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The paper describes a method for the separation of 18 nucleotides, nucleosides and nucleobases by isocratic solvent-generated ion-pair chromatography in 55 min. It has been developed for pharmacodynamic monitoring of mycophenolic acid, the active metabolite of the immunosuppressant mycophenolate mofetil. The method was applied for the detection of mycophenolic acid-induced changes in inosine 5′-monophosphate dehydrogenase (IMPDH) activity in the lysate of human peripheral blood mononuclear cells.

Background: Inosine 5′-monophosphate dehydrogenase (IMPDH) catalyses the oxidation of inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP). Thus, this enzyme plays an important role in the rate-limiting step of the de novo guanine nucleotide biosynthesis, making it a potent target for immunosuppressive drugs. Mycophenolic acid (MPA) is the most potent and specific inhibitor of IMPDH. Method: IMPDH activity is determined via evaluation of XMP formation and the inhibitory influence of MPA in human peripheral blood mononuclear cells (PBMCs) is assessed by means of high-performance liquid chromatography (HPLC). For this objective, we have optimised a method based on solvent-generated ion exchange chromatography by cautiously varying mobile phase parameters. Results: The optimised method renders it possible to separate 18 analytes in 54 min in a single isocratic experiment and to measure the IMPDH activity in the lysate of human PBMCs in dependence on incubation time, substrate, co-substrate and inhibitor concentrations. In this way, we have determined the Michaelis–Menten constants KM and Vmax for IMP and β-NAD+ and the inhibitor constant Ki for MPA. Conclusions: The chromatographic method presented in this report allows a rapid, reliable and reproducible quantification of IMPDH activity in PBMCs and therefore represents an attractive tool for the pharmacodynamic monitoring of the effects of MPA in patients under immunosuppressive therapy.

Background: Among the different factors playing crucial roles in endothelial cell activation, cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) have been reported to demonstrate profound effects on this cell type. It has been shown that the increased release of IFN-α/γ and TNF-α causes structural and functional modulations of the endothelial cell. These cytokines participate in the recruitment and activation of the immune system. CsA is an immunosuppressive drug that is necessary at high levels in human recipients of vascularised xenografts. This drug could contribute to a prolonged graft survival by modulation of endothelial cell activation. Methods: The present study deals with the effects of cyclosporin A on adhesion molecule expression (i.e. ICAM-1, VCAM-1, E-selectin, P-selectin, PECAM-1 and the L-selectin ligand CD 34) on the surface of cytokine stimulated HUVECs. The in vitro model described herein mimics the stimulation of endothelial cells by cytokines as seen during inflammatory processes after transplantation. Therefore, HUVECs were activated either with TNF-α, IL-1β or with a cytokine mixture consisting of those stimulants present at an elevated level in sera of patients during allograft rejection (i.e. IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ). Results: The results obtained show that the immunosuppression of CsA is not only achieved by inhibiting lymphocyte proliferation, but also by decreasing the expression of adhesion molecules on endothelial cells, which are the first target of the cellular rejection process. Conclusion: Co-incubation of stimulated endothelial cells with a final CsA concentration of 5 μg/ml revealed a significant down-regulating influence on the surface expression of E-selectin and VCAM-1.

The immunosuppressant mycophenolic acid (MPA) selectively inhibits proliferation of T- and B-lymphocytes by blocking inosine 5'-monophosphate-dehydrogenase (IMPDH), the key enzyme for de-novo-synthesis of guanine nucleotides. In an in vitro study the effects of MPA on human peripheral blood lymphocyte activation markers and on cell cycle characteristics were investigated. Mononuclear cells from healthy volunteers were incubated with phytohaemagglutinin (PHA) and increasing doses of MPA. After 72 h incubation an aliquot of the cells was stained with propidium iodide and measured by FACS analyses to assess the DNA shape. In addition, the expression of the activation markers HLA-DR and CD25 on T- and B-lymphocytes was determined by flow cytometry analysis.PHA stimulation led to a significant increase of the S-phase of cell cycle. PHA stimulation clearly increased mean fluorescence intensity (MFI) of HLA-DR expression on B-lymphocytes. PHA stimulation also elevated the number of CD25 positive B-lymphocytes. Expression of HLA-DR on T-lymphocytes was not influenced by PHA, whereas CD25 expression and MFI significantly increased. All the observed PHA induced effects were reduced by co-incubation with increasing doses of MPA. The data presented show that in vitro the immunosuppressive effect of MPA can be demonstrated using FACS technology on a cellular level. MPA leads to an inhibition of cell cycle proliferation in peripheral blood lymphocytes.

