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W P Bryant

Publications and source records attributed to W P Bryant.

6 recordsLinked to original sources
PubMed1997

Binding of immunoglobulin G from patients with autoimmune thyroid disease to rat sodium-iodide symporter peptides: evidence for the iodide transporter as an autoantigen.

The recent cloning of the rat sodium-iodide symporter (rNIS) from FRTL-5 cells makes possible studies of the role of this thyroid-specific protein as an antigen in autoimmune diseases of the thyroid (AITD). We generated 21 synthetic peptides replicating the entire sequence of the extramembranous domains (ExMD) of rNIS. Each was synthesized by automated chemistry, purified by high-pressure liquid chromatography (HPLC), and characterized by mass spectroscopy. Immunoglobulins were purified using protein A from serum of 27 patients with Graves' disease (GD), 27 patients with autoimmune hypothyroidism (HT), and 20 normal controls. Binding of IgG from patients and controls to each of the rNIS peptides was measured by enzyme-linked immunosorbent assay (ELISA). Binding of patient IgG significantly greater than control was observed with six peptides: peptide 262-280 (representing ExMD 8 between transmembrane [TM] domains VII and VIII), peptide 437-444 (ExMD 11), peptides 468-487, 483-602, and 498-517 from ExMD 12, and peptides 560-579 from the proximal portion of the carboxyl terminus (ExMD 13). 63% of GD patients and 26% of HT patients immunoglobulin G (IgG) bound peptide 498-517 compared to zero controls. Similarly, 59% of GD were positive against peptide 468-487 versus zero controls. Peptide 262-280 bound IgG from 44% of GD patients, 15% of HT patients, and none of the controls. The remaining peptides showed little or no binding of patient IgG. These data indicate that patients with GD and HT possess antibodies that recognize rNIS significantly greater than do normal individuals, suggesting that the iodide transporter represents an important autoantigen in AITD. They further suggest that the incidence of the antibodies is higher in GD than HT, and that the antigenic epitopes involve ExMD 8, 11, 12, and 13.

Amino Acid Sequence↗
PubMed1995

Normal function in vivo of a homozygotic polymorphism in the human thyrotropin receptor.

We have demonstrated previously and association between a polymorphism in the human thyrotropin receptor gene and an increased prevalence of autoimmune thyroid disease in individuals bearing this polymorphic allele. The polymorphism involves the nucleotide base substitution of a cytosine for the wild-type adenine at the first position of codon 52 and is found generally in the heterozygotic state. Such change results in the substitution of a threonine for the wild-type proline at this position in the receptor protein sequence. The resulting protein would lack a beta turn (at position 52) in a potential loop conformation, and thus would have a significantly altered three-dimensional conformation. The biologic consequences of this conformational change in the receptor are unknown, but may involve altered function or immunogenicity. We report here two individuals with normal thyroid function who are homozygous for the thyrotropin receptor polymorphism, suggesting that the altered receptor is able to respond normally to thyrotropin with respect to the maintenance of the euthyroid state.

Adenine↗
PubMed1995

Identification of thyroid blocking antibodies and receptor epitopes in autoimmune hypothyroidism by affinity purification using synthetic TSH receptor peptides.

