POPULARITY
BUFFALO, NY- February 7, 2024 – A new #research paper was #published in Aging (listed by MEDLINE/PubMed as "Aging (Albany NY)" and "Aging-US" by Web of Science) Volume 16, Issue 2, entitled, “PROX1 interaction with α-SMA-rich cancer-associated fibroblasts facilitates colorectal cancer progression and correlates with poor clinical outcomes and therapeutic resistance.” The tumor microenvironment (TME) plays a vital role in tumor progression through intricate molecular interactions. Cancer-associated fibroblasts (CAFs), notably those expressing alpha-smooth muscle actin (α-SMA) or myofibroblasts, are instrumental in this context and correlate with unfavorable outcomes in colorectal cancer (CRC). While several transcription factors influence TME, the exact regulator causing CAF dysregulation in CRC remains elusive. Prospero Homeobox 1 (PROX1) stands out, as its inhibition reduces α-SMA-rich CAF activity. However, the therapeutic role of PROX1 is debated due to inconsistent study findings. In this new study, researchers Shiue-Wei Lai, Yi-Chiao Cheng, Kee-Thai Kiu, Min-Hsuan Yen, Ying-Wei Chen, Vijesh Kumar Yadav, Chi-Tai Yeh, Kuang-Tai Kuo, and Tung-Cheng Chang from Taipei's National Defense Medical Center, Taipei Medical University, Taipei Medical University Shuang-Ho Hospital, and National Taitung University used the ULCAN portal and noted an elevated PROX1 level in advanced colon adenocarcinoma, linking to a poor prognosis. Their assays determined the impact of PROX1 overexpression on CRC cell properties, while co-culture experiments spotlighted the PROX1-CAF relationship. Molecular expressions were validated by qRT-PCR and Western blots, with in vivo studies further solidifying the observations. “Our study emphasized the connection between PROX1 and α-SMA in CAFs.” Elevated PROX1 in CRC samples correlated with increased α-SMA in tumors. PROX1 modulation influenced the behavior of specific CRC cells, with its overexpression fostering invasiveness. Kaplan-Meier evaluations demonstrated a link between PROX1 or α-SMA and survival outcomes. Consequently, PROX1, alone or with α-SMA, emerges as a CRC prognostic marker. Co-culture and animal experiments further highlighted this relationship. PROX1 appears crucial in modulating CRC behavior and therapeutic resistance within the TME by influencing CAFs, signifying the combined PROX1/α-SMA gene as a potential CRC prognostic marker. The concept of developing inhibitors targeting this gene set emerges as a prospective therapeutic strategy. However, this study is bound by limitations, including potential challenges in clinical translation, a focused exploration on PROX1/α-SMA potentially overlooking other significant molecular contributors, and the preliminary nature of the inhibitor development proposition. “As we advance in this field, the development and clinical validation of small-molecule inhibitors targeting PROX1/α-SMA become imperative, paving the way to refine and optimize CRC therapeutic interventions.” DOI - https://doi.org/10.18632/aging.205447 Corresponding author - Tung-Cheng Chang - 09432@s.tmu.edu.tw 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. Visit our website at https://www.Aging-US.com. MEDIA@IMPACTJOURNALS.COM
A new research paper was published in Aging (listed by MEDLINE/PubMed as "Aging (Albany NY)" and "Aging-US" by Web of Science) Volume 15, Issue 16, entitled, “Copy number variation as a tool for implementing pregnancy as an aging model.” Copy number variations (CNV) are a major contributor to genome variability. CNVs have been linked to aging and other degradable phenotypes such as pregnancy physiology. In this new study, researchers Mariana Andrawus, Lital Sharvit, Noga Touitou, Batia Lerrer, Haim Y. Cohen, and Gil Atzmon from University of Haifa and Bar-Ilan University used CNVs from pregnant mice to demonstrate how pregnancy can be used as a model of aging. “We hypothesize that pregnancy can serve as a model for aging by demonstrating similar biomarkers, pathologies, and genetic and epigenetic effects [3]. To test this hypothesis, we designed a study that assesses CNVs associated with human longevity (unpublished results) in pregnancy.” Candidate CNVs were selected by applying case-control analysis in human centenarians compared with control groups. These CNVs were aligned with the mouse genome and their copy variation was assessed using qRT-PCR in liver and blood tissue samples from pregnant mice throughout pregnancy (baseline; first, second, and third trimester; post-partum). Eight of the ten selected CNVs demonstrated a significant decline/increase trend throughout the pregnancy followed by opposite direction soon after delivery in the liver and blood of the mouse tissues. Furthermore, significant differential expression was detected among the candidate CNVs' close vicinity genes (APA2A, LSS, RBDHF1, PLAAT1, and SCL17A2), but not in the WSCD2 gene. Establishing a genetic link between longevity and pregnancy is a significant step toward implementing the pregnancy process as a model for aging. These results in pregnant mice highlight the mechanism and similarities between pregnancy and aging. “Investigating the mechanisms that cause such rejuvenation after labor could change our aging treatment paradigm.” DOI - https://doi.org/10.18632/aging.204936 Corresponding author - Gil Atzmon - gatzmon@univ.haifa.ac.il Video short - https://www.youtube.com/watch?v=82466m-S-tU Sign up for free Altmetric alerts about this article - https://aging.altmetric.com/details/email_updates?id=10.18632%2Faging.204936 Subscribe for free publication alerts from Aging - https://www.aging-us.com/subscribe-to-toc-alerts Keywords - aging, pregnancy, copy number variation, gene expression 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/2023.07.20.549847v1?rss=1 Authors: Aarts, M. T., Wagner, M., van der Wal, T., van Boxtel, A. L., van Amerongen, R. Abstract: Progesterone receptor (PR) signaling is required for mammary gland development and homeostasis. A major bottleneck in studying PR signaling is the lack of sensitive assays to measure and visualize PR pathway activity both quantitatively and spatially. Here, we develop new tools to study PR signaling in human breast epithelial cells. First, we generate optimized Progesterone Responsive Element (PRE)-luciferase constructs and demonstrate that these new reporters are a powerful tool to quantify PR signaling activity across a wide range of progesterone concentrations in two luminal breast cancer cell lines, MCF7 and T47D. We also describe a fluorescent lentiviral PRE-GFP reporter as a novel tool to visualize PR signaling at the single-cell level. Our reporter constructs are sensitive to physiological levels of progesterone. Second, we show that low background signaling, and high levels of PR expression are a prerequisite for robustly measuring PR signaling. Increasing PR expression by transient transfection, stable overexpression in MCF7 or clonal selection in T47D, drastically improves both the dynamic range of luciferase reporter assays, and the induction of endogenous PR target genes as measured by qRT-PCR. We find that the PR signaling response differs per cell line, target gene and hormone concentration used. Taken together, our tools allow a more rationally designed approach for measuring PR signaling in breast epithelial cells. 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/2023.07.10.548366v1?rss=1 Authors: Hayman, D. J., Lin, H., Prior, A., Charlesworth, G., Johnson de Sousa Brito, F. M., Hao, Y., Patel, K., Soul, J., Clark, I. M., Pirog, K. A., Barter, M. J., van 't Hof, R. J., Young, D. A. Abstract: microRNAs (miRNAs) are non-coding RNAs which modulate the expression of other RNA molecules. One miRNA can target many transcripts, allowing each miRNA to play key roles in many biological pathways. miR-324 is a miRNA previously implicated in bone and cartilage maintenance, defects of which result in common age-related diseases, such as osteoporosis or osteoarthritis (OA). In global miR-324-null mice cartilage damage was increased in both surgically and ageing-induced OA, despite minimal changes to the cartilage transcriptome, with few predicted miR-324 targets dysregulated. However, micro-computed tomography and histology demonstrated that global miR-324-null the mice had an increase in bone mineral density, trabecular thickness and cortical thickness, with many parameters increasing with age. The bone marrow of miR-324-null mice also had reduced lipid content while and in vivo TRAP staining revealed a decrease in osteoclasts, with histomorphometry demonstrating an increased rate of bone formation in miR-324-null mice. Ex vivo assays revealed that the high bone mass phenotype of the miR-324-null mice resulted from increased osteoblast activity and decreased osteoclastogenesis. RNA-seq and qRT-PCR followed by miR-324 target prediction and validation in osteoblasts, osteoclasts and bone marrow macrophages identified the osteoclast fusion regulator Pin1 as a miR-324 target in the osteoclast lineage and the master osteogenic regulator Runx2 as a target of miR-324-5p in osteoblasts, the in vitro overexpression of which recapitulated the increased osteogenesis and decreased adipogenesis phenotype observed in vivo. These data point to important roles of miR-324 in skeletal biology with altered bone homeostasis in miR-324-null mice potentially causal for the increased cartilage damage observed during OA and ageing. Elucidation of pathways regulated by miR-324 offer promise for the treatment of bone diseases such as osteoporosis. 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/2023.04.19.537409v1?rss=1 Authors: Matsumoto, T., Higaki, T., Takatsuka, H., Kutsuna, N., Ogata, Y., Hasezawa, S., Umeda, M., Inada, N. Abstract: ACTIN DEPOLYMERIZING FACTOR (ADF) is a conserved protein that regulates the organization and dynamics of actin microfilaments. Eleven ADFs in the Arabidopsis thaliana genome are grouped into four subclasses, and subclass I ADFs, ADF1-4, are all expressed throughout the plant. Previously, we showed that subclass I ADFs function in the regulation of the response against powdery mildew fungus as well as in the regulation of cell size and endoreduplication. Here, we report a new role of subclass I ADFs in the regulation of nuclear organization and gene expression. Through a microscopic observation of epidermal cells in mature leaves, we found that the size of chromocenters in both adf4 and transgenic lines where expression of subclass I ADFs are downregulated (ADF1-4Ri) was reduced compared with that of wild-type Col-0. A. thaliana possesses eight ACTIN genes, among which ACT2, -7, and -8 are expressed in vegetative organs. The chromocenter size in act7, but not in the act2/8 double mutant, was enlarged compared with that in Col-0. Microarray analysis revealed that 1,818 genes were differentially expressed in adf4 and ADF1-4Ri. In particular, expression of 22 nucleotide-binding leucine-rich repeat (NLR) genes, which are involved in effector-triggered plant immunity, was reduced in adf4 and ADF1-4Ri. qRT-PCR confirmed the altered expressions shown with microarray analysis. Overall, these results suggest that ADF regulates various aspects of plant physiology through its role in regulation of nuclear organization and gene expression. The mechanism how ADF and ACTIN regulate nuclear organization and gene expression is discussed. 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/2023.04.20.537643v1?rss=1 Authors: Babylon, L., Limbeck, M. T., Eckert, G. P. Abstract: Alzheimer disease (AD) is an emerging medical problem worldwide without any cure yet. By 2050, more than 152 million people will be affected. AD is characterized by mitochondrial dys-function (MD) and increased amyloid beta (A{beta}) levels. Coffee is one of the most commonly consumed beverages. It has many bioactive and neuroprotective ingredients of which caffeine (Cof), kahweohl (KW) and cafestol (CF) shows a variety of pharmacological properties such as anti-inflammatory and neuroprotective effects. Effects of Cof, KW, and CF were tested in a cel-lular model of AD on MD and A{beta}. SH-SY5Y-APP695 cells were incubated with 50M Cof, 1M CF and 1M KW for 24h. The energetic metabolite ATP was determined using a luciferase-catalyzed bioluminescence assay. The activity of mitochondrial respiration chain complexes was assessed by high-resolution respirometry using a Clarke electrode. Expression levels genes were deter-mined using quantitative real-time polymerase chain reaction (qRT-PCR). The levels of amyloid {beta}-protein (A{beta}1-40) were measured using homogeneous time-resolved fluorescence (HTRF). ROS levels, cAMP levels, and peroxidase activity were determined using a fluorescence assay. The combination of Cof, KW and CF significantly increased ATP levels. The combination had neither a significant effect on MMP, on activity of respiration chain complexes, nor on A {beta}1-40 levels. cAMP levels were slightly increased after incubation with the combination, but not the peroxi-dase activity. Pyruvate levels and the lactate-pyruvate-ration but not lactate levels were signifi-cantly enhanced. No effect was seen on the expression level of lactate dehydrogenase and py-ruvate dehydrogenase kinase. In some experiments we have tested the single substances. They showed significant results especially in ATP, lactate and pyruvate values compared to the con-trol. The combinations have a lesser effect on mitochondrial dysfunction in cells and none on A{beta} production. Whereas ATP levels and pyruvate levels were significantly increased. This suggests a change in glycolysis in neuronal cells harbouring human genes relevant for AD. 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/2023.04.03.535461v1?rss=1 Authors: Dong, K., He, X., Hu, G., Yao, Y., Zhou, J. Abstract: Objective: Vascular smooth muscle cells (VSMCs) are the primary contractile component of blood vessels and can undergo phenotypic switching from a contractile to a synthetic phenotype in vascular diseases such as coronary artery disease (CAD). This process leads to decreased expression of SMC lineage genes and increased proliferative, migratory and secretory abilities that drive disease progression. Super-enhancers (SE) and occupied transcription factors are believed to drive expression of genes that maintain cell identify and homeostasis. The goal of this study is to identify novel regulator of VSMC homeostasis by screening for SE-regulated transcription factors in arterial tissues. Approach and Results: We characterized human artery SEs by analyzing the enhancer histone mark H3K27ac ChIP-seq data of multiple arterial tissues. We unexpectedly discovered the transcription factor PRDM16, a GWAS identified CAD risk gene with previously well-documented roles in brown adipocytes but with an unknown function in vascular disease progression, is enriched with artery-specific SEs. Further analysis of public bulk RNA-seq and scRNA-seq datasets, as well as qRT-PCR and Western blotting analysis, demonstrated that PRDM16 is preferentially expressed in arterial tissues and in contractile VSMCs but not in visceral SMCs, and down-regulated in phenotypically modulated VSMCs. To explore the function of Prdm16 in vivo, we generated Prdm16 SMC-specific knockout mice and performed histological and bulk RNA-Seq analysis of aortic tissues. SMC-deficiency of Prdm16 does not affect the aortic morphology but significantly alters expression of many CAD risk genes and genes involved in VSMC phenotypic modulation. Specifically, Prdm16 negatively regulates the expression of Tgfb2 that encodes for an upstream ligand of TGF-{beta} signaling pathway, potentially through binding to the promoter region of Tgfb2. These transcriptomic changes likely disrupt VSMC homeostasis and predispose VSMCs to a disease state. Conclusions: Our results suggest that the CAD risk gene PRDM16 is preferentially expressed in VSMCs and is a novel regulator of VSMC homeostasis. Future studies are warranted to investigate its role in VSMCs under pathological conditions such as atherosclerosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.03.530736v1?rss=1 Authors: Kobori, C., Takagi, R., Yokomizo, R., Yoshihara, S., Mori, M., Takahashi, H., Javaregowda, P. K., Akiyama, T., Ko, M. S. H., Kishi, K., UMEZAWA, A. Abstract: Background: Melanocytes are an essential part of the epidermis, and their regeneration has received much attention because propagation of human adult melanocytes in vitro is too slow for clinical use. Differentiation from human pluripotent stem cells to melanocytes has been reported, but the protocols to produce them require multiple and complex differentiation steps. Method: We differentiated human embryonic stem cells (hESCs) that transiently express JMJD3 to pigmented cells. We investigated whether the pigmented cells have melanocytic characteristics and functions by qRT-PCR, immunocytochemical analysis and flow cytometry. We also investigated their biocompatibility by injecting the cells into immunodeficient mice for clinical use. Result: We successfully differentiated and established a pure culture of melanocytes. The melanocytes maintained their growth rate for a long time, approximately 200 days, and were functional. They exhibited melanogenesis and transfer of melanin to peripheral keratinocytes. Moreover, melanocytes simulated the developmental processes from melanoblasts to melanocytes. The melanocytes had high engraftability and biocompatibility in the immunodeficient mice. Conclusion: The robust generation of functional and long-lived melanocytes are key to developing clinical applications for the treatment of pigmentary skin disorders. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
