gouty arthritis drug

daniel kastner:well, gene [spelled phonetically], thank you very much for that kind introduction, andthank you all for the opportunity to be here this morning. it's really fantastic beingable to get acquainted with you all and talk with you about a topic that's near and dearto my heart, that being "horror autoinflammaticus: the adventures and the genomics of inflammation."and of course, the term "horror autoinflammaticus" is sort of a takeoff on the term "horror autotoxicus,"which was a term that was proposed by paul ehrlich back at the beginning of the 20thcentury. he was one of the great early immunologists, who recognized the severe consequences whenthe immune system turns against its host, and so ehrlich coined the term "horror autotoxicus,"and what we're going to be talking about this

morning, horror autoinflammaticus, is justbasically the turning against of the innate immune system of a part of the immune systemagainst the host, and so we'll focus on that particular aspect of the immune system. butanyway, i hope that over the course of the next three or four hours that i have withyou, that we can really get down to some details in terms of these fascinating autoinflammatorydiseases. so, in any event, i have nothing to disclosein terms of commercial relationships, but probably the first thing, at least for someof you, is to get down to the question of the systemic autoinflammatory diseases. whatare they, and why should you care? so, in any case, first of all, just in terms of thedefinition, they are a group of disorders

in which there are episodes of seemingly unprovokedinflammation in the absence of high-titer auto-antibodies, antigen-specific t-cells,or other features, cardinal features of the adaptive immune system, and no evidence ofinfection. despite the fact that there isn't any strong evidence for the adaptive immunesystem being involved in these diseases, they do really manifest dramatic systemic inflammation,and i'll just illustrate this on this slide. first of all, in the left-hand side, you cansee a laparoscopic view of the peritoneal cavity of a 7-year-old girl that we saw atthe nih a few years ago who has traps, the tnf receptor-associated periodic syndrome,and this is one of the diseases that we will be discussing this morning. this child hadepisodes of intermittent sterile peritonitis,

and what you can see here are basically adhesionsthat have formed because of the repeated episodes of sterile peritonitis. the next image isthe forearm of a young man from kansas city who has papa syndrome. papa syndrome is pyogenicarthritis with pyoderma gangrenosum and acne, and what you see here on his forearm is, infact, pyodermic gangrenosum, basically the breakdown of the skin and the infiltrationof the area with polymorphonuclear leukocytes. and this particular lesion, in the case ofthis patient, actually, it took us a year to resolve this lesion in this patient. andthen, finally, the image on the right is from a patient with the -- one of the newer autoinflammatorydiseases, the more newly recognized autoinflammatory diseases, and this is a patient with dira,the deficiency of the il-1 receptor antagonist,

something that we published in the new englandjournal about three years ago, and i'll be telling you about that in a little bit aswell. so anyway, as i mentioned, one of the importantaspects of these diseases is the fact that they are disorders of the innate immune system,and just to remind those of you who aren't thinking about immunology every day -- ifthere is any such person in this auditorium -- but in any case, the adaptive immune system,of course, is that part of the immune system where the players are lymphocytes. these area subset of the white blood cells and the receptors for various pathogens, receptorsthat rearrange in the genome and somatically mutate, whereas the adaptive immune systemis that part of the immune system that's a

little bit more ancient in terms of its historyin organisms, and it's the part of the immune system in which the myeloid lineage of cellsplays a more important role, and in which the receptors are actually hardwired in thegenome and do not somatically rearrange or mutate. now, this slide here is just a table of atleast a number of the autoinflammatory diseases. it's probably -- the print is probably toosmall for you to read, but i will just highlight the fact that there are a bunch of differentclasses of diseases now that have been put under this rubric of autoinflammatory. thefirst ones that were recognized were disorders that are hereditary periodic fever syndromes,as jean was alluding to in the introduction

-- diseases like familial mediterranean fever.but there are a host of other diseases that are also categorized as autoinflammatory,such as the idiopathic febrile syndrome, still's disease in children, adult still's diseasein adults, various pyogenic disorders like papa syndrome that i mentioned earlier, granulomatousdiseases like blau syndrome, and some would regard crohn's disease as being autoinflammatory.autoinflammatory disorders of the skin and bones such as dira; we'll be talking abouta few of them. and then a host of other things, such as metabolic diseases like gout, thatwe will talk about in a little bit. in any case, my exposure to autoinflammatorydiseases, and sort of the dawning of my interest in these diseases, actually happened wheni was a beginning fellow in rheumatology at

the nih back in 1985. i was 5 at the time,of course, and i happened to see in our new-patient clinic this man, sarkis, who was a man whowas referred to us with a mystery illness. and basically, at the time that we saw him,he was in his early 20s, of armenian ancestry, and he presented with a history of episodicattacks of monoarticular knee or ankle arthritis since infancy. these would usually occur onthe order of once a month or so and would last for several days at a time. they wouldbe accompanied by fever and an erythematous rash over the involved joint, and he wouldhave massive effusions of his joints at the times that he would have these attacks. betweenthe attacks, he was totally normal, and the attacks would resolve spontaneously. so, thiswas someone who, in his early 20s, had probably

had a couple hundred of these attacks overthe course of his life but had had actually no lasting damage of his joints, and whenhe walked in to see us, he was between attacks and looked totally normal. and the question was, what does he have? well,actually, at the time that i saw him, i didn't know what he had, either, just as a numberof other physicians who had seen him over the course of his life. but fortunately, therewas a fellow in the lab that i was working in who was from israel, and he said, "dan,it's obvious what this patient has. he has familial mediterranean fever." and sure enough,we witnessed an attack, we aspirated some fluid from his joint, from his knee. he had,like, a hundred thousand polys per cubic millimeter

in the synovial fluid, which is typical forthe arthritis attacks of fmf. these patients will have an arthritis that looks like a septicarthritis, essentially, so this is something that's a cardinal feature of fmf, and at thetime that we saw him, it was already recognized. the colchicine is a fairly effective treatmentin preventing the attacks of fmf. we put him on colchicine, and essentially, he has donewell ever since, for the 27 or so years that it's been since we first saw him. in any case, just to illustrate some of thefeatures of fmf, it's a recessively inherited disease. it's a disorder that is seen, asthe name implies, in individuals of mediterranean ancestry. that means jewish, arab, armenian,turkish, and italian people. recessive disease,

attacks of fever that last on the order ofusually one to three days, sometimes a little bit longer with the arthritis. they can havesevere abdominal pain from sterile peritonitis; they can have sharp pleuritic chest pain frompleurisy; they can have arthritis, as i described to you; they can have a skin rash as well.so, these are -- these things are illustrated on this slide. this is an upright film ofthe abdomen of a patient having a peritoneal attack of fmf, showing the air fluid levels.my pointer doesn't -- isn't very strong, but you can imagine that there are air fluid levelsthere. a left pleural effusion in this chest radiograph on the lower left. here, in thecenter, you have a posterior pericardial effusion, and actually, asymptomatic pericardial effusionsare relatively common in patients with fmf.

