TY - JOUR A1 - Piteau, Marianne A1 - Papatheodorou, Panagiotis A1 - Schwan, Carsten A1 - Schlosser, Andreas A1 - Aktories, Klaus A1 - Schmidt, Gudula T1 - Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) JF - PLoS Pathogens N2 - The Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin which is a major virulence factor of pathogenic Escherichia coli strains. Here, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as cellular receptor for CNF1 by co-precipitation of cell surface molecules with tagged toxin. The CNF1-Lu/BCAM interaction was verified by direct protein-protein interaction analysis and competition studies. These studies revealed amino acids 720 to 1014 of CNF1 as the binding site for Lu/BCAM. We suggest two cell interaction sites in CNF1: first the N-terminus, which binds to p37LRP as postulated before. Binding of CNF1 to p37LRP seems to be crucial for the toxin's action. However, it is not sufficient for the binding of CNF1 to the cell surface. A region directly adjacent to the catalytic domain is a high affinity interaction site for Lu/BCAM. We found Lu/BCAM to be essential for the binding of CNF1 to cells. Cells deficient in Lu/BCAM but expressing p37LRP could not bind labeled CNF1. Therefore, we conclude that LRP and Lu/BCAM are both required for toxin action but with different functions. Author Summary We study a crucial virulence factor produced by pathogenic Escherichia coli strains, the Cytotoxic Necrotizing Factor 1 (CNF1). More than 80% of urinary tract infections (UTIs), which are counted among the most common bacterial infections of humans, are caused by Uropathogenic Escherichia coli (UPEC) strains. We and others elucidated the molecular mechanism of the E. coli toxin CNF1. It constitutively activates Rho GTPases by a direct covalent modification. The toxin enters mammalian cells by receptor-mediated endocytosis. Here, we identified the protein receptor for CNF1 by co-precipitation of cell surface molecules with the tagged toxin and subsequent Maldi-TOF analysis. We identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as receptor for CNF1 and located its interaction site to the C-terminal part of the toxin. We performed direct protein-protein interaction analysis and competition studies. Moreover, cells deficient in Lu/BCAM could not bind labeled CNF1. The identification of a toxin's cellular receptor and receptor binding region is an important task for understanding the pathogenic function of the toxin and, moreover, to make the toxin accessible for its use as a cellbiological and pharmacological tool, for example for the generation of immunotoxins. KW - laminin receptor KW - factor-I KW - toxin KW - RHO KW - cells KW - activation KW - protein KW - domain KW - translocation KW - membrane Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117987 SN - 1553-7374 VL - 10 IS - 1 ER - TY - THES A1 - Metzger, Alexandra T1 - Molekulargenetische Charakterisierung tumorigener Chromosomenaberrationen in extranodalen Marginalzonenlymphomen vom MALT-Typ der Lunge T1 - Moleculargenetical characterising of tumorigenic chromosomal aberrations in pulmonary extranodal marginalzone b-cell lymphomas of the MALT type N2 - MALT-Lymphome der Lunge gehören als extranodale Marginalzonen-B-Zell-Lymphomen zu den malignen Non-Hodgkin-Lymphomen. Zahlreiche Untersuchungen haben gezeigt, dass rekurrenten genetischen Translokationen eine wichtige Rolle bei der Tumorgenese zukommt. Eine zentrale Rolle nimmt die Aktivierung des Transkriptionsfaktors NFkappaB ein, die Folge solcher rekurrenter Translokationen (z.B. t(11;18)(q21;q21), t(14;18)(q32;q21), t(1;14)(p22;q32)) und numerischer Chromosomenaberrationen (Trisomie 3, Trisomie 18) ist. In der vorliegenden Arbeit haben wir 60 Fälle extranodaler Marginalzonen-B-Zell-Lymphome der Lunge bezüglich der genannten genetischen Translokationen untersucht. Es ist dies die größte bisher in der Literatur beschriebene Serie von MALT-Lymphomen in dieser Lokalisation. Die Untersuchung der t(11;18) ergab in der vorliegenden Arbeit eine geringere Häufigkeit als in der Literatur beschrieben, wobei zu berücksichtigen ist, dass die bisher vorgestellten Studien deutlich geringere Fallzahlen aufwiesen. Bezüglich der Translokationen t(14;18), t(1;14), t(3;14) und der Trisomie 3 waren in der vorliegenden Studie ähnliche Häufigkeiten zu finden, wie sie in der Literatur beschrieben sind. Als möglichen