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Biomedical subjects

E Claassen

Publications and source records attributed to E Claassen.

At least 109 records · Page 6Linked to original sources
PubMed1990

Double immunocytochemical staining for in vivo detection of epitope specificity and isotype of antibody-forming cells against synthetic peptides homologous to human immunodeficiency virus-1.

Many infections evoke a strong humoral immune response. Some (e.g., HIV-1, EBV, CMV) also lead to disorders of the B-cell system. Data concerning cell dysfunction are largely derived from in vitro studies, which necessarily exclude all microenvironmental influences. The aim of this study was to develop a tool for the investigation of epitope specific humoral immune responses in vivo. Mice were immunized with one of two synthetic peptides, both 21 amino acids long and homologous to regions of the HIV-1 gp160. Cryostat sections of spleen and lymph nodes were incubated with the corresponding peptide coupled to alkaline phosphatase and simultaneously incubated with peroxidase-conjugated rabbit antisera specific for mouse immunoglobulin isotypes. We were able to show simultaneous detection of epitope specificity, isotype, and localization of antibody-forming cells and immune complexes in tissue sections. It should prove useful for in vivo investigation of the development of specific (e.g., anti-HIV-1) humoral immune response, the determination of B-cell specificity in lymph node infiltrates, and the role of immune complexes in lymph node pathology.

Alkaline Phosphatase↗
PubMed1990

Mechanism of follicular trapping: localization of immune complexes and cell remnants after elimination and repopulation of different spleen cell populations.

The role of marginal zone macrophages, marginal metallophilic macrophages (marginal metallophils) and marginal zone lymphocytes in the follicular trapping of immune complexes was investigated in a detailed elimination and repopulation study. Intravenous injection of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP) resulted in a complete and lasting elimination of marginal zone macrophages and marginal metallophils, while the number of marginal zone B-lymphocytes was temporarily reduced. By means of image analysis of light-microscopic images we quantified the repopulation of the above cell types and the presence of immune complexes during the repopulation process. Trapping of peroxidase-anti-peroxidase complexes was reduced up to Day 3 after administration of Cl2MDP-liposomes, but reached control values on Day 5, before reappearance of the different cell types. Therefore, marginal zone macrophages and marginal metallophils are neither directly nor indirectly involved in the transport of immune complexes to splenic follicles. It is unlikely that marginal zone B cells play a role in the transport of complexes, as a substantial reduction in B-cell number did not impair follicular trapping. At different time-points after treatment with Cl2MDP-liposome treatment, three macrophage markers (acid phosphatase, ligand for ERTR-9 and ligand for MOMA-2) were found in splenic follicles of several animals, but not in control animals. The presence of these macrophage markers in splenic follicles implies that soluble and particulate cell remnants migrate to the follicle and are retained there without the involvement of specific antibody and complement. Collectively, the data showing trapping of immune complexes despite the absence of several candidate transporter cell types and the localization of cellular remnants to splenic follicles provide evidence against a cell-mediated transport of immune complexes. The data argue in favour of diffusion as a transport mechanism of both immune and non-immune compounds to the follicle.

Animals↗
PubMed1989

Cognate interactions between helper T cells and B cells. II. Dissection of cognate help by using a class II-restricted, antigen-specific, IL-2-dependent helper T cell clone.

