Novel mutation and expanding phenotype in IRF2BP2 deficiency.

OBJECTIVES: Inborn errors of immunity manifest with susceptibility to infection but may also present with immune dysregulation only. According to the European Society for Immunodeficiencies Registry about 50% of inborn errors of immunity are classified as common variable immunodeficiencies (CVID). In only few CVID patients are monogenic causes identified. IFN regulatory factor-2 binding protein 2 (IRF2BP2) is one of 20 known genes associated with CVID phenotypes and has only been reported in two families so far. We report another IRF2BP2-deficient patient with a novel pathogenic variant and phenotype and characterize impaired B cell function and immune dysregulation. METHODS: We performed trio whole-exome sequencing, determined B cell subpopulations and intracellular calcium mobilization upon B cell receptor crosslinking in B cells. T cell subpopulations, T cell proliferation and a type I IFN signature were measured. Colonoscopy and gastroduodenoscopy including histopathology were performed. RESULTS: The 33-year-old male presented with recurrent respiratory infections since childhood, colitis and RA beginning at age 25 years. We identified a novel de novo nonsense IRF2BP2 variant c.1618C>T; p.(Q540*). IgG deficiency was detected as consequence of a severe B cell differentiation defect. This was confirmed by impaired plasmablast formation upon stimulation with CpG. No serum autoantibodies were detected. Intracellular cytokine production in CD4+ T cells and CTLA4 expression on FOXP3+ Tregs were impaired. Type I IFN signature was elevated. CONCLUSION: The identified loss-of-function variant in IRF2BP2 severely impairs B cell development and T cell homeostasis, and may be associated with colitis and RA. Our results provide further evidence for association of IRF2BP2 with CVID and contribute to the understanding of the underlying pathomechanisms.

Anti-Proteinase 3 Antibodies as a Biomarker for Ulcerative Colitis and Primary Sclerosing Cholangitis in Children.

OBJECTIVES: Anti-neutrophil cytoplasmic antibody (ANCA) directed against proteinase 3 (PR3) is a marker for granulomatosis with polyangiitis, but is also found in patients with inflammatory bowel disease (IBD), mainly ulcerative colitis (UC). The aim of our study was to investigate ANCA and PR3-ANCA in paediatric IBD. METHODS: We tested 326 paediatric IBD patients and 164 controls for anti-Saccharomyces cerevisiae antibodies (ASCA), ANCA (indirect immunofluorescence, IIF) and PR3-ANCA (chemiluminescence immunoassay). We applied the Paris classification for paediatric IBD and documented liver manifestations such as primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH). RESULTS: We found PR3-ANCA in 49/121 (40%) of UC, 20/187 (11%) of Crohn disease (CD) and 2/18 (11%) of IBD-unclassified (IBD-U) patients but in none of the controls. 54% UC and 12% CD patients were positive for ANCA (IIF). PR3-ANCA positive UC patients were characterised by more extensive disease (P = .070). Fourteen of 21 (67%) of UC patients with backwash ileitis were anti-PR3 ANCA-positive (P = .011). We diagnosed PSC or PSC/AIH in 19 UC and 3 IBD-U patients. Fifteen of 22 (68%) patients with PSC or PSC/AIH were anti-PR3-ANCA positive in contrast to 36 of 117 (32%) patients without PSC (P = .001). PR3-ANCA positive patients showed higher levels of gamma-glutamyl transferase, alanine transaminase and aspartate transferase (P < 0.001, 0.001, 0.006, respectively). Forty-seven percent of CD and 6% of UC patients were ASCA-IgA positive. PR3-ANCA-positive and -negative patients showed no significant differences concerning age at diagnosis, disease activity, need for drugs, and number of hospitalisations. CONCLUSIONS: Our study provides data for PR3-ANCA as a potential serological marker for paediatric UC and PSC.

Multiparametric Analysis of Connective Tissue Disease Specific Autoantibodies Using a Spot Immunoassay.

