Podcasts about pbmcs

Lung cancer is a leading cause of cancer-related deaths worldwide, with treatment responses varying widely among patients.Dr. Carminia Maria Della Corte from the University of Texas MD Anderson Cancer Center is at the forefront of research into using peripheral blood mononuclear cells (PBMCs) to identify immune biomarkers. Her work explores the cGAS/STING pathway, DDR gene mutations, and advanced 3D tumour models, offering insight into predicting immunotherapy responses and tailoring treatments for non-small and small cell lung cancer.Read the original research: doi.org/10.3390/biomedicines12040809

A new research paper was published by Aging (listed by MEDLINE/PubMed as "Aging (Albany NY)" and "Aging-US" by Web of Science) in Volume 15, Issue 15, entitled, “Associations between klotho and telomere biology in high stress caregivers.” Aging biomarkers may be related to each other through direct co-regulation and/or through being regulated by common processes associated with chronological aging or stress. Klotho is an aging regulator that acts as a circulating hormone with critical involvement in regulating insulin signaling, phosphate homeostasis, oxidative stress, and age-related inflammatory functioning. In this new study, researchers Ryan L. Brown, Elissa E. Epel, Jue Lin, Dena B. Dubal, and Aric A. Prather from the Department of Psychiatry and Behavioral Sciences, University of California, San Francisco, Department of Biochemistry and Biophysics, University of California, San Francisco, and the Department of Neurology and Weill Institute of Neurosciences, University of California, San Francisco discuss the association between klotho levels and telomere length of specific sorted immune cells among a healthy sample of mothers caregiving for a child with autism spectrum disorder (ASD) or a child without ASD - covarying age and body mass index - in order to understand if high stress associated with caregiving for a child with an ASD may be involved in any association between these aging biomarkers. “Here we examine the relationship between two important biomarkers of aging, klotho and telomere length, in a healthy sample stratified into groups based on a combination of (a) stressor exposure and (b) level of perceived stress (i.e., high-stress mothers of children with ASD compared to low-stress mothers of neurotypical children).” In 178 caregiving women, the researchers found that klotho levels were positively associated with telomere length in PBMCs (an effect driven by CD4+ and CD8+CD28− T cells) among high-stress mothers of children with an ASD, but not among low-stress mothers of neurotypical children. There were no significant associations between klotho and telomerase activity in either group, across cell types assessed here. “Our results suggest that klotho levels and telomere length may be associated through a coordinated downregulation of longevity factors occurring under higher stress caregiving conditions.” DOI - https://doi.org/10.18632/aging.204961 Corresponding author - Ryan L. Brown - ryan.brown@ucsf.edu Video short - https://www.youtube.com/watch?v=y0P4vsf1IIk Sign up for free Altmetric alerts about this article - https://aging.altmetric.com/details/email_updates?id=10.18632%2Faging.204961 Subscribe for free publication alerts from Aging - https://www.aging-us.com/subscribe-to-toc-alerts Keywords - aging, aging biology, stress, klotho, telomeres, telomerase About Aging-US Launched in 2009, Aging-US publishes papers of general interest and biological significance in all fields of aging research and age-related diseases, including cancer—and now, with a special focus on COVID-19 vulnerability as an age-dependent syndrome. Topics in Aging-US go beyond traditional gerontology, including, but not limited to, cellular and molecular biology, human age-related diseases, pathology in model organisms, signal transduction pathways (e.g., p53, sirtuins, and PI-3K/AKT/mTOR, among others), and approaches to modulating these signaling pathways. Please visit our website at https://www.Aging-US.com​​ and connect with us: SoundCloud - https://soundcloud.com/Aging-Us Facebook - https://www.facebook.com/AgingUS/ Twitter - https://twitter.com/AgingJrnl Instagram - https://www.instagram.com/agingjrnl/ YouTube - https://www.youtube.com/@AgingJournal LinkedIn - https://www.linkedin.com/company/aging/ Pinterest - https://www.pinterest.com/AgingUS/ Media Contact 18009220957 MEDIA@IMPACTJOURNALS.COM

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.09.545884v1?rss=1 Authors: Teranishi, K., Wagatsuma, K., Toda, K., Nomaru, H., Yanagihashi, Y., Ochiai, H., Akai, S., Mochizuki, E., Onda, Y., Nakagawa, K., Sugimoto, K., Takahashi, S., Yamaguchi, H., Ota, S. Abstract: Automation and quality control (QC) are critical in manufacturing safe and effective cell and gene therapy products. However, current QC methods, reliant on molecular staining, pose difficulty in in-line testing and can increase manufacturing costs. Here we demonstrate the potential of using label-free ghost cytometry (LF-GC), a machine learning-driven, multidimensional, high-content, and high-throughput flow cytometry approach, in various stages of the cell therapy manufacturing processes. LF-GC accurately quantified T cells in human peripheral blood mononuclear cells (PBMCs) and discriminated between live and dead cells, non-apoptotic live cells and early apoptotic/dead cells, T cells and non-T cells, activated T cells and quiescent T cells, and particulate impurities in PBMCs. The data support that LF-GC is a non-destructive label-free cell analytical method that can be used to monitor cell numbers, assess viability, identify specific cell subsets or phenotypic states, and remove impurities during cell therapy manufacturing. Thus, LF-GC holds the potential to enable full automation in the manufacturing of cell therapy products with reduced cost and increased efficiency. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.08.527670v1?rss=1 Authors: Escudero, B., Moya, M., Lopez-Valencia, L., Arias, F., Orio, L. Abstract: Background: Human apolipoprotein (APO)-E4 has been related to neuropsychiatric disorders such as Alzheimer disease and cognitive decline. Reelin and Clusterin share the VLDLR and ApoER2 receptors with APOE4. Here we checked the role of these components in Alcohol Use Disorder (AUD)-induced cognitive decline. Methods: This is a cross-sectional study with AUD-diagnosed patients (DSM-5) (n=24) recruited from an outpatient Alcohol Programme and matched controls (n=34). Participants were assessed by the validated Test of Detection of Cognitive Impairment in Alcoholism (TEDCA). APOE4 presence in plasma (distinguishing APOE4 carriers and no carriers subjects) and its levels were performed by e4Quant technique. The rest of biological markers were tested by Enzyme-Linked Immunosorbent Assay kits. Results: Plasma APOE4 isoform was present in 37.5% and 58.8% of patients and controls, respectively. Quantification analyses revealed that APOE4 reached similar plasma levels in carriers independently if they were AUD subjects or controls. Circulant plasma APOE4 had a negative effect on AUD cognition, specifically affecting Memory/Learning (p less than 0.01; eta2=0.15). Plasma Clusterin and Reelin increased in patients but, interestingly, Reelin plasma levels peaked in patients expressing APOE4 (p less than 0.05, lower case Greek eta2=0.09), who showed reduced VLDL and ApoER2 expression in peripheral blood mononuclear cells (PBMCs). Reelin was a good predictor of cognitive loss in patients, accounting for the 42.3% and 54.0% of general intelligence and executive function impairments, respectively. Conclusions: Reelin plasma levels are increased in AUD patients who express the APOE4 isoform, predicting cognitive deterioration to a great extent. Remarkably, plasma Reelin helps to differentiate between AUD patients with and without cognitive decline. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.22.521564v1?rss=1 Authors: Christensen, I. B., Ribas, L., Buch-Larsen, K., Marina, D., Larsen, S., Schwarz, P., Dela, F., Gillberg, L. Abstract: Background: Adjuvant chemo- and radiotherapy cause cellular damage not only to cancerous but also to healthy dividing cells. Antineoplastic treatments have been shown to cause mitochondrial respiratory dysfunction in non-tumorous tissues, but the effects on circulating human peripheral blood mononuclear cells (PBMCs) remain unknown. Aim: We aimed to identify changes in mitochondrial respiration of PBMCs after adjuvant chemo- and radiotherapy in postmenopausal early breast cancer (EBC) patients and relate these to metabolic parameters of the patients. Methods: Twenty-three postmenopausal women diagnosed with EBC were examined before and shortly after chemotherapy treatment often administered in combination with radiotherapy (n=18). Respiration (O2 flux per million PBMCs) was assessed by high-resolution respirometry of intact and permeabilized PBMCs. Clinical metabolic characteristics were furthermore assessed. Results: Respiration of intact and permeabilized PBMCs from EBC patients was significantly increased after adjuvant chemo- and radiotherapy (p=6x10-5 and p=1x10-7, respectively). The oxygen flux attributed to specific mitochondrial complexes and respiratory states increased by 17-43% compared to before therapy commencement. Leukocyte counts (p=1x10-4), hemoglobin levels (p=0.0003), and HDL cholesterol (p=0.003) decreased while triglyceride (p=0.01) and LDL levels (p=0.02) increased after treatment suggesting a worsened metabolic state. None of the metabolic parameters correlated significantly with PBMC respiration. Conclusion: This study shows that mitochondrial respiration in circulating PBMCs is significantly increased after adjuvant chemo- and radiotherapy in postmenopausal EBC patients. The increase might be explained by a shift in PBMC subpopulation proportions towards cells relying on oxidative phosphorylation rather than glycolysis or a generally increased mitochondrial content in PBMCs. Both parameters might be influenced by therapy-induced changes to the bone marrow or vascular microenvironment wherein PBMCs differentiate and reside. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.28.514304v1?rss=1 Authors: Sirkis, D. W., Warly Solsberg, C., Johnson, T. P., Bonham, L. W., Sturm, V. E., Lee, S. E., Rankin, K. P., Rosen, H. J., Boxer, A. L., Seeley, W. W., Miller, B. L., Geier, E. G., Yokoyama, J. S. Abstract: Background: Emerging evidence from mouse models is beginning to elucidate the brain's immune response to tau pathology, but little is known about the nature of this response in humans. In addition, it remains unclear to what extent tau pathology and the local inflammatory response within the brain influence the broader immune system. Methods: To address these questions, we performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) from carriers of pathogenic variants in MAPT, the gene encoding tau. Results: Analysis of ~181,000 individual PBMC transcriptomes from MAPT pathogenic variant carriers (n = 8) and healthy non-carrier controls (n = 8) demonstrated striking differential expression in monocytes and natural killer (NK) cells. We observed a marked reduction in the expression of CX3CR1 - the gene encoding the fractalkine receptor that is known to modulate tau pathology in mouse models - in monocytes and NK cells. We also observed a significant reduction in the abundance of nonclassical monocytes and dysregulated expression of nonclassical monocyte marker genes, including FCGR3A. Finally, we identified reductions in TMEM176A and TMEM176B, genes thought to be involved in the inflammatory response in human microglia. We confirmed differential expression of select biologically relevant genes dysregulated in our scRNA-seq data using droplet digital PCR as an orthogonal technique for quantitative validation. Conclusions: Our results suggest that human peripheral immune cell expression and abundance are modulated by tau-associated pathophysiologic changes. CX3CR1 and nonclassical monocytes in particular will be a focus of future work exploring the role of these peripheral signals in additional tau-associated neurodegenerative diseases. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.24.513576v1?rss=1 Authors: Sheridan, S. D., Horng, J. E., Yeh, H., McCrea, L., Fu, T., Perlis, R. H. Abstract: Background: The CYFIP1 gene, located in the neurodevelopmental risk locus 15q11.2, is highly expressed in microglia, but its role in human microglial function as it relates to neurodevelopment is not well understood. Methods: We generated multiple CRISPR knockouts of CYFIP1 in patient-derived models of microglia to characterize function and phenotype. Using microglia-like cells reprogrammed from peripheral blood mononuclear cells, we quantified phagocytosis of synaptosomes (isolated and purified synaptic vesicles) from human iPSC-derived neuronal cultures as an in vitro model of synaptic pruning. We repeated these analyses in human iPSC-derived microglia, and characterized microglial development and function through morphology and motility. Results: CYFIP1 knockout using orthogonal CRISPR constructs in multiple patient-derived cell lines was associated with statistically significant decrease in synaptic vesicle phagocytosis in microglia models derived from both PBMCs and iPSCs (p less than 0.0001). Morphology was also shifted toward a more ramified profile (p less than 0.0001), and motility was significantly reduced (p less than 0.0001). However, iPSC-CYFIP1 knockout lines retained the ability to differentiate to functional microglia. Conclusion: The changes in microglial phenotype and function from loss of CYFIP1 may contribute to pruning abnormalities observed in CYFIP1-associated neurodevelopmental disorders. Investigating risk genes in a range of CNS cell types may be required to fully understand the way in which common and rare variants intersect to yield neuropsychiatric disorders. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.08.23.505001v1?rss=1 Authors: Sharma, S., Fallgren, C., Weil, M. M., Chatterjee, A., Nagpal, P. Abstract: Galactic cosmic rays (GCR) in space induce increase in cerebral amyloid-{beta} levels and elevated levels of microgliosis and astrocytosis, causing accelerated neurodegeneration from this increased neuroinflammation. Even exposure to low-levels of high-Z high-energy (HZE) radiation (50 cGy) has been shown to induce biochemical and immunohistochemical changes in short-term leading to degradation in cognition, motor skills, and development of space-induced neuropathy. There is lack of effective neuroinflammation countermeasures, and current experimental therapies require invasive intracerebral and intrathecal delivery due to difficulty associated with therapeutic crossover between blood-brain barrier. Here, we present a new countermeasure development approach for neurotherapeutics using high-throughput drug-discovery, target validation, and lead molecule identification with nucleic acid-based molecules. These NanoligomerTM molecules are rationally designed using a bioinformatics and AI-based ranking method and synthesized as a single-modality combining 6-different design elements to up- or down-regulate gene expression of target gene at will, resulting in elevated or diminished protein expression of intended target. This platform approach was used to perturb and identify most effective upstream regulators and canonical pathways for therapeutic intervention to reverse radiation-induced neuroinflammation. The lead NanoligomerTM and corresponding target granulocyte-macrophage colony-stimulating factor (GM-CSF) were identified using in vitro cell-based screening in human astrocytes and donor derived peripheral blood mononuclear cells (PBMCs) and further validated in vivo using a mouse model of radiation-induced neuroinflammation. GM-CSF transcriptional downregulator Nanoligomer 30D.443_CSF2 downregulated proinflammatory cytokine GM-CSF (or CSF2) using simple intraperitoneal injection of low-dose (3mg/kg) and completely reversed expression of CSF2 in cortex tissue, as well as other neuroinflammation markers. These results point to the broader applicability of this approach towards space countermeasure development, and potential for further investigation of lead neurotherapeutic molecule as a reversible gene therapy. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

