Progenipoietins: biological characterization of a family of dual agonists of fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor.

The progenipoietins, a class of engineered proteins containing both fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor receptor agonist activities, were functionally characterized in vitro and in vivo. Four representative progenipoietins were evaluated for receptor binding, receptor-dependent cell proliferation, colony-forming unit activity, and their effects on hematopoiesis in the C57BL/6 mouse.The progenipoietins bound to fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor with affinities within twofold to threefold of the native ligands, and each progenipoietin bound simultaneously to both fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor. The progenipoietins exhibited different levels of activity in receptor-dependent cell proliferation assays. The fetal liver tyrosine kinase-3-dependent cell proliferation activity of three of four progenipoietins was decreased sixfold to 33-fold relative to native fetal liver tyrosine kinase-3 ligand, while granulocyte colony-stimulating factor receptor-dependent activity of the progenipoietins was within twofold to threefold of native granulocyte colony-stimulating factor. At nonsaturating concentrations, the progenipoietins stimulated colony formation to a greater extent than the equimolar combination of fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor. Treatment of mice with the progenipoietins yielded dramatic increases in peripheral blood and splenic white blood cells, polymorphonuclear leukocytes, and dendritic cells. These preclinical results demonstrate that the progenipoietins are potent hematopoietic growth factors that stimulate cells in a receptor-dependent manner. When administered in vivo, the progenipoietins effectively promote the generation of multiple cell lineages. Thus, in both in vitro and in vivo settings, the progenipoietins as single molecules exhibit the synergistic activity of the combination of fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor.

Promegapoietin, a family of chimeric growth factors, supports megakaryocyte development through activation of IL-3 and c-Mpl ligand signaling pathways.

OBJECTIVE: The signaling pathways induced by promegapoietin (PMP), a family of chimeric growth factors that activate the human IL-3 and c-Mpl receptors, were investigated. METHODS: The biological activity of PMP was examined by receptor binding, cell proliferation, ex vivo expansion of hematopoietic progenitor cells, and in vivo production of platelets. The activation of signaling pathways was examined by Western blot and Northern blot analyses. RESULTS: Two PMP molecules, PMP-1 and PMP-1a, induced proliferation of cells expressing the IL-3 receptor, c-Mpl, or both receptors and bound to the IL-3 receptor and c-Mpl with high affinity. Ex vivo expansion assays using human bone marrow CD34(+) cells suggested that PMP-1 induced greater total cellular expansion as well as expansion of CD41(+) megakaryocytic precursor cells than IL-3 or c-Mpl ligand alone. Subcutaneous administration of 50 microg/kg of PMP-1 for 10 days to rhesus monkeys resulted in increased platelet production in vivo from a baseline of 357 +/- 45 x 10(3) cells/mL to 1376 +/- 151 x 10(3) cell/mL. PMP-1 induced phosphorylation of the beta(c) subunit of IL-3 receptor and c-Mpl, JAK2, and STAT5b, but not STAT3. PMP-1 induced greater expression of Pim-1, c-Myc, and cyclin D2 than did either an IL-3 receptor agonist or c-Mpl receptor agonist alone. The magnitude of induction of early response genes was similar for PMP and the coaddition of IL-3 receptor agonist and c-Mpl receptor agonist. CONCLUSION: PMP combines the biological activities of IL-3 and c-Mpl ligand in a single molecule that can simultaneously activate signaling pathways induced by both these ligands.

The preparation of electronically sorted hematopoietic cells for visual analysis.

The authors describe a simple method of collecting and preparing electronically sorted hemic cells for visual analysis. Wright-stained preparations were obtained easily by this method and offered excellent preservation of morphologic features. Flow cytometer-derived data thus can be correlated with the morphologic characteristics of immunologically distinctive subsets of hemic cells.

Understanding and treating arm movement impairment after chronic brain injury: progress with the ARM guide.

