Tight Junction-bindendes C-terminales Clostridium perfringens Enterotoxin für Goldnanopartikel-vermittelte Optoperforation von Zellen : neue Methode für die Krebstherapie

Tight Junctions sind apikale interzelluläre Proteinverbindungen zwischen Epithelzellen. Die Überexpression von bestimmten Proteinen der Tight Junctions wie z.B. Claudin-3, -4 und -7 ist für einige Krebsarten charakteristisch. In der Krebsdiagnose und -therapie könnten diese Claudine als Marker verwendet werden, um Krebszellen zielgerichtet zu detektieren und behandeln zu können. Das C-terminale Fragment vom Clostridium perfringens Enterotoxin (C-CPE) kann spezifisch an diese Claudine binden. In dieser Arbeit wurden Goldnanopartikel (AuNP) mit dem rekombinanten C-CPE (Aminosäuren 194 bis 319) funktionalisiert, um die spezifische Ablation von Claudin-exprimierenden Krebszellen mit der Goldnanopartikel-vermittelten Laserperforation (GNOME-LP) Technik zu etablieren. Mit der Analyse der Claudin-Expression von verschiedenen Krebszellen mittels quantitativer real-time PCR und Immunfluoreszenz konnten Claudin-3, -4 und -7 positive humane Darm- (Caco-2), Brust- (MCF-7) und Ösophaguskrebszelllen (OE-33) sowie Hundebrustkrebszellen (TiHoDMglCarc1305) identifiziert werden. Die transepithelialen elektrischen Widerstandsmessungen und Lokalisationsstudien zeigten, dass das rekombinante C-CPE194-319 spezifisch an die Claudin-3, -4 und -7 positiven Krebszellen binden konnte. Die Optoperforation von mit 5 µg/ml C-CPE funktionalisierten AuNP behandelten Krebszellen mittels der GNOME-LP Technik tötete über 70 % der Claudin-positiven Krebszellen bei einer Laserfluenz von 60 mJ/cm² und einer Scangeschwindigkeit von 5 mm/s. Nicht-funktionalisierte AuNP verursachten keinen Zelltod. Zusätzlich konnte mit dieser optischen Methode die Struktur von Sphäroiden aus Ösophagus- und Brustkrebszellen in Matrigel geschädigt werden. Für eine zukünftige in vivo Anwendung ist es wesentlich die Zelltod-induzierenden physikalischen und biologischen Mechanismen zu verstehen. Bezogen auf die C-CPE-Funktionalisierung der AuNP, konnte mit einer höheren C-CPE-Konzentration von 20 µg/ml die Tötungseffizienz der Krebszellen mit einer reduzierten Laserfluenz auf 30 mJ/cm² maximiert werden. Unabhängig von der verwendeten Laserfluenz wurde eine maximale Tötungseffizienz bei einer Scangeschwindigkeit von 5 mm/s erreicht. Die Optoperforation von aus Darmkrebszellen bestehenden Sphäroiden im Matrigel zerstörte nachhaltig die Sphäroidstruktur. Sphäroide bestehend aus Ösophaguskrebszellen wurden stärker beschädigt als die optoperforierten Sphäroide mit 5 µg/ml C-CPE funktionalisierten AuNP. Die Betrachtung der biologischen Mechanismen zeigte, dass die GNOME-LP von mit C-CPE-AuNP behandelten Zellen eine Bindung von Annexin V an die Membran und eine reduzierte Aktivität der Mitochondrien aufwiesen. Die Aktivität der Caspase-3/-7 war in den Zellen nicht erhöht. Die gelelektrophoretische Analyse der DNA nach der Behandlung zeigte keine Apoptose-vermittelte Fragmentierung der DNA. Die Ergebnisse deuten vielmehr auf einen durch die GNOME-LP/C-CPE-AuNP Applikation hervorgerufenen nekrotischen als auf einen apoptotischen Zelltod hin. Damit ist die GNOME-LP mit C-CPE funktionalisierten AuNP eine vielversprechende effiziente Methode zur Ablation von Claudin-positiven Krebszellen. Die