>> OKAY, GOOD AFTERNOON AND WELCOME TO CLINICAL CENTER GRAND ROUNDS. OUR FOCUS TODAY IS IMMUNOLOGIC DISORDERS BUT SINCE THE 2 TALKS ARE NOT REALLY THE SAME EXCEPT THAT THEY DEAL WITH BLOOD, WE'LL HAVE THEM SEQUENTIALLY. AND PRESENTING FIRST WILL BE DR. RICHARD CHILDS WHO'S THE ACTING CLINICAL DIRECTOR OF NHLBI AND CHIEF OF THE SECTION OF OF TRANSPLANTATION IMMUNE O THERAPY IN HIS INSTITUTE'S INSTITUTE HEMEATOLOGY BRANCH AND HIS TOPIC IS NOVEL TRANSPLANT APPROACHES FOR PATIENTS WITH TREATMENT-REFRACTORY SEVERE APLASTIC ANEMIA. DR. CHILD SYSTEM A GRADUATE OF GEORGETOWN UNIVERSITY AND EARNED HIS MD AT GEORGE TOWN SCHOOL OF MEDICINE. COMPLETED AN INTERNSHIP IN RESIDENCE SCHEINTERNAL MEDICINE AT THE UNIVERSITY OF FLORIDA MEDICAL CENTER AND HE CAME TO THE NIH IN 1995 FOR FELLOWSHIPS IN MEDICAL ONCOLOGY AND HEMEATOLOGY. HE RECEIVED THE NIH DISTINGUISHED TEACHERS AWARD IN 2009. AND IS A MEMBER OF A NUMBER OF SOCIETIES INCLUDES THE AMERICAN SOCIETY OF CLINICAL INVESTIGATION AND THE AMERICAN ASSOCIATION FOR CHANCER RESEARCH AND THE INTERNATIONAL SOCIETY FOR CELLULAR THERAPY. HIS RESEARCH HAS FOCUSED ON TUMOR IMMUNOLOGY AND ALOE GENERATED AIC IMMUNO THERAPY AND ACTIVITIES AND PROJECTSIC TO TREAT NONMALIGNANT HEMEATOLOGICAL DISORDERS, MALIGNANCYS AND SOLID TUMORS. HE WAS THE FIRST TO ESTABLISH A EXISTENCE OF GRAPH VERSES TOLL-LIKE SOLID TUMOR EFFECT BY MEDIATED DONOR T-CELLS THAT COULD CURE PATIENTS WITH METASTATIC RENAL CELL CARCINOMAS. THIS SEMINOLE OBSERVATION DEFINED A NEW THERAPEUTIC ALLOCATION FOR IMMUNO GENERIC THERAPY. IT'S A PLEASURE TO WELCOME RICH TO THE TOADIUM. --PODIUM. [ APPLAUSE ] >> THANK YOU JOHN, IT'S A PLEASURE TO BE HERE. GOOD AFTERNOON. I HAVE NO FINANCIAL DISCLOSURES. SO THE OBJECT JUDGIVES OF MY TALK WILL BE 3 FOLD: FIRST, TO CHARACTERIZE THE NOVEL TRANSPLANT APPROACH DESIGNED TO PROVENT GOVERNMENT REJECTION IN PATIENTS WITH ABRAOF THETIC ANEMIA THAT HAVE FAILED TO RESPOND TO IMMUNO SUPPRESSIVE THERAPY AND SECOND TO TRANSPLANT APPROACH THAT USES UMBILICAL CORD BLOOD TO IMPROVE INGRAFTMENT AND SURVIVAL PATIENTS WITH TREATMENT REFRACTORY APLASTIC ANEME SOMEWHAT AND FINALLY TO IDENTIFY PATTERNS THAT CONSIDER GURRING INGRAFTMENT THAT OCCUR FOLLOWING A TRANSPLANT APPROACH THAT USES SEPARATE SOURCES OF DONOR STOLE CELLS. SO APLASTIC ANEME IS A RARE DISORDER CHARACTERIZED BY RECURSORS IN THE BONE MARROW AND MOST CASES IT CAN BE THOUGHT OF AS AN AUTOIMMUNE DISORDER, MEDIATED BY PATIENT T-CELLS WHICH ATTACK AND KILL HEMATOPOIETIC STEM CELLS IN THE BONE MARROW LEADING TO APLASIA. SEVERE APLASTIC ANEMIA RESULTS IN CYTOPENIA, PATIENT K'S HAVE THROMBOW SIGHT APENIA, ANEMIA THEY CAN HAVE VARYING DEGREES OF NUTRIPENIA, AND IF LEFT UNTREATED IT'S USUALLY FATAL. THE 2 CONVENTIONAL AND HIGHLY EFFECTIVE TREATMENTS FOR SEVERE APLASTIC ANEMIA, INCLUDE ALOE GENERIC BONE MARROW TRANSPLANTATION AND INUME O SUPPRESSIVE THERAPY. THIS IS FOR PATIENTS UNDER THE AGE OF 40 WHO HAVE AN HLA IDENTICAL SIBLING. THE RESULT OF TRANSPLANTATION FOR SEVERE APLASTIC ANEMIA USING AN HLA ADENTICAL 8 HOURS SIGNALING HAVE IMPROVED SIGNIFICANTLY OVER THE PAST 30 YEARS OVER THE MOST CONVENTIONAL SERIES REPORTS SURVIVAL RATES THAT APPROACH 80% OR BETTER WHEN AN ALOE GENIC TRANSPLANT IS USED AS UPFRONT THERAPY. IMMUNO SUPPRESSIVE THERAPY AND ACTIVITIES AND PROJECTS UTILIZING HORSE ATG AND CYCLOSPORIN REPRESENTS PATIENT WHO IS ARE OVERTHE AGE OF 40, GIVEN THE HIGHER RISK OF TRANSPLANT RELATED MORTALITY IN THE OLDER AGE GROUP. OR FOR PATIENTS UNDER THE AGE OF 40 WHO LACK A SIBLING OR URPD GO NOT TO UNDERGO A TRANSPLANT FOR PERSONAL PREFERENCE. NEAL YOUNG PIONEERED THE USE OF IMMUNO SUPPRESSIVE THERAPY FOR ALMOST 30 YEARS NOW AND RESPONSE RATES WITH CONVENTIONAL THERAPY ARE TYPICALLY IN THE RANGE OF 60-70%. FOR PATIENT WHO IS RESPOND TO IMMUNO SUPPRESSIVE THERAPY PROGNOSEIS IS EXCELLENT WITH LONG-TERM SER VIVAL GREATER THAN 90% IN 5 YEARS. IN CONTRAST FOR THE 35 PERCENT OR SO, PATIENTS THAT FAIL TO RESPOND TO THE THERAPY, PROGNOSIS IS SIGNIFICANTLY WORSE SO AS YOU CAN SEE FROM THESE DATA FROM THE HEMEATOLOGY BRANCH, PATIENTS WHO FAIL IMMUNO SUPPRESSIVE THERAPY HAVE AN IMPROVEMENT IN THE SURVIVE OVER THE LAST 20 YEARS THIS IS MOSTLY BECAUSE OF OUR ABILITY TO SUPPORT NEWT ROW PENIC MUTATIONS WITH EFFECTIVE ANTIMICROBIAL AND ANTIFUNGAL THERAPY BUT STILL ALMOST 50 OF THE PATIENTS WILL END UP DYING FROM COMPLICATIONS RELATED TO THEIR DISEASE. ONLY A MINORITY OF PATIENTS THAT FAIL IMMUNO SUPPRESSIVE THERAPY WILL RESPOND TO SECOND LINE AGENTS. ALOE GENERATED AIC BONE TRANSPLANTATION FROM AN HLA DONOR CAN KILL PATIENTS IF THEY FAIL TO RESPOND TO THE THERAPY, HOWEVER SINCE THEY HAVE A DELAY FROM THE DIAGNOSIS TO THE TRANSPLANT ARE HEAVILY TRANSFUSED AND IRON OVERLOADED AND HAVE A HIGHER INCIDENCE OF BEING HLA ALOE IMMUNIZED AS A CONSEQUENCE OF HEAVY TRANSFUSION BURDEN. THESE PATIENTS HAVE A SIGNIFICANTLY HIRE RISK OF GRAFT REJECTION, WHICH IS IN THE 10 TO 20% OF CASES WHICH REDUCES PROBABILITY OF LONG-TERM SURVIVAL. AND IN THE SETTING MANY GROUPS REPORTED MORTALITY RATES, THE RANGE OF 20-30%. SO SINCE NEAL REFERS OUR GROUP, A NUMBER OF PATIENTS THAT HAVE FAILED IMMUNOSUPPRESSION, WE WERE INTERESTED IN DESIGNING A TRANSPLANT APPROACH THAT WOULD BE TAYLORED SPECIFICALLY TO THESE HIGH RISK PATIENTS THAT HAVE A PROCLIVITY TO REJECT BONE MARROW TRANSPLANTS. SO WE SOUGHT TO DEVELOP A REGIMEN THAT WOULD TEST THE HYPOTHESIS WITH THE RISK OF GRAFT FAILURE COULD BE REDUCED BY THE FOLLOWING 2 MANEUVERS, THE FIRST WAS ADDING FLU VERA--FLUDARABINE, TO PSYCHE LOW SPORRIC ATG, NOW SINCE THEY'RE REJECTING THE GRAFT, THE MORE YOU CAN GIVE THEM, THE BETTER TO PREVENT THIS COMPLICATION. SECONDLY WE WANT TO USE A TRANSPLANT APPROACH THAT USED PERIPHERAL BLOOD STEM CELLS INSTEAD OF BONE MARROW AS A GRAFT SOURCE BECAUSE THESE CONDITION DANE PROGENITORS WHICH PREVENT GRAFT REJECTION THIS THIS POPULATION. SO THIS IS THE PROTOCOL THAT WE