Quantitative Real‐Time Polymerase Chain‐Reaction Assay Allows Characterization of<i>Pneumocystis</i>Infection in Immunocompetent Mice

Vestereng, Vibeke H.; Bishop, Lisa R.; Hernandez, Beatriz; Kutty, Geetha; Larsen, Helle Kiellberg; Kovacs, Joseph A.

doi:10.1086/382486

Cited by 48 publications

(76 citation statements)

References 15 publications

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“…This cohousing infection model results in infection of animals with predictable kinetics; infection of healthy animals peaks at ca. 35 to 42 days and is cleared by ϳ70 days (18,44). For each experiment, one to two cages housed exposed mice, and one cage housed unexposed mice.…”

Section: Methodsmentioning

confidence: 99%

“…For these studies, approximately equal numbers of IL-17A KO and C57BL/6 mice were cohoused with an infected seeder and sacrificed at days 35, days 56 to 58, and days 84 to 86, at which time the lungs, spleen, and serum were collected. Q-PCR was used to quantitate Pneumocystis organism load, and ELISA and proliferation assays were used to evaluate antibody and cell-mediated responses to Pneumocystis (44,45). There were 15 animals per strain in experiment 1 and 6 to 7 animals per strain in experiment 2.…”

Section: Methodsmentioning

confidence: 99%

“…DNA extraction from lung homogenates and subsequent quantitation of P. murina dihydrofolate reductase gene (dhfr) copies per mg of lung by means of a real-time PCR were performed as described previously (44). Since dhfr is a single-copy gene, the number of copies reflects the number of nuclei.…”

Section: Methodsmentioning

confidence: 99%

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Pulmonary Interleukin-17-Positive Lymphocytes Increase during Pneumocystis murina Infection but Are Not Required for Clearance of Pneumocystis

2017

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Pneumocystis remains an important pathogen of immunosuppressed patients, causing a potentially life-threatening pneumonia. Despite its medical importance, the immune responses required to control infection, including the role of interleukin-17 (IL-17), which is important in controlling other fungal infections, have not been clearly defined. Using flow cytometry and intracellular cytokine staining after stimulation with phorbol myristate acetate and ionomycin, we examined gamma interferon (IFN-␥), IL-4, IL-5, and IL-17 production by lung lymphocytes in immunocompetent C57BL/6 mice over time following infection with Pneumocystis murina. We also examined the clearance of Pneumocystis infection in IL-17A-deficient mice. The production of both IFN-␥ and IL-17 by pulmonary lymphocytes increased during infection, with maximum production at approximately days 35 to 40, coinciding with peak Pneumocystis levels in the lungs, while minimal changes were seen in IL-4-and IL-5-positive cells. The proportion of cells producing IFN-␥ was consistently higher than for cells producing IL-17, with peak levels of ϳ25 to 30% of CD3 ϩ T cells for the former compared to ϳ15% for the latter. Both CD4 ϩ T cells and ␥␦ T cells produced IL-17. Administration of anti-IFN-␥ antibody led to a decrease in IFN-␥-positive cells, and an increase in IL-5-positive cells, but did not impact clearance of Pneumocystis infection. Despite the increases in IL-17 production during infection, IL-17A-deficient mice cleared Pneumocystis infection with kinetics similar to C57BL/6 mice. Thus, while IL-17 production in the lungs is increased during Pneumocystis infection in immunocompetent mice, IL-17A is not required for control of Pneumocystis infection.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Pulmonary Interleukin-17-Positive Lymphocytes Increase during Pneumocystis murina Infection but Are Not Required for Clearance of Pneumocystis

2017

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“…This has been previously shown to result in infection of co-housed animals with predictable kinetics and infection of healthy animals peaking at 35-42 days [8]. We used this approach rather than intratracheal inoculation of organisms, as the latter, although commonly used in studies of Pneumocystis, will deliver a substantially larger organism load as a bolus and may induce immune responses that differ qualitatively or kinetically from those seen during naturally acquired infection [14].…”