up in the upper right, you can see a radiographof the hip in a patient with chronic arthritis of the hip. usually, the arthritis of fmfis a non-deforming, non-erosive arthritis, but in about 5 percent of untreated patients,you can get this picture of a destructive arthritis. and then down in the lower right,you have erysipeloid erythema, which is basically a reddish raised rash, usually on the dorsumof the foot, the ankle, or the lower leg that occurs in these patients, a lot of times mistakenas being an insect bite. now, histologically, as i mentioned, thesepatients have lots of polys in their synovial fluid or in their skin, if you were to biopsythe skin, and really, the thing that was the most devastating manifestation of fmf beforecolchicine therapy was systemic amyloidosis.

now, amyloidosis is a term that refers tothe ectopic deposition of protein in a number of different tissues in the body, and thereare different forms of amyloidosis, as many of you know, so that there is aa amyloidosis,al amyloidosis, transthyretin amyloidosis, and a host of other amyloidoses in which onecan have mutations and various proteins, a lot of them that are serum proteins. in theamyloidosis of fmf, what is being deposited is serum amyloid a, which is an acute-phasereactant which is produced by the liver during the inflammatory attacks of fmf, and a cleavageproduct of saa is what deposits in the kidneys and several other vital organs. and beforethe advent of colchicine therapy in fmf, amyloidosis was actually a major cause of death in fmfpatients.

now, back in the mid-1980s, this was a fascinatingdisease. it was a disease we didn't know what caused it. it was a disease with dramaticinflammation, and this was really at the advent, at the dawning of the human genome project.and so at that time, it was just becoming possible to map genes that cause human diseasesby basically comparing the inheritance of those diseases in families with the inheritanceof dna markers, which were just being discovered at that time, of known chromosomal location.so i thought that if others could be mapping and cloning the gene for diseases like cysticfibrosis, why couldn't dan kastner find the gene that causes fmf? and of course, the naivetã©of youth is a good thing. when you're 5 years old, you know, these kinds of things are great.

and so, fast-forward a little bit. this isa hipaa-approved photograph of a family that i visited in israel. so, basically, in thesummer of 1989, i spent the summer with this guy here, dr. mordechai pras, who ran a verylarge fmf clinic in tel aviv. and he made available to me patients with fmf as wellas unaffected family members. in some cases, they couldn't make it to the clinic, so wewent to them. and you can see, after getting informed consent -- there's a notebook withthe informed consent documents -- everyone would roll up their sleeves and give blood.we would have lunch, and it was a great thing. and here, this happens to be a family fromakko, which is a northern coastal town in israel. they are of moroccan-jewish ancestry,and actually a consanguineous family. the

parents in the family were first cousins toone another, and you may note the strong intrafamilial resemblance between mom and dad in this family.and then, also pictured here are several members of the family affected with fmf as well asone of the members of the family up here in the upper left, our upper left, who's totallyunaffected and turned out later, once we had the gene, not even to be a carrier for fmf. so, in any case, we did do what we set outto do, which was to map the gene for fmf, and it turned out to be on the short arm ofchromosome 16, and then we became sort of the genome project for that area of the humangenome. this was back at the time when things were just really getting underway in termsof the genome project, and so we developed

fairly high-resolution maps of this region,of -- oh, that's where it is -- of -- in the middle -- high-resolution maps of this areaof chromosome 16, narrowed things down to about a 200 kv interval. there were 10 genesthat we had to figure out were encoded in that region, and of course, as our luck wouldhave it, it was the 10th of the 10 genes that we looked at that had mutations in it thatwere, in fact, associated with inheritance of fmf. so, in any event, we did find, then,a gene depicted here, mefv, mutations in which cause fmf, and it encodes what was then apredicted protein, shown here, which we call "pyrin," after pyrexia. now, at the time that we were actually atthe point of finding the gene, we were in

a fight to the death with a french group,a race to the finish line, and so this was actually in july of 1997, so 15 years ago.and so we, and they, found the same gene. fortunately, it was the same gene at the sametime, and we named the encoding -- encoded protein "pyrin," after "pyrexia" for fever.the french group, being much more erudite than we, called it "mare nostrum" after "marenostrum" for the mediterranean sea. that was the latin for the mediterranean sea. we chosea name that would be relatively short, easy to pronounce, and perhaps easy to remember,hoping that then -- we didn't know for sure that the french had something, but we figuredif they did, it would be good, at least in terms of what name would finally stick, thatour name would be one that would be easier

to remember. so anyway, we called it "pyrin,"after pyrexia. and at the time, it was a novel protein. it was a protein that hadn't beenrecognized before. and it turns out that the n-terminal 90 or so amino acids -- at thattime, again, it was not known, but that domain turns out to be a domain that's found in some20 different proteins in humans that are involved in the regulation of inflammation and apoptosis.and so this actually became something that was more or less a key to understanding awhole branch of regulation of the innate immune system. and that domain, i will tell you,everyone refers to as "the pyrin domain," not "the mare nostrum domain" -- [laughter]

