Circulating monocytes are reduced by sphingosine-1-phosphate receptor modulators independently of S1P3.

Sphingosine-1-phosphate (S1P) receptors are critical for lymphocyte egress from secondary lymphoid organs, and S1P receptor modulators suppress lymphocyte circulation. However, the role of S1P receptors on monocytes is less clear. To elucidate this, we systematically evaluated monocytes in rats and mice, both in naive and inflammatory conditions, with S1P receptor modulators FTY720 and BAF312. We demonstrate that S1P receptor modulators reduce circulating monocytes in a similar time course as lymphocytes. Furthermore, total monocyte numbers were increased in the spleen and bone marrow, suggesting that S1P receptor modulation restricts egress from hematopoietic organs. Monocytes treated ex vivo with FTY720 had reduced CD40 expression and TNF-α production, suggesting a direct effect on monocyte activation. Similar reductions in protein expression and cytokine production were also found in vivo. Suppression of experimental autoimmune encephalomyelitis in mice and rats by FTY720 correlated with reduced numbers of lymphocytes and monocytes. These effects on monocytes were independent of S1P3, as treatment with BAF312, a S1P1,4,5 modulator, led to similar results. These data reveal a novel role for S1P receptors on monocytes and offer additional insights on the mechanism of action of S1P receptor modulators in disease.

Low dose nicotine and antagonism of ß2 subunit containing nicotinic acetylcholine receptors have similar effects on affective behavior in mice.

Nicotine leads to both activation and desensitization (inactivation) of nicotinic acetylcholine receptors (nAChRs). This study tested the hypothesis that nicotine and a selective antagonist of ß2*nAChRs would have similar effects on affective behavior. Adult C57BL/6J male mice were tested in a conditioned emotional response (CER) assay which evaluates the ability of an aversive stimulus to inhibit goal-directed behavior. Mice lever-pressed for a saccharin reinforcer according to a variable schedule of reinforcement during sessions in which two presentations of a compound light/tone conditioned stimulus (CS) co-terminated with a 0.1 or 0.3 mA, 0.5 s footshock unconditioned stimulus (US). During testing in the absence of the US, mice received doses of i.p. nicotine (0, 0.0032, 0.01, 0.032, 0.1 mg/kg) or a selective ß2 subunit containing nAChR (ß2*nAChR) antagonist dihydro-beta-erythroidine (0, 0.1, 0.3, 1.0, 3.0 mg/kg DHßE). There was a dose-dependent effect of nicotine revealing that only low doses (0.01, 0.032 mg/kg) increased CER suppression ratios (SR) in these mice. DHßE also dose-dependently increased SR at the 3 mg/kg dose. In ethological measures of fear-/anxiety-like behavior, these doses of nicotine and DHßE significantly reduced digging behavior in a marble burying task and 0.3 mg/kg DHßE promoted open-arm activity in the elevated plus maze. Doses of nicotine and DHßE that altered affective behavior had no effect on locomotor activity. Similar to previous reports with anxiolytic drugs, low dose nicotine and DHßE reversed SR in a CER assay, decreased digging in a marble burying assay and increased open arm activity in the elevated plus maze. This study provides evidence that inactivation of ß2*nAChRs reduces fear-like and anxiety-like behavior in rodents and suggests that smokers may be motivated to smoke in part to desensitize their ß2*nAChRs. These data further identify ß2*nAChR antagonism as a potential therapeutic strategy for relief of negative affect and anxiety.

Preliminary comparison of quantity, quality, and microarray performance of RNA extracted from formalin-fixed, paraffin-embedded, and unfixed frozen tissue samples.

Microarrays have been used to simultaneously monitor the expression of thousands of genes from biological samples, an approach that can potentially uncover previously unrecognized functions of genes. Microarray analyses can rarely be conducted retrospectively because of the requirement for RNA to be obtained from fresh or unfixed frozen tissues. Archived pathology specimens would need to be used for retrospective analyses, and these are typically preserved as formalin-fixed, paraffin-embedded (FFPE) tissue. Formalin-fixed tissues have been shown to yield compromised RNA compared with that obtained from frozen tissue. To begin to assess the performance of RNA extracted from FFPE samples on a microarray format, we compared RNA from a model system of pelleted lipopolysaccharide-stimulated human bone marrow stromal cells that were snap frozen with RNA from FFPE cells. RNA integrity and Affymetrix quality control parameters were assessed, and differentially regulated genes were analyzed with Ingenuity Pathway Analysis software. Results demonstrate that both snap-frozen and FFPE samples yielded intact RNA suitable for amplification prior to Affymetrix GeneChip analysis. Although some transcriptional information was lost with RNA extracted from the FFPE samples, Ingenuity Pathway Analysis revealed that the major pathways identified as affected by drug treatment were similar. Results show that FFPE samples are amenable to Affymetrix GeneChip analysis, expanding the possibility for expression profiling on archived tissue blocks in pathology laboratories.