I WANT TO INTRODUCE MY
COLLEAGUE, WHO HELPED ME WITH A
LOT OF THIS, SHE'S GOING TO
INTRODUCE ALL THE SPEAKERS AS
THEY COME UP.
>> THANK YOU FOR COMING.
BEFORE WE START, I WANT TO
MENTION TOMORROW MORNING WE HAVE
A POSTER SESSION AT THE FAES
TERRACE WITH REFRESHMENTS
FOLLOWED BY WALS LECTURE WITH
DR. IRENE GEORGAKODI.
WE'RE EXCITED TO INTRODUCE AN
AWESOME SPEAKER, DR. KAITLIN
SADTLER, CHIEF OF SECTION 4 AT
NIBIB, HER LAB FOCUSES ON
DEVELOPMENT OF IMMUNE ACTIVE
BIOMATERIALS FOR REGENERATIVE
MEDICINE USING MECHANISM-BASED
NODOLOGY AND AND -- METHODOLOGR
OUR OPENING TALK.
[APPLAUSE]
>>THANK YOU.
THANKS FOR THE NICE
INTRODUCTION, ALSO, STEVE, FOR
THE EFFORT OF ORGANIZING THIS
TOGETHER.
I KNOW IT'S NO SMALL FEAT.
HUGE THANK YOU FOR THE
INVITATION, A I'M A TENURE-TRACK
INVESTIGATOR AT NIBIB PRESENTING
ON WORK OUR LAB HAS BEEN DOING
IN THE PAST FEW YEARS ON MUSCLE
TRAUMA AND THE WAY IMMUNE CELLS
ARE IMPORTANT AND REACTING TO
MATERIALS WE PUT IN AND
SUBSEQUENT EFFECT ON TISSUE
GROWTH.
AS A BIT OF A BACKGROUND AS TO
WHERE I CAME FROM AND HOW I
ENDED UP BACK HERE AT THE NIH AS
A P.I., I'M A LOCAL.
I WENT THROUGH THE FREDERICK
COUNTY PUBLIC SCHOOL SYSTEM
PRIOR TO DEGREE AT UMBC, A YEAR
AT POSTBAC BEFORE MY Ph.D. AT
JOHNS HOPKINS TELLING MYSELF I
COULD NOT LIVE IN MARYLAND AND
ONLY MARYLAND, POPPED TO M.I.T.,
POSTDOC, FOR RETURNING AS A
STADTMAN INVESTIGATOR AT NIBIB,
FALL OF 2019 GETTING MY LAB
STARTED, EARLY 2020, A BRILLIANT
TIME TO OPEN THE LAB.
THE MICS ALSO DON'T LIKE COVID.
UP FRONT, QUICK ACKNOWLEDGMENTS.
WE'RE GOOD?
QUICK ACKNOWLEDGMENTS.
I'M A NEW P.I., AS A NEW P.I.
I'M BUILDING THE LAB, AS ALWAYS,
A HUGE TEAM EFFORT, SO ANYTHING
DONE HERE I DID MOST OF THE
PAPERWORK ON, THE FOLKS IN THE
PHOTO ARE THE ONES THAT DID MOST
OF THE PIPETTING.
HUGE THANKS TO EVERYBODY
INVOLVED, ESPECIALLY DR.
LACKWANI, AND ANOTHER
POSTDOCTORAL FELLOW AND ANOTHER
MD/PHD STUDENT, AND WE ALSO HAVE
SABRINA, A TECHNICIAN, AN
INDEPENDENT RESEARCH SCHOLAR,
AND A POSTBAC NOW AN MD/PHD
STUDENT AS WELL.
THESE FOLKS AND FOLKS THAT
AREN'T IN THIS PICTURE THAT I'LL
MENTION LATER HELPED BUILD THE
LAB, SO MANY THANKS TO THEM.
SO, JUST AS A QUICK OVERVIEW IN
TERMS OF WHAT WE THINK OF WHEN
WE'RE TALKING ABOUT IMMUNE
RESPONSES TO TRAUMATIC INJURY
AND BIOMATERIALS, PREVIOUSLY THE
IMMUNE SYSTEM WAS VIEWED AS A
BIT OF A BADDIE WHEN IT CAME TO
IMPLANTED MATERIALS, BUT
ULTIMATELY THERE'S GOOD AND BAD
THINGS THAT HAPPEN.
IF YOU THINK OF BIOMATERIAL
SCAFFOLD, WHERE WE WANT THEM TO
REMODEL AND INCORPORATE INTO THE
TISSUE, THE IMMUNE SYSTEM PLAYS
AN IMPORTANT ROLE THERE, THE
LOCATION OF THAT IMPLANTATION,
PRESENCE OF INJURY, THE TYPE OF
MATERIAL WE IMPLANT CHANGES THE
TYPES OF IMMUNE CELLS THAT COME
IN.
THAT CAN ALSO THEN AFFECT THEIR
ACTIVATION AND POLARIZATION,
CREATING SCAFFOLD IMMUNE
ENVIRONMENT AND ALTER STEM CELL
DIVERSITY,
-- DIFFERENTIATION WITH
DIFFERENT OUTCOMES.
IF WE THINK OF MEDICAL DEVICES,
COSMETIC IMPLANT NOT NECESSARILY
MEANT TO INTEGRATE WITH
SURROUNDING TISSUE BUT WORK MORE
IN THE STANDALONE WAY, WE HAVE
THE CANONICAL FOREIGN BODY
RESPONSE, IN THIS BOTTOM RIGHT
HERE.
WITH THAT, WHAT WE SEE IS IF YOU
THINK OF A BIG PLASTIC DEVICE,
WE HAVE PROTEIN ABSORPTION,
IMMUNE CELLS TRYING TO
INTEGRATE, THEY CAN'T, SO WHEN
THEY CAN'T DEGRADE IT WE GET
FIBROTIC CAPSULATION.
I WAS ABLE TO COLLABORATE WITH
AN ARTIST WHO PUT TOGETHER A
VIDEO, THE ARTIST IS MORE
TALENTED THAN I, HILARIOUS
VIDEO.
THERE IS THIS LARGE DISTURBANCE
IN HOMEOSTASIS, AND THIS
PERSISTENT INFLAMMATION, SO HOW
DO OUR CELLS NOT START TO ATTACK
ITSELF, SO IF WE HAVE CONSTANT
INFLAMMATION AROUND DAMAGED
TISSUE HOW COME THAT SWITCH
DOESN'T HAPPEN AND WE GET A
NEGATIVE RESPONSE?
SO OUR GOAL TO TO VIEW
ENGINEERING AS AN IMMUNOLOGIST,
AS MENTIONED, SO WE'RE USING
THIS MECHANISTIC BIOLOGY TO
OUNCE MECHANISMS THAT OF GO EARN
IMMUNE RESPONSES TO MATERIALS
AND SUBSEQUENT EFFECT ON TISSUE
GROWTH AND DEVELOPMENT.
THAT WAY WE CAN IDENTIFY TARGETS
FOR RATIONALLY DESIGNED
THERAPEUTICS, BUILT FROM THE
BOTTOM UP.
SO, IN TERMS OF MODELS, OUR
LABORATORY USES VML MOD MODEL A
MOUSE, 30 MILLIGRAMS OF TISSUE
REMOVED BILATERALLY FROM
QUADRICEPS, RESULTS IN 30%
PERMANENT DEFECT IN MUSCLE
FUNCTION.
IN TERMS OF THE DIFFERENT
MATERIALS THAT WE IMPLANT WE
LEAVE THE INJURY ALONE FOR
CONTROL BUT ALSO HAVE CHOSEN TWO
POLES OF IMMUNE RESPONSE TO
MATERIALS, A POWDERED
DECELLULARRIZED EXTRACELLULAR
MATRIX, AND A POWDERED
POLYETHYLENE, OUR PRO FIBROTIC
MATERIAL.
ECM IS USED FOR TREATMENT OF
SKIN WOUNDS, HERNIAS, ALSO HAS
GONE UNDERCLINICAL TRIALS FOR
VOLUMETRIC INJURIES, IT'S USED
IN ORTHOPEDIC IMPLANTS BUT WHEN
YOU GET THOSE SMALL WEAR
PARTICLES AND THOSE WIND UP
EMBEDDING IN LOCAL TISSUE YOU
GET STRONG INFLAMMATION AND
FIBROSIS, AND IT'S REALLY A
NASTY EFFECT.
SO, WHEN WE GOT KICK STARTED, WE
WOUND UP DEVELOPING A FEW FLOW
CYTOMETRY PANELS TO EVALUATE THE
DIFFERENT IMMUNE CELLS COMING IN
AND RESPONDING TO THE INJURY AND
TO THE MATERIAL.
I WON'T GET INTO ALL OF THE
CELLS THAT WE LOOKED AT, BUT WE
WERE ABLE TO ANALYZE THESE I
HAVE A STANDARD MANUAL GAIN AND
APPLYING COMPUTATIONAL METHODS
AS WELL, AND IF YOU WANT TO
AVOID THE TROUBLESHOOTING WE
HAVE A COMMON ARTICLE IN NATURE
REVIEWS METHODS IN COLLABORATION
WITH THE WOLFE LAB FROM NCI ON
SOME TIPS TO LOOK OUT FOR WHILE
DESIGNING THESE PANELS BECAUSE
THEY WERE PAINFUL WHEN YOU GET
HIGHLY AUTOFLUORESCENT SAMPLES.
SO, SINCE WE WERE INTERESTED IN
THIS ASPECT OF AUTOIMMUNITY WHAT
WE WOUND UP DIGGING INTO HERE IN
TERMS OF THE MYELOID IMMUNE
CELLS WERE THE
ANTIGEN-PRESENTING CELLS,
APCs.
DENDRITIC CELLS IN TERMS OF WHAT
COMES INTO THIS INJURY SPACE,
THEY PEAK AND COUNT BY ABOUT
SEVEN DAYS POST INJURY AND
QUICKLY CLEAR, PERSIST IN LOW
NUMBERS THROUGH 42 DAYS
POST-INJURY.
SO, WHAT WE WIND UP SEEING IS
THAT ECM TREATMENTS, SO
TREATMENT WITH THIS
PRO-REGENERATIVE MATERIAL WHICH
YOU CAN SEE IN THE TEAL COLOR,
POPULATION OF CONVENTIONAL
DENDRITIC CELL TYPE ONES.
SORRY ABOUT THE NAMES, IT'S HOW
IMMUNOLOGY GOES.
I LEARN THEM ON A DAILY BASIS.
WE ENRICHED THIS POPULATION THAT
EXPRESS HIGH LEVELS, LACKED
CANONICAL RECEPTOR.
THESE ARE ASSOCIATED WITH
ANTIGEN CROSS-PRESENTATION
CHARACTERIZED BY TWO MARKERS,
EXTERIOR ONE AND CD103, NOT SO
MUCH WITH POLYETHYLENE
TREATMENT.
IF WE LOOK IN THE LITERATURE
ABOUT OTHER MARKERS EXPRESSED ON
THEM DENDRITIC CELLS INTEREST
BEEN DESCRIBED, Th2 IMMUNITY
IS IMPORTANT, AND ANTIGEN
CROSS-PRESENTATION AND CD8 AND
INDUCE THE TREGS BEEN IMPLICATED
IN TOLERANCE IN A MOUSE MODEL BY
MARK DAVIS' GROUP, WE'RE GETTING
CURIOUS WHETHER THEY PLAY AN
IMMUNOREGULATORY ROLE.
WE'RE GOING TO FOCUS MOSTLY ON
THIS ECM TREATMENT,
PRO-REGENERATIVE MATERIAL.
FURTHERMORE WHEN WE LOOK AT
MATERIAL TYPE OR THE DIFFERENT
FORMS OF MATERIAL, IN ADDITION
TO POWDERED MATERIAL IF WE PLACE
ECM SHEETS, USED IN THE CLINIC,
AS WELL AS LARGER POLYETHYLENE
PARTICLES WE SEE THE SAME
PATTERN.
SO IT SEEMS TO BE AGNOSTIC TO
THAT FORMULATION OF THE
MATERIAL.
SO IT STILL MAINTAINS THE SAME
PATTERN WHERE THE SHEETZ ARE
RECRUITING DENDRITIC CELLS, PE
PARTICLES ARE NOT.
WE WANTED TO KNOW WHETHER OR NOT
THEY WERE THESE CROSS-PRESENTING
DENDRITIC CELLS IN THE
LITERATURE, SO WHAT WE LOOKED AT
WAS A TRANSCRIPTION FACTOR
REQUIRED FOR MATURATION OF CDC
1s.
GOOD DATA DAY, WHEN WE LOOK IN A
THREE KNOCKOUT MICE IN RED, WE
LOSE THEM.
THESE DO APPEAR TO BE SAME
CROSS-PRESENTING DEPENDENT CD1S
IN THE LITERATURE, JUST A LITTLE
WEIRD, WHICH IS EVERY IMMUNE
CELL.
NEXT QUESTION WAS ABOUT ADAPTIVE
IMMUNITY, SO WE CHARACTERIZED
THE IMMUNE INFILTRATE FOCUSING
ADAPTIVE IMMUNE CELLS WITH
ANOTHER FLOW PANEL.
WE'VE KNOWN IN THE PAST, MY
THESIS WORK WAS FOCUSED ON
HELPER T CELLS AND WANTED TO
KNOW THE LYMPHOID PROFILE AND
POTENTIAL OF REGULATORY IMMUNE
CELLS TO CALM THE IMMUNE
RESPONSE.
LOOKING AT THE DIFFERENT TYPES
OF ADAPTIVE IMMUNE CELLS PRESENT
YOU SEE HIGH LEVELS OF BOTH
ALPHA BETA T CELLS AS REPORTED
PREVIOUSLY BY OUR LAB AND
OTHERS, AS WELL AS HIGH LEVELS
OF NATURAL KILLER CELLS IN ECM
TREATMENT.
THIS IS INTERESTING BECAUSE
NATURAL KILLER CELLS SECRETE
CHEMOKINE THAT RECRUITS CDC
1s.
WE ALSO SEE THAT THESE -- THERE
ARE CD8s EXPRESSING HELIOS,
WE'RE PURSUING THAT FURTHER.
WHEN WE LOOK AT GENE EXPRESSION
OF THE T CELLS IN THE WOUND
VERSUS DRAINING LYMPH NODE OF
THE SAME ANIMAL WE SEE THAT
THESE T CELLS IN LOCAL AREA HAVE
HIGHER LEVELS OF IL, NOTHING
EVER FITS ON A BEAUTIFUL BINARY
SCALE BUT WE DO SEE THAT
ENRICHMENT OF REGULATORY IL-10
MARKER IN THE LOCAL TISSUE.
NOW, WHEN WE'RE TALKING ABOUT
TREATMENT SPECIFICALLY AND LOOK
AT THE LYMPH NODE WE CAN SEE
HIGH UPREGULATION OF -- THIS IS
THE MUSCLE TISSUE IN COMPARISON
TO OTHER TREATMENT GROUPS.
WE CAN SEE ETM TREATMENT DOES
UPREGULATE IL-HAD AND IL-13,
AGAIN CANONICAL Th2 CYTOKINES,
IL-10 AND IL-2RA FOR T-CELL
ACTIVATION AND ODDLY AIR, TOSSED
IN FOR FINE LIKE ANYONE DOES,
AUTOIMMUNE REGULATORY ELEMENT,
FOR T CELL DEVELOPMENT AND
SELF-TOLERANCE, GOES UP
PERIPHERALLY WHICH IS WEIRD.
WE DON'T KNOW WHAT WE'RE DOING
BUT IT HAPPENS.
SO, INTERESTING ASSOCIATION WITH
KIND OF SOME TOLERANCE.
SO, NOW WE'VE TALKED ABOUT THE
APCs, ADAPTIVE IMMUNE SYSTEM,
WHAT ABOUT CONNECTION BETWEEN
THE TWO?
WHAT HAPPENS WITH IN TERMS OF
REGULATING INACTIVATION AND
POLARIZATION OF IMMUNE CELLS
AFTER INJURY?
SO WE TOOK A LOOK BACK AT THE
BATTA 3 KNOCKOUT MODELS, T CELLS
IN TRAINING LYMPH NODE SEVEN
DAYS AFTER INJURY, RED BARS ARE
THE BATA 3 KNOCKOUT MOUSE,
INCREASE IN T-CELL ACTIVATION
AND SEE THAT IN THE BLOOD.
THIS IS BASED OFF THE EXPRESSION
OF CD44 AND LACK OF CD62 LIGAND.
IF WE LOOK AT MACROPHAGES,
PREVIOUSLY IMPLICATED AS AN
IMPORTANT PLAYER IN SCAFFOLD WE
MODELING AND WOUND HEALING WE
CAN SEE THEY ARE ALSO DISRUPTED
IN THE BATF3 KNOCKOUT MODEL,
WEIRD THINGS HAPPENING WITH
MACROPHAGES.
WHAT WE GET IS WE HAVE AN
INITIAL LAG IN MACROPHAGE
RECRUITMENT THAT PICKS UP, AND
MORE RECRUITED TO MUSCLE
INJURIES AT 21 DAYS IN THE BATF3
KNOCKOUT MODEL, DIFFERENT
SURFACE MODELINGS AND LOSE A
MACROPHAGE MARKER IN MICE, I HAD
TO GOOGLE WHAT IT DID, FIRST
PAPER WAS ASSOCIATED WITH
TOLERANCE IN MICE, ALSO GOOD
DATA TO HAVE.
CD301B MINUTEALLY CHANGED.
CD206 HAS BEEN UTILIZED AS A
MARKER OF KIND OF, YOU KNOW,
MORE HELPFUL MACROPHAGES IN THE
SCAFFOLD REMODELING PROCESS,
CANCER LITERATURE ASSOCIATED
WITH PHAGOCYTOSIS.
OUR QUESTION IS WHAT'S BRINGING
THEM THERE? SO WE START TO LOOK
AT THE TWO RECEPTORS THAT
CHARACTERIZE THESE CELLS, CD103
103
BINDS E-CADHERIN.
WE LOOKED AT EXPRESSION AFTER
INJURY, PCR AND
IMMUNOHISTOCELLTY SEE INCREASES
IN CADHERIN 1 GENE EXPRESSION,
WHICH IS E-CADHERIN, AND WE SEE
E-CADHERIN HISTOCHEMICALLY
AROUND THE ECM AND GRANULOMAS AS
WELL AS THINGS THAT LOOK LIKE
TERTIARY LYMPHOID STRUCTURES,
CO-LOCATING WITH CDPOSITIVE
CELLS.
THERE'S RECENT PAPER THAT SHOWED
THAT ACTIVATION OF BETA-CATENIN
WAS LINKED WITH MUSCLE ATROPHY,
MUSCLE WASTING.
E-CADHERIN ENGAGEMENT PREVENTS
TRANSLOCATION TO THE NUCLEUS,
THIS MIGHT BE A WAY FOR OUR
BODIES TO HELP TRY AND SAVE SOME
MUSCLE DURING AN INJURY AND
PREVENT ATROPHY.
SO WE LOOKED AT CD103, WHAT
ABOUT CCR 1?
BINDS THE CHEMOKINE IN MICE AND
HUMANS, HUMANS ALSO HAVE XCL2,
ASSOCIATED WITH CHEMOTAXIS OF
THE CELLS TO TUMORS.
WHEN WE LOOK AT PROTEIN AND GENE
EXPRESSION, TOP ROW LOCAL
TISSUE, WE SEE IN THAT MUSCLE WE
CAN UPREGULATION WITH THE ECM
TREATMENT AND WE ALSO SEE THAT
WITH THE TISSUE, THE WHOLE
MUSCLE HOMOGENIZED AT ONCE, IF
WE ISOLATE SINGLE CELLS WE SEE
THAT UPREGULATION.
THE LACK OF UPREGULATION AND
POLYETHYLENE TREATMENT IS A
POSSIBLE EXPLANATION WHY WE
AREN'T SEEING SEEING CDC1s,
NOT GETTING THE CHEMOKINE SIGNAL
TO GET RECRUITED IN.
IF WE LOOK IN THE BLOOD
TREMENDOUS WE SEE PEAK EARLY ON,
ASSOCIATED WITH THE RECRUITMENT
OF THESE CELLS, AND THEN
INTERESTINGLY RAG KNOCKOUT MODEL
WITHOUT T CELLS OR B CELLS WE
DON'T SEE CHANGES WITH XCL-1,
SUGGESTING IT'S SOURCED FROM
INDEPENDENT SUBTYPE, PRODUCED BY
T CELLS AND NK CELLS.
T CELLS ARE RAG DEPENDENT SO WE
LEANED TOWARD IT BEING THESE NK
CELLS.
AS MENTIONED THERE ARE
LITERATURE REPORTS OF NC CELL
MEDIATED CDC-1 TUMORS, AND SO WE
SORTED OUT LYMPHOCYTES AND BY WE
I MEAN 5:00 IN THE MORNING,
STARTING AN EXPERIMENT WE, I WAS
THERE, I PROMISE.
AND HAD SOME GREAT TEAM WORK TO
GET THIS DATA.
AGAIN, ANOTHER GOOD DATA DAY SO
WE SAW 300-FOLD HIGHER XCL-1, SO
IT APPEARS NATURAL KILLER CELLS
ARE PRODUCING THIS.
A POST BACK STIMULATED NC CELLS
IN VITRO WITH FRAGMENT
EXTRACELLULAR MATRIX AND DAMAGE
ASSOCIATED MOLECULAR PATTERN,
LOW MOLECULAR WEIGHT HYALURONIC
ACID.
AND WE DID SEE WITHIN 24 HOURS A
MODEST UPREGULATION IN THESE NC
CELLS, IT DOES APPEAR NATURAL
KILLER CELLS ARE GETTING TISSUE
DAMAGE SIGNALS INDUCING TO
SECRETE THIS.
DO THEY REGULATE TISSUE
REGENERATION AFTER INJURY?
WE SEE ALL OF THESE CHANGES TO
IMMUNE CELLS, WHAT ABOUT THE
TISSUE ITSELF?
SO IN OUR WILD TYPE MICE
NORMALLY YOU GET INQUINNAL FAT
BED MIGRATION, DENSE
INFILTRATION AROUND THE
PARTICLES, INTO THE ECM SCAFFOLD
BUT NO DENSE FIBROTIC CAB CYLE.
WITH BATF3 WE SEE DEPOSITION
SUGGESTING ISSUES WITH
PHAGOCYTOSIS, MORE WHITE FAT
WHERE IT SHOULDN'T BE, AND WE
ALSO SAW MORE DISTAL FROM THE
INJURY SIDE MUSCLE FIBER
NECROSIS AND CALCIFICATION,
CALCIUM IS STAINED BLACK ON THE
RIGHT.
WE FOLLOWED THIS WITH PCR, HAD
UPREGULATION ASSOCIATED WITH
WHITE FAT, ALSO INCREASE IN
ACVR1, A RECEPTOR THAT'S MUTATED
IN A HUMAN DISEASE ASSOCIATED
WITH SEVERE POSTTRAUMATIC SOFT
TISSUE OSSIFICATION.
INTERESTINGLY, WE ACTUALLY WOUND
UP SEEING INCREASES IN MUSCLE
REGENERATION MARKERS AS WELL.
THIS WAS ODD FOR US BUT IF WE
LOOK BACK AT OUR HISTOLOGY AND
LOOK BACK AT THAT MUSCLE MOST
MUSCLE FIBERS DO APPEAR TO HAVE
AN INCREASE IN MUSCLE FIBER
CROSS-SECTIONAL AREA.
SO WE MIGHT BE SEEING DISRUPTION
IN THE REGULATION OF OVERALL
TISSUE DEVELOPMENT AFTER INJURY.
IN GENERAL WE HAVE THIS CIRCUIT
HERE THAT'S SURROUNDED BY
DENDRITIC CELLS WORKING WITH
MACROPHAGES, EOSINOPHILS AND
TREGS ALL WORKING TOGETHER TO
ULTIMATELY DETERMINE WHAT THE
OUTCOME IS IN TERMS OF TISSUE
DEVELOPMENT.
NOW, WHAT'S OUR CURRENT
HYPOTHESIS REGARDING THIS KIND
OF SELF-REGULATION TO
AUTOIMMUNITY?
IF WE THINK ABOUT IT AFTER
INJURY WE HAVE THE RELEASE OF
DAMAGE ASSOCIATED MOLECULAR
PATTERNS, SELF ANTIGEN PRESENT
OUTSIDE HOMEO TASTE IS SO --
HOMEOSTASIS.
WE GET THIS RELEASE OF XCL-1
FROM THE INJURY SITE,
RECRUITMENT OF CDC1s, WOULD BE
ABLE TO CROSS-PRESENT THIS
ANTIGEN TO TREGS, AND GET
IMMUNOREGULATION AND
SELF-TOLERANCE IN THE MORE
REGULATORY ENVIRONMENT
CHARACTERIZED BY IL-10
EXPRESSION.
IF YOU THINK ABOUT CANCER,
CANCER IS USING PATHWAY TO
PROTECT ITSELF DURING DISRUPTED
HOMEOSTASIS.
SO THIS TISSUE INJURY AS MANY
HAVE SAID THAT CANCER IS THIS
WOUND THAT NEVER HEALS SO WE CAN
THINK OF TISSUE INJURY ACTUALLY
CREATING A PATHWAY THAT COULD
ALLOW CANCER TO SURVIVE.
AND SO SUBJECT TO CHANGE, BUT
THAT'S OUR CURRENT THINKING.
AND SO JUST SOME QUICK
ACKNOWLEDGMENTS, THOSE
HIGHLIGHTED IN TEAL HELPED OUT
WITH THIS PROJECT A LOT, THANK
YOU, AS WELL AS OUR LEADERSHIP
FROM NIBIB, RICHARD AND BRUCE
HAVE BEEN SUPPORTIVE OF OUR LAB,
AND THEN OUR ADMINISTRATIVE
OFFICERS THAT HAVE HELPED OUT AS
WELL AS MY OWN MENTORS AND
ADVOCATES OUTSIDE NIH THAT HAVE
BEEN HELPFUL IN MY CAREER
JOURNEY SO I MASSIVELY THANK
THEM.
I'M HAPPY TO TAKE QUESTIONS NOW
OR VIA E-MAIL IF YOU'D LIKE.
THANK YOU VERY MUCH.
[APPLAUSE]
>> TOO MUCH IMMUNOLOGY?
>> THANKS FOR THIS.
I TRY TO STAY AWAY FROM
IMMUNOLOGY.
A QUICK QUESTION.
SOME OF THE DIFFERENT -- AT
IMMUNOLOGY TALKS, MOUSE
IMMUNOLOGY, A QUESTION ABOUT
HUMAN, COULD YOU LET US KNOW
WHICH CELL TYPES HAVE BEEN
IDENTIFIED IN HUMANS, EVEN
NATURAL MATERIAL, CLINICAL
TRIALS INTO THE CLINIC?
>>YEAH, THE INTERESTING THING
ABOUT THIS CASCADE IS THAT IT IS
CONSERVED IN HUMANS.
THAT WAS SOMETHING THAT WAS
INTERESTING FOR US.
ONE OF THE REASONS I DIDN'T
KNOW, IT WAS A MOUSE MARKER, I
DIDN'T EVER FOLLOW INTO ITS
FUNCTION.
CD103 IN HUMANS, E-CADHERIN IN
HUMANS, XCR 1, XCL 1, WE'RE
PLANNING ON FOLLOWING IN HUMANS
TO CROSS-CHECK AND HAVE
INDEPENDENT RESEARCH SCHOLAR IN
THE GROUP WHO IS COLLABORATING
WITH NICOLE MORGAN'S GROUP ON
DEVELOPING MORE MICROFLUIDIC
MODELS AND SO HER LAB IS GOING
TO BE FOCUSED ON KIND OF GETTING
SOME MORE OF THOSE HUMAN MODELS
ESTABLISHED THAT WE CAN START
PLAYING AROUND WITH.
SO WE'RE EXCITED BECAUSE IT DOES
APPEAR TO BE -- IT'S THE SAME
PATHWAYS IN HUMANS, AND WE'RE
CURRENTLY WORKING ON GETTING A
COURIER SERVICE TO ACTUALLY GET
THE HUMAN SAMPLES, SO BITS OF
PAPERWORK LEFT BUT WE'RE
CHECKING ON THAT.
>>YOU TALK IN TERMS OF CANCER
RESEARCH, ARE YOU ALSO LOOKING
AT AUTOIMMUNE DISEASES IN
GENERAL?
(INAUDIBLE).
>> I KNOW NOTHING ABOUT CANCER.
WE FOCUS IN ON THIS TRAUMATIC
INJURY SPACE.
IT'S INTERESTING TO SEE CANCER
MANIPULATES SOME OF THOSE
PATHWAYS.
AND SO I THINK THAT THESE SORT
OF EXPERIMENTS, THIS SORT OF
INVESTIGATION, COULD HELP LEND
SOME INSIGHT INTO EVEN
AUTOIMMUNE DISEASES THAT ARE NOT
NECESSARILY SOMETHING YOU THINK
ARE ASSOCIATED WITH AN INJURY.
WE KNOW THAT PERSISTENT
INFLAMMATION SUCH AS COVID-19
CAN TRIGGER AUTOIMMUNE-LIKE
CONDITIONS.
