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    Öğe
    In vitro investigation on extracellular traps formation of cat polymorphonuclear leucocytes against Toxoplasma gondii
    (Tubitak Scientific & Technological Research Council Turkey, 2021) Simsek, Neslihan Sursal; Cakmak, Ayse; Yildiz, Kader
    Neutrophil granulocytes are one of the most important defenders of the innate immune system in the host. Moreover, neutrophils are able to reach the inflammation area and kill the pathogens using various defense strategies including neutrophil extracellular traps (NETs). However, there is still not enough information available regarding the innate immunity against Toxoplasma gondii in cats that are both definitive and intermediate hosts of this parasite. Therefore, we investigated the in vitro NETs formation which is induced by cat polymorphonuclear leucocytes (PMNs) against T. gondii tachyzoites. Firstly, PMNs were isolated from cat venous blood samples by using discontinues Percoll dilutions (72%, 63%, 54%, and 45%). Afterward, MN-tachyzoites cocultures were stained against histone (H3), neutrophil elastase (NE), and myeloperoxidase (MPO) by using monoclonal antibodies and were examined under a fluorescence microscope. The effect of different parasite doses (1:1, 1:3, and 1:5) and incubation times (30, 60, 90, and 120 min) on NETs formation was also evaluated. The presence of the extracellular DNA content was measured using a fluorometer. Confluent Vero cell cultures were used to assess the effect of NETs on the tachyzoites viability. The classical structures of NETs, such as extracellular DNA, NE, H3, and MPO were microscopically observed in the NETs formation released from cat PMNs. The amount of extracellular DNA increased in parallel with the incubation time (p < 0.001). The influence of the tachyzoites dose on the NETs formation was not statistically significant (p > 0.05). Zymosan was used as a positive control in the experiments and it was shown to be an important inducer for the NETs formation. In conclusion, as mentioned previous studies and considering our results, the NETs may be a conserved strategy to control T. gondii infection in hosts because of the immobilization and lethal effects.
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    Öğe
    Neutrophil isolation from feline blood using discontinuous Percoll dilutions
    (Georg Thieme Verlag Kg, 2018) Sursal, Neslihan; Cakmak, Ayse; Yildiz, Kader
    Objective: Some studies have performed in vitro neutrophil isolation from feline blood. The major limiting factor for these studies is the small volume of blood that can be collected without development of potentially life-threatening complications. In the present study we attempted neutrophil isolation from feline venous blood samples using discontinuous Percoll gradients. Material and methods: Blood was collected from the cephalic vein of clinically healthy adult cats. The blood samples were layered on Percoll dilutions (72 %, 63%, 54% and 45%). After centrifugation, the feline polymorphonuclear leukocytes (PMN) accumulated as a band between 72-63% Percoll dilutions. The total cell count was calculated using light microscopy counts. The percentage of the neutrophils was determined microscopically after staining with Diff-Quik stain. Neutrophil viability was evaluated with a 0.01 % Trypan blue assay. The activation was determined based on intact cell morphology in the isolated neutrophils. Results: The mean PMN number was 22 x 10(5) per ml (minimum-maximum: 20-26 x 10(5)ml). Neutrophil homogeneity was > 95% in the cell suspensions. The viability of isolated neutrophils was > 98%. The technique did not result in neutrophil activation. Conclusion and clinical relevance: Discontinuous Percoll gradients (72%, 63 %, 54% and 45%) can be used to isolate neutrophils from blood samples of cats. The technique was simple to perform and neutrophil activation was minimal.