SummaryGuinea pigs are highly susceptible to Legionella pneumophila infection and therefore have been the preferred animal model for studies of legionellosis. In this study guinea pig infections revealed that the Legionella virulence factor Mip (macrophage infectivity potentiator) contributes to the bacterial dissemination within the lung tissue and the spread of Legionella to the spleen. Histopathology of infected animals, binding assays with components of the extracellular matrix (ECM), bacterial transmigration experiments across an artificial lung epithelium barrier, inhibitor studies and ECM degradation assays were used to elucidate the underlying mechanism of the in vivo observation. The Mip protein, which belongs to the enzyme family of FK506-binding proteins (FKBP), was shown to bind to the ECM protein collagen (type I, II, III, IV, V, VI). Transwell assays with L. pneumophila and recombinant Escherichia coli HB101 strains revealed that Mip enables these bacteria to transmigrate across a barrier of NCI-H292 lung epithelial cells and ECM (NCI-H292/ECM barrier). Mipspecific monoclonal antibodies and the immunosuppressants rapamycin and FK506, which inhibit the peptidyl prolyl cis/trans isomerase (PPIase) activity of Mip, were able to inhibit this transmigration. By using protease inhibitors we found that the penetration of the NCI-H292/ECM barrier additionally requires a serine protease activity. Degradation assays with 35 Slabelled ECM proteins supported the finding of a concerted action of Mip and a serine protease. The described synergism between the activity of the collagen binding Mip protein and the serine protease activity represents an entirely new mechanism for bacterial penetration of the lung epithelial barrier and has implications for other prokaryotic and eukaryotic pathogens.

Staphylococcus aureusis the most common cause of nosocomial infections. Multiple antibiotic resistance and severe clinical outcomes provide a strong rationale for development of immunoglobulin-based strategies. Traditionally, novel immunological approaches against bacterial pathogens involve antibodies directed against cell surface-exposed virulence-associated epitopes or toxins. In this study, we generated a monoclonal antibody targeting the housekeeping protein IsaA, a suggested soluble lytic transglycosylase ofS. aureus, and tested its therapeutic efficacy in two experimental mouse infection models. A murine anti-IsaA antibody of the IgG1 subclass (UK-66P) showed the highest binding affinity in Biacore analysis. This antibody recognized allS. aureusstrains tested, including hospital-acquired and community-acquired methicillin-resistantS. aureusstrains. Therapeutic efficacyin vivoin mice was analyzed using a central venous catheter-related infection model and a sepsis survival model. In both models, anti-IsaA IgG1 conferred protection against staphylococcal infection.Ex vivo, UK-66P activates professional phagocytes and induces highly microbicidal reactive oxygen metabolites in a dose-dependent manner, resulting in bacterial killing. The study provides proof of concept that monoclonal IgG1 antibodies with high affinity to the ubiquitously expressed, single-epitope-targeting IsaA are effective in the treatment of staphylococcal infection in different mouse models. Anti-IsaA antibodies might be a useful component in an antibody-based therapeutic for prophylaxis or adjunctive treatment of human cases ofS. aureusinfections.

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Topical application of elastase to induce arterial aneurysm formation is an emerging murine model of vascular disease. In the context of aortic abdominal aneurysm (AAA), angiotensin II infusion and porcine pancreatic elastase perfusion models are commonly used today. This study, therefore, compares matrix remodeling, inflammation, and angiogenesis as distinct features of aneurysms in two models treated with intra-/extraluminal elastase. C57BL/6 mice underwent intra-/extraluminal elastase application via laparotomy and were followed up for 4 weeks. Basic histology and immunohistochemistry were performed at different time points along with transmission electron microscopy, PCR analysis, TUNEL assays, and blood analysis. Both models did not differ in aneurysm growth rate, but they showed distinct features and results depending on the way of elastase application. Extraluminal aneurysm induction preserved endothelial cell function and elastic fibers but showed ongoing acute inflammation, mainly in the adventitia. The destruction of elastic layers followed by chronic inflammation was a characteristic of intraluminal elastase perfusion, as well as medial angiogenesis, a key feature in human AAA. Different animal models harbor different features of human AAA and must, therefore, be chosen wisely. External elastase application mimics an acute inflammatory aneurysm, whereas intraluminal elastase perfusion shows chronic inflammation with angiogenesis and endothelial destruction, thus better mimicking human disease.

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Heterogeneity of AAA vessel walls based on inflammatory morphology does not correlate with AAA diameter yet harbors specific implications for basic research and possible aneurysm detection.

Limited comprehension of aneurysm pathology has led to inconclusive results from clinical trials. miRNAs are key regulators of post-translational gene modification and are useful tools in elucidating key features of aneurysm pathogenesis in distinct entities of abdominal and popliteal aneurysms. Here, surgically harvested specimens from 19 abdominal aortic aneurysm (AAA) and 8 popliteal artery aneurysm (PAA) patients were analyzed for miRNA expression and histologically classified regarding extracellular matrix (ECM) remodeling and inflammation. DIANA-based computational target prediction and pathway enrichment analysis verified our results, as well as previous ones. miRNA-362, -19b-1, -194, -769, -21 and -550 were significantly down-regulated in AAA samples depending on degree of inflammation. Similar or inverse regulation was found for miR-769, 19b-1 and miR-550, -21, whereas miR-194 and -362 were unaltered in PAA. In situ hybridization verified higher expression of miR-550 and -21 in PAA compared to AAA and computational analysis for target genes and pathway enrichment affirmed signal transduction, cell-cell-interaction and cell degradation pathways, in line with previous results. Despite the vague role of miRNAs for potential diagnostic and treatment purposes, the number of candidates from tissue signature studies is increasing. Tissue morphology influences subsequent research, yet comparison of distinct entities of aneurysm disease can unravel core pathways.