Anti-triiodothyronine antibodies in patients with rheumatoid arthritis associated with Hashimoto's thyroiditis

Anti-triiodothyronine antibody was found in a case of rheumatoid arthritis associated with Hashimoto's thyroiditis. The patient was a 40 year-old woman who had complained of polyarthralgia, joint-swelling and stiffness for seven years. She had a rheumatoid nodule and showed a positive RA test. Radiographic changes of hands and wrists showed osteoporosis, erosions and narrowing of joint space. Nonsteroidal anti-inflammatory drugs had been used for seven years. The diagnosis of Hashimoto's thyroiditis had been made by open biopsy of the thyroid gland seven years before. Serum T4, TSH, TBG, free T4, free T3 and r-T3 were all normal. On the other hand, serum T3 level was almost unmeasurable by radioimmunoassay. Binding of 125I-T3 to the patient's serum was studied by using polyethylene glycol (PEG) and column chromatography. By using the PEG method, the binding of 125I-T3 to the patient's serum was tenfold compared to control serum. Sephadex G-25 column chromatography (0.9 X 1.5 cm) of 125I-T3 with the patient's serum in the presence of 0.1% ANS showed an early radioactive peak, while control serum did not show an early peak. In the next experiments, the patient's serum was labelled with 125I-T3, mixed with human anti-IgG, IgM, IgA, lambda, kappa, incubated at 4 degrees C for 20 hours and centrifuged for 20 min. Strong binding to the anti-IgG and anti-lambda was detected. The presence of this abnormal T3-binding globulin in the patient's serum may have produced an undetectable T3 level.     

A case report of Hashimoto's disease with anti-thyroxine autoantibody (author's transl)]

In one case of untreated Hashimoto's disease, serum thyroxine (T4) value by radioimmunoassay (RIA) was significantly lower than that by competitive protein binding analysis (CPBA). The discrepancy was found to be due to the presence of antithyroxine autoantibody in the serum. This phenomenon was considered to be of practical importance in interpreting the T4 value by RIA in cases with autoimmune thyroid diseases. The patient was 59-year-old woman with a 30-year history of goiter. A diagnosis of Hashimoto's thyroiditis had been established by open biopsy of the thyroid ten years ago. The patient was judged to be euthyroid on the basis of clinical and laboratory evaluation (mean serum T4 by CPBA (Tetrasorb and Tetratab kit), 5.0 mug/100 ml; serum T3, 165 ng/100 ml; T3 resin uptake, 31.8%; and serum TSH, 2.0 muU/ml). TBG binding capacity was 24 mug/100 ml. Anti-thyroglobulin antibodies (anti-Tg), once positive ten years before, was negative at this time. But the mean T4 in the serum measured by T4 RIA and RIA-Mat T4 kit were 1.7 and 2.9 mug/100 ml, respectively. Recovery of the T4 added to the patient's serum evaluated by RIA-Mat T4 kit, was 71.2%, although the recovery using a control serum was 108%. Binding of 125I-T4 to the serum or fractions of the serum was studied by using polyethylene glycol (PEG) method, column chromatography, and double antibody precipitation. The results were as follows: 1) The binding of 125I-T4 to the patient's serum was detected by using RIA kit system without addition of anti-T4 serum. 2) On Sephadex G-200 chromatography of 125I-T4 incubated with the serum or the rabbit anti-T4 antibody in the presence of ANS, an early radioactive peak was observed by using the patient's serum as in the case of the anti-T4 antibody. When the serum after thermal inactivation of TBG, was incubated with 125I-T4, and was applied to the Sephadex G-200 column, a radioactive peak was observed in the area where 7S fraction was detected by protein peak. 3) The binding of 125I-T4 to the patient's IgG was 9.0% by using double antibody method when the binding to a control IgG was 0.5%. 4) The binding of 125I-T4 to IgG fractions was also proved by PEG method. 5) The binding of 125I-T4 was competitively inhibited by the addition of unlabeled T4. The affinity constant was 1.9 X 10(8) L/mol and its binding capacity was 0.8 mug/100 ml serum. From these data this T4 binding IgG was considered to be anti-T4 autoantibody. The cross reaction with T3 was approximately 8.3%. MIT and DIT did not displace labeled T4 when tested in amounts varying from 0.1 to 100 ng/assay. By using the paper electrophoresis, the binding of 125IT4 to the serum or IgG was not detectable. Therefore this method was considered unsuitable for detecting such anti-T4 antibody. As we couldn't find any significant binding of 125I-T4 to sera in 37 other patients with Hashimoto's disease by using the PEG method, the incidence of this phenomenon was considered to be low...     