Tötungseffizienz sollte zukünftig in Xenotransplantaten erforscht werden, um die optobasierte Methode präziser an in vivo Bedingungen anzupassen.Tight Juctions are apical intercellular protein connections between epithelial cells. The over expression of specific tight junction proteins like claudin-3, -4 and -7 is characteristic for some cancer types. In cancer diagnosis and therapy these claudins can be used as markers to target, detect, and treat cancer cells. The non-cytotoxic C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds specifically on these claudins. In this thesis, gold nanoparticles (AuNPs) were functionalized with the recombinant C-CPE (amino acids 194 to 319) as a tool to establish a new method for a specific ablation of claudin expressing cancer cells by using the gold nanoparticle-mediated laserperforation (GNOME-LP) technique. Analysis of the claudin expression in different cancer cells with quantitative real-time PCR and immunofluorescence experiments revealed the human colon (Caco-2), breast (MCF-7) and esophagus (OE-33) cell lines as well as the canine mammary cell line (TiHoDMglCarc1305) as claudin-3, -4 und -7 positive cancer cells. Transepithelial electrical resistance measurements and localization experiments showed binding of the recombinant C-CPE194-319 on the claudin-3, -4 and -7 positive cancer cells. The optoperforation of 5 µg/ml C-CPE functionalized AuNPs treated cancer cells by the gold nanoparticle-mediated laser perforation (GNOME-LP) technique killed over 70 % of the claudin-positive cancer cells at a laser fluence of 60 mJ/cm² and a scanning speed of 5 mm/s. The same laser fluence did not kill the cells when incubated with non-functionalized AuNPs. Furthermore, by using the optical method the formation of spheroids composed of esophagus cancer cells or breast cancer cells in Matrigel was reduced. For future in vivo application, it will be crucial to know the physical and biological mechanisms induced by cell death. Regarding the C-CPE functionalization of the AuNPs, increased C-CPE concentration of 20 µg/ml for the C-CPE-AuNP complex formation maximized the killing efficiency with a reduced laser fluence of 30 mJ/cm². Independently of the applied laser fluence, the maximal killing efficiency was achieved at a scanning speed of 5 mm/s. The optoperforation of spheroids composed of colon cancer cells in Matrigel permanently destroyed the complete spheroid formation. The formation of spheroids composed of esophagus cancer cells was more effectively reduced compared to optoperforated spheroids with 5 µg/ml C-CPE functionalized AuNPs. At the biological level, GNOME-LP of C-CPE-AuNPs treated cells showed annexin V binding to the cell membrane and a reduced mitochondria activity. The activity of caspase-3/-7 in the cells was not increased. Gel electrophoretic analysis of the DNA after optical treatment demonstrated no apoptosis-related DNA fragmentation. The results suggest that the GNOME-LP/C-CPE-AuNPs application induced rather necrotic than apoptotic reaction in cancer cells. Finally, the GNOME-LP with C-CPE functionalized AuNPs is a promising efficient approach for ablation of caludin-positive cancer cells. The killing efficiency should be investigated on xenograft in future to rationally adapt the opto-based method for future in vivo conditions

