Polymorphisms of interleukin (IL)-4 receptor alpha and signal transducer and activator of transcription-6 (Stat6) are associated with increased IL-4Ralpha-Stat6 signalling in lymphocytes and elevated serum IgE in patients with Graves' disease.

Activated interleukin (IL)-4Ralpha stimulates production of IgE through signal transducer and activator of transcription-6 (Stat6) activation in lymphocytes. Genetic studies have shown an association between polymorphisms in the genes encoding IL-4Ralpha and Stat6 and elevated serum IgE in patients with atopic disease. Some authors, including us, have reported an association of Graves' disease and elevated serum IgE. To analyse the relationship between IL-4Ralpha and Stat6 polymorphisms and elevated serum IgE in patients with Graves' disease, 169 patients with Graves' disease were studied. We investigated whether these polymorphisms affect IL-4Ralpha-Stat6 signalling in cultured human lymphocytes. A high frequency of both the Ile50 polymorphism in IL-4Ralpha and 13GT repeat variants of the Stat6 gene was observed in patients with Graves' disease and elevated serum IgE (Ile50 allele; P < 0.05, 13GT allele; P < 0.01 versus controls) but not in subjects with normal IgE. Cultured human lymphocytes with the Ile50 IL-4Ralpha polymorphism and the 13GT repeat variant of Stat6 showed increased IL-4 (and/or IL-13)-induced Stat6 activation (2.7-fold; P < 0.05 and 2.2-fold; P < 0.05, respectively). These findings suggest that polymorphisms in the IL-4Ralpha and Stat6 genes play an important role in elevation of serum IgE through increased Stat6 action in patients with Graves' disease.

Rifampin-induced hypothyroidism.

Three euthyroid patients with Hashimoto's thyroiditis developed hypothyroidism after the administration of rifampin. We studied 67 patients with tuberculosis. All of them were treated with rifampin. Of the 67 patients, 42 had negative tests for anti-thyroid antibodies (ATA) and 25 had positive tests for ATA. The diagnosis of Hashimoto's thyroiditis was made on the basis of positive tests for ATA. After the administration of rifampin, TSH levels were not significantly altered in all of the former 42 ATA-negative patients and in 22 of the latter 25 ATA-positives, but TSH levels increased in the other three (Patients 1, 2 and 3) of the latter 25 ATA-positives. Three euthyroid Hashimoto's patients (Patients 1, 2 and 3) developed hypothyroidism after the administration of rifampin. This rifampin-induced hypothyroidism resolved in each, once rifampin was discontinued. A) Patient 1: a 62-yr-old man with lymphoma had pulmonary tuberculosis. After the administration of rifampin, serum TSH increased to 170 mU/l; B) Patient 2: a peritoneal-biopsy specimen containing Langhans' giant cells led to a diagnosis of tuberculous peritonitis in a 66-yr-old woman with ascites. After the administration of rifampin, TSH increased to 12.4 mU/l; C) Patient 3: a 56-yr-old woman with a liver abscess and lymphadenopathy underwent lymph-node biopsy that showed Mycobacterium tuberculosis with caseating granulomas. After the administration of rifampin, TSH increased to 21.3 mU/l. After its administration, Patients 1, 2 and 3 developed hypothyroidism, and received T4. When rifampin was discontinued, the hypothyroidism resolved. After the course of rifampin-therapy had been completed, T4 was discontinued. At-risk patients who receive rifampin may become hypothyroid.

Low-dose metformin improves hyperglycaemia related to myotonic dystrophy.

