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Although a number of growth factors have been shown to be involved in neurogenesis,the role of inflammatory cytokines remains relatively unexplored in the normal brain. Here we investigated the effect of interferon gamma (IFNgamma) in the regulation of neural precursor (NP) activity in both the developing and the adult mouse brain. Exogenous IFNgamma inhibited neurosphere formation from the wild-type neonatal and adult subventricular zone (SVZ). More importantly,however,these effects were mirrored in vivo,with mutant mice lacking endogenous IFNgamma displaying enhanced neurogenesis,as demonstrated by an increase in proliferative bromodeoxyuridine-labeled cells in the SVZ and an increased percentage of newborn neurons in the olfactory bulb. Furthermore,NPs isolated from IFNgamma null mice exhibited an increase in self-renewal ability and in the capacity to produce differentiated neurons and oligodendrocytes. These effects resulted from the direct action of IFNgamma on the NPs,as determined by single-cell assays and the fact that nearly all the neurospheres were derived from cells positive for major histocompatibility complex class I antigen,a downstream marker of IFNgamma-mediated activation. Moreover,the inhibitory effect was ameliorated in the presence of SVZ-derived microglia,with their removal resulting in almost complete inhibition of NP proliferation. Interestingly,in contrast to the results obtained in the adult,exogenous IFNgamma treatment stimulated neurosphere formation from the embryonic brain,an effect that was mediated by sonic hedgehog. Together these findings provide the first direct evidence that IFNgamma acts as a regulator of the active NP pool in the non-inflammatory brain. View Publication

Caveolin 1 (Cav-1) is a major protein of a specific membrane lipid raft known as caveolae. Cav-1 interacts with the gp41 of the human immunodeficiency virus (HIV) envelope,but the role of Cav-1 in HIV replication and pathogenesis is not known. In this report,we demonstrate that HIV infection in primary human monocyte-derived macrophages (MDMs),THP-1 macrophages,and U87-CD4 cells results in a dramatic upregulation of Cav-1 expression mediated by HIV Tat. The activity of p53 is essential for Tat-induced Cav-1 expression,as our findings show enhanced phosphorylation of serine residues at amino acid positions 15 and 46 in the presence of Tat with a resulting Cav-1 upregulation. Furthermore,inhibition of p38 mitogen-activated protein kinase (MAPK) blocked phosphorylation of p53 in the presence of Tat. Infection studies of Cav-1-overexpressing cells reveal a significant reduction of HIV production. Taken together,these results suggest that HIV infection enhances the expression of Cav-1,which subsequently causes virus reduction,suggesting that Cav-1 may contribute to persistent infection in macrophages. View Publication

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Sonic hedgehog signaling pathway is important in developmental processes like dorsoventral neural tube patterning,neural stem cell proliferation and neuronal and glial cell survival. Shh is also implicated in the regulation of the adult hippocampal neurogenesis. Recently,nonpeptidyl Smoothened activators of the Shh pathway have been identified. The aim of this study was to investigate the effects of chlorobenzothiophene-containing molecule,Smo agonist (SAG),which has been shown to activate Shh signaling pathway,in neurogenesis and neuronal survival in in vitro and in vivo models. Our in vitro experiments showed that SAG induces increased expression of Gli1 mRNA,transcriptional target and mediator of Shh signal. In vitro experiments also demonstrated that SAG in low-nanomolar concentrations induces proliferation of neuronal and glial precursors without affecting the differentiation pattern of newly produced cells. In contrast to Shh,SAG did not induce neurotoxicity in neuronal cultures. The SAG and Shh treatment also promoted the survival of newly generated neural cells in the dentate gyrus after their intracerebroventricular administration to adult rats. We propose that SAG and similar compounds represent attractive molecules to be developed for treatment of disorders where stimulation of the generation and survival of new neural cells would be beneficial. View Publication

Changes in progenitor cell biology remain at the forefront of many theories of biologic aging,but there are limited studies evaluating this in humans. Aging has been associated with a progressive depletion of circulating progenitor cells,but age-related bone marrow-resident progenitor cell depletion has not been systematically determined in humans. Patients undergoing total hip replacement were consented,and bone marrow and peripheral progenitor cells were enumerated based on aldehyde dehydrogenase activity and CD34 and CD133 expression. Circulating progenitors demonstrated an age-dependent decline. In contrast,marrow-resident progenitor cell content demonstrated no age association with any progenitor cell subtype. In humans,aging is associated with depletion of circulating,but not marrow-resident,progenitors. This finding has impact on the mechanism(s) responsible for age-related changes in circulating stem cells and important implications for the use of autologous marrow for the treatment of age-related diseases. View Publication

