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Biomedical subjects

E Claassen

Publications and source records attributed to E Claassen.

At least 19 recordsLinked to original sources
PubMed1992

Monoclonal antibody mediated targeting of enzymes. A comparative study using the mouse spleen as a model system.

The aim of this study was to target enzymes specifically to cells in the murine spleen. Monomeric and polymeric conjugates of the enzymes horseradish peroxidase or alkaline phosphatase and monoclonal antibodies against cell surface determinants were prepared. Highly specific in vivo targeting of enzymes to macrophages was obtained only when monomeric MAb-enzyme conjugates were used.

Alkaline Phosphatase↗
PubMed1992

Post-formation fluorescent labelling of liposomal membranes. In vivo detection, localisation and kinetics.

A fast and simple method for the in vivo/in situ detection of liposomes is described. Utilizing lipophilic carbocyanine dyes, DiI and DiO, yellow (or red) and green fluorescent liposomes can be visualised with routinely available filters. The main advantages of the method are (i) the vesicles can be labelled after they are formed and (ii) the label does not interfere with proteins on the surface of the liposomes. Labelled liposomes were found in macrophages of spleen and liver (of mice) within 30 min of intravenous administration. In the spleen, labelled liposomes localised preferentially in the marginal zone macrophages, as confirmed by double staining with FITC-Ficoll. These data correlate well with the fact that empty or haptenated liposomes are thymus-independent antigens, and that other thymus-independent antigens are also specifically taken up by marginal zone macrophages. The immunological role of these macrophages in the processing and presentation of antigen-bearing liposomes can now be studied in more detail. Administration of high doses (1-3 mg lipid) of labelled liposomes showed that uptake occurred preferentially, but not exclusively, by marginal zone macrophages. After the marginal zone macrophages had been 'saturated', the red pulp macrophages took up the liposomes. DiI and DiO have also been successfully used for labelling lymphocytes and bacteria for in vivo homing studies. The fact that liposomes can be labelled after they have been formed is an advantage for retrospective (i.e. liposomes already in use/storage) studies in e.g. targeting of drugs by liposomes.

Affinity Labels↗
PubMed1992

Complement-mediated follicular localization of T-independent type-2 antigens: the role of marginal zone macrophages revisited.

In this study we demonstrate a hitherto undescribed phenomenon, namely that thymus-independent type-2 antigens (TI-2 Ag) localize in splenic follicles within 1 h after administration. The follicular localization of 2,4,6-trinitrophenyl (TNP)-Ficoll was not antibody mediated. In addition in case of high-dose administration we observed a relatively large amount of TI-2 Ag in marginal zone macrophages. However, after low-dose administration we observed a preferential localization of TNP-Ficoll in the splenic follicles. Detection of TNP-haptenated Ag in cryostat sections of murine spleens was performed with a high-affinity TNP-specific monoclonal antibody conjugated to beta-galactosidase. Within minutes after injection the TI-2 Ag localized in the marginal zone, attached to marginal zone macrophages and B cells. Twenty minutes after injection the Ag was also detected in the follicles and gradually accumulated there until 7 h after injection. Thereafter, the amount of follicular Ag gradually decreased but was still detectable up to 14 days after immunization. The follicular localization of TNP-Ficoll was complement dependent in contrast to the binding to and uptake by marginal zone macrophages. Double staining revealed that Ag was bound by macrophages, B cells and follicular dendritic cells. Haptenated thymus-dependent (TD) Ag localized exclusively in the red pulp macrophages. In vivo macrophage elimination drastically increased the amount of TNP-Ficoll in the follicles, and enhanced the humoral immune response at low doses of Ag. Moreover, complement deprivation of mice abrogated the localization of TI-2 Ag in the follicles, and led to a decreased humoral TI-2 immune response. In conclusion, we demonstrate for the first time that TI-2 Ag localize in follicles. Moreover, the presented results provide further evidence that B cells and follicular localized Ag play an important role in the induction of humoral TI-2 immune responses.

Animals↗
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↗
PubMed1992

Variant-specific monoclonal and group-specific polyclonal human immunodeficiency virus type 1 neutralizing antibodies raised with synthetic peptides from the gp120 third variable domain.

