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This study was designed to investigate one component of the Wnt/beta-catenin signaling pathway that has been implicated in stem cell self-renewal. Retroviral-mediated introduction of stable beta-catenin to primitive murine bone marrow cells allowed the expansion of multipotential c-Kit(low)Sca-1(low/-)CD19(-) CD11b/Mac-1(-)Flk-2(-)CD43(+)AA4.1(+)NK1.1(-)CD3(-)CD11c(-)Gr-1(-)CD45R/B220(+) cells in the presence of stromal cells and cytokines. They generated myeloid, T, and B lineage lymphoid cells in culture, but had no T lymphopoietic potential when transplanted. Stem cell factor and IL-6 were found to be minimal requirements for long-term, stromal-free propagation, and a beta-catenin-transduced cell line was maintained for 5 mo with these defined conditions. Although multipotential and responsive to many normal stimuli in culture, it was unable to engraft several types of irradiated recipients. These findings support previous studies that have implicated the canonical Wnt pathway signaling in regulation of multipotent progenitors. In addition, we demonstrate how it may be experimentally manipulated to generate valuable cell lines.

The Notch1-RBP-Jkappa and the transcription factor Runx1 pathways have been independently shown to be indispensable for the establishment of definitive hematopoiesis. Importantly, expression of Runx1 is down-regulated in the para-aortic splanchnopleural (P-Sp) region of Notch1- and Rbpsuh-null mice. Here we demonstrate that Notch1 up-regulates Runx1 expression and that the defective hematopoietic potential of Notch1-null P-Sp cells is successfully rescued in the OP9 culture system by retroviral transfer of Runx1. We also show that Hes1, a known effector of Notch signaling, potentiates Runx1-mediated transactivation. Together with the recent findings in zebrafish, Runx1 is postulated to be a cardinal down-stream mediator of Notch signaling in hematopoietic development throughout vertebrates. Our findings also suggest that Notch signaling may modulate both expression and transcriptional activity of Runx1.

Umbilical cord blood (CB) is a valuable source of stem cells for transplantation, but CB transplantations are frequently complicated by delayed platelet engraftment. The reasons underlying this are unclear. We hypothesized that CB- and peripheral-blood (PB)-derived megakaryocytes (MKs) respond differently to the adult hematopoietic microenvironment and to thrombopoietin (Tpo). To test this, we cultured CB- and PB-CD34(+) cells in adult bone marrow stromal conditioned media (CM) or unconditioned media (UCM) with increasing concentrations of recombinant Tpo and compared the effects of these conditions on CB-versus PB-MKs. PB-MKs reached highest ploidy in response to UCM + 100 ng/mL rTpo, and the addition of CM inhibited their maturation. In contrast, CB-MKs reached highest ploidy in CM without rTpo, and high rTpo concentrations (textgreater 0.1 ng/mL) inhibited their maturation. This is the first evidence that human neonatal and adult MKs have substantially different biologic responses to Tpo and potentially to other cytokines.

We recently reported the development of three monoclonal antibodies (MoAbs) to biologically active human erythropoietin (Ep). In the present study, we investigated the epitope specificity of these three antibodies, as well as their reactivity with Eps derived from species other than man. All three antibodies reacted with the Ep polypeptide itself, rather than with its carbohydrate moieties. Moreover, all three antibodies recognized separate nonoverlapping epitopes. Further studies with reduced/alkylated Ep and with sodium dodecyl sulfate-denatured Ep suggested that two of the MoAbs, anti-Ep-2 and anti-Ep-16, were specific for conformational, nonlinear determinants on the Ep molecule, whereas the third MoAb, anti-Ep-26, appeared to recognize a linear epitope. However, anti-Ep-26 did not react with synthetic peptides representing the 26 amino-, the 99-129 mid-region, or the 10 carboxy-terminal residues of Ep, nor with trypsin-, chymotrypsin-, or V8 protease-digested fragments of Ep. When tested with Ep from different species, the neutralizing capabilities of the three MoAbs were clearly different. Comparing their effectiveness against baboon, ovine and murine Ep, antibody 2 was most effective at neutralizing baboon Ep, antibody 16 was most effective against murine Ep, and antibody 26 showed little reactivity with any of these nonhuman Eps. Because these various Eps readily stimulate across species barriers, it is likely that the receptor binding domain on Ep has remained relatively conserved during evolution. Our results therefore suggest that the neutralizing capacity of our three anti-Ep MoAbs is caused not by binding directly to the Ep receptor binding domain on Ep, but by binding to distant regions, causing conformational changes in Ep, or by binding to regions close to the binding site, steric hindrance.

