Yersinia enterocolitica infection does not confer an increased risk of thyroid antibodies: evidence from a Danish twin study.

Understanding of the aetiological basis of thyroid autoimmunity may be gained by studying the early stages of the disease process. We aimed to (1) investigate the relationship between thyroid antibody status and Yersinia enterocolitica (YE) infection in euthyroid subjects and (2) explore the relative importance of genetic and environmental risk factors in the acquisition of YE infection. The association between thyroid antibody status and YE infection was explored using a case-control design. Furthermore, thyroid antibody-positive twins were compared with their thyroid antibody-negative co-twin. In 468 twins, IgA and IgG antibodies to virulence-associated outer-membrane proteins (YOPs) of YE were measured. Of these, 147 were thyroid antibody-positive (cases). A total of 147 age- and gender-matched twins were chosen as controls. The prevalence of YOP antibodies was lower among thyroid antibody-positive individuals than among controls. Yersinia infection was not associated with a positive thyroid antibody status: the odds ratio (with 95% CI) for YOP IgA-ab was 0.66 (0.42-1.05), P = 0.078 and for YOP IgG-ab it was 0.95 (0.60-1.50), P = 0.816. Within discordant twin pairs, the thyroid antibody-positive twin did not have an increased risk of Yersinia infection compared to the thyroid antibody-negative co-twin [odds ratio: YOP IgA-Ab: 0.94 (0.49-1.83), P = 0.866, and YOP IgG-Ab: 1.35 (0.72-2.53), P = 0.345]; 41% (95% CI 10-67% of the liability of being YOP antibody-positive was due to genetic effects. In conclusion, Yersinia infection does not confer an increased risk of thyroid antibodies. The genetic contribution in the acquisition of Yersinia infection is modest.

[Reduced radioiodine uptake at increased iodine intake and 131I-induced release of "cold" iodine stored in the thyroid]. / Verminderter Radioiod-Uptake durch erhöhte Iodzufuhr und 131I-induzierte Freisetzung von thyreoidal gespeichertem "kalten" Iod.

AIM: The extent of urinary iodine excretion (UIE) provides information about iodine supply and release. In the present study we investigated correlations between UIE and radioiodine uptake (RIU) as well as effects of radioiodine therapy on UIE in patients with autonomous goitre. PATIENTS, METHODS: In 197 consecutive patients with thyroid autonomy, UIE was measured twice during radioiodine test (RITe) and correlated with RIU. In 98 of these patients, thyroglobulin and thyroid volume (V) were determined prior to therapy. Individual changes in urinary iodine excretion (DeltaUIE) and TG (DeltaTG) could be investigated four weeks (4W) and six months (6M) after radioiodine therapy. Additionally, DeltaV was determined 6M after therapy. DeltaUIE, DeltaTG and DeltaV were correlated with target dose and target volume. RESULTS: Patients with higher iodine excretion exhibited significantly lower thyroidal radioiodine uptake values. Twofold increased UIE prior to therapy decreased radioiodine uptake by 25%. Compared with pretherapeutic values, UIE and TG were significantly increased four weeks after radioiodine therapy (p < 0.001). Median values of both parameters were found to be doubled. The product of target dose and target volume was not only correlated with a decrease of thyroid volume 6M after therapy, but also with an increase of UIE and TG in the early phase after therapy. CONCLUSIONS: It was confirmed that UIE during RITe is a measure for iodine intake and can be used to investigate the competition between stable iodine and radioiodine. The increase of UIE and TG four weeks after therapeutic administration of radioiodine can be explained by disintegrated thyroid follicles. The therapy-induced iodine release may be one important cause for the development of hyperthyroidism in some patients during the first weeks after radioiodine therapy. It may contribute to the known decrease of radioiodine uptake after preapplications of 131I in various thyroid diseases.

Expression of pendrin in benign and malignant human thyroid tissues.

