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Biomedical subjects

G Wagemaker

Publications and source records attributed to G Wagemaker.

At least 19 recordsLinked to original sources
PubMed1992

Flow cytometric analysis of peripheral blood erythrocyte chimerism in alpha-thalassemic mice.

A rapid and reliable method for longitudinal studies on the degree of red cell chimerism following bone marrow transplantation of alpha-thalassemic recipient mice is presented. Blood obtained by tail clipping from transplanted mice was analyzed by measuring forward light scatter (FLS) distribution of red cells using a flow cytometer. Amplification and threshold of FLS were specifically adjusted. For flow cytometric analysis, the red cells needed to be suspended in hypotonic saline (103 mmol/l NaCl). Osmotic fragility testing showed that lysis of erythrocytes did not significantly influence the measurements. Flow cytometric measurement allowed for a rapid determination of the degree of red cell chimerism.

Animals↗
PubMed1992

Validation of a serum-free growth factor-replenished in vitro culture system for hematopoietic progenitor cells in healthy donors and recipients of an allogeneic bone marrow graft.

The in vitro colony formation of hematopoietic progenitor cells of bone marrow samples, taken before and early after allogeneic bone marrow transplantation (BMT), was investigated prospectively. In order to circumvent culture-related and sample-related variations, a serum-free recombinant growth factor-replenished culture system was developed using T cell- and monocyte-depleted bone marrow samples. Samples of healthy bone marrow donors were used to validate the technique. The standardized culturing technique gave reproducible results, with numbers of colonies above those in conventional conditioned-medium technique. Colony formation in vitro of myelomonocytic precursor cells was found decreased in graft recipients, also after addition of growth factors, in comparison with healthy donors. The growth-promoting effect of the combination of IL-3 + GM-CSF was superior to that of either growth factor alone or conditioned medium. No effect was observed of T lymphocytes and monocytes on in vitro colony formation after bone marrow transplantation, probably as a result of functional impairment of these cells at that period after transplantation.

Bone Marrow Cells↗
PubMed1991

Receptor and antibody interactions of human interleukin-3 characterized by mutational analysis.

Human interleukin-3 (hIL-3) is a regulator of proliferation and differentiation of multipotent hemopoietic progenitor cells. Mutants of hIL-3 have been constructed by oligonucleotide-directed mutagenesis and expressed in Escherichia coli and Bacillus licheniformis. Purified muteins were assayed for induction of DNA synthesis in IL-3-dependent human cells and for binding to the IL-3 receptor. Residues at the NH2 and COOH termini together comprising one-quarter of the molecule could be removed without loss of biological function. Deletions of 6-15 residues within the central part of the molecule caused a large reduction (up to 5 logs) but no complete loss of activity. Substitution of evolutionary conserved residues resulted in a strong decrease of biological activity and demonstrated that the S-S bridge is an essential structural element in hIL-3. Interestingly, four muteins displayed a significantly higher potency of binding to the IL-3 receptor than in stimulating DNA synthesis. These results demonstrate that receptor binding may be (partly) disconnected from activation of DNA synthesis. Analysis of hIL-3 muteins demonstrated that the majority of monoclonal antibodies are directed against a small portion of the IL-3 molecule. The neutralizing potential of individual monoclonal antibodies could be increased by a combination of antibodies directed against nonoverlapping epitopes.

Amino Acid Sequence↗
PubMed1991

In vivo production of interleukin-5, granulocyte-macrophage colony-stimulating factor, macrophages colony-stimulating factor, and interleukin-6 during intravenous administration of high-dose interleukin-2 in cancer patients.

