALSO DO THIS BY OBLATION,. CPMS ON THE BRAIN SECTION AND AGAIN VERY GOOD LOCALIZATION OF THESE PARTICLES WITH INTERCRANIAL LEASHON AND WE STARTED TO ANALYZE GLIOUPTAKE IN SPONTANEOUS GBM MODELS GENETICALLY ENGINEERED MODEL THAT WE WORK IN COLLABORATION WITH A GFP CRE p53 FOX P10. [ INDISCERNIBLE ] ADMINISTRATION DEVELOP HYBRID GBM TUMORS AND WE CAN DO THE SAME EXPERIMENT INJECT PARTICLES TO THE TAIL VAIN AND SEE A VERY GOOD UPTAKE ON THESE PARTICLES WITHIN THE LESION AND AGAIN PARTICLE CONCENTRATIONS IN THE 10-13 RANGE SIMILAR TO WHAT WE SEE IN THE MODELS. WE THEN WENT AHEAD AND DESIGNED A SPECIFIC FORM FOR ONCOGENES WE THINK ARE CRITICALLY IMPORTANT FOR DISEASE AND THIS IS AN PEOPLER WHERE WE USE SMAs WITH siRNAs SPECIFIC FOR A FAMILY PROTEIN AND WE COULD SHOW THAT THIS IS VERY SIGNIFICANTLY UPREGULATED IN THE BEST MAJORITY OF GBM PATIENTS APPROXIMATELY 95% OF PATIENTS POSITIVE FOR THE PROTEIN. TO THE IS A M p53 INHIBITOR SO INTERACTS WITH SIGNALING PATH BASE WHEN WE TAKE THIS IN THE PICO MOLAR RANGE AND TREAT CELLS IN THE PARTICULAR PATIENT CELLS WE CAN SEE A VERY ROBUST KNOCKDOWN OF THIS GENE AND WE MADE SURE IT IS AN RNA BASED AFFECT NOT JUST SPECIFIC CYTOTOXIC AFFECT SO WHEN WE DO PCL WE CAN PRECISELY IDENTIFY THE CLEAVAGE SITE OF THE ENDOGENOUS TRANSCRIPT AND THE CLEAVAGE OCCURS AT THE PREDICTED SITE. THEN WE ALSO ASK THE QUESTION CAN WE USE THIS PLATFORM TO DELIVER siRNA AND MICRORNAs. WE DECIDED TO REALLY SPEND TIME AND TRY TO UNDERSTAND WHAT MICRORNAs ARE CRITICALLY IMPORTANT FOR THE DISEASE AND WE REALLY SPEND A LOT OF TIME IN MINING THE TCGA DATAET AND TO MAKE A SO LONG STORY SHORT, THE CRITICAL FILTERS ARE HERE SO WE WANTED TO BE SURE THAT OUR microRNA POSITIVELY CORRELATED WITH HOWEVER ALL SURVIVAL OF PATIENTS. AND OUR microRNA HAS THE ABILITY TO MOG LATE THERAPY INDUCED APOPTOSIS AND THE TOP MICRORNA WE IDENTIFIED IS 81 AND 82. SO WE DID A LOT OF BIOLOGY THIS microRNA AND FOUND OUT THAT IT IS ABLE TO TARGET GBM CRITICALLY IMPORTANT GBM ONCOGENES. AND CAN INFLUENCE MANY CANCER RELEVANT PROCESSES. SO WE WENT AHEAD AND DESIGNED THOSE THAT WE CAN FUNCTIONALIZE TO THE SEQUENCE AND VERY SIMILAR TO THIS THESE COUNTERPARTS THESE PARTICLES HAPPEN TO PENETRATE PATIENTS SO IT IS EFFECTIVELY THEY HAVE THE ABILITY TO TARGET GENE EXPRESSION EFFECTIVELY AND CAN INDUCE APOPTOSIS. SO WE WENT AHEAD AND DID A LOT OF IN-VIVO TUMOR REGRESSION STUDIES FOR EXAMPLE WE CAN TAKE PATIENT DERIVED MODEL SYSTEM THAT IS MODIFIED FOR THIS EXPRESSION. WE CAN INJECT. [ INDISCERNIBLE ] AND YOU CAN SEE THAT OVER TIME BIOLUMINESCENCE IS SIGNIFICANTLY DECREASED IN THE PRESENCE AND ALSO ON THE HISTOPATHOLOGICAL LEVEL WE SEE WHAT WE EXPECT TO SEE SO WE TOOK INCREASE IN APOPTOSIS AND EVALUATED BY CASPASE. SO, USING MODEL PRECLINICAL DATA AS A SPRINGBOARD, WE ARE NOW TRYING TO TRANSLATE THIS INFORMATION AND WE WERE ABLE TO FINALIZE TOXICOLOGY STUD -ES IN RODENTS AND NON HUMAN PRIMATES AND WE WERE ABLE TO SCALE THE PRODUCTION OF THESE SNS AND WE ARE LOOKING FORWARD NOW TO IND APPLICATION BY THE END OF THE MONTH AND PHASE III CLINICAL TRIAL OF THIS PARTICLES THAT WILL TAKE PLACE AT NORTHWESTERN TOWARDS THE END OF THIS YEAR. SO IN THE LAST COUPLE OF MINUTES, I WANT TO LEAVE YOU WITH SORT OF A VISION WHERE WE REALLY WANT TO TAKE THIS THING AND WHETHER WE THINK OUR NEXT CRITICAL STEPS ARE. I THINK ONE OF THE CRITICAL ISSUES IN THE NANOSPACE IS THERE IS NOT MUCH -- SO IF WE COMPARE TO SMALL MOLECULE DESIGN WHERE WE IDENTIFY A LEAD COMPOUND AND THEN SPEND A LOT OF TIME OPTIMIZING THIS CONSTRUCT THAT DOESN'T REALLY HAPPEN IN THE NANOSPACE. SO THAT IS SOMETHING WE ARE VERY ADAMANT ABOUT CHANGING. SO, AND THE IDEA IS THAT IT CAN TAKE ADVANTAGE OF THE MODEL STRUCTURE OF OUR SNA. SO WE CAN ASK QUESTION WHAT HAPPENS IF WE REPLACE CERTAIN DESIGN ELEMENTS OF THESE. FOR EXAMPLE, WHAT HAPPENS IF WE CHANGE THE CORE MATERIAL, THE CORE SHAPE, THE SURFACE CHEMISTRY, RNA DENSITY, PEGALATION, FUNCTIONALIZATION WITH TARGETING AND SO ON. WHAT HAPPENS TO THE PROPERTIES IN TERMS EVER BIODISTRIBUTION, PHARMACOKINETICS, EFFICACY IN TERMS OF KNOCKING DOWN A GENE EXPRESSION AND WE DECIDED TO ACTUALLY GO TO A MORE IN-VIVO-BASED SCREENING SYSTEM. YES, WE CAN SCREEN IN-VITRO AND WE CAN DO IN-VITRO BLOOD-BRAIN BARRIER MODELS AND WE HEARD A LOT ABOUT IT HERE BUT THE MOST RELEVANT SYSTEM IS AND AT THE MOST STRINGENT SYSTEM IS TO REALLY GO INTO IN-VIVO APPLICATIONS RIGHT AWAY AND TO DO THIS IT'S A VERY COMMON PROCESS. WE DECIDED TO GENERATE A MODEL SYSTEM AND OPTICAL SYSTEM THAT ALLOWS US TO SCREEN FOR GENE REGULATORY ACTIVITY OF THIS CONSTRUCTS IN REALTIME SO WHAT WE CAN DO IS TAKE OUR GENE OF INTEREST, CLONE THIS IN FRONT OF A PROTEIN WHICH IS INFRARED FLUORESCENCE PROTEIN AND TAKE THIS AND COEXPRESS INTO PATIENT CELLS WITH LUCIFERASE AND THEN TAKE THESE CELLS AND IMPLANT THEM IN IMMUNOCOMPROMISED ANIMALS SO WE HAVE A HANDLE ON MEASURING TUMOR VOLUME AS WELL AS FUSION POTENT COLLECTION REALTIME AND IN-VIVO. JUST TO GIVE YOU EXAMPLE OF HOW THIS CAN WORK, WE DID A STUDY USING MGMT AS A TARGET SO ANOTHER REALLY CRITICAL TARGET GENE FOR US. IT'S A METHYLTRANSFERASE THAT REMOVES METHYL ADD DUCTS FROM DNA THAT THE IS PLACED IN THE -- VERY IMPORTANT RESISTANCE FACTOR. SO WHEN WE CLONE MGMT IN FRONT OF IRFP670 WE CAN VISUALIZE EXPRESSION THROUGH IMMUNOFLUORESCENCE AND IMPORTANTLY, WE CAN INJECT THESE CELLS INTO THE BRAIN AND WE CAN VISUALIZE FUSION EXPRESSION IN-VIVO. SO WE THEN WENT AHEAD AND DESIGNED SNA SPECIFIC FOR MGMT AND THEY ARE ABLE TO DOWN REGULATE ENDOGENOUS AND THEY ARE ABLE TO DOWN REGULATE DIFFUSION PROTEIN IN GLIOMA CELLS AND WE CAN ALSO FOLLOW THIS BY CONFOCALLY PRY CROSS COPY IN THE PRESENCE OF SMAs WITH REDUCTION IN OVER ALL FLUORESCENCE. IN-VIVO APPLICATIONS WE CAN TAKE DUAL MODIFIED CELLS AND INJECT THEM INTO THE BRAIN OF ANIMALS AND THEN WE CAN MEASURE LUMINESCENCE AS WELL AS INFRARED FLUORESCENCE BEFORE INJECTION OF PARTICLES AND DOT SAME EXPERIMENT AFTER INJECTION OF THE PARTICLES AND THEN CALCULATE RATIO OF FLUORESCENCE OVER LUMINESCENCE SO IT IS NORMALIZED TUMOR VOLUME AND WE CAN DO THIS AS A FUNCTION OF SNA CONCENTRATIONS SO WE USE THREE DIFFERENT DOSAGES AND WE CAN ALSO DO THIS AS A FUNCTION OF TIME AND WE CAN REALLY DETERMINE WHAT SELL THE LEVEL OF KNOCKDOWN AND THE DEPENDENCY ON CONCENTRATION WHAT IS THE MOST IDEAL CONCENTRATION THAT WE CAN USE TO ACHIEVE THE MOST SIGNIFICANT AND MOST ROBUST KNOCK DOWN AND TAKE THIS INFORMATION THEN AND DEVELOP A PRECLINICAL PDX TRIAL WHERE WE TAKE PDX MODELS AND INJECT THEM EVERY OTHER DAY WITH SPECIFIC SNAS AND COTREAT USING CONCENTRATION AND DOSING REGIMENS DEVELOPED IN THE DUAL OPTICAL MODEL THAT I INTRODUCED TO YOU AND IF WE TAKE THIS COMBINATION HERE, YOU CAN SEE THE COMBINATION OF SNAs AND IT REALLY INDUCES MOST PROFOUND REDUCTION IN OVERALL TUMOR PROGRESSION. SO, BASICALLY WE CAN LEVERAGE THE SYSTEM AND REALLY ASK IMPORTANT FUNDAMENTAL QUESTIONS WHAT IS THE MOST OPTIMAL SNA ARCHITECTURE IN TERMS OF ACHIEVING THE MOST OPTIMAL AND MOST ROBUST TARGET GENOME IN-VIVO? WITH THAT I'M GOING THANK THE PEOPLE IN MY LABORATORY AND IN PARTICULAR, THE GROUP WITH WHOM WE ARE COLLABORATING FOR OVER SIX YEARS AND I WILL TAKE QUESTIONS AFTER THE SESSION. THANK YOU. [ APPLAUSE ] >> THANK YOU. WE DO HAVE A TIME FOR A QUESTION OR TWO BECAUSE THE NEXT SPEAKER STARTS IN 5 MINUTES. SO, YES? ANY QUESTIONS? OTHERWISE IF YOU WANT AN EXTRA FIVE MINUTES DURING YOUR VERY BRIEF BREAK BEFORE LUNCH THEN IF WE START 5 MINUTES EARLY THEN THAT MIGHT WORK AS WELL. SO THANK YOU. SO WITH THAT, WE CAN MOVE TO DR. EDWARD NEUWELT WHO WILL BE PRESENTING THREE AREAS WHERE STUDIES OF THE BLOOD-BRAIN BARRIER CHANGE PATIENT CARE. >> EDWARD NEUWELT: THANK YOU. FIRST I'D LIKE TO DR. RAPOPORT WHO TAUGHT ME HOW TO DO WHAT I'M GOING TO TALK ABOUT IS HERE. SO IN MY VIEW, HE IS ONE OF THE GODFATHER OF BLOOD-BRAIN BARRIER. SO IT'S A REAL HONOR TO HAVE HIM HERE AND BILL IS IN THE BACK AND THESE ARE BASICALLY ALL FUNDED STUDIES. THANK YOU BOTH. SO, BASICALLY WE ARE GOING TO TALK ABOUT CROSSING THE BRIDGE. THESE ARE THREE PARADIGMS WHICH BASICALLY STARTED WITH TEST TUBES, CELL CULTURE, ANIMALS, PHASE I, PHASE II, AND PHASE III TRIALS. CAN YOU HEAR ME? SO, BRAIN TUMOR THERAPY WITH OPENING THE BLOOD-BRAIN BARRIER, CHEMO PROTECTION TO PREVENTED THE TOXICITIES OF CHEMOTHERAPY AND NEUROIMAGING WITH NANOPARTICLES. WE HAVE OBTAINED FIVE OVER AN DRUG DESIGNATIONS -- ORPHAN DRUG -- FOR THESE THREE PROGRAMS, WHICH IS GREATLY FACILITATED WORKING WITH THE FDA TO TRY TO GET FDA APPROVAL WHICH WE ARE CLOSE TO ON THREE OF THOSE. THERAPEUTIC OPTIONS FOR THE TREATMENT OF PRIMARY METASTATIC TUMORS IS LIMITED AS WE TALKED ABOUT. FOR SENSITIVE TUMORS, THAT IS THE KEY ISSUE. DELIVERY IS NOT SUCH A PROBLEM AS I'LL TRY TO SHOW YOU. Y WOO CAN DO IT SAFELY BUT WE DON'T HAVE THE RIGHT REAGENTS. BUT WE HAVE PICKED REAGENTS WHERE WE DO HAVE THEM IN DIFFERENT PROOF OF PRINCIPLE. THIS IS A VERY OLD SLIDE BUT IT IS PROBABLY THE MOST IMPORTANT SLIDE. THIS IS A HIGH-GRADE GLIOMA, TWO NODULES, ONE VERY LITTLE WHICH IS ALL BROWN AND ONE THAT IS JUST BROWN RIGHT AT ONE CORNER. THE BLOOD-BRAIN BARRIER AND THE BLOOD TUMOR IS CONSISTENTLY INCONSISTENT WITH THE PRIMARY METASTATIC TUMORS AND WE CAN CUT OUT THE PART THAT ENHANCES AS AWE NEUROSURGEON WE SAY WE GOT IT ALL OUT BUT ALL THE LITTLE INFILTRATING CELLS THAT EVEN GO TO THE OTHER HEMISPHERE WE DON'T GET OUT. SO THESE ARE NOT REALLY SURGICAL DISEASES. THIS IS DR. RAPOPORT'S CARTOON. OPENING THE BLOOD-BRAIN BARRIER WITH INTRA-ARTERIAL HABEROS MOTTIC MAN I TOL. HE PUBLISHED RECENTLY FOUND BY PEOPLE IN SCANDINAVIA. HE BROUGHT IT BACK TO LIFE IN THE 1970s AND A PAPER IN SCIENCE AND THEN HE TOLD US TO DO A SMALL ANIMALS AND LARGE ANIMALS BEFORE WE DID IT IN PEOPLE. SO WE DID ALL KINDS OF SMALL AND LARGE ANIMALS USING EVANS BLUE, CT SCAN WITH CONTRAST, AND USPIO WHICH IS WE'LL TALK ABOUT WITH MR. I DIDN'T MEAN TO DO THAT -- I NEVER SEEN THAT HAPPEN. AND WE ALSO HOPE THE BLOOD CSF BARRIER. EVANS BLUE IS DARK AND THEN THE CSF TURNS BLUE SO WE OPENED BOTH BARRIERS. THESE WERE THE LARGE ANIMAL STUDIES WE DID THEN WE WENT IN, THIS WAS THE FIRST TIME IN 1978 AT THE VA WHEN I WAS THE UNIVERSITY TEXAS. AND THIS IS AN IMPORTANT -- THE METHOTREXATE DELIVERY HERE WE HAVE IV AND INTERARTERIALY WITH AND WITHOUT BLOOD-BRAIN BARRIER DISRUPTION. YOU SEE DISRUPTION TREMENDOUS INCREASE IN DELIVERY. WITH