>> WELL, GOOD MORNING,
EVERYBODY, AND THANK YOU SO MUCH
FOR JOINING US ON THIS RAINY
TUESDAY MORNING WITH A LOT GOING
ON ON THE CAMPUS.
THIS SYMPOSIUM IS PART 1 OF A
TWO-PART CELEBRATION OF THE FIVE
MEMBERS OF THE NIH FAMILY
ELECTED THIS YEAR TO THE
NATIONAL ACADEMY OF THE
SCIENCES.
DRS. THOMAS KUNKEL, KYUNG
KWON-CHUNG, GIORGIO TRINCHIERI,
SANDRA WOLLEN AND STEVEN
ROSENBERG.
SO TODAY WE'LL HEAR FROM DRS.
KWON-CHUNG, KUNKEL, AND
TRINCHIERI.
AND ON NOVEMBER 13TH, WE WILL
HEAR FROM DRS. WOLLEN AND
ROSENBERG.
SO FIRST I'LL TELL YOU A LITTLE
BIT ABOUT EACH OF TODAY'S THREE
SPEAKERS AND THEN ALLOW THEM TO
PROCEED WITH THEIR INSPIRATIONAL
TALKS UNINTERRUPTED.
HOPEFULLY WE'LL HAVE TIME FOR A
QUESTION OR TWO AFTER EACH OF
THE TALKS, AND PLEASE, IF YOU DO
HAVE A QUESTION WHEN EACH TALK
ENDS, PLEASE USE THE M THE MICRS
ON EITHER SIDE OF THE
AUDITORIUM.
SO KYUNG KWON-CHUNG IS A NATIVE
OF SOUTH KOREA, AND SHE RECEIVED
HER UNDERGRADUATE DEGREE FROM
ICHWA UNIVERSITY, WOMEN'S
UNIVERSITY IN SEOUL.
SHE RECEIVED A FULBRIGHT
FELLOWSHIP TO PURSUE HER
DOCTORAL STUDIES IN BACTERIOLOGY
AT THE UNIVERSITY OF WISCONSIN.
IN 1966, SHE JOINED NIAID AND
THE FIELD OF MEDICAL MYCOLOGY.
SHE HAS BEEN THE CHIEF OF THE
MOLECULAR MICROBIOLOGY SECTION
IN THE LABORATORY OF CLINICAL
IMMUNOLOGY AND MICROBIOLOGY
SINCE 1995.
SHE RECEIVED THE LIFETIME
ACHIEVEMENT AWARD FROM THE
AMERICAN SOCIETY FOR
MICROBIOLOGY IN 2017.
AND HER TALK TODAY IS ENTITLED
IN STEP WITH SCIENCE, A HALF
CENTURY OF JOURNEY IN MEDICAL
MYCOLOGY.
DR. THOMAS KUNKEL RECEIVED HIS
DOCTORATE IN 1977 FROM THE
UNIVERSITY OF CINCINNATI.
FOLLOWING A POSTDOCTORAL
FELLOWSHIP AT THE UNIVERSITY OF
WASHINGTON, HE JOINED NIEHS IN
1982.
DR. KUNKEL CURRENTLY LEADS THE
DNA REPLICATION FIDELITY GROUP,
WITHIN THE GENOME INTEGRITY AND
STRUCTURAL BIOLOGY LABORATORY.
HIS LAB STUDIES DNA REPLICATION
AND HOW ENVIRONMENTAL STRESSES
PERTURB THIS PROCESS.
HIS TALK TODAY IS ENTITLED
"ACCURACY MATTERS."
DR. GIORGIO TRINCHIERI IS A
NATIVE OF ITALY WHO RECEIVED HIS
M.D. AT THE UNIVERSITY OF
TURINO.
HE JOINED THE NCI CENTER FOR
CLINICAL RESEARCH IN 2006, AND
IS CURRENTLY CHIEF OF THE
LABORATORY OF INTEGRATIVE CANCER
IMMUNOLOGY.
PRIOR TO JOINING NCI,
DR. TRINCHIERI WAS THE DIRECTOR
OF THE LABORATORY FOR
IMMUNOLOGICAL RESEARCH IN FRANCE
AND AN NIH FOGARTY SCHOLAR IN
THE LABORATORY FOR PARASITIC
DISEASES AT NIAID.
HIS TALK TODAY IS ENTITLED
"IMMUNITY TO INFECTIONS, CANCER,
AND IN BETWEEN."
CONGRATULATIONS TO ALL OF YOU,
AND DR. KWON-CHUNG, THE PODIUM
IS YOURS.
[APPLAUSE]
>> THANK YOU.
CAN YOU HEAR ME?
IT'S QUITE A CHALLENGE TO TALK
ABOUT 55 YEARS OF CAREER
TRAJECTORY IN 30 MINUTES.
SO I WILL START.
AS A ROAD MAP FOR THIS TALK, WE
INCLUDE PERSONAL BACKGROUND
FIRST.
AND THEN FOR THE MENTORS OF
MYSELF.
SINCE I SPENT -- I HAD ABOUT 70%
OF MY WORK WAS IN CRYPTOCOCCUS,
I'LL HAVE A BRIEF INTRODUCTION
TO CRYPTOCOCCOSIS, AND THEN
MILESTONE ACHIEVEMENTS AND THEIR
IMPACTS ON THE PROGRESS OF
MEDICAL MYCOLOGY.
WHAT IS MILESTONE ANYWAY?
I DEFINE THIS AS IF THAT WORK
LAY THE FOUNDATION SO THAT THE
PROGRESS CAN BE MADE IN NEW
DIRECTION IN MEDICAL MYCOLOGY.
AND THEN CONCLUSIONS AND
ACKNOWLEDGMENT.
FIRST BACKGROUND AND MENTORS.
IN 1960 I APPLIED FOR GRADUATE
STUDY IN THE U.S. BECAUSE I
RECEIVED THE FULBRIGHT SCHOLAR
SHIP, AND I WAS TEACHING AT
UNIVERSITY IN SOUTH KOREA, SEOUL
KOREA, BECAUSE THIS WAS AFTER MY
MASTER'S DEGREE IN BACTERIOLOGY
FROM THAT UNIVERSITY.
I WAS ALREADY A MOTHER OF TWO
LITTLE BOYS AT THAT TIME, AND
THIS PERSON HERE IS NOW THAT
TINY TODDLER, BUT NOW HE HAVE A
P.I. AT NHLIB -- NATIONAL HEART,
LUNG AND BLOOD INSTITUTE, AND HE
IS A P.I. AND HE'S WORKING THREE
FLEURS DFLOORS DOWN FROM MY OFF.
MY PH.D. WORK STARTED AT THE
UNIVERSITY OF WISCONSIN IN 1961.
IF YOU WANT TO FIGURE OUT WHAT
IS -- WHAT DOES 1961 LOOK LIKE?
THIS IS EIGHT YEARS AFTER DNA
STRUCTURE WAS FOUND OR AT LEAST
BECAME CLEAR.
SO WHAT'S DNA STRUCTURE AND
EIGHT YEARS AFTER THAT, AND 10
YEARS BEFORE, THE FIRST
RESTRICTION ENDONUCLEASE BECAME
AVAILABLE.
SO FIRST YEAR GRADUATE SCHOOL
WAS SUPPORTED BY THE FULBRIGHT,
AS I SAID, AND FELLOWSHIP WAS
MANAGED BY U.S. STATE
DEPARTMENT.
AND THE REMAINING YEARS, THE NSF
RESEARCH GRANT FOR THREE YEARS,
BECAUSE DR. RAPER HAD AN NSF
GRANT TO MAKE A NEW MONO GRAPH.
THEN I COMPLETED DOCTORATE IN
1965.
AND I'M VERY SORRY FOR THIS
FUZZY PICTURE, BUT DR. RAPER AND
I WERE STANDING IN FRONT OF THE
BUILDING OF BACTERIOLOGY IN
UNIVERSITY OF WISCONSIN IN '61.
WHO IS PROFESSOR KENNETH RAPER
ANYWAY?
MOST OF YOU MAY HAVE NOT HEARD
ABOUT HIM.
HE IS FATHER OF CYST AT SYSTEMN
THE MOST COMMON MOLD LIKE
ASPERGILLUS, PENICILLUM AND
CELLULAR SLIME MOLD.
HE DISCOVERED VERY HIGH
PENICILLIN YIELDING STRAIN OF
PENICILLUM CRISOJENUM AND PFIZER
USED THIS STRAIN AND AS A
RESULT, WHOLESALE COST OF 10,000
UNITS OF PENICILLIN WAS $20
THEN, BECAME 3 CENTS BY WORLD
WAR II BECAUSE OF HIS STRAIN.
HE DISCOVERED AND DESCRIBED THE
LIFECYCLE OF CELLULAR SLIME MOLD
IN 1933.
WHICH BECAME MODERN ORGANISM FOR
CELL BIOLOGY RESEARCH.
MY CAREER IN MYCOLOGY TOOK OFF
AFTER THE D THE DISCOVERY WHICHI
DISCOVERED WHEN I WAS IN
GRADUATE SCHOOL BECAUSE
DR. RAPER GAVE ME THE RESEARCH
PROJECT SAYING ISOLATE AS MANY
NEW SPECIES OF ASPERGILLUS AND
DESCRIBE PHENOTYPICALLY,
BIOCHEMICALLY.
SO THAT WAS MY PROJECT, AND
WHERE WAS THIS ASPERGILLUS
COMING FROM?
IT WAS COMING FROM SOIL
COLLECTED IN COSTA RICA.
WHY COSTA RICA?
BECAUSE MOST OF THE ASPERGILLUS
AND PENECILLUM WERE FROM TREM
PRATT ZONE CLIMATE AND VERY
LITTLE WAS KNOWN ABOUT THOSE
ASPERGILLUS POPULATION IN
TROPICAL SOIL.
BECAUSE HE GOT NSF GRANT, HE HAD
TO INCLUDE AS MANY SPECIES AS HE
COULD.
AND I DISCOVERED NINE NEW
SPECIES DISCOVERED FROM COSTA
RICA SOIL AND THEN ONE OF THEM
TURNED OUT TO BE THE FIRST
HETEROTHALLIC SPECIES IN THE
GENUS ASPERGILLUS WHICH IS
COMPOSED OF 132 SPECIES.
NOW, WHAT IS THE HETEROTHAT
WILLISM?
FIRST TIME IS HOMOTHALLISMTHAT E
SEXUAL STATE IN PURE CULTURE.
IN OTHER WORDS, IT'S A
HERMAPHRODITE.
SECOND TYPE IS CALLED
HETEROTHALLISM.
IT'S SELF STERILE BUT CROSS
FERTILE.
NOW, IT REQUIRES MATING WITH
OPPOSITE MATING TYPE.
AND TO COMPLETE SEXUAL
LIFECYCLE.
SO I FOUND THIS HETEROTHALLIC AS
PASPERGILLUS FROM COSTA RECAN
SOIL AND THAT REALLY BECAME THE
LAUNCHING PAD FOR MY MYCOLOGICAL
CAREER.
WHERE DID I GET MEDICAL MYCOLOGY
TRAINING AT NIH?
DR. CHESTER EMMONS, WHO USED TO
BE THE SECTION CHIEF OF MEDICAL
MYCOLOGY AT THE NIAID, HE IS A
LEGENDARY PIONEER IN PATHOGENIC
MYCOLOGY IN THE UNITED STATES.
AND HE HIRED ME AS A VISITING
FELLOW IN MEDICAL MYCOLOGY
SECTION.
AND HE DISCOVERED THE ECOLOGICAL
NATURE OF ALMOST EVERY IMPORTANT
SYSTEMIC FUNGAL DISEASE SUCH AS
HISTOPLASMOSIS, CRYPT
TOECOCCOSIS AND HE'S THE ONE
THAT FOUND THE ASSOCIATION OF
COCCIDIOIDES WITH DESERT
RODENTS.
HIS THREE EDITIONS OF MEDICAL
MYCOLOGY SERVED AS THE REFERENCE
FOR MEDICAL MYCOLOGY IN GRADUATE
SCHOOL AND MEDICAL SCHOOL FOR
MORE THAN THREE DECADES.
TO MY DISAPPOINTMENT, HE
ANNOUNCED RETIREMENT AND GONE
AFTER SIX MONTHS AFTER MY
ARRIVAL, AND SO I WAS FORCED TO
FUNCTION AS AN INVESTIGATOR ON
MY OWN AND DIDN'T REALLY GET THE
BENEFIT OF BEING TRAINED BY
DR. EMMONS.
IN 1974, I JOINED CLINICAL
MYCOLOGY SECTION HEADED BY JOHN
BENNETT IN THE LEVEL THREE OF
CLINICAL INVESTIGATION.
AS YOU CAN SEE HERE, WHEN IT
COMES TO DIVERSITY, ZERO.
[LAUGHTER]
ZERO DIVERSITY IN RACE, GENDER,
AND ACADEMIC DEGREE.
EVERYONE WAS M.D., EVERYONE WAS
WHITE MALE, AND THEN I POINTED
MYSELF WITH YELLOW ARROW.
CAN YOU SEE IT?
SO MY PRESENCE MADE TO LCI AS
98% HOMOLOGY INSTEAD OF 100%.
WELL, IT WASN'T A VERY
COMFORTABLE ENVIRONMENT, BECAUSE
I FELT LIKE AN ODD BALL.
BUT THE SAVIOR WAS JOHN BENNETT.
HE GAVE ME THE CHANCE TO BUILD A
PROJECT PROGRAM ON CRYPTO COCCIS
AND CRYPTO COCCOSIS.
WHEN I JOINED, HE JUST OPENED
HIS RESOURCES FOR ME TO USE.
HE HAD NEARLY 1,000 STRAINS OF
ZESEROTYPED CRYPTOFORMENS IN HIS
STOCK AND HE HAD ALL THE
INFORMATION ABOUT WHERE THIS IS
COMING FROM, WHETHER IT'S A
CLINICAL SOURCE OR ENVIRONMENT
SOURCE, IF IT IS, WHAT KIND OF A
SAMPLE OR CLINICAL SAMPLE DOES
THE ISOLATE COME FROM.
SO SINCE I WORKED 70% OF MY
CAREER ON CRYPTO COCCUS, I'D
LIKE TO INTRODUCE BRIEFLY ABOUT
THE FUNGUS AND THE DISEASE.
AS YOU CAN SEE HERE, THE DISEASE
STARTS FROM INHALATION, AND IT
WILL MULTIPLY IN THE LUNG IF THE
INDIVIDUAL HAS SOME IMMUNE
DEFECT ISSUE.
AND THEN IT WILL SPREAD AND
CROSS THE BLOOD BRAIN BARRIER
AND CAUSE FULL FULMINATING BRAN
DISEASE, WHICH IS 100% FATAL
UNLESS TREATED.
AND THE RISK FACTORS,
HIV-POSITIVE AIDS PATIENTS AT
THE HIGHEST RISK.
IDIOPATHIC CD4 CYTOPENIA AND
CORTICOSTEROID TREATMENT, OR
ORGAN TRANSPLANTATION OR EVEN
CANCER, THESE ARE THE RISK
FACTORS FOR CRYPTOCOCCISIS.
AND ITS MOST COMMON CAUSE OF
DEATH IS MENINGOENCEPHALITIS.
ACTUALLY THIS IS THE MOST COMMON
CAUSE OF MENINGITIS, NOT VIRAL,
IF YOU REMOVE THE -- THIS IS THE
MOST COMMON MENINGITIS CAUSE IN
THE U.S.
HERE I'M SHOWING VERY
INTERESTING LOOKING BRAIN, OF
COURSE.
THE LEFT, YOU CAN SEE THE BRAIN
IS COVERED WITH A WHITE.
THIS MEANS A CRYPTOCOCCIS IS
GROWING VERY WELL AND THE OTHER
ONE IS A BRAIN SECTION, AND THIS
GLISTENING AREA MEANS -- AS WELL
AS FUNGAL GROWTH.
NOW, THIS FUNGUS IS NEUROTROPIC
BECAUSE IT HAS THE ABILITY TO
CROSS THE BLOOD BRAIN BARRIER
FIRST, AND THEN IT CAN SURVIVE
IN VERY LOW GLUCOSE ENVIRONMENT.
AND THEN IT HAS ENZYME CALLED
LACCASE, WHICH OXIDIZES DOPAM
DOPAMINE -- AND NOREPINEPHRINE,
NEUROTRANSMITTERS.
NOW I'M GOING TO SHOW CHRONOLOGY
OF MILESTONE RESEARCH.
AS I SAID, MILESTONE IF IT LAY
THE FOUNDATION FOR A NEW
DIRECTION OF A STUDY IN
CRYPTCOCCUS AND CRYPTOCOCCOSIS.
IF YOU LOOK AT THIS SLIDE, THE
LOWER PART IS THE DEVELOPMENT
OF -- TECHNOLOGICAL DEVELOPMENT
WHICH IS RELEVANT FOR OUR STUDY,
AND THEN I HIGHLIGHTED THE PARTS
WHICH ARE ALL CRYPTOCOCCAL
RESEARCH.
AND THE MOST IMPORTANT PART IS I
DISCOVERED HETEROTHALLISM IN TWO
AGENTS OF CRYPTOCOCCOSIS IN 1975
AND 1976.
SO THIS IS A YEAR AFTER I
ARRIVED AT THE BENNETT
LABORATORY, I ASKED HIM, CAN YOU
COLLECT -- JUST PICK 20 ISOLATES
FROM HALF CLINICAL AND HALF
ENVIRONMENTAL FROM YOUR 1,000
STOCK CULTURES, AND HE PICKED
20, AND THEN I WAS THINKING
MAYBE IT'S A HETEROTHALLISM AND
STUDIED FROM THERE.
