WELCOME EVERYONE TO THIS WEEK'S IIG SEMINAR AND IT'S REAL LY A VERY SPECIAL PLEASURE FOR ME TO INTRODUCE TODAY'S SPEAKER AND THAT'S DR. PHIL EPIGENETIC SCOTT, MANY OF YOU KNOW--PHILLIP SCOTT, MANY OF YOU KNOW. AS A FORMER NIH IMMUNOLOGY POST DOC WHO LEFT US TO ESTABLISH A DISTINGUISHED AND HIGHLY SUCCESS FUL CAREER IN ACADEMIA, PHIL IS A GREAT ROLE MODEL FOR YOU TRAINEES IN THIS AUDIENCE, SO PHIL RECEIVED HIS Ph.D. FROM THE VET SCHOOL AT THE UNIVERSITY OF PENNSYLVANIA WORK ING ON LEESH LEISHMANIASIS WHICH IS AN EXCITING STYLE OF STUDYING T-CELL REGULATION.--SO AS PHIL WILL EXPLAIN TO YOU IN HIS TALK, I'M SURE, L-MAJOR IS NOTICEABLE--NOTABLE FOR ITS DIFFERENT DISEASE OUTCOMES IN MICE WITH THE PARASITE CAUSING EITHER SELF-HEALING LESIONS OR EXACERBATED INFECTION, AND AT THAT TIME IS THAT BOTH OF THESE OUTCOMES WOULD BE DETERMINED BY CDFOUR RISM O SIGHTS. AND SO IN COLLAB COLLABORATION WITH BOB CATCH MAN AND DR. PIERCE, PHIL SOLVED THIS PARADOX BY SHOWING THAT THESE POLEAR APPOSITE FEKS INFECTION OUTCOMES WERE DETERMINED BY DIFFERENT THONE VERSUS TH-TWO CD FOUR SUBSETS AND THIS IS REAL LY ONE OF THE FIRST DEMONSTRATEISTRATIONS OF THE DIS TINCT FUNCTIONAL ROLE OF T- HELPER SUBSETS IN INFECTION AND WAS CONFIRM INDEED A LATER RELATED STORY. ON LEISHMANIASIS MODEL FROM RICHARD LOCKSLY WHICH WAS PUBLISHED SEVERAL MONTHS LATER. SO AFTER HIS SUCCESSFUL POST DOC HERE AT NIH, PHIL WAS RECRUITED BACK TO HIS OLD DEPARTMENT AT PE NN, WHERE MIRACULOUSLY WITHIN A FEW YEARS HE ADVANCED FROM ASSISTANT TO FULL PROFESSOR AND THEN CHAIR OF HIS DEPARTMENT. SO DURING THIS PERIOD, PHIL'S LAB PUBLISHED FRONT LINE TODAYS IN THE THONE-IMMUNITY AGAINST LEERK--LEISHMANIASIS. IN THIS WORK, PHIL AND GEORGEIO DEMONSTRATED THAT IL12 CAN SERVE AS A POTENT STATANT ADJUVANT FOR INDUCING HIGHLY POLARIZED TH-ONE VACCINE RESPONSES AND IN MORE REENLT TIMES PHIL'S GROUP HAS FOCUSED ON THE NATURE OF CDFOUR T-CELL MEMORY IN THE LEISHMANIASIS DEMONSTRATING THAT THE EXISTENCE OF LONG LIVED MEMORY CELLS IN THE ABSENCE OF PARASITES AND OF A POPULATION OF HOST PROTECTIVE SKIN RESIDENT PEOPLERY CD-FOUR T-CELLS AND IN THEIR CURRENT WORK WHICH FILL WILL TALK ABOUT TODAY, IS SCOTT LAB AND THEIR CLINICAL COLLABORATORS IN BRAZIL HAVE IDENTIFIED A MAJOR ROLE FOR T- CELLS INDUCED CUE TAINIOUS PATHOLOGY AND UNCOVERED AN UN EXPECTED FUNCTION FOR INFLAMMA STUDIES OF MULTIPLE ENDOCRINE DEPENDENT ILONE BETA IN THIS RE SPONSE. SO IN ADDITION TO HIS FRONT LINE RESEARCH, PHIL AS A PENN VET SCHOOL DEPARTMENT CHAIR AND NOW VICE DEAN HAS RECUTED A HIGHLY PRODUCTIVE AND INFLUENTIAL GROUP OF FACULTY MEMBERS INCLUDING CHRIS HUNTER WHO MANY OF US KNOW HERE. AND THESE FOLKS OVER THE YEARS HAVE MADE MAJOR CONTRIBUTIONS TO PARISATOLOGY AND FUNDAMENTAL IMMUNOLOGY AT BOTH PENN AND NATIONALLY. HE AND HIS COLLEAGUES HAVE ALSO BEEN ENTHUSIASTIC PARTICIPANTS IN THE U PENN NIH GRADUATE PARTNERSHIP PROGRAM. FINALLY I WANT TO SAY THAT ALTHOUGH PHIL IS BEST IMROAN FOR HIS WORK ON EFFECTOR MEMORY T- CELL BIOLOGY, HE IS DEEPLY COMMITTED TO APPLYING THIS KNOWLEDGE TO ALLEVIATING HUMAN DISEASE AND AS YOU'LL SEE, IN HIS TALK, HE DEVOTES A MAJOR EFFORT OF HIS GROUP'S WORK TO CLINICAL STUDIES IN THE NORTHEAST OF BRAZIL, AND TO THE DEVELOPMENT OF HOST DIRECTED THERAPEUTIC APPROACHES THAT COULD BE USED IN PATIENTS. SO PHIL, ON BEHALF OF ALL OF US, WELCOME BACK TO THE NIH AND WE LOOK TO YOUR TALK WHICH IS ENTITLED--IT'S COMING UP. DEFINING MECHANISMS--NO, PATHWAY S LEADING--NO. [LAUGHTER] NO. IT'S REALLY IT DOESN'T MATTER BUT IT'S DEFINING MECHANISMS OF PATHOGENESIS IN CUE TAINIOUS LEISHMANIASIS TO DEVELOP NEW APPROACHES TO THERAPY. >> THAT WAS GREAT. I THINK I WILL STOP NOW. IT'S REALLY, REALLY FUN TO BE BACK. I HAVE BEEN BACK SEVERAL TIMES SINCE I LEFT BUT THIS HAS BEEN A GREAT DAY AND TALKING TO A LOT OF PEOPLE AND JUST HAD A GREAT TIME. SO ALAN TOLD ME SOMETHING LAST NIGHT. SO I USED THIS QUOTE MANY TIMES AND IT'S A QUOTE THAT I THOUGHT WAS ORIGINAL WITH ALAN, WHICH IS THAT LEISHMANIASIS IS A NEGLECT ED DISEASE THAT HAS DONE FOR IMMUNOLOGY THAN IMEULOG IOLOGY HAS DONE FOR IT. IT'S TRUE. IT TURNS OUT HE STOLE IT FROM SOMEBODY ELSE AFTER A FEW DRINKS BUT IT IS TRUE AND I WILL TALK TODAY A BIT ABOUT SOME OF THE THINGS THEY THINK THAT I HOPE MAY ACTUALLY DO SOMETHING FOR THE PATIENTS. LET ME START OUT AND INTRODUCE THIS IDEA ABOUT WHY DID IMMUN OLOGYSTS EVER CARE ABOUT LEISHMANIASIS AT THE TIME? THEY DIDN'T CARE ABOUT IT BECAUSE OF PATIENTS BUT THEY CARED ABOUT IT THAT YOU COULD INFECT THE MICE AND DEPENDNG ON THE MOUSE STRAIN, HAD A HEALING INFECTION OR NONLATELYING INFECTION AND THAT WAS BASED ON WHETHER THE ANIMAL DEVELOPED A T H-ONE OR TWO RESPONSE AND AS AL AN MENTIONED THIS IS THE LAST PAPER WE PUBLISHED TOGETHER BEFORE I LEFT 30 YEARS AGO. SO 30 YEARS AGO, WE KNEW ABOUT T H-ONE AND TWO LEISHMANIASIS BUT YOU WOULD THINK WE HAVE A VACCINE BY NOW, BUT NO, NO. THIRTY YEARS LATER WE STILL DON'T HAVE A VACCINE EVEN THOUGH WE UNDERSTAND WHAT IMMUNITY IS ABOUT. I WILL MENTION THAT IN A MINUTE. SO I'M GOING TO TALK ABOUT CUE TAINIOUS LEISHMANIASIS AND IF YOU'RE THINKING ABOUT--LIVES IN MACROFAJS, DENDRITIC CELLS, NUTRIFILLS AND IL12'S CRITICAL IN PRIMING THONE CELLS AND PRIMING T-CELLS AND THEN ACT IVATING THESE CELLS TO KILL THE PARASITE AND AGAIN AS I KIND OF POINT OUT IN SPITE OF THAT ALL THIS INFORMATION THAT WE KNOW ABOUT TH-ONE CELLS AND CYTO KINES, ET CETERA, ALL OF THAT HAS NOT LEAD TO THE VACCINE FOR LEISHMANIASIS SO WE'RE IN MY LAB STUDYING MEMORY T-CELLS AND YOU MIGHT ASK WHY THAT IS. WHY DON'T WE HAVE A VACCINE? SO MY VIEW IS PRIMARILY IT'S BECAUSE WE DON'T UNDERSTAND HOW TO GENERATE LONG-TERM MEMORY CELLS WE DON'T