WELCOME TO THE CLINICAL CENTER GRAND ROUNDS, A WEEKLY SERIES OF EDUCATIONAL LECTURES FOR PHYSICIANS AND HEALTH CARE PROFESSIONALS BROADCAST FROM THE CLINICAL CENTER AT THE NATIONAL INSTITUTES OF HEALTH IN BETHESDA, MD. THE NIH CLINICAL CENTER IS THE WORLD'S LARGEST HOSPITAL TOTALLY DEDICATED TO INVESTIGATIONAL RESEARCH AND LEADS THE GLOBAL EFFORT IN TRAINING TODAY'S INVESTIGATORS AND DISCOVERING TOMORROW'S CURES. LEARN MORE BY VISITING US ONLINE AT HTTP://CLINICALCENTER.NIH.GOV >> GOOD AFTERNOON AND WELCOME TO CLINICAL CENTER GRAND ROUNDS. THE HOP RINSE CLOUD ACTIVITY CODE FOR TODAY IS 11462. IN ADDITION, EVALUATION FORMS ARE AVAILABLE FOR TODAY'S LECTURES AND ARE LOCATED AT THE BACK OF THE LIPSETT AMPHITHEATER. ALSO SUGGESTIONS FOR FUTURE TOPICS. SO PLEASE COMPLETE AN EVALUATION FORM BEFORE YOU DEPART. TODAY TWO COLLEAGUES FROM THE NATIONAL CANCER INSTITUTE AND THE NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES WHO REALLY NEED NO EXHIBIT DUCKS SO I WILL BE BRIEF. I'LL SPEAK ONTHEY'LL SPEAK ON THE BROADER TOPICS OF GENOMICS. OUR FIRST SPEAKER IS DR. THOMAS WALDMANN, DISTINGUISHED INVESTIGATOR AND CHIEF OF THE LYMPHOID MALIGNANCIES BRANCH AT THE NATIONAL CANCER INSTITUTE. HE WILL SPEAK ON INTESTINAL LYMPHANGIECTASIA, ALSO KNOWN AS WALDMANN'S DISEASE, REMEMBRANCES OF THINGS PAST. DR. WALDMANN EARNED HIS MEDICAL DEGREE FROM HARVARD MEDICAL SCHOOL, AND INTERNED AT THE MASSACHUSETTS GENERAL HOSPITAL IN INTERNAL MEDICINE. HE CAME TO THE NATIONAL CANCER INSTITUTE AS A CLINICAL ASSOCIATE IN THE METABOLISM BRANCH IN 1956. FROM 1958 TO 1959, HE CONTINUED HIS TRAINING AS A FELLOW IN THE METABOLISM BRANCH, AND HE WAS NAMED CHIEF OF THE METABOLISM BRAN OF, WHICH HAS SUBSEQUENTLY BEEN RENAMED THE LYMPHOID MALIGNANCIES BRANCH IN 1973. DR. WALDMANN HAS SIGNIFICANTLY ADVANCED BIOMEDICAL SCIENCE OVER HIS DISTINGUISHED 61-YEAR CAREER AS AN NIH SENIOR INVESTIGATOR. HIS RESEARCH ACCOMPLISHMENTS INCLUDE THE IDENTIFICATION AND CHARACTERIZATION OF THE INTERLEUKIN 2 RECEPTOR AS A TARGET FOR MONOCLONAL ANTIBODY MEDIATED THERAPY OF LEUKEMIA, LYMPHOMA AND MULTIPLE SCLEROSIS. THE CO-DISCOVERY OF INTERLEUKIN 15 AND ELUCIDATION OF ITS ROLE IN THE DEVELOPMENT OF NATURAL KILLER AND CD8 MEMORY T-CELLS, A FIRST IN HUMAN CLINICAL TRIAL OF IL15 TO TREAT PATIENTS WITH MALIGNANCY, AND THE ELUCIDATION OF MOLECULAR ABNORMALITIES IN THE COMMON GAMMA CYTOKINE JACK STAT SIGNALING PATHWAY, IN HTLB IS ASSOCIATED ADULT T-CELL LYMPHOMA CULMINATING IN A TRIAL OF THE JACK INHIBITOR TO TREAT PATIENTS WITH THE DISORDER. DR. WALDMANN IS A MEMBER OF THE NATIONAL ACADEMY OF SCIENCES, THE AMERICAN ACADEMY OF ARTS AND SCIENCES, THE INSTITUTE OF MEDICINE OF THE NATIONAL ACADEMY OF SCIENCES, AND THE ROYAL SOCIETY OF THE MEDICAL SCIENCES IN THE UNITED KINGDOM. AMONG HIS MANY HONORS ARE THE EHRLICH MEDAL AWARDED BY THE PAUL EHRLICH INSTITUTE IN GERMANY, THE RALPH M. STEINMAN AWARD IN HUMAN IMMUNOLOGY RESEARCH FROM THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS AND THE BRISTOL-MYERS SQUIBB AWARD FOR DISTINGUISHED ACHIEVEMENT IN CANCER RESEARCH. OUR SECOND SPEAKER IS DR. MICHAEL LEONARDO, A SENIOR INVESTIGATOR AND CHIEF MOLECULAR DEVELOPMENT OF THE IMMUNE SYSTEM SECTION IN THE LABORATORY OF IMMUNOLOGY AT THE NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES. DR. LENARDO ALSO DIRECTS THE CLINICAL GENOMICS PROGRAM AT NIAID. HE HOLDS POINT OF PROFESSOR AND IS VISITING FELLOW AT CAMBRIDGE UNIVERSITY. DR. LENARDO WILL SPEAK ON NEW MOLECULAR DISEASE, PATHOGENESIS, TREATMENT THROUGH GENOMICS. DR. LENARDO EARNED HIS MEDICAL DEGREE FROM WASHINGTON UNIVERSITY IN ST. LOUIS, MISSOURI. HE COMPLETED AN INTERNSHIP AND THEN FELLOWSHIP IN HEMATOLOGY AND ONCOLOGY AT THE UNIVERSITY OF IOWA HOSPITAL AND CLINICS, AND THEN BECAME A RESEARCH FELLOW AT THE WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY, WHERE HE CARRIED OUT MOLECULAR BIOLOGY RESEARCH UNDER THE MENTOR SHP OF NOBLE LAWYER LAUREATE DAVID BALTIMORE. HE CAME TO THE NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IN 198 EYE NINE AS AN INVESTIGATOR IN THE LABORATORY OF IMMUNOLOGY AND BECAME A TENURED SENIOR INVESTIGATOR IN 1995. HIS RESEARCH FOCUSES ON T LYMPHOCYTE REGULATION, MAGNESIUM METABOLISM IN IMMUNITY, AND GENETIC DISEASES OF THE IMMUNE SYSTEM. DR. LENARDO HAS FOUNDED OR COFOUNDED SEVERAL JOINT RESEARCH PROGRAMS AT THE NIH INCLUDING THE NIH OXFORD CAMBRIDGE BIOMEDICAL RESEARCH SCHOLARS PROGRAM AND THE NATIONAL MD/PH.D. PARTNERSHIP PROGRAM. HIS MEMBERSHIPS INCLUDE THE AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS, THE AMERICAN SOCIETY OF HUMAN GENETICS, AND THE AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY. DR. NARDO SERVED ON THE EDITORIAL BOARDS, JOURNAL OF EXPERIMENTAL MEDICINE, SCIENCE MAGAZINE AND BIOLOGY DIRECT. HE HAS CONTRIBUTED AT LEAST AT LAST COUNT 250 PEER REVIEWED PUBLICATIONS TO THE BIOMEDICAL LITERATURE. NOW LET'S BEGIN TODAY AND WELCOME OUR FIRST SPEAKER, DR. WALDMANN. [APPLAUSE] >> WELL, THANK YOU AND WELCOME. I WAS ASKED TODAY TO REFLECT BACK 60 YEARS, ONE YEAR AFTER I JOINED THE NIH, WHEN WE WERE INVOLVED IN THE DEFINITION OF A NEW CAUSE OF