Search PubMed⌕ Search

Biomedical subjects

C G Figdor

Publications and source records attributed to C G Figdor.

At least 163 records · Page 9Linked to original sources
PubMed1987

Role of p150,95 in adhesion, migration, chemotaxis and phagocytosis of human monocytes.

The leukocyte function-associated antigen-1 (LFA-1), the C3bi receptor (CR3) and the p150,95 antigen belong to a family of leukocyte surface molecules consisting of bimolecular complexes with alpha chains of 170 kDa, 165 kDa and 150 kDa, respectively, and a common beta subunit with a mol. mass of 95 kDa. In order to determine the function of the p150,95 antigen on human monocytes and U937 cells, and to study the functional relationship between this antigen and LFA-1 or CR3, we investigated the influence of monoclonal antibodies (mAb) directed against these cell surface molecules on the adhesive properties of these cells. The observation that anti-beta chain mAb strongly inhibited migration, chemotaxis, adhesion and phagocytosis of monocytic cells indicates a major role for LFA-1 family antigens in monocyte functions. Detailed analysis with a panel of anti-alpha chain antibodies demonstrated that both p150,95 and LFA-1 mediate random migration whereas in contrast, p150,95 and CR3 were shown to be involved in the directed migration of monocytes to f-Met-Leu-Phe. Furthermore, adhesion of monocytes to plastic surfaces or monolayers of endothelial cells as well as phagocytosis of latex particles was mediated by p150,95. The results demonstrate that, in spite of its relative low expression, the p150,95 glycoprotein is a major adhesion-associated molecule expressed by human monocytic cells.

Antigens, Surface↗
PubMed1987

Computer-assisted centrifugal elutriation. I. Detection system and data acquisition equipment.

To optimize cell separations by centrifugal elutriation, we constructed an on-line computer-controlled multiparametric light-scatter system. A bypass sample flow, at the outlet of an elutriation rotor, is hydrofocussed and three scatter parameters of each cell are determined up to a maximum of 15,000 cells/second. The 18-bit representation of the parameter values are cumulatively stored by means of a direct memory access interface. The histogram memory is continuously displayed to provide information on the number and type of cells that are elutriated. A special purpose operating system, implemented on a stand-alone computer configuration, allows a high data-acquisition rate and ample data processing capacity. In addition, a local network driver was constructed to facilitate off-line detailed analysis of the data. The equipment is well suited to monitor the centrigural elutriation process. The flexibility of the system allows an extension of the monitor to computer-controlled elutriation.

Animals↗
PubMed1987

Differential function of LFA-1 family molecules (CD11 and CD18) in adhesion of human monocytes to melanoma and endothelial cells.

Human peripheral blood monocytes from normal, healthy donors express the leucocyte function-associated antigen (LFA)-1, CR3 and p150,95. These heterodimeric antigens are members of a glycoprotein family sharing a common beta subunit but endowed with distinct alpha chains. They have been shown to play an important role in cell-cell interactions. In the present study we have investigated the role of these molecules in the interaction of monocytes with endothelial cells and melanoma (tumour) cells. Heterotypic cell-cell interactions were studied in single cell conjugate assays and by adhesion of monocytes to monolayers of cells. The results demonstrate that monoclonal antibodies directed against LFA-1 alpha, CR3 alpha, p150,95 alpha and the common beta chain strongly reduce the number of conjugates (71, 50, 60 and 89% inhibition, respectively), formed between monocytes and melanoma or endothelial cells in a single cell assay. In contrast, adhesion of monocytes to monolayers of the same cells seems only to depend on p150,95, since only antibodies directed to the alpha chain of this molecule and to the common beta chain inhibited adhesion. Interestingly, the number of conjugates formed with melanoma cells in single cell assays was at least twice the number of conjugates formed between monocytes and endothelial cells, whereas no differences were observed in the adhesion of monocytes to monolayers of these cells. However, the basis for this phenomenon is not yet clear. These results indicate that not only LFA-1 but also CR3 and p150,95 can mediate adhesion to target cells in suspension, but that monocyte adhesion to monolayers is caused by a different mechanism in which the p150,95 molecule seems to play a prominent role.