-- but "the pyrin domain." so, the pyrin domain, it turns out, formsthe six alpha-helical structure, shown here in the upper left, and that structure is sometimesreferred to as a "death fold," because it's seen in death domains, death effector domains,caspase recruitment domains, and pyrin domains. now, pyrin domains are actually the most numerousof these four families of domains. the interesting thing about this structure is it allows theformation of a dipole, with positive charges being shown in blue here and negative chargesbeing shown in red. and the idea is that by forming this dipole, what happens is thatyou can get, then, cognate interaction, self-self-interactions between pyrin domains. and so pyrin domainsof one protein can interact with pyrin domains

of another protein, basically to allow forintermolecular interactions and for various regulatory processes to happen in the cell. so, the pyrin domain of pyrin interacts witha protein that is sometimes known as asc: apoptosis-associated speck-like protein witha card domain, which is why most people call it "asc." and asc is a fairly small proteinthat has a pyrin domain at its n-terminus and a card domain, which is also a domainin the same death fold configuration, at its c-terminus. the card domain of asc interactswith the card domain of caspase one. and caspase one, some of you may know, is actually theenzyme that catalyzes the conversion of pro-interleukin-1 beta to interleukin-1 beta. and interleukin-1beta, il-1, is one of the major mediators

of fever and inflammation in humans. and sothis basically ties pyrin to the regulation of this process of il-1 activation. we have generated mice over the course ofthe years that have actually -- that harbor mutations in them, in the mouse pyrin, thatare the same mutations as what we see in humans with fmf. and you can see on the left, here'sa wild-type mouse, and then a littermate that has the v726a mutation. that's one of thefmf-associated mutations, substitution of the alanine for baline [spelled phonetically]at position 726, and you may see here, that, in fact, this mouse has arthritis of its hindpaw, and if you section the joint, there's lots of polymorphonuclear leukocytes in thesynovial fluid. moreover, if you compare the

peripheral blood leukocytes in the v726a bredonto a wild-type background, there's lots of -- there's a granular cytosis in thesemice. but if you breed it onto an il-1 receptor knockout so that you're blocking il-1 signalingin the mouse, that goes away. now, you may say, well, we don't treat mice,so, so what? well, so, i'll tell you so what. so, in any case, back in, i think it was 2005or something like that, we had this patient who was sent to us from the mayo clinic, whowas a man from baghdad, iraq. he was 18 years old at the time. he's homozygous for the m694vmutation at the fmf locus. now, that's the most severe, that's the mother of all mutationsat the fmf locus, and patients that have that mutation, if they're not treated aggressively,can develop amyloidosis. and so at the age

of 18, he actually did have systemic amyloidosis.he had amyloid in his kidneys and had a creatinine, at the time that he came to us, of 3.5. hehad amyloid in his heart, which is actually relatively unusual for aa amyloid, but hehad it, and he had an ejection fraction of 37 percent. he had amyloid in his gastrointestinaltract, which led him to have malabsorption and chronic diarrhea, and this is just allillustrated on the images here. so, this is the glomerulus of the kidney, and you cansee when -- it's stained with congo red. looked at under regular light, it looks like this.under polarizing light, you can see the apple-green birefringence that's typical for amyloidosis.stained with anti-aa monoclonal antibody, it shows up this way. here is amyloid in hisduodenum, causing chronic diarrhea, malabsorption.

here's amyloid in his heart; this is an anti-aastain. so anyway, given the fact that he had amyloidin his gi tract, that he had chronic diarrhea, and that -- actually, what happened was thati went to give a talk up in connecticut at a wharton conference, and i got this messageat the end of the talk that i should call the icu at the nih as soon as possible. so,i called the icu. this guy had, while i was away, gone into renal failure and heart failureand was in the icu, and there was the question even as to whether we should support him becauseof the fact that he had already such advanced amyloidosis, and what were we going to dofor him, and could we do anything like a kidney transplant for such a patient as this, becausethis is a process that seemed irreversible.

but, at that time, there was just beginningto be the thought that amyloid is actually a dynamic process in which you have depositionof whatever is the protein that's being deposited as the form of amyloid, but there's also aresorptive process, and that if you could block the deposition of amyloid, that theresorptive process would eventually lead to improvement in the patient. well, we couldn't treat him aggressively withcolchicine because he had diarrhea, and as many of you know, colchicine causes diarrhea.so, what to do? well, we were just beginning to see the light with regard to the connectionof pyrin with il-1. so, we thought, well, maybe we should treat him with an il-1 inhibitor,which we did. and here, this is just on the

y axis, acute-phase reactancy, that the serumamyloid a or the crp [spelled phonetically], while he was on -- at least in this image-- while he was on anakinra, which is the il-1 receptor antagonist, you can see that,in fact, his acute-phase reactants were well-controlled. we had to stop it for a period of time becausehe was septic. but in any case, we continued the treatment with him, and actually, hisamyloid has not totally gone away, but certainly much improved, so that at this point, hisejection fraction is 55 percent; he's able to eat pizza for lunch; he's had a kidneytransplant; and here's a picture of him, a recent picture of him. so, in fact, this hasbeen, for some patients, a lifesaving kind of thing. and there's actually an articlethat's going to be coming out in the annals

of internal medicine, a study that we wereinvolved in at the clinical center, using a different il-1 inhibitor, rilonacept, ina randomized placebo-controlled trial showing that rilonacept is effective in the treatmentof fmf. that's another il-1 inhibitor. all right. so, in any case, let's move onto another disease. i think you've heard enough fmf for the morning. so, let's talk aboutanother patient: christina. now, christina was a patient that was referred to us at thenih while we were looking for the gene for fmf. and she was not of mediterranean ancestry.instead, she was irish. she was actually referred -- her husband worked at the irish embassy.there was an irish anesthesiologist at the nih who called me up one day and said, "iheard you're working on familial mediterranean

fever." i said, "yes, that's true." "well,i've got something for you. i've got a patient with familial hibernian fever -- -- irish fever." so, i said, "all right." so anyway, she came to the nih, we saw her,27 years old at the time. she had a 14-year history going back to age 13, i guess, ofthree to five-week febrile episodes. now, remember, i told you that the episodes offmf last on the order of one to three days, so this is way too long for attacks of fmf.she had abdominal pain with her attacks, which of course, you can have with fmf, but shehad a couple of other things that you usually don't see with fmf: periorbital edema anda migratory rash. we saw her about one week

