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Biomedical subjects

A Coutinho

Publications and source records attributed to A Coutinho.

At least 217 records · Page 12Linked to original sources
PubMed1986

Polyclonal lymphocyte responses to murine Trypanosoma cruzi infection. I. Quantitation of both T- and B-cell responses.

Lymphoid activity was studied in spleen and lymph node cells from Trypanosoma cruzi-infected mice. Blast transformation in each lymphocyte class was assessed by dual parameter analysis for size and surface markers by both FACS and conventional immunofluorescence, while proliferative activity was measured by tritiated thymidine uptake, autoradiography, and analysis of DNA content in single cells. Acute infection results in rapid blast transformation and proliferative activity of all three lymphocyte classes (Ig+, L3T4+, and Lyt 2+). At 2 weeks of infection most cells in these organs are enlarged and more than half are dividing. By 2 and 6 months after infection (chronic phase of resistant strains), large numbers of activated B lymphocytes and, to a lesser extent, of Lyt 2+ T cells are still detected. Similar results were obtained in C57BL/6 (resistant) and C3H/HeJ (susceptible) mouse strains. The implications of this massive polyclonal lymphocyte response to the parasite for the physiopathology of acute and chronic infection are discussed.

Animals↗
PubMed1986

Polyclonal lymphocyte responses to murine Trypanosoma cruzi infection. II. Cytotoxic T lymphocytes.

Intraperitoneal infection of young adult C57BL/6 males with 10(5) blood or cloned culture forms of Trypanosoma cruzi (CL strain) induced the appearance in spleen, blood, and lymph nodes of cytotoxic effector cells detectable in a lectin-dependent 51Cr-release assay. The effector cells were conventional cytotoxic T lymphocytes (CTL), since they were Thy 1+ and Lyt 2+, and the lysis of tumour target cells was strictly dependent on the presence of lectin. CTL activity is already detectable in spleen 2 days after infection, reaches a peak at 2 weeks, and returns to normal levels during the chronic phase (1 month onwards). Increased levels of CTL activity were also detected in lymph nodes with similar kinetics, even in animals that were splenectomized prior to infection. In contrast to spleen, significant levels of CTL activity persisted in lymph nodes in the chronic phases. This functional variable correlates with the appearance of high numbers of large Lyt 2+ lymphocytes in the same organs (50 to 100-fold higher than in control, uninfected mice). Very similar responses are detected in a T. cruzi sensitive mouse strain (C3H/HeJ). It appears, therefore, that T. cruzi infection results in a large polyclonal activation of Lyt 2+ lymphocytes, some of which differentiate to effector, cytolytic functions.

Animals↗
PubMed1986

Quantitative evidence against inactivation of self-reactive B-cell clones.

The frequencies of murine B-cell precursors developing into clones secreting antibodies which bind to autologous (mouse) or heterologous (rabbit or human) forms of the same protein antigen (myosin and albumin) were determined in an attempt to directly test the hypothesis of higher decay rates of B lymphocytes exposed to self-antigens. The results exclude, on a quantitative basis, any form of inactivation or deletion of such cells.

Age Factors↗
PubMed1986

Strong mitogenic effect for murine B lymphocytes of an immunosuppressor substance released by Streptococcus intermedius.

A noncytotoxic protein substance, produced by Streptococcus intermedius, with very potent immunosuppressive properties (F3'EP-Si) was tested for lymphocyte mitogenic activity. Although devoid of T-cell mitogenicity, F3'EP-Si stimulated proliferation and led to high numbers of plaque-forming cells in cultures of normal or T-cell-depleted, small or large splenic B cells from both lipopolysaccharide-responding and -nonresponding mice. The B-cell mitogenic activity of F3'EP-Si was quantitatively comparable to that of lipopolysaccharide, and the simultaneous exposure to both mitogens stimulated additive B-cell responses. Injection of F3'EP-Si into normal mice resulted in increased numbers of spleen cells, higher rates of mitotic activity, and very large numbers of plaque-forming cells, predominantly of the immunoglobulin G2a and -b isotypes. In preliminary experiments, the analysis of surface markers among the lymphocytes participating in the blastogenic response in vivo revealed a T-cell component in the response to F3'EP-Si. These observations are discussed in the context of the immunosuppressive activity of this and other microbial substances.

Animals↗
PubMed1986

Functional and biochemical evidence for the recognition of T cell receptors by monoclonal antibodies to an immunoglobulin idiotype.

