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Recently, in vitro production of interleukin-2 receptor induced by mitogens have been shown to be impaired in autoimmune disorders including organo-specific autoimmune diseases. The aim of this study was to investigate serum levels of soluble interleukin-2 receptor in 20 untreated patients with Graves' disease and to follow up their changes in relation to clinical picture and TSH-receptor-, anti-thyroglobulin-, anti-microsomal as well as anti-eye muscle antibodies. Soluble interleukin-2 receptor level was significantly increased in newly-diagnosed Graves' patients compared to controls (667 +/- 270 vs. 205 +/- 45 U/ml) (P less than 0.001). Among the patients sera those with active infiltrative ophthalmopathy had higher soluble interleukin-2 receptor levels than those without eye symptoms (810 +/- 313 vs. 525 +/- 180 U/ml). Soluble interleukin-2 receptor level was normalized in Methimazole-treatment-induced remission in the majority of patients except those with ophthalopathy. In five patients the soluble interleukin-2 receptor levels were studied after interruption of thyrostatic therapy; an increase was observed in three patients; thereafter hyperthyrosis relapsed in two cases. Furthermore, a correlation was found between soluble interleukin-2 receptor levels and TSH-receptor antibodies, however, the association with other immune parameters examined was not significant. In conclusion, an enhanced level of soluble interleukin-2 receptor was detected in patients with untreated Graves' disease. This finding might play a significant role in regulation of impaired cell-mediated immune mechanism and has a prognostic value for relapse of autoreactive processes.

Radionuclide thyroid scanning of patients with Hashimoto’s thyroiditis (HT) may mimic other thyroid disorders including cold nodules, multinodular goiter and rarely hot nodules. The association of single hot nodules in such patients in the face of primary hypothyroidism has not been previously reported. We describe 6 female patients with HT who presented either with symptoms of overt thyroid failure or a sensation of lump in the neck (and later found to have mild thyroid failure) who had single firm thyroid nodules. These nodules were hot by both 99mTc pertechnetate and radioiodine thyroid scans. In three of 4 patients followed up for longer than 6 months on adequate thyroid replacement therapy the nodules regressed by up to 60%. Given “Best practice” recommendations patients with thyroid failure and single thyroid nodules would not be submitted to radionuclide scanning and this presentation of HT would have gone undetected.

We describe a woman with complete hypogonadotropic hypogonadism and a new compound heterozygous mutation of the GnRH receptor (GnRHR) gene. A null mutation L314X leading to a partial deletion of the seventh transmembrane domain of the GnRHR is associated with a Q106R mutation previously described. L314X mutant receptor shows neither measurable binding nor inositol phosphate production when transfected in CHO-K1 cells compared to the wild-type receptor. The disease is transmitted as an autosomal recessive trait, as shown by pedigree analysis. Heterozygous patients with GnRHR mutations had normal pubertal development and fertility. The present study shows an absence of LH and FSH response to pulsatile GnRH administration (20 microg/pulse, sc, every 90 min). However, GnRH triggered free alpha-subunit (FAS) pulses of small amplitude, demonstrating partial resistance to pharmacological doses of GnRH. FSH, LH, and FAS concentrations were evaluated under chronic estrogen treatment and repeat administration of GnRH. Not only were plasma FSH, LH, and FAS concentrations decreased, but FAS responsiveness was reduced. This new case emphasizes the implication of the GnRH receptor mutations in the etiology of idiopathic hypogonadotropic hypogonadism. We also have evidence for a direct negative estrogen effect on gonadotropin secretion at the pituitary level, dependent on the GnRHR signaling pathway.