Recombinant human interleukin-2 (IL-2), administered to cancer patients by continuous intravenous (IV) infusion (3 x 10(6) U/m2/d), was found to induce the in vivo production of colony-stimulating factors (CSF). Plasma obtained from patients during IL-2 treatment stimulated in vitro colony formation of normal human bone marrow cells, depleted of mononuclear phagocytes and T lymphocytes. This colony-stimulating activity (CSA) was identified as IL-5, granulocyte-macrophage CSF (GM-CSF), and macrophage CSF (M-CSF), by the ability of specific antibodies against these factors to neutralize their effects. The presence of IL-2-induced GM-CSF and M-CSF was also demonstrated by specific radioimmunoassays. During IL-2 treatment, plasma also contained detectable levels of IL-6, which was measured in a bioassay. Using a cDNA-polymerase chain reaction (PCR) with specific primer sets for the various CSF, we showed that IL-2 treatment induced the expression of mRNA for M-CSF, GM-CSF, IL-3, and IL-5, but not for granulocyte CSF (G-CSF) in peripheral blood mononuclear cells, suggesting differential expression of CSF in vivo in response to IL-2. Furthermore, no negative regulators of hematopoiesis, such as interferon gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha), were found in plasma. These data illustrate that in vivo administration of high-dose IL-2 may result in a stimulatory effect on hematopoiesis. The induction of detectable levels of IL-5 and GM-CSF in the circulation may explain the eosinophilia and neutrophilia observed in these patients.

Carcinoma, Renal Cell↗
PubMed1991

Production of human interleukin-3 using industrial microorganisms.

We expressed a cDNA encoding the multicolony stimulating factor interleukin-3 in a variety of cell types, including bacteria, yeast and mammalian cells. After evaluation of the advantages and disadvantages of each potential system, we designed a production and purification scheme using Bacillus licheniformis. The purification consists of hydrophobic interaction chromatography, two steps of ion exchange chromatography and gel filtration. The purified and formulated product entered clinical trials in November 1989.

Animals↗
PubMed1991

Impaired response of myelodysplastic marrow progenitors to stimulation with recombinant haemopoietic growth factors.

The regulation of haemopoiesis in myelodysplastic syndromes (MDS) was evaluated by measuring and comparing the in vitro response of marrow progenitors from 18 MDS patients to stimulation with recombinant haemopoietic growth factors (HGFs), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF) and interleukin-3 (IL-3). A similar pattern of colony growth was detected with all three HGFs in most MDS patients, exhibiting subnormal growth of GM-CFU and markedly poor to absent growth of BFU-E and CFU-GEMM. A common severe impairment in the growth of all colony types with all three HGFs was observed in five patients, four of whom presented with pancytopenia. The stimulation of MDS marrow progenitors with a five-fold higher than control saturating dose of HGFs induced a significant increase in the frequency of one, two, or all three colony types in cultures of 14 patients, whereas colony numbers in control (n = 8) marrow cell cultures were not significantly changed. All four of the non-responders were pancytopenic and three exhibited markedly impaired colony growth. Supersaturating GM-CSF, G-CSF and IL-3 increased GM-CFU numbers in six, three, and three patients, respectively. The values for BFU-E were three, six, and seven and for CFU-GEMM two, one, and five. The enhancement of MDS marrow colony numbers by supersaturating HGFs which exert their effects directly or via the action of marrow accessory cells, suggests that the progenitor cell growth abnormalities in these disorders may involve a defect in the capacity of accessory and/or progenitor cells to respond to stimulation with specific haemopoietic growth regulators.

Aged↗
PubMed1990

Species specificity of human interleukin-3 demonstrated by cloning and expression of the homologous rhesus monkey (Macaca mulatta) gene.