HYPOPROTEINEMIA AND IMMUNODEFICIENCY, LOSS OF PROTEINS INTO THE INTESTINAL TRACT, AND ONE OF THE MANY CAUSES OF THIS INTESTINAL LYMPHANGIECTASIA. I HOPE, TODAY, TO DEFINE THE CLINICAL AND IMMUNOLOGICAL FEATURES OF INTESTINAL LYMPHANGIECTASIA. BEFORE THAT, I HOPE YOU ALLOW ME A FEW MOMENTS OF REFLECTION OF WHAT WAS GOING ON 60 YEARS AGO OR 62 YEARS AGO IN MY LIFE. IN THAT TIME, I WAS A HARVARD MEDICAL STUDENT, AND AT THAT TIME, THE TURBULENCE WAS TRUE THEN AS IT IS NOW. JOE MCCARTHY AND THE HOUSE ON AMERICA ACTIVITIES GROUP, HOAAG, WERE INVESTIGATING HARD VARD MEDICAL SCHOOL AND ACADEMIA FOR COMMUNISTS IN AK ACADEMIA AND SET UP AN INVESTIGATIVE SESSION ON OUR CAM CAMPUS. ONE OF THE MEDICAL STUDENTS IN VANDERBILT HALL, WHERE I WAS, TOOK UMBRAGE AT THIS, AND PAINTED ALL THE TOILET SEATS RED. [LAUGHTER] IN THE MIDDLE OF THE NIGHT, I USED THE FACILITY, AND I DIPPED MY TENNIS SHOE IN THE RED PAINT. I SOON WAS DEMANDED TO COME TO THE DEAN'S OFFICE FROM THE MASS GENERAL, WHERE AN ARRAY OF ADMINISTRATORS INCLUDING THE DEAN SAID, THOMAS WALDMANN, YOU ARE MORALLY AND FINANCIALLY RESPONSIBLE FOR THIS REPREHENSIBLE ACT. I WAS, HOWEVER, TOO MUCH OF A GRIND TO BE PUNISHED AND WAS EXONERATED. MY NEXT TRIP TO THE DEAN'S OFFICE WAS MORE ENCOURAGING. SHERM WISEMAN AND I RECEIVED A $50 GRANT TO STUDY ERYTHRO.TIN IN A NICHE OF HARVARD DENTAL SCHOOL. WE WENT ON AND I WENT ON TO AN INTERNSHIP AT THE MASS GENERAL HOSPITAL. THAT WAS THE YEAR OF THE LAST MAJOR POLIO EPIDEMIC IN OUR COUNTRY. MORE THAN 300 PATIENTS WITH POLIO ENTERED THE MGH AND K. FRANK AUSTIN AND I, BEING ON LESSER ROTATIONS, WERE BROUGHT IN TO HANDLE THE 70 RESPIRATOR PATIENTS THAT WERE THERE. IN THE NEXT YEAR, THERE WAS THE DEVELOPMENT OF THE SLOCK VACCINE, AND I BECAME ENORMOUSLY IMPRESSED WITH THE CAPACITY OF THE IMMUNE SYSTEM TO PREVENT ACUTE INFECTIOUS DISEASES THROUGH VACCINES, AND THUS ENTERED A 60-YEAR LOVE AFFAIR WITH THE FIELD OF IMMUNOLOGY. AT THAT TIME, VERY LITTLE WAS KNOWN ABOUT THE IMMUNE SYSTEM. MAXWELL WINPE.TRAUB WAS THE MAJOR HEMATOLOGY TEXT, HUNDREDS OF PAGE, YET THE TOTALITY ABOUT A LYMPHOCYTE WAS: THE FUNCTION OF LYMPHOCYTE IS STILL OBSCURE. BECAUSE OF THEIR STRATEGIC POSITION IN LYMPH NODES AND BECAUSE THEY'RE RICH IN -- IT'S BEEN SUGGESTED LYMPHOCYTE IS INSTRUMENTAL IN THE DESTRUCTION OF TOXIC PRODUCTS OF PROTEIN METABOLISM. A ROLE IN TRANSFERENCE OF FAT FROM INTESTINAL EPITHELIUM TO THE LACTEALS HAS BEEN DENIED. THAT CERTAINLY DOESN'T DO FULL JUST TI OF WHAT WAS KNOWN THEN, BUT IT REVEALS THE ENORMITY OF WHAT HAS DEVELOPED IN A PARTICULAR INDIVIDUAL SCIENTIFIC LIFETIME. BUT WE DID NOT HAVE THE TECHNIQUES WE HAVE NOW. VERY LITTLE IN THE WAY OF MOLECULAR BIOLOGICAL TECHNIQUES WERE THERE. WE DID NOT KNOW T-CELLS OR B CELLS OR RECEPTORS OR SIGNALING PATHWAYS OR CYTOKINES. AND SO ONE HAD TO USE ALTERNATIVE APPROACHES. WE USED THE STUDY OF EXPERIMENTS OF NATURE, THE PRIMARY GENETIC IMMUNODEFICIENCY DISEASES AND LEUKEMIAS OF LYMPHOCYTES WITH RETAINED FUNCTIONS. THIS WAS THE TIME BETWEEN THE THEKOREAN AND VIETNAM WAR, THE DOCTORS' DRAFT WAS STILL GOING ON AND I WAS ONE OF THE YELLOW BERETS THAT CAME TO THE NIH FOR A TWO-YEAR STAY. AS I SAID, WE WERE STUDYING THE PRIMARY IMMUNODEFICIENCY DISEASES. ONE OF THE WAYS WE STUDIED THEM WITH JESSI STEIN FELD AND BOB GORDON WAS WITH PROTEIN TURNOVER, THE PEACEFUL USE OF RADIONUCLIDES. AND SO AS YOU MIGHT GUESS, THE MAJORITY OF PATIENTS WITH GENETIC LOW GAMMA GLOBULIN, IT WAS DUE TO DECREASED SYNTHESIS. HOWEVER, AS WE DID THESE TURNOVER STUDIES WITH ALBUMEN AND IGG, THERE WERE GROUPS OF PATIENTS THAT WERE NOT THE SYNTHESIS BUT OF SURVIVAL. SO IF ONE LOOKS AT DISORDERS OF IMMUNOGLOBULIN KA TAB LISM OR LOSS, WE FIRST DEFINED THAT HUMANS AND CHICKENS WITH -- DYSTROPHY HAVE A VERY SHORT SURVIVAL OF IGG ALONE. PEOPLE WHO HAD A CRYOGLOBULIN ANEMIA THAT WAS MIXED AND IGM ANTIIGG HAD A SHORT IGG SURVIVAL SURVIVAL, AND THOSE WHO MADE ANTIBODIES TO IGA HAD A VERY SHORT SURVIVAL OF THIS IMMUNOGLOBULIN CLASS. THERE WAS ALSO HYPERCATABOLISM OF SEVERAL PROTEIN CLASSES. WE HAD ONE WHERE A BROTHER AND SISTER OF AN INBRED MARRIAGE HAD VERY LOW ALBUMEN AND IGG. AND IT TURNED OUT THAT THEY WERE MISSING A PROTECTION SYSTEM THAT IS NORMALLY INVOLVED IN THE TRANSFER OF IMMUNOGLOBULINS FROM THE MOTHER TO THE FETUS AND IN POSTPARTUM INVOLVED IN THE PROTECTION OF THE SURVIVAL OF IG IGG AND, IT TURNS OUT, ALBUMIN. USING SERUM AND PCR, ONE COULD DEFINE WITH COLLABORATORS THAT THERE WAS A MUTATION IN THE -- BOTH ELEMENTS OF BETA 2 GLOBULIN OF THE RECEPTOR NEONATE. BUT THERE WERE ALSO GI LOSS. WELL-KNOWN, OF COURSE, WAS LOSS OF PROTEIN INTO THE URINE. BUT WITH JESSI STEINFELD AND BOB GORDON, WE DEFINED ANOTHER CAUSE OF LOSS AND SHORT SURVIVAL THAT IS LOSS OF PROTEIN INTO THE INTESTINAL TRACT. WHEN WE USED IODINE ATED PROTEINS AS I SHOWED YOU BEFORE, WE COULD TELL THAT THE SURVIVAL OF THE PROTEINS WAS SHORT. BUT WE COULD NOT DETERMINE WHERE THEY WERE BEING DEGRADED OR LOST LOST. EYE OWE NATEED PROTEINS -- WHICH GETS EXCRETED IN THE EUROPE, IT DOES NOT URINE, IT DOES NOT REVEAL LOSS INTO THE INTESTINAL TRACT, SO WE DEVELOPED ANOTHER ALTERNATIVE, CHROMIUM 