Epitope mapping of protein antigens by expression-PCR (E-PCR).
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Antigens↗
Biomedical subjects
H B Burch
Publications and source records attributed to H B Burch.
Immunoglobulins from Graves' disease patients stimulate phospholipase A2 and C systems in FRTL-5 and human thyroid cells.
We have studied the effects of immunoglobulin G from Graves' disease patients on phospholipase A2 (PLA2) and C(PLC) systems in FRTL-5 and human thyroid cells. Immunoglobulin G (IgG) from Graves' disease patients stimulated arachidonic acid (AA) release in a time- and dose-dependent manner. In FRTL-5 thyroid cells, removal of external calcium had no significant effect on the IgG (20 micrograms/ml)-induced AA release in FRTL-5 thyroid cells. U-73122 (3 mumol/l), a PLC inhibitor, and quinacrine (100 mumol/l) but not U-26384 (5 mumol/l), PLA2 inhibitors, blocked the IgG-induced (20 micrograms/ml) AA release in FRTL-5 thyroid cells. Immunoglobulin G (100 micrograms/ml) also stimulated accumulation of inositol-1,4,5-triphosphate (IP3) in a time- and dose-dependent (20-300 micrograms/ml) manner in FRTL-5 cells. Immunoglobulin G from Graves' disease patients induced a significant increase of IP3 production (p = 0.01) compared to IgG from normal subjects. Removal of external calcium had no significant effect on the IgG-induced IP3 production. The PLC inhibitor U-73122 completely blocked IgG-induced IP3 production from FRTL-5 thyroid cells. Also, in human thyroid cells, IgG from Graves' disease patients induced a significant increase of AA release (p = 0.001) and IP3 production (p = 0.004) compared to the IgG from normal subjects. These data indicate that IgG from Graves' disease patients induced PLA2 activity that was PLC dependent, a pattern referred to as sequential activation. Our studies suggest that IgG from Graves' disease patients activates PLA2 and PLC systems in FRTL-5 and human thyroid cells. These signal transduction pathways could be involved in the pathogenesis of Graves' disease and future studies are warranted to investigate this area.
Adult↗
Antithyroid drugs and radioiodine therapy.
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Antithyroid Agents↗
Thyrotropin receptor T cell epitopes in autoimmune thyroid disease.
The human TSH receptor represents the primary target of thyroid-stimulating immunoglobulins responsible for the hyperthyroidism of Graves' disease. In the present series of investigations, the distribution of T cell epitopes has been mapped using synthetic peptides spanning the entire extracellular region of the human TSH receptor. In vitro proliferative responses of the mononuclear cells were measured using flow cytometric analysis of bromodeoxyuridine incorporation into nuclei. In 8 of 11 samples from patients with Graves' disease, at least one (and up to 9) regions of the human TSH receptor induced proliferation, with the mean stimulation index being 39.8 +/- 47.3. No single universal stimulatory peptide was identified. In contrast, stimulation was not observed in three control subjects, while one control subject showed minimal stimulation (index of 5.7) to peptides encompassing a limited area (amino acids 31-65). The immunodominant epitope of patients with recent-onset Graves' disease was localized between amino acids 271 and 365, whereas the immunodominant epitope of patients with disease duration greater than 1 year localized between amino acids 91 and 215. We conclude that the bromodeoxyuridine incorporation method is a useful and important tool for detecting antigen-induced lymphocyte proliferation. The TSH receptor-specific T cells from different Graves' disease patients recognize variable distinct sites within the extracellular region of the TSH receptor, and the immunodominant epitope apparently shifts toward the N-terminus of the receptor protein during the course of treated Graves' disease.
Bromodeoxyuridine↗
Evaluation and management of the solid thyroid nodule.