Circulating immunoglobulin E (IgE) antibodies to L-thyroxine in a euthyroid patient with multinodular goiter and allergic rhinitis

A 30-yr-old woman with allergic rhinitis and multinodular goiter developed atopic manifestations on different desiccated thyroid extract treatment. Urticaria was observed when the patient was on L-T4 treatment; no atopy was experienced during L-T3 regimen. Serum total immunoglobulin E (IgE) concentration was 390 +/- 7kU/liter (mean +/- SD) prior to any treatment and rose to 850 +/- 7.5 kU/liter when the patient developed urticaria, but declined to baseline figures while she was on L-T3. Intracutaneous testing was positive for desiccated pork thyroid powder, L-T4 and D-T4, but negative for L-T3, DIT and L-tyrosine. Immunoradioligand analyses of mixtures of patient's serum or precipitated immunoglobulin fraction and of 125I-T4 or of 125I-T3 revealed binding of radiolabeled thyroxine to the patient's serum IgE, in turn bound to anti-human-IgE serum covalently coupled to paper discs. This binding was completely inhibited by the preincubation of immunoglobulin fraction with excess unlabeled L-T4 and D-T4, but not with excess nonradioactive L-T3, thus proving the specificity of the binding. Preadsorption experiments performed with desiccated pork thyroid powder solution mixed with the patient's immunoglobulin fraction suggested binding of some unknown component(s) of desiccated thyroid which was apparently not thyroglobulin. This study provides evidence of IgE antibodies to L-T4 cross reacting with D-T4 and capable of binding 125I-T4 in serum. It also suggests a model for the detection of circulating IgE antibodies to thyroid hormones.     

Triiodothyronine(T3) autoantibodies in a woman with nonthyroidal disorder: a study on preparation of serum IgG fraction employing protein A column chromatography

A 48-year-old non-goitrous woman, who had undergone cardiac surgery for mitral stenosis under the extracorporeal circulation, showed high levels of serum T3 and free T3 in a recent follow-up study, employing antibody coated-bead RIA for T3 and -Amerlex M particle RIA for free T3. However, other thyroid function tests (T4, free T4, TSH and TBG) were normal. We suspected that thyroid hormone autoantibodies (THAA) in her serum interfered with T3 and free T3 analyses. The presence of THAA was demonstrated by the use of various procedures as follows. Firstly, the patient's serum was directly incubated with 125I-T3 or -T4 analog which did not bind to TBG, followed by B/F separation with polyethyleneglycol, counting the precipitates. Secondly, after the serum was treated with an acid-charcoal solution to remove circulating thyroid hormone, the measurement of THAA was made as stated above. Normal sera were used as controls. Both the non- and acid-charcoal-treated sera showed much higher percentages of 125I-T3 analog precipitation as compared with controls. In the case of 125I-T4 analog, there was no difference between them. In the third study, the presence of IgG antibodies that bound T3 but not T4 was investigated. The IgG fraction of the patient's serum was separated employing a Protein A-Sepharose CL-4B column chromatography. Then, the prepared IgG fraction was purified by a technique of gel filtration chromatography (Sephacryl S 200). Non-purified and purified-IgG fractions both revealed higher binding percentages of 125I-T3 analog than the control IgG fraction and non-IgG fraction of the patient. Furthermore, a good dose response was observed between the binding percentage of 125I-T3 analog and each dose of the patient's serum or IgG fraction. From these observations, it was clarified that this woman had anti-T3 IgG autoantibodies using a Protein A column chromatography with confirmation of gel filtration chromatography.     