alternativen Aktivierungsweg des Zellzyklus zeigte sich in dieser Studie neben den genannten Translokationen sowohl eine Trisomie 3 als auch eine Amplifikation der Genkopienzahl von MALT1. Im Vergleich der genetischen und immunhistochemischen Ergebnisse bezüglich der FOXP1- und der BCL10-Expression zeigte sich für FOXP1 eine hohe Korrelation zwischen immunhistologischer Expression und genetischem Nachweis einer Genaktivierung, während für BCL10 eine starke Diskrepanz bezüglich Sensitivität und Spezifität gefunden wurde, so dass die immunhistologische Analyse nur einen Hinweis auf das Vorliegen einer genetischen Translokation zu geben vermag, aber nicht als Surrogatmarker zu verwenden ist. N2 - Pulmonary MALT-lymphomas as extranodal marginalzone b-cell lymphomas belong to the non hodgkin lymphomas. Many analyses showed that recurrent genetical translocations figure in the tumorgenesis. A central part takes the activation of the transkription factor NFkappaB resulted from such recurrent translocations (e.g. t(11;18)(q21;q21), t(14;18)(q32;q21), t(1;14)(p22;q32)) and numeric chromosomal aberrations (3+, 18+). In this paper, 60 cases of extranodal marginalzone b-cell lymphomas were analysed regarding the named genetical translocations. This is the extensivest up to now described serie of MALT-lymphomas in this location. KW - Chromosomenaberration KW - B-Zell-Lymphom KW - Non-Hodgkin-Lymphom KW - Fluoreszenz-in-situ-Hybridisierung KW - Translokation KW - b-cell lymphoma of the MALT type KW - non-hodgkin-lymphoma KW - chromosomal aberration KW - translocation Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-28676 ER - TY - JOUR A1 - Gaiser, Timo A1 - Geissinger, Eva A1 - Schattenberg, Torsten A1 - Scharf, Hanns-Peter A1 - Dürken, Matthias A1 - Dinter, Dietmar A1 - Rosenwald, Andreas A1 - Marx, Alexander T1 - Case report: a unique pediatric case of a primary CD8 expressing ALK-1 positive anaplastic large cell lymphoma of skeletal muscle JF - Diagnostic Pathology N2 - Primary involvement of skeletal muscle is a very rare event in ALK-1 positive anaplastic large cell lymphoma (ALCL). We describe a case of a 10-year old boy presenting with a three week history of pain and a palpable firm swelling at the dorsal aspect of the left thigh. Histological examination of the lesion revealed a tumoral and diffuse polymorphic infiltration of the muscle by large lymphoid cells. Tumor cells displayed eccentric, lobulated "horse shoe" or "kidney-shape" nuclei. The cells showed immunohistochemical positivity for CD30, ALK-1, CD2, CD3, CD7, CD8, and Perforin. Fluorescence in situ hybridization analysis revealed a characteristic rearrangement of the ALK-1 gene in 2p23 leading to the diagnosis of ALK-1 positive ALCL. Chemotherapy according to the ALCL-99-NHL-BFM protocol was initiated and resulted in a complete remission after two cycles. This case illustrates the unusual presentation of a pediatric ALCL in soft tissue with a good response to chemotherapy. KW - pediatric lymphoma KW - psoas muscle KW - classification KW - translocation KW - features KW - gene KW - ALK-1 KW - anaplastic large cell lymphoma KW - CD30 Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-135381 VL - 7 IS - 38 ER - TY - JOUR A1 - Dimopoulos, Meletios A. A1 - Weisel, Katja C. A1 - Song, Kevin W. A1 - Delforge, Michel A1 - Karlin, Lionel A1 - Goldschmidt, Hartmut A1 - Moreau, Philippe A1 - Banos, Anne A1 - Oriol, Albert A1 - Garderet, Laurent A1 - Cavo, Michele A1 - Ivanova, Valentina A1 - Alegre, Adrian A1 - Martinez-Lopez, Joaquin A1 - Chen, Christine A1 - Spencer, Andrew A1 - Knop, Stefan A1 - Bahlis, Nizar J. A1 - Renner, Christoph A1 - Yu, Xin A1 - Hong, Kevin A1 - Sternas, Lars A1 - Jacques, Christian A1 - Zaki, Mohamed H. A1 - San Miguel, Jesus F. T1 - Cytogenetics and long-term survival of patients with refractory or relapsed and refractory multiple myeloma treated with pomalidomide and low-dose dexamethasone JF - Haematologica N2 - Patients with refractory or relapsed and refractory multiple myeloma who no longer receive benefit from novel agents have limited treatment options and short expected survival. del(17p) and t(4;14) are correlated with shortened survival. The phase 3 MM-003 trial demonstrated significant progression-free and overall survival benefits from treatment with pomalidomide plus low-dose dexamethasone compared to high-dose dexamethasone among patients