ANTIBODIES AND THIS IS A MONOCLONAL ANTIBODY, 160,000 MOLECULAR WEIGHT, THE BARRIERS OPEN FOR THE FIRST FEW MINUTES BUT VERY QUICKLY THE TIGHT JUNCTIONS REFORM SO UNLIKE SMALL MOLECULES YOU HAVE TO GET THE ANTIBODIES IN QUICKLY BY IN FUSING THEM OR YOU WON'T GET THIS TREMENDOUS INCREASE IN DELIVERY. WE CAN DELIVER PARTICLES AS LARGE AS 270 NANOMETERS WHICH IS THE SIZE OF A HERPES VIRUS. NOW EFFICACY. WITH METHOTREXATE-BASED THERAPY, THIS IS SURVIVAL. WHEN WE FIRST STARTED. THE FIRST PATIENT WE DID WITH LYMPHOMA, SURVIVAL WAS ABOUT LIKE GLIOBLASTOMA 13 MONTHS. MAINLY TREATED WITH RADIATION. WE STARTED TREATING WITHOUT RADIATION SO WE DIDN'T HAVE COGNITIVE LOSS BACK IN THE LATE 1970s. WE RECENTLY REPORTED THIS IN THE JOURNAL OF CLINICAL ONCOLOGY WHICH WE HAD SEVERAL ARTICLES ON AND THE MEDIAN SURVIVAL FOR REASONABLE RISK PATIENT IS ABOUT 12 YEARS MEDIAN SURVIVAL, WHICH IS A TRAUMATIC INCREASE FROM WHAT IS SEEN WITH STANDARD DELIVERY. WE HAVE BEEN FANATICAL WITH REGARD TO GETTING BASELINE NEUROSITE TESTING BEFORE WE TREAT THE PATIENTS AND THEN YEARLY IF WE CAN, BUT THESE ARE PATIENTS WE FOLLOWED UP FOR AT LEAST TWO YEARS AFTER THERAPY AS LONG AS 26 YEARS AFTER THERAPY AND WILL NONE OF THEM WAS THERES ANY SIGNIFICANT LOSS OF SIGNIFICANT COGNITIVE FUNCTION AND IN FACT MOST OF THEM, COGNITIVE FUNCTION WAS IMPROVED. NOW IN MORE RECENT YEARS, WE HAVE BEEN USING ROW TUX IN. IT'S A CD20 ANTIGEN ANTIBODY EM EVER. PEOPLE SAY THAT ANTIBODIES DON'T CROSS THE BLOOD-BRAIN BARRIER BUT THEY DO. EVERYBODY IN THIS ROOM WHICH HAS A MOLECULAR WAVE COMES FROM THE SYSTEMIC CIRCULATION BUT NOT MUCH GETS IN. BUT THEY HAVE A VERY LONG PLASMA HALF LIVES AND IGG HAS AIRPLAYS HALF-LIFE OF 21 DAYS. SO IF YOU EVEN GET AN IV AND THERE IS A SMALL LEAK IT SLOWLILY LEAKS ACROSS AND IT HAS A DRAMATIC INCREASE IN OUR ANIMAL MODELS OF SURVIVAL AS YOU CAN SEE HERE. YOU CAN ONLY OPEN THE BLOOD-BRAIN BARRIER IN A RAT ONCE SO IT DOESN'T -- YOU CAN GET VERY GOOD INITIAL RESPONSE BUT IT DOESN'T REALLY INCREASE SURVIVAL VERY MUCH. UNLIKERI TUX IN. AND YOU CAN SEE WITH THESE IN FILLITATING TUMORS IN THESE TUMORS GO ALL THE WAY TO THE OTHER SIDE. IT HAS A DRAMATIC IMPACT WITH AND WITHOUT DISRUPTION WITH REGARD TO TREATING THE TUMOR. AND IN FACT, THE MEDIAN -- WE TAKE PATIENTS THAT ARE VERY SICK. INCREASED OUR MEDIAN SURVIVAL BY ADDING THIS TO THE REGIMEN FROM 3 YEARS TO 5 YEARS OVER ALL. INTERESTINGLY, WHEN THESE TUMORS REOCCUR, WE ARE UP TO ABOUT AN 80% COMPLETE RESPONSE RATE. THEY REOCCUR A LOT AND THAT IS ONE OF THE BIG PROBLEMS. BUT THEY DON'T REOCCUR AT THE ORIGINAL SITE. THEY -- 85% OF THEM REOCCUR SOMEWHERE ELSE UNLIKE GLIOMAS. AND ONE OF MY FELLOWS PUBLISHED A PAPER WHEREBY REALTIME PCR, WHEN THEY ARE INCOMPLETE RESPONSE, THERE IS STILL THE CLONE OF NUCLEIC ACID THAT YOU CAN SEE IN THE BLOOD. SO WE ARE THINKING THAT A LOT OF THESE REOCCURRENCES ARE RECEDING THE BRAIN. SO WE HAVE MORE RECENTLY INTRODUCED MAINTENANCE RETUX IN WHICH DOESN'T NORMALLY WORK FOR LARGE B-CELL LYMPHOMA OUTSIDE THE BODY BUT WE THINK WHAT HAPPENS IS THE CELLS COMING FROM THE BONE MARROW WE CAN CLIP OFF BECAUSE THE ANTIBODY HAD A LONG HALF-LIFE AND THE ANTIBODY IS SAFE TO GIVE EVERY OTHER MONTH. AND THAT IS WHAT WE HAVE BEEN DOING. AND SO, WE JUST OPENED DR. DO LITTLE IN OUR GROUP, A RANDOMIZED PROSPECTIVE STUDY THAT ONCE YOU GET THE PATIENTS INTO COMPLETE RESPONSE TO RANDOMIZE IF THEY NEED MAINTENANCE OR NOT AND 9 PATIENTS WE TREATED NOW LIKE THAT, THE EARLIEST REOCCURRENCE WAS NOT 1 1/2 YEARS LIKE IT WAS BEFORE. BUT IT'S 5 YEARS. SUMMARY. MAINTENANCE OBTUSE MAB MAY BE OPTION TO EXTEND DISEASE BECAUSE WE THINK IT IS HIDING IN PLACES THATRI TUX IN CAN'T GET TO. WE ARE HAPPY WITH THAT. SO THE SECOND THING IS NOT OPENING THE BLOOD-BRAIN BARRIER BUT TAKING ADVANTAGE OF THE BLOOD-BRAIN BARRIER IN TREATING CHILDREN WITH -- 80% OF ALL CHILDREN WITH ALL SOLID TUMORS, GET CISPLATIN. CISPLATIN IS VERY TOXIC IN CHILDREN. SO 80% OF THE KIDS ARE CURED. IF THEY HAVE A LITTLE VISION LOSS PEOPLE SAY THAT IS A BIG DEAL. BUT THEY SAY IF THAT IS THERE A A LITTLE HEARING LOSS IT ISN'T A BIG DEAL. BUT IT IS A BIG DEAL. THEY CAN'T LEARN IN SCHOOL. THEY CAN'T COMMUNICATE ON THE PLAYGROUND. THEY CAN'T IMITATE THEIR MOTHER'S VOICE AND SPEECH AND THEIR QUALITY OF LIFE IS TREMENDOUSLY NEGATIVELY IMPACTED. SO, WHAT DO WE DO? WELL, THERE IS A TYPICAL AUDIO GRAM WITH HIGH FREAKY IS. THEY ARE NOT DEAF, HIGH FREQUENCY HEARING LOSS IN MEASURABLE BLASTOMA THE MOST COMMON BRAIN TUMOR, IT IS BAKELY 100% OF THE KIDS THAT HAVE THIS. DOSE LIMITING. AND THE TWO DRUGS THAT WE FOUND FROM STUDIES OF ADULTS WERE GIVEN TO US BY SOMEONE IN THE OFFICE OF SCIENTIFIC MISCONDUCT HERE AT THE NIH WHOSE WIFE HAD A TUMOR WHICH THEY COULDN'T RESECT AND SHE CAME OUT WEST TO HAVE HER BLOOD-BRAIN BARRIER OPENED AND WE GAVE HER ERLOTNIB BASED CHEMOTHERAPY AND SHE GOT SIGNIFICANT HIGH FREQUENCY HEARING LOSS. HER HUSBAND WASN'T HAPPY SO HE INSISTED ON HIS LUNCH HOUR HE HEARD ON THE INTERNET A DRUG FOR CYANIDE POISONING CAN PREVENT DRUG INDUCED HEARING LOSS AND CAME OUT AND WANTS US TO GIVE IT TO HIS WIFE. WE SAID WE HAVE NO IRB APPROVAL WE CAN'T DO THAT. ONE OF OUR COLLEAGUES IN PHYSIOLOGY HAD A MODEL FOR IMMUNOGLYCOSIDE HEARING LOSS AND WE TRIED IN HIS GUINEA PIG MODEL AND IT ABSOLUTELY WORKED. AND WE THEN TRIED A SECOND FILE AND I'LL SHOW YOU DATA FROM THOSE. YOU CAN SEE IF YOU HAVE NO AN SEATAL CYSTEIN THERE IS TREMENDOUS HEARING LOSS WITH THESE THRESH HOLDS. BUT IF YOU GIVE EITHER BEFORE OR FOUR HOURS AFTER SEPARATING THE THIGH OL FROM THE CISPLATIN ACTIVE, WHICH IS 45 MINUTES, WE HAD TREMENDOUS OTOPROTECTION WITHOUT ANY PROTECTION OF THE TUMOR. THIS WAS A PNET BIG TUMOR CHEMOTHERAPY WITHOUT ANY FILES SODIUM THIGH OL SULPHATE AND THE TUMORS EQUALLY RESPONSIVE AND YOU CAN SEE THE HISTOLOGY. SO OTOPRO TECHNICIAN AND SODIUM THIGHO SULPHATE AND ASEATIL CYSTEIN WAS INTRODUCED AND THE OTOTOXICITY COMPARED TO HISTORICAL CONTROLS WAS DRAMATICALLY IMPROVED AND WE FOUND OR SINCE USED SODIUM THIGH OL SULFADE IN OUR ADULT POPULATION 3200 TIMES WHEN USING PLATINUM-BASED CHEMOTHERAPY AND WENT FROM 50% HEARING AIDS TO ZERO. THE IRB WOULD NOT LET DOUSE A PHASE III TRIAL BECAUSE OF THAT. AND THEN WE BEGAN STUDYING THIS IN CHILDREN AT THE REQUEST OF THE CHILDREN'S ONCOLOGY GROUP. BECAUSE WE HAD ALREADY DONE THE PHASE I AND PHASE II STUDIES, WE WENT TO A PHASE THROUGH STUDIES ONE IN COG, ALL THE KIDS IN THE UNITED STATES AND ONE IN SIOP, ALL THE KIDS OUTSIDE OF THE US. THOSE STUDIES BASED UPON THIS ANTI-IC DOTE BY THIS GUY FROM THE OFFICE OF SCIENTIFIC MISCONDUCT WAS THEN BEING TESTED IN TWO PHASE III TRIALS AND THE PRIMARY AIM FOR THE COG STUDY WAS TO SEE IF WE COULD DECREASE OTOTOXICITY WHICH HAD NEVER BEEN DONE BEFORE. AND YOU CAN SEE THIS WAS REPORTED THIS PAST YEAR AT ASCO. OTOTOXIC PIT CUT STS AND WITH STS MET THE PRIMARY ENDPOINT. OLDER KIDS CLEAR IMPROVEMENT BUT NOT AS DRAMATIC. IT WAS MONITORED BY OVERALL SURVIVAL AND EVENT-FREE SURVIVAL WHICH WERE NOT VERY DIFFERENT. INTERESTINGLY, UNPLANNED POST-HOC ANALYSIS SUGGESTED THAT THERE MAY BE SOME TUMOR PROTECTION IN KIDS AT DIAGNOSIS HAD DISSEMINATED DISEASE. THE PROBLEM IS THIS WAS NOT DESIGNED TO DO THIS AND THERE WERE FIVE PATIENTS WHO DIDN'T OR WERE NOT ANY ANY OF THE FIVE MAJOR CATEGORIES. THEY HAD UNRESPONSIVE TUMORS AND THEY HELPED TO GET STS. SO THIS IS WORRISOME BUT BUT IT'S HYPOTHESES GENERATED. BUT THE OTHER TRIAL IS VIRTUALLY NO EVIDENCE OF TUMOR PROTECTION AND THE DRAMATIC OTOPROTECTION. SO THAT WAS PRESENTED THIS PAST WEEKEND IN CHICAGO WITH ASCO AND FDA IS VERY INTERESTED IN NOW GOING AND GETTING FDA APPROVAL WHICH WILL PROBABLY TAKE ABOUT A YEAR TO CLEAN UP EVERYTHING. SO THAT IS THE SECOND AREA. WE TAKEN AN ANTIDOTE IN TIME AND SPACE AND THE THIGH OLE'S DON'T ACROSS THE BLOOD-BRAIN BARRIER VERY WELL. BUT BICEP RATING TIME AND SPACE, IF YOU GIVE THOSE AT THE SAME TIME AS THE CHEMOTHERAPY YOU MIGHT AS WELL NOT GIVE CHEMOTHERAPY. BUT IF YOU SEPARATE, WE CAN PROTECT THE HEARING DRAMATICALLY AND NOT PROTECT THE CHILD'S RESPONSE TO THE CHEMOTHERAPY. I'M GOING TO DONE WITH THIS. THESE ARE REFERRED -- WE HEARD GREAT TALKS THIS MORNING BUT GETTING FDA APPROVAL FOR NANOPARTICLES IS NOT A WALK IN THE PARK. SO, WE HAVE BEEN WORKING WITH THIS ONE THAT IS FDA APPROVED FOR IRON REPLACEMENT. IT'S CALLED FER MOCKS TOL. AT EARLY TIME POINTS IT'S A BLOOD POOL AGENT. SO YOU CAN LOOK AT HOW MUCH VASCULARITY THERE IS IN A TUMOR. NOW INFLAMMATION CAN BE TREMENDOUS. THAT IS CALLED PSEUDOPROGRESSION. WHEN YOU GIVE CHEMO RADIO THERAPY. AND WHEN THAT HAPPENS, THE SURVIVAL IN GLIOBLASTOMA TRIPLES. BUT THE SCAN BECAUSE THERE IS INFLAMMATION, LOOKS LIKE THERE IS OPEN BLOOD-BRAIN BARRIER AND YOU SHOULD STOP THERAPY BUT BY USING A BLOOD POOL AGENT, YOU CAN DO BLOOD VOLUMES. AND SHOW THAT THE BLOOD VOLUMES ARE ROCK BOTTOM AND THEREFORE THE INNATE IMMUNE SYSTEM IS WORKING. NOW LOOK AT THIS -- CAN YOU TURN THE LIGHTS DOWN? [ OFF MIC ] AFTER INJECTION OF NANOPARTICLE. SO TREMENDOUS RESOLUTION ALMOST DOWN TO THE ARTERIAL LEVEL. AND IF YOU DO THIS WITH T2 STAR, YOU CAN QUANITATE BY DOING A SUBTRACTION THE RELATIVE BLOOD VOLUME DIFFERENTIATE INFLAMMATION WHICH HAS LOW BLOOD VOLUME WITH PRO DRESSIVE TUMOR WHICH HAS HIGH BLOOD VOLUME. YOU CAN SEE THE MAP SHOWING THE RED IS THE HIGH BLOOD VOLUME IN THE CORTEX. THIS IS A CASE OF LYMPHOMA. GAD LIN YUM SHOWS YOU A LITTLE BIT OF THE TUMOR BUT MUCH MORE OF THE TUMOR PARTICULARLY BACK HERE IS MUCH GREATER BECAUSE AND ON T2, IT'S A T2 *NEGATIVE ENHANCEMENT WHICH THERE IS NO OTHER T2 *ENHANCEMENT AGENT AROUND. SO, HERE IS A PATIENT WITH A TUMOR THAT WAS ENLARGING BUT THAT IS WITH GATLIN YUM. SO WE SAW THAT THE BLOOD VOLUME WAS ROCK LOW RIGHT HERE. SO THIS WAS PSEUDOPROGRESSION, IN OUR VIEW. THAT PATIENT'S TUMOR GOT BIGGER AND ONE MONTH AFTER CRT TO 3 MONTHS AND NOW SHE IS MORE THAN 58 MONTHS OUT WITH NO REOCCURRENCE. HAD I NOT HAD THIS BIOMARKER I PROBABLY WOULD HAVE STOPPED THERAPY BECAUSE SHE WAS CLINICALLY WORSE. PSEUDOPROGRESSION. AND IN ALL OUR CASES, IF THEY HAD PSEUDOPROGRESSION AS MEASURED BY THESE NANOPARTICLES, IF THEY HAD PSEUDOPROGRESSION, MEDIAN SURVIVAL WAS 34 MONTHS F THEY DIDN'T HAVE PSEUDOPROGRESSION, 13 MONTHS. 