Our concepts and understanding of the etiology, evolution, and propagation of Graves' ophthalmopathy have become much more sophisticated than they were 10 years ago. Given our current state of knowledge, the following scheme for the pathogenesis of Graves' ophthalmopathy can be proposed. Circulating T cells in patients with Graves' disease, directed against an antigen on thyroid follicular cells, recognize antigenic epitopes that are shared by tissues contained in the retroorbital space. Of the cell types residing in these tissues, fibroblasts are most likely to act as both target and effector cells of the retroorbital immune process. This includes those fibroblasts present in the perimysium of extraocular muscles, which do not appear to be immunologically different from fibroblasts located in the retroorbital connective tissue. By contrast, convincing evidence implicating the human extraocular myocyte itself (rather than the tissue conglomerate of extraocular muscle) as a primary target in GO remains to be demonstrated. Together with adipocytes, fibroblasts may also serve as target and effector cells in pretibial myxedema. How autoreactive T cells escape deletion by the immune system and come to be directed against a self-antigen presented by cells residing in the thyroid gland and extrathyroidal locations is unknown. T cells are recruited to and infiltrate the orbit via certain adhesion receptors, which may also play a costimulatory role in T cell activation and facilitate antigen recognition. Analysis of variable region gene usage of the T cell antigen receptors in retroorbital T cells of patients with active GO reveals limited variability, suggesting that antigen-driven selection and/or expansion of specific T cells may occur early in the evolution of GO. Although the relative contributions of cellular and humoral immunity to the pathogenesis of GO are still uncertain, it is likely that both are important for full clinical expression and propagation of the autoimmune process within the orbit. T cells and macrophages populating the retroorbital space are now known to release certain cytokines (most likely a Th1-type spectrum) into the surrounding tissue. Cytokines and growth factors released both from infiltrating inflammatory and residential cells act upon fibroblasts in a paracrine and autocrine manner to stimulate the expression of immunomodulatory molecules, glycosaminoglycan synthesis, and cell proliferation in retroorbital fibroblasts. The existence of a thyroid cross-reactive antigen within the retroorbital tissues has long been postulated to explain the localized infiltration of autoreactive lymphocytes into the orbit. The recent detection, in fibroblasts and possibly other cellular components of the retroorbital space, of mRNA transcripts encoding the human TSH receptor, together with evidence of TSHr immunoreactivity in these cells, further highlight this intriguing possibility. However, presence of functionally active and/or immunogenic TSH receptor protein within the retroorbital tissues remains to be further substantiated. Taken together, a number of important steps in the complex pathogenesis of GO have been elucidated in recent years. Nevertheless, before the enigma of GO will be resolved, many important problems remain to be tackled, of which the nature of the primary antigen is only one.

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) regions gene usage in tyroid, orbital, pretibial tissue and peripheral blood mononuclear cells of patients with GD, GO and PTD was assessed using RP-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(b)eta 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.

Aim: Serum osteocalcin was shown in a previous study on first trimester pregnant women to correlate with bone density and to distinguish between fast and slow bone losers. The objective of the present study is to examine whether serum osteocalcin is related to vitamin D receptor (VDR) BsmI polymorphism in pregnant women.Study design: We determined osteocalcin serum levels and VDR BsmI genotype in 97 healthy first trimester pregnant women consecutively recruited during six months.Results: BB (21%), Bb (38%) and bb (41%) genotypes showed similar osteocalcin serum levels. However, in primigravidas (n = 38) the BB genotype was significantly associated with higher mean osteocalcin level (9.67 ng/mL) than the Bb (8.07 ng/mL) and the bb genotype (8.14 ng/mL), respectively (P < 0.05). The VDR genotype was the only independent parameter to correlate with serum osteocalcin (P < 0.05).Conclusion: Only primigravidas show in the first trimester a relation between the bone formation parameter serum osteocalcin and the VDR genotype BB which indicates a higher risk of fractures. For further clinical applications serum osteocalcin and VDR genotype should be tested on a cohort of primigravidas including measurements of bone density.

