A monoclonal antibody against the nucleus reveals the presence of a common protein in the nuclear envelope, the perichromosomal region, and cytoplasmic vesicles.

A monoclonal antibody that recognizes antigenic determinants on the nucleus of cultured mammalian cells was isolated. Immunofluorescence studies using this antibody showed that the recognized antigen was present not only on the nucleus but also in cytoplasmic vesicles of interphase cells and in the perichromosomal region of mitotic cells. Premature chromosome condensation analysis showed that the reactive site for this monoclonal antibody could be detected in the perichromosomal region during the G2 and M phases, but not during the G1 and S phases. Finally, immunoblot analysis showed that this monoclonal antibody prepared against the nucleus recognized a protein of approximately 40 kD both in the cytoplasm and in the perichromosomal regions.

Epidermal growth factor receptors on cultured neoplastic human thyroid cells and effects of epidermal growth factor and thyroid-stimulating hormone on their growth.

Epidermal growth factor (EGF) receptors on primary-cultured human thyroid cells from 27 neoplasias (nine adenomas and 18 differentiated carcinomas) were analyzed and compared with those on the cultured nonneoplastic part of human thyroid cells. Total binding of 125I-EGF to the nonneoplastic part, adenoma, and carcinoma cells did not differ significantly. Scatchard analysis showed that the neoplastic human thyroid cells, like their adjacent nonneoplastic counterparts, consistently possessed EGF receptors with two components. In a paired study of five patients, the association constant of the carcinoma cells' high-affinity component (Ka1) was found to be significantly lower than that of adjacent nonneoplastic thyroid cells (P less than 0.05). Furthermore, a study of the cells from 18 carcinomas revealed that overall their Ka1s (4.15 +/- 0.82 x 10(9) M-1, mean +/- SEM) were significantly lower than those of adenoma cells (10.34 +/- 1.51 x 10(9) M-1, n = 9) and of nonneoplastic cells adjacent to them (8.32 +/- 0.84 x 10(9) M-1, n = 23). The difference in Ka1s for adenoma and nonneoplastic thyroid cells was not statistically significant. The number of receptor sites (Cmax) per cell was not significantly different in any of the three. Incorporation of [3H]thymidine (dThd) increased significantly in all kinds of thyroid cells examined following the addition of 10 nM EGF, and the paired study showed that the size of this increase was not significantly different in neoplastic and adjacent nonneoplastic cells. The addition of 300 microunits/ml of thyroid-stimulating hormone caused a significant increase in dThd incorporation by adenoma cells but not by carcinoma or nonneoplastic cells. Furthermore, combined treatment with EGF and thyroid-stimulating hormone additively promoted adenoma cell growth only. A close inverse relationship was observed between Ka1 and the stimulatory effect of EGF on the dThd uptake in both nonneoplastic thyroid cells and adenoma cells. Carcinoma cells also showed similar profiles, but Ka1 relative to dThd increases were much smaller than the other two.

Differences in the electrophysiological response to I- and the inhibitory anions SCN- and ClO-4, studied in FRTL-5 cells.

The electrophysiological properties of the Na+/I- symporter (NIS) were examined in a cloned rat thyroid cell line (FRTL-5) using the whole-cell patch-clamp technique. When the holding potential was between -40 mV and -80 mV, 1 mM NaI and NaSCN induced an immediate inward current which was greater with SCN- than with I-. The reversal potential for I- and SCN- induced membrane currents was +50 mV. This is close to the value of +55 mV calculated by the Nernst equation for Na+. These results are consistent with I- and SCN- translocation via the NIS that is energized by the electrochemical gradient of Na+ and coupled to the transport of two or more Na+. There was no change in the membrane current recording with ClO-4 indicating that ClO-4 was either not transported into the cell, or the translocation was electroneutral. ClO-4 addition, however, did reverse the inward currents induced by I- or SCN-. These effects of I-, SCN- and ClO-4 on membrane currents reflect endogenous NIS activity since the responses duplicated those seen in CHO cells transfected with NIS. There were additional currents elicited by SCN- in FRTL-5 cells under certain conditions. For example at holding potentials of 0 and +30 mV, 1 mM SCN- produced an increasingly greater outward current. This outward current was transient. In addition, when SCN- was washed off the cells a transient inward current was detected. Unlike SCN-, 1-10 mM I- had no observable effect on the membrane current at holding potentials of 0 and +30 mV. The results indicate FRTL-5 cells may have a specific SCN- translocation system in addition to the SCN- translocation by the I- porter. Differences demonstrated in current response may explain some of the complicated influx and efflux properties of I-, SCN- and ClO-4 in thyroid cells.

