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Biomedical subjects

R H Beelen

Publications and source records attributed to R H Beelen.

At least 73 records · Page 4Linked to original sources
PubMed1994

In vitro purging of clonogenic leukaemic cells from human bone marrow by interferon-gamma-activated monocytes.

With a view to the immunologically mediated purging of autologous bone marrow transplants in acute myeloid leukaemia, the efficacy of cytotoxic monocytes to eradicate leukaemic cells has been studied using clonogenic assays. U937 cells were found to be sensitive to highly purified and interferon-gamma-activated human monocytes whereas HL60 cells were rather resistant as measured in an MTT-based cytotoxicity assay under liquid conditions. A spectrophotometric clonogenic assay measured almost complete inhibition of clonogenic activity for U937 cells at low effector-to-target cell (E/T) ratios of at least 0.1. Limiting dilution analysis detected a 2-3 log10 unit reduction in clonogenic activity. In an experimental mixture of U937 cells with a 20-fold excess of normal bone marrow nuclear cells a maximum 2-log10-unit killing could be measured at E/T = 10. Only at high E/T ratios could a reduction in granulocyte/macrophage-colony-forming units (cfu) be observed with only marginal effects on erythroid cfu and erythroid burst-forming. In conclusion, cytotoxic monocytes are highly potent anti-leukaemic effector cells, as measured in clonogenic assays, that do not compromise normal human progenitors.

Acute Disease↗
PubMed1994

Enhanced killing capacity of human Kupffer cells after activation with human granulocyte/macrophage-colony-stimulating factor and interferon gamma.

In this study we investigated the effect of the cytokines human granulocyte/macrophage-colony-stimulating Factor (hGM-CSF) and interferon gamma (IFN gamma) on human Kupffer-cell-mediated cytotoxicity against the SW948 colon carcinoma cell line. Kupffer cells were isolated from small liver wedge biopsies, taken from 14 patients who had had abdominal surgery for colon carcinoma or partial hepatectomy. The cells were incubated with hGM-CSF (100 ng/ml), or with IFN gamma (100 U/ml) or with their combination and the percentage cytotoxicity was determined using a recently described modified assay. Additional experiments were performed with tumour-necrosis-factor-alpha (TNF alpha)-sensitive U937 cells as target. The TNF alpha secretion of Kupffer cells was measured and we evaluated the effect of TNF alpha on colon tumour targets. We performed human-Kupffer-cell-mediated cytotoxicity blocking experiments with anti-TNF alpha and used paraformaldehyde-fixed Kupffer cells to demonstrate lysis of TNF alpha-sensitive WEHI-164 cells and of SW948 cells. The overall cytotoxicity against SW948 caused by unactivated Kupffer cells (n = 14), and by Kupffer cells activated with hGM-CSF (n = 14), IFN gamma (n = 6) or their combination (n = 6) was respectively: 19.5 +/- 2.6%, 25.3 +/- 2.9%, 41 +/- 9.4% and 45.6 +/- 8% at E/T = 1 and 28.2 +/- 2.9%, 35.6 +/- 3.2%, 55.6 +/- 9.7% and 62.8% at E/T = 5. All differences were statistically significant (P < 0.05). No growth-promoting activity by hGM-CSF on the SW948 tumour cells was observed. U937 cells were highly susceptible to Kupffer-cell-mediated cytotoxicity. The TNF alpha secretion by human Kupffer cells increased in parallel to their cytotoxicity after incubation with these cytokines. Soluble TNF alpha had only a slight anti-proliferative effect on SW948 cells, while specific anti-TNF alpha blocked Kupffer cell cytotoxicity by up to 80%. Finally, paraformaldehyde-fixed Kupffer cells were able to lyse WEHI-164 and SW948 cells. This indicates that expression of cell-associated TNF alpha is the main cytolytic mechanism of human-Kupffer-cell-mediated cytotoxicity. The implications for the use of hGM-CSF and IFN gamma in vivo are discussed.

Cell Death↗
PubMed1994

Macrophage subpopulations in the mouse spleen renewed by local proliferation.

