Specific antibody-receptor interactions trigger InlAB-independent uptake of listeria monocytogenes into tumor cell lines

<p>Abstract</p> <p>Background</p> <p>Specific cell targeting is an important, yet unsolved problem in bacteria-based therapeutic applications, like tumor or gene therapy. Here, we describe the construction of a novel, internalin A and B (InlAB)-deficient <it>Listeria monocytogenes </it>strain (Lm-spa⁺), which expresses protein A of <it>Staphylococcus aureus </it>(SPA) and anchors SPA in the correct orientation on the bacterial cell surface.</p> <p>Results</p> <p>This listerial strain efficiently binds antibodies allowing specific interaction of the bacterium with the target recognized by the antibody. Binding of Trastuzumab (Herceptin^®) or Cetuximab (Erbitux^®) to Lm-spa⁺, two clinically approved monoclonal antibodies directed against HER2/neu and EGFR/HER1, respectively, triggers InlAB-independent internalization into non-phagocytic cancer cell lines overexpressing the respective receptors. Internalization, subsequent escape into the host cell cytosol and intracellular replication of these bacteria are as efficient as of the corresponding InlAB-positive, SPA-negative parental strain. This specific antibody/receptor-mediated internalization of Lm-spa⁺is shown in the murine 4T1 tumor cell line, the isogenic 4T1-HER2 cell line as well as the human cancer cell lines SK-BR-3 and SK-OV-3. Importantly, this targeting approach is applicable in a xenograft mouse tumor model after crosslinking the antibody to SPA on the listerial cell surface.</p> <p>Conclusions</p> <p>Binding of receptor-specific antibodies to SPA-expressing <it>L. monocytogenes </it>may represent a promising approach to target <it>L. monocytogenes </it>to host cells expressing specific receptors triggering internalization.</p

Shigella Mediated Depletion of Macrophages in a Murine Breast Cancer Model Is Associated with Tumor Regression

A tumor promoting role of macrophages has been described for a transgenic murine breast cancer model. In this model tumor-associated macrophages (TAMs) represent a major component of the leukocytic infiltrate and are associated with tumor progression. Shigella flexneri is a bacterial pathogen known to specificly induce apotosis in macrophages. To evaluate whether Shigella-induced removal of macrophages may be sufficient for achieving tumor regression we have developed an attenuated strain of S. flexneri (M90TΔaroA) and infected tumor bearing mice. Two mouse models were employed, xenotransplantation of a murine breast cancer cell line and spontanous breast cancer development in MMTV-HER2 transgenic mice. Quantitative analysis of bacterial tumor targeting demonstrated that attenuated, invasive Shigella flexneri primarily infected TAMs after systemic administration. A single i.v. injection of invasive M90TΔaroA resulted in caspase-1 dependent apoptosis of TAMs followed by a 74% reduction in tumors of transgenic MMTV-HER-2 mice 7 days post infection. TAM depletion was sustained and associated with complete tumor regression

Use of a recombinant Salmonella enterica serovar Typhimurium strain expressing C-Raf for protection against C-Raf induced lung adenoma in mice

BACKGROUND: Serine-threonine kinases of the Raf family (A-Raf, B-Raf, C-Raf) are central players in cellular signal transduction, and thus often causally involved in the development of cancer when mutated or over-expressed. Therefore these proteins are potential targets for immunotherapy and a possible basis for vaccine development against tumors. In this study we analyzed the functionality of a new live C-Raf vaccine based on an attenuated Salmonella enterica serovar Typhimurium aroA strain in two Raf dependent lung tumor mouse models. METHODS: The antigen C-Raf has been fused to the C-terminal secretion signal of Escherichia coli α-hemolysin and expressed in secreted form by an attenuated aroA Salmonella enterica serovar Typhimurium strain via the α-hemolysin secretion pathway. The effect of the immunization with this recombinant C-Raf strain on wild-type C57BL/6 or lung tumor bearing transgenic BxB mice was analyzed using western blot and FACS analysis as well as specific tumor growth assays. RESULTS: C-Raf antigen was successfully expressed in secreted form by an attenuated Salmonella enterica serovar Typhimurium aroA strain using the E. coli hemolysin secretion system. Immunization of wild-type C57BL/6 or tumor bearing mice provoked specific C-Raf antibody and T-cell responses. Most importantly, the vaccine strain significantly reduced tumor growth in two transgenic mouse models of Raf oncogene-induced lung adenomas. CONCLUSIONS: The combination of the C-Raf antigen, hemolysin secretion system and Salmonella enterica serovar Typhimurium could form the basis for a new generation of live bacterial vaccines for the treatment of Raf dependent human malignancies

Autoantibodies to BRAF, a new family of autoantibodies associated with rheumatoid arthritis