Sharing of "idiotypes" by T and B cells with similar nominal specificities has been extensively reported in functional assays. The recent molecular characterization of T cell receptors has led to the suggestion that such idiotypic mimicries could result from "network" selection of available T cell repertoires. Alternatively, the validity of the conclusions taken from those functional assays could be questioned. We have now used an experimental system where recurrent expression of antibody idiotypes by T helper cells requires "learning" from the B cell/antibody compartment, and show here that the idiotypic determinants in question are indeed associated with T cell receptor molecules. A monoclonal antibody (F6(51)) directed to an idiotope of the TNP-binding BALB/c myeloma protein MOPC460 specifically inhibits antigen-dependent proliferation and helper activity of BALB/c anti-TNP-BALB/c helper T cells. The anti-idiotypic antibodies also induce IL-2 production by these helper cells and precipitate a surface molecule with characteristics of T cell receptor. We conclude that, in this particular system, T cell receptors and antibodies of similar nominal specificities share idiotypic determinants.

Animals↗
PubMed1985

Very large and isotypically atypical polyclonal plaque-forming cell responses in mice infected with Trypanosoma cruzi.

Normal C3H/HeJ mice, acutely infected with T. cruzi, develop large numbers of splenic Ig-secreting plaque-forming cells (PFC). IgG2a, IgG2b and IgG1 PFC account for over 90% of all PFC, while the numbers of IgG3- and IgA-secreting PFC are lower than in normal animals. These effects appear to be due to both T helper-dependent regulation and to a mitogenic activity associated with the parasites themselves.

Animals↗
PubMed1985

Segregation of reactivity to the mitogens lipopolysaccharide, lipoprotein and dextran sulfate in clones of the B cell lymphoma line WEHI 279.1.

The B cell lymphoma line WEHI 279.1 (surface IgM+, I-A+ and I-E+) was used as a model system to analyze functional reactivity of B lymphocytes to mitogens. Among the mitogens tested, lipopolysaccharide (LPS), lipoprotein (LP), and dextran sulfate (DxS) all induced a strong, dose-dependent growth inhibition of tumor cells. The analysis of independently derived clones, however, revealed that mitogen sensitivity could be lost in the absence of detectable alterations in the expression of those surface markers. Moreover, reactivity to LPS, LP and DxS segregated independently in these clones, suggesting distinct triggering mechanisms and functional receptors for these mitogens on the B cell membrane.

B-Lymphocytes↗
PubMed1985

Interactions of small B lymphocytes with unprimed noncytolytic T cells: dissociation between "presentation" and growth induction.

The accessory cell requirements in lectin-dependent triggering and growth of unprimed Lyt-2-T lymphocytes were analyzed by quantitatively comparing the ability of small B cells and peritoneal macrophages to either induce reactivity to growth factors or support growth. Lightly or nonirradiated small B cells were 15 to 30-fold less efficient as compared to T cell-depleted peritoneal cell populations, in the support of the lectin-stimulated Lyt-2-T cell proliferation. In contrast, lightly irradiated small B lymphocytes were quantitatively as efficient as macrophages in mediating lectin-driven Lyt-2-T cell proliferation, if relevant supernatants were added into culture. Finally, supernatants derived from cultures where T-small B cell ratios were optimal for growth of responder Lyt-2-lymphocytes were two orders of magnitude less efficient than conditioned medium obtained from cultures containing optimal T-macrophage ratios, in their ability to support growth of activated T cells. We conclude from these experiments that: in contrast to cytolytic T cell precursors, lectin-dependent induction of unprimed Lyt-2- T lymphocytes requires accessory cells; small B cells and macrophages are equally competent in this respect; and growth support by small B cell populations is due to contamination by macrophages which are the only cell type performing this function. We therefore interpret reports on Lyt-2- T cell proliferation upon stimulation with high numbers of small B cells as a two-step process: "presentation" and induction of T cells which is essentially B cell dependent, and factor production ensured by contaminating macrophages.

Animals↗
PubMed1985

Activation of Lyt-2+ T cells by antibodies towards brain-associated antigens. II. Antibody-dependent induction of "nonspecific" cell-mediated cytotoxicity.

