Charakterisierung der Funktion von Smyd1 und PML bei LPS-abhängigen Entzündungsreaktionen in Endothelzellen

Introduction: Sepsis is a global life-threatening condition associated with destructive effects on the organs due to exaggerated immune response. It commonly occurs after the exposure to Lipopolysaccharide (LPS) following infection with Gram-negative bacteria particularly in severely ill patients. LPS leads frequently to endothelial cell dysfunction and plays an important role in the progression of sepsis leading to one or multiple organs failure. Smyd1 is a histone methyltransferase that affects chromatin remodeling and is involved in regulation of cellular development and cancer. PML protein is involved in the oncogenesis of acute promyelocytic leukemia and has a prominent role in many physiological and pathological processes such as tumor suppression, angiogenesis and inflammatory responses. Both Smyd1 and PML were found to be expressed in endothelial cells but little is known about their role in sepsis and inflammation. The aim of this study was to determine the contribution of Smyd1 and PML to the immune response under septic conditions in ECs. Methods: EA.hy926 cells were treated with LPS. The expression of Smyd1 and PML was detected, after reverse transcription of mRNA, via Real time semi-quantitative PCR (RT-qPCR) and on the protein level using Fluorescence-activated cell sorting (FACS). Then, EA.hy926 cells were transfected with Smyd1 and PML carrying plasmids to study their effect on the expression of IL-1, IL-6, IL-8 and NF-κB on the mRNA level with RT-qPCR and/or on the protein level via immunoblotting, Enzyme-linked immunosorbent assay (ELISA) and FACS. Results: Incubation with LPS increased Smyd1 and PML, while incubation with IL-6 increased PML only. Overexpression of Smyd1 increased the expression of IL-1, IL-6, and IL-8, while PMLIV did not induce IL-8. Although Smyd1 and PMLIV appeared to increase the amount and activity of NF-κB, the Smyd1-induced expression of IL-6 was not completely dependent on NF-κB. Conclusions: It was shown in this work that Smyd1 and PML are involved in inflammatory reactions of the endothelial cells. Thereby, we reported an increase of IL-6, IL-8 and IL-1 by Smyd1 which could be in part due to Smyd1 dependent activation of NF-κB signaling pathway. We also confirmed the stimulatory effect PML on IL-6 expression in endothelial cells and revealed an IL-6-dependent increase of PML as well. A heuristic model is being developed in which, after an amplification phase of IL-6 production, the degradation of Smyd1 after its PML-dependent SUMOylation helps to end the acute inflammatory reaction.Einleitung: Die Sepsis als ein Erkrankungszustand mit überschießenden Abwehrreaktionen und daraus resultierendem Multiorganversagen ist weltweit verbreitet und lebensbedrohlich. Hervorgerufen wird sie besonders häufig durch die Freisetzung von Lipopolysacchariden (LPS) aus Gram-negativen Bakterien. Neben ihrer Wirkung auf Abwehrzellen im engeren Sinne wirken LPS auf vaskuläre Endothelzellen. Es kommt zur charakteristischen, inflammatorischen Veränderung des Endothels, die für den Krankheitsverlauf entscheidend ist. Smyd1 ist eine H3K4 Histon-Methyltransferase mit SET Domäne und spielt u.a. eine Rolle bei der Proliferation von Karzinomen. PML-Proteine, ebenfalls im Zusammenhang mit Karzinomen untersucht, bilden Kernkörperchen, in denen die Aktivität von Histonmethyltransferasen wie auch von Histon- Deacetylasen durch SUMOylierungsreaktionen reguliert wird. Sowohl Smyd1 wie PML konnten in Endothelzellen nachgewiesen werden. Ob sie bei inflammatorischen Reaktionen des Endothels eine Rolle spielen können, ist bisher unbekannt. Es war das Ziel dieser Arbeit, Smyd1 und PML in LPS-exponierten Endothelzellen zu untersuchen. Methoden: EA.hy926 Zellen wurden mit LPS oder IL-6 inkubiert oder es wurde Smyd1 oder PMLIV mit Hilfe von Expressionsplasmiden überexprimiert. Für Smyd1, PML, IL-1, IL-6, IL-8 und NF-κB kodierende mRNA wurde durch semi-quantitative real time RT-PCR bestimmt. Zugehörige Proteine wurden durch Immunoblot, FACS oder ELISA bestimmt. Ergebnisse: Inkubation mit LPS erhöhte Smyd1 und PML, während Inkubation mit IL-6 lediglich PML erhöht wurde. Überexpression von Smyd1 erhöhte IL-1, IL-6 und IL-8, während durch PMLIV IL-8 nicht erhöht wurde. Obwohl Smyd1 und PMLIV Menge und Aktivität von NF-κB zu erhöhen schienen, war die Smyd1-induzierte Expression von IL-6 nicht vollständig von NF-κB abhängig. Schlussfolgerungen: Es wurde in dieser Arbeit gezeigt, dass Smyd1 und PML an den inflammatorischen Reaktionen von Endothelzellen beteiligt sind. Dabei berichteten wir über einen Anstieg von IL-6, IL-8 und IL-1 durch Smyd1, der zum Teil auf eine Smyd1-abhängige Aktivierung des NF-κB-Signalwegs zurückzuführen sein könnte. Wir bestätigten auch die stimulierende Wirkung von PML auf die IL-6-Expression in Endothelzellen und zeigten ebenfalls einen IL-6-abhängigen Anstieg von PML. Es wird ein heuristisches Modell entwickelt,in dem nach einer Amplifikationsphase der IL-6 Produktion die Degradation von Smyd1 nach seiner PML- abhängigen SUMOylierung die akute Entzündungsreaktion zu beenden hilft

