POPULARITY
2 episodes with cd40
2 episodes with cd40
2 episodes with cd40
4 episodes with cd40
2 episodes with cd40
2 episodes with cd40
Description: Hosts Roz and Josh are joined by Maria Alejandra Mendoza, MD to discuss the key articles of the August issue of American Journal of Transplantation. [01:57] Impact of deceased organ donor marijuana use on donor culture positivity and solid organ transplant recipient outcomes https://doi.org/10.1016/j.ajt.2024.02.022 [13:11] China Issued Rules on Human Organ Donation and Transplantation https://doi.org/10.1016/j.ajt.2024.04.002 [21:10] Advancing Mouse Models for Transplantation Research https://doi.org/10.1016/j.ajt.2024.01.006 [30:05] Differential induction of donor-reactive Foxp3+ Treg via blockade of CD154 vs. CD40 https://doi.org/10.1016/j.ajt.2024.03.033 [40:27] Total Robotic Liver Transplant: The Final Frontier of Minimally Invasive Surgery https://doi.org/10.1016/j.ajt.2024.03.030
Sometimes, we learn more from what isn't achieved than when we achieve an expected result. Such is the case for our next guest, Dr. David R. Jayne, who is the first author of Clinical and Biomarker Responses to BI 655064, an Antagonistic Anti-CD40 Antibody, in Patients With Active Lupus Nephritis: A Randomized, Double-Blind, Placebo-Controlled, Phase II Trial , a study designed to characterize a dose-response relationship between an anti-CD40 monoclonal antibody known as Bi 655064 and complete renal response (CRR) in patients with active lupus nephritis (LN). Dr. Jayne and his research team's study may not have shown this dose-response relationship, but what was gained along the way surely shows the power of the negative study!
In this week's episode we'll discuss the findings from a study exploring the combination of concurrent pembrolizumab, adriamycin, vinblastine, and dacarbazine in newly diagnosed classical Hodgkin lymphoma, learn more about the effects of targeting the CD40/CD40-ligand axis in Waldenström Macroglobulinemia, and review the findings from a study aimed at improving the bone marrow homing of CAR-cytokine induced killer cells in AML.
Most oncology drugs turn things off—Alligator Bioscience prefers to turn things on, specifically, the immune system, by way of stimulation with CD40 and 4-1BB agonists. In this podcast, Alligator CEO, Søren Bregenholt, discusses the latest clinical data for lead anti-CD40 asset, mitazalimab, as well as two distinct 4-1BB agonists in development. Additionally, he answers the critical question: How did you tame the toxicity of these signaling molecules when so many others have failed?
This week, join authors Maryjane Farr and Josef Stehlik as they discuss their Perspective article "Heart Xenotransplant: A Door That Is Finally Opening." Dr. Carolyn Lam: Welcome to Circulation On the Run, your weekly podcast summary and backstage pass to the Journal and its editors. We're your co-hosts. I'm Dr. Carolyn Lam, Associate Editor from the National Heart Center in Duke National University of Singapore. Dr. Greg Hundley: And I'm, Dr. Greg Hundley, Associate Editor, Director of the Pauley Heart Center, at VCU Health in Richmond, Virginia. Dr. Greg Hundley: Well, Carolyn, this week's feature, very interesting, xenotransplantation, where organs from other species are transplanted into humans. And it's a perspective piece. And so, we're going to get a weighted conversation from two different individuals that have a different perspective on the topic. Dr. Greg Hundley: But, before we get to that, how about we grab a cup of coffee, and start with some of the other articles in the issue? Would you like to go first? Dr. Carolyn Lam: Absolutely, Greg. Although man, that is a big hook you just gave us. Xenotransplantation is seriously, seriously, a hot topic. Can't wait to learn more. Dr. Carolyn Lam: But, for this first paper I want to talk about, well, we know that sequencing Mendelian arrhythmia genes in individuals without an indication for arrhythmia genetic testing, can identify carriers of pathogenic, or lightly pathogenic, variants. However, to what extent do these variants associate with clinically meaningful phenotypes, and what do we know about variants of uncertain significance? Dr. Carolyn Lam: So to answer this question, Dr. Dan Roden, from Vanderbilt University, and his colleagues, looked at 10 arrhythmia susceptibility genes, that were sequenced in more are than 20,000 participants without an indication for arrhythmia genetic testing in the eMERGE III study, which is a multi-center prospective cohort. Variants, previously designated pathogenic, or likely pathogenic, were identified in 120 individuals, or 0.6% population. And electronic health records revealed an over-representation of arrhythmia phenotypes. Some variants of uncertain significance were also found in individuals with arrhythmias and patch clamping, confirmed reclassification, to likely pathogenic. Dr. Greg Hundley: Really interesting results from this eMERGE III study, Carolyn. So what's the take home message? Dr. Carolyn Lam: As genetic testing becomes more common, the combination of electronic health records and in vitro testing, will help classify variant pathogenicity. Population screening has the potential to identify patients with undiagnosed Mendelian rhythm disorders. However, we need to consider the pros and cons of such an approach. And this is discussed in an accompanying editorial by doctors, Walsh, and Bezzina, and Wilde, from Amsterdam University Medical Center. Dr. Greg Hundley: Very nice, Carolyn. Well, my first paper comes to us from Professor Karl Heusler from the University of Wurzburg. Carolyn, this study was a pre-specified analysis of the anticoagulation using the direct factor Xa inhibitor, apixaban, during atrial fibrillation catheter ablation comparison to vitamin K antagonist therapy, or the AXAFA–AFNET 5 trial. And it randomized 674 patients with atrial fibrillation, in a one-to-one fashion, to uninterrupted apixaban, or vitamin K antagonist therapy, prior to first time ablation, with a goal to assess the prevalence of magnetic resonance imaging detected ischemic brain lesions, and their association with cognitive function, three months after first time ablation, using the continuous oral anticoagulation in patients with paroxysmal atrial fibrillation. Dr. Carolyn Lam: Huh. Nice. So what did they find, Greg? Dr. Greg Hundley: Right, Carolyn. They found that brain MRI detected chronic white matter damage, as well as, acute ischemic lesions, were frequently found after first time ablation for paroxysmal atrial fibrillation, using uninterrupted oral anticoagulation. Including, 27.2% of those receiving apixaban, and 24.8% of those receiving the vitamin K antagonists. So Carolyn, no difference there. MRI detected acute ischemic brain lesions were not associated with cognitive function at three months after ablation. And then, Carolyn, the lower Montreal Cognitive Assessment scores, both before and after ablation, were associated with older age only, highlighting the safety of atrial fibrillation ablation on uninterrupted oral anticoagulation. Dr. Carolyn Lam: Oh, thank you, Greg. Well, my next paper talks about basilar artery occlusion, which we know is a devastating condition without definitive evidence to guide treatment. Now, while we do know that faster treatment times with endovascular therapy is associated with better outcomes in the anterior circulation of the brain. What about this relationship for basilar artery occlusion? See? So that's the question that this paper sought to answer, and it's led by Dr. Smith from University of Calgary in Alberta, Canada, and colleagues. They used individual level patient data from the Get With The Guidelines-Stroke nationwide US registry, prospectively collected from January 2015 to December 2019, and identified 3015 patients with basilar artery occlusion treated with endovascular therapy. Dr. Greg Hundley: Ah, Carolyn. And so what did they find here? Dr. Carolyn Lam: So, here are the results. Treatment of basilar artery occlusion with endovascular therapy, within six hours of last known well, is associated with better outcomes, compared to treatment after six hours. Including, lower odds of mortality and higher odds of reperfusion, independence, and discharge home. Dr. Carolyn Lam: There was a non-linear association between, faster treatment with endovascular therapy for basilar artery occlusion, and better outcomes, with the greatest per hour improvement in outcomes seen within six hours of the last known well. In summary, results indicate that, faster treatment with endovascular therapy may improve outcomes in basilar artery occlusion. Efforts should therefore be made, to optimize workflow, including pre-hospital, inner-hospital, intra-hospital processes, to achieve rapid treatment with endovascular therapy in acute stroke with basilar artery occlusion. Dr. Greg Hundley: Very nice, Carolyn. Well, my next paper comes to us from the world of pre-clinical science. And Carolyn, as we know, pulmonary hypertension can be caused by chronic hypoxia, leading to hyperproliferation of pulmonary arterial smooth muscle cells, and apoptosis-resistant pulmonary microvascular endothelial cells. And then, upon re-exposure to normoxia chronic hypoxia induced pulmonary hypertension in mice, is reversible. So in this study, the authors led by Dr. Christine Veith, from Justus Liebig University in Giessen, aimed to identify novel candidate genes involved in pulmonary vascular remodeling, specifically, in the pulmonary vasculature. Dr. Carolyn Lam: Ah, a very interesting and important topic. So what, or how, did they do this, Greg? Dr. Greg Hundley: Right, Carolyn. So following a microarray analysis, the investigative team assessed the role of secreted protein, acidic, and rich in cysteine, or SPARC, using lung tissue from idiopathic pulmonary arterial hypertension patients, as well as from chronic hypoxic mice. In this experiment, the mice were exposed to normoxia, chronic hypoxia, or chronic hypoxia with subsequent re-exposure to normoxia, at different time points Dr. Carolyn Lam: Okay, so what were the results? Dr. Greg Hundley: Okay, Carolyn, the big drum roll. So the microarray analysis of the pulmonary vascular compartment, after laser micro dissection, identified SPARC as one of the genes down-regulated at all reoxygenation time points that were investigated. Intriguingly, SPARC was vice versa, up-regulated in lungs, during development of hypoxia induced pulmonary hypertension in mice, as well as in idiopathic pulmonary hypertension. Although, SPARC plasma levels were not elevated in pulmonary hypertension. Dr. Greg Hundley: Transforming growth factor, or TGF-beta 1, or hypoxia induced factor to a signaling pathways, induced SPARC expression in human pulmonary arterial smooth muscle cells. In loss of function studies, SPARC silencing enhanced apoptosis, and reduced proliferation. And so Carolyn, in conclusion, these authors provide evidence for the involvement of SPARC in the pathogenesis of human pulmonary hypertension, and chronic hypoxia induced pulmonary hypertension in mice, most probably, by affecting vascular cell function. Dr. Carolyn Lam: Wow. Thanks for that, Greg. Well, let me give a tour of what else there is in today's issue. There's a letter from Dr. Ng on could cardiologists support, improve, the cardiovascular risk of GnRH agonists. There's a Case Series, by Dr. Blumer, on [entitled] Hemophagocytic Lymphohistiocytosis Associated with Endocarditis: A Case Years in the Making.” There's a Perspective piece by Dr. Hillis on [entitled], Is Asymptomatic Severe Aortic Stenosis Still a Waiting Game?” Dr. Greg Hundley: And Carolyn, from the mailbag, we have a Research Letter, from Professor McFadyen entitled, Inherited Thrombophilias are Associated with a Higher Risk of COVID-19 Associated Venous Thromboembolism, a Prospective Population Based Cohort Study. Dr. Greg Hundley: Well, now onto that perspective and discussion from two viewpoints on xenotransplantation. Dr. Carolyn Lam: Xenotransplantation. Cool. Let's go. Dr. Greg Hundley: Well welcome everyone, to this feature discussion. And today, we're taking a little bit of a, different tact, and we are going to discuss a perspective piece. As you know, usually we will discuss an original article, but we have a perspective. And we have with us, the two authors that created this perspective. Dr. Jane Farr from UT Southwestern, in Dallas, Texas, and Dr. Josef Stehlik, from University of Utah. Welcome to you both. Dr. Greg Hundley: And listeners, our discussion today is on cardiac xenotransplantation, taking a heart from another species and implanting it in a human subject. So Josef, we'll start with you. Could you tell us a little bit about the history of cardiac xenotransplantation, and what are some of the obstacles that have to be overcome, if we're considering performing this procedure in a patient? Dr. Josef Stehlik: Greg, thank you for that question. The concept of xenotransplantation has been around for a long time, with the biggest attraction being, a large and ideally safe source of organs for our patients. As far as cardiac xenotransplantation, the first human art xenotransplant was done in 1964, in a man with terminal heart failure, who received a chimpanzee heart at the University of Mississippi. Dr. Josef Stehlik: The patient didn't survive the surgery, and the way it was done back then, brought up a number of ethical issues, and other issues as well. And so, the next xenotransplant was not done until 1984, in a neonate with hypoplastic left heart syndrome, at Loma Linda University. You might have heard the term, Baby Fae, before. And this infant survived about 20 days, and so we couldn't consider it, long term success. However, these two first xenotransplant brought up some important issues that would be studied for years to come. And I think, that the biggest lesson was that, the intra-species immune barriers were a formidable obstacle, and that really, new technologies, and then new medications, would probably have to come into the clinical arena, before we could do it again. Dr. Greg Hundley: Very nice. Well listeners, now we're going to turn to our second author on this particular paper. And Jane, can you describe some of the circumstances pertaining to this most recent cardiac xenotransplantation? What transpired, and what's been the outcome with that individual? Dr. Maryjane Farr: Thanks, Greg. And thanks for having us here on this program today. So the circumstance around this particular groundbreaking transplant was such that, there was a critically ill patient. This man who was in cardiogenic shock. Both sides of his heart were not working. He was on life saving temporary mechanical support with VA ECMO. And he unfortunately, despite his cardiogenic shock, he was not eligible for standard allotransplantation. Dr. Maryjane Farr: Part of that story was really about, not meeting standard criteria for organ transplantation, probably just about anywhere, in terms of a long history of, maybe not taking his meds, or taking care of himself. And there's, certain criteria that he didn't fit into. And he actually had been assessed, as I understand it, by a number of programs, before the University of Maryland approached him with this possibility. Dr. Maryjane Farr: One other option that could have been taken, was a mechanical circulatory assist device. But as I say, both sides of his heart were not working, and so really, total cardiac replacement was really his only option. Dr. Greg Hundley: And so Jane, do we know anything about what happened? How did the surgical procedure go? Do we know anything about the outcomes? Dr. Maryjane Farr: This is of course, patient privacy. So what we know is really, what's in the public arena. And it's actually, there's been a lot of transparency, which has been terrific, by the patient, and the family, and the doctors, because this is such groundbreaking information. But this patient was truly critically ill. There was some paperwork done to try to get FDA approval for emergency experimental surgery, with xenotransplantation. And of course, all the research at University of Maryland, and in many other centers, nationally, and internationally, have been done over the years. And so finally, there was an approval to do this, and it was basically a scheduled surgery. Dr. Maryjane Farr: And as I understand it, it went just like any other transplant surgery. There was obviously, a procurement team for the genetically modified pig. There was cold storage of the device. Transport, at least as far as to the next operating room, or however it went. And then, standard implantation, and release of cross clamp, and perfusion. And at least by what you can read about, the heart started to work almost immediately. And then of course, I think that's the easy part. It was really all the intense and multi blockade immunosuppressive therapy, which is really, the challenge of this type of therapy. Dr. Greg Hundley: Very nice. Well, Josef, Jane's alluded to this a little bit, but who would be a candidate for this therapeutic, this form of therapy? Dr. Josef Stehlik: Greg, so that's an excellent question. And I would like to address it. Before I do that, maybe we should also mention, very briefly, a little bit of the science behind the genetically engineered pig, that Jane mentioned. Dr. Josef Stehlik: There were three main things that have been done, and what enabled that is gene editing. And here, I would like to actually mention Dr. Mario Capecchi, who received a Nobel Prize in 2007, for his groundbreaking work at the University of Utah, by describing mouse gene knockout. That has been part of what has been used for engineering, of course, in newer approaches, like CRISPR. Dr. Josef Stehlik: Some of the things that have been done is that, the highly antigenic carbohydrates that pigs have on their cell surface, have been edited out. There have been genes that have been edited out and in, connected to coagulation and compliment, to prevent clotting and bleeding in the organ and the recipient after transplant. Dr. Josef Stehlik: And of course, one thing that it's very relevant also to our COVID pandemic, there has always, with xenotransplantation, been a question. Could there be trans-species infection? And pigs do have endogenous retroviruses that are parts of their genome, and those have been edited out as well. And so in this way, some of the previous obstacles have been removed. Dr. Josef Stehlik: So to your question, who might be a candidate? And I absolutely agree with Jane, that in the first step, it should really be patients who are not candidates for other clinically approved approaches, like allotransplantation from human donors, or mechanical assist, that can be durable, and those are the characteristics that the patient met. And I think, the next patients that will come now, hopefully, will probably be in the same category. Dr. Josef Stehlik: Now, I believe, and again, this is a little bit of a speculation, that the next step will be patients who are not eligible for transplant, but who may be eligible for durable ventricular assist devices. And our goal will be to show, that survival and quality of life after xenotransplantation can approach survival and quality of life, on LVADs. And of course, LVADs are evolving, as well. Dr. Josef Stehlik: And then, to some degree, it might be the choice of the recommendation of the team, of the multidisciplinary team. What is the best match for the patient? And to some degree, I think patient preference, to really share decision making in patient preference. Dr. Josef Stehlik: And in the next step, I believe, that's what we are hoping for, that at some point, we will achieve is that, xenotransplant will rival the outcomes of human allotransplantation. And so, that will be probably, the next group of patients. How long this will take is to be seen. But I think, that it addresses your question, who could be the candidates for xenotransplant in the future? Dr. Greg Hundley: Very good. And Jane, Josef was touching on a topic here. How do the anti-rejection treatments differ in xenotransplantation, as compared to allograft transplantation? Dr. Maryjane Farr: And so, that's been the thing for all these decades. And so, the first thing is, genetically engineered xenotransplant organs, that can mitigate some of the anticipated xenoantigenic responses. Dr. Maryjane Farr: So first, these carbohydrates that we do not see, so they are foreign to us, so there can be acute fulminant rejection. So that's, one step, and the gene knockout can take care of that mostly, but not completely. And then there's humeral rejection, and then, cellular rejection. Dr. Maryjane Farr: The cocktail that gets put together for a xenotransplant includes, some of the things that we standardly use, like steroids, ATGAM, or antithymocyte globulin, which is a generalized T and B-cell depleting therapy. What's nuanced, and there's also some role for anti-CD20 B-cell therapy, but what it is nuanced in xenotransplant is anti-CD40 monoclonal antibody therapy. And that was specifically developed, and then studied in heterotopics, or non-human primate pig transplant. Because what turns out is that, the robust T-cell responses, by what's called the indirect pathway, really requires significant costimulatory blockade, where anti-CD40 therapy has been critically important, and well studied by these scientists and others at the University of Maryland, and elsewhere. Dr. Maryjane Farr: And as I understand it, anti-CD40 was really, is the basis, the backbone, of this therapy. And then there's