Local immune microenvironment of skin may play an important role in the development of pretibial myxedema.

Pretibial myxedema (PTM), characterized by the accumulation of glycosaminoglycans in dermis is an autoimmune skin disorder, which is almost always associated with Graves' disease (GD). Although fibroblast stimulated by thyroid-stimulating hormone receptor (TSHR) antibody, cytokines and growth factors have been postulated as target of the autoimmune process in the dermopathy, the pathogenesis of PTM remains unclear. We hypothesize that the local immune microenvironment of the skin including the antigens and antibodies, T cells, B cells, plasma cells and fibroblasts may play an important role in the development of PTM. Results obtained on PTM patients indicate increased thyroid-stimulating hormone receptor antibodies (TRAb) in the blood positively correlate with the dermal thickness of the lesions. Further analysis shows that there were more CD3+ T cells and CD20+ B cells in the skin lesions. These T and B cells are in close contact, indicating that inducible skin-associated lymphoid tissue (iSALT) may be formed in the area. In addition, we found that the infiltrating plasma cells can secrete TRAb, proving that B cells in the skin other than the thyroid are an additional source of TSHR antibodies. Meanwhile, the T and B cells in the skin or skin homogenate of patients can promote the proliferation of pretibial fibroblasts. In conclusion, our results provide evidence that the local immune microenvironment of the skin may play an important role in the development of PTM.

Treatment of pretibial myxedema with dexamethazone injected subcutaneously by mesotherapy needles.

Pretibial myxedema (PTM) is a rare extrathyroidal manifestation of Graves' disease that requires treatment when the clinical picture is markedly evident. In addition to topical treatment with steroid ointments, there have been previous reports of subcutaneous injections of steroids. This procedure may cause nodular degeneration of the skin due to fat atrophy when standard needles are used. In the present study, we have tried a novel modality of treatment of PTM by injecting a solution of dexamethasone in the subcutaneous tissue using needles employed for mesotherapy. These needles are ≤4 mm long and deliver the medication within the dermis or the first layer of the subcutaneous fat. We have treated five patients, four with diffuse and one with elephanthiasic PTM. We utilized multiple injections of a solution of dexamethasone, lidocaine, and saline in the PTM plaque and in the pretibial area, both in the PTM plaque and in the area surrounding the lesions, once a week for three consecutive weeks. Two patients with a more severe form of PTM underwent another two cycles four to six weeks after initial treatment. Patients were studied before and after treatment by clinical assessment and ultrasound of the pretibial skin. The treatment was well-tolerated, with only moderate pain upon injection of the solution. One month after treatment, all patients showed improvement of PTM at clinical assessment and a reduction of the thickness of the lesions at ultrasound of ∼15%, involving mostly the dermis. Moreover, all patients reported amelioration of the leg appearance. The present study, although preliminary, shows that intralesion steroid injection with mesotherapy needles in PTM is effective and well tolerated, and does not cause undesired long-term modifications of the skin. More studies are warranted to standardize such treatment in larger groups of patients.

Thyrotrophin receptor antibody characteristics in a woman with long-standing Hashimoto's who developed Graves' disease and pretibial myxoedema.

CONTEXT: Sequential conversion of Hashimoto's thyroiditis (HT) to Graves' disease (GD) is uncommon. Distinct immune paradigms, paucity of functioning tissue in long-standing HT, and infrequent conversion of blocking (TBAb) to stimulating (TSAb) thyrotrophin receptor antibody (TRAb) may account for this. Molecular and crystal structure analysis helps delineate TSH receptor (TSHR)/TRAb interactions in detail. Such 'fingerprinting' helps determine the behaviour and characteristics of TRAb in longitudinal studies. PATIENT: An 80-year-old woman taking thyroxine for long-standing HT became hyperthyroid. This persisted despite thyroxine withdrawal - free T3 was 7·3 pmol/l (2·6-5·7) and TSH < 0·01 mU/l (0·2-4·5) and TRAb highly positive. She had a goitre (ultrasound - HT), pretibial myxoedema, with mild inactive Graves' orbitopathy. She had RAI treatment and is on thyroxine replacement. MEASUREMENTS AND RESULTS: Blood samples at presentation (A) and 1 year (B) showed high TSAb and TPOAb activity but no TBAb. Experiments involving TSHR mutations confirmed that (i) TRAb had stable characteristics over 1 year; (ii) TSHR mutation R255D caused complete inhibition and (iii) R109A caused marked reduction of cAMP production by M22 (TSHR-stimulating human monoclonal antibody) and A and B; (iv) mutations R80A, E107A and K129A while affecting M22 had little effect on A and B. CONCLUSIONS: The reasons for an immunological paradigm shift in this elderly woman remain speculative. We believe that de-novo TSAb synthesis occurred converting her long-standing HT to GD although the mechanisms responsible remain unexplained. TRAb analysis confirmed stable autoantibody characteristics over 1 year and variable effects of TSHR mutations on TRAb and M22 function.