Parameters for Optoperforation-Induced Killing of Cancer Cells Using Gold Nanoparticles Functionalized With the C-terminal Fragment of Clostridium Perfringens Enterotoxin

Recently, we used a recombinant produced C-terminus (D194-F319) of the Clostridium perfringens enterotoxin (C-CPE) to functionalize gold nanoparticles (AuNPs) for a subsequent specific killing of claudin expressing tumor cells using the gold nanoparticle-mediated laser perforation (GNOME-LP) technique. For a future in vivo application, it will be crucial to know the physical parameters and the biological mechanisms inducing cell death for a rational adaptation of the system to real time situation. Regarding the AuNP functionalization, we observed that a relationship of 2.5 × 10−11 AuNP/mL to 20 µg/mL C-CPE maximized the killing efficiency. Regardingphysical parameters, a laser fluence up to 30 mJ/cm2 increased the killing efficiency. Independent from the applied laser fluence, the maximal killing efficiency was achieved at a scanning velocity of 5 mm/s. In 3D matrigel culture system, the GNOME-LP/C-CPE-AuNP completely destroyed spheroids composed of Caco-2 cells and reduced OE-33 cell spheroid formation. At the biology level, GNOME-LP/C-CPE-AuNP-treated cells bound annexin V and showed reduced mitochondria activity. However, an increased caspase-3/7 activity in the cells was not found. Similarly, DNA analysis revealed no apoptosis-related DNA ladder. The results suggest that the GNOME-LP/C-CPE-AuNP treatment induced necrotic than apoptotic reaction in tumor cells

Precursor B-ALL cell lines differentially respond to syk inhibition by entospletinib

Background: Impaired B-cell receptor (BCR) function has been associated with the pro-gress of several B-cell malignancies. The spleen tyrosine kinase (SYK) represents a potential therapeutic target in a subset of B-cell neoplasias. In precursor B-acute lymphoblastic leukemia (B-ALL), the pathogenic role and therapeutic potential of SYK is still controversially discussed. We evaluate the application of the SYK inhibitor entospletinib (Ento) in pre-and pro-B-ALL cell lines, character-izing the biologic and molecular effects. Methods: SYK expression was characterized in pre-B-ALL (NALM-6) and pro-B-ALL cell lines (SEM and RS4;11). The cell lines were exposed to different Ento concentrations and the cell biological response analyzed by proliferation, metabolic activity, apop-tosis induction, cell-cycle distribution and morphology. BCR pathway gene expression and protein modulations were further characterized. Results: Ento significantly induced anti-proliferative and pro-apoptotic effects in NALM-6 and SEM, while barely affecting RS4;11. Targeted RNAseq revealed pronounced gene expression modulation only in NALM-6, while Western Blot analyses demonstrated that vital downstream effector proteins, such as pAKT, pERK, pGSK3β, p53 and BCL-6, were affected by Ento exposure in the inhibitor-sensitive cell lines. Conclusion: Different acting modes of Ento, independent of pre-BCR dependency, were characterized, unexpected in SEM. Ac-cordingly, SYK classifies as a potential target structure in a subset of pro-B-ALLs. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

Probing Ligand-Receptor Interaction in Living Cells Using Force Measurements With Optical Tweezers

This work probes the binding kinetics of COOH-terminus of Clostridium perfringens enterotoxin (c-CPE) and claudin expressing MCF-7 cells using force spectroscopy with optical tweezers. c-CPE is of high biomedical interest due to its ability to specifically bind to claudin with high affinity as well as reversibly disrupt tight junctions whilst maintaining cell viability. We observed single-step rupture events between silica particles functionalized with c-CPE and MCF-7 cells. Extensive calibration of the optical tweezers’ trap stiffness and displacement of the particle from trap center extracted a probable bond rupture force of ˜ 18 pN. The probability of rupture events with c-CPE functionalized silica particles increased by 50% compared to unfunctionalized particles. Additionally, rupture events were not observed when probing cells not expressing claudin with c-CPE coated particles. Overall, this work demonstrates that optical tweezers are invaluable tools to probe ligand-receptor interactions and their potential to study dynamic molecular events in drug-binding scenarios

Longitudinal claudin gene expression analyses in canine mammary tissues and thereof derived primary cultures and cell lines

Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies

Implantation of a colorectal stent as a therapeutic approach in the treatment of esophageal leakage