UNDERSTAND WHICH OF THE ONES ARE GOING TO BE PRO TECTIVE. SO I'M NOT GOING TO TALK ABOUT TODAY BECAUSE I DON'T HAVE TIME IS OUR WORK ON RESIDENT MEMORY T -CELLS AND YOU I WILL SAY THAT OUR VIEW AT THE MOMENT IS S&P THIS IS THE TARGET POPULATION WE SHOULD BE LOOKING AT IN THE VACCINE. SKIN CELLS THAT ARE LONG LIVED IN THE ABSENCE OF PERSISTENT PARASITES SO I DON'T HAVE TIME TO TALK ABOUT THAT BUT I WILL GIVE A SHOUT OUT TO THAT IDEA. SO I THINK THAT THE REAL REASON IMMUNOLOGYSTS SHOULD BE INTEREST ED IN LEISHMANIASIS IS SUMMED UP THAL--THAL SLIDE. THIS IS SUCH AN INTERESTING DISEASE WHEN YOU LOOK AT THE CLINICAL PRESENTATIONS THAT TR OCCUR. AND THIS SPECTRUM OF CLINICAL PRESENTATION SYSTEM SOMEWHAT SIMILAR TO WHAT YOU SEE WITH TUBERCULOSIS AND LEPROSY, COUPLE OTHER DISEASES AS WELL, AND WHAT 'S ALWAYS BEEN VERY INTERESTING IS THAT DEPENDING UPON THE HOST IMMUNE RESPONSE, CAN YOU GET A VARIETY OF DIFFERENT CLINICAL PRESENTATIONS AND AT THE SWEET SPOT YOU GET LESIONS THAT ARE RELATIVELY BENIGN THAT WILL RESOLVE OVER SOMEWHERE BETWEEN THREE-SIX- EIGHT TO NINE NONTHS. BUT AT ONE END OF THE SPECTRUM, YOU HAVE A VERY POOR CELL IMMUN ITY RESPONSE, LOTS OF PARASITES, LITTLE LYMPHOCYTIC IN FILTRATION AND LOTS OF ANTI BODIES AND THESE PATIENTS ARE VERY DIFFICULT TO DRUG TREAT BUT THE OTHER INTERESTING END OF THE SPECTRUM IS THIS END WHERE YOU HAVE DISEASES EITHER SINGLE LESIONS THAT ARE REALLY QUITE SEVERE OR THE MORE SEVERE FORMS OF MUCOSAL DISEASE WHERE YOU HAVE TONS OF INFLAMMATORY CELLS, VERY FEW PARASITES, AND THIS DISEASE IS ALSO EXTREMLY DIFFICULT TO DRUG STREET. --TREAT. SO THIS KIND OF SHOWS THE SPECTRUM FROM INFLAMMATION AT THIS END TO VERY LITTLE INFLAMM ATION ABOUT LOTS OF PARASITES. SO WHAT WE'VE BEEN INTERESTED IN FOR QUITE A WHILE HAS BEEN DEFINING THE MECH MECHANISMS THAT ARE ASSOCIATE WIDE PROMOT ING THIS INFLAMMATORY RE SPONSE, WITH THE IDEA OF IDENTIFYING THOSE MECHANISMS AND THINKING ABOUT WHAT WAYS WE MIGHT BLOCK THAT PATHOLOGY. SO LET ME JUST SAY ONE THING BEFORE I GO FORWARD. I HAD ALWAYS THOUGHT THAT THIS END OF THE SPECTRUM WAS BECAUSE THERE WERE JUST TOO MANY TH-ONE CELLS, TOO MUCH GAMMA INTERFERON AND THAT WASENTIOUS SENTIALLY WHAT WAS CAUSING THE PATHOLOGY AND IF THAT WAS THE CASE, IT MIGHT BE DIFFICULT TO THINK ABOUT BLOCKING GAMMA INTERFERON BECAUSE AT THE SAME TIME YOU'RE BLOCKING GAMMA INTERFERON YOU MAY WELL BE ALLOWING PARASITES TO GROW UP AND OBVIOUSLY CAUSE DISEASE. SO IF THAT WAS THE CASE, IT WAS GOING TO BE A REAL DIFFICULT THING TO THINK ABOUT HOST DIRECT ED THERAPIES. AND IT TURNS OUT THAT'S NOT THE CASE AND SO WE'VE BEEN INTEREST ED IN THIS END OF THE SPECTRUM AND THAT'S WHAT I WILL TALK ABOUT TODAY. SO I WILL TALK ABOUT KNEE THINGS TODAY. THE FIRST I WILL TALK ABOUT HOW CYTOLYTIC CD8 T-CELLS RATHER THAN CDFOUR THONE CELLS PROMOTE DISEASE. I WILL THEN TALK ABOUT SOME UN PUBLISHED DATA THAT WE HAVE BASED ON RNASEQ DATA FROM THESE PATIENTS AND HOW VARIATIONS IN COMEGZ EXPRESSION OF CYTOLYTIC GENES, INFLUENCE THESE OUTCOME AND ASSUMING I HAVE TIME I WILL TALK ABOUT HOW THE SKIN MICRO BIOME MAY ALSO INNERCLUED THE PATHOLOGY OF THE CUTANEOUS LEISHMANIASIS. SO FIRST I WANT TO THANK THE PEOPLE WHO HAVE DONE THE WORK. THIS HAS BEEN A PHENOMENAL COLLABORATION BETWEEN MY LAB AND EDGAR'S LAB DOWN IN BRAZIL, AS WELL AS HIS SON LUCAS AND A VERY SUCCESSFUL BRAZILIAN POST DOC IN MY LAB WHO CAME SEVERAL YEARS AGO AND IS RESPONSIBLE FOR THE DEVELOPMENT OF THIS WHOLE PROJECT THAT WE'RE WORKING ON. IN ADDITION I HAVE LITTLER PHENOMENAL COLLABORATORS. ONE HAS BEEN DAN BITING WHO IS RESPONSIBLE FOR THE RNA SEQ ANALYSIS, I'M NOT DOING THE BIO-INFORMATICS, LET ME MAKE THAT VERY CLEAR. AND SO THIS IS WORK THAT DAN'S BEEN DOING AND REALLY HELPING TRAIN A VERY TALENTED POST DOC FROM BRAZIL, CAMILA WHO IS DOING MOST OF THE BIO-INFORMATICS WORK I WILL BE TALKING ABOUT. I ALSO HAVE A COLLABORATION WITH ELIZABETH GREIS WHO WAS AT NIH AND HE'S BEEN CRITICAL IN ANY OF THE SKIN MICROBIOME WE'RE DOING AND [INDISCERNIBLE] AS WELL AND FINALLY ANOTHER BRAZILIAN POST DOC WHO RECENTLY CAME AND IS INVOLVED IN THIS PROJECT. YOU MIGHT NOTICE A THEME HERE, NOTICE LIKE A LITTLE BIT OF A THEME, BRAZILIAN POST DOCKERS, VERY SUCCESSFUL AND CRITICAL FOR MY LAB, SO WHERE IS THIS PROJECT , THIS IS A PROJECT AS I SAID IN SALVADOR, THEREYA A FIELD SITE THAT'S ABOUT THREE AND HALF HOURS FROM SALVADOR. WE SEE BETWEEN 151800 PATIENTS AT THIS HEALTH POST PER YEAR WITH CUTANEOUS LEISHMANIASIS AND THE ISSUE REALLY WHEN WE STARTED WORKING ON THIS WAS THAT THERE REALLY WERE NO EXPERIMENTAL MODELS THAT REALLY STUDIED THIS TYPE OF INFECTION. THE EXPERIMENTAL MODELS I TOLD YOU IN THE BEGINNING, THEY DIDN'T REALLY EXPLAIN THE DISEASE WE WERE SEEING. SO THE FIRST THING WE DID, SEVERAL YEARS AGO WAS WE WENT TO BRAZIL AND WE STARTED BY LOOKING AT WHAT'S BEING EXPRESSED IN THE LESIONS AND SO, THE FIRST THING WE DO IS THE MICROARRAY STUDY WITH 27 PATIENTS, SUBSIDIARY QUEBTLY THOSE SAME SAMPLES WENT TO DAVID MOSSIER'S LABORATORY AND THEY DID RNA SEQ, AND STEVE CHRISTIANSEN WHO DID THAT WORK HERE WITH DAVID AND I WILL TELL YOU WHAT THAT INITIALLY SHOWED US. SO THIS IS OLD DATA BUT IT IND IGATEICATE--INDICATES SOMING WE WERE NOT EXPECTING AND IT'S SHOWN HERE. THAT THE GENES THAT WERE MOST HIGHLY UPREGULATED IN THESE LESIONS FROM CUTANEOUS PATIENTS WERE GENES ASSOCIATED WITH SIGNIFY TOLL-LIKE RECEPTOR SIS, GRANZYME B, PERFORIN AND THEY WEREN'T WHAT I WAS SPECTING AT ALL. I KNEW TNF WOULD BE UP AND GAMMA WOULD BE UP AND THEY ARE BUT THESE I DIDN'T EXPECT. SO THAT RAISED THE QUESTION OF WHETHER THE CYTOLYTIC RESPONSE CONTRIBUTED TO THE PATHOLOGY WE'RE SEEING IN CUTANEOUS