Parameters of Hemostasis in Sheep Implanted with Composite Scaffold Settled by Stimulated Mesenchymal Stem Cells-Evaluation of the Animal Model.

Implantation of composite scaffolds could be potentially associated with the risk of hemostatic disturbances in a recipient. However, there is a lack of information on possible alterations in clotting mechanisms resulting from such a procedure. The aim of the present work was to investigate changes in hemostatic parameters in sheep implanted with a scaffold composed of poly(ε-caprolactone) and hydroxyapatite and tricalcium phosphate (9:4.5:4.5), settled previously with mesenchymal stem cells stimulated by fibroblast growth factor-2 and bone morphogenetic protein-2. Nine Merino sheep were examined for 7 days, and measurements of clotting times (PT, aPTT), activities of antithrombin, protein C and clotting factors II-XII, and concentrations of fibrinogen and D-dimer were carried out before and 1 h, 24 h, 3 days and 7 days after scaffold implantation. The introduction of scaffold initially resulted in a slowdown of the clotting processes (most evident 24 h after surgery); PT and aPTT increased to 14.8 s and 33.9 s, respectively. From the third day onwards, most of these alterations began to return to normal values. The concentration of fibrinogen rose throughout the observation period (up to 8.4 g/L), mirroring the ongoing inflammatory reaction. However, no signals of significant disturbances in hemostatic processes were detected in the sheep tested.

Involvement of hemostasis in pathophysiology of RAO in horses.

Recurrent Airway Obstruction (RAO), also called severe asthma or heaves, is a chronic disease in adult horses caused by aeroallergens from straw or hay. Disturbances in hemostasis (intensified coagulation and depressed fibrinolysis) are considered one of the prominent reasons of inflammatory process, injury and dysfunction of the lungs. The aim of the study was to evaluate chosen parameters of hemostasis in horses with active form of RAO. Ten RAO-horses (group R) and ten healthy horses (group C) were exposed to straw and hay allergen challenge. The prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), fibrinogen concentration (Fb), stabilized fibrin degradation product (d-dimer), antithrombin (AT), protein C and coagulation factors II through XII were assessed in plasma obtained from blood of all the horses. Exposure to aeroallergens resulted in prolongation of aPTT in both groups of animals; it was evident in the group R and moderate in the group C. There were no differences in PT and TT. Concentrations of fibrinogen and d-dimer and activity of protein C in both groups were increased but lay within or near to reference values. The activity of AT was depressed in RAO-horses. All exposed horses showed increased activity of coagulation factors II, VIII and X but they had no changes in activity of factor V. Factors VII and XII displayed a reduction in activity. The decrease in factor IX activity was noted in the group C only. Various changes were observed in activity of factor XI; in horses with RAO it was elevated but in healthy horses it was declined. The changes of the parameters tested in RAO-horses indicate the involvement of coagulation and fibrinolysis which apparently remained under control of efficient and active mechanisms of general hemostasis.

Publisher Correction: Low-level light therapy reduces platelet destruction during extracorporeal circulation.

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Low-level light therapy reduces platelet destruction during extracorporeal circulation.

Extracorporeal circulation causes many deleterious effects on blood cells. Low-level light therapy (LLLT) in the red/near-infrared spectral range is known for its cytoprotective properties but its use during cardiopulmonary bypass (CPB) has not yet been studied. We aimed to assess whether LLLT protects platelets during CPB. 24 pigs were connected to 1-hour-CPB and observed for the next 23 hours. In 12 animals, blood circulating through the oxygenator was treated with LLLT. Platelet count and function were monitored throughout the experiment. The decrease in platelet count was greater in the control group, especially during CPB and after 24 hours. In LLLT group CD62P expression remained quite stable up to the 12th hour of the experiment, whereas in the control group it continuously decreased till the end of observation. Platelets in the control group were more prone to aggregation in the postoperative period than at the beginning of the experiment, whereas platelets in the LLLT group aggregated similarly or less intense. Limitation of platelet loss, pattern of aggregation and CD62P expression suggest that LLLT may stabilize platelet function during CPB and diminish the negative effects associated with the interaction of cells with an artificial surface.

