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2 episodes with cd45
2 episodes with cd45
2 episodes with cd45
Tonix Pharmaceuticals CEO Dr. Seth Lederman joined Steve Darling from Proactive to share exciting news about a significant achievement for the company. Tonix Pharmaceuticals has been awarded a potential contract worth up to $34 million over five years by the Defense Threat Reduction Agency (DTRA), an agency within the U.S. Department of Defense. This contract aims to develop small molecule broad-spectrum antiviral agents for the prevention or treatment of infections, thereby enhancing the medical readiness of military personnel in biological threat environments. Dr. Lederman explained that Tonix's program will concentrate on the optimization and development of its TNX-4200 program. The goal is to develop an orally available CD45 antagonist with broad-spectrum efficacy against various viral families through extensive preclinical evaluation. The program is expected to establish essential physicochemical properties, pharmacokinetics, and safety attributes to support an Investigational New Drug (IND) submission, ultimately funding a first-in-human Phase 1 clinical study. The agreement with DTRA is a strategic move to address the DoD's objective of protecting U.S. Joint Forces from potential biological weapon threats. The DoD announced in December 2022 that it aims to move beyond the traditional ‘one bug, one drug' approach and is seeking broad-spectrum drugs, as predicting which viruses or how many may be deployed in a biological threat scenario is challenging. Dr. Lederman emphasized that the collaboration with DTRA underscores Tonix Pharmaceuticals' commitment to advancing medical readiness and protection for military personnel. By developing a broad-spectrum antiviral agent, Tonix aims to provide a versatile and robust solution to potential viral threats, enhancing the preparedness and resilience of the U.S. military in diverse and unpredictable biological threat environments. The TNX-4200 program's focus on creating an effective, orally available antiviral agent highlights Tonix's innovative approach to addressing complex medical challenges. The successful development of this broad-spectrum antiviral could significantly impact the way viral infections are managed in military and potentially civilian populations, offering a proactive measure against a wide array of viral threats. Tonix Pharmaceuticals continues to be at the forefront of medical innovation, dedicated to developing cutting-edge solutions that meet the evolving needs of the healthcare and defense sectors. The support from the DoD through this substantial contract is a testament to the potential and importance of Tonix's TNX-4200 program in safeguarding the health and readiness of military personnel against biological threats. #proactiveinvestors #tonixpharmaceuticalsholdingcorp #nasdaq #tnxp #BroadSpectrumAntiviral, #USDepartmentOfDefense, #BiologicalWarfare, #DrugDevelopment, #ImmuneSystem, #SyntheticBiology, #VirusResearch, #InfectiousDisease, #R&D, #FrederickMaryland, #PharmaceuticalInnovation, #HealthcareTechnology, #Biotech, #MedicalResearch, #DefenseContract, #AntiviralAgent, #Healthcare, #PandemicPreparedness, #ScientificResearch#invest #investing #investment #investor #stockmarket #stocks #stock #stockmarketnews
This week, Catriona tells us about exciting new developments in using chimaeric antigen receptor (CAR) T cells against all forms of blood cancer; and Chris shares what physics can tell us about hand clapping, particularly the best way to clap for measuring acoustics.Wellhausen et al. 2023 “Epitope base editing CD45 in hematopoietic cells enables universal blood cancer immune therapy”: https://doi.org/10.1126/scitranslmed.adi1145(See also https://www.pennmedicine.org/news/news-releases/2023/august/an-immunotherapy-strategy-against-all-blood-cancers)Papadakis & Stavroulakis 2020 "Handclap for Acoustic Measurements: Optimal Application and Limitations: https://doi.org/10.3390/acoustics2020015Fletcher 2013 “Shock waves and the sound of a hand-clap – A simple model”: http://www.acoustics.asn.au/journal/2013/2013_41_2_Fletcher_paper.pdfMann et al. 2013 “The dynamics of audience applause”: https://doi.org/10.1098/rsif.2013.0466
A new research paper was published in Oncotarget's Volume 14 on July 20, 2023, entitled, “Development of a multiplex assay to assess activated p300/CBP in circulating prostate tumor cells.” Reduced SIRT2 deacetylation and increased p300 acetylation activity leads to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18) in castration-resistant prostate cancer (CRPC). In this new study, researchers Mikolaj Filon, Bing Yang, Tanaya A. Purohit, Jennifer Schehr, Anupama Singh, Marcelo Bigarella, Peter Lewis, John Denu, Joshua Lang, and David F. Jarrard from the University of Wisconsin examined whether circulating tumor cells (CTCs) identify patients with altered p300/CBP acetylation. “In the current study, employing a novel Exclusion-based Sample Preparation (ESP) technology (9) (Supplementary Figure 1) to isolate CTCs, we evaluated p300 activity, SIRT2 expression and H3K18 acetylation in CTCs in a series of patients with sensitivity or resistance to ADT.” CTCs were isolated from 13 advanced PC patients using Exclusion-based Sample Preparation (ESP) technology. Bound cells underwent immunofluorescent staining for histone modifying enzymes (HMEs) of interest and image capture with NIS-Elements software. Using the cBioPortal PCF/SU2C dataset, the response of CRPC to androgen receptor signaling inhibitors (ARSI) was analyzed in 50 subjects. Staining optimization and specificity revealed clear expression of acetyl-p300, acetyl-H3K18, and SIRT2 on CTCs (CK positive, CD45 negative cells). Exposure to A-485, a selective p300/CBP catalytic inhibitor, reduced p300 and H3K18 acetylation. In CRPC patients, a-p300 strongly correlated with its target acetylated H3k18 (Pearson's R = 0.61), and SIRT2 expression showed robust negative correlation with a-H3k18 (R = −0.60). A subgroup of CRPC patients (6/11; 55%) demonstrated consistent upregulation of acetylation based on these markers. To examine the clinical impact of upregulation of the CBP/p300 axis, CRPC patients with reduced deacetylase SIRT2 expression demonstrate shorter response times to ARSI therapy (5.9 vs. 12 mo; p = 0.03). A subset of CRPC patients demonstrate increased p300/CBP activity based on a novel CTC biomarker assay. “With further development, this biomarker suite may be used to identify candidates for CBP/p300 acetylation inhibitors in clinical development.” Read the full study: DOI: https://doi.org/10.18632/oncotarget.28477 Correspondence to: David F. Jarrard - jarrard@urology.wisc.edu Keywords: prostate cancer, circulating tumor cells, p300/CBP About Oncotarget: Oncotarget (a primarily oncology-focused, peer-reviewed, open access journal) aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science. To learn more about Oncotarget, visit Oncotarget.com and connect with us on social media: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Sign up for free Altmetric alerts about this article: https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28477 For media inquiries, please contact: media@impactjournals.com.
Auch wenn Adaptogene seit Jahrhunderten in der chinesischen Medizin und Ayuverda genutzt werden, sind sie erst seit wenigen Jahren auch bei uns im Fokus. Was sind Adaptogene? Was können Adaptogene leisten? Welche Adaptogene gibt es? Wie wirken Adaptogene? Was muss bei der Einnahme beachtet werden? Viel Spaß mit der Folge, deine Larry Blutbild-Analyse: https://calendly.com/larissareinelt/blutbild-analyse Studien-Auswahl: Evidence-based efficacy of adaptogens in fatigue, and molecular mechanisms related to their stress-protective activity, Panossian/Wikman, 2009 Understanding adaptogenic activity: specificity of the pharmacological action of adaptogens and other phytochemicals, Panossian, 2017 Chandrasekhar, K. et al. (2012): A Prospective, Randomized Double-Blind, Placebo-Controlled Study of Safety and Efficacy of a High-Concentration Full-Spectrum Extract of Ashwagandha Root in Reducing Stress and Anxiety in Adults. In: Indian Journal of Psychological Medicine, 34 (3), p. 255-262. Cooley, K. et al. (2009): Naturopathic Care for Anxiety: A Randomized Controlled Trial ISRCTN78958974. In: PLoS One, 4 (8), p. e6628. Andrade, C. et al. (2000): A double-blind, placebo-controlled evaluation of the anxiolytic efficacy of an ethanolic extract of withania somnifera. In: Indian Journal of Psychiatry, 42 (3), p. 295-301. Withania somnifera (Indian ginseng) in diabetes mellitus: A systematic review and meta-analysis of scientific evidence from experimental research to clinical application, Durg/Bavage/Shivaram, 2022. Protective effect of Astragalus membranaceus and Astragaloside IV in sepsis-induced acute kidney injury, Tang/Xin/Thang, 2022. Qi, Y. et al. 2017. Anti-Inflammatory and Immunostimulatory Activities of Astragalosides. Am J Chin Med. 45(6):1157–67. Wan, C. P. et al. 2013. Astragaloside II triggers T cell activation through regulation of CD45 protein tyrosine phosphatase activity. Acta Pharmacol Sin. 34(4):522–30.
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.17.537109v1?rss=1 Authors: Meza Guzman, L. G., Hyland, C. D., Bidgood, G. M., Leong, E., Shen, Z., Goh, W., Kratina, T., Rautela, J., Vince, J., Nicholson, S., Huntington, N. Abstract: The clinical development of Natural Killer (NK) cell-mediated immunotherapy marks a milestone in the development of new cancer therapies and has gained traction due to the intrinsic ability of the NK cell to target and kill tumour cells. To fully harness the tumour killing ability of NK cells, we need to improve NK cell persistence and overcome suppression of NK cell activation in the tumour microenvironment. The trans-membrane, protein tyrosine phosphatase CD45, regulates NK cell homeostasis, with genetic loss of CD45 in mice resulting in increased numbers of mature NK cells [1-3]. This suggests that CD45-deficient NK cells might display enhanced persistence following adoptive transfer. However, here we demonstrated that adoptive transfer of CD45-deficiency did not enhance NK cell persistence in mice, and instead, the homeostatic disturbance of NK cells in CD45-deficient mice stemmed from a developmental defect in the common lymphoid progenitor population. The enhanced maturation within the CD45-deficient NK cell compartment was intrinsic to the NK cell lineage, and independent of the developmental defect. CD45 is not a conventional immune checkpoint candidate, as systemic loss is detrimental to T and B cell development [4-6], compromising the adaptive immune system. Nonetheless, this study suggests that inhibition of CD45 in progenitor or stem cell populations may improve the yield of in vitro generated NK cells for adoptive therapy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534715v1?rss=1 Authors: van Deventer, S. J., Hoogvliet, I. A., van de Voort, M., Arnold, F., van Spriel, A. Abstract: Tetraspanin proteins play an important role in many cellular processes as they are key organizers of different plasma membrane receptors. Most tetraspanins are highly glycosylated, but the function of this post-translational modification remains largely unstudied. In this study we investigated the glycosylation of CD37 and CD53, two tetraspanins important for cellular and humoral immunity. Broad and cell-specific repertoires of N-glycosylated CD37 and CD53 were observed in human B cells. We generated different glycosylation mutants and analyzed their localization, nanoscale organization and protein interactions. Abrogation of glycosylation in CD37 revealed the importance of this modification for CD37 surface expression, whereas neither surface expression nor nanoscale organization of CD53 was affected by its glycosylation. CD37 interaction with its known partner proteins, CD20 and IL-6R, was not affected by glycosylation, other than via its changed subcellular localization. Surprisingly, glycosylation was found to inhibit the interaction between CD53 and its partner proteins CD45 and CD20. Together, our data show that tetraspanin glycosylation affects their function in immune cells, which adds another layer of regulation to tetraspanin-mediated membrane organization. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
This month on Episode 28 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the August 20th and September 3rd issues of Circulation Research. This episode also features an in-depth conversation with Dr Scott Cameron from the Cleveland Clinic and Dr Milka Koupenova from the University of Massachusetts Medical Center about their study, SARS-CoV-2 Initiates Programmed Cell Death in Platelets. Article highlights: Gupta, et al. Electronic Cigarettes and Oxidized Lipids Bartosova, et al. Glucose Derivative Induced Vasculopathy in CKD Atmanli, et al. DMD Correction Attenuates Cardiac Abnormalities Ma, et al. Length Dependent Activation in Porcine Myocardium Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast for 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 will be highlighting articles presented in our August 20th and September 3rd issues of Circulation Research. I also will speak with Dr Scott Cameron from the Cleveland Clinic and Dr Milka Koupenova from the University of Massachusetts Medical Center about their study, SARS-CoV-2 Initiates Programmed Cell Death in Platelets. Cindy St. Hilaire: The first article I want to share is titled Electronic and Tobacco Cigarettes Alter Polyunsaturated Fatty Acids and Oxidative Biomarkers. The first author is Rajat Gupta and the corresponding author is Jesus Araujo from UCLA. E-cigarettes have surged in popularity in the last decade and while many people switching from traditional cigarettes to smokeless ones view the latter as a safe alternative to smoking tobacco, emerging data shows that E-cigarettes cause adverse effects such as oxidative stress, inflammation and endothelial dysfunction in users. The aerosols produced during vaping contain similar levels of reactive oxygen species, also called ROS, as the vapors of tobacco smoke. However, data on the extent to which E-cigarettes, E-cigarette ROS, influences cardiovascular health is lacking. Cindy St. Hilaire: To address this, this group recruited 32 chronic users of E-cigarettes, 29 chronic tobacco smokers, and 45 individuals that used neither and they measured their plasma levels of oxidative biomarkers. The team found both similarities and differences between the E-cigarettes and the tobacco users. For example, both smoking groups had increased plasma antioxidant capacity and decreased levels of oxidized linoleic acid compared with the levels seen in non-users, while arachidonic acid levels were raised in tobacco smokers and reduced in E-cigarette users. Overall, however, the biomarker levels were deemed to be intermediate for E-cigarette users between the non-users and the tobacco users. This study suggests that while E-cigarettes carry a lower health risk than tobacco, they are by no means safe. Cindy St. Hilaire: The second article I want to share is titled Glucose Derivative Induced Vasculopathy in Children on Chronic Peritoneal Dialysis. The first author is Maria Bartosova and the corresponding author is Claus Schmitt and they're from the University of Heidelberg. Diabetes, high blood pressure and obesity are risk factors for both cardiovascular disease and chronic kidney disease. Worse still, loss of kidney function and even dialysis itself are thought to exacerbate cardiovascular issues. In the case of dialysis, it's thought that high levels of glucose degradation products, or GDPs, in the dialysis fluids