after she had delivered a healthy baby boy,and she was just going into an attack. during her pregnancy, she was totally attack-free,and this is actually quite typical for the disease that i'm going to be telling you about.she had a high white count, elevated acute-phase reactants, and had a history of respondingto corticosteroids, but not colchicine. so, she was not of mediterranean ancestry. shehad these prolonged attacks. the attacks had manifestations that aren't manifestationsthat you usually see in fmf. and she responds to steroids, but not colchicine. and hereshe is in the pedigree. and you can see that this looks more like a dominant pattern ofinheritance. she's got three sisters who are affected, her mother is affected -- well,the maternal aunt isn't, but then there's

a maternal cousin who is. so, what is this? and this actually had beencalled -- in the literature, there were a couple of cases reported, a family reported-- it had been called familial hibernian fever because it had been described amongst theirish. and there even had been a hypothesis that perhaps the irish are actually descendedfrom jewish sailors who were a part of the spanish armada, which was shipwrecked, andthat they swam ashore in ireland and actually intermarried with the irish population andintroduced a dominant form of fmf into the irish population. this was the thinking thatwas going on at that time. well, in any case, so, we had this patient,and for a while we just took care of her and

didn't know what it was, and we didn't havethe gene for fmf at the time. once we found the gene for fmf, then we screened that genefor mutations to see if there was some different kind of mutation that would cause a dominantlyinherited form of periodic fever. nothing there. in the meantime, my former fellow,mike mcdermott, a good irishman, actually finished his fellowship at the nih, took ajob over in london, and tracked down the original hibernian fever family, and mapped the genein that family to the short arm of chromosome 12. in the meantime, we had approved severalother families with dominantly inherited fever, and so it did appear that there was this regionon chromosome 12, the short arm of chromosome 12 -- and of course, the fmf gene is on chromosome16, so it can't be that gene -- some gene

on chromosome 12 that might be causing this.now, the region that mike had mapped the gene to was much too large for us to, you know,just look at a few candidates, and actually, mike came back to my lab to do a sabbaticalto try to figure out what the gene was. so, at first, while we were trying to findmore families to narrow down the region, we subjected this interval of the genome to avery important test: the embarrassment test. so the embarrassment test is, you look atall the genes that are known in a given candidate region. you think about the phenotype. andyou think, well, what gene would it be that would be the most embarrassing that if wespent five years looking for it, and then we found it by some, you know, positionalapproach or whatever, and then people would

say, well, we could've told you that at thebeginning. so, the gene in that interval that seemed to be -- would be the most embarrassingif it turned out to be it, was this one here: tnfrsf1a. and it's the gene that encodes the55-kilodalton receptor for tumor necrosis factor. now, tumor necrosis factor is anothermediator of fever and inflammation in humans. there are three major mediators of fever inhumans: il-1, tnf, and il-6. so, this is tnf, the tnf receptor. there's actually two tnfreceptors in humans: a 55-kilodalton tnf receptor that's encoded here, and the 75-kilodaltonreceptor that's encoded on chromosome 1. the protein that's encoded by this receptor isshown here. it has four cysteine-rich domains, a transmembrane domain, and intracellularly,a death domain. so, it's actually a cousin

of pyrin, you know, because remember, deathdomains and pyrin domains are similar in structure. so, in any case, mike mcdermott and ivonaaksentijevich, one of the people in my lab, set out, then, to screen this gene for mutations.they started, actually, in october of 1998, and on thanksgiving day -- i have a very hardworkinggroup in my lab -- they came in to check their electropherograms of their sequences, andthey found, on thanksgiving day -- thanksgiving day -- mutations in seven different familieswith dominantly inherited fever in this gene. that was the discovery of this disease onthanksgiving day, 1998. we had thanksgiving dinner as a lab afterwards, at ivona's house,actually. anyway, the mutations that they found are mutations that disrupt this loopty-loopstructure. see, there's a fancy folding structure

of these cysteine-rich domains that basicallyinvolves the formation of disulfide bonds. and the disulfide bonds essentially form betweencysteines. and if you have a mutation that substitutes something else for a cysteine,the disulfide bond can't form, and if the disulfide can't -- bond can't form, it doesn'tfold right. so, this thing doesn't fold right because you have mutations that substitutesomething else for the cysteines, such as, for example, c52f here, where you have a phenylalanineinstead of a cysteine at position 52. so, in any case, that, then, leads to this disease. now, in the original -- now, this is justsweet irony -- in the original hibernian fever family, it was actually a family of mixedancestry -- the one side of the family was

irish, the other side of the family was scottish.they were being seen at a center in nottingham, england, and i guess that the group in nottinghamfigured that the fever must come from the hot-blooded irish side of the family. butin point of fact, when we knew what the gene was and what the mutation was, turns out itcame from the scottish side of the family. so, it should've been caledonian fever, nothibernian fever. but actually, at that time, with the seven families that we had, we had,like, a finnish family, so should it be, you know, finnish fever or something like that?well, we decided, probably best, you know, just as a matter of international diplomacy,to take the ethnic attribution out of the name, and so we came up -- again, thinkingof short names that would be easy to remember

and that people would quote -- we came upwith the name traps: tnf receptor-associated periodic syndrome. and so that's what this disease is callednowadays, and here are just some clinical images of patients with traps. i already showedyou this one. this is the adhesions in the 7-year-old girl with repeated episodes ofperitoneal inflammation. this is pleural thickening in a middle-aged man with recurrent episodesof pleurisy. this is the migratory rash of traps, which is quite interesting. it's arash that starts proximal and moves distally, oftentimes on an extremity. in this case,this man has the rash on his inner thigh on this particular day that the picture was taken,and then it might be on the knee the next

day, the calf the next day, the foot the nextday. so, it moves down. it's not spreading; it's moving. and if you look by magnetic resonanceimaging, you can see that the inflammation actually goes down into the muscle compartment.it's not a myositis, though; it's a fasciitis that these patients have. you can see there'sconjunctivitis these patients have; they can have periorbital edema, and they can developamyloidosis. this is a kidney biopsy stained with an anti-aa monoclonal antibody. so, in any case, what causes this? we hadthought, at first, that the mutations led, and they do lead, to a problem of sheddingof tnf receptors off the cell's surface. retention of the tnf receptors would then lead to repeatedsignaling through the receptors. that does

