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Biomedical subjects

A Coutinho

Publications and source records attributed to A Coutinho.

At least 73 records · Page 4Linked to original sources
PubMed1995

Thymic epithelium induces full tolerance to skin and heart but not to B lymphocyte grafts.

Athymic nude mice reconstituted at birth with allogeneic thymic epithelia (TE) from day 10 embryos (E10), show life-long specific tolerance to skin and heart grafts, but eliminate B lymphocytes of the TE donor haplotype, nearly as well as those from a third strain. Previous immunizations with B cells do not alter the state of tolerance to skin grafts, but specifically accelerate elimination of lymphocytes. In contrast, transplantation of E15 allogeneic thymuses already seeded by hematopoietic cells resulted in chimeras tolerant to both skin and B lymphocytes. In vitro reactivities towards stimulator spleen cells of the haplotype of the thymus were observed in both E10 TE and E15 thymus chimeras. We conclude that induction of full in vivo tolerance to B cells requires hematopoietic cells, while this is not the case for induction of tolerance to skin and heart tissues; furthermore, in vitro reactivity to stimulator spleen cells of the tolerized haplotype is independent of in vivo tolerance.

Animals↗
PubMed1995

Studies on the T cell dependence of natural IgM and IgG antibody repertoires in adult mice.

The present experiments address the thymic dependence of IgM and IgG natural antibody repertoires in adult euthymic and athymic BALB/c mice, as well as in athymic animals reconstituted with a fixed number of syngeneic T cells. Within 3 weeks of the transfer of 10(7) syngeneic splenic T lymphocytes to athymic mice, the T cell compartment is essentially reconstituted in the peritoneal cavity (up to 80% of the numbers in euthymic animals), but is only 10-20% of controls in the spleen and lymph nodes. Early after transfer, there is an increase in the numbers of activated B cells and of immunoglobulin-secreting cells in the spleen, and within 1-2 weeks, the serum concentrations of IgG1 and IgG2a are fully reconstituted to control levels (30-40-fold increased). Multiparametric analyses of serum IgM and IgG repertoires revealed that euthymic and athymic mice share essentially all natural antibody reactivities towards syngeneic extracts of liver and muscle. When tested at the same immunoglobulin concentrations, however, nude sera consistently show higher values of reactivity in all detectable bands. The transfer of 10(7) splenic T cells into athymic mice results in a general decrease of serum IgM reactivities, some of which become undetectable, and in alterations of the serum IgG repertoire as early as 1 week, and for at least 4 weeks after transfer. T cell transfer, however, fails to restore the euthymic IgM and IgG repertoires within 4 weeks. The present observations demonstrate that, after limited T cell reconstitution of nude mice, there is a rapid and quantitatively important increase of serum IgG1 and IgG2a production; the serum IgM reactivity repertoire is qualitatively similar in euthymic and athymic animals, but is generally decreased by T cell activity; and the serum IgG repertoire, which is qualitatively similar in euthymic and athymic animals, is amplified by T cell activity and partially altered by T cell transfer into athymic animals. These results raise questions on the mechanisms of B cell activation and natural antibody repertoire selection in T cell-deficient adult individuals.

Animals↗
PubMed1995

Regulatory T cells in thymic epithelium-induced tolerance. I. Suppression of mature peripheral non-tolerant T cells.