can promote the addition of sugar moieties to vascular proteins and lipids causing vascular damage. To investigate this theory, Bartosova and colleagues studied vascular tissue from children with chronic kidney disease receiving dialysis fluids with either high levels or low levels of glucose degradation products and compared these to tissues from children not on dialysis at all. Cindy St. Hilaire: Proteome and transcriptome analysis of the vessel tissues revealed that compared with patients or no to low GDP fluids, patients receiving high GDP fluids had higher levels of damaging glycation, increased transcription of genes involved in cell death, and decreased transcription of genes involved in cell survival and cytoskeletal reorganization. In line with these findings, vessels from high GDP patients displayed considerable evidence of damage, such as markers of apoptosis, skeletal disintegration and thickened intimas. The results confirmed GDPs can cause vasculopathy and suggest low GDP fluids should be used for dialysis patients. Cindy St. Hilaire: The next article I want to share is titled Cardiac Myoediting Attenuates Cardiac Abnormalities in Human and Mouse Models of Duchenne Muscular Dystrophy. The first author is Ayhan Atmanli and the corresponding author is Eric Olson from UT Southwestern. Duchenne Muscular Dystrophy, or DMD, affects one in 5,000 baby boys and is caused by mutations in gene for dystrophin, an architectural protein essential for muscle cell integrity. Patients display profound muscle degeneration and weakness, with respiratory and heart muscle dysfunction being a major cause for death. With the recent improvements in respiratory medicine that extend the lives of patients, this group now focused on heart dysfunction and specifically, whether gene editing could mitigate it. The team created induced pluripotent stem cells, or iPSCs, from Duchenne Muscular Dystrophy patient and his healthy brother and showed that gene editing from the DMD cells enabled their development into normal-looking cardiomyocytes with normal contractile function and calcium handling, equivalent to that seen in healthy control cells. The unedited DMD cells, by contrast, did not develop normally. For great clinical relevance, the team edited DMD cells after cardiomyocyte differentiation showing that this reduced their propensity for arrhythmia, compared with that of unedited cells. Cindy St. Hilaire: Lastly, the team provided evidence to suggest gene editing may improve heart abnormalities in mice with the same mutation. All together the results are proof of principle and support of the development of gene editing therapy as treatment for DMD. Cindy St. Hilaire: The last article I want to share is titled The Super-Relaxed State and Length Dependent Activation in Porcine Myocardium. The first authors are Weikang Ma and Marcus Henze and the corresponding author is Thomas Irving and they're from the Illinois Institute of Technology. Myofilament length-dependent activation or LDA is the fundamental mechanism coupling the force of the heart's contraction to it's proceeding diastolic volume. In other words, LDA ensures that the more the heart fills, the stronger it contracts. Studies of rodent hearts have given insights into LDA mechanics. However, how it operates in large mammalian hearts is unknown. Using structural and biochemical analysis of pig myocardial fibers, this group found that compared with small stretches of the fibers which were equivalent to small diastolic volumes, long stretches induced greater ATP turnover and greater numbers of cross bridges between myosin and actin filaments which are critical contractile machinery proteins. Cindy St. Hilaire: Myosin motors can be found in three stages, engaged with actin, unengaged in a disordered, relaxed state but ready to engage, or super-relaxed state where they are essentially switched off. The team showed that as muscle stretch increased, the amount of super-relaxed myosin motors diminished with more myosin motors becoming engaged to enable a stronger contraction. When the fibers were treated with a myosin motor inhibitor, these stretch effects were impaired. In revealing the mechanisms of myofilament length-dependent activation, this study provides a platform for studying cardiomyopathies in which this system goes awry. Cindy St. Hilaire: So today, Dr Scott Cameron from the Cleveland Clinic and corresponding author of the paper, Dr Milka Koupenova from the University of Massachusetts Medical Center, are both with me to discuss their study, SARS-CoV-2 Initiates Programmed Cell Death in Platelets. And this article is in our September 3rd issue of Circ Research and for full disclosure, the editor of Circ Res, Dr Jane Freedman is also an author on this manuscript. And for full double disclosure, I know Dr Koupenova quite well as we were both graduate students together back in the Ravid Lab at Boston University. However, the full Editorial Board selects these articles, not just me alone and this one is timely, novel, and an amazing story. So thank you both for joining me today. Milka Koupenova: Thank you for having us. Scott Cameron: Privileged to be here. Cindy St. Hilaire: So before we jump into the story that is your paper, can you give us a little bit of background about platelets? I know for years, I guess certainly before Katya's lab, I just thought of platelets as little nucleus-free particles that clot. But we know they are so much more than that. So why are they so important? And how do they function to do more than just stop a bleed? Milka Koupenova: So this is a great question, Cindy, and I am happy that you alluded exactly to the anucleated nature of platelets. So platelets are cell fragments. They're precursors in the bone marrow, the megakaryocyte. They are the second most abundant blood component after the red blood cells. And traditionally, platelets have been known, as what you pointed out, as these little units that change their conformation once there is some form of a problem with either the vascular, which we have a cut, they come together, they form this clot, and bleeding is prevented. But as we have learned perhaps in the past 20 years that platelets have a profound immune role during various immune processes and infections for different kind of microbes. And particularly relevant to this paper is that we understand that platelets have clearly a role responding to the viruses and activating the immune system. Cindy St. Hilaire: Yeah, and that was actually my next question. You and Jane are the world-leading experts on platelets and viral responses. So what was known about that interaction, I guess before we started looking at SARS-CoV-2, what was known about that platelet virus or even type of virus interaction? Milka Koupenova: So SARS-CoV-2 is a RNA virus--respiratory virus that we actually thought similarly to influenza that it mostly stays in the lower respiratory tract where it becomes problematic. However, from our work with influenza, when we saw that in certain patients you actually can detect the virus in platelet. In the beginning of the pandemic, we hypothesized that perhaps, in some people, the virus crosses over into the circulation. And based on our previous studies with influenza, we wanted to see if that indeed is the case. Hence we initiated a study here at UMass with the department head who is also on the paper, Dr Finberg, who is a leading expert in influenza and novel virus and we collected platelets from people to see if we can detect it. And so in the beginning, we were not able to detect SARS-CoV-2 in platelets. So we collected platelets from 17 patients and by qPCR with the primers that the CDC has, for whatever reason I couldn't detect anything. And I was really frustrated because previous reports have shown that about 25%, in some people even 35% of the study population, SARS can be detected. So very interesting observations. Milka Koupenova: I could see it by immunofluorescence but I couldn't detect the RNA. And the story goes, that I attended a seminar on SARS-CoV-2 and the person was actually referencing a company that started from University of Pitt where you are. Cindy St. Hilaire: Oh, very nice. Milka Koupenova: And they do specific, it's called amplicon ARTIC v3 sequencing so they enrich for the SARS-CoV-2 RNA and screen by sequencing. And when we did that, we were able to detect it in all patients. So I freaked out and I said, "Oh my gosh, something is wrong." Milka Koupenova: And so I sent plasma, and I sent controls, and actually RNA from the virus and you can see beautifully that it's only in platelets. Four of the 17 people actually had RNA in the plasma, but what you can observe in all these people is that the virus is fragmented, meaning it's not infectious. And in a way what this tells us, it suggests that platelets are super important in the removing it from the circulation and they probably serve as a dead-end for the virus because you cannot find virus coming out of platelets and the RNA is chopped off. So what I would say, is that platelets are these amazing little units that serve as removal of the viral RNA for these particular viruses, respiratory viruses that are RNA viruses. Cindy St. Hilaire: I think that is so interesting. So essentially, they're almost like little composters that are chewing it up and preventing it from spreading in the organism. Milka Koupenova: Yes, and as a result there is a response. Cindy St. Hilaire: Scott, probably the most common thing that people know with SARS is that loss of smell, or taste, and things like that, but really that doesn't send anybody to the hospital. So really what are the symptoms of COVID-19 patients that tie in with platelets specifically? I feel like that's a lot of things that we maybe in the public, or on Twitter, and things didn't hear as much about. So really what are those big symptoms linking COVID and platelets and what are the implications of platelet death in the pathogenesis of COVID? Scott Cameron: So certainly I think several investigators are in the world of now showing that platelets are hyperactivated, Robbie Campbell and Matt Rondina put a really nice paper in Blood last year showing that platelets are hyperactive and there are other investigators who found something similar. And so the question is, what are the symptoms of hyperactive platelets in the SARS-CoV-2 patient? So what most of them would find is shortness of breath or dyspnea, and when they present to the emergency department, and certainly we saw this, the oxygen saturation which should be in the mid to high 90s on room air on an average person, was quite often low. It was in the 80s or 70s, sometimes even the 60s. Scott Cameron: And the real surprising thing was those are patients that would normally immediately be on a ventilator, but yet they could still be talking to you. And so if you have a platelet that's activated in a hyperthrombotic condition, like SARS-CoV-2, COVID-19, and then that forms a blood clot, you have a situation where the amount of oxygen the patients taking in and the amount of oxygen you're measuring in the artery is quite discrepant and we call that the alveolar arterial or oxygen gradient. So if you've got lots of platelet plugs through the microvasculature, it's going to take up some space the oxygen should be using for diffusing in. And so that would be manifested as shortness of breath and that's certainly one of the biggest tip-offs that a patient might have a blood clot, particularly in the lung. Cindy St. Hilaire: Some of these symptoms of COVID-19 are really worse in patients with comorbidities, diabetes, obesity and heart failure. Are platelets central to kind of the pathogenesis of those disease or the symptoms of those diseases? I guess the root of my question is, why do the comorbidities of diabetes, obesity, and heart failure make COVID worse? Is it something about those disease states themselves or is there a role for platelet? Scott Cameron: That's a brilliant question, no one's ever asked that before. And as Dr Koupenova said, I'm a little bit biased too because I firmly believe that in different disease states, the disease educates the platelets so you've got a different platelets phenotype. So focusing on diabetes, we know the platelet phenotype is different in diabetic patients. We know that platelet reactivity seems to be higher through the P2Y12 receptor. In terms of obesity, it is true, we know that, and this has been published also, and we know that the platelet phenotype is hyperactive in a patient with obesity and so that tells me that, that's a comorbidity that might affect platelet function and also vice versa for that case. And then in terms of why is it affecting males more prominently and more severely than females, well one of the beefs, I guess, that I had is that we treat diseases in women the same as we do in men assuming that the platelet phenotype in disease must be the same, but that's absolutely not true. And that's actually a theme that we have in our lab right now, we know that the behavior of platelets, and how platelets are educated in diseases is not all the same in women as in men and I think it's a huge disservice that we really had to have a pandemic that would make that quite clear to us. Cindy St. Hilaire: You kind of hit onto something that's really, I think it's now becoming more recognized certainly in the cardiovascular field and that is so many studies are really only on male mice, or only younger or older men, and we are missing not only a huge patient population, but probably some really interesting biology that is distinct. Milka Koupenova: So expanding on that, we know that in platelets, the toll-like receptors, and we've looked at the expression of all 10 in a study that we published in ATVB in 2015, actually, significantly if you look at Farmingham Heart Study data and the expression of these toll-like receptors they are increased in women versus men. And also, an interesting observation that never got published, once upon a time when I was doing studies with TLR7 mice is that if you inject TLR7 agonists, male mice would have a higher level of reduced platelet count than female mice at the same time points, right? And at that time it wasn't published. Definitely there are differences, but I also want to extrapolate a little bit on what was said at the beginning. We have to understand that when it comes to these comorbidities, everything affects a unit that doesn't have a nucleus, right? And diabetes and obesity have the so called profound, chronic inflammation of cytokines, such as IL6, that keep circulating. These things have effect on platelets. So we have two responses, we have the environment that affects platelets and we have the direct response of the virus that affects platelets. And that cumulative response truly can exhaust them and once they become exhausted, once they release their contents, as we show in this paper, then you're compromising their function and you will be compromising taking out the virus from one side and from the other side you're going to be compromising the environment because all of the content that comes out from a unit that already has free form proteins, it exhibits a true insult on what's being surrounded. So these clots that form in the lung or the platelets that circulate they no longer can be resolved properly. Cindy St. Hilaire: Yeah. Milka Koupenova: It's a balance. Cindy St. Hilaire: Yeah, so really it's like destroying the platelet not only are you destroying the vacuum that has to suck up those particles, you're then just dumping a whole bunch of pro-inflammatory things on all of the endothelial cell vasculature that those platelets are nearby. Cindy St. Hilaire: Actually that