Autosomal dominant inheritance of the tendency of develop thyroid autoantibodies.
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Adult↗
Biomedical subjects
B Rees Smith
Publications and source records attributed to B Rees Smith.
Severe combined immunodeficient (SCID) mice: a model for investigating human thyroid autoantibody synthesis.
We have studied the ability of lymphocytes from the blood, thyroid and lymph nodes of patients with autoimmune thyroid disease (AITD) to produce autoantibodies to thyroglobulin (Tg) and/or thyroid peroxidase (TPO) in SCID mice. Human IgG class Tg and/or TPO antibodies were detectable in plasma from SCID mice 7 days after transfer of 15-25 x 10(6) cells/mouse and the highest levels were recorded 2-3 weeks later. In contrast, Tg and/or TPO antibodies were undetectable in recipients of lymphocytes from thyroid antibody negative controls. AITD thyroid lymphocytes produced the most antibody in recipient mice and lower levels were observed in recipients of AITD blood and lymph node lymphocytes. The amounts of Tg and/or TPO antibody detected were in accordance with the ability of thyroid and lymph node lymphocytes to secrete these autoantibodies spontaneously in culture (indicating the presence of cells activated in the patient) and with the capacity of blood lymphocytes (probably B memory cells) to secrete Tg and/or TPO antibodies in culture in response to pokeweed mitogen. Tg antibodies in plasma from SCID recipients of thyroid lymphocytes were of subclasses IgG1, IgG2 and IgG4 and the proportions closely resembled those of the donor's serum Tg antibodies. Blood lymphocytes transferred to SCID recipients were also able to produce Tg antibodies of subclasses 1, 2 and 4 but the subclass distribution varied between mice and the reason for this is not clear at present. Since SCID mice provide an environment in which B lymphocytes from patients with AITD can be activated without mitogen to secrete thyroid antibodies, this model will provide a powerful system for elucidating the mechanisms regulating the secretion of human antibodies to Tg and TPO.
Animals↗
Cloning and expression of a human thyroglobulin autoantibody.
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Amino Acid Sequence↗
Human monoclonal thyroglobulin autoantibodies of high affinity. I. Production, characterisation and interaction with murine monoclonal thyroglobulin antibodies.
Four hybridomas secreting human thyroglobulin (Tg) autoantibodies of different IgG subclasses and light chain types (IgG1 lambda, IgG1 kappa, IgG2 lambda and IgG2 kappa) were obtained by direct fusion of Hashimoto thyroid lymphocytes with the mouse myeloma X63-Ag.653. The autoantibodies were specific for human Tg and the functional affinities were high (only 2.6-3.9 log10 pM Tg required to give 50% inhibition of binding in ELISA). Using thyroid lymphocytes, 4 lines secreting Tg autoantibodies were obtained from 11 fusions compared with 1 line from 32 fusions of Epstein Barr virus infected blood lymphocytes, which emphasises the importance of using lymphocytes derived from a tissue known to be enriched in thyroid autoantibody secreting precursor B cells. These 4 human Tg autoantibodies, as well as an IgG2 lambda Tg antibody previously derived from Hashimoto blood B cells and an IgG4 kappa monoclonal Tg antibody present in a Hashimoto serum, were used in attempts to probe the interaction between human Tg autoantibodies and the Tg molecule (2 polypeptides of 330 KD). The binding to 125-I Tg by 3/7 murine monoclonal antibodies was inhibited (36-78%) by an IgG2 lambda and an IgG4 kappa human monoclonal Tg autoantibody, indicating an overlap between the epitopes recognised by these 3 murine monoclonal Tg antibodies and 2 monoclonal human Tg autoantibodies. None of the human Tg autoantibodies (or the murine monoclonal Tg antibodies) bound to Tg denatured by reduction and alkylation. Although the number of observations is limited, our study demonstrates that high affinity human monoclonal Tg autoantibodies, like polyclonal serum Tg autoantibodies, recognise non-linear B cell epitopes on conformationally intact human Tg.
Animals↗
Human monoclonal thyroglobulin autoantibodies of high affinity. II. Interaction between thyroglobulin and thyroglobulin autoantibodies of different IgG subclasses.