in whom bortezomib and lenalidomide treatment had failed. At an updated median follow-up of 15.4 months, the progression-free survival was 4.0 versus 1.9 months (HR, 0.50; P<0.001), and median overall survival was 13.1 versus 8.1 months (HR, 0.72; P=0.009). Pomalidomide plus low-dose dexamethasone, compared with high-dose dexamethasone, improved progression-free survival in patients with del(17p) (4.6 versus 1.1 months; HR, 0.34; P < 0.001), t(4;14) (2.8 versus 1.9 months; HR, 0.49; P=0.028), and in standard-risk patients (4.2 versus 2.3 months; HR, 0.55; P<0.001). Although the majority of patients treated with high-dose dexamethasone took pomalidomide after discontinuation, the overall survival of patients treated with pomalidomide plus low-dose dexamethasone or highdose dexamethasone was 12.6 versus 7.7 months (HR, 0.45; P=0.008) in patients with del(17p), 7.5 versus 4.9 months (HR, 1.12; P=0.761) in those with t(4;14), and 14.0 versus 9.0 months (HR, 0.85; P=0.380) in standard-risk subjects. The overall response rate was higher in patients treated with pomalidomide plus low-dose dexamethasone than in those treated with high-dose dexamethasone both among standard-risk patients (35.2% versus 9.7%) and those with del(17p) (31.8% versus 4.3%), whereas it was similar in patients with t(4; 14) (15.9% versus 13.3%). The safety of pomalidomide plus low-dose dexamethasone was consistent with initial reports. In conclusion, pomalidomide plus low-dose dexamethasone is efficacious in patients with relapsed/refractory multiple myeloma and del(17p) and/or t(4;14). KW - translocation KW - plus dexamethasone KW - deletion 17P KW - bortezomib KW - therapy KW - abnormalities KW - stem-cell transplantation KW - growth-factor receptor 3 KW - high-risk cytogenetics KW - intergroupe francophone Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-140349 VL - 100 IS - 10 SP - 1327 EP - 1333 ER - TY - THES A1 - Beitzinger, Christoph T1 - Binding-, Blocking- and Translocation-Processes Concerning Anthrax-Toxin and Related Bacterial Protein-Toxins of the AB7-Family T1 - Charakterisierung von Bindungs-, Blockierungs- und Translokationsprozessen am Anthrax-Toxin und verwanten Toxinen der AB7-Gruppe N2 - Bacterial protein toxins belong to the most potent toxins which are known. They exist in many different forms and are part of our every day live. Some of them are spread by the bacteria during infections and therefore play a crucial role in pathogenicity of these strains. Others are secreted as a defense mechanism and could be uptaken with spoiled food. Concerning toxicity, some of the binary toxins of the AB7-type belong to the most potent and dangerous toxins in the world. Even very small amounts of these proteins are able to cause severe symptoms during an infection with pathogen species of the genus Clostridium or Bacillus. Apart from the thread the toxins constitute, they exhibit a unique way of intoxication. Members of the AB7-toxin family consist of a pore-forming subunit B, that acts as a molecular syringe to translocate the enzymatic moieties A into the cytosol of target cells. This complex mechanism does not only kill cells with high efficiency and therefore should be studied for treatment, but also displays a possibility to address certain cells with a specific protein cargo if used as a molecular delivery tool. Concerning both issues, binding and translocation of the channel are the crucial steps to either block or modify the system in the desired way. To gain deeper insight into the transport of binary toxins the structure of the B subunit is of great importance, but being a membrane protein, no crystal could be obtained up to now for either protective antigen (PA) of Anthrax toxin or any other AB7-type binding domain. Therefore, the method of choice in this work is an electro-physical approach using the so-called black-lipid-bilayer system for determination of biophysical constants. Additionally, diverse cell based assays serve as a proving method for the data gained during in vitro measurements. Further information was gathered with specially designed mutants of the protein channel. The first part of this thesis focuses on the translocation process and its possible use as a molecular tool to deliver protein cargo into special cell types. The task was addressed by measuring the binding of different effector proteins related and unrelated to the