Purified IgG from a rabbit anti-mouse brain antiserum (RaMB), previously shown to activate most Lyt-2+ T cells, was probed for lectin-like effects on cell-mediated cytolysis (CML) and found to induce "nonspecific" killing of syngeneic B cell targets by allospecific cytotoxic T lymphocytes (CTL) as effectively as concanavalin A, and at levels comparable to antigen-specific cytolysis of target cells. Interestingly, RaMB-mediated "nonspecific" CML of syngeneic targets, by polyclonally or specifically activated CTL, was restricted to B cell targets and cold target inhibition experiments indicated that syngeneic target cell blasts do not functionally interact with effector CTL in the presence of RaMB. The role of target cell Fc receptors was demonstrated by the competitive inhibition of RaMB-dependent CML by normal rabbit IgG. We conclude that RaMB both activates and bridges the effector CTL to target cells, RaMB-mediated "nonspecific" CML being a similar phenomenon to lectin-facilitated "nonspecific" cytolysis, and the mirror image of classical ADCC. These observations allowed us to interpret the effects of RaMB on allo-major histocompatibility complex-specific CML. Since RaMB stimulates specific CML of B cell targets while selectively blocking that of T cell targets, we conclude that RaMB antibodies interact with structures associated with the CTL antigen receptor, explaining the contradictory effects previously reported with monoclonal antibodies against the CTL receptor complex, which can both stimulate and inhibit T cell functions.

Animals↗
PubMed1985

Ontogenic development of "natural" and induced plaque-forming cell isotypes in normal mice.

The numbers of cells and background plaque-forming cells (PFC) in the spleen of C3H/HeJ mice increase exponentially during the first 2 weeks after birth, but much slower in bone marrow (BM). IgG1 and IgG2a PFC are the first non-IgM PFC detectable, while IgG3 and IgA PFC appear only around weaning. Adult-type PFC numbers and isotype pattern are present in spleen and BM at 4 and 15 weeks, respectively. Neonatal splenic C3H/Tif B cells produce non-IgM Ig classes in vitro in response to polyclonal activation by lipopolysaccharide or by helper T cells. These responses are of low magnitude during the first 2 weeks of life, but both secreted and membranebound IgG1 and IgG3 isotypes are detectable already a few days after birth, in a pattern that is identical to that typical of T cell-dependent or independent responses of adult cells. These results indicate full maturity of B cells in "switch" abilities already from birth, in spite of a general deficiency in terminal maturation. In addition, they demonstrate the complexity of isotype regulation in "background" antibody production in vivo.

Age Factors↗
PubMed1985

Natural effector T lymphocytes in normal mice.

The "natural" T-cell activity in normal unimmunized mice was studied. By double-parameter fluorescence-activated cell sorter analysis, it was found that 5-10% of all splenic Lyt-2+ and L3T4+ lymphocytes are large, of which more than half are in mitotic cycle. In contrast with small resting cells of the same phenotype, activated (large) T cells isolated from normal mice are functional effector cells: L3T4+ large cells induce normal B lymphocytes into proliferation and antibody secretion, while large Lyt-2+ cells efficiently suppress B-lymphocyte responses. No effector cell cytolytic activity could be detected among naturally activated T cells. The significance of these findings for the internal activity in the normal immune system is discussed.

Animals↗
PubMed1985

Mycoplasma mimicry of lymphokine activity in T-cell lines.

During screening of T-T hybridomas for B-cell-stimulating factors (BSF) we found in the conditioned medium of such a line (TUH-14) an activity that turned out to cosediment with a contaminating strain of Mycoplasma arginini on centrifugation. Since it only affected B blasts and only to a minor extent small cells and since it did not induce immunoglobulin secretion, the mycoplasma strain seemed to mimic a BSF. M. arginini was also found in two other T-T hybridoma producer lines but not in a clone of the EL-4 line. Three other M. arginini isolates were not stimulatory for B cells. The presented results point towards the importance of careful control for mycoplasma contamination in established cell lines.

Animals↗
PubMed1985

Functional analysis of pokeweed mitogen-dependent cell interactions in murine spleen cells. I. Lack of B-cell mitogenicity and low frequency of effector helper T cells.

The nature of lymphocyte responses on addition of pokeweed mitogen (PWM) to normal murine spleen cells was studied in low cell density cultures. PWM, over a wide range of concentrations, stimulated proliferation in a set of cells roughly 10-fold smaller than the lymphocyte populations responding to either concanavalin A or lipopolysaccharide. PWM also induced a relatively small number of B lymphocytes in these cultures to mature to Ig-secreting plaque-forming cells (PFC). Proliferative and PFC responses were completely abrogated by T-cell removal from normal spleen cell cultures. Moreover, cell mixture and irradiation experiments demonstrated that B lymphocytes do not proliferate in response to PWM, even in the presence of an excess of normal T cells, suggesting that PFC development results from terminal maturation without proliferation. Finally, parallel titrations of cloned helper cells, normal splenic T cells or T-cell blasts induced by PWM showed that the poor B-lymphocyte responses in normal spleen cell cultures is due to the very low frequency of competent helper cells in these populations. PWM, however, was competent to activate and expand this set of helper lymphocytes in primary cultures.