51 LABELED AL ALBUMIN. HERE THE CHROMIUM IS NEITHER SECRETED NOR ABSORBED ONCE LOST INTO THE INTESTINAL TRACT. NORMALS LOSE .2 TO 1.6% OF THE ADMINISTERED I.V. CHROMIUM INTO THE STOOLS. PATIENTS WITH G.I. LOSS CAN LOSE UP TO 1600-MILLILITERS OF PLASMA CLEAR OF THEIR PROTEINS PER DAY. THAT IS, ON YOUR RIGHT, YOU'RE SEEING THE EXCESSIVE EXCRETION OF CHROMIUM 51 IN THE STOOL FOLLOWING I.V. ADMINISTRATION. SO THUS WE HAD A WAY OF DEFINING PROTEIN-LOSEING ENTEROPATHY. ONE CAN LOSE ALPHA 1 ANTITRIPSIN IN THE STOOL AND THERE ARE OTHER WAYS OF DEFINING THIS CATEGORY. I DON'T ASK YOU TO READ THIS SLIDE, ONLY TO BE OVERWHELMED BY THE NUMBER OF CONDITIONS WHERE LOSS OF PROTEINS OCCUR INTO THE INTESTINAL TRACT. SO JUST AS THE ARRAY OF CAUSES OF PROTEINURIA, THERE ARE AN ARRAY OF CAUSES OF LOSS OF PROTEIN INTO THE INTESTINAL TRACT. IF WE FOCUS ON DISORDERS OF THE LYMPHATICS, THAT IS, ABNORMALITIES OF THE LYMPHATICS ASSOCIATED WITH LOSS INTO THE INTESTINAL TRACT, THE EQUIVALENT OF A THORACIC DUCT FISTULA INTO THE BOWEL, WE AGAIN SEE A NUMBER OF CAUSES THAT ARE SECONDARY. WHIPPLE'S DISEASE, CONSTRICTIVE PERICARDITIS WHERE THE -- THORACIC DUCT -- WHERE PAIR CAR DECK THOME WILL REVERSE THE CONDITION. BUT THEN AN IDIOPATHIC HYPOPROTEIN I'MEMA ALSO HAD INTESTINAL LYMPHANGIECTASIA. THE FIRST SUCH FAULT, FULLER ALBRIGHT, IN THE LATE 40s, IN PATIENTS WITH IDIOPATHIC HYPOPROTEINEMIA NOTED THAT ALBUMIN INFUSIONS WERE ALMOST IMMEDIATELY SEEN AS EXCESS NITROGEN IN THE URINE, AGAIN SUGGESTING SUGGING DEGRADATION OR LOSS. IN 1961, THE STUDIES WERE COMPLETED, AND WE DEFINED IN EAB PATIENTS 18 PATIENTS, INTESTINAL LYMPHANGIECTASIA. HERE WAS THE PAPER IN "GASTRONEUROLOGY." WHAT WE NOTED WAS THE PROTEINS WITH A LONG SURVIVAL, ALBUMIN AND IGG, T ONE HALF OF 17 IN 24 LOWEST LEVELS, BUT ONE HAD LOW LEVELS OF ALL REASONABLY LONG SURVIVING PROTEINS. THE FRACTIONAL CATABOLIC RATE OVER NORMAL ON YOUR LEFT IS ESSENTIALLY THE SAME IN ALL OF THEM. THAT IS, IT IS HIGH NOT ONLY FOR IGG, BUT ALSO FOR THE VERY LARGE PROTEIN IGM. THIS CONTRASTS WITH A SITUATION WITH THE KIDNEY. HERE THE SIEVVING FUNCTION OF THE KIDNEY IS RETAINED, IGM IS LOST MUCH LESS THAN THE SMALLER MOLECULES IGG AND THAT, IN TERN, TURN, LESS THAN ALBUMIN. HERE THERE IS BULK LOSS SYNTHESIS. HOWEVER, ON YOUR RIGHT IS REASONABLY NORMAL. THE PATIENTS CAN MAKE IMMUNOGLOBULINS. SO INTESTINAL LYMPHANGIECTASIA HAS A NUMBER OF CHARACTERISTICS. ITS AGE OF ONSET IN OVER 90% OF THE CASES IS FROM BIRTH TO 28 YEARS. IT IS USUALLY SPORADIC, BUT WE KNEW OF AT LEAST SEVEN FAMILIES WHERE TWO TO FOUR MEMBERS WERE INVOLVED. ALMOST 20%. THERE IS GENERALIZED EDIE MA, GENERALIZED EDEMA AND CHYLOUS EFFUSIONS. MILD GASTROINTESTINAL IN SYMPTOMS, NOT AT ALL IN 20%. THERE ARE VERY LOW LEVELS OF ALBUMINS AND GAMMA GLOBULINS DUE TO THIS LOSS. AND DUE TO THE LOSS OF RECIRCULATING LYMPHOCYTES, NAIVE CD44RA LYMPHOCYTES ARE PROFOUNDLY REDUCED. THE CHARACTERISTIC FEATURE IS A DILATED LYMPHATIC CHANNEL ON BIOPSY OF THE SMALL BOWEL, AND DUE TO THE LYMPHOCYTOPENIA, THERE'S SKIN ANERGY AND ABNORMAL ALLOGRAFT REJECTION. SO HERE IN THIS YOUNG LAD, YOU CAN SEE THE LYMPHEDEMA INVOLVED, WHICH IS TYPICAL, OUR CASES IN GENERAL ARE MORE SEVERE THAN THOSE YOU'RE GOING TO HEAR FROM MIKE LENARDO. AS I SAID, 45% OF THE CASES, THE DISORDERED LYMPHATICS POUR THEIR LYMPH FLUID IN EITHER THE THORACIC OR ABDOMINAL CAVITY, AND SO CHYLOUS OR MILKY EFFUSIONS ARE QUITE COMMON. THERE ARE -- THE X-RAYS OF THE SMALL BOWEL ARE ABNORMAL IN OVER 75% OF THE PATIENTS. IN IS THICKENING ENLARGEMENT OF THE VALVE YAR CHONDRANEITIES. PEDAL LIMB FAN JOE GRAMS WERE ABNORMAL IN ALMOST ALL CASES. IN SOME CASES IF YOU INJECT DYE INTO THE FOOT, INTO THE LYMPHATIC, IT WOULD APPEAR IN THE BOWEL LUMEN IN A NUMBER OF THE CASES. SO HERE YOU WILL NOTE THE WHITE STREAKS COURSING OVER THE BOWEL LYMPHATICS. THIS WOULD BE A TYPICAL CASE. AT THE EXTREME CASE, YOU MIGHT HAVE A COCOON OF GUY FIBROUS TISSUE COVERING THE BOWEL, AND HERE IN THIS UNFORTUNATE PATIENT, YOU SEE THE ENORMITY OF THE ABNORMALITY. THE CHARACTERISTIC FEATURE IS THE DILATED LYMPHATIC ON INTESTINAL BIOPSY THAT YOU SEE HERE. OR HERE. OR HERE. OR HERE. AND THROUGH THESE DISORDERED LYMPHATICS, ONE LOSES LYMPHOCYTES. SO IF THE NORMAL CONTROL LYMPHOCYTE NUMBER IS 2500, THE MEAN VALUE OF THE PATIENT'S IS 700. AND YET THERE ARE PATIENTS WITH 200 LYMPHOCYTES. YOU WOULD ANTICIPATE IF YOU THOUGHT OF THE CONDITION AIDS THAT A LOW IGG AND A LOW LYMPHOCYTE WOULD BE ASSOCIATED WITH PROFOUND INFECTIONS. AND THAT IS LARGELY NOT THE CASE. THERE ARE SOME THAT HAVE INFECTIONS, TB FUNGUS, BUT FOR THE MOST PART, THEY DID NOT HAVE, AND THE REASON FOR THAT IS IN CONTRAST TO GENETIC IMMUNODEFICIENCY WITH LACK OF SYNTHESIS OR AIDS, THE PATIENTS CAN MAKE ANTIBODY AND CELLULAR IMMUNE RESPONSES TO A NEW ANTIGEN, ESPECIALLY CAN MAKE ANTIBODY RESPONSES. HOWEVER, WHEN IT CAME TO CELLULAR RESPONSES, NORMAL INDIVIDUALS COULD RESPOND TO AT LEAST ONE OF THE FOUR ANTIGENS YOU SEE BY SKIN TEST IN 95% OF THE CASES. HOWEVER, ONLY 17% OF THE PATIENTS WITH INTESTINAL LYMPHANGIECTASIA THAT WARREN STROBER AND I STUDIED COULD MAKE A