11/14/19 - Andrew Stewart, MD

Transcript

• 00:01Good morning, and welcome to medical grand rounds, so if you want credit for grand rounds, please text 13147.
• 00:10So today, we have a special guest for special lecture and John Rauser Muskie will introduce the lectureship and the guests.
• 00:20Could just a couple of announcements before that next week will be a talk by James Lewis.
• 00:26For the Henry J been the lectureship in gastroenterology why to believe in diet as a therapy for immune mediated diseases and next November 28, is Thanksgiving, so there will be no grand rounds.
• 00:40It reminded that we have no commercial support for green runs in the conflicts of interest and now for introducing the lectureship and the Speaker John.
• 00:50So thank you. I carry so it's really a distinct pleasure and honor to be able to introduce to you. Today, the 2019 Arthur brought us lecturer and Andy Stewart from outside high school. Madison icon schooled Medison outside I guess. Technically, who is the Fishberg Professor Medison and director of diabetes obesity and Metabolism Institute at Mount Sinai.
• 01:21So before introducing Andy I wanted to just tell you a little bit about the broadest lectureship so the broadest lectureship is in honor of Arthur. It brought us here are some little sign posts from his career. So Arthur was if you don't know Arthur. He was born in Knoxville, TN. He received his bachelors degree from Washington and Lee University, MD. pH D from Vanderbilt, where he was first in his class.
• 01:53He went on to internship at residency at Massachusetts General Hospital in Boston and are in Fellowship at the NIH and then from there was recruited to Yale in 1976. He served as the Enercon section chief from 1984 to 2005 an associate chair for research food Department. Madison from 1994 to 2002. So Arthur started his career and his PhD thesis working with Earl Sutherland and grant little at Vanderbilt.
• 02:27To use the discovery of cyclic amp and develop measurements of the renal production of cyclic amp in response to GPCR activation in the kidney and specifically the parathyroid hormone receptor when he came to Yale. He used this measurement is a bioassay to study various aspects of mineral metabolism, including hypercalcemia in cancer patients and with one of his 1st Fellows.
• 02:57Andy Karl and Sonia and others at Yale and discovered parathyroid hormone related protein cloned. It's Jean created an immunoassay to measure it that we all use clinically now and then study went on to study the normal Physiology of PTHRP and several organs that are listed here. So Appan Arthur's retirement in 2014. The faculty and friends of the endocrine section A stab liszt.
• 03:27A lectureship in Arthur's honor and the broadest fun. Both contributes to this lectureship every other year as well as generally to the educational.
• 03:43Activities of the section so the lectureship honors prominent physician. Scientists have been leaders in mineral metabolism or related fields and Arthur Anandi Skews, Maine is the 3rd lecture. You can see the other people, there so moving on to Doctor Stewart. So he got his undergraduate degree at Trinity College in Hartford went to medical school at Columbia did his residency at Roosevelt Hospital in New York.
• 04:14And then came to Yale as an endocrine fellow and stayed through 1997 rising through the ranks to become a tenured professor. Ann chief of endocrine at the West Hayden, VA. He was recruited to become chief of endocrinology at the University of Pittsburgh in 1997, where he stayed until 2:20. Twelve when he was recruited to Mount Sinai to become a directive diabetes obesity Metabolism Institute at Mount Sinai so Andy.
• 04:45Is recipient of many honors I won't take too much of his time but just to mention a few American Society for clinical investigation. American Society of Association of American professors. He received the General Auerbach Award for Outstanding Scientific Achievement from the endocrine society. The Ray Kroc Scientific Award from the University of Upsala Diabetes Center. Ann Harold Rifkin Professorship from Albert Einstein College of Madison and now the broadest lectureship the Top off these honors.
• 05:18So Andy is had a very, very career. I think he's going to review some of that, starting out in mineral. Mataba lism and then pivoting to doing some very exciting work now about Reactivating. Calliphora oracion islet cells as a way to cure or reconstitute the beta cells in patients with diabetes so without further ado Andy.
• 05:56Alright so first let me just say wow. This is amazing today. You know, I spent many of the best years of my life here. Certainly the longest stretch of any place that I lived in as I look around the room. I know 50% of the people in the audience. This is amazing to me, so Yale is an incredible place. It's incredible place in my heart and my history. And so I had planned to give a talk on sort of the stuff that we did when I was at Yale with Arthur and this makes it even more special that is what I'm going to do. I'm going to tell the story of Arthur Broad Assessment? Can you see that? Yeah.
• 06:31So this is a story of funeral hypercalcemia malignancy from from following through to drug discovery and it really is. All attributable to Arthur everything I've learned I learned from Arthur. So I do have a disclosure. We filed a bunch of patents on some of the stuff that I'm going to show at the end related to drugs that block this kinase, Dirt 1A. This is my mentor art brought us and so Arthur Dan. I'm going to sort of dwell on this a little bit during the talk. Arthur is has been a phenomenal mentor to me, I work you know you hear so often that you need to get away.
• 07:06Fermenter establish your own independent career. I finally got the gumption. Delete my mentor after 19 years working with him and they will remain friends. For years and he did brilliant thing. He put me out at the VA's chief of Endocrine. So I could actually say I was independent. I worked in a completely different city, 7 miles away or whatever it is so obviously. Obviously, I'm independent right. But the truth is, we had joint liabilities that we came over and I continue to drink at the fountain of knowledge for another tour 19 years, so.
