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Biomedical subjects

G Wagemaker

Publications and source records attributed to G Wagemaker.

At least 55 records · Page 3Linked to original sources
PubMed1995

Surface markers and growth factor receptors of immature hemopoietic stem cell subsets.

The regenerative potential of bone marrow following exposure to relatively high doses of ionizing radiation, as well as the efficacy of hemopoietic growth factor treatment, are dependent on the residual number of hemopoietic stem cells. From studies in mice in particular, evidence has been obtained that immature hemopoietic stem cells are heterogenous with respect to repopulating capacity, with one subset being capable of short-term, transient hemopoietic reconstitution and another subset of sustained reconstitution. In rhesus monkeys, CD34+, RhLA-DRdull cells were identified as the small fraction of a bone marrow cell that contains reconstituting hemopoietic stem cells. The growth factor receptor phenotype of this immature cell fraction has been determined for granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and IL-6 as well as for kit-ligand, making c-kit an especially strong growth factor receptor marker for reconstituting stem cells. In addition, it is demonstrated that CD34+ cells appear in peripheral blood after exposure to radiation and are correlated to numbers of CD34+ cells in bone marrow. This finding suggests that circulating CD34+ cells may be used as a cellular marker with prognostic significance for both the number of residual stem cells as well as regeneration of immature hemopoietic cells in bone marrow.

Animals↗
PubMed1995

Conditions for engraftment of human acute myeloid leukemia (AML) in SCID mice.

Transplantation of human AML into severe combined immunodeficient (SCID) mice provides a useful experimental model but graft failures have been reported. We investigated the influence of a number of factors on the outgrowth of AML in the SCID mouse bone marrow (BM). The transplantation route and total body irradiation (TBI) were examined using the cell line HL-60 as a model for AML. The role of graft size and recombinant human IL-3 (IL-3) were investigated with patient samples of AML cells. Intravenous transplantation was demonstrated to be superior to intraperitoneal transplantation. Pretransplant conditioning resulted in a dose-dependent increase of AML growth in the SCID mouse. Cell dose titrations ranging from 3 x 10(7) - 3.6 x 10(5) AML cells i.v. per mouse revealed a minimum of 1.1 x 10(6) required for reproducible engraftment. Earlier and more extensive infiltration by human AML cells was seen following injection of greater cell numbers. IL-3 given post-transplantation SCID mouse recipients, promoted AML growth in three cases, whereas a fourth AML cell specimen also grew without support of IL-3. In vitro growth factor responsiveness of AML cells to IL-3 did not predict IL-3 dependence of AML growth in vivo.

Acute Disease↗
PubMed1994

Distribution of receptors for granulocyte-macrophage colony-stimulating factor on immature CD34+ bone marrow cells, differentiating monomyeloid progenitors, and mature blood cell subsets.

Biotin-labeled granulocyte-macrophage colony-stimulating factor (GM-CSF), in combination with phycoerythrin-conjugated streptavidin, enabled flow cytometric analysis of specific cell-surface GM-CSF receptors on rhesus monkey bone marrow (BM) and peripheral blood (PB) cells. GM-CSF receptors were readily detected on PB monocytes and neutrophils, but not on lymphocytes. In BM, GM-CSF receptors were identified on monocyte and neutrophil precursors and on subsets of cells that expressed the CD34 antigen. CD34+ cells with high GM-CSF-receptor expression coexpressed high levels of the class II major histocompatibility antigen RhLA-DR, whereas CD34+/RhLA-DRlow cells, which represent developmentally earlier cells, were either GM-CSF-receptor negative or expressed GM-CSF receptors at very low levels. The fluorescence histogram of CD34bright/RhLA-DRdull cells stained with biotin-GM-CSF showed that at least a fraction of these cells expressed low levels of GM-CSF receptors. CD34+ cells with high GM-CSF-receptor expression, purified by cell sorting, did not form colonies in culture or proliferate in response to GM-CSF. Instead, GM-CSF stimulation resulted in terminal differentiation into adherent cells, showing that these cells represented monocyte precursors. A distinct subset of CD34+ cells expressed GM-CSF receptors at low-to-intermediate levels and proliferated strongly in the presence of GM-CSF during short-term culture, but produced very few erythroid or monomyeloid colonies after longer culture periods. Most colony-forming cells, also those responsive to GM-CSF alone, were recovered in the subset of CD34+ cells on which GM-CSF receptors were virtually undetectable. These cells showed weaker proliferation in short-term proliferation assays than the CD34+/GM-CSF-receptor-intermediate cells, consistent with an immature phenotype. The results show that GM-CSF-receptor expression is initiated in a subset of immature, CD34bright/RhLA-DRdull cells and is progressively increased during differentiation into mature granulocytes and monocytes. The method used provides a new way to deplete developmentally early CD34+ cell of differentiating granulocyte and monocyte precursor cells.