Thyroid disease with monoclonal (immunoglobulin G lambda) antibody to triiodothyronine and thyroxine

A man with previous Graves' disease spontaneously developed hypothyroidism. He became euthyroid with T4 therapy, but developed inappropriately elevated serum levels of T3 and, to a lesser extent, T4. Gel filtration analysis (Sephadex G-150) of serum trace-labeled with [125I]T3 revealed binding to a high molecular weight fraction, distinct from normal T3-binding proteins. This abnormal activity cochromatographed with serum immunoglobulin G (IgG) by DEAE-cellulose chromatography and gel filtration, and was retained by the F(ab)2 fragment of IgG, indicating its true antibody nature. By isoelectric focussing, there was restricted heterogeneity of the [125I]T3-antibody complex (pI 9.0-9.1), and the antibody was identified as an IgG (lambda) monoclonal Ig by immune precipitation. Antigenic cross-reactivity with T4 was demonstrated by inhibition of hapten binding. The affinity of the antibody for T3 was high (Ka = 0.9 x 10(9) liter mol-1), and the T3 binding capacity of the antibody in serum was estimated as 1132 ng/dl, equivalent to 1.39 mg T3-specific IgG/liter (0.014% of the total serum IgG). This binding capacity was similar to the serum T3 values (1100-1300 ng/dl) at which transition from hypothyroid to euthyroid states was observed, as judged by clinical examination and measurement of serum TSH levels.     

Studies on thyroid hormone autoantibody (THAA) in 2 cases of Graves' disease with spuriously high free thyroxine values

Two patients with Graves' disease treated with methimazole (MMI) showed a discrepancy between serum free T4 (FT4) values and other hormone values (especially total T4) which was due to the presence of potent binding activity to labelled T4 analogue (125I-aT4) in their serum. This activity was demonstrated to be in immunoglobulin G (IgG) with kappa light chain isotype in both patients. The binding of 125I-aT4 to their serum was inhibited by unlabelled T4 in a dose-dependent manner. Autoantibodies had almost identical binding affinity to T4 and aT4, although they precipitated more radioactivity when 125I-aT4 was used. The binding of IgG purified from patients' sera to labelled T4 or aT4 was not greater than the corresponding sera, suggesting that the thyroxine binding proteins did not interfere with the assay. Since the specific radioactivity of 125I-aT4 is almost 10 times higher than that of 125I-T4, autoantibodies can precipitate almost 10 times more radioactivity in the FT4 assay than the total T4 assay, thus leading to the spuriously high FT4 values and large discrepancy between FT4 and TT4 values.     

Studies on thyroid hormone autoantibody in two euthyroid cases with spuriously high value of serum free triiodothyronine]

Spuriously high value of serum free triiodothyronine (FT3: Amerlex free T3 kit, Amersham, UK.) was noted accidentally on routine laboratory examination of two clinically euthyroid patients (case 1: FT3; 18.5 pg/ml, FT4; 1.1 ng/dl, T3; 103 ng/dl, T4; 8.2 micrograms/dl, TSH; 1.74 microU/ml, case 2: FT3; 8.5 pg/ml, FT4; 1.1 ng/dl, T3; 137 ng/dl, T4; 8.9 micrograms/dl, TSH; 1.45 microU/ml), the former with poorly controlled diabetes (FBG 253 mg/dl, HbA1c 12.1%) and the latter with essential hypertension (184/108 mmHg). Although the hypertensive patient showed mild diffuse goiter, there was no evidence that the patients had autoimmune thyroid diseases because anti-thyroglobulin antibody tests measured by radioimmunoassay and MCHA, TGHA or TBII were all negative. Their serum levels of TBG were within the normal range. Further studies revealed that both patients' sera had unusual binding activity to labelled polyaminocarboxy T3 (125I-aT3) but not labelled T3 (125I-T3). Furthermore, this binding protein was precipitated by goat anti-human immunoglobulin G (IgG). The IgG purified from both patients' sera also showed strong binding activity to 125I-aT3, which was inhibited by unlabelled T3 in a dose dependent manner. In conclusion, we found anti-T3 antibody in two clinically euthyroid patients with no apparent evidence of complicating autoimmune thyroid diseases. The stronger binding activity to polyaminocarboxy T3 rather than T3 may lead to the spuriously high value of serum FT3. The mechanisms of the production of such autoantibodies in our cases should be further investigated.     