Several uncontrolled studies suggest octreotide is beneficial in thyroid-associated ophthalmopathy (TAO); however, the natural tendency of TAO to improve mandates randomized, controlled trials. We report results of a double-blind, placebo-controlled trial of octreotide long-acting repeatable (LAR). Fifty euthyroid patients (11 males, 39 females; age 22-74 yr, median 50 yr) with active TAO [clinical activity score (CAS) > or =3, NOSPECS (no signs or symptoms; only signs, no symptoms; signs only; proptosis; eye muscle involvement; corneal involvement; sight visual acuity reduction) 2a-5a] of median duration 0.9 yr received either 30 mg LAR or placebo every 4 wk for 16 wk; both groups then received 30 mg LAR for wk 16-32 and were followed up without treatment for a further 24 wk. Objective assessments included all individual parameters of TAO, CAS, and derived scores for soft tissue inflammation (STI) and ophthalmopathy index (OI). During wk 0-16 there was significant reduction in STI, subjective diplopia, and CAS in LAR-treated patients; STI and CAS were also reduced with placebo. The OI reduced by -1.12 in LAR (P = 0.0017) vs. -0.23 in placebo (P = 0.33), giving a barely significant treatment effect by Wilcoxon (P = 0.043), but analysis of covariance failed to confirm this (P = 0.16). During wk 16-32 there was no significant change in OI in either group. The overall results (wk 0-32) showed reduction in STI and CAS in both groups. In this double-blind, placebo-controlled trial, no significant therapeutic effect of octreotide LAR was seen in patients with moderately severe TAO. The improvements in both treated and placebo groups emphasize that the results of open studies must be viewed with caution.

What causes GO is still a mystery, but the disease process results from a complex interplay of genetic and environmental factors. Genes such as those for HLA genes may determine a patient's susceptibility to the disease and its severity, but environmental factors, often unknown, may determine its course. Once established, the chronic inflammatory process within the orbital tissues appears to take on a momentum of its own. Given our current state of knowledge, the following working scheme for the pathogenesis of GO can be proposed (Fig. 1): On the background of a permissive immunogenetic milieu, circulating T cells in patients with GD, directed against certain antigens on thyroid follicular cells, recognize antigenic epitopes that are shared by tissues contained in the orbital space. Of the cell types residing in these tissues, preadipocytes and fibroblasts, most likely act as target and effector cells of the orbital immune process, respectively. This includes preadipocyte fibroblasts present in the perimysium of extraocular muscles, which do not appear to be immunologically different from those located in the orbital connective tissue. Orbital preadipocyte fibroblasts may be stimulated by unknown circulating or locally produced factors to differentiate into mature adipocytes that express increased levels of TSHr. How autoreactive T cells escape deletion and control by the immune system and come to be directed against a self-antigen presented by cells residing in the thyroid gland and extrathyroidal locations, is still unknown. Proliferation and expansion of autoreactive T cell clones may be due to mimicry of a host antigen by a microorganism, but this remains speculative. T cell recruitment into the orbital tissues is facilitated by certain chemokines and cytokines, which help to attract T cells by stimulating the expression of certain adhesion molecules (e.g., ICAM-1, VCAM-1, CD44) in vascular endothelium and connective tissue cells. These adhesion receptors are known to also play an important costimulatory role by activating T cells and facilitating antigen recognition, which amplifies the cellular immune process. Analysis of variable region genes of T cell antigen receptors in orbital T cells of patients with active GO has revealed their restricted TcR V gene usage, suggesting that antigen-driven selection and/or expansion of specific T cells may occur early in the evolution of GO. T cells and macrophages populating the orbital space are known to synthesize and release a [figure: see text] number of cytokines (most likely a Th1-type spectrum) into the surrounding tissue. Cytokines, oxygen free radicals and fibrogenic growth factors, released both from infiltrating inflammatory and residential cells, act upon orbital preadipocytes in a paracrine and autocrine manner to stimulate adipogenesis, fibroblast proliferation, glycosaminoglycan synthesis, and the expression of immunomodulatory molecules. Smoking, a well-known aggravating factor in GO, may aggravate tissue hypoxia and exert important immunomodulatory effects. The long held hypothesis of a thyroid cross-reactive antigen within the orbital tissues has recently gained significant support by an animal model of GO, and by in vitro and ex vivo studies. If confirmed in immunological studies, these data may well explain the localized infiltration of the orbital tissues by autoreactive lymphocytes that share intriguing molecular features with intrathyroidal lymphocytes. Local release of particular cytokines, TSHr-directed antibodies, or other factors might further enhance adipogenesis, glycosaminoglycan synthesis and expression of immunomodulatory proteins within the orbit. Other factors, including inflammatory cytokines, might act as counterbalancing inhibitors of these effects. However, if the net effect of these changes is to increase the volume of the fatty connective tissues within the orbit, then proptosis, extraocular muscle dysfunction, and periorbital congestion will ensue. Whether this hypothetical sequence of events will finally explain the involvement of the orbit in GD is unknown. Future studies will be aimed at identifying factors that might modulate adipogenesis in orbital cells and clarifying the link between adipogenesis and TSHr expression in the orbit. Taken together, a number of important details in the complex pathogenesis of GO have been resolved in recent years, but many challenges are still ahead. Elucidation of the primary antigen and how it is recognized by the immune system will be key issues.