A tremendous effort has been applied to the determination of optimum management strategies in patients with solid thyroid nodules. The systematic acquisition of experience with historical and physical examination features suggestive of malignancy and the careful crafting of noninvasive techniques to assist in this determination have each contributed to improvements in the safety and cost-effectiveness of management in these patients. Despite this progress, significant difficulties await resolution, including the preponderance of patients still subjected to thyroidectomy for benign follicular lesions and the reluctance of clinicians to abandon the use of diagnostic techniques that offer little useful information, such as routine scintillation scanning and diagnostic trials of suppressive therapy. An exciting area now in its infancy is the identification of useful molecular markers for the selection of the few malignant thyroid nodules from among the multitudes of benign lesions. As is true in diagnostic evaluation, uncertainty continues to be experienced by clinicians charged with proper nonsurgical management of the solid thyroid nodule. The use of thyroid hormone suppressive therapy, once considered a cornerstone of conservative management, has been cast in a new light revealing both the limited utility and potential harm associated with this approach. These uncertainties should not be viewed as impediments, but rather, as opportunities for growth, as it is controversy rather than complacency that stimulates new investigation and fresh approaches to old problems.
Aging↗
Discontinuing antithyroid drug therapy before ablation with radioiodine in Graves disease.
OBJECTIVE: To determine the relative effects on thyroid hormone levels of discontinuing antithyroid drug therapy and subsequent ablation with radioiodine in patients with hyperthyroid Graves disease. DESIGN: A clinical trial with a prospective analysis of the relative change in thyroid hormone levels over time in response to therapy in two study groups. SETTING: An outpatient endocrine clinic at a tertiary care hospital. PATIENTS: 21 patients with a clinical diagnosis of hyperthyroid Graves disease scheduled to receive ablation therapy with radioiodine (131I): 17 patients were pretreated with antithyroid drugs, and 4 were not. METHODS: Antithyroid drugs were stopped 6 days before radioiodine therapy. Patients were monitored clinically and biochemically with measurement of free and total levels of thyroxine (T4) and triiodothyronine (T3) on days -6, -3, -1; the day of radioiodine therapy; and days 1, 2, 3, 4, 5, 7, and 14. RESULTS: Before radioiodine treatment and compared with baseline measurement, the mean increase in free T4 levels after discontinuation of antithyroid therapy was 86% (95% CI, 16.1% to 156%), with a concurrent mean increase in free T3 levels of 71.6% (CI, 31% to 112%). Radioiodine therapy resulted in a mean decrease in free T3 levels of 28.7% (CI, -44.1% to -13.2%), a mean decrease in total T3 levels of 22.9% (CI, -39.4% to -6.4%), and stability in free and total T4 levels rather than aggravation of thyrotoxicosis. A smaller group of patients not receiving antithyroid drugs experienced a course qualitatively similar to that of pretreated patients after 131I treatment, with a mean reduction in free T4 levels of 39.8% (CI, -69.9% to -9.7%) and a mean decrease in free T3 levels of 49.4% (CI, -93.7% to -5.1%). CONCLUSION: Short-term increases in thyroid hormone levels in patients with Graves disease receiving radioiodine ablation occur primarily as a result of discontinuing antithyroid therapy rather than as a result of treatment with 131I. Stability or decrease in thyroid hormone levels, rather than further elevation, occurs during the 2-week interval after ablation therapy with 131I. Antithyroid drug therapy before radioiodine ablation may have little effect on the short-term biochemical course after 131I therapy for Graves disease. The homogeneity of our sample regarding age, diagnosis, and general health may prevent application of these findings to other populations without further study.
Adult↗
TSH receptor gene expression in retroocular fibroblasts.
RNA was isolated from fibroblasts from the retroocular area, from endomysial fibroblasts obtained from orbital lateral rectus muscle, and from abdominal skin fibroblasts. The RNA was reverse transcribed into cDNA which was then used as a template for PCR with primers encompassing a portion (nucleotides 989-1235) of the extra-cellular domain of the human TSH receptor (hTSH-R). A definite 247 BP product was detected from fibroblast RNA by ethidium bromide staining, and was confirmed by hybridization with labelled hTSH-R cDNA. The product had homology with the known TSH-R cDNA. These studies indicate that human fibroblasts can express hTSH-R, and they suggest that a cross reactive immunologic response between anti-hTSH-R and these fibroblast TSH receptors may play a role in the genesis of Graves' ophthalmopathy.
