GOOD AFTERNOON. WELCOME TO
THE 26TH ANNUAL ASTUTE CLINICIAN
LECTURE. I'M COLLEEN HAIGAN
CHIEF MEDICAL OFFICER AND
DIRECTOR AT THE NIH CLINICAL
CERTAINTY. THE LECTURE IS NIH
DIRECTOR'S WEDNESDAY AFTERNOON
LECTURE SERIES, EVENT HOSTED BY
THE CLINICAL CENTER. WE ARE
PLEASED TO WELCOME BOTH IN
PERSON ATTENDEES AND ALL OF OUR
PARTICIPANTS WHO ARE WATCHING
THROUGH THE NIH VIDEO WEBCAST.
DR. TABAK UNFORTUNATELY WAS
UNABLE TO JOIN US TODAY DUE TO
OTHER COMMITMENTS. TODAY'S A
LITTLE HOUSEKEEPING, TODAY'S CME
CODE IS 50097. PLEASE TEXT THIS
CODE TO THE JOHNS HOPKINS CME
PHONE NUMBER ON THE SLIDE TO
RECEIVE YOUR CME CREDITS FOR
PARTICIPATION AT THIS LECTURE.
FINALLY, LAST PIECE OF
HOUSEKEEPING. DURING THE LECTURE
IF YOU HAVE QUESTIONS PLEASE
SUBMIT THEM FOR DR. SEARS ANY
TIME DURING THE LECTURE BY
CLICKING THE LIVE FEEDBACK
BUTTON LOCATED ON THE VIDEOCAST
WEBSITE. AND DR. SEARS WILL HAVE
AN OPPORTUNITY TO ANSWER THOSE
QUESTIONS AS TIME PERMITS AT THE
CONCLUSION OF THE LECTURE. FOR
INDIVIDUALS IN THE ROOM THEY CAN
GO TO ONE OF THE MICS WHEN
ASKING A QUESTION. SO FIRST, THE
ASTUTE CLINICIAN LECTURESHIP IS
ESTABLISHED THROUGH A GIFT FROM
THE LATE ROBERT MILLER, AND HIS
WIFE. IT HONORS A U.S. SCIENTIST
WHO HAS OBSERVED AN UNUSUAL
CLINICAL OCCURRENCE AND THEN BY
INVESTIGATING IT HAS OPENED AN
IMPORTANT NEW AVENUE OF
RESEARCH. IT IS AN HONOR TO
INTRODUCE CYNTHIA SEARS, AN
INFECTIOUS DISEASE EXPERT IN GUT
AND FOOD BORNE INFECTIONS. DR.
SEARS' PRESENTATION TITLED
SLEUTHING THE MICROBIOME,
REVEALS UNDERCOVER AGENTS OF
ONCOGENESIS. THIS WILL PROVIDE
CONTEXT FOR MICROBE PROMOTION OF
COLON CARCINOGENESIS AS
ILLUSTRATED BY TWO VIGNETTES.SS
ONE DEFINED BY A SINGLE
BACTERIAL GENE AND SECOND
COMMUNITY POTENTIAL TO MOLD
DISEASE. SHE EARNED HER M.D.
FROM THOMAS JEFFERSON MEDICAL
COLLEGE AND COMPLETED RESIDENCY
AND FELLOWSHIP AT NY HOSPITAL
WEILL CORNELL MEDICAL CERTAINTY.
IN 1985 DR. SEARS BEGAN HER
TEACHING JOURNEY AS ASSISTANT
PROFESSOR OF MEDICINE AT
UNIVERSITY OF VIRGINIA BEFORE
TRANSITIONING TO JOHNS HOPKINS
UNIVERSITY SCHOOL OF MEDICINE
WHERE SHE IS STILL TODAY. DR.
SEARS IS A PHYSICIAN, MENTOR,
AND ALSO PROFESSOR OF MEDICINE
AND ONCOLOGY AT JOHNS HOPKINS
UNIVERSITY SCHOOL OF MEDICINE
AND PROFESSOR OF MOLECULAR
MICROBIOLOGY AND IMMUNOLOGY AT
THE HOPKINS BLOOMBERG SCHOOL OF
PUBLIC HEALT HEALTH. SHE LEADS E
MICROBIOME PROGRAM IF THE CANCER
INSTITUTE FOR CANCER
IMMUNOTHERAPY AT JOHNS HOPKINS
AND DIRECTOR OF THE MURINE
FACILITY AT JOHNS HOPKINS. THE
LAB IS CONDUCTING HUMAN AND
MURINE STUDIES TO DETERMINE IF
AND HOW MICROBIOME IMPACTS
CLINICAL RESPONSES TO CANCER
IMMUNOTHERAPY P. AS INFECTIOUS
DISEASE EXPERT DR. SEARS STUDIED
GUT INFECTIONS INCLUDING
DIARRHEAL AND FOOD BORNE
ILLNESSES SUCH AS C DID C DIFF D
HELICOBACTER. IN LABORATORY AND
CLINICAL SETTINGS SHE STUDIED
PATHOGENESIS FRAGILLIS OVER THE
PAST 25 YEARS. THE CURRENT
FOSSAS OF THE SEARS LABORATORY
IS DETERMINE HOW THE MICROBIOTA
AND SPECIFIC BACTERIAL PATHOGEN
HEARSAY CONTRIBUTE TO COLON
CARCINOGENESIS. THE LABORATORY
INTEGRATES STUDIES OF HUMAN AND
MOUSE MODELS, EMPLOYING
MICROBIOLOGY, BIOINFORMATICS,
IMMUNOLOGY, AND OTHER SCIENTIFIC
METHODS, DR. SEARS HAS WORKED
ABROAD IN THAILAND BRAZIL,
HAITI, BANGLADESH AND MAY LA
SHAH. SHE ALSO CERTAINED AS THE
EDITOR OF THE ASSOCIATE AD E OF
ED SR. EDITOR OF JOURNAL
INFECTIOUS DISEASE BETWEEN 2000
AND 2016 APPOINTED EDITOR IN
CHIEF OF JOURNAL OF INFECTIOUS
DISEASE IN JANUARY OF 2022.
SHE'S MEMBER OF THE E TERM
ADVISORY BOARD OF DIGESTIVE
DISEASE RESEARCH CENTER AT
VANDERBILT AND COLUMBIA
UNIVERSITY, HAS BEEN AN ACTIVE
MEMBER OF IDSA MORE THAN 20
YEARS, SERVING THE SOCIETY IN
NUMEROUS CAPACITY AND CURRENTLY
PRESIDENT OF THAT ORGANIZATION.
IN 2021 SHE WAS AWARDED THE
JOHNS HOPKINS CANCER CENTER
MENTORING AWARD. DR. SEARS IS
RECOGNIZED GLOBALLY FOR HER
SCIENTIFIC EXPERTISE AND IS A
TRUE ROLE MODEL AS A CARING
COMPASSIONATE CLINICIAN TO ALL
PATIENTS. DR. SEARS EXEMPLIFIES
THE TRAITS THAT FOR THE HONOR
RES UPON THIS LECTURESHIP WHICH
WAS CREATED 26 YEARS AGO. WITH
NO FURTHER ADIEU, I WOULD LIKE
TO INTRODUCE DR. SEARS. THANK
YOU.
[APPLAUSE]
GOOD AFTERNOON, EVERYONE. I WILL
CORRECT ONE THING I WAS
PRESIDENT OF IDSA IN 2019 SO NOT
THE CURRENT PRESIDENT, NOT TO
TRY TO TAKE THAT HONOR TODAY. SO
I WANT TO FIRST THANK DR. MILLER
AND HITS FAMILY FOR SUPPORTING
THIS LECTURE AND THEN DR.
GILLMAN AND THE NIH COMMITTEE
FOR SELECTING ME FOR THE HONOR
OF SPEAKING TODAY AND TO ALL OF
YOU, HERE IN -- AND ONLINE WHO
JOINED TO LISTEN. SO THE TITLE
AS YOU HAVE ALREADY HEARD IS
SLEUTHING THE MICROBIOME REVEALS
UNDERCOVER AGENTS OF
ONCOGENESIS. AND FEW DISCLOSE
SURES NOT PARTICULARLY DOMAIN. I
HOPE TO GIVE A FRAMEWORK HOW WE
CONSIDER MICROBES IN THE
PATHOGENESIS OF HUMAN COLON
CANCER, AND TALK TO YOU A LITTLE
BIT ABOUT SPECIFIC BACTERIA AS
WELL AS COMMUNITIES THAT MAYBE
CONTRIBUTING TO THE DISEASE. SO
THE OVERARCHING HYPOTHESIS IS
THE GUT MICROBIOME IS A CRITICAL
ENVIRONMENTAL FACTOR IMPACTING
DISEASE AND THERAPEUTIC
OUTCOMES. THAT CERTAINLY IS NOT
UNIQUE TO OUR GROUP. BUT IF WE
STEP BACK TOP THE FIELD OF
INFECTIOUS DISEASES, I THINK WE
NOW MUST CONSIDER THAT
ANTIBIOTIC EXPOSURE OVER LIFE
THROUGH ITS MICROBIOME IMPACTS
IS AN EVER-EVOLVING DISEASE
MODIFIER POTENTIALLY WITH HIGH
IMPACT ON CHRONIC DISEASE
DEVELOPMENT. SO AS COLLEEN JUST
SAID I'M GOING TO PRESENT DATA
TODAY WHERE WE TOGGLE BETWEEN
HUMAN STUDIES AND MURINE STUDIES
TRYING TO UNDERSTAND HOW THE
MICROBIOME IS AFFECTING DISEASE.
