Hello, my name is Megan and I'm a librarian at the University of Alabama. This video will demonstrate how to use the various advanced search features of JSTOR, a database of scholarly resources for the social sciences, humanities, and modern languages. JSTOR's advanced search is a powerful tool that can be very helpful for finding resources for your papers. You navigate to JSTOR's advanced search by selecting Advanced Search on the JSTOR home page. Like other advanced searches, JSTOR allows you to search by author and title, though it also allows you to search by abstract or caption. An abstract is a summary of an academic article and searching via abstract can help you narrow down to resources that mention your keyword within their abstracts. A caption is the short description of an image, graph, or chart and can help you find an article with a specific image. For example, if I run a caption search for American photographer Ansel Adams, I get a manageable 140 results, though JSTOR does warn that not all of their captions are searchable. A quick click-through of the first few results show the reprint of one of Adams' photographs within the article. If you use a field limiter, such as caption, abstract, title, or author, JSTOR's advanced search feature has a quirk. You will have to restart a search instead of changing it within the search results. Most of you have run a Boolean search before, using the Boolean of AND to link two keywords together, such as Alabama and football. For this next example, I'm going to use the OR Boolean, which allows me to have two search terms but results a return for either of them. Think with OR without you. I'm researching the history of Ukraine, so I'm going to search for journal articles on the capital city, Kyiv. Because of Kyiv's history, the city can be spelled as K-I-E-V, Kyiv is the Russian transliteration and was the official transliteration until Ukraine's independence in 1991. Kyiv is the Ukrainian transliteration and is the current official transliteration. Since this search is to find out what JSTOR holds, I'm going to change the content I can access to all content. This means that I will be able to see everything that JSTOR has on Kyiv. However, I might not be able to access all of it and I will have to request something from Interlibrary Loan or ILLIAD via the full text finder icon in the results. In the basic searching in JSTOR video, I cover the date and content type limiters. Under advanced search, there are a few more content types as JSTOR is expanding their holdings in primary sources. Before we get to the subject limiters, you can limit by language or the journal title if you know it. Speaking of journals, an advanced search page is a long list of disciplines, which contains specific journal titles. While they are referred to by JSTOR as disciplines, they appear to follow the Library of Congress subject headings. You can select an entire discipline or any number of journals within a discipline. Please remember that when narrowing by discipline, you run the risk of narrowing down your results to the point of exclusion, such as removing journals in one subject area that could be useful. JSTOR has a feature that allows you to see the topics associated with an article or book chapter. When you click on one, it takes you to what appears to be a curated collection of articles and book chapters about the topic. These topics are very useful for finding similar resources on the same topic, as you can search within the topics to narrow down your results. That's all for now. Thank you for watching. If you have further questions, you can call us, text us, or visit ask.lib.ua.edu to ask a librarian.


What's up everybody, Jayz here for JaspysCaseBricks.com. It is a new release day. Happy Opeachy Platinum Hockey release day. This is 2019-20 Upperedego Opeachy Platinum Hockey. This is a 16 box master case. It's gonna be a little bit guys, but we're gonna have a lot of fun throughout these 16 boxes. One of my favorite releases in hockey every year besides Cup Hockey. I love Opeachy Platinum. The colors, everything about it. Again, you get one autograph per box, which is on card. And again, we're giving away a free Opeachy Platinum box. So if you bought two or more teams, you get entered into winning a free box. You bought four teams. You get two entries. You bought six teams. You get three entries, etc, etc. And let's go. Good luck. Last but not most you're going to David. I forgot to put a little star right there. I don't have a pen. Put a star over here. Here's your last but not most of David. And good luck guys. Again, we have a couple more bigger teams on the website. And if you guys really want more, email Nick, say, I want more Platinum.  I think we have more coming over. I'm gonna open up both inner cases. That way we have all 16 boxes on the table. Remember, this is a master case. Yeah, email them whatever you guys want. We have some 0304 stuff in the back. We have some 0304 stuff. Maybe just not for sale yet. Not sure, Luke. Maybe, maybe some like two, four box breaks, but I think the Picker teams have moved pretty well. So it might just stick to PYTs. But I mean, I'll never say never though. No, we never say no. Sorry. That we will not do one of those. All right, now I'll rip open four boxes at a time to make this break go by a little quicker. But I feel like I can get this done in about an hour. Maybe an hour and 15. So, rip open four of them.  No, did you? Ah, it's got the Red Wings too. That's a good team right there. There you go. And get number two going guys. Again, it'd be awesome to sell out all three cases today. I would remind that hometown of Brett Fabred. You guys probably cringe seeing all these packs. I just get so excited. Come on, platinum. Don't be so rough ripping open packs. Be like 15, 16. Or it's easy. We got a Seisman Gold coming. No, this is, this would look more like Donner's Optic baseball. So they come out with Opeechee hockey every year. So you can think of that as like Donner's baseball and all the other ones that are just plain Donner's basketball. But then Opeechee platinum comes out and then you're looking at like Donner's Optic. You're looking at like Chrome in baseball. So basically that Chrome feel. That's why a lot of people love it. And then the beautiful colors you get. I like how they numbered Violet Pixels though. A couple years back or was it last year that they introduced Violet Pixels? I believe it was two years ago. They were just an insert. But you know, it's more odds than anything. Now I love that they numbered them. And then Arctic Freeze is now like out of 99 instead of 79. Which is a little weird when it was out of 79. Ooh, the sunsets look awesome. And if I had a good player, a favorite player to collect in this set, like rookie wise, I'd do it. I'll be into King's Hockey next year though for sure. They put Gabriel Vellardi in hockey products finally in NHL. Upper deck I mean. I'm all in. Yeah, you want Phillips to the Dina. I think there's other ones in there for him too. And Cayden Falscher is another one. Taro heroes I think is one of them as well. But definitely to the Dina. And then of course, you know, I mean, I don't know what else Red Wing's have as veterans, but I'm sure. I'm sure there's like Larkin and some other stuff. And then we should have persons of this stuff on the IG as well. It's just a little tricky because due to upper deck like rules, we can't give you guys the price over our website or in the air or weren't when we're on air. If you ever come into our shop, of course, all our prices are there. So you'd have to like email us to give you the price. So we'll have some of these for personal as I believe as well. All right, guys, let's go with the first box again. Here's the rainbow sets, of course, as you can see. Rainbow version. There's a retro. Teddy Bolger, that's a rookie. Victor Olufsen. And you got Anisimov, seismic gold out of 50, of course. Oh, look at this. Sunsets look beautiful. Tarot. These look awesome. Look at that. That's sick. What up, Pizzle? I know, man. It does look good, right? Carl Grunstrom. Rainbow for the Kings. Pettersen. It's a second year rainbow, but still nice. Mark Shifley. Koneckny, Sunset. Jormfront. Best in the world, Crosby. Dominic. Oh, there's a guy ahead of us. I've only pulled like three golden treasures. They're so hard to pull. Nylander, Violet Pixels. Again, these are numbered now to $3.99. Nice. Nick Suzuki, rainbow. Poeing. Blake Lazat. And we got Julian Gouter out of $1.99. Red Prisms. Gallagher. And Libor Hajak. Rangers. Cal. Carter Hart. That's the pink inserts now. So the pinks are not numbered. Neon Drysidle, Sunset. Jack Hughes. Best of the world, John Gibson. Crosby. Quinn Hughes. Gorno. Zadina. Oliver Wallstrom. Barbertson. Sunset. Suzuki Rookie. Sagan. Dahling. Gusev. Morgan Reilly. Drysidle. Adam Fox. Gibson for the Ducks. Going to Chris. 