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Biomedical subjects

G Gaudernack

Publications and source records attributed to G Gaudernack.

At least 109 records · Page 6Linked to original sources
PubMed1988

Immunomagnetic isolation of cells for serological BoLA typing.

This paper describes a totally new immunomagnetic (IM) technique adapted to serological BoLA typing. The basic technique has recently been developed by Vartdal et al. (1986) for serological HLA typing. The main advantage is that bovine mononuclear cells (e.g. T-cells and possibly their subsets, B-cells and monocytes) can be quickly and specifically isolated with high yield and viability from whole blood in a one-step procedure. This is achieved by magnetic separation of rosettes formed between the cells and superparamagnetic monosized polystyrene microspheres (Dynabeads TM) coated with cross-species reactive monoclonal antibodies (MAbs) specific for various human T-cell antigens or for HLA class II monomorphic epitopes. The cells are isolated within 5 min after a 5-min incubation at 4 degrees C. Magnetic separation of rosettes with a strong cobalt-samarium magnet eliminates all the laborious centrifugation steps necessary with conventional procedures. The isolated cells, still attached to the particles, are available for microcytotoxic assay. This is carried out within 55 min, including a two-step application of alloantiserum and complement and addition of acridine orange/ethidium bromide for the staining of viable (green) and dead (red) cells. The high viability of isolated cells gives a very low background kill compared with the conventional cytotoxic assay. The IM typing technique is also superior in sensitivity to the conventional technique as standardized for the international BoLA comparison test. The IM technique is likely to have its greatest impact on class II typing; class II positive cells being separated very efficiently. Polymorphic HLA class II MAbs detected likely polymorphic BoLA class II epitopes.

Animals↗
PubMed1988

Characterization of a monoclonal antibody ITI-Pl 1 directed against human platelet membrane glycoprotein IIb using extracts of whole platelets and platelet surface and intracellular membranes.

A monoclonal antibody (mAb) termed ITI-Pl 1 has been prepared by the hybridoma procedure. Using immuno-absorption and crossed immunoelectrophoresis of Triton X-100 extracts of untreated and EDTA-treated human platelets it was shown to be directed against the surface membrane glycoprotein IIb (GP IIb). This mAb binds to whole platelets independently of ADP-stimulation and the presence of Ca2+-ions. It saturates at around 870 ng/10(8) cells corresponding to approximately 35,800 molecules/platelet. ITI-Pl 1 did not significantly inhibit GP IIb-IIIa dependent functions such as platelet aggregation or fibrinogen binding. Immunofluorescence could be demonstrated using ITI-Pl 1 and intact normal platelets, but not with platelets from a Glanzmann's thrombasthenia patient. Crossed immuno-electrophoresis with platelet extracts from four different thrombasthenic patients gave a line precipitate in the intermediate gel with 125I-labelled ITI-Pl 1 and autoradiography indicating trace amounts of free GP IIb or the GP IIb-IIIa complex. The epitope on GP IIb detected by ITI-Pl 1 is not destroyed by neuraminidase treatment. Thus the mAb also interacts with neuraminidase-treated GP IIb-IIIa complex in highly purified platelet surface membrane fractions as well as with GP IIb-IIIa from untreated internal membranes isolated by continuous flow electrophoresis.

Animals↗
PubMed1988

Accessory cell-dependent T-cell activation via Ti-CD3. Involvement of CD2-LFA-3 interactions.

The activation of resting T cells to interleukin 2 (IL-2) production and DNA synthesis via Ti-CD3 is dependent on accessory cells (AC). Using positively selected, resting T cells activated with particle-bound anti-CD3, we investigated the ability of various cell lines to function as AC. We found that cell lines able to act as AC all expressed LFA-3, while cell lines not expressing LFA-3 were unable to provide AC signals. This applied to CD3+, CD4+, and CD8+ T cells. Sheep red blood cells (SRBC), which express LFA-3-like molecules, also had a weak, but significant AC function in this test system. Both CD4+ and CD8+ T cells activated with particle-bound anti-CD3 could be induced to enter DNA synthesis in the absence of AC when monoclonal antibodies reacting with CD2 were present instead of AC. IL-2 production could be detected in the latter cultures but not when positively selected CD3+ or CD2+ T cells were cultured alone. Our data suggest that activation of resting T cells via CD3 will lead to IL-2 receptor expression, while the interactions between LFA-3 and its ligand CD2 provide the necessary secondary signals for IL-2 production and induction of DNA synthesis.

