PubMed Health⌕ Search

Biomedical subjects

R H Beelen

Publications and source records attributed to R H Beelen.

At least 55 records · Page 3Linked to original sources
PubMed1996

Identification of the major chemokines that regulate cell influxes in peritoneal dialysis patients.

To investigate which members of the recently discovered family of chemotactic cytokines (chemokines) are important in leukocyte recruitment to a bacterial inflammation site, four different chemokines in the effluent of peritoneal dialysis patients suffering from acute bacterial peritonitis were measured. The presence of two neutrophil-attracting chemokines, interleukin-8 and human melanoma growth-stimulating activity (huGRO alpha), and two monocyte-attracting members of the chemokine superfamily, monocyte chemotactic protein-1 (MCP-1) and regulated on activation normal T cell expressed and secreted (RANTES), was investigated in patient effluents just before, during, and after a peritonitis episode. This was studied in seven peritonitis effluents of five patients by using chemokine-specific enzyme-linked immunosorbent assays. Cell populations in the dialysates were differentiated on cytocentrifuge preparations. The contribution of the detected chemokines to neutrophilic and monocytic cell influxes in the inflamed peritoneal cavity was analyzed by correlating concentrations of chemokines to the relevant cell numbers present in the dialysates of these patients. The detection of the neutrophil-attracting chemokine interleukin-8 during peritonitis was in accordance with other studies. Moreover, a second neutrophil chemoattractant, huGRO alpha, was identified in vivo. Both were elevated during inflammation (P < 0.02) and contributed significantly to the neutrophilic cell influx (P < 0.05). One of the monocyte-attracting chemokines, RANTES, could not be detected in any of the effluents, whereas the other, MCP-1, was significantly elevated during peritonitis (P < 0.02). In contrast to the other chemokines measured, MCP-1 concentration was relatively high in steady-state peritoneal dialysates. An absolute correlation between dialysate MCP-1 concentration and the number of macrophages in these effluents was absent. However, in a 48-well chemotaxis assay, monocyte migration toward peritonitis, as well as steady-state patient dialysates, could be blocked with antibodies to MCP-1. It was concluded, therefore, that MCP-1 is the most important monocyte chemoattractant in peritoneal dialysis steady-state and peritonitis patients; whereas, besides interleukin-8, huGRO alpha was identified as a major neutrophil-attracting chemokine in the peritonitis situation.

Acute Disease↗
PubMed1996

Renal function influences interleukin-8 background production by cultured human mesothelial cells.

Previous studies have demonstrated that mesothelial cells (MC) are important in the local host defense system of the peritoneal cavity. Most studies on the function of MC are performed on MC derived from material of patients with normal renal function (NRF). The aim of the present study was to examine differences in interleukin (IL)-8 expression by MC from patients with NRF and from patients with end-stage renal disease (ESRD). Therefore, MC were isolated from the omentum and pleural exudate of patients with NRF, from spent effluent of stable peritoneal dialysis (PD) patients, and from omentum obtained during catheter implantation prior to PD treatment. MC were stimulated with increasing doses of IL-1 beta or tumor necrosis factor-alpha for 24 hours, after which the supernatant was analyzed for IL-8 content. The IL-8 background level of MC isolated from patients with NRF was significantly lower than the IL-8 background level of MC derived from patients with ESRD. Although IL-8 production appeared to be higher in the ESRD MC, this difference was not significant after stimulation. While the overall immunity is depressed in uremia, MC are activated. The relatively high background of IL-8 might lead to an insensitivity of neutrophils by blocking the receptors and explain their impaired chemotaxis in uremia.

Adult↗
PubMed1996

The role of BCL-2 and bax protein in monocyte-mediated apoptosis in human leukemic cell lines.

