>>THIS IS MICHAEL GOTTESMAN,
NIH'S DEPUTY DIRECTOR FOR
INTRAMURAL RESEARCH.
I'M DELIGHTED TO INTRODUCE
TODAY'S NIH LECTURER, NIKI
MOUTSOPOULOS, WHO IS SENIOR
INVESTIGATOR AND CHIEF OF THE
ORAL IMMUNITY AND INFECTION
SECTION, NATIONAL INSTITUTE OF
DENTAL AND CRANIOFACIAL
RESEARCH.
SHE RECEIVED HER DDS FROM
ARISTOTLE UNIVERSITY OFS THEY
IN GREECE, IN 2003 CERTIFICATE
IN PERIODONTICS, UNIVERSITY OF
MARYLAND, Ph.D. FROM
DEPARTMENT OF DIAGNOSTIC
SCIENCES IN PATHOLOGY AT THE
BALTIMORE COLLEGE OF DENTAL
SURGERY AS WELL, BUT SHE WORKED
AT THE NIH IN THE LABORATORY OF
SHARON WALL, ALSO IN NIDCR,
RESEARCH FELLOW FOLLOWING THAT
IN NIDCR, AND THEN CLINICAL
FELLOW WORKING IN NIDCR WITH THE
MENTORSHIP OF STEVE HOLLAND, WHO
IS OF COURSE THE SCIENTIFIC
DIRECTOR IN NIAID.
ABOUT A DOZEN YEARS AGO BECAME
ASSISTANT CLINICAL INVESTIGATOR,
ALSO AT NIDCR, AND THEN RECENTLY
BECAME TENURED SENIOR
INVESTIGATOR, AFTER HER PERIOD
AS A TENURE-TRACK INVESTIGATOR.
SHE'S ALSO -- HAS A DEAN'S
FACULTY APPOINTMENT UNIVERSITY
OF MARYLAND SCHOOL OF DENTISTRY,
WELL RECOGNIZED IN HER FIELD,
THE FIELD OF ORAL IMMUNITY, BY
QUITE A NUMBER OF INVITATIONS TO
SPEAK AND PARTICIPATION AND
SERVICE COMMITTEES AT THE NIH
AND ELSEWHERE.
DR. MOUTSOPOULOS' RESEARCH IS
FOCUSED ON ORAL BARRIER IMMUNE
SYSTEM, GOAL TO UNDERSTAND
MOLECULAR AND CELLULAR BASIS OF
ORAL IMMUNITY IN HEALTH AND ALSO
IN THE RATHER COMMON DISEASE
PERIODONTITIS WHICH MANY OF YOU
ARE BOTH SUFFERING FROM AND
FAMILIAR WITH.
IT'S COMMON IN HUMANS AND LEADS
EVENTUALLY TO THE DESTRUCTION OF
TOOTH-SUPPORTING STRUCTURES WITH
LOSS OF TEETH.
BUT THE INFLAMMATION ASSOCIATED
WITH IT IS ALSO A PROBLEM
RESULTING IN SYSTEMIC
INFLAMMATORY CONDITIONS,
UNDERSCORING POSSIBILITY OF
COMMON MECHANISMS OF
SUSCEPTIBILITY TO DISEASE
TRIGGERING ASSOCIATED WITH
INFLAMMATION.
SO UNDERSTANDING THESE
MECHANISMS OF INFLAMMATION THAT
COME FROM THE PERIODONTITIS HAS
LED TO A ROBUST RESEARCH
PROGRAM.
WITHOUT FURTHER ADO LET ME
INTRODUCE DR. MOUTSOPOULOS'S
TALK, MUCOSAL IMMUNITY AT THE
ORAL BARRIER FROM BEDSIDE TO
BENCH.
NIKI, YOU'RE ON.
>> THANK YOU, DR. GOTTESMAN.
IT'S REALLY AN HONOR TO BE PART
OF THE NIH DIRECTOR SEMINAR
SERIES.
A FANTASTIC OPPORTUNITY FOR ME
TO DESCRIBE THE RESEARCH PROGRAM
I BUILT OVER THE YEARS AT NIDCR.
HOWEVER, BEFORE I GO INTO THE
SCIENTIFIC PART OF MY TALK, I
WANTED TO MAKE SOME
INTRODUCTIONS.
ON THIS SLIDE YOU'LL SEE ALL THE
MEMBERS THAT HAVE BEEN PART OF
THE ORAL IMMUNITY AND
INFLAMMATION SECTION THROUGHOUT
THE YEARS, IN THE PAST REALLY
DECADE.
THIS IS A DIVERSE GROUP OF
INDIVIDUALS, FROM MANY PARTS OF
THE WORLD THAT HAVE COME TO THE
NIH FOR THEIR TRAINING.
THEY BROUGHT TOGETHER A LOT OF
TALENT, AND THEY HAVE ALL, EACH
ONE OF THEM, BEEN TREMENDOUSLY
DEVOTED TO SCIENTIFIC INQUIRY.
THEY HAVE BEEN VERY COLLEGIAL
BUNCH, TREMENDOUS TEAM MEMBERS,
AND HAVE ENRICHED MY LIFE
PROFESSIONAL AND PERSONALLY.
I WANTED TO ACKNOWLEDGE THEIR
CONTRIBUTION AND THANK EACH ONE
FOR EVERYTHING THAT THEY HAVE
DONE SO FAR.
AND SO WITHOUT FURTHER ADO I
WILL GO INTO THE SCIENTIFIC PART
OF THIS.
WHAT DO WE STUDY?
WE'RE BROADLY INTERESTED IN
BARRIER IMMUNITY.
BARRIERS ARE PARTS OF THE HOST
EXPOSED TO ENVIRONMENT.
SKIN, MOUTH, LUNGS,
GASTROINTESTINAL TRACT THAT COME
IN CONTACT WITH THE OUTSIDE
WORLD.
WE'RE INTERESTED IN
UNDERSTANDING HOW THE LOCAL
IMMUNE SYSTEM CAN REGULATE
RESPONSES WITH THE OUTSIDE.
MANY OF THE FUNDAMENTAL
QUESTIONS FOR BARRIER IMMUNITY
ARE REALLY SHARED AMONG ALL OF
THE STRUCTURES.
SOME OF THEM INCLUDE HOW DO
TISSUES MAINTAIN INTEGRITY AND
FUNCTION DESPITE BEING
CONSTANTLY BOMBARDED WITH
ENVIRONMENTAL EXPOSURES.
HOW DOES THE LOCAL IMMUNE SYSTEM
PROTECT US FROM PATHOGENS THAT
WE COME IN CONTACT WITH?
AND HOW ARE WE PROTECTED FROM
THESE PATHOGENS BUT AT THE SAME
TIME WE CAN TOLERATE EVERYTHING
THAT'S INNOCUOUS, COMMENSAL
MICROBIOME, FOOD, HOW DO WE NOT
OVERINFLAME?
HOWEVER, WHEN WE START THINKING
OF HOW TO ANSWER THESE
QUESTIONS, IN AS MANY IN THE
FIELD OF BARRIER IMMUNITY HAVE
DONE SO, WE START TO REALIZE
THAT THE ANSWERS BECOME VERY
TISSUE SPECIFIC, AND THAT IT'S
VERY IMPORTANT TO UNDERSTAND
EACH ENVIRONMENT SEPARATELY TO
UNDERSTAND HOW IMMUNITY IS
REGULATED.
EACH ONE OF THESE TISSUES HAS A
VERY UNIQUE FUNCTIONALITY, HAS
UNIQUE ENVIRONMENTAL EXPOSURES,
THE SKIN COMES IN CONTACT WITH
THE OUTSIDE AND SUNLIGHT, MOUTH
COMES IN CONTACT WITH FOOD.
SO UNDERSTANDING THE EXPOSURES
IS VERY IMPORTANT, ULTIMATELY
FOR THOSE THAT ARE CLINICIANS
EACH TISSUE SIGHT IS
SUSCEPTIBILITY TO DIFFERENT
TYPES OF DISEASE SUSCEPTIBILITY.
NOW, THE AREA YOU'VE HEARD FROM
DR. GOTTESMAN WE'RE INTERESTED
IN BOTH BECOMES MANY OF OUR
CLINICAL BACKGROUNDS, THE ORAL
MUCOSA.
AND I WITH A LOT OF BIAS WILL
SAY ALTHOUGH THIS AREA THAT BE
RELATIVELY UNDERSTUDIED PAIRED
TO OTHER BARRIER TISSUES, IT'S
AN EQUALLY IMPORTANT ONE.
MOUTH COMES IN CONTACT WITH A
LOT OF ENVIRONMENTAL EXPOSURES,
AND ACTUALLY IS THE PLACE OF THE
HUMAN AND MAMMALIAN BODY WHERE A
LOT OF THESE OUTSIDE ELEMENTS
ARE ENCOUNTERED FOR THE FIRST
TIME FROM OUR BODY.
WE BREATHE THROUGH OUR NOSE AND
THROUGH OUR MOUTH TO SOME DEGREE
SO A LOT OF AIR PARTICLES AND AL
ALLERGENS COME THROUGH THE
MOUTH, AND WE EAT THROUGH MOUTH,
PATHOGENS FROM MICROBIOTA,
BEFORE THEY ENTER THE
RESPIRATORY SYSTEM BUT ALSO
GASTROINTESTINAL TRACT.
HOW FIRST EXPOSURES REGULATE OUR
IMMUNE RESPONSIVENESS LOCALLY
AND SYSTEMICALLY WE DON'T
UNDERSTAND VERY WELL.