Animals↗
PubMed1988

Induction of interleukin-2 production in CD4+ and CD8+ T-cell subsets after activation via CD3 and CD2.

The activation signals necessary for interleukin-2 (IL-2) receptor induction, IL-2 production, and DNA synthesis in resting T cells were investigated. IL-2 receptors were induced after activation via CD2 or CD3 alone, while IL-2 production in both CD4+ and CD8+ T cells required activation via both CD3 and CD2. The sequence of activation signals via CD3 and CD2 was shown to be important since DNA synthesis was induced when the primary activation signal was delivered via CD3, and the CD2 signal within 8 h. In contrast, no DNA synthesis was demonstrated when the primary activation signal was delivered via CD2 and the CD3 signal later. Ciclosporin A (CyA) inhibited T-cell DNA synthesis after activation via CD2 and CD3. The inhibition seemed to be due to the prevention of IL-2 synthesis.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Role of accessory cells in the activation of pure T cells via the T cell receptor-CD3 complex or with phytohaemagglutinin.

The ability of different subpopulations of blood mononuclear cells to serve as accessory cells in the activation of CD4+ and CD8+ T cells via Ti-CD3 or with phytohaemagglutinin (PHA) was studied. Pure CD4+ or CD8+ T cells did not respond to particle-bound anti-CD3 monoclonal antibodies (MoAb) or PHA, whereas responses were seen when non-T cells served as accessory cells. Removal of class II-positive cells from peripheral blood mononuclear cells (PBMC) or from non-T cells diminished, but did not completely abolish, the responses in both T cell subsets, indicating that the accessory cells are mainly found among the class II-positive cells. However, the class II molecules themselves were not involved, as demonstrated in antibody-blocking experiments. Removal of monocytes decreased the ability of non-T cells to serve as accessory cells for both CD4+ and CD8+ cells in PHA activation. In contrast, the removal of monocytes resulted in an enhanced activation by anti-CD3 MoAb in CD4+ T cells, while the activation of CD8+ T cells was less affected. Positively selected B cells were effective accessory cells in anti-CD3 and PHA activation. Furthermore, Epstein-Barr virus (EBV)-transformed B cell lines were very potent accessory cells both in anti-CD3 and PHA activation of T cells, and showed the strongest accessory cell function observed in this system on a per cell basis.

Antigen-Presenting Cells↗
PubMed1988

Requirements for phytohaemagglutinin activation of resting pure CD4+ and CD8+ T cells.

We have utilized a new method for obtaining highly purified cells using positive selection by immunomagnetic separation to study the conditions required for phytohaemagglutinin (PHA) activation of pure T4 and T8 cells. In the presence of accessory cells (AC), a comparable proliferative response was obtained in the two subsets. In the absence of AC, PHA induced low levels of interleukin 2 (IL-2) receptor expression as well as responsiveness to IL-2 in both T4 and T8 cells. If AC or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were also present, IL-2 production and DNA synthesis were seen in both subsets. A short preincubation with PHA 'primed' T cells for subsequent responsiveness to IL-2 or TPA, while preincubation with TPA did not induce response to PHA. Thus, PHA alone is sufficient for the first step of T cell activation leading to IL-2 receptor expression. The second step leading to IL-2 production, is dependent on direct interaction with AC in the presence of PHA. While T8 cells are dependent on help by T4 cells for proliferation to occur during stimulation with antigen, in PHA stimulation the requirements for activation and proliferation seem to be identical for T4 and T8 cells.

Antigen-Presenting Cells↗
PubMed1988

HLA-DQw3.1 and DQw3.2 associated exon polymorphisms detected by oligonucleotide probes.

HLA class II polymorphisms have been analyzed on the genomic level by two oligonucleotide probes corresponding to the DNA sequence coding for the amino acids 23-30 in the first domain of the beta chain of DQw3.1 and DQw3.2. Specific hybridization to single endonuclease fragments of DNA from some HLA homozygous cells was detected. Here we report the distribution of these DNA exon sequences among different DQ alleles, and demonstrate that the probes may be used to type for DQw3.1 and DQw3.2 respectively.