A RECENT PAPER CAME OUT I THINK
YESTERDAY, DAY BEFORE, DOUBLE
INCREASED RISK FOR TYPE 1
DIABETES IN PATIENTS WITH
COVID-19, INFLAMMATION CAN
INDUCE THOSE, THOUGH IT'S FRAMED
WITHIN TRAUMATIC INJURY AND
TISSUE RECONSTRUCTION COULD
HOPEFULLY OFFER INSIGHT INTO
OTHER AREAS.
>> ONE MORE THING.
(INAUDIBLE).
>> WE HAVEN'T MEASURED
BIOMECHANICAL PROPERTIES OF IT.
WHAT WE DID WAS TOOK THOSE DENSE
PLANTAR SHEETS, GOING TO HAVE
THE SAME FORMULATION, BUT
PROVIDED IN A DIFFERENT WAY,
VERSUS THE PARTICULATE, AND SO
WE DID LOOK AT THAT.
WE DIDN'T SEE A DIFFERENCE,
THOUGH MECHANICS ARE REALLY
IMPORTANT IN IMMUNE ACTIVATION
AND POLARIZATION SO PREVIOUS
STUDIES ASSOCIATED WITH MORE
SYNTHETIC HYDROGELS WHERE WE
MODIFIED STIFFNESS OF THAT
MATERIAL, YOU CAN SEE
STIFFNESS-DEPENDENT CHANGE IN
RESPONSE.
AND SO WE CERTAINLY HAVE DONE A
FEW OF THOSE EXPERIMENTS WITH
SYNTHETIC HYDROGELS AND MANY
OTHERS OUT THERE THAT ARE
BIOMATERIAL MECHANICS AND HAVE
DONE WONDERFUL WORK ON THAT.
>> (INAUDIBLE).
>> YES, WE LOVE TO BE ABLE TO
DEVELOP MORE SYNTHETIC KIND OF
CONTROLLED HYDROGELS, WHERE IT'S
X, Y AND Z AND WHAT'S IN EVERY
SINGLE BATCH SO HOPEFULLY TRYING
TO LEARN THE BASICS OF HOW
SOMETHING LIKE AN EXTRACELLULAR
MATRIX SCAFFOLD WHICH HELPS WITH
THIS IMMUNE RECRUITMENT AND MORE
POSITIVE WOUNDING OUTCOMES, SO
IF WE CAN LEARN FROM THAT TO
THEN APPLY TO THE DESIGN HONING
IN ON WHAT ARE THE GOOD THINGS
ABOUT THAT MATERIAL, SO, FOR
EXAMPLE, ONE OF THE THINGS WE
SEE, THIS REALLY HIGH EOSINOPHIL
RECRUITMENT EARLY ON, DO WE WANT
THAT MEAN EOSINOPHILS? WE DON'T
KNOW YET.
RAVI IS WORKING ON THAT
CURRENTLY.
SO WE'RE TRYING TO TEASE OUT THE
GOOD AND BAD OF THOSE MATERIALS
SO WE CAN GO AHEAD AND TAKE
THOSE PUZZLE PIECES AND PUT THEM
INTO SOMETHING THAT'S CONTROLLED
AND HAS MINIMAL BATCH-TO-BATCH
VARIABILITY.
THANK YOU SO MUCH.
[APPLAUSE]
>> IF ANYONE HAS QUESTIONS --
(INAUDIBLE).
>> THANK YOU.
NEXT SPEAKER IS DR. MATTHEW
WOLFE, STADTMAN INVESTIGATOR,
HEAD OF CANCER BIOMATERIALS
ENGINEERING SECTION FROM NCI.
LAB IS INVESTIGATING THE
IMMUNOMODULATORY BIOMATERIALS TO
USE IN NEXT GENERATION CANCER
IMMUNOLOGY.
THANK YOU.
>> ALL RIGHT.
THANK YOU VERY MUCH FOR INVITING
ME, FOR THAT INTRODUCTION.
OKAY.
SO, DID THE SLIDES COME UP?
I'M FROM THE NCI, FREDERICK,
MARYLAND.
I'M ALSO A STADTMAN
INVESTIGATOR, STARTED AUGUST
2020.
AND I ALSO USE BIOMATERIALS AND
ALSO USE EXTRACELLULAR MATRIX
FROM DECELLULARRIZED TISSUES.
WHAT I'M GOING TO TALK ABOUT
TODAY -- OH -- IS USING THESE
MATERIALS IN A VERY DIFFERENT
WAY.
LET'S SEE IF THIS WORKS.
ANYWAYS ...
>> (INAUDIBLE).
>> OH, I HOPE NOT.
THAT'S MISERABLE.
THERE WE GO.
THANK YOU.
ALL RIGHT.
SO LIKE I MENTIONED, WE ARE
BIOMATERIALS LAB.
AND WHAT YOU KNOW IN ADDITION TO
SOME THINGS THAT CAITLIN POINTED
OUT, MATERIALS PLAY AN IMPORTANT
ROLE IN CANCER.
SCAFFOLD, POROUS CELLS CAN BE
USED TO DELIVER DRUGS, IN A
CONTROLLED FASHION.
THEY CAN BE USED IN MODELING
DIFFERENT TISSUE SYSTEMS IN DRUG
DEVELOPMENT.
AND THEY CAN BE USED HOW CAITLIN
DESCRIBED IN A TISSUE
REPAIRATIVE CONTEXT, AND THEY
CAN BE IMMUNE MODULATORY, THAT'S
DEFINITELY TRUE IN THE PREVIOUS
TALK.
THE DRUG DELIVERY ASPECT AND
IMMUNE MODULATORY ASPECT WILL BE
THE THEMES I HIGHLIGHT TODAY.
DECELLULARIZATION TURNS A
TRANSPLANT INTO A MEDICAL
DEVICE.
REMOVE THE CELLS, PREVENT ACUTE
REJECTION.
AND WE VERIFY THAT A NUMBER OF
WAYS, AS DO OTHERS IN THE FIELD.
LET ME SEE IF I CAN GET A
POINTER UP HERE.
LOOKING AT REMOVAL OF ALL THESE
NUCLEI AND SMOOTH MUSCLE ACTIN,
SMALL INTESTINE, SAME THAT
CAITLIN MENTIONED, WE VERIFY DNA
REMOVAL, QUANTITATIVELY, AND
MAKING SURE THAT WHATEVER IS
LEFT DNA-WISE IS EXTREMELY
SMALL.
AND IT'S NOT JUST INTESTINE,
THOUGH THAT'S WHAT I'M GOING TO
FOCUS ON TODAY.
CAN YOU DO THIS WITH VIRTUALLY
ANY SOLID TISSUE FROM ANY ANIMAL
OR HUMAN SOURCE, IT'S NOT JUST
ACADEMICS USING THIS.
THEY ARE USED ALL THE TIME IN
THE CLINIC, SO BOTH HUMAN AND
ANIMAL TISSUES ARE USED AS
SOURCE MATERIALS FOR THESE ECM
SCAFFOLDED FOR TISSUE REPAIR IN
THE CLINIC, THESE ARE JUST
HANDFUL OF EXAMPLES CURRENTLY ON
THE MARKET.
AND WHAT WE ARE DOING IS WE'RE
LEVERAGING MATERIALS AND
VACCINES, ESPECIALLY CANCER
VACCINE.
AND SO IN THIS APPROACH, YOU
TAKE BIOMATERIAL SCAFFOLD AND
LOAD IT OR INFUSE IT WITH CANCER
VACCINE COMPONENTS, ANTIGENS,
ADJUVANTS.
AND WITH ANY DRUG DELIVERY
APPROACH LIKE THIS THERE ARE
CERTAIN ADVANTAGES WHEN YOU
IMPLANT THAT IN VIVO.
ONE IS THAT YOU PROLONG EXPOSURE
OF THE BODY OF THE IMMUNE SYSTEM
TO VACCINE EXPONENTS RATHER THAN
IT GETTING FLUSHED AWAY, WASHED
AWAY IMMEDIATELY.
THE MATERIAL ITSELF IS ALSO
IMMUNOSTIMULATORY, THAT'S ONE OF
THOSE FEATURES WE TRY TO
CAPITALIZE ON.
IT'S ATTRACTING LEUKOCYTES TO
WHERE THAT CANCER VACCINE
EXISTS.
YOU KNOW, TO THE THERAPEUTIC YOU
WANT IT TO RESPOND TO.
ULTIMATELY THIS PRODUCED A
POLARIZED IMMUNE RESPONSE THAT
IF DEVELOPED IN APPROPRIATE WAY
CAN TRAVEL TO OTHER SITES TO
ATTACK CANCER CELLS.
AND TO DATE THE MAJORITY OF
MATERIALS USED IN THIS SCAFFOLD
DELIVERY APPROACH HAVE BEEN
THOSE FOREIGN BODY REACTION TYPE
OF INFLAMMATORY RESPONSES, LIKE
PLGA, OR ALGINATE.
AND AS CAITLIN MENTIONED, ECM
SCAFFOLD ARE CERTAINLY IMMUNE
STIMULATORY.
AND DO MANY OF THESE SAME
THINGS.
BUT POLARIZE DIFFERENT IMMUNE
RESPONSE.
REALLY THIS IS REHASHING WHAT
WAS MENTIONED ALREADY, A LOT OF
TYPE 2 BIASED IMMUNE FEATURES,
CD206 POSITIVE MACROPHAGE, AND
LOW BUT NOT INSIGNIFICANT CD8 T
CELLS.
SO SINCE THESE MATERIALS ARE
USED EVERY DAY TO REPAIR TISSUE
FOLLOWING TUMOR RESECTION CAN WE
LEVERAGE THIS SCAFFOLD IMPLANT
IN A CANCER VACCINE
IMMUNOTHERAPY?
AND A QUESTION THAT COMES UP ON
THE WAY IS THIS IDEA OF TYPE 2
IMMUNITY IN CANCER.
REMEMBER THOSE IL-4-DRIVEN
CD4-DRIVEN MACROPHAGE
206-EXPRESSING MACROPHAGE
RESPONSES, CLASSICALLY THOUGHT
OF AS IMMUNE SUPPRESSIVE IN THE
CONTEXT OF CANCER.
BUT I WOULD CHALLENGE THAT
SIMPLY BECAUSE IT DOESN'T MATCH
WHAT WE FIND IN CLINICAL OR
PRE-CLINICAL DATA.
IN FACT, SUCH ECM MATERIALS
SOMETIMES CALLED BIOLOGICS ARE
ACTUALLY PREFERABLY USED IN
CONTAMINATED FIELDS WHETHER
THERE'S A HIGH RISK OF
INFECTION.
NOT SOMETHING WHETHER YOU WANT
BLANKET IMMUNE SUPPRESSION.
I POSIT IT'S A DIFFERENT TYPE OF
INFLAMMATION THAT IS NOT BLANKET
IMMUNE SUPPRESSION AND WE CAN
USE THIS TYPE OF LEUKOCYTE
ATTRACTION TO SYNERGIZE WITH
LOCALIZED CANCER VACCINE.
WE WANTED TO REALLY ANSWER THE
QUESTION OF WHETHER THESE ECM
SCAFFOLDS CAN MODULATE CYTOTOXIC
IMMUNITY.
WE WANTED TO STUDY INDUCING
EFFECT ON OF -- FROM CANCER
VACCINE.
LOADING A FORM OF ECM WITH TUMOR
ANTIGEN AND WITH A
CYTOTOXIC-INDUCING ADJUVANT,
HIGHLIGHTED HERE BECAUSE WE
DON'T GET THAT TYPE OF RESPONSE
INHERENTLY FROM ECM SO WE ADD
ADDITIONAL COMPOUNDS TO INDUCE
THAT.
CAN WE USE IT TO TREAT
ESTABLISHED TUMORS?
WE'LL USE A MOUSE MODEL.
ALONG THE WAY WE CAN ASK SEVERAL
QUESTIONS.
SO, IN ADDITION TO FUNDAMENTAL
ONE DOES THIS ECM SCAFFOLD
MICROENVIRONMENT AFFECT
CYTOTOXIC C TELL FUNCTION?
HOW DOES THE IMMUNE ADJUVANT
THAT WE'RE ADDING AFFECT HOST
RESPONSE AND HOW DOES THIS
COLLABORATE TO CREATE A
PRODUCTIVE ANTITUMOR RESPONSE
WITH CANCER VACCINE.
WE'RE ALSO USING A MUCOSA ECM
PARTICULATE, CLINICALLY
RELEVANT, WELL CHARACTERIZED, A
GOOD PLACE TO START.
SO WE TAKE THAT ECM, GRIND INTO
PARTICLES, THAT ARE ABOUT 50
MICRONS IN SIZE, IMPORTANTLY ARE
INJECTABLE AND SOMETHING WE CAN
MIX WITH OUR CANCER VACCINE OF
TUMOR ANTIGEN AND IMMUNE
ADJUVANTS.
AND AS A QUICK OVERVIEW, ANY
GOOD CANCER VACCINE HAS AT
MINIMUM TWO COMPONENTS.
THAT TUMOR ANTIGEN THAT PROVIDES
THE FINGERPRINT THAT IDENTIFIES
CANCER CELLS FROM YOUR NORMAL
CELLS AND THEN IMMUNE ADJUVANT
TO ATTRACT AND POLARIZE
RESPONSES AGAINST THAT ANTIGEN.
AND SO WHEN YOU DELIVER A CANCER
VACCINE YOU'RE INITIATING THE
CASCADE OF IMMUNE INTERACTIONS,
THIS IS JUST ONE BRANCH OF
WHAT'S GOING ON, BUT ULTIMATELY
WHAT I'M GOING TO BE FOCUSING ON
TODAY IS ABILITY TO INDUCE
ANTIGEN-SPECIFIC TUMOR ANTIGEN
SPECIFIC CD8 CELLS TO LYSE TUMOR
CELLS.
SO THE FIRST QUESTION, WHAT'S AN
APPROPRIATE ADJUVANT TO INDUCE
CYTOTOXIC RESPONSE WHEN
DELIVERED WITH ECM SCAFFOLD?
I WON'T GO THROUGH ALL THESE IN
A LOT OF DETAIL OTHER THAN TO
SAY THEY EACH STIMULATE A
DIFFERENT PATHWAY AND DIFFERENT
RESPONSE ON THE WAY TO
INITIATING CONTRIBUTING
CYTOTOXIC RESPONSE. .
WE CAST A WIDE NET BECAUSE NO
ONE HAS DONE THIS BEFORE WITH
ECM SCAFFOLD MATERIAL.
THIS IS BASELINE
CHARACTERIZATION TO STUDY EFFECT
OF WHAT ADJUVANTS WITH DOING TO
THE SCAFFOLD HOST RESPONSE
ITSELF.
SO HERE IS THE SIS IMPLANT IN A
SUBCUTANEOUS ENVIRONMENT, NO
INJURY HERE, JUST A SUB Q
INJECTION IN A MOUSE.
YOU GET IMMUNE ACTIVATION AND
INFILTRATION ON ITS OWN, YOU CAN
SEE A BUNCH OF CELLS, MAJORITY
OF IMMUNE CELLS INFILTRATING THE
SAS IMPLANT ON ITS OWN.
IN CONTRAST, SOMETHING LIKE
MPLA, A POTENT IMMUNE ADJUVANT,
CREATES A MUCH MORE ROBUST
CELLULAR INFILTRATE SO EVEN
AFTER 14 DAYS, WHICH IS WHEN
THESE RESPONSES SHOULD BE DYING
DOWN, WE SEE SOMETHING THAT'S
VERY DENSELY PACKED WITH CELLS,
ALMOST INFECTION LIKE, QUANTIFY
FROM HISTOLOGY ACROSS A TIME
COURSE, AND INDEED THAT'S WHAT
WE SEE, BOTH HAVE THIS AFTER ONE
DAY, CD8 DECREASED CURIOUSLY
THAT INITIAL INFILTRATION,
EVERYTHING CAUGHT UP BY DAY 7,
AND ONLY MPLA ARE THE CHRONIC
INFLAMMATORY ENVIRONMENT AFTER
14 DAYS.
THE ADJUVANTS ARE CHANGING AND
BOTH INFILTRATION NUMBER AND
KINETICS OF THAT RESPONSE.
THERE WE GO.
SO WE ALSO CHARACTERIZE WHAT
THOSE IMMUNE CELLS ARE.
SO THERE ARE CERTAIN IMMUNE
HALLMARKS ASSOCIATED WITH UCM
IMPLANTATION, SO CD206
MACROPHAGES BEING A BIG PART OF
THAT.
SO THOSE ARE INDUCED HIGHLY WHEN
ECM IS IMPLANTED ALONE.
AND SOMETIMES CALLED AN M2
MARKER IF YOU FOLLOW THAT
PARADIGM.
WE ALSO LOOK AT CD8 6, WHICH IS
IMPORTANT IN VACCINE RESPONSES
AND T CELL PRIMING.
AND WHAT WE FIND HERE ONLY THE
CD8 GROUP INDUCE APPRECIATABLE
UPREGULATION OF THAT CD86 THAT'S
SO IMPORTANT FOR T CELL PRIMING
AND ACTIVATION.
AND REALLY ONLY MPLA WAS
TRENDING TO DECREASE IN HIGH 206
REGULATION.
WE ALSO LOOK AT LYMPH NODES AS
SENTINELS OF WHAT'S GOING ON IN
THE REGIONAL ENVIRONMENT, NOT
JUST LOCALLY WITHIN THE
SCAFFOLD.
IL-4 BEING ANOTHER HALLMARK OF
ECM RESPONSE, SIS MATERIAL ALONE
INDUCES A LARGE UPREGULATION OF
IL-4, SURPRISINGLY ADJUVANTS
DIDN'T DIMINISH THAT EVEN THOUGH
THEY ARE CYTOTOXIC INDUCING, IN
SOME CASES ENHANCED IT IN FACT.
OKAY.
AND SO AFTER CHARACTERIZING
WHAT'S GOING ON LOCALLY AND
REGIONALLY THE GOAL IS TO
ULTIMATELY LOOK FOR FUNCTION SO
ARE WE GETTING CYTOTOXIC T
CELLS, ANTIGEN SPECIFIC T CELLS
FOLLOWING THIS APPROACH?
SO, TO DO THAT WE USE WHAT'S
KNOWN AS IN VIVO CTL ASSAY, A
QUANTITATIVE METRIC OF CD8 T
CELL KILLING, ANTIGEN SPECIFIC
FASHION.
AND SO WE TOOK OUR ECM, SIS ECM,
COMBINED WITH A MODEL ANTIGEN
USEFUL FOR TRACKING ANTIGEN
SPECIFICITY AND ADJUVANT.
EVEN WITH SALINE ALONE, THE
SOLUBLE DELIVER WRITE OF OCA AND
CDA WERE MAXING OUT ASSAY BUT
NOT LOSING ANYTHING BY
DELIVERING ARE OUR ECM.
AND THAT'S NOT THE CASE FOR SOME
OF THESE OTHER ADJUVANTS WHERE
MPLA BECOMES VARIABLE IN A
DECREASE.
WE HAVE A COMPATIBLE ADJUVANT
FOR INDUCING SKY TOE CYTOTOXIC
IMMUNITY.
ANOTHER IS TO PROLONG DELIVERY
OF THESE COMPOUNDS, PREVENT THEM
FROM GOING AWAY TOO QUICKLY AND
GETTING THEM TO STICK AROUND.
WE WANT TO SEE IF WE'RE GETTING
A BENEFIT FROM USING AN ECM
SCAFFOLD, WHEN DELIVERED IN
VIVO, FOR THAT WE TOOK THAT
OVALBUMIN MODEL, CONJUGATED TO
NEAR-INFRARED DYE FOR
FLUORESCENCE IMAGE, TRACKED MICE
OVER TIME, QUANTIFIED THE
RESPONSE, AND WHAT WE FOUND WAS
INDEED WITH RESPECT TO JUST
DELIVER THE OVA WITH SALINE
SUBCUTANEOUSLY, WE'RE ABLE TO
GET MORE SUSTAINED RELEASE AT
INJECTION SITE WITH SIS.
ALUM WAS OLD AS POSITIVE
CONTROL, AN OLD VACCINE ADJUVANT
THAT'S GOOD AT BINDING PROTEIN
AND KEEPING IT AT THE INJECTION
SITE.
WHAT I WANT TO POSIT HERE, TOO
GOOD IN THIS INSTANCE, APPEARS
TO BE LOCKED UP AT THAT
INJECTION SITE, RATHER THAN
BEING TAKEN UP BY THE IMMUNE
SYSTEM SO THAT IT CAN THEN BE
USED FOR ANTIGEN PROCESSING.
WHEREAS WE'RE GETTING EXTENDED
RELEASE WITH SIS OVER TIME LINE
THAT'S RELEVANT FOR VACCINATION
APPROACH.
SO WHERE IS THAT OVA GOING?
WELL, WE FIND THAT IT'S BEING
INTERNIZED BY MYELOID CELLS,
TAKING THAT FLUORESCENT OVA, CAN
VACCINATE, COLLECT MYELOID CELLS
EXPRESSING CD11B, AND WHAT WE
FIND IS THAT WE DO INDEED FIND
THOSE OVA-POSITIVE MYELOID CELLS
THAT HAVE EATEN ANTIGEN AND MADE
THEIR WAY TO THE LYMPH NODE IN
ORDER TO PRESENT IT TO T CELLS,
AND THIS INCREASES SUBSTANTIALLY
WHEN CDA IS USED AS ADJUVANT.
THE CONTRALATERAL LYMPH NODE,
JUST AS OUR NEGATIVE CONTROL.
OKAY.
SO, THE PUNCHLINE IS DOES THIS
WORK IN TREATING ESTABLISHED
TUMORS?
SO WHEN USING OUR VACCINE, OUR
SIS AS ECM SCAFFOLD COMPONENT,
CDA ADJUVANT WITH MOST EFFICACY
IN CTLA ASSAY INDUCING ANTIGEN
SPECIFIC CD8 RESPONSE AND
OVALBUMIN, THESE ARE CELLS THAT
FORM TUMORS WHEN THEY ARE
INJECTED SUBCUTANEOUSLY AND
EXPRESS THAT OVA ANTIGEN,
SOMETHING WE CAN VACCINATE
AGAINST IN OUR MODEL SYSTEM
HERE.
SO, ONCE TUMORS ARE ESTABLISHED
WE THEN THERAPEUTICALLY
VACCINATE ONCE AND AGAIN SEVEN
DAYS LATER.
AND SO WHAT YOU CAN SEE HERE IS
THAT UNTREATED ANIMALS TUMORS
GROW VERY RAPIDLY AS EXPECTED.
WHEN THE VACCINE IS PROVIDED IN
A SOLUBLE FORM JUST CDA AND OVA
SUSPENDED IN SALINE, PARTIAL
RESPONSE, ALL TUMORS EVENTUALLY
GROW AND REQUIRE SACRIFICE.
HOWEVER, WHEN DELIVERING WITH
SIS ECM SCAFFOLD WE GET GREATER
AN 50% RESPONSES SO TUMORS
REGRESS UPON TREATMENT, AND
ABOUT HALF OF THEM OR A LITTLE
MORE THAN HALF DON'T COME BACK.
AND SO THIS MANIFESTS AS
SURVIVAL BENEFIT COMPARED TO
OTHER GROUPS.
WHEN WE LOOK AT THESE ANIMALS
THE ONES THAT HAVE FULLY
REJECTED THEIR TUMORS UPON
VACCINATION, WE LET THEM GO FOR
A FEW MONTHS, FOR A COUPLE
MONTHS HERE TO MAKE SURE THEY
DON'T COME BACK AND PERFORM A
RECHALLENGE EXPERIMENT.
WHERE WE INJECT THE VERY SAME
CANCER LINE ON THE OTHER SIDE.
SO THAT WILL TELL US WHETHER WE
HAVE SYSTEMIC PROTECTION,
WHETHER THE MOUSE HAS BEEN
SUCCESSFULLY VACCINATED WITH
MEMORY RESPONSE.
INDEED THAT'S WHAT WE FIND HERE.
SO, AFTER THAT RECHALLENGE, OUR
NAIVE AGE MATCHED CONTROLS
RAPIDLY FORMED TUMORS AS
EXPECTED AND NOT A SINGLE MOUSE
WAS LOST FROM THE VACCINATED
GROUP SO WE HAVE IMMUNE MEMORY
AND PROTECTION IN THE LONG TERM
AND WE'RE CUTTING IT OFF HERE
BUT THEY WENT FOR A WHILE AFTER
THIS AS WELL.
AND FINALLY WE WANTED TO DO A
LITTLE VALIDATION THAT WHAT WE
THINK IS HAPPENING IS ACTUALLY
HAPPENING.
SO FROM OUR CTL ASSAY WE KNEW
THIS GENERATED CYTOTOXIC T CELLS
SPECIFIC FOR OVA BUT ARE THEY
RESPONSIBLE FOR TUMOR
REGRESSION?
THERE'S STILL STEPS INVOLVED
WHERE THE T CELLS HAVE TO
INFILTRATE AND BECOME EFFECTIVE,
IN THAT ENVIRONMENT AS WELL.
SO WE CONFIRMED THAT CD8 T CELLS
WERE DOING THIS USING CELL
DEPLETION EXPERIMENT, MONOCLONAL
ANTIBODIES, WE CAN DEPLETE
DIFFERENT IMMUNE SUBTYPES FROM
MICE BEARING EG 7 TUMORS, AND BY
APPLYING ANTI-CD8 TREATMENT WE
EFFECTIVELY REDUCE CIRCULATING
CYTOTOXIC CD8 T CELLS TO ZERO.
AND SO IN THIS EXPERIMENT, AS
EXPECTED, THE UNTREATED MICE
AGAIN GREW VERY RAPIDLY, THOSE
THAT WERE VACCINATED BUT GOT THE
CONTROL ANTIBODY AGAIN 50% OR
OVER 50% RESPONSE RATES, AS
BEFORE.
AND WHEN DEPLETING CD8 T CELLS
THAT EFFECT IS COMPLETELY LOST.
SO, THESE CD8 T CELLS ARE
ABSOLUTELY CRUCIAL AND NECESSARY
FOR FUNCTIONAL TUMOR REGRESSION
SO WE ARE INDUCING THIS SYSTEMIC
ANTITUMOR RESPONSE IN A
CD8-DEPENDENT FASHION FOLLOWING
THIS SIS ECM VACCINE.
SO, I'LL STOP THERE AND OFFER
SOME CONCLUDING THOUGHTS.
THOUGH THE TYPE 2 IMMUNE
RESPONSE BIASED ECM SCAFFOLD,
THOUGH THE ECM SCAFFOLD IS TYPE
2 BY AS DOES NOT SUPPRESS CD8 T
CELLS, THE CYTO TOXICS IMMUNE
ADJUVANTS DID NOT NEGATE
HALLMARK FEATURES OF THE IMMUNE
RESPONSE AND ECM SCAFFOLD
SYNERGIZED TO CURE ESTABLISHED
TUMORS, SO REMEMBER SOLUBLE
DELIVERY OF THE SAME COMPONENTS
WAS ONLY A DELAY RATHER THAN A
CURATIVE RESPONSE.
AND SO I'LL CONCLUDE SAYING THAT
CYTOTOXIC T CELL IMMUNITY CAN BE
BIOORTHOGONAL TO A
PRO-REGENERATIVE ECM SCAFFOLD
IMMUNE ENVIRONMENT, WE CAN
INDUCE ARM OF IMMUNITY WITHOUT
DAMAGING OR DISRUPTING THE
BENEFICIAL ELEMENTS OF THAT ECM
RESPONSE, THAT ARE IMPORTANT FOR
HEALING, FOR EXAMPLE.
LET ME THANK MY TEAM FOR MAKING
THIS ALL POSSIBLE, ESPECIALLY
SANJAY, WHO DID THE MAJORITY OF
THIS WORK THAT YOU SAW HERE
TODAY.
WE'RE EXPANDING AND HIRING IF
YOU KNOW POSTDOCS TO JOIN THE
LAB AND WORK IN THIS AREA, SEND
ME A NOTE.
THANK YOU, AND ARE THERE ANY
QUESTIONS?
[APPLAUSE]
YES?
>> GREAT TO SEE THE WORK YOU
PUT TOGETHER.
YES, THANK YOU, MATT.
GREAT TO SEE YOUR WORK PUT
TOGETHER THIS WAY.
SO, YOU KNOW, ANTI-COLLAGEN
ANTIBODIES ARE GENERATED IN
PEOPLE WITH RHEUMATOID
ARTHRITIS.
I'M CURIOUS, USING ECM AS A
DELIVERY METHOD TO RAMP UP THE
IMMUNE SYSTEM FOR A VACCINE
APPROACH, ARE YOU CONCERNED OR
HAVE YOU CHECKED DID YOU GET
ANTI-ECM PROTEIN ANTIBODIES THAT
MIGHT CAUSE SOME OTHER SIDE
EFFECTS THAT WE WEREN'T LOOKING
FOR?
>> YEAH, THAT'S A GOOD
QUESTION, ONE THAT WE'VE THOUGHT
ABOUT.
NOW, HAVEN'T LOOKED FOR -- I
BELIEVE IT'S ANTI-COLLAGEN TYPE
2 THAT IS IN RHEUMATOID, BUT,
NO, WE HAVEN'T SEEN ANY EVIDENCE
AND WE'VE TAKEN THESE MICE OUT
FOR ABOUT SIX MONTHS.
THEY ARE STILL GOING NOW
ACTUALLY.
AND THERE'S NO EVIDENCE OF
DEGENERATION.
