Untersuchung des Infektionsverhaltens verschiedener respiratorischer Viren in humanem ex vivo kultiviertem Lungengewebe

Mechanismen, die zur unterschiedlichen Pathogenität niedrig- und hochpathogener aviärer und humanpathogener Influenzaviren im Menschen beitragen, sind bisher nur ansatzweise verstanden. Auch sind Pathomechanismen, die der Middle East Respiratory Syndrome (MERS)-CoV-Infektion zu Grunde liegen, bisher weitgehend unbekannt. In dieser Arbeit wurde ein humanes ex vivo Lungenkulturmodell für die Untersuchung der Influenzavirus- und der MERS-CoV-Infektion etabliert. Dabei wurde die Replikationsfähigkeit und der Zelltropismus der Viren systematisch verglichen. Während hochpathogene aviäre, saisonale und pandemische Influenzaviren effizient in der humanen Lunge replizierten, konnten sich niedrigpathogene aviäre Viren und ein porcines Virus kaum vermehren. Alle Viren zeigten jedoch den gleichen Zelltropismus und infizierten im Alveolarepithel ausschließlich Typ II Zellen. Die unterschiedliche Pathogenität dieser Viren lässt sich daher nicht durch Unterschiede im Zelltropismus erklären. Gleichwohl konnte der humane und der aviäre Influenzavirusrezeptor sowohl auf Typ II als auch auf Typ I Zellen nachgewiesen werden. Niedrigpathogene aviäre Influenzaviren sind in der humanen Lunge durch die Freisetzung von zum größten Teil nicht infektiösen Viruspartikeln restringiert. MERS-CoV replizierte in humanem Lungengewebe mit ähnlicher Kinetik wie ein hochpathogenes H5N1 Virus. MERS-CoV Antigen war sowohl im Bronchialepithel, Alveolarepithel sowie im Endothel nachweisbar. Der funktionelle Rezeptors des MERS-CoV, die Dipeptidylpeptidase 4, wurde in allen infizierten Zelltypen sowie in Alveolarmakrophagen nachgewiesen. Die mikroskopische Analyse infizierter Gewebeproben weist zudem auf einen infektionsbedingten Alveolarschaden hin. Die Studie trägt wesentlich zum pathophysiologischen Verständnis pulmonaler Influenzavirus- und MERS-CoV-Infektionen bei. Das humane ex vivo Lungenkulturmodell stellt ein klinisch relevantes Modell zur Untersuchung respiratorischer Infektionen im Menschen dar.Mechanisms contributing to the different pathogenicity of low and highly pathogenic avian and seasonal influenza viruses in humans are currently only partially understood. Furthermore, underlying pathological mechanisms of the Middle East Respiratory Syndrome (MERS)-CoV infection in humans are widely unknown. In this study a human ex vivo lung culture model was established, which allowed investigating influenza virus and MERS-CoV infection. Replication and cellular tropism of the different viruses were compared systematically. While highly pathogenic avian, seasonal and pandemic influenza viruses replicated efficiently, low pathogenic avian viruses and a porcine virus propagated poorly. However, all viruses showed the same cellular tropism and infected only type II cells within the alveolar epithelium. Therefore, the different pathogenicity of these viruses cannot be attributed to different cellular tropisms. Nevertheless, the human and the avian influenza virus receptor could be detected on type II and type I cells. Low pathogenic avian influenza viruses are restricted in the human lung by the release of mostly non-infectious progeny virus particles. The MERS-CoV replicated with similar kinetics to a highly pathogenic H5N1 virus in the human lung. MERS-CoV antigen was detected in the bronchial epithelium, alveolar epithelium and endothelium. The functional receptor of MERS-CoV, dipeptidylpeptidase 4, was found in all infected cell types and in alveolar macrophages. Microscopic analysis of infected tissue samples indicated an alveolar damage provoked by MERS-CoV infection. This study contributes substantially to the pathophysiological understanding of the pulmonary influenza virus and MERS-CoV infection. The human ex vivo lung culture model represents a clinically relevant model to investigate human respiratory infections

