Podcasts about ccl5

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.21.521419v1?rss=1 Authors: Mitchell, J. M., Mabin, J. W., Muehlbauer, L. K., Annis, D. S., Mathur, S. K., Johansson, M. W., Hebert, A. S., Fogerty, F. J., Coon, J. J., Mosher, D. F. Abstract: IL5 and IL33 are major activating cytokines that cause circulating eosinophils to polarize, adhere, and release their granule contents. We correlated microscopic features of purified human blood eosinophils stimulated for 10 min with IL5 or IL33 with phosphoproteomic changes determined by multiplexed isobaric labeling. IL5 caused phosphorylation of sites implicated in JAK/STAT signaling and localization of pYSTAT3 to nuclear speckles whereas IL33 caused phosphorylation of sites implicated in NF{kappa}B signaling and localization of RELA to nuclear speckles. Phosphosites commonly impacted by IL5 and IL33 were involved in networks associated with cytoskeletal organization and eosinophil adhesion and migration. Many differentially regulated phosphosites were in a diverse set of large proteins-RAB44, a "large RAB" associated with crystalloid granules; NHSL2 and VIM that change localization along with the nucleus during polarization; TNFAIP3 vital for control of NF{kappa}B signaling, and SRRM2 and PML that localize, respectively, to nuclear speckles and PML bodies. Gene expression analysis demonstrated differential effects of IL5 and IL33 on IL18, CCL5, CSF1, and TNFSF14. Thus, common effects of IL5 and IL33 on the eosinophil phosphoproteome are important for positioning in tissues, degranulation, and initiation of new protein synthesis whereas specific effects on protein synthesis contribute to phenotypic heterogeneity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

In episode 3 of Exploration Science, Professor Ingrid Dijkgraaf and Dr. Amine Jmel discuss the potential of tick salivary proteins as lead molecules to develop therapeutics, vaccines, and imaging agents for applications ranging from cardiac disease to livestock immunization. DOI Links: Inhibition of platelet adhesion, thrombus formation, and fibrin formation by a potent αIIbβ3 integrin inhibitor from ticks: 10.1002/rth2.12466 Immunomodulatory Proteins in Tick Saliva from a Structural Perspective: 10.3389/fcimb.2021.769574 Exogenous Integrin αIIbβ3 Inhibitors Revisited: Past, Present and Future Applications: 10.3390/ijms22073366 Structural characterization of anti-CCL5 activity of the tick salivary protein evasin-4: 10.1074/jbc.RA120.013891 Tick Saliva Protein Evasin-3 Allows for Visualization of Inflammation in Arteries through Interactions with CXC-Type Chemokines Deposited on Activated Endothelium: 10.1021/acs.bioconjchem.0c00095 Tick saliva protein Evasin-3 modulates chemotaxis by disrupting CXCL8 interactions with glycosaminoglycans and CXCR2: 10.1074/jbc.RA119.008902 Watch on YouTube: https://youtu.be/EZBAt7Sw-e4