Background: Cytokines influence the expression of adhesion molecules and hence, regulate the passage of leucocytes from the blood to the site of inflammation causing leucocyte accumulation and the modulation of the nature and progression of inflammatory responses. They form a complex communication network causing results which are not determined by the effects of a single cytokine but especially by the interaction of several cytokines. Method: For the determination of adhesion molecule expression on the surface of enzymatically detached endothelial cells, flow cytometry is applied. Fluorescence-conjugated mouse monoclonal antibodies directed against VCAM-1, ICAM-1, PECAM-1, CD34, E- and P-selectin are used. Results: We clearly demonstrate that ICAM-1, PECAM-1, P-selectin and CD34 are-in relation to an incubation cocktail containing solely TNF-α, IL-1β and IFN-γ-altered antagonistically by the supplementary addition of the inflammatory cytokines IL-2 and IL-6 as well as the anti-inflammatory cytokines IL-4 and IL-10, whereas VCAM-1 is synergistically enhanced under the same test conditions. Conclusion: The results of our in vitro investigations show that the effects of a single cytokine within a multi-component cytokine combination on endothelial adhesion molecule expression are strongly influenced by the nature of the other cytokines present in the combination tested.

Immunosuppressive drugs are needed to prevent the rejection of transplanted organs by the immune system. Immunosuppressive antimetabolites act by interrupting cell metabolism. Their mechanism of action can be studied in vitro by measuring the inhibition of biochemical activities which is reflected by changes in the nucleotide content. In our experiments, human peripheral blood mononuclear cells (PBMC) isolated from healthy volunteers were used. After PBMC stimulation with phytohaemagglutinin (PHA) to mimic activation occurring at a rejection crisis, cells were exposed to varying concentrations of different immunosuppressants (i.e., mycophenolic acid, cyclosporin A and prednisolone) for 68 h at 37 degrees C. Changes in nucleotide content were observed by determining the concentrations of 15 nucleotides using a newly developed HPLC method. The results obtained for mycophenolic acid (MPA; final concentrations in a range between 0.1 and 5 micromol/l), cyclosporin A (CsA; final concentrations between 100 ng/ml and 1 microg/ml) and prednisolone (final concentrations between 0.5 and 10 micromol/l) are given as percentage changes in nucleotide content versus controls and are expressed as mean +/- confidence interval. The possibility of synergistic effects was investigated by incubating the cells with mixtures of all three immunosuppressive drugs varying the amount of mycophenolic acid. In addition, we have shown the effects of MPA/guanosine co-incubation on the intracellular nucleotide levels. Stimulation of peripheral blood mononuclear cells with phytohaemagglutinin led to a significant increase of pyrimidine and purine nucleotides versus control values (100%). Pyrimidine (CTP, UDP, UTP) and purine nucleotides (GDP, GTP, ADP, ATP) were elevated up to 153+/-14% and 142+/-17%, respectively. Under co-incubation of cells with MPA, the GTP level decreased in a dose-related manner to 56+/-3% of control at a MPA final concentration of 5 micromol/l. Concomitantly, an increase of UTP values to 203+/-18% versus control was observed under co-incubation with 1 micromol/l MPA. Co-incubation of mononuclear cells with guanosine (50 micromol/l) compensated for the effects of MPA on intracellular GTP levels. Combination of MPA, CsA and prednisolone did not alter intracellular nucleotide profiles of PBMC compared to those under MPA incubation alone. The depletion of the guanine nucleotide pool and concomitant increase of uridine nucleotides under the influence of the immunosuppressive drug mycophenolic acid is caused by its inhibitory effects on the key enzyme of de novo purine biosynthesis, inosine 5'-monophosphate dehydrogenase (IMPDH).