I FORMULATED CGB MEDIA THAT CAN
SEPARATE THE TWO CRYPT TOECOCCAL
PATHOGENS, BECAUSE AT THAT TIME
YOU REMEMBER THERE WAS NO DNA
SEQUENCE OR -- AND NO MOLOTOV
FOR SURE.
SO FOR THE IDENTIFICATION OF
THESE TWO SPECIES WAS QUITE
LABORIOUS, BECAUSE YOU HAD TO
HAVE EITHER ANTIBODIES SPECIFIC
TO EACH SEROTYPE, AND YOU KNOW
HOW IT COST AND THE TIME THAT'S
SPENT TO GET THE ANTIBODIES.
SO THIS MEDIA REALLY LAID THE
FOUNDATION FOR LOTS OF THINGS.
AND THEN DISCOVERED MOST
IMPORTANT BASIC VIRULENCE
FACTORS OF CRYPT COCCUS IN '82.
AND THEN TRANSFORMATION OF
CRYPTOCOCCUS, DNA MEDIATED
TRANSFORMATION IN 1990, AND THEN
CREATED A CONGENIC SET AT ONE OF
THE STRAIN THAT I CREATED WAS
USED FOR THE FIRST GENOME
SEQUENCING STRAIN OF
CRYPTOCOCCUS.
AND ONCE FRANCE FORMATION
TECHNIQUE WAS ACHIEVED AND FIRST
THING WE DID WAS MOLECULAR
KOCH'S POSTULATE WAS FULL FIELD
IN 1994.
AND WE IDENTIFIED THE FIRST
FUNGAL QUORUM SENSES PEPTIDE
BECAUSE THEY WERE ALL ORGANIC
ASSAY AND PEPTIDE WAS ONLY FOUND
IN -- BUT WE FOUND THE
CRYPTOCOCCUS HAS THE FIRST
PEPTIDE QUORUM SENSING MATERIAL.
AND THEN AZOLE HETERORESISTANCE
IN GM-CSF AUTOANTIBODIES AS A
MAJOR RISK FACTOR FOR C. GATTII,
BUT I CANNOT GO THROUGH ALL THIS
AND I'M JUST GOING TO
CONCENTRATE ON THE FIRST FIVE
SUBJECTS, BECAUSE THAT'S REALLY
THE MAJOR FOUNDATION WE LAID FOR
THE PROGRESS.
SO HOW DID THIS WORK WHICH I
CALL MILESTONE WORK LAY THE
FOUNDATION FOR ADVANCEMENT OF
WHAT FIELD OF RESEARCH?
FIRST CRYPTOCOCCUS WAS NOT EVEN
CLASSIFIED PROPERLY BECAUSE IT
DIDN'T HAVE A SEXUAL STATE
BEFORE.
AND THEN PHYLOGENY OF THE FUNGAL
KINGDOM COULD BE STUDIED
INCLUDING CRYPTOCOCCUS, AND
PATHOBIOLOGY FIELD
IDENTIFICATION OF ESSENTIAL
VIRULENCE FACTORS, AND THE
FULFILLMENT OF MOLECULAR COST
POSTULATE FOR VIRULENCE FACTOR,
AND THEN ECOLOGY IDENTIFIED THE
NEW SOURCE OF ECOLOGICAL NICHE
OF CRYPTO COCCI.
WHEN IT COMES TO EPIDEMIOLOGY,
EPIDEMIOLOGY OF THE TWO SPECIES
WHICH CAUSE CRYPTOCOCCOSIS WAS
QUITE DIFFERENT.
IN GENETICS -- AND IN CELL
BIOLOGY, IDENTIFICATION OF THE
FIRST FUNGAL DENSITY-DEPENDENT
QUORUM SENSING PEPTIDE AND THE
HETERORESISTANCE TO AZOLE DRUGS.
SO SINCE I CANNOT COVER ALL
THIS, I'M GOING TO JUST TALK
ABOUT THIS IN DETAIL, AS I SAID
BECAUSE THIS WAS REALLY THE VERY
BASIC FOUNDATION OF KNOWLEDGE
THAT WAS NEEDED FOR
CRYPTOCOCCUS.
NOW WHEN I JOINED DR. BENNETT'S
OFFICE/LABORATORY IN '74, I WAS
GIVEN A CHANCE TO STUDY KR CRYP,
BUT I HAD TO HAVE A GOAL FIRST.
SO MY FIRST GOAL WAS UNCOVERING
THE COMPLETE LIFECYCLE OF
CRYPTOCOCCUS BY INVESTIGATING
THE POSSIBILITY OF
HETEROTHALLISM BECAUSE UNTIL
THIS TIME, CRYPTOCOCCUS WAS
KNOWN AS A BUDDING YEAST, THAT'S
ALL.
AND DISCOVERED THE
HETEROTHALLISM LIFECYCLE IN
NEOFORMENS IN 1975 AND THE OTHER
SPECIES IN 1976, AND RESOLVE THE
TAXONOMY POSITION OF
CRYPTOCOCCUS WITHIN -- AND
ESTABLISHED THE FOUNDATION FOR
THE PHYLOGENETIC STUDY.
NOW, THIS IS THE WAY THE FUNGUS
WAS KNOWN FROM THE TIME OF
DISCOVERY WAS 1894 UNTIL 1975,
WHEN I DISCOVERED THE SEXUAL
STATE.
NOW THIS IS JUST A SCANNING
ELECTRON MICROSCOPY SHOWING THAT
IT'S COVERED WITH POLICY
SACCHARIDE CAPSULE.
WHEN I FOUND THE LIFECYCLE, I
WAS NOT ONLY HAPPY BUT WHOLE
THINGS START OPENING UP.
FIRST, LIFECYCLE HERE, THIS
IS -- I CALL MATTA AND MATT
ALPHA BECAUSE THERE WERE TWO
SEX, ALPHA AND A.
I JUST NAMED IT AS ALPHA AND A.
THERE'S NO REASON WHY IT CANNOT
BE PLUS OR MINUS OR SOMETHING
ELSE.
AND WHEN YOU MIX THESE YEAST
CELLS, ON THE MEDIA, WHICH IS
CONDUCIVE FOR THE SEXUAL
REPRODUCTION, THEN THIS -- YOU
CAN SEE THE ZONE HERE COMPLETELY
DIFFERENT MORPHOLOGY, AND THIS
IS WHAT IT WAS.
FIRST MATING TYPE A AND ALPHA
JUST BUD AND BUD, BUT WHEN THEY
MEET TOGETHER, THEY CON JEW GATE
AND PRODUCE THIS FLUFFY WHITE
AREA.
AND THE NUCLEAR FUSION OCCURS AT
THE TIP OF BASIDIA, AND THEN ME
IOS IS AND MEITIC NUCLEI
REMAIN -- BUD OUT THE SPECIES IN
THE LONG CHAIN.
THIS IS A SCANNING ELECTRON
MICROSCOPY.
SO COMPLETELY DIFFERENT
MORPHOLOGY OF ASEXUAL AND SEXUAL
STAGE OF CRYPTOCOCCUS.
HOW DID IT AFFECT TAXONOMY?
WELL, WE ALREADY SHOWED THAT
THERE MUST BE TWO SPECIES.
WE KNEW CRYPTOCOCCUS WAS
HETEROGENEOUS BUT NOT QUITE AS
TWO SPECIES.
SO THE TWO SPECIES CONCEPT WAS
EMERGED AFTER I SHOWED THIS
PERFECT STATE.
AND BEFORE THIS PERFECT STATE,
CRYPTOCOCCUS WAS ALWAYS
IDENTIFIED AS A SEROTYPE A OR
DOB OR C.
THIS IS ALL DR. BENNETT'S WORK.
NOW WHEN I DID THE MATING, AD
MATING TYPE PRODUCED THIS KIND
OF SEXUAL STATE, WHEREAS B AND C
SEROTYPE PRODUCE COMPLETELY
DIFFERENT MORPHOLOGY OF SPORES.
AND IT'S REALLY SURPRISING
BECAUSE OF THE 20 STRAINS HE
PICKED RANDOMLY, THERE WERE TWO
MATING TYPES, AND WE'LL LOOK AT
WHY I SAY THIS.
SO IF YOU DISCOVER THE
HETEROTHALLISM, HOW DID IT
IMPACT THE FIELD?
EPIDEMIOLOGICAL SIGNIFICANCE OF
MATING TYPE.
WE JUST THINK OH, THERE'S GOT TO
BE META, METAL FA, HALF AND
HALF, AMONG CLINICAL
ENVIRONMENTAL SOILS.
WHAT WE WERE SO SURPRISED WAS
METAL FA WAS 99.8%, MATTA IS
ONLY .2%.
IF YOU THINK ABOUT THE
PROBABILITY OF MYSELF GETTING 20
STRAINS FROM DR. BENNETT IN
WHICH TWO EYES LATS WERE META,
CAN YOU IMAGINE, SO I
CALCULATED, IT LOOKED LIKE
PROBABILITY IS 1 IN 1500
CHANCES.
1 IN 1500.
SO I WAS VERY LUCKY THAT HE
SELECTED 20 ISOLATES AMONG WHICH
THERE WERE TWO MATING TYPE A
STRAIN.
THAT WAS ALL HE HAD, TWO MATING
TYPE A STRAIN OUT OF 1,000.
CONSTRUCTION, CONGENIC SET OF
MATTA AND MATT ALPHA, BECAUSE
WHEN YOU SEE THAT MAT ALPHA
PREDOMINATES, THEN YOU'RE
CURIOUS ABOUT WHAT IS MATALPHA
DIFFERENT FROM MATA AND WHY IS
IT SO MUCH MORE COMMON?
NEXT QUESTION OF COURSE YOU WILL
ASK IS, WHAT IS THE ROLE OF
MATING TYPE IN VIRULENCE?
MAT ALPHA WAS FOUND TO BE
SIGNIFICANTLY MORE VIRULENT THAN
MATA IN THE ANIMAL MODEL.
WE DON'T KNOW WHY, BECAUSE
THERE'S JUST SO FEW MAT A OUT
THERE.
AND GENOMIC ANALYSIS OF MAT
LOCI, AND EVOLUTION OF FUNGAL
SEX CHROMOSOME PROJECT EMERGED.
MITOCHONDRIA INHERITANCE, UNO
PARENTAL INHERITANCE FORMAT A
PARENT JUST LIKE MAMMALIAN CASE,
THE MITOCHONDRIA COMES FROM THE
MOTHER, BUT MITOCHONDRIA FROM
CRYPTO COMES FROM MAT A ISOLATE.
NOW, WHO BENEFITED FROM ALL THIS
DISCOVERY OF HETEROTHALLISM AND
BUILT THEIR CAREER ON THE MAT
MATING?
JOE HEITMAN IN DUKE, HE HAD ALL
THIS COMPLETING THE MATING TYPE
LOCI GENETIC MAP OF MATING TYPE
LOCI.
AND PHEROMONE SIGNALING CASCADE
AND CELLULAR CIRCUITS REQUIRED
FOR MATING.
AND HE SPENT PROBABLY NO LESS
THAN 10 YEARS TO ACCOMPLISH ALL
THIS.
AND X. LIN NOW IN GEORGIA, BUT
SHE USED TO BE AT TEXAS A & M,
SHE DISCOVERED THE SAME SEX
MATING AMONG CRYPTOCOCCUS.
AND PHEROMONE INDEPENDENT
UNISEXUAL MATING.
AND THEN DR. XU IN CANADA, HE'S
THE ONE STUDIED THE MAT A
DEPENDENT MITOCHONDRIAL
INHERITANCE.
SO ALL THESE PEOPLE BUILT THEIR
CAREER BENEFITING FROM
HETEROTHALLISM LIFECYCLE OF
CRYPTOCOCCUS.
THIS IS 2000, SO THIS IS LONG
BEFORE CRY CRYPTOCOCCAL GENOME
SEQUENCING WAS ESTABLISHED.
WHEN MARVIN KAROS FROM GERMANY
WAS WITH US, -- WAS TEACHING HOW
TO ISOLATE THE MATING TYPES AND
WE WERE SO SU SURPRISED THAT ITS
AT LEAST 50KB LONG, AND IF YOU
LOOK AT -- THERE'S ONLY TWO
GENES AND THEY ARE LIKE LESS
THAN 8KB.
COMPARED TO THAT, 50KB AND ALL
THE MAT KINASE CASCADE GENES
WERE SITTING ON THE MAT LOCUS.
AND AS I SAID, AND WHEN GENOME
SEQUENCING WAS DONE IN 2004,
HEITMAN AT DUKE COMPLETED AND
BOTH MATING TYPE, THE MATING
TYPE LOCUS WAS 100KB OR 117KB,
IT'S ALMOST SEX CHROMOSOME.
ABOUT PHYLOGENY, HOW DID THEY
BENEFIT, HOW DID THE AREA OF
PHYLOGENY BENEFIT FROM
HYPOTHALLISM?
IF YOU SEE THIS MORPHOLOGY, THIS
IS THE SEXUAL STATE.
NOW WE CAN COMPARE ABOUT THE
REMAINING FUNGAL SPECIES IN THE
KINGDOM FUNGI.
THE NEAREST ONE WAS THIS
STRUCTURE, PRODUCED BY TREMELLA.
THAT IS WOOD EAR MUSHROOM.
THEY ALL GROW ON TREE BARK.
SO FINALLY, WE FL WE NOTICE THIS
CRYPTO COCCUS SHOULD BELONG TO
THE TREMELLA GROUP.
WHEN DNA SEQUENCING BECAME
AVAILABLE, PHYLOGENETIC TREE
BASED ON ITS INDEED SHOWED THAT
CRYPTOCOCCUS GROUP AND THEN
TREMELLA SPECIES WERE IN THE
SAME ORDER.
SO NOW WE COULD PLACE THE
CRYPTOCOCCUS IN THE TREMELLALES
IN MICROBIOTA.
BECAUSE OF ALL THIS DIFFERENCE,
FAY LOW GENPHYLOGENETIC STUDY WD
BY MANY PEOPLE AND EVENTUALLY
FOUND THAT CRYPTOCOCCUS GATT
GATTII -- THIS PERSON,
DR. MITCHELL, IS IN DUKE, AND
MARIANA IS IN ITALY, AND
BOEKHOUT IS IN POLAND, AND MEYER
WAS IN AUSTRALIA.
SO IT'S LIKE WHOLE INTERNATIONAL
TEAMS START STUDYING THE
PHYLOGENY.
SO THE FIRST GOAL WAS DISCOVERED
IN THE FULL LIFECYCLE.
NOW THAT'S DONE, AND THE SECOND
GOAL WAS TO FIND THE VIRULENCE
FACTOR.
AND HOW DO YOU FIND THE VIEWER
LANCE FACTOR?
VIRULENCE FACTOR?
SO WHAT I DID WAS A DICHOTOMOUS
DIAGNOSTIC APPROACH OF -- THAT
TIME THERE WERE ONLY 27 SPECIES
IN THE GENUS CRYPT COCCUS.
SO THEIR ABILITY TO UTILIZE
CARBOHYDRATES AND NITROGEN
SOURCE AND THE GROWTH ABILITY AT
37 DEGREE, AND THEN THE
PHENOTYPE OF COLONY THAT WAS
GROWN ON THE MEDIA THAT HAS
DIPHENOLIC COMPOUNDS.
SO I MADE THIS DIAGRAM,
DICHOTOMOUS DIAGRAM, AND WHAT
STOOD OUT -- I HAVE CIRCLED HERE
BUT IT DOESN'T SHOW.
AT THE TOP LEFT, IT SAYS COLONY
IS BROWN, LECTOSE IS
NON-UTILIZED AND GROWTH AT
37 DEGREE.
I LOOKED AT ALL 27 SPECIES,
CRYPTOCOCCUS WAS UNIQUE
DISTINGUISHING CHARACTER AS
THIS.
WHY BROWN COLONY ON THE
DIPHENOLIC MEDIA?
PROFESSOR STIVE, WHO USED TO BE
WORKING AT ROBERT KOCH'S
INSTITUTE IN BERLIN, AT THAT
TIME, OF COURSE, THE ECOLOGICAL
NICHE OF CRYPTOCOCCUS NEOFOR
MENS BEST KNOWN IS THE PIGEON
DROPPING, SO HE THINK MAYBE
PIGEONS EAT THE BIRD SEED AND
SOMEHOW BIRD SEED IS SELECTIVELY
LETTING CRYPTOCOCCUS GROW MUCH
BETTER.
SO HE EXTRACTED THE BIRD SEED,
PUT IT INTO THE AUGER, AND THEN
PLATED THE CLINICAL YEAST, ALL
THE CANDIDA SPECIES REMAIN WHITE
AND KN NEOFORM CRYPTOCOCCUS IS E
ONLY ONE THAT PRODUCED THE ROUND
COLONY, THE MELANIN.
AND SO REMEMBER 37 DEGREE
GROWTH, MELANIN INFORMATION ON
MEDIA, AND THE NEGATIVE
UTILIZATION LECTOSE, SO THE NEXT
THING WE DID WAS MADE A MU TAN,
AT THAT TIME OF COURSE THERE'S
NO GENETIC MANIPULATION, YOU DO
UV RADIATION AND GET THE MELANIN
NEGATIVE AND THEN LEAVE THE
MELANIN POSITIVE PARENT, CROSS
THEM, AND THEN ISOLATED ALL THE
F1 PROGENY AND THERE'S NO
QUESTION IT'S MAN DALIAN
INHERITANCE OF THE MELANIN FOR
PLAITION EXACTLY OUT OF 25
COLONIES, 12 TO 13.
AND THEN 37 DEGREE GROWTH.
AND THEN CAPSULE.
THESE WERE BY FAR THE ESSENTIAL
VIRULENCE FACTOR OF CRYPTOCO
CRYPTOCOCCUS.
