[Fine specificity of anti-ß2glycoprotein I antibodies in systemic autoimmune diseases is mostly directed against domain 1]. / La fine specificità degli anticorpi anti-ß2 glicoproteina I nelle malattie autoimmune sistemiche è prevalentemente diretta verso il dominio 1.

OBJECTIVE: Anti-ß2 GPI are a formal laboratory criterion for the antiphospholipid syndrome (APS). They were demonstrated to be a risk factor for thrombosis and fetal losses but can also be detected in patients with systemic autoimmune disease (SAD), in healthy adults individuals and pre-school children. It has been suggested that different subpopulations of anti-ß2GPI may carry different pathogenetic potential: autoantibodies against Domain1 seem to be associated with thrombosis; autoantibodies against Domain4/5 have been identified in patients with non-thrombotic conditions. METHODS: We studied 48 patients with SAD (32 systemic lupus erythematosus, 16 undifferentiated connettive tissue disease), 64 patients with APS, 57 one-year-old healthy children born to mother with SAD, 33 children with atopic dermatitis. All subjects were IgG anti-ß2 GPI positive. The specificity of anti-ß2 GPI was investigated using ELISA research products containing recombinant ß2 GPI D1 and D4/5 antigens. Cut-off values are calculated as 95th percentile on 100 NHD. IgG anti-ß2 GPI were tested at a validated home-made ELISA routinely performed in our laboratory. No thrombotic events were recordered in patients with SAD and in both groups of children. RESULTS: Patients with SAD and APS showed prevalent reactivity for D1 while children in both groups preferentially recognize D4/5. CONCLUSIONS: IgG anti-ß2 GPI against D1 seem to cluster in patients with systemic autoimmune conditions. Their pathogenic potential in determine APS manifestations may be mitigated by adequate prophylaxis.

'Non-criteria' aPL tests: report of a task force and preconference workshop at the 13th International Congress on Antiphospholipid Antibodies, Galveston, TX, USA, April 2010.

Abstract: Current classification criteria for definite APS recommend the use of one or more of three positive standardized laboratory assays, including anticardiolipin antibodies (aCL), lupus anticoagulant (LA), and antibodies directed to ß(2)glycoprotein I (anti-ß(2)GPI) to detect antiphospholipid antibodies (aPL) in the presence of at least one of the two major clinical manifestations (i.e., thrombosis or pregnancy morbidity) of the syndrome. Several other autoantibodies shown to be directed to phospholipids and/or their complexes with phospholipids and/or to proteins of the coagulation cascade, as well as a mechanistic test for resistance to annexin A5 anticoagulant activity, have been proposed to be relevant to APS. A task force of worldwide scientists in the field discussed and analyzed critical questions related to 'non-criteria' aPL tests in an evidence-based manner during the 13th International Congress on Antiphospholipid Antibodies (APLA 2010, 13-16 April 2010, Galveston, Texas, USA). This report summarizes the findings, conclusions, and recommendations of this task force.

[Subpopulations of anti-ß2glycoprotein I antibodies with different pathogenic potential: fine specificity against the domains of ß2glycoprotein I]. / Sottopopolazioni di anticorpi anti-ß2glicoproteina I con diverso potenziale patogenetico: fine specificità nei confronti dei domini della ß2glicoproteina I.

