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.

Analysis of anti-ganglioside antibodies by a line immunoassay in patients with chronic-inflammatory demyelinating polyneuropathies (CIDP).

BACKGROUND: Unlike for acute immune-mediated neuropathies (IN), anti-ganglioside autoantibody (aGAAb) testing has been recommended for only a minority of chronic IN yet. Thus, we used a multiplex semi-quantitative line immunoassay (LIA) to search for aGAAb in chronic-inflammatory demyelinating polyneuropathy (CIDP) and its clinical variants. METHODS: Anti-GAAb to 11 gangliosides and sulfatide (SF) were investigated by LIA in 61 patients with IN (27 typical CIDP, 12 distal-acquired demyelinating polyneuropathy, 6 multifocal-acquired demyelinating sensory/motor polyneuropathy, 10 sensory CIDP, 1 focal CIDP and 5 multifocal-motoric neuropathy), 40 with other neuromuscular disorders (OND) (15 non-immune polyneuropathies, 25 myasthenia gravis), 29 with multiple sclerosis (MS) and 54 healthy controls (HC). RESULTS: In contrast to IgG, positive anti-GAAB IgM against at least one ganglioside/SF was found in 17/61 (27.9%) IN compared to 2/40 (5%) in OND, 2/29 MS (6.9%) and 4/54 (7.4%) in HC (p=0.001). There was a statistically higher prevalence of anti-sulfatide (aSF) IgM in IN compared to OND (p=0.008). Further, aGM1 IgM was more prevalent in IN compared to OND and HC (p=0.009) as well as GD1b in IN compared to HC (p<0.04). The prevalence of aGM1 IgM in CIDP was lower compared to in multifocal motor neuropathy (MMN) (12% vs. 60%, p=0.027). Patients showing aSF, aGM1 and aGM2 IgM were younger compared to aGAAb negatives (p<0.05). Patients with aSF IgM positivity presented more frequently typical CIDP and MMN phenotypes (p<0.05, respectively). CONCLUSIONS: The aGAAb LIA revealed an elevated frequency of at least one aGAAb IgM in CIDP/MMN patients. Anti-SF, aGM1 and aGM2 IgM were associated with younger age and anti-SF with IN phenotypes.

Next-Generation Autoantibody Testing by Combination of Screening and Confirmation-the CytoBead® Technology.

Occurrence of autoantibodies (autoAbs) is a hallmark of autoimmune diseases, and the analysis thereof is an essential part in the diagnosis of organ-specific autoimmune and systemic autoimmune rheumatic diseases (SARD), especially connective tissue diseases (CTDs). Due to the appearance of autoAb profiles in SARD patients and the complexity of the corresponding serological diagnosis, different diagnostic strategies have been suggested for appropriate autoAb testing. Thus, evolving assay techniques and the continuous discovery of novel autoantigens have greatly influenced the development of these strategies. Antinuclear antibody (ANA) analysis by indirect immunofluorescence (IIF) on tissue and later cellular substrates was one of the first tests introduced into clinical routine and is still an indispensable tool for CTD serology. Thus, screening for ANA by IIF is recommended to be followed by confirmatory testing of positive findings employing different assay techniques. Given the continuous growth in the demand for autoAb testing, IIF has been challenged as the standard method for ANA and other autoAb analyses due to lacking automation, standardization, modern data management, and human bias in IIF pattern interpretation. To address these limitations of autoAb testing, the CytoBead® technique has been introduced recently which enables automated interpretation of cell-based IIF and quantitative autoAb multiplexing by addressable microbead immunoassays in one reaction environment. Thus, autoAb screening and confirmatory testing can be combined for the first time. The present review discusses the history of autoAb assay techniques in this context and gives an overview and outlook of the recent progress in emerging technologies.

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.

Detection of anti-dsDNA antibodies by computer-aided automated immunofluorescence analysis.

INTRODUCTION: NOVA View is a computer aided fluorescence microscope that is used for the automated reading and interpretation of indirect immunofluorescent tests in diagnostic immunology. The objective of the present study was to evaluate the performance of the NOVA View® system for the measurement of anti-dsDNA antibodies using the Crithidia luciliae indirect immunofluorescence test (CLIFT) technology. METHODS: Analytical performance of NOVA View CLIFT was assessed in repeatability (within run) and reproducibility (between runs and instruments) studies. Two hundred-fifty patient samples (N=200 consecutive samples and N=50 samples from systemic lupus erythematosus patients) were tested to evaluate the agreement between results generated with NOVA View CLIFT, and those obtained with manual microscopic reading of the same slides. Positivity rate in SLE was assessed on the 50 SLE samples. RESULTS: The NOVA View system showed high level of repeatability and reproducibility within runs, between runs, and between instruments. Agreement of NOVA View software interpretation and digital image reading results with manual microscopic reading results was 96.0%, and the same positivity rate was obtained on SLE samples by NOVA View digital image reading as that of manual microscopic reading (36.0% vs. 38.0%, respectively). CONCLUSION: Results generated by NOVA View CLIFT were equivalent to those obtained by manual microscopic reading on a large routine sample set. NOVA View demonstrated consistency within and between runs, and between instruments. Automation of CLIFT provides reliability and is a suitable alternative for routine clinical laboratories.

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.

Assessment of modulated cytostatic drug resistance by automated γH2AX analysis.

The efficacy of many chemotherapeutic agents relies on the preferential destruction of rapidly dividing cancer cells by inducing various kinds of DNA damage. The most deleterious type of DNA lesions are DNA double-strand breaks (DSB), which can be detected by immunofluorescence staining of phosphorylated histone protein H2AX (γH2AX). Furthermore, γH2AX has been suggested as clinical pharmacodynamic biomarker in chemotherapeutic cancer treatment. A great challenge in treating neoplastic diseases is the varying response behavior among cancer patients. Thus, intrinsic or drug-induced overexpression of efflux pumps often leads to multiple drug resistance (MDR) and treatment failure. In particular, inter-individual differences in expression levels of efflux pumps, such as the permeability glycoprotein (P-gp), were shown to correlate with cancer progression. Several efficient cytostatic drugs, including the DSB-inducing agent etoposide (ETP) are known P-gp substrates. In this respect, modulation of MDR by P-gp inhibitors, like the immunosuppressives cyclosporine A (CsA) and rapamycin (Rapa) have been described. Here, we investigated the application of γH2AX focus assay to monitor the impact of CsA and Rapa on ETP-induced cytotoxicity in human peripheral blood mononuclear cells. Evaluation of γH2AX foci was performed by the automated fluorescence microscopy and interpretation system AKLIDES. Compared to ETP treatment alone, our results revealed a significant rise in γH2AX focus number and percentage of DSB-positive cells after cells have been treated with ETP in the presence of either CsA or Rapa. In contrast, DSB levels of cells incubated with CsA or Rapa alone were comparable to focus number of untreated cells. Our results successfully demonstrated how automated γH2AX analysis can be used as fast and reliable approach to monitor drug resistance and the impact of MDR modulators during treatment with DSB-inducing cytostatics..