Thyroglobulin and microsomal antibodies in patients with insulin dependent diabetes mellitus and their relatives.

The sera for 88 parents and 9 siblings of 73 patients with insulin dependent diabetes mellitus in childhood and 437 controls matched in age and sex, were tested by the thyroglobulin and microsome-coated tanned red cell hemagglutination test (Fuji-Zoki Co. Tokyo). None of 73 children with diabetes mellitus had antithyroglobulin antibodies, whereas twelve (16.4%) had antimicrosomal antibodies compared with the incidence of 0.4% and 1.1%, respectively, in 437 controls. In the parents and siblings of these probands, thyroid antibodies were also found in increased incidence. The incidence of antimicrosomal antibodies in the 68 mothers was significantly higher than in controls matched for age and sex, but the incidence of the positive thyroid antibodies in the 20 fathers and 9 siblings was not significantly different from that in control populations. The incidence of thyroid antibodies tended to be higher, though not significant, in parents and siblings of diabetic children with positive thyroid antibodies than in those of diabetics with negative ones. These findings suggest that immunogenetic factors may be responsible for the pathogenesis of some cases of diabetes mellitus in childhood.     

Circulating thyroglobulin and antithyroglobulin antibodies after fine needle aspiration of thyroid nodules

Circulating thyroglobulin and antithyroglobulin antibodies were assayed in fifteen patients affected by solitary "cold" nodules of the thyroid who had undergone fine needle aspiration for cytologic diagnosis. The aim of the study was to investigate whether a minor insult to thyroid tissue such as fine needle aspiration is able to release thyroglobulin into the circulation and to induce the formation of antithyroglobulin antibodies. The results obtained are as follows: 1. Circulating thyroglobulin increased substantially after fine needle aspiration in 11 out of 15 patients. 2. Antithyroglobulin antibodies did not appear in any patient during a follow-up period of two months. 3. The magnitude of basal thyroglobulin and thyroglobulin increase after needle aspiration were unrelated to cytologic diagnosis.     

Human leukocyte antigen (HLA) complex and anti-thyroidal antibodies in Graves' disease

The study aimed at: 1) assessing occurrence of HLA-A, HLA-B and HLA-C antigens in patients with Graves' disease in comparison with control group of healthy individuals; 2) determining relationship between circulating serum antimicrosomal and antithyroglobulin antibodies and selected HLA complex antigens. Human leukocyte antigens A, B, and C were detected with serological technique using cytotoxicity test. Thyroidal antimicrosomal and antithyroglobulin antibodies were titrated with radioimmunological solid phase technique while anti-membrane antibodies with immunoenzyme technique. The study involved 50 patients with Graves' disease and 50 healthy individuals. HLA-B8, HLA-B15, HLA-B35, and HLA-Cw3 antibodies were detected more frequently in patients with Graves' disease than in the healthy individuals. Antimicrosomal and antithyroglobulin antibodies were detected in the same group in 76% and 58% of patients, respectively whereas anti-membrane antibodies in 92% of patients. Comparison of the occurrence of thyroidal antimicrosomal and antithyroglobulin antibodies with the presence of HLA-B8, HLA-B35, HLA-B15, and HLA-Cw3 antigens did not show statistically significant correlation between these two parameters.     

Measurement of autoantibodies against human eye muscle plasma membranes in Graves' ophthalmopathy.

Antibodies that reacted with plasma membranes of human eye muscle but showed no binding to plasma membranes of human skeletal muscle were identified in serum of patients with Graves'' ophthalmopathy. Rabbit antithyroglobulin serum at a dilution of 1 X 10(-3) to 20 X 10(-3) had no effect on the binding of these antibodies to eye muscle membrane antigens. There was no correlation between antihuman eye muscle plasma membrane antibodies and antihuman thyroid membrane antibodies or antibodies against thyroglobulin. It is suggested that specific antibodies against eye muscle membranes are present in Graves'' ophthalmopathy and that this disease might represent a distinct autoimmune disorder.     