AB7 toxin family. These proteins were tested in titration experiments for the blockage of the ion current through a membrane saturated with toxin channels. Especially the influence of positively charged His-tags has been determined in detail for PA and C2II. As described in chapter 2, a His-tag transferred the ability of being transported by PA, but not by C2II, to different proteins like EDIN (from S. aureus) in vitro and in cell-based experiments. This process was found to change the well-known voltage-dependency of PA to a huge extend and therefore is related to membrane potentials which play a crucial role in many processes in living cells. Chapter 3 sums up findings, which depict that binding partners of PA share certain common motives. These could be detected in a broad range of substrates, ranging from simple ions in an electrolyte over small molecules to complex protein effectors. The gathered information could be further used to design blocker-substrates for treatment of Anthrax infections or tags, which render PA possible as a molecular syringe for cargo proteins. The deeper insight to homologies and differences of binary toxin components is the core of chapter 4, in which the cross-reactivity of Anthrax and C2-toxin was analyzed. The presented results lead to a better understanding of different motives involved in binding and translocation to and via the B components PA and C2II, as well as the enzymatically active A moieties edema factor (EF), lethal factor (LF) and C2I. In the second part of the thesis, the blockage of intoxication is the center of interest. Therefore, chapter 5 focuses on the analysis of specially designed blocker-substrate molecules for PA. These molecules form a plug in the pore, abolishing translocation of the enzymatic units. Especially, if multi-resistant strains of Anthrax (said to be already produced in Russia as a biological weapon) are taken into consideration, these substrates could stop intoxication and buy time, to deal with the infection. Chapter 6 describes the blockage of PA-channels by anti-His antibody from the trans-side of the porin, an effect which was not described for any other antibody before. Interestingly, even mutation of the estimated target amino acid Histidine 310 to Glycine could not interfere with this ionic strength dependent binding. N2 - Bakterielle Protein-Toxine gehören zu den wirksamsten bekannten Toxinen. In vielfältigen Variationen findet man sie in allen Bereichen des Lebens. Einige werden von den Bakterien während einer Infektion freigesetzt und übernehmen einen wichtigen Part in der Pathogenität. Andere werden zu Verteidigungszwecken sekretiert und können in verdorbenen Lebensmitteln gefunden werden. Was die Wirkung binärer Toxine der AB7-Gruppe angeht, so gehören diese zu den potentesten und gefährlichsten Giften weltweit. Selbst kleine Mengen dieser Proteine können schwerste Symptome während einer Infektion mit Bakterien der Gattung Clostridium oder Bacillus verursachen. Abgesehen von der Bedrohung die durch die Toxine ausgeht, zeichnen sie sich durch einen einzigartigen Intoxikationsmechanismus aus. AB7-Toxine sind aus einer porenformenden Domäne B, die als eine Art molekulare Injektionskanüle fungiert, und enzymatisch aktiven Proteinen A zusammengesetzt. Der komplexe Wirkmechanismus ermöglicht es nicht nur Zellen in höchst effektiver Weise abzutöten und sollte deswegen zu Behandlungszwecken untersucht werden, sondern könnte auch als molekulares Werkzeug umfunktioniert werden, um spezielle Zellen mit gewünschten Proteinen zu beladen. Für beide Zwecke (Blockierung und gezielter Transport) ist die Bindung an, und der Transport durch die porenformende Domäne von größter Bedeutung. Die Struktur der B-Domäne ist wichtig um tiefere Einsicht in den Transportprozess der binären Toxine zu ermöglichen. Leider ist es bisher nicht gelungen die Kristallstruktur des Membranproteins protective antigen (PA) von Anthrax oder irgendeiner anderen Bindedomäne eines AB7-Toxins zu lösen. Deshalb wurde in dieser Arbeit ein elektrophysiologischer Ansatz zur Bestimmung der biophysikalischen Konstanten des Prozesses gewählt, die Black-lipid-Bilayer Methode. Zusätzliche Versuche an Zellen und mit Mutanten der Proteine dienen zur Absicherung der in vitro Ergebnisse. Im ersten Teil der Arbeit wird der Translokationsmechanismus, und die mögliche Nutzung dessen als molekulares Werkzeug näher