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Biomedical subjects

K Welte

Publications and source records attributed to K Welte.

At least 91 records · Page 5Linked to original sources
PubMed1995

The protein tyrosine kinase JAK2 is activated in neutrophils from patients with severe congenital neutropenia.

Severe congenital neutropenia (SCN; or Kostmann syndrome) is an autosomal recessive disorder characterized by a maturation arrest of myelopoiesis at the level of promyelocytes. Myeloid precursor cells from patients with SCN require pharmacological dosages of recombinant human granulocyte colony-stimulating factor (r-metHuG-CSF; Filgrastim; Amgen, Thousand Oaks, CA) to differentiate to normal neutrophils. Thus, it is hypothesized that the underlying defect responsible for SCN is based on an abnormal G-CSF-induced signal transduction pathway. Because JAK2, a nonreceptor tyrosine kinase, is involved in the signaling pathway of G-CSF, we examined the expression and activity of JAK2 in neutrophils from SCN patients during r-metHuG-CSF treatment. The immunoprecipitated JAK2 protein showed increased tyrosine phosphorylation in neutrophils from SCN patients as compared with that in neutrophils from healthy donors, suggesting that this kinase is activated. In vitro kinase assays of immunoprecipitated JAK2 confirmed that neutrophils from SCN patients show an increased autophosphorylation of JAK2 in comparison with that of neutrophils from healthy volunteers. These findings suggest that JAK2 is activated in SCN patients.

Adolescent↗
PubMed1995

Mutations in the gene for the granulocyte colony-stimulating-factor receptor in patients with acute myeloid leukemia preceded by severe congenital neutropenia.

BACKGROUND: In severe congenital neutropenia the maturation of myeloid progenitor cells is arrested. The myelodysplastic syndrome and acute myeloid leukemia develop in some patients with severe congenital neutropenia. Abnormalities in the signal-transduction pathways for granulocyte colony-stimulating factor (G-CSF) may play a part in the progression to acute myeloid leukemia. METHODS: We isolated genomic DNA and RNA from hematopoietic cells obtained from two patients with acute myeloid leukemia and histories of severe congenital neutropenia. The nucleotide sequences encoding the cytoplasmic domain of the G-CSF receptor were amplified by means of the polymerase chain reaction and sequenced. Murine myeloid 32D.C10 cells were transfected with complementary DNA encoding the wild-type or mutant G-CSF receptors and tested for their responses to G-CSF. RESULTS: Point mutations in the gene for the G-CSF receptor were identified in both patients. The mutations, a substitution of thymine for cytosine at the codon for glutamine at position 718 (Gln718) in one patient and at the codon for glutamine at position 731(Gln731) in the other, caused a truncation of the C-terminal cytoplasmic region of the receptor. Both mutant and wild-type genes for the G-CSF receptor were present in leukemic cells from the two patients. In one patient, the mutation was also found in the neutropenic stage, before the progression to acute myeloid leukemia. The 32D.C10 cells expressing mutant receptors had abnormally high proliferative responses but failed to mature when cultured in G-CSF. The mutant G-CSF receptors also interfered with terminal maturation mediated by the wild-type G-CSF receptor in the 32D.C10 cells that coexpressed the wild-type and mutant receptors. CONCLUSIONS: Mutations in the gene for the G-CSF receptor that interrupt signals required for the maturation of myeloid cells are involved in the pathogenesis of severe congenital neutropenia and associated with the progression to acute myeloid leukemia.

Acute Disease↗
PubMed1995

Absence of c-kit receptor and absent proliferative response to stem cell factor in childhood Burkitt's lymphoma cells.