This month on Episode 39 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the August 5th and 19th issues of the journal. This episode also features an interview with Dr Annet Kirabo and Dr Ashley Pitzer from Vanderbilt University on their article, Dendritic Cell ENaC-Dependent Inflammasome Activation Contributes to Salt-Sensitive Hypertension.   Article highlights:   Jain, et al. Role of UPR in Platelets   Orlich et al: SRF Function in Mural Cells of the CNS   Xue et al: Gut Microbial IPA Inhibits Atherosclerosis   Wang et al: Endothelial ETS1 on Heart Development   Cindy St. Hilaire:        Hi, welcome to Discover CircRes, the podcast of the American Heart Association's journal Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to be highlighting articles from our August 5th and August 19th issues of Circulation Research. I'm also going to have a chat with Dr Annet Kirabo and Dr Ashley Pitzer from Vanderbilt University about their study, Dendritic Cell ENaC-Dependent Inflammasome Activation Contributes to Salt-Sensitive Hypertension.   But before I get to the interview, I first want to share an article from our August 5th issue, and that article is titled, Unfolded Protein Response Differentially Modulates the Platelet Phenotype. The first author of this study is Kanika Jain and the corresponding author is John Hwa from Yale University. Self-stress can lead to protein misfolding, and the accumulation of misfolded proteins can lead to a reduction in protein translation and may alter gene transcription, a process collectively known as the unfolded protein response, or UPR. UPR is well documented in nucleated cells; however, it has not been studied in platelets, which are anuclear, but do have a rapid response to cellular stress. In this study, they investigated the UPR in anucleate platelets and explore its role, if any, in platelet physiology and function.   They found that treating human and mouse platelets with various stressors caused aggregations of misfolded proteins and induction of UPR-specific factors. Oxidative stress, for example, induced the UPR kinase PERK, while an endoplasmic reticulum stressor induced the transcription of the UPR factor XBP1. The team went on to study the UPR in platelets from people with type II diabetes, which is a population in which platelet mediated thrombosis is a major complication. They showed that protein aggregation and upregulation of the XBP1 pathway in diabetic patient platelets correlated with disease severity. Furthermore, treating the diabetic patient platelets with a chemical chaperone that helps to correct protein misfolding reduced protein aggregations and prevented the cells prothrombotic activation. This work confirms that even without transcription, platelets display stress-induced UPR, and that targeting this response may be a way to reduce thrombotic risk in diabetic patients.   Cindy St. Hilaire:        The second article I want to share with you is from our August 5th issue and is titled, Mural Cell SRF Controls Pericyte Migration, Vessel Patterning and Blood Flow, and it was led by Michael Orlich from Uppsala University in Sweden. Blood vessels are lined with endothelial cells and surrounded by mural cells. Vascular smooth muscle cells are the mural cells in the case of veins and arteries, and pericytes are the mural cells in the case of capillaries. In the capillaries, pericytes maintain blood-brain and blood-retina barrier function and can mediate vascular tone, similar to smooth muscle cells. While these pericytes and smooth muscle cells are related, they have distinct roles and characteristics.   To learn more about the similarities and the differences between pericytes and smooth muscle cells, this group examined how each would be affected by the absence of SRF in the other. SRF is a transcription factor, essential for nonvascular or visceral smooth muscle cell function. In visceral smooth muscle cells, SRF drives expression of smooth muscle actin and other smooth muscle genes. Using mice engineered to lack SRF in mural cells, they show that SRF drives smooth muscle gene expression in these pericytes and smooth muscle cells, and its loss from smooth muscle cells causes atrial venous malformations and diminishes vascular tone. In pericytes, loss of SRF impaired cell migration in angiogenic sprouting. In a mouse model of retinopathy, activation of SRF drove pathological growth of pericytes. This work not only highlights the various functions of SRF in mural cell biology, but it also suggests that it has a role in pathological capillary patterning.   Cindy St. Hilaire:        The third article I want to share is from our August 19th issue of Circulation Research and is titled, Gut Microbially Produced Indole-3-Propionic Acid Inhibits Atherosclerosis by Promoting Reverse Cholesterol Transport and its Deficiency Is Causally Related to Atherosclerotic Cardiovascular Disease. The first authors are Hongliang Xue and Xu Chen, and the corresponding author is Wenhua Ling from Sun Yat-Sen University in Guangzhou, China. Recent studies provide evidence that disorders in the gut microbiota and gut microbiome derived metabolites affect the development of atherosclerosis. However, which and how specific gut microbial metabolites contribute to the progression of atherosclerosis and the clinical relevance of these alterations remain unclear. Gut microbiome derived metabolites, such as short-chain fatty acids and trimethylamine N-oxide, or TMAO, have been found to correlate with atherosclerotic disease severity.   This study has now found that serum levels of indole-3-propionic acid, or IPA, are lower in atherosclerosis patients than controls. The team performed unbiased metagenomic and metabolomic analyses on fecal and serum samples from 30 coronary artery disease patients and found that, compared with controls, patients with atherosclerosis had lower gut bacterial diversity, depletion of species that commonly produce IPA and lower levels of IPA in their blood. Examination of a second larger cohort of atherosclerosis patients confirmed this IPA disease correlation. The team also showed serum IPA was reduced in a mouse model of atherosclerosis, and that supplementing such mice with dietary IPA could slow disease progression. Analysis of the macrophages from these mice showed that IPA increased cholesterol efflux, and the team went on to elucidate the molecular steps involved. The results of this study not only unraveled the details of IPA's influence on atherosclerosis, but suggest boosting levels of this metabolite could slow atherosclerotic disease progression.   Cindy St. Hilaire:        The last article I want to share is also from our August 19th issue, and it's titled, Endothelial Loss of ETS1 Impairs Coronary Vascular Development and Leads to Ventricular Non-Compaction. The first author is Lu Wang and the corresponding author is Paul Grossfeld, and they are at UCSD. Congenital heart defects, or CHDs, are present in nearly 1% of the human population. In some cases, the heart defects result from a genetic error, which can give researchers clues to its etiology. Jacobson syndrome is a complex condition caused by deletions from one end of chromosome 11, and the occurrence of a congenital heart defect in this syndrome has been associated with the loss of the gene ETS1. ETS1 is an angiogenesis promoting transcription factor, but how ETS1 functions in heart development was not known.   Wang and colleagues now show that both global or endothelial-specific loss of ETS1 in mice caused differences in embryonic heart development that ultimately led to a muscular wall defect known as ventricular non-compaction. The mice also had defective coronary vasculogenesis associated with decreased abundance of endothelial cells in the ventricular myocardium. RNA sequencing of ventricular tissue revealed that, compared with controls, mice lacking ETS1 had reduced expression of several important angiogenesis genes and upregulation of extracellular matrix factors, which together contributed to the muscular and vascular defects.   Cindy St. Hilaire:        Today I have with me, Dr Annet Kirabo and Dr Ashley Pitzer, both from Vanderbilt University, and we're going to talk about their paper, Dendritic Cell ENaC-Dependent Inflammasome Activation Contributes to Salt-Sensitive Hypertension. This article is in our August 5th issue of Circulation Research. Thank you both so much for joining me today.   Annet Kirabo:             Yeah, thank you so much for having us.   Ashley Pitzer:              Yeah, thank you for having us.   Cindy St. Hilaire:        Yeah, it's a great paper. I think we're all familiar with hypertension and this idea that too much salt is bad for our cardiovascular system. When I was a kid, my grandparents had those salt replacements on their kitchen table, Mrs. Dash and whatever. But, like you said in the start of your paper, the exact mechanism by which salt intake increases blood pressure and also increases cardiovascular risk, it's not really well understood, and you guys are focusing on the contribution of immune responses in this process or in this pathogenesis. Before we dig into the details of your paper, I was wondering if you could give us a little bit of background about what's known regarding the role of inflammation in this salt-sensitive hypertension pathogenesis.   Annet Kirabo:             Yeah. It's difficult to know where begin to from, but the role of inflammation in cardiovascular disease have been known for many, many decades. Right now, Dr David Harrison showed more than 10 years ago that T cells contribute to hypertension, but the mechanisms were not known. Back when I was a post doc in David Harrison's lab, we discovered a new mechanism, how immune cells are activated in inflammation and hypertension, whereby we found that there is increased oxidative stress in antigen-presenting cells. This leads to formation of oxidative products known as arachidonic acid or lipid products known as isolevuglandin, or IsoLGs. These IsoLGs are highly, highly reactive and they adapt to lysines on proteins. This is a covalent binding, which leads to permanent alteration of proteins, and so these proteins act as neoantigens that are presented as self-antigens to T cells, leading to an autoimmune-like state in hypertension.   Annet Kirabo:             We found that these antigen-presenting cells are activated and they start producing a lot of cytokines that paralyze T cells to IL-17 producing T cells that contribute to hypertension. And so, when I started my lab back in 2016, we discovered that excess dietary salt profoundly activates this pathway, and we found for the first time that these antigen-presenting cells, they express ENaC, the epithelial sodium channel, and sodium goes into these antigen-presenting cells and activates the NADPH oxidase, which is an enzyme which produces this reactive oxygen species, leading to this IsoLG formation, which I've talked about, and leading to inflammation.   So, three years ago when Ashley joined my lab, she had extensively studied the inflammasome in her PhD program, and she suggested why don't we look at the role of the inflammasome in this pathway and how IsoLG may contribute to this. In her paper that we are discussing right now, she found that in a dependent manner, sodium enters the cell and activates this pathway, and the NLRP3 inflammasome is involved in this process.   Cindy St. Hilaire:        That's such a wonderful story that fits together so many pieces. One of the things you talk about, which I guess I didn't even appreciate myself is, there are certain individuals out there who are more salt-sensitive than others.   Annet Kirabo:             Yeah.   Cindy St. Hilaire:        What is that difference? Do we know the root cause of that? And then also, how many individuals are we talking about are salt-sensitive?   Annet Kirabo:             Salt-sensitive blood pressure, it is a variable trait and it's normally distributed in the population, but it happens more in some individuals than others. It happens even in 25% of people without any hypertension. These people go to that doctor, that doctor thinks they're normal, they don't have any hypertension, but these people can be at a risk of sudden heart attack or cardiovascular risk or even a stroke, simply because when they eat a salty meal, their blood pressure will go up.   Cindy St. Hilaire:        Yeah, that's one of my questions. How much salt are we talking about here? And not only how much in a meal, but a sustained amount? How bad is a miso soup a day?   Annet Kirabo:             Yes. The American Heart Association and the World Health Organization have recommendations. American Heart Association recommends one spoon per day. We have refused to adapt to this recommendation, but that is the recommendation that they have recommended per day to eat. But this is difficult because most of the salt, as you know, is already in our food through processing in our processed foods and we don't have any control over how much salt we have, and there's also a lot of adding of salt at a table.   Cindy St. Hilaire:        Ashley, your background was more the inflammasome. What were your thoughts entering into this project? Did you have much of a hypertension background?   Ashley Pitzer:              No. My graduate thesis focused mainly on endothelial dysfunction and cardiovascular disease, and so it was a pretty easy segue. But it was just with Annet, so excited about the project and showing me all the data and this robust IL-1 beta production that she was seeing after these immune cells being exposed to high salt, I, with my inflammasome background, was immediately like, this could be playing a role. And so it was, like I said, a pretty easy transition and, as is in the paper, we're doing human studies. All of my research back in grad school was very basic research, so it was very exciting to see how our research was being translated with people having this condition and potentially finding mechanisms where we can target this to help actual people.   Cindy St. Hilaire:        I think a lot of us who are not in the hypertension field, and maybe this was you before you joined Annet's lab, we really only kind of think of the kidneys and the blood vessels when we think about hypertension, but studies like this are changing that. And I think a lot of Annet's earlier work, as well as the work of others, have shown a role for this epithelial sodium channel as an important player in this salt-induced hypertension. New to me, it's not just found in the kidney, which I totally did not appreciate that. And it's this channel sensing the salt that can trigger this IL-1 beta production that does a whole bunch of other things.   Cindy St. Hilaire:        What are those other things? What are those cells that are affected and where is this happening? Obviously it's not just kidney cells, but is it only in the kidney or are these systemic cells? What do we think is happening?   Ashley Pitzer:              That's the question, is, where is this happening? There's been studies at Vanderbilt by Jens Titze and his lab showing, where are these immune cells sensing the salt? And so they've shown that sodium accumulates in the skin, a huge argument is for they're sensing the sodium in the kidney because that's where a lot of it is being processed. But these immune cells travel through the whole body, so they're seeing it where there are the highest amounts of sodium concentration, and so I would argue it's in the kidney.   Annet Kirabo:             Indeed, because we're now collaborating with Tina Kon, and we have recently published with her a paper in the International Journal of Science, where we have done sodium MRI and we find this accumulation of sodium in the kidney even much more than in the skin. And we know that the kidney is where sodium is highly concentrated. So the working hypothesis in the lab is that these immune cells can be activated wherever they are, in the lymph nodes or not, in other tissues, but they can travel to the kidney.   We find that in high salt, if you feed high salt to the mouse, the endothelium in the kidney becomes dysfunctional and it expresses molecules, chemoattractants, that attract these immune cells in the kidney. We think that the high salt accumulation in the kidney can activate these, and then these immune cells are activated and they produce cytokines. Dr Steve Crowley showed that they can produce IL-1 beta, which induces activation of sodium channels that can be induced. We have also actually found that even IL-17 can be produced by these immune cells in the kidney and they can activate sodium channels in the kidney, leading retention of sodium and water and hypertension.   Cindy St. Hilaire:        Very cool. You used a lot of mice in this paper. Can you tell us, I just want to know a little bit about the models you chose to use, but also how similar is hypertension in mouse and humans? Obviously for atherosclerosis, we have to do lots of things to get them to form a plaque. Is hypertension similar in a mouse and do mice also show this salt-sensitive phenotype?   Annet Kirabo:             That is an extremely important point. If you read our paper, we use a slightly different approach. Most people do benchside to bed approach. We did the opposite. We did a bed to benchside approach.   Cindy St. Hilaire:        Always smart.   Annet Kirabo:             Yeah. We first started humans, and then with some references, we went to the mice, because I think when it comes to salt-sensitive blood pressure, mice are different from humans. In fact, if we look in the lab, we find that female mice are protected from salt-sensitive blood pressure, but we find that in the humans, it's the opposite. Females are more prone to salt-sensitive hypertension. Those are studies that we are doing right now. We haven't published. But we know that it can be different.   The model we use most of the time in the lab, the C57 mice, are resistant to salt-sensitive hypertension. These C57 mice would rather die before they raise their blood pressure in response to salt. We can induce salt-sensitivity in these mice like in the paper that we are discussing. When we induce the endothelial dysfunction using L-NAME and we wash it out, then these mice, when you give them, subsequently, salt, suggests that they become salt-sensitive. But we also have a salt-sensitive mouse model that we use, the 129/SV mouse. So we use several models to kind of prove the same thing over and over again with the findings that we found in humans.   Cindy St. Hilaire:        And you used a technique, which I'm a little bit familiar with, but I'd like to hear, A, about it from you, but also your experience in using it, and that is CITE-seq. So, how does that work?   Ashley Pitzer:              That was with our human study where we actually had patients come in, who were hypertensive, took them off medication for 2 weeks. They come in, we get baseline samples, we give them a salt load on one day, and then the next day we completely salt deplete them.   Cindy St. Hilaire:        How much is a salt load? Like a Big Mac? What's a salt load?   Ashley Pitzer:              Yeah, it's pretty much just like eating Lays chips all day. It's a lot of salt. It's a very salty meal.   Annet Kirabo:             And then in addition, we also infuse saline too.   Cindy St. Hilaire:        Oh, wow.   Annet Kirabo:             Because these people, when they come into the hospital, some them have already eating high salt. This approach is to just maximize the whole system so that then when we sort deplete everybody, it's at the same level and it's just to unify the whole process. But sorry, Ashley, you go ahead.   Ashley Pitzer:              With the CITE-seq, we're able to take different patients on different days. So we take samples each day, and we can give each sample a barcode, basically. Give them a barcode, we can pool them all together, process them, and we can sequence their RNA, we can probe for a certain amount of protein expression as well. So then when we analyze, we can look at protein expression, so you get the translation and the transcription for each person on each day, and then you're able to compare. And so you get this huge picture and it's a lot of data.   Cindy St. Hilaire:        How long did it take you to sort through?   Ashley Pitzer:              Well, we have a statistician who does all of that, because my wheelhouse is here and it is on a different planet. So we have somebody who helps us with that who does an unbiased approach. And then once he does an analysis, gives us back what are the things that are changing the most, and one of those was IL-1 beta.   Annet Kirabo:             As you can see, our list is huge, this is a massive input of so many collaborators. We have computational people on there that help us with this. I can't even begin to learn these techniques, but with all this collaboration and the resources at Vanderbilt, these things are possible. And so, this is a really powerful approach where you can combine protein expression and you get the specific cells that express the genes and you couple the channel type to the gene expression.   Annet Kirabo:             We actually found that not all monocytes are the same. There's a specific class that of monocytes, A small class of monocytes that is so angry, and the inflammasome is activated and producing this IL-1 beta, and that is enough to contribute to this phenotype of salt-sensitive hypertension, which dynamically changed according to blood pressure, suggesting that this is a targetable salt-sensitive blood pressure, even in normotensive people, is a targetable trait. And because these monocytes are in blood, can we get a blood sample and routinely diagnose salt-sensitive blood pressure so that doctors are aware and they can appropriately advise patients.   Cindy St. Hilaire:        This was samples obviously taken from a blood draw, right? So they're circulating.   Annet Kirabo:             It was a blood draw, yes.   Cindy St. Hilaire:        What do you think about these immune cells, perhaps, native in the kidney? Do you think the small population of angry cells, like you said, is escaping from the kidney environment? What do you think?   Annet Kirabo:             When I was a post-doc in David Harrison's lab, we found that the most angry dendritic cells that contribute to this inflammation and hypertension are monocyte-derived. So that's why in the human study we focused on monocytes, because there are so many subtypes of dendritic cells, plasmacytoid dendritic, classical dendritic cells. We have studied all of these subtypes, and we have focused on monocyte-derived dendritic cells because they're the ones that seem to be contributing to this phenotype the most.                                     Cindy St. Hilaire:        You guys focused in on the NLRP3 inflammasome, which, obviously it's a really critical component broadly for the innate immune system. Do you think that this is going to be a targetable approach that can be leveraged for hypertension? Or do you think it's too broad? What do you think about that as a therapeutic potential?   Ashley Pitzer:              Even when you look in our paper, and we use a knockout model, where we use a completely global knockout model, put them on high salt, and we give them back only dendritic cells that are from wild-type mice, so they have that NLRP3, that have been exposed to high salt. We were able to increase blood pressure, but I also did, in mice, where I gave them an IL-1 beta neutralizing antibody, similar to canakinumab, which is the CANTOS trial, and there's not much of a difference. There is, but it's minor. It's very minor.   Ashley Pitzer:              So, to be able to target in specific cell types in humans one thing, it's very difficult, and maybe one day we can get there. But I think it at least gives us a better idea of what is the full picture, what's the big mechanism going on with immune cells? In part of our human study, we are looking at something to try and be able to identify who is salt sensitive. So if anything, we're able to sit here and potentially have a way of identifying salt-sensitive patients, where, right now, all we can do is have them come in like we do and do a 3-day study, and not everybody can do that.   Annet Kirabo:             To add onto that, perhaps you know, we are talking about precision medicine. This is an era of precision medicine where you need to really tailor treatments if we can get there, and I think this is one way. CANTOS trial. They had no way of knowing who is salt-sensitive and who is not, it was a global approach, and the lack of differences in blood pressure might be explained that this IL-1 beta pathway is targetable in a specific population whose blood pressure is probably driven by inflammation. There are so many, many mechanisms that drive hypertension, and so perhaps we need to focus this on salt-sensitive people, and maybe we can really use this approach to target. Plus, this is ENaC-dependent.   As you know, amiloride has lost favor in the clinic as a treatment of hypertension, because in the majority, it's not effective. But studies have shown that in Black men, for example, who had been categorized salt-resistant, when they give them amiloride, their blood pressure went down, and yet it's not effective in the majority of the people.   So, can we bring back, can we take another look at amiloride. As our studies indicate that blockade of ENaC is anti-inflammatory and it's also antioxidant agent, can we at least bring back amiloride and look at it again and we focus it for specific populations of people that may be more prone to salt-sensitive hypertension?   Here we have so many targets for potential precision treatment of salt-sensitive potential in this paper. You can target SGK1, which we know is possible, we listed a number of clinical trials that they have used NLRP3 inflammasome inhibitors, you can use amiloride for these people, and you can also potentially scavenge IsoLGs.     Cindy St. Hilaire:        What was the most challenging aspect of this study? There's a lot of moving parts, so what was the biggest challenge? And then, also, what was the most surprising part or the most pleasantly surprising part?   Ashley Pitzer:              You have to think, most of this was going on right when the pandemic hit. And right before that, we had started our human recruitment for the human study. And so that put a little bit of a time damper on it.   Ashley Pitzer:              Other than that, it was just, we were finding one thing, developing a new experiment, doing it again, doing it again. And honestly, what was the most surprising and rewarding was just seeing the same thing in, because we took just PBMCs from normotensive patients, treated them with high salt, and saw the changes that we did with the inflammasome. And to see that exactly again in an in vivo model of giving patients high salt and seeing the same thing, it was very rewarding and confirmed that, okay, we're on the right path. Seeing the same thing over and over and over again, it kind of reaffirms that you had a good idea.   Annet Kirabo:             I might add, one of the most challenging was, initially, the computational. Oh, part of the pandemic I was, the pandemic hit, I had a baby during the pandemic, and it was my time to leave my home, and then all these things were going on. We had a clinical trial where patients had to come in. Vanderbilt was so super supportive ,even checking for COVID-19. Our patients could not have COVID-19. We needed to check them.   Cindy St. Hilaire:        Yeah.   Annet Kirabo:             They also had to check for COVID-19. And so during that time, I realized, wait, I need learn computation analysis. I realized I cannot learn, and then reached out to collaborators that helped. That was extremely challenging. And then the other challenging thing that we faced later during the pandemic is vaccinations. In our criteria, these people cannot be vaccinated for reasons. We've studied inflammation, hypertension, and so vaccination was confounding. And even COVID-19 is even more for confounding. So we had this exclusion criteria where we could not recruit anyone.   Annet Kirabo:             Everybody was having COVID, everybody was being vaccinated, and everybody was in that exclusion criteria, so it was difficult to get people. We have had some slow down, but right now it's beginning to build up.   Cindy St. Hilaire:        So, what's next? What's the next question?   Annet Kirabo:             We have so many.   Cindy St. Hilaire:        That means it was a great study. If you have more, that means it was a great study.   Annet Kirabo:             Yeah. This study and us, it kind of warms. The inside seat just opened up, we have primary data in the genetic regulation of ENaC, we have primary data where we found. We are trying to figure out the specific ENaC channel in these antigen-presenting cells. We don't know. We found that ENaC delta, for example, it's not found in a kidney or you talked about a kidney contribution versus immune cells. ENaC delta is not found in the kidney, but we have primary data that show that ENaC delta is the most correlated with cardiovascular risk, is the most correlated with kidney disease and all forms of hypertension. So now we're like, ENaC delta expressed in the immune cells, not in the kidney, it is the one that is most involved in cardiovascular disease, so how are we going to tell the world that.   Cindy St. Hilaire:        Yeah, very cool.   Annet Kirabo:             Those cells, not necessarily the kidney. The kidney plays a part because the cells are going there, but it's very, very exciting. Plus a number of other lines that we are investigating.   Cindy St. Hilaire:        It's great. Well, congratulations, again, on this publication, on just getting all this done with what sounds like extremely difficult patient recruitment. So, Dr Kirabo and Dr Pitzer, thank you so much for joining me today and I'm looking forward to these next studies on maybe ENaC delta.   Annet Kirabo:             Thank you. Thank you so much.   Ashley Pitzer:              Thank you for having us.   Cindy St. Hilaire:        That's it for the highlights from the August 5th and August 19th issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and hashtag Discover CircRes. Thank you to our guests, Dr Annet Kirabo and Dr Ashley Pitzer.   This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on the go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2022. Opinions expressed by speakers in this podcast are their own, and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.  