• 07:37This is Arthur Arthur. Thank you so John showed a slide like this. I just want to say that I arrived around the time. Arthur had just arrived as an assistant professor and he'd been recruited by Howard Ruston Rasmussen, who is the chief and John had a slide like this too. and I just wanted to tell you in case you don't know Arthur literally wrote the book on disorders of mineral metabolism. So I arrive in his lab with no knowledge and no training so the next part of this thing is the story of the definition of racial hypercalcemia.
• 08:08Legacy story and the identification of PTHRP and this, if you're a yield person. You need to know this is a Yale story. The subtitle of this is how Arthur taught me to think so. I arrived here in 1978. I was a new fellow I was well suited for a career in academia had been a GP on a little island called Fishers Island had no experience in science. No particular interest in Science, except when it came to tie a time for my interview as a fellow and so I arrived and said you know this is what I'm here Arthur and.
• 08:42You know, and so Arthur says, Well, you know what do you want to work on so Arthur is you've seen I had developed this in the project cycle game PSA that allowed him to study and understand every disease of mineral metabolism. The only one they weren't working on was hypercalcemia malignancy and Arthur had all the tools to explore that it turns out, I didn't really know that so, so I arrive. I had been a resident at Roosevelt in New York and I've seen lots of people with hypercalcemia malignancy and I thought it was incredibly interesting and fun too.
• 09:14Frieden see people wake up and then of course, there's maybe not so happy after that, but it was dramatic and so I thought it was interesting so Arthur said, OK, So what do you want to work on and I said well let's say you're doing osteoporosis you're going to Austin Malaysia. You're doing rickets. You're doing hyperparathyroidism. You're doing hypoparathyroidism doing nephrolithiasis? What else is or I'll do hyper calcium. Lignin see you said, OK, what do you know about that and I said well not so much and he said well.
• 09:45What do you know how to do I said well doctor Bronner's I actually don't really know how to do anything and he said, OK, Stewart can you can you collect blood now I can do that we should be able to get urine samples 'cause you needed them for Yeah, I can do that. Arthur can you do bone biopsy's well? We had done bone biopsies, but not the kind you do in mineral that piggyback rides. Bobo biopsies right. So so but you know you can learn so he said, OK, I want you to run around collect a bunch of samples on people with hypercalcemia and relevant controls.
• 10:17And then bring them to me will run them in our lab, and then let's see what comes out of that and so I did that ran around got a bunch of samples from lots of people all with the extensive two page. IRB consent form that was popular in 1978 and and then brought these samples to Arthur Arthur random, he would. Give me the data and then I would plot them and so it looked like this so Arthur said measures in a fragile cyclic amp.
• 10:47The real phosphorus threshold 125 vitamin D calcium excretion and these were sort of cutting edge assays in 1970. Eight 1979 and so he said. You know, so wow. This is pretty amazing. He took one look at this and said this is amazing and I said, Yeah, I mean, like it's a lot of dots and he said. You know what this means I said Arthur. I had no idea what it means we said he said. Well, you've discovered a new hormone and what we thought was that this hormone would be like PTH stimulates cycle gampi. This is the group of people who.
• 11:20At the time were called Ectopic hyperparathyroidism, but in essays at the time. There was no PTH so these people with ectopic hyperparathyroidism have elevated in fragile cyclic amp just like people with hyperparathyroidism and they also are phosphate. Rick just like hyperparathyroidism on the other hand, the distal tubule where calcium is handled in the proximal tubule where 125 is synthesized behave differently so this is saying there's a hormone out there. That's like parathyroid hormone, but can't be parathyroid hormone.
• 11:50And I was like wow. You know like wow. I mean, like that's all I can think of is wow. So Arthur said. This is a big deal. You need to, you need to think about pursuing this and I said, Well, you know, I'm in for another 6 months and my fellowship, so he said. Well, first of all let's write this up. Let's admit this is a paper and so this guy submitted to the New England Journal. An was accepted so this date December 11th 1980. I'm going to come back to in a moment but a couple things. I want to tell you about this one is so here it is on the cover.
• 12:23This is like beginning of my sort of middle of my second year is fellow an annoying Journal was important. I'd never even dreamed of publishing anything. So I'm first author and that's a sign of Arthur's generosity because I wrote a draft of this that then was completely replaced with meaningful thought and and and Arthur senior author but an important thing. That's not on here is the original final author was Howard Rasmussen, so we used to meet down fit in.
• 12:55Every week and talk about various projects that were going on and I saw this thing gets accepted. We have our weekly lab meeting. I had gotten a letter in the Mail. So I showed this at the group and said look at this, this thing has been accepted. You believe that Howard said. Let me see the letter. He looked at the letter, he said. I want my name off that paper.
• 13:15I'm like well, I mean, like is there something wrong. Did am I lying or is this fraudulent or what's the problem. He said no or 3rd? I have lots of papers. This is Howard. Speaking Arthur's new assistant professor. He's actually been your mentor. He helped you write this thing, he did all the assays orther needs to be first author and I mean last author and I need to disappear, so for me. That was, I mean. Now it's way more meaningful to me that it was then 'cause I didn't really understand the generosity, but think about that. That's so rare.
• 13:46Or so Howard had a fantastic mentor and then that trickled down to me as well. So anyway back to bone biopsies. Arthur said well these syndromes seem to be different? Can you do bone biopsies? Yes, so actually Carlin Sonia Peggy be a an Bob Lang taught me how to do bone biopsies and so we do these trans iliac Crest biopsies. And so this is a biopsy from a person with primary hyperparathyroidism in the message is there's a lots of osteoid and these are osteoblasts on Top of the osteoid there's also a lot of.
