Tomasz Maślanka

In vitro effects of dexamethasone on bovine CD25+CD4+ and CD25-CD4+ cells.

This paper investigates the in vitro effect of dexamethasone on bovine CD25highCD4+, CD25lowCD4+ and CD25-CD4+ T cells. Only a small percentage of bovine CD25highCD4+ (2-4%) and CD25lowCD4+ (1-2%) cells expressed Foxp3. Dexamethasone caused considerable loss of CD25-CD4+ cells, but it increased the relative and absolute numbers of CD25highCD4+ and CD25lowCD4+ lymphocytes, while at the same time reducing the percentage of Foxp3+ cells within the latter subpopulations. Considering all these, as well as the intrinsically poor Foxp3 expression in bovine CD25+CD4+, it can be concluded that the drug most probably increased the number of activated non-regulatory CD4+ lymphocytes. It has been found that changes in cell number were at least partly caused by proapoptotic effect of the drug on CD25-CD4+ cells and antiapoptotic effect on CD25highCD4+ and CD25lowCD4+ cells. The results obtained from this study indicate that the involvement of CD4+ lymphocytes in producing the anti-inflammatory and immunosuppressive effect of dexamethasone in cattle results from the fact that the drug had a depressive effect on the production of IFN-γ by CD25-CD4+ cells. Secretion of TGF-β and IL-10 by CD4+ lymphocytes was not involved in producing these pharmacological effects, because the drug did not affect production of TGF-β and, paradoxically, it reduced the percentage of IL-10+CD4+ cells.

In vitro effects of meloxicam on the number, Foxp3 expression, production of selected cytokines, and apoptosis of bovine CD25+CD4+ and CD25-CD4+ cells

The purpose of this study was to evaluate the effect of meloxicam (MEL) on selected immune parameters of bovine CD25highCD4+, CD25lowCD4+, and CD25-CD4+ cells. Peripheral blood mononuclear cells (PBMCs) collected from 12-month-old heifers were treated with MEL at a concentration corresponding to the serum level of this medication following administration at the recommended dose (MEL 5 × 10-6 M) and at a concentration 10 times lower (MEL 5 × 10-7 M). After 12 and 24 h of incubation with the drug, the percentage of CD25highCD4+ cells decreased; however, this disturbance was quickly reversed. Furthermore, the absolute number of CD25highCD4+ cells in the PBMC populations treated with MEL 5 × 10-6 M for 48 and 168 h was increased. Prolonged (168 h) exposure to the drug increased the percentage of Foxp3+ cells in the CD25highCD4+ cell subpopulation. The higher dose of MEL was found to significantly increase the percentage of IFN-γ+ cells among the CD25-CD4+ cells. These results indicated that MEL does not exert an immunosuppressive effect by depleting CD4+ cells and suppression of IFN-γ+ production by these cells. Furthermore, IL-10 and TGF-β production was not changed following exposure to MEL.

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.

In vitro studies on the influence of dexamethasone and meloxicam on bovine WC1+ γδ T cells.