The cytokine stem cell factor (SCF) synergizes with interleukin-7 (IL-7) to enhance the proliferation of pre-B cells. To examine the role of SCF and its receptor, c-kit, in the pathogenesis of pediatric Burkitt's lymphomas (BL), we investigated the expression of SCF and c-kit in BL cells and the mitogenic activity of SCF on BL cells. A panel of 13 BL cell lines and 7 fresh biopsy tumors was investigated. BL cells were stimulated either by Epstein-Barr virus (EBV) infection or by different reagents and cytokines, and expression of SCF and c-kit was studied on the mRNA level by Northern blot analysis and reverse-transcriptase polymerase chain reaction (RT-PCR), followed by Southern blotting. c-kit expression was also studied by fluorescence-activated cell sorting and by crosslinking of digoxigenin-labeled recombinant human SCF to the cell surface. Proliferation of BL cell lines was measured by 3H-thymidine incorporation. Low-level expression of c-kit mRNA was detected in 2 of 13 unstimulated BL cell lines and in 1 fresh BL tumor. One cell line showed upregulation of c-kit mRNA with A23187 and downregulation with phorbol myristate acetate. Neither c-kit nor SCF could be detected in any other cell line under any condition of stimulation as analyzed by Northern blot analysis, RT-PCR followed by Southern blot analysis, crosslinking, and immunofluorescence. No response to SCF was seen in 3H-thymidine incorporation assays. We conclude that most BL cells express neither SCF nor c-kit and that the low-level expression of c-kit in some BL cells most likely has no biologic significance.

Adolescent↗
PubMed1995

Coexpression of stem cell factor and its receptor c-Kit in human malignant glioma cell lines.

Stem cell factor (SCF), a hematopoietic growth factor, is the ligand of the tyrosine kinase receptor encoded by the c-kit proto-oncogene. Beside the important role of this receptor-ligand complex in hematopoiesis, gametogenesis and melanogenesis, SCF and its receptor have been shown to be expressed in the brain. We have studied the expression of SCF and c-kit in 20 human malignant glioma cell lines at the mRNA as well as at the protein level. In addition, recombinant human (rh) SCF was tested in [3H]thymidine uptake assays for a mitogenic effect on these cells. SCF and c-Kit proteins were detected in the cytoplasm of glioma cells by alkaline phosphatase-monoclonal anti-alkaline phosphatase immunostaining and Western blot analysis. However, neither SCF nor c-Kit were seen on the cell surface by flow cytometry. Furthermore, none of the proliferation assays showed a mitogenic effect for exogenously added rhSCF. Blocking studies using an anti-SCF antibody failed to demonstrate modulating effects on the growth of selected cell lines. These results suggest that SCF and c-Kit may mediate non-proliferative signals or may employ intracellular mechanisms for autocrine growth regulation of glioma cells.

Blotting, Western↗
PubMed1995

Absence of G-CSF receptors and absent response to G-CSF in childhood Burkitt's lymphoma and B-ALL cells.

The expression of the granulocyte colony-stimulating factor (G-CSF) receptor in childhood Burkitt's lymphoma (BL) cells, and the mitogenic effect of G-CSF on these cells, was studied in a panel of 13 Epstein-Barr virus (EBV) positive and negative BL cell lines derived from nine children. G-CSF receptor mRNA expression was investigated by Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). Binding of G-CSF to BL cell lines was measured by chemical crosslinking of 125I-G-CSF, and proliferation by thymidine incorporation. Inducibility of the G-CSF receptor was studied by stimulation with interleukin-1 beta, tumour necrosis factor-alpha, Staphylococcus aureus Cowan A, anti-human IgM, phorbol myristate acetate, calcium ionophore A23187, and by infection in vitro by immortalizing and non-immortalizing strains of EBV. BL cell lines, unstimulated or stimulated by biological reagents or EBV infection, did not bind radioionated G-CSF in crosslinking experiments. No stimulation by recombinant human G-CSF was observed in 3H-thymidine incorporation assays. No G-CSF receptor mRNA was detected by Northern blot analysis or RT-PCR in BL cell lines. It is concluded that G-CSF plays no direct stimulatory role in the growth of these malignant B-cells, making a deleterious influence of G-CSF in the clinical treatment situation unlikely.

Adolescent↗
PubMed1995

Extramedullary hematopoiesis and intratumoral production of cytokines in childhood hepatoblastoma.

