Hubert Ziółkowski

Prostaglandin E2 down-regulates the expression of CD25 on bovine T cells, and this effect is mediated through the EP4 receptor

A crucial event in the initiation of an immune response is the activation of T cells, which requires IL-2 binding to its high-affinity IL-2 receptor for optimal signaling. The IL-2 receptor α-chain (CD25) is needed for the high affinity binding of IL-2 to effector cells and is potently induced after T cell activation. The aim of this research has been to determine whether prostaglandin E2 (PGE2) affects the CD25 expression on bovine T cells, and if it does, then which of the PGE2 receptor (EP) subtype(s) mediate(s) this effect. Herein, we report that exposure of peripheral blood mononuclear cells (PBMC) to PGE2 considerably reduces the percentage and absolute counts of CD25(+)CD4(+), CD25(+)CD8(+) and CD25(+)WC1(+) T cells, significantly increases the value of these parameters with respect of CD25(-)CD4(+), CD25(-)CD8(+) and CD25(-)WC1(+) T cells, and does not affect counts of the total populations of CD4(+), CD8(+) and WC1(+) T cells. These results indicate that PGE2 down-regulates the CD25 expression on bovine T cells. Moreover, we show that the selective blockade of EP4 receptor, but not EP1 and EP3 receptors, prevents this effect. Interestingly, the exposure of PBMC to a selective EP2 receptor agonist leads to a substantial increase in the percentage and absolute number of CD25(+)CD4(+), CD25(+)CD8(+) and CD25(+)WC1(+) T cells. In conclusions, the PGE2-induced down-regulation of CD25 expression on bovine CD4(+), CD8(+) and WC1(+) T cells should be considered as immunosuppressive and anti-inflammatory action, because these lymphocytes primarily represent effector cells and adequate CD25 expression is essential for their correct functioning. The PGE2-mediated down-regulation of the CD25 expression on bovine T cells is mediated via the EP4 receptor, although selective activation of the EP2 receptor up-regulates the CD25 expression on these cells. Thus, with respect to the effect of PGE2 on the CD25 expression on bovine T cells, EP4 receptor serves as an inhibitory receptor, whereas EP2 receptor functions as a stimulatory receptor. The fact that non-selective stimulation of EP receptors, i.e. triggered by PGE2, leads to weaker CD25 expression proves that inhibitory actions prevail over stimulatory ones. These results indicate the possibility of pharmacological manipulation of the CD25 expression on T cells via selective antagonists and agonists of EP2 and EP4 receptors.

Effects of dexamethasone and meloxicam on bovine CD25+ CD8+ and CD25- CD8+ T cells--in vitro study.

The aim of undertaken research was an in vitro evaluation of the effects of dexamethasone and meloxicam on selected bovine CD8(+) T lymphocyte subpopulations. Dexamethasone induced a fast-occurring and lasting depletion of CD25(-)CD8(+) cells. This was primarily the result of the proapoptotic effect of dexamethasone, but the antiproliferative effect of the drug was clearly responsible for the deepening of this disturbance. Dexamethasone transiently increased the relative and absolute CD25(high)CD8(+) and CD25(low)CD8(+) cell numbers. This effect was not a consequence of increased proliferation, but at least partly resulted from the antiapoptotic effect of the drug on these cells. The obtained results indicate that induction of CD8(+) lymphocyte depletion and impairment of IFN-γ production by these cells participate in the production of the anti-inflammatory and immunosuppressive effect of dexamethasone in cattle. An increase in Foxp3, IL-10 and TGF-β production by CD8(+) lymphocytes is not involved in the production of these effects because the drug did not affect the percentage of TGF-β(+)CD8(+) cells, while paradoxically reducing the percentage of cells with the suppressive phenotype, i.e. IL-10(+)CD25(low)CD8(+) and Foxp3(+)CD25(low)CD8(+) cells. Meloxicam did not substantially affect CD8(+) lymphocytes as to their percentage, absolute number, apoptosis, proliferation, Foxp3 expression and IFN-γ, IL-10 and TGF-β production. Thus, in the context of the parameters being estimated, meloxicam seems a relatively safe anti-inflammatory drug to be used in infectious diseases in cattle.

