In sepsis, platelets may become activated via toll-like receptors (TLRs), causing microvascular thrombosis. Megakaryocytes (MKs) also express these receptors; thus, severe infection may modulate thrombopoiesis. To explore the relevance of altered miRNAs in platelet activation upon sepsis, we first investigated sepsis-induced miRNA expression in platelets of septic patients. The effect of abnormal Dicer level on miRNA expression was also evaluated. miRNAs were profiled in septic vs. normal platelets using TaqMan Open Array. We validated platelet miR-26b with its target SELP (P-selectin) mRNA levels and correlated them with clinical outcomes. The impact of sepsis on MK transcriptome was analyzed in MEG-01 cells after lipopolysaccharide (LPS) treatment by RNA-seq. Sepsis-reduced miR-26b was further studied using Dicer1 siRNA and calpain inhibition in MEG-01 cells. Out of 390 platelet miRNAs detected, there were 121 significantly decreased, and 61 upregulated in sepsis vs. controls. Septic platelets showed attenuated miR-26b, which were associated with disease severity and mortality. SELP mRNA level was elevated in sepsis, especially in platelets with increased mean platelet volume, causing higher P-selectin expression. Downregulation of Dicer1 generated lower miR-26b with higher SELP mRNA, while calpeptin restored miR-26b in MEG-01 cells. In conclusion, decreased miR-26b in MKs and platelets contributes to an increased level of platelet activation status in sepsis.

In the process of sepsis, activated platelets shed microvesicles containing microRNAs (miRNAs), which can be internalized by distinct recipient cells in circulation, consequently eliciting a potent capability to regulate their cellular functions in different diseases. In the present study, activated human platelets transferring miR-223 into endothelial cells via platelet-derived microparticles (PMPs) was investigated in vitro during septic conditions with a proposed mechanism involving in downregulation of the enhanced expression of intercellular adhesion molecule-1 (ICAM-1). The uptake of PMPs encasing miR-223 and the adhesion of peripheral blood mononuclear cells (PBMCs) on human coronary artery endothelial cells (HCAECs) were observed by immunofluorescence microscopy upon co-culture with PMPs isolated from sepsis or control plasma. The expression of miR-223-3p and its gene target ICAM1 in HCAECs were quantified by RT-qPCR and ELISA after the cells were incubated with septic or control PMPs, whose levels were induced with thrombin-receptor activating peptide (TRAP). Leukocyte-depleted platelets (LDPs) from septic patients showed a decreased miR-223 level, while septic plasma and PMPs revealed an elevated miRNA level compared to control samples. Similarly, TRAP-activated LDPs demonstrated a reduced intracellular miR-223 expression, while increased levels in the supernatant and PMP isolates were observed vs. untreated samples. Furthermore, TNF-α alone resulted in decreased miR-223 and elevated ICAM1 levels in HCAECs, while PMPs raised the miRNA level that was associated with downregulated ICAM1 expression at both mRNA and protein levels under TNF-α treatment. Importantly, miR-223 was turned out not to be newly synthesized as shown in unchanged pre-miR-223 level, and mature miR-223 expression was also elevated in the presence of PMPs in HCAECs after transfection with Dicer1 siRNA. In addition, septic PMPs containing miR-223 decreased ICAM1 with a reduction of PBMC binding to HCAECs. In conclusion, septic platelets released PMPs carrying functional miR-223 lower ICAM1 expression in endothelial cells, which may be a protective role against excessive sepsis-induced vascular inflammation.

MicroRNAs (miRNA) are short, non-coding RNAs consisting of 18–25 nucleotides that regulate posttranscriptionally the gene expression involved in the regulation of physiological processes of the cells. Their key role is to modulate the translation of target mRNAs via binding to complementary sequences within the 3’ UTRs of mRNAs resulting in altered protein synthesis or even the degradation of mRNAs. miRNAs are carried not only by cells with nucleus, but also in platelets, red blood cells, and they are present in the circulation, in urine and in other body fluids as well. The fact about functional miRNAs in platelets without nucleus having a half-life of 8–12 days was questioned for a long time, thus it was also obscure whether platelets are able to produce proteins de novo when being exposed to different challenges. In the last few years, several publications have described the expression and function of certain platelet mRNAs with their regulatory miRNAs in terms of regulation of cell activation, especially in diseases in which platelet activation status is elevated, such as in type 2 diabetes mellitus or in sepsis. Apart from their pathophysiological role, miRNAs may be applied as potential new biomarkers in the investigation or differential diagnosis of these clinical conditions. This review article sought to summarize the recent findings about platelet miRNAs focusing on their altered expression in diabetes and sepsis. Orv Hetil. 2018; 159(47): 1962–1970.

