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M Zanetti

Publications and source records attributed to M Zanetti.

At least 199 records · Page 11Linked to original sources
PubMed1987

Bovine leukocyte phagocytosis and bacteria killing monitored by intracellular acridine orange fluorescence and extracellular fluorescence quenching.

The time course of phagocytosis and intracellular killing of serum-opsonized Escherichia coli K12 and Staphylococcus aureus SG511 by glass-adherent bovine peripheral blood polymorphonuclear leukocytes (PMNLs) and cultured monocytes (macrophages) was monitored by fluorescence microscopy of single cells using the acridine orange (AO)/crystal violet (CV) technique. After interaction of glass-adherent leukocytes (20, 40, 60 min, 37 degrees C) with opsonized bacteria, cells were stained with the fluorescent dye AO. Living bacteria stained green, dead bacteria stained orange. The addition of CV to AO-stained bacteria quenched the fluorescence of extracellular bacteria only. CV does not penetrate living bovine PMNLs which allows the discrimination of ingested (fluorescent) and extracellular (nonfluorescent) bacteria during attachment and phagocytosis of bacteria by adherent PMNLs. We investigated quantitatively phagocytosis and intracellular killing of serum-opsonized bacteria by bovine PMNLs from 22 bulls of 4 different Swiss dairy breeds. Within 60 min maximum uptake (approximately 12 bacteria/PMNL) and killing (approximately 80%) of serum-opsonized Escherichia coli K12 and Staphylococcus aureus SG511 were achieved. The AO/CV technique was also used to quantify the uptake and intracellular killing of serum-opsonized Escherichia coli K12 by cultured monocytes (macrophages). Within 60 min maximum uptake of bacteria (approximately 16/MO) was achieved; approximately 83% of bacteria were killed.

Acridine Orange↗
PubMed1987

Idiotype regulation of self responses, autoantibody V regions and neonatal B cell repertoire.

The role of autoantibodies themselves in immune regulation is still unknown. There is evidence that some autoantibody idiotypes (Id) may play a regulatory role in physiologic and possibly pathologic situations of the immune system. Here we present evidence that (1) certain autoantibody Id modulate the immune response to self antigens (regulatory Id), and (2) that the neonatal preimmune repertoire is primarily devoted to self recognition. The humoral and cellular events following immunization with regulatory Id suggest an active participation of T cells in the regulatory events. Based on immunochemical, structural and molecular genetics analysis, it appears that this regulatory Id is of germline origin as it exists within the neonatal preimmune repertoire. B cell hybridomas generated from unstimulated splenocytes of neonatal mice are primarily self-reactive, suggesting that the immune system essentially begins as a self-recognizing system. The findings are discussed with regard to the relationship between germline repertoire, autoantibodies, and regulatory idiotypes.

Animals↗
PubMed1986

Perturbation of the autoimmune network. II. Immunization with isologous idiotype induces auto-anti-idiotypic antibodies and suppresses the autoantibody response elicited by antigen: a serologic and cellular analysis.

We report on the humoral and cellular events following autologous immunization against an idiotype (Id62) borne on a murine monoclonal autoantibody to thyroglobulin, and their impact on the autoantibody response to thyroglobulin. BALB/c mice with a state of active auto-anti-idiotypic immunity and challenged with thyroglobulin in complete Freund's adjuvant 2 wk after the last immunization with idiotype were found to have a suppressed autoantibody response. This suppression could be adoptively transferred to syngeneic x-irradiated recipients by using whole spleen cells from idiotype-primed mice. Transfer of separate T and B lymphocyte populations proved instrumental in disecting humoral from cellular events and in establishing that whereas B cells were required for transferring an intact anti-idiotype antibody response, T cells from idiotype-primed mice were necessary to transfer suppression. These findings contribute to our understanding of the interrelationship between antigen, idiotype, and anti-idiotype in the immune response to self-antigens, and the role of certain idiotypes in regulating autoimmune responses.

Animals↗
PubMed1986

Detection of a regulatory idiotype on a spontaneous neonatal self-reactive hybridoma antibody.