Pretibial myxedema: pathophysiology and treatment options.

Pretibial myxedema or localized myxedema or thyroid dermopathy is an autoimmune manifestation of Graves' disease. It also occasionally occurs in Hashimoto's thyroiditis. Lesions of thyroid dermopathy are usually asymptomatic and have only cosmetic importance. Advanced forms of dermopathy are associated with elephantiasis or thyroid acropachy. Almost all cases of thyroid dermopathy are associated with relatively severe ophthalmopathy. Usually ophthalmopathy appears first and dermopathy much later. All patients with localized myxedema have high serum concentrations of thyroid-stimulating hormone receptor antibodies, indicating the severity of the autoimmune condition. Occurrence of thyroid dermopathy in areas other than pretibial skin indicates a systemic process. Similar to Graves' ophthalmopathy, thyroid-stimulating hormone receptors in the connective tissue may be the antigen responsible for the immune process. Both humoral and cellular immune mechanisms are involved in the stimulation of fibroblasts and the production of large amounts of glycosaminoglycans. Localization in the pretibial area relates to mechanical factors and dependent position. Diagnosis of thyroid dermopathy is based on signs and typical pretibial skin lesions in association with a history of Graves' hyperthyroidism and ophthalmopathy. In some cases, skin biopsy is needed for confirmation. The lesions are usually mild and are overshadowed by more symptomatic ophthalmopathy. Most cases of thyroid dermopathy do not require any therapy. In mildly severe symptomatic cases and when there is cosmetic concern, topical corticosteroids applied under occlusive dressing are beneficial. In more severe cases, systemic immunomodulation may be necessary; however, conclusive evidence for long-term efficacy of these modalities is lacking. When significant edema and elephantiasis are present, local compressive therapy may have added benefit. In mild cases that do not require treatment, 50% of patients achieve complete remission after several years. Severe cases that receive topical corticosteroids or other therapies do not have a better outcome than untreated milder cases. Current treatment modalities for thyroid dermopathy and acropachy are at best palliative. Better and safer means of immunomodulation are needed.

Thyrotropin receptor antibodies: new insights into their actions and clinical relevance.

The thyrotropin receptor (TSHR) is a G-protein-coupled receptor with a large ectodomain. TSH, acting via TSHR, regulates thyroid growth and thyroid hormone production and secretion. The TSHR undergoes complex post-translational processing involving dimerization, intramolecular cleavage, and shedding of its ectodomain, and each of these processes may influence the antigenicity of the TSHR. The TSHR is also the major autoantigen in Graves' disease, as well as a leading candidate autoantigen in both Graves' ophthalmopathy and pretibial myxedema. The naturally conformed TSHR is most effectively presented as an autoantigen to the immune system, causing the production of stimulating TSHR-Abs. There are also autoantibodies which block the TSHR from TSH action, and neutral TSHR-Abs which have no influence on TSH action. TSHR-Abs can be detected by competition assays of TSHR-Abs for labeled TSH, or monoclonal TSHR-Ab binding to solubilized TSHRs, or by bioassays using thyroid cells or mammalian cells expressing recombinant TSHRs.

Autoimmune thyroid disease and pregnancy: relevance for the child.