BACKGROUND: One of the clinical features of myotonic dystrophy is insulin resistance with non-obese diabetes mellitus (DM). Recently, the mechanism of insulin resistance in patients with myotonic dystrophy was revealed. The optimal treatment of DM with myotonic dystrophy has not been established. We report the effect of metformin in a patient with myotonic dystrophy without obesity. CASE REPORT: A 58-year-old woman (BMI = 22.1 kg/m2) with myotonic dystrophy and DM was followed at our clinic. She had been treated with glimepiride for DM for the last 6 months, without achieving good control (HbA(1c) 9.3%). She was admitted with congestive heart failure and cholecystitis. She was treated with diuretics, antibiotics and insulin. As her blood glucose fell, we discontinued insulin and started glimepiride, but her glycaemic control had worsened. We started metformin instead of glimepiride. After 4 weeks of metformin, HbA(1c) was decreased to 7.4%, while HOMA-IR during glimepiride treatment was 4.9, and 3.7 with metformin. Three months later, HbA(1c) was maintained (7.5%). CONCLUSION: It is important to choose the optimal treatment for DM in myotonic dystrophy, because the patients have hyperinsulinemia caused by specific mechanism and could not reduce the insulin resistance. Metformin improved hyperglycemia through increased insulin-independent glucose uptake in peripheral muscle. We believe metformin is the optimal agent for these patients.

Multiple fragments of human TG are capable of inducing oral tolerance to whole human TG.

Oral tolerance is the mechanism by which the immune system remains unresponsive to orally administered soluble antigens. Mice immunized with human TG (hTG), resulting in the induction of experimental autoimmune thyroiditis (EAT), provide an ideal in vivo system in which to examine oral tolerance to hTG. In the present study, we characterize epitopes of hTG that are capable of inducing oral tolerance. hTG is a large homodimeric protein, 660 Kd. The limited proteolysis of hTG using trypsin (TR) generates several smaller fragments of hTG ranging in size from 29 Kd to 145 Kd. Using hTG fragments h1TR (residues 1-521), h4bisTR (residues 2513-2713), h6TR (residues 522-1626), and h7TR (residues 1627-2512), prepared from both iodine rich and iodine poor hTG, we investigated the ability of these fragments to induce oral tolerance. The oral administration of iodine rich h6TR or h7TR suppresses hTG specific immune responses in a manner similar to whole hTG. In contrast, the oral administration of iodine rich h1TR or h4bisTR exacerbates hTG specific immune responses. Unlike iodine rich h1TR or h4bisTR, the oral administration of iodine poor h1TR or h4bisTR fails to augment hTG specific immune responses. In fact, h4bisTR suppresses hTG specific immune responses. These results indicate that hTG contains multiple epitopes that differentially affect oral tolerization. Tolerogenic epitopes reside within fragments h6TR and h7TR. The removal of iodine, and presumably hormone, from h4bisTR converts an immunogenic epitope to a tolerogenic epitope.

Relation of three polymorphisms of the CTLA-4 gene in patients with Graves' disease.

Graves' disease is an autoimmune disease believed to be caused by a combination of environmental and genetic factors. One of the candidate genes is CTLA-4, a negative regulator of T cell activation. Three polymorphisms of the gene have been described, in the promoter at position -318, at position 49 in exon 1, and an (AT)n repeat within the 3'-untranslated region of exon 4. Many studies describe the association between a polymorphism of the CTLA-4 gene and autoimmune disease. To investigate the association of these CTLA-4 gene polymorphisms with each other, we analyzed the combined frequencies of each polymorphism and calculated the disequilibrium coefficients. We studied DNA samples from 120 Graves' disease (GD) patients and 80 healthy donors (NC). The exon 1 position 49 A/G polymorphism and promoter polymorphism at position -318, were typed using a PCR-restriction fragment length polymorphism method (PCR-RFLP). The polymorphic (AT)n repeat in exon 4 was determined by PCR amplification of genomic DNA, resolution of the amplified products on sequencing gels, and detection by autoradiography. There was a significant difference between GD and NC patients and occurrence of the polymorphism in exon 1 and exon 3, but not for the polymorphism in the promoter region. Furthermore, we found that the genotype with both the G allele in exon 1 and the 106 bp allele of the AT repeat in exon 4 occurred with much higher frequency in GD than NC (p<0.01), and that these polymorphisms are in linkage disequilibrium with each other. These results support the concept that CTLA-4 plays a critical role in the autoimmune process in GD, and that GD depends on multiple genetic susceptibility factors. Because the exon 1 and exon 4 polymorphisms are in strong linkage disequilibrium. It is not possible at this time to determine their unique relation to CTLA-4 function. Studies relating each polymorphism to CTLA4 function are required to determine whether one, or both, polymorphism(s) promote autoimmune disease.