Background:In sepsis, platelet activation by lipopolysaccharide (LPS) via TLR4 can result in microvascular thrombosis. However, megakaryocytes (MKs) also express TLRs, thus severe infection can modulate thrombopoiesis. Both MKs and platelets are rich in microRNAs (miRNAs) to regulate messenger RNA (mRNA) function and different cellular events. Murine MKs were previously described to produce platelets with altered mRNA profile in septic mice.Aims:We characterized sepsis‐induced expression of mRNAs with their regulatory miRNAs in ex vivo and in vitro stimulated platelets as well as in MK cell cultures. The contribution of Dicer function to abnormal miRNA levels was also investigated.Methods:Leukocyte‐depleted platelets of 22 septic and 26 healthy individuals were analyzed for P2RY12 and SELP (P‐selectin) mRNAs. TaqMan Open Array was performed for miRNAs, and RT‐qPCR were used for verification. The effects of sepsis were also studied in isolated platelets and MEG‐01 cells in vitro after treatment with recombinant TNF‐α (100 ng/mL) or LPS (O55:B5, 100 ng/mL) with lipoprotein binding protein (100 ng/mL) and soluble CD14 (150 ng/mL) for 1–24 h. Dicer level was observed by western blotting and fluorescence microscope, while its function was tested by calpain inhibitor (calpeptin, 10 μmol/L) in septic MKs and silencing by Dicer siRNA (40 pmol) in MEG‐01 cells in comparison to control samples with NEG‐01 siRNA for P2RY12 and SELP expression. To prove the effect of LPS, TLR4 expression was observed on platelets and MKs by flow cytometry. Induced inflammatory conditions were evaluated by miR‐155 expression and IL‐1β/IL‐6 mRNA levels by RT‐qPCR and via nuclear translocation of p65 subunit of NF‐κB pathway by fluorescence microscope.Results:There was augmented platelet activation as surface and soluble P‐selectin were increased in septic patients (P < 0.001). Platelets and MKs upregulated P2RY12 and SELP gene expression (P < 0.01) accompanied with elevated inflammation‐specific miR‐155 and decreased miR‐223/26b. Additionally, 64 other platelet miRNAs (e.g. miR‐150, let‐7e) indicated ≥2‐fold decrease, while 37 (e.g. miR‐191) were increased at the same degree in sepsis vs. controls. Septic conditions resulted in substantial p65 translocation into nuclei causing at least 2‐fold elevated miR‐155 and IL‐1β or IL‐6 mRNA levels (P < 0.05) in platelets and MKs in the ex vivo and in vitro experiments via TLR4 receptors that could be detected on the surface of platelets (3–7%) and MEG‐01 cells (8–12%). After a transient induction of Dicer and miRNAs at 1 h, reduced Dicer mRNA and protein levels were seen in LPS/TNF‐α treated platelets/MKs after 4–24 h causing attenuated miR‐223/26b. Calpain inhibition restored miRNA levels, while downregulation of Dicer generated higher P2RY12 and SELP levels in MEG‐01.Summary/Conclusion:Alteration in Dicer‐dependent miRNAs contribute to elevated mRNA levels in MKs and reactive platelets during sepsis.This study is supported by the GINOP‐2.3.2–15–2016–00043‐IRONHEART project.

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Az egyházak finanszírozására vonatkozóan többféle modell alakult ki az egyes államokban, melyet döntően meghatároz az adott ország állam–egyház viszonyrendszere. Közös pont azonban, hogy valamilyen módon, közvetve vagy közvetlenül valamennyi állam nyújt költségvetési támogatásokat a vallási közösségeknek. Az egyházak finanszírozási modelljei a legtöbb esetben, így magyarországon is komplex, sokcsatornás rendszert alkotnak. Hazánkban az egyházak támogatása számos költségvetési előirányzatból áll, az állam finanszírozza az egyházak hitéleti és közcélú tevékenységét is.

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Hungary is a country of favourable conditions with agropotential, for this reason the regulations regarding agriculture is constantly the bone of legislative contention. Thepurpose of this study is to present the actual agropotential contradictions concerning the agricultural producers, specifically focusing on certain problems without any detailed representation of the valid rules of law. The agrofinancial anomalies referring to agricultural producers can be found mostly in the system of the personal income tax (allowance, tax immunity), the value added tax (special agricultural legal position) as well as the social insurance.The severity of the problem is supported by the agricultural producers’ behavioral types of paying taxes, globalisation process of these days and our place in the European Union. The resolution of the contradictions in agriculture does not lie by any means in the proper way of making the rules of the law of taxation, but in a comprehensive economic policy, which is to be waited for.