IL-17 Expression in the Time Course of Acute Anti-Thy1 Glomerulonephritis

Background Interleukin-17 (IL-17) is a new pro-inflammatory cytokine involved in immune response and inflammatory disease. The main source of IL-17 is a subset of CD4+ T-helper cells, but is also secreted by non-immune cells. The present study analyzes expression of IL-17 in the time course of acute anti- thy1 glomerulonephritis and the role of IL-17 as a potential link between inflammation and fibrosis. Methods Anti-thy1 glomerulonephritis was induced into male Wistar rats by OX-7 antibody injection. After that, samples were taken on days 1, 5, 10 (matrix expansion phase), 15 and 20 (resolution phase). PBS-injected animals served as controls. Proteinuria and histological matrixes score served as the main markers for disease severity. In in vitro experiments, NRK-52E cells were used. For cytokine expressions, mRNA and protein levels were analyzed by utilizing RT-PCR, in situ hybridization and immunofluorescence. Results Highest IL-17 mRNA-expression (6.50-fold vs. con; p<0.05) was found on day 5 after induction of anti-thy1 glomerulonephritis along the maximum levels of proteinuria (113 ± 13 mg/d; p<0.001), histological glomerular-matrix accumulation (82%; p<0.001) and TGF-β1 (2.2-fold; p<0.05), IL-6 mRNA expression (36-fold; p<0.05). IL-17 protein expression co-localized with the endothelial cell marker PECAM in immunofluorescence. In NRK-52E cells, co-administration of TGF-β1 and IL-6 synergistically up-regulated IL-17 mRNA 4986-fold (p<0.001). Conclusions The pro-inflammatory cytokine IL-17 is up-regulated in endothelial cells during the time course of acute anti-thy1 glomerulonephritis. In vitro, NRK-52E cells secrete IL-17 under pro-fibrotic and pro-inflammatory conditions

MTLA in the field of tension between educational and professional reality using the example of molecular biology : a triangulation study

Der Beruf der Medizinisch-technischen Laboratoriumsassistent*innen (MTLA) unterliegt schon immer einer starken, am Fortschritt der biomedizinischen Forschung orientierten, Progression. Aktuell werden immer mehr klassische Analysen durch molekularbiologische Methoden ergänzt bzw. ersetzt. Dies bringt eine starke Erhöhung der Sensitivität und Spezifität mit sich. Zudem ermöglichen diese Methoden einen schnelleren und gezielteren Therapiestart, was einen starken Effekt auf die Patient*innensicherheit hat. In den der MTLA-Ausbildung zugrunde liegenden Ordnungsmitteln sind molekularbiologische Inhalte derzeit jedoch nicht verortet. Ziel dieser Forschungsarbeit war es, eine vermutete Diskrepanz zwischen Ausbildungs- und Berufsrealität in Bezug auf molekularbiologische Methoden empirisch zu belegen und deren Effekte auf berufstätige MTLA in ihrem Tätigkeitsfeld aufzudecken. Methodisch wurde hier triangulativ vorgegangen, in dem zunächst problemzentrierte, leitfadengestützte Expert*innen-Interviews geführt wurden. Nach deren Auswertung auf Basis der Grounded Theory flossen die Ergebnisse in die Erstellung eines Online-Fragebogens ein. Dieser richtete sich an berufstätige MTLA, die ihren Abschluss in Deutschland absolviert haben. Durch die Verschränkung der Methoden konnte einerseits sehr deutlich eine hohe Relevanz molekularbiologischer Techniken, mit einem Anteil von etwa einem Drittel aller laboratoriumsmedizinischen Analysen, gezeigt werden. Zugleich wurde offengelegt, dass auch aktuell noch eine inadäquate molekularbiologische Ausbildung der berufstätigen MTLA stattfindet. Es konnte gezeigt werden, dass heute immerhin etwa 97% aller Absolvent*innen molekularbiologische Inhalte während ihrer Ausbildung vermittelt bekommen. Allerdings beträgt der Anteil nur knapp 1,6% der laut MTLA-Ausbildungs- und Prüfungsverordnung für technische Assistenten in der Medizin (MTLA-AprV) geforderten 3170 Unterrichtsstunden. Nach dem Konzept der „Institutionen“ von Berger und Luckmann ist dies de facto auf die zugrunde liegenden starren und nicht mehr der Berufsrealität entsprechenden Ordnungsmittel zurückzuführen. Die defizitäre MTLA-Ausbildung wird durch die Handelnden auf informellem Weg überbrückt. So erlangen die MTLA ihr molekularbiologisches Wissen und die zugehörigen Kompetenzen derzeit über Kolleg*innen, das Selbststudium, über weiterführende Studien und Weiterbildungen, um in der Berufsrealität handlungsfähig zu bleiben. Dieser Prozess ist ein deutliches Indiz für eine aktuell stattfindende Professionalisierung. Auf Grundlage dieser Studie wird eine weitreichende Novellierung der MTLA-Ausbildung und der zugrunde liegenden Ordnungsmittel empfohlen. Vorausgehend sollte dringend eine längst überfällige, umfassende Berufsfeldanalyse stattfinden. Nur ein derartiger Novellierungsprozess kann zu einer Sicherung der Profession beitragen, auf deren Handeln 70% aller ärztlichen Diagnosen beruhen.The Medical-technical laboratory assistants (MTLA) occupation has always been subject to a strong progression based on the evolution of biomedical research. More and more classic analyzes are currently being supplemented or replaced by molecular biological methods. This leads to a strong increase in sensitivity and specificity. In addition, these methods enable a faster and more accurate start of therapy, which has a remarkable effect on patient safety. However, molecular biological content is currently not located in the regulatory means on which the MTLA training is based. The aim of this research work was to empirically prove a suspected discrepancy between training and occupational reality in relation to molecular biological methods and to uncover their effects on working MTLA in their field of activity. In terms of methodology, a triangulative approach was used, in which initially guide-based, problem-centered expert interviews were conducted. After the interview-analyzes, based on the Grounded Theory, the results were incorporated into the creation of an online questionnaire. This was aimed at working MTLA, who graduated in Germany. Firstly, by interlinking the methods, a high relevance of molecular biological techniques was shown, with a share of about a third of all laboratory medical analyzes. At the same time, it was disclosed that the molecular biological training of the working MTLA is still inadequate. It could be shown that around 97% of all graduates today receive molecular biological content during their training. However, the share is just under 1.6% of the 3170 teaching hours required by the Ausbildungs- und Prüfungsverordnung für technische Assistenten in der Medizin (MTLA-AprV). According to the concept of the “Institutions” by Berger and Luckmann, this is de facto due to the underlying rigid means of organization that no longer correspond to occupational reality. The deficient MTLA training is, at the present time, bridged by the actors in an informal way. For example, the MTLA currently acquire their molecular biology knowledge and related competences through colleagues, self-study, further studies and supplementary training in order to remain able to act in the occupational reality. This process is a clear indication of the currently ongoing professionalization. On the basis of this study, a comprehensive amendment of the MTLA training and the underlying means of organization are recommended. Beforehand, a long overdue, profound occupational field analysis should urgently take place. Only such an amendment process can contribute to securing the profession, on whose actions 70% of all medical diagnoses are made