DNA↗
PubMed1988

Human granulopoiesis in vitro--medium-dependent growth regulation by a granulocyte-derived inhibitor.

A number of reports have indicated that mature blood granulocytes produce regulators that inhibit proliferation of progenitor cells in the bone marrow. However, this concept of negative feedback of granulopoiesis is still controversial. To examine whether conflicting results may depend upon the experimental set-up, we have compared colony formation by human bone marrow cells in different growth media. Unmodified McCoy's medium, which in feeder layer cultures supports the formation of large numbers of colonies, was a poor growth medium in cultures supplied with crude or recombinant colony stimulating factor (CSF). The colony formation improved when the medium was supplemented with defined additives. In CMRL 1066 cultures, granulocyte extract (GRE) consistently caused a strong inhibition of colony formation. In contrast, with unmodified McCoy's medium, granulocyte extract enhanced colony formation in a dose-dependent manner. The enhancing effect of granulocyte extract coincided with low colony numbers in the control cultures. The stimulatory effect of granulocyte extract in McCoy's medium, switched to strong inhibition when thymidine, a component of CMRL 1066 medium, was added. The inhibitory and stimulatory activities were found in the same molecular weight fractions (30-60 kD) after gel filtration. Both modulators in granulocyte extract appeared to be independent of monocytes and T lymphocytes in the bone marrow, as shown by removal of these cells with magnetic microspheres coated with specific monoclonal antibodies. The present work shows that regulation of cell proliferation in vitro depends strongly on culture conditions, such as choice of medium. It appears that thymidine acts as co-factor for the inhibitor in granulocyte extract.

Bone Marrow Cells↗
PubMed1988

Direct immunomagnetic quantification of lymphocyte subsets in blood.

A method is described where superparamagnetic polymer microspheres coated with monoclonal antibodies (MoAb) are used for the direct and fast quantification of the absolute number of cells of various lymphocyte subsets in blood. Blood samples were incubated with microspheres coated with a subset specific MoAb. Using a magnet the microsphere-rosetted cells were isolated and washed. Following lysis of the cell walls to detach the microspheres, the cell nuclei were stained with acridine orange and counted in a haemocytometer using an immunofluorescence microscope. With MoAb specific for CD2, CD4, CD8 and CD19, reproducible absolute counts of the corresponding lymphocyte subsets were obtained which correlated closely with those obtained by an indirect quantification method.

Cell Separation↗
PubMed1987

Identification of platelet antigens by monoclonal antibodies using crossed immunoelectrophoresis with immunoblotting of the monoclonal antibody.

A method is described for the identification of antigens by monoclonal antibodies. This is applicable whenever precipitating antibodies to the same antigens from a different species are available. The method is based upon: Separation and immunoprecipitation of cellular proteins with a polyspecific antiserum in crossed immunoelectrophoresis in the presence of the non-denaturing detergent Triton X-100 and the monoclonal antibody. Coprecipitation of the monoclonal antibody with its antigen. Subsequent passive transfer of the monoclonal antibody in the antibody-antigen complex onto a nitrocellulose membrane. Visualization of the blotted antibody using an enzyme-linked secondary antibody and a chromogenic substrate. Identification of the corresponding antigen by comparisons to the immunoprecipitate pattern of the original immunoplate. To test this method we have analyzed the detection of the antigens recognized by six previously described monoclonal antibodies against platelet membrane proteins and von Willebrand factor. Specific immunoblots were obtained in each case using small amounts of monoclonal antibodies. Thus, the technique provides an alternative when epitopes are denatured by SDS, and avoids the use of radioactively labelled monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1987

Induction of various HLA class II molecules in a human colonic adenocarcinoma cell line.

The colonic carcinoma cell line HT-29 had no constitutive expression of HLA class II molecules. Gamma interferon (IFN-gamma) induced expression of HLA class II molecules in a dose-dependent manner with 100 U/ml as an optimal dose. The expression of HLA-DR, HLA-DP, and HLA-DQ molecules seemed to follow different kinetics. While DR and DP molecules were maximally induced after 2 days, DQ molecules appeared later with maximum percentage positive cells after 8 days. Treatment with a prostaglandin synthesis inhibitor (indomethacin) neither induced class II expression nor altered the dose-response curve for IFN-gamma; this indicated that possible endogenous production of prostaglandins in this cell line did not interfere with its class II expression. The lectins phytohaemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), and wheat germ agglutinin (WGA) did not induce class II expression.