The sodium iodide symporter (NIS) is the key regulator of iodine access to the thyroid gland and several other specialized tissues. It serves important functions both in thyroid physiology and pathophysiology and in the integrity and function of various extrathyroidal tissues.

A male type-2 diabetic, treated with the peroxisome proliferator-activated receptor (PPAR) agonist, Pioglitazone, experienced exacerbation of his thyroid eye disease (TED), which had been stable and inactive for more then 2 yr. Expansion of the orbital fat developed, and we have investigated the effects of PPAR gamma agonists, including Pioglitazone and, subsequently, an antagonist on the adipogenesis of preadipocytes from TED orbits and Graves' neck fats. The percentage of differentiating cells, assessed by oil red O staining, morphological changes, and PPAR gamma transcript levels, was determined for preadipocytes in hormone/agonist-induced models of adipogenesis, supplemented or not with PPAR gamma agonists or antagonist. The PPAR gamma agonists resulted in a 2- to 13-fold increase, and a PPAR gamma antagonist produced a 2- to 7-fold reduction in adipogenesis in vitro. Effects were dose dependent and maximal at 1 or 10 micro M. We suggest that care should be exercised when selecting patients for treatment with PPAR gamma agonists and that such agonists may be contraindicated in individuals with a previous history of autoimmune thyroid or eye diseases. Our work also suggests that PPAR gamma antagonists could provide a novel therapy for TED patients in the active stage of disease.

UNLABELLED:To improve management of patients with Graves' orbitopathy, a multi-center collaborative approach is necessary in order to have large enough sample sizes for meaningful randomized clinical trials. This is hampered by a lack of consensus on how to investigate the eye condition. The European Group on Graves' Orbitopathy aims to overcome this and has designed a preliminary case record form (CRF) to assess Graves' orbitopathy patients. This form was used in this first multi-center study.AIM:To investigate patient characteristics and treatment strategies in 152 new consecutively referred patients with thyroid eye disease seen in nine large European referral centers.METHODS:Newly referred patients with Graves' orbitopathy were included who were seen between September and December 2000. Demographic data and a complete ophthalmological assessment were recorded.RESULTS:One-hundred and fifty-two patients (77% females) were included. Diabetes was present in 9%, and glaucoma or cataract in 14% of patients. Forty percent were current smokers, 9% also had dermopathy, and only 33% reported a positive family history of thyroid disease. Mild eye disease was seen in 40%, moderately severe eye disease was seen in 33% and severe eye disease was seen in 28% of patients. Soft tissue involvement was the most frequent abnormality (seen in 75%), proptosis > or =21 mm was found in 63%, eye motility dysfunction in 49%, keratopathy in 16% and optic nerve involvement was found in 21% of patients. According to the clinical impression, 60% had active eye disease. Immunosuppressive treatment was planned more frequently in active patients (57/86; 66%) than in inactive patients (5/57, 9%; Chi-square 46.16; P<0.02). There were no important differences among the eight centers regarding the severity and the activity of their patients.CONCLUSIONS:In view of the large number of patients recruited in only 4 months, multi-center studies in the eight EUGOGO centers appear to be feasible.