Effect of antigen challenge on dynamics of CD62P and CD41/61 expression on platelets in horses with recurrent airway obstruction (RAO).

Recurrent airway obstruction (RAO), also known as heaves, is an allergic respiratory condition that develops in horses following an exposure to aeroallergens in hay and straw. This is manifested by airway hyperreactivity, inflammation, bronchoconstriction, as well as a leukocyte and platelet infiltration into the airways. Platelet activation and an increase in circulating platelet-leukocyte aggregates may lead to airway remodeling. The aim of this study was to explore the effect of seven-day antigen challenge on dynamics of platelet indices and CD41/61 and CD62 P expression on platelets in horses with RAO. Ten RAO-affected horses and ten healthy horses were included in this study. All horses were exposed to 7 days hay and straw challenge. Blood samples were collected prior to the challenge (Pre-challenge) and 1, 2, 3, 7 and 14 days after the initiating the antigen challenge. Blood samples were obtained to determine the platelet count (PLT), mean platelet volume (MPV) and platelet large cell ratio (P-LCR). Expression of CD62 P and CD41/61 was detected by flow cytometry on activated platelets. Antigen challenge resulted in a significant gradual decrease of PLT in RAO horses, but not in controls. MPV and P-LCR in control and RAO-affected horses remained unchanged after antigen challenge. The expression of CD62 P and CD41/61 in RAO horses was significantly higher compared to control horses. The antigen challenge resulted in an increase expression of CD62 P and CD41/61 on the platelets of RAO-affected horses, while did not lead to significant changes in the control group. An increased expression of CD62 P and CD41/61 indicates platelet activation what may contribute to the formation of platelet aggregates in their respiratory system.

Investigation of the immune effects of Scutellaria baicalensis on blood leukocytes and selected organs of the chicken's lymphatic system.

BACKGROUND: The health of chickens and the welfare of poultry industry are central to the efforts of addressing global food security. Therefore, it is essential to study chicken immunology to maintain and improve its health and to find novel and sustainable solutions. This paper presents a study on investigation of the effect of Scutellaria baicalensis root (SBR) on the immune response of broiler chicken, especially on lymphocytes and heterophils reactivity, regarding their contribution to the development of immunity of the chickens. METHODS: The 121-day-old Hubbard Hi-Y male broiler hybrids were randomly assigned to four treatment groups, three SBR supplemented groups (0.5, 1.0, and 1.5% of SBR) and one control group. Each treatment was replicated five times with six birds per replicate pen in a battery brooder. Blood was collected after 3rd and 6th wk of the experiment, and hemoglobin and hematocrit values were determined, as well as total leukocyte count and differential count were performed. Nitroblue tetrazolium test and phagocytosis assay as nonspecific immune parameters and humoral immune responses to the antigenic challenge by sheep red blood cells were performed. Moreover, the ability of peripheral blood lymphocytes to form radial segmentation (RS) of their nuclei was analyzed. Body weight and relative weight of spleen, liver, and bursa of Fabricius were recorded. RESULTS: Results showed that mean heterophile/lymphocyte ratio increased in the SBR groups compared to the control group and the blood of the chickens showed lymphocytic depletion. The results also demonstrated that the relative weight of bursa of Fabricius and spleen in groups fed with SBR significantly decreased compared to the control group. This study also showed that the addition of SBR significantly inhibited the formation of RS of nuclei compared to some cytotoxic substances. CONCLUSION: We found that SBR supplementation should be carefully evaluated when given to poultry. The excess intake of SBR supplementation may cause immunologic inhibition and may negatively affect the development of immune organs. SBR has inhibited the formation of radial segmentation nuclei showing antimetastatic properties and also the phagocytosis of chicken heterophils.