Coexistence of autoantibody to human thyrotropin (TSH) and autoanti-idiotypic antibody to antihuman TSH antibody in a case with simple goiter

A 70-yr-old woman with simple goiter showed normal serum levels of T4, T3, free T4, TSH receptor antibody (TRAb) and increased TBG. Discrepancy in serum hTSH level was observed by different assay methods. Coexistence of both autoantibodies for hTSH and for anti-hTSH antibody were demonstrated by the reaction of the patient's antibody with both 125I-hTSH and 125I-anti-hTSH (monoclonal antibody; mAb). These two autoantibodies belong to the polyclonal immunoglobulin G (IgG). The autoantibody for hTSH recognized only beta-subunit of hTSH. Neither stimulating type of TRAb in Graves' disease nor blocking type of TRAb in primary hypothyroidism interfered with the binding of the patient's antibody to 125I-hTSH or 125I-anti-hTSH. Anti-idiotypic antibody (anti-ID antibody) for anti-hTSH antibody was purified by anti-hTSH antibody affinity chromatography. The binding reaction of 125I-anti-hTSH (mAb) with this anti-ID antibody could be inhibited by the unlabeled hTSH. This anti-ID antibody might represent the internal image of the nonbiological active site of TSH molecule, because of absence of thyroid stimulating activity. Goiter in this patient may have occurred by the unbound TSH with IgG (free TSH) and the bound TSH with IgG, because TSH levels in both the whole serum and the IgG free serum (the unbound TSH with IgG) were decreased significantly by T4 treatment. Coexistence of these antibodies may participate in the autoimmune mechanism of an idiotype-anti-idiotype network.     

Further evidence for the presence of thyroxine binding globulin-like protein in human breast adipose tissue. Deiodination of thyroxine and triiodothyronine by the microsomal fraction

The object of the study was to obtain information on how adipose tissue of normal subjects processes thyroid hormones. L-125I-thyroxine(125I-T4) is bound by the cytosol fraction of normal human female breast adipose tissue with high affinity. Computer analysis of the binding data revealed the presence of two saturable systems with Kd values of 3.9 and 29.1 nM and binding capacities of 1.7 and 8.7 pmol/mg of cytosol protein, respectively; a third binding system was non-saturable. The binding of the iodothyronines to the cytosol fraction indicated that L-triiodothyronine (T3) possessed two-fold higher affinity as compared to L-thyroxine (T4) whereas other iodothyronines had relative affinities of less than 3%. Binding of 125I-T4 was optimal at pH 7.0 and was sensitive to the action of pronase and neuraminidase. Affinity chromatography of the cytosol fraction using T3-epoxy-sepharose 6B and con A-sepharose 4B, yielded a 125I-T4 binding component that was purified 150-fold. Isoelectric focusing of the purified fraction yielded six major brands of protein which had pI values comparable to those of human serum thyroxine-binding-globulin (TBG); however, the pattern of separation was different. Incubation of the fraction with 125I-T4 followed by isoelectric focusing and autoradiography revealed that the protein bands bound radioactivity. The microsomal fraction of the adipose tissue deiodinated 125I-T4 to L-125I-triiodothyronine (125I-T3) to an extent of 0-0.8 fmol/(mg of protein X min) which corresponded to about 0.2%; T4 was not deiodinated to L-3,3',5'-triiodothyronine (r-T3).(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of 125I-triiodothyroacetic acid to measure nuclear thyroid hormone receptor

125I-Triac was employed to measure hepatic thyroid hormone nuclear receptor (RT) in the rat. The binding properties of 125I-Triac and 125I-T3 were compared in a 0.4 M KCl extract of a liver nuclear preparation. The order in which the stable compounds, Triac, T3, T4 and rT3, competed for 125I-Triac and 125I-T3 binding in liver nuclear extract was similar (Triac greater than T3 greater than T4 greater than rT3), suggesting association of both radioligands with RT. Scatchard plot analysis of specific 125I-Triac and 125I-T3 binding in nuclear extract gave approximately equal estimates of the maximum binding capacity (MBC). However, the binding affinity, as represented by the equilibrium association constant (KA), was higher for 125I-Triac than for 125I-T3 (7-10 X 10(9)M-1 vs 1-3 X 10(9)M-1). To determine the effect of contaminating serum proteins on estimates of MBC and KA, a small amount of dilute rat serum was added to the same nuclear extract preparation. Addition of serum decreased the KA value and markedly increased the MBC values estimated by analysis of 125I-T3 binding data. In contrast, KA and MBC values derived from 125I-Triac binding data were not influenced appreciably by the addition of serum. These data indicate that: 1) both 125I-Triac and 125I-T3 bound to RT in rat liver nuclear extract, 2) the affinity of RT for 125I-Triac is appreciably greater than for 125I-T3, and 3) estimates of RT concentration (MBC) made with 125I-Triac are less sensitive to serum protein contamination than those made with 125I-T3. These properties of 125I-Triac may be useful in efforts to demonstrate RT in tissues that have low RT levels and/or when serum contamination is present.     