Application of ultra-performance columns in high-performance liquid chromatography for determination of albendazole and its metabolites in turkeys

Methods for determination of albendazole (ALB), albendazole sulfoxide (SOX) and albendazole sulfone (SON) in turkey blood plasma, using high-performance liquid chromatography (HPLC) with fluorescence detection, were developed. Moreover, comparison of HPLC columns with ultra-performance liquid chromatography (UPLC) columns was performed. Albendazol was administered orally in 5-week-old birds (n = 18) at a dose of 25 mg/kg b.w. Accuracy and precision of the developed method were satisfactory and stability studies showed acceptable variation (below 15%) in ALB, SOX and SON concentrations when the samples were stored at -75°C for 15 days. UPLC(®) columns gave higher peaks from typical HPLC columns retaining high quality of analysis. Pharmacokinetic analysis indicated quick elimination of ALB from turkey blood plasma. The mean residence time of SON was at least two times longer than that of SOX and four times longer than that of ALB. The elimination half-lives for ALB, SOX and SON were 0.7 ± 0.27, 5.37 ± 6.03, 9.17 ± 5.12 h, respectively. The obtained results indicate that the described method allows for precise determination of albendazole and its metabolites in turkey plasma. Moreover, using UPLC columns in HPLC apparatus results in higher sensitivity as compared with the classical HPLC columns.

Pharmacokinetics of oxytetracycline in broiler chickens following different routes of administration

The aim of this study was to determine the pharmacokinetics of oxytetracycline (OTC) in broiler chickens following intravenous (IV), intramuscular (IM), subcutaneous (SC) and oral (PO) administrations at a dose of 15 mg/kg bodyweight. Plasma concentrations of OTC were determined using liquid chromatography-tandem mass spectrometry and non-compartmental pharmacokinetic analysis was then conducted. The absorption half-life time was 1.23 ± 0.36 h, 1.19 ± 0.52 h, and 0.49 ± 0.38 h after IM, SC and PO administration, respectively. The elimination half-life time was 27.41 ± 6.06 h, 10.23 ± 4.20 h, 7.83 ± 0.56 h, and 14.86 ± 9.23 h, and the mean residence time was 9.67 ± 1.7 h, 11.45 ± 1.76 h, 11.38 ± 0.59 h, and 10.37 ± 3.91 h after IV, IM, SC and PO administration, respectively. Bioavailability was 76.88 ± 12.90%, 92.20 ± 10.53% and 12.13 ± 4.56% after IM, SC and PO administration, respectively, which indicated that OTC is poorly absorbed from the gastrointestinal tract in broiler chickens.

Prostaglandin E2 exerts the proapoptotic and antiproliferative effects on bovine NK cells

The aim of this research was to determine whether prostaglandin E2 (PGE2) affects bovine NK cells in respect of their counts, apoptosis and proliferation, and if it does, then which of the PGE2 receptor (EP) subtype(s) mediate(s) these effects. We here report that long-term, but not short-term, exposure of bovine peripheral blood mononuclear cells to PGE2 at 10(-5)M, 10(-6)M and 10(-7)M, but not at 10(-8)M, caused a significant increase in the percentage of early apoptotic cells among NK cell subset. Moreover, PGE2 at 10(-5)M and 10(-6)M, but not at 10(-7)M and 10(-8)M, induced a considerable decrease in the absolute count of NK cells. The magnitude of these effects increased with an increasing concentration of PGE2. The blockade of EP1, EP2, EP3 and EP4 receptors did not prevent the PGE2-induced apoptosis and depletion of NK cells. The results suggest that the proapoptotic effect of PGE2 is secondary in character and the induction of this effect is not mediated through EP receptors. Furthermore, the studies demonstrated that PGE2 at 10(-5)M and 10(-6)M, but not at 10(-7)M and 10(-8)M, highly significantly reduced the percentage of proliferating NK cells. The EP1, EP1/2 and EP3 receptor antagonists were unable to block this effect significantly, whereas the selective blockade of EP4 receptors prevented the PGE2-induced inhibition of NK cells proliferation. These results indicate that PGE2 at certain concentrations may impair the proliferation of NK cells and this effect is mediated via the EP4 receptor.