Antigens, Neoplasm↗
PubMed1986

Sensitive and quantitative determination of monocyte adherence.

A new sensitive and highly reproducible one-step ELISA is described to quantitatively determine the adherent capacity of monocytes and related cell lines. Cells were labelled with a monoclonal antibody/peroxidase conjugate which did not affect the adhesive properties of these cells. The labelled cells were allowed to adhere for 1 h and subsequently stained by the addition of substrate. The results demonstrate that there is a good correlation between the number of peroxidase-labelled adherent cells and the absorbance measured at 450 nm. Furthermore the assay permits the use of very low cell numbers since adherent cells could be measured efficiently at a level of only 100-500 cells/well. The method may be very useful in the selection of hybridomas that secrete antibodies which inhibit adherence of cells. In addition it can be applied to study the adhesive properties of any cell type, provided that appropriate monoclonal antibodies are available.

Cell Adhesion↗
PubMed1986

Characterization of melanoma-associated surface antigens involved in the adhesion and motility of human melanoma cells.

The functional properties of the melanoma-associated antigens detected by monoclonal antibodies (MAbs) AMF-6 and AMF-7 were investigated. These MAbs were selected previously because of their capacity to block the anti-melanoma reactivity of cytotoxic T-lymphocyte clones AMF-6 and AMF-7 detect a melanoma-associated proteoglycan (MW greater than 450-250 kDa) and a molecular complex, which under reducing conditions consists of 4 compounds of 120, 95, 29 and 25 kDa respectively. AMF-6 reacted strongly with all 30 cultured melanomas and all 41 melanomas in frozen tissue sections. Significant cross-reactivity was only observed with nevi and perineurium, whereas normal skin melanocytes were negative. AMF-7 reacted with all 25 cultured melanomas and all 34 melanomas in frozen sections. AMF-7 cross-reacted with a proportion of nevi and endothelial cells from small vessels. The antigen detected by AMF-6 and AMF-7 could not be modulated by retinoic acid or recombinant gamma-IFN, which induced or enhanced the expression of HLA-DR, HLA-DQ and Class-I MHC antigens. In addition, the antigens were not readily modulated when cells were incubated in excess amounts of AMF-6 and AMF-7. Interestingly, the antigen detected by AMF-7 was strongly associated with the adhesion and cytoplasmic spreading of melanoma cells to plastic surfaces and monolayers of vascular endothelial cells. AMF-6 did not block the adhesion of melanoma cells but delayed cytoplasmic spreading. Both AMF-6 and AMF-7 blocked fibronectin-induced chemotaxic motility and chemokinesis of melanoma cells. In addition to their membrane localization, the antigens detected by AMF-6 and AMF-7 were also abundant in extracellular adhesion plaques deposited by cultured melanoma cells. Our results indicate that the high-MW melanoma-associated proteoglycan and the antigen detected by AMF-7 are associated with adhesion and/or cytoplasmic spreading and motility of human melanoma cells, suggesting that these antigens are associated with the (hematogenic) dissemination of human melanoma. This is supported by the finding that AMF-7 stained primary tumors heterogeneously, whereas metastases were homogeneously stained.

Animals↗
PubMed1985

Biochemical and functional characteristics of the human leukocyte membrane antigen family LFA-1, Mo-1 and p150,95.