Age-related changes of platelet and plasma coagulation parameters in young pigs.

The literature on hemostatic processes in swine is sparse and often fragmentary; hence, we conducted our study to characterize age-related changes in selected parameters of primary and secondary hemostasis in 50 growing pigs between day 2 and week 24 of age. We measured platelet count (PLT), mean platelet volume, platelet-to-large cell ratio, prothrombin time, activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen concentration. Among primary hemostasis parameters, PLT underwent the largest fluctuation with the animals' age, ranging from 340 to 730 × 10(9)/L. However, statistical significance was only detected for 4-week-old piglets compared to 18-week-old animals. Of the secondary hemostasis parameters measured, TT and aPTT were the most changeable. Activated partial thromboplastin time displayed a characteristic biphasic course, being relatively short before week 5 of age (17.8-19.9 s) and then becoming much longer (28.7-52.5 s). The aPTTs measured in animals 6 weeks of age and older were statistically different (p < 0.01) from those in younger piglets. The 2 main components of hemostasis, platelet hemostasis and plasma coagulation, did not develop at the same time. It took much longer for secondary hemostasis to stabilize, whereas platelet parameters were stable early in life.

Expression of surface platelet receptors (CD62P and CD41/61) in horses with recurrent airway obstruction (RAO).

Recurrent airway obstruction (RAO) is an allergic disease of horses similar to human asthma, which is characterized by airway inflammation and activation of neutrophils, lymphocytes and platelets. Platelet activation and an increase in circulating platelet-leukocyte aggregates may lead to airway remodeling. The aim of this study was to investigate platelet status in RAO-affected horses based on the platelet morphology and platelet surface expression of CD41/61 and CD62P. Ten RAO-affected horses and ten healthy horses were included in this study. Blood samples were obtained to determine the platelet count (PLT), mean platelet volume (MPV) and platelet large cell ratio (P-LCR). Expression of CD62P and CD41/61 was detected by flow cytometry on activated platelets. The median PLT was significantly reduced in horses with RAO compared to the controls. The MPV and the P-LCR values were significantly higher in RAO horses than controls. Expression of CD41/61 on platelets was increased in RAO horses, while CD62P expression was reduced. This study demonstrated the morphological changes in platelets and expression of platelet surface receptors. Despite the decrease of CD62P expression, the observed increased surface expression of CD41/61 on platelets in horses with RAO may contribute to the formation of platelet aggregates in their respiratory system.

[Influence of gelatin-alginian matrixes on morphological and functional changes of blood leukocytes]. / Wplyw matryc zelatynowo-alginianowych na zmiany morfologiczne i czynnosciowe leukocytów krwi.

BACKGROUND: Knowledge of the relation of biomaterials and living tissues constitutes necessary information which should be used when composing a set of optimal carriers, e.g. for drugs or preparations supporting blood clotting. OBJECTIVES: This paper presents an assessment of the influence of contact of gelatin-alginian matrixes with blood on leukocyte reactivity: the ability of mononuclear cells (lymphocytes and monocytes) to create a radial segmentation of nuclei--RS (the morphological change), and the ability of leukocytes to phagocytosis of yeast Saccharomyces cerevisiae cells and to produce active oxygen derivatives (functional changes). MATERIAL AND METHODS: After having contact with the matrixes, the test of induced and spontaneous RS, the phagocytic test and nitroblue tetrazolium reduction test for blood leukocytes were performed. RESULTS: The obtained results showed a decrease in the ability of mononuclear cells to form RS and in the ability of granulocytes to reduce nitroblue tetrazolium--NBT, but an increase in their phagocytic activity. CONCLUSIONS: Temporary contact of gelatin-alginian matrixes with blood did not cause any morphological changes in the leukocytes. However, changes of their reactivity were observed.