• 14:17Phone resourcing from Ostia class and these are osteoclast down here, so in hyperparathyroidism. There's a lot of bone Resour Petion. There's also a lot of bone formation and there couple. They both go up. They both go down at the same level and then these cysts are places where osteoclast have eaten holes in bone. That's why it's called Osteitis Fibrosa Cystica, OK, so that's what you see in hyper para and then here's what you see in people with which is now called HM which by the way is the term minted by Arthur. This is the humoral hypercalcemia malignancy or invented that so you should know that seal.
• 14:48He'll turn so here you can see a person human hypercalcemia. No tumor in the marrow. So it has to be humoral osteoclast are reserving bone all over the place. But there's no bone formation going on at all, so this has to be a different disease to this is the relationship between osteoblastic bone formation and Osteoclastic Bonera sorption. They're tightly coupled in normal people and people with hyper para of various kinds and other kinds of high turnover.
• 15:15But in people with HM there completely young, coupled so more evidence that the skeleton is seeing something different in PTHRP in HM than it is in hyperparathyroidism, so Arthur is like you know you really need to think about trying to purify this thing and so I guess it Arthur. You understand what you're dealing with here right. I don't have to do anything. I'm like a GP. He said, OK, well, you can learn so Arthur used to write these.
• 15:46Uh these extensive daily menus recipes for me to do so. Here's what you're going to do and E so just to blow it up here. We are, and by the way I'm one of the few people in the world who can read authors or there's handwriting in and actually he says, I can read it better than a secretary could.
• 16:05Which is another story so so this is January 24th 1979? I'm in new fellow making my debut in the lab and you can see Arthur is written. All this stuff out in detail right and so here's what I'm going to do that day and the next day. This June 25th 1979. Obviously, I still have this notebook is OK. Now we're going to learn how to count tritium and P32. You're going to do it on this thing called the Packard. That's assimilation counter. It's out in the whole on LMP.
• 16:35On the 2nd floor and you're going to identify it because it's model number 3385, so like you see what he's working with and by the way it was also the only piece of equipment out in the Hall so, so and you're going to count tritium and you're going to do that in the Red Channel annual notice. The tritium channel because it's labeled tritium so you put your samples in this thing it spits out numbers and then you're going to get this thing and Andy it's called a print out.
• 17:05So I just want you to know this is Omar is the Arthur. I mean, Arthur taught me everything so. So now you know, there's a syndrome. There's this new hormone. If you had some tumors and extracted them you could set up a nasse and so I met people in Cardiology, like Larry and arrange to get dog kidneys. So I could make kidney membranes to do a deadly cyclist asses in. We've got tumors from people with this syndrome and then showed that they could stimulate daylight cyclists and that then allowed us to Purify.
• 17:36PTHRP and actually another Arthur sidebar here is that you know, Arthur has repeatedly stepped out of my way to make sure I get credit for stuff, he thought of and then wrote etc. Cetera so at this point, he steered me to Bill Konigsberg, who I don't know if the older people. Remember Bill Konigsberg think it's his name. His name, but he steered meeting chemistry people and I sort of developed an identity is the protein verification person in the lab while off Arthur went off to learn molecular biology, which was.
• 18:07New England at the MGH and Whitehead. So so I then labored on an 8 years later terrified PTHRP and this was exciting. This was a real Eureka moment. We were looking for something that was like PTH but wasn't PTH. It came off the sequencer and we could see it was very much like PTH in the first 13 amino acids. But it wasn't the same as PTH so it was PTH like this was known to be the business end of the molecule that could bind PTH and activate PTH signaling so we knew it was the right thing and we were an intense competition with other groups.
• 18:40We were trying to do the same thing and make sure through the Grapevine that they had purified it. They sequenced it. When I got there sequence that turns out, they provide the album. And so we were. We knew that we had something in that purified but we didn't know what it was when we saw this sequence. We were like this is clearly it and then the two sequences diverge completely so the reason that its PTH like is that it's like PTH only it's different so that evolved into this name. PTH parathyroid hormone related protein or PTHRP, so I was sort of protein chemistry guy at that point.
• 19:11Thanks Arthur getting out of my way, and making sure I got credit for everything and by then, he was the molecular guy, so he used that to clone the PTH C DNA from CDA libraries 'cause that's how it's done in that era and Arthur could see that coming and could see that he needed to learn how to do that, so he did together with Marguerite Moshannon. This is the PTHRP gene on a Locust Syntenic to the parathyroid hormone gene on chromosome 11 and 12. So then I turned with Terrence Wood, particularly I don't know if Terrance is here or not I hope he's here, but
• 19:41In any case, a purified protein and then do this post. Translational processing so think of prohormones like Propio Malana cordon. PTHRP is a prohormone. This is the active piece. That's as the sequence homology. This will wonder 36 with PTH so this is the biologically active form PTHRP and also there's a longer form also this president. The circulation so now we're into fulfilling kochs postulates and so I say to Arthur.
• 20:12Yeah, but who is Coke and water coax postulates right so I'm telling you learn everything from this man, so the first thing we did together with Carl and Sonia and John roll off his show the PTHNPTHRP bound to the same. PTH Receptor That's present in bone, and kidney and that signals and binzen signals equivalently and activates the same downstream, signaling pathway. So sure enough, it is biologically just like PTH. We also infused it into animals so these are studies. We did at the VA.
• 20:43Under under Oscars to delish wherever you win if I lost you so admitted to go. So so now. This is in building 5 out at the VA infusing PTHRP and the animals or in rats over 3 days trying to prove that this was the hyper calcium in principle, and sure enough, they became severely hypercalcemia can died and less. We stopped the infusion in which case they normalized so here's another sidebar. My son, also worked in the lab that summer. He was a sophomore at Yale. So his job was to measure calcium measure phosphate and collect rat urine and measure calcium phosphate in rat urine.