UTILIZED THAT WAS OPEN UNTIL ABOUT A YEAR AGO. PATIENTS WOULD BE CONDITIONS WITH CYCLOPHOS MID AND HORSE ATG, THE CYCLOPHOS MID DOSE WAS A TOTAL OF 120-MILLIGRAMS WHICH WAS LOWER THAN THE CONVENTIONAL 200-MILLIGRAM PER KILOGRAM DOSE, IT WAS GIVEN 25-MILLIGRAMS PER METERED SQUARED OVER 5 DAYS, WE GET RELATIVELY HIGH DOSES OF ATG, 40-MILLIGRAMS PERKILOGRAM PER DAY, AND PATIENT JUST RECEIVE IMMUNOSUPPRESSION ALONE. PATIENTS RECEIVE CYCLOSPORIN AS GBHD PROPHYLAXIS AND AN ALOE GRAPH WAS INFUSED ON DAY INTERIOR AND IT WAS GCSF FROM EITHER HLA IDENTICAL OR SINGLE HLA ANTIGEN MISMATCHED RELATIVE AND THE TARGET DOSE OF CD34 POSITIVE PROGENITOR CELLS WAS 5 MILLION CELLS PERKILOGRAM. AND THIS REPRESENTS A 2-4 FOLD HIGHER CD34 STEM CELL DOSE THAN YOU WOULD GET WITH A BONE MARROW TRANSPLANT. SO, TO DATE 56 PATIENTS WERE FRANZ PLANTED, THE MAR JORRITY HAD SEVERE APLASTIC ANEMIA, THEY WERE ASSOCIATE WIDE MARROW APLASIA. THESE PATIENTS HAD MULTIPLE RISK FACTORS THAT PLACE THEM AT RISK FOR GRAFT FAILURE INCLUDING HEAVY A HEAVY TRANSFUSION BURDEN AND MEDIAN TRANSPLANT ELEVATED OVER 2000. ALL THESE SEVERE APLASTIC ANEMIA PATIENTS HAD FAILED, PRIOR TO IMMUNOSUPPRESSION WITH ATG BECAUSE THESE PATIENTS WERE HEAVILY TRANSFUSED MORE THAN 40% OF THEM HAD DETECTABLE HLA ANTIBODIES IN THE CIRCULATION PRIOR TO UNDER GOING TRANSPLANTATION. PERIPHERAL BLOOD STEM CELL THAT WE USE WIDE THE TRANSPLANT WAS RICH IN CD34 POSITIVE PROGENITOR CELLS CONTAINING 3 TIMES MORE CDPOSITIVE CELLS THAN YOU WOULD TYPICALLY SEE IN A BONE MARROW TRANSPLANT. AND THE FOLLOW UP DATA ON THIS COHORT IS NOW APPROACHING 5 YEARS. SO TRANSPLANT RELATED MORTALITY HAS BEEN LOW, AND SURVIVAL AS BEEN EXCELLENT WITH THIS APPROACH OVERALL 10% OF PATIENTS DIED FRIDAY TRANSPLANT RELATED MORTALITY AND MOST OF THESE OCCURRED AS A CONSEQUENCE OF GRAFT VERSES HOST DISEASE, SURVIVAL IS EXCELLENT OVER KETCH% WITH ALMOST A 5 YEAR FOLLOW UP, AND WHAT REALLY HAS BEEN MOST REMARKABLE IS THAT 100% OF THESE PATIENTS HAD SUSTAINED INGRAFTMENT OF DONEAR STEM CELLS. WE HAD NO ACUTE OR LATE GRAFT FAILURES AND THAT'S QUITE REMARKABLE IF YOU CONSIDER THAT THIS TYPE OF POPULATION GETTING CONVENTIONAL BONE MARROW TRANSPLANT WOULD BE EXPECTED TO HAVE GRAFT FAILURE RATES IN THE RANGE OF 10 TO 25%. SO THE TRANSPLANT DID ITS JOB FOR GETTING THE GRAFT IN. THE MAJOR SIDE EFFECT OF THIS APPROACH AND THE CONSEQUENCE OF ESTABLISHING RAPID DONOR GRAFTMENT THE IS THAT WE SAW VERY HIGH INCIDENCE OF CHRONIC GRAFT VERSES HOST DISEASE. 72% OF PATIENTS DEVELOPED CHRONIC GRAFT HOST DISEASE, THIS IS HIGHER THAN WHAT YOU WOULD SEE WITH A CONVENTIONAL TRANSPLANT WITH THE NUMBERS BEING AROUND 50%. OUR CHIMERISM STUDIES REVEAL A NOVEL FINDING AND THAT WAS WHEN WE LOOKED AT T-CELL INGRAFTMENT, WE FOUND THAT PATIENTS HA HAD RAPID DONOR T-CELL INGRAFTMENT WHICH WAS DEFINED AS DONOR T-CELLS BEING MORE THAN OR EQUAL TO 95% OF T-CELL CHIMERISM BY DAY 30 OR EARLIER WAS SIGNIFICANTLY MORE LIKELY TO DEVELOP CHRONIC GRAFT VERSES HOST DISEASE. ALL CHRONIC GRAFT VERSES HOST DISEASE AS WELL AS EXTENSIVE CHRONIC GRAFT VERSES HOST DISEASE WHICH CONSIDERED AT A 5 FOLD HIGHER INCIDENCE IN THE RAPID INGRAFTMENT IN COMPARED WITH PATIENTS WITH DELAYED INGRAFTMENT AND IN A MULTIVARIANT ANALYSIS, RAPID CELL ANALYSIS REMAINED A PREDICTIVE VARIABLE FOR AN INCREASED RISK OF CHRONIC GBHD. REMARKABLY AS A CONSEQUENCE OF A HIGHER INCIDENCE OF CHRONIC GRAFT HOST DISEASE, EXTENSIVE LIE CHRONIC GRAFT VERSES HOST DISEASE, PATIENT WHO IS HAD DONEAR INGRAFTMENT HAD A MEDIAN 3 YEARS LONGER THAN PATIENTS THAT HAD MORE DELAYED DONOR T-CELL INGRAFTMENT. SO THE CONCLUSION FOR THE FIRST PART OF MY TALK IS THAT FLEW DERRA BINE TRANSPLANTATION USING GCSF T-CELLS WHICH APPEARS TO PREVENT RAPID DONOR T-CELL INGRAFTMENT IS ASSOCIATED WITH POOR TRANSPLANT OUTCOME. IT APPEARS TO BE ASSOCIATE WIDE AN INCREASE OF RISK OF GVHD, AND IT WAS AN APPROACH WITH A HIGH RISK POP EULOGISTS, A HIGH INCIDENCE OF GVHD COMPLICATES THAT TYPE OF STRATEGY OUR DATA WOULD SUGGEST THAT OUR HIGHER TRANSPLANTED T-CELL DOSES THAT COME WITH MOBILIZED GRAFTS WITH TRANSPLANTS T-CELLS THAT HAVE UNDERGONE POLARIZATION CHANGE RELATED TO G-MOBILIZATION, ARE FACTORS THAT MAY HAVE INFLUENCED THIS INCREASE INCIDENCE OF CHRONIC GRAFT VERSES HOST DISEASE. SO BASED ON THESE DATA WE REEBTLY--RECENTLY INIT INITIATED A NEW PROTOCOL FOR PATIENTS WITH SEVERE APLASTIC ANEMIA THAT'S DESIGNED TO TRY TO SLOW THE RATE OF DONOR T-CELL INGRAFTMENT IN AN EFFORT TO AVOID CHRONIC GRAFT VERSES HOST DISEASE. SO FOR THIS PROTOCOL WHICH OPENED ABOUT A YEAR AND HALF AGO, PATIENTS RECEIVED THE SAME CONDITIONING REGIMEN OF CYCLOPHOS MID, FLU DERBINE AND EQUITE ATG, THEY RECEIVE CYCLOSPORIN AND MEX O TREKSATE AS A PROPHYLAXIS AND THEN ON DAY 0, THESE PATIENTS RECEIVE AN ALLOGRAFT THAT'S BEEN ENGINEERED TO CONTAIN CD34, POSITIVE CELLS THAT HAVE BEEN SELECTED IN T-CELL DEPLETED FOLLOWING GCSF MOBILIZATION, COMBINE WIDE 2 TIMES 10 TO THE SEVENTH, NONMOBILIZED T-CELLS THAT ARE COLLECTED PRIOR TO GCSF MOBILIZATION. SO THE IDEA BEHIND THIS PROTOCOL IS TO TRY TO INFUSE A GRAFT THAT CONTAINS A SIGNIFICANTLY HIGHER NUMBER OF STEM CELLS THAN WHAT YOU WOULD FIND IN A BONE MARROW TRANSPLANT BUT CONTAINS A LOWER NUMBER OF T-CELLS THAT WOULD COME IN A PERIPHERAL BLOOD STEM CELL AND 20 FOLD NUMBER AND THESE ARE THE NUMBERS WE WOULD TRANSPLANT WITH A CONVENTIONAL BONE MARROW TRANSPLANTS AND THEN THESE HAVING NOT BEEN EXPOSED WILL NOT UNDERGO THAT TH2 POLARIZATION THAT MAY LEAD TO CHRONIC GRAFT OR HOST DISEASE. SO THE PRIMARY END POINT WILL BE THE INCIDENCE OF CHRONIC GRAFT VERSES HOST DISEASE, AT 1 YEAR, WE'RE TESTING THE HYPOTHESIS THAT THIS ENGINEERED ALLO GRAFT WILL RESULT IN A 50% REDUCTION IN THE RATE OF CHRONIC GBHD COMPARED TO OUR HISTORICAL CONTROLS WHILE MAINTAINING AN INGRAFTMENT RATE OF 90%. SO WE'VE ENROLLED 6 PATIENTS ON TO THIS STUDY TO DATE AND I'M HAPPY TO REPORT ALL 6 HAVE INGRAFTED