The totipotent embryonic stem cell generates various mesodermal cells when stimulated with BMP4. Among the resulting cells, those expressing flk-1 and/or PDGFRα displayed chondrogenic activity in the presence of TGFβ3 and expressed cartilage-specific genes in 7 to 16 day pellet cultures. Depositions of cartilage matrix and type II collagen were detected by day 14. TGFβ-stimulated chondrogenesis was synergistically enhanced by PDGF-BB, resulting in a larger cartilage particle filled with a cartilaginous area containing type II collagen, with a surface cell layer expressing type I collagen. In contrast, noggin inhibited both the TGFβ-and TGFβ+PDGF-stimulated cartilage formation, suggesting that a BMPdependent pathway is involved. In fact, replacement of TGFβ3 with BMP4 on days 10 to 12 markedly elevated the cartilage matrix deposition during the following 7 to 8 days. Moreover, culture with TGFβ3 and PDGF-BB, followed by the incubation with BMP4 alone, resulted in a cartilage particle lacking type I collagen in the matrix and the surface layer, which suggests hyaline cartilage formation. Furthermore, such hyaline cartilage particles were mineralized. These studies indicate that the PDGFRα + and/or flk-1 + cells derived from embryonic stem cells possess the full developmental potential toward chondrocytes, in common with embryonic mesenchymal cells.

We have identified and cloned a novel human cytokine with homology to cytokines of the interleukin-17 (IL-17) family, which we have termed human IL-17E (hIL-17E). With the identification of several IL-17 family members, it is critical to understand the in vivo function of these molecules. We have generated transgenic mice overexpressing hIL-17E using an apolipoprotein E (ApoE) hepatic promoter. These mice displayed changes in the peripheral blood, particularly, a 3-fold increase in total leukocytes consisting of increases in eosinophils, lymphocytes, and neutrophils. Splenomegaly and lymphoadenopathy were predominant and included marked eosinophil infiltrates and lymphoid hyperplasia. CCR3 ؉ eosinophils increased in the blood and lymph nodes of the transgenic mice by 50-and 300-fold, respectively. Eosinophils also increased 8-to 18-fold in the bone marrow and spleen, respectively. In the bone marrow, most of the eosinophils had an immature appearance. CD19 ؉ B cells increased 2-to 5-fold in the peripheral blood, 2-fold in the spleen, and 10-fold in the lymph nodes of transgenic mice, whereas CD4 ؉ T lymphocytes increased 2-fold in both blood and spleen.

Optimal T cell activation requires the interactions of co-stimulatory molecules, such as those in the CD28 and B7 protein families. Recently, we described the co-stimulatory properties of the murine ligand to ICOS, which we designated as B7RP-1. Here, we report the co-stimulation of human T cells through the human B7RP-1 and ICOS interaction. This ligand-receptor pair interacts with a K:(D) approximately 33 nM and an off-rate with a t((1/2)) > 10 min. Interestingly, tumor necrosis factor (TNF)-alpha differentially regulates the expression of human B7RP-1 on B cells, monocytes and dendritic cells (DC). TNF-alpha enhances B7RP-1 expression on B cells and monocytes, while it inhibits it on DC. The human B7RP-1-Fc protein or cells that express membrane-bound B7RP-1 co-stimulate T cell proliferation in vitro. Specific cytokines, such as IFN-gamma and IL-10, are induced by B7RP-1 co-stimulation. Although IL-2 levels are not significantly increased, B7RP-1 co-stimulation is dependent on IL-2. These experiments define the human ortholog to murine B7RP-1 and characterize its interaction with human ICOS.

Osteoclast precursors (OCPs) circulate in the mononuclear fraction of peripheral blood (PB), but their abundance and surface characteristics are unknown. Previous studies suggest that the receptor activator for NF‐κB (RANK) on cytokine‐treated OCPs in mouse bone marrow interacts with osteoprotegerin ligand (OPGL/TRANCE/RANKL/ODF) to initiate osteoclast differentiation. Hence, we used a fluorescent form of human OPGL (Hu‐OPGL‐F) to identify possible RANK‐expressing OCPs in untreated peripheral blood mononuclear cells (PBMCs) using fluorescence‐activated cell sorting analysis. Monocytes [CD14‐phycoerythrin (PE) antibody (Ab) positive (+) cells, 10–15% of PBMCs] all (98–100%) co‐labelled with Hu‐OPGL‐F (n > 18). T lymphocytes (CD3‐PE Ab+ cells, 66% of PBMCs) did not bind Hu‐OPGL‐F; however, B cells (CD19‐PE Ab+ cells, 9% of PBMCs) were also positive for Hu‐OPGL‐F. All Hu‐OPGL‐F+ monocytes also co‐labelled with CD33, CD61, CD11b, CD38, CD45 and CD54 Abs, but not CD34 or CD56 Abs. Hu‐OPGL‐F binding was dose dependent and competed with excess Hu‐OPGL. When Hu‐OPGL‐F+, CD14‐PE Ab+, CD33‐PE Ab+, Hu‐OPGL‐F+/CD14‐PE Ab+ or Hu‐OPGL‐F+/CD33‐PE Ab+ cells were cultured with OPGL (20 ng/ml) and colony‐stimulating factor (CSF)‐1 (25 ng/ml), OC‐like cells readily developed. Thus, all freshly isolated monocytes demonstrate displaceable Hu‐OPGL‐F binding, suggesting the presence of RANK on OCPs in PB; also, OCPs within a purified PB monocyte population form osteoclast‐like cells in the complete absence of other cell types in OPGL and CSF‐1 containing medium.

