Ig Fractions Research Articles

Detection of Antitrophoblast Antibodies in the Sera of Patients With Anticardiolipin Antibodies and Fetal Loss

AbstractWomen with anticardiolipin antibodies (ACLA) are at increased risk for fetal loss. One potential explanation for this outcome is that sera from these individuals contain antibodies reactive with trophoblast cells, which are involved in the establishment of the uteroplacental vasculature and maintenance of placental blood fluidity. To examine this hypothesis, we compared the incidence of trophoblast-reactive antibodies in 27 patients with AC LA and a history of fetal loss with that in 29 normal pregnant women. Sera from 20 patients, but only one control, contained trophoblast-reactive antibodies (P < .001). These antibodies were not directed against major histocompatibility class I antigens, and reacted with both term and first-trimester trophoblast cells. In most cases, sera from which AC LA were adsorbed by cardiolipin-containing liposomes maintained reactivity against cells. In addition, patient Ig fractions immunoprecipitated an ~62-kD protein from the trophoblast cell surface, stimulated the release of arachi-donic acid and thromboxane A2 by trophoblasts, and inhibited the binding of prourokinase to trophoblast urokinase receptors. These observations show that sera from women with AC LA and a history of fetal loss contain antitrophoblast antibodies. These antibodies may be serologically distinct from ACLA, and may contribute to the pathogenesis of fetal demise.

Detection of antitrophoblast antibodies in the sera of patients with anticardiolipin antibodies and fetal loss

Women with anticardiolipin antibodies (ACLA) are at increased risk for fetal loss. One potential explanation for this outcome is that sera from these individuals contain antibodies reactive with trophoblast cells, which are involved in the establishment of the uteroplacental vasculature and maintenance of placental blood fluidity. To examine this hypothesis, we compared the incidence of trophoblast-reactive antibodies in 27 patients with ACLA and a history of fetal loss with that in 29 normal pregnant women. Sera from 20 patients, but only one control, contained trophoblast-reactive antibodies (P &lt; .001). These antibodies were not directed against major histocompatibility class I antigens, and reacted with both term and first-trimester trophoblast cells. In most cases, sera from which ACLA were adsorbed by cardiolipin- containing liposomes maintained reactivity against cells. In addition, patient Ig fractions immunoprecipitated an approximately 62-kD protein from the trophoblast cell surface, stimulated the release of arachidonic acid and thromboxane A2 by trophoblasts, and inhibited the binding of prourokinase to trophoblast urokinase receptors. These observations show that sera from women with ACLA and a history of fetal loss contain antitrophoblast antibodies. These antibodies may be serologically distinct from ACLA, and may contribute to the pathogenesis of fetal demise.

Isolation and partial characterization of turbot ( Scophthalmus maximus) immunoglobulins

1. 1. An IgM-like immunoglobulin was isolated from the serum of turbot ( Scophthaimus maximus) by both fast protein liquid chromatography (FPLC) on Superose® 6HR columns and affinity chromatography on Mensep® protein A cartridges. 2. 2. Molecular weight of the native Ig was estimated to be 835 kD by SDS-PAGE and 820 kD on the basis of gel filtration. 3. 3. Purity was verified by SDS-PAGE (under non-reducing and reducing conditions) and by immunoelectrophoresis and immunoblotting of antisera raised against the isolated Ig fraction in mice: only minor protein contaminants were observed. 4. 4. Both SDS-PAGE under reducing conditions and immunoblotting also indicated that the heavy and light chains of the isolated turbot Ig are of about 78 and 27 kD, respectively. 5. 5. Total circulating Ig level was estimated by single radial immunodiffusion using a polyclonal anti-turbot Ig serum to be about 4.8 mg/ml. 6. 6. The same antiserum was used to verify that the purified proteins were Igs, in an ELISA test using serum from Vibrio mguillarum -immunized turbot as primary antibody and immobilized V. anguillarum cells as antigen. 7. 7. Finally, our data indicate that about 25% of turbot Igs bind to Mem-Sep® protein A.