Monocytes or monocyte-derived supernatants are able to kill leukemic cells via apoptosis, thereby preferentially effecting more mature leukemic cells. In the present study, the relationship between apoptosis and the apoptosis related proteins, bcl-2 and bax, was investigated in a number of human leukemic cell lines. Monocyte-derived supernatant induces extensive apoptosis in U937 myeloid leukemia cells and minor apoptosis in HL60 cells. No apoptosis was seen in four other cell lines (THP1, HL60-D3, KG1, and K562). The expression of bcl-2 and bax protein was determined in both groups of leukemic cell lines by flow cytometry (bcl-2 and bax) and Western blotting (bcl-2) at baseline level and after incubation with monocyte supernatant after different time periods. No clear relation was found between baseline bcl-2 or bax protein expression and the occurrence of apoptosis after incubation with monocyte supernatant. After different incubation time periods, no change was found in bcl-2 protein expression in U937 and K562 cells, whereas in KG1, HL60, and especially in THP1 cells, a significant decrease could be noticed. On the other hand, there was an increase in bcl-2 expression in HL60-D3 cells. Bax protein expression, measured at the same time points, remained essentially unchanged in HL60-D3 cells, decreased significantly in U937, HL60, and THP1 cells and slightly in K562 cells, and increased significantly in KG1 cells. Also, the ratio bax/bcl-2 decreased in HL60D3, but especially in U937 and HL60 cells, increased slightly in THP1 and KG1 cells, and remained essentially unchanged in K562 cells. Rh-tumor necrosis factor-alpha (TNF-alpha), the main mediator of monocyte mediated cytotoxicity, induced apoptosis in U937, HL60, and THP1 cells, thereby showing changes in bcl-2 expression similar to those found for monocyte derived supernatants. We concluded that in human leukemic cell lines, there is no relation between either bcl-2 or bax protein expression or the ratio of both, and apoptosis mediated by monocyte derived supernatant or TNF-alpha.

Apoptosis↗
PubMed1995

Enhanced human Kupffer cell-mediated cytotoxicity after activation of the effector cells and modulation of the target cells by interferon-gamma: a mechanistic study at the cellular level.

In this study we demonstrate enhanced Kupffer cell (KC) cytotoxicity against several colorectal cell lines by activation of KC and by modulation of the targets (SW948, WiDR, HT29, and SW620) with IFN-gamma. We demonstrated that soluble TNF-alpha had no effect on these tumor cells, while cytotoxicity against SW948 and WiDR was blocked by anti-TNF-alpha. Experiments using a transwell system stressed the importance of close intercellular contact for this process. Anti-IL-1 did not inhibit cytotoxicity against SW948. Modulation of HT29, WiDR, and SW948 by IFN-gamma (500 U/ml) induced a significant increase in cytotoxicity. We conclude that cell-associated TNF-alpha may be responsible for KC cytotoxicity against SW948, a process requiring close intercellular contact. WiDR is only partly lysed by a TNF-alpha-dependent mechanism, whereas HT29 is not. Furthermore, IFN-gamma is involved in the regulation of tumor susceptibility.

Cytotoxicity, Immunologic↗
PubMed1995

Novel isolation and purification method permitting functional cytotoxicity studies of macrophages from milky spots in the greater omentum.

Milky spots in the greater omentum are well organized perivascular infiltrates of leukocytes which are probably involved in the clearance of tumor cells from the peritoneal cavity. In milky spots, macrophages are the predominant cell type forming a distinct population of cells. To investigate whether these macrophages have a function in the control of metastatic spread in the peritoneal cavity, a novel isolation and purification method was developed in order to study the functional cytotoxicity of macrophages from milky spots in the greater omentum against tumor cells in vitro. In order to obtain a cell suspension, greater omenta of unstimulated healthy male WAG/RIJ rats were incubated in collagenase/DNase suspension and filtered. Subsequently, macrophages were isolated and purified using flow cytometry by sorting unstained cells on the basis of size and internal complexity. Macrophages and other cells were identified by routine May-Grünwald-Giemsa staining and by immunophenotyping with the specific macrophage monoclonal antibody ED 1. Furthermore, macrophage subtypes were characterized by ultrastructural analysis. Functional cytotoxicity of the isolated macrophages was assayed against the syngeneic CC 531 tumor cell line in a colorimetric MTT assay. From three greater omenta of healthy rats 1.16 +/- 0.16 x 10(6) macrophages were isolated with a purity of 83 +/- 2% and a viability of > or = 96%. The macrophages were of the exudate (monocytic), exudate-resident and resident cell type and were in equal proportions. The contaminating cells were mainly mesothelial. A maximum cytotoxicity of approximately 30% was reached with the macrophage fraction at an effector-to-target ratio of 10. Furthermore, it was established that the mesothelial cells did not exhibit cytotoxicity.

Animals↗
PubMed1995

Cellular composition of milky spots in the human greater omentum: an immunochemical and ultrastructural study.