Characteristics of antibody produced during chronic treatment with LHRH.

In a patient with hypogonadotropic hypogonadism treated with luteinizing hormone releasing hormone (LHRH), secondary failure of both subjective and hormone responses occurred at the time of appearance of binding of 125I-LHRH by the patient's serum. On electrophoresis of the patient's serum with 125I-LHRH, label was found only in the gamma globulin region. 125I-LHRH added to the patient's serum was precipitated by sheep anti-human immunoglobulin G (anti-IgG) but not by sheep anti-human immunoglobulin M (anti-IgM). Competitive displacement of 125I-LHRH by unlabeled LHRH was demonstrated while TSH releasing hormone (TRH), somatostatin and rat pituitary hormones showed no displacement when tested at concentrations 5 X 10(6) greater than that of LHRH. Studies using 14 different analogs of LHRH revealed that those with changes at the carboxy terminus showed binding similar to LHRH. It is concluded that IgG antibody to LHRH was produced in this patient by repeated administration of synthetic LHRH. It is further concluded that antibody specificity is directed toward the N terminus region.     

Interference with thyrotropin receptor antibody determination by a spuriously occurring anti-bovine TSH antibody

Abnormally negative values of thyrotropin binding inhibitor immunoglobulin (TBII) were found in the sera from a patient with Graves' disease. This was due to the presence of potent bovine TSH (bTSH) binding activity in the sera. This activity was demonstrated to be in immunoglobulin G (IgG) with a lambda light chain isotype, which was shown to have an affinity for bTSH with a Ka value of 3.5 X 10(10) M-1 and a maximum binding capacity of 1.1 X 10(-14) M/mg IgG. F(ab')2 fragments obtained through pepsin digestion from the patient's IgG retained bTSH binding activity. [125I] bTSH binding to this IgG was inhibited by the TSH receptor. The inhibition was not completely competitive, suggesting the presence of different binding sites for this IgG and the TSH receptor on the TSH molecule. This IgG, however, could not bind labelled human TSH (hTSH). Since neither TSH nor other pituitary derivatives had ever been given to the patient, this bTSH binding activity was considered to be due to a spuriously occurring anti-bTSH antibody.     

Familial dysalbuminemic hypertriiodothyroninemia in a Japanese kindred

A 56-year-old Japanese housewife had been diagnosed as having Graves' disease and was treated with methimazole. When she was referred to our hospital, the serum T3 level was high irrespective of high TSH level. High serum T3 levels were also observed in two out of her three sisters. Electrophoresis revealed that binding of 125I-T3 to serum albumin was markedly increased whereas the binding of 125I-T4 to serum albumin was slightly increased in the three sisters whose serum T3 levels were high. These data indicate that the presence of an albumin variant is the cause of hypertriiodothyroninemia in this family.     

Nuclear thyroid hormone receptors in cultured bone cells

Thyroid hormones influence bone metabolism, but a direct interaction of triiodothyronine with nuclear T3 receptors in bone cells has not yet been reported. We investigated 125I-T3 binding to nuclei isolated from the cloned osteoblastlike rat osteosarcoma cells ROS 17/2.8. At 37 degrees C, saturable 125I-T3 binding to isolated nuclei reached equilibrium by 30 minutes and was completely displaced upon the addition of 500 nmol/L unlabeled T3. Nonsaturable binding represented about 0.5% of the radioactivity added (20% of the total binding). Thyroxine and 3,3',5'triiodothyronine competed with 125I-T3 with a 20-fold and 400-fold lower affinity than T3, respectively. Analysis of equilibrium competition experiments revealed the presence of a single class of homogeneous binding sites with an association constant of 5.0 +/- 0.3 X 10(9) mol/L-1 and a maximum nuclear binding capacity of 0.13 +/- 0.02 ng/mg DNA. A twofold increase of bone Gla protein (BGP) secretion was observed with T3 treatment suggesting that these T3 nuclear receptors are coupled with a biological response.     