Base Sequence↗
Thyrotropin receptor antisera for the detection of immunoreactive protein species in retroocular fibroblasts obtained from patients with Graves' ophthalmopathy.
Autoimmunity against the TSH receptor (hTSH-R) is known to be the proximate cause of thyroidal activation in Graves' disease, but has not been definitively linked to extrathyroidal manifestations of this disorder, such as ophthalmopathy and pretibial myxedema. In an effort to increase our knowledge concerning mechanisms responsible for Graves' ophthalmopathy, we used antiserum directed against a highly immunogenic portion of the hTSH-R (amino acids 352-367; P1) to assess the presence of this receptor or immunologically related protein in cultured human retroocular fibroblasts obtained from patients with Graves' ophthalmopathy. Immunoenzymatic and immunofluorescent studies revealed specific staining of both cytoplasmic and cell membrane-associated protein in discrete vesicles. To further evaluate the immunoreactive species present in these cells, immunoblotting experiments were performed using hTSH-R-specific antisera (anti-P1) and sera obtained from patients with Graves' disease. Several protein bands were identified using both anti-P1 and Graves' disease patient sera, including species at mol wt of 95, 71, and 18 kilodaltons, the possible significance of which is discussed. The results support the hypothesis that immunity against the hTSH-R or related proteins contributes to the ophthalmopathy of Graves' disease.
Amino Acid Sequence↗
Expression polymerase chain reaction for the in vitro synthesis and epitope mapping of autoantigen. Application to the human thyrotropin receptor.
The clinical applicability of a newly described polymerase chain reaction directed protein expression system was assessed for the in vitro synthesis and partial epitope mapping of large radiolabeled human thyrotropin receptor (hTSH-R) protein segments. PCR amplification of targeted regions within the hTSH-R cDNA followed by in vitro transcription and translation permitted rapid synthesis of protein segments ranging in size from 18 to 62 kDa. Initial epitope mapping was directed at a 52 amino acid segment unique to the hTSH-R compared to otherwise homologous glycoprotein hormone receptors. Sera from Graves' disease patients known to have autoantibodies against the hTSH-R were used to immunoprecipitate two protein fragments differing only by the presence of the unique region in the larger fragment (E5) but not in the smaller fragment (E4). Dense precipitation bands were obtained using Graves' sera to immunoprecipitate E5 whereas little or no specific immunoprecipitation of E4 occurred. Normal sera gave only weak immunoprecipitation bands of E5. The technique provides significant advantages over conventional cloning methods and should have general applicability in the study of other protein targets of autoimmune disease.
Amino Acid Sequence↗
Graves' ophthalmopathy: current concepts regarding pathogenesis and management.
Referring to the hyperadrenergic theory on the pathogenesis of Graves' ophthalmopathy, a 1934 JAMA editorial (513) stated "the mechanism of exophthalmos is well understood though the knowledge would seem to be poorly disseminated." Several subsequent theories on pathogenesis, stated equally emphatically, have experienced a similar fate to that prompting this remark, but not before negatively impacting upon the management of patients suffering from this disorder. Thus, it is with an element of caution that we conclude that the sequence of events contributing to the pathogenesis of Graves' ophthalmopathy has become progressively more lucid, due to the assiduous efforts of many investigators in this field. Demonstration of an inextricable link between the eye and the thyroid in Graves' ophthalmopathy seems limited only by our ability to detect subtle involvement of one or the other of these two organs in exceptional cases, although not all authors share this viewpoint (514). The factors modulating the degree of expression of the thyroid component, such as concurrent lymphocytic thyroiditis or qualitative differences in TRAb seem more tangible than those affecting the clinical expression of eye involvement. Environmental factors such as smoking are associated, to a degree that matches or surpasses that known for genetic predisposition to this disorder. Local anatomy, including such factors as vulnerability to obstruction of venous drainage, must play a role as evidenced by asymmetric eye involvement and rapid relief of inflammatory changes after orbital decompression surgery. The transition from palliative to curative measures in Graves' ophthalmopathy will require further advances in our understanding of the putative shared thyroid-eye antigens, demonstration that these antigens are etiologically important and concomitant advances in antigen-specific immune therapy.
