Detection of GAD65 antibodies in diabetes and other autoimmune diseases using a simple radioligand assay.

Autoantibodies to glutamic acid decarboxylase (GAD) are frequent at or before the onset of insulin-dependent diabetes mellitus (IDDM). We have developed a simple, reproducible, and quantitative immunoprecipitation radioligand assay using as antigen in vitro transcribed and translated [35S]methionine-labeled human islet GAD65. By using this assay, 77% (77 of 100) of serum samples from recent-onset IDDM patients were positive for GAD65 antibodies compared with 4% (4 of 100) of serum samples from healthy control subjects. In competition analysis with unlabeled purified recombinant human islet GAD65, binding to tracer was inhibited in 74% (74 of 100) of the GAD65-positive IDDM serum samples compared with 2% of the control samples. The levels of GAD antibodies expressed as an index value relative to a standard serum, analyzed with or without competition, were almost identical (r = 0.991). The intra- and interassay variations of a positive control serum sample were 2.9 and 7.6%, respectively (n = 4). The frequency of GAD antibodies was significantly higher with IDDM onset before the age of 30 (80%, 59 of 74) than after the age of 30 (48%, 10 of 21) (P < 0.01). The prevalence of islet cell antibodies showed a similar pattern relative to age at onset. Because simultaneous occurrences of multiple autoimmune phenomena are common, we analyzed sera from patients with other autoimmune diseases. The frequency of GAD antibodies in sera positive for DNA autoantibodies (8% [2 of 25] and 4% [1 of 25] in competition analysis) or rheuma factor autoantibodies [12% (4 of 35) and 3% (1 of 35) in competition analysis] was not different from that in control samples. In contrast, in sera positive for ribonucleoprotein antibodies the frequency of GAD antibodies was significantly increased (73% [51 of 70] and 10% [7 of 70] in competition analysis [P < 0.025]). In conclusion, even large numbers of serum samples can now be tested for GAD65 antibodies in a relatively short time, allowing screening of individuals without a family history of IDDM for the presence of this marker.

Autoantibodies to lamins A and C in sera of patients showing peripheral fluorescent antinuclear antibody pattern on HEP-2 cells.

Lamins A, B, and C are the major proteins of a polymeric structure called nuclear lamina, which is intercalated between chromatin and the inner membrane of the nuclear envelope. Using immunofluorescence on HEp-2 cells, specific enzyme-linked immunosorbent assay, and Western blotting performed against nuclear lamina preparation from Ehrlich ascites tumor cells, we characterized three patients, whose sera contained antibodies to nuclear lamins. The reaction pattern observed in two of the patients may result from single or combined occurrence of anti-lamin A and C antibodies. The third patient had antibodies that probably recognized an epitope in the carboxy-terminal region of lamin C. The sera were donated by a heterogeneous group of patients, and no common clinical or laboratory signs seemed to link them together.

An enzyme-linked immunosorbent assay for autoantibodies against the nuclear protein Scl-70.

This paper describes the development of an enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of autoantibodies against the nuclear protein Scl-70. The isolation of Scl-70 from rat livers and the conditions for the ELISA are described. Compared with the already established double diffusion in gel for detection of anti-Scl-70 antibodies this ELISA has advantages.

A new radioimmunoassay for IgM and IgG rheumatoid factors, based on a double antibody method.

A new radioimmunoassay has been developed for measuring IgM and IgG rheumatoid factors. Diluted sera from donors and patients were incubated with immunoprecipitates prepared from sheep serum and rabbit anti-sheep IgG antiserum. The precipitates were washed, and radioiodinated rabbit F(ab')2 fragments specific from human IgM or IgG were added. The precipitates were isolated from filtration and measured in a gamma counter. With this assay IgM rheumatoid factors were detected in sera from all patients with seropositive rheumatoid arthritis and in sera from 40% of patients with seronegative rheumatoid arthritis. IgG rheumatoid factors were found in sera from 68% of the seropositive and 40% of the seronegative patients. Gel filtration experiments demonstrated that it is possible to detect monomeric IgG rheumatoid factors and IgM rheumatoid factors of molecular weight smaller than pentameric IgM. Furthermore it has been shown that IgG rheumatoid factor activity is still present after reduction of IgM rheumatoid factors with dithiothreitol.