Animals↗
PubMed1994

Stable multilineage hematopoietic chimerism in alpha-thalassemic mice induced by a bone marrow subpopulation that excludes the majority of day-12 spleen colony-forming units.

We have investigated the contribution of highly purified day-12 spleen colony-forming units (CFU-S-12) as well as more primitive cells to sustained blood cell production using in vivo and in vitro assays that allow frequency analysis. Normal or day-6 post-5-fluorouracil light-density bone marrow (BM) was sorted on the basis of differences in rhodamine-123 (Rh123) retention or wheat germ agglutinin (WGA) affinity and tested in vivo using a recently developed alpha-thalassemic chimeric mouse model. In addition, short-term and long-term clonal activity was assessed in vitro using a limiting dilution-type long-term BM culture, the cobblestone area forming cell assay. When sublethally irradiated alpha-thalassemic mice were transplanted with as many as 281 purified WGAbright CFU-S-12, derived from a fraction containing 95% of all CFU-S-12 from day-6 post-5-fluorouracil light-density BM of wild-type mice, detectable chimerism was not observed at 6 months posttransplantation. In contrast, only three CFU-S-12 were included in the Rh123dull and WGAdim subpopulations that induced 29% to 58% and 21% to 31% stable multilineage donor-type chimerism of erythrocytes and leukocytes, respectively. The Rh123dull and WGAdim cells were up to 240-fold enriched for long-term repopulating ability (LTRA) as compared with unseparated BM. A comparable level of chimerism was found in the different hematopoietic organs and at the level of BM CFU-S-12. The frequency of the LTRA unit capable of inducing a 10% sustained level of donor-type erythrocytes was calculated to be 1 to 2 per 10(5) BM cells. Several reports have suggested that LTRA and spleen colony formation could be capacities of the same stem cell subset. However, the present results show that the majority of CFU-S-12 have only short-term repopulating ability and are physically separable from more primitive stem cells with long-term multilineage reconstituting capacities.

Animals↗
PubMed1994

Cloning and expression of interleukin-3 genes of chimpanzee and New World monkeys.

Interleukin-3 (IL-3) genes were cloned from chimpanzee (Pan troglodytes), tamarin (Saguinus oedipus) and marmoset (Callithrix jacchus) and expressed in COS cells. Although the IL-3 gene structure is well conserved in these primate species, sequence analysis revealed extensive base substitutions. The chimpanzee IL-3 protein, which is highly homologous (98.5% identity) to human IL-3, stimulated proliferation of human cells dependent on IL-3. In contrast, due to the numerous amino acid substitutions in the New World monkey IL-3 species, no stimulation of human cells was observed, illustrating the extensive evolutionary divergence of IL-3.

Amino Acid Sequence↗
PubMed1994

Molecular evolution of interleukin-3.

Chimpanzee, tamarin, and marmoset interleukin-3 (IL-3) genes were cloned, sequenced, and expressed. Western blot analysis demonstrated that functional genes were isolated. IL-3 sequences were compared with those of mouse, rat, rhesus monkey, gibbon, and man. Multiple alignment of the IL-3 coding regions showed that only a few regions had been conserved during mammalian evolution, which are likely associated with functional domains of the IL-3 protein. Substitution rates for the various lineages were calculated and the numbers of synonymous and nonsynonymous substitutions were estimated separately. Distance matrices of the IL-3 coding regions were used to construct phylogenetic trees which revealed large differences in IL-3 evolution rate as well as a more rapid substitution rate for rodents and a rate slowdown during hominoid evolution. Extremes were rhesus monkey IL-3, which accumulated few synonymous substitutions, and gibbon IL-3, which had almost exclusively synonymous substitutions. In rhesus monkey IL-3, nonsynonymous substitutions outnumbered synonymous substitutions, which could not be readily explained by a random process of substitutions. We assume that during evolution of IL-3, the majority of the amino acid replacements and the impaired interspecies functional cross-reactivity originate from selection mechanisms with the most likely selective force being the structure of the heterodimeric IL.3 cell-surface receptor. Insight into IL-3 architecture and structural analysis of the IL-3 receptor are needed to analyze the unusually fast evolution of IL-3 in more detail.

Animals↗
PubMed1994

Bone marrow transplantation has a significant effect on enzyme levels and storage of glycosaminoglycans in tissues and in isolated hepatocytes of mucopolysaccharidosis type VII mice.