33 out of $1.99. And we got Oliver Wallstrom. Look at that. Violet Pixels Rookie Autograph for the Islanders. It's going to Dylan. Jack Hughes Retro. Nylander. Bertuzzi. Jonathan Quick. $2.37 out of $3.99. And Amberley. These ones I'll pay me sleep when I can. I'll put those over there. Next box. We got Joe Lesperance out of $1.49. Retro. Blue Wave. Or Blue Rainbow, sorry. Nice. Tobias Jormfront. Sunset. Man, they really hit it with that sunset. It looks beautiful. Faraby. Nathan McKinnon Retro. Makar. Quick Sunset. Jack Hughes Rookie. Suzuki Rookie. Adam Fox. Rainbow Rookie. Calder Front Runners. Drew Dowdy. $21 out of $3.99. Quinn Hughes. And look at that. That is an update. Sebastian Ajo. The future is now auto. From $18.19. Josh Cruz. You got the Hurricanes. There you go. Little Sebastian Ajo. Kerry Price. Pierre-Luc Dubois. All right. Last box. Then we'll move on to the next four. Zadina. Rookie Retro.  Yeah, right? Great car for a $20 team. Exactly. And that's $20 throughout the whole master. You know, a PG problem is not really a pricey product. So that's what makes it even more fun. Poeing. Gensel. To $3.99. Ruinsky. Kirby Dodge. And we got Faraby. To $3.99. And Zach Meck. Ewan. For the next. Going to Joseph. Kusev. Giordano. And Nick Suzuki. Look at that. Nice blue rainbow. $145. Out of $149. All right, guys. A lot of color. A lot of color. So far. No surge. No checkers. Or no golden treasures yet. All right. Let's go to the next four, guys. All right, guys. Let's go to the next four. All right, you guys. So again, this is a 16 box master case. It will take me about an hour to break each one of these cases. So again, I do see that there's like 26 orders on the website. And I got to go through and keep on coming. Orange checkers. Nice. Our first orange checkers. Orange checkers are only hobby exclusive. And I believe they're only out of 25. All right. Again, going through the orders. We're done a four left. And that's 2017 flawless round of number block. But it looks like the orders that have been coming in is for more platinum. We're done a six and seven left in the next two cases. So again, if you want your break to sell out that you're in, spread the word out. Maybe some other customers can help out. If everybody wants to do platinum, that's all good with me, too. Again, we're live early today. Joe will be here today. He'll be here for a half day. He'll be streaming at six o'clock. So I'll still be here for the next six hours with you guys. You may see him around before six here, but he was doing a half day today. And he should be back to normal tomorrow, Friday, Saturday. Let's get some Barrett Hayton. Had some magic in the world, juniors. Man, do a breath fire. It looks like he can still play football today. He looks more yoked than he did when he was playing. You know what they should do, guys? Do you guys remember that back in the day when it had the Pro Bowls and they'd have the veteran Hall of Famers that have retired from the league play against each other, flight football style? They should bring that back. I love that they brought back the skills competition for the Pro Bowl, for football. But it would be so cool to see a lot of these old-timers play football again, the flight football style.  That's crazy. All right, good luck, guys. Next box. There's your Dominic right there, Josh. Little Sunset. Jack Hughes called her front runners. The Brinkat. Miko Hescher. Kale Fleury. Suriella to 399. Noah Dobson. And Connor Clifton. Rookie Autos for the Bruins. Kirby Dutch. And look at this. Kale McCarr, little pink. That's nice. Ian, who got the avalanche. Whoa, did we get a surge? Wow, we did. Tobias Jornfront. 2 out of 10 for my Kings, Joseph. There you go. 2001, still 19 years old for my Kings. Nice. Collect the rainbow now. You got one of the hardest ones besides the Golden Treasures. That was a good box. Zadina Rainbow. Eric Carlson, 2199. Yeah, the Kings do have a lot of young prospects in this year's products. They got about like five rookies, I think. I mean, they're definitely top three in the farm system. I mean, some say they're number one, number two. And they do have a lot of young talent, so that's why they're kind of getting rid of a lot of these players. I don't, sorry, Tony Pinarin, Orange Checkers.  It's like if they're that talented in the minors, and you got to give them a chance. Carlson. And Blake Lazat for my Kings. Rainbow autograph. There you go. Kings Mojo. Kapokako, Jonathan Quick Pink. Jamie Ben. Next box. Ooh, look at that. Arctic Free is looking awesome this year. Out of 99. Jeff Skinner. Really does look like a frozen pond. Lafferty. Ryan Johansson. Nikita Goose of Rainbow. Tara Vainen. Kapokako. We got Matt Duchain. To 199. P.K. Subban. Barrett Hayton. And a Lucas Walmart. That is an update autograph for the Hurricanes to 50. From 1718. Red Prism autographs. Nice. Another update for you, Josh. And the Carolina Hurricanes. And Tarot. Pink. Red Wings. Buena John. Next box. Lean Bergman. Rookie Autograph. That's a pink rookie auto. Out of 99. Matt Pink. Sharks. Buena Michael Baluchi. Matthew Kuchuk. Eichel Sunset. Zadina Calder. And we got Kyle Connor. Seismic Gold. 25 out of 50. Jett's going to Chris. Brady Kuchuk. Violet Pixels. To 399. Stamkos. 192 out of 399. Hughes Rookie. And another Lucas Walmart. Update. That's crazy. I think this one had two autographs there, didn't it? This one had the Lean and the Lucas. So it's probably like 15, 16. Sometimes there is two autos in a box. Johansson. Morgan Frost. Copacaco Retro. Next half-inner. We're halfway through guys. Probably today at one point Luke don't know when, but number two is already down to four and number three is at seven. So then everything else is still not sold out. So we might be doing three OPG bottom cases back to back to back guys. Getting it all out of the way. And again, Joe still will be here. We'll be streaming from six to ten the last four hours with you guys. So I guess we can knock out the Platinum with me. All right, our next one guys. Is that you Brad? Is that you Matt? What's up man? And I know man, I know. The good thing is I do hockey here though. And I'll see if we can get some more guys.  Watch everybody that's watching that doesn't like hockey. Be like, no. All right guys, next four boxes. Good luck. Barzel to $199. All of a sudden sunset. It's my favorite favorite set this year. Sunsets awesome. Quinn Hughes rainbow. What up Nathan? I type in, but I can't. Someone wants to type in that I said hi to him. Cody Gloss, Antony. Matt Carr. Look at that. A Quinn Hughes and a Jack Hughes rainbow in the same box. There you go. Mario. Ferraro. $3.99. Whoa, we got a redemption. First redemption I've seen. Capococco. Truba. It's just a pink. All right, Nico. Let's use you as a little filler. We got a PG Platinum rookie. Autograph. Matt Pink. Number to $99. And it's Victor Olufsen. There you go. Buffalo Saber is going to Michael. Dobson to $3.99. Brady Keeper. Matt Murr. Capococco. Clouderue. And Carl Grunstrom. Oliver Ackman Larson. No one for the Kings. All right, next box, guys. I know. Well, they do have the most autos rookies in here, Nicholas, but you want to send me a Yelts jersey? Maybe I'll get some Yelts in there. Kizuki. Ladiskov. Liza. And Connor Hallibog. Ooh, that's a beautiful looking card right there. Retro autograph. Red Rainbows. Jett's going to Chris. I'm not sure, Luke. To my knowledge, we only got three cases for group breaks today. I'm not sure if we're going to get more, but that's all we got right now. If you guys really want more platinum, I can see if we can get some more soon. Ooh, look at this one. Matt. Arctic Freeze. They're just so nice this year. Look at that card. Nikita Gusev. 26 out of 99. Devils win a Matt. Capococco. Look at that. Very nice. Out of 399. Boca Sunset. Quinn Hughes. Jack Hughes. I feel like Quinn Hughes and Jack Hughes are linked together, guys. You get one in one box, you're going to get the other. Anders Lee. Suzuki. Trayor. Jacob Slavin. Zidina. Anisima. To 99. Arctic Freeze. This is one, Dom. These breaks are taking about an hour and 15 minutes or so. And Rampitlik. National Predators. Red Rainbow Retro Autos. So I'm not sure if the number two I already sell out. I don't even know if it's sold out yet. No, number two is down to four left. So it hasn't not sold out yet. And Boquests. Matt Pink. Alrighty, guys. Last four boxes. So I'm at 55-minute marks. Like I said, yeah, these will take me about an hour and 15 or so. It's a rip. So if you guys really want to just get all this old peachy platinum done. So, number two and sell at number three. All right, guys. Last four boxes. What are the base variations, Nathan? Like the numberings. Like the ones that are X number and above. Sorry, I can't really type right now, Nathan. But if you can listen. Oh, really? Well, I'll double check that in the sorting process. But thank you. So it says it on the back. Like right here. Or it says it on the front. But I'll double check those though. When we're in the sorting process, if that's the case. Nice. I didn't realize that happened though. That's awesome. We'll check that out in the sorting process though. In case I don't see any. I feel like they always have some type of unannounced Easter eggs in there. You know what I mean? I always have something. But yeah, we will. My wife obviously helped me out sorting when I did my own hockey stuff. She knows all about platinum. I believe so. I thought I had seen them in previous years, but I don't think it was OPG platinum. I think Nathan. No more. I started doing them more in 15-16, but I have done 14-15. I think OPG has just been around forever. The platinum, yeah. I really started breaking more hockey when it was 15-16 product. I mean, that class now is like really loaded. And especially if I'm sure 14-15 cup is really, really expensive now. Man, there's people that just love platinum, Joseph. And you're chatting with one of them that I know of.  