Determination of antithyroid antibodies by an immunoenzymatic method; an adaptation for expressing results in international units]

Solid phase immunoenzymatic methods (ELISA) have been developed for the determination of antithyroglbulin (ATG), antimicrosomal (AMc) and antimembrane (ATMA) antibodies in blood serum. The results have been expressed in international units (IU). The level of nonspecific reaction was determined on the basis of 30 samples of blood serum obtained from healthy donors. The double standard deviation values amounted to 8 IU for antithyroglobulin antibodies, 17 IU for antimicrosomal antibodies and 53 IU for antimembrane antibodies at the serum dilution of 1:100. The values of double standard deviation obtained for the healthy donors correspond to the borderline between the positive serum samples and those containing no autoantibodies. The level of autoantibodies in patients with autoimmune diseases of the thyroid varied considerably ranging from complete absence to several thousand units per milliliter in single cases. Antithyroglobulin antibodies were determined simultaneously by using the described method and the commercial kit (Walker, Cambridge) and the results obtained by the two methods were compared. A linear correlation with the correlation coefficient r = 0.93, p < 0.001 was obtained. A good but nonlinear correlation was demonstrated with the methods expressing the results in titre values.     

The use of monoclonal antibodies against thyroglobulin in immunoperoxidase techniques

A monoclonal mouse antibody to human thyroglobulin was used in a modified biotin-peroxidase-complex method to demonstrate thyroglobulin in different tissues and tumors of the thyroid gland. Mouse ascites fluid containing monoclonal antibodies could be diluted up to 1:1,600 for detection of thyroglobulin, whereas no staining was observed in a medullary carcinoma. Nonspecific background staining was negligible. These results demonstrate that monoclonal antibodies against thyroglobulin are suitable in the immunohistochemical localisation of thyroid hormones.     

Immunoenzymatic method for determining thyroglobulin levels in human blood serum]

An inexpensive enzyme immunoassay method was designed for the determination of thyroglobulin concentration in human blood serum. The range of concentrations of thyroglobulin which can be measured by the method is between 6 and 800 ng/ml. The sensitivity of the method is comparable to that of the commercial test kits. The values of thyroglobulin concentration obtained with the use of the described method are strongly correlated (r = 0.946) with those obtained by using the reference method (IRMA kit of Byk, Sweden). The intraassay coefficient of variation ranged from 5.5 to 10.2% and interassay coefficient of variation from 9.5 to 13.2% depending on the thyroglobulin concentration. The upper limit of blood serum thyroglobulin concentration in healthy subjects was 70 ng/ml. The results of thyroglobulin determination obtained with the described method are falsely lowered in the presence of antithyroglobulin antibodies; simultaneous determination of these antibodies is thus necessary in such a case. It seems that the described method may be used for monitoring the patients after surgical treatment of differentiated thyroid cancer aimed at early detection of metastases.     

The thyroid "microsomal" antigen is an epitope on the thyrotropin receptor

Antimicrosomal antibodies are present in the sera of most patients with autoimmune thyroiditis, and Graves' disease. It has, in general, been difficult to separate antimicrosomal activity from that directed against the thyrotropin (TSH) receptor in Graves' IgG preparations. The "microsomal" antigen has been localized to the endoplasmic reticulum and microfollicular aspect of thyrocytes; its structure is however unknown. In an attempt to identify the thyroid microsomal antigen, we studied the interaction of Hashimoto's IgG with high microsomal antibody titre and negative for thyroglobulin with purified thyroid plasma and light microsomal membranes. We allowed Hashimoto's, Graves', and control IgGs to bind to protein blots of thyroid plasma membranes resolved on SDS-PAGE under non-reducing conditions. All seven Hashimoto's IgG at a concentration of 2 mg/ml interacted with an M approximately 197,000 polypeptide corresponding to the TSH holoreceptor. By contrast to Graves' IgG (which were positive at 1 mg/ml), however, this binding was not blocked by pretreatment of the protein blots with TSH. Normal IgGs showed no binding at concentrations of up to 2 mg/ml. Both Hashimoto's and Graves' IgG interacted with TSH-affinity column-purified receptor preparations. Two of the Hashimoto's IgGs induced adenylate cyclase activation in thyroid plasma membranes, three inhibited TSH-stimulated enzyme activation, and two were without effect. Two classes of autoantibodies, other than TSH receptor directed, were encountered; one class raised to antigens common to all seven patients and another class unique to individual patients, eg, Mr 210,000 and Mr 20,000 polypeptides. We propose that the TSH receptor has multiple epitopes (functional domains), and the one to which antimicrosomal antibody bind is likely to be spatially separated from that with which Graves' IgG and TSH interact. Differences in affinity or number of sites allows for the demonstration of Graves' IgG against a background of antimicrosomal antibody.     