Prolonged effects of recombinant human interleukin-1 alpha on mouse thyroid function.

Rapid and prolonged effects of recombinant human interleukin-1 alpha (IL-1 alpha) on mouse thyroid were studied. After daily administration of 15 micrograms or 1 microgram IL-1 for 7 consecutive days, serum T4 concentrations rapidly fell to undetectable levels but returned to near control level after the cessation of IL-1. On the 31st day, 3 weeks after the drug cessation, a significant depression of serum T4 was observed again. In addition, the IL-1-treated mouse thyroid showed an in vitro unresponsiveness to TSH, with an increase of pituitary TSH (2.24-fold by 15 micrograms IL-1). To understand underlying mechanisms further, serial observations were performed. Thyroidal T4 contents increased initially, decreased to a low level at day 14, and returned to approximately the control level. IL-1 administration induced an increase in the basal thyroidal cAMP level for a prolonged period. Its response to TSH showed a gradual decline to a level approximately 30% of the control by the 31st day. Pituitary TSH contents on the 22nd and 31st days showed significant elevations. Slight decreases in thyroidal TSH binding and T4 contents also were seen concomitantly. These studies indicate that an administration of a large dose of IL-1 results in a dramatic decrease in serum T4 primarily through the inhibition of T4 release from the thyroid. The results also indicate the induction of a prolonged hypothyroid state due to the unresponsiveness to TSH via a postreceptor mechanism.

Preparation and characterization of monoclonal antithyrotropin receptor antibodies obtained from peripheral lymphocytes of hypothyroid patients with primary myxedema.

Anti-TSH receptor antibodies (TSH-R Ab), which have been detected in the serum of some patients with primary myxedema, are themselves considered to induce hypothyroidism. These are termed blocking-type TSH-R Ab (TSH-R BAb), because they inhibit adenylate cyclase stimulation by TSH on thyrocytes or nonthyroidal cells transfected with TSH-R complementary DNA. We prepared monoclonal TSH-R BAb and characterized them. Peripheral lymphocytes from three patients with primary hypothyroidism and potent TSH-R BAb were transformed by Epstein-Barr virus, and the culture supernatants were screened by TSH binding inhibitor immunoglobulin (TBII) assay. Twenty positive and 7 negative lymphocyte clones were obtained; their monoclonality was confirmed by Southern blot analysis, using an immunoglobulin (Ig) JH probe. These monoclonal antibodies were then tested for TSH-R BAb activity. TSH-R BAb activity ranged from 24.1-58.5% (normal range, < 24%) in all 20 TBII-positive clones and in 2 of 7 TBII-negative clones. An enzyme-linked immunosorbent assay showed that the Ig isotypes of these clones with TBII and/or TSH-R BAb activity were IgG in 8 and IgM in 14. Another enzyme-linked immunosorbent assay and Southern blot analysis of the light chains revealed that 13 clones had kappa-chains, whereas the light chains could not be determined in the other 9 clones. To summarize, 1) we obtained 22 clones that produced monoclonal TSH-R BAb, including 8 IgG-type clones. 2) The clones exhibited dominant usage of the kappa-chain. 3) Although all TBII clones had TSH-R BAb activity, their TBII and TSH-R BAb activities were not significantly correlated, and two TSH-R BAb clones did not show TBII activity.