Athymic mice grafted at birth with allogeneic thymic epithelium (TE) display life-long tolerance to tissue grafts of the TE donor strain, in spite of harboring peripheral T cells capable of rejecting those grafts. Tolerance is maintained in these chimeras by TE-specific regulatory CD4 T cells. We presently address the quantification and the mechanisms of this dominant tolerance process. C57BL/6 mice containing variable but defined numbers of peripheral, resident T cells received cell transfers of graded numbers of peripheral T cells from B6(BALB E10) chimeras (C57BL/6 nude mice grafted with TE from 10-day-old BALB/c embryos), resulting in a series of animals containing a wide range of donor (tolerant) versus host (non-tolerant) T cell chimerism. Increasing the relative representation of donor T cells results in a progressive delay in the rejection of BALB/c skin grafts, life-long tolerance being achieved at a ratio of tolerant and non-tolerant T cell populations of 1. In recipients displaying full tolerance, graft-reactive non-tolerant T cells were not deleted, anergized or committed to noninflammatory functions. Thus, sorted host T cells from tolerant recipients readily rejected BALB/c skin grafts upon transfer to immunodeficient animals. Finally, measurements of "helper" and inflammatory activities, as well as interleukin-4 and interferon-gamma production, failed to discriminate between T cell populations from tolerant and non-tolerant animals after specific in vitro stimulation. We conclude that: (a) TE-selected regulatory T cells can suppress, in a quantitative manner, in vivo T cell responses against major and minor histocompatibility antigens expressed by the TE and, (b) this suppressive activity neither inactivates mature non-tolerant T cells, nor does it seem to drive their differentiation along noninflammatory pathways.

Animals↗
PubMed1995

Analysis of the natural human IgG antibody repertoire: life-long stability of reactivities towards self antigens contrasts with age-dependent diversification of reactivities against bacterial antigens.

We used a quantitative immunoblotting technique to analyze the repertoires of IgG antibody reactivities in the serum of healthy young children, young adult males and aged males with self and non-self antigens. Densitometric patterns of reactivity of purified IgG with self antigens were highly conserved between individuals within a given age group and across age groups. Inter-individual differences were observed, however, upon analysis of self reactivities of IgG in whole serum. A striking heterogeneity between individuals within a given age group and across age groups characterized the reactivity of purified IgG and of IgG in whole serum with bacterial antigens. Inter-individual differences were more marked among aged individuals than among individuals of other age groups. Analysis of variances of reactivities of IgG with bacterial antigens further demonstrated an increased diversity of repertoires of aged donors compared with those of young adults and children. Our results document the stability of the self-reactive repertoires of IgG throughout life, which contrasts with the diversification of the repertoire of IgG antibody reactivities directed toward foreign antigens with aging. These findings support the concept that self-reactive antibody repertoires are positively selected throughout life by a restricted set of self antigens shared by all individuals.

Adult↗
PubMed1995

Natural autoantibodies.

Autoantibodies of the IgM, IgG and IgA classes, reactive with a variety of serum proteins, cell surface structures and intracellular structures, are 'naturally' found in all normal individuals. Present in human cord blood and in 'antigen-free' mice, their variable-region repertoire is selected by antigenic structures in the body and remains conserved throughout life. Encoded by germline genes with no, or few, mutations, natural autoantibodies are characteristically 'multireactive' and do not undergo affinity maturation in normal individuals. Natural autoantibodies may participate in a variety of physiological activities, from immune regulation, homeostasis and repertoire selection, to resistance to infections, transport and functional modulation of biologically active molecules.

Animals↗
PubMed1995

Self-association of the polyene antibiotic nystatin in dipalmitoylphosphatidylcholine vesicles: a time-resolved fluorescence study.

The interaction between Nystatin and small unilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, both in gel (T = 21 degrees C) and in liquid-crystalline (T = 45 degrees C) phases, was studied by steady-state and time-resolved fluorescence measurements by taking advantage of the intrinsic tetraene fluorophore present in this antibiotic. It was shown that Nystatin aggregates in aqueous solution with a critical concentration of 3 microM. The enhancement in the fluorescence intensity of the antibiotic was applied to study the membrane binding of Nystatin, and it was shown that the antibiotic had an almost fivefold higher partition coefficient for the vesicles in a gel (P = (1.4 +/- 0.1) x 10(3)) than in a liquid-crystalline phase (P = (2.9 +/- 0.1) x 10(2)). Moreover, a time-resolved fluorescence study was used to examine Nystatin aggregation in the membrane. The emission decay kinetics of Nystatin was described by three and two exponentials in the lipid membrane at 21 degrees C and 45 degrees C, respectively. Nystatin mean fluorescence lifetime is concentration-dependent in gel phase lipids, increasing steeply from 11 to 33 ns at an antibiotic concentration of 5-6 microM, but the fluorescence decay parameters of Nystatin were unvarying with the antibiotic concentration in fluid lipids. These results provide evidence for the formation of strongly fluorescent antibiotic aggregates in gel-phase membrane, an interpretation that is at variance with a previous study. However, no antibiotic self-association was detected in a liquid-crystalline lipid bilayer within the antibiotic concentration range studied (0-14 microM).

