Ultrastructural Study of Pneumocystis carinii in Explant Cultures of Rabbit Lung and in Cultures with and without Feeder Cells

Pneumocystis carinii-parasitizcd lung explains were obtained from corticoid-lreated rabbits and maintained in vilro. Twenty-one days after the beginning of explant cultures, the ullrastructura! morphology of trophozoite, precyst and cyst forms was normal as compared to the in vivo ui-trastrucuirc of P. carinii from infected rabbit. However, after the 36th day, only altered forms of P. carinii were observed. Lung tissue showed only minor alterations. fntracytoplasmic lamellar inclusions were observed in type 2 alveolar cells from which they were released. While the total number of parasites increased approximately 4-fold from day 0 to day 41, trophozoite counts increased approximately 6 times. Pneumocystis cells from inocula and supcmaics of cultures with and without Vera cells showed important ullrastructural alterations.     

Ultrastructural localization of epitopes recognized by monoclonal antibodies produced to rat Pneumocystis carinii.

The binding sites of five monoclonal antibodies (MAb) developed against rat Pneumocystis carinii were examined at the ultrastructural level by using a post-embedding labeling method. Although all five MAb reacted with the pellicle of P. carinii, they were divided into two groups by localization of binding sites. The MAb 168.2.1, 174.2.1, and 215.2.1 reacted mainly with the electron-dense outer layer, whereas MAb 227.1.1 and 228.1.1 labeled both the outer dense layer and the middle lucent layer. With in the first group of MAb, no significant differences were observed in the reactivity patterns seen with the different stages of P. carinii. In the second group, however, the intensity of labeling of the electron-dense layer was higher in the precyst, cyst, and ruptured cyst stages than in the trophozoite stage. These latter results indicate that there may be an increase in antigen accumulation during development from the trophozoite to the cyst stages, or that antigens may be modified the development.     

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.     

Ultrastructural morphology and staining characteristics of Pneumocystis carinii in situ and from bronchoalveolar lavage

The ultrastructure of Pneumocystis carinii obtained from rats by bronchoalveolar lavage (BAL) was compared with organisms in situ. All developmental forms of the organism as seen in situ were present in the lavage fluid. Trophozoites in situ were adhered to type I epithelium, had smooth surfaces, and were interdigitated with the underlying epithelium. Nonadherent trophozoites in situ and trophozoites in lavage fluid were more pleomorphic and irregular in shape with tubular projections extending from all surfaces. Microtubular and nuclear details not reported elsewhere were observed. To enhance the ultrastructural detail of P. carinii obtained by lavage, phosphotungstic and tannic acid fixation, uranyl acetate en bloc staining, and acid phosphatase staining were performed. These techniques enhanced the visibility of membranes, mitochondria, nuclei, and vacuoles. With tannic acid, increased contrast of the organism's cell coat was obtained and differences in staining intensity and thickness related to developmental stages were observed. In lavage samples with few pneumocystis organisms or those specimens heavily contaminated with macrophages, erythrocytes, or other cellular debris, tannic acid allows for easier recognition as other lung materials do not show the same distinctive staining reaction. Lung sections observed after BAL showed intact but damaged epithelial surfaces devoid of organisms. No intracellular organisms were observed. BAL removes organisms from the alveolar lumen as well as adhered organisms and is a useful method for concentrating the various morphologic forms of P. carinii.     

Cultured Rat and Purified Human Pneumocystis carinii Stimulate Intra-but not Extracellular Free Radical Production in Human Neutrophils

The production of free radicals in human neutrophils was studied in both Pneumocystis carinii derived from cultures of L2 rat lung epithelial-like cells and Pneumocystis carinii purified from human lung. Using the cytochrome C technique, which selectively measured extracellular superoxide generation, hardly any free radical production was observed after stimulation with cultured rat-derived P. carinii. A chemiluminescence technique, which separately measured intra- and extracellular free radical production, was subsequently employed to differentiate the free radical generation. It was established that 1) P. carinii stimulated intra- but not extracellular free radical production in human neutrophils. 2) opsonized cultured rat-derived P. carinii stimulated human neutrophils to a strong intra-cellular response of superoxide production, and 3) opsonized P. carinii. purified from human lung also stimulated human neutrophils to produce intracellular free radicals.     