BACKGROUND: Based on advances over several decades, a significant number of autoantibodies are aiding the diagnosis, differential diagnosis and even prognostic estimates of patients with connective tissue diseases (CTD). Based on cost and time constraints, multiplex assays are used more and more in routine practice. Here we describe the evaluation results of a novel spot immunoassay (SeraSpot® ANA) for multiplex analysis of the main CTD specific autoantibodies, which is based on autoantigens immobilized on microtitration plates. METHODS: For evaluation of the ANA spot immunoassay, comprising dsDNA, histone, nucleosome, Scl-70, U1-RNP (mixture of U1-RNP-A, C, and 70k), Sm (SmD), RibP (P0), Ro52/TRIM21, Ro60, La/SS-B, CENP-B, Jo-1, PM/ Scl-100, and Ku as target antigens, sera of 489 patients with systemic autoimmune rheumatic disease (SARD), sera of 54 patients with herpes virus infections, and sera of 202 apparently healthy individuals (AHI) were tested. RESULTS: The concordance between the SeraSpot and EIA results of disease specific autoantibodies (AABs) was high (94 - 97% for CENP-B, Jo-1, Scl-70, Sm, La, Ro52 and Ro60 antibodies) to moderate (86.7% for dsDNA antibodies), and no major differences in the frequency of these AABs in patients with CTD were observed. In SLE patients, the SeraSpot results of dsDNA antibodies correlate better with CLIFT results and HEp-2 cell pattern than the EIA results. The diagnostic specificities of SLE, SSc, SjS, and myositis specific AABs are very high (96.5 - 100%) compared to apparently healthy individuals, but lower with regard to serological differentiation of the SARD entities (86.6 - 99.1%). However, very high positive likelihood ratios (10 up to infinite) could be obtained by disease specific AAB profiles. CONCLUSIONS: The SeraSpot® ANA assay allows the detection of 14 AAB specificities used for the diagnosis and dif-ferentiation of CTD in one approach. Although the concordance between the SeraSpot® assay and EIA is moderate for most of the tested AAB specificities, defined AAB profiles are suitable for the correct diagnosis of the CTD entities. If time matters, the authors suggest the parallel employment of immunofluorescence on HEp-2 cells and the SeraSpot® ANA assay for screening and specific AAB determination of patients with suspected CTD.

Identification of Chitinase-3-Like Protein 1 as a Novel Neutrophil Antigenic Target in Crohn's Disease.

BACKGROUND AND AIMS: There is an increasing incidence of inflammatory bowel disease [IBD]. Autoimmune responses are involved in the pathophysiology of IBD, but their underlying pathways and target antigens have not yet been fully elucidated. METHODS: Autoantigenic targets in IBD were identified after separation of whole cell proteins isolated from neutrophils using two-dimensional electrophoresis and matrix assisted laser desorption ionization - time of flight mass spectrometry-based protein identification of the spots that displayed Western blotting signals with anti-neutrophil cytoplasmic antibody-positive sera. The prevalence of IgG, IgA and secretory IgA [sIgA] to chitinase 3-like protein 1 [CHI3L1] was analysed by enzyme-linked immunosorbent assays using recombinant CHI3L1 in 110 patients with Crohn's disease [CD], 95 with ulcerative colitis [UC], 126 with coeliac disease [CeD] and 86 healthy controls [HCs]. RESULTS: The 18-glycosylhydrolase family member CHI3L1 was identified as a neutrophil autoantigenic target. CD patients displayed significantly higher levels of IgG to CHI3L1 than patients with UC and CeD (p < 0.0001, respectively). IgA and sIgA to CHI3L1 was significantly higher in CD than in UC, CeD and HCs [p < 0.0001, respectively]. IgA and sIgA to CHI3L1 demonstrated the highest prevalence in CD [25.5%, 28/110; and 41.8%%, 46/110] compared to HCs [2.3%, 2/86; and 4.7%%, 4/86; p = 0.0015 and p < 0.0001] and are associated with a more complicated progression of CD. CONCLUSION: CHI3L1 is a novel neutrophil autoantigenic target in CD. IgA and sIgA to CHI3L1 may serve as novel markers for CD and may facilitate the serological diagnosis of IBD.

Autoantibodies Against Glycoprotein 2 Isoforms in Pediatric Patients with Inflammatory Bowel Disease.