AND TWO FELLOWS WORKED ON THESE
PROJECTS, DR. POLACHECK AND JUDY
ROSE, BOTH OF THEM ARE RETIRED
NOW, BUT THIS CHART SHOWS THE
WILD TYPE AND THEN REV REVERTAN,
BUT MELANIN NEGATIVE OR CAPSULE
NEGATIVES ARE ALMOST AVIRULENT
DEPENDING ON THE STRAIN.
AND THIS IS A MOUSE STUDY.
SO WE ESTABLISHED THAT BUY YOE
CHEMICAL STEPS IN MELANOGENESIS
DESCRIBED AND INVOLVEMENT OF
ONLY ONE ENZYME, BECAUSE IT WAS
CLEAR THAT ONLY ONE ENZYME WAS
INVOLVED.
AND THAT WAS LACCASE, WE CALLED
PHENOL OXIDASE.
THAT POSITION OF MELANIN IN THE
CELL WALL WE NOTICED, AND THEN
PETER WILLIAMSON, WHO CAME HERE,
HE'S SITTING HERE, BUT HE CAME
HERE AS A CLINICAL FELLOW, AND
THE FIRST 20 YEARS OF HIS CAREER
WAS TOTALLY FOCUSED ON HOW
CRYPTOCOCCUS PRODUCED MELANIN.
AND HOW IS IT SYNTHESIZED, WHAT
KINDS OF GENES ARE REGULATED,
HOW IS IT BEING
SHIPPED/TRANSPORTED TO THE CELL
WALL AND EVENTUALLY SECRETED?
ALL OF THESE PICTURES, THERE ARE
JUST TONS OF GENETIC FACTORS
INVOLVED TO MAKE THE MELANIN
FORMATION.
SO I'M GLAD PETER STARTED
WORKING ON MELANIN AS SOON AS WE
KNEW IT WAS A VIRULENCE FACTOR.
AND THEN, RIGHT NOW AT HOPKINS,
PREVIOUSLY HE WAS AT NEW YORK,
HE PRODUCED MELANIN GHOST MADE
FROM MELANIZED CRY -- MELANIN
STAINED GHOST, AND THE
INTERESTING PART IS THIS
ANTIBODY IS CURRENTLY IN
EVALUATION FOR THE TREATMENT OF
HUMAN MELANOMA, BECAUSE HUMAN
MELANIN, CRYPT CO CRYPTOCOCCAL N
ARE BOTH U MELANINS.
DISCOVERY OF THIS CGV DIAGNOSTIC
MEDIA AND THE IMPACT ON THE
PROGRESS OF CRYPT CO CRYPTOCOCCY
AND EPIDEMIOLOGY.
BEFORE MY MEDIA WAS MADE, WHAT
WE KNEW WAS JUST ABOUT THIS
NATURAL SOURCE OF CRYPTOCOCCUS
WAS PIGEON DROPPINGS, AND THEN
THE SOIL CONTAMINATED BY AVEEN
GUANOS.
SECOND SPECIES, CRYPTOCOCCUS
GAATII WAS NEVER ISOLATED FROM
ENVIRONMENT UNTIL 1990.
BECAUSE GAATII AND NEOFORMANS
WERE DISCOVERED ONE YEAR APART,
1994 AND ONE IN 1995, SO IT'S
ALMOST LIKE 100 YEARS THAT WE
DIDN'T KNOW WHERE GATTII WAS
COMING FROM.
OUR DIAGNOSTIC MEDIA BECAME
INSTRUMENTAL FOR THE STUDY OF
C.GATTII FROM EUCALYPTUS TREE IN
1990 WHICH LED TO RESEARCH FOR
ARBORIA FORM, BECAUSE TREMELLA
AND CRYPTOCOCCUS ARE VERY
CLOSELY RELATED.
TREMELLA GROWS ON TREE BARK AND
WHY NOT CRYPTOCOCCUS?
BUT EVERYONE WAS FOCUSING ON THE
PIGEON DROPPINGS.
THAT'S WHY WE NEVER FOUND, UNTIL
1990.
SO THIS IS IN MEDIA.
NEOFORMANS DOES NOT UTILIZE --
CANNOT GROW IN CGB -- WHEREAS
C.GATTII BECOMES BLUE WITHIN 24
HOURS.
WE'WHILE USING THIS MEDIA, FELLW
IN AUSTRALIA, DAVID ELLIS, HE
SAMPLED EUCALYPTUS TREE DROP THE
LEAVES AND TREE BARKS SWABBED
AND TONS OF YEAST WERE ISOLATED,
BUT HE HAD TO LOOK FOR GATTII.
HE USED MY MEDIA, AND HE
ISOLATED THE FIRST GATTII FROM
EUCALYPTUS TREE, AND HUNDREDS OF
OTHER PEOPLE STARTED LOOKING FOR
TREES, OF COURSE.
SO THIS AN ECOLOGICAL IMPACT,
BUT WHAT ABOUT EPIDEMIOLOGY?
C. NEOFORMANS, AT LEAST 70% OF
THEM ARE FROM HIV-POSITIVE
PATIENT, CLINICAL SOURCE.
AND THEN ONLY ABOUT 25% COMES
FROM OTHERS.
AS I SAID, WHAT THE RISK FACTOR
IN THE BEGINNING.
BUT INTERESTINGLY, IF YOU LOOK
AT THE GATTII, VERY FEW COMES
FROM AIDS PATIENTS, AND THE
REMAINDERS OFTEN SAY PREVIOUSLY
HEALTHY PATIENTS.
SO THEY THOUGHT IT WAS AN
IMMUNOCOMPETENT PATIENT, UNTIL
WE FOUND THAT GM CSF AUTO
ANTIBODY WAS A RISK FACTOR,
WHICH HAS NEVER BEEN TESTED IN
THE LABORATORY WHEN THEY WERE
LOOKING FOR PATIENTS'
IMMUNOLOGICAL -- SO GATTII WAS
FOUND TO INFECT PREVIOUSLY
HEALTHY PATIENTS, SO
EPIDEMIOLOGY AND ECOLOGY BOTH
DEVELOP THE VERY FAST AFTER THIS
MEDIA HAS BEEN FORMULATED.
NOW I LOOKED AT ALL THE TREES
FROM WHICH THE CRYPTOCOCCUS
COMES FROM BUT I JUST PICKED 12
HERE BECAUSE THESE NAMES LIKE
ACACIA, PINE AND OAK TREE AND
PLATANUS, ALMOST EVERYONE KNOWS
WHAT THEY ARE.
BUT THERE ARE ABOUT MORE THAN
100 TREES THAT GATTII AND
NEOFORMANS ARE INHABITING.
AND BY 1985, THAT PCR BECAME
AVAILABLE.
AND ALL OF MY WORK SO FAR HAS
BEEN CLASSICAL, AND THEN THE
HANDWRITING ON THE WALL OF MY
LAB, KEEP ON SHOWING THAT YOU
NEED TO CHANGE THE RESEARCH
DIRECTION.
REORIENT YOUR DIRECTION TO
MOLECULAR GENETICS.
IT WAS VERY INCONVENIENT, ALMOST
SCARY TO STEP OUT OF MY COMFORT
ZONE, BUT I DECIDED TO CHANGE
IT, THAT WAS IN 1985.
AT THAT TIME I WAS ALREADY EARLY
50s, AND IT WAS A REALLY HARD
DECISION I HAD TO MAKE.
SO WHAT DID I DO AFTER CHANGING
THE DIRECTION?
FIRST THING I DID WAS DEVELOPING
THE MOLECULAR TECHNIQUE OF
CRYPTOCOCCUS AND COME UP WITH A
TRANSFORMATION METHOD.
AND THEN THE VIRULENCE FACTOR,
MOLECULAR KOCH POSTULATE WAS
FULL FIELD.
SO I'M GOING TO TALK ABOUT THIS
TOO BECAUSE THIS, TOO, COMES
AFTER MY REORIENTATION, AND I
HAVE NO TIME TO GO THROUGH HERE.
SO WHAT DID I DO?
I ENROLLED FOR EACH MOLECULAR
BIOLOGY COURSE OFFERED BY
COLDSPRING HARBOR AND I LEFT
HOME TO STAY THREE WEEKS IN THE
LAB, AND I FOUND I WAS THE ONLY
ONE WHO ARE IN 50s, THE REST
OF THEM WERE IN THEIR 20s AND
30s AT THE MOST.
SO DEVELOPED MOLECULAR TOOLS
UPON ARRIVAL, AND WORKED IN
COLLABORATION WITH JEFF EDMAN
FROM UC SAN FRANCISCO.
NOW WE HAD DEVELOPED
TRANSFORMATION TECHNIQUE
DEVELOPED.
AND THAT MADE US TO DO MOLECU
MOLECULAR -- POSTULATE FOR
VIRULENCE FACTOR.
AND BY DOING CLONING CAPSULE
GENES AND TRANSFORMING CAP MINUS
MUTANT, CAP POSITIVE DISRUPTION
AND ALL OF THESE, MADE -- JUN
CHANG, STAFF SCIENTIST IN OUR
LAB, TOOK THIS PROJECT.
AND USED THE CAP MINUS MUTANT,
IT LOOKED LIKE -- YOU DON'T SEE
ANY CAP -- COLONY IS VERY DRY,
SO WHEN HE COMPLEMENTED WITH THE
GENOMIC DNA, IT BECAME NOT ONLY
CAPSID POSITIVE OF -- COLONY,
BUT THE MOST IMPORTANT PART IS
ALL THIS VIRULENCE CAME BACK.
THE GREENS ARE THE CAP MINUS
STRAINS, THERE'S FOUR DIFFERENT
STRAINS.
FOUR DIFFERENT MUTANTS.
AND THEN THE RED IS THE WILD
TYPE, AND BLUE IS THE ONE WHICH
IS COMPLEMENTED THIS MUTANT, AND
THEY ALL BECAME VIRULENT.
SO AFTER THIS HUGE CHANGE
OCCURRED, CRYPTOCOCCUS RESEARCH
FIELD EXPERIENCES SUDDEN
TURNABOUT FROM MID 1990s.
WHY?
BECAUSE AIDS CAME ON, AND THEN
LOW LEVEL OF LAB HAZARD, SO SHEY
SHOULDN'T BE AFRAID TO MOVE IN
FROM OTHER FIELD, AND GENETIC
AMENABILITY DUE TO THE CROSSING
AND TRANSFORMATION AND ALL OF
THIS, AVAILABILITY OF MOLECULAR
TOOLS, AND CONFIRMED VIRULENCE
FACTORS SO THEY CAN GO AFTER
VIRULENCE, AND GENETIC
DIVERSITY, AND EXCELLENT ANIMAL
MODELS.
BECAUSE OF ALL THESE, PEOPLE
START MOVING IN, INFLUX FROM ALL
THESE AREAS WITH A STRONG
BACKGROUND IN MOLECULAR GENETICS
OF SACK ROW MY CYST OR OTHER
MICROORGANISM.
AND GLYCOBIOLOGY, BECAUSE THE
CAPSULE BEING THE MAY YOUR
VIRULENCE FACTOR AND KNEW VERY
LITTLE ABOUT CAPSULE SYNTHESIS
MICROBIOLOGY CAME IN AND
EVOLUTION BIOLOGISTS BECAUSE OF
THE DIVERSITY OF CRYPTOCOCCAL
POPULATION, AND GENETICS
BIOINFORMATICS AND CELLULAR AND
MOLECULAR IMMUNOLOGY ESPECIALLY
BECAUSE EXCELLENT ANIMAL MODELS.
EVENTUALLY BY YEAR 2011,
CRYPTOCOCCUS BECAME A MODEL
YEAST FOR THE STUDY OF FUNGAL
PATHOGENICITY AND PUBLISHED A
BOOK BY ASN.
I TRIED TO HAVE AS MANY PAPERS
PICTURE BUT THIS IS ALL I COULD
GET.
BY THE YEAR 2017, ALL OF THESE
PEOPLE I CALL A VILLAGE, PLUS
THERE WERE ABOUT 400 AUTHORS,
CO-AUTHORS/COLLABORATORS, AND
WORKED SO THAT I COULD
ACCOMPLISH TO THIS DAY.
CONCLUSION.
NIAID PLAYED A KEY ROLE IN
LAYING THE FOUNDATION FOR
PROGRESS IN THE CRYPTOCOCCAL
RESEARCH STUDY IN 1975, AND
RE-ORIENTING THE RESEARCH
DIRECTION TO STAY ALIGNED WITH
SCIENTIFIC ADVANCEMENT WAS
INCONVENIENT AND CHALLENGING,
BUT IT WAS CRUCIAL FOR
SUSTAINING A LONG AND PRODUCTIVE
CAREER.
OUR CONTRIBUTION TRANSFORMED
WHAT WAS ONCE CONSIDERED A
OBSCURE PATHOGEN, THERE WAS JUST
300 CASES A YEAR, NEW CASES,
UNTIL AIDS CAME IN.
INTO A MODEL ORGANISM IN
BIOMEDICAL RESEARCH.
AS HIV EPIDEMIC HEIGHTENED THE
IMPORTANCE OF CRYPTO COCCOSIS.
I AM DEEPLY INDEBTED TO MY
MENTORS AND DEDICATED CO-WORKERS
AND SUPPORTIVE COLLEAGUES WHO
NUMBER MORE THAN 400 TODAY DATE.
THANK YOU.
[APPLAUSE]
>> LOOKS LIKE WE MIGHT HAVE A
QUESTION OVER HERE.
>> I HAVE A NAIVE QUESTION.
>> PLEASE.
>> HOW DOES THE MELANIN
CONTRIBUTE TO VIRULENCE?
IS THAT KNOWN?
>> EXCUSE ME?
>> HOW DOES THE MELANIN
CONTRIBUTE TO THE VIRULENCE?
>> OH.
MANY, MANY DIFFERENT WAYS.
I THINK THAT THE BEST WAY WILL
BE PETER WILLIAMS WILL ANSWER,
BUT IT PROTECTS THE CRYPTOCOCCUS
BECAUSE IT'S ANTIOXIDANT, AND
THEN OF COURSE IT'S ACCUMULATED
IN THE CELL WALL, SO ONCE -- THE
MEL NIEZED CELLS ARE MUCH MORE
DIFFICULT TO BE KILLED.
>> THANK YOU.
>> ACTUALLY I HAVE A QUESTION
FOR YOU.
YOU KNOW, I'M THINKING BACK ON
WHEN I WAS DOING CLINICAL WORK
AND MOST OF THE PEOPLE I SAW
WITH CRYPT CO CRYPTOCOCCAL MENIS
A NEUROLOGIST WERE PEOPLE WITH
DIABETES THAT WAS OUT OF
CONTROL.
>> RIGHT.
>> AND YET THE CRYPTOCOCCUS
APPARENTLY CAN LIVE IN LOW
GLUCOSE ENVIRONMENTS.
IS IT THE IMMUNE SYSTEM OF THE
UNCONTROLLED DIABETIC THAT IS
SOMETHING UNRELATED TO THE
GLUCOSE CONTENT OF THEIR BLOOD?
>> MAYBE I DIDN'T MENTION IT,
BUT THIS CRYPTOCOCCOSIS IS
PRIMARILY FOR IMMUNODEFICIENT
PATIENTS.
NUMBER ONE.
SO I'M SURE DIABETES PATIENTS
MUST BELONG THERE TOO.
BUT THE REASON WHY IT CAN THRIVE
IN THE BRAIN IS BECAUSE THIS
ORGANISM CAN THRIVE ON LOW
GLUCOSE MEDIA.
NOT THAT CRYPTO -- JUST LIKE THH
GLUCOSE BUT THEY CAN SURVIVE
VERY WELL IN LOW GLUCOSE.
>> THANK YOU.
THANK YOU VERY, VERY MUCH.
[APPLAUSE]
OUR NEXT SPEAKER IS DR. THOMAS
KUNKEL.
[APPLAUSE]
>> IT'S A GREAT PLEASURE TO BE
HERE.
I'VE NEVER HAD TO GIVE A TALK
THAT WAS DESCRIBED AS IT WAS TO
ME TO TRY TO COVER 42 YEARS OF
RESEARCH IN 30 MINUTES.
SO I'M GOING TO TRY DO THAT WITH
MINIMAL NEW DATA, BUT TO
DESCRIBE SOME OF THE THINGS THAT
HAVE REALLY STRUCK ME IN MY TIME
AS A SCIENTIST.
SO I WAS AS A CHILD A MEMBER OF
A LARGE FAMILY, 11 OF US, SIX
BROTHERS AND TWO SISTERS AND MY
PARENTS, AND WE GREW UP IN A
TINY LITTLE TOBACCO FARM IN THE
HILLS OF KENTUCKY.
AND THE FURTHEST THING FROM MY
MIND AS A CHILD WAS TO BECOME A
SCIENTIST.
AND WHAT LED TO THAT WAS THE
FACT THAT MY FATHER HAD A HEART
CONDITION AND PASSED AWAY AT THE
AGE OF 60, WHEN I WAS VERY
YOUNG, AND HAD TO -- AND WE HAD
TO MOVE INTO THE CITY, AND THAT
ACTUALLY BROKE MY HEART BECAUSE
UNTIL I WAS ABOUT 25 YEARS OLD,
I THOUGHT I WAS GOING TO BE A
FARMER, AND THAT WAS WHAT I
DEARLY LOVED TO DO.
BUT AT THE SAME TIME, I ENJOYED
OTHER THINGS, LIKE VERY MUCH
ENJOYED SCIENCE FICTION.
AND I WOULD READ BOOKS ABOUT
SCIENCE FICTION FROM THE TIME I
WAS ABOUT SEVEN YEARS OLD UNTIL
TODAY.
AND I STILL ENJOY THAT.
