Mitochondrial antibodies in primary biliary cirrhosis. I. Localization of the antigen to mitochondrial membranes.

The antigen reacting with complement-fixing antibodies in the sera of patients with primary biliary cirrhosis was localized predominantly in the mitochondrial fraction of tissue homogenates obtained by differential centrifugation. Purified mitochondrial preparations had a high content of the antigen whereas purified lysosomes failed to fix complement with PBC sera. Analysis of a number of fractionation experiments showed a high correlation between antigen content and the mitochondrial enzyme succinic dehydrogenase in all fractions. There was much poorer correlation with lysosomal and micrososomal enzyme markers. The patterns of staining obtained with a fluorescein conjugate of IgG from a PBC patient closely paralleled those obtained with a histochemical method for the demonstration of succinic dehydrogenase, further confirming the mitochondrial localization of the antigen. Staining was brightest in cells containing mitochondria with well-developed cristae. Studies on mitochondria fragmented by osmotic lysis, hexane, lysolecithin, and ultrasound suggest that the antigen is associated with the mitochondrial inner membranes.

Islet cell antibodies identify latent type I diabetes in patients aged 35-75 years at diagnosis.

One hundred fifty-four selected patients with nonketotic diabetes diagnosed between the ages of 35 and 75 yr and treated with diet or oral hypoglycemic agents for at least 1 yr were investigated for parameters of glycemic control (weight loss, blood glucose, and glycosylated hemoglobin), islet cell function (fasting and glucagon-stimulated C-peptide responses), and immunologic markers of insulitis (total ICA and CF-ICA) or autoimmunity (thyroid and gastric antibodies). These parameters were all repeated in 9 of 22 ICA-positive patients after a 2-yr follow-up and correlated with secondary drug failure. The antibody tests were also done on 51 nondiabetic controls matched for age and body weight. The 22 (14%) diabetic subjects having positive islet cell antibodies (ICA) included more women than men with a shorter duration of symptoms, lower body weight, more associated thyroid autoimmunity, and a tendency to have more type I diabetes in their families, although glycemic control, age at onset, and family history of type II diabetes were the same as in the 132 ICA-negative cases. Patients with ICA had lower initial C-peptide levels and showed little rise after glucagon stimulation. Beta cell function deteriorated significantly during the 2-yr follow-up in 9 of 22 positive patients and more ICA-positive patients required insulin. It is suggested that these latent type I diabetic patients are characterized by persistent ICA, progressive loss of beta cells, and a high frequency of thyrogastric autoimmunity. The determination of ICA may be of clinical value in the diagnosis and treatment of nonketotic diabetes with onset in later life.

Secondary failure to treatment with oral antidiabetic agents in non-insulin-dependent diabetes.

To study the etiopathogenesis of secondary drug failure to treatment with oral antidiabetic agents in patients with non-insulin-dependent diabetes (NIDD) we compared 60 "nonresponders" with 60 "responders" to treatment with oral drugs. Secondary drug failure was defined as mean diurnal blood glucose greater than 12 mmol/L after an initial good response of greater than or equal to 2 yr. The nonresponders were characterized by 50% lower C-peptide concentrations than the responders (P less than 0.001). We could not, however, define a critical C-peptide level to discriminate between patients requiring and not requiring insulin therapy. There was a wide overlap of individual C-peptide values between responders and nonresponders that attenuates the clinical value of single C-peptide measurements in predicting therapy. Only by serial measurements over a period of time was it possible to achieve information about changes in beta cell function. The nonresponders showed increased frequency of islet cell (P less than 0.01), thyroid antimicrosomal (P less than 0.01), and gastric parietal cell antibodies (P less than 0.02). In nonresponders, HLA-antigen B8 was increased (P less than 0.05) and HLA-B7 decreased (P less than 0.01) compared with frequencies of responders. In conclusion, impaired beta cell function is a characteristic feature of many, but not all, NIDD patients who fail on treatment with oral antidiabetic drugs. The presence of islet cell and thyrogastric antibodies can unmask a distinct group of NIDD patients with a high risk of secondary drug failure and subsequent insulin dependency. HLA typing may further help to predict secondary failure in NIDD.

Further studies on thyroid growth-stimulating immunoglobulins in euthyroid nonendemic goiter.

Sixty-two consecutive patients with sporadic euthyroid goiter (57 women and 5 men) from noniodine-deficient areas, including 15 patients with diffuse goiter, 39 patients with multinodular goiter, and 8 patients with a single nodule, were studied for the presence of serum thyroid growth-stimulating immunoglobulins (TGI) by the ultrasensitive cytochemical bioassay based on DNA cytophotometry (Feulgen-cytochemical bioassay). Using strictly specified conditions, 43 patients (67%) were positive. Values tended to be high in diffuse goiter, nodular goiters reccuring after partial thyroidectomy, and those with recent growth. Thirty-seven individual immunoglobulin (Ig)-rich fractions obtained by ammonium-sulfate precipitation from 20 normal subjects, 13 atrophic thyroiditis patients, and 4 dyshormonogenetic goiter patients were tested similarly, and only 3 gave positive growth assays. These results lend further support to our concept that a majority of patients with sporadic nontoxic simple goiters have a variant of thyroid autoimmune diseases separate from lymphocytic thyroiditis. With regard to assays thought to reflect activities of TSH receptor antibodies, none of 20 tested Ig preparations stimulated thyroid cAMP production. The TSH binding inhibition assay gave weak positive activity, but failed to correlate with either TGI or TSH unresponsiveness to TRH. These findings suggest that sporadic goiter TGI is not directed to the TSH-binding site. Dose-response studies performed with Igs of patients with nontoxic goiters and with human TSH standard and goitrous hyperthyroid Graves Igs as controls all revealed bell-shaped responses. Similar maximal values were reached regardless of the growth stimulus applied. However, approximately 10 times more Ig was needed to reach maximal responsiveness in sporadic goiter than in goitrous Graves' disease (i.e. 125-500 micrograms vs. 15-125 micrograms Ig/ml culture fluid). The optimal dose of human TSH ranged from 0.01-1.0 microU/ml. The assays in the present series of the 62 consecutive patients with nontoxic diffuse or nodular goiter were all carried out with a fixed amount of 125 micrograms Ig/ml and considering a value above 5% of cells in the S-phase as a positive assay. Some Ig preparations negative for TGI at this concentration may contain TGI when tested using other doses, and these are a prerequisite to assess the potency of growth antibodies in individual patients.