• 21:16And so that was part of this paper that turned out to be a JC I paper and so this was like again high five. We had synthesized PTHRP were squirting it into animals. It was doing exactly we thought, This is filling at least 1 of coax postulates and so this is very exciting and my son, Charlie was so excited about this that. He decided to go into investment banking, so then we moved to people and this is this is another Yale story.
• 21:46This is Maria ever heard K, who is a real feel fellow and Maria, who some of you will remember basically was doing infusion studies on the geeked up on 105 in that era and show that in fact, you confuse PTHRP in the normal people and reproduce. The elevation in the project projected game. Pete that Arthur had predicted, and we went on, and then did infusion studies with calcium and this is more recent actually. Not that recent but just making the point that you confuse PTH European red or PTH and blue.
• 22:17And make normal human beings volunteers IE grad students hypercalcemia too. Reasonable degree therapeutically safe to create one of trying to say by infusing PTHRP. So now one more thing remained to fulfill coax postulates and that was this developing an asset, so bill. Burgess was a fellow at the time and build decided that he wanted to raise antibodies to PTHRP and so he developed a close relationship with the format and definite if I remember right.
• 22:47So he would go out there and immunized chickens, and rats and rabbits and pigs and sheep and cows and he finally got rabid antibodies that were good that allowed him to develop a two sight assay for PTHRP so for Cokes Pasolas, you wanted, something that was elevated in the blood of people with HM but not in controls not a normal controls and not in people with other kinds of cancer with or without hypercalcemia. Not in hyperparathyroidism, which is this group and so sure enough, the only time PTHRP is elevated.
• 23:18Is in the serum in serum is in people with humor hyper calcium legacy? This also ended up being a New England Journal Paper. It became the basis for the classes that are currently in use. One other side bar is as an additional controlled by then we knew the PTHRP was in milk, which John Wesley Mirsky has now studied extensively so it took us 8 years to purify PTHRP and sequence it once we knew that it was in milk at concentrations 1000 fold higher than.
• 23:49Blood of patients dying of HM we went down to Wawa. There's one at the bottom of the Hill at the VA right went under Wawa bought a quart of milk purified it sequenced in a week. If we had only known.
• 24:01Alright so life lessons for more through AEP by the way in our lab. Everybody had initials. Howard was HR iOS. AFS he's JW etc, etc. Arthur is ATB except his word bigger capitals. So this is life lessons to me from a EB so generosity Arthur. I thought telling other story well when we first had these tumor extract stimulating cyclase activity. We had a bioassay. I was maybe I think maybe I was new assistant professor or
• 24:31Delete fellow In other words, second fellow because in those days. It was different so Arthur comes up to me and said Andy I've just been invited to give a talk at this prestigious conference called Laurentian Hormone Conference. That was the most prestigious conference in the chronology. It was yours. In Banff Lake Louise and he said. You know, I've been excited talked about this work. It's quite the world's attention it's very important, it's very exciting and I said, You know, I'm sitting there, thinking to myself, I'm gonna say anything 'cause my boss, so I kept my mouth shut.
• 25:05But I'm thinking it's kind of stealing your work and then I'm thinking. But that's really cool. Somebody thinks this is interesting. But maybe I should present it, but I'd screw it up. It's a great honor better let him do it. So then he completed his sins, which was to say, and this meeting is in September. This coming September. It was now around March. I'm going to call them and tell them I have Cholera 2 days before and you'll be presenting the work and so this is this is generosity. Howard had done it for him. Arthur did it repeatedly for Maine in our world.
• 25:35We're able to collaborate because he sent me the VA. So obviously I'm independent which was a complete line and and then he allowed me to carve out the protein chemistry space that was my expertise. He carved out. The molecular space. That, clearly, is his expertise and so that generosity and Truth or respect and connecting made important people in the world bone mineral metabolism? Who then would write letters for promotion and for grants etc.
• 26:02Another thing I learned from Arthur is that science is hard right. I mean, Arthur. If those of you know him know that he's committed deeply interested in serious music, opera, and Symphony and he also is interested in visual arts, so and actually as it collection. Let's go ahead and impressive so I don't know anything about this stuff and I still don't know nothing about opera. I still don't quite get opera, but that's me, but Sciences or it's very creative and I sort of I meet.
• 26:32You know, most of my friends are retired they're all in Florida. They know him an endocrinologist. They don't know what that is so they called me in entomologist and they can't. They can't figure out what's so fascinating about entomology that I wouldn't be retired and it's just this Sciences and art. It's incredible incredibly creative. It's incredibly fun and that's what it's all about and that's directly attributable chords and learn as many technique. I remember being terrified of learning new techniques in order. It was like no learning this learn this learn that those are your vocabulary those are your cultures.
• 27:03And you'll be able to bring them into your toolbox, and broaden your ability to do things and then there aren't any barriers between disciplines there. Artificial don't think of yourself as a cell biologist or a protein chemistry person or a clinician or as as are at Doctor or mouse doctor think of yourself as a detective and follow the story at the story says. You gotta do rats. Do rats at the story says you have some cells do cells. The story says. You gotta do something people do people. It's all it's all the barriers or artificial and you need to follow the story.
• 27:36Alright so let me just wiggle water here. I'm having fun. I hope you guys are having as much fun as I am.
• 27:47So everybody now doesn't even know that I was in bone in the old days and so I want to tell you a little story about how we move from bone from HM into diabetes so.
