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

Aufklärung der Rolle von DFS70/LEDGF/p75 bei der DNA-Schadensantwort mit Hilfe der CRISPR/Cas9-Technologie

Lens epithelium derived growth factor (LEDGF), is known to be overexpressed in different solid cancers and cancer cell lines but is also involved in acquired immunodeficiency syndrome (AIDS) and diverse inflammatory diseases. Due to its chromatin-binding ability, it acts as a transcriptional coactivator, promoting anti-apoptotic pathways leading to increased tumor aggressiveness and chemotherapy resistance. In this thesis, the role of LEDGF in laryngeal cancer cells was investigated. Thus, a knock-out of LEDGF in HEp-2 cells was successfully generated by CRISPR/Cas9 technology. Moreover, LEDGF-deficient HEp-2 cells exhibited a decreased proliferation and migration, as well as an increased chemotherapy sensitivity towards topoisomerase II inhibitor etoposide. The DNA damage response was in more detail investigated, where LEDGF depleted cells showed a drastic reduction in the recruitment of downstream damage-response related proteins, more precisely replication protein A subunit of 32kDa (RPA32), and an increased amount of DNA fragmentation, highlighting the role of LEDGF in homology-directed DNA repair. In addition, a efficient method was established to allow genomic tagging of LEDGF at its C-terminus with EGFP which enables the monitoring of LEDGF in living cells. Therefore, an efficient Cas9-mediated DNA DSB induction was supplemented with a DNA template containing homology arms to the C-terminus as well as the EGFP sequence to allow homology-directed repair. Subsequently, EGFP-expressing cells were FACS sorted and single cells expanded. Almost all test clones showed a homozygous genotype for LEDGF-EGFP fusion which exhibited the typical nuclear pattern of LEDGF.Der von Linsenepithel abgeleitete Wachstumsfaktor (LEDGF) ist dafür bekannt, dass er in verschiedenen Krebsarten und Krebszelllinien überexprimiert ist, aber auch am erworbenen Immundefizienzsyndrom (AIDS) und verschiedenen entzündlichen Erkrankungen beteiligt ist. Aufgrund seiner chromatin-bindenden Wirkung agiert er als transkriptioneller Coaktivator, der anti-apoptotische Wege fördert, welche wiederum zu erhöhter Tumoraggressivität und Chemotherapie-Resistenz führen. In dieser Arbeit wurde die Rolle von LEDGF in Kehlkopfkrebszellen untersucht. Mit Hilfe der CRISPR/Cas9-Technologie wurde LEDGF in HEp-2 Zellen erfolgreich ausgeschalten. Darüber hinaus zeigten LEDGF K.O. HEp-2-Zellen eine verminderte Proliferation und Migration sowie eine erhöhte Chemotherapie-Sensitivität gegenüber dem Topoisomerase-II-Inhibitor Etoposid. Bei der Untersuchung der DNA-Schadenreaktion zeigten LEDGF K.O. Zellen eine signifikante Verringerung der Rekrutierung von Downstream-Proteinen, hier im speziellen von der Replikationsprotein A 32kDa-Untereinheit (RPA32), und eine erhöhte Menge an DNA-Fragmentierung, was die Rolle von LEDGF bei der Homologie-spezifischen DNA-Reparatur unterstreicht. Darüber hinaus wurde eine effektive Methode etabliert, um den C-Terminus von LEDGF genomisch mittels EGFP zu markieren, um so die Beobachtung von LEDGF in lebenden Zellen zu ermöglichen. Hierbei wurde ein effizienter Cas9-induzierter DNA-Doppelstrangbruch herbeigeführt und durch eine DNA-Vorlage ergänzt. Diese enthält homologe Bereiche zum LEDGF C-Terminus sowie die EGFP-Sequenz, um eine Homologie-spezifische Reparatur zu ermöglichen. Anschließend wurden EGFP-exprimierende Zellen mittels FACS sortiert und Einzelzell-Kolonien gebildet. Fast alle Testklone zeigten einen homozygoten Genotyp für das LEDGF-EGFP-Fusionsprotein, mit dem typischen nukleären Expressionsmuster von LEDGF

