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Biomedical subjects

A Coutinho

Publications and source records attributed to A Coutinho.

At least 325 records · Page 18Linked to original sources
PubMed1976

Spleen cells from animals tolerant to a thymus-dependent antigen can be activated by lipopolysaccharide to synthesize antibodies against the tolerogen.

Immunological tolerance was induced in adult mice by the injection of 5 mg of deaggregated hapten-protein conjugate. The tolerant state was confirmed 4-19 days later by the failure of such animals to mount an immune response against an aggregated form of the same thymus-dependent hapten-protein conjugate as well as by the inability of spleen cells from tolerant animals to respond to a thymus-independent hapten-carrier conjugate. Even though the animals were fully tolerant, their spleen cells were activated by lipopolysaccharide (LPS) in vitro to produce normal numbers of plaque-forming cells against the hapten. The finding that spleen cells from tolerant animals could be activated by LPS into synthesis of antibodies against the tolerogen indicates that tolerance to thymus-dependent antigens does not affect B cells, but presumably only T cells. It is suggested that the only stringent test for the existence of B-cell tolerance is the inability of polyclonal B-cell activators to activate antibody synthesis against the tolerogen. The findings make it unlikely that B-cell tolerance to autologous thymus-dependent antigens exists and further indicate that such antigens cannot deliver activating or tolerogenic signals to B cells, although they are competent to combine with and block the Ig receptors.

Animals↗
PubMed1976

Association between mitogenicity and immunogenicity of 4-hydroxy-3,5-dinitrophenacetyl-lipopolysaccharide, a T-independent antigen.

Polymyxin B, which is a basic polypeptide produced by various strains of Bacillus Polymyxa, has previously been shown to prevent the lethal effect of LPS and to neutralize the Schwartzmann reaction. In this study we have investigated the interactions between polymyxin B and lipopolysaccharide (LPS) and hapten LPS conjugates. Polymyxin B was found to suppress mitogenicity of LPS and also to inhibit immunogenicity of the hapten conjugate 4-hydroxy-3,5-dinitrophenacetyl (NNP)-LPS. Inhibition was not due to interference with the expression of NNP determinants nor to cross-reactivity between PB and the hapten. Since mitogenicity and immunogenicity decreased in parallel, we conclude that B-cell activation in specific thymus independent responses does not take place in the absence of a nonspecific (non-Ig-mediated) signal.

Animals↗
PubMed1976

Hapten-induced B cell paralysis. II. Evidence for trivial mechanisms of tolerance.

Injection into mice of a free reactive form of the hapten (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP), induces a state of specific unresponsiveness to the hapten, upon its challenge with thymus-cependent and independent carriers. This unresponsiveness is maintained in vitro. Both induction and expression of the unresponsive state were found to be independent of T cells. Analysis of the mechanisms responsible for the B cell "tolerance" demonstrated that the major cause of unresponsiveness in this system is the blockade of specific surface receptors on B cells by the hapten, abolishing the focusing function of these receptors. "Tolerant" B cells, though they were unresponsive to the antigen, could be activated to anti-hapten antibody secretion by the B cell mitogen lipopolysaccharide (LPS), which does not require Ig-mediated binding. They also became fully responsive to the antigen NNP-LPS in specific thymus-independent responses after 24 hours of in vitro incubation in the absence of tolerogen, conditions which allow "deblocking" of Ig surface receptors, and, thus, restoration of the focusing function of these receptors. These results demonstrate that great care is required for a clearcut definition of B cell tolerance. In this example, the tolerant B cells were perfectly responsive to a competent ligand, and no indication of an altered triggering or effector processes was found. It appears that in this, as in many other cases of B cell tolerance, the systems or the animals are tolerant, whereas B cells maintain a resting nonactivated state and are fully responsive to the triggering signal. Thus, the existence of tolerance-inducing signals resulting in B cell unresponsiveness is questioned.

Antibody Formation↗
PubMed1976

A plaque assay for all cells secreting Ig of a given type or class.