• 28:00OK, so now PTHRP is the genus Clone. We all have it probably wasn't given to us so that we get hypercalcemia and die in the last 2 weeks of life are probably had other purposes. So Arthur with Kyoji Acadia, who was a postdoc decided using more than blasted survey all kinds of normal tissues in rodents and people to the extent possible and it turns out the PTHRP is expressed all over the body and so then it became well. This is getting pretty long list. We can't really remember all these things so it turns out there's an easy way to remember where it's not where it's expressed this is the list of tissues that don't express' PTHRP.
• 28:34So now the group is pretty large there's a West Haven Group. There's a Yale group completely independent except for the fact that we worked intensely together all the time we had our joint landings, but were independent right not really so. So so then we said, OK, well, let's now. It's time to divvy up the body who is going to work on what? So here's the list. Arthur wanted to work on brain. We were so relieved because none of us knew anything about brain and we knew or were smarter than all of us and so it seemed like natural that Arthur would want to work on the brain.
• 29:07And then coral wanted to work on the skeleton and focus on osteoclast and osteoblast and so like that's good and then John wasn't Mirsky wanted to work on epidermis an hair follicles, which I had wanted and and mammary gland and it turns out, he's done amazing stuff with that and I hope I hope he has talked or will talk to all of these have really interesting stories and John is or leader in that.
• 29:37And then I said, OK, well, I want to do skeleton, but I want to continue sort of in vivo CRC kinds of studies with that and so we did that Neil Soifer was a fellow with me at the VA. He was a real fellow he wanted to do stuff and kidney. Another post doc that I had who is French. It was a vascular smooth muscle person and there's a vasodilatory. Beiser relaxant story with PTH repeat. But since I was chief invented print at the VA and was going to be a clinic and seeing lots of diabetes and since we knew by then that one of the tissues. That makes PTHRP is pancreatic. I that's I said let me work on that.
• 30:12So we knew it was and I let's we didn't know what it did. We knew it. the OR in a level. We knew it. At the protein level. So we turned to Bill Philbrick and Bill had made trans jeans with Arthur and John West Shore Mirsky expressing the PTHRPC DNA under the control of various tissues. Specific promoters and had the 3 prime end that stabilized it. So when it was expressed it wouldn't get degraded rapidly and in our case, we said. Can you make a transgene that would allow us to express this thing and beta cells and at that point, the rat insulin promoter ripped was well known.
• 30:45And so we made these mice, it, trying to see what PTHRP might be doing in islands.
• 30:50Our hypothesis was its molecular biology is new, Transgenics Renew knockouts hadn't been invented yet and our hypothesis that we were trying to disprove is that we were too stupid to make mice and so we did that and the phenotype that we got was completely unexpected. So this is the ripped it repeat mouse that the driver of this was rupee. Basava, who was a postdoc with Arthur and then with me out at the VA and the phenotype that you can see is in the transgenic animals, they got these gigantic eyelids.
• 31:21And when we looked at proliferation markers their beta cells were proliferating the animals were getting hypoglycemic and even severely hypoglycemic if you let them if you fasted them long enough an their PTA their insulin levels were elevated so you know, I'm seeing diabetics that VA. I realize this is important every diabetic needs more beta cells and at that point. It turns out nobody had ever made a mouse never know never had a model of a million model where basil mass could be increased, and lead to hypoglycemia hyperinsulinemia, so that seemed pretty important and rupee went on.
• 31:56And pursued that So what I want to talk about now is so weird. That's gone and I'm going to say a lot more about this in the in the conference. Tomorrow then driven conference. If you're interested so that basically for me was career. Changing we decided we begin to beta server generation and see see what we could do with that, so this is from a paper published in 2005 by Peter Butler, who is chief of endocrine at UCLA and Peter has done lots of studies. Autopsy studies in the pancreas of people with diabetes and related disorders.
• 32:30And so it's possible to publish in 2005 that beta cell mass is reduced in people in the pancreas that people with type. One diabetes and you might think well like you know, so what's new about that, then have we known that for a century because insulin was discovered. You know like 100 years ago and it turns out that's all true except that everybody thought this number was zero. Everybody thought that if you had autoimmune diabetes for 20 years 50 years. All of your beta cells are gone and now study after study had had confirmed this and in fact, this is probably an underestimate there's probably something down too.
• 33:02Maybe 20% of normal, it might be the average all the existing literature, so the point is people with type one diabetes still have beta cells. The beta cells, they in theory, could be regenerated So what do they look like so this is a picture from that paper so insulin is Brown. This is insulin stating out in the periphery of the pancreas. These are acinar cells out here. These are insulin positive cells so visually beta cells. This is a sick looking. I lit but 2 things are important. One is even after 20 years of diabetes. You had to have had type one diabetes for 20 years to be in a study.
• 33:34There's still instant positive cells in the eye lid and also this is supposed to be an autoimmune disease. But there's no there's no immune infiltration here and then in the wall of the pancreatic duct their insulin positive cells. It's not known right now, what the stem. Cells are for beta cells, but they are believed to be either ductal cells or Perry. Ductile sales for example, like these cells might be might be adult stem cells for beta cells. So it looks like. These cells are trying to regenerate in human beings with longstanding decades of type 2 diabetes.
• 34:06In the old days, the definition was type, one diabetes was you had no C peptide. You didn't make that you couldn't measure the connecting peptide of insulin. You couldn't measure insulin because they were taking insulin out of a bottle so you could measure C peptide so the definition of type. One diabetes was you have no C peptide, but essays have gotten better and now they're actually pretty sensitive and so this is a more recent paper showing that in fact, even after you've had type one diabetes for 1020 thirty forty 5070 years.