AND NONE OF THOSE PATIENTS HAVE DEVELOPED CHRONIC GBHD AT THIS POINT. WE'VE ALSO SEEN THAT T-CELL CHIMERISM IS COME NOTHING SLOWER THAN WHAT WE SAW WITH THE OTHER TRANSPLANT REGIMEN, FOLLOW UP IS OBVIOUSLY SHORT ON THIS APPROACH, BUT IT LOOKS LIKE WE'RE OFF TO A GOOD START. SO WE'VE ALSO BEEN INTERESTED IN EXPLORING ALTERNATIVE GRAFT SOURCES FOR PATIENTS WHO LACK HLA MATCHED DONOR, EITHER A SIB DONOR OR A MATCHED UNRELATED DONOR. SO ABOUT 40% OF PATIENTS THAT HAVE ANEMIA, THAT'S REFRACTORY TO IMMUNOSUPPRESSION PRECIOUSES ITCH THERAPY WILL NOT HAVE AN HLAMATCHING DONOR AVAILABLE TO SERVE FOR TRANSPLANT. THESE PATIENTS ARE POTENTIALLY CANDIDATES FOR AN HLA MISMATCHED TRANSPLANT USING A NUMBER OF DIFFERENT GRAFT SOURCES INCLUDING UMBILLICICAL CORD BLOOD THAT'S TIP CALIFORNIALY MISMATCHED OR HAPPEN LO IDENTICAL OR HALF MATCHED STEM CELLS FROM A RELATIVE. UMBILICAL CORD TRANSPLANTATION HAS BEEN EXPLORED NOW FOR 20 YEARS AS AN ALTERNATIVE GRAFT SOURCE FOR PATIENTS WITH HEMOTO LOGICAL MALIGNANCIES THAT LACK AN HLA-MATCHED DONOR WHO REQUIRE A TRANSPLANT. THESE HAVE DESPITE BEING HLA-MISMATCHED HAVE SLOWER INCIDENCE OF GRAFT VORCES HOST DISEASE THAN WHAT YOU WOULD FIND A PERIPHERAL BLOOD STEM CELL TRANSPLANT THAT'S FULLY MATCHED. THAT THE POOR BLOOD. THE MAJOR DISADVANTAGE IS THAT THEY CONTAIN A RELATIVELY SMALL NUMBER OF HEMATOPOIETIC STEM CELLS THAN WHAT YOU GET FOR THE STEM CELLS, NUTRIFILL RECOVERY IS SIGNATURES THIEF CANTILY DELAYED, NUTRIFILL RECOVERY CAN OCCUR ANYWHERE FROM 25 TO 40 DAYS AFTER TRANSPLANTATION, SO THESE PATIENTS ARE SITTING AROUND AFTER THE CONDITIONING REGIMEN NEAR A LONG TIME, AND THEY'RE SUSEPTIBLE TO INFECTION. THEY ALSO HAVE DELAYS IN PLATELET INGRAFTMENT AND INCREASED RISK FOR GRAFT FAILURE. MOST STUDIES FOR PATIENTS WITH HEMATO LOGICAL STUDIES HAVE GRAFT RATES IN THE RANGE OF 20-15%. SO PUBLISHED DATA SO FAR HAVE SHOWN THAT THE OUTCOME FOR SEVERE APLASTIC ANEMIA HAS BEEN DISAPPOINTING. DAT FRA THE NEW YORK PLOD CENTER HAS REPORTED TRANSPLANT RELATED MORTALITY RATES OF 60%, WITH MOST OF THESE PATIENTS DYING AS A CONSEQUENCE OF HAVING PROLONGED NUTRIPENIA AFTER THE CONDITIONING REGIMEN, COMING TO INFECTION, OR REJECTING THE TRANSPLANT. LANCHEST SERIES THAT'S BEEN PUBLISHED TO DATE WAS PUBLISHED FROM THE EUROPEAN BONE MARROW TRANSPLANT CONSORTIUM. 71 PATIENTS WITH APLASTIC ANEMIA RECEIVED A SINGLE CORE TRANSPLANT OR DUAL CORE TRANSPLANT OVER ALL SURVIVAL IN THAT COHORT WAS 3 YEARS IT WAS ONLY 38% AND WHEN THEY LOOKED AT SURVIVE AT BASED ON THE TOTAL NUMBER OF CELLS INFUSED THEY FOUND A HIGHER NUMBER OF T& C SAYS HAD BETTER SURVIVAL AROUND 48%. BUT PATIENTS THAT GOT LESS THAN 39 MILLION T& Cs PERKILOGRAM HAD A ABYSS MALSURVIVAL OF ONLY 18% AND WHAT'S IMPORTANT IS THAT WHEN WE DO CORE TRANSPLANTS IN ADULTS BECAUSE THE CELL NUMBERS ARE BASED ON THE WAY OF THE--WEIGHT OF THE RECIPIENT, IT'S TYPICAL TO HAVE LOW CELL NUMBERS LIKE THIS WHEN TRANSPLANTING AN ADULT PATIENT. SO BEING ABLE TO ACHIEVE THESE NUMBERS EVEN THOUGH THESE ARE NOT PARTICULARLY IMPRESSIVE, IS NOTGpG x SO WHEN YOU THINK THE DATA CAN'T GET WORSE, THIS ARTICLE COMES ALONG FROM CHIN A18 PATIENTS WITH SEVERE APLASTIC ANEMIA THAT DID NOT HAVE AN HLA MATCH DONOR THAT WENT TO CORE BLOOD TRANSPLANTATION BEFORE IMMUNO SUPPRESSIVE THERAPY AND THEY THOUGHT THAT MAYBE THE REASON THAT CORE BLOOD TRANSPLANTATION WAS SO BAD WAS THAT THESE PATIENTS WERE FAILING IMMUNOSUPPRESSION, THEY WERE BEING TRANSFUSED AND BECOME ALOE IMMUNIZED AND IF THEY WON'T TO CORE TRANSPLANT EARLIER, THE RESULTS WOULD BE BETTER SO 18 PATIENTS TRANSPLANTED HERE, REMARKABLY 65% OF THE PATIENTS NEVER HAD DETECTABLE CORE BLOOD INGRAFTMENT AND ONLY 1 OF THEM HAD SUSTAINED CORE BLOOD INGRAFTMENT SO I THINK THE STUDY HIGHLIGHTS HOW DIFFICULT IT CAN BE TO GET A CORE TRANSLABORATORY TO TAKE IN THESE PATIENTS THAT HAVE AN OVERACTIVE CELLULAR IMMUNE SYSTEM. SO TO OVERCOME THESE POOR RESULTS WE DECIDE TO INVESTIGATOR A TRANSPLANT APPROACH THAT COMBINE A SINGLE CORE BLOOD UNIT WITH ENRICHED STEM CELLS FROM A LAP LO IDENTICAL RELATIVE AND OUR INTEREST WAS SPARKED BY A PAPER THAT WAS PUBLISHED BY MANY FERNANDEZ FROM SPAIN WHO SHOWED THAT THESE DUAL CORD OR STEM CELL SOURCE TRANSPLANTS WERE ASSOCIATED WITH MORE RAPID NUTRIFILL INGRAFTMENT WITH MEDIAN NUTRIFILLS COMPARED TO 11 DAYS OVER 30 DAYS WITH THE UNIT AND IMPORTANTLY THAT OVER ALL INGRAFTMENT RATES WITH THESE, NONE OF THESE PATIENTS HAD APLASTIC ANEMIA, THAT INGRAFTMENT RATES WERE THE QUITE GOOD. SO, OUR GOAL WAS TO TEST 2 HYPOTHESIS, THE FIRST THAT CO TRANSPLANTATION OF A SINGLE CHORD UNIT COMBINE WIDE ENRICHED APPLICATIONS LO IDENTICAL STEM CELLS WOULD SHOTTEN A PERIOD OF NUTRIFILL RECOVER CHE IS QUITE IMPORTANT BECAUSE MANY OF THEM COME INTO THE TRANSPLANT WITH OPPORTUNISTIC INFECTIONS INCLUDING INVASIVE FUNGAL INFECTIONS WHICH ARE DIFFICULT TO HOLD AND CHECK WITH THESE PATIENTS ARE NUTRIPENIC. SO WE HOPE NUTRIFILL INGRAFTMENT WOULD BE EXPEDITED BECAUSE WE'RE TRANSPLANTING A HIGHER NUMBER OF CELLS FROM THE DONOR AND THAT MIGHT DECREASE THE RISK OF OPPORTUNISTIC INFECTION AND MORBIDITY IN THAT INFECTION AND SECONDLY THE HAPPEN LO IDENTICAL SEMESTER STELLS COULD SERVE AS A BACK UP STEM CELL SOURCE IN THE CORD UNIT IN THE EFFECT THE CORD UNIT FAILED TO INGRAFT AND 1 OF THE PREDICTABLE THINGS ABOUT TRANSPLANTATION IS THAT THE QUALITY OF THE CORD SOURCE CAN VARY FROM CENTER TO CENTER THAT COLLECTS THEM AND SOMETIMES THINGS WILL FOLLOW A CORD UNIT DOWN AFTER YOU CONDITION THE PATIENT AND IT WILL TURN OUT THAT THE VIABILITY IS QUITE POOR AND THERE'S NO TURNING BACK AT THAT POINT. THE CORE UNIT IS THE CARD YOU WERE DEALT AND YOU HAVE TO DEAL WITH IT AND THESE PATIENTS OFTEN WILL NOT INGRAFT AND THE CHANCES OF SALVAGING THEM WITH A SECOND TRANSPLANT IS QUITE LOW. SO THIS IS A CONDITIONING REGIMEN THAT WE UTILIZE. THE SAME PLATFORM OF PSYCHOPHOS FOCUSED ON MID AND TOTAL EQUINE ATG AS PRIOR