3 TIMES THE SURVIVAL F THEY MGMT, MEDIAN SURVIVAL IS 54 MONTHS WITH GLIOBLASTOMA. THOSE ARE THOSE NUMBERS. HERE WE HAVE A TYPICAL PATIENT T1, T2, T1 PLUS GAD, BLOOD VOLUME WITH STEADY STATE, WHICH IS MUCH MORE SENSITIVE THAN DSE DYNAMIC SUSCEPTIBILITY CONTRAST, THAT IS WHAT THAT IS. AND FOR SURGEONS LOOKING FOR A PLACE -- [ OFF MIC ] AND THE REST OF IT YOU MIGHT A GET A DIAGNOSIS OF GRADE 2 OR 3. AT 24 HOURS THE ION PARTICLES ARE TAKEN UP BY INFLAMMATORY CELLS NOT BY TUMOR CELLS AND THAT CORRELATES WITH THE DIAGNOSIS OF SUICIDEO PROGRESSION. SO IN SUMMARY -- PSEUDOPROGRESSION -- VISUALIZATION OF ABNORMAL VASCULATURE AS WELL AS BIOMARKER OF RESPONSE, THESE PARTICLES ARE UNIQUE AND WE NOW USED THEM IN 600 IMAGING SESSIONS WITH THE HEAD-TO-HEAD GAD LIN YUM. IN CONCLUSION N HEALTH AND DISEASE, IN EXTENDING TO OLD AGE, ENDOTHELIAL CELLS NEURONS, GLIA CONSTITUTE THE -- [ READING ] AND THESE THREE EXAMPLES ARE EXAMPLES WHERE WE WENT FROM THE TEST TUBE TO ANIMALS, TO PATIENTS, AND IN ALL THREE CASES IT'S DRAMATICALLY CHANGED THE WAY I SPENT HALF OF MY LIFE TAKING CARE OF PATIENTS WITH MALIGNANT BRAIN TUMORS. THANK YOU. [ APPLAUSE ] >> THANK YOU FOR YOUR TALK. WE WILL TAKE ADDITIONAL QUESTIONS DURING THE OPEN MIC DISCUSSION AND WE'LL MOVE FORWARD WITH THE NEXT PRESENTATION BY DOCTOR JUSTIN HANES, DRUG AND GENE DELIVERY TO THE BRAIN. >> JUSTIN HANES: I GUESS WHILE THAT IS COMING UP, I WANT TO THANK MARGARET FOR THE INVITATION. HAPPY TO BE HERE TO TALK ABOUT THE WORK WE ARE DOING AT HOPKINS ON DRUG AND GENE DELIVERY THROUGH THE BLOOD-BRAIN BARRIER INTO THE BRAIN. FIRST OF ALL, I WANT TO ACKNOWLEDGE THE TWO OTHER PIs A COLLABORATE WITH, RICH PRICE FROM THE UNIVERSITY OF VIRGINIA WHERE HE DIRECTS THE ULTRASOUND CENTER AND JOHN, AN ASSISTANT PROFESSOR IN THE CENTER I DIRECT AT HOPKINS. SO I'M GOING TO TELL YOU ABOUT OUR APPROACH WHICH IS BASICALLY TAKING ADVANTAGE OF TWO RELATIVELY NEW OR RECENT ADVANCES. ONE IS OPENING UP THE BLOOD-BRAIN BARRIER USING FOCUSED ULTRASOWN -- ULTRASOUND YOU HEARD IN THE FIRST TALK YESTERDAY THAT REACHED HUMAN CLINICAL TRIALS NOW. RICH HAS BEEN A LEADER IN THIS FIELD AT THE UNIVERSITY OF VIRGINIA. WE HAVE BEEN WORKING WITH HIM FOR MORE THAN 10 YEARS. AND WE ARE FOCUSED ON DELIVERING DRUGS AND GENES THAT ARE PACKAGED IN NANOPARTICLES THROUGH VASCULATURE AND USING FOCUSED ULTRASOUND. SO THE SEPARATE INNOVATION THAT OUR LAB HAD MADE SEPARATELY WAS WE DISCOVERED WAYS TO MAKE NANOPARTICLES MORE RAPIDLY PENETRATE THROUGH TISSUES INCLUDING THROUGH THE EXTRACELLULAR MATRIX OF THE BRAIN OR IN TUMORS AND SO THEY CAN SPREAD MORE UNIFORMLY AND REACH ALL THE VARIOUS CELLS. SO NOW AT THE END OF MY TALK, I'LL SHOW YOU SOME RESULTS WE ARE COMBINING THESE TWO THINGS. SO I'LL START WITH THE FIRST THING WHICH IS THE CREATING THE NANOPARTICLE AND FOR LACK OF TIME I'M GOING TO GIVE YOU SOME HIGHLIGHTS BUT THIS IS A PAPER THAT WE PUBLISHED A FEW YEARS AGO NOW WHERE WE SHOWED THAT QUITE LARGE NANOPARTICLES WE SHOWED IN THIS PAPER UP TO 114 NANOMETERS CAN FAIRLY RAPIDLY MOVE THROUGH THE PREJUDICE MA OF THE BRAIN ONLY IF THEY ARE WELL COATED WITH NONADHESIVE POLYMERS. WHICH IS ALSO USED FOR COATING OF PARTICLES TO MAKE THEM IN THE BLOOD BUT IF YOU CAN GET TO DENSE COATINGS, THOSE PARTICLES CAN SPREAD. THIS WAS JUST A STUDY WHERE WE COINJECTED UNCODED POLY-SCI REASON PARTICLES ALONG WITH COATED ONES. AND THE COATED ONES IN GREEN SPREAD FURTHER IN A SHORT PERIOD OF TIME THAN THE CODED ONCE -- UNCOATED ONCE WHICH STAY AT THE NEEDLE TRACK. SO, IN THIS PAPER, WE DESCRIBED THE REQUIREMENT TO GET TO EXTREMELY HIGH SURFACE DENSITIES OF PEG WHERE THE PEG GOES FROM A MUSHROOM SHAPE WHERE THERE IS PLENTY OF ROOM IN BETWEEN THEM TO A BRUSH-TYPE SHAPE WHERE THEY ARE CONSTRAINED AND START TO STRAIGHTEN OUT AND THEY CAN PROTECT THAT SURFACE WHICH NORMALLY ALLOW THE PARTICLE TO GET STUCK AS IT MOVES THROUGH THE EXTRACELLULAR MATRIX. SO WHETHER YOU GET TO THE HIGH DENSITIES AND MEASURE THIS IN VARIOUS WAYS BUT WE SHOWED POTENTIAL ONCE YOU GET TO NEUTRAL ZETA POTENTIALS THE PARTICLES THAT ARE FILLED IN ARE THE ONLY PARTICLES THAT ARE ABLE TO DIFFUSE AND MOVE AND THOSE ARE ALL SOMEWHERE IN THE RANGE OF ZERO TO MINUS 4 SO VERY NEUTRAL TYPES SURFACE CHARGES. WE TRIED TO QUANTIFY THIS AND WE ESTIMATED THAT ABOUT -- YOU NEED SOMETHING AROUND 9 OR MORE PEG CHAINS PER NANOMETER SQUARED ON THE SURFACE. SO WELL INTO THE BRUSH REGIME OF PEG. THIS IS JUST A LITTLE COINJECTION EXPERIMENT WE DID IN ANIMALS AND BASICALLY ALSO GLOSSING OVER A LOT OF THIS BUT WE DID THIS IN MICE, RATS AND THEN IN HUMAN BRAIN THAT WAS FRESHLY EXCISED IN THE OR. AND ALL SPECIES THE MESH SIZE SEEMED ABOUT THE SAME AND THE PARTICLES ARE THE SAME SIZES COULD MOVE. THEY GOT TOO LARGE THEY COULDN'T MOVE AND THEY NEEDED THE COATINGS IN ORDER TO MOVE. BUT THIS IS JUST LOOKING AT A FAIRLY LARGE PARTICLE SIZE, 100 NANOMETERS WITH THE COATINGS 114. HERE IS THE INJECTION TRACK, ENCODED PARTICLES ARE IN THE MIDDLE. THE COATED PARTICLES ARE MOVING AWAY FROM THERE AND IF YOU LOOK AT A REGION FAR AWAY FROM THE INJECTION SITUDING THE INTERCRANIAL WINDOW, YOU CAN WASH THESE PARTICLES STILL MOVING AROUND DIFFUSING SO THEY CAN POTENTIALLY SPREAD OVER TIME, THE QUESTION IS HOW FAR? SO, THE NEXT STEP WE DID WAS WE TOOK JUST A CHEMO THERAPEUTIC DRUG IN THIS CASE PACK LA TAXOL AND LOADED INTO PLGA PARTICLES THAT HAD THE DENSE PEG COATINGS OR DIDN'T. WE SHOWED THEY HAD THE SAME SIZES AND SAME RELEASE KINETICS OF THE DRUG AND THEY ARE IDENTICAL EXCEPT FOR ONE PARTICLE COULD PENETRATE AND OTHER COULDN'T. WE DID THE COINJECTION EXPERIMENTS AND SHOWED THE GREEN ONES ARE NON PENETRATING AND HERE IS AN HOUR AND 44 HOURS AND THIS IS TUMOR AREA AND THIS ISN'T SHOWING TOO MUCH YET BUT I'LL TRY TO SHOW YOU MORE DETAILS IN A SECOND. BUT IN THIS INITIAL PAPER WHERE WE ARE JUST ADMINISTERING IN THE BRAIN, JUST LOOKING AT THE AFFECT IF YOU COULD MAKE THE THINGS PENETRATE THE PARTICLES PENETRATE AND CARRY DRUGS TO SPREAD THE DRUG OUT, YOU GET A MUCH BETTER AFFECT AGAINST THE TUMOR SO HERE ISANCE MALL WITH A 9L GLIOMA WITH NO TREATMENT. IF YOU JUST DIRECTLY INJECT PACK LA TAXEL INSIDE OF A PLGA PARTICLE WHICH I'LL CALL A CP CONVENTIONAL PARTICLE, CONVENTIONAL NANOPARTICLE, OR THE SAME LOADING OF PACLITAXEL EXCEPT YOU HAVE THE SURFACE COATING SO A BRAIN PENETRATING NANOPARTICLE GIVES YOU A BETTER AFFECT. SO WE HAVE GONE ON FROM SMALL MOLECULES AND ALSO LOOKING AT NEW CLAYIC ACIDS. I WANT TO SHOW A LITTLE BIT OF THAT. AND WE HAVE SHOWN IF YOU GET VERY DENSE PEG COATINGS ON VERY SMALL GENE CARRIERS WE ARE TALKING ABOUT 50 NANOMETERS OR SO THAT THEY CAN DISTRIBUTE QUITE WELL. SO HERE IS SEIKO INJECTION OF THE YELLOW IS AN UNPEG LATED NON VIRAL POLYMER NANOPARTICLE WITH DNA AND THEN THE RED IS THE PEG LATED VERSION OF THE GENE CARRIER. SO THIS IS JUST INJECTED INTO THE STRIATUM OF RATS. AND YOU LOOK AT THE DISTRIBUTION. HERE IS THE TRANSFECTION. YOU CAN SEE WITH NO PEG IT DOESN'T GO VERY FAR AWAY FROM THE INJECTION SITE W SOME PEG WHAT WE CALL CPN CONVENTIONALLY PEG LATED NANOPARTICLE YOU GET BETTER SPREAD WITH THE DENSE PEG YOU GET A BETTER SPREAD. HERE IS A THREE-DIMENSIONAL RENDERING OF THIS AND YOU GET TRANSFECTION THROUGHOUT THE STRIATUM OF THE RAT FROM A SINGLE DELIVERY OF THIS PARTICLE AT ONE SITE. AND THERE IS QUANTIFICATION OF THAT. WE ALSO DONE THIS IN TUMORS. THE TUMOR EXTRACELLULAR MATRIX CAN BE MORE DENSE AND HAVE A SMALLER PROSSITY. BUT WITH THESE VERY SMALL NANOPARTICLES IT DOESN'T MATTER. THEY PENETRATE WELL. HERE IS THE DISTRIBUTION IN RED OF THE HEAVILY PEG LATED AND THEN THE TRANSFECTION IS IN GREEN. SO YOU CAN SEE WHEREVER THEY DISTRIBUTE THEY ALSO TRANSFECT THE TISSUE. IF YOU HAVE AN UNPEG LATED NANOPARTICLE THEY TRANSFECT POORLIY AND DON'T SEE THAT MANY CELLS. SO WE WERE ABLE TO QUANT BY THIS IN THE TUMOR. I WANTED TO SHOW YOU FROM A SINGLE INFUSION. WE WERE ABLE TO QUANTIFY THE PERCENTAGE IN THIS CASE OF TUMOR CELLS THAT ARE GETTING THE TRANSGENE SO WE SHOWED THAT ABOUT 70% OF THE TUMOR CELLS HAVE PARTICLES IN THEM. IN ABOUT ALMOST 55 OR SO PERCENT, YOU CAN SEE MEASURABLE LEVELS OF TRANSFECTION. IF YOU'RE USING HEAVILY PEG LATED SYSTEM. IF YOU HAVE UNPEG LATED SYSTEM, YOU SEE MUCH LOWER LEVELS OF BOTH CELL UPTAKE AND TRANSFECTION. WE HAVE PUT IN A COUPLE OF DIFFERENT GENES IN THIS CASE WE ARE LOOKING AT A p53 TRANSGENE EXPRESSION IN ORTHTOPIC 9L RAT TUMOR AND WE WERE ABLE TO SEE IN THIS AND IN ANOTHER SUICIDE GENE ALMOST A DOUBLING IN THE SURVIVAL TIME FROM SINGLE ADMINISTRATION. WE ARE NOT HITTING ALL OF THE CELLS WITH THE SINGLE ADMINISTRATION. WE MIGHT HAVE TO DO MULTIPLE ADMINISTRATIONS TO DO BETTER AND OBVIOUSLY THERE COULD BE OTHER WAYS TO APPROACH THIS AND I'LL TALK ABOUT ONE OF THEM. THIS IS WHERE I WANTED TO GET INTO THE MOVING BEYOND THE DIRECT ADMINISTRATION INTO THE BRAIN AND BEING ABLE TO PUT THE PARTICLES INTO THE BLOOD AND CAN WE GET THEM INTO THE BRAIN AND IF SO, WHAT CAN THEY DO AFTER THAT STAGE? AND AGAIN, SINCE WE ARE USING THIS VERY DENSE PEG COATING TO MAKE THE PARTICLES MOVE THROUGHOUT THE BRAIN, THAT ALSO HELPS WHEN YOU PUT THEM IN THE BLOOD. IT ALLOWS THEM TO CIRCULATE. SO THEN INJECT THEM INTO THE BLOOD AND THEN PUT THE PATIENT UNDER THE MAGNET AND UNDER THE FOCUS ULTRASOUND IN THE MAGNET AND DO YOUR MR GUIDEED TARGETING AND OPEN THE BLOOD-BRAIN BARRIER WHERE YOU LIKE USING MICROBUBBLES WITH FOCUSED ULTRASOUND. HOPEFULLY YOU SAW THE TALK YESTERDAY. WHEN YOU DO THIS AND HIT A TARGETED AREA WHERE FOCUSED ULTRASOUND WITH MICROBUBBLES GOING IN CIRCUMSTANCEILANCE, YOU CAN DISRUPT THE BLOOD-BRAIN BARRIER AND OPEN IT FOR A SHORT PERIOD OF TIME AND THEN IT RESEALS. THIS IS JUST WORK THAT RICH HAD BEEN DOING TO SHOW THAT YOU CAN FIND WAY THAT IS LOOK SAFE TO DO THIS IN RODENTS. OF COURSE AT HARVARD THEY SHOWED IT SAFE IN MONKEYS AND NOW BEEN STARTED HUMAN TRIALS THAT YOU HEARD ABOUT YESTERDAY OR SO FAR WHAT I UNDERSTAND IS LOOKING QUITE SAFE. BUT THIS IS JUST SHOWING FOR EXAMPLE AT OPTIMAL PRESSURES SOMEWHERE BETWEEN .5 AND .6, YOU'RE NOT GETTING MUCH AT ALL TEMPERATURE RISE SOMETHING LIKE MAYBE A DEGREE OR SO LOCALLY AT THE SITE. AT THE IDEAL PROBLEM YOU'RE NOT GETTING HISTOLOGICAL DAMAGE. AT HIGHER PRESSURES YOU CAN SEE DAMAGE. SO THIS IS JUST SHOWING THE PARTICLES THAT WORK THE BEST ARE THE ONES THAT CAN CIRCULATE A LONG TIME, THAT HAVE A LOT OF PEG. SO IF YOU PUT THE PEG LATED PARTICLES INTO THE BLOOD AND THEN HIT WHEREVER IN THE BRAIN YOU LIKE TO SEE THE PARTICLES GO, FOCUS ULTRASOUND, YOU CAN GET ACCUMULATION OF THE PARTICLES INTO THE BRAIN. AND THIS IS JUST VERY EARLY STUDIES THAT WHERE