happen, but it appears to have a rather minoreffect in terms of the inflammation. what actually is the problem in traps is constipatedmonocytes. so, in any case, what happens is that when these receptors misfold, there'sa problem with the trafficking of the receptors from the endoplasmic reticulum to the golgiapparatus and then to the cell's surface. so, if you compare what happens with wild-typereceptors in this transfection system, you can see that you get -- the green is the receptor,the red is just a marker for the golgi, and you can see that there is colocalization ofthe wild-type with the golgi apparatus. but in the case of mutant receptor, you can seethat it just gets stuck in the endoplasmic reticulum. and you can see, actually, in cellsfrom patients -- these are human patients

with traps -- and you can see that there'sa reception of tnf receptor intracellularly compared with wild-type. what that does is shown here on this slide.so, when you signal through the tnf receptor, when tnf signals through the tnf receptor,what happens is tnf is actually a trimer in the bloodstream. the trimer of tnf binds tothree of the tnf receptors. it induces trimerization of the receptors. and when that happens, itbrings together, in close apposition, three of these death domains on the intracellularside of the cell membrane, and that, then, engages a signaling complex that leads tocytokine activation in the cell. when you have these mutant receptors, they actuallyaggregate in the endoplasmic reticulum. and

so there is actually then constitutive aggregationof these death domains intracellularly, which leads to constitutive activation of the pathwaysthat lead to inflammation through the tnf receptor. so, that's at least the major mechanismof inflammation. let's now turn from fmf, from traps, to threeother diseases -- this is a threefer -- that are caused by mutations in the same gene.and this is one that's really near and dear to my heart, because in fact, it turns outthat this gene encodes a protein that's a cousin of pyrin. so anyway, so these threediseases are sometimes known as caps: cryopyrin-associated periodic syndromes. so, the common featurein these diseases is that these patients have fever, recurrent fever, with a hives-likeskin rash. it's not true hives. they don't

have mast cells in these lesions. they don'thave elevated levels of histamine in their bloodstream. it's neutrophils, actually, thatare in these skin lesions. and there are three diseases. one of them is called fcas: familialcold autoinflammatory syndrome, or urticaria. it's cold-induced hives and fever that thesepatients will get. it's dominantly inherited. the person, if they go out in the cold foran hour or so, they'll break out in hives and have a fever. if they walk into an air-conditionedroom, if they live in the south -- and a lot of these people have moved to the south becauseof avoidance of cold weather, basically -- if they go into an air-conditioned room, they'llbreak out in hives, after an hour or so. and they feel lousy, and they have to actuallygo to bed in order to recover.

second disease, that's also caused by mutationsin the same gene, is a disease called muckle-wells syndrome. it's not cold-induced, but the patientsget fever, they get the same hives-like rash. actually, this patient here has muckle-wells.they get arthritis, they can develop sensorineural hearing loss, and they can develop amyloidosis.and then the most severe is a disease called nomid: neonatal onset multi-system inflammatorydisease. in europe, it's called cinca syndrome -- chronic infantile neurologic cutaneousand articular syndrome -- and it is a disease in which there's fever, hives-like rash, bonyovergrowth of the epiphyses of the long bones, and most devastating, cns disease. these patientsdevelop basically a chronic, aseptic meningitis that leads to blindness, and deafness, andmental disability. so, it's a very severe

illness and actually wasn't thought to begenetic at first because most of the patients who develop it have it as a spontaneous denovo mutation and never have children of their own, so it was thought to be a sporadic diseasea few years ago. so, in any case, hal hoffman at the universityof california, san diego, looking at some families with cold urticaria and muckle-wells,mapped the causative gene to the long arm of chromosome 1. and in the candidate interval-- this was around 2000, 2001 -- he found a predictive gene that had a pyrin domain.so, he applied the time-honored embarrassment test to this region and decided that he wouldscreen the gene for mutations associated with these two diseases, and lo and behold, hefound that there were mutations in this gene

in the so-called nacht domain, which is justa acronym and has nothing to do with falling asleep at night or anything like that. butin any case, this protein has a pyrin domain at its n-terminus, it has a nacht domain,which is a protein interaction domain, in the middle, and a leucine-rich repeat domainat its c-terminus. it can interact with asc, that same protein that pyrin can interactwith, and it also can have a role in activating il-1. now, at the time that hal was doing thesestudies, we were seeing a patient with muckle-wells, and my colleague, raphaela goldbach-mansky,was seeing this young man from north carolina named jonathan. and jonathan had been sentto the nih with possible still's disease,

systemic-onset jia, and here's his pictureback 10 years ago or something like that. and here he is. i don't know that you canmake the diagnosis of systemic-onset jia from this picture. but there were some other featuresthat didn't seem typical for systemic-onset jia. he had a hives-like rash. he had papilledema.he had some element of ventriculomegaly. and he had these knobby looking knees, which arepathognomonic for nomid. this appearance of the knees is what nomid knees look like, orcinca, if you're in europe. so, raphaela correctly diagnosed this patient as having nomid, neonatalonset multisystem inflammatory disease. and it was the two fellows who were on servicethat actually catalyzed this discovery. so, these fellows had been seeing my patient,with muckle-wells, and had been seeing raphaela's

patient, with nomid. and they said, well,the skin rash of these two diseases looks very similar, are you sure that they're notthe same disease? we said no, they're not the same disease, why do you -- haven't youbeen reading, you know? but they insisted, and so we thought, well, maybe they're right.maybe there is some connection there. and, of course, the gene for muckle-wells had justbeen identified by hal hoffman, so we knew what that was. so we decided, well, we'd checkit and see whether or not jonathan, this patient with nomid, in fact had mutations in thisgene, the gene that encodes this protein, cryopyrin. and so here's raphaela, the personthat saw jonathan, and ivona, who did the sequencing, and low and behold, what theyfound was that, in fact, there was a mutation

in cryopyrin in nomid. and then, you know, this is one of these greatnih stories. so, you know, we were telling people about this, you know, wasn't it interesting?and it happened that there was this guy, sergio, from argentina, who was a fellow up on the11th floor, two floors up from us, who had brought a couple of dna samples with him fromargentina of nomid patients, in the hopes there would be someone at the nih doing studiesof the genetics of nomid that he could then collaborate with. so, he gave us these samples,after appropriate paperwork was done, and sure enough, they had mutations in this gene,too. and it turns out that about half of the patients with nomid have mutations in thisgene. the mutations are clustered in the nacht

