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

New Platform Technology for Comprehensive Serological Diagnostics of Autoimmune Diseases

Antibody assessment is an essential part in the serological diagnosis of autoimmune diseases. However, different diagnostic strategies have been proposed for the work up of sera in particular from patients with systemic autoimmune rheumatic disease (SARD). In general, screening for SARD-associated antibodies by indirect immunofluorescence (IIF) is followed by confirmatory testing covering different assay techniques. Due to lacking automation, standardization, modern data management, and human bias in IIF screening, this two-stage approach has recently been challenged by multiplex techniques particularly in laboratories with high workload. However, detection of antinuclear antibodies by IIF is still recommended to be the gold standard method for antibody screening in sera from patients with suspected SARD. To address the limitations of IIF and to meet the demand for cost-efficient autoantibody screening, automated IIF methods employing novel pattern recognition algorithms for image analysis have been introduced recently. In this respect, the AKLIDES technology has been the first commercially available platform for automated interpretation of cell-based IIF testing and provides multiplexing by addressable microbead immunoassays for confirmatory testing. This paper gives an overview of recently published studies demonstrating the advantages of this new technology for SARD serology

Toll-like receptor 9 controls anti-DNA autoantibody production in murine lupus

Systemic autoimmune disease in humans and mice is characterized by loss of immunologic tolerance to a restricted set of self-nuclear antigens. Autoantigens, such as double-stranded (ds) DNA and the RNA-containing Smith antigen (Sm), may be selectively targeted in systemic lupus erythematosus because of their ability to activate a putative common receptor. Toll-like receptor 9 (TLR9), a receptor for CpG DNA, has been implicated in the activation of autoreactive B cells in vitro, but its role in promoting autoantibody production and disease in vivo has not been determined. We show that in TLR9-deficient lupus-prone mice, the generation of anti-dsDNA and antichromatin autoantibodies is specifically inhibited. Other autoantibodies, such as anti-Sm, are maintained and even increased in TLR9-deficient mice. In contrast, ablation of TLR3, a receptor for dsRNA, did not inhibit the formation of autoantibodies to either RNA- or DNA-containing antigens. Surprisingly, we found that despite the lack of anti-dsDNA autoantibodies in TLR9-deficient mice, there was no effect on the development of clinical autoimmune disease or nephritis. These results demonstrate a specific requirement for TLR9 in autoantibody formation in vivo and indicate a critical role for innate immune activation in autoimmunity

SLE, An Overlooked Disease: Possibilities for Early Rescue by Early Diagnosis

Systemic lupus erythematosus (SLE) is a progressive autoimmune disease associated with widespread organ damage that can eventually cause death. Worldwide prevalence of SLE is difficult to report mainly due to difficulty in diagnosis as a result of its heterogeneous nature and nonspecific protean manifestations. Currently, circulating anti-DNA antibodies are the most specific diagnostic biomarkers for SLE where many detection assays are being employed in clinical practice. However, the diagnostic value of these techniques is challenged by the detection of only subpopulations of these antibodies with varying sensitivity and specificity. This is mainly attributed to differences in the antigen source and presentation and in the employed reaction conditions. This chapter will thoroughly discuss the technology, advantages, and limitations of each assay in addition to a special focus on the recently developed diagnostic technologies and novel biomarkers. Moreover, SLE will be presented as a disease model highlighting the importance of personalized medicine

B cells at the interface of innate and adaptive immunity in systemic lupus erythematosus: requirements for the activation of autoreactive B cells in autoimmune disease

Systemic autoimmune disease is characterized by loss of immunologic tolerance to a restricted set of self-nuclear antigens. These macromolecular complexes can be grouped into two categories: DNA-containing autoantigens such as chromatin, and RNA containing autoantigens such as Smith antigen (Sm) and related ribonucleoprotein complexes. Elucidating the mechanism for selective targeting of these molecules in systemic lupus erythematosus (SLE) may provide clues to the etiology of disease. We hypothesized that Toll-like receptors (TLRs), germline-encoded pattern-recognition receptors of the innate immune system, could dictate target antigen specificity in SLE. Using genetic ablation of various TLRs in murine models of SLE, we have demonstrated that TLRs are critical for directing the autoimmune response against canonical nuclear autoantigens.In the absence of TLR9, a receptor for CpG sequence motifs in DNA, the generation of autoantibodies to DNA-containing antigens was specifically inhibited. Other autoantibodies specific for RNA-containing antigens were maintained or even increased in TLR9-deficient autoimmune mice. We then investigated whether TLR3, a receptor for double-stranded RNA, or TLR7, a receptor for single-stranded RNA, were required for the generation of autoantibodies to RNA-containing antigens. While TLR3 did not appear to affect autoantibody production, the absence of TLR7 led to a reduction in anti-ribonucleoprotein antibodies. Genetic deletion of these receptors also had dramatic, but opposing, effects on disease progression. TLR9-deficient mice developed exacerbated disease and systemic inflammation with accelerated mortality, while TLR7-deficient mice had ameliorated clinical disease and decreased immune activation. A critical component of disease pathogenesis in these mice appeared to be the activation of type I interferon-producing plasmacytoid dendritic cells (pDCs), which was increased in the absence of TLR9, but decreased in the absence of TLR7.Further studies on the mechanism of autoantibody production and pDC activation revealed that TLR9 expression within B cells was required for anti-DNA antibody production, and that circulating serum factors generated in the absence of TLR9 could induce interferon production by pDCs. We have also shown that costimulation by CD4+ helper T cells contributes to autoantibody production in SLE. Integration of innate and adaptive activation signals is thus central to B cell autoantibody production and clinical disease progression in SLE