The Pendred syndrome gene (PDS) encodes a transmembrane protein, pendrin, which is expressed in follicular thyroid cells and participates in the apical iodide transport. Pendrin expression has been studied in various thyroid neoplasms by means of immunohistochemistry (IHC), Western blot and RT-quantitative real-time PCR. The expression was related to the functional activity of the thyroid tissue. Follicular cells of normal, nodular goitre and Graves' disease tissues express pendrin at the apical pole of the thyrocytes. In follicular adenomas, pendrin was detected in cell membranes and cytoplasm simultaneously in 10 out of 15 cases. Pendrin protein was detected in 73.3 and 76.7% of the follicular (FTC) and papillary (PTC) thyroid carcinomas, respectively, where pendrin was solely localised inside the cytoplasm. An extensive intracellular immunostaining of pendrin was observed in six out of 11 (54.5%) of positive FTCs and 19 out of 23 (82%) of PTCs. Focal reactivity was detected in one follicular- and three papillary carcinomas, whereas pendrin protein was absent in three of 15 FTC and four of 30 PTC; mRNA of pendrin was detected in 92.4% of thyroid tumours. The relative mRNA expression of pendrin was lower in cancers than in normal thyroid tissues (P<0.001). The pendrin protein level was found to parallel its mRNA expression, which was not, however, related to the tumour size and tumour stage. In conclusion, pendrin is expressed in the majority of differentiated thyroid tumours with high individual variability but its targeting to the apical cell membrane is affected.

Increased prevalence of antibodies to enteropathogenic Yersinia enterocolitica virulence proteins in relatives of patients with autoimmune thyroid disease.

Infections have been implicated in the pathogenesis of a number of autoimmune diseases, and Yersinia enterocolitica (YE) might play a role in the development of autoimmune thyroid disease (AITD). Clinical evidence in support of this hypothesis has been inconclusive. We reasoned that looking earlier in the natural course of AITD might enhance chances of finding evidence for YE infection. Consequently, we determined seroreactivity against YE in subjects at risk of developing AITD, i.e. in 803 female relatives of AITD patients in self-proclaimed good health. As a comparison group we used 100 healthy women who participated in a program for reference values. IgG and IgA antibodies to virulence-associated outer membrane proteins (YOPs) of YE were measured by a specific assay. Serum thyroid peroxidase antibodies (TPO-Ab) as indicators of AITD were considered to be positive at levels of> 100 kU/l. The prevalence of YOP IgG-Ab was higher in AITD relatives than in controls (40.1% vs. 24%, P = 0.002), and the same was true for YOP IgA-Ab (22% vs. 13%, P < 0.05). Of the 803 AITD relatives, 44 had an increased or decreased plasma TSH, and 759 were euthyroid as evident from a normal TSH; the prevalence of YOP-Ab did not differ between these three subgroups. TPO-Ab were present in 10% of controls and in 27% of the AITD relatives (P < 0.001). The prevalence of TPO-Ab in the euthyroid AITD relatives was not different between YOP IgG-Ab positive and negative subjects (23.3% vs. 24.7%, NS), nor between YOP IgA-Ab positive and negative subjects (21.2% vs. 24.9%, NS). In conclusion, healthy female relatives of AITD patients have an increased prevalence of YOP antibodies, which, however, is not related to the higher prevalence of TPO antibodies in these subjects. The findings suggest a higher rate of persistent YE infection in AITD relatives. Susceptibility genes for AITD may also confer a risk for YE infection.

Enhanced intestinal expression of heat shock protein 70 in patients with inflammatory bowel diseases.

Heat shock protein (Hsp) 70 is stress-inducible and exhibits both protective and antigenic properties. This study investigated the mucosal expression of the constitutive (Hsp70c) and inducible form (Hsp70i) as well as antibodies against human Hsp70 in inflammatory bowel disease and controls. Biopsies were assessed by immunoblot and immunofluorescence, resection specimens by immunohistochemistry, and mucosal antibody content by isoelectric focusing. Compared to controls, expression of Hsp70 was enhanced in ulcerative colitis (P<0.05), less so in Crohn's disease and infectious colitis. Strong epithelial staining was found for Hsp70c and Hsp70i in both diseases. Mucosal and submucosal mononuclear cells showed enhanced Hsp70c expression in Crohn's disease and to a lesser degree in ulcerative colitis. Antibodies of isotypes A or M were detected in nearly all patients and controls. The different pattern of Hsp70 expression in Crohn's disease compared to ulcerative colitis points to a distinct protective and immunological function, whereas a role in autoimmunity seems unlikely.