Tryptophan (Trp) catabolism mediated by indoleamine 2,3-dioxygenase (IDO) plays a central role in the regulation of T-cell-mediated immune responses. In this study, we also demonstrate that natural killer (NK)-cell function can be influenced by IDO. Indeed, l-kynurenine, a Trp-derived catabolite resulting from IDO activity, was found to prevent the cytokine-mediated up-regulation of the expression and function of specific triggering receptors responsible for the induction of NK-cell-mediated killing. The effect of l-kynurenine appears to be restricted to NKp46 and NKG2D, while it does not affect other surface receptors such as NKp30 or CD16. As a consequence, l-kynurenine-treated NK cells display impaired ability to kill target cells recognized via NKp46 and NKG2D. Instead, they maintain the ability to kill targets, such as dendritic cells (DCs), that are mainly recognized via the NKp30 receptor. The effect of l-kynurenine, which is effective at both the transcriptional and the protein level, can be reverted, since NK cells were found to recover their functional competence after washing.

Many studies have demonstrated a role for netrin-1-deleted in colorectal cancer (DCC) interactions in both axon guidance and neuronal migration. Neogenin, a member of the DCC receptor family, has recently been shown to be a chemorepulsive axon guidance receptor for the repulsive guidance molecule (RGM) family of guidance cues [Rajagopalan S, Deitinghoff L, Davis D, Conrad S, Skutella T, Chedotal A, Mueller B, Strittmatter S (2004) Neogenin mediates the action of repulsive guidance molecule. Nat Cell Biol 6:755-762]. Here we show that neogenin is present on neural progenitors, including neurogenic radial glia, in the embryonic mouse forebrain suggesting that neogenin expression is a hallmark of neural progenitor populations. Neogenin-positive progenitors were isolated from embryonic day 14.5 forebrain using flow cytometry and cultured as neurospheres. Neogenin-positive progenitors gave rise to neurospheres displaying a high proliferative and neurogenic potential. In contrast, neogenin-negative forebrain cells did not produce long-term neurosphere cultures and did not possess a significant neurogenic potential. These observations argue strongly for a role for neogenin in neural progenitor biology. In addition, we also observed neogenin on parvalbumin- and calbindin-positive interneuron neuroblasts that were migrating through the medial and lateral ganglionic eminences, suggesting a role for neogenin in tangential migration. Therefore, neogenin may be a multi-functional receptor regulating both progenitor activity and neuroblast migration in the embryonic forebrain.

In herpesvirus infections, the virus persists for life but is contained through T-cell-mediated immune surveillance. How this immune surveillance operates is poorly understood. Recent studies of other persistent infections have indicated that virus persistence is associated with functional deficits in the CD8(+) T-cell response. To test whether this is the case in a herpesvirus infection, we used a mutant murine gammaherpesvirus that is defective in its ability to persist in the host. By comparing the immune response to this virus with a revertant virus that can persist, we were able to dissect the changes in the antiviral CD8(+) T-cell response that are induced by virus persistence. Surprisingly, persistently infected mice controlled a secondary challenge infection more rapidly than nonpersistently infected mice, indicating enhanced rather than diminished effector functions. Consistent with this, virus-specific CD8 T cells from these mice exhibited faster upregulation of the cytotoxic mediator granzyme B. Another unexpected finding was that CD8(+) T cells from neither infection responded efficiently to homeostatic cytokines. The unresponsiveness of the memory cells from the nonpersistently infected mice appears to be linked to the prolonged replication of virus within the lungs. Other changes seen in different chronic infection models were also observed, such as changes in Bcl-2 levels, interleukin-2 production, and the immunodominance hierarchy. These data show persistence of gammaherpesvirus type 68 alters the properties of CD8(+) T cells and illustrates that immune surveillance does not require CD8 T cells with the same attributes as classical" memory CD8(+) T cells."

A new monoclonal antibody, Ber-EP4, directed against a partially formol resistant epitope on the protein moiety of two 34 kilodalton and 39 kilodalton glycopolypeptides on human epithelial cells is described. Immunostaining of a wide range of normal and neoplastic human tissues and cell lines showed that all carcinomas and all non-neoplastic epithelial cells, except hepatocytes, parietal cells, and apical cell layers in squamous epithelia, homogeneously expressed Ber-EP4 antigen. As Ber-EP4 does not detect any normal or neoplastic non-epithelial cells, this antibody might prove valuable for the differentiation of the following (i) non-epithelial tumours from undifferentiated carcinomas; (ii) hepatocytes from bile duct cells in certain liver diseases; (iii) mesothelial cells from carcinoma cells in lung biopsy specimens; and (iv) reactive mesothelial cells from carcinoma cells in smears of serous effusions.