LIKEWISE, WE PERFORMED THIS
VACCINATION IN MATURE MICE, NOT
QUITE OLD AGE, WE DIDN'T SEE ANY
DIFFERENCE OR LONG-TERM
MORBIDITIES THERE.
WE DIDN'T LOOK AT JOINTS BUT
COLLECTED TISSUES ALONG THE WAY
TO LOOK FOR EVIDENCE OF
INFLAMMATION IN OTHER TISSUES,
AND WE JUST DON'T SEE THAT.
THOUGH FOR THE MOUSE, IT'S A
CONCERN, BUT KIND OF WHAT MAKES
ME FEEL BETTER ABOUT THAT TOO IS
IT'S HARD TO INDUCE THOSE MODELS
IN THAT YOU NEED A SPECIFIC
ADJUVANT AND SPECIFIC TYPE OF
COLLAGEN DELIVERED IN A CERTAIN
WAY TO MAKE THAT MODEL WORK.
TO MY KNOWLEDGE.
AND I THINK THAT THAT MAKES
SENSE THAT WE'RE ALL VERY
TOLERANT TO OUR VERY BASE
STRUCTURAL ECM PROTEINS, THINGS
IF WE INDUCED AN AUTOIMMUNE
RESPONSE AGAINST WE'D HAVE A
DYSTROPHY AND BE IN REALLY BIG
TROUBLE.
SO, WE DON'T SEE -- LONG WAY OF
SAYING WE DON'T SEE IT HAPPENING
AND SOME THOUGHTS OF WHY THAT
MIGHT BE.
>> FOLLOWING UP ON HIS
QUESTION, SOMETIMES YOU SEE
COLLAGEN FORMED AROUND THE
IMPLANT, THINGS WALLED OFF.
MY QUESTION RELATES TO YOU HAVE
MICE THAT SURVIVED AND MICE THAT
KILL THE TUMORS, T CELLS KILL
THE TUMORS
WHAT DO YOU SEE WITH DIFFERENCE
IN EXTRACELLULAR MATRIX AROUND
THESE IMPLANTS THAT YOU PUT IN
AS FAR AS SURVIVAL?
>>VERY GOOD QUESTION.
SO, THE BASELINE WITHOUT ANY
MANIPULATION DIFFERENCE BETWEEN
THESE MATERIALS AND SOMETHING
LIKE POLYPROPYLENE MESH IS THAT
YOU TEND NOT TO GET THAT
FIBROTIC DEPOSITION WITH THESE
ECM BIASED MATERIALS, SO THAT'S
ONE OF THE REASONS THEY ARE USED
IN THE CLINIC TO AVOID FIBROTIC
SIDE EFFECTS.
WE DON'T HAVE LONG-TERM
EVALUATION OF THE MATERIAL PLUS
ADJUVANT YET SO THAT'S AN
INTERESTING QUESTION TO SEE IF
THAT'S BEING ALTERED, AND IT'S
ON THE LIST OF THINGS WE'RE
WORKING ON.
YEAH, BY DEFAULT, IT SHOULDN'T
HAPPEN AND WE'RE GOING TO SEE IF
WE'RE SCREWING THAT UP BY ADDING
THESE OTHER COMPOUNDS.
OKAY.
THANK YOU.
[APPLAUSE]
>> TEN-MINUTE BREAK.
>> WE'RE GETTING BEHIND TIME SO
LET'S GET GOING AGAIN.
THANK YOU.
>> ALL RIGHT.
NEXT SPEAKER, DR. CHEN, IF
YOU'RE HERE.
KNOW, NICOLE MORGAN, MY BAD.
ACTIVE CHIEF OF TRANS-NIH SHARED
RESOURCE ON BIOMEDICAL
ENGINEERING AND PHYSICAL
SCIENCES, RESEARCH FOCUSING ON
MICROFABRICATION WITH BIOMEDICAL
RESEARCH, PLEASE WELCOME DR.
MORGAN.
[APPLAUSE]
>> THANKS FOR THE OPPORTUNITY
TO TALK AND INTRODUCTION.
I'M GOING TO MOSTLY NOT TALK
ABOUT MOST OF WHAT WE DO TODAY
BUT I'M GOING TO TALK ABOUT A
FEW COLLABORATIONS FOCUSED ON
DEVELOPING TOOLS FOR CONTROLLING
AND MEASUREMENT OF OXYGEN IN
TISSUE CULTURE FOR IN VITRO
SYSTEMS.
HOW DO I DO THE THING?
PERFECT.
OKAY.
SO I'M HEAD OF THE SHARED
RESOURCES, HEAD UP
MICROFABRICATION AND
MICROFLUIDICS, WE RUN
MULTI-USER, IT HELPS TRAINEES
WITH CURRENT RESEARCH AND FUTURE
CAREER PROSPECTS TO DEVELOP SOME
OF THE TECHNIQUES AND TOOLS OF
BIOMEDICAL ENGINEERING TO TAKE
WITH THEM.
IT HELPS US FIGURE OUT A LITTLE
BIT MORE CLOSELY WHAT PEOPLE
WANT TO DO REALLY.
AND IN TERMS OF THE DEVICE
DESIGN.
LATER WE'LL TALK MORE ABOUT THE
FACILITY, I'M GOING TO SKIP TO
TALKING AND GIVING ORIENTATION
FOR PROJECTS WE'VE BEEN WORKING
ON AROUND OXYGEN DELIVERY AND
CONTROL.
FOR ORIENTATION, WE'RE BREATHING
21% OXYGEN RIGHT NOW, THAT'S NOT
WHAT CELLS ARE EXPERIENCING.
PHYSIOLOGICAL LEVELS OF OXYGEN
IN THE CIRCULATION, 3 TO 8%
PARTIAL PRESSURE, AND 200
MICRONS, SO IN BETWEEN
INTERCAPILLARIES LET'S THINK
ABOUT ADHERENT CELL CULTURE.
WE HAVE MONOLAYER OF CELLS UNDER
MEDIA, IN ROOM AIR, 18.6% OXYGEN
ONCE YOU TAKE CARBON DIOXIDE AND
WATER VAPOR.
IT'S SUBSTANTIALLY LOWER THAN
ATMOSPHERE BECAUSE TRANSPORT IS
LIMITED IN BY MIXING AND
DIFFUSION, SUBSTANTIAL DEPLETION
OF OXYGEN AT THAT CELL SURFACE
BY CONSUMPTION.
WE HAVE RECIPES FOR CULTURING.
THERE ARE OTHER FACTORS AT PLAY
BUT A LOT HAS TO DO WITH KEEPING
THEM AT OXYGEN LEVELS THEY ARE
HAPPY WITH.
3D THINGS GET COMPLICATED.
YOU HAVE SPHEROIDS, OTHER 3D
STRUCTURES IN MATRIGEL OR ECM
HYDROGEL WITH NO BULK
CONVECTION, TRANSPORTS DIFFUSION
ONLY, THE POSITION OF THE 3D
STRUCTURE IN THE MATRIX, IF YOU
HAVE A HANGING THAT'S UNIFORM
BUT YOU HAVE A SITUATION WHERE
THE OUTSIDE OF THE SPHEROID IS
PROBABLY AT HIGHER OXYGENATION
THAN PHYSIOLOGICAL
CONCENTRATIONS AND INSIDE
BECOMES HYPOXIC AND NECROTIC.
ALL SPHEROIDS, YOU DON'T LET
THEM GET TOO BIG, 500 MICRONS
CENTERS BECOME NECROTIC.
YOU HAVE A CULTURE, THE OUTSIDE
IS LEAST MECHANICALLY
CONSTRAINED, MOST OXYGENATED,
CAN MULTIPLY WITHOUT BOUND WHILE
CELLS IN THE MIDDLE STARVE.
SO PEOPLE HAVE BEEN WORKING ON
STRATEGIES FOR OXYGEN DELIVERY
IN 3D, FASCINATING AND NECESSARY
WITH TISSUE ON A CHIP, ECM IN
THE MIDDLE WITH PERFUSION
CHANNELS, OTHER STRATEGIES WHERE
PEOPLE WILL DEVELOP SORT OF
INDUCED VASCULARIZATION INSIDE
AN ECM, CELL-BEARING ECM.
AND THESE ARE GREAT BUT HARD TO
SCALE UP.
YOU NEED FLOW CONTROL FOR
MULTIPLE CHANNELS PER DEVICE,
YOU NEED TO -- IF YOU DON'T
INDUCE VASCULARIZATION YOU NEED
PATTERN CHANNELS 200 MICRONS
APART IN HYDROGEL, STILL HARD.
YOU HAVE A LARGE PARAMETER
STAYS, CHOOSE USUAL ECM, CELL
TYPE, FLOW CONDITIONS, SO IT'S
HARD TO BE CONFIDENT THAT THE
PARTICULAR CHOICES YOU'VE MADE
ALONG THOSE DIMENSIONS ARE GOING
TO BE RELEVANT FOR THE BIOLOGY
YOU WANT.
SO, WHEN WE LOOKED AT THIS
PROBLEM, WHAT IS THE SIMPLEST
WAY THAT'S SCALABLE THAT WE CAN
GET CONTROLLED OXYGEN DELIVERY
TO 3D CULTURE VOLUMES.
SO THE SYSTEM WE HAVE IS THIS
OXYGEN PERMEABLE MEMBRANE WITH
PILLARS SPACED BY INTERCAPILLARY
DISTANCE, 200 MICRONS APART,
SEED CELLS AROUND THESE PILLARS
IN MATRIGEL, PUT THE WHOLE
VESSEL INSIDE THIS BIOREACTOR
THAT HAS -- HOW DOES THE POINTER
WORK?
INTERESTING.
WE PUT THE WHOLE SYSTEM IN A
BIOREACTOR WITH TWO
CONTROLLED -- TWO CHAMBERS WITH
CONTROLLED ATMOSPHERE, SOURCE
GAS ON THE BOTTOM, 3% OXYGEN IF
WE'RE LOOKING FOR PHYSIOLOGICAL,
LOWER IF WE WANT HYPOXIA.
THERE'S MEDIA ON TOP.
ON TOP OF THAT SPACE IT'S
ANOXIC, 5% CO2 FOR PH BALANCE
AND NITROGEN.
THIS IS WHAT THE BIOREACTOR
LOOKS LIKE FIRST GENERATION FOR
MULTI-WELL PLATES, AGAIN TWO
CHAMBERS, SIX SIX-WELL PLATES,
GET TO HIGHER DENSITY OF PLATES
OR HIGHER DENSITY OF WELLS.
THERE'S LIKE TWO MACHINE TO
PLASTIC PIECES SANDWICH BE
MEMBRANE IN BETWEEN.
THERE'S A CONTROL CHAMBER BELT
IN -- BUILT IN, YOU CAN USE IT
AT THE SAME TIME YOU RUN THE
EXPERIMENT.
WHAT DO WE GET HERE?
WHAT WE'LL SAY IS CONVENTIONAL
IS LEFT TWO COLUMNS, CELLS GROWN
WITH 21% OXYGEN FROM THE TOP AND
3% FROM THE TOP, SORT OF THE
HYPOXIA CHAMBER, IF YOU WILL.
AND YOU SEE WHAT YOU EXPECT,
GROWING MORE HAPPILY IN 21% THAN
3%.
WHEN YOU PUT CELLS IN THE
GRADIENT CONFIGURATION IN
BETWEEN CHAMBERS, 3% AND 0%
OXYGEN, THEY GROW AS WELL AS 2%
TO A WEEK, SEE MORE HYPOXIA IN
21% CELLS, WHAT'S HAPPENING IS
THE CELLS NEAR THE OXYGEN HAVE
PROLIFERATED TO THE POINT THEY
ARE STARVING.
AND SO YOU ARE -- I'M GOING TO
MOVE ON BUT YOU'RE LOOK TO LOOK
AT MORE GENERAL EXPRESSION
ANALYSIS IN THE PUBLISHED
RESULTS BUT, AGAIN, WHAT YOU
WOULD EXPECT FROM FROM PRINCIPAL
COMPONENT ANALYSIS WHERE THE
GRADIENT CELLS, 21% CLUSTER MORE
TIGHTLY, 3% CELLS ARE WHAT WE
WOULD THINK OF MORE AS A HYPOXIC
SAMPLE, AND THEN AS A PLUG TO --
PLEASE DO CULTURE EXPERIMENTS IN
3D, 2D IS IN LEFT FIELD.
SO, WHAT ARE WE TRYING TO DO
NOW?
WE'RE LOOKING AT NOW BIOLOGICAL
QUESTIONS LIKE DRUG RESISTANCE,
FUNCTION OF OXYGENATION, MONITOR
GROWTH SO WE HAVE DEVICES THAT
DELIVER OXYGEN IN SIMILAR
CONFIGURATIONS COMPATIBLE WITH
LONG-TERM IMAGING MICROSCOPES
AND LOOKING FOR SPATIAL
RECEIPTOUTS.
WHAT I WANT TO TALK ABOUT NOW IS
WHETHER WE CAN ALSO IN REAL TIME
CHARACTERIZE OXYGEN
CONCENTRATION EXPERIENCED BY
CELLS AS FUNCTION OF DELIVERING
CONSUMPTION.
WE STARTED WITH A COLLABORATION
WITH NHLBI, WITH A PROBE FOR
MEASURING INTRACELLULAR OXYGEN,
A FUSION, THE OVERLAP OF
mCHERRY AND MYOGLOBIN DEPENDS
ON OXYGENATION STATE OF
MYOGLOBIN.
SO, HOW WE MEASURE THIS IS WITH
LIFETIME.
WE COULD MEASURE A CHANGE IN
INTENSITY, THAT'S GOING TO
DEPEND ON EXPRESSION OF THE
PROBE AND CELLS, THIS IS A MESS.
WE'RE GOING TO MEASURE LIFETIME,
INDEPENDENT OF CONCENTRATION.
HOW THIS WORKS, WE EXCITE WITH
LASER PULSE, MEASURE THE PHOTONS
EMITTED AS A FUNCTION OF TIME,
DO THIS MANY TIMES, BUILD UP A
DISTRIBUTION OF PHOTONS EMITTED,
AND WE GO AHEAD AND WE FIT THAT
DISTRIBUTION, AS EXPONENTIAL
DECAY.
AND WE'RE IMAGING SO REPEAT FOR
EACH PIXEL.
WE'RE MEASURING INTRINSIC
FLUORESCENCE, COMBINED WITH
OXYGEN CONCENTRATION
MEASUREMENTS.
AND QUICKLY WE'RE USING TWO
PHOTON EXCITATION TO MAKE SURE
THAT WE'RE GETTING MINIMAL
BACKGROUND FLUORESCENCE AND LESS
PHOTO DAMAGE.
THIS IS WHAT WE SEE.
THIS IS CELLS, ADHERENT CELLS
MEASURED WITH ATMOSPHERIC OXYGEN
AND THEN LOWEST OXYGEN THE
CHAMBER WILL GET TO, 5%.
WITH THE STANDARD MYOmCHERRY
PROBE, RIGHT PANELS TARGETED TO
MITOCHONDRIA.
WHAT WE DO TO CONVERT THE
LIFETIME, EVENTUALLY I'LL FIGURE
THIS OUT, BUT MAYBE NOT TODAY.
WHAT WE SEE WITH THE LIFETIME --
OH.
WHAT WE SEE WITH THE LIFETIME IS
HOW WE GET -- LET ME TRY AGAIN.
HOW WE GO FROM THE LIFETIME TO
OXYGEN CONCENTRATION IS WE HALT
IN THE CELLS, THE TOP PANEL,
ASSUMPTION THAT INSIDE IS THE
SAME AS OUTSIDE MEASURED WITH
FIBEROPTIC PROBE.
USING THAT DATA WE'RE ABLE TO
GET THE COMPARTMENTAL OXYGEN AS
A FUNCTION OF THE EXTRACELLULAR
OXYGEN IN THE RESPIRING CELLS.
WE'RE THE MICROFABRICATION
GROUP.
WHAT IF WE WERE ABLE TO GET
MEASUREMENTS OF THE OXYGEN
CONCENTRATION IN THESE CELLS AS
FUNCTION OF SUPPLIED OXYGEN
CONCENTRATION FOR A RANGE, MADE
A SUBSTRATE WITH A SINGLE GAS
CHANNEL WE FEED 8% OXYGEN
THROUGH AND THIN LAYER OF PDMS,
SO THE OTHER BLUE BOXES ARE JUST
A RULE TO USE THE MICROSCOPE,
FIGURE OUT WHERE WE ARE,
MICROSCOPIC IMAGES.
SO, WHAT WE GET IF WE ARE THEN
LOOKING AT THE OXYGEN
CONCENTRATION AS A FUNCTION OF
DISTANCE FROM THE SOURCE CHANNEL
WE HAVE MEASURED ON THE LEFT,
MODELED ON THE RIGHT.
DON'T HAVE CONVECTION.
THIS IS PRETTY GOOD AGREEMENT.
MEASUREMENTS WITH FIBEROPTIC
PROBE AND GET REASONABLE
AGREEMENT, A GOOD GRADIENT AND
CAN PUT CELLS IN THIS AND
MEASURE OUR MYO-mCHERRY AND
AUTOFLUORESCENCE AND SEE HIGHER
OXYGENATION CLOSE TO SOURCE
CHANNEL, LESS OXYGENATION AWAY
FROM SOURCE CHANNEL, MORE
GLYCOLYSIS FARTHER FROM THE
SOURCE CHANNEL.
THIS IS CONSISTENT THROUGH
MULTIPLE MEASUREMENTS.
AND THEN WHAT I'LL SAY WE CAN DO
WITH THIS TECHNIQUE, THIS IS NOT
A GREAT EXAMPLE, WE ALSO GET
NON-EQUILIBRIUM DATA.
WHAT I SHOWED UP TILL NOW WHAT
HAPPENS IF YOU WAIT TWO HOURS
AND START MEASURING.
THIS IS WHAT HAPPENS IF YOU
START MEASURING RIGHT AWAY.
SO YOU CAN ACTUALLY -- THIS IS
SOMETHING WHERE WE JUST CHANGE
THE CONCENTRATION IN ATMOSPHER,
ALSO COULD DO IN RESPONSE TO
ADDING A DRUG OR CHANGING MEDIA
AND MEASURE RESPONSES IN REAL
TIME.
THE OTHER THING WE WOULD LIKE TO
DO BUT NEED TO UNDERSTAND
SPATIAL AVERAGING SOFTWARE
BETTER, SO IF PEOPLE HAVE
SUGGESTIONS THAT WOULD BE GREAT,
WE WITH LOOK AT HETEROGENEITY.
IF YOU LOOKED AT IMAGES BEFORE,
WHAT WE SAW WAS THAT THEY
WEREN'T ALL THE SAME COLOR SO WE
HAVE OTHER TECHNIQUES THAT WE
MEASURE ENSEMBLE AVERAGES OF
CONSUMPTION RATE OXYGEN
CONCENTRATION BUT DON'TS HAVE
THE SORT OF GRANULAR DATE WE
HAVE GET FROM THAT.
WE'RE INTERESTED IN LOOKING AT
HETEROGENEITY WITHIN A LAYER AND
REALLY WHAT WE WANT TO DO OF
COURSE IS TO SEE WHAT HAPPENS
WHEN WE GO INTO 3D.
THESE ARE PRELIMINARY
MEASUREMENTS ON SPHEROIDS,
CHANGED COLOR SCALE ON YOU, WE
CAN GET A FEW HUNDRED MICRONS
INTO THE SPHEROID WITH
SINGLE-CELL RESOLUTION AND
MEASURE OXYGEN CONCENTRATION IN
THAT SYSTEM AS WELL.
AND WHAT WE WANT TO DO NOW IS
SORT OF PUT THAT -- COMBINE THAT
WITH THE OTHER SYSTEM THAT WE
HAVE.
OKAY.
SO, ONE QUICK DETOUR ON THIS,
MYO-mCHERRY SYSTEM, WE HAD TWO
PROBES, ONE TARGETED TO
CYTOPLASM, ONE TO MITOCHONDRIA.
AND WE SEE A MEASURABLE
DIFFERENCE IN OXYGEN
CONCENTRATION BETWEEN THE TWO
COMPARTMENTS.
AND OUR COLLABORATORS LAB KEPT
GETTING DINGED WITH THIS
ASSERTION, WELL, YOU CAN'T HAVE
THESE SUBCELLULAR DIFFERENCES IN
OXYGEN CONCENTRATION, THIS
DOESN'T HAPPEN.
AND SO WE KEPT LOOKING AND
SEEING IT.
AND SO WE DECIDED TO TAKE A LOOK
AT IT WITH FINITE ELEMENT
MODELING.
I HAD A POSTBAC DURING COVID AND
BEYOND THAT WHO WENT AHEAD AND
USED MICROSCOPIC IMAGE OF
MITOCHONDRIAL NETWORK IN A MOUSE
MYOBLAST TO CONSTRUCT GEOMETRIC
MODEL SIMILAR IN SCALE AND
COMPLEXITY AND CONNECTIVITY AND
PUT IT IN A QUARTER CELL AND
WENT AHEAD AND USED THAT WITH
LITERATURE VALUES TO SEE IF HE
COULD LOOK AT ON THE FORMATION
OF SUBCELLULAR OXYGEN GRADIENTS,
CELLS GROWING ON IMPERMEABLE
SURFACE, GLASS.
THE COLOR SCALE IS NORMALIZED
BUT IF YOU LOOK AT THE CELLS
BEING GROWN IN THE LOW OXYGEN
CONDITION, SORRY, GO BACK.
IF YOU LOOK AT THE CELLS IN THE
LOW OXYGEN CONDITION, THERE'S A
MUCH -- THERE'S DEPLETION OVER A
GREATER SPATIAL EXTENT THAN
ATMOSPHERIC OXYGEN.
CELL DENSITY HAS A MAJOR EFFECT.
THIS IS THE DIAGONAL OF A
COMPUTATIONAL CELL, CELLS ON A
SQUARE ARRAY, AND IF YOU HAVE
CELLS THAT ARE CLOSELY SPACED
THEY DEVELOP SUBSTANTIAL
GRADIENT IN OXYGEN
CONSTELLATION, FARTHER APART
THAN CELL DIAMETER YOU'RE VERY
CLOSE TO EQUILIBRIUM ATMOSPHERE
VALUES, JUST A LITTLE DISTANCE
AWAY FROM THE CELLS.
AND THEN FINALLY THE -- RELEVANT
TO BOTH CAN YOU HAVE SUBCELLULAR
GRADIENTS, CAN YOU HAVE
SUBSTANTIAL GRADIENTS IN YOUR
CELLS AND EARLIER BIOREACTOR
WORK?
WHETHER THE SURFACE YOU'RE
GROWING CELLS ON IS PERMEABLE TO
OXYGEN OR NOT MAKES A HUGE
DIFFERENCE, SO WE HAVE ON THE
LEFT CELLS GROWING ON GLASS, ON
THE RIGHT ON PDMS WITH OXYGEN
FROM BELOW AS WELL, AND YOU'LL
NOTICE ONE IS THE GRADE WENTZ
SEEN IN THE PDMS SITUATION
DEVICE ARE MUCH SMALLER, YOU CAN
SEE THE NUMBERS IN THE CHART
BELOW.
YOU CAN SEE THAT IN THE NUMBERS
IN THE CHART BELOW AS WELL,
DIFFERENCES BETWEEN TWO
COMPARTMENTS, BUT ALSO THE
OXYGEN CONCENTRATION THE CELL IS
EXPERIENCING IN PDMS IS THE
SOURCE CON CONCENTRATION,
CONSISTENT WITH RESULTS WHERE WE
HAVE A SOURCE OXYGEN
CONCENTRATION OF 3%, CELLS
GROWING HAPPILY.
SO I THINK THAT IS MY TIME.
AND I JUST WANT TO THANK
EVERYONE WHO HELPED WORK ON THIS
SO IT'S A COLLABORATION BETWEEN
OUR GROUP AND MICROFABRICATION
UNIT, AND MICHAEL GOTTESMAN'S
GROUP, AND OTHERS.
THANKS.
[APPLAUSE]
>> THANK YOU.
THIS IS REALLY EXCITING.
I HAVE TWO QUESTIONS.
ONE, SINCE YOU TAKE THE
mCHERRY LABELED CELLS, HAVE YOU
BEEN ABLE TO DO CORRELATIONS IN
THE SIGNAL?
SO IF YOU SEE CHANGES IN
mCHERRY DO YOU SEE --
>>IN THE ONE SLIDE I HAD ON
HETEROGENEITY WE CAN TALK LATER,
BUT THE ONE SLIDE ON
HETEROGENEITY THAT WAS
CORRELATION, SORT OF LOOKING AT
THE TWO AXES, MYO-mCHERRY AND
AUTOFLUORESCENCE FOR DIFFERENT
REGIONS.
IT'S -- I THINK -- IT'S OKAY.
YEAH, WHAT WE -- I THINK I WOULD
LIKE TO BE A LITTLE BIT MORE
CONFIDENT IN THE SPATIAL
AVERAGING OF THE SOFTWARE, SO I
NEED TO DO A BIT OF DEEP DIVE
INTO THAT I THINK.
>>OKAY, GREAT.
AND THEN THE OTHER QUESTION IS I
WAS TRYING TO UNDERSTAND THE
SIMULATIONS THAT YOU'RE DOING.
ARE THE SIMULATIONS EXPLAINING,
TRYING TO EXPLAIN OXYGEN
GRADIENTS FROM MITOCHONDRIA INTO
THE CYTOPLASM?
I THOUGHT YOUR SIMULATIONS WERE
FOR OXYGEN GRADIENTS ACROSS
CELLS.
>> APOLOGIES FOR THAT.
SO, I THINK WHAT I WOULD SAY IS
I'M INTERESTED IN OXYGEN
GRADIENTS CAUSED BY CELLS WITHIN
3D STRUCTURES, WHAT HAVE YOU,
THE PARTICULAR THING WE WERE
GOING AFTER WITH THE DETAILED
STRUCTURE OF THE NETWORK WAS
ACTUALLY EXTENT TO WHICH YOU CAN
GET OXYGEN GRADIENTS WITHIN A
CELL.
SO, IF YOU LOOK AT THE
MITOCHONDRIAL NETWORK IT'S LIKE
THIS BASKET AROUND THE NUCLEUS,
AND IT SPREADS.
AND SO IF YOU LOOK -- IN THE
IMAGES OF SINGLE CELLS, THERE'S
VARIATION IN COLOR, VARIATION IN
LIFETIME, IN OXYGEN
CONCENTRATION.
WE CLEARLY SEE IT.
AND THERE WERE -- MAYBE WE'RE
BEARING THE SCARS OF REVIEWS
WHERE PEOPLE WERE SAYING, NO,
THESE DON'T HAPPEN.
THESE OXYGEN GRADIENTS, THIS
CAN'T HAPPEN.
AND SO PART OF THE GOAL WITH
COMPUTATIONAL TOOLS TO SET OUT
CONDITIONS UNDER WHICH IT CAN
HAPPEN AND EXPLAIN WHY PEOPLE
THINK IT MIGHT NOT HAPPEN.
AND PART OF THE ANSWER THERE IS
THAT IF YOU HAVE -- I MEAN, IF
YOU HAVE CELLS THAT ARE GETTING
OXYGEN IN 3D, THOSE GRADIENTS
ARE MUCH LESS PRONOUNCED.
IF YOU'RE UNABLE TO MEASURE AT
THE SAME SCALE AS
OXYGEN-CONSUMING STRUCTURES IT'S
HARDER TO SEE GRADIENTS, BUT NOW
WE CAN.
>> GREAT, THANK YOU.
>> ANY OTHER QUESTIONS?
THANK YOU.
>> NEXT IS DR. CHEN,
CO-DIRECTOR EVER METABOLIC
CLINICAL RESEARCH UNIT CORES AND
SUPPORT SERVICES AND SECTION
CHIEF FOR ENERGY METABOLISM
SECTION, DIABETES,
ENDOCRINOLOGY, OBESITY BRANCH.
AND RESEARCH ON UNDERSTANDING
HOW HUMAN ENERGY METABOLISM AND
PHYSICAL ACTIVITY ARE REGULATED
AND IMPACT DISEASE SUCH AS
OBESITY.
>> THANK YOU SO MUCH.
I DIDN'T KNOW WHAT TO EXPECT
WITH THE ENCLAVE, CONCLAVE, I
DIDN'T WEAR MY RED ROBE OR MY
RED CAP.
GREAT ARRANGEMENTS OF DR.
MORGAN, THAT'S WHAT I'M GOING TO
TALK ABOUT, EXCEPT NOT THE WHOLE
BODY LEVEL.
LET'S SEE HOW THIS GOES.
I APOLOGIZE, THE TITLE, TRYING
TO PUT BME AND BMR TOGETHER.
HOW DO WE MEASURE ENERGY
METABOLISM VIA PRINCIPLES VIA
BIOMEDICAL ENGINEERING?
I DO MEASURING OF METABOLIC
RATE, ONE LETTER DIFFERENCE,
BME, BMR, SOMETIMES I GET
CONFUSED WITH THOSE.
THIS IS A DIFFERENT TALK.
THERE WILL BE MORE PRESENTED ON
THE HUMAN RESEARCH AND SO WE DO
CLINICAL RESEARCH HERE IN THE
CLINICAL CENTER, MY LAB ON FIFTH
FLOOR IN THE CLINICAL CENTER.
