>> good afternoon. i think we'll begin. let though who arrive late will join us. we have a slightly, but more discriminating perhaps audience this afternoon. these are very informal by design so we encourage you to
ask questions, even while people are speaking. and if something is said that you don't understand, sometimes clinical terminology is baffling to the more basic scientists and the reverse is true, too. so for those who haven't been here before, is there anybody
who doesn't know what this is a picture of? what is it? >> new york city. what's the bridge? >> that's right. so this is the most famous bridge in the history of the world, particularly inyou grew
up near new york. the reason it's the logo for demystifying medicine sta it's a pretty good example of trying to link the big advances in physical and biological sciences with human disease. and that's really been the purpose of this course, which is
now in its 12th year. you're welcome to attend any or all of the sessions. they are all, also live. and so when you ask questions, if you raise your hand we'll give you a microphone. every week there are between 200 and up to 400 people just on the
campus here who are watching live, and then a few days later it goes into the nih video ar chive and goes out to the world. its aactually replicated or most of the material is used now in 18 different countries and over 2 dozen major north american
teaching institutions. any criticisms, comments? suggestions? please send them along because we try to accommodate what the interests are, both people with more column training, and -- clinical training and those who have more bench training.
so because inflammation, which is old, is having an unbeliev able -- i wouldn't use the word renaissance, explosion is more like it. spinning off into a huge number of disease categories that previously were not labeled inflammation.
we a sear aways of 4 of these sessions over the next several weeks that are dealing with different aspects of so if we jump back to the first century, the word, to set on fire because what was recognized inflammation in those days were things like abscesses and really
hot bulging stuff. and so cellsis gave us the initial description of heat, readiness, swell, pain, and loss of function which are the hallmarks of infection, sepsis, and for the next 100 years or more, the focus was really on wound healing because people
were businessing killing each other throughout that period of time. much as today. but, of course, they didn't have antibiotics and fancy surgery. wound healing, infection and sepsis, e-mailed epidemics all became part of the inflammation.
it's fascinating to read about this. it isn't until the 20th century that the reaction to inflammation begins to appear in the form of immunity. and in 1882, while studying star fish in which he made an incision and update a crude
micro scope, looked at these fun ny looking cells that accumulated around the wound, he actually discovered the innate immune system. and there is a reference here to a most remarkable readable and exciting book, all of this stuff , by the way, is on our web
page, if you google demystifying medicine, you can see all the power points, the references, all the background from today and the previous 12 years. then we get into the 20th century with the discovery of the adaptive immune system. and of course we're off to the
races with andgen mediated cell responses, t cells, b cells, and so forth. but that's not the end of it. so now in the 21st century, suddenly, inflammation is reborn and it's reborn in the context of almost every disease you can think of.
and some of these are going to be the topics of these sessions. today, for example -- whoops, i put a misspelling in there. it's the role of inflammation as involved in cardiovascular disease. we'll get back to that in a minute.
and later a session on inflammation regarding immuno suppression like with hiv and drugs that suppress the immune system as well as disease s. it's assuming an ever increasing role in cancer, interestingly enough.
which will also be discussed. as well as in autoimmune disease s, and more recently, neuro degenerative diseases like alzheimer's, which would also be discussed, as well as obesity. and one might legitimately ask what's next? and what it means, of course, is
that inflammation as seen in the first century was just the tip of an iceberg. and the mechanisms of inflammation are what really set the ball rolling for everything that happened. and that brings us to today's topic.
an important part in the mechanisms of inflammation are people are inheritable diseases who manifest frequent next infections of one kind or another. john gallin, who i'm sure you all know, is going to lead a discussion today with two of his
colleagues on new approaches to arterial sclerosis, coming from a study of an inheritable inflammatory type of disease, granulomatous disease. so dr. gallon is went to cornell medical school, trained in new york, also at the nih. and then came back to the nih in
1976 as a senior investigator. he's been director of the niaid intramural program, chief of the laboratory of host offenses, and best known since 1994 as the director of the nih clinical center. if there are any basic scientist s here who have never
been in the clinical v i urge you to go. it's an important part of your education. it's the other part of the bridge, but two parts of the bridge become somewhat synonomous. so today, dr. gallin has arrang
ed for tom, got his ph. d. at the university of virginia, did a post-doc in cyto skeletal research at yale, came to niaid in 1988, and is a senior investigator and collaborator and very closely in volved to work together on topics that are going to be
presented today. and then gale walsh, is that right, in post -- on his way to medical school, working in the laboratory, and he's going to contribute to this discussion. we're also very grateful to dr. gallin for arranging to a patient.
very variable we try, whenever possible, to have a live patient previously tell the story of their disease in their own terms why? because almost every basic scientist whom i talk to without about this course, one of the things they highly talk about is
putting a human face on a so dr. gallin. >> okay. well, thank you. 12 years ago when you came to me and said you wanted to do this course called demystifying medicine, and explained to me the idea of creating this bridge
between the basic scientists and the clinical investigator's, i got very excited about that. and i had long known that if you're a clinician and you want to do good science, you dam well better have connective to basic scientists if you want to have some success.
and i think when you hear the conversations we're going to have this afternoon with tom and myself, and -- you'll get a feeling for how, in our program, we've tried to bring this together. so what i wanted to do was to tell you a story about some
patients that i've had the privilege of carrying for, for a long part of my career, and in particular, tell you how sometimes the unexpected observations in patients give you some insights that might be relevant to some common diseases so this is a picture of the
clinical center. if any of you want a tour. call me up. i'd be thrilled to take you around and show you some of the secrets and places that nobody else gets to see because this key i got gets my into all sorts of special places.
so at the clinical center, we do 3 things. we study the pathophysiology of we conduct first in human studies with new therapeutics. probably more than anywhere else in the world. we have about 1600 protocols, the engine that drive the place,
clinical protocols. and about 730 of those are clinical trials, 93% of those are called phase 1 or phase 2, which are first in human with new drugs. the other thing we do is woe study a lot of patients with rare diseases, over 530 cohorts
of patients with rare diseases are being studied today at the and that's clearly more than anywhere else. why do we study patients with rare diseases? well, first of all, they're not so rare. it's estimated 18 to 25 million
citizens in the united states have a rare disease. that grows every day as we get more and more delinuation of the genotype of common diseases. we realize there are more and more rare diseases. breast cancer is now a series of genetic mutations characterizing
some subsets of disease. so it's not so rare. 2, we can provide hope to these patients which is phenomenally important. but third, we can bring patients here, more easily than people can bring them to a hospital anywhere else in the world
because we travel patients, there is no cost for a patient to be here, and patients can -- we can keep them as long as we want, because we never bill patients. we've never billed a patient since the hospital opened. even though a few years ago
congress tried to get us to start billing. we protested and they surprising ly listened and said okay. but it often is true that study ing patients with rare diseases find insights into common diseases.