one last thing. And that is, temsirolimus, which is a pro drug of proliferation signal inhibitor therapy, that we standardly use in transplant. That's utilized to arrest the further growth of the xenotransplant. So that sounds like it's the cocktail, and there's some published reports, on these scientists using just such cocktail in their non-human primate transplant models. Dr. Greg Hundley: Well, listeners, we've heard a really interesting story here. But now, let's ask these experts, first, Josef, and then, Jane. Josef, moving forward, what are the concerns that you really see in this aspect of research? Dr. Josef Stehlik: Greg, I think, one of the issues that will have to be addressed, are ethical considerations. And we've seen, that after the news of xenotransplant was made public, there has been a lot of discussion among public about ethics of xenotransplant. I think it will be important to really proactively address that. Dr. Josef Stehlik: One aspect from the past is, we knew that primate xenotransplant have not been embraced by the public, just because of the closeness of primates to humans. I think, some of that will be mitigated, now that we are using pigs. But of course, there are many who feel strongly about humane treatment of animals. And so I think, regulation will need to be established that will address that, and that will make both the professionals and the wary public, comfortable with this approach. Dr. Josef Stehlik: And another thing that will need to be addressed, and Jane talked about it a little bit is, what parts of care for xenotransplant will be different from human allotransplant. Right? So how do the assessment of the biopsies differ? Right? We'll probably have a new grading scheme looking at xenotransplant. Should the antimicrobial prophylaxis be different? So we do prevent the possibility of trans-species infections we haven't seen before, et cetera. So there would be a lot of work for the transplant teams to do, as well. Dr. Greg Hundley: And, Jane. Dr. Maryjane Farr: Yes. One thing that's hard, this is amazing science, and this is a huge opportunity to transplant more patients, many of whom die on the wait list every year. But what really needs to be understood also, as we move into this area, and this is where us, as clinicians, get involved in some of these conversations in particular, is that this patient actually wasn't eligible for transplant. And these are very, very difficult decisions that centers are tasked to make. Dr. Maryjane Farr: It can get really tricky, and there's lots of patients who say, "Okay, I'm not a transplant candidate.", because of this or that, or the other reason. And there's, some reasons that are more important than others. They'll say, "Transplant me anyway. Give me a heart that you might turn down. Just give me a chance." And we don't do that. And insurance companies don't pay for that. And we have to actually find a way to be rational in our approach. Dr. Maryjane Farr: But truly, acknowledging that, if we had more resources, we could probably expand transplant even with the organs that we do have, because we turn down about, probably about 40% of organs, and maybe even more, every year, because we want to match the best organs. So it's really important that xenotransplant, in centers that can do this, demonstrate that this therapy works, and it provides a good quality and quantity of life, for at least, to be reasonable. And once you get there, then you can start to talk about, whether you need to think about allocation, and all that. So you can see how the conversation's going to go on for the next 10 years, about how this fits in. Dr. Greg Hundley: You both alluded to the fact, we need more research. And so, incrementally, for maybe each of you in 30 seconds. What do you see as the next research study that needs to be performed in this space? First, Josef, and then again, Jane. Dr. Josef Stehlik: That's a tough question, but I'll try to address it. I think, it will be a little bit in parallel to the first human allotransplant. Now that we've figured out the procedure and the organ that we can use, I think, it will be research focused on the care of the transplant recipient. And the task, number one, will be to identify immunosuppression that will be safe and effective, to protect this heart from dysfunction for many years after transplant. Dr. Greg Hundley: And Jane? Dr. Maryjane Farr: Yeah. You need to do a case series. The handful of centers in this country, and maybe the world, but I only know about this country, that have been studying and working towards this day, should take the lead. University of Maryland has taken the lead, and there are other centers who have been thinking hard about this, and preparing for this time for a long time, and they should lead the way, and try to do this with all the expertise that they've already built. And then as time passes, we can see what their outcomes are, and then we can start to think about, should there be a randomized controlled clinical trial? What should we compare it against? Who should be offered the opportunity? But at first, we need to find that there's safety and efficacy in the patients that are selected, and also, they themselves select, to go through this operation and therapy. Dr. Greg Hundley: Well listeners, we want to thank Dr. Jane Farr and Dr. Josef Stehlik, for providing their perspective on a recent procedure, involving the xenotransplantation of a genetically engineered porcine heart, into a human subject with advanced biventricular heart failure, that was not well suited for human heart allograft transplantation. Dr. Greg Hundley: Well, on behalf of Carolyn and myself, we want to wish you a great week, and we will catch you next week On the Run. Dr. Greg Hundley: This program is copyright of the American Heart Association, 2022. The opinions expressed by speakers in this podcast are their own, and not necessarily, those of the editors, or of the American Heart Association. For more, please visit ahajournals.org.
In this episode we talk with Richard Hull the Founder and CEO of Pongalo, formerly know at Latin Everywhere. Rich Hull is an American media and entertainment executive, and film and television producer, who's companies control one of the largest libraries of Spanish content in the world.
In this episode we talk with Hollywood producer Lori McCreary. Lori is best know for producing the movie Invictus, and the TV series Madam Secretary. With her Data Science background from UCLA, Lori has been a pioneering the movement of bring an analytical approach to Hollywood film. Creative Disruption Podcast with Ricky Ray Butler and Derral Eves Insights and stories from leading creators, writers, producers and marketers on how the worlds of advertising, entertainment, and data science are converging.
My Set From Chris Chettys Birthday Raid Train! Presented By: ZEN Ajax! Enjoy!
My Set From Chris Chettys Birthday Raid Train! Presented By: ZEN Ajax! Enjoy!
Hey Watch Fans,Kev's Back! He wants to talk Casio! This is a brand that always can challenge how we perceive horology. Regardless, how we felt before about this brand...we will now use words like innovative, tech, and nostaglia to describe Casio. Here are some of the watches we discuss: AQ500 a cool hybrid Casiotron their first digital watch C80 and CA53W calculator watches CD40 and CD80 databanks AT-552 and TC-600 true touchscreen calculator watches GSTB 100GC - 1A Kevs new favourite two tone FKT100L first radio wave watch GB - 6900 their first blue tooth watchEnjoy the show,Follow Kevin @WatchFanatics.ca on InstagramFollow David @watchfanaticdavid on InstagramLike WatchFanatics.ca on Facebook Subscribe to www.watchfanatics.ca
This month on Episode 18 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the October 23 and November 6 issues of Circulation Research. This episode features an in-depth conversation with Drs Eric Boilard from the Université Laval in Quebec, Canada, and Fadila Guessous from Mohammed VI University of Health Sciences in Casablanca, Morocco regarding their study titled Platelets Can Associate with SARS-Cov-2 RNA and Are Hyperactivated in COVID-19. Article highlights: Feng , et al. No Contribution of EMPs to Endothelium Lin, et al. Step Count and Predicted CVD Risk González-Hernández, et al; Sox17 in Developmental Coronary Arteriogenesis Khawaja, et al; HIV Antiretrovirals Alter Endothelial Activation Dr Cindy St. Hilaire: Hi, welcome to Discover CircRes, the podcast of the American Heart Association's Journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh and today, I'll be highlighting four articles selected from the late October and early November issues of Circ Res. Dr Milka Koupenova, who is a platelet expert at the University of Massachusetts Medical School, will join me to interview Drs. Eric Boilard from the Université Laval in Quebec, Canada, and Fadila Guessous from Mohammed VI University of Health Sciences in Casablanca, Morocco. They're here to discuss their study, Platelets Can Associate with SARS-CoV-2 RNA and are Hyperactivated in COVID-19. Dr Cindy St. Hilaire: The first article I want to share is titled No Evidence for Erythro-Myeloid Progenitor-Derived Vascular Endothelial Cells in Multiple Organs. The first authors are Teng Feng and Zibei Gao, and the corresponding author is Hui Zhang from ShanghaiTech University in Shanghai, China. Dr Cindy St. Hilaire: In the early stages of mammalian embryogenesis, a population of cells called mesoderm-derived angioblasts, gave rise to self-renewing intraembryonic endothelial cells, that go on to populate the entire vasculature of the growing fetus. Recent studies have suggested that there is an additional embryonic source of vascular endothelial cells that is derived from erythro-myeloid progenitor cells, also called EMPs, that are found in organs such as the liver, the lung, the heart, and the hindbrain. Dr Cindy St. Hilaire: Evidence of an EMP cell source for vascular endothelial cells stemmed from the use of mirroring cell lineage tracking models. However, the authors of this study argue that these former conclusions were based on flawed genetic tools. To mark erythro-myeloid progenitor cells, the initial study had used a set of supposedly EMP-specific transgenes. However, transgenes are notoriously leaky, meaning, that cells other than EMPs may have been marked. To more accurately mark EMP derived cells, this study used a reporter that they integrated into the endogenous loci of a gene, CSFR1, which is specifically expressed in EMPs. Using this model, they failed to find any evidence of EMP derived vascular endothelial cells in any organ, thus they consider it unlikely that EMPs give rise to vascular endothelial cells. Knowing the true origin of vascular endothelial cells is essential, as it informs the design, development of treatments, and regenerative medicine approaches for vascular diseases. Dr Cindy St. Hilaire: The second article I want to highlight is titled Association of Habitual Physical Activity with Cardiovascular Disease Risk. First author is Mayank Sardana and the corresponding author is Honghuang Lin, and they're at Boston University School of Medicine. Everybody knows that exercise is very good for overall cardiovascular health and current government guidelines suggest a minimum of 150 minutes of moderate to vigorous exercise per week is necessary to keep hearts healthy. There's an ever-growing interest in the use of smart watches and fitness trackers to assess an individual's activity, and many people believe that 10,000 steps a day is a good health goal. However, it's unknown whether the data obtained from smartwatches actually aligns with cardiovascular health prediction. Dr Cindy St. Hilaire: To address this gap, this study recruited participants from the Framingham Heart Study to wear Apple smart watches for a month. The participants had also undergone a 10 year atherosclerotic cardiovascular disease risk assessment based on their age, sex, cholesterol levels, blood pressure, diabetes diagnosis, and smoking history. The step count and activity data from 903 participants found that for every 1,000 average daily step increases, there was an associated 0.18% reduction in predicted cardiovascular disease risk. This association was weaker but still significant after adjusting for body mass indices. The authors conclude that like other measures of activity, those recorded by smart watches are correlated with better cardiovascular health. Dr Cindy St. Hilaire: The third article I want to share is titled, SOX17 Controls Emergence and Remodeling of Nestin-Expressing Coronary Vessels. The first author is Sara González-Hernández and the corresponding author is Joan Isern, and they're from Centro Nacional de Investigaciones Cardiovasculares in Madrid, Spain. The exact signals and mechanisms that regulate coronary vascular development are not fully characterized, yet defining these pathways could provide valuable insights into both life threatening congenital coronary abnormalities as well as the neovascularization process that occurs after myocardial injury. To examine coronary vasculature development in more detail, this team created a transgenic reporter system, which used an enhancer region within the Nestin gene to specifically label mouse coronary artery endothelial cells. Dr Cindy St. Hilaire: Cells were isolated from developing embryos at stages of vessel sprouting and vessel remodeling, embryonic days 13.5 and 17.5 respectively. And transcriptional differences were assessed between coronary and endocardial endothelial cells. They found that the transcription factor SOX17 was more highly expressed in these coronary endothelial cells compared with the endocardial endothelial cells, and that expression of SOX17 increased between these two time points that hearts were collected. Coronary specific deletion of SOX17 in genetically engineered mice lead to severe defects in arterial remodeling, confirming the transcription factor's role in coronary vessel development. The team suggested that perhaps SOX17 could be a future therapeutic target for conditions, where promoting coronary artery regeneration or remodeling might be of clinical benefit. Dr Cindy St. Hilaire: The last article I want to share with you before we switch to our interview is titled, HIV Antivirals Affect Endothelial Activation and Endothelial-Platelet Crosstalk. The first author is Akif Khawaja, and the corresponding author is Michael Emerson from the Imperial College, London. Infection with HIV is not the death sentence that it once was. With a regime of continuous anti-retroviral medications, the disease can be managed for the long term. Now that people with HIV are living longer, it is clear that they are at a greater risk of developing cardiovascular disease, possibly due to the off-target effects of these drugs. One HIV treatment, Abacavir Sulfate or ABC, was found to cause a 90% increase in the relative risk of myocardial infarction. ABC has been proposed to cause endothelial dysfunction, however a mechanism by which this may occur has not been established. To see if anti-retrovirals effect endothelial cell function, this group tested three drugs; ABC, as well as tenofovir alafenamide or tenofovir disoproxil fumarate, TAF and TDF respectively, on human endothelial cells in culture. Dr Cindy St. Hilaire: They found that treatment with ABC, but not TDF or TAF, caused endothelial cells to produce more cell adhesion protein, ICAM-1, and also tissue factor, both of which promote blood clotting. Endothelial-derived microparticles, or EMPs, are a biomarker of vascular dysfunction, and these were produced in the ABC treated cells, not in the TDF or TAF treated cells. These EMPs also promoted the increase of ICAM-1 and tissue factor expression, as well as the activation of platelets which can induce blood clotting. These results suggest that altering HIV treatments to avoid or minimize endothelial damage could help to reduce the cardiovascular disease risk in HIV patients. Dr Cindy St. Hilaire: Today, I'm excited to have with me Drs. Eric Boilard from Université Laval in Quebec, Canada, Fadila Guessous from Mohammed VI University of Health Sciences in Casablanca, Morocco, and they are here to discuss their study, Platelets Can Associate with SARS-Cov-2 RNA and Are Hyperactivated in COVID-19. Also, with me today is Dr Milka Koupenova from the University of Massachusetts Medical Center in Worcester Mass, and she's an expert on platelet virus interactions, and she also wrote the editorial that's accompanying this article in our November 6th issue. So thank you, everyone, for joining me today. Dr Eric Boilard: Thank you for inviting us. I look forward to reading the editorial. Dr Milka Koupenova: I hope you like it Eric. Dr Cindy St. Hilaire: Yeah, we're spanning a lot of time zones and we're actually having a little bit of technical difficulties so hopefully Fadila is going to be able to pop in. We can certainly see her on our Zoom call right now. But with that, we're many months into this pandemic now and it is blatantly clear that COVID does not just present with pulmonary symptoms. Many organ systems can be affected, such as the circulatory and neurological systems, but one thing that connects all the parts of the body is the blood system, and before we get into the details of the study, I would love to hear how this collaboration between Quebec and Morocco happened. And then, could you summarize the major findings? Dr Eric Boilard: We both, in Morocco and in Quebec, we had pretty much the same working hypothesis that platelets may be contributing to the overwhelming inflammation in COVID-19 and why. As you know, in Morocco the pandemic hit before us in Canada, and they had a committee that studied patient samples and studies were going on. Whereas us in Quebec, in fact to be frank, in Quebec City, we didn't really have the first wave even. So we were very excited to evaluate our hypothesis but we had no clues other than the actual virus. We actually were working with the virus and human platelets from healthy individuals, but no samples from patients. Clinicians in Morocco were very willing to contribute and to perform working on patients so that's how Younes Zaid, the first author of the manuscript, and Fadila then contacted us given our past work on platelets and viruses and the collaboration was initiated. Dr Cindy St. Hilaire: That's wonderful. I love hearing about these multi-institute collaborations. Team science is really the future. I think it really elevates everything. Dr Eric Boilard: Despite the fact that the distance and I mean, it was through Zoom, but we've been doing weekly meetings with the two labs since March. Dr Cindy St. Hilaire: So, can you maybe give us a quick summary of the major findings of your study? Then we'll dig into the details of it. Dr Eric Boilard: Sure. When we actually started the work, what we knew about COVID-19 was that the severity was likely due to the overwhelming inflammation due maybe to a cytokine storm, so we knew that. It was reported that patients with COVID-19 had a lower platelet count and although thrombocytopenia in the patients was very mild or modest, and did not require transfusion, at that time, our hypothesis was that platelets could contribute to inflammation and therefore could release cytokines and other molecules from their granules in COVID-19. So it's only a couple of weeks after that became more of use that thrombosis and cardiovascular manifestations could also contribute to morbidity and mortality. Dr Eric Boilard: Our design was to look at platelet activation in vitro. So there were studies where we performed aggregation assays with platelets from COVID-19 patients, some were severe and non-severe. There were assays where we actually evaluated a number of molecules and plasma from the patients. We looked at granular components, like PF4 and serotonin, that were greatly increased in COVID-19, both in non-severe and severe patients. We looked at vesicles, or microparticles, released from platelets that were also increased, and cytokines. We look at up to 42 cytokines, if I remember well, in the plasma of the patients and we also look at these cytokines in the platelets from the patients and we found less cytokines in the platelets when there was inflammations, which may suggest that platelets have released their cytokine cargo. I mentioned that platelets were hyperactivated so they bound better on collagen and they aggregated faster with suboptimal concentration of thrombin. Dr Milka Koupenova: I find this dysregulation of cytokines in the platelets specifically very interesting and the fact perhaps that you see that certain cytokines are decreased in platelets and increased in plasma, basically can suggest that maybe platelets are the ones that are contributing to the overall cytokine storm, as you said. My question for you is, what do you think is the contribution of platelets to the dysregulated immune cell response in immune cell activation during this particular infection? Dr Eric Boilard: That's a good question. There are studies that show that platelets could interact with leukocytes. The leukocytes migrate to organs through lungs in this case, so one possibility is that molecules derive from platelets and cytokines but also their interaction with leukocytes could further promote their migration to lungs or adhesion to vasculature. There are studies that suggest that NETs form in COVID-19 and could contribute thrombosis and NETs can implicate interaction between platelets and neutrophils. Or molecules derived from platelets that are neutrophils