Untersuchungen zur Einführung der Selektiven Anthelminthischen Therapie beim Pferd im Raum Salzburg

From March until November 2008 faecal samples of 281 horses and 5 donkeys in an area of 40 km around Salzburg were analysed with a modified McMaster method for Strongyle eggs. Each time a faecal egg count (FEC) exceeded 250 eggs per gram faeces (EPG) the horse was treated with either pyrantel, ivermectin or moxidectin and FEC reduction test was done on day 14 and 21. Additionally 259 horse owners participated in an online questionnaire survey about worm control. In 52 (40.3 %) horses no strongyle eggs were detected in any of nine samples. In 39 horses (30.2 %), eggs were detected in at least one sample, but the egg count did not exceed 250 EPG at any of the nine sampling occasions. The remainder of the horses (29.5 %) had to be treated at least once due to the FEC exceeding 250 EPG. The number of treatments was reduced to 46 % of the number of treatments in the year before. The maximal and mean FEC both dropped significantly after the start of the study. There was a slight statistically significant negative correlation between the age of the horses and the maximal and mean FEC of each horse. The first FEC had a positive statistically significant correlation with the maximal FECs in the following eight months. Furthermore horses with more treatments had a higher first FEC than horses with less or no treatments. The efficacy of ivermectin and moxidectin was 100 %. On one farm resistance to pyrantel was detected. 80,2 % of the participants of the questionnaire survey knew selective anthelmintic treatment and 85,7 % chose the correct definition of resistance. The avoidance of the development of anthelmintic resistance was very important for 82,6 % of the participants. These results suggest that selective anthelmintic treatment can reduce the pasture contamination with strongyle eggs as well as the number of anthelmintic treatments. While using pyrantel the efficacy of this drug should be monitored. Furthermore these data can be helpful to design selective anthelmintic treatment programs, which can be applied by equine practitioners. The participating horse owners prefer this method of worm control, as it is the only one that avoids the development of resistance

Untersuchungen zur Einführung der Selektiven Anthelminthischen Therapie beim Pferd im Raum Salzburg

From March until November 2008 faecal samples of 281 horses and 5 donkeys in an area of 40 km around Salzburg were analysed with a modified McMaster method for Strongyle eggs. Each time a faecal egg count (FEC) exceeded 250 eggs per gram faeces (EPG) the horse was treated with either pyrantel, ivermectin or moxidectin and FEC reduction test was done on day 14 and 21. Additionally 259 horse owners participated in an online questionnaire survey about worm control. In 52 (40.3 %) horses no strongyle eggs were detected in any of nine samples. In 39 horses (30.2 %), eggs were detected in at least one sample, but the egg count did not exceed 250 EPG at any of the nine sampling occasions. The remainder of the horses (29.5 %) had to be treated at least once due to the FEC exceeding 250 EPG. The number of treatments was reduced to 46 % of the number of treatments in the year before. The maximal and mean FEC both dropped significantly after the start of the study. There was a slight statistically significant negative correlation between the age of the horses and the maximal and mean FEC of each horse. The first FEC had a positive statistically significant correlation with the maximal FECs in the following eight months. Furthermore horses with more treatments had a higher first FEC than horses with less or no treatments. The efficacy of ivermectin and moxidectin was 100 %. On one farm resistance to pyrantel was detected. 80,2 % of the participants of the questionnaire survey knew selective anthelmintic treatment and 85,7 % chose the correct definition of resistance. The avoidance of the development of anthelmintic resistance was very important for 82,6 % of the participants. These results suggest that selective anthelmintic treatment can reduce the pasture contamination with strongyle eggs as well as the number of anthelmintic treatments. While using pyrantel the efficacy of this drug should be monitored. Furthermore these data can be helpful to design selective anthelmintic treatment programs, which can be applied by equine practitioners. The participating horse owners prefer this method of worm control, as it is the only one that avoids the development of resistance

