Analyse der funktionellen Bedeutung der aktinbindenden Proteine VASP und α-Actinin-4 [Alpha-Actinin-4] in Helicobacter-pylori-kolonisierten Epithelzellen

Helicobacter pylori (H. pylori) ist ein gram-negatives Bakterium, das die menschliche Magenmukosa kolonisieren kann. Ca. 50 % der Weltbevölkerung sind mit diesem Erreger infiziert, wobei er ohne medizinische Behandlung über Jahrzehnte in seinem Wirt persistieren kann. Das Bakterium gilt als eine der häufigsten Ursachen bei der Entwicklung von schweren gastrointestinalen Erkrankungen wie chronischer Gastritis und Gastral- oder Duodenalulkus. Darüber hinaus kann eine Infektion aber auch zu einem gastralen Adenokarzinom oder dem MALT- („mucosa-associated lymphoid tissue“) Lymphom führen. Bestimmte H. pylori-Stämme können über ein Typ IV Sekretionssystem (T4SS) das bakterielle Protein CagA („cytotoxin-assoziiertes Gen A“) in die Wirtszelle injizieren und dadurch Signaltransduktionswege stören, die die Morphologie und Mobilität der infizierten Wirtszelle drastisch verändern. In diesem Zusammenhang wurde die putative Phosphorylierung der Proteine VASP („Vasodilator-stimuliertes Phosphoprotein“) und α-Actinin-4 untersucht, welche beide regulatorische Funktionen im Zytoskelett der Wirtszelle ausüben. Im Rahmen dieser Doktorarbeit sollte der Einfluss von H. pylori auf diese Proteine analysiert werden, sowie die daraus resultierenden zellulären Auswirkungen. Es konnte verifiziert werden, dass H. pylori die Phosphorylierung der drei bekannten Phosphorylierungsstellen von VASP, bzw. eine Tyrosin-Phosphorylierung von α-Actinin-4 (ACTN4) induziert. Weiterhin zeigte sich, dass beide Proteine nach einer Infektion mit dem Bakterium in fokalen Kontaktstellen der Zellen lokalisieren. Zusätzlich lies sich zeigen, dass VASP und α-Actinin-4 eine entscheidende Rolle bei der durch H. pylori-induzierten Zellelongation spielen, da eine Herunterregulation der Genexpression von beiden Proteinen über siRNA zu einer Inhibition der morphologischen Veränderungen führte. Die genaueren Studien über VASP zeigten, dass hauptsächlich die Phosphorylierungsstelle VASPSer239 für die H. pylori-induzierte Zellelongation verantwortlich ist und die Phosphorylierung durch die Proteinkinase G (PKG) vermittelt wird. Für α-Actinin-4 konnte in dieser Arbeit des Weiteren gezeigt werden, dass die Kinase c-Abl eine Tyrosin-Phosphorylierung vermitteln kann, wobei die genaue Phosphorylierungsstelle im Protein noch ermittelt werden muss. Durch den Einsatz von H. pylori-Mutanten lies sich darüber hinaus noch zeigen, dass die Phosphorylierung von VASPSer239 und VASPThr278 durch das bakterielle Protein CagA deutlich verstärkt wird. Für die Tyrosin-Phosphorylierung von α-Actinin-4 war CagA sogar ausschlaggebend. Diese neu entdeckten zellulären Zielproteine bei einer H. pylori-Infektion ermöglichen weitere Einblicke in die Deregulation des eukaryotischen Zytoskeletts und möglichen Mechanismen bei der Krebsentstehung.Helicobacter pylori (H. pylori) is a gram-negative bacterium which colonizes the human stomach. Approximately 50 % of the world´s human population is infected with the pathogen and it can persist for decades without medical treatment. It is considered to be the common cause for the development of severe gastrointestinal diseases like chronic gastritis, and gastro- or duodenalulcer. Beyond that, an infection can lead to gastric cancer or MALT („mucosa-associated lymphoid tissue“)-lymphoma. Certain H. pylori-strains inject the bacterial protein CagA („cytotoxine-associated gene A”) via a type IV secretion system (T4SS) directly into the host cell leading to a deregulation of signalling pathways that can induce drastic changes of the morphology and mobility of host cells. In this context it was aimed at a more detailed insight into the influence of H. pylori on the regulation of the actin binding proteins VASP („Vasodilator-stimulated phosphoprotein”) and α-Actinin-4 and the resulting effects on H. pylori-induced cell migration. It could be verified, that H. pylori induced phosphorylation of three phosphorylation sites of VASP and a tyrosine phosphorylation of α-Actinin-4, respectively. Furthermore, it was shown that after an infection with the bacterium both proteins were localised into focal adhesions. Additionally, it was discovered that VASP and α-Actinin-4 played crucial roles during H. pylori-induced cell elongation, because knock-down of the protein expression via siRNA inhibited H. pylori-dependent morphological changes. Detailed studies of VASP showed that the phosphorylation site VASPSer239 was mainly responsible for the H.pylori-induced cell elongation and that this phosphorylation was mediated through the protein kinase G (PKG). Furthermore it could be shown for α-Actinin-4 that the kinase c-Abl mediated a tyrosine-phosphorylation, whereas the precise site in the protein is still unknown. Using H. pylori mutants it was found, that the phosphorylation of VASPSer239 and Thr278 was enhanced through the bacterial protein CagA. For tyrosine phosphorylation of α-Actinin-4, CagA was also crucial. These new identified cellular target proteins during an infection with H. pylori permit further insights in the deregulation of the eukaryotic cytoskeleton and possible mechanisms in cancer development