AND THAT'S ILLUSTRATED BY THIS
TO YOUR LEFT-HAND SIDE SIDE, THE
MIBGYTHEMILKY WAY AS IT MIGHT AR
FROM 10 BILLION LIGHT YEARS
AWAY, AND THAT'S 10 TO THE 23RD
METERS AND WE'RE ALL SCIENTISTS
AND KNOW THAT THAT IS A HUGE
DISTANCE.
AND ON THE RIGHT IS WHAT I DO,
WHICH IS TO STUDY THE FIDELITY
OF DNA REPLICATION AND IT'S
ILLUSTRATED THERE BY THE X-RAY
CRYSTAL STRU STRUCTURE OF A DNA
POLYMERASE, CORRECTLY PAIRING A
INCOMING OPPOSITE A T.
AND THE DISTANCE THERE IS
ABSOLUTELY HUGE BETWEEN THE LEFT
AND THE RIGHT.
IN IS THE EVOLUTION OF LIFE ON
EARTH AS PRESENTED TO ME BY MY
EXCELLENT COLLEAGUE, ROELLE
SHOPPER.
THE FORMATION OF THE MOON IS ON
THE LEFT AND THAT WAS
4.5 BILLION YEARS A AND THAT'S
ALMOST UNIMAGINABLE FOR US TO
THINK ABOUT WHAT A BILLION YEARS
IS.
AND ON THE RIGHT IS WHAT THE NIH
DOES, WHICH IS TO UNDERSTAND THE
IMPACT OF THE ENVIRONMENT ON
HUMAN HEALTH.
AND YOU CAN SEE OVER THERE, HOME
SAIPIANS HAVE ONLY BEEN AROUND
FOR A VERY, VERY, VERY TINY
SLICE OF TIME.
SO ONE OF THE THINGS THAT HAS
ALWAYS INTRIGUED ME IS HOW DID
LIFE EVOLVE ON EARTH OVER 4 1/2
BILLION YEARS?
SO TIME IS A VERY CRITICAL
DETERMINANT OF THE ANSWER TO
THAT QUESTION.
THIS IS ANOTHER IMAGE PROVIDED
TO ME BY ANOTHER GOOD COLLEAGUE
OF MINE AT THE NIEHS,
ENVIRONMENTAL HEALTH SCIENCES
INSTITUTE.
WE LIVE IN NORTH CAROLINA, SO I
DON'T GET TO WALK DOWN YOUR
BEAUTIFUL HALLWAYS HERE.
I WAS VERY IMPRESSED WITH ALL OF
THE STUFF THAT YOU GUYS PROBABLY
SEE EVERY DAY AND MAYBE DON'T
EVEN PAY ANY ATTENTION TO
ANYMORE, WHICH IS THE LIST OF
PREVIOUS COLLEAGUES OF YOURS WHO
WON PRIZES AND DISCOVERED
BIOLOGY AS WE KNOW IT TODAY, AND
I TOOK QUITE A WHILE COMING DOWN
THE HALLWAY HERE TO LIPSETT TO
READ SOME OF THAT STUFF LIKE
RIGHT OUTSIDE IS MARSHAL
NUREMBERG AND MARSHAL NUREMBERG
WAS THE MENTOR FOR ONE OF MY
BEST FRIENDS IN THE WORLD WHO
RECENTLY PASSED AWAY, SO I'M NOT
THAT FAR REMOVED BIOLOGICALLY
FROM THE TIME FRAME THAT MADE
NIH GREAT, AND THE BASIC
RESEARCH THAT DETERMINES LIFE ON
EARTH.
JEFF NUELER PROVIDED ME THIS,
HE'S AN NMR SPECTROSCOPIST.
THE TOOLS USED ON THE BOTTOM TO
DISCOVER PROTEIN DYNAMICS AND
MOLECULAR MOTIONS USING NUCLEAR
MAGNETIC RESONANCE, AND THE TIME
FRAME HERE IS VERY, VERY
DIFFERENT THAN THE 4 BILLION
YEARS OF EVOLUTION THAT I
DESCRIBED.
IN FACT, IF YOU WANT TO
UNDERSTAND REPLICATION FIDELITY,
THEN YOU NEED TO UNDERSTAND WHAT
GOES ON IN THE X-RAY CRYSTAL
STRUCTURE OF A DNA POLYMERASE
WHERE THE LENGTH OF A HYDROGEN
BOND BETWEEN AN A AND A T BASE
PAIR IS 2 ANGSTROMS, TO ROUGHLY
THE SIZE OF THE HUMAN BEING, AS
WE COULD ROUGHLY DESCRIBE AS
BEING 2 METERS TALL, AND IT
COVERS A TIME FRAME OF 10 TO THE
MINUS 14TH SECONDS TO GREATER
THAN 4 BILLION YEARS.
AND IN ORDER TO COVER THAT
IMMENSE AMOUNT OF TERRITORY TO
UNDERSTAND DNA REPLICATION
FIDELITY, WE NEED FOUR BASIC
TECHNOLOGIES, WHICH I DESCRIBE
AS STRUCTURAL BIOLOGY,
BIOCHEMISTRY, GENETICS, AND
GENOMICS.
IF YOU WANT TO TYPE THE HUMAN
GENOME, IT'S 6 BILLION
NUCLEOTIDES, AND IF YOU WERE
TYPING ON A WORD PROCESSOR, YOU
WOULD USE -- SPELL SELECTING THE
CORRECT BET LETTER OF THE
ALPHABET, YOU WOULD USE BACK
SPACE, AND YOU WOULD USE SPELL
CHECK TO ULTIMATELY GET AN
ERROR-FREE DOCUMENT.
NOW I HAD A SLIDE THAT I TOOK
OUT THAT WAS 500 LETTERS, AND I
HAD STUDENTS COME IN TO THE LAB
OVER THE YEARS WHO USED ONE, TWO
OR THREE OF THOSE METHODS TO
TYPE THAT 500 LETTERS, AND
THERE'S THEIR ERROR RATES.
ABOUT 20 MISTAKES FOR EVERY 500
NUCLEOTIDES IF ALL YOU USE IS
THE FIRST STEP.
IF YOU ADD THE SECOND STEP, YOU
IMPROVE FIDELITY, AND IF YOU ADD
SPELL CHECK, YOU GET AN
UNBLEMISHED DOCUMENT.
SAME HOLDS FOR REPLICATION
FIDELITY.
WHERE THE DNA POLYMERASE THAT
SELECTS THE CORRECT NUCLEOTIDE
HAS A CERTAIN SELECTIVITY FOR A
CORRECT OVER AN INCORRECT
NUCLEOTIDE.
PROOFREADING, THAT IS, THE
EXO -- REMOVAL OF A MISMATCH IS
ROUGHLY EQUIVALENT TO BACKSPACE,
AND SPELLCHECK COULD BE ROUGHLY
EQUIVALENT TO TWO MAJOR REPAIR
PROCESSES THAT I WOULD LIKE TO
BRIEFLY DESCRIBE IN THE NEXT FEW
MINUTES.
ONE IS DNA MISMATCH REPAIR AND
THE OTHER IS RIBONUCLEOTIDE
EXCISION REPAIR.
THE WORLD'S EXPERT ON THAT
SUBJECT IS SOMEWHERE IN THIS
AUDIENCE LISTENING TO ME.
THAT'S BOB KROUCH.
AND WHAT WE HAVE INTN
INVESTIGATING FOR THE 42 YEARS
THAT I'VE BEEN AT THE NIEHS WAS
TO TRY TO UNDERSTAND HOW
REPLICATION FIDELITY CAN BE WHAT
I CONSIDER ONE OF THE MOST
AMAZINGLY ACCURATE BIOLOGICAL
PROCESSES THAT OCCUR, AND I'M
GOING TO GIVE YOU A LITTLE BIT
ABOUT THAT, AS A PERSON WHO WAS
ORIGINALLY TRAINED AS A
BIOCHEMIST AND PICK UNDER
STRUCTURAL BIOLOGY, GENETICS AND
GENOMICS AS TIME WENT BY.
SO THIS IS WHAT WE BELIEVE
SUMMARIZES WHAT IT TAKES TO
REPLICATE THE 6 BILLION
NUCLEOTIDES OF DNA IN THE
NUCLEAR GENOME OF A HUMAN BEING.
ONE, TWO, THREE AND FOUR ARE THE
PROCESSES THAT WE HAVE BEEN
VIGOROUSLY INVESTIGATING FOR THE
42 YEARS THAT I'VE BEEN AT THE
NIEHS, AND MOST OF THAT WAS ON
THE SELECTIVITY OF THE DNA POE
LPOLYMERASES THEMSELVES, NUMBER
TWO, IN WHICH YOU HAVE A CORRECT
PROPERLY ALIGNED DNTP INCOMING
OPPOSITE A CORRECT TEMPLATE
BASE, AND YOU ALSO HAVE THE
CORRECT SUGAR MOIETY,
DEOXYRIBONUCLEOTIDES, INSTEAD OF
RIBONUCLEOTIDES.
BUT OVER TIME, WE HAVE
INVESTIGATED ALL THOSE
PROCESSES, AND I DON'T HAVE TIME
TO DESCRIBE TO YOU EVERYTHING
THAT WE'VE LEARNED ABOUT THOSE,
SO I PICKED A COUPLE OF SELECTED
SUBJECTS THAT HAVE HAD AN IMPACT
ON HOW WE THINK ABOUT
REPLICATION FIDELITY, AND BY
THAT, I MEAN HOW THE WORLD
THINKS ABOUT IT BASED OP WH ON S
NOW IN MOLECULAR BAYOLOGY
TEXTBOOKS, PARTLY BASED ON WHAT
MY LAB HAS CONTRIBUTED TO THIS
AREA OF INVESTIGATION.
AND THE REASON WE'RE INTERESTED
IS THE RESPONSIBLE MECHANISMS
THAT DETERMINE REPLICATION
FIDELITY, THEIR CONNECTIONS TO
HUMAN HEALTH WHICH IS SIMILAR TO
WHAT THE NIH DOES AS AN
INSTITUTION OR A SET OF
INSTITUTIONS, AND THE
EVOLUTIONARY CONSERVATION OF
THOSE PROCESSES, WHICH IS MORE
OF A PERSONAL INTEREST ON MY
PART, BUT NONETHELESS, IMPORTANT
TO HUMAN HEALTH.
AND THE PROCESSES THAT WE THINK
ABOUT ARE THE ELONGATION OF DNA
ON AN OPEN TEMPLATE WITHOUT A
DOWNSTREAM DOUBLE STRANDED
NATURE.
SO IT'S ADDING NUCLEOTIDES TO A
TEMPLATE PRIMER FOR 6 BILLION
NUCLEOTIDES AND DOING IT WITHOUT
MAKING A MISTAKE.
AND THEN THERE'S OKAZAKI
FRAGMENT MATURATION WHICH IS THE
PROCESS WHICH ON THE LAGGING
STRAND OF REPLICATION ON WHICH
OKAZAKI FRAGMENTS ARE MADE, YOU
HAVE TO GET RID OF THE RNA
PRIMER THAT WAS USED TO INITIATE
THOSE OKAZAKI FRAGMENTS AND YOU
DO THAT BY A STRAND DISPLACEMENT
REACTION.
AND THAT OCCURS ABOUT 25 MILLION
TIMES PER REPLICATION OF THE
HUMAN GENOME.
AND THEN THE POST REPLICATION
REPAIR PROCESSES, THERE ARE TONS
OF REPLICATION -- OR REPAIR
PROCESSES THAT OCCUR IN CELLS.
THEY'RE DONE BY THE 17 NORMAL
HUMAN DNA POLYMERASES, WHICH MY
LAB INVESTIGATE WOULD FOR MORE
THAN 25 YEARS.
AND THEY OCCUR BY MISMATCH OF
THOSE MANY DNA REPAIR PROCESSES,
TWO OF THEM ARE PARTICULARLY
IMPORTANT TO THE FIDELITY OF
REPLICATION OF THE HUMAN NUCLEAR
GENOME.
SO THESE ARE THE 17 DNA
POLYMERASES THAT WE KNOW ABOUT
TO DATE.
IN MY LAB OVER TIME, WE'VE
INVESTIGATED THE FIDELITY WITH
WHICH THEY CAN REPLICATE DNA
WITHOUT MAKING A MISTAKE.
AND I'M GOING TO LEAVE ABOUT
HALF OF THAT WORK BEHIND AND
FOCUS ON ONE ASPECT, THE
FIDELITY OF NUCLEAR DNA
REPLICATION, AND OUR CURRENT
WORK IS PRIMARILY DONE IN
BUDDING YEAST AS A LEGITIMATE
MOLECULAR MODEL FOR WHAT GOES ON
IN HUMANS.
SO THESE ARE THE THREE NORMAL
REPLICATIONS THAT REPLICATE THE
6 BILLION NUCLEOTIDES.
THEY'RE POLYMERASES ALPHA, DELTA
AND EPSILON OR 1, 2 AND 3, AND
THIS SLIDE IS JUST TO SHOW YOU
THAT THEY'RE HEAVILY CONSERVED
IN MAMMALS AND IN OTHER
EUKARYOTES INCLUDING THE TWO
YEAST ORGANISMS THAT WE HAVE
SOME INFORMATION ON S.CEREVIIAE
AND POMBE.
AT THE VERY BOTTOM, YOU'LL SEE
THEY HAVE A FIDELITY OF DNA
REPLICATION DONE BY OUR
BIOCHEMICAL MEASUREMENTS OVER
THE YEARS THAT ARE DIFFERENT,
POL ALPHA LACKS A
PROOFREADING -- AND ITS ACCURACY
IS ABOUT ONE MISTAKE FOR EVERY
2,000 NUCLEOTIDES PRELIMINARY -E
CURRENT WORK SUGGESTS THAT ALPHA
ONLY INITIATES SHORT CHAINS BY
LAYING DOWN AN RNA PRIMER AND
THEN EXTENDING IT BY A FEW MORE
NUCLEOTIDES WITH DNA, WHEREAS
DELTA AND EPSILON DO THE
MAJORITY OF LEADING AND --
STRAND REPLICATION.
SO HOW DO WE KNOW ABOUT THE
FIDELITY OF LEADING AND LAGGING
STRAND REPLICATION?
WELL, HERE'S ONE WAY TO ADDRESS
THAT QUESTION.
WE CAN USE MUTATOR ALLELES OF
THOSE THREE REPLICATIONS WHICH
HAVE AN ACTIVE SITE POINT
MUTATION IN WHAT'S CALLED
LEUCINE 612 IN THE X-RAY CRYSTAL
STRUCTURE OF YEAST POL DELTA.
AND THAT -- WE CAN MAKE MUTANT
IN WHICH WE CHANGE THAT
LEUCINE -- OR ITS HOMOLOGUE IN
THE B FAMILY REPLICASE FAMILY IN
POL ALPHA IS AN EQUIVALENT
LEUCINE THAT WE CHANGED FROM ME
THOI NEEN OR IN EPSILON, THAT WE
CHANGED TO GLYCINE, AND WHAT WE
KNOW FROM YEARS OF WORK ON THOSE
PROTEINS IS THAT THEY HAVE
PREFERENCES.
YOU CAN MAKE 12 POSSIBLE
MISMATCHES IN ORDER TO GET BASE
SUBSTITUTION ERRORS, AND WE KNOW
THAT THEY ARE SELECTIVE IN
MAKING ONE PARTICULAR MISMATCH
OVER ANOTHER IN ORDER TO EXPLAIN
A BASE SUBSTITUTION MUTATION
THAT OCCURS IN A CELL.
SO FOR EXAMPLE, POL DELTA L TO M
HAS A 28 FOLD HIGHER PROBABILITY
OF INSERTING G OPPOSITE T THAN
IT HAS OF INSERTING C OPPOSITE
A, WHEN THE COMPLEMENTARY
STRAPPED IS STRANDIS BEING REPL.
SO WE CAN USE THAT KIND OF
REPLICATION FIDELITY ASYMMETRY
FOR EACH OF THESE MUTATOR
POLYMERASES AND ASK WHAT HAPPENS
WHEN YOU SEQUENCE A HUMAN
GENOME -- OR A YEAST GENOME
THAT'S BEEN THROW 30 PASSAGES ON
A PETRI DISH, WHICH IS
EQUIVALENT TO ABOUT 900 CELLULAR
GENERATIONS OR 900 REPLICATION
CYCLES.
AND WHAT YOU REALIZE WHEN YOU
SEQUENCE THE WHOLE GENOME OF A
POL DELTA L TO M MUTATOR ALLELE,
IS THAT THE KINDS OF MUTATIONS
THAT APPEAR IN THE CELL DEPEND
ON WHERE THE MUTATION IS AND
WHICH OF THE REPLICASES IS DOING
THE REPLICATION, SO IN THIS
EXAMPLE, POL DELTA MAKES ERRORS
ON THE LEFT-HAND SIDE THAT ARE
CONSISTENT WITH G OPPOSITE T,
AND ON THE RIGHT-HAND SIDE, WITH
ERRORS THAT ARE CONSISTENT WITH
G OPPOSITE T ON THE OPPOSITE
STRAND.
YOU CAN USE THIS KIND OF LOGIC
FOR ALL THREE REPLICASES AND ALL
12 BASE SPACED MISMATCHES AND
ARRIVE AT A MODEL THAT SUGGESTS
THAT PO POL ALPHA IS INITIATING-
CONTINUES TO DO THE BULK OF THE
FRAGMENTS AND POL EPSILON IS THE
MAJOR LEADING STRAND REPLICASE.
THAT'S A QUESTION THAT HAS
POSED -- HAS BEEN POSED FOR
YEARS, EVER SINCE WATSON AND
CRAIG DESCRIBED THE ASYMMETRIC
CONFIGURATION OF DNA, AND THE
DATA THAT WE GENERATED ON THESE
MUTATOR ALLELES WAS CONSISTENT
WITH THAT MODEL ON THE LOWER
RIGHT-HAND SIDE.