Natural killer cell activity in patients with Graves' disease and Hashimoto's thyroiditis.

Natural killer (NK) cell activity of peripheral blood lymphocytes (PBL) against k562 human tumor cell targets was studied in patients with Graves' disease and Hashimoto's thyroiditis. NK activity was measured in a standard 4-hour 51chromium (Cr) release assay. Cytotoxicity was expressed as lytic units (LU)/10(6) PBL. Significantly decreased NK cell activity was demonstrated in both groups of patients, with mean (+/- SE) lytic units of 10.3 (+/- 9.1) and 13.3 (+/- 10.3) for patients with Graves' disease and Hashimoto's thyroiditis, respectively, compared with 36.0 (+/- 26.3) for age- and sex-matched normal subjects. When patients with Graves' disease were analyzed according to their thyroid status; NK activity was significantly depressed in (1) hyperthyroid patients before treatment; (2) hyperthyroid patients receiving antithyroid therapy; and (3) euthyroid patients receiving antithyroid therapy, compared with normal subjects. Graves' disease patients who were hypothyroid after radioactive iodine therapy or thyroidectomy had normal NK activity. No significant differences between hyperthyroid and euthyroid patients or between hypothyroid patients and normal subjects were demonstrated. NK activity in patients with Graves' disease did not correlate with serum levels of thyroxine, the presence or severity of ophthalmopathy, or titers of serum thyroid antibodies. In patients with Hashimoto's thyroiditis there was no correlation between NK activity and goiter size, titers of antithyroid antibodies, or thyroid status. These findings suggest that depression of NK activity in both disorders is secondary to abnormalities of thyroid hormone secretion, although an effect of the underlying autoimmune reactions has not been excluded.

Antigen receptor variable region repertoires expressed by T cells infiltrating thyroid, retroorbital, and pretibial tissue in Graves' disease.

To date, it has remained unclear whether T cells infiltrating thyroid, retroorbital, and pretibial tissue of patients with Graves' ophthalmopathy and pretibial dermopathy represent a primary immune response that is directed against certain antigenic determinants shared among these involved tissues. To characterize these T cells at the molecular level, we compared the T cell antigen receptor (TcR) variable (V) region gene usage in thyroid, retroorbital, pretibial tissue, and peripheral blood mononuclear cells of two patients with Graves' disease, ophthalmopathy, and pretibial dermopathy. Ribonucleic acid was extracted, reverse transcribed, and amplified using the PCR and 22 V alpha and 23 V beta gene-specific oligonucleotide primers. The resulting TcR V alpha and V beta transcripts were verified by Southern hybridization analysis using TcR C region-specific, digoxigenin-labeled oligonucleotide probes. In addition, complementarity determining regions 3 and junctional regions of TcR V beta genes were sequenced. Marked similarities of intrathyroidal, retroorbital, and pretibial TcR V alpha and V beta gene repertoires were noted with respect to the degree of TcR V gene restriction and the patterns of individual V genes expressed. Sequence analysis of junctional domains of V beta families revealed oligoclonality of intrahyroidal, retroorbital, and pretibial T cells. In addition, certain conserved junctional motifs were shared by T cells derived the thyroid gland and the extrathyroidal sites. Our results suggest that in the two patients with Graves' disease and extrathyroidal manifestations studied, similar antigenic determinants may have contributed to the recruitment and oligoclonal expansion of T cells both within the thyroid gland and at the involved extrathyroidal sites.

Transgenic mice models in autoimmunity--discussion.