NMR Relaxometry Accessing the Relaxation Spectrum in Molecular Glass Formers

It is a longstanding question whether universality or specificity characterize the molecular dynamics underlying the glass transition of liquids. In particular, there is an ongoing debate to what degree the shape of dynamical susceptibilities is common to various molecular glass formers. Traditionally, results from dielectric spectroscopy and light scattering have dominated the discussion. Here, we show that nuclear magnetic resonance (NMR), primarily field-cycling relaxometry, has evolved into a valuable method, which provides access to both translational and rotational motions, depending on the probe nucleus. A comparison of ¹H NMR results indicates that translation is more retarded with respect to rotation for liquids with fully established hydrogen-bond networks; however, the effect is not related to the slow Debye process of, for example, monohydroxy alcohols. As for the reorientation dynamics, the NMR susceptibilities of the structural (α) relaxation usually resemble those of light scattering, while the dielectric spectra of especially polar liquids have a different broadening, likely due to contributions from cross correlations between different molecules. Moreover, NMR relaxometry confirms that the excess wing on the high-frequency flank of the α-process is a generic relaxation feature of liquids approaching the glass transition. However, the relevance of this feature generally differs between various methods, possibly because of their different sensitivities to small-amplitude motions. As a major advantage, NMR is isotope specific; hence, it enables selective studies on a particular molecular entity or a particular component of a liquid mixture. Exploiting these possibilities, we show that the characteristic Cole–Davidson shape of the α-relaxation is retained in various ionic liquids and salt solutions, but the width parameter may differ for the components. In contrast, the low-frequency flank of the α-relaxation can be notably broadened for liquids in nanoscopic confinements. This effect also occurs in liquid mixtures with a prominent dynamical disparity in their components

Naturschutzleistungen des Ökologischen Landbaus: Wiederansiedlung seltener und gefährdeter Ackerwildpflanzen naturräumlicher Herkünfte auf Ökobetrieben

Die Intensivierung der Landwirtschaft hat zum Rückgang vieler Ackerwildpflanzen geführt. Der Ökologische Landbau bietet günstige Voraussetzungen für ihren Schutz. Wie entsprechende Populationen etabliert werden können, untersuchte ein Verbundprojekt der Bayerischen Landesanstalt für Landwirtschaft, der Technischen Universität München und der Universität Kassel. Die AG Freising untersuchte drei seltene winterannuelle Arten (Consolida regalis, Legousia speculumveneris, Lithospermum arvense) in mehrfaktoriellen Feldversuchen, in Praxisaus-saaten auf Bio-Betrieben sowie in Gewächshausexperimenten. Messgrößen waren Individuendichte, Samenproduktion und Bodensamenvorrat der Zielarten, zudem wurde der Ertrag der Feldfrüchte bestimmt. Frühe Herbstsaaten und geringe Konkurrenz durch Kulturen brachten beste Erfolge. Zur erfolgreichen Ansiedlung der Ackerwildkräuter wird eine Aussaat in Blanksaat oder in reduziert gesäten Winterungen, wie Dinkel oder Roggen, bis spätestens Mitte Oktober empfohlen. Klee-Gras und Sommerungen wie Erbsen ermöglichten kaum bzw. kein Auflaufen der Zielarten, die jedoch teils im Bodensamenvorrat überdauern. Die AG Witzenhausen untersuchte die Wiederansiedlung von Ackerwildkräutern auf Praxis-betrieben. Dazu wurden artenreiche Spenderflächen identifiziert und autochthones Saatgut gefährdeter Arten entnommen. Samenmischungen wurden in Blühfenster und den benachbarten Getreidebestand ausgebracht. Zudem wurde die Übertragung von Oberboden arten-reicher Flächen getestet. Im Anlagejahr konnte sich bei beiden Verfahren ein Teil der eingebrachten Arten reproduzieren. Dies gelang bei Konkurrenz mit Getreide tendenziell schlechter. In den Folgejahren konnten bei Anbau von Getreide wiederum einige Arten nachgewiesen werden; die meisten Samen gelangten bei Bodenbearbeitung in tiefere Bodenschichten und reicherten die Samenbank an. Praxisempfehlungen zur Wiederansiedlung von Ackerwildkräutern auf ökologisch bewirtschafteten Äckern wurden als Broschüre veröffentlicht