REGIMEN, WE ADDED 1 SHOT OF LOW DOSE TOTAL BODY RADIATION JUST TO TRY TO IMMUNO SUPPRESS THE PATIENT A BIT MORE KNOWING THAT GRAFT REJECTION WAS A MAJOR ISSUE WITH THIS TYPE OF APPROACH. WE USE TACK ROUGH ATOM LIMITS AND MICROPHILIC ACID AS GHD PROPHYLAXIS AND ON DAY 0, A SINGLE CORE BLOOD UNIT WAS TRANSPLANTED IN COMBINATION WITH A GCSF MOBILIZED CD34 SELECTED HAPPEN LO IDENTICAL STEM CELLS. SO PATIENTS THAT WERE ELIGIBLE FOR THESE APPROACH WERE BETWEEN THE AGES OF 6 AND 55, THEY HAD TO BE PROFOUNDLY NUTRIPENIC WITH LESS THAN 500 CELLS THAT WIRE THAT BECAUSE WE KNEW WE WERE GOING INTO AN ARENA WHERE MORTALITY MAY HAVE BEEN AS HIGH AS 60% OR HIGHER AND THESE ARE THE PATIENTS THAT ARE REALLY SUSCEPTIBLE TO OPPORTUNISTIC INFECTIONS AND DEATH. THEY HAD TO FAIL AT LEAST 2 PRIOR STANDARD IMUME O SUPPRESSIVE THERAPIES, THEY HAVE NO AVAILABLE HLA MATCH DONOR OR NONRELATED DONOR AND THEN IMPORTANTLY THEY HAD THE AVAILABILITY OF AT LEAST 1, 4 OUT OF 6 OR BETTER MATCH CORE BLOOD UNIT WITH A MINIMUM OF T& T C, WITH TNC, AND THAT'S A LOW NUMBER AND THEY HAD AVAILABLE H LA HAPPEN LO IDENTICAL DONOR. FOR THE STEM CELL SOURCE THE GRAFT WAS MOBLINGIZED FROM A RELATIVE USING GCSF, AND THEY UNDERWENT THE SELECTION USING MILTENYI, AND THOSE ARE CD34S PER KILOAND LESS THAN T-CELL DOSE, LESS THAN 5 TIMES 10 TO THE THIRD AND AT THE RISK OF GETTING GVHD WITH THE CELLS, EVEN THE HAPPEN LO IDENTICAL IS LOW. SO TO DATE, THEY'VE BEEN TRANSPLANT OFFICE OF DIVERSITY THIS REGIMEN, INCLUDING APLASTIC ANEMIA 2 PATIENT THAT HAD APLASTIC ANEMIA, THAT EVOLVED AND ALL THESE PATIENTS WERE ROUGH ATOM FOUNDLY NEWT ROUGH ATOM PENIC AND THE MEDIAN A& C IS 111. AND PATIENTS HAD FAILED A MEDIAN OF 3 PRIOR IMMUNO SUPPRESSIVE REGIMENS IN THESE PATIENTS WERE HEAVILY TRANSFUSED RECEIVED A MEDIAN PLATELET TRANSFUSIONS, AND RED CELL TRANSFUSIONS, AS A CONSEQUENCE OF THESE TRANSFUSIONS, MORE THAN 50% OF THESE PATIENTS HAD DETECTABLE HLA ALLO ANTIBODIES IN THEIR CIRCULATION AND THIS BECAME A REAL CHALLENGE. JENNIFER WILDER WHO PICKED THE CORD UNITS FOR US, WOULD OFTEN HAVE TO SPEND SIGNIFICANT TIME PICKING CORD UNITS THAT THE PATIENT DID NOT HAVE AN HLA ANTIBODY TO A MISMATCHED ALLELE ON THAT DONEAR. --DONOR. WITH REGUARD TO CHARACTERISTICS, THE TNC STUDY WAS ASSOCIATED WITH A VERY HIGH MORTALITY. 82% MORTALITY WITH THOSE KIND OF CELL NUMBERS. MAJORITY OF THESE PATIENTS 83% RECEIVED A CORE BLOOD TRANSPLANT THAT WAS MISMATCHED. IT WAS MATCHED ONLY 4 OUT OF 6 HLA ANTIGENS AND THE MAJORITY OF THE HAPPEN LO DONORS TURNED OUT TO BE PARENTS. SO REMARKABLY ALL 12 OF THESE PATIENTS HAVE INGRAFTED THE MEDIAN TIME TO NUTRIFILL RECOVERY HAS BEEN VERY SHORT, ONLY 10 DAYS. THE MEDIAN TIME TO PLATELET RECOVERY HAS BEEN 10 DAYS THAT'S SHORTER THAN A TRANSPLANT AND MEDIAN TIME IS 4 OR 5 DAYS AND THE DATES OF CORE BLOOD AAND I C OVER 500 HAS BEEN 35 DAYS AND THATY QUITE TYPICAL FOR A CORE BLOOD TRANSPLANT AND WE WERE ABLE TO CALCULATE THAT CORD A AND C OVER 500 BY MULTIPLYING THE PERCENTAGE OF MYELOID CHIMERS AND FROM THE CORD TIMES THE TOTAL AMC BUT HAVING NUTRIFILLS COME BACK, MORE THAN 20 DAYS EARLIER IS WHAT WE BELIEVE AN IMPORTANT FACTOR FOR OUR OUTCOME WHICH I'LL SHOW NUCLEOTIDES A SECOND. ONLY A MINORITY OF PATIENTS DEVELOP GBHD. THREE OF THE 12 WHICH WAS MILD IN MOST CASES, NO PATIENTS DEVELOP GRADE 4 BO GBHD WE HAD NO CASES OF STEROID REFRACTORY, AND I MEAN IT'S REALLY REMARKABLE WHEN WE CONSIDER THAT WHEN WE'RE DOING PERIPHERAL STEM CELLS FROM AN HLA THAT WE SAW A SIGNIFICANTLY HIGHER INCIDENCE OF BOTH SEVERE AND STEROID FRACTURED GBHD. FIVE PATIENTS HAVE DEVELOPED BUT IN MOST CASES CHRONIC GBHD HAS BEEN MILD AND EASY TO TREAT WITH LOW DOSES OF CORTICO STEROID THERAPY. SO THE MAJOR MORBIDITY WE'VE SEEN WITH THIS APPROACH HAS BEEN VIRAL REACTIVATION. NINE PATIENTS REACTED CMB VIRUS, UNFORTUNATELY 1 OF THOSE PATIENTS DEVELOPED NEWELONNITE AND I GUESS THE CMB BECAME RESISTANT AND THE PATIENT DIED FROM NEWELLENNITEIS AND WE HAVE 3 PATIENTS THAT DEVELOPED ADENOVIRATION, AND 1 THAT DEVELOPED DEC AND 1 THAT GOT PNEUMONIA THAT ENDED UP SURVIVING BUT--AND WE'RE ABSOLUTELY GRATEFUL FOR THE SUPPORT WE GOT FROM THE INFECTIOUS DISEASE SERVICE TO GET THIS PATIENT THROUGH AS WELL AS FROM THE CRITICAL CARE MEDICINE SERVICE. NINE PATIENTS REACTIVATED WITH THE VIRUS, ALL THESE PATIENTS WERE TREATED WITH IMPERRIC OR PREEMPTIVE RETUXAMAB AND NONE OF THOSE DEVELOPED PROLIFERATIVE DISORDER ALL PATIENTS RECEIVED TRANSFUSION AND NOW OUR MEDIAN FOLLOW UP IS JUST PASSED 1 YEAR AND I'M HAPPY TO REPORT THAT 11 OF THESE 12 PATIENTS ARE 92% SURVIVE. SO HERE CAN YOU SEE THE NUTRIFILL RECOVER SCHEINGRAFTMENT KINETICS THAT ARE TYPICAL FOR PATIENTS THAT ARE TREATED ON THIS REGIMEN. THIS PATIENT HAD RAPID NUTRIFILL RECOVER WEB CONNECTED THE ANC 500 OVER BY DAY TAKEN 10 WHEN WE LOOK AT THE CHIMERISM STUDIES YOU CAN SEE THAT EVERYTHING BACK EARLY ON IS FROM THE HAPPEN LO DONOR. AND WE DON'T SEE INGRAFTMENT OF THAT CORD UNTIL SEVERAL WEEKS LATER AND THEN GRADUALLY SUE AN INCREASE IN HEMEAT O POETICESIS FROM THE CORE UNIT AND THEN WE HAVE THIS PERIOD OF TRANSIENT PERIOD OF DUAL MYELOID CHIMERISM FROM BOTH GRAFT SOURCES AND THEN EVENTUALLY MYELOID CHIMERRICISM IS 100% CHORD IN ORIGIN. AND IN CONTRAST WE SEE A COMPLETE DIFFERENCE IN THE T-CELL LINEAGE SO THE CHIMERISM IS PREDOMINANTLY FULL CORD TRUSTEES THE GET GO, SO THESE ARE NOT T-CELL PLATED, THE HAPLO IS DEPLETED, AND LOOKING AT THIS DATA, 1 MIGHT SUSPECT THAT THE REASON THAT OUR HAPLO CELLS DISAPPEARED IS THAT THESE INGRAFTED T-CELLS MAY BE KILLING THEM OFF AND INDEED, STUDIES IN THE LAB BY NICOLE GOURMLY HAS SHOWN THAT VERY EARLY ON, AFTER THE TRANSPLANT, THESE CORE BLOOD T-CELLS APPEAR TO BE TOLERANT TO THE HAPLOCELL, BUT WITH TIME WE START TO SEE AN INCREASE IN REACTIVITY AGAINST THE HAPPEN LO CELLS APPROACH IN GREEN THAT SHOW THE REACKIVITY AGAINST COMPLETELY„3u8 MISMATCHED THIRD PARTY CELLS. SO THESE DATA