A dual origin, by both immigration of blood monocytes and local proliferation has been reported for macrophages, which are heterogeneous in several aspects. Until now few studies have focussed on renewal of macrophages. Therefore, in this study macrophage renewal by local proliferation has been studied in the spleen. Spleen tissue of mice which had received BrdU intravenously was investigated immunohistochemically to detect BrdU incorporated by macrophages. BrdU incorporation by splenic macrophages was studied under steady state conditions as well as during repopulation after experimental macrophage depletion. Under steady state conditions, BrdU incorporation was found in 3.0 +/- 0.5% of the white pulp macrophages and in approximately 1% of the metallophilic macrophages only. Comparable results were found during repopulation after experimental macrophage depletion. The data suggest that renewal of macrophages by local proliferation is restricted to certain subsets in the spleen and that the macrophage subsets of the spleen are renewed by different mechanisms, or belong to different lineages of macrophage differentiation.

Animals↗
PubMed1994

Analysis of the peritoneal cellular immune system during CAPD shortly before a clinical peritonitis.

We analysed the peritoneal cellular immune system 24-48 h before the onset of a clinical peritonitis. Peritoneal cells were obtained from the overnight dialysis effluents 1 or 2 days (day-1 and day-2) preceding the day of peritonitis, the last overnight effluent before the peritonitis effluent (day P), and the first peritonitis effluent. Nine peritonitis episodes of six patients were studied. The number of Fc receptor positive cells, the chemotactic activity, and immunophenotype of the peritoneal cell population at day-2 and day-1 were similar to the postperitonitis control effluent. However, immunophagocytosis and phagocytosis capacity of the peritoneal macrophages was decreased in five of six episodes at day-2 and -1 compared to control peritoneal macrophages. The overnight effluents of day P revealed a moderate influx of neutrophilic granulocytes and an increase of bacterial killing capacity and chemotactic activity. Activation of the peritoneal T cells at day P was shown by the increase in MHC class II positive T cells and an increase in the CD4/CD8 ratio. Bacterial cell cultures of the effluents were positive in three episodes 24-48 h before peritonitis, and of all overnight effluents at day P. These results indicate that malfunctioning of phagocytosis by peritoneal macrophages may contribute to the development of a CAPD peritonitis.

Adult↗
PubMed1994

Enrichment and characterization of dendritic cells from human bronchoalveolar lavages.

In the present study about 0.3% to 1.6% of human bronchoalveolar lavage (BAL) cells were identified as typical dendritic cells (DC), having an irregular outline, lobulated nucleus, and clear distinguishable acid phosphatase activity or EBM11 (anti-CD68) reactivity in a spot near the nucleus. After DC enrichment, using transient adherence to plastic, FcR-panning, and a density metrizamide gradient, a population containing 7-8% typical DC was obtained. This DC-enriched low density fraction, containing the highest percentages of DC, very strongly induced T cell proliferation in an allogeneic mixed leucocyte reaction (MLR), which was significantly higher than that induced by other partly (un)fractionated BAL cells. These data indicate that DC seem to be the major accessory cells in the BAL fluid, and therefore may be important in the regulation of T cell immune responses in the lung.

Acid Phosphatase↗
PubMed1994

Maturation-dependent susceptibility to monocyte-mediated cytotoxicity in acute myeloid leukemia.