To enable preclinical studies on homologous interleukin-3 (IL-3) in primate species, we isolated the gene encoding Rhesus monkey IL-3 (RhIL-3). The nucleotide sequence of the RhIL-3 gene displayed 92.9% homology with that of the human IL-3 (hIL-3) gene. The isolated RhIL-3 gene encodes a 143-amino acid (aa) precursor polypeptide, nine C-terminal residues shorter than the human protein. Protein homology was found to be 89.5% for the signal peptide (19 aa) and 80.5% for the mature protein (124 aa). Comparison of the human and RhIL-3 coding sequences showed that the majority of substitutions had occurred at amino acid replacement sites indicating a rapid evolution of the IL-3 protein. After expression of a genomic fragment in COS cells, RhIL-3 cDNA was constructed, which enabled large-scale production of the RhIL-3 polypeptide, RhIL-3 produced by Bacillus licheniformis and purified to homogeneity appeared to be approximately 100-fold more effective in stimulating Rhesus monkey hematopoietic progenitors than hIL-3, whereas RhIL-3 and hIL-3 showed comparable stimulatory activity on normal as well as malignant human hematopoietic cells. Thus, the rapid evolution of hIL-3 has resulted in a unidirectional species specificity, which most likely restricts the in vivo effects of hIL-3 in Macaca species.

Amino Acid Sequence↗
PubMed1990

Highly increased production of bone marrow-derived blood cells by administration of homologous interleukin-3 to rhesus monkeys.

Recombinant rhesus monkey interleukin-3 (IL-3) was administered to normal rhesus monkeys in graded doses ranging from 3 to 30 micrograms/kg/d subcutaneously for 30 consecutive days or given as a continuous intravenous infusion at a dose of 30 micrograms/kg/d for 16 days. After a lag phase of about 1 week, a highly increased, dose-dependent production of bone marrow-derived blood cells was observed, preceded by amplification of bone marrow hematopoietic progenitor cells. Simultaneously, peripheral blood progenitor cells rose. The increase included basophilic, eosinophilic and neutrophilic granulocytes, monocytes, and the erythrocyte and platelet lineages. Characteristically, a T-lymphocyte response was absent. It is concluded that IL-3 in vivo stimulates blood cell production from an immature, multipotent progenitor cell.

Animals↗
PubMed1990

Interleukin-3.

Interleukin-3 (IL-3) is a hemopoietic growth factor involved in the survival, proliferation and differentiation of multipotent hemopoietic cells. In five mammalian species, including man, the gene encoding IL-3 has been isolated and expressed to yield the mature recombinant proteins. The human IL-3 gene encodes a protein of 133 amino acids with two conserved cysteine residues and 2 potential N-linked glycosylation sites; human native IL-3 has not been characterized. Comparison of the IL-3 genes revealed a more rapid evolutionary divergence than has been observed for other hemopoietic growth factors, and, hence, a more pronounced species specificity of the functional proteins was found. In agreement with its stimulatory action on immature multipotent cells, the in vivo actions of homologous recombinant IL-3 in nonhuman primates include a highly increased production of blood cells along the neutrophilic, eosinophilic and basophilic granulocyte as well as the monocyte, red cell and platelet lineages.

Animals↗
PubMed1990

Factors influencing reconstitution by bone marrow transplantation.

Reconstitution of levels of pheripheral blood cells following bone marrow transplantation is dependent on the engraftment of the pluripotent hemopoietic stem cells of the transplant. In the case of allogeneic transplants, a successful engraftment carries a high risk of the development of GvHD, which is a serious and often life-threatening complication. T cell depletion of the allogeneic bone marrow graft can completely prevent GvHD provided a sufficient degree of depletion is achieved. Experiments with laboratory animals and clinical experience have shown that the higher rate of graft failure that occurs with T cell depleted marrow grafts can be avoided by increasing the conditioning dose of TBI with approximately 2 Gy. Experiments with T cell depleted autologous bone marrow grafts and with grafts of autologous purified stem cells in monkeys demonstrate that the decreased engraftment of T cell depleted bone marrow cannot be ascribed to a trophic function of T lymphocytes, e.g. via production of hemopoietic growth factors. Using sublethally irradiated Rhesus monkeys, the effect of post-irradiation treatment with GM-CSF or with Rhesus monkey r IL-3 was studied. Enhancement of blood cell regeneration was only recorded within a relatively small TBI dose range. When the dose of TBI induces more than approximately a 3-log stem cell kill, treatment with growth factors becomes ineffective. Comparable results are obtained when supralethally irradiated monkeys given relatively small grafts of T cell depleted autologous bone marrow were treated with the hemopoietic growth factors.