Characterization of the T lymphocyte subsets and lymphoid populations involved in the induction of low-dose oral tolerance to human thyroglobulin.

Using mice deficient in CD8alpha, TCRdelta, CD4, or CD120a, as well as adoptive transfer experiments in wild-type and RAG-1-deficient mice, we characterized the T lymphocyte subsets and lymphoid populations involved in the induction of low-dose oral tolerance to human thyroglobulin (hTg). The oral administration of hTg, but not the intraperitoneal (ip) administration of hTg, generates lymphocytes that can transfer tolerance. Purified CD8alpha+ lymphocytes successfully transfer tolerance, while the depletion of CD8alpha or TCRdelta lymphocytes prevents the transfer of tolerance. Oral tolerance can be induced in CD4-deficient mice and RAG-1-deficient mice reconstituted with cells from CD120a-deficient mice, but not in CD8alpha-, TCRdelta, or CD120a-deficient mice. These findings indicate that CD8alpha and TCRdelta T lymphocytes are necessary for the oral induction and transfer of tolerance to hTg. Additionally, functional Peyer's patches are necessary for the induction of low-dose oral tolerance to hTg.

Remission and recurrence of hyperthyroid Graves' disease during and after methimazole treatment when assessed by IgE and interleukin 13.

We analyzed the relationship between serum IgE concentrations and the remission or recurrence of Graves' disease. One hundred seven patients with Graves' disease were treated with methimazole (MMI). Serum IgE concentration greater than 170 IU/ml was found in 41 of 107 untreated patients (38.3%). However, the presence of TSH-binding inhibiting immunoglobulin or thyroid-stimulating antibody did not correlate with the IgE concentrations. Remission was found in 20 of 41 patients with elevated IgE concentrations (48.8%) after 18 months of MMI treatment, as opposed to 53 of 66 patients with normal concentrations (80.3%) (P = 0.0014). MMI treatment was discontinued in 73 patients who were followed for 26-48 months. The recurrence of Graves' disease was found in 13 patients, whereas the remaining 60 were still in remission. The rate of long-standing remission was lower in patients with elevated than normal IgE concentration (34.1% vs. 69.7%, P = 0.0007). We also analyzed serum levels of interleukin (IL)-13. Although IL-13 was not detected in all patients, the detection rate was higher in patients without remission and in those with recurrence than in those with long-standing remission (47.1%, 38.5%, and 13.3%, respectively; P = 0.0012). More patients with elevated IgE were positive for allergic diseases and for family history of allergic diseases in their first-degree relatives. We conclude that the elevation of IgE and the higher detection rate of IL-13 are associated with both remission and recurrence of Graves' disease.

Developmental origin of the rat adenohypophysis prior to the formation of Rathke's pouch.

In amphibians, it has already been shown that the adenohypophysis originates from the anterior neural ridge. During the migration and morphogenesis of this organ, the anterior neural ridge transiently forms a Rathke's pouch-like structure by attaching itself to the rostral tip of the foregut, and finally gives rise to the adenohypophysis by detaching from the foregut and becoming connected to the infundibulum of the hypothalamus. In order to identify the origin of the adenohypophyseal cells in mammalian embryos prior to the formation of Rathke's pouch (RP), we labeled the rostral end of the neural plate and the adjacent area focally with DiI at the open neurula stage (9.5 dpc). After a 48-hours culture of the whole embryos, strongly labeled cells were detected in the RP only when DiI was applied to a small area situated just anterior to the rostral end of the neural plate. By explanting the labeled RP for a further 7 days, we confirmed immunohistochemically that the labeled cells developed into the secretory cells of the adenohypophysis. The developmental origin of the adenohypophysis is identified for the first time in the early mammalian embryo before the formation of RP.

CTLA-4 gene polymorphism at position 49 in exon 1 reduces the inhibitory function of CTLA-4 and contributes to the pathogenesis of Graves' disease.