SO, OUR LAB HAS BEEN VERY
INTERESTED TO UNDERSTAND HOW
HEALTH IS REGULATED IN THE ORAL
MUCOSA, HOW THIS ENVIRONMENT AND
HOST BALANCE IS MAINTAINED, BUT
ALSO HOW DOES THE IMBALANCE
BETWEEN EXPOSURES AND HOST LEAD
TO DISEASE.
WE'VE PARTNERED WITH NIH
HOSPITALS INTERESTED IN ORAL
MUCOSA MANIFESTATIONS AND
DISEASES BUT I WON'T TALK ABOUT
MANY OF THESE TODAY.
I WILL FOCUS ON THE DISEASE THAT
WE HAVE PARTICULARLY BEEN
INTERESTED IN IN OUR GROUP,
WHICH IS PERIODONTITIS DISEASE.
IT'S A PREVALENT GENERAL HUMAN
DISEASE, ONE OF THE MOST
PREVALENT INFLAMMATORY DISEASES
IN FACT IN HUMANS.
YOU'LL SEE HERE FROM AN OLD CDC
SLIDE THAT THE PREVALENCE OF
PERIODONTITIS DISEASE IN GENERAL
POPULATION IN SEVERE FORMS IS
ABOUT 8% OF THE GENERAL
POPULATION, THAT IS VERY HIGH.
WHAT HAPPENS IS PERIODONTITIS
DISEASE, WE FIND MANY HAVE
DOCUMENTED THIS, DYSBIOTIC
MICROBIAL COMMUNITY PRESENT ON
THE ROOT SURFACE OF TEETH.
THIS MICROBIOME IS THOUGHT TO
TRIGGER INFLAMMATORY RESPONSES
WITHIN MUCOSAL TISSUES
PARTICULARLY DESTRUCTIVE IN
SUSCEPTIBLE INDIVIDUALS, LEADING
TO DESTRUCTION BOTH OF THE SOFT
TISSUES MUCOSA SURROUNDING
DENTITION AS WELL AS SUPPORTING
BONE, LEADING TO FUNCTIONAL
ISSUES, LEADING TO A LOT OF
HEALTH COSTS, ALSO TO TOOTH LOSS
IN SEVERE CASES.
NOW, WHAT ARE OUR QUESTIONS AS
WE TRY TO UNDERSTAND
PERIODONTITIS DISEASE
SUSCEPTIBILITY AND PATHOGENESIS?
AS FOR MANY OTHER COMPLEX
INFLAMMATORY DISEASES WHERE BOTH
HOST AND EXPOSURE TO IMMUNE
ENVIRONMENTAL EXPOSURES ARE
IMPORTANT, IN PERIODONTITIS IT'S
BEEN DIFFICULT TO DISSECT OUT
THE COMPLEX GENETICS OF
SUSCEPTIBILITY.
WHILE WE KNOW THAT PERIODONTITIS
DOES SEGREGATE WITHIN FAMILIES,
ESPECIALLY ITS MORE AGGRESSIVE
FORMS IN YOUNGER INDIVIDUALS,
WE'LL SEE IN HIGHLY PREVALENT IN
TWINS, IN FAMILIES, IT'S BEEN
VERY HARD TO DISSECT OUT
SPECIFIC GENES AND PATHWAYS THAT
PREDISPOSE TO US THIS CONDITION.
SECONDLY, WE DON'T COMPLETELY
UNDERSTAND PATHOPHYSIOLOGY AND
BECAUSE OF THAT DON'T HAVE
TARGETED THERAPY OF
INTERVENTION.
SO WHAT WE TYPICALLY DO
CLINICALLY WILL REMOVE DYSBIOTIC
MICROBIOME BY DENTAL CLEANINGS,
AS MANY OF YOU HAVE EXPERIENCED,
ALSO BY COMBINED CLEANING AND
SURGICAL APPROACHES WHICH WILL
REMOVE ALSO SOME DISEASED
TISSUE.
HOWEVER, THESE ARE NOT TARGETED
APPROACHES TO SPECIFIC
MICROBIOTA OR SPECIFIC
INFLAMMATORY RESPONSES.
THESE ARE MANY TIMES PROCEDURES
THAT WILL HAVE TO BE
CONTINUOUSLY REPEATED, IT
BECOMES A LIFE-LONG TREATMENT
RATHER THAN INTERVENTION THAT
TARGETS DISEASE ALSO ON AND WILL
STOP IT.
WE WANT TO UNDERSTAND
PATHOPHYSIOLOGIC MECHANISMS TO
START TO EDUCATE OURSELVES AND
THINK OF TARGETED THERAPY.
NOW, PERIODONTITIS HAS BEEN
ASSOCIATED CLINICALLY WITH
NUMBER OF DISTAL AND SYSTEMIC
OTHER DISEASES, AND IT'S STILL
HARD TO DISSECT OUT WHETHER
THERE'S A CAUSATIVE LINK BETWEEN
PERIODONTITIS OR PERIODONTAL
MICROBIOTA AND ILLNESS OR SHARED
SUSCEPTIBILITY, ALSO QUESTIONS
THAT BECOME RELEVANT NOT ONLY TO
THE ORAL FIELD BUT ALSO TO THE
GENERAL MEDICAL AUDIENCE.
AND FINALLY, FOR THOSE OF US
THAT ARE INTERESTED IN BASIC
BIOLOGY, WE THINK THAT
PERIODONTITIS DISEASE ALSO
PRESENTS IDEAL MODEL TO START TO
LOOK AT CONCEPTS THAT RELATE TO
HOST-MICROBE INTERACTION AT
MUCOSAL SITES AT THE SETTING OF
HEALTH AND SETTING OF DISEASE.
AND SO WHILE THESE ARE QUESTIONS
THAT ARE BROAD AND VERY MUCH
SHARED WITHIN THE WHOLE
COMMUNITY, INTERESTING IN THIS
DISEASE, WHAT WE TRY TO DO
DIFFERENTLY WITH MY PROGRAM,
BECAUSE OF THE NIH ENVIRONMENT,
TAILOR THE WAY WE WILL LOOK AT
THESE QUESTIONS AND TRY TO TAKE
ADVANTAGE OF THE UNIQUE SETTING
OF THE INTRAMURAL PROGRAM.
AND AS YOU ALL KNOW, THE NIH
CLINICAL CENTER AS MANY OF US DO
CONSIDER THE CROWN JEWEL OF THE
NIH, IT IS THE BIGGEST RESEARCH
HOSPITAL IN THE WORLD AND VERY
MUCH SPECIALIZES ON RARE AND
GENETIC DISEASES.
AND SO BY BRINGING AT THE NIH A
LOT OF THESE COHORT OF RARE
DISEASES WE HAVE THE OPPORTUNITY
TO TREAT PATIENTS THAT DON'T
HAVE ACCESS TO TREATMENT OUTSIDE
BECAUSE OF THE RARITY OF DISEASE
SOMETIMES BUT ALSO WE CAN STUDY
THEM HOW SPECIFIC GENES AND
RELATED PATHWAYS LINK TO
PARTICULAR DISEASE
SUSCEPTIBILITY.
THROUGH THESE CLINICAL
PHENOTYPING STUDIES WE CAN START
TO LINK GENOTYPES TO CLINICAL
PHENOTYPES.
AND MANY OF US DO MANY OF THESE
STUDIES UNDER THE LENS OF
DISEASE WE CARE ABOUT.
IN OUR CASE IT'S PERIODONTITIS
DISEASE.
THROUGH THE YEARS WE'VE BEEN
SEEING A LOT OF PATIENTS WITH
GENETIC DESTRUCTIONS IN THE
IMMUNE SYSTEM THAT ARE LINKED TO
PERIODONTITIS DISEASE.
AND THAT HAS STARTED TO GIVE US
A LITTLE BIT OF MAP OF TIMES OF
IMMUNE CELLS AND IMMUNE PATHWAYS
THAT MAY RELATE.
WE EXTEND THESE STUDIES BEYOND
CLINICAL PHENOTYPING TO STUDY
ORAL TISSUES, ORAL CELLS, TRY TO
UNDERSTAND IMMUNE RESPONSIVENESS
OF ORAL CAVITY, FURTHER EXTEND
TO ANIMAL MODELS WHEN WE HAVE
ANIMAL MODELS THAT WILL
PHENOCOPY HUMAN DISEASE AND TRY
TO DO THIS WITH THE IDEA OF
TURNING THIS FULL CIRCLE BACK
INTO HUMAN INTERVENTION WHEN WE
CAN, TO TARGET THESE RARE SEVERE
DISEASES WHEN POSSIBLE.
AND SO THROUGH THE YEARS IT
BECAME OBVIOUS TO US THAT THERE
IS A SPECIFIC IMMUNE PATHWAY
THAT BECAME OF IMPORTANCE FOR
PERIODONTITIS STABILITY AND
IMMUNOPATHOLOGY, AND THAT IS THE
Th17-NEUTROPHIL AXIS THAT I
WILL INTRODUCE SUPER OFFICIALLY.
