Prolonged impairment of hematopoiesis after high-dose therapy followed by autologous bone marrow transplantation

Hematopoietic reconstitution has been studied in 180 patients after autologous bone marrow transplantation based on peripheral blood cell (PBC) recovery time and marrow progenitor counts sequentially tested for up to 4 years. Several factors that could influence hematopoietic reconstitution have been analyzed including sex, age, diagnosis, disease status, conditioning regimen, graft progenitor content, graft in vitro purging, and postgrafting administration of growth factors. Before transplantation, marrow progenitor values were normal only for colony-forming unit granulocyte macrophage (CFU-GM) in contrast to colony-forming unit-erythroid (CFU-E), burst-forming unit-erythroid (BFU-E), and colony-forming unit-megakaryocyte (CFU-Meg). After transplantation, as described with allogenic grafts, these values remained low for several years, although PBC counts were nearly normalized within a few weeks. Pregraft values were reached after 2 years for CFU-GM and BFU-E, and after 4 years for CFU-E, while CFU-Meg failed to reach pregraft values after this time. Normal levels were reached after 4 years only by CFU-GM. On univariate and multivariate analysis, the following factors appeared to delay both PBC and marrow progenitor reconstitution: underlying disease (particularly acute myeloid leukemias), graft characteristics such as low stem cell content and in vitro purging, conditioning regimens with total body irradiation or busulfan, and lack of postgraft administration of growth factors. In conclusion, high-dose therapy followed by bone marrow transplantation induces a deep and prolonged impairment of hematopoiesis irrespective of any alloimmune reaction or postgraft immunosuppressive therapy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma/serum levels of flt3 ligand are low in normal individuals and highly elevated in patients with Fanconi anemia and acquired aplastic anemia

The flt3 ligand is a growth factor that stimulates the proliferation of hematopoietic progenitor and stem cells. We established a sensitive enzyme-linked immunosorbent assay (ELISA) to measure the concentration of flt3 ligand in plasma or serum from normal individuals, as well as in patients with hematopoietic disorders. Concentrations of flt3 ligand in plasma or serum from normal individuals were quite low: only 12% (7 of 60) of normal individuals had flt3 ligand levels above 100 pg/mL (the limit of detection). In contrast, 86% (19 of 22) of samples from patients with Fanconi anemia and 100% (eight of eight) of samples from patients with acquired aplastic anemia had plasma or serum levels above 100 pg/mL. Mean plasma or serum concentrations (calculated by assigning a value of 0 pg/mL to any sample reading below the level of detection) were as follows: normal volunteers, 14 pg/mL; patients with Fanconi anemia, 1,331 pg/mL; and patients with acquired aplastic anemia, 460 pg/mL. Concentrations of flt3 ligand in blood are, therefore, specifically elevated to a level that may be physiologically relevant in hematopoietic disorders with a suspected stem cell component. The elevated flt3 ligand concentrations in these individuals may be part of a compensatory hematopoietic response to boost the level of progenitor cells.     

Identification of a novel thrombin receptor sequence required for activation-dependent responses

Thrombin receptor (TR) activation by alpha-thrombin requires proteolytic cleavage, although synthetic peptides modeled after the new N-terminus directly effect receptor activation without cleavage, presumably by interacting with an unidentified region of the receptor. To further define critical residues responsible for receptor activation, we performed epitope mapping of anti-TR1-160, a previously described polyclonal antibody that inhibits peptide ligand-induced receptor activation in various cell types expressing a functional TR. An enzyme-linked immunosorbent assay (ELISA) using overlapping decapeptides derived from the TR extracellular domains identified four immunodominant peaks within the long N-terminal extension centered between amino acids 34-44, 48-67, 65-79, and 87-94. Soluble peptides derived from regions 83-94, but not those from other regions of the receptor, neutralized the ability of anti-TR1-160 to inhibit peptide ligand-induced platelet aggregation, suggesting that antibodies directed against this region of the TR are important in ligand-mediated activation. Thrombin receptor mutants lacking discrete regions of the TR were subsequently evaluated using microinjected Xenopus oocytes. Whereas a TR mutant lacking amino acid residues Thr67-Lys82 (TR delta 67-82) showed normal to exaggerated responses to either alpha-thrombin or synthetic peptide ligands, only TR mutants with limited deletions spanning the residues Gln83-Ser93 exhibited dysfunctional responses to either agonist (200 nmol/L alpha-thrombin or 200 mumol/L TR42-47). These data provide a model for receptor activation that implicates a discrete and previously uncharacterized sequence within the TR N- terminal extension that is necessary for initiation of signal transduction events independent of the initiating agonist.     

A monoclonal antibody against an erythrocyte ontogenic antigen identifies fetal and adult erythroid progenitors

A murine monoclonal antibody (MoAb) designated FA6-152 has been obtained by immunizing mice with fetal erythrocytes. This antibody agglutinates fetal but not adult erythrocytes. Among blood cells, this antibody bound to both adult and fetal monocytes, platelets, and reticulocytes, but did not react with lymphocytes and granulocytes. Fluorescent labeling of marrow cells and of in vitro BFU-E, CFU-GM, and CFU-MK-derived colonies has shown that the antigen defined by FA6-152 MoAb was absent from the granulocytic precursors and was detected on the megakaryocytic lineage at a later stage of differentiation than the platelet-specific markers. In contrast, the antigen appeared as a very early marker of the erythroid differentiation since all erythroblasts, including proerythroblasts, were labeled even before the expression of glycophorin A. Cells from adult marrow and fetal liver were sorted with the FA6-152 MoAb and studied by electron microscopy and cell culture. The negative fraction contained granulocytic, monocytic, and megakaryocytic precursors, whereas the positive fraction was devoid of these precursors and contained monocytes, erythroblasts at all stages of maturation, and a homogeneous population of blasts. Cultures have shown that the only hematopoietic progenitors present in this positive fraction were CFU-E and some BFU-E. The antigenic density was related to the differentiation stage of the erythroid progenitors. In conclusion, this antibody is similar to the previously described 5F1 MoAb (Bernstein and Andrews, J Immunol 128:876, 1982; and Andrews et al, Blood 62:124, 1983) and provides a useful probe for studies leading to improved understanding of normal and malignant erythroid differentiation.     