Beta 2-glycoprotein I is a requirement for anticardiolipin antibodies binding to activated platelets: differences with lupus anticoagulants

Antiphospholipid (aPL) antibodies are of major interest not only because the lupus anticoagulant (LA) causes an inhibition of in vitro blood coagulation, but also because the presence of aPL antibodies confers a risk of thrombosis. The inhibition of in vitro phospholipid- dependent coagulation (LA) is thought to be caused by the binding of LA to procoagulant phospholipid surfaces, thus impeding the clotting process. Another class of aPL antibodies are those originally described to be directed against negatively charged phospholipids, in particular cardiolipin (ACA). ACA are usually directed against a complex antigen consisting of negatively charged phospholipid and a plasma protein, beta 2-glycoprotein I (beta 2-GPI). Further, there is antibody heterogeneity even within individual patients so that ACA and LA are separable using physicochemical techniques such as ion exchange chromatography and chromatofocusing. Using such techniques we have enriched Ig fractions for LA and ACA from two patient plasmas. The majority of Ig with LA activity had a pI of 7.2 to 7.3 whereas ACA had a pI of 5.0 to 5.2. Using these enriched fractions labeled with [125I]- iodine we have shown that LA binds to platelets in a specific and saturable manner. Binding is dependent on thrombin activation. [125I]- ACA behaves differently. Like LA, binding is specific and dependent on thrombin activation but in this case requires the presence of beta 2- GPI. ACA, in the presence of beta 2-GPI, competes for binding with LA suggesting the same or contiguous site. There is no cross-reactivity of these antibodies with GPIIb/IIIa and the most likely binding site is phospholipid. In neither case does LA nor ACA have an effect on thrombin-induced release of serotonin or beta-thromboglobulin nor do they affect platelet aggregation induced by a number of agonists. This antibody binding may play an etiological role in thrombocytopenia associated with aPL, but does not explain thrombosis on the basis of hyperaggregability or increased platelet release.

β2-Glycoprotein I Is a Requirement for Anticardiolipin Antibodies Binding To Activated Platelets: Differences With Lupus Anticoagulants

Antiphospholipid (aPL) antibodies are of major interest not only because the lupus anticoagulant (LA) causes an inhibition of in vitro blood coagulation, but also because the presence of aPL antibodies confers a risk of thrombosis. The inhibition of in vitro phospholipid-dependent coagulation (LA) is thought to be caused by the binding of LA to procoagulant phospholipid surfaces, thus impeding the clotting process. Another class of aPL antibodies are those originally described to be directed against negatively charged phospholipids, in particular cardiolipin (ACA). ACA are usually directed against a complex antigen consisting of negatively charged phospholipid and a plasma protein, beta 2-glycoprotein I (beta 2-GPI). Further, there is antibody heterogeneity even within individual patients so that ACA and LA are separable using physicochemical techniques such as ion exchange chromatography and chromatofocusing. Using such techniques we have enriched Ig fractions for LA and ACA from two patient plasmas. The majority of Ig with LA activity had a pI of 7.2 to 7.3 whereas ACA had a pI of 5.0 to 5.2. Using these enriched fractions labeled with [125I]-iodine we have shown that LA binds to platelets in a specific and saturable manner. Binding is dependent on thrombin activation. [125I]-ACA behaves differently. Like LA, binding is specific and dependent on thrombin activation but in this case requires the presence of beta 2-GPI. ACA, in the presence of beta 2-GPI, competes for binding with LA suggesting the same or contiguous site. There is no cross-reactivity of these antibodies with GPIIb/IIIa and the most likely binding site is phospholipid. In neither case does LA nor ACA have an effect on thrombin-induced release of serotonin or beta-thromboglobulin nor do they affect platelet aggregation induced by a number of agonists. This antibody binding may play an etiological role in thrombocytopenia associated with aPL, but does not explain thrombosis on the basis of hyperaggregability or increased platelet release.

The appearance in thyroidectomized mice of immunoglobulins that bind TSH and stimulate FRTL-5 thyrocytes.