Th17 CELLS ARE CD4 POSITIVE,
CELLS THAT TYPICALLY FOUND AT
BARRIER SITES, UNDERNEATH THE
MUCOSA OR THE SKIN, AND THEY ARE
THOUGHT TO BE THERE BECAUSE THEY
PLAY A ROLE IN BARRIER IMMUNITY,
THROUGH THE CYTOKINE THEY
STIMULATE CONNECTION BETWEEN
EPITHELIAL CELLS IN ORDER TO
HAVE BARRIER INTEGRITY, THEY
STIMULATE EPITHELIAL CELLS TO
SECRETE ANTIMICROBIAL PEPTIDES
SO THEY MEDIATE EPITHELIAL THIS
WAY BUT HAVE PRIMARY ROLE IN
RECRUITING INNATE CELLS
NEUTROPHILS, AND THAT'S
CONNECTION THAT WE HAVE VERY
MUCH ZEROED IN ON THROUGH THE
YEARS.
AGAIN, FOR THE PURPOSES OF THIS
TALK, A VERY SHORT INTRODUCTION
ON NEUTROPHIL IMMUNE RESPONSES.
NEUTROPHILS ARE THE MOST
ABUNDANT IMMUNE CELLS IN THE
BODY, INNATE CELLS THAT COME
RAPIDLY INTO A TISSUE WHEN
THERE'S INJURY, WHEN THERE'S
INFECTION, AND HAVE PRIMARILY OR
MAJORLY ANTIMICROBIAL
CAPACITIES.
THEY WILL TRY TO TARGET
MICROBIAL INFECTIONS EITHER BY
PHAGOCYTOSING MICROBES, BY
SECRETING GRANULES WITH ENZYMES
THAT WILL BE DESTRUCTIVE TO
MICROBES, BUT ALSO BY RELEASING
THEIR DNA CONTENT INTO THE FORM
OF NETS TO CAPTURE LARGER
MICROBIOTA.
WHEN THEY ARE OVERACTIVATED
BECOME PATHOGENIC.
THE PARADIGM SET FOR NEUTROPHIL
RESPONSE WHEN YOU DON'T HAVE
NEUTROPHILS OR HAVE DEFICIENT
NEUTROPHILS YOU END UP WITH
INFECTION.
WHEN HAVE YOU TOO MANY
NEUTROPHIL YOU END UP WITH
INFLAMMATION.
I WILL SAY THAT THROUGH THE
YEARS WE'VE CHALLENGED THE
PARADIGM, NOT THAT IT DOESN'T
HOLD TRUE, BUT TO REALIZE
SOMETIMES NEUTROPHIL WILL ALSO
LEADS TO INFLAMMATION.
SO THROUGH THE YEARS WE'VE DONE
A LOT OF WORK BOTH ON BASIC
Th17 BIOLOGY AT THE ORAL
CAVITY, BUT ALSO TO UNDERSTAND
GENETIC SUSCEPTIBILITY TO
PERIODONTITIS THROUGH STUDY OF
NEUTROPHIL DEFECT.
IN THE SETTING OF BASIC STUDIES
IN OUR LAB WE VERY MUCH FOCUSED
ON THE BIOLOGY OF Th17 CELLS
AND THEIR REGULATION IN HEALTH
AND DISEASE, AND A LOT OF THESE
FUNDAMENTAL STUDIES WERE DONE
FROM MY FIRST TWO POSTDOCAL
FELLOWS IN THE LAB, BOTH NOW
INDEPENDENT INVESTIGATORS,
UNIVERSITY OF CHILE.
WE'VE COMPETE COMPLETED A LARGE
EFFORT IN THE LAB SPEARHEADED BY
ANOTHER TALENTED POSTDOCTORAL
FELLOW DRAKE WILLIAMS WHO
COMPILED SINGLE CELL ANALYSIS,
FUNDAMENTAL STUDIES THAT HAVE
GIVEN US UNDERSTANDING OF TISSUE
PHYSIOLOGY, WITHIN THE ORAL
MUCOSA, BUT I WILL NOT BE ABLE
TO COVER ANY OF THESE TODAY.
I THOUGHT THAT FOR THIS, FOR THE
PURPOSE OF THIS TALK, IT MADE
MORE SENSE TO FOCUS ON MORE
CLINICAL TRANSLATIONAL STUDIES
IN GENETIC FORMS OF
PERIODONTITIS, AND I THOUGHT
THAT WAS APPROPRIATE BECAUSE IN
SOME WAYS THIS TYPE OF SEMINAR
IS CELEBRATION OF THE INTRAMURAL
PROGRAM SO I WANTED TO HIGHLIGHT
THE TYPES OF STUDIES THAT REALLY
CAN ONLY UNIQUELY ALMOST BE DONE
WITHIN THE INTRAMURAL
ENVIRONMENT, AND BECAUSE OF
COLLABORATIONS WITH MANY GROUPS
IN ORDER TO MAKE THIS POSSIBLE.
SO, WHAT OUR CLINICAL STUDIES
WITHIN GENETIC COHORTS HAVE
SHOWN US IS THAT NOT HAVING
NEUTROPHILS OR SETTINGS OF
NEUTROPENIA LINKED TO
PERIODONTITIS DISEASE
SUSCEPTIBILITY, BUT ALSO THAT
THE OVERABUNDANCE OF NEUTROPHILS
ALSO IS LINKED TO PERIODONTITIS
IMMUNOPATHOLOGY, TELLING US THIS
BALANCE OF NEUTROPHILS BECOMES
IMPORTANT FOR THIS SITE.
SO, TODAY I WILL IN A LITTLE BIT
MORE DETAIL PRESENT WORK WE'VE
DONE ON LESS SPECIFIC MONOGENIC
NEUTROPENIA, THIS IS WORK THAT I
STARTED EARLY ON AS AN ASSISTANT
CLINICAL INVESTIGATOR WORKING
WITH DR. HOLLAND IN NIAID, IN
COLLABORATION WITH GEORGE GALLES
AT THE UNIVERSITY OF MARYLAND
TWO SUPPORTED US THROUGH STUDIES
OF AND CLINICAL MODELS OF THIS
DISEASE.
I WILL ALSO PRESENT ON THE FLIP
SLIDE MONOGENIC DISORDER THAT IS
LINKED TO NEUTROPHIL
HYPERACTIVATION,
IMMUNODEFICIENCY, COLLABORATIVE
WORK, COMBINED PROJECT OF OUR
GROUP, AND OUR INSTITUTE, AND
LED BY A TALENTED FELLOW THAT
WAS CO-MENTORED BY THE TWO LABS.
I'LL START WITH NEUTROPHIL
IMMUNODEFICIENCIES, IN GENERAL
THEY HAVE BEEN LINKED TO
PERIODONTITIS, AND HERE LIKE
YEARS AGO PUT THIS CARTOON
TOGETHER TO LOOK AT NEUTROPHIL
DEVELOPMENT IN THE BONE MARROW,
NEUTROPHIL EGRESS, TRAFFICKING
THROUGH THE BLOODSTREAM, AND
ATTACHMENT TO GO INTO TISSUES.
AND AS WE'VE MAPPED THIS PATHWAY
OF NEUTROPHILS OUT WE REALIZE
DEFECTS IN MANY PARTS OF THIS
PATHWAY ARE ASSOCIATED WITH
SEVERE PERIODONTITIS IN HUMANS.
IF YOU DON'T HAVE GOOD
APPROPRIATE NEUTROPHIL
DEVELOPMENT YOU HAVE TREMENDOUS
PERIODONTITIS.
IF NEUTROPHILS GET STUCK IN BONE
MARROW, CANNOT TRAFFIC OR ENTER
TISSUES, ALL OF THESE ARE LINKED
WITH PERIODONTAL PATHOLOGY.
WE ZEROED IN VERY MUCH ON
LEUKOCYTE ADHESION DEFICIENCY,
WE HAD A COHORT AT THE NIH, AND
IT IS CAUSED -- IS A PROTOTYPIC
MENDELIAN DEFACT IN THE CD18
GENE, THE COMMON CHAIN FOR BETA2
INTEGRIN USED TO FORM TIGHT
ADHESION ON EPITHELIAL CELL
WALL, TRANSMIGRATE INTO TISSUES.
IF YOU HAVE DISRUPTION OF CD18
THEY CAN NOT GO INTO THE TISSUE.
THIS IS A MODEL DISEASE TO
UNDERSTAND WHAT HAPPENS IN THE
SETTING OF TISSUE NEUTROPENIA.
CLINICALLY WHAT HAPPENS TO THE
PATIENT, THEY HAVE RECURRENT
INFECTION, NON-HEALING WOUNDS
AND SPONTANEOUS LESIONS IN THE
SKIN THAT WILL NOT HEAL, MANY
TIMES.
THEY HAVE A DOMINANT ORAL
PHENOTYPE, THEY WILL PRESENT
WITH SEVERE PERIODONTITIS AT A
VERY YOUNG AGE, AND RECURRENT
ORAL ULCERS THAT WILL IMPEDE
THEM FROM EATING.
NOW, I TOOK A LEAP HERE AND
DECIDED TO SHOW A CLINICAL
DENTAL RADIOGRAPH, I'LL WALK YOU
THROUGH IT FOR THOSE NOT
DENTISTS ON THE CALL.
THIS IS A 13-YEAR-OLD FEMALE
THAT HAD BEEN SEEN AT THE NIH
PRETTY MUCH THROUGHOUT HER LIFE.
WHAT I'VE DONE HERE, PANORAMIC
X-RAY, I DREW YELLOW LINE WHERE
I DEPICT WHERE BONE LEVELS
THEORETICALLY WOULD BE IN A
PATIENT OF THIS AGE.
AND THE WHITE LINE IS WHERE THE
BONE IS IN THIS PATIENT.
SO ALL OF THIS DARK SPACE IS HOW
MUCH BONE HAS BEEN LOST AROUND
THE DENTITION.