This week, please join authors Mikhail Kosiborod and Christian Schulze and Editorialist Stefan Anker as they discuss the original articles "Effects of Empagliflozin on Symptoms, Physical Limitations and Quality of Life in Patients Hospitalized for Acute Heart Failure: Results From the EMPULSE Trial" and "Effects of Early Empagliflozin Initiation on Diuresis and Kidney Function in Patients With Acute Decompensated Heart Failure (EMPAG-HF)" and the editorial "SGLT2 Inhibitors: From Antihyperglycemic Agents to All-Around Heart Failure Therapy." Dr. Carolyn Lam:             Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to the journal and its editors. We're your co-hosts. I'm Dr. Carolyn Lam, Associate Editor from the National Heart Center and Duke National University of Singapore. Dr. Greg Hundley:           And I'm Dr. Greg Hundley, associate editor, director of the Pauley Heart Center at VCU Health in Richmond, Virginia. Dr. Carolyn Lam:             I'm so excited about the feature discussion this week. It is a paired feature along with their editorial and it's all focused on SGLT2 inhibitors. The first, results from the EMPULSE trial, Effects of Empagliflozin on Symptoms, Physical Limitations and Quality of Life in Patients Hospitalized for Acute Heart Failure; and the second, the EMPAG-heart failure trial, The Effects of Early Empagliflozin Initiation on Diuresis and Kidney Function in Patients with Acute Heart Failure. Incredibly important topics, incredibly important discussion. Wait up for it. We're just going to tell you a little bit more about two other original papers in today's issue, and I'm going to go first, Greg. Is that okay? Dr. Greg Hundley:           You bet. Dr. Carolyn Lam:             Now, really interesting topic here. We have strong evidence supporting the effective blood pressure and cardiovascular disease risk lowering properties of healthy diet such as the DASH diet, Mediterranean diet, and so on and so on. But what about the diet consumed by a fifth of the entire world's population? The Chinese cuisine. Interestingly, today's paper addresses just that. This is from authors, Dr. Wu, from Peking University Clinical Research Institute and colleagues who performed a multicenter patient and outcome assessor blind randomized feeding trial among 265 participants with baseline systolic blood pressure of 130 to 159 in four major Chinese cuisines. And these are the Shandong, Huaiyang, Cantonese, and Szechuan cuisines, and here's how they did it. After a seven day run in period on a control diet matching the usual local diets, participants were randomized to continue with the control diet or the cuisine based Chinese heart healthy diet for another 28 days. The primary outcome was systolic blood pressure. The study developed the first heart healthy Chinese diet that fits Chinese food culture and emphasizes its palatability by involving master shifts in developing the recipes. Dr. Greg Hundley:           Oh wow. Carolyn, this is really interesting, especially one fifth of the world's population in studying a heart healthy diet. So did it work? I can't wait to hear the results. Dr. Carolyn Lam:             Well, the change in systolic and diastolic blood pressure from baseline to the end of the study in the control group was five millimeters mercury and 2.8 millimeters mercury reduction, respectively. The net difference of change between the two groups in systolic and diastolic blood pressure were a reduction of 10 and almost four millimeters mercury, respectively. The effect size did not differ among cuisines, and so in summary, with a patient and assessor blind randomized feeding trial, this study really demonstrated that the blood pressure lowering effect of the Chinese heart health diet could indeed be substantial, and importantly, be compatible with medications while palatable and affordable in Chinese adults with high blood pressure, and so these results support the idea that food is medicine and will give many patients with high blood pressure the confidence to adopt heart healthy diets in their lifestyle treatment. Dr. Greg Hundley:           Wow, Carolyn, that is really an interesting article. So many of these articles today could all be features in and of themselves. That was just outstanding. Well, my next paper comes to us from the world of preclinical science, and it's from Dr. Sean Wu from Stanford University School of Medicine. So Carolyn, immune checkpoint inhibitors are monoclonal antibodies that are used to activate the immune system against tumor cells. Now, despite their therapeutic benefits, immune checkpoint inhibitors have the potential to cause immune mediated adverse events such as myocarditis, a rare but serious side effect with up to 50% mortality in affected patients. Now histologically, patients with immune checkpoint inhibitor of myocarditis have lymphocytic infiltrates in the heart implicating T-cell mediated mechanisms. However, the precise pathologic immune subsets and molecular changes in immune checkpoint inhibitor myocarditis are unknown. Dr. Carolyn Lam:             Wow. So insights into the etiology of these immune checkpoint associated myocarditis cases must be very important. So what did they find? Dr. Greg Hundley:           Right, Carolyn? So clonal cytotoxic, TEMRA CD8+ cells were found to be significantly increased in the blood of patients with immune checkpoint inhibitor myocarditis corresponding with an analogous increase in effector cytotoxic CD8+ cells in the blood and hearts of PD-1 deficient mice with myocarditis. These expanded effector CD8+ cells had unique transcriptional changes, including upregulation of the chemokines CCL5, CCL4, and CCL4L2, and they may serve as attractive diagnostic therapeutic targets for reducing life threatening cardiac immune related adverse events in immune checkpoint inhibitor treated cancer patients, and Carolyn, just like so many of our articles, there's a very nice accompanying editorial by Professor Gianluigi Condorelli that also offers an update on current research pertaining to non-systemic steroid therapy to treat immune mediated myocarditis. Well, Carolyn, how about we jump to some of the other articles in the issue? Dr. Carolyn Lam:             Oh, you bet, Greg. There's an exchange of letters between Drs. Madias and Knops regarding the article “Efficacy and Safety of Appropriate Shocks and Antitachycardia Pacing in Transvenous and Subcutaneous Implantable Defibrillators: The Analysis of All Appropriate Therapy in the PRAETORIAN Trial.” Dr. Greg Hundley:           And also in the mail bag, Professor Mark has a Research Letter entitled “Effect of Empagliflozin on Kidney Biochemical and Imaging Outcomes in Patients with Type 2 Diabetes, or Prediabetes, and Heart Failure with Reduced Ejection Fraction, The SUGAR-DM-HF Study,” and our own Tracy Hampton has several synopses from articles published elsewhere in our piece on cardiovascular news. Well, how about we get onto that feature forum discussion, two papers, two editorialists. I can't wait. Dr. Carolyn Lam:             Me too. Let's go, Greg. Dr. Greg Hundley:           Welcome, listeners to this July 26th feature forum discussion. So remember, listeners, for forum discussions, we have several manuscripts that focus on a singular topic and we bring together the authors, our associate editors, and also an editorialist, and today, I want to introduce, we have with us Dr. Mikhail Kosiborod from Mid America Heart Institute in Kansas City, Missouri, Dr. Christian Shults from University Hospital Jena in Germany, Stefan Anker from Charité in Berlin, Germany, and our Associate Editors, Brendan Everett from Brigham and Women's Hospital in Boston, Massachusetts, and Justin Grodin from University of Texas Southwestern Medical Center in Dallas, Texas. Welcome, gentleman, and we'll start with you, Mikhail. Could you describe for us the background information that went into the preparation of your study and what was the hypothesis that you wanted to address? Dr. Mikhail Kosiborod:   Well, thanks very much, Greg. The background for the study, which was the secondary analysis of the EMPULSE trial was patients that are hospitalized with acute decompensated heart failure represent a very high risk group. We know that they have high risk of death and hospitalizations, and we also know that they have very poor health status that's very high burden of symptoms, physical limitations, and poor quality of life, and so addressing those treatment goals, trying to reduce the risk of clinical events like death and hospitalizations and improve the symptoms and physical limitations in this patient population are very important treatment goals. Now we previously demonstrated in the main results of the EMPULSE trials that using empagliflozin initiating empagliflozin SGLT2 inhibitor in this patient population as compared with placebo provided a significant total clinical benefit, which was a composite of total death, repeat hospitalizations for heart failure, or a change in a Kansas City cardiomyopathy questionnaire, which is a kind of a gold standard measure of patient's health status. What we tried to do in a much more granular fashion in this study is to understand the effects of empagliflozin as compared with placebo on this very important outcome, the Kansas City cardiomyopathy questionnaire, and we actually evaluate all of the key domains and composite symptoms, physical limitations, as well as quality of life. Dr. Greg Hundley:           Very nice, and Mikhail, can you describe for us what study population specifically, and then what was your study design? Dr. Mikhail Kosiborod:   Well, this was a population of patients that were hospitalized with heart failure and that EMPULSE was unique in its design because first of all, previous SGLT2 inhibitor trials mostly focused on patients with chronic heart failures that were in an outpatient setting, including prior trials of empagliflozin, and EMPULSE really focused on acutely hospitalized patient population, but it included patients regardless of ejection fraction. So as they were hospitalized with decompensated heart failure and reduced or preserved ejection fraction. They were enrolled regardless of if they had type 2 diabetes, they were enrolled essentially, regardless of kidney function, only patients with EGFR of less than 20 were excluded, and also importantly, was this study and a unique feature of the study in particular was that we enrolled patients whether they had acute de novo heart failure. That means that was a new diagnosis of heart failure that was bad enough for them to be hospitalized or worsening chronic heart failure requiring hospitalization. So it was really an all-comer trial for patients acutely hospitalized for heart failure. So we had just over 500 patients and they were randomized in the hospital. After a brief period of stabilization, we use empagliflozin, 10 milligrams daily or placebo and treated for 90 days, and the primary outcome at 90 days was a total clinical benefit that I described that was a composite, hierarchical composite of total death hospitalizations, repeat hospitalizations for heart failure and changing KCCQ. In this study, again, we focused predominantly on KCCQ, trying to understand the effects on health status, again, symptoms, physical limitations, and quality of life. Dr. Greg Hundley:           Excellent. And Mikhail, what were your study results? Dr. Mikhail Kosiborod:   Well, what we observed, a couple of things. One is we first examined the effects of empagliflozin on the primary endpoint across the range of KCCQ and baseline, and what we found was that regardless of the degree of symptomatic impairment and baseline, empagliflozin was consistent in providing them total clinical benefits that I described previously, and then kind of shifting to what I think is the most interesting findings, the effects of empagliflozin versus placebo on KCCQ, what we found was that as you would imagine in this population of patients that were acutely hospitalized with heart failures, that had very poor health status, very low KCCQ at baseline, and within the first 90 days, which was observation period, both groups of patients had substantial improvements in KCCQs. As one would expect after acutely decompensated episode of heart failure and treatment in a hospital, everyone got better. But patients treated with empagliflozin had significantly greater improvement in KCCQs than those that were treated with placebo, and that was first of all, a very substantial difference between the two groups. It was more than five points in favor of empagliflozin already at 15 days and was highly statistically significant, and it was maintained throughout the 90 day treatment period. So the fact that we saw both a clinical meaningful and statistically significant improvement in just 15 days, I think is a very important clinical message, and then finally, I guess what I will mention is these benefits of empagliflozin while main outcome we looked at was KCCQ total symptoms, we're focusing on the symptoms, but it was consistent when we looked at physical limitations as well as quality of life. So really, all key domains of KCCQ were impacted in a similar way. Dr. Greg Hundley:           Very nice. So in acute heart failure, marked symptomatic improvement after the administration of the SGLT2 inhibitor empagliflozin at 10 milligrams per day. Well, now listeners, we're going to turn to our associate editor, Dr. Brendan Everett, and Brendan, again, you have many papers come across your desk. What attracted you to this particular manuscript? Dr. Brendan Everett:      Well, thanks, Greg, and I think this manuscript caught my eye because of the importance of the clinical question, and Mikhail outlined why I think that was really relevant. So we understand that this class of medications or SGLT2 inhibitors have important effects on outcomes like re-hospitalization in patients with heart failure, and what was particularly striking about this paper is that it took patients rather than those with chronic heart failure, but as Mikhail mentioned, enrolled a patient population that was actually in the hospital, and I think this was an important frontier for this particular question about when to start the SGLT2 inhibitor and what kind of benefits there might be. Furthermore, I think the fact that they did not select the population based on ejection fraction was particularly striking, and of course, I think is remarkable, but now old news, they did not select on the presence or absence of diabetes as well. And so those three components really attracted me to the paper. I also think the outcome is one that really is valuable and worth exploring, and specifically, I'm talking about how patients feel on the medication after a hospitalization for heart failure. Appropriately, we focused on re-hospitalization for heart failure and cardiovascular death in prior trials in this space, and I think we need to embellish those findings or further deepen those findings with a perspective on how patients actually feel when they get the medication, and of course, it goes without saying that what's particularly important here also is that it was a randomized placebo controlled trial, and so the results have some element of internal validity that I think is really important. So those were the things, Greg, that really attracted my attention as I read the paper for the first time. Dr. Greg Hundley:           Thank you so much, Brendan. Well, listeners, we've got a second paper today and we're next going to hear from Dr. Christian Shults, and he also is focusing on really another aspect of the administration of empagliflozin in patients with acute heart failure and that pertains to the renal function of the patients. So Christian, could you describe for us the background pertaining to your study and what was the hypothesis that you were intending to address? Dr. Christian Schulze:     Thanks, Greg. Well, it's great to introduce all study here in this running. So our study impacted those in acute decompensated heart failure. The impact HF trial was a study based on the hypothesis that we wanted to test, whether empagliflozin has effects in acute decompensated heart failure, and we focused on the patient population that was not addressed in EMPULSE, patients that came to the ER and needed to be treated right away, and we wanted to know and this was our main hypothesis, but are the diuretic and [inaudible 00:17:11] effects of the SGLT2 inhibitor on this case, empagliflozin, actually had an impact on diuretic regimens and kidney functions since this is one of the main end points that limits treatment, and also is one of the outcomes of patients with acute decompensated heart failure in the hospital. Dr. Greg Hundley:           Very nice. And so Christian, what study design did you implement and who was included in your study population? Dr. Christian Schulze:     So we also used the randomized two arm study design. We included patients with acute decompensated heart failure independent of left ventricular ejection fraction. Patients needed to have an NT-proBNP of more than 500. The average NT-proBNP in fact was 4,300 in our entire patient population, and we included patients within 12 hours of presentation. So many of these patients have been recruited in the ER, they presented two hour cardiology heart failure service, and then were immediately randomized to the trial in the two arms, and we tested not 10 milligrams of empagliflozin. We actually tested 25 milligrams of empagliflozin based on in-house data that 25 milligrams potentially had a stronger diuretic effect compared to 10 milligrams. Dr. Greg Hundley:           And what did you find? Dr. Christian Schulze:     So we followed patients for five days. It was a relatively short period of time. It was designed to address the in-house phase of patients with acute decompensated heart failure. The mean duration of stay was 6.3 days in the hospital so this was exactly the time that we wanted to test. We had a 30 day endpoint for safety issues, and what we could see is that patients on 25 milligrams on empagliflozin on top of standard diuretic regimens and medical care had 25% higher diuretic outputs compared to patients in the placebo group. We also found no differences in markers of renal injury dysfunction, and could in fact confirm that after 30 days, patients in the empagliflozin group had a better EGFR compared to patients in the placebo group. On top, we saw a more rapid decrease in body weight and also a more profound decrease in NT-proBNT values. Dr. Greg Hundley:           And Christian, just for our listeners to put a little bit of this in perspective, what was the range of serum creatinine for the patients that were enrolled in your study? Dr. Christian Schulze:     So the main EGFR in the entire population was around 60 and the creatinine values were around 107 on average in the entire cohort. So this is a very typical population. We had around 30% of the population with de Novo heart failure, around 20 to 30% of the population was pre-treated for preexisting heart failure. So very typical population of patients with heart failure presenting to the emergency room. Dr. Greg Hundley:           And did you have any kind of lower level EGFR cutoff, I mean, for enrollment into this study? Dr. Christian Schulze:     So when we designed the trial, we actually still had the sub classification of diabetes or impaired glucose or homeostasis as an inclusion criteria. We dropped it before we started the trial because the data came out that this is actually, in fact, not a critical issue for patients with heart failure. So diabetes was not a subgroup in our trial and the lower limit of EGFR was actually a thoroughly defined protocol. Dr. Greg Hundley:           Very nice. Well, listeners, now we're going to turn to our second associate editor, Dr. Justin Grodin from University of Texas Southwestern Medical Center in Dallas, Texas, and Justin, similar to Brendan, and you see many papers come across your desk and so what attracted you to this particular paper by Christian and his colleagues? Dr. Justin Grodin:            Well, Greg, I think first and foremost, and I think very similar to Brendan, but I think what's always striking is if I may just take a step back, decompensated heart failure in the United States is the number one cause for hospitalization among Medicare beneficiaries. So I think really, the brunt and really the truly public health message of the disease is very important in the applicability, and even though that decompensated heart failures is one of the most common things that we ever encounter when we practice, internists, cardiologists, et cetera, we have very, very little clinical trial guidance that tells us how to decongest individuals when they're hospitalized with swelling and heart failure and a lot of these individuals can be quite ill, and we have some clinical trial data, but largely, we have a lot of negative studies or inconclusive studies in this space. So certainly, what drew me to this trial was definitely that context, and obviously, based on the mechanistic data with SGLT2 inhibitors, I think one of the natural questions, which Christian addresses, is that we know that up front, they do augment natriuresis. So I think it's very compelling to marry those two together because this is what many of us that use these medications regularly have been asking is whether or not they would have some efficacy in that regard, and then another thing that caught my eye and me as a cardiorenal investigator was, just as Christian highlighted, was we have a clinical trial that randomly assigned individuals, really that were ill and many of whom were not stabilized within 12 hours of presentation, and we're talking about patients that are coming into the hospital at all times during the day in and I think that's very remarkable that we have something with standard... We have a study with standardized assessments where we're really trying to ask a very practical, pragmatic question, which is do these therapies lower the sodium balance in individuals with decompensated heart failure, and I think what's important is largely, we've got a lot of medications that supplement loop diuretics, which are the class of drugs that the majority of us use, and we have a lot of other therapies that we use that really have very little data or poor data that guide us such as thiazide diuretics, carbonic and hydrase inhibitors, mineralocorticoid receptor antagonists, and so here, we have a clinical trial that asks a question that's on many people's minds. And then we do have very compelling, at least short term pragmatic and mechanistic data that does tell us that these individuals do have a greater natriuretic effect when empagliflozin is used as an adjunct to standardized loop diuretic therapy. So it's a very practical clinical question, and I think what's very important, and we could debate probably all day about the implications of GFR change and kidney function change while we're decongesting somebody with diuretics, but I think what's reassuring to all the clinicians is we really didn't see an effect on kidney function despite a greater natriuretic effect or enhanced diuretic effect, if you will, with the use of empagliflozin. Dr. Greg Hundley:           Very nice. Well, thank you, Justin. Listeners, now we have an editorialist and as you know, editorialists really help us put the scientific presentation of an original manuscript into the perspective of really the global theme of a topic, and we have Stefan Anker from Berlin, and Stefan, can you describe for us how do we put these two manuscripts and results that we've heard about really in the context now of moving forward with the use of SGLT2 inhibitors in the management of patients with acute decompensated heart failure? Dr. Stefan Anker:            Thank you so much. Really, I think these two papers, on the one hand, enhance our certainty about early use, and on the other hand, possibly show us that there might be even more to achieve by, on the one hand, moving even earlier with the application of SGLT2 inhibitors or possibly consider the higher dose. Now let's take one step back. These drugs were developed in type two diabetes and the first successful trial was the [inaudible 00:25:42] outcome trial. Many people have forgotten that this trial tested two doses and not only one, the 10 and the 25 milligram dose, and of course, with the success for improving kidney outcomes and heart failure hospitalization outcomes, we move forward into these two specialist areas, on the one hand, broadening it to the non-diabetic communities, but on the other end, narrowing it by focusing on the 10 milligram dose regardless of whether there is [inaudible 00:26:12]. And we basically now learn A, to use these drugs even earlier than we did in the big trials and we can now be sure to start their use in the hospital, and if you take the average change in quality of life results seen, you actually get a better result for the patient on quality of life when you start earlier than when you start late in the ambulatory studies where basically, in the chronic setting, maybe you have one and a half to two points difference. Here, you now have four and a half points in the study shown by Mikhail, and of course, it's also good to know that you can start this in any type of patient, regardless of their quality of life. The impact study from Christian, they basically moved it now even earlier, moving into the hospital space is possible based on EMPULSE. Moving it into the acute admission space is at least a consideration now based on what Christian here has shown. And he is actually addressing the one question I hear very often in my presentations about SGLT2 inhibitors, what about this 25 milligram dose? Is there a place for this in cardiology as well, and a possible place is shown here, not only that this is a safe thing to do, but also you get urinary output. Of course, we may in the future, want to see this compared, directly compared to the 10 milligram dose, but of course, the world is not created in one day, but needs more than one and so really, I think these two studies, on the one hand, address an important issue, when to start using them. On the other hand, show us a little bit of a glimpse to the future. Dr. Greg Hundley:           Very nice, Stefan, and listeners, we get to take advantage of having these authors, editors, and editorialists together and ask them what they see as the next study to be performed in this sort of sphere of research. So Mikhail, we'll start with you. In 30 seconds or less, what do you see as the next study to be performed in this arena of research? Dr. Mikhail Kosiborod:   I think, Greg, what we've learned recently, including from the EMPULSE trial, we have this population of patients in a hospital with heart failure's a huge issue as Justin mentioned, and until recently, we had very little [inaudible 00:28:31] for them beyond the usual kind of decongestion with loop diuretics and trying to make them feel better, but you look at outcome data. It really was a dearth of effective therapies that have meaningful impact on important outcomes. Now that's changing, SGLT2 inhibitors is one example. There are some other recent examples in this patient population, like a firm HF and iron deficient patients with heart failure. But the bottom line is it's no longer kind of a desert, if you will, of positive trials. We now have something we can do and I think what this proves is that we need to actually invest more, both in terms of resources and time to really do what we we're being able to do in other areas of heart failure and those patients with chronic, half and half where we can start developing pillars of therapy that can actually truly improve outcomes with this patient population and there is a lot going on that makes me optimistic that's going to be the case in the coming years. Dr. Greg Hundley:           Very nice. And Brendan? Dr. Brendan Everett:      Well, I think both trials mentioned today really pushed our understanding of this population forward. I think the biggest clinical question that I face when I'm caring for these patients is that we have four, at least, guideline directed therapies, right? We have beta blockers, we have ARBs, ACE inhibitors and ARNIs. We have mineral receptor antagonists and we have SGLT2 inhibitors. So which do we use in what order and how do we start them, and what kind of parameters do we use to guide us if we're limited either by renal function or by blood pressure or by some other factor. And we often, if not always, have one of those constraints that we're dealing with and so I would say the next step for me is trying to sort out which of these therapies and what order ought to be our highest priority for patients with acute decompensated heart failure as we move quickly from the acute decongestion stage towards discharge and a chronic therapy that will then be followed as an outpatient over the ensuing days and months. Dr. Greg Hundley:           Very nice. And Christian. Dr. Christian Schulze:     Thank you again, and Brandon pointed out very nicely. I mean, we have good evidence now for chronic heart failure treatment. We have the four columns of heart failure medical therapy. Questions that remain open is what do we do with all these patients that are now guideline medicated, come to the hospital with an acute decompensation? Should we carry on with the medication? Should we terminate and in particular, should be carry on with full dose, 50% dose of SGLT2 inhibitors, and the next question is, what dose should we use, in fact, for SGLT2 inhibitors? Is it in group effects or is sotagliflozin comparable to empagliflozin, and then is there a role for a step by scheme that we initially have in high dose therapy that we then downgrade to 10 milligrams on the chronic heart failure treatments, and then of course, quality of life is very important. We should ask this question also in this patient population that is early on treated, do we see benefits that carry on in the outpatient setting and do we see an effect of early treatment on long term benefits? Dr. Greg Hundley:           Justin? Dr. Justin Grodin:            Well, I would have to agree with all of my colleagues here on this call. I think all have raised really good points, but I think one very simple, and I'll echo some of Brendan's statements, but one very simple question is we know that when we decongest people and initiate a negative salt balance in the hospital for decompensated heart failure, we cause neurohormonal activation and there are a lot of downstream untoward effects from chronic decongestive therapies, and I think one of the more compelling things is we still yet have defined what is the best way to decongest individuals with swelling or volume overload in the hospital. Here, we have compelling studies with SGLT2 inhibitors for quality of life and really, the way patients feel. And this is really what's important to them, and then something very pragmatic to clinicians and let's make people pee more, but I think one of the compelling questions, and I don't know if it will be answered, is we have a lot of choices for supplemental therapies and different diuretic strategies when patients come in the hospital for decompensated heart failure, and I do think that these studies do move the needle with SGLT2 inhibitors. I think that's abundantly clear, but we still don't know what is the best way to dry out my patient or make my patient pee so that they feel better, but I do think that these studies do at least set the stage that there's some compelling advantages to SGLT2 inhibitors. Dr. Greg Hundley:           And then lastly, Stefan. Dr. Stefan Anker:            Thank you. Besides the detailed points mentioned by many, and Christian, totally support 25 versus 10 milligram, how long 25 milligram, if at all in the future. Besides this, I'm interested in the big picture question. So what about the post myocardial infarct congestion/heart failure situation, and there will be two trials in the next 18 to 24 months that report on this, and my pet kind of area is actually to treat heart failure where nobody thinks it is heart failure, and what I mean is for instance, advanced cancer patients, cardiac wasting cardiomyopathy. So the heart failure in sick cancer patients, and indeed, we are planning to do exactly that now in a study focusing on hospice care patients to really improve the quality of life, the very thing focus here on the EMPULSE trial. Dr. Greg Hundley:           Well, listeners, we want to thank our authors, Dr. Mikhail Kosiborod from Mid America Heart Institute in University of Missouri, and Christian Shults from the University Hospital in Jena, Germany. Also, our associate editors, Dr. Brendan Everett from Brigham and Women's Hospital in Boston, and Dr. Justin Grodin from University of Texas Southwestern in Dallas, Texas, and also, our editorialist, Dr. Stefan Anker from Charité in Berlin, Germany for bringing us these two manuscripts pertaining to two randomized clinical trials regarding the administration of the SGLT2 inhibitor, empagliflozin in acute heart failure, demonstrating first, marked improvement in heart failure symptoms and health related quality of life. And second, in those with estimated GFRs greater than 30 mls per minute, an augmentation of natriuresis in the setting of the co-administration of diuretics without deterioration in renal function. Well, on behalf of Carolyn and myself, we want to wish you a great week and we will catch you next week on the run. This program is copyright of the American Heart Association, 2022. The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more, please visit ahajournals.org.