Antimicrosomal antibodies, gastric parietal cell antibodies and antinuclear factors in insulin dependent diabetes mellitus.

Thyroid antimicrosomal antibodies, gastric parietal cell antibodies (PCA) and antinuclear factors were studied in 208 insulin dependent diabetic (IDD) according to the duration of diabetes and patient's age at the time of testing. Antimicrosomal antibodies were found in 11 out of 47 (23.4%) IDD with the duration of less than one year, however this value declined to 13.1% at 1 to 3 years, 15.3% at 4 to 5 years, 10.8% at 6 to 10 years and 5.8% at more than 10 years. Of the 47 IDD, 7 (14.8%) were positive for gastric parietal cell antibodies. The prevalence of PCA declined with increasing duration of diabetes. However, this decrease in the prevalence of antimicrosomal antibodies and PCA was not so extreme as that of pancreatic islet cell antibodies. Antinuclear factors did not reveal a significant correlation with the duration of diabetes. In normal controls, the prevalence of antimicrosomal antibodies, PCA and the antinuclear factors increased progressively with age. In IDD, the prevalence of the antinuclear factors was also progressively greater with age. However, the prevalence of antimicrosomal antibodies in IDD decreased with age and those of PCA showed the lowest percent in the 40-69 year-age group.     

Adipocyte-TSH-receptor-related antibodies in Graves' disease detected by immunoprecipitation

Fat cell TSH receptor-related antibodies were detected by immunoprecipitation of 125I-TSH-receptor complexes and the nature of the antibodies was analyzed. To 125I-TSH prebound to Triton-solubilized receptors from guinea pig fat tissues, 50 micrograms of immunoglobulin G (IgG) was added and precipitation was effected by the addition of antihuman IgG. Immunoprecipitation values in 13 patients with Graves' disease were significantly (p less than 0.05) higher than those in 11 normal subjects. No significant increase in the values was seen in 8 patients with Hashimoto's disease. No correlation was observed between immunoprecipitation values and titers of antimicrosomal and antithyroglobulin antibodies. Neither was there any correlation between the values and TSH-binding inhibitor immunoglobulins (TBII) detected by the radioreceptor assay. The IgG fractions positive for the immunoprecipitation antibody were found to be poor human thyroid stimulators (HTS) relative to their TBII activities. And a highly significant correlation was observed between TBII and HTS activities among IgGs without detectable antibody by immunoprecipitation (r=0.907; p less than 0.005; n=7). These findings 1) demonstrate that immunoprecipitation assay using fat cell TSH receptor may detect TSH receptor-related antibodies different from TBII in patients with Graves' disease and 2) suggest the antibodies may recognize determinants on the receptor or its vicinity that do not participate in the binding of TSH or thyroid stimulating antibody, and may interfere with thyroidal response to these stimulators.     

Eye muscle antibodies in endocrine exophthalmos. Clinical and serological observations

Serum samples were obtained from 65 patients with endocrine exophthalmos class I-V. In 33/65 patients who were treated either with prednisone or with ciclosporin, blood was sampled before, during and after therapy. Antibodies against eye muscle were determined during the course of immunosuppressive therapy in order to have an objective parameter of the therapeutic effect. To ascertain the specificity of the reaction both eye and abdominal muscles were used as antigens in an ELISA system. Both IgG and IgM antibodies were detected. In 45/65 patients (71%) eye muscle antibodies were positive before starting therapy. Antibodies were mostly detected in patients with active disease. Patients with exophthalmos of recent onset always had IgM antibodies whereas patients with chronic exophthalmos were mostly IgG positive. Patients with relapse showed mostly IgG but also IgG and IgM positivity in 2 cases. In 58% of cases only IgG antibodies were found whereas in 34% both IgG and IgM were detected and in 8% only IgM antibodies. There was no association between antibodies directed against eye muscle and thyroid microsomal and thyroglobulin antibodies or with the state of thyroid function. Furthermore there was no correlation between exophthalmos classes and eye muscle antibody binding activity. The antibody level declined during therapy with prednisone or with ciclosporin but rose again 8-12 weeks after stopping the drug in patients with progressive disease.     