domain, just as they are for the other twodiseases. and, in fact, the balls, the different colored balls, represent mutations associatedwith the different diseases, and you can see they're all clustered in the same region.we have no idea why one mutation causes one of these diseases, and another mutation theother disease. the pyrin domain is almost invariant, andleucine-rich repeat domain seldom has mutations either. and so this -- the gene encodes thisprotein cryopyrin, pyrin because it has a pyrin domain, cryo, because at least someof the patients have cold-induced symptoms. and cryopyrin forms a macromolecular complex,and this is actually something that if you're taking boards or whatever, you probably oughtto know. the macromolecular complex is called

the inflammasome. and the inflammasome, youdon't need to know all the components of the inflammasome, but it is, basically, a complexthat's involved in the activation of il-1 beta. it's one of several complexes that canactivate il-1 beta. so you have this inflammasome, and basically the mutations that are associatedwith these diseases are in the nacht domain, and they're activating mutations that turnon this process all the time, constitutively. so, we reasoned that if il-1 is turned onall the time in these patients, just like i told you about the patient from bagdad,iraq, that we decided to treat with anakinra, we decide that we would do a trial of anakinrain nomid, because this is a devastating disease. and we thought that if there was somethingthat really deserved some attention, it was

this disease. on the left hand side of theimage here, you can see how il-1 ordinarily signals. you can think of il-1 as blue bubbles,just for purposes of this discussion. and il-1 has to bind the two chains of its receptorin order to signal. the green, type one il-1 receptor, and the purple, il-1 receptor accessoryprotein. it has to engage both of those receptors in order to deliver a signal. in all of us,we have something called il-1 receptor antagonist, which is basically a protein that can bindto the type 1 receptor, but doesn't bind to the accessory protein. so, it competes withil-1 to bind to its receptor, and basically, it can bind but it doesn't signal. so, it'sbasically a way of turning off signaling by il-1. and it's something that normally happensduring inflammation in people, is that you

get il-1 receptor antagonist levels goingup in the bloodstream, at least, in part, as a homeostatic mechanism to tone down theinflammation. there's a recombinant form of this that'sknown as anakinra, or kineret, the trade name. and so, anyway, we did a trial of anakinrain nomid, and, essentially, the results are shown here; it was published in the new englandjournal in 2006. within two or three days, the hives-like skin rash goes away completely.the conjunctivitis goes away completely. within three months, this white here, this is thechronic aseptic meningitis. this is a mri, with a flare image, and, basically, all ofthe white is inflammation and you can see it's gone, basically, within three months.the arrow points to the cochlea, this is a

fiesta image of the head, and this is cochleitis.this is what leads to deafness in these patients, and cochlear inflammation goes away as well.so, anyway, this has been a very effective treatment for nomid. so now we have a little quiz here, and we'llgo a little bit more quickly through the other diseases, because we have several other diseasesto talk about, and only 14 minutes to do it in. so anyway, here's your quiz. so, couldthis be nomid? so, here is a patient, a 9-month-old child from canada, who is referred to us withthese total -- this total body pustular rash. and here's the hair, so this is the fold ofthe neck; it's pustulars all over the body. the patient had a multifocal osteomyelitis,aseptic osteomyelitis, and you can see here

some of the punched-out lesions the arrowsare pointing to throughout the body. and then the patient also had evidence of vasculitis.so, from what i told you about nomid, is this nomid? no, of course not, because the skinlesions of nomid are hives-like, not pustulars. because the bone lesions of nomid are overgrowthof the epiphysis of the long bones, the knobby looking knees, not multifocal recurrent osteomyelitis,and i didn't say anything about vasculitis in nomid. so, this was not nomid. we wereasked is this nomid? just given the pictures, we said no, it's not. we actually did sequencefor mutations and cryopyrin, didn't find any. but, the referring physician from canada wasan obstinate character, and so he treated the patient with anakinra anyway, and thisis what happened. so, here's the child before

treatment, and you can see pustulars on theface. within three days, this child is starting to shed his skin, you can see he's kind ofsmiling here. and within a week he'd shed nearly all his skin, pustulars went away completely,and the multifocal osteomyelitis resolved within two or three months. so, what is this?what could this be that basically responds to an il-1 receptor antagonist, but it's notnomid? well, again, this important test, the embarrassment test, once again, comes to therescue. so we were thinking, well, okay, so here's a patient who responds to the il-1receptor antagonist. so what gene would be the most embarrassing, that if it turned outto be it, and we hadn't looked at it first, which one should we look at first? well, ofcourse it's the gene that encodes the endogenous

il-1 receptor antagonist. so, we looked at that, and lo and behold,what we found was that this patient was homozygous for a two-base-pair deletion in the codingregion of the il-1 receptor antagonist gene. that's almost too good to be true. homozygousfor the same two-base-pair deletion, how could that be, you know? so, we sequenced the parents;sure enough, the parents were carriers for it. the kid really is homozygous for the two-base-pairdeletion. and then of course, we took a better history, and it all became clear when we learnedthat the patient was from newfoundland. and so, basically, the explanation there, of course,is that newfoundland is an island off the eastern coast of canada, and many of the currentresidents of newfoundland are descendants

of settlers who came to newfoundland, actually,200 years ago. and they are at least, you know, distantly related to one another, inthe sense that there's a founder population there. and so probably one of the early settlersto newfoundland had this mutation, just as a heterozygous would have no symptoms associatedwith it, but it just happened that the two parents both were carriers for this, and thenthe child was as well. we now know that there are other mutationsin this gene, for example, a stop codon amongst people living in the bible belt of the netherlands.there's another mutation that we see in the middle east, yet another mutation in northeasternpuerto rico that are associated with this phenotype. and so we, again, thought thatbecause there are mutations in the same gene

associated with a particular phenotype, wewould give this disease a name, and the name that we have given it is dira, the deficiencyof the il-1 receptor antagonist. again, adhering to the naming conformity of short, easy topronounce, and easy to remember. so, in any case, this table just summarizes the comparisonof nomid with dira. different genes are involved. the functional consequences for nomid, it'sactivation of the inflammasome. for dira, it's decreased inhibition of il-1. differentskin rashes, different bone manifestations, different cns involvement as well. and then, finally, the last of these monogenicdiseases, and maybe we'll curtail things a little bit, so as not to get into the lunchhour, but, in any case, this disorder that

