Histochemical observations on Pneumocystis carinii: selective demonstration of honeycomb forms.

Histochemical investigations of pulmonary lesions indicated selective coloration of membranes of honeycomb stages of Pneumocystis carinii by the periodic acid--sodium bisulfite--resorcin-fuchsin reaction for basement membranes; mucus, fibrin and other deposits in respiratory pathways did not react. These membranes were colored selectively also by the picro-Sirius Red F3BA method for collagens; fungi in tissues from patients with candidiasis remained unstained. For simultaneous demonstration of honeycomb and cyst forms of Pneumocystis carinii, sections were prestained with Grocott's modification of Gomori's methenamine-silver nitrate technic and then treated with the periodic acid-Schiff (PAS) or picro-Sirius Red F3BA reaction. In contrast to other Gram-positive microorganisms, cysts of Pneumocystis carinii were immediately decolorized by acetone-ether mixtures; this indicates differences in the mode of dye binding. Frequently, only one stage of Pneumocystis carinii was found in a given area. Hence a combination of reactions showing different stages is recommended for studies of small tissue samples.     

Ultrastructural observations on the attachment of Pneumocystis carinii in vitro.

Pneumocystis carinii trophozoites grow in vivo in close contact with host cells. The attachment of Pneumocystis to the lung cells seems to be a critical step in the parasite's development. Up to now, the contact of Pneumocystis with mammalian tissue culture cells was shown using light and scanning electron microscopy. The methods are not sufficient to observed in detail the parasite-feeder cell area of contact. In this work, the attachment of Pneumocystis trophozoites to feeder cells was examined in serial sections using transmission electron microscopy. When the contact of a trophozoite with a feeder cell took place, the development of filopodia penetrating deeply into invaginations of the feeder cell plasma membrane was observed. Then, the apical tips of filopodia become bulged anchoring the parasite to the feeder cell. The behaviour of Pneumocystis in feeder cell cultures is compared to that of the parasite in other in vitro or in vivo experimental models.     

Ploidy of cell-sorted trophic and cystic forms of Pneumocystis carinii

Once regarded as an AIDS-defining illness, Pneumocystis pneumonia (PcP) is nowadays prevailing in immunocompromised HIV-negative individuals such as patients receiving immunosuppressive therapies or affected by primary immunodeficiency. Moreover, Pneumocystis clinical spectrum is broadening to non-severely-immunocompromised subjects who could be colonized by the fungus while remaining asymptomatic for PcP, thus being able to transmit the infection by airborne route to susceptible hosts. Although the taxonomical position of the Pneumocystis genus has been clarified, several aspects of its life cycle remain elusive such as its mode of proliferation within the alveolus or its ploidy level. As no long-term culture model exists to grow Pneumocystis organisms in vitro, an option was to use a model of immunosuppressed rat infected with Pneumocystis carinii and sort life cycle stage fractions using a high-through-put cytometer. Subsequently, ploidy levels of the P. carinii trophic and cystic form fractions were measured by flow cytometry. In the cystic form, eight contents of DNA were measured thus strengthening the fact that each mature cyst contains eight haploid spores. Following release, each spore evolves into a trophic form. The majority of the trophic form fraction was haploid in our study. Some less abundant trophic forms displayed two contents of DNA indicating that they could undergo (i) mating/fusion leading to a diploid status or (ii) asexual mitotic division or (iii) both. Even less abundant trophic forms with four contents of DNA were suggestive of mitotic divisions occurring following mating in diploid trophic forms. Of interest, was the presence of trophic forms with three contents of DNA, an unusual finding that could be related to asymmetrical mitotic divisions occurring in other fungal species to create genetic diversity at lower energetic expenses than mating. Overall, ploidy data of P. carinii life cycle stages shed new light on the complexity of its modes of proliferation.     

Immunopathogenesis of Pneumocystis carinii pneumonia

Pneumocystis carinii pneumonia (PCP) is a life-threatening infection that occurs in immunocompromised individuals, particularly those with advanced human immunodeficiency virus (HIV) infection. Interestingly, morbidity and mortality is related to the underlying cause of immunosuppression, with AIDS patients faring better than oncology patients for example. In addition, the prognosis of PCP has been correlated with markers of inflammation rather than with organism numbers. There is now increasing evidence that lung damage occurring during PCP is a result of the type and extent of the host inflammatory response to P. carinii rather than a result of direct damage by the organism. This review will discuss the experimental and clinical data demonstrating how the host-mediated inflammatory response to infection with P. carinii determines the ultimate outcome of PCP. A better understanding of the pathophysiology of PCP should lead to the development of improved therapies for the treatment of PCP.     

Diagnosis of Pneumocystis carinii pneumonia

Pneumocystis carinii is the prime opportunistic pathogen of our time, the leading cause of fatal pneumonia in the increasing number of immunosuppressed subjects encountered on oncology and transplant programmes' and in subjects with the acquired immuno-deficiency syndrome (AIDS).     

Uptake and Metabolism of L-Serine by Pneumocystis carinii carinii

ABSTRACT. Serine is an important amino acid that is utilized in the biosyntheses of proteins and lipids. It is directly incorporated into the head group of phosphatidylserine, which in turn can be converted to other phospholipids. Also, it is required for the formation of long chain bases, precursors of sphingolipids. Uptake and incorporation of radiolabeled serine into both lipids and acid-precipitable material were demonstrated in Pneumocystis carinii carinii organism preparations freshly isolated from infected rat lungs. Radioactivity in proteins was about double that observed in lipids. Liquid scintillation spectrometry of metabolically radiolabeled lipids separated by thin-layer chromatography showed 53% of the total radioactivity were in phosphatidylserine, 12% in phosphatidylethanolamine, 24% in ceramides, and 11% in long chain bases and other compounds. Four long chain bases were detected by thin-layer chromatography in hydrolyzed P. carinii ceramides metabolically labeled with radioactive serine. Phytosphingosine and dihydrosphingosine were tentatively identified by their migrations on thin-layer plates. Radiolabeled ethanolamine was incorporated into P. carinii phosphatidylethanolamine, but relatively low incorporation of radiolabeled choline into phosphatidylcholine occurred. The observations made in this study indicated that P. carinii has the biosynthetic capacity to metabolize phospholipid head groups and to de novo synthesize sphingolipids. L-Cycloserine and β-CI-D-alanine, inhibitors of long chain base synthesis, reduced the incorporation of serine into P. carinii long chain bases and ceramides, which supported the conclusion that the pathogen synthesizes sphingolipids.