Vom Linsenepithel abgeleiteter Wachstumsfaktor (LEDGF) als Hilfsmittel für die Diagnose der gutartigen Prostatahyperplasie (BPH) und die molekularen Grundlagen von LEDGF in menschlichen Krebszellen

Benign prostatic hyperplasia (BPH) is considered an age-related disease of men with unknown etiopathophysiology. Chronic inflammation represents one of the most important pathophysiological mechanisms in this context, and there is also evidence for the involvement of autoimmune responses in an inflammatory environment in the prostate. Conventional therapeutic approaches for BPH are limited; however, immunosuppression by TNF (tumor necrosis factor)- blockers seems to reduce symptoms in BPH patients due to the reduction of epithelial hyperplasia and macrophage-triggered inflammation. Therefore, this cumulative dissertation addresses biomarker-based BPH diagnosis by lens epithelium-derived growth factor splice variant 75 kDa (LEDGF/p75). LEDGF/p75 is an autoantigen that is overexpressed in solid tumors and functions as a stress-related transcriptional co-activator. It also plays an important role in cancer and DNA damage repair (DDR). The generation of CRISPR/Cas9-modified HEp-2 LEDGF deficient (KO) and HEp-2 LEDGF/p75 overexpressing (OE) cell lines provided new insights for the diagnosis and therapy of cancer and genetic disorders. Herein, IgG autoantibody reactivity to LEDGF/p75 was detected 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) linear immunoassay (LIA) and Chritidia luciliae immunofluorescence test (CLIFT). In addition, proliferation and migration capacity as well as chemosensitivity of genome-edited cells were determined by optimized all-in-one approach, followed by investigation of DDR signaling pathways by Western blot and immunofluorescence. Surprisingly, no increased binding of autoantibodies (autoAbs) to LEDGF/p75 was detected in cancer patients, but autoAb reactivity to LEDGF/p75 O/E cells was unexpectedly increased in approximately 50% of patients with BPH. At the molecular level, LEDGF-deficient cells exhibit decreased proliferation and migration and increased sensitivity to the chemotherapeutic agent etoposide. A significantly Decreased DNA damage response could also be visualized. New diagnostic capabilities using HEp-2 cells overexpressing LEDGF/p75 or mitochondrial DNA as autoimmune targets could be used to identify BPH patients with autoimmune responses. Furthermore, this Thesis shows that LEDGF is not only involved in the recruitment of DNA damage response proteins, but also affects the proteosomal regulation of DDR signaling molecules.Die benigne Prostatahyperplasie (BPH) gilt als altersbedingte Erkrankung des Mannes mit unbekannter Etiopathophysiology. Chronische Entzündungen stellen in diesem Zusammenhang eine der wichtigsten pathophysiologischen Mechanismen dar, zudem gibt es Beweise für die Beteiligung von Autoimmunreaktionen in einem entzündlichen Umfeld in der Prostata. Konventionelle Therapiekonzepte für BPH sind begrenzt, eine Immunsuppression durch TNF--Blocker scheint jedoch die Symptome bei BPH-Patienten aufgrund der Verringerung der Epithelhyperplasie und der durch Makrophagen ausgelösten Entzündung zu verringern. Die vorliegende kumulative Dissertation befasst sich daher mit der Biomarker-basierten BPH-Diagnostik durch die vom Linsenepithel-abgeleitete Wachstumsfaktor Splicevariante mit 75 kDa (LEDGF/p75). LEDGF/p75 ist ein Autoantigen, das in soliden Tumoren überexprimiert wird und als stressbedingter transkriptioneller Co-Aktivator fungiert. Darüber hinaus spielt es eine wichtige Rolle bei Krebs und der DNA-Schadensreparatur. Durch die Erzeugung von CRISPR/Cas9-modifizierten HEp-2 LEDGF defizienten und HEp-2 LEDGF/p75 überexprimierenden Zelllinien konnten neue Erkenntnisse für die Diagnostik und Therapie von Krebs und genetischen Störungen gewonnen werden. Hierfür wurde die IgG-Autoantikörperreaktivität auf LEDGF/p75 bei Patienten mit Prostatakrebs (PCa, n=89), Blasenkrebs (BCa, n=116), benigner Prostatahyperplasie (BPH, n=103) und Blutspendern (BD, n=60) mittels indirektem Immunfluoreszenztest (IFA) linearem Immunoassay (LIA) und Chritidia luciliae Immunfluoreszenztest (CLIFT) nachgewiesen. Zudem wurde die Proliferations- und Migrationskapazität sowie die Chemosensitivität der genom-editierten Zellen mittels optimiertem All-in-One Ansatz bestimmt, gefolgt von der Untersuchung der DDR-Signalwege durch Western Blot und Immunfluoreszenz. Überraschenderweise konnte bei Krebspatienten keine erhöhte Bindung von Autoantikörpern gegen LEDGF/p75 festgestellt werden, aber die Autoantikörperreaktivität auf LEDGF/p75 O/E-Zellen war bei etwa 50 % der Patienten mit BPH unerwartet erhöht. Auf molekularer Ebene zeigen LEDGF-defiziente Zellen eine verringerte Proliferation und Migration sowie eine erhöhte Empfindlichkeit gegenüber dem Chemotherapeutikum Etoposide auf. Auch eine signifikant verringerte DNA-Schadensantwort konnte sichtbar gemacht werden. Neue diagnostische Möglichkeiten unter Verwendung von HEp-2-Zellen mit Überexpression von LEDGF/p75 oder mitochondrialer DNA als Autoimmunziele könnten zur Identifizierung von BPH-Patienten mit Autoimmunreaktionen eingesetzt werden. Darüber hinaus zeigt diese Thesis, dass LEDGF nicht nur an der Rekrutierung von Proteinen der DNA-Schadensantwort beteiligt ist, sondern auch die proteosomale Regulierung von DDR-Signalmolekülen beeinflusst

