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.

[Analysis of the results obtained in fertilization in vitro according to the degree and type of teratospermia]. / Analyse des résultats obtenus en fécondation in vitro selon le degré et le type de tératospermie.

The aim of this study was to evaluate the consequences of sperm morphology abnormalities on fertilization capacity. We selected 115 couples who consulted for in vitro fertilization at the Salengro Maternity Ward (Lille, France). Teratospermia was > 40% in all. The mean rate of cleavage was 23% and was significantly lower than in a population which had had in vitro fertilization for a tube indication (63%, p < or = 0.001). In addition, this level was significantly correlated with teratospermia (p < or = 0.05). The ROC method demonstrated that above the threshold of 74% teratospermia, fertilization decreases significantly. We distinguished several types of abnormal morphology and demonstrated a weak correlation between head abnormalities and the rate of cleavage, a stronger correlation between abnormalities of the intermediary piece and rate of cleavage and finally the absence of any correlation between flagella abnormalities and the rate of fertilization. Teratospermia is therefore an important factor in evaluating fertilization capacity. A better selection of normal spermatoids could improve the chance of success in cases of male sterility.

[The rabbit, experimental host of Pneumocystis carinii]. / Le lapin, hôte expérimental de Pneumocystis carinii.

Pneumocystis carinii can be collected from experimentally immunodepressed rats. However, development of experimental pneumocystosis in this host requires corticoid-treatment during 8 to 12 weeks while P. carinii can be obtained from immunodepressed rabbits after 2 to 4 weeks. In 400 to 650 g body weight-white rabbits normally fed, two immunodepression protocols were used: (a) daily subcutaneous injections of hydrocortisone acetate (10 mg/kg body weight) and G penicillin (25,000 IU)-streptomycin (3 mg); (b) continuous oral administration of prednisolone (20 mg/l/day) and amoxicillin (25 mg/l/day) both diluted in drinking water. Between 55 and 78% of rabbits treated by any immunodepression protocol died within 5-20 days after a terminal important diarrhoea. Seventy and seventy-seven per cent of the young rabbits respectively treated by protocols (a) or (b), showed the presence of Pneumocystis by the direct microscopic examination, but all of them were found positive after lung concentration. Fifty-five per cent of the older rabbits (950-1,400 g) were positive by pulmonary smear examination when treated with protocol (a). Pneumocystis obtained were good antigens for indirect immunofluorescence and immunoblot assays using human and rabbit sera.

Ultrastructural study of Pneumocystis carinii in explant cultures of rabbit lung and in cultures with and without feeder cells.

Pneumocystis carinii-parasitized lung explants were obtained from corticoid-treated rabbits and maintained in vitro. Twenty-one days after the beginning of explant cultures, the ultrastructural morphology of trophozoite, precyst and cyst forms was normal as compared to the in vivo ultrastructure 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. Intracytoplasmic 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 supernates of cultures with and without Vero cells showed important ultrastructural alterations.

High osmotic pressure enables fine ultrastructural and cytochemical studies on Pneumocystis carinii. I. Epon embedding.

High osmotic pressure was used to preserve the ultrastructure of rabbit-, SCID mouse-, and rat-derived Pneumocystis carinii organisms from osmotic stress during fixation. Organelles and cytosol were well preserved within the tonicity range of 850-1,300 mosmol. Under these experimental conditions, we determined that the endoplasmic reticulum was well developed in all parasite stages and could observe the Golgi complex, autophagic vacuoles, dense bodies, type II endoplasmic saccules, and the recently described outer surface membrane, which was found in all parasite stages. The biological implications of these findings are discussed.

High osmotic pressure for Pneumocystis carinii London Resin White embedding enables fine immunocytochemistry studies: I. Golgi complex and cell-wall synthesis.

A method for embedding Pneumocystis carinii in hydrophilic resin (London Resin White) has been developed for immunocytochemistry studies. Using high osmotic pressure (about 850 mosmol) from fixation to embedding, this method improved the preservation of the fine structure as well as the antigenicity of rabbit- and SCID mouse-derived P. carinii. Cytochemistry studies were performed using colloidal gold-conjugated lectins (concanavalin A, glycine max, Ulex europaeus) that reacted with the cytoplasmic components (endoplasmic reticulum, Golgi vesicles). Colloidal gold-conjugated streptavidin was also tested and was found to be reactive with the parasite cell wall and cytoplasmic components, which precludes its indiscriminate use in P. carinii immunocytochemistry studies.

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.