A modification of the hemolytic plaque assay using protein A-coated red cells is described which makes use of the fact that the Fc portion of IgG binds to protein A. A number of murine plasmacytomas secreting different classes of Ig have been tested for plaque formation with these indicator red cells. In the presence of complement-binding antibodies specific for the corresponding class of secreted Ig, between 10 and 70% of all plated myeloma cells formed plaques. The assay shows a prozone effect in excess of antibody, suggesting that complexes of antibody and secreted Ig effect lysis of the target cells. This assay can be used to enumerate cells secreting any molecules for which complement-binding antibodies are available.

Bacterial Proteins↗
PubMed1976

Characterization of dextran-sulfate-sensitive cells.

The cell type participating in the mitotic response to the polyclonal B-cell activator (PBA) dextran-sulfate (DS) was investigated. Cells from murine fetal liver, adult bone marrow, and spleen were studied; only a limited number of all cells present in each organ responded to DS. Morphological studies of the activated cells showed the major population of activated cells in spleen to have the appearance of lymphoblasts. In bone marrow, several classes of hematopoietic cells were mitotically active, including mononuclear cells (lymphoblasts and monocytes), megakaryocytes, and myeloblasts. In these cultures, however, it was not possible to differentiate between DS-activated and spontaneously proliferating cells. Bone marrow and, to some extent, spleen cell cultures activated with DS contained a relative increase in numbers of phagocytic cells, whereas stimulation of spleen cells with lipopolysaccharide did not result in an increased phagocytosis. However, adherent cells were not necessary for activation of DNA synthesis by DS in spleen, and this cell type did not contribute to a measurable degree to the DNA synthetical response. DS cannot be regarded as a general stem cell mitogen for bone marrow cells since it failed to promote colony growth of hematopoietic cells in an in vitro system. However, supernatants from DS-activated spleen and bone marrow cell cultures did stimulate colony growth of murine bone marrow cells, indicating that stem cells of nonlymphoid origin might be indirectly activated by DS. In conclusion, the major cell population activated by DS in spleen is lymphocytes. In bone marrow, other cell types seem to participate in the response as well, but the activation mechanism may be indirect and not primarily the result of DS interaction with these cells.

Animals↗
PubMed1976

Heterogeneity of B cells: direct evidence of selective triggering of distinct subpopulations by polyclonal activators.

This investigation was undertaken to assess directly the previous postulate that distinct subsets of B cells in the adult mouse spleen are selectivity triggered by different polyclonal B-cell activators (PBA). Several strains of mice and lipopolysaccharide (LPS), purified protein derivative of tuberculin (PPD), and dextran sulphate (DS) were used in this study. Two experimental approaches were used: stimulation after addition of two PBAs simultaneously to cell cultures and eliminating the responding population to one PBA, by a hot pulse of radioactive thymidine, on a later response to another PBA. The results of these experiments indicated that DS stimulated a cell population completely different from that stimulated by the LPS- and PPD-sensitive cells. When LPS and PPD stimulations were compared, it was found that the cells responding to these PBAs were largely distinct, although some cells were sensitive to both these PBAs. The extension of the overlaps in these subsets (the number of cells that could be activated by either PBA) was found to vary from one strain to another. These experiments gave direct evidence of the existence of subsets that can be activated by different PBAs. The present results also provided indications of the functional performance of distinct subsets of B cells on activation.

Animals↗
PubMed1976

Inhibition of in vitro immune responses by a fraction from seminal plasma.

