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Antigen presentation determines immunologic outcome, and by modifying the presentation of allergen to the host one can prevent an allergic response. Under certain conditions, covalent linkage, of ovalbumin to rat IgG, a molecule already tolerated by the host, can make a protein-IgG conjugate which down-regulates the immune response to this food allergen. The suppression is allergen specific. It affects both T and B cell immune responses. Administration of allergens linked to isologous IgG may provide a novel strategy for allergy prevention.

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Serum antibody concentrations to two viral, five bacterial, and two food antigens were investigated in 307 elderly Swiss subjects, and the hypothesis of whether serum antibody titers decreased with age was tested. The cross-sectional part of the study consisted of 216 unselected consecutive patients hospitalized in one geriatric hospital. The patients were divided into two age groups (65 to 84 and 85 to 102 years old), and their antibody titers were compared. No age-related decreases in antibody titers were observed. The members of the two age groups were well matched for medical diagnosis and nutritional and inflammatory status. The prospective part of the study consisted of 91 healthy elderly subjects living in the community; they were 71 to 76 years old when they were enrolled in the study. Their serum antibody status was measured at the beginning of the study and 4 years later. We observed a significant decrease in diphtheria antitoxin levels and a significant increase in antibody titer to the capsular polysaccharide of Streptococcus pneumoniae. No change in antibody titer to rotavirus, respiratory syncytial virus, lipopolysaccharide of Escherichia coli, C polysaccharide of S. pneumoniae, or the polyribosyl-ribitol phosphate of Haemophilus influenzae was observed. Thus, no signs of B-cell immunosenescence were seen in these two groups of elderly Swiss people.

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Intravenous administration of trinitrophenyl-modified isologous immunoglobulin-induced nonresponsiveness to subsequent epicutaneous painting of sensitizing doses of trinitrochlorobenzene. Isologous immunoglobulin with various degrees of trinitrophenyl substitution (11.2, 14.3, 27 and 47.3) prevented sensitization. The suppression of contact hypersensitivity was dependent on the dose of tolerogen and was hapten specific. Tolerance was inducible in mice of the strains CBA (H-2k), C57BL/6 (H-2b), and DBA/2 (H-2d) but not in Balb/C (H-2d) mice, suggesting that this trait maps outside the murine major histocompatibility complex. Tolerance induced by trinitrophenyl-modified immunoglobulin was associated with decreased hapten-induced proliferation of draining lymph-node cells. Unlike in other models of tolerance in which a decreased interleukin-2 to interleukin-4 ratio can be observed, administration of tolerizing trinitrophenylated immunoglobulin was associated with deficient hapten-induced release of both interleukin-2 and interleukin-4.

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Previous studies have shown that both nucleosides and oligonucleotides linked to isologous gammaglobulin suppress anti-nucleic acid antibody production both in vivo and in vitro. The aim of this study was to determine whether one can make a DNA-human gammaglobulin (HGG) conjugate which can inhibit anti-double stranded DNA (dsDNA) antibodies obtained from a heterogeneous population of systemic lupus erythematosus (SLE) sera. To do so, we constructed conjugates of sonicated dsDNA fragments of 100-400 base pairs covalently linked to HGG with varying degrees of substitution of DNA:HGG. An ELISA inhibition assay was used to determine which conjugate best inhibits the binding of anti-dsDNA antibodies. Conjugate 2, prepared with monomeric HGG (150 kD) with a high degree of substitution (3.72 DNA:HGG) inhibited the binding of anti-dsDNA antibodies from 27 of 31 SLE sera. In addition, this conjugate inhibited the spontaneous formation of anti-dsDNA in vitro by cultured lymphoid cells from selected SLE patients. Together, this data suggests that a 'generic' tolerogen may provide an antigen specific therapy for SLE.

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We have shown that, under certain conditions, a single dose of a conjugate of beta-lactoglobulin linked to isologous IgG suppresses specific antibody responses (IgE, IgG, and IgA) in rats immunized with beta-lactoglobulin. This suppression is allergen specific. It also appears to be effective when animals are first immunized and then tolerized. In addition, administration of the beta-lactoglobulin-IgG tolerogen abolishes mast cell mediator release. This has been shown both in vivo and with passively sensitized mast cells in vitro. We propose that the construction of a tolerogen made of whole protein allergen linked to IgG could provide a novel specific therapy for allergic reactions.

