Biomedical subjects

J Charreire

Publications and source records attributed to J Charreire.

At least 37 records · Page 2Linked to original sources

PubMed1995

Molecular heterogeneity of antigen- or idiotype-induced anti-thyroglobulin monoclonal autoantibodies.

To define the molecular basis of the cognitive interaction in experimental autoimmune thyroiditis (EAT), we sequenced the variable regions of monoclonal autoantibodies to thyroglobulin (Tg), specific or not for the F40D peptide, a Tg peptide capable of inducing EAT in CBA/J mice. Three MoAbs were obtained by immunization with syngeneic Tg of CBA/J (3B8G9, 2F6F2) or C57Bl/6 (4D11F4) mice. 3B8G9 was specific for F40D peptide, whereas 2F6F2 and 4D11F4 were not. Two others were raised in CBA/J mice by manipulation of idiotypic pathways: B12 resulted from the immunization with one Ab2 beta, bearing the internal image of one F40D epitope, and TA2 from the immunization with F40D-specific cytotoxic HTC2 T cells. B12 and TA2 were both specific for F40D. All hybridomas expressed different members of the J558 VH family, except 3B8G9 which expressed a Q52 VH gene segment. These data led us to hypothesize that regulatory anti-id autoantibodies used members of one VH family located in the 5'-end of the VH locus, whereas EAT-associated autoantibodies used a member of one of the most D-proximal VH family. As expected, no homologies were found when anti-F40D monoclonal autoantibodies were compared with two other monoclonal autoantibodies displaying a different epitopic specificity. Among the anti-F40D monoclonal autoantibodies, one histidine residue located in position 35 of the CDR1 region was constantly found. Moreover, TA2 and B12 exhibited two common amino acids in their CDR3 regions, one glycine and one tyrosine, in positions 98 and 99, respectively. Striking homologies were found between TA2 and one anti-polyGAT MoAb, and between 3B8G9 and some anti-phenyloxazolone (phOx) monoclonal autoantibodies. Lastly, the VK sequence from 4D11F4 was identical at the amino acid level to the VK sequence from another monoclonal autoantibody, 81B1, which was previously raised towards syngeneic Tg in CBA/J mice. Our data imply that anti-idiotypic regulatory circuits in EAT might be generated by a heterogeneous population of B cells rather than obtained by a single dominant B cell population.

Amino Acid Sequence↗

PubMed1994

Induction of autoimmunity by immunization of mice with human thyrotropin receptor.

The development of autoimmunity was investigated after repeated immunizations with human thyrotropin receptor (hTSH-R) of five congenic strains of female and male mice. After each immunization, free T3 levels and antibodies to hTSH-R and to six peptides of the hTSH-R were assayed. Our results showed that H-2s and H-2q female mice developed features of autoimmunity such as antibody responses to hTSH-R and to hTSH-R peptides, transient variations in the levels of free T3 thyroid hormone, and lymphocytic infiltrations in their thyroid glands. Concerning the antibody responses to hTSH-R peptides, we found that peptide P1 (352-366) contained a major B cell epitope. Furthermore, strain-specific B cell epitope was exemplified by peptide 92 (12-30) and two male- and female-specific B cell epitopes were located in peptides 91 (32-46) and 93 (316-330), respectively. These features appeared rather related to hyperthyroidism.

Amino Acid Sequence↗

PubMed1994

Antibodies specific for human thyrotropin receptor induce MHC antigen expression in thyroid cells.

Autoantibodies (AAbs) to hormone receptors are found in autoimmune diseases such as Graves' disease (GD) or myasthenia gravis. A structural link between hormone receptor and MHC genes has been documented, suggesting a possible co-regulation of MHC and hormone receptor genes. Thus, in vitro experiments were designed to search for a pathologic role for AAbs. In a model study, we investigated whether adding murine anti-human thyrotropin receptor mAbs would affect MHC gene expression in either cloned human thyroid epithelial cell or primary murine thyroid epithelial cell cultures. We found that two anti-human thyrotropin receptor monoclonal AAbs, 11E7 and 34A, induced, with an intensity comparable to that of IFN-gamma, transcription and expression of class I and class II/Ii chain proteins in human and murine thyroid epithelial cells. Two other anti-human thyrotropin receptor mAbs, 12E3 and 243-3, were ineffective. These data suggest a new role for autoantibodies in the pathology of autoimmune endocrinopathies.