LEISHMANIASIS. THE PROBLEM WAS WE DIDN'T HAVE EXPERIMENTAL MODELS AT THAT TIME TO REALLY DISSECT WHAT WAS HAPPENING. WE SUMMIZED FROM SOME OF OUR WORK IN PATIENTS THAT IT WAS CD- EIGHT MEDIATED AND SO WHAT FER NANDA DID IS SHE DEVELOPED TWO MODELS TO BEGIN TO UNDERSTAND EXPERIMENTALLY IN MICE WHAT MIGHT BE HAPPENING AND ONE WAS REALLY A VERY EXTREMELY REDUCTIONIST MODEL WHERE WE TOOK RAG MICE. SHE TOOK RAG MICE AND TRANSFER RED INTO THOSE MICE CD- EIGHT T-CELLS AND LOOKED AT THE DISEASE AND I'LL SHOW YOU WHAT THAT DISEASE LOOKS LIKE AND THE OTHER MODEL WAS ONE WHERE WE EITHER INFECTED MICE WITH LCMV AND WAITED TOO MONTHS AND THEN CHALLENGED WITH LEISHMANIASIS OR CO INFECTED AND IN EITHER CASE WE GOT EXACERBATED DISEASE AND IT WAS CD8 T-CELL DEPENDENT. I WON'T TALK A LOT ABOUT THAT BUT I WILL MENTION IT A BIT. SO THAT WAS--MODEL THAT WE DECIDED TO ASK ARE CD8 T-CELLS REALLY PATHOLOGIC, WHY ARE THEY CAUSING PATHOLOGY IF THEY ARE? THIS IS AGAIN DATA FROM A WHILE AGO NOW, IT WAS ACTUALLY ONE OF FER NANDA'S FIRST PAPERS IN THE LAB AND AGAIN I SHOWED YOU THE MODEL HERE AND THIS IS THE COURSE OF INFECTION THIS, IS EAR THICKNESS, SIZE OF THE LESION AS AN INDICATION OF THE DISEASE, OVER TIME AND WHAT YOU SEE IS IF YOU INFECT RAG MICE WITH LEISHMANIASIS YOU DON'T SEE MUCH OF A LESION AND THAT ALREADY TELLS YOU SOMETHING INTERESTING AND THAT IS THAT ALTHOUGH THERE'S A LOTS OF PARAICIDE SIGHTS THERE--PARASITES THERE THOSE LESIONS ARE ABOUT INFLAMM ATORY RESPONSE, NOT SURPRISING BUT THAT'S WHAT THAT SAYS, IF YOU TRANSFER IN CDFOUR AND CD8 T-CELLS YOU SEE A SMALL LESION WHICH IS CONTROLLED BUT THE INTERESTING ONE IS IF YOU TRANSFERRED IN CD8 T-CELLS ONLY, YOU SEE THIS REALLY SEVERE LESION SHOWN HERE AND YOU ALSO SEE METASTASIS TO THE OTHER EAR THAT WAS NOT INFECTED TO THE TAIL, THE NOSE, ET CETERA AND IN THE PRESENCE OF CD8 T-CELLS YOU SAW THIS SEVERE DISEASE. IMPORTANTLY THE NUMBER OF PARASITES IN THIS EAR, THAT A MOUSE THAT GOT CD8 T-CELLS AND THIS RAG MOUSE WAS THE SAME. SO THIS HAD NOTHING TO DO WITH PARASITE NUMBERS, ONLY TO DO WITH THE FACT THAT THERE ARE CD8 T-CELLS PRESUMABLY CAUSING THIS PATHOLOGY. SO IT TURNS OUT THAT THOSE CD8 T -CELLS WERE CYTOLYTIC, FER FERNANDO WENT ON TO SHOW BY IMAGERY AND SIGNIFY TOMETRY AND TRANSFER EXPERIMENTS THAT THIS IS THE GREEN CD8 T-CELL, THESE ARE MACROPHAGES INFEBLGHTED WITH LEISHMANIASIS THAT'S RED AND STAINED FOR LAMP ONE. IT'S SHOWING AT THE SYNAPSE THAT THESE ARE CD8 T-CELLS ARE BIND ING THERE AND IN MAY MOVIE I HAD TO TAKE OUT BECAUSE IT WAS N'T WORKING SHE WAS ABLE TO SHOW THAT IN FACT THIS LEADS TO LYSIS OF THE TARGET CELL. SO THAT'S STILL DOESN'T TELL YOU WHETHER IT'S A GOOD OR BAD THING SO THE OBVIOUS EXPERIMENT TO DO IS TO TRANSFER IN CD8 T-CELLS THAT CAN'T BE CYTOLYTIC WHICH WOULD BE PERFORIN KNOCK OUT CD8 T-CELLS AND THAT'S SOME EXPERIMENT AND WHAT YOU SEE IS THESE ARE WILD TYPE CD8 T-CELLS, THESE ARE PERFORIN KNOCK OUT CD8 T-CELLS TO BE CYTOLYTIC THERE ISN'T ANY DISEASE. SO FROM THAT, SHE DEVELOPED A MODEL SHOWN HERE, CD8 T-CELLS LIESING TARGETS, AND WE HAVE IDEAS THAT THAT MAY LEAD TO METASTASIS, I HAVE TO SAY WE HAVEN'T TRACKED THAT DOWN, BUT PERFORIN GRAND ZYME B CELL DEATH SOMEWHERE ALONG THIS PATHWAY IT LED TO INFLAMMATION AND AT THE SAME TIME WE FOUND THAT BI STANDARD CD8S AND THAT WOULD BE THE CD8S THAT WERE LC AND CD SPECIFIC COULD INFILTRATE TRAIT LESIONS AND ALTHOUGH THEY WERE NOT SPECIFIC, THEY WERE ABLE TO KILL TARGETS BECAUSE THEY EXPRESS NKDTWO D, AND WE SHOWED THAT THAT ALSO LED TO--OR THAT INTERACTION ALSO LED TO LYSIS OF PATHOLOGY. SO NOW, IN A VARIETY OF STUDIES BOTH FROM OUR LAB AS WELL AS OTHERS, IT'S PRETTY CLEAR THAT CD8 T-CELLS AND LEISHMANIASIS CAN BE PATHOLOGIC. BUT THE QUESTION THAT THAT RAISE S ARE SEVERAL. THE QUESTIONS THAT RAISES ARE SEVERAL. ONE THING THAT WE'RE INTERESTED IN IS HOW IMPORTANT ARE THESE BI STANDARD CD8 T-CELLS SO THOSE ARE EXPERIMENTS THAT ARE ONGOING IN BRAZIL PARTICULARLY LOOKING AT NKT-TWO D CELLS COMING INTO LESIONS. THE OTHER, THERE WE GO, THE OTHER QUESTION WAS WHAT REGULATE S THESE CD8 T-CELLS? SO IT TURNS OUT THE CD8 T-CELLS THAT GO INTO THE SKIN ARE CYTO LYTIC BUT THEY DON'T MAKE INTERFERON GAMMA SO IN THAT TISSUE ENVIRONMENT THERE ARE SIGNALINGS THAT THAT DETERMINE WHETHER THE CD8S CAN BE GAMMA PRODUCERS OR WHETHER THEY'RE GOING TO BE CYTOLYTIC AND ACTUAL LY FERNANDEZ NOW TRYING TO TRACK DOWN THOSE SIGNALINGS IN VOLVED IN CD8 T-CELL FUNCTION. WHAT I WANT TO SHOW YOU IS DATA ON HOW THIS SIGNIFY TOLL-LIKE RECEPTOR SIS MIGHT LEAD TO INFLAMMATION SO THERE'S A LOGICAL, VERY LOGICAL TARGET HERE OR LOGICAL POSSIBILITY OF WHAT THAT WILL BE AND IT'S ILONE BETA AND SO GOING BACK TO THE HUMAN DATA AND LOOKING AT SOME OF THE GENE EXPRESSION, WHAT WE FOUND WAS THAT ILONE BETA GENE EXPRESSION CORRELATED WITH GRAN ZYME B, SWRAN ZYME A, PERFOR IN, IF WE LOOKED AT THE FOLD CHANGE, ILONE BETA HAD A GREATER FOLD CHANGE THAN ANYTHING ELSE WE THOUGHT WOULD BE IMPORTANT--IF WE BLOCK ILONE BETA, YOU BLOCK THE PATHOLOGY BUT ANTIONE-ALPHA DIDN'T REALLY DO THAT, FINALLY THAT LED TO THE OBVIOUS QUESTION OF GHEE IS THIS POTENTIALLY SOMETHING THAT WOULD BE THERAPEUTIC AND THEN OUR INITIAL EXPERIMENTS WE USED A DRUG CALLED ANTIKIN RAWHICH BLOCKS THE ILONE RECEPTOR, AND EXPERIMENTS WERE DONE AGAIN IN THE RAG MOUSE OR IN THIS CO INFECTION IN MOUSE, WHERE WE TREATED TWO WEEKS AFTER INFECTION WITH LEISHMANIASIS OR TEN DAYS AFTER THE LCMV CLEARED AND THE VIRUS SHOULD BE GONE BY THEN AND THE RESULTS ARE SHOWN LEERK--LEISHMANIASIS, AGAIN THIS IS THE LARGE LESIONS YOU SEE IN CD8 RECONSITUTED RAG MICE. IN THE LCMV MICE YOU SEE INCREASED PATHOLOGY. IF YOU TREAT WITH ANAKINRA, YOU