Platelets form complexes with neutrophils during inflammatory processes. These aggregates migrate into affected tissues and also circulate within the organism. Several studies have evaluated platelet-neutrophil complexes as a marker of cardiovascular diseases in human and mouse. Although multiple publications have reported platelet-neutrophil complex counts, we noticed that different methods were used to analyze platelet-neutrophil complex formation, resulting in significant differences, even in baseline values. We established a protocol for platelet-neutrophil complex measurement with flow cytometry in murine and human whole blood samples. In vitro platelet-neutrophil complex formation was stimulated with ADP or PMA. We tested the effect of different sample preparation steps and cytometer settings on platelet-neutrophil complex detection and noticed false-positive counts with increasing acquisition speed. Platelet-neutrophil complex formation depends on platelet P-selectin expression, and antibody blocking of P-selectin consequently prevented ADP-induced platelet-neutrophil complex formation. These findings may help generating more comparable data among different research groups that examine platelet-neutrophil complexes as a marker for cardiovascular disease and novel therapeutic interventions.
High mobility group box 1 (HMGB1) acts as both a nuclear protein that regulates gene expression, as well as a pro-inflammatory alarmin that is released from necrotic or activated cells. Recently, HMGB1-expression in human atherosclerotic plaques was identified. Therapeutic blockade of HMGB1 reduced the development of diet-induced atherosclerosis in ApoE knockout mice. Thus, we hypothesised an interaction between HMGB1 and activated platelets. Binding of recombinant HMGB1 to platelets was assessed by flow cytometry. HMGB1 bound to thrombin-activated human platelets (MFI 2.49 vs 25.01, p=0.0079). Blood from wild-type, TLR4 and RAGE knockout mice was used to determine potential HMGB1 receptors on platelets. HMGB1 bound to platelets from wild type C57Bl6 (MFI 2.64 vs 20.3, p< 0.05), and TLR4-/- mice (MFI 2.11 vs 25.65, p< 0.05) but failed to show binding to platelets from RAGE-/- mice (p > 0.05). RAGE expression on human platelets was detected by RT-PCR with mRNA extracted from highly purified platelets and confirmed by Western blot and immunofluorescence microscopy. Platelet activation increased RAGE surface expression (MFI 4.85 vs 6.74, p< 0.05). Expression of HMGB1 in human coronary artery thrombi was demonstrated by immunohistochemistry and revealed high expression levels. Platelets bind HMGB1 upon thrombin-induced activation. Platelet specific expression of RAGE could be detected at the mRNA and protein level and is involved in the binding of HMGB1. Furthermore, platelet activation up-regulates platelet surface expression of RAGE. HMGB1 is highly expressed in platelet-rich human coronary artery thrombi pointing towards a central role for HMGB1 in atherothrombosis, thereby suggesting the possibility of platelet targeted anti-inflammatory therapies for atherothrombosis.
Objective To date, no biomarkers have been established to predict haematological complications and outcomes of extracorporeal membrane oxygenation (ECMO). The aim of this study was to investigate the expression of a panel of microRNAs (miRNAs), which are promising biomarkers in many clinical fields, in patients before and after initiating ECMO. Methods Serum miRNA levels from 14 patients hospitalized for acute respiratory failure and supported with ECMO in our medical intensive care unit were analysed before and 24 hours after ECMO. In total, 179 serum-enriched miRNAs were profiled by using a real-time PCR panel. For validation, differentially expressed miRNAs were individually quantified with conventional real-time quantitative PCR at 0, 24, and 72 hours. Results Under ECMO support, platelet count significantly decreased by 65 × 103/µL (25th percentile = 154.3 × 103/µL; 75th percentile = 33 × 103/µL). Expression of the 179 miRNAs investigated in this study did not change significantly throughout the observational period. Conclusions According to our data, the expression of serum miRNAs was not altered by ECMO therapy itself. We conclude that ECMO does not limit the application of miRNAs as specific clinical biomarkers for the patients’ underlying disease.
ZUSAMMENFASSUNGDie amerikanische Trypanosomiasis oder auch Chagas-Krankheit wird durch das Protozoon Trypanosoma cruzi verursacht. Vor allem unerkannt und unbehandelt kann sie zu schwerwiegenden Organschäden bis hin zum Tod führen. Weltweit geht man aktuell von 6–7 Mio. Infizierten aus. Initial eher auf ländliche Gebiete in Lateinamerika beschränkt, breitete sich die Infektion durch Populationsbewegungen und nichtvektorielle Übertragungswege auf große Städte sowie in nichtendemische Regionen weltweit aus. Die WHO zählt die Chagas-Krankheit zu den sog. vernachlässigten Tropenerkrankungen und nur ein kleiner Bruchteil der Betroffenen erhält eine adäquate Versorgung. Im Herbst 2022 wurden erstmals Handlungsempfehlungen für Deutschland veröffentlicht. Sie sollen der Vernachlässigung entgegenwirken, nichtvektorielle Übertragungswege unterbinden und durch adäquates Management die assoziierte sozio-ökonomische Belastung, Morbidität und Mortalität verringern.
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