was one thing that I thought you spent a decent portion of the discussion on, and that is the method by which the blood is collected really impacts the outputs you observe in quote unquote platelets. Can you talk about the importance of that because I think that's one thing, certainly as a PhD who's just like, "Oh, yeah. I'm just going to collect blood from my mice and do this thing," how critical is that point in the experiment, in the blood collection? Milka Koupenova: So I am very adamant when it comes to platelets for the blood to be drawn in citrate. And I have to say that a lot of the studies that you would see in the literature are done using EDTA blood or serum. They all have their importance. I'm not going to dismiss it, but if you want to truly measure what's inside in plasma, versus what's inside in platelets, or what's inside in any cell for that matter, you got to go for citrate. You have to be very careful not to shake the blood. You have to be very careful not to cool down the blood. So the nurses probably hated me because often I would be like, "You can't do this. You can't put it on ice. You can't warm it up to above certain degrees. Everything has to be controlled and done correctly." Milka Koupenova: And so I had done in the past studies in which I would take plasma from the same patient in EDTA, in citrate and then isolate the RNA, have my tech isolate the RNA, and we send it to a fragment analyzer, and you can see how much more RNA you will get in the EDTA plasma. I'm not even talking about serum. Milka Koupenova: Serum is a very different thing, then you're definitely going to get platelet content in it, in the serum, right? So it's important to distinguish that perhaps when you're getting EDTA plasma you are looking at a content that could have been inside in platelet and I can't stress enough that when it comes to these particular studies, citrate, dextrose, phosphate is your place to go and be. Cindy St. Hilaire: So in terms of translational potential, what do your findings suggest about future therapies or targets to investigate as therapy? And is modulating platelets a potential for combating viral infections or mitigating their severity? Milka Koupenova: Well, Scott and I actually talk a lot about that. Scott Cameron: That's right. Milka Koupenova: I personally would say, control the inflammation, never let it go to platelet. Let me back up a little bit, if you have to, you have to, right? But your go to method should be inflammation, if you don't get to the point that you need to control platelets then you're in a better place because it becomes very fickle. From everything that you hear me say, you push it to one side and the balance is destroyed. You deactivate platelets or inhibit platelets well, are they now not able to pick up the virus and then you're now having the virus circulating somewhere. Now, if you don't treat platelets that's also not good. So you're in the very fickle situation if you get to the point that you need to control the activation of platelets and there are trials currently that are trying to look at those things. Scott, I'm going to refer this a little bit more to you because you have done some interesting things with that particular point. Scott Cameron: No, it's a great question, Milka, and I think that as platelet biologists, nobody more than I wanted it to be true that platelets would be the ultimate target. I mean, clearly patients with SARS-CoV-2 have thrombosis, clearly platelets are activated, so should we inactivate them? That was the whole point of the RECOVERY trial and one of the benefits I'll tell you before I sort of go into that is, working in a large organization like the Cleveland Clinic and we have access to data and lots of it extremely quickly, and so because of that I of course could see how many patients were coming into our hospital with thrombotic events. And I could see what the independent predictors of thrombotic events was and it wasn't the platelet count, sometimes platelet count was low, sometimes it's high in the SARS-CoV-2 patient. And if you took those individuals that were on aspirin, comparing them to those that are not in a propensity match study, one of the things that we find is that aspirin doesn't seem to affect or improve mortality or the number of blood clots in the patient with SARS-CoV-2. Scott Cameron: We compared that to all non-steroidal anti-inflammatory medications that patients may have been taking also in a propensity match study just in case it was the mechanism action of the drug, rather than the drug itself, and we found that NSAIDs not only did not protect patients, but they were not necessarily harmful either, which was one of the things that came out at the start of the pandemic. Among, I'll add, the absence of evidence based medicine and a lot of cases where naturally people, including clinicians, were scared and so they were going off label and they were trying a lot of different medications with really not a shred of randomized controlled data. Scott Cameron: But now that we're 18 months into it, the first and biggest study that came back was the RECOVERY trial, which we were all waiting on, where patients were given aspirin and short term mortality was examined over an observational period of one month. And just like we found in a propensity match study, which is as close as you'll get to a clinical trial in a retrospective manner, the prospect of RECOVERY trial actually showed the curves were almost super imposeable, those that got aspirin versus those that didn't. So I think low dose aspirin clearly is not going to be enough for those patients, but I'll also add that over the observational period of one month they also didn't see a higher incidence of death in those patients. And I think Milka's point is really well taken that you have to remember that as well being an entity of thrombosis, platelets are immunological entities and so you've got to really consider should we be inhibiting them and if you are inhibiting them, I think the time point at which you should inhibit them is what we should examine, not just an all or nothing, inhibited or not. Milka Koupenova: It's just in our linear brains we prefer to think of it as one straight, linear pathway, but it isn't, and I think platelets are actually a great example of how many pathways are feeding into one tiny fragment and that particular blood cell is inducing this profound response during these infections. Cindy St. Hilaire: I think most people have heard that angiotensin-converting enzyme 2, also called ACE2 is the receptor of SARS-CoV-2. The virus itself uses it to bind and become internalized into the cell, but there's been some discussion or even some discrepancy of data as to whether platelets truly express ACE2 and if that is the means for the virus to enter the platelets. So can you share with us what is the current state of knowledge about that? Scott Cameron: Yeah, just as a segue of some of the things that Milka said, I think the preparation of your sample is part of the answer. If you draw in the incorrect tube, if you the tube is not completely filled, and the ratio of citrates to whole blood isn't correct you're going to have discrepant results. If you biomechanically activate the platelets by drawing through a short needle, in a small-bore needle for example, that's going to activate the platelets. If you cool them, it's going to activate them. But then also, depending on how you decide to separate them, we always washed platelets in my lab, we wash them two or sometimes three times, and I can tell you if you use flow cytometer we get one white blood cell for every 12,000 platelets. Scott Cameron: And some investigators might go one step further and they'll a CD45 depletion set, which is certainly important if you're studying RNA. But one of the issues, as you well know, a CD45 is also on the surface of platelets, so if you start with a low expressing protein and you CD45 deplete them, you are actually going to get a decrease in your platelet yields. I've seen it, I think Milka's seen it, various other investigators have, and you might find yourself at the threshold of what your antibody can detect. It's also variably expressed. If you look at even healthy individuals, some of them have almost none. So if you look at 10 individuals, you might actually find none, but then if you look at another 10, the amount of expression that we see is kind of all over the place. It's not like other receptors where one tends to express a certain amount and that's the way it is in health. ACE2 doesn't seem to be that way for whatever reason. Milka Koupenova: We were able to detect in some of the people by qPCR, but what was interesting is that from the three primers that I used there was never the same person who we were able to detect all three primers with for that receptor. That tells you that maybe they are changes of one base that is not enough for the primer to detect it, right? That becomes another possibility of not being able to detect. Milka Koupenova: And so I go to confocal microscopy where I use 100 lens and tons of hours in the microscope room, and Scott is completely right, it's really hard to see it particularly in healthy people. And it starts to pick a little bit more in people with cardiovascular disease or people with COVID that are old. So it's a bit complicated, but the important thing here is, besides the fact that we are detecting ACE2 and we're detecting proteins and I use controls, biological controls to prove that this is the case and it's not just an antibody problem, is that the virus will get picked up by platelets even if you don't have ACE2. That is the take home message from this paper is that the platelet has evolved various mechanisms by which is utilizes getting it inside. It is that important for this virus. This type of virus is not recirculating. In this case, what we observed is that the virus is attached to microparticles that are of platelet origin for that matter. Cindy St. Hilaire: So really what you're saying, what I'm hearing is the platelet is the superhero of the body. Milka Koupenova: Definitely. Absolutely. No bias, absolutely. Cindy St. Hilaire: Unbiasedly, it is a superhero. Well, Dr Cameron and Dr Koupenova, thank you so much not only for this amazing discussion, but for really an elegant, elegant paper that is really bringing to light the complex interaction between SARS-CoV-2 and platelets. So thank you so much for joining me and keep publishing amazing stories like this. Milka Koupenova: Thank you for having us. Scott Cameron: Thank you, an honor to be here. Thanks again. Cindy St. Hilaire: That's it for the highlights from August 20th and September 3rd 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, Dr Scott Cameron and Dr Milka Koupenova. This podcast is produced by Ashara Ratnayaka, 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 Cynthia St. Hilaire, and this is Discover CircRes, your on-the-go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association, 2021. 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 information, please visit ahajournals.org.
Tricha Kumar is a professional dancer and filmmaker who also happens to be the Bollywood film industry's #1 dance choreographer of Bhangra. Join the Carolina Desis this week as Tricha sits down with us to discuss current projects, her philosophy on dance as a career and an artform, the state of Bhangra in Bollywood, the value of labor as a backup dancer, adjusting to Covid, and so much more! IG: @tricha.k Twitter: @trichakumar Website: trichakumar.com MOON: facebook.com/moonthefilm MOON: https://moonthefilm.com/ Witness to a Devouring Monster: https://www.instagram.com/wtadm_film/ WTADM Fundraising: https://seedandspark.com/fund/wtdamfilm#story
This month on Episode 23 of Discover CircRes, host Cindy St. Hilaire highlights the topics covered in the April 2nd Compendium on Hypertension issue, as well as discussing two articles from the April 16 issue of Circulation Research. This episode also features an in-depth conversation with Dr Kathryn Moore from the New York University School of Medicine, discussing her study, miR-33 Silencing Reprograms the Immune Cell Landscape in Atherosclerotic Plaques. Article highlights: Compendium on Hypertension Mustroph, et al. CASK Regulates Excitation-Contraction Coupling Ward, et al. NAA15 Haploinsufficiency and CHD Cindy St. Hilaire: Hi, and 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. Cindy St. Hilaire: Today, I'm going to be highlighting the topics presented in our April 2nd Compendium on Hypertension, as well as two articles from the April 16th issue of Circ Res. I also will speak with Dr Kathryn Moore from New York University School of Medicine about her study, miR-33 Silencing Reprograms the Immune Cell Landscape in Atherosclerotic Plaques. So the April 16th issue of Circulation Research is a compendium on hypertension. As introduced by Rhian Touyz and Ernesto Schiffrin, there are over 10,000 articles in PubMed related to hypertension. Hypertension is a major cause of morbidity and mortality worldwide, and data trends suggest that fewer and fewer patients are able to control their blood pressure medically. Further, the recent Sprint trial showed us that lowering blood pressure to levels below previously recommended values strongly correlated with significantly reduced rates of cardiovascular events and risk of death. Cindy St. Hilaire: As such, the April 2nd issue of Circ Res provides an extensive and expansive review on the current knowledge in the field. The series starts with an article on hypertension in low and middle-income countries by Aletta Schutte and colleagues. There they present the stark differences in the trajectory, healthcare, inequality, and established and emerging risks that are specific to low and middle-income countries. Cindy St. Hilaire: Robert Carey and colleagues present an evidence-based update in their article titled Guideline-Driven Management of Hypertension. In Pathophysiology of Hypertension, David Harrison and colleagues present the concept of the mosaic theory of hypertension originally proposed by Dr Irvine Page in the 1940s, which proposes that hypertension is the result of multiple factors that in some, raise blood pressure and induce end-organ damage. This article further refines this theory by incorporating what is known regarding the role of things like oxidative stress, inflammation, genetics, sodium homeostasis, and the microbiome in hypertension pathogenesis. Cindy St. Hilaire: Phil Chowienczyk and Jay Humphrey and colleagues cover the contribution of Arterial Stiffness and Cardiovascular Risk in Hypertension and identify steps required for making arterial stiffness measurements a keystone in hypertension management, and cardiovascular disease prevention as a whole. In Renin Cells, The Kidney, And Hypertension, Maria Luisa Sequeira Lopez and Ariel Gomez cover the major mechanisms that control the differentiation and fate of renin cells, the chromatin events that control the memory of the renin phenotype, and the major pathways that determine the cells’ plasticity. Cindy St. Hilaire: Meena Madhur and Annet Kirabo and colleagues penned the article, Hypertension: Do Inflammation and Immunity Hold the Key to Solving this Epidemic? In this Teview, they covered the emerging concepts of how environmental, genetic, and microbial-associated mechanisms promote both innate and adaptive immune cell activation and help lead to hypertension. Cindy St. Hilaire: In the article, The Gut Microbiome in Hypertension. Dominik N. Müller and colleagues present insights into the host-microbiome interaction and summarize the evidence of its importance in the regulation of blood pressure