Intrasexual cuticular hydrocarbon dimorphism in a wasp sheds light on hydrocarbon biosynthesis genes in Hymenoptera

Cuticular hydrocarbons (CHCs) cover the cuticle of insects and serve as desiccation barrier and as semiochemicals. While the main enzymatic steps of CHC biosynthesis are well understood, few of the underlying genes have been identified. Here we show how exploitation of intrasexual CHC dimorphism in a mason wasp, Odynerus spinipes, in combination with whole-genome sequencing and comparative transcriptomics facilitated identification of such genes. RNAi-mediated knockdown of twelve candidate gene orthologs in the honey bee, Apis mellifera, confirmed nine genes impacting CHC profile composition. Most of them have predicted functions consistent with current knowledge of CHC metabolism. However, we found first-time evidence for a fatty acid amide hydrolase also influencing CHC profile composition. In situ hybridization experiments furthermore suggest trophocytes participating in CHC biosynthesis. Our results set the base for experimental CHC profile manipulation in Hymenoptera and imply that the evolutionary origin of CHC biosynthesis predates the arthropods’ colonization of land.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Transient receptor potential ion channels V4 and A1 contribute to pancreatitis pain in mice

The mechanisms of pancreatic pain, a cardinal symptom of pancreatitis, are unknown. Proinflammatory agents that activate transient receptor potential (TRP) channels in nociceptive neurons can cause neurogenic inflammation and pain. We report a major role for TRPV4, which detects osmotic pressure and arachidonic acid metabolites, and TRPA1, which responds to 4-hydroxynonenal and cyclopentenone prostaglandins, in pancreatic inflammation and pain in mice. Immunoreactive TRPV4 and TRPA1 were detected in pancreatic nerve fibers and in dorsal root ganglia neurons innervating the pancreas, which were identified by retrograde tracing. Agonists of TRPV4 and TRPA1 increased intracellular Ca2+ concentration ([Ca2+]i) in these neurons in culture, and neurons also responded to the TRPV1 agonist capsaicin and are thus nociceptors. Intraductal injection of TRPV4 and TRPA1 agonists increased c-Fos expression in spinal neurons, indicative of nociceptor activation, and intraductal TRPA1 agonists also caused pancreatic inflammation. The effects of TRPV4 and TRPA1 agonists on [Ca2+]i, pain and inflammation were markedly diminished or abolished in trpv4 and trpa1 knockout mice. The secretagogue cerulein induced pancreatitis, c-Fos expression in spinal neurons, and pain behavior in wild-type mice. Deletion of trpv4 or trpa1 suppressed c-Fos expression and pain behavior, and deletion of trpa1 attenuated pancreatitis. Thus TRPV4 and TRPA1 contribute to pancreatic pain, and TRPA1 also mediates pancreatic inflammation. Our results provide new information about the contributions of TRPV4 and TRPA1 to inflammatory pain and suggest that channel antagonists are an effective therapy for pancreatitis, when multiple proinflammatory agents are generated that can activate and sensitize these channels

Plasma cell leukemia: A multicenter retrospective study of 150 patients