Activation of T cells requires at least two signals transduced by the Ag-specific TCR and a costimulatory ligand such as CD28. CTLA-4, expressed on activated T cells, binds to B7 present on APCs and functions as a negative regulator of T cell activation. Our laboratory previously reported the association of Graves' disease (GD) with a specific CTLA-4 gene polymorphism. In theory, reduced expression or function of CTLA-4 might augment autoimmunity. In the present study, we categorized autoimmune thyroid disease patients and normal controls (NC) by genotyping a CTLA-4 exon 1 polymorphism and investigated the function of CTLA-4 in all subjects. PBMCs and DNA were prepared from GD (n = 45), Hashimoto's thyroiditis (HT) (n = 18), and NC (n = 43). There were more GD patients with the G/G or A/G alleles (82.2% vs 65.1% in NC), and significantly fewer patients with the A/A allele (17.8% vs 34.9% in NC). In the presence of soluble blocking anti-human CTLA-4 mAb, T cell proliferation following incubation with allogeneic EBV-transformed B cells was augmented in a dose-dependent manner. Augmentation induced by CTLA-4 mAb was similar in GD and NC (GD, HT, NC = 156%, 164%, 175%, respectively). We related CTLA-4 polymorphism to mAb augmentation of T cell proliferation in each subgroup (GD, HT, NC). Although PBMC from individuals with the G/G alleles showed 132% augmentation, those with the A/A alleles showed 193% augmentation (p = 0.019). CTLA-4 polymorphism affects the inhibitory function of CTLA-4. The G allele is associated with reduced control of T cell proliferation and thus contributes to the pathogenesis of GD and presumably of other autoimmune diseases.

Production of a recombinant newt growth hormone and its application for the development of a radioimmunoassay.

Complementary DNA (cDNA) encoding newt (Cynops pyrrhogaster) growth hormone (nGH) was cloned from a cDNA library constructed from mRNAs of newt pituitary glands and was expressed in Escherichia coli. Based on Northern blot analysis using the cDNA as a probe, the nGH mRNA was estimated to be 940 bases in length. The recombinant nGH (nGHr) had a molecular mass of 22 kDa as determined by SDS-PAGE and possessed considerable bioactivity as determined in a Xenopus cartilage assay. Using the nGHr, we produced a polyclonal antibody against nGHr. Western blot analysis of newt anterior pituitary gland homogenates revealed that this antiserum specifically detected a single 22-kDa band, and histological studies of newt pituitary gland sections showed that the cells that reacted immunologically by the anti-nGHr antiserum corresponded to those stained by an antiserum against rat GH. A radioimmunoassay (RIA) that is specific and sensitive for nGH was developed, employing the antiserum thus produced. The sensitivity of the RIA was 57 +/- 7 pg/100 microl assay buffer. Interassay and intraassay coefficients of variation were 1.22 and 2.70%, respectively. Serial dilutions of plasma and pituitary homogenate of C. pyrrhogaster yielded dose-response curves that were parallel to the standard curve. Plasma from hypophysectomized newts showed no cross-reactivity. Moreover, displacement curves obtained using pituitary homogenates of the sword-tailed newt (C. ensicauda) and the crested newt (Triturus carnifex) were also parallel to the standard curve. Mammalian and frog GHs and prolactins (PRLs), as well as newt PRL, showed no inhibition of binding, even at relatively high doses, in this RIA. The RIA was used to measure GH released from newt pituitaries in vitro. Enhancement of GH release by 10(-7) M thyrotropin-releasing hormone was observed in cultures of newt pituitaries.

Clinical usefulness of TSAb assay with high polyethylene glycol concentrations.