UNLABELLED: To examine the interaction of immunoglobulins from patients with newly diagnosed hypothyroidism with the TSH receptor (TSHr), we tested protein-A purified IgG in an ELISA assay with a series of peptides representing the entire extracellular domain (ECD) of human TSHr. Antibodies bound, on average, 4.1 peptides (range 0-16) per patient, and antibodies from 26 of 30 patients (86.6%) demonstrated binding to at least one peptide. Six of the 20-mer peptides (61, 151, 181, 301, 361, 376) were most frequently recognized. These were used to construct affinity columns and separate IgGs from 10 patients into bound and unbound fractions. All fractions were tested for their ability to stimulate and inhibit cAMP generation in FRTL-5 cells. Inhibitory IgGs were purified from 9 patients (90%), suggesting that the incidence of blocking antibodies (TBAb) in autoimmune hypothyroidism is higher than previously reported. 7 of 10 patients had antibodies that recognized peptide 361 further supporting the importance of this epitope in TBAb binding. Anti-microsomal and anti-thyroglobulin antibodies did not co-purify with inhibitory antibodies, and were always in the unbound fractions. We found no correlation between the pattern of antibody binding or bioactivity with clinical manifestations of hypothyroidism. CONCLUSIONS: (1) The majority of patients with autoimmune hypothyroidism have antibodies against the TSHr-ECD that recognized linear epitopes. Most have antibodies directed at more than one site and the pattern is quite heterogeneous. (2) Six sites (noted above) are most frequently recognized. (3) Inhibitory antibodies are distinct from anti-microsomal and anti-thyroglobulin antibodies.

Adolescent↗
PubMed1994

Delineation of amino acid residues within hTSHr 256-275 that participate in hormone binding.

The amino acid sequence 256-275 of the human thyrotropin (TSH) receptor extracellular domain has previously been shown to participate in a high affinity TSH binding site by a synthetic peptide approach as well as by site-directed mutagenesis. To further investigate this binding site, we synthesized a series of peptides with alanine substitutions for each residue in the native sequence. Peptides were also synthesized containing truncations or deletions of the native sequence. Each peptide was tested for its ability to inhibit 125I-bTSH binding to porcine thyroid membrane preparations, and the concentration at which 50% inhibition of binding occurred was determined (EC50). Alanine substitution at residues Tyr258, Cys262, Cys263, Phe265, Lys266, Asn267, Lys269, Lys270, and Arg272 all resulted in statistically significant decreases in activity when compared to the native sequence (p < 0.05). Alanine substitution of the remaining residues did not alter their activity. Comparison of this sequence with the corresponding sequences of the remaining glycoprotein hormone receptors (human lutropin and human follitropin receptors) reveals that these residues lie within one of the most highly conserved regions of the extracellular domain. We conclude that 9 specific amino acids within the sequence 256-275 of hTSHr (-Y--CC-FKN-KK-R--) participate in the interaction of the hTSHr-extracellular domain with TSH. This may represent a site in which the nonconserved residues are involved in the binding of the beta-subunit and the conserved residues are involved in the binding of the common alpha-subunit or a region of the beta-subunit that is common to all glycoprotein hormones.

Amino Acid Sequence↗
PubMed1994

Aqueous vasopressin infusion during chemotherapy in patients with diabetes insipidus.

BACKGROUND: Patients who have suprasellar germinomas in childhood often present with central diabetes insipidus (CDI). The authors investigated the use of aqueous vasopressin (AVP) by continuous infusion to control the fluid and electrolyte balance in germinoma patients with CDI during aggressive fluid hydration as a part of a preirradiation chemotherapy protocol. METHODS: Three patients with suprasellar germinomas and CDI were treated with four courses of preirradiation chemotherapy. Two patients were treated with a continuous AVP infusion at an initial rate of 0.08-0.10 mU/kg per hour during hydration. Fluid intake, urine output, body weight, urine specific gravity, and serum electrolyte concentrations were monitored closely, and the infusion rate was adjusted accordingly. RESULTS: Very low dose AVP infusion controlled fluid balance while allowing appropriate diuresis during chemotherapy. Fluid intake and output were markedly less in the AVP-treated patients (3.8 L/m2 per day) than in the untreated patient (20 L/m2 per day). CONCLUSIONS: The use of very low dose AVP infusion at an initial rate of 0.08-0.10 mU/kg per hour during hydration therapy allowed easily titratable control of fluid and electrolyte balance in the patients studied and avoided the complications associated with desmopressin acetate antidiuresis or withholding antidiuretic treatment altogether.

Adolescent↗