POSITIVE SKIN TEST RESPONSE. DIE NITROCHLOROBENZENE, WHICH WE DO NOT USE THESE DAYS, COULD NOT SENSITIZE THE PATIENTS AS IT CONNOR MALLS. FURTHERMORE, OF THE FIVE PATIENTS STUDIES FOR OVER TWO YEARS, THEY DID NOT REJECT MISMATCHED SKIN GRAFTS. THAT IS, THEY WERE ANER JIK IN THE ABILITY TO MAKE AN ARRAY OF CELL MEDIATED IMMUNE RESPONSES. WELL, THE THERAPY OF SECONDARY INTESTINAL LYMPHANGIECTASIA WAS OKAY. WHIPPLE'S DISEASE, YOU CAN TREAT WITH ANTIBIOTICS OR ANTIBIOTICS AND STEROIDS, CONSTRICTIVE PERICARDITIS, YOU CAN TREAT WITH PERICARDIECTOMY, AND WITH THAT, YOU WILL NOW RETURN THE IMMUNE SYSTEM TO ITS FUNCTION. THE PATIENTS WITH TUBERCULOSIS, CONSTRICTIVE PERICARDITIS, HAS A NEGATIVE TEU BERKLIN TEST UNTIL YOU REVERSE THE LYMPHOCYTE LOSS, BUT THEN THE TESTS ARE POSITIVE. BUT IN IDIOPATHIC INTESTINAL LYMPHANGIECTASIA, THERE WAS VERY LITTLE. ONE USED MIDDLE CHAIN TRIGLYCERIDES RATHER THAN -- THE MIDDLE CHAIN UPTAKE IS IN THE VEINS RATHER THAN IN THE LYMPHATICS, BUT IT REALLY WAS A DIFFICULT CHALLENGE. SO THE NEXT CHAPTER IN THE STORY, I'M GOING TO TURN TO MIKE LENARDO, AND AT LEAST A SUBSET OF PATIENTS, I THINK THERE ARE DIFFERENT MOLECULAR ERRORS BUSH A SUBSET OF PATIENTS, HE'LL TALK TO YOU ABOUT WHAT ARE THE MOLECULAR ABNORMALITIES AND WHAT ARE THE IMPLICATIONS OF THESE ABNORMALITIES TO NEW APPROACHES TO THERAPY. MIKE? [APPLAUSE] >> HERE'S MY FINANCIAL DISCLOSURES. AND OUR LEARNING OBJECTIVES. SO I'D LIKE TO BEGIN JUST BY TALKING ABOUT HOW WE APPROACH THESE KINDS OF DISEASES, PARTICULARLY IF THEY'RE REFERRED TO AS A CONGENITAL AND LIKELY GENETIC ABNORMALITY. AND WITHIN NIAID OVER THE LAST FEW YEARS, A GROUP OF US HAVE PUT TOGETHER A CLINICAL GENOMICS PROGRAM THAT'S REALLY BEEN SPEARHEADED BY SOME OF THE INITIATIVES OF OUR SCIENTIFIC DIRECTOR, STEVE HOLLAND, TO TAKE CHILDREN THAT ARE REFERRED WITH GENETIC ABNORMALITIES AND THEN TRY TO REALLY GET DOWN NOT ONLY TO A GENE THAT WE THINK IS CAUSATIVE, BUT ALSO DEFINE THE PATHOGENETIC MECHANISM WITH THE IDEA OF FIRST OF ALL TRYING TO DO SOMETHING TO HELP THESE INDIVIDUALS, BUT ALSO TO UNDERSTAND MORE ABOUT HOW THE HUMAN IMMUNE SYSTEM WORKS. AS YOU CAN IMAGINE, THIS WOULD BEGIN WITH A CLINICAL WORKUP IN THE CLINIC, BUT THEN IT WOULD RAPIDLY GO INTO THE LABORATORY WHERE WE WOULD ANALYZE IN THIS CASE LYMPHOCYTES OR OTHER IMMUNE CELLS FOR ABNORMALITIES AND TRY TO DEFINE SOME CLEARCUT ABNORMALITY OR CELLULAR PHENOTYPE THAT THEN WE CAN LATER USE TO ASSAY FOR THE KINDS OF GENES THAT COME OUT OF FURTHER ANALYSIS. THIS IS THEN FOLLOWED, OF COURSE, BY THESE MASSIVELY PARALLEL DNA SEQUENCING TECHNOLOGIES, EITHER WHOLE EXOME OR -- COMPUTATIONAL ANALYSIS AND THEN GOES INTO THE PROCESS OF GENE VALIDATION. THIS TURNS OUT TO REALLY BE THE MOST CHALLENGING, THE MOST INTERESTING AND THE MOST NECESSARY PART OF THE PROCESS BECAUSE ONE OF THE BIG THINGS WE'VE LEARNED FROM THE HIGH THROUGHPUT SEQUENCING OF LARGE NUMBERS OF GENOMES IS THAT WE ARE JUST RIDDLED WITH VARIANTS AND DIFFER DRAMATICALLY FROM EACH OTHER AT THE GENETIC LEVEL SO YOU DON'T JUST GET A SINGLE GENE IN MOST INSTANCES, YOU GET A VARIETY OF POSSIBLE CANDIDATE GENES AND I'LL GO INTO A LITTLE BIT MORE OF THAT IN A SECOND. BUT ONCE THIS PROCESS HAS BEEN COMPLETED, THEN WE GO BACK TO THE CLINIC THROUGH THE GENETIC COUNSELORS TO ADVISE THE PATIENT AND THE FAMILY WHAT THE IMPLICATIONS ARE OF THE GENETIC LESION, AND HOPEFULLY GET A CONCEPT FOR A NOVEL THERAPY FOR THIS INDIVIDUAL. AND SO THE CIRCLE GOES AROUND AND AROUND, AND I JUST WANT TO MAKE TWO POINTS ABOUT THIS, FIRST OF ALL, THAT IN ORDER TO GET THIS PART DONE, WHICH AS YOU CAN SEE BY THIS LITTLE PIE CHART HERE, THE MOST TIME-CONSUMING OF STEPS IN THE RIGHT HALF OF THIS CIRCLE, YOU NEED ONE INDIVIDUAL WHO'S GOING TO BE THE QUARTERBACK FOR THAT, USUALLY A CLINICAL FELLOW, POSTDOC, SOMETIMES EVEN A GRADUATE STUDENT. BUT SURROUNDING THAT INDIVIDUAL, THIS IS THE PURPOSE OF THE PROGRAM, YOU NEED EVERYONE ELSE WHO KNOWS ABOUT THE PATIENT OR KNOWS ABOUT THE FAMILY OR KNOWS ABOUT THE DISEASE, AND THAT CAN RANGE FROM THE CASE MANAGER OR THE STUDY COORDINATOR NURSE ALL THE WAY THROUGH THE P.I., INCLUDING THE GENOMICS FOLKS, BIOINFORMATICS FOLKS, AND BIOCHEMISTS AND MOLECULAR BIOLOGISTS. SO IT'S REALLY A TEAM APPROACH IN THAT WE FOUND TO BE MOST EFFECTIVE, SO THE GROUP WITHIN NIGH NIAID IS HIGHLY INTERACTIVE, EVEN WITH GROUPS DOING THIS OUTSIDE OF OUR INSTITUTE AND ACROSS THE CLINICAL CENTER. THE GOAL, OF COURSE, IS TO UNDERSTAND THE PATHOGENETIC MECHANISM AS HE AS I DESCRIBED AND HOPEFULLY COME UP WITH AN IDEA FOR TREATMENT. SO IT'S HARD TO BELIEVE NOW, BUT IT WAS ONLY A GENERATION AGO THAT HARD CORE MOLECULAR BIOLOGISTS DIDN'T WANT TO HAVE MUCH TO DO WITH CLINICAL MEDICINE. CLINICAL RESEARCH WAS CONSIDERED SORT OF SOFT, ALTHOUGH I THINK WE SAW SOME OF THE BEST KINDS OF WORK DONE IN THE TALK THAT WE JUST HEARD, BUT IN GENERAL, PARTICULARLY GENETICISTS BASICALLY WENT FOR EVERY OTHER ORGANISM OTHER THAN HUMANS BECAUSE OF THE PRESUMED COMPLEXITY, SO E. COLI, PHAGES AND VIRUSES, MICE, FLIES, WORMS, AND IT'S ONLY BEEN IN THE LAST DECADE OR SO THAT REALLY A HUGE FOCUS HAS TURNED TOWARDS