As a group, the autoimmune thyroid diseases, including Graves' disease, Hashimoto's thyroiditis, and primary myxedema, are among the most common endocrine disorders encountered during pregnancy. Therefore, a substantial number of offspring will grow and develop in utero under conditions of maternal autoimmune thyroid disease and may be exposed to abnormal maternal thyroid function, maternal thyroid antibodies, and/or numerous therapeutic agents used to manage maternal thyroid dysfunction. This article reviews the effects that these various aspects of maternal autoimmune thyroid disorders can have on pregnancy outcome, as well as on the physical growth, neuropsychological development, and thyroid status of the developing fetus and neonate.

Recombinant monoclonal thyrotropin-stimulation blocking antibody (TSBAb) established from peripheral lymphocytes of a hypothyroid patient with primary myxedema.

Anti-TSH receptor antibodies (TRAbs) have been known to be involved in Graves' disease and primary hypothyroidism. We previously isolated and reconstituted immunoglobulin (Ig) genes of Epstein-Barr virus-transformed B cell clones producing monoclonal TRAbs obtained from Graves' patients. In the present study, we performed a similar experiment using a B cell clone, 32A-5, derived from a patient with primary hypothyroidism. The variable region genes of Ig heavy (H) and light (L) chains were isolated and sequenced from the 32A-5 clone. A significant number of somatic mutations were found in variable regions of H and L chain gene segments. Each pair of H and L chain cDNAs was ligated into an expression vector for IgG1 production and stably introduced into myeloma cells. The transfectants were injected ip into BALB/c mice to yield ample volume of the antibody for following applications. Interactions of recombinant 32A-5 with Graves' sera with varying thyroid-stimulating antibody (TSAb) activities were studied. The recombinant antibody tended to suppress TSAb activities in 10 of 15 Graves' sera, in which four were significantly inhibited. In summary, this is the first study to analyze human monoclonal TSH-stimulation blocking antibodies (TSBAb) at the molecular level. Use of human recombinant monoclonal TSBAb may be an analytical tool for molecular-basis etiology and an alternative therapeutic path for Graves' disease.

Pretibial myxoedema associated with Hashimoto's thyroiditis.

Pretibial myxoedema is a cutaneous mucinosis typically associated with Graves' disease, although it may also develop in subjects with non-thyrotoxic thyroid pathologies. This report presents a rare case of pretibial myxoedema occurring in a 58-year-old woman with biopsy-proven Hashimoto's thyroiditis. The hypothetical pathogenetic link between the two disorders is discussed with particular attention to the role of thyroid stimulating hormone receptor antibodies.

[Extracorporeal photopheresis--treatment option in scleromyxedema?]. / Extrakorporale Photopherese--Therapieoption beim Skleromyxödem?

Scleromyxedema is an uncommon disease of unclear etiology. Therapy is difficult. Two patients with scleromyxedema were treated with extracorporeal photopheresis (ECP). The first patient has been treated unsuccessfully for 3 months with PUVA-bath-therapy and for one year with cyclophosphamide and prednisolone. Thus supplementary treatment with ECP was initiated, as the cyclophosphamide and prednisolone were gradually reduced. After 29 cycles of ECP, the skin lesions had almost disappeared and the associated myopathy also resolved. In the second patient initial monotherapy with ECP was started after PUVA-bath-therapy for 3 months did not show any effect. After temporary improvement with ECP every four weeks, the skin lesions relapsed, so oral cyclophosphamide was added. These two cases confirm the effect of ECP in scleromyxedema, but probably combination therapy is at least initially more successful.

Myxedema accompanied by huge portal-systemic shunt without portal hypertension.

A 43-year-old woman with a huge portal-systemic shunt accompanied by myxedema showed slow speech and behavior. Several imaging studies revealed a bold portal-systemic shunt from the splenic vein to the left renal vein. In addition, hypothyroidism caused by chronic thyroiditis was diagnosed, and synthesized thyroxine replacement was effective for the symptoms. However, the serum ammonia and indocyanin green retention remained in the abnormal range, nevertheless the portal vein pressure was normal and findings of liver cirrohsis were not recognized histologically. Surgical shunt closure was performed, resulting in normalized serum ammonia levels and serum branched chain amino acids /aromatic amino acids ratio, and improvement of the ammonia tolerance test.

Autoantibodies to the flavoprotein subunit of succinate dehydrogenase: analysis of specificity in autoimmune thyroid disease.