Eye Diseases↗
Life-threatening thyrotoxicosis. Thyroid storm.
Although important strides in recognition and therapy have significantly reduced the mortality in this disorder from the nearly 100% fatality rate noted by Lahey, survival is by no means guaranteed. More recent series have yielded fatality rates between 20% and 50%. Although some authors have attributed this improvement, in part, to a relaxation of the diagnostic criteria for thyroid storm, it more likely represents improvements in early recognition and the beneficial effects of the serial addition of antithyroid, corticosteroid, and antiadrenergic therapies to the treatment of this disorder. Thyroid storm is a dreaded, fortunately rare complication of a very common disorder. Most cases of thyroid storm occur following a precipitating event or intercurrent illness. Effective management is predicated on a prompt recognition of impending thyroid storm which is, in turn, dependent on a thorough knowledge of both the typical and atypical presentations of this disorder. An unwavering commitment to an aggressive, multifaceted therapeutic intervention as outlined herein is critical to the obtainment of a satisfactory outcome.
Humans↗
Graves' IgG recognizes linear epitopes in the human thyrotropin receptor.
Twenty-nine peptides covering the full extracellular domain of the human thyrotropin receptor have been synthesized and tested for reactivity with Graves' patients' and normal sera in ELISA. Two peptides, amino acids 331-350 and the second extracellular loop of the transmembrane segment, bound IgG-s from 5 and 4 of 10 Graves' disease patients' sera, respectively. Neither of these two peptides showed enhanced binding to normal IgG. There were no apparent differences between the Graves' disease and normal group with respect to the other 27 peptides. We conclude that peptide 331-350 and the second extracellular loop carry important linear epitopes which may contribute to the disease process in selected Graves' patients.
Enzyme-Linked Immunosorbent Assay↗
Reactive hypoglycemic coma due to insulin autoimmune syndrome: case report and literature review.
Recurrent episodes of postprandial hypoglycemic symptoms culminated in hypoglycemic coma in a hypertensive but otherwise healthy man while he was taking hydralazine. The patient was found to have an extreme elevation in the immunoreactive insulin level, leading to the discovery of insulin antibodies in the absence of prior exposure to exogenous insulin. Negative results of an anatomic study of the pancreas and an inability to reproduce hypoglycemia during a prolonged fast helped to exclude insulinoma. In contrast, symptomatic hypoglycemia developed in response to oral glucose loading and was associated with an elevation in total and free insulin as well as C-peptide levels. The patient was diagnosed with insulin autoimmune syndrome, which, although a common source of hypoglycemia in Japan, has been well documented in only 15 cases from other countries. HLA typing revealed the patient to be positive for groups Cw4 and DR4, a combination that has been preliminarily associated with insulin autoimmune syndrome in Japan. Unlike the majority of cases previously reported, this patient had no clinical or serologic evidence of an underlying autoimmune disorder and had not been exposed to drugs containing sulfhydryl groups. This case adds to the world literature on insulin autoimmune syndrome, lends support to a postulated HLA association, and documents the presence of insulin autoantibodies in the absence of another underlying autoimmune disorder.
Adult↗
Analysis of human TSH receptor gene and RNA transcripts in patients with thyroid disorders.
Human TSH receptor (hTSH-R) gene and RNA transcripts were analyzed by Southern and Northern blots in patients with various thyroid disorders, and in tissue cell lines. A 1.4 Kb cDNA encoding the extracellular human TSH-R domain was used as a probe. Southern analysis revealed two constant bands of 11.0 and 5.0 Kb (hTSH-R) in the thyroid and human white cell samples studied, regardless of the disease process. Northern analysis showed a predominant band at about 4.4 Kb in the thyroid tissues but not in non-thyroid tissue or cell lines tested. There were no gene rearrangements or abnormal transcripts in Graves' disease or multinodular goiter samples. In contrast, the labelled cDNA TSH-R probe did not bind to RNA isolated from 1 of 2 papillary cancer samples. A portion of the unique area of the h-TSH receptor (approximately nucleotides 1100-1230) was directly sequenced in thyroid glands from patients with Graves' disease, multinodular goiter, and differentiated thyroid cancer. No mutations or polymorphisms were identified in these samples, as compared to normal thyroid or control placenta, although further definition of sequence variation in other areas of the TSH receptor, as well as in more samples, needs to be performed. The present study indicates the normal patterns of DNA and RNA hybridization in a variety of thyroid tissues and disease states, and demonstrates that pathologic thyroid samples, with the possible exception of thyroid cancer, were not associated with specific nucleotide abnormalities in the unique area of the TSH receptor that was studied.