The effect of bone marrow transplantation (BMT) on enzyme and glycosaminoglycan levels of various tissues and isolated parenchymal cells of lethally irradiated gusmps/gusmps mice was studied. These mice have an inherited deficiency of the lysosomal enzyme beta-glucuronidase with less than 1% of normal enzyme activity present in all tissues and represent a model of human mucopolysaccharidosis type VII. Tissues were evaluated 200 d after BMT and liver parenchymal cells 300 d after BMT. Normal levels of beta-glucuronidase activities were present in spleen and peripheral blood leukocytes of gusmps/gusmps mice that underwent transplantations. Intermediate activities were found in lung (73%), kidney (4%), liver (10%), heart (53%), muscle (55%), brain (6%), and liver parenchymal cells (10% of normal controls). A concomitant decrease in activity of the secondarily increased enzyme beta-hexosaminidase was observed. BMT also led to a substantial reduction in storage of glycosaminoglycans in lung (130 to 100%), heart (350 to 106%), kidney (439 to 217%), brain (177 to 91%), liver (613 to 125%), and liver parenchymal cells (443 to 161% of normal controls). These findings were supported by electron microscopy. A normalization of the storage process was seen in the visceral organs spleen and liver and in the histiocytes of the heart. The kidney showed variable improvement depending on the cell type. In the brain, a substantial improvement of neuronal storage was observed, but BMT apparently had no effect on storage in glial cells. The subcellular localization of beta-glucuronidase was investigated in liver parenchymal cells of mice that underwent transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Identification of functional domains of interleukin-3 by construction of primate interspecies chimera.

Interleukin-3 (IL-3) is involved in regulation of proliferation and differentiation of multipotent hemopoietic cells and stimulates the production of most blood cell types. The observed functional specificity across species concurs with an extreme rate of IL-3 amino acid substitutions during mammalian evolution. Tamarin IL-3 exhibited 70.5% sequence identity with human IL-3 and was severely impaired in supporting proliferation of human IL-3-dependent cells. In contrast, chimpanzee IL-3 displayed high amino acid sequence homology (98.5%) and could substitute for human IL-3. A panel of interspecies chimera between the chimpanzee and tamarin IL-3 genes has been constructed and expressed in Escherichia coli and eukaryotic cells to investigate the role of substitutions in different protein domains on the functional species specificity. Our analyses show that substitutions at residues encoded by the first two exons appear crucial in the functional species specificity, whereas C-terminal alterations show only moderate effects.

Amino Acid Sequence↗
PubMed1994

Interleukin-3 (IL-3) receptors on rhesus monkey bone marrow cells: species specificity of human IL-3, binding characteristics, and lack of competition with GM-CSF.

The relative affinity of recombinant human interleukin-3 (IL-3) binding to normal rhesus monkey bone marrow cells was found to be 25- to 50-fold less than that of homologous IL-3, which explained the species specificity of human IL-3 observed when tested in Macaca species. In contrast, only a small difference was found between human and rhesus monkey IL-3 in relative binding affinity for receptors on human acute myelogenous leukemia (AML) cells, which confirmed that the species specificity of IL-3 is largely unidirectional. The biological significance of the findings was demonstrated by direct in vivo comparison of the effects of high-dose recombinant rhesus monkey and human IL-3. The binding characteristics of IL-3 receptors on rhesus monkey bone marrow and peripheral blood cells were further characterized by specific binding of radiolabeled rhesus monkey IL-3. Scatchard analysis of two bone marrow samples demonstrated high-affinity IL-3 receptors (25 and 80 sites/cell, respectively; equilibrium dissociation constants [Kd] of 8 and 3 pM/L) as well as low-affinity receptors (1070 and 1290 sites/cell; equilibrium dissociation constants of 2600 and 1200 pM/L). In addition, IL-3 receptor expression was also detected on purified CD34-positive bone marrow cells. Competition by human granulocyte-macrophage colony-stimulating factor (GM-CSF) of IL-3 binding to high- or low-affinity receptors on rhesus monkey peripheral blood and bone marrow cells could not be demonstrated, which may indicate that the growth factor-specific alpha-subunits of the GM-CSF and IL-3 receptors are expressed predominantly on different cell types.

Animals↗
PubMed1994

Neutralizing antibodies during treatment of homologous nonglycosylated IL-3 in rhesus monkeys.

