Lipid rafts are essential for release of phosphatidylserine-exposing extracellular vesicles from platelets.

Platelets protect the vascular system during damage or inflammation, but platelet activation can result in pathological thrombosis. Activated platelets release a variety of extracellular vesicles (EVs). EVs shed from the plasma membrane often expose phosphatidylserine (PS). These EVs are pro-thrombotic and increased in number in many cardiovascular and metabolic diseases. The mechanisms by which PS-exposing EVs are shed from activated platelets are not well characterised. Cholesterol-rich lipid rafts provide a platform for coordinating signalling through receptors and Ca2+ channels in platelets. We show that cholesterol depletion with methyl-β-cyclodextrin or sequestration with filipin prevented the Ca2+-triggered release of PS-exposing EVs. Although calpain activity was required for release of PS-exposing, calpain-dependent cleavage of talin was not affected by cholesterol depletion. P2Y12 and TPα, receptors for ADP and thromboxane A2, respectively, have been reported to be in platelet lipid rafts. However, the P2Y12 antagonist, AR-C69931MX, or the cyclooxygenase inhibitor, aspirin, had no effect on A23187-induced release of PS-exposing EVs. Together, these data show that lipid rafts are required for release of PS-exposing EVs from platelets.Isaac Newton Trust/ Wellcome Trust ISSF/University of Cambridge Joint Research Grant British Heart Foundation grant SP/15/7/3156

The influence of experimental administration of low zearalenone doses on the expression of Th1 and Th2 cytokines and on selected subpopulations of lymphocytes in intestinal lymph nodes

The aim of this study was to characterize the immune response taking place in ileocecal lymph nodes (ICLN) in control (n=15) and zearalenone (ZEN)-treated (n=15) pigs. The experiment was carried out over 42 days; a dose of 0.1 mg kg⁻¹ feed day⁻¹ of ZEN was administered to the animals. The dose used in the experiment was at a level where no adverse effects are observed (NOAEL) in the ovaries, uterus and vagina. ICLN samples for analysis were collected on the 14th, 28th and 42nd day of the experiment. The analysis of cytokine concentration in the tissues showed that pigs treated with ZEN had an increased level of cytokines produced by helper Th1 lymphocytes (IL-2, IL-12 and IFN- γ) on the 28th day of the experiment. The level of cytokines produced by helper Th2 lymphocytes (IL-4 and IL-10) was characterized by a statistically non-significant upward trend, as compared with the control group. Flow cytometry showed a linear decrease in the percentage of CD21+ B, CD2+ T and CD4+CD8- T cells and an increase in the percentage of CD8+CD4- and TCRγδ + T cells in pigs treated with ZEN. Both ZEN and α-ZEL (α-zearalenone) concentrations increased over time in the liver, but only ZEN concentration increased in ICLN. The results obtained demonstrate that a NOAEL concentration of ZEN shifts the immune response in pig ICLN towards Th1/Th17, probably with a simultaneous activation of M1 macrophages. Moreover, we observed an increase in humoral cytokine secretion; this can be explained by a negative feedback loop and a phenotypic switch of macrophages from M1 to M2, as well as a switch of immune response from Th1 to Th2 type. ZEN can therefore influence the process of cytokine secretion and the percentage of lymphocytes in ileocecal lymph nodes

The effect of T-2 toxin on percentages of CD4plus, CD8plus, CD4plusCD8plus and CD21plus lymphocytes, and mRNA expression levels of selected cytokines in porcine ileal Peyer’s patches

The immune system is one of the main toxicity targets of the T-2 toxin. In view of scant research data demonstrating the effect of T-2 on cellular and humoral responses in gut-associated lymphoid tissue (GALT), this study set out to investigate the effects of chronic exposure to low doses of the T-2 toxin (200 μg T-2 toxin kg⁻¹ feed) on percentages of CD4⁺ and CD8⁺ T lymphocytes, CD4⁺/CD8⁺ double-positive T lymphocytes, CD21⁺ B cells, and IL-2, IFN-γ, IL-4 and IL-10 mRNA expression levels in porcine ileal Peyer’s patches. The investigated material comprised ileum sections sampled from piglets (aged 8-10 weeks, body weight of 15-18 kg) on days 14, 28 and 42 of the experiment. After 42 days of exposure to T-2, a significant drop in the quantity of the IL-10 product was observed (R=0.94; S.E. 0.49-0.79; p<0.001). A gradual decrease in the amount of IL-4 and IFN-γ cytokine transcripts was found throughout the experiment, but the reported trend was not significant. On experimental days 14 and 42, a significant increase in the percentage of CD8⁺ T lymphocytes was observed in comparison with the control (p=0.04 and p=0.05, respectively), whereas on day 28, a significant decrease in the percentage of the above subpopulation was noted (p=0.00). The percentage of CD21⁺ B cells in the experimental group decreased steadily in comparison with the control, and the observed drop was significant on days 28 and 42 (p=0.06 and p=0.00, respectively). On days 14 and 28, the percentages of CD4⁺ and CD8⁺ T lymphocytes were lower in the experimental animals than in the control group, and the drop reported on day 28 was statistically significant (p=0.03)

Tannic acid-modified silver nanoparticles for wound healing: the importance of size

Piotr Orlowski,1 Magdalena Zmigrodzka,2 Emilia Tomaszewska,3 Katarzyna Ranoszek-Soliwoda,3 Monika Czupryn,1 Malgorzata Antos-Bielska,1 Janusz Szemraj,4 Grzegorz Celichowski,3 Jaroslaw Grobelny,3 Malgorzata Krzyzowska1 1Military Institute of Hygiene and Epidemiology, Warsaw, Poland; 2Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences (WULS-SGGW), Warsaw, Poland; 3Department of Materials Technology and Chemistry, Faculty of Chemistry, University of Lodz, Lodz, Poland; 4Bionanopark, Lodz, Poland Introduction: Silver nanoparticles (AgNPs) have been shown to promote wound healing and to exhibit antimicrobial properties against a broad range of bacteria. In our previous study, we prepared tannic acid (TA)-modified AgNPs showing a good toxicological profile and immunomodulatory properties useful for potential dermal applications.Methods: In this study, in vitro scratch assay, antimicrobial tests, modified lymph node assay as well as a mouse splint wound model were used to access the wound healing potential of TA-modified and unmodified AgNPs.Results: TA-modified but not unmodified AgNPs exhibited effective antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli and stimulated migration of keratinocytes in vitro. The tests using the mouse splint wound model showed that TA-modified 33 and 46&nbsp;nm AgNPs promoted better wound closure, epithelialization, angiogenesis and formation of the granulation tissue. Additionally, AgNPs elicited expression of VEGF-&alpha;, PDGF-&beta; and TGF-&beta;1 cytokines involved in wound healing more efficiently in comparison to control and TA-treated wounds. However, both the lymph node assay and the wound model showed that TA-modified AgNPs sized 13&nbsp;nm can elicit strong inflammatory response not only during wound healing but also when applied to the damaged skin.Conclusion: TA-modified AgNPs sized &gt;26&nbsp;nm promote wound healing better than TA-modified or unmodified AgNPs. These findings suggest that TA-modified AgNPs sized &gt;26&nbsp;nm may have a promising application in wound management. Keywords: hydrolyzable tannin, split wound, silver, antimicrobials, inflammation, fibroblasts, monocyte