AND THAT'S NOW, I THINK, YOU
NEVER PROVE ANYTHING IN SCIENCE,
YOU ONLY TEST HYPOTHESES AND TRY
TO DISPROVE THEM, BUT IF YOU
CAN'T DISPROVE THEM WITH ANY
OBSERVATIONS YOU MAKE, THEN YOU
PUT FORWARD THE HYPOTHESIS THAT
YOU BELIEVE IS TRUE, AND I
BELIEVE THAT THAT MODEL DEFINES
THE ROLES OF THE THREE MAJOR
REPLICASES IN REPLICATING THE
LEADING AND THE LAGGING
STRAPPED.
AND THAT'S ONE OF THE
OBSERVATIONS THAT LED TO MY
ELECTION TO THE NATIONAL ACADEMY
OF SCIENCES IN MAY.
THAT'S NOT NEARLY ENOUGH.
SO WE ALSO, IN 2010, AND THIS IS
A STATEMENT ADDRESSED TO ANY OF
THE STUDENTS IN THE AUDIENCE, OR
THE YOUNGER STUDENTS, I'M STILL
A STUDENT, BUT -- PEOPLE TRYING
TO FIGURE OUT WHAT THEY WANT TO
BE WHEN THEY GROW UP, THEY MAY
WANT TO THINK ABOUT THIS.
RIBONUCLEOTIDES ARE NOT NORMALLY
CONSIDERED PART OF DNA BUT I GOT
A STUDENT IN THE LAB IN 2009 WHO
WORKED WITH MY POSTDOC AND FOUND
THAT RIBONUCLEOTIDES, THE WRONG
SUGAR, NOT THE WRONG BASE UP
THERE IN GREEN, ARE FREQUENTLY
INCORPORATED INTO DNA.
IN FACT, FOR YEARS, PEOPLE HAD
THOUGHT THAT DNA POLYMERASES
INCORPORATE -- TRIPHOSPHATES BUT
THEY'RE VERY, VERY POOR AT
INCORPORATING RIBONUCLEOTIDES.
BASED ON X-RAY CRYSTAL
STRUCTURE, YOU COULD IMAGINE
WERE THAT'S SEW BASED ON
CALCULATING THE DNTP AND THE
RNTP CONCENTRATIONS IN YEAST
CELLS BY OUR COLLEAGUE ANDREI
CHABES, WE DISCOVERED THAT DNTP
CONCENTRATIONS ARE MUCH, MUCH
LOWER THAN ARE RIBONUCLEOTIDE
INCORPORATIONS CONSISTENT WITH
MUCH MORE FREQUENT OR MUCH MORE
ABUNDANT RIBONUCLEOTIDE
SYNTHESIS TO MAKE THE REST OF
THE GENETIC CODE WORK THAN ARE
DEOXIES, BUT DNA POLYMERASES USE
MUCH LOWER DNTP CONCENTRATIONS.
THIS CAN BE PUT INTO THE FIELD
OF DNA REPAIR BY THE FOLLOWING.
THIS IS A SLIDE TAKEN FROM --
GIVEN TO ME BY THOMAS LINDELL
ABOUT 20 YEARS AGO DESCRIBING --
THOMAS WON THE NOBEL PRIZE FOR
HIS WORK ON BASE EXCISION
REPAIR, BUT HE WAS ALSO A VERY
STRONG POE PO PROPONENT OF DNA E
AND HOW IT AFFECTS REPAIR
PROCESSES, REPLICATION FIDELITY,
AND THIS IS A LIST OF THE
LESIONS IN DNA THAT HAD BEEN
STUDIED FOR ABOUT 50 YEARS AT
THAT POINT.
AND HOW FREQUENTLY THEY OCCUR IN
A HUMAN CELL IN THE THIRD -- OR
IN THE SECOND FROM THE RIGHT
COLUMN.
SO THAT'S THE NUMBER OF ABASIC
SITES, WHICH IS THE MOST
ABUNDANT LESION PRODUCED IN A
HUMAN CELL, AT LEAST AS FAR AS
WE KNEW 20 YEARS AGO, AND THE
OTHERS ARE LESS FREQUENT THAN
THAT BUT THEY'RE ALL HIGHLY
MUTAGENIC AND VERY, VERY
INTERESTING TO PEOPLE INTERESTED
IN MUTAGENESIS LIKE I AM.
AND SO WE GAVE ALL THOSE
LITTLE -- THOSE NUMBERS █OVER
THERE, LITTLE DOTS, DESCRIBING
THE ABUNDANCE OF THAT KIND OF
DAMAGE IN A CELL, AND WHEN MY
POSTDOC AND HER STUDENT WORKED
ON HOW FREQUENTLY THE REPLICASES
INCORPORATED RIBONUCLEOTIDE
TRIPHOSPHATES INTO THE NUCLEAR
GENOME DURING NORMAL REPLICATION
IN YEAST, NO DAMAGE, THAT'S WHAT
WE SAW.
OF ALL THE KINDS OF
NON-CANONICAL NUCLEOTIDES PUT
INTO THE GENOME IN EUKARYOTES,
THE MOST ABUNDANT ARE
RIBONUCLEOTIDE TRIPHOSPHATES.
SO THAT'S LED TO A LOT OF WORK
THAT WE AND OTHERS HAVE DONE AND
NOW THE NUMBER OF PEOPLE WORKING
ON RIBONUCLEOTIDE INCORPORATION
DURING REPLICATION IS MUCH, MUCH
LARGER THAN IT WAS IN 2010, WHEN
WE PUBLISHED THIS RESULT IN THE
PNAS, AND WE'VE DONE TWO THINGS
WITH IT.
WE'VE USED RIBONUCLEOTIDE
INCORPORATION IN A YEAST CELL
THAT COULD NOT REPAIR THOSE
RIBONUCLEOTIDE, AND WE FOUND
THAT USING THIS NEW PROCEDURE
THAT WE DEVELOPED A LONG TIME
AGO, HYDEN-SEQ, WHEN WE MAP
WHERE RIBONUCLEOTIDES ARE PUT
IN, THEY'RE ENTIRELY CON SITION
TENT WITH OUR MODEL FROM THE
MUTAGENESIS STUDIES WHICH SHOW
THAT RIBONUCLEOTIDES ARE
FREQUENTLY PUT IN BY POL ALPHA,
LESS FREQUENTLY BY POL DELTA,
AND LESS FREQUENTLY STILL BY POL
EPSILON, BUT THEY'RE STILL
ABUNDANTLY MISINCORPORATED INTO
THE NUCLEAR GENOME, AND WHERE
THEY ARE PUT IN IS ENTIRELY
CONSISTENT WITH OUR MODEL THAT'S
NOW IN TEXTBOOKS OF WHICH ROLES
THOSE THREE REPLICASES PERFORM
DURING DNA REPLICATION.
WE'VE ALSO ASKED WHAT ARE THE
CONSEQUENCES OF RIBONUCLEOTIDES
IN DNA.
YOU CAN GO TO THAT ANNUAL REVIEW
OR BIOCHEMISTRY REVIEW WE
PUBLISHED A COUPLE YEARS AGO AND
IT DESCRIBES A LOT OF BIOLOGY,
GENETICS, AND NOW GENOMICS THAT
DESCRIBES HOW MANY RIBOS ARE PUT
IN DURING REPLICATION AND HOW
THEY ARE REPAIRED, AND THEY ARE
REPAIRED BY A PROCESS WHICH WE
NOW CALL RIBONUCLEOTIDE EXCISION
REPAIR, WHICH IS IN ADDITION TO
MISMATCH REPAIR, A STRONG
PROTECTOR OF PUTTING THE WRONG
THING INTO THE GENOME.
AND WHEN WE DO THAT, AND I
FORGOT WHAT I DID HERE, IF YOU
KNOCK OUT RNA H2 IN HUMANS OR
YEAST, YOU HAVE LOTS OF
RIBONUCLEOTIDES THAT WERE
INADVERTENTLY INCORPORATED
DURING REPLICATION, AND WHEN
THEY'RE NOT REMOVED, BECAUSE OF
THE RNA STAGE II DEFECT, THEY
CAUSE MUTAGENESIS, WHICH IS ONE
OF THE MAIN THINGS THAT MY LAB
IS INTERESTED IN.
SO IF YOU'RE INTERESTED, YOU CAN
READ THAT ARTICLE.
AND WE'RE VERY PROUD OF THIS
WORK BECAUSE FOR 50 YEARS, IT
WAS BIOCHEMISTS I TELLING US
RIBONUCLEOTIDES WERE
INCORPORATED DURING REPLICATION
BUT ONLY RARELY, TO IN 2010,
DISCOVERING THAT THEY'RE NOT
THAT RARE AND THAT IMPLIES THERE
MAY BE SOME IMPORTANT SELECTIVE
ADVANTAGES TO INCORPORATING
RIBONUCLEOTIDES INTO DNA, ONE OF
WHICH WE PROPOSE AS A STRAND
SIGNAL DISCRIMINATION FOR DNA
MISMATCH REPAIR.
SO THIS IS EUKARYOTIC MISMATCH
REPAIR, AND YOU CAN SEE GOING
FROM THE TOP, WHERE A MISMATCH
IS IN THAT LITTLE HEX AGOAL BOX,
THAT'S LEFT BEHIND BY THE
REPLICATION FORK AND IT NEEDS TO
BE REPAIRED OR YOU GET MUTATIONS
THAT AMONG OTHER THINGS CAUSE
CANCER, AND THE STEPS THAT ARE
INVOLVED ARE A NUMBER OF
PROTEINS ULTIMATELY LEADING TO A
CORRECT MOLECULE AT THE BOTTOM
IN WHICH THAT MISMATCH REPAIR
HAS BEEN REMOVED.
SO WE'VE BEEN STUDYING THAT AS
WELL FOR QUITE SOME TIME, EVEN
BEFORE THE NOBEL PRIZE FOR
STUDYING MISMATCH REPAIR BACK IN
2015.
AND WE'VE BEEN STUDYING IT BASED
ON AN ARTICLE PUBLISHED IN THE
PNAS BY MAT MESSELSON AT MIT WHO
WROTE IN 1976 THAT IF YOU WANT
TO UNDERSTAND THE RELAWTION SHIP
BETWEEN REPLICATION AND DNA
MISMATCH REPAIR IN VIVO, IT MAY
BE POSSIBLE THAT MISMATCH REPAIR
ACTS IN A DIRECTED M MANNER,
POSSIBLY BECAUSE OF THE SPECIAL
RELATIONSHIP TO THE REPLICATION
COMPLEX.
MY LAB, WHICH IS VERY INTERESTED
IN MECHANISMS THAT DETERMINE
REPLICATION FIDELITY, HAS BEEN
INVESTIGATING THOSE PROCESSES.
DOES MISMATCH REPAIR EFFICIENCY
DEPEND ON ANYTHING DESCRIBED IN
BLACK BELOW THAT.
AND WE'VE DONE KUWAI QUITE A BIF
WORK ON THAT OVER THE YEARS, AND
I JUST WANT TO END BY SHOWING
YOU JUST A COUPLE OF THINGS.
THIS IS A SUMMARY SLIDE OF WHAT
I'VE SAID.
WHERE WE BELIEVE THE REPLICATION
FIDELITY OF THE NUCLEAR
REPLICASES ARE THOSE NUMBERS
COLORED IN COLORS UP THERE FOR
BASE SUBSTITUTIONS.
WE ALSO HAVE DONE A LOT OF WORK
ON INSERTION/DELETION MUTATIONS.
WE ALSO BELIEVE THAT
RIBONUCLEOTIDES ARE ABUNDANTLY
INCORPORATED DURING REPLICATION.
BUT BOTH OF THOSE KINDS OF
LESIONS CAN BE REPAIRED BY
EITHER MISMATCH REPAIR OR
RIBONUCLEOTIDE EXCISION REPAIR,
AND WE PUBLISHED A PAPER IN
MOLECULAR CELL A WHILE AGO
SHOWING THAT NUCLEOTIDE NUCLEON
REPAIR -- TO CREATE A NIC DNA
AND THAT NIC CAN POSSIBLY BE
USED TO REPAIR REPLICATION
ERRORS BY MISMATCH REPAIR.
THAT'S A SUMMARY OF WHAT WE'VE
DONE.
NOW I WANT TO SHOW YOU JUST A
LITTLE BIT MORE.
IN THE COURSE OF THIS WORK, BACK
IN 1985 WHEN I HAD BEEN RUNNING
MY OWN SHOW, WHICH IS ME AND ONE
TECHNICIAN TO BEGIN WITH, AT THE
NIEHS, I DISCOVERED A METHOD
THAT COULD BE USED TO ENGINEER
ANY DNA SEQUENCE TO CHANGE ITS
NUCLEOTIDE COMPOSITION TO MAKE
MUTATIONS IN GENES.
AND I PUBLISHED THAT IN THE PNAS
IN 1985, RIGHT AFTER MY LAB GOT
STARTED.
AND I PUT THAT UP THERE NOT TO
BRAG, ALTHOUGH THAT'S ONE OF THE
REASONS THAT I THINK I GOT
ELECTED TO THE NATIONAL ACADEMY,
BUT IT'S BECAUSE WITHIN ABOUT
FIVE YEARS, I DID A LITERATURE
SEARCH AND IT HAD ALREADY
RECEIVED MORE THAN 10,000
CITATIONS.
SO IT'S ONE OF THE TOP FEW
PUBLICATIONS THE NIH HAS EVER
PUBLISHED IN TERMS OF THE NUMBER
OF CITATIONS, AND THAT WAS
BECAUSE IT WAS -- THAT PROCEDURE
WAS SO APPLICABLE TO MANY, MANY
QUESTIONS IN BIOLOGY WHERE YOU
WANT TO MAKE A MUTATION IN THE
GENE, BUT IT WAS DONE IN 1985,
WHEN IT WAS VERY DIFFICULT TO DO
UNLESS YOU HAD SOME PHENOTYPIC
SLEK SHUP.
SELECTION.
THIS ALLOWED ME TO DO THAT AS
ONE EVENT OUT OF EVERY TWO
GENOMES THAT I SEQUENCED.
WITHOUT ANY PHENOTYPIC
SELECTION.
YOU CAN READ THAT PAPER IF
YOU'RE INTERESTED.
BUT IT MADE ME THINK VERY
STRONGLY LAST WEEK WHEN A
COLLEAGUE OF MINE SENT ME A
PUBLICATION AND SAID YOU'VE GOT
TO READ THIS, AND IT WAS APPLIED
RESEARCH WON'T FLOURISH WITHOUT
BASIC SCIENCE, AND THIS WAS BY
THE THREE AUTHORS LISTED THERE,
WHO DESCRIBED THE PORTFOLIO OF
RESEARCH THAT NIH HAS FUNDED
SINCE ITS EXISTENCE, AND DECIDED
THAT ABOUT 51% OF THAT RESEARCH
IS CURRENTLY BASIC RESEARCH,
WHICH IS WHAT MY LAB STUDIES,
VERY BASIC QUESTIONS, AND ABOUT
49% IS CURRENTLY IN WHAT I CALL
ARTIFICIALLY TRANSLATIONAL
RESEARCH AS BEAUTIFULLY
ILLUSTRATED BY THE FIRST TALK WE
HEARD HERE.
SO WITH THAT THOUGHT IN MIND, I
WANTED TO END THIS BY DESCRIBING
TO YOU ONE THING THAT WE'RE
REALLY EXCITED ABOUT NOW IN MY
LAB.
I'M INTERESTED IN THE QUESTION
OF DOES SIMPLE CHAIN ELONGATION
COPY DNA WITH VERY, VERY HIGH
FIDELITY, WHEREAS STRAND
DISPLACEMENT, WHICH OCCURRED
DURING OKAZAKI FRAGMENT
MATURATION, THAT REQUIRES PRIMER
B BE REMOVED FROM THE DNA AND
THAT'S DONE, WELL, I'LL SHOW YOU
HOW THAT'S DONE IN THE NEXT
SLIDE, BUT GOING BACK TO ONE OF
THOSE SLIDES I SHOWED IN THE
MIDDLE, THE ELONGATION ON AN
OPEN TEMPLATE HAS TO OCCUR FOR
EVERY NUCLEOTIDE THAT'S
INCORPORATED OR MOST EVERY
NUCLEOTIDE, BUT OKAZAKI FRAGMENT
MATURATION USES THE LOWER THING,
STRAND DISPLACEMENT.
SO WE'RE VERY INTERESTED IN THAT
QUESTION, ENERGETICALLY,
BIOCHEMICALLY, STRUCTURALLY, BUT
WE'RE ALSO INTERESTED IN THAT
FROM THE STANDPOINT OF APPLYING
WHOLE GENOME SEQUENCING GENOMICS
APPROACH TO THE QUESTION.
SO WE DID THAT IN A YEAST STRAIN
THAT LACKS OKAZAKI FRAGMENT
MATURATION PROCESSING BECAUSE IT
LACKS FLAP ENDO NUCLEASE 1,
WHICH DISRUPTS THAT FLAP, SO
THAT IT CAN BE EXCISED BY FEN1,
AND WHEN WE DID THAT MONTHS AGO,
WE FOUND SOMETHING THAT TOTALLY
CONFUSES ME.
BUT IN A GOOD WAY.
IT'S ALWAYS GREAT TO BE
CONFUSED, BECAUSE EVENTUALLY IF
YOU WORK HARD ENOUGH ON IT, YOU
CAN FIND OUT SOMETHING
INTERESTING.
SO -- I APOLOGIZE FOR THAT.
I JUST WANT TO SHOW YOU TWO
SLIDES.