This month on Episode 45 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the February 3rd and February 17th issues of Circulation Research. This episode also features an interview with Dr Hind Lal and Dr Tousif Sultan from the University of Alabama at Birmingham about their study Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation. Article highlights: Pi, et al. Metabolomic Signatures in PAH Carnevale, et al. Thrombosis TLR4-Mediated in SARS-CoV-2 Infection Cai, et al. Macrophage ADAR1 in AAA Koide, et al. sEVs Accelerate Vascular Calcification in CKD Cindy St. Hilaire: Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cynthia St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to be highlighting the articles from our February 3rd and 17th issues of Circulation Research. I'm also going to have a chat with Dr Hind Lal and Dr Tousif Sultan from the University of Alabama at Birmingham about their study, Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation. But before I get to the interviews, here are a few article highlights. Cindy St. Hilaire: The first article I want to highlight comes from the laboratory of Dr Peter Leary at the University of Washington, and the title is Metabolomic Signatures Associated With Pulmonary Arterial Hypertension Outcomes. Pulmonary Arterial Hypertension or PAH is a rare but life-threatening disease in which progressive thickening of the walls of the lung's blood vessels causes increased blood pressure and that increased blood pressure ultimately damages the heart's right ventricle. Interestingly, progression to heart failure varies considerably among patients, but the reasons why there is variability are not well understood. To find out, this group turned their attention to patient metabolomes, which differ significantly from those of healthy people and thus may also change with severity. Blood samples from 117 PAH patients were analyzed for more than a thousand metabolites by mass spectrometry and the patient's progress was followed for the next three years. 22 patients died within a three-year period and 27 developed significant right ventricle dilation. Other measures of severity included pulmonary vascular resistance, exercise capacity and levels of BNP, which is a metric of heart health. Two metabolic pathways, those relating to polyamine and histidine metabolism, were found to be linked with all measures of severity suggesting a key role for them in disease pathology. While determining how these pathways influence disease as a subject for further study, the current findings may nevertheless lead to new prognostic indicators to inform patient care. Cindy St. Hilaire: The next article I want to discuss is coming from our February 3rd issue of Circulation Research and this is coming from the laboratory of Dr Francisco Violi at the University of Rome and the title is Toll-Like Receptor 4-Dependent Platelet-Related Thrombosis in SARS-CoV-2 Infection. Thrombosis can be a complication of COVID-19 and it is associated with poor outcomes, including death. However, the exact mechanism by which the virus activates platelets, which are the cells that drive thrombosis, is not clear. For one thing, platelets do not appear to express the receptor for SARS-CoV-2. They do however, express the TLR4 receptor and that's a receptor that mediates entry of other viruses as part of the immune response. And TLR4 is ramped up in COVID-19 patient platelets. This group now confirms that, indeed, SARS-CoV-2 interacts with TLR4, which in turn triggers thrombosis. The team analyzed platelets from 25 patients and 10 healthy controls and they found that the platelet activation and thrombic activity were both boosted in the patient samples and could not be blocked using a TLR4 inhibitor. Additionally, immunoprecipitation and immunofluorescent experiments further revealed colocalization between the virus protein and the TLR4 receptor on patient platelets. The team went on to show that the signaling pathway involved reactive oxygen species producing factors p47phox and Nox2, and that inhibition of phox 47, like that of the TLR4 receptor itsel,f could prevent platelet activation. As such, this study suggests that inhibiting either of these proteins may form the basis of an antithrombotic treatment for COVID-19. Cindy St. Hilaire: The third article I want to highlight is coming from the lab of Shi-You Chen at University of Missouri and the title of this article is ADAR1 Non-Editing Function in Macrophage Activation and Abdominal Aortic Aneurysm. Macrophage activation plays a critical role in abdominal aortic aneurysm development, or AAA development. Inflammation is a component of this pathology; however, the mechanisms controlling macrophage activation and vascular inflammation in AAA are largely unknown. The ADAR1 enzyme catalyzes the conversion of adenosine to inosine in RNA molecules and thus this conversion can serve as a rheostat to regulate RNA structure or the gene coding sequence of proteins. Several studies have explored the role of ADAR1 in inflammation, but its precise contribution is not fully understood, so the objective of this group was to study the role of ADAR1 in macrophage activation and AAA formation. Aortic transplantation was conducted to determine the importance of nonvascular ADAR1 in AAA development and dissection and angiotensin II infusion of ApoE knockout mice combined with a macrophage specific knockout of ADAR1 was used to study the role of ADAR1 macrophage specific contributions to AAA formation and dissection. Allograft transplantation of wild type abdominal aortas to ADAR1 haploinsufficient recipient mice significantly attenuated AAA formation. ADAR1 deficiency in hematopoietic stem cells also decreased the prevalence and the severity of AAA and it also inhibited macrophage infiltration into the aortic wall. ADAR1 deletion blocked the classic macrophage activation pathway. It diminished NF-κB signaling and it enhanced the expression of a number of anti-inflammatory microRNAs. Reconstitution of ADAR1 deficient but not wild type human monocytes to immunodeficient mice blocked the aneurysm formation in transplanted human arteries. Together these results suggest that macrophage ADAR1 promotes aneurysm formation in both mouse and human arteries through a novel mechanism of editing the microRNAs that target NF-κB signaling, which ultimately promotes vascular inflammation in AAA. Cindy St. Hilaire: The last article I want to highlight is also from our February 17th issue of Circulation Research and it is coming from the lab of Shintaro Mandai at Tokyo Medical and Dental University and the title of the article is Circulating Extracellular Vesicle Propagated MicroRNA signatures as a Vascular Calcification Factor in Chronic Kidney Disease. Chronic Kidney Disease or CKD accelerates vascular calcification in part by promoting the phenotypic switching of vascular smooth muscle cells to osteoblast like cells. This study investigated the role of circulating small extracellular vesicles or SUVs from the kidneys in promoting this osteogenic switch. CKD was induced in rats and in mice by an adenine induced tubular interstitial fibrosis and serum from these animals induced calcification in in vitro cultures of A-10 embryonic rat smooth muscle cells. Intraperitoneal administration of a compound that prevents SEV biosynthesis and release inhibited thoracic aortic calcification in CKD mice under a high phosphorus diet. In Chronic Kidney Disease, the microRNA transcriptome of SUVs revealed a depletion of four microRNAs and the expression of the microRNAs inversely correlated with kidney function in CKD patients. In vitro studies found that transected microRNA mimics prevented smooth muscle cell calcification in vitro. In silico analyses revealed that VEGF-A was a convergent target of all four microRNAs and leveraging this, the group used in vitro and in vivo models of calcification to show the inhibition of the VEGF-A, VEGFR-2 signaling pathway mitigated calcification. So in addition to identifying a new potential therapeutic target, these SUV propagated microRNAs are a potential biomarker that can be used for screening patients to determine the severity of CKD and possibly even vascular calcification. Cindy St. Hilaire: Today I have with me Dr Hind Lal who's an associate professor of medicine at the University of Alabama Birmingham and his post-doctoral fellow and the lead author of the study Dr Tousif Sultan. And their manuscript is titled Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation. And this article is in our February 3rd issue of Circulation Research. So thank you both so much for joining me today. Tousif Sultan: Thank you. Hind Lal: Thank you for taking time. Cindy St. Hilaire: So ponatinib, it's a tyrosine kinase inhibitor and from my understanding it's the only treatment option for a specific group of patients who have chronic myelogenous leukemia and they have to harbor a specific mutation. And while this drug helps to keep these patients alive essentially, it's extremely cardiotoxic. So cardiotoxicity is somewhat of a new field. So Dr Lal, I was wondering how did you get into this line of research? Hind Lal: So I was fortunate enough to be in the lab of Dr Tom Force and he was kind of father of this new area, now is very developed, it's called cardio-oncology. On those days there were basically everything started in cardio-oncology. So I just recall the first tyrosine kinase approved by FDA was in 2000 and that was... Imagine and our paper came in Nature Medicine 2005 and discovering there is... so to elaborate it a little bit, the cancer therapy broadly divided in two parts. One is called non-targeted therapy like chemotherapy, radiations, et cetera, and then there are cytotoxic drugs. So those cytotoxic drugs because they do not have any targeted name on it so they are, cardiotoxic are toxic to any organ was very obvious and understanding. When these targeted therapy came, which is mainly kinase inhibitor are monoclonal antibodies. So these are targeted to a specific pathway that is activated only in the cancer cells but not in any other cells in the body so they were proposed as like magic bullets that can take off the cancer without any cardiotoxity or minimal side effects. But even in the early phase like 2005 to 2010, these came out, these so-called targeted, they are not very targeted and they are not also the magic bullets and they have serious cardiotoxicity. Cindy St. Hilaire: And so what's the mechanism of action of ponatinib in the leukemia and how does that intersect with the cardiovascular system? Hind Lal: Yeah, so this is very good question I must say. So what we believe at this point because, so leukemia if you know is driven by the famous Philadelphia chromosome, which is a translicational gene, one part of human chromosome nine and one part of human chromosome 22 and they translocate make a new gene which is BCR-ABL gene. And because it was discovered in Philadelphia UPENN, is named that Philadelphia chromosome, which is very established mechanism, that's how CML is driven. But what we have discovered that the cardiotoxicity driven by totally, totally different from the ponatinib is one of the inflammatory So it's kind of goodening. So this question is so good. One kind of toxicity is called on-target, when toxicity is mediated by the same mechanism, what is the mechanism of the drug to cure the cancer? So in that case your absolute is minimal because if you manipulate that, the drug's ability to cure the cancer will be affected but if the toxicity and the efficacy is driven by two different mechanism, then as in case of ponatinib seems like it's NLRP3 and inflammasome related mechanism. So this can be managed by manipulating this pathway without hampering the drug efficacy on the cancer. Cindy St. Hilaire: So what exactly is cardiotoxicity and how does it present itself in these patients? Hind Lal: So these drugs like ponatinib, they call broader CVD effects. So it's not just cardiac, so they also in hypertensives and atherosclerosis and thrombosis, those kind of thing. But our lab is primarily focused on the heart. So that's why in this paper we have given impresses on the heart. So what we believe at this point that ponatinib lead to this proinflammatory pathway described in this paper, which is just 108A9-NLRP3-IL-1β and this inflammatory pathway lead to a cytokine storm very much like in the COVID-19 and these cytokine storms lead to excessive myocarditis and then finally cardiac dysfunction. Cindy St. Hilaire: Is the cytokine storm just local in the cardiac tissue or is it also systemic in the patients? Is cardiotoxicity localized only or is it a more systemic problem? Tousif Sultan: I would like to add in this paper we have included that we look this cytokine things and explain blood circulation, bone marrow. So the effect is everywhere, it's not local. So we didn't check other organs, maybe other organs also being affected with the ponatinib treatment. Cindy St. Hilaire: And what's the initial phenotype of a patient has when they start to get cardiotoxicity, what's kind of like a telltale symptom? Hind Lal: So good thing that in recent years cardio-oncology developed. So initially the patient that were going for cancer treatment, they were not monitored very closely. So they only end up in cardiology clinic when they are having some cardiac events already. So thanks to the lot of development and growth in the cardio-oncology field, now most patients who going for a long-term cancer treatment, they are closely monitored by cardiology clinics. Cindy St. Hilaire: Got it. So they can often catch it before a symptom or an event. That's wonderful. Hind Lal: Yeah, so there's a lot of development in monitoring. Cindy St. Hilaire: Wonderful. So you were really interested in figuring out why ponatinib induces cardiotoxicity and you mentioned that really up until now it's been very difficult to study and that's because of the limitation of available murine models. If you just inject a wild type mouse with ponatinib, nothing happens really. So what was your approach to finding relatively good murine models? How did you go about that? Hind Lal: So this is the top scientific question you can ask. So like science, the field is try and try again. So initially this is the first paper with the ponatinib toxicity using the real in vivo models. Any paper before this including ours studies published, they were done on the cellular model in hiPSC, that isolated cardiomyocytes. So you directly putting the ponatinib directly the isolated cells. So this is first case when we were trying to do in vivo, maybe other attempt in vivo but at least not published. So first we also treated the animals with ponatinib and that failed, we don't see any cardiotoxic effect. And then when we going back to the literature, the clinical data is very, very clear from pharmacovigilance that ponatinib is cardiotoxic in humans. So when we're not able to see any phenotype in mouse, we realize that we are not mimicking what's happening in the humans. So we certainly missing something. Now once again I quote this COVID-19, so many people get infected with COVID-19 but people are having preexisting conditions are on high risk to developing