Transgenic mice are an important in vivo model for studying the function of single genes. Specific induction and tissue specific expression of the inserted genes are the great advantages of this system. However, there a risks in constructing transgenic mice and the interpretation of the experimental data. To prevent artefacts and to optimize the transgenic model, the experimental systems have to fulfill the following presets: 1. Experiments has to be done with a stable transgenic line and not with the first heterogeneous transgeniced generation, because they differ in their site of gene insertion and in the cellular response. 2. The number of experiments have to be statistically sufficient. Often only a few number of experiments are done, due to the bad reproduction of transgenic mice. 3. Health of transgenic mice has to be proven, since the transgenic animals are under laboratory conditions and not under professional breeding conditions. 4. As a control there should be a transgenic mouse with a comparable pseudogene insertion beside the normal littermate control, to exclude artefacts by the simple gene insertion. If all of these presets are fulfilled, a transgenic mice model will be one of the best experimental systems.

Bacterial virulence antigens and the pathogenesis of autoimmune thyroid diseases (AITD).

There is now substantial evidence that autoimmune thyroid diseases (AITD) coincide with a subclinical persisting infection with Yersinia enterocolitica (YE) which manifests through humoral and cellular immune reactions against YE at the onset of AITD. The humoral and cellular crossreactivities of YE with thyroid autoantigens are exclusively directed against conformational epitopes of YE membrane associated antigens and of YE plasmid encoded virulence proteins (YOPs). Especially, the outer membrane domain of the TSH-Receptor (THSR) appears to have conformational homologies with YE antigens. Immunological- and molecular findings, however, do not allow definite conclusions about a potential role of YE-infection in AITD, although the evidence is suggestive. Recent investigations on the effect of YE-superantigen (Sag) on T-cells from patients with AITD as well as AITD like manifestations in YE immunized mice and rats may yield more conclusive information about the role of YE infection in the pathogenesis of AITD.

Molecular analysis of antigen receptor variable region repertoires in T lymphocytes infiltrating the intrathyroidal and extrathyroidal manifestations in patients with Graves' disease.

To determine whether T cells infiltrating thyroid, orbital and pretibial tissue of patients with Graves' ophthalmopathy (GO) and pretibial dermopathy (PTD) represent a primary immune response that is directed against certain antigenic determinants shared between these involved tissues, we characterized these T cells at the molecular level. T cell antigen receptor (TcR) variable (V) region gene usage in thyroid, orbital, pretibial tissue and peripheral blood mononuclear cells of patients with GD, GO and PTD was assessed using RT-PCR and 22 V alpha and 23 V beta gene-specific oligonucleotide primers, followed by Southern hybridization analysis using TcR C-region-specific, digoxigenin-labelled oligonucleotide probes. In some instances, CDR3- and junctional regions of TcR V beta genes were sequenced. Marked restriction and similarities of V alpha and V beta gene usage were detected in samples derived from patients with active GO and PTD of recent onset. Moreover, sequence analysis of junctional domains of V beta families revealed oligoclonality of some intrathyroidal, orbital and pretibial T cell populations as well as the presence of conserved junctional motifs shared by T cells derived the thyroid gland and the extrathyroidal sites. These data suggest that similar antigenic determinants may be responsible for the recruitment and oligoclonal expansion of T cells both within the thyroid gland and at the involved extrathyroidal sites in Graves' disease.

Glucocorticoids modulate the synthesis and expression of a 72 kDa heat shock protein in cultured Graves' retroocular fibroblasts.