i couldn't help but share a quote per a letter from william harvey, rip written in 1657. i want to read it to you. he wrote nature is nowhere ac customed more oply to display her secret mysteries than in cases where she shows traces of her workerings apart from the
beaten path. no there any better way to advance the proper practice of medicine than to give our minds to the discovery of the usual law of nature, by the careful investigation of cases of rarer forms of disease. 1657 by the man who discovered
the circuitry system. i think it's pretty amazing that he had that insight. so for today, what we're going to tell you about is that we have got some insights into the common disease atherosclerosis from studying patients with in the last 20 years, we have
learned a tremendous amount what's called the nox family of nadph oxidases, which is the family of enzymes, one of which, nox2, is effective in cdg. tom leto has done a tremendous amount of work covering this. and lastly, we want to take you on a journey that we're pursuing
to discover knox inhibitors. we think that may be surprising ly useful. let me start with the story of chronic granulomatous disease, a rare disease of fagocytes. this story happens practices its origins in 1933, a very shatter paper we cw -- short paper which
appear in the american journal of physiology. studying dog leukocytes that were stimulated with bacteria, using a war burg apparatus. some of you may have used in your life. where you put the leukocytes in a vessel and the leukocytes and
attached arm, you tip it and then you mix them together. and that occurs at times 0. he was measuring oxygen consumption and showed there was a burst of oxygen consumption when he mixed the bacteria with the dog leucocytes. and it lasted about 140, 190
minutes to 140 minutes that. bust of oxygen is what we call the respiratory burst that occur s in leukocytes. and this was the patient that showed that. 20 years or so later, the first patient who had an obnormalty of this system was described, from
the laboratory of charles janeway. who was, i believe, at yale. describing a child who had in creased gamma globulin associated with severe recurrent and chronic non specific infection. he didn't realize at the time
that this patient had disease of the oxidase, but described the clinical map fessations. and the second patient was described in the laboratory of robert good by [indiscernible] in 1957. a few year years hatter. that cities was called a fatal
granulomatous of childhood. the patients died very early. in 1967 one of robert good's fellows, paul, went on to become a very famous paediatric researcher at the university of minnesota, realized he couldn't stand the name fatal granulomatous disease of child
hood. he said how do you go to parents and tell them their child has that disease, so he changed it. he renamed it chronic he described, in his paper, there was an abnormality in the killing of act by the neutrophil s or leucocytes obtain
ed from those patients. his lab went on and described that there was some metabolic activity defects in patients with this abnormality. and bob and david at harvard in 1967, defined and acnormalty of a leucocyte oxidase in these and bob boehner and david, then,
went on to develop a simple as say that could demonstrate this abnormality called the mbt die test. this gets converted into a blue color when the respiratory burst occurs. and on the left of your slide is a normal cell that's blue, the
middle is a patient with granulomatous disease, no blue. and in the right, an excellent carrier where you see as a result of lionization, or enact vation of some of the cells exchromosome hybrid of normal and acnormal cells. an normal cells.
so initially, it was vellet that cd -- cgd was a single disease of a single enzyme defect. it occurred about 1 in 200,000 live births in the united states the mortality was about 2% per year. in the patient population. and there was an abnormality of
the nadph oxidase. now, the clinical manifestations of nadph oxidase are shown in this slide. the patient on the top -- he gave me permission to use his name, phillip carp. and he -- this was a long time ago. this was 20 years ago.
his swollen lymph node is a granuloa, as we call it. and these patients can also get granulomas in vital structures in the body look like the esophagus and this cobble stone appearing esophagus is a collection of granulomas. it can be so severe the patients
can't swallow their food. they can get these in the bladder, and that can obstruct the know of urine and cause kidney failure. under the microscope shown on the right, these patients get this accumulation of these granulomas, which are fusions of
macrophages or mononuclearer phagocytes to form these giant cells which lead to obstruction. now, in addition to this funny abnormality of inflammation, these patients get infections because i'll show you in a few minutes, they can produce some of the essential products of
post defense. the second patient is the brother of the first patient. his name is robert carp. when he was 11 years old, when i saw him in the clinic one day, i asked him ho he was. he said i'm fine. then he says, well, i got this
little thing on my finger. he says what do you think? i said i don't know. let's take a look at it. and we took a needle and we lanced it. and we looked at it under the microscope. and this is what we saw.
we saw these branch elements, studiod with neutrophils, which are attacking it. not destroying it. this turned out to be aspergill us. a nasty fungal infection which it turns out these patients get. a few days later, robert develop
ed an abnormal gate, an normal walking. and we did a brain scan which was something we used to do in those days. we don't do it much anymore. and he had some lesions in his brain. and then he developed lesions on
his back. his skin. and he had metastatic fungal infection of aspergillus. and the community of clinicians at the clinical center were very worried as to whether or not he would survive. some of them said he won't
survive because it's in the i said i don't believe that. and we gave him phenomenally aggressive treatment with the only antifungal agent at the time, and daily normal white cells for a white. and he got better. and he's here today so let me
introduce robert carp. why don't you come here? have a seat. so robert, i don't know if you can remember what it was like when you were 11 years old to come to this place with you and your brother having this funny problem.
tell us what you can remember. what it was like back then. >> well, i've like to thank dr. gallin for inviting me. i really appreciate it. i always enjoy the opportunity to speak to families, patients, and of course physicians and doctors and scientists.
so thank you for having me. it was a long time ago. i was looking at my finger, and my brother, and i don't have much memory of that. coming to nih was overwhelming. it was scary. understanding this severity of my infection was difficult for
an 11-year old boy. but i also had a very, very phenomenal group of physicians and doctors, and my head doctor at the time back in 1983 was this guy. c he was my primary physician and he was the host of -- were you the host of -- director of
the host of laboratory defenses at that time? >> it happened quite -- hadn't quite started. >> he was the head of something because i called him the big cheese. and i drew. i drew pictures of him and his
staff and his doctors that help ed me. but it was scary. and it was difficult. i remember the treatments very well. every 3 days a new iv. they didn't have central lines. we didn't do substantial lines
because of fear of infection. every 3 days this 11-year old would get a new iv. this is a very difficult medicine. every day i would get a spike -- i would spike a fever. first chills, really uncontrol led, then a fever, high
hundreds, 102, 103. and you'd get nauseous. i went through this for -- i believe i was treated for 4 months like this. but i came around. and here i am turning 46 this didn't think i'd get past 11. >> tell us about your children.