HUMAN GENETICS. AND PART OF THE REASON IS THAT THE DEVELOPMENT OF NEW TECHNOLOGY. SO WHEN I WAS AN UNDERGRADUATE AT JOHNS HOPKINS WORKING IN THE CHROMOSOME LAB AT VICTOR MAKUSIQR.K, THERE WERE DISEASES BEING DEFINED BUT ESSENTIALLY NOTHING KNOWN ABOUT WHAT THE CAUSATIVE GENES WERE OR THE PATHOGENETIC MECHANISMS. NOW AS THIS TALK I HOPE WILL EXEMPLIFY, THIS IS NOT THE CASE. SO OF COURSE AT THE TOP OF THE LIST IS THESE MASSIVELY PAIR PARALLEL TECHNIQUES THAT HAVE COME OUT OF A DRIVE FROM THE HUMAN GENOME PROJECT TO GET TO THE THOUSAND DOLLAR OR NOW BELOW WHOLE GENOME, BUT LET'S NOT FORGET ABOUT A WHOLE OTHER NUMBER OF ALLIED TECHNOLOGIES THAT ARE NOW AT OUR DISPOSAL IN THE LAB TO DO THIS KIND OF GENETIC ANALYSIS, INCLUDING HIGH EFFICIENCY TRANSFEKTION METHODS FOR HUMAN CELLS THAT I ALLOW YOU TO PUT GENES BACK ON, RNAI AND NOW CRISPR, SO IF YOU HAVE A CELLULAR PHENOTYPE, YOU CAN GO BACK BACK AND KNOCK OUT THE GENE, INDUCE PLURIPOTENT STEM CELLS ALLOWING US TO MODEL ALL SORTS OF DIFFERENT TISSUES IN THE BODY, AND ALSO OFTEN OVERLOOKED IS JUST THE SHEER DESKTOP COMPUTATIONAL POWER WE HAVE TO BE ABLE TO INTERROGATE AN ENTIRE GENOME ESSENTIALLY ON OUR LAPTOP. SO WHEN YOU PUT ALL THESE TOGETHER, IT ALLOWS US NOW TO DO HUMAN GENETIC RESEARCH WITH ALMOST THE SAME PRECISION AS ANIMAL MODELS, LACKING, OF COURSE, THE ONE INTERVENTION WHICH IS INTENTIONAL BREEDING OF DIFFERENT MUTANTS TOGETHER. WE HAVEN'T BEEN ABLE TO DO THAT OBVIOUSLY. ONE OF THE THINGS ALSO THAT'S GONE ON IN THE FIELD THAT'S IMPORTANT TO REMEMBER DRIVEN LARGELY BY THESE SEQUENCING TECHNOLOGIES IS AN EVOLUTION OF THE APPROACH TO HUMAN DISEASE. SO WHEN THE FIRST GENOME WAS DONE AND THERE WAS AN IDEA THAT WE NEEDED TO COMPARE VARIANTS FROM DIFFERENT INDIVIDUALS IN THE DISEASE SETTING, THIS SORT OF SHORTHAND METHOD FOR DOING THAT WAS TO JUST TYPE INDIVIDUALS FOR COMMON POLYMORPHISMS WITH THE IDEA THAT COMMON GENETIC CHANGES WOULD UNDERLIE COMMON DISEASES. SO YOU COULD DO HAPLOTYPES MUCH LESS EXPENSIVELY IN THOSE DAYS THAN DOING A GENOME, AND THE IDEA WAS TO TRY TO ASSOCIATE THOSE WITH DISEASES. HOWEVER, WE HAVE CHOSEN THE APPROACH OF GOING AFTER MENDELIAN DISORDERS. THIS DIAGRAM THAT CAME FROM THE REVIEW THAT JIM LUPSKI WROTE WILL GIVE YOU A VERY CLEAR UNDERSTANDING OF WHY WE THINK THIS IS AN IMPORTANT AND WORTHY APPROACH, AND THAT IS WHEN YOU HAVE A DE NOVO MUTATION THAT' RISES DURING MEIOSIS IN A GERM CELL AND POPS UP IN AN INDIVIDUAL, AND WHETHER THAT CAN BE THEN PASSED ON TO THE NEXT GENERATION, THE MOST DELETERIOUS AND HIGHLY PENETRANT ARE GOING TO BE RESTRICTED POSSIBLY TO THAT INDIVIDUAL IF THEY'RE VERY SICK AND DIE BEFORE REPRODUCTION OR, TO A SMALL GROUP WHERE IT HAS A VERY DA MATIC EFFECT ON FITNESS. BY CONTRAST, IF YOU LOOK AT COMMON SNPS THAT ARE OUT IN THE POPULATION AT THE LEVEL OF 1% OR GREATER, THOSE HAVE NOW BEEN PURIFIED BY GENERATIONS OF SELECTION AND, THEREFORE, THE IMPACT ON DISEASE IS MUCH, MUCH, MUCH LESS BY DEFINITION. AND IN FACT, THAT'S EXACTLY WHAT MOST GWAS STUDIES AT LEAST THE ONES I'M FAMILIAR WITH IN THE IMMUNOLOGY SPACE SHOW WITH THE EXCEPTION PERHAPS OF THE MHC GENES, WHICH HAVE A VERY HIGH LINKAGE DISEQUILIBRIUM WITH A LOT OF DIFFERENT AUTOIMMUNE DISEASES BUT FOR THE MOST PART, YOU GET VERY SMALL EFFECTS FROM THESE. ON THE OTHER HAND, WHEN YOU LOOK FOR THESE VERY HIGHLY PENETRANT EARLY MUTATIONS THAT ARE RESTRICTED WITHIN FAMILIES THAT YOU FIND VERY POTENT EFFECTS ON IMMUNE FUNCTION. JIM CALLS THIS CLAN GENOMICS, BECAUSE OF THE UNDERSTANDING NOW THAT THE ONLY PERSON, THE ONLY PEOPLE ON EARTH THAT ARE LIKE EACH ONE OF YOU IS -- ARE YOUR SIBLINGS, YOWSH PARENTS AND YOUR CHILDREN. ONCE YOU BEGIN TO GO BEYOND THAT, THEN SELECTION BEGIN TO HAVE AN EFFECT AND CERTAIN GENE ABNORMALITIES ARE NEVER GOING TO BE SEEN AGAIN. BUT THERE'S A MASSIVE NUMBER OF VARIANTS THAT WE FIND IN EACH GENOME, SO IT'S A VERY COMPLICATED SCIENCE, AND IN A WAY, WE'VE DISTILLED THAT COMPLEXITY DOWN, PERHAPS OVERSIMPLIFIED IT IN STUDYING THESE DISORDERS. SO I WANT TO TELL YOU ABOUT PATIENTS THAT ARE VERY, VERY SIMILAR TO WHAT TOM JUST SAID AND POINT OUT A COUPLE OF FEATURES. ONE IS THEY CAME TO OUR ATTENTION BECAUSE OF THE LOW IMMUNOGLOBULINS AND THE IMMUNODEFICIENCY THAT THEY HARBORED, BUT THEY HAVE THE SAME FEATURE THAT HE DESCRIBED, WHICH IS VERY LOW PROTEIN AND A CONSTANT G.I. UPSET AND DIARRHEA AND LOSS OF PROTEIN IN THE STOOL STOOL. THE CONSEQUENCE OF THAT FOR THESE CHILDREN IS QUITE SEVERE. THESE ARE GROWTH CHARTS, TYPICAL HEIGHT/WEIGHT GROWTH CHARTS, YOU CAN SEE THE RED LINE INDICATES PATIENTS WITH THIS DISEASE, THEY'RE JUST REALLY -- DON'T DO WELL. AS TOM DESCRIBED, IF YOU LOOK IN THE INTESTINE WITH BIOPSIES AND USE MARKERS OF LYMPHATIC SURFACE MARKERS, YOU CAN SEE THE DISTENDED LIMB LYMPHATICS THAT HE DESCRIBED. ONE OTHER FEATURE WE'VE SEEN IN THIS PARTICULAR FORM OF WALDMANN'S DISEASE IS ALSO A HIGH PROPENSITY FOR THROM THROMBOCYST. THIS SHOWS AN ECHO CARD GRAM WITH A HUGE THROMBUS IN THE RIGHT AKREE UM, THESE ARE SO BIG THEY HAVE TO BE ACTUALLY SURGICALLY REMOVED, THEY CAN'T BE MELTED AWAY WITH TYPICAL ANTI-COAGULATION. SOME OF THE OTHER FEATURES