NOW, IT'S IMPORTANT TO
APPRECIATE THAT THIS IS ONLY A
13-YEAR-OLD.
PERMANENT DENTITION HAS ONLY
BEEN HERE FOR A FEW YEARS, IN
THE PATIENT'S MOUTH.
THIS PATIENT HAD BEEN ON BROAD
SPECTRUM ANTIBIOTICS ALL THEIR
LIFE, RECEIVED DENTAL CARE.
HOWEVER, THIS TYPE OF DISEASE IS
RECALCITRANT, LARGELY
RECALCITRANT TO STANDARD OF CARE
TREATMENT.
WE HAVE SOMETHING VERY SEVERE
THAT WE CAN'T TREAT WITH MEANS
WE KNOW.
THESE PATIENTS END UP LOSING
THEIR TEETH EARLY IN LIFE
BECAUSE THEY ARE NOT SUPPORTED
BY BONE.
THEY BECOME VERY MOBILE.
AND WE EITHER EXTRACT THEM
BECAUSE THEY ARE UNCOMFORTABLE
OR THEY EXFOLIATE.
SO, WHEN WE EXTRACT TEETH FROM
THESE PATIENTS, WE SEE THAT ALL
OF THE BONE HAS BEEN REPLACED BY
INFLAMMATORY SOFT TISSUE,
THERE'S TONS OF INFLAMMATION.
WHEN WE LOOK AT INFLAMMATORY
INFILL STRAIGHT WE SEE A SEA OF
CELLS AND CHARACTERIZE THEM AS
Th17 CELLS.
IF WE LOOK AT CYTOKINE
EXPRESSION IN THESE REGIONS WE
SEE CYTOKINES RELATED TO
DIFFERENTIATION NEUTROPHIL
RECRUITMENT, ONE OF THE MAIN
PATHWAYS Th17 REGULATES.
THIS AXIS IS SUPER UPREGULATED
IN THESE PATIENTS BUT DON'T KNOW
WHETHER THESE TYPES CELLS OF
CELLS, OR DIDN'T KNOW, WHETHER
THEY PLAY A ROLE IN DISEASE
PATHOGENESIS.
THE WAY WE WERE THINKING ABOUT
THIS, AT LEAST HYPOTHETICALLY,
IS THAT AS I MENTIONED IL-17 AND
Th17 ARE IMPORTANT FOR
NEUTROPHIL CHEMO ATTRACTION.
IF NEUTROPHILS HAVE IMPAIRMENT
AND CANNOT MAKE IT BACK INTO THE
TISSUE WE SUSPECT THEY AXIS
AMPLIFIES TO COMPENSATE AND MAKE
NEUTROPHILS COME BACK IN AND
SUSPECT THIS AMPLIFICATION LEADS
TO DISRUPTIVE IMMUNOPATHOLOGY.
HOWEVER, IN THE BEGINNING THIS
WAS MORE OF A HYPOTHESIS.
WE REALLY DIDN'T KNOW WHETHER
Th17 CELLS AND THEIR RELATED
CYTOKINES DRIVE IMMUNOPATHOLOGY.
SO, THAT WAS THE TIME THAT WE
REACHED OUT TO GEORGE, WHO HAD
BEEN -- WAS AN EXPERT IN THE
FIELD, ALREADY WORKING ON THE
LFA KNOCKOUT MOUSE, THE MODEL
FOR DISEASE.
WHAT GEORGE HAD SEEN PRIOR TO
WORKING WITH US ALREADY THESE
MICE HAVE SPONTANEOUS
PERIODONTITIS DISEASE AND THEIR
TISSUES HAVE A LOT OF
UPREGULATION OF IL-17-RELATED
RESPONSES, SO WHEN WE GOT
TOGETHER WE FELT THIS WAS A
TREMENDOUS FORTUNE FOR US TO
ALREADY HAVE A MODEL WHERE WE
COULD DO PRE-CLINICAL TRIALS,
INHIBIT DIFFERENTIAL OR
FUNCTION, SEE IF THEY ARE
IMMUNOPATHOGENNIC.
SO WHAT YOU'LL SEE HERE IS BONE
LOSS MEASUREMENT, AMOUNT OF
DESTRUCTION THAT THE LFA
KNOCKOUT MICE HAVE,
SIGNIFICANTLY HIGHER FROM
CONTROL.
HOWEVER, WHEN -- OOPS, SORRY.
WHEN GEORGE'S GROUP TREATED
THESE MICE, EITHER WITH IL-17 OR
23, THEY COULD INHIBIT DISEASE
PROGRESSION ALMOST COMPLETELY,
WHILE WHEN HE USED ANTIBODIES
COULD NOT INHIBIT DISEASE
PROCESS.
SO THIS MADE US KIND OF ALMOST
COME FULL CIRCLE WITH THIS
PROCESS, STARTING FROM CLINICAL
OBSERVATIONS TO STUDIES IN HUMAN
TISSUES TO CLINICAL STUDIES IN
MOUSE MODELS.
AND STARTING TO THINK ABOUT
HUMAN INTERVENTION.
AND WE STARTED TO THINK ABOUT
HUMAN INTERVENTION ALSO BECAUSE
THERE WAS ALREADY AGE AVAILABLE
AND FDA APPROVE THAT WERE
TARGETED RELEVANT PATHWAYS.
WITH THE P 40 ANTIBODY THAT
INHIBITS PART OF THE IL-23
CYTOKINE, AND THUS INHIBITS
Th17 DIFFERENTIATION, WAS
ALREADY FDA APPROVED FOR
PSORIASIS AND PSORIATIC
ARTHRITIS, LATER FOR CROHNS, HAD
A PRETTY GOOD SAFETY PROFILE, AT
LEAST FOR DISEASE THAT IT WAS
USED BEFORE.
AND IT WAS SOMETHING THAT WE
COULD START CONSIDERING FOR LAD.
THESE STUDIES AGAIN WERE DONE IN
A VERY CLOSE PARTNERSHIP WITH
DR. HOLLAND AND HIS GROUP, AND
BETTY MACIANO WAS INSTRUMENTAL
PUTTING TOGETHER A CLINICAL
PROTOCOL FOR LAD, USE IN LAD.
PRIOR TO PUTTING TOGETHER A
PROTOCOL WE FIRST HAD ONE
PATIENT THAT CAME TO US WHERE
USING THIS AGENT AS A ONE-CASE
PROOF OF CONCEPT BECAME
IMPORTANT BECAUSE OF CLINICAL
NEED.
THIS WAS A 19-YEAR-OLD MALE
FROM -- ACTUALLY FROM ITALY,
REFERRED TO US BY DR. ANGELO,
ORAL DISEASE, 100% OF SITES HAD
SEVERE INFLAMMATORY MARKERS BUT
THE PATIENT ALSO HAD A
TREMENDOUS SACRAL WOUND BECAUSE
OF AN ACCIDENT THAT HAD BEEN
NON-HEALING FOR OVER TWO YEARS,
DESPITE SURGICAL APPROACHES,
DESPITE ANTIBIOTICS, AND
STEROIDS.
AND WHEN WE LOOKED AT THE ISSUES
OF THESE AND OTHER PATIENTS THAT
HAD LESIONS WE RELATESSED THE
IL-17 HYPER RESPONSIVENESS IS
NOT JUST A FEATURE OF ORAL
CAVITY BUT OF OTHER
IMMUNOPATHOLOGICAL REGIONS IN
LAD.
SO THIS ALSO JUSTIFIED US TO
THINK OF SYSTEMIC TREATMENT WITH
SUCH AN AGENT.
AND SO WHEN WE TREATED THIS
PATIENT, WITHIN WEEKS WE STARTED
TO SEE REDUCTION IN ORAL
INFLAMMATION, WE ALSO SAW
REDUCTION IN CYTOKINE RESPONSES,
RELATED TO IL-17, WITHIN THE
TISSUES.
AND MAYBE MOST SIGNIFICANTLY FOR
THIS PATIENT, WITHIN A FEW
MONTHS HE STARTED HAVING THE
WOUND BECOME TO HEAL, AND WITHIN
A YEAR THIS WOUND BECAME TOTALLY
CLOSED, WITH VERY MINIMAL
SCARRING COMPARED TO HOW OTHER
LAD PATIENTS OF COURSE HEAL.
AND SO THIS WAS AN EXCITING
CLINICAL CASE FOR US.
IT WAS PUBLISHED AS A SINGLE
CASE, INDEX CASE, BUT BECAME
IMPORTANT TO US BECAUSE ALSO IT
BECAME THE IMPETUS FOR US TO
CONSIDER PUTTING A FORMAL
PROTOCOL TOGETHER, TO TREAT LAD
PATIENTS.
WE PUT THIS PROTOCOL TOGETHER
WITH DR. HOLLAND'S GROUP TO
BRING IN FIVE TO TEN LAD
PATIENTS WITH VERY RARE DISEASE,
MOSTLY TO LOOK AT SAFETY, BUT TO
ALSO START TO GET SOME INSIGHT
ABOUT THE POTENTIAL OF THIS
AGENT TO LEAD TO RESOLUTION OF
INFLAMMATION.
AND I WILL SAY THAT SO FAR, THE
PROTOCOL'S BEEN UP FOR 2 1/2 OR
3 YEARS, WE'VE RECRUITED FIVE
PATIENTS, HOPING FOR A COUPLE
MORE.
AND SO WHERE ARE WE WITH LAD?