Significant potential exists for enhancing physical rehabilitation following neurologic injury through the use of robotic and mechatronic devices (or "rehabilitators"). We review the development of a rehabilitator (the "ARM Guide") to diagnose and treat arm movement impairment following stroke and other brain injuries. As a diagnostic tool, the ARM Guide provides a basis for evaluation of several key motor impairments, including abnormal tone, incoordination, and weakness. As a therapeutic tool, the device provides a means to implement and evaluate active assist therapy for the arm. Initial results with three stroke subjects demonstrate that such therapy can produce quantifiable benefits in the chronic hemiparetic arm. Directions for future research regarding the efficacy and practicality of rehabilitators are discussed.

Identification of a mouse anti-rat IgE monoclonal antibody, 44.7b, which inhibits IgE binding to RBL cells.

We have identified a monoclonal antibody specific for rat IgE, 44.7b, which blocks binding of rat IgE to the mast cell-like line RBL. Other monoclonal anti-rat IgE antibodies (MARE1 and B5) did not inhibit IgE binding to these cells. Furthermore, 44.7b did not react with IgE previously bound to these cells. These results indicate that 44.7b binds to an epitope on the IgE molecule which is within the binding site of the mast cell IgE Fc receptor (FcERI). The 44.7b monoclonal antibody did recognize IgE bound to a B lymphocyte cell line indicating that mast cell and B lymphocyte FcERs recognize different regions of the IgE molecule.

Circular polysomes predominate on the rough endoplasmic reticulum of somatotropes and mammotropes in the rat anterior pituitary.

We have studied the shape and size distribution of membrane-bound polysomes in somatotropes and mammotropes, which are the sources, respectively, of growth hormone and of prolactin in the rat pituitary. The observations were made in conventional electron micrographs of these cells in situ, where occasional surface or en face views of the rough endoplasmic reticulum allow the polysomes to be seen as rows of ribosomes arranged in distinctive patterns on the membranes. It is possible by this means to characterize the shape and number of ribosomes for the total population of bound polysomes in the respective cell types. The great majority of membrane-bound polysomes in these two cell types (81% in somatotropes, 78% in mammotropes) have an approximately circular shape and contain an average of 6.8 (somatotropes) or 6.5 (mammotropes) ribosomes, which is an appropriate size for translation of the polypeptide hormones produced by these cells. About 17% of the membrane-bound polysomes in somatotropes and 20% in mammotropes have a spiral shape, resembling somewhat the letter "G," and contain about eight to nine ribosomes in both cell types. The preponderance of circular polysomes on the rough endoplasmic reticulum of somatotropes and mammotropes suggests the possibility that ribosomes (or the 40S ribosomal subunit) may recycle on the polysome after the translation of growth hormone or of prolactin.

Adaptive assistance for guided force training in chronic stroke.

This work describes a novel form of robotic therapy for the upper extremity in chronic stroke. Based on previous results, we hypothesized that a training task that encourages subjects to consciously guide endpoint forces generated by the hemiparetic arm will result in significant gains in functional ability of the arm, superior to more conventional methods of therapy. In addition, since stroke survivors present with varying degrees of arm movement ability, we developed an adaptive algorithm that tailors the amount of assistance provided in completing the guided force training task. The algorithm adapts a coefficient for velocity-dependent assistance based on measured movement speed, on a trial-to-trial basis. The training algorithm has been implemented with a simple linear robotic device called the ARM Guide. One participant completed a two month training program with the adaptive algorithm, resulting in significant improvements in the performance of functional tasks.

A method for transplantation of luteinizing granulosa cells: evidence for progesterone secretion.

A method is described to enable the investigation of luteal tissue that is formed in the absence of ovarian theca lutein cells. Immature rats were given a s.c. injection of pregnant mare's serum gonadotropin (PMSG) followed 48 h later by an i.v. injection of human chorionic gonadotropin (hCG). Both ovaries were removed 8 to 10 h after the injection of hCG, the preovulatory follicles punctured, and the granulosa cells expressed into medium using gentle pressure with a dissecting scalpel. The cells were centrifuged at low speed (50 X g) for 5 min, resuspended and recentrifuged. The cells were aspirated into PE 90 polyethylene tubing, the tubing sealed at one end and then placed into a microcapillary hematocrit tube. The cells were packed by centrifugation in a microhematocrit centrifuge for 2 min, after which they were autotransplanted by injecting them beneath the kidney capsule. Progesterone, determined in blood samples obtained 4 through 12 days after transplantation, was elevated on most days compared to ovariectomized controls without cell transplants. In a second experiment, serum progesterone in ovariectomized-adrenalectomized rats with autotransplants was also elevated (4 to 6 ng/ml) compared to controls without cell transplants in which progesterone was about 0.1 ng/ml. Morphology of transplants revealed luteal cells with considerable vascularity and with the typical appearance of cells observed in in situ corpora lutea. This procedure, with modifications to include homotransplants in inbred strains, holds promise for investigation of the endocrine characteristics of granulosa lutein cells devoid of cells of thecal origin.