Aurora kinases are key regulators of cell mitosis and have been implicated in the process of tumorigenesis. In recent years,the Aurora kinases have attracted much interest as promising targets for cancer treatment. Here we report on the roles of Aurora A and Aurora B kinases in clear cell renal cell carcinoma (ccRCC). Using genomewide expression array analysis of 174 patient samples of ccRCC,we found that expression levels of Aurora A and B were significantly elevated in ccRCC compared to normal kidney samples. High expression levels of Aurora A and Aurora B were significantly associated with advanced tumor stage and poor patient survival. Inhibition of Aurora kinase activity with the drug VX680 (also referred to as MK-0457) inhibited ccRCC cell growth in vitro and led to ccRCC cell accumulation in the G2/M phase and apoptosis. Growth of ccRCC xenograft tumors was also inhibited by VX680 treatment,accompanied by a reduction of tumor microvessel density. Analysis of endothelial cell lines demonstrated that VX680 inhibits endothelial cell growth with effects similar to that seen in ccRCC cells. Our findings suggest that VX680 inhibits the growth of ccRCC tumors by targeting the proliferation of both ccRCC tumor cells and tumor-associated endothelial cells. Aurora kinases and their downstream cell cycle proteins have an important role in ccRCC and may be potent prognostic markers and therapy targets for this disease. View Publication

Selumetinib (AZD6244; ARRY-142886) is a tight-binding,uncompetitive inhibitor of mitogen-activated protein kinase kinases (MEK) 1 and 2 currently in clinical development. We evaluated the effects of selumetinib in 31 human breast cancer cell lines and 43 human non-small cell lung cancer (NSCLC) cell lines to identify characteristics correlating with in vitro sensitivity to MEK inhibition. IC(50) textless1 micromol/L (considered sensitive) was seen in 5 of 31 breast cancer cell lines and 15 of 43 NSCLC cell lines,with a correlation between sensitivity and raf mutations in breast cancer cell lines (P = 0.022) and ras mutations in NSCLC cell lines (P = 0.045). Evaluation of 27 of the NSCLC cell lines with Western blots showed no clear association between MEK and phosphoinositide 3-kinase pathway activation and sensitivity to MEK inhibition. Baseline gene expression profiles were generated for each cell line using Agilent gene expression arrays to identify additional predictive markers. Genes associated with differential sensitivity to selumetinib were seen in both histologies,including a small number of genes in which differential expression was common to both histologies. In total,these results suggest that clinical trials of selumetinib in breast cancer and NSCLC might select patients whose tumors harbor raf and ras mutations,respectively. View Publication

BACKGROUND AIMS Heart failure therapy with human embryonic stem cell (hESC)-derived cardiomyocytes (hCM) has been limited by the low rate of spontaneous hCM differentiation. As others have shown that p38 mitogen-activated protein kinase (p38MAPK) directs neurogenesis from mouse embryonic stem cells,we investigated whether the p38MAPK inhibitor,SB203580,might influence hCM differentiation. METHODS We treated differentiating hESC with SB203580 at specific time-points,and used flow cytometry,immunocytochemistry,quantitative real-time (RT)-polymerase chain reaction (PCR),teratoma formation and transmission electron microscopy to evaluate cardiomyocyte formation. RESULTS We observed that the addition of inhibitor resulted in 2.1-fold enrichment of spontaneously beating human embryoid bodies (hEB) at 21 days of differentiation,and that 25% of treated cells expressed cardiac-specific α-myosin heavy chain. This effect was dependent on the stage of differentiation at which the inhibitor was introduced. Immunostaining and teratoma formation assays demonstrated that the inhibitor did not affect hESC pluripotency; however,treated hESC gave rise to hCM exhibiting increased expression of sarcomeric proteins,including cardiac troponin T,myosin light chain and α-myosin heavy chain. This was consistent with significantly increased numbers of myofibrillar bundles and the appearance of nascent Z-bodies at earlier time-points in treated hCM. Treated hEB also demonstrated a normal karyotype by array comparative genomic hybridization and viability in vivo following injection into mouse myocardium. CONCLUSIONS These studies demonstrate that p38MAPK inhibition accelerates directed hCM differentiation from hESC,and that this effect is developmental stage-specific. The use of this inhibitor should improve our ability to generate hESC-derived hCM for cell-based therapy. View Publication