Clinical correlates and substrate specificities of antibodies exhibiting neutrophil nuclear reactivity--a methodological study.

In a serological laboratory with a routine service for determining autoantibodies to human neutrophils, antibodies giving a selective or preferential reaction with the nucleus or perinuclear area of neutrophils are not uncommon. The aim of this study was to look for clinical correlates with the presence of such neutrophil-reactive autoantibodies. The specificity of such antibodies for nuclear or cytoplasmic antigens was studied in 65 consecutive sera displaying nuclear/perinuclear reactivity at a titre of at least 80 using the indirect immunofluorescence technique (IIF) on ethanol-fixed leucocytes. The sera were also investigated by IIF on formalin-acetone fixed leucocytes and on HEp-2 cells. ELISA techniques were used to measure antibodies to azurophil granule constituents (ANCA), purified myeloperoxidase (MPO-ANCA), and lactoferrin (LF-ANCA). Furthermore a qualitative spot immunoassay was used for the detection of antibodies to alpha, beta, and gamma fractions, and the nuclear fraction of neutrophils, purified proteinase 3 (PR3), MPO, enolase, lysozyme, elastase, lactoferrin, and cathepsin G. The diagnoses linked to such GS-ANA/pANCA positivity were arthritides, vasculitides, inflammatory bowel disease and chronic hepatic conditions. MPO was the main antigen recognized in the vasculitis group, but apart from that, rather limited antigen reactivity was demonstrable by these techniques, lysozyme being the most frequently recognized autoantigen in patients with arthritides. Human lymphocytes served as a suitable control substrate when distinguishing between GS-ANA/pANCA and ANA, whereas HEp-2 cells usually could not be used if both classes of antibodies were present in a sample. Furthermore, formalin-acetone fixation is not recommended for routine use.

Screening for autoantibodies to the nucleolar U3- and Th(7-2) ribonucleoproteins in patients' sera using antisense riboprobes.

In this study we report the detection of autoantibodies to the nucleolar U3- and Th(7-2) ribonucleoprotein (RNP) particles in sera from patients with connective tissue diseases. The method described employs radioactively labelled antisense U3- and Th RNA which are hybridized to immunoprecipitated U3- or Th RNA from a HeLa cell extract. Of the 66 sera that were screened with this method seven sera (11%) precipitated only Th RNP, 16 sera (24%) precipitated only U3 RNP and 4 sera (6%) precipitated both U3- and Th RNP. Both anti-U3 RNP and anti-Th RNP activity appeared to be mostly associated with scleroderma or scleroderma-associated diseases. Using this method we also showed that some of the Th RNP particles in a cell extract are associated with the La autoantigen. We conclude that for the identification of immunoprecipitated RNAs this method is very sensitive and provides unambiguous data.

A prospective study of thyroid function, morphology and autoimmunity in young patients with type 1 diabetes.

OBJECTIVE: Thyroid autoantibodies (TA) and thyroid ultrasonography are widely used in the diagnosis of autoimmune thyroid disease (AITD). However, we know little of the significance of having ultrasonographic abnormalities (USabn) without having any other signs of AITD. In a previous population-based study of 105 young patients with type 1 diabetes (T1DM) we found a high prevalence (42%) of USabn. In the present study we evaluate the development of both USabn and TA in a 3-Year follow-up of this cohort. DESIGN: Of the 105 previously investigated children and adolescents with T1DM (aged 5-21 Years), 101 were re-examined. Serum concentrations of tri-iodothyronine (T(3)), thyroxine (T(4)), TSH, thyroid peroxidase antibodies (TPOab) and thyroglobulin antibodies (Tgab), as well as thyroid size and morphology were determined in all patients. RESULTS: During the 3 Years follow-up period, the prevalence of thyroid dysfunction increased from 5 to 8%, the prevalence of TPOab was unchanged at 13%, while the prevalence of Tgab decreased from 14 to 7%. The prevalence of USabn increased from 42 to 49%. Most patients presented USabn at both examinations. Patients with USabn had a higher prevalence of TA than those without USabn (P=0.038) and higher serum levels of TSH (P=0.027). All patients with thyroid dysfunction presented with USabn. However, many patients with USabn had no other signs of AITD. CONCLUSIONS: A high prevalence of thyroid dysfunction, TA and thyroid USabn were found in young patients with T1DM. Thyroid USabn was a sensitive but non-specific marker of AITD and is therefore unsuitable for screening purposes. Instead, we recommend regular screening using serum TSH in the follow-up of young diabetic patients.