>> i have 2 beautiful children, 8 and 11. i have with a college degree, working. i live a very, very normal life. and i owe my life to nih and scientists like yourself. and to doctors. so i feel very fortunate to be
part of nih and to come to nih and find it. >> great. are there any questions anybody wants to ask mr. carp? okay. well -- >> so what's happened in the interval from when you were very young?
>> sure. sure. >> you're still young, but -- >> youngish. >> 35 years, i guess. >> it's 35 years. so i've had infections periodically, especially when i was younger.
you wouldn't be able to tell it. i've had multiple surgeries. some very invasive, especially in the 80s. they were very aggressive. multiple surgeries, multiple hospital stays. i have been very fortunate at nih to be part of clinical trial
i was among the first to be on the clinical trials of gamma interferon and other groundbreaking studies. in and out of hospitals. my last major infection was 2009 where i was diagnosed with a very rare bacteria that was scoffed here, thusly named
bethesda densis. i had ivs for another four months. since then i have been lucky. i'm involved with outreach when i can. but for the most part i've lived a very, very good life with the exception of being in the
hospital now and then and being a very sick patient. >> are you still on the interferon therapy? >> i am not on interferon. i am pro fell latic ikes, ain't fungal, backram, which i have been all my life. i was born in south africa.
and my older brother, who is 18 months older, backram wasn't even around them. so we were both serious ill. i have been on it my whole life. i made the decision not to continue with interferon for my own personal wellbeing. i didn't like the injections.
but i think i've done pretty well that. but i do advocate for it as a treatment. i must say that one of the reasons he's done so well was because of his parents, his mom, in particular, and his dad was so supportive, helpful when you
were little. >> absolutely. my parents were phenomenal, especially my mother who was with me in '83, 4 months by my bedside every fever i spiked. so she was there. so i really encourage you, we have ph.d. candidates in the
room. if you're thinking about going to this practice, and becoming part of nih, i want you to know it does impact us directly and it does make a difference. i would literally not be alive if it wasn't forever dr. gallin, his team, and the scientists at
nih who i'm immensely grateful for. thank you. [applause] well, thank you, robert. and he's brought up a lot of things that i'm not going to tell you about today but we participated over this journey
on a number of advances in the treatment of these patients, first with the ain't biotic back tremendous. everybody said you can't put somebody on aches daily, screw up their microbiome. we didn't know about that then. we helped put that on the map as
a successful way to reduce the number of infections. then we did studies with an anti fungal drug which we tested in 25 patients. it took us ten years to complete that study. and the reason was that we did what's called a double blind
cross over trial where you would put half the patients on drug, half the patients on placebo, cross them over, and compare the number of infections and the 2 limbs. one of the things came to me one day with his pills. he said i didn't take any of the
pills. but i got an infection. but you don't have to count me. well, i went to the statistician nope, you got to call him. it's what they call intent to treat. so he had a relapse on the drug because he didn't take the drug.
and that meant we had to go on and on and people said you shouldn't do the study. you're never going to complete it. it did take ten years. we completed it. it was a new england journal of medicine article, changed the
care for these patients and is now used regularly. interestingly, the editorial about the study wasn't about the study, but about the statistical design. and that was kind of interesting then we did the first drug study using a immune adjuvant in a
population of patients who have immune deficiency, interferon gamma. and we had done some in vitro studies where my technician, who was doing the studies -- i was looking to see if we could in duce these granulomas with interferon gamma.
we couldn't. she said i think you're fixing these cells in some of these patients, because she is very compulsive. she's now diocesesed. and -- deceased. she made this observation which was published.
there was a paper from harvarden edy we got again tech to do a blind study of the team of investigators were convened in california. all they wanted to do was the bio chemical measurement as the outcome. i said -- i won't participate.
i want a clinical outcome in addition. and since we had two-thirds of the patients, we had a fair amendment of influence. and it turned out that the only difference was the clinical outcome, we couldn't show the effect bio chemly.
it dramatically reduced the so it became the standard of care for these patients. but the trouble with interferon gamma and with robert -- what robert didn't tell you, it makes patients feel like they have a flew-like syndrome all the time. in addition to taking injections
, they don't feel so good. but a lot of the patients choose to take it. and then this last thing he mentioned, this bethesda densis organism, the full name is granu lo bethesda denysis, discovered in steve hollands'
lab here, which is an organization that's only been seen in chronic granulomatous but it's a very sea breezious very serious infection. it stands for chronic that's where it got its name. bethesda densis is bethesda, maryland, hence the name.
we're spending a lot of time de fining that organism and trying to understand why it's pathogenic. so going back to the history in 1986, stewart's laboratory at harvard discovered the gene for the most common form of chronic granulomatous disease which is
on the x chromosome that i will tell you about in a minute, doing substrategics technology, it was the first time that was used. and then a few years later, he and his team defined a b cyto chrome as being a critical product of the gene.
the first gene was for -- it's a dimer. or it's a two molecular complex, gp91 molecular phagocyte oxidase called gp91 fox. and that links to a smaller molecular weight protein of 22 kiladaltons, p22 fox. and then our lab, with tom harry
malik, karen, and [indiscernible] and i defined a -- some cytosolic proteins, 47 kiladalton and 67 kilo dalton protein. it game clear that the concept, one enzyme, one protein, the basis of disease was wrong, this is a whole family of prepares.
they all must interact for this system to work. this slide depicts some of the key elms, not all of them, but the main key ones. this gp91 phagocyte oxidase or fox is bound to a a 22 fox and i'll show you more about the structure in a minute.