we'll talk about, just briefly, is papa syndrome.so, here is very severe cystic acne on the back of one of our patients with papa syndrome.and it's caused by mutations in this gene, pstpip1, which actually encodes a proteinthat's a pyrin binding protein. and so it just goes to show how mutations in all differentaspects of this pathway of regulation of il-1 can actually lead to different inflammatorydiseases. so, in this case, actually pstpip1 binds to pyrin, and, in fact, the disease-associatedmutations are associated with increased binding of pstpip1 to pyrin, which leads to increasedil-1 production and other cytokine production. so this schematic is just a depiction of il-1activation, some of the steps in il-1 activation. and what i've shown you is that, dependingon where in the pathway you're looking, if

you're mutating pyrin, you can have this erysipeloiderythema skin rash. if you're mutating pstpip1, you can get pyoderma gangrenosum. if you'remutating a nlrp3, you get these urticarial-like skin rashes. if you're mutating the il-1 receptorantagonist, you get this total body hives-like rash. so, in any case, there's a number of differentdiseases that are all caused by mutations in this pathway. and, in fact, dira is theprototype for a group of diseases in which receptor antagonist are mutated, and justone that was published in the new england journal a year or so ago, is a disease nowcalled ditra, deficiency of the il-36 receptor antagonist. and basically, il-36 signals ina similar way to il-1, with a binding of two

chains of a receptor, and a receptor antagonistthat only binds to one chain, and those patients get a form of pustular psoriasis. now maybe i'll just finish up by indicatingto you that, in fact, these pathways that i've told you about, that we've learned aboutthrough these monogenic diseases, are pathways that are important in some much more commongenetically complex diseases. so that we now know that monosodium urate, for example, activatesthe inflammasome. and that at least some of the inflammation in gout is due to excessiveil-1 production, and, in fact, there have been successful studies of il-1 inhibitorsin gout. type 2 diabetes, actually is another disorder, genetically complex, that has anil-1 component to it. it turns out that islet

cells of the pancreas synthesize il-1 beta,induced by hyperglycemia. il-1 beta is actually toxic to islet cells, so hyperglycemia causesislet cells to make il-1, which causes them, basically, to commit suicide, which then leadsto further hyperglycemia. so that, actually, if you treat patients with type 2 diabeteswith an il-1 inhibitor, as shown in this paper in the new england journal from a while ago,glycemic control is actually improved. and then, probably the most common of thesediseases, atherosclerosis. so, atherosclerosis has, as i think many of you recognize, aninflammatory component, and if one looks at mouse models of atherosclerosis, here is cholesteroldeposition in a wild-type mouse, but if one knocks out various components of the inflammasomenlrp3, which is cryopyrin, asc, or il-1 knockouts,

these mice do not develop atherosclerosis.now, you might say that's great for the mice, but, in fact, there is a trial, the cantostrial, that is going on right now. it's a trial that novartis, the maker of monoclonalantibody against il-1, canakinumab or ilaris. and this is a trial 17,200 patients, who havehad myocardial infarction, treating them either with placebo, or with three different dosesof this anti il-1 antibody. and the outcome, what they're looking for as the primary outcome,is the number of second cardiac events, effectively, in these patients, with the idea that blockingil-1 will prevent recurrent cardiac events. just the drug for each patient that's gettingactive drug, is about $100,000 dollars a year, so that for 17,200 patients, you're talkingabout a trial that's a billion-dollar trial.

so, definitely, these pathways are important,or thought to be important, we believe they're important, in common diseases. well, we don't have time to talk about candle,which is another interesting new disease that we're working on, or about plaid; this isanother disease we've published in the new england journal earlier this year. we'll justflip through these slides. or about a disease that's coming out next month in the americanjournal, or about behcet's disease, either. but you see, these are all things that maybewould get you to invite me back some other time for another talk, for part two of this.so i'll just, you know, go through these slides, you know, some very interesting associationsalong marco polo's silk root, but you'll have

to hear the next installment to know aboutthat. and here's just a, maybe to finish up, a piediagram of some almost 1,900 patients that we have studied genetically at the nih, inour autoinflammatory diseases clinic. and the interesting thing is that in only abouta third of them, do we have a genetic explanation; in two-thirds of them we don't. now, not allof them probably have a mendelian disease, but this just highlights the point that there'splenty more to be found amongst these patients, and that there's, i think, still a rich sourceof patients for study, and that we can learn a lot from these patients. so just to summarize, the autoinflammatorydiseases manifest constitutive or easily triggered

innate immune activation. mendelian autoinflammatorydiseases provided important insights into the regulation of inflammation. il-1 betaactivation protein misfolding, and well, we didn't talk about proteasome dysfunction,but take my word for it, are three mechanisms of mendelian autoinflammatory disease. basedon the demonstration of an important role for the inflammasome and their pathophysiology,a number of common disorders such as gout, type 2 diabetes, and atherosclerosis havebeen shown to have an autoinflammatory component. and again, for the next talk, genome-wideassociation, and next-gen sequencing studies allow the identification of susceptibilityloci for the more common but genetically complex autoinflammatory disorders. here is just thecast of characters that really made all this

happen. and, of course, the clinical centerof the nih, where we carried out most of these studies. so, anyway, it is now one minute to 9, andi apologize for talking a little bit over, but hopefully you've learned at least a littlebit. thanks a lot. [applause] male speaker:i hope you come back for part two. i wanted to ask you a question about amyloidosis. asa rheumatologist, i treat a lot of inflammatory disease and i rarely see amyloidosis. so,number one, am i preventing amyloidosis by treating inflammation, and number two, howdo you tell who's at risk for amyloidosis