Cerebral microvessels and derived cells in tissue culture: isolation and preliminary characterization.

Microvessels isolated from mouse forebrain were used as the source material for the derivation of cerebral vascular endothelium and smooth-muscle cells in culture. The microvessels were isolated by a mechanical dispersion and filtration technique, and were maintained in vitro as organoid cultures. A microvessel classification system was developed and proved to be useful as a tool in monitoring culture progress and in predicting the type(s) of microvessel(s) that would give rise to migrating and/or proliferating cells. The isolated cerebral microvessels were heterogeneous in diameter, size of individual vascular isolate, and proliferative potential. The isolated microvessels ranged in diameter from 4 micron to 25 micron and in size from a single microvascular segment to a large multibranched plexus with mural cells. The initial viability, determined by erythrosin B exclusion, was approximately 50% on a per cell basis. All microvessel classes had proliferative potential although the rate and extent of proliferation were both microvessel class- and density-dependent. The smaller microvessels gave rise to endothelial cells, whereas the large microvessels gave rise to endothelial and smooth-muscle cells. The viability and progress of a microvessel toward derived cell proliferation seemed to be directly proportional to the number of mural cells present.

Regulation of human IgE synthesis by soluble factors. Papain treatment of a FcE receptor-positive B-cell line (RPMI-1788) releases regulatory factors for IgE synthesis.

A novel mechanism for the release of helper and suppressor factors for human IgE synthesis is described. When FcE receptor-positive RPMI-1788 cells are treated with papain, a helper factor(s) for human IgE synthesis is released. At the same time a significant decrease in the number of cell surface FcE receptors is observed. The immunoglobulin synthesis-enhancing activity is IgE isotype-specific inasmuch as the same supernatant suppresses the synthesis of human IgA myeloma cells. When the FcE receptor-positive RPMI-1788 cells are treated with tunicamycin and then with papain, a suppressor factor(s) for human IgE synthesis is released. The mechanism by which these factors affect human myeloma IgE synthesis is unclear at present. Our results indicate that enhanced IgE synthesis is not due to increased numbers of secreting cells nor to an increased release of presynthesized IgE. In summary, papain treatment of FcE receptor-positive, but not FcE receptor-negative cells, generates a factor that regulates IgE synthesis. These results also provide evidence for the close relationship between the IgE regulatory factors and the low affinity receptors for IgE present on lymphocytes.

Regeneration of cerebral microvessels: a morphologic and histochemical study after local freeze-injury.

The sequential changes that occur during regeneration of cerebral microvessels was studied in young mice after a local freeze-injury. The immediate breakdown in the blood-brain barrier to horseradish peroxidase was followed by dissolution of cells in the vessel wall although the basal lamina was preserved. This residual basal lamina formed the pathway for the orderly regeneration of microvessels. Three days after injury, reactive endothelial cells with prominent pinocytosis and patent interendothelial channels were present at the edge of the lesion. By 5 days, endothelialization of necrotic vessels was in progress with patent vascular channels and tight junction formation. Establishment of astrocytic contacts or perivascular fibrosis by 7 days was generally followed by reconstitution of normal vascular morphology and integrity at 15 to 35 days after injury.

Megakaryocyte ex vivo expansion potential of three hematopoietic sources in serum and serum-free medium.

