Infections caused by Streptococcus pneumoniae, a commensal of the human nasopharynx, have accounted historically for more morbidity and mortality than any other bacterium. This bacterium is a major cause of community-acquired pneumonia leading in a multitude of cases to pneumococcal sepsis, and is also a significant cause of meningitis, bacteraemia, and otitis media in children. Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are sentinel features of septic organ failure in patients with pneumogenic sepsis.
The major objective of this study is the description of cellular and molecular events that regulate local and systemic inflammatory responses to pneumococcus and its haemolytic toxin, pneumolysin, during infection in mice of resistant and susceptible phenotypes.
In this project, infection studies with S. pneumoniae and pneumolysin using resistant and susceptible mice to identify cell types that initiate and control septic infections due to pneumococcal infection, will be performed. Gene expression profiles of these relevant cells and of the infecting bacteria will be generated to provide clues as to the nature of these cells and type of immune response induced, and the pneumococcal genes expressed during infection. These studies will pinpoint additional genetic loci that control the course of pneumococcal infection. Pharmacological intervention therapies with the mTNF-alpha-derived peptide (mLtip), which has been shown to improve dynamic lung compliance and airway resistance as well as a resolution of alveolar edema will be conducted and therapeutic outcome monitored using whole genome microarrays. Finally, genes identified in this study will be evaluated as potential biomarkers for S. pneumoniae-induced ALI.
Results/Project Status
The mouse model of intranasal/intratracheal pneumoccal infection was established together with attendant techniques that examine for edema formation and physiological correlates of lung failure. We have also established techniques for the isolation of eukaryotic and bacterial RNA from infected lung tissue and blood for microarray analysis.
In NGFN-2, an in-vitro model for the application of bacteria and / or bacterial toxins such as listeriolysin or pneumolysin to bone marrow macrophages was established. Gene expression profiles following exposure to several different toxin concentrations and varying duration of treatment were assessed by microarray analyses of total RNA with the CodeLink-Bioarray-system. The analyses have revealed an unexpectedly large adaptive program to toxin treatment that has both common and distinctive features for the respective toxin applied. Biological processes that are most strongly affected include a plethora of transcriptional factors that modulate immunity, signal transduction pathways and cellular transport processes.
Another experimental model created in NGFN-2 allowed the ex vivo infection of isolated peripheral (human) monocytes with gram positive bacterial pathogens (Streptococcus pneumoniae, Staphylococcus aureus, Listeria monocyto¬genes) from patient isolates. Total RNA from these cells was also processed with the CodeLink-Bioarray-system and first expression profiles have been gained. This approach may bring the possibility of an early detection of a specific response to a local infection at a timepoint when application of serologic diagnostic methods is not possible.
The effects of the intratracheal instillation and the intravenous application of pneumolysin were studied in the ex-vivo ventilated / perfused mouse lung model. Intratracheal application induced a prompt vasoconstrictory response and lung edema formation, whereas the intraveneous application had no effect on these parameters. These effects were found to be reversed by the pharmacological intervention with the mTNF-alpha-derived peptide (mLtip) that improved dynamic lung compliance and airway resistance after alveolar flooding. There was also a significant reduction in lung edema. In collaboration with Professor R. Lucas (University of Applied Sciences, Krems, Austria) the therapeutic role of the mLtip-recombinant protein will be studied in a mouse model of pneumococcal sepsis. We have initiated studies to examine for the recuperative gene expression signature that is induced by application of the therapeutic recombinant protein mLtip to lung epithelial cells.
Outlook
The studies presented here use a combination of gene expression profiling and molecular analysis to investigate the molecular cross talk between bacterial toxins and host defence mechanisms at the cellular level and in infection models. The analysis of these data will provide the basis for further experiments designed to monitor the changes in functional pathways both in tissue and cell preparations of the lung and periphery in order to understand the coordination of the immune (inflammatory) reactions. Along with the comparison of these expression profiles from tissues of resistant and susceptible animals, these data should point to relevant genes suitable for pharmaceutical therapy. Moreover, the existing in vitro and in vivo models provide an optimal platform for testing new hypothetical drug substances, expected to prevent the hyperinflammatory effects of streptococcal infection and to enhance the resistant phenotype.
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