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A number of lactose-binding lectins have recently been identified in the rat and mouse intestine, one of which corresponds to the C-terminal domain of IgE-binding proteins, originally identified in rat basophilic leukemia (RBL) cells and mouse 3T3 fibroblasts. In the present report, we describe the affinity purification of a rat intestinal lactose-specific lectin which binds murine IgE antibodies. This binding most likely occurs via the immunoglobulin carbohydrate chains, as it is inhibited by lactose. This intestinal lectin molecule is also immunologically related to the previously described IgE-binding protein (epsilon BP) isolated from RBL cells, since it is recognized by antibodies raised against recombinant epsilon BP. This intestinal form of epsilon BP has a molecular mass of 17.5 kDa, which is much lower than that of its RBL cell analogue (31 kDa). The attachment of IgE to the mouse intestinal epithelium was demonstrated by immunohistochemistry, along with the presence of a corresponding mouse intestinal epsilon BP. The carbohydrate-dependent nature of this attachment was established by demonstrating that IgE binding to mouse epithelium was specifically abolished by lactose (4 mM) and by a blood-group-A-active tetrasaccharide (0.2 mM), but not by mannose (10 mM). Finally, the association of IgE with the mouse intestinal epithelium was prevented by competition with the purified IgE-binding lectin isolated from rat intestine. Although the physiological function of this intestinal protein is still unknown, the finding that IgE binds to a lectin in the intestinal epithelium pinpoints a possible novel mechanism for the regulation of IgE-mediated disorders, such as food allergy.

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A novel strategy to prevent an allergic immune response is presented. We tested the idea that a potent allergen such as cow's milk Beta lactoglobuline (ßLG) can be rendered tolerogenic by covalent linkage to isologous gammaglobuline. We found that, under certain conditions, a single intravenous dose (of 2 mg) of ßLG conjugated to rat gammaglobuline prevent both IgE and IgG antibodies to ßLG. This was allergen specific. It appears to be also effective in sensitized animals. In addition, it prevents serotonine release by rat mastocytes both in vivo and in vitro. We suggest that this approach could provide a novel strategy to specifically prevent allergy.

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In order to extend the concept of constructing tolerogens (i.e., compounds which induce immunologic tolerance), we developed a novel method to covalently link either protein or peptide to isologous gammaglobulin. We used disuccinimidyl suberate (DSS) for preparing protein conjugates in solution in a novel use of this reagent. We tested the efficacy of this method in two different experimental models: in the first, we found that administration of pigeon cytochrome C conjugated to mouse IgG in vivo induces T cell unresponsiveness in vitro. In the second, we induced unresponsiveness to factor VIII light chain both in newborn and, more importantly, in adult mice already immune to factor VIII. We hope that this simple method will provide a powerful tool to construct tolerogens useful in the specific treatment of either allergic or autoimmune diseases.

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We examine whether B cell lines enriched for DNA specificity from either autoimmune (BWF1) or normal mice (Balb/c) can be rendered unresponsive to autoantigen in terms of the specific suppression of direct antibody-forming cells to DNA. These B cell lines were both Lyt-1 positive and negative. Preincubation with oligonucleotide, covalently linked to mouse gamma-globulin, specifically suppressed the antigen-driven response elicited by DNA horse red blood cells in B cell lines from both strains of mice. There is a 5-fold difference in susceptibility to DNA-specific tolerance induction between B cell lines of BWF1 and Balb/c mice. Thus, B cells from autoimmune mice do not appear to have an inherent absolute defect in being rendered tolerant to autoantigen, but are relatively less susceptible to DNA-specific tolerance than nonautoimmune cell lines.

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C5-deficient mice grafted with thymus from C5-sufficient donors and immunized with C5 failed to make humoral antibody to C5, suggesting that the transfer of thymus had induced tolerance. Irradiated C5-deficient hosts repopulated with lymphoid cells from thymectomized C5-deficient mice grafted with C5-sufficient thymus also failed to respond to immunization with C5, thus showing that the state of tolerance can be adoptively transferred. These results demonstrate that natural tolerance to self-protein antigen is "learned" in the thymus.

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In vitro studies were undertaken to determine whether the level of anti-DNA antibody can be modulated in humans with systemic lupus erythematosus (SLE). DNA fragments of different sizes, i.e., oligonucleotide (N20-30) or oligonucleotide (N10-100), were covalently linked either to human gammaglobulin (HGG) and used as tolerogens or to keyhole limpet hemocyanin and used as immunogens. Experiments were done to determine whether such tolerogens specifically diminish antibodies to denatured DNA, native DNA, or both. PBL were obtained from 87 patients with SLE, 55 of whom spontaneously produced anti-DNA antibodies in vitro. Furthermore, of these 55 test subjects 23 made anti-DNA antibodies in response to antigen challenge in vitro. Exposure of PBL to tolerogenic oligonucleotide-HGG reduced spontaneous antibody formation in 34 of the 55 patients' PBL and abrogated the in vitro-induced response in all instances. The suppression was tolerogen specific. In some SLE patients lymphoid cells were suppressed by both (N10-100)-HGG and (N20-30)-HGG, while in others lymphoid cells were suppressed by only one. Longitudinal studies of spontaneous antibody production showed that the same tolerogens consistently reduced anti-DNA antibody formation in lymphoid cells of 12 patients on several occasions over a 2-yr interval, but in 8 others the results were either variable or inconsistent. In contrast, tolerogens consistently abrogated the antigen-induced response in all 23 patients' PBL. These results obtained in humans in vitro suggest that the principle of carrier-determined tolerance could be applied as a specific therapy for SLE in vivo.