Megakaryocytes (MK) were expanded from purified human CD34+ cells obtained from three sources, bone marrow (BM), mobilized peripheral blood progenitor cells (PB), and umbilical cord (UC) blood. CD34+-selected cells were cultured for 12 days with 10 ng/ml thrombopoietin (TPO), 10 ng/ml IL-3, 10 ng/ml TPO + 10 ng/ml IL-3, or 200 ng/ml promegapoietin (PMP), a chimeric dual agonist of the c-Mpl and human IL-3 receptors. MK production was compared in serum-free versus human serum-supplemented liquid media. PMP and the combination of TPO and IL-3 (TPO + IL-3) increased MK production similarly. Culturing CD34+ cells with PMP in serum-free medium resulted in a twofold increase in MK yield compared with serum-supplemented medium. CD34+ cells from UC proliferated more than those from either BM or PB in liquid culture, resulting in much greater MK production under all conditions. Phenotypic analysis of the uncultured CD34+ cells showed that BM had a higher frequency of CD34+/CD41+ cells than PB or UC. TPO + IL-3 or PMP produced larger and greater numbers of BFU-MK and CFU-MK per seeded CD34+/CD41+ cell from UC than from either BM or PB. Thus, although uncultured CD34+-selected BM cells contained a higher frequency of committed mature MK progenitors, UC CD34+ cells had a greater proliferative capacity and, therefore, were more productive. PMP induced megakaryocytopoietic activity comparable to that achieved with TPO + IL-3 and may be useful for ex vivo expansion of MK for clinical trials.

T cell activation is regulated by voltage-dependent and calcium-activated potassium channels.

Membrane potential (Vm) is tightly controlled in T cells through the regulated flux of ions across the plasma membrane. To investigate the functional role of voltage-dependent (Kv) and calcium-activated (KCa) potassium channels in T cell activation, we compared the effects of two K+ channel blockers, namely kaliotoxin (KTX) and charybdotoxin (CHTX), on Vm, calcium influx, and cell proliferation. KTX potently inhibited Kv (ID50 = 3 nM) but not KCa (ID50 = 5 microM) currents in T cells. Resting T cells exposed to KTX (300 nM) depolarized from -56 mV to -50 mV. KTX had no effect on the transient membrane hyperpolarization that characteristically follows receptor-mediated T cell stimulation. However, T cells stimulated in the presence of KTX subsequently depolarized to -40 mV. KTX also reduced the steady state intracellular free calcium concentration ([Ca2+]i) in stimulated cells by 19% and inhibited T cell proliferation by 35%. CHTX potently inhibited both Kv and KCa currents (ID50 = approximately 1 nM). CHTX (300 nM) depolarized resting T cells to -48 mV, equivalent to the effect observed for KTX. In stimulated T cells, 300 nM CHTX completely blocked the induced hyperpolarization and subsequently depolarized the cells to -21 mV. These effects were associated with a 45% reduction in peak [Ca2+]i, a 60% decrease in steady state [Ca2+]i, and 63% inhibition of T cell proliferation. These results suggest that both Kv and KCa conductances contribute to the underlying mechanisms of T cell activation.

Dendritic cells generated in vivo by a chimeric hematopoietic growth factor, progenipoietin-4, demonstrate potent immunological function.

Recently, a dual receptor agonist for human Flt3 and G-CSF receptors, progenipoietin-4 (ProGP-4), was shown to be highly effective in expanding DC in vivo. In this study, we examined the immunological activity of ProGP-4-generated dendritic cell (DC) in an HLA-A2.1 transgenic mouse system. ProGP-4 DC were found to be approximately equivalent in presenting a cytotoxic T lymphocyte (CTL) peptide to a CTL line in vitro compared with bone marrow (BM)-derived DC and >20-fold more efficient than macrophages or B cells, and >100-fold better than BM-DC, macrophages, or B cells at presenting PADRE, a universal helper T cell epitope, to a T cell clone. The heightened epitope presentation by ProGP-4 DC was paralleled in vivo inasmuch as a >6-fold increase in CTL induction was observed compared with other APC populations following ex vivo loading with peptide. The in vitro and in vivo CTL responses stimulated by ProGP-4 DC could be further augmented by either culturing with tumor necrosis factor-alpha (TNF-alpha) or co-loading with PADRE. Collectively, our results indicate that peptide-loaded ProGP-4-generated DC demonstrate potent antigenicity and immunogenicity for CTL, making them an attractive component of epitope-based vaccines.