Since it started, he'll tell you. He probably doesn't even know. He still has so much. I don't know, man. I mean, at one point, even with me, you're ripping a ton. Not just with me, just all throughout the years. But when they introduced that violet pixel, it was so beautiful. I really love the Arctic freeze now. And the Sunset, for sure. I think creating a whole retro set of Sunsets or a whole rookie set of Sunsets would be awesome. All right. Let's go. Good luck. Barkoff out of $3.99. Svechnikov, Arctic freeze to $9.99. Yeah, I'd like to see a checkered autograph, for sure. Pro-Horken? Not too much to ask for. Ben Bishop for the stars. Joe. Red Rainbow Autos. There you go. Arborsen, that's just a matte pink. And we got Nathan McKinnon, size of a gold. $33 out of $50. Carter Heart Sunset. Matt Carr. Calder. Barrett Hayton, retro. Jack Hughes. Look, I'm telling you guys, Jack Hughes and Quinn Hughes are always paired together. I already caught it in this first case. Heinola, $249. Blue Rainbow, retro. Jets. Zadina. Ooh, Sunsets. I wonder if these are actually going to sell for, honestly. Again, very nice set. Jimmy Shulks, Frederick, Dodge, Souter. I mean, that'd be great if it was a $20, $30 card. That'd be awesome. I'd take that. Cody Glass. I guess we'll find out soon. Once people start selling these online. Bobrovsky, to $149. And Caden Fulcher. Rookie Autos for the Detroit Red Wings. Going to John. Rainbow. Goalie. Out of Ontario. Born 1998. All of a sudden. All right, two more boxes, guys. Max Domi. Couple of Cockle, Jack Hughes. Kale. Zadina. Rookie there for the Washington Capitals. 399 for the Pixels. BullQuest. Rainbow. And Ryan Pohling for the Montreal Canadiens. Going to Ian. All right, last box, guys. Good luck. Cerella. Little Arctic Freeze to 99. Corporal Cockle. Sandin. Dodge. Nathan McKinnon. Oh, that's one of them right here. Pond Hockey. So you know what? They're like a color, then. That one you're talking about, Pond Hockey? So they're colored, then. Oh, so good. Oh, I get it. The background is a pond. There you go. Yeah, the frozen pond. So they're colored, then. That's cool. So I guess unannounced. Variation. So yeah, the background is like a little pond. It'll say Pond Hockey. That's cool. There you go. Avalanche. I think I actually might have pulled one earlier, honestly. I thought those were Arctic Freeze, but I didn't realize in the back I might have said Pond. But I'll double check, but there you go. Gwyn Hughes. Goudreau. Pohling. Pedersen. All right, last little run right here, guys. Good luck. Should be at least an odd over here. And it's Tarot. Red Wings. John. All right, guys. I'm also going to pen and see all these rookie stuff. There's a huge stack, so a lot of other teams are going to get some nice color, or sorry, nice rainbows and retros and stuff like that. Here are some of the numbered stuff that I'll go through. Or rookies. So Goudreau. These are all the colors. Or rainbow big rookies. Cockle. Redemption to Olufsen. Those are Otto. Jacky's, Gwyn Hughes, rainbows, Olufsen, Sunsets. A couple of nice suns in there. Size of my gold. Connor. Cal Connor. Again, a lot of beautiful colors in this Opiechi platinum this year. I'm digging it. Again, more known for the color, but of course getting color Otto's is always great too. And then here were some Otto's, some of the bigger hits. So there's that Olufsen to 99. Matt Pink, Redemption, Tarot. There's that Pond dancer, which is pretty cool. Pohling. Fulcher. Mekkar, Sunset. Bishop. Pitlick. Gusev to 99. Hullabuck. Grunstrom. Bergman. Matt Pink. Walmart. Walmart to 50. Lozat. Panarin orange checkers. For my Kings, Jornfront. Surge to 10. Clifton. Mekkar. Matt Pink. Zack. MacEwen. Sebastian Ajo. The future is now insert, update, Otto, Walsstrom, and Hayshack. There you go, guys. Opiechi platinum. 16 box master case break. Pikachu number one just sold out. On number two and three are on the website and about to sell out as well. Get it going. Jaspyscasebreaks.com. And actually, sorry, got to give away a box. Remember, if you bought two or more teams, you have a chance to win a free box.  Six teams, three entries, et cetera, et cetera. So, alpha ties by customer name, Chris. You had two spots, you get one entry. David just won. Don, you had two. You get one entry. Dylan with one. Ian, two. You get one entry. Joe with one. John, two. You get one entry. Joseph, one, two, three, four. So you get two entries. Josh, X-Line with just one. But Josh, Prusya, two. You get one entry. Karen with two. You get one entry. Mark with two. You get one entry. Matt Buckler with one. Michael Gallucci with two. You get one entry. Michael Tuckley with one. Nick with one. Richard one. Rick with one. Scott with one. Willing with one. So there you go. Just these customers here. That makes sense. Michael Gallucci was the last one. Michael with one. Nick, Rich, Rick, Scott. Will, there you go. Customer number one gets that free box. And I'm not sure if our other case came in for the free box. So we'll just ship those two sealed. And we got a six and a four. Ten times. Good luck. One, two, three, four, five, six, seven, eight, nine, ten.  There you go. After ten times. Congratulations, Karen. You're going to get a free LPG climbing box. Again, we'll ship that to you sealed. And there you go, guys. Jasperyscasebricks.com.


of a recession which is two consecutive quarters of negative growth I can tell you this is not the technical definition of a recession it is not it is not I will say that the textbook definition of recession is not is not two negative quarters of GDP well a recession is two quarters in row negative growth I don't think we're remember way way back during the old-timey sepia tone days of 2018 when everyone knew the definition of a woman and a recession Donald Trump was president and oddly the media and Democrats were way more eager to declare a recession Thursday that first reading of second-quarter GDP there's a possibility this is a negative number two consecutive quarters of negative growth the definition of a recession is a decline in output for two or about six months a recession is just two consecutive quarters of economic decline when we talk about the possibility of a recession what is a recession a recession is two consecutive quarters of two consecutive quarters two consecutive quarters of declining GDP because as you know it's two consecutive quarters of down GDP that signals it is actually a definition of a recession I mean the most common definition of a recession to consecutive quarters of negative growth that's Richard said even if we don't have two consecutive quarters of negative growth, we might have one quarter of growth so deep that it's classified as a recession. And you know, one of the reasons that we should all be very careful about even using the word recession, you can't really even officially use that. You know, one of the reasons that we should all be very careful about even using the word recession. The White House has been eager to push back on this notion that two consecutive quarters of negative GDP growth automatically equals a recession. Yes, it is a rule of thumb, but the White House is really emphasizing here and trying to educate the public essentially over the last week. And to simply say, we have a good economy, we have a bad economy. Come on, Willie, we have to remember what's actually causing inflation. You've got a war in Ukraine. You have the challenge of getting the American people positive about the economy. It's very psychological. How do you change the way people think? Because we are in an economic recovery. A recession is just two consecutive quarters of economic decline. Isn't it more important that the carbon emissions will be reduced by 40% by 2030? OK, inflation comes and goes, but the air we breathe is more important. Well, I'm not concerned about a recession.  Doesn't sound like a recession to me. Thank you very much.