Animals↗

PubMed1993

The effects of a monoclonal antibody to interferon-gamma on experimental autoimmune thyroiditis (EAT): prevention of disease and decrease of EAT-specific T cells.

CBA/J mice immunized with thyroglobulin (Tg) develop an experimental autoimmune thyroiditis (EAT) with lymphocytic infiltration of the thyroid glands, autoantibodies to Tg and occurrence of EAT-specific T cells. When these mice were treated for 4 weeks after immunization with 1 mg/week of a monoclonal antibody (mAb) that neutralizes the activity of interferon-gamma (IFN) a beneficial effect on the onset of EAT was observed. Characteristic features of EAT were significantly reduced, including the lymphocytic infiltrations of the thyroid glands and the serum levels of autoantibodies to Tg. Moreover, in lymphoid organs, mAb to IFN-gamma significantly reduced the percentages of Tg-specific CD8+ cells, labeled by the anti-clonotypic mAb AG7. These Tg-specific T cells seem responsible for thyroid damages and disease development, since EAT was simultaneously abrogated. These results show that IFN-gamma plays an essential role in the pathophysiology of EAT and suggest the possibility to treat autoimmune thyroid diseases with mAb to IFN-gamma or drugs able to antagonize the production and/or the action of this cytokine.

Animals↗

PubMed1993

Immunization with thyroglobulin-specific cytotoxic T cell hybridoma induces anti-thyroglobulin antibodies: characteristics of monoclonal anti-thyroglobulin auto-antibody.

We have produced five monoclonal autoantibodies (mA-Abs) to thyroglobulin (Tg) and more precisely to one epitope located within the < 10-kDa pTg tryptic fragment suspension capable of inducing experimental autoimmune thyroiditis (EAT). They were selected from spleen cells from CBA/J mouse immunized with the syngeneic cytotoxic T cell hybridoma HTC2. HTC2 cells are specific for one Tg epitope located within the EAT inducer pTg tryptic fragments and are able to prevent EAT induction by pTg. The restricted specificity of the humoral response previously observed in vivo was further demonstrated and defined in vitro at the single cell level. Competitive studies for binding to pTg or to the < 10-kDa pTg tryptic fragments demonstrated that HTC2-induced anti-Tg mA-Abs recognized an epitope(s) located in the < 10-kDa pTg tryptic fragment (as did 3B8G9, one conventional anti-Tg mA-Ab we selected). We ruled out the possibility that HTC2-induced anti-Tg A-Abs belong to the group of the natural A-Abs due to the lack of recognition of actin, dsDNA, TNP-ovalbumin, tubulin, their isotypes (IgG1 or Ig2a), and their affinities (in the 10(-7) M order of magnitude). The results strengthen the hypothesis that T and B cells sharing the same specificity can express similar idiotopes on their respective receptors for antigen. They also demonstrate the existence of a regulatory idiotypic network that could explain the protection from EAT after injection of inactivated HTC2 cells or its anti-clonotypic mAb.

Animals↗

PubMed1993

Production of Ab2 beta, anti-idiotypic monoclonal antibodies, specific for human thyrotropin receptor.

We had previously selected five monoclonal antibodies (mAb1) upon their specific binding to human thyrotropin (hTSH). These mAbs1, which are specific for two distinct epitopes of the hTSH beta-chain, also inhibit the hTSH binding to human thyroid membrane preparations. We then immunized BALB/c mice with the various mAbs1 in attempt to generate anti-idiotypic Abs (Ab2) directed to the hTSH receptor (hTSHR). Five hybridomas secreting monoclonal antibodies (mAb2) to the hTSHR were selected from one mouse immunized with the mixture of the five mAbs1. These monoclonal antibodies to the hTSHR were selected upon their ability to specifically bind to the hTSHR. Moreover, we showed that binding of mAbs2 to the individual mAbs1 used for immunization was specifically inhibited by hTSH and not by insulin or by human chorionic gonadotrophin, a highly related glycoprotein hormone. The five mAbs2 inhibited the binding of iodine-labeled hTSH to its receptor by 8 to 41%; moreover, two of them stimulated the adenylate-cyclase system of the thyroid cells. With respect to their properties, mAbs2 were classified as mAb2-beta.