The influence of prostaglandin E2 on the production of IFN-γ by bovine CD4+, CD8+ and WC1+ T cells

The aim of these studies was to assess the influence of prostaglandin E2 (PGE2) on the production of interferon-gamma (IFN-γ) by bovine CD4(+), CD8(+), and WC1(+) T cells and furthermore, should this effect exist, to identify the E-prostanoid (EP) receptor subtype(s) responsible for this influence. We here report that exposure of bovine peripheral blood mononuclear cells (PBMCs) to PGE2 significantly and dose-dependently decreased the percentage of IFN-γ-producing CD4(+) and CD8(+) T cells. It was also shown that PGE2 reduced IFN-γ production by WC1(+) T cells, but this effect was not dose dependent. The impairment of IFN-γ production should be recognized as an anti-inflammatory and immunosuppressive action, thus the obtained results confirm the paradoxical status of PGE2 as a proinflammatory factor with immunosuppressive activity. The blockade of EP1, EP2, EP3, and EP4 receptors did not prevent PGE2-induced reduction of IFN-γ production by CD4(+) and CD8(+) T cells, indicating that this effect of PGE2 is not mediated through EP receptors. On the contrary, the blockade of either EP2 or EP4 receptors, but not EP1 or EP3 receptors, prevented the PGE2-induced reduction of percentage of IFN-γ-producing WC1(+) T cells. These findings indicate that the ability of PGE2 to impair IFN-γ production by WC1(+) T cells is mediated via EP2 and EP4 receptors. These results suggest the possibility of pharmacological manipulation of IFN-γ production by WC1(+) T cells via selective antagonists and agonists of EP2 and EP4 receptors.

Feline Infectious Peritonitis: Immunohistochemical Features of Ocular Inflammation and the Distribution of Viral Antigens in Structures of the Eye

Feline infectious peritonitis (FIP) is a serious, widely distributed systemic disease caused by feline coronavirus (FCoV), in which ocular disease is common. However, questions remain about the patterns of ocular inflammation and the distribution of viral antigen in the eyes of cats with FIP. This study characterized the ocular lesions of FIP including the expression of glial fibrillary acidic protein and proliferating cell nuclear antigen by Müller cells in the retina in cases of FIP and to what extent macrophages are involved in ocular inflammation in FIP. Immunohistochemistry for FCoV, CD3, CD79a, glial fibrillary acidic protein, calprotectin, and proliferating cell nuclear antigen was performed on paraffin sections from 15 naturally occurring cases of FIP and from controls. Glial fibrillary acidic protein expression was increased in the retina in cases of FIP. Müller cell proliferation was present within lesions of retinal detachment. Macrophages were present in FIP-associated ocular lesions, but they were the most numerous inflammatory cells only within granulomas (2/15 cats, 13%). In cases of severe inflammation of the ciliary body with damage to blood vessel walls and ciliary epithelium (3/15, 20%), some macrophages expressed FCoV antigens, and immunolabeling for calprotectin on consecutive sections suggested that these FCoV-positive macrophages were likely to be recently derived from blood. In cases of severe and massive inflammation of most ocular structures (4/15, 26%), B cells and plasma cells predominated over T cells and macrophages. These results indicate that gliosis can be present in FIP-affected retinas and suggest that breakdown of the blood–ocular barrier can allow FCoV-bearing macrophages to access the eye.

