Sandra M. McLachlan

Carbimazole and the Autoimmune Response in Graves' Disease

Microsomal antibodies and antibodies directed toward the receptor for thyroid-stimulating hormone (TSH) decreased in parallel while patients with Graves disease were taking carbimazole, whereas no significant changes were observed during treatment with placebo or propranolol. The changes in autoantibody levels during carbimazole treatment were independent of changes in serum thyroxine and could have been due to a direct effect of the drug on autoantibody synthesis. Evidence for this suggestion was provided when low doses of methimazole (the active metabolite of carbimazole) were found to inhibit thyroid-autoantibody production in cultured lymphocytes. Since thyroid lymphocytes are probably a major site of thyroid-antibody synthesis in Graves disease and methimazole is concentrated in the thyroid during treatment, a local action of the drug on antibody production seems likely. This possibility could be important in the use of carbimazole to control hyperthyroidism.

An enzyme-linked immunoassay for thyroid microsomal antibodies

An enzyme-linked immunoassay (ELISA) for microsomal antibody is described. The method was found to be rapid, sensitive and precise and analysis of 115 serum samples showed good correlation between the ELISA and the conventional tanned red cell haemagglutination test. The presence of thyroglobulin antibody, rheumatoid factor, antinuclear factor or gastric parietal cell antibodies did not interfere in the microsomal antibody ELISA but some sera with mitochondrial antibody activity appeared to cause a non-specific effect. The ELISA was particularly useful for analysing microsomal antibody production by Hashimoto lymphocyte cultures and in some cases antibody synthesis could be studied in the absence of mitogen. The high capacity of the ELISA combined with its sensitivity suggest that it will be a valuable technique for studying microsomal autoantibody activity both in serum and in lymphocyte cultures.

Thyrotropin receptors in adipose tissue, retro-orbital tissue and lymphocytes.

Thyrotropin (TSH) receptors on retro-orbital muscle and fat have been implicated in the pathogenesis of Graves exophthalmos and it has been suggested that TSH has a direct effect on human fat metabolism. We have therefore investigated the interaction of biologically active 125I-labelled TSH with membranes prepared from human adipose, retro-orbital and thyroid tissue. Since lymphocytes contain receptors for several polypeptide hormones, TSH binding to lymphocyte membranes was also studied. We were unable to demonstrate TSH receptors in adult human adipose tissue, retro-orbital muscle and fat, or peripheral blood lymphocytes. In contrast, adult and neonatal guinea pig adipose tissue membranes showed similar TSH binding characteristics to guinea pig thyroid membranes.

Studies of thyroglobulin autoantibody synthesis using a micro-ELISA assay

Thyroglobulin autoantibody synthesis by Hashimoto lymphocyte cultures has been studied using an ELISA, a plaque assay and tanned red cell haemagglutination. The ELISA system was found to be the most suitable and using this method IgG-class thyroglobulin antibody synthesis was detectable in cultures of mitogen-stimulated lymphocytes from all 10 Hashimoto patients studied, but not in cultures of lymphocytes from 4 normal donors. The ELISA was also sufficiently sensitive to detect thyroglobulin antibody synthesis in mitogen-free lymphocyte cultures from 4 out of the 10 Hashimoto patients and consequently it should be possible to use this system to study the effect of various pathophysiological factors on autoantibody synthesis which would otherwise be masked by mitogenic stimulation.

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 Hashimotos disease in seven patients taking thyroxine. The autoantibodies were principally of subclasses IgGl 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 13lIodine treatment or subtotal thyroidectomy. Similar observations were made for thyroglobulin antibodies in Hashimoto patients studied over 2½‐4 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 individuals response to these thyroid autoantigens.

Kinetics of immunoglobulin production by cultured human peripheral blood lymphocytes

A study of immunoglobulin synthesis by human peripheral blood lymphocytes cultured in Marbrook flasks is described. Maximal amounts of immunoglobulin were produced by 3 weeks and most of this was synthesised between 6 and 21 days. In the region of 90% of the immunoglobulin was secreted into the medium. Pokeweed mitogen (PWM) stimulated cultures produced about 10 times as much immunoglobulin as cells cultured in medium only. After 21 days stimulated cultures produced about 150 microgram of IgG, 125 microgram of IgM and 50 microgram of IgA per 10(7) cells.

