Understanding why LPS is unable to mobilize the MyD88-independent pathway in human neutrophils

Background: Lipopolysaccharide (LPS) activates both MyD88-dependent and -independent signaling via TLR4, but the extent to which each cascade is operative in different cell types remains unclear. Materials and methods: Human neutrophils and monocytes, isolated from buffy coat, were stimulated with LPS and then disrupted in a nitrogen bomb to prepare protein extracts with preserved integrity and association. Lysates were then subjected to WB, immunoprecipitation or co-immunoprecipitation using specific antibodies raised against TBK1, TRIF, TRAF3,NAP1, HSP-90 and SHP-2. Results: In a previous study (Tamassia N. et al., J Immunol.2007; 178(11):7344-56), we have demonstrated that human neutrophils are unable to mobilize the MyD88-independent path-way, as revealed by the lack of inducibility neither of IFNb(the principal MyD88-independent gene) nor of IFNb-dependent genes in LPS-treated cells. Consistent with these latter results, we have also found that IRF3, a critical transcription factor for IFNb gene induction, and TBK1, an IRF3-phosphory-lating kinase, are both not activated by LPS in human neutrophils. We are currently investigating the molecular mechanisms that, ultimately, may explain why in neutrophils the TLR4-mediated, MyD88-independent pathway is impaired. Specifically, we are testing whether the interactions of TBK1with its various regulatory proteins, such as TRIF, TRAF3,NAP1, HSP-90 and SHP-2, appropriately occur or not inhuman neutrophils. Conclusions: Our results will elucidate the molecular bases of the disconnected activation of the signaling pathways down-stream of TLR4 in key cellular components of the inflammatory and immune responses. They will also help to better understand the primordial role of neutrophils in host defence against non-viral infections

IFN-\u3b2 Expression Is Directly Activated in Human Neutrophils Transfected with Plasmid DNA and Is Further Increased via TLR-4-Mediated Signaling.

Upon LPS binding, TLR4 activates a MyD88-dependent pathway leading to the transcriptional activation of proinflammatory genes, as well as a MyD88-independent/TRIF-dependent pathway, responsible for the transcriptional induction of IFN-\u3b2. Previous findings delineated that human neutrophils are unable to induce the transcription of IFN-\u3b2 in response to TLR4 stimulation. Because neutrophils do not express protein kinase C \u3b5, a molecule recently reported as essential for initiating the MyD88-independent/TRIF-dependent pathway, we optimized an electroporation method to transfect PKC\u3b5 into neutrophils with very high efficiency. By doing so, a significant IFN-\u3b2 mRNA expression was induced, in the absence of LPS stimulation, not only in PKC\u3b5-overexpressing neutrophils but also in cells transfected with a series of empty DNA plasmids; however, LPS further upregulated the IFN-\u3b2 transcript levels in plasmid-transfected neutrophils, regardless of PKC\u3b5 overexpression. Phosphoimmunoblotting studies, as well as chromatin immunoprecipitation assays targeting the IFN-\u3b2 promoter, revealed that IFN-\u3b2 mRNA induction occurred through the cooperative action of IRF3, activated by transfected DNA, and NF-\u3baB, activated by LPS. Additional immunoblotting and coimmunoprecipitation studies revealed that neutrophils constitutively express various cytosolic DNA sensors, including IFN-inducible protein 16, leucine-rich repeat (in Flightless I) interacting protein-1, and DDX41, as well as that IFN-inducible protein 16 is the intracellular receptor recognizing transfected DNA. Consistently, infection of neutrophils with intracellular pathogens, such as Bartonella henselae, Listeria monocytogenes, Legionella pneumophila, or adenovirus type 5, promoted a marked induction of IFN-\u3b2 mRNA expression. Taken together, these data raise questions about the role of PKC\u3b5 in driving the MyD88-independent/TRIF-dependent response and indicate that human neutrophils are able to recognize and respond to microbial cytosolic DNA

IFN-β expression is directly activated in human neutrophils transfected with plasmid DNA and is further increased via TLR-4-mediated signaling

