Anti-phospholipid IgG antibodies detected by line immunoassay differentiate patients with anti-phospholipid syndrome and other autoimmune diseases.

PURPOSE: Anti-phospholipid antibodies (aPL) analyzed by line immunoassay (LIA) can recognize beta2-glycoprotein I (ß2GPI) domain 1 (D1) epitopes depending on ß2GPI binding to distinct phospholipids. The aPL LIA was compared with consensus ELISA to investigate whether both techniques can discriminate anti-phospholipid syndrome (APS) patients from aPL-positive, systemic autoimmune rheumatic diseases (SARD) patients without clinical symptoms of APS and controls. METHODS: Thirty-four APS patients (14 arterial/venous thrombosis, 16 pregnancy morbidity, and 4 both), 41 patients with SARD lacking clinical APS criteria but demonstrating positivity for anti-ß2GPI (aß2GPI) IgG, and 20 healthy subjects (HS) were tested for aPL to cardiolipin (aCL), phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol (aPG), phosphatidylinositol, phosphatidylserine, ß2GPI, prothrombin, and annexin V by LIA. Samples were also tested for aCL, aß2GPI, aß2GPI-domain 1 (aD1), and aß2GPI-domains 4-5 (aD4-5) by ELISA and for lupus anti-coagulant. RESULTS: Comparison of LIA with ELISA revealed a good agreement for the consensus criteria aPL aß2GPI and aCL IgG (kappa = 0.69, 0.68, respectively) and a moderate agreement for IgM (kappa = 0.52, 0.49, respectively). Regarding ELISA, aD1/aD4-5 demonstrated the best performance of differentiating APS from asymptomatic SARD [area under the curve (AUC): 0.76]. aPG IgG had the best performance by LIA (AUC: 0.72) not significantly different from aD1/aD4-5. There was a good agreement for aPG IgG with aD1/aD4-5 (kappa = 0.71). CONCLUSIONS: aD1/aD4-5 (ELISA) and aPG IgG (LIA) differentiate APS from SARD patients. PG appears to interact with ß2GPI of APS patients and exposes D1 thereof for disease-specific aPL binding in LIA.

Simultaneous comprehensive multiplex autoantibody analysis for rapidly progressive glomerulonephritis.

Rapidly progressive glomerulonephritis (RPGN) is mainly caused by anti-glomerular basement membrane (GBM) antibody-mediated glomerulonephritis, immune-complex or anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides and leads to rapid loss of renal function. Detection of ANCA and autoantibodies (autoAbs) to GBM and dsDNA enables early diagnosis and appropriate treatment of RPGN aiding in preventing end-stage renal disease.Determination of ANCA on neutrophils (ANCA) as well as autoAbs to myeloperoxidase (MPO-ANCA), proteinase 3 (PR3-ANCA), GBM, and dsDNA was performed by the novel multiplex CytoBead technology combining cell- and microbead-based autoAb analyses by automated indirect immunofluorescence (IIF). Forty patients with granulomatosis with polyangiitis (GPA), 48 with microscopic polyangiitis (MPA), 2 with eosinophilic GPA, 42 with systemic lupus erythematosus (SLE), 43 with Goodpasture syndrome (GPS), 57 with infectious diseases (INF), and 55 healthy subjects (HS) were analyzed and findings compared with classical single testing.The CytoBead assay revealed for GPA, MPA, GPS, and SLE the following diagnostic sensitivities and for HS and INF the corresponding specificities: PR3-ANCA, 85.0% and 100.0%; MPO-ANCA, 77.1% and 99.1%; anti-GBM autoAb, 88.4% and 96.4%; anti-dsDNA autoAb, 83.3% and 97.3%; ANCA, 91.1% and 99.1%, respectively. Agreement with classical enzyme-linked immunosorbent assay and IIF was very good for anti-GBM autoAb, MPO-ANCA, PR3-ANCA, and ANCA, respectively. Anti-dsDNA autoAb comparative analysis demonstrated fair agreement only and a significant difference (P = 0.0001).The CytoBead technology provides a unique multiplex reaction environment for simultaneous RPGN-specific autoAb testing. CytoBead RPGN assay is a promising alternative to time-consuming single parameter analysis and, thus, is well suited for emergency situations.