BACKGROUND: Milky spots in the greater omentum of some animals are well organized perivascular infiltrates of leucocytes, and are considered to have characteristics of secondary lymphoid tissue. To determine whether milky spots in the human greater omentum can also be regarded as secondary lymphoid tissue, we studied milky spots in an unstimulated state. METHODS: Patients were selected on the basis of absence of disease in the peritoneal cavity that might influence the state of the milky spots. Using monoclonal antibodies against macrophages, B-lymphocytes and T-lymphocytes, and immunoperoxidase labeling, the number of these cells and their location in milky spots were studied by light microscopy. However, the stromal components of the greater omentum, especially those within the milky spots, were studied by electron microscopy. RESULTS: Milky spots in the human greater omentum are relatively uniform vascularized accumulations of mononuclear cells comprising macrophages (67.9% +/- 9.4, mean +/- standard deviation), B-cells (10.1% +/- 3.4), T-cells (10.2% +/- 3.7), and mast cells. However, no special B-cell and T-cell areas could be distinguished. On the ultrastructural level it was demonstrated that macrophages are present in different stages of maturation and can enter or leave the milky spots. Furthermore, no cells characteristic of secondary lymphoid organs, such as interdigitating cells or follicular dendritic cells, were seen. CONCLUSIONS: These data indicate that unstimulated milky spots in the human greater omentum are to a great extent just a preformed specific accumulation of primarily macrophages within the stroma of the greater omentum, and therefore, cannot be regarded as true secondary lymphoid tissue. Milky spots could serve as a gateway for, as well as a provider of peritoneal macrophages when the intra-abdominal status so requires. Finally, the data from this study are compared with the data of other studies of human milky spots and those in animals.

Adolescent↗
PubMed1995

Fresh colorectal tumor cells isolated from individual patients differ in their susceptibility to monocyte mediated cytotoxicity.

Studies on monocyte/macrophage mediated cytotoxicity usually pertain to the use of cell lines that are liable to antigenic and structural changes. Therefore we compared monocyte mediated cytotoxicity against colorectal tumor cell lines (WiDR, HT29, SW620, and SW948) with fresh colorectal tumor cells from patients. Fresh tumor cells were isolated from surgical specimens by a short enzymatic treatment (Collagenase/DNAse). Monocytes were obtained from one healthy donor. Cytotoxicity was determined using the MTT-assay. Fresh colorectal tumor cells displayed a similar differential susceptibility to cytotoxic monocytes as cell lines. Cytotoxicity against fresh tumor cells ranged from 4.9% to 50.4% at E/T ratio 5 (n = 9). Activation of monocytes with Interferon-gamma (100 U/ml) induced an increase of 6.2% +/- 1.6 (n = 4, P = 0.06). In this study we demonstrate monocyte mediated cytotoxicity against colorectal tumor cells isolated from individual patients. This may be important in view of the development of adoptive immunotherapy and cell-directed immunotherapy.

Cell Separation↗
PubMed1995

The combination 5-fluorouracil/levamisole induces enhanced rat Kupffer cell-mediated cytotoxicity in vitro against the syngeneic colon adenocarcinoma cell line CC531.

The mode of action of the combination treatment 5-fluorouracil (5-FU) and levamisole in colorectal cancer patients is unknown. It is postulated that the beneficial effect may be explained by an immunomodulatory effect on Kupffer cell (KC) cytotoxicity. We evaluated the effect of levamisole (200 micrograms/ml) and 5-FU (10 microM) on rat KC cytotoxicity against syngeneic CC531 tumor cells. Viability of KCs was unaffected by 5-FU and/or levamisole. The combination did not enhance growth inhibition of CC531 compared to 5-FU alone. A significant increase in KC cytotoxicity was observed after 24-hr incubation with 5-FU/levamisole especially at an effector/target ratio of 10 (P < 0.05). 5-FU alone had no effect on KC cytotoxicity, while levamisole induced only a slight increase. Our in vitro data suggest that the additive effect of the combination 5-FU/levamisole on KC cytotoxicity may attribute to the beneficial effect of the adjuvant treatment in colorectal cancer patients.

Adenocarcinoma↗
PubMed1995

Effect of glucose in dialysis fluid on antibacterial defence in the peritoneal cavity.

