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Biomedical subjects

W S Bont

Publications and source records attributed to W S Bont.

At least 19 recordsLinked to original sources
PubMed1985

The geometric progression of nuclear volumes.

Vesicles formed by the fragmentation of biomembranes form discrete size classes. Their surface areas can be represented by two geometric series with a common ratio of 2. The surface areas of membrane vesicles found in intact cells, including nuclear envelopes are terms of the same two series. These observations can be interpreted as indicating that nuclear volumes have physically determined, discrete values, which do not vary in a continuous way. Therefore an increase of nuclear volume, for any reason, will result in a jump to the next larger term in the series.

Animals↗
PubMed1985

Concentration of hematopoietic progenitor cells from human bone marrow by a new type of blood component separator.

A new type of blood component separator (BCS) was used for the isolation of hematopoietic progenitor cells from human bone marrow aspirates. The BCS was filled with 100-150 ml bone marrow and centrifuged to prepare a buffy coat. This buffy coat was isolated in 10-15% of the original bone marrow volume and contained 64 +/- 8% of the nucleated cells (NC). Morphological examination revealed that the buffy coat was highly enriched for myeloblasts, promyelocytes, lymphocytes and monocytes, whereas the contamination with granulocytes was reduced to 46 +/- 8% of the granulocytes initially present in the bone marrow suspension. In addition the contamination with red blood cells (RBC) was very low; the buffy coat contained only 6 +/- 2% of the RBC. Furthermore it was demonstrated by means of colony assays that the buffy coat was highly enriched for hematopoietic progenitor cells. It contained 91 +/- 6% of the granulocyte/monocyte progenitor cells (CFU-GM) and 87 +/- 9% of the erythroid progenitor cells (BFU-E). These results are comparable to those obtained with continuous or semicontinuous blood cell processors. The advantages of the BCS is that it is a simple and inexpensive apparatus which fits in a normal blood bank centrifuge. It permits efficient preparation and isolation of a buffy coat from human bone marrow without substantial loss of hematopoietic progenitor cells.

Bone Marrow Cells↗
PubMed1984

A centrifugal elutriation system of separating small numbers of cells.

One of the major disadvantages of centrifugal elutriation (CE) is the relatively large volume (150 ml) of the various fractions, especially if small numbers of cells have to be separated and the fractions contain few cells. To reduce the volume of the fractions 2 elutriator rotors were coupled in series. Since the rotor speed of the second rotor was always kept 750 rpm higher than that of the first rotor, cells elutriated from rotor 1 were collected in rotor 2. After elutriation of a complete fraction from rotor 1, and collection in rotor 2, the cells were harvested from rotor 2. This was achieved by means of a flow distribution unit (FDU), which made it possible to disconnect the flow of both rotors and simultaneously reverse the flow of the second rotor. It is demonstrated that 40-95 X 10(6) mononuclear leukocytes may be fractionated without loss of resolution in fractions of only 9 ml. The lymphocyte (greater than 99%) and monocyte subpopulations (88-94%) obtained were as pure as with CE carried out with only 1 rotor. In addition, the cells in rotor 2 could be washed and suspended in culture medium prior to harvesting by means of the FDU. In this way loss of cells by additional centrifugation steps was avoided. Erythrocytes (RBC) present in certain lymphocyte fractions were lysed with NH4Cl and after lysis of the RBC and elution of ghosts and debris, the cells were washed and harvested. This procedure did not affect cell viability and the PHA response of the lymphocytes. The versatile system described made it possible to apply CE for the separation of small numbers of cells without loss of resolution, and demonstrated that CE is ideally suitable for concentration and washing of cells, and removal of contaminating RBC, not affecting the recovery, viability and function of the cells.

Air Pressure↗
PubMed1983

Inhibition of amino acid incorporation in a cell-free system and inhibition of protein synthesis in cultured cells by reaction products of selenite and thiols.