Expression and activity of soluble guanylate cyclase in injury and repair of anti-thy1 glomerulonephritis

Expression and activity of soluble guanylate cyclase in injury and repair of anti-thy1 glomerulonephritis.BackgroundActivation of soluble guanylate cyclase and generation of cyclic 3′,5′-guanosine monophosphate (cGMP) is the main signal transducing event of the L-arginine-nitric oxide pathway. The present study analyzes the expression and activity of the nitric oxide-cGMP signaling cascade in and the effect of the specific soluble guanylate cyclase stimulator Bay 41-2272 on the early injury and subsequent repair phase of acute anti-thy1 glomerulonephritis.MethodsAnti-thy1 glomerulonephritis was induced by OX-7 antibody injection in rats. In protocol 1 (injury), Bay 41-2272 was given starting 6 days before antibody injection. One day after disease induction, parameters of mesangial cell injury (glomerular cell number and inducible nitric oxide synthesis) were analyzed. In protocol 2 (repair), Bay 41-2272 treatment was started one day after antibody injection. On day 7, parameters of glomerular repair [glomerular matrix score, expression of transforming growth factor (TGF)-β1, fibronectin, and plasminogen-activator-inhibitor (PAI)-1, infiltration with macrophages and fibrinogen deposition (indicating platelet localization)] were determined. In both protocols, tail bleeding time, systolic blood pressure, plasma cGMP levels, glomerular mRNA expression of endothelial nitric oxide synthase (eNOS), α1 and β1 soluble guanylate cyclase, and basal and nitric oxide-stimulated glomerular cGMP production were analyzed.ResultsBay 41-2272 prolonged bleeding time, reduced blood pressure, and increased plasma cGMP levels in both protocols. In the injury experiment, disease induction increased inducible nitric oxide synthesis and reduced glomerular cell number, while expression and activity of soluble guanylate cyclase was almost completely diminished. Bay 41-2272 did not affect parameters of mesangial cell injury and glomerular soluble guanylate cyclase expression and activity. In the repair protocol, expression and activity of soluble guanylate cyclase was markedly increased by disease. Bay 41-2272 further enhanced soluble guanylate cyclase expression and activity. This went along with significant reductions in proteinuria, glomerular matrix accumulation, expression of TGF-β1, fibronectin, and PAI-1, macrophage infiltration and fibrinogen deposition as compared to the untreated anti-thy1 animals.ConclusionGlomerular nitric oxide signaling via cGMP is markedly impaired during injury of anti-thy1 glomerulonephritis, while it is highly up-regulated during subsequent repair. Further pharmacologic soluble guanylate cyclase stimulation limits glomerular TGF-β overexpression and matrix expansion, suggesting that the soluble guanylate cyclase enzyme represents an important antifibrotic pathway in glomerular disease

TGF-β1, IL-6 and IL-17 mRNA expression of NRK-52E after glucose, IL-6, TGF-β1 or IL-17 stimulation.

<p>TGF-β1, IL-6 and IL-17 mRNA expression in NRK-52E cells 15 min after stimulation by 25 mM glucose, 10 ng/ml IL-6, 5 ng/ml TGF-β1 or 25 ng/ml IL-17. Analysis utilized real-time PCR and was normalized to β-actin as housekeeping gene. (# p <0.05 vs. nc; * p<0.01 vs. nc).</p

IL-17 and synaptopodin protein expression of an anti-thy1 glomerulonephritic animal on d5.

<p>Representative images showing glomerular IL-17 and synaptopodin protein expression in renal tissue sections of an anti-thy1 glomerulonephritic animal on d5 after disease induction, in paraffin-embedded immunostained sections at x400 magnification. IL-17 green, synaptopodin red, nucleus blue, merge yellow.</p

IL-17 and PECAM-1 protein expression of an anti-thy1 glomerulonephritic animal on d5.

<p>Representative images showing glomerular IL-17 and PECAM-1 protein expression in renal tissue sections of an anti-thy1 glomerulonephritic animal on d5 after disease induction, in paraffin-embedded immunostained sections at x400 magnification. IL-17 green, PCAM-1 red, nucleus blue, merge yellow.</p