CVD. So there was some literature on that line. So we use this very, very same concept that if there is preexisting conditions, so likely who'd have developing future cardiac event will be more. So we use two model in this paper one atherosclerosis model which is APoE null mice mice, another is tag branding which is pressure overload model for the heart and as soon as we start using what we call comorbidity model like patient is having some preexisting conditions and we very clearly see the robust defect of ponatinib on cardiac dysfunction. Cindy St. Hilaire: Yeah, it's really, really well done and I really like that you use kind of two different models of this. Do you think it's also going to be operative in maybe like the diabetic mirroring models? Do you think if we expand to other comorbidities, you might also recapitulate the cardiotoxicity? Hind Lal: So you got all the best questions. Cindy St. Hilaire: Thank you. I try. Hind Lal: So because this is CML drug and lot of the risk factor for cardiovascular and cancer are common and even metabolic disease. So most of the time these patients are elderly patients and they're having metabolic conditions and most of the time they have blood pressure or something CVD risk factors. So I agree with you, it'll be very relevant to expand this to the diabetes or metabolic models, but these were the first study, we put all our focus to get this one out so news is there then we can expand the field adding additional models et cetera. But I agree with you that will be very logical next step to do. Cindy St. Hilaire: Yeah. And so I guess going back to what you know from the human study or the clinical trials or the human observations, are different populations of patients with CML more predisposed to cardio toxicity than others or is that not known yet? Hind Lal: So one other area called pharmacovigilance. So what pharmacovigilance does patient all over the world taking these drugs. So WHO have their own vigilance system and FDA have their own, so it's called BG-Base for the WHO and it's called the FAERS for the FDA. So one can go back in those data sets and see if X patient taking this Y drug and what kind of symptoms or adverse effect they are seeing and if these symptoms are associated with something else. So there is data that if patients having CVD risk factor, they are more prone to develop ponatinib induced cardiac events. But it needs more polish like you asked the just previous question, diabetes versus maybe blood pressure means hypertension, atherosclerosis, or thrombosis. So it has not been delineated further but in a one big bucket if patients are having CVD risk factor before they are more prone and more likely to develop the cardiac events. Cindy St. Hilaire: So after you established that these two murine models could pretty robustly recapitulate the human phenotype, what did you do next? How did you come upon the S100A8/A9-NLRP3-IL-1β signaling circuit? How did you get to that? Hind Lal: So in basic science work, whenever we do mouse is called until we get there is cardiac dysfunction, it's called phenotype, right? So mouse had a cardiac phenotype. So next step is, "Why? What is leading to that phenotype?" That's what we call mechanism. So there the best idea to fit the mechanism is using one of the unbiased approaches like you do unbiased proteomics, unbiased RNC analysis, something like this that will analyze the entire transcript like RNC and say, "Okay, these pathway are," then you can do further analysis that will indicate these pathway are different, are altered. So in this case we used RNC analysis and it came out that this yes A8 and yes A9, 100A8 and nine, they were the most upregulated in this whole set. And thereafter we were very lucky. So we started this study at Vanderbilt, where my lab was and thereafter we very lucky to move here and found Sultan who had a lot of experience with this inflammation and immune system and then Sultan may add something on this so he'll be the better person to say something on this. Tousif Sultan: So after our RNC analysis, so we got this S100A8 and nine as top hit with the ponatinib treatment. So then we validated this finding with our flow cytometric, qRT PCR aand then we started which pathway is going to release cytokine and all that. So we found that is NLRP3 inflammasome. Cindy St. Hilaire: Yeah and well and I guess maybe step back, what is S100A8/A9? What are those? Tousif Sultan: Yeah, S10A8/A9 is a calcium binding protein. So that's also called alarmin and they basically binds with the pathogen associated pattern and other TLR2 like receptors and then start inflammatory pathway to release cytokine and all that and it's stable in heterodimer form. So S100A8 heterodimer with A9 and then bind with TLR and a start in this inflammatory pathway. Cindy St. Hilaire: And what type of cell is that happening in? Is that happening in the immune cells only or is it also in the cardiomyocyte, or...? Tousif Sultan: Yeah, we have included all this data. So from where this alarmin is coming with ponatinib treatment, so literature also suggested that neutrophils and monocytes, those cells are the potential to release the alarmin. So here we also found these two type of cells, neutrophils and monocytes. They release huge alarmin with the treatment of ponatinib. Cindy St. Hilaire: And so really taking this really neat mechanism to the next level, you then tried attenuating it by using broad anti-inflammatory steroid dexamethasone but also by targeting these specific components, the NLRP and the S100A specific inhibitors and they worked well. It worked really nicely. Does your data show that any of these therapies work better than the other and then are these viable options to use in humans? Hind Lal: Yeah, we have some data in the paper. Are very broad which help a lot in COVID patients, far very acute infections. So in this case, situation is very different cause most of CML patients will going to take ponatinib for lifelong, there is no remission, right? So in those case, its certainly not a very attractive option. We have shown data in the paper that dexamethasone help with the heart but lead to some metabolic changes. So we have compared those with the NLRP3 inhibitors, those metabolic alterations, dexa versus the NLRP3 inhibitors, CY-09. And we demonstrated that targeting is specifically with paquinimod, our NLRP3 inhibitor CY-09, feel better. It can still rescue the cardiac phenotype without having those adverse effect on metabolic parameters. Cindy St. Hilaire: That's wonderful. Do you think though that because you have to take ponatinib for life, that long-term NLRP inhibition would also cause problems or...? Hind Lal: So because not every patient who taking ponatinib would develop the cardiac phenotype, right? Which is like a 10%, 12%, patient developing cardiac dysfunction. So I think someone like I strongly believe paquinimod, which is inhibitor of S100A9, will be really good option or at least we have enough data that make us nail for at least a small clinical trial. And we quickly moving on that. At UAB we have our clinical cardio-oncology program and we are already in touch with the director for the clinical cardio-oncology program. So what we trying to do in that small trial is if one of the standard therapy for heart like beta blocker or ARBs inhibitor, is there any preference like one work better than the other in the standard care? So first we doing that project, then we obviously looking forward if one small clinical trial can be done with paquinimod. I strongly believe it should be helpful. Cindy St. Hilaire: That is wonderful. And so do you think... There's other chemotherapeutic agents or probably even other non-cancer drugs that cause cardiotoxicity, do you think this mechanism, this pathway, this S100A-NLRP-IL-1β axis is operative in all cardiotoxicities or do you think it's going to be very specific to the ponatinib? Hind Lal: So it's certainly not all, but it'll be certainly more than ponatinib. So in our lab we are using another kinase inhibitor, which is osimertinib and it's not published yet, but now we know that it's also cardiotoxic because it's taking metabolic root or energetics disruption but not this pro-inflammatory part, but we're doing another project which is strep pneumonia induced cardiac dysfunction, which is called pneumonia. So strep pneumoniae, which leads to the pneumonia ,and lot patient die because of the failing heart we see here in the hospitals and we see these pathways operational over there and we gearing up to do clinical trial on that aspect as well, but it's not generalized like all kind of heart will have the same mechanism. Cindy St. Hilaire: It's wonderful to see you're already taking those next steps towards really kind of bringing this to a translational/clinical study. So what was the most challenging aspect of this study? Tousif Sultan: The challenging aspect, ponatinib is a kinase inhibitor and that was surprising for us how it's activating immune cells. Generally kinase inhibitors, inhibits all the cells like that. So that was challenging. So we repeated it many times did in vitro experiment to confirm that. So we just added, just treated in vitro immune cells with the ponatinib and confirmed it. So that was little challenging. Cindy St. Hilaire: So what's next? You mentioned you're going to try some clinical trials, early stage clinical trials. What's next mechanistically, what do you want to go after? Hind Lal: So what we are doing next and we are very, very eagerly trying to do that. So what it was done, we used the cardiac comorbidity models, but as you know, anybody who will take ponatinib will have cancer, right? So we strongly believe that we miss one factor. There was no cancer on these. So that is very logical next step. What that will allow us to do, what rescue experiment we'll have done in this paper. So we saw, "Okay, this rescue the cardiac phenotype, which is taken care of now," but very same time, we not able to demonstrate that this is happening without hurting the cancer efficacy. So if we have the dual comorbid mouse, which have CML a real thing and we have cardiac thing, then that will allow us to demonstrate, "Okay, we got something that can take care of the cardiac problem without hurting the efficacy on the cancer." And it will be best if you also help little bit to more potentiate the cancer efficacy. Cindy St. Hilaire: Yes. Excellent. Well, congratulations on a beautiful study, really exciting findings. Dr Lal and Dr Sultan, thank you so much for taking the time to talk with me today. Tousif Sultan: Thank you so much. Hind Lal: Well thank you, Cynthia. We really appreciate your time. Thank you for having us. Cindy St. Hilaire: Yeah, it was great. Cindy St. Hilaire: That's it for our highlights from the February 3rd and February 17th issues of Circulation Research. Thank you so much for listening. Please check out the Circulation Research Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Dr Hind Lal and Dr Tousif Sultan. This podcast is produced by Ishara Ratnayake, edited by Melissa Stoner and supported by the editorial team at Circulation Research. Some of the copy text for the highlighted articles was provided by Ruth Williams. I'm your host, Dr Cynthia St. Hilaire, and this is Discover CircRes, you're on-the-go source for most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2023. And the opinions expressed by the speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahajournals.org.
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.15.516629v1?rss=1 Authors: Qi, W., Fang, Z., Luo, C., Hong, H., Long, Y., Dai, Z., Liu, J., Zeng, Y., Zhou, T., Xia, Y., Yang, X., Gao, G. Abstract: Objective: Non-alcoholic fatty liver disease (NAFLD), characterized by hepatic steatosis, is one of the most common causes of liver dysfunction. ATGL is closely related to hepatic steatosis as the speed-limited triacylglycerol lipase. Nevertheless, the expression and regulation of ATGL in NAFLD remain unclear. Methods: Using immunohistochemistry and qRT-PCR to detect the expression of ATGL and BTRC in different models with hepatic steatosis. Co-IP evaluated the binding of ATGL and BTRC. Knockdown of BTRC employed by adenoviruses and then analyzed the ATGL expression, triglyceride levels, and lipid droplets accumulation. Results: Our results revealed that ATGL protein level was decreased in animal and cellular models of hepatic steatosis and the liver tissues of cholangioma/hepatic carcinoma patients with hepatic steatosis, while the ATGL mRNA level had hardly changed; which means the decreased ATGL mainly degraded through the proteasome pathway. BTRC was identified as the E3 ligase for ATGL, up-regulated, and negatively correlated with ATGL level. Moreover, adenovirus-mediated knockdown of BTRC ameliorated hepatic steatosis via up-regulating ATGL level. Conclusions: Our study demonstrates a crucial role of elevated BTRC in hepatic steatosis through promoting ATGL proteasomal degradation as a new ATGL E3 ligase and suggests BTRC may serve as a potential therapeutic target for NAFLD. 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/2022.09.07.506981v1?rss=1 Authors: Parks, S. Z., Rutter, G. A., Leclerc, I. Abstract: Background: Soluble Resistance Related Calcium Binding Protein (sorcin) is a calcium (Ca2+) binding protein which has been shown to play a role in maintaining intracellular endoplasmic reticulum (ER) Ca2+ stores and lowering ER stress. Recently, our lab has demonstrated that sorcin expression was downregulated in the islets of Langerhans of mice fed a high-fat diet or in human islets incubated with the saturated fatty acid palmitate. We also showed that overexpression of sorcin under control of the rat insulin promoter (RIP7) in C57BL/6J mice, or whole body sorcin deletion in 129S1/SvImJ mice, improves or impairs insulin secretion and pancreatic {beta}-cell function respectively. The mechanisms behind this beneficial role of sorcin in the pancreatic {beta}-cell might depend on protection against lipotoxic endoplasmic reticulum (ER) stress through improved ER Ca2+ dynamics and activation of the Activating Transcription Factor 6 (ATF6) branch of the unfolded protein response (UPR). Whether sorcin is also implicated in hypothalamic ER stress during the progression of obesity is unknown. This could potentially contribute to the diminished satiety typically observed in overweight individuals. Aim: To investigate a potential role of sorcin in hypothalamic ER stress, leptin resistance, hyperphagia and obesity. Methods: Whole-body sorcin null mice, backcrossed onto the C57BL/6J genetic background, were used. Body weight, food intake and EchoMRI body composition were measured in vivo whereas qRT-PCR analysis of sorcin and ER stress markers expression were performed on the arcuate nucleus of the hypothalamus. Leptin signalling through STAT3 phosphorylation was measured by Western blots on sorcin-null HEK293 cells, engineered by CRISPR/Cas9, and transfected with leptin receptor (LepRb). Results: Sorcin expression was not influenced in the arcuate nucleus (ARC) of the hypothalamus by diet-induced obesity. Whole-body sorcin ablation did not cause ARC ER stress nor changes in body weight, body composition or food intake in C57BL/6 male mice exposed to a high-fat, high-sugar diet. STAT3 phosphorylation (Y705) in response to leptin was not impaired in sorcin-null HEK293 cells. Conclusion: In our model, whole body sorcin ablation did not increase hypothalamic ER stress nor influenced food intake or body weight. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer
1. Tucker: Does Fauci believe the vaccine is ineffective? 3. Perspectives on the Pandemic Leemon McHenry 4. Dr. Ryan Cole Blows The Whole COVID-19 Propaganda Away Study identifies specific antioxidants that may reduce oncogenic HPV infection in women Louisiana State University, April 12, 2021 A study led by Hui-Yi Lin, Ph.D., Professor of Biostatistics, and a team of researchers at LSU Health New Orleans Schools of Public Health and Medicine has found that adequate levels of five antioxidants may reduce infection with the strains of the human papillomavirus (HPV) associated with cervical cancer development. Findings are published in the Journal of Infectious Diseases. Although previous studies have suggested that the onset of HPV-related cancer development may be activated by oxidative stress, the association had not been clearly understood. This study evaluated associations between 15 antioxidants and vaginal HPV infection status -- no, low-risk, and oncogenic/high-risk HPV (HR-HPV) -- in 11,070 women aged 18-59 who participated in the 2003-2016 National Health and Nutrition Examination Survey. Study results showed that lower levels of serum albumin and four dietary antioxidants - vitamins A, B2, E, and folate -- were associated with a higher risk of HR-HPV infection. Albumin is the most bountiful circulating protein in plasma, and decreased serum albumin was found to be associated with increased systemic inflammation and impaired immune response. Based on the four dietary antioxidants, the researchers developed a nutritional antioxidant score. "Our results showed that the women with the lowest quartile of the nutritional antioxidant score had a higher chance of both high-risk and low-risk HPV infection compared with the women with the highest quartile score after adjusting for other factors such as age, race, smoking, alcohol, and the number of sexual partners in past 12 months," notes the paper's lead author Hui-Yi Lin, PhD, Professor of Biostatistics at LSU Health New Orleans School of Public Health. Human Papillomavirus is a well-known risk factor for cervical cancer, which is the fourth most common female cancer and contributed to 7.5% of cancer deaths for women worldwide in 2018. Certain HPV strains are more likely to trigger precursor events leading to cancer development. These strains are called oncogenic or high-risk [HR] HPV strains. Almost all cervical cancers are directly linked to previous infection with one or more HR-HPV infections. "Currently, there is no effective antiviral therapy to clear genital HPV infection," adds Dr. Lin. "It is important to identify modifiable factors, such as antioxidants, associated with oncogenic HPV infection in order to prevent HPV carcinogenesis onset." Exercise benefit in breast cancer linked to improved immune responses Tumors grew more slowly and responded better to immunotherapy in mice that exercised compared with sedentary mice. Massachusetts General Hospital, April 12, 2021 Exercise training may slow tumor growth and improve outcomes for females with breast cancer - especially those treated with immunotherapy drugs - by stimulating naturally occurring immune mechanisms, researchers at Massachusetts General Hospital (MGH) and Harvard Medical School (HMS) have found. Tumors in mouse models of human breast cancer grew more slowly in mice put through their paces in a structured aerobic exercise program than in sedentary mice, and the tumors in exercised mice exhibited an increased anti-tumor immune response. "The most exciting finding was that exercise training brought into tumors immune cells capable of killing cancer cells known as cytotoxic T lymphocytes (CD8+ T cells) and activated them. With more of these cells, tumors grew more slowly in mice that performed exercise training," says co-corresponding author Dai Fukumura, MD, PhD, deputy director of the Edwin L. Steele Laboratories in the Department of Radiation Oncology at MGH. As Fukumura and colleagues report in the journal Cancer Immunology Research , the beneficial effects of exercise training are dependent on CD8+ T cells; when the researchers depleted these cells in mice, tumors in mice that exercised no longer grew at a slower rate. They also found evidence that recruitment of CD8+ T cells to tumors was dependent on two chemical recruiters (chemokines) labeled CXCL9 and CXCL11. Levels of these chemokines were increased in mice that exercised, and mice that were genetically engineered to lack the receptor (docking site) for these chemokines did not recruit CD8+ T cells and did not have an anti-tumor benefit. "Humans whose tumors have higher levels of CD8+ T cells tend to have a better prognosis, respond better to treatment, and have reduced risk of cancer recurrence compared with patients whose tumors have lower levels of the immune cells, effects that were echoed by a reduced incidence of metastasis, or spread, of the cancers in mice that exercised," says co-corresponding author Rakesh K. Jain, PhD, director of the Steele Labs at MGH and Andrew Werk Cook Professor of Radiation Oncology at HMS. CD8+ T cells are also essential for the success of drugs known as immune checkpoint inhibitors, such as Keytruda (pembrolizumab), Opdivo (nivolumab) and Yervoy (ipilimumab), which have revolutionized therapy for many types of cancer, but have to date had only limited success in breast cancer. The researchers found that exercise-trained mice displayed a much better response to immune checkpoint blockade, while the drugs did not work at all in sedentary mice. "We showed that daily sessions of a moderate-to-vigorous intensity, continuous aerobic exercise training, lasting 30-45 minutes per session, induces a profound reprogramming of the tumor microenvironment that rewires tumor immunity, recruiting and activating CD8+ T cells to an unprecedented level with a non-pharmacological approach. Similar exercise training could be prescribed to a patient referred to an exercise oncology program," says Igor L. Gomes-Santos, PhD, lead author and exercise physiologist and post-doctoral fellow in the Steele Labs. He notes that current clinical guidelines focus on general wellness, improved fitness levels and quality of life, but not necessarily on improved cancer treatment, especially immunotherapy, and that this lack of evidence limits its application in clinical practice. More convincing, mechanism-based data are needed to motivate oncologists to discuss exercise training with their patients, to motivate patients to become more active and to expand implementation of outpatient exercise oncology programs, the investigators say. Higher dietary total antioxidant capacity associated with lower risk of cognitive impairment National University of Singapore, April 12 2021. The results of a study reported on April 7, 2021 in The Journals of Gerontology® Series A revealed a lower risk of cognitive impairment among older individuals who consumed more antioxidants. The study included 16,703 participants in the Singapore Chinese Health Study, which enrolled men and women aged 45 to 74 years between April 1993 and December 1998. Questionnaires completed upon enrollment provided information concerning dietary and supplement intake that was evaluated for antioxidant content using the Comprehensive Dietary Antioxidant Index and the Vitamin C Equivalent Antioxidant Capacity. Disease status and lifestyle factors were updated during follow-up visits conducted every five to six years. Cognitive function was evaluated 20.2 years after the beginning of the study. Cognitive impairment was detected among 14.3% of the participants. Among those whose Comprehensive Dietary Antioxidant Index Scores placed them among the top 25% of participants, the risk of having developed cognitive impairment was 16% lower than that of participants among the lowest 25%. Those whose Vitamin C Equivalent Antioxidant Capacity was among the top 25% experienced a risk that was 25% lower. When antioxidant nutrients were individually analyzed, greater daily intake of vitamin C, vitamin E, carotenoids and flavonoids was associated with a reduction in the risk of cognitive impairment. Among carotenoids, alpha carotene and beta cryptoxanthin were found to be protective and among flavonoids, anthocyanins, flavan-3-ols, flavones and flavonols were associated with lower risk. “These findings suggested that higher total antioxidant capacity of midlife diet was associated with lower odds of cognitive impairment in later life,” authors Li-Ting Sheng and colleagues concluded. “The generalizability of results to other populations remains to be confirmed, and future studies with repeated measures of dietary variables and cognitive functions are still needed. Beneficial effect of quercetin on ovalbumin-induced rhinitis Xian Jiao-tong University (China), April 8, 2021 According to news reporting originating in Shaanxi, People’s Republic of China, research stated, “Asthma is a chronic inflammatory airway disease, characterized by reversible goblet cells, smooth muscle hyperplasia, airflow obstruction, hyperactivity enhanced, ultra-structural remodeling, and airway mucus production. The current experimental study was aimed at scrutinizing quercetin’s inhibitory effect on airway inflammation in mice and its possible mechanism of action.” The news reporters obtained a quote from the research from Xi’an Jiaotong University, “The mice received varying doses of quercetin from 22-30 days (1, 10 and 50 mg / kg, p.o.) and montelukast (10 mg / kg, p.o.). Intranasal OVA has been instilled on the 21 days. Biochemical parameters, spleen weight, physiological parameters, interleukin (IL-113 and IL-6) parameters and immunoglobin-E (IgE) were calculated at the end of the experimental study. To investigate the potential mechanism of action, Paw edema and mast cell de granulation are estimated. Used to measure immune and inflammatory mediators, qRT-PCR technique. Quercetin significantly (P
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.11.292730v1?rss=1 Authors: Newman, T., Chang, H. F. K., Jabbari, H. Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus spreading across the world causing the disease COVID-19. The diagnosis of COVID-19 is done by quantitative reverse-transcription polymer chain reaction (qRT-PCR) testing which utilizes different primer-probe sets depending on the assay used. Using in silico analysis we aimed to determine how the secondary structure of the SARS-CoV-2 RNA genome affects the interaction between the reverse primer during qRT-PCR and how it relates to the experimental primer-probe test efficiencies. We introduce the program DinoKnot (Duplex Interaction of Nucleic acids with pseudoKnots) that follows the hierarchical folding hypothesis to predict the secondary structure of two interacting nucleic acid strands (DNA/RNA) of similar or different type. DinoKnot is the first program that utilizes stable stems in both strands as a guide to find the structure of their interaction. Using DinoKnot we predicted the interaction of the reverse primers used in four common COVID-19 qRT-PCR tests with the SARS-CoV-2 RNA genome. In addition, we predicted how 12 mutations in the primer/probe binding region may affect the primer/probe ability and subsequent SARS-CoV-2 detection. While we found all reverse primers are capable of interacting with their target area, we identified partial mismatching between the SARS-CoV-2 genome and some reverse primers. We predicted three mutations that may prevent primer binding, reducing the ability for SARS-CoV-2 detection. We believe our contributions can aid in the design of a more sensitive SARS-CoV-2 test. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.21.262279v1?rss=1 Authors: Wurlitzer, J. M., Stanisic, A., Wasmuth, I., Jungmann, S., Fischer, D., Kries, H., Gressler, M. Abstract: Fungi are traditionally considered as reservoir of biologically active natural products. However, an active secondary metabolism has long not been attributed to early diverging fungi such as Mortierella spec. Here, we report on the biosynthesis of two series of cyclic pentapeptides, the malpicyclins and malpibaldins, as products of Mortierella alpina ATCC32222. The molecular structures of malpicyclins were elucidated by HR-MS/MS, Marfey's method, and 1D and 2D NMR spectroscopy. In addition, malpibaldin biosynthesis was confirmed by HR-MS. Genome mining and comparative qRT-PCR expression analysis pointed at two pentamodular nonribosomal peptide synthetases (NRPS), malpicyclin synthetase MpcA and malpibaldin synthetase MpbA, as candidate biosynthetic enzymes. Heterologous production of the respective adenylation domains and substrate specificity assays proved promiscuous substrate selection and confirmed their respective biosynthetic roles. In stark contrast to known fungal NRPSs, MpbA and MpcA contain bacterial-like dual epimerase/condensation domains allowing the racemization of enzyme-tethered L-amino acids and the subsequent incorporation of D-amino acids into the metabolites. Phylogenetic analyses of both NRPS genes indicate a bacterial origin and a horizontal gene transfer into the fungal genome. This is the first report of nonribosomal peptide biosynthesis in basal fungi which highlights this paraphylum as novel and underrated resource of natural products. Copy rights belong to original authors. Visit the link for more info
While next generation sequencing enables researchers to unveil expression levels of the entire genome, qRT-PCR remains the gold standard for measuring transcript levels of individual genes for functional studies and for the purposes of publication. In this webinar, you will learn: • Low (1-5 genes) vs medium (~300 genes) throughput experimental design • Pros and cons of self-designed vs “off the shelf” assays • How to set up your wet lab experiments start to finish • Downloadable example step-by-step experiments with real data analysis and tutorial • Biological considerations (time series data, cell population frequency changes + more) • Examples of these techniques in publications • Common pitfalls and how to avoid them • Limitations of the technique • MIQE and publication standards Whether you are interested in a few genes or a few hundred, join Matthew Mule as he takes you through the necessary steps to validate expression levels of target genes using qRT-PCR with single gene assays and other medium-throughput platforms.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 18/19
Gestationsdiabetes mellitus (GDM) ist eine erstmalig in der Schwangerschaft aufgetretene oder diagnostizierte Glukosetoleranzstörung, die 3-8% aller Schwangerschaften betrifft (Metzger et al. 1998). Pathophysiologisch besteht eine große Ähnlichkeit zwischen GDM und Diabetes mellitus Typ 2, die genauen Mechanismen sind aber noch nicht bekannt (Metzger et al. 2007). Die Diagnose eines GDM bringt für Mutter und Kind verschiedene akute und langfristige Komplikationen mit sich. Veränderungen in der Plazenta mit ihren wichtigen metabolischen, endokrinen und immunologischen Funktionen während der Schwangerschaft (Gätje et al. 2011; Gude et al. 2004), könnten dabei eine wichtige Rolle spielen. Ziel dieser Studie war es daher, Expressionsveränderungen verschiedener wichtiger Rezeptoren, darunter nicht-steroidale Kernrezeptoren, wie der Vitamin D-Rezeptor (VDR), Peroxisome proliferator-activated receptor-gamma (PPARγ) sowie die Estrogen-Rezeptoren α und β (ERα und β)und Human leukocyte antigen-G (HLA-G), in der Plazenta bei GDM zu untersuchen, um dadurch die pathophysiologischen Vorgänge in der Plazenta bei GDM besser verstehen zu können. Für diese Studie wurden die Plazenten von 40 Patientinnen mit GDM und von 40 gesunden Frauen verwendet. Beide Gruppen enthielten jeweils 20 Plazenten von männlichen Feten und 20 von weiblichen. Zunächst wurde die Expression der einzelnen Rezeptoren mittels immunhistochemischen Färbungen untersucht. Signifikante Unterschiede in der Expression einzelner Rezeptoren wurden mit Hilfe weiterer Methoden, wie Doppelimmunfluoreszenz, qRT-PCR, Zellkultur oder rt-MSP bestätigt. In unserer Studie konnten wir zeigen, dass bei GDM eine gesteigerte Expression von VDR vorliegt. Im Gegensatz zum VDR fanden wir eine Inhibition der PPARγ-Expression bei GDM. Mit Hilfe unserer in vitro-Versuche konnten wir zusätzlich die konzentrationsabhängige Regulation der Expression von VDR und PPARγ durch ihre Liganden belegen. Bei GDM lag weiterhin eine erhöhte Expression von ERα vor. Zudem konnten wir zeigen, dass der ERα-Promotor in GDM-Plazenten demethyliert vorliegt. GDM positive Plazenten wiesen zudem eine verminderte Expression von ERβ und HLA-G auf. Abschließend konnten wir für den VDR und ERβ geschlechtsspezifische Unterschiede in der Kontrollgruppe identifizieren: Die Plazenten männlicher Feten exprimierten mehr VDR bzw. ERβ als die der weiblichen. Die besonders ausgeprägten Expressionsveränderungen im EVT an der maternalen-fetalen Grenzzone könnten dafür sprechen, dass diese Teil der Regulation der Immunantwort und der Insulinresistenz in GDM-Plazenten sind. Weiterhin scheinen die Expressionsveränderungen einiger Rezeptoren eine Folge der bei GDM veränderten Konzentrationen bzw. Zusammensetzung der Liganden zu sein. Unsere Studie liefert wichtige Vorkenntnisse, um mit Hilfe klinischer Studien die allgemeinen Empfehlungen zur Nahrungszusammensetzung und Supplementation in der Schwangerschaft zu klären.