Attempts at in vitro cultivation of Pneumocystis carinii from human bronchoalveolar lavage fluids without feeder cells.

We attempted to cultivate Pneumocystis carinii obtained from two bronchoalveolar lavage fluids of AIDS patients with P. carinii pneumonia, in a system wherein cysteine and 2-mercaptoethanol were substituted for the feeder cells. The presence of P. carinii cysts was monitored for 11 days under conditions of continuous culture. Moderate increase in cyst forms was observed until day 11. Further study with this system would be required to determine if the observed increase in cyst numbers is reproducible and whether the cyst form is a response to adverse in vitro conditions or is a manifestation of growth.     

Pneumocystis carinii of the Common Shrew, Sorex araneus, Shows a Discrete Phenotype

ABSTRACT. We carried out an immunohistological and morphological study on Pneumocystis carinii originating from the common shrew, Sorex araneus . Immunologic properties were studied by applying two commercially available immunofluorescence staining kits with differing developmental form specificity to a lung homogenate. The cyst form-specific staining kit reacted with cysts originating from S. araneus . Ultrastructurally this particular antigen epitope specifically deposited on the electron-lucent middle layer of the cyst pellicle. The immunohistochemical staining kit reacting with both cyst and trophozoite forms from human and rat origin did not react with any developmental forms of P. carinii originating from S. araneus . Both kits demonstrated P. carinii in the lung homogenate of a field vole, Microtus agrestis. In morphologic examination, the methenamine silver-stained cyst forms of P. carinii from S. araneus and from M. agrestis differed in size from each other and from those originating from laboratory rats. Ultrastructurally P. carinii from S. aruneus did not differ from organisms of rat origin.     

Pneumocystis carinii in pleural fluid. The cytologic appearance

We describe the cytologic appearance of Pneumocystis carinii in pleural fluid of a patient with acquired immunodeficiency syndrome and a rapidly accumulating pleural effusion. The diagnosis of P carinii infection was made by examination of air-dried, Diff-Quik-stained Cytospin preparations of the pleural fluid. The diagnostic appearances of P carinii organisms stained by this method and by the Papanicolaou stain are reviewed. The unusual predominance of the trophozoite forms of the organism in this case made Diff-Quik an ideal special stain for identifying the organisms. Furthermore, this case illustrates a novel presentation of P carinii infection and suggests that P carinii should be considered an etiologic agent in the differential diagnosis of pleural effusion in an immunocompromised host.     

Morphological evaluation of Pneumocystis carinii after extraction from infected lung

Changes in Pneumocystis carinii induced by the extraction of the parasite from rabbit lung have been investigated. Samples obtained using 4 extraction methods were evaluated by light and transmission electron microscopy. Light microscopic evaluation was insufficient to give a measure of the P. carinii viability or to detect parasitic cellular alterations. In contrast, ultrastructural evaluation provided information on host and P. carinii cell integrity, which is a critical condition for viability. None of the tested methods was ideal. How thoroughly and in what shape P. carinii need to be extracted from tissues will determine which extraction technique is of best use.     

Golgi complex and lysosomes in rabbit derived Pneumocystis carinii.

The ultrastructural morphology of Pneumocystis carinii obtained from nonimmunosuppressed rabbit is described in details. Golgi complex and primary lysosomes of P carinii are described here for the first time. They are easily revealed by the zinc iodide-osmium tetroxide cytochemical reagent. Thiamine pyrophosphatase and beta-glycerophosphatase activities are found in the parasite but cytidine 5' monophosphatase activity is not observed. A weak thiamine pyrophosphatase activity is detected in Golgi vesicles. An endomembranous saccular structure, present from the intracystic body stage to the precystic stage, apparently plays the role of secondary lysosome. A second type of endomembranous saccular structure, only present in the well developed trophozoitic and precystic forms is also described. The presence of carbohydrates in the cell wall of the parasite was demonstrated by periodic acid-thiosemicarbazide-silver proteinate staining and lectin concanavalin A labeling. The development of Golgi vesicles preceded the transition from double-layered to three-layered parasite stages.     