Is Pancrelipase Delayed Release Capsule (Creon) Safe for Use in Patients’ ≥7 Years Old Who Suffer From Pancreatic Insufficiency Due to Cystic Fibrosis?

Objective: The objective of this selective EBM review is to determine whether or not Pancrelipase Delayed Release Capsule (Creon) is safe for use in patients’ ≥7 years old who suffer from pancreatic insufficiency due to cystic fibrosis. Study Design: Systemic review of 3 English language primary studies, published between 2000-2010. Data Sources: Two double blind randomized controlled trials (RCTs) as well as an open label phase study with a double blind phase comparing the safety of Pancrelipase Delayed Release Capsule (Creon) in treating pancreatic insufficiency in those suffering from Cystic Fibrosis. These studies were found using Cochrane Systematic Reviews and PubMed. Outcomes Measured: The outcomes of each study measured the safety and tolerability of Creon by monitoring vital signs, weight, BMI, safety laboratory values, and adverse events. Adverse events were tracked in all studies. Results: Graff et al demonstrated that for every three participants who took Creon, there was one fewer incidence of Treatment Emergent Adverse Events (TEAE’s) than in the group of participants taking the placebo. TEAE’s were reported in 5 patients (29.4%) while taking Creon and in 9 patients (53.6%) while receiving the placebo. There were also no discontinuations due to treatment adverse events and no serious adverse events noted during the trial. Trapnell et al demonstrated that for every four participants who took Creon there was one fewer incidence of TEAE’s than in the group of participants taking the placebo. Stern et al demonstrated that during the double blind phase in both the adult and pediatric/adolescent studies, higher percentages of placebo-treated patients (67% and 70% respectively) reported treatment emergent adverse events than Creon treated patients (39% and 61% respectively). Conclusion: Creon does appear to be safe and tolerable for those over the age of 7 suffering from pancreatic insufficiency due to cystic fibrosis. The TEAE’s reported in each study proved the tolerance of Creon to surpass that of other treatment modalities. These studies also address the other benefits of Creon in allowing for significant improvements in stool fat, weight, and nitrogen and a significant reduction in daily stool frequency. Although these are promising results more RCTs must be conducted with wider age groups in order to compare the safety of Creon

Effect of prolonged stimulation of the heme oxygenase/carbon monoxide system by hemin on blood pressure and penile erection of spontaneously hypertensive rats

Essential hypertension (EH) is a risk factor for many cardiovascular disorders. Treatment of established EH, especially for prolonged control of this pathogenic process, represents a great challenge. Moreover, hypertension is considered an important risk factor for the development of many other diseases, e.g. erectile dysfunction. Hemin and other heme derivatives, e.g. heme-L-lysinate (HLL) and heme-L-arginate, have been used extensively to upregulate expression of heme oxygenase (HO) and production of endogenous carbon monoxide (CO). Short-term hemin administration for 4-5 days has been shown to markedly decrease high blood pressure (BP) in spontaneously hypertensive rats (SHR), but not in normotensive Wistar-Kyoto (WKY) or Sprague Dawley (SD) rats. This short-term therapy was effective in treating young, but not adult SHR. In the present study, hemin (15 mg/kg/day) was administered to 12-week old adult SHR through subcutaneously implanted osmotic minipumps for 3 consecutive weeks (the hemin protocol). Into the second week of the hemin protocol, BP of SHR was normalized from 203.2 ± 2.5 to 123.4 ±1.9 mmHg (n=20,