Autoantibodies in highly aged humans.

The presence of 14 different autoantibodies was determined in 65 persons, aged 95 years and older, without overt disease. The prevalence of positive anti-immunoglobulin latex tests, of autoantibodies against nuclear components and against thyroid microsomes was significantly increased. This selective increase of autoantibodies of low titre and without cluster formation is considered to be the result of a loss of control within the immune system due to ageing, rather than as a sign of latent disease.

Types of 'reticulin' antibodies detected in human sera by immunofluorescence.

Reticulin antibodies have been classified by immunofluorescence into five types reacting with distinct antigens of intra- and extracellular components in mesenchyme. Two types of fibrillar antigens can be distinguished on the basis of the staining patterns, anatomical distribution, and species specificity. A third antibody reacts with either small fibres, amorphous proteins, or mucopolysaccharides lining the hepatic sinusoids (ground substance antigens). In addition, at least two kinds of intrasinusoidal cells show cytoplasmic fluorescence, ie, Kupffer cells and glass-adherent, blood-borne cells antigenically related to peritoneal macrophages. Some sera may contain antibodies reacting with sinusoidal endothelial cells though this has not yet been proven. It has been confirmed that all these distinct antibodies related to reticulin antigens are most frequent in dermatitis herpetiformis and coeliac disease, but they are also found with increased frequency in chronic heroin addicts and in rheumatoid and Sjögren's syndromes. About 5% of normal individuals had such antibodies and no significant increase could be demonstrated in autoimmune disorders or in liver cirrhosis. The antibodies appear to be stimulated by bacterial or nutritional antigens and are likely to represent anamnestic responses rather than direct cross reactions with a multiplicity of foreign antigens.

An evaluation of two new haemagglutination tests for the rapid diagnosis of autoimmune thyroid diseases.

Two haemagglutination tests using preserved turkey erythrocytes are described for the detection of thyroglobulin and microsomal antibodies, respectively. Comparative studies with the more traditional sheep cell techniques show good correlation of titres when testing sera from patients with autoimmune thyroid disorders.

Immunological and histological studies in primary biliary cirrhosis.

Thirty-one patients with primary biliary cirrhosis in whom adequate histological liver material was available were studied by immunological and histological methods. There was no statistically significant correlation between individual histological features and the level of serum mitochondrial antibodies. A relationship between the duration of symptoms and histological stage of the disease supports the present concept of its evolution. However, several stages were often identified in the same specimen. Four cases with negative mitochondrial antibodies were similar to the other 27 clinically and histologically.

Classification of smooth muscle autoantibodies detected by immunofluorescence.

Three hundred and twelve sera containing antibodies to smooth muscle (SMA) wer analysed for the immunofluorescence patterns they produced in various tissues. A classification is described based on the three main appearances in rat kidney. Some sera, mainly of low titre, reacted only with vessel walls (SMA-V), some stained vessels and renal glomeruli (SMA-G) and high titre sera, mainly from patients with chronic active hepatitis stained vessels, glomeruli, and intracellular fibrils in renal tubules (SMA-T). Peripheral staining in hepatocytes or thyroid cells was not a regular feature. 41/43 polyclonal SMA-T and -G sera were absorbed out completely by actin, and this also removed the pericullular staining in liver and thyroid when present. High titre SMA-V antibodies could not be absorbed by actin, and the antigen remains to be identified.

[Can growth-stimulating immunoglobulins (TGI) explain sporadic euthyroid goiters?]. / Les immunoglobulines stimulantes de croissance (TGI) peuvent-elles expliquer certains goitres sporadiques euthyroïdiens?

IgGs capable of stimulating thyroid growth have been demonstrated (thyroid growth stimulating IgG-TGI) using a cytochemical bioassay based upon measurement of DNA synthesis in slices of guinea-pig thyroid. This activity has been confirmed by measurement of 3H-thymidine incorporation into DNA, in rat dispersed thyroid follicles cultured in suspension. Circulating TGI has been observed in patient with "simple" goitre, Graves' disease and more rarely in Hashimoto's thyroiditis. TGI titers are correlated with goitre size or lack of suppressibility by exogenous thyroid hormones rather than with thyroid function as assessed by circulating hormone levels. In patients with primary myxoedema and thyroid atrophy, existence of TGI-blocking IgGs has also been demonstrated. Existence of natural TGI has been corroborated by experimental production of a monoclonal anti-TSH receptor antibody with thyroid growth activity. These observations raise several points: --relationship of TGI to other thyroid antibodies, specially to anti-TSH receptor antibodies; --is "simple" goitre, either diffuse or nodular, an auto-immune thyroid disorder? How to explain the usual functional and structural heterogeneity of these goitres?; --metabolic relationship between DNA synthesis and activity of glucose-6-phosphate deshydrogenase involved in hormonogenesis; mechanism of the apparent uncoupling between cell multiplication and secretory activity.