TOM MENTIONS ARE HERE, BUT THIS IS THE PHENOTYPE THAT WE OBSERVED IN A VARIETY OF INDIVIDUALS, AND IT TURNS OUT THAT THE GENE THAT WE FOUND THAT WAS COMMON TO A NUMBER OF FAMILIES WITH THIS DISORDER WAS A HOMOZYGOUS LOSS OF MUTATIONS IN THE CD55 GENES OR DECAY ACCELERATING FACTOR. STUDIED A LOT BY JOHN ATKINSON, WHO ACTUALLY HAD ALSO BEEN A CLINICAL FELLOW AT NIH BUT IS AT WASHINGTON UNIVERSITY IN ST. LOUIS FOR MANY YEARS. THESE ARE A VARIETY OF MUTATIONS THAT CREATE PREMATURE STOPS OR AMINO ACID CHANGES THAT DESTABILIZE THE PROTEIN, AND ALL ARE LOSS OF FUNCTION, LOSS OF PROTEIN EXPRESSION, AND THAT'S SHOWN BY THESE FLOW CYTOMETRY PLOTS HERE. HERE'S A NORMAL CONTROL, YOU CAN SEE IT'S A SURFACE PROTEIN, YOU CAN EASILY SEE EXPRESSED ON A VARIETY OF DIFFERENT KINDS OF CELL TYPES, AND HERE ARE PATIENT SAMPLES AND ACCEPT FOR PATIENT 6 THAT HAS A SMIDGEON OF IT LEFT, THEY'RE ALL PRETTY MUCH NULL, AND YOU CAN SEE IF YOU DO A WESTERN BLOT THAT VERIFIES THAT. AND CORRELATING WITH THAT IS THE FACT THAT YOU CAN SEE WE HAVE UNAFFECTED PARENTS HERE THAT HARBOR A SINGLE ALLELE, AND THE CHILDREN THAT ARE AFFECTED ALL HAVE EITHER COMPOUND HETEROZYGOUS OR HOMOZYGOUS RECESSIVE MUTATIONS. SO THAT CAUSED US TO GO BACK TO OUR TEXTBOOKS AND GO TO THE LITERATURE AND SORT OF RE-LEARN COMPLEMENT, AND IT TURNS OUT NOW COMPLEMENT IS MUCH MORE COMPLEX AND MUCH MORE IMPORTANT, PARTICULARLY FOR THE INNATE FORM OF IMMUNITY. AND THE REASON FOR THAT IS THAT THERE'S A VARIETY OF DIFFERENT WAYS TO ACTIVATE THE CENTRAL MEDIATOR OF COMPLEMENT, THE ENZYME C3 CON VER TASTE, THAN THE CLASSIC IMMUNE COMPLEX DRIVING C1Q INTO THE PATHWAY. THESE INVOLVE A NUMBER OF THINGS OF INTEREST, THIS IS DAMAGE ASSOCIATED MOLECULAR PATTERN, APOPTIC CELL ASSOCIATED MOLECULAR PATTERN AND PATHOGEN ASSOCIATED MOLECULAR PATTERN, AND THESE ARE PICKED UP BY SPECIFIC PROTEINS THAT THEN CAN ALSO DRIVE THE ACTIVATION OF C3 CONVERTASE. ONE OF THE EFFECTS OF THIS IS OF COURSE THE WELL-KNOWN ACTIVATION OF THE REST OF THE CASCADE OF PROTEASES THAT RESULT IN THE MEMBRANE ATTACK COMPLEX THAT CAN BIND TO A MICROORGANISM AND POP IT, ESSENTIALLY, BUT IT CAN ALSO DEPOSIT ON OUR OWN CELLS AND CAUSE DAMAGE AS I'LL SHOW YOU IS THE CASE IN THIS DISEASE. SO CD55 ACTS AS A DIRECT BLOCKER. THIS IS JUST A MODEL OF THE PROTEIN STRUCTURE ACTUALLY PHYSICALLY BINDING AND INHIBITING C3 CONVERTASE. ONE THING I'D LIKE TO MENTION THAT TIES THIS WITH IMMUNOLOGY IS THAT THE C3 CONVERTASE AND C5 CONVERTAS E, ONE OF THE SPLIT PRODUCTS OF THOSE PROTEINS ARE THE SO CALLED A FRAGMENTS WHICH ARE KNOWN AS ANTIPHYLOTOXINS BECAUSE THEY CAN ACTUALLY HAVE ANTISTIMULATORY EFFECTS ON A RANGE OF CELLS, MAINLY MACROPHAGES BUT ALSO T-CELLS, AND MODULATE THEIR ACTIVITY AS I'LL SHOW YOU THROUGH SPECIFIC RECEPTORS. SO THERE'S THIS INTERMARRIAGE THAT HAS BECOME OBVIOUS AS WE'VE UNDERSTOOD THE FUNCTIONS OF THESE PROTEINS AND IT REMINDS ME OF AN ANECDOTE, ONE DAY -- AS YOU KNOW MY CHIEF FOR OVER 25 YEARS WAS BILL PAUL, WHO PASSED AWAY A COUPLE YEARS AGO SADLY, ALSO ONE OF THE GREAT GIANTS OF NIH INTRAMURAL SCIENCE. BILL EDITED THIS LARGE TEXTBOOK CALLED "FUNDAMENTAL INEU NOLG." I REMEMBER WALKING INTO HIS OFFICE ONE DAY WHEN HE WAS EDITING THE CHAPTERS AND HE SAID, YOU KNOW, I USED TO ALWAYS HATE READING THE COMPLEMENT CHAPTER BECAUSE IT WAS SO BORING. BUT HE SAYS NOW WITH ALL THESE GENE KNOCKOUTS, WE'RE FINDING ALL THIS WAYS IT INFLUENCES THE IMMUNE SYSTEM, AND I THINK THIS DISEASE ACTUALLY EXEMPLIFIES HIS STATEMENT. SO HOW DO WE KNOW THAT THIS IS, IN FACT, WHAT'S GOING WRONG IN THESE PATIENTS? WE LOOK AT THEIR CELLS AND TRY TO FIND EVIDENCE THAT COMPLEMENT IS DERANGED. ONE OF THE WAYS TO DO THAT IS LOOK' ONE OF THE PRODUCTS OF C3 WHICH DEPOSITS AND STABLY RESIDES ON CELLS ONCE COMPLEMENT HAS BEEN ACTIVATED. YOU CAN SEE WHEN YOU LOOK AT PATIENT CELLS HERE IN BLUE OR RED, COMPARED TO NORMAL CONTROLS, YOU CAN ACTUALLY SEE BY STAINING THE SURFACE OF THE CELLS THAT THERE IS DEPOSITION OF THIS COMPLEMENT FRAGMENT. AND IF YOU LOOK AT A VARIETY OF PATIENTS DIRECTLY EX VIVO COMPARED TO NORMAL CONTROLS USING EITHER NORMAL MEDIA OR ACIDIFIED MEDIA WHICH PROMOTES THIS PROCESS CALLED THE HAM TEST, YOU CAN ACTUALLY SEE THERE IS SIGNIFICANTLY MORE DECH DEP SITION DEPOSITION OF THIS, ALTHOUGH THERE'S MORE CONSTITUTIVE DEPOSITION OF THIS IN NORMAL PEOPLE, IN ALL OF US, AND WE THINK THAT THAT'S OBVIOUSLY REGULATED OR LIMITED BY THE PRESENCE OF THE CD55 PROTEIN. ONE OF THE FASCINATING THINGS ABOUT THIS THAT I LEARNED IN THIS PROJECT WAS THAT THESE COMPLEMENT COMPONENTS, UNLIKE WHAT I LEARNED IN MEDICAL SCHOOL, THESE WERE JUST SYNTHESIZE BID THE LIVER AT PUT INTO CIRCULATION, ACTUALLY ARE MADE BY IMMUNE CELLS, IN PARTICULAR, T-CELLS, SO YOU CAN TAKE THE LEUKEMIA CELL LINE WHICH WE USE ALL THE TIME IN THE LAB AS SORT OF A REAGENT CELL LINE AND YOU CAN ACTUALLY SEE THAT IT MAKES COMPLEMENT PROTEINS, AND ALSO HAS CD55. IF CD IF FIVE IS KNOCKED DOWN BY CRISPR, YOU ACTUALLY NOW SEE DEPOSITION OF THE C3D COMPLEMENT COMPONENT ON THE SURFACE OF JERCAD, SO IN THE SINGLE LINE ITSELF, YOU CAN SEE WHAT'S GOING ON IN THE IMMUNE CELLS IN SORT OF A