Three-dimensional reconstruction of rabbit-derived Pneumocystis carinii from serial-thin sections. II: Intermediate precyst.

Three-dimensional reconstruction of a binucleate intermediate precyst of Pneumocystis carinii was performed from serial-thin sections using the CATIA (Conception Assistée Tridimensionnelle Inter Active) Dassault system program. The presence of a mitochondrion, complex well-developed endoplasmic structures, and numerous Golgi vesicles was established. A better understanding of the ultrastructure of rabbit-derived P. carinii stages made it possible to formulate hypotheses on the evolution and physiology of the endomembrane system. Thus, the presence of the well-developed endoplasmic saccular structure and more than 230 Golgi vesicles in its vicinity might be implicated in the differentiation of the parasite surface structures and might also be related to nuclear division and individualization of intracystic bodies.

Introduction of Pneumocystis carinii in a colony of SCID mice.

Pneumocystis carinii-free SCID mice were housed closely exposed to corticosteroid-treated non-SCID mice in a conventional area of our laboratory animal facilities. A one-day exposure was sufficient for P. carinii transmission. The lung infection increased thereafter. Irradiation or splenectomy of SCID mice at the beginning of the exposure resulted in a marked increase of parasite multiplication. Extrapulmonary foci of pneumocystosis were detected in heart and spleen of SCID mice infected by P. carinii via air transmission.

Production of a monoclonal antibody using lymphocytes from Pneumocystis infected mice.

A colony of BALB/c mice maintained in a protected area of our laboratory was not infected with Pneumocystis carinii. During corticosteroid treatment, animals became infected by exposure to infected mice. After four months of corticosteroid treatment, BALB/c mice developed severe pneumocystosis. Stopping of treatment was associated with: (i) high mortality of mice, (ii) decreased lung parasite level and (iii) appearance of anti-P. carinii antibodies in survivors. A monoclonal antibody (MAb) 4F2 was obtained by immortalisation of spleen lymphocytes of a female BALB/c mouse 3 months after the cessation of corticosteroid treatment. The MAb 4F2 recognized a 210-220 kDa mouse P. carinii antigen, but did not react with rat-, rabbit- or human-derived P. carinii. This MAb reacted with all stages of mouse P. carinii.

In vitro attachment of Pneumocystis carinii from mouse and rat origin.

The attachment of Pneumocystis carinii to lung cells could play a role in the pathophysiology of P carinii pneumonia. The trophozoite attaches to type I alveolar epithelial cells. Physical, chemical, and extracellular matrix factors, involved in the mouse-or rat-derived P carinii attachment to fibroblastic cells in culture, were examined using a new model of in vitro adherence. The development of parasite filopodia penetrating deeply the host cell cytoplasm was observed using transmission electronic microscopy. Killed P carinii organisms were unable to attach to cultured cells. Also, parasites were unable to attach to killed target cells. The P carinii in vitro attachment was partially inhibited by cytochalasin B. In contrast, the parasite attachment was not affected when the target cell cytoskeleton was altered. In our work conditions, sialic acids were not involved in the attachment process. Present results showed that fibronectin (Fn) plays a role in the parasite attachment, and suggest that a specific Fn-binding receptor is present at the surface of mouse-derived P carinii organisms.

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.

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.

Is Pneumocystis carinii a deep mycosis-like agent?

Pneumocystis carinii is a widespread eukaryotic microorganism found in the lungs of healthy mammals, including humans. It is able to proliferate extensively in the alveoli, becoming an important agent of severe pneumonitis in immunosuppressed hosts, especially in persons suffering from AIDS. The taxonomic position of P. carinii is uncertain. Typical cytoplasmic organelles of eukaryotic cells have been found and described in the parasite. Biochemical research is hindered by the lack of an efficient in vitro culture system. Results of comparative study of nucleic acid sequences suggest that Pneumocystis is a fungus. However, ultrastructural, biochemical and nucleic acid homology insights appear as clearly insufficient to class Pneumocystis. Pneumocystis infection might be acquired, as deep mycoses, from environmental sources through the respiratory tract. Thus, the hypothesis of an environmental stage of the parasite must be considered. Pneumocystis might be seen as a widespread pathogenic dimorphous fungus. As fungal agents, P. carinii is able to disseminate from the infected lung to other organs. However, deep mycoses and pneumocystosis induce different histopathological changes in the host. Furthermore, deep fungal infections, unlike pneumocystosis, cannot be transmitted from one infested host to another one. Beside these two aspects, pneumocystosis shares many features with deep mycoses. Research on the epidemiology of pneumocystosis is needed.