The purpose of these studies was to examine whether thyroid stimulating antibodies in Graves' patients could arise as auto-antiidiotypic antibodies to endogenous anti-TSH antibodies. The model system chosen was the thyroidectomized mouse, exhibiting an elevated level of endogenous, circulating TSH. Mice were thyroidectomized by 131I administration. Sera samples were drawn 1 to 14 months later. The following activities were measured in the immunoglobulin (Ig) fractions prepared: (a) TSH binding by elisa techniques, (b) iodide pump activity (as measured by 99mTcO4 uptake) and (c) increased [3H]thymidine incorporation into the DNA of FRTL-5 cells. TSH binding Igs were detected in 29/98 mice thyroidectomized for 7-14 months. Stimulation of technetium uptake was observed in 59/110 mice and stimulated labeled thymidine uptake in 37/102 mice, beginning eight and nine months after thyroidectomy, respectively. Of the positive animals, 51 showed a single stimulating activity. The incidence and the serum titers of Igs that stimulate technetium uptake increased significantly with time. Indeed, in the group tested 14 months post-thyroidectomy, 75% of the sera were positive for this antibody with a mean titer eightfold higher than the controls. Hybridomas were prepared from the spleen lymphocytes of thyroidectomized mice. Of these, 18 produced 99mTcO4 uptake stimulating Igs, 12 [3H]thymidine-uptake stimulating Igs and 18 TSH binding Igs. Most of the hybridomas secreted Igs with a single bioactivity. One monoclonal antibody was isolated which neutralized the bioactivity of bTSH on FRTL-5 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid assay for immune complex bound antigens from urine of cancer patients.

Concentrated urine from patients with various types of cancer was fractionated by S-500 gel filtration chromatography to yield high molecular weight (mw) immune complexes (IC) and lower mw immunoglobulin (Ig) fractions. Column fractions were assayed for the presence of IgM and IgG by immunoblot probing using anti-human IgM and anti-human IgG alkaline phosphatase conjugates. These results were used to define IC fractions (those from high mw S-500 fractions which were positive for immunoglobulin) and Ig fractions (those from medium mw S-500 fractions which were positive for immunoglobulin). Antigen components of high mw S-500 IC fractions were then determined by immunoblot probe using medium mw S-500 Ig fractions as the antibody probe (i.e. autoantibody). This method for identification of immune complex antigens has the potential to probe for tumor-associated antigens, autoantigens, or foreign antigens from starting material which contains both immune complex and free immunoglobulin.

Effect of B lymphocytes from different organs on hematopoietic colony formation in the spleen by bone marrow cells

The influence of B-lymphocytes from various sources on splenic colony formation was studied in the syngeneic system. B-lymphocytes were obtained by panning with IgG-fraction of rabbit anti-mouse Ig, absorbed on Petri dishes. In addition, adherent cells, Thy-1+ and SC-1+ were eliminated from the fraction of Ig(+)-cells. SC-1- and SC-1+ fractions, containing, respectively, stem cells and T-lymphocyte precursors, were obtained by panning with IgG-fraction of rabbit anti-SC-1 serum. SC-1- cells transferred to irradiated syngeneic mice did not induce colony formation in the spleen. Introduction of SC-1- and SC-1+ cells induced formation of colonies. A similar helper effect occurred when SC-1(-)-cells were introduced with bone marrow or lymph node B-cells, but not with splenic B-cells. Splenic, but not bone marrow and lymph node B-cells inhibited colony formation by combination of SC-1- and SC-1+ cells. All effects of Ig+ cells were abolished by treatment of cells with rabbit anti-MBLA serum. Thus, B-cells of various origin can either enhance or inhibit colony formation. The enhancing of inhibitory effect after B (MBLA+)-cells elimination from suspension of bone marrow and lymph node (but not spleen) Ig(+)-cells resulted from the activity of B-contrasuppressors.