Comparative sensitivity evaluation for 122 CE-marked rapid diagnostic tests for SARS-CoV-2 antigen, Germany, September 2020 to April 2021

INTRODUCTION: Numerous CE-marked SARS-CoV-2 antigen rapid diagnostic tests (Ag RDT) are offered in Europe, several of them with unconfirmed quality claims. AIM: We performed an independent head-to-head evaluation of the sensitivity of SARS-CoV-2 Ag RDT offered in Germany. METHODS: We addressed the sensitivity of 122 Ag RDT in direct comparison using a common evaluation panel comprised of 50 specimens. Minimum sensitivity of 75% for panel specimens with a PCR quantification cycle (Cq) ≤ 25 was used to identify Ag RDT eligible for reimbursement in the German healthcare system. RESULTS: The sensitivity of different SARS-CoV-2 Ag RDT varied over a wide range. The sensitivity limit of 75% for panel members with Cq ≤ 25 was met by 96 of the 122 tests evaluated; 26 tests exhibited lower sensitivity, few of which failed completely. Some RDT exhibited high sensitivity, e.g. 97.5 % for Cq < 30. CONCLUSIONS: This comparative evaluation succeeded in distinguishing less sensitive from better performing Ag RDT. Most of the evaluated Ag RDT appeared to be suitable for fast identification of acute infections associated with high viral loads. Market access of SARS-CoV-2 Ag RDT should be based on minimal requirements for sensitivity and specificity

Breast Cancer Management During the COVID-19 Pandemic: The Senologic International Society Survey

Objective: In early 2020, the spread of coronavirus disease-2019 (COVID-19) led the World Health Organization to declare this disease a pandemic. Initial epidemiological data showed that patients with cancer were at high risk of developing severe forms of COVID-19. National scientific societies published recommendations modifying the patients' breast cancer (BC) management to preserve, in theory, quality oncologic care, avoiding the increased risk of contamination. The Senology International Society (SIS) decided to take an inventory of the actions taken worldwide. This study investigates COVID-19-related changes concerning BC management and analyzes the will to maintain them after the pandemic, evaluating their oncological safety consequences. Materials and Methods: SIS network members participated in an online survey using a questionnaire (Microsoft® Forms) from June 15th to July 31st, 2020. Results: Forty-five responses from 24 countries showed that screening programs had been suspended (68%); magnetic resonance imagines were postponed (73%); telemedicine was preferred when possible (71%). Surgeries were postponed: reconstructive (77%), for benign diseases (84%), and in patients with significant comorbidities (66%). Chemotherapy and radiotherapy protocols had been adapted in 28% of patients in both. Exception for telemedicine (34%), these changes in practice should not be continued. Conclusion: The SIS survey showed significant changes in BC's diagnosis and treatment during the first wave of the COVID-19 pandemic, but most of these changes should not be maintained. Indeed, women have fewer severe forms of COVID-19 and are less likely to die than men. The risk of dying from COVID-19 is more related to the presence of comorbidities and age than to BC. Stopping screening and delaying treatment leads to more advanced stages of BC. Only women aged over 65 with BC under treatment and comorbidities require adaptation of their cancer management