Lens epithelium-derived growth factor splice variant of 75 kDa (LEDGF/p75) is an au- toantigen 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 ther- apy. 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.

Antiphospholipid antibodies (aPL), assumed to cause antiphospholipid syndrome (APS), are notorious for their heterogeneity and detect phospholipids and phospholipid-binding proteins. The persistent presence of Lupus anticoagulant and/or aPL against cardiolipin and/or β2 glycoprotein I have been shown to be independent risk factors for vascular thrombosis and pregnancy morbidity in APS. Among 30 others, viral infections have been proposed to trigger the production of aPL while mostly being considered non-pathogenic. Yet, the potential pathogenicity of infection-associated aPL has gained momentum since an increasing number of patients infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been described with coagulation abnormalities and hyperinflamma-tion, together with the presence of aPL. Here, we present data from a multicentric, mixed-severity 35 study including three cohorts of individuals who contracted SARS-CoV-2 as well as non-infected blood donors. We simultaneously measured 10 different criteria and non-criteria aPL (IgM and IgG) by using a line immunoassay. Further, IgG antibody response against three SARS-CoV-2 proteins was investigated using tripartite automated blood immunoassay technology. Our analyses revealed that select non-criteria aPL are enriched concomitant to or after an infection with SARS-CoV-2. Linear mixed-ef-40 fect models suggest an association of aPL to prothrombin (PT) with the strength of the antibody response against SARS-CoV-2 and further influenced by SARS-CoV-2 disease severity and sex of the individuals. In conclusion, our study is the first to report an association between disease severity, anti-SARS-CoV-2 immunoreactivity and aPL against PT in patients with SARS-CoV-2.

Aim TSH-receptor (TSHR)-autoantibody (TRAb) is the serological hallmark of Graves’ disease (GD). Recently, 3rd-generation radioimmunoassays (RIA) employing monoclonal TRAb such as M22 or T7 instead of TSH for the inhibition of human TRAb binding with solid-phase TSHR (coated tubes) have been introduced into laboratory routine. Methods As current assays typically employ a consecutive incubation of patient serum and labelled monoclonal TRAb, automation of TRAb RIA is a challenge. Thus, the assay procedure using human TSHR-coated tubes and the mouse monoclonal TRAb T7 was modified by combining both steps. The novel one-step method was compared with its corresponding consecutive 3rd-generation RIA by investigating 304 individuals encompassing 102 patients with active GD (GDa), 43 patients with GD after successful therapy (GDt), 31 with Hashimoto’s disease (HD), 28 with non-autoimmune thyroid diseases (NAITD) and 100 healthy subjects (HS). Results With the new method, the incubation time was shortened by approximately one hour. Both 3rd-generation RIAs did not reveal a significantly different assay performance by comparing areas under the curve (AUC) with receiver operating characteristics curve analysis (AUC one-step: 0.94, AUC two-step: 0.96, p > 0.05, respectively). The two-step TRAb RIA demonstrated sensitivity and specificity values of 87.5 % and 96.2 %, respectively, whereas the one-step revealed 84.6 % and 96.2 %, respectively. Conclusion One-step 3rd-generation RIA may be used for the reliable detection of TRAb. The shorter and easier assay design may improve its use and enable automation in routine nuclear medicine laboratories.

Autoimmunity to chitinase 3-like 1 (CHI3L1) has recently been reported in hepatic and bowel inflammatory conditions. Considering that CHI3L1 plays a role as prognostic biomarker in multiple sclerosis (MS), here we investigated CHI3L1 as potential autoantigenic target in the disease. We determined serum CHI3L1 autoantibodies by enzyme-linked immunosorbent assay (ELISA) in a cohort of 60 untreated MS patients with different clinical forms of the disease and 20 healthy controls (HC). IgG levels to CHI3L1 were similar between patients with relapsing-remitting MS, primary and secondary progressive MS, and HC. These findings do not support a role for CHI3L1 autoantibodies in MS pathogenesis.

Background Lens epithelium derived growth factor splice variant of 75 kDa (LEDGF/p75), is overexpressed in different solid cancers and cancer cell lines and various autoinflammatory diseases. Due to its ability to bind chromatin, it acts as a transcriptional co-activator and promotes anti-apoptotic signalling pathways that lead to increased tumour aggressiveness and resistance to chemotherapy. The role of LEDGF/p75 in DNA-damage repair (DDR) is still not completely elucidated particularly regarding the ubiquitin-dependent regulation and degradation of DDR signalling molecules.Methods Different LEDGF model cell lines were generated, a complete knock-out of LEDGF (KO) as well as the re-expression of LEDGF/p75 or LEDGF/p52 using CRISPR/Cas9 technology. Then, various assays were performed to determine their proliferation and migration capacity as well as their chemosensitivity. Moreover, DDR signalling pathways were investigated by western blot and immunofluorescence.ResultsLEDGF-deficient cells exhibited a decreased proliferation (d t (WT) = 21 h, d t (KO) = 26 h) , 60 % decreased migration, as well as an 30-50 % increased sensitivity towards the topoisomerase II inhibitor etoposide. Moreover, LEDGF depleted cells showed a significant reduction by 65 % in the recruitment of downstream DDR-related proteins like replication protein A 32 kDa subunit (RPA32) after exposure to etoposide. Re-expression of LEDGF/p75 rescued all knock-out effects, while re-expression of LEDGF/p52 had no effect.Surprisingly, untreated LEDGF KO cells showed an increased amount of DNA fragmentation combined with an increased formation of γH2AX and Breast cancer type 1 susceptibility protein (BRCA1). In contrast, the protein levels of ubiquitin-conjugating enzyme UBC13 and nuclear proteasome activator PA28γ were substantially reduced upon LEDGF KO. Conclusions This study provides evidence that LEDGF is not only an important player in the DDR after chemotherapeutic treatments but is also involved in the maintenance of the general genome integrity. Moreover, this study provides for the first time an insight into the possible role of LEDGF in the ubiquitin-dependent regulation of DDR signalling molecules and highlights the involvement of LEDGF/p75 in homology-directed DNA repair.