Animals↗
PubMed1988

Donor-derived cells in the central nervous system of twitcher mice after bone marrow transplantation.

The twitcher mouse is an animal model of galactosylceramidase deficiency, comparable to Krabbe's disease, a lysosomal storage disease in humans. As in most lysosomal storage diseases, neurological deterioration is a prominent feature of the disease in these mice. Transplantation of enzymatically normal congenic bone marrow was earlier found to result in prolonged survival and increased levels of galactosylceramidase in the visceral organs of twitcher mice. It is now reported that bone marrow transplantation results in increased galactosylceramidase levels in the central nervous system (CNS). Concomitantly, the levels of psychosine, a highly toxic lipid that progressively accumulates in the CNS of untreated twitcher mice, stabilized at much lower levels in the CNS of treated twitcher mice. Histologically, a gradual disappearance of globoid cells, the histological hallmark of Krabbe's disease, and the appearance of foamy macrophages capable of metabolizing the storage product were seen in the CNS. By immunohistochemical labeling it was demonstrated that these foamy macrophages were of donor origin. The infiltration of enzymatically competent, donor-derived macrophages was accompanied by extensive remyelination in the CNS. It is concluded that after bone marrow transplantation, donor-derived macrophages infiltrate the affected brain tissue and are capable of inducing a partial reversal of the enzyme deficiency.

Animals↗
PubMed1988

The repopulation capacity of bone marrow grafts following pretreatment with monoclonal antibodies against T lymphocytes in rhesus monkeys.

Complement-mediated lysis of (subsets of) T lymphocytes in bone marrow grafts is increasingly used to prevent acute graft-versus-host disease in human bone marrow transplant recipients, especially in case of major immunogenetic disparity between donor and recipient. Since T lymphocyte depletion has resulted in an increased frequency of allogeneic engraftment failures, its effect on hemopoietic reconstitution was measured in rhesus monkeys. The reactivity patterns of commonly used types of antihuman T lymphocyte monoclonal antibodies (MCAs) with rhesus monkey lymphocytes was analyzed using a double-label cytofluorometry technique and found to be very similar to those with human lymphocytes. The antibodies investigated included CAMPATH-1 (recognizing an antigen present on virtually all lymphocytes and monocytes), OKT4 + 4a (CD4, helper/inducer T lymphocytes), B9 (CD8, suppressor/cytotoxic T lymphocytes), WT-1 (CD7, pan-T), and anti-DR MCAs as stem cell toxic controls. Their possible toxicity to hemopoietic stem cells was studied by using a semiquantitative autologous regeneration assay. Cytotoxic lysis of cells in the bone marrow grafts reacting with the T lymphocyte purging MCAs did not result in delayed regeneration compared to untreated autologous grafts. It is concluded that T lymphocyte depletion using anti-T-lymphocyte MCAs does not influence the repopulating capacity of an autologous bone marrow graft.

Animals↗
PubMed1988

Effect of bone marrow transplantation on enzyme levels and clinical course in the neurologically affected twitcher mouse.

The effect of allogeneic bone marrow transplantation (BMT) was investigated in the neurologically affected twitcher mouse, a model for human Krabbe's disease. Twitcher mice have a hereditary deficiency of the lysosomal enzyme galactosylceramidase, which causes growth delay, tremor, and paralysis of the hind legs. Death occurs at 30-40 d of age. After BMT galactosylceramidase activity increased to donor levels in hemopoietic organs. In lung, heart, and liver, galactosylceramidase activity rose to levels intermediate between those of twitcher and normal mice. Increased galactosylceramidase activity in liver parenchymal cells indicated uptake of the donor enzyme by recipient cells of nonhemopoietic origin. Enzyme activity also increased in kidney tissue. BMT resulted in a gradual increase in galactosylceramidase activity in the central nervous system to 15% of normal donor levels. A 5-6-fold increase in galactosylceramidase activity was found in the peripheral nervous system. This increase in enzyme activity was accompanied by a partial alleviation of neurological symptoms. In particular, paralysis of the hind legs was prevented by BMT. BMT led to a modest restoration of growth and prolonged survival. In several cases, the mice survived for more than 100 d, but eventually all animals died with severe neurological disease.