8014 Background: Despite the use of novel induction regimens, stem cell transplantation (SCT), and maintenance therapy, plasma cell leukemia (PCL) remains a challenging disease with a dismal prognosis. Currently, there is no agreed standard of care management for PCL. We conducted a multicenter retrospective analysis of the clinical presentation, treatment, and outcomes of 150 patients with PCL. Methods: Data of patients diagnosed with pPCL or sPCL between 01/2010 and 01/2021 were entered into a study-specific REDCap database from 7 different U.S. academic sites. PCL was defined as ≥ 5% circulating plasma cells. Clinical data included baseline patient characteristics, clinical presentation, treatment, therapeutic response, and survival outcomes. Overall survival (OS) curves were plotted using the Kaplan-Meier method. Cox proportional hazards regression tested associations between patient characteristics and OS, generating hazard ratios (HR) and 95% confidence intervals (CI), adjusted for PCL type (primary or secondary) and SCT. Results: The analytical cohort included 93 pPCL and 57 sPCL patients. Median age at diagnosis was 60 years. High-risk cytogenetics were found in 56.7% of the patients where it was documented. Of the 79 patients with a documented induction regimen, 58.2% received a proteasome inhibitor triplet, 22.8% received a VTD-Pace like conventional chemotherapy, and 3% received a daratumumab quadruplet regimen. SCT (autologous or allogeneic) was done in 56.1% of the patients. The median OS for all patients, those with pPCL, and those with sPCL was 20.3, 36.6, and 3.2 months, respectively. Secondary PCL was associated with worse survival outcomes compared with pPCL (HR, 2.46; 95% CI, 1.51-3.98; p<0.001) (Table). Median OS was better in patients treated with a proteasome inhibitor triplet regimen vs VTD PACE-like combination (28.2 versus 12.6 months). OS was prolonged among patients who underwent any type of SCT compared with those who did not undergo SCT (44.0 versus 5.7 months, p<0.001). Conclusions: This multicenter retrospective study is one of the largest PCL analyses performed to date and reveals the clinical practice patterns of treatment and survival of PCL patients across the U.S. in the novel treatment era. The survival analysis reinforces the poor prognosis in sPCL patients and the continued need for novel treatment approaches in this patient population. While limited by retrospective design, this analysis suggests prolonged survival with transplantation in both pPCL and sPCL. [Table: see text

Participation as a research approach in academia: a converging field

Citizen science, transdisciplinary research, dialogic forms of science communication or public engagement: these and other research approaches and fields, often subsumed under participatory research, have in common that they enable people outside of academia to actively engage in the production of scientific knowledge. However, each of these fields sets its own goals, uses different formats and has a different scope and impact. The conference 'Opportunities and Limitations of Participation in Academia' held in September 2022 as part of the German Science Year 'Participate!' aimed to connect the various participation communities in Germany and to explore commonalities and success factors. Through intensive discussions in four working groups, a keynote speech and a panel discussion, the conference initiated an exchange of ideas and experiences amongst researchers in a converging field. This report is a summary of the key questions and outcomes of the conference