Adenylyl Cyclases↗

PubMed1993

Ig VH gene family usage in spleen cells of CBA/J mice immunized with experimental autoimmune thyroiditis (EAT) inducer antigens.

Experimental autoimmune thyroiditis (EAT) is an autoimmune disorder of the thyroid gland induced in susceptible strains of mice by thyroglobulin (Tg). We recently showed that low Mr (< 10 kDa) Tg tryptic fragments and a 40 amino-acid peptide (F40D) from Tg could induce EAT as well as native Tg. Because it has been reported that autoantibodies (A-Abs) express VH families preferentially located in the D-proximal VH gene segment, we investigated whether A-Abs specific for one pathogenic peptide from Tg were also skewed towards D proximal VH gene segment. In that respect, we immunized CBA/J mice with EAT inducer antigens of decreasing sizes: Tg (660 M(r)), < 10 kDa Tg trypic fragments or F40D peptide (4.9 kDa M(r)) from Tg. The VH gene segments utilized by immune spleen cells were determined by hybridization to total spleen cell RNA previously deposited onto nylon membranes and densitometric scans. This study was conducted on days 7 and 9 after determination of the maximum amounts of mRNA coding for immunoglobulins and on day 28 when A-Ab levels are the highest. Results were compared to VH gene segment expression both in normal and adjuvant-injected mice. We found that immunization of CBA/J mice with EAT inducer antigens stimulate B cells the restriction of which, in terms of VH family usage, depends on the size of the immunizing antigen: the larger the antigen, the higher the numbers of VH families used. Moreover, we found that B cell stimulation consecutive to immunization with the peptidic antigen inducing EAT occurs in VH Q52 family, a VH encoded by D-proximal gene segment.

Animals↗

PubMed1992

Characterization and sequencing of a 40-amino-acid peptide from human thyroglobulin inducing experimental autoimmune thyroiditis.

We previously demonstrated that: a) a cytotoxic T cell hybridoma (HTC2) was able to induce lysis of syngeneic macrophages pulsed with either porcine thyroglobulin (pTg) or the tryptic fragments (TF) from pTg less than 10 kDa (M(r)) and that b) these low M(r) pTg TF included pathogenic epitopes because their injection into CBA/J mice induces thyroid lymphocytic infiltration typical of experimental autoimmune thyroiditis. Therefore the biochemical analysis of the TF preparation from pTg less than 10 kDa M(r) was undertaken and the characterized peptides were tested for their ability to be recognized or not by HTC2 cells. The sequencing of the selected peptides showed a 70% sequence homology with a portion of human thyroglobulin (hTg). The lack of a published sequence of pTg led us to synthesize a 40-amino acid peptide (F40D) similar to that portion of hTg. This F40D peptide was able to generate lymphocytic infiltrations in CBA/J mice thyroid glands, as was the native pTg molecule. Although the lymphocytic infiltrations were similar in the pTg or F40D-immunized mice, auto-antibodies to pTg or to hTg were only detectable in mice immunized with pTg. In contrast, autoantibodies levels to F40D peptide were significantly increased in serum from mice in which EAT had been induced by the F40D peptide. This highly hydrophobic peptide shows a M(r) of 4,492 kDa; it is located at the end of the second-third of the thyroglobulin molecule and up to now represents a unique sequence from the hTg molecule inducing experimental autoimmune thyroiditis.

Amino Acid Sequence↗

PubMed1992

Protection from experimental autoimmune thyroiditis conferred by a monoclonal antibody to T cell receptor from a cytotoxic hybridoma specific for thyroglobulin.