Section: Methodsmentioning

confidence: 99%

Immune responses toPneumocystis murinaare robust in healthy mice but largely absent in CD40 ligand-deficient mice

Hernández-Novoa

Bishop

Logun

et al. 2008

Journal of Leukocyte Biology

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Pneumocystis is a pathogen of immunocompromised hosts but can also infect healthy hosts, in whom infection is rapidly controlled and cleared. Microarray methods were used to examine differential gene expression in the lungs of C57BL/6 and CD40 ligand knockout (CD40L-KO) mice over time following exposure to Pneumocystis murina. Immunocompetent C57BL/6 mice, which control and clear infection efficiently, showed a robust response to infection characterized by the up-regulation of 349 primarily immune response-associated genes. Temporal changes in the expression of these genes identified an early (Week 2), primarily innate response, which waned before the infection was controlled; this was followed by primarily adaptive immune responses that peaked at Week 5, which coincided with clearance of the infection. In conjunction with the latter, there was an increased expression of B cell-associated (Ig) genes at Week 6 that persisted through 11 weeks. In contrast, CD40L-KO mice, which are highly susceptible to developing severe Pneumocystis pneumonia, showed essentially no up-regulation of immune response-associated genes at Days 35-75. Immunohistochemical staining supported these observations by demonstrating an increase in CD4+, CD68+, and CD19+ cells in C57BL/6 but not CD40L-KO mice. Thus, the healthy host demonstrates a robust, biphasic response to infection by Pneumocystis; CD40L is an essential upstream regulator of the adaptive immune responses that efficiently control infection and prevent development of progressive pneumonia.

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“…Detection of nucleic acids by real-time PCR has emerged as an alternative approach to measurement of microbial burden in host tissues. This method is particularly valuable with microorganisms that cannot be propagated in artificial media or in tissue culture, such as Pneumocystis, for which PCR has replaced quantitative microscopy for enumeration of cysts (2,131). Nucleic acid amplification techniques also can be less cumbersome than tissue culture methods for enumerating viruses and chlamydiae (80,132) and for quantitation of organisms that grow very slowly on artificial media, such as Mycoplasma and Mycobacteria.…”

Section: The Next Practical Considerations: How To Get Information Outmentioning

confidence: 99%

Animal models of human pneumonia

Mizgerd¹,

Skerrett

2008

American Journal of Physiology-Lung Cellular and Molecular Phys

126

118

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Pneumonia is a medical and public health priority, and advances against this disease will require improved knowledge of biological mechanisms. Human pneumonia is modeled with experimental infections of animals, most frequently mice. Mouse models are leading to important discoveries relevant to pneumonia, but their limitations must be carefully considered. Several approaches to establishing pneumonia in mice have been developed, and each has specific strengths and weaknesses. Similarly, procedures for characterizing microbial and host responses to infection have unique advantages and disadvantages. Mice are not small humans, and the applicability of results from murine models to human disease depends on understanding the similarities and differences between species. Additional considerations such as mouse strain, microbe strain, and prior mouse-microbe interactions also influence the design and interpretation of experiments. Results from studies of pneumonia in animals, combined with complementary basic and translational studies, are elucidating mechanisms responsible for susceptibility to and pathophysiology of lung infection.

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Quantitative Real‐Time Polymerase Chain‐Reaction Assay Allows Characterization ofPneumocystisInfection in Immunocompetent Mice

Cited by 48 publications

References 15 publications

Pulmonary Interleukin-17-Positive Lymphocytes Increase during Pneumocystis murina Infection but Are Not Required for Clearance of Pneumocystis

Pulmonary Interleukin-17-Positive Lymphocytes Increase during Pneumocystis murina Infection but Are Not Required for Clearance of Pneumocystis

Immune responses toPneumocystis murinaare robust in healthy mice but largely absent in CD40 ligand-deficient mice

Animal models of human pneumonia

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