Okay, thank you, Gene, for that kind introduction. So what I'll do over the course of the next 50-55 minutes is to give you some information on three discrete subjects, moving from the specific to the general. One is the recent advances in our treatment of aplastogonemia, not a disease that I know that you will likely see a patient or have to make treatment decisions, but a remarkable example of success in hematology and in medicine, and looking at the number of gray heads in the audience, or bald heads in the audience, I'm going to be able to tell you that a disease that most of you saw as being uniformly and rapidly fatal in training is now one that is manageable in almost all patients. The second is to tell you about the new telomiropathies, that's a much more general and internal medicine interest of their patients that you undoubtedly have seen in your own clinics who have telomiropathies that are not rare, and they're hard to recognize because they involve multi-organs and can be quite subtle. So you either will retrospectively or prospectively be able to make this diagnosis. The third point is also of general interest, which is the relationship between telomiratrition, which is a normal phenomenon, but also can be accelerated genetically or nitrogenically and cancer. So I'm starting with aplastogonemia, it's a fantastic disease, first described at the end of the 19th century by this man, Paul Ehrlich, in his younger days when he was a dozent at Charite Hospital in Berlin, did an autopsy on a young woman who had died after a catastrophic brief illness, bleeding, clearly anemic, probably infected, and when he broke open her bone, he saw, well not this, he didn't have a confocal microscope, but he saw instead of the plump, juicy marrow of megaloblastic anemia, because pernicious anemia at the end of the 19th century was common in Germany and fatal, instead of seeing the rich bone marrow of B12 deficiency, he saw fat, and that was the basis of his case report, probably not publishable in the New England Journal these days. So this is a confocal image, I don't think I'm going to be able to get it to move for some reasons or a variety of reasons, but this picture shows you the marrow under the most modern techniques, this is a marrow that is devoid of normal hematopoiesis, you'll see that more clearly in a mouse model image in a moment, it's filled with fat, that's what's shown in green, and CD34, which is stained with red here, and which is the compartment of hematopoietic progenitor and stem cells, is only staining the endothelium, that's the vasculature that you're seeing in red. This is a patient with aplaskin, I'm going to return to her in the middle of my talk, she's somewhat older than typical, but she's got most of the clinical manifestations, she's presenting with anemia, she's pale, she's bleeding in multiple sites, so this is very alarming to the patient obviously, and to the emergency room physician or primary health care provider, ascii etymoses, and shortly afterwards became infected, so it can be a very dramatic presentation. Bone marrow is really an important organ, if it's not functioning, if the blood counts are really low as in severe disease, it's really uniformly fatal. These survival curves are from the 60s and 70s when, for example, Jean and I were in training, and patients with severe disease defined by blood counts would be dead a year or a year and a half after presentation, even with modern blood transfusions. This is a disease of young people, which makes it even more troubling to deal with, usually young people who have been previously quite well. You see that the peak is in the late teenage years and early 20s and 30s. It's a disease also that is interesting, environmental connections, shown on the left is a clipping from a 1930s newspaper, a 1920s newspaper in New York, which made the association between benzene exposure and the leather factories of northern New Jersey and bone marrow failure, it actually did not lead to any changes in benzene use, despite a great campaigning on the part of the Essex County Coroner. That's not a problem in the United States anymore, it is a problem in countries like China where there are actually epidemics of benzene poisoning that arise in industrial sectors. On the right though is the more common phenomena, which is idiosyncratic bone marrow failure due to medical drug use. This also is a terrible occasion for the patient, who may be only taking an anesthetic or an antibiotic and for the treating physician and obviously the consulting hematologist. It also has a major impact on drug development and this clipping from the New York Times shows not only the stock share price but paralleling that would be the number of people doing research in this company with only two dozen cases of bone marrow failure, apoloscenemia resulting from the introduction of a new drug into the market. This is a ward in the northern part of Vietnam in Hanoi, a picture of a patient a few years ago. Apoloscenemia is a very common disease in hematology services in East Asia and China, Southeast Asia and India. This is the female service at that time. There are two or three patients stacked to a bed and a third to a third to a half of the patients in a hematology service have bone marrow failure whereas most of you will not see a patient in a year and even a hematologist may only see one per year. We've done formal epidemiologic studies in Southeast Asian. The rate of the disease, the incidence of the disease is two or three times higher than it is in very similarly performed studies done in Europe and Israel and probably also in the United States. This is a slide I took just recently on the trip to India and shows the same thing. You can just note as the physician giving this talk he's shown in the upper left noted that in this large hematology service apoloscenemia is actually a more common diagnosis than is acute myeloid leukemia in the Bengali area of India. Now in the historic period that Gene was alluding to in my CV we thought of apoloscenemia really in isolation. It was one of these oddities separated from other hematology diseases, other medical diseases. The focus was entirely on the etiology that came from history and supposition. It was endless I think in retrospect rather foolish questioning of the patient for possible exposures. None of which were relevant to what happened to the patient afterwards was usually sent home to die with minimal transfusions. Now we have a modern, not final, but a modern view of apoloscenemia that looks something like this. It's a very simple outline which is that there are two major components. I'm going to tell you about a third which is the genetics but the major components are the hematopoietic system which is gone but not completely in patients with apolasia and the immune system which is doing the killing of those hematocarp genitor and stem cells. So in the first phase we do have some inciting event but we don't worry about that. We think that in almost all cases it disturbs the immune system. There are some ideas of how it might do that. Leads to almost total destruction of the hematopoietic compartment because the bone marrow like a lot of other organs is capable of pretty good function until almost all the cells are gone. Then there's recovery which can occur I think rather uninterestingly as a result of transplant more provocatively when we give immunosuppressive drugs from a biological point of view. And then what I want to stress at the end of my talk is this late phenomena of the development of leukemia or myelotysplastic syndrome in a subset of patients about 15% who have responded in some cases to anti-immune system therapy. So this is an updated view of the Venn diagram in which we now can relate aplastic anemia on the one side to other immune mediated diseases that affect a single organ or almost invariably T cell mediated and also to leukemia and myelotysplasia. This is adding the genetics and I'm going to talk a bit about that especially as far as the inherited mutations are concerned in hematopoietic stem cells as we go along. So this is a dramatic case that we treated back in the 1980s so we have a long follow of a young man with hepatitis, seronegative, non-A, non-B, non-C, two or three months later very stereotypically, develops very severe pancytopenia, invariably fatal in these patients according to the literature, comes to the NIH from Colorado, is treated with just four days of rather mild immunosuppressive called anti-thymocyte globulin and six months of cyclosporin. You see the dramatic rise in the blood counts which were sustained and have been sustained as he now has entered his 30s. This is a summary of many trials of anti-thymocyte globulin, a polyclonal preparation made from horse serum of antibodies directed against thymic cells. You see that the NIH actually has the largest or among the largest studies as a single center institution. I also think the most careful studies since we're able to have long follow up to Dubonmero, cytogenetics, morphology, histochemistry without physical constraint, we have at least up until recently the most detailed information on our patients as they enter and go through therapy. There are consistent results between 60 and 70% of patients who have a single round of this immunosuppressive therapy to recover marrow function and blood hub. So we're losing these nice images. I don't know if I can go back and show you. This is a mouse model which you're not going to be able to see completely, but I'll just tell you using words that when we take very small numbers of lymphocytes that are mismatched either for the breast and the embattability loci and infuse those into a mouse that's mismatched, we produce very specific bone marrow failure and can demonstrate the potency of small numbers of lymphocytes. They're far more effective than any chemotherapy that we can give in specifically destroying the bone marrow and that's destruction as a result again using mouse models of CD8 cells as the effectors, type 1 cytokines and a major bystander effect. In other words, once the destructive process begins it affects all the cells that are present even if only a few are targeted at the outset, including cells at that point that are matched either for H2 or minor histic embattability. Now I want to just tell you briefly about two major trials that were done across the street, both of which have been very informative of treatment and also give you an update on where we are. Now the first is rather banal. We did a complicated study that was intended actually to test a drug that's not shown here which turned out to be a bust. These two studies actually have told me that I don't really know how to do clinical research because I keep getting results exactly the opposite of the ones we hypothesize. Rabbit antithymocyclobulin is more potent than horse. When it came on the market in the United States it was likely, based on results for example in renal allograft rejection, transplantation in general, that rabbit would be better than horse because it was more effective and we did a study that incorporated a comparison of these two similarly named and similarly labeled antithymocyclobulins. And the reason it ended up in the New England Journal as opposed to some obscure hematology subspecialty venue is that the results were the opposite of those predicted which is that we saw the usual 60 to 70 percent response rate. In this case I think it was 68 percent at six months for the horse ATG. That's a very good response rate meaning patients are transfusion independent, good neutrophil numbers and about half of that with the rabbit ATG. The New England Journal insisted on some sort of mechanism what I think they really meant is that they wanted to see some biological difference between these two ATGs. They sound the same and the most likely difference that even though the rabbit ATG is far more potent as we expected. You see that in pink is the very dramatic reduction in total lymphocyte count with rabbit ATG compared to the more transient