Modulation of the Host Interferon Response and ISGylation Pathway by B. pertussis Filamentous Hemagglutinin

Bordetella pertussis filamentous hemagglutinin (FHA) is a surface-associated and secreted protein that serves as a crucial adherence factor, and displays immunomodulatory activity in human peripheral blood mononuclear cells (PBMCs). In order to appreciate more fully the role of secreted FHA in pathogenesis, we analyzed FHA-induced changes in genome-wide transcript abundance in human PBMCs. Among the 683 known unique genes with greater than 3-fold change in transcript abundance following FHA treatment, 125 (18.3%) were identified as interferon (IFN)-regulated. Among the latter group were genes encoding several members of the IFN type I response, as well as 3 key components of the ISGylation pathway. Using real-time RT-PCR, we confirmed FHA-associated increases in transcript abundance for the genes encoding ubiquitin-like protein, ISG15, and its specific protease USP18. Western-blot analysis demonstrated the presence of both, free ISG15 and several ISGylated conjugates in FHA-stimulated PBMC lysates, but not in unstimulated cells. Intracellular FACS analysis provided evidence that monocytes and a natural killer-enriched cell population were the primary producers of ISG15 in PBMCs after FHA stimulation. Our data reveal previously-unrecognized effects of B. pertussis FHA on host IFN and ISGylation responses, and suggest previously-unsuspected mechanisms by which FHA may alter the outcome of the host-pathogen interaction

Entwicklung von Strategien zur Kontrolle von Lupinenblattrandkäfern (Sitona spp.) im integrierten und ökologischen Lupinenanbau

Das Ziel des Projekts SILU war es, Strategien zur Regulierung der Lupinenblattrandkäferarten zu erarbeiten und zur Praxisreife zu führen, die sowohl im integrierten als auch im ökologischen Landbau zur Anwendung kommen können. Dadurch soll die Anbausicherheit von Lupinen (Lupinus angustifolius), die durch Schäden der Lupinenblattrandkäfer (Sitona gressorius und S. griseus) stark eingeschränkt wird, verbessert werden. In vier Versuchsjahren wurden Daten zur Blattrandkäferaktivität sowie den auftretenden Schäden in Lupinen gesammelt. Aus den Sammeldaten wurde ein Entsscheidungshilfemodell (SIMSILU) entwickelt, welches erstmals einen optimierten Behandlungszeitpunkt zur Bekämpfung der Käfer definiert. Der richtige Zeitpunkt der Insektizidapplikation ist die Grundlage für eine effektive Kontrolle der Blattrandkäfer. In zukünftigen Versuchen wird das Modell erprobt, dabei werden unterschiedliche Insektizide getestet, u.a. biologische Präparate. In Fütterungsversuchen unter Laborbedingungen konnte gezeigt werden, dass NeemAzal eine fertilitätseinschränkende Wirkung auf S. gressorius hat. In Freilandversuchen muss diese Wirkung bestätigt werden

Entwicklung von Strategien zur Kontrolle von Lupinenblattrandkäfern (Sitona spp.) im integrierten und ökologischen Lupinenanbau

Das Ziel des Projekts SILU war es, Strategien zur Regulierung der Lupinenblattrandkäferarten zu erarbeiten und zur Praxisreife zu führen, die sowohl im integrierten als auch im ökologischen Landbau zur Anwendung kommen können. Dadurch soll die Anbausicherheit von Lupinen (Lupinus angustifolius), die durch Schäden der Lupinenblattrandkäfer (Sitona gressorius und S. griseus) stark eingeschränkt wird, verbessert werden. In vier Versuchsjahren wurden Daten zur Blattrandkäferaktivität sowie den auftretenden Schäden in Lupinen gesammelt. Aus den Sammeldaten wurde ein Entsscheidungshilfemodell (SIMSILU) entwickelt, welches erstmals einen optimierten Behandlungszeitpunkt zur Bekämpfung der Käfer definiert. Der richtige Zeitpunkt der Insektizidapplikation ist die Grundlage für eine effektive Kontrolle der Blattrandkäfer. In zukünftigen Versuchen wird das Modell erprobt, dabei werden unterschiedliche Insektizide getestet, u.a. biologische Präparate. In Fütterungsversuchen unter Laborbedingungen konnte gezeigt werden, dass NeemAzal eine fertilitätseinschränkende Wirkung auf S. gressorius hat. In Freilandversuchen muss diese Wirkung bestätigt werden

Separate assessment of gluteus medius and minimus: B-mode or M-mode ultrasound?