During administration of homologous nonglycosylated IL-3 to rhesus monkeys, reversal of hematologic effects and disappearance of side effects suggested a neutralizing anti-IL-3 antibody response. Among a total of 20 monkeys treated with IL-3, ELISA of serial serum samples revealed anti-IL-3 antibodies in ten animals. Antibody production tended to be dose dependent. Triplicate subcutaneous injections and i.v. administration provoked earlier appearance of antibodies than single s.c. injection. Prolonged continuous intravenous IL-3 administration (63 and 93 days) at a dose of 1 microgram/kg/day did not result in antibody production. Among a total of eight animals with sufficiently high titers to allow for antibody purification, seven appeared to have generated antibodies that neutralized the biologic activity of IL-3 in vitro. In six monkeys, the response to IL-3 decreased while antibody titers rose, strongly suggesting neutralization of IL-3 in vivo. It is concluded that recombinant, nonglycosylated IL-3 as used in this study may elicit a neutralizing antibody response.

Animals↗
PubMed1994

Selective advantage of normal erythrocyte production after bone marrow transplantation of alpha-thalassemic mice.

Anemia resulting from alpha-thalassemia in mice was corrected by transplantation of normal bone marrow cells following sublethal total body irradiation, resulting in partial hematopoietic chimerism with a preponderance of normal peripheral blood red cells. Peripheral blood red cell chimerism in recipients of graded numbers of bone marrow cells from sex-mismatched donors, determined by cytometric analysis, was directly compared with immature hematopoietic cell (CFU-S) chimerism and peripheral blood white cell chimerism. The latter two were assessed by fluorescent in situ hybridization with a murine Y-chromosome-specific probe. Peripheral blood white cell chimerism consistently corresponded with immature hematopoietic cell chimerism, emphasizing the selective advantage of normal red cell production in partially chimeric alpha-thalassemic mice.

Animals↗
PubMed1993

Occurrence of a soluble nonspecific suppressor factor in the serum early after birth.

A nonspecific suppressor factor has been identified in serum of newborn rats and calves. This factor, designated SUF-s, was shown to interfere--across species barriers--with lymphocyte responses in vitro and in vivo. SUF-s interferes in vitro with T- and B-cell proliferation induced by different mitogens and IL-2. Our findings indicate that the activity of SUF-s in vitro, which is of a reversible nature, is directed at an early event in the cascade of T-cell activation. SUF-s does not affect intrinsically regulated proliferation, such as that of tumor cells or established cell lines. In vivo, SUF-s prevents graft-vs-host disease induced by transplantation of allogeneic bone marrow cells in lethally irradiated mice. Using of affinity chromatography, hydrophobic interaction chromatography, and gel filtration, a 15,000-fold purification of the suppressive factor was attained. The moiety engaged in suppression was identified as a 20- to 40-kDa protein. The biological activity is destroyed at temperatures above 70 degrees C, by proteolytic enzyme digestion and under alkaline conditions but was resistant to acidic and reducing conditions. Judged by its biological activity and some of its physical properties, SUF-s is most likely distinct from other described suppressor factors or known cytokines with suppressor activity, such as IL-4, IL-10, interferon-gamma, transforming growth factor-beta or alpha-fetoprotein.

Aging↗
PubMed1993

Flow cytometric detection of receptors for interleukin-6 on bone marrow and peripheral blood cells of humans and rhesus monkeys.

The expression of receptors for interleukin-6 (IL-6) on human and rhesus monkey peripheral blood and bone marrow (BM) cells was examined by multiparameter flow cytometry after staining with biologically active, biotin-labeled human IL-6 and phycoerythrin-conjugated streptavidin. Consistent with the multiple biologic effects of IL-6 in stimulating immune functions and hematopoiesis, IL-6 receptors were detectable on a wide variety of cell types. In peripheral blood, IL-6 receptors were detectable on monocytes, granulocytes, and on CD4+ T lymphocytes but not on resting, CD19+ B lymphocytes and CD56+ natural killer (NK) cells. CD8+ T lymphocytes also expressed IL-6 receptors but at lower levels than CD4+ cells. The IL-6 receptors on granulocytes were only detectable after staining with high concentrations of biotin-IL-6, suggesting that most IL-6 receptors on these cells represent low-affinity sites. In contrast, IL-6 receptors on both CD4+ and CD8+ T lymphocytes were detectable at biotin-IL-6 concentrations as low as 10 pmol/L, indicating that these cells bind IL-6 with high affinity. IL-6 receptor expression patterns on rhesus monkey and human blood cells were very similar except that receptor levels on granulocytes were lower in humans than in rhesus monkeys. Similar differences in expression levels were observed for IL-6 receptors that were detectable on most granulocyte precursors in the mononuclear fraction of rhesus monkey and human bone marrow. In addition to these relatively mature cell types, IL-6 receptors were detectable on a large fraction of human and rhesus monkey BM blast cells that express the CD34 antigen. The presence of IL-6 receptors on CD34+ BM blast cells, which are the precursor cells of most, if not all, BM-derived blood cells, is consistent with the ability of IL-6, in conjunction with other cytokines, to stimulate immature hemopoietic cells in vitro and to promote blood cell production when administered in vivo.