Antiphospholipid antibodies detected by line immunoassay differentiate among patients with antiphospholipid syndrome, with infections and asymptomatic carriers.

BACKGROUND: Antiphospholipid antibodies (aPL) can be detected in asymptomatic carriers and infectious patients. The aim was to investigate whether a novel line immunoassay (LIA) differentiates between antiphospholipid syndrome (APS) and asymptomatic aPL+ carriers or patients with infectious diseases (infectious diseases controls (IDC)). METHODS: Sixty-one patients with APS (56 primary, 22/56 with obstetric events only, and 5 secondary), 146 controls including 24 aPL+ asymptomatic carriers and 73 IDC were tested on a novel hydrophobic solid phase coated with cardiolipin (CL), phosphatic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, beta2-glycoprotein I (ß2GPI), prothrombin, and annexin V. Samples were also tested by anti-CL and anti-ß2GPI ELISAs and for lupus anticoagulant activity. Human monoclonal antibodies (humoAbs) against human ß2GPI or PL alone were tested on the same LIA substrates in the absence or presence of human serum, purified human ß2GPI or after CL-micelle absorption. RESULTS: Comparison of LIA with the aPL-classification assays revealed good agreement for IgG/IgM aß2GPI and aCL. Anti-CL and anti-ß2GPI IgG/IgM reactivity assessed by LIA was significantly higher in patients with APS versus healthy controls and IDCs, as detected by ELISA. IgG binding to CL and ß2GPI in the LIA was significantly lower in aPL+ carriers and Venereal Disease Research Laboratory test (VDRL) + samples than in patients with APS. HumoAb against domain 1 recognized ß2GPI bound to the LIA-matrix and in anionic phospholipid (PL) complexes. Absorption with CL micelles abolished the reactivity of a PL-specific humoAb but did not affect the binding of anti-ß2GPI humoAbs. CONCLUSIONS: The LIA and ELISA have good agreement in detecting aPL in APS, but the LIA differentiates patients with APS from infectious patients and asymptomatic carriers, likely through the exposure of domain 1.

Autoantibodies to asialoglycoprotein receptor (ASGPR) in patients with autoimmune liver diseases.

BACKGROUND: The liver asialoglycoprotein receptor (ASGPR) is the only organ-specific autoantigenic target in autoimmune hepatitis (AIH) patients and corresponding autoantibodies (Abs) have been suggested aiding in the serology of autoimmune liver diseases (ALD). METHODS: A novel enzyme-linked immunosorbent assay (ELISA) employing purified rabbit ASGPR was used to detect ASGPR Abs in patients with ALD and controls. ASGPR Ab was determined in sera from 172 patients with AIH type 1, AIH type 2 (n=42), primary biliary cirrhosis (PBC) (n=113), cryptogenic liver disease (n=30), toxic liver disease (n=11), primary sclerosing cholangitis (PSC) (n=27), HCV infection (n=25), non-alcoholic steatohepatitis (n=43) and 100 blood donors. ASGPR Ab positivity was compared with AIH-related Abs (ANA, ASMA, Abs to LKM-1, LC-1, and SLA/LP) in patients with AIH. RESULTS: Patients with AIH-1 and AIH-2 demonstrated an ASGPR Ab prevalence of 29.1% and 16.7%, respectively. ASGPR Ab positivity in patients with AIH-1 and AIH-2 was not significantly different to those in patients with PSC and HCV (p>0.05, respectively). ASGPR Ab levels in all study cohorts were significantly different with the highest medians in patients with AIH, PSC, and HCV infection (p<0.0001). ASGPR Ab can be found as only AIH-specific Ab determined by LIA and ELISA in 24.4% of AIH patients (48/197). CONCLUSIONS: The novel ASGPR Ab ELISA is a specific diagnostic tool for ASGPR Ab detection in AIH. In addition to AIH, patients with PSC can demonstrate elevated ASGPR Ab amongst those with ALD suggesting a tolerance break to ASGPR in PSC.