Thyroid function, morphology and autoimmunity in young patients with insulin-dependent diabetes mellitus.

OBJECTIVE: An association between insulin-dependent diabetes mellitus (IDDM) and autoimmune thyroid disease is well recognized. We have studied the prevalence of thyroid dysfunction, autoimmunity and morphological abnormalities by ultrasonography in young diabetics. SUBJECTS AND METHODS: Among young IDDM patients less than 18 years old and living in the county of Funen, Denmark, 105 of 116 eligible patients participated. They were compared with 105 healthy children matched for sex and age. Routine thyroid function parameters (thyroxine (T4), tri-iodothyronine (T3), T3 resin uptake and TSH) and thyroid autoantibodies (anti-thyroid peroxidase, TPOab, and thyroglobulin antibodies, Tgab) were measured. Thyroid size and morphology were determined by ultrasonography. RESULTS: Two of the diabetics had previously diagnosed hypothyroidism and three new cases of subclinical hypothyroidism were found. There were no significant differences in thyroid function variables or thyroid volume between diabetics and controls. Thyroid volume correlated significantly with age and weight in both groups. Among diabetics, 17 had thyroid autoantibodies (13 with TPOab, 14 with Tgab and 10 with both) compared with 2 children in the control group (P<0.001). Forty-four with IDDM as opposed to 11 of the controls (P<0.001) had morphological abnormalities at ultrasonography. Most of them had various degrees of hypoechogenicity thought to be a marker of thyroid autoimmunity. Among the 17 diabetics with autoantibodies, 10 had morphological abnormalities at ultrasonography. CONCLUSIONS: A high proportion of young IDDM patients without any clinical signs of thyroid disease have markers of thyroid autoimmunity. Many have thyroid autoantibodies, but even more have abnormalities by thyroid ultrasonography.

Pituitary-cell autoantibody diversity in sera from patients with untreated Graves' disease.

Sera from 22 untreated patients with recently diagnosed Graves' disease (GD) were screened in an immunocytochemical tissue assay for presumptive pituitary IgG autoantibodies, as defined by the presence of immunoreaction with rat and swine pituitary cell types. Forty four patients with Hashimoto's thyroiditis (HT) and 97 healthy subjects were also studied. Anti-pituitary antibodies were found in 14 of the 22 GD sera (64%). Of these, 6 sera reacted with cytoplasmic components of growth hormone (GH) cells, 3 with prolactin (PRL) cells, and 5 with both GH and PRL cells. Yet, none of the immunoreactive sera reacted with human GH, bovine PRL or TSH in dot-blot assays and absorption studies. Anti-pituitary antibodies also occurred in 4 of the 44 HT patients (9.1%) and in 9 of the 97 healthy subjects (9.2%). The frequency of sera revealing anti-pituitary antibodies was significantly higher in patients with GD compared to the groups of HT patients (P less than 0.00005), and healthy subjects (P less than 0.00005). Healthy subjects and patients with HT had a similar frequency of anti-pituitary antibodies (P = 1.0000). These data demonstrate that in thyroid autoimmune conditions antibodies reactive with cytoplasmic components of pituitary GH/PRL cells, may be present in sera from patients with GD. The pathological importance of this observation is at present unknown.

Anti-thyroid peroxidase antibodies during pregnancy and postpartum. Relation to postpartum thyroiditis.

The frequency of autoantibodies to thyroid microsomes (MAb), thyroid peroxidase (TPOAb) and thyroglobulin (TgAb) was studied in 736 women during and after pregnancy. The aim was to study the relationship between TPO Ab and post-partum thyroid disease, and to compare their behaviour with MAb. Seventy-five (10%) were either MAb or TgAB positive, of whom 36 were sampled serially at 18, 30 and 36 weeks of pregnancy and 1, 6 and 12 months post partum. Twenty of the antibody negative mothers were selected at random for controls. Twelve of the 36 antibody positive mothers developed post-partum thyroid dysfunction (group 1), 24 did not (group 2) compared with none of the 20 controls (group 3). Six months post partum, TPO Ab titres rose overall in both groups 1 and 2, but only in those with measurable TPO Ab during the first trimester. A TPO Ab was less frequently positive than MAb, but the difference was not statistically significant. TPO Ab are not superior to MAb for predicting post partum thyroid disease in pregnant women.