Antibody populations in the antiphospholipid syndrome

Of immunoglobulins directed against self-antigens which arise in autoimmune disorders such as systemic lupus erythematosis (SLE), the antiphospholipid (aPL) antibodies are of major interest to haematologists not only because the lupus anticoagulant (LA) causes an inhibition of in vitro blood coagulation, but also because the presence of certain aPL antibodies confers a risk of thrombosis. The inhibition of jn vitro phospholipid-dependent coagulation (LA) is thought to be due to the binding of LA to procoagulant phospholipid surfaces, thus impeding the clotting process. It is likely that LA interacts with lipid structures formed in the presence of hexagonal phase phosphatidyl-ethanolamine and thus that the antibody is directed against a complex phospholipid epitope. Another class of aPL are those originally described to be directed against negatively charged phospholipids, in particular cardiolipin. We have recently shown that ACL antibodies are usually directed against a complex antigen consisting of negatively charged phospholipid and a plasma protein, β 2 -glycoprotein-I (β 2 -GPI) (McNeil et al, Proc. Natl. Acad. Sci. 87:4120, 1990). Further, we have found a major degree of antibody heterogeneity even within individual patients so that ACL and LA are separable using physicochemical techniques such as ion exchange chromatography and chromatofocusing. Using such techniques we have enriched Ig fractions for LA and ACA. The majority of Ig with LA activity had a PI of 7.3-7.4 whereas ACA had a PI of 5-5.3. Using these enriched fractions labelled with 125 I-iodine we have shown that LA binds to platelets in a specific and saturable manner. Binding is dependent on thrombin activation. 125 I-ACA behaves differently. Like LA-IgG, binding is specific and dependent on thrombin activation but in this case requires the presence of β 2 -GPI. In neither case does LA or ACA have an effect on thrombin-induced release of 5-HT or β-thromboglobulin nor do they affect platelet aggregation induced by a number of agonists. This antibody binding may play an aetiological role in thrombocytopenia associated with aPL, but does not explain thrombosis.

Antibody in sera of patients infected with Trichomonas vaginalis is to trichomonad proteinases.

A recent report demonstrated the immunogenic character of the cysteine proteinases of Trichomonas vaginalis. It was of interest, therefore, to examine for the presence of serum anti-proteinase antibody among patients with trichomoniasis. An immunoprecipitation assay was used involving protein A-bearing Staphylococcus aureus first coated with the IgG fraction of goat anti-human Ig and then mixed with individual sera of patients to bind human antibody. These antibody-coated bacteria were then added to detergent extracts of T vaginalis. Bound immune complexes on S aureus were washed and solubilised for electrophoretic analysis on acrylamide copolymerised with gelatin for detection of proteinase activity. Sera from patients (50/50), but none from sera of normal, uninfected women, possessed IgG to numerous trichomonad cysteine proteinases. The presence of this serum anti-proteinase antibody disappeared after drug treatment and cure of patients of the T vaginalis infection. The commonality of the anti-proteinase antibody in the sera of patients with trichomoniasis provided evidence for the expression of the same repertoire of parasite proteinases during infection. These observations have important implications for the in vivo relevance of the proteinases and indicate that strategies to use a specific serum antibody response for diagnosis of this infection may be possible.

Generation and specificity of monoclonal anti-idiotypic antibodies against human HIV-specific antibodies. I. Cross-reacting idiotopes are expressed in subpopulations of HIV-infected individuals.

In this study we have generated monoclonal anti-idiotypic antibodies against human monoclonal and polyclonal anti-HIV antibodies in seropositive sera. A human anti-gp41 mAb (H2, IgM kappa) was used to immunize BALB/c mice and to prepare hybridoma anti-antibodies that react with H2 and not with normal human IgM. Similar monoclonal anti-antibodies were made in BALB/c mice immunized with Ig fraction prepared from a pool of HIV-seropositive sera. Both kinds of anti-idiotypic antibodies reacted with antibodies in pools of seropositive sera and with individual seropositive sera but not with normal human Ig or seronegative sera. The Id-positive Ig from single donors were isolated on two different anti-Id immunoabsorbents and shown to bind to p24 and gp120, respectively. The detection and isolation of idiotypically cross-reactive human anti-HIV antibodies from seropositive donors demonstrated, for the first time, the existence of shared Id expressed by antibodies against HIV Ag. The utility of cross-reacting anti-idiotypic antibodies as tools to dissect the network regulation of the anti-viral immunity in AIDS is discussed.

