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Bone is accrued and maintained primarily through the coupled actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Cumulative in vitro studies indicated that proline-rich tyrosine kinase 2 (PYK2) is a positive mediator of osteoclast function and activity. However, our investigation of PYK2؊/؊ mice did not reveal evidence supporting an essential function for PYK2 in osteoclasts either in vivo or in culture. We find that PYK2؊/؊ mice have high bone mass resulting from an unexpected increase in bone formation. Consistent with the in vivo findings, mouse bone marrow cultures show that PYK2 deficiency enhances differentiation and activity of osteoprogenitor cells, as does expressing a PYK2-specific short hairpin RNA or dominantly interfering proteins in human mesenchymal stem cells. Furthermore, the daily administration of a small-molecule PYK2 inhibitor increases bone formation and protects against bone loss in ovariectomized rats, an established preclinical model of postmenopausal osteoporosis. In summary, we find that PYK2 regulates the differentiation of early osteoprogenitor cells across species and that inhibitors of the PYK2 have potential as a bone anabolic approach for the treatment of osteoporosis.human mesenchymal stem cell ͉ osteoclast ͉ osteoblast P roline-rich tyrosine kinase 2 (PYK2) and focal adhesion kinase (FAK) are nonreceptor tyrosine kinases, and together they constitute the focal adhesion kinase subfamily (1). Unlike FAK, PYK2 expression is relatively restricted, with highest levels in the brain and the hematopoietic system. PYK2Ϫ/Ϫ mice have been described previously and appear normally developed (2, 3). Characterization of the immune system of PYK2Ϫ/Ϫ animals revealed the absence of marginal zone B cells along and abnormal T cell-independent type II responses (2), as well as altered macrophage morphology, adhesion, and migration (3).Although PYK2 is expressed in both bone-forming osteoblasts and bone-resorbing osteoclasts, the skeletal phenotype of PYK2Ϫ/Ϫ mice has not been described. In vitro studies pointed to a positive role for PYK2 in osteoclast maturation and bone resorption. PYK2 localizes to the podosomes of osteoclasts (4), and, upon integrin binding, cell attachment, and actin ring formation, PYK2 associates with a variety of proteins including p130 CAS (5), Src (4), Cbl (6), integrins (4), gelsolin (7), and paxillin (8). Antisense depletion of PYK2 (9), but not the expression of a kinase inactive dominant negative mutant (10), blocked osteoclast spreading and bone resorption, indicating that PYK2 catalytic activity may be dispensable. The in vitro effects of bone anabolic stimuli suggested that PYK2 might have a positive role in osteoblasts as well. Treatment of osteoblast cells with fluoroaluminate led to increased PYK2 autophosphorylation, Src association, and kinase activity (11) and was associated with increased cell attachment and spreading (12). Likewise, in an anabolic model of mechanical loading, PYK2 autophosphorylation and kinase activity were stimulated in o...

Protective immunity to leishmaniasis has been demonstrated in murine models to be mediated by T cells and the cytokines they produce. We have previously shown that resistance to experimental Leishmania infantum infection in the dog, a natural host and reservoir of the parasite, is associated with the proliferation of peripheral blood mononuclear cells (PBMC) to parasite antigen and to the production of interleukin-2 and tumour necrosis factor. In this study we show that PBMC from asymptomatic experimentally infected dogs produce interferon-gamma upon parasite antigen-specific stimulation, whereas lymphocytes from symptomatic dogs do not. In addition, we report for the first time the lysis of L. infantum-infected macrophages by PBMC from asymptomatic dogs and by parasite-specific T cell lines derived from these animals. These T cell lines were generated by restimulation in vitro with parasite soluble antigen and irradiated autologous PBMC as antigen-presenting cells. We show that lysis of infected macrophages by T cell lines is major histocompatibility complex restricted. Characterization of parasite-specific cytotoxic T cell lines revealed that the responding cells are CD8+. However, for some animals, CD4+ T cells that lyse infected macrophages were also found. In contrast to asymptomatic dogs, lymphocytes from symptomatic dogs failed to proliferate and produce interferon-gamma after Leishmania antigen stimulation in vitro and were not capable of lysing infected macrophages. These results suggest that both the production of interferon-gamma and the destruction of the parasitized host cells by Leishmania-specific T cells play an important role in resistance to visceral leishmaniasis.

A compact and stable bicyclic bridged ketal was developed as a ligand for the asialoglycoprotein receptor (ASGPR). This compound showed excellent ligand efficiency, and the molecular details of binding were revealed by the first X-ray crystal structures of ligand-bound ASGPR. This analogue was used to make potent di- and trivalent binders of ASGPR. Extensive characterization of the function of these compounds showed rapid ASGPR-dependent cellular uptake in vitro and high levels of liver/plasma selectivity in vivo. Assessment of the biodistribution in rodents of a prototypical Alexa647-labeled trivalent conjugate showed selective hepatocyte targeting with no detectable distribution in nonparenchymal cells. This molecule also exhibited increased ASGPR-directed hepatocellular uptake and prolonged retention compared to a similar GalNAc derived trimer conjugate. Selective release in the liver of a passively permeable small-molecule cargo was achieved by retro-Diels-Alder cleavage of an oxanorbornadiene linkage, presumably upon encountering intracellular thiol. Therefore, the multicomponent construct described here represents a highly efficient delivery vehicle to hepatocytes.