so these might be some of their contributions. Dr Milka Koupenova: You are reporting changes of IL-7 in platelets which is particularly interesting because they're responsible for T and B cell development and activation. I was wondering if you could comment on that particular fact and how that may be practically responsible for these responses in these patients that come and present with symptoms. Dr Eric Boilard: Yeah, it's a good question. If we look at the different cytokines that were in platelets, we found that many were reduced. That pointed to their release in COVID-19 and some we had expected, such as some broad factors and there was TGF, CD40 ligands inside the cytokine but we expect that its stored in platelets that was released. That made sense to us. IL7, like you said, it was not really reported in platelets and when we look at the 40... broad area of cytokines using the multiplex, that increased in platelets during COVID-19. So this one was increased. Does it mean that platelets translated but some of it is released but some remain in platelets? We have no explanation for this. Does it impact lymphocytes or B cells? Dr Fadila Guessous: If we go further and in terms of seeing the front they know what was downstream, IL7 production in general does not look like for psychometry, what happens to B cells and T cells in our complex. So we just look at the panel of the 48 cytokines, but we didn't go really further. Dr Cindy St. Hilaire: I kind of have a little bit of a more basic question regarding cytokines and where they're coming from. I'm going to guess it's definitely not known for COVID but in maybe any viral infection that can lead to a cytokine storm, is it known what the relative contributions of cytokines are from the platelets, from the leukocytes, and from inflammatory cells? Is there any evidence of which is the bigger player? I mean I know all the different camps have ideas, but has anyone looked at that? Dr Fadila Guessous: We were talking about this actually with Eric. If I had to rephrase your question, like what's the starting point, right in terms of cytokine storms? Dr Fadila Guessous: We don't know. We don't know because when it started in China, everyone was talking about the big cytokine storm. Everyone was talking about monocytes, about neutrophils, so on and so forth but the platelets we were just saying there was thromboembolism in the lungs, right? But no one was talking about cytokine production by the platelets. I think we were the first ones, Eric please correct me if I'm mistaken, we were the first ones to show, when we started, to show that there was the cytokine release by platelets. How big of the extent of the contribution of platelets to the cytokine storm, we don't know, but we have to figure out first what's the key player? The first one to respond, right? Dr Cindy St. Hilaire: Lots of projects. Dr Fadila Guessous: Well the big question, actually we have this big contribution in terms of the cytokine release and activation of platelets, but is it the starting point? We don't know. Dr Cindy St. Hilaire: I guess related to that, you noted that you saw very little ACE2 expression in the platelets which most people appreciate is the entry mechanism of the virus into the platelets. Can you discuss that a little bit more, maybe explain it a little bit more and what that might mean? Your findings versus what other people have seen versus potential other means of entry? Dr Eric Boilard: Yeah sure. As you mentioned, ACE2 is the putative receptor for the virus so that made sense to us to examine whether the receptor may be expressed by platelets. I don't think we mentioned it but we found that there were some... at least for some patients there were SARS-CoV-2 RNA associated with platelets and that's why in part we look at ACE2 RNA expression by platelets. It seems simple but it wasn't that simple. We were actually using through PCR and different approaches and primers we got different answers using intron-spanning and non-intron-spanning primers from that, the receptor was presented so that in fact we ended up concluding that there was little or no ACE2 RNA in platelets of COVID-19 individuals that we examined but that there was some DNA that could be contaminating, potentially due to extracellular DNA maybe provided by extracellular vesicles[inaudible 00:19:30] or something but either way we're speculating. Dr Eric Boilard: One intriguing aspect of the study is that the SARS-CoV-2 RNA was not found on platelets from every patient, it was only in 20% of them and we unfortunately were not able to look at the RNA of these positive patients to determine whether these patients were positive for ACE2 RNA and potentially in some donor cells in some patients, there might be some levels of expression and we are not excluding this. Dr Eric Boilard: So yeah, you ask whether it was consistent with other studies? There was the study by Manny that was published, in Blood where they also could not find the receptor but our studies contrast with the one that was more recently reported by Zhang in Journal Of Hematology & Oncology. In this study they found in fact that a robust expression of ACE2, both protein and RNA, and it was both in humans and mouse so clearly there's still work to do to determine whether ACE2 is present or not, but there could be other means of interaction between the SARS-CoV-2 and platelets if there is indeed an interaction. We speculated that maybe in some patients there could be antibodies against other coronaviruses, you know the cold, the viruses that give the minor cold symptoms and that in some of the patients these cross-reacting antibodies could promote the interaction with platelets through their receptor for IgG , that could be a means. Dr Eric Boilard: There are other receptors that are expressed by platelets, there's probably several. I'm sure we have a list, but one could be the CD147. It's a receptor that is known to be expressed by platelets and that was suggested to interact with SARS-CoV-2, at least it does interact with SARS-CoV, the first one, CoV-1. So these are potential candidates. So there's work to do. Dr Fadila Guessous: What we found was that in Eric's lab, that you have this 20% of aged people that have this SARS-CoV RNA in place, in 20% of aged people. So should we look only at aged people for this ACE2? If they have the receptor for ACE2, maybe? It's like 20%, only aged. And this 20% of people we investigated, they were only aged. Not other people. So do we... probably, we have to go further and investigate more of this age group for ACE2. . Dr Milka Koupenova: So if I can make a comment, despite the fact if ACE2 was expressed or not, right? Could it explain the profound thrombotic response that you see? What's your take on it? Dr Eric Boilard: Personally, I doubt it. I think the disease starts in the lungs and this is where the damages are made, and that it may expand to blood vessels and then multiple players can then from a place of activation damages themself, the lack of O2 in some patients that are in ICU. And then of course the activation of the other cells, the cytokines themselves. So there are numerous ways I will say that can be to play for activation. In fact, the study by us, the one in Blood, they use plasma from the COVID-19 patient and I would assume there was no actual virus particle in this plasma. And they incubated it with platelets, and platelets were then activated. So there are molecules in COVID-19 that circulate in blood that can activate platelets, and that adds to the vasculature damage trauma. I would think that that could come from principle factors. Dr Milka Koupenova: Which would explain why is it so dysregulated, which brings it to the next question now with a favorite question in the literature. Should we be using antiplatelet drugs? Your opinion, Eric and Fadila? Dr Fadila Guessous: Well with my collaborator, Younes Zaid you know, and first author on this paper, actually they are going for that. They are having small trial giving antiplatelets treatment to a few patients in our lab. You will hear the story. I don't know if Eric will allow me to disclose this? I don't know because Younes is not here, but they are using heparin for now and for the treatment, but now they are trying antiplatelet treatment as a small trial. Dr Milka Koupenova: So when do they give antiplatelet drugs? At what stage of the disease? Dr Cindy St. Hilaire: I think maybe the way to ask the question is: If you were going to use antiplatelet therapy for changing the course of the disease, where do you think is the best time point within the disease, from the day you get infected to symptom presentation and well after that, what time point do you think it's most critical, the platelet function? Dr Fadila Guessous: It's a good question. It's a good question because the trial is done in in our lab and I didn't have insight. What time is the time point. Honestly, I don't have an answer for that in time point. Because I have a... they started the trial- Dr Cindy St. Hilaire: No, but I guess, based on what we know and what you've found in your study, where would you think it's more critical, if we even can speculate on that? Dr Fadila Guessous: Yeah, the symptoms. I would give them from the beginning of the symptoms for me. Because otherwise, when you are in the cytokine storm and you have all this [thromboembolism in the lungs and everything, it's too late. Dr Eric Boilard: And if I can add something. When we think of immune cells or B cells, D cells, we don't say anti-lymphocytes or anti-neutrophil therapy. But when we come to platelets, we think that the molecules that are known to impact some of the platelet activities, we think of aspirin, clopidogrel, coagulation, and we call it anti-platelets but. But platelets, I mean, they can do a lot independently of from vaccine, independently of EDP, and sometimes we forget that they are more than just these poor thrombotic cells and that there are different studies that has been published by different groups and us that found that you can use, for example, COX-1-deficient platelets. So they won't make thromboxane yet, they can make lot of IL1 and be pro inflammatory, although they will not make thromboxane. So we actually use mice where were we use a variety of anti-platelet therapies but yet their platelets were able to come out inflammation so there's no one drug that can get all the platelet functions at once, so it's important to remember that. Dr Cindy St. Hilaire: It sounds like maybe some of them could possibly make things like a cytokine storm almost worse, if we're targeting the wrong anti-activity? Dr Eric Boilard: All depends on how platelets are activated. Whether we are activating a GP pathway, or a nighttime pathway or both at the same time, and the environment where platelets are localized and activated so that they all back the response to this. Dr Cindy St. Hilaire: So you're saying it's complicated. Dr Eric Boilard: Yeah, I think it's not simple. Dr Milka Koupenova: Anything complicated, it's platelets. So in a way I think that perhaps what we need to acknowledge is targeting platelets for whatever response. I completely agree that platelets should be targeted at one point but what drugs should be used, is the question? What specific receptor or what specific response? And if we manage to figure some form of controlling the inflammatory response, that specific, what you see, that huge amount of cytokine dysregulation that will be great. But if you're targeting platelet-specific function when it comes to thrombosis, I'm a bit concerned that there has to be an exact time in which that's important. And the reason why is because if you have this damage in the endothelium, as you see reproducing the virus in the endothelial cells, and if platelets are not doing their own function then perhaps a lot more virus is leaking into the circulation. And that could be why you see some patients that have it, some patients that don't have it, which is a question that I want to ask. Dr Milka Koupenova: Why do you think some patients have it and some patients don't have it? And do you think that perhaps as the virus gets processed into a platelet and digested, we are missing a time point? Because we're not taking platelets from patients at the same time after infection. We have no way of doing that. So what is your take on the fact that you have virus in some platelets? And from your study, perhaps, if we rely on the numbers, the people who have more severe symptoms seem to have a little bit more. Correct me if I'm wrong. Dr Eric Boilard: Actually, the older people have more chance of having it but it was not in the more severe people. In fact, even among the non-severe patients, there was 20% of them that had platelets with positive RNA. So that didn't correlate with severity or any outcomes. The only correlation we could identify was age. Dr Milka Koupenova: So do you think that perhaps in older individuals there are problems would be degradation of the virus, and hence they are not initiating the proper response? Dr Eric Boilard: Yeah, potentially. Maybe there's somehow an indication of the virus from the organs and it's a destination or it's not captured fast enough on the first varrier of the immune cells in the lungs. Right now we don't have the evidence that it's actually the virus that is in platelets, right? The RNA and there's still work to determine if the virus is there. Dr Milka Koupenova: So practically, in summary, we don't know what it is. Although, I mean, with a bias, me probably like you, and you're very nice, you're cautious, but I also believe that the virus... and it's a belief. That the virus gets inside of platelets. Dr Fadila Guessous: How, Milka? How? Dr Milka Koupenova: There could be some issue that we don't see, and as Eric says, it's some form of an age factor. There could be other receptors. Platelets are tricky, they don't act like every regular cell so they may have evolved to have other receptors that maybe they are not as functional in the ither cells but they're platelet specific. There is the process that from all these cytokines perhaps, platelets are just sucking in things by micropinocytosis, which has happened. The thing that's important to think and mention is that if a virus is not going into a platelet by receptor-specific mechanism, perhaps it's locating to the wrong compartment, and then not introducing the proper immune response. Perhaps that's the reason of why you're catching it only in 20%. This is all very, very speculative. Dr Eric Boilard: Yeah. And are you putting in a contribution of megakaryocytes] in the lungs where they will burst … Dr Milka Koupenova: Yes. That it. Absolutely, it's quite possible. That's a collaboration then you should perhaps consider with Dr Craig Morrell. Which is very interesting, right? We don't know. We were never able to figure, even with our full studies, we also saw, what is it, four out of 18? So it's like 25% of the platelets had RNA for a flu but when you look what the antibody of the virus you find that a lot more platelets have it. So is it that we're missing some form of a process that the virus quickly gets digested and it initiates a response? I don't know until we get the right tools. But currently we are struggling with tools, practically, to assess that question. Dr Eric Boilard: Yet it's impressive what's been done since March with the Covid. Dr Milka Koupenova: Oh, some of them is good. Some of them is really important science and some of them is questionable that raises more questions. Dr Eric Boilard: Absolutely, yeah. Dr Milka Koupenova I personally think it's very important what your study is showing because it gives a base of truly thinking about how platelets maybe truly acting as an immune cell at the beginning. I know that Eric and I might be a little bit biased when it comes to that, but when does the thrombotic response stop? And when does the immune response stop of a platelet? And is it necessary to stop and to be all together? How do we push one and doesn't stop the other? These are the questions that we in the field truly need to assess and that's what I really like about your study, is that it raises these important questions. Dr Fadila Guessous: Before the call, I was looking at the paper from University of Verona. I mean, I was hoping they will find more answers to the same question that is a favorite from University of Verona, and actually they are also more speculative. They didn't answer the question, the same questions we are asking. Dr Milka Koupenova: Well, until we have the virus on hand, the actual virus, it's a little hard for us. And then the other point is even if you have the virus on hands, you're doing all of these in vitro studies, how do you assess the entire immune response? Can we think of platelets just as platelets without being in cells, right? What is your take on that, Eric or Fadila? Can you think of platelets just as platelets when it comes to mediating a response to a viral infection? Because we think okay we do this particular thing with platelets and then they mediate this and they mediate that, but perhaps the interaction between platelets and all the immune cells at the beginning of the response is super important. And I do struggle sometimes with the assessment of what amount is just platelets and what is the communication between each immune cell and platelet. It's very hypothetical. Dr Eric Boilard: I agree that I see platelets as communicators to other cells. I mean, they're so numerous and there are bags with cytokines and other molecules and RNA and micro RNAs and it can impact for different organs, tissues, cells. The fact that they were activated, even in the non-severe patients, all the non-severe patients we analyzed, platelets were there, they were activated. So there was something going on, although thrombosis was not of use in these patients, so they made plates front row and the front part of the disease and the pathogen as a cell. Dr Milka Koupenova: So is there a way of somehow, as you perceive in your future studies, to get platelets from people who get the virus but don't have any symptoms, and compare their cytokine profile of platelets to what you see? Because we focus so much on the people who get infected, logically, right? But truly to assess what is the difference, if we can figure why those patients are not having this profound response, right? Dr Eric Boilard: Something that we dream of. The fact that collecting platelet from someone who has no symptoms is…Because we, if they have no symptoms, then they don't come to us. They don't stop- Dr Fadila Guessous: They don't show up at the hospital. Dr Milka Koupenova: And so you have to recruit them by tests, right? Because we screen, for instance, our institution screens. But it's hard, right? No one wants to take blood from a… Dr Fadila Guessous: You get this kind of calls, people are asymptomatic, they don't have any symptom, they won't show up at the hospital. We used whatever we had in our recruitment from the hospital. Dr Eric Boilard: In fact, you can maybe go through the transfusion work, the studies on transfusion where they actually collect blood from people and people sign that they have no symptoms when they do give blood. And there are studies from China and more recent ones in France where they then went back to this bags of... these were plasma, but maybe one could do it with platelet concentrates. And then go back to these bags where people had signed they had no symptoms when they gave blood, and screened platelets for RNA and then... but you have dozens of dozens of samples to test, but they are available. And maybe someone could find that many were positive but they were asymptomatic, using transfusion studies, maybe. But recruiting people from outside, asking we want people with no symptoms for… Dr Cindy St. Hilaire: I know we're doing sentinal testing of our students, so. Somehow the group doing that needs to couple with platelets studies. But that was wonderful. Thank you Dr Boilard, Geussous, and Koupenova for joining me today. This has been an amazing interview. It's a wonderful study. Good luck on the future research and thank you for moving the field forward on this. Dr Eric Boilard: Thank you very much for inviting us. It was great and we are pleased to interact with you. Thank you. Dr Milka Koupenova: Very nice to meet you Fadila, Eric. Dr Fadila Guessous: Thank you so much for having us. It's really it's a pleasure to have this interview with you and of course I have to say that really it has been a wonderful collaboration with Eric and Younes in our lab. I am from Casablanca so we had really many people involved in this project and everyone, we worked really, really hard to get this very... I mean, you can see the time, it's from March to now, to get this paper out it was really a big effort. A big effort from the Moroccan side and from Eric's team in Quebec City and so we are really very pleased to have this wonderful collaboration. Thank you so much for having us. Dr Cindy St. Hilaire: That's it for the highlights from the late October and early November issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Drs. Eric Boilard and Fadila Guessous, as well as Dr Milka Koupenova for co-hosting the interview with me. Dr Cindy St. Hilaire: This podcast is produced by Rebecca McTavish and Ishara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles was provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, you're on-the-go source for the most exciting discoveries in basic cardiovascular research.