The IgG Subclass Distribution of Thyroglobulin Antibody Synthesized in Culture

Thyroglobulin autoantibodies synthesized by Hashimoto lymphocytes in culture and present in serum have been analysed in terms of their IgG subclass distribution. The autoantibodies produced in vitro were frequently IgG4 or IgG1. whether pokeweed mitogen or Epstein‐Barr virus was used to stimulate the cultures, and the subclass distribution of these thyroglobulin antibodies was similar to that observed in the patientsserum. It appears therefore that the antibodies synthesized in vitro in response to polyclonal B‐cell activators resemble those produced in vivo, and it seems likely that both pokeweed mitogen and Epstein‐Barr virus influence the same B‐cell precursors of autoantibody‐synthesizing celts, albeit by different mechanisms.

HLA CLASS II DNA GENOTYPES IN GRAVES' DISEASE: CLUES TO INHERITANCE OF THE HLA‐LINKED COMPONENT OF SUSCEPTIBILITY

Restriction fragment length polymorphism analysis using DQα, DQβ and DRβ cDNA probes was performed in Graves disease patients and control subjects. The following restriction fragment patterns were increased in frequency in patients compared with control subjects: 10 + 70 + 4.0kb DRβ/Taq1 fragments (66%vs32%; P < 0.01;corrected P< 0.06),7.0+4.0 kbDQβ/BamHI fragments (55%vs 15%; P< 0.001; corrected P<0006), and a DQα/TaqI 4.6kb fragment (75%vs 36%; P< 0.005; corrected P < 0.02). These associations could be accounted for by the known association of the B8‐DR3 haplotype with the disorder. No non‐DR3‐related restriction fragment pattern was associated with the disease using any of the probes with restriction enzymes TaqI and BamHI.

The development of large primordia inVicia faba L.: Some cytological and anatomical changes

SummaryVarious cytological and anatomical measurements have been made on large primordia (LP) and newly emerged lateral roots (JE) ofVicia faba. The results indicate that two distinct periods of quiescence occur during the development of LP. The first of these takes place just prior to the formation of a cavity in the cortex of the primary root adjacent to the LP, while the second occurs shortly before the LP emerges as a lateral root. It is suggested that both periods of quiescence are induced as a result of pressure built up between the expanding LP and the adjacent cells of the primary root. LP growth was a result of both cell division and cell expansion, both of these parameters being greater at some stages of development than at others. The formation of a root cap began before LP emergence as a lateral root, but the number of cells in the cap remained small until the latter event took place.

Lateral root elongation inVicia faba L.

SummaryMitotic index (MI) was determined in the cap, epidermis, cortex and stele at successive intervals along the apical mm of lateral roots of various specific lengths. This parameter was found to decrease basally along roots of each length examined and to decline as the lateral root elongated. Moreover, MI was different in the various tissues investigated. These results have been discussed with respect to changes in the other parameters of cell proliferation and to root growth by elongation. It is suggested on the basis of the data obtained, and other results in the literature, that epidermal initial cells could be differentiated from those of the cortex and stele before the quiescent centre appeared in these roots, but the initial cells of the latter two tissues were indistinguishable until after the quiescent centre began to form.Mitotic index (MI) was determined in the cap, epidermis, cortex and stele at successive intervals along the apical mm of lateral roots of various specific lengths. This parameter was found to decrease basally along roots of each length examined and to decline as the lateral root elongated. Moreover, MI was different in the various tissues investigated. These results have been discussed with respect to changes in the other parameters of cell proliferation and to root growth by elongation. It is suggested on the basis of the data obtained, and other results in the literature, that epidermal initial cells could be differentiated from those of the cortex and stele before the quiescent centre appeared in these roots, but the initial cells of the latter two tissues were indistinguishable until after the quiescent centre began to form.