Multiple Sclerosis Discovery: The Podcast of the MS Discovery Forum
[intro music] Host – Dan Keller Hello, and welcome to Episode Thirty-Seven of Multiple Sclerosis Discovery, the podcast of the MS Discovery Forum. I’m your host, Dan Keller. This week’s podcast features an interview with Jeanne Loring, who works with human induced pluripotent stem cells in a mouse model of MS. But to begin, we’d like to tell you about one of the most useful features of the MS Discovery Forum. Each week somewhere between 30 and 110 papers related to multiple sclerosis are published in the scientific literature. At MSDF, we endeavor to list them all, publishing links to a curated set of each week’s new papers every Friday at msdiscovery.org/papers. The first step in curating this list is an automated PubMed query that pulls all papers containing the terms multiple sclerosis, myelin, optic neuritis, acute disseminated encephalomyelitis, neuromyelitis optica, transverse myelitis, experimental autoimmune encephalomyelitis, cuprizone, neurodegeneration, microglia, and several related terms. This query returns many false positives. MSDF editors read all the titles and most of the abstracts and make judgments about which papers are directly relevant to MS or related disorders. Last week, for example, the query returned 139 papers and, in our judgment, only 58 of them – 42% – were truly MS-related. Some weeks the proportion is even lower than that. The query terms neurodegeneration, myelin, and microglia are responsible for most of the false positives. Neurodegeneration, in particular, returns many references related to other neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, stroke, and hypoxia, to name a few. Editorial judgments on which articles are relevant are often subjective, and we frequently struggle with those decisions. It’s easy to decide relevance when an article actually mentions multiple sclerosis. It’s harder when it mentions only myelin or only Th17 cells. If you think we’ve missed an important MS-related article—or if you think we’ve included an irrelevant article—I hope you’ll let us know by emailing us at editor@msdiscovery.org. And we’re open to suggestions on how to adjust our PubMed query to decrease false positives and false negatives. Once we’ve chosen which of the articles to include in the week’s list, we select between two and four of them as Editors’ Picks. Those are the week’s articles that seem to us to be the most important or interesting or intriguing. Once again, we invite readers to take issue with our choices. We’d love to hear about important articles that we have not designated Editors’ Picks or, on the contrary, Editors’ Picks that don’t deserve the honor. [transition music] Now to the interview. Dr. Jeanne Loring is Professor of Developmental Neurobiology and Director of the Center for Regenerative Medicine at the Scripps Research Institute in San Diego. She and her collaborators have been testing human neural precursor cells derived from embryonic stem cells in a mouse model of MS. The cells are injected into the spinal cords of immunocompetent mice with a model of MS induced by a neurotropic hepatitis virus. The cells are rejected within a week, but in that time they suppress the immune system and induce remyelination. Interviewer – Dan Keller In terms of how you came upon your most recent finding about human pluripotent stem cells in the mouse model of MS, could you give me a little bit of the back story? Interviewee – Jeanne Loring Oh yeah, sure. It was really interesting. So Tom Lane and I set out to try to develop a stem cell therapy for MS using human cells. So as a control experiment, we took human pluripotent stem cells, in this case embryonic stem cells, and turned them into neural precursors; differentiated them just a little bit. And then we transplanted them into Tom’s mouse model of MS. These mice were not immunosuppressed, and so we expected the cells to be rejected. And this was just our first experiment. But the results were not what we expected. After the cells were rejected, the mice started getting better, and their clinical scores improved. And then after several months, these mice were almost indistinguishable from normal mice. The first thing we thought was that we’d gotten the cages mixed up, and we were looking at something different. But we’ve repeated the experiment now more than a hundred times, and about 75% of the time we get the same result. So what this tells us is that these cells that we put into the animals are having some effect during the seven days that they exist in the animals that leads to both immunosuppression and remyelination and a clinical improvement which is quite remarkable. MSDF When you say 75% of the time, does that mean you get almost no effect 25 % of the time? Or does it mean that 75% or the mice? Because that would say whether you’re making your stem cells right or not. Dr. Loring It’s 75% of the mice. MSDF How do you explain it at this point, or where do you go from here to find a way to explain it? Dr. Loring So once we’d realized that we had a phenomenon that was repeatable, we realized that there was something special about these cells. And we tested other cell types, like the pluripotent stem cells that they were derived from, and human fibroblasts, and discovered that neither one of those was effective. And since then we’ve also tried other ways of making neural precursor cells, and those cells aren’t effective either. So it’s something extremely special about the cells that we used in these experiments, which is very lucky when you think about it. So we’ve now, both Tom and I – even though we’re not in the same place – we’ve set out to try to find out what it is about these cells that gives them these properties. Our first sort of cut on this – our hypothesis – is that the cells are secreting something that has a lasting effect. Our sort of big picture idea is that there are probably more than one protein or glycoprotein being secreted. And together they suppress the immune system so they act on the inflammatory response so that they increase the number of regulatory T cells that are produced and decrease the other T cell types. And they induce remyelination. So Tom is now working on trying to identify what factors these cells make that are inducing the T regulatory cells. And on my side, we’re trying to identify what it is that makes them remyelinate. MSDF When you make these cells, how do you know you got a good batch? Can you characterize them? Are there biomarkers, and you can say, “We did it right this time?” Dr. Loring Yes. In fact, that turns out to be really important because we did it wrong a few times. And we have a gene expression signature. It’s essentially diagnostic assay for this particular cell type. We’ve boiled it down to a set of qRT-PCR markers. And, because we have collaborators in Australia, we had to be able to transfer this quality control assay to them. So far it seems like those markers, I think it’s a group of 10 or 12 markers, seem to be predictive of the cells’ working in the animals. MSDF And just to clarify, that’s real time quantitative polymerase chain reaction? How are you going about characterizing what they’re doing? I mean, are you doing cytokine measurements or you’re looking at cells that get produced in the mice? Dr. Loring Tom is really handling the cells that get produced in the mouse. He’s doing the T cell analysis. What we’re doing, we developed an in vitro cell culture method to look for the effect of these cells on maturing oligodendroglia in culture. And we found that something secreted by these cells which shows up in their culture medium actually induces maturation of oligos – of OPCs – in vitro. I guess that’s another result that we didn’t expect to be quite so clear. So that shows that there’s something that is secreted by the cells. I mean, that’s the most likely idea. And on Tom’s side, he’s shown that the conditioned medium from the cells induces T-reg generation. And on our side, we’ve shown that conditioned medium from the cells induces oligo maturation. So now we’re trying to figure out what it is in that conditioned medium because now we think we can do a cell-free therapy for MS if we can identify what the factors are. It would be much simpler for us to do even a protein therapy for MS than it is to do a cell therapy. So both sides are taking sort of a candidate gene approach in which we’re identifying the proteins that are most highly specifically expressed in the cells that work in the mice. We have a list of those proteins, and we’ve sort of snatched a few candidates out of that group, and we’re testing to see whether each one of those proteins in purified form has the same effect as the conditioned medium. The other approach, which is more tedious but more likely to actually tell us what’s going on is for us to fractionate the medium into different sized proteins and then test each one of those fractions. We’re in the process of doing that right now. MSDF But it sounds like these cells are pluripotent. Not pluripotent in the normal sense of a stem cell leading to different lineages, but they have a couple of effects. One is the immunomodulatory, the other is regenerating oligodendrocytes. Do you think it really requires the gamish of proteins? If you fractionate them, will you possibly lose the signal? And that’s a big matrix to put back together again. Dr. Loring Yes, it is. And obviously, if we get no signal from our fractions, we’ll put our fractions back together again and try to find out whether – there are only three fractions, really, right now. So we’ll try different combinations of these fractions to try to find out if we can reproduce the effect. The effect is quite robust. We essentially get no maturation in medium conditioned by other cell types, but we get very strong maturation when we use conditioned medium from this particular neural precursor cell. MSDF If you only have three fractions now, is it because you just have chosen not to fractionate it even more until you know what’s going on? Dr. Loring Yes, we’re trying to hone in on it. So we don’t want too many different things to look at right away. I’m hoping that we find that only one of those fractions works, and that we can discover everything is within that fraction, but I really can’t predict what’s going to happen. MSDF It sounds like the approach would be to put everything in except one each time as opposed to keep adding back. You’ve got to find the one critical one missing that makes the thing not work. Dr. Loring Yes, and eventually we will do that with specific antibodies, but right now that is, since we don’t really have our candidates narrowed down enough, that isn’t a viable approach. But you’re absolutely right. We want to find out if that’s missing, whatever the things that are that are missing. And I’m hoping it’s not so complex that it’s five or six or seven proteins, because that’ll make it much harder for us. MSDF How do characterize the condition of the mice? Dr. Loring So that’s Tom’s area of expertise. It’s essentially an observation of the mice over time. We have a movie which I can show you, but I can’t actually do it in a recording. It’s quite obvious when the mice – they’re blind scored so the person who looks at the mice and sees how well they’re walking around doesn’t know whether they’re controls or experimentals. If you just see the movies that are selected at particular times after the cells have been transplanted, it’s quite dramatic. They have a much better clinical score. Essentially, they’re almost normal after six months. MSDF And how are you sure that the cells you injected into the spinal cord are gone, that they’ve been rejected completely? Dr. Loring That’s a good question. We used a method for live imaging of cells in which we use luciferase to label the cells. And then we used an instrument which allows us to image the cells in mice – in living mice – over time. So we did this in individual mice and saw that they disappeared over time. And after eight days we couldn’t detect them anymore. That doesn’t mean there isn’t one or two left because the resolution isn’t that high. We will go back eventually and look through sections of the spinal cords and see whether we can detect any. The other thing we can do is (skip 13:37) a human-specific markers. So we can just take a section of the spinal cord and find out if there’s any human cells in it at all, or any human genes in it at all. But we haven’t done that yet. MSDF Do the cells have to be gone? Have you tried injecting a second time? Dr. Loring No, we haven’t. We don’t know. We really don’t know. It would be very interesting if it reversed the effects. Then we’d really have a problem to solve. MSDF What else is there important to add or that we’ve missed that’s important to this kind of research? Dr. Loring So our dream is that we will identify a group of proteins and the concentration of those proteins necessary to have these two effects in this mouse model. And then we will do some biological engineering. We’ll be putting the cells into these little spheres and matrix that allows slow release of these proteins or controlled release of these proteins. And then, instead of putting cells in, we’ll put these beads in. And I don’t know whether that would end up being the final product or not, but there are lots of ways to deliver proteins, and this one I find rather attractive because it doesn’t require pumps or syringes. And I think that’s certainly the direction we’re going to try to go in. And so Tom Lane and I have just gotten an NIH grant for five years of funding, which seems like a very long time to me. So in five years we will have discovered the best way to deliver these things. We’ll discover what they are and the best way to deliver them. Tom has put conditioned medium into the mice, and it also works. MSDF Because I was going to ask, had you encapsulated the cells just to see that the supernatant does it without cell contact? Dr. Loring It turns out that the conditioned medium itself, you inject that into spinal cord, it’s not as dramatic an effect, but you have a clear clinical improvement. MSDF Have you tried injecting it either IV or intraperitoneally? Dr. Loring Yes. Well, we didn’t inject the conditioned medium. We did try injecting the cells, and they pretty much stayed where we injected them. These cells, unlike mesenchymal stem cells, they aren’t very migratory. So they don’t really have the receptors that cause them to move to areas of inflammation like CXCR4, for example; they don’t express that on their surfaces. So that does not seem to be a good delivery method for these cells. They don’t go anywhere. MSDF I was also thinking that if something they secrete is important, whether it circulates. Maybe they’re not making enough concentration if you inject them outside of the central nervous system, but it seems like you’re going to be faced with a little cumbersome problem in a clinical situation years and years ahead from now if you have to keep injecting proteins into the spinal cord as opposed to more peripheral. Dr. Loring I agree. And the solution to that is generally to look for peripheral effects and then try to suppress those when you do a therapy like this. That’s a long time, and we could certainly imagine how we would do it. But we need to know what those proteins are before we can decide on whether we expect them to have effects peripherally or not. But I agree with you; delivering them intravenously would be far easier. MSDF I know you have a lot of work ahead of you now with this, but is there another animal model of MS – or even another mouse model of MS – where you can see if it works even in a mouse model different from this one? Dr. Loring Yes, we’re actively pursuing that with our collaborators in Australia. And it’s interesting because they’ve gotten some positive preliminary results using the EAE model, but the approach to the EAE model I’ve realized is quite different. Generally, what people do is they provide the therapy at the time that the pathology is developing, and they try to prevent it, which is a really different idea than what we had using the mice that are already paralyzed. So they have found that if you can deliver the cells at least close to the spinal cord, then you can see some effects. The problem is that in Australia, and this is one of those technical things we had not anticipated, they don’t have permission to inject cells into the spinal cord. So they have to go through their animal rights people or their animal protection groups and try to get permission to do so. So with Craig Walsh at UC Irvine we have started doing parallel experiments with the EAE model. I’m not necessarily sure that it’s going to have similar effects. I mean, I really don’t know. MSDF Can you describe how these mice in your experiments were made to have MS? Dr. Loring Yes, they were given a virus, a neurotropic virus, which kills off the oligodendroglia. They become demyelinated, and there is a secondary inflammatory response. So the mice are actually paralyzed in their hind quarters at least by the time we put the cells in. They have to be fed by hand. So this is not a trivial thing to do. But we’re trying to reproduce the effects during the progressive form of MS, for example, or during an attack of MS. So we’re trying to repair the mice or cure the mice that are in a condition which would be similar to the worst case scenario for people with MS. MSDF Do you think this may also have effects not only on the myelin, but also on damaged neurons? Dr. Loring We don’t know, because the mice haven’t really had enough time to get a lot of neuronal damage, but that’s a very good question. We don’t know yet. MSDF I appreciate it. Thank you. Dr. Loring You’re welcome. It was a pleasure. [transition music] Thank you for listening to Episode Thirty-Seven of Multiple Sclerosis Discovery. This podcast was produced by the MS Discovery Forum, MSDF, the premier source of independent news and information on MS research. MSDF’s executive editor is Robert Finn. Msdiscovery.org is part of the non-profit Accelerated Cure Project for Multiple Sclerosis. Robert McBurney is our President and CEO, and Hollie Schmidt is vice president of scientific operations. Msdiscovery.org aims to focus attention on what is known and not yet known about the causes of MS and related conditions, their pathological mechanisms, and potential ways to intervene. By communicating this information in a way that builds bridges among different disciplines, we hope to open new routes toward significant clinical advances. We’re interested in your opinions. Please join the discussion on one of our online forums or send comments, criticisms, and suggestions to editor@msdiscovery.org. [outro music]
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 05/06
Chemotherapeutic treatment of hepatocellular carcinoma often leads to chemoresistance during therapy or upon relapse of tumors. For the development of better treatments, a better understanding of biochemical changes in the resistant tumors is needed. Therefore, especially in vivo models are very important tools to generate standardized cell-material, which can be examined by high throughput techniques. Thus, it should be possible to find new targets for therapy or even for diagnostic. This thesis focusses on the characterization of the in vivo chemoresistant human hepatocellular carcinoma HUH-REISO established from a metronomically cyclophosphamide (CPA) treated HUH7 xenograft mouse model. First step of the work was the establishment of the xenograft mouse model. SCID mice bearing subcutaneous HUH7 tumors were treated i.p. with 75 mg/kg CPA every six days. After 10 weeks of response to the therapy, the tumor growth relapsed and tissue grew with very fast doubling time again, despite of ongoing treatment. This aggressive manner of growth under therapy could be also observed in a re-implantation study where the reisolated CPA chemoresistant HUH-REISO tumors grew without a lag phase, indicating an endogenous imprinted component. To evaluate this, tumors were examined by immunohistochemistry, a functional blood-flow Hoechst dye assay, and qRT-PCR for ALDH-1, Notch-1, Notch-3, HES-1, Thy-1, Oct-4, Sox-2 and Nanog mRNA levels. Histochemical analysis of HUH-REISO tumors revealed significant changes in host vascularization of tumors and especially in expression of the tumor-derived human endothelial marker gene PECAM-1/CD31 in HUH-REISO in comparison to parental HUH-7 cells and in vivo passaged HUH-PAS cells (in vivo grown without chemotherapeutic CPA pressure). The pronounced network of host murine vascularization in parental HUH-7 tumors was completely substituted by a network of human and murine vessel-like structures in HUH-REISO tumors under therapy. In addition, cell lines of these tumors were analyzed in endothelial trans-differentiation studies on matrigel. In those studies with limited oxygen and metabolite diffusion, followed by a matrigel assay, only the chemoresistant HUH-REISO cells exhibited tube formation potential and expression of human endothelial markers ICAM-2 and PECAM-1/CD31. Such a trans-differentiation capacity requires a lineage of cells with pluripotent capacities like so called tumor stem cells. Indeed, I could show in a comparative study on stemness and plasticity markers that Thy-1, Oct-4, Sox-2 and Nanog were upregulated in resistant xenografts. Furthermore, under therapeutic pressure by CPA, tumors of HUH-PAS and HUH-REISO displayed regulations in Notch-1 and Notch-3 expression, which I could also show by qRT-PCR. Notch-1 raised in HUH-PAS under therapeutic pressure, meanwhile it was conversely regulated in comparison to Thy-1, Oct-4, Sox-2 and Nanog in HUH-REISO. In both groups Notch-3 was inducible by 2 times CPA treatment and fell back on base level after further four therapeutic cycles in HUH-REISO. To conclude all these finding: chemoresistance of HUH-REISO was not manifested under standard in vitro, but only under in vivo conditions. HUH-REISO cells showed increased pluripotent capacities and the ability of trans-differentiation to endothelial like cells in vitro and in vivo. These cells expressed typical endothelial surface marker and functionality. Although the mechanism behind chemoresistance of HUH-REISO and involvement of plasticity remains to be clarified, we hypothesize that the observed Notch regulations and upregulation of stemness genes in resistant xenografts are involved in the observed cell plasticity.