The interaction between leukocytes and endothelial cells plays the essential role in inflammation. Endothelial cells express a variety of adhesive receptors that regulate their adhesion to leukocytes and also to the extracellular matrix. These interactions are complex phenomena that require multiple recognition mechanisms, and include the first rolling and later the stationary adhesion and transmigration of leukocytes. It is known that cytokines have regulatory effects on cell adhesion molecules expression. In the present study we investigated the influence of cytokines (IL-1b, IL-2, IL-4, IL-6, IL-8 IL-10, TNF-a, IFN-g) and the combined use of IL-2, IL-4, IL-6, IL-8 and IL-10 with IL-1b, TNF-b or IFN-g on the expression of adhesion molecules of cultured human umbilical vein endothelial cells (HUVECs) after stimulation for 16 hours. Likewise, in vitro model described herein is designed to mimic the activation of endothelial cells by cytokines as seen during inflammatory processes. This process is mediated by specific cell adhesion molecules being crucial for the generation of immune and inflammatory responses. Therefore, HUVECs are treated with two different cytokine combinations consisting of either IL-2, IL-6, IL-8 IFN-g and TNF-a or IL-1b, IL-2, IL-4, IL-6, IL-10, IFN-g and TNF-a. Endothelial cells were collected from hunam umbilical vain using collagense type II, and cell cultures in complete medium were kept in the incubator (37.4?C, 5% CO2). After stimulation cells were prepared for analysis using tripsinisation procedure. The surface expression of the following adhesion molecules was determined in cultured human umbilical vein endothelial cells (HUVECs) by means of flow-cytometric analysis: CD 62P (P-selectin), CD 62E (E-selectin, ELAM-1) CD 106CD 34 (L-selectin ligand). The highest CD 62E expression on the surface of HUVECs was found when endothelial cells were stimulated with TNF-a alone. Also they were increased after stimulation with IL-1b, while IL-4 led to down-regulation of CD 62E. Incubation of HUVEC monolayers with IL-1b, IL-4 as well as TNF-a and IFN-g, statistically significant, reduced the surface expression of CD 34 while other cytokines did not affect CD 34 expression. Incubation of HUVECs with a single cytokine caused no statistically significant changes in CD 62P expression compared to controls. The most potent effect on CD 54 expression was found under TNF-a stimulation; IL-1b and IFN-g had also amplifying effects, while all other tested cytokines caused no significant changes in surface molecule expression. Surface expression of CD 106 was amplified during incubation with IL-1b, IL-4, TNF-a and IFN-g. Single stimulation of tested cytokines did not significantly alter the cell surface expression of CD 31. Concomitant stimulation with IL-2, IL-4, IL-6, IL-8 or IL-10 with IL-1b, TNF-a or IFN-g led to different effects compared with effects of single cytokine stimulation: CD 62E were up-regulated under co-stimulation with combination of IL-1b and IFN-g, IL-6 and IL-1b, and also in all combinations with TNF-a. Statistically significant differences were found in CD 62P surface expression after concomitant stimulation with IL-1b and IFN-g, and in combinations with TNF-a. Co-stimulation with IL-10 and IL- 1b, TNF-a or IFN-g, or IL-8 with IL-1b or IFNg, IL-6 with IFN-g, IL-4 with TNF-a or IFN-g and IL-2 with IFN-g significantly decreased the level of CD 34 surface expression. (VCAM-1), CD 54 (ICAM-1), CD 31 (PECAM-1) and CD 54 expression was up-regulated after stimulation with IL-1b and IFN-g, and under concomitant stumulation with TNF-a. Surface expression of molecule CD 106 was higher after co-stimulation of cytokines with TNF-a, and IL-4 or IL-10 with IL-1b. These effects indicate modulation of single cytokine effects. Intracellular mechanisms included in those effects need to be investigated. Also there were found modulatory effects of cytokine combinations. Some effects of cytokine combinations were different in comparison to single cytokine effect. This finding indicates that intracellular mechanisms are present and responsible for signal modulation of single cytokine. The application of these two cytokine combinations mimicing inflammation reactions results in effects of comparable dimensions significantly increasing the mean fluorescence intensity of E-selectin, VCAM-1 and ICAM-1 surface expression accompanied by the induction of P-selectin expression. The experiments reveal a strong up-regulation of these cell surface antigens under conditions mimicing inflammation. This is an essential finding stressing the importance of endothelial cells during inflammatory processes.