To study the effect of glucose concentration and dwell time of dialysis fluid on peritoneal antibacterial defence, an experimental infection with Staphylococcus aureus was induced in rats. For this purpose rats were inoculated intraperitoneally with Staphylococcus aureus at different intervals after the administration of various dialysis fluids. Twenty-four hours later the numbers of bacteria and cells in the peritoneal cavity were determined. The number of bacteria was correlated positively with the glucose concentration. Furthermore, an inverse correlation between dwell time and the number of bacteria was observed. Neither finding could be attributed to a glucose-dependent growth of the bacteria or disruption of the killing capacity of peritoneal cells in vitro. A glucose-dependent increase in the volume of the peritoneal fluid could partially explain the differences found in vivo. It is concluded that the glucose in dialysis fluid impairs antibacterial defence in the peritoneal cavity and that longer dwell times enhance this defence.

Animals↗
PubMed1995

Interleukin-8 production by human mesothelial cells after direct stimulation with staphylococci.

Mesothelial cells (MC) are able to produce interleukin-8 (IL-8) after stimulation with IL-1 beta or tumor necrosis factor alpha. The aim of our study was to investigate whether MC are able to produce IL-8 after direct stimulation with clinically relevant bacteria. We observed a significant IL-8 response by the MC which were directly stimulated with viable staphylococci.

Cells, Cultured↗
PubMed1995

Effect of interferon-gamma in dialysis fluid on peritoneal defence in rats.

BACKGROUND: A major drawback of continuous ambulatory peritoneal dialysis (CAPD) is the occurrence of peritoneal infection. This might be explained by a non-optimal phagocytic capacity of peritoneal cells which can be improved by stimulating factors. AIM: To investigate the effect of addition of interferon-gamma (IFN) to dialysis fluid with various glucose concentrations or to saline (as control) on the peritoneal defence against Staphylococcus aureus in an experimental dialysis model in rats. METHODS: Twenty-four hours after the administration of either dialysis fluid containing various glucose concentrations or saline with or without IFN, bacteria were injected intraperitoneally. At the time of the bacterial infection and 24 h later cellular and bacterial parameters were studied. RESULTS: The addition of IFN to dialysis fluid or saline resulted in a significant (P < 0.01) increase in the number of peritoneal macrophages at the time of infection; this was accompanied by a significant increase in both the number of Ia-positive peritoneal macrophages (P < 0.01) and the production of nitrite by macrophages (P < 0.05) at the time. IFN in dialysis fluid as well as in saline significantly (P < 0.01) reduced the recovery of bacteria from the peritoneal cavity 24 h after infection. Only the absence of IFN glucose increased the recovery of bacteria from the peritoneal cavity at the same time. CONCLUSION: In this experimental model the addition of IFN to dialysis fluid lowered the recovery of staphylococci from the peritoneal cavity by means of activation of an increased number of macrophages.

Animals↗
PubMed1995

Cancer antigen 125: a bulk marker for the mesothelial mass in stable peritoneal dialysis patients.

Mesothelial cells that line the peritoneal cavity are capable of producing several proinflammatory cytokines such as interleukin-6 and interleukin-8. Since they are the most numerous cell in the peritoneal cavity when the lining mesothelial cells are included, they may play a major role in the local antibacterial defence mechanism. Cancer antigen (CA)125 is expressed by mesothelial cells (as by other coelomic epithelium-derived cells) and might therefore be considered a marker of the mesothelium. The aim of this study was to determine whether CA125 is a bulk or an activation stage mesothelial cell marker. A positive correlation was found between the mesothelial cell number and the CA125 concentration in dialysate of stable PD patients (P = 0.03). CA125 release by mesothelial cell cultures during confluence showed that the release per cell was constant in time. Stimulation of mesothelial cells in a confluential phase with IL1 beta, TNF alpha, IFN gamma and TGF beta did not result in an increase in CA125 release. Cell lysis showed that CA125 is also present intracellularly. This implies that release of intracellular CA125 can be a disturbing factor in interpreting the CA125 concentration of dialysate in situations where mesothelial cell death may occur, such as in peritonitis. It can be concluded, that our data show that dialysate CA125 is a bulk marker for the mesothelial cell mass in stable PD patients and can thus provide data on the state of the peritoneal membrane in the follow-up of the individual patient.

Adult↗
PubMed1995

Isolation and purification of large quantities of fresh human Kupffer cells, which are cytotoxic against colon carcinoma.