BACKGROUND: Anti-Glycoprotein 2 (GP2) antibodies are associated with a more complicated course of Crohn's disease (CD) in adults. Four different GP2 isoforms with different length and antibody-binding sites have been identified so far but not been explored in serological studies. We aimed to investigate the diagnostic utility of autoantibodies against all 4 isoforms of GP2 in an exclusively pediatric population for the first time. METHODS: We included 278 children and adolescents with inflammatory bowel disease: 164 with CD, 114 with ulcerative colitis, 83 disease controls (acute gastrointestinal infection, nonspecific gastrointestinal functional disorders), and 219 healthy controls. Sera were tested for anti-GP2 antibodies using 4 different isoforms of GP2 for anti-Saccharomyces cerevisiae antibodies, antineutrophil cytoplasmic antibodies, and pancreatic antibodies. RESULTS: Anti-GP2 antibodies were significantly more prevalent in patients with CD than in ulcerative colitis and controls. We found a sensitivity of 38% (with a specificity of 95%) for anti-GP2 IgG against isoform 4 in CD. Anti-GP2 IgA against isoform 1 and anti-GP2 IgG against isoform 4 possessed the best diagnostic values for identification of CD. For the differentiation of CD from ulcerative colitis anti-GP2 IgG against isoforms 3 and 4 proved to be most accurate markers. Anti-GP2 antibodies were associated with a more complicated disease behavior and bowel surgery in CD. In a subgroup of patients with CD, anti-GP2 IgG against isoform 4 proved to be a relatively stable marker over time independent of disease activity. CONCLUSIONS: Anti-GP2 antibodies against different isoforms are specific markers for CD and for different phenotypes in pediatric inflammatory bowel disease.

Simultaneous detection of celiac disease-specific IgA antibodies and total IgA.

PURPOSE: Celiac disease (CD) serology requires analysis of tissue transglutaminase type-2 (TG2autoAbs), deamidated gliadin (DGAbs), and as reference endomysial autoantibodies (EmA). Total IgA assessment helps to determine IgA-deficient CD patients. The novel multiplex indirect immunofluorescence (IIF) technique CytoBead was used to develop the first quantitative one-step serological CD assay comprising both simultaneous IgA autoAb and total IgA testing. METHODS: CytoBead CeliAK detecting TG2autoAb, DGAb, EmA, and simultaneously total IgA uses fluorescent microparticles for antigen and antibody immobilization along with monkey-esophagus tissue sections on glass slides. The assay was interpreted visually by classical fluorescent microscopy and digital IIF using AKLIDES(®). Overall, 380 samples (155 CD patients, 5 with IgA deficiency, 68 with cystic fibrosis, 59 with eye disease, 93 blood donors) were run for performance analysis. Data were compared with classical IgA autoAb analysis by ELISA and IIF. RESULTS: Comparing CD-specific IgA autoAb testing by CytoBead with classical IIF and ELISA, very good agreements for EmA, TG2autoAb, and DGAb were determined (Cohen's κ = 0.98, 0.96, 0.85, respectively). The difference between multiplex and single testing revealed a significant difference for TG2autoAb testing only (McNemar, p = 0.0078). Four CD patients and 4 controls demonstrated TG2autoAb positivity by ELISA but were negative by CytoBead. Further, 140/155 (90.9 %) CD patients demonstrated TG2autoAb levels above ten times the upper normal and all five IgA-deficient samples IgA levels <0.2 g/L by CytoBead. CONCLUSIONS: The novel multiplex CytoBead CeliAK enables simultaneous CD-specific autoAb and IgA deficiency analyses comparable with classical testing by single-parameter assays. Thus, comprehensive CD serology by CytoBead can alleviate the workload in routine laboratories.

Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing.