Saw palmetto boosts testosterone synthesis Kyung Hee University (South Korea), June 30 2021.   The June 2021 issue of the Journal of Medicinal Food reported the finding of a beneficial effect for saw palmetto against symptoms of andropause in rats.  "Andropause, the male equivalent of menopause, is the set of symptoms caused by the age-related deficiency in male hormones that begins to occur in men in their late 40s to early 50s," Jeong Moon Yun and colleagues explained. "The symptoms of andropause include physical, psychological, and sexual problems, such as fatigue, increased body fat, decreased muscle strength and sexual function, depression, and memory loss." Dr Yun and associates evaluated the effects of an extract of saw palmetto in Leydig cells (in which testosterone biosynthesis occurs) subjected to oxidative stress and in aged rats. In Leydig cells, the administration of testosterone lowered 5 alpha-reductase (which converts testosterone to dihydrotestosterone) and increased total testosterone.  In rats, one of three doses of saw palmetto extract was administered for four weeks. A control group of animals received no treatment. At the end of the treatment period, saw palmetto supplemented rats had significantly less fat tissue weight gain and total weight gain compared to the controls, without a gain in other tissue weight. Serum triglycerides, total cholesterol and the LDL to VLDL cholesterol ratio were also lower in the supplemented groups. Serum total and free testosterone and sperm counts were higher, and sex hormone binding globulin (SHBG) and 5 alpha-reductase levels were lower in all supplemented groups in comparison with the controls. In tests of muscle endurance, rats that received saw palmetto had longer swimming times compared to the control group.  "We suggest that supplementation of saw palmetto may relieve the symptoms of andropause syndrome, including decreased spermatogenesis and muscle endurance and metabolic syndrome by increasing testosterone biosynthesis and bioavailability," the authors concluded.         Diet rich in omega 3 fatty acids may help reduce headaches Trial provides 'grounds for optimism' for many people with persistent headaches and those who care for them University of North Carolina, July 1, 2021 Eating a diet rich in omega 3 (n-3) fatty acids reduces the frequency of headaches compared with a diet with normal intake of omega 3 and omega 6 (n-6) fatty acids, finds a study published by The BMJ today. Modern industrialised diets tend to be low in omega 3 fatty acids and high in omega 6 fatty acids. These fatty acids are precursors to oxylipins - molecules involved in regulating pain and inflammation. Oxylipins derived from omega 3 fatty acids are associated with pain-reducing effects, while oxylipins derived from omega 6 fatty acids worsen pain and can provoke migraine. But previous studies evaluating omega 3 fatty acid supplements for migraine have been inconclusive. So a team of US researchers wanted to find out whether diets rich in omega 3 fatty acids would increase levels of the pain-reducing 17-hydroxydocosahexaenoic acid (17-HDHA) and reduce the frequency and severity of headaches. Their results are based on 182 patients at the University of North Carolina, USA (88% female; average age 38 years) with migraine headaches on 5-20 days per month who were randomly assigned to one of three diets for 16 weeks.  The control diet included typical levels of omega 3 and omega 6 fatty acids. Both interventional diets raised omega 3 fatty acid intake. One kept omega 6 acid intake the same as the control diet, and the other concurrently lowered omega 6 acid intake. During the trial, participants received regular dietary counseling and access to online support information. They also completed the headache impact test (HIT-6) - a questionnaire assessing headache impact on quality of life. Headache frequency was assessed daily with an electronic diary. Over the 16 weeks, both interventional diets increased 17-HDHA levels compared with the control diet, and while HIT-6 scores improved in both interventional groups, they were not statistically significantly different from the control group.  However, headache frequency was statistically significantly decreased in both intervention groups.  The high omega 3 diet was associated with a reduction of 1.3 headache hours per day and two headache days per month. The high omega 3 plus low omega 6 diet group saw a reduction of 1.7 headache hours per day and four headache days per month, suggesting additional benefit from lowering dietary omega-6 fatty acid.  Participants in the intervention groups also reported shorter and less severe headaches compared with those in the control group. This was a high quality, well designed trial, but the researchers do point to some limitations, such as the difficulty for patients to stick to a strict diet and the fact that most participants were relatively young women so results may not apply to children, older adults, men, or other populations.  "While the diets did not significantly improve quality of life, they produced large, robust reductions in frequency and severity of headaches relative to the control diet," they write.  "This study provides a biologically plausible demonstration that pain can be treated through targeted dietary alterations in humans. Collective findings suggest causal mechanisms linking n-3 and n-6 fatty acids to [pain regulation], and open the door to new approaches for managing chronic pain in humans," they conclude. These results support recommending a high omega 3 diet to patients in clinical practice, says Rebecca Burch at the Brigham and Women's Hospital, in a linked editorial. She acknowledges that interpretation of this study's findings is complex, but points out that trials of recently approved drugs for migraine prevention reported reductions of around 2-2.5 headache days per month compared with placebo, suggesting that a dietary intervention can be comparable or better.  What's more, many people with migraine are highly motivated and interested in dietary changes, she adds. These findings "take us one step closer to a goal long sought by headache patients and those who care for them: a migraine diet backed up by robust clinical trial results."     The Southern diet - fried foods and sugary drinks - may raise risk of sudden cardiac death University of Alabama, June 30, 2021  Regularly eating a Southern-style diet may increase the risk of sudden cardiac death, while routinely consuming a Mediterranean diet may reduce that risk, according to new research published today in the Journal of the American Heart Association, an open access journal of the American Heart Association. The Southern diet is characterized by added fats, fried foods, eggs, organ meats (such as liver or giblets), processed meats (such as deli meat, bacon and hotdogs) and sugar-sweetened beverages. The Mediterranean diet is high in fruits, vegetables, fish, whole grains and legumes and low in meat and dairy. "While this study was observational in nature, the results suggest that diet may be a modifiable risk factor for sudden cardiac death, and, therefore, diet is a risk factor that we have some control over," said James M. Shikany, Dr.P.H., F.A.H.A., the study's lead author and professor of medicine and associate director for research in the Division of Preventive Medicine at the University of Alabama at Birmingham. "Improving one's diet - by eating a diet abundant in fruits, vegetables, whole grains and fish such as the Mediterranean diet and low in fried foods, organ meats and processed meats, characteristics of the Southern-style dietary pattern, may decrease one's risk for sudden cardiac death," he said. The study examined data from more than 21,000 people ages 45 and older enrolled in an ongoing national research project called REasons for Geographic and Racial Differences in Stroke (REGARDS), which is examining geographic and racial differences in stroke. Participants were recruited between 2003 and 2007. Of the participants in this analysis, 56% were women; 33% were Black adults; and 56% lived in the southeastern U.S., which is noteworthy as a region recognized as the Stroke Belt because of its higher stroke death rate. The Stroke Belt states included in this study were North Carolina, South Carolina, Georgia, Tennessee, Alabama, Mississippi, Arkansas and Louisiana. This study is the latest research to investigate the association between cardiovascular disease and diet - which foods have a positive vs. negative impact on cardiovascular disease risk. It may be the only study to-date to examine the association between dietary patterns with the risk of sudden cardiac death, which is the abrupt loss of heart function that leads to death within an hour of symptom onset. Sudden cardiac death is a common cause of death and accounted for 1 in every 7.5 deaths in the United States in 2016, or nearly 367,000 deaths, according to 2019 American Heart Association statistics. Researchers included participants with and without a history of coronary heart disease at the beginning of the study and assessed diets through a food frequency questionnaire completed at the beginning of the study. Participants were asked how often and in what quantities they had consumed 110 different food items in the previous year. Researchers calculated a Mediterranean diet score based on specific food groups considered beneficial or detrimental to health. They also derived five dietary patterns. Along with the Southern-style eating pattern, the analysis included a "sweets" dietary pattern, which features foods with added sugars, such as desserts, chocolate, candy and sweetened breakfast foods; a "convenience" eating pattern which relied on easy-to-make foods like mixed dishes, pasta dishes, or items likely to be ordered as take-out such as pizza, Mexican food and Chinese food; a "plant-based" dietary pattern was classified as being high in vegetables, fruits, fruit juices, cereal, bean, fish, poultry and yogurt; and an "alcohol and salad" dietary pattern, which was highly reliant on beer, wine, liquor along with green leafy vegetables, tomatoes and salad dressing. Shikany noted that the patterns are not mutually exclusive. "All participants had some level of adherence to each pattern, but usually adhered more to some patterns and less to others," he explained. "For example, it would not be unusual for an individual who adheres highly to the Southern pattern to also adhere to the plant-based pattern, but to a much lower degree." After an average of nearly 10 years of follow-up every six months to check for cardiovascular disease events, more than 400 sudden cardiac deaths had occurred among the 21,000 study participants. The study found: Overall, participants who ate a Southern-style diet most regularly had a 46% higher risk of sudden cardiac death than people who had the least adherence to this dietary pattern. Also, participants who most closely followed the traditional Mediterranean diet had a 26% lower risk of sudden cardiac death than those with the least adherence to this eating style. The American Heart Association's Diet and Lifestyle recommendations emphasize eating vegetables, fruits, whole grains, lean protein, fish, beans, legumes, nuts and non-tropical vegetable cooking oils such as olive and canola oil. Limiting saturated fats, sodium, added sugar and processed meat are also recommended. Sugary drinks are the number one source of added sugar in the U.S. diet, according to the Centers for Disease Control and Prevention, and the American Heart Association supports sugary drink taxes to drive down consumption of these products. "These findings support the notion that a healthier diet would prevent fatal cardiovascular disease and should encourage all of us to adopt a healthier diet as part of our lifestyles," said Stephen Juraschek, M.D., Ph.D., a member of the American Heart Association's Nutrition Committee of the Lifestyle and Cardiometabolic Health Council. "To the extent that they can, people should evaluate the number of servings of fruit and vegetables they consume each day and try to increase the number to at least 5-6 servings per day, as recommended by the American Heart Association. Optimal would be 8-9 servings per day. "This study also raises important points about health equity, food security and social determinants of health," he continued. "The authors describe the "Southern Diet" based on the U.S. geography associated with this dietary pattern, yet it would be a mistake for us to assume that this is a diet of choice. I think American society needs to look more broadly at why this type of diet is more common in the South and clusters among some racial, ethnic or socioeconomic groups to devise interventions that can improve diet quality. The gap in healthy eating between people with means and those without continues to grow in the U.S., and there is an incredible need to understand the complex societal factors that have led and continue to perpetuate these disparities." This current research expands on earlier studies on participants from the same national stroke project, REGARDS. In a 2018 analysis, Shikany and colleagues reported that adults ages 45 and older with heart disease who had an affinity for the Southern diet had a higher risk of death from any cause, while greater adherence to the Mediterranean diet was associated with a lower risk of death from any cause. And in a 2015 study, the Southern diet was linked to a greater risk of coronary heart disease in the same population. The large population sample and regional diversity, including a significant number of Black participants, are considered strengths of the REGARDS research project. However, potential limitations of this study include that that dietary intake was based on one-time, self-reported questionnaires, thus, it relied on the participants' memory. Self-reported diet can include inaccuracies leading to bias that could reduce the strength of the associations observed. One usual association that remains unexplained is that among individuals with a history of heart disease, those who most adhered to the sweets dietary pattern had a 51% lower risk of sudden cardiac death than participants who followed that pattern the least. Researchers note that they found "no viable explanation for the inverse association of the sweets dietary pattern with risk of sudden cardiac death in those with a history of coronary heart disease."     5-minute workout lowers blood pressure as much as exercise, drugs 'Strength training for breathing muscles' holds promise for host of health benefits University of Colorado, July 2, 2021 Working out just five minutes daily via a practice described as "strength training for your breathing muscles" lowers blood pressure and improves some measures of vascular health as well as, or even more than, aerobic exercise or medication, new CU Boulder research shows. The study, published June 29 in the Journal of the American Heart Association, provides the strongest evidence yet that the ultra-time-efficient maneuver known as High-Resistance Inspiratory Muscle Strength Training (IMST) could play a key role in helping aging adults fend off cardiovascular disease - the nation's leading killer.  In the United States alone, 65% of adults over age 50 have above-normal blood pressure - putting them at greater risk of heart attack or stroke. Yet fewer than 40% meet recommended aerobic exercise guidelines. "There are a lot of lifestyle strategies that we know can help people maintain cardiovascular health as they age. But the reality is, they take a lot of time and effort and can be expensive and hard for some people to access," said lead author Daniel Craighead, an assistant research professor in the Department of Integrative Physiology. "IMST can be done in five minutes in your own home while you watch TV." Developed in the 1980s as a way to help critically ill respiratory disease patients strengthen their diaphragm and other inspiratory (breathing) muscles, IMST involves inhaling vigorously through a hand-held device which provides resistance. Imagine sucking hard through a tube that sucks back.  Initially, when prescribing it for breathing disorders, doctors recommended a 30-minute-per-day regimen at low resistance. But in recent years, Craighead and colleagues have been testing whether a more time-efficient protocol--30 inhalations per day at high resistance, six days per week--could also reap cardiovascular, cognitive and sports performance improvements. For the new study, they recruited 36 otherwise healthy adults ages 50 to 79 with above normal systolic blood pressure (120 millimeters of mercury or higher). Half did High-Resistance IMST for six weeks and half did a placebo protocol in which the resistance was much lower.  After six weeks, the IMST group saw their systolic blood pressure (the top number) dip nine points on average, a reduction which generally exceeds that achieved by walking 30 minutes a day five days a week. That decline is also equal to the effects of some blood pressure-lowering drug regimens.  Even six weeks after they quit doing IMST, the IMST group maintained most of that improvement. "We found that not only is it more time-efficient than traditional exercise programs, the benefits may be longer lasting," Craighead said. The treatment group also saw a 45% improvement in vascular endothelial function, or the ability for arteries to expand upon stimulation, and a significant increase in levels of nitric oxide, a molecule key for dilating arteries and preventing plaque buildup. Nitric oxide levels naturally decline with age.  Markers of inflammation and oxidative stress, which can also boost heart attack risk, were significantly lower after people did IMST. And, remarkably, those in the IMST group completed 95% of the sessions. "We have identified a novel form of therapy that lowers blood pressure without giving people pharmacological compounds and with much higher adherence than aerobic exercise," said senior author Doug Seals, a Distinguished Professor of Integrative Physiology. "That's noteworthy." The practice may be particularly helpful for postmenopausal women. In previous research, Seals' lab showed that postmenopausal women who are not taking supplemental estrogen don't reap as much benefit from aerobic exercise programs as men do when it comes to vascular endothelial function. IMST, the new study showed, improved it just as much in these women as in men.  "If aerobic exercise won't improve this key measure of cardiovascular health for postmenopausal women, they need another lifestyle intervention that will," said Craighead. "This could be it." Preliminary results suggest MST also improved some measures of brain function and physical fitness. And previous studies from other researchers have shown it can be useful for improving sports performance. "If you're running a marathon, your respiratory muscles get tired and begin to steal blood from your skeletal muscles," said Craighead, who uses IMST in his own marathon training. "The idea is that if you build up endurance of those respiratory muscles, that won't happen and your legs won't get as fatigued." Seals said they're uncertain exactly how a maneuver to strengthen breathing muscles ends up lowering blood pressure, but they suspect it prompts the cells lining blood vessels to produce more nitric oxide, enabling them to relax. The National Institutes of Health recently awarded Seals $4 million to launch a larger follow-up study of about 100 people, comparing a 12-week IMST protocol head-to-head with an aerobic exercise program. Meanwhile, the research group is developing a smartphone app to enable people to do the protocol at home using already commercially available devices. Those considering IMST should consult with their doctor first. But thus far, IMST has proven remarkably safe, they said. "It's easy to do, it doesn't take long, and we think it has a lot of potential to help a lot of people," said Craighead.   Research suggests atheroprotective role for chrysin Fu Jen Catholic University (Taiwan), July 1, 2021 According to news reporting originating from New Taipei, Taiwan, research stated, “Atherosclerosis and its related clinical complications are the leading cause of death. MicroRNA (miR)-92a in the inflammatory endothelial dysfunction leads to atherosclerosis.” Our news editors obtained a quote from the research from Fu Jen Catholic University, “Kruppel-like factor 2 (KLF2) is required for vascular integrity and endothelial function maintenance. Flavonoids possess many biological properties. This study investigated the vascular protective effects of chrysin in balloon-injured carotid arteries. Exosomes were extracted from human coronary artery endothelial cell (HCAEC) culture media. Herb flavonoids and chrysin (found in mint, passionflower, honey and propolis) were the treatments in these atheroprotective models. Western blotting and real-time PCRs were performed. In situ hybridization, immunohistochemistry, and immunofluorescence analyses were employed. MiR-92a increased after balloon injury and was present in HCAEC culture media. Chrysin was treated, and significantly attenuated the miR-92a levels after balloon injury, and similar results were obtained in HCAEC cultures in vitro. Balloon injury-induced miR-92a expression, and attenuated KLF2 expression. Chrysin increased the KLF2 but reduced exosomal miR-92a secretion. The addition of chrysin and antagomir-92a, neointimal formation was reduced by 44.8 and 49.0% compared with balloon injury after 14 days, respectively. Chrysin upregulated KLF2 expression in atheroprotection and attenuated endothelial cell-derived miR-92a-containing exosomes.” According to the news editors, the research concluded: “The suppressive effect of miR-92a suggests that chrysin plays an atheroprotective role.” This research has been peer-reviewed.     False-positive mammogram results linked to spike in anxiety prescriptions Penn State University, July 2, 2021 Women who experience a false-positive mammogram result are more likely to begin medication for anxiety or depression than women who received an immediate negative result, according to a study led by Penn State researcher Joel Segel. The finding highlights the importance of swift and accurate follow-up testing to rule out a breast cancer diagnosis. The study found that patients who receive a false-positive mammogram result are also prescribed anxiety or depression medication at a rate 10 to 20 percent higher than patients who receive an immediate negative result. These prescriptions are new and not continuations of previously prescribed medicines. A false-positive result is one where a suspicious finding on the screening mammogram leads to additional testing that does not end up leading to a breast cancer diagnosis. Additionally, within that group of patients who required more than one test to resolve the false-positive there was a 20 to 30 percent increase in those beginning to take anxiety or depression medications. The increase was particularly noticeable among women with commercial insurance who required multiple tests to rule out a breast cancer diagnosis. "The results suggest that efforts to quickly resolve initially positive findings including same-day follow-up tests may help reduce anxiety and even prevent initiation of anxiety or depression medication," said Segel, assistant professor of health policy and administration at Penn State. This study demonstrates that some women who experience a false-positive mammogram may need additional follow-up care to effectively handle the increased anxiety that may accompany the experience, Segel said. More importantly, from a practitioner standpoint, the study identifies sub-populations who may be most at risk of increased anxiety following a false-positive mammogram, Segel said. Specifically, women whose false-positive result requires more than one follow-up test to resolve, women with commercial insurance who undergo a biopsy, women who wait longer than one week to receive a negative result, and women who are under age 50 may all be at higher risk of experiencing clinically significant anxiety or depression. "Regular breast cancer screening is critical to early detection," Segel said. "Patients should continue to work with their providers to ensure they are receiving guideline-appropriate screening and should follow up with their providers if they experience either anxiety or depression following screening or any type of care." Researchers studied commercial- and Medicaid-claims databases to identify women ages 40 to 64 who underwent screening mammography with no prior claims for anxiety or depression medications. The findings recently appeared in Medical Care.     Thymoquinone in Black Seed oil increases the expression of neuroprotective proteins while decreasing expression of pro-inflammatory cytokines Florida A&M University, June 29, 2021    According to news originating from Tallahassee, Florida, research stated, "Neuroinflammation and microglial activation are pathological markers of a number of central nervous system (CNS) diseases. Chronic activation of microglia induces the release of excessive amounts of reactive oxygen species (ROS) and pro-inflammatory cytokines." Our news journalists obtained a quote from the research from Florida A&M University, "Additionally, chronic microglial activation has been implicated in several neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Thymoquinone (TQ) has been identified as one of the major active components of the natural product Nigella sativa seed oil. TQ has been shown to exhibit anti-inflammatory, anti-oxidative, and neuroprotective effects. In this study, lipopolysaccharide (LPS) and interferon gamma (IFN gamma) activated BV-2 microglial cells were treated with TQ (12.5 mu M for 24 h). We performed quantitative proteomic analysis using Orbitrap/Q-Exactive Proteomic LC-MS/MS (Liquid chromatography-mass spectrometry) to globally assess changes in protein expression between the treatment groups. Furthermore, we evaluated the ability of TQ to suppress the inflammatory response using ELISArray ™ for Inflammatory Cytokines. We also assessed TQ's effect on the gene expression of NFKB signaling targets by profiling 84 key genes via real-time reverse transcription (RT2) PCR array. Our results indicated that TQ treatment of LPS/IFN gamma-activated microglial cells significantly increased the expression of 4 antioxidant, neuroprotective proteins: glutaredoxin-3 (21 fold; p< 0.001), biliverdin reductase A (15 fold; p< 0.0001), 3-mercaptopyruvate sulfurtransferase (11 fold; p< 0.01), and mitochondria] Ion protease (> 8 fold; p< 0.001) compared to the untreated, activated cells. Furthermore, TQ treatment significantly (P < 0.0001) reduced the expression of inflammatory cytokines, IL-2 = 38%, IL-4 = 19%, IL-6 = 83%, IL-10 = 237%, and IL-17a = 29%, in the activated microglia compared to the untreated, activated which expression levels were significantly elevated compared to the control microglia: IL-2 = 127%, IL-4 = 151%, IL-6 = 670%, IL-10 = 133%, IL-17a = 127%. Upon assessing the gene expression of NFKB signaling targets, this study also demonstrated that TQ treatment of activated microglia resulted in > 7 fold down-regulation of several NFKB signaling targets genes, including interleukin 6 (IL6), complement factor B (CFB), chemokine (C-C motif) ligand 3 (CXCL3), chemokine (C-C) motif ligand 5 (CCL5) compared to the untreated, activated microglia. This modulation in gene expression counteracts the > 10-fold upregulation of these same genes observed in the activated microglia compared to the controls. Our results show that TQ treatment of LPS/IFN gamma-activated BV-2 microglial cells induce a significant increase in expression of neuroprotective proteins, a significant decrease in expression inflammatory cytokines, and a decrease in the expression of signaling target genes of the NF kappa B pathway. Our findings are the first to show that TQ treatment increased the expression of these neuroprotective proteins (biliverdin reductase-A, 3-mercaptopyruvate sulfurtransferase, glutaredoxin-3, and mitochondrial Ion protease) in the activated BV-2 microglial cells. Additionally, our results indicate that TQ treatment decreased the activation of the NF kappa B signaling pathway, which plays a key role in neuroinflammation." According to the news editors, the research concluded: "Our results demonstrate that TQ treatment reduces the inflammatory response and modulates the expression of specific proteins and genes and hence potentially reduce neuroinflammation and neurodegeneration driven by microglial activation."