To investigate the physiologic relevance of an idiotype-driven regulation of the immune system, we began a search for spontaneous self-reactive hybridomas in neonatal BALB/c mice. We sought hybridoma antibodies reactive with thyroglobulin (Tg) and expressing Id62, a recurrent idiotype with regulatory properties borne on induced adult autoantibodies to Tg. We describe herein such a neonatal Tg binding/Id62-positive monoclonal antibody (mAb) B10H2, and compare it with prototype mAb 62, an adult Tg-binding/Id62-positive mAb. Both mAb react with the same Tg epitope, as demonstrated by their abilities to totally inhibit the binding of the other to Tg. Moreover, as assessed by a double-reciprocal plot of their binding to Tg, their relative affinities for Tg are comparable. Likewise, mAb B10H2 appears idiotypically similar to mAb 62 because it totally inhibits the binding of mAb 62 to homologous anti-idiotype. Finally, as previously shown for mAb 62, mAb B10H2 expresses Id62 independently on both heavy (H) and light (L) chains, as evidenced by the immunoblot binding of a specific anti-Id62 probe to separated H and L chains. By molecular genetic analysis both antibodies appear to have made use of a member of the same VH 7183 gene family. Altogether, these findings suggest that neonatal mAb B10H2 and adult mAb 62 are very similar if not identical, and regulatory idiotype Id62 may be germline encoded. Furthermore this observation supports, in general, the concept of an idiotype-driven regulation for autoimmune responses.

Animals↗
PubMed1986

B-lymphocyte regulation of the immune system. III. Preparation and characterization of rabbit antibodies that inhibit the biological activity of the lymphokine, B-cell-derived enhancing factor (BEF).

Antibodies that inhibit the biological activity of B-cell-derived enhancing factor (BEF) were produced in a rabbit immunized with BEF obtained from a non-immunoglobulin-producing B-cell line. Immunoglobulin G (IgG) fractions purified from the preimmune or the immune rabbit serum were compared by testing for their capacity to inhibit regulatory activities of BEF. It was found that the enhancing activity exerted by BEF on the in vitro antibody response to sheep red blood cells was inhibited when anti-BEF IgG was incorporated in the cultures. Moreover, anti-BEF IgG abolished the inhibitory activity exerted by BEF on the expression of Fc receptor by lymphoid cells. Comparable concentrations of preimmune IgG were ineffective in inhibiting BEF activity. The passage of supernatant fluid containing BEF activity through an anti-BEF IgG-coupled Sepharose column yielded an 84-fold purified product that exhibit BEF immunoregulatory properties. These results will accelerate further studies aimed at better characterization of the BEF molecule(s).

Animals↗
PubMed1986

A role for antiidiotypic antibodies in immunologically mediated nephritis.

In summary, evidence exists that immunologically mediated renal diseases can be modulated by interfering with the mechanism of production of autoantibodies by specific antiidiotypic immunity. However, more studies are necessary to establish efficient and safe parameters from which to suggest such treatment in human immune nephritides. Although the evidence is still circumstantial, it is necessary to keep in mind that antiidiotypic antibodies may also add to glomerular immune deposits and potentially could contribute to the chronicity of some forms of immunologic nephritis. The fate of the majority of immunologic nephritides is a slow progression toward end-stage renal insufficiency. Although in most cases the etiology remains to be determined, it is generally felt that a better understanding of the initial immune dysregulation may lead to a greater possibility of control and cure. In this perspective, new directions of research, such as the one reviewed here, should be considered. Modulation of the damaging autoimmune responses in SLE or Goodpasture's Syndrome might be possible. For instance, the identification of a cross-reacting idiotype among anti-GBM antibodies would be the first step for a possible future use of natural or synthetic idiotypes for autovaccination purposes. Antiidiotype antibodies, particularly in the form of monoclonal chimaeric (mouse/human) hybridomas, could be utilized for passive immunosuppression, particularly in combination with plasmapheresis, with or without conventional immunosuppressive drugs. Clearly, this is part of a long-term process that may or may not be realized. The success of new forms of specific immunologic treatment for kidney diseases may ultimately depend upon ongoing efforts in the study of the new molecular aspects of regulation of potential nephritogenic immune responses.

Animals↗
PubMed1986

Shared idiotypes and restricted immunoglobulin variable region heavy chain genes characterize murine autoantibodies of various specificities.