In this episode from the series “Key Decisions in HIV Care,” Latesha Elopre, MD, MSPH, and Karine Lacombe, MD, PhD, discuss important considerations for PrEP in cisgender women, including: Discussion of the PrEP gap and racial and gender disparities in PrEP accessCDC, EACS, and WHO guidance on PrEP eligibilityPrEP regimens currently recommended for adults and adolescents including FTC/TDF daily, FTC/TAF daily, and long-acting cabotegravirData from phase III trials on the efficacy of FTC/TDF daily for women including Partners PrEP, TDF, VOICE, and FEM-PrEPPharmacokinetic data of FTC/TDF and FTC/TAF in the female genital tract and discussion of why on-demand dosing of FTC/TDF is not recommended in cisgender womenData and recommendations for the use of the dapivirine ring from ASPIRE and The Ring Study and their open-label extensions, DREAM and HOPEData from the HPTN084 study on the use of long-acting cabotegravir as PrEP for cisgender womenRecommendations for the use of PrEP during pregnancy and breastfeedingClinical monitoring and considerations during PrEP usePresenters:Latesha Elopre, MD, MSPHAssociate ProfessorDivision of Infectious DiseasesAssistant Dean of Diversity and InclusionGeneral Medical EducationUniversity of Alabama at BirminghamBirmingham, Alabama Karine Lacombe, MD, PhDProfessorUMR-S1136Sorbonne UniversityHead, Infectious Diseases DepartmentSt Antoine Hospital, AP-HPParis, France Content based on an online CME program supported by educational grants from Gilead Sciences, Inc.; Janssen Therapeutics, Division of Janssen Products, LP; and ViiV Healthcare.Follow along with the slides at: https://bit.ly/36s2ovBLink to full program:https://bit.ly/3q2DlGd

Listen to summaries of the latest oncology-focused research published in this week's issue of Oncotarget, Volume 12, Issue 20. https://www.oncotarget.com/archive/v12/i20/ Priority Research Paper (Cover) - “Identification of Smurf2 as a HIF-1α degrading E3 ubiquitin ligase” https://doi.org/10.18632/oncotarget.28081 Research Paper - “A high-throughput customized cytokinome screen of colon cancer cell responses to small-molecule oncology drugs” https://doi.org/10.18632/oncotarget.28079 Research Paper - “Opposing effects of BRCA1 mRNA expression on patient survival in breast and colorectal cancer and variations among African American, Asian, and younger patients” https://doi.org/10.18632/oncotarget.28082 Research Paper - “Pan-drug and drug-specific mechanisms of 5-FU, irinotecan (CPT-11), oxaliplatin, and cisplatin identified by comparison of transcriptomic and cytokine responses of colorectal cancer cells” https://doi.org/10.18632/oncotarget.28075 Research Paper - “Comparative microsomal proteomics of a model lung cancer cell line NCI-H23 reveals distinct differences between molecular profiles of 3D and 2D cultured cells” https://doi.org/10.18632/oncotarget.28072 Research Paper - “Tissue biodistribution and tumor targeting of near-infrared labelled anti-CD38 antibody-drug conjugate in preclinical multiple myeloma” https://doi.org/10.18632/oncotarget.28074 Research Paper - “Multipeptide stimulated PBMCs generate TEM/TCM for adoptive cell therapy in multiple myeloma” https://doi.org/10.18632/oncotarget.28067 Research Paper - “NEDD8-activating enzyme inhibition induces cell cycle arrest and anaphase catastrophe in malignant T-cells” https://doi.org/10.18632/oncotarget.28063 Research Paper - “Safety and initial efficacy of ablative radioembolization for the treatment of unresectable intrahepatic cholangiocarcinoma” https://doi.org/10.18632/oncotarget.28060 Review - “Switching Hedgehog inhibitors and other strategies to address resistance when treating advanced basal cell carcinoma” https://doi.org/10.18632/oncotarget.28080 Editorial - “Fibrocytes in primary myelofibrosis” https://doi.org/10.18632/oncotarget.27971 (PDF Download) Research Perspective - “Ras-p53 genomic cooperativity as a model to investigate mechanisms of innate immune regulation in gastrointestinal cancers” https://doi.org/10.18632/oncotarget.27983 Keywords - cytokine profiling, breast cancer, colorectal cancer, colon cancer, lung cancer, multiple myeloma (MM), cellular therapy, T-cell lymphoma, cholangiocarcinoma, basal cell carcinoma, myelofibrosis, gastrointestinal cancer, cancer, science, research, oncology About Oncotarget Oncotarget is a bi-weekly, peer-reviewed, open access biomedical journal covering research on all aspects of oncology. To learn more about Oncotarget, please visit https://www.oncotarget.com/ or connect with: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget LinkedIn - https://www.linkedin.com/company/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/oncotargetyoutube Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget Oncotarget is published by Impact Journals, LLC. Please visit https://www.impactjournals.com/ or connect with @ImpactJrnls Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.13.379982v1?rss=1 Authors: Thiel, G., Tandl, D. H., Sponagel, T., Fuck, S., Smit, T., Hehlgans, S., Jakob, B., Fournier, C., Roedel, F., Roth, B., Moroni, A. Abstract: Radiation therapy is efficiently employed for eliminating cancer cells and reducing tumor growth. To further improving its therapeutic application it is mandatory to unravel the molecular effects of ionizing irradiation and to understand whether they support or counteract tumor therapy. Here we examine the impact of X-ray irradiation on immune activation of human T cells with single doses typically employed in tumor therapy. We discover that exposing cells to radiation triggers in a population of leukemic Jurkat T cells and in peripheral blood mononuclear cells (PBMCs) a canonical Ca2+ signaling cascade, which elicits immune activation of these cells. An early step in the signaling cascade is the initiation of sustained oscillations of the cytosolic Ca2+ concentration, an event mediated by store operated Ca2+ entry (SOCE) via an X-ray induced clustering of the Calcium Release-Activated Calcium Modulator 1 with the stromal interaction molecule 1 (Oari1/STIM1). A functional consequence of the Ca2+ signaling cascade is the translocation of the transcription factor nuclear factor of activated T cells (NFAT) from the cytosol into the nucleus where it elicits the expression of genes required for immune activation. These data imply that a direct activation of blood immune cells by ionizing irradiation has an impact on toxicity and therapeutic effects of radiation therapy. Copy rights belong to original authors. Visit the link for more info