In the pathomechanism of the thyroid associated ophthalmopathy (TAO) the inflammatory cytokines produced by infiltrating lymphocytes of the retroorbital tissues are involved. The activated lymphocytes have been shown to secrete a number of cytokines including tumour necrosis factor-alpha, interleukin-1 and interferon-gamma. The widely used immunosuppressive therapies have potential serious side effects. The pentoxifylline (Ptx) is known to have effect on production of cytokines. The aim of this study was to investigate the effect of Ptx on expression of HLA-DR molecules and production of glycosaminoglycan of human retroorbital tissue cultures and potential efficacy in patients with TAO. It was found that pentoxifylline (Ptx) was able to inhibit significantly the HLA-DR expression and glycosaminoglycan synthesis induced by inflammatory cytokines including TNF-alpha, IFN-gamma and IL-1. Ten patients with untreated moderate severe ophthalmopathy (8 female and 2 male) were excluded from steroid treatment due diabetes mellitus and psychiatric disease. Classification of eye changes was made by NOSPECS categories and total eye score. All patients were euthyroid during the study and was no remarkable difference in thyroid function and eye symptoms. Before and during Ptx therapy the laboratory parameters were also determined including glycosaminoglycan. TNF-alpha, anti-TSH-receptor, anti-eye muscle, anti-thyroglobulin and anti-thyroid peroxidase antibodies in the patients'sera. It was found a remarkable improvement in the eye symptoms in eight of ten patients. The levels of glycosaminoglycan (uronic acid) and TNF-alpha gradually decreased in eight patients who considered to be responders. The levels of uronic acid in plasma of the responders were found to be significantly lower after Ptx treatment. Before Ptx therapy the TNF-alpha in the sera was not different remarkably in non-responders and responders. After 4 weeks Ptx treatment the TNF-alpha decreased significantly in responders compared to non-responders (20.9 +/- 4.8 pg/ml v. s. 28.3 +/- 6.1 pg/ml) (p0.01). The titre of anti-eye muscle antibodies were found to be lower at the end of observation, however, the anti-thyroid antibodies were not changed remarkably. It was concluded that Ptx in the majority of patients (8/10) has a beneficial effect on inflammatory symptoms of TAO and laboratory parameters and suggested to use as an additive therapy, however, further comparative studies are required for final evaluation of Ptx in the treatment of TAO.

75 patients with Graves' disease (54 with ophthalmopathy) were investigated using the tests of leucocyte adherence inhibition and immune adsorption with 125I-labelled Staphylococcus Protein A, against human eye muscle "crude" membrane antigen. The results of positive leucocyte adherence inhibition (10 out of 26 vs. 1 out of 28, P less than 0.05) and anti-human eye muscle membrane antibody index (mean +/- S.D.) (1.89 +/- 1.20 vs. 0.84 +/- 0.38, P less than 0.001) showed a correlation with the patients with clinically active eye disease and the HLA-B8 antigen in Graves' ophthalmopathy (P less than 0.01). Positive leucocyte adherence inhibition was observed in 9 out of 21 cases of Graves' disease without ophthalmopathy, but its prognostic relevance has to be confirmed in the development of ophthalmopathy.

In view of the reported variation in the association between HLA antigens and Juvenile Diabetes Mellitus (J.D.M.) among different Caucasian populations, we have undertaken a study of these antigens among 44 Caucasian Newfoundlanders and 135 matched controls. We have also studied the allotypic markers for Immunoglobulin G (Gm) and variants of C3 among 36 of these patients. We found that both HLA--B8 and B15 were increased among the patient group, resulting in a relative risk of 3.9 and 4.4 respectively. While these values are the highest to be described for J.D.M. among Caucasians, and fell outside the 95% confidence intervals for the combined relative risk calculated from published series, it is still possible that they can be accounted for by sampling. The combination of the two antigens increased the relative risk for J.D.M. in an additive fashion. Additionally, we also found that the combination of HLA B8 and B18, but not B15 and B18, also appear to act in an additive manner. The incidence of Gm allotypes and variants of C3 were not different in the J.D.M. group from those observed among controls.

We assessed the efficacy of a new thyroxine radioimmunoassay kit (Abott) in which polyethylene glycol is used to separate bound from free hormone. Mean serum thyroxine was 88 +/- 15 (+/-SD) microgram/liter for 96 normal persons. Results for hypothyroid and hyperthyroid persons were clearly separated from those for normal individuals. Women taking oral contraceptive preparations showed variable increases in their serum thyroxine values. The coefficient of variation ranged from 1 to 3% within assay and from 5.4 to 11% among different assays. Excellent parallelism was demonstrated between thyroxine values estimated by this method and those obtained either by competitive protein binding or by a separate radioimmunoassay for the hormone.