Antiinsulin receptor antibodies in an insulin-dependent diabetic may arise as autoantiidiotypes

An insulin-requiring diabetic patient with intermittent periods of increased insulin requirements and insulin resistance was studied. The patient was found to have high titers of antiinsulin antibodies; subfractionation of the patient's serum revealed several populations of antiinsulin antibodies with differing affinities and titers for insulin. The ability of one of the insulin antibody fractions to bind [125I]iodoinsulin was markedly inhibited by the patient's serum (insulin depleted) and by purified total immunoglobulin G from which antiinsulin antibodies and insulin were removed. These findings suggested an antiidiotypic antibody in the patient's immunoglobulin G fraction reacting specifically with the antiinsulin antibody subfraction. Finally, the patient's serum contained an antiinsulin receptor antibody, as demonstrated by the ability of serum to specifically immunoprecipitate covalently labeled soluble insulin receptors. In conclusion, these results suggest that this patient generated a widespread polyclonal response to insulin, with the development of several populations of antiinsulin antibodies. An antiidiotypic antibody to a specific insulin antibody subfraction was present in the patient's serum which we believe had structural similarity to the binding site of the insulin molecule, accounting for the reactivity of the antiidiotypic antibody with the insulin receptor.     

Enhanced thyroxine metabolism in hexachlorobenzene-intoxicated rats

The effect of hexachlorobenzene (HCB) (1 g/kg bw) administration for 4 weeks, on thyroxine (T4) and triiodothyronine (T3) metabolism was studied in Wistar rats. The effect on serum binding of T4 has also been studied. Animals were injected with a tracer dose of either labeled hormone and by examining serum L-125I-T4 and L-125-I-T3, kinetics of radiolabeled hormones metabolism were calculated. The T4 metabolic clearance (MCI) as well as the distribution space, were increased by 6 fold. Decreased serum T4 levels result from an increase both in deiodinative and fecal disposal in HCB-treated rats. 125I-T3 metabolism was slightly affected. The enhanced peripheral disposition of thyroxine appears to lead to increased thyroid function, as measured by augmented TSH serum levels and 125I-thyroidal uptake. Serum binding of T4 was not affected.     

Characteristics of an antiluteinizing hormone isoantibody produced during gonadotropin induction of ovulation.

Production of an antihuman LH antibody was suspected in a woman with isolated LH deficiency who received human pituitary gonadotropin (hPG) to induce ovulation and who developed secondary drug failure associated with very high "serum LH' values. Binding of [125I]LH to various dilutions of the patient's serum was demonstrated by precipitation with polyethylene glycol or sheep antihuman immunoglobulin G (anti-IgG) but not by precipitation with sheep antihuman immunoglobulin M (anti-IgM). Unlabeled LH competitively displaced [125I]LH from a 1:200 final dilution of the patient's serum, and indicated a single class of binding sites with a binding affinity of 1.5 X 1011 M-1 and a binding capacity of 84 ng LH/ml serum. The isoantibody was reactive against antigenic determinants in hPG, LH, and hCG but not against human FSH. Further examination showed binding to the beta, but not the alpha, subunit of two LH preparations and to beta hCG. It is concluded that repeated administration of hPG to this patient with isolated LH deficiency evoked IgG isoantibody formation against the beta subunit of LH.     

Demonstration of anti-TSH antibody in TSH binding inhibitory immunoglobulin-positive sera of patients with Graves' disease