Microenvironments appear important in stem cell lineage specification but can be difficult to adequately characterize or control with soft tissues. Naive mesenchymal stem cells (MSCs) are shown here to specify lineage and commit to phenotypes with extreme sensitivity to tissue-level elasticity. Soft matrices that mimic brain are neurogenic, stiffer matrices that mimic muscle are myogenic, and comparatively rigid matrices that mimic collagenous bone prove osteogenic. During the initial week in culture, reprogramming of these lineages is possible with addition of soluble induction factors, but after several weeks in culture, the cells commit to the lineage specified by matrix elasticity, consistent with the elasticity-insensitive commitment of differentiated cell types. Inhibition of nonmuscle myosin II blocks all elasticity-directed lineage specification-without strongly perturbing many other aspects of cell function and shape. The results have significant implications for understanding physical effects of the in vivo microenvironment and also for therapeutic uses of stem cells.

The considerable therapeutic potential of human multipotent mesenchymal stromal cells (MSC) has generated markedly increasing interest in a wide variety of biomedical disciplines. However, investigators report studies of MSC using different methods of isolation and expansion, and different approaches to characterizing the cells. Thus it is increasingly difficult to compare and contrast study outcomes, which hinders progress in the field. To begin to address this issue, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human MSC. First, MSC must be plastic-adherent when maintained in standard culture conditions. Second, MSC must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules. Third, MSC must differentiate to osteoblasts, adipocytes and chondroblasts in vitro. While these criteria will probably require modification as new knowledge unfolds, we believe this minimal set of standard criteria will foster a more uniform characterization of MSC and facilitate the exchange of data among investigators.

CONTEXT: Endothelial dysfunction seems to be the first step of the atherosclerotic process. In the past few years, it has been demonstrated that injured endothelial monolayer is restored by a premature pool of circulating progenitor cells (PCs) and a more mature one of circulating endothelial PCs (EPCs). Even though there is increasing evidence that estrogens play a beneficial role on EPCs and, even if debated, on the cardiovascular system, less is known about androgens. OBJECTIVE: Our objective was to evaluate the levels of circulating PCs and EPCs in men with hypogonadotropic hypogonadism (HH) and the effect of prolonged testosterone (T) replacement therapy on these cells. DESIGN AND SETTING: We conducted a prospective study on males with HH at a university andrological center. PATIENTS: The study included 10 young HH patients (28.6 +/- 3.1 yr) and 25 age-matched controls. INTERVENTIONS: Idiopathic HH patients were treated with T gel therapy, 50 mg/d for 6 months. MAIN OUTCOME MEASURES: We assessed circulating PC and EPC concentrations and immunocytochemistry for androgen receptor expression on cultured EPCs. RESULTS: At baseline, HH patients showed a significant reduction of both PCs and EPCs with respect to controls. T replacement therapy induced a significant increase of these cells with respect to baseline. Immunocytochemistry on cultured EPCs showed strong expression of the androgen receptor. CONCLUSIONS: Hypotestosteronemia is associated with a low number of circulating PCs and EPCs in young HH subjects. T treatment is able to induce an increase in these cells through a possible direct effect on the bone marrow.

The influence of membrane potential on the effects of the enantiomers and the racemate of Bay K 8644 [1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluor-methylphenyl)-p yri dine-5-carboxylate] on force of contraction and on action potentials were studied in guinea-pig papillary muscles in order to detect possible changes in the direction of drug action or in potency. Membrane potential was varied by changing the potassium concentration ([K+]o) in the bathing solution. At normal resting potential, (-)-Bay K 8644 enhanced force of contraction and prolonged the action potential duration measured at 50% of repolarization (APD) to the same extent as the racemate and with similar pD2 values. After membrane depolarization by raising [K+]o from 5.4 to 17.4 mmol/l, the (-)-enantiomer and the racemate prolonged the APD to a similar degree but enhanced force to a lesser extent. The maximum rate of depolarization of slow action potentials, Vmax, was increased at the highest concentrations (10(-5) mol/l). The effects of (+)-Bay K 8644 were more complicated. At high concentrations (10(-5) mol/l) it decreased force of contraction and APD, the pD2 values were one order of magnitude lower than for the (-)-enantiomer and the racemate. A high concentration (+)-Bay K 8644 (10(-5) mol/l) virtually abolished contractile activity at all membrane potentials, the extent of shortening in APD increased with membrane depolarization in elevated [K+]o. Vmax of slow action potentials was decreased.(ABSTRACT TRUNCATED AT 250 WORDS)