We previously demonstrated the stimulatory effect of polyethylene glycol (PEG) on thyroid-stimulating antibody (TSAb)-IgG-stimulated cAMP production (thyroid stimulating (TS) index) in porcine thyroid cell (PTC) assay. In the present study the clinical usefulness of the practical method using high PEG concentrations was examined. TS activity using PEG 22.5% precipitated fraction (PF) was significantly higher compared to standard TSAb activity using 12.5% PF from TSAb-positive serum, but the maximum TS activity was observed with PEG 12.5% PF + 4% PEG or PEG 22.5% PF + 2% PEG. In all cases of untreated Graves' patients, TSAb activity determined by PEG 22.5% PF was higher compared to standard TSAb activity using PEG 12. 5% PF from test serum, but the highest TSAb activity was observed by PEG 12.5% PF + 4% PEG without increased cAMP production to normal serum. TSAb was positive in 85% (40/47), 98% (46/47) and 100% (47/47) of untreated Graves' patients by the method of PEG 12.5% PF, PEG 22.5% PF and PEG 12.5% + 4% PEG, respectively. Increased TSAb activity by PEG 12.5% PF + 4% PEG method was also observed even if the standard TSAb activity using PEG 12.5% PF method was negative in the euthyroid states of Graves' patients during antithyroid drug therapy. The stimulatory effect of PEG on TS activity was not found in other thyroidal diseases [thyroiditis chronica (with high serum TSH), thyroid stimulation-blocking antibody (TSBAb)-positive sera (with low serum TSH), adenomatous goiter, subacute thyroiditis, and thyroid cancer]. The stimulatory effect of 5% PEG on TS activity produced directly by small amounts of Graves' serum (50 microl) was also found, although the sensitivity was lower than with PEG-precipitated IgG from 0.2 ml serum. The clinical usefulness of the sensitive TSAb assay using PEG-precipitated IgG or direct serum assay in the presence of high PEG concentrations was demonstrated.

Polyethylene glycol augments thyroid cAMP responses by fragments from protease-digested TSAb or TSBAb-IgG.

In the previous reports, we have demonstrated (1) that polyethylene glycol (PEG)(5%) augmented TSAb (thyroid stimulating antibody)-stimulated cAMP responses of porcine thyroid cells, and (2) that fragments from papain-digested TSBAb (thyroid stimulation blocking antibody) could stimulate thyroid cAMP synthesis. Thus, we studied the effect of 5% PEG on cAMP responses stimulated by the protease-digested TSAb- or TSBAb-fragments. Stimulatory effect of 5% PEG on cAMP production by Fab fragment (Mr 50 KDa) and the retarded fraction (Mr 20 KDa) from the gel-filtration on Sephadex G-100 using papain-digested TSAb-IgG unbound to Protein A-Sepharose was observed. Similar stimulatory effect of 5% PEG on the second fraction (Fc with trace amounts of Fab) in the gel-filtration on Sephadex G-100 using papain digested TSAb-IgG bound to Protein A-Sepharose was observed. Stimulatory effect of PEG on the second fraction was derived from Fab fragment. PEG (5%) also showed stimulatory effect on cAMP production by F(ab')2 fragment (Mr 100 KDa) from the gel-filtration on Sephadex G-100 using pepsin-digested TSAb-IgG unbound to Protein A-Sepharose. PEG (5%) augmented cAMP responses by both Fab and the retarded fractions from the gel-filtration using papain-digested TSBAb-IgG unbound to Protein A when these fractions could stimulate cAMP synthesis. In conclusion, PEG (5%) augments cAMP responses stimulated by F(ab')2, Fab and the smaller molecular components (Mr 20 KDa) separated from protease-digested TSAb-IgG. PEG also augments cAMP responses stimulated by Fab and the smaller molecular components with thyroid stimulating activity separated from papain-digested TSBAb-IgG.

Augmentation of thyroid-stimulating antibody-stimulated cyclic adenosine monophosphate response by polyethylene glycol, polyvinyl alcohol, and dextran; highly sensitive porcine thyroid cell thyroid-stimulating antibody assay.