this is where the electronics pass through to cause a re duction of oxygen to super oxide, by super obscried gets converted to hydrogen perrox i'd [indiscernible] gets converted to hypocloseris acid, which is bleach, converted to chlorine. so obviously, these are potent
antimicrobials. if the system doesn't work as it doesn't in cgd, you got a it's also linked to the glucose 6 fox fate dehydrogenase. the mechanism to replenish of the the oxidase and is a few -- a few patients have been described with g6pd deficiency,
who also have cgd. some over these other proteins, like the p40 fox, recently described component which has a regulatory function as well as as the rag proteins. you know now that it can occur with mutations in genes coding for a lot of these proteins or
essentially all of them. the most common. the x linked version which is what robert carp has, about two-thirds of the patients on the exchromosome. the 22 fox is rare. it's less than 5%. and the cytosolic elements, p47
foxes, the second most common in about a third of the patients. and then this p67 fox is rare. and then you can see g6pd is very rare, and p40 fox mutation is even rarer, or just as rare. so tom will tell you a little bit about some of these oxidases , and there is a family
of oxidases that are related to this that exist in different tissues, but these are the ones that are in the phagocytes. and at nih, this is current data depicting the number of families patients and carriers. we've had the privilege of caring for over the years.
and the ratios that i just told you about are shown. you'll see we have a lot of car riers which is proving to be a valuable cohort of patients to have the opportunity to study. now, about 7 years ago or -- we published this paper in the new england journal of medicine
which i feel -- essential helped us in understanding these patients, and provided some guidance into the care. and this is looking at the re sidual oxidase, the little bit that's left in some of these patients, and it's because in some patients, the mutation
causing -- you get no oxidase produces. in some you get a little bit. and this slide depicts the structure of the components of the oxidase shown with the gp91 fox shown on the left, and all these circles represent points of mutations which are defined
in the code on the upper right. the binding domain is where the iron is. and that's where electrons have to pass. one of the amazing things, in this fox molecule. mutations throughout the molecule can cause cgd.
there is an fad and nadph bind ing domains. mutations in those regions tend to be more serious. the p22 fox mutation is rare. and you can see where the mutations have been defined to date. and tom perhaps can tell you a
little bit about the relationship of these sar com ology domains and building of the scaffold of this protein during activation, since he did a lot of the pioneering work. the p47 fox mutation is interest ing. in general, there is a hot spot.
when this slide was made, there was only one location of mutations. there is now a few others. for this gene mike pence and then the p67 fox mutation -- gene so what this article did is we looked at all these mutations and we made a graph plotting 2
different assays for production of these reactive oxygen species , one using dhr, and one looking at super oxide pro duction directly. and plotted one against the other. you can see there is a line. but the important point is these
red dots are patients that died. and the -- so the first observation that i want to tell you is that we can detect a collection of patients with the least amount of super oxide who are more susceptible, the least amount of residual. normal would be off the slide.
and -- what this paper enabled -- i'm not showing you all the analysis. it enabled stratification of cgd patients based on the residual respiratory burst activity and risk of mortality. based on those data, we could de fine at birth, we thought,
high risk patients seen with lower absence residual phagocyte ros and we are now using that to guide us in treatment of these patients aggressively, particularly early in life. so the patients who produce no super oxide at all, who've had serious infections, are
candidates for bone marrow transplants early. and that can be a curative procedure. and harry malik, who ink has spoken here previously, he's now developed tools for doing gene therapy to correct patients which is very valuable, will be
very valuable for patients who a good match, a good donor. >> [inaudible] >> they're very close. that's obviously a critical question. if i tested a normal from one day to another, there may be more variability in the small
amounts of the cdg patients. for example, with robert carp an his brother phillip, we just herd the data today, they were tested on 90 indications when we-- 9 occasions when we made thisafter. interestingly, they were the only two family members that fell into what we called
different quadrants. but the variation was like -- the numbers we used was the number of super oxide produced per hour. it was like 1.6 verses 1.4, or -- so they statistically were no different. so what i want to do now is
shift to an observation that is really the subject of this talk, having given this background. that is observations that suggest loss of nadph function is productive of atherosclerosis this story for me started when i , unfortunately, had to go to autopsies on these patients.
and i noticed that one of the early signs of atherosclerosis, which is fatty streaking in the aority eye, i didn't think it was there. i kept asking the pathologist, what do you think of that? we can't really be convinced of that.
i said, well -- so we -- i just filed it in the back of my mind. we couldn't prove it. there were interesting mouse studies which i'm going to show you that renewed my interest. and then a colleague from italy, franciso, you have in your pack age a short mini review that
he and i just published this month about this story. he published some things that really perked my interest. so the mouse study is shown here, the one that i got first interested in and published in the journal of clinical investigation in 2001 by barry
lang, who is here at the nih. who took advantage of crossing apoe deficientant mice, very prone for atherosclerosis with c gd mice, using a p47 knockout mice that have been developed in our laboratory. steve and i and sharon jackson did.
and first looking at mice who were fed a high fat diet, who did not have the cgd gene, you remove the aorta and you stain it, with lipid, you get this red deposit the. you can see there is a lot of red depose the. on the right is the mouse that
had both the apoe knockout and c gd knockout. there was protection. so in this animal model, this time, this experiment, it looked like the cgd protected the development of lipid deposits in the aorta of these animals. unfortunately, this observation
did not consistently get re produced in the literature. there was some studies which particularly using the gp91 fox knockout by mary, who had initially cloned the gp91 -- i guess the p22 fox gene, didn't confirm. there was enough in the
literature that was of interest to us. this colleague, francisco, from rome, published a series of papers suggesting that this cgd animals -- humans, excuse me, were protected from atherosclerosis. and these are all in that review
article. i'm not going to go into full depth here. so we designed a protocol to -- at the clinical center titled non invasive assessment of atherosclerosis in patients with cgd and other disorders. immune system.
taking vac of new newt gingrichs which had -- of new meteorologist -- [technical difficulties]. taking advantage of new technologies. and the question was, does loss of nadph function and cgd result undecreased atherosclerosis?
