Włodzimierz Markiewicz

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.

Influence of β2- and β3-adrenoceptor agonists on contractile activity of the porcine myometrium in the luteal phase and the first days of pregnancy

This study analysed the relaxant properties of salbutamol (β2-adrenoceptors agonist) and BRL 37344 (β3-adrenoceptors agonist) regarding the contractility of porcine myometrium on days 10-14 of the oestrous cycle (cyclic group; n = 10) and on days 3-5 of pregnancy (early pregnant group; n = 6). The activity of myometrial strips (tension, frequency and amplitude) was recorded under isometric conditions using force transducers. The contractility was assessed further following the administration of increasing concentrations of the agonists (10-9-10-4 M), both with and without β-adrenoceptor antagonists (butaxamine - a selective β2- adrenoceptor antagonist, propranolol- a non-selective β1- and β2-adrenoceptor antagonist and bupranolol - a non-selective β1-, β2- and β3-adrenoceptor antagonist) at a concentration of 10-4 M. Although neither salbutamol nor BRL 37344 caused changes in the tension, at the highest concentrations they decreased the frequency and amplitude of contractions. These changes were more evident after salbutamol treatment and in the early pregnant group. Antagonists given alone did not cause changes in the parameters examined but changed some activity of the agonists. Butoxamine reduced the decrease in frequency and amplitude induced by salbutamol and produced a decrease in the tension after BRL 37344 treatment in the early pregnant group. Propranolol reduced the decrease in frequency and amplitude induced by salbutamol in both examined groups and did not cause significant changes in BRL 37344 activity. The administration of bupranolol before salbutamol treatment caused an increase in the tension and reduced the decrease in the frequency in the cyclic group. Moreover, bupranolol eliminated a decrease in frequency and induced an increase in amplitude caused by BRL 37344 in both groups and these changes were more evident in the early pregnant group. The data indicates that both β2- and β3-adenoreceptors are involved in the regulation of the contractility in both groups, but the changes after agonists and antagonists treatment are more evident in the early pregnant myometrium.

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 doxazosin on the contractility of the urinary bladder in female pigs with experimentally induced cystitis

The present in vitro study investigated the influence of doxazosin on the contractility of the urinary bladder in female pigs with experimentally induced cystitis. Fifteen juvenile female piglets (18-20 kg body weight) were randomly assigned into three groups (n=5 animals each): i) control (clinically healthy animals, without doxazosin treatment), ii) animals with induced inflammation of the urinary bladder, but without doxazosin treatment (experimental group I) and iii) animals with inflamed bladder, treated orally with doxazosin (0.1 mg/kg body weight for 30 days; experimental group II). Thereafter, the pigs were sacrificed and strips of the bladder trigone were suspended in organ baths. The tension and amplitude of the smooth muscles was measured before and after exposition to 5-hydroxytryptamine (5-HT; 10-6-10-4 M), acetylocholine (ACh; 10-5-10-3 M) and norepinephrine (NE; 10-9-10-7 M). 5-HT caused an increase in the tension of contractions in all the groups and the amplitude in the experimental groups, however, the effect was higher in the experimental group I than in group II as compared to that found in the pre-treatment period. ACh caused an increase in the tension in the control group and a decrease in the amplitude in both experimental groups; these changes significantly differed between the control and doxazosin-treated group. NE caused a decrease in the tension in both experimental groups and amplitude in all the groups, however, the effect was most strongly expressed in doxazosine-treated group. The present study has revealed that long-term administration of doxazosin causes a desensitization of the detrusor smooth muscle to in vitro applied mediators in the autonomic nervous system.