The interaction of human thyroglobulin (Tg) autoantibodies of different IgG subclasses with Tg was investigated using four high affinity human monoclonal thyroglobulin (Tg) autoantibodies, secreted by human-mouse hybridomas, of subclasses IgG1 (kappa and lambda) and IgG2 (kappa and lambda) and an IgG4 kappa serum monoclonal Tg antibody. With exception of a low level of interference in binding between one IgG1 lambda Tg antibody and one IgG2 kappa Tg antibody (27% decrease), binding by human monoclonal Tg antibodies of one IgG subclass was unaffected by pre-incubation of 125-I Tg (or Tg on an ELISA plate) with a human monoclonal Tg antibody of a different IgG subclass. Furthermore, preincubation of Tg-coated ELISA plates with an IgG1 human monoclonal Tg antibody had little effect on binding to Tg by IgG2, IgG3 and IgG4 Tg antibodies present in the sera of 6 Hashimoto patients. Comparable observations were made using an IgG2 monoclonal Tg antibody and serum Tg antibodies of subclasses IgG1, IgG3 and IgG4. Binding of an IgG1 kappa Tg antibody was inhibited (> 80%) by pre-incubation of Tg with an IgG1 lambda Tg antibody derived by fusion of lymphocytes from the same Hashimoto patient. In contrast, pre-incubation of Tg with an IgG2 kappa Tg antibody had little effect on subsequent binding by an IgG2 lambda Tg antibody derived from lymphocytes of a different Hashimoto patient.(ABSTRACT TRUNCATED AT 250 WORDS)
Antibodies, Monoclonal↗
Cytokines, thyroid autoantibody synthesis and thyroid cell survival in culture.
In autoimmune thyroid disease lymphoid cells infiltrating the thyroid gland occur in conspicuous aggregates or as a diffusely distributed population invading the thyroid follicles. Consequently cytokines secreted by activated T cells or macrophages could influence neighbouring thyroid cells as well as other lymphocytes. We have investigated this possibility using recombinant cytokines. Thyroid cell survival was assessed in terms of mitochondrial dehydrogenase activity in monolayers exposed to tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-1 (IL-1 alpha and beta) and interleukin-2 (IL-2) in the presence or absence of thyroid-stimulating hormone (TSH). Neither TNF-alpha nor IL-2 affected thyroid cell survival, IFN-gamma was usually inhibitory and IL-1 alpha slightly enhanced cell survival in some experiments. However, the effects were small and variable and were not enhanced by potentially synergistic combinations of cytokines, longer periods of exposure, or different culture conditions. In contrast, IFN-gamma, IL-2 and TNF-alpha inhibited the ability of thyroid lymphocytes from patients with Graves' disease and Hashimoto's thyroiditis to synthesize autoantibodies to thyroid peroxidase (TPO) and thyroglobulin (Tg). Comparison of lymphoid populations isolated by digestion and/or mechanical disaggregation indicated that a population of activated B cells, plasma cells and T cells, intimately associated with thyroid cells since they could only be extracted by digestion, was influenced by cytokines. Our studies suggest that in addition to its well-recognized ability to induce MHC class II antigens on thyroid cells, IFN-gamma may inhibit thyroid cell proliferation and TNF-alpha, IFN-gamma and IL-2 may down-regulate thyroid autoantibody synthesis.
Autoantibodies↗
Thyroid peroxidase and the induction of autoimmune thyroid disease.
Animal models of autoimmune thyroid disease are associated with thyroglobulin (Tg) as autoantigen whereas in man the autoimmune response to microsomal antigen/thyroid peroxidase (TPO) appears to play a major role in thyroiditis. Consequently, we have compared the ability of TPO and Tg to induce thyroid autoantibodies and thyroid damage in mice known to be susceptible (CBA/J) or resistant (BALB/c) to thyroiditis induced using murine Tg. Groups of three to five mice were immunized twice using Freund's complete adjuvant with 80-100 micrograms highly purified porcine (p) TPO, pTg, rat (r) Tg, human Tg, bovine serum albumin (BSA) or BSA + 0.2 micrograms pTg (the level of Tg contamination of TPO). Four weeks after immunization with TPO, plasma from CBA/J (but not BALB/c) mice contained IgG class antibodies which bound to TPO-coated tubes in the presence or absence of excess Tg (and could therefore be clearly distinguished from Tg antibodies) but there was no evidence of thyroiditis in either strain of mice. In contrast, in CBA/J mice immunized with rTg and, to a lesser extent in mice that had received pTg, thyroid tissue was infiltrated with lymphoid cells and/or neutrophils and antibodies to pTg (but not pTPO) were present. Our observations demonstrate that induction of TPO antibody alone is insufficient to lead to thyroiditis in CBA/J mice. Further, these studies emphasize the complex interactions between MHC and different thyroid antigens in the processes leading to thyroid destruction.