Prevalence of thyroid peroxidase, thyroglobulin and thyrotropin receptor antibodies in a long-term follow-up of juvenile Graves disease.

OBJECTIVE: To relate the presence of thyroid peroxidase antibodies (TPO ab), thyroglobulin antibodies (Tg ab) and thyrotropin receptor antibodies (TSH-R ab) to the clinical course in a long-term follow-up of patients with juvenile Graves' disease (JGD). DESIGN: Patients with JGD were drawn from a Danish retrospective study and reexamined. RESULTS: A number of 105 patients were reexamined 4-21 years (median 10 years) after diagnosis. Three groups were formed: Gr.1: euthyroid patients with anti-thyroid drug (ATD) cessation more than 12 months before reexamination (n=41). Gr.2: patients still on ATD (n=24). Gr.3: subtotally thyroidectomized patients (n=40). Positive TPO ab titers were found in 75% of the patients. In 13% of the patients the titers were very high: >10,000 U/ml. Positive Tg ab were found in 51%. The prevalence of TPO ab and Tg ab was the lowest in group 3. Stimulating TSH-R ab titers were found in 13%. No patients had blocking TSH-R ab. The prevalence of TSH-R ab was 3% in the euthyroid patients, without surgery performed. 15% in the surgical patients, and 25% in the patients still on ATD. CONCLUSIONS: Many JGD patients were euthyroid at the long-term follow-up in spite of high TPO ab and Tg ab titers. Stimulating TSH-R ab were rare (13%). According to this presence of TPO ab, Tg ab or TSH-R ab does not predict the final outcome of JGD. Further studies are needed.

Anti-thyroid peroxidase antibodies in thyroid disorders and non-thyroid autoimmune diseases.

A new commercial method for measurement of anti-thyroid peroxidase (anti-TPO DYNOtest, Henning, Berlin) was evaluated in normal subjects and in patients with autoimmune thyroid and non-thyroid diseases, and compared to an immune fluorescence method for measurement of anti-microsomal antibodies (MicAb), and a radioimmunological method for quantifying thyroglobulin antibodies (TgAb). The majority of normal subjects had anti-TPO levels below 52 U/ml and patients with Hashimoto's thyroiditis had levels above 200 U/ml, with a good correlation to MicAb. In other autoimmune thyroid diseases the correlation was less pronounced. In non-thyroid autoimmune diseases MicAb showed falsely positive reactions in the presence of other autoantibodies, e.g. mitochondrial antibodies. The present study indicates that the anti-TPO method should probably replace measurements of MicAb for routine clinical use, thus providing a sensitive, precise, antigen specific method with the ability to reveal quantitative fluctuations. The study also indicates that TgAb could be abolished in routine diagnosis of autoimmune thyroid diseases and be reserved for special clinical situations, research purposes as well as measurement in sera before evaluation of serum thyroglobulin levels.

Serological and clinical findings in patients with serological evidence of reactivated Epstein-Barr virus infection.

IgM directed against Epstein-Barr virus (EBV) early antigen (IgM-EA) has been established as an early marker of EBV infection and IgG directed against Epstein-Barr virus nuclear antigen I (IgG-EBNA-1) as a late marker. Simultaneous seropositivity to IgM-EA and IgG-EBNA has therefore been proposed as indicating reactivation of latent EBV infection. We have studied 191 patients with serological evidence of reactivated EBV infection with regard to clinical presentation, antibodies directed against EBV viral capsid antigen (IgM-VCA, IgG-VCA), cytomegalovirus (IgM-CMV), human herpesvirus 6 (IgM-HHV-6). IgM rheumatoid factor (IgM-RF), anti-nuclear or anti-cytoplasmic antibodies (ANA/ACA), and total IgM. The clinical manifestations varied considerably, but a diagnosis was established in 121 of the patients. The diversity of the clinical diagnosis probably reflects common reasons for requesting an EBV serological test rather than clinical manifestations of reactivated EBV infection. Only 5.8% of the patients with "serological EBV reactivation" gave a positive result for IgM-VCA. We conclude that "serological EBV reactivation" does not represent an entity relating to clinical manifestations, but probably reflects a non-specific activation of the immune system.