WE COUNTIFIED THAT WORK. MORE RECENTLY, RICH AND HIS GROUP HAVE BEEN DOING REALLY ELEGANT WORK TAKING OUR GENE CARRIERS THAT ARE VERY SMALL AND HIGHLY STABLE AND WE SHIP THEM TO HIM AND HE HAS BEEN PUTTING INTO THE BLOOD AND THEN USING FOCUSED ULTRASOUND AND SEEING IF WE CAN GET GENE TRANSFECTION. NON-INVASIVE DELIVERY PARTICLE IN THE BLOOD AND THEN DELIVERY INTO THE BRAIN. HERE IS A DOSE ESCALATION STUDY GOING FROM RIGHT TO LEFT. 50 MICOGRAMS OF DNA ON THE RIGHT AND THEN ALL THE WAY UP TOP 350 ON THE LEFT. YOU CAN SEE THAT WITH LIVE ANIMAL OR TAKE THE BRAIN OUT AND LOOK AGAIN AND YOU CAN SEE IT MORE CLEARLY THAT YOU DO GET THIS DOSE KEYSALATION AFFECT AND YOU CAN GET THE TRANSFECTION WHERE YOU WANT IT USING FOCUSED ULTRASOUND. INTERESTINGLY, I SHOWED YOU THAT QUANTIFICATION OF THE CELLS TRANSFECTING BY PUTTING OUR PARTICLES DIRECTLY INTO THE BRAIN THEY DID THE SAME THING WHERE THEY DID STAINING TO LOOK AT DIFFERENT CELL TYPE AND SHOWED THESE PARTICLES LIKE TO GO INTO NEURONS AND ASTROCYTES AND THIS IS LOOKING AT TRANSFECTED CELLS. CELLS PRODUCING A TRANSGENE. AND FROM A SINGLE DOSE OF PARTICLES INTO THE BLOOD, AND THEN FOCUSED ULTRASOUND TO GET THEM INTO THE BRAIN, WE ARE TRANSFECTING ABOUT 40% OF NEURONS AND ALMOST 60% OF ASTROCYTES. SO THIS IS SOMETHING THAT WE HOPE WE CAN DO OVER AND OVER IN PATIENTS MULTIPLE TIMES AND MAYBE GET MOST OF THE CELLS. ONE AREA WHERE WE HAVE BEEN APPLYING THIS IS PARKINSON'S DISEASE AND SO WE HAVE A PARKINSON'S DISEASE MODEL WHERE A DRUG IS GIVEN THAT WILL KILL THE DOPE NERGIC NEURONS AND SO UNLESS YOU PROTECT THEM SOMEHOW. AND SO WE HAVE BEEN USING OUR DNABPN TO DELIVER A GENE THAT WILL ENCODE FOR GDNF AND THEN RICH TAKES THOSE AND PUTS THEM INTO THE ANIMALS AND GETS THEM INTO THE BRAIN USING FOCUSED ULTRASOUND. HERE IS THE LEVELS OF GENE EXPRESSION AFTER 14 DALES IT IS ALREADY QUITE HIGH AND STILL GROWING UP TO 28 DAYS FROM A SINGLE ADMINISTRATION. BUT THEN THEY LOOKED AT HISTOLOGY OF THIS AND THESE ARE NORMAL ANIMALS SO YOU SEE THE DOTS AND THE STAINING HERE IS THAT DOPA NERGIC NEURONS BOTH UNTREATED AND NOTHING HAPPENING IN THIS ANIMAL OR JUST BEEN GIVEN NANOPARTICLES SYSTEMICALLY. IF YOU HAVE ANIMAL WITH THE PARKINSON'S DISEASE MODEL SEE THE NEURONS HAVE BEEN DEPLETED. AND THESE ANIMALS THAT ARE TREATED AFTER THEY ALREADY STARTED TO SHOW SIGNS OF THE DISEASE THEY CAN PROTECT THOSE NEURONS AND APPARENTLY MAKE SOME OF THEM GROW BACK OR COME BACK IN. SO THIS IS JUST THE QUANTIFICATION OF THE ROTATION TEST THESE ANIMALS BECAUSE YOU KILL ALL THOSE NEURONS ON ONE SIDE OF THE BRAIN THEY START TO SPIN IN THE CAGE. AND SO THEY ARE TREATED -- NOT SHOWING WHERE THEY ARE TREATED BUT TREATED AROUND THE TWO WEEK POINT WHERE ALL THE ANIMALS ARE SPIN BEING 7 TIMES PER MINUTE IN THEIR CAGE SO THEY ARE EVEN GIVEN NO TREATMENT AT ALL, THAT'S THE BLUE. THE THING THAT KILLS THE NEURONS, THE TREATMENT WITHOUT FOCUS ULTRASOUND DOESN'T HELP IT AT ALL. IT DOESN'T HURT NOR HELP. BUT IF YOU GIVE THE TREATMENT AND HELP IT GET INTO THE BRAIN WITH FOCUSED ULTRASOUND, THESE ANIMALS START TO STABILIZE AND MAYBE EVEN IMPROVE SO THEY ARE SPINNING IN A FAIRLY STABLE WAY ONLY A FEW TIMES PER MINUTE SEVERAL WEEKS AFTER THE ADMINISTRATION OF A SINGLE DOSE. THIS IS ALSO SOME VERY EARLY WORK THAT WE ARE DOING ALONG WITH RICH AND ROGER'S GROUP AND BJ'S GROUP AT UBA AND IN THIS CASE, WE ARE USING A microRNA THAT IS A TUMOR SUPPRESSIVE microRNA 34A AND WE ARE USING OUR NANOPARTICLES PUT INTO THE BLOOD TO DELIVER THIS INTO BRAIN. AND I NOTED TO SHARE THIS EARLY RESULT. SO, IF YOU LOOK AT THIS AT DAY 20 YOU START TO SEE A DIFFERENCE. THE GRAY BARS ARE UNTREATED ANIMALS AND TUMORS ARE GLOWING RAPIDLY. THE LIGHT GRAY BARS ARE IF THEY ARE GIVEN THE microRNA BPN BUT WITHOUT ANY FOCUSED RESULT SOUND. THE GREEN BARS THEY ARE GIVING BPN BUT WITH A SUB OPTIMAL FOCUSED RESULT SOUND OF PRESSURE .4 MEGAPAS CALLS AT .FIVE WITH THE microRNA, YOU SEE SIGNIFICANT DECREASE IN TUMOR SIZE USING THIS microRNA TUMOR SUPPRESSOR. SO I WANT TO ACKNOWLEDGE SOME PEOPLE AND START WITH THE UBA GROUP -- UVA GROUP. HERE IS RICH HEREAFTER, AND BRIAN HAS BEEN DOING A LOT OF THE WORK IN PARKINSON'S MODEL. KELSEY DID EARLY WORK WITH ELIZABETH TO SEE THAT NANOPARTICLES COULD SAFELY GET INTO THE BRAIN USING FOCUSED ULTRASOUND AND COLLEEN HAS BEEN DOING A LOT OF THE WORK WITH THE TUMOR SUPPRESSOR microRNA. AND THEN FINALLY ACKNOWLEDGING ALL OF OUR COLLABORATORS AT THE UNIVERSITY OF VIRGINIA AND QUITE A FEW DIFFERENT COLLABORATORS AND PEOPLE IN MY LAB AT HOPKINS. THANK YOU VERY MUCH. [ APPLAUSE ] >> THANK YOU VERY MUCH DR. HAINES. WE'LL TAKE ADDITIONAL QUESTIONS AT THE OPEN MIKE DISCUSSION FOR YOUR SESSION. RIGHT NOW WE HAVE TIME FOR A BRIEF BREAK. >> SO, LET'S BEGIN THE DISCUSSION OF IN-VITRO MODELS. SO, WE HEARD A RANGE OF TALKS THIS MORNING SPANNING ORGAN ON A CHIP MODELS TO ORGAN GENESIS. WE HEARD ABOUT STEM CELL TECHNOLOGY AND WE ALSO HEARD ABOUT INSILICO MODELS. SO, THESE ARE SOME OF THE THOUGHTS WE HAD IN TERMS OF KEY CHALLENGES. SO JUST BRIEFLY GO THROUGH THEM. AS YOU PROBABLY GOT A SENSE THIS MORNING, INTEGRATION IS ONE OF THE KEY CHALLENGES SO WE SAW EVERYONE IS DEVELOPING DIFFERENT COMPONENTS AND INTERGREATING DIFFERENT SUBCOMPONENTS BUT ULTIMATELY ALL COMPONENTS NEEDS TO BE INTEGRATED. HOW TO DO THAT AND HOW TO VALIDATE IT WILL BE AN IMPORTANT ISSUE. SIMILARLY CLUEING THE RIGHT MODEL. SO FIGURING OUT AND PROVIDING A TOOLKIT IF YOU LIKE OR TOOLBOX OF MODELS FOR SPECIFIC APPLICATIONS THAT CAPTURE THE PHYSIOLOGICAL FEATURES YOU NEED FOR THE PARTICULAR QUESTION YOU'RE ASKING. HUMAN CELL SOURCING. WE HEARD THIS MORNING IN PARTICULAR ABOUT BRAINING MICROVASCULAR ENDOTHELIAL CELLS DERIVED FROM STEM CELLS. THIS HAS BEEN DONE TO SOME DEGREE IN EARLY STAGES FOR ASTROCYTES AND PERICYTES. THIS NEEDS TO BE DONE AND VALIDATION NEEDS TO BE IMPROVED TO GET TO THE POINT THAT THESE TOOLS CAN BE PUT IN THE LABS, AT EVERYONE'S LAB TO USURY PRODUCIBLEY. STANDARDIZATION, VALIDATION ARE OBVIOUSLY VERY KEY. ONE OF THE CHALLENGES WE FACE IN BUILDING IN-VITRO MODELS IS VERY OFTEN WE DON'T KNOW EXACTLY WHAT THE IN-VIVO CRITERIA ARE. SO, IN TERMS OF -- FOR EXAMPLE, WITH PERMEABILITY. SIMPLE MEASUREMENTS LIKE PERMEABILITY. IF WE MEASURE A PERMEABILITY IN OUR MODEL, HOW DO WE KNOW IF IT IS PHYSIOLOGICAL OR NOT? WE DON'T HAVE IN MANY CASES GOOD BENCHMARKS TO COMPARE TO AND THIS IS AS I SEE ONE OF THE MAJOR CHALLENGES WE HAVE. HUMAN RELEVANCE. WE HEARD A LITTLE BIT THIS MORNING IN TERMS OF DISEASE MODELS OF ENDOTHELIAL CELLS. THIS IS GOING TO BE OBVIOUSLY THE NEXT WAVE I THINK OF INCREASING COMPLEXITY TO BE ABLE TO ADDRESS SPECIFIC DISEASES. AND IN TERMS OF MODELING, WE HEARD A LITTLE BIT THIS MORNING ABOUT HOW INCREASING AND COMPUTING POWER WILL ENABLE US TO DEAL WITH HUNDREDS OF THOUSANDS OF ATOMS, CRITAL STRUCTURES FOR MANY OF THE EFLUX PUMPS ARE KNOWN. SOME ARE NOT KNOWN. WITH THOSE CRYSTAL STRUCTURES, WE WILL ENABLE THE MODELERS TO BE ABLE TO ASSEMBLE THOSE PROTEINS, TRANSPORTERS AND SO ON AND PUT US IN A POSITION TO BE ABLE TO TEST ON THE COMPUTER FOR SMALL MOLECULE DRUGS, FOR EXAMPLE, WHETHER THEY ARE LIKELY TO BE SUBSTRATES OF EFLUX PUMPS. SO IN TERMS OF OPPORTUNITIES, I THINK WHAT WE FELT COLLECTIVELY AT LEAST BEFORE THE MEETING WAS THAT OVER THE NEXT FIVE YEARS OR SO, THERE ARE TREMENDOUS OPPORTUNITIES FOR INTEGRATING WHAT EVERYONE INDIVIDUALLY AND OTHERS ARE LEARNING IN TERMS OF BUILDING MORE COMPLEX MODELS, IN TERMS OF IMPROVING PHYSIOLOGICAL RELEVANCE, DISEASE RELEVANCE, BEING ABLE TO DO DISEASE MODELING, THERAPEUTIC TESTING AND SO ON AND AS WE ALREADY MENTIONED ON A SIMULATION SIDE, TAKING ADVANTAGES OF IMPROVING IN COMPUTING POWER AND GETTING THOSE TOOLS IN THE HANDS OF NOT JUST PEOPLE LIKE SNYDER BUT MORE LADLE IN EXPERIMENTALIST LABS AS WELL. TO BE ABLE TO PERFORM THOSE KINDS OF STIPULATIONS. SO I THINK THAT WAS OUR SUMMARY AT LEAST BEFORE THE MEETING. SO, I GUESS NOW I INVITE OTHER COMMENTS AND QUESTIONS. [ OFF MIC ] AND I THINK THAT THE FIELD IS MISSING A LOT ON QUANTITATION OF PERMEABILITY. WE TALK A LOT -- I HEARD A LOT OF WORDS LIKE, IT'S OPEN OR IT'S CLOSED. I DON'T THINK IT'S AS SIMPLE AS THAT. I THINK WE NEED TO HAVE MORE QUANTITATIVE MEASURES ABOUT WHAT PERMEABILITY IS, NOT OPEN AND CLOSED. BUT PERMEABILITY OF VARIOUS SUBSTANCES, VARIOUS DRUGS, VARIOUS PHYSIOLOGICAL COMPONENTS. SO, WITH THAT IN MIND, THE PERMEABILITY IS REALLY RELATED TO THE TYPE JUNCTIONS AND THE WAY, THE TIGHTNESS THAT WE HEARD ABOUT THE TIERS AND SO ON, THE ELECTRICAL RESISTANCE, AND SO, THOSE MOLECULAR ON A MOLECULAR BASIS, ARE RELATED TO THE OCCLUDE IN, CLOUD IN 5, AND JUNCTIONAL ADHESION MOLECULES. SO MY QUESTION IS, ARE THOSE THE ONLY MOLECULES THAT WE KNOW OR THAT ENGTHEY ARE THE ONES WE KNOW BUT WHAT DO YOU THINK ARE THE OTHER -- ARE THERE OTHER MOLECULES OR OTHER GENE THAT IS EXPRESS PROTEINS THAT ARE ESSENTIAL FOR MAKING THIS VARIABILITY IN PERMEABILITY? DO WE KNOW ALL THE MOLECULES THAT WE NEED TO KNOW FOR MAKING TIGHT JUNCTIONS? >> I THINK YOUR QUESTION IS SEVERAL-FOLD. THE FIRST REGARDING THE TIGHT JUNCTION MOLECULES CERTAINLY THERE ARE VARIOUS COMPONENTS TO THE TIGHT JUNCTIONS WHICH YOU MENTIONED. BUT ADDITIONAL THINGS LIKE LSR AND TRISELL LIN AND OTHER COMPONENTS OF TIGHT JUNCTIONS THAT YOU CAN LOOK FOR TO TRY AND VALIDATE THE MODELS. I THINK ONE OF THE BASELINE KEYS TO AN INVITE ROW BLOOD-BRAIN BARRIER MODEL FOR APPLICATIONS FOR PERMEABILITY-TYPE MEASUREMENTS, YOU WANT TO RESTRICT THE PARACELLULAR DIFFUSION AS MUCH AS POSSIBLE SO YOU'RE NOT MEASURING THAT AS AN ARTIFACT. AND I MENTIONED THIS MAYBE A LITTLE BIT EARLIER TODAY BUT IF YOU ARE LOOKING AT A HYDROPHILIC MOLECULE AND YOU DON'T CLOSE DOWN THE PARACELLULAR PORE OR WEEKLY LYPOPHILIC MOLECULE, WHAT YOU WILL MEASURE IN THE PETRE DISH IS PARACELLULAR TRANSPORT RATHER THAN THE TRANSCELLULAR TRANSPORT WHICH IS MOST RELEVANT. IN TERMS OF TRANSCELLULAR TRANSPORT, IN ADDITION TO THE PARACELLULAR PATHWAY, IT'S VERY MUCH MORE RECOGNIZED NOW AFTER A FEW RECENT PAPERS ON THE AFFECTS OF PERICYTES ON THE BLOOD-BRAIN BARRIER FUNCTION THAT VESICLE TRAFFICKING IS SERIOUSLY DOWN REGULATED AT THE IN-VIVO BBB LARGELY THROUGH PERISITE INTERACTIONS. THAT'S ANOTHER THING YOU CAN LOOK AT IN YOUR IN-VITRO MODEL IS QUANTITATION OF WHAT I WOULD CALL GENERICALLY TRANSCELLULAR TRAFFICKING LIKELY THROUGH MACKRA PIANO CYTOSIS AND THINGS OF THAT NATURE. THEY SHOULD BE