Detection and expression of a cDNA clone that encodes a polypeptide containing two inhibitory domains of human calpastatin and its recognition by rheumatoid arthritis sera.

RA is the most frequent and most destructive inflammatory arthropathy. Rheumatoid factors, in spite of their lack of disease specificity, are important serological markers for RA and appear important in its immunopathogenesis as well. In search of more disease-specific autoimmune systems, we have screened a human placenta lambda gt11 cDNA expression library using selected sera from patients with classical erosive RA. We have identified one clone (RA-1) that is recognized by three of five screening sera. The 950-bp insert shows a complete nucleotide sequence homology to the cDNA encoding the two COOH-terminal domains of calpastatin. The deduced open reading frame of the RA-1 cDNA predicts a 284-amino acid protein, with a calculated mol wt of 35.9 kD. Calpastatin is the natural inhibitor of calpains, which are members of the cysteine proteinases recently implicated in joint destruction in rheumatic diseases. The two domains encoded by the RA-1 clone each contain the structural features associated with the inhibitory activity of human calpastatin. By Western blotting, 45.5% or 21/44 RA sera specifically recognized both the fusion and the cleaved recombinant protein. This is in contrast to 4.7% (2/43) in nonrheumatoid sera and 0/10 in normal sera. Anticalpastatin autoantibodies could represent a disease-associated marker in chronic erosive arthritis of the rheumatoid type and could hypothetically play a dual pathogenic role, directly via an immune interference and indirectly through an immune complex mechanism.

Immunopathological and immunochemical analysis of autoimmune enterocolitis in mice.

BALB/c (H-2d) and C57BL/6 (H-2b) mice were immunized with one or two doses of allogeneic or syngeneic mucosal antigen (MA) from small intestinal mucosa. This antigen was also characterized by immunoenzymatic assay (ELISA), by SDS-PAGE and by Western blotting. Single-dose immunized mice of either strain, sacrificed 30 days after immunization, synthesized marginal levels of anti-MA antibodies, as assessed by ELISA tests. BALB/c mice receiving two doses of the immunogen produced antibodies at a highly significant level (p less than 0.001) when compared to C57BL/6 mice belonging to the same experimental group. BALB/c mice immunized with syngeneic MA gave a humoral anti-MA response similar to that of BALB/c immunized with allogeneic MA. All experimental mice, irrespective of their genetic background or the number of doses of MA received, develop a cell-mediated immune response to MA. BALB/c mice immunized with two doses of allogeneic or syngeneic MA developed lesions mainly located in the small intestine, characterized by macroscopical and microscopical vascular congestion and hemorrhaging, epithelial cell loss, mononuclear cell infiltration of the lamina propria, cryptal degeneration and granuloma formation. Immunochemical analysis showed varying degrees of cross-reactivity between MA, liver and kidney saline extracts when heterologous anti-MA sera were tested by ELISA. Absorption of these sera with mouse liver or kidney saline extract lyophilizates reduced reactivity of anti-MA antibodies, although the degree of residual activity remained high. SDS-PAGE of mouse MA, kidney, and liver extracts revealed the presence of two polypeptidic bands of less than 17 kD molecular weight belonging to MA. Immunoblotting (Western blot) analysis revealed that heterologous or isologous anti-MA antibodies incubated with mouse MA reacted with the same epitopes as well as with others shared by other organs. Absorption of these sera with liver or kidney saline extracts revealed two organ-specific epitopes belonging to MA. Data presented here support the possibility of a genetic control of the susceptibility of BALB/c mice to the development of an acute autoimmune enterocolitis (AEC). This response appears to be a function of the amount of immunogen administered as well as the genetic background of immunized mice. The presence of a cell-mediated immune response to MA alone, however, is not sufficient for lesions to appear the histopathological alterations characteristic of AEC are present only in experimental groups where detectable levels of anti-MA antibodies are found. The antibody response seems to be directed against two unique epitopes belonging to MA.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibody interference in thyroid assays: a potential for clinical misinformation.

Measurements of thyrotropin and of total and free thyroxine and triiodothyronine are widely used diagnostic methods for thyroid function evaluation. However, some serum samples will demonstrate a nonspecific binding with assay reagents that can interfere with the measurement of these hormones. Several recent case reports have described the presence of such interferences resulting in reported abnormal concentrations of thyroid hormones inconsistent with the patient's thyroid state. Circulating thyroid hormone autoantibodies, described in thyroid and nonthyroid disorders, are an important class of interference factor and can bind to hormone tracers used in various immunoassays. Two additional categories of interfering antibodies may particularly interfere within two-site immunoassays for thyrotropin. These include heterophile antibodies, especially human anti-mouse antibodies, and rheumatoid factors, which can cause interferences by immunoglobulin aggregation and (or) cross-linking of both capture and signal antibodies. Here we review the nature of these disturbances; their occurrence, prevalence, and detection; and the clinical consequences of the failure to recognize such interference.