AS I MENTIONED BEFORE, WE HAVE
COME FULL CIRCLE, WE'RE HERE TO
EVALUATE REALLY THE VALUE OF
THIS SPECIFIC INTERVENTION WE'VE
CHOSEN, WE'RE IN THE MIDST OF
THE TRIAL.
WE KNOW AT THE NIH THAT THIS IS
A RARE DISEASE, WE WILL NOT BE
ABLE TO GET TOO MANY MORE
PATIENTS BUT WE'VE BEEN TALKING
TO CENTERS AROUND THE WORLD TO
SEE IF WE CAN PARTNER TO EXTEND
CLINICAL STUDIES IN LAD WITH A
MORE MULTI-CENTERED TYPE STUDY.
WE'RE INTERESTED IN OTHER
NEUTROPHIL THAT ARE SEEN IN
OTHER CENTERS TO SEE WHETHER
THIS Th17 HYPERCOMPENSATION
LEADS TO PERIODONTITIS IN ALL OF
THE TYPES OF NEUTROPHIL
DEFICIENCY OR OR WHETHER
ADDITIONAL BIOLOGICAL MECHANISMS
ARE IMPORTANT.
WE HAVE A LOT TO DO AND WE'RE --
THIS IS PART OF OUR FOCUS.
WITHIN THE Th17 BIOLOGY SPHERE
THERE'S A LOT OF QUESTIONS FOR
US TO CONTINUE THINKING ABOUT
WHEN IT COMES TO REGULATION OF
Th17 PATHWAYS.
I WILL MOVE ON FROM THE SETTING
OF NEUTROPENIA TO ACTIVATION AND
RECRUITMENT, AND JUST SAY THAT
UP TO NOW WE'VE BEEN THINKING OF
OVERABUNDANCE OF NEUTROPHIL
SETTING OF COMMON FORMS, THOSE
SEEN IN YOUNGER ADULTS.
WE CAME TO STUDY UNDER THE
PRISM, THE LENS OF ANOTHER
GENETIC DEFECT, WHICH IS
PLASMIOGEN DEFICIENCY, IT'S AN
ENZYME WE SEE REDUCED ENZYMATIC
ACTIVITY AND WIDESPREAD
IMMUNOPATHOLOGY.
HOW THEY PRESENT, THEY HAVE
CONJUNCTIVE LESIONS, THEY HAVE
GASTROINTESTINAL LESIONS, LUNG
DISEASE, VAGINAL DISEASE, AND
TREMENDOUS PERIODONTITIS EARLY
IN LIFE, SIMILAR TO WHAT
CLINICALLY WE'VE SEEN WITH LAD.
AND SO JUST A COUPLE WORDS OF
PLASMINOGEN AS AN ENZYME, IT'S A
PROTEASE ACTIVATED INTO THE
ACTIVE FORM PLASMIN, MAJOR ROLE
IN DEGRADING FIBRIN CLOTS, THE
KEY COMPONENT OF HEMOSTATIC
PLUG, NECESSARY FOR HEMOSTASIS.
HOWEVER, AFTER HEMOSTASIS IS
ACHIEVED IT'S IMPORTANT TO
REMOVE FIBRIN TO ALLOW FOR WOUND
HEALING TO OCCUR.
WHEN PLASMINOGEN IS NOT THERE,
FIBRIN CONTINUOUSLY DEPOSITS,
AND IN FACT WHAT IS SEEN IN
THESE PATIENTS WITH PLASMINOGEN
DEFICIENCY THERE ARE DEPOSITS IN
THE LESIONS MENTIONED HERE.
THIS DISEASE BECAME OF INTEREST
BECAUSE OF ANOTHER ANOTHER
COLLABORATION WITHIN OUR
INSTITUTE.
THOMAS BOOGA, NIDCR, HAD BEEN
WORKING ON PLASMINOGEN AND LYSIS
FOR DECADES AND BROUGHT THIS
DISEASE TO MY ATTENTION.
HE'S THE REASON WE ENGAGED IN
THESE STUDIES.
THOMAS HAD MADE THE PLASMINOGEN
KNOCKOUT MOUSE DECADES AGO AND
HAD SEEN THESE MICE PHENOCOPY
CLOSELY HUMAN DISEASE,
SPONTANEOUSLY HAVE A LOT OF
PHENOTYPES SEEN IN HUMAN
DISEASE, CONJUNCTIVITIS,
SYSTEMIC INFLAMMATION SHOWN BY
SPLENOMEGALY, ALSO ENLARGED
LYMPH NODES THAT DRAIN
GASTROINTESTINAL TRACT, AND MORE
RECENTLY SHOWN TO HAVE
SPONTANEOUS PERIODONTITIS
DISEASE.
WE THOUGHT THIS IS A GOOD PLACE,
GOOD MODEL TO ANSWER THE
QUESTION OR TO START TRYING TO
ANSWER THE QUESTION OF HOW DOES
PLASMINOGEN DEFICIENCY LEAD TO
MUCOSAL IMMUNOPATHOLOGY, AND IN
OUR CASE PERIODONTITIS DISEASE.
LAKMLAI SAW A SPONTANEOUS
PHENOTYPE, TREMENDOUS BONE LOSS.
SHE'S DONE MICRO CT SCANS, CAN
YOU SEE THE TEETH AND THE BONE
LEVELS, AND IT'S EASY TO
APPRECIATE EVEN WITH NAKED EYE
THAT THERE'S A LOT MORE BONE
LOSS IN THE DEFICIENT MICE
VERSUS WILD TYPE.
WHEN SHE QUANTIFIES SHE SEES
THERE'S SIGNIFICANT BONE LOSS BY
11 WEEKS OF AGE WHICH BECOMES A
LOT MORE DRAMATIC BY 24 OR SO
WEEKS OF AGE.
AND SO SHE THEN LOOKS AT THE
MUCOSAL TISSUE AND SEES
DESTRUCTION, FIBRIN DEPOSITION
IN THE MUCOSAL COMPARTMENT
REPLACING COLLAGEN, DESTRUCTION
OF EPITHELIUM, AND INFLAMMATORY
INFILTRATE.
AND SO BECAUSE OF THIS DOMINANT
FIBRIN AFFILIATION WE ASKED THE
QUESTION, WHETHER FIBRIN IS WHAT
DRIVES IMMUNOPATHOLOGY.
AND THE CLEAR GENETIC WAY OF
ANSWERING THIS QUESTION IS THAT
WE CAN CROSS PLASMINOGEN
DEFICIENT MICE WITH MICE ALREADY
AVAILABLE THAT DON'T HAVE FIBRIN
OHGEN AND FIBRIN AND ASK WHETHER
THIS WOULD RESCUE DISEASE.
INDEED, COMPLETE RESCUE OF
PHENOTYPE WHEN YOU SEE IT
QUANTIFIED YOU WILL APPRECIATE
THAT THIS IS THE LEVEL OF BONE
LOSS IN THE PLASMINOGEN
DEFICIENT HOUSE AND WHEN SHE'S
CROSSED TO THE FIBRINOGEN
KNOCKOUT HAS RESCUE THAT'S 100%.
SO, FIBRIN DOES DRIVE
IMMUNOPATHOLOGY BUT WE WANTED TO
KNOW HOW.
SO WE STARTED TO CHARACTERIZE
MUCOSAL LESIONS IN THESE MICE.
AND WE'VE DONE A NUMBER OF
STUDIES.
I WILL SHOW YOU THIS AS AN
EXAMPLE.
HERE YOU SEE GINGIVAL TISSUES
STAYED FOR FIBRINOGEN.
THERE'S RECUTE.
IN TISSUES THAT NEUTROPHILS COME
IN CONTACT WITH FIBRIN BUT
SURROUND DEPOSITS AND VALIDATED
BY FLOW CYTOMETRY, RNA
SEQUENCING, ALSO LOOKED AT
INFILTRATION OF OTHER IMMUNE
CELL SUBSETS AND SAW ONLY
NEUTROPHILS, ESPECIALLY EARLY
ON, THESE ARE THE ONLY CELLS
THAT SIGNIFICANTLY INCREASE IN
THE LESION.
WHICH LED US TO THE QUESTION OF
NEUTROPHIL-FIBRIN INTERACTION
WHAT DRIVE BIOLOGY?
FIBRIN HAS A BINDING SITE FOR
NEUTROPHIL CD11B, THIS BINDING
SITE IS A CRYPTIVE DOMAIN.
IT DOESN'T EXPOSE THIS DOMAIN
BUT WHEN IT'S LAID DOWN INTO A
CLOT OR WITHIN THE TISSUES THIS
DOMAIN IS EXPOSED IN MYELOID
CELLS INCLUDING NEUTROPHILS
COMBINED.
WE WERE FORTUNATE THAT A
COLLABORATOR OF THOMAS HAD MADE
A MOUSE THAT HAS A SPECIFIC
MUTATION IN THIS SITE.
THESE MICE HAD NORMAL FIBRIN,
NORMAL FIBRIN ACCUMULATION,
NORMAL CLOTTING BUT FIBRIN
CANNOT BIND MYELOID CELLS.
COULD CROSS PLASMINOGEN KNOCKOUT
MOUSE TO KNACKOUT AND SEE
WHETHER THIS COULD RESCUE
DISEASE, THIS WOULD TELL US
WHETHER THIS SPECIFIC BINDING
SITE IS WHAT DRIVES
IMMUNOPATHOLOGY.