this was published a few years ago in circulation. and let me show you the bottom line. so the approach is to take an mri of the core corroted artery. there is an external and encore roded artery. you can measure the difference
between the inside and outside of that vessel. measuring the in this caseness of the car ootid artery -- and to show you how dramatically this can appear, who are 3 people that do not have cgp who do not have atherosclerosis and you can see the thickness of the
vessel is easily detected. we did, in a blinded fashion, looked at all our patients who were -- i think it was about 30 years of age. and as the group, you can see the cgds can have less thicken ing of the carotid artery wall than normals.
we then looked at two of the main types of cgd that we had in our cohort, the p47 fox women, and you can see it was -- there was a difference between normals and them. we looked at the males, both the p47 and the gp91 fox deficient males.
it held for all the groups. interestingly, when we looked at the different measure, something called the coronary plaque index , we didn't see a difference between the normal and cgd, and to make a long story short, there seems to be an emerging difference in
atherosclerosis in the coronary arteries verses the major blood vessels in the body. that's the subject of another study. now, this observation of decreased thickening of the caro tid artery occurred despite increased risk of
atherosclerosis in these patients of the one measure of inflammation associated with atherosclerosis is something called c eactive protein. you can see it's increased in in the cgd populations. another, an enzyme released from neutrophils southed with in
creased risk of increased in cgd. another is hdl, which if you need a simple way to remember what that means, h stands for healthy. this is your healthy lipoprotein and they have a reduced level so they were at increased risk.
and although ldl, which you can think of as lousy lipoproteins, they were normal but oxidized ld l, which is associated with in creased risk, was increased in in the cgd patients. so, the summary of the paper was that cgd patients are protected from carotid artery thickening,
suggesting absec of nox 2 which is the name of the oxidase with cgd is protected of carotid art ery. and the increased risk factors of elevated mpo, crp, ox diced l dl and low hdl in counting de links nox 2 from those observations.
here is a question that comes out. would reactive osgenspecies products missing in cgd be good therapeutic targets for prevent ing and possibly treating atherosclerosis and other inflammatory diseases is this so that's a question we have been
interested in. and at this point, to tell you more about the nox proteins, and some potential targets, i wanted to introduce tomleteo who is going to tell you about nox family and nadph oxidases, mediators ofnation intersection fibromyalgia rosis and cancer
progression. if there are any questions for me, before we move on ... testing. yes. thank you, dr. gallin. so you can appreciate from dr. gallin's discussion that scientists and clinicians have
recognized that fago sitic cells have this robust capacity for generation of high levels of reactive oxidants, and the react ive oxidants are -- down stream products are very potent microbe sightal agents. so this has been appreciated for 5, 6 decades.
and it's only in this post genomics era with the flood of information from the genome efforts in the late 90s that workers in this field, including myself, have, then, come to recognize that the phagocyte oxidases is but one representative member of -- one
representative member of this -- there we go. so the fago sidic oxidases is just one of 7 members now that have been identified since 16 years ago. so now they all have in common structural features that are already seen in the gp91 fox or
nox 2 component. and in the human genome, now, we know that this tick tech passes try the membrane, and donates to molecular oxygen giving rise to this super oxide free radical, which is the primary product of most of these nox enzymes. so knobs 1-4, very similar in
size. they have on the order of 35 to 55 sequence identity. you have these dual opposition dices, 1, 2, and they have a do main with another pro oxidase similarity, so they're called do you oxes. they have both [indiscernible]
like structure and pro oxidase like structure. this domain seems to be -- concerned more with the generation of perrox i'd rather than utilization. unable to -- nox 1 and 3 are closest homo lox to nox 2. they are multiple component,
rack dependent, dependent on cytosol factors like p47 fox and-- secondly, you have nox. then these 3 enzymes are calcium dependent, their activity goes up with elevations in cytosolic calcium levels. so they've regulated in 3 different ways. so here, there is an outline so
you can appreciate the range of functional activities of these enzymes where they're located, and the consequences of gene defects that have now been identified in these other nan fago sitic oxidases. i've flagged knobs components mutated in man with a asterisk.
those mutated knockout models, those being underlined. and so you can appreciate, there is a range of oxidases that, when knocked out or mutated, defective, give a variety of phenotypes. you already heard about phenotypes of cgd including
protective vascular phenotypes, such as against atherosclerosis. and -- atherogenesis. turns out 2 of these obsoxidases , nox 2 and 4, animal s with defects in those oxidases also can have vascular phenotypeds. in nox 1 it's expressed in
smooth muscle cells, they are protected from a hypertensive effects of chronic angio tensen. and in the case of nox 4, they're all protected from re profusion injury such as in the brain. and then they also have protection againstify broughtic
disease in liver, lung, and -- ify broughtic disease in liver, lung and kidney. so it turns out that the knobs 4 is probably the most widely ex pressed, particularly high in the kidney. the nox 1 is particularly high in colin epithelium.
and there are recent reports there is epithelial phenotype, open wound healing. and then there is a report from the lab showing that one variant in man is associated with very early on set inflammatory bowel likewise, identified a very do you duox, early on set.
so knobs 3 is only in the inner ear, has -- its knockout has the effect of causing the mice torose their sense of cravety and balance. the duox 1 and 2 were first recognized in thyroid tissue. and later, highlighted their expression also in nasal tissues
, such as respiratory. and the to you ox 1 are protect ed from inflammatory asthmatic phenotypes. also has the function in urinary bladder, bladder contractions, and has some kind of signaling function. and then in the case of do you
ox 2, it's primarily in thyroid tissue. and mutations in man will cause severe congenital hyper thyroid ism. so you can appreciate that in these oxidases, many different functions and tissues, some of these effects of knockouts are
-- suggest that maybe you might want to inhibit these other oxidases for perhaps suppressing inflammatory disease s in these other sites as so i -- i wanted to focus on inflammatory but i divert for 2 slides to show you the most remarkable phenotypes, and just
illustrate the deverty of their functions. one with fox 1 and duox 2 knock outs. we see here hire is the case of the nox 3 mutant mice. they called it the head tilt phenotype. this house looks a little sea
sick. it has no sense of balance. what happens in these mice, they lack these crystal bony structures, the so-called auto lifts that touch the hair cells. in the inner ear. so they don't know which end is up or down.
when airdrop them in a beaker, they don't know which direction to swim to save themselves from drowning. in the case of the duox mutant animals, you have this remark able congenital hypo thyroidism. this is because thyroid tissue
makes a lot of hydrogen peroxide to support the activity in the process of [indiscernible] to make thyroid hormone. so these animals have all the hallmarks of create nism. they have a smaller growth rate, shorter bones, lower density in their bones.