OBJECTIVE: Anti-ß2glycoprotein I antibodies (a-ß2GPI) are a laboratory criterion for the antiphospholipid syndrome (APS) and were demonstrated to be involved in the pathogenesis of APS. However, they can also be detected in asymptomatic subjects. It has been suggested that a-ß2GPI against Domain1 (D1) associate with thrombosis, while those recognizing Domain4/5 (D4/5) have been identified in non-thrombotic conditions. We evaluate the specificity of a-ß2GPI in different clinical situations. METHODS: We studied 39 one-year-old healthy children born to mothers with systemic autoimmune diseases (SAD) (15 (38.4%) were born to mothers who were a-ß2GPI positive), 33 children with atopic dermatitis (AD) and 55 patients with APS (50 adults and 5 paediatrics). All subjects were IgG a-ß2GPI positive. IgG a-ß2GPI were performed by homemade ELISA, while IgG a-ß2GPI D1 and D4/5 were tested on research ELISAs containing recombinant ß2GPI domains antigens. RESULTS: One-year-old children and AD children displayed preferential reactivity for D4/5; patients with APS recognized preferentially D1. We also found a good correlation between a-ß2GPI and D4/5 in one-year-old (r=0.853) and AD children (r=0.879) and between a-ß2GPI and D1 in the APS group (r=0.575). No thrombotic events were recorded in both groups of children. CONCLUSIONS: A-ß2GPI found in non-thrombotic conditions (healthy children born to mothers with SAD and AD children) mostly recognize D4/5, in contrast to the prevalent specificity for D1 in the APS group. The different specificity could at least partially explain the "innocent" profile of a-ß2GPI in children.

A case of dermatitis herpetiformis with IgA endomysial antibodies but negative direct immunofluorescent findings.

A patient with clinical findings of dermatitis herpetiformis (DH), negative direct immunofluorescent (DIF) findings for junctional IgA deposits in 2 biopsy specimens, and positive for IgA endomysial (AEmA) and tissue transglutaminase (tTG) antibodies responded initially to dapsone. After dapsone had to be discontinued because of side effects, a gluten-free diet and supportive therapy controlled the disease; the AEmA and tTG antibodies became negative. Our data on 10 consecutive DH cases examined by DIF and by serum studies for AEmA and antibodies to tTG, point to frequencies of 90% DIF positive and 70% AEmA and tTG positive cases. The use of both DIF and serum tests for AEmA and tTG reveals DH cases not detected by DIF alone that respond to gluten-free diet. Findings on autoantibodies to tTG, an enzyme that metabolizes gliadin, points to a role of tTG in the immunopathology of gluten-sensitive enteropathy and helps to explain the need for a gluten-free diet in the management of DH cases.

Immunoglobulin A (IgA) deficiency and alternative celiac disease-associated antibodies in sera submitted to a reference laboratory for endomysial IgA testing.

Immunoglobulin A (IgA) deficiency occurs more frequently in patients with celiac disease (CD) than in the general population and can lead to false-negative results in the best serologic test for CD, endomysial IgA (EMA). To evaluate the impact of IgA deficiency on serologic detection of CD in a reference laboratory setting, IgA levels were measured in 510 consecutive serum specimens submitted for testing for EMA; 510 consecutive serum specimens submitted for Helicobacter pylori IgG testing served as a gastrointestinal symptom control group. The frequency of IgA deficiency was significantly higher among the specimens submitted for testing for EMA (5.1%) than among the specimens from the symptom control group (1.4%). Three subsets of sera from the group of specimens submitted for testing for EMA were then tested by additional serologic assays for CD; these subsets were EMA-positive sera (n = 25), EMA-negative, IgA-deficient sera (n = 26), and control sera (from EMA-negative, IgA-nondeficient patients age matched to IgA-deficient patients; n = 26). The proportions of EMA-positive sera positive by other assays for CD were 92% for transglutaminase IgA (TG-IgA), 80% for gliadin IgA, 84% for gliadin IgG, 60% for endomysial IgG (EMG), and 32% for transglutaminase IgG (TG-IgG). Very low proportions (0 to 8%) of IgA-deficient sera and control sera were positive for TG-IgA, gliadin IgA, EMG, and TG-IgG. Eight of 26 (31%) IgA-deficient serum samples were positive for gliadin IgG, whereas 3 of 26 (12%) control serum samples were positive for gliadin IgG, but this difference was not statistically significant. Physicians supplied clinical data for 18 of 26 patients with IgA deficiency; only 4 patients had undergone small-bowel biopsy, and 0 of 4 patients showed villous atrophy. These findings show that IgA deficiency is found more frequently among sera submitted for testing for EMA in a reference laboratory setting, but there was no clear-cut serologic or clinical evidence of CD in EMA-negative, IgA-deficient patients.

Relevance of complement fixing antinuclear antibodies.

