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Myocarditis offers a unique opportunity to study the factors contributing to its transition from a viral infection to an autoimmune disease. In this article, we review recent studies on the role of nitric oxide (NO), gamma interferon (IFN-gamma) and interleukin 12 (IL-12) in the progression from early (viral) to late (autoimmune) phases of myocarditis induced by Coxsackievirus B3 (CB3) in highly susceptible (A.CA) and moderately susceptible (B10.M) mice. NO plays a paradoxical role, being protective in early stages but detrimental later in the course of disease. Treatment with antibody to IFN-gamma reduced early disease, but had little effect on the severity of cardiac lesions at later times. Treatment with recombinant (r) IL-12 significantly reduced the autoimmune cardiac lesions in moderately susceptible B10.M mice, but had no measurable effect in highly susceptible A.CA animals. These studies provide evidence that the profile of inflammatory mediators produced early in the course of viral infection determines the later development of autoimmune disease.

Previously we showed that autoimmune myocarditis could be induced in mice by immunization with purified murine cardiac myosin (MCM). In this study, we found that identical disease could also be induced in genetically susceptible mice by immunization with porcine cardiac myosin (PCM). The cardiac lesions induced by both antigens were characterized by extensive infiltration of the myocardium accompanied by myocyte necrosis. A novel finding was the presence of multinucleated giant cells and eosinophils in the cardiac infiltrates, in addition to a mixture of mononuclear cells and polymorphonuclear cells described previously. Immunohistochemical staining demonstrated that the mononuclear cells consisted predominantly of macrophages, CD4+ T cells and, to a lesser extent, CD8+ T cells and B cells. In addition, increased cardiac expression of adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) were demonstrated in mice that developed myocarditis as compared with those that did not develop disease upon immunization with either PCM or MCM. The levels of TNFalpha detected in spleen cell culture supernatant were found to be higher in mice that developed myocarditis than in those that did not develop the disease. Mice immunized with PCM generated T cells and B cells reactive not only with PCM but also with MCM, and vice versa. In addition, the serum levels of IgG1 anti-MCM antibodies produced in mice immunized with PCM as well as MCM were found to correlate positively with the development of myocarditis. Such a detailed characterization of the murine model of autoimmune myocarditis induced by PCM or MCM allowed us to compare the disease process induced by homologous self and foreign antigens.

Here we describe for the first time that recognition by human T cells of human thyroglobulin depends upon its iodine content. We have examined the proliferation of lymphocytes from blood of autoimmune thyroiditis patients and normal individuals to thyroglobulin preparations containing different amounts of iodine. A minimal degree of iodination was required to elicit the proliferative response of both patients and normal individuals since thyroglobulin preparations containing no detectable iodine did not induce proliferation. A non-iodinated thyroglobulin preparation that was iodinated in vitro produced significant proliferation of both patient and normal lymphocytes. Addition of IL-2 to the culture medium enhanced proliferation but did not change the pattern of response.

Autoantibodies to human thyroglobulin (hTg) are found in the sera of many patients with thyroid diseases. To localize epitopes recognized by these autoantibodies, hTg was incubated with tryspin for 4 hours at 37 degrees C under non-reducing conditions. Releasing peptides from hTg in their natural conformation. These peptides were then analyzed by western immunoblot using either autoantibodies from patients with autoimmune thyroiditis or murine monoclonal antibodies (mAb) produced against hTg. The autoantibodies reacted primarily with two low molecular weight peptides with apparent molecular weights (MWap) of 15 and 20 kDa. The pattern of tryptic peptides recognized by these autoantibodies resembled that of one of the mAbs (137C1), as shown by immunoblots in either one or two dimensional SDS-PAGE. To characterize these peptides further, they were separated by a high performance liquid chromatography (HPLC). The column separated the 4-hour tryptic digest of hTg into multiple peptide peaks. Further analysis by SDS-PAGE showed that one of these peaks contained the 15 kDa peptide. The 15 amino acid sequence at the amino-terminus of this peptide was determined. This amino acid sequence (KVPTFATPWPDFVPR) corresponds to a unique sequence near the carboxyl-terminal end of hTg, starting with amino acid 2657. The size of the peptide indicates that it extends to the carboxyl-terminal end of hTg. This fragment contains one of the antigenic sites of hTg that binds autoantibodies from patients with autoimmune thyroid disease.

Thyroglobulin antibodies (TgAbs) are typically found in autoimmune thyroid diseases and, more rarely, in nonautoimmune thyroid diseases and healthy subjects. To determine whether TgAbs associated with different conditions recognize different epitopes on the thyroglobulin molecule, we studied 28 patients with Hashimoto's thyroiditis, 30 with Graves' disease, 21 with thyroid carcinoma, 18 with nontoxic goiter, and 25 healthy subjects. All patients were selected for the presence of TgAbs; 4/25 healthy subjects also had TgAbs. The sera were assayed for the their ability to inhibit the binding of monoclonal antibodies to thyroglobulin in an ELISA assay. We found that: 1) TgAbs in Hashimoto's patients preferentially recognized three clusters of epitopes (II, III and typically VI), with no difference between the goitrous and the atrophic variants; 2) TgAbs in Graves' patients were directed toward cluster II, with no difference between the presence or the absence of ophthalmopathy; 3) TgAbs in thyroid carcinoma patients recognized the same clusters as Hashimoto's patients; 4) TgAbs in nontoxic goiter patients and in the four healthy subjects showed no restriction in epitope recognition. We suggest that in individuals with no overt clinical or biochemical thyroid abnormalities but with TgAbs, the finding that these TgAbs recognize particular immunodominant clusters may be utilized to predict full-blown thyroid disorders. Longitudinal studies are needed to evaluate the possible clinical application of this methodology.

Genetic susceptibility is an important issue in understanding the mechanism of the autoimmune endocrinopathies and in assessing the risk of these conditions in pediatric patients. To this end, we evaluated autoantibodies to thyroid antigens, thyroglobulin (TgA) and microsomal antigen (TMA), in white and in American black juvenile patients with Type I diabetes mellitus (DM) to determine the predictive value of thyroid autoantibodies for the development of autoimmune thyroid disease. Sera from 159 patients (77 black and 82 white) with Type I DM were evaluated. A greater number of whites (41/82 or 50%) than blacks (12/72 or 16%) had thyroid autoantibodies (p less than 0.01). Fourteen patients (4 black and 10 white) exhibited hypothyroidism, and all had both TgA and TMA. Three patients (all black) had Graves' disease, one of whom had both TgA and TMA. Families of each racial group that had a diabetic child (proband) with thyroid autoantibodies (seropositive) or without thyroid autoantibodies (seronegative) were assessed for TgA and TMA as well as autoimmune thyroid disease. The prevalence of thyroid autoantibodies among siblings of seropositive probands was significantly greater than among the siblings of seronegative probands (p less than 0.01). The white sibling population showed a closer association of thyroid autoantibody prevalence with increasing age (p less than 0.05) than the blacks. Significantly more parents of probands than control parents exhibited thyroid autoantibodies (p less than 0.01). The general pattern of inheritance of either racial group showed that if one or both parents had thyroid autoantibodies, their progeny developed a significantly higher prevalence of thyroid autoantibodies than those of the seronegative parents. While there was no increase in overt thyroid disease among siblings of seropositive probands, a risk of developing autoimmune thyroid disease is probably imparted to these siblings by virtue of the thyroid autoantibodies.