WE WERE EXCITED WITH THE RESULT,
BECAUSE WHEN SHE DID MAKE THIS
CROSS, WE REALIZED THAT NOT ONLY
IS THE RESCUE COMPLETE, BUT
ACTUALLY THE RESCUE OF THIS
PHENOTYPE WAS EVEN ABOVE AND
BEYOND WHAT WE SEE IN WILDTYPE
MICE, INDICATING THAT SOME OF
THIS AGE-RELATED PERIODONTITIS
BONE LOSS THAT HAPPENED WITH AGE
IN MICE COULD BE RESCUED BECAUSE
OF THE GAMMA ABLATION.
SO THIS THEN ALSO WAS VERY
IMPORTANT TO US, MORE STRIKING
BECAUSE WHEN WE LOOKED AT THESE
MICE FIBRIN WOULD STILL
ACCUMULATE IN THE TISSUES TO THE
SAME DEGREE APPROXIMATELY, AND
NEUTROPHILS WOULD STILL
ACCUMULATE TO ALMOST THE SAME
DEGREE, TELLING US THAT YOU
REALLY NEED THIS FUNCTIONAL
INTERACTION BETWEEN FIBRIN AND
NEUTROPHIL TO CD11B AND FIT
GAMMA TO DRIVE IMMUNOPATHOLOGY,
OTHERWISE EVEN THOUGH YOU HAVE
THE SAME AMOUNT OF FIBRIN AND
NEUTROPHILS, THEY ARE NOT
ENGAGED IN A WAY THAT CAUSES A
PROBLEM.
AND SO, THEN OF COURSE THE NEXT
QUESTION WAS TO UNDERSTAND WHAT
HAPPENS AS A RESULT OF THE
ENGAGEMENT, AND FOR THIS LAKMLAI
HAS DONE TREMENDOUS AMOUNT OF
WORK I'LL TRY TO SUMMARIZE IN A
FEW SLIDES.
SHE'S DONE A LOT OF EXPERIMENTS
IN VITRO AND IN VIVO, BUT SHE
FIRST DEVELOPED THIS IN VITRO
MODEL WHERE SHE WOULD CREATE
PLATES OF FIBRIN, FIBRIN WOULD
BE FROM A WILDTYPE MOUSE OR A
MOUSE THAT HAD A FIT GAMMA
MUTATION, AND WOULD THEN PUT
NEUTROPHILS IN FROM HUMAN OR
MOUSE, DID EXPERIMENTSES BOTH
WAYS, AND LOOKED AT NEUTROPHIL
BINDING AND FRACTIONALITY IN THE
SET OF THE TWO FIBRINS.
SO, THIS WOULD BE ABLE TO TELL
US WHAT TYPES OF NEUTROPHIL
FUNCTIONS ARE RELATED TO THE FIB
GAMMA ENGAGEMENT.
FIRST OF ALL, THE FIRST SET OF
DATA THAT SHE GOT WAS VERY CLEAR
THAT FIB GAMMA CD11B BINDING IS
IMPORTANT FOR NEUTROPHILS TO
EVEN ADHERE TO FIBRIN.
SO IF YOU HAD THE MUTATION A LOT
LESS NEUTROPHILS COULD WOULD
STICK.
ATTACHMENT OF NEUTROPHILS SEEMS
TO OCCUR MAJORLY THROUGH THIS
DOMAIN.
SHE ALSO BY DOING DIFFERENT
TYPES OF STIMULI REALIZED THAT
WHEN YOU HAVE THIS ENGAGEMENT,
NEUTROPHILS WILL SIGNIFICANTLY
PRODUCE MORE REACTIVE OXYGEN
SPECIES AND ALSO MAKE A LOT MORE
NET.
I'M NOT GOING TO SHOW MUCH OF
THE RAW DATA BUT I THOUGHT THAT
SHARING V BILL MIGHT BE A BETTER
WAY TO GIVE OVERVIEW OF THE
DATA.
IN THIS VIDEO YOU WILL SEE,
YOU'LL SEE NEUTROPHILS PLATED ON
WILDTYPE VERSUS KNOCK-IN FIBRIN,
WHAT IS SHOWN HERE IS STAINING
FOR BLUE FOR NUCLEI OF EVERY
CELL THAT'S PRESENT.
CELLS BECOME GREEN WHEN THEY ARE
PERMEALJZED.
WILDTYPE SIDE MORE NETTING
NEUTROPHILS AS OPPOSED TO
KNOCK-IN SIDE YOU HAVE MANY
FEWER SIGNIFICANTLY FEWER CELLS
FORMING NET.
AND SO THIS LED US TO CONCLUDE
THAT THIS ENGAGEMENT OF
NEUTROPHILS IS IMPORTANT NOT
ONLY FOR ADHESION BUT ALSO FOR
THEIR ACTIVATION AND PRODUCTION
OF ROS AND NETs.
NEXT WE WANTED TO ASK WHETHER
SOME OF THESE EFFECTOR FUNCTIONS
OF NEUTROPHILS ARE IMPORTANT FOR
IMMUNOPATHOLOGIST.
SHE SAW WITH VARIOUS EXPERIMENTS
WHEN SHE REMOVED NETs COULD
INHIBIT IMMUNOPATHOLOGY NOT
TOTALLY, BUT SIGNIFICANTLY,
INDICATING THAT THE PROCESS IS
AN IMPORTANT ONE DRIVING
IMMUNOPATHOLOGY BUT MAY NOT BE
THE COMPLETE STORY OF HOW
NEUTROPHILS AND FIB GAMMA
ENGAGEMENT LEADS TO DISEASE
HERE.
NOW, ALL OF THIS DATA HAS BEEN
VERY EXCITING TO US BECAUSE --
FIRST BECAUSE IT STARTS TO
EXPLAIN POTENTIALLY WHY
IMMUNOPATHOLOGY IS OCCURRING IN
THE ORAL CAVITY, IN PATIENTS
WITH FIBRINOLYTIC DEFECTS.
WE ALSO STARTED CONSIDERING
THESE ARE BROADER -- PATHWAYS
THAT MIGHT BE IMPORTANT IN A
MORE BROAD SENSE.
WE'RE THINKING THAT IN THE
SETTING OF ANY WOUND, ANY
INJURY, YOU NEED FIBRIN OF
COURSE TO COME IN AND FORM A
CLOT, AND IT'S IMPORTANT THEN
THAT FIBRIN WILL BE ABLE TO
ATTACH NEUTROPHIL LOCALLY AND
ACTIVATE THEM LOCALLY IN ORDER
FOR NEUTROPHILS TO MEDIATE A
LOCAL FUNCTION THERE OF
PRODUCING ROS, NORMING
NORMING -- FORMING NET, TO CLEAR
THE WOUND FROM PATHOGEN.
WHEN IT'S CLEARED HEALING CAN
BEGIN TO OCCUR.
HOWEVER, ONE CAN SPECULATE THAT
IF FIBRIN IS CONSISTENTLY
DEPOSITED OR NOT REMOVED, THIS
CAN ALSO BECOME A PATHWAY THAT
BECOMES IMMUNOPATHOLOGIC AND
LEADS TO CHRONIC WOUNDS OR
TISSUE IMMUNOPATHOLOGY WHICH IS
WHAT WE THINK WE'RE SEEING HERE.
NOW, BEYOND THE BASIC BIOLOGICAL
CONCEPTS HERE WE ALSO WANTED TO
TAKE ANOTHER STEP AND SEE HOW
THIS PATHWAY COULD POTENTIALLY
FIT WITH GENERAL FORMS OF OUR
DISEASE OF INTEREST, WHICH IS
PERIODONTITIS DISEASE.
AND THE REASON WE STARTED
THINKING OF GENERAL FORMS OF
PERIODONTITIS, IT'S BEEN
APPRECIATED FOR YEARS THAT
NEUTROPHILS ARE ABUNDANT IN THE
LESIONS OF PERIODONTITIS,
ESPECIALLY YOUNGER ADULTS.
THIS IS DATA FROM OUR LAB,
LESIONS OF PERIODONTITIS, NUMBER
OF NEUTROPHILS BY FLOW
CYTOMETRY, THIS DATA IS
CONSISTENT WITH MANY MORE IN THE
FIELD FOR DECADES THAT HAVE SEEN
BY HISTOLOGY, TONS OF
NEUTROPHILS IN PERIODONTITIS,
SPECULATED NEUTROPHIL ACTIVATION
DRIVES DISEASE BUT HOW THIS
COULD BE HAPPENING IS SOMETHING
WE HAD NOT BEEN ABLE TO DELVE IN
SO FAR.
HOWEVER, FIBRIN IS ALSO
SOMETHING THAT WE APPRECIATE IS
PRESENT IN THE LESION OF THE
PERIODONTITIS.
WE KNOW INFLAMMATION CAUSES A
LOT OF BLEEDING SO THE IDEA THAT
FIBRIN AND NEUTROPHILS COME IN
CONTACT IN LESIONS OF
PERIODONTITIS IS ONE NATURALLY
VERY LOGICAL TO US BUT WE WANTED
TO GO FARTHER TO START TO THINK
OF.
SO WHAT GOT US EVEN MORE EXCITED
ABOUT THIS PROSPECT IS WHILE WE
WERE WORKING ON THE BASIC ENDS
ON THIS PROJECT THERE WAS A
LARGE GWAS STUDY THAT WAS
PERFORMED IN GERMANY WHERE THEY
LOOKED AT WHAT WAS THEN CALLED
AGREESIVE FORMS OF PERIODONTITIS
DISEASE, YOUNG ADULTS WITH
SEVERE DISEASE, AND THEY DID A
LARGE GWAS STUDY AND THE RESULT
OF THAT STUDY WAS THAT THE
GENETIC TRAIT THAT WAS MOST
CLOSELY ASSOCIATED WITH
AGGRESSIVE FORMS OF
PERIODONTITIS WAS PLASMINOGEN,
AROUND THE PLASMINOGEN GENE.