SUGGEST THAT INGRAFTED CORE BLOOD T-CELLS ARE IMOWNOLOGICALLY MILE O OBLATTING THE STEM CELLS WHICH ARE NOT A PROBLEM SINCE WE HAVE HEMEAT O POETICESIS THAT THEN ENSUE FROM THE CORD UNIT. HERE YOU CAN SEE, CHIMERISM DATA THAT SUMMARIZE FOR PATIENTS THAT HAVE A TYPICAL PATTERN OF INGRAFTMENT WHERE WE SEE RAPID AND SUSTAINED BLOOD, CORE BLOOD T-CELL INGRAFTMENT AND RAPID MYELOID INGRAFTMENT FROM THE HAPPEN LO DONOR AND THEN WE SEE WITH TIME A TRANSITION FROM HAPLO HEMEAT O POETICESIS TO FULL CHORD DONOR HEMEAT O POETICESIS. AND WE'VE ALSO SEEN 2 PATIENTS THAT HAVE GONE THROUGH THIS PROTOCOL THAT HAVE HAD FAIL TOWER INGRAFT WITH THE CORD UNIT. --EXAMPLE I'LL SHOW HERE IS A 14 YEAR-OLD BOY WHO WAS HEAVILY PRETREATED, FAILED 3 LINES OF PRIOR IMMUNO SURVEYS PRECIOUSESSIVE THERAPY, HE HAD MULTILE OPPORTUNIST IRK BACTERIAL INFECTIONS AND PULMONARY HLA ALON IMMUNIZED AND JULY IN 2009, HE UNDERWENT A CORD HAPPEN LO TRANSPLANT ON THIS PROTOCOL, USED THE BROTHER AS THE HAPLO DONOR AND WE USE A CORD UNIT THAT WAS 4 OUT OF 6 AND DESCENT AND VERY, VERY, HIGH DOSE OF CD34 POSITIVE CELLS SO WE WERE QUITE HAPPY ABOUT THIS CHORD UNIT, HAPPY UNTIL WE THOUGHT IT AND WE SAW THE VIABILITY WAS 6%. AS WEs BEFORE THERE,'S NO TURNING BACK AT THIS POINT. SO REMARKABLY THIS PATIENT HAD RAPID RECOVERY FROM HIS NUTRIFILL ACCOUNT. HIS NUTRIFILLS WERE OVER 500 BY DAY 10. AND WHEN WE LOOKED AT HIS CHIMERISM STUDIES WE WERE SURPRISED TO SEE, MYELOID CHIMERISM IS FULLY HAPPEN LO EARLY ON AND WE NEVER SAW, INGRAFTMENT OF THAT CORD UNIT AT ALL WHEN WE LOOK AT THE T-CELL LINEAGES WE NEVER PICKED UP THE CORD, AND THE T-CELLS EARLY ON WERE 100% RECIPIENT IN ORIGIN. THIS WAS A NERVE WRACKING PERIOD FOR US BECAUSE WE WERE VERY CONCERNED THAT THIS PATIENT WAS AT VERY HIGH RISK TO REJECT THOSE HAPPEN LO CELLS PHYSICAL THE PATIENT WAS READMITTED FOR CMV REACTIVATION AND I REMEMBER SITTING AND DISCUSSING WITH COLLEAGUES ABOUT WHAT WE SHOULD DO FOR THIS PATIENT. AND REALLY HAVING NO DATA TO GUIDE US EITHER WAY WE DECIDED TO DO THE VERY BOLD THING OF DOING ABSOLUTELY NOTHING. SUBSEQUENTLY NICOLE GOURMLY DID AN MRI ASSAY, WE WANTED TO SEE WHAT WERE THE T-CELLS ALL ABOUT, WERE THEY GOING TOEE ACCOUNTIVE AND KILL OFF THE HAPLO CELLS OR BE TOLERANT AND THE DATA SHE GENERATED WAS REASSURING. SO WHILE THE T-CELLS WERE ALLO REACTIVE TO THE MISMATCHED CELLS THAT APPEARED TO HAVE NO ALOE REACTIVITY TO THE HAPPEN LO DONOR HAVING THE SAME AMOUNT OF PROLIFERATION IS WHAT YOU WOULD SEE CELLS. SO THIS SO THIS ARESURED US THERE WAS A STATE OF TOLERANCE IN THIS PATIENT AND INDEED WITH TIME WE STARTED TO SEE T-CELLS BEING GENERATED PRESUMABLY FROM THE STEM CELL COMPARTMENT FROM THE HAPPEN LO DONOR AND NOW THE PATIENT HAS MIXED T-CELL CHIMERISM AND HE'S NOW 3 YEARS POST TRANSPLANT AND REMAINS TRANSFUSION INDEPENDENT. SO IN CONCLUSION OUR PRELIMINARY RESULTS SUGGEST THAT THE CO INFUSION OF A HAPLOIDENTICAL GRAFT COMBINE WIDE CORD CELL SYSTEM A FEASIBLE TRANSPLANT OPTION FOR PATIENTS WITH SAA, WHO FAIL IMMUNO SUPPRESS ANT THERAPY AND LACK A DONOR. THIS TRAIT STRATEGY APPEARS TO SHORTEN THE TIME WITH NUTRIFILL RECOVERY, PROVIDES BACK UMKC STEM CELL COURSE IN THE EVENT OF UCB GRAFT FAIL AND YOU ARE MAY IMPROVE THE OUTCOME OF UCB TRANSPLANTATION IN HIGH RISK PATIENTS WITH SAA. OUR FUTURE EFFORTS WILL BE AIMED AT TRYING TO ENGINEER THESE CORE BLOOD TRANSPLANTS TO RESTORE CELLULAR IMMUNITY MUCH EARLIER SO THAT WE CAN TRY TO PREVENT THESE NUMEROUS VIRAL INFECTIONS THAT HAVE BEEN THE MAJOR MORBIDITY ASSOCIATED WITH THIS TRANSPLANT APPROACH. SO I WOULD LIKE TO ACKNOWLEDGE ALL THE NUMBERS OF OUR TRANSPLANT TEAM THAT CONTRIBUTED TO THESE STUDIES AS WELL AS MEMBERS OF THE HEMEATOLOGY BRANCH AND DR. YOUNG AND HIS COLLEAGUES FOR FAITH AND ALLOWINGITOUS DO THIS HIGH RISK TRANSPLANT PROCEDURE ON THESE PATIENTS. I ALSO WANT TO ACKNOWLEDGE THE MEMBERS OF THE NIH CENTER WHO SUPPORTED THE EFFORTS TO TRANSPLANT THESE PATIENTS AND TO GET THEM THROUGH AND THEY ARE REALLY LARGELY RESPONSIBLE FOR THE EXCELLENT RESULTS THAT WE'VE SEEN. I CAN TELL YOU THAT THE ICU STAFF HAS PLAYED A CRITICAL ROLE, THE INFECTIOUS DISEASE STAFF HAS ALSO PLAYED A CRITICAL ROLE AND THE NURSING STAFF ON BOTH NORTHEAST AND NORTHWEST HAS BEEN ABSOLUTELY EXCEL SPENT LAST I'D LIKE TO ACKNOWLEDGE THE NATIONAL MARROW DONOR PROGRAM WHICH IS DONATED THESE CORD UNITS FOR US TO ALLOW US TO CONDUCT WHAT WE BELIEVE IS A VERY IMPORTANT STUDY. THANK YOU. [ APPLAUSE ] >> WE'RE A LITTLE OVER, IF THERE'S 1 OR 2 BURNING QUESTIONS WE CAN TAKE IT BUT OTHERWISE WE'LL MOVE ON. NONE, SO WE'LL MOVE ON. HOLD YOUR QUESTIONS TILL LATER. DID. >> OKAY, OW SECOND SPEAKER IS DR. JEFFREY MILLER, SENIOR INVESTIGATOR AND CHIEF OF THE CHIEF OF THE SECTION OF MOLECULAR GIVING--YOU NATIONAL LIBRARY OF MEDICINICS AND THERAPEUTICS AT NIDDK'S MOLECULAR MEDICINE BRANCH. HE WILL DISCOVER THE RETICULOCYTE FROM THE BRANCH--HE CAME HERE 921995 NEAR AND WERE CLINICAL FELLOWSHIPS AT NHBBI. HE'S A MEMBER OF THE ASCI, AMERICAN SOCIETY OF HEMEATOLOGY AND AMERICAN SOCIETY OF HUMAN GENETICS. AND HIS CURRENT RESEARCH FOCUSES ON ARITHROADWAY CELL GENOMICS AND ARITHROUGH ATOM POETICESIS AND HIS GROUP HAS DEVELOPED METHODS FOR STUDYING HUMAN ARITHROUGH ATOM POEIES AND I GUESS REALTIME WITH AN EMPHASIS ON EXPERIMENTAL MANIPULATIONS THAT OFFER CLINICAL INSIGHTS. THE WORK HAS LED TO SEVERAL ADVANCES OVER THE LAST DECADE INCLUDING UNDERSTANDING AND PREVENTING DON BROCK MEDIATED HEMOLYSIS AND ASEMIA IRON RELATED OVERLOADING AND INEFFECTIVE ARITHROUGH ATOM POETICESIS. PLEWS WELCOME DR. MILLER. [ APPLAUSE ] OKAY, SO I'M GOING TO TALK ABOUT RETICULOCYTES TODAY BECAUSE THEY'RE WONDERFUL AND I HAVE NO CONFLICTS AT ALL AND I HAVE NOTHING TO DISCLOSE. BASED ON ALL THE HELP I GOT FROM THE LIBRARY AND FINDING SOME VERY OLD ARTICLES AND TRANSLATING THOSE I'LL GIVE A BRIEF HISTORY OF SCIENTIFIC RESEARCH OVER THE LAST 150 YEARS AND I'LL TALK ABOUT OUR