• 34:37Most people still have C peptide and so these beta cells that people have are still able to make insulin and they still will kick out more insulin more C peptide if you give him a glucose load.
• 34:50So this is also an old study. This is from Milan and it's a It's a group of renal transplant, surgeons who are trying to make middle transplants better. Most of the patients they were treating had type one diabetes and we're in renal failure for that reason because they said. You know here's our usual protocol. You get a call in the middle of the night. You can then we bombard you with immunosuppressives and the transplant outcomes are very good, but they're not perfect. So maybe we could make them better by doing immune immunosuppressive preconditioning with rapper Myson.
• 35:21So let's take a group of patients in our dialysis unit of type one diabetes who by definition of type one diabetes cause. Their C peptide is low. This is the old sorry. This is the old limit of normal. The newer one is closer to this. But even buy that asset values are low and then will randomize them receive. Randomized rapper Myson for a month or so until they get a kidney transplant, and then we'll see how they transplant outcomes are unanticipated. Lee and surprisingly they found that actually when you take people with type one diabetes and put 'em on immunosuppressive suddenly.
• 35:55Initial markers C peptide insulin goes up so that suggests that these cells that are sitting in the city that I lots of people with type one diabetes or actually able to regenerate and perform better, so the other half of this slide is Type 2 diabetes, so in the United States, there about 2, 1/2 million people with type one diabetes. That's not enough historically that hasn't been enough to get the former world excited about doing anything, it's sort of like call us when you have a real market so.
• 36:25It turns out that Type 2 diabetes is obviously much more common in the USA. 30,000,000 people and so this is another Peter Butler study. He's getting a little reputation as an ambulance chaser, but these are autopsy studies on people with Type 2 diabetes. Tittied does Type 2 diabetes who are either lean or obese and it turns out that beta cell mass and non diabetic. Normal people is about 1, 1/2% of the pancreas and it's reduced in TH in people with Type 2 and it's reduced in obese people with Type 2.
• 36:56So it turns out that beta cells. Inadequacy beta cell deficiency is an important part of type 2, so these papers again are published in the early part of this century.
• 37:07In retrospect just like that's hardly surprising. What everybody thought and by the way which person and yellow at Mount Sinai discovered is that that Type 2 diabetes actually isn't insulin resistance to disease and that became dogma. That's the only kind of that's the only cause of type 2 diabetes, but you know that can't be true. Everybody is overweight is insulin resistance. So it must be insulin resistance. But something else. So it turns out that there's something else is in adequate numbers that beta cells. So it turns out now that type one and Type 2 diabetes are both.
• 37:39Related to both associated with results from in adequate numbers of functioning beta cells.
• 37:46So everybody with diabetes needs more beta silt that's why you hear about stem cells were going to stem cells were gonna make beta cells. We're going to transplant them. But in the world. There are 400 million people with with IDs. I don't think we're doing stem cell transplants on the mall.
• 38:00So we got into this through PTHRP as I mentioned so this is a list of growth factors nutrients. Small molecules and other things that make Rd in beta cells replicate so PTHRP was actually number one on the list. There was nothing else, and then rupee did placental lactogen. Adolfo Garcia Okonja, who's a postdoc here. He's from Spain. He arrived at the VA in 6 months later. I told her moving to Pittsburgh and he said he was very happy to get out of jail. So so these are actually the first three models of things that would make beta cells mammalian beta cells replicate in Vivo.
• 38:34And again nobody believed us were a bunch of Boneheads and we're in Pitts Burg so like what could we possibly new because everybody new beta cells don't replicate so in fact that was that was great for us because that gave us a lot of freedom to operate so now the list is very long there a lot of things that make data cells replicating rodents. So here's the list of ones of those that translate into humans right. So none of the things on this list. Make human beta social replicate so that you know, Arthur says follow story where it goes the answer here is obvious.
• 39:05So, in the human eye lids, so it turns out that human eye, Ladson Road. Niles are different. This is what am outside. It looks like everything we know about diabetes comes from Nelson Rad. I'll it's so there a bunch of beta cells in the middle of that make insulin and then there's a core. I mean, a rim mantle of Alpha cells that make Glucagon and Delta cells that makes it matter. Staten so surprise surprise. You can publish in PNAS in 2006 that this is what the human eye that looks like it doesn't look anything like a Rd in island and so this is.
• 39:35Sure, that example one of about 50 different ways in which human eyelets and now sawdust avert so we want to make human beta cells regenerate So what do we know about that see how we doing timewise I'm going to finish early chance you're going to have to do a lot of talking So what do we know about human Beta Server Generation? So it turns out that it's pretty easy to get rodent based social replicate. But here's what happens in people so this is beta cell replication. Human beta cell replication from autopsy studies here and this is age.
• 40:06This is embryonic life, no basis or replication during the first year of life. There is beta cell replication. It's around 2% and then it tails off during early childhood and by the time you're an adolescent. They're pretty much not much. There's pretty much no better cell replication going on and in in also full adulthood. Full maturity there is no basis replication going on and no one has ever been able to make beta cells replicate so this is good news because now we know.
• 40:36The rates of replication that we all did when we were one year old. We also know the beta cells of the people in this room have now were made when they were one year old and there aren't they're not coming back. If you lose them and so the bad news is that the very tissues. We study categoric islands from humans distributed by NIH very tissues that we want to study to learn how to make better cells replicate by definition don't and won't replicate and Moreover. If you're going to develop a therapy to make beta cells replicate you're not going to be using it in one year olds in your first clinical trials are going to be using it in people.