The study of the Ig variable region heavy chain (VH) genes used to encode antibodies specific for self-epitopes from murine hybridomas showed that three VH families are primarily utilized: VH J558, the largest family, and VH QPC52 and VH 7183, the families most proximal to the Ig joining region heavy chain genes. These monoclonal autoantibodies express cross-reactive idiotopes shared by rheumatoid factors and antibodies specific for Sm. The expression of these idiotypes is independent of major histocompatibility complex and Ig constant region heavy chain haplotypes, self-antigen specificity, and even the VH gene family utilized. Though the experiments described here are limited to murine autoantibodies, similarities exist between murine and human autoimmune diseases. Studies that aim to investigate the relationship between VH gene expression and the presence of cross-reactive idiotypes among human autoantibodies should enable us to better understand the mechanisms of autoimmunity and self-tolerance.

Animals↗
PubMed1986

Idiotypic regulation of autoantibody production.

The manuscript will deal with various aspects of the origin of autoantibody formation in relationship with idiotypy. The proposed analysis will cover three major aspects: (1) overview of the published data on idiotypic characterization of autoantibodies in various systems by conventional serologic investigation; (2) presentation of the structural and genetic data available for autoantibodies; and (3) description of the most significant experiment of in vivo manipulation of autoantibody production using idiotypy as the central element for immune regulation. The manuscript will also discuss some theoretical aspects of regulation by idiotype and its possible relevance to improve our understanding of autoreactivity as well as the pathogenesis of certain autoimmune diseases.

Animals↗
PubMed1985

Two subpopulations of differentiated chondrocytes identified with a monoclonal antibody to keratan sulfate.

We have prepared a monoclonal antibody, named MZ15, that specifically binds keratan sulfate. Immunofluorescence studies showed that the distribution of keratan sulfate in articular cartilage was not uniform: the amount of keratan sulfate increased with distance from the articular surface. Two subpopulations of chondrocytes could be distinguished after isolation from cartilage by the presence or absence of cell surface keratan sulfate. Keratan sulfate-negative chondrocytes were shown to come from the upper cartilage layers. There was therefore a direct correlation between biochemical heterogeneity of cartilage matrix and heterogeneity within the chondrocyte population. During growth in monolayer culture, superficial chondrocytes began to synthesize keratan sulfate, but the cells could still be distinguished from cultures of deep or unfractionated chondrocytes by their reduced substrate adhesiveness and tendency to remain rounded.

Animals↗
PubMed1985

B lymphocyte regulation of the immune system. II. Inhibition of Fc receptor expression of lymphocytes by BEF, a lymphokine of B cell origin.

Recently, we described a new lymphokine of B cell origin, capable of selectively preventing the differentiation of T suppressor cells from the precursor into the effector stage. As a result, antibody production against various antigens is markedly increased. We termed this lymphokine B cell-derived enhancing factor (BEF). To discern the mechanism(s) by which BEF interferes with the activation of T suppressor cells, experiments were undertaken to explore the effect of BEF on the induction of Fc receptors (FcR). The induction of FcR on T cells has been implicated in the down-regulation of antibody synthesis, and it has been suggested that the expression of FcR for a given immunoglobulin precedes the release of factors with regulatory functions for the corresponding isotype. In the experiments reported here, murine spleen cells were incubated for 24 hr in the presence of IgG1 or IgA monoclonal antibodies, were washed, and the number of FcR gamma 1+ and FcR alpha+ cells were calculated by a rosette assay. The effect of BEF was studied either during the inductive phase or before, i.e., by pretreating the cells with BEF for 18 hr at 37 degrees C before the inductive phase. Our results show that BEF abolishes, in a dose-dependent manner, the expression of isotype-specific FcR in spleen cells when present during the inductive phase, as well as when cells are pretreated with it. In successive experiments, we tested the effect of BEF on the induction of FcR on T cell-enriched or B cell-enriched spleen cells. The results show that BEF is effective in selectively inhibiting FcR expression on T lymphocytes, but not on B lymphocytes, once isolated from the total spleen cell population. These findings provide further insight into the mechanism by which BEF modulates the immune response, and suggest that different mechanisms may be involved in the induction of FcR on T and B lymphocytes, respectively.

Animals↗
PubMed1985

Heavy and light chains of a mouse monoclonal autoantibody express the same idiotype.