Reaction products of selenite with thiols were tested for an inhibitory effect on amino acid incorporation in a cell-free system derived from rat liver and on protein synthesis in intact P815 and L1210 cells. In the cell-free system maximum inhibition, up to 96%, was reached at about 10 microM selenium. In intact cells inhibitory effect varied depending on which reaction product or cell line was used. Maximum inhibition was obtained after 30 min of incubation with selenium concentrations ranging from 0.25 microM to over 7 microM. Selenite itself also inhibited protein synthesis of L1210 cells, but only after 90 min of incubation and starting at selenium concentrations of 2 microM. Inhibition of protein synthesis in intact cells was followed by cell death. Pre-incubation of the reaction products of a monothiol (2-propanethiol) and of a vicinal dithiol (2,3-dimercapto-1-propanol) in culture medium showed a rapid decrease of the inhibitory capability of the product from the monothiol, but not of the product from the dithiol. The results indicate that selenite and a thiol react to form products which have differential toxic effects to cells in vitro.

Amino Acids↗
PubMed1983

Theory and practice of centrifugal elutriation (CE). Factors influencing the separation of human blood cells.

Centrifugal elutriation (CE) is currently a widely used preparative cell separation technique. In order to optimize the separation of cells that show only small differences in sedimentation velocity, several conditions that might influence the resolution capacity, such as rotor speed, counterflow, jetstream, cell load, density, and viscosity of the elutriation medium, were analyzed. Experiments carried out with human red blood cells (rbc) indicated that selective losses of rbc from the rotor caused by the jetstream, could be prevented if the separations were carried out at high rotor speeds, as predicted by the theory. In addition, high cell loads (5 X 10(8) rbc) resulted in better separations than low cell loads (5 X 10(7) rbc). Human monocytes were separated into subpopulations that differed only about 0.003 g/mL in density, but have virtually the same size. The separation was carried out either by increasing the density or viscosity of the elutriation medium or by decreasing the rotor speed. In all cases similar results were obtained. These results indicated that under optimal conditions CE can be applied for the separation of cells that differ only slightly in sedimentation velocity.

Blood Cells↗
PubMed1983

Induction of phenotypic differentiation, interleukin 2 production, and PHA responsiveness of "immature" human thymocytes by interleukin 1 and phorbol ester.

Small human thymocytes (ST) representing 70% of the thymocytes were isolated according to size by centrifugal elutriation. Although these ST contained approximately 30% PNA-cells, they failed to respond to lectins, indicating the existence of a PNA-ST subset that can be considered to belong to the "immature" thymocyte population. The ST were induced to proliferate if, in addition to PHA, IL 1-containing supernatants of highly purified monocyte cultures or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were present. The incubation of the ST for 90 hr with TPA or IL 1 in the absence of PHA resulted in a strong reduction in the percentage of cells reacting with the immature thymocyte markers TdT and PNA. In addition, the OKT6+ cells were partially reduced after incubation with IL 1. Concomitantly, an increase in the percentage of cells reacting with the mature T cell markers OKT1 and OKT3 was observed, whereas HLA antigens became strongly expressed on all ST. Although IL 1 or TPA were unable to induce proliferation of the ST, these substances induced IL 2 production by these cells. These shifts to cells with more "mature" phenotypes that are able to produce IL 2 were not observed if the ST were incubated with PHA or culture medium only. The responder capacity of the ST to PHA plus TPA was not significantly affected by the depletion of the more "mature" OKT3+ and OKT1+ cells. In addition, in this situation OKT1+, OKT3+, OKT6- cells were found to be generated from OKT1-, OKT3-, OKT6+ cells. Therefore, it could be excluded that the proliferative responses were due to a selective expansion of a preexisting mature T cell population. Our results indicate that TPA mimics IL 1 in the induction of differentiation of the ST to a stage in which subpopulations of these cells are able to produce IL 2 and to respond to PHA. Because only the proliferating ST were found to react with a monoclonal antibody, which is thought to be directed at the IL 2 receptor (anti-Tac), our data suggest that PHA is required for the induction of expression of receptors for IL 2 in those ST subpopulations that are able to proliferate in the presence of IL 2 generated in situ.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1982

Rapid isolation of mononuclear cells from buffy coats prepared by a new blood cell separator.