The third variable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) external membrane glycoprotein gp120 is of crucial importance in eliciting neutralizing antibodies in infected persons. Polyclonal (PAb) and monoclonal (MAb) antibodies directed against selected epitopes in the V3 domain are valuable tools for analysis of the involvement of such sequences in neutralization and for definition of the relation between amino acid variability and immunological cross-reactions. The aim of this study was to obtain such site-specific antibodies. By using synthetic peptides derived from the V3 domain, a group-specific neutralizing PAb, two high-affinity HIV-1 IIIB neutralizing MAb, and two nonneutralizing MAb were raised. A 15-amino-acid peptide overlapping the tip of the V3 domain of HIV-1 MN was used to produce a rabbit PAb (W0/07). This PAb inhibited syncytium formation induced by HIV-1 IIIB and four field isolates. A similar IIIB-derived peptide was used to generate two murine immunoglobulin G1 (IgG1) MAb (IIIB-V3-13 and IIIB-V3-34). Pepscan analysis mapped the binding site of IIIB-V3-34 to the sequence IRIQRGPGR. The Kds of IIIB-V3-13 and IIIB-V3-34 for gp120 were 6.8 x 10(-11) and 1.6 x 10(-10) M, respectively. These MAb neutralized IIIB but not MN and inhibited syncytium formation induced by IIIB. They are applicable in enzyme-linked immunosorbent assays, immunocytochemistry, and flow cytometry. A peptide covering the left base of the V3 domain was used to generate two murine IgG1 MAb (IIIB-V3-21 and IIIB-V3-26). The binding site of IIIB-V3-21 was mapped to the sequence INCTRPN. These MAb did not neutralize HIV-1 and did not inhibit syncytium formation. This study supports the notion that HIV-1 neutralizing antibodies suitable for multiassay performance can be obtained with synthetic peptides and that high-affinity MAb can be generated. Such site-specific antibodies are useful reagents in the analysis of HIV-1 neutralization. In addition, the cross-neutralization of different viral strains by PAb generated through single-peptide immunization is directly relevant to vaccine development.

Amino Acid Sequence↗
PubMed1992

Immunological discrimination between the human apolipoprotein E2(Arg158----Cys) and E3 isoforms.

A specific anti-apoE2(Arg158----Cys) monoclonal antibody was raised by means of immunization of mice with a variant specific synthetic peptide. The peptide sequences used were homologous to apolipoprotein E of human and mouse. Consequently, the mouse immune system was tolerant to most of the selected sequences. Immunization with only one of selected peptides (amino acids 154-172) evoked an anti-peptide and anti-native protein response. Surprisingly, this peptide was predicted to have a low antigenicity index, in contrast to the other used peptides. The variant specific anti-peptide MAb that was generated with this sequence, recognizes apoE2(Arg158----Cys) and not apoE3. We here describe a sensitive, time saving, and simple immunoblot assay to detect apoE2(Arg158----Cys) in human sera without prior isoelectric focusing of serum proteins.

Amino Acid Sequence↗
PubMed1992

Immunological functions and in vivo cell-cell interactions of T cells in the spleen.

The spleen is an important lymphoid organ, involved in immune responses against all types of antigen that appear in the circulation. Its complex anatomical organization, with distinct compartments containing specialized cell types, provides a microenvironment which allows different cell-cell interactions and determines the direction of developing immune responses. In this review we evaluate the vast amount of in vitro data dealing with antigen presentation, cell-cell interactions, T and B cell activation, and the immunoregulatory role of cytokines, as suggested to be involved in immune responses. As a basis for understanding of in vivo processes, these in vitro data will be related to discrete phenomena of in vivo immune responses, such as antigen localization/trapping, cell migration patterns of immunocompetent cells, cytokine production, and antibody formation in the different compartments of the spleen. Finally, we try to bring order to the sequence of events that occur in the spleen after antigenic challenge by presenting an in vivo model for T cell dependent and T cell independent immune responses.

Animals↗
PubMed1992

Histological and functional differentiation of non-lymphoid cells in the chicken spleen.