In order to determine the involvement of T-B cell contact vs lymphokine production in mediating B cell cycle entry and progression, Th cell clones "defective" in lymphokine production were cloned. Th-3.1 is one such clone that required IL-2 to produce significant levels of IL-4 and IFN-gamma. Unlike conventional Th clones, Th-3.1 induced B cell proliferation only in the presence of Ag and IL-2. In contrast to the absolute requirement of IL-2 for Th-3.1-induced B cell proliferation, IL-2 was not required for the formation of stable Th-3.1-B cell conjugates or Th-3.1-induced B cell entry into the G1 phase of the cell cycle. In the absence of IL-2 and under conditions that promoted Th-B cell interactions, Th-3.1 induced 10 to 20% of resting B cells to enter G1. B cell entry into the cell cycle was not inhibited by anti-lymphokine mAb or promoted by exogenous lymphokines, suggesting that endogenous lymphokine activity was not required for Th-3.1-induced G0 to G1 transition. The data suggested that the IL-2-independent induction of B cells into G1 by Th-3.1 was a cell contact-dependent event. Direct proof that Th-3.1-B cell contact was necessary for B cell cycle entry was provided by comparative in situ analysis of the RNA synthetic activity and the RNA content of B cells that were in physical contact with Th-3.1 or not in contact with Th-3.1. In situ autoradiography of RNA synthesis illustrated that a high frequency of B cells in contact with Th-3.1 expressed heightened RNA synthetic activity, whereas "bystander" B cells were less frequently induced into cycle. In situ laser cytometry of B cell size and total RNA content showed that B cells in physical contact with Th-3.1 had a higher RNA content and were larger than "bystander" B cells present in the same microcultures. This model system has allowed the dissection of T cell help into IL-2-dependent and IL-2-independent phases. Early cell contact-dependent events and B cell cycle progression into G1 were IL-2 independent, whereas the production of lymphokines (IL-4, IFN-gamma) by Th-3.1 and Th-3.1-induced B cell proliferation was IL-2 dependent.

Animals↗
PubMed1989

Primary in situ immune response in popliteal lymph nodes and spleen of mice after subcutaneous immunization with thymus-dependent or thymus-independent (type 1 and 2) antigens.

Mice were immunized subcutaneously with thymus-independent (TI)-type 1 antigen trinitrophenylated lipopolysaccharide (TNP-LPS), TI-type 2 antigen TNP-Ficoll or thymus-dependent (TD) antigen TNP-keyhole limpet haemocyanin (TNP-KLH) in order to study the primary in situ immune response in popliteal lymph nodes (PLN) and spleen. The spleen responded more rapidly in developing specific antibody-forming cells (AFC) than the lymph nodes did, in spite of the fact that antigens reach the spleen only after passing several lymph node stations. This difference between lymph nodes and spleen in developing AFC was particularly significant with respect to the responses to TI (both type 1 and type 2) antigens. No differences in the distribution of specific AFC in PLN and spleen were observed after immunization with TI and TD antigens. Results are discussed with respect to the relative contributions of lymph nodes and spleen to immune responses to antigens injected subcutaneously.

Animals↗
PubMed1989

Marginal zone macrophages and their role in the immune response against T-independent type 2 antigens: modulation of the cells with specific antibody.

Marginal zone macrophages are strategically positioned in the marginal zone of the spleen and are thought to play an important role in the initiation of the immune response to T-independent type 2 responses. The cells are characterized by high phagocytic activity and by the selective uptake of neutral polysaccharides. In the mouse marginal zone macrophages react specifically with the monoclonal antibody ER-TR9. Injection of the antibody resulted in a complete abrogation of the uptake of neutral polysaccharides by the cells in vivo, although the cells were still capable of taking up latex and carbon particles. The complete blockade of the polysaccharide uptake did not result in an altered humoral immune response against this antigen. When the antibody ER-TR9 was coupled to the toxin gelonin a complete elimination of the marginal zone macrophages could be established in vivo. However, complete elimination did not result in changes of the immune responses against 2,4,6-trinitrophenylated Ficoll, suggesting that the marginal zone macrophages are either not involved in this type of response, or that their function can be taken over by other cells. The possible role of these cells and the importance of the spleen in the immune response against bacterial antigens is discussed.

Animals↗
PubMed1989

In situ analysis of antibody-forming cells from the BALB/c CRIc idiotype family: idiotopic heterogeneity among clustered cells.

Immunocytochemical staining methods were applied toward defining the in situ expansion of the BALB/c mouse antibody-forming cells (AFC) that express the CRIc idiotype (id) family associated with the antibody response against the p-azophenylarsonate (Ar) hapten. CRIc+ AFC dominated the early primary anti-Ar response but represented a decreased fraction of the anti-Ar AFC during secondary responses. Two subsets of the CRIc family, CRIc1 and CRIc2, differed in their relative expression, with CRIc1 more pronounced during primary responses and CRIc2 better expanded in secondary responses. Idiotopic differences among AFC in very close proximity suggested convergent migration among B cells having similar but not identical V regions or possibly V region mutations among clonally derived cells. The approach also allowed in situ idiotypic analysis of polyclonally activated B AFC.

Adjuvants, Immunologic↗
PubMed1989

Characterization of polyclonal antibodies against the N-terminal domain of the human androgen receptor.