Previously, we reported that 5% polyethylene glycol (PEG) (6000) augmented thyroid-stimulating antibody (TSAb)-stimulated cyclic adenosine monophosphate (cAMP) production in porcine thyroid cell (PTC) assay. This augmentation by PEG was specific to TSAb-stimulation. In this study we examined the effects of nonionic hydrophilic polymers such as PEG, polyvinyl alcohol (PVA), and dextran (DEX) on TSAb-stimulated cAMP production. We demonstrated that graded doses of PEG, PVA, and DEX augmented TSAb-stimulated cAMP productions; the prominent augmentations were observed with 5% PEG (20,000), 5% PEG (6000), 6% PEG (4000), 10% PVA, 14% DEX T-250, and 14% DEX T-70. PVA did not augment thyrotropin (TSH)-stimulated cAMP synthesis. Five percent PEG (20,000), 14% DEX T-250, and 14% DEX T-70 augmented TSH-stimulated cAMP synthesis very slightly. PEG, PVA, and DEX had no effects on the cAMP synthesis stimulated by GTPgammaS, forskolin, or pituitary adenylate cyclase activating polypeptide (PACAP), which stimulated adenylate cyclase. We also demonstrated that PEG, PVA, and DEX augmented the cAMP responses stimulated by small amounts (50 microL) of sera from Graves' patients; small amounts (50 microL) of sera could be used instead of purified immunoglobulin G (IgG). This may simplify the TSAb assay. We developed a highly sensitive simplified TSAb assay. PEG weakly augmented TSAb binding to isolated TSH receptor (thyrotropin-binding inhibitor immunoglobulin [TBII] increased slightly). The mechanisms of the augmentations of TSAb-stimulated cAMP productions by PEG, PVA, and DEX is not simply explained by increased binding of TSAb to the receptors. Some factors that enhance TSAb action at the receptor site are suggested.

Mechanism of the augmentative effect of high polyethylene glycol (PEG) concentrations on the thyroid stimulating activity in TSAb-IgG using a porcine thyroid cell assay.

We previously demonstrated that high polyethylene glycol (PEG) concentrations (5% PEG) significantly augmented cAMP production in response to TSAb-IgG using the porcine thyroid cell (PTC) assay. The mechanism of the stimulatory effect of 5% PEG on cAMP production was examined by a two-step incubation with PTC. TSAb-IgG was preincubated with or without addition of 5% PEG in the PTC assay for 2.5 hr (1st incubation) and separated PTC was re-incubated with fresh Hank's buffer for 5 hr (2nd incubation). cAMP production in the 1st incubation medium by co-incubation of TSAb-IgG and 5% PEG for 2.5 hr was significantly increased (3.3-fold) compared to that without 5% PEG. When the cAMP content in PTC and the incubation medium were compared in the same volume of incubation medium after co-incubation of TSAb-IgG and 5% PEG for 2.5 hr, cAMP contents in PTC were about 7-fold higher than that in the incubation medium, and this ratio did not change in the incubation medium of TSAb-IgG without 5% PEG. Similar increases in cAMP contents in PTC (6.6-fold) compared to the incubation medium were also observed with bTSH, although there was no augmentative effect of 5% PEG on cAMP production by bTSH in either the incubation medium or PTC. When PTC, which had been preincubated with normal-IgG and 5% PEG in the 1st incubation, was re-incubated with TSAb-IgG in the 2nd incubation medium, cAMP production by TSAb-IgG was not stimulated by 5% PEG. The augmentative effect of 5% PEG on cAMP production by TSAb-IgG was observed whenever 5% PEG and TSAb-IgG were co-incubated in either the 1st or 2nd incubation. However, no stimulatory effect of 5% PEG on bTSH was observed. These results suggested the stimulatory effect of 5% PEG on TSAb-IgG-stimulated cAMP production may be due to the increase of binding or incorporation of TSAb-IgG into the membranes of PTC compared to TSH.

Developmental studies for identification of the inhibitory center of melanotropes in the toad, Bufo japonicus.