Apparent genetic difference between hypothyroid patients with blocking-type thyrotropin receptor antibody and those without, as shown by restriction fragment length polymorphism analyses of HLA-DP loci.

HLA types in Japanese patients with primary hypothyroidism were analyzed to see whether those with blocking-type TSH receptor antibody (TSH-R BAb M) differed genetically from those with idiopathic myxedema (IM). HLA typings of -A, -B, -C, -DR, and -DQ (73 antigens) were performed serologically, and those of -D and -DP (29 antigens) were analyzed by the restriction fragment length polymorphism method. Thirty patients were studied with TSH-R BAb M, and 28 with IM. The data were analyzed and compared with our previous results from 88 Graves' patients, 46 Hashimoto patients, and 186 control subjects. Overall, 192 patients with 4 autoimmune thyroid disorders showed a decrease in -Aw19 and an increase in -DQw4 (corrected P < 0.05) and significant associations of -Aw33, -Bw46, -Cw3, -DRw8, -DR9, and -DQw3. In TSH-R BAb M patients, increases in -B35, -Bw60, and -Dw8 and decreases in -DR4 and -DPw2 were seen, whereas IM patients showed increased -DPw2, -Bw61, and -Dw23. In comparisons between TSH-R BAb M and IM, the difference in -DPw2 was highly significant. HLA-B35 differed significantly in these 2 types of hypothyroidism. In conclusion, TSH-R BAb M patients have decreased frequency of -DPw2 and are genetically similar to Graves' disease, whereas IM patients are characterized by high frequency of -DPw2 and are genetically similar to Hashimoto's thyroiditis.

A monoclonal antibody to alpha-human atrial natriuretic polypeptide.

A monoclonal antibody to alpha-human atrial natriuretic polypeptide (alpha-hANP), KY-ANP-I, has been produced by fusion of a nonproducing mouse myeloma cell line, X63-Ag8.653, with spleen cells from BALB/c mice immunized with synthetic alpha-hANP conjugated to bovine thyroglobulin using the carbodiimide coupling procedure. Hybridomas were screened for antibody production by radioimmunoassay using culture media and 125I-alpha-hANP. They were cloned by the limiting dilution technique, expanded in culture, and injected intraperitoneally into BALB/c mice. The obtained antibody belonged to the immunoglobulin G1 subclass. Analysis by a Scatchard plot revealed a high affinity for alpha-hANP, with an association constant of 3.1 x 10(10) M-1. With this monoclonal antibody, a specific radioimmunoassay for alpha-hANP has been established. The antibody in mouse ascites was available for radioimmunoassay at a final dilution of 1:10(6). Values of IC10 and IC50 in this radioimmunoassay were 3 and 30 fmol/tube, respectively. The radioimmunoassay showed a cross-reactivity of 0.9% with alpha-rat ANP. alpha-hANP-(8-22) and alpha-ANP-(1-6) exhibited less cross-reactivity than alpha-rat ANP on a molar basis. There was no cross-reaction with alpha-ANP-(17-28). Thus, the recognized epitope must be located in the N-terminal half of the ring structure of alpha-hANP including Met12 residue. This radioimmunoassay could detect gamma-hANP and beta-hANP as well as alpha-hANP. The monoclonal antibody was also useful for immunohistochemical studies. ANP-positive cells were finely stained in the human atrium using the avidin-biotin-peroxidase complex technique.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartate-474 in the first exoplasmic loop of the thyrotropin receptor is crucial for receptor activation.

Asp-474 in the first exoplasmic loop of the thyrotropin receptor (TSHR), which is conserved among all glycoprotein hormone receptors, was mutated to Glu which is similarly charged but is longer by one methylene group and expressed in Cos-7 cells. Cells expressing this mutant receptor showed markedly impaired TSH- and TSAb (thyroid stimulating antibody)-stimulated cAMP responses with no effect on TSH binding affinity when compared with cells expressing a similar number of wild-type receptors. These results suggest the importance of Asp-474 in TSHR in receptor activation as demonstrated for LHR (lutropin receptor), but this, unlike LHR, is not due to the electrostatic interaction of this Asp residue with the alpha-subunit Lys-91 of the hormone.