and they have grossly elevated thyroid stimulating hormone levels which cause them to have these hyper trophic thyroid glands. the defect is a mutation that interferes with the duox trans location to the plasma membrane where it has to do this
hormone synthesis. getting back to our theme about inflammatory disease. i just want to highlight 3 avenues of investigation that got us and others thinking about whether these other non fago sit ic oxidases could have roles in innate immunity and
inflammation and stuff i'll describe later. maybe cancer progression. several of these oxidases accumulate at their highest levels on epithelial surfaces, and it's not in several cases that they have this barrier and in the case of duox, for
example, in major airways, the enzyme is disposed on the plasma membrane. they put hydrogen peroxide into the lumen. it could have a direct mike -- you're ventilating your lungs 20,000 times a day. somehow they stay clean.
we like to think this generator may have a role in that. do i ox 1, tracts, do i ox 2, gi tract, exocrine glands. pancreas. several of these oxidases show a responsiveness to microbial pathogen or danger associate associated molecular patterns in
the colon, here, formal pep side which also stimulates phagocytic cells. lps, lipo poly sacried, in the case of nox 4. in the respiratory epithelium, you have this poly ic that triggers activation, and higher expression of duox 2 in airways.
then you have these dangerous signals in airways, history mean or atm that will -- atp that will active in reconstituted cell models. thirdly, each of those noxes has a unique responsiveness to different cytokines, knobs 1 and gut epithelium induced by gamma
interfere p or tnf alpha. tgf beta, inducer of knobs 4, and 2 cytokines and airways in duce duox 1. gamma interfere p, duox 2, in airways. and there is a good evidence coming out now that at least 3 of these oxidases, knobs nox 1
and knobs 4, they all shoal roll s in sell migration promoting cell migration and wound healing. tgf beta is a well-known cytokine wound heal in a variety of contexts. to mention about the nox 1 based oxidase, in colin epithelium,
you have, in this case, the closest homo log to the phagocytic prototype. you have this p67 like nox activator and p47 like nox organizer that work all in the same way. it extends at a functional level in that all of these inducers or
activators are already recogniz ed as affecting the phagocyte system. and some interesting work out of andrew's lab in the last couple years is indicated that a formal peptide from normal [indiscernible] species will trigger activation of nox 1.
in in the mouse with the formal pep side receptor knockout or nox 1 knockout, it appears that wound healing and colon epidemiology epithelium is di minished. in the case of nox 4 -- i'm going to dwell on nox 4 for the rest of the talk.
it's a very big interest in our lab right now. and actually, we were the first to describe nox 4. atthe time we called it re nox because of its very high express in kidney. we and other labs have now accumulated large body of
evidence to indicate that nox 4 is induced in a variety of tissues that are particularly susceptible to fibrosis which is triggered by tgf beta. and there are now actually clinical trials on nox 4 inhibitors to assess the benefit in patients with diabetic neff
ropethy, and also there is a orphan drug trial for very rare idepathic pulmonary fine fibrosis. in river, fatty liver conversion toify broughtic disease is also abated with nox 4 knockout or with these nox 4 inhibitors. in our own lab, we got into this
work with howard that hepatosite s infected with hepatitis c virus show an enhancement of nox 4. we suggested that with chronic infection, with hcv, you could have oxidative stress from nox 4 that triggers cirrhosis and transformation to cancer.
we have other studies where we looked at my mifrayings, metabolism activity of breast cancer tumor models. we see tgp beta induces nox 4. to illustrate that, the whole process of cell migration and progression fibrosis involves this phenomenon called
epithelial to transition. this is a process where polarized cells become disassociated and take on a mesen cheapal phenotype that gives them enhanced motility. they make more protein. the cytoexcelen rearranges. this process of emt is the basis
for normal development, normal also in the case of fibrosis, which is an unresolved prolong wound heal process. then you have process of metabolism also involving -- metastases also involving emt. it's been said by any authors that cancer is a wound that
won't heal. so tgf beta has a big role. it's a master switch in this emt and to investigate that, howard in my lab has used two breast cancer models in these scratch wound assays. and showing here that in this normal mortalized line, we see
only nox 4 transcripts are in duced by tgf beta. likewise, in this metastases line, it induce early nox 4 transcripts. promotes scratch wound closure within 24 hours here. that can be blocked by rna silencing of nox 4.
rather than 100% closure, we maintain the gap by suppressing nox 4. likewise, in this metabolism line, the wound closure is much faster, they migrate faster than metabolism cells. we can -- et cetera et cetera we can block that with dominant
so we propose this model that nox 4 is a target gene of tgf beta activation, transcription factor binds directly to the nox 4 promoter. and the enhanced levels of nox 4 mrna in protein, then, is a requisite for all of these emt related events.
it's published. i won't show you the data but nox 4 also enhances hikebro effecten expression and act ivation. we then asked what factors might account for the enhanced mi gration of the metastatic line they migreat faster.
the answer is the mutation status of p53. turns out that there is a convergence of pathways. wild type p53 has different effects than mutant p53. the tgf beta can do many things in many settings. unthe presence of wildtype, it
has these dna damage response effects of pro eptotic responses and suppression. and in cells with p53 mutations, you have migratory response to t gf beta. it turns out that the effects are translated at the level of wildtype suppresses nox 4 and
the mutants enhance it. and we can show that here in this p53 lung model in which here the background, p53 null cells by western blotting. if you treat with tgf beta, you see nox 4 goes way back. if you transeffect in the wild type, you suppress nox 4 levels
here. you fail to induce nox 4 levels the mutant forms enhance nox 4 levels either with or without tg p beta. in parallel with these changes we see that the migration act ivity is stunted by trans fection with wildtype and
supported or enhanced by the mu tant forms. and migration, this is a invasive assay, can be blocked by nominant negative nox 4. and so we have this represented schematically here. while type suppression presses the mutant supports nox 4 in
duction, excess ross, and the whole pro migratory process. we then asked, after publishing that paper 2 years ago in the last couple years, asked whether we could generalize this model and whether this applies to many tumor types and sure enough, we looked at some 6 different --
these are representing some of the most common mutations in p53 seen in many tumor types. we've tested these mutant forms in at least 5 different tumor models, not only lung and breast , but also pain creatic tumors, help pat site, and coal electronic tumors.