OBJECTIVES: The presence of anti-TSH antibodies in Graves' patients with unusually low TSH binding inhibitory immunoglobulin (TBII) has been reported. Recently, we found the first case of an anti-TSH antibody in TBII-positive sera of patients with Graves' disease. The prevalence and immunological specificity of this anti-TSH antibody were examined. DESIGN: The presence of 125I-bovine(b) TSH binding antibody in TBII positive serum was examined by prolonged incubation of more than 1 day because only weak binding occurred after 1 h incubation at 37 degrees C. The clinical course of these patients and binding characteristics of anti-bTSH antibody were examined. RESULTS: The corrected method-TBII activity (%)[1 - (a - b)/(c - d)] x 100 and the standard method-TBII activity (%) [1 - (a - d)/(c - d)] x 100 [a, 125I-bTSH binding with TSH receptor (R) in the presence of test serum; b, 125I-bTSH binding with test serum; c, 125I-bTSH binding with TSH R in the presence of normal serum; d, 125I-bTSH binding with normal serum] were calculated. The corrected method-TBII activity was always higher than the standard method-TBII activity in anti-bTSH antibody-positive serum. Anti-bTSH antibody-positive cases in TBII-positive Graves' disease were found in approximately 1% of Graves' patients. Anti-bTSH antibodies were confirmed as IgG from the increase of precipitated radioactivity by adding rabbit antihuman IgG antibody after the incubation of 125I-bTSH with test serum. These antibodies bind with not only bTSH, bTSH(alpha) and bLH, but also porcine (p)TSH, pTSH(alpha) and pFSH. However, these antibodies did not bind with human TSH. Binding of 125I-bTSH with patient's serum was neither inhibited by other Graves' thyroid stimulating antibody (TSAb), nor thyroid blocking antibody (TBAb) in primary hypothyroidism. CONCLUSIONS: The presence of anti-bTSH antibody in TBII-positive serum of high titre means that TBII-positive sera cannot rule out the absence of anti-bTSH. Thus, determination of 125I-bTSH binding with test serum in TSH receptor assays is necessary to determine the precise TBII activity and to detect anti-bTSH antibody.     

Triiodothyronine binding immunoglobulin in a euthyroid man without apparent thyroid disease; its properties and effects on triiodothyronine metabolism

A 54 year old man with markedly elevated serum T3, but without an apparent thyroid disease, was found to have a specific antibody to T3. His serum thyroxine, TBG and TSH were in normal range, but T3-RSU was markedly low. Antibodies to thyroglobulin and microsome were negative. He was judged euthyroid because of a normal basal metabolic rate and a normal thyroidal 123I uptake which was suppressed by T3 administration. When serum was extracted with ethanol prior to assay, serum T3 was found to be in the upper border of normal range. Several experiments revealed the presence of an antibody to T3 in his serum with an affinity constant of 3.3 X 10(9) M-1. The binding capacity of the antibody was 7.6 ng/mg of IgG. The binding of [125I]T3 was almost specific to T3, and potencies of T4 and fT3 in displacing [125I]T3 binding were only 1.0 and 0.3%, respectively, of that of T3. The antibody contained both kappa and lambda chains and was therefore polyclonal. The T3 metabolic clearance rate, which was determined by disappearance of injected [125I]T3 from serum, was lower in this patient (7.44 1/day) than in normal. The T3-production rate was decreased to 14.9 micrograms/day, and serum free T3 concentration as well as urinary T3 excretion rate were also reduced. Since both serum total and free T4 concentrations were normal, the supply of T4 to peripheral tissues would be sufficient to keep this patient in a euthyroid state in spite of the anti-T3 antibody.     

Selective cytotoxicity of 125I-labeled monoclonal antibody T101 in human malignant T cell lines

The radiolabeled anti-T cell antibody T101 can be used for specific tumor localization, but unlabeled T101 produces limited cytotoxicity in patients. We thus studied the in vitro cytotoxic effects of T101 labeled with 125I, a radionuclide known for its short-range, high-linear-energy electrons. We showed that 125I-T101 could be readily prepared at high specific activity with high immunoreactivity. Human malignant T cell lines HUT 102, MOLT-4, and HUT 78 were found to differ in the number of T65 determinants (the antigen recognized by T101) and the sensitivity to external x-ray radiation, which were of significance for the cytotoxicity of 125I-T101 in vitro. The cytotoxic effects of 125I-T101 were also found to be dose dependent and increased with exposure time under frozen conditions. As controls, unlabeled T101 had no cytotoxic effect, while free Na 125I or the 125I-labeled irrelevant antibody 9.2.27 exerted minor cytotoxicity. In HUT 102 and MOLT-4, more than 3 logs' cell killing was achieved within four weeks. Because considerable cytotoxicity was demonstrated in vitro by 125I-T101 on T65-positive malignant cells, and because low-dose 111In-T101 can be used successfully for tumor localization, future trials using 125I-T101 at high specific radioactivity may improve therapeutic results in patients with T65-positive malignancies.