Inflammatory cytokines provide a third signal for activation of naive CD4+ and CD8+ T cells

The effects of inflammatory cytokines on naive T cells have been studied using MHC protein/peptide complexes on microspheres, thus avoiding the use of APCs whose functions may be affected by the cytokines. IL-1, but not IL-12, increased proliferation of CD4+ T cells in response to Ag and IL-2, which is consistent with effects on in vivo priming of CD4+ cells. In contrast, proliferation of CD8+ T cells to Ag and IL-2 required IL-12, and IL-12 replaced adjuvant in stimulating an in vivo response to peptide. These results support a model in which distinct inflammatory cytokines act directly on naive CD4+ and CD8+ T cells to provide a third signal, along with Ag and IL-2, to optimally activate differentiation and clonal expansion.     

Cdk5 regulates STAT3 activation and cell proliferation in medullary thyroid carcinoma cells

The biological behaviors of thyroid cancer are varied, and the pathological mechanisms remain unclear. Some reports indicated an apparent aggregation of amyloid accompanying medullary thyroid carcinoma (MTC). Amyloid aggregation in neurodegeneration leads to hyperactivation of Cdk5 and subsequent neuronal death. Based on the connection with amyloid, the role of Cdk5 in MTC is worthy of investigation. Initially, the expression of Cdk5 and its activator, p35, in MTC cell lines was identified. Cdk5 inhibition by specific inhibitors or short interfering RNA decreased the proliferation of MTC cell lines, which reveals the importance of Cdk5 in MTC cell growth. Although p35 cleavage has been considered as an important element in neurodegeneration, it seems that p35 cleavage was not a major cause in Cdk5 activity-dependent MTC cell proliferation because neither Cdk5 activity nor cell growth was affected by the inhibition of p35 cleavage. Clearance of amyloid by antibody neutralization indicated that MTC cell proliferation was supported by calcitonin-derived extracellular amyloid and subsequent Her2 and Cdk5 activation. Significantly, the STAT3 pathway was involved in Cdk5-dependent proliferation of MTC cells through Ser-727 phosphorylation. In addition, Cdk5 inhibition reduced nuclear distributions of both the Cdk5-p35 complex and phospho-STAT3 in MTC cells. Finally, Cdk5 inhibition retarded tumor formation in vivo accompanying the reduction of phospho-STAT3. Our findings suggest the first demonstration of a novel and specific role for Cdk5 kinase in supporting the proliferation of the medullary thyroid carcinoma cells and could shed light on a new field for diagnosis and therapy of thyroid cancer.     

Constitutive STAT3 activation in intestinal T cells from patients with Crohn's disease

Via cytoplasmic signal transduction pathways, cytokines induce a variety of biological responses and modulate the outcome of inflammatory diseases and malignancies. Crohn's disease is a chronic inflammatory bowel disease of unknown etiology. Perturbation of the intestinal cytokine homeostasis is believed to play a pivotal role, but the pathogenesis of Crohn's disease is not fully understood. Here, we study intestinal T cells from Crohn's disease and healthy volunteers. We show that STAT3 and STAT4 are constitutively activated in Crohn's patients but not in healthy volunteers. The activation is specific, because other STAT proteins are not constitutively activated. Furthermore, the STAT3 regulated protein, SOCS3, is also constitutively expressed in Crohn's patients but not in healthy volunteers. Taken together, these data provide evidence of abnormal STAT/SOCS signaling in Crohn's disease. This aberrant activation, so far noted only in malignant cells, establish a new critical approach for better understanding the immunopathogenesis of Crohn's disease.     

Stromal cells provide the matrix for migration of early lymphoid progenitors through the thymic cortex

During steady state lymphopoiesis in the postnatal thymus, migration of precursors outward from the deep cortex toward the capsule is required for normal differentiation. Such migration requires, at a minimum, expression of adhesive receptors on the migrating lymphoid cells, as well as a stable matrix of their ligands persisting throughout the region of migration. In this study, we address the nature of this adhesive matrix. Although some precursor stages bound efficiently to extracellular matrix ligands, a specific requirement for the cell surface ligand VCAM-1 was also found. In situ analysis revealed that early precursors are found in intimate contact with a matrix formed by stromal cells in the cortex, a proportion of which expresses VCAM-1. In vivo administration of an anti-VCAM-1 Ab resulted in decreased thymic size and altered distribution of early precursors within the cortex. These results indicate that precursors migrating outward through the cortex may use a cellular, rather than extracellular, matrix for adhesion, and suggest that the VCAM-1(+) subset of cortical stroma may play a crucial role in supporting the migration of early precursors in the steady state thymus.     

Analysis of Ras-dependent signals that prevent caspase-3 activation and apoptosis induced by cytokine deprivation in hematopoietic cells

In hematopoietic cells, Ras has been implicated in signaling pathways that prevent apoptosis triggered by deprivation of cytokines, such as interleukin-3 (IL-3). However, the mechanism whereby Ras suppresses cell death remains incompletely understood. We have investigated the role of Ras in IL-3 signal transduction by using the cytokine-dependent BaF3 cell line. Herein, we show that the activation of the pro-apoptotic protease caspase-3 upon IL-3 removal is suppressed by expression of activated Ras, which eventually prevents cell death. For caspase-3 suppression, the Raf/extracellular signal-regulated kinase (ERK)- or phosphatidylinositol 3-kinase (PI3-K)/Akt-mediated signaling pathway downstream of Ras was required. However, inhibition of both pathways did not block activated Ras-dependent suppression of cell death-associated phenotypes, such as nuclear DNA fragmentation. Thus, a pathway that is independent of both Raf/ERK and PI3-K/Akt pathways may function downstream of Ras, preventing activated caspase-3-initiated apoptotic processes. Conditional activation of c-Raf-1 also suppressed caspase-3 activation and subsequent cell death without affecting Akt activity, providing further evidence for a PI3-K/Akt-independent mechanism.     