We report on the detection of the same idiotype (Id62) on the heavy and light chains of a mouse monoclonal autoantibody to thyroglobulin. This result was obtained by a simple, sensitive, and reproducible technique, namely immunoblot on nitrocellulose paper of electrophoretically separated heavy and light chains by using a panel of 125I-labeled anti-idiotype probes. These included polyclonal heterologous and syngeneic affinity-purified antibodies and a syngeneic monoclonal antibody. The immunoblot findings were confirmed through the solid-phase radioimmunoassay binding of the anti-idiotype probes to isolated H and L polypeptide chains prepared by conventional chromatography methods. Because anti-idiotype probes define a putative regulatory idiotype of the BALB/c autoimmune response to thyroglobulin, it is suggested that the simultaneous presence of the same idiotype on H and L chains may confer to antibody regulatory properties within the idiotypic network. The significance of the existence of the same idiotype on H and L chains and the implication of this finding for the regulation of the immune response to self antigens are discussed.

Animals↗
PubMed1984

Autoreactive rat T-cell lines: establishment and cellular characteristics.

Stable self-reactive T-cell lines were established from the draining lymph nodes of Buffalo rats primed with rat thyroglobulin (Tg) in complete Freund's adjuvant. Following two rounds of in vitro selection with Tg or with purified protein derivative (PPD) of Mycobacterium tuberculosis, Tg- or PPD-specific lines were selected. Self-reactivity, manifested by a potent proliferative response of these lines to syngeneic stimulators in the absence of the selecting antigen (Tg or PPD), emerged by 3-4 weeks after the establishment of the lines. This proliferation could not be attributed to reactivity against heterologous serum components. Despite this syngeneic mixed-leukocyte reaction (SMLR) which was readily detectable, specific stimulation by Tg or PPD over the level of the SMLR could still be demonstrated. These lines also responded to concanavalin A but not to allogeneic stimulator cells. More than 95% of T cells of these lines displayed the rat T-helper-cell-specific (W3/25) marker and they secreted a defined lymphokine, T-cell growth factor (TCGF), upon stimulation with syngeneic stimulator cells, independent of the presence of soluble antigen. T-Cell lines such as the ones described here will be useful in elucidating the biological significance of the AMLR/SMLR and its relation to nominal antigen reactivity.

Animals↗
PubMed1984

Mesangial immune deposits induced in rats by antibodies to fibronectin.

Immune deposits in the glomerular mesangium were induced in rats by injection of rabbit antibodies to rat plasma fibronectin (FN). By direct immunofluorescence and electron microscopy, deposits of immunoglobulins were detected in the mesangium of all rats injected with anti-FN IgG but not of control rats injected with normal rabbit IgG. By light microscopy, kidneys obtained 20 days after the antibody injection appeared to be normal. No proteinuria was detected during the experiment. Tissue uptake studies combined with direct immunofluorescence examination suggest that the initial accumulation of rabbit immunoglobulins in the glomerular mesangium is probably due to direct local binding of anti-FN antibody rather than trapping of immune complexes formed in the circulation. Quantitation of the direct binding using an in vivo perfused kidney system indicate that only a small fraction of the injected antibodies (less than 1 microgram/kidney) could bind. These studies indicate that (1) mesangial immune deposits may be induced by injection of antibodies to a glycoprotein, fibronectin, which is a normal structural component of the mesangium; (2) the initial accumulation of immunoglobulins in the mesangium is probably related to an in situ binding; and (3) mesangial antigens might be involved, in certain cases, in autoimmune reactions.

Animals↗
PubMed1984

Induction of autoantibodies to thyroglobulin by anti-idiotypic antibodies.

We report on the induction of autoantibodies to thyroglobulin (Tg) and idiotype-positive molecules (Id') in naive BALB/c mice and Buffalo (BUF) rats after immunization with affinity-purified rabbit anti-Id antibodies directed against the Id of a mouse monoclonal antibody ( mAb62 ) specific for a hormone-containing epitope of Tg. Autoantibodies to Tg were produced in about one-half of anti-Id immunized mice, whereas Id' was detected in all but one mouse. Autoantibodies to Tg and Id' were not produced in control mice similarly immunized with non-anti-Id Ig. To clarify the relationship between molecules with Tg-binding activity and those referred to as Id', Tg-specific monoclonal antibodies were generated from an anti-Id-immunized mouse. By competitive inhibition experiments, we could determine that they were idiotypically very similar to the monoclonal antibody ( mAb62 ) at the origin of the immunologic cascade. Thus, autoantibodies induced by anti-idiotypic immunization alone are likely to be of the Ab1' type. Similarly, autoantibody to Tg and Id' molecules were induced in young normal rats of the autoimmune-prone BUF strain. Together, these results suggest that antibodies to Id directed against autoantigens are sufficient per se to activate self-reactivity.

Animals↗