SUMMARY We have examined the combined usefulness of TSH-receptor antibody detection and HLA status on the therapeutic response to standard doses of radioactive iodine (RAI) in forty-three hyperthryoid Graves’patients. Twenty-three patients had detectable TSH-receptor antibodies as measured by 125I-TSH binding-inhibition (TBI) prior to administration of 7 mCi RAI. Eighteen (78%) of these patients were rendered euthyroid within 3 months. In contrast, twenty patients were TBI negative prior to RAI and sixteen (80%) of these individuals remained hyperthyroid at 3 months and required two, or more, doses of RAI to control their thyroid function. DR3 status alone was not strongly associated with resistance to RAI. However, of sixteen patients without detectable TBI activity and who required two or more doses of RAI, ten patients were DR3 positive (62%) compared with 25% in a control population. Only one patient who was both TBI and DR3 negative required more than one dose of RAI. These data indicate that TBI may be an accurate predictor of the therapeutic response to RAI in patients with hyperthyroid Graves’disease. Investigation of HLA status alone was of limited value, although it appeared that the combination of TBI and DR3 may be of additional help in developing an overall strategy for the treatment of hyperthyroid Graves’disease.

Three mAb to variable region determinants of the alpha/beta-chain TCR were used to detect discrete populations of peripheral blood T cells. T cells sharing a TCR determinant defined by such an antibody presumably use the same or similar TCR V or J genes for their alpha- or beta-chains. Thus analysis with these mAb provides a tool to investigate TCR gene usage and expression. Since autoantigen specific T cells may play an important role in initiating autoimmune diseases, TCR were analyzed in different autoimmune diseases and control groups including rheumatoid arthritis, Graves disease, idiopathic thrombocytopenic purpura, psoriasis, SLE, insulin-dependent diabetes mellitus, and in nonautoimmune control diseases and normals. Purified T cells were stained by indirect immunofluorescence with three mAb to TCR variable regions: mAb S511 stains 1.8 +/- 0.9% (mean +/- 2 SD), mAb C37 stains 3.4 +/- 1.5% and mAb OT145 stains from 0 to 6% of T cells from normal donors. Several individuals were identified with expanded subsets of positive T cells. One patient with adult ITP followed during a 12-mo period consistently had elevated percentages of T cells staining with the mAb OT145 (15.9 to 24.5%). These cells were found to be exclusively CD8+. By Southern blotting DNA prepared from these OT145+, CD8+ cells, but not DNA from the patient's OT145- T cells, revealed a clonal rearrangement using a beta-chain C region probe. Thus this patient had a monoclonal expansion of CD8+, OT145+ cells. Hyperexpression of a TCR variable region, as defined by the available mAb, could not be associated with any of the diseases studied. Examination of T cells at the site of autoimmunity, such as T cells from rheumatoid arthritis synovial fluid, revealed normal percentages of cells staining with these mAb. Immunoperoxidase staining of psoriatic lesional skin showed no striking enrichment of T cells bearing one or the other TCR type.

Graves' disease is associated with HLA-DR3 in Caucasoids. We have now demonstrated, on the basis of disease-associated MHC haplotypes (A1, Cw3, B8, DR3 and fragments thereof) from 38 families in which more than one member had Graves' disease compared with MHC haplotypes from 56 healthy families, that the risk was highest with the DR locus (relative risk for A1, B8, DR3 = 2.3, for B8, DR3 = 5.3, and for DR3 = 6.8). We further used the sib-pair method to explore linkage of Graves' disease liability to the MHC in 67 affected sib-pairs. The data were consistent with an MHC-linked recessive gene with a frequency of 0.2 to 0.3 and a penetrance of 7.2%; the data, however, accommodated penetrance of up to 16.3%. A recessive model was also consistent with the HLA-B8 genotype distribution in 286 unrelated patients. As the effect of the marker alleles on the course of the disease had been debated several times, we applied a cluster analysis method using 49 clinical and laboratory characteristics, including the HLA-A and HLA-B antigens of 196 patients. Three groups were identified, corresponding to patients with mild disease, Hashitoxicosis and severe (relapsing) disease. The prevalence of HLA-B8 was 8.9%, 21% and 87%, respectively (compared to 18.8% in 380 controls). This suggests the existence of an underlying continuum of genetic liability, apparently related to that for Graves' disease severity, associated with the MHC and mediated through immunoregulatory disturbances.