There’s more than one way to measure how fast you’re ageing. There’s chronological age - the number of years you’ve been alive - and then there’s biological age, which you can think of as the total damage your body has accumulated over the years. Your chronological age may differ from your biological age, in which case it’s interesting to understand why. The good news is you can reduce your biological age by improving your lifestyle, which in turn can lengthen lifespan and healthspan. The question is, then, how to quantify biological age? On this podcast, NBT Scientific Director Megan Hall talks about PhenoAge: a measure of biological age that can be determined by analyzing a shortlist of common blood markers. We talk about why PhenoAge is important and valid as a reliable measure of biological status, and how you can get your PhenoAge score. Megan also offers tips for improving your PhenoAge once you’ve got your baseline. This episode has a ton of information, so be sure to follow along with Megan’s outline. Here’s the outline of this interview with Megan Hall: [00:00:25] Arden Pope, PhD; Studies on the effects of air pollution on human health. [00:01:15] Puppy update. [00:05:54] Is ageing a disease? Article: Bulterijs, Sven, et al. "It is time to classify biological aging as a disease." Frontiers in genetics 6 (2015): 205.   [00:06:35] Primary vs secondary ageing. [00:08:02] Book: Lifespan: Why We Age - and Why We Don't Have To, by David A. Sinclair PhD. [00:08:16] Ken Ford; STEM-Talk Podcast. Ken Ford on the NBT Podcast: Optimal Diet and Movement for Healthspan, Amplified Intelligence and More. [00:09:19] Measuring ageing. [00:13:09] Theories of ageing - more than 300 theories; Articles: Tosato, Matteo, et al. "The aging process and potential interventions to extend life expectancy." Clinical interventions in aging 2.3 (2007): 401. 2. da Costa, Joao Pinto, et al. "A synopsis on aging—Theories, mechanisms and future prospects." Ageing research reviews 29 (2016): 90-112. 3. Jin, Kunlin. "Modern biological theories of aging." Aging and disease 1.2 (2010): 72.  [00:13:34] Grandmother hypothesis; Podcast: The Postmenopausal Longevity Paradox and the Evolutionary Advantage of Our Grandmothering Life History, with Kristen Hawkes, PhD. [00:14:48] Program Theories and Damage Theories. [00:17:45] Epigenetic clock theory of aging; Steven Horvath; Study: Horvath, Steve, and Kenneth Raj. "DNA methylation-based biomarkers and the epigenetic clock theory of ageing." Nature Reviews Genetics 19.6 (2018): 371.  [00:19:02] Steven Horvath's TEDx talk: Epigenetic Clocks Help to Find Anti-Aging Treatments. [00:20:47] Book: Masters of Doom: How Two Guys Created an Empire and Transformed Pop Culture, by David Kushner. [00:21:43] DNA methylation; Article: Horvath, Steve. "DNA methylation age of human tissues and cell types." Genome biology 14.10 (2013): 3156. [00:23:13] Offspring of semi-supercentenarians have lower epigenetic age; Study: Horvath, Steve, et al. "Decreased epigenetic age of PBMCs from Italian semi-supercentenarians and their offspring." Aging (Albany NY) 7.12 (2015): 1159.  [00:23:36] Methylation based biological age associated with: 1.  breast cancer risk: Kresovich, Jacob K., et al. "Methylation-based biological age and breast cancer risk." JNCI: Journal of the National Cancer Institute 111.10 (2019): 1051-1058. 2. Frailty: Breitling, Lutz Philipp, et al. "Frailty is associated with the epigenetic clock but not with telomere length in a German cohort." Clinical epigenetics 8.1 (2016): 21; 3. All-cause mortality: Marioni, Riccardo E., et al. "DNA methylation age of blood predicts all-cause mortality in later life." Genome biology 16.1 (2015): 1-12 and Christiansen, Lene, et al. "DNA methylation age is associated with mortality in a longitudinal Danish twin study." Aging cell 15.1 (2016): 149-154. [00:24:46] PhenoAge as a biomarker of ageing for lifespan and healthspan; Study: Levine, Morgan E., et al. "An epigenetic biomarker of aging for lifespan and healthspan." Aging (Albany NY) 10.4 (2018): 573. [00:29:06] Nine blood markers that make up PhenoAge. [00:29:57] PhenoAge related to COVID-19; Study: Kuo, Chia-Ling, et al. "COVID-19 severity is predicted by earlier evidence of accelerated aging." medRxiv (2020).  [00:30:34] Combining PhenoAge with DNA methylation data as a predictor of mortality. [00:33:28] Episode 59 of HumanOS podcast: Are You Biologically Older or Younger Than Your Chronological Age? [00:33:58] Dr. Josh Turkett’s 4-quadrant model. [00:34:00] Lifestyle factors that accelerate ageing: Sleep: Li, Xiaoyu, et al. "Association between sleep disordered breathing and epigenetic age acceleration: Evidence from the Multi-Ethnic Study of Atherosclerosis." EBioMedicine 50 (2019): 387-394; Socioeconimic status, childhood and adult adversity: Liu, Zuyun, et al. "Associations of genetics, behaviors, and life course circumstances with a novel aging and healthspan measure: Evidence from the Health and Retirement Study." PLoS medicine 16.6 (2019): e1002827; Education: Zhao, Wei, et al. "Education and lifestyle factors are associated with DNA methylation clocks in older African Americans." International journal of environmental research and public health 16.17 (2019): 3141. [00:35:59] Protein; Podcast: Why You’re Probably Not Eating Enough Protein (How to Know for Sure), with Megan Hall. [00:36:50] Book: The Good Gut: Taking Control of Your Weight, Your Mood, and Your Long-term Health, by Justin Sonnenburg and Erica Sonnenburg. [00:37:23] Bloodsmart.ai. [00:38:35] Patreon: nbt.link [00:39:33] Age reversal possible in humans? Study: Fahy, Gregory M., et al. "Reversal of epigenetic aging and immunosenescent trends in humans." Aging cell 18.6 (2019): e13028.  [00:40:15] Simon Marshall, PhD. [00:41:00] Interpreting your blood markers to understand PhenoAge. [00:46:11] PhenoAge vs Predicted Age.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.08.288316v1?rss=1 Authors: Schiffer, L., Bossey, A., Taylor, A. E., Akerman, I., Scheel-Toellner, D., Storbeck, K.-H., Arlt, W. Abstract: Context: Androgens are important modulators of immune cell function impacting proliferation, differentiation and cytokine production. The local generation of active androgens from circulating androgen precursors is an important mediator of androgen action in peripheral androgen target cells of tissue. Objective: To characterize the activation of classic and 11-oxygenated androgens in human peripheral blood mononuclear cells (PBMCs). Methods: PBMCs and natural killer cells were isolated from healthy male donors and incubated ex vivo with precursors and end products of the classic and 11-oxygenated androgen pathways. Steroid concentrations were quantified by liquid chromatography-tandem mass spectrometry. The expression of genes encoding steroid metabolizing enzymes was assessed by quantitative PCR. Results: The enzyme AKR1C3 is the major reductive 17{beta}-hydroxysteroid dehydrogenase in PBMCs and has higher activity for the generation of the active 11-oxygenated androgen 11-ketotestosterone than for the generation of the classic androgen testosterone from their respective precursors. Natural killer cells are the major site of AKR1C3 expression and activity within the PBMC compartment. Steroid 5-reductase type 1 catalyzes the 5-reduction of classic but not 11-oxygenated androgens in PBMCs. Lag time prior to the separation of cellular components from whole blood sample increases 11KT serum concentrations in a time-dependent fashion, with significant increases detected from two hours after blood collection. Conclusions: 11-oxygenated androgens are the preferred substrates for androgen activation by AKR1C3 in PBMCs, primarily conveyed by natural killer cell AKR1C3 activity, yielding 11KT the major active androgen in PBMCs. Androgen metabolism by PBMCs can affect the levels of 11-oxygenated androgens measured in serum samples, if samples are not separated in a timely fashion. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.05.135392v1?rss=1 Authors: Duong, A., Evstratova, A., Sivitilli, A., Hernandez, J. J., Gosio, J., Wahedi, A., Sondheimer, N., Wrana, J. L., Beaulieu, M., Attisano, L., Andreazza, A. Abstract: Mitochondrial health plays a crucial role in human brain development and diseases. However, the evaluation of mitochondrial health in the brain is not incorporated into clinical practice due to ethical and logistical concerns. As a result, the development of targeted mitochondrial therapeutics remains a significant challenge due to the lack of appropriate patient-derived brain tissues. To address these unmet needs, we developed cerebral organoids (COs) from induced pluripotent stem cells (iPSCs) derived from human peripheral blood mononuclear cells (PBMCs) and monitored mitochondrial health from the primary, reprogrammed and differentiated stages. Our results show preserved mitochondrial genetics, function and treatment responses across PBMCs to iPSCs to COs, and measurable neuronal activity in the COs. We expect our approach will serve as a model for more widespread evaluation of mitochondrial health relevant to a wide range of human diseases using readily accessible patient peripheral (PBMCs) and stem-cell derived brain tissue samples. Copy rights belong to original authors. Visit the link for more info