The Sa system: a novel antigen-antibody system specific for rheumatoid arthritis.

OBJECTIVE: To describe a novel autoimmune system (Sa/anti-Sa) specific for rheumatoid arthritis (RA). METHODS: Antibodies were detected in immunoblots using human spleen and placenta extracts as antigens. Sera from 482 patients with various rheumatic diseases as well as from healthy controls were evaluated to define the disease associations of anti-Sa antibodies. RESULTS: Sera from 88 of 206 (42.7%) unselected patients with RA recognized specific protein bands (the Sa antigen) in immunoblots of spleen or placenta extracts, including 9 of 31 (29%) patients seen in the first few months after disease onset. Anti-Sa antibodies were found both in rheumatoid factor (RF) negative (17/63 or 27%) and in RF positive patients with RA (71/143 or 50%). They were nevertheless absent in RF positive patients with other connective tissue diseases (0/39). Antibodies to Sa were essentially found in sera from patients with RA (specificity 98.9%) being found only in 3 patients whose arthritides did not fulfill the ACR criteria. The positive predictive value of anti-Sa antibodies for RA was 96.7%, while its negative predictive value was 69.8%. Anti-Sa antibodies were predominantly of the IgG isotype, with titers varying from 1/50 to > 1/1000. The Sa antigen was characterized as a poorly soluble protein that is present in normal human tissues and that is distinct from all previously described RA associated autoimmune systems. CONCLUSION: Anti-Sa antibodies are a novel serological marker highly specific for RA. Since anti-Sa antibodies occur independently of RF, they can be used as an additional diagnostic tool. The molecular nature of the Sa antigen as well as its potential pathogenic role in a significant proportion of patients with chronic articular inflammation of the rheumatoid variety merit further definition.

Data collection and information presentation for optimal decision making by clinical managers--the Autocontrol Project.

The Autocontrol methodology has been developed in order to support the optimisation of decision-making and the use of resources in the context of a clinical unit. The theoretical basis relates to quality assurance and information systems and is influenced by management and cognitive research in the health domain. The methodology uses population rather than individual decision making and because of its dynamic feedback design promises to have rapid and profound effect on practice. Most importantly the health care professional is the principle user of the Autocontrol system. In this methodology we distinguish three types of evidence necessary for practice change: practice based or internal evidence, best evidence derived from the literature or external evidence concerning the practice in question, and process based evidence on how to optimise the process of practice change. The software used by the system is of the executive decision support type which facilitates interrogation of large databases. The Autocontrol system is designed to interrogate the data of the patient medical record however the latter often lacks data on concomitant resource use and this must be supplemented. This paper reviews the Autocontrol methodology and gives examples from current studies.

Comparative multicentre study of a panel of thyroid tests using different automated immunoassay platforms and specimens at high risk of antibody interference.

The introduction of automation for immunoassays in recent years has brought about important and evident improvements in assay precision. Increasing standardization and comparability between platforms should enable the development of clinical guidelines and diagnostic algorithms for appropriate clinical decision making. A continuing source of variation between different automated immunoassay platforms is the sporadic effect of interfering antibodies or substances, thus causing aberrant results not supporting the patient's clinical status. The aim of this study was to describe current thyroid panel variation between automated immunoassay platforms including population specimens at risk of antibody interference. A multisite design with laboratories in three different countries using four different automated immunoassay platforms (Roche-Boehringer Mannheim Elecsys (Italy), Roche-Boehringer Mannheim ES300 (Wales), Bayer Immuno 1 and the Bayer ACS:180 evaluated the thyroid panel of thyrotropin (TSH), triiodothyromine (T3), free thyroxine (FT4) and free triiodothyronine (FT3). A common set of 158 randomly selected patient samples of non-thyroid and thyroid disorders, with and without treatment, was tested. Included were 62 patient samples at risk for endogenous antibody interference with high antimicrosomal antibody, anti-TSH receptor antibody and increased rheumatoid factor sub-populations. Across all controls and between platforms, precision measurements were comparable and varied between 0.7% and 12.8% for TSH, 2.8% and 13% for FT4, 1.8% and 10.5% for FT3 and 3.1% and 16% for T3 assay. Acceptable correlation and reproducibility were found between the three Bayer Immuno 1 platforms at each country's site with all four thyroid panel assays demonstrating r-values of 0.989 to 1.000 and slopes of 0.915 to 1.078. Comparisons between the different platforms showed acceptable correlation for all thyroid panel assays. Specimens containing rheumatoid factor were associated with a significantly increased variation between systems for the FT4 and FT3 assays (p < 0.01). This effect did not appear to be selective for a given platform. For specimens with raised autoimmune antibodies and therefore at risk of assay antibody interference, no variation could be observed between the platforms.