BLOCK THAT IN BOTH OF THESE MODELS. SO THAT SUGJOAOF THES THAT RIERKS LONE BETA IS CRITICAL AND IF WE CAN BLOCK ILONE BETA WE MAY BE ABLE TO BLOCK PATHOLOGY IN THE EXPERIMENTS I'M NOT GOING TO SHOW YOU, WE ASKED WHETHER OR NOT NLRP-THREE AND INVOLVED IN THE PATHWAY AND WE FOUND THAT INDEED IN CASPACE 111 KNOCK OUTS THESE ANIMALS DIDN'T DEVELOP DISEASE. SO THAT KIND OF LEADS YOU TO THE IDEA THAT THERE'S A SERIES OF PATHWAYS OR I SHOULD SAY POTENTIAL TARGETS FOR HOST DIRECTED THERAPY. AND I KIND OF POINTED OUT SOME OF THEM HERE, THESE ARE THE OBVIOUS ONES FROM WHAT I TALKED ABOUT AND WE'VE GONE ON TO SHOW THAT NLRPTHREE INHIBITORS CAN BLOCK THIS PATHWAY IN THE EXPERIMENTAL MODELS AND THAT ANA KIIN, RA, AS WELL AS IL-ONE BETA CAN BLOCK THE PATHWAY IN THE EXPERIMENTAL MODELS SO ONE THING WE'RE INTERESTED IN NOW IS TALKING TO NOVARTIS, ABOUT TRY ING TO DO A CLINICAL TRIAL OF KINKUNA MAB WITH THIS DRUG, WHICH IS A TERRIBLE, BUT VERY IN ENTIOUS FICIENT AT CONTROLLING DISEASE, AND FRANKLY IN NORTHEAST BRAZIL, THE NUMBER OF PATIENTS THAT UNDERGO THIS TREATMENT THAT FAIL THE FIRST ROUND OF TREATMENT IS IN THE ORD ER OF 40-60%. IT'S 21 DAYS OF IV INJECTION OF ANTIMONEY. DEVELOPED 70 YEARS AGO, THAT'S WHAT WE'RE TREATING THESE PATIENTS WITH SO IT'S PRETTY HORRIFIC, SO WHAT HAPPENS WHEN YOU FAIL TREATMENT? YOU G--GO BACK AT 90 DAYS AND THEY EVALUATE YOU AND IF YOU FAIL TREATMENT THEY DO ANOTHER 21 DAYS OF TREATMENT SO THEN IF THAT FAILS THEY MAY GO TO A SECOND LINE DRUG BUT THAT'S KIND OF WHAT THE STATE-OF-THE-ART IS. THE OTHER THICK THAT I--THING I DIDN'T HAVE TIME TO GO INTO IN DEPTH AND THE OTHER THERAPY THAT WE'RE PARTICULARLY INTERESTED IN AS WELL IS BLOCKING JACK 13, AND WE FOUND OUT TAUPA FISCHERIN BLOCKS THE PATHOLOGY SO WE'RE INTERESTED IN WHETHER OR NOT THIS OR SOME OF THE OTHER JACK INHIBITORS MIGHT BE USEFUL IN PATIENTS PARTICULARLY IF THEY'RE TOPICAL, IF WE CAN USE THEM TOP ICALLY. SO THAT'S WHERE WE ARE ON KIND OF THINK OF TARGETS FOR CONTROL LING--CONTROLLING THE DISEASE IN THESE PATIENTS. SO THE SECOND THING I WANT TO TALK ABOUT, IS SOMETHING THAT IS UNPUBLISHED AT THE MOMENT AND IT ECHOES BACK TO THE RNA SEQ DATA AND IT'S THE QUESTION THAT WE HADN'T THOUGHT ABOUT INITIALLY IS GHEE, WE KNOW THAT THESE GENE S ARE UPREGULATED IN PATIENT S AND NOT IN THE NORMAL SKIN BUT WE ALSO THOUGHT, YOU KNOW HOW ARE THEY VARIABLE IN TERMS OF THEIR EXPRESSION AND WOULD THAT VARIABILITY MEAN ANYTHING? SO YOU HAVE SOME PATIENTS THAT HAVE HIGH LEVEL OF EXPRESSION, ARE THEY GOING TO HAVE A DIFFERENT PHENOTYPE THAN THOSE WITH A LOW LEVEL, EVEN THOUGH EVERYBODY HAS MORE THAN NORMAL SKIN. SO THESE EXPERIMENTS WERE DONE WITH ANOTHER SET OF PATIENTS WHICH YOU COLLECTED LAST YEAR. THIS GROUP HAS 21 PATIENTS. WE'VE LOOKED AT LESIONS, LOOKED AT BLOOD AND QPC R FOR PARASITES AS WELL, AND SO, IN THIS STAD,-- STUDY WHAT WE FIND AND THIS IS ALL RNA-SEQ DATA SO INDEED THESE FOUR GENES THAT WE'RE PARTICULARLY INTERESTED IN , THERE IS A LOT OF VARIATION, ALTHOUGH ALL OF THEM ARE GREATER IN TERMS OF EXPRESSION COMPARED TO HEALTHY SKIN, WHAT YOU SEE IS A SIGNIFICANT VARIATION IN THEIR EXPRESSION. YOU SAID AH, OKAY, SO MAYBE THAT 'S IMPORTANT, RIGHT? SO INSTEAD OF JUST ASSUMING THESE ARE THE MOST VARIABLE GENE S WHAT WE NEXT DID WAS ASK, WELL ARE THESE THE ONLY VARIABLE GENES ARE OR ARE THERE OTHER GENES THAT ARE VARIABLE THAT MIGHT BE IMPORTANT AS WELL. BECAUSE WE'RE BI TAC WE THINK THESE WILL BE THE IMPORTANT GENE S AND SO WHAT WE DID IS WE FOCUSED ON GENES THAT HAD A HIGH COENTIOUS FICIENT OF VARIATION AND A LARGE FOLD CHANGE AND THAT 'S REALLY ROUGHLY SEEN IN THIS CIRCLE HERE. AND SO THERE WERE CERTAIN GROUPS OF GENES, FIRST OF ALL FORTUNATE LY AS WE WOULD HAVE EXPECTED BASED ON WHAT I JUST SHOWED YOU, THIS INCLUDED THE FOUR GENES WE'RE INTERESTED IN AND INCLUDED A VARIETY OF CHEMO KINES THAT ARE IN RED, DAVID MOS SIER AND STEVE, YEAH, AND A LOT OF GENES ASSOCIATED WITH B- CELLS, COMPLEMENT FC RECEPTORS AND DAVID AND STEVE HAVE SHOWN THIS IS ONE OF THE PHENOTYPES IN THESE PATIENTS AND THEN OTHER GENES THAT FRANKLY WE DIDN'T KNOW WHAT THEY DID. SO THIS RAISES ANOTHER QUESTION AND THAT IS WHAT'S DRIVING THIS EXPRESSION OF THESE DIFFERENT LEVELS OF GENES? SO THAT'S THE QUESTION. WHAT WERE THE POSSIBILITIES? ANYBODY HAVE A GUESS? I TELL YOU IT'S NOT SOMETHING I THOUGHT IT WOULD BE. I HAD ALWAYS ASSUMED THESE LESIONS HAD SUCH A LOW NUMBER OF PARASITES THAT THE PARASITES WERE NOT PART OF THIS STORY. BUT I WAS WRONG. THE PARASITES ARE A PART OF THIS STORY. SO WHAT WE LOOKED AT, FIRST OF ALL THIS JUST SHOWS EXPRESSION OF LEISHMANIASIS, TRANSCRIPTS FROM THE RNA-SEQ AND DAVID AND STEVE DID THIS IN THE OTHER SAMPLES AS WELL WHICH I WILL COME TO IN A MINUTE OF COURSE THIS IS HEALTHY AND THERE SHOULDN'T BE ANYTHING THERE. THESE ARE THE PATIENTS AND WE ALSO DID PC R AND YOU CAN SEE THAT WITH PC R WE GOT PRETTY MUCH WE CAN IDENTIFY PARASITES IN ALMOST ALL OF THESE PATIENTS. IN THE RNA-SEQ PATIENTS SOME WE COULDN'T DETECT ANY PARASITE TRANSCRIPTS AND DAVID AND STEVE SAW THE SAME THING WITH THAT. OUR HYPOTHESIS WAS THOUGH, THIS WOULD BE HIGH LEVELS OF PARASITE S AND THAT'S WHY WE'RE SEEING TRANSCRIPTS AND INDEED, IT TURNS OUT IF YOU COMPARE THESE TWO ASIGNIFYS OF PC R VERSUS THE BRAZILIAN TRANSCRIPTS , IT REALLY IS AN IND ICATION OF WHETHER OR NOT YOU CAN SEE THOSE TRANSCRIPTS, WHICH IS VUBS BUT IT'S IMPORTANT TO SHOW THAT. SO THAT ALLOWED US TO ASK, DO PARASITES DICTATE THE DIFFERENT IAL EXPRESSION OF THESE GENES WE THINK ARE IMPORTANT AND SO, THE ANSWER'S YES AND THIS SHOWS QUANTITATION OF PARASITES BY PC R OF THE GENES THAT WE'RE INTERESTED IN, PERFORIN, ILONE B AND THERE'S A