Major advances in understanding and treating breast cancer have been made in the last two decades, yet it remains a significant problem with breast cancer being the most commonly identified cancer and the leading cause of cancer death among women worldwide. For many years breast cancer research has mainly focused on genetically changed cancer cells. However, recently the importance of the stromal compartment surrounding cancer cells in facilitating tumor growth, invasion and metastasis has been widely recognized. Cumulating evidence suggests that in particular carcinoma-associated myofibroblasts play a key role within the tumor stroma and influence many aspects of carcinogenesis. Nevertheless, the cell type of origin as well as the precise mechanisms by which these cells develop has not been conclusively established and remains controversial. The role of human adipose tissue derived stem cells (hASCs) in this context has not been studied so far. hASCs are locally adjacent to epithelial breast cancer cells and might represent early response cells within the tumor stroma. Hence, the aim of this study was to investigate whether carcinoma-associated myofibroblasts may originate from hASCs. The present study revealed that a significant percentage of hASCs differentiate into myofibroblast-like cells expressing alpha smooth muscle actin (α-SMA) and tenascin-C when exposed to conditioned medium from the human epithelial breast cancer cell lines MDMAB231 and MCF7. This process is induced by transforming growth factor beta 1 (TGFβ1) secreted from breast cancer cells. It was shown that conditioned medium from MDMAB231 and MCF7 contains significant amounts of TGFβ1. It could further be demonstrated that the differentiation of hASCs towards myofibroblasts is dependent on TGFβ1 signaling via phosphorylation of Smad2 and Smad3 in hASCs. The induction of myofibroblasts can be abolished using a neutralizing antibody to TGFβ1 as well as by pretreatment of hASCs with SB431542, a selective inhibitor of the TGFβ1 activin receptor-like kinases 4, 5 and 7. Additionally, hASC-derived myofibroblasts exhibit functional properties of carcinoma-associated myofibroblasts such as the increased secretion of the tumor-promoting soluble factors SDF-1α and CCL5. Furthermore hASC-derived myofibroblasts as well as conditioned medium from these cells promote the in vitro invasion of MDAMB231 breast cancer cells. Moreover inhibition of the TGFβ1 signaling pathway in hASCs reduces the potential of these cells to enhance the invasion of breast cancer cells. Overall, the data of the present study suggest that human adipose tissue derived stem cells can differentiate into carcinoma-associated myofibroblast under the influence of TGFβ1 secreted from breast cancer cells in vitro. The differentiation of hASCs towards these tumor-promoting cells can be abolished by targeting the TGFβ1 signaling pathway. Hence, inhibition of the TGFβ1 signaling pathway may prove to be an interesting target for breast cancer therapies. In vivo studies on the cancer microenvironment under special consideration of the interactions between hASCs and cancer cells should be of interest for breast cancer research in the future.