The human leukocyte function-associated (LFA-1) antigen, the monocyte differentiation antigen Mo-1 which is characterized as the C3bi receptor and the glycoprotein p150,95 are characterized biochemically. Immunoprecipitations carried out with 6 different monoclonal antibodies (mAb) against LFA-1 indicated that four mAb (SPV-L1, SPV-L5, SPV-L7 and SPV-L11) were directed against the alpha chain, whereas mAb CLB54 and MHM-23 were found to react with the common beta chain of LFA-1, Mo-1 and p150,95. LFA-1 and Mo-1 expressed on KG-1 cells or lymphocytes, monocytes and granulocytes from one donor were homogeneous. Interestingly the alpha chain of p150,95 showed heterogeneity. The molecular weight of the alpha chain expressed on monocytes was consistently higher than that of the alpha chain on granulocytes. The beta subunits of LFA-1 and Mo-1 (as detected by mAb Bear-1) are not only similar in molecular weight and isoelectric focusing patterns, but it is demonstrated here that they are also identically glycosylated and have similar protein backbones as judged by tryptic peptide mapping. In spite of their structural similarities. LFA-1 and Mo-1 differ completely in some of their biological functions. Anti-LFA-1 mAb strongly inhibited monocyte-dependent T cell proliferation induced by tetanus toxoid or Helix pomatia hemocyanin and pokeweed mitogen-driven specific antibody production in vitro, whereas the anti-Mo-1 antibody Bear-1 was ineffective. These results suggest that the differences in these biological functions of LFA-1 and Mo-1 may be related to their different alpha subunits, which may recognize specific counter structures.

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↗
PubMed1984

Development of human connective tissue mast cells from purified blood monocytes.

Highly purified subfractions of human peripheral blood monocytes, when cultured in the presence of 30% L cell supernatant and 30% horse serum, assumed all the characteristics that define human connective tissue mast cells. After three weeks of culture, 75% of the cells developed metachromasia and granular chloroacetate esterase staining, and their intracellular histamine levels increased from 0.0 to 50.5 ng/10(6) cells. On electron microscopy, the cells developed intracytoplasmic granules with all the features typical for mature and immature mast cells. Cultured cells bound 55 pg 125I-IgE/10(6) cells, while labelling was negligible with cells prior to culture and with heat-denatured 125I-IgE. Fluorescent staining with anti-IgE increased slightly as well, while staining with monoclonal anti-monocyte and anti-HLA-Dr markers decreased. Purified lymphocytes did not assume mast cell characteristics, and lymphokines did not induce or enhance in vitro mast cell development or IgE binding. The data therefore further support the concept that connective tissue mast cells arise from the monocytoid lineage.

Binding Sites, Antibody↗
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

Lymphocyte maturation in the human thymus. Relevance of purine nucleotide metabolism for intrathymic T cell function.

The combination of centrifugal elutriation as an efficient and reproducible method to separate thymocytes by size, micromethods to assess purine interconversion enzymes, and assessment of purine (deoxy)nucleoside inhibition of mitogen responses enabled us to study purine metabolism at the intrathymic level. Out of six fractions, four (nos. 3-6), containing medium- and large-sized lymphocytes, showed a proliferative response after stimulation with phytohaemagglutinin (PHA). In fractions 1-6 the number of cells with an immature immunological phenotype gradually decreased, and cells with the phenotype of mature cells gradually increased. The enzyme activity ratio of adenosine deaminase to purine nucleoside phosphorylase gradually decreased from 21 in fraction 1 to 7 in the last fraction (blood T-cell value, 0.7). We conclude that this enzyme activity ratio is a useful marker for intrathymic T-cell maturation stages. In PHA-responsive cell fractions (3-6), the sensitivity to inhibition of the PHA response by (deoxy)adenosine and deoxyguanosine was inversely related to the enzyme activity ratio of ecto-5'-nucleotidase to deoxycytidine kinase. These findings are compatible with the hypothesis that intracellular concentrations of phosphorylated (deoxy)nucleosides are related to this inhibition. We conclude that the differences in purine metabolism among the various (mitogen-responsive) human thymocyte fractions are related to lymphoid cell function. Since the number of cells contributing to the enzyme activities and the number of cells contributing to the proliferative response (about 15% of unseparated cells) differ considerably, it is not possible to evaluate enzyme activities in unseparated thymocytes in terms of relationships between purine metabolism and lymphocyte function.

Adenosine Deaminase↗
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↗