Reliability of mycophenolic acid monitoring by an enzyme multiplied immunoassay technique.

BACKGROUND: For mycophenolic acid (MPA), substantial inter- and intra-individual variability and drug interactions have been observed and therapeutic drug monitoring is now recommended. In this study, a MPA commercial Enzyme Multiplied Immunoassay Technique (EMIT) was evaluated and compared with the HPLC-UV reference method which is easily practicable in a routine laboratory. METHODS: Plasma samples (n = 117) were collected from adult renal graft patients treated by mycophenolate in combination with either cyclosporin A (CyA) (n = 32) or tacrolimus (n = 85). RESULTS: Considering all samples, correlation was excellent (p < 0.0001). However, significant MPA overestimation was observed with EMIT in the early post-transplant period (30%, n = 32) or when combined with cyclosporin (45%). CONCLUSIONS: In the early post-transplant period, or in cases where CyA is used in combination with MPA, the EMIT cannot be recommended. HPLC or LC/MS are here the method of choice.

Modulation of murine dendritic cell function by adenine nucleotides and adenosine: involvement of the A(2B) receptor.

Adenosine triphosphate has previously been shown to induce semi-mature human monocyte-derived dendritic cells (DC). These are characterized by the up-regulation of co-stimulatory molecules, the inhibition of IL-12 and the up-regulation of some genes involved in immune tolerance, such as thrombospondin-1 and indoleamine 2,3-dioxygenase. The actions of adenosine triphosphate are mediated by the P2Y(11) receptor; since there is no functional P2Y(11) gene in the murine genome, we investigated the action of adenine nucleotides on murine DC. Adenosine 5'-(3-thiotriphosphate) and adenosine inhibited the production of IL-12p70 by bone marrow-derived DC (BMDC). These inhibitions were relieved by 8-p-sulfophenyltheophylline, an adenosine receptor antagonist. The use of selective ligands and A(2B) (-/-) BMDC indicated the involvement of the A(2B) receptor. A microarray experiment, confirmed by quantitative PCR, showed that, in presence of LPS, 5'-(N-ethylcarboxamido) adenosine (NECA, the most potent A(2B) receptor agonist) regulated the expression of several genes: arginase I and II, thrombospondin-1 and vascular endothelial growth factor were up-regulated whereas CCL2 and CCL12 were down-regulated. We further showed that NECA, in combination with LPS, increased the arginase I enzymatic activity. In conclusion, the described actions of adenine nucleotides on BMDC are mediated by their degradation product, adenosine, acting on the A(2B) receptor, and will possibly lead to an impairment of Th1 response or tolerance.

Gene expression profiling defines ATP as a key regulator of human dendritic cell functions.

Extracellular ATP and PGE2 are two cAMP-elevating agents inducing semimaturation of human monocyte-derived dendritic cells (MoDCs). We have extensively compared the gene expression profiles induced by adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) and PGE2 in human MoDCs using microarray technology. At 6 h of stimulation, ATPgammaS initiated an impressive expression profile compared with that of PGE2 (1125 genes compared with 133 genes, respectively) but after 24 h the number of genes regulated by ATPgammaS or PGE2 was more comparable. Many target genes involved in inflammation have been identified and validated by quantitative RT-PCR experiments. We have then focused on novel ATPgammaS and PGE2 target genes in MoDCs including CSF-1, MCP-4/CCL13 chemokine, vascular endothelial growth factor-A, and neuropilin-1. ATPgammaS strongly down-regulated CSF-1 receptor mRNA and CSF-1 secretion, which are involved in monocyte and dendritic cell (DC) differentiation. Additionally, ATPgammaS down-regulated several chemokines involved in monocyte and DC migration including CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL8/MCP-2, and CCL13/MCP-4. Interestingly, vascular endothelial growth factor A, a major angiogenic factor displaying immunosuppressive properties, was secreted by MoDCs in response to ATPgammaS, ATP, or PGE2, alone or in synergy with LPS. Finally, flow cytometry experiments have demonstrated that ATPgammaS, ATP, and PGE2 down-regulate neuropilin-1, a receptor playing inter alia an important role in the activation of T lymphocytes by DCs. Our data give an extensive overview of the genes regulated by ATPgammaS and PGE2 in MoDCs and an important insight into the therapeutic potential of ATP- and PGE2-treated human DCs.