and provide recommendations for ongoing and future research. Cindy St. Hilaire: Paul Cohen, James Sowers, and colleagues cover Obesity, Adipose Tissue, and Vascular Dysfunction in which they discuss the abnormal remodeling of specific adipose tissue depots during obesity and how this contributes to the development of hypertension, endothelial dysfunction, and vascular stiffness. Cindy St. Hilaire: Clinton Webb, Satoru Eguchi, Rita Tostes, and colleagues cover Vascular Stress Signaling in Hypertension. In this Review, they discuss common adaptive signaling mechanisms against stresses, including the unfolded protein response, antioxidant response element signaling, autophagy, mitophagy, mitochondrial fission and fusion, STING-mediated responses, and activation of pattern recognized receptors. And how all of these responses contribute to vascular stress and ultimately hypertension. Cindy St. Hilaire: Rhian Touyz and colleagues then specifically dig into the topic of Oxidative Stress and Hypertension, focusing in on recent advances in delineating the primary and secondary sources of reactive oxygen species, the posttranslational oxidative stress modification ROS induces on protein targets important for redox signaling, their interplay between ROS and endogenous antioxidant systems, and the role of inflammation activation and endoplasmic reticular stress in the development of hypertension. Cindy St. Hilaire: Curt Sigmund and then colleagues cover the Role of the Peroxisome Proliferator Activated Receptors in Hypertension. In this Review, they discuss the tissue- and cell-specific molecular mechanisms by which PPARs in different organ systems modulate blood pressure and related phenotypes, such as endothelial cell dysfunction. Importantly, they also discuss the role of placental PPARs in preeclampsia which is a life-threatening form of hypertension that accompanies pregnancy. Cindy St. Hilaire: Daan van Dorst, Stephen Dobbin, and colleagues provide the Review, Hypertension and Prohypertensive Antineoplastic Therapies in Cancer Patients. Many cancer therapies have prohypertensive effects. And this Review covers some of the mechanisms by which these antineoplastic agents lead to hypertension and details the current gaps in knowledge that future clinical studies must investigate, to identify the exact pathophysiology and the optimal management of hypertension associated with anticancer therapy. Cindy St. Hilaire: In Hypertension, a Moving Target in COVID-19, Massimo Volpe, Reinhold Kreutz, and Carmine Savoia, review available data on the role of hypertension and its management in COVID-19. Cindy St. Hilaire: Melvin Lobo and colleagues review Device Therapy of Hypertension. In this Review, they discussed the newer technologies, which are predominantly aimed at neuromodulation of peripheral nervous system targets, and discuss the preclinical data that underpin their rationale and the human evidence that supports their use. Cindy St. Hilaire: Last but not least, in Artificial Intelligence in Hypertension: Seeing Through a Glass Darkly, Anna Dominiczak and colleagues cover a clinician-centric perspective on artificial intelligence and machine learning as applied to medicine and hypertension. In this Review, they focus on the main roadblocks impeding implementation of this technology in clinical care and describe efforts driving potential solutions. Cindy St. Hilaire: This is an expansive set of Reviews written by the leading experts in the field and provides an up-to-date assessment of all aspects of hypertension. The graphics, and the articles are absolutely beautiful. And I'm sure we will be seeing a lot of them in upcoming presentations. Hopefully at AHA and the other sub-meetings when we're all back in person. Cindy St. Hilaire: In the April 16th issue, I want to highlight the article, Loss of CASK Accelerates Heart Failure Development. The first author is Julian Mustroph, and the corresponding authors are Lars Maier and Stefan Wagner from the University Medical Center in Regensburg, Germany. Despite advances in cardiovascular medicine, heart failure takes the lives of tens of thousands of Americans each year. To develop novel treatments, a better understanding of the conditions of molecular pathology is needed. One contributing factor in heart failure is increased activity of the Ca/calmodulin-dependent kinase II (CaMKII). Cindy St. Hilaire: In this paper, the authors suggest a way to get CaMKII levels under control. Ca/CaM-dependent serine protein kinase or CASK, suppresses CaMKII neurons and the team showed that CASK is also expressed in human heart cells, where it associates with CaMKII. Next, they engineered mice to CASK specifically in cardiomyocytes, finding that when these animals are subjected to beta-adrenergic stimulation, cardiomyocyte like CaMKII activity was significantly greater than that seen in control animals. Calcium spark frequency and the propensity for arrhythmia were also increased. Furthermore, in a mouse model of heart failure, mice lacking CASK fared worse and had reduced survival compared to the wild type control animals while boosting CASK expression in wild type animals reduced the elevated CaMKII activity and calcium sparks associated with heart failure. The author suggests that increasing CASK activity might be a heart failure treatment strategy worthy of further study. Cindy St. Hilaire: The last article I want to share from the April 16th issue is titled, Mechanisms of Congenital Heart Disease Caused by NAA15 Haploinsufficiency. The first author is Tarsha Ward, and the co-senior authors are Kris Gevaert, Christine Seidman, and JG Seidman from Harvard University in Boston, Massachusetts. A number of genetic variants are associated with congenital heart disease, including loss of function variants of the gene encoding NAA15, a sub N-terminal acetyltransferase complex called NatA, which acetylates a large portion of newly forming proteins. To find out how these variants contribute to defective heart development, the authors performed genome editing on human pluripotent stem cells to convert one or both copies of NAA15 gene into congenital heart disease linked to variants. The team then examined cardiomyocyte differentiation, protein acetylation, and protein expression in the edited and unedited cells. Cindy St. Hilaire: They found that while NAA15 haploinsufficiency cells were able to develop into cardiomyocytes seemingly normally, the cell's contractile ability was significantly impaired. Cells homozygous for NAA15 variants failed to differentiate and had poor viability. The team also found that while only a small number of proteins had reduced end terminal acetylation in NAA15 haploinsufficiency cells, over 500 proteins had altered expression levels, four of which were encoded by congenital heart disease-linked genes. This work provides the first insights into the effects of NAA15 variants in human cells and sets the stage for analyzing other congenital heart disease-linked variants in this manner. Cindy St. Hilaire: Today, Dr Kathryn Moore from NYU School of Medicine is with me to discuss her study, miR-33 Silencing Reprograms the Immune Cell Landscape in Atherosclerotic Plaques, which is in our April 16th issue of circulation research. So thank you so much for joining me today, Kathryn. Kathryn Moore: My pleasure. Cindy St. Hilaire: Atherosclerosis is the result of lipid-induced chronic inflammation, and while lipids are kind of thought to be an initial driver, therapies that target lipids alone, such as statins, they're not sufficient. They can obviously bring things down and improve things a lot, but a lot of research now is focused on uncovering the nuances of the inflammatory component of atherosclerosis to help identify new targets for therapies. One specific arm of this research has focused on resolving atherosclerotic inflammation. And my first question to you is, what exactly does resolving inflammation mean in the context of an atherosclerotic plaque? And maybe could you give us a little primer on some of those key cell types or processes involved in that. Kathryn Moore: I'm really fascinated by the resolution of inflammation and in particular, in the atherosclerotic plaques. So inflammation used to be thought of as an active process, almost a one-way process, which in order to resolve had to stop. But actually, the pro-inflammatory and anti-inflammatory responses are a continuum. And so inflammation resolution, we now recognize is an active process, and it's not just a matter stopping the influx of immune cells but these cells take on new phenotypes and different functions. And the immune cells themselves are required for resolution of inflammation and tissue repair. And so we're really interested in looking at what those pathways are, that tip the balance between pro-inflammatory responses and pro-resolving responses and how to incite them in the plaque so that you can start to remodel the plaque to be more stable or have a more favorable phenotype, or even to regress the plaque, to shrink the plaque in size. Cindy St. Hilaire This study specifically focused on microRNA-33, and I believe your lab was one of the very first to look at this specific, but also other micro RNAs in atherosclerosis. And the prior research that you and others have shown is that this microRNA modulates a variety of genes that control lipid metabolism. You found this in mice, but also in monkeys. And really by using anti-miRs against this microRNA, you can induce cholesterol efflux and that cholesterol will leave the liver and the macrophage cells, and it's incorporated into the protective HDL particles and excreted. Cindy St. Hilaire: And so it has this really nice protective effect. However, the effects seen in these animal studies were suggested that microRNA-33 had HDL independent action, which I think is where your story starts. So could you tell us some of the premises or the gaps in knowledge between those first initial findings of miR-33 that led you to conduct this study and then kind of what the design of the study was? Kathryn Moore: So, as you mentioned, we discovered miR-33 as an inhibitor of cholesterol efflux and the pathways that lead to the generation of HDL, the so-called good cholesterol. And when you inhibit miR-33 in mice and monkeys, you can raise plasma levels of HDL. But we also saw that in mice that had been fed a Western diet continuously, we saw favorable changes in the atherosclerotic plaque under conditions where we didn't see the increase in HDL. So if the mice are on a Western diet, the levels of miR-33 in the liver are very low, and inhibiting it doesn't cause the increase in HDL cholesterol. But we still saw this 25% regression in atherosclerotic plaques. And that got us thinking about the other things that miR-33 could be doing and around the same time, I was also very interested in immunometabolism and how the metabolic state of macrophages influences their function. Kathryn Moore: And Mihail Memet, who is a former postdoc in my lab made the discovery that miR-33 could inhibit fatty acid oxidation in macrophages and that this polarized the cells to a more inflammatory phenotype. So when we give the miR-33 inhibitors, we're raising a level of fatty acid oxidation in the macrophages and they become more tissue reparative. And so we suspected that could be the mechanism going on in the plaque but those studies, those initial studies were done over five years ago. And that was before the advent of single-cell technologies, which have really revolutionized how we're studying the atherosclerotic plaque. So in this study, we were able to apply some of these more high dimensional analyses of all of the immune cells in the plaque. And really look at how inhibiting miR-33 was altering their transcriptome and their phenotype. Cindy St. Hilaire: Yeah, so that is a perfect segue to my next question, which is you're doing this single-cell RNA-sequencing on tissue, but it's not just any tissue. It's not like a nice spleen that you can kind of pop open and all the cells fall out nicely and you can fax them or whatever. This is from an aorta, which itself is fibrous and tough on top of the atherosclerotic plaque, which is also difficult. So can you discuss maybe some of the challenges regarding doing this exact kind of analysis with this tissue and maybe some of the limitations or controls that you used to help really refine your result? Kathryn Moore: It is a little bit challenging to learn how to digest the aorta to release the immune cells, so to isolate the CD45+ immune cellsthat then go on to the sequence that takes some trial and error to get the right conditions. But actually, once you've done that a couple of times, it's not as difficult as it seems but I think that one of the challenges of doing these types of studies is integrating the results that we get from the single-cell RNA-sequencing with the other technologies that we've used in the past to analyze atherosclerosis. Kathryn Moore: So, previously when we were analyzing atherosclerotic plaque size or immune cell content, we are doing this through histology and immunostaining. And single-cell RNA-sequencing has identified all these new immune cell subsets based on transcriptomic signatures. And they don't really match up nicely with the protein signatures that we've used in the past. Cindy St. Hilaire: Yeah. Kathryn Moore: I saw this as a great opportunity to try to integrate all these techniques. And see if we could come to some middle ground. To understand how maybe the new subsets that we're identifying with single-cell RNA-seq from the aortic immune cells matched some of the things that we were able to do by looking at histology and tracing monocytes and macrophage entry and retention in the plaque. Cindy St. Hilaire: How did it line up? What's the nice Venn diagram of this study and what we've all been doing previously? Kathryn Moore: Well, it's a challenge, but what I thought was really really fascinating was we did monocyte-macrophage tracing experiments. Because one of the things we find when we inhibit miR-33 is we have a 50% decrease in the macrophage content of the plaque, but how is that happening? And what we found was there was an increase in the recruitment of monocytes into the plaque which may sound surprising if the plaque is shrinking, but they are the cells that are needed. They're the cleanup crew that are being introduced. But we saw a decrease in retention of macrophages and a decrease in proliferation and an increase in macrophage death and clearance of the apoptosis cells. And then through the single-cell RNA-sequencing, we were able to look at the different macrophage subsets. We had resident macrophages, Trem2hi metabolic macrophages, and MHCIIhi inflammatory macrophages. Kathryn Moore: We were able to look at their transcriptomes and say, "Which of these subsets are most likely to be performing those functions that we saw before?" And that was fun because that was like piecing together a puzzle. And what we saw, what it leads us to believe is that the Trem2hi metabolic macrophages are the ones that are undergoing aptosis. They have an increase in aptosis genes and eat-me signals and the MHCIIhi, having an increase in athoscoertic genes like mirTK that will help them clear the dying cells and the MHCIIhi macrophages also have decreased markers of proliferation. So although we used to think about macrophages as this one big pool, now we're able to say that these different subsets are performing different functions. And to me that's really exciting. Cindy St. Hilaire: Oh, that is exciting. And it's also extremely complicated because I was having enough trouble with just the two types of macrophages of a couple of years ago. The