Expression of chimeric HCV peptide in transgenic tobacco plants infected with recombinant alfalfa mosaic virus for development of a plant-derived vaccine against HCV

Hepatitis C virus (HCV) is the major etiologic agent of blood transfusion–associated and sporadic non-A non-B hepatitis affecting more than 180 million worldwide. Vaccine development for HCV has been difficult and there is no vaccine or effective therapy against this virus. In this paper, we describe the development of an experimental plant-derived subunit vaccine against HCV. Our subunit vaccine originates from a consensus HCV-HVR1 epitope (R9) that antigenically mimics many natural HVR1 variants. This HVR1 sequence was cloned into the open reading frame of a plant virus, Alfalfa Mosaic Virus (ALMV) coat protein (CP). The chimeric ALMV RNA4 containing sequence-encoding R9 epitope was introduced into full-length infectious ALMV-RNA3 that was utilized as an expression vector. The recombinant chimeric protein is expressed in transgenic tobacco plants (P12) expressing ALMV RNA1 and 2. Plant–derived HVR1/ALMV-CP reacted with HVR1 and/or ALMV-CP specific monoclonal antibodies and immune sera from individuals infected with HCV. Using plant-virus based transient expression to produce this unique chimeric antigen will facilitate the development and production of an experimental HCV vaccine. A plant derived recombinant HCV vaccine can potentially reduce expenses normally associated with production and delivery of conventional vaccine. Key Words: Hepatitis C virus (HCV), transgenic tobacco plants (P12), consensus HCV HVR1 epitope (R9), and chimeric ALMV-RNA4. African Journal of Biotechnology Vol.3(11) 2004: 588-59

The Methyltransferase Smyd1 Mediates LPS-Triggered Up-Regulation of IL-6 in Endothelial Cells

The lysine methyltransferase Smyd1 with its characteristic catalytic SET-domain is highly enriched in the embryonic heart and skeletal muscles, participating in cardiomyogenesis, sarcomere assembly and chromatin remodeling. Recently, significant Smyd1 levels were discovered in endothelial cells (ECs) that responded to inflammatory cytokines. Based on these biochemical properties, we hypothesized that Smyd1 is involved in inflammation-triggered signaling in ECs and therefore, investigated its role within the LPS-induced signaling cascade. Human endothelial cells (HUVECs and EA.hy926 cells) responded to LPS stimulation with higher intrinsic Smyd1 expression. By transfection with expression vectors containing gene inserts encoding either intact Smyd1, a catalytically inactive Smyd1-mutant or Smyd1-specific siRNAs, we show that Smyd1 contributes to LPS-triggered expression and secretion of IL-6 in EA.hy926 cells. Further molecular analysis revealed this process to be based on two signaling pathways: Smyd1 increased the activity of NF-kappa B and promoted the trimethylation of lysine-4 of histone-3 (H3K4me3) within the IL-6 promoter, as shown by ChIP-RT-qPCR combined with IL-6-promoter-driven luciferase reporter gene assays. In summary, our experimental analysis revealed that LPS-binding to ECs leads to the up-regulation of Smyd1 expression to transduce the signal for IL-6 up-regulation via activation of the established NF-κB pathway as well as via epigenetic trimethylation of H3K4

Distribution Of Peach Latent Mosaic Viroid In Commercial Orchards Of Peach In The North Of Tunisia

peer reviewe

Molecular features of new Peach Latent Mosaic Viroid variants suggest that recombination may have contributed to the evolution of this infectious RNA.