Administration of active vitamin D sterols to treat secondary hyperparathyroidism in patients with chronic kidney disease receiving dialysis has been associated with elevated serum calcium and phosphorus levels, which may lead to increased risk of vascular calcification. However, calcimimetics, by binding to the parathyroid gland calcium-sensing receptors, reduce serum parathyroid hormone, calcium, phosphorus, and the calcium-phosphorus product. Using cultured bovine aorta vascular smooth muscle cells (BASMCs), an in vitro model of vascular calcification, we compared calcification levels and gene expression profiles after exposure to the phosphate source ss-glycerolphosphate (BGP), the active vitamin D sterols calcitriol and paricalcitol, the calcimimetic R-568, or BGP with the active vitamin D sterols or R-568. Cells exposed to BGP (10 mM) alone or with calcitriol or paricalcitol showed dose-dependent BASMC calcification. No change in calcification was observed in cultures exposed to BGP with R-568, consistent with the observed lack of calcium-sensing receptor expression. Microarray analysis using total cellular RNA from cultures exposed to vehicle or BGP in the absence and presence of 10(-8) M calcitriol or paricalcitol for 7 days showed that cells exposed to BGP with calcitriol or BGP with paricalcitol had virtually identical gene expression profiles, which differed from those of cells treated with BGP or vehicle alone. Several osteoblast- and chondrocyte-associated genes were modulated by BGP and vitamin D exposure. In this study, exposure of BASMCs to phosphate and active vitamin D sterols induced calcification and changes in expression of genes associated with mineralized tissue.

Osteoclast precursors (OCPs) circulate in the mononuclear fraction of peripheral blood (PB), but their abundance and surface characteristics are unknown. Previous studies suggest that the receptor activator for NF-kappaB (RANK) on cytokine-treated OCPs in mouse bone marrow interacts with osteoprotegerin ligand (OPGL/TRANCE/RANKL/ODF) to initiate osteoclast differentiation. Hence, we used a fluorescent form of human OPGL (Hu-OPGL-F) to identify possible RANK-expressing OCPs in untreated peripheral blood mononuclear cells (PBMCs) using fluorescence-activated cell sorting analysis. Monocytes [CD14-phycoerythrin (PE) antibody (Ab) positive (+) cells, 10-15% of PBMCs] all (98-100%) co-labelled with Hu-OPGL-F (n > 18). T lymphocytes (CD3-PE Ab+ cells, 66% of PBMCs) did not bind Hu-OPGL-F; however, B cells (CD19-PE Ab+ cells, 9% of PBMCs) were also positive for Hu-OPGL-F. All Hu-OPGL-F+ monocytes also co-labelled with CD33, CD61, CD11b, CD38, CD45 and CD54 Abs, but not CD34 or CD56 Abs. Hu-OPGL-F binding was dose dependent and competed with excess Hu-OPGL. When Hu-OPGL-F+, CD14-PE Ab+, CD33-PE Ab+, Hu-OPGL-F+/CD14-PE Ab+ or Hu-OPGL-F+/CD33-PE Ab+ cells were cultured with OPGL (20 ng/ml) and colony-stimulating factor (CSF)-1 (25 ng/ml), OC-like cells readily developed. Thus, all freshly isolated monocytes demonstrate displaceable Hu-OPGL-F binding, suggesting the presence of RANK on OCPs in PB; also, OCPs within a purified PB monocyte population form osteoclast-like cells in the complete absence of other cell types in OPGL and CSF-1 containing medium.

Amino-terminal fragments of huntingtin, which contain the expanded polyglutamine repeat, have been proposed to contribute to the pathology of Huntington's disease (HD). Data supporting this claim have been generated from patients with HD in which truncated amino-terminal fragments forming intranuclear inclusions have been observed, and from animal and cell-based models of HD where it has been demonstrated that truncated polyglutamine-containing fragments of htt are more toxic than fulllength huntingtin. We report here the identification of a region within huntingtin, spanning from amino acids 63 to 111, that is cleaved in cultured cells to generate a fragment of similar size to those observed in patients with HD. Importantly, proteolytic cleavage within this region appears dependent upon the length of the polyglutamine repeat within huntingtin, with pathological polyglutamine repeatcontaining huntingtin being more efficiently cleaved than huntingtin containing polyglutamine repeats of nonpathological size.

BackgroundMonoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor are efficacious for the prevention of migraine headaches. The downstream molecular mechanisms following ligand-receptor blockade by which these antibodies prevent CGRP signaling through CGRP receptors have not been demonstrated.MethodsHere we produced tool monoclonal functional antagonist antibodies against CGRP and its canonical receptor and developed a novel cellular model using fluorogen-activated protein technology that allows detection of CGRP receptor internalization by flow cytometry and, for an extended time course, visualization by confocal microscopy.ResultsUsing this cell model we showed that these antagonist antibodies block both CGRP-induced cAMP signaling and CGRP receptor internalization. At least 10-fold higher concentrations of either antibody are necessary to block CGRP receptor internalization compared with cAMP accumulation in our cell model.ConclusionThese data reinforce our understanding of how monoclonal functional antagonist antibodies interfere with CGRP signaling.