study showed that inhibiting this miR-33 using these anti-miR-33 oligos, and you're just kind of injecting oligos against it. And you're doing this in mice with established atherosclerosis. This helped to alter these monocyte and macrophage populations in the plaque itself. Cindy St. Hilaire: Do you think a function of the success of this study and essentially this therapy in the mouse is really dependent on the fact that it's targeting these circulating cells that are then going to the plaque? And I guess part of that question is, do you think part of this is because it's a circulating cell that can take it up, and then change and be delivered to the location it's going to, as opposed to that oligo targeting the plaque itself and the cells that are already residing there. Do you have any sense of that? Kathryn Moore: So it's interesting because one of the things that we did with our single-cell RNA-seq was to look at all immune cells in the plaque and say, "How many miR-33 target genes are changing in the ones from the treated mice?" And in the monocytes, you see very little change in miR-33 target genes. And that's consistent with what we know from Regulus Therapeutics who designed the anti-miR-33 antisense oligonucleotides. So we don't think that the ASO are being taken up in the circulation. I think they're actually being taken up by the macrophages in the plaque. And one of the great things about trying to target macrophages is they're very phagocytic. So they're going to be the ones that take up these ASOs, and the single-cell really allowed us to see whether it was just macrophages that were being affected or whether there were other immune cell populations that also seemed to have miR-33 induce changes. And of course it's hard from the single-cell to infer whether this is direct or indirect. Cindy St. Hilaire: Yeah. Kathryn Moore: But it seemed as if T-cells also were targeted by the anti-miR-33, definitely macrophages. We saw some changes in dendritic cells, very little changes in K cells, for example. And no changes in monocytes. And so it also begins to tell us how many different cell types are being affected and who's driving the bus when it comes to these changes. But by far the most miR-33 target genes change were the macrophage populations. And I think that's really due to their phagocytic ability. Cindy St. Hilaire: So I know there's a great divergence generally in microRNAs between mice and humans or really any species, but there are homologs to this in humans. What is the same and what is different between, I guess, this particular targeting micro RNA or what we know about it in mice and humans? Kathryn Moore: So mice have only one copy of miR-33, whereas humans and monkeys have two copies but those two copies are very similar in sequence. They differ only by two nucleotides. So you can use the same antisense oligonucleotides to target in mice and in non-human primates, for example. It's never been tried in humans. Cindy St. Hilaire: Yeah, of course. Not yet. Kathryn Moore: But it has been tried in monkeys, and we were able to effectively inhibit both miR-33a and miR-33b in the non-human primates. But the different variants of miR-33 have different transcriptional regulation. So they're induced under different conditions. And I think that's one way that mice and humans will really differ-the conditions where you'd have high levels of miR-33 will be different. Cindy St. Hilaire: Got it. Yeah. And the mice has that in the SREBP gene and humans. Kathryn Moore: And miR-33a is an SREBP-2 gene, which is SREBF2. And in humans there's an additional copy, which is SREBF. So it's in both of the SREBP genes in humans. Cindy St. Hilaire: Interesting. So I wonder, we need to ask the evolutionary biologist. Did they segregate together? I mean, I guess they must have. That's really interesting. That's cool. Kathryn Moore: One of the things that I love about miR-33 is that the SREBP-2 gene is turned on when cholesterol levels are low and it acts to increase the pathways involved in cholesterol synthesis and uptake. And miR-33 is transcribed at the same time. And what it does is it blocks the exits for cholesterol from the cell and from the body. And so it's just this hidden gem in the locus that sort of boosts SREBP-2 function. Cindy St. Hilaire: Its amazing stuff works out like that. I love it. So if we were going to leverage this inflammation resolution as atherosclerotic therapy, wherein the continuum of the disease, should we target? You know, we have obviously atherosclerotic plaque does not happen overnight. Teenagers can even have evidence of a fatty streak. If we were going to leverage antisense oligos as therapy, especially specifically against miR-33, where do you think would be a good place to target? And do we know, or have the kind of imaging capabilities to maybe identify that window right now in patients? Kathryn Moore: That's an interesting question. So lipid-lowering therapies will remain the first line of treatment for atherosclerosis, but lipid-lowering alone is insufficient to regress the plaque. It can stabilize plaques, but it doesn't really cause them to shrink. And when you think about the patient population that presents with cardiovascular disease, it's adults, for the most part. These are people in their fifties and sixties, and we've missed the chance to stop the early events. And so those are the majority of the people that are being treated. And I think there is room there to treat inflammation at the same time in the hopes of tipping that balance between pro-inflammatory events and then inflammation resolution. So we know surprisingly little about that tipping point. And now I think when miR-33 inhibition is fascinating in that it can affect both lipid metabolism and inflammation. And so I think that as an add-on therapy with lipid-lowering, it would be interesting, but of course, I'm not ready. Cindy St. Hilaire: We're not there yet. Cindy St. Hilaire: So I guess what's next for this line of research? What are kind of the next questions that the single-cell RNA-seq discovered for you? Was there anything kind of surprising or really exciting that you want to pursue next? Kathryn Moore: One of the things that I thought was really interesting was that the different macrophage subpopulations had different miR-33 target genes being de repressed. And that's probably not surprising, but I didn't initially think that would happen, but of course, the subpopulations are identified based on their unique transcriptomes. So they're not all the same, which means that they'll have different levels of miR-33, and they'll have different levels of the miR-33 target genes. And so Abca1, which we think about all the time as a miR-33 target gene that's involved in cholesterol efflux, it went up in Trem2hi macrophages and the resident macrophage population, but not in the MHCIIhi. The target genes and the MHCIIhi were different than the other two populations. And I think this now gives us a chance to sort that out. Kathryn Moore: And some of the targets in the MHCIIhi macrophages were ones that are involved in chromatin reorganization- Cindy St. Hilaire: Oh, interesting. Kathryn Moore: ... and inscriptional regulation. And when I looked across the other subsets, I could see that common pattern in T-cells and B-cells that were changing. And I think that's one way that miR-33 could have a broad impact. MiR-33 is a little bit of a unique microRNA. It has a very potent impact on these pathways. Other microRNAs often can change gene expression by 10 to 20%, but miR-33, when we inhibit it, we see really powerful effects. And I think that if it is involved in targeting genes that mediate chromogenic reorganization or transcriptional complex formation, that gives us a hint of how it could be having additional impact. Cindy St. Hilaire: That's really cool. And this was an absolutely beautiful story, not only in kind of dissecting out the mechanisms at play, but you know, those beautiful tisney plots and the nice graphics of the single-cell stuff. Kathryn Moore: The first author of the paper, Milessa Afonso, is a postdoc that just left the lab, and she worked so hard on this and did such a beautiful job. Cindy St. Hilaire: Well, it's a wonderful story and I'm really happy we were able to publish it. So, Dr Moore, thank you so much for joining me today. Kathryn Moore: My pleasure. Thank you. Cindy St. Hilaire: That's it for the highlights from the April 2nd and 16th issues of Circulation Research. Thank you so much for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @circres and hashtag DiscoverCircRes. Thank you to our guest, Dr Kathryn Moore. This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the Editorial Team of Circulation Research. Copy text for the highlighted articles was provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire and this is Discover CircRes, your on-the-go source for the most exciting discoveries in basic cardiovascular research.
Hello everyone! As we close 2020, I'd like to announce the next chapter of the podcast: We would like to introduce Fuerza Creativa! This new podcast will be the Spanish version of Creative Drive, where we will showcase more amazing poetry and short fiction from all over the world! Disfruten el primer episodio en Fuerza Creativa! Título: El enigma de los tornillos Autor: Antonio Rolando Arenas Obtuvo el título de Master of Arts with a Major in Modern Languages en Almeda University (USA, 2010), es Diplomado en Gestión Pública de la Provincia de Mendoza por la Escuela de Gobierno y Administración de la provincia de Mendoza – FCPyS, UN de Cuyo (Mendoza, 2012), es Profesor de Lengua y Literatura para el tercer ciclo de la EGB de la Educación Polimodal por el Instituto Superior del Profesorado PT-14 Escuela Italiana (Mendoza, 2013) y es Diplomado en Historia Argentina en Latinoamérica por la Escuela de Gobierno y Administración de la provincia de Mendoza-Instituto de Revisionismo Histórico Argentino e Iberoamericano Manuel Dorrego (Mendoza, 2015). Recibió el diploma del Curso Profesional de Analista Químico. Neptunos Formación. España, 2018. Publicó el libro Interrogantes, el libro La página del buffet; una publicación literaria diferente y el libro Diálogos internos. El Juego de los errores disponible en Barnes & Noble!
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.23.310144v1?rss=1 Authors: Aramesh, M., Mergenthal, S., Issler, M., Weber, F., Plochberger, B., Qin, X.-H., Liska, R., Duda, G., Huppa, J., Ries, J., Schuetz, G., Klotzsch, E. Abstract: When T-cells probe their environment for antigens, the bond between the T-cell receptor (TCR) and the peptide-loaded major histocompatibility complex (MHC) is put under tension, thereby influencing the antigen discrimination process. Yet, the quantification of such forces in the context of T-cell signaling is technically challenging. Common approaches such as traction force microscopy (TFM) employ a global readout of the force fields, e.g. by measuring the displacements of hydrogel-embedded marker beads. Recent data, however, indicated that T-cells exert tensile forces locally via TCR-enriched microvilli while scanning the surface of antigen-presenting cells. Here, we developed a traction force microscopy platform, which allows for quantifying the pulls exerted via T-cell microvilli, in both tangential and normal directions, during T-cell activation. For this, we immobilized specific T-cell activating antibodies directly on the marker beads used to read out the hydrogel deformation. Microvilli targeted the functionalized beads, as confirmed by superresolution microscopy of the local actin organization. Moreover, we found that cellular components, such as actin, TCR and CD45 reorganize upon interaction with the beads, such that actin forms a vortex-like ring structure around the beads and TCR is enriched at the bead surface, whereas, CD45 is excluded from bead-microvilli contacts. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.01.278853v1?rss=1 Authors: Lupardus, P. J., Rokkam, D. Abstract: CD45 is an abundant and highly active cell-surface protein tyrosine phosphatase (PTP) found on cells of hematopoietic origin. CD45 is of particular importance for T-cell function, playing a key role in the activation/inactivation cycle of the T-cell receptor signaling complex. The extracellular domain of CD45 is comprised of an N-terminal mucin-like domain which can be alternatively spliced to a core domain (RO) consisting of four domains with fibronectin 3 domain (FN3)-like topology. The study of CD45 has been hampered by a small set of publicly available antibodies, which we characterized as specific to the N-terminal FN3 domains of CD45 RO. To broaden the human CD45 reagent set, we identified anti-CD45 single domain VHH antibodies from a post-immune llama phage display library. Using a yeast display domain mapping system and affinity measurement we characterized seven unique clonotypes specific for CD45 RO, including binders that target each of the four FN3-like domains. These VHH molecules are important new tools for studying the role of CD45 in T-cell function in vitro and in vivo. Copy rights belong to original authors. Visit the link for more info
Dr Carolyn Lam: Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to the journal and its editors. I'm Dr Carolyn Lam, associate editor from the National Heart Center and Duke National University of Singapore. Dr Greg Hundley: And I'm Dr Greg Hundley, associate editor, Director of the Pauley Heart Center from VCU Health in Richmond, Virginia. Dr Carolyn Lam: Our feature paper today discusses trans-ethnic genome-wide association studies and the insights in the genetic architecture and heritability of long QT syndrome, a massive study that we will be digging into, but only after we talk a little bit about the other papers in this week's issue. And I'm going to start, Greg. Are you ready with your coffee? Dr Greg Hundley: I am. Dr Carolyn Lam: The first original paper really represents seminal work, showing that the endothelium can directly regulate obesity and insulin resistance. Now, as obesity develops, there is a decline in adipose tissue vascularity, which seems counterintuitive, and an increase in fibrosis. So authors, led by Dr Chen from the Irell and Manella Graduate School of Biological Sciences in the City of Hope, speculated that the reduction in vascularity in this adipose tissue might have an adverse effect on adipose tissue function. Now, these authors previously identified Argonaute-1, or AG01, a key component of microRNA-induced silencing complex, as a crucial regulator in hypoxia-induced angiogenesis. So in the current study, they aim to determine the AG01-mediated endothelial cell transcriptome, the functional importance of AG01-regulated endothelial function in vivo, and the relevance to adipose tissue function and obesity. A new mouse model with genetic deletion of AG01 in the endothelium was useful to investigate the importance of endothelial regulation of adipose tissue function. The findings were that in mice fed high fat, high sucrose diet, the suppression of endothelial AG01 promoted adipose tissue browning, and led to an anti-obesity phenotype. Endothelial cell AG01 thrombospondin-1 pathway was induced in the endothelium from human donors with insulin resistance. In total, this study suggests a novel mechanism, by which endothelial cells through AG01 thrombospondin-1 pathway controls vascularization and function of adipose tissues, insulin sensitivity, and whole-body metabolic state. Dr Greg Hundley: Interesting, Carolyn. So tell me about this clinically. Where do we take this from here? Dr Carolyn Lam: I thought you would ask. So endothelial dysfunction, per se, can