Engineered vascular beds provide key signals to pancreatic hormone-producing cells

The mechanisms underlying early islet graft failure are not entirely clear, but are thought to involve ischemic injury due to delayed vascularization. We hypothesize that blood vessels play an active role in cell-cell communications supporting islet survival and engraftment. To test this hypothesis and to uncouple endothelial cell (EC)-generated signaling stimuli from their nutritional and gas exchange functions, we developed three dimensional (3D) endothelial vessel networks in engineered pancreatic tissues prepared from islets, fibroblasts and ECs. The tri-culture setup, seeded on highly porous biocompatible polymeric scaffolds closely mimics the natural anatomical context of pancreatic vasculature. Enhanced islet survival correlating with formation of functional tube-like endothelial vessels was demonstrated. Addition of foreskin fibroblasts to islet-endothelial cultures promoted tube-like structure formation, which further supported islet survival as well as insulin secretion. Gene expression profiles of EC growth factors, extracellular matrix (ECM), morphogenes and differentiation markers were significantly different in 2D versus 3D culture systems and were further modified upon addition of fibroblasts. Implantation of prevascularized islets into diabetic mice promoted survival, integration and function of the engrafted engineered tissue, supporting the suggested role of ECs in islet survival. These findings present potential strategies for preparation of transplantable islets with increased survival prospects.     

Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization

Adults maintain a reservoir of hematopoietic stem cells that can enter the circulation to reach organs in need of regeneration. We developed a novel model of retinal neovascularization in adult mice to examine the role of hematopoietic stem cells in revascularizing ischemic retinas. Adult mice were durably engrafted with hematopoietic stem cells isolated from transgenic mice expressing green fluorescent protein. We performed serial long-term transplants, to ensure activity arose from self-renewing stem cells, and single hematopoietic stem-cell transplants to show clonality. After durable hematopoietic engraftment was established, retinal ischemia was induced to promote neovascularization. Our results indicate that self-renewing adult hematopoietic stem cells have functional hemangioblast activity, that is, they can clonally differentiate into all hematopoietic cell lineages as well as endothelial cells that revascularize adult retina. We also show that recruitment of endothelial precursors to sites of ischemic injury has a significant role in neovascularization.     

Thymosin activation of T-killer precursors for hematopoietic stem cells]

Lymph node cells of normal CBA mice, syngeneic radiation chimerae CBA leads to CBA and B-mice after incubation with thymosin (fraction 5) were transplanted to sublethally irradiated recipients (CBA X C57BL) F1; the number of endogenic colonies in the recipient's spleen was then recorded. Thymosin was shown to increase the killer activity of the lymph node cells of normal mice CBA, syngeneic chimerae CBA leads to CBA, but not of B-mice. As suggested, TU-cells' subpopulation served as target cells for thymosin.     

Extraction of near-field fluorescence from composite signals to provide high resolution images of glial cells

The subdiffraction optical resolution that can be achieved using near-field optical microscopy has the potential to permit new approaches and insights into subcellular function and molecular dynamics. Despite the potential of this technology, it has been difficult to apply to cellular samples. One significant problem is that sample thickness causes the optical information to be comprised of a composite signal containing both near- and far-field fluorescence. To overcome this issue we have developed an approach in which a near-field optical fiber is translated toward the cell surface. The increase in fluorescence intensity during z-translation contains two components: a far-field fluorescence signal when the tip of the fiber is distant from the labeled cell, and combined near- and far-field fluorescence when the tip interacts with the cell surface. By fitting a regression curve to the far-field fluorescence intensity as the illumination aperture approaches the cell, it is possible to isolate near-field from far-field fluorescent signals. We demonstrate the ability to resolve actin filaments in chemically fixed, hydrated glial cells. A comparison of composite fluorescence signals with extracted near-field fluorescence demonstrates that this approach significantly increases the ability to detect subcellular structures at subdiffraction resolution.     

Detection of cytokines in supernatant from hematopoietic stem/progenitor cells co-cultured with mesenchymal stem cells and endothelial progenitor cells

This study aimed to investigate the significance of cytokine expression in supernatant from hematopoietic stem/progenitor cells (HSCs/HPCs) co-cultured with mesenchymal stem cells (MSCs) or endothelial progenitor cells (EPCs). Mononuclear cells (MNCs) were isolated from normal human umbilical cord blood and then cultured solely or co-cultured with MSCs or EPCs. Changes in the number of MNCs and HSCs/HPCs were observed, and MNC proliferation was tested by carboxyfluorescein diacetate succinimidyl ester. The cultured supernatants of the treated MSCs and EPCs were collected at 24 h after co-culture and used to determine the concentrations of IL-3, IL-6, stem cell factor (SCF), TPO, Flt3l, and VEGF. The total number and proliferation of MNCs increased significantly when co-cultured with MSCs or EPCs than when cultured alone, particularly when MNCs were co-cultured with EPCs. The differences in IL-3 and Flt3l concentrations between groups were not significant. However, IL-6 in the MSC group was significantly higher than that in the two other groups. The SCF and TPO concentrations were highly expressed in the EPC group. The VEGF concentrations in the MSC group and the EPC group were higher than those in the control group. These results indicated that MSCs and EPCs possibly favor the proliferation of MNCs and HSCs/HPCs. IL-6 and VEGF may be related to hematopoietic reconstitution and homing ability of HSCs/HPCs. TPO may have a specific relationship with the promotion of HSCs/HPCs differentiation.