Over-Expression of LEDGF/p75 in HEp-2 Cells Enhances Autoimmune IgG Response in Patients with Benign Prostatic Hyperplasia-A Novel Diagnostic Approach with Therapeutic Consequence?

Lens epithelium-derived growth factor splice variant of 75 kDa (LEDGF/p75) is an autoantigen over-expressed in solid tumors and acts as a stress-related transcriptional co-activator. Participation of autoimmune responses in the pathophysiology of benign prostatic hyperplasia (PBH) and a corresponding immunosuppressive therapy by TNFalpha antagonists has been recently suggested. Thus, autoAb testing could aid in the diagnosis of BPH patients profiting from such therapy. We generated CRISPR/Cas9 modified HEp-2 LEDGF knock-out (KO) and HEp-2 LEDGF/p75 over-expressing (OE) cells and examined IgG autoantibody reactivity to LEDGF/p75 in patients with prostate cancer (PCa, n = 89), bladder cancer (BCa, n = 116), benign prostatic hyperplasia (BPH, n = 103), and blood donors (BD, n = 60) by indirect immunofluorescence assay (IFA). Surprisingly, we could not detect elevated binding of autoAbs against LEDGF/p75 in cancer patients, but autoAb reactivity to LEDGF/p75 OE cells in about 50% of patients with BPH was unexpectedly significantly increased. Furthermore, a line immunoassay enabling the detection of 18 different autoAbs revealed a significantly increased occurrence of anti-dsDNA autoAbs in 34% of BPH patients in contrast to tumor patients and BD. This finding was confirmed by anti-mitochondrial (mDNA) autoAb detection with the Crithidia luciliae immunofluorescence test, which also showed a significantly higher prevalence (34%) of anti-mDNA autoAbs in BPH. In summary, our study provided further evidence for the occurrence of autoimmune responses in BPH. Furthermore, LEDGF/p75 over-expression renders HEp-2 cells more autoantigenic and an ideal target for autoAb analysis in BPH with a potential therapy consequence.

Human glycoprotein-2 expressed in Brunner glands - A putative autoimmune target and link between Crohn's and coeliac disease.

Glycoprotein 2 (GP2) is an autoantigen in Crohn's (CD) and coeliac disease (CeD). We assessed GP2-isoform (GP21-4)-expression in intestinal biopsies of paediatric patients with CD, CeD, ulcerative colitis (UC), and healthy children (HC). Transcription of GP21-4 was elevated in proximal small intestine in CeD and CD patients (only GP22/4) compared to jejunum (CeD/CD) and large bowel (CD). CeD patients demonstrated higher duodenal GP22/4-mRNA levels compared to HC/UC patients whereas CD patients showed higher GP24-mRNA levels compared to UC patients. Duodenal synthesis of only small GP2 isoforms (GP23/4) was demonstrated in epithelial cells in patients/HC and in Brunner glands (also large isoforms) with a more frequent apical location in CD/CeD patients. All four GP2 isoforms interacted with gliadin and phosphopeptidomannan. Gliadin digestion improved binding to GP2 isoforms. GP21-4 binding to CeD/CD-related antigens, elevated duodenal GP21-4-mRNA transcription, and GP2-protein secretion in Brunner glands of CeD/CD patients suggest an autoimmune CeD/CD link.

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.

Mucosal Autoimmunity to Cell-Bound GP2 Isoforms Is a Sensitive Marker in PSC and Associated With the Clinical Phenotype.

Introduction: Zymogen granule glycoprotein 2 (GP2) was demonstrated as first autoimmune mucosal target in primary sclerosing cholangitis (PSC) associated with disease severity. Autoantibodies to four GP2 isoforms (aGP21-4) were found in patients with inflammatory bowel diseases but reactivity against specific GP2 epitopes has not been investigated in PSC yet. Hence, the prevalence of aGP21-4 and their association with the PSC phenotype for risk prediction were examined. Methods: GP2 isoforms were stably expressed as glycosylphosphatidyl - inositol-anchored molecules in the membrane of HEp-2 cells and used as autoantigenic targets in indirect immunofluorescence assay (IFA). aGP21-4 IgA and IgG were detected by IFA in 212 PSC patients of four European university hospitals and 145 controls comprising 95 patients with cystic fibrosis and 50 healthy subjects. Results: Combined aGP21 and aGP24 IgA testing with a sensitivity of 66.0% and a specificity of 97.9% resulted in the best diagnostic performance (Youden index: 0.64) regarding all aGP2 and combinations thereof. aGP24 IgA positivity is significantly associated with the presence of cirrhosis in PSC (p = 0.0056). Logistic regression revealed the occurrence of aGP21 IgA (odds ratio [OR] 1.38, 95% confidence interval [CI]: 1.03-1.86) and aGP24 IgA (OR 1.52, 95%CI: 1.07-2.15) along with male gender (OR 0.51, 95%CI: 0.27-0.97) and older age (OR 1.03 95%CI: 1.01-1.05) as significant risks for the concomitant presence of cirrhosis in PSC. Conclusions: Combined aGP21 and aGP24 IgA analysis is preferred to single aGP2 isoform analysis for sensitive PSC autoantibody testing. Positivity for aGP21 and aGP24 IgA is associated with cirrhosis in PSC and could be used for risk stratification.

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.

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.