BACKGROUND: For the serological diagnosis of systemic autoimmune rheumatic diseases, a two-tier approach starting with sensitive antinuclear antibody (ANA) detection by indirect immunofluorescence (IIF) on HEp-2 cells followed by characterization of positive findings with different immunoassays is recommended. To overcome drawbacks of this approach, we developed a novel technique allowing the combination of screening and simultaneous confirmatory testing. For the first time, this creates the basis for second generation ANA testing. METHODS: ANA and autoantibodies (autoAbs) to double-stranded DNA (dsDNA), CENP-B, SS-A/Ro52, SS-A/Ro60, SS-B/La, RNP-Sm, Sm, and Scl-70 were determined by IIF and enzyme-linked immunosorbent assay (ELISA), respectively, and compared to simultaneous analysis thereof by second generation ANA analysis in patients with systemic lupus erythematosus (n=174), systemic sclerosis (n=103), Sjögren's syndrome (n=46), rheumatoid arthritis (n=36), mixed and undetermined connective tissue diseases (n=13), myositis (n=21), infectious disease (n=21), autoimmune liver disease (n=93), inflammatory bowel disease (n=78), paraproteinemia (n=11), and blood donors (n=101). RESULTS: There was very good agreement of second generation ANA testing with classical one by IIF and ELISA regarding testing for ANA and autoAbs to dsDNA, CENP-B, SS-B, RNP-Sm, Scl-70, SS-A/Ro52, and SS-A/Ro60 (Cohen's κ>0.8). The agreement for anti-Sm autoAb was good (κ=0.77). The differences of both approaches were not significant for autoAbs to SS-B/La, RNP-Sm, Scl-70, SS-A/Ro60, and SS-A/Ro52 (McNemar's test, p>0.05, respectively). CONCLUSIONS: Second generation ANA testing can replace the two-tier analysis by combining IIF screening with multiplex confirmative testing. This addresses shortcomings of classical ANA analysis like false-negative ANA findings and lack of laboratory efficiency and standardization.

Stable expression of human muscle-specific kinase in HEp-2 M4 cells for automatic immunofluorescence diagnostics of myasthenia gravis.

Muscle-specific kinase (MuSK) belongs to the nicotinic acetylcholine receptor complex which is targeted by pathogenic autoantibodies causing Myasthenia gravis. While up to 95% of patients with generalized Myasthenia gravis were shown to be positive for acetylcholine receptor-specific autoantibodies, up to 70% of the remaining patients develop autoantibodies against MuSK. Discrimination of the autoantibody specificity is important for therapy of Myasthenia gravis. Recently, the new automatic fluorescence assessment platform AKLIDES has been developed for immunofluorescence-based diagnostics of autoimmune diseases. In order to establish an AKLIDES procedure for the detection of MuSK-specific autoantibodies (anti-MuSK), we developed a recombinant HEp-2 cell clone expressing the human MuSK cDNA. Here we show at the mRNA and protein level that the cell clone HEp-2 M4 stably expresses human MuSK. We provide evidence for a localization of MuSK at the cell membrane. Using cell clone HEp-2 M4 on the AKLIDES system, we investigated 34 patient sera that were previously tested anti-MuSK positive by radioimmunoassay as positive controls. As negative controls, we tested 29 acetylcholine receptor-positive but MuSK-negative patient sera, 30 amytrophic lateral sclerosis (ALS) patient sera and 45 blood donors. HEp-2 M4 cells revealed a high specificity for the detection of MuSK autoantibodies from 25 patient sera assessed by a specific pattern on HEp-2 M4 cells. By using appropriate cell culture additives, the fraction of cells stained positive with anti-MuSK containing sera can be increased from 2-16% to 10-48%, depending on the serum. In conclusion, we provide data showing that the novel recombinant cell line HEp-2 M4 can be used to screen for anti-MuSK with the automatic AKLIDES system.

GD3-7-aldehyde is an apoptosis inducer and interacts with adenine nucleotide translocase.

We prepared GD3-7-aldehyde (GD3-7) and determined its apoptotic potential. GD3-7 proved to be more efficient to induce pro-apoptotic mitochondrial alterations than GD3 when tested on mouse liver mitochondria. GD3-7-induced mitochondrial swelling and depolarization was blocked by cyclosporin A (CsA) supporting a critical role of the permeability transition pore complex (PTPC) during GD3-7-mediated apoptosis. In contrast to GD3, GD3-7 was able to induce channel formation in proteoliposomes containing adenine nucleotide translocase (ANT). This suggests that ANT is the molecular target of GD3-7. Using a specific antiserum, GD3-7 was detected in the lipid extract of the myeloid tumor cell line HL-60 after apoptosis induction, but not in living cells. Therefore, GD3-7 might be a novel mediator of PTPC-dependent apoptosis in cancer cells.