• 41:09And in this age group, so this is a problem for us and we began to think about this and say look why won't why won't pay their cells replicate let's try and understand why they don't, it turns out, they will replicate if you do if you do the right things. So here's how I thought about it. Arthur told told me it across disciplines and so I began to think about why won't beta cells replicate and like John my family is from Fermont and so when your car breaks down like they were older were from Vermont. So you don't call AAA with car breaks down you take the car apart and put it back together again, so if your car won't start you pop the hood. You look at the engine and see if you got spa.
• 41:44Mark plugs ignition wires, etc, etc. Carburetor know blah blah blah so if your beta cell won't start. Here's an idea? Why don't you look and see what's going on with cell cycle and so we thought maybe they would have lots of cell cycle inhibitors and have no cell cycle activators, so in this slide. This is sort of the list of engine parts in the nucleus that drive cell cycle progression in any cell type. The green ones are cell cycle activators. the Reds or the brakes and so we did an inventory.
• 42:15And this was also with a with a postdoc Natalie Theos Key Tish, who now actually is running a lab at Regenerx on.
• 42:21And So what this shows is that all of the cells So what Natalie showed us that all of beta cells contain every single cell cycle molecule known to man and they're all expressed at high levels. So all the cell cycle inhibitors? Are there that's not typical most cells expressed one of these one of these one of these but they don't express all of them. It sells express all of them at high levels so in the automotive analogy. The brakes are on and the brakes are on hard. The emergency brake the handbrake every other break it's in Park.
• 42:52On the other hand of the cell cycle activators are there so there's gas in the carburetor the car should go but it can't because the brakes are on so we said. Well let's let's play around with this and see if we can make beta cells replicate either by adding gas in the carburetor, adding cyclins or taking taking the brakes off. It's easier to overexpress than silence things so we started out just by overexpressing cycling so automotive Lee speaking gas in the carburetor and So what Natalie found is that if you measure BDU that's a marker of proliferation in beta cells that are staying.
• 43:24I'm assuming so there's no replication going on normally inhuman. I'll ask these are dispersed human eye. Let's like categoric. I list from NIH. If you put in viruses that so this is Alexi control virus nothing happens. But if you put in D cyclins alone or decide cleanse plus CD case that's these guys suddenly you can see prodigious rates of replication. And if you quantify them you can turn the normal basically infinitesimal rate of data so replication into.
• 43:55It's a striking strikingly high levels of beta cell replication. So it turns out you can really make you a better cells. Replicating you can replicate that make them replicated high rates. So this was this was published in 2010. Another one just before it had been 29. So then we thought thinking of my cancer. Life with Arthur well. This is probably going to be bad. If you make them really proliferate rapidly. They're going to differentiate and they're going to become terrible. Beta cells and they won't work. So let's develop a model and this was done by Adolfo Garcia Okonja and Natalie.
• 44:27The ASCII test, let's take human eyelets, which we can get from NIH transplant them into the kidney capsule of skid mice. We can make them diabetic by giving them Streptase, Oh descent and let's put in just enough islets to make their blood sugar come down a little bit. So we make sure the outlets are working and then will treat them will give them ideas that are transfected with control virus or with cycling's and CD case so that's what this slide shows so we can take skin. Mice make them diabetic strep. This ODIs and stress disorders in their blood sugars or 405 hundred.
• 44:59OK and if we do nothing else for the next 6 weeks. They stay diabetic. If we put in 500 islets or actually someone remember 1500 lives in these experience put in 1500 islets and don't do anything else that blood sugar comes down a little bit, but doesn't normalize if we put in 4000 islands, the blood Sugar Humanae. Let's rap normalizes rapidly and so the question was what happens if we put in 1500 islets, but also transduced them or infected with viruses overexpressing CD case and Cyclones that make them proliferate.
• 45:32And you can see that you can turn 1500 islets, which is a marginal mass and doesn't control sugar into a fully therapeutic Mass that stays the maintains normal blood glucose for 6 weeks by simply overexpressing these molecules and then to prove that it's the human allograft transplant in the kidney. That's making the diabetes better you can remove the graph where unilateral refracta me the animal lives and then you can see that they become diabetic again. So this proves you can have it both ways. You can have crazy rates of human beta separation.
• 46:05You can do that in vivo with human eyelets and you don't get dedifferentiation. You actually get enhanced function, so that was in the diabetes world that was that was big news.
• 46:16So I'm going to skip that one. So so now. Now it's 1974 and or maybe a little bit before, though anyway. We had shown sorry. I'm losing my years 2004 or thereabouts. Just a decade. You know they slip by so. So now we know that using gene therapy approaches. We can fix diabetes and enhance human autographs and by then people were doing human eye transplants in people with diabetes.
• 46:45But then we said well can, we find a drug that would make beta cells replicate and so we basically developed a high throughput screen and this is based on something I learned on 105 at Yale with from a person Richard Lerner. That's now sort of everybody does this, but to me, it was new when I was here, so we basically did a high throughput screen with and found this molecule called harmony that blocks this kinase called Dirk 1A and leads to human based so replication. So our goal was to find a drug a small molecule that we could get.
• 47:16Give the people that make better cells replicate and this work by the way it was done by Plumb Long, who's who was with me in Pittsburgh and now is it Mount Sinai. He spectacular so the idea was let's set up a mic promoter driven luciferase Reporter screen so luciferase Reporter for reasons. I think you all know we pick the mic promoter 'cause every cell requires mic you can't proliferate in any cell type if you don't have mics so it's a universal governor of proliferation if you look at the Net Promoter.
