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As described in the preceding paper, monoclonal antibodies have been raised by immunization of rats with mouse hematopoietic cells which detect a major cell-surface glycoprotein (Mr = 95 000) of mouse bone-marrow cells of the granulocytic series. While most of the monoclonal antibodies detect this molecule one bone-marrow and spleen cells of all mouse strains, two antibodies recognize alternative allelic forms of the molecule. One alloantigen is expressed in all the remaining inbred strains examined. The alloantigens are codominantly expressed on the cells of F1 mice. Backcrosses of DBA/2 and C57BL/6 with F1 mice (B6D2F1) confirmed that a single genetic locus is involved in the expression of the two antigenic forms and demonstrated linkage to Ly-m11 which has previously been mapped to mouse chromosome 2. These genetic mapping experiments and the biochemical properties of the glycoprotein suggested that it might be identical to a glycoprotein first identified on murine fibroblasts by Hughes and August and designated Pgp-1. This has been firmly established by exchange of monoclonal antibody reagents and sequential immunoprecipitations.

Studies of repertoires of mouse monoclonal CD4(+) T cells have revealed several mechanisms of self-tolerance; however, which mechanisms operate in normal repertoires is unclear. Here we studied polyclonal CD4(+) T cells specific for green fluorescent protein expressed in various organs, which allowed us to determine the effects of specific expression patterns on the same epitope-specific T cells. Peptides presented uniformly by thymic antigen-presenting cells were tolerated by clonal deletion, whereas peptides excluded from the thymus were ignored. Peptides with limited thymic expression induced partial clonal deletion and impaired effector T cell potential but enhanced regulatory T cell potential. These mechanisms were also active for T cell populations specific for endogenously expressed self antigens. Thus, the immunotolerance of polyclonal CD4(+) T cells was maintained by distinct mechanisms, according to self-peptide expression patterns.

Identification of agents that target human leukemia stem cells is an important consideration for the development of new therapies. The present study demonstrates that rocaglamide and silvestrol, closely related natural products from the flavagline class of compounds, are able to preferentially kill functionally defined leukemia stem cells, while sparing normal stem and progenitor cells. In addition to efficacy as single agents, flavaglines sensitize leukemia cells to several anticancer compounds, including front-line chemotherapeutic drugs used to treat leukemia patients. Mechanistic studies indicate that flavaglines strongly inhibit protein synthesis, leading to the reduction of short-lived antiapoptotic proteins. Notably though, treatment with flavaglines, alone or in combination with other drugs, yields a much stronger cytotoxic activity toward leukemia cells than the translational inhibitor temsirolimus. These results indicate that the underlying cell death mechanism of flavaglines is more complex than simply inhibiting general protein translation. Global gene expression profiling and cell biological assays identified Myc inhibition and the disruption of mitochondrial integrity to be features of flavaglines, which we propose contribute to their efficacy in targeting leukemia cells. Taken together, these findings indicate that rocaglamide and silvestrol are distinct from clinically available translational inhibitors and represent promising candidates for the treatment of leukemia.

Immunomodulatory derivative (IMiD) CC-4047, a new analog of thalidomide, directly inhibits growth of B-cell malignancies in vivo and in vitro and exhibits stronger antiangiogenic activity than thalidomide. However, there is little information on whether CC-4047 affects normal hematopoiesis. Here we investigated the effect of CC-4047 on lineage commitment and differentiation of hematopoietic stem cells. We found that CC-4047 effectively inhibits erythroid cell colony formation from CD34+ cells and increases the frequency of myeloid colonies. We also demonstrate that development of both erythropoietin-independent and erythropoietin-dependent red cell progenitors was strongly inhibited by CC-4047, while terminal red cell differentiation was unaffected. DNA microarray analysis revealed that red cell transcription factors, including GATA-1, GATA-2, erythroid Kruppel-like factor (EKLF), and growth factor independence-1B (Gfi-1b), were down-regulated in CC-4047-treated CD34+ cells, while myeloid transcription factors such as CCAAT/enhancer binding protein-alpha (C/EBPalpha), C/EBPdelta, and C/EBPepsilon were induced. Analysis of cytokine secretion indicated that CC-4047 induced secretion of cytokines that enhance myelopoiesis and inhibit erythropoiesis. In conclusion, these data indicate that CC-4047 might directly influence lineage commitment of hematopoietic cells by increasing the propensity of stem and/or progenitor cells to undergo myeloid cell development and concomitantly inhibiting red cell development. Therefore, CC-4047 provides a valuable tool to study the mechanisms underlying lineage commitment. View Publication