Basic fibroblast growth factor counteracts the suppressive effect of transforming growth factor-beta 1 on human myeloid progenitor cells

We have previously shown that basic fibroblast growth factor (bFGF) is mitogenic for human bone marrow stromal cells and enhances myelopoiesis in human long-term bone marrow culture. In the present study, we examined the mechanism by which bFGF enhances granulopoiesis. We observed that bFGF significantly abrogated the inhibitory effect of transforming growth factor-beta 1 (TGF-beta 1) on granulocyte- macrophage colony-stimulating factor (GM-CSF)-supported progenitor cell growth (P = .009). The partial reversal of TGF-beta 1-mediated suppression was dependent on the dose of bFGF used. In addition, we noted that the inclusion of neutralizing antibody to TGF-beta 1 significantly augmented the clonogenic response to GM-CSF. We have also shown that 10 ng/mL or 100 ng/mL of bFGF resulted in a 30% to 100% increase in GM-CSF-mediated progenitor cell growth (P = .0001). These data suggest that bFGF may enhance myelopoiesis by modulating the inhibitory response to TGF-beta 1.     

Characterization of human marrow stromal cells: role in progenitor cell binding and granulopoiesis

Adherent cell layers and their associated extracellular matrices form when human marrow is incubated in cultures containing hydrocortisone and horse serum. These stromal layers contain cells positive for alkaline phosphatase; secrete collagens types I and III and fibronectin, bind the anti-actin monoclonal antibodies (MoAbs) HHF and CGA-7; stain with oil red O, and express the acetylated LDL receptor. Highly purified CD34 (My10)-positive progenitor cells attach to these stromal layers, and a 16-fold enrichment of CFU-GM in both stromal attachment and semisolid agar assays was observed. Granulopoiesis persisted up to 40 days (mean duration 25 days) after passaged stroma were recharged with stromal cell-depleted target cells in a two-stage liquid marrow culture system. Although equal to marrow fibroblasts in their ability to bind CD34+ myeloid progenitors, stromal layers were better at supporting granulopoiesis. This system provides an in vitro model to characterize the components of stroma and stroma-cytomatrix that enhance marrow progenitor cell localization and maintenance.     

Reversal of acute ("malignant") myelosclerosis by allogeneic bone marrow transplantation

A 28-yr-old woman with acute malignant myelosclerosis received, as primary treatment, ablative chemotherapy and total body radiation therapy followed by bone marrow transplantation from her histocompatible brother. The patient is now well more than 15 mo after bone marrow transplantation, with normal peripheral blood counts, a normal bone marrow, no evidence of graft-versus-host disease, and is on no therapy. In light of the poor results obtained with conventional chemotherapy in this disease, bone marrow transplantation may represent the treatment of choice for patients who have an appropriate donor.     

Monoclonal antibodies to the 140,000 mol wt glycoprotein of B lymphocyte membranes (CR2 receptor) initiates proliferation of B cells in vitro

Several mouse monoclonal IgG antibodies (AB1, AB2, AB3, and AB5) were developed that reacted with a 140,000 mol wt glycoprotein on the surface of cultured RAJI B lymphoid cells. The antibodies reacted with purified normal human peripheral blood B cells and CLL Ig+ B cells and showed specific germinal center and mantle zone staining in tissue sections of secondary lymphoid organs. Immunodepletion studies using 125I surface-labeled Raji cell membrane antigens demonstrated that the antigen identified by AB5 is the same 140,000 mol wt glycoprotein detected by anti-B2 that has recently been shown to react with the C3d fragment or CR2 receptor. (Iida et al: J Exp Med 158:1021, 1983). Addition of the AB series and anti-B2 monoclonal antibodies to cultures of purified human peripheral blood B cells resulted in the uptake of 3H- thymidine at two to six times background control levels provided that irradiated autologous T cells were added to the culture. Stimulation was not evoked by other monoclonal antibodies to B cell surface molecules (ie, B1, BA-1, BA-2, and HLA-DR). Pepsin-generated F(ab')2 fragments of anti-CR2 antibodies were essentially as effective as the intact IgG molecule in stimulating B cells. Induction of B cell proliferation by antibody binding to CR2 suggests that the C3d receptor may have an integral role in regulation of humoral immune response.     

健康成人血浆中氧化还原巯基水平与早期动脉粥样硬化之间的关系

背景与目的氧化应激是血管疾病发病机制中重要的病原学因素。我们假设:氧化应激可以预测健康人群中的早期动脉粥样硬化。为此,本研究调查了氧化应激标志物和早期动脉粥样硬化之间的关系。方法用超声检测140个不吸烟、无动脉粥样硬化的健康者颈动脉内膜中膜厚度。通过测量血浆中