Extramedullary hematopoiesis is a characteristic feature of hepatoblastoma (HB). We investigated 15 HB to characterize intratumoral hematopoietic foci and to find clues to the pathophysiology of their formation. By conventional histology and immunohistochemistry, we found erythroblasts in all and megakaryocytes in 10 of the HB, whereas granulocyte and monocyte precursor cells could not be identified in hematopoietic foci of any tumor. Only a minority of erythropoietic cells in these foci contained fetal Hb (HbF). We recently found that HB cells produce IL-1 beta and thus stimulate stromal cells to secrete IL-6. We therefore searched for other hematopoietic cytokines in HB. Supernatants of primary HB cultures were subjected to ELISA, bioassayed, and immunoblotted. We detected erythropoietin (EPO) in 11 of 15, stem cell factor (SCF) in 7 of 11, granulocyte colony-stimulating factor (G-CSF) in 4 of 15, granulocyte/macrophage colony-stimulating factor (GM-CSF) in 6 of 15, IL-3 in 1 of 12, leukemia inhibitory factor (LIF) in 1 of 9, and macrophage colony-stimulating factor (M-CSF) in 1 of 8 conditioned media. With immunoenzymatic labeling we localized EPO and SCF to the cytoplasm of epithelial HB cells, whereas stromal cells and cells of immature fibrous tissue of mixed HB expressed SCF, G-CSF, GM-CSF, LIF, and M-CSF. EPO and SCF could also be detected in extracts of epithelial HB cells. We conclude that, in HB, erythropoiesis and megakaryopoiesis but not the granulocyte-macrophage lineage is induced by fetal and embryonal tumor cells in cooperation with stromal cells by locally secreted cytokines.

Carcinoma, Hepatocellular↗
PubMed1995

Effects of bone marrow fibroblasts on the proliferation and differentiation of myeloid leukemic cell lines.

The effects of normal bone marrow fibroblasts (BM FB) on proliferation and differentiation of 10 myeloid leukemic cell lines were investigated in a serum-free co-culture system. The proliferation of three of the cell lines was supported by BM FB. Three of the myeloid cell lines were inhibited 40-70%. The co-culture supernatants were tested for the secretion of hematopoietic cytokines by bioassays. Except for IL-6, which was already produced constitutively by BM FB, only little amounts of interleukin-1 (IL-1), granulocyte colony-stimulating factor (G-CSF), or granulocyte-macrophage colony-stimulating factor (GM-CSF) could be detected in several co-culture supernatants. It could be shown that, according to cytologic and functional criteria, the myeloid leukemic cell lines ML-2 and PLB-985 differentiate along the monocyte-macrophage pathway after co-culture with BM FB. They revealed a histiocytic phenotype and could be induced to produce reactive oxygen intermediates (ROI) after stimulation with zymosan or phorbol-myristate-acetate (PMA). Additional proof for differentiation was obtained from flow cytometric analysis of surface differentiation antigens and adhesion molecules. The neutralization of IL-6 activity in the co-cultures by antibodies resulted in prevention of differentiation of PLB-985 cells, while differentiation of ML-2 cells in the co-cultures was not affected by addition of anti-IL-6 antibodies. Furthermore, in co-culture experiments with fibroblasts from skin and foreskin, we found a differentiation of PLB-985 cells comparable to that in co-cultures with BM FB, but poor differentiation of ML-2 cells. These data suggest that different mechanisms are involved in the differentiation of ML-2 and PLB-985 cells.

Antibodies, Monoclonal↗
PubMed1995

G-CSF in the long-term treatment of cyclic neutropenia and chronic idiopathic neutropenia in adult patients.

