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Biomedical subjects

D M Boorsma

Publications and source records attributed to D M Boorsma.

At least 37 records · Page 2Linked to original sources
PubMed1993

Increased adherence to keratinocytes of peripheral blood mononuclear leucocytes of a patient with drug-induced erythema multiforme.

The pathogenetic mechanisms involved in the development of drug-induced erythema multiforme (EM) are still largely unknown. The observation that epidermal keratinocytes (KC) in EM express intercellular adhesion molecule-1 (ICAM-1) points to a putative role for T-cell/KC adhesion in the pathogenesis of EM. In this study, the binding of peripheral blood mononuclear leucocytes (PBML) from a patient with carbamazepine-induced EM and of normal control PBML to autologous and heterologous KC was investigated, using two different binding assays. Patient PBML obtained at the time of disease (t0) showed an increased binding to ICAM-1-positive heterologous KC, which could be inhibited completely by anti-LFA-1. Adhesion of patient PBML-t0 to autologous KC, and to carbamazepine-pretreated heterologous KC in sections of skin biopsies, was also increased, but was found to be only partially LFA-1-dependent. These findings support the view that PBML/KC adherence plays an important role in the pathogenesis of this drug-induced EM.

Adult↗
PubMed1992

Production of monomeric antigen-enzyme conjugate to study requirements for follicular immune complex trapping.

Studies concerning the localization of immune complexes in lymphoid follicles and the involvement of these trapped immune complexes in the regulation of the immune response have thus far been performed with poorly defined complexes in terms of size and composition. For that reason, the minimum requirements for trapping in terms of number of antigen- and antibody molecules present in immune complexes could not be determined. We here describe the production and in vivo use of a monomeric HSA-HRP antigen-enzyme conjugate, readily demonstrable in cryostat sections and ELISA. This conjugate was obtained by combining the glutaraldehyde coupling-method with chromatography to fractionate monomeric and multimeric constituents. SDS-PAGE analysis showed that the conjugate consisted of a single molecular species of 109 kDa, whereas the often used periodate oxidation coupling method yielded a heterogeneous population of multimeric, oligomeric and monomeric molecules. We investigated the minimal size requirements for the composition of immune complexes to be trapped in murine spleen follicles using three different conjugates (monomeric HSA-HRP, multimeric HSA-HRP and multimeric HSA-HRP-Penicillin) and a panel of anti-HSA and anti-Penicillin monoclonal antibodies. We demonstrate that the smallest immune complexes, consisting of one antibody and two conjugate molecules, do not localize in splenic follicles. Immune complexes prepared with a single monoclonal antibody localize in follicles only if the epitope recognized occurs repeatedly on the antigen. The relevance of these results for physiological follicular trapping of protein antigens is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Spectophotometric quantification of cell-cell adherence by an enzyme-linked immuno-cell adhesion assay.

In the present study a spectophotometric method was developed to determine the number of T cells binding to human epidermal keratinocytes (KC). In this cell adhesion assay the adherent T cells were detected with a cell-specific monoclonal antibody conjugated to horseradish peroxidase and the coloured substrate quantified in an ELISA reader at 492 nm. A correlation was demonstrated between the number of T cells and the extinction values measured. The enzyme-linked immuno-cell adhesion assay (ELICAA) was used to quantify KC/T lymphocyte adherence in a series of experiments designed to evaluate its reliability and reproducibility. Compared with the 51Cr-labelled adherence assay, the ELICAA was a safe, rapid and accurate method avoiding the use of radioactive material.

Antibodies, Monoclonal↗
PubMed1990

The significance of Leu 8 negative T cells in lymphoid skin infiltrates: malignant transformation, selective homing or T-cell activation?

The expression of Leu 8 was studied on skin biopsies from a large group of patients with benign and malignant skin disorders and correlated with the expression of T-cell differentiation antigens and activation markers. The effect of in vitro stimulation of peripheral blood T cells and T-cell subsets on the expression of Leu 8 antigen was also determined. In all the skin diseases studied an inverse relationship was found between the proportions of cells expressing Leu 8 and HLA-DR. A deficiency of Leu 8 positive cells was not specific for mycosis fungoides, but was also found in several reactive dermatoses. Stimulation of peripheral blood cells with phytohaemagglutinin (PHA), concanavalin A (Con A), and anti-CD3-PMA resulted in a considerable decrease of Leu 8 antigen expression on day 3 in both CD4+ and CD8+ T cells. These data suggest that the low proportion of Leu 8+ T cells in mycosis fungoides and several reactive skin disorders is not related to malignant transformation or selective homing of Leu 8- T cells, but probably results from local T-cell activation.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

Chemotaxis of the peritoneal cells and the detection of a chemo-attractant in the effluent from peritoneal dialysis patients.

