Oxidation-Specific Epitopes (OSEs) Dominate the B Cell Response in Murine Polymicrobial Sepsis

In murine abdominal sepsis by colon ascendens stent peritonitis (CASP), a strong increase in serum IgM and IgG antibodies was observed, which reached maximum values 14 days following sepsis induction. The specificity of this antibody response was studied in serum and at the single cell level using a broad panel of bacterial, sepsis-unrelated as well as self-antigens. Whereas an antibacterial IgM/IgG response was rarely observed, studies at the single-cell level revealed that IgM antibodies, in particular, were largely polyreactive. Interestingly, at least 16% of the IgM mAbs and 20% of the IgG mAbs derived from post-septic mice showed specificity for oxidation-specific epitopes (OSEs), which are known targets of the innate/adaptive immune response. This identifies those self-antigens as the main target of B cell responses in sepsis

In vitro studies of the activity of flavan-3-ols and proanthocyanidins: neuraminidase inhibiting and vasorelaxant effects

Die vorliegende Arbeit betrachtet das antigrippale, vasorelaxierende und vasoprotektive Potential einer Reihe von Flavan-3-olen und Proanthocyanidinen. Im ersten Teil wurde das Hemmpotential der Testsubstanzen gegenüber einer bakteriellen (V. cholerae) und einer viralen Influenzavirus A Neuraminidase (NA) (N1) mittels eines MuNANA-Fluoreszenz-Assays überprüft. Im Vergleich zu den Positivkontrollen Zanamivir und Oseltamivircarboxylat inhibierten proanthocyanidinreichen Fraktionen (Salix spp., Nelia meyeri, Cephalophyllum spp., Betula spp., Potentilla erecta, Rhus leptodictya, Diospyros kaki, Pelargonium sidoides) und Ellagitannine (Geraniin, Granatin A, Carpinusin, Terchebin, Casuariin, Vescalagin, Paeonianin C) die bakterielle NA signifikant effektiver. Im Gegensatz dazu hemmten alle polyphenolischen Substanzen die virale NA bei Weitem weniger als die NA-Referenzinhibitoren. Die Struktur- Wirkungsbeziehungen zeigten, dass der Polymerisationsgrad, die 2,3-Stereochemie, das Hydroxylierungsmuster und die 3-O-Galloylierung Einfluss auf die inhibitorische Aktivität der untersuchten Flavan-3-ole und Proanthocyanidine nehmen. In der Reihe der Ellagitannine ist der Grad der Galloylierung entscheidend. In Ergänzung dazu wurde ein synergistischer Hemmeffekt einer Zanamivir/EPs® 7630-Kombination (1:10) gegenüber der V. cholerae NA nachgewiesen. Die Auswertung der Röntgenstrukturdaten von Ko- Kristallisaten der V. cholerae NA bzw. Influenzavirus A NA (N1) mit entweder Zanamivir oder Oseltamivircarboxylat ergab, dass aufgrund der Wechselwirkungen mit dem lediglich viral vorkommenden 150-loop beide NA-Inhibitoren das virale Enzym effektiver hemmen als die bakterielle NA. Im zweiten Teil wurde mittels Organbadstudien das vasorelaxierende Potential überprüft. Koronararterienringe des Schweins wurden mit U46619 kontrahiert und durch die kumulative Zugabe polyphenolischer Testsubstanzen relaxiert. In der Reihe der Flavan-3-ole und der proanthocyanidinhaltigen Fraktionen induzierten galloylierte Substanzen eine konzentrationsabhängige Vasodilatation. Die Experimente zeigten, dass Diospyros-Proanthocyanidine sowie Procyanidin-B2-3-O-gallat die Phosphoinositid-3-Kinase/Proteinkinase B, die endotheliale NO-Synthase und die löslichen Guanylylcyclase stimulierten. Beide Testsubstanzen induzierten eine endothelabhängige Relaxation über den NO/cGMP-Signalweg. Zudem wurde die Beteiligung der Na+/K+-ATPase am Relaxationsmechanismus nachgewiesen. Des Weiteren wurde die ROS-Produktion in Abhängigkeit von der Testsubstanz untersucht und für Procyanidin B2-3-O-gallat die mitochondriale Atmungskette als potentielle ROS-Quelle identifiziert.The present study was undertaken to evaluate the anti-influenza, vasorelaxant and vasoprotective potential of a series of flavan-3-ols and proanthocyanidins and to gain insight into the mode of action. The first part of this thesis addressed the inhibitory potential of the tested substances against a bacterial (V. cholera) and a viral influenza A (California/04/2009[H1N1]) neuraminidase (NA), using a (MuNANA)-fluorescence-based assay. The present study verified that the substrate MuNANA is recognized by both NAs. Compared to the positive controls zanamivir und oseltamivir carboxylic acid, the studied proanthocyanidin enriched fractions (Salix spp., Nelia meyeri, Cephalophyllum spp., Betula spp., Potentilla erecta, Rhus leptodictya, Diospyros kaki, Pelargonium sidoides) and ellagitannins (Geraniin, Granatin A, Carpinusin, Terchebin, Casuariin, Vescalagin, Paeonianin C) were significantly more effective against the bacterial NA. In contrast, all polyphenolic samples were far less effective inhibitors of the viral NA than the reference samples. Structure-activity relationships indicated that the degree of polymerization, the 2,3-stereochemistry, hydroxylation patterns and the presence of a 3-O-galloyl group largely affected the inhibitory potential of the tested flavan-3-ols and proanthocyanidins. The degree of galloylation appeared crucial in the series of ellagitannins. In addition, the combination of Zanamivir with EPs® 7630 (1:10) showed a synergistic inhibitory effect in studies against V. cholerae NA. The evaluation of co-crystal data of the bacterial respectively viral NA and either Zanamivir or oseltamivir carboxylate showed due to the interactions with the only viral occurring 150-loop both drugs inhibit the viral enzyme more effectively than the bacterial NA. In the second part of this thesis the vasorelaxant potential was investigated using a tissue bath protocol. Porcine coronary arterial rings were contracted with U46619 and relaxed by cumulative addition of the polyphenolic samples. Within the series of flavan-3-ols and proanthocyanidin- enriched fractions, galloylated compounds induced a concentration-dependent vasodilatation. The experiments showed that Diospyros-proanthocyanidins and procyanidin B2-3-O-gallate stimulated phosphoinositide-3-kinase/protein kinase B, endothelial NO synthase, and soluble guanylyl cyclase, respectively. Both samples evoked an endothelium-dependent relaxations via the NO/cGMP pathway. In addition, Na+/K+-ATPase was involved in the relaxant response to these polyphenols. Intracellular ROS formation in response to the proanthocyanidin samples was verified and the mitochondrial respiratory chain identified as a potential source of reactive oxygen species for the procyanidin B2-3-O-gallate