BACKGROUND: While the mortality of esophageal surgery has decreased due to technological advancements, there is still a complication rate of about 30%. One of the main complications is the anastomotic leakage associated with a significant rate of morbidity and mortality. To close the leakage the efficacy of self-expanding stents (SES) has been shown in different studies. However, the high rate of stent migration limits the use of commercial available stents. In our case we were faced with the problem that the diameter of all available stents was too small to attach tightly to the mucosal wall of the esophagogastric anastomosis. CASE PRESENTATION: We used, for the first time to our knowledge, a metal stent designed for colorectal application in an extensive anastomotic leak after esophageal resection in a patient with an esophageal cancer. After primary surgery with subtotal esohagectomy the anastomotic leak was stented endoscopically with a Polyflex self-expanding covered plastic stent after no response to intensive conventional management. Even though the stent was placed correctly, the diameter of the Polyflex stent was too small to attach onto the wall of the esophagogastric anastomosis. Again surgery was performed with a thoracal resection of the esophageal remnant and a hand made anastomosis. Unfortunately, again an anastomotic leak was detected soon after. To close the leak we decided to use a covered colorectal stent (Hanarostent) with an inner diameter of 30 mm. Sixteen weeks later the stent was extracted and complete mucosal healing of the esophageal leak was observed. CONCLUSION: The stent implantation with a large wide diameter offers a good chance to close more extensive leaks and prevent stent migration

Microbiological airway colonization in COPD patients with severe emphysema undergoing endoscopic lung volume reduction

Background: Endoscopic lung volume reduction (eLVR) is a therapeutic option for selected patients with COPD and severe emphysema. Infectious exacerbations are serious events in these vulnerable patients; hence, prophylactic antibiotics are often prescribed postinterventionally. However, data on the microbiological airway colonization at the time of eLVR are scarce, and there are no evidence-based recommendations regarding a rational antibiotic regimen. Objective: The aim of this study was to perform a clinical and microbiological analysis of COPD patients with advanced emphysema undergoing eLVR with endobronchial valves at a single German University hospital, 2012–2017. Patients and methods: Bronchial aspirates were obtained prior to eLVR and sent for microbiological analysis. Antimicrobial susceptibility testing of bacterial isolates was performed, and pathogen colonization was retrospectively compared with clinical parameters. Results: At least one potential pathogen was found in 47% (30/64) of patients. Overall, Gram-negative bacteria constituted the most frequently detected pathogens. The single most prevalent species were Haemophilus influenzae (9%), Streptococcus pneumoniae (6%), and Staphylococcus aureus (6%). No multidrug resistance was observed, and Pseudomonas aeruginosa occurred in <5% of samples. Patients without microbiological airway colonization showed more severe airflow limitation, hyperinflation, and chronic hypercapnia compared to those with detected pathogens. Conclusion: Microbiological airway colonization was frequent in patients undergoing eLVR but not directly associated with poorer functional status. Resistance testing results do not support the routine use of antipseudomonal antibiotics in these patients

Review article: Assessing the costs of natural hazards - state of the art and knowledge gaps

Efficiently reducing natural hazard risks requires a thorough understanding of the costs of natural hazards. Current methods to assess these costs employ a variety of terminologies and approaches for different types of natural hazards and different impacted sectors. This may impede efforts to ascertain comprehensive and comparable cost figures. In order to strengthen the role of cost assessments in the development of integrated natural hazard management, a review of existing cost assessment approaches was undertaken. This review considers droughts, floods, coastal and Alpine hazards, and examines different cost types, namely direct tangible damages, losses due to business interruption, indirect damages, intangible effects, and the costs of risk mitigation. This paper provides an overview of the state-of-the-art cost assessment approaches and discusses key knowledge gaps. It shows that the application of cost assessments in practice is often incomplete and biased, as direct costs receive a relatively large amount of attention, while intangible and indirect effects are rarely considered. Furthermore, all parts of cost assessment entail considerable uncertainties due to insufficient or highly aggregated data sources, along with a lack of knowledge about the processes leading to damage and thus the appropriate models required. Recommendations are provided on how to reduce or handle these uncertainties by improving data sources and cost assessment methods. Further recommendations address how risk dynamics due to climate and socio-economic change can be better considered, how costs are distributed and risks transferred, and in what ways cost assessment can function as part of decision support