THIS IS THE BIG PICTURE FOR THE
FIELD. ON ONE END OF THE
SPECTRUM WE ARE CONSIDERING THE
IMPACT OF SINGLE SPECIES. THE
THREE THAT GET DISCUSSED MOST
OFTEN ARE LISTED HERE,
FUSOBACTERIUM, ESCHERICHIA
CO-LIE AN INTERTOX GENIC
BACTEROIDES FRAGILIS. WE CAN
TAKE A MOUSE AND ADD ONE BUG TO
ITS MICROBIOME AND LEAD TO
COMPLETE CHANGE OF PHENOTYPE
DEPENDING ON TOXIN IT MAKES AND
IL 17 IN THE RIGHT GENETIC
CONTEXT. THAT GENETIC CONTEXT IS
A MOUSE THAT IS HETEROZYGOUS FOR
THE GENE APC, WHICH STANDS FOR
AD NO, MA'AMTUS POLYCOSIS COLI
AND IS THE GENE HEREDITARY
CANCER SYNDROME KNOWN AS
FAMILIAL ADENOMITUS AND IT IS
MUTATED OVER 80% OF SPORADIC
COLON CANCER. EACH OF THE
BACTERIA I JUST LISTED HAS
SUBSTANTIVE CONNECTIONS, TO
COLON CANCER IN HUMANS IN THE
LITERATURE. ON THE OTHER SIDE OF
THE EQUATION IS THE IDEA THAT
MICROBIAL COMMUNITY IS IMPORTANT
TO DISEASE DEVELOPMENT. AND IN
THIS CARTOON WHAT I'M SHOWING
YOU IS THE ENTIRE COLON IS
COVERED WITH TWO LAYERS OF
MUCOUS, ONE IS A LOOSE LAYER OF
MUCOUS, READILY INVADED BY
BACTERIA, THE SECOND IS THIS
DENSE LAYER OF MUCOUS THAT
STRETCHES THE AXIS OF THE COLON,
LARGELY STERILE, EXCEPT WHEN THE
RIGHT BUGS COME ALONG AND ARE
ABLE TO PENETRATE THAT AND
PIERCE IT AND THEN SIT RIGHT ON
TOP OF THE EPITHELIAL CELLS. AND
STUDYING THESE BIOFILMS HAS LED
US TO CONCEPTS AND
CARCINOGENESIS AS WELL AS
SURPRISING POTENTIAL CONTRIBUTOR
TO COLON CANCER. SO TWO
VIGNETTES AS MENTIONED ALREADY
TODAY. ONE IS THE POWER OF ONE,
IT USES A MODEL ENTEROTOXIGENIC
BACTEROIDES WHICH WE HAVE BEEN
STUDYING A LONG TIME. THE SECOND
IS POWER COMMUNITY, OBSERVATIONS
OF BIOFILM, RETURN TO THE FIRST
VIGNETTE, AND TALK ABOUT
INTERTOXIGENIC BACTEROIDES
FRAGILIS OR EBF. WHERE IN THE
WORLD DOES ETBF COME FROM? THESE
ARE KNOT NOT A HOUSEHOLD NAME,
IN IN CLINICAL MEDICINE. AND IT
TURNS OUT THESE ORGANISMS WERE
DISCOVERED BY DR. A LITTLE
MEYERS, VETERINARY SCIENTIST AT
MONTANA STATE UNIVERSITY IN THE
MID 1980s. AND HE TOOK ON THIS
PERPLEXING PROBLEM OF DIARRHEAL
DISEASE IN NEWBORN LAMBS WHICH
APPEARED NOT TO FOLLOW THE USUAL
PATH OF OTHER DIARRHEAL DISEASE.
IN AN ELEGANT SERIES OF
EXPERIMENTS TRYING TO FIGURE OUT
WHAT WAS GOING ON AT THE END OF
THE DAY, HE CAME TO THE
CONCLUSION THAT IT WAS THIS
BACTEROIDES FRAGILIS STRAIN IN
THE LOOP STIMULATING THIS
SECRETION. AND IN FACT, WHEN YOU
PUT THE SUPERNATANTS FROM THAT
BUG INTO ADDITIONAL LIGATED
LOOPS THEY BLEW UP SO MUCH WITH
FLUID THEY WOULD BURST SIMILAR
TO WHAT YOU CAN SEE IN ALCOHOL
CHOLERATOXIN. DR. BRAD SACK, ATS
HOPKINS MEDICINE WAS A SLEUTH OF
CHILDHOOD DIE REARIA. AT THAT
TIME CHILDHOOD DIARRHEA WAS THE
NUMBER ONE CAUSE OF MORBIDITY
AND MORTALITY, ONLY HALF COULD
BE EXPLAINED. SO HE NOTICED DR.
MEYERS WORK AND TOOK IT TO THE
FIELD WHERE HE WENT TO THE
INDIAN HEALTH INDIAN HOST
SERVICE HOSPITAL IN WHITE RIVER,
ARIZONA AND DID EPIDEMIOLOGY
STUDY LINKING ETBF TO HUMAN
DIARRHEAL DISEASE. WHEN I FIRST
WENT TO HOPKINS DR. SACK CALLED
ME UP AND LITERALLY CAME TO MY
OFFICE WITH THIS TUBE OF 20 FOLD
CONCENTRATED CULTURE
SUPERNATANTS FROM AN ETBF STRAIN
THAT DR. MYERS SENT HIM AND SAID
ALL THE ASSAYS WE TYPICALLY DO
IN DIARRHEAL DISEASE TO DETECT
TOXINS HAVE FAILED. I HEAR YOUR
WORK WITH COLON EPITHELIAL
CELLS, TAKE THIS AND SEE WHAT
YOU CAN DO. SO FORTUNATELY WE
WERE WORKING WITH THESE HUMAN
COLON CANCER CELLS CALLED HT 29C
1 CELLS. HAVING THIS TOOL YOU
CAN SEE TOP THE CONTROL SELLS
AND SEE WHAT HAPPENS AFTER YOU
ADD IN YOUR PURIFIED BRFT OR THE
CULTURE SUPERNATANT, WE
CERTAINLY HAD A TOOL. AND WHICH
IT ALLOWED US TO GO AND FIND THE
BFT GENE AND PURIFIED THE TOXIN.
LEARNING THAT IN FACT IT WAS
ACTIVE AT VERY LOW
CONCENTRATIONS. THIS IS AN ASSAY
WE STILL USE TODAY. SO STEPPING
FORWARD IT TURNS OUT ETBF IS
CHARACTERIZED BY A SINGLE
VIRULENCE FACTOR, BACTEROIDES
FRAGILIS GENE, IT IS A TOXIN
DEVELOPED BY THE ORGANISM AND
MOLDED BY THE ORGANISM AND
SECRETED AS A MATURE
20-KILODALTON PROTEIN. IT IS A
ZINC DEPENDENT PROTEASE TOXIN
DUE TO A SINGLE CHROMOSOME GENE
IN THE ORGANISM, OCCASIONAL LAY
TWO COPIES. ON THE RIGHT SHOWS
YOU THE SPECTRUM OF WHAT HAPPENS
WITH BACTEROIDES FRAGILIS, CLOSE
TO 90% OF US LISTENING ARE
COLONIZED WITH B FRAG. AT ONE
END ARE THE NON-TOX GENIC BACK
TROY DEES FRAGILIS THOUGHT
IMPORTANT TO MUCOSAL IMMUNE
SYSTEM AND WORK DONE BY DR.