Different Inhibitory Potencies of Oseltamivir Carboxylate, Zanamivir, and Several Tannins on Bacterial and Viral Neuraminidases as Assessed in a Cell-Free Fluorescence-Based Enzyme Inhibition Assay

Neuraminidase is a key enzyme in the life cycle of influenza viruses and is present in some bacterial pathogens. We here assess the inhibitory potency of plant tannins versus clinically used inhibitors on both a viral and a bacterial model neuraminidase by applying the 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA)-based activity assay. A range of flavan-3-ols, ellagitannins and chemically defined proanthocyanidin fractions was evaluated in comparison to oseltamivir carboxylate and zanamivir for their inhibitory activities against viral influenza A (H1N1) and bacterial Vibrio cholerae neuraminidase (VCNA). Compared to the positive controls, all tested polyphenols displayed a weak inhibition of the viral enzyme but similar or even higher potency on the bacterial neuraminidase. Structure–activity relationship analyses revealed the presence of galloyl groups and the hydroxylation pattern of the flavan skeleton to be crucial for inhibitory activity. The combination of zanamivir and EPs® 7630 (root extract of Pelargonium sidoides) showed synergistic inhibitory effects on the bacterial neuraminidase. Co-crystal structures of VCNA with oseltamivir carboxylate and zanamivir provided insight into bacterial versus viral enzyme-inhibitor interactions. The current data clearly indicate that inhibitor potency strongly depends on the biological origin of the enzyme and that results are not readily transferable. The therapeutic relevance of our findings is briefly discussed