Animals↗
Thyroid autoantigens and human T cell responses.
We investigated the ability of T cells from patients with Hashimoto's thyroiditis and with Graves' disease as well as control donors to proliferate in response to thyroid peroxidase (TPO) and thyroglobulin using (i) lymphoid cells from different lymphoid organs; (ii) unfractionated or CD8- depleted lymphoid suspensions or T cells + autologous low density cells (LDC); (iii) 200-microliters well cultures and 20-microliters hanging-drop microcultures; and (iv) intact TPO and thyroglobulin, denatured thyroglobulin and 12 synthetic peptides predicted on the basis of the amino acid sequence of TPO to be T cell epitopes. In 200-microliters well cultures, proliferative responses (assessed in terms of 3H-thymidine uptake) to intact TPO or thyroglobulin, digested thyroglobulin or synthetic TPO peptides were not significantly different in unfractionated or CD8-depleted lymphoid suspensions from blood, thyroid or lymph nodes of TPO/thyroglobulin autoantibody-positive patients, autoantibody-negative patients or control donors. In contrast, blood T cells from some high titre patients with Hashimoto's thyroiditis (but not from healthy individuals) proliferated in response to intact thyroglobulin or TPO presented by autologous LDC in hanging-drop microcultures. Heat denatured thyroglobulin (with which thyroglobulin autoantibodies do not interact) did not stimulate proliferation and this observation, together with the ability of T cells from some patients to respond to intact thyroglobulin in the absence of LDC, indicated that thyroglobulin-specific B cells may be involved in antigen presentation. As we were unable to demonstrate proliferation by blood T cells + LDC from all thyroglobulin antibody-positive patients with Hashimoto's thyroiditis, our studies suggest that the presence of sufficient precursor T cells, as well as the number and type of antigen-presenting cells, are critical for T cell proliferative responses to human TPO and thyroglobulin.
Antigen-Presenting Cells↗
Structural analysis of the TSH receptor.
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Animals↗
Immunoprecipitation of human adrenal microsomal antigen.
Human adrenal microsomes have been labelled with 125I and immunoprecipitated with sera from patients with Addison's disease. The immunoprecipitates were then analysed by SDS-PAGE and autoradiography. 13 of the 23 sera from the Addison patients studied contained antibodies which reacted with a 55 kDa adrenal microsomal protein. The same 13 sera were also positive for adrenal antibodies as judged by immunofluorescence. The 55 kDa protein was not immunoprecipitated from placenta or thyroid microsomes by Addison sera. Furthermore, patients with Graves' disease or rheumatoid arthritis did not immunoprecipitate the 55 kDa protein from adrenal microsomes. Our studies suggest therefore that Addison sera contain antibodies to a 55 kDa adrenal specific protein which may well be the antigen observed on immunofluorescence.
Addison Disease↗
Analysis of TSH receptors and microsomal antigen in different human thyroid tissue specimens.
Affinity labelling with a 125I-labelled photoactive derivative of TSH (HSAB-TSH) was used to analyse TSH receptor size in the following specimens of human thyroid tissue: (1) cold nodules; (2) autonomous nodules; (3) papillary carcinoma; (4) medullary carcinoma; (5) metastasis of papillary carcinoma to lymph node; (6) anaplastic carcinoma, and (7) Graves' thyroid. In addition, a sample of histologically normal thyroid tissue surrounding specimens 1-4 was analysed in each case. Thyroid microsomes were also prepared from the tissue samples, solubilized using 1% deoxycholate and labelled with 125I. The preparations were immunoprecipitated using microsomal autoantibodies and protein A and analysed by SDS-PAGE and autoradiography. These studies indicated that no differences in the characteristics of the TSH receptor or of microsomal antigen were observed in the tissue samples 1-3 and 7. Neither protein was detected in tissue specimens 4-6.
Autoantigens↗
Autoantibodies to the thyrotropin receptor.