Analysis of Fas system in pulmonary injury of graft-versus-host disease after rat intestinal transplantation.

The lung is one of the primary targets of acute graft-versus-host disease (GVHD), which is the principal complication that occurs after allogeneic intestinal transplantation. The purpose of this study is to investigate the involvement of Fas/Fas ligand system in pulmonary injury after rat semi-allogeneic intestinal transplantation. The lungs were serially harvested from LEW x BN F1(LBNF1) recipients of either LEW heterotopic intestinal allografts or LBNF1 isografts, on days 1, 3, 5, 9, and 13 posttransplant. In light microscopy, pulmonary injury became apparent on day 13 in the allogeneic combination, showing a thickening of the alveolar septa. The incidence of apoptosis, examined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) biotin nick end-labeling, was observed to increase steadily in the alveolar cells accompanied by a progression of GVHD. In an immunohistochemical study, Fas was constitutively expressed in the lung, although Fas ligand was expressed most extensively on day 9. The immunoreactivity of both Fas and Fas ligand were observed in alveolar cells, in addition to leukocytes. An analysis by reverse transcription polymerase chain reaction also revealed that the expression of Fas mRNA was constitutive without any significant change, although that of Fas ligand mRNA increased substantially and peaked on day 9, which was significant compared to the isogeneic combination. In conclusion, transcriptionally up-regulated Fas ligand and increased number of apoptosis suggests that the Fas system may play a role in the pathophysiology of GVHD-induced pulmonary injury.

Improved hepatic microcirculation by human soluble urinary thrombomodulin in the xeno-perfused porcine liver.

PURPOSE: Both the protein C/thrombomodulin system and the heparin/anti-thrombin III system are major physiological anticoagulant systems, which may also play a major role in preserving the hepatic microcirculation in xenogeneic liver transplantation. To compensate for the functional incompatibilities of the porcine thrombomodulin (TM)-cofactor activity beyond species for human thrombin, soluble human TM protein was tested in xenogeneic perfusion of the porcine liver. MATERIALS AND METHODS: The livers were harvested from adult female pigs and perfused through the portal vein (PV) and hepatic artery (HA) for 2 hr, with fresh human blood in group 1 (n=5), fresh porcine blood (10 units/ml) in group 2 (n=5), and fresh human blood with TM (50,000 units/1.5 l) in group 3 (n=5). The tissue PO2 level, tissue blood flow, PV and HA pressures were all continuously monitored. Circulating perfusate and liver tissue samples were periodically obtained for blood chemistry and histologic analyses. RESULTS: The activated protein C (aPC) level was significantly elevated in the TM-treated group 3 (47.5%+/-3.5% at preperfusion and 51%+/-2.8% after 120 min of perfusion) in comparison to group 1 (32.3%+/-7.2% and 35.3+/-12.0%). The hepatocyte enzyme release of aspartate aminotransferase (AST) was suppressed significantly more in group 3 (238.2+/-107 IU/l), than in group 1 (672.3+/-160 IU/l) at 2 hr after reperfusion. In group 3, the tissue PO2 levels and tissue blood flow also remained significantly higher throughout the perfusion. The platelet counts in the perfusate remained significantly higher in group 3 (37.1% to 74.3% of the preperfusion level) than in group 1 (4.4% to 14.7%), after 0 to 80 min of perfusion. According to the histologic findings, the degree of interlobular hemorrhaging and congestion decreased remarkably more in group 3 than in group 1. CONCLUSION: These findings thus indicated that soluble thrombomodulin protein extracted from human urine remarkably improved hepatic microcirculation in the xenoperfused porcine liver. The thrombomodulin/protein C system might, thus, play an important role in restoring the physiological anticoagulant system in the xenoperfused porcine liver.

Establishment of 8-azaguanine-resistant mutants from rat thyroid cell line FRTL-5.