Exoproteomic profiling uncovers critical determinants for virulence of livestock-associated and human-originated Staphylococcus aureus ST398 strains

Staphylococcus aureus: with the sequence type (ST) 398 was previously associated with livestock carriage. However, in recent years livestock-independent S. aureus ST398 has emerged, representing a potential health risk for humans especially in nosocomial settings. Judged by whole-genome sequencing analyses, the livestock- and human originated strains belong to two different S. aureus ST398 clades but, to date, it was not known to what extent these clades differ in terms of actual virulence. Therefore, the objective of this study was to profile the exoproteomes of 30 representative S. aureus ST398 strains by mass spectrometry, to assess clade-specific differences in virulence factor secretion, and to correlate the identified proteins and their relative abundance to the strains' actual virulence. Although the human-originated strains are more heterogeneous at the genome level, our observations show that they are more homogeneous in terms of virulence factor production than the livestock-associated strains. To assess differences in virulence, infection models based on larvae of the wax moth Galleria mellonella and the human HeLa cell line were applied. Correlation of the exoproteome data to larval killing and toxicity toward HeLa cells uncovered critical roles of the staphylococcal Sbi, SpA, SCIN and CHIPS proteins in virulence. These findings were validated by showing that sbi or spa mutant bacteria are attenuated in G. mellonella and that the purified SCIN and CHIPS proteins are toxic for HeLa cells. Altogether, we show that exoproteome profiling allows the identification of critical determinants for virulence of livestock-associated and human-originated S. aureus ST398 strains

Extracellular Proteome and Citrullinome of the Oral Pathogen Porphyromonas gingivalis

Porphyromonas gingivalis is an oral pathogen associated with the inflammatory disease periodontitis. Periodontitis and P. gingivalis have been associated with rheumatoid arthritis. One of the hallmarks of rheumatoid arthritis is the loss of tolerance against citrullinated proteins. Citrullination is a post translational modification of arginine residues, leading to a change in structure and function of the respective protein. This modification, which is catalyzed by peptidylarginine deiminases (PADs), plays a role in several physiological processes in the human body. Interestingly, P. gingivalis secretes a citrullinating enzyme, known as P. gingivalis PAD (PPAD), which targets bacterial and human proteins. Because the extent of P. gingivalis protein citrullination by PPAD was not yet known, the present study was aimed at identifying the extracellular proteome and citrullinome of P. gingivalis. To this end, extracellulai proteins of two reference strains, two PPAD-deficient mutants, and three clinical isolates of P. gingivalis were analyzed by mass spectrometry. The results uncovered substantial heterogeneity in the extracellular proteome and citrullinome of P. gingivalis, especially in relation to the extracellular detection of typical cytoplasmic proteins. In contrast, the major virulence factors of P. gingivalis were identified in all investigated isolates, although their citrullination was shown to vary. This may be related to post-translational processing of the PPAD enzyme. Altogether, our findings focus attention on the possible roles of 6 to 25 potentially citrullinated proteins, especially the gingipain RgpA, in periodontitis and rheumatoid arthritis.</p

Quadratic forms of dimension 8 with trivial discrimiand and Clifford algebra of index 4

Izhboldin and Karpenko proved in 2000 that any quadratic form of dimension 8 with trivial discriminant and Clifford algebra of index 4 is isometric to the transfer, with respect to some quadratic \'etale extension, of a quadratic form similar to a 2-fold Pfister form. We give a new proof of this result, based on a theorem of decomposability for degree 8 and index 4 algebras with orthogonal involution