EFFORTS TO FIND THE RETICULOCYTE TRANSCRIPT OHM AND HOW WE'VE BEEN EXPLORE TAG FOR ANEMIA RESEARCH AND FINALLY I'LL HIT ON THIS NEW AREA OF RESEARCH WHICH IS TO TRY TO ENGINEER RETICULOCYTE AND RED CELLS IN THE LABORATORY. SO IT ALL STARTS HERE. 150 YEARS AGO, WITH WILHELM HERB, AND HE HAD BLOOD SMEARS AND HE APPLIED DILUTE VINEGAR TO THOSE SMEARS AND HE NOTICED THERE WAS A SUBSET OF RED CELLS THAT WOULD GET THE GRANULES WITH THE VINEGAR BUT HE DEPARTMENT KNOW IT BUT HE HAD JUST DISCOVERED THE RETICULOCYTE. WE THEN JUMP AHEAD TO THE TURN OF THECEPTORY, WHEN--CENTURY WHEN ARTHUR PEPPENHYM, WHICH COINED THE TERM STEM CELL, HE LOVED HEMEAT O POEESIS AND HE DIED BEFORE HIS TEXTBOOK WAS PUBLISHED AND IN THE BACK OF HIS TEXTBOOK, YOU CAN SEE HIS IDEA OF HEMATOPOIESIS RIGHT AROUND 1913 OR 1915 AND HE REALLY HAD MOST OF IT CORRECT. WHERE HE THOUGHT THERE WERE STEMMISH TYPE CELLS, THE CELL LOOKS LIKE A CD34 CELL BUT WOULD DIFFERENTIATE INTO LYMPHOID CELLS OR MONOCYTES OR MOST IMPORTANTLY ALONG GRANUE LOW POEESIS ON THE LAST AND ARITHROUGH ATOM POETICESIS ON THE RIGHT. AND HAPPEN HYM IS THE FIRST GUY WHO SAID THERE IS A TRANSITION CELL BETWEEN THE NUCLEATED RED CELL AND THE RED CELL AND HAD IS A RETICULOCYTE WITH A GEL BODY ON THE SIDE. ANOTHER 30 YEARS WE FIND LUDWIG, HE LIKED TO AIR AT RETIC CUE LOWITES. AND BEFORE HE DID THIS, WAS THE DISCOVERY OF A BLUE STAIN WHICH WAS A SUPER VITAL STAIN AND THIS STAIN HAD THE SAME EFFECT ON RETIC LO SIGHTS AS THIS DILUTE VINEGAR AND IT CAUSED CONDENSATION AND ALMOST A NET LIKE STRUCTURE HENS THE NAME RETIC LO SIGHT. HE THEN WENT ON BEYOND PAPPEN HYM'S DISCOVERIES TO SAY THAT THIS WAS IN THIS A NUCLEATED RED CELL, AGAIN BLUE BECAUSE OF THE STAIN AND ONCE THE CELL LOST THE NUCLEUS, YOU WERE LEFT WITH A NET LIKE STRUCTURE WHICH OVERTIME WOULD DISSOLVE IN THEwT.Ww CELLS. BECOME A MATURE RED CELL. AT THE END OF WORLD WAR II, THEY FIGURED OUT WHAT THE RETICULUM WAS, I COULD NOT FIND A LARGER IMAGE THIS, IS MARKED BY THE SMALL YELLOW ERROR, HE ALSO LIKED THESE SMALL BLUE GRANULES, NOBODY KNEW WHAT THEY WERE AND HE CALLED THEM BASOPH ILIES, AND HE NOTICED IF YOU ADD RIBONUCLEACE TO THE CELLS THEY DISAPPEAR COMLITELY SO HE'S KRET EDUCATIONAL WITH THE KNOWLEDGE THAT A RETIC CUE LOW SIGHT IS AN RNA CELL. THIS LEAD TO A YOUNG BIY CHEMIST REREALIZE TAG YOU COULD USE THESE TO DO TRANSLATIONAL PROTEIN BIOCHEMISTRY AND ALAN SHECTER DIDDED STUDIES BEFORE COMING TO THE NIH AND CONDUCTED THIS STUDY WHICH IS A REPRODUCTION OF MARSHAL NUREMBERG'S STUDY USING POLYURIA DEAN AND HE DEMONSTRATED THE UNIVERSALITY OF THE GENETIC CODE AS A MEDICAL STUDENT. FINALLY SAM RAPPA PORT, IN 1986 PUBLISHED PROBABLY THE FIRST AND POSSIBLY OHM BOOK ON RETICULOCYTE WHERE HE WAS TRYING TO DESCRIBE WHAT WAS ENCODED BY THE RETICULOCYTE MRNA. AND IT WAS LIKE THE RED CELL WAS A BAG OF GLOBINGIN AND RETICULOCYTE WAS A BAG OF ENCODED M RNA. AND HE ALSO SAID THERE MAY BE OTHER PROTEINS AND MUST BE MEMBRANE PROTEINS ENCODED BUT REALLY 95% WAS DEEMED TO BE MOSTLY GLOBIN. THAT LEFT US IN 1986 WITH THE IDEA THAT THE RETIC CUE LOW SIGHT HAS LOTS OF MRNA, IT ENCODES ALMOST ALL GLOBIN AND ONLY 5% OF IT IS LEFT TO MYSTERY. SO WHEN I STARTED WORKING ON THIS THAT MYSTERY WAS SORT OF BOTHERING US A BIT AND THE QUESTION WAS: REALLY IS THE 5% ONLY 2 ORALLY OTHER PROTEINS ENCODED OR WAS THERE A LOT OF PROTEINS ENCODED AT A SMALL OR LOWER LEVEL. WAS THE RETIC CUE LOW SIGHT A SMOKING GUN WHERE YOU GET A LOT OF INFORMATION AND A LOT OF SECRETS ON ARITHROUGH ATOM POETICESIS FROM THESE CELLS. WE TRIED TO INVESTIGATOR THIS, THIS WOULD BE SASHA GIEWBEN'S WORK WHERE WE MADE LIBRARYS AND WITH THE GENOME AND WE DID HIGH THROUGH PUT SEQUENCING AND OF COURSE WE SEQUENCED A LOT OF GLOBIN AND THEN SASHA TRIED TO SUBTRACT THE GLOBIN MRNA AND THAT WORKED IN THE LESS MATURE CELLS BUT IN THE RETIC CUE LO SIGHTS WE STILL SEQUENCE A LOT OF GLOBIN AND THEN THE ARRAYS CAME ALONG. AND SO, HERE, DR. GO AN IN THE LAB AND DR. NOTE IN THE LAB WORKED ON WHAT THE TRANSCRIPT OHM WAS COMPOSED OF AND THESE ARRAYS ALLOWED US TO SEE WHAT WAS THERE BESIDES GLOBIN AND GOH, WORKED ON THE MRNA AND NOH, WORKED ON THE MicroRNA PROJECTS WE USE THE RETICULOCYTES BECAUSE WE WANT TO SEE IF THERE WERE DEVELOPMENT ALT CHANGES AND IT WAS A TREASURE TROVE BECAUSE THERE WERE OVER A THOUSAND MRNAs AND OVER 200 MICRORNAs AND WE WERE TRYING FOCUSED ON FIGURE OUT WHAT TO DO WITH ALL OF THIS INFORMATION. SO WHAT WE DID WE SPENT CONSIDERABLE TIME JUST STARING AT THESE ARRAYS AND NOT KNOWING QUITE WHAT TO DO, SIMILAR TO THIS ARRAY IF YOU STAIR AT IT, AND YOU TRYw3coœ TO COUNT THE NUMBER OF GRAY OR BLACK DOTS YOU'LL END UP GETTING A BIT FRUSTRATED. IT'S A LESSON IN FUTILITY SO WHAT WE DEEICIDE TO DO INSTEAD WAS TO GO FOR CANDIDATE PROGECS. WE KNEW WHAT WAS THERE. WE WEREN'T SURE WHY IT WAS ALL THERE TOGETHER. BUT WE IMU SOME OF IT MIGHT BE IMPORTANT FOR BASIC SCIENCE AND CLINICAL SCIENCE. SO OF COURSE THERE WERE A GROUP OF MEMBRANE PROTEINS ENCODED. SIGNAL TRANSDUCTION, IRON REGULATION, A LOT OF MITOCHONDRIAL BIOLOGY IS ENCODED IN THESE ARRAYS. AND OUR FAVORITE HEMOGLOBIN. THERE WAS A HEMOGLOBIN THAT HAD BEEN OVERLOOKED IN THE PAST THAT WAS DISCOVERED FROM THIS. BUT THIS IS CLINICAL GRAND ROUNDS SO WE ALSO LOOKED FOR THINGS THAT MIGHT FILL IN GAPS IN CLINICAL KNOWLEDGE, NOT JUST BASIC SCIENCE KNOWLEDGE AND FOR THIS, THE REST OF THE TALK I'LL TALK ABOUT OUR EFFORTS TO BETTER UNDERSTAND THALASSEMIA, IT IS DERIVED FROM THE ROOT WORDS, THE SEA AND BLOOD BECAUSE THALASSEMIA, WAS DISCOVERED AROUND PEOPLE WHO LIVE AROUND THE MEDITERRANEAN SEA, AND MOSQUITOES LIVE AROUND THE SEA, SO SICKLE CELL PROTECTS PEOPLE FROM MALARIA. THIS IS A SELECT ADVANTAGE WHERE IT CAME FROM. THAT WILLASEMIA IS A GENETIC DISEASE, BETA SALIVA--SALIVA WHICH YOU'VE HEARD ABOUT OR SEEN IN THE CLINIC INVOLVES MUTATIONS IN THIS THE BETA GLOBIN GENES WHICH YOU'VE SEENOT CHROMOSOME 11. ALPHA