Glucocorticoids modulate numerous proliferative, metabolic and immunological functions in human fibroblasts, some of which appear to be mediated via glucocorticoid receptors. We studied the influence of glucocorticosteroids on the synthesis and expression of a 72 kDa heat shock protein that is thought to play a role in thyroid autoimmunity. Experiments were performed using orbital fibroblasts derived from patients with Graves' ophthalmopathy and normal individuals. Cell monolayers were exposed to various concentrations of dexamethasone, the specific glucocorticoid agonist RU 28362, the glucocorticoid antagonist RU 38486, or combinations thereof, prior to heat stress or exposure to hydrogen peroxide. Heat shock protein 72 expression was assessed using sodium dodecylsulfate polyacrylamide-gel electrophoresis of cellular extracts, followed by autoradiography or immunoblotting with a mouse monoclonal antibody against the 72 kDa heat shock protein and quantitative scanning densitometry. In addition, cellular distribution of the immunoreactivity for the 72 kDa heat shock protein was studied using indirect immunofluorescence on parallel cultures. In other experiments, aimed at studying heat shock protein synthesis, cell cultures were pulse-labeled with [35S]-methionine prior to harvesting. Treatment with dexamethasone or RU 28362 markedly attenuated the heat stress-enhanced synthesis and expression of the 72 kDa heat shock protein and several other heat shock proteins both in normal and in Graves' retroocular fibroblasts (p < 0.001). In addition, either treatment reduced baseline expression of the 72 kDa heat shock protein in Graves' retroocular fibroblasts (p < 0.01). These effects were dose-dependent and appeared to be mediated via the glucocorticoid receptor, because combined exposure to dexamethasone or RU 28362 plus RU 38486 completely restored synthesis and expression of the 72 kDa heat shock protein. Baseline or stress-enhanced expression of the 72 kDa heat shock protein was not altered by treatment of monolayers with RU 38486 alone. As demonstrated by immunofluorescence, the characteristic intracellular shifting of the 72 kDa heat shock protein in response to cellular stress was partially inhibited by glucocorticoid agonists and restored by simultaneous exposure to glucocorticoid agonists and RU 38486. These results demonstrate that dexamethasone and the specific glucocorticoid agonist RU 28362 can modulate baseline- and stress-induced synthesis and expression of the 72 kDa heat shock protein, as well as its subcellular distribution, in cultured retroocular fibroblasts. Our studies suggest that these compounds exert these effects via the glucocorticoid receptor.

Discontinuous and continuous stimulation of FRTL-5 thyroid cells with bTSH cause different cAMP and nuclear proliferation antigen responses.

Pulsatile TSH secretion has been described in man. We investigated the effect of discontinuous TSH stimulation on FRTL-5 thyroid cells. FRTL-5 monolayers were pulsed with TSH in 4 h incubation periods with alternate 4 h TSH-free intervals, or continuously incubated with TSH. The cAMP production of cells was measured in the supernatant of monolayers. Expression of a nuclear proliferation antigen in FRTL-5 monolayers was determined by a monoclonal antibody (Ki-67) using the alkaline phosphatase-anti-alkaline-phosphatase staining method. The TSH concentration in the stimulation series ranged from 0.01 to 1.0 U/l medium. Rhythmic cAMP production was observed in both discontinuous and continuous stimulation. With discontinuous stimulation cAMP production peaked after about 24 and 48 h, while in the continuous presence of TSH peaks were observed at 32-40 and 48 h. At all TSH concentrations the effect of discontinuous stimulation was higher than in continuously stimulated cultures. The discontinuous incubation stimulated nuclear proliferation antigen expression of FRTL-5 more intensely and there was a positive correlation with TSH concentration. We conclude that the rhythmic pattern of cAMP production after TSH stimulation is independent of the TSH pulse. The amplitude of stimulation and proliferation of FRTL-5, however, is increased by discontinuous TSH application.

Methimazole and propylthiouracil inhibit the oxygen free radical-induced expression of a 72 kilodalton heat shock protein in Graves' retroocular fibroblasts.