Upon LPS binding, TLR4 activates a MyD88-dependent pathway leading to the transcriptional activation of proinflammatory genes, as well as a MyD88-independent/TRIF-dependent pathway, responsible for the transcriptional induction of IFN-β. Previous findings delineated that human neutrophils are unable to induce the transcription of IFN-β in response to TLR4 stimulation. Because neutrophils do not express protein kinase C ε, a molecule recently reported as essential for initiating the MyD88-independent/ TRIF-dependent pathway, we optimized an electroporation method to transfect PKCε into neutrophils with very high efficiency. By doing so, a significant IFN-β mRNA expression was induced, in the absence of LPS stimulation, not only in PKCε-overexpressing neutrophils but also in cells transfected with a series of empty DNA plasmids; however, LPS further upregulated the IFN-β transcript levels in plasmid-transfected neutrophils, regardless of PKCε overexpression. Phosphoimmunoblotting studies, as well as chromatin immunoprecipitation assays targeting the IFN-β promoter, revealed that IFN-β mRNA induction occurred through the cooperative action of IRF3, activated by transfected DNA, and NF-κB, activated by LPS. Additional immunoblotting and coimmunoprecipitation studies revealed that neutrophils constitutively express various cytosolic DNA sensors, including IFN-inducible protein 16, leucine-rich repeat (in Flightless I) interacting protein-1, and DDX41, as well as that IFN-inducible protein 16 is the intracellular receptor recognizing transfected DNA. Consistently, infection of neutrophils with intracellular pathogens, such as Bartonella henselae, Listeria monocytogenes, Legionella pneumophila, or adenovirus type 5, promoted a marked induction of IFN-β mRNA expression. Taken together, these data raise questions about the role of PKCεin driving the MyD88-independent/TRIF-dependent response and indicate that human neutrophils are able to recognize and respond to microbial cytosolic DNA. Copyright © 2012 by The American Association of Immunologists, Inc

a comparison between 2004 and 2005 for a specific search in the field of biotechnology

Aim of this work was the analysis of the changes occurring in hit lists of selected search engines at two different points of time.The same search query was applied to the search engine Google and to the science search engine Scirus in August 2004 and in April 2005. The first 40 hits of each search have been analysed on the basis of predefined qualitative and quantitative parameters. Further an analysis of overlapping between search hits at the above mentioned time points has been carried out for each search engine separately.The results of this study has shown on one hand the existence of positive trends in the result lists of both search engines (less dead links, a higher precision in results) and on the other hand the role played by search engines themselves in the dynamic of result lists. The method applied here has shown to be useful for describing and measuring trends and changes in the search results of search engines over a time span.Das Ziel dieser Arbeit war die Identifizierung und Quantifizierung der Veränderungen in den Suchergebnissen aus ausgewählten Suchmaschinen im Jahresabstand.Eine Recherche mit der gleichen Suchabfrage ist mit der allgemeinen Suchmaschine Google und mit der wissenschaftlichen Suchmaschine Scirus im August 2004 und April 2005 durchgeführt worden. Die ersten 40 Treffer sind nach vordefinierten qualitativen und quantitativen Parametern analysiert worden. Weiters ist die Überlappung der Suchergebnissen für die oben genannten Abfragezeitpunkte je nach Suchmaschine getrennt bewertet worden.Die aus dieser Studie resultierenden Ergebnisse zeigen einerseits die Existenz von positiven Tendenzen in den Ergebnislisten der untersuchten Suchmaschinen (Verbesserungen in der Relevanz der Treffer, eine Verringerung der Anzahl an nicht abrufbaren Links, sowie ein Zuwachs an erzielter Information) und andererseits die Rolle der Websuchinstrumente selbst hinsichtlich den Veränderungen von Suchergebnissen bei einer Recherche im Web

Mechanical Recanalization in Basilar Artery Occlusion: The ENDOSTROKE Study

ObjectiveA study was undertaken to evaluate clinical and procedural factors associated with outcome and recanalization in endovascular stroke treatment (EVT) of basilar artery (BA) occlusion. MethodsENDOSTROKE is an investigator-initiated multicenter registry for patients undergoing EVT. This analysis includes 148 consecutive patients with BA occlusion, with 59% having received intravenous thrombolysis prior to EVT. Recanalization (defined as Thrombolysis in Cerebral Infarction [TICI] score 2b-3) and collateral status (using the American Society of Interventional and Therapeutic Neuroradiology/Society of Interventional Radiology collateral grading system) were assessed by a blinded core laboratory. Good (moderate) outcome was defined as a modified Rankin Scale score of 0 to 2 (0-3) assessed after at least 3 months (median time to follow-up=120 days). ResultsThirty-four percent had good and 42% had moderate clinical outcome; mortality was 35%. TICI 2b-3 recanalization was achieved by 79%. Age, hypertension, National Institutes of Health Stroke Scale scores, collateral status, and the use of magnetic resonance imaging prior to EVT predicted clinical outcome, the latter 3 remaining independent predictors in multivariate analysis. Independent predictors of recanalization were better collateral status and the use of a stent retriever. However, recanalization did not significantly predict clinical outcome. InterpretationBeside initial stroke severity, the collateral status predicts clinical outcome and recanalization in BA occlusion. Our data suggest that the use of a stent retriever is associated with high recanalization rates, but recanalization on its own does not predict outcome. The role of other modifiable factors, including the choice of pretreatment imaging modality and time issues, warrants further investigation. Ann Neurol 2015;77:415-42