GOOD CORRELATION WITH INCREASED NUMBERS OF PARASITES, AND INCREASED EXPRESSION OF THESE GENES, SO NOW WHAT? OKAY, THIS IS GREAT, WE WILL CORRELATE THIS WITH SOME PHENO TYPE IN THE PATIENTS. WHAT ARE THE KIND OF PHENOTYPES WE WOULD LOOK AT? LESION SIZE, THAT WOULD BE GOOD ONE TO LOOK AT. DTH BECAUSE WE HAVE THAT DATA. WE WERE EXCITED ABOUT THAT. VERY EXCITED BUT THERE'S NO CORRELATION. ALL RIGHT? SO WHY IS THAT? RIGHT? WELL, ACTUALLY I THINK ONE OF THE REASONS THE LESION SIZE DOESN'T CORRELATE, IT'S A PRETTY CRUDE MEASURE OF THE PATHOLOGY OF THE LESION. BECAUSE BASIC IT'S JUST SIZE, IT DOESN'T REALLY MEASURE ANYTHING OTHER THAN SIZE. SO I THINK THAT'S PART OF THE REASON WE'RE NOT REALLY SEEING MUCH OF A CORRELATION THERE. BUT THERE'S SOMETHING ELSE THAT 'S REALLY HAPPENING IN THESE PATIENTS THAT WE THOUGHT WE WOULD LOOK AT AND THAT'S RE SPONSIVENESS TO TREATMENT. SO LET ME JUST TELL YOU WHAT HAPPENS IN THESE PATIENTS. THEY COME INTO THE CLINIC. THIS IS IN A VERY RURAL REGION AND THEY KNOW LEISHMANIASIS IS OCCURRING AND THEY KNOW WHAT IT IS AND THEY ARE GOING TO THE HEALTH FOCUS, BECAUSE THEY ULTIMATELY KNOW WHAT IT IS, IT'S SO COMMON THERE, THEY GO TO THE CLINIC, THEY'RE GETTING THE STUDY, OBVIOUSLY THEY'RE CONSENT ED, A VARIETY OF METADATA COLLECTED AND AT THE TIME THAT THEY COME INTO THE CLINIC, THERE'S A BIOPSY DONE AND THEN THEY'RE TREATED. AND THEN AT 90 DAYS THEY EITHER CURE OR THEY DON'T AND THAT'S WHERE YOU HAVE EITHER CURE OR FAILURE AND SO WE BEGIN TO ASK, GHEE, IS THERE A DIFFERENCE IN CURE AND FAILURE DEPENDING UPON EITHER PARASITE NUMBER OR THESE GENES. AND SO, THIS IS AGAIN NOW, LOOK ING AT THOSE PATIENTS THAT FAILED OR CURED. AND SO WE HAVE THAT ON A P-VALUE OF FAILURE VERSUS CURE ON THIS AXIS AND FOLD CHANGE ON THIS AXIS AND WHAT WE FOUND WAS FIRST OF ALL WE FOUND OUR FOUR GENES OF INTEREST IN VARYING DEGREES OF SIGNIFICANCE, WE'RE ALL LARGE LY--LARGE FOLD CHANGES AND THEN WE FOUND A SERIES OF OTHER GENES AS WELL THAT WE HAD NO IDEA WHY THEY WERE INVOLVED AND I'LL JUST MENTION A TLEDZ BIT ABOUT THAT, BUT IMPORTANTLY, THESE GENES DID ASSOCIATE WITH TREATMENT FAILURE. SO IF THEY SEESHT WITH TREATMENT FAILURE, YOU MIGHT THINK THEY WILL ASSOCIATE WITH PARASITE NUMBERS, AND THIS IS THE DATA SUGGESTING IN FACT THAT'S THE CASE, THESE ARE THE PATIENTS THAT CURED, THESE ARE THE PATIENTS THAT FRAIL FAILED--FAIL ED AND THIS IS PARASITE NUMBERS AND WE ALSO LOOKED AT THIS WITH THE RNA SEQ IF OF THE LEISHMANIASIS TRANSCRIPTS AND THIS IS OUR DATA SHOWING AGAIN THE DIFFERENCE BETWEEN FAIL AND YOU ARE CURE. FAILURE HAVING LARGER NUMBER OF TRANSCRIPTS AND THIS IS THE FIRST SET OF DATA FROM STEVE AND DAVE SHOWING IN FACT THAT AGAIN, THE NUMBER OF TRANSCRIPTS CORRELATED WITH TREATMENT FAIL URE AS WELL. AND FINALLY, IF WE LOOKED AT THE ACTUAL NUMBER OF PARASITES BASED ON THE PC R, WE FOWBD WE COULD DIVIDE THESE INDIVIDUALS INTO THE TWO POPULATIONS BASED ON TIME TO CURE, PATIENTS THAT HAD LESS THAN 32,000 PARASITES IN THE BIOPSY, FOUR MILL MILLIMETER BIOPSY, CURED SIGNIFICANTLY FASTER THAN PATIENTS THAT HAD GREATER THAN 32,000 PARASITES. I HAVE NO IDEA WHAT THOSE NUMBER S, WHY THAT--IT'S REALLY QUITE STRIKING. IT'S ALMOST A CUT OFF AT THAT POINT. WE DON'T REALLY UNDERSTAND THAT YET BUT IT CERTAINLY SEEMS TO BE IMPORTANT AS TO WHETHER OR NOT YOU'RE GOING TO RESPOND TO THIS TREATMENT. AND THE DIFFERENCE IS NOT THAT GREAT BETWEEN RESPONSIVENESS AND FAILURE. SO FINALLY IN A MODEL SYSTEM, WE THINK AGAIN THERE'S THIS MODEL OF CD8 T-CELLS MEANING PATHOLOGY , CELL DEATH AND NLRP- THREE IL-ONE DATA AND THE ONES THAT CAME UP WITH ASSOCIAT ED WITH TREATMENT FAIL URE WERE CCL-THREE AND CCL FOUR. AND THAT'S CHEMO KINES WE'RE INTERESTED IN PARTICULARLY IN THE MOUSE MODEL OF CCREFRESH YOUR RECOLLECTION FIVE AND EIGHT T-CELLS ARE INVOFFED IN THE--IN VOLVED IN THE PATHOLOGY. THE OTHER GENE THAT CAME UP WAS IL33 RECEPTOR. THIS WAS ALSO A BIT OF A SURPRISE. WE DIDN'T HAVE A REASON TO REAL LY FOCUS ON IL33 RECEPTOR EXCEPT HAVING IT COME UP, WE'RE FORCED TO ACTUALLY LOOK AT WHAT IT'S DOING AND IT TURNS OUT THAT THERE ARE SEVERAL PAPERS OUT THERE SUGGESTING THAT IL33--IL33 PROMOTES INCREASE SAID POSITIVE FEEDBACK AND SIGNIFY TOLL-LIKE RECEPTOR SIS OF CD8 T-CELLS SO HYPOTHESIS IS THAT MIGHT BE WHERE IT'S ACTING AND FINALLY IN DATA I'M NOT GOING TO TALK ABOUT NOW THAT'S ONGOING IN THE LAB, IS WE'RE INTERESTED IN ONE ARE THOSE SIGNALS THAT ARE ASSOCIAT ED WITH ACTIVATION OF NL RPTHREE. SO ANOTHER GENE THAT'S HIGHLY UP REGULATED IN THESE LIEWGZS IS THE PTWO X SEVEN RECEPTOR WHICH IS THE RECEPTOR FOR EXTRA CELL ULAR IEE, AUDIENCE TP AND WHEN YOU FIND EXTRA SEMESTERULAR ATP TO THE RECEPTOR, IT ACTIVATE S THE NLRPTHREE AS AN INFLAMMA STUDIES OF MULTIPLE ENDOCRINE SO THAT'S A POTENTIAL TARGET THAT WE CAN LOOK AT. SO THOSE ARE IMPORTANT COMPONENT S WE THINK IN DRIVING THE PATHOLOGY AND KIND OF THE LAST THING THAT--LAST GROUP OF THINGS I WANT TO SHOW YOU WAS WHAT ARE THE OTHER THINGS THAT MIGHT BE IMPORTANT? AND SO, WHAT ELSE MIGHT BE IMPORTANT IN PROMOTING THIS PATH OLOGY? BECAUSE WE KNOW BACTERIA ARE ASSOCIATED A LOT WITH ILONE BETA WE THOUGHT TO LOOK AT THE SKIN MICROBIOME, IT'S NOT HOW IT WENT BUT IT'S HIGHWAY I TELL THE STORY. IT'S MUCH MORE COMPLICATED. I SAID I WOULD NEVER WORK IN THE MICROBIOME FOR A VARIETY OF REASONS BUT WE ENDED UP THERE. SO I WILL TELL YOU THAT DATA AND IT DOES THE SKIN MICROBIOME INFLUENCE THE DEVELOPMENT OF PATHOLOGY AND CUTANEOUS LEISHMANIASIS. THIS IS TRUE, ONE OF THE REASONS WE THOUGHT IT WOULD BE DOING IS FIRST OF ALL, YOU ASK ME BELL INDICATES DATA FROM LEISHMANIASIS MAJ ORIN--MAJOR IN MICE SUGGESTED THE SKIN MICROBIOME WOULD BE IMPORTANT