PRETTY LOY IN TERMS OF THE AMOUNT OF MATERIAL THAT CAN GO THROUGH THAT PATHWAY. AGAIN IF YOU HAVE LEAKINESS BETWEEN YOUR JUNCTIONS YOU WILL NEVER SEE THOSE AFFECTS BECAUSE THAT THROUGH PUT OF THAT PATHWAY IS LOWER THAN FREE MOLECULAR DIFFUSION. >> SO I AGREE THE FIRST THING TO REALIZE IS THAT THERE IS THE TRANSSIGH TO THETIC PATHWAYS AS WELL AND PROBABLY PREDOMINATE IN MOST KINDS OF DISEASE STATES. AND WE ALSO KNOW A LOT ABOUT THE MECHANISMS THAT ARE CONTROLLING BOTH OF THOSE. MCP1 SEEMS TO BE HAVING SOME AFFECT ON THE TRAFFICKING OF THE TIGHT JUNCTION PROTEINS AND PKC SEEMS TO UNDERLIE SOME OF THE MECHANISMS THAT THE BLOOD-BRAIN BARRIER AND EITHER MECHANISM AND WE CAN QUANTIFY AND SIZE SO WE CAN MEASURE THE RATES FOR EXAMPLE, SIMULTANEOUSLY INJECT SEVERAL SUBSTANCES LIKE SUCROSE AND ALBUMIN AND MEASURE THEIR THE RATE OF ENTRY. WE CAN DO THOSE IN ANIMAL MODELS AND IN HUMANS AS WELL. AND I THINK THAT IS VERY CRITICAL TO DO. I THINK THE ONE THING THAT MAYBE WE SHOULD REALLY, REALLY TRY TO BACK AWAY FROM IS AS I FIELD IS USING THE DYES PARTICULARLY TRIPAN BLUE AND I'M SURE MOST OF THE BBB PEOPLE READ NORM SANDERS REVIEW ON NOT USING EVIDENCE BLUE TRIPAN BLUE OR ANY OF THOSE DYES. THEY ARE JUST NOT QUANTITATIVE. I THINK THEY ARE GIVING US ERRONEOUS INFORMATION. HE GOES THROUGH SOME OF THE WAYS THAT COULD HAVE OCCURRED. >> I THINK I'LL MIKE JUST ONE OTHER COMMENT RELATED TO PARACELLULAR TRANSPORT. WE HEARD YESTERDAY MANY EXAMPLES OF BOTH SPACIAL AND TEMPORAL CHANGES IN PERMEABILITY WHERE FROM IN-VITRO POINT OF VIEW, CERTAINLY HISTORICALLY WE TEND TO THINK FROM MAY TRANSWELL ASSAISE AS BEING PERMEABILITY BEING A PARAMETER THAT APPLIES TO A MONOLAYER UNDER SOME SET OF CONDITIONS. FROM A MODELING POINTED OF VIEW, WE NEED TO COME CLOSER TO THE PHYSICAL REALITY AND THINK ABOUT THE ISSUES THAT DICTATE SPACIAL AND TEMPORAL CHANGES AND THIS INVOLVES QUANTIFYING THINGS LIKE ENDOTHELIAL CELL DYNAMICS, HOW THEY ARE MOVING, THEIR ACTIVITY, PROLIFERATION, TURNOVER, ALTHOUGH LOW IS GOING TO CONTRIBUTE AND ALSO MOLECULES THAT INDUCE QUIESCENCE. SO IT IS ANOTHER CHARACTERISTIC THAT IS BANDED AROUND A LOT IN THE VASCULAR ENGINEERING FIELD BUT VERY FEW OR VERY DIFFICULT TO PIN DOWN QUANTITATIVELY PARAMETERS OR HOW TO DEFINE WHEN YOU HAVE QUIESCENCE WHEN YOU DON'T. SO THE ROLE OF MODULATORS AND QUIESCENCE INDUCERS IN SPATIALLY AND TEMPORALLY MODULATING PERMEABILITY I THINK IS SOMETHING THAT WE ALL NEED TO TAKE INTO ACCOUNT AND TO FIGURE OUT HOW TO MAKE CONNECTIONS TO IN-VIVO. >> SO I'LL ADDRESS TO THE BROADER PANEL BECAUSE I HAVE TWO QUESTIONS THAT ARE SOMEWHAT RELATED. AND YOU MENTIONED THE DIFFICULTY OF AT LEAST IN YOUR MODELS OF ISOLATING ENOUGH BRAIN MICROVASCULAR ENDOTHELIAL CELLS SO YOU'RE STARTING WITH HUVEX. AND OBVIOUSLY ALLUDED TO THE FACT THAT OBVIOUSLY THAT ENDOTHELIAL CELLS ARE NOT CREATED EQUAL AND THAT YOU ULTIMATELY ARE GOING TOWARDS TRYING TO GET THE MICROVASCULAR BRAIN MICROVASCULAR CELLS INTO YOUR MODEL. SO, ONE OF THE QUESTIONS I MEAN OBVIOUSLY THAT WOULD ALLOW A DIFFERENT KIND OF CROSS TALK BBB CROSS TALK TO BE ASSESSED COMPARED TO HUVEX. THAT'S A COMMENT. BUT I THINK WHAT I'M WONDERING IS IS THAT EVEN PRESUMABLY AND I'M INFERRING HERE, I DON'T KNOW IF THIS IS RIGHT AND YOU MAY BE ABLE TO CORRECT ME. BUT ACROSS THE DEVELOPMENTAL PATHWAY EVEN MICROVASCULAR BRAIN MICROVASCULAR ENDOTHELIAL CELLS ARE NOT THE SAME. PRESUMABLY BECAUSE DURING TIME OF LATER GESTITION ALL THE WAY TO THE FIRST YEAR OF LIFE, YOU HAVE HIGH PLASTICITY AND EYE HIGHER GROWTH RATE BECAUSE YOU DEFINED STANDARD ADULT BRAIN MICROVASCULAR AND ONE OF YOU DID, AS SAYING VERY PRETTY YOU MUCH STAT NICK TERMS OF NOT EXTENDINGS WOULD THAT NOT BE DIFFERENT IN SAY NEONATES? AND IF SO, IF YOU DID RNA-SEQ TO DIFFERENTIATE BETWEEN ADULT MICROVASCULAR AND LET'S SAY LATE GESTATIONAL AND AGAIN I KNOW THAT IS A CHALLENGE IN TERMS OF SAMPLING BUT NONETHELESS, IF YOU COULD DO THAT, WOULD IT HELP INFORM NEXT GENERATION REGENITIVE CONCEPTS ON THE BLOOD SIDE? BECAUSE YOU PRESENTED PHENOMENAL DATA ON THE BRAIN SIDE AND GLIAL NEURONAL AND REGENITIVE CAPACITY AND I'M WONDERING FROM THE BLOOD SIDE IF WE COULD PUSH THAT BY UNDERTAKING SOME OF THESE -- JUST A BROAD AND PROBABLY STUPID IDEA BUT I THOUGHT I WOULD PUT TO THE OUT THERE. >> I THINK IT'S THE HOLY GRAIL IN SOME DEGREE AND HOPEFULLY, THIS IS WHERE PEOPLE LIKE SERGIO, EVEN THOUGH HE IS NOT WORKING IN THE BLOOD-BRAIN BARRIER AREA BUT THE ORGAN GENESIS PEOPLE, THE HOLY GRAIL IS TO ASSEMBLE AND MIMIC ASPECTS OF BLOOD-BRAIN BARRIER DEVELOPMENT. >> WE ARE CERTAINLY AT VANDERBILT BEGINNING TO TRY AND MODEL THE DEVELOPING FETAL BLOOD-BRAIN BARRIER SO THAT IS ONE ASPECT OF MODELING THAT WE ARE BEGINNING TO DO. BUT I WOULD SAY FOR THE PRELIMINARY DATA, WHILE AS ADULTS AND OLDER ADULTS, WE WANT TO POTENTIALLY BE ABLE TO REPAIR OUR BLOOD-BRAIN BARRIER. WE WOULD NOT WANT A FEELS BLOOD-BRAIN BARRIER BECAUSE OUR BRAINS WILL NOT BE ABLE TO RESPOND OR RECOVER THE WAY A FETAL BRAIN CAN AND THEREFORE CAN HANDLE THE VERY LEAKY SORT OF NATURE OF THAT DEVELOPING ONE. SO I'M SURE IT WILL GIVE US SOME HINTS BUT I THINK THAT WE ARE REALLY GOING TO HAVE TO LOOK AT REPAIR MECHANISMS VERSUS DEVELOPING MECHANISMS AND I WOULD SAY THAT FROM OUR PRELIMINARY WORK WHERE WE HAVE SHOWN INSTANCES WHERE WE DISRUPTED THE BLOOD-BRAIN BARRIER AND SEEN RECOVERY AND REPAIR THOSE MECHANISMS LOOK VERY DIFFERENT FROM THE MECHANISMS IN DEVELOPING THE FETAL BLOOD-BRAIN BARRIER. SO I THINK THAT IN TERMS OF HOPING ADULT PATIENTS WE WANT TO PUSH AND NUDGE THE REPAIR MECHANISMS MORE THAN TRY TO GO BACK TO A LESS DIFFERENTIATED STATE. [ OFF MIC ] >> AND I WOULD SAY WE WANT TO FOR ADULT PATIENTS REALLY WANT TO FOCUS ON REPAIR BECAUSE WE STILL HAVE TO GET REGENERATION FOR ALL THE CELLS IF WE WENT BACK TO SORT OF THE FETAL AND THERE ARE MECHANISMS OF REPAIR FOR THE BLOOD-BRAIN BARRIER THAT AREN'T FUNCTIONING QUITE AS WELL. MY OWN BET. >> SO IN TERMS OFS TEMPORAL PROCESS AT LEAST IN RODENTS, BARRIER FORMATION STARTS ABOUT 11 OR 12 AND PROGRESSES THEN THROUGH POSTNATAL DEVELOPMENT. RNA-SEQ ANALYSIS IS AT GENENTECH SO THOSE DATA EXIST. AND REALLY WHERE THEY -- WE ARE COMBINING THE CONCEPT OF DIFFERENTIATION OF STEM CELLS IN THE PEE TREAT DISH BUT TRYING TO FIND KEY PATHWAYS. AND MODULATORS SITUATIONS. SO I AGREE WE DON'T WANT TO TURN THEM BACK INTO A FETAL BRAIN ENDOTHELIAL CELL BUT IF YOU LOOK AT THE DEVELOPMENTAL TIMELINE AS YOU SUGGEST YOU CAN FIND MAYBE KEY PATHWAYS THAT CAN TURN ON TO TITAN THINGS AND MAY RECOVER SOME OF THESE DEFICITS. JUST MY OPINION. >> MY QUESTION IS WITH RESPECT TO THE SUITABILITY OF MODELS THAT YOU MENTIONED. SO AS I MENTIONED, I AM INTERESTED, WE ARE MAKING MULTIFUNCTIONAL EXOSOMES THAT HAVE THE CAPABILITY OF CROSSING THE BLOOD-BRAIN BARRIER TARGETING ONE COMPONENT OF IT AND GO BEYOND IT AND THEN TARGET SPECIFICALLY HERT2 POSITIVE CANCER, FOR INSTANCE. SO, WHAT EXISTING MODEL IN-VITRO MODEL AT THE MOMENT YOU THINK WOULD BE MOST SUITABLE TO TEST THIS KIND OF POSSIBILITY. [ OFF MIC ] >> I'M SORRY? >> THAT IS EASY. YOU WANT TO USE THE MODEL TOL EXPRESS THE CHARACTERISTICS THAT YOU WANT. BUT THAT IS EXACTLY WHAT YOU HAVE TO DEFINE. YOU HAVE TO -- THE MODELS THAT ARE AVAILABLE YOU HAVE TO FIND OUT AND THEY MAY NOT -- >> I THINK THAT ANY OF THE MODELS CURRENTLY THAT WERE PRESENTED HERE WITH POTENTIALLY THE EXCEPTION OF THE COMPUTER MODELING, WOULD CERTAINLY WORK FOR TESTING EXOSOMES. BUT YOU WOULD GET DIFFERENT DATA BACK FROM THEM. YOU CAN CERTAINLY PUT IN IN THE TRANSWELL AND SEE IF IT CROSSES ON THE OTHER SIDE HAVE YOU GOT CANCER CELLS, YOU CAN CERTAINLY SEE HOW IT AFFECTS CANCER CELLS FOR THAT. BUT THEN YOU WOULDN'T HAVE INFORMATION ABOUT SORT OF FLOW AND HOW FLOW DYNAMICS MIGHT AFFECT EXOSOMAL UPTAKE SO THEN IF YOU WANTED MORE INFORMATION ABOUT FLOW AND SHEAR STRESS AND HOW THAT MIGHT AFFECT THAT, YOU WANT TO MOVE INTO POTENTIALLY A MICROFLUIDICS THEN. [ OFF MIC ] >> GO BACK TO ONE OF THE THINGS WE OUTLINED PREVIOUSLY. AND I THINK AS PROFESSOR BANKS SAID, IT DEPENDS ON WHAT YOU WANT TO DO. [ OFF MIC ] >> YOU DIDN'T TELL US WHAT THE RECEPTOR TARGET WAS FIRST OF ALL. SO THAT IS ONE THING TO THINK ABOUT. IS THE RECEPTOR EXPRESSED AND THEN LET'S SAY IT IS YOU SEE TRANSCELLULAR TRAFFICKING THEN YOU HAVE TO UNDERSTAND THE IN-VIVO EXPRESSION OF THAT PARTICULAR RECEPTOR MAY BE DIFFERENT SO IF YOU COMPARED TWO RECEPTORS OR VARIOUS TARGETING MOLECULES AGAINST A GIVEN RECEPTOR, THOSE ARE THE TYPES OF THINGS THE IN-VITRO MODELS COULD BE USEFUL FOR BUT THEN REMEMBER THAT SHOULD NARROW MAYBE A LARGER SUBSET OF EXPERIMENTS SO THAT YOU GO IN-VIVO AND DETERMINE WHICH OF YOUR SUBSET IS IMPORTANT. >> BUT I THINK THIS IS THE CRUCIAL PROBLEM WITH STARTING WITH IN-VITRO MODEL. BECAUSE YOU CAN GET AN ANSWER. NOW IS THAT ANSWER THE CORRECT ANSWER? YOU CAN USE SEVERAL MODELS AND GET SEVERAL ANSWERS. THEY ARE A LITTLE BIT LIKE THE ECONOMIST WHERE YOU ASK THREE ECONOMISTS A QUESTION AND YOU GET FOUR OPINIONS. YOU CAN GO TO WITH THE BLOOD-BRAIN BARRIER MODELS AS WELL. SO I START IN-VIVO. IT'S NOT THAT HARD. USUALLY FEWER MICE TO DO IN-VIVO EXPERIMENT THAN IN-VITRO EXPERIMENT AND IT'S USUALLY SET UP. YOU'RE GOING TO HAVE TO GO THERE ANYWAY SOONER OR LATER. [ OFF MIC ] >> IT DEPENDS ON WHETHER YOUR QUESTION NEEDS HUMAN CELLS AS WELL WOULD SAY. ANIMAL MODELS THEY HAVE A GREATER COMPLEXITY. IT'S A FULL ANIMAL, FULL INTACT BLOOD SYSTEM. IT'S REAL BLOOD. BUT MICE ARE NOT MEN AND THERE ARE SEVERAL CASES WHERE IF YOU HAD TESTED IT IN A MOUSE YOU WOULD HAVE GOTTEN THE WRONG ANSWER BECAUSE MICE DO NOT RESPOND THE SAME WAY. THE CLASSIC ONE IS VITAMIN C. MICE PRODUCE THEIR OWN. THEIR CELLS DON'T NEED IT AND THEY DON'T HAVE THE SAME METABOLIC REQUIREMENTS THAT OUR BLOOD-BRAIN BARRIER DOES. SO IF YOU'RE TRYING TO ANSWER QUESTIONS THAT HAVE ESPECIALLY METABOLIC ONES THAT HAVE SPECIFIC HUMAN NEED, AND AGAIN I GUESS PROBABLY NOT ALLOWED TO JUST GO STRAIGHT TO IN-VIVO IN HUMANS, THAT IS WHEN I THINK THE IN-VITRO MODELS THAT A LOT OF THE MEMBERS UP HERE ARE DEVELOPING ARE REALLY CRITICAL BECAUSE THEN YOU HAVE THOSE HUMAN CELLS TO TEST INSTEAD OF THE ANIMALS. ALSO FOR PEOPLE WHO ARE GOING INTO THAT DRUG TESTING MODE, YOU HAVE YOUR SMALL AND YOUR LARGE ANIMAL. YOU GET A DIFFERENT RESULT FROM YOUR SIGNAL AND YOUR LARGE AND YOU'RE TRYING TO DECIDE IS THIS SOMETHING I SHOULD STILL PURSUE? PERHAPS MOVING INTO A HUMAN CELL MODEL MIGHT