Background: Radiotherapy, administered in fractionated as well as ablative settings, is an essential treatment component for breast cancer. Besides the direct tumor cell death inducing effects, there is growing evidence that immune mechanisms contribute - at least in part - to its therapeutic success. The present study was designed to characterize the type and the extent of cell death induced by fractionated and ablative radiotherapy as well as its impact on the release of monocyte migration stimulating factors by dying breast cancer cells. Methods: Cell death and senescence assays were employed to characterize the response of a panel of breast cancer cell lines with different receptor and p53 status towards.-irradiation applied in a fractionated (daily doses of 2 Gy) or ablative setting (single dose of 20 Gy). Cell-free culture supernatants were examined for their monocyte migration stimulating potential in transwell migration and 2D chemotaxis/chemokinesis assays. Irradiation-induced transcriptional responses were analyzed by qRT-PCR, and CD39 surface expression was measured by flow cytometry. Results: Fast proliferating, hormone receptor negative breast cancer cell lines with defective p53 predominantly underwent primary necrosis in response to.-irradiation when applied at a single, ablative dose of 20 Gy, whereas hormone receptor positive, p53 wildtype cells revealed a combination of apoptosis, primary, and secondary (post-apoptotic) necrosis. During necrosis the dying tumor cells released apyrase-sensitive nucleotides, which effectively stimulated monocyte migration and chemokinesis. In hormone receptor positive cells with functional p53 this was hampered by irradiation-induced surface expression of the ectonucleotidase CD39. Conclusions: Our study shows that ablative radiotherapy potently induces necrosis in fast proliferating, hormone receptor negative breast cancer cell lines with mutant p53, which in turn release monocyte migration and chemokinesis stimulating nucleotides. Future studies have to elucidate, whether these mechanisms might be utilized in order to stimulate intra-tumoral monocyte recruitment and subsequent priming of adaptive anti-tumor immune responses, and which breast cancer subtypes might be best suited for such approaches.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 16/19
Die vorliegend präsentierten Experimente hatten zum Ziel, die hypothetische Rolle von Gq/11- bzw. G12/13-koppelnden heptahelikalen Transmembrandomänenrezeptoren (7TMR) als Mechanosensoren für die Initiation des myogenen Tonus zu untersuchen. Um festzustellen, welche 7TMR hierfür besonders relevant sind, wurden Leitungsgefäße mit Widerstandsgefäßen in ihren RNA-Leveln für eine Reihe von 7TMR verglichen. Dies geschah auf der Grundlage, dass Leitungsgefäße einen geringeren myogenen Tonus aufweisen als Widerstandsgefäße. Eine aus höheren RNA-Leveln ableitbare größere Anzahl an putativen Sensormolekülen sollte also über eine größere Mechanosensitivität des Gefäßes zu einem höheren Ausmaß an myogener Vasokonstriktion führen. Die RNA-Level wurden mittels quantitativer RT-PCR (qRT-PCR) bestimmt. Die Quantifizierung erfolgte relativ zum geometrischen Mittel dreier Haushaltsgene. Die untersuchten Widerstandsgefäße umfassten kleine Mesenterialarterien, Nierenarterien und Gehirnarterien. Die untersuchten Leitungsgefäße umfassten die A. mesenterica superior, Bauchaorta, A. carotis communis und die Pulmonalarterie. Folgende Rezeptoren stellten sich in der qRT-PCR aufgrund ihres Expressionsprofils als vielversprechende molekulare Sensorproteine in Widerstandsgefäßen heraus: AT1B-Angiotensinrezeptor, ETA-Endothelinrezeptor, V1A-Vasopressinrezeptor, �1A-Adrenozeptor. In einem zweiten Schritt wurde versucht, über pharmakologische Inhibition der vorgenannten Rezeptoren eine Reduktion des myogenen Tonus zu erreichen. Die eingesetzte Methode war die isobare Konstriktionsmessung (Arteriographie) an isolierten kleinen Mesenterialarterien. Die Methode erforderte vor der eigentlichen Applikation der Pharmaka die Registrierung eines myogenen Tonus in Abwesenheit jeglicher Pharmaka. Dann erst wurde der myogene Tonus unter Anwesenheit von Pharmaka ein zweites Mal registriert. Bei der genaueren Analyse des ersten myogenen Tonus fiel dessen bisigmoider Verlauf auf. Möglicherweise liegt dieser charakteristischen Form eine zeitversetzte Aktivierung der an die putativen mechanosensitiven 7TMR koppelnden G-Proteine zugrunde: Zunächst werden wahrscheinlich Gq/11-Proteine aktiviert, dann G12/13-Proteine. Bei der Analyse des Kurvenverlaufs zum zweiten myogenen Tonus zeigte sich unter Kontrollbedingungen, d.h. unter Abwesenheit von Pharmaka, eine Linksverschiebung relativ zum ersten myogenen Tonus. Darüberhinaus änderte sich die Kurvenform von bisigmoid zu monosigmoid. Wahrscheinlich sind auch für diese Charakteristika Eigenheiten der an die putativ mechanosensitiven 7TMR koppelnden G-Proteine verantwortlich: Die im Zuge des ersten myogenen Tonus aktivierten G12/13-Proteine inaktivieren möglicherweise langsamer durch GTP-Hydrolyse als die Gq/11-Proteine. Deshalb könnten zu Beginn des zweiten myogenen Tonus beide G-Protein-Spezies aktiv sein und so die Mechanosensitivität der glatten Muskelzellen drastisch erhöhen, was die Linksverschiebung erklären würde. Die nun konzertiert erfolgende G-Protein-Aktivierung könnte ferner den monosigmoiden Kurvenverlauf erklären. Die Applikation der Pharmaka erfolgte zunächst als Kombination von antagonistischen Substanzen an AT1-, ETA-, V1A- und �1-Rezeptoren. Eingesetzt wurden Candesartan, BQ-123, Relcovaptan und Prazosin. Diese Kombination reduzierte den myogenen Tonus signifikant in seiner Amplitude. Anschließend wurde Prazosin als Monosubstanz getestet. Der Hemmeffekt unterschied sich nicht von der ursprünglichen Viererkombination. Schließlich erfolgte eine Testung der ursprünglichen Kombination unter Auslassung von Prazosin. Auch hier war der hemmende Effekt derselbe. Zur Erklärung dieser Befunde wurde das Konzept der negativen Interferenz herangezogen: Dabei konkurrieren 7TMR um einen limitierten Pool an G-Proteinen. Inverse Agonisten (wie sie die Substanzen Candesartan und Prazosin darstellen) führen zu einer Sequestrierung von G-Proteinen an den jeweiligen Rezeptoren ohne folgende Signaltransduktion. Dabei könnte die Applikation eines inversen Agonisten dieselben Effekte erzielen wie eine kombinierte Applikation. Letztlich konnte durch den hemmenden Effekt von Prazosin für �1-Adrenozeptoren bestätigt werden, dass sie eine mechanosensitive Funktion ausüben. Unter Berücksichtigung der Ergebnisse des qPCR-Teils handelt es sich wahrscheinlich um �1A-Adrenozeptoren. Deren Mechanosensitivität kann einen Teil der Kontraktion glatter Muskelzellen auf einen steigenden intravasalen Druck vermitteln und damit einen entsprechenden Anteil am myogenen Tonus erklären.
Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 06/07
In dieser Studie wurde die Kompetenz des Immunsystems von Turopolje (TxT), Deutsche Landrasse x Pietrain (LxP) und Deutsche Landrasse x Turopolje (LxT) verglichen. Die verschiedenen Rassen sind Vertreter einer alten und einer modernen Rasse und einer Kreuzung von beiden. Hauptziel war es zu untersuchen, ob sich die verschiedenen Rassen in ihrer Immunabwehr gegenüber einer Infektion unterscheiden und wie das Immunsystem durch Stressoren belastet wird. Außerdem wurde untersucht, ob sich LxT zur kommerziellen Mast eignet. Unterschiede in der Sekretion von Immunglobulin G und M im Kolostrum und reifen Milch der Deutsche Landrasse und Turopolje Sauen, sowie deren Aufnahme durch die Ferkel wurde mittels ELISA untersucht. Nach dem Absetzen der Ferkel wurden zwei getrennte Gruppen gebildet: Die erste Gruppe wurde mit einem attenuierten Lebendimpfstoff gegen das Porzine Reproduktive und Respiratorische Syndrom Virus (PRRS MLV) immunisiert, um eine Infektion zu simulieren. Die Fragmente des PRRS MLV wurden aus dem Serum, den Leukozyten, den Tonsillen und dem Lymphonodus tracheobronchale extrahiert und mittels qRT-PCR gemessen. Durch ELISA wurden die Konzentrationen der Interleukine-1β, 6, 10 und 12 gemessen. Die Genexpression von CD163, SIGLEC1, Mx1, TLR7 und TLR8, TRAF6, Myd88, Interleukin 1, 6, 8, 10, 12, TNFα, TGFβ und CXCL12 wurde näher untersucht. Innerhalb der nicht geimpften Gruppe untersuchte man den Einfluss von Stress auf das Immunsystem. Hierbei wurde die Konzentration von Interleukin 6, 10, 12 im Plasma mittels ELISA, die Genexpression in den Lymphozyten durch qRT-PCR von Interleukin 1β, 6, 10, 12 und TNFα bestimmt. Außerdem wurde eine mitogenstimulierte Lymphozytenproliferation mittels Lumineszenzmessung durchgeführt. Bei beiden Gruppen wurde ein Differentialblutbild angefertigt, um Veränderungen im weißen Blutbild untersuchen zu können. Weiterhin wurde mittels ELISA die Immunglobulinkonzentration G und M im Serum untersucht. Es wurde in der Gruppe der immunisierten Tiere sichtbar, dass die Rassen unterschiedlich auf die Vakzination reagierten. TxT zeigt keine Konzentrationsveränderung von Interleukin 1β im Plasma. Durch die unveränderte Konzentration des Interleukins könnten vermehrt zytotoxische T Zellen gebildet werden. Als Folge wird TNFα aufreguliert. TNFα inhibiert CD163, daher wird nur eine geringe Anzahl von B-Zellen aktiviert und es werden spezifische Antikörper gebildet. Im Gegensatz dazu reagieren die beiden anderen Rassen mit einer Immunantwort des Typs 2. Die oben beschriebene Inhibierung kann nicht stattfinden und es kommt zur Synthese der B-Zellen und zu einer erhöhten Konzentration an Immunglobulinen und spezifischen Antikörpern. Die Ergebnisse meiner Studie können tendenziell den Einfluss des Stresses auf das Immunsystem bestätigen. So deuten bei TxT die geringere Immunglobulinkonzentration und das Differentialblutbild darauf hin, dass die Immunreaktion auf Stress eher auf T-Zellen basiert (Immunreaktion Typ 1). Auch bei LxT und LxP scheint es, dass die Immunantwort Typ2 und eine Hochregulation der Genexpression von IL6 und die Konzentration im Plasma dominieren. Weiterhin besteht eine Tendenz, dass TxT auf Stress robuster reagieren als die beiden anderen Rassen. Nach der Schlachtung wurden die Schweinehälften aller Rassen und Gruppen mittels der SEUROP-Klassifizierung eingeteilt und bewertet. Bei Schweinen, die in der 25. Lebenswoche geschlachtet wurden, untersuchte man zusätzlich den Tropfsaftverlust und das intramuskuläre Fett. Im Vergleich der Schachtkörper und Fleischqualität schnitten die Tiere der Kreuzungsrasse (LxT) qualitativ am besten ab. Schlussfolgernd ist die Kreuzungsrasse (LxT) zur Mästung als Nutzungsrasse geeignet. Sie stellt eine Bereicherung innerhalb der kommerziellen Schweinefleischproduktion dar.
Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 06/07
This work employed a mouse model of liver specific depletion of the gene Cdh1 and its respective protein E cadherin to study the role of this protein in liver homeostasis and pathophysiology. The experiment was done with specific focus on the effects concerning hepatocellular carcinoma (HCC) development. Background: Cadherins are present in all higher organisms, and have been studied rigorously in the past. The cadherin family is huge, encompassing more than 400 (known) members. E cadherin is the name-giver of that family and is considered to be of great importance to a broad range of physiological and pathophysiological functions. Known functions include cell-cell adhesion and deregulation of E-cadherin (in almost all cases a down-regulation) is associated with increased aggressiveness in both human and animal tumors. Aside from that, E-cadherin is of great importance during embryogenesis. Worldwide, HCC is an important disease in humans, especially in certain countries (mostly developing countries). While females are only occasionally affected by HCC, it ranks among the top 3 tumor-related death causes in males. The difficulties in treating this tumor curatively make research of genes or proteins relevant to HCC important for human medicine improvement. The existence of a connection between Cdh1 or E Cadherin and HCC has been suggested, but more research is still required. Methods: Employing Cre/loxP technology, a mouse model of liver specific E cadherin depletion was created (L Cdh1del/del). The mice were compared to littermates with normal Ecadherin levels (L Control). Mice body and organ weight was documented at different ages, and liver tissue was analyzed using qRT-PCR (cDNA), Western blot, histochemistry and immunohistochemistry. To test effects of the reduced E-cadherin on tumor development, a cohort of male mice was injected with a chemical carcinogen (DEN) at two weeks of age to induce HCC, and mice were analyzed 4, 8 or 12 months later. Results: Aside from a slight retardation in weight gain, L Cdh1del/del did not suffer from severe health effects or spontaneous tumor development. Histology showed some alterations around the small bile ducts in the liver (in the periportal fields) and RNA analysis showed that mice underwent a phase of considerably altered RNA activity (429 significantly regulated genes at 3 weeks of age), but later only a few up/down-regulated genes remained (28 genes at 6 weeks of age). Aside from Cdh1, no genes considered cadherin family members were regulated. Western blot analysis, qRT-PCR and IHC confirmed that E cadherin was down regulated on RNA level and on protein level in this animal model. All mice injected with DEN developed tumors, but L Cdh1del/del were affected more heavily, with tumors reaching large diameters faster. If mice were kept longer than 8 months, L Cdh1del/del had to be euthanized significantly earlier than L Control. A spin-off of the model was the establishment of a permanent cell line, developed from a liver tumor of a L Cdh1del/del mouse. PCR requiring a functional primer binding site on exon 10 of Cdh1 could not produce DNA product, indicating that the cell line was a derivative of an E-cadherin negative liver cell. Conclusion: Liver specific E cadherin reduction had a surprisingly small effect in the present mouse model (compared to the effects of E cadherin loss in organs like the skin or intestine, as documented in the literature) if mice were not challenged with a chemical carcinogen. If mice were challenged with experimental HCC induction, lack of E cadherin had a strong effect on the tumor growth. These findings attest, by an experimental animal model, the importance of E cadherin for tumor development in the liver. This data reinforces previous observations concerning E cadherin effects on tumors in studies working with resected human tumors of the liver or with conditional organ specific mouse models studying carcinoma in other organs (like the mammary gland, for example). Therefore, this animal model could help improve the understanding of mechanisms regulating aggressiveness in human tumors.
Background: Chemotherapeutic treatment of hepatocellular carcinoma often leads to chemoresistance during therapy or upon relapse of tumors. For the development of better treatments a better understanding of biochemical changes in the resistant tumors is needed. In this study, we focus on the characterization of in vivo chemoresistant human hepatocellular carcinoma HUH-REISO established from a metronomically cyclophosphamide (CPA) treated HUH7 xenograft model. Methods: SCID mice bearing subcutaneous HUH7 tumors were treated i.p. with 75 mg/kg CPA every six days. Tumors were evaluated by immunohistochemistry, a functional blood-flow Hoechst dye assay, and qRT-PCR for ALDH-1, Notch-1, Notch-3, HES-1, Thy-1, Oct-4, Sox-2 and Nanog mRNA levels. Cell lines of these tumors were analyzed by qRT-PCR and in endothelial transdifferentiation studies on matrigel. Results: HUH-REISO cells, although slightly more sensitive against activated CPA in vitro than parental HUH-7 cells, fully retained their in vivo CPA chemoresistance upon xenografting into SCID mice. Histochemical analysis of HUH-REISO tumors in comparison to parental HUH-7 cells and passaged HUH-PAS cells (in vivo passaged without chemotherapeutic pressure) revealed significant changes in host vascularization of tumors and especially in expression of the tumor-derived human endothelial marker gene PECAM-1/CD31 in HUH-REISO. In transdifferentiation studies with limited oxygen and metabolite diffusion, followed by a matrigel assay, only the chemoresistant HUH-REISO cells exhibited tube formation potential and expression of human endothelial markers ICAM-2 and PECAM-1/CD31. A comparative study on stemness and plasticity markers revealed upregulation of Thy-1, Oct-4, Sox-2 and Nanog in resistant xenografts. Under therapeutic pressure by CPA, tumors of HUH-PAS and HUH-REISO displayed regulations in Notch-1 and Notch-3 expression. Conclusions: Chemoresistance of HUH-REISO was not manifested under standard in vitro but under in vivo conditions. HUH-REISO cells showed increased pluripotent capacities and the ability of transdifferentiation to endothelial like cells in vitro and in vivo. These cells expressed typical endothelial surface marker and functionality. Although the mechanism behind chemoresistance of HUH-REISO and involvement of plasticity remains to be clarified, we hypothesize that the observed Notch regulations and upregulation of stemness genes in resistant xenografts are involved in the observed cell plasticity.