The Novel Crohn's Disease Marker Anti-GP2 Antibody Is Associated with Ileocolonic Location of Disease.

Crohn's disease (CD) is an inflammatory bowel disease (IBD) that can affect the whole gastrointestinal tract. The ileocolonic variant of CD, an inflammation of both the ileum and the large intestine, accounts for up to 50% of the cases with CD, whereas Crohn's ileitis affecting the ileum is diagnosed in about 30%. Crohn's colitis, which is confined to the large intestine and accounts for the remaining 20%, is difficult to distinguish from the large bowel inflammation seen in patients with ulcerative colitis (UC). The pathogenesis of CD is not yet completely understood. Autoimmunity is one factor that can partake in the triggering or modulation of inflammatory processes in IBD. The major zymogen-granule membrane glycoprotein 2 (GP2) has been recently identified as a major autoantigenic target in CD. Interestingly, GP2 is mainly expressed in the pancreas and has also been demonstrated to be a membrane-anchored receptor of microfold cells in the follicle-associated epithelium. Remarkably, GP2 is overexpressed at the site of CD inflammation in contrast to the one in UC. By utilizing novel enzyme-linked immunosorbent assays for the detection of GP2-specific IgA and IgG, the loss of tolerance to GP2 has been associated with a specific clinical phenotype in CD, in particular with the ileocolonic location of the disease.

Erosion of telomeric 3'-overhangs in white blood cells of aged subjects with high frequency of very short telomeres.

After extended proliferation, cells enter a state of replicative quiescence that is probably due to progressive telomere shortening. It is supposed that changes in telomere structure eventually expose the chromosome ends to undesired recombination events and thus promote cell senescence. The telomeric 3'-overhang is crucial for efficient chromosome capping, but its specific role in telomere shortening and in triggering the senescence program is uncertain. We have addressed this issue by measuring the 3'-overhangs of a human tissue cells aging in vivo. The 3'-overhangs were analyzed in blood samples from 41 individuals aged 91-106 years and 89 individuals ranging from 6 months to 85 years. We found that the overall 3'-overhang length did not significantly change with age, but did, however, find extensively eroded 3'-overhangs in 3 subjects of the 91-106 years cohort and one 61 years old subject affected with Down syndrome. These subjects had 3'-overhang length distributions skewed towards shorter tails, the shortest overall telomere lengths and the highest frequencies of very short telomeres. These data raise the possibility that during ageing very short telomeres with very poor 3'-overhangs can reach a critical point for functional telomeres.

The role of DNA damage response pathways in chromosome fragility in Fragile X syndrome.

FRAXA is one of a number of fragile sites in human chromosomes that are induced by agents like fluorodeoxyuridine (FdU) that affect intracellular thymidylate levels. FRAXA coincides with a >200 CGG*CCG repeat tract in the 5' UTR of the FMR1 gene, and alleles prone to fragility are associated with Fragile X (FX) syndrome, one of the leading genetic causes of intellectual disability. Using siRNA depletion, we show that ATR is involved in protecting the genome against FdU-induced chromosome fragility. We also show that FdU increases the number of gamma-H2AX foci seen in both normal and patient cells and increases the frequency with which the FMR1 gene colocalizes with these foci in patient cells. In the presence of FdU and KU55933, an ATM inhibitor, the incidence of chromosome fragility is reduced, suggesting that ATM contributes to FdU-induced chromosome fragility. Since both ATR and ATM are involved in preventing aphidicolin-sensitive fragile sites, our data suggest that the lesions responsible for aphidicolin-induced and FdU-induced fragile sites differ. FRAXA also displays a second form of chromosome fragility in absence of FdU, which our data suggest is normally prevented by an ATM-dependent process.