SO THAT EXCITED US VERY MUCH
BECAUSE IT HAPPENED AT THE SAME
TIME THAT WE WERE WORKING, AND
SO THAT ALSO INSTIGATED US TO GO
AND REACH OUT TO A FANTASTIC
COLLEAGUE, WHO HAS BEEN FOCUSING
ON GENOME IS OF ORAL DISEASES
FOR YEARS, INVOLVED IN THE GWAS
STUDY FOR PERIODONTITIS IN THE
U.S.
AND SO WE ASKED KIMON AND HIS
GROUP TO LOOK AT VARIATIONS
WITHIN THEIR COHORT, IN THE U.S.
COHORT WE HAD A MUCH SMALLER
NUMBER OF PATIENTS WITH
SEVERE -- WITH DIFFERENT TYPES
OF PERIODONTITIS, IN A WAY, SO
THEY DID TARGETED LOOKUP FOR
GENETIC POLYMORPHISMS AROUND THE
PLASMINOGEN LOCUS AND WHAT KIMON
AND TEAM REPORTED BACK WAS THAT
PLASMIOGEN VARIATIONS WERE VERY
MUCH ASSOCIATED WITH SEVERE
FORMS OF PERIODONTITIS BUT EVEN
VERY INTERESTINGLY ALSO VERY
MUCH AASSOCIATED WITH CARRIAGE
OF SPECIFIC ORAL MICROBE CALLED
AA HERE SIMILAR TO WHAT WAS
REPORTED IN THE GERMAN
POPULATION.
AT THIS POINT THERE WAS MORE FOR
US TO DO BUT WE'RE EXCITED WE
HAVE THIS INTERESTING GENOMIC
DATA IN THE COMMON FORMS OF
PERIONDONTIIS, HOW CAN MENDELIAN
SYNDROMES INFORM OUR
UNDERSTANDING.
USING VARIATION AS EXAMPLE IN A
WAY NOW THROUGH STUDY OF
MENDELIAN DISEASES WE HAVE A
TRAIT THAT WE CAN GO LOOK AT, AT
THE GENERAL POPULATION, THAT
COULD POTENTIALLY HELP US
STRATIFY DISEASE POPULATION.
NOW, THERE'S MORE FOR US TO DO.
WE HAVE TO REALLY SEE WHETHER
THE GENETIC VARIATIONS WE HAVE
IDENTIFIED HAVE A FUNCTIONAL
OUTPUT, AND THIS IS SOMETHING WE
NEED TO BE DOING.
BUT WE'RE HOPING THAT THESE KIND
OF STUDIES MAY HELP US STRATIFY
PATIENTS AND POTENTIALLY LEAD TO
THERAPEUTIC INTERVENTIONS THAT
ARE DIFFERENT FOR DIFFERENT
TYPES OF DISEASE SUBSETS.
IN CLOSING, I WANT TO LEAVE YOU
WITH THIS MESSAGE OF HOW WE'VE
TRIED TO DO THESE STUDIES WITHIN
THE NIH ENVIRONMENT, STUDYING
FROM MENDELIAN DISEASE, TRYING
TO DO BASIC STUDIES, FUNDAMENTAL
BIOLOGY AT THE ORAL CAVITY,
PAIRING BOTH HUMAN TISSUES AND
CELLS WITH PRE-CLINICAL AND
MECHANISTIC STUDIES IN MICE,
ALWAYS WITH THE HOPE OF TURNING
BACK INTO HUMAN INTERVENTION,
AND EVEN BETTER POTENTIALLY
EDUCATING AND INFORMING OF
UNDERSTANDING OF COMMON FORMS OF
DISEASE.
NOW, MAYBE THE MOST IMPORTANT OR
SECOND MOST IMPORTANT SLIDE
AFTER THANKING MY OWN GROUP,
WHICH HERE I DIDN'T HAVE ROOM TO
RETHANK, TO ACKNOWLEDGE
TREMENDOUS PARTNERSHIPS THAT WE
HAVE THROUGH THE YEAR.
AND SO THIS SLIDE DEPICTS REALLY
OUR MAJOR COLLABORATORS THAT
HAVE BEEN LONG-TERM PARTNERS,
HAVE ENRICHED OUR PROGRAM,
REALLY HELPED US SET UP AND
CONTINUE THROUGH THE YEARS BOTH
WITHIN NIDCR, WITHIN NIH, AND
YOU'VE HEARD OF MANY OF THE
SELECT ONES SPECIFICALLY.
AND IN THE EXTRAMURAL PROGRAM
AND INTERNATIONALLY.
I WANT TO ALSO DO A SPECIAL
THANK YOU TO MY MENTORING TENURE
TRACK COMMITTEE WHICH STARTED AS
A MENTORING COMMITTEE AND TURNED
INTO LONG-TERM COLLABORATORS,
PARTNERS, I WOULD SAY FRIENDS.
ALSO, I HAVE TO FINISH BY
THANKING THE TREMENDOUS SUPPORT
FROM THE NIDCR LEADERSHIP
STARTING WITH LARRY TABAK EARLY
ON, WHO ENCOURAGED ME TO GO THIS
DIRECTION, PREVIOUS SD AND
INSTITUTE DIRECTORS, AND OF
COURSE CURRENT LEADERSHIP, MATT
HOFFMAN, JANICE LEE, AND MARINA
D'SOUZA.
THANK YOU FOR YOUR ATTENTION.
I WILL STOP SHARING NOW.
>> NIKI, THANK YOU VERY MUCH,
FOR A FANTASTIC TALK.
FOR THOSE ON THE VIDEOCAST, THIS
IS MATT HOFFMAN, SCIENTIFIC
DIRECTOR OF NIDCR, HOSTING
QUESTIONS.
IF YOU WANT TO ASK A QUESTION,
JUST CLICK ON THE BUTTON ON THE
VIDEOCAST THAT SAYS SEND LIVE
FEEDBACK, THAT WILL COME
DIRECTLY TO ME.
NIKI, I'LL START WITH ONE OF THE
QUESTIONS THAT CAME UP THROUGH
YOUR TALK FROM HOWARD YOUNG WHO
IS ASKING HAVE YOU EVER
CONSIDERED USING IL-17 IN A
TOOTHPASTE FORMULATION TO TREAT
THE DISEASE, AND I GUESS THERE
WOULD BE A ROLE RANGE OF TYPES
OF ACTIMERS TO CONSIDER.
>> FOR LAD, THIS IS WHERE WE
STARTED, IT MADE SENSE TO USE
SYSTEMIC INTERVENTION BECAUSE
OBVIOUSLY ORAL MANIFESTATIONS
ARE NOT THE ONLY MANIFESTATIONS
THAT WE HAVE.
WHILE WE STARTED OUR STUDIES IN
THE ORAL CAVITY REALIZED
DYSREGULATION WAS WIDESPREAD.
FOR LAD HAVEN'T BEEN THINKING
THAT MUCH AT LOCAL FORMULATIONS.
HOWEVER, AS HOWARD IS STATING,
FOR COMMON FORMS OF
PERIODONTITIS A MORE LOCALIZED
APPROACH IS WHERE WE NEED TO
START AND THINK ABOUT, AND WE
HAVEN'T -- YOU KNOW, THIS IS
WHERE WE KNOW WE NEED TO BE.
BUT WE HAVEN'T DONE THE SPECIFIC
STEPS OF THINKING ABOUT THIS
PRACTICALLY FOR GENERAL FORMS OF
PERIODONTITIS.
YES, I APPRECIATE VERY MUCH THE
POINT.
>> ANOTHER QUESTION FROM PETER
YOOON.
CAN PROTEIN THAT LACKS CD11 SITE
BE APPLIED AS TREATMENT FOR
PERIODONTITIS.
>> THANK YOU FOR THE QUESTION.
ACTUALLY, ONE OF THE REASONS
THAT EXCITED US ABOUT THE
PROSPECT OF TARGETING THIS
PATHWAY IS THAT THERE IS
PRE-CLINICAL WORK USING FIBRIN
ANTIBODY THAT TARGETS
SPECIFICALLY THE FIB GAMMA
DOMAIN, THAT'S BEEN USED FOR
CLINICALLY FOR MULTIPLE
SCLEROSIS AND FOR RHEUMATOID
ARTHRITIS.
SO THE IDEA OF TARGETING THAT
SPECIFIC BINDING IS VERY
INTERESTING, EXCITING TO US.
>> OKAY.
SO, OBVIOUSLY EVERYONE'S
THINKING ABOUT TREATMENT.
WE HAVE ANOTHER QUESTION HERE
FROM STEVE HOLLAND WHO IS
SAYING, GREAT TALK, IN VIEW OF
THE ROLE OF NETs AND
PERIODONTAL DISEASE AND
PLASMINOGEN IS THERE ROOM FOR IN
GENERAL OR PLG IN PARTICULAR?
>> YEAH, SO THIS IS SOMETHING
THAT WE'RE THINKING OF AS WELL.
AGAIN, TRYING TO UNDERSTAND THE
COHORTS OF GENERAL POPULATION
PATIENTS WHERE THIS PATHWAY IS
IMPORTANT IS THE FIRST STEP.