The phenotypes and functions of various populations of non-lymphoid cells in the chicken spleen were investigated with monoclonal antibodies and after in vivo administration of antigens. Monoclonal antibody CVI-ChNL-68.1 was used to detect red pulp macrophages, interdigitating cells, and monocytes, whereas CVI-ChNL-68.2 was used to detect ellipsoid-associated non-lymphoid cells (EANC). Two new monoclonal antibodies were developed: CVI-ChNL-74.2, which specifically recognized red pulp macrophages and a ring of macrophages surrounding the peri-ellipsoid lymphocyte sheath; and CVI-ChNL-74.3, which specifically recognized follicular dendritic cells (FDC) in germinal centres and small clusters of stromal cells in T-cell areas. After intravenous injection of lipopolysaccharide (LPS), the number of 68.1+ and 74.2+ macrophages decreased dramatically, whereas 68.2+ EANC and 74.3+ FDC were unaffected. After intravenous injection of heat-inactivated Brucella abortus, the numbers of both macrophages and EANC decreased. In contrast, a significant increase of 74.3+ cells was observed in the T-cell areas outside the germinal centres. As expected, intravenous injection of non-mitogenic antigens, such as keyhole limpet haemocyanin and Ficoll, and carrageenan did not affect the non-lymphoid cell populations. At least six subpopulations of non-lymphoid cells in the chicken spleen can now be discriminated with monoclonal antibodies. Our results show that mononuclear phagocytes are sensitive for mitogenic stimulators such as LPS and Brucella abortus. In contrast, stromal non-lymphoid cells are only sensitive to the particulate mitogen Brucella abortus. We conclude that the complex formed by ellipsoid cells, the peri-ellipsoid B-cell sheath, and the surrounding macrophages, is the functional equivalent of the mammalian marginal zone.

Animals↗
PubMed1991

Synthetic peptide conjugates with horseradish peroxidase and beta-galactosidase for use in epitope-specific immunocytochemistry and ELISA.

Synthetic peptide-alkaline phosphatase conjugates can be used to detect the epitope specificity of (i) antibody-forming cells in vivo by immunocytochemistry; (ii) of antibody secreting cells in vitro by spot-ELISA; and (iii) antibodies in solution by capture ELISA. The availability of synthetic peptide-enzyme conjugates using detector enzymes other than alkaline phosphatase would offer several important advantages, for example in double staining approaches. This paper reports the production of synthetic peptide-horseradish peroxidase conjugates and synthetic peptide-beta-galactosidase conjugates. A peptide of 21 amino acids (SP 29) was coupled to peroxidase in seven differing molar ratios of peptide over peroxidase, ranging from 1:3.4 to 1:575, using periodate oxidation of the enzyme. SP 29 was coupled to beta-galactosidase in four molar ratios ranging from 1.25 to 10, using glutaraldehyde pre-activation of the enzyme. The enzyme activity of the different conjugates was determined, the conjugates were tested in direct capture-ELISA with peptide-specific monoclonal antibodies, and the conjugates were tested in immunocytochemistry to detect peptide-specific B cells. The results show that the conjugates perform best if the peptide is coupled to the enzyme at relatively low molar ratios (1-30). The availability of these new peptide-enzyme conjugates broadens the applicability of synthetic peptides for detection purposes in several assay systems.

Animals↗
PubMed1991

In vivo kinetics and characterization of IFN-gamma-producing cells during a thymus-independent immune response.

Using immunohistochemical techniques, we studied IFN-gamma-producing cells (IFN-gamma-PC) in vivo during immune responses to thymus-independent type-2 (TI-2) Ag. Detection of IFN-gamma-PC in cryostat sections of spleen-tissue was performed with an enzyme labeled mAb directed against IFN-gamma. After TNP-Ficoll immunization, IFN-gamma-PC and TNP-specific antibody-forming cells (TNP-AFC) displayed similar kinetics reaching a maximum number at day 5 to 7. The IFN-gamma-PC were localized in the same compartment as TNP-AFC and a part of them in juxtaposition to TNP-AFC. Immunization with other TI-2 Ag resulted also in a significant increase of the number of IFN-gamma-PC. In a parallel experiment we found both in vivo and in an ELISA-spot assay a significant increase of the number of IFN-gamma-PC and IFN-gamma-spot-forming-cells, respectively, in spleens of mice 6 to 7 days after TNP-Ficoll immunization. Double staining of spleen sections for IFN-gamma and surface Ag revealed that 5 to 7 days after TNP-Ficoll immunization, +/- 40% of the IFN-gamma-PC expressed the MT4 Ag (CD4), +/- 50% the Lyt-2+ Ag (CD8) and +/- 10% the asialo-GM1 Ag (NK cell). This study represents the first description of the in vivo activity and characterization of IFN-gamma-PC during a TI-2 immune response. Moreover, the presented data confirm suggestions from in vitro investigations that IFN-gamma and T cells may play a direct role in the in vivo regulation of a primary immune response against a TI-2 Ag.

Animals↗