SO, AND IT'S BEEN MENTIONED, I'M
INTERESTED IN BODY WEIGHT
REGULATION, AND AS IT TURNS OUT
IT'S A SIMPLE LAW OF
THERMODYNAMICS, THE ENERGY
BALANCE, IT'S REALLY THE ENERGY
IN VERSUS ENERGY OUT.
WE'VE GOT THE ENERGY IN, WE HAD
COOKIES AND WHATEVER SNACKS YOU
HAD, AND THAT WOULD BE THE
ENERGY IN.
IF I HAD TO ASK YOU HOW MANY
CALORIES IS THAT, MOST OF YOU
DON'T EVEN KNOW.
IT DEPENDS ON WHAT YOU ATE AND
HOW MUCH IS DIGESTED.
FORTUNATELY I CAN MEASURE ENERGY
OUT MORE PRECISELY.
THAT'S WHAT WE FOCUS ON.
WE CAN MEASURE BODY COMPOSITION,
THAT'S ANOTHER BIG PART OF MY
LAB, MEASURE OF BODY
COMPOSITION.
IF YOU HAVE TWO OF THE THREE
COMPONENTS YOU CAN DETERMINE
ENERGY IN.
THIS IS WHAT WE'RE GOING TO TALK
ABOUT.
PARTICULARLY DIFFERENT
COMPONENTS OF ENERGY
EXPENDITURE, I'LL SHRINK DOWN TO
BMR, BUT THERE ARE OTHER
COMPONENTS WE ALSO TRY TO
MEASURE.
AS HISTORY GOES THE CONCEPT OF
HEAT HAS BEEN REALLY PROPOSED BY
ARISTOTLE, THE FOOD MUST BE
CAPABLE OF BEING DIGESTED AND
THOSE ACTIVITIES OF DIGESTION,
HEAT, DIRECT CALORIEMETRY, YOU
MEASURE THE HEAT, RIGHT?
BUT THE -- I KEEP GOING TO THE
WRONG ONE.
SO, 1770s IN PARIS, THIS
INVESTIGATOR LAVASIER MEASURED
BY RABBIT GENERATING HEAT AND
ICE BATH IN THE PREVIOUS SLIDE,
BUT HE DISCOVERED, HE AND HIS
PARTNER DISCOVERED IF YOU DON'T
FEED AIR INTO THIS SYSTEM, THE
RABBIT DIES.
THE HEAT DOESN'T MATTER OR NOT.
IN HUMANS, YOU START TO FOCUS ON
MEASURING GASES EXCHANGES, AND
THERE BY MEASURING IN THIS
UNITS, WHICH I LOVE THE MOST,
THE PUBLISHED AND DOCUMENTED BY
SCIENCE AND NUTRITION GRAHAM
LUSK, RESTING MAN REQUIRED 1200
AT 26 DEGREES C, 1400 AT 12
DEGREES C, WITH DIFFERENCE IN
METABOLISM CONSUMPTION.
ADVANCE 100 YEARS, A GERMAN
SCIENTIST DEVISED A ROOM WHICH
WE CALLED THE ROOM CALORIEMETER,
REGULATE TEMPERATURE BY OPENING
WINDOWS OR DOORS, SEALED IN
ONE-WAY FLOW MATTER, OPEN
CIRCUIT MATTER, WHICH HE
MEASURED THE CO2 PRODUCTION, SO
NOW YOU HAVE OXYGEN CONSUMPTION
AND CO2.
WHY ARE THOSE IMPORTANT?
I'LL SHOW YOU IN THE NEXT SLIDE
OR SO.
SO, ANOTHER 100 YEARS LATER,
1900s, METABOLIC CHAMBER CAME
IN, BUT IT IS -- THE REASON IT'S
A CHAMBER, BUILT IN THE BASEMENT
OF A UNIVERSITY BUILDING, METAL
ROOM, COLD AND DARK, THEY CALLED
IT THE CHAMBER.
THE FIRST OF ITS KIND MEASURING
HUMANS THAT DOES TWO PARTS.
ONE IS DIRECT, OR HEAT
MEASUREMENT, BY INSULATING WITH
COPPER WATER AND WATER
CIRCULATING THROUGH IT, MEASURE
THE TEMPERATURE CHANGE OF THE
WATER, THE VOLUME AND THE
CONDUCTIVITY OF THE WATER,
MEASURES THE DIRECT HEAT
EXCHANGE.
SIMULTANEOUSLY THEY MEASURE GAS
EXCHANGE, BY CIRCULATING AIR AND
MEASURE THE CO2 PRODUCTION BY
THE ABSORPTION, SODA LIME,
SULFURIC ACID, YOU CAN MEASURE
BOTH DIRECT, INDIRECT, HEAT
EXCHANGE, DIRECTLY BY THE HEAT,
AND INDIRECTLY BY GAS EXCHANGE.
AND THUS BY VALIDATED PRINCIPAL
OF INDIRECT CALORIEMETRY, WHEN
YOU HAVE A MOLECULE OF FAT YOU
NEED MORE OXYGEN THAN YOU
PRODUCE IN CO2.
PROTEIN, MIXED PROTEIN,
DIFFERENT EXCHANGE.
BY THE DIFFERENT EXCHANGES OF
CARBOHYDRATE, FAT, PROTEIN,
MACRONUTRIENTS, SO WHEN YOU EAT
THE COOKIES, CARBOHYDRATES AND
FAT WITH A LITTLE BIT OF
PROTEIN, REMEMBER YOUR LUNCH,
THAT'S THE THINGS WE ALSO BURN.
WE AS AN ENGINE, FLEX FUEL
BURNER, AS WE DO, SO WE MEASURE
THESE THREE COMPONENTS, NAMELY
CO2 PRODUCTION, OXYGEN
CONSUMPTION, AND URINARY
NITROGEN PRODUCTION, PROTEIN
TURNOVER, WE CAN CALCULATE
EVERYTHING.
THAT'S EXACTLY WHAT WE DO
INDIRECTLY.
WE HAVE ANOTHER REPRESENTATION
OF THE WHAT FUEL WE'RE BURNING,
THE RATIO OF OXYGEN CONSUMPTION
AND CO2, SIMPLE RATIO OF CO2
PROSECUTION OVER OXYGEN
CONSUMPTION, IF IT'S ONE,
CARBOHYDRATE, 6: 6, WESTERN
DIET IS 0.85.
HIGHER THAN 1 IS LIPOGENESIS,
BELOW 7 WHICH MOST PEOPLE DON'T,
ALCOHOL OXIDATION PROCESS,
SOMETIMES WE BURN ALCOHOL AS A
VALIDATION PROCESS.
SO THEN YOU HAVE THE STANDARD
EQUATION, DEVELOPED 100 YEARS
AGO.
THAT YOU CAN ESSENTIALLY
CALCULATE THE ENERGY EXPENDITURE
OR METABOLIC RATE, A MINUTE,
HOUR, DAY, CALCULATED BY THIS
EQUATION, OXYGEN CONSUMPTION,
CO2 PRODUCTION.
ALL THAT'S BEING SAID, HOW DOES
IT DO IT?
WELL, WE HAVE TO CALCULATE AN
OPEN CIRCUIT MATTER, INCORRECT
CALORIEMETRY, WHAT IS OXYGEN
CONSUMPTION, VOLUME OF INSPIRED
OXYGEN AND FRACTIONAL
CONCENTRATION OF OXYGEN TIMES
VOLUME OF WHAT'S INSPIRED, MINUS
THE EXPIRED OXYGEN, SORT OF
CONTENT.
SIMILARLY YOU DO THIS WITH CO2.
SO, THIS IS THE SIMPLE EQUATION,
BEING BIOMEDICAL ENGINEERS WE
HAVE TO SHOW EQUATIONS, RIGHT?
THIS IS THE SCHEMATIC OF HOW
IT'S DONE.
ANOTHER 100 YEARS, NOT BY
CHANCE, BUT JUST THE FACT THESE
THINGS ARE ADVANCED IN 100-YEAR
CYCLES.
THIS IS A METABOLIC CHAMBER
BUILT IN SWITZERLAND, SINCE
MOVED TO THE U.S., WITH CHAMBERS
IN THE RESEARCH FACILITIES AND
NIDDK, PHOENIX, AND PENNINGTON
BIORESEARCH CENTER, INDIRECT
MEASUREMENT OF OXYGEN AND CO2
NOW, YOU CAN SEE SMALL AIR ROOM
AT THE TIME 17 CUBIC METERS,
DESK, SINK, TOILET, A BED ON THE
SIDE OR SOFA, THEY HAVE A RADAR
SYSTEM TO MEASURE MOVEMENT, AIR
LOCKED, FOOD IN, WHAT NOT, I'LL
SHOW YOU THE REAL PICTURE.
AND THEN THEY DRAW THE AIR OUT
AND SAMPLE THE FLOW RATE WHICH
HAS STATIC VOLUME AND DYNAMIC
VOLUME, WHICH IS OPEN CIRCUIT
DESIGN NOW, AIR CONDITIONING AND
FILTERING, MOISTURE, MEASURE
OXYGEN BY PARAMAGNETIC SENSOR,
CONTINUOUS SENSOR AND CO2 BY
NEAR-INFRARED SENSOR, MODERNIZED
CALORIEMETRY BY ITSELF.
THIS IS THE CHAMBER I BUILT IN
THE CLINICAL CENTER.
WE RECRUITED FROM VANDERBILT,
WE, MY POSTDOC AND I, CAME HERE
2006, BUILT THREE CHAMBERS,
7th FLOOR CLINICAL CENTER,
THEY STILL OPERATE MOST OF THE
TIME.
SO IT'S A VOLUME OF 27,000
LITERS NOW.
APPLY DR. MORGAN'S PRINCIPLE,
THINK ABOUT 21% OF THE AMBIENT
AIR IS OXYGEN, AND SO WE MEASURE
DYNAMIC RANGE OF 1%, 20 TO 21%
OXYGEN, 0 TO 1% CO2.
YOU CAN DO THE MATH, BUT IF IT'S
1/5 OF THE AIR IS OXYGEN, YOU
END UP WITH MAYBE 70,000 LITERS
OF OXYGEN, AND BUT WE MEASURE
OXYGEN CONSUMPTION RATE PER
MINUTE BETWEEN 200 AND 300
MILLILITERS, REALLY THE
PRECISION MEASUREMENT OF THESE
THINGS THAT MATTERS.
TURNS OUT THERE ARE ONLY A FEW
OF THESE FACILITIES AROUND THE
WORLD.
AND THIS IS SOME OF THE PICTURES
SHOWING DIFFERENT DESIGNS, BY
AND LARGE SIMILAR IN VOLUME,
SMALLEST IS PROBABLY A
UNIVERSITY CHAMBER, 17 CUBIC
METERS, OURS IS 27, ANSCHUTZ IS
25 CUBIC MEETERS AND PENNINGTON
30 CUBIC METERS, FOUR CHAMBERS,
FACILITIES AROUND THE WORLD, WE
KEEP TRACK OF THEM SO WE JUST
HAD A MEETING AND FIGURED OUT
HOW MANY METABOLIC CHAMBERS, WE
NEED TO UPDATE BECAUSE CHINA
ADDED FOUR CHAMBERS IN THE
PANDEMIC ERA.
AND WE LOST TWO CHAMBERS
INCLUDING MY OLD CHAMBERS IN
VANDERBILT IN NASHVILLE,
TENNESSEE.
SO, THERE'S SOME UPDATES ON
THESE THINGS BUT ROUGHLY 40 OF
THESE FACILITIES AROUND THE
WORLD, THAT CAN DO THESE
METABOLIC CHAMBER MEASUREMENTS.
HUMAN CHAMBERS.
WITH ANY CHAMBER WE TALK ABOUT
TESTS, REPRODUCIBILITY, WE DID
THE STUDY WHEN WE FIRST HAD THE
FACILITIES RUNNING, 10 SUBJECTS
IN THE CHAMBER, THIS IS WHEN IRB
ALLOWED INVESTIGATOR ITSELF IN
THE INVESTIGATION.
I'M HERE SOMEWHERE.
THIS IS A BLUNT ALMOND PLOT, 24
HOURS, VERSUS AVERAGE OF THE
TWO.
YOU CAN SEE THE CURVE IS PRETTY
FLAT, MEANING THERE'S REALLY NOT
MUCH BIAS, DIFFERENCE ACCORDING
TO SIGNAL.
AND WE CAN ALSO DO THAT WITH THE
RQ, RESPIRATORY QUOTIENT, .85 IS
AVERAGE OF RQ OF THE WHOLE DAY.
THERE'S A VARIATION.
SOME PEOPLE CLOSE TO .8, SOME
ACTUALLY CLOSE TO .9.
BUT THE ENERGY EXPENDITURE,
KILOCALORIES TODAY, 1500 TO
4,000 PER DAY, BODY SIZE
MATTERS, WHICH I'LL SHOW ANOTHER
SLIDE OF THAT.
SO WHEN WE LOOK WHAT'S IMPORTANT
IN THE SLIDE, THIS NUMBER RIGHT
HERE, STANDARD DEVIATION, PLUS
OR MINUS 63.
THE DIFFERENCE OBVIOUSLY IS ALSO
IMPORTANT, 50 KCALS PER DAY,
BETWEEN DAY 1 AND DAY 2, WE'LL
CALL IT FIRST DAY EFFECT,
DIFFERENT ENVIRONMENT, FIGURE
OUT WHERE TO PUT YOUR CHOOSE,
LAPTOPS, BECAUSE IT'S INDIRECT
MEASUREMENT, YOU CAN BRING LAP
TOPS AND WHAT NOT BECAUSE WE'RE
NOT MEASURING HEAT, WE'RE
MEASURING GASES.
YOU SEE THE CHAMBER, THERE'S NO
PLANTS, NO THINGS THAT CONSUME
OXYGEN OR PRODUCE OXYGEN.
SO, NEVERTHELESS WE CAN ACTUALLY
HAVE A REPRODUCIBILITY OF ABOUT
3% OF THE 24 HOUR ENERGY
EXPENDITURE, PROBABLY AS HIGH AS
IT'S GOING TO GO.
AND RQ IS ALSO 2%, AND PLUS OR
MINUS 2%.
SO LET'S TAKE A LOOK AT VERY
QUICK WHAT INDIVIDUAL DATA LOOKS
LIKE, TAKING TWO DATA POINTS,
ONE WITH HIGHEST ENERGY
EXPENDITURE AND ONE WITH LOWEST,
HERE IS THE LOWEST.
THESE ARE NOW EXPRESSED IN A
24-HOUR MINUTE-BY-MINUTE CYCLE,
THIS IS MINUTE-BY-MINUTE CHANGES
IN KCALS PER MINUTE NOW.
SO SLEEPING, THIS LEAN HEALTHY
FEMALE SUBJECT IS BURNING ABOUT
A LITTLE LESS THAN 1 KCAL PER
MINUTE, THIS IS HOW EFFICIENT WE
ARE, 200 MILLILITERS OF OXYGEN
USED PER MINUTE.
WE WANT TO MEASURE WITHIN 5% OF
THAT.
TALK ABOUT PRECISION, SQUEEZING
ANY SIGNAL OUT OF THAT.
AND THE BLUE AND BLACK LINE
SHOWS REPRODUCIBILITY OF DAY 1
AND DAY 2, AND THAT'S TOTAL OF
1%.
YOU'LL SEE THERE'S SOME BUMPS
DURING THE DAY, AND IT'S NOT
EXERCISE AT ALL, SO WE HAVE
MOTION SENSOR IN THERE, THESE
ARE PURELY DUE TO MEAL WHICH
I'LL TELL YOU ABOUT MEAL INDUCED
OR THERMO EFFECT OF FEEDING.
THE DYNAMIC RANGE COMES IN,
DIFFERENT SUBJECT LIKES TO RUN.
WE HAD A TREADMILL IN THE ROOM,
YOU CAN SEE IT HERE.
HE WOULD RUN 10 MILES PER HOUR,
30 MINUTES IN THE MORNING, 30
MINUTES IN THE AFTERNOON.
THERE BY SORT OF DEMONSTRATING
THE DYNAMIC RANGE OF MOST
INDIVIDUALS CAN RANGE FROM 10
TIMES OF THE BASIC METABOLIC
RATE.
IT DOESN'T LAST VERY LONG IN HIS
CASE AN HOUR, BUT MOST SUBJECTS
CAN'T DO THAT, RIGHT?
SO WHAT IT DEMONSTRATES IS NOT
ONLY REPRODUCIBILITY,
MINUTE-BY-MINUTE LEVEL, BUT ALSO
SHOWS WE CAN TEASE OUT
COMPONENTS, SUCH AS EXERCISE,
SLEEP, SUCH AS DIFFERENT MEALS,
RIGHT?
AND SO HOW DO WE DO THAT?
YOU KNOW, YOU CAN DRAW THE
LINES, THE WAY WE DO THAT IS
CORRELATE THE ENERGY EXPENDITURE
IN MINUTE-BY-MINUTE FASHION
VERSUS ACTIVITY LEVEL, EITHER BY
SORT OF PORTABLE OR WEARABLE
SENSORS, OR BY RADAR SYSTEM
WHICH WE PUT IN THERE, THERE ARE
TWO SMALL BLACK DOTS, YOU CAN
PROBABLY SEE ON TOP OF THE
CEILING, THESE ARE MOTION
SENSORS, MUSEUM GRADE MOTION
SENSORS DETECTED, SO THAT'S WHAT
WE USE.
ENGINEERS, WE WIRE THEM, RIGHT?
AND WE MEASURE THE ACTIVITY.
SO WITH THE ACTIVITY INCREASES
YOU'LL SEE THAT ACTIVITY GOES
UP, ENERGY EXPENDITURE GOES UP
DUE TO ACTIVITY RELATED TO THE
MOVEMENT.
THEN YOU DRAW THIS INTERCEPT,
ZERO INTERCEPT, ZERO MOVEMENT,
THAT INTERCEPT IS HIGHER THAN
MEASURED BASIC METABOLIC, I SHOW
YOU HOW TO MEASURE THAT.
THE DIFFERENCE, THERMIC EFFECT
OF FEEDING, CLASSIFY ALL
MEASUREMENTS OF COMPONENTS WHICH
THESE ARE THREE MAJOR COMPONENTS
OF TOTAL ENERGY EXPENDITURE,
RESIDENT ENERGY EXPENDITURE,
THERMIC EFFECT OF FEEDING AND
YOUR ACTIVITY ENERGY
EXPENDITURE.
OKAY.
SO IT TURNS OUT IN OUR WESTERN
LIFESTYLE THE RESTING ENERGY
EXPENDITURE OR BASIC METABOLIC
EXPLAINED 60 TO 75% OF A TOTAL
ENERGY EXPENDITURE.
I.E. IT'S OBLIGATORY COMPONENT,
YOU HAVE TO SURVIVE, TO HAVE 60,
70% OF YOUR BASIC METABOLIC
RATE.
KNOW, THAT TURNS OUT TO BE
CLINICALLY MEANINGFUL RESEARCH
AND UNRESEARCHED SETTING, SO WE
DO THAT ALMOST EVERY DAY,
DIFFERENT WAYS TO MEASURE.
IT DOESN'T HAVE TO BE IN
METABOLIC CHAMBER, TURNS OUT.
WE DID THAT IN THE CLINIC,
WHETHER A PEDIATRIC UNIT OR
ADULT UNIT, USING THIS METABOLIC
CART.
THIS IS A CUTE PICTURE.
LITTLE BABY, WE MEASURED A
2-YEAR-OLD THREE WEEKS AGO.
THIS IS A VENTILATED HOOD OVER
AN INFANT, THEY CAN BE ASLEEP
HOPEFULLY, BECAUSE OTHERWISE
THEY WOULD BE IN CRYING IN THERE
AND NOT A METABOLIC RATE AT ALL,
OR ADULT UNIT, ESSENTIALLY THIS
IS THE INSTRUMENT THAT WE
MEASURE.
SAME PRINCIPLE EXCEPT WITH THE
SMALL DEAD SPACE, 7 LITERS OF
DEAD SPACE RATHER THAN 27,000
LITERS.
THE RATE IS DONE FASTED
OVERNIGHT, SUPINE, DIM LIGHT,
THERMONEUTRAL, WHAT I SPENT THE
LAST SIX YEARS TRYING TO FIGURE
THAT OUT, HAS TO BE QUIET,
MEASURING 25 TO 35 MINUTES BUT
IN A BABY I'M HAPPY WITH 10.
THERE ARE DIFFERENT
CONFIGURATION, WITH THE BABY WE
DIDN'T USE A VENTILATOR BUT CAN
WITH PATIENTS.
THE BIGGEST QUESTION THAT COMES
OUT, WE HAVE THE MEASUREMENT OF
BASIC METABOLIC RATE, IS IT
NORMAL, HYPER, OR HYPO.
CLINICALLY WE HAVE TO DETERMINE
THAT STATUS OF METABOLIC RATE.
TURNS OUT IT'S ONE OF THE LAST
LINEAR SYSTEMS LEFT THAT YOU
ASSOCIATE THESE 88 INDIVIDUALS,
RESTING ENERGY EXPENDITURE,
BASIC METABOLIC RATE, VERSUS
FAT-FREE MASS OR LEAN BODY MASS
MEASURED BY BODY COMPOSITION
METHODS, DEXA OR UNDERWATER, IT
HAS AMAZING LINEAR ASSOCIATION,
WHETHER YOU'RE LEAN, OBESE,
MALES AND FEMALES.
THEY ALL FOLLOW THIS LINEAR
LINE.
THERE'S OBVIOUSLY STANDARD
DEVIATIONS, WE CAN CALL THESE
THREE INDIVIDUALS HYPER
METABOLIC AND THEN MAYBE ONE OR
TWO INDIVIDUALS MAY BE HYPO
METABOLIC, THAT'S
CROSS-SECTIONALLY WE CAN
DETERMINE THAT, THIS IS MY
DATABASE AT VANDERBILT BEFORE I
CAME HERE.
WE HAD OVER 200 SUBJECTS.
SO THIS DEMONSTRATES TWO THINGS.
ONE IS THAT FAT-FREE MASS HAS
MUCH HIGHER CONTRIBUTION PER
KILOGRAMS VERSUS FAT MASS, ABOUT
4:1, REPRODUCED BY MANY LABS.
CROSS-SECTIONALLY.
THE OTHER THING THAT ALWAYS USES
AS EXAMPLE FOR MY PATIENTS, IF
I'M OVERWEIGHT I MUST HAVE A
LOWER METABOLIC RATE, AND THIS
SHOWS PER KILOGRAM OF FAT MASS
BY AND LARGE YOU HAVE HIGHER
METABOLIC RATE, FAT MASS
CONTRIBUTES TO METABOLIC RATE,
NOT NEGATIVELY, OKAY?
AND SO WE CAN ALSO APPLY TO
CLINICAL COMPUTATION, OLDER
STUDY WE DID THAT COMPARED
PATIENTS UNDER DIALYSIS,
PERITONEAL OR CHRONIC RENAL,
VERSUS CHRONIC RENAL FAILURE
PATIENTS, NON-DIALYSIS, CAN PLOT
RESTING METABOLIC RATE VERSUS
FAT-FREE MASS, CHRONIC RENAL
FAILURE HAS LOWER RESTING
METABOLIC RATE PER KILOGRAM
VERSUS PATIENTS RECEIVING
DIALYSIS, PRESUMABLY DUE TO
PROTEIN LEAK THAT THEY HAVE TO
REPLENISH, HIGHER PROTEIN
TURNOVERS.
THIS IS NON-DIALYSIS DAY, YOU
CAN PLOT THE GROUP-WISE
DIFFERENCE.
FIRST STUDY WITH CLINICAL TRIAL,
RECEPTOR AGONIST, APPROVED BUT
NOT FOR OBESITY RESEARCH, OR FOR
OBESITY TREATMENT BUT FOR
ANOTHER INDICATION, BUT THIS IS
WHEN WE TOOK A COMPOUND FROM
RHYTHM PHARMACEUTICAL AND WE
KNOW THE MECHANISM MAY AFFECT
METABOLISM AND DID STUDY IN
METABOLIC CHAMBER, 48 HOUR
RANDOM CROSSOVER STUDY IN I
THINK 12 INDIVIDUALS, MEASURED
RESTORATIONING METABOLIC RATE IN
THE DAY, COMPARED DRUG VERSUS
PLACEBO, AND CROSSOVER FASHION
WERE ABLE TO SHOW THE CHAMBER
MEASUREMENT, RESTING METABOLIC
RATE WAS SLIGHTLY HIGHER, ABOUT
6% HIGHER WITH THE DRUG VERSUS
PLACEBO, THIS IS, AGAIN, WITH
INDIVIDUAL DIFFERENCE OF 6% WITH
12 INDIVIDUALS, SHOWS YOU THE
PRECISION OF THE MEASUREMENT AND
LEVERAGE, BE ABLE TO STUDY
PHYSIOLOGY, WHOLE HUMANS.
AND THIS IS THE SPAGHETTI PLOT.
AND WE ALSO DID A HOOD
MEASUREMENT.
RIGHT HERE.
YOU CAN SEE BY GROUP DIFFERENCE
NOT MUCH, BUT THE PRECISION OF
THE CHAMBER MEASUREMENT IS
HIGHER, THEREFORE WE HAVE A
BETTER P-VALUE.
ANOTHER THING THAT GETS A LOT OF
INTEREST IS HOW THE BASIC
METABOLIC RATE CHANGES, IT'S NOT
STATIC.
BODY COMPOSITION CHANGES AS
WELL.
RESIDENT ENERGY EXPENDITURE
CHANGES WITH WEIGHT LOSS.
BEE DID THIS STUDY BY COMPARING
GASTRIC BYPASS SURGERY,
TREATMENT FOR SEVERE OBESITY,
VERSUS BIGGEST LOSER SORT OF
CONTESTANTS, WELL DOCUMENTED IN
THE SORT OF POPULAR SCIENCE
LITERATURE.
BUT WHEN WE DO THAT, YOU MAY NOT
SEE.
BIGGEST LOSER, 13 SUBJECTS, THEY
FOLLOW UP 7 MONTHS, MATCHED WITH
THE BARIATRIC SURGERY PATIENTS
WE FOLLOWED AT VANDERBILT, 13,
MATCHED FOR AGE AND BODY WEIGHT.
THEY CAME IN AT 140 KILOGRAMS,
135 KILOGRAMS, WELL MATCHED.
AND PERCENT BODY FAT WAS 50/50,
SEVEN MONTHS THE BIGGEST LOSER
LOST 35% OF THE BODY WEIGHT.
FAIRLY WELL MATCHED BUT WHEN YOU
MEASURE ENERGY EXPENDITURE,
VERSUS CHANGES IN BODY
COMPOSITION, THERE'S AN
ADAPTATION OF 400 CALORIES PER
DAY, THEY DROP BELOW PREDICTED
ENERGY EXPENDITURE ACCORDING TO
NO BODY COMPOSITION COMPARED TO
HALF OF THAT IN THE GASTRIC
BYPASS SURGERY.
TWELVE MONTHS LATER THE GASTRIC
SURGERY RETURNED TO ORIGINAL
LEVEL IN RESTING METABOLIC RATE,
METABOLIC ADAPTATION REDUCED TO
ALMOST NOTHING.
WE DIDN'T MEASURE IN BIGGEST
LOSERS BECAUSE THEY HAVE GONE
HOME AND WE CAN'T FOLLOW THEM
UP.
SO, THIS SHOWS WHAT I SHOWED
BEFORE.
IT TURNS OUT THAT THE
INDIVIDUAL, THIS IS INDIVIDUAL
METABOLIC ADAPTATION VERSUS THE
CHANGES IN THE ENERGY IMBALANCE
OR CALCULATED ENERGY INTAKE
VERSUS BODY COMPOSITION, AND
LEPTIN LEVEL, WHICH IS FAT
SECRETED, WHITE FAT SECRETED
HORMONE, SENSED BY THE BRAIN.
SO INDIVIDUAL LEVELS OF
METABOLIC ADAPTATION IS
CORRELATED TO BOTH MEASURES
SUGGESTING IT IS A PHYSIOLOGIC
MEASURE WE CAN MEASURE AND
PREDICT.
I WON'T TELL YOU THE OTHER
COMPONENTS, ALSO VERY IMPORTANT
BUT SMALLER IN SCALE, PHYSICAL
ACTIVITY, USING A LOT OF
WEARABLES TO DO THAT, AND
ADAPTIVE THERMOGENESIS WHICH
INCLUDE DIET INDUCED AND AS WELL
AS COLD INDUCED, WHEN WE TALK
ABOUT COLD WE MEASURE BROWN FAT,
SO I'M NOT GOING TO TELL YOU ANY
OF THAT BECAUSE THAT'S GOING TO
TAKE A LOT OF TIME.
HOPEFULLY THESE ARE MY NOTES,
AND CHEAT SHEET, TAKE HOME, THE
RESIDENT ENERGY EXPENDITURE IS A
MAJOR COMPONENT OF TOTAL ENERGY
EXPENDITURE, HIGHLY CORRELATED
WITH BODY COMPOSITION, FAT FREE
MASS, NOT STATIC, IT'S ACUTE AND
RAPID CHANGES IN BODY WEIGHT CAN
RESULT IN METABOLIC ADAPTATION,
PHARMACEUTICALS OR
NUTRICEUTICALS CAN ALTER BASIC
METABOLIC RATE IN ACUTE AND
CHRONIC CONDITIONS, THIS IS A
TEXTBOOK FROM 1970s SHOWING
METABOLIC RATE DO ADJUST TO
CLINICAL CONDITIONS BOTH IN
TERMS OF FEVER AS WELL AS
PARTIAL STARVATION, WHICH IS
REALLY NOT MUCH DIFFERENT THAN
THE BIGGEST LOSER CASE, RIGHT?