This review considers recent developments in our understanding of the properties of TRAb, particularly measurement of the antibodies and their sites of action and synthesis. Two new assay methods have allowed considerable improvements in the sensitivity, specificity, precision, and ease of measuring TRAb. In particular: 1) receptor assays based on inhibition of receptor-purified labeled TSH binding to detergent-solubilized TSH receptors and 2) bioassays based on stimulation of cAMP release from monolayer cultures of isolated thyroid cells. Detailed studies with the two assays indicate that TSH receptor antibodies nearly always act as TSH agonists in patients with a history of Graves' hyperthyroidism. Studies in areas of dietary iodine sufficiency suggest that measurement of the antibodies at various stages in the course of treating Graves' disease can be of value in predicting the outcome of therapy. However, in areas of iodine deficiency, difficulties in the ability of patients' thyroid tissue to recover from the effects of antithyroid drugs may prevent the receptor antibodies from causing a relapse of thyrotoxicosis. Consequently, the predictive value of receptor antibody measurements would be expected to be lower in these geographical areas. Although patients with a history of Graves' hyperthyroidism nearly always have TRAb which act as TSH agonists, about 20% of patients with frank hypothyroidism due to autoimmune destruction of the thyroid have TRAb which act as TSH antagonists (blocking antibodies). There is some evidence that these blocking antibodies can cause hypothyroidism particularly in the neonate. With regard to the site of synthesis of TRAb, there is now direct evidence that they are synthesized by thyroid lymphocytes, particularly the lymphocytes in close proximity to thyroid follicular cells. This is consistent with the well established effects of antithyroid treatment (drugs, radioiodine, or surgery) on TRAb levels in addition to their effects on thyroid hormone synthesis. Recent studies using affinity labeling with 125I-labeled TSH have enabled elucidation of the structure of the TSH receptor. TSH receptors in human, porcine, and guinea pig thyroid tissue have a two-chain structure in which the TSH binding site is formed on the outside surface of the cell membrane by a water-soluble A subunit (Mr approximately 50 K). The A subunit is linked by a disulfide bridge and weak noncovalent bonds to the amphiphilic B subunit (Mr approximately 30 K). This subunit, which penetrates the lipid bilayer, probably forms the site for interaction of the receptor with the regulatory subunits of adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)
Antigen-Antibody Reactions↗
TSH receptor antibodies in autoimmune thyroiditis.
Out of 2,322 patients attending a thyroid clinic in north west Germany over a three year period, 123 were found to have evidence of autoimmune thyroiditis (hypothyroid, latent hypothyroid or euthyroid) and 96 were available for further analysis. TSH receptor antibodies (TRAb) were detectable by receptor assay in five of these patients (and a further two who attended the clinic later) and all the TRAb positive sera showed TSH blocking activity by bioassay. All of the patients with blocking activity were hypothyroid (on treatment) and represented 15% of this group of 34 patients. This suggests that in hypothyroid patients with autoimmune thyroiditis, the prevalence of TSH receptor antibodies with blocking activity is similar in northern Europe and Japan (21% of 43 patients; (1]. In the present study, no relationship between thyroid volume as assessed by sonography and the presence or absence of blocking antibodies was apparent. As blocking antibodies were undetectable in patients at early stages of the disease (i.e., in the euthyroid or latent hypothyroid groups) it seemed unlikely that these antibodies were a major causative factor in the development of hypothyroidism.
Adult↗
The subunit structure of the thyrotropin receptor.
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Animals↗
IgG subclass distribution of thyroid autoantibodies: a 'fingerprint' of an individual's response to thyroglobulin and thyroid microsomal antigen.
The IgG subclass distribution of autoantibodies to thyroglobulin and thyroid microsomal antigen was studied in 21 patients with Graves' disease during fluctuations in total IgG class autoantibody levels induced by various forms of therapy. In addition, changes in autoantibody subclass distributions were investigated during the natural course of Hashimoto's disease in seven patients taking thyroxine. The autoantibodies were principally of subclasses IgG1 and/or IgG4 in Graves' patients although IgG2 contributed significantly to thyroglobulin antibodies in 5/7 Hashimoto sera. In Graves' disease the distribution of microsomal and thyroglobulin antibodies among the IgG subclasses remained essentially unchanged over periods of 6 months-2 years whether autoantibody levels decreased during carbimazole therapy or increased transiently following 131Iodine treatment or subtotal thyroidectomy. Similar observations were made for thyroglobulin antibodies in Hashimoto patients studied over 2 1/2-4 years; furthermore, the IgG subclass distribution of microsomal antibodies was usually different from that of thyroglobulin antibodies in the same patient. These observations suggest that the microsomal and/or thyroglobulin antibody subclass distribution is characteristic for a particular individual and may be regarded as the 'fingerprint' of an individual's response to these thyroid autoantigens.