This study evaluates the possibility of treating Bon1 and QGP pancreatic neuroendocrine tumor cells with radioactive iodide ((131)I) after stable transfection with the thyroid sodium iodide symporter (NIS). NIS expression was driven either by the strong viral cytomegalovirus promoter or by the tissue-specific chromogranin A promoter. Using either approach, NIS expression was confirmed by reverse transcription-PCR and Western blotting. Uptake of radioactive iodide was increased approximately 20-fold by chromogranin A promoter-driven NIS expression and approximately 50-fold by cytomegalovirus promoter-driven NIS expression. Maximal uptake was reached within 15 min in QGP cells and 30 min in Bon1 cells. Effective half-life was 5 min in QGP and 30 min in Bon1 cells. No evidence of organification was detected by high-performance liquid chromatography and gel filtration chromatography. (131)I was a highly effective treatment in NIS-expressing QGP and Bon1 cells, reducing clone formation by 99.83 and 98.75%, respectively, in the in vitro clonogenic assay. In contrast, clone formation was not reduced in QGP and Bon1 cells without NIS expression after incubation with the same activity concentration of (131)I as compared with mock treated cells. Absorbed doses to QGP and Bon1 cells are up to 150 and 30 Gy, respectively. In addition, a direct cytotoxic effect of radioiodide was demonstrated in NIS-expressing Bon1 cells after (131)I incubation. In conclusion, radioiodide treatment after NIS gene transfer appears to be a promising novel approach in the therapy of neuroendocrine tumors if its highly encouraging in vitro effectiveness can be transferred to the in vivo situation.

Receptor activator of NF‐κB (RANK) ligand (RANKL) and osteoprotegerin (OPG) play essential roles in bone metabolism and immune responses. RANKL activates RANK, which is expressed by osteoclasts and dendritic cells (DC), whereas OPG acts as its decoy receptor. The role of RANKL and OPG in thyroid physiology is unclear. Northern analysis revealed pronounced OPG mRNA levels in normal human thyroid. By contrast, RANKL mRNA levels were most abundant in lymph node and appendix, and low in the thyroid. In the human thyroid follicular cell line XTC and in primary human thyroid follicular cells, OPG mRNA levels and protein secretion were upregulated by interleukin (IL)‐1β ?(33‐fold), tumor necrosis factor (TNF)‐α (eightfold), and thyrotropin (TSH) (threefold). RANKL mRNA was stimulated in XTC by IL‐1β? and TNF‐α, but inhibited by TSH. Conditioned medium harvested from IL‐1β‐treated XTC (containing high concentrations of OPG) inhibited RANKL‐induced CD40 upregulation and cluster formation of DC. OPG mRNA levels were three times more abundant in surgical thyroid specimens of Graves' disease as compared to other thyroid diseases. Our data suggest that RANKL and OPG are produced in the thyroid gland by thyroid follicular cells, are regulated by cytokines and TSH, and are capable of modulating dendritic cell functions. Thus, these cytokines may represent important local immunoregulatory factors involved in the pathogenesis of autoimmune thyroid diseases. J. Cell. Biochem. 86: 642–650, 2002. © 2002 Wiley‐Liss, Inc.