The migration of peritoneal cells from 25 continuous ambulatory peritoneal dialysis patients and eight healthy women undergoing laparoscopy were studied. Peritoneal cells of continuous ambulatory peritoneal dialysis patients migrated to commonly used chemoattractants, like N-formyl-methionyl-leucyl-phenyl-alanine-methyl- ester and casein, but they also migrated to high concentrations of recombinant interleukin-1 alpha and to endotoxin (lipopolysaccharide). In the peritoneal effluent from continuous ambulatory peritoneal dialysis patients a rather heat stable chemoattractant was found with a molecular weight of 40-200 kDa with an optimal activity at approximately 125 kDa. The chemoattractant is a protein and is not complement factor 5a or interleukin-1 and was only found in peritoneal effluent from continuous ambulatory peritoneal dialysis patients, but not in peritoneal fluid from healthy women undergoing laparoscopy. Therefore, peritoneal dialysis might induce the generation of a chemoattractant. The optimal chemotactic response of peritoneal cells from continuous ambulatory peritoneal dialysis patients to N-formyl-methionyl-leucyl-phenyl-alanine-methyl- ester in medium could be enhanced by replacing the medium by peritoneal effluent. So the chemotaxis of peritoneal cells to the factor in the peritoneal effluent is caused by another mechanism, which might involve different cell surface receptor populations, than the chemotactic response to N-formyl-methionyl-leucyl-phenyl-alanine-methyl-ester.

Adult↗
PubMed1987

A rapid and simple hapten conjugation method for monoclonal antibodies to be used in immunoenzyme single and double staining procedures.

A rapid and simple method was developed for the haptenization of monoclonal antibodies (MAbs), to be used in immunoenzyme single and double staining techniques. Using this method minute amounts of MAbs can be haptenized without purification of the antibody or removal of the excess hapten. The haptenized MAb can be ready for use with 2.5 h. The method consists of a direct incubation of the antibody with the haptenizing agent and subsequent addition of an amino acid solution to stop the reaction. Commonly available reagents were tested, of which dinitrofluorobenzene, trinitrobenzene sulfonic acid and an oxazolone derivative gave the best results. The procedure was evaluated by using MAbs directed against lymphoid cell surface membrane antigens in an indirect immunoenzyme staining on frozen sections using peroxidase or alkaline phosphatase-conjugated anti-hapten antibodies as second step antibody. It was found that those MAbs, which show good staining results in conventional indirect immunoenzyme procedures, can also be used successfully after haptenization in single as well as double staining procedures. Combination of haptenized and biotynilated MAbs gave good results when weakly reactive MAbs had to be included in immunoenzyme double staining.

Antibodies, Monoclonal↗
PubMed1987

The effect of occlusion of the skin with transdermal therapeutic system on Langerhans' cells and the induction of skin irritation.

Sensitization to drugs in transdermal therapeutic systems is a common unwanted event. We investigated whether prolonged occlusion of the skin causes skin irritation, and in this way might play a role in the possible induction of sensitization to drugs from these systems. Occlusion was effected with a placebo transdermal therapeutic system and silver-patch test in a time span ranging from eight hours to seven days in five groups of five volunteers. Skin irritation was judged on clinical aspects, histopathologic and immunofluorescence findings, and changes in the Langerhans' cell systems. The results indicate that occlusion with the systems used in this experiment provokes only slight or no skin irritation.

Administration, Cutaneous↗
PubMed1986

Avidin-HRP conjugates in biotin-avidin immunoenzyme cytochemistry.

Avidin-HRP conjugates were prepared, analysed and tested for avidin-biotin immunocytochemistry. Suitable biotinylation of enzymes, antigens and antibody was obtained by reacting biotin at equimolar ratio to epsilon aminogroups in proteins. The avidin-biotin interaction was used for immunocytochemical detection of phenomena in the field of immunology, i.e. immune complex trapping, specific antibody forming cells and in serology for the cytochemical detection of human auto-antibodies to basement membrane components. Avidin-HRP conjugation using the two step glutaraldehyde method gave a very small amount of monomeric, low molecular weight conjugate with excellent performance. Avidin-HRP conjugation using the periodate method was modified at two points. The first modification concerns the molar ratio of avidin to HRP in the reaction mixture which was brought to about equimolarity. The second modification concerns the periodate concentration which was decreased five fold, ten fold and twenty fold. Decreasing the periodate concentration decreased the amount of polymeric conjugate. Optimal amounts of monomeric, low molecular weight conjugate were obtained with a ten fold decrease of the periodate concentration. Comparable cytochemical results were obtained with monomeric conjugates obtained using both preparation methods.