Eleven adult patients with chronic neutropenia have been treated with recombinant human granulocyte colony-stimulating factor (G-CSF). Seven patients had idiopathic sporadic neutropenia, 2 idiopathic congenital neutropenia and 2 cyclic neutropenia. Treatment was started with 3 micrograms/kg s.c. daily and was modified according to response. All patients had a rapid increase of neutrophils. The cycle length shortened from 21 to 14 days in patients with congenital cyclic neutropenia. Doses required for maintenance ranged from 0.1 to 8 micrograms/kg. One patient with idiopathic sporadic neutropenia recovered after 151 days with an absolute neutrophil count (ANC) of > 2000/microliters and had no need for further treatment. The overall efficacy of the treatment was good with abrogation of severe infections. Treatment has been continuously given to a maximum of 4.5 years. No loss of efficacy was observed during long-term treatment. There was no evidence for lineage drain or stem cell exhaustion with prolonged treatment. Cytogenetic analyses in seven of eleven patients did not reveal the development of abnormal cell-clones. G-CSF is safe and efficient in long-term treatment of adult patients with chronic neutropenias.

Adult↗
PubMed1994

Differential regulation of stem cell factor mRNA expression in human endothelial cells by bacterial pathogens: an in vitro model of inflammation.

Production of hematopoietic growth factors by endothelial cells plays a pivotal role during inflammatory processes. Stem cell factor (SCF) is known to be expressed constitutively in endothelial cells. To investigate the regulation of this cytokine expression by inflammatory stimuli, we cocultured human umbilical vein endothelial cells (HUVEC) with various gram-negative bacterial strains (Escherichia coli, Yersinia enterocolitica, Chlamydia trachomatis, and Neisseria meningitidis, respectively). Experiments were performed with bacterial concentrations ranging from 10(2) to 10(7) bacteria/mL for 3 hours. SCF-specific mRNA expression was studied using Northern blot analysis. Stimulation with the enteropathogenic bacterial strains Y enterocolitica and E coli resulted in a significant concentration-dependent increase of SCF mRNA expression. Similar results were obtained in coculture experiments with N meningitidis. As shown in experiments with E coli, the accumulation of SCF transcripts was also time-dependent. In contrast, coculture of HUVEC with the intracellular gram-negative strain C trachomatis had no effect on SCF mRNA expression. To elucidate the role of the gram-negative bacterial cell wall components, we stimulated HUVEC with bacterial lipopolysaccharide (LPS). LPS induced a maximal SCF mRNA accumulation within 2 hours followed by decrease of SCF-specific transcripts to the basal level after 24 hours. In addition, we exposed HUVEC to the classical inflammatory cytokine interleukin-1 alpha (IL-1 alpha). Kinetic experiments showed a similar pattern of regulation with an increase of SCF mRNA within 2 hours, persisting up to 12 hours, and a decrease to basal transcription after 24 hours. From these data, we conclude that SCF expression is regulated by inflammatory stimuli, such as IL-1 alpha and bacterial pathogens, suggesting an important role of SCF during inflammation.

Cells, Cultured↗
PubMed1994

Immunolocalization of granulocyte-colony-stimulating factor in human glial and primitive neuroectodermal tumors.

Granulocyte-colony-stimulating factor (G-CSF) is a hematopoietic cytokine that regulates the differentiation of myeloid progenitors and the function of mature neutrophils. It is produced in vitro by monocytes/macrophages, mesothelial cells, fibroblasts and endothelial cells after appropriate induction by inflammatory mediators like IL-1 and TNF. Normal as well as tumorous glial cells can also be induced to produce CSFs in vitro. However, little is yet known about the in vivo expression of G-CSF as a mediator in inflammation and malignancy within the human central nervous system. The aim of the present study was to investigate by immunostaining the expression of the G-CSF protein within non-tumorous and tumorous glial tissues, and primitive neuroectodermal tumors. Using the murine monoclonal anti-G-CSF TM 82/60 antibody, we found high G-CSF expression in astrocytoma WHO grades I and II and reactive brain tissue, low expression in astrocytoma WHO grade III, and none in glioblastoma, oligodendroglioma WHO grades II and III, and medulloblastoma. In consecutive sections of the tissue samples, G-CSF protein was localized in GFAP-positive glial cells, but not in macrophages/microglial cells, which expressed HLA-DR, detected by the antibody CR3/43. Computer-assisted microdensitometric evaluation of the intensity of immunostaining for G-CSF and statistic analysis of the data revealed significant differences between the diagnostic entities studied (p < 0.0001). We conclude that in vivo expression of G-CSF is a characteristic of reactive as well as tumorous astrocytes, with the latter losing this feature at higher degrees of dedifferentiation.

