The TSH receptor (TSHR) is constitutively active and is further enhanced by TSH ligand binding or by stimulating TSHR antibodies (TSHR-Abs) as seen in Graves' disease. TSH is known to activate the thyroid epithelial cell via both Galphas-cAMP/protein kinase A/ERK and Galphaq-Akt/protein kinase C coupled signaling networks. The recent development of monoclonal antibodies to the TSHR has enabled us to investigate the hypothesis that different TSHR-Abs may have unique signaling imprints that differ from TSH ligand itself. We have, therefore, performed sequential studies, using rat thyrocytes (FRTL-5, passages 5-20) as targets, to examine the signaling pathways activated by a series of monoclonal TSHR-Abs in comparison with TSH itself. Activation of key signaling molecules was estimated by specific immunoblots and/or enzyme immunoassays. Continuing constitutive TSHR activity in thyroid cells, deprived of TSH and serum for 48 h, was demonstrated by pathway-specific chemical inhibition. Under our experimental conditions, TSH ligand and TSHR-stimulating antibodies activated both Galphas and Galphaq effectors. Importantly, some TSHR-blocking and TSHR-neutral antibodies were also able to generate signals, influencing primarily the Galphaq effectors and induced cell proliferation. Most strikingly, antibodies that used the Galphaq cascades used c-Raf-ERK-p90RSK as a unique signaling cascade not activated by TSH. Our study demonstrated that individual TSHR-Abs had unique molecular signatures which resulted in sequential preferences. Because downstream thyroid cell signaling by the TSHR is both ligand dependent and independent, this may explain why TSHR-Abs are able to have variable influences on thyroid cell biology.

No abstract

TSH receptor (TSHR) antibodies (Abs) may be stimulating, blocking, or neutral in their functional influences and are found in patients with autoimmune thyroid disease, especially Graves' disease (GD). Stimulators are known to activate the thyroid epithelial cells via both Gs- and Gq-coupled signaling pathways, whereas blockers inhibit the action of TSH and may act as weak agonists. However, TSHR neutral Abs do not block TSH binding and are unable to induce cAMP via Gsα. The importance of such neutral Abs in GD remains unclear because their functional consequence has been assumed to be zero. We hypothesized that: 1) neutral TSHR Abs are more common to GD than generally recognized; 2) they may induce distinct signaling imprints at the TSHR not seen with TSH itself; and 3) these signaling events may alter cellular function. To evaluate these hypotheses, we first confirmed the presence of neutral TSHR Abs in sera from patients with GD and then, using mouse and hamster neutral TSHR monoclonal Abs (N-mAbs) performed detailed signaling studies, including a proteomic Ab array, with rat thyrocytes (FRTL-5) as targets. This allowed us to examine a battery of signaling cascades and their downstream effectors. Neutral TSHR Abs were indeed frequently present in sera from patients with GD. Sixteen of 27 patients (59%) had detectable neutral TSHR Abs by competition assay with N-mAbs. On examining signaling cascades, we found that N-mAbs induced signal transduction, primarily via the protein kinase A II cascade. In addition to the activation of phosphatidylinositol 3K/Akt, N-mAbs, unlike TSH, had the ability to exclusively activate the mammalian target of rapamycin/p70 S6K, nuclear factor-κB, and MAPK-ERK1/2/p38α signaling cascades and their downstream effectors p90 ribosomal kinase/MAPK-interacting kinase-1/mitogen and stress-activated kinase-1 and N-mAbs activated all forms of protein kinase C isozymes. To define the downstream effector mechanisms produced by these signaling cascades, cytokine production, proliferation, and apoptosis in thyrocytes were investigated. Although N-mAbs produced less cytokines and proliferation compared with TSH, they had the distinction of inducing thyroid cell apoptosis under the experimental conditions used. When dissecting out possible mechanisms of apoptosis, we found that activation of multiple oxidative stress markers was the primary mechanism orchestrating the death signals. Therefore, using oxidative stress-induced apoptosis, N-mAbs may be capable of exacerbating the autoimmune response in GD via apoptotic cells inducing antigen-driven mechanisms. This may help explain the inflammatory nature of this common disorder.

To circumvent the long-term effects of papillary ablation for extracting common bile duct stones (< 12 mm in diameter) in endoscopic sphincterotomy (EST), endoscopic papillary dilation (EPD) was attempted in 20 patients. To evaluate papillary function before and after the procedures, manometry of the sphincter of Oddi was carried out in 13 with EPD and 10 of 20 patients with EST. Extraction of all stones was successful (100%) in both groups at an equal rate. Repeated numbers of procedures were common in both groups. However, the mean duration of the procedure was high in EPD compared to EST (63 min vs 42 min, P < NS). Adjunctive therapies like mechanical lithotripsy (ML), nasobiliary drainage, and choledochoscopy were included in EPD, while EST required a basket catheter and ML. There was no significant difference on manometry before and after the procedures (P = NS), although papillary function was found to have decreased after the EPD. In contrast, all patients in the EST group lost papillary function after the procedure. Thirty-day morbidity and mortality rate were absent in both groups. Immediate and 2.5-year follow up complications were uncommon in both groups. As a simple method, EPD may be an effective and safe alternative to EST in the management of patients with bile duct stones who require maintenance of papillary function.

The immunologic processes involved in autoimmune thyroid disease (AITD), particularly Graves’ disease (GD), are similar to other autoimmune diseases with the emphasis on the antibodies as the most unique aspect. These characteristics include a lymphocytic infiltrate at the target organs, the presence of antigen-reactive T and B cells and antibodies, and the establishment of animal models of GD by antibody transfer or immunization with antigen. Similar to other autoimmune diseases, risk factors for GD include the presence of multiple susceptibility genes, including certain HLA alleles, and the TSHR gene itself. In addition, a variety of known risk factors and precipitators have been characterized including the influence of sex and sex hormones, pregnancy, stress, infection, iodine and other potential environmental factors. The pathogenesis of GD is likely the result of a breakdown in the tolerance mechanisms, both at central and peripheral levels. Different subsets of T and B cells together with their regulatory populations play important roles in the propagation and maintenance of the disease process. Understanding different mechanistic in the complex system biology interplay will help to identify unique factors contributing to the AITD pathogenesis.

No abstract

Autoantibodies to thyroglobulin and thyroid peroxidase are common in the euthyroid population and are considered secondary responses and indicative of thyroid inflammation. By contrast, autoantibodies to the TSH receptor are unique to patients with Graves' disease and to some patients with Hashimoto's thyroiditis. Both types of thyroid antibodies are useful clinical markers of autoimmune thyroid disease and are profoundly influenced by the immune suppression of pregnancy and the resulting loss of such suppression in the postpartum period. Here, we review these three types of thyroid antibodies and their antigens and how they relate to pregnancy itself, obstetric and neonatal outcomes, and the postpartum.

The results are consistent with an autoimmune etiology in a subset of cases of Tourette's syndrome.