Two series of experiments were performed to identify the inhibitory center of the melanotropes in the intermediate lobe of hypophysis of the toad, Bufo japonicus. First, developmental changes in the distribution of dopaminergic neurons were examined from hatching stage to postmetamorphosis using an antiserum against dopamine synthase (tyrosine hydroxylase, TH). In the postmetamorphic toads, TH-positive cell bodies were localized in three clusters. One was the preoptic recess organ (PRO) in the prechiasmatic area, the other two were the paraventricular organ (PVO) and infundibular nucleus (IN) in the postchiasmatic area. Each of them exhibited different ontogenetic changes. During larval development, TH-positive cell bodies were first detected in the PVO and IN at a premetamorphic stage. The number of immunoreactive cells increased rapidly in both loci as metamorphosis proceeded, although the two nuclei showed different growth profiles. By contrast, in the PRO, a very small number of immunoreactive cells were observed before the onset of the prometamorphic period. Although the number of immunoreactive neurons increased as metamorphosis progressed, early neurons were confined to the caudal area of the PRO (cPRO), the rostral area of the PRO (rPRO) being devoid of TH-positive cells. Immunoreactive TH neurons appeared in the rPRO for the first time at the end of metamorphic climax. This timing coincided well with the development of TH-positive nerve endings in the pars intermedia (PI) and median eminence. In the second series of experiments, the embryonic primordium of the PRO was surgically extirpated from open neurulae to examine the effects of PRO-ectomy. In 75% of the operated animals, background adaptation was not observed, their dermal melanophores remained permanently dispersed even on the white background. Dopaminergic neurons in the rPRO and the immunoreactive nerve endings in the PI and median eminence were scarcely observed in these animals. It was concluded that the present data strongly support the hypothesis that rPRO is the center of white-background adaptation.

Increase of thyroid stimulating activity in Graves' immunoglobulin-G by high polyethylene glycol concentrations using porcine thyroid cell assay.

Cyclic adenosine monophosphate (cAMP) production during a 5-hour incubation using porcine thyroid cells (PTC) was stimulated significantly more by polyethylene glycol (PEG) 22.5% precipitated fractions (ppt frs) than by PEG 12.5% ppt frs from almost all Graves' sera. However, the thyrotropin (TSH) binding inhibition (TBI) activities of the PEG 12.5% and 22.5% ppt frs using porcine thyroid membranes were similar, and did not change in the 5-hour incubation. When the PEG 12.5% ppt fr from Graves' serum and the PEG 22.5% ppt fr from normal human serum (NHS) were coincubated, cAMP production was also stimulated as much as by the PEG 22.5% ppt fr from Graves' serum. When purified thyroid stimulating antibody (TSAb)-immunoglobulin G (IgG) and the PEG 22.5% ppt fr from NHS were coincubated, increased cAMP production was also observed, whereas bovine thyrotropin (bTSH) did not produce this effect. When purified TSAb-IgG and PEG solutions were coincubated, maximum increases in cAMP production (approximately 10-fold) with 5% PEG were found, whereas no increase was observed using bTSH. The stimulatory effect of high PEG concentrations on thyroid stimulating activity was observed by TSAb-IgG in salt-free or salt-containing medium (<0.15 mol/L NaCl concentration) but not by either TSAb-IgG conjugated to protein A-sepharose 4B or the inactivated TSAb-IgG by the treatment of 70 degrees C for 10 minutes. No stimulatory action by PEG was found with the thyroid stimulating substances such as GTPgammaS, forskolin, or pituitary adenylate-cyclase activating polypeptide (PACAP). The increased thyroid stimulating activity of Graves (IgG) at high PEG concentrations suggests the existence of some factors influencing the ability of TSAb to stimulate thyroid cells, although the exact mechanism remains to be clarified.

Separation method of IgG fragments using protein L.

Protein L (IgG kappa-chain-binding bacterial protein) showed a precipitate line(pseudo-immuno-reaction) with IgG and F(ab')2 fragment, but did not show any line with the Fab fragment, the Fc fragment and free kappa-chains in the micro-Ouchterlony method. The IgG and Fab fraction obtained from pa-pain-digested IgG (from the sera of patients with chronic thyroiditis), followed by Protein A-Sepharose, were separated by Protein L-Sepharose affinity chromatography. The unbound fraction (UF) consisted of IgG(lambda) or Fab(lambda) and the bound fraction (BF) consisted of IgG(kappa) or Fab(kappa) were obtained. Anti-thyroglobulin and anti-thyroid peroxidase antibody activities were found equally in both the UF and the BF. When Fab(kappa) was reduced with dithiothreitol (DTT), the Fd fragment in the UF could be separated from the free kappa-chain and the unreduced Fab(kappa) in the BF with a Protein L-Sepharose column. A separation method of human IgG fragments such as free kappa-chain, combined forms of kappa-chain [Fab or F(ab')2], and the Fd region, using Protein L, is described.