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1995

The ontogeny of class-regulation of CD4+ T lymphocyte populations.

The differential class-regulation of CD4+ T lymphocyte populations is believed to play a major role in determining the qualitative behaviour of the immune system, and in the fate of immune responses in particular. In this article we propose a model for the dynamics of the Th1 and Th2 subpopulations. We put forward the concept of an 'antigenic niche' which allows us to postulate that the key feature underlying the regulation of Th differentiation pathways is the population dynamics of the lymphocytes themselves. Using this model we are able to account for a number of well established experimental observations which were hitherto apparently unrelated and poorly understood. This suggests that our simplified model might be capturing some essential features of the immune system.

Animals↗
PubMed1995

The network theory: 21 years later.

The network theory was proposed 21 years ago, attracting then much interest as applied to the regulation of (clonal) immune responses. The first 10 years of 'idiotypic network' research have thus addressed questions that were already appropriately solved by the clonal selection theory, leading to a justifiable loss of its impact. In contrast, 'second generation networks', concentrate on systemic properties that emerge from a network organization, thus providing a framework for several major questions that seem to supersede clonal solutions: the developmental 'learning' of self antigenic composition and the maintenance of the respective 'memory' (self-tolerance), repertoire selection and the homeostatic regulation of lymphocyte numbers, natural immune activities that are independent of external antigens, the physiology of autoreactivity. The immune network may well contribute solutions to autoimmune diseases, where clonal approaches, classical and modern alike, have failed.

Allergy and Immunology↗
PubMed1994

Isolation of peritoneal precursors of B-1 cells in the adult mouse.

Two weeks of daily peritoneopheresis of adult mice result in the selective depletion of B-1 cells, followed by the appearance of a population of B220+IgM-lymphocytes in the peritoneal cavity. These cells share with bone marrow (BM) pre-B cells expression of lambda 5, VpreB, and RAG-1 genes and a higher fraction of unrearranged V to DJ heavy (H) chain immunoglobulin (Ig) gene segments, when compared with mature B lymphocytes. Upon transfer to SCID recipients, sorted peritoneal B220+IgM- cells fail to colonize the BM, repopulate very few B cells in the spleen, but entirely reconstitute the B-1 cell compartment in the peritoneal and pleuropericardial cavities. In contrast, parallel transfers of sorted BM and pleuropericardial cavities. In contrast, parallel transfers of sorted BM B220+IgM- cells result in reconstitution of the BM and spleen B lineage cell compartments, but in no coelomic B cell repopulation. Both types of pre-B cells reconstitute splenic plasma cells of donor origin, but with markedly distinct efficiencies: the ratio of IgM-plasma cell/B cell numbers in the spleens of peritoneal pre-B cell recipients is more than 500-fold higher than that of recipients reconstituted by BM pre-B cells. We take these data to indicate that (1) differentiative commitment to the B-1 cell population occurs before selection events on mature cells; (2) B-1 precursors exist or may be locally produced in the adult mouse; (3) there is a lineage-related differential ability of mature B cells to undergo terminal differentiation to high-rate Ig secretion.

Animals↗
PubMed1994

Differential contribution of thymic outputs and peripheral expansion in the development of peripheral T cell pools.

The number of peripheral T cells in mice increases up to 100-fold in the first few weeks of life. We have followed the fate of Thy-1 congenic T cells transferred into newborn recipients, to evaluate the relative contribution of thymic output versus peripheral expansion in the constitution of peripheral T cell pools during post-natal development. The results show that in normal animals there is essentially no peripheral expansion of T cells, which show slow turnover rates (1 to 2 months) along that time period. The rates of cell accumulation in the periphery require, therefore, an average of 1 x 10(6)-2 x 10(6) mature thymic emigrants/day for the first 3 weeks of life.