Introduction: Circulating microRNAs (miRNAs) are easily accessible and have already proven to be useful as prognostic markers in cancer patients. However, their origin and function in the circulation is still under discussion. In the present study we analyzed changes in the miRNAs in blood plasma of head and neck squamous cell carcinoma (HNSCC) patients in response to radiochemotherapy and compared them to the changes in a cell culture model of primary HNSCC cells undergoing simulated anti-cancer therapy. Materials and methods: MiRNA-profiles were analyzed by qRT-PCR arrays in paired blood plasma samples of HNSCC patients before therapy and after two days of treatment. Candidate miRNAs were validated by single qRT-PCR assays. An in vitro radiochemotherapy model using primary HNSCC cell cultures was established to test the possible tumor origin of the circulating miRNAs. Microarray analysis was performed on primary HNSCC cell cultures followed by validation of deregulated miRNAs via qRT-PCR. Results: Unsupervised clustering of the expression profiles using the six most regulated miRNAs (miR-425-5p, miR-21-5p, miR-106b-5p, miR-590-5p, miR-574-3p, miR-885-3p) significantly (p = 0.012) separated plasma samples collected prior to treatment from plasma samples collected after two days of radiochemotherapy. MiRNA profiling of primary HNSCC cell cultures treated in vitro with radiochemotherapy revealed differentially expressed miRNAs that were also observed to be therapy-responsive in blood plasma of the patients (miR-425-5p, miR-21-5p, miR-106b-5p, miR-93-5p) and are therefore likely to stem from the tumor. Of these candidate marker miRNAs we were able to validate by qRT-PCR a deregulation of eight plasma miRNAs as well as miR-425-5p and miR-93-5p in primary HNSCC cultures after radiochemotherapy. Conclusion: Changes in the abundance of circulating miRNAs during radiochemotherapy reflect the therapy response of primary HNSCC cells after an in vitro treatment. Therefore, the responsive miRNAs (miR-425-5p, miR-93-5p) may represent novel biomarkers for therapy monitoring. The prognostic value of this exciting observation requires confirmation using an independent patient cohort that includes clinical follow-up data.
Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 05/07
Die Mastitis des Rindes ist die wirtschaftlich bedeutendste Einzeltiererkrankung in der Milchwirtschaft. Staphylococcus aureus (S. aureus) und Escherichia coli (E. coli) zählen zu den wichtigsten Mastitiserregern, die jedoch zumeist sehr unterschiedliche Krankheitsbilder hervorrufen. So verursacht E. coli hauptsächlich transiente, akute klinische Mastitiden, während S. aureus als bedeutendster Verursacher chronischer bis subklinischer Mastitiden mit persistierendem Verlauf angesehen wird. In den letzten Jahrzehnten wurde die Pathophysiologie der entzündeten Milchdrüse eingehend wissenschaftlich bearbeitet. Frühe Zeitpunkte einer intramammären Infek-tion, zu denen die Pathogene zwar bereits erkannt wurden, aber noch keine klinischen Symptomen in Erscheinung getreten sind, wurden bislang in vivo noch nicht untersucht. Zu den ersten wirtsseitigen Ereignissen nach dem Eindringen und Erkennen der Patho-gene zählen Veränderungen in der Abundanz von mRNA-Transkripten immun-relevanter Gene. Vertreter dieser differentiell exprimierten Gene gehören z.B. zu den Zytokinen, Chemokinen und antimikrobiellen Peptiden. Diese sollen es dem Wirt ermöglichen, eine adäquate Immunantwort zu initiieren, welche als entscheidend für den klinischen Verlauf der Erkrankung gilt. In der vorliegenden Arbeit wurde ein Tiermodell zur Simulation der ersten 3 h nach dem Eindringen der Pathogene E. coli und S. aureus in das Euter etabliert. Das Hauptaugenmerk lag hierbei auf der Untersuchung von Expressionsänderungen ausgewählter Kandidatengene nach unterschiedlich langer Erregerexposition (1-3 h) in verschiedenen Kompartimenten der Milchdrüse. Dabei wurden die Lokalisationen Zitzenzisterne (ZZ), Drüsenzisterne (DZ) und das ventrale Euterparenchym (EU) näher untersucht. Großes Augenmerk lag auf einer strengen Standardisierung der Versuchs-tiere, um die Vergleichbarkeit der erzielten Ergebnisse sicherzustellen. Der Versuch umfasste 12 erstlaktierende Kühe der Rasse Holstein-Friesian mit makelloser Allgemein- und Eutergesundheit und einer Zellzahl < 50.000/ml Milch in allen 4 Eutervierteln. Bei den Tieren wurden 3 Euterviertel jeweils sequentiell über einen Zeitraum von 1, 2 und 3 h entweder mit 5*106 CFU E. coli1303 (n = 6) oder S. aureus1027 (n = 6) intrazisternal inokuliert. Ein Kontrollviertel blieb unbehandelt und diente der Ermittlung von Basisexpressionswerten. Mit Hilfe der unterschiedlich lange inokulierten Viertel konnte ein zeitlicher Verlauf der Expressionsänderungen ausgewählter Kandidatengene extrapoliert werden. Wie erwartet traten innerhalb des dreistündigen Versuchszeitraumes keinerlei klinische Effekte bei den Versuchstieren auf. Die innere Körpertemperatur, der Leukozytengehalt im Blut, der SCC und verschiedene Milchinhaltsstoffe erfuhren keine Veränderungen, die auf die Pathogen-Exposition zurückzuführen waren. Durch wiederholte Unter-suchung von Milchproben im Versuchsverlauf wurde die bakterielle Vermehrung analysiert. Hierbei konnte eine Vervielfachung von E. coli um durchschnittlich das 30-fache festgestellt werden, wohingegen sich S. aureus innerhalb der ersten 3 h p. inoc. vergleichsweise schwach vermehrte (durchschnittlich 1,8-fach). Die beiden im Tiermodell verwendeten Bakterienstämme wurden parallel in vitro in Milch und in Nährmedium kultiviert, um deren Wachstum und Adaptationsfähigeit zu untersuchen. E. coli erreichte nach 14-stündiger Inkubation in Milch ein Maximum von 1,2*109 ± 2,5*108 CFU/ml (MW ± SD), während S. aureus mit 5,3*108 ± 8,9*108 CFU/ml (MW ± SD) eine deutlich schwächere und heterogenere Vermehrung zeigte. Im Anschluss an diese Vorinkubation in Vollmilch wurde untersucht, ob sich die Pathogene an das spezifische Wachstumsmedium Milch anpassen konnten. Nach Umsetzen in frische Vollmilch zeigten die Pathogene jedoch kein verbessertes und beschleunigtes Wachstum im Vergleich zu in standardisiertem Medium vorkultivierten Bakterien. Um die Vergleichbarkeit mit früheren Experimenten sicherzustellen, wurden E. coli und S. aureus deshalb mit Nährmedium für die in vivo-Versuche präpariert. Drei Stunden nach Inokulation des ersten Euterviertels wurden die Tiere getötet und Gewebeproben innerhalb von 20 min aus den drei zu untersuchenden Lokalisationen (ZZ, DZ, EU) der einzelnen Euterviertel gewonnen. Die Proben wurden unmittelbar nach Entnahme in Stickstoff schockgefroren und bis zur Aufarbeitung bei -80°C tiefgefroren. Im Rahmen weiterer Untersuchungen fand dann die mRNA-Extraktion sowie die Analyse der Transkript-Abundanzen entzündungsrelevanter Kandidatengene (IL6, TNF, CXCL8, CCL20, S100A9, LAP, LCN2, MX2 und CYP1A1) mittels qRT-PCR statt. Die Auswahl geeigneter Gene erfolgte sowohl anhand eines orientierenden Vorversuchs in vivo als auch anhand bekannter, in vergleichbaren in vitro-Experimenten regulierten Genen der Milchdrüsenepithelzelle. Es konnte gezeigt werden, dass die Expressionsänderungen nach Kontakt mit E. coli deutlich stärker und homogener ausfielen als nach Kontakt mit S. aureus. Bereits nach 1-stündiger Erregerexposition konnten signifikante Steigerungen der mRNA-Expression einiger Gene verzeichnet werden. Dies galt vor allem für die Chemokine CCL20 und CXCL8 sowohl nach Exposition mit E. coli, als auch mit S. aureus. Für die Zytokine IL6 und TNF konnte eine rasche mRNA-Expressionssteigerung nach 1-stündiger intramammärer Inokulation von E. coli nachgewiesen werden, während eine Regulation im Euter mit S. aureus inokulierter Tiere erst nach 2 h und vergleichsweise schwächer eintrat. Die antibakteriell wirkenden Faktoren S100A9 und LAP waren den Chemo-kinen und Zytokinen zeitlich nachgeschaltet, wurden aber nur nach Exposition mit E. coli deutlich hochreguliert. Im Gegensatz zu in vitro-Untersuchungen mit Milch-drüsenepithelzellen konnte für die Gene LCN2, MX2 und CYP1A1 keine nennenswerte Regulation nach Inokulation mit E. coli und S. aureus festgestellt werden. Des Weiteren fiel auf, dass die untersuchten Kandidatengene invariant stärker in ZZ und DZ heraufreguliert wurden, als im EU. Meist ähnelten sich ZZ und DZ in Stärke und Verlauf der Expressionsänderung. Im ventralen Euterparenchym dagegen konnte nach Inokulation von E. coli nur für die Gene IL6, TNF, CXCL8 und S100A9 eine vergleichsweise schwache, aber statistisch signifikante Steigerung der mRNA-Expression aufgezeigt werden. Nach Inokulation mit S. aureus konnte in dieser Lokalisation keine Hochregulation der untersuchten Kandidatengene festgestellt werden. Das in dieser Arbeit etablierte Tiermodell zeigt erstmalig in vivo die frühe patho-genspezifische und kompartimentabhängige Regulation immunrelevanter Gene im Eutergewebe der Milchkuh auf. Es bietet damit eine gute Basis für holistische Ansätze zur Untersuchung sehr früher Ereignisse bei der Wirt-Pathogen-Interaktion. Mittelfristig soll hiermit aufgeklärt werden, welche wirts- und pathogenseitigen Mechanismen zur Entstehung akuter und chronischer Mastitiden führen und welche Faktoren persistente Infektionen der Milchdrüse fördern oder verhindern. Detaillierte Kenntnisse über solche frühen Ereignisse ebnen den Weg, Ansätze für eine verbesserte Mastitis-Diagnostik, -Prophylaxe und -Therapie bei der bovinen Mastitis zu finden.
Introduction. Dendritic cells (DCs) and oxLDL play an important role in the atherosclerotic process with DCs accumulating in the plaques during plaque progression. Our aim was to investigate the role of oxLDL in the modulation of the DC homing-receptor CCR7 and endothelial-ligand CCL21. Methods and Results. The expression of the DC homing-receptor CCR7 and its endothelial-ligand CCL21 was examined on atherosclerotic carotic plaques of 47 patients via qRT-PCR and immunofluorescence. In vitro, we studied the expression of CCR7 on DCs and CCL21 on human microvascular endothelial cells (HMECs) in response to oxLDL. CCL21- and CCR7-mRNA levels were significantly downregulated in atherosclerotic plaques versus non-atherosclerotic controls 90% for CCL21 and 81% for CCR7 (P < 0.01)]. In vitro, oxLDL reduced CCR7 mRNA levels on DCs by 30% and protein levels by 46%. Furthermore, mRNA expression of CCL21 was significantly reduced by 50% (P < 0.05) and protein expression by 24% in HMECs by oxLDL (P < 0.05). Conclusions. The accumulation of DCs in atherosclerotic plaques appears to be related to a downregulation of chemokines and their ligands, which are known to regulate DC migration. oxLDL induces an in vitro downregulation of CCR7 and CCL21, which may play a role in the reduction of DC migration from the plaques.
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 04/06
Die vorliegende Dissertation befasst sich mit den durch S-Lost-verursachten Symptomen in der Haut, die zunächst durch starke Blasenbildung und später durch eine verzögerte Wund-heilung charakterisiert sind. Bei S-Lost handelt es sich um einen chemischen Kampfstoff, der erstmals im ersten Weltkrieg zum Einsatz kam und bis heute in vielen internationalen Kon-flikten großen Schaden anrichtete, obwohl der Gebrauch schon 1925 durch die Genfer Konvention verboten wurde. Aktuell stellt S-Lost zudem eine Bedrohung durch terroristische Aktivitäten dar. Da für S-Lost-induzierte Verletzungen bislang keine spezifisch wirksamen Behandlungs-methoden verfügbar sind, besteht großes Interesse an der Aufklärung der dem Krankheitsbild zugrunde liegenden molekularen Pathomechanismen, um daraus Rückschlüsse auf besser ge-eignete therapeutische Maßnahmen ziehen zu können. In unseren ersten Experimenten wurden als mögliche Auslöser der Blasenbildung die Expression und Sekretion ausgewählter Matrix-Metalloproteinasen (MMPs) und deren endogenen Inhibitoren, den tissue inhibitors of matrix metalloproteinases (TIMPs) in einem 3D-Haut-modell und in verschiedenen Zelltypen der Haut (Keratinozyten, Fibroblasten, mikrovaskuläre Endothelzellen, mesenchymale Stammzellen, monozytäre Zellen, PMN-Granulozyten) sowohl in Mono- als auch in Mischkultur untersucht. Unter Verwendung von molekularbiologischen und proteinbiochemischen Methoden wie qRT-PCR, Zymographie und Western Blot gelang der Nachweis, dass MMPs - insbesondere MMP-9 - nach Exposition der Zellen (v.a. Fibroblasten und monozytäre Zellen) mit S-Lost deutlich hochreguliert wurden. Zu Erhärtung der Annahme, dass MMP-9 durch Degradation der Basalmembran zwischen Epidermis und Dermis zur Blasenbildung beiträgt, konnte als Pathomechanismus nun erstmals eine parakrine Stimulation von Fibroblasten durch S-Lost-behandelte Keratinozyten identifiziert werden, als deren Folge eine vermehrte MMP-9-Sekretion resultierte. Darüber hinaus zeigte sich in weiteren Versuchen unter Verwendung des sog. Scratch-Assays und eines Transwell-basierten Invasionsassays, dass das Migrations- und Invasionsverhalten der Fibroblasten in Gegenwart des konditionierten Mediums der S-Lost-behandelten Keratinozyten positiv beeinflusst wurde. Aus klinischer Sicht sprechen diese Erkenntnisse für neue therapeutische Ansätze, die darauf beruhen sollten, die S-Lost-induzierte, auf proteolytischer Aktivität basierende Blasenbildung der Haut durch Applikation spezifischer MMP-Inhibitoren zu behandeln. In einem weiteren Projekt wurde die verzögerte Wundheilung als spätes Symptom der S-Lost-Vergiftung auf zellulärer Ebene untersucht, bei der eine eingeschränkte Re-Epithelialisierung der betroffenen Hautstellen beobachtet wird. Sowohl für den Prozess der Wundheilung als auch für die stetige Erneuerung der Haut werden epidermale Stammzellen benötigt, die für die Bildung von Keratinozyten verantwortlich sind. Diese unipotenten Progenitorzellen befinden sich in der basalen Schicht der Epidermis und sind in der Lage zu proliferieren und anschließend terminal zu differenzieren. Um eine Beeinflussung dieser Prozesse durch S-Lost zu untersuchen, wurden primäre unreife Keratinozyten (NHEK) verwendet und hinsichtlich ihres Differenzierungspotenzials untersucht. Dabei erwies sich S-Lost als potenter Induktor der Differenzierung von NHEK, was durch Bestimmung der Expression typischer Markerproteine wie Keratin-1, Involucrin und Loricrin gezeigt wurde. Die Induktion des Reifungsprozesses war sowohl von einem Rückgang der Proliferation als auch von einer verminderten Migrationsrate der Zellen begleitet. Die eingehende Analyse von mitogen-activated protein kinase (MAPK)-Signaltransduktionswegen führte zu der Erkenntnis, dass die Aktivitäten von p38 und ERK1/2 gegenteilige Rollen im Differenzierungsprozess einnehmen. Studien mit spezifischen Inhibitoren der MAPK be¬legten, dass p38 für den Reifungsvorgang in NHEK essentiell ist, während ERK1/2 diesem entgegen wirkt. So konnte durch Blockade von p38 die von S-Lost ausgelöste Differenzierung der Zellen verhindert werden. Ebenso war es durch diese Behandlung möglich, die von S-Lost stark beeinträchtige Migrationsfähigkeit der Keratinozyten wiederherzustellen, welche mit einer erhöhten MMP-1-Expression einherging. Davon abgeleitet erscheint es therapeutisch sinnvoll, selektive p38-Inhibitoren für die Behandlung von Wundheilungsstörungen nach Exposition der Haut mit S-Lost einzusetzen. Zusammenfassend erbrachten unsere Studien also den Nachweis, dass der S-Lost-induzierten Blasenbildung (als frühes Symptom) die spezifische Induktion der MMP-9 zugrunde liegt. Darüber hinaus konnte erstmals eine verfrühte Differenzierung in unreifen Keratinozyten der Haut (als mögliche Ursache für die verzögerte Wundheilung) nachgewiesen werden, wobei die MAPK p38 bei der Initiierung des Prozesses von entscheidender Bedeutung ist. Aufgrund dieser Resultate empfiehlt sich eine kombinierte Applikation von Inhibitoren der Aktivitäten von MMP-9 und p38, wobei der Einsatz jedoch zeitlich abgestimmt erfolgen sollte, um pathologische Effekte (Blasenbildung bzw. Differenzierungsinduktion in Keratinozyten) zu blockieren, ohne die positiven Auswirkungen von MMP-9 und p38 auf die Heilung (Migration von Immunzellen und Keratinozyten bzw. Reepithelialisierung) zu hemmen.