reduction with horse is that unfortunately that reduction is directed at CD4 cells and especially at T regulatory cells. So T regulatory cells are simply gone in patients who will get rabbit ATG for six months whereas they recover quickly to actually better than previous numbers in patients who receive horse. Now is that absolutely no, we don't know that but that's the strongest association that we were able to observe with all lymphocyte subsets, multiple cytokines and microarrays. Now the long-term effects of this effective anti-thymocycline therapy are obvious and these are the results of long-term survival curts. You see they go out for a decade or longer and patients who actually respond to that first round of immunosuppression have virtually 90, almost 100% long-term survival and that's been consistent for multiple decades. We've also improved the treatment of patients who fail that first round of horse ATG and we now have results in terms of survival in these failed patients that are not as similar from what we saw at the very outset when we were treating patients in total at NIH back in the 1980s. Most of them will be alive five to ten years later and the reasons for that are varied. I think mostly the credit of the pharmaceutical industry which has developed much more effective and easily administered antifungal drugs, our aggressiveness in giving second courses of immunosuppression which I'm not going to go into detail and also the ability to go to alternative donor transplantation patients who fail. So multiple reasons. So this is the sort of story overall, there's great improvement in overall response rates. Now what does the failure do? I actually don't think it's due to a non-immune path of physiology with perhaps rare instances and again for the generalists in the audience we know that we have patients with multiple sclerosis who can present with a single episode of optic neuropathy or some other neurologic syndrome. We call it multiple sclerosis and they're fine. We have other patients who have progressive disease that kills them in just a few years.  We have patients who present with megacol. We don't think of those as having differences in their path of physiology. We believe that that's simply a black box in terms of the immune system and differences in the target organ. So I don't think it's a question of path of physiology. We don't understand our immunological therapy as I've shown you. ATG, it's more potent, should work better. Cytoxin should work better. Adding rapamycin, adding other drugs to cyclosporine should work better. If you don't, horse ATG and cyclosporine remains the standard and gives us the best results. And I think that the more likely explanation for why some patients do not respond is that they don't have a stem cell reserve that's adequate. That's something that we felt hopeless and concerning up until recently. And I wanted, this is the simple diagram that if you don't have any stem cells you've got nothing to work on when you remove the immune factors. And the desire, of course, would be to treat patients or to shift the curve to the right where we're treating patients who have better blood counts and better stem cells at the outset. We know that stem cells are low in patients by indirect methods. This is a particular type of bone marrow and peripheral blood assay for very primitive progenitor cells. And what I want you to appreciate is that there are very reduced numbers in all patients. This is per mononuclear cell in the bone marrow. This is per volume of blood. These are severe aplastics compared to normals. If you multiply this by the 3 or 4%, which is the number of cells that are left in a plastic bone marrow, you've got 95 to 99% elimination of bone marrow stem and progenitor cells in patients where they place your shear down at the very bottom of the curve. And we know that patients who either have good blood counts to begin with or are shown here who have a complete recovery after they're treated with antithymocyclinocyclosporin do better long-term. They have better survival and they have fewer long-term complications. And this is just one of many examples suggesting that the initial stem cell reservoir reserve dictates how patients do long-term. So, we didn't know how to address this. And again, more or less accidentally, we were able to discover what we think is the first effective stem cell stimulator that's available in clinical practice. And it's this small molecule called ultrombopagin. The hematologist in the audience will have familiarity with this in the context of idiopathic thrombocytopenic perperor. It's licensed for use in patients with a refractory ITP. And my colleague, Cindy Dunbar, and I undertook a study really with the premise that we would avoid unnecessary use of this expensive agent in patients with low platelet counts. That simply having a drug that was thought to influence platelet counts didn't mean that you should use it in a patient who's thrombocytopenic as a result of bone marrow failure because other growth factors don't work very well in bone marrow. And EPO, patients have sky-high levels of either orthroponin. Even EPO doesn't do much. Nupogen, GC-SF. And we knew from our own studies over the course of several decades of just showing results from two papers that TEPO levels, thrombopoietin levels, are really quite high in patients with aplastic anemia. So, ultrombopagin should not work. Now, this also ended up in the New England Journal because the results were, again, counter to our hypothesis and expectations. We took patients who had extremely refractory aplastic anemia. It failed immunosuppression, failed other growth factors, failed male hormones, treated them with ultrombopagin. About half of them showed responses. That was totally unexpected. And even more surprising was the quality of the responses. First, they were either buy or try lineage, not just in platelets. So, that suggested a broad effect or a specific effect on stem cells. Second, they were very robust. It wasn't a matter of the platelet counts creeping up by 10 or 20,000. This shows you plate responses, some of them into the normal range in the course of six months. Hemoglobin, even more impressive. These patients who have responded are now receiving or undergoing phlebotomy to remove excess iron. I won't share the neutrophils, but they also comparably went up.  So, when we looked at six, nine, 12 months after treatment, and I'm showing you three pre and post-apairs, you see the three of these four patients actually have normal bone marrow cellulite, which we actually don't see when we treat with immunosuppression. The better the bone marrow remains hypocytum. So, this really suggested that we had shifted the stem cell number to the right and that we now had more operating stem cells in these patients. We have a current trial that's combining, which I think would be logical, combining upfront in patients with aplausic anemia, immunosuppression, and ultrombopagin. I'll just show you the sorts of results we're obtaining. This is a young Navy Petty Officer who also had coincidentally eosinophilic fasciitis. It's one of the rheumatologic syndromes associated with aplausic anemia. You see, not only that his bone marrow fills up when he's assayed in this instance that three months, but shown on the right are CD34 cells, which are also now visible, not excessively, but visible in this patient. These are the early results of this study. Very high response rate to date at six months. Ninety percent of patients have responded, and the robustness of the response is also very striking. Patients' blood counts begin to go up in the first month. They're very atypical for immunosuppression alone. This is the CD34 number. You can ignore the scientific data, but in the early stages, it looks like we're getting really remarkable increases in CD34 cells in the bone marrow, I think shown here, 30 to 40-fold increases in CD34s. And these are the blood count increases. Again, you see this very striking slope to the curves, very rapid increases in reticulocytes, platelets, and neutrophils. Now there are problems with this type of therapy that will be obvious to many of you, so I speak, which is that we are stimulating stem cells, but there's also the risk that we may be stimulating abnormal cells, or indeed, in the process of causing bone marrow cells to turn over lead to problems. So this is the big worry that we have, is that we may elicit clonal evolution, and I'll show you some data related to that as we finish. I want to make the point in this slide also that the obvious question is, why does ultrombopag work when patients have very high levels of thrombopoietin? And I think this is a feature, obviously hypothetical, a feature of the fact that it's a small molecule, it escapes binding like plasma proteins, such as thrombopoietin. There aren't other cells to compete with their receptors, there's only a few stem cells left in the bone marrow of a patient with aplastic anemia, and as a small molecule, we really can flood the hematopoietic niche with very high concentrations, and the magnitude greater than achieved with thrombopoietin and physiologic or even pathophysiologic circumstances. Now I want to return to this patient, I showed you early on, who ten years after she was successfully treated, so in clinic every year she's doing great, comes back again bleeding, now with a very malignant appearing bone marrow, blasts that you can appreciate, abnormal cytogenetics, and this is the problem of malignant or clonal evolution in patients with aplastic anemia, which is shown in our large series, occurs in about 12 to 15% of patients long term, very troubling. It's really awful to have a patient like this come back after you've had great success with them, think that they're home free, and have them come back with a virtually impossible to treat disease except for trans. So this is the problem of clonality, which is really raised by the great American hematologist Bill Damashek in the mid-20th century, put together aplastic anemia, what we would now call myelodysplasia, leukemia, and hypothesize some relationships, I and others have followed up on this problem, but we really have not understood where the problem, what the problem is, but the problem actually I think now has been solved, as to why these patients do go on to develop leukemia. And it has to do with a very fundamental aspect of cell biology, which are the telomeres, the ends of the chromosome, and with the stability of chromosomes in general. Information that's so fundamental has led to a whole series of Nobel prizes and really dates back even prior to the discovery of DNA experiments that were done in the 1940s and 50s by Mueller and McClintock, and then the later discovery of DNA led to this understanding that replication of DNA, the characteristics of replication of DNA would lead to inevitable loss at the ends of the chromosomes as a result of the requirement to produce a fragment in the lagging strand. That was not going to be feasible when the DNA replication apparatus reached the termini. There would inevitably be loss, as Alexei Levnikov called it, a marginotomy, a problem with loss of the genetic information that's transmitted from cell to cell with every replication. So nature's solution to that problem, the end replication problem, has been the telomere. The telomere really is not that complicated to structure. It's DNA, it's hundreds to even thousands of repeated hexanucleotides as shown here with specific proteins that coat these hexanucleotides. And this provides many advantages to the cell. First of all, it's nonsense material, so its loss doesn't impact on the transmission of genetic information. Second, this complex forms a stable and recognized end to the chromosome that avoids it being recognized as, for example, a DNA or chromosome fragment or a DNA virus or DNA that needs to be repaired. And the third is it provides a platform, a template for an active process of repair with every mitosis which is affected by this complex, the telomerase complex. So