Ninety-eight members of a large Newfoundland family, seven of whose members over three generations suffered from Graves' disease, were studied with respect to the mode of transmission of the disease and its association with HLA. Compared to Newfoundland communities of similar size and geographical location, very little consanguinity was documented in this family. The susceptibility to Graves' disease appeared to be inherited as a dominant with a variable degree of expressivity; the degree of expressivity being determined by the female sex. In part of the pedigree, the susceptibility to Graves' disease entered the family with a wife. Three of her offspring who subsequently developed Graves' disease shared with her the haplotype A9, Bw16. Of the three remaining affected family members, two shared the haplotype A1, B8, whereas the third carried the haplotypes Aw32,b8; a9,bw16. Graves' disease could be associated with either of these two haplotypes in the last individual. This study shows that the susceptibility to Graves' disease is inherited associated with HLA and that whereas the disease susceptibility gene for Graves' disease is in linkage disequilibrium with HLA-B8 in Caucasians, it can be randomly associated with other HLA-B antigens.

We have extended our study of an incomplete variant of multiple endocrine neoplasia Type I (MEN IBurin). In this syndrome, primary hyperparathyroidism and prolactin-secreting adenoma are common, with hormone-secreting pancreatic tumors being rarely seen. The recent localization of the prolactin structural gene to chromosome 6 made further investigation of linkage to HLA of particular interest. Results in 2 multigeneration families exclude close linkage to HLA. We cannot at this time draw any inference regarding linkage of MEN IBurin to the prolactin structural gene.

Taking advantage of high prevalence of prolactinomas in Newfoundland families with variant multiple endocrine neoplasia Type I (MEN I), we studied the control of prolactin secretion by dopamine in family members with prolactinomas (n = 6), family members who have hyperparathyroidism but no prolactinomas (n = 6), as well as healthy members (n = 8) in unaffected sibships. Four unrelated patients with empty sella syndrome were also studied as sella size controls for the prolactinoma group. Dopamine (2 micrograms/kg/min) was less effective in reducing serum prolactin in the prolactinoma group than in the other three groups studied; serum prolactin also showed little rebound in prolactin comparable to controls. Family members with hyperparathyroidism but no prolactinoma (and to a lesser degree the empty sella syndrome group) showed much less post-infusion rebound than controls. We conclude that family members genetically predisposed to prolactinoma (and patients with empty sella syndrome) may exhibit subtle abnormalities in dopaminergic control of prolactin secretion.

In order to further elucidate the genetics of Graves' disease, we studied two families with several affected members, as well as tested the degree to which HLA haplotypes were shared in affected sibpairs. Further, we sought to identify the disease related haplotypes by determining the haplotypes shared among affected parent-child combinations. In one family, two affected sibs differed at four possible parental HLA haplotypes; no evidence of recombination was observed which could account for the result. In the other family, five siblings were affected. Four out of the five affected sibs shared the maternal haplotype HLA-A11, Bw51, Cw5, Cw-, DRw5, Bfs, GLO1, whereas three shared the paternal haplotype HLA-A1, B8, Cw-, DRw3, BfS and GLO1. Looking at haplotype sharing, two pairs of sibs were found to be HLA identical, whereas the fifth sib shared one haplotype with one of these pairs but not with the other. Out of 14 (eight of our own and six from the literature) affected sibpairs examined, nine were found to be HLA identical and four shared one haplotype, suggesting that the contribution of both paternal haplotypes may be necessary for the susceptibility to the disease. Fourteen parent-child combinations were studied; in only three out of 13 in which the shared haplotype could be ascertained was the haplotype B8 positive; this distribution is similar to controls. However, of the remaining 10 combinations which did not share a B8 positive haplotype, five were B8 positive at one or the other of the non-shared haplotypes.

T lymphocyte subsets were prospectively examined in the peripheral blood and thyroid aspirates of 10 patients with hyperthyroid Graves' disease before and after treatment with methimazole and attainment of euthyroidism. T lymphocyte subsets were identified with monoclonal antibodies and pattern of alpha-naphthyl-acetate esterase (ANAE) staining pattern in the case of peripheral blood and ANAE staining pattern with thyroid aspirate smears. Before treatment, OKT8+ lymphocytes were significantly decreased (18.4% +/- 4.8) (S. D.) in the patients compared to control (28.8 +/- 6.7%, p less than 0.05), the OKT4/OKT8 ratio was increased (2.92 vs 2.11). Percent OKT8+ lymphocytes were not different from the controls when the ten patients had been rendered euthyroid. ANAE mononuclear cells with a diffuse pattern (presumed suppressor cells) were 4.2% +/- 1.8 before treatment and 8.3 +/- 2.4 (p less than 0.05) after treatment and 11.5% +/- 2.2 in controls. ANAE mononuclear cells with diffuse pattern represented 4.2% +/- 1.8 of the mononuclear cells infiltrating the thyroid gland of untreated patients and rose to 8.3% +/- 2.4 after the patients had become euthyroid. ANAE negative cells (B cells and some T cells) were increased in the thyroid of untreated patients. It is concluded that mononuclear cells with presumed suppressor T cell phenotype are decreased in the blood and thyroid glands of patients with active Graves' disease and that this defect is corrected when euthyroidism has been established.