THEY DO DECREASE METABOLIC RATE
WITH PARTIAL STARVATION,
INCREASES WITH BURNS OR FEVER.
SO WE'RE VERY INTERESTED IN
DISEASE CONDITIONS, CANCER, HIV,
COVID-19, WHETHER WEIGHT LOSS
CONDITIONS OR AGE-SPECIFIC, YOU
KNOW, INTERVENTIONS IN THAT, AND
AS WELL AS DIFFERENT MEDICATIONS
USED FOR DIABETES, OBESITY,
SSRIs, VACCINES.
SO OUR PRINCIPLE IS TO MEASURE
EVERY COMPONENT POSSIBLE AND
WHAT I'VE SHOWN IS ONLY ONE
ASPECT OF WHAT WE DO.
WE MEASURE BODY COMPOSITION, WE
ALSO MEASURE PET-CT CAN BROWN
FAT AND USE ACCELEROMETER AND
WEARABLES TO MEASURE PHYSICAL
ACTIVITY AND STILL HAVE TROUBLE
MEASURING PHYSICAL ACTIVITY.
I WANT TO ACKNOWLEDGE THE PEOPLE
IN MY LAB, CURRENT LAB, AND
PREVIOUS MEMBERS HERE.
WITH THAT, I'D BE HAPPY TO
ANSWER ANY QUESTIONS.
[APPLAUSE]
>> DO YOU USE OPTICAL IMAGING
DEVICES -- (INAUDIBLE).
>> SO LET ME REPEAT THE
QUESTION FOR PEOPLE ONLINE.
YOU DIDN'T USE THE MICROPHONE.
YOU'RE THE HOST, YOU CAN BREAK
THE RULES.
THE QUESTION IS DO I USE ANY
OPTICAL MEASUREMENTS
SIMULTANEOUSLY IN THE CHAMBER.
WE DO HAVE OPTICAL CAMERA, AND
ACTUALLY WITH SLIGHT INFRARED TO
IT, MOSTLY FOR SAFETY.
WE DON'T RECORD IT FOR SUBJECT
PRIVACY REASONS.
BUT THEY ARE METABOLIC CHAMBERS
WITH OPTICAL IMAGING FOR, YOU
KNOW, RECORD THE TYPE OF
ACTIVITIES AND INTENSITY OF
ACTIVITIES.
AND LAST BUT NOT THE LEAST, WE
DO HAVE WHEN WE DO BROWN FAT
STUDIES, WE DO AN IR IMAGING OF
SURFACE BODY TEMPERATURE, BUT
STATIC IMAGE.
>> I'M CURIOUS ABOUT THE RADAR
MEASUREMENT OF ACTIVITY AND HOW
IT'S ONLY A SINGLE PARAMETER
BECAUSE I WONDER IF YOU LOOKED
AT THE DIFFERENCES IN ENERGY
EXPENDITURE AS A FUNCTION OF --
>>YES, YES.
ANOTHER VERY GOOD QUESTION.
SO, THE RADAR SENSOR HAS REALLY
BEEN IN THERE FOR HISTORICAL
REASONS BECAUSE A LOT OF THE
TECHNIQUE TRYING TO SEPARATE THE
DIFFERENT COMPONENTS IS RADAR
BASED, BUT WE HAVE A LOT MORE
WEARABLE SENSORS TO GET MORE
QUANTITATIVE MEASUREMENTS, IT'S
A QUANTITATIVE/QUALITATIVE
MEASUREMENT, RIGHT?
BUT ACTUALLY BEFORE I CAME HERE
IN 2006 I HAD TWO R01s AT
VANDERBILT, DEVELOPING WEARABLE
SENSORS.
SO IF YOU WONDER HOW YOUR APPLE
WATCH ESTIMATES YOUR CALORIES,
WE HAD A PART IN THAT.
>> IN TERMS OF BODY
COMPOSITION, HOW MUCH EFFECT
DOES MUSCLE MASS HAVE ON EITHER
ENERGY EXPENDITURE OR BMR?
>>VERY GOOD QUESTION.
SO, IT TURNS OUT MUSCLE MASS AT
RESTING LEVEL CONTRIBUTES TO
VERY LITTLE OF YOUR BASIC
METABOLIC RATE BUT ORGAN SIZE IS
HIGHLY CORRELATED WITH MUSCLE
MASS.
IF WE COULD MEASURE ORGAN SIZE
AND ORGAN-SPECIFIC METABOLIC
RATE, THAT'S THE HOLY GRAIL OF
MEASURING THE METABOLIC
DIFFERENCES, THAT HAS MUCH
HIGHER CONTRIBUTION, FOR EXAMPLE
BRAIN, LIVER, HEART, AND KIDNEY
CONTRIBUTES TO ABOUT 60% OF
METABOLIC RATE, BUT ON THE
INDIVIDUAL LEVEL IT'S ACTUALLY
VERY DIFFICULT TO DETERMINE.
P. .
>> OZEMPIC, METABOLIC RATE?
>> ANOTHER INTERESTING
QUESTION.
WE'RE DOING A FEW OF THOSE
STUDIES, BUT IT TURNS OUT MOSTLY
REGULATING ON THE ENERGY INTAKE
SIDE, NOT SO MUCH ENERGY
EXPENDITURE BUT HOW DOES THAT
IMPACT METABOLIC ADAPTATIONS?
WE DON'T KNOW.
OKAY, THANK YOU.
[APPLAUSE]
INVENTION, DESIGN, DEVELOPMENT
AND APPLICATION OF BIOMEDICAL
OPTICS AND INSTRUMENTATION TO
BROAD RANGE OF INTERDISCIPLINARY
RESEARCH PROGRAMS.
>> THANKS YOU FOR THE
INTRODUCTION AND INVITING ME TO
PRESENT THE WORK IN DEVELOPING
ADAPTIVE OPTIC TECHNIQUES TO
EXPLORE RETINAL FUNCTION AND
DYSFUNCTION.
I'D LIKE TO START BY RECOGNIZING
COLLABORATORS, ESPECIALLY HERE
AT NATIONAL EYE INSTITUTE,
INCLUDING KATHY KUKROS AND WORK
WORKING WITH RETINAL DISEASES
AND JOHNNY TAM WHO WE'RE HELPING
BUILD SOME A.O. SYSTEMS.
HE'S DONE SOME NICE WORK USING
ICG TECHNIQUE TO LOOK AT
METABOLISM AND RPE CELLS.
SO, I WORK FOR THE OFFICE OF
SCIENCE AND ENGINEERING LABS,
AND OUR PRIMARY OUTPUT IS
REGULATORY SCIENCE TOOLS,
REGULATORY SCIENCE IS DEFINED BY
FDA AS DEVELOPMENT OF NEW TOOLS,
STANDARDS, APPROACHES TO ASSESS
QUALITY AND PERFORMANCE OF
FDA-REGULATED PROCESS.
IN THE RESEARCH ARM, OUR OFFICE,
WE HAVE ESTABLISHED A CATALOG OF
REGULATORY SCIENCE TOOLS THAT
INCLUDE PHANTOMS, MODELS, USEFUL
IN THE INDUSTRY.
IF YOU CERTAIN REGULATORY
SCIENCE TOOLS CATALOG FROM THE
FDA.GOV WEBSITE YOU'LL ACCESS
THIS AND LOOK AT TOOLS WE'VE
DEVELOPED IN THE OFFICE.
WE BELIEVE THESE TOOLS WILL
EXPAND THE SCOPE OF INNOVATEIVE
SCIENCE-BASED APPROACHES TO
IMPROVE DEVELOPMENT OF EMERGING
MEDICAL TECHNOLOGIES.
WE'VE ESTABLISHED ADAPTIVE
OPTICS PROGRAM AT FDA, TO ALLOW
CELLULAR RESOLUTION OF RETINA BY
CORRECTING FOR OCULAR
ABERRATIONS THAT OCCUR IN ALL OF
US.
THE PROGRAM GOALS, ONE TO
DEVELOP PHANTOM-BASED EYE MODELS
AND NON-CLINICAL TOOLS FOR
PERFORMANCE ASSESSMENT, AND
THESE RETINAL IMAGING DEVICES
THAT USE ADAPTIVE OPTICS INCLUDE
DEFORMABLE MIRROR, PERSONALIZED,
EVERY PATIENT SUBJECT IN FRONT
OF THEM THEY ADAPT TOP.
AND THAT CAUSES SOME CHALLENGE
FOR PERFORMANCE ASSESSMENT.
THE SECOND PART OF OUR PROGRAM
IS TO INVENT, DEVELOP, VALIDATE
A.O. BASED STRUCTURAL AND
FUNCTIONAL BIOMARKERS FOR EARLY
DISEASE DIAGNOSIS FOR USE AS
PRIMARY CLINICAL ENDPOINTS.
I'D LIKE TO TAKE YOU ON A TOUR
OF THE RETINA AND NOTE A.O.
TECHNIQUES WE'RE USING TO
EXPLORE THE RETINA.
THIS IS A CROSS-SECTION TAKEN
FROM AN A.O. SYSTEM.
YOU CAN SEE THE LAYERS THERE,
THE MAIN CELLULAR COMPONENTS OF
RETINA INCLUDE GANGLION CELLS,
IMPORTANT IN GLAUCOMA, BIPOLAR
CELLS AND LIGHT SENSITIVE
PHOTORECEPTORS AND RPE, WHICH IS
A MONOLAYER, AND THROUGH WHICH
NUTRIENTS FROM THE CHORIOCAP
LARULARIS PASS TO PHOTO
PHOTORECEPTORS.
WE'RE GOING TO EXPLORE
MACROPHAGE CELLS AT THE TOP OF
THE RETINA, FURTHER IN THE
RETINA, WE'LL LOOK AT GLAUCOMA.
A NEW TECHNIQUE, YOU CAN MEASURE
FUNCTION OF INDIVIDUAL
PHOTORECEPTORS, RPE CELL, I
NOTED WE'LL LOOK AT ORGANELLE
MOTILITY AND LATEST WORK IN
IMAGING CHORIOCAPILLARIS.
MACROPHAGES MAKE UP 15% OF BRAIN
CELLS, PRIMARY FUNCTION IS IN
IMMUNE RESPONSE TO INJURY.
MY UNDERSTANDING, WONG DID SOME
NICE WORK AT NIH, AND NEI, IN
STAINING THESE CELLS IN THE
RETINA WHICH OCCUR PRIMARILY IN
THE PLEXIFORM LAYERS AS YOU CAN
SEE HERE IN THIS FLUORESCENT
IMAGE, A PORTION OCCUR AT THE
MEMBRANE.
UNTIL RECENTLY CELLS HAVE BEEN
ONLY VISUALIZED WITH FLUORESCENT
MARKERS, EXOGENOUS DYES OR
TRANSGENIC LABELING.
ANOTHER RECENT STUDY FOUND THAT
IN THE RESTING STATE WITHIN 10
MICRONS OF THE RETINAL SURFACE,
82% ARE MICROGLIA, COMPARED TO
69% VITREOUS MACROPHAGE CELLS,
9% PARA VASCULAR MACROPHAGES.
YOU'LL SEE THEM CALLED
MACROPHAGE-LIKE CELLS, BUT THEY
ARE PREDOMINANTLY MICROGLIA.
WE UNDERTOOK A STUDY USING
ADAPTIVE OPTICS TO RESOLVE CELLS
IN THE RETINA AND WE WERE ABLE
TO DO THAT AND MAP THEIR
DISTRIBUTION, IN LIVE HUMAN
EYES.
YOU CAN SEE DENSITY OF CELLS
INCREASES US A MOVE AWAY FROM
THE FOVEA, CORRELATING WITH
THICKNESS OF THE NERVE FIBER
LEVEL WHICH WE THINK THEY HAVE A
ROLE IN CLEARING AND DISEASES.
WE'RE ALSO ABLE TO LOOK AT TIME
LAPSE VIDEOS OF THESE -- AND
THIS IS A 25-MINUTE TIME LAPSED
VIDEO OF THE CELLS, YOU CAN SEE
IN THE RESTING STATE PROBING
ENVIRONMENTS, THESE IMAGES ARE
FROM LIVE HUMAN EYES, NOW THAT
WE CAN EXPLORE THAT, WE
UNDERTOOK A FEW STUDIES TO LOOK
AT CHANGES.
THE FIRST WAS WITH AGING, WE
FOUND HUMANS LOSE 2% OF
MICROGLIA PER YEAR, CELLS AREN'T
REPLENISHED FROM UNDERLYING
PLEXIFORM LAYERS.
WE FOUND THE MICRO MICROGLIA AE
DENSER IN EARLIER STAGES OF
DISEASE, SLIGHT INCREASE IN
PROCESS VELOCITY WITH GLAUCOMA.
ANOTHER STUDY WE RECENTLY
FINISHED, PUBLISHED, MICROGLIA
DENSER IN M.S. SUBJECTS THAN
HEALTHY VOLUNTEERS AND CONFIRMED
EARLIER WORK THAT THE PROCESS
VELOCITY IS SLIGHTLY HIGHER WITH
DISEASE.
SO IMMUNE CELLS PLAY A ROLE IN
RETINAL HOMEOSTASIS AND
NEUROLOGICAL AND OPHTHALMIC
DISEASE, IMAGING IN LIVING HUMAN
EYES, EXPLORED IN A NUMBER OF
DIFFERENT CONDITIONS INCLUDING
TRAUMATIC BRAIN INJURY, OPTIC
NEUROPATHY, RETINAL VAIN
OCCLUSION, DIABETIC RETINOPATHY
AND OTHERS.
NOW MOVING FURTHER INTO THE
RETINA WE'LL LOOK AT RETINAL
VESSEL AUTOREGULATION IN
GLAUCOMA.
SO THE RETINA IS CENTRAL NERVOUS
SYSTEM TISSUE, IT SHARES
CHARACTERISTICALLY HIGH
METABOLISM OF THE BRAIN,
HYPEROXIA IS A CHANGE IN BLOOD
OXYGEN PARTIAL PRESSURE THAT
ACTIVATES AUTOREGULATION, SO OUR
HYPOTHESIS ABERRANT BLOOD KNOW
PRECEDES STRUCTURAL CHANGES AND
CAN BE USED AS BIOMARKER AND TO
DETERMINE RETINAL VAST VASCULAR
FUNCTION.
WE HAVE AN OSLO SCAN, WHICH IS
SCANNING LASER, IN A LINE SCAN
MODE WHERE AT TENS OF KILOHERTZ
RATES TRACK INDIVIDUAL RED BLOOD
CELLS FLOWING THROUGH VESSELS.
YOU CAN SEE THE STREAKS, THE
ANGLE THE STREAK MAKES WITH
RESPECT TO THE SCANNER AND TO
VESSEL, GIVES QUANTITATIVE
MEASURE OF FLOW VELOCITY.
AND WE CAN PAIR THAT WITH OUR
ABILITY TO COLLECT VERY HIGH
RESOLUTION AOOCT VOLUMES WHERE
WE CAN EXTRACTS VESSEL DIAMETER
SO YOU CAN SEE HERE, WE CAN
RESOLVE THE VESSEL WALLS HERE
AND GET PRECISE MEASURES OF
VESSEL DIAMETER AND PUTTING
THOSE TWO TOGETHER ALLOWS US TO
DO QUANTITATIVE BLOOD FLOW IN
OUR SUBJECTS.
SO WE DO AN OXYGEN CHALLENGE
EXPERIMENT, WHERE WE GIVE THEM
OXYGEN SUPPLEMENTATION DURING
IMAGING AND RECORD VITALS AND
SPO2.
THE FIRST VALIDATION WE DID OF
THIS EXPERIMENT WAS A BRANCHED
VESSEL EXPERIMENT WHERE WE FOUND
A SUBJECT THAT HAD AN AREA WHERE
THEY HAD A PARENT AND TWO
DAUGHTER VESSELS, WE COULD DO
THE LINE SCAN APPROACH ON THIS
TO MEASURE THE VELOCITIES, AND
MEASURE THEIR DIAMETERS, AND WE
FOUND THAT THE PARENT, MEAN FLOW
RATE IN THE PARENT, WAS THE SUM
OF THE TWO DAUGHTER VESSELS TO A
HIGH DEGREE, WHICH SHOWED
CONSERVATION OF MEAN FLOW RATES.
NOW WE'RE TAKING THIS TO A
HEALTHY COHORT AND THEN TO OUR
GLAUCOMA SUBJECTS.
WE CAN SEE CARDIAC CYCLE AND
PARABOLIC FLOW ACROSS
VASCULATURE, PUTTING THOSE
TOGETHER WERE ABLE TO MEASURE,
ACCOMPANIED WITH
VASOCONSTRICTION IN ARTERIALS
AND VENTROLES, NO CHANGE IN MEAN
FLOW RATES WITH OXYGEN
SUPPLEMENTATION INDICATING
EFFICIENT AUTOREGULATION, ONLY
HAVE DONE ONE GLAUCOMA SUSPECT,
20% DECREASE IN MEAN FLOW RATE
SO WE'RE WORKING WITH A FULL
GLAUCOMA COHORT AND WE SHOULD
HAVE RESULTS THERE SOON.
NEXT I'D LIKE TO MOVE TO THE
PHOTORECEPTOR, NEW FUNCTIONAL
MEASURE IS CALLED
OPTORETINOPATHY, COROLLARY OF
MULTI-FOCAL ERG, TWO VARIETIES
OF OSLO OR AOOCT BUT MEASURING
THE SAME THING.
THE MORE RECENT DEVELOPMENT, YOU
HOOK AT PHASE CHANGES IN
SPECIFIC LAYERS OF THE RETINA,
ENTER SEGMENT, OUTER SEGMENT
JUNCTION, OPTI RETINOPATHY IS AN
ESTABLISHED TECHNIQUE.
A PROCESS WE'RE USING, WE
COLLECT VOLUMES ACROSS STIMULUS,
FROM AND REGISTER TO EACH OTHER,
CAN TRACK PHASE DYNAMICS AND
INDIVIDUAL CONES, SO AGAIN PHASE
CHANGE BETWEEN INNER AND OUTER
SEGMENT AND TIPS WE GET A
MEASURE OF THE OPTICAL PATH
LENGTH CHANGES, ELONGATION OF
PHOTORECEPTORS.
WE CAN CLASSIFY.
IF WE IMAGE A REGION WITH NO
OVERLYING VASCULATURE, APPLY
STIMULUS, SEE PHASE CHANGE WHICH
WE CAN EQUATE WITH OPTICAL PATH
LENGTH CHANGE FROM THE
650-NANOMETER STIMULUS LIGHT.
SO, USING THE PRINCIPLE
COMPONENT ANALYSIS CAN SEPARATE
L, M, S-CONES IN THIS PHASE
RESPONSE.
AND YOU CAN DO THIS WITH HIGH
ACCURACY, VERY LOW UNCERTAINTY.
SO THEN YOU CAN START TO APPLY
OTHER STIMULUS SOURCES.
WE HAVE A SUPER CONTINUUM LASER
SOURCE CHANNEL THAT'S COUPLED
INTO ADAPTIVE OPTICS SYSTEM, AND
YOU CAN LOOK AT DIFFERENCES IN
THESE WHERE IF YOU PROBE WITH
526 LIGHT AS EXPECTED YOU'D SEE
HIGHER RESPONSE IN M VERSUS L
CONES FOR 650-NANOMETER LIGHT.
CAN YOU PLOT THESE ON A
PRINCIPAL COMPONENT CURVE AND
SEE CLUSTERING OF INDIVIDUAL
CONES AND CONE CLASSIFICATION IS
FINE BUT WHAT WE WANT TO USE
THIS FOR IS LOOKING AT RETINAL
DEGENERATION AND CONE
PHOTORECEPTOR FUNCTION.
WE CAME ONE A MODEL TO SORT OF
REPRESENT THIS IN A VECTOR
REPRESENTATION WHERE THE ANGLE
FROM THE -- VECTOR FROM ORIGIN
TO CENTROID OF THOSE M, L,
S-TYPE CONES GIVE A MEASURE OF
AMPLITUDE, AND ANGLE IS SPECTRAL
RESPONSE.
IF WE LOOK AT A SUBJECT THAT HAS
HEALTHY EYES, NO DIAGNOSED
RETINAL DISEASE, AND LOOK AT
AREAS OF DRUSEN, YOU CAN SEE ALL
THE WAY ACROSS THE MACULA, NICE
RESOLUTION OF PHOTORECEPTORS, IN
DRUSEN LOOK AT CROSS-SECTIONAL B
SCAN, DRUSEN IS EVIDENT IN BOTH
VIEWS, WORKING WITH DUKE
UNIVERSITY ON A MACHINE LEARNING
ALGORITHM TO MEASURE THE CONE
OUTER SEGMENT LENGTHS, HAVE DONE
THAT AS WELL, BUT THE IMPORTANT
THING IS THE CONES ABOVE THE
DRUSEN RETAIN THEIR PAIRED
REFLECTION SO WE'RE ABLE TO
MEASURE PHASE CHANGES IN THESE
DRUSEN REGIONS.
IF WE LOOK IN THE VOLUME AT RPE
LAYER, WE CAN SEE REGIONS HERE
WHERE THE RPE CELLS ARE
RETAINED, BUT THE MAJORITY OF
THE DRUSEN AREA HAS THIS
HETEROGENEOUS SCATTERING
FEATURE.
AND WE CAN ALSO LOOK AT THE
CHORIOCAPILLARIS, YOU CAN SEE IN
THE TEMPORAL PART OF THE MACULA,
RELATIVELY HEALTHY NORMAL
VASCULATURE AND CLOSER TO THE
NASAL REGION YOU SEE DISRUPTION,
MOST IMPORTANTLY IN THE REGION
AROUND THE DRUSEN YOU CAN ALSO
SEE DISRUPTION OF THE RETINAL
VASCULAR NETWORK THAT SUPPLIES
PHOTORECEPTORS AND RPE.
LOOKING AT THE CHANGES IN THESE
LOCATIONS NEAR THE DRUSEN, THIS
IS THE FIRST LOCATION, AND IF
YOU LOOK AT THE ADJACENT
NON-DRUSEN REGION YOU CAN PLOT
OUT THE VECTORS AND THEN WHEN
YOU APPLY OUR ANALYSIS AND LOOK
AT THE REGIONS AND FURTHER APPLY
THE VECTORS THERE YOU SEE
THERE'S A TENDENCY OF A WEAKER
RESPONSE IN THE DRUSEN REGION
AND ALSO SMALLER ANGLE, AND THIS
MAY INDICATE THERE'S A SPECTRAL
SENSITIVITY SHIFT THAT'S CAUSED
BY A CHANGE IN THE WAVE GUIDING
PROPERTIES IN THESE CELLS.
WE'VE DONE THIS FOR ANOTHER
REGION IN THE SAME EYE, AND THEN
IN A FEW SUBJECTS FOR REGIONS
WE'VE SEEN SIMILAR RESULTS,
WEAKER RESPONSE IN DRUSEN
REGION, MEASURABLE REDSHIFT IN
THE DRUSEN REASON, SO WE THINK
WE'RE UNDERWAY EXPLORING THIS IN
ADDITIONAL SUBJECTS WITH DRUSEN
AND WE THINK THIS IS A SUITABLE
FOR A BIOMARKER OF EARLY AMD.
LET'S MOVE INTO THE RPE CELLS
AND LOOK AT ORGANELLE MOTILITY.
FIRST WITH OUR ADAPTIVE OPTICS
SYSTEMS WE'RE ABLE TO DO
HIGH-SPEED IMAGING AND COLLECT
VOLUMES ACROSS THE TEMPORAL
MACULA, IN THIS SUBJECT HERE
THIS HEALTHY VOLUNTEER, THE
FOVEA IS HERE, GOING INTO THE
TEMPORAL MACULA, YOU CAN SEE
PHOTORECEPTOR CHANGES US A RECEN
THE PERIPHERY, ALSO THE DOTS,
ABLE TO QUANTIFY CONE DENSITY
AND CAN YOU DO THE SAME THING
WITH RPE CELLS SO WE GET A NICE
PICTURE OF THE RPE CELLS ACROSS
THE MACULA AND FOUND THAT THEIR
DENSITY CHANGES THOUGH NOT AS
MUCH AS CONE PHOTORECEPTORS, THE
RATIO OF CELLS THAT SERVES EACH
CONE PHOTORECEPTOR GOES DOWN
APPRECIATABLY AS YOU MOVE IN
ECENTRICITY.
IN THESE RPE CELLS, THERE'S
MITOCHONDRIA.
MELANOSOMES MOVING AROUND, A
SOURCE OF CONTRAST.
LOOKING AT INDIVIDUAL SINGLE
VOLUME FROM PHOTORECEPTORS CAN
SEE PHOTORECEPTOR LAYER BUT IN
THE RPE CELLS WE JUST SEE THIS
SPECKLE NOISE, BUT WHAT WE CAN
DO IS LOOK AT THIS OVER TIME AND
THEN WE GET A VERY CONSTANT
PHOTORECEPTOR PICTURE BUT THIS
BOILING OF THE TISSUE, NOT
REALLY BOILING BUT LOOKS LIKE
IT'S BOILING, DECORRELATED
LAYINGS IN SPECKLE PATTERN WHICH
WE CAN USE TO CREATE AVERAGE
IMAGES, DECOR DECOR -- DECORRE,
LOOKING AT INDIVIDUAL CELL OVER
TIME, WE'RE ABLE TO CALCULATE
THE CROSS-CORRELATION FUNCTION,
COMPARED TO LAYERS THAT ARE MORE
STABLE OR THAT DON'T HAVE ANY
DE-CORRELATION.
WHEN WE DO THIS WE GET A
CROSS-CORRELATION COEFFICIENT
THAT WE CAN FIT TO EXPONENTIAL
CURVE AND TIME CONSTANT OF WHICH
IS THE BIOMARKER, MOTILITY TIME
CONSTANT, AND SEVEN SUBJECTS AND
EARLY STUDY WE FOUND TIME
CONSTANT WAS ABOUT FIVE SECONDS.
AND THAT ALSO PUTS A LIMIT ON
HOW WE DO OUR IMAGING, WE HAVE
TO SEPARATE OUR VOLUMES BY FIVE
SECONDS IN ORDER TO ALLOW
DE-CORRELATION TO OCCUR TO GET
THE RPE-ENHANCED CONTRAST.
SO, WE'RE USING THIS TECHNIQUE
IN SOME KATHY KUKRAS' SUBJECTS
BUT IN INITIAL STUDY IN HEALTHY
SUBJECTS WANTED TO EXPLORE
SOURCE OF ORGANELLE
DE-CORRELATION MORE AND LOOKED,
WITH THE VERY HIGH CAPABILITIES
OF OCT LOOK AT APICAL, MEDIAL,
BASAL REGIONS, YOU CAN SEE THESE
REGIONS SEPARATED BY ABOUT 4
MICRONS, IF YOU DO AN AVERAGE OF
THESE VOLUMES YOU SEE THE RPE
CELLS ARE CLEARLY DELINEATED,
BUT IF YOU LOOK AT SINGLE
VOLUMES THERE'S DIFFERENT
SPECKLE PATTERN IN EACH OF THEM
AND COMPARING THAT TO THE SIGNAL
FROM BROOKS MEMBRANE YOU CAN SEE
THE DIFFERENCES THERE.
WHEN WE DID THIS AND DID OUR
MEASUREMENT WE FOUND IN APICAL
REGION WE GET LONGER TIME
CONSTANT, IN BASAL REGION WHERE
THERE'S MITOCHONDRIA WE GET MUCH
SHORTER TIME CONSTANT, MORE
ACTIVITY IN THE MITOCHONDRIA AND
BASAL REGION.
AND THAT'S JUST HISTOOF THE RPE
CELL, YOU CAN SEE THE
MELANOSOMES, NUKE NUCLEUS,
MITOCHONDRIA.
WE'LL MOVE TO THE
CHORIOCAPILLARIS.
THIS IS A COLLECTION OF SINGLE
FOLLOWS AND COLLECTED IN 10
MINUTES, THIS IS AN AOOCTA SCAN
FROM HEALTHY VOLUNTEER, WE CAN
RESOLVE THE CHORIOCAPILLARIS, A
DENSE CAPILLARY NETWORK JUST
BELOW THE PHOTORECEPTORS.
AND THEN YOU CAN TAKE A LOOK AT
THE VASCULAR NETWORK ACROSS THIS
REGION, NICE DELINEATION OF
VOIDS AND VESSELS.
WE STARTED TO EXPLORE THIS IN
KATHY'S SUBJECTS, THIS IS A
SUBJECT WITH CONE ROD DYSTROPHY.
YOU CAN SEE THEY HAVE A CENTRAL
LESION OF ABOUT 7 OR 8 DEGREES
WHERE THERE'S A DISRUPTION IN
THE PHOTORECEPTOR AND RPE CELLS.
THE PATIENT INTERESTINGLY HAD
VERY GOOD CENTRAL VISUAL ACUITY.
SO IF WE TAKE A LOOK AND DO THE
OCT SCANS ACROSS THE MACULA IN
THIS SUBJECT, YOU CAN SEE THE
BROAD OUTLINES OF THE LESION
HERE, AND IN THE PARAGRAPH RAIL
D. - PERIPHERAL REGION, WE CAN
SEE HYPER REFLECTIVE
PSEUDODRUSEN, WERE ABLE TO
MEASURE PHOTORECEPTORS IN THE
CENTRAL LESION WHICH EXPLAINS
THEIR GOOD VISUAL ACUITIY.