Die von intrinsichen renalen Zellen und infiltrierenden Leukozyten exprimierten Zytokine sind zentrale Vermittler entzündlicher Nierenerkrankungen. Tumor Nekrose Faktor-α (TNF) ist ein solches proinflamatorisches Zytokin, das in der glomerulären Entzündungsreaktion involviert ist. Die funktionelle Rolle von TNF wurde in Tiermodellen der Glomerulonephritis belegt. Die biologischen Effekte von TNF werden durch die beiden funktionell eigenständigen TNF-Rezeptoren TNFR1 (CD120a) und TNFR2 (CD120b) vermittelt. Neuere Daten zeigen, dass in Modellen einer Immunkomplex-Glomerulonephritis wie der nephrotoxische Serumnephritis die beiden TNF-Rezeptoren in vivo unterschiedliche Funktionen bei der glomerulären Entzündung vermitteln können. Der vorliegenden Arbeit liegt die Hypothese zugrunde, dass Tnfr1 und Tnfr2 unterschiedliche inflammatorische TNF-Effekte in Glomeruli vermitteln. Daher war das Ziel dieser Arbeit, Expression und Funktion der beiden TNF-Rezeptoren in Maus-Glomeruli zu charakterisieren und die Tnfr-abhängig exprimierten Entzündungsmediatoren in Maus-Glomeruli zu identifizieren. Aufbauend auf den Ergebnissen dieser Arbeit könnten selektive, Tnfr-spezifische Therapien zur Hemmung der glomerulären Entzündungsreaktion entwickelt werden. Zudem wurde in dieser Arbeit die funktionelle Rolle der beiden TNF-Rezeptoren im MRL/lpr-Mausmodell der Lupusnephritis untersucht, um eine selektive Tnfr-Blockade als mögliche Therapiestrategie zu charakterisieren. Hierfür war eine Rückkreuzung von Tnfr1- und Tnfr2-defizienten C57BL/6J-Mäusen in den MRL/lpr-Hintergrund erforderlich. Um TNF-Rezeptor-1- und 2-vermittelte inflammatorische Signalwege in Glomeruli zu identifizieren wurde die Expression und die Funktion der beiden TNF-Rezeptoren in Mausnieren, in isolierten Glomeruli ex vivo und murinen glomerulären Endothel- und Mesangialzellen in vitro untersucht. In normaler Mausniere konnte eine Tnfr1- und Tnfr2-mRNA- und Protein-Expressionen präferentiell in Glomeruli im Vergleich zum Tubulointerstitium nachgewiesen werden. Die Expression von beiden TNF-Rezeptoren und die TNF-induzierte Induktion von Tnfr2-mRNA-Expression wurde auch in vitro sowohl in murinen glomerulären Endothel- als auch Mesangialzelllinien bestätigt. Die prominente glomeruläre TNF-Rezeptor-Expression korrelierte mit einer konstitutiven glomerulären mRNA-Expression von Adhäsionsmolekülen wie Icam-1, Vcam-1, E- und P-Selektin und Chemokinen wie Ccl2, Ccl3 und Ccl5. Eine intraperitoneale TNF-Injektion induzierte die Expression dieser Mediatoren präferentiell in Glomeruli. Diese in vivo TNF-Exposition führte zu einer raschen glomerulären Akkumulation von Leukozyten einschließlich Neutrophilen und mononukleären Phagozyten, die mittels einer kompartimentspezifischer Durchflußzytometrie analysiert wurden. Um Tnfr-abhängige inflammatorische Effekte in intrinsischen glomerulären Zellen unabhängig von infiltrierenden Leukozyten zu untersuchen, wurde eine Microarray-Gene-Expressionsanalyse an intakten Glomeruli durchgeführt, die aus Wildtyp und Tnfr-defizienten Mäusen isoliert und anschließend mit TNF ex vivo stimuliert wurden. Die meisten TNF-Effekte wurden ausschließlich durch Tnfr1 vermittelt, unter anderem die induzierte mRNA-Expression von Adhäsionsmolekülen, proinflammatorischen Chemokinen, Komplement-Faktoren und proapoptotischen Molekülen. Im Gegensatz dazu fanden wir nur vier Tnfr2-abhängig exprimierte Gene, einschließlich einer kleinen GTPase der Rab-Familie (Rab6b). Diese Ergebnisse wurden durch quantitative RT-PCR-Analysen von TNF-stimulierten Glomeruli und primären Mesangialzellen bestätigt. Weitere Untersuchungen zeigten allerdings auch einen Beitrag von Tnfr2 bei der gesteigerten glomerulären Expression von Adhäsionsmolekülen und Chemokine nach Stimulation mit niedrigen TNF-Konzentrationen auf. Im Gegensatz zur Wildtyp-Kontrolle fehlte in TNF-stimulierten Tnfr1-defizienten Glomeruli die Sekretion verschiedener proinflammatorischer Chemokine beinahe vollständig. Interessanterweise war die Proteinexpression auch in Tnfr2-defizienten Glomeruli signifikant herunterreguliert. Folglich sind die meisten inflammatorischen TNF-Effekte in Glomeruli via Tnfr1 durch die induzierte Expression von proinfammatorischen Mediatoren wie Adhäsionsmolekülen und Chemokinen vermittelt. Darüber hinaus dürfte Tnfr2 zu dieser inflammatorischen Antwort beitragen, wenn Glomeruli niedrigen TNF-Konzentrationen ausgesetzt sind. Ferner scheint Tnfr2 posttranskriptionell die Chemokinsekretion in Glomeruli nach einer TNF-Exposition zu beeinflussen, möglicherweise durch die Tnfr2-abhängig exprimierte Rab GTPase Rab6b, die am intrazellulären Transport und der Sekretion von inflammatorischen Molekulen beteiligt sein könnte. In Bezug auf Tnfr-spezifische, anti-inflammatorische Therapien weisen die hier präsentierten Ergebnisse somit darauf hin, dass eine selektive Tnfr1-Blockade eine glomeruläre, insbesondere durch Granulozyten und Makrophagen vermittelte Entzündung verbessern könnte, möglicherweise bei geringer Hemmung immunregulatorischer und antimikrobieller Funktionen von TNF, die redundant durch Tnfr2 vermittelt werden könnten. Dagegen erscheint aufgrund der erhobenen Daten im MRL/lpr-Mausmodell eine Blockade von TNF oder beider Rezeptoren bei der Lupusnephritis, in der glomeruläre Neutrophileninfiltrate keine entzündliche Rolle spielen, weniger erfolgversprechend. Gleichzeitig weisen die vorliegenden Ergebnisse auf eine immunsuppressive, die systemische Immunreaktivität beim SLE begrenzende Funktion von Tnfr2 hin.