HELP YOU MAKE THAT DECISION. SO I THINK THAT IS WHEN YOU HAVE TO ASK THE QUESTION OF WHAT YOU WANT TO USE. [ OFF MIC ] >> PROBABLY MOST PEOPLE WOULD -- MOST PEOPLE AT THIS POINT WOULD GO WITH THE TRANSWELL MODEL. IT'S THE GOLD STANDARD. IT'S VERY -- THE LEVEL OF IN-VITRO WORK SHE IS TALKING ABOUT IS NOT FOR THE FAINT OF HEART. JUST ACQUIRING HUMAN BRAIN ENDOTHELIAL CELLS PRIMARY END THEY'LLIAL CELL IS A BIG ADVENTURE. USE THE HUMAN D3 WHICH ARE MANY PEOPLE ENDOTHELIAL CELLS ADVOCATE THEY ARE NOT BRAIN ENDOTHELIAL CELLS. THEY CERTAINLY DON'T RESPOND IN OUR ASSAYS THE WAY THAT PRIMARIES DO. SO, THIS IS NOT AN AREA FOR THE FAINT OF. >> THE BUT THOSE WHO HAVE FAINT HEARTS SHAMELESS PLUG, VANDERBILT IS OPENING ITS CORE CENTER WHERE YOU DON'T WANT TO TOOL UP TO DO THIS YOURSELF, SEND US YOUR DRUG, YOUR COMPOUND AND WE'LL RUN IT THROUGH OUR BLOOD-BRAIN BARRIER MODEL FOR YOU. BECAUSE HE IS RIGHT. IT IS A LOT OF TOOLING UP. IT'S A LOT OF CELL CULTURE AND A LOT OF SPACE TO DEDICATE TO SOMETHING ESPECIALLY IF YOU ONLY WANT ONE EXPERIMENT AND YOU WANT TO SEE THE OUTAND THE I THINK A LOT OF PLACES WILL PROBABLY GO TO THIS CORE SYSTEM SINCE NOT EVERY LAB WILL WANT TO ADOPT THE TECHNOLOGIES WE ARE DEVELOPING HERE. [ OFF MIC ] >> GENENTECH IS CALLED TDM ONE. THAT OUGHT TO BE THE CONTROL. IT DOESN'T REQUIRE COMPLIMENT. THE CURRENT THERAPY FOR HERT2 POSITIVE IS HERCEPTIN BUT HAS NO IMPACT IN THE BRAIN BUT TDM1 IS AN ANTIBODY DRUG COVERAGE GAT WHICH WOULD BE A PERFECT CONTROL FOR YOUR SYSTEM. [ OFF MIC ] >> I THINK IT'S ITERATIVE PROCESS THAT YOU HAVE TO START SOME PLACE. YOU PICK YOUR POISON AND YOU TRY TO VALIDATE IT WITH THE OTHER MODELS AND AS THINGS BECOME AVAILABLE SUCH AS HUMAN CELLS OR HUMAN IMAGING, YOU WILL SORT OF KNOW FOR SURE IN 10-15 YEARS BUT IT SOUNDS TRITE BUT THAT IS SORT OF WHAT WE -- IT IS WHAT WE HAD TO DO EARLY ON WHEN WE STARTED MAPPING OUT CYTOKINE TRANSPORTERS. WE HAD TO START WITH IN A REVERSE WAY OF THE WE HAD TO START WITH THE CYTOKINES THAT WERE AVAILABLE. SOMETIMES INJECTING HUMAN CYTOKINES IN THE NICE AND THEY OFTEN CROSS BY SPECIFIC SYSTEM AND WE GOT THE MOUSE CYTOKINES LATER AND WE WERE ABLE TO VALIDATE THINGS LIKE THAT. WHEN WE GO THROUGH THE IN-VITRO SYSTEM, SOMETIMES THEY ARE THERE AND SOMETIMES THEY ARE NOT. SOMETIMES THEY APPEAR WHEN THEY START TO TRICULTURE OR CO-CULTURE. IT'S AN AREA WHERE YOU HAVE TO BE COMFORTABLE WITH UNCERTAINTY AND YOU FIND LEADS AND YOU FOLLOW THEM UP. SO, IF YOU FIND SOMETHING THAT INCREASES THE TRANSPORT AND IF YOU GO TO EVENTUALLY GET AROUND TO -- AS ED HAS DONE, GET AROUND TO ADDRESSING IN PATIENCE AND IT WAS PRETTY SMART. AND IF IT DOESN'T WORK YOU BLAME IT ON THE CELLS. >> ONE OTHER COMMENT I'D LIKE TO MAKE COMING BACK TO MODELS IS I'M A FIRM BELIEVER THAT THE MODELS WE GENERATE NEED TO BE GOTTEN INTO SOME OF YOUR LABS. THAT SHOULDN'T BE SOMETHING THAT ONLY A SMALL GROUP OF PEOPLE ARE DEVELOPING. I THINK IT IS REALLY IMPORTANT THAT WE TRANSLATE THEM TO NEUROSCIENTISTS LABS TO PHARMACOLOGIST LABS AND SO ON. TO THE EXTENT THAT THOSE OF YOU WHO ARE INTERESTED IN DOING IT I THINK WE ARE ALL MORE THAN HAPPY TO PROVIDE TRAINING SESSIONS. WE RETUNELY PROVIDE ONE WEEK WORKSHOPS FOR RESEARCHERS TO COME TO OUR LAB TO LEARN TECHNOLOGY WE HAVE AND I THINK IF WE ARE GOING TO HAVE IMPACT, THEN THESE TOOLS HAVE TO BE PUT IN THE HANDS OF MORE THAN JUST A SMALL GROUP OF PEOPLE. >> ANYMORE QUESTION? WE, MOVE ON TO THE NEXT SESSION. ... >> WE HAVE GENERAL QUESTIONS WHICH WERE PREPARED TODAY FOR OUR ROUNDTABLE AND I DON'T WANT TO THE READ THIS FOR YOU. YOU CAN BRIEFLY LOOK AT OUR SCREEN. AND I BELIEVE THE PEOPLE DEVELOP THEIR OWN QUESTIONS TO OUR PANEL AND WE CAN START THE DISCUSSION. >> SO YOUR SESSION TALKED A LOT ABOUT TARGETING AND I NOTICED IF I GOT THE SLIDES CORRECTLY, MOST OF THE TARGETING WAS USING ALREADY KNOWN TARGETING REAGENTS. THAT MAY WORK WELL IN RODENTS MAYBE SUB OPTIMAL IN SOME WAYS. YESTERDAY WE TALKED ABOUT LOTS OF OMICS, GENES, PROTEINS, AND WE TALKED ABOUT LIP KIDS AND METABOLITES. ONE THING WE EVAPORATE TALKED ABOUT THAT MAY BE IMPORTANT WITH THE INTERACTION OF BLOOD FLUIDS WITH THE BLOOD-BRAIN BARRIER INTERFACE AND ALSO FROM THE CONTEXT OF TARGETING IS GLYCOBIOLOGY. AND I'M WONDERING HOW IMPORTANT THAT IS IN EXOSOMES AND EXOSOME FUNCTION BECAUSE THEY DO REPRESENT SOME SNAPSHOT OF THE CELL SURFACE AND THEN ALSO IT'S BEEN WELL RECOGNIZED FOR SOMETIME THAT THERE IS A SUBSTANTIAL GLYCOKAY LICKS OR SUGAR HYDROGEL THAT LINES THE LUMEN NETWORK IN THE BRAIN AND THAT CAN CHANGE IN DISEASE STATES. SO, I'M WONDERING HOW IMPORTANT IS TARGETING, NEW TARGETING REAGE AND HOW IMPORTANT IS THIS GEIKO KAY LEX THROUGH MATERIALS AND TISSUES -- GLYCO. >> I START TO SPEAK ABOUT TARGETING. SO TARGET ABILITY IS STILL VERY, VERY IMPORTANT BECAUSE WE DO NOT INJECT TONSE OF DRUGS AND DRUGS SHOULD BE SELECTIVE. IT REDUCES TOXICITY AND INCREASE EFFICACY OF TREATMENT. THIS IS WHY TARGET ABILITY IS VERY IMPORTANT, PLUS TODAY WE DEMONSTRATE SEVERAL SPEAKERS SHOWED THAT STEP-BY-STEP TARGET ABILITY AND PENETRATION OF DIFFERENT BIOLOGICAL BARRIERS. NOTED ONLY BBB. IT'S A ASSEMBLE FOR DISCUSSION BUT THIS IS NOT ENOUGH FOR ENDPOINT WHICH IS TREATMENT. SO, I STILL BELIEVE TARGETTABILITY AND DRUG DESIGN, NANODRUG DESIGN OR ANY KIND OF DRUG DESIGN SHOULD BE VERY PRECISE. I UNDERSTAND YOUR QUESTION ABOUT EXOSOMES. PERSONALLY I DO NOT HAVE THIS EXPERIENCE. I LEARN A LOT YESTERDAY AND A LITTLE BIT TODAY. HOWEVER, I HAVE HAD SOME DISCUSSIONS YESTERDAY WITH EXPERTS WHO WORKED WITH EXOSOMES AND THE MESSAGE I RECEIVED OUR WORK TOGETHER WITH EXPERTS WHO KNOWS BIOLOGY OF NATIVE EXOSOMES AND US WHO WORK IN INCH BY ENGINEERING, NOT TECHNOLOGY FIELD WOULD BE AVAILABLE BECAUSE WE HAVE TO MAKE THEM A LITTLE BIT SMALLER, MORE PRICE AND MORE TARGETABLE. THEY WILL BE DIFFUSED AND ENDPOINT TREATMENT WILL BE NOT VERY EFFECTIVE. [ OFF MIC ] >> WHAT HAPPENED WITH FARAH DECKS IT WAS COMPLETELY COVERED WITH A CARBOHYDRATE AND IT WAS A DISASTER. I THINK THE CARBOHYDRATE COVERAGING NANOPARTICLES OR OTHER THINGS ON THE SURFACE SO THEY DON'T GET STICKY IS VERY IMPORTANT. BUT YOU CAN, AS JULIA NICELY SHOWED, YOU CAN CONJUGATE THINGS TO THEM TO MAKE THEM VERY TARGET-SPECIFIC. HERT2, EGFR. >> SO I THINK WE'LL GET FREE TARGETING LIKE FOR EXAMPLE WITH THE -- PRETTY MUCH GO TO THE MESSENGER RNA YOU'RE TRYING TO BLOCK. AND I REMEMBER STANLEY'S WORK WITH FREE FATTY ACIDS THAT HE WAS ABLE TO SHOW THE BRAIN THAT HAD DAMAGE AND WAS REPAIRING BECAUSE THAT'S WHERE IT WAS. I THINK THAT THERE IS A LOT OF PHYSIOLOGICAL TARGETING. WE KNOW THAT FOR EXAMPLE REGULATORY PROY TEENS GO TO VARIOUS REGIONS DEPENDING ON WHICH ONE IT IS SO PERHAPS THAT COULD BE USED AS A SORT OF TROJAN HORSE TARGETING. BUT I THINK YOU'RE RIGHT. THE GLYCOPROTEINS THESE ARE PROBABLY GOING TO BE KEYS FOR THE FIRST GENERATION OF TARGETING I WOULD THINK. AND SO YOU NEED TO FIND MORE OF THESE TRANSPORTERS AND WHERE THEY ARE. AND I GUESS IT GOES BACK TO THE PREVIOUS QUESTION ABOUT THE BLOOD-BRAIN BARRIER BEING DIFFERENT NOT AT DIFFERENT AGES BUT DIFFERENT REGIONS OF THE BRAIN SO THAT AS WE PERHAPS DO SINGLE CELL GENETICS ON THE CELL WE CAN FIGURE OUT WHAT IS BEING EXPRESSED WHERE. AND WITH THAT, WE MIGHT BE ABLE TO GET PRETTY PRECISE. >> MAYBE ONE ADDITIONAL COMMENT. I WOULD AGREE THAT I THINK TARGETING A HUMAN BEING IS GOING TO BE EXTREMELY CHALLENGING. WE CANNOT FORGET THE FACT THAT ALL OF THE MOUSE MODELS THAT WE HAVE SHOWN TODAY OR WE HAVE BEEN CONTINUING WORKING WITH THESE MONOCLONAL DISEASES. SO IF YOU FOR EXAMPLE LOOK AT RECEPTOR EXPRESSION, PRETTY MUCH EVERY SINGLE TUMOR CELL IN A MOUSE IS EXPRESSED SO TARGETING IS NOT AS DIFFICULT. AN ISSUE BEING WHERE YOU HAVE A VERY HIGHLY INTERTUMORAL HETEROGENEITY AND INTERTUMORAL EXPRESSION IN THOSE RECEPTORS. UNLESS YOU HAVE A MULTI-VALIANT TARGETING STRATEGY THAT TARGETS MULTIPLE RECEPTORS, I THINK IT WILL BE VERY DIFFICULT TO TRANSLATE THE SUCCESSES YOU SEE IN MOUSE MODELS INTO HUMAN BEING. >> YES, I WOULD LIKE TO AGREE WITH ALEXANDER IN THE -- FOR SUCCESSFUL DRUG APPROVAL THIS IS WHY FDA REQUIRES SEVERAL ANIMAL MODELS AT THE SAME TIME TO DEMONSTRATE THIS IS STILL THE BEST TOOL WE HAVE TODAY AND IT GAVE SEVERAL MODELS FOR ONE DRUG T IS ALWAYS BETTER THAN ONLY ONE. THIS IS WHY FDA WANTS IT. REGARDING DIFFERENCES BETWEEN ANIMALS, HUMAN, I WAS SURPRISED TO READ THE PAPER THAT 85% OF DRUGS WHICH WERE DEVELOPED MODELS CANCER MODELS, ON ANIMALS WERE 1-1 SIMILAR TO HUMANS AND THIS IS WHY SO FAR WE HAVE SEVERAL SUCCESSFUL DRUGS INCLUDING CHIP POINT INHIBITORS OR HERCEPTIN. YOU KNOW? WE KNOW ABOUT DRUG RESISTANCE AND MANY OTHERS. IT'S A SINGLE TARGET. WE PUBLISHED GOOD PAPERS AND CELL. MILLION TARGETS. BUT AVERAGE TIME TO EMULATE ONE TARGET IS ABOUT 10 YEARS. HOW MULTIMILLION DOLLARS WE HAVE TO SPEND UNTIL BENCH TO BEDSIDE EVERYONE KNOWS THESE NUMBERS. SO IT IS A BIG PROBLEM. STILL WE CANNOT BLOCK 5 TARGETS AT THE SAME TIME FOR ONE PATHOLOGICAL CONDITION PER PATIENT. >> DR. EDWARD NEUWELT HAS A COMMENT. >> WITH REGARDS WHAT WAS SAID YESTERDAY, I THINK IN DISEASES LIKALS HEIMERS DISEASE, YOU CAN SEE -- ALZHEIMER'S -- YOU CAN SEE THIS IS WITHOUT FER MOCKS TOL AND THIS IS WITH. YOU CAN SEE THE SPACES THROUGHOUT THE CORTEX GOING DOWN TO -- [ OFF MIC ] THOSE SPACES ARE -- WHAT DOES JEFF CALL IT? THE GLYCO-- WHETHER YOU USE A GLYCOLYMPHATTIC OR THE PERIVASCULAR -- THEY ARE GOING TO BE DAMAGED AND I JUST WANTED YOU TO SEE THAT YOU CAN IMAGE IT IN-VIVO WITH TREMENDOUS RESOLUTION. YOU SEE THOSE VERTICAL LINES? PUT IT IN THE SEPTEMBER KEL AND WITHIN 15 MINUTES YOU SEE -- IN THE VENTRICLE AND WITHIN 15 MINUTES YOU SEE THESE -- [ OFF MIC ] I THOUGHT ESPECIALLY FROM YESTERDAY, I THINK THIS WOULD BE A GOOD MODEL. WE THINK CLOUD TWO BUT IN OTHER NEURODEGENERATIVE DISEASES THERE IS A BARRIER OPENING, THERE MEMBER INFLAMMATION AND SCARRING. >> MINE WILL BE PROBABLY EQUALLY BROAD. FOR TREATMENT OF SOME CANCER TYPES IN THE PERIPHERY, WE HAVE BEGUN TO ATTEMPT TO TRAIN THE IMMUNE SYSTEM TO TARGET THOSE CANCERS. WITHOUT TARGETING THE MICROFULLYIA IN THE BRAIN AND ACTIVATING THAT SINCE IT IS ALREADY WHERE IT IS SUPPOSED TO BE. >> DO YOU KNOW SEATS GENETICS? THAT IS EXACTLY WHAT THEY SAY -- SEATTLE GENETIC. TARGETING -- 40% OF ALL METASTATIC AND PRIMARY BRAIN TUMORS ARE MACROPHAGES AND TARGETING THE SC RECEPTOR IS A NON-SPECIFIC WAY TO SUPPLEMENT DIRECT TARGETING ANTIGENS AND MOLECULES