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 03/06
PC4 is a small protein with unique DNA-binding properties that affects transcription and has presumptive roles in DNA repair and genome stability. It was originally isolated from a cofactor fraction termed the ‘‘upstream stimulatory activity’’ (USA) of HeLa cell nuclear extracts. The cofactor has been shown to broadly enhance RNA polymerase II-driven gene transcription in the presence of activators (e.g., hormone receptors, viral activators, cell-specific and ubiquitous activators). Although such data imply that PC4 is a very important factor in vivo, human tumor cell lines with PC4 knockdowns are without obvious phenotypes. To further study the in vivo role of PC4, we constructed constitutive and conditional knockout mouse models as well as knockout embryonic stem cells. Mammalian PC4 is here shown to be an essential factor during early embryogenesis. PC4-/- embryos develop normally until E5.5, but then degenerated around E7.5. PC4 knockout ES cell lines were generated from PC4-/- blastocysts (E3.5), which develop normally from 2-cell stage embryos. All PC4 knockout ES cell lines displayed a severe proliferation deficit phenotype, which could be partially rescued by re-expression of human PC4. The reduced proliferation was not due to an increase in cell apoptosis. Occasionally, PC4 knockout ES cells undergo tetraploidy apparently as a survival mechanism to circumvent the loss of PC4. Knocking down PC4 in mouse embryonic fibroblasts also resulted in reduced proliferation rates. These data indicate that PC4 is important for cell proliferation in embryos. Moreover, in vitro embryoid body formation and in vivo teratoma formation assays provided preliminary evidence for an important role of PC4 in differentiation. Differentiated ES cells displayed alterations in germ-layer specific gene expression, that are in agreement with morphological abnormalities observed in histological analyses of PC4-/- embryos at E6.5 and E7.5. Thus, depletion of PC4 results in reduced proliferation and impaired differentiation, the consequence of which appears to be of gastrulation arrest in early embryos. In an attempt to understand the underlying mechanisms of this phenotype, differential gene expression in ES knockout and wild-type cells was studied. Microarray and qRT-PCR analyses revealed more than 2 fold alterations in expression of many genes in knockout ES cells as compared with wild-type cells. These include enhanced expression of p21, Rb1, and Ddit4l, and lower expression of Sfmbt2, Tdrd12, and Dppa3, suggesting a specific direct or indirect physiological role of the cofactor. Nevertheless, the previously proposed role of PC4 in p53 expression and function was not confirmed using the knockout model. Taken together, this work represents the first description of the physiological functions of PC4 during mammalian embryogenesis.
Background: Idiopathic Pulmonary Fibrosis (IPF) is an unresolved clinical issue. Phosphodiesterases (PDEs) are known therapeutic targets for various proliferative lung diseases. Lung PDE6 expression and function has received little or no attention. The present study aimed to characterize (i) PDE6 subunits expression in human lung, (ii) PDE6 subunits expression and alteration in IPF and (iii) functionality of the specific PDE6D subunit in alveolar epithelial cells (AECs). Methodology/Principal Findings: PDE6 subunits expression in transplant donor (n = 6) and IPF (n = 6) lungs was demonstrated by real-time quantitative (q)RT-PCR and immunoblotting analysis. PDE6D mRNA and protein levels and PDE6G/H protein levels were significantly down-regulated in the IPF lungs. Immunohistochemical analysis showed alveolar epithelial localization of the PDE6 subunits. This was confirmed by qRT-PCR from human primary alveolar type (AT)II cells, demonstrating the down-regulation pattern of PDE6D in IPF-derived ATII cells. In vitro, PDE6D protein depletion was provoked by transforming growth factor (TGF)-beta 1 in A549 AECs. PDE6D siRNA-mediated knockdown and an ectopic expression of PDE6D modified the proliferation rate of A549 AECs. These effects were mediated by increased intracellular cGMP levels and decreased ERK phosphorylation. Conclusions/Significance: Collectively, we report previously unrecognized PDE6 expression in human lungs, significant alterations of the PDE6D and PDE6G/H subunits in IPF lungs and characterize the functional role of PDE6D in AEC proliferation.
Glomerular diseases account for the majority of cases with chronic renal failure. Several genes have been identified with key relevance for glomerular function. Quite a few of these genes show a specific or preferential mRNA expression in the renal glomerulus. To identify additional candidate genes involved in glomerular function in humans we generated a human renal glomerulus-enriched gene expression dataset (REGGED) by comparing gene expression profiles from human glomeruli and tubulointerstitium obtained from six transplant living donors using Affymetrix HG-U133A arrays. This analysis resulted in 677 genes with prominent overrepresentation in the glomerulus. Genes with 'a priori' known prominent glomerular expression served for validation and were all found in the novel dataset (e.g. CDKN1, DAG1, DDN, EHD3, MYH9, NES, NPHS1, NPHS2, PDPN, PLA2R1, PLCE1, PODXL, PTPRO, SYNPO, TCF21, TJP1, WT1). The mRNA expression of several novel glomerulus-enriched genes in REGGED was validated by qRT-PCR. Gene ontology and pathway analysis identified biological processes previously not reported to be of relevance in glomeruli of healthy human adult kidneys including among others axon guidance. This finding was further validated by assessing the expression of the axon guidance molecules neuritin (NRN1) and roundabout receptor ROBO1 and -2. In diabetic nephropathy, a prevalent glomerulopathy, differential regulation of glomerular ROBO2 mRNA was found.In summary, novel transcripts with predominant expression in the human glomerulus could be identified using a comparative strategy on microdissected nephrons. A systematic analysis of this glomerulus-specific gene expression dataset allows the detection of target molecules and biological processes involved in glomerular biology and renal disease.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 08/19
Hintergrund Humane mesenchymale Stammzellen sind ein viel versprechendes Ziel für die ex vivo Gentherapie, und Lentiviren sind exzellente Vehikel für den Gentransfer in hMSCs, da sie hohe Transduktionsfrequenzen mit langfristiger Genexpression erreichen. Dennoch könnte die Seneszenz von hMSCs die therapeutische Anwendung, infolge von zeitaufwendiger Zellselektion und Virus Titration, limitieren. Diese Arbeit beschreibt optimierte Protokolle für hoch effizienten ex vivo lentiviralen Gentransfer in hMSCs und eine schnelle und verlässliche Methode, um den funktionellen lentiviralen Titer mittels quantitativer Polymerase-Kettenreaktion (qPCR) zu bestimmen. Methoden EGFP wurde als Markergen/-protein verwendet, um verschiedene lentivirale Expressionsvektoren herzustellen. Die Produktion von Lentiviren wurde mit verschiedenen Verpackungssystemen getestet. Der Prozentsatz transduzierter Zellen wurde durch Polybrene und Blasticidinselektion erhöht. hMSCs von verschiedenen Spendern wurden mittels PCR und Western Blot analysiert. Regulierte Genexpression wurde durch Herstellung eines Tet-On selbstregulierten Expressionsvektors erreicht. Mit einem p24 ELISA-Test wurden übrig gebliebene virale Partikel im Zellkulturüberstand detektiert. Die Effizienz des lentiviralen Gentransfers wurde mittels Fluoreszenz-Mikroskopie beobachtet und mittels qRT-PCR und FACS-Analyse quantifiziert. Die lentiviralen Titer wurden mit qRT-PCR der exprimierten Transgene bestimmt. Die hMSC Differenzierung wurde histologisch untersucht. Ergebnisse Selbstinaktivierende lentivirale Vektoren der dritten Generation zeigten hoch effizienten Gentransfer in hMSCs bei der Verwendung von Polybrene. Die Blasticidinselektion hat den Prozentsatz der transgenen Zellen weiter erhöht unter Selektion von Zellen die mehrere Transgenkopien tragen. Die positiven Effekte von Polybrene und der Blasticidinselektion sind nicht von hMSCs eines speziellen Spenders abhängig. Präzise Regulation der Genexpression wurde durch Herstellung eines selbstregulierten Tet-On-Expressionssystems erreicht. Keine viralen Antigene wurden im Zellkulturüberstand nach aufeinander folgenden Medienwechseln detektiert, was auf die Abwesenheit von infektiösen Partikeln nach einigen Tagen hindeutet. In dieser Arbeit wurde ein starker linearer Zusammenhang zwischen der Virusverdünnung und der Stärke der Transgenexpression mittels qPCR Analysen beobachtet, wodurch die Virustitration durch Quantifizierung der Transgenexpression ermöglicht wird. Abschließend wurde durch Differenzierung in die adipogene, osteogene und chondrogene Richtung gezeigt, dass transduzierte hMSCs ihren Stammzellcharakter beibehalten haben und dass die Transgenexpression durch die Differenzierung nicht beeinflusst wurde. Schlussfolgerungen Die Quantifizierung der Transgen-Kopienanzahl durch qRT-PCR ist eine schnelle und verlässliche Methode, um den funktionellen lentiviralen Titer nach dem ex vivo Gentransfer in hMSCs zu bestimmen. Die in dieser Arbeit optimierte und charakterisierte Methode für die effiziente lentivirale Transduktion von humanen mesenchymalen Stammzellen, in Verbindung mit regulierbarer Transgenexpression, ist ein sicheres, verlässliches und leistungsstarkes Verfahren und bildet eine aussichtsreiche Grundlage für zukünftige Gentherapie und Tissue Engineering Anwendungen in hMSCs.
Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 02/07
The aim of the study was to further investigate the importance of local other and to detect possible differences of expression and localisation in poly- and monoovulatory species. Therefore ovaries of sexually mature gilts were collected at the local abattoir and follicles, which were determined to be in the follicular phase of oestrous cycle, ovarian stromal tissue and corpus luteum (CL) were prepared. In two experiments mRNA expression of gonadotropin receptors (LHR, FSHRregulatory factors during the final follicle growth in pig, to clarify relations among each), Aromatase (P450 Aro) and growth factors (fibroblast growth factor (FGF-1, FGF-2, FGF-7) and their receptors (FGFR-1IIIc, FGFR-2IIIb, FGFR-2IIIc), vascular endothelial growth factor (VEGF) and receptors (VEGFR-1, VEGFR-2), angiopoietin-tie-system (Angp-1, Angp-2, Tie-1, Tie-2) and nitric-oxid-synthase (eNOS, iNOS) were measured by qRT-PCR. An immunohistochemically determination and localisation was carried out for FGF-2, VEGF and VEGFR-1. Molecular size of FGF-2 was determined by westernblot. mRNA expression of the selected factors in pig total follicles and expression intensity in comparison to stromal tissue and CL was checked in experiment 1. Experiment 2 was concerned with the exact regulation of growth factors in the follicle compartments granulosa cells (GC) and theca interna (TI) during final follicular growth. A classification of follicles was performed according to follicular fluid oestradiol-17β (E2)-, progesterone and prostaglandin F2α content into four groups (2-3mm, 4-6mm, >7mm, post LH). Expression of FSHR decreased during follicle growth, while LHR and Aromatase increased. mRNA of FGF-family members was strongly expressed in stromal tissue. This was confirmed immunohistochemically by dominant localisation of FGF-2 protein in stromal cells. 18kDa and 22kDa isoforms of FGF-2 were overriding determined in stromal tissue. VEGF was located in GC and TI but not in stromal tissue. The receptor, VEGFR-1 was found in the endothelial- and smooth muscle cells of blood vessels. VEGF mRNA expression decreased after LH-peak. VEGFR-2 was upregulated in large follicles, VEGFR-1 after LH-peak. Angp-2/Angp-1 ratio was highest in small follicles, lowest in large follicles. After LH-peak the ratio rose again. Expression of eNOS was opposite with iNOS, which was downregulated in large follicles. FSH plays an important part in recruiting a folliclepool at time of luteolysis, while the further developing follicles become LH dependent. The number of LHR is an important selection criteria for the follicles. LH and E2 cause GC proliferation by mediators like FGF-7 and enhance angiogenesis by stimulating VEGF. While the follicle growth-up fast the pressure of oxygen in the follicular antrum decreases, whereby angiogenetic factors like VEGF and angiopoietins get stimulated and provide an adequate blood supply for the follicles. Activated by LH and VEGF eNOS is producing NO, which controls E2 synthesis and leads to a better blood flow in the TI through vasodilatation. Increasing E2 concentrations in large follicles stimulate Angp-1, which stabilise the now sufficient bloodvessel network in the TI. After the LH-peak Angp-2 destabilises the blood vessels, which is important for the process of ovulation and the succeeding angiogenesis in CL buildup. For follicle selection an important parameter seems to be the localisation of VEGF and FGF in the follicle. VEGF acts in the follicle and therefore follicles which express more VEGF and receptors have a developmental advantage. In cows this fact is suggested as one basic in selection of the one dominant follicle. FGF-2 in the pig is mainly expressed in stromal tissue and able to activate FGFR in the TI and increases angogenesis in a synergistic way with VEGF and its receptors. FGF-2 in stromal tissue consequently has influence on a few follicles and is maybe an important component in selection of follicles during final follicular growth in polyovulatory species.