OBJECTIVE: Thyroid-associated ophthalmopathy is a progressive eye disorder affecting the extraocular muscle and orbital connective tissue and is considered to have an autoimmune aetiology. A recent study reported a close relationship between serum antibodies against the flavoprotein subunit of succinate dehyhdrogenase (SDHFp) and active thyroid-associated ophthalmopathy involving eye muscle damage. The aim of the present study was to develop a sensitive and quantitative radiobinding assay for the detection of antibodies to the flavoprotein subunit of succinate dehydrogenase and to use this to determine the distribution of antibodies in different patient groups. DESIGN AND PATIENTS: Serum samples from the following patient groups were analysed: 20 systemic lupus erythematosus; 20 Addison's disease; 26 autoimmune hypothyroidism; 28 Graves' hyperthyroidism; 12 pretibial myxoedema; 25 thyroid-associated ophthalmopathy. Sera from 20 healthy subjects were used as controls. [35S]-labelled succinate dehydrogenase flavoprotein was produced in an in vitro transcription-translation system and subsequently used in immunoprecipitation experiments with sera from patient and control groups to test for the presence of antibodies to the flavoprotein. RESULTS: Succinate dehydrogenase flavoprotein antibodies were detected in five of the 20 (25%) patients with Addison's disease, six of the 20 (30%) with systemic lupus erythematosus, five of the 26 (19%) with autoimmune hypothyroidsm, six of the 28 (21%) with Graves' hyperthyroidism, two of the 12 (17%) with pretibial myxoedema and three of the 25 (12%) with thyroid-associated ophthalmopathy. The frequencies of flavoprotein antibodies were significantly greater than controls (P-value < 0.05) for patients with systemic lupus erythematosus (P = 0.02), but not for patients with either Addison's disease (P = 0.05), pretibial myxoedema (P = 0.13), Graves' hyperthyroidism (P = 0.07), autoimmune hypothyroidism (P = 0.06) or thyroid-associated ophthalmopathy (P = 0.24). For the patients with thyroid-associated ophthalmopathy, the frequency of SDHFp antibodies did not appear to be related to the length of time from diagnosis: the group containing samples taken less than one year from diagnosis showed no increased frequency of SDHFp antibodies when compared to controls (P = 0.10), with three of the 18 (17%) patients being positive. With respect to seven patients with thyroid-associated ophthalmopathy diagnosed for more than a year, SDHFp antibodies were not detected in any of their serum samples. In addition, the clinical severity of the disease, as recorded by the NOSPECS classification, did not correlate with the frequency of SDHFp antibodies: P = 0.13, 0.33 and 0.38, respectively, for patients with Grade II, III and IV ophthalmopathy. Similar results were also found in the case of patients with pretibial myxoedema and eye disease: P = 0.06 for patients with Grade III ophthalmopathy and, SDHFp antibodies were not detected in any of the sera taken from patients with Grade IV ophthalmopathy. In addition, no association was found between disease duration and the frequency of antibodies to the flavoprotein in this patient group. CONCLUSIONS: Our results indicate that succinate dehydrogenase flavoprotein antibodies are not a suitable marker for thyroid-associated ophthalmopathy, at least with the assay system used, as they can be found in patients who do not have eye disease and therefore lack the disease specificity required of a diagnostic tool.

Regulation of thyrotropin receptor protein expression in insect cells.

Expression of large quantities of conformationally intact thyrotropin receptor (TSHR) is essential to understand the structure-function relationship of the receptor. We expressed three different constructs of full-length human TSHR in insect cells: (a) a TSHR cDNA lacking signal sequence (TSHR-ns), (b) a TSHR cDNA containing human TSHR signal sequence (TSHR-hs) and (c) a TSHR cDNA with baculovirus envelope protein encoded signal sequence gp-67 (TSHR-gp). No unique protein band, corresponding to any of these recombinant proteins, was visible upon Coomassie Blue staining after SDS-PAGE. However, Western blot using TSHR specific monoclonal antibody showed unique bands around 80, 100 and 100 kDa in TSHR-ns, TSHR-hs and TSHR-gp virus infected insect cells respectively. All three full-length TSHR proteins could neutralize the TSH binding inhibitory immunoglobulin (TBII) activity from sera of experimental animals. However, only glycosylated proteins (TSHR-hs and TSHR-gp) neutralized the TBII activity of sera from autoimmune thyroid patients, confirming the importance of glycosylation for patient autoantibody reactivity. Expression levels of full-length TSHR proteins were much lower than the levels of similarly produced corresponding ectodomains of TSHR proteins. Southern blot and Northern blot analyses showed that DNA and RNA levels in full-length TSHR virus infected insect cells were comparable to the levels found in cells infected with viruses encoding only the ectodomain of TSHR. These data suggest that full-length TSHR expression is very low and is regulated at the translational level.