there's a telomere which is the end of the DNA and telomerase which is an enzymatic complex that will add on hexanucleotides at the end of every mitosis. It doesn't completely repair the loss of telomeres, but almost does. So it maintains telomere length in replicating cells. Now, telomerase is made up of two major components which I'm going to talk of. One is the enzyme itself, telomerase, encoded by a gene called TURT with a T at the end. The other is the RNA template. So this is a gene that produces an RNA that is the template for the reverse transcription to telomeres. And that's called TURC, shown here. This is TURC, and this is the product telomerase. Now, when cells get critically short telomeres, they are not able to continue to replicate. So under those circumstances they either go to sleep, undergo senescence, or they undergo apoptosis. For an organ, that's the appropriate response. If you've got a liver and a liver cell has been replicating and replicating and replicating and it reaches the ends of a develops a critically short telomere in one of the chromosomes, the cell just disappears from the population. And that's a benign phenomena as far as the liver is concerned. But if there's suppression of the DNA damage or DNA response, DNA repair responses, that's for example in artificial systems in which P53 is eliminated, those cells can continue to replicate. And at that point the chromosomes have become unstable. Those short telomeres allow end to end fusions and non-reciprocal translocations and other phenomena that result in aneuploidy and in frank malignancy. So those are the two phenomena. Now if we just take cells and put fiberblaster example with them with the cell culture, we can't culture them indefinitely. That's the Haiflich phenomena. That's due to shortening of telomeres. It can be overcome by inducing or transfecting with a telomere telomerase gene. But we haven't been very clear on what this actually means in human beings until recently. This is a laboratory but it's among many papers that have demonstrated that short telomeres and defective telomerase underlies some patients with aplasticinemia that's inherited, presents either in the pediatric population as a rare syndrome called dyskertosis, congenital, or in adults more subtly as aplasticinemia or other types of bone marrow failure. This is our initial paper and it's the number of points I want to make from this slide. This is the description of the first mutations in telomerase, in the tert gene. That's the most important component of the enzyme complex. That's the enzyme itself and that's what's regulated. First I want you to appreciate, unfortunately, that this is the pattern of telomere attrition in normals and telomeres do get short as we age. Not at our age, it's always people that are older than us up here and most of the shortening of telomeres occurs early on. If you have teenage children you can tell them they're actually aging much faster than you are. Of course, the fact that kids are growing and their organs are growing so there's a lot of replication going on. The second point is that patients who have a telomeropathy, to the extent that we know almost always will have extremely short telomeres. It's the basis of a commercial blood test. Again, this is something that can be ordered or you can send a patient over to our clinic. We have a CLIA laboratory that does telomere length. You see that wherever the mutations were in these patients they result in extremely short telomeres. This is really fun to work with because this is not a gene with some funny combination of letters and numbers and you're not really sure what the function is and there's a yeast analog blah blah blah and you think it's related to a 10% increase in Alzheimer's disease. We have an in vivo assay which is the telomere length and we even can look at telomerase activity both transcription and protein in research laboratories. Now I want to show you the spectrum of telomere disease as we see it in the clinic with some I think dramatic cases. This is again a military officer really a remarkable story. He's airlifted out of Afghanistan not because he has a blood problem but because he has tongue cancer which is a diagnosed while he's there. It comes back. He already has metastatic disease shown here in the CT scan. Now I wish I could say that I was a physician who made the diagnosis but a various dude army hematologist looked at his nails and his skin and said this looks like stuff that I heard about in medical school this is dyskeratosis congenital. So this patient we showed had a mutation in the DKC1 gene which that protein product stabilizes the telomerase complex really critical and that's what we see in children with X-linked very severe dyskeratosis congenital. So this is the pediatricians dream. This is the late presentation of a clearly pediatric disease presenting in adulthood. By the way this patient's bone marrow disappeared when he got a single round of chemotherapy so he had underlying severe bone marrow failure but it wasn't apparent at the time of diagnosis of his cancer. Now this is the other end of the spectrum here's a patient who presented to our clinic with a fairly modest history a little bit of thrombocytopenia over the course of about a decade not terribly severe. He had big red cells which is very typical and elevated MCV is a very good sign of bone marrow failure and is often seen in these patients with telomerase. He had hair that had grade when he was in his early 20s. This is a very easy thing to look for and to ask for in the clinic. It's a good ice breaker to ask a male or female patient whether they've died their hair you know they always wonder why you're intruding but in fact this is a very typical history in the patient and in the family. Not pathenomonic but suggestive of a telomer problem. This guy took a picture of his hair and we identified that he had extremely short telomeres and a mutation in Turk in our laboratory. So this is the more typical presentation of a patient with a telomereopathy and you can see why this has been missed because he's a fairly subtle hematologic manifestations. He also had cryptic cirrhosis I'm going to get to that in a moment. This is a fantastic family that we study they live about an hour and a half from NIH they're farming people and the Mennonites and Amish are terrific for genetic studies that's familiar of course to those who are listening in from Johns Hopkins. This family is big. This picture was taken after lunch when half of them had gone back to their tasks they keep excellent genealogic records. So we can trace them back at least six generations. Everyone shown in green had a novel mutation in the telomerase gene those in white of wild type telomerase. That we saw had presented he's a dairy farmer presented again with a story of very modest bone marrow problems and progressive thrombocytopenia and anemia but had a striking family history in retrospect although he didn't recognize it at the time he did indeed have gray hair it was easy to make the diagnosis when we walked into the room and saw him he had failed previous therapy given elsewhere he was transplanted by my colleague Rick Childs an excellent example of the value of genetic testing because he matched siblings one of whom was a young woman with macrocytosis who was HLA matched she had the mutation that's a disaster you don't want to actually transplant from somebody in the family as I'll show you in a moment even if they have normal blood counts and he's done quite well with really minimal problems following transplant this is the problem in families is that everyone who carries a mutation has a defect in the amount of poisons even if their blood counts are normal these are a number of family members from another family showing hypoplastic bone marrow normal blood counts but if you look at measures of a amount of poesis colony formation CD34 cell number and so on they're much decreased and the growth factors are elevated so we think that patients can maintain normal blood counts for a lifetime and some other factor immunologic presumably is what overlays and causes disease this is the same family showing a second manifestation very important to remember of telomere disease which is cirrhosis or liver failure so four women in this family ultimately suffered fatal liver failure including a young woman who died just recently who had had a liver transplant when she was 19 for liver failure had done well for 20 years and then finally succumbed I'm going to show you some of these patients but I want to stress that this is something we've not appreciated explicitly I published a paper that was in one of the lesser of the hematology journals several decades ago recognizing a relationship between cirrhosis and aplastic no idea why when we began to understand the telomere diseases I remember the patients that's already a triumph to remember the patients name I hadn't seen them in 20 years but we couldn't reach them the doctor died the phone number that we had didn't answer I finally got a phone call quite accidentally from Susan Lightman in our transfusion medicine department who said I have a young woman here who wants to donate to the National Marrow Donor Program because of the terrible story in her family and she says that you had seen her brother and her father some years ago which is the case so the story was bad I saw the young man who had very modest thrombocytopenia he progressed outside of our hands and went to transplant and died his father also had aplastic anemia he ended up with esophageal varices due to severe cirrhosis and blood to death from his gastrointestinal tract terrible story so he saw this young woman and actually she had short telomeres which was awful you know she's got a couple of kids there looks great but she did not have a telomere apathy because her genes was normal so why is her telomere short? she inherited those from her father so the father's sperm also had very short telomeres and the mean telomere length was reduced but if you can repair your telomeres work that Richard Hodes has done in the mouse and we've done in humans it appears that if you can repair your telomeres even if you start with them being short that doesn't lead to disease she has a brother who was having problems with alcohol because he had obviously the sense of being faded to have a terrible disease he also ended up having normal telomeres and I'm sorry having a normal telomerase genes so he didn't need to worry about that so we've also shown as has a German group that telomere telomerase gene mutations occur rather frequently in patients with cirrhosis independent of any prior history family or otherwise so this may be actually one of the most common manifestation of the telomeropathies which is cirrhosis often in the context of hepatitis infection or steatosis with diabetes or with alcohol so this is an underlying risk factor in about 5 to 8% of patients as in our study and in the concurrent German study now the third manifestation that you need to remember for the telomeropathies is pulmonary fibrosis this is one of the members of the Mennonite family she actually made the diagnosis of pulmonary disease at an NIH conference she turned to her husband instead of in short of breath for the last month or two and this is her CT scan and she actually died a couple of months later with a combination of pulmonary fibrosis cryptic cirrhosis which was diagnosed on liver biopsy and bone marrow failure this is the documentation of pulmonary fibrosis familial and telomerase mutations this is from our own hematology service there's a very wide range of hematologic disease that's associated with telomere gene mutations from moderate aplastognomia, MDS aplastognomia and even leukemia a few subtle points about the telomereopathies here's a family where we could not identify a mutation in TURT or TURT obviously not in DKC1 there was a female patient and it was only until we looked at the promoter region that we found a mutation in the cat shown here in the cat gene in the cat