CASPER AT HARVARD. THER END OF
THE SPECTRUM ARE THESE B FRAG
STRAINS PRO INFLAMMATORY AND
TUMORIGENIC. IN HUMANS DATA HAS
ALSO ACCRUED ASSOCIATING ETBF
WITH INFLAMMATORY DIE RELEASE
DISEASE AND INFLAMMATORY BOW WE
WOULD DBOWELDISEASE AND COLON CS
IS ONE SLIDE I SUMMARIZED BY
LIFE AND MANY OTHERS ON ONE
SLIDE SO LET ME WALK YOU THROUGH
THIS. THE FIRST THING I WANT TO
DO IS TO REVIEW THE TIME LINE.
THIS IS NOW GETTING APC
HETEROZYGOUS MOUSE WHICH WITH
THE SINGLE INOCULATION TO THE
MOUSE WE CAN GET COLONIZATION
AND TURNS OUT WE GET INITIATION
OF TUMOR FORMATION QUICKLY SO
THAT BY ONE WEEK YOU CAN SEE
THESE MICROSCOPIC TUMORS IN THE
COLON MUCOSA OF THE MOUSE DUE TO
LOSS OF HETERO ZYGOSITY. AND OF
COURSE OVER TIME THEY GROW UNTIL
THEY ARE BIGGER TUMORS. I WANT
TO POINT OUT HOW IMPORTANT THESE
STUDIES DR. LU AND AND DR.
(INDISCERNIBLE) WHERE THEY
WALKED WITH ME THE LAST 25 YEARS
IN DEVELOPING THESE DATA. SO THE
FIRST THING I WANT TO TELL YOU
IS ABOUT THE TOXIN, THEN INDEED
I MENTIONED THIS IS THE
VIRULENCE FACTOR OF NOTE. HERE
IS -- SEE IF I CAN DO IT. THERE
WE GO. HERE IS THE EVIDENCE FOR
THAT. IF E WE TAKE THE ORGANISM
AND DO AND IN FRAME DELETION
MUTANT SO THE TOXIN GENE IS NO
LONGER THERE WE OBLITERATE ALL
THAT TUMORIGENESIS AND
INFLAMMATION. ONLY WHEN THAT
GENE IS THERE DO YOU SEE ANY OF
THESE BIOLOGICAL EFFECTS IN THE
MICE. NOW, THE NEXT STEP IN THIS
PROCESS THAT WE STUDIED WAS E
CAD HEREIN CLEAVAGE, ACTIVATION
OF CMIC AND CELL PROLIFERATION.
HOW DID THAT COME TO POE BE?
EARLY IN THE 1990 ET CETERA I
WAS PRESENTING AT A CONFERENCE
AND AS I WALKED AWAY FROM THE
PODIUM DR. JAMES NELSON CHAIR OF
THE DEPARTMENT OF CELL BIOLOGY
AT STANFORD STOPPED ME AND SAID,
YOU KNOW, THERE IS SOMETHING
ABOUT THOSE CELLS THAT MAKES ME
THINK OF E CAD HEREIN. I HAVE
THREE AGENTS, WHY DON'T I SEND
THEM TO YOU AND SEE WHAT YOU
THINK. SO INDEED HE DID AND THIS
WESTERN BLOT SHOWS THAT IN FACT
DR. NELSON WAS SPOT ON. SO IN
THE FIRST LANE IS BLOT SHOWING E
CAD HEREIN, 120-KILODALTON
PROTEIN THAT IS KEY TO THE
COLONIC ENTHELIAL CELLS
TOGETHER. LANES 2 AND FORWARD
SHOW WHAT HAPPENS AFTER ADDITION
OF BFT TO THE HD 29 CELLS AND
WHAT YOU SEE IS THIS RAPID
CLEAVAGE OVER 30 TO 45 MINUTES
OF THE E CAD HEREIN AND WHAT THE
BLOT HERE SHOWING RESIDUAL
INTRACELLULAR DOMAIN. WHEN E CAD
HEREIN GETS CLEAVED IT RELEASES
IT RELEASES THIS EXTRA FRAGMENT
WHICH I WILL SHOW YOU IN A
MOMENT. THERE IS MY CARTOON. SO
WHAT HAPPENED NEXT WAS TWO
THINGS. PART ONE BURT VOGELSTEIN
AND KEN KENSLER PUBLISHED
SIMILAR PAPERS SAYING THE WINT
PATHWAY IS CRITICAL TO THE
DEVELOPMENT OF COLON CANCER.
SIMULTANEOUSLY I DID EXPERIMENT
I FORGOT TO STOP AND CAME BACK
IN THE NEXT MORNING AND LOOKED
IN THE INCUBATOR AND LOW AND
BEHOLD THE TISSUE CULTURE FLASK
WAS BRIGHT YELLOW. SO I THOUGHT
I NOT ONLY MESS UP THE
EXPERIMENT I CONTAMINATED THE
FLASK. WHEN I LOOKED UNDER THE
MICROSCOPE, IN FACT THE MENU WAS
CLEAR. WHAT HAPPENED IS OVER
NIGHT THE CELLS HAD PROLIFERATED
MADLY AND HAD USED UP ALL THE
GLUCOSE IN THE MEDIA. SO WE HAD
NEVER DONE LONGER EXPERIMENTS,
IT WAS A NEW OBSERVATION. SO
HERE IS A CARTOON OF WHAT WE
THINK HAPPENED. SO I MENTIONED E
CADHERIN IS A STRUCTURAL PROTEIN
IN THE PART OF THE JUNCTIONAL
COMPLEX IN THE COLON AND
TETHERED TO THE INTRACELLULAR
DOMAIN OF E CADHERIN IS A LITTLE
POOL OF BETA CATENIN. WHICH AT
THE TIME WAS THOUGHT TO BE
BIOLOGICALLY INERT. HOWEVER WE
KNEW BFT BOUND TO A SPECIFIC
RECEPTOR, WE DIDN'T KNOW WHAT IT
WAS ON THE CELL, WE SHOWED THAT
E CADHERIN GOT CLEAVED AND IN
FACT WE WENT ON TO SHOW THAT
BETA CATENIN TRANSLOCATED TO THE
NUCLEUS ACTIVATED CMIC AND LED
TO CELL PROLIFERATION. AS WE
CONTINUED FORWARD TRYING TO
UNDERSTAND THIS PROCESS, IN THE
MOUSE WE LEARNED THAT THE TOXIC
DNA DAMAGE SHOWN HERE BY GAMMA H
2A STAINING WHICH IS A MEASURE
OF DOUBLE STRANDED DNA BREAKS
INDICATIVE OF DNA DAMAGE. WITH
(INDISCERNIBLE) HERE IN THE
AUDIENCE TODAY WE STUDIED
ADDITIONAL TRANSDUCTION PATHWAYS
AND IDENTIFIED THAT P STAT 3 WAS
ACTIVATED AMONG ALL THE STATS
AND THAT ACTIVATION
PHOSPHORYLATION THAT PROTEIN IS
CRITICAL TO THE PRODUCTION IL 17
WHICH PROVED TO BE PRO
CARCINOGENIC IN THIS MODEL.
FRANK AND HIS TEAM HELPED
FURTHER PULL THIS APART AND WHAT
WE ULTIMATELY LEARNED IS THAT
THE IL 17 RECEPTOR ON THE
COLOLLIC EPITHELIAL CELL IS
CRITICAL TO THE CARCINOGENESIS.
ONCE THAT RESELL TORR IS ENGAGED
BY IL 17 THERE IS NF KAPPA B
ACTIVATION, RELEASED FROM THE
EPITHELIAL CELLS, CXCL
CHEMOKINES THEN THAT PULLS IN
ALL THESE IMMATURE MYLOAD CELLS
JUST TO THE DISTAL COLON WHERE
WE SEE THE TUMORS THAT IS SO
IMPORTANT TO THE TUMORIGENESIS
THAT THE PICTURES SHOW. WE TRIED
FOR YEARS TO FIGURE OUT BFT
RECEPTOR WAS AND THROUGH AN SI
RNA LIBRARY WE ZEROED IN ON GPR
35 GTB BINDING PROTEIN THAT IS
PREVALENT AND HAS HIGH
EXPRESSION LEVEL IN THE GUT.