Maligne Zellen wachsen in einem komplexen zellulären und extrazellulären Umfeld, welches die Initiierung und Aufrechterhaltung des malignen Phänotyps bedeutend beeinflusst. Tumore bestehen zum einen aus den Tumorzellen, zum anderen aus dem unterstützenden Stroma, das Fibroblasten, Endothelien, Perizyten, Lymphgefäße, ein mononukleäres Infiltrat und die Extrazellulärmatrix einschließt. Dieses Tumor-Mikromilieu hat einen großen modulierenden Einfluss auf das Tumorwachstum, die Invasivität und das Metastasierungspotential. Mesenchymale Stammzellen (MSC) sind pluripotente Vorläuferzellen, die an der Aufrechterhaltung und Wiederherstellung der Gewebeintegrität beteiligt sind. Geschädigtes Gewebe führt zur Mobilisation von MSC und deren Rekrutierung an den Ort der Schädigung. Tumore werden vom Organismus als nicht-heilende Wunden angesehen, so dass MSC in das tumorassoziierte Stroma rekrutiert werden. Dort tragen die Zellen zu verschiedenen Aspekten des Tumorwachstums bei, indem sie als Progenitorzellen für die Tumorgefäße und stromale fibroblastenartige Zellen dienen. Im Rahmen dieses Ausdifferenzierungsprozesses werden gewebespezifische Gensets wie das CC-Chemokin CCL5 in den mesenchymalen Stammzellen aktiviert und zur Expression gebracht. Das Pankreasadenokarzinom ist eines der aggressivsten soliden Malignome des Menschen und ist gekennzeichnet durch eine ausgeprägte Proliferation des Stromas. Ziel der vorliegenden Arbeit war es, zum einen die Rolle mesenchymaler Stammzellen im Stroma des Pankreaskarzinoms zu evaluieren und zum anderen eine gewebespezifische stammzellbasierte und promoterkontrollierte Suizidgentherapie mit dem Stroma als Angriffspunkt zu etablieren. Mesenchymale Stammzellen wurden aus dem Knochenmark von C57BL/6 p53-/- Mäusen isoliert und sowohl mit den Reportergenen des rot fluoreszierendem Proteins (RFP) und des grün fluoreszierenden Proteins (eGFP) als auch mit dem Suizidgen der Herpes Simplex I Thymidinkinase (HSV-TK) unter Kontrolle des CCL5-Promoters transfiziert. Die HSV-TK führt zu einer Phosphorylierung und Aktivierung der Prodrug Ganciclovir, welches zytotoxisch auf Thymidinkinase-positive (TK+) Zellen und über den sogenannten „Bystandereffect“ auf umgebende Thymidinkinase-negative (TK-) Zellen wirkt. Diese Stammzellen wurden C57BL/6 Mäusen intravenös injiziert, die orthotope und syngene panc02- Pankreastumore trugen. Die i.v. Applikation von nativen MSC führte zu einer Verdopplung der Tumormasse und einer gesteigerten lokalen Aggressivität im Sinne einer Peritonealkarzinose im Vergleich zur Kontrollgruppe. Dabei zeigten sich erhöhte Ccl5-Expressionsniveaus im Tumorgewebe von Tieren, die MSC erhalten hatten. In-vitro konnte gezeigt werden, dass MSC bei adäquater Stimulation zur Ccl5 Expression angeregt werden und somit als Quelle des beobachteten Ccl5-Anstiegs in Frage kommen. Die stromale Aktivierung des CCL5-Promoters in den mesenchymalen Stammzellen konnte durch Verwendung von CCL5-Promoter/Reportergen Stammzellen direkt nachgewiesen werden. Dabei zeigten sich spezifisch im Tumorgewebe Fluoreszenzsignale, die sich in der Immunhistochemie morphologisch genauer darstellen ließen. Eine Reportergenexpression war spezifisch im stromalen Kompartiment der panc02- Tumore nachweisbar, andere untersuchte Organe mit Ausnahme der Milz zeigten keine Reportergenexpression. Der Einsatz der therapeutischen CCL5-Promoter/HSV-TK MSC in Kombination mit der intraperitonealen Gabe von Ganciclovir führte zu einer Tumormassenreduktion um 50%. Darüberhinaus konnte die Therapie die Metastasierungsrate in Milz, Leber und Peritoneum signifikant senken. Es wurden keine systemischen Nebenwirkungen beobachtet. Bei der Untersuchung von humanen Pankreaskarzinomen und korrespondierenden Pankreasnormalgeweben aus den gleichen Patienten zeigte sich bei der Mehrheit eine Hochregulation von CCL5-mRNA. Im immunhistochemischen Nachweis konnte die CCL5 Expression auf Proteinebene im Tumorstroma gezeigt werden, entsprechendes Normalgewebe zeigte bis auf vereinzelte Zellen keine CCL5 Produktion. Das Tumorstroma stellt aufgrund seiner vitalen Bedeutung für die Tumorprogression einen vielversprechenden Ansatzpunkt künftiger therapeutischer Interventionen dar. Mesenchymale Stammzellen eigenen sich hierbei im Rahmen einer Suizidgentherapie als zellbasierte Vehikel. Dank der gezielten Migration und des Einsatzes gewebespezifischer Promoter kann dabei eine hohe Selektivität der Genexpression im Tumorgewebe mit Minimierung der systemischen Nebenwirkungen erreicht werden. Der CCL5-Promoter wird im stromalen Kompartiment des murinen pankreatischen Adenokarzinoms aktiviert und eignet sich daher für die selektive und spezifische Expression von therapeutischen Genen wie der HSV-TK. Dieser Ansatz kann eine mögliche Therapieoption des ansonsten therapieresistenten humanenPankreaskarzinoms darstellen.