IN THE PATHOLOGY SO YOU SEE IT WAS A GOOD INDICATION IT MIGHT BE WORTH LOOKING AT AND THEN SPECIFICALLY, FER NANDA WENT AND DID GERM-FREE EXPERIMENTS IN THIS RAG MODEL WITH CD8 T-CELLS SO IF YOU REMEMBER, IF YOU TAKE CD8 T-CELLS AND PUT THEM INTO A RAG MOUSE, THEY DEVELOP SEVERE LESIONS. IF YOU TAKE CD8 T-CELLS AND PUT THEM INTO A GERM-FREE RAG MODEL, THERE'S NO DISEASE. AND THAT'S SHOWN HERE, WE CAN'T MEASURE THE LESION AS IT DEVELOP S BECAUSE THEY'RE INSIDE THE ISOLITTOR SO THIS IS WHEN THE MICE WERE SACRIFICED WHICH I THINK WAS ABOUT FIVE OR SIX WEEK S AND YOU SEE THAT THIS IS THE CONTROL AND THESE ARE THE MICE THAT WERE RAG MICE THAT WERE IN GERM-FREE CONDITIONS AND YOU CAN SEE THAT REALLY YOU DON'T GET PATHOLOGY. SO THAT SUGGESTED, YEAH, MAYBE, MAYBE, BECAUSE THE SKIN, OBVIOUS LY RIGHT, BUT IT SAYS SOMETHING ABOUT GERM-FREE MICE MIGHT BE IMPORTANT. SO AGAIN, I DON'T NEED TO SHOW THIS. LET ME GO ON. SO WE JUST DID THE SIMPLE EXPERIMENT AND ASKED WELL WHAT DOES THE--WHAT DOES THE MICROBIOME LOOK LIKE IN MICE INFECT WIDE LEISHMANIASIS. THIS IS COURSE OF INFECTION WITH LEISHMANIASIS MAJ OR, SKIN SWAP- -SWABS AND YOU SEE INFECTION DEPRIVATIONING OVER SIX WEEKS AND AS THE LESION RE SOLVES IT GOES AWAY. WHAT HAPPENS IN MICE THAT DON'T HEAL? WELL, IN MYSELF--MICE THAT DON'T HEAL EITHER ANTI12 MICE OR ANTII L12 TREATED B-SIX MICE OR B ALB NICE, YOU SEE THE NONHEAL ING LESION AND IN THESE MICE IT'S A DOMINANT STREP.& OUR QUESTION WE'RE INTERESTED IN , DOES IT MATTER? SO ONE OF THE PROBLEMS OBVIOUSLY IN THIS FIELD IS YOU CAN SHOW, CAN YOU DESCRIBE WHAT'S HAPPEN ING, BUT THEN DOES IT REAL LY HAVE AN EEIVELGT ON DISEASE AND SO, ONE OF THE WAYS THAT SIERRA WHO DID THESE EXPERIMENTS WAS ABLE TO SHOW THAT IS DID WE DO CO HOUSING WHERE SHE CO HOUSED NAIVE MICE WITH THE INFECTED MICE AND SHE FOUND THAT THESE NAIVE MICE WOULD DEVELOP A STAFF DISBIOSEIS SO THAT DISBIOSEIS TRANSFERRED TO THE NAIVE MICE AND THAT ALLOW ED US TO THINK ABOUT WELL, GHEE IF WE INFECT THOSE MICE WILL THAT HAVE A MORE SEVERE DISEASE. SO AGAIN THIS IS THE--MAKEUP OF THE EXPERIMENT. WE HAD DISBIOTIC MICE THAT WE INFECTED AND WE HAD CONTROL MICE THAT WERE INFECT EXPTD I SHOULD SAY THAT THE DISBIOTIC MICE EXPRESSED HIGHER LEVELS OF IL- ONE BETA. MAYBE IT'S OBVIOUS BUT IT'S IMPORTANT TO SHOW. AND THEN WHEN SIERRA DID THE COURSE OF INFECTION, SHE FOUND THAT THE DISBIOTIC MICE HAD A MORE SEVERE DISEASE COMPARED TO CONTROLS, YOU SEE THIS SLIDE HERE SHOWING HISTOLOGY OF THOSE LESIONS AND PROBABLY IMPORTANTLY THE PARASITE NUMBERS WERE THE SAME. SO AGAIN WE'RE NOT TALKING ABOUT ANYTHING DOING TO CHANGE THE MAG NITUDE OF THIS INFECTION, IT'S SIMPLY THE DISBIOSEIS IS SO THIN MORE SEVERE DISEASE. NOW WHY IS THAT? WELL, I'M NOT SURE WE COMPLETELY KNOW BUT ONE OF THE CANDIDATES HAT WE WERE OBVIOUSLY REALLY INTERESTED IN WAS ILONE BETA. I ALREADY TOLD YOU THAT IT WAS INCREASED IN THESE PATIENTS, IN THESE MICE. AND SO THE OBVIOUS EXPERIMENT WAS TO DO THAT THE DISBIOSEIS EXPERIMENT, CO HOUSING EXPERIMENT BUT NOW TREAT WITH IL ONE BETA. AND THE EXPERIMENT THAT AUGUSTA HAS DONE RECENTLY IN THE LAB WHERE YOU HAVE CONTROL MICE, PATHOLOGY SCORE, CONTROL MICE, OVER TIME, THESE ARE THE CONTROL S, THESE ARE THE DISBI OTIC MICE WITH MORE SEVERE LESIONS AND THESE ARE THE DISBI OTIC MICE WITH ILONE BETA AND IT TURNS OUT AGAIN ILONE BET A IN THIS CASE IS ANOTHER BAD ACTOR IN TERPS OF PROMOTING INCREASED PATHOLOGY. SO FINALLY DOES THE SKIN MICROBIOME CHANGE DURING LEISHMANIASIS PATIENTS? AND THAT WAS SOMETHING THAT WE WERE VERY INTERESTED IN BASED ON WHAT WE HAD ALREADY DONE IN PATIENTS AND SO WE WENT BACK TO BRAZIL, AND WE TOOK A VARIETY OF SKIN SAMPLES, SWABS, AND I'M JUST GOING TO SHOW YOU, SWABS FROM THE LESION AND IN 44 PATIENTS, THEY SEGREGATED INTO THREE GROUPS. THEY SEGGREATIGATE INTO PATIENTS THAT HAD A DISBIOSEIS DOMINATED BY STAFF, DOMINATED BY STREP AND A GROUP OF PATIENTS THAT HAD NO DOMINANT BACTERIA GROWING UP IN TERMS OF THEIR LESIONS. SO THE QUESTION, LAST QUESTION REALLY IS DOES THIS TYPE OF DIS BIOSEIS HAVE ANY EFFECT ON THE DISEASE? AND THE ANSWER IS WE DON'T KNOW THAT YET. BUT WE DO KNOW IS WE CAN GO BACK TO THE RNAR SEQ DATA ASK AND THIS IS FAIRLY PRELIMINARY AND WE'VE DONE MAPPING NOT ONLY TO THE PARASITE BUT ALSO TO STAFF LO CAUCUS AND STREPT O CAUCUS AND WE FIND A GOOD CORRELATION IN TERMS OF GRANZYME B PERFORIN, GRANUE LIESIN, GRANZYME BETA IS CLOSE AND IL-ONE BETA WHETHER OR NOT THEY HAVE EITHER OF THESE OR NONDETECTIBLE READS SO THAT'S VERY PRELIMINARY DATA. WHAT WE'RE DOING NOW IS WE'RE GOING BACK TO ANOTHER GROUP, ANOTHER--ACTUALLY PANEL OF PATIENT SAMPLES THAT ARE BEING SHAPED TO US--SHIPPED TO US RIGHTED ABOUT NOW, THEY WILL BE 50 PATIENTS WHERE WE WILL HAVE SWAPS, RNA, BIOPSIES FROM THE LESIONS, WE WILL HAVE BLOOD AND WE WILL ALSO HAVE PC R FOR THE PARASITE NUMBERS. SO WE THINK WE SHOULD BE ABLE-- WE WILL HAVE OUTCOME OF WHETHER THEY FAILED TREATMENT OR NOT AND HOPEFULLY THAT WILL GIVE US A CHANCE TO UNDERSTAND WHETHER OR NOT DISBIOSEIS MATTER S WHETHER YOU HAVE STREP, STAFF OR NEITHER. SO KIND OF IN SUMMARY, WE THINK THAT WHAT I'VE SHOWN YOU AND TR IED TO SHOW YOU IS THAT IL-ONE BETA, CLEARLY IS INVOLVED IN THE PATHOLOGY IN THESE PATIENTS, IT'S INVOLVED IN PATHOLOGY WE SEE IN OUR EXPERIMENTAL MODELS, THAT CAN COME FROM THIS CELL DEATH SIGNIFY TOLL-LIKE RECEPTOR SIS PATHWAY, ALSO IT CAN COME FROM A DISBIOSEIS AND IN DATA I DIDN'T HAVE TIME TO SHOW YOU, WE ALSO KNOW THAT TH17 CAN BE INVOLVE INDEED DRIVING PALGT OLOGY AND SOME OF THE MODEL S IN CUTANEOUS LEISHMANIASIS AND OBVIOUSLY IL- ONE BETA IS IMPORTANT IN THAT PATHWAY AS L. SO STEPPING BACK, LIKE OKAY, WE HAVE THESE