BACKGROUND: Connective tissue diseases (CTDs) are a heterogeneous group of disorders defined by the association of a variety of clinical manifestations with immunologic and other laboratory findings. Overlap of syndromes and aberrant findings appear rather frequently. METHODS: Sera of eight antinuclear antibody (ANA) negative, cases of subacute cutaneous lupus erythematosus (SCLE) with antibodies to Ro (SS-A) and a ninth case with clinical and laboratory signs of Sjögren's syndrome and systemic lupus erythematosus (SLE) were tested for complement (C') fixing antinuclear antibodies (C-ANAs). The ninth case was examined in depth by direct immunofluorescence (DIF) and a two-step "C + DIF" test of biopsies for C' fixation to in vivo bound ANAs, as well as serum tests for C-ANA, ANA, and SCLE markers. RESULTS: Sera of five of the eight ANA negative, Ro(SS-A) positive SCLE cases had C-ANAs. The ninth case, a 50-year-old woman with clinical and laboratory signs of Sjögren's syndrome and SLE, gave a strong positive C + DIF reaction in the skin biopsy for in vivo bound ANAs that fix C', but negative ANAs and C-ANAs in routine serum tests; they revealed antimitochondrial antibodies. Serum tests on normal skin, however, revealed weak ANA and strong C-ANA reactions with in vitro fixed C'. CONCLUSIONS: ANA negative cases of SCLE or Sjögren's syndrome may have C-ANAs. A case with Sjögren's syndrome and signs of SLE had both in vivo and in vitro C' fixing ANAs. C-ANA tests can aid in the identification of such cases.

Standardized measurement of major immunoglobulin class (IgG, IgA, and IgM) antibodies to beta2glycoprotein I in patients with antiphospholipid syndrome.

Patients with the antiphospholipid syndrome (APS) have autoantibodies directed against epitopes on beta2 glycoprotein I (beta2GPI). We describe herein the performance characteristics of standardized enzyme-linked immunosorbent assays (ELISAs) for anti-beta2GPI of the three major immunoglobulin classes: IgG, IgA, and IgM. All three assays generated highly linear standard curves (5 points, r > or = 0.993 for each); precision was excellent both intra-assay and run-to-run, with coefficients of variation (CV) ranging from 2.3% to 6.6%. Values for IgG anti-beta2GPI correlated strongly with those obtained by an earlier method (r = 0.80, P< 0.0001). A study group consisting of 203 healthy subjects was used to generate percentile-based reference intervals for all three classes of anti-beta2GPI. APS subjects' anti-beta2GPI were found to differ significantly (P <0.0001 for each) from those of the control population. All three assays correlated well with beta2GPI-dependent anticardiolipin antibody (aCL) measurements; IgG: r = 0.94 (P <0.0001), for IgA: r = 0.82 (P<0.001) and for IgM: r = 0.84 (P<0.0001). We suggest that these ELISAs may provide valuable standardized measurements of IgG, IgA, and IgM anti-P2GPI.

Antinuclear antibodies (ANA) and complement fixing ANA in systemic connective tissue diseases.

Screening for antinuclear antibodies (ANA) with parallel tests for complement fixing ANA (C-ANA) reveal that C-ANA react either as strongly as or more strongly than ANA in most cases of systemic lupus erythematosus (SLE) and related disorders including CREST syndrome. But sera of drug induced LE and other ANA positive subjects have weak or no C-ANA. (P < 0.0005). Titrations with parallel C-ANA/ANA tests of two cases reveal primarily ANA and less C-ANA reactions in a case of drug induced LE but in CREST syndrome both ANA and C-ANA tests yield elevated titers with stronger C-ANA reactions. These findings point to distinct immunochemical mechanisms in C-ANA and ANA reactions.

Current concepts and diagnostic evaluation of autoimmune disease.