cause metabolic dysregulation, rendering targeting dysfunctional endothelium, a potential therapeutic strategy to counteract obesity, and metabolic disorders. So this study really opens a door to that. Dr Greg Hundley: Very nice. Well, I've got another basic science paper, and it evaluates single-cell RNA sequencing to dissect the immunological network of autoimmune myocarditis. And it comes from Dr Jiangping Song from the State Key Laboratory of Cardiovascular Disease of Fuwai Hospital, and the National Center for Cardiovascular Disease, Chinese Academy of Medical Sciences, and Peking Union Medical College. So Carolyn, the study aimed to investigate the immunological network during the transition from myocarditis to cardiomyopathy, and to identify the genes contributing to the inflammatory response to myocarditis. So mice were treated with myosin heavy chain alpha-peptides to generate an experimental autoimmune myocarditis model. The investigators performed single-cell RNA sequencing analysis of CD45 plus cells extracted from mouse hearts during different experimental autoimmune myocarditis phases, including normal control, acute inflammation, subacute inflammation, and then in the myopathy phase. Also, human heart tissues were collected from surgically removed hearts of patients who had undergone heart transplantation. Dr Carolyn Lam: So what did they find, Greg? Dr Greg Hundley: Well, Carolyn, a comparison of the single-cell RNA sequencing data from different experimental autoimmune myocarditis phases suggested that some cell clusters, such as macrophage cluster 2 and Th17 cells, were associated with the inflammatory response in the experimental autoimmune myocarditis model. The HIF1A expression level correlated with the extent of the inflammatory response, and PX-478, a HIF1A inhibitor, alleviated the inflammation during the different experimental autoimmune myocarditis phases. Immunohistochemical staining revealed that HIF1A expression was upregulated in autoimmune myocarditis from the tissue samples from the explanted hearts. Thus, the HIF1A inhibitor alleviated inflammatory cell infiltration, and that may serve as a potential therapeutic target in clinical practice. Dr Carolyn Lam: Wow. That is some serious clinical implications. Well, my next paper is really the first systematic echocardiographic evaluation of consecutive patients requiring hospitalization due to COVID-19, and it comes from Dr Topilsky and colleagues from Tel Aviv Medical Center. Dr Greg Hundley: So Carolyn, what did they find in this series? Dr Carolyn Lam: So among a hundred consecutive patients diagnosed with COVID-19 infection who underwent complete echocardiographic evaluation, within 24 hours of admission, only 32% had a normal echocardiogram at baseline. The most frequent abnormality was right ventricular dilatation or dysfunction. Among patients developing clinical deterioration during follow-up, which were 20% of these hospitalized patients, repeated echocardiograms showed further deterioration of the right ventricular parameters, probably related to increased pulmonary resistance. Five of these patients had deep vein thrombosis. Dr Greg Hundley: Carolyn, my next study comes from Dr Stephen Fremes, and it's a modeling study out of the University of Toronto. It modeled TAVR versus SAVR valve durability to determine the effects on life expectancy across a broad range of age. Dr Carolyn Lam: Interesting. And what were the results? Dr Greg Hundley: Well, based on their simulation models, the durability of TAVR valves must be 70% shorter than that of surgically replaced valves to result in reduced life expectancy in patients with similar demographics to recent trials. However, in younger patients, the threshold for TAVR valve durability was substantially higher. In younger patients, life expectancy was reduced when TAVR durability was 30%, 40% and 50% shorter than surgical valves in 40, 50 or 60-year-old patients, respectively. So Carolyn, these findings suggest that durability concerns should not influence the initial treatment decision regarding TAVR versus SAVR in older low-risk patients, based on current evidence supporting TAVR valve durability. However, in younger low-risk patients, valve durability must be weighed against other patient factors, such as life expectancy. Dr Carolyn Lam: Thanks Greg, for that summary. Well, let me tell you about other papers in this issue. There are a pair of letters to the editor by Dr Opotowsky, and a response by Dr Goldberg regarding the paper results of the Fontan Udenafil Exercise Longitudinal, or FUEL trial. There's a research letter by Dr Strik, Validating QT-Interval Measurement Using the Apple Watch ECG to Enable Remote Monitoring During the COVID-19 Pandemic. There are two On My Mind papers, the first, Telemedicine and Forgotten America by Dr Julien, and the second, The COVID-19 Pandemic: Ethical and Scientific Imperatives for "Natural" Experiments by Dr Lewis. Dr Greg Hundley: Very nice. Well, Carolyn, I've got a research letter evaluating the effect of evolocumab on atherogenic lipoproteins during the peri and early post-infarction period. It's a placebo-controlled randomized trial from Dr Gary Gerstenblith. Sarah Cuddy also worked through a tough case of cardiac amyloid when a fat biopsy was negative, but imaging studies of the heart suggested cardiac amyloid. Carolyn, I've also got an On My Mind piece, and it's entitled, Can Old Ally Defeat a New Enemy? And it's by Dr Paul Gurbel, and he discusses the use of inhaled aspirin to treat patients with COVID-19. And then finally, Carolyn, I have a prospective piece from Dr Robert Lefkowitz who discusses β-arrestin-biased angiotensin II receptor agonists for treatment of COVID-19. Well, Carolyn, what a great issue, and let's get onto that feature discussion. Dr Carolyn Lam: Yay. Let's go, Greg. Dr Greg Hundley: Well, listeners. Now we're turning to our feature discussion, and we are very fortunate to have Professor Connie Bezzina from Amsterdam University Medical Center to talk to us about her paper related to long QT syndrome. Welcome, Connie. And I was wondering, before we get started in discussing your paper, could you tell us a little bit about the background in this area? And then, what was the hypothesis that you wanted to address? Prof Connie Bezzina: So over the last 20 to 30 years, we've learned a lot about the genetic underpinnings of inherited cardiac disorders associated with sudden cardiac arrest. And basically, we've learned a lot about mutations in specific genes that co-segregate with these disorders within families. However, two outstanding features have remained unresolved. Essentially, the first unresolved issue is the fact that we observe, oftentimes, a low disease penetrance and variable disease expression within families, which means that not everybody within a family that carries a familial mutation is affected by the disorder. But two, so among those that are affected, some are affected more severely than others. So some people would have only the ECG abnormality, whereas other people, for instance, would have the ECG abnormality and arrhythmic events. And you could also have individuals, indeed, who don't even manifest any disease manifestations. This is one of the outstanding challenges. The other outstanding challenge is the fact that, despite extensive genetic testing of the known genes in some probands and some families, they remain genetically lucid, in that we don't find a likely genetic defect in a minority of families. And of course, that hinders genetic testing and implementation of genetic testing in such families. Dr Greg Hundley: What was the question you were going to answer with your study? And tell us a little bit about your study design and your study population. Prof Connie Bezzina: Yeah, so essentially, we figured that assigning these disorders to one large genetic defect might be an oversimplification of biological phenomenon. So we hypothesized that even in these Mendelian disorders, the inheritance of additional genetic factors alongside the familial mutation could contribute to risk. Of course, there will be other factors such as environmental factors, which we did not tackle in the study. The central hypothesis of the study was that common genetic variation, which is present in the germ population, could modulate the effect of the familial genetic defect of the Mendelian mutation. So in order to do this, we assembled a large consortium of investigators from multiple centers in Europe, in North America and Japan, worldwide, to bring together about 1700 probands with the long QT syndrome. So we tested this hypothesis in the long QT syndrome because we figured, among the rare inherited rhythm disorders, it's one of the more common disorders. Also, because each individual center has too few patients. To do this locally, we put this group of investigators together to come up with 1700 probands. The study design was a genome-wide association study with a case-control design, where we tested the association of millions of SNPs littered across the genome with susceptibility for the disorder. So this led us to identify three single-nucleotide polymorphisms that are associated with susceptibility for the long QT syndrome. What we immediately saw is that, actually, these three SNPs, perhaps not surprisingly at all, had been previously associated with the extent of the QT interval, with QT interval in the general population. This is not surprising, of course, because repolarization is a central part of physiological mechanism in the long QT syndrome. So this basically indicated overlap between genetic control of the QT interval in the germ population and susceptibility to the long QT syndrome. So the fact that the three SNPs that we identified as long QT syndrome susceptibility SNPs had been associated with QT interval duration in the germ population, we felt that that was pointing to assure genetic underpinnings between these two phenotypes. So we went on to investigate that by looking at the correlation between the odds ratio for long QT syndrome susceptibility and the effect that these SNPs have on QT interval in the germ population. And in fact, we found a very high correlation between those. So essentially, this pointed to sure genetic factors between QT interval in the germ population and long QT syndrome susceptibility. Of course, we wanted to look for disease variability. The next thing we wanted to do was whether these SNPs could actually explain disease variability. Now, this was perhaps the most disappointing part of the study, because when we constructed a polygenic risk score based on SNPs that impact on the QT in the germ population, we found no relation to QT interval among patients, and also no relation to life-threatening arrhythmic events among the patients. We think that this is because our patients... or probrands. They're primarily probands, so they are all more sick. So we didn't have enough variability in our patient set to identify an association with disease variability. And in fact, this is at variance with previous studies that tested individual SNPs, and even our own studies with smaller polygenic risk scores that did find an association between a polygenic risk score based on QT SNPs and QT prolongation and events among patients. So we think that this is certainly something to study further in the future, in larger patient sets where we not only have the probands, but also their relatives, their mutation-carrying relatives, which will give us a bigger variability to actually test this hypothesis. So we think that looking at probands actually was a very good design to find susceptibility variance but was not maybe a good design to find SNPs or polygenic risk scores to test their effect on disease variability. Dr Greg Hundley: It sounds like you've found certain gene low PSI that indicate a predilection for prolongation of the QT interval, but not necessarily are those gene low PSI consistent with who's going to experience an adverse cardiovascular event as a result of their genetic constitution. Is that a fair statement? Prof Connie Bezzina: Well, I think that the setting, because we had probands, they were the most sick people in their families. I think to have stronger conclusions on that, we need to test the polygenic risk scores in families where there are people who are differentially affected. Dr Greg Hundley: I see. I- Prof Connie Bezzina: We had too-narrow of a variability in a probands-only design, as opposed to a study where we would have probands who are severely affected and mutation-carrying relatives who are less severely affected. Dr Greg Hundley: Very nice. So that puts that clearly into context. This was a massive effort. You have quite a list of investigators, and you mentioned you had to gather so many sites. How would you conduct that next study? Would you need another large collection of individuals and many sites to take that on? Prof Connie Bezzina: Yes. I'm a geneticist, and geneticists always want larger, larger numbers, and I'm also one of those. So I'm interested in explaining as much as possible into individual variability. And I think to do that properly, I think we should go preferably for a similar design where we will approach the same centers. And hopefully, we can organize the next study, which will have these probands and their relatives. Dr Greg Hundley: Now, just quickly, for us working in the clinic, how should we approach genetic testing in patients with long QT? Prof Connie Bezzina: At the moment, I think our findings don't have an immediate impact. I think our findings tell us about the genetic architecture of the disorder. And actually, one thing I haven't gone into yet is the fact that what we also found is that patients who do not have mutations in the no-long QT genes, which were called mutation-negative, which are about 20% of all long QT syndrome probands, actually have a higher burden of these common variants that prolong the QT interval. So we think, actually, that mutation-negative long QT syndrome probands will not have a Mendelian large effect variant but will have perhaps a higher burden of these QT-prolonging alleles. Therefore, I think this has direct implications for clinical genetics of these patients, because if you have a proband in whom you don't find a mutation in the known genes, you could think that maybe it is not monogenic, which has implications because you don't have a single genetic defect to test on that family. One would need to keep follow-up of more family members until we understand more about the genetics of those individuals. Dr Greg Hundley: So Connie, this has been just a wonderful discussion. Any additional studies examining the genetic architecture of individuals that we need to think about for the future? Prof Connie Bezzina: Sure. So for long QT syndrome in particular, as additional SNPs that modulate the QT interval in the germ population are identified, it will be very important to incorporate these into larger polygenic risk scores, and see whether we could have a better discriminative capacity of such polygenic risk scores in discriminating between severely affected and less severely affected people, or who is more at risk for an arrhythmic event. Outside of long QT syndrome, I think there's a lot of work to be done with respect to the likely complex inheritance of many of these disorders that we previously considered to be Mendelian. So for instance, ongoing work in our group concerns Brugada syndrome, where we're seeing the same kind of thing, and hypertrophic cardiomyopathy, where we're seeing the same kind of inheritance. Dr Greg Hundley: Well listeners, on behalf of both Carolyn and myself, we look forward to catching you on the run next week. Take care. This program is copyright the American Heart Association 2020.
