The current study was done to assess if heterogeneity existed in the degree of adipogenesis in stromal cells (preadipocytes) from multiple donors. In addition to conventional lipid-based methods, we have employed a novel signal amplification technology, known as branched DNA, to monitor expression of an adipocyte specific gene product aP2. The fatty acid binding protein aP2 increases during adipocyte differentiation and is induced by thiazolidinediones and other peroxisome proliferator activated receptor gamma ligands. The current work examined the adipogenic induction of aP2 mRNA levels in human adipose tissue stromal cells derived from 12 patients (mean age +/- SEM, 38.9 +/- 3.1) with mild to moderate obesity (mean body mass index +/- SEM, 27.8 +/- 2.4). Based on branched DNA technology, a rapid and sensitive measure of specific RNAs, the relative aP2 level in adipocytes increased by 679 +/- 93-fold (mean +/- SEM, n=12) compared to preadipocytes. Normalization of the aP2 mRNA levels to the housekeeping gene, glyceraldehyde phosphate dehydrogenase, did not significantly alter the fold induction in a subset of 4 patients (803.6 +/- 197.5 vs 1118.5 +/- 308.1). Independent adipocyte differentiation markers were compared between adipocytes and preadipocytes in parallel studies. Leptin secretion increased by up to three-orders of magnitude while measurements of neutral lipid accumulation by Oil Red O and Nile Red staining increased by 8.5-fold and 8.3-fold, respectively. These results indicate that preadipocytes isolated from multiple donors displayed varying degrees of differentiation in response to an optimal adipogenic stimulus in vitro. This work also demonstrates that branched DNA measurement of aP2 is a rapid and sensitive measure of adipogenesis in human stromal cells. The linear range of this assay extends up to three-orders of magnitude and correlates directly with independent measures of cellular differentiation.

IFN-␤, an effective therapy against relapsing-remitting multiple sclerosis, is naturally secreted during the innate immune response against viral pathogens. The objective of this study was to characterize the immunomodulatory mechanisms of IFN-␤ targeting innate immune response and their effects on dendritic cell (DC)-mediated regulation of T cell differentiation. We found that IFN-␤1a in vitro treatment of human monocyte-derived DCs induced the expression of TLR7 and the members of its downstream signaling pathway, including MyD88, IL-1R-associated kinase 4, and TNF receptor-associated factor 6, while it inhibited the expression of IL-1R. Using small interfering RNA TLR7 gene silencing, we confirmed that IFN-␤1a-induced changes in MyD88, IL-1R-associated kinase 4, and IL-1R expression were dependent on TLR7. TLR7 expression was also necessary for the IFN␤1a-induced inhibition of IL-1␤ and IL-23 and the induction of IL-27 secretion by DCs. Supernatant transfer experiments confirmed that IFN-␤1a-induced changes in DC cytokine secretion inhibit Th17 cell differentiation as evidenced by the inhibition of retinoic acid-related orphan nuclear hormone receptor C and IL-17A gene expression and IL-17A secretion. Our study has identified a novel therapeutic mechanism of IFN-␤1a that selectively targets the autoimmune response in multiple sclerosis. The Journal of Immunology, 2009, 182: 3928 -3936. M ultiple sclerosis (MS)3 is a chronic inflammatory CNS disease initiated by sensitization of the immune system to CNS myelin Ags (1). IFN-␤1a has been used for many years as a first-line therapy for releasing relapsing (RR) MS. Although its efficacy in suppressing disease activity is well documented in large randomized placebo-controlled clinical trials (2), its in vivo operative mechanisms of action are not completely elucidated. IFN-␤1a's reported mechanisms of action include inhibition of MHC class II expression on monocytes and microglia, suppression of T cell proliferation, regulation of IL-12/IL-10 cytokine transcription, and inhibition of inflammatory cell migration into the CNS via interference with VLA-4-mediated cell adhesion and matrix metalloproteinase 9 activity (3). However, IFN-␤'s effects are complex and cellular responses to IFN-␤ involve Ͼ500 genes (4), suggesting that some of the relevant mechanisms of action may still not be identified. Although IFN-␤ is secreted during the antiviral innate immune response, the physiological role of endogenous type I IFNs in the regulation of the adaptive immune response is poorly understood. We propose that the administration of high-dose recombinant IFN-␤1a, which provides a strong therapeutic effect, may enhance the naturally operative IFN-␤-mediated control of the autoimmune response. We studied the effects of IFN-␤ in the context of recent advances in our understanding of the role of the innate immune response (5) and Th17 cells (6) in the development of the autoimmune response.The discovery of the Th17 cell lineage marked a new era in the studies of the autoimmune res...

TCR degeneracy may facilitate self-reactive T cell activation and the initiation of an autoimmune response in multiple sclerosis (MS). MHC class II alleles of the DR2 haplotype DR2a (DRB5*0101) and DR2b (DRB1*1501) are associated with an increased risk for MS in Caucasian populations. In order to selectively expand and characterize T cells with a high degree of TCR degeneracy that recognize peptides in the context of disease-associated DR2 alleles, we developed DR2-anchored peptide mixtures (APM). We report here that DR2-APM have a high stimulatory potency and can selectively expand T cells with a degenerate TCR (TCR deg ). Due to the low concentration of individual peptides in the mixtures, T cell clones' proliferative response to DR2-APM implies that multiple peptides stimulate the TCR, which is a characteristic of TCR deg . The frequency of DR2-APM-reactive T cells is significantly higher in MS patients than in healthy controls, suggesting that they may play a role in the development of the autoimmune response in MS. DR2-APM-reactive cells have a dual DR2 restriction: they recognize DR2-APM in the context of both DR2a and DR2b molecules. The DR2-APM-reactive cells' IL-17 secretion, together with cross-reactivity against myelin peptides, may contribute to their role in the development of autoimmune response in MS.