Animals↗
PubMed1993

Acute side effects of homologous interleukin-3 in rhesus monkeys.

Interleukin-3 treatment of juvenile rhesus monkeys elicits a dose- and time-dependent syndrome that includes urticaria, palpable lymph nodes, splenomegaly, thrombocytopenia, anemia, vomiting, diarrhea, intestinal bleeding, edema, and arthritis, apart from a strong stimulation of hemopoiesis. Arthritis was found to occur significantly more often in animals expressing the major histocompatibility complex alleles B9 and Dr5. Histological analysis revealed an abundance of mast cells in urticaria and, to a lesser extent, in lungs and synovia of arthritic joints. Active osteoclasts were abundant in ribs and arthritic joints. Extramedullary hemopoiesis was encountered in liver, spleen, and kidneys. The spleen showed deposits of hemosiderin, and in the liver, Kupffer cells were loaded with iron, indicating enhanced turnover of hemoglobin. Lymph nodes and bone marrow showed macrophages involved in hemophagocytosis, which probably contributed to the development of anemia and thrombopenia. Biochemical parameters in sera were indicative of parenchymal liver damage, with cholestasis and increased erythrocyte destruction. The side effects were strongly reduced in monkeys subjected to total body irradiation just before interleukin-3 treatment. Histamine antagonists were not significantly effective in preventing side effects, which is explained by the perpetual stimulation of basophilic granulocytes by exogenous interleukin-3. The nature of the side effects indicates that interleukin-3 may be involved in the pathogenesis of acute type hypersensitivity reactions and arthritis.

Anemia↗
PubMed1993

Correction of murine beta-thalassemia by partial bone marrow chimerism: selective advantage of normal erythropoiesis.

beta-Thalassemic mice were transplanted with normal congeneic BM cells after sublethal total body irradiation, which resulted in partial RBC chimerism and correction of anemia. Enumeration of donor-type early hemopoietic progenitor cells (CFU-S) demonstrated that the correction of anemia originated from a minority of normal immature BM cells. It is concluded that successful BMT in beta-thalassemia does not necessarily require ablation of endogenous BM.

Animals↗
PubMed1993

Pharmacokinetic basis for optimal hemopoietic effectiveness of homologous IL-3 administered to rhesus monkeys.

To design an interleukin-3 (IL-3) administration schedule for optimal hemopoietic effectiveness, serum half-life (t1/2) was determined after intravenous (i.v) and subcutaneous (s.c.) bolus injections. The initial t1/2 in serum after i.v. injection was about 10 minutes and the terminal t1/2 close to 2 hours. Subcutaneous administration resulted in plateau levels after 2 to 4 hours, while the apparent terminal t1/2 was similar to that after i.v. infusion. The bioavailability of IL-3 following subcutaneous administration was only about 40% of that following i.v. administration. Hemopoietic effects of continuous i.v. infusion of IL-3 was then compared to s.c. administration in either one, two, or three daily injections. Doses chosen ranged from 1 to 30 micrograms/kg per day. In agreement with the more limited bioavailability of IL-3 following s.c. administration, continuous i.v. infusion was much more effective in stimulating hemopoiesis than s.c. administration. Two or three daily s.c. injections did not improve the hemopoietic response compared to a single s.c. injection, which is in agreement with the apparent terminal t1/2 of 101 min. It is concluded that IL-3 is more effective by continuous i.v. infusion than by subcutaneous administration.

Animals↗
PubMed1993

Compensatory splenic hemopoiesis in beta-thalassemic mice.

beta-Thalassemic mice, homozygous for the deletion of the beta major-globin gene, were investigated for compensatory hemopoiesis in bone marrow and spleen. Apart from characteristic severe anemia, these mice have a marked granulocytosis, monocytosis and lymphocytosis. A large compensatory expansion of late (CFU-E) erythroid progenitor cells was demonstrated, predominantly in the spleen. Immature hemopoietic cells (CFU-S) were also expanded, as were early progenitor cells of erythroid (BFU-E), as well as granulocyte/macrophage (GM-CFU) and megakaryocytic (CFU-Meg) lineages. It is concluded that the persistent erythropoietic stress results in a selective expansion of immature hemopoietic cells and inappropriate production of nonerythroid blood cells from excess production of progenitor cells.

Animals↗