Demonstration of thyroid stimulating activity within H chain fragments of TSAb-IgG by protease digestion and reduction.

OBJECTIVE: Whether or not the distribution of biologically active fragments in TSAb-IgG molecules parallels antigen-binding activity in other anti-thyroidal antibodies was examined. DESIGN: Both the thyroid stimulating (TS) activity (cAMP production in thyroid cells) and TSH binding inhibition (TBI) activity (determined by TSH receptor assay) were examined by measuring the reduction in TSAb-IgG followed by gel-filtration on Sephadex G-100. Two forms of IgG [IgG(kappa) and IgG(lambda)] were separated from TSAb-IgG by column chromatography using Protein L-Sepharose (specifically binds to kappa chain). The IgG(kappa) was reduced with dithiothreitol (DTT) and the unbound fraction (UF) (the free heavy (H) chain) and the bound fraction (BF) (the free kappa chain and the non-reduced IgG(kappa)) were separated using Protein L-Sepharose. The Fab fragment was separated by Protein A-Sepharose after papain hydrolysis of TSAb-IgG, then separated into two Fab(kappa) and Fab(lambda) forms by Protein L-Sepharose. The Fd fragment (or fragment containing Fd) was prepared from Fab(kappa) by DTT reduction followed by Protein L column. RESULTS: The free H chain fraction showed TS and TBI activity, but neither anti-thyroglobulin (Tg) nor anti-thyroid peroxidase (TPO) antibody activities. The free light (L) chain was not biologically active. Similar TS and TBI activities were found not only in IgG(kappa) and IgG(lambda) but also in the Fab(kappa) and Fab(lambda) fractions. Fd fragment that was not contaminated with free kappa chain had TS and weak TBI activities. CONCLUSIONS: The thyroid stimulating activity in 5 TSAb-IgG samples was found in IgG(kappa), IgG(lambda), Fab(kappa), Fab(lambda), H chain and Fd separated by papain digestion and reduction. These results showed that TSAb was polyclonal and that the Fd fragment was important as the biologically active site.

Calmodulin purified from human and porcine thyroids inhibits thyrotropin binding to porcine thyroid cells.

A thyrotropin (TSH) binding inhibiting protein (TBIP) that inhibits TSH binding to the TSH receptor, as determined by the TSH receptor assay, was purified from human and porcine thyroid. The soluble fraction (100,000 x g supernatant of Graves' thyroid homogenate) was precipitated with ammonium sulfate between 1.75 to 2.5 mol/L. TBIP was eluted by 0.5 mol/L sodium chloride (NaCl) containing 20 mmol/L Tris buffer, pH 7.5 from a Q-sepharose column. The unbound fraction from concanavalin A (Con A) and blue-sepharose was gel-filtered using sephadex G-100, and finally purified by Resource Q column chromatography. Purified TBIP was confirmed as a single protein band of 17 kDa. The TBI activity in the purified TBIP was significantly decreased by either etnylene glycol tetraacetate (EGTA) (1 mmol/L) or antibody to calmodulin (CaM) in the TSH receptor assay. The TBIP was confirmed immunologically as CaM by the Ouchterlony method using antibody for CaM. These findings demonstrated that the TBIP purified from human and porcine thyroids was, in fact, CaM. We examined the effects of TBIP purified from human thyroid on bovine TSH (bTSH) or thyroid stimulating antibody (TSAb)-stimulated cyclic adenosine monophosphate (cAMP) production in porcine thyroid cells (PTC). TBIP itself did not increase basal levels of cAMP production, but inhibited bTSH (100 mU/L)-stimulated cAMP production. However, TBIP did not inhibit cAMP production stimulated by TSAb-IgG and various thyroid stimulators (GTPgammaS, forskolin and pituitary adenylate cyclase-activating polypeptide [PACAP, 27 and 38 amino acids]). Authentic CaM purified from bovine brain behaved in a manner similar to that of TBIP. These data showed that CaM differentially affects thyroid stimulation by TSH and TSAb in intact thyroid cell experiments.