THAL INVOLVES MUTATIONS OR DELETIONS IN 1, BOTH THE ALPHA GENES IN CHROMOSOME 16. BUT HOW DOES IT CAUSE THE DISEASE THALASSEMIA. THIS WAS DISCOVERED RIGHT UP STAIRS HERE IN BUILDING 10OT SEVENTH FLOOR BY THIS GENTLEMAN, ART NIENHUIS, AND AGAIN USING RETICULOCYTES WHAT HE WAS ABLE TO DISCOVER AND DESCRIBE IN A SIMPLE WONDERFUL ARTICLE, MOST LIKELY EXPLANATION FOR THE MOLECULAR BASIS, WOULD APPEAR TO BE THE THALASEMMIA, IS PRODUCED AND REDUCED VERY SIMPLY. DO YOU DON'T MAKE ENOUGH MRNA, SO YOU DON'T MAKE ENOUGH HEMOGLOBIN. THALASSEMIA, CAUSES ANEMIA AND MAKES SOME SENSE, YOU'RE NOT MAKING A LOT OF HEMOGLOBIN AND THIS IS A TYPICAL BLOOD SMEAR WHERE THESE CELLS, IT'S NOT JUST A SLIDE, THE CELLS ARE REALLY WASHED OUT. YOU CAN SEE THE CENTRAL PALERS IS FAIRLY LARGE AND INTERESTINGLY THERE'S A LOT OF THESE FUNNY LITTLE CELLS, THESE ARE NUCLEATED RED BLOOD CELLS AND IN THAL, THEY CONTAIN THIS LITTLE TINY BIT OF CYTOPLASM ON THE OUTSIDE. THEY ARE NOT HAPPY. CLINICALLY, THL-TRAIT WHICH PROTECTS YOU FROM MALARIA DOES NOT CAUSE ANEMIA OR CAUSE DISEASE, BUT IF IN YOUR FAMILY YOU'RE INHERENT A THAL TRAIT FROM MOM AND DAD OR HOMOZYGOUS OR HETEROGENEOUS ROW ZYGOUS MUTATIONS CAN YOU GET THAL MEDIA OR MAJOR, MAJOR YOU'RE TRANSFUSION DEPENDENT FOR ALL OF YOUR LIFE. AND WHAT THAT MEAN SYSTEM YOU GET UNITS OF BLOOD FOR THE REST OF YOUR LIFE AND THIS HAS SAVED THESE PATIENTS LIVES BUT OF COURSE THE UNITS OF BLOOD HAVE TROUBLES. FIRST, THE CROSS MATCHING AFTER A PERIOD OF TIME, YOU RUN INTO DIFFICULTIES BECAUSE OF ALL THE ANTIGENS ON THE BLOOD SO YOU HAVE DIFFICULTIES FINDING THE RIGHT DONOR. SECOND, RED BLOOD CELLS HAVE A LOT OF IRON. SO, THE PATIENTS GET IRON OVERLOADED AND THESE THAL PATIENTS GET IRON OVERLOADED EVEN IF THEY'RE NOT TRANSFUSED. AND THEN THIRDLY, YOU KNOW JUST HAVE THIS ANEMIA THIS IS LIFE LONG SO YOU'RE CONSTANTLY STUCK WITH THIS. IT'S A CHRONIC DISEASE. SO FOR US WE WANTED TO ASK HOW COULD WE USE ALL THIS WONDERFUL NEW INFORMATION TO FILL IN THE GAPS OF KNOWLEDGE FOR THALASSEMIA, WE LOOKED FOR PROJECTS THAT HAD SOMETHING TO DO WITH TRANSFUSION MEDICINE, IRON OVERLOAD AND OF COURSE WE'RE ALWAYS LOOKING FOR A WAY TO PREVENT THE ANEMIA OR RESCUE THE ANEMIA. ONE OF THE EARLY PROJECTS WITH THESE ARITHROADWAY TRANSCRIPT OHMS WAS FIND THANKSGIVING CANDIDATE WHICH WE CALL DOC 1. NOBODY ELSE CALLED IT 1. NOBODY ELSE CALL TODAY THAT, IT'S CALLED ART 4 NOW. THIS IS A SIMPLE GENE, WE THOUGHT IT MIGHT BE THIS DOMBROCK BLOOD GROMBECAUSE IT HAD A MOTIF AND IT'S KNOWN THAL GROUP CARRIER CARRIES SEVERAL ANTIGENS THAT AREN'T THERE IN PATIENTS WITH P& H THAT LACK THAT PARTICULAR TYPE OF SURFACE PROTEIN ON THE RED CELLS. AND IN COLLABORATION WITH MARRIAN REED, NEW YORK BLOOD CENTER, SHE HELPED US CONFIRM THAT THIS INDEED WAS DOMBROCK AND BASED ON HER EXPERTISE, WE WERE ABLE TO MAP OUT THE 2 MAIN ANTIGENS THAT ARE CARRIED BY THIS PROTEIN, AND MARRIAN'S GONE ON WITH A SERIES OF WONDERFUL PROJECTS TO MAP OUT 5 ADDITIONAL ANTIGENS AND PEOPLE LIKE WILLIE FLAGLE IN OUR OWN DEPARTMENT OF TRANSFUSION MEDICINE NOW USE MOLECULAR METHODS RATHER THAN HEMAGLUTEN TO MATCH THE BLOOD SORRY SICKLE CELL AND MATCH THE BLOOD FOR OTHERS WHO REQUIRE CHRONIC TRANSFUSIONS. A SECOND CANDIDATE, THIS IS IN THE IRON FIELD, IS THAT GENE AND PROTEIN IN A PROTEIN CALLED GDF 15, GROWTH AND DIFFERENTIATION FACTOR 15, HERE WE WERE LOOKING FOR SOMETHING THAT MIGHT BE SECRETED FROM A ARITHROADWAY CELLS AND TRAVEL TO THE LIVER TO REGULATE IRON. AND PERHAPS ADD TO THE IRON OVERLOAD. WE FOUND THIS CANDIDATE IN THE PILE. WE THOUGHT IT WAS PARTICULARLY INTERESTING BECAUSE OF IT'S PROMOTER REGION AND IT HAS THESE 3 EGR MOTIFS WHICH SUGGEST IT'S TURNED ON AND STRESS, HAS P53 MOTIF, SUGGEST IT MIGHT BE RELATED TO APOPTOSIS. BOTH OF THOSE'RE ACTIVE IN THAT WILLSEMIA, ORYTH ROUGH ATOM POETICEEIS. AFTER PROCESSING YOU END UP WITH THE PROTEIN AND WHEN WE TESTED THIS, IT'S DR. TAN O'S GROUP IN MY AND WHEN WE TESTED THIS, USING SERUM FROM PATIENTS WITH THALASSEMIA, OR HEALTHY VOLUNTEERS, CAN YOU SEE THEY HAVE INCREDIBLY HIGH LEVELS OF GDF 15 AND HAVE HEPC IDIN AND SUPPRESSED SO IT'S FELT THAT THE SECRETION OF GDF-15 ADDS TO THE IRON OVERLOOM SYNDROME AND ACTUALLY THERE'S A BIT OF A DOUBLE WHAMMY IN THAT THOSE PATIENTS HAVE SO MUCH IRON IN THE MACOFADGES FROM TRANSFUSION THAT THIS HIGH LEVEL OF TRANSFUSION RELEASES THAT TOXIC IRON WHICH WOULD OTHERWISE BE RELATIVELY SAFE FOR THESE PATIENTS. MORE RECENTLY THIS IS FROM LAST YEAR, A GROUP FROM LEBANON, LOOKED AT GDF 15 IN THE CONTEXT OF DISEASE SEVERITY IN THAL, AND BASED ON THE HUGE RANGE OF EXPRESSION FROM LEVELS OF ABOUT A HUNDRED PICO GRAMS PERMILL AND ALL OF US, MOST OF US TO LEVELS IN THE 50,000 OR MORE IN THALASEMIA, IT TURNS OUT TO BE A GOOD DISEASE SEVERITY MARKER. BUT OF COURSE WHAT WE'VE BEEN LOOKING FOR FOR MANY YEARS NOW, IS A CLUE AS TO HOW WE CAN JUST SOLVE THE PROBLEM AND THAT IS TO RESCUE THE INEFFECTIVE ARITHROUGH ATOM POETICESIS AND TO UNDERSTAND WHAT EFFECTIVE ARITHROUGH ATOM POETICESIS IS, AND YOU HAVE TO UNDERSTAND THAT EREGROPYIS IS MUCH LIKE THE AISLE ELF TOWER, THEY'RE VERY WELL ENGINEERED, THEY CONTAIN 8 LOT OF IRON AND THEY BOTH MAKE SOMETHING VERY BEAUTIFUL. SO INCOMPLETE ERYTH ROUGH ATOM POETICESIS WOULD BE 2/THIRDS, IT'S NOT COMPLETION, AESTHETTICS NOT THERE, AND IT'S NOT FUNCTIONAL AND THE RED CELLS ARE NOT FUNCTIONAL IF WE TRANSLATE THAT IDEA OR VISUAL INTO THE CELLS THEMSELVES WE CAN NOW CULTURE CELLS QUITE EASILY IN THE LABORATORY, THIS IS A CD34 CELL, MUCH LIKE THAT CELL IN PAPPEN HYM'S ILLUSTRATION AND WHAT HAPPENS IN THAT WILLSEMIA, IS AT THE SEMESTER CELL LEVEL THERE'S A GENETIC MUTATION. THE CELLS COMMIT TO ARITHROUGH ATOM POETICESIS AND THEY DIFFERENTIATE JUST FINE. BUT THEY DON'T MAKE MUCH BETA GLOBIN. SO INSTEAD OF BECOMING A RED CELL, AS THEY SHOULD, THEY APOPITOSE. SO YOU END UP WITH THESE FUNNY RED CELLS THAT ARE VERY LOW IN NUMBER. PATIENTS DEVELOP