AND SO THEY THINK THAT IS WHAT YOU'RE SAYING IS RIGHT. THE INNATE IMMUNE SYSTEM AGAIN IS FOCUSED ON THE MACROPHAGE. SO I THINK TARGETING THE MACROPHAGE IS A NEW AREA THAT PEOPLE ARE STARTING TO TALK ABOUT. >> AND I THINK -- I DON'T KNOW THE PERSON WHO DID THE FOCUSED ULTRASOUND IN THE AD MOUSE MODEL, BUT I THINK THAT THE WAY THERE WAS CLEARANCE IN IMPROVING COGNITIONS BECAUSE ACTIVATING MICROGLIA. IS THAT RIGHT? I THINK THAT WAS THEIR THEORY AND EXPLANATION. >> WHEN THEY WERE HAVING THE PSEUDO-- >> SO I THINK IT WAS THE FIRST TALK, SECOND TALK. THEY WERE USING FOCUSED ULTRASOUND IN ALZHEIMER'S DISEASE MOUSE MODEL AND WILL GOT IN PROVED COGNITION. HOW IS THAT WORKING? THEY WERE SPECULATING THAT BECAUSE THE ULTRASOUND -- SOMETHING WAS ACTIVATED IN THE MICROGLIA -- [ INDISCERNIBLE ] SO, I THINK THEY DID SHOW SOME MICROGLIAL ACTIVATION. >> WE CAN GO DOWN THE QUESTION LIST. SO WE TALKED ABOUT MOST PREVALENT BRAIN DISEASE THAT CAN PUSH THE BBB DELIVERY STRATEGY. SO I HEAR PERHAPS ALZHEIMER'S? >> I THINK IT DEPENDS BECAUSE IF IT IS -- IF YOU GO FOR EXAMPLE FOR HIGHER MORTALITY DISEASE YOU CAN PUSH A DRUG DELIVERY SYSTEM IN CLINICAL TRIAL MUCH QUICKER THAN A DISEASE THAT SIDE AFFECTS MAY NOT BE AS FORGIVEN AS IT IS FOR EXAMPLE IN CANCER. HOW APPLICABLE THAT TECHNOLOGY CAN BE GENETICALLY IN DISEASES OF THE CENTRAL NERVOUS SYSTEM IS QUESTIONABLE BUT -- >> ALSO QUESTION NUMBER 3, IT IS KIND OF TOXICITY AND I SAW THIS ANSWER FROM OUR DR. NEW WELT SO CAN I SEE IF YOUR ANSWER, WHAT IS POTENTIAL HARM OF THE BBB IS ALTERED? CAN POST BBB DISRUPTION RECOVERY AN ISSUE? >> WE DO IT 24 TIMES OVER A PERIOD OF A YEAR. WE SEEN ABSOLUTELY NO EVIDENCE OF INFLAMMATION OR INJURY AND WE DO CAREFUL COMPREHENSIVE NEUROSITE TESTING BEFORE AND AFTERWARDS. WE FIND IT TO BE VERY SAFE. IT'S INVASIVE, THOUGH. YOU PUT PEOPLE TO SLEEP. THERE IS DISCUSSIONS SOMETIMES THAT THERE ARE SEIZURES. THERE IS NO SEIZURES. IT'S WHEN YOU USE METHOTREXATE AS A KEY DRUG. LIKE IF YOU HAVE PENICILLIN IN THE BLOOD AND YOU OPEN IT, YOU'LL GET FOCAL SEIZURES. SO THERE ARE USUALLY FOCAL SEIZURES WITH NO CONSEQUENCE. BUT WITH OPENING, WE NOW HAVE FOLLOW-UP UP TO NEUROCOMPREHENSIVE NEUROSITE TESTING UP TO 26 YEARS AFTER THEY HAD A COMPLETE RESPONSE AND THEIR FUNCTIONING AS TAX COLORS DETROIT AND ORTHOPAEDIC SURGEON IN -- [ OFF MIC ] NOO. >> IT'S VERY SIMPLE. HOW EASY IS IT TO CROSS DEPARTMENT CELL LINES? YOU HAVE TO GET INVOLVED WITH MULTIPLE DEPARTMENTS. AND CROSSING THOSE LINES IS NOT SO EASY. YOU HAVE TO GET A GROUP OF PEOPLE THAT ARE WILLING TO DO. NOTHING COMPLICATED ABOUT IT. WE DO IT ROUTINELY FOUR DAYS A WEEK IN OREGON AND HAVE FOR -- ANYBODY THAT WANTS TO COME SEE IT IS WELCOME TO COME. >> SO I'M AFRAID PEOPLE MIGHT GET CONFUSED AND I THINK YOU'RE ADVOCATING WE CAN DO THIS FOR DAILY MEDICATION SNOOZE THIS IS INVASIVE PROCEDURE BUT IF YOU HAVE A DEADLY DISEASE, AND YOU HAVE AN AGENT THAT -- LYMPHOMA IS VERY SENSITIVE F YOU CAN GET THE AGENT TO THE TARGET. GLIOBLASTOMA IS NOT. WE ONLY DO BLOOD-BRAIN BARRIER DISRUPTION WHEN WE ARE GOING IF CURE. SO THAT IS TUMORS BECAUSE THEY ARE VERY SENSE TESTIFY ALKYLATING AGENTS OR LYMPHOMA BUT WE USE STANDARD DOSE CHEMOTHERAPY. THE COMPETITION WHICH IS MUCH MORE AVAILABLE IS BONE MARROW TRANSPLANT. AND THEY ONLY INCREASE DRUG DELIVERY 2-3 FOLD. THEIR TREATMENT RELATED MORTALITY WHEN THEY ARE GOING FOR CURE IS 10%. OUR IS LESS THAN 1%. BUT IT IS AVAILABLE. >> IT MEANS THAT FOR BBB OPENING, CLOSING, WE MEANING FDA, HAS A VERY PRICE GUIDELINE HOW TO EMULATE ALL KINDS OF DRUGS NOT ONLY FOR NEUROTOXICITY, WE KNOW THEY HAVE A A GUIDELINE BUT FOR NOVEL DRUGS AND NANOTECHNOLOGICAL FORMULATIONS. THIS IS MY QUESTION BUT I KNOW -- >> WE HAVE AN IND. WE DO EVERYTHING ON PROTOCOL. >> IT'S REGARDING YOUR IND AND YOUR PARTICULAR TREATMENT BUT IN ADDITION QUESTION IS BROADER. SO, HOW BBB REACTS AND WHAT KIND OF TESTS, GUIDELINE WE HAVE TO BE SURE THAT WE DO NOT DAMAGE THE VASCULATURE NORMAL VASCULATURE IN PARTICULAR, FOR PATIENTS WHEN WE OPEN BBB. >> WE DO ALL KINDS OF FANCY DYNAMIC IMAGING, DCE, DSC TO MAKE SURE THERE IS NO EVIDENCE OF DAMAGE. >> BUT I HAD A QUESTION ABOUT THAT, THOUGH. YOU'RE SAYING I CAN OPEN AND CLOSE THE BARRIER AND THEY ARE DESIGN NORMAL BUT THEN WE HAVE TRAUMATIC BRAIN INJURIES WHERE THEY ARE SUB CONCUSSIVE FORCES NOT EVEN REALLY BIG CONCUSSIONS BUT SEEING A LOT OF NEURONAL DAMAGE BY THIS CONTINUE TAP TAP TAPPING WE THINK OPENS AND CLOSES THE BLOOD-BRAIN BARRIER LOTS OF TIMES. SO, I'M HAVING TROUBLE RECONCILING YOUR DATA WITH THAT DATA. >> SO CAN I GO FIRST? I AM HAVING AN EPIPHANY. SO WHAT ED'S WORK SHOWS US IS THAT OPENING THE BLOOD-BRAIN BARRIER MANY TIMES IS NOT THE PATHOLOGY. OPENING OF THE ENDOTHELIAL CELL WHICH MAY BE THE TIGHT JUNCTIONS YOU ARE ALSO THINKING MAYBE DOING TRANSSIGH TO THETIC AS WELL. WE CAN DO THAT TO THE ENDOTHELIAL CELL SO IT DOESN'T GET ANGRY AND START TO CAUSE TROUBLE. SO, THE TOXICITY MUST BE PRETTY MUCH THEN FROM THE THINGS THAT ARE IN THE BLOOD GETTING INTO THE BRAIN, WHICH IS WHAT THE BLOOD-BRAIN BARRIER IS DESIGNED TO KEEP OUT. BECAUSE WHEN YOU OPEN IT, YOU HAVE GOT THE PROFUSION GOING THROUGH SO I ASSUME BLOOD PRODETECTIVES ARE NEVER ENTERING THE BRAIN. SO IT MUST BE THOSE THINGS IN CIRCULATION CAUSING THE TROUBLE OF COURSE IN TBI ARE THERE. ALONG WITH GOD KNOW WHAT IS ELSE IF YOU'RE IN A WAR ENVIRONMENT AND BREATHING IN. >> I THINK THESE ION PARTICLES WOULD BE GREAT FOR THE DEPARTMENT OF DEFENSE BECAUSE THEY CAN -UE CAN INFLAMMATION WITH REPETITIVES HEAD INJURIES BUT YOU CAN'T SEE WITH GAD LIN YUM. AND YOU CAN LOOK AT THE VASCULATURE DOWN TO 15 MICRONS WHICH IS WITH A 3T MEG AND IT'S JUST AN INTERVENOUS INJUNCTION. SO I THINK WITH REGARD TO THE ISSUE OF TRAUMATIC BRAIN INJURY, AS A BIOMARKER LOOKING AT THE PERMEABILITY, BLOOD VOLUME AND FLOW AND LOOKING AT INFLAMMATION FROM CD11BC AND CD68 AND THE CD45 CELLS, THAT WOULD BE A GOOD -- BECAUSE IT HAS A LONG PLASMA HALF-LIFE YOU WON'T SEE IT IF YOU DO THE SCAN RIGHT AWAY BUT IF YOU WAIT 24-48 HOURS IT LEAKS ACROSS MINIMALLY OPEN BLOOD-BRAIN BARRIER AND TARGETS THOSE PHAGOCYTES. >> SO I THINK THE OTHER THING WE SHOULD ALL BE CLEAR ABOUT IS THAT THE PATHOLOGIES THAT OFTEN OPEN THE BLOOD-BRAIN BARRIER DON'T DO IT ONE TIME. THEY OFTEN SET UP CASCADES THOUGHT TO BE NEURAL-IMMUNE BASED SO THE BARRIER WILL OPEN AND CLOSE AND OPEN AND CLOSE AND I DON'T THINK ANYONE FIGURED OUT HOW LONG THAT CAN GO ON. BUT IT SEEMS TO GO ON FOR QUITE SOMETIME. SO AND THE OTHER WIERD THING YOU CAN DAMAGE ONE PART OF THE CENTRAL NERVOUS SYSTEM AND THEN OPENING MAY BE ELSEWHERE. STUDIES SHOW THAT SPINAL CORD INJURIES FROM A LAB THAT SHOW THAT ACTUALLY DAMAGING SPINAL CORD YOU CAN GET OPENING 12 TO 24 HOURS LATER IN THE BRAIN PROPER. SO, AGAIN I THINK THAT UNDERLIES PATHOLOGIES ARE PROBABLY MEDIATED AND REPETITIVE, OR SORRY, ITERATIVE. >> I WANTED TO FOLLOW-UP ON THE DR. BANKS EPIPHANY. THAT WAS ONE OF THE PURPOSES OF THE WORKSHOP IS TO CONSIDER THE BLOOD COMPONENTS NOT JUST THE BRAIN SIDE, BRAIN TO BLOOD. BUT WHAT ARE THE COMPONENTS LEADING TO THE BRAIN. SO THANK YOU FOR GOING THERE. >> SO I THINK THE MOST IMPORTANT ONE IS 6. THE COLLABORATIONS ACROSS FIELDS SIMULATING THERAPEUTIC DEVELOPMENT. THE CONFERENCE IS BEAUTIFUL FOR THAT. THAT IS PEOPLE HERE FROM ALL THESE DIFFERENT AREAS AND EVEN AT THE TABLE HERE. EVERYBODY IS FROM GREAT BACKGROUNDS. I THINK BLOOD-BRAIN BARRIER IS A TOUGH AREA AND IT REALLY WILL ONLY GO FORWARD WHEN THERE IS COLLABORATIONS ACROSS FIELDS. >> SO LONG THESE LINES JULIA MENTIONED SHE HAD A CONVERSATION ABOUT EXOSOMES AND THE EXPERTS COULD WORK WITH NANOTECHNOLOGY EXPERTS. ARE THERE OTHER INSTANCES YOU CAN SORT OF THINK OF AS WELL AS PART OF THIS MEETING. >> IT WAS A SHORT DISCUSSION BUT I FORGOT THE NAME OF THIS WOMAN. BLONDE. [ OFF MIC ] [ OFF MIC ] >> SHE FROM -- I FORGOT, OH, MY GOD. PONYTAIL. OKAY. DOESN'T MATTER. ALSO RUSSIAN. KAREENA. YES. SHE TOLD ME WE HAVE TO FABRICATE EXOSOMES WHICH ARE MUCH SMALLER AND THAT IT WOULD BE NICE TO HAVE LIMITED CIRCLES WITH LIMITED MEMBRANE RECEPTORS OR SOMETHING WHICH WE CAN EVEN SEND TO THE CELLS OR ACCEPT OR TARGET. YES. [ OFF MIC ] >> YES. RUSSIAN. IT'S VERY INTERESTING LIKE LEAVING OUR BLOOD OR ORGANSINGS WHO PRODUCE THEM VERY IMPORTANT IF A TISSUE-SPECIFIC OR CELL-SPECIFIC. SO WE HAD A VERY SHORT CONVERSATION JUST EXCHANGE OUR BUSINESS CARDS BUT AGAIN IT IS TISSUE ENGINEERING BY ENGINEERS. THEY HAVE TO SIT DOWN AND DESIGN. THIS IS WHAT WE DO WHEN WE DESIGN A NEWNANO DRUGS FOR IMAGING OR TREATMENT. BUT VERY, VERY IMPORTANT HOW MUCH WE KNOW ABOUT THIS NEW FIELD EXOSOMES TODAY. AFTER INFLAMMATION, WE CAN DESIGN SOMETHING WHICH WILL BE SUITABLE IN ARTIFICIAL EXOSOMES. THOUGH THIS WAS MY CONVERSATION WITH HER YESTERDAY. >> I THINK MOST OF US -- MOST OF THE PEOPLE THAT DO WORK ON NANOTECHNOLOGY OR DRUG DELIVERY SYSTEM PRIMARILY FOCUS ON BRAIN TUMORS IN GENERAL. SO, MANY OF THE LESSONS WE LEARN WE NEVER HAD THE CHANGES OF APPLYING THEM TO THE OTHER NEURODEGENERATIVE DISEASES. AND THIS MORE AND MORE THERAPEUTIC MOLECULES WILL BECOME AVAILABLE FOR ALZHEIMER'S AND PARKINSON'S T WILL BE GREAT IF WE CAN WORK WITH A DRUG DELIVERY PEOPLE AND -- BECAUSE I THINK THAT THEY DON'T CROSS TALK AS WELL. WE ARE VERY GOOD AT TALKING TO CANCER BIOLOGISTS BUT NOT VERY GOT AT TALKING TO THE NEUROSCIENTISTS AS OF NOW. >> THAT WAS MY POINT. A LOT OF THE TALKS TODAY WERE VERY CANCER HEAVY AND IF YOU LOOK AT DISEASES THAT INVOLVE THE GOODBYE AND AFFECT PEOPLE OTHER THAN NEURODEGENERATIVE DISEASES -- BLOOD-BRAIN BARRIER -- STROKE IS ONE OF THE HUGE, HUGE DILEMMAS VERY LITTLE IMPROVEMENT AND IT'S AN AREA THAT WOULD BE RIPE FOR THIS THING BECAUSE OF BLOOD-BRAIN BARRIER IS DISRUPTED. ANOTHER AREA WHERE COLLABORATION WOULD HELP IS LEVERAGING CLINICAL SAMPLES AND ACCESS TO CLINICAL SIMPLES AND CLINICAL TOOLS WE HAVE WITH THE MODEL SYSTEM THAT IS YOU FOR EXAMPLE DEVELOPED TO STUDY USING PATIENT SAMPLES. FOR EXAMPLE DR. NEUWELT'S TECHNIQUE USING A MICROICALITY TERCOULD BE USED TO SAMPLE BLOOD AT THE SITE OF THE DELIVERY AT THE AGENTS IN THE BLOOD-BRAIN BARRIER SO OPENS UP TO COLLABORATION AND CLINICIAN SCIENTISTS THAT WAY. >> I WOULD LIKE TO DEFENSE NANOTECHNOLOGY SITTING NANOSTUDY SECTION FOR MANY YEARS. WE INTENSIVELY WORK FOR CARDIOVASCULAR DISEASES. WE WORK FOR DIABETES, INFLAMMATION SO NOT ONLY CANCER. CANCER IS HISTORICALLY WAS MAYBE THE FIRST FIELD BUT MANY OTHER