A clonotypic mAb, AG7, has been prepared from splenocytes of CBA/J mice immunized with a cytotoxic T cell hybridoma, HTC2, specific for a pathogenic epitope of the thyroglobulin molecule in association with class I MHC Ag. AG7 binds to HTC2 cells but not to the other T cell hybridomas tested. Moreover, when used in functional studies, AG7 blocks the HTC2 capacity of specific target lysis. It also reacts with a determinant that comodulates with the CD3 Ag present on the surface of HTC2 cells. Immunoprecipitation of 125I-labeled solubilized HTC2 membranes demonstrated two bands located at 90 and 72 kDa under nonreducing conditions, which became a 46-kDa band under reducing conditions. Finally, when AG7 is injected into CBA/J mice, on day -1 before immunization with the pathogenic tryptic fragments of the thyroglobulin molecule, experimental autoimmune thyroiditis is abrogated. Thus, one of the multiple potential mechanisms of the protective immunity against EAT induced by HTC2 cells that we previously proposed, i.e., the generation of anti-clonotypic antibodies to HTC2 TCR, seems apparent.

Animals↗

PubMed1992

Characterization of monoclonal antibodies to the human thyrotropin receptor.

We have produced four monoclonal antibodies (mAbs), 34A, 49G, 11E7, and 12E3, which bind the human TSH receptor (hTSH-R) when expressed on a human thyroid cell line (GEJ), freshly dissociated human and murine thyroid cells, or Chinese hamster ovary cells stably transfected with the hTSH-R gene. These mAbs were obtained after immunization of DBA/1 mice with affinity-purified TSH-binding sites from GEJ cells. Biochemical studies, including sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, Western blot, and immunoprecipitation of solubilized GEJ cell membranes or human thyroid cells showed that most of the mAbs recognized two bands: one located at 46-48 kilodaltons and the other at 86-88 kilodaltons. Inhibition of [125I]hTSH binding to solubilized porcine membranes (TSH-receptor auto-antikörper assay) or Chinese hamster ovary cell membranes previously transfected with hTSH-R gene showed that mAb 34A recognizes the hTSH-binding site of both receptors. In contrast, mAbs 49G, 11E7, and 12E3 recognize a structure located near the hTSH-binding site. Lastly, the ability of these mAbs to stimulate murine thyroid function was investigated by measuring cAMP production and iodide accumulation. The 34A mAb, which fully competes with [125I]TSH for binding to hTSH-R, was able to induce both functions. Conversely, the 12E3 mAb, which was the least potent inhibitor of [125I]TSH binding to hTSH-R-transfected cells had no effect. A relationship was, therefore, established between the capacity of mAb to hTSH-R to inhibit [125I]hTSH binding and their ability to induce thyroid functions.

Animals↗

PubMed1992

T cell mapping of one epitope from thyroglobulin inducing experimental autoimmune thyroiditis (EAT).

These data collect the advance made in the last few years in our laboratory in defining one epitope from the thyroglobulin (Tg) molecule (660 KDa) inducing Experimental Autoimmune thyroiditis (EAT) in CBA/J mice. We achieved the characterization of one EAT-inducer Tg peptide by combining "in vitro" biochemical and immunological approaches and "in vivo" studies. Since T cells recognize degraded forms of the antigen and since endogenous antigens preferentially activate class I-restricted T cells, we hypothesized that one cytotoxic T cell hybridoma, named HTC2, which prevents further EAT induction in mice injected with Tg would be specific for one EAT inducer peptide. In order to identify one Tg epitope inducing EAT, enzymatic treatment of the protein by trypsin, HPLC purification and sequence analysis were performed. Simultaneously, tryptic digests were used to pulse CBA/J macrophages and tested for their ability to be recognized by HTC2 cells. Lastly, when digests were recognized by HTC2 cells their capacity to induce EAT in CBA/J mice was evaluated. To further assess the pathogenicity of the sequenced Tg peptide, one synthetic peptide was made and its capacity to induce EAT verified. By this procedure we identified for the first time one 40 amino-acid peptide from human thyroglobulin inducing EAT in CBA/J mice.

Amino Acid Sequence↗

PubMed1991

Improved amphotericin B activity by a monoclonal anti-Cryptococcus neoformans antibody: study during murine cryptococcosis and mechanisms of action.