Animals↗
PubMed1986

Detection of specific anti-hapten antibody-producing hybridoma cells in tissue sections of spleen and liver by hapten-enzyme conjugates using an immunoenzyme approach.

A method is presented for the detection of hybridoma cell growth in vivo by making use of the specific antigen (hapten) recognition properties of the produced monoclonal antibodies. Horseradish peroxidase (HRP)-labeled antigen was used for demonstration of intracellular specific antibodies in hybridoma cells. It appeared that intravenously injected anti-penicilloyl-producing hybridoma cells developed mainly in the red pulp of the spleen; to a lesser extent they were found in the liver.

Animals↗
PubMed1986

Simultaneous immunoenzyme double labelling using two different enzymes linked directly to monoclonal antibodies or with biotin-avidin.

A novel and rapid method for immunoenzyme double staining with monoclonal antibodies (McAb) is described. The principles of this method are the simultaneous application of primary antibodies and the unrelatedness of the two detection systems. One McAb is directly labelled with horseradish peroxidase and the other McAb is labelled with biotin. This second McAb is thereafter detected using an avidin-alkaline phosphatase conjugate. This conjugate was prepared by a new method using a heterobifunctional reagent. Double staining of cell surface membranes of human tonsil was studied in cryosections using various combinations of McAbs to lymphoid cell markers. As expected, suppressor T cells were found to be contained within the pan T cell population on the basis of the distinguishable intermediate colour produced. Similarly, it was shown that most of the suppressor T cells were not HLA-DR activated in this tonsil. In cryostat sections of human skin T6 positive Langerhans cells in the epidermis were shown to carry the HLA-DR antigen.

Alkaline Phosphatase↗
PubMed1986

Double-enzyme conjugates, producing an intermediate color, for simultaneous and direct detection of three different intracellular immunoglobulin determinants with only two enzymes.

A new double-enzyme conjugate was synthesized by coupling alkaline phosphatase (AP) to horseradish peroxidase (HRP). After AP (blue) and subsequent HRP (red) cytochemistry, this new conjugate produced a stable intermediate-colored (violet) product. By coupling this double-enzyme conjugate to an antigen (trinitrophenyl, TNP) or an antibody (anti-mouse immunoglobulin G2a), anti-TNP or -IgG2a-producing cells could be demonstrated as violet cells in spleen sections. This led to the development of a rapid one-step incubation--two-step cytochemical procedure for simultaneous detection of three different determinants in a single tissue section. To demonstrate this novel triple staining method, we coupled three different antigens to, respectively, AP, HRP, and AP-HRP. When spleen sections of immunized animals were incubated with a mixture of these three antigen-enzyme conjugates, we could distinguish antibody-forming cells against each of these three antigens simultaneously as red (HRP), blue (AP), and violet (AP-HRP) cells. The simultaneous detection of three different classes of intracellular antibodies in a single section also proved to be possible with this method. With this study we provide a new direct method for detection of three different intracellular immunoglobulins after a one-step incubation and a two-step standard cytochemical procedure.

Alkaline Phosphatase↗
PubMed1985

Binding of different antigen-enzyme and antibody-enzyme conjugates by intracellular antibodies in cytoplasm and Golgi complex of plasma cells. A double immunocytochemical study.

Intracellular immunoglobulins in plasma cells were characterized by antigen-enzyme conjugates and anti-immunoglobulin antibody-enzyme conjugates applied in a double immunocytochemical approach. After their assemblage, immunoglobulins in the cytoplasm of anti-TNP anti-body producing plasma cells can be demonstrated both by TNP-enzyme conjugates and by anti-immunoglobulin (mu or gamma chain specific) antibody-enzyme conjugates. Once arrived in the Golgi complex (GC) detection with TNP-enzyme conjugates remains possible, but anti-immunoglobulin anti-body-enzyme conjugates did not bind to a detectable degree. Similar results were obtained in experiments where immunoglobulin-enzyme conjugates were used both as an antigen-enzyme conjugate and as an antibody-enzyme conjugate.

Alkaline Phosphatase↗