An ancient family of mobile genomic islands introducing cephalosporinase and carbapenemase genes in Enterobacteriaceae

The exchange of mobile genomic islands (MGIs) between microorganisms is often mediated by phages, which may provide benefits to the phage's host. The present study started with the identification of Enterobacter cloacae, Klebsiella pneumoniae and Escherichia coli isolates with exceptional cephalosporin and carbapenem resistance phenotypes from patients in a neonatal ward. To identify possible molecular connections between these isolates and their β-lactam resistance phenotypes, the respective bacterial genome sequences were compared. This unveiled the existence of a family of ancient MGIs that were probably exchanged before the species E. cloacae, K. pneumoniae and E. coli emerged from their common ancestry. A representative MGI from E. cloacae was named MIR17-GI, because it harbors the novel β-lactamase gene variant blaMIR17. Importantly, our observations show that the MIR17-GI-like MGIs harbor genes associated with high-level resistance to cephalosporins. Among them, MIR17-GI stands out because MIR17 also displays carbapenemase activity. As shown by mass spectrometry, the MIR17 carbapenemase is among the most abundantly expressed proteins of the respective E. cloacae isolate. Further, we show that MIR17-GI-like islands are associated with integrated P4-like prophages. This implicates phages in the spread of cephalosporin and carbapenem resistance amongst Enterobacteriaceae. The discovery of an ancient family of MGIs, mediating the spread of cephalosporinase and carbapenemase genes, is of high clinical relevance, because high-level cephalosporin and carbapenem resistance have serious implications for the treatment of patients with enterobacteriaceal infections

Connections between Exoproteome Heterogeneity and Virulence in the Oral Pathogen Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterial pathogen associated with severe periodontitis and nonoral diseases. Clinical isolates of A. actinomycetemcomitans display a rough (R) colony phenotype with strong adherent properties. Upon prolonged culturing, nonadherent strains with a smooth (S) colony phenotype emerge. To date, most virulence studies on A. actinomycetemcomitans have been performed with S strains of A. actinomycetemcomitans, whereas the virulence of clinical R isolates has received relatively little attention. Since the extracellular proteome is the main bacterial reservoir of virulence factors, the present study was aimed at a comparative analysis of this subproteome fraction for a collection of R isolates and derivative S strains, in order to link particular proteins to the virulence of A. actinomycetemcomitans with serotype b. To assess the bacterial virulence, we applied different infection models based on larvae of the greater wax moth Galleria mellonella, a human salivary gland-derived epithelial cell line, and freshly isolated neutrophils from healthy human volunteers. A total number of 351 extracellular A. actinomycetemcomitans proteins was identified by mass spectrometry, with the S strains consistently showing more extracellular proteins than their parental R isolates. A total of 50 known extracellular virulence factors was identified, of which 15 were expressed by all investigated bacteria. Importantly, the comparison of differences in exoproteome composition and virulence highlights critical roles of 10 extracellular proteins in the different infection models. Together, our findings provide novel clues for understanding the virulence of A. actinomycetemcomitans and for development of potential preventive or therapeutic avenues to neutralize this important oral pathogen. IMPORTANCE Periodontitis is one of the most common inflammatory diseases worldwide, causing high morbidity and decreasing the quality of life of millions of people. The bacterial pathogen Aggregatibacter actinomycetemcomitans is strongly associated with aggressive forms of periodontitis. Moreover, it has been implicated in serious nonoral infections, including endocarditis and brain abscesses. Therefore, it is important to investigate how A. actinomycetemcomitans can cause disease. In the present study, we applied a mass spectrometry approach to make an inventory of the virulence factors secreted by different clinical A. actinomycetemcomitans isolates and derivative strains that emerged upon culturing. We subsequently correlated the secreted virulence factors to the pathogenicity of the investigated bacteria in different infection models. The results show that a limited number of extracellular virulence factors of A. actinomycetemcomitans have central roles in pathogenesis, indicating that they could be druggable targets to prevent or treat oral disease