Hello all! Trying out some video for the Creative Drive Podcast! Here's a short story I wrote a few months ago, I'm working my way back into writing fiction, so here it goes! If you're a writer with a day-job, I'd love to know what you're up to and how you're holding up in the throes of every day chaos. Submit your poetry and flash fiction today and I'd love to share it on the podcast! Send in your short works today, especially the rejected ones at creativedriveshow (at) gmail (dot) com, or through our website! https://idlewy.blog/you/ Much love, j
Seth Lederman is a physician, scientist and founder and executive officer of innovative biopharmaceuticals companies. Prior to founding Tonix, among the companies Dr. Lederman founded was Targent Pharmaceuticals, which developed late-stage oncology drugs, including pure-isomer levofolinic acid (levoleucovorin). Targent’s assets were sold to Spectrum Pharmaceuticals, which marketed levoleucovorin as Fusilev® for advanced colorectal cancer, where it gained significant market acceptance. Dr. Lederman served as an Associate Professor at Columbia University from 1996 until April 13, 2017. He joined the faculty of Columbia University's College of Physicians and Surgeons in 1985, became Assistant Professor of Medicine in 1988, and Associate Professor with tenure in 1996 and Director of the Laboratory of Molecular Immunology in 1997. From 1988 to 2002, Dr. Lederman directed basic science research at Columbia in molecular immunology, infectious diseases and the development of therapeutics for autoimmune diseases. Dr. Lederman is author of numerous scientific articles, and inventor of technologies recognized by a number of issued patents. His fundamental work on the CD40-Ligand (CD154) elucidated the molecular basis of T cell helper function and has led to the development of therapeutic candidates for autoimmune diseases and organ transplant rejection in collaboration with Biogen and UCB. The successful defense of his CD154 patents has led to important precedents in defining the relationship of therapeutics and molecular targets. In collaboration with Prof. David Baltimore (then at Rockefeller University and later MIT), Dr. Lederman identified and functionally characterized the CD40 signaling molecule, TRAF-3. His early work on HIV contributed to the understanding of how the V3 loop of HIV gp120 was involved in fusion with CD4 cell membranes, an early and essential event in viral entry and infection. In addition to his research, Dr. Lederman served as attending physician in the Edward Daniels Arthritis and Autoimmunity Clinic on the Medical Service at Columbia Presbyterian Hospital from 1988-1996. Dr. Lederman represented U.S. Biotechnology at a Beijing Summit in September 2015 with China’s President Xi Jinping. The Summit was organized by the U.S. Chamber of Commerce and involved a delegation of U.S. CEOs, former U.S. Cabinet officials and leading academic experts. Dr. Lederman earned an AB from Princeton in Chemistry cum laude in 1979 and an MD from Columbia University's College of Physicians and Surgeons in 1983. Dr. Lederman trained in internal medicine and rheumatology at Columbia's Presbyterian Hospital. He was an NIH Physician-Scientist 1985-1990 at Columbia.
On today's episode, we had the great pleasure of speaking with Duke professor, economist, author, and self-described nerd Ronnie Chatterji on his 2020 political campaign as the Democratic candidate for North Carolina State Treasurer. Join us as we learn more about his vision for North Carolina, his background working for the Obama administration, the significance of the office of NC State Treasurer, his own experiences as a Carolina Desi, and much more. Remember to VOTE on or before November 3rd. To learn more about Ronnie and his platform, visit www.ronniechatterji.com.
The advent of cancer immunotherapy has radically changed the field of oncology by improving the way many malignancies, including several aggressive and orphan diseases, are being treated with subsequent major improvement of patients’ prognosis. The first crucial and successful step in the field was the development of agents able to inactivate inhibitory immune receptors resulting in a subsequent increased anti-tumor response. Among them, antibodies blocking CTLA-4 (ipilimumab) and PD-1/PD-L1 (nivolumab, pembrolizumab, atezolizumab and durvalumab) are already widely available in clinical practice. More recently, to further improve the ability of the immune system to eradicate cancer cells, several other stimulatory or inhibitory molecules have been recognized as possible targets. ESMO Open has launched a special series of mini-reviews aiming to provide an update of the most interesting and upcoming targets in cancer immunotherapy including LAG3, TIM3, CD40, B7x, OX40, ICOS, VISTA, CD27, GITR and neoantigens. All these mini-reviews contain information on biological background (i.e. what the target is, where it is expressed and what is the physiological role as well as the expected effect when targeting it), drugs under development for targeting that specific molecule as well as current on-going clinical trials with targeted agents (including those in combination with other immune checkpoint inhibitors). In this podcast, Anna Berghof talks to Matteo Lambertini - Department of Medical Oncology, U.O.C. Clinica di Oncologia Medica, IRCCS Ospedale Policlinico San Martino, Genova, Italy. Read the Abstract: https://esmoopen.bmj.com/content/4/Suppl_3/e000795
This month on Episode 5 of the Discover CircRes podcast, host Cindy St. Hilaire highlights five featured articles from the September 27 and October 11, 2019 issues of Circulation Research and talks with Sarvesh Chelvanambi and Matthias Clauss about their article HIV-Nef Protein Transfer to Endothelial Cells Requires Rac1 Activation and Leads to Endothelial Dysfunction: Implications for Statin Treatment in HIV Patients. Article highlights: Stamatelopoulos, et al. Reactive Vasodilation in AL Amyloidosis Cao, et al. Miro2-Mediated Cardiac Mitochondrial Communication Georgakis, et al. Circulating MCP-1 Levels and Incident Stroke Sun, et al. Body Mass Index and DNA Methylation Tan, et al. Yy1 Suppresses DCM Through Bmp7 and Ctgf Transcript Cindy St. H: Hi. Welcome to Discover CircRes, the monthly podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, and I'm an assistant professor at the University of Pittsburgh. My goal as host of this podcast is to share with you highlights from recent articles published in the September 27th and October 11th issues of Circulation Research. We'll also have an in-depth conversation with Drs Matthias Clauss and Sarvesh Chelvanambi, who are the lead authors in one of the exciting discoveries from our October 11th issue. The first article I want to share with you is titled, Reactive Vasodilation Predicts Mortality in Primary Systemic Light Chain Amyloidosis. The first authors are Drs Kimon Stamatelopoulos, Georgios Georgiopoulos, and the corresponding author is Dr Efstathios Kastritis. And the studies were conducted at the National Kapodistrian University of Athens School of Medicine in Athens, Greece. So we hear about amyloids a lot in things like Alzheimer's, but amyloids are really just aggregates of protein that fold into shapes. And the nature of these shapes allows these individual protein molecules to bind and form many copies that form these fibers that are rather sticky. And the fibers then aggregate into larger and larger globules. And light chain amyloidosis is the most common type of amyloidosis. It's a rare but deadly disease, and it's caused by antibody-producing cells that are aberrantly churning out parts of antibodies called light chains. And it's these light chains that will aggregate and form sticky fibers. So these fibers aggregate and form amyloid deposits, and these deposits build up and damage the organs and the tissue in which they're accumulating. And because it's dependent on where the aggregates are accumulating, AL amyloidosis can present with a wide variety of symptoms. However, symptoms of heart dysfunction and low blood pressure correlate with poor prognosis. And because vascular dysfunction can contribute to hypotension or low blood pressure, this group decided to examine the vascular health of patients by conducting a measurement called flow-mediated vasodilation. And so this is a measurement where the diameter of the brachial artery, which is located in your arm, is measured before and then after a brief period of lower arm ischemia. And they formed a cohort of 115 newly diagnosed AL patients and another cohort of 115 matched controls. This study found that in AL patients, flow-mediated vasodilation was higher than in aged, sex, and cardiovascular risk factor-matched controls. The mean follow-up time for this study was 54 months, and in that time, the authors went on to find that high values of FMD in the amyloidosis patients was strongly predictive of mortality. In fact, high FMD values were more predictive of death than some measures of cardiovascular health. These results suggest that flow-mediated vasodilation may be a superior means of identifying AL patients most at risk and for assessing potential benefits of therapeutic interventions. The next article I'd like to highlight is titled, Miro2 Regulates Inter-Mitochondrial Communication in the Heart and Protects Against TAC-Induced Cardiac Dysfunction. The first author is Yangpo Cao, and the corresponding author is Ming Zheng. And the work was conducted at Peking University, Beijing, China, Key Laboratory of Molecular Cardiovascular Science at the Ministry of Education, also in Beijing, China. Beating heart cells have very high energy requirements, and thus they need lots of fully functioning mitochondria. And as we all know from our good old high school biology days, mitochondria are the powerhouse of the cell. Mitochondrial health and performance is directly dependent on the ability of individual mitochondria to be able to communicate with each other. In many cells, this mitochondrial communication occurs via the fusion of mitochondria into a giant network. However, in cardiomyocytes, the mitochondrial movement is much more constrained. In cardiomyocytes, mitochondria communicate by briefly connecting with neighboring mitochondria, which is often called kissing, mitochondrial kissing, or by nanotunneling, which is when the mitochondria create a sustained connection by means of long nanometer-sized tubular protrusions called nanotubes. And it's thought that the proper health of the cell is dependent on proper mitochondrial communication. Miro2 is a Rho GTPase on the outer mitochondrial membrane and it harbors a calcium sensing domain. Miro2 can interact with transport proteins to promote mitochondrial transport along microtubules in a calcium-dependent manner. This group wanted to investigate whether Miro2 regulates cardiac inter- mitochondrial communication. To do this, they used transverse aortic constriction or TAC or they used an Ang II infusion model to induce hypertrophy in murine hearts. Using these two models, they found Miro2 expression was decreased via Parkin-mediated ubiquitination, and they also found that inter-mitochondrial communication was disrupted. By contrast, transgenic mice over-expressing Miro2 were more resistant to hypertrophy, and they were able to do this by maintaining proper cardiac function than their wild type counterparts. Together these results reveal a novel role for Miro2 in mitochondrial communication and show that maintaining such communication may mitigate effects of hypertrophy. The next paper I want to highlight is titled, Circulating Monocyte Chemoattractant Protein-1 or MCP-1 and the Risk of Stroke: A Meta-Analysis of Population-Based Studies Involving 17,180 individuals. That is a huge study. The first author is Marios Georgakis, and the corresponding author is Martin Dichgans. And they are from the University of Munich in Munich, Germany. A major component of atherosclerosis is chronic inflammation and inhibiting the activity of proinflammatory cytokines has been identified as a potential therapeutic strategy to help slow the disease progression. One such cytokine under study is monocyte chemoattractant protein-1 or MCP-1, and animal studies have shown that blocking MCP-1 limits, or boosting MCP-1, accelerates atherosclerosis. However, large scale observational studies of MCP-1 in humans are lacking. To address this gap in knowledge, this group performed a meta-analysis of previously unpublished data from six population cohorts, which totaled over 17,000 individuals. These individuals were followed for an average of 16 years, which when you think about it, this is an absolutely huge study. So in looking at this cohort of patients, the team identified a significant association between high baseline MCP-1 levels and the likelihood of suffering a future ischemic stroke. Interestingly, this effect was not seen with hemorrhagic stroke, which is typically not associated with atherosclerosis. These findings not only support the previous animal studies, but also support a recent study in humans in which a genetic predisposition for high levels of MCP-1 was associated with an increased risk of coronary artery disease and stroke. This study also suggests that future studies should explore the potential of lowering MCP-1 levels as a possible prevention strategy. Perhaps there could be another CANTOS-like trial where we use something to block MCP-1 signaling. Maybe that would have much broader effects. I guess we'll have to wait and see what the data says. The next paper I want to highlight is titled, Body Mass Index Drives Changes in DNA Methylation, a Longitudinal Study. The first authors are Dianjianyi Sun, Tao Zhong and Shaoyong Su, and the corresponding authors are Shengxu Li and Wei Chen. And they're from the Children's Minnesota Research Institute, Children's Hospitals and Clinics of Minnesota in Minneapolis, Minnesota and The Peking University Health Science Center in Beijing, China, respectively. So it's well appreciated that obesity is increasing worldwide. And obesity contributes to a whole host of cardiovascular morbidity, and ultimately contributes to mortality. It's also well known that environmental factors such as the food we eat and the air we breathe, as well as genetic factors, can influence a person's risk of obesity. And recently there have been studies that suggest that perhaps epigenetic factors also contribute to obesity. And just to remind you what epigenetics is, DNA is the genetic code, and mutations can happen on DNA that can alter either gene expression or maybe protein folding or whether a protein is made at all. But epigenetic factors are not as permanent as DNA mutations. Epigenetic factors are alterable modifications that can happen to DNA itself or that can happen to the proteins on which the DNA is wrapped around. And epigenome-wide association studies have shown that DNA methylation at certain loci is linked to an increase in body mass index, or BMI. However, it's unknown whether these methylations are a cause or consequence of obesity. So to get to the bottom of this, this group performed a large-scale longitudinal study. They examined thousands of DNA methylation sites in 995 white individuals and 490 black individuals. And they also determined the subjects' BMIs. They did this at a baseline measurement and then approximately six years later, they collected the same data in the same patient cohort. What they found was that only a handful of methylation sites were shared between the two ethnicities. And in both groups, however, there was a similar unidirectional link between BMI and methylation. Very interestingly, baseline BMI could predict methylation at a number of genetic loci. However, the team found that none of those baseline methylation sites could predict future BMI. From this data, the authors are able to conclude that it's obesity driving the methylation at certain genetic loci as opposed to certain genetic loci driving obesity, which I think is just extremely interesting. Really nice study. The last article I want to highlight for you is a paper titled, Yin Yang 1 Suppresses Dilated Cardiomyopathy and Cardiac Fibrosis Through Regulation of Bmp7 and Ctgf. The first author is Chia Yee Tan, and the corresponding author is Jianming Jiang, and they're from the National University of Singapore. Dilated cardiomyopathy or DCM is characterized by left ventricle enlargement and associated contractile dysfunction and fibrosis. Patients with DCM are at risk of arrhythmia and also of sudden death. And there's actually a huge number of genetic variants that have been linked to DCM, but the most common one or the most well-studied are mutations that affect the nuclear lamin gene or LMNA. So LMNA knockout mice are used to study the role of this gene in DCM, and these animals exhibit not only cardiac defects but also systemic defects. And those systemic defects include things like shorter lifespan, growth retardation, muscular dystrophy, neuropathy, and lipodystrophy. Recently, LMNA-related dilated cardiomyopathy was linked to the deregulation of cardiac cell cycle. Meaning there was issues in how these cardiac cells are proliferating. So in this study, Tan and colleagues showed that boosting expression of a protein involved in cell cycle regulation, this protein is called Yin Yang 1, so boosting this gene's expression actually reversed the dilated cardiomyopathy symptoms in mice with heart-specific LMNA deficiency. Compared with untreated mice, mice receiving Yy1 via an adenoviral vector exhibited improved cardiac function and also reduced fibrosis after four weeks. The team then went on to show that Yy1 treatment prompted suppression of the extracellular matrix factor, Ctgf, and the upregulation of the growth factor, Bmp7. Now, neither of these factors alone could rescue the symptoms of LMNA lacking mice. However, when both of these factors were manipulated together, they mimicked Yy1 treatment. These results highlight that Yin Yang 1 and its downstream targets Bmp7 and Ctgf are key players and potential therapeutic targets that can be harnessed for tackling LMNA-driven dilated cardiomyopathy. Okay, so now we're going to have our interview with Drs Matthias Clauss and Sarvesh Chelvanambi. And they are from Indiana University School of Medicine in Indianapolis, Indiana. And their title of their paper is, HIV-Nef Protein Transfer to Endothelial Cells Requires Rac1 Activation and Leads to Endothelial Dysfunction: Implications for Statin Treatment in HIV Patients. So thank you both very much for joining me. Sarvesh C: Thank you so much, Cindy. Matthias C: Thanks for having us here. Cindy St. H: Could you both introduce yourselves and tell us a little bit about your background? Sarvesh C: My name is Sarvesh Chelvanambi. I grew up in Chennai, India. I did my undergraduate degree