box of the promoter very important binding site for transcription factors this is actually the first pathogenic mutation in this region ever described in humans so the regulation of the telomere repair complex is complex including not only the promoter and other regulatory regions for the genes but other genes that impact on telomerase activity this is a very unusual, very striking and important example of telomere disease in the clinic got a separate disease gets an umbilical cord transplant very prolonged period of engraftment and donor cell leukemia a year and a half later this is a patient of our colleagues in the cancer institute and the umbilical cord blood that had been infused into this patient, the telomere is half of the length of normal so deficient product for reasons that are not clear all these balls represent the telomere length in that used umbilical cord blood in the patient after infusion of these cells and the patient ultimately succumbed to their donor leukemia and finally we can model this disease in inducible pluripotent stem cells as shown here a paper that's actually just appeared in JCI and showing that again in inducible in IPS you see this reduction in telomere repair that occurs in vitro as it appears to occur also in evil so this is the diagram to remember that first this disease can manifest in three organs we think that um telomere repair is the substrate on which environmental factors like alcohol or hepatitis virus for liver smoking for patients with pulmonary fibrosis and the immune system for marrow failure and the way of thinking about this keratosis congenital as it relates to the penetrant set of genes almost always resulting in disease in the first decade of life associated with skin and mucosal membrane problems and these mutations occurring more subtly in the adult population now I want to return to this patient to complete my talk with the link between telomere attrition and cancer so in this patient there was a remarkable feature which is that his father had died in Baltimore many years ago early onset myelodysplastic syndrome and acute myeloid leukemia not only was he young when he presented with this in his 30's but he had an unusual course which is that he died after a single round of chemotherapy and almost always we can get patients even adult or older patients through one round of remission inducing chemotherapy this patient died never recovered his blood counts so he also we learned on testing of archival bone marrow had the same mutation as did our patient and that led to a larger study by my colleague Rodrigo Collado showing that again in between 5 and 10% of patients with new onset acute myeloid leukemia this is non-APL we can find mutations in the telomerase gene complex again germline mutations that are the risk factor for the development of leukemia and almost always associated with chromosome abnormalities that's not expected because about 50% of patients have normal chromosomes that they present with AML when we look at our patients with aplastic anemia and ask the question is there a relationship between telomeratrician and outcome again we see this very strong link with short telomers in this instance not related to genetic mutations but simply to the pathophysiology of aplastic anemia the requirement of limited numbers of stem cells to replicate in order to compensate for stem cell loss and low blood counts so this work a couple of years ago now shows that the major risk factor for malignant clonal evolution mainly monosomia 7 is having short telomeres even in the normal range shown here so patients with the lowest quartile of telomeres about 5 to 7 fold higher rate of clonal evolution and of evolution to monosomia 7 then do those patients who have longer telomeres and we can see that in patients months to years before they manifest with chromosome abnormalities we take their bone marrow that we've stored out of the freezer and grow them with the normal growth factors those patients with short telomeres which you can see here here's just a slide of the chromosomes you see that many of these let me see if I can get that back many of these chromosomes are lacking a telomere you should have seen like double headed worms as we see on the left side with longer telomeres when we take those cells out and culture them we see abnormalities in the chromosomes in the area typing of a variety of bone marrow showing you chromosome rearrangements in their bone marrow is occurring X vivo years before they present this is more recent data showing that those patients who undergo clonal evolution have extraordinarily accelerated telomere attrition so the normal telomere loss is in the order 40 to 60 nucleotides per year in a normal person and that's also the loss that occurs in patients with stable aplastic anemia in those patients who progress it's about 5 to 10 times higher so in the order 400 versus 60 base pairs per year that's an extraordinary acceleration in telomere loss again not because we think they have mutations but because limited numbers of stem cells are attempting to compensate for the total and we can also see that at the level of the individual chromosome I'm just showing you this is an example this is a method that's called Stella or single telomere length analysis we're looking here at the X and Y chromosomes this is a stable disease this is a southern hybridization and over the course of many months you see this nice peak in telomere length no changes here's a patient who's undergoing telomere attrition which we can see in this individual chromosome that telomere gets short that telomere gets short over the course of 2 years and that is the typical pattern in patients who go to malignant transformation I'm just showing you some summary slides here I'm going to continue to do or take advantage of the ability to do the first direct comparison of genetics genomics and chromosome genomics by this telomere attrition so our patients are going on to severe pancytopenia, MDS and leukemia and there are now about 60 candidate genes some of which have just recently again been described in patients with acute leukemia and two of these patients had those 50 or 60 candidate genes asking whether mutations and comparing that with this telomere attrition that I've shown you which is a regular occurrence prior so these are the genes and this is an example of the data that we've obtained so in two of these patients indeed we did detect mutations in two of the genes that have been implicated in leukemia and MDS one of them is DNMT3A which is a gene that affects and we're present in our patients for years beforehand and apparently stable, this is a patient actually who was successfully treated with immunosuppression as a DNMT3 large clone remains stable as she goes in and out of remission, second patient shown here but in six of our eight patients there were no mutations in any of the 60 candidate genes that have been implicated in MDS and AML and in a much larger series of 30 to 40 patients with aplastic anemia we've seen no mutations in DNMT3A or the other major mutations so at least the hypothesis for now is that this progression to chromosome instability which predisposes to leukemia, to severe pancytopenia to MDS occurs independent of selective mutations in these candidate genes and as a result, not surprisingly of this accelerated telomere attrition due to limited stem cell number and that by the way would appear to explain patients who have better stem cell numbers when they present to treatment do better long term and patients who can get a complete recovery that's indirectly evidence of the fact that they've got sufficient stem cells to avoid this rapid telomere attrition so this is just telomere lengths in those patients who have now undergone L-Trombopag therapy and I think you can appreciate that in patients who have treatment, naive disease shown in red, they've got much longer telomeres in general than those patients two, three, four, five years before they get L-Trombopag and if we look at those patients who undergo clonal evolution when their bone marrow are stimulated with L-Trombopag that's shown in red, again you appreciate that they're in general at the lower portion of the telomere length and indeed the patients who are up here at the top tend to have rapid and successful reconstitution of their bone marrow shown here so this is the summary of the cancer story of course genetic mutations that are easy to look at but we also know that the major risk factor for virtually all cancers is aging and as I've shown you, telomere attrition is just a normal phenomena that occurs with aging I think is likely to underlie all of those aneuploid cancers that we see in other organs in addition to the bone marrow and it is in fact in this middle group of immune and inflammatory diseases where there's been this length that we've been looking at for well over 100 years between the state of chronic inflammation or of immune destruction and the later development of cancer that I think telomere biology actually provides the link and there are many examples everyone in a subspecialty can think of a disease like ulcerative colitis in its predisposition to colon cancer, esophageal cancer following on barits esophagitis, graft versus host disease and late cancers, many links and I think that these are explicable by telomere biology probably before any of us were born by this brilliant young guy, Teodor Bovary very sad story, he published this fantastic monograph just at the beginning of the First World War in German and it really attracted far too little attention he died a little bit after the war very sad story but he's really a brilliant brilliant book in terms of describing the importance of chromosomes and their instability I want to finish with at least one hopeful note so can we do anything about this one of our Mennonite patients holding up her handmade chart she had aplastic anemia her handmade chart of her hematocrit when she was treated with a very old fashion therapy for aplastic anemia one we know works in a subset of patients which are male hormones so here she got down and she got a decaturabalin, hematocrit goes to normal and actually stays up on decaturabalin for a decade so we've shown recently that the mechanism of action for male hormones acting on the bone marrow is almost certainly through telomerase we've not really known what the mechanism is and I think this is the most likely one so this is just the effect of a variety of sex hormones male and female on telomere activity, telomerase activity in vitro in hematopoietic cells you can also show the same thing in lymphocytes and this is the model based on many enzyme inhibition experiments it is actually through the female a sex hormone estradiol and a binding site estrogen response elements in the promoter of the TIRT gene of that critical telomerase gene that these hormones act and up-regulate telomerase and maybe that's in fact why our telomeres are stable through much of our adult life is that we've got the sustenance as a result of sex hormone activity we can even model this in animals so if you take TIRT or TIRT deficient animals we can avoid telomer length as telomere attrition as for example after transplant with limiting numbers of cells you can see the difference between experiments between animals treated with testosterone and those not if they there's no telomerase that doesn't occur here's animals that are exposed to repeated doses of total body radiation young animals, longer telomeres older animals even more striking avoiding telomere attrition so finishing we have a protocol that's up and running actually at NIH looking at long course of dinosaur inpatients who have short telomeres with or without telomerase gene mutations this just shows you the characteristics of the protocol and this is the effect on telomere length which dinosaur appears to stabilize or actually allow elongation in these patients with short telomeres and leads to hematopoietic recovery in the majority when we select these patients based on telomere length