Aging↗
PubMed1994

Negative selection of multireactive B cell clones in normal adult mice.

In the absence of intentional immunizations, normal mice produce natural antibodies that react with a variety of self and foreign antigens. We have now addressed the putative physiological selection of such reactivities and some of their clonal characteristics, by analyzing antibodies produced by B cells at different stages of differentiation. Using an antigen-specific spot-enzyme-linked immunosorbent assay (ELISA) with a panel of self and foreign antigens, we found that newly formed B cells, either from adult bone marrow or from newborn spleen, contain the highest frequencies of IgM antibodies with reactivities towards the panel. Resting peripheral B cells show lower frequencies of such antibodies, that are lowest among naturally activated splenic plasma cells. Analyses of monoclonal IgM antibodies derived from lipopolysaccharide-stimulated bone marrow and spleen cell hybridomas in normal mice show that the majority of reactivities scored in spot-ELISA originate from multireactive IgM clones. In Western blots against a large number of self antigens, each multireactive IgM antibody studied shows a unique and specific pattern of reactivity. We conclude that multireactive B cell clones are very frequent in the emergent repertoires of newborns and adults, but are subsequently negatively selected from bone marrow to periphery, and from the available repertoire to that of natural plasma cells. It, thus, seems that multireactivity of natural antibodies is not a positively selected property, but represents the sum of unique multireactive clones that have escaped inactivation or deletion.

Animals↗
PubMed1994

Regulation of VH-gene expression is a lineage-specific developmental marker.

We have previously shown that in IgH congenic mice VH-gene family usage in neonatal spleen B cells and adult Ig-secreting cells is entirely determined by the IgH locus, while in adult resting B cells it is regulated by genetic element(s) located outside the IgH locus. Two observations reported here demonstrate that the differential expression of VH genes is an intrinsic property of the respective cell populations, determined by both the IgH locus and by a cis element(s) operating independently in the same animal. First, the study of F1 hybrids between the IgH congenic B6a and CB.20 strains demonstrates that cis elements control VH-gene family expression. Second, studies in irradiation chimeras showed that the environment in which cell differentiation proceeds is unable to overcome those controls. In chimeras of IgH congenic donors, VH-gene expression in fetal liver-derived splenic B cells and Ig-secreting cells is dictated by the IgH haplotype, while in bone marrow-derived B cells is entirely determined by the cis element(s). These results show a developmental and cell lineage-related restriction in VH-gene expression, and suggest that most adult splenic Ig-secreting cells may originate from precursors originally present in fetal liver, but which are rare among adult bone marrow precursors and CD5+ B cells.

Aging↗
PubMed1994

Developmental shift in the patterns of interleukin production in early post-natal life.

In mice, immunological tolerance to self is established in the perinatal period, when tolerance susceptibility to allogenic tissues is higher than in adults. We have now investigated whether this could result from developmental regulation of effector functions of T cells exposed to specific antigens, by studying the "natural" or T cell receptor-induced expression of several interleukin genes. We used qualitative and quantitative polymerase chain reaction methods to study interleukin (IL)-2, IL-4, IL-10 and interferon-gamma mRNA expression by splenic cells at different ages. The results show that newborn peripheral cells (up to day 7), in contrast to the T lymphocytes of adult mice, express high levels of IL-4 and interferon-gamma, and very low levels of IL-2 messenger spontaneously and upon specific T cell activation. This characteristic phenotype depends on intrinsic T cell properties, as it is not due to the newborn environment.

Animals↗
PubMed1994

Selectivity of recognition of variable (V) regions of autoantibodies by intravenous immunoglobulin (IVIg).

In the present study, we demonstrate that intravenous immunoglobulin (IVIg) is capable of binding to variable (V) regions of anti-endothelial cell antibodies (AECA) of healthy donors and patients with systemic lupus erythematosus (SLE). Among V regions of AECAs, IVIg selectively recognized certain idiotypes expressed by the autoantibodies of a given individual, in the case of both natural and SLE-associated AECAs. These observations provide new and direct evidence that IVIg interacts idiotypically with V regions of autoantibodies and that the efficacy of such interaction depends on individual autoantibody specificity. Our findings may be relevant for the understanding of the mechanisms that control expression of natural autoantibody activity in serum and for that of the differences in response to IVIg therapy that are seen between patients with autoimmune disease.