In view of the lack of data on the effect of meloxicam (non-steroidal anti-inflammatory drug) on bovine γδ T cells (WC1(+) cells) and very poorly recognized effects of dexamethasone (steroidal anti-inflammatory drug) on these cells, the purpose of the present study has been to determine the in vitro influence of these drugs on CD25(high)WC1(+), CD25(low)WC1(+) and CD25(-)WC1(+) lymphocytes of the peripheral blood of cattle. Peripheral blood mononuclear cells were treated with the drugs in concentrations reflecting their plasma levels achieved in vivo at therapeutic doses (dexamethasone 10(-7)M; meloxicam 5×10(-6)M) and at ten-fold lower concentrations. It was found out that percentages and absolute counts of CD25(high)WC1(+) and CD25(low)WC1(+) cells increased in the presence of dexamethasone, and this effect was at least partly attributable to lower mortality of these cells, whose apoptosis was depressed by exposure to dexamethasone. It seems certain that this effect was not a result of increased multiplication of CD25(high)WC1(+) and CD25(low)WC1(+) cells because their proliferation was reduced in the presence of dexamethasone. Exposure to this drug caused a rapidly occurring and lasting depletion of CD25(-)WC1(+), which was at least partly due to their higher apoptosis. The results seem to suggest that impaired proliferation of these cells was responsible for a more profound expression of this disorder. Paradoxically, the percentage of cells producing IFN-γ, a proinflammatory cytokine, increased in the presence of dexamethasone, whereas the count of cells secreting the key anti-inflammatory and immunosuppressive cytokine, i.e. IL-10, declined. This effect was observed in all analyzed subpopulations of cells. Meloxicam did not interfere so drastically as dexamethasone with the functioning of WC1(+) lymphocytes because it did not affect their apoptosis, proliferation, percentage or absolute count. With respect to the effect of meloxicam on counts of particular WC1(+) lymphocyte subpopulations, it was only demonstrated that exposure to the drug was correlated with a transient and very weakly expressed decrease in the relative and absolute counts of CD25(high)WC1(+) and CD25(low)WC1(+) cells, which was most probably a result of a temporary down-regulation of the expression of the CD25 molecule. In the presence of meloxicam, percentages of IFN-γ(+)CD25(-)WC1(+) cells as well as cells producing IL-10 declined, an effect observed in all analyzed cell populations. These results suggest that care should be taken when administering this medication to animals with bacterial or viral infections, and we should avoid giving it to patients suffering from allergic or autoimmune disorders.

IκB kinase β inhibitor, IMD-0354, prevents allergic asthma in a mouse model through inhibition of CD4(+) effector T cell responses in the lung-draining mediastinal lymph nodes.

IκB kinase (IKK) is important for nuclear factor (NF)-κB activation under inflammatory conditions. It has been demonstrated that IMD-0354, i.e. a selective inhibitor of IKKβ, inhibited allergic inflammation in a mouse model of ovalbumin (OVA)-induced asthma. The present study attempts to shed light on the involvement of CD4(+) effector (Teff) and regulatory (Treg) T cells in the anti-asthmatic action of IMD-0354. The animals were divided into three groups: vehicle treated, PBS-sensitized/challenged mice (PBS group); vehicle treated, OVA-sensitized/challenged mice (OVA group); and IMD-0354-treated, OVA-sensitized/challenged mice. The analyzed parameters included the absolute counts of Treg cells (Foxp3(+)CD25(+)CD4(+)), activated Teff cells (Foxp3(-)CD25(+)CD4(+)) and resting T cells (CD25(-)CD4(+)) in the mediastinal lymph nodes (MLNs), lungs and peripheral blood. Moreover, lung histopathology was performed to evaluate lung inflammation. It was found that the absolute number of cells in all studied subsets was considerably increased in the MLNs and lungs of mice from OVA group as compared to PBS group. All of these effects were fully prevented by treatment with IMD-0354. Histopathological examination showed that treatment with IMD-0354 protected the lungs from OVA-induced allergic airway inflammation. Our results indicate that IMD-0354 exerts anti-asthmatic action, at least partially, by blocking the activation and clonal expansion of CD4(+) Teff cells in the MLNs, which, consequently, prevents infiltration of the lungs with activated CD4(+) Teff cells. The beneficial effects of IMD-0354 in a mouse model of asthma are not mediated through increased recruitment of Treg cells into the MLNs and lungs and/or local generation of inducible Treg cells.

Effect of dexamethasone and meloxicam on counts of selected T lymphocyte subpopulations and NK cells in cattle – In vivo investigations

The purpose of these investigations has been to assess the in vivo effect of dexamethasone (DEX) and meloxicam (MEL) on percentages and absolute counts of cells within selected T lymphocyte subsets and NK cells in cattle. DEX application caused substantial loss of NK, CD4(+), CD8(+) and WC1(+) T cells, but the drugs influence on T-cell count was selective, that is it varied according to the presence and intensity of CD25 expression. Reduced counts of T lymphocytes were due to the depletion of CD25(-)CD4(+), CD25(-)CD8(+) and CD25(-)WC1(+) T cells. The loss of CD25(-)CD8(+) and CD25(-)WC1(+) T cells was a deep and lasting disorder, whereas the depletion of CD25(-)CD4(+) T cells was manifested less strongly and regressed promptly. The administration of DEX did not affect absolute counts of CD25(low)CD4(+) and CD25(low)CD8(+) T cells, but induced an increase in percentages and absolute counts of CD25(high)CD4(+), CD25(high)CD8(+), CD25(low)WC1(+) and CD25(high)WC1(+) T cells. In respect of the effect on counts of CD4(+), CD8(+) and WC1(+) T cells, MEL proved to be a safe medication, because it did not alter counts of these lymphocytes. The administration of MEL led to an increase in the absolute count of NK cells, but the effect did not appear quickly and its development required time.