Antibodies against the N-terminal domain of the human androgen receptor (hAR) were prepared by two different approaches. Firstly, rabbits were immunized with a beta-galactosidase-hAR (amino acids (aa) 174-353) fusion protein. Secondly, two synthetic peptides corresponding to potentially antigenic sites located within this fragment (aa 201-222 and 301-320) were used as immunogens. The obtained antisera contained high titer anti-hAR antibodies as was established with several independent methods (e.g. sucrose gradient centrifugation, immunoprecipitation, Western blotting). The two anti-peptide antisera specifically stained nuclei of glandular epithelial cells in frozen sections of human prostate tissue. Progesterone, estradiol and glucocorticoid receptors were not immunoprecipitated with these antisera. The specific hAR antibodies provide new tools for the characterization of this steroid receptor as well as for diagnostic purposes in pathology of the human prostate and androgen resistance.

Animals↗
PubMed1989

Antibodies to a short synthetic peptide related to the hinge segment of human IgG3 recognizes thermally or fixative induced conformational changes in the human IgG3 molecule.

A synthetic decapeptide (SP) was used to produce a murine monoclonal antibody specific for the human IgG3 molecule. Recognition of the IgG3 determinant is heat- and fixation-sensitive in ELISA and immunoenzyme cytology, respectively. The antibody specifically recognizes a sequence from the hinge region of IgG3, but only when subtle alterations in the conformation are induced by mild heating (greater than 40 degrees) and subsequent stabilization by means of electrostatic interactions in solid-phase assays or by fixation with formalin acetic acid mercury chloride. The structure of the human IgG3 molecule is especially sensitive to microenvironmental influences, as can be concluded from its behaviour under various physicochemical conditions. To this, we add that the immunogenic determinants in the structure of relatively flexible parts of this protein can be severely altered by the routine application of fixation methods. It is shown that these changes can also be of importance in the recognition of antigen by monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1989

Marginal zone of the murine spleen in autotransplants: functional and histological observations in the response against a thymus-independent type 2 antigen.

Splenic tissue from mice was autotransplanted; after initial necrosis, a rapid restoration of implants into a structure histologically indistinguishable from splenic tissue was observed. The development of the marginal zone in these autotransplants, as determined with monoclonal antibodies against different splenic cell types and routine histological stains, was compared with the local and systemic response against a thymus-independent (TI) type 2 antigen. Full restoration of time course and peak of anti-trinitrophenyl (TNP) serum titres against TNP-Ficoll was observed at 4 weeks after autotransplantation. Anti-TNP antibody-forming cells were observed in subnormal and normal numbers in 2- and 4-week old autotransplants, respectively. The appearance of normal numbers of antibody-forming cells, and the restoration of antibody titres at week 4 correlated with the return of newly formed B cells in a normal marginal zone. An unexpected observation was that marginal zone macrophages did not return until 10 weeks after transplantation, thereby making the necessity for these cells in the normal TI-2 response unlikely. We conclude that normal anti-TI-2 responses (onset and peak titres) can be restored by autotransplantation of splenic tissue. B cells and marginal zone organization are responsible for this response, for which marginal zone macrophages seem expendable. The partial protection against overwhelming post-splenectomy infections, given by autotransplants, can thus be explained by restorative capabilities of these implants on antigen presentation and antibody formation against TI-2 antigens, and not by an increase (compared with splenectomized individuals) of phagocytosis by marginal zone macrophages.

Animals↗
PubMed1989

Cellular basis of an auto-anti-allotypic mechanism for the maintenance of chronic allotype suppression in the rabbit.