HLA/Bf haplotypes were examined in a large three-generation Newfoundland family with a high incidence of Graves' disease. In that family Graves' disease was inherited in association with the haplotype HLA Aw24, Bw39 in some instances and with HLA B8-containing haplotypes in other instances. As all seven members of the family who suffered from Graves' disease were homozygous for the Bf S allele, the study for Bf was uninformative. However, the examination of other HLA/Bf haplotypes disclosed some interesting associations. One-hundred-and-five out of 168 HLA-A, -B, -Bf haplotypes were Bf S. Although numerically deviant, no unusual HLA B/Bf associations were observed. Bf F entered the family only once. A new finding is the association between HLA B27 and Bf S1; the haplotype entered the family once and was passed on to eight family members over three generations. Bf S1 was previously reported in association with HLA B12 or W21. None of these family members had ankylosing spondylitis. The Bf allele F1 entered the family three times, always in association with HLA B18.

We typed patient groups with type I diabetes (n = 78), Graves' disease (n = 81), goitrous autoimmune (n = 52), "silent" (n = 18) and postpartum thyroiditis (n = 15) for human leucocyte antigens (HLA) A, B, C, DR and DQ. The results were compared to those obtained from 256 healthy controls typed for HLA-A, -B, -C and 140 typed for -DR. All these 140 controls were genotyped. Previously described associations of DR3 (OR (odds ratio) = 2.68, p less than 0.005) and DR4 (OR = 3.26, p less than 0.0001) in type I diabetes is confirmed. In this series, however, HLA-DR3/DR4 heterozygotes were apparently at no greater risk for type I diabetes than DR3 or DR4 homozygotes. The relative risk conferred by DR3/DR4 heterozygotes (6.48) was less than that for DR3 homozygosity (2.8), suggesting a recessive major histocompatibility complex-related susceptibility to type I diabetes. Graves' disease was associated with DR3 (OR = 3.02, p less than 0.0005); the increased frequency of DR3 homozygotes in this series is consistent with recessive HLA-linked susceptibility to Graves' disease proposed on the basis of family data. Hashimoto's thyroiditis, on the other hand, was associated with HLA-DR4 (OR = 3.08, p less than 0.0001), the latter finding confirming our earlier report on 21 patients. The increase of HLA-DR4 in both post-partum and silent thyroiditis suggests that these conditions are immunogenetically related, and may well represent variants of chronic autoimmune thyroiditis.

In a previous study we were able to separate, using cluster analysis, 196 patients with Graves' disease evaluated for a large number of clinical and laboratory characteristics, including HLA-A and HLA-B typing into one subset with recurring disease and a high prevalence of ophthalmopathy and another subset with mild disease and little ophthalmopathy. Prevalence of HLA-B8 was much higher in the first as compared to the second group. The present study was undertaken in 117 new patients with Graves' disease, typed for HLA-A, HLA-B, HLA-C and DR antigens and IgG heavy chain markers, to determine whether these characteristics could be used to segregate patients into clinically relevant subsets. There was a greater proportion of Gm fb homozygotes among patients than among controls (chi2 = 4.71, p less than 0.05) as well as individuals with HLA-B8 and DR3, previously documented for this disease. Two patient clusters were identified. In one (C1), there is a high incidence of exophthalmos, recurrence of hyperthyroidism after drug treatment, high titres of anti-thyroglobulin antibody, and an association with other autoimmune (including thyroid) diseases, a tendency for the disease to be familial and the presence of larger goitres. The incidence of HLA-B8 was greater in C1, while HLA-B12 was more frequent in the mild cluster, C2. HLA-DR3 was found to be associated with patients in the severe cluster and HLA-DR2 with patients in the mild cluster.(ABSTRACT TRUNCATED AT 250 WORDS)