at Miami University in Oxford, Ohio. I got a Bachelor of Arts in Zoology with a minor in Finance. I then went to the Pennsylvania State University where I got my Masters in Biotechnology before coming over to Indiana University in 2014 to do my PhD work. And then I joined the lab of Dr Matthias Clauss, and in 2016, I got an American Heart Association predoctoral fellowship to study this project specifically. Cindy St. H: Wow! Congratulations. That's wonderful. Sarvesh C: Thank you so much. Cindy St. H: And now you completed the circle by publishing your AHA grant in Circulation Research. Sarvesh C: Exactly. Cindy St. H: And Matthias, how about you? Matthias C: I'm a Research Professor at IU School of Medicine, and my research interests focus in understanding how stressors connected with endothelium in this way contribute to vascular disease. These stressors include cigarette smoke and viral infections. Regarding viral agents, we are studying both acute infections and chronic infections and that is HIV. This HIV interest started actually 12 years ago in collaboration with Dr Samir Gupta who is also of course on this paper. We started off with a simple question, why are there so many cardiovascular events in patients, in HIV patients, with interrupted antiretroviral therapy? Cindy St. H: So it's not just the fact that they're HIV positive, it's that they were on therapy and then went off it? Matthias C: Yes. And this was part of this SMART study and this study was then actually halted because of the safety issues. Cindy St. H: So you're starting with the idea that patients with HIV who go off this antiviral therapy are more prone or get more cardiovascular events. So what did you start with, with this particular study? Matthias C: Well, our overarching idea was that the HIV virus could also do damage in the era of the combined antiretroviral therapy. And we started up with two questions, one was, is there an HIV protein which is persistent? And the other question, how is this HIV protein, if there's any one which is persistent, performing this? And this may be then leading over to your specific way to address these questions. Sarvesh C: That's kind of where we are starting with this project. Because back in 2016, the START trial came out saying, "We need to change the way we treat HIV patients," because initially the previous regimen of our drugs had a lot of metabolic side effects, but the current regimen of integrase inhibitors is actually really good and has very low metabolic effects. So there was a New England Journal Of Medicine paper that said, "Well, if a patient walks into the clinic, they're diagnosed as being HIV positive, put them on antiretroviral therapy right away." But even in this era when everybody is on ART and there's almost no viral replication, you still see the persistence of a lot of comorbidities. And especially those associated with vascular events, whether it's peripheral arterial disease, coronary arterial disease, and a lot of other vascular diseases in the lung, or the kidney or the brain. So that kind of is what set us up, is there an element in the blood of these patients that is contributing towards vascular dysfunction? Cindy St. H: And so the protein that you are talking about in this paper is a protein called Nef, and is that where you come in, Sarvash? Sarvesh C: Yes, because the project before I joined the lab, that's kind of where it led off, saying that Nef can get to the endothelium and it's very good at killing endothelial cells, but the mechanism through which it transfers into endothelial cells and the signaling pathways that Nef hijacks to induce this apoptosis was not clearly elucidated. A lot of work is done in Nef in monocytes and macrophages because as an HIV protein, it was studied in CD40 cells and the whole immune system as a whole, but we were the first to leverage all of those findings within an endothelial context and answer the questions, so what does Nef do and how does it get there? Cindy St. H: All right, so tell us a little bit what does it do and how does it get there? Sarvesh C: So we started doing some experiments with starting with HIV patient blood. So we took two fragments, we took the PBMC fraction, that Dr Clauss was talking about, which we knew had Nef within many of those cells. We also took the extracellular vesicle fraction, and we chose to look at this because there's a lot of literature out there saying that this fraction could not only disseminate particles throughout the body but also help signal through that. So in both of these fractions we added to the endothelial cells, we found increased apoptosis in HIV patients when compared to HIV negative patients. And we were excited, but then we went and asked which of these patients had HIV Nef positivity in their blood, and then using that information when we stratified our apoptosis results, we made the surprising observation that the HIV positive, Nef positive patients were more prone to endothelial cell apoptosis. And this sparked a lot of conversation, so how do we target this and what is the signaling pathway it gets into? And that is kind of what led to most of the work in this paper, where you're showing that the transfer is mediated by extracellular, because this is such a nice tool, for HIV I guess, to spread itself into literally every cell type. Because while the HIV virus can only infect very few cell types, extracellular vesicles can be taken up by anything. And the second observation we made was within endothelial cells, we found the signaling pathways that Nef was able to hijack to induce cell death. And that became the focus of this paper. Cindy St. H: That was one thing I wanted you to clarify, because I think what a really interesting aspect of this study is that it's the immune cells that are infected. The endothelial cells themselves are healthy and really they're getting this damage from the vesicles. That is,…wow! I don't know. It's just a really, really neat study. So can you tell us a little bit about the techniques you used in this paper? Sarvesh C: Yes, so we did a lot of assays to evaluate endothelial cell stress. So we started by looking at apoptosis, and a lot of those studies were done by looking at caspase-3 activity, which is a classic marker for cell death. We also did a lot of microscopy work where we took out extracellular vesicles out of those vesicles on the endothelial cells to show the uptake of Nef protein and thereby hammer that extracellular vesicles are indeed a mechanism of transfer for this protein in particular. Now, one of the interesting experiments that we actually ended up doing, which was not a part of this paper really, was we wanted to see if chemotaxis was being affected by this. So we took an endothelial monolayer and separated T-cells that are expressing Nef using a Transwell membrane. And I had this huge problem where I couldn't read for a week because instead of using the 4-micron filters that allow T-cells to transfer, I was using 0.4 micron filters that T-cells cannot transfer through. But I still went about it and did my whole experiment because I didn't make that realization until a week later, because when I looked at the bottom of these chambers, there were no T-cells at all. But when I looked at the endothelial cells, I observed cytoplasmic transfer and Nef transfer, and we had a couple of conversations going, why is this happening? Did the T-cells all die or did they disappear? And that's when we went back and looked in literature and found that Nef is very good at making virion particles. And those are the similar pathways that extracellular vesicle trafficking comes from. And so that was a huge shift in the way this project was designed and where we then started looking into the modes of transfer, the protein and the subsequent apoptosis that that transfer can cause. Cindy St. H: I love this story. So essentially your mistaken filter created this paper and this finding of the vesicles affecting the endothelial cells. Matthias C: Yeah, that's a typical finding for practitioner Chelvanambi, because he has this gift to turn negative things into positive things. So we have a lot of fun, and this mistake was really the beginning of a great study. Cindy St. H: That's wonderful. Really beautiful images, as well. So a little bit digging into, I guess, the next step. So first off, how were the endothelial cells getting damaged? They're getting damaged from these extracellular vesicles, but then what's Nef doing in the endothelial cell? What's happening there? Sarvesh C: So that was a very big question because if you look at it, Nef is a very small protein with almost no known enzymatic function. And yet it is able to interact with a lot of host proteins, which I guess makes it a very good viral protein. So when I went back and looked at literature, there were a host of studies in the 90s to show that Nef interacts with this kinase and that small GTPases, and there was a giant list for us to go after. And we were kind of left a bit fuddled, because we were like, which signaling pathway do we start with? Cindy St. H: Right. It's almost like there's too many. Sarvesh C: Exactly. And so what we ended up doing was we started looking into one of the various mutants of Nef that we had access to. And one of these mutants was a mutant that was incapable of PAK2 activation, and we showed that that doesn't have a lot of these stress damages. So we asked, "What is directly upstream of PAK2?" And that is where Rac1 came into the picture. And the small GTPase Rac1 is a nice candidate because it is also a master of many, many trades. Cindy St. H: I love this because it's such an interesting multidisciplinary approach to addressing the question, why are patients with HIV getting more cardiovascular events? What do you think evolutionarily is going on? Why would this be beneficial? Why would damaging the endothelium be beneficial? What are your thoughts on that? Sarvesh C: Personally, I think this is a side effect because HIV is never meant to exist in the era of ART. One of the analogies I always like to use is from Harry Potter, where HIV is Voldemort, which is the big bad villain. And what we have done is a really good job of banishing Voldemort. But what we have failed to do as a field is target its Death Eater, Nef. And I think with what we are suggesting, this paper with additional statins and other strategies that focus this, we can get to that point where we not only block HIV expansion but also the expansion of its minions, Nef. Cindy St. H: I love this analogy. I think you should redo your graphical abstract in a Harry Potter theme. Matthias C: Yeah, but I like your question. But also in this regard, I think it may be an example of a novel mechanism, how viral infections work in a different way than just infecting cells. And there's evidence from lots of viruses that they make toxic proteins, and why they are doing this, we don't know. But we noticed that the systemic effect of Nef may have some advantage for the infectious agent, because it makes T-cells more sticky, it makes them stick and transmigrate through the endothelium, and that is also shown in our paper. Cindy St. H: You have evidence that perhaps statins would be beneficial to give to these HIV patients on ART therapy. Can you tell us a little bit about that and how that would work? Sarvesh C: So based on what we did on our mouse studies that was a part of this paper, even after there is endothelial dysfunction, treatment with statins was able to restore endothelial function. Currently, there is a study going on called The Reprieve Trial where they're giving a statin called pitavastatin to HIV patients. The interesting part here is that these are HIV patients who don't have dyslipidemia. And the long-term goal is that statin treatment can help prevent the development of cardiovascular events. We're eagerly awaiting the results of this trial. Cindy St. H: Well done. Well thank you so much for speaking with me today. It was a pleasure to speak with you, Dr Chelvanambi and Dr Clauss. And congratulations again on this beautiful project, this beautiful story. And really, the implications for helping patients with HIV is really profound. HIV used to always be in the news and now that we have the ART therapy it's not talked about as much, but these patients are still in danger and I think your study is really doing a lot to highlight that and maybe even help them. So thank you very much and congratulations. Matthias C: Thank you. Sarvesh C: Thank you so much for the opportunity. Cindy St. H: So that's it for highlights from the September 27th and October 11th issues of Circulation Research. Thank you so much for listening. This podcast is produced by Rebecca McTavish, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Sidney St. Hilaire, and this is Discover CircRes, your source for the most up-to-date and exciting discoveries in basic cardiovascular research.
Commentary by Dr. Valentin Fuster
Dr David Bajor - University of Pennsylvania, Philadelphia, USA Dr Bajor talks to ecancertv at AACR 2015 about combining the immunostimulatory anti-CD40 monoclonal antibody CP- 870,893 with the immune checkpoint inhibitor tremelimumab in advanced melanoma. This approach was found to be safe, with clinical evidence of response. Read the news story for more.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 16/19
Zystische Fibrose ist die häufigste vererbbare letale Erkrankung in Europa, die trotz Fortschritten in Diagnostik und Therapie weiterhin mit einer verkürzten Lebenserwartung einhergeht. Einer der Hauptgründe verfrühter Sterblichkeit betroffener Patienten sind persistierende pulmonale Infektionen mit Pseudomonas aeruginosa. Der Keim bedient sich des Quorum Sensing (QS), eines interbakterielles Kommunikationssystems, um die Ausbildung von Virulenzfaktoren zu regulieren und die Immunantwort des Patienten zu beeinflussen. In dieser Arbeit wurde die Auswirkung des P. aeruginosa QS Moleküls 3oxoC12-HSL auf die Reifung humaner Dendritischer Zellen (DZ) untersucht. DZ vermitteln als professionelle Antigen-präsentierende Zellen zwischen angeborenem und erworbenem Immunsystem. Eine Infektion wurde simuliert, indem humane DZ mit Lipopolysaccharid oder Zytokin-Cocktail aktiviert wurden. Anschliessend wurde die Expression von Maturations- und Migrationsmarkern sowie Zytokinsekretion in Anwesenheit von 3oxoC12-HSL untersucht. Bei LPS-stimulierten DZ kam es in Anwesenheit von 3oxoC12-HSL zu einer erniedrigten Expression der Reifungsmarker CD80, CD86, CD83, CD40, HLA-DR, sowie der Migrationsmarker CD184 (CXCR4) und CD197 (CCR7). Die Coinkubation mit Zytokin-Cocktail und 3oxoC12-HSL ergab eine Herabregulierung der Maturationsmarker CD80, CD86 und HLA-DR. Auf unstimulierte DZ zeigte 3oxoC12-HSL keinen Effekt, das Oberflächenmarker-Expressionsprofil dieser Zellen glich dem unreifer DZ. 3oxoC12-HSL inhibierte auch die Sekretion der pro-inflammatorischer Zytokine IL-12, IFN-gamma, MIP-1alpha, TNF-alpha durch LPS- bzw. Zytokin-Cocktail-gereifte DZ. Insgesamt zeigen unsere Ergebnisse, dass 3oxoC12-HSL die Reifung von DZ unterdrückt und somit das Zustandekommen einer effektiven Immunantwort verhindert wird.
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 05/06
Mon, 9 Dec 2013 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18105/ https://edoc.ub.uni-muenchen.de/18105/1/Draeseke_Anne.pdf Draeseke, Anne ddc:570, ddc:500, Fakultät für Biologie
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 05/06
Wed, 20 Nov 2013 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17070/ https://edoc.ub.uni-muenchen.de/17070/1/Stojanovic_Kristina.pdf Stojanovic, Kristina ddc:570, ddc:500, Fakultät für
Numerous epidemiological studies have consistently demonstrated the strong association between type 2 diabetes mellitus (T2DM) and an increased risk to develop cardiovascular disease. The pathogenesis of T2DM and its complications are characterized by pancreatic, adipose tissue and vascular inflammation. CD40 and CD40L, members of the tumour necrosis factor (receptor) TNF(R) family, are well known for their role in immunity and inflammation. Here we give an overview on the role of CD40-CD40L interactions in the pathogenesis of T2DM with a special focus on pancreatic, adipose tissue and vascular inflammation. In addition, we explore the role of soluble CD40L (sCD40L) as a potential biomarker for the development of cardiovascular disease in T2DM subjects. Finally, the therapeutic potential of CD40-CD40L inhibition in T2DM is highlighted.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 13/19
Dendritic cells (DCs) play a key role in the initiation of adaptive immune responses and the maintenance of self-tolerance. Due to their therapeutic potential, understanding the mechanisms that guide DC differentiation and effector functions is important. DC differentiation and activation depends on transcription factor control of stage-specific gene expression. The recent identification of posttranscriptional control of gene expression by microRNAs (miRNAs) has added another layer of gene regulation that might be important in DC biology. We analyzed the miRNA expression profiles of different DC subsets and identified several miRNAs differentially expressed between plasmacytoid DCs (pDCs) and conventional DCs (cDCs). In terms of miRNA expression, pDCs were more closely related to CD4+ T cells than to cDCs. We also observed that pDCs and cDCs preferentially expressed miRNAs associated with lymphoid or myeloid lineage differentiation, respectively. By knocking down miR-221 or miR-222 during in vitro DC differentiation, we obtained a higher pDC frequency. While p27kip1 and c-kit are confirmed miR-221/222 targets, we additionally identified the pDC cell fate regulator E2-2 as a potential miR-221/222 target. Thus, our analysis points to a role for miRNAs in directing and stabilizing pDC and cDC cell fate decisions. To assess the general influence of miRNAs on DCs, we generated mice with a DC-specific conditional knockout of the key miRNA-producing enzyme Dicer. Dicer-deficient mice dis- played no alterations in short-lived spleen and lymph node DCs. However, long-lived epidermal DCs, known as Langerhans cells (LCs), showed increased turnover and apoptosis rates, leading to their progressive loss. Upon stimulation, Dicer-deficient LCs were able to properly upregulate the surface molecules MHC class I and CCR7, but not MHC class II, CD40 and CD86. In consequence, they were incapable of stimulating CD4+ T cell proliferation. The work presented in this thesis indicates a role for miRNAs in DC regulation not covered by transcription factors. Having demonstrated a role for miRNAs in DC lineage fate decisions, as well as in LC homeostasis, maturation and function, we conclude that miRNAs regulate various aspects of DC biology and thereby contribute to the control of adaptive immune responses.