AND WE CAN APPLY OUR AO-OCTA
PROCESSING, AND YOU NOTICE
IMMEDIATELY THE CHANGE IN
VESSELS IN THE LESION REGION
ABOUT THINNER CHORIOCAPILLARIS
VESSELS, UNCLEAR WHETHER THESE
ARE ACTUALLY THINNER VESSELS OR
FROM SCATTERING CHANGES.
WE'VE QUANTIFIED ALL OF THIS AND
COMPARED THE CONE-ROD DYSTROPHY
TO HEALTHY VOLUNTEER, FURTHER
QUANTIFICATION, HEALTHY
VOLUNTEER DENSITY IS CONSISTENT
ACROSS THE MACULA, YOU SEE
CHANGES IN VESSEL DENSITY WHERE
THE LESION IS IN CONE-ROD
DYSTROPHY, SIMILARLY WITH THE
VESSEL DIAMETER THERE MAY BE
SOME CHANGES IN VESSEL
TORTUOSITY, AND THEN WE CAN LOOK
AT REGIONS IN BETWEEN
VASCULATURE AND SEE CLEAR
DIFFERENCES IN THE DISEASE EYE
COMPARED TO HEALTHY EYE.
HEALTHY VOLUNTEERS HAVE
CONSISTENT VALUES, CONSISTENT
VASCULAR NETWORK AND THESE
CHORIOCAPILLARIS METRICS ARE
EXTREMELY SENSITIVE TO VASCULAR
CHANGES SO THEY MAKE VERY GOOD
BIOMARKERS.
I'LL STOP THERE AND THE FDA
MISSION IS TO ENABLE GREATER
PATIENT ACCESS TO INNOVATIVE
TECHNOLOGIES WHICH YOU AT THE
NIH COMPLETELY UNDERSTAND.
WE'VE ESTABLISHED ADAPTIVE
OPTICS REGULATORY SCIENCE
PROGRAM IN OUR OFFICE AND OUR
LABS ARE EQUIPMENT WITH TWO
STATE OF THE ART MULTI-MODAL AO
SYSTEMS THAT SEARCH AS PLATFORM
FOR CLINICAL STUDIES WITH A
NUMBER OF COLLABORATORS AT NIH
NEI, DEMONSTRATING AND DEVELOP
AO-BASED BIOMARKERS IMPORTANT IN
VISION SCIENCE STUDIES TO BETTER
UNDERSTAND RETINAL METABOLISM
AND FUNCTION, BUT ALSO FOR
LOOKING AT OPHTHALMIC AND
NEUROLOGICAL DISEASE DIAGNOSIS
AND TREATMENT.
I'LL STOP AND TAKE ANY
QUESTIONS.
[APPLAUSE]
>>HAVE YOU LOOKED AT
CORRELATION BETWEEN VESSEL
DIAMETER OR DENSITY AND
MITOCHONDRIAL FUNCTION?
>>NOT YET.
BUT THAT'S A GREAT QUESTION AND
A GREAT IDEA.
THAT'S SOMETHING WE WANT TO
BEGIN TO EXPLORE.
BUT WE HAVEN'T DONE THAT YET.
YOU CAN TELL WE'RE ON THE CUSP
OF GREAT ADVANCES BUT JUST GOT
ALL THESE TECHNIQUES PERFECTED,
STILL IN THE INITIATION STAGE
AND SORT OF EARLY DEVELOPMENT
STAGE OF THEM, AND THERE'S A LOT
OF WORK THAT GOES INTO DOING --
VALIDATING A BIOMARKER AND DOING
REPRODUCIBILITY STUDIES BUT
THAT'S A GREAT IDEA.
>> GOING BACK TO THE BEGINNING,
YOU MENTIONED A NEED FOR
PHANTOMS, I WONDERED IF YOU ARE
COMBINING MEASUREMENTS ON
PHANTOMS WITH ANY OF THESE
PATIENT MEASUREMENTS OR TO
CHARACTERIZE THE SYSTEMS AND
WHAT YOUR NEEDS ARE AROUND
THOSE.
>>SO, WE'RE DOING A COUPLE OF
THINGS.
ONE, SO THE PHANTOMS THAT WE'RE
DEVELOPING FIRST LOOKING AT
RESOLUTION.
I HAVE A SORT OF A VISION TO
MAKE THE PHANTOMS A LITTLE MORE
DYNAMIC AND BE ABLE TO BE LIKE
STRESS TEST FOR OUR AO SYSTEMS,
I TALKED ABOUT HOW IT'S A
PERSONALIZED DEVICE THAT'S
CHANGING WITH EACH SUBJECT
THAT'S IN FRONT OF IT.
RIGHT NOW WE HAVE A NUMBER OF
RESOLUTION PHANTOMS THAT
BASICALLY MIMIC THE
PHOTORECEPTOR DENSITY AND WE'RE
USING THOSE ON A NUMBER OF
SYSTEMS INCLUDING THE ONES IN
JOHNNY'S LAB BUT ALSO IN SOME
MULTI-SITE CLINICAL STUDIES SO
THE IDEA THERE TO GET
STANDARDIZED MEASUREMENTS ACROSS
ALL OF THE INSTRUMENTS THAT ARE
IN A MULTI-SITE CLINICAL STUDY,
IN COLLABORATION WITH JACKIE
DUNCAN UC-SAN FRANCISCO AND JOE
CARROLL AT MARQUETTE, SO THEY
HAVE A LARGE STUDY AT A
NUMBER -- I THINK FIVE OR SIX
SITES, WE SEND OUR PHANTOMS
THERE, THEY TEST THEIR DEVICES,
MAKE SURE THEY ARE STANDARDIZED
IN TERMS OF WHAT THE
MEASUREMENTS AND QUANTIFICATION
YOU GET OUT OF THEM, AND THEN
THEY HAVE MORE CONFIDENCE IN THE
NUMBERS THEY GET FROM THEIR
CLINICAL STUDIES THAT CAN BE
COMPARED ACROSS SITES.
>>WHEN YOU THINK ABOUT DYNAMIC
ARE YOU THINKING ABOUT SOFT
MATERIALS YOU CAN STRESS IN REAL
TIME TO GET THEM TO FLEX?
>>WELL, THERE'S A NUMBER OF
DIFFERENT ADAPTIVE LENSES, SO
IT'S PROBABLY SIMILAR TO WHAT
YOU'RE TALKING ABOUT, SENSES YOU
CAN SHAPE AND FOCUS, AND PUTTING
AN ARBITRARY FOCUS PATTERN OR
ANY ABERRATIONS ON A LENS AND
THEN SEEING HOW THE SYSTEM
RESPONDS TO THAT AND IS ABLE TO
CORRECT FOR THAT, THAT'S THE
IDEA.
>> THANK YOU.
>> YOU'RE PROBABLY MORE
FAMILIAR WITH FORMAL LENSES THAN
I AM, THE MATERIAL THEY USE.
>> I WAS THINKING ABOUT THE
PHANTOMS, HOW YOU MAKE A DYNAMIC
PHANTOMS FOR STRESS TEST.
>> THERE'S IDEAS WILL YOU TO
MAKE FLOW PHANTOMS TOO, THAT'S
MORE DIFFICULT.
AGAIN, THAT'S SOMETHING WE'RE
EXPLORING.
ALL RIGHT.
THANK YOU VERY MUCH.
[APPLAUSE]
>> DR. MANU PLATT IS DIRECTORY
OF NIH-WIDE CENTER, ALSO CHIEF
OF A LAB, I'LL LET HIM DECOMPOSE
THAT ACRONYM.
>> THANKS FOR HAVING ME.
THANKS TO ALL THE ORGANIZERS AND
THANKS TO MY NEW COLLEAGUES AT
THE NIH.
LOOK, MA, I'M PRESENTING AT THE
NIH!
NO, IT'S BEEN WONDERFUL GETTING
THE CENTER OFF THE GROUND AND
TALKING TO PEOPLE ABOUT WHAT
WE'RE TRYING TO DO AND WHAT YOU
ALL DO.
IT'S GREAT TO COME HOME AND TALK
ABOUT MY OWN RESEARCH WHICH
FEELS REALLY GREAT.
YES, I'M THE DIRECTOR OF THE NEW
BETA CENTER, RUNNING MY OWN LAP
HERE.
MATRICES LAB, THERE'S GREAT
SICKLE CELL RESEARCH AT NIH.
TO PLUG MY LAB WE DO MORE THAN
SICKLE CELL DISEASE BUT I SPEND
TIME TRYING TO UNDERSTAND WHY
CHILDREN ARE AT INCREASED RISK
OF STROKES, BUT WE CARE ABOUT
THE EXTRACELLULAR MATRIX ME
MODELING AND PROTEOLYTIC
CHANGES, TO ALSO UNDERSTAND SOME
OF THESE THINGS HAPPENING IN
THESE INDIVIDUALS WITH SICKLE
CELL, MY LAB IS USING LABEL-FREE
MAGNETIC RESONANCE IMAGING, BUT
WE ALSO MERGED THAT WITH
COMPUTATIONAL FLUID DYNAMICS AND
HEMO DYNAMICS ALSO LINKED TO
BIOLOGICAL RESPONSES.
CHECK IT OUT IF THIS IS
INTERESTING.
TODAY WE'LL TALK ABOUT SINGLE
CELL DISEASE, MULTI-FACTOR
ZEALS.
THERE'S ONE NUCLEOTIDE CHANGE
FROM A TO T, THAT CHANGES ONE
AMINO ACID, GLUTAMIC TO VALENE,
HEMOGLOBIN ONLY EXPRESSED IN ONE
TYPE OF CELL, RED BLOOD CELLS.
WHAT'S THE PROBLEM?
RED BLOOD CELLS GO EVERYWHERE.
AND THAT'S WHERE YOU GET THIS
SYSTEMIC DAMAGE.
EVEN TODAY SICKLE CELL IS NOT
CURED, LIFE EXPECTANCY IS LOWER,
36 FOR MEN, 42 FOR WOMEN,
THERE'S EVEN A SEX DIFFERENCE IN
LIFE EXPECTANCY, AND A HEALTH
DISPARITY IN THE UNITED STATES.
ONE IN 400 AFRICAN AMERICANS IS
HOMOZYGOUS, AND 1 IN 12 ARE
CARRIERS.
THERE'S 100,000 AMERICANS IN THE
U.S. LIVING WITH SICKLE CELL
DISEASE BUT GLOBAL LIP MILLIONS.
YOU CAN SEE THAT SUB-SAHARAN
AFRICA IS MAJOR, INDIAN, ARABIC
WORLD, NORTH AND SOUTH AMERICA
AS REMNANTS OF CHATTEL SURGERY.
90% OF GLOBALLY DIE IN THE FIRST
FIVE YEARS OF LIFE, MAKING IT A
DIFFICULT PROBLEM TO QUESTION,
TUGGING AT MY HEART STRINGS.
MOLECULAR ORIGINS WE'VE KNOWN
FOR YEARS, IDENTIFIED CLINICALLY
IN 1910, WE'VE BEEN STUDYING
THIS FOR MORE THAN 113 YEARS.
FROM THE HYDROPHILIC TO VALENE
IT EXPOSES THAT VALENE TO
SURFACE OF MOLECULE, VALENE IS
HYDROPHOBIC, ALL THE HEMOGLOBINS
SEQUESTER AND SELF-ASSEMBLE.
IF YOU'RE A POLYMER CHEMIST,
GREAT QUESTIONS, CREATING A
STIFF FIBER THAT DEFORMS THE RED
CELLS AS YOU CAN SEE IN THE
VIDEO, WE TOOK IN MY LAB UNDER
THE OXYGENATION CONDITIONS.
AND BECAUSE THEY ARE RED BLOOD
CELLS, EFFECTED NUCLEUS, RIGID
PART, THIS IS DEFORMABLE.
DEOXYGENATED CONDITIONS WE SEE
THIS HAPPENING.
YOU IN THE CAPILLARIES WHERE THE
CELLS RELEASE OXYGEN AND START
TO TAKE ON CO2 YOU BEGIN TO GET
SICKLING AND THESE
VASOOCCLUSIONS AND BLOCKAGES IN
POST-CAPILLARY VENULES, LEADING
TO CRISIS AND PAIN.
WHEN I STARTED MY LAB AT GEORGIA
TECH, I FOUND OUT CHILDREN WITH
SICKLE CELL WERE AT INCREASED
RISK OF STROKE.
MY Ph.D. WAS CARDIAC DISEASE,
ATHEROSCLEROSIS, IN OXYGENATED
BLOODS CREST ALSO.
DEOXYGEN RATED WHEN THE GET RED
CELL SICKLING, WHAT'S HAPPENING
HERE?
THERE ARE STUDIES SHOWING THE
TIME FOR THE SICKLE POLYMER TO
MELT IS LONGER THAN THE TIME TO
TRAVERSE THROUGHOUT THE
OXYGENATED BLOODSTREAM SO
STARTED LOOKING AT PUTTING
TOGETHER SOME INTERESTING
PARADIGMS ABOUT WHAT SICKLING
CELLS AND WHAT DAMAGE TO LARGE
ARTERIES.
11% OF CHILDREN WILL HAVE A
STROKE, A MAJOR STROKE, BEFORE
AGE 16, BUT UP TO A THIRD OF
THESE WILL HAVE WHAT WE CALL
SILENT STROKE, IT DOESN'T
PROGRAM BUT UNDER MR, YOU SEE
DAMAGE TO THE BRAIN.
SILENT STROKES AFFECT QUALITY OF
LIFE, LOWER ACADEMIC ACHIEVEMENT
FOR CHILDREN WITH SICKLE CELL,
PARTIALLY BECAUSE OF THE STROKES
AND DAMAGES TO THEIR LESIONS,
HELD BEHIND.
BUT AS THE PATIENTS AGE, AGAIN
HUGE RISK FOR CHILDREN TO HAVE
ISCHEMIC STROKE BUT AS THEY AGE
THEN THE RISK INCREASES FOR THEM
TO GO FROM ISCHEMIC TO
HEMORRHAGIC, BY TWENTIES AND
30s BACK TO INFARCTIVE AGAIN.
WHAT'S HAPPENING WITH THIS
AGE-RELATED DIFFERENCES?
WE START TO EQUATE WITH MOUSE
AGE FOR INTERVENTIONAL STUDIES.
MY OLD WORK WAS ENDOTHELIAL
BIOLOGY, SHEER STRESS, LOOKING
AT SICKLE CELL THERE WAS AMAZING
CLINICAL TRIAL CALLED THE STOP
TRIAL BY BE ON BOB ADAMS,
TRANSCRANIAL DOPPLER, INTERNAL
CAROTID ARTERY, MIDDLE CEREBRAL
AND INTERIOR CEREBRAL ARTERY IF
GREATER THAN 200 CENTIMETERS PER
SECOND AT INCREASED RISK OF
STROKES, PUT THEM ON MONTHLY
BLOOD TRANSFUSIONS AND THIS IS A
CLINICAL TRIAL THAT ENDED EARLY
BECAUSE IT WAS OBVIOUS CHILDREN
WITH BLOODED TRANSFUSIONS WERE
BEING PROTECTED AND THOSE
WITHOUT WERE HAVING STROKES
AND/OR DYING, A GREAT FINDING.
YOU CAN IDENTIFY WHICH CHILDREN
ARE AT GREATEST RISK FOR
STROKES.
WELL, IF YOU START TO HAVE
INCREASED BLOOD FOE YOU CAN HAVE
STENOSIS, WHY MAY BE THERE
STENOSIS EXCEPT WHEN THEY WERE
DOING MAGNETIC RESONANCE
ANGIOGRAMS YOU DIDN'T ALWAYS
DETECT STENOSIS WHERE YOU WOULD
GET INCREASED BLOOD FLOW, LOT OF
CELLS ABOUT WHAT ARE THE RED
CELLS DOING, THEY ARE ADHESIVE
AND CAN STICK TO THE WALL OF THE
ARTERY, AND MORE TRANSIENT IN A
LUMINAL REMODELING FOR PLAQUE
AND SICKLE CELL.
YOU WOULD GET THIS DOWNSTREAM
REGION OF FLOW REVERSAL
EXACERBATING PATHWAYS LEADING TO
SICKLE CELL CAUSING THIS
ARTERIAL DAMAGE.
FOR THOSE THAT DON'T STUDY
CARDIOVASCULAR AND HEMODYNAMICS,
PATHOLOGIST WAY BACK IN THE
'60s IDENTIFIED WHERE PLAQUES
FORM, REGIONS OF BIFURCATIONS,
CALLED HIS ENGINEER BUDDY, MY
FORMER DEAN DOWN AT GEORGIA
TECH, THEY SHOWED AT THESE
REGIONS OF BIFURCATION YOU GET
REGIONS OF FLOW SEPARATION AND
FLOW REVERSAL WITH CARDIAC
CYCLE.
THIS IS WHERE A PLAQUE WOULD
FORM IN THE ARTERY.
WHEN YOU GO INTO THE BRAIN,
INTERNAL CAROTID ARTERY BRANCHES
LIKE CRAZY INSIDE THE BRAIN,
THERE'S THESE POINTS WHERE YOU
COULD HAVE REGIONS OF BLOOD
FLOW.
IN AUTOPSY, IN THIS 5-YEAR-OLD
YOU CAN SEE THE LUMINA IS CLOSED
OFF, FRAGMENTED ELASTIC LAMINA,
DAMAGED LAYERS.
WHICH WE KNOW WHEN YOU BREAK
ELASTIN THEY CHANGE PHENOTYPE,
BECOME PROLIFERATIVE, ALL THESE
THINGS COULD HAPPEN DUE TO
DAMAGE TO THE EXTRACELLULAR
MATRIX.
I LOVE THESE ENZYMES, PROTEASES.
CYSTEINE CATHEPSINS, THE ARTERY
WANTS TO KEEP THAT LUMEN OPEN AS
LONG AS POSSIBLE UNTIL THAT
PLAQUE ENCROACHES TOO FAR.
WE START LOOKING AT CATHEPSINS,
REGULATED BY SHEER STRESS
PUTTING ON FLOW REMODELING
ISSUES HAPPENING IN SICKLE CELL
DISEASE, AND WE GOT A STUDY,
RIGHT?
A LOT OF BIOMEDICAL FRIENDS
STUDY MATRIX PROTEINASES, I'M
HAPPY FOR YOU, GREAT WORK THAT'S
BEEN HAPPENING, BUT THE CYSTEINE
CATHEPSINS ARE MORE POWERFUL AS
THEY TURN OVER SUBSTRATES FASTER
AND ARE TURNED OFF OR LOW LEVELS
BUT GET TURNED ON AND UP UNDER
DISEASE.
BIG PHARMA HAS BEEN CHASING
INHIBITORS, BUT 16 MADE IT, 16
FAILED.
WE'RE STILL LOOKING FOR DRUGS TO
INHIBIT THESE ENZYMES TO STOP
THAT REMODELING.
ALL OF THAT IS BACKGROUND TO
SHOW WE WERE TAKING THIS
MULTI-SCALE APPROACH WHERE WE
CARE ABOUT ENZYMES BEING
ELEVATED, HOW ARE THEY BEING
INFLUENCED, AS WELL AS
INFLAMMATION IN THE BIOCHEMISTRY
THAT LEADS TO THEIR
UPREGULATION, THAT WILL REMODEL
THAT ARTERY THAT COULD LEAD TO
THIS PATHOPHYSIOLOGY OF A
STROKE.
HOPEFULLY WE FIND OTHER
BIOMARKERS ALONG THE WAY FOR
OTHER DRUG TARGETS.
SO, WORKING WITH RUSSELL AT
SAINT ST.
JUDE'S MY FORMER STUDENT WAS
ABLE TO TAKE ANGIOGRAMS FROM
CHILDREN WITH SICKLE CELL,
RECREATED IN SILICO,
COMPUTATIONAL FLUID DYNAMICS TO
LOOK AT WHERE ARE THESE POSSIBLE
REGIONS OF STENOSIS OR
FLOW-MEDIATED CHANGES CAUSING
INCREASED BLOOD VELOCITIES, IT
WASN'T OBVIOUS.
AND IN DOING SO WE IDENTIFIED
THAT THE GEOMETRY ALONE, ANATOMY
OF THE CHILDREN IS QUITE
DIFFERENT.
YOU CAN SEE THE SAME INPUT IN
THE ICA BUT IN THE SICKLE
VESSELS MCA, IT'S GOING UP.
THERE'S THESE BUMPS IN THE
VESSELS, WE CALL THEM BUMPS,
MICROFEATURES TO MAKE IT SEEM
FANCY, YOU CAN IMAGINE FOR
MAGNETIC RESONANCE ANGIOGRAM
THIS IS THE NEGATIVE OF THE
BLOOD VESSEL.
I WAS UPSET.
CHRIS, DID YOU NOT SMOOTH IT?
HE FOLLOWED THE SAME ALGORITHMS
FOR NON-SICKLE AND SICKLE BUT
WHEN WE THEN LOOKED AT REGIONS
OF FLOW SEPARATION AND FLOW
CIRCULATION WE SAW IN THE SICKLE
CELL INDIVIDUALS, FROM HUMANS,
AGAIN THOSE ARE THE SITES WHERE
WE ACTUALLY SAW REGIONS OF FLOW
SEPARATION AND RECIRCULATION
WITH THE CARDIAC CYCLE AND DID
NOT SEE THAT IN INDIVIDUALS
WITHOUT SICKLE CELL AT THAT
CRITICAL JUNCTION.
THE ANATOMY IS DIFFERENT, WE
CARE ABOUT SUBSTRUCTURAL
CHANGES.
WE CAN'T LOOK IN HUMANS, I
APPLAUD ALL OF YOU, BUT
UNIVERSITY OF ALABAMA BIRMINGHAM
DEVELOPED THIS SICKLE CELL
TOWNES TRANSGENIC MOUSE, KNOCKED
OUT HOUSE HEMOGLOBIN AND KNOCKED
IN THE HUMAN, FOLLOWING
MENDELIAN GENT IS.
SS IS HOMOZYGOUS.
YOU'LL SEE WE NORMALLY COMPARE
WITH AS FOR CONTROL BECAUSE
THESE ARE LITTER MATE CONTROLS.
THE FIRST THING WE WANTED TO DO
EARLY ARTERIAL DAMAGE TO THE
CAROTID ARTERIES IN THESE MICE,
WE DID CAROTID BECAUSE MICE ARE
SMALL, SO RIGHT AT ONE MONTH AT
WEANING WE ISOLATED CAROTID
ARTERIES, WE WERE ASTOUNDED,
ALREADY SEEING A NUMBER OF
ELASTIN BREAKS BY THREE WEEKS,
NO INTERVENTION, JUST SICKLE
CELL DISEASE.
AND THEN I'M SKIPPING THROUGH
THINGS, ALSO LOOKED AT PATHWAYS
UPREGULATING CATHEPSIN
REGULATION.
SO JNK, WE BEGAN TO INJECT MICE
FROM ONE TO THREE MONTHS WITH
JNK INHIBITOR TO STOP EXPRESSION
AND STOP ARTERIAL REMODEL.
THIS IS AN EXCITING RESULT,
SEVERAL YEARS AGO, THERE'S A
GENE DOSING EFFECT, SS,
IMMUNOHISTOCHEMISTRY, INCREASED
CATHEPSIN K IN CAROTID ARTERY
WALLS, WHEN THEY RECEIVED IP
INJECTIONS OF THE JNK INHIBITOR
THAT PRESERVED ARTERY STRUCTURE,
OUT TO FIVE MONTHS, INJECTED
DAILY, ISCHEMIC STROKES EARLY
ON, HEMORRHAGIC STROKES, BACK TO
ISCHEMIC, WE'RE SEEING THIS
INCREDIBLE REMODELING OF THE
SICKLE CELL ARTERIES BY FIVE
MONTHS THAT, AGAIN, JNK
INHIBITION WAS ABLE TO BLOCK
THAT REMODELING.
WE CARED ABOUT ELASTIC BREAKS
FROM THE AUTOPSIES IN CHILDREN
BUT THE QUESTION WAS WHAT IS
HAPPENING?
MY WONDERFUL STAFF SCIENTIST
HANNAH SUNG ACTUALLY BEGAN TO DO
NASCENT STAINING, BY FIVE MONTHS
COLLAGEN INDICATED BY BLUE
STAINING IS KIND OF GONE IN THAT
SICKLE CELL CAROTID ARTERY,
HOMOGENIZED TO QUANTIFY COLLAGEN
IN THE WALL, IF YOU GIVE THEM
THE JNK INHIBITOR YOU CAN
PROTECT COLLAGEN IN THE VESSEL
WALL.
SO THERE'S A PICTURE OF HER,
BECAUSE THERE'S SO MUCH
VARIABILITY, 11% OF CHILDREN
HAVING A MAJOR STROKE, A THIRD
SILENT STROKE, THAT WAS
REFLECTED IN FLUCTUATION AMONG
OUR MICE.
HANNAH FOUND THIS METHOD OF
USING LABEL-FREE MAGNETIC
RESONANCE ANGIOGRAM, WE WANTED
TO DO LONGITUDINAL SCANS, SCAN
OVER A PERIOD OF TIME.
SOMETIMES THEY DIE BUT WE WANTED
TO MINIMIZE VARIABILITY AND BE
ABLE TO TRACK IT FOR INDIVIDUAL
ANIMALS.
SO AGAIN THE BENEFITS,
NON-INVASIVE, GOOD SIGNAL
WITHOUT CONTRAST, BETTER WITH
CONTRAST, WE'RE HOPE TO THINKING
ABOUT IT.
I WOULD SAY I'VE BEEN DOING IT
FOR 13 YEARS, BUT SOME REVIEWS I
USED TO GET FROM MY WONDERFUL
NIH REVIEWERS WAS THAT MICE
NEVER SHOWN TO HAVE A STROKE, NO
ONE SHOWS IT HAVING A STROKE,
WHY SHOULD I BELIEVE YOU'RE
SEEING A STROKE IN THE MICE?
THAT'S NOT FAIR.
THAT'S WHETHER WE WENT WITH
LONGITUDINAL SCANS TO START
EXACERBATED ARTERIAL DAMAGE AND
SHOW PROGRESSING OVER TIME.
LOOKING AT TEN BRAINS THREE
MIGHT HAVE ABNORMALITIES IN
SCAN, BRAIN SCAN.
HOW CAN WE FOLLOW INDIVIDUAL
ANIMALS?
SAME MICE AT ONE AND THREE
MONTHS.
AS THEY GROW THE CROSS-SECTIONAL
AREA IS LARGER, BUT YOU SEE
VARIATION AMONG OUR SICKLE CELL
ANIMALS, WIDE VARIABILITY.
A NEW GRAD STUDENT HAS TAKEN
THIS ON, DOING SCANS OF OUR
SICKLE CELL MICE, AND WILL
EXPORT DATA AND RECREATE THE
VESSELS USING MIMICS.
WE'RE FOLLOWING THE SAME ANIMAL
ONE, THREE, FIVE MONTHS, SHE
FINISHED SEVEN MONTH SCANS AS
WELL, NOW QUANTIFYING FOR SINGLE
MOUSE.
I'M SHOWING AGGREGATED DATA
BECAUSE WE'RE STILL FINDING BEST
WAYS TO TRACK INDIVIDUAL MICE.
THIS IS N OF 18 WHERE WE'RE
SEEING THAT THERE'S AN INCREASE
IN THE CAROTID ARTERY SIZE IN
SICKLE CELL ANIMALS, MORE
SIGNIFICANT IN MALES THAN
FEMALES, EVEN AS WE SEE IN DATA
HERE.
WHAT WE'VE BEEN THINKING ABOUT
IS THE ONLY FUNCTIONAL CURE FOR
SICKLE CELL IS BONE MARROW
TRANSPLANT OR HEMATOPOIETIC STEM
CELL TRANSGENIC PLANT, STILL
WITH SOME COMPLICATION OF
CEREBRAL INFARCTION, WONDERING
IF WE COULD SEE THAT IN THE
MOUSE MODEL EVEN AFTER
CORRECTING FOR THE GENE MUTATION
BY REPLACING THE CELLS WITH
HEALTHY CELLS COULD WE STILL OR
TRACK DAMAGE TO ARTERY WALL,
GREAT WORK AT NIH CLINICAL
TRIALS FOR SICKLE CELL DISEASE,
HOPING TO GET INVOLVED WITH THAT
WORK.
HYPOTHESIS IF WE DO EARLY SCD
WILL THAT PROTECT SS ARTERIES
FROM ARTERIAL MODELING?
WE COMPARED TWO MONTHS AND FOUR
MONTHS, ARTERIAL DIAMETERS,
SEEING THE EXPANSIVE REMODELING.
AND WE SWITCHED , HANNAH SWITCHD
TO BUSULFAN, TO MONITOR THE
BLOOD VESSELS.
AND WOULD CONFIRM HSCT WAS
SUCCESSFUL RUNNING PCR, THE TAIL
SHOULD STILL CARRY THE SICKLE
CELL MUTATION, COLLECT
PERIPHERAL BLOOD FROM
HEMATOPOIETIC STEM CELLS AND
MAKE SURE THEY WERE AT LEAST
HETEROZYGOUS FOR WILD TYPE
ALLELE.