Anti-Helicobacter Pylori, anti-thyroid peroxidase, anti-thyroglobulin and anti-gastric parietal cells antibodies in Czech population

Autoimmune thyropathies are frequently linked to many infections, such as Helicobacter pylori, which are also supposed to play a role in their pathogenesis. The aim of this study was to evaluate the relationships between thyroid and gastric autoimmunity and H. pylori infection on a large sample of Czech population (n=1621) by monitoring the autoantibodies against thyroglobulin (anti-Tg) and thyroid peroxidase (anti-TPO) and gastric parietal cell (anti-GPC, representing thyrogastric syndrome) in correlation with antibodies against Helicobacter pylori (anti-H. pylori) of classes IgG and IgA. The interrelation between autoantibodies and H. pylori antibodies was assessed by H. pylori seropositivity. In H. pylori seropositive persons as compared to seronegative irrespective of age and sex, a higher occurrence of anti-TPO (10.4 % vs. 5.8 %, p=0.001) and anti-GPC (6.1 % vs. 1.7 %, p<0.001) was found. Differences in anti-TPO occurrence were significant in both men (7.0 % vs. 3.3 %, p=0.03) and women (12.7 % vs. 8.0 %, p=0.02), differences in anti-GPC occurrence were significant only in women (7.2 % vs. 1.7 %, p<0.001). Results of this study support the idea of a connection between infection of H. pylori and the occurrence of anti-TPO autoantibodies representing thyroid autoimmunity and gastric parietal cells autoantibodies representing the thyrogastric syndrome.     

Synthesis of thyroglobulin in the polyribosome cell-free system of the thyroid gland]

Protein synthesis (in particular, thyroglobulin) is studied in a cell-free system containing polyribosomes of thyroid gland. After the incubation with radioactive label, ribosomes were centrifugated, supernatant was dialyzed against 0.05 M Tris-HCl buffer, pH 7.2 for a night, concentrated by ficol and centrifugated in 5-20% linear sucrose gradient. 14C-radioactivity of 19S fraction (thyroglobulin) was shown to comprise about 25% of total radioactivity. The rest radioactivity was found in 3--8S fractions. The distribution of the radioactivity as it was shown by polyacrylamide gel electrophoresis is as follows: 27S--11.0%; 19S--14.2%; 12S--5.8% and 4S--7.6% of total radioactivity in polyacrylamide gel. It is supposed that two successive processes take place in thyroid gland: a synthesis of thyroglobulin subunits and an exchange of new synthesized subunits with presynthesized ones.     

Autoimmune disorders in liqvidators 11 years after the Chernobyl accident

We studied long-lasting consequences of the low-doses irradiation on the immune system of 71 clean-up workers who participated in the emergency work after the Chernobyl Plant accident in 1986 and 25 healthy donors from Belarus. In sera of the workers the level of autoantibodies to thyroid gland antigens (thyroglobulin and microsomal fraction of thyroid gland) was increased in 48% of cases, the level of autoantibodies to lens oculis antigen was increased in 44% of cases; the level of circulating immune complexes was elevated in 55%, and the serum level of thyroglobulin in 60% of people. Immunological disorders were found without any definite clinical evidences of diseases and this allows us to consider the examined contingent as a group of risk for the development of autoimmune pathology in the future.     