Cripto is a developmental oncoprotein that signals via mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K)/Akt and Smad2/3 pathways. However, the molecular basis for Cripto coupling to these pathways during embryogenesis and tumorigenesis is not fully understood. In this regard, we recently demonstrated that Cripto forms a cell surface complex with the HSP70 family member glucose-regulated protein-78 (GRP78). Here, we provide novel functional evidence demonstrating that cell surface GRP78 is a necessary mediator of Cripto signaling in human tumor, mammary epithelial and embryonic stem cells. We show that targeted disruption of the cell surface Cripto/GRP78 complex using shRNAs or GRP78 immunoneutralization precludes Cripto activation of MAPK/PI3K pathways and modulation of activin-A, activin-B, Nodal and transforming growth factor-beta1 signaling. We further demonstrate that blockade of Cripto binding to cell surface GRP78 prevents Cripto from increasing cellular proliferation, downregulating E-Cadherin, decreasing cell adhesion and promoting pro-proliferative responses to activin-A and Nodal. Thus, disrupting the Cripto/GRP78 binding interface blocks oncogenic Cripto signaling and may have important therapeutic value in the treatment of cancer.

Central issues in intracoronary infusion (ICI) of bone marrow (BM)-cells to damaged myocardium for improving cardiac function are the cell number that is feasible and safe to be administrated as well as the retention of cells in the target area. Our study addressed these issues in eight patients with chronic ischemic cardiomyopathy undergoing ICI of selected BM-progenitors. We could immunomagnetically isolate 0.8 +/- 0.32 x 10(7) CD133(+) cells and 0.75 +/- 0.24 x 10(7) CD133(-)CD34(+) cells from 310 +/- 40 ml BM. After labeling these cells with (99m)Tc-hexamethylpropylenamineoxime, they were infused into the infarct-related artery without any complication. Scintigraphic images 1 (eight patients) and 24 hours (four patients) after ICI revealed an uptake of 9.2% +/- 3.6 and 6.8% +/- 2.4 of the total infused radioactivity in the infarcted area of the heart, respectively; the remaining activity was distributed mainly to liver and spleen. We conclude that through ICI of CD133(+) and CD133(-)CD34(+) BM-progenitors a significant number of them are preferentially attracted to and retained in the chronic ischemic myocardium.

FUS/TLS (fused in sarcoma/translocated in liposarcoma) and TDP-43 are integrally involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We found that FUS/TLS binds to RNAs from textgreater5,500 genes in mouse and human brain, primarily through a GUGGU-binding motif. We identified a sawtooth-like binding pattern, consistent with co-transcriptional deposition of FUS/TLS. Depletion of FUS/TLS from the adult nervous system altered the levels or splicing of textgreater950 mRNAs, most of which are distinct from RNAs dependent on TDP-43. Abundance of only 45 RNAs was reduced after depletion of either TDP-43 or FUS/TLS from mouse brain, but among these were mRNAs that were transcribed from genes with exceptionally long introns and that encode proteins that are essential for neuronal integrity. Expression levels of a subset of these were lowered after TDP-43 or FUS/TLS depletion in stem cell-derived human neurons and in

Butein has been reported to exert anti-inflammatory effect but the possible mechanism involved is still unclear. Here, we report the inhibitory effect of butein on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression. Butein also inhibited the induction of tumor necrosis factor-alpha and cyclooxygenase 2 by LPS. To further investigate the mechanism responsible for the inhibition of iNOS gene expression by butein, we examined the effect of butein on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. The LPS-induced DNA binding activity of NF-kappaB was significantly inhibited by butein, and this effect was mediated through inhibition of the degradation of inhibitory factor-kappaB and phosphorylation of Erk1/2 MAP kinase. Furthermore, increased binding of the osteopontin alphavbeta3 integrin receptor by butein may explain its inhibitory effect on LPS-mediated NO production. Taken together, these results suggest that butein inhibits iNOS gene expression, providing possible mechanisms for its anti-inflammatory action.

BACKGROUND: Fetal alcohol spectrum disorders (FASD) are a leading cause of neurodevelopmental disability. The mechanisms underlying FASD are incompletely understood, and biomarkers to identify those at risk are lacking. Here, we perform metabolomic analysis of embryoid bodies and neural lineages derived from human embryonic stem (hES) cells to identify the neural secretome produced in response to ethanol (EtOH) exposure. METHODS: WA01 and WA09 hES cells were differentiated into embryoid bodies, neural progenitors, or neurons. Cells along this progression were cultured for 4 days with 0, 0.1, or 0.3% EtOH. Supernatants were subjected to C18 chromatography followed by ESI-QTOF-MS. Features were annotated using public databases, and the identities of 4 putative biomarkers were confirmed with purified standards and comparative MS/MS. RESULTS: EtOH treatment induced statistically significant changes to metabolite abundance in human embryoid bodies (180 features), neural progenitors (76 features), and neurons (42 features). There were no shared significant features between different cell types. Fifteen features showed a dose-response to EtOH. Four chemical identities were confirmed: L-thyroxine, 5'-methylthioadenosine, and the tryptophan metabolites, L-kynurenine and indoleacetaldehyde. One feature with a putative annotation of succinyladenosine was significantly increased in both EtOH treatments. Additional features were selective to EtOH treatment but were not annotated in public databases. CONCLUSIONS: EtOH exposure induces statistically significant changes to the metabolome profile of human embryoid bodies, neural progenitors, and neurons. Several of these metabolites are normally present in human serum, suggesting their usefulness as potential serum FASD biomarkers. These findings suggest the biochemical pathways that are affected by EtOH in the developing nervous system and delineate mechanisms of alcohol injury during human development.