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 04/06
Fri, 1 Oct 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13337/ https://edoc.ub.uni-muenchen.de/13337/1/Hampel_Franziska.pdf Hampel, Franziska ddc:57
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 11/19
Dendritic cells (DC) play a central role in connecting innate with specific adoptive immunity resulting in target specific activation T-cells. As professional antigen presenting cells (APC) DC specifically stimulate T-effector cells, especially tumor-cytotoxic T-cells. Therefore they are regarded as interesting candidates for anti-tumor or anti-leukemic vaccination strategies. The insufficient expression of costimulatory antigens, MHC molecules and tumor-associated antigens (TAA) on the surface of cancer cells and disturbed mechanisms of apoptosis are the main reason for an ineffective immune response in oncologic diseases. It was shown that acute myeloid leukemic cells can be differentiated to leukemia-derived DC (DCleu ), regaining the stimulatory capacity of professional DC while potentially presenting the whole leukemic antigen repertoire. Thus, vaccination strategies, using ex vivo or in vivo generated DC, might induce a highly specific anti-leukemic T-cell response circumventing the cumbersome identification of leukemia-associated antigens. In this thesis DC antigen (DCA) expression profiles of mononuclear cells (MNC) and dendritic cells (DC) generated from these MNC should be analyzed. The generated MNC and DC should be compared with respect to their DC antigen (DCA) expression profiles and the DCAs value to detect and quantify (leukemia-derived) DC in different AML/MDS subtypes and under different culture conditions. Therefore MNC and DC were generated from 137 patients with acute myeloid leukemia (AML) and 49 patients with myelodysplastic syndromes (MDS) under 6 different serum free culture conditions. DCA studied were: CD1a/1b/1c, CD206, CD25, CD137L, CD83, CD86, CD80 and CD40. DC-generating media were chosen according to their different mechanisms of inducing DC-differentiation: 1. ‚Basic method‘: TNF/GM-CSF/IL-4, 2. MCM-Mimic, 3. Ca Ionophore, 4. Picibanil, 5. Poly I:C and 6. Cytokines. Quality and quantity of generated DC was estimated by Flow cytometry applying a specified, ‘DC-based’ gating-strategy. Expression and coexpression profiles of 10 different DCA as well as various costimulatory molecules, maturation markers and blast antigens were evaluated. Only those DCA qualified for the quantification of leukemia-derived DC that were not expressed on uncultured MNC fractions. AML patients presented with an average of 58 % blasts, MDS patients with 13 % blasts in MNC fractions. DCA were expressed on average on less than 7% of uncultured MNC, however some of the markers could be expressed on up to 77% of uncultured cells in single AML cases. Consequently these DCA did not qualify for detection of DC in those cases. Highest expression rates were found for CD86 and CD40 in naïve AML and for CD137L and CD40 in naïve MDS samples. Other DCA (e.g. CD1a, 1b, 1c) were only rarely found on naïve blasts. DCA expression on uncultured AML and MDS MNC varied with FAB types and cytogenetic risk. After culture in different DC-differentiating media, on average 28% DC could be generated from AML MNC and 30% from MDS MNC, depending on methods used, with an average DC viability of more than 60% and an average DC maturity of 49% (AML) and 56% (MDS). On average 36% of leukemic blasts could be converted to DC. Proportions of DCleu in the total DC fraction varied from 40-58% and were on average 49% (AML) and 43% (MDS) after culture. Average results of all culture methods tested were comparable, however every method failed to create DC in some individual cases. The most important results of this thesis are: 1. It could be shown that DCA are expressed on naïve blasts in AML and MDS in individual patients. That means that the individual patients’ DCA-profiles have to be evaluated before DC-culture to find suitable DCA to detect and quantify (leukemia-derived) DC after culture. 2. Different methods of DC-generation qualify with varying individual efficiency to generate leukemic, mature, migratory and viable DC in individual cases. 3. To select the best DC-generating method the best DC-marker (no expression on naïve blasts, high expression on DC) has to be chosen to quantify DC in individual samples. 4. The use of only one method is not sufficient to create DC in every single AML and MDS sample. However, a successful, quantitative DC/DCleu -generation is possible in every case of AML and MDS by the combination of 3 different DC-generating media, but not every blast is convertible to DC leu . 5. There is a need for new, specific DC-markers that are not expressed on naïve blasts.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 10/19
Die protektive Wirkung von Immunsuppressiva auf den hepatischen I/R-Schaden deutet darauf hin, dass T-Zellen bei diesem alloantigen-unabhängigen Ereignis eine Rolle spielen. Die Mechanismen der Aktivierung bzw. der mikrovaskulären Rekrutierung von CD4+ T-Zellen bei alloantigen-unabhängiger I/R der Leber sind jedoch weitgehend ungeklärt. Ziele der vorliegenden Arbeit waren daher (1) die Rekrutierung von T-Zell-Subopulationen in den postischämischen hepatischen Mikrogefäßen in vivo zu untersuchen, (2) die Mechanismen einer Interaktion von CD4+ T-Zellen und Thrombozyten während hepatischer I/R zu analysieren, (3) die Rolle von CD4+ T-Zellen an der Ausbildung des hepatischen I/R-Schadens zu beurteilen, (4) zu untersuchen, ob die postischämische Rekrutierung von CD4+ T-Zellen MHC Klasse II-abhängig stattfindet und (5) zu analysieren, ob CD4+ T-Zellen während hepatischer I/R mit Kupffer-Zellen interagieren. In der vorliegenden Studie konnte erstmals in vivo der Typ, die mikrovaskuläre Lokalisation und die Kinetik der Lymphozyten-Endothelzell-Interaktion während hepatischer I/R intravitalmikroskopisch charakterisiert werden. So konnte gezeigt werden, dass insbesondere CD4+ T-Zellen, und nicht CD8+ T-Zellen, während I/R in der hepatischen Mikrozirkulation akkumulieren. Diese Akkumulation tritt hauptsächlich in den Sinusoiden auf, nur zu einem geringeren Teil in den postsinusoidalen Venolen. Bereits nach 30-minütiger Reperfusion ist gegenüber der schein-operierten Gruppe eine signifikante Zunahme der Anzahl akkumulierter CD4+ T-Zellen in den Mikrogefäßen der Leber zu beobachten, die Anzahl emigrierter CD4+ T-Zellen nimmt im Verlauf der Reperfusionszeit signifikant zu. Im Rahmen der Untersuchungen konnte gezeigt werden, dass CD4+ T-Zellen an der Ausbildung des hepatischen I/R-Schadens beteiligt sind. Über CD40L- und CD28-abhängige Signalwege ist die postischämische Akkumulation von Thrombozyten und Leukozyten in der hepatischen Mikrozirkulation von CD4+ T-Zellen abhängig. Darüber hinaus wird die Ausbildung des mikrovaskulären Schadens, gemessen anhand des sinusoidalen Perfusionsdefizites, sowie die Ausbildung des hepatozellulären Schadens, gemessen anhand der hepatischen Transaminasen, CD40L- und CD28-abhängig über CD4+ T-Zellen mediiert. Mittels simultaner Visualisierung zweier Zellpopulationen in vivo konnte in dieser Dissertations¬schrift erstmals nachgewiesen werden, dass CD4+ T-Zellen und Thrombozyten während hepatischer I/R kolokalisieren. Unter Verwendung P-Selektin- und CD40L-defizienter Mäuse konnte in vivo nachgewiesen werden, dass eine feste Adhärenz zwischen Thrombozyten und CD4+ T-Zellen über P-Selektin und PSGL-1 vermittelt wird, während die kostimulatorischen Moleküle CD40 und CD40L eine reziproke Aktivierung unter Thrombozyten und CD4+ T-Zellen bedingen. In einem weiteren Abschnitt dieser Studie konnte unter Verwendung von blockierenden Antikörpern schließlich erstmals in vivo gezeigt werden, dass die im Rahmen der hepatischen I/R stattfindende Aktivierung von CD4+ T-Zellen MHC-Klasse II-unabhängig abläuft. Schließlich wurde in einem weiteren Abschnitt dieser Dissertationsschrift erstmals in vivo nachgewiesen, dass eine reziproke Aktivierung von Kupffer-Zellen und CD4+ T-Zellen während hepatischer I/R vorliegt. Die Anzahl postischämisch akkumulierter CD4+ T-Zellen ist nicht nur nach vollständiger Depletion von Kupffer-Zellen, sondern auch nach selektiver Unterbindung der Signalwege über TNF-α und IL-6 sowie des Abfangens freier Sauerstoffradikaler signifikant vermindert. Vice versa konnte hier Anhand der Untersuchung der Phagozytoseaktivität von Kupffer Zellen mittels Latex-Beads gezeigt werden, dass CD4+ T-Zellen die Aktivität von Kupffer-Zellen beeinflussen. Weitergehende Untersuchungen zur reziproken Aktivierung von Kupffer-Zellen und CD4+ T-Zellen konnten unter Verwendung von Durchflusszytometrie zeigen, dass proinflammatorische Mediatoren wie TNF-α und IL-6, vornehmlich freigesetzt durch Kupffer-Zellen während hepatischer I/R, nicht nur direkt aktivierend auf CD4+ T-Zellen wirken, sondern auch sinusoidale Endothelzellen aktivieren können. Eine Aktivierung der sinusoidalen Endothelzellen mit entsprechender Alteration der Expression von Adhäsionsmolekülen, wie z.B. ICAM-1, VCAM-1 und VAP-1 stellt wiederum einen pathophysiologischen Mechanismus dar, der mit einer konsekutiven Verstärkung der Akkumulation von CD4+ T-Zellen nach I/R verbunden ist. Zusammenfassend weisen diese in vivo Daten darauf hin, dass hepatische I/R die Akkumulation und Emigration von CD4+ T-Zellen, jedoch nicht von CD8+ T-Zellen induziert. Adhärente CD4+ T-Zellen sind in Sinusoiden mit Thrombozyten kolokalisiert; dies lässt eine gegenseitige Aktivierung beider Zelltypen durch direkten Zellkontakt oder über die Aktivierung des Endothels vermuten. Eine CD4 T-Zell-Defizienz geht mit einer Verminderung der postischämischen Thrombozytenakkumulation und mit einer Reduktion des mikrovaskulären I/R-Schadens einher. Die postischämische Rekrutierung von CD4+ T-Zellen in hepatischen Mikrogefäßen wird durch Kupffer-Zellen, wahrscheinlich über die Freisetzung von Sauerstoffradikalen, TNF-α und IL-6, vermittelt.
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 03/06
Die adoptive T-Zelltherapie ist eine attraktive Alternative zu konventionellen Therapien zur Behandlung von malignen Erkrankungen. So konnten bereits Tumorremissionen bei Melanompatienten nach adoptivem T-Zelltransfer erreicht werden (Dudley et al, 2002b; Morgan et al, 2006). Während im autologen System jedoch oft nur unzureichende Antitumorantworten zu generieren sind, zeigt der Erfolg der allogenen Stammzelltransplantation, dass im allogenen System T-Zellen hoch effektiv Tumorzellen bekämpfen können. Die allogene Stammzelltransplantation konnte auch bei B-Zell-Non-Hodgkin-Lymphomen, wie beispielsweise der chronischen lymphatischen Leukämie (CLL), mit Hilfe eines Transplantat-gegen-Leukämie-Effektes (Graft-versus-Leukemia, GvL) lang andauerndes, krankheitsfreies Überleben bewirken. Sie birgt aber ein sehr hohes Morbiditäts- und Mortalitätsrisiko auf Grund der Transplantat-gegen-Wirts-Erkrankung (Graft-versus-Host-Disease, GvHD) in sich. Die im Transplantat enthaltenen T Zellen sind hierbei sowohl für den erwünschten GvL-Effekt verantwortlich, gleichzeitig aber auch für die unerwünschte GvHD (Horowitz et al, 1990; Kolb et al, 2004). Zur Minimierung des Risikos einer GvHD könnten T Zellen eingesetzt werden, die spezifisch und allorestringiert Peptide von tumorspezifischen Antigenen erkennen und somit bevorzugt Tumorzellen angreifen. Die Reaktivität der T Zellen kann durch einen T Zellrezeptor (TZR)-Transfer auf sekundäre Zellen übertragen werden. Diese transgenen Zellen können dann mittels adoptivem T Zelltransfer im Patienten zur selektiven Bekämpfung von Tumorzellen zum Einsatz kommen. In Vorarbeiten wurde FMNL1 (formin related protein in leukocytes 1) als hoch attraktives tumorassoziiertes Antigen identifiziert, das in der chronischen lymphatischen Leukämie (CLL) und in anderen Lymphomen, sowie in Zelllinien solider Tumoren stark überexprimiert wird, während es in gesunden Zellen fast ausschließlich in hämatopoetischen Zellen vorkommt. Ziel der vorliegenden Arbeit war es, allorestringierte FMNL1-peptidspezifische T-Zellen zu isolieren, zu charakterisieren und den T-Zellrezeptor dieser T-Zellen in sekundäre Zellen zu transduzieren. Hierzu wurden Peptide des tumorassoziierten Antigens FMNL1 mit Hilfe von Prädiktionsalgorithmen vorhergesagt und in T Zell-Stimulationsansätzen eingesetzt. Unter Einsatz von HLA-A2-positiven T2-Zellen als antigenpräsentierende Zellen, die mit dem prädizierten synthetischen Peptid FMNL1-PP2 beladen waren, ist es gelungen allorestringierte, FMNL1-PP2-spezifische T Zellen eines gesunden HLA-A2-negativen Spenders zu isolieren. Von 67 T-Zellklonen bzw. oligoklonalen T-Zellen konnte bei neun T-Zellklonen Allorestriktion und FMNL1-PP2-Peptidspezifität nachgewiesen werden. Der T-Zellklon SK22 war für diese neun T-Zellklone, die auf Sequenzebene einen identischen T-Zellrezeptor aufwiesen, repräsentativ. Der T-Zellklon SK22 zeigte in Reaktion auf peptidbeladene T2-Zellen eine hohe Peptidspezifität für FMNL1-PP2 im Kontext mit dem für SK22 allogenen HLA-A2. Nach Zielzellerkennung sezernierte der T-Zellklon Zytokine wie IFNγ, TNFα, GM-CSF und teilweise IL2. Der T Zellklon zeigte eine hohe Aktivität und mittlere Avidität gegen FMNL1 PP2-beladene T2-Zellen. Des Weiteren wurde die Reaktivität gegen unbeladene native Zellen getestet. Der T-Zellklon SK22 erkannte verschiedene Zellen, wenn sie HLA-A2-positiv waren und gleichzeitig FMNL1 exprimierten. Hierzu zählten zum einen maligne Zellen, darunter verschiedene Epstein-Barr-Virus (EBV)-positive und EBV-negative Lymphomzelllinien und die Nierenzellkarzinomzelllinie RCC26, die gut erkannt wurden sowie CD40-aktivierte CLL-Zellen, die schwächer erkannt wurden. Bei der Untersuchung von gesundem Gewebe wurden FMNL1-exprimierende HLA-A2-positive periphere Blutleukozyten (PBL) schwach und B-Zellen in mittlerer Stärke erkannt. HLA-A2-positive Zellen, die FMNL1 nicht exprimieren, wie beispielsweise Lungenfibroblasten, wurden vom T-Zellklon SK22 nicht erkannt. Der T Zellklon zeigte Kreuzreaktivität gegen neun verschiedene lymphoblastoide Zelllinien (LCL), die Allelvarianten von HLA-A2 exprimierten. Zusätzlich wurden 4 von 18 HLA-A2-negativen LCL-Zelllinien erkannt. Jeweils zwei dieser vom T Zellklon SK22 erkannten HLA-A2-negativen LCL-Zelllinien trugen ein gemeinsames MHC-Klasse-I-Molekül. Eines davon war HLA-A*3303, welches durch die Erkennung der HLA-A*3303-positiven Transfektante der C1R-Zelllinie bestätigt werden konnte. Das andere war HLA-A*6802, welches zur HLA-A2-Superfamilie gehört. Der T-Zellrezeptor des T-Zellklons SK22 wurde identifiziert, sequenziert und kloniert, sowie mit Hilfe von Retroviren in sekundäre Zellen eingebracht. Durch den Transfer des T Zellrezeptors von SK22 in sekundäre Zellen konnte nachgewiesen werden, dass dieser T Zellrezeptor für die spezifische Reaktivität des T-Zellklons SK22 verantwortlich war. Dies zeigte sich in der T-Zellrezeptor-Oberflächenexpression nach Transduktion in Jurkat76-CD8α-Zellen und in der Übertragung der Funktionalität des T-Zellklons in PBL. Der T Zellrezeptor von SK22 ist ein „schwacher“ Rezeptor, da er in der Konkurrenzsituation mit einem weiteren Rezeptor nur in geringem Grade an der Zelloberfläche von PBL exprimiert wurde. Durch einen Austausch der jeweiligen konstanten Regionen der T-Zellrezeptor-SK22-Sequenzen durch die konstanten Bereiche eines murinen T-Zellrezeptors konnten in der Summe verbesserte Expressionswerte in Jurkat76-Zellen und eine verbesserte Funktionalität in PBL erreicht werden. Der T-Zellklon SK22 zeigte Allorestriktion, FMNL1-PP2-Peptidspezifität und Zytotoxizität gegen FMNL1-exprimierende Zellen, insbesondere gegen Tumorzellen. Die beobachtete Kreuzreaktivität ist Fokus weiterführender Untersuchungen. Im Fall des T-Zellrezeptors von SK22 bedeutet es, dass Spender und Patienten sorgfältig nach Analyse des gesamten MHC-Klasse-I-Expressionsmuster ausgewählt werden müssen. Im Rahmen einer haploidentischen Stammzelltransplantation ist jedoch der klinische Einsatz dieses spezifischen T-Zellrezeptors zur Behandlung von B-Zell-Non-Hodgkin-Lymphomen und anderen FMNL1-überexprimierenden Tumorerkrankungen vielversprechend.
Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 03/07
Effect of B-cell activating factor of the tumor necrosis factor family (BAFF) on the expression of pro- and anti-apoptotic bcl-2 family members in chicken B cells The recently discovered chicken cytokine BAFF (B-cell activating factor of the tumor necrosis factor family) was characterised as an important regulator of chicken B-cell homeostasis. Besides regulating B-cells in secondary lymphatic organs BAFF seems to have a significant impact on chicken B-cell development in the Bursa of Fabricius, too. Past studies already showed that chicken BAFF plays a vital role in the survival of B-cells both in vitro and in vivo. Yet molecular correlation for this effect has still to be established in the chicken. In mouse and man, the antiapoptotic effect of BAFF was linked to a regulation of certain bcl-2 family members. Thus this study focused on the identification of bcl-2 family members in the chicken and their regulation by BAFF at transcriptional level. By means of RT-PCR the expression of both anti-apoptotic (e.g. bcl-2, bcl-xL and Nr13) as well as pro-apoptotic (e.g. bak, bid, bim and bok) transcripts was shown in bursa, spleen and heart muscle at various developmental stages. To enable further studies quantitative RT-PCR assays were established for both anti-apoptotic (e.g. bcl-2, bcl-xL and Nr13) and pro-apoptotic (e.g. bak, bid, bim and bok) bcl-2 family members as well as for the B-cell specific marker chB6 and chBAFF. During bursal development, transcripts for pro-apoptotic bok and anti-apoptotic bcl-xL are increased while the level of bcl-2 mRNA is decreased. Considering the vast raise in B-cell number within the developing bursa, a means of correlating this with characterised changes in transcription levels had to be established. This was done based on expression levels of the B-cell marker chB6. Thus it could be shown that transcription of both anti-apoptotic genes like bcl-2 and Nr13 and pro-apoptotic genes such as bak and bim were decreased based on the amount of B-cell. In contrast, levels of bok transcript remained unchanged in B-cells during bursal development. Isolated lymphocytes taken from the spleen were used for inital studies on the impact of BAFF in vitro. In agreement with published data, the anti-apoptotic effect of BAFF could be demonstrated in this study, too. At transcriptional level, this was linked to a decrease in the transcription of pro-apoptotic bim. In contrast, incubation of spleen cells with chicken CD40-ligand resulted in the vast proliferation of B-cells from both juvenile and mature birds. However, age-related differences in the survival of lymphocytes were observed in this study, which correlated with a lower increase of anti-apoptotic bcl-xL in mature cells than in juvenile in response to CD40-ligand stimulation. To further analyse the effect of BAFF on bursal B-cell development in vivo a previously published retroviral vector system (RCAS) was utilized. Both the effect of overexpression of BAFF as well as its neutralization using a soluble decoy receptor (BCMA) were characterised at transcriptional level. Overexpressing BAFF led to insignificant changes during the development of the bursa. Since BAFF is expressed at high levels during all stages of bursal development, gene overexpression may not exert additional effects. Neutralization of BAFF on the other hand caused distinct changes among bcl-2 family members at the transcriptional level. It again proofed necessary to correlate these changes with B-cell numbers represented by the level of chB6 transcription. By this method genes highly expressed within the remaining B-cell population were characterised. Anti-apoptotic bcl-2 along with Nr13 was shown to be significantly increased in comparison to control cells.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 08/19
In dieser Arbeit wurden immunhistologische Charakteristika der Nickel-induzierten (Epikutantest-) Ekzemreaktion im Vergleich mit Nickel-exponierter, reaktionsloser Haut analysiert. Weiterhin wurden immunhistologische Untersuchungen an bei Revisionsoperationen erhaltenen Gewebeproben aus der Umgebung nicht-zementierter CrCOMo-basierter Hüftendoprothesen durchgeführt. Die Analysen wurden aus 10 Biopsien aus Nickelepikutantestarealen (5 Personen mit, 5 Personen ohne Nickelallergie) und an 6 periimplantären Gewebeproben durchgeführt. Für Nickel-allergische Personen (Ekzem im Testfeld) waren im Vergleich mit Nickel-exponierter, klinisch reaktionsloser Haut deutliche Infiltrate CD3+ T-Lymphozyten mit überwiegend CD4+ Zellen sichtbar. Das entzündliche Infiltrat wurde anhand der Expression von IL-2-Rezeptor und CD69 (als früher Aktivierungsmarker) als Hinweis auf aktivierte bzw. dem Wachstumsfaktor IL-2 empfängliche Zellen hinterfragt. Hier zeigte sich eine – wenn auch individuell schwankende – erhöhte Expression im Vergleich zu den nicht-allergischen Personen. Dazu passt auch die über Expression des Markers Ki-67 erkennbare, gesteigerte Proliferation innerhalb der Infiltratzellen in Gruppe 1. Daneben war die „physiologische“ proliferative Aktivität in den basalen Epidermisanschnitten erkennbar. Bei der Untersuchung von Adhäsionsstrukturen fiel in den mit Ekzem reagierenden Nickel-Testfeldern eine vergleichsweise deutliche Expression von ICAM-1 und E-Selektin (CD62E) auf. Dem könnte die bei einer Kontaktallergie unter TH1-Dominanz vorherrschende IFN-γ-Produktion zugrunde liegen. In einem TNF-α/IFN-γ-reichen Umfeld wird auch die Expression von CD40 als kostimulatorische Struktur für aktivierte T-Zellen gefördert. Eine CD40-Hochregulation war speziell in Proben der Gruppe 1 zu beobachten. Wir haben nun innerhalb der Biopsien nickelallergischer Personen auch CD45RO-tragende Zellen nachweisen können. Dies deutet auf die Anwesenheit von (Antigen-erkennenden) Memory-T-Zellen hin. An Gewebeproben aus periimplantärem Gewebe – erhalten bei Revisionsoperationen nicht-infektiöser unzementierter CrCoMo-Hüftgelenke – wurde in unserer Arbeit gefragt, ob sich Elemente wie bei der kutanen Spättypüberempfindlichkeitsreaktion auf Nickel finden lassen. Auch wenn diese Gewebe verschiedenen Legierungsmetallen und Partikeln noch dazu über Jahre hinweg exponiert waren, so fanden sich auch hier individuell schwankend: CD3+ Lymphozyten, meist CD4-dominiert; Hochregulation von Adhäsionsmolekülen; Expression der kostimulatorischen Struktur CD40; IL-2-R+ Zellen. Speziell im Präparat E2 waren auch deutlich CD45RO-tragende Zellen im Sinne von Memory-Zellen sichtbar. Bei diesem Patienten hatten Schmerzen, Lockerung und Ergussbildung zum Wechsel seiner Metall-Metall-Hüftendoprothese geführt. Zusammenfassend konnten mit den beschriebenen Methoden Charakteristika des Nickel-Kontaktekzems untersucht werden. Periimplantäre Gewebe aus revidierten Metall-Metall-Hüftendoprothesen können ebenfalls wie hier beschrieben lymphozytäre Entzündungsphänomene aufweisen. Die Ergebnisse sollen Ausgangspunkt zur Untersuchung einer möglichen metallallergischen Komponente bei nicht-infektiöser Endoprothesenunverträglichkeit sein.
It is generally assumed that human differentiated cells have a limited life-span and proliferation capacity in vivo, and that genetic modifications are a prerequisite for their immortalization in vitro. Here we readdress this issue, studying the long-term proliferation potential of human B cells. It was shown earlier that human B cells from peripheral blood of healthy donors can be efficiently induced to proliferate for up to ten weeks in vitro by stimulating their receptor CD40 in the presence of interleukin-4. When we applied the same stimuli under conditions of modified cell number and culture size, we were surprised to find that our treatment induced B cells to proliferate throughout an observation period of presently up to 1650 days, representing more than 370 population doublings, which suggested that these B cells were immortalized in vitro. Long-term CD40-stimulated B cell cultures could be established from most healthy adult human donors. These B cells had a constant phenotype, were free from Epstein-Barr virus, and remained dependent on CD40 ligation. They had constitutive telomerase activity and stabilized telomere length. Moreover, they were susceptible to activation by Toll-like receptor 9 ligands, and could be used to expand antigen-specific cytotoxic T cells in vitro. Our results indicate that human somatic cells can evade senescence and be conditionally immortalized by external stimulation only, without a requirement for genetic manipulation or oncoviral infection. Conditionally immortalized human B cells are a new tool for immunotherapy and studies of B cell oncogenesis, activation, and function.
Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 03/07
Die B-Zellentwicklung der Vögel zeigt im Vergleich zu Maus und Mensch grundsätzliche Unterschiede. Davon ausgehend konnte in neuerer Zeit auch für die meisten Haustierspezies gezeigt werden, dass sie für die Reifung ihrer B-Zellen darmassoziiertes lymphatisches Gewebe (GALT) verwenden. Da Hühner-B-Zellen in einem einzigartigen GALT-Organ, der Bursa fabricii reifen, stellt das Huhn ein exzellentes Modell dar, um die zugrunde liegenden Mechanismen der B-Zellreifung zu studieren. Zahlreiche Mausmodelle zeigen, dass TNF-TNF-R- Familienmitglieder wichtige Regulatoren der B-Zellreifung und –funktion darstellen. Um die Struktur und die Funktion des CD40-CD40L-Systems im Huhn zu untersuchen, wurde zuerst das CD40-Expressionsmuster auf hämatopoetischen Zellen und verschiedenen Zellinien mittels durchflusszytometrischer Untersuchungen unter Verwendung des monoklonalen Antikörpers AV79 analysiert. Alle B-Zellen aus Blut, Milz, Zäkaltonsillen und der Bursa exprimierten das CD40-Antigen. Im Gegensatz dazu konnte CD40 nur auf einer Subpopulation der T-Zellen gefunden werden. Bei der Analyse von Zellinien konnten sowohl eine B-Zellinie als auch eine T-Zellinie sowie embryonale Fibroblasten als CD40+ Zellen identifiziert werden. Um die funktionelle Rolle von CD40 im B-Zellsystem zu studieren, wurden B-Zellen aus Bursa, Milz und Zäkaltonsillen mit einem rekombinanten CD40L-Konstrukt stimuliert. Die Zugabe von rChCD40L verlängerte die Lebensspanne von B-Zellen signifikant und induzierte sowohl eine Proliferation der B-Zellen als auch einen Klassenwechsel der Immunglobuline. Die Aktivierung der B-Zellen durch rChCD40L führt zu einer verstärkten Expression von MHCII-Molekülen sowie zur Sekretion von IL-6. Zusätzlich konnten durch rChCD40L erstmals Langzeitkulturen primärer Hühner-B-Zellen etabliert werden. In diesen Langzeitkulturen war rChCD40L in der Lage, die antigenspezifischen Antikörpertiter in in vitro-Kulturen von Milz-B-Zellen immunisierter Tiere signifikant zu erhöhen. Ausgehend von diesen Daten kann auf eine essentielle Rolle des CD40-CD40L-Systems in der Entwicklung und der Funktion der B-Zellen in einem nicht zu den Säugetieren gehörenden Wirbeltier geschlossen werden. Somit stellt das CD40-CD40L-System ein phylogenetisch konserviertes System dar. Darüber hinaus bietet die Etablierung von Langzeitkulturen primärer Hühner-B-Zellen ein neues Werkzeug für Studien zur Wirt-Pathogen-Interaktion.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 05/19
Thu, 13 Jul 2006 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/5595/ https://edoc.ub.uni-muenchen.de/5595/1/Bongers_Martina.pdf Bongers, Martina
Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 02/07
Myokardiale Blockade des CD40-Liganden durch adenoviralen Gentransfer von CD40-Fc zur Steigerung der kardialen Kontraktionskraft und Verminderung des Remodeling im insuffizienten Kaninchenherz.
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 01/06
GDNF (glial cell line-derived neurotrophic factor) und NTN (Neurturin), die zwei zuerst beschriebenen Liganden der GDNF-Familie, fungieren als Überlebens- und Entwicklungsfaktoren für definierte Populationen von zentralen und peripheren Neuronen. GDNF ist darüber hinaus für die Nierenentwicklung erforderlich. Für die Vermittlung ihrer biologischen Wirkung benutzten GDNF und NTN einen Rezeptor, der aus zwei Ketten besteht: Die Signal-transduzierende Komponente RET wird sowohl von GDNF als auch von NTN benutzt. RET wird von 21 Exonen kodiert und kommt in multiplen Spleiß-Varianten vor. Für die Liganden-Spezifität ist eine zweite Rezeptorkomponente verantwortlich, ein Mitglied der GFR-Familie. GFRa-1 bindet präferentiell GDNF, während GFRa-2 NTN stärker als GDNF bindet. Ziel dieser Arbeit war es, mögliche wechselseitige Interaktionen zwischen dem Nerven- und Immunsystem durch die GDNF-Familie zu untersuchen. Zu diesem Zweck wurde zunächst die Expression von GDNF, NTN und ihrer Rezeptoren in gereinigten Immunzell-Subtypen untersucht. Dabei zeigte sich, dass der Prototyp dieser Liganden-Familie, GDNF, von keiner der untersuchten Immunzellen exprimiert wurde. Hingegen wurde das verwandte NTN von T-Zellen, B-Zellen und Monozyten exprimiert wie mit RT-PCR, Western Blot und Immunzytochemie gesehen wurde. Transkripte für das zu NTN und GDNF verwandte Persephin (PSP) wurden in Monozyten und mononukleären Zellen des peripheren Blutes gefunden. Der Transmembran-Rezeptor RET wurde von allen untersuchten Immunzell-Subtypen exprimiert. B-Zellen und T-Zellen exprimierten unterschiedliche Isoformen von RET, sowohl im extrazellulären Liganden-bindenden als auch im intrazellulären Signal-transduzierenden Teil. Die Expression der Isoformen von RET wurde zudem in T-Zellen und B-Zellen noch stark durch Aktivierung reguliert. In CD8+ T-Zellen wurde auch eine bislang noch nicht beschriebene Spleiß-Variante am 5` Ende beobachtet. Im Gegensatz zu T-Zellen und B-Zellen exprimierten Monozyten nur die volle Länge von RET. Auch die Liganden-bindenden Ketten GFRa-1 und GFRa-2 wurden von Immunzellen exprimiert wie mit RT-PCR und FACS gesehen wurde. GFRa-2 war deutlich abundanter als GFRa-1. Von GFRa-2 wurden verschiedene Isoformen in Immunzellen gefunden. In der in T-Zellen und B-Zellen am stärksten exprimierten Isoform ist Exon 2 und 3 nicht enthalten. Dem resultierenden Protein fehlen die N-terminale Cystein-reiche Domäne und eine N-Glykosylierungsstelle, eine Region, die allerdings für die Bindung von NTN und die Interaktion mit RET entbehrlich ist. Mögliche Effekte von GDNF und NTN auf Immunzellen wurden untersucht. Dabei zeigte sich, dass GDNF und NTN an der Regulation von TNF-alpha beteiligt sind. Wenn GDNF oder NTN nach 5 oder 6 Tagen zu LPS+IFN-g stimulierten Blutzellen oder zu ConA aktivierten T-Zellen gegeben wurde, dann war nach weiteren 24 h der TNF-a-Gehalt im Überstand reduziert. Weitere Experimente wiesen daraufhin, dass diese Reduktion des TNF-a-Gehalts auf eine verstärkte Aufnahme oder Verbrauch zurückzuführen ist. Proliferation, Expression von Aktivierungsmarkern (HLA-DR, CD38, CD40, CD69, CD86) oder Produktion von IFN-g und IL-4 wurden durch GDNF und NTN nicht beeinflusst. Zusammenfassend zeigt diese Arbeit, dass Immunzellen den neurotrophen Faktor NTN produzieren und Rezeptoren für GDNF und NTN besitzen. Multiple Isoformen der Signal-transduzierenden Kette RET wurden exprimiert und durch Aktivierung reguliert. NTN und GDNF regulierten in aktivierten T-Zellen und Monozyten die Aufnahme oder den Verbrauch von TNF-a. Diese Befunde weisen daraufhin, dass Immunzellen miteinander und auch mit dem Nervensystem mit Hilfe der GDNF-Familie interagieren können.
Pathogenic events leading to in-stent restenosis (ISR) are still incompletely understood. Among others, inflammation, immune reactions, deregulated cell death and growth have been suggested. Therefore, atherectomy probes from 21 patients with symptomatic ISR were analyzed by immunohistochemistry for pathogen burden and compared to primary target lesions from 20 stable angina patients. While cytomegalovirus, herpes simplex virus, Epstein-Barr virus and Helicobacter pylori were not found in ISR, acute and/or persistent chlamydial infection were present in 6/21 of these lesions (29%). Expression of human heat shock protein 60 was found in 8/21 of probes (38%). Indicated by distinct signals of CD68, CD40 and CRP, inflammation was present in 5/21 (24%), 3/21 (14%) and 2/21 (10%) of ISR cases. Cell density of ISR was significantly higher than that of primary lesions ( 977 +/- 315 vs. 431 +/- 148 cells/mm(2); p < 0.001). There was no replicating cell as shown by Ki67 or PCNA. TUNEL+ cells indicating apoptosis were seen in 6/21 of ISR specimens (29%). Quantitative analysis revealed lower expression levels for each intimal determinant in ISR compared to primary atheroma (all p < 0.05). In summary, human ISR at the time of clinical presentation is characterized by low frequency of pathogen burden and inflammation, but pronounced hypercellularity, low apoptosis and absence of proliferation. Copyright (C) 2004 S. Karger AG, Basel.