Current therapy of cryptococcosis is unsatisfactory, particularly in patients with AIDS. Experimental cryptococcosis models in DBA/2 mice were used to determine whether the murine monoclonal anticryptococcal antibody (designated E1) might potentiate the chemotherapeutic effect of amphotericin B (AmB). According to the inoculum size, these mice died spontaneously from acute pneumonia (high inoculum) or from brain swelling (lower inoculum). AmB and E1 together significantly improved the survival of mice in both models compared with AmB alone. The mechanisms by which E1 might potentiate AmB activity were investigated in vitro. When cryptococci were preincubated with AmB or another polyene antibiotic, nystatin, there was an augmented binding of E1. AmB enhanced phagocytosis by unstimulated peritoneal macrophages in the presence of E1 or normal rabbit immunoglobulins. Normal and immune IgG deserve further study to determine under what circumstances the chemotherapeutic effect of AmB can be enhanced.

Amphotericin B↗

PubMed1990

Production and characterisation of monoclonal antibodies directed against different epitopes of human thyroid stimulating hormone.

We have produced monoclonal antibodies (mAbs) to human thyroid stimulating hormone (hTSH) and selected five that specifically recognize hTSH and do not cross-react with the other human glycoprotein hormones such as luteinizing hormone (LH), chorionic gonadotropin (CG), follicle stimulating hormone (FSH). All of the antibodies were of the IgG1 subclass with affinities ranging from 5.3 X 10(8) to 1.9 X 10(10) mol-1.l; they could be assigned to two subgroups on the basis of their epitope specificity.

Antibodies, Monoclonal↗

PubMed1990

Prevention of experimental autoimmune thyroiditis through the anti-idiotypic network.

The central goal in the therapy of autoimmune diseases is to develop potent tools able to exert specific control of the immune response to self Ag. Anti-Id may provide such specific immunodulators because the relevance of the idiotypic network in autoimmunity is well documented. We now describe the protective immunity against experimental autoimmune thyroiditis induced exclusively by a thyroglobulin (Tg)-specific cytotoxic T cell clone and show that this down-regulation occurs through the generation of anti-Id antibodies (Ab) (Ab2Beta) which recognize the paratope of a anti-Tg mAb (Ab1) specific to the pathogenic epitope of the Tg molecule. We further analyze the various steps of the Ab responses (Ab1, Ab2, and Ab3) in terms of poly-, mono-, and autospecificities for the pathogenic epitope of the Tg molecule and for the idiotope of the related Ab.

Animals↗

PubMed1990

Autoantibody specific for a thyroglobulin epitope inducing experimental autoimmune thyroiditis or its anti-idiotype correlates with the disease.

In order to elucidate the role of anti-thyroglobulin (Tg) autoantibodies (A-Ab) in experimental autoimmune thyroiditis (EAT), a kinetic study was conducted in EAT-susceptible (CBA/J) and non-susceptible (C57BL/6) strains of mice. From day 0 to 70 post-Tg immunization, titers of A-Ab to Tg and to the linear 5-10-kDa Tg tryptic fragment inducing EAT as well as anti-idiotypic A-Ab representing the internal image of the thyroidogeneic antigen were measured. EAT onset, development and recovery correlate with the presence of two subsets of A-Ab only in susceptible strains of mice. First, with the presence of anti-Tg A-Ab to one determinant borne by the linear 5-10-kDa Tg tryptic fragment, and second with the presence of anti-idiotypic A-Ab specific for the monoclonal anti-Tg A-Ab (3B8G9) paratope which binds to Tg determinant inducing EAT.

Animals↗

PubMed1990

In vivo beneficial effects of cyclosporin A and 1,25-dihydroxyvitamin D3 on the induction of experimental autoimmune thyroiditis.