The hip abductors gluteus medius (Gmed) and minimus (Gmin) differ slightly in function and how they are affected by hip joint pathology. A separate assessment of Gmed and Gmin is feasible by ultrasound (US) imaging. B-mode and M-mode US can be used to measure muscle thickness. Two B- and two M-mode scans of Gmed and Gmin thickness were taken in relaxation on 16 asymptomatic volunteers, repeated within 4 days on 11 subjects. Three types of intra-rater reliability of muscle thickness measurements were examined: (1) within-session reliability comparing two scans from the same session, (2) between-days reliability comparing thickness from two scanning occasion within 4 days and (3) reliability of taking thickness measurements by re-measuring the same US scans after 1 week. Thickness measurements on B- and M-mode images provided ICC3,1 >0.96 for within-session reliability. ICC3,k >0.89 for between-days reliability and ICC3,1 >0.85 for re-reading the same scans were estimated. Minimal detectable changes >1.0 mm within-session, >2.4 mm between-days and >1.7 mm for re-reading scans indicated that small thickness changes are not detectable. The investigation suggests a slight advantage for fascia recognition in B-mode and the advantage of visual control of muscle relaxation in M-mode

Muscle thickness measurements to estimate gluteus mediusand minimus activity levels

The clinical assessment of gluteus medius and minimus force sharing requires non-invasive measurements of individual activity levels. Do ultrasound measurements of change of muscle thickness substitute invasive electromyography (EMG)? Isometric hip abduction in 20–80% maximal voluntary isometric contraction (MVIC) was measured using dynamometry, M-mode ultrasound for gluteus medius and minimus thickness and EMG using (1) surface electrodes on gluteus medius, n = 15, (2) fine-wire electrodes in deep gluteus medius and minimus, n = 6. Gluteus medius thickened by 5.0 (SD 2.5) mm at 80% MVIC while gluteus minimus thickness was constant in the surface EMG study and decreased by 1.6 (SD 1.6) mm at the more ventral location in the fine-wire EMG study. Thickness change of gluteus medius enabled prediction of torque (r2 0.66) and of surface EMG amplitude (r2 0.57). Surface EMG enabled higher torque prediction (r2 0.84) than thickness change. Thickness change of gluteus minimus did not enable a practically relevant estimation of torque production. Ultrasound examination revealed a differential thickening behaviour of gluteus medius and minimus which enabled estimation of isometric torque production only for gluteus medius but with lower precision than surface EMG

Gene Expression Analysis Reveals New Possible Mechanisms of Vancomycin-Induced Nephrotoxicity and Identifies Gene Markers Candidates

Vancomycin, one of few effective treatments against methicillin-resistant Staphylococcus aureus, is nephrotoxic. The goals of this study were to (1) gain insights into molecular mechanisms of nephrotoxicity at the genomic level, (2) evaluate gene markers of vancomycin-induced kidney injury, and (3) compare gene expression responses after iv and ip administration. Groups of six female BALB/c mice were treated with seven daily iv or ip doses of vancomycin (50, 200, and 400 mg/kg) or saline, and sacrificed on day 8. Clinical chemistry and histopathology demonstrated kidney injury at 400 mg/kg only. Hierarchical clustering analysis revealed that kidney gene expression profiles of all mice treated at 400 mg/kg clustered with those of mice administered 200 mg/kg iv. Transcriptional profiling might thus be more sensitive than current clinical markers for detecting kidney damage, though the profiles can differ with the route of administration. Analysis of transcripts whose expression was changed by at least twofold compared with vehicle saline after high iv and ip doses of vancomycin suggested the possibility of oxidative stress and mitochondrial damage in vancomycin-induced toxicity. In addition, our data showed changes in expression of several transcripts from the complement and inflammatory pathways. Such expression changes were confirmed by relative real-time reverse transcription–polymerase chain reaction. Finally, our results further substantiate the use of gene markers of kidney toxicity such as KIM-1/Havcr1, as indicators of renal injury

Chlorination and oxidation of the extracellular matrix protein laminin and basement membrane extracts by hypochlorous acid and myeloperoxidase

Basement membranes are specialized extracellular matrices that underlie arterial wall endothelial cells, with laminin being a key structural and biologically-active component. Hypochlorous acid (HOCl), a potent oxidizing and chlorinating agent, is formed in vivo at sites of inflammation via the enzymatic action of myeloperoxidase (MPO), released by activated leukocytes. Considerable data supports a role for MPO-derived oxidants in cardiovascular disease and particularly atherosclerosis. These effects may be mediated via extracellular matrix damage to which MPO binds. Herein we detect and quantify sites of oxidation and chlorination on isolated laminin-111, and laminin in basement membrane extracts (BME), by use of mass spectrometry. Increased modification was detected with increasing oxidant exposure. Mass mapping indicated selectivity in the sites and extent of damage; Met residues were most heavily modified. Fewer modifications were detected with BME, possibly due to the shielding effects. HOCl oxidised 30 (of 56 total) Met and 7 (of 24) Trp residues, and chlorinated 33 (of 99) Tyr residues; 3 Tyr were dichlorinated. An additional 8 Met and 10 Trp oxidations, 14 chlorinations, and 18 dichlorinations were detected with the MPO/H2O2/Cl- system when compared to reagent HOCl. Interestingly, chlorination was detected at Tyr2415 in the integrin-binding region; this may decrease cellular adhesion. Co-localization of MPO-damaged epitopes and laminin was detected in human atherosclerotic lesions. These data indicate that laminin is extensively modified by MPO-derived oxidants, with structural and functional changes. These modifications, and compromised cell-matrix interactions, may promote endothelial cell dysfunction, weaken the structure of atherosclerotic lesions, and enhance lesion rupture. Keywords: Extracellular matrix, Hypochlorous acid, Laminin, Protein oxidation, 3-chlorotyrosine, Myeloperoxidas