Adolescent↗
PubMed1994

Matched unrelated transplants.

The National Marrow Donor Program (NMDP) has a computerized registry of more than 900,000 volunteer bone marrow donors for patients in need of a transplant. The donors available through the NMDP offer an option for the 70% of patients who could benefit from a marrow transplant but who do not have a suitable related donor. The complex process of finding a compatible unrelated donor involves seven steps, and the median length of time to find a donor through the NMDP is approximately 6 months. Each month, approximately 50 marrow transplants are facilitated by the NMDP.

Bone Marrow Transplantation↗
PubMed1994

Altered surface marker expression and function of G-CSF-induced neutrophils from test subjects and patients under chemotherapy.

We have previously reported an altered surface marker expression and chemotaxis of G-CSF-induced neutrophils from patients with severe congenital neutropenia. However, effects of G-CSF and influence of the underlying disease on neutrophils could not be discerned. In this study we have evaluated the effects of G-CSF on neutrophil phenotype and function in patients under chemotherapy and in healthy test subjects. We found a significantly enhanced expression of Fc gamma RI, CD14 and CD54 and a decrease in the level of Fc gamma RIII during G-CSF treatment. In addition, motility of G-CSF-induced neutrophils was significantly decreased. The effects were seen in patients under cytotoxic chemotherapy and in healthy test subjects. Surface marker alterations and neutrophil motility were affected by G-CSF administration in a dose-dependent manner. Kinetic studies on neutrophils from healthy test subjects demonstrated that all effects could be seen after a single administration of 300 micrograms G-CSF and began to appear within 4 h. Release of partially immature neutrophils from the bone marrow and indirect activation of these cells by G-CSF are discussed as possible reasons for the findings presented. They demonstrate that G-CSF has profound effects on neutrophil phenotype and function in vivo which might have clinical implications.

Antigens, Surface↗
PubMed1994

Changes in light-scatter profile, membrane depolarization and calcium mobilization of neutrophils induced by G-CSF in vivo.

To extend our studies about phenotypical and functional alterations of G-CSF-induced neutrophils we have evaluated their light-scatter profile, mobilization of intracellular calcium ([Ca2+]i) and membrane depolarization after stimulation. A significant increase in the forward scatter signals could be demonstrated in such neutrophils from patients with neutropenias of various origin and from healthy test subjects. This increase began 4 h and returned to normal 96 h after G-CSF injection in the latter group. We found an impairment of [Ca2+]i mobilization in neutrophils from patients with glycogen storage disease type IB after stimulation of these cells with fMLP. It was even more pronounced than in severe congenital neutropenia (SCN). However, [Ca2+]i fluxes were normal when ionomycin was used. Neutrophils from patients with cyclic neutropenia (cyNP) and chemotherapy-induced neutropenia (chNP) mobilized [Ca2+]i similar to those from healthy donors. Furthermore, we found a decreased percentage of neutrophils depolarizing after stimulation with fMLP and PMA in patients with SCN, whereas membrane depolarization was normal in patients with chNP and cyNP. All the alterations found here are suggested to be caused by a partial immaturity of the neutrophils, although in vivo activation and a direct effect of G-CSF on myeloid precursors might be involved.

Calcium↗
PubMed1994

Effects of granulocyte colony-stimulating factor in children with severe neutropenia.