Antiidiotypic antibodies to HLA class I alloantibodies in normal individuals: A mechanism of tolerance to noninherited maternal HLA antigens

Recent reports indicate that 25%–50% of transplant patients exhibit B-cell nonresponsiveness to their noninherited maternal HLA antigens (NIMAs). To test the hypothesis that tolerance of NIMAs is mediated by antiidiotypic antibodies, sera from seven normal human subjects were tested for the capacity to inhibit the reactivity of HLA alloantisera directed to NIMAs. Five of seven sera inhibited (50%–100%) the cytotoxicity of monospecific alloantisera directed to their NIMAs. This inhibition was specific in that antisera directed to third-party HLA antigens were not inhibited. Cytotoxicity inhibition by normal sera was selective for antisera directed to HLA-B locus antigens. Absorption with an antibody specific for an HLA class I framework determinant eliminated the inhibitory activity of three of the five sera, suggesting that the inhibition was mediated by soluble HLA antigens in these cases. However, two of the sera retained inhibitory activity following soluble antigen depletion, suggesting that, in these cases, inhibition is mediated by antiidiotypic antibodies. This hypothesis was confirmed by purifying the immunoglobulin (Ig) fraction of one of these sera by anti-Ig affinity chromatography; the column eluate (Ig fraction) but not the effluent (Ig-depleted serum) was capable of inhibition. These data are consistent with the hypothesis that tolerance of NIMAs is mediated, at least in part, by antiidiotypic antibodies.

Human and murine anti-DNA antibodies induce the production of anti-idiotypic antibodies with autoantigen-binding properties (epibodies) through immune-network interactions.

To examine the potential role of immune-network interactions in the production of lupus autoantibodies, normal NZW rabbit antibody responses were analyzed after immunization with one of the following Ig preparations: human lupus serum anti-dsDNA antibodies, human lupus serum anti-ssDNA antibodies, a mixture of human lupus serum anti-dsDNA and anti-ssDNA antibodies, the MRL-lpr/lpr anti-dsDNA mAb H241, and the MRL-lpr/lpr anti-ssDNA mAb H130. Four of five rabbits produced Ig typical of lupus autoantibodies: individual rabbit Ig cross-reacted with multiple autoantigens including nucleic acids, cardiolipin, SmRNP, glomerular extract, laminin, and exogenous Ag. Rabbit anti-Id against human anti-dsDNA antibodies were highly specific for dsDNA. Notably, in each serum the autoantibody activity was confined to the anti-Id Ig fraction. A similar spontaneously occurring Id-anti-Id interaction was also found between anti-ssDNA and anti-dsDNA antibodies isolated from an individual lupus patient. These results indicate that lupus autoantibodies which share Ag binding properties with pathogenic Ig, including both cross-reactive and anti-dsDNA antibodies, can induce the production of Ig with similar autoantigen binding properties through immune-network interactions. This phenomenon, if unregulated, could lead to the amplification of pathogenic autoantibody production in individuals with systemic lupus.

Monoclonal antibody to Theiler's murine encephalomyelitis virus defines a determinant on myelin and oligodendrocytes, and augments demyelination in experimental allergic encephalomyelitis.

Theiler's murine encephalomyelitis virus (TMEV) causes a chronic demyelinating disease in mice. The mechanisms underlying the demyelination have not been fully elucidated. We have raised a mAb to TMEV (DA strain), H8, that reacts both with TMEV VP-1 and galactocerebroside (GC). In mouse brain cultures, cells positive for the mAb H8 epitope were double labeled with antibody to myelin basic protein, indicating that those cells were oligodendrocytes. Further, mAb H8 could immunostain myelin structures in frozen sections from mouse brains. When injected intravenously into mice with acute allergic encephalomyelitis, mAb H8 increased by 10-fold the size of demyelinated areas within the spinal cords. This is the first report demonstrating that an antibody to virus can enhance demyelination of a central nervous system disease. Ig fractions from the sera of mice with chronic TMEV infection had antibody(s) to GC, as well as to TMEV, as determined by ELISA. Furthermore, a competition ELISA for TMEV or GC antigen revealed that sera from these infected mice contained antibody(s) with the same specificity as mAb H8. Our results indicate that antibodies generated by immune response to TMEV can react with myelin and oligodendrocytes, and contribute to demyelination through an immune process.