We studied the inhibitory effect of a fraction of bovine seminal plasma on in vitro lymphocyte responses to specific and nonspecific ligands. Seminal plasma was fractionated by Sephadex G-100 chromatography, and the inhibitory component was found to have a molecular weight of above 100,000 daltons. This fraction was devoid of lymphocytotoxicity and could inhibit, in a dose-dependent mode, both the primary antibody response of normal mouse spleen cells to the thymus-dependent antigen sheep erythrocytes and the one-way mixed lymphocyte reactions. Concanavalin A-induced proliferative T-cell responses were also inhibited by the same fraction. The inhibition of the T-lymphocyte responses to Con A was complete at suboptimal concentrations of the mitogen and could only partially be overcome at supraoptimal concentrations. Four other fractions in seminal plasma were also studied in this particular system, three being without effect and one being stimulatory. The inhibitory fraction was also found to inhibit the polyclonal B-cell responses induced by fetal calf serum. The importance of the present findings for the immunophysiology of reproduction are discussed.

Animals↗
PubMed1976

Genetic control of B-cell responses. II. Identification of the spleen B-cell defect in C3H/HeJ mice.

The responsiveness of spleen cells from C3H/HeJ and C3H/Tif mice to lipopolysaccharide (LPS) was compared. Around 1,000-fold higher concentration of LPS were required to minimally activate HeJ cells, as compared with Tif high-responder cells, to both proliferation and polyclonal antibody secretion. However, HeJ cells did respond to higher LPS concentrations (100 and 1000 mug/ml). This selective pattern of responsiveness to LPS was also observed in the polyclonal responses of strains to LPS in vivo. Furthermore, the unresponsiveness of HeJ spleen cells was found to depend on a pure B-cell defect in the capacity to interact and/or generate triggering signals on interaction with LPS. Thus, adherent cells, thymus-derived lymphocytes, serum factors, and other non-specific conditions inherent in spleen cell suspensions of low-responder mice were not responsible for suppressing a putative B-cell response to LPS. The present findings are compatible with the possibility that the defect in C3H/HeJ B cells reflects the absence of a structure on the cell surface membrane that is functionally responsible for mediating LPS triggering.

Animals↗
PubMed1975

Genetical control of B-cell responses. IV. Inheritance of the unresponsiveness to lipopolysaccharides.

The inheritance of B-cell responsiveness to lipopolysaccharide (LPS) was studied in 55 crosses between mice of the low-responder strain C3H/HeJ and the high-responder strains B10.5M and C3H/Tif. F1 hybrid mice between the low-and the high-responder strains, showed in every case responses which were intermediate between the responses obtained with each parent. The responsiveness among F2 hybrid and backcross mice to either high- or low-responder parents, segregated into intermediate, high, or low categories, respectively. The present results are compatible with the hypothesis that responsiveness to LPS is determined by one single, codominantly expressed, autosomal gene. The capacity to develop a specific thymus-independent response to a hapten-LPS conjugate, also under genetical control, was found to segregate together with the capacity to develop polyclonal responses to LPS.

Animals↗
PubMed1975

Genetical control of B-cell responses. III. Requirement for functional mitogenicity of the antigen in thymus-independent specific responses.

Spleen cells from C3H/HeJ mice fail to respond with polyclonal antibody synthesis to mitogenic concentrations of lipopolysaccharide (LPS) which are optimal for activating spleen cells from a high-responder strain (B10.5M). This unresponsiveness is selective for LPS, since C3H/HeJ cells respond as normals to another B-cell mitogen, purified protein derivative of tuberculin. Spleen cells from low-responder mice also fail to mount a specific anti-NNP plaque-forming cell (PFC) response, when challenged in vitro by NNP-LPS. However, C3H/HeJ cells develop normal responses to another thymus-independent hapten conjugate, DNP-AECM-Ficoll. C3H/HeJ mice fail to mount a specific anti-LPS antibody response, when challenged in vivo with doses of soluble LPS which are fully immunogenic for the high-responder strain. However, C3H/HeJ mice develop normal direct and indirect PFC responses to LPS, when challenged with a thymus-dependent form of the immunogen. These results are interpreted as indicating as absolute requirement for functional mitogenicity of the antigen, in the induction of specific thymus-independent antibody responses.

Animals↗
PubMed1975

Role of C'3 and Fc receptors in B-lymphocyte activation.