Jane Ferguson:                  Hi there. Welcome to Getting Personal: Omics of the Heart, the podcast from Circulation: Genomic and Precision Medicine. I'm Jane Ferguson, and this is Episode 36 from February 2020.                                                 First up, we have “Identification of Circulating Proteins Associated with Blood Pressure Using Mendelian Randomization” from Sébastien Thériault, Guillaume Paré, and colleagues from McMaster University in Ontario. They set out to assess whether they could identify protein biomarkers of hypertension using a Mendelian randomization approach. They analyzed data from a genome-wide association study of 227 biomarkers which were profiled on a custom Luminex-based platform in over 4,000 diabetic or prediabetic participants of the origin trial.                                                 They constructed genetic predictors of each protein and then used these as instruments for Mendelian randomization. They obtained systolic and diastolic blood pressure measurements in almost 70,000 individuals, in addition to mean arterial pressure and pulse pressure in over 74,000 individuals, all European ancestry with GWAS data, as part of the International Consortium for Blood Pressure.                                                 Out of the 227 biomarkers tested, six of them were significantly associated with blood pressure traits by Mendelian randomization after correction for multiple testing. These included known biomarkers such as NT-proBNP, but also novel associations including urokinase-type plasminogen activator, adrenomedullin, interleukin-16, cellular fibronectin and insulin-like growth factor binding protein-3. They validated all of the associations apart from IL-16 in over 300,000 participants in UK Biobank. They probed associations with other cardiovascular risk markers and found that NT-proBNP associated with large artery atherosclerotic stroke, IGFBP3 associated with diabetes, and CFN associated with body mass index.                                                 This study identified novel biomarkers of blood pressure, which may be causal in hypertension. Further study of the underlying mechanisms is required to understand whether these could be useful therapeutic targets in hypertensive disease.                                                 The next paper comes from Sony Tuteja, Dan Rader, Jay Giri and colleagues from the University of Pennsylvania and it's entitled, “Prospective CYP2C19 Genotyping to Guide Antiplatelet Therapy Following Percutaneous Coronary Intervention: A Pragmatic Randomized Clinical Trial”.                                                 They designed a pharmacode genomic trial to assess effects of CYP2C19 genotyping on antiplatelet therapy following PCI. Because loss of function alleles in CYP2C19 impair the effectiveness of clopidogrel, the team were interested in understanding whether knowledge of genotype status would affect prescribing in a clinical setting. They randomized 504 participants to genotype guided or usual care groups and assessed the rate of prasugrel or ticagrelor prescribing in place of clopidogrel within each arm. As a secondary outcome, they assessed whether prescribers adhere to genotype guided recommendations. Of genotyped individuals, 28% carried loss of function alleles. Within the genotype guided group overall, there was higher use of prasugrel or ticagrelor with these being prescribed to 30% of patients compared with only 21% in the usual care group. Within genotype individuals carrying loss of function alleles, 53% were started on prasugrel or ticagrelor, demonstrating some adherence to genotype guided recommendations.                                                 However, this also meant that 47% of people whose genotype suggested reduced effectiveness were nevertheless prescribed clopidogrel. This study highlights that even when genotype information is available, interventional cardiologists consider clinical factors such as disease presentation and may weight these more highly than genotype information when selecting antiplatelet therapy following PCI.                                                 The next paper is about “Deep Mutational Scan of an SCN5A Voltage Sensor and comes to us from Andrew Glazer, Dan Roden and colleagues from Vanderbilt University Medical Center. In this paper, the team aim to characterize the functional consequences of variants and the S4 voltage sensor of domain IV and the SCN5A gene using a high throughput method that they developed. SCN5A encodes the major voltage gated sodium channel in the heart and variants in SCN5A can cause multiple distinct genetic arrhythmia syndromes, including Brugada syndrome, long QT syndrome, atrial fibrillation, and dilated cardiomyopathy, and have been linked to sudden cardiac death.                                                 Because of this, there's considerable interest in understanding the functional and clinical consequences of different variants, but previous approaches were time consuming and results were often inconclusive with many variants being classified as uncertain significance. This newly developed deep mutational scanning approach allows for simultaneous assessment of the function of thousands of variants, making it much more efficient than low throughput patch clamping. The team assessed the function of 248 variants using a triple drug assay in HEK293T cells expressing each variant and they identified 40 putative gain of function and 33 putative loss of function variants. They successfully validated eight of nine of these by patch clamping data. Their study highlights the effectiveness of this deep mutational scanning approach for investigating variants in the cardiac sodium channel SCN5A gene and suggests that this may also be an effective approach for investigating putative disease variants and other ion channels.                                                 The next article is a research letter from Connor Emdin, Amit Khera, and colleagues from Mass General Hospital in the Broad Institute entitled, “Genome-Wide Polygenic Score and Cardiovascular Outcomes with Evacetrapib in Patients with High-Risk Vascular Disease: A Nested Case-Control Study”. In this study, the team set out to probe the utility of using polygenic risk scores to predict the risk of major adverse cardiovascular events within individuals already known to be at high cardiovascular risk and to assess whether genetic scores can identify individuals who would benefit from the use of a CETP inhibitor such as Evacetrapib. They analyze data from the ACCELERATE trial which had tested Evacetrapib in a high risk population, and they found no effect on the incidents of major adverse cardiovascular events overall. Within a nested case-control sample of individuals experiencing major CVD events versus no events, they applied a polygenic risk score and found that the score predicted major cardiovascular events.                                                 Patients in the highest quintile of the risk score were at 60% higher risk of a major cardiovascular event than patients in the lowest quintile. There was no evidence of any interaction between the genetic risk score and Evacetrapib. These data suggest that genetic risk scores may have utility in identifying individuals at high risk events but may not have utility in identifying individuals who may derive more benefit from CETP inhibition. The next letter concerns “Epigenome-Wide Association Study Identifies a Novel DNA Methylation in Patients with Severe Aortic Valve Stenosis” and comes from Takahito Nasu, Mamoru Satoh, Makoto Sasaki and colleagues from Iwate Medical University in Japan. They were interested in understanding whether differences in DNA methylation could underlie the risk of aortic valve stenosis. They conducted an EWAS or epigenome-wide association study of peripheral blood mononuclear cells or PBMCs from 44 individuals with aortic stenosis and 44 disease free controls.                                                 They collected samples at baseline before a surgical intervention in the individuals with aortic stenosis and collected a follow-up sample one year later. They found that DNA methylation at a site on chromosome eight mapping to the TRIB1, or tribbles homolog one gene, was lower in the aortic stenosis group than in the controls at baseline. They replicated the association in an independent sample of 50 cases and 50 controls. TRIB1 MRNA levels were higher in the aortic stenosis group than the controls. When they looked at methylation status one year after aortic valve replacement or a transcatheter aortic valve implantation in patients with stenosis, they found that DNA methylation had increased in the cases while TRIB1 MRNA decreased. These data suggests that methylation status of TRIB1 and expression of TRIB1 may relate to the disease processes in aortic stenosis such as hemodynamic dysregulation and they can be reversed through surgical intervention. Changes in the methylation status of TRIB1 could be a novel biomarker of response to aortic valve replacement.                                                 The next letter comes from Niels Grote Beverborg, Pim van der Harst, and colleagues from University Medical Center Groningen and is entitled, “Genetically Determined High Levels of Iron Parameters Are Protective for Coronary Artery Disease”. Their study addresses the conflicting hypotheses that high iron status is either deleterious or protective against cardiovascular disease. The team constructed genetic predictors of serum iron status using 11 previously identified snips and tested the genetic association with CAD in UK Biobank data from over 408,000 white participants. Overall, the genetic score for higher iron status was associated with protection against CAD. Ten of the snips suggested individual neutral or protective effects of higher iron status on CAD, while one iron increasing snip was associated with increased risk of disease but this was thought to be likely through an iron independent mechanism. Overall, these data suggest that a genetic predisposition to higher iron status does not increase risk of CAD and is actually protective against disease.                                                 The final letter is entitled, “Confidence Weighting for Robust Automated Measurements of Popliteal Vessel Wall MRI” and comes from Daniel Hippe, Jenq-Neng Hwang, and colleagues from the University of Washington. They were interested in assessing whether images of popliteal artery wall incidentally obtained during knee MRI as part of an osteoarthritis study could be used to study the development and progression of atherosclerosis. They developed an automated deep learning based algorithm to segment and quantify the popliteal artery wall in images obtained over 10 years in over 4,700 individuals. Their approach, which they named FRAPPE, or fully automated and robust analysis technique for popliteal artery evaluation, was able to reduce the average time required for segmentation analysis from four hours to eight minutes per image. They applied weights based on confidence for each segment to automatically improve the accuracy of aggregate measurements such as mean wall thickness or mean lumen area. Their data suggest that this automated method can rapidly generate useful information on atherosclerosis from MRI images obtained as part of other studies. When combined with other data. This approach may facilitate novel discovery in secondary analyses of existing studies in an efficient and cost effective way.                                                 And that's all for issue one of 2020. Come back next time for more of the latest papers from Circulation: Genomic and Precision Medicine. Speaker 2:                           This podcast is copyright American Heart Association 2020.  

This podcast evaluates results from a phase II clinical trial of pembrolizumab for relapsed Mycosis Fungoides and Sezary Syndrome in the context of the current systemic treatment landscape for this disease. TRANSCRIPT This JCO Podcast provides observations and commentary on the JCO article "Pembrolizumab in Relapsed and Refractory Mycosis Fungoides and Sezary Syndrome: A Multicenter Phase II Study" by Khodadoust et al. My name is Jennifer Amengual, and I am an Assistant Professor of Medicine at the Columbia University Irving Medical Center in New York, New York, USA. My oncologic specialty is lymphoma. Mycosis fungoides, otherwise known as MF, and Sezary Syndrome, its leukemic variant, are rare subtypes of cutaneous T-cell lymphoma. Although most patients with MF have indolent disease, those with advanced stage MF often experience resistance to systemic therapy with a persistent and progressive disease course, which has a negative impact on overall well-being and survival. Patients may have intense pruritus, which can then put them at increased risk for staphylococcus infection, sepsis and in turn can trigger disease flare. Advanced-stage MF is a chronic disease that is not considered curable; therefore, it is necessary to consider management strategies that provide symptom relief and can be maintained over a long period of time. Most patients cycle through a multitude of therapies, often including skin-directed and, later, systemic therapies. Many of the acceptable therapies are derived from retrospective studies or single-arm trials; therefore, it is not possible to objectively compare outcomes and toxicity. In order to put the outcomes of pembrolizumab in context of other acceptable treatment modalities, I will discuss some of these treatments now. Systemic retinoids, such as bexarotene, are taken orally and can induce an overall response of 45-55%. The drug is well tolerated but can lead to hypertriglyceridemia and hypothyroidism, requiring monthly monitoring and frequent treatment with lipid-lowering agents and thyroid hormone replacement. Low-dose methotrexate, administered either orally or by intravenous infusion weekly, can lead to response rates between 30 and 50%. Common side-effects include mucositis, cytopenias and gastrointestinal upset. Pralatrexate is a novel antifolate drug that when studied using half the dose of that which is approved for PTCL , induced a 45% response rate for patients with MF. The treatment is also well tolerated, with the most common side-effects being mucositis and thrombocytopenia. There are 2 histone deacetylase inhibitors approved for use in MF and Sezary Syndrome. Both romidepsin and vorinostat have overlapping toxicity profiles, including gastrointestinal disturbances, fatigue, anorexia, and cytopenias. Romidepsin is also associated with ECG abnormalities. In patients with cutaneous T cell lymphoma, romidepsin has demonstrated a response rate of 34% and is associated with a median duration of response of 15 months. Vorinostat, an oral drug, has demonstrated a response rate of 30%. Recently, 2 biologics have been in used for patients with MF. Brentuximab vedotin is an antibody drug conjugate which targets the surface marker CD30 and brings monomethyl auristatin E into the cell. It is associated with a response rate of 70% in MF. Responses are significantly better in those with greater than 5% of CD30 expression. The most common side-effect is peripheral neuropathy. Mogamulizumab is a humanized antibody directed against the chemokine receptor CCR4.  This agent was studied in a randomized trial compared to vorinostat, where it led to a PFS of 7.7 months and response rate of 28%. The most common adverse events were attributed to infusion reactions, rash, diarrhea, and fatigue. There was also an increased risk of graft-versus-host disease in patients who subsequently went on to allogenic transplant, which needs to be considered when using this treatment strategy. As you can see, there are many different approaches to the treatment of advanced-stage MF and Sezary Syndrome, and the drugs I have discussed do not encompass an exhaustive list. Immune checkpoint antibodies against Programmed cell death protein 1 otherwise known as PD-1 interfere with inhibitory signaling pathways expressed on exhausted T-cells and wake up these cells allowing them to perform anti-tumor activities. The investigators rationalize the use of PD-1 inhibitors in MF and Sezary syndrome given PD-1 dysfunction and altered expression on MF cells and  the skin microenvironment. The Cancer Immunotherapy Trials Network performed a multicenter phase II single-arm study of pembrolizumab in 24 patients across 8 centers with relapsed or refractory MF or Sezary Syndrome. Of the 24 patients enrolled, 9 had MF and 15 had Sezary syndrome. Most had advanced disease represented by tumor stage disease, erythrodermic presentation, or were stage IIIB or higher. Patients received a median of 4 prior lines of systemic therapy. In addition, 4 patients had evidence of histologic large cell transformation. These represent some of the most challenging clinical situations. Pembrolizumab was administered every 3 weeks for 24 months or until withdrawal of consent, adverse reactions or investigator’s decision. The therapy was well tolerated, with no grade 4 or 5 immune-related adverse events. There were, however, 11 immune-related adverse events observed among 9 patients. This led to discontinuation of treatment in 4 patients, all due to grade 3 events including pneumonitis, duodenitis, hepatitis, and a corneal ulcer. Interestingly, over half of the patients with Sezary Syndrome experienced a flare of their symptoms early in their treatment course. This manifested as increased erythema, pruritus, and peripheral edema. Patients were managed with topical steroids and close observation. Only 1 patient had a prolonged reaction requiring systemic therapy, and no patients were discontinued from therapy due to flare of their disease. Patients with MF did not experience any skin toxicity. The authors investigated the relationship of this reaction to response and found that 3 of 8 Sezary patients who experienced a skin flare went on to achieve a response, whereas only 1 of the 7 Sezary patients who did not have a flare achieved a response. Using mass cytometry profiling of PBMCs, the authors observed a sevenfold increase in PD-1 expression on circulating Sezary cells in patients who experienced a flare. Although these observations were made across a small number of patients, it will be worthwhile to pay attention to this in future studies to determine if this is a true predictor for flare reaction or response. Pembrolizumab demonstrated significant clinical responses in 9 of 24 patients, leading to an overall response rate of 38%. This falls right into the range with other acceptable treatment options. Patients were heavily pretreated, yet the number of prior lines of therapy did not impact the response to pembrolizumab, possibly owing to non-overlapping mechanism of action. In addition, responses were seen in both MF and Sezary patients and those with erythrodermic and tumor stage presentations. There was a trend toward better outcomes in the MF patients. Responses were durable, and the median duration was not reached within a follow-up period of 58 weeks. The 1-year PFS was 65%, and OS was 95%. Two responding patients relapsed 8 to 12 weeks after discontinuation of treatment. The authors discuss that this may indicate the need for continuous treatment in this patient population, which is contrary to the general treatment strategy used for checkpoint inhibitors in other malignancies. Although this study only enrolled 24 patients, pembrolizumab appears to have similar outcomes as other drugs used in advanced-stage MF and Sezary Syndrome, with an acceptable toxicity profile. It produces a long duration of response, making it an attractive addition for patients suffering from this disease. Pembrolizumab does not have overlapping mechanism of action with most of the drugs used today and can therefore be easily integrated into the sequence of treatment for patients with MF and Sezary Syndrome. Finally, it is possible that pembrolizumab’s effects will enhance those seen with other agents. We should look forward to confirmatory clinical trials of pembrolizumab for the management of patients with MF or Sezary Syndrome. This concludes this JCO Podcast. Thank you for listening.

Background: The capacity of patient's Natural Killer cells (NKs) to be activated for cytolysis is an important prerequisite for the success of antibody-derived agents such as single-chain triplebodies (triplebodies) in cancer therapy. NKs recovered from AML patients at diagnosis are often found to be reduced in peripheral blood titers and cytolytic activity. Here, we had the unique opportunity to compare blood titers and cytolytic function of NKs from an AML patient with those of a healthy monozygotic twin. The sibling's NKs were compared with the patient's drawn either at diagnosis or in remission after chemotherapy. The cytolytic activities of NKs from these different sources for the patient's autologous AML blasts and other leukemic target cells in conjunction with triplebody SPM-2, targeting the surface antigens CD33 and CD123 on the AML cells, were compared. Methods: Patient NKs drawn at diagnosis were compared to NKs drawn in remission after chemotherapy and a sibling's NKs, all prepared from PBMCs by immunomagnetic beads (MACS). Redirected lysis (RDL) assays using SPM-2 and antibody-dependent cellular cytotoxicity (ADCC) assays using the therapeutic antibody Rituximab (TM) were performed with the enriched NKs. In addition, MACS-sorted NKs were analyzed for NK cell activating receptors (NCRs) by flow cytometry, and the release of TNF-alpha and IFN-gamma from blood samples of both siblings after the addition of the triplebody were measured in ELISA-assays. Results: Patient NKs isolated from peripheral blood drawn in remission produced comparable lysis as NKs from the healthy twin against the patient's autologous bone marrow (BM) blasts, mediated by SPM-2. The NCR receptor expression profiles on NKs from patient and twin were similar, but NK cell titers in peripheral blood were lower for samples drawn at diagnosis than in remission. Conclusions: Peripheral blood NK titers and ex vivo cytolytic activities mediated by triplebody SPM-2 were comparable for cells drawn from an AML patient in remission and a healthy twin. If these results can be generalized, then NKs from AML patients in remission are sufficient in numbers and cytolytic activity to make triplebodies promising new agents for the treatment of AML.