[Idiopathic myxedema and blocking type antibodies to TSH receptor].

The pathogenetic role of thyrotropin-stimulation blocking antibodies (TSBab) in idiopathic myxedema has been described. Idiopathic myxedema is characterized as atrophic thyroid gland and positive autoantibodies to thyroid gland, including TSBab. TSBab is positive in 20-30% cases of idiopathic myxedema. TSBab is disappeared in some cases of TSBab positive idiopathic myxedema during treatment. In some of these cases, thyroid function recovered after the disappearance of TSBab. Transient neonatal hypothyroidism with TSBab positive was born in mothers of TSBab positive with autoimmune thyroid diseases. In these cases, thyroid function recovered with the disappearance of TSBab. Form these findings, it is suggested that TSBab may play a certain pathogenetic role in idiopathic myxedema.

[Immunology in medical practice. XX. Organ specific autoimmune diseases]. / Immunologie in de medische praktijk. XX. Orgaanspecifieke auto-immuunziekten.

Organ specific autoimmune diseases are predominantly diseases of the endocrine glands and involve amongst others the thyroid (Hashimoto's disease, primary myxoedema, Graves' disease), the islets of Langerhans (type I diabetes mellitus) and the adrenals (Addison's disease). Over the past fifty years the knowledge on the pathogenesis of these diseases has considerably increased, leading to a large number of newly developed diagnostic tools, particularly determination of autoantibodies. Most of these autoimmune diseases have a (long) subclinical latency period. During this latency period a reliable prediction of later clinical manifestation is feasible. Preventive interventions during the latency period to correct underlying abnormalities of the target endocrine gland and/or the immune system are currently being tested in experimental animal models.

[Significance of histopathologic analysis of skin lesions in scleromyxedema. Light microscopy, electron microscopy, immunohistochemistry and immunofluorescence microscopy]. / Bedeutung der histopathologischen Analyse der Hautläsionen bei Skleromyxödem. Lichtmikroskopie, Elektronenmikroskopie, Immunhistochemie und Immunfluoreszenzmikroskopie.

Scleromyxedema (SM) may be considered as a possible disease entity in the differential diagnosis of scleroderma. Clinical data and the results of light, immunohistochemical, immunofluorescence and electron microscopic study of skin biopsies taken from a 53-year old patient with SM are reported. In the patient with SM in which abnormal serum paraprotein was not identified, the skin biopsy showed mucinous material in the dermis and proliferation of fibroblasts accompanied by mild dermal sclerosis. Immunofluorescence showed scanty granular IgG along the epidermal basement membrane and IgG and C1q focally along the connective tissue fibres in the dermis of clinically involved skin. In addition to clinical findings, detailed skin biopsy studies including contemporary techniques can contribute to the diagnosis of the disease.

Modulation of Fas-mediated apoptosis of human thyroid epithelial cells by IgG from patients with Graves' disease (GD) and idiopathic myxoedema.