Adenocarcinoma↗
PubMed1987

Activation of resting, pure CD4+, and CD8+ cells via CD3. Requirements for second signals.

We studied the requirements for secondary activation signals in pure CD4+ and CD8+ T cells after stimulation with anti-CD3 antibodies. Stimulation of CD4+ or CD8+ cells with anti-CD3 monoclonal antibodies (MoAb) bound to polystyrene monosized particles never resulted in a proliferative response. However, DNA synthesis was observed when recombinant interleukin 2 (IL-2) or other secondary signals, such as those provided by phorbol myristate acetate (PMA) or autologous accessory cells (AC), were also added. These secondary signals were not in themselves capable of inducing DNA synthesis in the absence of particle-bound anti-CD3. We also found that the signals provided by AC may be dependent on the activation state of these cells. Thus, the effects of accessory cells were enhanced by a factor present in fetal calf serum (FCS), most likely endotoxin or lipopolysaccharide (LPS), which alone, however, were not able to activate T cells, even in the presence of particle-bound anti-CD3. Recombinant IL-1 over a broad dose range was unable to replace PMA or activated AC after stimulation with particle-bound anti-CD3. Purified CD4+ and CD8+ T cells behaved identically in all the experiments, indicating that the basic mechanisms for activation in the two T-cell subsets are identical.

Antibodies, Monoclonal↗
PubMed1987

Effects of endotoxins from Bacteroides intermedius and Escherichia coli on human monocytes in vitro.

Endotoxins (lipopolysaccharides, LPSs) from the strictly anaerobic bacterium Bacteroides intermedius and from Escherichia coli were compared with respect to effects on human monocytes. Exposure of monocyte monolayer cultures to the structurally different endotoxins caused morphological changes, demonstrated by large rounded cells containing cytoplasmic granules with the B. intermedius LPS, and smaller cells with several cytoplasmic protrusions with the E. coli LPS after 10 days of culture. The B. intermedius LPS was, at some doses, able to induce an increased level of the lysosomal enzyme acid phosphatase and increased C3b-receptor-mediated phagocytosis, while other doses gave no effect or weak suppression compared to control cultures. The stimulatory effects were donor-dependent. The E. coli LPS had predominantly suppressive effects on acid phosphatase level and C3b phagocytosis. Both endotoxins resulted in enhanced expression of the activation related cell surface antigen, characterized by the mononuclear phagocyte specific monoclonal antibody 1D5. Biphasic dose-response relationships were observed, particularly pronounced with B. intermedius LPS, giving stimulation of enzyme activity with low (1 ng/ml) and high (0.5 microgram/ml) doses, and suppressed enzyme activity with intermediate doses (0.01-0.10 microgram/ml). The results imply that the effects of endotoxins on different aspects of human monocytes vary considerably; they also suggest that the balance between stimulatory and suppressive properties of a particular endotoxin may influence its net effect on mononuclear phagocyte functions.

Acid Phosphatase↗
PubMed1987

Depletion of T lymphocytes from human bone marrow. Use of magnetic monosized polymer microspheres coated with T-lymphocyte-specific monoclonal antibodies.

A new technique for depletion of T cells from bone marrow is presented. Bone marrow cells (BMC) were rosetted with magnetic monosized polystyrene microspheres coated with monoclonal antibodies (MAbs) specific for T cell CD2 and CD3 antigens. Rosetted T cells were subsequently removed from non-T cells with the aid of a magnet. This immunomagnetic separation procedure was carried out in less than 40 min and reproducibly removed T cells, leaving a maximum of 0.025% sheep-red-blood-cell (SRBC) rosette-forming cells and less than 0.02% T cells as detected by a T cell limiting dilution assay. The efficacy of the depletion procedure was further shown by flow cytometry data, by effective removal of cells from a T cell line added to the BMC prior to immunomagnetic separation, and by abrogation of interleukin 2 (IL-2)-producing capacity in T-cell-depleted BMC (BMC-T). The T cell depletion procedure provided a 43-74% recovery of non-T cells present in the Isopaque-Ficoll-isolated bone marrow mononuclear cell fraction and did not disturb the growth potential of stem cells, as assayed by hematopoietic stem cell assays.

Antibodies, Monoclonal↗