Blutgerinnung und Entzündung sind zwei stark miteinander verknüpfte Vorgänge im menschlichen Körper. Es ist gängige Meinung, dass während einer Sepsis die systemische Entzündung unweigerlich zu einer Aktivierung des Gerinnungssystems und einer gleichzeitigen Inhibition des blutgerinnungshemmenden Systems sowie der Fibrinolyse führt. Durch die massive Aktivierung der Gerinnung kommt es zu einer sogenannten Verbrauchskoagulopathie, bei der vor allem die antikoagulatorische Kapazität stark reduziert ist. So ist die Aktivität von ATIII und APC, zwei der wichtigsten körpereigenen Gerinnungsinhibitoren, während der Sepsis erheblich vermindert. Rekombinant hergestelltes APC hat als Xigris® seit 2002 die europäische Zulassung zur Behandlung der schweren Sepsis. In einer klinischen Studie der Phase 3 wurde eine Reduktion der 28-Tage Sterblichkeit durch die Gabe von APC bei Patienten mit schwerer Sepsis festgestellt. Im Gegensatz zu APC zeigte ATIII in einer Studie der Phase 3 an Patienten mit schwerer Sepsis keine Reduktion der 28-Tage Sterblichkeit. Es ist generell akzeptiert, dass das septische Mehrorganversagen durch die systemische Immunantwort des Organismus auf eine Infektion und die daraus resultierende überschießende systemische Freisetzung inflammatorischer Mediatoren vermittelt wird. Die Freisetzung dieser Mediatoren erfolgt unter anderem aus Monozyten. Durch den entzündlichen Stimulus während einer Verletzung oder Sepsis wird auf Monozyten neben der Freisetzung von Zytokinen auch TF induziert, der Rezeptor und Aktivator von FVII. Der Komplex aus TF und FVIIa stellt den Anfangspunkt der exogenen Gerinnung dar. Seit einigen Jahren wird rFVIIa als „rescue therapy“ zur Kontrolle schwerer traumatisch verursachter Hämorrhagien diskutiert. Dabei soll durch die Gabe von rFVIIa die Gerinnung spezifisch am Ort der Verletzung verstärkt werden. Da durch das Trauma und die damit verbundene Entzündungsreaktion Monozyten vermutlich TF exprimieren, stellen auch diese Zellen einen potentiellen Angriffspunkt für rFVIIa dar. Das Ziel der vorgelegten Arbeit war, mögliche gerinnungsunabhängige immunmodulatorische Eigenschaften dieser, in der Intensivmedizin verwendeten, körpereigenen Substanzen zu untersuchen. Im Speziellen sollte die Auswirkung der Pro- bzw. Antikoagulantien auf die LPS-induzierte Zytokinfreisetzung von Monozyten untersucht werden, ein wichtiger Bestandteil in der Entstehung von Sepsis und MODS. Dazu wurde ein Versuchsmodell mit einer humanen, monozytären Zelllinie, MonoMac6, etabliert. Die Produktion von IL-1β, IL-8 und TNFα wurde intrazellulär mit Hilfe der Durchflusszytometrie bestimmt. Im Zellkulturüberstand wurden die Konzentrationen von IL-1β, IL-8, IL-10 und TNFα mit einem Luminex-100 System gemessen. Zusätzlich wurde für rFVIIa der Einfluss auf die LPS-induzierte IL-1β-, IL-6-, IL-8- und TNFα-Synthese von CD14+ Monozyten in PBMCs durchflusszytometrisch erfasst. In der vorgelegten Arbeit konnte eine limitierende Wirkung von APC und ATIII auf die LPS-induzierte Zytokinproduktion von Monozyten festgestellt werden. APC verminderte die IL-1β-, IL-10- und TNFα-Ausschüttung signifikant, wobei Überstandsmessungen die eindeutigsten Ergebnisse zeigten. Der Effekt von ATIII ließ sich durchflusszytometrisch besser bestimmen, als aus dem Überstand. Es zeigte sich eine signifikant verminderte LPS-induzierte IL-1β- und TNFα-Produktion der Monozyten. Zusätzlich wurde der Effekt von Heparin auf die ATIII-Wirkung untersucht, da es mehrere Hinweise auf eine negative Beeinflussung der ATIII-Therapie in der Sepsis durch gleichzeitige Gabe von Heparin gibt. In der vorgelegten Arbeit konnte interessanter Weise kein antagonisierender Effekt von Heparin auf die immunologische Wirkung von ATIII festgestellt werden. Neben dem positiven Effekt durch die Wiederherstellung der Antikoagulation während der Sepsis, kann APC auch zur Wiederherstellung des Gleichgewichts zwischen Pro- und Antiinflammation beitragen. APC vermindert sowohl die Synthese pro-, als auch die antiinflammatorisch wirkender Mediatoren, was eine Erklärung für die Wirksamkeit bei dem sehr heterogenen Kollektiv der Sepsispatienten sein könnte. ATIII reduziert nach unseren Ergebnissen lediglich die Synthese der proinflammatorischen Zytokine TNFα und IL-1β, nicht aber die von IL-10. Geht man davon aus, dass APC deshalb wirksam ist, weil es sowohl eine überschießende Pro- als auch Antiinflammation dämpfen kann, wäre das eine Erklärung für das Fehlen des klinischen Wirksamkeitsnachweises für ATIII bei Patienten mit schwerer Sepsis. Für rFVIIa ergab sich ein messbarer, aber nicht eindeutig charakterisierbarer immunmodulatorischer Effekt auf die LPS-induzierte Zytokinproduktion von Monozyten. Bei MonoMac6 Zellen zeigte sich ein leicht begrenzender Effekt auf die IL-8- und TNFα-Produktion. Die Behandlung von PBMCs mit rFVIIa ergab keine veränderte LPS-induzierte Zytokinfreisetzung von CD14+ Monozyten. In verschiedenen Versuchsabläufen wurden MonoMac6 Zellen zur Erhöhung der TF-Expression vor der Inkubation mit rFVIIa mit TNFα oder LPS behandelt. Dabei zeigte sich je nach Messmethode und Vorbehandlung außer einer geringen Steigerung der TNFα-Synthese LPS-vorbehandelter Monozyten kein Effekt. Zusätzlich wurde eine Transfektion der MonoMac6 Zellen mit TF durchgeführt. In Versuchen mit dieser Zelllinie ergab sich eine erhöhte TNFα-Synthese unter rFVIIa-Einfluss. Dieses Ergebnis ist jedoch mit Vorsicht zu betrachten, da die Reaktion der transfizierten Zellen auf LPS alleine im Kontrollexperiment nicht der des Wildtyps entsprach. Eine rFVIIa-induzierte Bildung von Thrombin kann ebenfalls in die Immunreaktion eingreifen, dies illustriert die in der vorgelegten Arbeit unter Thrombineinfluss gemessene, stark verminderte LPS-induzierte IL-10 Ausschüttung. Allerdings konnte der direkte immunmodulatorische Effekt von rFVIIa weder mit der Komplexbildung aus TF und rFVIIa noch mit der von rFVIIa vermittelten Produktion von Thrombin in Verbindung gebracht werden. Die immunmodulatorische Wirkung von rFVIIa auf die LPS-induzierte Zytokinproduktion von Monozyten konnte mit den in der vorgelegten Arbeit verwendeten Modellen nicht abschließend geklärt werden und lässt noch viel Raum für weitere Untersuchungen. Transfektionsversuche mit anderen monozytären Zelllinien oder die Selektion von TF-exprimierenden Immunzellen aus kritisch kranken Patienten wären mögliche Versuchsansätze für die Zukunft. Auch ein geeigneter Stimulus zur Induktion von TF auf isolierten humanen Monozyten, der nicht gleichzeitig die Zytokinsynthese anregt, würde vielversprechende Möglichkeiten bieten.

EBV ist ein Herpesvirus, welches in über 90% aller Erwachsenen nachgewiesen werden kann und in den wenigsten Fällen Beschwerden verursacht. Unter bestimmten Umständen kann es aber zur Ausbildung einer Infektiösen Mononukleosekommen und auch an der Entstehung einer Reihe von Krebserkrankungen, allenvoran die PTLD (posttransplant lymphoproliferative disease), das Burkitt- und das Hodgkin-Lymphom, ist EBV ursächlich beteiligt. Trotz zahlreicher Bemühungen und einiger vielversprechender Ansätze ist bis heute kein wirksamer Impfstoff gegen das Epstein-Barr Virus vorhanden. Im Bereich Exosomen als Mittel zur Induktion von Immunantworten wird seit gut 10 Jahren geforscht und ihre Wirksamkeit konnte bereits in klinischen Studien zur Behandlung mehrerer Krebsarten getestet werden. In dieser Doktorarbeit wurden HEK 293-Zellen und ein auf diesen Zellen basierendes Verpackungssystem für virale Vektoren auf ihre Eignung hin untersucht, rekombinante Exosomen und Virus-like-Particles (VLPs) zu produzieren, welche eventuell als DNA-freies Vakzin gegen EBV eingesetzt werden könnten. In EBV-positiven Verpackungszelllinien konnte durch Induktion des lytischen EBV-Zyklus die Freisetzung DNA-freier VLPs erreicht werden. Genau wie Exosomen aus 293-Zellen, die zuvor mit Expressionsplasmiden für EBV-Antigene transfiziert worden waren, konnten sie aus dem Zellkulturmedium aufgereinigt werden. Ihr großes immunogenes Potential zeigte sich bei der Reaktivierung von EBV-spezifischen TZellklonen und Gedächtnis-T-Zellen aus PBMCs, wo bereits geringe Mengen für eine Stimulation ausreichten. Zu der hohen Effizienz der Partikel trug ihr Tropismus bei, der auf virale Glykoproteine, vor allem gp350, zurückzuführen war. Die Partikel besaßen dadurch eine Affinität zu B-Zellen, über die effizient die Präsentation der Exosomen und Virus-like-Particles erfolgte. Auch in in vivo-Versuchen, bei denen mit dem hu-PBMC-Rag-Mausmodell und dem MHV-68-Mausmodell gearbeitet wurde, konnten durch Immunisierung mit Exosomen bzw. Virus-like-Particles virusspezifische humorale wie zelluläre Imunantworten ausgelöst werden. In Stimulationsexperimenten von malignen Zellen aus Patienten mit chronisch lymphatischer B-Zellleukämie (B-CLL) konnte ich weiterhin zeigen, dass Exosomen und VLPs auch als Überträger funktioneller Moleküle wie den CD40L, einem Mitglied der Tumor-Nekrose-Faktor-Familie, fungieren können. Dieser bewirkte in den sonst nicht immunogenen und deshalb vom Immunsystem nicht erkannten CLL-Zellen eine verstärkte Expression kostimulatorischer, Adhäsions- und apoptoseassoziierter Moleküle. Auf diese Weise war es möglich, autologe Tumor- und EBV-spezifische T-Zellen zu reaktivieren. Exosomen und Virus-like-Particles könnten deshalb bei der Behandlung der B-CLL eine vielversprechende Alternative zur Gentherapie darstellen.

Background/Aims: Inflammation plays an important role in atherosclerosis and stroke. Acute infections are recognized as trigger factors for ischemic stroke. Methods: In this whole genome expression profile study of 15 patients and 15 control subjects, we tested the hypothesis that patients with a history of atherothrombotic stroke show enhanced transcription of inflammatory genes in circulating leukocytes. RNA from unstimulated or lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) was analyzed with Affymetrix U133A GeneChips using a pooling design. Expression of single genes and functional groups of genes was analyzed by global statistical tests. Results: A total of 10,197 probe sets showed positive calls. After correction for multiple testing no single probe set revealed significant differences either without or with LPS stimulation. However, significant global expression differences were found upon LPS stimulation for the group of genes that are involved in cell-cell signaling. Conclusion: LPS stimulation of PBMCs, a condition mimicking bacterial infection, induces differential expression of a group of cell-cell signaling genes in patients with previous atherothrombotic stroke. This finding can be caused by genetic differences between both groups, but acquired risk factors, medication and technical factors may also have contributed to the result. Copyright (C) 2009 S. Karger AG, Basel

Objectives: To study the clinical outcome, treatment response, T-cell subsets and functional consequences of a novel tumour necrosis factor (TNF) receptor type 1 (TNFRSF1A) mutation affecting the receptor cleavage site. Methods: Patients with symptoms suggestive of tumour necrosis factor receptor-associated periodic syndrome (TRAPS) and 22 healthy controls (HC) were screened for mutations in the TNFRSF1A gene. Soluble TNFRSF1A and inflammatory cytokines were measured by ELISAs. TNFRSF1A shedding was examined by stimulation of peripheral blood mononuclear cells (PBMCs) with phorbol 12-myristate 13-acetate followed by flow cytometric analysis (FACS). Apoptosis of PBMCs was studied by stimulation with TNFa in the presence of cycloheximide and annexin V staining. T cell phenotypes were monitored by FACS. Results: TNFRSF1A sequencing disclosed a novel V173D/ p.Val202Asp substitution encoded by exon 6 in one family, the c.194–14G.A splice variant in another and the R92Q/p.Arg121Gln substitution in two families. Cardiovascular complications (lethal heart attack and peripheral arterial thrombosis) developed in two V173D patients. Subsequent etanercept treatment of the V173D carriers was highly effective over an 18-month follow-up period. Serum TNFRSF1A levels did not differ between TRAPS patients and HC, while TNFRSF1A cleavage from monocytes was significantly reduced in V173D and R92Q patients. TNFa-induced apoptosis of PBMCs and T-cell senescence were comparable between V173D patients and HC. Conclusions: The TNFRSF1A V173D cleavage site mutation may be associated with an increased risk for cardiovascular complications and shows a strong response to etanercept. T-cell senescence does not seem to have a pathogenetic role in affected patients.