In children with all types of severe neutropenia the development of G-CSF for therapeutic use changed the quality of their life dramatically. Missing the most important cells in the defense against bacterial infections the neutrophilic granulocytes, these patients suffered from episodes of severe, often life-threatening bacterial infections. They spent numerous days in hospital, requiring intravenous antibiotic treatment. Recurrence of bacterial infections at the same site led to irreversible tissue damage, for example in the lung, requiring often disabling surgical interventions. In most patients G-CSF treatment induced an increase of blood and tissue neutrophils to a level high enough to guarantee a normal defense against bacterial infections. The quality of life improved substantially in these children. The fact that they have to inject themselves daily does not cause any problems. Overall, taken in consideration all possible adverse events during our short observation period, all patients who responded to G-CSF benefit from this treatment to a degree never considered to be possible before.

Adolescent↗
PubMed1993

Severe congenital neutropenia: abnormal growth and differentiation of myeloid progenitors to granulocyte colony-stimulating factor (G-CSF) but normal response to G-CSF plus stem cell factor.

Several mechanisms have been proposed to explain the pathogenesis of severe congenital neutropenia (SCN); however, the mechanism(s) still remains unknown. In particular, clinical observations suggest that abnormal responsiveness of myeloid progenitors to hematopoietic growth factors (HGFs) is a possible mechanism. Therefore, to better define the status of hematopoietic progenitors in the bone marrow (BM) of patients with SCN, the responsiveness of myeloid progenitors to HGFs from two SCN patients was compared with the responsiveness of progenitors from healthy individuals. BM cells (BMCs) from the first SCN patient required higher (10- to 100-fold) concentrations of granulocyte colony-stimulating factor (G-CSF) to achieve maximal and half-maximal colony growth in vitro compared with BMCs from controls. In contrast, the dose-response of interleukin-3 (IL-3) and granulocyte-macrophage-CSF (GM-CSF) in colony formation was normal. Interestingly, IL-3, GM-CSF, and G-CSF at optimal doses showed reduced ability to induce neutrophil differentiation of BMCs from a SCN patient compared with BMCs from controls. Despite an abnormal responsiveness of mature myeloid progenitors to G-CSF in this SCN patient, myeloid progenitors responsive to the combination of stem cell factor (SCF) and G-CSF showed normal dose-response. In contrast to G-CSF alone, the combination of G-CSF and SCF induced the formation of neutrophils almost to the same extent compared with cultures of normal BMCs. Furthermore, also on BM progenitor cells obtained from the second patient with SCN, SCF highly synergized with G-CSF to promote neutrophil progenitor cell growth and differentiation in vitro. Thus, these results indicate that one mechanism of the pathogenesis in SCN patients is reduced responsiveness of neutrophil progenitor cells to G-CSF and that SCF can enhance the responsiveness of these cells to G-CSF.

Bone Marrow↗
PubMed1993

Production of interleukin-1 beta and interleukin-6 in hepatoblastoma.

Thrombocytosis and fever are frequent symptoms in children with hepatoblastoma. Interleukin-6 (IL-6) has been shown to mediate thrombocytosis and an acute-phase reaction including fever. We therefore investigated samples from 14 untreated patients with hepatoblastoma for this cytokine and in addition for interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), all of which are known to induce IL-6 production. High serum levels of IL-6 were only found in 3/14 patients; the other cytokines were not detectable. In contrast, 12/14 tumors produced substantial amounts of IL-6 in primary cell culture, while IL-1 beta was found in 3/14 supernatants; IL-1 alpha and TNF-alpha were always negative. Immunoenzymatic staining of fresh tumors revealed that IL-6 is not produced by the tumor cells, but rather by surrounding fibroblasts and endothelial cells. In tumor cells only IL-1 beta, but neither IL-1 alpha, TNF-alpha nor IL-6, could be detected. In co-culture experiments with fibroblasts and endothelial cells, addition of hepatoblastoma cells enhanced IL-6 production. Including an IL-1 receptor antagonist abolished this effect incompletely. Our results suggest that tumor cells in hepatoblastoma induce IL-6 production in surrounding fibroblasts and endothelial cells by virtue of their endogenous secretion of IL-1 beta and supposedly some other, as yet unidentified, mediator.

Carcinoma, Hepatocellular↗