Quantitation of ovine cytokine mRNA by real-time RT-PCR.

In this study we describe for the first time the dynamics of the expression of the cytokines, IL-1beta, IL-12p40, TNFalpha in ovine dendritic cells and macrophages after LPS stimulation. Real time RT-PCR was used for the quantitation of these cytokines and IL-4 and IFNgamma as well as two potential housekeeping genes (HKG), ATPase and GAPDH, in mRNAs from ovine leucocyte populations. Both dual-labelled probes (TAMRA/FAM) and SYBR Green assays were utilised, using a Corbett Research RotorGene and ABI 7700 machine. In order to quantitate each cytokine in our assays all C(T) values were compared to a standard curve generated using plasmid DNA containing the cytokine of interest. To validate our assays, concanavalin A-stimulated peripheral blood mononuclear cells (PBMCs) and LPS-stimulated monocyte-derived dendritic cells (MoDC) and monocyte-derived macrophages (MDMØ) were examined. We found that peak cytokine mRNA expression was between 3 and 6 h for the cytokines examined except for IL-12p40 where peak cytokine release was around 12 h post-stimulation in MDMØ and PBMCs. However, in MoDCs, peak IL-12p40 mRNA expression was observed within 3-6 h. We have identified a sensitive and reliable method for the identification of ovine cytokine mRNAs.

The soluble form of CD83 is present at elevated levels in a number of hematological malignancies.

Recombinant forms of soluble CD83 (sCD83) inhibit anti-tumor responses. In this analysis of circulating sCD83 levels we report that although >95% of acute myeloid leukemia (AML) and multiple myeloma (MM) patients have normal or only weakly elevated sCD83 levels, 20% of chronic lymphocytic leukemia (CLL) and 5/7 mantle cell lymphoma (MCL) patients have significantly elevated levels (>1 ng/ml). Isolated CLL cells both weakly expressed membrane CD83 (mCD83), and released sCD83 during in vitro culture. We conclude that malignant cells are a potential source of sCD83 and that it may have functional and/or prognostic significance in hematological malignancies, particularly CLL and MCL.

The clinical significance of soluble CD86 levels in patients with acute myeloid leukemia and myelodysplastic syndrome.

BACKGROUND: Levels of the soluble form of CD86 (sCD86) are elevated in a proportion of patients with leukemia. Although it is a potential modulator of antitumor responses, the significance of sCD86 in patients with hematologic malignancies is unknown. METHODS: The authors evaluated sCD86 levels by enzyme-linked immunosorbent assay in patients with acute myeloid leukemia (AML) (n = 57 patients) and patients with myelodysplastic syndrome (MDS) (n = 40 patients) and analyzed the relation between sCD86 levels and various clinical parameters. RESULTS: Levels of sCD86 were elevated (> 2.32 ng/mL) relative to normal donors (0.22-2.32 ng/mL; n = 51 patients) in 25% of patients with AML and in 27% of patients with MDS. Patients with AML who had elevated sCD86 levels had significantly lower complete remission (CR) rates compared with patients with AML who had normal sCD86 levels. In multivariate analysis using sCD86 as a continuous variable and including the interaction of age and sCD86 as a variable, sCD86 was a significant prognostic factor (P = 0.014) independent of cytogenetics. Further analysis demonstrated that, in patients with AML age 60 years and younger, but not in patients older than 60 years, elevated sCD86 levels were associated with significantly shorter survival (P = 0.04). There was no correlation between sCD86 levels and CR rates or survival in patients with MDS. CONCLUSIONS: The presence in patients with AML of elevated levels of circulating sCD86 were associated with lower CR rates and poor survival. The prognostic significance of sCD86 was independent of cytogenetics but was modulated by age, in that it was independently significant only in younger patients. The results suggest that sCD86 may play a role in modulating immune responses associated with the progression of AML.