8-Azaguanine (AG)-resistant mutant clones were isolated from FRTL-5 rat thyroid cells, by treating them with ethylmethane sulfonate for 48 h and then, after the recovery period, by selecting them in AG-containing medium. Isolated mutants were further selected in 6-thioguanine-containing medium, and two mutant clones were finally obtained (AG1 and AG2). The mutants retained several thyroid stimulating hormone (TSH) responsiveneses following acquisition of AG resistance: they responded to bovine TSH (bTSH), by producing cyclic 3',5'-adenosine monophosphate (cAMP) and thyroglobulin, and taking up iodine. However, TSH-responsive profiles were slightly different from each other in several points. They were killed in hypoxanthine-aminopterin-thymidine (HAT)-selective medium and applied to cell hybridization study with human thyrocyte from a Graves' patient: the hybrid turned out to produce both rat and human thyroglobulin even 12 months after the hybridization.

A thyroid cancer specific monoclonal antibody which recognizes cryptic epitope(s) of human thyroglobulin.

A monoclonal antibody (TCM-9) specific for human thyroid cancer but not for Graves' disease, adenoma or normal thyroid tissue was shown to recognize a 300 K protein but not to bind to native or mature human thyroglobulin (Tg). In this study, we investigated further the antigen recognized by TCM-9. When purified Tg was treated with periodate, dithiothreitol (DTT) and sodium dodecyl sulfate (SDS), immunoblotting of treated Tg with TCM-9 revealed an apparent enhancement of the staining only with DTT-treated Tg. Furthermore, the DTT-treated Tg was shown to bind dose-dependently to plates coated with TCM-9. We performed SDS-PAGE and immunoblot analyses on the immune complex obtained from a homogenate of thyroid cancer tissue which was preincubated with an excess of anti-Tg monoclonal antibody. When the protein was autoradiographed with [125I]TCM-9, a definite band was observed. The results indicate that TCM-9 is likely to be directed against a masked epitope of thyroglobulin which can be exposed after treatment with a reducing agent.

A novel activating mutation in the thyrotropin receptor gene in an autonomously functioning thyroid nodule developed by a Japanese patient.

OBJECTIVE: A number of activating mutations of the thyrotropin receptor (TSHR) have been found in autonomously functioning thyroid nodules (AFTNs) in European patients. We aimed to study TSHR mutation in AFTNs in Japanese patients because no TSHR activating mutation has been found by previous incomplete studies. DESIGN: A typical AFTN developed in a 69-year-old Japanese woman was studied. METHODS: The entire exon 10 of the TSHR cDNA was sequenced. Functional studies were done by site-directed mutagenesis and transfection of a mutant construct into COS-7 cells. RESULTS: We identified a novel heterozygous TSHR gene mutation, Leu512-->Arg (L512R; CTG-->CCG), from the AFTN. The mutation was not detected in the adjacent normal thyroid tissue. COS-7 cells transfected with L512R mutant TSHR expression vector exhibited a 3.3-fold increase in basal cAMP level compared with that of cells transfected with wild-type TSHR DNA, confirming that the mutation was the direct cause of the AFTN. TSHR activating mutations involving the third transmembrane helix reported to date are S505R/N and V509A as well as L512R. An in vitro site-directed mutagenesis study encompassing residues 505-513 revealed that mutations involving residues other than these three did not show constitutive activation. CONCLUSION: This is the first TSHR activating mutation found in a Japanese patient, although true prevalence of TSHR activating mutations in AFTNs developed in Japanese patients remains to be elucidated. In addition, functional studies suggested that amino acid residues in the third transmembrane helix maintaining inactive conformation of the TSHR seem to be located on the same surface of the alpha-helix, possibly making interhelical bonds with another helix.

Presence of heterogeneous thyroid-stimulating antibodies in sera from individual Graves' patients as shown by synthesized thyrotropin receptor peptide application: evidence showing two independent epitopes and a possible recognition of two epitopic regions by one antibody molecule.

