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

Flow cytometric assessment of activation of peripheral blood platelets in dogs with normal platelet count and asymptomatic thrombocytopenia

Platelets play a crucial role in hemostasis. Their activation has not yet been evaluated in healthy dogs with a normal and low platelet count. The aim of this study was to determine the influence of activators on platelet activation in dogs with a normal platelet count and asymptomatic thrombocytopenia. 72 clinically healthy dogs were enrolled. Patients were allocated into three groups. Group 1 consisted of 30 dogs with a normal platelet count, group 2 included 22 dogs with a platelet count between 100 and 200×109/l and group 3 consisted of 20 dogs with a platelet count lower than 100×109/l. Platelet rich-plasma (PRP) was obtained from peripheral blood samples using tripotassium ethylenediaminetetraacetic acid (K3-EDTA) as anticoagulant. Next, platelets were stimulated using phorbol-12-myristate-13-acetate or thrombin, stabilized using procaine or left unstimulated. The expression of CD51 and CD41/CD61 was evaluated. Co-expression of CD41/CD61 and Annexin V served as a marker of platelet activation. The expression of CD41/CD61 and CD51 did not differ between the 3 groups. Thrombin-stimulated platelets had a significantly higher activity in dogs with a normal platelet count than in dogs with asymptomatic thrombocytopenia. Procaine inhibited platelet activity in all groups. In conclusion, activation of platelets of healthy dogs in vitro varied depending on the platelet count and platelet activator

Comparison of the influence of ADTA-K3 and sodium citrate on haematology analysis in healthy dogs

The study was carried out on 30 clinically healthy dogs of various breeds. Haemoglobin concentration, haematocrit, platelet count and platelet haematocrit were significantly lower in citrate blood than in tripotassium ethylenediaminetetraacetic acid (EDTA-K3) blood. The study confirmed the limited usage of sodium citrate in haematology analysis, unless canine EDTA-dependent thrombocytopenia is suspected

Evaluation of reticulated platelets in dogs with breed-related thrombocytopenia

The aim of this study was to evaluate the percentage of reticulated platelets in healthy dogs with breed-related thrombocytopenia. Seventy two dogs, clinically healthy, were enrolled in the study. Blood was collected from the patients and anticoagulated with tripotassium ethylenediaminetetraacetic acid (K3-EDTA) and sodium citrate. Platelet count was obtained by an impedance haematology analyser and platelet morphology was evaluated by examination of blood smears. Patients were allocated into two groups. Group 1 consisted of 30 dogs with normal platelet count, whereas group 2 was composed of 42 dogs with thrombocytopenia. Thrombocytopenia was present in both K3-EDTA and citrate blood samples. Patients with thrombocytopenia were divided into two subgroups: the first subgroup included dogs with platelet count in K3-EDTA anticoagulated blood from 100 to 200 x10⁹/L, patients in the second subgroup had a platelet count of less than 100 x10⁹/L. The percentage of young reticulated platelets (RPs) labelled with thiazole orange, and the percentage of platelets coated with platelet surface-associated IgG, were determined in platelet-rich plasma (PRP) by a flow cytometer. The mean percentage of RPs in K3-EDTA and citrate PRP was significantly higher in dogs with thrombocytopenia than in dogs with normal platelet count. The mean percentage of RPs was significantly higher in citrate PRP than in K3-EDTA PRP in all groups. The results suggest that idiopathic, asymptomatic thrombocytopenia is not caused by platelet surface-associated IgG. Dogs with breed-related thrombocytopenia have a competent bone marrow

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