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We examined whether C5-sufficient mice which are naturally tolerant to this antigen have suppressor T cells to C5 humoral immune response. Two congenic strains of mice B10.D2 (NSN) and B10.D2 (OSN) differing only in the presence or absence of C5 were used. Irradiated (760 rds) sufficient hosts were reconstituted with a nonadherent spleen cell suspension from either sufficient or deficient mice or a mixture of both. Hemolytic C5 levels were assayed. Sufficient spleen cells appeared to prevent the drop of C5 level caused by anti-C5 antibody made by deficient spleen cells. Spleen cell suspensions from sufficient mice primed with deficient spleen cells exhibited better anti-C5 activity than normal sufficient spleen cell suspensions. This anti-C5 activity is abrogated by treatment of the NSN spleen cell suspensions obtained from NSN primed with OSN spleen cells with anti-Thy-1.2 antiserum and complement. Suppression of the humoral response to C5 failed to affect the anti-sheep red blood cell immune response. Suppressor T cells are resistant to low-dose irradiation, cortisone treatment and adult thymectomy. In contrast, they are sensitive to high doses of irradiation and both high and low doses of cyclophosphamide treatment. Thus, C5-sufficient mice, in contrast to C5-deficient mice, appear to have antigen-specific suppressor T cells which downregulate the humoral immune response to C5. In addition, we examined the relationship of these suppressor T cells to the state of tolerance in helper T cells of C5-sufficient mice. This was done in irradiated deficient mice which were repopulated with spleen cell suspensions selectively depleted of either Lyt-1+ or Lyt-2+ T cell subsets. These chimeras were challenged with murine C5 and both the primary and secondary immune response was measured by inhibition of the C5 hemolytic activity. It was found that only spleen cell suspensions of the deficient mice selectively depleted from the Lyt-2+ subset of T cells responded to the antigen both in the primary and secondary response. In contrast, either subset of T cells from the sufficient mice failed to respond. Thus, it appears that in sufficient mice helper T cells to C5 are intrinsically tolerant or physically and/or functionally deleted. In conclusion, the data suggest that both T cell compartments are unresponsive and play a role in the mechanism of tolerance to a physiologic antigen.

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The immunogenicity of DNA fragments (either oligonucleotide (oligo) or total DNA digest) covalently linked to keyhole limpet hemocyanin (oligo-KLH or DNA-KLH) was tested with peripheral blood lymphocytes (PBL) from 63 systemic lupus erythematosus patients (SLE) in vitro. PBL from 10 normal individuals and 11 rheumatoid arthritis (RA) patients served as controls. Antibodies to three nucleic acid antigens (oligo, denatured DNA (d-DNA), and native DNA (n-DNA] were assayed in supernatants of cultured lymphoid cells by a sensitive solid-phase radioimmunoassay. More than 50% of SLE and RA patient lymphoid cells formed spontaneous antibodies to one or several nucleic acid antigens. In contrast, only two normals did. After in vitro challenge with oligo-KLH or DNA-KLH, cultured lymphocytes of more than 50% of SLE patients formed antibodies to one or several nucleic acid antigens. Similar results were obtained in PBL from RA patients. In SLE patients, the response to both antigens was either monospecific or polyspecific, but DNA-KLH appeared to raise a greater proportion of antibody to n-DNA than oligo-KLH. A greater proportion of patients with active disease responded in vitro compared with those with inactive disease. A mixture of oligo together with KLH was not immunogenic in vitro. Oligo-KLH or DNA-KLH did not raise antibody to an irrelevant antigen, ovalbumin. Of particular interest, PBL from seven of 10 normal subjects formed antibody to n-DNA after challenge in vitro with oligo-KLH. The data support the view that DNA fragments could be an important immunogen in SLE. Furthermore, this study provides an in vitro model to test the tolerogenicity of similar fragments of DNA linked to self carrier molecules such as gamma-globulin.

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The HLA genetic region was studied in 51 patients with systemic onset juvenile rheumatoid arthritis: 35 with childhood onset and 16 with adult onset (adult Still's disease). HLA genotypes were established by including family members, 261 of whom were also typed in the study. The most marked difference between patients and controls involved the HLA-DR4 gene, which occurred with a frequency of 0.348 in the childhood onset patients and 0.170 in the controls (chi 2 = 8.97, P = 0.0028, adjusted P = 0.017). In contrast, the adult onset patients showed a marginal increase in HLA-DR7, but were similar to controls with respect to HLA-DR4. HLA-Bw35 was increased in children with systemic onset disease, in accordance with earlier findings. The results suggest that patients with systemic onset juvenile rheumatoid arthritis have complex HLA associations which are different in childhood onset and adult onset disease.

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