Host-parasite relationship in Pneumocystis carinii infection: activation of the plasmalemmal vesicular system in type I alveolar epithelial cells.

Ultrastructural studies of the attachment zone between Pneumocystis carinii (Pc) and type I alveolar epithelial cells showed a new aspect of the host-parasite relationship, i.e. an activation of the plasmalemmal vesicular system in the alveolar cells associated with Pc trophozoites in close apposition. This phenomenon may be involved in the nutrition of the trophozoite.     

Three-dimensional reconstruction of rabbit-derived Pneumocystis carinii from serial-thin sections. I: Trophozoite.

The highly complex ultrastructural morphology of the endomembrane system in Pneumocystis carinii led us to perform three-dimensional reconstruction from serial-thin sections using the CATIA (Conception Assistée Tridimensionnelle Inter Active) Dassault system program. The three-dimensional reconstruction of a small trophozoite made it possible to better understand the morphological relationship among organelles and to suggest cytophysiological hypotheses. By reconstructing other parasite stages, we gathered information about the evolution of organelles during the life cycle and about their physiology.     

The effects of extracellular matrix (ECM) proteins on the attachment of Pneumocystis carinii to lung cell lines in vitro.

Pneumocystis carinii cells labeled with fluorescein isothiocyanate were co-cultured with tissue culture cells. Measurements of attachment was determined by the tissue culture cell fluorescence after washing out the P. carinii organisms. The effects of the extracellular matrix proteins, laminin and fibronectin, on the binding of P. carinii onto the monolayer of cultured cells were investigated for better understanding of organism-cell interactions. The internalization of P. carinii by MRC5 cells was observed.     

Freeze-fracture localization of filipin-sterol complexes in plasma- and cyto-membranes of Pneumocystis carinii

The polyene antibiotic, filipin, was used as the probe for demonstrating sterols in the freeze-fractured plasma- and cytomembranes of Pneumocystis carinii. The distribution of filipin-sterol complexes was homogeneous on the plasma membrane throughout all developmental stages from trophozoite to cyst; however, the density of the complexes gradually decreased with the progress of development. In the trophozoite, the density of the complexes was 485 +/- 42/micron2 on the P face and 341 +/- 27/micron2 on the E face. It was 249 +/- 50 on the P face and 132 +/- 48 on the E face in the precyst and 138 +/- 24 and 59 +/- 20, respectively, in the cyst. The membranes of nucleus, mitochondria, and small round bodies showed more or fewer complexes while no complexes were found in the membranes of one endoplasmic reticulum. In nuclear and mitochondrial membranes, some small scattered clusters of complexes were observed. Two types of vacuoles were distinguished: one having many complexes in its membrane and the other having none at all.     

Cell surface protease PRT1 identified in the fungal pathogen Pneumocystis carinii

The subtelomeric regions of the chromosomes of many organisms contain gene families that allow adaptation to a changing environment. In a number of parasites, these subtelomeric gene families encode cell surface proteins that undergo antigenic variation. Proteases are another important virulence determinant in pathogenic microorganisms. We report the localization of the PRT1 protease of the pathogenic fungus Pneumocystis carinii sp. f. carinii, encoded by a subtelomeric gene family, to the cell surface of both the trophozoite and the cyst phase of the organism. Using anti-PRT1 antiserum, we demonstrated specificity to P. carinii sp. f. carinii in sections of infected rat lungs and, using immunofluorescence, we showed that the PRT1 protease has the characteristic distribution of a surface protein. The anti-PRT1 antiserum showed cross-reactivity with a number of P. carinii sp. f. carinii proteins migrating between 185 kDa and 28 kDa, the majority migrating between 42 kDa and 52 kDa, a region that has been shown by serological studies to contain important immunodominant P. carinii proteins. Cross-reactivity was also observed with P. carinii sp. f. hominis proteins. We have also cloned a portion of the catalytic domain of PRT1 from P. carinii sp. f. hominis, P. carinii sp. f. muris and P. carinii sp. f. rattus. Our data suggest that the PRT1 protease plays an important role in the pathogenicity of P. carinii.     