HOWEVER, THAT WAS NOT THE
RECEPTOR. WHEN YOU KNOCK OUT
THAT PARTICULAR PROTEIN YOU GET
MORE TUMORS NOT LESS TUMORS. SO
IT REMAINED A PUZZLE WHAT THE
BFT TOXIN WAS BINDING TO. ENTER
MAX WHITE AN MST TALENTED MSTP
STUDENT AT JOHNS HOPKINS. FROM
THIS FIRST TIME I MET MAX, HOE
TOLD HIM ABOUT THIS PROBLEM, HE
GOT FASCINATED BY IT AND I WAS
FORTUNATE TO HAVE HIM JOIN THE
LAB. HE POSITED THAT BFT NEEDED
HELP FROM SOME COMPONENT OF AN
INTACT CELL TO CLEAVE E
CADHERIN. SO IN TODAY'S WORLD HE
TOOK ON A DIFFERENT TYPE OF
GENETIC SCREEN AND IN THIS CASE
IT WAS A CRISPER CAS 9 LIBRARY
IN HT 9 CELLS, TURNED OUT THAT
MATT WILDER AT HARVARD HAD
ALREADY MADE THIS LIBRA LIBRARYT
IS A DIE RELEASE DISEASE
INVESTIGATOR. WE CALLED HIM AND
HE INVITED MAX TO HIS LAB WHO
WORKED WITH (INDISCERNIBLE) TO
TEST THE LIBRARY. SO IN THE
FIRST RUN THE ASSAY USED TO
SCREEN THE LIBRARY WAS LOOKING
FOR LOSS OF E CADHERIN. WHAT MAX
CHOSE TO DO IS TO SORT OUT THE
TOP 10 PERCENT OF CELLS THAT
WERE STILL EXPRESSING E CADHERIN
THINKING THAT IN FACT THOSE
CELLS MUST HAVE LOST A GENE
THROUGH THE LIBRARY THAT WAS
CRITICAL TO THE ACTION OF BFT.
THEN ON DEEP SEQUENCING THOSE
CELLS THAT VERY FIRST RUN, ONE
GENE POPPED OUT AS SIGNIFICANT
AND THAT WAS (INAUDIBLE) 4 H.
MAX WENT BACK AND REPEATED THE
SCREEN AND SECOND TIME THROUGH
ADDITION SAL GENES CAME UP BUT
CLOUD AND 4 BECAME FURTHER
ENRICHED. NOW I WILL SHOW YOU A
SUBSET OF THE THE EXPERIMENTS
THAT MAX HAS DONE TO TRY TO
UNDERSTAND CLOUD AND 4 MAYBE A
RECEPTOR FOR THE BFT PROTEIN P.
SO IN THE FIRST FIGURE HERE WHAT
I AM SHOWING YOU IS A CLAUDIN 4
KNOCK OUT CELLS, HEK 29 CELLS
LOSE STABLE BFT BINDING. IN THE
BLOTS HERE YOU CAN SEE IN THE
WILD TYPE CELLS YOU CAN DETECT
CLAUDIN 4 AND BINDING OF BFT. IF
YOU KNOCK OUT CLAUDIN 4 IN THOSE
CELLS YOU LOSE THE SIGNAL. IF
YOU KNOCK OUT CLAUDIN 3 AS
POSSIBLE CONTROL THOSE SIGNALS
ARE STILL THERE BECAUSE CLAUDIN
4 IS STILL THERE. IN THE FLOW
PLOTS YOU CAN SEE WHEN BFT BINDS
CELLS IT SHIFTS CELLS TO THE
RIGHT BECAUSE THIS PROTEIN HAS
BEEN@ED TO THE CELLS. IF YOU USE
-- ADDED TO THE CELLS. IF YOU
USE THE C THE CLAUDIN 4 KNOCK
OUT CELLS DON'T MOVE BECAUSE THE
PROTEIN WON'T BIND. IF YOU USE
THE CLAUDIN 3 KNOCK OUT CELLS
THEY MOVE SIMILAR TO WILD TYPE.
THE SECOND TYPE OF PROTOCOL MAX
USED WAS TO TEST IF THE TOXIN
WOULD CO-IMMUNOPRECIPITATE WITH
CLAUDIN 4. THAT IS WHAT THIS
PLOT SHOWSS. IF U YOU USED AN
ANTI-CLAUDIN 4 ANTIBODY, GOT THE
CLAUDIN 4 AND BFT AT THE SAME
TIME IF YOU USE ANTI-CLAUDIN 2
ANTIBODY HE DIDN'T GET THE THE
SIGNALS. THE NEXT PROTOCOL WAS
HEK 293T CELLS. THESE HAS NO
ENDOGENOUS E CADHERIN OR
CLAUDIN, IS. SO WHAT HE DID, IS
HE TRANSFECTED IN CLAUDIN 4 AND
THIS PLOT SHOWS WHETHER THE
PROTEIN CLAUDIN 4 WAS
FLUORESCENTLY LABELED OR NOT,
THAT IN FACT THAT RESTORED
BINDING OF BFT TO THE CELLS
WHEREAS TRANSFECTING IN CLAUDIN
2 DIDN'T RESTORE THAT BINDING.
LASTLY, HE PUT THE WHOLE SYSTEM
TOGETHER. HERE HE TOOK THE HEK
293P CELLS, AND HE LABELED E
CADHERIN WITH M CHERRY AS A
FLUORESCENT PROBE. AND
TRANSFECTED THAT IN TO THESE
CELLS. HERE SHOWS THE FLOW PLOT
SHOWING YOU THE POSITION OF THE
E CADHERIN ON THE SURFACE OF THE
CELLS. IF IF HE ADDS BFT TO THAT
JUST EXPERIENCING E CADHERIN,
THAT SIGNAL DOESN'T MOVE AT ALL.
IF YOU CO-TRANSFECTS IN THE
CLAUDIN 4 HE IN FACT AND THEN
ADDS BFT YOU CAN SEE THE SIGNAL
MOVE BECAUSE THE E CADHERIN HAS
NOW BEEN CLEAVED AND THERE IS
THAT 80-KILODALTON EXTRA
CELLULAR DOMAIN THAT'S BEEN
RELEASED. SO WHAT HAPPENS NEXT?
MAX IS WORKING ON CLAUDIN 4
MUTATIONAL ANALYSES, AND IN THIS
CASE THIS IS A OF CLAUDIN 4 A
TRANSMEMBRANE PROTEIN. HE SO FAR
HAS TAKEN OFF THIS C TERMINUS
SIGNALING DOMAIN, THAT IS VERY
LITTLE EFFECT ON THE BINDING OF
THE BFT. WE THOUGHT THAT THE
LARGER EXTRA CELLULAR DOMAIN
WOULD PROBABLY BE VERY IMPORTANT
TO THE BINDING OF BFT BUT IN
FACT THAT IS NOT CORRECT EITHER.
TURNS OUT THAT LITTLE EXTRA
CELLULAR DOMAIN, THE SMALLER NUB
IS CRITICAL TO THE BFT THOUGH WE
SUSPECT THE TWO DOMAINS ARE
INTERACTIVE. THE OTHER THING WE
ARE DOING IS WE ARE DISCUSSING
WITH DR. XAVIER RUTH AND
COLLEAGUE WOOLRICH ECHART,
INSTITUTE OF BARCELONA, MODELING
SYSTEM. DR. GOMEZ RUTH IS THE
ONLY ONE WHO CRYSTALIZED BFT
EVER. IN THE WORLD. SO WE SET UP
A ZOOM CALL AND HAD A WONDERFUL
TIME WORKING THROUGH THIS
PROBLEM WITH THEM AND IF YOU
HAVE QUESTIONS ABOUT THAT, I
WOULD BE HAPPY TO ANSWER THEM AT
THE END. SO STEPPING BACK, MAX
HAS WRITTEN A VERY NICE REVIEW
THAT GOT PUBLISHED THIS MONTH IN
NATURE REVIEW MICROBIOLOGY, THIS
IS WHAT WE THINK IS THE LIST OF
CREDIBLE BACTERIAL CONTRIBUTORS
TO COLON CANCER PATHOGENESIS.
THERE IS 11 OF THEM ON THIS
LIST. AS WE LOOK AT THE LINKAGE
FOR ALL BUT SALMONELLA, THE
LINKAGE IS HUMAN LINKAGE
EPIDEMIOLOGICAL DATA SUGGESTS
THESE RELEVANT TO COLON CANCER.