[inaudible]? dan kastner:yeah, those are both great questions. so, we do believe that the more effective treatmentsfor inflammatory disease have led to a reduced frequency of amyloidosis. certainly, chronicinfectious diseases, things like tuberculosis, were common causes of amyloidosis back inthe age before there were effective treatments for tuberculosis. and we see less amyloidosisassociated with things like rheumatoid arthritis, as the biologics have become more widely used.so, i do think that aggressive treatment, and that's certainly what we do with the periodicfever syndrome patients, is that we really aggressively treat their underlying inflammationto the point that we want to normalize their

acute phase reactants. now, in terms of who's at risk for amyloidosis,that's also an excellent question. now, it's probably been looked at most systematicallyin patients with fmf. and so, in fmf, certainly the mutations that are associated with moresevere disease, as you might expect, are associated with a higher probability of amyloidosis.males, for some reason, are associated with a higher risk of amyloidosis. noncompliancewith treatment, associated with a higher risk of amyloidosis. there is a polymorphism inthe amyloid locus actually, that is associated with a higher risk of amyloidosis, probablybecause it prevents the normal degradation of the amyloid protein.

and then, the most captivating of all association,is that it depends on where you grow up as to what your chances of getting amyloid are,at least with fmf. armenians who grow up in armenia, for example, have about a 25 percentrisk of developing amyloidosis by the time they're 30. armenians in the united states,with the same spectrum of mutations, even if not treated with colchicine, have lessthan 1 percent risk of developing amyloidosis. and there was a big study done by isabelletouitou, published in "arthritis and rheumatism" a few years ago, that looked at country oforigin as being really one of the major predictive factors in whether or not you get amyloid.and people who come from countries where infant mortality is higher, and therefore, we thinkthe health care availability may be lower,

have a higher risk of developing amyloid forreasons unknown, but that seems to be the case. male speaker:[inaudible] dan kastner:say it again? male speaker:so populations that use [inaudible], if they were to move away from it, how long wouldmother nature take to extinguish those [inaudible]? dan kastner:well, let's -- so, we do think, and i didn't have time to talk about this, that, at leastat one time, there may have been a selective advantage for mutations at the fmf locus.and, in fact, if you look at the carrier frequency

for mutations in this gene, in mediterranean/middleeastern populations, it's incredibly high. it's like one in three to one in five. now,if you contrast that with the carrier frequency for cystic fibrosis, which is the most commonlethal recessive disease in caucasians in north america, one in 20; so this is incrediblyhigh, one in three to one in five. and there's been a lot of speculation as to whether theremight be, or might have been, some infectious agent that was selecting for these mutationsover the centuries. so far, at least in various epidemiologic studies and studies of experimentalanimals, we haven't figured out what that agent would be. dan kastner:apparently not, no.

male speaker:is the amyloid you have referenced to anything related to what you see in outsiders? [inaudible]my second question is that colchicine is so inexpensive; does it have use in the broadspectrum of the diseases that you mentioned? dan kastner:yes. so, both excellent questions. with regards to the amyloid of alzheimer's disease, it'sa different protein that is being deposited. a beta, as opposed to aa, so it is a differentchemical process, although it does appear, there are some studies that would suggestthat il-1 does play some role in the pathogenesis of even the amyloidosis in alzheimer's disease.so, that's an area still under study, as to whether or not maybe that would help in someway. but, the thing is, all amyloid looks

the same under the microscope when you stainit with congo red. it all gets this, if you look at it under polarizing light, this apple-greenbirefringent appearance to it. but it's, you know, different proteins that are being deposited,but probably there's some final common pathway that makes them, you know, misfold and depositin that way, so that's an interesting question. now the question as to whether or not colchicinewould have a role in treating some of the other amyloidoses, that's something that onecould consider. there was the thinking, back in the old days, that colchicine might beeffective in fmf, even if you can't prevent the attacks, that it might still prevent theamyloidosis. colchicine does prevent the amyloidosis of fmf. but, it appears that that's relatedto its ability to prevent the inflammatory

attacks of fmf. so, it's not that it has ananti-amyloidagenic effect; it has an anti-inflammatory effect in that disease. which then leads toless burden of saa in the blood, and less to deposit, so we don't think that it's becauseit has a direct effect on amyloid deposition. as far as the cost of colchicine, just a wordabout that, as some of you may know, colchicine used to be available as several generic formsin the united states, and then, because of some well-intentioned legislation, it turnedout that if a maker of a drug like that, which had never undergone appropriate clinical trials,if a maker of the drug went through certain tests with the fda, they could then get exclusivelicense and put all of the other companies out of business. so there's a company, unionpharmaceuticals, that did just that, using

gout as the prototype, and so, essentially,at this point, there's only one form of colchicine that is available in the united states, thetrade name of it is colcrys, and it's made by this company, union pharmaceuticals. andthe cost of colchicine has now gone up from roughly 10 cents a tablet to $5 a tablet,which will persist for the length of time that they have an exclusive license on this. and again, this was something that was well-intentioned,you know, in the idea that this would encourage further rigor in terms of the testing of agentsthat had never been subjected to the scrutiny of modern trials. but, you know, it's endedup sort of causing this issue with regard to cost of the drug, and actually, colcrysitself, we've seen in some of our patients,

is perhaps milligram per milligram, or .1milligram to .1 milligram, a little bit less effective than the -- some of the other genericforms. and so, in fact, one has to make dose adjustments in the patients when they switchfrom their generic to colcrys. so, an interesting thing, sort of a quasi- political, medical-politicalkind of issue i guess. male speaker:is there a dark side to [inaudible] in terms of risk and infection? dan kastner:that's a great question, gene. so we don't see a lot of problems, but, certainly, thetnf inhibitors are a lot more associated with opportunistic infections, with microbacterialinfections, with fungal infections, than il-1

inhibitors. we do see some increase in riskof upper respiratory illnesses, but at least at the doses that we give for these diseases,no, we do not see -- and part of the problem is, the thing with these patients, is thatthey have a very hyperactive innate immune system, and so what we're doing, treatingthem with the il-1 inhibitors, is sort of bringing it back to normal. in a lot of cases,parents of kids with these diseases will say that everybody else in the family will geta cold or the flu, and this child, who's not been treated yet, doesn't. they may get theirrecurrent fever syndrome, but they don't get colds and flus. when we treat them with il-1inhibitors, or whatever other biologic, then they no longer have their periodic fever episodes,but they, like the rest of us mortals, begin

to get colds and flus like anyone else. male speaker:that's going to be very hard to follow. thank you very much for coming in today. dan kastner:thanks, gene.

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