behandelt. Dies erfolgte durch Bindungsstudien an PA und C2II mit Effektoren (sowohl aus der AB7-Familie, als auch nicht näher verwandt). In Kapitel 2 wird beschrieben, dass ein His-Tag es EDIN (von S. aureus) und anderen Effektoren ermöglicht, dass ein Transport durch PA-Poren in vitro und in vivo stattfindet (nicht aber durch C2II). Ebenfalls konnte eine starke Abweichung in der bekannten Spannungsabhängigkeit von PA festgestellt werden, die den Prozess eindeutig mit den Membranpotentialen in Verbindung bringt, die häufig eine wichtige Rolle im Metabolismus spielen. Kapitel 3 fasst zusammen, dass Bindungspartner von PA bestimmte Motive beinhalten, die von Substraten wie Ionen in Elektrolyten, über kleine Moleküle, bis hin zu komplexen Proteinen reichen. Diese Erkenntnisse könnten genutzt werden um Blockersubstanzen zur Behandlung von Anthrax, oder Tags zur Aufnahme durch Anthrax zu designen. Neueste Befunde zu Homologien und Unterschieden zwischen den Komponenten der binären Toxine sind der Kern von Kapitel 4, in dem die Kreuzreaktivität von Anthrax und C2-Toxin analysiert wurde. Die enthaltenen Daten ermöglichen einen tieferen Einblick in die verschiedenen Stufen der Bindung und Translokation des edema factor (EF), des lethal factor (LF) und von C2I an und durch PA und C2II. Im zweiten Teil rückt die Blockierung der Intoxikation in den Fokus. Die Analyse speziell designter Blockersubstanzen für PA wird in Kapitel 5 behandelt. Diese formen einen Porenverschluss, der weiteren Transport von Toxinkomponenten verhindert. Eine besondere Bedeutung könnten diese Substanzen im Zusammenhang mit Multiresitenz bei Anthrax Stämmen (vermutlich in Russland als biologische Waffe hergestellt) zur Verhinderung von Symptomen und der Verlängerung der Zeit spielen, die man hat um neue Antibiotika zu erzeugen. Kapitel 6 beschreibt zum ersten Mal die Blockierung von PA-Poren mittels eines Anti-His Antikörpers von der trans-Seite aus. Interessanterweise trat diese Ionenstärke abhängige Blockierung, auch bei einer Histidin zu Glycin Mutation an der Stelle 310 (vermutete Bindeposition) auf. KW - Bacillus anthracis KW - Translokation KW - Toxin KW - Protective antigen KW - Bindungsprozess KW - Black-lipid-bilayer KW - Bacillus anthracis KW - protective antigen KW - Anthrax Toxin KW - translocation Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-70052 ER - TY - JOUR A1 - Alzheimer, Mona A1 - Svensson, Sarah L. A1 - König, Fabian A1 - Schweinlin, Matthias A1 - Metzger, Marco A1 - Walles, Heike A1 - Sharma, Cynthia M. T1 - A three-dimensional intestinal tissue model reveals factors and small regulatory RNAs important for colonization with Campylobacter jejuni JF - PLoS Pathogens N2 - The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host. Here, we report the development and application of a human three-dimensional (3D) infection model based on tissue engineering to study host-pathogen interactions. Our intestinal 3D tissue model is built on a decellularized extracellular matrix scaffold, which is reseeded with human Caco-2 cells. Dynamic culture conditions enable the formation of a polarized mucosal epithelial barrier reminiscent of the 3D microarchitecture of the human small intestine. Infection with C. jejuni demonstrates that the 3D tissue model can reveal isolate-dependent colonization and barrier disruption phenotypes accompanied by perturbed localization of cell-cell junctions. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D model deviated from those obtained with 2D-monolayers, but recapitulated phenotypes previously observed in animal models. Moreover, we demonstrate the involvement of a small regulatory RNA pair, CJnc180/190, during infections and observe different phenotypes of CJnc180/190 mutant strains in 2D vs. 3D infection models. Hereby, the CJnc190 sRNA exerts its pathogenic influence, at least in part, via repression of PtmG, which is involved in flagellin modification. Our results suggest that the Caco-2 cell-based 3D tissue model is a valuable and biologically relevant tool between in-vitro and in-vivo infection models to study virulence of C. jejuni and other gastrointestinal pathogens. KW - in vitro KW - stem cells KW - invasion KW - host KW - adhesion KW - epithelial cells KW - translocation KW - virulence KW - responses KW - microenvironment Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229454 VL - 16 IS - 2 ER -