WHEN WE MOVE TO ANIMAL MODELS IT
GETS SLIMMER BECAUSE MANY OF
THESE ORGANISMS DON'T READILY
COLONIZE MICE. . BUT YOU CAN
SEE A FEW DO. SO THERE IS
OPPORTUNITIES TO STUDY IN MORE
DETAIL THAN MECHANISMS OF THOSE
PARTICULAR ORGANISMS. SO NOW WE
WILL TURN TO VIGNETTE 2. HERE WE
WILL TALK ABOUT POWER OF
COMMUNITY AND DISCOVERIES WITH
BIOFILMS. SO GOING TO START WITH
A CLINICAL STUDY THAT WE DID
WORKING WITH (INDISCERNIBLE) AND
THE QUESTION WE WERE ASKING THIS
WAS PUBLISHED IN GUT, WAS IS
THERE AN ASSOCIATION BETWEEN ANY
ORAL ANTIBIOTIC USE AND THE
DEVELOPMENT OF COLON CANCER? SO
WE WERE FORTUNATE SNUFF TO GET
FUNDING FROM DR. PAUL ATWATER
WHO RUNS AN ENVIRONMENTAL
PROGRAM AT HOPKINS AND ABLE TO
BUY DATA FROM THE CLINICAL
PRACTICE RESEARCH DATA WHICH HAS
DATA FROM 1989 TO 2012 AT THE
TIME WE DID THIS. SO
LONGITUDINAL DATA. THIS IS THE
LARGEST CLINICAL DATABASE IN THE
WORLD. WORKING WITH THEIR
SCIENTISTS, WE DID A MATCHED
DESIGN WHERE THE CONTROLS AND
CANCER, COLON CANCERS ARE
MATCHED BY YEAR OF BIRTH GENDER
PRACTICE SITE AND CRPD
REGISTRATION DATE. SO WE HAD THE
OPPORTUNITY TO LOOK AT ALMOST
29,000 COLON -- COLORECTAL
CANCER AND 138,000 CONTROLS. THE
CORE RESULTS ARE SHOWN HERE. ON
THE HORIZONTAL AXIS ARE TOTAL
DAYS OF EXPOSURE TO ORAL
ANTIBIOTICS. THE STRIKING AND
UNEXPECTED FINDING WAS THAT BY
DAY 15 TOTAL ANTIBIOTIC EXPOSURE
OF ANTIBIOTIC IN DATABASE, THERE
WAS INCREASE ASSOCIATION WITH
THE INCIDENCE OF COLON CANCER.
IN CONTRAST AT RECTAL CANCER,
EXPOSURE TO ANTIBIOTICS LED TO
DECREASE RISK THAT SEEMED TO
LEVEL OUT ABOUT DAY 60. WHICH
ARE ABLE TO TAKE SOME OF THOSE
COLON CANCERS AND FIGURE OUT
WHERE THEY WERE FROM THE
DATABASE AND SOME WERE PROXIMAL
AND SOME WERE DISTAL. YOU CAN
SEE 100% OF THE SIGNAL FOR COLON
CANCER TRACED TO THE PROXIMAL
COLON WITH NO EFFECT. IN THE
DISTAL COLON. ONE OTHER PIECE OF
INFORMATION WE FOUND CONVINCING
WAS THAT THIS ASSOCIATION WITH
ORAL ANTIBIOTIC EXPOSURE ONLY
MATTERED FOR ORAL ANTIBIOTICS
TAKEN MORE THAN 10 YEARS
PREVIOUSLY. SO THIS IS NOT
CONFOUNDING BY INDICATION AND
THAT IS THE MAGIC NUMBER USED IN
THE FIELD FROM THE TIME YOU GET
THE START OF COLON CANCER UNTIL
IT BECOMES A POLYP AND
GASTROINTERROLOGIST SEES IT.
SUBSEQUENTLY ACTUALLY THE
HARVARD NURSES HEALTH STUDY WAS
PUBLISHED JUST BEFORE OURS AND
SUBSEQUENT STUDY OUT OF SWEDEN
ALSO VALIDATED THIS IDEA THAT
THE ORAL ANTIBIOTIC EXPOSURE AND
SUBSEQUENT COLON CANCER WOULD BE
DISTANT IN TIME. SO FOR ME THIS
HIGHLIGHTS HOW ANTIBIOTIC
MEDIATED MICROBIOME MAY
CONTRIBUTE TO CHRONIC DISEASE
DEVELOPMENT. SO NOW LET ME TELL
YOU MORE ABOUT BIOFILMS. SO THIS
IS THE WORK OF CHRISTINE
(INAUDIBLE) AN SUBSEQUENTLY
JULIA DREWAS. IN THE TOP FIGURE
HERE, THIS IS A HEALTHY COLON,
THAT LIGHT BLUE STAIN YOU ARE
SEEING IS THAT DENSE INNER
MUCOUS LAYER ON THE RIGHT OF THE
EPITHELIAL CELLS. TO DO THIS
PROJECT, WE SORT OF ROCKED BACK
AND SAID THERE IS IS A LOT OF
BUGS IN THE GUT. MAYBE WE
SHOULDN'T PUT ALL OUR MONEY IN
ONE BUCKET. LET'S BACK OFF AND
TRY TO LOOK BROADER WAY AT HOW
BACTERIA MAY BE AFFECTING COLON
CANCER, WE WENT TO THE PATHOLOGY
SUITE AND GOT PIECES OF TUMOR
FROM THE SURFACE AND WE GOT
PIECES OF TUMOR FROM THE FAR
DISTAL MARGIN OF SURGICAL
RESECTION, USUALLY HALF THE
COLON, AND BROUGHT THEM BACK O
THE LAB. AND THEN WE HYBRIDIZED
THEM WITH BACTERIAL R RNA PROBE
TO DETECT ALL BACTERIA THAT WERE
THERE. TO SUR SURPRISE WE CAN
SEE THAT PINK, THAT PINK IS A
MASS OF BACTERIA SITTING ON TOP
OF THE COLON TUMOR AND ON TOP OF
THE EPITHELIAL CELLS IN THE
NUCLEI IN THE CELLS STAINED IN
BLUE BY DAPI. IN ADDITION IF YOU
TAKE THAT NORMAL FLANKING IT
ALSO HAD THESE DENSE AGGREGATES
INVASIVE BIOFILM PRESENT AS
WELL. ALMOST ALWAYS IF THE TUMOR
SHOWS A BIOFILM THE FLAGGING
NORMAL WAY AWAY FROM THE TUMOR
IS ALSO GOING TO SHOW THAT
LEADING US TO BELIEVE THAT
PROBABLY THOSE BACTERIA ARE ALL
ALONG THE SWATH OF THE TISSUE.
WE WANTED TO KNOW WHAT THOSE
BACTERIA WERE. SO WE USED A
VARIETY OF PROBES IN AND THE
BOTTOM LINE IS ALL OF THESE
BIOFILMS ARE POLYMICROBIAL. WHEN
WE DID THIS STAINING, WHAT WE
COULD SEE IS THAT THE BACTERIA
INVADED TUMOR. SO EVERY TUMOR
WITH POSITIVE BIOFILM HAS AN
INTRATUMOR BACTERIAL POPULATION.
THE NEXT IS TO GO TO COLONOSCOPY
SUITE TO SEE PATIENTS AVERAGE
RISK FOR COLON CANCER AND WE
LEARN SUBSEQUENTLY 10 TO 15%
INDIVIDUALS UNDERGOING
COLONOSCOPY HAVE BIOFILMS BUT
SUNNY AND POLYMICROBIAL SITTING
ON TOP OF THE CELLS. ADDITION
WORK OVER TIME WE CAME TO
UNDERSTAND THIS WAS A SPECTRUM
OF BACTERIA BUT WITH THREE
FLAVORS IF YOU WILL. MOST ARE
POLYMICROBIAL THAT SHOW
PREDOMINANCE OF BACTEROIDES,
THERE IS LARGE SUBSET THAT ALSO
HAVE FUSO BACTERIUM BLOOMS SHOWN
IN YELLOW AND TINY SUBSET THAT
IS MADE UP OF A COMPLEX OF
DIFFERENT PROTEOBACTERIA GRAM
NEGATIVE RODS. I'M DISAPPOINTED
TO SAY THAT IN FACT WE STILL
DON'T KNOW WHO EXACTLY IS
SITTING IN THOSE INVASIVE
BIOFILMS BUT CAROLINE WENTZEL,
CELL MOLECULAR GRADUATE
PROFESSOR IS ANALYZING THE DATA
NOW SO HOPEFULLY WE WILL KNOW
SOON. WE WERE DOING THIS STUDY
AT HOPKINS AND DECIDED TO SET UP
A PARALLEL STUDY WITH SOME
FUNDING IN MALAYSIA. SO IN THIS
CARTOON ON THE LEFT OF THE
COLON, ALL THE RED DOTS,
REPRESENT BIOFILM POSITIVE
TUMORS WE EXAMINE AND THE BLUE
DOTS ARE BIOFILM NEGATIVE TUMORS
WHAT YOU CAN SEE IN THE
SUMMATION HERE IS THAT ALMOST
90% OF RIGHT COLON TUMORS WILL
HAVE BIOFILMS. REMEMBER
ANTIBIOTIC SLIDE. IN CONTRAST
ONLY ABOUT HALF OF THE TUMORS IN
THE LEFT COLON ARE POSITIVE.
WHEN YOU GO TO THE COLONOSCOPY
SUITE, IT IS AGNOSTIC TO
SCIENCE, 10 TO 15% SHOW
BIOFILMS. IN DATA I DON'T HAVE
TIME TO SHOW YOU BUT I WISH TO
TELL YOU IS WE LEARNED THESE
BIOFILMS CAN BE CARCINOGENICICI.