Reactive oxygen species are generated in tissues by activated mononuclear cells and macrophages. These cells infiltrate the thyroid gland in Graves' disease (GD), as well as the retroocular and pretibial space in Graves' ophthalmopathy and pretibial myxedema (PTM). Because a 72 kilodalton heat shock protein (HSP 72) is associated with autoimmune thyroid disease and is selectively expressed in fibroblasts derived from involved sites of patients with Graves' ophthalmopathy and pretibial myxedema, we studied the influence of oxygen free radicals (OFR), oxygen radical scavangers (ORS), and antithyroid drugs on HSP 72 expression in Graves' retroocular fibroblasts. Fibroblast monolayers were exposed to hydrogen peroxide (H2O2) or heat stress with simultaneous treatment, or pretreatment, with the ORS diaminobenzidine, nicotinamide, glutathione, propylthiouracil (PTU), or methimazole (MT). HSP 72 expression was determined by sodium dodecylsulfate polyacrylamide-gel electrophoresis, followed by immunoblotting with an anti-HSP 72 monoclonal antibody and densitometric quantitation of HSP 72 immunoreactivity. Baseline HSP 72 expression in Graves' retroocular fibroblasts was strongly enhanced by H2O2 and heat stress. Both pretreatment and simultaneous treatment with the ORS and any of the antithyroid agents significantly reduced the abundance of H2O2-induced (P less than 0.01), and to a lesser degree heat-induced (P less than 0.05), HSP 72 expression. These results demonstrate that, in Graves' retroocular fibroblasts, H2O2-induced HSP 72 expression is diminished both by classical ORS and by the antithyroid agents PTU and MT. In addition, heat-induced HSP 72 expression appears to be mediated in part by OFR. Stimulation of immunogenic 70 kilodalton HSPs by OFR, derived from immunocompetent cells infiltrating the affected tissues in GD, may play a role in the autoimmune process. The beneficial effect of MT and PTU on the clinical course and immune status of patients with GD may be related to their OFR-scavanging properties.

Immunohistochemical detection and localization of a 72-kilodalton heat shock protein in autoimmune thyroid disease.

Recently described immunological functions for heat shock proteins (HSPs) and our previous demonstration of site-selective HSP-72 expression in cultured fibroblasts derived from extrathyroidal manifestations of Graves' disease (GD) prompted us to determine whether expression of the inducible 72-kilodalton HSP can be detected in human thyroid tissues. Immunohistochemistry was performed on formalin-fixed paraffin-embedded thyroid tissue specimens from patients with GD, Hashimoto's thyroiditis (HD), and multinodular goiter (MNG) as well as on normal thyroid tissue. A mouse monoclonal anti-HSP-72 antibody and an ultrasensitive avidin-biotin-peroxidase complex detection system were used for these studies. Striking differences in HSP-72 immunoreactivity were detected both between tissues from GD and HD compared with MNG and normal thyroid and between GD thyroid glands treated preoperatively with antithyroid medication and untreated GD glands. Strong HSP-72 reactivity in GD and HD tissues was detected in thyroid follicles as well lymphocytic infiltrates. No HSP-72 reactivity was detected in MNG or normal thyroid tissue. HSP-72 immunoreactivity was markedly reduced in GD glands that received preoperative antithyroid drug treatment. In conclusion, high levels of HSP-72 expression in autoimmune thyroid disease may reflect a state of chronic cellular stress, but could also represent an immunomodulatory factor of relevance in the autoimmune process in GD.

Cell surface localization of a 72 kilodalton heat shock protein in retroocular fibroblasts from patients with Graves' ophthalmopathy.

Heat shock proteins (HSPs) have been implicated in autoimmune disease, although they are generally considered to be intracellular in location. We demonstrate that, under certain circumstances, the inducible intracellular 72 kilodalton HSP can also be detected on the surface of cells. We used cultured retroocular fibroblasts derived from patients with severe Graves' ophthalmopathy (GO) and normal individuals in our studies. Sodium dodecylsulfate polyacrylamide-gel electrophoresis of immunoprecipitated cell lysates, derived from surface-radioiodinated GO cell monolayers, resulted in a single band of appropriate molecular weight on the autoradiogram. Further, a bright cell surface staining pattern was observed when indirect immunofluorescence was performed using the same anti-HSP 72 monoclonal antibody on parallel cell cultures. No cell-surface HSP 72 reactivity was detected in normal retroocular fibroblast monolayers by either method. These results are the first demonstration, by immunoprecipitation of surface-labeled proteins, of HSP expression on the surface of cells. This localization of HSP 72 on the surface of affected cells obtained from patients with an autoimmune disease may have implications concerning the role of this molecule in autoimmunity in general, and particularly in the immune process of GO.

Enhanced induction of a 72 kDa heat shock protein in cultured retroocular fibroblasts.