The chemokine RANTES (regulated on activation, normal T-cell expressed and secreted)/CCL5 is involved in the pathogenesis of cardiovascular disease in mice, whereas less is known in humans. We hypothesised that its relevance for atherosclerosis should be reflected by associations between CCL5 gene variants, RANTES serum concentrations and protein levels in atherosclerotic plaques and risk for coronary events. We conducted a case-cohort study within the population-based MONICA/KORA Augsburg studies. Baseline RANTES serum levels were measured in 363 individuals with incident coronary events and 1,908 non-cases (mean follow-up: 10.2±4.8 years). Cox proportional hazard models adjusting for age, sex, body mass index, metabolic factors and lifestyle factors revealed no significant association between RANTES and incident coronary events (HR [95% CI] for increasing RANTES tertiles 1.0, 1.03 [0.75-1.42] and 1.11 [0.81-1.54]). None of six CCL5 single nucleotide polymorphisms and no common haplotype showed significant associations with coronary events. Also in the CARDIoGRAM study (>22,000 cases, >60,000 controls), none of these CCL5 SNPs was significantly associated with coronary artery disease. In the prospective Athero-Express biobank study, RANTES plaque levels were measured in 606 atherosclerotic lesions from patients who underwent carotid endarterectomy. RANTES content in atherosclerotic plaques was positively associated with macrophage infiltration and inversely associated with plaque calcification. However, there was no significant association between RANTES content in plaques and risk for coronary events (mean follow-up 2.8±0.8 years). High RANTES plaque levels were associated with an unstable plaque phenotype. However, the absence of associations between (i) RANTES serum levels, (ii) CCL5 genotypes and (iii) RANTES content in carotid plaques and either coronary artery disease or incident coronary events in our cohorts suggests that RANTES may not be a novel coronary risk biomarker. However, the potential relevance of RANTES levels in platelet-poor plasma needs to be investigated in further studies.

Mon, 10 May 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/11512/ https://edoc.ub.uni-muenchen.de/11512/1/Kumar_Dilip.pdf Kumar, Dilip

Die Rekrutierung von Zellen ist ein komplexer, in mehreren Schritten ablaufender Mechanismus, der eine zentrale Bedeutung für zahlreiche biologische Prozesse, wie z.B. Entzündung, Transplantatabstoßung, Tumormetastasierung und Stammzell¬migration hat. Die Migration von Zellen aus dem Blutstrom oder einem Reservoir in ein Zielgewebe bzw. Zielorgan und umgekehrt wird durch zahlreiche spezifische und unspezifische Reize ausgelöst und orchestriert. Dies erfolgt zu einem großen Teil durch von Chemokinen regulierte Mechanismen. Chemokine sind chemotaktische Zytokine, welche an spezifische auf der Zelloberfläche exprimierte Chemokinrezeptoren (CCR) binden. Zellen mit entsprechenden Chemokinrezeptoren wandern entlang eines Chemokingradienten zum jeweiligen Ziel, z.B. einem Entzündungsherd oder einem Zielorgan. Erstes Ziel dieser Arbeit war die Analyse der Chemokinrezeptorexpression im kutanen T-Zell Lymphom (CTCL), einem Non-Hogkin-Lymphom mit primärer kutaner Manifestation. Der Nachweis von Chemokinrezeptoren erfolgte in vitro mit der Polymerasekettenreaktion (PCR), der Durchflusszytometrie und mit Migrations-versuchen. Der Chemokinrezeptornachweis auf Hautschnitten von CTCL-Patienten erfolgte mit der Immunhistochemie. Erstmals konnte der hautassoziierte Chemokinrezeptor CCR10 im Rahmen des CTCL nachgewiesen werden. Außerdem gelang der Nachweis der Chemokinrezeptoren CCR4, CCR7 und CXCR3 in Hautschnitten und Lymphknotenbiopsien. CXCR3 wurde erstmals im Sezary Syndrom, einer fortgeschrittenen und aggressiven CTCL-Unterform, beschrieben. In der Immunhistochemie wurde die stärkste CCR10-Expression in Sezary Syndrom-Hautschnitten festgestellt. In Biopsien von befallenen Lymphknoten zeigte sich ein auffälliges CCR10-Verteilungsmuster: CCR10-positive Zellen wurden im Lymphsinus nachgewiesen, drangen aber nur vereinzelt in den Lymphknoten ein. In peripheren, nicht-kutanen Lymphomen wurde CCR10 nicht nachgewiesen und ist somit vermutlich exklusiv auf dem primär kutanen CTCL exprimiert. Es ist davon auszugehen, dass CCR10 den Epidermotropismus vor allem in aggressiveren Stadien reguliert. Die Bedeutung von CCR10 für die lymphatische Metastasierung des CTCL ist noch nicht geklärt. CCR10 könnte in der Zukunft als Faktor für die klinische Einstufung des CTCL oder als Ziel für eine gezielte Tumortherapie dienen. Die gezielte Tumortherapie ist u.a. mit Chemokinantagonisten möglich. Sie erlauben die gezielte Beeinflussung der chemokingesteuerten Rekrutierung von Leukozyten, Stammzellen oder Tumorzellen. Deshalb wurde ein membranbindender Antagonist des Chemokins CCL5, als potentielles Agens für die lokale Therapie von Tumoren oder von Transplantatabstoßungen, generiert. Das Chemokin CCL5 und seine Rezeptoren spielen in der akuten Transplantatabstoßung und in der Tumorprogression, z.B. im Mammakarzinom, eine zentrale Rolle. Der CCL5-Antagonist Met-RANTES inhibiert in Transplantatabstoßungsmodellen die Rekrutierung von Leukozyten. Der akute Entzündungsprozess und der daraus resultierende chronische Gefäßschäden werden so vermindert. Auch in einem Tumormodell ist ein Effekt auf die lokale Tumorprogression wahrscheinlich. Der in dieser Arbeit hergestellte CCL5-Antagonist Met-RANTES(Dimer)-GPI soll eine lokale Therapie ohne systemische Nebenwirkungen ermöglichen. Durch die erstmals beschriebene Bindung eines Chemokins oder Chemokinderivats an einen Glykosylinositolphosphatidyl (GPI)-Anker soll der Antagonist effektiv in die Zellmembranen von Endothelzellen inkorporiert werden, länger auf dem Endothel verbleiben und die benötigte Proteinmenge vermindern. Zunächst wurde durch die Erweiterung des signalgebenden N-Terminus von CCL5 der CCL5-Antagonist Met-RANTES generiert. Ein Aminosäureaustausch erzeugte ein dimerisierendes Molekül, welches einfacher als die zur Polymerisierung neigende Wildform zu isolieren war. Das Protein wurde mit der PCR mit einem GPI-Anker fusioniert und in Chinese Hamster Ovary (CHO)-Zellen subkloniert. Met-RANTES(Dimer)-GPI wurde erfolgreich aus den CHO-Zellen isoliert und mit der Säulenchromatographie gereinigt. In in vitro-Versuchen wurde Met-RANTES(Dimer)-GPI effektiv in die Oberfläche von humanen Endothelzellen reinkorporiert und hemmte die transendotheliale Migration von Monozyten, welche bei der Transplantat¬abstoßung und bei der Tumorprogression eine wichtige Rolle spielen. Mit Met-RANTES(Dimer)-GPI präperfundierte Transplantate zeigen möglicherweise einen geringeren vaskulären Schaden bei der akuten Transplantatabstoßungsreaktion. Im Tumormodell soll eine Hemmung der Tumorinfiltration durch Monozyten, welche eine beschleunigte Tumorprogression verursachen, erreicht werden. Im Vergleich zu nicht GPI-gebundenen CCL5-Antagonisten würde eine lokale fokussierte Therapie ermöglicht und eine eventuell geringere zu applizierende Proteinmenge bei längerer Verweildauer erzielt. Die Ergebnisse dieser Arbeit erlauben zunächst einen genaueren Einblick in die Pathogenese des CTCL. Der Chemokinrezeptor CCR7 wird vor allem von fortgeschrittenen Formen mit lymphatischer Infiltration exprimiert. CCR10 wird erstmals im Zusammenhang mit dem CTCL beschrieben und vor allem von fortgeschrittenen Unterformen exprimiert. Desweiteren wurde ein membranbindender Chemokinantagonist hergestellt. Erstmals wird die Kombination eines Chemokins oder Chemokinderivats mit einem GPI-Anker beschrieben. Der Antagonist erlaubt eine hohe lokale Applikation ohne systemische Zirkulation des Agens. Mögliche Einsatzgebiete sind die gezielte Tumortherapie oder die Behandlung der Transplantatabstoßung.