Noradrenergic and peptidergic innervation of the mammary gland in the immature pig

The presence and pattern of coexistence of some biologically active substances in nerve fibres supplying the mammary gland in the immature pig were studied using immunohistochemical methods. The substances studied included: protein gene product 9.5 (PGP), tyrosine hydroxylase (TH), somatostatin (SOM), neuropeptide Y (NPY), galanin (GAL), calcitonin gene-related peptide (CGRP) and substance P (SP). The mammary gland was found to be richly supplied by PGP-immunoreactive (PGP-IR) nerve fibres that surrounded blood vessels, bundles of smooth muscle cells and lactiferous ducts. The vast majority of these nerves also displayed immunoreactivity to TH. Immunoreactivity to SOM was observed in a moderate number of nerve fibres which were associated with smooth muscles of the nipple and blood vessels. Immunoreactivity to NPY occurred in many nerve fibres associated with blood vessels and in single nerves supplying smooth muscle cells. Solitary GAL-IR axons supplied mostly blood vessels. Many CGRP-IR nerve fibres were associated with both blood vessels and smooth muscles. SP-IR nerve fibres richly supplied blood vessels only. The colocalization study revealed that SOM, NPY and GAL partly colocalized with TH in nerve fibres supplying the porcine mammary gland.     

Autoantibodies to thyroid gland antigens in chronic relapsing urticaria

The content of total IgE, antibodies to thyroglobulin (TG-Ab), antibodies to thyreoid peroxidase (TPO-Ab) in the blood serum and skin reaction to autologous serum were detected in patients with chronic relapsing urticaria (CRU). The skin test to autologous serum yielded positive results in 42% of the patients. The elevated levels of TG-Ab and TPO-Ab were detected in 30.7% and 35.4% of the patients, the elevated level of total IgE was detected in 60% of the patients. At the same time the detection rates of antithyreoid antibodies and the elevated level of IgE were not linked with skin reaction to autologous serum. Apparently, in addition to autoantibodies to IgE or its receptor (the positive skin test to autologous serum), thyroid gland antibodies may take part in the mechanism of the CRU formation.     

Nerve-muscle networks in the accessory gland tubules of a male insect: Structural and physiological properties

The accessory gland tubules of the cockroach Leucophaea maderae, (F.) are innervated by the phallic nerves which arise from the terminal ganglion. Tubule preparations stained with methylene blue showed that a fine network of nerve fibres covers the entire surface of the tubule with putative nerve cells at various points. Muscle fibres of the tubules were arranged in an irregular lattice and appeared as flattened elipses approx 8–10 μm across and 1.5 μm in thickness. These muscles were monomyofibrillar with a poorly developed T-tubule system and a diffuse Z band. Innervation of the muscles showed abundant evidence of neurosecretomotor-type terminals. Clear evidence was also obtained for a multiterminal-type innervation. Accessory gland tubules had a basic wave-like pattern of motion in vitro which showed a wide variation in the time-course of each swing (1–50 s). A twisting and curling motion of the tubules was also recorded. Spontaneous junctional potentials were observed in accessory gland preparations isolated from the terminal ganglion. The duration for junction potential ranged between 30–140 ms. Action potentials recorded from muscles had a slower rise time and a longer duration (230–530 ms).     

Immunochemical evidence for a role of complex carbohydrate chains in thyroglobulin antigenicity

Thyroglobulin secreted by porcine thyroid cells in serum-free culture was previously found (Ronin, C., Fenouillet, E., Hovsepian, S., Fayet, G., and Fournet, B. (1986) J. Biol. Chem. 261, 7287-7293) to contain more highly branched complex carbohydrate chains than the thyroid-derived molecules. When assayed for their ability to react with polyclonal antibodies directed against the natural prohormone in competitive radioimmunoassays, using the porcine antigen as iodinated tracer and standard competitor, in vitro thyroglobulin appeared to be 4-fold less immunoreactive than the in vivo molecule. However, both types of thyroglobulin exhibited superimposable incomplete displacement curves after peripheral deglycosylation using a mixture of neuraminidase, alpha-, and beta-galactosidases while they behave as good competitors as the native antigen after removal of the majority of the carbohydrate chains by Endo-beta-N-acetylglucosaminidase F. Solid-phase binding assays revealed that in vitro thyroglobulin was able to bind less antibodies than its thyroid-derived counterpart before as well as after treatment with exo- or endoglycosidases. Furthermore, only 20% of thyroglobulin biosynthetically labeled with glucosamine could be precipitated with specific antibodies followed by addition of staphylococcal-protein A, whereas up to 61% of the labeling was specifically bound when the antibodies were preincubated with protein A. After pronase digestion, both thyroglobulin-like material displayed different carbohydrate structures as judged by concanavalin A-Sepharose analysis. Thus, substituting multiantennary complex carbohydrate chains to the usual biantennary and high mannose structures present on thyroid-derived thyroglobulin had a profound effect on the prohormone immunoreactivity.     