ANEMIA AND THEY DEVELOP IRON OVERLOAD. WHAT WE'VE BEEN DOING IN THE LAB IS WE'VE BEEN WATCHING OTHER GROUPS AND THERE'S TABOO GROUPS-OF 2 GROUPS GLOBALLY, DR. DUA IN PARIS AND THE ANC GROUP IN ENGLAND AND WHAT THEY'RE DOING FOR THE DEPARTMENT OF TRANSFUSION MEDICINE FOR THEIR DEPARTMENTS IS THEY'VE BEEN TRYING TO ENGINEER OR GROW RED CELLS FROM DONORS THAT HAVE VERY UNUSUAL PHENOTYPES, WITH THE IDEA THAT MAYBE YOU CAN GROW BLOOD FOR TRANSFUSION RATHER THAN GETTING IT FROM ANOTHER HUMAN. AND WITH THIS IDEA IN MIND, WE THOUGHT WELL IF THEY CAN DO THAT, MAYBE WE CAN ENGINEER THALASSEMIA, AND THE REASON WE WANT TO ENGINE THALASSEM IA IN THE LABORATORY IS BECAUSE THERE'S NO WAY TO STUDY THIS ERYTH ROUGH ATOM POETICESIS AND PATIENTS ARE HARD TO COME BY AND THEY T MIGHT BE A RISK EVER THE CD34 SES AND THEY IS ALL THESE DIFFERENT FEIGNEE TIMES. SO A LAB IN HERE, VERSES A LAB IN INDIA OR THE iPAD LAND MAY GET 3 DIFFERENT RESULTS BASED ON THE DONOR. SO WHAT WE DECIDEDDED TO DO WAS TO ASK OURSELVES WELL, INSTEAD OF ENGINEERING THE GOODNESS IN THESE CELLS, MAYBE WE CAN ENGINEER A BAD OUTCOME. WE WENT FORWARD WITH THIS SHORT HAIR PIN RNA TECHNOLOGY WHICH COME OUT, IT COME OUT THESEULENTY VIRAL VECTORS WORKED VERY WELL AND WE TRY TO ASK OURSELVES, WELL, MAYBE WE CAN JUST KNOCK DOWN ALL THAT BETA GLOBIN AND WE TRANSDUCED THE CELLS HERE, THE HOPE WAS THAT THEY WOULD MAKE LESS BETA GLOBIN. AND HAVE AN INEFFECTIVE ARITHROUGH ATOM POEESIS OR A FEIGNEE TYPE. THAT WAS THE IDEA. THIS IS WORK DONE BY MOSTLY TERRY LEE IN THE GROUP. AND TERRY LEE STARTED ADDING THESE S, R NA LENTIVIRUSES WHICH WE CALL HPV AND REMARKABLY DESPITE THIS INCREDIBLY HIGH LEVEL OF BETA GLOBIN EXPRESSED IN THESE CELLS, IT WAS KNOCKED DOWN 90% BY SHRNA: THE GAMMA GLOBIN WHICH IS THE FETAL GENE SEEMS TO BE INCREASED BUT WE'RE NOT SURE IF THAT'S THE CASE OR IF JUST THERE WAS A SELECTIVE ADVANTAGE IN THE ARITHROUGH ATOM POETICESIS BECAUSE SOME CELLS EXPRESSED THAT MPLET DELTA IS KNOCKED DOWN BECAUSE IT TARGETS THE HPV DOES TARGET DELTA AND EPISALON WITH THE EMBRYONIC GENE IS ALREADY LOW AND IT DOESN'T GET KNOCKED DOWN. AND IN CONTRAST, WE LOOKED AT THE ALPHA GLOBIN CLUTTER AND IN THE ALPHA GLOBIN CLUSTER THERE WAS NO CHANGE SUGGESTING THAT WEFkœ1— HAD SPECIFICALLY KNOCKED DOWN THE BETA GENE. WE THEN LOOKED AT THE PROTEIN, SO OF COURSE IN BETA THAT WILLSEMIA IF WE WERE RECAPITULATING THIS, WE WOULD EXPECT A LOW LEVEL OF HEMOGLOBIN A, BETA GLOBIN GENE, IS INTRODUCED OR INCORPORATED WITH THE ALPHA GLOBIN GENE TO MAKE HEMOGLOBIN A IN OUR ADULTS. AND IN OUR CONTROLS WE SAW HIGH LEVELS OF HEMOGLOBIN A AND RATHER TINY LEVELS OF HEMOGLOBIN F, THE HEMOGLOBIN YOU WOULD EXPECT FROM ADULT CELL AND WHEN WE KNOCK DOWN THE BETA WE ORIGINALLY THOUGHT, UH-OH, THE A LOOKS THE SAME BUT IF YOU LOOK AT THE MILLI VOLTS THERE'S HARDLY ANY HEMOGLOBIN BEING PRODUCED O THE F-PEEK IS VERY SIMILAR TO THE LEVEL WITH THE CONTROL THAT THE A-PEAK CAME FROM 60 DOWN TO 2, BUT THAT WAS VERY GOOD. WE THEN ASKED IS ARITHROUGH ATOM POETICESIS INEFFECTIVE AND HERE YOU USE FLO CYTOMETRY DONE BY COLLINE IN MY GROUP AND WHAT HAPPENS IN ARITHROUGH ATOM POETICESIS IS THEY DON'T MEDE THEIR IRON ANYMORE, SO CD71 IS LOST, THAT'S THE RECEPTOR FOR TRANSFER ENSEL, SO THEY DON'T NEED TO IMPORT ANYMORE IRON SO THIS RECEPTOR IS LOST, AND IN OUR CONTROLS ABOUT 40% OF THE CELLS, QUICKLY LOSE THEIR CD71 AND WHEN WE KNOCKED ON THAT BETA GLOBIN GENE, THEY DIDN'T UNDERGO THE FINAL STAGE OF DIFFERENTIATION. AND THIS REALLY CLENCHED IT FOR US THAT WE INDEED NOW HAVE THE FIRST MODEL OF THAL USING PRIMARY CELLS. LOOKING AT THE CELLS, CONTROLS ON THE LEFT, THAL ON THE RIGHT, OR HPV KNOCK DOWN AND YOU CAN SEE THE CELLS MATURE, LEAVE LATE BUT IN ALL THE CELL THERE IS' PLENTY OF HEMOGLOBIN. THEY DO NOW ENUCLEATE IN THE LABORATORY LEAVING CELLS ALA HAPPEN HYM WITH THE JOLLY BODY HERE AND ALA HYL MEYER WITH THE A NET-LIKE STRUCTURE WHEN STAINED WITH BLUE, SO WE'RE VERY PLEASED WITH HOW THE CULTURES ARE GOING. WHEN WE KNOCK ON THE BETA GLOBIN GENE IT'S QUITE DIFFERENT. THE CELLS DON'T MATURE QUITE RIGHT THEY'RE VERY PALE OR BLUE. MANY OF THE CELLS WILL EITHER APOPITOSE BY FLOW CYTOMETRY AND VISUALLY. AND WE DO GET AN OCCASIONALLY CELL THAT ENUCLEATES THAT THESE CELLS CONTAIN HARDLY ANY HEMOGLOBEIN, MANY OF THEM ARE BLUE RATHER THAN RED AND THERE IS AN OCCASIONALLY RED CELL WHICH MAY BE A CELL CURRENTLY INVESTIGATORRING THAT. SO WHAT TO DO WITH THIS. THIS IS WHAT'S ON GOING NOW IN THE GROUP. WE NOW HAVE A MODEL FOR INEFFECTIVE ARITHROUGH ATOM POEESIS AND WHAT WE STARTED DOING A SERIES OF SCREENS TO TRY TO ENGINEER A RESCUE. SO TRY TO RESCUE THESE CELLS WHERE AGAIN THEY'LL MAKE A RED CELL AND THAT ENUCLEATES AND WE'RE USING GENES IN THESE SCREENS FROM BCL11 A WHICH IS RECENTLY DISCOVERED, GLOBIN ITSELF, GENES FROM OUR TRANSCRIPT OHMS, CYTOKINES, THAT WE STUDIED PREVIOUSLY THAT I DID NOT HAVE TIME TO TALK ABOUT VERSES OTHERS BASED ON KNOWING IN THE TRANSCRIPT OHMOT SURFACE OF THE CELLS AND DRUGS AND WE'RE HOPING WE CAN GET A COMBINATION OF THESE OR THESE ALONE THAT WILL RESCUE THAT WILLSEMIA IN THE LAB AND TRY TO TRANSLATE THAT INTO THE CLINIC. SO IN SUMMARY: THERE'S BEEN A WIDE RANGE OF DISCOVERIES THAT HAVE HELPED DEFINE THE RETIC CUE LOW SIGHT OVER THE LAST 50 YEARS AND THEY WERE RAIRKS DENTIFY AS A ROBUST SOURCE OF GENETIC INFORMATION, DIFFUSE MEDICINE, IRON BIOLOGY AND ARITHROUGH ATOM POETICESIS AND THIS IDEA YOU CAN ENGINEER A NUCLEATED RED CELLS IN YOUR LAB, LEADING TO ALL KINDS OF NEW TOOLS THAT WE'RE TRYING TO USE TO COME UP WITH NEW IDEAS FOR THE CLINIC. I THINK IT'S FAIR TO SAY THAT I'M GOING WITH HEALTHY FOR THE FUTURE. AND YOU KNOW OF COURSE, THANK YOU, ALAN SCHECTER, ALL OF THE POST DOCS WHO HAVE COME THROUGH MY GROUP. MANY OTHERS HERE AT NIH AND IN THE AREA SPECIAL THANKS TO DTM WITH ALL THE COLLABORATORS AND OTHERS AROUND THE WORMED. THANK YOU. --WORLD. THANK YOU. [ APPLAUSE ]