Immunocytochemical identification of antibody-forming cells (AFCs) in situ was used to test the hypothesis that the maintenance of chronic allotype suppression in heterozygous rabbits is the result of an autoimmune B-cell-mediated response. Appreciable numbers of B cells with antibody activity directed against the suppressed allotypic determinant were found in spleen and bone marrow sections of all chronically suppressed rabbits examined. Appropriate double-staining was used to determine that such cells were of the non-suppressed allotype. These cells were indistinguishable from anti-allotypic AFCs found in larger numbers in spleens of normal heterozygous rabbits that had been immunized against a heterologous allotypic determinant. Auto-anti-allotypic AFCs were not found in suppressed rabbits less than 8 week old, nor were they found in normal (non-suppressed) heterozygous rabbits or chimeric rabbits formed by the injection of histocompatible but allotype-mismatched lymphoid cells at birth. The findings reported here support the hypothesis that the long-term maintenance of allotype suppression in the rabbit may result from the suppressive activities of autoimmune B cells. It is suggested that the suppression of an allotype during the first few weeks of life could result in a loss of tolerance to a self-determinant. The kinetics of auto-anti-AFC production support this idea in showing that such cells are generated following the decline of the antibody used to induce suppression. The triggering event may be the emergence of B cells expressing the previously suppressed gene product.

Aging↗
PubMed1988

Detection of antibody, idiotype, and anti-idiotype forming cells by in situ immunocytochemical staining.

Methods are described for the immunocytochemical staining of cryostat sections of lymphoid tissue with enzyme conjugates of antigen, idiotype (Id) and anti-idiotype. Results established this as a useful approach, for simultaneously detecting Id and anti-Id antibody forming cells (AFC) in situ. As a model, the 5AF6 Id family associated with the BALB/c mouse antibody response against the p-azophenyl-arsonate (Ar) epitope was examined by two-color immunocytochemical staining, allowing the simultaneous detection of both Id+ and Id- anti-Ar AFC. Spleens from mice secondarily immunized with Ar antigen but not normal mice contained anti-Id AFC stained with the 5AF6 Id but not with another immunoglobulin of the same isotype. A sequential staining method was developed which allowed the detection of both Id and anti-Id AFC in the same tissue, thus providing a means of examining Id and anti-Id antibody networks in intact lymphoid tissues.

Animals↗
PubMed1988

In vitro and in vivo elimination of macrophage tumor cells using liposome-encapsulated dichloromethylene diphosphonate.

It is shown in the present study that RAW 264 tumor cells can be killed by liposome-entrapped dichloromethylene diphosphonate (DMDP), both in vitro and in vivo. DMDP is ingested by phagocytic cells when entrapped in liposomes. Once phagocytized the liposomal membranes are digested and the drug is released into the cell and is ready for action. In vitro, even low doses of liposome-entrapped DMDP caused an significant reduction in cell numbers. In vivo, liposome-encapsulated DMDP markedly reduced tumor formation in the liver, when given 1 day after injection of 1 x 10(6) RAW 264 tumor cells. Liposome-encapsulated DMDP, given 4 or more days after injection of the tumor cells had no significant effect. We concluded that tumor formation by RAW 264 cells is only susceptible to in vivo treatment with liposome-entrapped DMDP during a short period of time after injection of the cells. Obviously, phagocytosis of the tumor cells is reduced after this period making the cells less susceptible to treatment with the liposome-entrapped drug.

Animals↗
PubMed1988

Marginal metallophilic macrophages in the mouse spleen: effects of neonatal injections of MOMA-1 antibody on the humoral immune response.

To investigate the role of the marginal metallophilic macrophages in the humoral immune response, mice were injected from birth with the monoclonal antibody MOMA-1, which reacts specifically with these cells. Multiple injections led to a drastic reduction in the number of cells but not to a complete elimination of the macrophage subpopulation. In these animals the humoral immune response against a Thymus-independent (TI) type 1 antigen was not disturbed but the responses against Thymus-dependent (TD) and T1 type 2 antigens were reduced as determined by specific antibody forming cells and serum titers. The results are discussed in relation to earlier findings that in the adult situation elimination of all macrophages in the marginal zone using DMDP encapsulated liposomes led to a reduction in the response to only TI type 2 antigens.

Animals↗
PubMed1988

Sequential double immunocytochemical staining for in situ identification of an auto-anti-allotype immune response in allotype-suppressed rabbits.