CD25+CD127+Foxp3- Cells Represent a Major Subpopulation of CD8+ T Cells in the Eye Chambers of Normal Mice

The aim of this study has been to determine whether eye chambers constitute part of the normal migratory pathway of naive CD4+ and CD8+ T cells in mouse and if natural CD4+CD25+Foxp3+ and CD8+CD25+Foxp3+ regulatory T cells are present within these eye compartments. To this aim, the cells obtained from aqueous humor (AH) of normal mice were phenotyped in terms of the expression CD4, CD8, CD25, CD127 and transcription factor Foxp3. The mean percentage of CD8+ T cells in the total AH lymphocyte population was as high as 28.69%; the mean percentage of CD8high and CD8low cells in this population was 34.09% and 65.91%, respectively. The presence of cells with the regulatory phenotype, i.e. CD25+Foxp3+ cells, constituted only 0.32% of CD8+ T cell subset. Regarding the expression of CD25, AH CD8+ T cells were an exceptional population in that nearly 85% of these cells expressed this molecule without concomitant Foxp3 expression. Despite having this phenotype, they should not be viewed as activated cells because most of them co-expressed CD127, which indicates that they are naive lymphocytes. With regard to the markers applied in the present research, CD8+CD25+CD127+Foxp3- T cells represent the most numerous subset of AH CD8+ cells. The results suggest that eye chambers in mice are an element in the normal migratory pathway of naive CD8+ T cells. The study presented herein demonstrated only trace presence of CD4+ cells in the eye chambers, as the mean percentage of these cells was just 0.56. Such selective and specific homing of CD8+ and CD4+ cells to the eye chambers is most clearly engaged in the induction and maintenance of ocular immune privilege.

Distribution of CD4+CD8+ double positive T cells in a mouse model of allergic asthma.

The present study describes the distribution of CD4+CD8+ double-positive (DP) T cells in various immune compartments of mice with ovalbumin (OVA)-induced allergic asthma. It was found that the absolute number of DP T cells was considerably increased in the mediastinal lymph nodes and lungs of asthmatic mice as compared with that determined in the healthy subjects. On the contrary, the absolute counts of DP T cells was significantly decreased in the head and neck lymph nodes, and in peripheral blood of OVA-immunized mice. These results suggest that DP T cells may be involved in the pathogenesis of allergic asthma.

A comparison of the efficacy and pharmacokinetics of ivermectin after spring and autumn treatments against Cyathostominae in horses

The aim of the present study was to determine the efficacy of ivermectin against Cyathostominae infections and to describe the drugs pharmacokinetic parameters during two seasonal deworming treatments in horses. The study was performed on warm-blooded mares aged 3-12 years weighing 450-550 kg. A single bolus of an oral paste formulation of ivermectin was administered at a dose of 0.2 mg/kg BW in spring and autumn. Fecal samples were tested before treatment and 1, 2, 3, 4, 6, 10, 20, 30, 40, 50, 60, 75 days after treatment. Ivermectin concentrations in blood samples collected before treatment, 0.5, 1, 2, 3, 4, 6, 12, 24, 36 and 48 hours after treatment, and 3, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60 and 75 days after drug administration were determined by high pressure liquid chromatography. Drug absorption was significantly (p<0.05) slower (tmax: 21.89±11.43 h) in autumn than in spring (tmax: 9.78±8.97 h). Maximum concentrations (Cmax) of ivermectin in the blood plasma of individual horses (8.40-43.08 ng/ml) were observed 2-24 h after drug administration during the spring treatment and 2-36 h (6.43-24.86 ng/ml) after administration during the autumn treatment. Significantly higher (p<0.05) ivermectin concentrations were found during the first 4 hours after administration in spring in comparison with those determined after the autumn treatment. The administration of the recommended dose of ivermectin resulted in 100% elimination of parasitic eggs from feces in spring and autumn treatment.