peer reviewe

Anabolic androgenic steroid abuse in the United Kingdom: An update

© 2020 The British Pharmacological Society. This is the peer reviewed version of the following article: Mullen, C, Whalley, BJ, Schifano, F, Baker, JS. Anabolic Androgenic Steroid Abuse in the United Kingdom; An Update The increasing popularity of anabolic androgenic steroids. Br J Pharmacol. 2020, which has been published in final form at https://doi.org/10.1111/bph.14995. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.Anabolic androgenic steroids (AASs) are prescribed for medical conditions related to low testosterone. Abuse of AASs has surged as they become recognised as potent image enhancement drugs. The primary goal of most abusers is to obtain a more attractive outward appearance. Abuse is complex. There are a vast range of AAS substances illegally available, the nature of their true composition is difficult to evaluate. Users follow dosing patterns which incorporate a number of different AASs, in addition to other pharmaceutical substances believed to complement the desired physical effects or manage unwanted effects. Animal work and medical case reports suggest potential to cause serious hepatotoxicity, plus possible neurotoxicity, nephrotoxicity and damage to the cardiovascular and reproductive systems. As the long-term AASs users reach maturity, further controlled experimentation, with larger sample sizes, is required. Data gathering should be directed towards the most vulnerable group of AAS users, females and adolescent boys.Peer reviewedFinal Accepted Versio

Maca (L. meyenii) for improving sexual function: a systematic review

<p>Abstract</p> <p>Background</p> <p>Maca (<it>Lepidium meyenii</it>) is an Andean plant of the brassica (mustard) family. Preparations from maca root have been reported to improve sexual function. The aim of this review was to assess the clinical evidence for or against the effectiveness of the maca plant as a treatment for sexual dysfunction.</p> <p>Methods</p> <p>We searched 17 databases from their inception to April 2010 and included all randomised clinical trials (RCTs) of any type of maca <it/>compared to a placebo for the treatment of healthy people or human patients with sexual dysfunction. The risk of bias for each study was assessed using Cochrane criteria, and statistical pooling of data was performed where possible. The selection of studies, data extraction, and validations were performed independently by two authors. Discrepancies were resolved through discussion by the two authors.</p> <p>Results</p> <p>Four RCTs met all the inclusion criteria. Two RCTs suggested a significant positive effect of maca on sexual dysfunction or sexual desire in healthy menopausal women or healthy adult men, respectively, while the other RCT failed to show any effects in healthy cyclists. The further RCT assessed the effects of maca in patients with erectile dysfunction using the International Index of Erectile Dysfunction-5 and showed significant effects.</p> <p>Conclusion</p> <p>The results of our systematic review provide limited evidence for the effectiveness of maca in improving sexual function. However, the total number of trials, the total sample size, and the average methodological quality of the primary studies were too limited to draw firm conclusions. More rigorous studies are warranted.</p

Treatments for people who use anabolic androgenic steroids: a scoping review.

BACKGROUND: A growing body of evidence suggests that anabolic androgenic steroids (AAS) are used globally by a diverse population with varying motivations. Evidence has increased greatly in recent years to support understanding of this form of substance use and the associated health harms, but there remains little evidence regarding interventions to support cessation and treat the consequences of use. In this scoping review, we identify and describe what is known about interventions that aim to support and achieve cessation of AAS, and treat and prevent associated health problems. METHODS: A comprehensive search strategy was developed in four bibliographic databases, supported by an iterative citation searching process to identify eligible studies. Studies of any psychological or medical treatment interventions delivered in response to non-prescribed use of AAS or an associated harm in any setting were eligible. RESULTS: In total, 109 eligible studies were identified, which included case reports representing a diverse range of disciplines and sources. Studies predominantly focussed on treatments for harms associated with AAS use, with scant evidence on interventions to support cessation of AAS use or responding to dependence. The types of conditions requiring treatment included psychiatric, neuroendocrine, hepatic, kidney, cardiovascular, musculoskeletal and infectious. There was limited evidence of engagement with users or delivery of psychosocial interventions as part of treatment for any condition, and of harm reduction interventions initiated alongside, or following, treatment. Findings were limited throughout by the case report study designs and limited information was provided. CONCLUSION: This scoping review indicates that while a range of case reports describe treatments provided to AAS users, there is scarce evidence on treating dependence, managing withdrawal, or initiating behaviour change in users in any settings. Evidence is urgently required to support the development of effective services for users and of evidence-based guidance and interventions to respond to users in a range of healthcare settings. More consistent reporting in articles of whether engagement or assessment relating to AAS was initiated, and publication within broader health- or drug-related journals, will support development of the evidence base