Characterization of a folate receptor in parotid gland and a folate binding protein in saliva from humans. Epitope relatedness to human milk folate binding protein.

The present study was performed to establish the antigenic identity and origin of the folate binding protein in human saliva. We identified a folate receptor in human parotid and submandibular gland which immunoreacted with antibodies against human milk folate binding protein, as evidenced by ELISA and immunostaining of ductal epithelium and secretory glandular material. The receptor concentration was 0.4-1.4 nmol 3H-folate bound/g protein. Ligand binding was of a high-affinity (K=10(10) M(-1)) type, exhibited positive cooperativity, a slow radioligand dissociation at pH 7.4, and inhibition by folate analogues. The concentration of immunoreactive folate binding protein in saliva as determined by ELISA with antibodies against human milk folate binding protein was several fold higher than that determined by radioligand binding (nil - 1 nM). This indicates that a major fraction of the immunoreactive material does not bind 3H-folate, and could represent a precursor form of the protein. In conclusion, the folate binding protein in human saliva seems to be a secretory product of the salivary glands. The protein is also epitope-related to folate binding proteins in other human mucosal secretions.

A high-affinity soluble folate receptor in fluids of non-neoplastic ovarian cysts: radioligand binding, molecular size, hydrophobic residue, and immunological properties.

The presence of a soluble folate receptor in fluids of non-neoplastic ovarian cysts was demonstrated. Radioligand binding exhibited characteristics typical of high-affinity folate-binding proteins. These included positive cooperativity, a tendency to increased binding affinity with decreasing receptor concentration, a slow ligand dissociation at pH 7.4 and inhibition by folate analogues. The folate receptor was probably synthesized in the lining epithelial cells of the cysts which showed positive immunostaining with antibodies against human milk folate-binding protein. The gel filtration profile of cystic fluid contained two radioligand-bound peaks, 25 and 100 kDa, whereas a single band of 70 kDa was seen on SDS-PAGE immunoblotting. Treatment with the enzyme phosphatidylinositol-specific phospholipase C resulted in a partial conversion of the 100 kDa peak to the 25 kDa peak. This suggests that insertion of a hydrophobic glycosylphosphatidylinositol tail into Triton X-100 micelles could give rise to large molecular size forms of the receptor on gel filtration.

The high-affinity folate receptor of normal and malignant human colonic mucosa.

The hypothesis that folate depletion is a risk factor for development of colonic neoplasia prompted us to study the presence of a putative folate receptor in human colon mucosa. Binding of 3H-folate to normal and malignant mucosa studied by equilibrium dialysis was of high-affinity type (K = 10(10) L/mol) and displayed apparent positive cooperativity. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. As compared to methotrexate, 5-formyltetrahydrofolate was a potent inhibitor of binding. Gel filtration revealed a 25 kDa and a 100 kDa peak of folate-binding activity. Immunoreactivity studies were performed with rabbit antibodies against human 25 kDa milk folate-binding protein. Immunoblotting showed a single band at 65 kDa, and tissue sections exhibited immunostaining of mucosal areas. The present folate receptor with characteristics similar to those of other high-affinity folate-binding proteins may serve as a regulator of intracellular folate concentration in colon mucosa.

Folate receptor of human mammary adenocarcinoma.

Binding of 3H-folate to human mammary tumor homogenate was of a high-affinity type (K = 10(10) M-1) and displayed apparent positive cooperativity. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. As compared to methotrexate, 5-formyltetrahydrofolate acted as a strong inhibitor of radioligand binding. Gel chromatography of radioligand-labeled homogenate of tumor tissue revealed three peaks: a small > or = 110 kDa peak and two major peaks of folate-binding activity (M(r) approximately 25 kDa and M(r) approximately 100 kDa). Mammary tumor tissue showed immunostaining with rabbit antibodies against human milk folate binder. A parallel elevation in the concentrations of folate-binding protein and triglyceride in tumor tissue as compared to normal tissue adjacent to the tumor was compatible with the localization of folate-binding protein in the triglyceride-rich fraction of mammary gland homogenate.