P . b . b . G Z 0 2 Z 0 3 1 1 0 8 M , V e r l a g s p o s t a m t : 3 0 0 2 P u r k e r s d o r f , E r s c h e i n u n g s o r t : 3 0 0 3 G a b l i t z Indexed in SCOPUS/EMBASE/Excerpta Medica www.kup.at/mineralstoffwechsel Österreichische Gesellschaft für Orthopädie und Orthopädische Chirurgie Österreichische Gesellschaft für Rheumatologie Offizielles Organ der Österreichischen Gesellschaft zur Erforschung des Knochens und Mineralstoffwechsels Member of the HIV-Infektion und Knochengewebe

BACKGROUND:The human sodium iodide symporter (hNIS) is a transmembrane protein that mediates the active transport of iodide in the thyroid gland. Following cloning of NIS, NIS expression has been detected in a broad range of nonthyroidal tissues, suggesting that iodide transport in these tissues is conferred by the expression of functional NIS protein.METHODS:The aim of this study was to examine functional hNIS expression in kidney by reverse transcription-polymerase chain reaction (RT-PCR), ribonuclease protection assay (RPA), immunohistochemistry, and Western blot analysis accompanied by iodide accumulation studies in kidney cells.RESULTS:Using a pair of full-length hNIS-specific oligonucleotide primers, RT-PCR followed by Southern hybridization revealed hNIS mRNA expression in normal human kidney tissue. The PCR products were subjected to automated sequencing and revealed full identity with the published human thyroid-derived NIS cDNA sequence. Furthermore, positive protected bands indicating the presence of hNIS mRNA were apparent in RPA gel lanes corresponding to human kidney cells as well as Chinese hamster ovary (CHO) cells stably transfected with hNIS cDNA and Graves' thyroid tissue. Immunohistochemical analysis of normal human kidney tissue using a mouse monoclonal hNIS-specific antibody showed marked hNIS-specific immunoreactivity confined to tubular cells, while no hNIS-specific immunoreactivity was detected in the glomeruli. NIS protein expression in human kidney cells was further confirmed by Western blot analysis. In addition, accumulation of (125)I was detected in human kidney cells in vitro and was shown to be sodium dependent and sensitive to perchlorate.CONCLUSIONS:Functional hNIS expression was demonstrated in the renal tubular system, suggesting that renal iodide transport may be, at least in part, an active process driven by NIS.

Receptor activator of nuclear factor (NF-kappaB) ligand (RANKL), its cellular receptor, receptor activator of NF-kappaB (RANK), and the decoy receptor osteoprotegerin (OPG) constitute a novel cytokine system. RANKL produced by osteoblastic lineage cells and activated T lymphocytes is the essential factor for osteoclast formation, fusion, activation, and survival, thus resulting in bone resorption and bone loss. RANKL activates its specific receptor, RANK located on osteoclasts and dendritic cells, and its signaling cascade involves stimulation of the c-jun, NF-kappaB, and serine/threonine kinase PKB/Akt pathways. The effects of RANKL are counteracted by OPG which acts as a soluble neutralizing receptor. RANKL and OPG are regulated by various hormones (glucocorticoids, vitamin D, estrogen), cytokines (tumor necrosis factor alpha, interleukins 1, 4, 6, 11, and 17), and various mesenchymal transcription factors (such as cbfa-1, peroxisome proliferator-activated receptor gamma, and Indian hedgehog). Transgenic and knock-out mice with excessive or defective production of RANKL, RANK, and OPG display the extremes of skeletal phenotypes, osteoporosis and osteopetrosis. Abnormalities of the RANKL/OPG system have been implicated in the pathogenesis of postmenopausal osteoporosis, rheumatoid arthritis, Paget's disease, periodontal disease, benign and malignant bone tumors, bone metastases, and hypercalcemia of malignancy, while administration of OPG has been demonstrated to prevent or mitigate these disorders in animal models. RANKL and OPG are also important regulators of vascular biology and calcification and of the development of a lactating mammary gland during pregnancy, indicating a crucial role for this system in extraskeletal calcium handling. The discovery and characterization of RANKL, RANK, and OPG and subsequent studies have changed the concepts of bone and calcium metabolism, have led to a detailed understanding of the pathogenesis of metabolic bone diseases, and may form the basis of innovative therapeutic strategies.