Localization of cytoskeletal proteins in Pneumocystis carinii by immuno-electron microscopy

Pneumocystis carinii causes serious pulmonary infection in immunosuppressed patients. This study was undertaken to observe the cytoskeletal proteins of P. carinii by immuno-electron microscopy. P. carinii infection was experimentally induced by immunosuppression of Sprague-Dawley rats for seven weeks, and their lungs were used for the observations of this study. The gold particles localized actin, tropomyosin, and tubulin. The actin was irregularly scattered in the cytoplasm of the trophic forms but was much more concentrated in the inner space of the cell wall of the cystic forms called the inner electron-lucent layer. No significant amount of tropomyosin was observed in either trophic forms or cystic forms. The tubulin was distributed along the peripheral cytoplasm and filopodia of both the trophic and cystic forms rather than in the inner side of the cytoplasm. Particularly, in the cystic forms, the amount of tubulin was increased and located mainly in the inner electron-lucent layer of the cell wall where the actin was concentrated as well. The results of this study showed that the cell wall of P. carinii cystic forms is a structure whose inner side is rich in actin and tubulin. The location of the actin and tubulin in P. carinii suggests that the main role of these proteins is an involvement in the protection of cystic forms from the outside environment by maintaining rigidity of the cystic forms.     

Three-Dimensional Reconstruction of Rabbit-Derived Pneumocystis carinii from Serial-Thin Sections I: Trophozoite

The highly complex ultrastructural morphology of the endomembrane system in Pneumocystis carinii led us to perform three-dimensional reconstruction from serial-thin sections using the CATIA (Conception Assistée Tridimensionnelle Inter Active) Dassault system program. The three-dimensional reconstruction of a small trophozoite made it possible to better understand the morphological relationship among organelles and to suggest cytophysiological hypotheses. By reconstructing other parasite stages, we gathered information about the evolution of organelles during the life cycle and about their physiology.     

Relationship between partial inhibition of glycolysis and hemolysis after induction of gametocytogenesis in synchronous cultures of Plasmodium falciparum

In the present study, the low molecular-weight fraction of the culture supernatant of anti-Plasmodium falciparum antibody-producing hybridoma cells (HybSL) was used in synchronous culture with P. falciparum FVO strain. When synchronous cultures were treated with HybSL solution on day 5, gametocytogenesis was also induced. Gametocytes were consistently found from the third day after treatment and reached a peak on the fourth day. An increase in pH and hemoglobin concentrations and decrease in lactate concentrations were observed on the first day after treatment. These phenomena suggested that HybSL solution partially inhibited glycolysis of erythrocytes parasitized with schizonts and resulted in hemolysis of infected erythrocytes. On the other hand, the production of gametocytes did not increase in cultures treated with HybSL solution on day 4 of synchronous cultures in which ring forms were plentiful. Most ring forms were not killed by HybSL solution and quickly developed to trophozoites and schizonts rather than gametocytes. Consequently, it is assumed that ring forms on day 4 of synchronous cultures have finished differentiation into the asexual stage. The conversion of asexual parasites to gametocytes may be triggered only when late-stage trophozoites or early-stage schizonts are treated with HybSL solution.     

Serial propagation of Pneumocystis carinii in cell line cultures.

Pneumocystis carinii was propagated on three cell lines routinely cultured in many laboratories; the method is practical and convenient. Organisms produced were found to be reactive to Pneumocystis antisera. Studies of antigenic relationships, life cycles, and diagnostic methods will be made easier by these cultures.     

Serial propagation of Pneumocystis carinii in cell line cultures.

Pneumocystis carinii was propagated on three cell lines routinely cultured in many laboratories; the method is practical and convenient. Organisms produced were found to be reactive to Pneumocystis antisera. Studies of antigenic relationships, life cycles, and diagnostic methods will be made easier by these cultures.