Animals↗
PubMed1985

Functional recognition of bacterial mitogens by B lymphoma cells: reactivity of WEHI 279.1 to lipopolysaccharide and selection of nonreactive variants.

To analyze functional mitogen recognition by reactive B lymphocytes, we studied the effects of bacterial lipopolysaccharide (LPS) on the growth of the WEHI 279.1 B lymphoma line (W279). We found that LPS inhibits, in a dose-dependent manner, the growth of W279 cells in culture and that it reduces the frequency of cells growing as clones under limiting dilution conditions. Furthermore, we show that differential reactivity of "wild-type" cells to increasing LPS concentrations reflects the heterogeneity in the lymphoma cell population and the frequencies of "resistant" variants to each mitogenic concentration. This allowed us to derive variant tumor cell lines and clones, no longer LPS sensitive, either from mass cultures or, in a single-step selection, under limiting dilution conditions in the presence of low and high concentrations of LPS. Although mitogen reactivity is progressively lost upon prolonged culture, resistance to LPS was found to be a stable trait in selected variants, suggesting that it results from loss of functional mitogen recognition by the reactive cells. The specificity of mitogen reactivity or resistance was shown by the fact that some of the variant clones are still reactive to T helper cell-derived factors and others are not. Thus reactivity to LPS and to T cell factors can be separated, suggesting that the cell lines described here provide new tools for the biochemical analysis of B cell activation.

Animals↗
PubMed1985

Absence of immunoglobulin heavy chain expression results in altered kappa/lambda light chain ratios.

A few hundred hybridoma cell lines derived from spleen cells of normal, nonimmunized, 6-day-old BALB/c or BALB.B10 mice were screened for H- and L-chain production. Roughly half of these hybridomas produced no Ig chain into the culture supernatants. Of the Ig-secretors, 80% produced IgM molecules carrying either kappa or lambda chains in a ratio of 9:1. A surprisingly high frequency of clones (roughly 20%) were found to produce only light chains and in this group, the kappa/lambda ratio was altered to around 1. The indications from the primary screening were confirmed by isolating a large number of these hybrid cell lines and extensive testing of both culture supernatants and ascitic fluids in solid phase RIA and ELISA assays. In some cases, the absence or presence of heavy chain synthesis and the isotype of the light chain was also confirmed by biosynthetic labeling of the hybridoma clones, specific immuno-precipitation of cell lysates or supernatants, followed by analysis in SDS polyacrylamide gels. We conclude from these results that the absence of heavy chain expression is accompanied by frequencies of lambda chain expression, which are much higher than those observed in normal immunoglobulin producing cells, and equal to those of kappa chains. This conclusion can be interpreted as to suggest that the low frequency of lambda producing cells in mouse is in part determined by restrictions in the heavy-light chain interactions at the protein level.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1985

High frequency of natural autoantibodies in normal newborn mice.

Spleen cells from 6-day-old nonimmunized BALB/c and BALB.B10 mice were fused with the nonsecreting hybridoma cell line Sp2/0. Three hundred and eighty-four immunoglobulin-secreting hybrids were screened for antibody activity against mouse actin, tubulin, and myosin, and against TNP, peroxidase, renin, DNA, and neurofilaments. At least 24 hybridomas in the collection (6.25%) exhibited antibody activity against this panel of antigens. Ten of these hybrids were cloned, were propagated, and the corresponding monoclonal IgM protein was isolated from ascitic fluids and was further characterized. At least four groups of antibody specificities were identified: 1) one clone reacting with TNP only; 2) one clone reacting with both actin and tubulin; 3) two clones which bound to both TNP and actin; and 4) a fourth group, comprising the six other clones, which all exhibited widespread reactivity and bound to actin, tubulin, myosin, and TNP. These results indicate: 1) B cell clones directed against self antigens are activated in the internal environment and are recovered consequently by somatic cell hybridization; 2) the widespread antibody specificities found for these newborn mouse antibodies are very similar to those previously characterized with human natural antibodies and human monoclonal Ig; and 3) the frequency of B cells binding to cytoskeletal proteins and TNP is very high (at least 6.25%).

Animals↗