• 47:47It's impacted by every single signaling pathway. That's mitogenic so yes. Nick is a knock a gene but normally it's expressed in very low levels physiologically to dry proliferation. If you expressed it crazy high levels in a tumor. It causes tumors. So our screen was in hep C2 cells because they are human and liver and pancreas aren't that far apart. Nick promoter luciferase screen. We screened 100,000 drugs. We ended up with one drug that makes beta cells replicate.
• 48:18And that was that was harming and again this is all fun long, so it turns out harming is able to make beta cells replicate and it binds to this kinase called dark money that I'll say more about.
• 48:31It turns out that it activates beard. You incorporation and dispersed human eyelets, whereas the control doesn't do anything you can see it with BRD. You you can see with KS 67 and it replicates the rates of replication that we all had when we were one year old, so harming treatment of human eyelets in vitro gives you therapeutically relevant rates of human beta so replication. The pathway that it works through is shown here, so just just just sort of summarize harming.
• 49:01Is hitting this molecule called Dirk 1A Dirk One? Is a kinase that phosphorylates in fact transcription factors and other things so the paradigm is this calcium and who sells beta cells or calcium address because glucose GOP one so funny. Oreos lots of other things. Calcium calcium goes into beta cells that activates calmodulin that activates calcineurin. Coulson's a phosphatase and calcineurin. D phosphorylates in fats that are sitting inside a plasm stranded and unable to get the nucleus.
• 49:32When there D phosphorylated they can go to the nucleus. They can drive jeans that dry cell cycle like Nick like CK1, like cyclins, etc. And they repress repress cell cycle inhibitors, so if that's all that happened everytime. Calcium entered a cell in response to glucose you had beta cell proliferation, so with Dirk Monedas is it's the brake on, beta cell proliferation. It takes it removes the phosphorylation. Phosphate groups on these guys kick them out of the nucleus and that's the termination signal for proliferation.
• 50:04So it makes perfect sense if you hit Dirk one day with drugs. This block its activity. It would be mitogenic So what I've shown you then so far is here's this sort of physiological rate of replication. Here's our problem. Adult beta cells won't replicate. Here's what you get from harming harming gives about 1, 1/2 percent, is pretty similar to what we all did when they were babies.
• 50:25So then the next board is this thing is published and we're all excited and were thinking. But you know, we're not the first people who claimed to have discovered a drug that would make their cells replicate there are other things out there that people have tried and then nobody could reproduce them. So we were relieved now to learn that lots of other people have these guys right now for artist. These guys were at Joslyn and Brode Institute and Al Powers is a Vanderbilt Justin Anasis at Stanford class ketchup in a bunch of Biotechs. So now we're in a phase where we have molecules that make beta cells replicate its highly reproducible in harmony is going from you never heard of it.
• 51:00Now it's sort of the industry standard for the fries in a cyclic amp for Bissell Replication Arthur think of it that way.
• 51:08So OK, so nice that you can all do this in a dish. Can you do it in vivo so again we turned again this is Adolfo Garcia Okonja and turned our transplant models with marginal massive human eyelids, so if you put in this case. 'cause we get better at this 500 islets into a strip does odaesan diabetic mouse blood sugar comes down a little bit if you take those same identical animals with identical. I'll it's from the same donor and treat them with harming it basically normalizes their postprandial glucose.
• 51:38In a normalizer glucose tolerance test so not only does it drive proliferation enhancement cell function in a dish. It actually works with human eyelets in vivo if you'll accept human eyelets in this kid mouse as in vivo and it dries proliferation in vivo as well. These are shown as fold changes for BRD U for perforation for 1067. In fact, the numbers aren't that high so if you actually look at the percent proliferation. They're not as high as in vitro so if you think of this slide, then it sort of looks like this.
• 52:11This is what you get in vitro from harming this is what you get in vivo still in the right direction, but not as high as we'd like it to be so yeah, so we're getting close to stopping is this we know that based on mass is reduced in type one and Type 2 if your proliferated 1% per day, which is we're harming doesn't devo. You'll probably get there in 12 months in Type 2 diabetes. But you're going to need more proliferation. If you want to store Basil mask in a reasonable time frame and Type 1.
• 52:41So here's an advertisement for the next talk, but I'll just tell you the bottom line and it basically now using other things that we've learned through techniques that Arthur Arthur taught me thinking techniques. We can now routinely get human based so replication up into the 5 to 8% range. That's absolutely not for type. Monotype, too, so this is again Oh Maj to Arthur. I mean, Arthur taught me to take ideas from the bedside do things that you need to do take them to the bench do what follows what you need to do there and it doesn't matter what the barriers are you just need to do that figured out how to do it.
• 53:16Find someone to collaborate with who knows how to do it and then take these things back to the bedside. So now we know how to diagnose HM we can do stuff with diabetes. There's a Vasco recent Gnosis story. There's osteoporosis treatment story. There's a lactation Physiology story. This is John waste. Mirsky so Arthur. This is to honor you Arthur Arthur by the way is very shy and hates attention. So I just want to pour it on a little bit more so Arthur. This is thanks to you for teaching me how to do everything. It thanks to Yale for putting me in a position to do this.
• 53:48I still think of Yale is home, I'm glad to see that nothing has changed not even not any exchange of this room. So so please keep it that way. And then and then just one last thing for Arthur. So when I came to Yale at topic hyperparathyroidism was that was what we call this disease is no longer that it's doing it now. Now it's HM so In addition to Arthur. I need to think a lot of people. I'm not going to mention all these people. I just want to mention punk who's been simply fantastic to work with and Adolfo.
• 54:18Garcia, who was here for 6 months and then was able to escape from jail.
• 54:23So I'm going to stop there. I'm happy to answer questions.