To define the epitope(s) of stimulating thyrotropin receptor antibody (TSH-R-Sab), we synthesized 19 oligopeptides covering almost all amino acids of the extracellular domain of the human TSH-R and studied these effects on the inhibition of one TSH-R-Sab activity. Four of the 19 peptides encompassing residues 31-50 (P31-20), 91-119 (P91-29), 287-304 (P287-18) and 354-367 (P354-14) were found to show significant TSH-R-Sab inhibition and to have similar effects on the other three Graves' immunoglobulins. When these peptides were applied in combination with P354-14 only P287-18 revealed additional effects but the other two combinations did not. Furthermore, sequential addition of these peptide pairs confirmed the additional effects of P287-18 and P354-14. Sequential peptide-affinity gel studies were then performed. Most of the TSH-R-Sab activity in the unabsorbed fraction from P287-18 gel was absorbed to a subsequent P354-14 gel and the eluted fraction from P287-18 mostly remained unabsorbed by the P354-14 gel. On the other hand, most of the unabsorbed fraction from P91-29 gel remained unabsorbed even by the subsequent P354-14 gel. When a P354-14 affinity gel-purified TSH-R-Sab immunoglobulin was labeled and evaluated for its binding to FRTL-5 cells, additions of original immunoglobulin, P354-14 and P91-29 resulted in significant inhibition of the binding but P287-18 did not affect either. From these results, it was concluded that most of the individual Graves' immunoglobulins contain at least two heterogeneous moieties with TSH-R-Sab activity, one of which binds P354-14 and the other binds P287-18. Further, P354-14 and P91-29 were indicated to bind the same molecule of TSH-R-Sab immunoglobulin.

Thyroid-stimulating antibodies in sera from patients with Graves' disease are heterogeneous in epitope recognition.

Two synthetic peptides, P354-14 (amino acid nos. 354 to 367) and P338-16 (nos. 338 to 353), corresponding to the partial amino acid sequences of the hTSH receptor structure were studied for their ability to bind specifically serum IgGs from patients with Graves' disease and to inhibit thyroid stimulating TSH receptor antibody (TSH-R SAb) activity. IgG binding was measured by an ELISA using sera from 102 Graves', 20 Hashimoto patients, and 9 normal subjects. Both peptides showed significantly increased IgG binding of Graves' sera compared with those of Hashimoto and normal sera. There was a significant correlation (r = 0.529) between the amount of IgG bound by the two peptides, but neither of these values correlated well with their TSH-R SAb activity nor thyrotropin-binding inhibitor TSH receptor antibody (TSH-R IAb) activity. TSH-R SAb inhibiting effects of these peptides were then analysed by measuring TSH-R SAb activity after incubation with the peptides. Among eight Graves' IgGs tested the TSH-R SAb activity of three was inhibited by both peptides, two were inhibited only by P354-14 and three were not affected by either. These TSH-R SAb inhibiting effects were dose-dependent and reproducible. To confirm these findings, a peptide-sepharose gel affinity absorption study was performed. Eleven Graves' IgGs were applied to both peptide gels and the TSH-R SAb activity of the unabsorbed fraction was measured. The TSH-R SAb activity of five IgGs was strongly absorbed only by P354-14 and five others were absorbed by both peptides to an almost similar extent.(ABSTRACT TRUNCATED AT 250 WORDS)

Expansion of helper T-cell recognition in mice immunized with a synthetic peptide corresponding to the C-terminal thyrotropin receptor-specific insert.

We previously reported that immunization with a synthetic peptide of human thyrotropin receptor (TSH-R) expanded humoral autoimmunity to TSH-R (Sugawa H, Ueda Y, Ueda M, Kosugi S, Ichiyama S, Mori T. Immunization with the 'immunogenic Peptide' of TSH receptor induces oligoclonal antibodies with various biological activities. Peptide 1998;19:1303-7.). In the present study, we examined this phenomenon at the T-cell level. Balb/c mice were immunized with a synthetic peptide corresponding to the C-terminal-specific insert of human TSH-R. Spleen cells were collected and subjected to antigen-specific ELISPOT assay. The number of interleukin 4-secreting cells specific to P354-367 increased within 3 weeks. Cells responding to the other peptides increased 7 weeks after immunization. This phenomenon was not observed in mice immunized with bovine serum albumin alone. During immunization, numbers of interferon-gamma-secreting lymphocytes were not changed significantly. These results indicated that immunization with C-terminal TSH-R-specific insert peptide causes fluctuation in the type 2 helper T-cell population but not type 1 Th cells against the TSH-R, and the recognition repertoire of type 2 helper T cell was expanded by the peptide.