they all support higher expression bottom line in nox 4 and nox 4 dependent cell mi gration. in one other separate investigation, we went to the cancer genome atlas database, and just surveyed those tumors have documented mutations in p53
, the so-called hot spot and sure enough, we found a variety of tumors, pancreatic, head, neck, breast tumors, and they also show higher nox 4 expression than the tumors that have wildtype p53. so i'll end with this overview and just point out that there
are a number of inflammatory fine rotic metastatic disease processes that may involve a variety of these other noxes, and that they may also be target ed to suppress inflammatory disease orify broughtic or metastatic disease. the key is specific inhibitors
that don't suppress all the nox es, and there by maybe inhibit the desirable effects such as most offense. i'll stop there and -- well, site the people who have done the work in my lab. [list off names] questions? >> [inaudible question]
>> yang of any data on that, no. >> well, thank you. that's really a brave new world. >> so i hope you have a sense of-- start with cgd, and you -- who knows where you're going to end up in terms of these nox proteins. nox 2 is called nox 2, but it was the first described.
tom can explain why it was named 2 instead of 1. >> politics. so -- [inaudible] [inaudible] so what we wanted to do was end with a short present ation by gal wald, spending a year at nih between college, graduated from cornell
last summer and going to middle school -- medical school. he's trying to discover new nox inhibitors with the concept that , gee, if we could find such things, maybe they would have potential therapeutic use. so i'll let him -- >> thank you very much for the
introduction. it really is an honor and privilege being here and presenting along with side dr. gallin and dr. leto. my goal sue explain early effort s in identifying a nox 2 specific inhibitor and explain how the basic science can be
more broadly translated into clinical medicine. the drug development process begins with the screening of thousands of compounds as potential candidates for medical those compounds that appear promising update go further test ing for their properties,
efficacy, mechanism of action, among other studies before they enter a preclinical animal model and subsequent clinical trials. it is estimated that the cost to develop a drug can at times exceed over $2 billion by the time it reaches the market. our labs insight to nox 2 in
atherosclerosis precipitations the search for identifying a nox 2 inhibit inhibitor. in collaboration with the national center at -- after advancing translational science we developed a screening assay that protected the production of reactive oxygen species using an
artificial cell line. these are k562 cellings that ex press the nox 2 and cytosolic components. we stimulated the cells using acetate, known as pma, and then measured the production of ros using liminal instanced assay. as a proof of concept, we screen
ed over 4400 compounds that were derived from 2 librar ies. many were approved for clinical use by the fda and other regulatory authorities, such as those in the eu, canada and japan. and these drugs were ideally
suited for repurposing as they have already passed major regulatory hurdles. in our high throughput screening assay, we utilized the work day and night to finance fill with reaction mixture. what happens, the ros produces oxidized liminal to generate a
like product shown in the white walls. inhibitory act activity is shown in the black walls. it is note worth eto mention that pma activates cell line via pkc dependent mechanism, meaning that known compounds were considered to be false positives
, such as this in the bottom right. and n cats generated a list of 102 compounds for us for subsequent screenings. and a first screen was counter screen using xanthinoxidase. and this is an enzyme involved in metabolism and with the
addition of substrates, you can generate -- and this assay was quite robust. it had a 3 fold purpose. we were able to screen for interference with signal detection. and also screen for any caven jures of ros, and third, we
will rule out non specific inhibition in another oxidase so we monitored our reaction over the course of 900 seconds in our xanthinoxidase assay. so in blue here is our vehicle control. these are some of the other select compounds.
red, these are compounds that have comparable activity to our and to a certain extent, some of these other compounds show interference with the assay itself. in particular those at the bottom are clear cut false positives, since they are
inhibiting the signal detection. there is basically no count. we expressed each compound as a percent control using an area under the curve analysis for the first 900 seconds, and establish ed that using a 50% cut off of the 100 given to us, 44 compounds had more than 50% act
ivity. in other words, 44 compounds had less than 50% inhibitory act ivity in our xanthine oxidase aorta say. that is that's 58, acted as anti -oxidants or otherwise interfered with the assay itself let me turn your attention, in
our primary screen we used an we next wanted to evaluate and confirm inhibitory activity. using a liminal enhanced assay. instead of using pma, we used e. coli as a stimulus. nox 2 is endogenously expressed on neutrophiles and monosites, the main cell type in disease.
pmn shows robust response, those cells that are derived from cgd show barely if any activity. blue flatted. it's known that in -- nox 2 is -- it pro produces suggesting that r.os production is nox 2 dependent and relies on the aorta semly and translocation of
the factors to the membrane com plex. we monitored the assay for the first 2 hours after the initiation of the respiratory burst using genomic. here is our positive control. the negative control that has no stimulus is the blue line here
near the x axis. we expressed pressed every com pound as percent control using area under the curve forever for the first two hours and established a 10% cut off. of the 44 compounds that passed our xanthine counter screen, 36 compounds showed more than 90%
inhibition. i would like to point that we did screen all 102 compounds. this is just those 36 that passed our counter screen as one major concern with both our primary screen and also the -- the whole sale neutrophile assay , any inhibitory activity
that we see cannot be necessar ily attributed to inhibitory activity on knobs 2. we, therefore, developed broken cell nox assay that allows us to buy pass membrane receptor stimulation, and we fractionated neutrophiles into the membrane and added sds, inducing a -- dis
rupts a intermolecular bond that induces a change in p47 fox that allows us to directly activate the system. instead of using the end point, we measures super oxide. it reduces cytocream c, results absorption peak. super oxide is -- sod.
yes, catilouses the conversion of super oxide into hydrogen per oxide, and results in -- is -- sorry, served to validate super ox -- so if you add sod, you see no obsorption point. orange color instead of pink when it is reduced. we monitored our broken cell as
say for the first 30 minutes after the addition, and vehicle control here is in brown. in the middle of the curves. our sod is actually super impos ed by the red compound. we found interesting that there were several compounds that had symmetry activity over the
positive control. that suggests either that the compound had inherent reaccusing ability, was able to reduce cyto chrome c, or that instead of being inhibitor of knobs 2, it was potent activator of the system itself. we, then, expressed each com
pound, and found that of the 36 compounds past both the counter screen and whole sale neutrophilia say, ten compounds had more than 50% inhibition in the broken cell assay. and this summary table outlines our 4 screening assays to identify a nox 2 specific
inhibitor. we received 102 compounds from n cats, and ran at 100 micro molar on 3 different assays. the set criteria, in fact, quite arbitrary. we could very well change in the future, was set at 50%, xanthine oxidase assay.