Polymerase chain reaction-based identification of clinically relevant Pasteurellaceae isolated from cats and dogs in Poland.

A set of polymerase chain reaction (PCR) assays for identification of the most important Pasteurellaceae species encountered in cats and dogs were developed. Primers for Pasteurella multocida were designed to detect a fragment of the kmt, a gene encoding the outer-membrane protein. Primers specific to Pasteurella canis, Pasteurella dagmatis, and Pasteurella stomatis were based on the manganese-dependent superoxide dismutase gene (sodA) and those specific to [Haemophilus] haemoglobinophilus on species-specific sequences of the 16S ribosomal RNA gene. All the primers were tested on respective reference and control strains and applied to the identification of 47 canine and feline field isolates of Pasteurellaceae. The PCR assays were shown to be species specific, providing a valuable supplement to phenotypic identification of species within this group of bacteria.

Genetic diversity of Pasteurella dagmatis as assessed by analysis of the 16S rRNA and rpoB gene sequences.

A total of 16 Pasteurella dagmatis strains, including 11 feline and 4 canine isolates as well as one strain isolated from a tiger, were analyzed using partial 16S rRNA and rpoB gene sequence comparison. Phylogenetic studies based on both genes revealed that the population of P. dagmatis recovered from cats in Poland differs markedly from canine strains, constituting a well-separated cluster within Pasteurella sensu stricto species group. The isolate from a tiger seems to represent yet another evolutionary lineage within P. dagmatis.

An acidic glycoconjugate from Lythrum salicaria L. with controversial effects on haemostasis.

AIM OF THE STUDY: Lythrum salicaria L. belongs to the small Lythraceae family of 22 genera, which range in habit from herbs to shrubs and trees found with worldwide distribution (Heywood, 1993). The generic name of Lythrum derived from Greek "luthron"--blood, possibly referring to the color of the flowers or to the one of its herbal use as an astringent to stop bleeding (Thompson et al., 1987; Mountain, 1994; Pawlaczyk and Pacula, 2002). The flowering parts and the flowering branch tips are used in traditional medicine and pharmaceuticals internally in a form of decoctions or as extracts for treatment of diarrhea, chronic intestinal catarrhs, hemorrhoids and eczema, or externally to treat varicose veins, venous insufficiency and gums (Mantle et al., 2000; Rauha et al., 2000). The aim of this study was to isolate the plant glycoconjugate from flowering parts of Lythrum salicaria, and to verify its influence on blood coagulation process. MATERIALS AND METHODS: From the air-dried flowering parts of this plant a water-soluble glycoconjugate has been isolated by hot alkaline extraction followed by neutralization and purification by multi-steps extraction with organic solvents, dialysis and concentration. The plant isolate was tested in vitro on anticoagulant activity on human plasma, and on Wistar rats blood system in vivo as well as ex vivo. RESULTS: A dark brown isolate was obtained in the yield of 8% of starting material (w/w) as a macromolecular compound with M(w) approximately 12,500. Chemical analysis revealed the presence of carbohydrates (30%), phenolics (1g contained 1.2mM of gallic acid equivalent) and proteins (0.8%). The result of compositional analyses of carbohydrate part revealed the predominance of uronic acids (approximately 66%), galactose (approximately 12%), rhamnose (approximately 10%) and arabinose (approximately 9%) residues indicating thus the presence of pectic type of polymers, i.e. galacturonan and/or rhamnogalacturonan associated with arabinogalactan in Lythrum glycoconjugate. In vitro and ex vivo experiments showed complete inhibition of plasma clot formation, however, the application of Lythrum glycoconjugate in vivo showed controversial effect on animal blood system in comparison with in vitro ones, i.e. pro-coagulant activity. CONCLUSION: The in vivo results give a scientific explanation for the traditional use of Lythrum salicaria as a styptic agent. It seems that pro-coagulant activity of this complex could be probably connected with the other factors in blood circulation system, like platelets.