November is National Family Caregiver Month, and while I was at the 4th Annual National Caregiving Conference in Chicago, I met this week's guest, Caregiver Strategist Penny Patnaude. Penny and I talked about some of the things that caregivers want to be thinking about in planning their own strategy for providing great care to their loved one without losing themselves in the process. I'll also introduce you to Dr. Lindsey Knowles, a young MS rehabilitation researcher who will give us an insider's look at what the Tykeson Fellows Conference is all about, and why it's so important for the future of MS research. We're also talking about the cannabis-based treatment for MS spasticity that was approved in the U.K. You'll hear about last week's International Progressive MS Alliance meeting in Amsterdam, and we'll tell you about an investigational therapy that can reset the immune system in the mouse model of MS in a single dose. We'll remind you that if you're a young adult, between 18 and 45 years old, First Descents is offering you a week-long ski trip to Crested Butte, Colorado. And it's FREE! (Don't worry, we'll tell you how and where to begin the application process!) You'll hear about the research team in Montreal that may have found a way to stop MS progression. And we'll tell you how easy it is to share this episode of RealTalk MS with family and friends! We have a lot to talk about! Are you ready for RealTalk MS??! ___________ Thanks for all the kind words! :22 The National MS Society & RealTalk MS Partnership :52 Join the Conversation! Get In Touch with RealTalk MS 2:21 The Progressive MS Alliance Meeting in Amsterdam 3:32 The Tykeson Fellows Conference & My Conversation with Dr. Lindsey Knowles 5:26 Sativex Approved in U.K. for MS Spasticity 11:12 Single Dose of Investigational Therapy Resets the Immune System in Mouse-Model of MS 13:19 First Descents Offers a FREE Ski Trip to Crested Butte to Young Adults Living with MS 15:39 Canadian Research Team Says They May Be Able to Stop MS Progression 17:51 My Interview with Caregiver Strategist Penny Patnaude 19:51 Share This Episode 27:11 How to Subscribe to RealTalk MS, Download the RealTalk MS App, or Listen with Alexa 27:36 ___________ SHARE THIS EPISODE OF REALTALK MS Just copy this link & paste it into your text or email: https://realtalkms.com/117 ___________ ADD YOUR VOICE TO THE CONVERSATION I've always thought about the RealTalk MS podcast as a conversation. And this is your opportunity to join the conversation by sharing your feedback, questions, and suggestions for topics that we can discuss in future podcast episodes. Please shoot me an email or call the RealTalk MS Listener Hotline and share your thoughts! Email: jon@realtalkms.comPhone: (310) 526-2283 ___________ LINKS If your podcast app doesn't allow you to click on these links, you'll find them in the show notes in the RealTalk MS app or at www.RealTalkMS.com Give RealTalk MS a Rating & Review The National MS Society & RealTalk MS Announce a Partnership The International Progressive MS Alliance Administration of a CD45 Antibody Drug Conjugate as a Novel, Targeted Approach to Achieve Immune System Reset: A Single Dose of CD45-targeted ADC Safely Conditions for Autologous Transplant and Ameliorates Disease in Multiple Models of Immune Disease First Descents Skiing at Crested Butte Activated Leukocyte Cell Adhesion Molecule Regulates B Lymphocyte Migration Across Central Nervous System Barriers The Caregiver Strategist Give RealTalk MS a Rating & Review Download the RealTalk MS App for iOS Download the RealTalk MS App for Android ___________ Follow RealTalk MS on Twitter, @RealTalkMS_jon, and subscribe to our newsletter at our website, RealTalkMS.com. RealTalk MS Episode 117 Hosted By: Jon Strum Guests: Penny Patnaude Tags: MS, MultipleSclerosis, MSResearch, MSSociety, Caregiving, Sativex, Stemcells, FirstDescents, RealTalkMS Privacy Policy
The TWiVodrome considers the intestinal tract as an alternative infection route for MERS coronavirus, and how reduced accumulation of defective viral RNAs might lead to severe influenza. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler Become a patron of TWiV! Links for this episode HaHa influenza virus (mBio) MERS coronavirus intestinal transmission (Sci Adv) Simulated gastric and intestinal fluids Stomacher Defective genomes and severe influenza (PLoS Path) Image credit Letters read on TWiV 481 Weekly Science Picks Kathy - CD45.1 and CD45.2 mice may not be functionally equivalent Rich - Relative humidity versus dewpoint (video) Dickson - Underwater photography winners Alan - Photo of a single atom Vincent - Proposed Federal science budget cuts and The Blog is Dead Listener Pick Jess - Louis Tompkins Wright Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 16/19
Beim kolorektalen Karzinom treten Biomarker zunehmend in den Fokus. Ziel muss daher die Etablierung von effektiven Markern sein, um den Patienten eine möglichst effektive, auf das Individuum zugeschnittene Diagnostik und Therapie anbieten zu können. In unserer Studie zum kolorektalen Karzinom sind nach Ausschlusskriterien n=108 Patienten mit Primärtumoren und n=59 Patienten mit Lebermetastasen vom KRK in die statistische Analyse eingegangen. Aus postoperativ entnommenem, N2-schockgefrorenem Gewebe wurde ein Biomarker-Profil aus molekulargenetischen und immun-histologischen Markern erstellt. Genetische Mutationen auf dem KRas- und BRaf-Gen wurden mittels Mikrodissektion/PCR/Pyrosequenzierung detektiert und mittels immunhistochemischer Färbungen die Moleküle EGF-R, Her2/neu, IGF1-R, c-Met, CD44v6, Ki67, CD45 und HLA-DR analysiert. Die statistische Analyse erfolgte univariat mittels Chi-Quadrat-Test, T-Test, Mann-Whitney-U-Test und Kruskal-Wallis-Test. Überlebensanalysen erfolgten mittels Kaplan-Meier-Schätzer und Cox-Regressionsanalyse. Hierbei ergaben sich in der univariaten Analyse zwischen den einzelnen Parametern der Primärtumoren folgende statistisch signifikante Korrelationen: Eine Mutation/der Wildtyp im KRas-Gen korrelierte mit dem L-Stadium, einer Mutation/der Wildtyp im BRaf-Gen und IGF1-R (p=0,007; 0,003; 0,034). Der um die Mutation G13D erweiterte KRas Wild-typ bzw. mutiert (ohne G13D) korrelierte hierunter mit dem L-Stadium, der Histologie, sowie ebenfalls mit BRaf (p=0,038; 0,039; 0,011). Eine Mutation im BRaf-Gen (Exon 15) war mit dem Geschlecht, der Lokalisation und dem Grading des Primärtumors signifikant verbunden (p=0,042; 0,003; 0,002). Der EGF-R korrelierte mit der Lokalisation, dem Grading, dem L-Stadium und CD44v6-Positivität (p=0,031, 0,020, 0,006, 0,021). Bei Her2/neu fand sich für das Geschlecht, die Lokalisation, CD45 und für HLA-DR eine Ver-knüpfung (p=0,005, 0,021, 0,032, 0,006). Zudem konnte ein Zusammenhang zwischen Her2/neu (Score) und dem Geschlecht (p=0,009) sowie der Tumorlokalisation (p=0,010) nachgewiesen werden. Für c-Met und IGF1-R (p=0,021) und für IGF1-R mit der Lokalisation des Primarius (p=0,027) bestand eine positive Korrelation. Des Weiteren korrelierten CD45 mit HLA-DR (p=0,046) und Ki67 mit dem Alter der Patienten (p=0,015). Hinsichtlich der Lebermetastasen konnte eine Verbindung von EGF-R mit der Histologie, mit IGF1-R und c-Met (p=0,046; 0,004; 0,007) nachgewiesen werden. Zudem konnte für c-Met und der Tumor-größe, dem löslichen präoperativen Tumormarker CA19-9 und CD44v6 (p=0,004; 0,003; 0,044) eine positive Korrelation nachgewiesen werden. HLA-DR und der BMI wiesen einen statistisch signifikanten Wert (p=0,022) auf und für Ki67 und CD44v6 bestand ebenfalls eine signifikante Verbindung (p=0,007). Ki67 und das Grading (p=0,017) sowie Her2/neu (Score) und die Histologie der Lebermetastasen (p=0,018) wiesen ebenfalls Signifikanz auf. Für das Gesamtüberleben im Kollektiv der Primärtumoren konnte im Kaplan-Meier-Schätzer ein Zusammenhang von N-Stadium (p
Purpose: To describe new details of epiretinal cell proliferation in flat-mounted internal limiting membrane specimens. Methods: One hundred nineteen internal limiting membrane specimens were removed en bloc with epiretinal membranes from 79 eyes with macular pucker (MP) and 40 eyes with vitreomacular traction syndrome. Intraoperatively, posterior vitreous detachment was assessed as complete or incomplete. Whole specimens were flat-mounted on glass slides and processed for interference and phase-contrast microscopy, cell viability assay, and immunocytochemistry. Results: Mean cell viability percentage was higher in MP than in vitreomacular traction syndrome. Two cell distribution patterns were found. Anti-CD163 labeling presented predominantly in MP with complete posterior vitreous detachment. CD45 expression was similar in all groups of diagnosis. Anti-glial fibrillary acidic protein (GFAP) labeling was found in MP irrespective of the extent of posterior vitreous detachment. Alpha-SMA (alpha-smooth muscle actin) labeling was mainly presented in MP with incomplete posterior vitreous detachment and in vitreomacular traction syndrome. Simultaneous antibody labeling included GFAP/CD45, GFAP/CD163, CD163/CD45, and CD163/alpha-SMA. Conclusion: Hyalocytes constitute a major cell type of epiretinal cell proliferation in eyes with MP and vitreomacular traction syndrome. Glial cells, notably retinal Muller cells, are involved as well. It appears that transdifferentiation of cells in vitreomacular traction might be more frequent than previously thought and that those cells possess a greater variability of immunocytochemical properties than expected. RETINA 33:77-88, 2013
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 14/19
Purpose. To describe new details of epiretinal cell proliferation in flat-mounted internal limiting membrane (ILM) specimens. Methods. One hundred and nineteen ILM specimens were removed en-bloc with epiretinal membranes (ERMs) from 79 eyes with macular pucker (MP) and 40 eyes with vitreomacular traction syndrome (VMTS). Intraoperatively, posterior vitreous detachment (PVD) was assessed as complete or incomplete. Whole specimens were flat-mounted on glass slides, and processed for interference and phase contrast microscopy, cell viability assay, and immunocytochemistry. Results. Mean cell viability percentage was higher in MP than in VMTS. Two cell distribution patterns were found. Anti-CD163 labeling presented predominantly in MP with complete PVD. CD45 expression was similar in all groups of diagnosis. Anti-GFAP labeling was found in MP irrespective of the extent of PVD. Alpha-SMA labeling mainly presented in MP with incomplete PVD and in VMTS. Simultaneous antibody labeling included GFAP/CD45, GFAP/CD163, CD163/CD45, and CD163/α-SMA. Conclusions. Hyalocytes constitute a major cell type of epiretinal cell proliferation in eyes with MP and VMTS. Glial cells, notably retinal Müller cells, are involved as well. It appears that transdifferentiation of cells in vitreomacular traction might be more frequent than previously thought and that those cells possess even more different immunocytochemical properties than expected.
Olá, Nesta edição do podcast apresento-vos o artigo "A single Naive CD8+ T cell precursor can develop into diverse effector and memory subsets" por Stemberg et al Immunity 2007, 27:985. Este artigo demonstra que um único precursor T naïve OT-1 pode gerar um repertorio diverso de células T efectoras e T memória (memória efectoras e memórias centrais). Este artigo vem redescrever o nosso conhecimento sobre as origens das populações memória T. Os autores transferem uma única célula T naïve OT-1 (CD45.1 +) num ratinho naïve C57BL/6 (CD45.1 -) e estimulam as células com bacteria Listeria monocytogenes expressando o antigénio OVA. 12 dias após a estimulação e a transferência da célula precursora os autores observam a expansão celular das células CD45.1+ com formação de células memória efectoras (CD62L low, CD45RA +) e células memória centrais (CD62L high, CD45RA+). Estas células memória são capazes de desenvolver respostas memória após restimulação e mesmo quando a transferência das célula precursora ocorre após a estimulação antigénica. Até breve. Pedro
Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 02/07
Comparatively little is known about the structure and development of the lung associated immune system in the chicken, despite its important role in the defence numerous infectious diseases. In this study lung samples were obtained from line M11 leghorn chickens on days 1, 3 and 7 after hatch and of 2, 3, 4, 6, 8 and 13 weeks old birds for immunohistological, scanning as well as transmission electron microscopy (TEM). Monclonal antibodies reactive with T-cells (CD3, CD4, CD8), B cells (BU-1), myeloid cells (KUL1) or all leucocytes (CD45) were used in this study. To maintain the structural integrity of cyropreserved tissue samples for immunhistological studies lung tissue was perfused with low melting agarose by application through the trachea. An age depended development of the lung immune system was observed and found to show different developmental characteristics in the bronchus associated lymphoid tissue (BALT) and the immune system of the parachoncial tissue and the interparabronchial connective tissue. Roughly, three developmental stages could be discriminated. During the first week after hatch cells of the innate immune system dominated the picture while T-cells and B-cells were rarely found. Similarly, myeloid cells were much more abounded than lymphocytes in the second stage (2.-4. week). However, an increase of lymphocytes was found with T-cells in excess of B-cells. T-cells were found most frequently in the interparabronchial connective tissue where they formed small aggregates. In wall of the primary bronchus organized lymphoid aggregates were first observed in the 4th week after hatch. In 6-8 weeks old birds (stage 3) distinct B- and T-cell areas began to develop in these lymphoid aggregates with T-cells occupying the central area and B-cells located at the periphery. Increasing frequencies and sizes of T-cell aggregates were also seen in the wall of the parabronchus and the interparabronchial connective tissue at this stage of development. Finally, in birds older than 12 weeks the BALT structures showed a clear separation into B-cell follicles surrounded by a T-cell area formed by CD4+ lymphocytes. Similarly, distinct B-cell follicles with adjacent T-cell regions were observed in the interparabronchial connective tissue. Scanning electron microscopy revealed that the epithelium covering the BALT reagions at the openings of the secondary bronchi was largely unciliated at all developmental stages. By TEM a unique cell type was found intercalated into this epithelium which was in close contact with lymphocytes and macrophages located in cellular pockets. These structure properties are similar to M cells in the mammalian mucosa associated lymphoid tissue (MALT). In conclusion, the lung associated immune system in the chicken is largely made up of cells of the innate immune system during the first week after hatch. Subsequently, cells of the adaptive immune system start to appear and to form organized lymphoid structures in the primary bronchus and to disseminate in the remaining lung tissue. Mature morphology of these structures is seen in birds 12 weeks and older.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 03/19