Ziel der vorliegenden Arbeit war die Charakterisierung von Antikörpern und ihrer immunsuppressiven Wirkung im präklinischen Hundemodell. Dazu wurde eine T-Zelldepletionsmethode mit dem caninen CD6-Antikörper MT-606 und Kaninchen-komplement zur alleinigen GvHD-Prophylaxe bei der allogenen Knochenmark-transplantation entwickelt. Zusätzlich sollten die regulatorischen Eigenschaften von CD6-depletierten Knochenmarkzellen in vitro in der gemischten Lymphozytenkultur untersucht werden. Mit Hilfe des kreuzreagierenden Antikörpers MT-606, der das canine CD6-Antigen erkennt, konnte eine einfache und kostengünstige Depletionsmethode etabliert werden, welche die Fähigkeit des CD6-Antikörpers MT-606 zur Aktivierung der Komplement-kaskade ausnützt. Es wurde zunächst durch in vitro-Experimente gezeigt, dass die Methode mit MT-606 und Kaninchenkomplement eine effektive Depletion der CD6-positiven Zellen gewährleistet, wohin gegen die hämatopoetischen Stammzellen unangetastet bleiben. Die CD6-Depletion geht einher mit einer nahezu vollständigen Depletion der CD4-positiven Zellen, während die CD8-positiven Zellen nur teilweise depletiert werden. Durch drei Knochenmarktransplantationen im homo-hetero-System und zwei im DLA-identischen System wurde gezeigt, dass sich die CD6-Depletion als alleinige Methode zur GvHD-Prophylaxe eignet. Die Etablierung einer Toleranz gegenüber dem Spenderorganismus wurde durch eine Hauttransplantation vom Spender auf den Empfänger bei einem Hund gezeigt. Regelmäßige Analysen des Spenderchimärismus konnten zeigen, dass die Transplantation von CD6-depletiertem Knochenmark eine langfristige Erholung des hämatopoetischen Systems garantiert. In mehreren in vitro-Experimenten sollte die regulatorische Fähigkeit von CD6-depletierten Knochenmarkzellen in der gemischten Lymphozytenkultur (MLC) untersucht werden. Die MLC ist eine Kurzzeit-Suspensionskultur, die es erlaubt, die Reaktivität von T-Zellen eines Spenders gegen die durch Bestrahlung inaktivierten PBMCs eines anderen Spenders zu untersuchen. Die Lymphozytenproliferation oder ihre eventuelle Inhibition können durch den Einbau von radioaktiv markiertem Thymidin oder durch die Weitergabe des Farbstoffs CFSE von der Mutter- auf die Tochterzellen quantifiziert werden. Trotz Anwendung unterschiedlicher Analysesysteme konnte kein supprimierender Effekt von CD6-depletierten Knochenmarkzellen auf die Lymphozytenproliferation in der gemischten Lymphozytenkultur nachgewiesen werden. In einigen Fällen wirkten die CD6-depletierten Knochenmarkzellen sogar stimulierend auf die Proliferation der Lymphozyten. Um die generelle Anwendbarkeit des verwendeten MLC-Systems zum Nachweis von supprimierenden Eigenschaften einer Zellpopulation zu zeigen, wurden mesenchymale Stammzellen an Stelle von CD6-depletierten Knochenmarkzellen eingesetzt. Diese bewiesen eine hohe Potenz zur Unterdrückung der Lymphozytenproliferation in der MLC. Trotz offensichtlicher Wirkung bei der GvHD-Prophylaxe konnte ein regulatorischer Effekt von CD6-depletierten Knochenmarkzellen in der MLC nicht gezeigt werden, obwohl das System durchaus zum Nachweis von supprimierenden Eigenschaften anderer Zellen geeignet ist. In einer zweiten Reihe von Transplantationen wurde untersucht, ob durch zusätzliche Transfusion von Spenderlymphozyten (DLT) nach der Transplantation von CD6-depletiertem Knochenmark im Empfänger eine GvHD induziert werden kann. Der Zeitpunkt der DLT variierte bei den einzelnen Transplantationen, es wurde aber immer eine Zelldosis von ca. 1 ∙ 108 MNCs/kg Körpergewicht transfundiert. Bei je einem Hund konnte durch DLT an den Tagen 3, 14 oder 20 eine GvHD induziert werden, deren Hauptmanifestationsort bei zwei Hunden die Leber und bei einem Hund die Haut war. Bei einer Wiederholung des Transplantationsschemas mit DLT an Tag 20 konnte bei einem weiteren Hund keine GvHD induziert werden. Offensichtlich bestand zu diesem Zeitpunkt bereits eine Toleranz gegenüber dem Spenderorganismus. Das etablierte System mit Transplantation von CD6-depletiertem Knochenmark und DLT vor Tag 20 zur Induktion einer GvHD eignet sich hervorragend, um die Einsetzbarkeit von HSV-TK-transduzierten Zellen zur Gentherapie zu testen. Dies wird Gegenstand zukünftiger Untersuchungen sein.

Hintergrund und Ziele der Arbeit: Bakterien und DNA Viren werden anhand unmethylierter CpG-Motive innerhalb ihrer DNA von den TLR 9 tragenden PDCs und den B Zellen des humanen Immunsystems als Gefahrensignale erkannt. Mittels synthetischer, CpG-enthaltender ODN nutzt man diese Grundsatzmechanismen, um vergleichbare Immunantworten auszulösen. Auf Grundlage eines unterschiedlichen immunologischen Aktivierungsprofils wurden bislang drei CpG-Klassen definiert: CpG-A, CpG-B und CpG-C. Mit Hilfe von CpG-A war es erstmals möglich, IFN-α in PDCs (den endogenen Hauptproduzenten dieses Zytokins) in Mengen zu induzieren, wie es bislang nur mit Viren selbst möglich war. Auch CpG-C stimuliert PDCs zur Sekretion von IFN α und aktiviert darüber hinaus B Zellen - eine Eigenschaft, die CpG-A nicht besitzt. Die sequenzspezifischen und strukturellen Voraussetzungen für diese differenziellen Wirkprofile waren bislang unzureichend verstanden, auch weil die Struktur-Analysen nur begrenzt auf die tatsächlichen Vorgänge im physiologischen Milieu übertragbar waren. Um CpG-ODN für die therapeutische Anwendung zu optimieren, sind die genauen Kenntnisse der Struktur-Wirkungsbeziehungen jedoch unverzichtbar. Ein zweiter Ansatzpunkt zur Optimierung der Anwendung liegt in der Verbesserung der systemischen Stabilität von CpG-ODN. Die Bindung von CpG-ODN an partikuläre Trägersysteme (z.B. Gelatine-Nanopartikel) wurde bereits in unserer Abteiliung als mögliches drug-delivery-System etabliert. Eine Weiterentwicklung dieses Prinzips wären partikuläre Strukturen, die aus immunstimulatorischen Nukleinsäuren aufgebaut keiner weiteren Trägermaterialien bedürfen. Beide Ansatzpunkte führen zu den Zielen dieser Arbeit: 1) Die Aufklärung der Struktur-Wirkungsbeziehungen der CpG-Klassen A und C durch Etablierung geeigneter Methoden zur Untersuchung im physiologischen Milieu. 2) Die Entwicklung immunstimulatorischer partikulärer Strukturen auf Basis der in Teil 1) identifizierten wirksamen Strukturelemente beider CpG-Klassen. Ergebnisse: 1) Struktur-Wirkungsbeziehungen von ODN 2216 (CpG-A) und ODN M362 (CpG-C): CpG-A bildet im physiologischen Milieu spontan multimolekulare Strukturen, deren mittlere Durchmesser mit 24 40 nm im Größenbereich von Viren liegen. Es zeigte sich, dass für diese Multimerisierungen das Zusammenspiel aus flankierenden Poly-G-Motiven, palindromischem Zentrum und eingelagerten Natrium- oder Kaliumionen entscheidend ist. Physiologisches Milieu wirkt sich sowohl den Umgebungs-pH und die Na+/K+-Konzentrationen als auch die Temperatur (37 °C) betreffend optimal förderlich auf die Strukturbildung aus. Die Identifizierung dieser maßgeblichen Faktoren machte es möglich, den Strukturaufbau von CpG-A experimentell zu kontrollieren und die immunologischen Wirkungen der verschiedenen Strukturen direkt zu vergleichen. Für die rasche und hohe Induktion von IFN-α und anderen inflammatorischen Zytokinen durch PDCs sind große Partikel verantwortlich. Die Multimerisierungen von ODN 2216 werden bei pH < 6 zunehmend aufgehoben. Unterbindet man die Multimerisierungen durch Präinkubation der ODN bei Temperaturen > 60 °C oder durch Entzug der stabilisierenden Natriumionen (indem man sie zuvor in Aqua ad inj. löst), so verliert ODN 2216 seine immunstimulatorische Aktivität in Bezug auf PDCs. Die schwache Wirkung der CpG-A-Monomere kann jedoch durch Präinkubation von PDCs mit IFN β deutlich gesteigert werden. Im Gegensatz zu den ebenfalls einzelsträngig vorliegenden ODN 2006 (CpG-B) haben auch Monomere von ODN 2216 keine aktivierende Wirkung auf B Zellen. CpG-C hat durch die palindromische Sequenz die Möglichkeit, Hairpins und Duplices zu bilden. ODN M362 zeigt jedoch keine Hairpinstrukturen. Die Duplexformationen sind bei 37 °C in vitro nicht stabil und spielen keine Rolle bei der durch diese ODN initiierten B-Zell-Aktivierung. Duplices haben jedoch Anteil an der Induktion von IFN-α in PDCs. Die in dieser Arbeit etablierten Protokolle der Temperatur-Präinkubation ermöglichen erstmalig eine experimentelle Kontrolle der Strukturbildungen von CpG-A und CpG-C und dadurch den Vergleich von Struktur und Wirkung. Das Standardprotokoll für Gelelektrophorese wurde dahingehend modifiziert, dass ein physiologisches Milieu sowohl durch die anwesenden Ionen als auch durch die Umgebungstemperatur (37°C) simuliert werden konnte. 2)Design Nukleinsäure-basierter Nanopartikel: Zentrale Elemente von CpG-A und CpG-C (palindromische Sequenz, gerüstartige Verbindung mehrerer Nukleinsäuren) wurden eingesetzt, indem ODN M362-Sequenzen (CpG-C) an bi- und trivalenten Grundgerüsten (Linkern) für den Strukturaufbau optimiert wurden. Trivalente Linker ermöglichen die variierende Zusammenlagerung der palindromischen Nukleinsäuren in drei Richtungen des Raumes und dadurch die Bildung großer Partikel. Diese sind den bisher bekannten Maximalstimuli CpG-B und CpG-C hinsichtlich der Aktivierung von B-Zellen gleichwertig. Erstmalig konnten auf diese Weise B-Zellen durch partikuläre Strukturen stark aktiviert werden. Nach Vor-Komplexierung der Partikel mit Poly-L-Arginin wird die Aktivität bei B-Zellen nochmals verstärkt. Kurze, nicht-palindromische CpG-DNA-Sequenzen an trivalenten Grundgerüsten induzieren nach Vor-Komplexierung mit Poly-L-Arginin deutlich mehr IFN-α in PBMCs als CpG-A, obwohl sie selbst nicht multimerisieren. Wird die (palindromische) RNA-Sequenz von CpG-C an einem trivalenten Linker verwendet, so können ebenfalls große Strukturen generiert werden, die nach Transfektion vergleichbare Mengen IFN-α in PBMCs induzieren wie CpG-A. Ausblick: Die vorliegende Dissertation verbindet Fragestellungen der Immunologie und der pharmazeutischen Technologie mit den Möglichkeiten der Biochemie. Es werden nicht nur verschiedene Methoden zur strukturellen Untersuchung von CpG-ODN im physiologischen Milieu etabliert, sondern auch die experimentelle Kontrolle der Strukturbildung von CpG-A ermöglicht. Die entwickelte Technik der Generierung dreidimensionaler, über palindromische Nukleinsäuren aufgebauter Partikel ist nicht auf CpG-Motive in DNA begrenzt, sondern kann auf eine andere für Viren charakteristische Nukleinsäure (Einzelstrang-RNA) übertragen werden. Dadurch würde zusätzlich möglich, die immunologischen Profile von ssRNA, dsRNA und CpG in einem Partikel zu kombinieren und die Art der Immunantwort je nach Zusammensetzung der Partikel gezielt zu bestimmen. Die klinische Relevanz dieser Arbeit ergibt sich aus den neuen Erkenntnissen über die Multimerisierungen von CpG-A, welche dessen therapeutischen Einsatz optimieren und besser standardisierbar machen sollen. Außerdem werden neue Hinweise auf die unterschiedlichen Aufnahme- und Erkennungsmechanismen beider CpG-Klassen und deren Aktivierung der Synthese von IFN-α gewonnen. Darüber hinaus wurde durch die Entwicklung der Polyvalenten Linker eine grundsätzlich neue Technik im Stil eines Baukastensystems etabliert, welche als Grundstein einer neuen Generation von immunstimulatorischen Multimeren dienen soll. Die Koadministration von Adjuvans und Antigen in direkter räumlicher Nähe bietet neue Gestaltungsmöglichkeiten in der Vakzineentwicklung. Zudem ist zu erwarten, dass unter Einbeziehung der RNA basierten immunologischen Wirkprofile innerhalb eines Partikels der Einsatz von CpG-ODN zur Therapie von Virusinfektionen und Tumoren weiter verbessert werden kann.