Attempts were made to identify the non-Ig lymphocyte receptor responsible for B-cell induction by antigen and polyclonal B-cell activators (PBA). As a first step, the role of C'3 and Fc receptors was analyzed. It was shown that complement could be fixed onto B cells to such an extent that the lymphocytes could not bind complement-coated red cells, but this did not result in induction of polyclonal antibody synthesis, nor did it inhibit the lymphocytes response to PBA. However, the C'3 receptros possessed a passive focussing role in the induction of polyclonal antibody responses. Thus, PBA that had fixed complement activated polyclonal responses at lower concentrations than the same substances that had not fixed complement. Most likely the dual binding of PBA molecules to B cells by the PBA and the C'3 receptors caused more PBA molecules to be bound to each cell. However, the focussing function of the C'3 receptors was several orders of magnitude smaller than that of the Ig receptors. Analogous studies were carried out with Fc receptors. Binding of different types of antigen-antibody complexes did not cause activation of polyclonal or specific antibody synthesis, nor did it significantly interfere with induction of antibody synthesis by PBA substances.

Animals↗
PubMed1975

Factors influencing activation of B-cells in immunity.

The hypotheses so far advanced for B-cell activation by antigen are based on the assumption that the interaction between antigen and the Ig receptors delivers at least the initial triggering signal. There are few, if any, experimental findings to support this assumption. On the contrary, a variety of findings indicate that the Ig receptors do not deliver any signal to the cells, whereas activation can be regularly achieved without participation ofIg receptors. The available evidence forces us to suggest that antigen-induced B-cell triggering is always caused by one nonspecific signal, which is delivered to the cells by surface structures, which are not the Ig receptors. For distinctive features of this hypothesis see One Nonspecific Triggering Signal. Various competing hypotheses for B-cell activation have been analyzed, but none of them appears to satisfy the experimental findings.

Animals↗
PubMed1975

Mechanism of b-lymphocyte activation: failure to obtain evidence of a direct role of the Ig receptors in the triggering process.

Experiments were designed to test two hypotheses of B-cell activation by antigen: the cross-linking concept, postulating that a suitable degree of antigen-induced cross-linking of the Ig receptors is sufficient for immunocyte triggering, and the two-signal hypothesis, suggesting that a first signal delivered by antigen interacting with the Ig receptors followed by a second signal given by, for example, a polyclonal B-cell activator is necessary for activation. The results did not support either of these hypotheses. Thus, the hapten FITC coupled to human serum albumin and human gammaglobulin in different conjugation ratios failed to activate B cells, whether the hapten-protein conjugates were soluble or precipitated, whether the experiments were carried out in the presence or absence of different concentrations of sera from different species, and irrespective of the day of assay. Furthermore, the same FITC-protein conjugates or FITC itself coupled to Sepharose particles failed to induce a specific anti-FITC response, even though a range of 10-9-fold concentrations of FITC were used. In contrast, FITC coupled to lipopolysaccharide (LPS) regularly induced a primary anti-FITC response in all the above systems, whether FITC-LPS was soluble or coupled to Sepharose particles. The conjugation ratio of FITC to LPS was within the range of epitope densities used with FITC-protein conjugates. Analogous studies were performed with the above compounds and, in addition, NNP-cap and fowl gammaglobulin, added alone or together with LPS to lymphocyte cultures. In no case did the antigen plus LPS give a better specific anti-FITC response than LPS alone, irrespective of the culture conditions, the epitope densities, the physical form of the conjugates, and whether they were bound to Sepharose particles or not, although this would be expected in terms of the two-signal concept. The results are compatible with the one nonspecific signal hypothesis, ascribing a passive role to the Ig receptors and an active triggering function to thymus-independent antigens. Therefore, the ability to trigger B cells directly will depend on the nature of the carrier, triggering being achieved if the carrier is a polyclonal B-cell activator; the epitope density and the degree of cross-linking of Ig receptors are unimportant for delivering the triggering signal, although they can facilitate the binding of the conjugate to the specific B cells.

Animals↗