The Challenge of <i>Pneumocystis carinii</i> Culture1

Sloand, Elaine M.; Laughon, Barbara E.; Armstrong, Martine Y. K.; Bartlett, Marilyn S.; Blumenfeld, Walter; Cushion, Melanie T.; Kalica, Anthony R.; Kovacs, Joseph A.; Martin, William J.; Pitt, Elisabeth; Pesanti, Edward L.; Richards, Frederic M.; Rose, Richard M.; Walzer, Peter D.

doi:10.1111/j.1550-7408.1993.tb04902.x

Cited by 90 publications

(54 citation statements)

References 19 publications

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“…Other limitations of these studies include the possibility that the observed response of human AMs to rodent-derived Pneumocystis may reflect in part the origin of the organisms. Unfortunately, in the absence of reliable culturing methods, Pneumocystis of human origin are not available (Sloand et al, 1993). However, the recent observation that rat-derived Pneumocystis elicit similar chemokine and NF-B responses comparing human AMs to rat AMs in part validates the use of this model (Zhang et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

Cdc42 and RhoB Activation Are Required for Mannose Receptor-mediated Phagocytosis by Human Alveolar Macrophages

Zhang

Zhu

et al. 2005

MBoC

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Human alveolar macrophages (AMs) phagocytose Pneumocystis (Pc) organisms predominantly through mannose receptors, although the molecular mechanism mediating this opsonin-independent process is not known. In this study, using AMs from healthy individuals, Pc phagocytosis was associated with focal F-actin polymerization and Cdc42, Rac1, and Rho activation in a time-dependent manner. Phagocytosis was primarily dependent on Cdc42 and RhoB activation (as determined by AM transfection with Cdc42 and RhoB dominant-negative alleles) and mediated predominantly through mannose receptors (as determined by siRNA gene silencing of AM mannose receptors). Pc also promoted PAK-1 phosphorylation, which was also dependent on RhoGTPase activation. HIV infection of AMs (as a model for reduced mannose receptor expression and function) was associated with impaired F-actin polymerization, reduced Cdc42 and Rho activation, and markedly reduced PAK-1 phosphorylation in response to Pc organisms. In healthy AMs, Pc phagocytosis was partially dependent on PAK activation, but dependent on the Rho effector molecule ROCK. These data provide a molecular mechanism for AM mannose receptor-mediated phagocytosis of unopsonized Pc organisms that appears distinct from opsonin-dependent phagocytic receptors. Reduced AM mannose receptor-mediated Cdc42 and Rho activation in the context of HIV infection may represent a mechanism that contributes to the pathogenesis of opportunistic pneumonia.

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Section: Discussionmentioning

confidence: 99%

Cdc42 and RhoB Activation Are Required for Mannose Receptor-mediated Phagocytosis by Human Alveolar Macrophages

Zhang

Zhu

et al. 2005

MBoC

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“…Since the organism cannot be readily grown in vitro, the traditional quantification method for P. carinii is enumeration of organisms by microscopic examination, which is difficult and cumbersome due to the organism's growth in clusters and the presence of different morphological forms (14,19). Different stains vary in their sensitivities of detection as well as in their abilities to detect the different forms of P. carinii.…”

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confidence: 99%

“…carinii, the common subspecies infecting the immunosuppressed laboratory rat, which is the most commonly used in vivo model of PCP (2,19). We then assessed this new assay during studies attempting to establish the continuous axenic culture system recently described by Merali et al (14).…”

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confidence: 99%

Development of a Rapid Real-Time PCR Assay for Quantitation ofPneumocystis cariniif. sp.carinii

Larsen

Kovacs

Stock³

et al. 2002

J Clin Microbiol

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A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect <5 copies of a plasmid standard per tube. It was reproducibly quantitative (r ‫؍‬ 0.99) over 6 log values for standards containing >5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r ‫؍‬ 0.99). The assay was applied to a recently reported in vitro axenic cultivation system for P. carinii and confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease in P. carinii DNA that exceeded the expected decrease due to dilution of the inoculum upon transfer. In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical, such as in vitro antimicrobic susceptibility testing or in vivo immunopathological experiments.

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“…Recently, P. carinii has been detected by PCR DNA amplification of blood from rats and human patients with PCP and with disseminated pneumocystosis, thus helping to answer a number of important questions about the pathogenesis of P. carinii infection.2i , Considering the potential importance of detection of the organism in the blood, a culture method was used which has been employed extensively for the short-term culture of rat-or human-derived P. carinii. 17, 18 In the current study, the cell line was infected with a different inoculum source, which consisted of PBMC from human patients with P. carinii infection instead of conventional inocula.…”

Section: Discussionmentioning

confidence: 99%

Evidence of Pneumocystis carinii in cell line cultures infected with peripheral blood mononuclear cells isolated from AIDS patients with P. carinii pneumonia

Contini¹,

Mastrantoni²,

Romani³

et al. 1995

Journal of Medical Microbiology

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Summary. The detection of Pneumocystis carinii was investigated in an in-vitro system consisting of a human lung epithelial cell line (A-549) inoculated with infected peripheral blood mononuclear cells (PMBC) from HIV-infected patients with proven or suspected P. carinii pneumonia (PCP), and from HIV-negative patients with other lung infections. Supernates from cultures were sampled daily and evaluated for the presence of P. carinii by Giemsa and immunofluorescence staining. P. carinii was isolated from 98 (95.1 YO) of 103 culture supernate samples from patients with proven pneumocystosis and 45 (66.1 %) of 68 from patients with suspected PCP 40 or 72 h after PBMC inoculation. This system has been shown to support the growth of P. carinii but did not seem to be adequate for the production of large numbers of organisms, although long-term survival in vitro for up to 3 weeks was observed. Recovery of P. carinii from infected PBMC strongly supports previous observations about its ability to disseminate haematogenously and could represent a further advance in understanding the pathogenesis and diagnosis of PCP.

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The Challenge of Pneumocystis carinii Culture1

Cited by 90 publications

References 19 publications

Cdc42 and RhoB Activation Are Required for Mannose Receptor-mediated Phagocytosis by Human Alveolar Macrophages

Cdc42 and RhoB Activation Are Required for Mannose Receptor-mediated Phagocytosis by Human Alveolar Macrophages

Development of a Rapid Real-Time PCR Assay for Quantitation ofPneumocystis cariniif. sp.carinii

Evidence of Pneumocystis carinii in cell line cultures infected with peripheral blood mononuclear cells isolated from AIDS patients with P. carinii pneumonia

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