AREAS ORGANS AND PATHOLOGICAL CONDITIONS IS COVERED BY NANOTECHNOLOGY NOWADAYS. >> I LIKE TO COMMENT ON ONE OF THE QUESTIONS ABOUT THE BLOOD-BRAIN BARRIER OPENING SCENARIO TO DELIVER NANODRUGS OR SOMETHING. LIKE FOR EXAMPLE DR. LANES WORK DOES PENETRATING NANOPARTICLES THAT COULD BE USED FOR NEURODEGENERATIVE DISEASES TREATMENT. IT CAN BE -- THE DISTRIBUTION OF THE NANOPARTICLE IS ALSO IMPORTANT. SO IF IT IS THE EFFECTIVE IN DISTRIBUTING IT PERHAPS WE DON'T NEED TO OPEN THE ABOUT. BB THAT OFTEN. >> WE DON'T EVEN HAVE TO CROSS THE BLOOD-BRAIN BARRIER WHETHER YOU HAVE CAROTID STENOSIS THERE IS EITHER A SOFT PLAQUE OR A HARD PLAQUE AND A LOT OF THESE NANOPARTICLES ARE TAKEN UP BY INFLAMMATORY CELLS AND YOU CAN DIFFERENTIATE THE VASCULAR SURGEONS IF YOU CAN IDENTIFY THE PATIENTSES WITH THE SOFT PLAQUE ANDURE EXPERT AT THE BOTTOM OF THIS TABLE KNOWS MORE ABOUT THIS THAN WE DO, BUT I MEAN, THAT WOULD CHANGE THE CARE BECAUSE YOU CAN GO FROM AN 80% STENOSIS TO 50% IF THERE WAS A LOT OF INFORMATION BECAUSE THOSE ARE THE PATIENTS THAT EMBOLIES AND GET STROKES. >> AND I THINK WE NEED WHEN TALKING ABOUT CANCER AND BLOOD-BRAIN BARRIER WE NEED TO DIFFERENTIATE TALKING ABOUT METASTATIC CANCERS OF PRIMARY. PRIMARY BLOOD-BRAIN BARRIER IS A LOT CLOSER TO NON CANCER BLOOD-BRAIN BARRIER SO IF WE CAN -- DEALING WITH TRICKS TO GET THINGS ACROSS OTHER THAN DISRUPTION THAT MIGHT BE LESS TRANSLATABLE TO THE NORMAL BLOOD-BRAIN BARRIER BUT TALKING ABOUT TECHNOLOGIES WE ARE DEVELOPING FOR EXOSOAPS OR WHATEVER THAT MIGHT BE MORE IMMEDIATELY TRANSLATABLE THAN IF DEALING WITH METASTATIC TUMORS THAT RECAPITULATE A LOT OF PERIPHERAL VASCULATURE. AND QUIP TON SMITH SHOWED THAT THE BLOOD-BRAIN BARRIER OR THE BLOOD TUMOR BARRIER ALTHOUGH MORE OPEN, IS STILL PRETTY TIGHT COMPARED TO PERIPHERAL TISSUES. >> AND VERY VARIABLE. >> SO I THINK THAT IF THE TRICKS WE LEARN FROM BRAIN CANCER ARE A GREAT PLACE TO BEGIN FOR THE REST OF THE BLOOD-BRAIN BARRIER WE'LL HAVE. >> AND I THINK A LOT OF NIECE USES ARE ORPHAN DISEASES. AND IT'S NOT VERY HARD TO GET AN ORPHAN DRUG DESIGNATION AND THEN ITS RESEARCH IS -- COMPANIES ARE INVOLVED AND YOU GET A TAX DEDUCTION AND A TAX CREDIT. SO I THINK ORPHAN DRUG DESIGNATION IS SOMETHING THIS GROUP SHOULD PAY MORE ATTENTION TO. [ OFF MIC ] >> SO DO YOU HAVE COMMENT WITH REGARDING TO YOUR BLOOD PENETRATING NANOPARTICLE THAT CAN BE POTENTIALLY USED FOR GENETIC DISEASES IN. >> YES. RICH PRICE AND OTHERS ARE INTERESTED IN THAT AND WE HAVE GOT SOME MONEY FROM THE FOCUSED ULTRASOUND FOUNDATION TO LOOK TAT IN PARKINSON'S AND TAKINGS THE APPROACH OF GENE THERAPY PARTIAL I BECAUSE OF THE NEWER PLASMIDS THAT HAVE CLEANED UP AND HAVE A LOT OF CPG SEQUENCES TAKEN OUT, YOU CAN GET VERY LONG LASTING SUSTAINED TRANSGENE EXPRESSION FOR MONTHS FROM A SINGLE ADMINISTRATION. SO, WE STARTED THAT AND THE DATA I SHOWED WAS FROM OUR FIRST STUDY IN RATS. SO IT WAS PROMISING AND NOW WE ARE TRYING TO DO IT IN MORE SOPHISTICATED PARKINSON'S MODELS BUT WE ARE VERY INTERESTED IN NON CANCER APPLICATIONS AND VERY INTERESTED IN CANCER APPLICATIONS. IF THESE THINGS CAN REALLY SURVEILL MOST OF THE BRAIN, THEN YOU SHOULD BE ABLE TO ADMINISTER THEM, HAVE THE GENE ONLY TURNED ON IN CANCER CELLS USING SPECIFIC PROMOTORS AND THEREFORE DELIVER WITH THE GOAL OF DELIVERING IT EVERYWHERE IN THE BRAIN AND ONLY HAVING IT TURNED ON IN THE TUMOR SO VERY INTERESTED IN THAT. SO THESE THINGS, IT'S FUN TOY BEEN TARGETING BECAUSE YOU CAN'T TARGET THE CELLULAR YOU CAN NEVER GET TO. AND VERY FEW THINGS ONCE THEY GET IN ACROSS THE BLOOD-BRAIN BARRIER, DO THEY PENETRATE FURTHER THAN THAT. THEY STAY THERE AND THE BRAIN IS GOD AT STOPPING THINGS FROM SPREADING. EVEN IF YOU'RE A SMALL MOLECULE YOU DON'T GO VERY FAR BECAUSE YOU GO INTO THE FIRST CELL YOU SEE AND THERE ARE DRAINS EVERYWHERE. SO YOU REALLY NEED THESE KIND OF TROJAN HORSE-LIKE THINGS THAT CAN DIFFUSE AROUND AND SLOWLY DISBURSE THEIR MEDICATIONS IN ORDER TO ACHIEVE A UNIFORMED DELIVER TOW SUCH A HIGH PERCENTAGE OF THE CELLS WITHIN A TUMOR OR SPECIFIC REGION OF THE BRAIN. SO THOSE ARE THE AREAS WHERE THIS KIND OF TECHNOLOGY COULD BE VERY USEFUL. ED. >> I HAD A QUICK QUESTION FOR DR. PACIFICCA ABOUT YOUR THREE-DIMENSIONAL MODELS BECAUSE YOU -- PASCA -- SORRY. BECAUSE THREE-DIMENSIONAL MODELS OF HUMAN SEIZ RECALL CORTEX MODELING IS -- CEREBRAL CORTEX MODELING OR WONDER FIGURE THERE IS A POTENTIAL APPLICATION USING MICROFLUIDICS AND INTERFACE BETWEEN SESSION. THE FIRST SESSION OF TODAY AND THE SECOND SESSION. ARE THERE SOME OPPORTUNITIES THERE? >> I KNOW YOU FELT LIKE A FISH OUT OF WATER EARLIER TODAY. BECAUSE YOU HAVE THE ABILITY TO BUILD THREE-DIMENSIONAL MODELS OF CORTICAL DEVELOPMENT AND DO YOU SEE ANY POSSIBILITY ABOUT INTERFACING THAT WITH MICROFLUIDICS? >> ONE POSSIBILITY IS QUITE LARGE STRUCTURES SO DON'T REALLY FIT INTO MICROFLUIDIC DEVICE. BUT WORN OF THE THINGS THAT CAN BE DONE THE CELLS CAN BE ISOLATED AND THEY CAN BE ASSOCIATED IN THE CELLS OR THE SYSTEM CAN BE USED FOR MATURATION AND FOR LONG TERM CULTURE AND THEN AT THE APPROPRIATE TIME ONE CAN EITHER IMMUNOPAN OR EVEN USE FLORESCENT FACTS TO ISOLATE THE CELL TYPES OF INTEREST AND THEN REASSEMBLE THEM WITH MICROFLUIDIC DEVICES. THAT IS ONE POSSIBILITY. >> THANK YOU VERY MUCH. THIS WAS JUST ANOTHER EXAMPLE OF POTENTIAL COLLABORATIVE APPROACHES TO THINKING DIFFERENTLY. >> LET'S THANK OUR PANEL MEMBERS. >> THIS IS REALLY VERY INTERESTING AND TO SEE WHERE THEOS MOT MOBBIC METHOD HAS GONE. I THINK THE KEY THING WITH THES ON MONIC METHOD IS THAT IT TARGETS THE TUMOR BUT THE TUMOR FREQUENTLY IS AVASCULAR AND BUT IT TARGETS THE AREA AROUND THE TUMOR WHERE YOU HAVE THE INFILTRATING TUMOR BUT I WONDER, IT SOUNDS LIKE IT ACTUALLY HAS WORKED FOR ONE SPECIFIC TYPE OF BRAIN TUMOR. ED, HAS IT WORKED WITH ANY OTHER TUMORS OTHER THAN PRIMARY LYMPHOMA? >> IT'S WORKED FOR PRIMITIVE DERMA TUMORS BUT THEY ARE ALSO VERY RARE. IT WORKS FOR -- [ LOW AUDIO ] >> TO WHAT EXTENT HAS IT BEEN ACCEPTED BY OTHER PROGRAMS? >> LOW ODD AWE. >> NOT -- [ LOW AUDIO ] >> NOT WELL? >> IT TAKES A LOT OF PEOPLE TO DO IT. >> IT'S LIFE-SAVING. [ LOW AUDIO ] IT'S INVASIVE AND IT TAKES TIME. THE ONLY OTHER ALTERNATIVE IS IN CNS LYMPHOMA IS PEOPLE ARE DOING BONE MARROW TRANSPLANT AND YOU INCREASE DRUG DELIVERY 2-3 FOLD NOT 100. THE PROBLEM WITH BONE MARROW TRANSPLANT IS IN THE 3-4 RECENT REPORTS THEY DON'T HAVE THE DURATION OF SURVIVAL AND THEY HAVE A 10% DRUG INDUCED TOXICITY AND DEATH. >> AND COULD SOMEBODY COMMENTED ABOUT USING ALREADY ESTABLISHED TRANSPORTERS SUCH AS IMMUNOACID TRANSPORTER FOR DELIVERING AGONISTS TO THE CNS FOR TREATMENT? AMINO ACID TRANSPORTER. HAVE YOU USED THAT FOR DELIVERING DRUGS TO THE CNS? IT'S A HIGH DELIVERY TRANSPORTER FOR THE NEUTRAL AMINO ACID TRANSPORTER. [ OFF MIC ] SORT OF A DELIVERY AND UNFORTUNATELY, MANY OF THOSE EARLY ONES HUGE MOLECULES THAT WENT ON WITH GLUCOSE AND SO, IT WAS SORT OF LIKE ASKING A VOCES WAGON TO PULL THE SPACE SHUTTLE, I THINK. BUT THEN TOM DAVIS LAB REVISITED THAT AND HE FOUND THAT INDEED HE COULD GET MANY SUBSTANCES ACROSS THE BLOOD-BRAIN BARRIER CONSIDERABLY FASTER AND GET THERAPEUTIC AFFECTS HOWEVER, IT WASN'T THROUGH GLUT1. SO IT WAS PROBABLY MORE THAN HE WAS CREATING MOST OF THEM ARE PEPTIDES, SMALL PEPTIDES. SO PROBABLY CREATING SMALL VERSIONS OF GLYCOPROTEINS OR GLYCOPEPTIDES AND MAYBE USING ENDOCYTOSIS AND MECHANISMS. [ OFF MIC ] I THINK GWEN TON TRIED THE AMINO ACID TRANSPORTER BUT -- >> IT DIDN'T WORK. >> NOT VERY WELL. >> IS THERE ANY WAY TO DIRECTLY MODIFY THE JUNCTIONAL PERMEABILITY? >> YES. >> I KNOW THERE ARE TIGHT JUNCTIONS ALL OVER BUT WITH AMINO THERAPY OR A WAY TO DO THAT WITH A HIGH DOSE. >> MOST OF THEM ARE DISEASE STATES. OBVIOUSLY HYPOXIA IN BLOOD-BRAIN BARRIER DISRUPTION. LPS WILL OPEN THE BLOOD-BRAIN BARRIER ALTHOUGH IT IS HARDER TO DO IT THAN YOU MIGHT THINK WHEN YOU WANT TO. WHAT SELLS NEW ABOUT THE BLOOD-BRAIN BARRIER? [ LAUGHS ] SO, THERE IS ALSO SOME, AS WE ARE BEGINNING TO UNDERSTAND THE CELL PHYSIOLOGY, AS I SAID, PKC AND MCP1 ARE MAJOR AREAS IN THE TRANSLOCATION AT THE TIGHT JUNCTIONS OUT TO THEIR SITES OF ACTION THAT THERE IS HOPE THERE THAT ONE COULD MODIFY IF YOU WANTED TO. BUT, IT IS INTERESTING BECAUSE THERE IS A WHOLE OTHER LITERATURE THAT CLOSED THE BLOOD-BRAIN BARRIER AND THIS OTHER LITERATURE TRYING TO OPEN IT AND NO ONE READS EACH OTHER'S LITERATURE. IT'S KIND OF FUN TO CROSS-FERTILIZE. >> AND WE STARTED THIS GLIOBLASTOMA WITH THE MAJOR KILLER WITH A MEDIAN SURVIVAL OF 17 MONTHS AND IT STILL APPEARS TO BE THAT. [ OFF MIC ] >> WHAT? [ OFF MIC ] >> SO I HOPE IN THE NEXT 50 YEARS THAT IT SOUNDS LIKE PEOPLE HAVE IDENTIFIED SPECIFIC TYPES. [ OFF MIC ] >> NOBODY MENTIONED WATER FLOW HERE. EQUIPORE IN. OPENING THE BLOOD-BRAIN BARRIER, EDEMA AND LIMITATIONS OF EDEMA AND RESOLUTION. >> LYMPHATICS ARE A LITTLE BIT OF THAT -- >> WITH LYMPHATICS YOU -- EDEMA REALLY IS A SECONDARY CONSEQUENCE EVEN IF IT IS TRANSIENT AND NON -- DOESN'T HAVE A LONG TERM EFFECT OF TRANSIENT OPENING OF THE BLOOD-BRAIN BARRIER AS I REMEMBERED. >> ABSOLUTELY. [ OFF MIC ] >> THE REASON THEOS MOTTIC METHOD WORKS SO EFFICIENTLY AND INCREASED PERMIABILITY IS FIRST YOU WITHDRAW THE WATER FROM THE BRAIN WITH A HYPERTONIC SOLUTION THROUGH FLOW AND THEN AFTER THE BOLUS OF THE HYPERTONIC SOLUTION PASSES, THE WATER FLOWS BACK THROUGH THE DILATED OPEN TIGHT JUNCTIONS AND THE HIGH PERMEABILITY IS ESSENTIALLY ASSOCIATED NOT WITH DIFFUSION BUT WITH BULK FLOW. >> THANK YOU VERY MUCH FOR YOUR COMMENTS. YOU CAN CONTINUE THE TALK AFTER THE CONCLUSION OF THE MEETING. I WANTED TO TAKER THIS OPPORTUNITY TO THANK EVERYONE FOR PARTICIPATING IN THIS WORKSHOP. SO IT IS DRAWING TO A CLOSE NOW. WE ARE AT 1:30. TIME FLIES BY WHEN YOU'RE HAVING FUN. SO, WITH THAT, I WANTED TO THANK ALL THE SPEAKERS FROM ALL THE SESSIONS AND IT WAS AMAZING HOW THE VARIOUS GROUPS CAME TOGETHER AND SORT OF THOUGHT ABOUT COLLABORATIVE APPROACHES BECAUSE THAT WAS ONE OF THE KEY GOALS OF THE WORKSHOP. IN TERMS OF NEXT STEPS, REALLY WE WILL COAL 8 EVERYTHING THAT WE HEARD AT THE MEETING INTO A WORKSHOP REPORT WHICH WILL BE AVAILABLE IN SOMETIME T WILL TAKE A LITTLE BIT OF TIME TO PUBLISH. AND THEN WE HAVE A LOVELY SCIENCE WRITER WHO IS COVERING THE MEETING AND WE HOPE TO DEVELOP FURTHER COLLABORATIVE APPROACHES AFTER THIS MEETING. SO, THIS IS A GREAT VENUE FOR EVERYONE TO PARTICIPATE. THANK YOU VERY MUCH. [ APPLAUSE ]