Toxin-induced necroptosis is a major mechanism of Staphylococcus aureus lung damage.

Staphylococcus aureus USA300 strains cause a highly inflammatory necrotizing pneumonia. The virulence of this strain has been attributed to its expression of multiple toxins that have diverse targets including ADAM10, NLRP3 and CD11b. We demonstrate that induction of necroptosis through RIP1/RIP3/MLKL signaling is a major consequence of S. aureus toxin production. Cytotoxicity could be prevented by inhibiting either RIP1 or MLKL signaling and S. aureus mutants lacking agr, hla or Hla pore formation, lukAB or psms were deficient in inducing cell death in human and murine immune cells. Toxin-associated pore formation was essential, as cell death was blocked by exogenous K+ or dextran. MLKL inhibition also blocked caspase-1 and IL-1ß production, suggesting a link to the inflammasome. Rip3(-/-) mice exhibited significantly improved staphylococcal clearance and retained an alveolar macrophage population with CD200R and CD206 markers in the setting of acute infection, suggesting increased susceptibility of these leukocytes to necroptosis. The importance of this anti-inflammatory signaling was indicated by the correlation between improved outcome and significantly decreased expression of KC, IL-6, TNF, IL-1α and IL-1ß in infected mice. These findings indicate that toxin-induced necroptosis is a major cause of lung pathology in S. aureus pneumonia and suggest the possibility of targeting components of this signaling pathway as a therapeutic strategy.

Staphylococcus aureus protein A mediates invasion across airway epithelial cells through activation of RhoA GTPase signaling and proteolytic activity.

Staphyococcus aureus and especially the epidemic methicillin-resistant S. aureus strains cause severe necrotizing pneumonia. The mechanisms whereby these organisms invade across the mucosal epithelial barrier to initiate invasive infection are not well understood. Protein A (SpA), a highly conserved and abundant surface protein of S. aureus, activates TNF receptor 1 and EGF receptor (EGFR) signaling cascades that can perturb the cytoskeleton. We demonstrate that wild-type S. aureus, but not spa mutants, invade across polarized airway epithelial cell monolayers via the paracellular junctions. SpA stimulated a RhoA/ROCK/MLC cascade, resulting in the contraction of the cytoskeleton. SpA(+) but not SpA(-) mutants stimulated activation of EGFR and along with subsequent calpain activity cleaved the membrane-spanning junctional proteins occludin and E-cadherin, facilitating staphylococcal transmigration through the cell-cell junctions. Treatment of polarized human airway epithelial monolayers with inhibitors of ROCK, EGFR, MAPKs, or calpain prevented staphylococcal penetration through the monolayers. In vivo, blocking calpain activity impeded bacterial invasion into the lung parenchyma. Thus, S. aureus exploits multiple receptors available on the airway mucosal surface to facilitate invasion across epithelial barriers.

Differences in diagnosis and treatment using telemedicine versus in-person evaluation of acute illness.

OBJECTIVE: We designed a telemedicine model for diagnosis of common, acute illness to compare telemedicine and in-person evaluations on reproducibility of diagnosis and treatment. METHODS: Subjects were seen by usual physicians in ambulatory settings. Subjects were also evaluated separately by experienced general pediatricians (study physicians), either in person or via telemedicine, based on random assignment. The primary measure of reproducibility was study physician agreement with usual physician on primary diagnosis. Analysis compared reproducibility for telemedicine versus in-person evaluations. Relevance of agreement on primary diagnosis was measured by comparing agreement on prescribed medications. RESULTS: Agreement on diagnosis of study physicians with usual physicians for the 492 visits studied was 89%. The difference in the proportion of visits with disagreements between telemedicine study and in-person study evaluations (13.8% vs 8.3%, respectively) bordered on significance (P = .051). Disagreement proportions for prescriptions were similar (32.2% vs 27.4%), however. Telemedicine evaluation for children with upper respiratory tract (URI)-ear symptoms involved unique technical requirements and clinical judgments. For this largest subgroup of 202 visits, disagreement on diagnosis for telemedicine occurred more often than for in-person evaluation (17.6 vs 6.3%, P < .02). For the remaining 290 visits, telemedicine and in-person study physicians disagreed on diagnosis about equally (11.5 vs 9.9%). CONCLUSIONS: Excluding the URI-ear group, reproducibility of telemedicine diagnosis did not differ from that of in-person diagnosis. For the URI-ear group, reproducibility of diagnosis by telemedicine and in-person evaluation varied significantly.

Effectiveness of telemedicine in replacing in-person evaluation for acute childhood illness in office settings.

For the purpose of reducing the social and economic burden imposed by common acute childhood illness, we developed a telemedicine model to enable diagnosis and treatment of illness episodes presenting in pediatric office settings. The study objective was to assess the effectiveness of this telemedicine model in replacing illness visits to traditional healthcare settings and to compare effectiveness of this model (base model) with that of alternative models including simple office laboratory tests and albuterol administration (simple model) or a complete complement of tests and procedures (extended model). Eligible subjects had an acute problem and were seen in the pediatric primary care practice or pediatric emergency department of the University of Rochester Medical Center. All subjects were seen by the setting's usual physician. Subjects were also evaluated, based on random assignment, by a study physician in person or by a study physician via telemedicine. Effectiveness was defined as completion of the visit to the point that diagnosis was made. Forms completed by study physicians, and standard medical records indicating the tests and procedures requested for the purpose of completing the visit, were used to identify the model used in completing the visit. Effectiveness (proportion of visits completed) of the base model was assessed and its effectiveness was compared to that of simple and extended telemedicine models. Among 520 randomized visits, 492 were evaluated by study physicians in person (253) or via telemedicine (239). Using the base model, study physicians completed 74.1% of visits via telemedicine compared to 76.7% for study physicians in person and 76.0% for usual physicians. The simple model increased completion rates substantially. Using this model, study physicians completed 84.9% of visits via telemedicine compared to 86.6% for study physicians in person and 85.2% for usual physicians. The extended model increased effectiveness in completing visits still more, with telemedicine study physicians completing 97.1% of visits compared to 96.8% for in-person study physicians and 100% for usual physicians. Approximately 85% of illness visits presenting to primary care pediatric practice could be completed using a telemedicine model that included only simple office laboratory testing and albuterol administration.