we looked at compounds that show ed less than 50% activity, and more than 90% reduction in neutrophil, and more than 50% re duction in broken cell assay. this generated ten lead com pounds, which we determined the ic50 using the broken cell assay.
it was interesting that of these ten compounds, 3, in asterisks, had apparent stimulatory act ivity at certain concentrations. 3 were activating nox 2 at a concentration below 100 micro molar. and in conclusion, we were able
to establish proof of concept pipeline for identifying a nox 2 specific inhibitor, using 4 distinct assays. many of our potential candidates , those 102 tested com pounds, we found them to be false positives, due to ain't oxidant effects or inhibition in
pathways upstream of knobs 2. we will further study these ten lead compounds with dr. leto. we're under current development for developing cell lines that suppress iso forms such as nox 1 and 4 as a method to identify non discriminate inhibition of the nox family.
in trying to identify inhibiters , we're gearing up with n cats try screen larger libraries, containing over half a million compounds as potential candidates. it is in our not too distant future to move to a precounsel animal model predisposed to
atherosclerosis, and then test our compounds in vivo. thank you very much. [applause] >> that's his first presentation i just want to end by pointing out that as with all these projects, there is a lot of people that make this work.
and our lab [indiscernible] sitting here has been a key play er. and then tara, one of the nurses who helps to recruit the harry malik and steve, partners with me for decades. and nicole on the right is another nurse who is currently
working with us. statistician was [indiscernible] , then you see we've had 2 post-docs working with us recently, anthony is now an md ph.d. candidate at the university of connecticut. and [indiscernible], soon going to medical school.
and then the radiologists who helped us do the mri scan, david , who is chief of radiology , cardiologists [list of names] helps us interpret the can scannings and cared for some of the abnormalities we picked up in some normal hosts. and doug, up at -- in frederick,
with saic who helped us with the assays. so we're all open for questions. you mentioned that the cut offs you choose were was that mainly to narrow down the list of compounds you had? or was that assay based? i'm trying to figure out what
the goal is from your lab. is it to get a single lead com pound or to say here is what we got, maybe you can take this to phase one at some point or test in animals? that's a great question. i think ideally, we wanted to have compounds that are
interfering least with our say -- assay. so perhaps less than 90% re duction, and with our broken cell assay compounds, more than 90% inhibition. the issue with that, that would generate right now a 0 compound, just based on those arbitrary
cut offs. so this is why we're gearing up to a much larger library of small molecule repository that contained over 500,000 compounds of those that really ra leading our lead compounds, we can, then , chemically synthesize them further and find a more specific
inhibitor of nox 2. >> thank you. >> i have a question. maybe you can answer this. so if the objective of the inhibitor would be to inhibit nox activity, correct? are you concerned that you might be producing the phenotype of
chronic granulomatous disease? or other infection, what do you think about that? >> that is a concern. and really, what happens in patients with cgd, they're more susceptible toward infection. our goal here is to take the perspective from cv disease and
-- cardiovascular disease and obtake an compound that does not necessarily abolish all super oxide production, with you rather eliminates those to a certain extent. nox 2 is expressed on the surface in the plasma membranes also intracellular compartments.
perhaps an inhibitor, rather localized to those at the surface, and super oxide is still generated inside the cell and can kill off pathogens. >> we need the microphone. >> i think i have it. you -- you are probably sorry that we have a lot of carriers
in our cohort and the excellent carriers of cgd are interesting, thewoman who are caring the gene depending on lionization of when it occurs, can have a variable amount of normal cells and an normal cells. so we have over 200 such patients or subjects.
and they can be extremely lion ized, so that they are either essentially 99% of their cells are an normal, or 1% of their cells are an normal. one of the things we're doing now is studying all those women to see if the spectrum of lion ization correlates with
protection from atherosclerosis, so that the hope is that we will see that people are protected with, say, 50% loss of function and if that proves to be the case, then we would want a com pound that knocks you down 50 %. those people that are 50% abnorm
al, have a normal life in terms of infection. and in an actual fact, you probably only need about 5 to 10 % normal cells to be clinical ly protected from infections. you don't need a lot of them. now, there is a weakness in this
study that we're doing in that cgd, all the cells are suppressed. and in these women who are car riers, a percentage of them are -- an normal. and it could be that the normal cells complement the defect in the abnormal cells.
we won't see anything. away don't know that. but the question is, how much suppression would you need to get protection from the inflammatory -- exaggerated inflammatory process, we we're all subjected to every day of our lives.
>> cgd patients -- are cgd patients less susceptible to atherosclerosis? neoplastic disease? >> this measure of carotid art ery thickness is the marker we have. until recent years, they haven't been living normal lives like
robert carp and more and more of these patients are going to live we anticipate fully normal years so we don't have a historical base to really answer your question cleanly. i can't say whether they're protected from cancer. >> what about the mice?
if you have a knock down or knockout, nox 2, and subject them to some chemical or other stimulants that would produce neoplasm -- >> yeah. we haven't done. , nor has anyone else yet. a good experiment.
>> well. lots to do. the other thing that struck me was that 3 of your candidates showing effects are in the pi3 kinase, akt pathway. and mtor. so is there any connection between what we know about the
regulation of nox trafficking within a cell, and pi3 kinase pathway? there might be? i would guess. no? >> well, pi3 kinase inhibitors do inhibit the phagocyte oxidase >> but are they doing it because
nox is present both -- somewhere in the cytoplasm, and also, in the airways and the membrane. so they fet get there by trafficking. they may be cycling, i don't know, like some of our transport ers do. >> my concern is the inhibition
of the kinases, common place signal mediators will hit so many cellular systems. besides the noxes. so i'm less inclined to consider them as good drug candidates. >> well, are their any other questions? i want to thank you all very,
very much. this was really very exciting, very provocative. i trust there are a lot of people who are out there, who have benefited from this. so thank you again, john. and thank you very much.
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