THEY AREN'T ALWAYS BUT CAN BE
AND WE CAN TELL THAT BECAUSE MT.
HUMAN TISSUE WE SEE DECREASED
BARRIER FUNCTION BY LOSS OF E
CADHERIN AND WE SEE ACTIVATION
OF CARCINOGENIC MUCOSAL AND
COLONIC EPITHELIAL CELL PATHWAYS
LIKE IL 6 AND STAT 3. WE SEE
THAT THE BIOFILMS ARE ASSOCIATED
WITH COLON EPITHELIAL CELL
PROLIFERATION. THEN WHEN WE PUT
TUMORS INTO MICE, FLURRIES OF
TUMORS INTO MICE WE GET
INDUCTION OF TUMORS IN THOSE
MICE. (INDISCERNIBLE) TOOK ON
THE DIFFICULT CHALLENGE TRYING
TO FIGURE OUT IF BIOFILMS IN
EACH PERSON WERE
PROCARCINOGENNIC. IN HER INITIAL
ALNAL SEIZE SHE TRIPPED UPON
BASICALLY, A TUMOR, 3728T,
69-YEAR-OLD MAN WITH STAGE 1
CARCINOMA IN THE RIGHT COLON
THAT IN FACT WAS PLOTTED HERE
ARE NUMBERS OF TUMORS AND THIS
PARTICULAR SLURRY WAS PRO
CARCINOGENIC. HERE IS SHOWING
THE TUMORS IN THE DISTAL GUT.
SHE QUALITIERED IT TO FIGURE HOW
MANY BUGS WERE THERE. TO SUR
SURPRISE WE DIDN'T FIND BFRAG OR
MUCOBACTERIUM. WE DID NOTICE
THAT IT HAD A CLOSTRIDIUM
DIFFICILE IN THERE. SO IF YOU
DID -- SHE TESTED THIS ISOLATES
NOW AND IN FACT THAT REPLICATED
THE RESULT WITH THE SLURRY. HE
DID THE OBVIOUS NEXT EXPERIMENT
AND PULLED THE C DIPF OUT OF THE
ISOLATE AND LOOKED AT WHAT
HAPPENED AND INDEED WHAT YOU CAN
SEE HERE IS THAT WITHOUT THE C
DIFF, I CAN'T CONTROL MY
POINTER. WITHOUT THE C DIFF
ISOLATES DON'T LEAD TO TUMOR
FORMATION. THEY ONLY LEAD TO
TUMOR FORMATION IN THE PRESENCE
OF THE C DIFF. THESE ARE
ADDITIONAL EXPERIMENTS WE DID TO
TRY TO CONFIRM THESE OBSERVA
OBSERVATIONS. JULIA IDENTIFIED A
SLURRY FROM A PATIENT THAT WAS
NOT PARTICULARLY TUMORIGENIC.
WHAT SHE DID WAS ADDED THIS C
DIFF TO THAT PARTICULAR SLURRY,
INDEED IT DID CONVERT IT TO BE
CARCINOGENIC IN THE MOUSE MODEL,
NUMBERS OF TUMORS PLOTTED HERE.
JADA AT ASTRA ZENECA DOING AN
ENTIRELY DIFFERENT PROJECT IN
THE LAB ON BIOFILM NEGATIVE
TUMORS HAD ONE SAMPLE, THAT LED
TO A LARGE NUMBER OF TUMORS AS
SHOWN HERE. SHE DECIDED BECAUSE
OF WHAT JULIA OBSERVED IN THE
LAB TO LOOK FOR C DIFF AND THAT
TOO WAS C DIFFICILE POSITIVE.
ANGELA CHEN WHO IS OBTAINING HER
Ph.D. IN MOLECULAR
MICROBIOLOGY IMMUNOLOGY GROUP
TOOK ON THE CHALLENGE OF SEEING
IF THE SYSTEM WORKED IN SPECIFIC
PATHOGEN FREE MICE AND INDEED
SHE ORDERED TOX GENIC STRAINS
AND NON-TOX GENIC STRAINS OF C
DIFF FROM ATCC. THE TOXOXI GENIC
STRAINS INDUCED TUMORS THE
NON-TOXI GENIC DID NOT. SO MANY
OF YOU IN THIS ROOM AND ONLINE
KNOW CLOSTRIDIUM BACTEROIDES
DIFFICILE INFECTION IS A DISEASE
PRECIPITATED BY ANTIBIOTIC
EXPOSURE IN MICROBIAL SHIFTS.
THIS IS A OBLIGATE AN AROBE AND
IT PRODUCES TWO TOXIN, TOXIN A
AND B BUT IT IS TOXIN B THOUGHT
TO BE MOST RELEVANT TO HUMAN
DISEASE. WE NOW KNOW IN IS NOW
THE MOST COMMON HEALTHCARE
ASSOCIATED INFECTION IN THE
U.S., A LEADING CAUSE OF GASTROO
ENTERITIS IN THE U.S. BUT WE
DON'T KNOW MUCH OF ANYTHING
ABOUT ITS PERSISTENCE IN THE
GUT. WE KNOW C DIFF RECURS IN
ACUTE INFECTION BUT NO DATA
AFTER C DIFF HOW MANY PEOPLE
HANG ON TO IT FOR A PROTRACTED
PERIOD OF TIME. NOW, JEWEL YAP
PUBLISHED HER RESULTS WITH
ANGELA CHEN AND OTHERS IN CANCER
DISCOVERY AND SHE PROVIDED
EVIDENCE TO LINK FOUR MECHANISMS
TO THE CARCINOGENICITY OF C
DIFFICILE BUT TODAY I WILL ONLY
SHOW YOU DATA ABOUT THE BIOFILMS
AN LINKAGE TO TOXIN AND THEN I
WILL SHOW YOU UNPUBLISHED DATA
WORKING ON THE COMMUNITY WHICH I
THINK IS VERY INTERESTING. SO
HERE AGAIN, WE ARE BACK TO THE
MOUSE. . WHAT JULIA DID SHE
INOCULATED THE MICE TO ALL MICE
BUT NOT C DIFF THE PINK IS
MASSIVE BACTERIA IN THE LUMEN OF
THE GUT AND THE BLACK SPACE
BETWEEN PINK AND BLUE, NUCLEI OF
THE EPITHELIAL CELLS, IS THAT
BLACK SPACE, THAT IS AN INNER
MUCOUS LAYER LARGELY NOT
PENETRATED HERE THOUGH
OCCASIONALLY IS, EVEN WITH THE
ISOLATES AS YOU CAN SEE IN THIS
PICTURE. IF YOU LOOK AT THIS
PICTURE ON THE RIGHT WHICH IS
INCREASED MAGNIFICATION YOU
DON'T SEE BACTERIA IN THE CRIBS
OF THE COLON EPITHELIUM. LOOK AT
THE BOTTOM NOW, THIS IS THE
ISOLATES INOCULATED WITH C DIFF
AND THE STRUCTURE OF THE BIOFILM
IS DRAMATICALLY DIFFERENT. IN
ADDITION LOOK AT THE PHOTO ON
THE FAR RIGHT, NOW THESE
BACTERIA REACH DOWN INTO THE
CRYPTS WHICH IS WHERE THE
COLONIC STEM CELLS ARE WHICH IS
THE PROGENITOR CELL FOR COLON
CANCER.
I THINK OVER HERE YOU CAN SEE IT
THOSE YELLOW RODS, THOSE ARE THE
C DIFF. SO IT IS NOT BECAUSE
THERE IS AN OVERWHELMING AMOUNT
OF C D I F F THERE, IT IS
BECAUSE THEY CHANGED THE
FUNCTION OF THE COMMUNITY. WITH
THE HELP OF (INDISCERNIBLE) C
DIFF IS VERY HARD THE MUTATE WE
OBTAINED SOME STRAINS THAT HAD
THE TOXIN B KNOCKED OUT. WHEN
JEW LA AND I TREATED THE --
JULIA TREATED THE MICE,
COLONIZED THE MICE WITH TOXIN B
STRAINS THEY COLONIZED FINE BUT
DIDN'T INDUCE TUMORS LIKE THE
TOXIN B POSITIVE STRAINS. T SO C
DIFFICILE POSITIVE COMMUNITY
INDUCES TUMORS THAT AGAIN IS
APPEARS TO BE DRIVEN BY
VIRULENCE GENE OF C D I F F
WHICH IS TOXIN B. NOW I WILL
DESCRIBE WHAT WE ARE DOING
TODAY, THE WORK OF HADLY POUR
GUARD CELL AND MOLECULAR
GRADUATE STUDENT, EARLIER WORK
DONE BY REECE KNIPEL WHO IS A
POST DOC WORKING IN THE LAB. SO
WE GOT INTERESTED IN WHAT ARE
THE REST OF THOSE ISOLATES DOING
IN THAT BIOFILM? SO I WANTED TO
DESCRIBE THE TWO EXPERIMENTAL
PROTOCOLS THEN SHOW YOU THE
DATA. SO IN THE FIRST PROTOCOL
WE EXAMINE HOW CULTURE
SUPERNATANTS FROM SOME OF THE
BACTERIA IN THAT BIOFILM, MIGHT
INFLUENCE GROWTH OF C DIFFICILE,
DONE BY GROWING STRAINS FOR 72
HOURS FILTER STERILIZING THEM
GETTING RID OF THE BUG AND
PUTTING THE C D I F F BACK IN
AND SEEING IF IT GROWS OR NOT.