Animals↗
PubMed1988

Stimulating spectrum of human recombinant multi-CSF (IL-3) on human marrow precursors: importance of accessory cells.

Recently, human multi-CSF was obtained by molecular cloning. In the present study, the effects of multi-CSF in vitro were investigated by comparative culture of whole bone marrow or progenitor cells obtained by sorting the cell fraction that binds the monoclonal antibody (MoAb) B13C5 (CD 34). Multi-CSF stimulated erythroid (BFU-E), multipotential (CFU-GEMM) and eosinophil (CFU-Eo) colonies in cultures of the progenitor cell enriched fraction, whereas (besides BFU-E, CFU-GEMM, and CFU-Eo) granulocyte (CFU-G), granulocyte-macrophage (CFU-GM), and macrophage (CFU-M) colony-forming cells also were stimulated by multi-CSF when unfractionated bone marrow was cultured. Reconstitution of the progenitor cell fraction (B13C5 positive) with the B13C5-negative population restored the broad spectrum of progenitor cell stimulation. This suggested that accessory cells are required for expression of the full spectrum of progenitor cell stimulation by multi-CSF. Subsequently, specific marrow cell populations, including T lymphocytes, granulocytic cells, and monocytes, were prepared by using selected MoAbs in complement-mediated lysis or cell sorting, added to cultures of hematopoietic progenitors and tested for accessory cell function. The results demonstrate that small numbers of monocytes permit the stimulation of CFU-G, CFU-GM, and CFU-M by multi-CSF. These monocyte-dependent stimulating effects on CFU-G, CFU-GM, and CFU-M could also be achieved by adding recombinant GM-CSF as a substitute for monocytes to the cultures. Therefore, multi-CSF most likely has direct stimulative effects on BFU-E, CFU-GEMM, and CFU-Eo and indirect effects on CFU-G, CFU-GM, and CFU-M in the presence of monocytes.

Antigens, CD34↗
PubMed1988

Growth regulation of human acute myeloid leukemia: effects of five recombinant hematopoietic factors in a serum-free culture system.

The response of human acute myeloid leukemia (AML) cells to the distinct hematopoietic growth factors (HGFs), ie, recombinant interleukin-3 (IL-3), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF), and erythropoietin (Epo) was investigated under well-defined serum-free conditions. Proliferative responses to these factors, when added separately as well as in combinations, were analyzed in 25 cases of human AML using 3H-thymidine incorporation and colony assays. The 3H-thymidine uptake data revealed that IL-3, GM-CSF, G-CSF, and M-CSF were stimulators of AML proliferation in 19, 15, 13, and 4 cases, respectively. Epo only stimulated DNA synthesis in the cells of the single erythroleukemia case. GM-CSF stimulation was seen only in IL-3 reactive cases and GM-CSF, when combined with IL-3, could not further elevate the DNA synthesis evoked by IL-3 alone. On the other hand, in six cases, G-CSF enhanced the IL-3- or GM-CSF-stimulated thymidine uptake. These results suggest that subpopulations of AML cells that are activated by distinct CSFs (eg, IL-3/GM-CSF-responsive cells and G-CSF-responsive cells) coexist. The 3H-thymidine incorporation assay was more sensitive for measuring CSF responses than methylcellulose colony cultures, since activation of DNA synthesis was more frequently seen than induction of colony formation. DNA synthesis experiments revealed eight different CSF response patterns among these 25 cases. CSF phenotyping may be a useful addition to the morphologic classification of AML, since these patterns directly reflect the ability of the proliferating subsets of AML cells to respond to the CSFs.

Cell Division↗