WHEN WE INVESTIGATED MISMATCH
REPAIR, IF I CAN EVER GET BACK
TO THAT, ONE OF THE NUCLEASES
THAT'S THOUGHT TO BE INVOLVED IN
REMOVING A MISMATCH, ONCE THE
NEWLY SYNTHESIZED STRAND HAS
BEEN NICKED TO INCLUDE AN ENTRY
POINT FOR A NUCLEASE, ONE OF THE
NUCLEASES IS FEN1 THAT WORKS
DURING OKAZAKI FRAGMENT
MATURATION, AND THAT'S WHAT I
WAS SHOWING HERE.
THIS, HOWEVER, IS A VERY
COMPLICATED SLIDE SO I'M NOT
GOING TO TRY TO EXPLAIN IT TOO
MUCH, BUT TO TRY TO SAY THAT
THIS IS THE CHANGE IN LENGTH IN
BASE PAIRS, EITHER INSERTIONS ON
THE RIGHT OR DELETIONS ON THE
LEFT, WHICH OCCURS IN A STRAIN
THAT'S LACKING MISMATCH REPAIR
COMPLETELY BECAUSE YOU'VE
DISRUPTED THE FIRST SEMINAL
PROTEIN IN THE PATHWAY, THE MUSE
S ALPHA HETERODIMER.
AND IN GREEN YOU SEE ALL THE
SINGLE BASE MUTATIONS CREATED IN
AN MSH2 STRAIN, ONE OF THE
REASONS IT'S SUSPECTED IN BEING
HEAVILY INVOLVED IN TUMOR
FORMATION.
AND AS YOU GO UP AND OUT, YOU
SEE THERE'S MUCH LESS LOWER RATE
OF REPLICATION ERRORS IN A
MISMATCH REPAIR DEFECTIVE YEAST
CELL AS ILLUSTRATED BY THE RED,
WHICH HAVE -- IF YOU COULD READ
IT, YOU CAN'T, LOWER MUTATION
RATES.
SO THE PREDOMINANT CONSEQUENCE
OF KNOCKING OUT ALL MISMATCH
REPAIR IS POINT MUTATIONS.
AND WE PUBLISHED THIS QUITE A
WHILE AGO, BUT I PUT IT LIKE
THIS BECAUSE THIS IS WHAT
HAPPENED WHEN WE KNOCKED OUT RAD
27.
SO THERE ARE SO MANY THINGS
DIFFERENT ABOUT THIS SLIDE THAN
KNOCKING OUT MISMATCH REPAIR, IT
REALLY CONFUSES ME, BUT YOU
STILL SEE GREEN.
A LOT OF FRAME SHIFT MUTATIONS
RESULTING FROM LOSS OF DNA
MISMATCH REPAIR, SO RAD 27 IS
INVOLVED IN THAT PROCESS, BUT
YOU ALSO SEE A LOT OF OTHER
ERRORS IN VARIOUS ERRORS AROUND
THIS CURVE, THEY INVOLVE BOTH
DELETIONS AND INSERTIONS, SO WE
BELIEVE THERE'S AT LEAST ONE
OTHER MECHANISM THAT IS CRITICAL
THAT RAD 27 FLAP ENDONUCLEASE BE
THERE FOR BECAUSE OF THE HIGH
RATE OF MU TEATION MUTATIONS INR
REGIONS OF THAT PLOT.
WE DON'T UNDERSTAND THAT.
I THINK, AS DOES MY COLLEAGUE
WHO IS RESPONSIBLE FOR THIS
WORK, THAT THERE MAY BE A TOTAL
OF 30 DIFFERENT MECHANISMS
RESPONSIBLE FOR THIS, OF WHICH
ALL OF THEM ARE AFFECTED BY LOSS
OF RAD 27, WHICH ANOTHER WAY TO
SAY THAT IS IT'S DOING MUCH MORE
THAN CREATING POINT MUTATIONS,
WHICH IS THE MAJOR EFFECT OF
LOSS OF MISMATCH REPAIR.
I DON'T UNDERSTAND THAT EXCEPT,
I MEAN, I COULD GO ON FOR HALF
AN HOUR ABOUT THIS, BUT I'M NOT
GOING TO.
I AM GOING TO POINT OUT THAT IN
THE LAST FEW YEARS, MANY
ARTICLES HAVE DESCRIBED
SPECIFICITY IN TUMORS ISOLATED
FROM A WIDE VARIETY OF CANCERS.
EVERY TIME I OPEN YOU C UP CELL,
NATURE OR SCIENCE, I READ
ANOTHER VERY EXCITING ARTICLE ON
THAT SUBJECT.
THESE STUDIES FOCUS ON THE
IDENTITY OF POINT MUTATIONS THAT
DRIVE TUMOR FORMATION TOWARDS
DEFINING TARGETS FOR CLINICAL
INTERVENTION.
THE TRANSLATIONAL SIDE OF WHAT
NIH DOES.
THE RESULTS I JUST SHOWED YOU ON
THAT LAST SLIDE IN THE YEAST RAD
27 MUTATIONAL PROFILE SUGGESTS
THAT SUCH DRIVERS MAY INCLUDE
GENES, MUTATIONS IN GENES THAT
DRIVE LARGER MUTATIONAL EVENTS.
AND THAT SUBJECT HAS NOT YET
BEEN INVESTIGATED BY THOSE
MULTIPLE EXCITING TRANSLATIONAL
RESEARCH STUDIES.
SO THERE IS A WIDE OPPORTUNITY
FOR ANY OF YOU STUDENTS IN THE
AUDIENCE TO BECOME INVOLVED IN
THIS WHICH IS STILL A GROWTH
AREA.
AND LAST, I WANT TO SINCERELY
THANK THE MANY OUTSTANDING
COLLEAGUES THAT I'VE HAD OVER
THE YEARS FOR CONTRIBUTING TO
THIS RESEARCH FOR NOW 42 YEARS.
THANK YOU.
[APPLAUSE]
>> AGAIN, QUESTIONS?
I'M GOING TO CHECK ONLINE.
I KNOW THERE'S A LOT OF PEOPLE
WATCHING AND PERHAPS FROM NORTH
CAROLINA.
>> HELLO.
THANK YOU FOR YOUR INTERESTING
TALK.
I'M JUST WONDERING, ANY
PERSPECTIVE REGARDING TO AGING
AND TO FERTILITY?
>> YEAH, THERE ARE LOTS OF
STUDIES IN THE LITERATURE ON THE
ROLE OF MUTATIONS, OR FAILURE TO
CREATE MUTATIONS IN THE AGING
PROCESS.
VERY ROBUST SET OF LITERATURE.
IN FACT, NIH HAS AN AGING
INSTITUTE, AND SEVERAL OF MY
COLLEAGUES IN THAT INSTITUTE
COULD TELL YOU PLENTY MORE ABOUT
THAT SUBJECT THAN THAT BRIEF
ANSWER THAT I JUST GAVE.
>> IS THERE AN ANALOGOUS PROCESS
FOR WHEN A MISTAKE IS MADE IN
IRANIRNAELONGATION LIKE IN THE N
OF RNA?
>> YES, THERE ARE A LOT OF
ANALOGIES TO ERROR CORRECTION
DURING RNA SYNTHESIS AND ERROR
CORRECTION DURING DNA SYNTHESIS.
THERE'S A WORLD OF RESEARCH DONE
ON THAT SUBJECT.
OF COURSE ONE ERROR IN RNA
SYNTHESIS IS NOT AS SEVERE AS
ONE ERROR IN DNA SYNTHESIS
BECAUSE YOU MAKE A LOT MORE RNA
THAN YOU MAKE DNA, BUT
NONETHELESS, THERE ARE ANALOGIES
THAT HAVE BEEN DRAWN BETWEEN THE
TWO PROCESSES.
>> GREAT.
THANK YOU.
ALL RIGHT.
THANK YOU VERY, VERY MUCH.
[APPLAUSE]
I'VE BEEN ASKED BEFORE OUR FINAL
TALK JUST TO LET YOU KNOW THAT
THERE WILL BE A RECEPTION AFTER
THIS SESSION RIGHT IN THE LOBBY
DIRECTLY OUTSIDE.
AND OUR FINAL SPEAKER IS
DR. GIORGIO TRINCHIERI.
[APPLAUSE]
>> THANK YOU, THANK YOU VERY
MUCH.
I'M VERY HAPPY TO BE HERE,
REALLY EXCITED TO HEAR THE
PREVIOUS TWO TALKS.
I THINK MY TALK WILL HAVE
SOMETHING A LITTLE BIT DIFFERENT
THAT MY CAREER HAS BEEN NOT VERY
STABLE, I'VE BEEN MOVING AROUND,
I THINK A LOT MORE THAN THE TWO
PREVIOUS SPEAKERS.
AND I REALLY START IN ITALY,
THEN MOVED TO THE IMMUNOLOGY,
YOU CAME TO THE STATES IN
PHILADELPHIA.
I WENT BACK TO SWITZERLAND FOR A
COUPLE OF YEARS, AND THEN I
MOVED TO -- BACK TO WISTAR FOR
20 YEARS, I WENT TO A COMPANY
SCHERING-PLOUGH IN FRANCE, AND
IN 2004, COMING BACK FROM
INDUSTRY, I WAS VERY HAPPY TO
COME TO NIH.
FIRST I DID A SABBATICAL,
MENTIONED A LITTLE BIT OF THAT,
IN NIAID AND FOR ALMOST THE LAST
20 YEARS IN THE NATIONAL CANCER
INSTITUTE.
AND MY -- SO I'M GOING TO TRY TO
PUT TOGETHER MY HISTORY WITH
SOME OF THE SCIENCE THAT WAS
GOING AT THE SAME TIME, AND ONE
OF MY FIRST PROJECTS IN TORINO
BUT ALSO THE -- IMMUNOLOGY WAS
TO IDENTIFY WHAT THE CYTOTOXIC T
LYMPHOCYTE WOULD RECOGNIZE
WITHIN THE MAJOR
HISTOCOMPATIBILITY COMPLEX HLA
IN HUMAN.
SO OBVIOUSLY WE KNOW A LOT NOW
ABOUT HLA, CLASS 1, CLASS 2,
MANY DIFFERENT GENE, CLASS 3,
AND WE KNOW THE INVOLVEMENT IN
DISEASE AND ALL OF THAT.
BUT THAT WAS VERY DIFFERENT IN
THOSE YEARS, TALK ABOUT '68,
'72.
WE KNEW THAT THERE WERE ANTIGEN
THAT WERE RECOGNIZED BY SERA
FROM -- WOMAN AND THEY WERE
CALLED SEROLOGICAL DEFINE AND
JUST AT THAT TIME IT WAS
DISCOVERED THERE WERE ACTUALLY
TWO DIFFERENT LOCI, A AND B.
THESE ARE THE ANTIGEN THAT WERE
DESCRIBED ORIGINALLY BY SOME OF
THE PIONEER IN THE FIELD.
BUT ALSO FRITZ BARK AT THAT TIME
FOUND WHEN YOU MIX --
TOGETHER -- CELL CULTURE AND
DEFINE A SEPARATE LOCUS IN THE
HLA CHROMOSOME THAT HE DEFINED
AS LYMPHOCYTE DEFINE NOW W WE KW
IS CLASS 2 MHC.
SO WHAT WE DID IS WE MIXED UP --
CULTURE WITH STIMULATE --
UNIDIRECTIONAL DONOR LYMPHOCYTE
STIMULATED BY THE LYMPHOCYTE
FROM DONOR B.
WE KNEW THAT WAS REGULATED BY
THIS LI LYMPHOCYTE DEFINED LOCU.
BUT WHEN WE LOOKED AT THE
CYTOTOXIC T-CELL IN THIS CULTURE
THEY WERE KILLING NOT FROM THE
LOCUS FROM WHICH THEY WERE
ACTIVATED BUT THEY RECOGNIZE THE
SEROLOGIC ANTIGEN -- THEY WERE
RECOGNIZED BY THE ANTIBODY ON
CLASS I HLA ORGANIZED BY THE
CYTOTOXIC T LYMPHOCYTE.
SO WE IDENTIFIED THAT THE T
LYMPHOCYTE RECOGNIZE CLASS 1 --
CELL THAT OBVIOUSLY WAS
IMPORTANT FOR -- TRANSPLANT
AFTER GRAFT REJECTION.
A COUPLE OF YEARS LATER,
ZINGERNAGEL AND DOHERTY GOT
MAJOR FUNDING FOR WHICH THEY WON
A NOBEL PRIZE, THE RECOGNITION
OF VITAL ANTIGEN IS ACTUALLY
RESTRICTED BY SELF CLASS I MHC.
SO -- ALSO RECOGNIZE CLASS I
MHC.
WHAT WAS NOT KNOWN AT THE TIME,
THERE WAS A LOT OF DISCUSSION,
IS WHETHER A TUMOR ANTIGEN ALSO
NEEDED CLASS I FOR RECOGNITION.
SO WHEN I MOVED TO WISTAR, I
WORKED WITH BARB RECREATION AND
PARKS -- THAT WERE -- T ANTIGEN,
AND WE FIND OUT IF YOU IMMUNIZE
MICE -- TRANSFORM CELL, THEY
WILL KILL -- CYTOTOXIC T-CELL
BUT ONLY IF THEY WERE FROM THE
SAME STRAIN WE IMMUNIZED.
SO B6 MICE ONLY KILL B6 AS --
CELL -- CELL,, BALB/C MICE -- T-
DESCRIBE, BY THE WAY A SURPRISE
BECAUSE WE ALSO IMMUNIZE MICE
NOT ONLY -- TRANSFORM CELLS, BUT
ALSO WITH AL JO ALLOGENEIC -- AD
REGARDLESS OF WHICH CELL WE USE
TO IMMUNIZE THE MICE, STILL ALL
THIS ALLOGENEIC -- SO WE BA BASY
FOUND THE TUMOR ANTIGEN -- CELL
ARE CURRENTLY REPRESENTED IN
VIVO IN SUCCESSION WITH --
WHAT'S NOW KNOWN AS ANTIGEN
CROSS PRESENTATION.
NOW WE KNOW HOW THE T-CELL
RECEPTOR WORK AND WE KNOW ALL
THE MOLECULAR DETAIL OF THESE
PARTICULAR INTERACTIONS IN TERMS
OF ACTION.
BUT IN THE 70s THE T-CELL
RECEPTOR WAS DESCRIBED BY
REINHARDT AND MARK DAVIS IN THE
80s, SO AT THAT TIME WE REALLY
DIDN'T KNOW WHAT WAS GOING ON
AND WE HAD A LOT TO DISCUSS WITH
PETER DOHERTY WHEN HE WAS AT
WISTAR INTUESDAY AND WE CAME OUT
WITH THIS CONCEPTUAL PAPER AND
TRY TO FIGURE OUT WHAT WAS GOING
ON, AND WE BASICALLY -- DRAWING
FROM THE PAPER, WE SAID THAT MAY
BE THAT MH SEA CHANGED THE
VIRUS, SO THE VIRUS CHANGED MHC
OR MAYBE THERE'S TWO RECEPTORS,
MAYBE THERE'S A RECEPTOR THAT
SEE WHAT IS BETWEEN THE TWO, AND
ACTUALLY THE FIRST HYPOTHESIS
THAT WE HAVE, THE POSSIBILITY,
THE H2MHC WAS MULTIPLIED BY THE
VIRUS, WE KNOW NOW IT'S
MULTIPLIED BY THE PEPTIDE, IS
PROBABLY THE ONE CLOSE WITH
EVENTUALLY WAS DISCOVERED TO BE
THE REAL RECOGNITION.
NOW WHAT ABOUT CROSS
PRESENTATION?
EXACTLY AT THE SAME TIME, THE
SAME MONTH, MAY '76, MIKE --
DESCRIBED CROSS PRESENTATION, IN
THIS CASE IT WAS WORKING WITH
THE MINOR -- ANTIGEN, AND BEVAN
AND MANY OTHER REALLY DID ALL
THE MOLECULAR CHARACTERIZATION
ANTIGEN CROSS PRESENTATION.
WHAT WE KNOW NOW IS THAT THE
ANTIGEN CAN BE CROSS PRESENTED
BY SPECIALIZED CELL TYPES
MOSTLY, THIS -- MOSTLY ONE
SUBSTITUTE DENDRITIC CELL BUT
AGAIN WHEN WE WERE DOING THE
STUDY WAS IN '76, THE DENDRITIC
CELLS WERE ACTUALLY DESCRIBED BY
STEINMAN IN '73 AS FUNNY LOOKING
CELLS IN THE LYMPHONODE OF MICE.
IT WAS NOT ONLY REALLY AFTER
2004, YES, AFTER THE STUDY I'VE
BEEN DISCUSSING NOW, BEVAN'S
STUDY, THAT -- EVENTUALLY
IDENTIFIED THE DENDRITIC CELL AS
THE ANTIGEN PRESENTING CELL IN
CULTURE AND LYMPHOCYTE PRESENT
BEING IN GENERAL.
AND AT THAT TIME OBVIOUSLY WE
COULD NOT ATTRIBUTE -- NOR BEVAN
TO DENDRITIC CELL AND WHAT WE
WERE REALLY THINKING WAS WITH --
IT WAS -- A MAJOR ANTIGEN
PRESENTING CELL WERE A MACROPHA,
THE TUMOR CELL IN VIVO AND THEN
PRESENT ON THEIR H2 TO THE
IMMUNE SYSTEM OF THE HOST.
I'D LIKE TO MENTION EMIL UNANUE,
ACTUALLY PASSED AWAY A SHORT
TIME AGO, AND -- WAS THE ONE
THAT NOMINATED ME FOR THE
ACADEMY.
SO THE OTHER WHEN WE RECOGNIZE
THAT THE HUMAN -- HLA CLASS I,
WE THOUGHT THAT MAYBE IF WE USE
ANTIBODY AGAINST HLA CLASS 1,
REMEMBER THERE WERE NO
MONOCLONAL ANTIBODY AT THAT
TIME, THERE WERE SE SERA FROM A
WOMAN, MAYBE WE CAN BLOCK THE
KILLING.