Fibroblasts in the retroocular connective tissue appear to play a central role in the pathogenesis of Graves' ophthalmopathy (GO). We hypothesize that specific attributes, differentiating normal from GO retroocular fibroblasts, may render the latter more vulnerable to the ongoing immunopathological process in GO. We investigated whether GO fibroblasts differ from normal fibroblasts with respect to their sensitivity and capacity to express the inducible 72 kDa heat shock protein (HSP) in response to stressful environmental stimuli. Cultured retroocular fibroblasts derived from patients with GO and normal individuals were exposed to various changes in the culture medium that may simulate conditions in the affected retroocular space in vivo. HSP 72 reactivity was determined using sodium dodecyl sulfate polyacrylamide-gel electrophoresis of cellular extracts, followed by immunoblotting with a mouse monoclonal anti-HSP 72 antibody and quantitative scanning densitometry. In addition, indirect immunofluorescence was performed on parallel fibroblast monolayers. Following exposure to heat and acidic pH, deprivation from nutrients, and high monolayer density, GO fibroblasts expressed HSP 72 with significantly greater sensitivity and in significantly higher abundance than did normal fibroblasts. These results demonstrate that changes in the physiological environment induce HSP 72 expression in cultured fibroblasts. The enhanced sensitivity and capacity of GO retroocular fibroblasts to express the inducible HSP 72 in response to stressful stimuli may play a role in the autoimmune process affecting the retroocular space in GO.

Increased cellular growth of FRTL-5 thyroid cells mediated by discontinuous stimulation with bTSH.

Previous reports showed pulsatile secretion of TSH in man. Therefore, we investigated the effect of discontinuous versus continuous TSH stimulation on the cellular level of FRTL-5 thyroid cells, namely the expression of a nuclear proliferation antigen (NPAg). The expression of this antigen correlates linearly with the 3H-thymidine incorporation and is a marker for cellular growth. The FRTL-5 cells were stimulated for 4 hours with bTSH (0.01-1.0 U/l). Compared to continuously stimulated cell cultures the discontinuous stimulation of FRTL-5 cells with bTSH showed a significant higher rate of NPAg expression, i.e. cellular growth.

Detection, cellular localization, and modulation of heat shock proteins in cultured fibroblasts from patients with extrathyroidal manifestations of Graves' disease.

We hypothesize that fibroblasts obtained from the retroocular space and the pretibial skin, sites affected by the peripheral manifestations of Graves' disease, share unique characteristics that may in part explain the site specificity of Graves' ophthalmopathy (GO) and pretibial myxedema (PTM). Heat shock proteins (HSPs), synthesized by cells undergoing stress, function to maintain cellular homeostasis and are probably involved in the intracellular processing and cell surface presentation of antigens. We investigated possible differences in the expression of 70-kDa HSPs between cultured fibroblasts obtained from patients with severe GO and normal individuals. In addition, we compared HSP expression in fibroblasts derived from tissues involved in the extrathyroidal manifestation of Graves' disease (GO and PTM) with that in fibroblasts from uninvolved tissues. HSPs were detected by both immunoblotting and indirect immunofluorescence, using monoclonal antibodies that are directed against HSP72, HSP72/73 (termed HSP70), and HSP90. HSP expression at baseline and after treatment with various cytokines and heat stress was examined. At baseline, HSP72 reactivity was exclusively detected in retroocular and pretibial fibroblasts from patients with severe GO and PTM, but was not observed in abdominal fibroblasts from these patients and was not detectable in fibroblasts from any anatomical site of normal individuals. The abundance of HSP70 expression at baseline and after treatment with certain cytokines was significantly greater in retroocular and pretibial fibroblasts from patients with GO than in normal individuals. In addition, characteristic changes in the cellular localization of HSPs before and after exposure to heat stress and cytokines were observed; cell surface expression of HSP70 was detected at baseline in fibroblasts from patients, but not in normal fibroblasts. These data provide the first evidence that HSPs are differentially expressed by fibroblasts derived from tissues affected by the extrathyroidal manifestations of GD. These proteins may have a role in localized immune processes, leading to the development of GO and PTM.