MODEL SYSTEM WAY. SO WHAT EFFECTS DO THESE TOXINS HAVE IN THE PATIENTS? WELL, THEY MODULATE THE TYPES OF CYTOKINES THAT THEY MAKE OR DON'T MAKE. TNF ALPHA, SHORT OF SORT OF THE CHIEF INFLAMMATORY CYTOKINE WE STUDY, WE BELIEVE -- WE KNOW THIS IS DUE TO THE RECEPTORS BECAUSE IF YOU USE THEM, YOU CAN SEE THAT THAT SUPPRESSES NOT ONLY THE PATIENT ABNORMAL TNF SECRETION BUT ALSO SUPPRESSES TNF SECRETION IN THE NORMAL CONTROLS. ALSO IL10 PROTECTION IS REDUCED IN THE PATIENTS, AND YOU CAN SEE THAT LOOKS -- IT'S HARD TO TELL BECAUSE THERE'S NOT MUCH CHANGE, BUT IT LOOKS LIKE IT MAY ALSO BE AN EFFECT OF THE TOXIN PROPERTIES SO THERE'S NOT SIMPLY GENERATION OF THE MEMBRANE ATTACK COMPLEX BUT A WHOLE HOST OF EVENTS THAT TAKE PLACE IF COMPLEMENT ACTIVATION IS NOT PROPERLY INHIBITED BY CD55. IF WE LOOK AT THE GUT TO TRY TO TIE THIS DIRECTLY WITH THE PATHOLOGY OF WALDMANN'S DISEASE, YOU CAN SEE THAT THERE IS -- YOU CAN SEE DEPOSITION OF C3 AND THE MEMBRANE ATTACK COMPLEX IN VESSELS WITHIN THE INTESTINE AND BIOPSIES FROM THESE PATIENTS AND ALSO THIS IS ACTUALLY A LYMPHATIC. AND IF YOU JUST SCAN WITH DIFFERENT BIOPSIES THE BOWEL OF SOME OF THESE PATIENTS YOU SEE FIRST OF ALL MASSIVE LYMPHOCYTE INFILTRATION IN CERTAIN FOCAL POINTS OF THE BOWEL, IN FACT MOST OF THE CASES HAVE BEEN ORIGINALLY DIAGNOSED AS INFLAMMATORY BOWEL DISEASE BECAUSE OF THIS KIND OF A PICTURE, BUT THEN YOU ALSO SEE THESE HUGELY DISTENDED LIMB LMPHATICS THAT WE THINK ESSENTIALLY BREAK AT THE SEAMS A BIT AND THAT'S WHAT'S DUMPING THE PROTEIN-RICH FLUID INTO THE GUT RATHER THAN RETURNING IT BACK THROUGH THE LYMPHATICS TO THE CIRCULATION. SO HOW DO WE -- WHAT WOULD WE DO ABOUT THIS NOW THAT WE KNOW THE GENE AND WE THINK WE KNOW THE PATHOGENIC MECHANISM? LUCKILY THERE HAS BEEN DEVELOPED AND CLINICALLY APPROVED BY THE FDA A COMPLEMENT INHIBITOR AND THERE'S SOME OTHERS THAT ARE OUT THERE THAT HAVEN'T YET REACHED CLINICAL APPROVAL SO WE WANTED TO TRY THOSE, SO FIRST OF ALL ONE IS NOT YET RELEASED FOR CLINICAL USE BUT YOU CAN SEE IT JUST PROFOUNDLY SUPPRESSES THIS ABNORMAL COMPLEMENT ON LYMPHOCYTES FROM THE PATIENTS IF YOU USE THAT. AND IF YOU USE THE ANTIBODY THAT IS CALLED ECOLIZAMAB, SOLIRIS, IT JUST FLAT LINES COMPLEMENT ACTIVATION, BOTH IN THE NORMAL CONTROLS AND IN PATIENTS. NOW ONE INTERESTING QUESTION, OF COURSE, THAT COMES OUT OF THIS IS FIRST OF ALL WILL IT AFFECT -- CAN YOU IMPROVE THE PATIENTS' DISEASE BY GIVING THIS ANTIBODY, NOW WE THINK WE KNOW THE BIOCHEMICAL MECHANISM, BUT ALSO WOULD THERE BE AN ADVERSE EFFECT OF SOME NORMAL EFFECT THAT YOU HAVE TO HAVE COMPLEMENT THERE, AND THOSE ARE DIFFICULT QUESTIONS THAT WOULD REQUIRE LARGE STUDIES IN THE FUTURE, BUT I'LL SHARE WITH YOU THAT AS OUR FINDINGS BECAME KNOWN A GROUP IN ISRAEL HAD IDENTIFIED A LARGE -- WITH CD55 DEFICIENCY WITH SEVERAL AFFECTED PATIENTS, SO THEY USED IT IN THESE PATIENTS AND THIS COULD FIND FIRST OF ALL THE DEPOSITION OF COMPLEMENT ON WHITE CELLS WENT DOWN SO BIOCHEMICALLY IT WAS DOING WHAT IT WAS SUPPOSED TO DO BUT WHAT WAS REALLY EXCITING IS IF YOU LOOKED AT ALBUMIN CONCENTRATION WITH A LONG ENOUGH TREATMENT, YOU COULD SEE ALL THREE AFFECTED MEMBERS THAT WERE TREATED GOT UP INTO THE LOWER LIMIT OF NORMAL RANGE OF ALBUMIN, TOTAL PROTEIN WAS UP AND IF YOU LOOK AT THEIR CLINICAL PHENOTYPE IN TERMS OF BOWEL MOVEMENTS, THESE WERE REDUCED, SUGGESTING THAT THE PATHOLOGICAL PROCESS WAS BEING REVERSED. SO THIS IS INCREDIBLY ENCOURAGING FOR THE ABILITY TO TAKE CARE OF THESE PATIENTS IN THE FUTURE. SO THE TAKEHOME MESSAGE, THIS IS CD55 DEFICIENCY WITH HYPERACTIVATION OF COMPLEMENT, ANGIOPATHIC THROMBOSIS, AND WE HAVE NOT YET -- THERE ARE A NUMBER OF CROSSOVERS THAT HAVE BEEN IDENTIFIED IN THE LITERATURE BETWEEN THE CLOTTING CASCADE AND THE COMPLEMENT CASCADE, WE HAVEN'T EXACTLY FIGURED OUT WHY THEY HAVE SUCH A PREDISPOSITION TO THIS, BUT IT SEEMS TO GO HAND IN HAND WITH CD55 DEFICIENCY, AND OF COURSE PROTEIN -- WE CALL THIS PARTICULAR VERSION OF WALDMANN'S DISEASE CHAPEL DISEASE WITH OUR NAMING CONVENTION FOR THESE NE GENIC DISORDERS AND I DESCRIBED TO YOU WHAT THIS IS DUE TO. THE DIAGNOSIS NOW BECOMES VERY EASY WHEN YOU GET A PATIENT WITH THIS DISEASE, YOU CAN STAIN FOR CD55 AND/OR CD3 DEPOSITION, AND THE IN VITRO AND CLINICAL STUDIES SUGGEST NOW THAT WE HAVE A TREATMENT FOR CHAPELLE DISEASE. I JUST WANT TO POINT OUT IN CLOSING THAT THIS IS NOT A ONE-TIME LUCKY HIT THAT WE GOT. IN FACT, THE WHOLE GENOMICS PROGRAM HAS BEEN SOLVING THESE WITH INCREASING EFFECTIVENESS OVER THE LAST FEW YEARS. I'LL JUST POINT OUT CHAI DISEASE, WE CAN DIAGNOSE AND TREAT THAT, LRBA DEFICIENCY SIMILARLY CAN BE TREATED BY THAT BECAUSE IT LEADS TO A DECREASE IN CTLA-4 SO BOTH THESE CAN BE DIAGNOSED, I'VE ALREADY DESCRIBED TO YOU KHAI DISEASE,, XMEN DISEASE, WEE EV FOUND ORAL MAGNESIUM CAN HELP THAT, AND WE'VE DISCOVERED -- STAFF CLINICIAN ARE SHOWED THAT THIS COULD BE TREATED WITH RAPAMYCIN, AND NOW WE HAVE A CLINICAL TRIAL IN THE CLINICAL CENTER OF A SPECIFIC P110 INHIBITOR FROM NOVARTIS TO TREAT THAT DISEASE. SO YOU CAN SEE THAT THESE NEW TECHNOLOGIES I'VE DESCRIBED TO YOU HAVE ALLOWED US TO TAKE PREVIOUSLY UNDEFINED