IF WE'RE USING TO TRY TO
COMPLETELY BLOCK, WE DID NOT
REALLY BLOCK THE KILLING BUT WE
GOT MORE KILLING AND THE REASON
WAS THAT WE'RE IN OUR CULTURE,
NATURAL KILLER CELLS THAT WERE
ABLE TO DO ANTIBODY
CYTOTOXICITY, REALLY BROUGHT FOR
SEVERAL YEARS IN MY GROUP TO
STUDY MANY OF THE BIOLOGY OF
THIS INNATE LYMPHOCYTE OF
NATURAL KILLER CELL.
SO WITH -- WE DESCRIBED FOR
EXAMPLE THE RECEPTOR RECOGNIZING
IMMUNOGLOBULIN ON THE NATIONAL
KILLER CELL, AND WE DISCOVER
THAT IT WAS A MAJOR ACTIVATOR
FOR NATURAL KILLER CELL
FUNCTION, BUT WE ALSO FIND OUT
THAT THE NK CELL THAT WERE KNOWN
TO KILL TUMOR CELL OR TEU NOR
CELL LINE, WERE ALSO VERY EE
EFFICIENT IN KILLING VIRUS
INFECTED CELL BUT NOT THE SAME
FIBROBLAST -- IN THIS CASE WERE
NOT INFECTED BY VIRUS.
AND WHEN WE LOOK AT WHAT WAS
HAPPENING THE AFFECTED CELL, WE
FOUND OUT THAT PRODUCTION OF
TYPE 1 INTERFERON WAS NEEDED TO
ACTIVATE NK CELLS IN VIRUS
INFECTED CELLS.
BUT WHEN WE TRY TO IDENTIFY
WHICH CELL IN THE CULTURE WAS
PRODUCING INTERFERON, THAT WAS
NOT NK CELLS, THEY WERE NOT THE
VIRUS INFECTED CELL BUT ACTUALLY
A VERY RARE CELL TYPE IN HUMAN
PERIPHERAL BLOOD THAT WE DEFINE
AS THE -- APOLOGIZE, THIS SEEMS
TO BE FROZEN.
OKAY.
LET'S GO BACK.
THAT WE RECOGNIZE -- THAT WE
DEFINE NATURAL TYPE 1 INTERFERON
PRODUCING CELL AND CELL TYPE NOW
IS VERY WELL STUDIED --
DENDRITIC CELL.
WE FOUND THESE CELLS WERE THE
MOST EFFICIENT PRODUCER OF TYPE
1 IN PERIPHERAL BLOOD.
THERE WERE SOME -- WITH
DENDRITIC CELL BUT ACTUALLY
QUITE DIFFERENT, HERE I SHOW THE
MORPHOLOGY OF HUMAN DENDRITIC
CELL IN THE BLOOD VERSUS THE
INTERFERON PRODUCING CELLS.
AND WE STUDIED THAT FOR A FEW
YEARS, BUT WHAT WAS SURPRISING
WAS UNTIL ABOUT 20 YEARS LATER,
IMMUNOLOGISTS IN MOUSE WERE
CLAIMING THAT THIS CELL DID NOT
EXIST IN THE MOUSE, THEY WERE
ONLY PRESENT IN HUMAN.
SO WE WENT TO TRY WHEN I WAS IN
ROSAN TO TRY TO IDENTIFY THE
CELL AND WE FOUND OUT ACTUALLY
THEY'RE VERY SIMILAR IN THE
MOUSE AS IN HUMAN.
THEY ARE MOUSE TYPE 1 PRODUCING
CELL AND THIS WAS DEFINED
PLASMA -- MORPHOLOGY AND THEY'RE
NOW -- DENDRITIC CELL.
WHEN I MENTIONED THAT THIS
REALLY BROUGHT ME TO COLLABORATE
FOR MANY, MANY YEARS WAS A VERY
CLOSE FRIEND OF MINE, CHRISTINE
BIRON, THAT WE DID THE MAJOR
PLAYER IN THE NK CELL FIELD AND
VIRUS INFECTION, AND WE ARE
HAPPY TO COLLABORATE IN SOME OF
THIS STUDY.
NOW, INTERFERON CAN INDUCE -- IN
CASE -- BECAME CYTOTOXIC -- WE
FOUND A FEW YEARS LATER THAT
OTHER CYTOKINE LIKE INTERLEUKIN
2 OR STIMULATION THROUGH THE --
RECEPTOR WERE NOT ONLY -- TO
BECOME MORE CYTOTOXIC, ALSO USE
THEM TO PROLIFERATE BUT MOST
IMPORTANT WAS INDUCE THEM TO
PRODUCE A CYTOKINE.
THE MAJOR ONE IS INTERFERON
GAMMA BUT ALSO THE CYTOKINE
LISTED HERE.
NOW THIS FINDING THAT -- NK CELL
AND -- T-CELL -- WITHOUT
SENSITIZATION WAS REALLY THE
FIRST IDENTIFICATION OF NK CELL
IS AN EXAMPLE OF -- TYPE I
SIMILAR TO NK CELL, TYPE 2 AND
TYPE 3.
BUT THE OTHER THING THAT WE WERE
VERY INTERESTED IN AT THAT TIME
WAS THE FACT NK CELLS WERE KNOWN
TO BE ABLE TO REGULATE
HEMOPOIESIS, INTERFERON GAMMA --
MACROPHAGE AND THE -- CYTOKINE
COULD INDUCE -- OF -- MAJOR
ROLE -- BUT ALSO TWO OTHER
CYTOKINE, WE ARE ALL WORKING
TOGETHER WITH INTERFERON GAMMA
IN HAVING THIS EFFECT OF MYELOID
DIFFERENTIATION AND INHIBITION
OF HEMATOPOIESIS.
SO THESE ARE THE CYTOKINES THAT
ARE THOUGHT TO BE GENE CLONE.
WE COULD NOT GET -- FOR TOXINS
SO WE DECIDE WE HAD TO PURIFY
LYMPHOTOXIN.
AND THE GOOD SOURCE OF
LYMPHOTOXIN IS FROM EBV
TRANSFORMED HUMAN B CELL LINE.
SO WE STARTED PURIFYING FROM ONE
OF THE CELL LINE, AND WHY WE ARE
DOING THAT AN WE'RE TESTING IN
FRACTIONATION SUPER NATANT --
BECAUSE WE KNEW THAT THE B CELL
LINE ALSO ACTIVATED AND CAN
INTRODUCE INTERFERON PRODUCTION
WITH -- IF WE'RE SEEING THE
SUPER NATANT OR SOME ABILITY TO
STIMULATE INTERFERON GAMMA OR
ACTIVATE NK CELLS.
THIS IS REALLY THE FIRST TWO
COLUMBUS WE DID.
THE FIRST COLUMN WAS -- WE'VE
FOUND A BROAD PEAK AND ALSO
INTERFATHER ON GAMMA PRODUCTIONS
STIMULATING NK CELL, BUT THEN
WHEN -- PEER FIED BY ION
EXCHANGE CHROME MAAING TO FEE,
WE COULD COMPLETELY SEPARATE
FROM THE ABILITY TO STIMULATE N
KSM CELL TO PRODUCE INTERFERON
GAMMA.
SO WE DEFINE THIS NEW ACTIVITY
AS A NATURAL KILLER CELL
STIMULATORY FACTOR THAT WAS --
ABILITY TO -- INDUCE INTERFERON
GAMMA PRODUCTION.
AND WHEN WE BEAUTIFY ESPECIALLY
THE WORK BY THAT TIME, WE FIND
OUT THERE IS A VAN AROUND 70 --
BUT WHETHER THIS WAS REDUCED, IT
WAS ACTUALLY FORMED BY TWO
CHAINS, ONE OF 40,000, P40, ONE
OF THE 35,000 OR P35.
SO THIS WAS ACTUALLY NOT A
SINGLE CHAIN CYTOKINE, WAS
ACTUALLY -- IN WHICH THE
ACTIVITY WAS FOUND.
SO THAT WAS VERY SURPRISING
BECAUSE ALL THAT CYTOKINE AT
THAT TIME WERE KNOWN TO BE
SINGLE CHAIN, BUT THEN WE
ACTUALLY FOUND OUT MAYBE WE
SHOULD NOT BE SURPRISED BECAUSE
IL-1, IL2, IL-8, IL10 ARE NOT
ONLY SITE KIN, BUT THE FIRST
AIRPLANE, DUAL ENGINE, WAS.
IL2.
FOUND STRUCTURE -- WHEN
HETERODIMER FORM BY IL-6 AND
FORM OF IL-6 RECEPTOR.
SO THE IL12 WAS STARTED BY US
AND MANY OTHER, THE MOLECULES
WERE CHARACTERIZED, THE RECEPTOR
WAS CHARACTERIZED, FROM JACK --
IF YOU'RE LISTENING TO TESTIMONY
VCH -- VERY FAMOUS MOLECULES,
AND THE SIGNALS PROVES THAT
SIGNALING MOST AT FOUR WOULD
INDUCE INTERFERON GAMMA AND
OTHER ACTIVATED MONIKERS.
SO INTERLEUKIN 12 ENDED UP TO BE
A CYTOKINE THAT PROVIDES THE
CRITICAL LINK BETWEEN INNATE
ADAPTIVE IMMUNITY THAT'S
PRODUCED BY -- MOSTLY BY MYELOID
CELL, DENDRITIC CELL IN RESPONSE
TO MANY STIMULI, INCLUDING
INFECTION, BACTERIAL AND VIRAL,
AND ACTIVATING ON NK CELL AND
T-CELL.
AND IN 1993, WE WORK WITH
SERGIO -- COULD ACTUALLY SEE
THAT IL12 IS THE MAJOR MOLECULES
THAT IS INVOLVED IN THE
PRIORITIZATION OF THE FUNCTIONAL
SUBSET OF T-CELL, THE TH1
T-CELL, THE TL -- 1 THAT ARE THE
T-CELL THAT PRODUCE INTERFERON
GAMMA.
AND AGAIN, LIKE FOR CROSS
PRESENTATION, EXACTLY IN THE
SAME MONTH, IN 1993, THEY
PUBLISHED EXACTLY THE SAME
RESULT IN THE MOUSE.
AT THAT TIME OBVIOUSLY -- WAS A
MAJOR COMPETITOR OF US AND NOW
BECAME A WONDERFUL FRIEND AND
MANY COLLABORATIONS WE'VE DONE.
ONE I'D LIKE TO MENTION ACTUALLY
ANNE WAS ELECTED TO THE ACADEMY
OF SCIENCE AS INTERNATIONAL
ACADEMY MEMBER.
SO WHEN WE STARTED LOOKING
BETTER AT THE INTERACTION OF THE
DIFFERENT CHAIN WITH DELTA, WE
ACTUALLY START SEEING WHERE
OTHER CHAINS THAT WERE
INTERACTING WITH BEFORE,
PARTICULARLY ONE IS CALLED P19,
THEN NOW WE KNOW THIS FORM
ANOTHER CYTOKINE THAT WAS NOT
REALLY THE WORK IF MY LAB, IT
WAS MOSTLY THE WORK AT THAT
TIME -- FERNANDO, ROB AND --
THAT REALLY DESCRIBE MANY IL23
AND OTHER CYTOKINE OF THIS
FAMILY AND THIS REALLY WHAT HAS
BECOME NOW AS A GROWING FAMILY
OF HETERODIMERIC CYTOKINE, AND
IL23 AT THE BEGINNING WITH SOME
OF THE ACTIVITY IL23 WERE
ASSIGNED TO US TO HAVE BUT WHAT
ACTUALLY DUE TO THE ABILITY OF
I23 NOT TO -- AND THEREFORE
IL-23 IS ACTUALLY A MAJOR ROLE
IN AUTOIMMUNITY.
THE WORK ON IL12 REALLY STARTED
WHAT WAS VERY CLOSE FOR ALMOST
10 YEAR COLLABORATION BETWEEN
PHILADELPHIA AND BETHESDA.
WITH THE LAB OF SHERRA AS WELL
AS ALUMNI WILL LIKE PHIL SCOTT
THAT WAS ALREADY IN PHILADELPHIA
DEFINITELY YA, IF THERE'S --
COME TO BETHESDA FOR LAB MEETING
AND WITH ALL THESE
COLLABORATIONS WE WORK ON
EFFECTIVE IL20, IN CANCER, IN
INFECTION, AND IN AUTOIMMUNITY.
AND IT WAS REALLY A WOU WONDERFL
TIME OF COLLABORATIVE WORK ON
ALL THESE DIFFERENT ASPECTS.
AND I MENTIONED TO YOU THAT IL12
IS INDUCED BY MOSTLY MYELOID
CELL AND DENDRITIC CELL
STIMULATION WITH PATHOGEN OR
WITH BACTERIA OR WITH VIRUS.
AND THAT REALLY BROUGHT THE
THINKING BACK OF WHAT IS THE
WORK OF WILLIAM COLEY WITH THE
COLEY TOXIN WHICH WAS ABLE TO
TREAT OSTEOSARCOMA WITH A
MIXTURE OF BACK YEAR YA, STRECT
COCCUS AND -- AND INDUCE
INFLAMMATION AND -- REME REMISSN
SOME PATIENT AND WHAT WAS
CONSIDERED TO BE PROBABLY THE
FIRST IN THE LATE 19TH CENTURY,
THE FIRST FORM OF IMMUNOTHERAPY.
AND AS I MENTIONED, INDEED A
COLEY TOXIN -- ACTIVATE -- THAT
KILLED THE TUMOR AND OUR WORK AN
MANY OTHER LAB -- IN TUMOR
KILLING.
THAT'S REALLY BROAD TO STEVE
BALDWIN, QUITE POPULAR IN THE
IMMUNOTHERAPY FIELD.
IN WHICH IL12 WAS DEFINED AS THE
NEXT GREAT MAGIC BULLET.
THAT TIME OF THIS INTERACTION
WITH -- IN THE TUMOR, TUMOR
IMMUNOTHERAPY, REALLY BROUGHT ME
VERY CLOSE TO THE CANCER
RESEARCH INSTITUTE IN WHICH I'M
STILL INVOLVED AFTER MANY YEARS,
AND THE CANCER RESEARCH
INSTITUTE, YOU MAY KNOW, WAS
FOUNDED BY DR. HELENE COLEY
NAUTS, AND THAT REALLY BROUGHT
MANY DISCUSSIONS -- THAT WAS --
OF THE CANCER RESEARCH
INSTITUTE.
SO THIS WAS THE NEXT GREAT MAGIC
BULLET, SO DID WE CURE CANCER?
IT'S GREAT IN THE MOUSE, IT'S
CONSIDERED THE MOST POTENT
CYTOKINE -- SO TOXIC THAT IT WAS
REALLY NEVER SUCCESSFULLY USED
IN THE CLINICS, AND THERE WERE
ACTUALLY MAJOR TOXIC EFFECT
DURING THE CLINICAL TRIAL.
THERE IS STILL WORK, THERE'S
STILL ATTEMPT TO USE IN A MORE
LOCALIZED TARGET WAY TO AVOID
TOXICITY.
WE DID SOME WORK WITH STEVE
ROSENBERG ON THAT AND ON CAR-T
CELLS AND SOME PEOPLE ARE STILL
WORKING ON THAT, AND FOR
EXAMPLE, ROSA KAPLAN AT NCI IS
REALLY GETTING VERY NICE WORK
WITH TRANSFECTING MYELOID CELLS
WITH -- AVENUES IN THERAPY.
SO THE ANTIBODY AGAINST THE
P4010IL12 WOULD BLOCK -- WAS
APPROVED BY FDA FOR VIE CYST,
ARTHRITIS AND THE DRUG -- IS
ACTUALLY SELLING MORE THAN
$10 BILLION PER YEAR.
AND I DON'T KNOW IF I'M PROUD OR
ASHAMED OF THAT BUT IT'S
ACTUALLY ONE OF THE 10 DRUGS BY
THE ADMINISTRATION TARGETED FOR
PRICE REDUCTION.
PROBABLY THIS DRUG IS NOT GOING
TO BE AROUND FOR LONG BECAUSE
NOW IT'S A SPECIFIC ANTIBODY
AGAINST IL20 THEY BUT MUCH
MORE -- AN MOST LIKELY THEY WILL
REPLACE THE LESS SPECIFIC
ANTI -- SO IN 2004, I WANT TO
LEAVE MY ADVENTURE IN BIG PHARMA
WHEN I WAS WORKING FOR
SCHERING-PLOUGH AND I WAS HAPPY
TO BE ABLE TO DO BASIC SAY ENS
THERI SCIENCETHERE BUT YOU DON'G
DOING BASIC SCIENCE IN A BIG
PHARMA COMPANY.
TWO PERSON I REALLY NEED TO
THANK FOR THAT, ONE IS MY OLD
FRIEND ALAN SHER THAT BROUGHT ME
FOR SIX MONTH SABBATICAL THAT
LASTING TWO YEARS IN HIS LAB,
AND KATHY ZOON AND TONY FAUCI
REALLY SUPPORTED THAT AND I HAD
A WONDERFUL YEAR AND A HALF, TWO
YEARS WORKING IN THE INFECTION
PARASITE LAB AND REALLY ENJOYING
MANY, MANY FRIEND, OLD FRIEND
AND NEW FRIEND I MET THERE.
THAT I ALSO KNEW FOR A LONG TIME
ENTICED ME TO MOVE TO FREDERICK
AND THEN EVENTUALLY TO BETHESDA,
AND -- FOR ALMOST 20 YEARS.