AFTER HSCT IT WAS QUITING, DATA
SHE JUST FINISHED COLLECTING,
AND THATs EARLY HSCT WERE ABLE
TO PROTECT ARTERIAL DIAMETER OF
BLOOD VESSELS, AT AGE 5 MONTHS,
SO THREE MONTHS AFTER.
IN THE SHAM MICE THEY STILL HAVE
SIGNIFICANT DIFFERENCE.
IN LATE MICE, HSCT AT FOUR
MONTHS, WE'RE STILL SEEING A
SIGNIFICANT DIFFERENCE BETWEEN
THE CONTROLS AND THE S S MICE.
WHAT DOES THAT SAY?
THE EARLIER YOU DO THE HSCT, YOU
PROTECT THE VESSELS MORE SO.
WE'LL KEEP FOLLOWING THESE AND
LOOK AT EXTRA TREATMENT OF JNK
IF YOU DO THE HSCT LATER.
WHAT IS GREAT ABOUT THE MAGNETIC
RESONANCE ANGIOGRAPHY LOOKING AT
FEATURE WHERE IS WE MIGHT SEE
STENOSIS OR BLOCKAGE, IN OTHER
STUDIES PEOPLE MANIPULATE LASERS
TO CAUSE A STROKE BUT THAT'S NOT
WHAT YOU SEE IN PEOPLE LIVING
WITH SICKLE CELL.
WE DIDN'T WANT BLOWOUT STROKES.
KNOW SHE CAN LOOK AT TORTUOSITY.
ARE THEY REALLY HAVING STROKES?
NOW WITH M.R. SHE CAN LOOK FOR
OCCLUDED VESSELS, THAT NOW COULD
GIVE A STRONG INDICATION THAT
ACTUALLY MAYBE A STROKE THESE
MICE ARE HAVING TO CONVINCE THE
COMMUNITY, TRADITIONAL BRAIN
SCANS SHOW ABNORMALITIES ON THE
BRAINS, NOT IN ALL THE MICE, AT
LEAST 10 TO 15%.
AND ONE MORE THING, WHAT IS THE
CHALLENGE HERE, WE SAID WE WERE
MEASURING ARTERIAL DIAMETER BUT
AS YOU GO THE LENGTH OF THAT
ARTERY, CHANGES FLUCTUATE, AND
EVEN THIS ONE MOUSE
INTERESTINGLY FROM THREE MONTHS
TO FIVE MONTHS SHE BEGAN TO
NOTICE WHEN WE LOOKED AT THE
PERCENT AREA CHANGE, OR FROM 3
TO 5 MONTHS, SOME MICE HAD A
REDUCTION IN THE AREA WHICH
DOESN'T MAKE SENSE IF THE MOUSE
IS GROWING OR AT LEAST SHOULD
MAINTAIN.
WE'VE BEEN TRYING TO CHANGE THE
WAIT WE ANALYZE ALONG THE ENTIRE
LENGTH, LOOKING AT VOLUME, NOW
TRYING TO IDENTIFY STENOSES SO
WE CAN SACRIFICE WHEN WE SEE THE
STENOSIS AND UNDERSTAND WHAT'S
HAPPENING.
IT INDUCES SEX AND AGE-RELATED
DIFFERENCE IN LARGE ARTERY
REMODEL AND SS MALES SHOW
GREATEST EXPANSION CORRELATING
WITH HUMANS IN THE SEX-SPECIFIC
DIFFERENCES IN LIFE EXPECTANCY.
CURATIVE HEMATOPOIETIC STEM CELL
TRANSPLANT DOES PROTECT THESE
ARTERIES FROM PATHOLOGICAL
REMODEL, ESPECIALLY AT EARLIER
AGE.
AND AGAIN THROUGH THIS
LONGITUDINAL MRA WE'RE ABLE TO
GET MORE OF A TIME LINE FOR THIS
DAMAGE AND NOW WE'LL LOOK AT
HISTOLOGICAL ANALYSES AND MATCH
WITH SUGGESTED THERAPEUTICS.
ALSO TO GET TO THE CASE MADE AND
HAVE DATA FOR CATHEPSIN KNOCKOUT
MICE, MTA HAS FINALLY MOVED
THROUGH, SO THE MICE CAN BE SENT
HERE.
NO MATTER WHAT YOU'RE STUDYING,
IT APPLIES TO SICKLE CELL
DISEASE.
INTERESTINGLY ENGINEERING
QUESTIONS, INTERESTING CHEMICAL
QUESTIONS AND NEW METHODOLOGIES.
THANK YOU FOR LISTENING.
I THANK NIBIB FOR FUNDING TO
CONTINUE THIS WORK AND THIS WAS
MY RESEARCH GROUP IN SEPTEMBER
AT THE SICKLE CELL WALK-RUN IN
GEORGIA.
THERE'S LEANNA WITH WONDERFUL
KIDS, HER HUSBAND RAN OFF
SOMEWHERE.
THERE WE ARE.
THANK YOU VERY MUCH.
[APPLAUSE]
>> WHAT DOES THE RETINAL
VASCULATURE LOOK LIKE?
>> DEFINITELY THERE'S DAMAGE,
YOU CAN SEE IN EARLY 20s, LATE
TEENS, BUT EVEN BEYOND THAT
SICKLE TRAIT WHICH PEOPLE ARE
TOLD IS BENIGN THERE'S RETINAL
DAMAGE, ONE OF THE COMPLICATIONS
OF SICKLE CELL TRAIT.
>> YOU COULD DO A LOT MORE WITH
THE RETINAL VESSELS, CEREBRAL
VESSELS ARE HARD TO GET TO I
GUESS AND HARDER TO IMAGE.
>> I WAS INTERESTED IN TALKING
WITH YOU ABOUT THAT.
REMEMBER THE A.S. IS A LARGER
POPULATION BUT THEY ARE ALL HERE
AT THE CLINICAL CENTER.
>> YOU'RE GOING TO GUESS THE
THEME OF MY QUESTION.
HAVE YOU LOOKED AT THE DIFFERENT
CELLS THAT ARE PARTICIPATING IN
THAT REMODELING WITHIN THE
VASCULATURE BECAUSE, YOU KNOW,
YOUNG EVEN IN UTERO VERSUS BIRTH
MICE WILL HAVE A DIFFERENT
IMMUNE RESPONSE TO TISSUE
DAMAGE, CATHEPSINS COME FROM
MYELOID CELLS, CURIOUS.
>> I DIDN'T SHOW THE HUMAN
WORK.
WE STARTED WITH GETTING BLOOD
FROM PEOPLE WITH SICKLE CELL,
USING WITH ENDOTHELIA CELLS IN
THE SHEER STRESS FLOW CHAMBER.
THERE'S CHRONIC AND SYSTEMIC
INFLAMMATION, HIGH LEVELS OF
CIRCULATING TNF ALPHA.
IF YOU TAKE PBMCs COMPARED TO
A.A., ADHERE BETTER, EVEN
WITHOUT PRE-TREATMENT WITH TNF
ALPHA AND TURN ON EXPRESSION OF
CYSTEINE CATHEPSINS, IT'S A
POSITIVE FEEDBACK, MORE
MONOCYTES, MOR ADHESIVE, THEY
AIN'T GOT NO CHANCE.
IN OTHER STUDIES WE BEGAN TO
LOOK AT INFLAMMATORY MONOCYTES
VERSUS ANTI-ININFLAMMATORY,
WE'VE SEEN THAT IN HUMANS AND
MICE, IT'S THE DIFFERENT MARKER,
CD16, THERE'S A GREATER NUMBER
OF INFLAMMATORY MONOCYTES IN
INDIVIDUALS WITH SS, EVEN
MONOCYTES ARE MORE
PROTEOLYTICALLY ACTIVE BEFORE
THEY BIND AND STICK TO CELLS.
HANNAH CARES ABOUT T CELLS, I
TELL HER TO GET THAT STUFF OUT
OF MY FACE BUT NOW WE'RE HERE
AND WE SHOULD TALK.
IF THERE'S OTHER DATA WE HAVE
ALREADY COLLECTED WE'D LOVE TO
SHARE AND SHOW INTERESTING
THINGS TO LOOK AT.
>> GREAT TALK.
>> ALL RIGHT, THANK YOU.
>> THIS IS NOT AN IMMUNOLOGY
QUESTION.
AM I CORRECT IT'S THE TURBULENT
FLOW AFTER THE STENOSIS CAUSING
THE DAMAGE AND SUBSEQUENT
REMODELING?
>>WE DON'T KNOW IF IT GOES
TURBULENT BUT DEFINITELY
DISTURBED FLOW, ENDOTHELIAL
CELLS.
>> CAN YOU ADD SOMETHING TO THE
CIRCULATION TO LAMINARIZE THE
FLOW, LIKE DRUG RESISTANT
POLYMERS.
>> OH, INTERESTING.
THE PROBLEM THAT -- MOST PEOPLE
CALL THIS SICKLE CELL ANEMIA,
HEMATOCRIT IS 45% WITHOUT, IN
SICKLE CELL IT'S 25%.
IN THE PROCESS OF THEM BREAKING
UP THE MEMBRANES EACH INDIVIDUAL
RED CELL MEMBRANE GETS FOR
VISCOUS, THAN HAVING THEM IN
AGGREGATE DOES NOT GET MORE
VISCOUS BECAUSE THERE'S LESS RED
BLOOD CELLS SO WHEN YOU ASK,
THERE'S STILL INDIVIDUAL RED
CELLS CREATING THEIR OWN
CHANGES.
YOU DON'T WANT TO THIN OUT RED
CELLS BECAUSE YOU'RE ALREADY AT
LIMITS HOW YOU TRANSPORT OXYGEN.
THERE HAVE BEEN OTHER DRUGS THAT
PEOPLE HAVE TRIED IN THE PAST
WHERE THIS HAS BEEN A PROBLEM,
POLYMERS WOULD PREVENT
HEMOGLOBIN MOLECULES FROM
POLYMERIZING, BUT EXCRETING
THROUGH THE KIDNEYS, YOU GOT TO
KEEP ENOUGH MOLECULES IN THE
BLOODSTREAM WITH THE HEMOGLOBIN
TO STOP THAT.
BUMMER.
THE ONE TREATMENT THAT DOES
WORK, WHERE CLINICAL TRIALS ARE
GOING NOW, ONE DRUG ON THE
MARKET THAT'S REALLY EFFECTIVE,
THERE ARE THREE BUT ONE REALLY
EFFECTIVE, TURNS BACK ON FETAL
HEMOGLOBIN, IN THE RED CELL
INTERFERES WITH SICKLE
POLYMERIZATION, VISCOSITY GOES
DOWN.
THAT'S WHY PEOPLE TARGET
PROTECTING THE RED CELL BUT I
HAVEN'T HEARD PEOPLE TALK ABOUT
HOW DO YOU MAKE THE FLOW MORE --
LESS VISCOUS, BECAUSE RED CELLS
ARE INCREASING VISCOSITY.
LONG ANSWER, SORRY.
>> THANK YOU.
>> OKAY.
>> YOU SAID PORT OF THE PROBLEM
WITH CLOTS AND THINGS FORMING
ARE CELLS ARE STICKING TO THE
BLOOD VESSEL WELL.
HAVE PEOPLE ATTACKED THAT FROM A
PHARMACOLOGICAL VIEWPOINT?
>> YEAH, THERE'S A DRUG OUT
NOW, I THINK IT'S BEING TESTED,
KRIZUNLAZIMAB, P SELECTIN
ANTIBODY, IT HAS, LAST PAPER I
READ, WAS EFFECTIVE IN REDUCING
NUMBER OF VASOOCCLUSIONS OR
VASOOCCLUSIVE CRISES.
>> THANK YOU.
>> ALL RIGHT.
THANK YOU VERY MUCH.
[APPLAUSE]
>> WE'LL HAVE FOUR SHORT
TEN-MINUTE TALKS.
I WANT TO ANNOUNCE TOMORROW
WE'RE GOING TO HAVE A LUNCH WITH
DR. GEORGAKOUDI FOR WALS, IF YOU
WOULD LIKE TO JOIN US FOR DINNER
LET US KNOW.
CONTACT STEVE.
LET US KNOW IF THERE'S ANY
DIETARY RESTRICTION.
THE LUNCH WILL BE FROM TWELVE TO
ONE P.M. TOMORROW.
LET US KNOW.
ALL RIGHT.
NEXT SPEAKER IS DR. LICHEN
XIANG, NINR, WE WOULD LIKE TO
WELCOME HIM TO GIVE HIS TALK.
THANK YOU.
>>I'M A RESEARCH FELLOW,
INSTITUTE OF NURSING RESEARCH,
DIVISION OF NURSING, MY TOMMIC
IS RAPID POINT-OF-CARE TEST,
I'VE BEEN DOING THIS FROM MY
GRADUATE YEARS TO NOW.
THIS IS THE FIRST TEN-MINUTE
TALK FOR TODAY, I'LL TRY TO MAE
IT ON TIME.
YOU ARE MISSION TO SAVE PROBLEMS
IN THIRD WORLD COUNTRY WHICH
THERE ARE MANY CHILDRENS DIE
LIKE OVER 500,000 CHILDREN DIE
EVERY YEAR BECAUSE OF THE
DIARRHEA, ACCORDING TO THE
W.H.O., EVEN NOW TODAY.
EVEN TODAY.
SO, THERE ARE MANY KIDS DIE
BECAUSE OF LACK OF RESOURCES TO
TREAT IT OR DIAGNOSE IT.
THE CURRENT METHOD FOR TESTING
THE DIARRHEA INFECTIONS ARE
EITHER BEING PCR BASED OR CELL
CULTURE BASED, WASTING TIME,
SLOW, AND REQUIRED HIGHLY
SKILLED TECHNICIANS, THEY DEMAND
DEDICATED LABORATORY RESOURCES,
SO THOSE ARE NOT VERY APPLICABE
IN THE FIELD OR POOR COUNTRIES.
AND SO THERE'S A NEED FOR RAPID
REALTIME LOW COST POINT OF CARE
THAT CAN DIAGNOSE THE PATHOGENS
THAT ARE CAUSING DIARRHEA, AND
ALSO IN THE REAL LOW COST
SETTINGS.
SO, AT THE VERY BEGINNING OF
THIS RESEARCH WE FACED BIG
CHALLENGES BECAUSE IF WE'RE
DEALING WITH STEW THERE'S A LOT
OF INTERFERENCE THAT CAN
INTERFERE YOUR DNA APPLICATIONS,
ALL SUCH THINGS.
THAT'S WHY MOST OF THE LABS
USING THE LIKE SAMPLE PREP KIDS
TO ISOLATE DNA FROM THE STOOL OR
USING THE CENTRIFUGE AT LEAST,
RIGHT?
SO THE VERY BEGINNING WE WANTED
TO TRY TO HAVE SOMETHING THAT IS
LOW COST AND DON'T NEED A LAB TO
DO BIOSEPARATION.
THIS IS THE METHOD WE DEVELOPED,
MICRO SPHERE BIO-SEPARATION
METHOD, IT HAS A REALLY, REALLY
LOW DENSITY, SO IF WE PUT THAT
INTO A LIQUID IT WILL FLOAT TO
THE TOP VERY FAST.
SO HERE'S THE REALTIME MEDIA
CLIP THAT WE JUST MIX IT UP WITH
THESE AND WATER, PBS, YES, AND
THEN JUST ESSENTIALLY FLOAT TO
THE TOP, RIGHT?
GENERALLY USING BUOYANCY FORCE.
THE IDEA IF WE FUNCTIONALIZE
THOSE BEADS WITH CAPTURING
MATERIALS THAT CAN SPECIFICALLY
CAPTURE THE DNA THAT I WANT OR
THE TOTAL DNA THAT I WANT, SO IT
CAN CARRY THIS DNA, FLOAT TO THE
TOP, SO THEN WE CAN USE THAT
MATERIAL TO SEPARATE FIRSTLY
LYSIS THE STEW, AND THEN
SEPARATE THE DNA THAT WE WANT.
AND THEN WE APPLY THEM TO THE
LATERAL FLOW ASSAY, WHICH IS WE
SEE THE PREGNANCY TEST BASED
LIKE STRIPS, THAT WE CAN SAY IF
THIS EXISTS OR NOT, RIGHT?
HERE IS THE RESULT.
BY APPLYING THOSE BIO-SEPARATION
METHOD WE SEPARATE DNA FROM
STEW, AND THEN WE SPIKE IN THE
PATHOGENS INTO THE STEW, AND
THEN WITH DIFFERENT
CONCENTRATIONS FROM LOW TO HIGH,
I WANT TO SEE LIMIT OF DETECTION
OF THIS METHOD AND WE ALSO
QUANTIFYING INTENSITY OF THE
TEST DOTS HERE, LATER WE MADE
THE LINE.
SO THE TEST DOT HERE, WE HAVE
QUITE A LINEAR REGRESSION ON
THAT.
IF WE INCREASE, KEEP INCREASE
THE CONCENTRATION OF THE
PATHOGEN, YOU WILL BE LIKE FLAT.
SO LATER ON WE TRIED, ALSO TRIED
SENSITIVITY AND SELECTIVITY OF
OUR METHOD.
THE PICTURE ON THE RIGHT IS THE
TEST.
WE USED THE RPA AMPLIFICATION
METHOD TO AMPLIFY THE
CRYPTOSPOREDIAN, IF WE SPIKE
DIFFERENT OTHER BACTERIAS AND
PATHOGENS, PARASITES IN THERE,
GRDSM, WHATEVER, NO SIGNALS,
THAT'S THE SENSITIVITY AND
SELECTIVITY.
WE THINK, OKAY, TEST ONE STEW
SAMPLE, TELL US MORE
INFORMATION, NOT CHRIS CRYPTO,
BUT ALSO LIKE C. DIFF, E. COLI,
WHATEVER, THAT WE WANT, WE WANT
A MULTIPLEX.
SO THE PICTURE AT THE LEFT IS WE
ACTUALLY HAVE DIFFERENT MASTER
MIX OF THE RPA AMPLIFICATION,
AND THEN WE AMPLIFY TWO OR THREE
PATHOGENS AT A TIME.
SO WE CAN HAVE THREE MULTIPLEX
LIKEY MULTIPLEX
GIARDIA, THREE AT ONE TIME.
WE TRY DIFFERENT COMBINATIONS
AND SEE SELECTIVITY IS PRETTY
GOOD.
SO, LATER ON, WE ALSO HAVE
ANOTHER QUESTION.
SO, THE TEST STRIP LIKE THE
PREGNANCY TEST STRIP, BECAUSE OF
THE COVID MAYBE EVERYONE USING
THAT FOR COVID TEST, SO IT'S
PRETTY SHORT.
AND WE TRY TO HAVE LIKE TEN OR
FIVE PATHOGENS AT A TIME FOR ONE
STRIP BUT DOESN'T WORK.
SO WE WANT TO TRY LIKE AS MUCH
AS WE CAN, LIKE WE WANT TO GET
ONE SAMPLE AND GET AS MUCH
ANSWER AS WE CAN.
SO WE TRIED ANOTHER PLATFORM,
CALLED THIS PAPER ORIGAMI
MULTIPLEX SENSOR.
SO WHAT WE DO IS WE CHANGE THE
IDEA, THE LOGIC BEHIND IT.
LATERAL FLOW IN THE LATERAL, AND
WHAT IF WE USE MICRO FLUID NOT
ONLY ON LATERAL BUT VERTICAL,
OKAY?
WE HAVE PRINT THOSE PATTERNS ON
A CHROMATOGRAPHY PAPER, AND THEN
WHAT YOU SEE HERE IN THE BLACK
COLOR ARE WAX, SO WAX ARE
HYDROPHOBIC, SO IF YOU HAVE A
LIQUID SAMPLE APPLIED TO THE
PAPER, STUCK TO THE PAPER, AND
TRAVELING FROM LAYER 1 TO 2 TO 3
TO 4.
AND ALL THE WAY DOWN, IT TRAVELS
LIKE NOT VERTICALLY BUT -- NOT
LATERALLY BUT VERTICALLY, RIGHT?
SO, WE VIEWED THIS KIND OF
VERTICAL ORIGAMI SENSOR BECAUSE
AT THE VERY BEGINNING WE PRINT
AS A PAPER, THEY WOULD FOLD IT
TO A SENSOR.
SO, THEN WE APPLIED THE SAMPLE
AT THE VERY FIRST LAYER, AND
WE'LL GO DOWN, DOWN, DOWN, DOWN,
ALL THE WAY TO THE BOTTOM, AND
IF WE HAVE THE C. DIFF APPEAR IT
WILL TURN COLOR, FLUORESCENT
GREEN, IF THERE'S NO, IT'S AN
ACTIVE CONTROL, BROWNISH.
OKAY?
SO, BY DOING THAT, WE CAN DO
EIGHT PATHOGENS AT A TIME
BECAUSE LOOK AT THE PATTERN IN
THE MIDDLE, SO EACH ARM IS ONE
PATHOGEN.
SO ONE SAMPLE INJECT IN THE
MIDDLE, WILL TRAVEL DOWN AND
SPREAD AND DOWN TO THE BOTTOM,
AND WE CAN GET EIGHT ANSWERS AT
A TIME.
ACCURACY AND SENSITIVITY OVER
98% FOR NOW.
WE'RE GOING TO IMPROVE THAT.
SO RIGHT NOW WE TRIED C. DIFF,
SHIGELLA, ETEC, EPEC, ROTAVIRUS,
NOROVIRUS, TYPE 1 AND 2,
CRYPTOSPORIDUM, GIARDIA.
ALL OF THOSE LIKE THIS IS -- WE
RECENTLY GOT THE PATENT ISSUED,
GLOBAL PATENT FOR THIS PAPER
ORIGAMI AND MICRO SPHERE TWO
YEARS AGO.
AND I AM THE PRIMARY MENTOR FOR
BOTH OF THE PAT -- PRIMARY
INVENTOR FOR BOTH OF THE
PATENTS, AND THAT IS MY
RESEARCH.
[APPLAUSE]
>> VERY CLEVER IDEAS.
I LOVE THEM.
FOURTH PANEL, LAST SLIDE,
INTERMEDIATE POSITIONS.
WHAT ARE THOSE FOR?
>> THAT'S A GOOD QUESTION, FOR
THE TWO DOTS, RIGHT?
SO, THE VERY FIRST IDEA FOR THAT
WAS TO INCUBATE, YOU KNOW, LIKE
EITHER FOR RPA OR LAMP
APPLICATION REQUIRES A HEATING.
EITHER TO 37 DEGREES C OR OVER
40 DEGREES C.
SO WE HAVE LIKE THICKER, YOU SEE
A THICKER DESIGN RATHER THAN ALL
THE OTHER LAYERS BECAUSE IT ACTS
AS THE RESERVOIR TO KEEP SOME OF
THE SAMPLES OVER THERE, AND WE
HEAT UP.
WE USE -- ACTUALLY AT THE VERY
BEGINNING WE TRIED THE FOOT
WARMER YOU PUT IN YOUR SHOES IN
THE WINTER TO KEEP YOU WARM.
AND PUT THAT LAYER UNDERNEATH
THAT.
WHEN YOU SQUEEZE IT, IT WOULD
GENERATE HEAT AND YOU REMOVE IT,
DRAIN DOWN SO SHOWS COLOR AND IT
REMAINS LOW COST AND ALSO CAN
HEAT IT UP.
LATER ON BECAUSE OF TECHNOLOGY
GOT MUCH BETTER, WE CAN DO A
ROOM TEMPERATURE APPLICATION, NO
NEED FOR HEATING AT THIS MOMENT.
BUT FOR ESPECIALLY LAMP, THERE'S
A ROOM TEMPERATURE LAMP,
REALTIME ROOM TEMPERATURE LAMP,
SO WE CAN APPLY THAT AND WE HAVE
FURTHER RESEARCH DOING OTHER
AMPLIFICATIONS THAT IS UNDER THE
PATENT REVIEW SO I CANNOT REVIEW
HERE.
SO, YEAH, THE TECHNOLOGY IS
MOVING FORWARD.
>> AGREED.
THIS IS REALLY COOL.
CURIOUS HOW TEMPERATURE STABLE
ARE THESE, LIKE LOW-RESOURCE
SETTINGS WITHOUT REFRIGERATION,
TEMPERATURE UP TO HOW LONG?
YOU SAID THIS WAS WAX?
CURIOUS ABOUT THAT.
>> SO THE WAX -- SO, WHEN WE
MADE THIS, WE ONLY PRINT WAX ON
ONE SIDE OF THE PAPER AND THEN
WE PUT IN THE OVEN AND MAKE THE
WAX WET AND IT WILL SINK THROUGH
THE PAPER, SO GENERATE ACTUALLY
THE REAL HYDROPHOBIC WALL IN THE
PAPER.
AND THEN THAT WILL -- THAT IS
OVER 100 DEGREES C, SO I DON'T
THINK ANY LIKE ENVIRONMENTAL
TEMPERATURE WILL HEAT IT UP
AGAIN TO DAMAGE THIS CHANNEL.
BUT INCLUDING THE HEATING THAT I
JUST MENTIONED, EITHER 37
DEGREES C OR OVER 45, SO IT
WON'T DAMAGE THE PATTERN THAT WE
DEVELOPED.
AND ALSO THAT'S A GOOD QUESTION
TOO, YOU SEE THIS TRANSPARENT
THING OVER HERE?
IT'S THE MEMBRANE THAT WE TRY TO
SEAL EVERYTHING INTO A SEALED
DEVICE THAT LIKE ONCE YOU INJECT
THERE'S ANOTHER SEAL AT THE TOP,
SO THE USER WON'T TOUCH THE STEW
BECAUSE, YOU KNOW, THEY APPLY
THE STEW SAMPLES, TO KEEP IT
SANITIZED.
>> SO, I SEE THAT LIKE THE
NEGATIVE CONTROL C. DIFF SHOW
GREEN FLUORESCENCE, WHAT ABOUT
OTHER SPECIES, DO THEY SHOW
DIFFERENT COLOR FLUORESCENCE ON
DIFFERENT PATTERN?
HOW CAN YOU TELL?
>> YEAH, FOR THIS, YES, THAT'S
A GREAT QUESTION.
SO AT THE VERY BOTTOM YOU SEE
THE -- YEAH, YOU SEE THE PICTURE
RIGHT HERE, RIGHT?
SO, ONE OF THE DOTS, ONE OF THE
TWO DOTS IS THE C. DIFF.
IT WILL BE THIS ONE OUTSIDE,
OUTSIDE IS CONTROL, INSIDE IS A
TEST.
WE WILL LABEL WHICH ARM IS WHICH
AT THE VERY BOTTOM.
SO THEY ESSENTIALLY HAVE THE
SAME SIGNAL BUT BECAUSE WE LABEL
IT, YOU WILL KNOW WHICH ARM
REPRESENTS WHAT.
>> THANK YOU.
>> YES.
>> THANK YOU SO MUCH.
>> ALL RIGHT, THANK YOU.
[APPLAUSE]
>> NEXT SPEAKER IS DR. ETHAN
COHEN FROM OFFICE OF SCIENCE AND
ENGINEERING LABORATORY AT FDA.
>> I'D LIKE TO THANK THE
ORGANIZERS FOR INVITING ME.
I'D LIKE TO TALK TODAY ABOUT --
COBALT CAUSES RAPID CHANGE IN
RAPID STRUCTURE, OF THE RABBIT
RETINA.
I WORK AT FDA SO WE HAVE THIS
DISCLAIMER.
GO PAST THAT.
SO I WORK AT MEDICAL DEVICE
COMPANY, MEDICAL DEVICE
GOVERNMENT AGENCY.
AND WE'RE ALWAYS DEALING WITH
TOXICOLOGY ISSUES, TOXICOLOGY OF
DRUGS, TOXICOLOGY OF DEVICES,
SOME DEVICES WORK WELL, SOME
DEVICES FAIL.
AS DAN POINTED OUT, WE NEED TO
DEVELOP A SERIES OF REGULATORY
SCIENCE TOOLS TO HELP INDUSTRY
BRAIN DEVICES THROUGH THEY CAN
DEMONSTRATE ARE SAFE, REDUCES
USE OF ANIMALS TO SOME DEGREE.
THREE, THAT GIVES SOME IDEA OF
HOW THESE SUBSTANCES ARE
AFFECTING LIVING TISSUE IN REAL
TIME.
SO, IN THE CASE OF -- I'M GOING
TO TALK ABOUT THE CASE OF
COBALT, AND THERE WERE ABOUT A
MILLION COBALT CHROMIUM HIP
IMPLANTS PRODUCED, AND A
SUBSTANTIAL NUMBER OF THESE
IMPLANTS PRODUCE HIGH LEVELS OF
COBALT IN THE PATIENT'S BLOOD,
AND IN A VERY LIMITED SUBSET OF
PEOPLE THIS LEVEL OF COBALT WAS
ENOUGH TO IMPAIR VISION OR CAUSE
TINNITUS OR BALANCE ISSUES.
WHAT ARE THE MECHANISMS OF
COBALT TOXICITY AND I'M USING
THE LIVING RETINA AS THE MODEL
SYSTEM TO EXAMINE THESE EFFECTS
IN REAL TIME.
AND I USE A THING CALLED OP --
OPTICAL TOMOGRAPHY.
COBALT HAS MANY EFFECTS ON
TISSUES, ONE WE DON'T UNDERSTAND
WELL ETHNICITY ALLOTHIONINE
RECEPTOR ACTIVATION, OF INTEREST
TO MITOCHONDRIAL PEOPLE BY
INTERFERING WITH REDOX SYSTEMS
AND WE KNOW IT CAN MIMIC
BACTERIAL CELL WALL ACTIVATING
TOLL 4 RECEPTORS, COBALT AND
NICKEL BOTH.
