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A pharmacodynamic assay has been previously developed to monitor ciclosporin treatment in dogs by assessing inhibition of cytokine transcription after whole blood stimulation with 12‐myristate 13‐1 acetate and ionomycin (PMA/I). In this study, whole blood stimulation with either PMA/I or lipopolysaccharide (LPS) was used to assess the effect of multiple drugs (azathioprine, ciclosporin, mycophenolate, leflunomide and prednisone) after a 7‐day treatment course on production of cytokines measured with a multiplex assay in healthy dogs (n = 4 for each treatment). Interleukin‐10 (IL‐10), interferon gamma (IFN γ) and tumour necrosis factor alpha (TNF α) were significantly activated by PMA/I stimulation and IL‐6, IL‐10 and TNF α by LPS stimulation, in the absence of immunosuppressive drugs. After ciclosporin treatment, IL‐10, IFN γ and TNF α production was significantly reduced after stimulation with PMA/I compared to pre‐treatment. After prednisone treatment, TNF α production was significantly reduced after stimulation with PMA/I or LPS compared to pre‐treatment. No significant change was observed after treatment with azathioprine, leflunomide or mycophenolate. This methodology may be useful to monitor dogs not only treated with ciclosporin, but also with prednisone or a combination of both. Further studies are needed to assess the use of this assay in a clinical setting.

Guanylic nucleotides are essential cellular players, and the critical enzyme in their tightly regulated synthesis in Saccharomyces cerevisiae is encoded by the IMD2 gene. The transcription of IMD2 is subject to general repression by nutrient limitation through the cis nutrient-sensing element. It is also subject to specific feedback regulation by the end products of the guanylic nucleotide synthesis pathway. The critical cis element for this latter mechanism is the guanine response element (GRE), a TATAATA sequence which is located 202 nucleotides upstream of the transcription initiation site and which functions as the IMD2 TATA box. We show that the GRE functions in conjunction with a 52-nucleotide stretch near the transcription start site. This very unusual promoter structure ensures low, basal expression of IMD2 and the recruitment of TFIID to the GRE in response to guanylic nucleotide limitation.

The yeast IMD2 to IMD4 and GUA1 genes, involved in GMP synthesis, are highly expressed in exponentially growing cells but are shut off when cells cease to grow upon nutrient limitation. We show for the IMD2 gene that this effect is not specific to certain carbon sources or to growth rate. Strikingly, the cis elements responsible for this nutritional response are contained within a 23-nucleotide sequence in the coding region of the IMD2 gene. Despite its very unusual location, this regulatory sequence mediates the repression of transcription initiation. From our data, we conclude that GMP synthesis is downregulated upon nutrient limitation through an active mechanism. We show that this transcriptional shutoff abolishes any possibility of the induction of IMD2, even under drastic conditions of guanylic nucleotide limitation. Taken together, these results indicate that low levels of guanylic nucleotides could be required for proper entry into stationary phase.