SO WHAT I WAS DESCRIBING TO YOU
WORK BETWEEN INFECTION DISEASE
AND CANCER REALLY START MAKE ME
AND SOME OF MY COLLABORATORS TO
REALLY PUSH THIS IDEA, AND
REALLY THINKING ABOUT LEARNING
FROM THE TWO SYSTEM HOW TO --
INFECTION DISEASE -- UNDERSTAND
CANCER IMMUNITY, THE FACT THAT
ANY INFECTION, ACUTE INFECTION
IS ACTIVE TO ELIMINATE THE
PATHOGEN, BUT THEN YOU SHOULD
MOVE THE INFLAMMATION RESOLUTION
INFECTION INFLAMMATION IMMUNITY
TO GO BACK TO TISSUE
HOMEOSTASIS.
IN TUMOR IT'S ACTUALLY THE
OPPOSITE.
THIS MECHANISM OR RESOLUTION OF
INFLAMMATION ARE THE ONE THAT
INDUCE CHRONIC INFLAMMATION,
INDUCE -- AND BLOCK IMMUNE
AGAINST THE TUMOR, AND IN TUMOR,
WE WANT TO DO THE OPPOSITE, WE
ACTUALLY WANT TO REACTIVATE THE
IMMUNE SYSTEM AND INFLAMMATION
IN A WAY TO KILL THE TUMOR
CELLS.
AND THE LAST 10 OR 12 YEARS WHAT
REALLY MY LAB REALLY STARTED
FOCUSING WAS A NEW ASPECT IN
MICROBIOLOGY INFECTIOUS DISEASES
REALLY WAS THE ROLE OF THE
COMMENSAL -- IS MICROBIOME.
AND REALLY WE'VE BEEN INTERESTED
TO START STUDYING CANCER AS A
DISEASE OF THE METAORGANISM, SO
THE ORGANISM LIKE OTHER ANIMAL
DPSH BY THEIR OWN CELLS TOGETHER
WITH THE COMMAND CELL MICROBE.
WE KNOW THIS MICROBIOME IS
REALLY IMPORTANT FOR THE
SURVIVAL OF THE METAORGANNISM.
THE CROSSTALK BETWEEN THE
MICROBIOTA REGULATE MANY
PHYSICAL FUNCTION, THIS INCLUDES
IMMUNITY, MECHANICAL FUNCTION
METABOLISM.
SO WE REALLY WANTED TO STUDY
THIS IN CANCER, AND WE WORK WITH
MANY OF THE INVESTIGATOR OF NIH,
I WOULD SAY THAT PARTICULAR
WHICH WITH JULIE SEGRA AND -- IN
REALLY TRYING TO PUSH THE STURDY
OF THE MICROBIOME AS NEW
FRONTIER OF RESEARCH.
I THINK THIS TRANS NIEH ATTEMPT
DIDN'T WORK VERY WELL BUT EASILY
THE CONTRIBUTION OF JULIE SAGER
TOGETHER WITH -- THE FIELD AS
REALLY BEING GREAT FUNDAMENTALLY
AS MAY EVENTUALLY BECOME -- OUR
WORK TOGETHER WITH -- IN
COLLABORATION WITH ME ON OUR OWN
RECEIVED SOME INTEREST AND I
ACTUALLY WAS QUITE EXCITED WHEN
NATIONAL GEOGRAPHIC REPORTED OUR
WORK.
I NEVER THOUGHT NATIONAL
GEOGRAPHIC WOULD BECOME
INTERESTED IN OUR WORK.
WHAT WAS NOT SO NICE, THAT THE
SECTION -- WAS NOT EXACTLY
ROCKET SCIENCE.
THEN I REMEMBERED THAT MY SON
GAVE ME A PRESENT A FEW YEARS
BEFORE AND I THINK HE WAS VERY
PROPHETIC AND HE GAVE ME THIS
COFFEE CUP, AND I STILL HAVE IT
ON MY DESK.
I THINK IT'S A GOOD REMINDER FOR
HOW WE SHOULD LOOK AT SCIENCE.
OKAY, SO MICROBIOME IS NOW SEEN
AS A MAJOR PLAYER IN CANCER,
IT'S -- INSTABILITY, DEFINITELY
IN FAVORING -- TUMOR GROW, BUT
ALSO AS A MAY YOUR ROLE IN
REGULATION OF CANCER THERAPY.
THIS HAS BEEN GOING FOR QUITE A
LONG TIME, IT WAS ONLY
'22 THAT -- FINALLY PUT THE
MICROBIOME AS ONE OF THE
CANCER -- SO WHAT WE DID WAS THE
WANT TO KNOW IF THE CANCER
THERAPY COULD BE AFFECTED BY THE
MICROBIOME.
AND WE ESTABLISHED THE GUT
MYOBIOME -- CANCER THERAPY
MOSTLY BY MODULATE ANTITUMOR
RESPONSE, MONITORING -- IMMUNITY
BUT DIRECTLY INDIRECTLY --
IMMUNITY.
AND OUR PAPER IN WHICH THE MAJOR
PLAYER -- NOT SHOWN IN THIS
PICTURE, HE WAS ALREADY BACK TO
JAPAN, BUT -- AND HIS -- IN 2013
WAS TO SHOW THAT IF YOU WANT
TO -- OR IMMUNOTHERAPY USING --
YOU NEED THE PRESENCE OF THE GUT
MICROBIOTA.
AND WITHIN GUT MICROBIOTA, YOU
NEED CERTAIN SPECIES THAT WERE
FAVORTIVE RESPONSE.
-- FEW YEARS BEFORE LIKELY A
DIFFERENT SITUATION -- SHOW THAT
LPS AND MICROBIOME -- SOME ROLE
BUT AT THE SAME TIME, WE PUBLISH
IN 2013LAURENCE ZI TV OGEL, LONG
TIME FRIEND OF MINE, IN
PITTSBURGH, PUBLISHED IN PARIS
THE ROLE OF THE MICROBIOME AND
OTHER CHEMOTHERAPY IN
CYCLOPHOSPHAMIDE.
THE MAJOR INTEREST IN THE
COMMUNITY REALLY COME WHEN
LAURENCE AS WELL AS -- SHOW IN
MICE THAT THE MICROBIOTA PLAY A
ROLE NOT ONLY IN CHEMOTHERAPY
BUT ALSO IN IMMUNOTHERAPY WITH
IMMUNE CHECKPOINT INHIBITORS IN
CTLA4 OR ANTIPD-1.
OUR PAPER AND LAURECE'S PAPER IS
ALSO LISTED AS IN SCIENCE 2013,
SO WE CONTINUE WORK IN THE LAST
10 YEARS IN MICE IN CLINICAL
WORK, AND WE STUDY TOGETHER WITH
MANY OTHER INVESTIGATORS SOME OF
THE M -- IN WHICH SOME BACTERIAN
THE LEFT MAY FAVOR RESPONSE TO
ANTI-PD-1 BUT -- WOULD PLAY A
MAJOR ROLE IS OUR OTHER TYPE OF
BACTERIA, MOSTLY GRAM-NEGATIVE,
SEEMS TO HAVE AN INHIBITORY
EFFECT ON THE RESPONSE OF PD-1
AND THE QUESTION IS WHAT DO WE
WANT TO TARGET FOR THERAPY, THE
POSITIVE EFFECT OR THE
ELIMINATING NEGATIVE EFFECT.
NOW, IF THE MICROBIOME EFFECT --
THE RESPONSE TO CANCER THERAPY
AND BECAUSE WE CAN CHARACTERIZE
THE MICROBIOME IN THE IF YOU
TELL OGUT OF THEPATIENT, CAN WEE
PATIENT WILL RESPOND BASED ON
THE BABBLING TIER YA IN THE GUT
BACTERIA IN THE GUT OF THE
PATIENT.
EACH INDIVIDUAL IS ABOUT
COUPLE -- MORE DIFFERENT BACK
FEAR YA SPECIES IN THE GUT AND
THERE ARE SEVERAL THOUSAND --
DISTRIBUTE IN DIFFERENT BACTERIA
AND EACH PERSON IS REALLY
DIFFERENT AND ITS GEOGRAPHIC
LOCALITY IS A DIFFERENT
MICROBIOME.
SO WHEN YOU TRY TO USE A MACHINE
LEARNING TO PREDICT A RESPONSE,
TRAIN THE MACHINE LEARNING WITH
THE DATA FROM ONE -- AND PREDICT
THE RESPONSE IN ANOTHER ONE IS
COMPLETELY FAIL.
BUT IF YOU DO TRAIN YOUR MODEL
USING ALL THE DIFFERENT CORE
TOGETHER LEAVING OUT ONLY ONE
CORE, THEN THINGS START TO BE
BETTER AND WE CAN GET A
REASONABLE HIGH ACCURACY IN
PREDICTION OF THE RESPONSE.
THERE ARE ALWAYS A LOT OF -- IN
THIS WORK DESPITE THE DIFFERENT
QUALITY OF MELANOMA PATIENT AND
THERE'S REALLY A LOT OF WORK IN
OUR LAB AND MANY OTHER LABS TO
TRY TO DEFINE MIC MICROBIOME
BIOMARKER FOR PREDICTION OF THE
RESPONSE.
BUT THE OTHER THINGS IS THE --
CAN WE USE -- IN THERAPY, CAN WE
TARGET THE MICROBIOME TO IMPROVE
THERAPY RESPONSE.
AND WHAT WE DID AND ALL OUR
CLINICAL WORK IS REALLY BEEN A
WONDERFUL -- MANY A
COLLABORATION NOW WITH SHOWING
HERE THE WORK OF DEVAR AND -- IN
PITTSBURGH, AND WHAT WE WERE
ABLE TO SHOW THAT IF WE GIVE A
FECAL MICROBIOTA TRANSPLANT FROM
A PATIENT RESPOND TO ANTIPD-1 TO
PATIENT THAT HAVE COMPLETELY
FAILED TO RESPOND TO ANTIPD-1,
WE GET A REVERSION OVERCOME THIS
PD-1 RESISTANCE IN APPROXIMATELY
40% OF THE PATIENTS.
THERE ARE NOW THREE OR FOUR
STUDY IN OTHER GROUP THAT'S
SHOWN ABOUT THE SAME RESULT THAT
IS VERY PROMISING.
BUT WHAT ACTUALLY WAS HAPPENING
JUST A FEW DAYS AGO, A
PRELIMINARY STUDY WAS REPORTED
BY GROUP IN ITALY THAT DID FIRST
RANDOMIZED TRIAL, IT IS ACTUALLY
QUITE SIGNIFICANT EFFECT OF THE
FECAL MICROBIOTA TRANSPLANT IN
IMPROVING THE RESPONSE IN
PATIENTS THAT FAILED THERAPY
BEFORE THE TRANSPLANT.
AND THE OTHER THING THAT WE WORK
AND THAT WE ARE TRYING TO FIGURE
OUT, ARE THERE WAY TO CHANGE THE
MICROBIOTA, AND ONE WAY THAT IS
POSSIBLE TO DO THAT IS WITH
DIET, AND TOGETHER WORKING WITH
JENNIFER WARGO, WE'VE BEEN ABLE
TO SHOW THAT PATIENT WITH HIGH
FIBER DIET ARE MUCH MORE
RESPONSE SIEVE TO ANTIPD-1
THERAPY, WE COMPLETELY
REPRODUCED THOSE RESULTS IN MICE
AND SHOWED THAT THE FIBER OF THE
DIET IS AFFECTING PD-1 RESPONSE
BY CHANGING THE MICROBIOTA
COMPOSITION IN THE -- AT LEAST
IN THE MOUSE ENVIRONMENT.
SO I'VE BEEN DISCUSSING LIKE MY
TWO COLLEAGUES BEFORE ME MY
YEARS IN SCIENCE.
I WOULD LIKE TO LIST EACH
PERSON, ITS CONTRIBUTION, I
DON'T HAVE A PICTURE OF EVERYONE
I'VE WORKED WITH, I TRY TO
COLLECT AS MANY AS POSSIBLE,
SOME OF YOU MAY KNOW SOME OF
THEM, ESPECIALLY THE NIH PEOPLE
BUT IT'S BEEN A LONG JOURNEY, A
REALLY REWARDING ONE AND I'M
THANKFUL TO THE NIH FOR THE LAST
20 YEARS AND BE ABLE TO DO THIS
TYPE OF WORK.
THANK YOU VERY MUCH.
[APPLAUSE]
>> THANK YOU.
KNOW THE ROUTINE.
IF YOU HAVE QUESTIONS, AT THE
MICS.
>> YOU KNOW, IT INTERESTING TO
ME THAT YOUR JOURNEY HAS TAKEN
YOU FROM THE ENDOGENOUS IMMUNE
SYSTEM AND THE CELLS THAT
MEDIATE IT, AND I THINK WE ALL
THINK OF AS BEING THERE TO FIGHT
THIS FOE, THE EXOGENOUS
MICROBIAL UNIVERSE, ALL THE WAY
ALMOST FULL CIRCLE TO THE POINT
WHERE YOU'RE STUDYING THE
PEACEFUL COEXISTENCE AND MAYBE
COLLABORATION WITH THE
MICROBIOME.
IS THERE A DANGER OF THE
ENDOGENOUS IMMUNE SYSTEM KILLING
OFF SOME OF THESE -- I MEAN, ARE
THERE DISORDERS THAT RESULT FROM
SEEING THE MICROBIOME AS FOE, OR
IS THERE SOMETHING THAT PROTECTS
THESE PARTICULAR ORGANISMS FROM
ATTACK BY THE HUMAN IMMUNE
SYSTEM?
>> OBVIOUSLY, I MEAN, WE EVOLVE
AS AN ECOLOGICAL SYSTEM AND WE
HAVE MOST OF THE TIME PRETTY
GOOD.
THERE ARE INNATE AND IMMUNE
MECHANISMS THAT REGULATE THAT.
THE EPITHELIAL BARRIER ALWAYS
PLAYS A MAJOR PHYSICAL ROLE IN
AVOIDING THAT.
BUT SOMETIMES THINGS GO WRONG
AND YOU HAVE TWO TYPE OF
PATHOLOGY.
ONE IS WHAT THEY CALL -- THEY
NORMALLY ARE OKAY, BUT IN
CERTAIN SITUATIONS, THEY MIGHT
BECOME A PATHOGEN.
-- TYPICAL ONE, WE ALL --
DIFFICILE, BUT IF IT GROW IS NOT
CONTROLLED IT BECOMES A MAJOR
PATHOGEN.
THE OTHER ONE IS THE OPPOSITE
WHEN OUR SYSTEM IS BECOMING TOO
ACTIVATED AGAINST THE COMMENSAL
AND THEN OBVIOUSLY THE MAJOR
DISEASE YOU CAN THINK IN THIS
CASE IS INFLAMMATORY BOWEL
DISEASE, CROHN'S DISEASE, AND
ULCERATIVE COLITIS.
>> WONDERFUL.
WONDERFUL.
YOU KNOW, I THINK ALL OF THE
TALKS WE'VE HEARD TODAY REALLY
GIVE US A SENSE THAT THERE'S
ALWAYS A NEXT QUESTION.
I MEAN, I THINK ESPECIALLY THE
WORK WE'VE JUST HEARD, YOU KNOW,
THERE IS SOMETHING ABOUT SCIENCE
THAT KEEPS ONE INTELLECTUALLY
CURIOUS FOR A LIFETIME, AND THAT
ALWAYS MAKES THERE BE A NEXT
QUESTION.
SO THANK YOU SO MUCH.
>> THANK YOU.
[APPLAUSE]
>> WELL, BEFORE WE GO OUT FOR
OUR RECEPTION, I HESITATE TO SAY
THIS IS A TRADITION, THIS IS
ONLY THE SECOND TIME I'VE DONE
THIS, BUT LAST YEAR, CHRIS
ACTUALLY COMMENTED THAT I DID A
SORT OF A SUPPLETIVE TOUR, THREE
SENTENCES, THAT TIED TOGETHER
THREE TALKS THAT SEEMED AT FACE
VALUE TO HAVE ABSOLUTELY NOTHING
TO DO WITH ONE ANOTHER.
AND THIS YEAR, WHAT STRUCK ME
WAS THE SORT OF CAREER TAKE-HOME
LESSONS THAT PERVADED REALLY ALL
THREE OF THE TALKS THAT WE'VE
HEARD SO I'VE COME UP WITH FIVE
SORT OF, I DON'T KNOW, LIFE AND
CAREER LESSONS THAT I WANT TO
SHARE WITH YOU, AT LEAST ONE OF
WHICH MAY GET ME IN A LOT OF
TROUBLE.
THE FIRST IS, ONE SHOULDN'T BE
AFRAID TO STICK ONE'S NOSE WHERE
SOME WOULD SAY IT DEFINITELY
DOESN'T BELONG.
I THINK THAT IS SOME OF THE
EXCITEMENT OF SCIENCE.
THE SECOND IS THAT ONE SHOULD
HASTEN TO JUXTAPOSE FIELDS THAT
NO SANE PERSON WOULD EVER PUT
TOGETHER.
THE THIRD IS THAT YOU SHOULD
ALWAYS CHECK YOUR WORK AND
PARTICULARLY YOUR SPELLING
BEFORE YOU HAND YOUR WORK IN.
THE FOURTH, AND THIS MAY BE THE
PROBLEMATIC ONE, IS THAT FOREIGN
POLITICAL INTERFERENCE EXTENDS
TO INTERLEUKIN NAMING
CONVENTIONS.
AND THE FIFTH, AND THIS IS THE
CRUX OF THE WHOLE THING, AND
AGAIN, PERVADED ALL THREE OF THE
TALKS TODAY, YOU SHOULD USE YOUR
SCIENCE TO FORM A BROAD AND DEEP
NETWORK OF FRIENDS AND A LEGACY
OF IMPACT.
SO PLEASE JOIN US FOR THE
RECEPTION OUTSIDE, AND JOIN ME
IN THANKING OUR SPEAKERS ONCE
AGAIN.
[APPLAUSE]