BUT INHIBITING MITOSIS AND
NEUTROPHIL FUNCTION LOCALLY AND
OBVIOUSLY IT IS USED ALREADY
(INDISCERNIBLE) LESIONS, THAT
WOULD MAKE A LOT OF SENSE.
>> CAN I FOLLOW UP?
WHEN YOU WERE LOOKING AT THE
ROLE OF NEUTROPHIL NETs YOU
TALKED ABOUT THE ROLE OF
REACTIVE OXYGEN SPECIES.
SO, IS THAT ANOTHER AREA THAT
YOU'RE CONTINUING TO STUDY, OR
WHAT'S THE ROLE THERE?
>> THOSE ARE TIED TOGETHER, CAN
BE TIED TOGETHER.
SO, PART OF THE PROCESS INCLUDES
REACTIVE OXYGEN SPECIES, SO
COULD BE A CONTINUUM OR COULD BE
SEPARATE.
AND I WILL SAY THAT OUTSIDE OF
THE SETTING OF PLASMINOGEN
DEFICIENCY WE'VE BEEN
COLLABORATING WITH MARIANA AND
HER GROUP TO THINK OF MITOSIS
PATHWAYS IN THE SETTING OF
INFLAMMATORY BONE DISEASE
INCLUDING PERIODONTITIS, TRYING
TO LOOK AT THE GENERAL
POPULATION IN THE ROLE OF NETs
AND NEUTROPHIL FUNCTIONAL IN
PERIODONTITIS DISEASE IN
GENERAL.
WE'RE TRYING TO GO DOWN THOSE
PATHS, SOMETIMES I WON'T HAVE A
VERY SATISFYING ANSWER QUITE
YET.
>> THE NEXT QUESTION THAT'S COME
THROUGH FROM KATHERINE MACKEY,
THANK YOU FOR A WONDERFUL
INFORMATIVE TALK.
I'M A NEW ACI MORE FAMILIAR WITH
GUT MICROBIOME COMMUNITY
CHARACTERIZATION, DOES THE
TREATMENT OF SOME OF THESE
CLINICAL INFLAMMATORY CONDITIONS
WITH ANTIBIOTICS HAVE NEGATIVE
IMPLICATIONS FOR THE COMMENSAL
ORAL MICROBES AND CARRY
DOWNSTREAM NEGATIVE EFFECTS, IN
ADDITION TO THE INTENDED
ELIMINATION OF THE PERIODONTAL
PATHOGENS?
>> AGAIN, MY ANSWER MAY NOT BE
SATISFYING.
FOR LAD, ANTIBIOTICS DO HELP.
ALTHOUGH WE USE VERY BROAD
SPECTRUM ANTIBIOTICS AND DEPLETE
A LOT OF COMMENSAL, I THINK NET
DECREASE IN MICROBIAL BURDEN
SEEMS TO PLAY A ROLE AND TO HELP
RATHER THAN HAVE AT LEAST
CLINICALLY AND NEGATIVE
CONSEQUENCE BUT POINT IS WELL
TAKEN HOW DISTURBANCES BECAUSE
OF ANTIBIOTIC TREATMENTS MIGHT
BE DETRIMENTAL LONG TERM AND
REALLY ANTIBIOTICS, AS WE ALL
APPRECIATE, ESPECIALLY FOR THESE
TYPE OF DISEASES THAT ARE NOT
INFECTIONS, ARE CAUSED BY
COMMENSALS THAT SHIFT IN
PROPORTION, MIGHT NOT BE THE WAY
TO GO AND WE MIGHT HAVE TO THINK
ON THE MICROENVIRONMENT ALSO AT
MORE TARGETED APPROACHES.
>> THEN, NIKI, CAN YOU ALSO TELL
US FROM THE MICROBIOME
PERSPECTIVE, YOU MENTIONED A.A.
ACTINONYSIS IN YOUR PLASMINOGEN
STUDIES.
IS THERE A THOUGHT THAT SOME OF
THOSE SPECIFIC MICROBIAL
COMMUNITIES THAT ARE ASSOCIATED
WITH PERIODONTAL DISEASE MAY BE
RESPONDING TO UNDERLYING GENETIC
CONDITION OR UNDERLYING
PATHOLOGY RELATED?
>> IT'S A BIG QUESTION, MATT.
IN A GOOD WAY.
BUT SO WHEN WE THINK OF
GENETICS, THERE'S A COUPLE WAYS
ONE CAN THINK ABOUT IT.
SO DOES -- ARE YOU SUSCEPTIBLE
BECAUSE OF GENETICS TO
DECOLONIZE OR OVER-- BECOME
DYSBIOTIC OR ARE YOU RESPONDING
IN A PRO-INFLAMMATORY WAY, NO
MATTER WHAT YOUR STIMULUS IS?
SO THERE COULD BE DIFFERENT WAYS
BY WHICH YOU BECOME SUSCEPTIBLE.
A.A. IS A UNIQUE BUG ASSOCIATED
WITH SPECIFICALLY FORMS OF
PERIODONTITIS DISEASE COMPARED
TO RELATED COMMENSALS,
INTERESTING TO ZERO IN.
OBVIOUSLY IN THE PLASMINOGEN
DEFICIENCY TRAIT CORRELATES
CLOSELY SO WE HAVE TO PAY
ATTENTION TO THAT SPECIFIC BUG
AND ITS ROLE.
>> AND WHAT ABOUT PG?
HAVE YOU LOOKED AT DISEASE
SUSCEPTIBILITY FOR PG?
>> IN PLASMINOGEN TRAITS DATA
AVAILABLE HAD A LOT OF THE
CLASSIC PERIODONTAL PATHOGENS.
A.A. CORRELATED.
PG IS NOT ASSOCIATED SO THERE WE
DIDN'T HAVE AN ASSOCIATION.
NOT TO SAY THAT IT'S, YOU KNOW,
OBVIOUSLY WE KNOW IT'S
IMPLICATED IN MANY FORMS OF --
CHRONIC FORM OF PERIODONTITIS.
>> CAN WE THINK ABOUT PATIENT
VARIABILITY?
YOU MENTIONED IN THE LAST SLIDE
ABOUT SUBCLASSIFYING PATIENTS.
HOW ARE YOU GOING TO DO THAT?
WHAT ARE FORESEEING IN THE
FUTURE CLINICALLY AS A WAY OF
DOING THAT?
>> I'LL TELL YOU WHAT OUR NEXT
STEPS ARE.
IT'S EASIER FOR ME TO THINK
SPECIFIC, I THINK IT'S EASIER TO
ANSWER WHEN YOU HAVE A SPECIFIC
PLAN.
WITH PLASMINOGEN, BECAUSE WE
HAVE GENETIC VARIATIONS WE'VE
BEEN TALKING TO EXTRAMURAL
COLLABORATORS TO LOOK ABOUT
GENOMIC DATA AVAILABLE AND TRY
TO LOOK AT THE PRESENCE OF THESE
GENOMIC -- GENETIC VARIATIONS,
HOW THEY -- WHAT TYPES OF
PATIENTS THEY SHOW UP IN.
BUT WE HAVE TO THEN ALSO SEE
WHETHER THE GENETIC VARIATION
HAS A FUNCTIONAL OUTPUT.
DOES THAT SNP REALLY END UP
LEADING TO PLASMINOGEN --
REDUCED PLASMINOGEN ENZYMATIC
ACTIVITY OR PLASMINOGEN LEVELS?
AND CAN THAT PROVIDE FOR US
GROUPS OF PATIENTS THAT HAVE
THIS PLASMINOGEN-RELATED PROBLEM
THAT WE WOULD THEN THINK OF
TARGETING THAT SPECIFIC PATHWAY.
AND THAT IS NOT GOING TO BE
WIDESPREAD FOR ALL FORMS OF
PERIODONTITIS BUT WE SHOULD GO
DOWN THE LINE OF PATHWAYS THAT
MIGHT ALLOW US TO SUBCLASSIFY
FURTHER THIS CONDITION.
>> SO, NIKI, IN THE FUTURE WILL
WE BE DOING HOME TESTS SIMILAR
TO WHAT WE'VE DOING FOR
SARS-COV-2 RECENTLY?
>> I DON'T KNOW.
>> WHERE WE PUT A BIT OF SALIVA
IN A -- YOU'VE OBVIOUSLY
IDENTIFIED A NUMBER OF POTENTIAL
TARGETS RELATIVELY EASY TO
TRANSLATE INTO SOME HAND HELD
DEVICE TO GIVE PATIENTS SOME
IDEA WHAT MIGHT BE APPROPRIATE
TARGETED THERAPIES?
>> WE CAN SPECULATE THAT COULD
HAPPEN.
WE'RE NOT THERE YET.
I DON'T WANT TO JUMP INTO THAT
CONCLUSION QUITE YET.
>> THANKS VERY MUCH.
I WANT TO THANK THANK YOU VERY
MUCH AGAIN.
FASCINATING TAKE, GREAT
QUESTIONING.
I HAND BACK TO MICHAEL GOTTESMAN
TO CLOSE THE SESSION.
MICHAEL?
>> LET ME ECHO WHAT YOU SAID,
MATT.
THIS WAS A FANTASTIC LECTURE,
LEARNED AN ENORMOUS AMOUNT, I'M
SURE OTHER LISTENERS DID AS
WELL.
THANK YOU.
WE WISH YOU THE BEST OF LUCK AND
ALL THE SUPPORT YOU NEED TO
CONTINUE YOUR WORK.
THANK YOU VERY, VERY MUCH.
>> IT'S PLAN A PLEASURE AND
HONOR.
THANK YOU.
>> THANKS.