Immunocytochemical staining has been used to detect putative autoimmune B-cells in rabbits undergoing chronic allotype suppression. This condition is seen in heterozygous rabbits exposed perinatally to antibody against the paternal immunoglobulin allotype. Such animals develop lifelong suppression for this allotype and have been used as models for study of antibody-induced disturbance of immune regulation. Normal rabbits deliberately immunized against a heterologous allotype were used to establish the feasibility of identifying cells forming anti-allotypic antibodies in cryostat sections of rabbit lymphoid tissues. Incubation and staining of tissue sections from suppressed rabbits then revealed the presence of autoimmune B-cells, with antibody specificity for the suppressed allotype, in all chronically suppressed adult rabbits tested. Sequential incubation and staining with allotype- and anti-allotype-enzyme conjugates established that such cells were of non-suppressed origin. Auto-anti-allotype antibody-forming cells were not found in normal heterozygotes or in chimeric rabbits. The immunocytochemical techniques described here permitted simultaneous detection of specificity (i.e., anti-allotype) and origin (allotype) of antibody-forming cells involved in an autoimmune response, as well as their anatomical correlation with other B-cells of suppressed or non-suppressed origin. Since the method described can be adapted to detection of alternate cell markers, we believe it to have potential application to the study of other autoimmune phenomena.

Animals↗
PubMed1988

The role of macrophages in LPS-induced lethality and tissue injury.

In the present study we investigated the role of mononuclear phagocytes in the pathogenesis of lipopolysaccharide (LPS)-induced lethality and tissue injury. Since hepatic and splenic macrophages are the primary sites of localization of i.v.-injected LPS, we selectively eliminated these macrophages using liposome-encapsulated dichloromethylene diphosphonate (DMDP). After double DMDP-liposome treatment the phagocytic cells in the liver and spleen were completely eliminated, except for the macrophages in the white pulp of the spleen which were affected to a lesser extent by this treatment. An i.v. injection of LPS into DMDP- and saline-pretreated mice showed that the latter animals exhibited febrile-associated symptoms such as lethargy and ruffled fur, but that macrophage elimination abrogated these symptoms. Although after double saline- or DMDP-pretreatment the LD50 appears to be 1 mg and 630 micrograms, respectively, the differences in lethality between both groups of mice were not statistically significant. Therefore, we concluded that hepatic and splenic macrophages are not necessary for LPS-induced lethality. The role of macrophages in LPS-induced local tissue damage was studied by comparing the histopathological changes in hepatic and splenic tissue between DMDP- and saline-pretreated mice. A sublethal dose of LPS induced similar hepatic lesions in macrophage-depleted and saline-pretreated mice, whereas the histopathological changes in the spleen were much more pronounced after DMDP-pretreatment. Particularly in the inner periarteriolar lymphocyte sheath (PALS) of these mice, the number of T cells was considerably reduced and extensive cellular necrosis could be found. These data strongly suggest that the local tissue damage resulting from LPS injection may not be due to its localization in mononuclear phagocytes but rather to interaction with other cell types.

Animals↗
PubMed1988

Effects of chronic injection of sphingomyelin-containing liposomes on lymphoid and non-lymphoid cells in the spleen. Transient suppression of marginal zone macrophages.

Mice were injected with sphingomyelin/cholesterol or phosphatidylcholine/cholesterol (PC/C) liposomes, from twice up to 10 times, on alternate days. Administration of sphingomyelin/cholesterol (SM/C) liposomes gave rise to hepato and splenomegaly, microgranulomatous infections and changes in macrophage numbers and activity in spleen and liver. Enzyme and immuno-cytochemical methods were used, to demonstrate the effect of liposomes on the lymphoid and non-lymphoid cell populations, on cryostat sections of the spleen. Routine histological staining, of sphingomyelin/cholesterol treated animals, showed no drastic changes in morphology or compartmentalization of the spleen, apart from a small enlargement (with some microgranulomas) of the red pulp. No significant differences were found in the presence or localization of T-helper, T-cytotoxic/suppressor, T-total-lymphocytes, B-total-lymphocytes, red pulp macrophages, marginal metallophils, or non-lymphoid dendritic cells. However, a transient suppression of cells expressing marginal zone macrophage surface marker ERTR-9, was observed between the second and eighth (intravenous) administration of sphingomyelin/cholesterol liposomes. Immunization of these animals with trinitrophenyl (TNP)-ficoll, a thymus-independent type-2 antigen which is specifically processed by marginal zone macrophages (MZM), showed that these cells were not suppressed with regard to their immunological function. We conclude that chronic administration of sphingomyelin liposomes influences macrophages, probably through a general phagocytic-system overload, but not permanent or damaging changes in splenic cell populations or immunological functions occur.

Animals↗