Cryptococcus neoformans is an environmental fungus and an opportunistic human pathogen. Previous studies have demonstrated major alterations in its transcriptional profile as this microorganism enters the hostile environment of the human host. To assess the role of chromatin remodeling in host-induced transcriptional responses,we identified the C. neoformans Gcn5 histone acetyltransferase and demonstrated its function by complementation studies of Saccharomyces cerevisiae. The C. neoformans gcn5Delta mutant strain has defects in high-temperature growth and capsule attachment to the cell surface,in addition to increased sensitivity to FK506 and oxidative stress. Treatment of wild-type cells with the histone acetyltransferase inhibitor garcinol mimics cellular effects of the gcn5Delta mutation. Gcn5 regulates the expression of many genes that are important in responding to the specific environmental conditions encountered by C. neoformans inside the host. Accordingly,the gcn5Delta mutant is avirulent in animal models of cryptococcosis. Our study demonstrates the importance of chromatin remodeling by the conserved histone acetyltransferase Gcn5 in regulating the expression of specific genes that allow C. neoformans to respond appropriately to the human host. View Publication

Osteoclasts are resident cells of the bone that are primarily involved in the physiological and pathological remodeling of this tissue. Mature osteoclasts are multinucleated giant cells that are generated from the fusion of circulating precursors originating from the monocyte/macrophage lineage. During inflammatory bone conditions in vivo,de novo osteoclastogenesis is observed but it is currently unknown whether,besides increased osteoclast differentiation from undifferentiated precursors,other cell types can generate a multinucleated giant cell phenotype with bone resorbing activity. In this study,an animal model of calvaria-induced aseptic osteolysis was used to analyze possible bone resorption capabilities of dendritic cells (DCs). We determined by FACS analysis and confocal microscopy that injected GFP-labeled immature DCs were readily recruited to the site of osteolysis. Upon recruitment,the cathepsin K-positive DCs were observed in bone-resorbing pits. Additionally,chromosomal painting identified nuclei from female DCs,previously injected into a male recipient,among the nuclei of giant cells at sites of osteolysis. Finally,osteolysis was also observed upon recruitment of CD11c-GFP conventional DCs in Csf1r(-/-) mice,which exhibit a severe depletion of resident osteoclasts and tissue macrophages. Altogether,our analysis indicates that DCs may have an important role in bone resorption associated with various inflammatory diseases. View Publication

Regulatory T cells (Tregs) are a suppressive subset of CD4(+) T lymphocytes implicated in the prevention of acute GVHD (aGVHD) after allo-SCT (ASCT). To determine whether increased frequency of Tregs with a skin-homing (cutaneous lymphocyte Ag,CLA(+)) or a gut-homing (α(4)β(7)(+)) phenotype is associated with reduced risk of skin or gut aGVHD,respectively,we quantified circulating CLA(+) or α(4)β(7)(+) on Tregs at the time of neutrophil engraftment in 43 patients undergoing ASCT. Increased CLA(+) Tregs at engraftment was associated with the prevention of skin aGVHD (2.6 vs 1.7%; P=0.038 (no skin aGVHD vs skin aGVHD)),and increased frequencies of CLA(+) and α(4)β(7)(+) Tregs were negatively correlated with severity of skin aGVHD (odds ratio (OR),0.67; 95% confidence interval (CI),0.46-0.98; P=0.041) or gut aGVHD (OR,0.93; 95% CI,0.88-0.99; P=0.031),respectively. This initial report suggests that Treg tissue-homing subsets help to regulate organ-specific risk and severity of aGVHD after human ASCT. These results need to be validated in a larger,multicenter cohort. View Publication

The receptor for advanced glycation end products (RAGE) is produced either as a transmembrane or soluble form (sRAGE). Substantial evidence supports a role for RAGE and its ligands in disease. sRAGE is reported to be a competitive,negative regulator of membrane RAGE activation,inhibiting ligand binding. However,some reports indicate that sRAGE is associated with inflammatory disease. We sought to define the biological function of sRAGE on inflammatory cell recruitment,survival,and differentiation in vivo and in vitro. To test the in vivo impact of sRAGE,the recombinant protein was intratracheally administered to mice,which demonstrated monocyte- and neutrophil-mediated lung inflammation. We also observed that sRAGE induced human monocyte and neutrophil migration in vitro. Human monocytes treated with sRAGE produced proinflammatory cytokines and chemokines. Our data demonstrated that sRAGE directly bound human monocytes and monocyte-derived macrophages. Binding of sRAGE to monocytes promoted their survival and differentiation to macrophages. Furthermore,sRAGE binding to cells increased during maturation,which was similar in freshly isolated mouse monocytes compared with mature tissue macrophages. Because sRAGE activated cell survival and differentiation,we examined intracellular pathways that were activated by sRAGE. In primary human monocytes and macrophages,sRAGE treatment activated Akt,Erk,and NF-kappaB,and their activation appeared to be critical for cell survival and differentiation. Our data suggest a novel role for sRAGE in monocyte- and neutrophil-mediated inflammation and mononuclear phagocyte survival and differentiation. View Publication