In view of cellular immunotherapy with cytotoxic monocytes in minimal residual leukemia we have studied the effects of monocytes on the growth and survival of leukemic cells from cell lines and from patients with acute myeloid leukemia (AML). Using highly purified and interferon-gamma (IFN gamma) activated human monocytes, monocyte-mediated cytotoxicity (MMC) was evaluated in an MTT-based colorimetric cytotoxicity assay against six human leukemic cell lines (U937, THP1, KG1, K562, HL60, and 1,25(OH)2D3 differentiated HL60 cells) and cells from AML patients. Leukemic cells from cell lines with an immature phenotype were found to be resistant to MMC, whereas leukemic cells with a more mature and monocytic phenotype were sensitive. This paralleled the sensitivity to tumor necrosis factor-alpha (TNF-alpha). AML cells from patients with an immature phenotype (FAB-M1/M5A) were significantly less sensitive to MMC as compared to more mature AML cells (FAB-M2/M4/M5B). The growth stimulatory effects of non-activated monocytes on immature AML cells could be abrogated in the presence of IFN gamma or IL-3 and GM-CSF. In addition, these cytokines further potentiated MMC, preferentially affecting cells with a more mature phenotype. AML cells with an immunologically immature phenotype (CD34(high), HLA-Dr(low), CD13(low), CD14(low)) were revealed as the least sensitive cells to MMC. The growth stimulatory effects of IL-3/GM-CSF with or without TNF-alpha on AML cells correlated with resistance to MMC. In addition, the cytolytic effects of TNF-alpha in the presence of IFN gamma correlated with an increased susceptibility of AML cells to MMC. In conclusion, our data strongly indicate that MMC is related to maturation in AML, which is correlated to the differential stimulatory and cytolytic effects of monocyte-derived cytokines such as IL-3, GM-CSF, and TNF-alpha.

Acute Disease↗
PubMed1993

Cell cycle specific effects of tumor necrosis factor alpha in monocyte mediated leukemic cell death and the role of beta 2-integrins.

Human monocytes are involved in host defense against neoplastic cells. In view of cellular immunotherapy with cytotoxic monocytes in minimal residual disease of acute myeloid leukemia we have studied the role of monocytes in cell cycle dependent leukemic cell death of U937, THP-1, and HL-60 cells in vitro. Leukemic cells separated in G1 of the cell cycle by countercurrent centrifugal elutriation were highly susceptible to monocyte mediated cytotoxicity, whereas cells in S and G2-M were less sensitive or completely resistant as compared to unfractionated control cells. HL-60 cells resistant to cytotoxic monocytes became sensitive to monocyte mediated cytotoxicity upon differentiation induction with 1,25-dihydroxyvitamin D3 which paralleled an accumulation of cells in G1 of the cell cycle. The differences in susceptibility of cell phase separated populations to monocyte mediated cytotoxicity paralleled differences in sensitivity to the cytotoxic effects of tumor necrosis factor alpha, as secreted by gamma-interferon activated monocytes. Furthermore, monocyte mediated cytotoxicity was markedly inhibited in the presence of anti-CD11/CD18 monoclonal antibodies recognizing the alpha and beta chains of the beta 2-integrin adhesion proteins. By fluorescence activated cell sorter immunofluorescence a marked increase in mean fluorescence density of the beta 2-integrins could be demonstrated on cells in G1 of the cell cycle as compared to unseparated leukemic cells. A decrease in mean fluorescence density was shown for cells in G2-M. By blocking experiments with anti-CD11/CD18 monoclonal antibodies, the differences in mean fluorescence density were functionally relevant since cells in G1 were shown to be the most sensitive cells to beta 2-integrin dependent monocyte mediated cytotoxicity. In conclusion these data show that differences in sensitivity to tumor necrosis factor and in the expression of beta 2-integrins may play a central role in cell cycle dependent monocyte mediated antileukemic activity.

Antibodies, Monoclonal↗
PubMed1993

Isolation of cytotoxic Kupffer cells by a modified enzymatic assay: a methodological study.

Kupffer cell (KC)-mediated cytotoxicity against tumor cells is of interest, since the liver is a major site of metastatic growth of primary colorectal cancer. KC isolation methods from rat livers, to study the tumoricidal properties of these cells, are based on perfusion of the liver and are therefore not suitable for human KC isolation from liver biopsies. In view of application to isolate KC from small wedge human liver biopsies, we have developed an isolation procedure for rat KC that does not require perfusion techniques. Liver tissue fragments were incubated with pronase with continuous pH registration and neutralization. KC were subsequently separated from other non-parenchymal cells by Nycodenz gradient centrifugation and purified by counterflow centrifugal elutriation. KC and other non-parenchymal cells were identified by immunophenotyping with a cytoplasmic monoclonal antibody ED1 and by ultrastructural analysis. About 3 x 10(6) KC per gram liver were isolated with a final purity of > 95% without loss of viability. To ensure that functionally competent KC were isolated, we assayed cytotoxicity against CC531 tumor cells in a recent developed cell-mediated MTT assay. Maximum cytotoxicity of KC was approximately 40% at an effector to target ratio of 10. In conclusion our approach seems to be a useful and simple method to isolate KC with good functional properties from rat livers, without the need for perfusion techniques.

