Name: characterization of inflammatory responses during intranasal colonization with streptococcus pneumoniae
Uploaded: 2014-01-17T13:00:00
Description: Watch this Scientific Journal Video about Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae at JoVE.com

The authors would like to thank Dr. Jeffery Weiser of the University of Pennsylvania for his gift of the clinical strains of Streptococcus pneumoniae. This work was funded by the Canadian Institutes for Health Research. CV was funded by a M. G. DeGroote fellowship and a fellowship from the Canadian Thoracic Society. This work was funded by the Ontario Lung Association and Canadian Institutes of Health Research (CIHR). Work in the Bowdish laboratory is supported in part by he Michael G. DeGroote Centre for Infectious Disease Research and the McMaster Immunology Research Centre.

Before you begin: all steps are done in a Biohazard Level 2 (BSL2) Biological Safety Cabinet (BSC) unless otherwise stated. Please ensure that you have obtained the appropriate Biohazard Approval for use of infectious bacterial pathogens as per institutional guidelines prior to initiation of the experiments. Additionally, please ensure that you have all the materials and reagents necessary to conduct the procedure prepared beforehand. Mice used in these experiments have included female C57BL/6 mice from ...

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In this study we presented detailed methods for the intranasal colonization of mice using a clinical isolate strain of Streptococcus pneumoniae and the subsequent isolation and characterization of the immune cells recruited to nasopharynx in response to the bacteria. We demonstrated how a bacterial inoculum can be cultured in nutrient-rich media and used to establish a colonization event in mice, which is initially restricted to the nasopharynx. We then showed how responding immune cell types that are recruited ...

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characterization of inflammatory responses during intranasal colonization with streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Figure 1 represents an overview schematic summarizing the main steps of the protocol. Figures 2-3 provide visualization of the microbiological methodology inherent to the protocols described herein. Figure 4 represents proper positioning of a mouse to perform an intranasal colonization, while Figure 5 depicts typically changes in weight of mice colonized with S. pneumoniae strain P1547. Figures 6-7 represent specific stages of t...

Watch this Scientific Journal Video about Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae at JoVE.com

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

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Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

A Murine Model of Group B Streptococcus Vaginal Colonization

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...