AND IN ADDITION, WHAT YOU MAY
NOT KNOW IS COBALT ACTIVATES
PHOSPHO-DIESTERASE.
IT BINDS TO PROTEIN ON THE
HYPOXIA INDUCED FACTOR THAT
MIMICS HYPOXIA, AND THIS CAUSES
CHANGES IN GENE TRANSCRIPTION.
AND FINALLY BEING A
PHYSIOLOGIST, YOU KNOW THE
COBALT INHIBITS CALCIUM
CHANNELS.
SO, WHAT HAPPENS TO A PATIENT
WHO HAS A VERY SEVERE CASE OF
COBALTERIA?
THEY COULD HAVE A LEVEL OF 900
MICROGRAMS PER ML, AND WHAT YOU
CAN SEE HERE OCT IMAGE WHICH DAN
HAS DISCUSSED, THIS IS WHAT A
MORE NORMAL CLINICAL OCT IMAGES
LOOKS LIKE OF A RETINA THAN
EARLIER GENERATION, AND LET'S
SEE, DOES THIS HAVE A LASER
POINTER?
OVER HERE?
HERE, I CAN USE THIS I GUESS.
WHAT YOU CAN SEE, EXCUSE ME,
WHAT YOU CAN SEE HERE, THE UPPER
LEFT-HAND PANEL A
CROSS-SECTIONAL IMAGE USING A
NORMAL CLINICAL OCT, FOVEA,
THERE'S A LOT OF GANGLION CELLS
BUT YOU CAN'T SEE A LOT WHAT'S
GOING ON IN THE PHOTORECEPTORS.
THESE PATIENTS WHEN THEY COME
INTO THE CLINIC IF YOU LOOK AT
VISUAL FIELDS, WHICH YOU CAN SEE
ON THE FAR RIGHT, YOU CAN SEE
VERY LARGE REGION, WHERE THEY
LOST CENTRAL VISION.
AND ALSO WHEN YOU PROBE THE
RETINA USING MULTI-FOCAL
ELECTRORETINOGRAMS REFLECTING
LARGELY OUTER RETINAL FUNCTION,
THIS IS WHAT A NORMAL RETINA
LOOKS LIKE WHEN YOU PROBE THE
SURFACE OF THE FUNDUS, SENSITIVE
IN OUR FOVEA, YOU SEE A PEAK OF
SENSITIVITY IN PATIENTS, THIS
PEAK IS COMPLETELY LOST.
AND THIS SUGGESTS THAT THERE'S A
SIGNIFICANT DEFICIT TO THE
PHOTORECEPTORS.
SO, LET'S SEE.
OKAY.
SO, WHAT WE'VE -- I'VE USED HERE
IS A VERY SPECIAL PREPARATION
WHERE WE CAN KEEP THE RETINA
ALIVE OUTSIDE THE BODY ATTACHED
TO PIGMENT EPITHELIUM, THIS IS
CALLED A RABBIT EYE CUP
PREPARATION HERE.
BASICALLY THE BACK HALF OF THE
RABBIT EYE IS EVERTED, LIKE A
BALL CUT IN HALF, EVERTED AND
MOUNTED ON THE SORT OF DOME
CHAMBER, AND THEN USING OPTICAL
CURRENT STEMMOGRAPHY CAN SEE ALL
THE LAYERS OF THE LIVING RETINA
IN REAL TIME, HERE IS THE
GANGLION CELL LAYER, AND HERE,
THESE LAYERS HERE ARE
PHOTORECEPTOR ASSOCIATED LAYERS.
REMEMBER I SAID THE MULTI-FOCAL
ELECTRORETINOGRAM IS SEVERELY
LOST IN THESE PATIENTS THAT HAVE
HIGH COBALTERUA.
NOW WE LOOK AT OCT IMAGES OF
RABBIT RETINA CAN PERFUSE IN
REAL TIME AND SEE EFFECT ON THE
RETINAL STRUCTURE.
THAT'S REALLY THE ADVANTAGE HERE
OF THIS RETINAL PREPARATION, WE
DON'T HAVE ADAPTIVE OPTICS, SO
WE DON'T HAVE TO DEAL WITH THE
DISTORTIONS OF THE EYE, AND WE
DON'T HAVE TO DEAL WITH THE
PHARMACOKINETICS AND PARTITION
COEFFICIENTS OF DRUGS, LIVER
PATHWAYS, KIDNEY ALLUTIAN.
HERE WE SEE TEN MINUTES AFTER,
YOU CAN SEE CLEARLY HERE THAT
THERE'S A LOSS OF OF THE FINE
LINE NEXT TO THE PIGMENT
EPITHELIUM AND INNER
SEGMENT/OUTER SEGMENT LINE.
IN ADDITION YOU CAN SEE THERE'S
CHANGES ALSO THE MEMBRANE WHERE
THE GANGLION CELLS ARE.
SO WHAT THESE TWO IMAGES, THEY
ARE FLIPPED OF THE SAME RETINA
BEFORE AND AFTER THE DRUG SO CAN
YOU DIRECTLY COMPARE THE
STRUCTURE.
SO YOU CAN SEE THERE'S A
SIGNIFICANT CHANGE IN THE OUTER
RETINA, IT'S VERY RAPID, WHICH
IS SOMEWHAT UNEXPECTED, AND WHAT
WE'RE TRYING TO FIGURE OUT IS
WHAT IS CAUSING THIS
DEGENERATION.
SO, WHAT WE CAN DO NOW IS WE CAN
LOOK AT HOW THE CHANGES IN THAT
RETINAL STRUCTURE CHANGE WITH
TIME, AND WE DO THAT BY
TAKING -- LET'S SEE HERE.
IT'S NOT GOING BACKWARDS.
HOLD ON FOR A SECOND.
WE CAN TAKE A SQUARE REGION HERE
OF THE RETINA, AVERAGE IT
LATERALLY, AND THEN TAKE
DIFFERENT TIME POINTS EVERY 15
SECONDS OF THE CHANGES IN THE
RETINAL STRUCTURE WITH THE
PRESENCE OF COBALT.
AND SO HERE YOU SEE A TEST WHERE
WE USED NO COBALT AND YOU CAN
SEE HERE WHEN WE PUT THE RINGER
WHICH HAS NOTHING IN IT,
BASICALLY THERE'S VERY ALSO
EFFECT -- VERY LITTLE EFFECT ON
THE RETINAL STRUCTURE, THIS IS A
CROSS-SECTION AS A LINE OF THE
RETINAL STRUCTURE, BEFORE AND
AFTER THE COBALT RIGHT HERE AND
RIGHT THERE.
AND YOU CAN SEE BASICALLY
THERE'S VERY LITTLE EFFECT.
NOW, IF WE DO THE SAME THING,
EXCUSE ME, IN THE PRESENCE OF
COBALT YOU CAN SEE HERE THAT
THERE'S A DROP IN THE INNER
SEGMENT, OUTER SEGMENT LINE AND
A DROP IN WHAT WE KNOW ARE THE
INTERDIGITATION ZONE, ON THE
ORDER OF TEN MINUTES.
IF THIS WAS ACTING THROUGH SOME
SORT OF HYPOXIA-INDUCED FACTOR
OR GENE TRANSCRIPTION YOU WENT
GET EFFECT OF THAT RAPIDITY.
AND SO ON AVERAGE, THE RETINA,
PHYSICAL DISTANCE OF THE RETINA
SHRINKS AN AVERAGE OF 3.3
MICRONS IN THE PRESENCE OF
COBALT BUT THE MAJORITY OF THE
SHRINKAGE IS HERE, SIGNIFICANT
PORTION OF THE OUTER SEGMENTS OF
THE PHOTORECEPTORS ARE BEING
AFFECTED BY THE COBALT.
AND -- HOLD ON ONE MOMENT.
HERE YOU CAN SEE ON THE RIGHT
COMPARISON OF THREE RETINAS
WHERE WE APPLIED COBALT BEFORE
AND AFTER, AND YOU CAN SEE IN
EVERY CASE THESE LINES
REPRESENTING THE PHOTORECEPTOR
AREA SHOW THE ACTUAL
PHOTORECEPTORS ARE SHRIVELED
SLIGHTLY IN THE PRESENCE OF
COBALT.
OKAY.
EXCUSE ME.
SO, NOW WE CAN LOOK AT THE
COMPARISON OF THE EFFECTS OF
COBALT, ON CROSS-SECTIONAL
PROFILE.
YOU CAN SEE AGAIN THE BEFORE AND
AFTER THE COBALT, BEFORE IS
BLUE, AFTER IS RED.
AND YOU CAN SEE THERE'S A LARGE
DROP IN THIS PEAK HERE ON THE
INTERDIGITIZATION ZONE IN
PRESENCE OF COBALT AND ALSO PEAK
OF INNER/OUTER SEGMENT ZONE.
SO THE QUESTION THAT WE HAVE,
THIS SHOWS A CROSS-SECTION WITH
TIME, OF THE EFFECT ON THE
INNER/OUTER SEGMENT LINE, DROP
IN REFLEXIVITY WITH TIME,
DECLINED AVERAGE OF NINE UNITS,
SO PRETTY CLEAR THIS IS EFFECT
ON OUTER RETINA, AND THEN WE
WANT TO KNOW WHAT IS THE DOSE
AND WHAT RELEVANCE DOES IT HAVE
TO HUMAN VALUES?
WELL, A THOUSAND PARTS PER
MILLION, ABOUT THE WORST DOSE
REPORTED IN PATIENTS, WORKS OUT
TO 17 MICROMOLAR COBALT.
SO THAT'S -- THINK ABOUT THAT,
THAT'S A PATIENT THAT WOULD HAVE
COBALT THREE, FOUR YEARS IN
THEIR IMPLANT, SO IT'S A LONG
EXPOSURE TIME.
AND SO WHAT WE'VE DONE HERE IS
WE'VE STARTED TO DO DOSIMETRY,
YOU CAN GET THE SAME EFFECT OF
COBALT ON THE PHOTORECEPTOR
LINES AT 200 MICROMOLAR AND
SMALLER EFFECTS AT 40
MICROMOLAR, NOT FAR OFF FROM THE
DOSES THAT ARE AFFECTING
PATIENTS.
SO, THOUSAND DOLLAR QUESTION WE
WANT TO KNOW IS HOW IS COBALT IN
FACT AFFECTING THE RETINA?
WE'VE PREVIOUSLY LOOKED AT A
SERIES OF DIFFERENT
NEUROTOXICANTS, A DIP 8, NMDA,
AND WE'VE LOOKED AT CYANIDE AND
CHLOROQUIN, A WELL-KNOWN DRUG
THAT AFFECTS MITOCHONDRIA, AND
WHAT YOU CAN SEE HERE IS HERE WE
HAVE THE RETINA BEFORE AND AFTER
COBALT.
AND YOU CAN CLEARLY SEE THIS
CHANGE IN THE INTERDIGITIZATION
ZONE AND ALSO CHANGE IN THE
BRIGHTNESS OF THE INNER/OUTER
SEGMENTS.
SAME RABBIT RETINA, DIFFERENT
EXPERIMENT, IN THIS CASE USING
CYANIDE, A WELL-KNOWN METABOLIC
UNCOUPLER MUCH MITOCHONDRIA,
SAME LOSS NEXT TO THE RPE AND
ALSO SEE A LOSS IN THE
BRIGHTNESS OF THE INNER/OUTER
SEGMENTS.
ON THE RIGHT YOU CAN SEE
HISTOLOGY PICTURE OF BEFORE AND
AFTER CYANIDE SHOWING THIS
DISRUPTION OF THE PHOTORECEPTOR
TIPS.
WE CAN SEE DISRUPTION OF THE
PHOTORECEPTOR TIPS, THE THOUSAND
DOLLAR QUESTION WE'RE WORKING ON
CURRENTLY IS TRYING TO
UNDERSTAND HOW COBALT IS CAUSING
THIS EFFECT.
SO WE'VE DEVELOPED A METHOD TO
IMAGE METAL IONS RELEASED ON
RETINAL STRUCTURE IN REAL TIME,
SO THIS SAVES ON SACRIFICING
ANIMALS AT MULTIPLE TIME POINTS.
COBALT CAUSES A RAPID THINNING
IN THE RABBIT RETINA ON THE
ORDER OF MINUTES, NOT ACCOUNTED
FOR HYPOXIA FACTOR TYPE, GENE
ALTERATION, AM OPT POOK ARE POCK
ARE R. L. -- ALTERED POSITION OF
IS/OS LINE.
THERE ARE A FEW SMALLER EFFECTS
ON THE OUTER SIDE OF THE RETINA,
AFFECTING GANGLION CELLS, SO THE
RABBIT EYE OFFERS RETIME
ASSESSMENT OF TOXICANTS ON THE
RETINAL STRUCTURE AND WE'RE
GOING TO OFFER IT ACTUALLY AS A
FREE REGULATORY SCIENCE TOOL
THAT YOU CAN USE FOR STUDIES.
I DON'T KNOW IF YOU CAN DO
ANYTHING WITH SICKLE CELL WITH
IT BUT YOU'RE WELCOME TO IT.
THIS IS WHAT IT LOOKS LIKE.
HERE ARE MY COLLABORATORS, AND I
ALSO WORK WITH PEOPLE AT THE EYE
INSTITUTE AS WELL.
SO JOHANNA, STEVE, ROSLYN, THANK
YOU.
>> IN THE INTEREST OF TIME,
SAVE YOUR QUESTIONS FOR THE VERY
END IF POSSIBLE.
WE ALSO HAVE A POSTER
PRESENTATION FROM ALL THE SHORT
TALK SPEAKERS TOMORROW.
MAKE SURE YOU ATTEND THEIR
POSTER AND ASK ANY QUESTIONS YOU
MIGHT HAVE.
>> QUESTIONS?
>>WE'LL SAVE IT FOR LATER.
NEXT DR. QU TIAN, NATIONAL
INSTITUTE OF AGING.
>> GREAT TO BE HERE.
THANK YOU FOR THE ORGANIZERS OF
THE EVENT AND OPPORTUNITY TO
PRESENT SOME OF OUR WORK.
MY TITLE IS MITOCHONDRIAL
FUNCTION PREDICT IT'S COGNITIVE
IMPAIRMENT, ASSOCIATED WITH
BIOMARKERS IN NEURODEGENERATION.
SO, MITOCHONDRIA CASCADE
HYPOTHESIS WAS FIRST PROPOSED IN
2004, STATES MITOCHONDRIAL
FUNCTION WAS GENETICALLY
DETERMINED AT BIRTH.
OVER TIME, MITOCHONDRIA FUNCTION
WILL DECLINE WITH AGING.
ONCE THE DECLINE PASSED CERTAIN
THRESHOLD IMAGE MAY TRIGGER
FUTURE ALZHEIMER'S PATHOLOGY.
SO OVER THE YEARS THERE HAS BEEN
RESEARCH DEMONSTRATING
MITOCHONDRIA DYSFUNCTION IS
IMPORTANT HALLMARK OF AGING.
AND THERE HAS ALSO BEEN EVIDENCE
SHOWING CONNECTION BETWEEN
MITOCHONDRIA DYSFUNCTION WITH
A-BETA AND TAU, HALLMARKS OF
ALZHEIMER'S DISEASE, MOST OF THE
EVIDENCE COMING FROM ANIMAL
MODELS, THERE'S BEEN LIMITED
EVIDENCE FROM HUMAN STUDIES.
AND ALONG THE LINE WE
HYPOTHESIZE MITOCHONDRIAL
DYSFUNCTION WOULD CONTRIBUTE TO
FUTURE DECLINE AND ALSO
IMPAIRMENT OF COGNITION,
POTENTIALLY THROUGH LOW ENERGY
AVAILABILITY, INCREASED STRESS
AND INFLAMMATION.
WE'RE USING DATA FROM THE
BALTIMORE LONGITUDINAL STUDY OF
AGING WHICH IS THE AMERICAN'S
LONGEST RUNNING STUDY, BEGAN IN
1958, PARTICIPANTS COMING FROM
THE COMMUNITY, SO WE'RE TESTING
THE HYPOTHESIS WHETHER
MITOCHONDRIAL FUNCTION WILL
PREDICT FUTURE DEVELOPMENT OF
COGNITIVE IMPAIRMENT OR
DEMENTIA.
WE IDENTIFIED 469 PARTICIPANTS,
INITIALLY COGNITIVE NORMAL,
AVERAGE AGE 70, DIVERSE SAMPLE
WITH MANY ETHNICITIES AND RACE,
AND FOLLOW-UP FIVE YEARS, 5%
MITOCHONDRIAL IMPAIRMENT OR
DEMENTIA.
IN SUBSET WE ALSO LOOK AT
ASSOCIATIONS BETWEEN IN VIVO
MITOCHONDRIAL FUNCTION WITH
BIOMARKERS ALZHEIMER'S DISEASE
OF THE BRAIN.
THE SPECIFIC MEASURE HERE WITH
THE SKELETAL FUNCTION IS MAXIMAL
CAPACITY, IN VIVO ASSESSMENT.
YOU SEE THE PART PANTS ARE IN
THE SCANNER, AND THEY ARE
INSTRUCTED TO PERFORM INTENSE
EXERCISE UNTIL A POINT OF
EXHAUSTION, AND THE GOAL OF THE
EXERCISE PROTOCOL TO DEPLETE
PHOSPHO-CREATINE IN THE MUSCLES.
SOLID LINE INDICATES CHANGE IN
THE MUSCLE.
WE'RE CAPTURING RECOVERY RATE IN
THE SKELETAL MUSCLE.
SO THE RATE OF RECOVERY
INDICATES A HIGHER -- FASTER
RECOVERY RATE INDICATES HIGHER
OXIDATIVE CAPACITY AND HIGHER
MITOCHONDRIAL FUNCTION.
HERE WE DENOTE AS KPCR.
WE'VE FOUND THIS HAS BEEN
ASSOCIATED WITH EX VIVO
MITOCHONDRIA RESPIRATION FROM
MUSCLE BIOPSY.
ACROSS TISSUES WE'VE SEEN
CORRELATION BETWEEN IN VIVO
OXIDATIVE CAPACITY WITH
DIFFERENT MITOCHONDRIA MARKERS.
AND FROM THE STUDY WE FOUND THAT
THE PARTICIPANT HAD HIGHER
MITOCHONDRIAL FUNCTION AT
BASELINE, THEY HAD A HIGHER
PROBABILITY OF BEING COGNITIVE
NORMAL, LOWER RISK OF DEVELOPING
COGNITIVE IMPAIRMENT OR DEMENTIA
COMPARED TO LOW MITOCHONDRIAL
FUNCTION AT BASELINE,
INDEPENDENT OF THE DEMOGRAPHIC
FACTORS AS WELL AS APOE 4
CARRIER STATUS.
WE FOUND THIS IS ASSOCIATED WITH
BIOMARKERS OF ALZHEIMER'S
DISEASE, HERE AMYLOID TRACER
UPTAKE BASED ON PET IMAGING,
ASSOCIATION PRESENT IN GRAY
MATTER AND WHITE MATTER.
AND WE ALSO FOUND THERE'S
ASSOCIATION WITH BIOMARKERS OF
NEURODEGENERATION INDICATED BY
THE GFAP, AND ALSO A TREND
TOWARDS LOWER NfL, INDICATOR
OF NEURONAL INJURY.
WE FOUND THE STRONGEST
ASSOCIATION HAS BEEN LOCALIZED
TO SPECIFIC BRAIN AREA INCLUDING
PRE-CENTRAL GYRUS, POST CENTRAL
GYRUS AND LOBE, IMPORTANT FOR
MOTOR FUNCTION AND EXECUTION.
ASSOCIATION BETWEEN
MITOCHONDRIAL FUNCTION AND
AMYLOID TRACER UPTAKE HAS BEEN
SPECIFIC TO SPECIFIC REGIONS.
IN SUMMARY THIS FINDING PROVIDES
THE FIRST EVIDENCE OF IN VIVO
MITOCHONDRIAL FUNCTION TO THE
FUTURE COGNITIVE IMPAIRMENT AND
FUNDING FROM THE BELL MARKERS
MAY INDICATE MITOCHONDRIAL
DYSFUNCTION MAY PLAY KEY ROLE IN
A.D. POTENTIAL
APPROACHESOLOGICAL CHANGES AND
NEUROINFLAMMATION.
FUTURE STUDIES SHOULD FOCUS ON
LONGITUDINAL EXAMINATION
ALSO INVESTIGATE OTHER IMPORTANT
MECHANISMS SUCH AS MITOCHONDRIAL
FUNCTION IN THE BRAIN WHICH CAN
BE ASSESSED BY M.R. SPECTROSCOPY
AS WELL AND PERFUSION, IRON
DEPRIVATION, ALSO ONE OF THE
IMPORTANT MECHANISMS OF
OXIDATIVE STRESS, ALSO WHITE
MATTER HEALTH IN THE BRAIN.
BASED ON THE OBSERVATIONAL
STUDIES IT'S IMPORTANT TO NOTE
THERE'S BEEN PRELIMINARY
EVIDENCE FROM THE LITERATURE
THAT MITOCHONDRIAL FUNCTION CAN
BE IMPROVED IN HUMANS THROUGH
BOTH NON-PHARMACOLOGICAL
APPROACH AND SUPPLEMENTS, AND
THERE'S SOME EVIDENCE FROM
TRIGLYCERIDES, SO OUR STUDIES
LAY A FOUNDATION FOR FUTURE EYE
INTERVENTION STUDIES,
EXPERIMENTAL STUDY TO TEST
WHETHER IMPROVING MITOCHONDRIAL
FUNCTION MAY IMPROVE BRAIN
HEALTH OR MUSCLE FUNCTION WITHIN
AGING.
SO WITH THAT, I'D LIKE TO
ACKNOWLEDGE THE STUDY
PARTICIPANTS AND STAFF AND MANY
CO-AUTHORS IN THIS WORK.
AND IN THE INTEREST OF TIME I'M
HAPPY TO TAKE ANY QUESTIONS.
WELL, WE'LL TAKE QUESTIONS
TOMORROW.
ALL RIGHT, THANK YOU.
[APPLAUSE]
>>LAST SPEAKER IS A A BIOMEDIL
UPPING NEAR.
TODAY I WOULD LIKE TO TALK TO
YOU ABOUT MARKER FABRICATION AND
MICROFLUIDICS FOR CELL BIOLOGY.
BACKGROUND, I WORK AT
MICROFABRICATION AND MICROFLUID
UNITS, UNDER BIOMEDICAL
ENGINEERING, NIBIB, IN BUILDING
13.
SO, LET ME START WITH
REINTRODUCING YOU TO MARKER
FABRICATION, DEVELOPED FOR
SEMICONDUCTOR INDUSTRY, CAPABLE
OF PATTERNING FEATURES DOWN TO
ONE MICRONS.
NOW, WHEN YOU GET TO MANIPULATE
FLUIDS AND GASES IN CHANNELS
THAT ARE IN MICROMETER RANGE
THAT'S CALLED MICROFLUIDICS,
USED AND APPLIED IN MANY FIELDS.
NOW, THE REASON IT IS IMPORTANT
FOR US IS BECAUSE WE CAN USE
MICROFLUIDICS AND
MICROFABRICATION TO REPLICATE
CELLULAR MICROENVIRONMENT AND
CREATE SMALL SCALE SYSTEMS.
IN THE NEXT FEW SLIDES I'LL TALK
ABOUT ONE OF THE PROJECTS WE
HAVE IN COLLABORATION WITH
RESEARCHERS, THEY WANTED TO
DEFINE NEUTROPHIL HETEROGENEITY
IN PATIENTS WITH SLE, BUT
UNDERSTANDING THE EFFECTS OF
MECHANICAL PRODUCTS OF
NEUTROPHILS AS THEY ARE
TRAFFICKING THROUGH PULMONARY
MICRO VATS -- MICROVASCULATURE,
COMPARING ROUGHNESS OF NORMAL
AND LOW DENSITY NEUTROPHILS IN
SLE PATIENTS WITH HEALTHY
CONTROLLED NORMAL DENSITY
NEUTROPHILS AND ONES THAT HAVE
THEM CHEMICALLY PRIMED.
THEY CAME WITH A ROUGH IDEA
ABOUT WHAT THEY WANTED TO DO.
AND HOW THEY WANTED TO QUANTIFY
THE NEUTROPHIL ROUGHNESS, AND
CORRELATE THAT WITH THE TIME IT
TAKES FOR THE CELL TO TRANSIT
THROUGH PULMONARY
MICROVASCULATURE, WE HAD
DISCUSSIONS, WE TOOK INTO
ACCOUNT THE READOUT REQUIREMENTS
AND CONSTRAINTS THEY HAD FOR THE
PROJECT, AND WE CAME UP WITH TWO
DIFFERENT PATTERNS, TWO
DIFFERENT DEVICE PATTERNS.
DESIGN 1, BRANCHING NETWORK
WHICH REPLICATES PULMONARY
VASCULATURE, DESIGN 2 MULTIPLE
CONSTRUCTIONS, SINGLE CHANNEL.
THAT MIMICS CAPILLARY BED.
USING THE CAD DRAWINGS WE
ORDERED A PHOTOMASK.
ONCE WE HAVE THE MASK WE USE IT
IN A PROCESS CALLED
PHOTOLITERATUROGRAPHY, WE COAT
THE SURFACE WITH A
PHOTORESISTANT MATERIAL, SHINE
UV LIGHT THROUGH THE PHOTO MASK,
IN THIS CASE AREAS OF
PHOTORESIST, WILL CROSS-LINK,
EVERYTHING ELSE IS WASHED OFF.
WHAT YOU END UP WITH IS SILICONE
VAPOR WITH FEATURES THAT CAN BE
USEDDED A TEMPLATE OR MOLD TO
MAKE DEVICES.
ONCE YOU HAVE THOSE TEMPLATES,
WE CAST CURE IT, UNMOLD, CUT
THEM, PUNCH IN OUTLETS AND
PREPARE THEM FOR THE
EXPERIMENTS.
OUR COLLABORATORS TRIED ALL THE
DIFFERENT DESIGN ITERATIONS WE
PROVIDED TO THEM AND THEY ENDED
UP USING THESE BRANCHING
NETWORKS FOR THEIR EXPERIMENTS.
AND WHAT THEY DID WAS THAT THEY
INTRODUCED ONE CELL AT A TIME,
TO THIS BRANCHING NETWORK, AND
THEY MEASURED HOW LONG IT WILL
TAKE FOR A CELL TO ENTER AND
THEN EXIT.
THERE'S A CORRELATION BETWEEN
CELL ROUGHNESS AND HOW LONG IT
WILL -- HOW LONG THE CELLS WILL
STAY IN THE BRANCHING SYSTEM.
TO CHANGE GEARS A LITTLE BIT I'M
GOING TO
BEADS AND CAPSULES, HERE ON THE
LEFT YOU'RE LOOKING TO PHASES
COMING TOGETHER MAKING DROPS IN
OIL, USUALLY FOR EXPERIMENTS
LIKE THIS WE RUN THE CELLS LIEU,
EACH DROP WILL HAVE MAXIMUM OF
ONE CELL, ONE OF THE
METHODS FOR SORTING IS FLOW
CYTOMETRY BUT THE DROPS AT THE
STAIN ARE NOT COMPATIBLE WITH
FLOW CYTOMETRY BECAUSE THEY ARE
SURROUNDED BY OIL.
THERE ARE A ANOTHER SET OF
DEVICES, YOU'RE LOOKING AT HALF
HERE, IN THE FIRST PART OF THE
DEVICE WE HAVE THE WATER AND OIL
DROPS, AND ON THE SAME DEVICE
THE DROPS ARE REENCAPSULATED,
NOW WE HAVE WATER AND OIL IN
WATER DROPS THAT ARE NOW
COMPATIBLE WITH FLOW CYTOMETRY.
THE SKY IS THE LIMIT WITH
FABRICATION, I HAVE MORE
EXAMPLES FOR YOU.
HERE YOU'RE SEEING AN EXAMPLE OF
DEVICES USED FOR CULTURE AND
ISOLATION OF AXONS.
IN THE MIDDLE WE HAVE AN EXAMPLE
OF TISSUE, ON A CHIP, HERE
FIBROSIS ON A CHIP IS STUDIED.
LASTLY USE OF PBMS DEVICES FOR
PATTERNING SURFACES, YOU CAN SEE
PILLARS ABOUT ONE MILLIMETER IN
DIAMETER, COATED WITH MATRIGEL,
STAMPED ON A GLASS, CELLS ARE
SEATED THERE, YOU CAN SEE
SPATIAL ORGANIZATIONS.
I'LL BE AVAILABLE TOMORROW IF
YOU HAVE QUESTIONS ON THE POSTER
SESSION.
YOU CAN GO INTO THE WEBSITE AND
SEE WHAT CAPABILITIES WE HAVE ON
CAMPUS.
LASTLY, BUT NOT LEAST, I THANK
MY MENTOR DR. MORGAN, THE
ORGANIZERS, FOR THIS SEMINAR,
AND GIVING ME THE OPPORTUNITY TO
TALK AND THANK YOU ALL
EVERYTHING.
>>I HAVE A BUNCH OF QUESTIONS.
I HAVE TO CLOSE THE MEETING FOR
RECORDING PURPOSES.
THANK YOU SO MUCH, EVERYONE.