MODELS, WHAT DOES THAT MEAN? AGAIN, I SHOWED YOU PARTLY PART OF THIS. I THINK IT MEANS WE HAVE SOME REAL POSSIBLE TARGETS FOR DOING SOMETHING FOR THESE PATIENTS AND SO, THEY'RE KIND OF OBVIOUS, I TALKED ABOUT KINAYA AS A WAY OF BLOCKING IL-ONE BETA, I TALKED ABOUT [INDISCERNIBLE], I AM EXCITED ABOUT THIS PATHWAY BECAUSE OF THE TOPICAL TREATMENT WE'VE TRIED SEVERAL NLRPTHREE INHIBITORS THAT ALSO BLOCK THIS PATHWAY AND OF COURSE YOU CAN EN VISION, THIS IS MABEL A BIT OUT THERE, WHAT YOU PROP PRIMATESSOTIC STRAINS MIGHT BE-- PROBIOTIC STRAINS MIGHT BE THERE, OR MORE SIMPLY GOING BACK TO THE LITERATURE OF ANTIBIOTIC TREATMENT IN THESE PATIENTS AND I HAVE TO SAY WE'VE DONE THAT AND THE LITERATURE IS A MESS WHICH IS MAYBE NOT SURPRISING AND SO I THINK WE NEED TO GO BACK AND SERIOUSLY THINK ABOUT ANTIBIOTIC TREATMENT WHICH WE'RE DOING FIRST OF ALL IN THE MOUSE BEFORE WE GO TO PATIENTS. SO IN TERMS OF TAKE HOME MESSAGE S. SO BY COMBINES STUDYING IN PATIENTS AND MIRRORING MODELS WE THINK A PIPELINE OF HOST DIRECT ED THERAPY SYSTEM AVAILABLE AND IL-ONE BETA IS CERTAINLY ONE OF OUR FIRST TARGETS BUT ONLY ONE OF SEVERAL. AND THE OTHER THING IS THAT--I THINK THIS IS IMPORTANT FROM AN IMMUNE O LOGIC POINT OF VIEW, BUT FROM A PRACTICAL POINT OF VIEW, WE CAN TELL AWE PATIENT WHETHER THEY'RE GOING TO RESPOND TO THERAPY WHEN THEY COME INTO THE CLINIC. SO THEY DO BIOPSIES ON THESE PATIENTS TO BE ABLE TO IDENTIFY THE FABLGHT THEY HAVE LEISHMANIASIS SO IF THEY DO THAT , WE CERTAINLY CAN LOOK FOR QUANTITATIVE PARASITES AS WELL AS THESE HOST GENES THAT WE KNOW ARE ASSOCIATE WIDE TREATMENT FAILURE AND IF WE CAN DO THAT, WE CAN ACTUALLY SAY, YOU KNOW WHAT, THIS WILL FAIL. SO WE NEED THAT SECOND LINE DRUG RATHER THAN STARTING THEM ON ANTIMONY. SO FINALLY AND THIS IS REALLY FINALLY, HOW COULD YOU DO THAT? THIS IS IN A HEALTH POST, IT'S THREE AND HALF HOURS OUTSIDE OF SALVADOR, IT'S VERY RURAL, WE HAVE A CENTRIFUGE, REFRIGERATOR, WE ACTUALLY HAVE A FLOW SIGNIFY TOM TERBUT THAT'S AMAZING BUT NOT MUCH MORE THAN THAT. AND SO, DAN BITING HAS BEEN IN VOLVED WITH THE COMPANY IN PHILADELPHIA, THAT MAKES SOMETHING CALLED BIO--COMPANY'S CALLED BIOAMINE AND IT MAKES THIS PORTABLE PC R MACHINE, THIS IS AN IPHONE SITTING IN THIS PORTABLE PC R MACHINE AND SO, YOU CAN TAKE THIS PORTABLE PC R MACHINE AND YOU CAN SAMPLE--CAN YOU PREP A SAMPLE IN LESS THAN A MINUTE AND YOU CAN GET A RESULT AN HOUR. SO WE WENT TO BRAZIL, DAN AND I AND HE USED THIS IN THE CLINIC TO BE ABLE TO IDENTIFY PATIENTS THAT HAD A CERTAIN AX MOUNT OF PARASITES. SO WE'RE NOW IN A SITUATION WHERE IF WE THINK ABOUT NOW MAKING A DIFFERENCE, IT'S REALLY GOING AND BEING ABLE TO USE SOMETHING LIKE THIS TO RIERKS-- IDENTIFY THE PATIENTS THAT WILL FAIL AND GET THEM ON A BETTER DRUG. WE SHOULD ALL BE WORKING ON BET TER DRUGS, BUT THAT'S NOT WHAT WE WORK BUT BAH THAT'S WHAT WE NEED IN THIS DISEASE. AGAIN, THESE ARE THE PEOPLE THAT DID THE WORK AND I APPRECIATE YOUR ATTENTION. THANK YOU. [ APPLAUSE ] >> YES? IT'S ME. I WAS INTRIGUED BY ISG15 AS ONE OF YOUR GENES THAT WERE CORRELATED WITH LACK OF RESPONSE AND I WAS WONDERING IF MORE GENERALLY DO YOU SEE AN INTERFER ON SIGNATURE IN GENES AND THEN SECOND PART OF THAT QUESTION IS AND YOU I WERE TALK ING ABOUT JACK INHIBITORS MIGHT WORK AND DO YOU THINK BLOCKING TYPE ONE INTERFERONS MIGHT BE EFFICACIOUS? >> YEAH, SO YOU DO SEE INTERFER ON RESPONSE, CLEARLY. WE HAVEN'T ACTUALLY THOUGHT ABOUT BLOCKING TIARAS ONE INTER FERONS. WE HAVEN'T REALLY MEASURED THEM. DAVEED DID YOU DO SOME THINGS WITH TYPE ONE? >> HARD TO MEASURE, EASIER TO MEASURE THEIR ACTION. >> YEAH SO WE HAVEN'T DONE THAT O THE ISG15 COMES UP VERY HIGH. WHAT IT'S DOING IS REALLY TOUGH. THE APOE-A IS TOUGH AND THERE'S THREE GENES THAT I DON'T KNOW WHAT THEY'RE DOING SO IT'S EN CLUEDING 15. >> SO ALL OF THESE EXPERIMENT INGS YOU NEVER LOOKED AT STAT TWO? >> HAS ANYBODY EVER LOOKED AT STAT TWO? I SHOULD, IS THAT WHAT YOU'RETAL LING ME? >> YEAH, YOU SHOULD. >> YEAH, YEAH. >> SO WHEN YOU BLOCK THIS ILONE BETA MEDIATED INFLAMMATION, THE LESION SIZE GOES DOWN, HAVE YOU EVER CAREFULLY LOOKED AT PARASITE BURDENS, TOO WHETHER IT GOES DOWN? >> SO THAT'S A REALLY IMPORTANT QUESTION, IT DOES NOT CHANGE THE PARASITE BURDEN. THERE IS NO ROLE THAT WE SEE FOR ILONE BETA BEING PROTECTIVE IN LEISHMANIASIS. BUT IT ALSO IF YOU GET--IF THAT PARASITE IF THE LESION GOES DOWN IN SIZE, IT ALSO DOESN'T DE CREASE THE NUMBER OF PARASITE. SO IT'S NOT IF YOU TAKE IT AWAY, YOU GET LESS PARASITES IF YOU TAKE IT AWAY IT GETS MORE PARASITES. IT DOESN'T CHANGE THE PARASITE NUMBER. >> DR. MOZIER. >> SO YOU SAID YOU CAN QUANT ITATE PARASITES AS A WAY TO PREDICT WHETHER YOU'RE GOING TO GET TREATMENT FAILURE OR CURE. AND YOU SAID THAT PARASITE QUANT ITATION ALSO CORRELATES WITH PARASITE TRANSCRIPT NUMBERS MY QUESTION IS, HAVE YOU LOOKED AT SPECIFIC PARASITE TRANSCRIPTS TO FIND OUT WHICH ONES CORRELATE WITH CURE OR FAILURE. >> SO WE HAVEN'T. I MEAN YOU KNOW THE LEVEL OF-- TRANSCRIPTS THAT ARE THERE. SO WE HAVEN'T REALLY TRIED. I MEAN I--WE REALLY SHOULD HAVE TALKED ABOUT THIS THE LAST TIME WE WERE TOGETHER, DID YOU EVER LOOK IN YOUR DATA? >> NO. >> BECAUSE IN THE CUTANEOUS PATIENTS YOU'RE LOOKING AT, YOU THERE YOU CAN LOOK AT IT BECAUSE YOU HAVE MUCH HIGHER LEVEL TRANSCRIPTS. >> BUT THERE'S NO CURE. >> I'M NOT SURE WE WILL SEE MUCH >> OKAY. >> THAT'S A GOOD QUESTION. >> HI, SO IN THE CO TRANSFER EXPERIMENTS WHERE YOU HAD THE MICE THAT WERE DISBIOTIC THAT WERE THEN MORE PREDISPOSED TO THE DISEASE, DID THEY HAVE INCREASED CD8 RESPONSES IN THOSE LESIONS, DID YOU MEASURE THAT? >> NOT OBVIOUSLY, NO. NO. IT DOESN'T SEEM TO BE--DOESN'T SEEM TO BE ASSOCIATE