dinosaur works that's it so I want to thank of course the people who did all of the bench work and clinical work especially Rodrigo Collado Phil Scheinberg and others for putting this all together and thank you for your attention so the answer to that is actually not entirely known we don't know in humans certainly in cell lines they're well demarcated telomere lengths that are not consistent with the cell continuing to replicate and one of the problems is that it's not the average telomere length it's a single chromosome once that single chromosome develops extremely short telomeres the cell will stop replicating it'll go to sleep it induces the appropriate responses for senescence or apoptosis so it's very dependent on in that particular cell which chromosome is critically short now the reasons for instability are not known in detail but what occurs and what can be easily seen in cell culture and animal models and I think we can see it in humans is end to end fusion so without the full telomere the chromosomes just stick to each other and are dragged across the anaphase plate yeah so that's actually a very interesting question I think if it's so let me give you the simpler you've got a patient with liver disease and they're anemic I wouldn't blow that off to their in fact there often are big red cells in that setting aren't there I wouldn't blow that off to just be a secondary effect of the cirrhosis which is what's been done since I was an intern that may well be evidence of an underlying telomereopathy I think the issue of chronic inflammatory disease or of chronic anemia is more complicated because what I've argued and which I think is important in this general setting of inflammation is that there's a regenerative stress in an organ lung we don't know 73 different cell types who knows which of them is involved bone marrow is pretty easy stem cells are just chugging it out and their telomeres are getting short because there are too few of them but the other mechanism that it for example there's a very strong link between short telomeres and atherosclerotic disease cardiac outcomes and so on now is that due to telomere shortening occurring in endothelial cells possibly but the hypothesis has been that it's actually a reflection of reactive oxygen species there's just more damage to the chromosome in general and the telomere is part of the chromosome so you're going to see that being affected and be short so telomeres may indeed be short in patients with with increased reactive oxygen species as part of chronic inflammatory or tumor or some other underlying problem it's really not been looked at I mean actually it would be interesting to see we've not looked and you know it's just hard to study those patients because usually their anemia is not their major problem but that would be an easy thing for us to do or our audience beyond the audience auditorium could you bear in question what is the definition of normal length actually it depends on the age so it also depends on the assay so the assay that we use is a gene amplification sorry the question was the normal what's normal and normal depends on how old you are and what the assay is so the normal for an umbilical cord blood as you can see is much larger much higher than the normal for elderly person in their 80s or 90s but there's actually some there actually is overlap between older humans with their longer telomeres and some children with relatively short telomeres so it's not an either or so it's always age adjusted and it depends also on the assay the gold standard or the standard is a southern hybridization but it's very cumbersome to do and not suitable for screening we use gene amplification so it's a reverse PCR and we have a range of kilobases of telomere length that we calculate for any particular age there's a commercial assay that also depends on fish and flow and they also have a range of normals but it's always age dependent yes it's the length of the telomere no we have not a chance to look at telomere length in perforia actually that's really a brilliant question I mean really sometimes you get a question that just makes you think sorry so there's a brilliant question because it has to do with chloramphenicol so there's been chloramphenicol was related in the 60s and 70s to actually epidemics of aplausconemia are actually occurring in the United States so let me say first I'm not sure of that relationship but then I'll assume that there is one I'm not sure because the epidemiology is really not very good and it's unfortunately one of those examples of somebody publishes one paper with a lot of now in retrospect defects in the methodology as for example the chloramphenicol being given after the aplausconemia actually occurred but that wasn't looked at very carefully I mean it's kind of not likely to be a cause and effect so we don't actually know and when you look at if you look either in East Asia we saw no relationship with chloramphenicol in aplausconemia it's freely available, it's still widely used if you look at countries like Sweden and others where they actually monitored the amount of chloramphenicol that they imported and also had rates of aplausconemia no relationship between the chloramphenicol going up and coming down so it's still a little bit left up in the air the second point I want to make is it's often hard to make the distinction between cause and effect the story with the early graying in telomeres is pretty illustrative of that so there's a little literature about hair dye causing aplausconemia if you or your wife uses a hair dye you know that it smells bad and it looks like it should cause all sorts of diseases so it seemed logical that hair dye could be put on your head, scalp it all gets absorbed, that it could cause bad things but I don't think that that's a relationship at all I think what we were detecting were those patients who were dyeing their hair gray early and they had a fundamental genetic lesion that they were obscuring when they looked at their hair but the last point of your question the part that I think is really interesting is that chloramphenicol does have a very regular effect on the bone marrow and these were done, actually I hesitate to even describe them because they were probably unethical and you know we're not really supposed to talk about unethical studies but since having brought it up we'll finish it and then we'll say it an unnamed institution on terminally ill cancer patients in which massive doses of chloramphenicol were given to them in their terminal phases and what happened in those patients very consistently is that they developed a profound anemia so you could speculate that what chloramphenicol was doing was creating a tremendous regenerative stress on the bone marrow and that it could in fact be linked by that mechanism to bone marrow failure but that's again speculative what's the point of going on a biotic and wouldn't it be interesting to know if there are some people that are focused on that? Yeah that was actually speculated about many years ago because one of the questions there's a famous hematologist whose name I'm blocking but you may remember Wash U who went down to Colombia he had this relationship with Colombia and what he observed was that there seemed to be this outbreak of chloramphenicol related aplastic anemia and then it went away so you actually the population in chloramphenicol could continue to be given Yeah well how do we know they're longer that's the problem so the studies that have been done this is also a very critical question there are some famous people even those who have run the Nobel Prize now who are promoting this idea of getting your telomeres measured and then you'll see how long you live because there are a lot of studies that have suggested some relationship with longevity I have pretty long telomeres so I don't have a I prefer to believe that that were the case but let me tell you what my reservations are and then I'll answer your question directly the question has to do with the telomere length and very old people and centenarians for example So I think that the problem is really one of selective publication that a lot of studies that are negative or don't show a relationship really just don't appear and I'm really skeptical of this relationship cause and effect that telomeres actually cause aging and again I mentioned that there's this overlap between kids with particularly short but normal telomeres and older people, 60, 70s and 80s who have telomeres that are particularly long and actually are on the same length as children so it's kind of hard to think that that's cause and effect when you've got that sort of overlap your question though is really a hard one to answer because what is the right telomere length for a person who's 90 years old and what has been looked at are just the telomere lengths per se they are a certain number like 50 or 20 but what is striking is that they're in a very narrow range but I think that that without getting into the complexities that only reflects the fact that the homeostasis of the hematopoietic system may lead to a regression to the mean it's complicated but the idea would be that those hematopoietic stem cells with particularly short telomeres get eliminated and you now have a population that's actually barely consistent in its telomere length that's what survived in the person that's 90 or 100 but there's no optimal length because we don't know who do we compare with you have to be 100, that presumably is the right telomere length for getting to be 100 so in cryptogenic cirrhosis it's your hypothesis that it's turnover in liver cells that really drive it and they're actually quite good animal models of Dupino, Lenhard Rudolf and others that suggest that that is in fact the mechanism for that and for epatocidal carcinoma they're called taking most of the liver out of red and the week later the thing was back the liver is quite regenerative is the change in length of telomeres over time consistent or does it alter? no it does change and that may be important as the rate of change so as you saw the curve is sigmoid so the rate of change in childhood is much rapiter than it is in most of our adult life where telomeres are relatively stable until we get whatever age I am one year older yes so that's really that's the problem I think really not I'm trying to be tactful I think that's the sort of information that is very easy to no of course they're all in scientific journals but it's even hard to define what a scientific journal is so the question has to do with the relationship between nutrition what you eat and telomere length so I don't know how you actually do such a study I mean as I've shown you there's very little telomere length that changes so you go back and you ask people whether they're eating a lot of red meat versus a lot of green vegetables really I just don't believe it and I think that I think it's a diversion from all the things when these sort of studies come out and then people are making money because they're measuring telomere length and you see something on the news I mean that's the point at which you should become not just skeptical but somewhat cynical yeah it's the red wine why don't we see how well that works it does so I can't say it does but the GWASs that have been done do show a link between short telomeres in telomeres and other telomere related genes in lung cancer so it's not a relationship that holds true for all cancer so this is where I have to be careful so you can make these broad statements that you know problems either genetic or otherwise always underlie cancer and they have to do with telomeres so for example in breast cancer the literature is all over the place you know some GWASs are showing negative associations showing positive but for lung cancer the relationship is pretty consistent that the steps for telomere length actually do relate to susceptibility to lung cancer so it's not different from liver well it seems in our sample of one but long telomeres are associated with excellent lectures thank you