Experimental autoimmune thyroiditis in mice chronically treated from birth with anti-IgM antibodies

The role of T lymphocytes in the pathogenesis of experimental autoimmune thyroiditis in mice is well established while the role of B lymphocytes is unclear. Mice with thyroid lesions have thyroglobulin antibodies whereas these antibodies can occur in mice immunized with Tg that do not develop thyroid lesions. To determine whether thyroglobulin antibodies are necessary for the development of the thyroid infiltrates with mononuclear cells, which are characteristic for experimental autoimmune thyroiditis, AKR mice chronically treated from birth with goat anti-mouse IgM antibodies were immunized with mouse thyroglobulin in Freund's complete adjuvant when they were 7 weeks old. Control mice, similarly immunized, were chronically injected from birth with normal goat gamma-globulin. Three weeks after immunization, all mice were sacrificed, thyroglobulin antibodies in the serum were measured by hemagglutination assay and enzyme-linked immunosorbent assay, and thyroid pathology was assessed. The serum concentration of IgG and IgM, the percentage of B and T lymphocytes in the spleen (flow cytometry), and the in vitro proliferative response of spleen lymphocytes to stimulation by PHA, LPS, and Tg were also measured. All mice treated with anti-IgM antibodies did not have detectable thyroglobulin antibodies but 63% of these mice and 88% of control mice (all of which had thyroglobulin antibodies) had thyroid lesions. Mice treated with anti-IgM antibodies that did not have thyroid lesions had a more pronounced depression of B lymphocytes than similarly treated mice that had thyroid lesions. These experiments suggest that thyroglobulin antibodies are not necessary for the development of thyroid infiltrates with mononuclear cells. B lymphocytes could still participate in the production of experimental autoimmune thyroiditis by presenting thyroglobulin to helper T lymphocytes.     

Skelemins: cytoskeletal proteins located at the periphery of M-discs in mammalian striated muscle

The cytoskeletons of mammalian striated and smooth muscles contain a pair of high molecular weight (HMW) polypeptides of 220,000 and 200,000 mol wt, each with isoelectric points of about 5 (Price, M. G., 1984, Am. J. Physiol., 246:H566-572) in a molar ratio of 1:1:20 with desmin. The HMW polypeptides of mammalian muscle have been named "skelemins," because they are in the insoluble cytoskeletons of striated muscle and are at the M-discs. I have used two-dimensional peptide mapping to show that the two skelemin polypeptides are closely related to each another. Polyclonal antibodies directed against skelemins were used to demonstrate that they are immunologically distinct from talin, fodrin, myosin heavy chain, synemin, microtubule-associated proteins, and numerous other proteins of similar molecular weight, and are not oligomers of other muscle proteins. Skelemins appear not to be proteolytic products of larger proteins, as shown by immunoautoradiography on 3% polyacrylamide gels. Skelemins are predominantly cytoskeletal, with little extractable from myofibrils by various salt solutions. Human, bovine, and rat cardiac, skeletal, and smooth muscles, but not chicken muscles, contain proteins cross-reacting with anti-skelemin antibodies. Skelemins are localized by immunofluorescence at the M-lines of cardiac and skeletal muscle, in 0.4-micron-wide smooth striations. Cross sections reveal that skelemins are located at the periphery of the M-discs. Skelemins are seen in threads linking isolated myofibrils at the M-discs. There is sufficient skelemin in striated muscle to wrap around the M-disc about three times, if the skelemin molecules are laid end to end, assuming a length-to-weight ratio similar to M-line protein and other elongated proteins. The results indicate that skelemins form linked rings around the periphery of the myofibrillar M-discs. These cytoskeletal rings may play a role in the maintenance of the structural integrity of striated muscle throughout cycles of contraction and relaxation.