Animals↗
PubMed1993

The cellular composition in the peritoneal cavity and the cytotoxic function of the peritoneal cells from patients with ovarian cancer; effect of tumor necrosis factor-alpha treatment.

In patients with epithelial ovarian cancer (EOC), the cellular composition in the peritoneal cavity and the functional capacities of the peritoneal cells (PC) are unknown. Especially the peritoneal macrophages (m phi) could play an important role in defense against tumor cells. To study the cellular composition in the peritoneal cavity and the functional capacities of PC, these cells were obtained from three patients with EOC. The PC were immunophenotyped and tested functionally in vitro in a cytotoxicity assay. One of the patients was treated intraperitoneally (i.p.) with a single dose of 0.06 mg/m2 tumor necrosis factor-alpha (TNF-alpha). PC were obtained before the treatment, after 24 h and after 1 week. PC from healthy women undergoing laparoscopic sterilization served as controls. It appeared that patients with EOC have a lower percentage of macrophages (m phi) in the peritoneal cavity than healthy persons. These m phi of patients were also less capable of killing U 937 tumor cells as compared to the peritoneal m phi of control persons. However, in the patient treated i.p. with TNF-alpha the cytotoxic capacities of the peritoneal m phi were strongly improved. The percentage cytotoxicity at an effector to target ratio of 10, increased from 17% to 80%. Thus, the peritoneal m phi in this patient were activated in vivo to a tumoricidal state. These findings indicate that PC in patients with EOC differ from controls, but further investigation is necessary to define the contribution of the disease and/or prior chemotherapy to this defect.

Adenocarcinoma↗
PubMed1993

Macrophage phenotype regulation by colony-stimulating factors at bone marrow level.

Macrophages (m phi s) can be divided into several subpopulations, which differ in phenotype, function, and localization patterns. However, little is known about the mechanisms that regulate this heterogeneity. We investigated whether m phi heterogeneity is regulated by colony-stimulating factors (CSFs) at the bone marrow level. By clonal expansion of bone marrow-derived precursor cells in the presence of CSF-1, granulocyte-macrophage CSF or multi-CSF (interleukin-3), phenotypic heterogeneity was observed between m phi colonies. Heterogeneity was found especially when different CSF culture conditions were used but also between m phi colonies derived under the same CSF culture condition. Our results illustrate that CSFs from the bone marrow hemopoietic microenvironment are important for the induction of phenotypic heterogeneity within the progeny of cloned m phi precursor cells during maturation and differentiation in vitro.

Animals↗
PubMed1993

Separation of alveolar macrophages and dendritic cells via autofluorescence: phenotypical and functional characterization.

In a previous study we demonstrated that an increase of monocytes and dendritic cells (MDC) was In the current study, the bright autofluorescence of alveolar macrophages (AMs) was used to separate them efficiently from the MDC. Sorting of freshly isolated BAL cells resulted in a high-autofluorescent fraction, consisting predominantly of AMs, and a low-autofluorescent fraction containing the MDC, lymphocytes, and granulocytes. Thus, a clear separation between suppressive (AM) and stimulating (MDC) activity was obtained as shown in antigen-specific T cell responses. Flow cytometric parameters, density fractionation, and a series of ED monoclonal antibodies raised against rat macrophage antigens showed that both AMs and MDC were diverse populations. After overnight culture, more than 80% of an MDC population with a density range of 1.065-1.079 changed to a lower density (< 1.056) and morphologically developed into DCs with many processes. Concomitantly, monoclonal antibody ED1 expression changed from a granular pattern to a discrete juxtanuclear spot localization.

Animals↗