A new rapid method is described for the isolation and purification of functional active human Kupffer cells without the need of in situ perfusion techniques. Liver wedge biopsies (3 to 5 g), obtained after laparotomy, were incubated with pronase under continuous pH registration. Human Kupffer cells were subsequently separated from other nonparenchymal cells by Nycodenz gradient centrifugation and purified by counterflow centrifugal elutriation. Kupffer cells, 1.7 +/- 0.4 x 10(6) per gram liver, were isolated with a purity of 95% +/- 3%. Cell-mediated cytotoxicity of Kupffer cells was assayed against a human colon carcinoma cell line (SW948). Kupffer cell cytotoxicity was 42% +/- 9% (mean +/- SD) at an effector-to-target cell ratio of 10 and significantly increased to 73 +/- 17% (P < .05) after activation of Kupffer cells with interferon-gamma. In conclusion, a reliable and relatively simple method is provided to isolate and purify fresh human Kupffer cells in large yields, which show spontaneous as well as gamma-interferon-induced cytotoxicity against a human colon carcinoma cell line.

Carcinoma↗
PubMed1994

Isolation of rat and human Kupffer cells by a modified enzymatic assay.

A new rapid method is described for the isolation and purification of rat and human Kupffer cells without the need of liver perfusion techniques. Rat livers or small human liver wedge biopsies obtained peroperatively were incubated with pronase under continuous pH registration. Kupffer cells were subsequently separated from other nonparenchymal cells by Nycodenz gradient centrifugation and purified by counterflow centrifugal elutriation. Identification of Kupffer cells was achieved on the basis of ultrastructural analyses and immunophenotyping.

Animals↗
PubMed1994

Methods for studying immuno-effector functions and antigen presenting activity of human macrophages.

Macrophages are now recognized as cells that perform a variety of functions making them important cells in both the specific and non-specific immune responses. In the inflammatory response mature monocytes leave the bloodstream to enter macrophages and then migrate to the site of infection. This process is usually co-ordinated by the production of so-called chemotaxins that direct the movements of the macrophages. This can be measured in vitro by a chemotaxis assay that separates macrophages from the chemotactic fluid by a micropore filter. After incubation migrated cells are counted at the other side of the filter and indicate the chemotactic response. At the inflammatory site phagocytosis and killing of invading bacterial micro-organisms is an important feature of macrophages. We describe an assay to measure phagocytosing capacity using IgG-coated sheep red blood cells and a bacterial killing assay. In addition a tumor killing assay is briefly introduced. Finally, the specific immune response is initiated by presentation of antigen to antigen specific T cells by specialized cells such as dendritic cells that are closely related to macrophages. An enrichment procedure for macrophages and dendritic cells is outlined and antigen presentation assays are described.

Antigen-Presenting Cells↗
PubMed1994

A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia.

The MTT-colorimetric monocyte mediated cytotoxicity assay, based upon the ability of living cells to reduce 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) into formazan, was evaluated using leukemic cells from five representative human leukemic cell lines and from 28 patients with acute myeloid leukemia (AML). An excellent linearity between absorbance and leukemic cell number was observed up to 5 x 10(4) cells/well and 50 x 10(4) cells/well for all cell lines and patients samples tested, respectively, in a 96-wells microtiter culture system. A huge variability in the susceptibility of leukemic cells to purified and IFN-gamma-activated human monocytes could be observed at effector-to-target cell (E:T) ratios of 1. The mean signal-to-noise ratio of the MTT assay for monocyte-leukemic cell mixtures from patients was 2.69 +/- 0.39 at E:T 1. In conclusion, the MTT based monocyte mediated cytotoxicity assay should be useful for studying the susceptibility of a variety of leukemic cells from cell lines and from patients with AML to monocytes in a rapid, sensitive and semi-automated manner.

Acute Disease↗
PubMed1994

An ultrastructural study of dopamine-immunoreactive nerve fibres in milky spots of the human greater omentum.

An ultrastructural study was performed to examine the presence and distribution of dopamine-immunoreactive nerve fibres in milky spots of the human greater omentum. Non-myelinated nerve fibres were located perivascularly as well as throughout the milky spots. Dopamine immunoreactivity was demonstrated in the nerve fibres and in a portion of the macrophage population. These results demonstrate a discrepancy between human and non-human milky spots.

Adult↗