Adolescent↗
The TSH receptor: structure and interaction with autoantibodies in thyroid disease.
Studies of the TSH receptor using affinity labelling with photoactive derivatives of TSH and analysis by SDS-PAGE have shown that the receptor contains 2 subunits (A and B), linked by a disulphide bridge. Similar results are obtained with TSH receptors from human, porcine and guinea pig thyroid tissue and from guinea pig fat. Analysis of affinity labelled receptors under non-denaturing conditions suggest that subunits additional to the A and B subunits are not present. Hydrodynamic measurements indicate that the receptor A subunit has an approximately spherical structure (Stokes' radius 70 A) and when this interacts with TSH (an elongated structure with Stokes' radius 56A) a very elongated complex (Stokes' radius 104A) is formed. Isoelectric focusing studies of the TSH receptor A subunit, TSH and TSH receptor antibodies indicate that charge-charge interactions are of considerable importance in the binding of hormone and antibody to the receptor.
Animals↗
Antibodies to membrane antigens in autoimmune thyroid disease.
The possibility that sera from patients with autoimmune thyroid diseases contain autoantibodies to thyroid membrane proteins distinct from microsomal antigen and the TSH receptor has been investigated using (a) solid phase assay system based on human thyroid membranes and 125I-labelled protein A and (b) immunoprecipitation of detergent solubilized 125I-labelled thyroid membranes followed by gel electrophoresis and autoradiography. In the solid phase assay binding to membranes showed a highly significant correlation with binding to microsomes (r = 0.82; P less than 0.001; N = 82) indicating that the interaction between the serum and the membranes was due principally to microsomal antibody binding to microsomal antigen contaminating the membrane preparations. However, there were some discrepancies suggesting that an additional antigen-antibody system was involved. This possibility was then investigated using immunoprecipitation of 125I-labelled thyroid membranes. A labelled protein with mol wt 54 K was specifically immunoprecipitated (relative to normal pool serum) by 3 out of 4 sera from patients with Graves' disease who showed high binding to thyroid membranes. A further 4 sera from such patients with low membrane binding affinity failed to immunoprecipitate the 54 K protein. Sera from some patients with Hashimoto's disease and some patients with rheumatoid arthritis and one patient with Addison's disease also immunoprecipitated the 54 K protein from solubilized thyroid membranes. These studies suggested that antibodies interacting with the 54 K protein contributed to the discrepancies between thyroid membrane and microsome binding. However, the 54 K protein was also immunoprecipitated from detergent solubilized membranes prepared from human placenta, skeletal muscle and adrenal tissue.(ABSTRACT TRUNCATED AT 250 WORDS)
Antigens↗
A technique for the isolation and mitogenic activation of thyroglobulin-specific human B lymphocytes.
In previous studies we demonstrated that Hashimoto peripheral blood lymphocytes enriched for thyroglobulin (Tg) binding activity could be activated to secrete increased amounts of Tg antibody by Epstein - Barr virus (EBV) but not by pokeweed mitogen (PWM). We now report an investigation into the requirements for the isolation of Tg receptor positive (TgR+) B cells capable of being stimulated by PWM. The interaction between Hashimoto lymphocytes and Tg coated erythrocytes followed by red cell lysis interfered with the ability of the population to synthesize Tg antibody. However, this could be overcome if the rosettes formed between Tg coated erythrocytes and the Tg receptors on B cells were dissociated by digestion followed by red cell lysis and overnight incubation before the addition of PWM. Using this approach, Hashimoto TgR+ B cells could be stimulated by the mitogen to secrete immunoglobulin with a higher Tg antibody specific activity than unfractionated lymphocytes. Consequently, enriched populations of antigen specific human B cells capable of responding to mitogenic signals can be prepared by a positive selection technique.
Antibody Formation↗