GROUPS OF PATIENTS THAT CAME WITH A SERIOUS PATHOLOGY BUT UNKNOWN MOLECULAR BASIS, AND ACTUALLY PUT A MOLECULAR IDENTITY TO IT, WHICH ALLOWS IMPROVED DIAGNOSIS AND TREATMENT. SO I'LL STOP THERE AND JUST PEOPLE INVOLVED AS YOU CAN IMAGINE, BUT I WANT TO POINT OUT THE GROUP THAT WORKED ON CHAI DISEASE, AND THOSE IN VIENNA, US AUSTRIA. SO THANK YOU, TOM, FOR DEFINING THIS DISEASE FOR US AND LEEGD THE WAY THAT WE CAN FOLLOW IN YOUR FOOTSTEPS AND ACCOMPLISH THIS. [APPLAUSE] >> SO PLENTY OF TIME FOR QUESTIONS. PLEASE USE THE MICROPHONES IN THE AISLES. >> TO BOTH OF YOU, BUT TOM, I'VE HEARD YOU TALK MANY TIMES OVER THE YEARS AND EACH TIME YOU DO, YOU HAVE SOMETHING ADDITIONALLY EXCITING TO SAY, AND I CAN'T THINK OF ANYONE WHO EMBODIES MORE OF THE SPIRIT OF THIS INSTITUTION OVER ALL THESE YEARS THAN YOU DO, AND DR. LENARDO, I'M SURE I'LL SAY THAT ABOUT YOU, BUT IT WILL TAKE A FEW MORE YEARS BEFORE THAT. [LAUGHTER] I. JUST SAW A PATIENT IN THE CLINIC, IN FACT, I TOOK CARE OF SOME OF THESE PATIENTS WITH YOU BUT FOR THE LIFE OF ME I CAN'T REMEMBER WHY. I SAW A PATIENT IN THE CLINIC THAT WAS REFERRED BY DR. HOLLAND, WHO THOUGHT THIS PATIENT MIGHT HAVE LIPODYSTROPHY. SHE HAD MASSIVE SWELLING OF THE -- MASSIVE ENLARGEMENT OF THE LEGS, WHICH LOOKED TO US LIKE LYMPHEDEMA, AND SHE HAS WHAT LOOKS LIKE A SYNDROME CALLED MELROY SYNDROME. SO MY QUESTION IS, IS THAT IF THAT DISEASE IS A DISEASE OF THE LYMPHATICS, SOMETHING RELATED ANATOMICALLY TO THE LYMPHATIC VESSEL, AND WHAT WE'RE TALKING ABOUT HERE IS THE LYMPHATICS BEING KIND OF SECONDARILY AFFECTED BY THESE IMMUNE MECHANISMS. HOW DO THESE TWO THINGS RELATE TO EACH OTHER? IN OTHER WORDS, IS THERE A PRIMARY ABNORMALITY IN THE LYMPHATIC SYSTEM ITSELF, AND WHY IS THAT PARTICULARLY IN THE INTESTINAL AREA VERSUS THE OTHER AREAS WHERE THE LYMPHATICS ARE SEEN? >> I DON'T KNOW, BUT CERTAINLY THERE HAVE BEEN THOSE THAT FELT THAT VEGFR3 AND VEGFC AND D IN MELROY'S AND SO FORTH, EMBERGER'S SYNDROME IN GERMANY, WHERE THESE ARE ALTERNATIVE MUTATIONS THAT ARE ASSOCIATED WITH MELROY'S, TO ME, MELROY'S JUST TELLS YOU WHERE THE LEGS ARE INVOLVED, AND THAT PERHAPS IF ONE REALLY LOOKS, THERE IS SORT OF OBSTRUCTION OF THE LYMPHATICS CENTRALLY WHEN THE G.I. TRACT IS INVOLVED, AND WITH THAT, THERE IS DILATATION OF THOSE BEYOND THAT. BUT I THINK ONE WOULD HAVE TO REFLECT, AGAIN, AS I SAY, 2011, THERE WAS A PAPER FROM GERMANY ON EMBERGER'S, SO I THINK ONE HAS TO BRING THE WHOLE TOGETHER AND THINK OF THESE DISORDERED LYMPHATIC -- THERE ARE A NUMBER OF GENES DEFINEED INVOLVED IN THE GENERATION OF A NORMAL LYMPHATIC, AND I THINK IF ONE LOOKS AT DISORDERED LYMPHATICS AND PERHAPS ONE WILL FIND MULTIPLE GENETIC ERRORS THAT CAN HAVE AS THEIR COMMON GROUND SOME DISORDER OF THE LYMPHATICS, AND WITH THAT, OBVIOUSLY THE THERAPY THERAPIES MAY BE DIFFERENT DEPENDING ON THE DISEASE, BUT AT THAT TIME WAS, YOU KNOW, A -- JUST A CHANCE OBSERVATION ON A GUT BIOPSY HAS EVOLVED. THINKING OF THE PAST, I CAME HERE FOR A TWO-YEAR STINT, AND AFTER I WAS HERE A FEW YEARS, MY WIFE WAS APPROACHED AT A PARTY AND SAYS, DON'T WORRY, TOM WILL GET A GOOD JOB IN ACADEMIA SOMETIME. [LAUGHTER] WHAT I DIDN'T TELL YOU ABOUT THE RED TOILET SEAT IS THAT FOR THE NEXT SIX MONTHS, EVERY DAY IN MY LOCKED MAILBOX WAS A RED JELLY BEAN. I REALLY THINK SHERM WEISSMAN WAS PLAYING A PRACTICAL JOKE, BUT ANYWAY, THAT ENDED WHEN AT A PARTY IN WASHINGTON AT A BREAK, A 2-YEAR-OLD LITTLE GIRL CAME AND SAYS, DR. WALDMANN, HERE'S YOUR RED JELLY BEAN. SO ANYWAY, THAT'S THE END OF THE RED TOILET SEAT STORY. THANK YOU FOR YOUR COMMENT, BY THE WAY. >> ALL RIGHT. OTHER QUESTIONS? I DO HAVE ONE QUESTION FOR YOU IN TERMS OF THE VARIABILITY AT PRESENTATION. I WASN'T QUITE SURE WHETHER CHAI PATIENTS WERE PRESENTING IN INFANCY OR THEY'RE PRESENTING AT DIVERSE TIMES IN EITHER CHILDHOOD OR EARLY ADULTHOOD. CAN YOU GIVE US A LITTLE BIT MORE -- >> IT'S VERY EARLY. IT'S A VERY EARLY DISEASE. I DON'T KNOW WHETHER I MYSELF KNOW WHETHER IT'S IN INFANCY, BUT IT'S VERY EARLY, AND THE VARIATION IN PHENOTYPE IS NOW WE RECOGNIZE THE NORM, NOT THE EXCEPTION. SO EVERY GENETIC DISEASE WE FIND, THERE'S A TREMENDOUS AMOUNT OF VARIABILITY, IN SOME CASE, INCOMPLETE PENETRANCE. SO IN ONE DISEASE, WE HAVE WORKED ON FOR A LONG TIME AT NIH, AUTOIMMUNE -- THERE'S ONLY 70% PENETRANCE, SO A MOM HAVING THE SAME PENETRANCE AS HER CHILD MAY BE NORMAL BUT THE CHILD IS AFFECTED. I THINK THE GENETIC DETERMINANTS ARE THE INTERESTING ONES FOR US TO CHASE OVER THE NEXT FEW YEARS WITH THE SYSTEMS BIOLOGY PROGRAM. >> AND JUST LYMPHANGIECTASIA AS A WHOLE, THERE ARE TWO MAJOR PERIODS. THERE ARE THOSE VERY EARLY, YOU MAY SEE THAT PROFOUND ONE, MIGHT HAVE BEEN AT BIRTH AND DURING THE FIRST YEAR OF LIFE. THE OTHER IS DURING ADOLESCENCE, AS A SECOND TIME THAT YOU SEE IT, BUT ESSENTIALLY ALL OF THEM WERE BEFORE THE AGE OF 28, SO IN OTHER WORDS, THIS IS A YOUNG INDIVIDUAL'S PRESENTATION DISEASE. >> THANK YOU VERY MUCH TO BOTH OF YOU. NEXT WEEK, THERE WILL BE NO GRAND ROUNDS BECAUSE OF THANKSGIVING HOLIDAY. WE'LL BE BACK HERE ON THE 29TH FOR OUR FIRST CLINICAL PATHOLOGICAL CONFERENCE, AND SO WE'LL SEE THEN.