In a recent work, we provided evidence that the in vitro inhibitory effect of cyclosporin A (CsA) was potentiated by the addition of another immunosuppressive molecule, the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). In the present study, we investigated the in vivo influence of the association of both drugs administered at infratherapeutic doses, using an experimental model of autoimmune thyroiditis in CBA mice. Treatment regimen of the animals was initiated at priming with thyroglobulin (Tg) and consisted of daily administration of CsA (10 and 20 mg/kg/day, intragastrically) and/or 1,25(OH)2D3 (0.1 and 0.2 microgram/kg/day, ip) for 21 days. Control mice that were given a placebo preparation orally and the vehicle of vitamin D3 metabolite ip developed a severe disease as assessed by histological examination on Day 28 postimmunization and detection of circulating anti-Tg antibodies. Treatment with either drug administered alone at the doses mentioned above did not affect the incidence of thyroiditis and only reduced by up to 26% the severity of histological lesions. In contrast, the mice treated simultaneously with both drugs exhibited a lower incidence of thyroid pathology and developed a significantly milder disease (P less than 0.001) as compared to controls. However, there was no alteration in the levels of anti-Tg antibodies. This in vivo beneficial effect of low doses of CsA and 1,25(OH)2D3 was not due to an accumulation of CsA in the blood of treated mice since the levels of CsA were similar, regardless of the administration of 1,25(OH)2D3. Our data suggest that these two immunomodulatory agents used together at low doses may be an effective therapy of autoimmune disorders with fewer side effects.

Animals↗

PubMed1990

Improvement of amphotericin B activity during experimental cryptococcosis by incorporation into specific immunoliposomes.

Cryptococcosis is an opportunistic infection that is responsible for increased morbidity and mortality in patients with the acquired immunodeficiency syndrome. The high toxicity of the antifungal agent that is mainly used against cryptococcosis, amphotericin B (AMB), accounts for the need for new treatments, especially in patients with the acquired immunodeficiency syndrome because of the high relapse rate of cryptococcosis. Drug targeting may be one of these alternate treatments. Since we have demonstrated that an immunoglobulin G1 (IgG1) anti-Cryptococcus neoformans serotype A monoclonal antibody (E1) was protective during experimental cryptococcosis in mice, we investigated whether specific targeting of AMB with liposomes that bear E1 would improve the therapeutic index of the drug. For that purpose, in vitro and in vivo experiments were designed to compare the specificities and activities of these liposomes with those of control immunoliposomes bearing a nonrelated IgG1 monoclonal antibody (CY34). The immunoliposomes were prepared by covalently linking E1 or CY34 and small unilamellar vesicles. When immunoliposomes were incubated with yeast cells, only E1-bearing liposomes recognized C. neoformans. In vivo, mice that were treated 24 h after infection with one injection of AMB (0.12 mg/kg of body weight) intercalated into E1-bearing liposomes survived significantly longer than did those given the same dose of AMB alone or AMB intercalated into nontargeted liposomes or control immunoliposomes. None of the mice that were given control treatments did statistically better than those that were given AMB. Keeping in mind that this kind of therapy requires knowledge of the antigenic type of the infecting organism, the results suggest that specific targeting of small doses of AMB improve the efficacy of AMB and might be an alternative to the use of larger doses of AMB.

Amphotericin B↗

PubMed1990

Normal suppressive T cell function of Epstein-Barr virus induced B cell activation in type 1 (insulin dependent) diabetes mellitus.

Several studies have demonstrated abnormalities of T cell regulation of Epstein-Barr virus-induced B cell activation in systemic autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematous, and systemic sclerosis. However, a normal suppressive peripheral T cell function was observed in Graves' disease. To investigate whether this abnormality is a common feature to other autoimmune diseases, we studied T cell regulation of Epstein-Barr virus induced B cell activation in 15 newly diagnosed type 1 (insulin dependent) diabetes mellitus patients and 10 normal control subjects. Peripheral B lymphocytes infected with Epstein-Barr virus were cultured for 20 days in the presence or absence of autologous T cells at different ratios (1:1 and 1:4). IgM and IgG secretions into the supernatants were determined using an enzyme-linked immunosorbent assay. The extent of suppression when T cells were added, as measured by a suppression ratio, was not significantly different in type 1 (insulin dependent) diabetes mellitus patients and normal subjects. We conclude that in type 1 (insulin dependent) diabetes mellitus, the autoimmune reactivity is not dependent upon a generalized suppression defect. It can be hypothesized, therefore, that in type 1 diabetes mellitus as well as in Graves' disease, a local or organ specific suppressor deficit may induce the autoimmune phenomena.

Adolescent↗