Eye Muscle Membrane Reactive Antibodies are not Detected in the Serum or Immunoglobulin Fraction of Patients with Thyroid-Associated Ophthalmopathy Using an Elisa and Crude Membranes

We tested sera and purified immunoglobulin (Ig) fractions from patients with autoimmune thyroid disorders (AITD), with and without ophthalmopathy, and normal subjects, for the presence of antibodies reactive with eye muscle membrane antigens in an optimized enzyme-linked immunosorbent assay (ELISA). We found no correlation between ELISA results and the presence or severity of ophthalmopathy in patients with AITD for either serum or Ig, and there were no significant differences between the mean values (+/- SE) for the three groups (AITD with ophthalmopathy, AITD without ophthalmopathy and normals) for either serum or Ig. In contrast Ig from 8 of 19 (45%) patients with thyroid-associated ophthalmopathy reacted with a 64 kDa eye muscle membrane antigen in SDS-polyacrylamide gel electrophoresis and Western blotting, while tests were positive in only one of the 8 patients with AITD without eye disease and in none of the 8 normal subjects. The presence of antibodies to a 64 kDa antigen in immunoblotting did not correlate with the levels of antibodies measured in ELISA. We conclude that the ELISA, incorporating a crude membrane fractions as antigen, is not useful as a clinical test for eye muscle autoantibodies.

STIMULATION OF CYCLIC AMP PRODUCTION IN FRTL‐5 THYROID CELLS BY CRUDE IMMUNOGLOBULIN FRACTIONS OF SERUM FROM PREGNANT WOMEN

In an assay for thyroid-stimulating antibodies, in which FRTL-5 thyroid cells were incubated with crude immunoglobulin (Ig) fractions precipitated from serum with 15% polyethylene glycol, significant increase in cAMP production was elicited by the samples from 25 (35.7%) out of 70 pregnant women. The highest value was 529.5%. There was a close correlation between thyroid stimulating activities and serum hCG concentrations (r = 0.708, P less than 0.001). When 125I-hCG was added to serum from pregnant women, about 20% of the radioactivity was incorporated into the Ig fractions. hCG preparations within a range of concentrations of 30-300 IU/m elicited 2.3-16.5 times increase in cAMP in a dose-dependent manner. Nine pregnant women with serum TSH concentrations less than the lower limit of the normal range (less than 0.25 mU/l) displayed significantly higher values for both thyroid stimulating activities and serum hCG concentrations (P less than 0.001, respectively) compared with those who had normal TSH levels in serum. These data suggest that hCG or its variant may stimulate the thyroid sufficiently to suppress secretion of TSH from the pituitary in some pregnant women.

Genetic polymorphism of rabbit V Ha region: A new allotype, a108

We report a new rabbit IgV H allotype, designated a108, which was detected following studies of the progeny of a wild rabbit ( Oryctolagus cuniculus) from Zembra Island (Tunisia). This allotype seems to be specific of this island, since it has not been detected in other wild rabbit populations (in Spain, Portugal or France). Its determinants are located on the Fd fragment of the immunoglobulin (Ig) heavy chain and it behaves like the product of an allele at the a locus. Allotype a108 was strongly related serologically to a1 and a107, and to a lesser extent to a101, a109 and a3 allotypes. We also detected determinants shared among the a1, a107, a108, a101 and a109 allotypes. These determinants were expressed by a large fraction of Ig molecules from rabbits of allotype a1, a107 and a108 and by a very small fraction of Ig from rabbits of allotype a101 and a109.

Comparative studies on the structure of biliary immunoglobulins of some avian species I. Physico-chemical properties of biliary immunoglobulins of chicken, turkey, duck and goose

In pooled bile of chicken, turkey, duck and goose immunoglobulins (Igs) + were found in relatively high amounts between 4.5 and 15.0 mg/ml (corresponding to 28 to 36% of the total protein contents). All biliary Ig fractions studied possess (H 2L 2) n structure estimated by SDS-PAGE. In chicken and turkey L chains are assumed to be non-covalently linked to the polymeric H chains. In duck and goose L chains are completely linked to H chains. The M r of H chains are rather similar: 64 kDa for the Igs of chicken and turkey bile and 67 kDa for the biliary Igs of duck and goose. There are slight differences in the electrophoretic mobility: Chicken and turkey Igs are β 1/ α 2-globulins and the corresponding Ig fractions from duck and goose are β 2-globulins.

