Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-11-05
2004-06-08
Pak, Michael (Department: 1646)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100
Reexamination Certificate
active
06747139
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of molecular biology and immunology. More particularly, the invention relates to human thyrotropin receptor compositions. The invention is also related to diagnostic and therapeutic applications using human thyrotropin receptor compositions according to the present invention.
BACKGROUND OF THE INVENTION
The most important of the diseases that cause thyrotoxicosis is Graves' disease, also known as Parry's or Basdow's disease. However, not all hyperthyroidism is a result of Grave's disease. Additionally, not all thyrotoxicosis is due to hyperthyroidism. Thyrotoxicosis is the clinical, biochemical and physiological result of sustained delivery of excessive quantities of thyroid hormones to the peripheral tissues. Hyperthyroidism is used to denote the situation where excess thyroid hormones stems from sustained thyroid hyperfunction.
There are a number of causes for thyrotoxicosis, the major ones include: Graves' disease, toxic adenoma, toxic multinodular goiter, thyrotoxicosis factitia, ectopic thyroid, and thyroiditis. A portion of these are hyperthyroid based whereas the remaining portion is nonhyperthyroid. Graves' disease is by far the most common cause of thyrotoxicosis, as well as the only autoimmune cause, it is therefore of extreme importance that an accurate assay be available to allow the differentiation between the auto immune and non-autoimmune varieties since the treatments, and pathogenesis differ so drastically. Bardin,
Current Therapy in Endocrinology and Metabolism,
B. C. Decker Inc. Toronto (1988).
Graves' disease is a relatively common disorder that occurs at any age, but most often in the third and fourth decades. The disease is more frequent in women and the ratio of predominance in women may be as high as 7:1. The manifestations of Graves' disease include one or more of the following: hyperthyroidism with diffuse goiter, ophthalmopathy, and dermopathy.
Thyrotropin, also known as thyroid stimulating hormone (TSH), is the primary hormone that regulates thyroid cell differentiated function and proliferation (Dumont et al., Adv. Cyclic Nucleotide Res. 14:479-489 (1981)). These effects are mediated by its interaction with the thyrotropin (TSH) receptor on the plasma membrane of thyroid cells. Thyroid stimulating auto-antibodies, the cause of thyrotoxicosis in Graves' disease, mimic the actions of TSH by their interaction with the TSH receptor (Rees Smith et al., Endocr. Rev. 9:106-121 (1988)). In Graves' disease, anti-TSH receptor auto-antibodies bind to the TSH receptor on the thyroid cell surface. Such binding causes unregulated stimulation of the thyroid cell which then produces excessive amounts of thyroid hormone.
The pathophysiological importance of the TSH receptor has motivated efforts directed to its characterization at both the protein as well as the genetic level. Hence, it has been found that it is a member of the G-protein-coupled receptor superfamily with glycoprotein hormone ligands, and that it is characterized by large, heavily glycosilated ectodomains with leucine-rich repeats encoded by several exons. (Gross et al., Biochem. Biophys. Res. Comm. 177:679-687 (1992); Tsai-Morris et al. J. Biol. Chem. 266:11355-11359 (1991); Koo et al., Endocrinol. 128:2297-2308 (1991)). Furthermore, characterization of cell bound TSH has identified two distinct functional forms. In one form, TSH is a single peptide receptor. In a second form, TSH is a heterodimer protein complex comprising subunit peptides A and B linked by disulfide bonds (Furmaniak et al., FEBS Letters 215:316-322 (1987), and Russo et al., Mol. Endocrinology 5:1607-1612 (1991)). Immunoprecipitation or immunoblotting analysis of mammalian cell extracts have shown variable proportions of single and two subunits forms, albeit the pathological significance of this finding is yet to be understood (Lossfelt et al., Proc. Natl. Acad. Sci. USA 895:3765-3769 (1992); Hata et al., Biochem. Biophys. Res. Comm. 164:1268-1273 (1989); Potter et al., BBRC 205:361-367 (1994); Chazenbalk et al., Endocrinology 137:4586-4591 (1996); Graves et al., Endocrinology 137:3915-3920 (1996)). Recent studies have suggested that subunits A and B are the product of intramolecular cleavage at two separate sites, with the concomitant loss of a TSH fragment, referred to in the literature as peptide C (expected to correspond to an approximately 5 KDa plypeptide fragment) (Chazenbalk, et al., Endocrinology 138:2893-2899 (1997)). To date however, the sites of cleavage, the kinetics of the cleavage as well as the enzyme(s) involved have yet to be elucidated. In addition, the physiological and/or pathological implications of peptide C, if any, have yet to be investigated.
The difficulties encountered in characterizing Graves' disease at the molecular level parallel the obstacles encountered in devising sensitive and specific diagnostic assays. Thyroid stimulating auto-antibodies, originally named “long-acting thyroid stimulators” or LATS, were measured with a bioassay system (McKensie) using mouse thyroid tissue. This assay was considered to be fairly specific for Graves' disease, although positive results were found in a few patients with Hashimoto's disease and occasionally, albeit in smaller titers, in other unrelated conditions. In addition, this assay could only detect 40-45% of patients with Graves' disease. Notably, there was no correlation between titer and the severity of the disease.
More recently, other related IgG antibodies were found that prevent adsorption of LATS onto human thyroid tissue and as a group were therefore called “LATS protector.” This abnormal IgG antibody (or antibodies) could be demonstrated by three different assay techniques. One technique used high-titer LATS antibody as the indicator system whose endpoint was to show that antibody in patient serum blocked uptake of the LATS antibody by human thyroid tissue. A second technique, also known as “TSH-binding inhibiting immunoglobulin” or TBII assay, measured, the ability of patient antibody to inhibit or block the binding of radiolabeled TSH to human or animal thyroid acinar cell membranes. A third technique measured the ability of the antibody to stimulate human or animal thyroid acinar cell membrane-bound adenylate cyclase, producing increased cyclic adenosine monophosphate (AMP) activity. The original assay method used human thyroid tissue, so the antibody was originally called “human thyroid-stimulating immunoglobulin.” Since animal thyroid tissue can be used, the assay now is simply called “thyroid-stimulating immunoglobulin” (TSI). For example, one modification of the TSI uses special TSH-dependent FRTL-5 rat tissue culture thyroid cells. The LATS-protector assay has been reported to detect that about 75%-80% (range , 60%-90%) of patients with Graves' disease. The LATS-protector assay is very complicated. The TBII technique is reported to detect only about 70%-80% (range, 39%-100%) of Graves' disease, and the TBI detects only about 75%-80% (range, 18%-100%). Neither the TBII nor the TBI are simple or easy.
In addition to the low sensitivity inherent to the systems used, most laboratories using either technique use “homemade” reagents, which accounts for much of the great variation in sensitivity reported in the literature. Furthermore, since many clinical laboratories base their test performance claims on data from one or more research laboratories using the same technique but separate reagents additional levels of variability are compounded upon standardization. Besides sensitivity, it is necessary to question specificity, since different laboratories may find different numbers of false positive results when patients with hyperfunctioning thyroid nodules, nonfunctioning nodules or goiter, thyroiditis, autoimmune disorders, and clincially normal status are tested.
At present, the TSI assay is used mainly for patients with borderline or conflicting evidence of Graves' disease, patients who have some
McLachlan Sandra
Rapoport Basil
Hollrigel Greg S.
Pak Michael
Quest Diagnostics Investments Inc.
Stout Donald E.
Stout, Uxa Buyan & Mullins, LLP
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