International audienceBRAF (v raf murine sarcoma viral oncogene homologue B1) is a serine-threonine kinase involved in the mitogen-activated protein kinase (MAPK) signalling pathway, known to be implicated in the production of pro-inflammatory cytokines.We have observed that sera from rheumatoid arthritis (RA) patients recognize the BRAF's catalytic domain, which encompasses amino acids 416 to 766. Here, we identify peptide targets of anti-BRAF autoantibodies and test whether anti-BRAF autoantibodies may interfere with BRAF kinase activity.METHODS:Anti-BRAF autoantibodies were detected by ELISA (enzyme-linked immunosorbent assay) in the serum of RA patients and controls, using 40 overlapping 20mer peptides encompassing the catalytic domain of BRAF as immunosorbents. To test whether autoantibodies to BRAF influence BRAF kinase activity, we developed an in vitro phosphorylation assay of MEK1 (mitogen extracellular regulated kinase), a major BRAF substrate. MEK1 phosphorylation by BRAF was tested in the presence of purified anti-BRAF autoantibodies from RA patients or control antibody.RESULTS:We found that one BRAF peptide, P25 (656 to 675), is specifically recognized by autoantibodies from RA patients. Of interest, anti-P25 autoantibodies are detected in 21% of anti-CCP (cyclic citrullinated peptides) negative RA patients. Anti-BRAF autoantibodies activate the in vitro phosphorylation of MEK1 mediated by BRAF.CONCLUSIONS:Anti-BRAF autoantibodies from RA patients preferentially recognize one BRAF peptide: P25. Autoantibody responses to P25 are detected in 21% of anti-CCP negative RA patients. Most anti-BRAF autoantibodies activate BRAF kinase activity

AB Toxins: A Paradigm Switch from Deadly to Desirable

To ensure their survival, a number of bacterial and plant species have evolved a common strategy to capture energy from other biological systems. Being imperfect pathogens, organisms synthesizing multi-subunit AB toxins are responsible for the mortality of millions of people and animals annually. Vaccination against these organisms and their toxins has proved rather ineffective in providing long-term protection from disease. In response to the debilitating effects of AB toxins on epithelial cells of the digestive mucosa, mechanisms underlying toxin immunomodulation of immune responses have become the focus of increasing experimentation. The results of these studies reveal that AB toxins may have a beneficial application as adjuvants for the enhancement of immune protection against infection and autoimmunity. Here, we examine similarities and differences in the structure and function of bacterial and plant AB toxins that underlie their toxicity and their exceptional properties as immunomodulators for stimulating immune responses against infectious disease and for immune suppression of organ-specific autoimmunity

Implementierung einer bidirektionalen Temperaturregelung für einen Metallhydrid-Reaktor

Im Kontext der weltweiten Bemühungen den Klimawandel aufzuhalten, gewinnen Brennstoffzellen im Mobilitätssektor zunehmend an Bedeutung. Technisch gesehen ist die Lebensdauer von Brennstoffzellen ein begrenzender Faktor für ihren Einsatz in der Mobilität. Die Lebensdauer wird von zwei Hauptfaktoren verringert: dem Kaltstart und den Temperaturschwankungen aufgrund von Lastzyklen. Eine bidirektionale und aktive Temperaturregelung könnte mittels Metallhydriden möglich sein, um die Temperaturschwankungen zu verhindern. Metallhydride sind die Verbindungen zwischen Metallen und Wasserstoff, die bei der Reaktion miteinander reversibel Wärme und Kälte freisetzen können, sodass eine schnelle, aktive, bidirektionale Temperaturregelung möglich ist. In der vorliegenden Arbeit wird eine aktive, bidirektionale Temperaturregelung mittels einem Metallhydrid-Reaktor entwickelt und implementiert. Als Grundlage für diese Regelung werden die Eigenschaften des Metallhydrid-Reaktors in experimentellen Versuchen ermittelt. Dabei zeigt sich, dass der Wasserstoffumsatz und die Reaktionskinetik temperatur- und druckabhängig sind. Zusätzlich wird die Reaktionskinetik vom Beladungszustand des Metallhydrids beeinflusst. Die übertragene thermische Leistung des Metallhydrids auf das Wärmeträgerfluid, das den Reaktor durchströmt, hängt von Druck, Temperatur und dem Volumenstrom des Wärmeträgerfluids ab. Basierend auf den Erkenntnissen aus den Versuchen am Teststand wird die Temperaturregelung entwickelt. Der Regelkreis wird als Kaskadenregelung realisiert. Im äußeren Regelkreis erfolgt die Temperaturregelung mit dem Druck als Stellgröße, während der innere Regelkreis den Druck regelt. Dabei werden der Proportional-Integral-Regler und der Proportional-Integral-Differential-Regler als Reglertypen untersucht. Die Parameteroptimierung der Regler erfolgt simulativ anhand eines definierten Gütefunktionals. Die Optimierung der Regelparameter und die Überprüfung der Qualität des Reglers erfolgen mit Hilfe von definierten Störtrajektorien. Die Simulation dient weiterführend zur Analyse des Regelkreises und zeigt, dass der Regelkreis nicht stabil ist. Dies spiegelt sich auch in den experimentellen Ergebnissen der Temperaturregelung wider. Die Ergebnisse der Temperaturregelung zeigen, dass der Temperaturregler die Temperatur mit einer mittleren Abweichung von 0,25 K für eine 5 K-Temperaturtrajektorie stabilisieren kann. Die Stellgröße Druck ist zur Temperaturregelung geeignet, da die Reaktortemperatur dem Druck mit einer Verzögerung von 1 s folgt. Allerdings weist die Regelgröße ein oszillierendes Verhalten auf, das durch das nichtlineare Verhalten des Reaktors bedingt ist. Zudem ist das Regelverhalten von dem Beladungszustand des Reaktors abhängig. Insgesamt verdeutlicht diese Arbeit, dass die Umsetzung einer Temperaturregelung mit einem Metallhydrid-Reaktor möglich ist. Zur Verbesserung des Regelverhaltens sollten zukünftige Konzepte Regler einschließen, die den Beladungszustand des Reaktors und das nicht-lineare Reaktorverhalten berücksichtigen