A newly developed cell separator for the preparation and fractionation of buffy coat cells from human peripheral blood is described. In this cell separator buffy coats (BC-1) as routinely obtained from blood banks were used for the preparation of a second buffy coat (BC-2) with a volume of only 5-6 ml. A special fractionation device allowed sterile isolation of almost pure platelets and mononuclear cells with recoveries of 75 +/- 10% and 89 +/- 4% respectively. The white blood cell contamination of the platelet suspension never exceeded 20 X 10(6) leukocytes (i.e., less than 1 leukocyte per 5000 platelets). Furthermore, the mononuclear cell suspensions were shown to be contaminated with only 3 +/- 2% granulocytes, whereas the white blood cell/red blood cell ratio was 2.6 +/- 1.6, so that they could therefore be directly used for further separation by means of centrifugal elutriation. These results indicate that this cell separator provides a rapid (+/- 1 h) isolation of both platelets and mononuclear cells without exposing the buffy coat cells to foreign substances like Ficoll or Percoll.

Blood Platelets↗
PubMed1982

Isolation of human thymocytes differing in maturation state and function by centrifugal elutriation.

A modified centrifugal elutriation technique was used to separate (up to 3 X 10(9)) human thymocytes, according to size in 6 different fractions. Eighty percent of the unfractionated thymocytes were recovered in fractions 1 and 2. The majority of these thymocytes appeared to be small and phenotypically immature as was determined by the high percentage of cells reacting with the monoclonal antibodies OKT-6, Mas-036 and peanut agglutinin. In addition, a relatively low percentage of the cells reacted with a monoclonal antibody directed against HLA-A, B and C determinants (Mas-015). The immaturity of these thymocytes was confirmed by their failure to respond to phytohemagglutinin (PHA) and their negligible responder capacity in mixed leukocyte cultures. Fractions 3-6, representing 20% of the unfractionated thymocytes, were collected arbitrarily and contained thymocytes of various maturation stages as judged by their phenotype. The PHA responsiveness and responder capacity in mixed leukocyte cultures of the thymocytes in these fractions were, in general, considerably higher than those of the unfractionated thymocytes. Our data indicate that centrifugal elutriation is a fast and reproducible method to separate large quantities of functionally inactive and phenotypically immature thymocytes from the more mature and functionally active thymocytes.

Antibodies, Monoclonal↗
PubMed1982

Isolation of functionally different human monocytes by counterflow centrifugation elutriation.

Human peripheral blood monocytes were isolated by counterflow centrifugation elutriation (CCE). This technique was modified in such a way that various monocyte fractions (viability greater than 99%) could be elutriated by increasing the density of the CCE-medium in steps of 0.0027 g/ml. All monocytes showed the same size distributions as determined by electronic sizing, which indicated that they differed in their density only. Both cytoplasmic esterase and peroxidase activity increased with the density of the cells. Furthermore, the monocytes with the highest density were 2.3-4 times more active in an antibody-dependent cellular cytotoxicity (ADCC) assay than those with the lowest density. In contrast, the monocyte with the highest density were less capable to induce the proliferation of lymphocytes in mixed leukocyte cultures (MLC) than those with the lowest density. This observation could not be attributed to differences in the expression of HLA-DR determinants, since a monoclonal antibody directed against HLA-DR antigens reacted equally well with the monocytes in different fractions. These results provide evidence for the existence of functionally different subsets of monocytes or different states of differentiation or maturation.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1981

Isolation of large numbers of highly purified lymphocytes and monocytes with a modified centrifugal elutriation technique.