Antibody Specificity↗
PubMed1994

Fluorescence study of a derivatized diacylglycerol incorporated in model membranes.

A fluorescence study of a diacylglycerol derivatized with the n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) chromophore (NBD) was carried out. Fluorescence self-quenching was observed for this probe in lecithin model membranes due to collisional interaction rather than to an aggregational behaviour of the probe. The efficient energy migration (Ro = 28 A) of the NBD fluorophore was studied via the fluorescence depolarization upon increase of probe concentration in membranes, and the results are compared with a model where a random distribution of the probes is assumed. A surface location of the chromophore was concluded for the NBD derivative of diacylglycerol, both from the fluorescence parameters and from the study of its fluorescence quenching by spin label probes. Very high lateral diffusion coefficients were obtained for these probes, both from the self-quenching (D = 2-6 x 10(-6) cm2 s-1) and from the spin probe quenching (D = 3.5 x 10(-6) cm2 s-1) studies. A concomitant fluorescence study of the related probe NBD-phosphatidylcholine revealed that its photophysical behaviour is similar to the derivatized diacylglycerol.

4-Chloro-7-nitrobenzofurazan↗
PubMed1994

Leukocytes of patients with Schistosoma mansoni respond with a Th2 pattern of cytokine production to mitogen or egg antigens but with a Th0 pattern to worm antigens.

Cytokine responses of peripheral blood mononuclear cells from humans infected with Schistosoma mansoni were assessed. By ELISA and ELISPOT, persons with acute and hepatosplenic infections produced higher levels of interleukin (IL)-4 and IL-5 and higher frequencies of IL-4-producing cells in response to mitogen than did uninfected persons. In contrast, mitogen-induced production of the Th1 cytokine interferon-gamma (IFN-gamma) did not differ from that of uninfected controls. Upon stimulation with egg antigens, many patients responded with elevated IL-4 mRNA levels but displayed no appreciable increases in Th1 (i.e., IFN-gamma and IL-2) cytokine transcripts. Nevertheless, in cells stimulated with adult worm antigen, a more mixed Th0-type response was observed with production of both Th1 and Th2 cytokines. These results support previous findings in laboratory mice that schistosome infection results in increased production of Th2 cytokines. Unlike mice, infected humans do not display a generalized down-modulation in Th1 responses but instead show a selective deficiency in IFN-gamma and usually IL-2 responses to egg antigens.

Adolescent↗
PubMed1994

Analysis of natural and disease-associated autoantibody repertoires: anti-endothelial cell IgG autoantibody activity in the serum of healthy individuals and patients with systemic lupus erythematosus.

The present study demonstrates that natural IgG with anti-endothelial cell activity is present in the serum of healthy individuals and in pooled normal human Ig. By using a novel method that allows for the simultaneous and quantitative assessment of reactivities of antibodies with a large number of antigens in tissues, we observed that natural anti-endothelial cell antibody (AECA) recognizes a restricted set of self antigens in endothelial cells that is conserved among healthy individuals. The extent to which natural AECA activity is expressed in serum and the pattern of reactivity of AECA with endothelial cell antigens showed little variability between individuals. Analysis of AECA in the serum of patients with systemic lupus erythematosus (SLE) revealed a higher amount of activity and a wider spectrum of antigenic specificities than that recognized by natural antibodies in endothelial cell extracts. AECA activity of IgG in whole serum was lower than that of purified IgG in the case of healthy individuals and showed little variation among individuals. In contrast, no difference was found between AECA activity of purified IgG and that of IgG in patients' serum suggesting that SLE sera lack the factors that control expression of AECA activity in the serum of healthy individuals. Our results indicate that natural autoantibodies recognize a restricted and conserved set of self antigens. Our observations further suggest that defective regulation of the expressed autoreactive B cell repertoire is the basis for expansion of novel clonal specificities and enhanced autoantibody activity in serum of patients with autoimmune disease.

Autoantibodies↗