The expression of two autoimmune thyroid diseases. GD and idiopathic myxoedema, is associated with antibodies to the thyroid-stimulating hormone (TSH) receptor. Thyroid stimulating antibodies (TSAb) in GD are TSH agonists and cause hyperthyroidism as well as goitre, whereas thyroid stimulation blocking antibodies (TSBAb) in idiopathic myxoedema are TSH antagonists and cause hypothyroidism and thyroid atrophy. We investigated the effect of antibodies to TSH receptor on Fas-mediated apoptosis of thyroid epithelial cells (thyrocytes). Human IgG was isolated from healthy donors, patients with GD and idiopathic myxoedema. Human thyrocytes were obtained from surgical specimens. Thyrocytes were cultured in the presence or absence of human IgG with or without interferon-gamma (IFN-gamma) or IL-1beta for a specified time. After incubation, we examined the level of cAMP in cultured supernatants and both Fas and Bcl-2 expression on thyrocytes. In addition, we examined anti-Fas-mediated apoptosis of thyrocytes. Fas expression on thyrocytes was significantly down-regulated by Graves' IgG and TSH, although idiopathic myxoedema IgG did not affect Fas expression on thyrocytes. Idiopathic myxoedema IgG abrogated the effect of TSH on both cAMP production and inhibition of Fas expression on thyrocytes. Treatment of thyrocytes with IL-1beta or IFN-gamma caused a marked augmentation of Fas expression on thyrocytes. The increase of Fas expression of thyrocytes induced by IL-1beta or IFN-gamma was significantly suppressed in the presence of TSH or Graves' IgG. Anti-Fas-induced apoptosis of thyrocytes was observed in thyrocytes treated with IL-1beta or IFN-gamma, but was markedly inhibited in the presence of TSH or Graves' IgG. Furthermore, idiopathic myxoedema IgG abrogated most of the inhibitory effect of TSH on Fas-mediated apoptosis of thyrocytes treated with IL-1beta or IFN-gamma. Bcl-2 expression of thyrocytes did not change after stimulation with TSH, Graves' IgG, idiopathic myxoedema IgG, IL-1beta or IFN-gamma. These results suggest that TSAb found in Graves' patients may be potentially involved in the development of goitre by inhibition of Fas-mediated apoptosis of thyrocytes. In addition, TSBAb inhibit the action of TSH and increase the sensitivity toward Fas-mediated apoptosis of thyrocytes, inducing thyroid atrophy seen in patients with idiopathic myxoedema.

Association of autoimmune thyroid disease with a microsatellite marker for the thyrotropin receptor gene and CTLA-4 in a Japanese population.

To examine the genetic contribution of the thyroid-stimulating hormone receptor (TSHR, or thyrotropin receptor) gene to autoimmune thyroid disease (AITD), we identified a dinucleotide repeat polymorphism near the TSHR gene that mapped to an 8.6 cM interval between D14S74 and D14S55 on the long arm of human chromosome 14. Association studies revealed a significant difference (p = 3.8 x 10(-5) between the TSHR microsatellite allele frequency distribution in 81 unrelated Japanese AITD patients and 113 Japanese controls, with a significant increase in the 180 pb allele (allele 1) of the microsatellite sequence (p = 5.8 x 10(-7). The risk for AITD with the 180 bp allele was 3.5, with association highly significant in female patients (p = 1.1 x 10(-5) and less dramatic, but still significant, in male patients (p = .02). These results suggest that the 180 bp allele of the TSHR microsatellite is associated with a susceptibility locus for AITD in Japanese patients. Two additional genetic markers have been evaluated for association in the Japanese AITD patients. The TSHR codon 52 (C52-->A52) transition mutation was not observed in the Japanese. A polymorphism for the CTLA-4 gene was genotyped and, while association with AITD was not observed (p = .15), a significant association was observed between CTLA-4 alleles of 110 bp (p = .01) and 106 bp (p = .004) and susceptibility to primary hypothyroidism or idiopathic myxedema, respectively.

Recombinant human TSH receptor expressed in E. coli.

We expressed the extracellular domain (20-408 aa, (T) of human TSH receptor (TSHR) in E. coli to detect TSHR autoantibodies (TRAb) and, moreover, we expressed the two portions (20-218 aa (5') and 217-408 aa (3')) of the extracellular domain thought to distinguish thyroid stimulating antibodies (TSAb) from blocking antibodies (TSBAb), using pGEX.3X as the expression vector. Using Western blotting analysis of the sera from patients with autoimmune thyroid disease, sera from Graves' patients and patients with idiopathic myxedema who had TSBAb reacted with the fusion protein (T), but none of the control sera reacted with it. We further evaluated whether or not the positive sear for T recognized fusion proteins (5') or (3'). The sera from Graves' patients reacted with both fusion proteins (5') and (3'). The sera from patients with idiopathic myxedema did not react with either of fusion proteins (5') or (3'). These findings suggest that these recombinant TSHR proteins could be used as antigens to detect TRAb, and differentiate TSABb from patients with idiopathic myxedema.