Die vorliegende Arbeit beschäftigt sich mit der funktionellen und molekularen Charakterisierung von humanen CD34- Zelllinien aus dem peripheren Blut (V54/1, V54/2) im Vergleich zu den aus dem Knochenmark etablierten Zelllinien (L87/4, L88/5). Die Klone V54/1 und V54/2 wurden aus dem peripheren Blut nach Stammzellmobilisierung und CD6 Depletion durch Zugabe eines Faktorengemisches aus IL-1b, IL-3, IL-6, IL-7, IL-8 und IL-11 erzeugt. L87/4 und L88/5 hingegen sind adhärente und wachstumsarretierte Stromazellen, die die Erhaltung und Differenzierung von hämatopoetischen Vorläuferzellen durch Mediatoren ermöglichen (Thalmeier et al. 2000). Das Ziel dieser Arbeit war die Untersuchung von Stammzelleigenschaften bei den Zelllinien L87/4, L88/5, V54/1 und V54/2. Dazu soll die Färbung mit den Farbstoffen Rhodamin 123 (Rh123) und Hoechst 33342 zeigen, ob Subpopulationen innerhalb der Klone mit unterschiedlichen Färbeeigenschaften, bestehen. Die biologische Bedeutung der beiden Farbstoffe liegt darin, dass Sie dazu geeignet sind frühe Stammzellen zu identifizieren. Als Substrat der P-Glykoproteinpumpe, die u.a. auf frühen Vorläuferzellen mit stark erhöhter Repopulationskapazität gefunden wird, werden diese Farbstoffe aus der Zelle gepumpt. Der Farbstoff-Efflux kommt durch die mdr-Gen-kodierte (multi-drug-resistance) und Kalzium-abhängige P-Glykoproteinpumpe zustande. Das P-Glykoprotein hat neben der Bedeutung in der Stammzellbiologie in der angewandten Medizin eine wichtige Funktion in der Resistenzentwicklung von Tumoren. Des weiteren wurden bei den Zelllinien stammzellrelevante Oberflächenantigene (CD10, CD34, CD14, CD105, SH3 und CD117) untersucht, um Unterschiede zwischen L87/4, L88/5 und den Klonen V54/1, V54/2 zu erkennen. Versuche zur Induktion der Differenzierung sollten Hinweise auf die Plastizität der Zelllinien geben. Experimente an den durch den Rh123-Efflux unterscheidbaren Subpopulationen der Zelllinie V54/2 dienen der Aufklärung von Unterschieden in Morphe, zellulären Transportfunktionen und Funktionseinheiten von Transkriptionsfaktor Netzwerken. Methodisch wurde für die Analyse der Epitope und der Färbungen mit Rh123 und Hoechst 33342 ein Durchflußzytometer verwendet. Die Analyse der Funktionseinheiten von Transkriptionsfaktor Netzwerken wurde mittels Reverse Transkriptase Polymerase Ketten Reaktion durchgeführt. Die Ergebnisse der Färbeexperimente zeigten, dass bei allen untersuchten Zelllinien durch eine unterschiedliche Anfärbbarkeit der Zellen mit dem Farbstoff Rh123 zwei Subpopulationen unterschieden werden können. Die jeweils größere Subpopulation der Zelllinien färbt sich mit Rh123 an und bleibt auch nach einer definierten Inkubationszeit, die den Rh123-Efflux ermöglichen soll, gefärbt. Sie wird Rh123high genannt. Die übrigen Zellen, die bei allen Zelllinien unter 10% der Gesamtpopulation betragen, sind in der Lage den Farbstoff aus der Zelle zu pumpen. Diese Subpopulation wird Rh123low genannt und ist mit Stammzelleigenschaften wie tausendfach erhöhter Repopulationsfähigkeit in NOD/SCID-Mäusen assoziiert. Es konnte also innerhalb der untersuchten monoklonalen Linien eine Rh123low Subpopulation identifiziert werden, die sich durch zahlreiche biologische Eigenschaften von der Gesamtpopulation unterscheidet. Da der Rh123 Efflux durch eine Kalzium-abhängige Pumpe zustande kommt, lässt sie sich durch den Kalziumantagonisten Verapamil hemmen. Eine Hemmung der Pumpe bewirkt, dass die Rh123low Zellen nicht mehr in der Lage sind Rh123 aus der Zelle zu pumpen, so dass sie nach einer definierten Inkubationszeit mit Rh123 gefärbt bleiben. Neben diesem funktionellen Beweis für die P-Glykoproteinpumpe konnte durch den strukturellen Nachweis der Pumpe mittels eines Antikörpers gegen P-Glykoprotein ein definitiver Beweis für das Vorhandensein der aktiven P-Glykoproteinpumpe bei der Rh123low Population erbracht werden. Mit dem anderen Farbstoff Hoechst 33342 können die jeweiligen Anteile der Zelllinien in den einzelnen Stadien des Zellzyklus nachgewiesen und zudem ein kleiner Anteil an Zellen bestimmt werden, der als „Side Population“ (SP-Zellen) definiert wird. Diesen SP-Zellen werden Eigenschaften von aktiven Stammzellen zugeschrieben. Hierbei besteht ein Unterschied zwischen den aus dem Knochenmark und den aus dem peripheren Blut etablierten Linien, da die Zellen aus dem peripheren Blut nicht nur ein anderes Zellzyklusmuster aufweisen, sondern auch einen höheren Anteil an SP-Zellen besitzen. Es wurden vergleichende Untersuchungen zwischen den Zelllinien und zwischen den Rh123high und Rh123low Subpopulationen innerhalb einer Zelllinie mit Antikörpern gegen die Epitope CD14, CD45, HLA-DR, CD10, CD117, CD105 und SH3 durchgeführt. Dabei waren CD14 und CD45 auf allen Zelllinien negativ, wobei alle Zelllinien eine positive Expression für den mesenchymalen Marker Endoglin (CD105) und für SH3 (CD73) zeigten. CD117 konnte nur auf den aus dem Knochenmark etablierten Zelllinien L87/4 und L88/5 nachgewiesen werden. CD34, ein charakteristischer Marker für hämatopoetische Vorläuferzellen, aber auch für Endothelzellen, konnte nur auf den Zellen der Rh123low Subpopulation nachgewiesen werden. Im Gegensatz dazu exprimieren die Rh123high Zellen kein CD34. Da es sich bei den Zelllinien um Klone handelt, ist der Unterschied in der Expression von CD34 zwischen der Rh123low und der Rh123high Population ein deutlicher Hinweis auf die Plastizität der Zelllinien und das Fließgleichgewicht zwischen Rh123low und Rh123high. Durch eine Zellsortierung der Zelllinie V54/2 wurde die Rh123low von der Rh123high Subpopulation getrennt, um sie dann bezüglich ihrer Morphologie, dem Wachstum in Methylzellulose und der Expression ausgewählter Funktionseinheiten von Transkriptionsfaktor Netzwerken zu untersuchen. Dabei erhärtete sich die Hypothese, dass es sich bei der Rh123low Subpopulation um aktivere Zellen mit einer gesteigerten Expression von erythroid/myeloischen und mesodermalen Eingaben (z.B. VEGF, BMP-4), Rezeptoren (z.B. tie-1), vernetzter Transkriptionsfaktoren (z.B. GATA, ETS) und letztendlich Ausgaben (z.B. PECAM) handelt. Diese fungieren in Netzwerken mit dem Ziel, stammzellrelevante Funktionen zu ermöglichen. Die Morphologie zeigte in den Zytozentrifugationspräparaten deutliche Unterschiede zwischen Zellen der Rh123low und der Rh123high Subpopulation. Die Rh123low Subpopulation besteht aus lymphoid-ähnlichen Zellen, was für Zellen mit Stammzellfunktion charakteristisch ist. Die Rh123high Subpopulation dagegen hat ein insgesamt größeres Zellvolumen und einen gebuchteten Kern mit perinukleärer Aufhellung. Untersuchungen des klonalen Wachstums in der Methylzellulose ergaben bei keiner der Subpopulationen eine wesentliche Koloniebildung. Durch die Inkubation der Zelllinie V54/2 mit dem Neurotropen Wachstumsfaktor (NGF) konnte eine morphologische Änderung in Richtung einer neuronalen/glialen Differenzierung nach 8-12 Stunden induziert werden. Der immunhistochemische Nachweis von Glial Fibrillary Acidic Protein (GFAP) bestätigte die mesenchymale Potenz zumindest in Richtung einer glialen Differenzierung. Das unterschiedliche Expressionsmuster ausgewählter, für die Differenzierung notwendiger Zusammenspieler innerhalb von Transkriptionsfaktor Netzwerken innerhalb der Rh123high und der Rh123low Population bei V54/2 war ein weiterer Hinweis, dass es sich bei der Rh123low Subpopulation um aktive Vorläuferzellen mit möglicher Stammzellpotenz handelt. In der Rh123low Subpopulation wurde im Gegensatz zur Rh123high Population eine Expression von BMP4, GATA1, GATA3 nachgewiesen, die essentiell für die Hämatopoese und für eine mesenchymale Differenzierung ist. Die Faktoren für GATA2, GATA3, beta globin, Elf-1 und PECAM1 wurden in einem stärkeren Maß in der Rh123low als in der Rh123high Population exprimiert. BMP-Rez., Myb, sowie die Endothel-assoziierten Faktoren Tie-1 und VEGF waren in beiden Subpopulationen gleich stark vorhanden. Bei den wenigen Funktionseinheiten der größeren und Rh123high Population handelt es sich vor allem um angiogenetische Faktoren, was auf eine limitierte Differenzierungseigenschaft der Rh123high Subpopulation und die enge Beziehung zwischen Blut- und Endothelzellen („Hämangioblast“) hinweist. Ein Nachweis für die Plastizität der Stammzellen innerhalb der von uns etablierten Zelllinien wurde dadurch erbracht, dass die zellsortierten Subpopulationen Rh123low und Rh123high nach dem Sortierexperiment getrennt rekultiviert wurden, wobei das Wachstum der Rh123low Subpopulation deutlich langsamer war als das der Rh123high Subpopulation. Nach zwei Wochen wurden die zellsortierten Subpopulationen erneut einer Rh123 Färbung unterzogen, wobei sich wiederum das ursprüngliche Verhältnis zwischen den Rh123low und Rh123high Subpopulationen einstellte. So kann man aus der Transdifferenzierung der Zelllinien von Rh123low in Rh123high und umgekehrt die Plastizität der hier untersuchten adulten Stammzelllinien ableiten. Die Ergebnisse sollen zum grundlegenden Verständnis der Biologie adulter (nicht embryonaler) Stammzellen beitragen und damit die Möglichkeit schaffen, adulte Stammzellen bzw. deren Subpopulationen gezielt für einen reparativen Gewebe- und Organersatz zu verwenden. Dabei liefern sie die Basis für weitergehende Untersuchungen zum besseren Verständnis der physiologischen und regenerativen Vorgänge, z.B. auch bei Alterung oder bei gesteigerter Funktion. Darüber hinaus kann aufgrund der vorliegenden Ergebnisse durch weitere Untersuchungen möglicherweise besser verstanden werden, ob es gelingen kann das Potential adulter Stammzellen zur therapeutischen Gewebereparation, z.B. zur Verhinderung oder Verringerung einer Narbenbildung, zu nutzen.
Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 01/07
Production and characterization of monoclonal antibodies specific for equine leucocytes This work was initiated to produce new monoclonal antibodies for the characterization of equine leucocyte-subpopulations. For immunisation, leucocytes isolated from the peripherial blood, lymph nodes and the intestinal epithelium of horses, have been used. Out of six cell-fusions 78 clons were isolated. To characterize the mAbs, the attention was focused on immunofluorescence analysis. Antibodies of the clones 4-13, 4-58, 1-19 and 6-39 have been purified by protein G-sepharoses chromatography and, except mAb 4-58, biotinylated. MAb 4-13, mAb 4-58 and mAb 5-50 are binding specifically to equine T-cells. Flow cytometer analyses demonstrated an increased frequency following the stimulation with Concanavalin A. The frequency of mAb 4-13- and mAb 4-58-positive cells matched the total frequency of CD4- and CD8-positive cells. The number of cells, marked by mAb 5-50, is slightly lower. Most likely these three mAbs recognize three different, as yet unknown antigens. MAb 4-58 stimulated the in vitro proliferation of leucocytes. Moreover mRNA for interleukin-4 and interferon gamma was detected in these cells after the stimulation. By contrast the antibodies 1-19, 2-52, 4-36, 4-55 and 6-39 are B-cell-specific, since they were present on cells that were Ig-positive. The monoclonal antibodies 6-5 and 6-17 stain all equine leucocytes. Possibly they are binding to an equine CD45-molecule. The antibodies of clone 4-18 recognize an antigen on granulocyctes. Preliminary data suggests that mab 4-39 is binding to a lektin, that can be found mainly on B-Lymphocytes and endothelial cells. No group of leucocytes and no antigen could be assigned to the other antibodies. The antibodies described here will be valuable tools to characterize different equine leucocyte-subpopulations by flow cytometry.
Aims: During pregnancy, the placenta produces a variety of steroid hormones and proteins. Several of these substances have been shown to exert immunomodulatory effects. Progesterone is thought to mediate some of these effects by regulating uterine responsiveness. The aim of this study was to clarify the effect of amniotic fluid transferrin and its N-glycans on the release of progesterone by first trimester trophoblast cells in vitro. Methods: Cytotrophoblast cells were prepared from human first trimester placentae by trypsin-DNAse dispersion of villous tissue followed by a percoll gradient centrifugation and depletion of CD45 positive cells by magnetic cell sorting. Trophoblasts were incubated with varying concentrations (50-300 mug/ml) of transferrin from human amniotic fluid and serum as well as with N-glycans obtained from amniotic fluid transferrin. Culture supernatants were assayed for progesterone by enzyme-immunometric methods. Results: The release of progesterone increased in amniotic fluid transferrin- and N-glycan-treated trophoblast cell cultures compared to untreated trophoblast cells. There was no stimulating effect of serum transferrin on the progesterone production of trophoblast cells. Conclusions: The results suggest that amnion-transferrin and especially its N-glycans modulate the endocrine function of trophoblasts in culture by up regulating progesterone secretion.
Memory T cells display phenotypic heterogeneity. Surface antigens previously regarded as exclusive markers of naive T cells, such as L-selectin ( CD62L), can also be detected on some memory T cells. Moreover, a fraction of CD45RO(+) ( positive for the short human isoform of CD45) memory T cells reverts to the CD45RA(+) ( positive for the long human isoform of CD45) phenotype. We analyzed patients with biopsy-proven localized Wegener's granulomatosis (WG) (n = 5), generalized WG (n = 16) and age- and sex-matched healthy controls ( n = 13) to further characterize memory T cells in WG. The cell-surface expression of CD45RO, CD45RA, CD62L, CCR3, CCR5 and CXCR3 was determined on blood-derived T cells by four-color flow cytometric analysis. The fractions of CCR5(+) and CCR3(+) cells within the CD4(+) CD45RO(+) and CD8(+) CD45RO(+) memory T cell populations were significantly expanded in localized and generalized WG. The mean percentage of Th1-type CCR5 expression was higher in localized WG. Upregulated CCR5 and CCR3 expression could also be detected on a fraction of CD45RA(+) T cells. CD62L expression was seen on approximately half of the memory T cell populations expressing chemokine receptors. This study demonstrates for the first time that expression of the inducible inflammatory chemokine receptors CCR5 and CCR3 on CD45RO(+) memory T cells, as well as on CD45RA(+) T cells ('revertants'), contributes to phenotypic heterogeneity in an autoimmune disease, namely WG. Upregulated CCR5 and CCR3 expression suggests that the cells belong to the effector memory T cell population. CCR5 and CCR3 expression on CD4(+) and CD8(+) memory T cells indicates a potential to respond to chemotactic gradients and might be important in T cell migration contributing to granuloma formation and vasculitis in WG.
Natural killer (NK) cell activity appears to be conserved throughout vertebrate development but NK cells have only been well characterized in mammals. Candidate NK cells have been identified in the chicken as cytoplasmic CD3+ and surface T cell receptor (TCR)/CD3- (TCRO) lymphocytes that often express CD8. The fact that the TCRO cells are abundant in the embryonic spleen before T cells enter this organ allowed us to cultivate the embryonic TCRO cells using growth factors derived from activated adult lymphocytes. These TCRO cells were cytotoxic for an NK target cell line. They expressed cell surface CD8, a putative interleukin-2 receptor, CD45 and a receptor for IgG, but did not express CD4, major histocompatibility complex class II or immunoglobulin. Biochemical analysis of the cytoplasmic CD3 antigen revealed two of the three CD3 , and homologues, and RNA transcripts for the third. The CD3 monoclonal antibody also precipitated a 32-kDa dimer that may represent a heterodimer of different CD3 constituents. TCR and gene transcripts were not detected in the TCRO cells. These results indicate that the avian TCRO cell is the mammalian NK cell homologue. The shared evolutionary features of T cells and NK cells in birds and mammals support the idea that they derive from a common progenitor
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