Die direkte Rekrutierung von Effektor-Leukozyten aus dem peripheren Blut in interstitielle Gewebe wird in Teilen von chemotaktischen Zytokinen (Chemokinen) und ihren Rezeptoren kontrolliert. Diesen Schritt zu blockieren stellt ein wichtiges Instrument der Kontrolle einer Entzündungsreaktion dar. Das proinflammatorische Chemokin CCL5/RANTES ist ein chemotaktisches Agens das über die CCR1-, CCR3- und CCR5-Rezeptoren von „Gedächtnis“-CD4+ T-Zellen, Monozyten und Eosinophile wirkt. Es wird von vielen Gewebetypen im Laufe einer Entzündungsreaktion produziert. Die CCR1- und CCR5-Aktivierung konnte als wichtiger Faktor für die Entstehung von akuten Abstoßungsreaktionen mittels in-vitro und in-vivo Experimenten identifiziert werden. Durch metRANTES, dem um ein Methionin verlängerten CCL5-Protein welches einen potenten CCR1 und CCR5 Rezeptor-Antagonisten darstellt, konnte eine Abstoßungsreaktion effektiv reduziert und in Kombination mit anderen Substanzen fast völlig unterdrückt werden. Weitere Modifizierungen am metRANTES-Protein verändern die für CCL5 charakteristische Multimerisierung und seine GAG-Bindungskapazität. Das „protein engineering“ genannte Verbinden eines Proteins mit einem GPI-Anker bietet die Möglichkeit, durch Reintegration in die Oberflächenmembranen unterschiedlicher Gewebe, Proteine an definierte Lokalisationen zu bringen. Dort können sie, dank der Fähigkeit, sich in die Membranen anderer Zellen wieder einzufügen und die ursprüngliche Funktion wieder anzunehmen (Premkumar, Fukuoka et al. 2001; Djafarzadeh, Mojaat et al. 2004), längere Zeit als soluble Chemokine verbleiben. In dieser Arbeit wurden unterschiedliche CCL5-Analoga, sowie ein virales Chemokin-Analogon, um eine GPI-kodierende Sequenz erweitert und in einen Expressionsvektor subkloniert. Mittels Transformation wurden sie in Chinese Hamster Ovarial-Zellen (CHO-Zellen) exprimiert. Ihre Expression an der Zelloberfläche konnte dank der FACS-Analyse ermittelt werden und in einem gleichen Schritt wurde die Fähigkeit verschiedener anti-RANTES Antikörper, an die N-terminal veränderten Proteine zu binden, analysiert. Diese membranverankerten Proteine wurden in höchstmöglicher Konzentration aus der Einheitsmembran extrahiert und in einem weiteren Schritt durch unterschiedliche Chromatographieverfahren isoliert. Dabei wurde die Heparin-Chromatographie gefolgt von einer Size-exclusion Chromatographie als Methode mit der besten Reinheit und Ausbeute identifiziert. Zuletzt wurden die gereinigten, GPI-verbundenen CCL5-Analoga mit „einfachen“ CHO-Zellen, sowie humanen mikrovaskulären Endothelzellen inkubiert und ihre Reintegration an der Zelloberfläche bewiesen. Die in dieser Arbeit hergestellten GPI-gebundenen RANTES-Antagonisten eröffnen die Möglichkeit, in weiteren in-vitro, sowie in-vivo Versuchen, die biologische Aktivität dieses Chemokins gezielt zu blockieren oder (in einem analogen Verfahren) zu verstärken. Dadurch kann die Bedeutung des RANTES-Proteins in der Transplantatabstoßung, sowie in weiteren Entzündungsreaktionen herausgearbeitet werden. Durch Perfusion des Organs vor der Transplantation mit dem GPI-verbudenen Chemokin-basierenden Antagonisten, erhofft man sich die Integration des GPI-Ankers in die mikrovaskuläre Endothelialzellmembran und somit die Präsentation des Antagonisten für die zirkulierenden Leukozyten. Ein solches Vorgehen könnte dem Gefäßsystem während der kritischen ersten Tage nach der Transplantation Schutz bieten und somit signifikant die akute vaskuläre Verletzung, die mit einer Verschlechterung der Überlebensprognose verbunden ist, vermindern (Notohamiprodjo, Djafarzadeh et al. 2005).

Thu, 15 Oct 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/10798/ https://edoc.ub.uni-muenchen.de/10798/1/Hosse_Judith.pdf Hosse, Judith

Die vorliegende Studie trägt zum besseren Verständnis des breiten klinischen Spektrums und des Fehlens universeller Serum- Marker der infektionsassoziierten Krankheitsaktivität der Lupus- Nephritis und möglicherweise anderer Formen der Immunkomplexnephritis bei. Exogener Kontakt mit TLR 7- Liganden hat die Entwicklung der Lupus- Nephritis bei jungen, gesunden MRL- Wildtyp und MRLlpr/lpr Mäusen nicht getriggert und hatte keinen signifikanten Effekt auf die Krankheitsaktivität bei diesen jungen Mäusen. Bei alten Lupus- Mäusen führte eine ähnliche Exposition jedoch zu einem merklichen Anstieg der Serumspiegel proinflammatorischer Zytokine und von IFNα, sowie zu einer Infiltration der Nierenglomeruli 18 Wochen alter MRLlpr/lpr Mäuse mit Makrophagen und einer (nicht signifikanten) Erhöhung der Autoantikörperspiegel. Diese Daten unterstützen die Theorie, dass dem Toll- like Rezeptor 7 eine Rolle bei den Mechanismen, die das Fortschreiten der autoimmunen Gewebsschädigung in MRLlpr/lpr Mäusen fördern, zukommt. Basierend auf den Ergebnissen der funktionellen Rolle von TLR 7 in Lupus- Mäusen und in Primärzellen, die aus Lupusmäusen isoliert wurden, sahen wir in der TLR 7- Blockade ein mögliches neues Ziel, um die schädlichen Effekte des Signalings über Immunkomplexe und endogene Liganden zu begrenzen. Es zeigte sich, dass die Blockade von TLR 7 und TLR 7 + TLR 9 die Gewebsschäden in Nieren und Lunge signifikant reduzieren konnte. Eine TLR 7 Antagonisierung mit dem Oligodeoxyribonukleotid IRS661 senkte die Menge an Autoantikörpern (insbesondere anti- SM, anti- dsDNA, IgG2a, IgG2b), entzündlichen Zytokine und Chemokinen im Serum, glomerulären Ablagerungen von IgG2a und Komplementfaktor C3c deutlich. Die Hemmung von TLR 7 reduzierte ebenfalls die CC- Chemokin- gesteuerten Makrophagen- und T- Zell- Infiltrate in den Nieren. Dies zeigte sich durch erniedrigte Spiegel von Ccr2, Ccr5, Ccl2 und Ccl5 in den Nieren behandelter Tiere. Diese Ergebnisse unterstützen das Konzept, dass endogene TLR 7- Liganden zur Pathogenese der Autoantikörperproduktion und der autoimmun vermittelten Gewebsschädigung des SLE beitragen. Die TLR 7- Blockade könnte ein neues therapeutisches Konzept beim Lupus erythematodes sein.

The global burden of chronic kidney diseases remains an ongoing medical challenge. Therapies that can halt or reverse advanced renal injury are not yet available. Increasing numbers of patients progress to the end-stage renal failure and require renal replacement therapy, the latter being associated with significant mortality, a lower quality of life, and high costs for national health systems. Thus, new treatment strategies that slow down, halt or even revert progressive renal damage are requested. Chemokines and their receptors are involved in the pathogenesis of renal diseases. They mediate leukocytes and macrophages recruitment and activation during initiation as well as progression of renal inflammation. Infiltrating leukocytes are the major source for proinflammatory and profibrotic cytokines and are therefore critical for mediating fibroblast proliferation, differentiation into myofibroblasts, matrix production, and tubular atrophy. Recent advances in the understanding of the molecular mechanisms that regulate renal leukocyte recruitment suggest chemokines and chemokine receptors as novel targets for specific pharmacological intervention. The aim of the present thesis was to investigate the role of chemokine receptor CCR1 for the progression of chronic kidney diseases, e.g. Alport disease and diabetic nephropathy. Two different animal models were used: Col4A3-deficient mice and type 2 diabetic db/db mice with advanced diabetic nephropathy. We blocked CCR1 in Col4A3-deficient mice with BX417, a small molecule CCR1 antagonist, and BL5923, a novel orally available antagonist with a high specificity for human and murine CCR1 in uninephrectomized type 2 diabetic db/db mice, respectively. Treatment with BX471 (25mg/kg) from weeks 6 to 10 of life improved survival of COL4A3- deficient mice, characterized by glomerulosclerosis and subsequent progressive tubulointerstitial injury, leading to fatal end-stage renal disease (ESRD). Improvement was associated with less interstitial macrophages, apoptotic tubular epithelial cells, tubular atrophy, interstitial fibrosis, and less globally sclerotic glomeruli. BX471 reduced total renal Ccl5 mRNA expression by reducing the number of interstitial CCL5-positive cells in inflammatory cell infiltrates. Intravital microscopy of the cremaster muscle in male mice identified that BX471 or lack of CCR1 impaired leukocyte adhesion to activated vascular endothelium and transendothelial leukocyte migration, whereas leukocyte rolling and interstitial migration were not affected. Furthermore, in activated murine macrophages, BX471 completely blocked CCL3-induced CCL5 production. When CCR1 was blocked with BL5923 (60mg/kg, b.i.d), the interstitial recruitment of ex vivo labeled macrophages was markedly decreased in uninephrectomized male db/db mice with type 2 diabetes. Similarly, BL5923 orally administered from month 5 to 6 of life reduced the numbers of interstitial macrophages in uninephrectomized db/db mice. This was associated with reduced numbers of Ki-67 proliferating tubular epithelial and interstitial cells, tubular atrophy, and interstitial fibrosis in uninephrectomized db/db mice. Glomerular pathology and proteinuria were not affected by the CCR1 antagonist. BL5923 reduced renal mRNA expression of Ccl2, Ccr1, Ccr2, Ccr5, Tgf-β1, and collagen I-α1 when compared to untreated uninephrectomized male db/db mice of the same age. Thus, we identified a previously unrecognized role for CCR1-dependent recruitment of interstitial macrophages for the progression of chronic kidney disease in Alport disease and diabetic nephropathy. These data identify CCR1 as a potential therapeutic target for Alport disease and late stage diabetic nephropathy or other progressive nephropathies associated with interstitial macrophage infiltrates.

Background: Chemokines immobilized on endothelial cells play a central role in the induced firm adhesion and transendothelial migration of leukocytes. Activation of platelets at sites of vascular injury is considered to support leukocyte adhesion and extravasation. However, activated platelets also secrete soluble glycosaminoglycans that can interfere with immobilization of chemokines. We therefore analyzed the impact of platelet derived glycosaminoglycans on the immobilization of the chemokine CCL5 (RANTES) on human microvascular endothelial cells and their influence on CCL5-CCR5 interactions. Results: We confirm that undiluted serum in contrast to plasma decreases binding of CCL5 to endothelial cells. However, when lower concentrations of serum were used, CCL5-presentation on endothelial cells was markedly enhanced. This enhancement was neutralized if serum was digested with chondroinitase ABC. Using different chondroitinsulfate-subtypes we demonstrate that chondroitinsulfate A mediates the enhanced presentation of CCL5 on endothelial cells, whereas chondroitinsulfate B/C even at low concentrations block CCL5 binding. CCR5 downregulation on CCR5-transfected CHO cells or human monocytes is increased by preincubation of CCL5 with serum or chondroitinsulfate A. Conclusion: We show that chondroitinsulfate A released from platelets increases the binding of chemokines to endothelial cells and supports receptor internalization in a dose dependent manner. These data help to understand the proinflammatory effects of activated platelets.

Thu, 4 Dec 2003 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/1711/ https://edoc.ub.uni-muenchen.de/1711/1/Frink_Michael.pdf Frink, Michael ddc:610, ddc:600, Medizinis