Das Auftreten von Resistenzen ist eines der Hauptprobleme der heute angewandten antiretroviralen Therapie. Resistenzen können diagnostisch mit dem sog. genotypischen oder dem phänotypischen Verfahren festgestellt werden. Die genotypische Diagnostik beruht auf der Nukleotid-Sequenzierung der viralen Target-Gene Reverse Transkriptase (RT) oder Protease (PR), die relativ einfach zu bewerkstelligen ist. Ein phänotypischer Resistenztest und damit direkter Aktivitätstest der Enzyme RT und PR ist sehr viel präziser. Dies ist z.B. beim Vorliegen komplexer Resistenzmuster oder einem Mangel an genotypischer Information von resistenzrelevanten Mutationen bei einem neu verwendeten antiviralen Medikament von großer Bedeutung. Die zu testenden Enzyme werden entweder aus DNA, isoliert aus PBMCs aus Patientenvollblut oder Plasma-RNA rekombinant hergestellt. Vor kurzem wurde nun die Frage erhoben, ob die diagnostisch gemessenen Werte Unterschiede aufweisen, je nachdem ob DNA oder RNA als Ausgangsmaterial verwendet wurde. Die Veröffentlichungen zu diesem Thema waren diskreptant in ihren Aussagen. Zur Klärung dieser Frage wurde in der vorliegenden Arbeit das phänotypische Resistenzverhalten der HIV-Protease von 12 Patienten gegenüber 5 verschiedenen Proteaseinhibitoren untersucht. Aus jedem der Patienten wurde ein "Protease-Paar" sowohl aus HIV-DNA als auch HIV-RNA (via cDNA) rekombinant produziert. Die Patienten wiesen eine unterschiedliche Höhe der Viruslast auf, sie lag zwischen 1.900 cp/ml und 230.000 cp/ml, und hatten unterschiedlich hohe CD4-Zellzahlen. Mit dieser Untersuchung sollte also erstens die Frage beantwortet werden, ob zu einem gegebenen Zeitpunkt Abweichungen im Resistenzprofil zwischen den beiden Formen genetischer Information von HIV im Körper vorliegen können. Im positiven Fall sollte festgestellt werden, welchen Einfluss die Virusvermehrung/Virusdynamik und der Immunstatus des betroffenen Patienten, ausgedrückt durch die Laborparameter Viruslast und CD4-Zellzahl, darauf haben. Zweitens sollte dasjenige Probenmaterial gefunden werden, welches für den phänotypischen Resistenztest das optimale Resultat ergibt: entweder HIV-DNA aus PBMCs im Vollblut oder HIV-RNA aus Blutplasma. Der hier verwendete phänotypische Resistenztest (PRT) basiert auf der direkten Messung der HIV-Proteaseaktivität, die durch rekombinante Expression der gesamten Population von HIV-Protease eines Patienten hergestellt wurde. Für die vorliegende Arbeit wurde parallel sowohl HIV-DNA aus PBMCs im Vollblut als auch cDNA aus viraler RNA im Blutplasma des Patienten als Ausgangsmaterial für die nested PCR verwendet. Nach erfolgter Expression des Enzyms in E. coli und effektiver Ein-Schritt-Aufreinigung wurde die Proteaseaktivität mit einem neuen und schnellen phänotypischen Testsystem mittels eines Fluoreszenzsubstrates in Ab- und Anwesenheit der verschiedenen Inhibitoren gemessen. Durch den Vergleich mit Wildtyp-Werten konnten die entsprechenden Resistenzfaktoren berechnet werden. Ergebnis: Von den 12 untersuchten Patienten zeigten sich in 3 "DNA/RNA-Paaren" signifikante Unterschiede in mindestens einem Proteaseinhibitor, während bei 9 Patienten übereinstimmende Resistenzmuster gefunden wurden. Schlussfolgernd wird festgestellt, dass die Verwendung von entweder DNA oder RNA als Ausgangssubstanz für den benutzten Resistenztest unterschiedliche Ergebnisse im Resistenzmuster erbringen kann, dies aber nicht notwendigerweise der Fall ist. Zwischen dem Auftreten bzw. Fehlen von unterschiedlichen Resistenzmustern und den virologischen und immunologischen Parametern Viruslast und Anzahl der CD4 positiven Zellen konnte keine klare Korrelation festgestellt werden. Ein Auftreten von Differenzen zwischen den Resistenzmustern von DNA und RNA kann die Folge eines Populationswechsels der HIV-RNA sein, die im Gegensatz zur archivarischen Natur der meist integrierten DNA aus ruhenden CD4 positiven T-Gedächtniszellen oder Monozyten erst kürzlich von aktiv infizierten Zellen produziert wurde. Ein Fehlen von Unterschieden kann bedeuten, dass die Hauptfraktion der DNA entweder erst kürzlich generiert wurde oder die RNA sich nicht verändert hat. Für die diagnostische Anwendung des PRT haben diese Ergebnisse folgende Konsequenzen: um die aktuelle Resistenzsituation abzubilden, ist virale RNA aus dem Blutplasma das bevorzugte Ausgangsmaterial. Um dagegen stille Mutationen aufzudecken, die z.B. für zukünftige Therapien oder eine adäquate Postexpositionsprophylaxe von Bedeutung sind, sollte virale DNA, stammend aus PBMCs des Vollblutes, verwendet werden.

EBV is a γ-herpes virus which is able to infect human resting B-cells and to transform them into permanently growing lymphoblastoid cell lines (LCLs). EBNA2 (Epstein-Barr virus nuclear antigen 2) is one of the first viral proteins expressed after in vitro infection and interacts with different cellular proteins like RBP-Jκ and PU.1. The EBNA2 protein acts as a transcriptional activator of the viral Latent Membrane Proteins 1 and 2 (LMP1 and LMP2) and the viral nuclear genes EBNA1, EBNA3A, -3B, -3C, EBNA-LP. Additionally EBNA2 is also able to transactivate cellular genes like CD21, CD23 or c-myc. To study the different EBNA2 target genes and the function of EBNA2 a LCL was established (ER/EB2-5 cells, Kempkes et al., 1995) harboring an estrogen-inducible EBNA2. In the presence of estrogen the ER/EBNA2 fusion protein (estrogen receptor binding domain) is located in the nucleus were EBNA2 can transactivate its target genes, whereas in the absence of estrogen the ER/EBNA2 fusion protein is kept in the cytoplasm and therefore inactive. The cells proliferate in the presence of estrogen and they arrest in the absence resulting in a phenotype similar to resting B-lymphocytes. By using the ER/EB2-5 cell line I could clearly show that the cell surface molecule CD83, belonging to the immunoglobuline superfamily (Zhou et al., 1992), is upregulated after the activation of EBNA2. By using a derivative ER/EB2-5 cell line that constitutively expressed LMP1 I could show that CD83 is still expressed even in the absence of functional EBNA2 suggesting that LMP1, the viral target gene of EBNA2, is responsible for the induction of CD83. Therefore I analysed the activation of the CD83 promoter by LMP1. LMP1 is a transmembrane protein with a short intracellular N-terminus, 6 hydrophobic transmembrane domains and a long intracellular C-terminus, containing C-terminal activator regions CTAR1, 2 and 3. The different CTAR regions are responsible for activating genes via NF-κB, ATF, AP1 and STAT signaling pathways. For the activation of its target genes LMP1 uses the same signaling molecules (TRAF, TRADD) as family members of the TNF-R family (CD40, TNF-R1, TNF-R2). The CD83 promoter was activated by LMP1 as shown by promoter luciferase reporter assays in 293-T cells. The induction was not observed in the absence of a NF-κB binding site in a CD83 promoter mutant. Furthermore LMP1 mutants which are mutated in the binding regions for TRAF2 (CTAR1) or TRADD (CTAR2) are not able to transactivate the CD83 promoter. By co-transfection of LMP1 and dominant/negative IκB the CD83 promoter could not be activated because of inactivation of NF-κB. These experiments clearly demonstrate that the CD83 promoter is transactivated by LMP1 via NF-κB. Additionally to the regulation of CD83 I was also interested in the functional role of CD83. Until now only little is known about the function of CD83. CD83 seems to have a specific role in the decision to single positive CD4+ T-cells in the thymus (Fujimoto et al., 2002). I have tested a possible co-stimulatory function of CD83 to CD4+ T-cells by retroviral expression of CD83 in non-professional antigen presenting cells (RCC). Indeed CD83 expression increased the CD4+ response in comparison to CD80 or GFP retroviral infected RCC cells. In mixed lymphocyte reactions this co-stimulatory effect could not be clearly demonstrated although a soluble CD83-Ig showed a small inhibitory influence. The identification of a CD83 ligand molecule could give new insights into the function of CD83. Therefore a CD83-Ig fusion protein as well as a CD83-tetramer construct were generated and used to screen for a potential ligand of CD83. First results showed that the CD83-Ig fusion protein and the CD83-tetramer construct bound to CD4+ and to CD8+ T-cells of isolated PBMCs as well as to activated T-cells in a culture of mixed T-cell populations.

Die vorliegende Arbeit stellt eine Charakterisierung der im Jahr 1995 von Wissenschaftlern der Firma Abbott Diagnostics (North Chicago, USA) entdeckten flaviähnlichen, im Tamarinen vorkommenden GB-Viren, GBV-A und GBV-B, sowie des humanen GB-Virus-C dar. GBV-A und GBV-B sind beide im sogenannten “GB-Agens H205”, das als Referenzmaterial dient, vorhanden. GBV-A und GBV-B wurden isoliert und charakterisiert. Da GBV-A im Gehirngewebe in höheren Konzentrationen als GBV-B vorhanden war, gelang die Isolierung von GBV-A durch eine Tamarinpassage mit verdünntem inokuliertem Gehirnmaterial. GBV-B wurde durch Tamarinpassage des Serums eines Affen, der nach Ausheilen der GBV-A-Infektion noch GBV-B-RNA positiv war, in reiner Form gewonnen. In Re- und Kreuzinfektionsstudien mit Tamarinen konnte eine homologe Immunität gegen den gleichen Erreger, aber das Fehlen einer Kreuzimmunität gegen den anderen Erreger gezeigt werden. Die Untersuchung von histologischen Schnitten der Organe infizierter Krallenaffen mit Hilfe der in situ-Hybridisierung gab Aufschluß über den Ort der Replikation dieser Viren. GBV-B ist der Erreger einer Hepatitis bei Krallenaffen und war mit einer für dieses Virus spezifischen Sonde durch Hybridisierung im Zytoplasma von Hepatozyten nachzuweisen. Mit einer für GBV-A spezifischen Sonde wurde dieses Virus nur in Gehirngewebe nachgewiesen. Für das humane GBV-C wurde der Lymphknoten, genauer das Zytoplasma der Lymphozyten, als Replikationsort ermittelt. Eine Leberpathogenität dieses Virus konnte nicht gefunden werden. Klinische Studien könnten eine Assoziation von GBV-C mit Erkrankungen des lymphatischen Systems untersuchen. Entsprechend der Lokalisation des Hybridisierungssignals im Bereich der Lymphknotenrinde (überwiegend B-Lymphozyten) konnten, nach Auftrennung von PBMCs mit Hilfe des MACS-Systems in einzelne Zellfraktionen, die B-Lymphozyten als hauptsächlicher Replikationsort wahrscheinlich gemacht werden. Weitere Hinweise auf die Replikation von GBV-C in Lymphozyten lieferte die in vitro-Infektion von PBMCs durch GBV-C mit einem anschließend beobachteten Anstieg der GBV-C-Konzentration im Kulturüberstand. In knochenmarktransplantierten Patienten wurde durch weitgehende Zerstörung des Knochenmarks ein Absinken der GBV-C-Konzentration gezeigt. Nach der Regeneration des lymphatischen Systems wurde häufig die ursprüngliche GBV-C-Konzentration wieder erreicht. Auch dieses Ergebnis läßt sich durch die Replikation von GBV-C in Zellen des lymphatischen Systems erklären. Zur Bestimmung der Prävalenz von GBV-C wurde ein Enzymimmuntest zum Nachweis von E2-Antikörpern mit rekombinanten Virusproteinen und monoklonalen Antikörpern gegen dieses Virus entwickelt. Ein 39 As langes Peptid im C-Terminus eines C-terminal verkürzten E2-Proteins war die antikörperbindende Domäne des monoklonalen Antikörpers. Für GBV-C hatten Hämophile, Homosexuelle und Prostituierte ein erhöhtes Infektionsrisiko. Deshalb ist neben dem parenteralen Übertragungsweg auch die sexuelle Übertragung des Virus bedeutend. Für eine sexuelle Übertragung des Virus spricht, daß bei Homosexuellen das Vorhandensein von Antikörpern gegen GBV-C mit der Anamnese einer durchgemachten Syphilis oder Gonorrhoe korreliert. Normalerweise führt die Produktion von E2-Antikörpern zu einer Elimination von GBV-C und einem Ausheilen der Infektion.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.11.021915v1?rss=1 Authors: Zhu, F., Wang, W., Ma, Q., Yang, Z., Ju, Y., Fan, Y., Guo, R., Wang, Q., Mu, X., Zhao, B., Gao, Y., He, X., Gao, F., Qian, L., Chen, C., Chen, Y., Gao, C., Ma, X.-c. Abstract: Objective: Gut microbiota dysbiosis and aberrant gut-brain functional modules including short-chain fatty acid (SCFA) production and long-lasting immune activation (IA) are presented in schizophrenia. Given the key roles of gut microbiota and SCFA in shaping immunity, we propose that dysbiosis-induced SCFA upregulation could contribute to IA and behavioral symptoms in schizophrenia. Design: Gut microbiota, SCFA, and IA biomarkers were compared between schizophrenic patients and healthy controls. The roles of SCFA in schizophrenia-related IA were analyzed in cultured peripheral blood mononuclear cells (PBMCs) and a mouse model of schizophrenia. The effects of SCFAs on schizophrenia-related phenotypes were analyzed in both human and mouse. Results: Both microbial-derived SCFA and SCFA-producing bacteria were elevated in the guts of schizophrenic patients, and this increased SCFA production in gut was associated with IA in schizophrenia. The microbiome signature underpinning schizophrenia-related IA includes increased diversity and increased SCFA-producing bacteria and inflammation-associated bacteria. The impact of SCFAs on immune responses of cultured PBMC depend on the diagnosis and IA status of donors. Small-molecule serum filtrates from immune-activated schizophrenic patients increased the inflammatory response of PBMCs from healthy volunteers, which can be enhanced and attenuated by SCFAs supplementation and inhibition of SCFA signaling, respectively. Chronically elevated SCFAs in adolescence induced neuroinflammation and schizophrenia-like behaviors in adult mice. Moreover, chronically elevated SCFAs in adult mice prenatally exposed to IA potentiated their expression of schizophrenia-like behaviors. Conclusion: microbiota-derived SCFAs are important mediators of dysregulated gut-brain axis and participant in pathogenesis via enhance IA in schizophrenia. Copy rights belong to original authors. Visit the link for more info