A modified centrifugal elutriation technique is described for the isolation of large numbers of lymphocytes and monocytes. Elutriation was carried out by lowering the rotor speed at a constant flow rate which was generated by hydrostatic pressure. The flow rate could be kept constant if the separation procedure was performed at high pressure and high systemic resistance. Up to 2.3 X 10(9) mononuclear cells derived from 2000 ml blood were separated in one single experiment in approximately 1 h. The lymphocytes and monocytes were isolated at purities of 98 +/- 1% and 94 +/- 1% respectively. The purity of the lymphocytes was increased to 99.8 +/- 0.1% by a second elutriation run. Additional advantages of the elutriation procedures are that the choice of medium is free, and that relatively large numbers of cells may be separated with high recoveries.

Cell Separation↗
PubMed1980

Requirement for monocytes in the spontaneous cytotoxic effects of human lymphocytes against non-lymphoid target cells.

Human lymphocytes have been shown to lyse in vitro a large variety of target cells derived from tumour as well as normal tissues. The effector cells involved in this spontaneous cell-mediated cytotoxicity (SCMC) have generally been characterised as lymphocytes which lack surface membrane immunoglobulin (sIg) and lack or express only low-affinity receptors for sheep red blood cells (SRBC), but possess receptors for the Fc portion of IgG (refs 3--7). We have previously shown that monocytes may be involved in SCMC, as strong depletions of adherent cells always resulted in a reduction in SCMC. In the present study, experiments were undertaken to determine the role of monocytes in SCMC. Evidence is presented that monocytes have a helper function in the lymphocyte-mediated SCMC against target cells growing in monolayer cultures, whereas effector cells in SCMC against lymphoid target cells growing in suspension cultures were lysed by lymphocytes in the absence of monocytes.

Cells, Cultured↗
PubMed1980

The requirement for monocytes in spontaneous cytotoxicity by lymphocytes from healthy donors and melanoma patients.

Lymphocytes from 12 healthy donors were depleted of monocytes by velocity sedimentation at unit gravity (average monocyte contamination 0.2%) and tested for spontaneous cytotoxicity (SC) against tumor target cells in 23 h microcytotoxicity assays. Sixty percent of all lymphocytes were recovered in this monocyte-depleted lymphocyte fraction (LF). In contrast, with the corresponding unfractionated lymphocytes (UL) the LF cells were not spontaneously cytotoxic on T24 bladder carcinoma, NKI-1, NKI-7, NKI-8, NKI-10 melanoma, and MC-1 mammary carcinoma cell line cells and target cells derived from two short-term melanona cultures (Me 215 and Me 223). SC of the LF could be restored by reconstitution with autologous monocytes, which were obtained > 80% pure in the same velocity sedimentation procedure. Addition of 4-8% monocytes was sufficient to restore the SC of the LF to the level achieved with the corresponding UL, whereas maximal induction of SC was observed after addition of 8-16% monocytes. Higher numbers of monocytes had suboptimal effects. SC of the LF could also be restored by monocyte culture supernatants. The spontaneous cytotoxic effector cells were characterized as non-E rosette-forming, membrane sIg-negative Fc-receptor-bearing lymphocytes, a proportion of which probably also bear C3-receptors. Since the LF was not spontaneously cytotoxic, in spite of the presence of Fc-receptor bearing lymphocytes, it is concluded that monocyte help, mediated via soluble factors, is required. Evidence is presented that the lack of SC by the LF might be attributable to the failure of these cells to make appropriate contact with the target cells. LF/target cell contact followed by target cell kill occurred after addition of monocytes. Effector/target cell contacts, artificially established by agglutinins, did not result in target cell kill. Thus the effector/target cell contacts observed in the presence of monocytes suggest recognition of particular membraner determinants involved in SC. An identical requirement for monocytes was observed with LF cells from 5 stage 1 and 5 stage 11 melanoma patients tested for SC against both melanoma and non-melanoma target cells. This indicates that either the cytotoxicity against melanoma cells with lymphocytes from melanoma patients is of the same nature as the SC of lymphocytes from healthy donors, or that specific melanoma-associated cellular cytotoxicioty also requires monocyte help.

Adult↗