Adult↗
Nucleotide and amino acid homology between the human thyrotropin receptor and the HIV-1 Nef protein: identification and functional analysis.
A comparison of the nucleotide sequence of the human thyrotropin receptor (hTSH-R) with that of HIV-1 revealed 61% homology between a 161 base pair region encoding a unique portion of the hTSH-R and an immunogenic HIV-1 regulatory protein, nef. Amino acid analysis of this region shows 27% homology, including a segment in which 7/10 consecutive amino acids are identical. Sera from rabbits successfully immunized with a 16 amino acid portion of the hTSH-R (352-367, p1) was assessed for reactivity against a partially homologous nef peptide (nef-1) by ELISA, with a finding five-fold higher post-immunization values compared to pre-immune sera. The specificity of this response was verified with Western blot, using recombinant nef protein. An ELISA using nef-1 gave 64% higher values with sera from Graves' disease patients than with normal controls. This homology and immunologic cross-reactivity suggests an avenue through which a shared immune response against an HIV-1 related retrovirus could play a role in the pathogenesis of Graves' disease.
Amino Acid Sequence↗
Lymphocytes express thyrotropin receptor-specific mRNA as detected by the PCR technique.
Human lymphocytes are known to play a critical role in autoimmune diseases both by producing antibodies and by participating in lymphokine-cellular interactions. TSH, a classic pituitary hormone, may be secreted by human lymphocytes, and controversy has existed whether a specific, authentic TSH receptor also was present on the surface of these cells. The objective of our study was to identify TSH receptor transcripts after designing specific oligonucleotides that would recognize a unique putative TSH binding area of the thyroidal TSH receptor. The existence of TSH receptor transcripts was probed by employing these primers in a PCR reaction with cDNA derived from normal peripheral human lymphocytes and human thyroid tissue, as well as with cDNA from a medullary cancer cell line and rat liver. Human lymphocytes and thyroid tissue, but not medullary cancer cells or rat liver, demonstrated specific TSH receptor amplification product both by ethidium bromide staining and by Southern blot hybridization with labeled TSH receptor cDNA. The lymphocyte cDNA was partially sequenced and found to be identical to the thyroid-derived cDNA. These findings indicate that normal, nonactivated, human lymphocytes produce transcript for a TSH receptor that appears identical to that in thyroid tissue. Future studies should focus on the regulation of this transcript, as well as on the role TSH and TSH receptor may play in modulating local lymphokine activation of T and B cells, both in normal conditions and in autoimmune thyroid disease.
Base Sequence↗
[Arthritis following osteosynthesis of supra-/intercondylar femoral fractures].
Of 104 supra-/intercondylar femur fractures in adults (AO/ASIF Classification types A 1-3 and C 1-3), who were treated by open reduction and internal fixation from 1975 to 1985 59 fractures in 57 patients could be traced and reviewed clinically and radiographically after a mean follow-up period of 5 years and 7 months (range: 2-11 years). Among these, 47 knees/patients without preexisting knee-joint pathology were analyzed for the development of degenerative arthritis. The incidence for grade 2 and 3 changes in the femoropatellar compartment were 23% for supracondylar fractures (A type) and 62% for intercondylar lesions (C type). In the femorotibial compartment the incidence for supracondylar fractures was 38% and 23% for intercondylar fractures. In 93% of the patients the arthritic changes were radiographical findings, which did not cause relevant symptoms. The development of radiographical degenerative changes depends mainly on the type of the fracture; it is favoured by axial malalignment of more than 5 degrees of varus or valgus and local complications.
Adolescent↗