Immunization with the "immunogenic peptide" of TSH receptor induces oligoclonal antibodies with various biological activities.

Balb/c mice were immunized with a synthetic peptide (P354-14) corresponding to "the immunogenic peptide" of human thyrotropin receptor (hTSH-R). Through screening for binding to the peptide, we obtained several monoclonal antibodies with various biological activities: thyroid stimulation (SAb), inhibition of TSH stimulation (BAb) and no significant effect on cAMP production. One of the stimulatory clones was further studied. This clone enhanced cAMP production in Cos-7 cells transformed with the truncated TSH-R cDNA deleting the immunogenic peptide. These results indicated that the immunogenic peptide of the TSH-R induces oligoclonal anti-TSH-R antibodies, although the region is not essential for the functional epitope.

Immunoglobulin G that interferes with thyroid-stimulating antibody measurements can be eliminated specifically by incubation with synthetic peptides corresponding to partial sequences of the human thyrotropin receptor.

Two IgG preparations out of more than 100 tested, distinct from the typical Graves' disease IgG, were shown specifically to enhance the cAMP production of FRTL-5 cells by the addition of a synthetic peptide, P-218, corresponding to the partial amino acid sequence from No. 354 to 367 of the h thyroid-stimulating hormone (TSH) receptor. IgG obtained from a patient with Graves' disease revealed a serial alteration of the enhancement; negative in July, 1989, potent in January, 1991, and weak in September 1991. During this time there was no remarkable change in the patient's serum protein components or TSH receptor antibody activities. A peptide with a completely reverse sequence of P-218 showed little effect, and P-218 in combination with bTSH or forskolin did not affect cAMP production by these ligands, and did not alter the inhibitory activity of thyroid-stimulation-blocking antibody. High concentrations of P-218 resulted in reduction of such enhancing effects of cAMP by thyroid-stimulating antibody. P-218 affinity chromatography showed almost complete absorption and recovery of thyroid-stimulating antibody and P-218 reactivity. In the 15 synthesized peptides with proximal sequences of P-218 (from 338 to 378), regions thought to be involved with the enhancement were defined as follows: 354-367 (P-218) is a critical unit; 354-357 and 364-367 are considered to be the essential sites; several amino acid extensions on both N- and C-terminal sides of P-218 show additional enhancement. In conclusion, evidence was shown to indicate the presence of IgG that interferes with thyroid-stimulating antibody measurements.

Incidence and characteristics of thyroid dysfunction following interferon therapy in patients with chronic hepatitis C.

Thyroid functions were analyzed before, during and after interferon (IFN) therapy in patients with chronic hepatitis C. According to the results of routine thyroid function tests and measurements of the levels of anti-thyroid autoantibody prior to the therapy, patients were divided into 2 groups; Group A (19 patients) had at least one abnormal finding related to the thyroid, and Group B (40 patients) did not show any abnormality. Five patients (26%) in Group A and 4 (10%) in Group B showed thyroid dysfunctions which were very clearly reflected by thyrotropin (TSH) measurements. Interestingly, the time of peak TSH elevation in Group A (mean +/- SD, 4.3 +/- 0.8 months) was significantly earlier than that in Group B (6.8 +/- 0.8). Most patients in Group B were diagnosed as having destructive thyroiditis. These findings may suggest that the pathogenesis of IFN-induced thyroid dysfunction consists not only of exacerbation of pre-existing thyroid autoimmunity but also of de novo destructive changes even in the intact thyroid before IFN therapy.