Folate receptor in malignant effusions of ovarian carcinoma.

Binding of 3H-folate in human ovarian adenocarcinoma tissue was of a high-affinity type (K approximately 10(10) M-1) and displayed apparent positive cooperatively. A high-affinity folate receptor was also present in ascitic fluid and pleural effusion. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. The folate analogues methotrexate and in particular 5-formyltetrahydrofolate acted as inhibitors of 3H-folate binding in ascitic fluid. Ovarian adenocarcinoma tissue showed immunostaining with rabbit antibodies against human milk folate-binding protein. The gel filtration diagram contained two peaks of radiolabelled folate (at 25 and 100 kDa). The 25 kDa peak was predominant in ascitic fluid and pleural effusion. A single band of 70 kDa was seen on SDS-PAGE immunoblotting of tissue and malignant effusions. The concentration of folate receptor in tissue and fluid specimens could be determined by an immunochemical method (ELISA) utilizing antibodies against human milk folate-binding protein.

Anti-corticosteroid antibodies in AIDS patients.

Patients suffering from AIDS tend to have symptoms that resemble those encountered in adrenocortical insufficiency. Serum sodium concentrations and blood pressure values were monitored and found to be subnormal, despite the fact that renin activity and aldosterone levels were either normal or elevated. We report the presence of autoantibodies directed against hydrocortisone among such AIDS patients. Indirect immunofluorescence technique using patients' sera and adrenal glands from AIDS patients as antigen showed antibodies to adrenocortical cells in sera from 9 of the 12 AIDS patients and in none from patients with autoimmune diseases and viral diseases or healthy blood donors. No similar reaction was seen in normal human or monkey adrenal glands. An ELISA technique was developed using hydrocortisone as antigen and using this technique 45% of the AIDS patients were found to have antibodies to hydrocortisone. It is possible that anti-corticosteroid antibodies (ACSA) may play a role in the pathophysiology of the Addison's like syndrome seen in terminal phases of AIDS.

Thyroid function, morphology and prevalence of thyroid disease in a population-based study of Danish centenarians.

OBJECTIVES: To investigate thyroid function, morphology, and autoimmunity in relation to physical function in an unselected population of centenarians. DESIGN: A population-based survey. SETTING: Denmark. PARTICIPANTS: All persons living in Denmark who celebrated their 100th anniversary during the period April 1, 1995 to May 31, 1996, a total of 276 persons. MAIN OUTCOME MEASUREMENTS: Thyroid hormones (TSH, T4, FT4I, T3, FT3I, and T3RU), thyroid autoantibodies (TPOab and Tgab), thyroid volume, activities of daily living according to the Katz Index of ADL. RESULTS: In all, 207 (75%) of the 276 eligible subjects participated, and 148 agreed to blood tests. Among the participants, 2.9% had previously known hyperthyroidism, and the same proportion had previously known hypothyroidism. The blood tests did not reveal any undiagnosed cases of overt thyroid dysfunction. However 7.2% had a subnormal serum TSH, and 2.9% had an elevated serum TSH; all had normal serum T3 and serum T4 levels. Thyroid autoantibodies were detected in 26 (17.6%) centenarians (11.5% had Tgab and 9.5% had TPOab). Among relatively independent centenarians, low serum T3 was significantly associated with high comorbidity (P = .029), whereas both low serum T3 and thyroid autoantibodies were significantly associated with ADL-dependency (P < .001 and P = .030, respectively). Ultrasonography (n = 50) revealed a small gland with a median volume of 8.3 mL (range 3.2-27.9) compared with an expected volume of 20 mL (14-26) (P < .001). There was no significant relationship to body weight. When examined by ultrasound, only 26% had significant morphological alterations. CONCLUSIONS: Thyroid dysfunction does not seem to be more prevalent among centenarians than among younger old people. Low serum T3 is related to poor physical function and co-morbidity, whereas thyroid autoimmunity is related only to poor physical functioning. Despite atrophy of the thyroid gland, these findings suggest that thyroid function is well preserved in centenarians.