Human bocavirus type-1 (HBoV1) has a high tropism for the apical membrane of human airway epithelia. The packaging of a recombinant adeno-associated virus 2 (rAAV2) genome into HBoV1 capsid produces a chimeric vector (rAAV2/HBoV1) that also efficiently transduces human airway epithelia. As such, this vector is attractive for use in gene therapies to treat lung diseases such as cystic fibrosis. However, preclinical development of rAAV2/HBoV1 vectors has been hindered by the fact that humans are the only known host for HBoV1 infection. This study reports that rAAV2/HBoV1 vector is capable of efficiently transducing the lungs of both newborn (3- to 7-day-old) and juvenile (29-day-old) ferrets, predominantly in the distal airways. Analyses of in vivo, ex vivo, and in vitro models of the ferret proximal airway demonstrate that infection of this particular region is less effective than it is in humans. Studies of vector binding and endocytosis in polarized ferret proximal airway epithelial cultures revealed that a lack of effective vector endocytosis is the main cause of inefficient transduction in vitro. While transgene expression declined proportionally with growth of the ferrets following infection at 7 days of age, reinfection of ferrets with rAAV2/HBoV1 at 29 days gave rise to approximately 5-fold higher levels of transduction than observed in naive infected 29-day-old animals. The findings presented here lay the foundation for clinical development of HBoV1 capsid-based vectors for lung gene therapy in cystic fibrosis using ferret models.

BACKGROUND Molecular subtyping has allowed for the beginning of personalized treatment in children suffering from medulloblastoma (MB). However, resistance inevitably emerges against these therapies, particularly in the Sonic Hedgehog (SHH) subtype. We found that children with SHH subtype have the worst outcome underscoring the need to identify new therapeutic targets. PROCEDURE High content screening of a 129 compound library identified agents that inhibited SHH MB growth. Lead molecular target levels, p90 ribosomal S6 kinase (RSK) were characterized by immunoblotting and qRT-PCR. Comparisons were made to human neural stem cells (hNSC). Impact of inhibiting RSK with the small molecule BI-D1870 or siRNA was assessed in growth assays (monolayer, neurosphere, and soft agar). NanoString was used to detect RSK in a cohort of 66 patients with MB. To determine BI-D1870 pharmacokinetics/pharmacodynamics, 100 mg/kg was I.P. injected into mice and tissues were collected at various time points. RESULTS Daoy, ONS76, UW228, and UW426 MB cells were exquisitely sensitive to BI-D1870 but unresponsive to SHH inhibitors. Anti-tumor growth corresponded with inactivation of RSK in MB cells. BI-D1870 had no effect on hNSCs. Inhibiting RSK with siRNA or BI-D1870 suppressed growth, induced apoptosis, and sensitized cells to SHH agents. Notably, RSK expression is correlated with SHH patients. In mice, BI-D1870 was well-tolerated and crossed the blood-brain barrier (BBB). CONCLUSIONS RSK inhibitors are promising because they target RSK which is correlated with SHH patients as well as cause high levels of apoptosis to only MB cells. Importantly, BI-D1870 crosses the BBB, acting as a scaffold for development of more long-lived RSK inhibitors.

Neural differentiation of human embryonic (ES) and induced pluripotent (iPS) stem cell lines has been used for research in early human development, drug discovery, and cell replacement therapies. It is critical to establish generic differentiation protocols to compare the neural specification potential of each individually derived pluripotent stem cell line and identify the efficacious lines for research and therapeutic use. Here, we describe a reproducible and quantitative protocol to assess the neural progenitor (NP) generation of human pluripotent stem cell lines. This method includes a robust and well-defined neural inducing platform for Pax6(+) neural rosette (neuroectodermal cells) generation, propagation, and subsequent differentiation into nestin(+) NPs. A side-by-side comparison under common culture conditions among three human ES cell lines, TE03, TE06, and BG01V, and one iPS cell line, HD02, showed highly variable efficiency in their differentiation into NPs. View Publication