WIDE NOW ALL OF A SUDDEN MORE CD8S. NO. >> THANKS. >> HI, GREAT TALK. THANKS. I WAS WONDERING WHAT COMES FIRST DISBIOSEIS AND THEN THE DISEASE OR THE DISEASE CAUSE DISBIOSEIS AND ENHANCE THE DISEASE? >> I'M REALLY GLAD YOU ASKED THAT QUESTION. >> SO I THINK, YOU KNOW SO THE WHOLE PROBLEM WAS, OKAY, SO, IF YOU INFECT, SO THIS IS MICE, RIGHT, INFEBLGHT MICE, THEY DON'T HAVE A DISBIOSEIS AND THEY DEVELOP ONE, AND SO, OKAY, YOU HAVE AN INFECTION SO YOU DEVELOP A DISBIOSEIS, THAT'S PRETTY COMMON, RIGHT, INFLAMMATION, SO THEN DOES IT MATTER, THAT'S WHY WE MADE MICE THAT WERE DISBIOTIC BEFORE WE INFECTED THE MICE. AND THEN WE FOUND THAT YEAH, IT DOES MATTER. SO I THINK THAT IT'S--I THINK OF IT AS A CIRCULAR POSITIVE FEEDBACK LOOP, DOES THAT WORK? YOU LIKE THAT? IS THAT OKAY? >> YES, THANK YOU. >> OKAY. >> SO I MIGHT NOT HAVE FOLLOWED YOUR CCR FIVE STORY BUT WHAT ABOUT MAR RAFFA ROCK, WOULD BLOCKING CCR-FIVE HELP? >> YEAH, AGAIN THIS IS REALLY RECENT DATA THAT'S WHY WE WERE LIKE, OKAY, IF THESE PARTICULAR CHEMO KINES ARE UP AND THEY'RE ASSOCIATED WITH TREATMENT FAIL URE AND THEY'RE BINDING TO C CR FIVE, CAN WE USE WELL KNOWN DRUGS THAT BLOCK CCR FIVE, WE HAVEN'T TRIED IT YET IN THE MOUSE MODEL. >> THE OTHER THING IF I REMEMBER CORRECTLY, THE ANTIMONY IS EXTREMELY TOXIC IN ADDITION TO BEING INEFFECTIVE IT SOUNDS LIKE SO WHAT ABOUT JUST A BETTER TREATMENT CAN YOU JUST USE AM FOCUSED ON TERRACEIN. >> WELL IT ALSO HAS ITS PROBLEM MINGS AS WELL, SO THAT IS THE SECOND LINE OF DRUG. SO WHY DO THEY USE ANTIMONY, IT'S FREE GIVEN TO THEM BY THE DRUG--BY THE COMPANY. SO, YEAH. >> SO ONE SUCCESSFUL HOST DIRECT ED THERAPY, THAT WE ACTUAL LY DO USE IN CUTANEOUS LEISHMANIASIS IS IMIKLMODSO WHAT TOPICALLY AND SO ONE OF THE THINGS THAT AT LEAST HAS BEEN PROVEN TO WORK QUITE REASONABLY WELL IN CONJUNCTION WITH TYPICAL LY LIPO STUDIES OF MULTIPLE ENDOCRINAL AMPOTER ASIN BUT PRESUMABLY WOULD PROVIDE BENEFIT TO THE ANTIMONY TREATED PATIENTS SO I GUESS MY QUESTION IS DO YOU AVOIDED DISCUSS AT ALL ABOUT TLR AGANISTS AND I GUESS I'M ASKING BROADLY WHAT YOU THINK THE ROLE IS OF TLR ENGAGE MENT OR LACK OF ENGAGEMENT IN DISEASE? >> SO, THIS IS COMPLICATED IN THIS SENSE THAT YOU REALLY--SO IF YOU HAVE A PATIENT THERE, ARE YOU TRYING TO CONTROL THE PARASITE NUMBERS BY ENHANCING THE IMMUNE RESPONSE? WHICH YOU MIGHT--I'M NOT QUITE SHOWER SURE HOW IT'S WORKING IN THAT SITUATION BUT IT'S NOT DAMP ENING THE IMMUNE RESPONSE, WHEREAS WHEN I TALK ABOUT HERE IS DAMPENING AN IMMUNE RESPONSE THAT IS A PATHWAY THAT IS NOT INVOLVED IN PARASITE PROTECTION. SO I THINK THAT'S MY NONANSWER. AND I'M STICKING WITH IT. [LAUGHTER] >> ALL RIGHT, I WILL MAKE ONE LAST COMMENT. >> IS IT A GOOD ONE? >> WHEN WE ACTUALLY SEE PATIENTS AND THIS GETS TO THE QUESTION OF THE MICROBIOME, IT IS NOT UN COMMON TO SEE EITHER STAFF OR STREP SECONDARY TRUE BACTERIAL AND SO I DO THINK THERE IS THE POSSIBILITY THAT AND THOSE LESIONS TYPICAL LYE LOOK WORSE IN THOSE THAT HAVE INFECTION THAN THOSE THAT DO NOT. SO THE REAL QUESTION IS IS IT SOMETHING ABOUT--A DISBIOSEIS OR IS IT REALLY INCREASED COLONIZ ATION WITH THE PATHOGENIC BACTERIA. >> YEAH, SO--YOU'RE ABSOLUTELY CORRECT AND IT'S VERY DIFFICULT TO TELL THE DIFFERENCE, SO WHAT I DIDN'T SHOW YOU IS THE DATA THAT WE DID THE LESION AND THEN WE ALSO DID ADJACENT TO THE LESION TO BE CONTRA LATERAL SKIN AND ADJACENT TO THE LESION, WE ALSO SAW MORE STAFF. BUT IT'S A REALLY DIFFICULT THING AND I TALK A LOT TO THE CLINICIANS THERE ABOUT THIS ISSUE OF FRANK BACTERIAL INFECTION VERSUS NOT. AND IT'S INTERESTING FROM THEIR POINT OF VIEW, MOST OF THESE LESIONS DON'T DEVELOP A BACTERIAL INFECTION WHICH MAKES WE WONDER WHETHER OR NOT TREAT ING WITH ANTIIL-ONE BETA WOULD BE A GOOD THING IF IT IN CREASESSED BACTERIAL LOAD BUT IT'S A GOOD QUESTION AND IT'S WITH THE DATAA AND IT'S SOMETHING WE WOULD NEED TO THINK ABOUT AND BE AWARE OF. YES? >> THANKS FOR THE NICE TALK, I WAS WONDERING THAT TALKING ABOUT BIOMARKERS HAVE YOU TRIED TO SEE SOMETHING IN THE SERUM LIKE SOME MICRO RNA SIGNATURES OR SOMETHING? >> YEAH, WE HAVEN'T DONE IT IN THE SERUM BUT I TOLD YOU WE'VE TAKEN BLOOD AND DONE WHOLE BLOOD , RNA-SEQ ON THE WHOLE BLOOD AND UNFORTUNATELY, SO FAR, WE DON'T SEE--WE DON'T SEE A GENE EXPRESSION IN THE BLOOD THAT'S ASSOCIATED WITH TREATMENT FAILURE. WE SEE SOME INTERESTING CHANGES AND SOME INTERESTING GENES AND SOME NONCODING GENES THAT ARE LIKE WOW, THAT COULD BE INTERESTING, BUT NOTHING THAT LOOKS LIKE A BIOMARKER YET. >> THANK YOU. >> I HAVE A QUESTION [INDISCERNIBLE]--THAT IS HOW DID YOU MAKE THE LEAP FROM CD-EIGHT CELLS [INDISCERNIBLE] TO IL-ONE AS THE FINAL EFFECTOR OF THAT RE SPONSE? >> SO IT WASN'T THAT--I'LL TELL YOU HONESTLY IT WASN'T THAT HARD AND THE REASON IT WASN'T THAT HARD, I DIDN'T SHOW IT HERE BUT THE FIRST MICRO ARRAY, IS LIKE THE GIFT THAT KEEPS ON GIVING. THAT MICRO ARRAY, WE USED FOR, I THINK WE PROBABLY GOT SEVEN OR EIGHT PAPERS OUT OF ONE SILLY LITTLE MICRO ARRAY. AND ONE OF THE THINGS IN THAT MICROARRAY, IS WE MADE A META-- METAPATHWAY, FROM THE CD8 T-CELL SIGNIFY TOLL-LIKE RECEPTOR SIS TO INFLAMMA STUDIES OF MULTIPLE ENDOCRINE ACTIVATION TO ILONE BETA BASED ON GENE EXPRESSION AND SO, THE MICRO ARRAY SAID WE DIDN'T KNOW IF THAT WAS THE CASE BUT THAT WAS HOW WE FOCUSED ON NLRPTHREE AND HOW WE FOCUSED ON ILONE BETA >> OTHER SAMPLES OF TYPE OF CONNECTION. >> WHAT DO YOU MEAN? IN TERMS OF CD8S, THERE'S ANOTHER SITUATIONS, THERE'S COUPLE SITUATIONS THERE'S A SITUATION WITH CEREBRAL MALARIA WHERE THE CD8S ARE PATHOLOGIC BUT THERE'S ANOTHER ONE ESCAPING ME BUT THERE ARE A COUPLE. >> ANY OTHER QUESTIONS? ALL RIGHT, WELL, THANK YOU VERY MUCH. >> ALL RIGHT. THANK YOU.