This article discusses autoimmune reactions and the numerous mechanisms by which an autoimmune response may be initiated, including genetic factors, T-cell bypass mechanisms, and idiotypes. Human autoimmune diseases are classified into three main groups, ie, organ-specific, non-organ specific, and disorders with non-organ-specific autoantibodies with lesions restricted to one or a few organs, that are examined in detail. General laboratory tests and interpretation of results in relation to state or treatment of the particular disease, age and sex of the patient, and the sensitivity of the test system used are reviewed.

Studies in immunodermatology. VIII. Immunofluorescence studies of stratum corneum antibodies.

Stratum corneum antibodies (SCAb) as detected by indirect immunofluorescence (IIF) can be absorbed from human sera with homogenates of normal human callus which contain stratum corneum antigen. Titers of concomitantly present antibodies remain unchanged in sera absorbed with callus, thus showing that this absorption is specific. SCAb reacting in a passive hemagglutination assay with an antigen derived from human callus by means of a trypsin-phenol-water extraction can be specifically absorbed with the homologous antigen. However, absorption with callus fails to remove the hemagglutinating antibodies and absorption with the trypsin-phenol-water antigen preparation fails to remove SCAb detected by IIF. These data indicate a nonidentity relationship between these antigen systems despite their similar histological and anatomical tissue distribution. Reactivity of immunoglobulins and complement only with areas of traumatized stratum corneum in skin explants cultured on sera containing SCAb provide some insight as to a possible normal physiologic role for these ubiquitous complement-fixing autoantibodies.

Quantitative studies on immunofluorescent staining. VII. Fractionation of conjugates by acid dialysis to reduce nonspecific staining.

Dialysis of fluorescein-conjugated antibodies at pH 5.8-6.0 can reduce unwanted nonspecific staining. Selective precipitation of highly fluoresceinated proteins in mildly acidic buffer is a simple technique which can produce a reagent with greatly improved nonspecific staining characteristics with little or no loss of antibody activity. This technique may be especially useful when fluorescent antibody tests are to be performed on tissues which have a high propensity for nonspecific staining such as skin.

Immunopathology of psoriasis.

Immunofluorescence (IF) studies by the direct and indirect methods demonstrate immunoglobulins and complement bound in vivo in psoriatic scales. The IF pattern is comparable to that of stratum corneum antibodies (SCAb) bound in vitro on specific substrate, as visualized by the indirect IF method. Formation of immune complexes can be responsible for the "squirting papilla" phenomenon, and conversion of the stratum corneum - which is normally an inaccessible antigen - into its reactive form seems to be brought about by proteases of polymorphonuclear leukocytes. Stimulation of protease production by polymorphonuclears appears to be an important factor in the pathogenesis of psoriasis. The stratum corneum of the epidermis is probably the target, and becomes an antigen for SCAb present in the circulation.

Coexistence of bullous pemphigoid and systemic lupus erythematosus.

A vesiculobullous eruption with clinical and histological features of bullous pemphigoid developed in a 28-year-old woman with proven systemic lupus erythematosus (SLE). Serum of this patient contained elevated titers of antinuclear antibodies but basement membrane antibodies could not be detected at first, though they did appear in blister fluid. Normal monkey skin explants cultured on this patient's sera gave positive direct immunofluorescence (IF) at the basement membrane zone (BMZ) for IgG deposits. The use of tissue culture methods may be helpful because of the capacity of this test system to reveal the presence of the antibodies to the BMZ despite the presence of the antinuclear antibodies that appear to interfere with their demonstration in standard indirect IF tests.

In vitro effects of pemphigus antibodies on skin.

Acantholysis occurring in rhesus monkey skin explants cultured on sera of 9 pemphigus patients was found to be largely dependent on the titre of intercellular antibody, and not on the participation of complement. Skin explants cultured on normal human sera and pemphigoid sera failed to give rise to intercullular staining or to develop lesions. Six of eight 'negative' pemphigus sera with intercellular antibody titres of less than 20 on skin (and titres ranging from 20 to 160 on monkey esophagus) reacted with the skin explants as revealed by direct immunofluorescence with an anti-IgG conjugate. The binding of antibodies from 3 of these 6 reactive sera resulted in some pathological changes in the explants. At least two of these 3 'negative' sera came from pemphigus patients with skin lesions.