IN THE SECOND PROTOCOL,
BASICALLY DOING THE SAME THING
BUT USING THE CULTURE
SUPERNATANT AT 72 HOURS AFTER
THE C D I F F WASSALLED AND IT
GREW -- ADDED AND IT GREW TO SEE
HOW MUCH TOXIN PRODUCED BY THE C
D I F F ON THAT CULTURE. AND
OVER HERE ARE VERO CELLS, A
CLASSIC ASSAY FOR C DIFFICILE
TOXINS. ON THE TOP NORMAL CELLS,
ON THE BOTTOM THEY ROUND UP WHEN
THEY SEE TOXIN A OR B IT TURNS
OUT. REECE HAD DONE THIS
PHENOMENALLY COMPLICATED SET OF
EXPERIMENTS TO FIGURE OUT WHICH
BUGS IN THE 30 MEMBER COMMUNITY
WERE IMPORTANT TO INTERACTING
WITH C D I F F. ONE THAT POPPED
OUT FROM HIS WORK WAS C CELLAELA
PNEUMONIA. SO PUT D C I F F IN
FRESH MEDIUM IT GROWS PINE. IF
YOU TAKE MEDIA AT 72 HOURS
FILTER STERILIZE AND REINOCULATE
IT WITH C DIFF RED LINE ON THE
BOTTOM, IT DOESN'T GROW. AT ALL
HOWEVER TAKE 72 HOUR CULTURE
FILTRATES OF A RANGE OF GRAM
NEGATIVE RODS INCLUDING CLUB
SEAL LA PNEUMONIAA AND
REINOCULATE WITH C DIFF THEY
GROW WELL AS SHOWN HERE. WE
THINK OR KNOW THAT CULTURE
SUPERNATANTS AND PROTEOBACTERIA
IN FACT RESTORE C DIFFICILE
GROWTH. THIS IS THE EXPERIMENT
DONE ADDITIONAL EXPERIMENT DONE
BY REECE AT ASTRA ZENECA AND IN
THE ZERO COLUMN IS WHETHER OR
NOT HE TOOK ALL THOSE ISOLATES
GREW THEM UP FOR 72 HOURS,
FILTER STERILIZED THEM, GREW THE
C DIFF AND TESTED THE TOXIN
PRODUCTION IN THAT CULTURE SOUP.
WITHOUT ADDITION AND JUST AS C
DIFF ALONE THE LEVEL OF TOXIN
PRODUCTION YOU SEE, AS YOU ADD
PROGRESSIVELY MORE OF THE
CULTURE SUPERNATANT FROM THE
OTHER BUGS YOU IN FACT GET
REDUCTION IN TOXIN PRODUCTION. I
WILL TELL YOU WHERE YOU KNOW
MORE THING. WE HAVE NOW THIS
FACTOR IN CULTURE SUPERNATANTS
APPEARS LESS THAN 4-KILODALTONS,
ITS HEAT LYPASE AND PROTEASE
RESISTANT. SO WE ARE SUSPICIOUS
OF A SMALL MOLECULE METABOLITE
MEDIATING THESE EFFECTS. HERE
IS WORKING HYPOTHESIS, THAT YOU
TAKE C DIFF IT IS HELPER BUGS IN
THOSE BIOFILMS AND YOU PUTS
THEMSELVE TOGETHER AND GET LESS
TOXIN PRODUCTION BY C DIFF BUT
SUSTAIN THE GROWTH A PERFECTED
STORM. NAMELY THE HELPER STRAINS
IN THE COLONIC MICROBIOME
PREVENT GROWTH OF THE ORGANISM
BUDDY MINISH TOXIN PRODUCTION
WHICH ENABLES IT TO REMAIN AS A
PERMANENT OR A CHRONIC COLONIZER
OF THE HUMAN GUT LIMITING THE
AMOUNT OF INFLAMMATION INDUCED
AND WE KNOW INFLAMMATION IS
CRITICAL TO THE DEVELOPMENT OF
COLON CANCER. NEXT STEPS, DOES C
DIFF HAVE A PLAUSIBLE LINKAGE TO
COLON CANCER, IT IS A COMPLETELY
NEW TOPIC. WE STARTED TO LOOK.
IF WE GO TO MALAYSIA COHORT, WE
HAVE NOW SEQUENCED 110 COLON
CANCER FROM THAT COHORT AND 29
OF THE 110 ARE POSITIVE FOR C
DIFFICILE IN THE SEQUENCE
ANALYSES. 25 OF THE 29 OF THOSE
ARE IN FACT BIOFILM POSITIVE.
WHEN WE LOOK AT OUR HOPKINS
COLON CANCER COHORT, PLEATED
OVER TIMES AND ONLY LOOK AT SIX
COLON CANCER TO DATE, FIVE OF
SIX WERE POSITIVE FOR C DIFFER
SILL AND TWO TOXIJENIC ANOTHER
POSSIBLE THING TO THINK ABOUT IS
C DIFFICILE DISEASE INCREASED
OVER TIME AND WE HAVE NEW
EPIDEMIOLOGY IN COLON CANCER
WITH ONSET OF EARLY ONSET
COLORECTAL CANCER WHICH WE ARE
SEEING IN INDIVIDUALS UNDER AGE
50 BEFORE SCREENING COLONOSCOPY
IS TYPICALLY RECOMMENDED.
LASTLY WE KNOW INFLAMMATORY
BOWEL DISEASE THERE IS A TON OF
C DIFFICILE COLONIZATION AND/OR
DISEASE, THAT IS A CONDITION
THAT ALSO HAS INCREASED RISK OF
COLON CANCER DEVELOPMENT. SO
WHAT ARE WE ACTUALLY DOING?
CYNTHIA SAY FELL IN THE LAB AND
SEAN ANDERSON FELL FELLOW IN THE
LAB ARE REEXAMINING HOW OFTEN WE
ARE ALL EXPOSED TO C DIFFICILE
LOOKING AT IGG ANTIBODIES TO
TOXIN B. WE HAVE COLLECTED 400
SAMPLES OF SERA AT HOPKINS,
PEDIATRIC AND ADULT AND IN THE
PROCESS OF TRYING TO GET THAT
DONE. WE HAVE ALSO INITIATED A
LISTENING TUESDAYNAL
COLONIZATION STUDY, 12 MONTH
STUDY IN ADULTS HOPE TO EXTEND
TO OUTPATIENTS AND PEDIATRICS
AGAIN LED BY SEAN ANDERSON TO
GET INITIAL C DIFF ISOLATE THAT
OVER TIME POTENTIALLY HOW LONG
IS THE ORGANISM CARRIED, WHAT IS
THE COMMUNITY ASSOCIATED WITH.
LASTLY WE ARE TRYING TO QUERY
LARGE DATABASES HOPKINS AND
ULTIMATELY NETWORK HOSPITALS TO
TRY TO LOOK AT THE INTERSECTION
OF C DIFFICILE INFECTION
COLONOSCOPY AND COLORECTAL
CANCER DETECTION. THIS IS LED BY
AND ALL STAR GROUP IN THE ID
DIVISION, I AM MOSTLY OBSERVER
LEARNER HERE BUT SEAN IS WORKING
ON THIS WITH MATTHEW ROBINSON,
AND KELLY AT HOPKINS. WITH THAT
I WANT TO TURN TO SOME TRIBUTES
OBVIOUSLY I AM STANDING HERE
BECAUSE OF THE SUPPORT
MENTORSHIP I RECEIVED OVER TIME
SO I WANT TO THANK DICK GRANT
WHETHER OR NOT WAS MY FIRST
MENTOR AT UNIVERSITY OF VIRGINIA
WHO TOOK ME INTO HIS LAB DESPITE
FACT I HAD NO IDEA HOW TO
PIPETTE. AND JULIE IN THE
DEPARTMENT OF PHARMACOLOGY THERE
OBVIOUSLY -- I WAS FIRST M.D.