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 01/06
Aufgrund der Zunahme an Organ- und Knochenmarkstransplantationen und der damit verbundenen Immunsuppression bzw. immunsuppressiven Therapie sowie der zunehmenden Zahl an AIDS-Patienten ist das Zytomegalovirus (CMV) als Pathogen in den letzten zwanzig Jahren trotz der Einführung wirksamer antiviraler Medikamente bis heute von großer klinischer Bedeutung. Während bei immunkompetenten Personen eine primäre CMV-Infektion durch das Immunsystem kontrolliert werden kann, führt eine Primärinfektion oder eine Reaktivierung einer latenten CMV-Infektion in immunsupprimierten Patienten zu lebensbedrohlichen Komplikationen. Die Pathogenese einer CMV-Infektion wird entscheidend von der Qualität der antiviralen Immunantwort des Wirtes beeinflusst und Kenntnisse über die Interaktion von CMV mit dem Immunsystem sind für die Prophylaxe und Behandlung einer CMV-Infektion von großer Bedeutung. Dendritische Zellen (DCs) sind die wichtigsten Antigen-präsentierenden Zellen des Immunsystems und spielen bei der Initiierung einer antiviralen Immunantwort eine zentrale Rolle. Die Stimulation von naiven T-Zellen durch DCs und die Auslösung einer zytotoxischen T-Lymphozyten-Antwort trägt entscheidend zur Eliminierung von viral-infizierten Zellen bei. Die Interaktion des Zytomegalovirus mit dendritischen Zellen gibt dem Virus eine Möglichkeit, seine Eliminierung durch das Immunsystem des Wirtes entscheidend zu beeinflussen. Zur Identifikation von Zielzellen für latente und lytische Infektionen durch MCMV und zur Untersuchung der Auswirkungen einer MCMV-Infektion auf den Phänotyp und die Funktion der Zellen wurde die murine hämatopoetische Stammzelllinie FDCP-Mix als Modellsystem verwendet. Definierte Differenzierungsstadien der Zellen entlang der dendritischen Reihe wurden hierzu mit einer GFP-exprimierenden MCMV-Mutante infiziert. Während undifferenzierte FDCP-Mix-Zellen und von FDCP-Mix-Zellen abgeleitete reife DCs nicht produktiv infizierbar waren, setzten unreife DCs infektiöse Virusnachkommen frei. In reifen DCs wurden nur virale Proteine der sehr frühen und frühen Phase der viralen Genexpression synthetisiert, während späte Genprodukte nicht nachgewiesen werden konnten. Die Infektion unreifer und reifer DCs resultierte anfänglich in deren Aktivierung, erkennbar an der vorübergehend verstärkten Expression der Oberflächenmoleküle CD80, CD86, CD40, MHC-Klasse-I und Klasse-II. Die verstärkte Expression der MHC- und ko-stimulatorischen Moleküle auf reifen DCs einige Stunden nach Infektion spiegelte sich in einer gesteigerten Stimulation naiver autologer T-Zellen durch infizierte DCs wider. In der späten Phase der Infektion war die Aktivierung von autologen T-Zellen beeinträchtigt. Dies korrelierte mit der reduzierten Oberflächenexpression der MHC- und ko-stimulatorischen Moleküle auf infizierten reifen DCs. Allogene T-Zellen konnten durch MCMV-infizierte DCs weder in der frühen noch in der späten Phase der Infektion stimuliert werden. Diese Ergebnisse sprechen dafür, dass DCs im Laufe einer MCMV-Infektion mehrere Rollen spielen: (1) unreife DCs produzieren MCMV-Nachkommen und können so zur Verbreitung des Virus im Wirt beitragen; (2) in einem frühen Stadium der Infektion aktivieren DCs naive T-Zellen und initiieren damit eine antivirale Immunantwort, die einer Ausbreitung der viralen Infektion entgegenwirkt. (3) Zu einem späteren Zeitpunkt der Infektion ist die Stimulation der T-Zell-Proliferation durch MCMV-infizierte DCs beeinträchtigt. Dies ist einer der Mechanismen, welche die Persistenz des Virus in seinem Wirt ermöglichen. Unabhängig vom Zeitpunkt der Infektion ist bei der allogenen Transplantation die Induktion der T-Zell-Antwort immer beeinträchtigt. Die Unfähigkeit der CMV-infizierten DCs, naive allogene T-Zellen zu stimulieren, trägt so zu einer reduzierten antiviralen Kontrolle bei, was CMV-verbundene Krankheiten nach allogenen Knochenmarkstransplantationen begünstigt und gravierende gesundheitliche Probleme zur Folge hat.
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 01/06
EBV is a γ-herpes virus which is able to infect human resting B-cells and to transform them into permanently growing lymphoblastoid cell lines (LCLs). EBNA2 (Epstein-Barr virus nuclear antigen 2) is one of the first viral proteins expressed after in vitro infection and interacts with different cellular proteins like RBP-Jκ and PU.1. The EBNA2 protein acts as a transcriptional activator of the viral Latent Membrane Proteins 1 and 2 (LMP1 and LMP2) and the viral nuclear genes EBNA1, EBNA3A, -3B, -3C, EBNA-LP. Additionally EBNA2 is also able to transactivate cellular genes like CD21, CD23 or c-myc. To study the different EBNA2 target genes and the function of EBNA2 a LCL was established (ER/EB2-5 cells, Kempkes et al., 1995) harboring an estrogen-inducible EBNA2. In the presence of estrogen the ER/EBNA2 fusion protein (estrogen receptor binding domain) is located in the nucleus were EBNA2 can transactivate its target genes, whereas in the absence of estrogen the ER/EBNA2 fusion protein is kept in the cytoplasm and therefore inactive. The cells proliferate in the presence of estrogen and they arrest in the absence resulting in a phenotype similar to resting B-lymphocytes. By using the ER/EB2-5 cell line I could clearly show that the cell surface molecule CD83, belonging to the immunoglobuline superfamily (Zhou et al., 1992), is upregulated after the activation of EBNA2. By using a derivative ER/EB2-5 cell line that constitutively expressed LMP1 I could show that CD83 is still expressed even in the absence of functional EBNA2 suggesting that LMP1, the viral target gene of EBNA2, is responsible for the induction of CD83. Therefore I analysed the activation of the CD83 promoter by LMP1. LMP1 is a transmembrane protein with a short intracellular N-terminus, 6 hydrophobic transmembrane domains and a long intracellular C-terminus, containing C-terminal activator regions CTAR1, 2 and 3. The different CTAR regions are responsible for activating genes via NF-κB, ATF, AP1 and STAT signaling pathways. For the activation of its target genes LMP1 uses the same signaling molecules (TRAF, TRADD) as family members of the TNF-R family (CD40, TNF-R1, TNF-R2). The CD83 promoter was activated by LMP1 as shown by promoter luciferase reporter assays in 293-T cells. The induction was not observed in the absence of a NF-κB binding site in a CD83 promoter mutant. Furthermore LMP1 mutants which are mutated in the binding regions for TRAF2 (CTAR1) or TRADD (CTAR2) are not able to transactivate the CD83 promoter. By co-transfection of LMP1 and dominant/negative IκB the CD83 promoter could not be activated because of inactivation of NF-κB. These experiments clearly demonstrate that the CD83 promoter is transactivated by LMP1 via NF-κB. Additionally to the regulation of CD83 I was also interested in the functional role of CD83. Until now only little is known about the function of CD83. CD83 seems to have a specific role in the decision to single positive CD4+ T-cells in the thymus (Fujimoto et al., 2002). I have tested a possible co-stimulatory function of CD83 to CD4+ T-cells by retroviral expression of CD83 in non-professional antigen presenting cells (RCC). Indeed CD83 expression increased the CD4+ response in comparison to CD80 or GFP retroviral infected RCC cells. In mixed lymphocyte reactions this co-stimulatory effect could not be clearly demonstrated although a soluble CD83-Ig showed a small inhibitory influence. The identification of a CD83 ligand molecule could give new insights into the function of CD83. Therefore a CD83-Ig fusion protein as well as a CD83-tetramer construct were generated and used to screen for a potential ligand of CD83. First results showed that the CD83-Ig fusion protein and the CD83-tetramer construct bound to CD4+ and to CD8+ T-cells of isolated PBMCs as well as to activated T-cells in a culture of mixed T-cell populations.
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 01/06
Epstein-Barr Virus (EBV) infiziert ruhende primäre humane B-Zellen und induziert deren unbegrenzte Proliferation. Dieser Prozess der B-Zell-Immortalisation ist ein Modellsystem, das die pathogenetischen Mechanismen bei der Tumorentstehung widerspiegelt. In vitro gilt das virale latente Membranprotein 1 (LMP1) für die Immortalisation von B-Zellen als essentiell. LMP1 ist ein integrales Membranprotein, das als konstitutiv aktivierter Pseudorezeptor verschiedene Signalwege in der B-Zelle induziert und dabei analoge Funktionen zum zellulären CD40-Rezeptor wahrnimmt. Das Genom des EBV ist in dem Maxi-EBV-System einer genetischen Manipulation zugänglich. Zuerst habe ich verschiedene Mutanten des LMP1 Gens im Kontext des EBVGenoms etabliert und auf ihren Phänotyp untersucht. Überraschenderweise war es möglich, mit allen LMP1 mutierten EBVs proliferierende B-Zellklone zu generieren, dies gelang sogar mit einer „knock out“ Mutante des kompletten LMP1 Gens. Die zehn verschiedenen LMP1- Mutanten unterschieden sich gravierend in ihrer Effizienz, B-Zellen zu immortalisieren. So wurden bis zu 100 mal mehr Virionen benötigt, um z.B. mit der LMP1-„knock out“-Mutante proliferierende B-Zellklone zu etablieren. Eine solche B-Zelllinie wies in einem in vivo Experiment mit SCID-Mäusen im Gegensatz zu B-Zelllinien mit Wildtypvirus kein onkogenes Potential auf. Im Widerspruch zu den bisherigen Veröffentlichungen einer anderen Gruppe zeigen meine Ergebnisse, dass LMP1 für den Prozess der B-Zell-Immortalisation in vitro zwar kritisch, aber nicht zwingend notwendig ist. Für die Onkogenität von EBV in vivo ist LMP1 dagegen absolut essentiell. Die Zielgene des LMP1, die die verschiedenen Effekte wie B-Zell-Immortalisation, Onkogenität und Tumorentstehung vermitteln, sind nicht vollständig bekannt. Ein zweites Ziel dieser Arbeit war deshalb die umfassende Katalogisierung dieser Zielgene. Da LMP1 und der CD40-Rezeptor analoge Funktionen und gemeinsame Signalmediatoren und -wege aufweisen, sollten vergleichende Untersuchungen von LMP1- und CD40-regulierten Genen durchgeführt werden. Zu diesem Zweck wurde ein konditionales LMP1-System in humanen B-Zellen etabliert, das es erlaubt, LMP1-Signaltransduktion innerhalb eines sehr kurzen, definierten Zeitraums zu induzieren und differentiell exprimierte Gene zu identifizieren. Da der CD40-Rezeptor auf humanen B-Zellen konstitutiv exprimiert ist und durch Interaktion mit seinem Liganden aktiviert werden kann, konnte die Analyse CD40-regulierter Zielgene im selben Zellsystem erfolgen. Unter Anwendung von ATLAS Array-Filtern und Affymetrix Chips wurden 144 LMP1- und 28 CD40-regulierte Gene identifiziert. Schließlich konnte in Zellzyklusanalysen gezeigt werden, dass LMP1-Signale in humanen B-Zellen echte proliferative Effekte vermitteln und nicht nur anti-apoptotische Funktionen erfüllen.
Dendritic cells (DC) constitute a heterogeneous leukocyte population having in common a unique capacity to induce primary T cell responses and are therefore most attractive candidates for immunomodulatory strategies. Two populations of blood DC (CD11c+ CD123(dim) and CD11c- CD123(high)) have been defined so far. However, their direct isolation for experimental purposes is hampered by their low frequency and by the lack of selective markers allowing large scale purification from blood. Here we describe the monoclonal antibody (mAb) M-DC8, which was generated by immunizing mice with highly enriched blood DC. This mAb specifically reacts with 0.2-1% of blood leukocytes and enables their direct isolation by a one-step immunomagnetic procedure from fresh mononuclear cells. These cells can be differentiated from T cells, B cells, NK cells and monocytes using lineage-specific antibodies. M-DC8+ cells express HLA class It molecules, CD33 and low levers of the costimulatory molecules CD86 and CD40. Upon in vitro culture M-DC8+ cells spontaneously mature into cells with the phenotype of highly stimulatory cells as documented by the upregulation of HLA-DR, CD86 and CD40; in parallel CD80 expression is induced. M-DC8+ cells display an outstanding capacity to present antigen. In particular, they proved to be excellent stimulators of autologous mixed leukocyte reaction and to activate T cells against primary antigens such as keyhole limpet hemocyanin. Furthermore, they induce differentiation of purified allogeneic cytotoxic T cells into alloantigen-specific cytotoxic effector cells. While the phenotypical analysis reveals similarities with the two known blood DC populations, the characteristic expression of Fc gamma RIII (CD16) and the M-DC8 antigen clearly defines them as a novel population of blood DC. The mAb M-DC8 might thus be a valuable tool to determine circulating DC for diagnostic purposes and to isolate these cells for studies of antigen-specific T cell priming. Copyright (C) 2000 S. Karger AG, Basel.
We have constructed a soluble chimeric fusion protein between the mouse CD8 alpha chain and the mouse CD40 T cell ligand. This protein binds to both human and mouse B cells. By itself it induced a modest degree of B cell proliferation, but together with anti-immunoglobulin (anti-Ig) antibody it greatly stimulated B cell proliferation, as determined by both [3H]thymidine uptake and increase in cell numbers. These data are evidence that the CD40 ligand on T cells provides a signal that drives B cell proliferation. This signal is synergistic with that delivered by anti-Ig antibody.
PeerView Family Medicine & General Practice CME/CNE/CPE Audio Podcast
Go online to PeerView.com/FFX860 to view the activity, download slides and practice aids, and complete the post-test to earn credit. In this activity, an expert in Sjögren's Syndrome reviews current concepts in the pathophysiology of pSS, and discusses the obstacles faced in recent clinical trials and how new approaches may overcome these obstacles in the future. Upon completion of this activity, participants should be better able to: Recognize the burden of disease and unmet needs experienced by patients with primary Sjögren's Syndrome, Describe key pathways involved in the pathology of primary Sjögren's Syndrome and implications for treatment, Evaluate recent clinical trial data related to emerging treatment options for primary Sjögren's Syndrome, Identify patients who might derive benefit from novel therapeutic options for primary Sjögren's Syndrome.
PeerView Family Medicine & General Practice CME/CNE/CPE Video Podcast
Go online to PeerView.com/FFX860 to view the activity, download slides and practice aids, and complete the post-test to earn credit. In this activity, an expert in Sjögren's Syndrome reviews current concepts in the pathophysiology of pSS, and discusses the obstacles faced in recent clinical trials and how new approaches may overcome these obstacles in the future. Upon completion of this activity, participants should be better able to: Recognize the burden of disease and unmet needs experienced by patients with primary Sjögren's Syndrome, Describe key pathways involved in the pathology of primary Sjögren's Syndrome and implications for treatment, Evaluate recent clinical trial data related to emerging treatment options for primary Sjögren's Syndrome, Identify patients who might derive benefit from novel therapeutic options for primary Sjögren's Syndrome.
Go online to PeerView.com/FFX860 to view the activity, download slides and practice aids, and complete the post-test to earn credit. In this activity, an expert in Sjögren's Syndrome reviews current concepts in the pathophysiology of pSS, and discusses the obstacles faced in recent clinical trials and how new approaches may overcome these obstacles in the future. Upon completion of this activity, participants should be better able to: Recognize the burden of disease and unmet needs experienced by patients with primary Sjögren's Syndrome, Describe key pathways involved in the pathology of primary Sjögren's Syndrome and implications for treatment, Evaluate recent clinical trial data related to emerging treatment options for primary Sjögren's Syndrome, Identify patients who might derive benefit from novel therapeutic options for primary Sjögren's Syndrome.
Go online to PeerView.com/FFX860 to view the activity, download slides and practice aids, and complete the post-test to earn credit. In this activity, an expert in Sjögren's Syndrome reviews current concepts in the pathophysiology of pSS, and discusses the obstacles faced in recent clinical trials and how new approaches may overcome these obstacles in the future. Upon completion of this activity, participants should be better able to: Recognize the burden of disease and unmet needs experienced by patients with primary Sjögren's Syndrome, Describe key pathways involved in the pathology of primary Sjögren's Syndrome and implications for treatment, Evaluate recent clinical trial data related to emerging treatment options for primary Sjögren's Syndrome, Identify patients who might derive benefit from novel therapeutic options for primary Sjögren's Syndrome.
Go online to PeerView.com/FFX860 to view the activity, download slides and practice aids, and complete the post-test to earn credit. In this activity, an expert in Sjögren's Syndrome reviews current concepts in the pathophysiology of pSS, and discusses the obstacles faced in recent clinical trials and how new approaches may overcome these obstacles in the future. Upon completion of this activity, participants should be better able to: Recognize the burden of disease and unmet needs experienced by patients with primary Sjögren's Syndrome, Describe key pathways involved in the pathology of primary Sjögren's Syndrome and implications for treatment, Evaluate recent clinical trial data related to emerging treatment options for primary Sjögren's Syndrome, Identify patients who might derive benefit from novel therapeutic options for primary Sjögren's Syndrome.
Go online to PeerView.com/FFX860 to view the activity, download slides and practice aids, and complete the post-test to earn credit. In this activity, an expert in Sjögren's Syndrome reviews current concepts in the pathophysiology of pSS, and discusses the obstacles faced in recent clinical trials and how new approaches may overcome these obstacles in the future. Upon completion of this activity, participants should be better able to: Recognize the burden of disease and unmet needs experienced by patients with primary Sjögren's Syndrome, Describe key pathways involved in the pathology of primary Sjögren's Syndrome and implications for treatment, Evaluate recent clinical trial data related to emerging treatment options for primary Sjögren's Syndrome, Identify patients who might derive benefit from novel therapeutic options for primary Sjögren's Syndrome.
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