Toll-like Receptor 7 and TLR9 Dictate Autoantibody Specificity and Have Opposing Inflammatory and Regulatory Roles in a Murine Model of Lupus

SummaryAntibodies (Abs) to RNA- and DNA-containing autoantigens are characteristic of systemic lupus erythematosus (SLE). We showed previously that Toll-like receptor (TLR) 9, recognizing DNA, is required for the spontaneous generation of DNA autoantibodies, but not for the development of lupus nephritis in susceptible mice. We report that lupus-prone mice deficient in TLR7, a receptor for ssRNA, failed to generate Abs to RNA-containing antigens (Ags) such as Smith (Sm) Ag. TLR9 and TLR7 also had dramatic effects on clinical disease in lupus-prone mice. In the absence of TLR9, autoimmune disease was exacerbated, lymphocytes and plasmacytoid DCs were more activated, and serum IgG and IFN-α were increased. In contrast, TLR7-deficient mice had ameliorated disease, decreased lymphocyte activation, and decreased serum IgG. These findings reveal opposing inflammatory and regulatory roles for TLR7 and TLR9, despite similar tissue expression and signaling pathways. These results have important implications for TLR-directed therapy of autoimmune disease

The scavenger receptor SCARF1 mediates apoptotic cell clearance and prevents autoimmunity

Clearance of apoptotic cells is critical for control of tissue homeostasis however the full range of receptor(s) on phagocytes responsible for recognition of apoptotic cells remains to be identified. Here we show that dendritic cells (DCs), macrophages and endothelial cells use scavenger receptor type F family member 1 (SCARF1) to recognize and engulf apoptotic cells via C1q. Loss of SCARF1 impairs uptake of apoptotic cells. Consequently, in SCARF1-deficient mice, dying cells accumulate in tissues leading to a lupus-like disease with the spontaneous generation of autoantibodies to DNA-containing antigens, immune cell activation, dermatitis and nephritis. The discovery of SCARF1 interactions with C1q and apoptotic cells provides insights into molecular mechanisms involved in maintenance of tolerance and prevention of autoimmune disease

Lung involvement in "stable" undifferentiated connective tissue diseases: a rheumatology perspective

Previous studies of the occurrence of interstitial lung disease (ILD) in undifferentiated connective tissue diseases (UCTD) were conducted in patients admitted to Respiratory Medicine Units. The aim of the present prospective study was to investigate lung involvement in UCTD patients admitted to a Rheumatology Unit. Eighty-one consecutive UCTD patients were enrolled in the study. Each patient underwent history and physical examination, routine laboratory investigations, antinuclear antibody (ANA) profiling, B-mode echocardiography, and lung function study according to previously reported methods. Lung high resolution computed tomography (HRCT) was performed in patients who provided informed consent. Six patients (7.4%) had a history of grade II dyspnea. Three of them had a DLCO ranging from 42 to 55% of the predicted value; and a HRCT-documented ILD with a non-specific interstitial pneumonia (NSIP) pattern. Symptoms in the other three patients were due to cardiac disease. None of the 75 asymptomatic patients, had relevant findings at physical examination, 26/75 had a DLCO <80% (<70% in 10 cases). Of these, 3 of the 30 patients who underwent lung HRCT were affected by NSIP-ILD. Six of the 81 enrolled were affected by ILD, which was symptomatic in three patients. A higher percentage of patients had a reduced DLCO. The latter finding may reflect a preradiographic ILD or a preechocardiographic pulmonary vascular disease

Automated evaluation of autoantibodies on human epithelial-2 cells as an approach to standardize cell-based immunofluorescence tests

INTRODUCTION: Analysis of autoantibodies (AAB) by indirect immunofluorescence (IIF) is a basic tool for the serological diagnosis of systemic rheumatic disorders. Automation of autoantibody IIF reading including pattern recognition may improve intra- and inter-laboratory variability and meet the demand for cost-effective assessment of large numbers of samples. Comparing automated and visual interpretation, the usefulness for routine laboratory diagnostics was investigated. METHODS: Autoantibody detection by IIF on human epithelial-2 (HEp-2) cells was conducted in a total of 1222 consecutive sera of patients with suspected systemic rheumatic diseases from a university routine laboratory (n = 924) and a private referral laboratory (n = 298). IIF results from routine diagnostics were compared with a novel automated interpretation system. RESULTS: Both diagnostic procedures showed a very good agreement in detecting AAB (kappa = 0.828) and differentiating respective immunofluorescence patterns. Only 98 (8.0%) of 1222 sera demonstrated discrepant results in the differentiation of positive from negative samples. The contingency coefficients of chi-square statistics were 0.646 for the university laboratory cohort with an agreement of 93.0% and 0.695 for the private laboratory cohort with an agreement of 90.6%, P < 0.0001, respectively. Comparing immunofluorescence patterns, 111 (15.3%) sera yielded differing results. CONCLUSIONS: Automated assessment of AAB by IIF on HEp-2 cells using an automated interpretation system is a reliable and robust method for positive/negative differentiation. Employing novel mathematical algorithms, automated interpretation provides reproducible detection of specific immunofluorescence patterns on HEp-2 cells. Automated interpretation can reduce drawbacks of IIF for AAB detection in routine diagnostics providing more reliable data for clinicians