PERSON SHE LET WORK IN HER LAB
AND I LEARNED A TREMENDOUS
AMOUNT, WE ARE STILL FAST
FRIENDS. IN PARTICULAR I LEARNED
A LOT ABOUT WRITING AS SHE
DUBBED ME THE QUEEN OF THE
MISPLACED MODIFIER. AT JOHNS
HOPKINS I WAS CO-HIRED BY MARK
(INAUDIBLE) THEN HEAD OF PI AND
JOHN BARTLETT HEAD OF ID AND
RECEIVED I NOR MOUSE SUPPORT
FROM THEM OVER THE LAST DECADES
I TOLD YOU HOW INVALUABLE
GETTING TO KNOW DR. SACK WAS AND
LASTLY DREW PERDOLE NOW ALMOST
20 YEARS SINCE I TOTAL HIM I
DIDN'T KNOW ANYTHING ABOUT THE
MUCOSAL IMMUNE SYSTEM AND COULD
HE HELP ME OUT AND INDEED HE
HAS, WE REMAIN WONDERFUL
COLLABORATORS. I ALSO WANT TO
REALLY THANK (INDISCERNIBLE) WU
AND (INDISCERNIBLE) THERE WITH
ME IN THE BEGINNING.
(INDISCERNIBLE) DECIDED TO
RETIRE THIS YEAR AND I'M LARGELY
HEART BROKEN OVER THAT
REVELATION AND AUGUSTA FRANKO
LOST HIS LIFE TO LYMPHOMA MANY
THE EARLY 2000s BUT THE WORK
IN GENETICS OF ETBF STAINS TODAY
-- SPANS TODAY. THERE ARE MANY
OTHER PEOPLE CURRENT LAB AND
PRIOR LABS THAT CONTRIBUTED TO
THIS PROJECT AS WELL AS NUMEROUS
PEOPLE WHO COLLABORATED WITH US
OVER TIME. I OWE A DEEP
GRATITUDE TO THE PEOPLE WHO
INSTITUTIONS THAT FUNDED US AND
THAT IT IS THE NIH IS THE
FOUNDATION OF THAT. IT IS MOSTLY
NIDDK AND CI I DID GET ONE AND
NAIA GRANT AT ONE TIM TIME. LAST
NOT LEAST, ALL THE GENEROUS
PATIENTS WHO HAVE WORKED WITH
US. ONE DAY I CAME BACK INDEED
MY DOOR HAD BEEN ADORING AND IT
IS NOW BEEN THERE AT LEAST A
DECADE AND OF COURSE WORK
REMAINS ABSOLUTELY TRUE. THANK
YOU VERY MUCH FOR LISTENING. BE
HAPPY TO ANSWER ANY QUESTIONS.
[APPLAUSE]
>> ANYONE FREE TO QUESTIONS,
PLEASE YOU CAN KICK US OFF.
>> THANK YOU SO MUCH.
>> WAITING FOR IT TO TURN ON.
>> MY QUESTION RELATES TO THE
END STIMULUS. . DO YOU THINK THE
BUGS AND TOXINS YOU DESCRIBED
ARE DIRECTLY STIMULATING THE
COLON CELLS TO TRANSFORM INTO
MALIGNANCY? OR AS YOU SUGGESTED
SEVERAL TIMES, ARE THEY CREATING
THIS MASSIVE INFLAMMATORY
REACTION WHOSE REACTIVE OXYGEN
SPECIES AND OTHER PRODUCTS ARE
INDUCING THE TRANSFORMATION?
>> I THINK THIS IS A BIG DEBATE
IN THE FIELD. SO THE ONE
ORGANISM ON THAT LIST BEING
CONSIDERED AS POSSIBLY TARGETING
THE DNA, THAT TRANSFORMING THE
CELLS IS THE COLON MADE BY
POLYSYNTHASE E. COLI. I HOWEVER,
DON'T THINK THE EVIDENCE HOLDS
UP FOR THAT CONCLUSION AS YET. I
THINK THESE ORGANISMS ARE
PROMOTIONAL. THEN IN FACT I
DON'T KNOW WHAT CAUSES THE
MUTATION OF APC THAT'S
FOUNDATIONAL TO COLON CANCER BUT
I REALLY THINK THE BUGS ARE
COMING IN CREATING AS YOU JUST
SAID. THE CONTEXT FOR
DEVELOPMENT OF CANCER.
>> IT WOULD BE EASY WITH USING
THE TOXINS TO SEE WHAT AFFECT
THEY HAVE FOR EXAMPLE ON
PHAGOSITIC CELLS AND OTHER CELLS
TO GENERATE A HUGE GENERATION OF
REACTIVE OXYGEN SPECIES.
>> WE DID THAT ONCE WITH JERRY
DONOWITS YOU PROBABLY KNOW
TRYING TO LOOK AT BFT EFFECT ON
POLYS. THERE WAS NO EFFECT THAT
WE COULD DETECT. IF TESTIFY
CLAUDIN 4 IS RECEPTOR FOR BFT I
DON'T THINK IT IS ON MOST OF
THOSE CELLS ANYWAY. SO AT LEAST
WITH THAT TOXIN, WE HAVEN'T
STUDIED THE OTHERS, I DON'T
THINK IT IS DIRECT ON THE IMMUNE
SYSTEM, I THINK IT IS A CASCADE
FROM THE LUMIN TO SUB MUCOSA
BACK TO EPITHELIAL CELLS, AT
LEAST WITH ETBF, IT IS THAT
FEEDBACK OF IL 17 ON THE
EPITHELIAL SIDE THAT SEEMS TO
TRIGGER THE EPIPEELIAL CELL TO
CHANGE ITS FUNCTION.
>> THANK YOU.
>> COUPLE OF QUESTIONS ONLINE.
SO DID THE ANTIBIOTIC DATA HAVE
SUFFICIENT DETAIL TO SEE IF SOME
ANTIBIOTICS ARE ASSOCIATED WITH
HIGHER COLON RISK COMPARED TO
OTHER ANTIBIOTICS?
>> I DIDN'T SAY THIS BUT WHEN WE
DRILLED DOWN IN TO THAT
QUESTION, IT TURNED OUT THAT THE
ASSOCIATED ANTIBIOTIC WAS
PENICILLIN OF ALL THINGS. I MUST
ADMIT I WAS SURPRISED BUT
PENICILLIN HAS A VARIETY OF
ACTIVITIES AGAINST AN AROBES AND
PARTICULARLY WHEN -- EVERY TIME
YOU TAKE AN ORAL ANTIBIOTIC IT
IS NOT COMPLETELY ABSORBED.
EVERY TIME IT HITS THE COLON AND
MODIFIES THE MICROBIOME. IT IS
AN IMPOSSIBLE QUESTION TO STUDY
BUT I THINK THOSE SERIAL SHIFTS
SOMEHOW CHANGE THE BIOLOGY OF
THE MUCOUS LAYER THERE, MAKE IT
MORE AMENABLE TO BIOFILM
INVASION IN SOME OF THE AFFECTS
THAT WE HAVE SEEN. THAT RESULT
GOT FURTHER REINFORCED WHEN WE
LOOKED AT PEOPLE THAT ONLY HAD
EVER TAKEN PENICILLIN VERSUS ANY
OTHER ANTIBIOTIC AND ACTUALLY
THE RESULTS HELD UP EVEN UNDER
THOSE CIRCUMSTANCES.
>> ANOTHER QUESTION, MAY HAVE
ANTICIPATED THIS ANSWER, WERE
THERE FUNGI IN THE BIOFILMS?
>> I HAPPEN TO LIKE BACTERIA, SO
WE REALLY FOCUSED BACTERIA, WE
HAVEN'T DEALT, THERE ARE FEW
PAPERS OUT THERE NOW, I THINK WE
HAVE A LOT TO LEARN ABOUT
VIRUSES PARASITES AND FUNGI HOW
THEY MAY INFLUENCE COLON CANCER.
>> THAT IS IT ONLINE. DR.
HADIGAN?
>> I JUST WANT TO THANK DR.
SEARS AGAIN FOR A FANTASTIC
LECTURE. THANKS EVERYBODY FOR
ATTENDING AND LET THOSE
INDIVIDUAL WHOSE ARE PRESENT
HERE KNOW THERE IS A RECEPTION
OUTSIDE LIPSETT, HOPE YOU WILL
BE ABLE TO JOIN US AND MAYBE
HAVE AN OPPORTUNITY TO MEET DR.
SEARS. THANK YOU, EVERYBODY.
[APPLAUSE]