Dexamethasone inhibits and meloxicam promotes proliferation of bovine NK cells

Abstract Due to the unrecognized effect of dexamethasone (DEX) and meloxicam (MEL) on bovine natural killer (NK) cells, studies have been undertaken in order to determine whether the above medications can affect these cells in respect of their counts, apoptosis, proliferation and production of selected cytokines. Peripheral blood mononuclear cells (PBMCs) were treated with the drugs in concentrations reflecting their plasma levels achieved in vivo at therapeutic doses and in 10-fold lower concentrations. The effect of DEX and MEL on percentages and absolute counts of NK cells was determined 6, 12, 48 and 168 h after the exposure of PBMCs to the drugs. At each time point, it was found out that DEX reduced the absolute count of NK cells, an effect attributed to the proapoptotic and anti-proliferative influence of the drug on these cells. DEX lowered the production of IFN-γ by the analyzed cells and raised the percentage of IL-10-producing cells. Thus, the above effects are important elements contributing to the complex mechanism responsible for the anti-inflammatory and immunosuppressive properties of the drug. MEL neither affects the apoptosis of NK cells nor did it reduce their count. Moreover, one-week exposure to MEL raised the absolute count of these cells, which was the result of their more intense proliferation in the presence of the drug. Thus, the influence of MEL with respect to the proliferation and count of NK cells was immunostimulating. On the other hand, MEL reduced the percentage of IFN-γ-producing NK cells, which in turn is an immunosuppressive effect.

Effect of inhaled and systemic glucocorticoid treatment on CD4+ regulatory and effector T cells in a mouse model of allergic asthma

&NA; To achieve a better understanding of mechanisms underlying the anti‐asthmatic action of inhaled and systemic glucocorticoids (GCs) and to provide more data regarding the risk of a negative effect of inhaled GCs on CD4+ T cells, a study was conducted on the effect of ciclesonide and methylprednisolone on CD4+ effector (Teff), regulatory (Treg) and resting (Trest) T cells within respiratory and extra‐respiratory tissues in a mouse model of allergic asthma. The study indicated that one, and possibly a key mechanism, underlying the anti‐asthmatic action of inhaled and systemic GCs is the prevention of the activation and clonal expansion of CD4+ Teff cells in the mediastinal lymph nodes (MLNs), which consequently prevents infiltration of the lungs with CD4+ Teff cells. The beneficial effects of GCs in asthma treatment were not mediated through increased recruitment of Treg cells into the MLNs and lungs and/or local generation of Treg cells. The results demonstrated that inhaled and systemic GCs induced comparable depletion of normal CD4+ Teff, Trest and Treg cells in the MLNs, head and neck lymph nodes and peripheral blood. Furthermore, inhaled, but not systemic GC therapy, led to the loss of these cells in the lungs. Thus, the study suggests that inhaled GC therapy may not be safer at all than systemic one with respect to the adverse effect on CD4+ T cells present within and outside the respiratory tract. Moreover, administration of inhaled GCs can produce negative effects on lung‐residing CD4+ T cells. HighlightsInhaled and systemic GCs prevented OVA‐induced proliferation of CD4+ T cells in MLNs.Anti‐asthmatic action of inhaled and systemic GCs was not mediated through Treg cells.Inhaled GCs induced depletion of CD4+ T cells in the lungs, MLNs, HNLNs and PB.Inhaled and systemic GC treatment led to the loss of CD4+ Treg, Teff and Trest cells.Inhaled and systemic GCs induced comparable depletion of normal CD4+ T cells.