Kappa/lambda immunoglobulin distribution in Graves' thyroid-stimulating antibodies. Simultaneous analysis of C lambda gene polymorphisms.

From patients with untreated Graves' disease 11 sera showing high cAMP release in the FRTL-5 cell assay were studied for relative proportions of kappa or lambda Ig molecules showing cAMP releasing activity. Immunoabsorption of gamma-globulins was performed using monoclonal murine anti-kappa or anti-lambda antibodies linked to cyanogen bromide-activated sepharose. Specific kappa- or lambda-adsorbed fractions were also eluted from immunoabsorbents using chaotrophic thiocyanate buffers and equilibrated with pH 7.4 low salt buffer by dialysis. Immunoabsorption and elution experiments showed that five Graves' sera contained predominant cAMP-releasing activity within lambda Ig fractions, whereas two Graves' sera showed predominant cAMP-releasing activity in kappa Ig fractions. Four sera showed cAMP release approximately equally divided between kappa and lambda Ig both after immunoabsorption and specific anti-kappa or anti-lambda eluates were studied. C lambda genotypes were examined by Southern blotting and restriction fragment length polymorphism analysis of Eco RI-digested genomic DNA from 158 patients with Graves' disease in parallel with 112 normal controls and 29 patients with autoimmune hypothyroidism. Notable shifts in proportions of 8/8 and 18/18 genotypes were present when Graves' patients were compared with normal controls. Allelic frequencies and ratios of genotype 8 to 18 were significantly different (P less than 0.05) when Graves' patients were compared either to normal controls or to patients with autoimmune hypothyroidism.

Inhibition of thyrotropin-stimulated iodide uptake in FRTL-5 thyroid cells by crude immunoglobulin fractions from patients with goitrous and atrophic autoimmune thyroiditis.

We studied the effects of crude immunoglobulin (Ig) fractions of serum from patients with goitrous and atrophic autoimmune thyroiditis on TSH-, thyroid-stimulating immunoglobulin (TSI)-, forskolin-, and dibutyryl cAMP-stimulated 125I uptake by FRTL-5 thyroid cells. TSH-stimulated 125I uptake was inhibited by the Ig fractions from 15 patients with atrophic thyroiditis who had serum TSH binding inhibitor Igs (TBII), 10 (62.5%) of 16 TBII-negative patients with atrophic thyroiditis, 7 (43.8%) of 16 hypothyroid patients with goitrous thyroiditis who had no TBII activity, and only 2 (15.4%) of 13 euthyroid patients with goitrous thyroiditis who were negative for TBII. The mean inhibition of TSH-stimulated 125I uptake produced by the crude Igs from the former 3 groups of hypothyroid patients was statistically significant (P less than 0.001, P less than 0.001, and P less than 0.01, respectively) and correlated closely with the ability of the Ig fractions to inhibit TSI-stimulated 125I uptake (r = 0.882) and TSH-stimulated cAMP accumulation (r = 0.929). The inhibition of TSH- or TSI-stimulated 125I uptake by Ig samples containing TBII correlated significantly with the TBII activities. On the other hand, in the presence of Igs from TBII-negative hypothyroid patients, the inhibition of TSH-stimulated 125I uptake correlated significantly with that of forskolin-stimulated 125I uptake (r = 0.685). Although 6 (12.8%) of 47 Ig samples from hypothyroid patients inhibited dibutyryl cAMP-stimulated 125I uptake, the activities were marginal. These findings suggest that at least 2 types of antibodies are involved in the inhibition of TSH- or TSI-stimulated 125I uptake: 1 being a competitive inhibitor of TSH binding to its receptors, and another exerting influence on a step subsequent to TSH or TSI binding, presumably through adenylate cyclase inhibition.