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First recognised as 'schizonts' of Trypanosoma cruzi, Pneumocystis organisms are now considered as part of an early-diverging lineage of Ascomycetes. As no robust long-term culture model is available, most data on the Pneumocystis cell cycle have stemmed from ultrastructural images of infected mammalian lungs. Although most fungi developing in animals do not complete a sexual cycle in vivo, Pneumocystis species constitute one of a few exceptions. Recently, the molecular identification of several key players in the fungal mating pathway has provided further evidence for the existence of conjugation and meiosis in Pneumocystisorganisms. Dynamic follow-up of stage-to-stage transition as well as studies of stage-specific proteins and/or genes would provide a better understanding of the still hypothetical Pneumocystislife cycle. Although difficult to achieve, stage purification seems a reasonable way forward in the absence of efficient culture systems. This mini-review provides a comprehensive overview of the historical milestones leading to the current knowledge available on the Pneumocystis life cycle.

The genus Pneumocystis comprises noncultivable, highly diversified fungal pathogens dwelling in the lungs of mammals. The genus includes numerous host-species-specific species that are able to induce severe pneumonitis, especially in severely immunocompromised hosts. Pneumocystis organisms attach specifically to type-1 epithelial alveolar cells, showing a high level of subtle and efficient adaptation to the alveolar microenvironment. Pneumocystis species show little difference at the light microscopy level but DNA sequences of Pneumocystis from humans, other primates, rodents, rabbits, insectivores and other mammals present a host-species-related marked divergence. Consistently, selective infectivity could be proven by cross-infection experiments. Furthermore, phylogeny among primate Pneumocystis species was correlated with the phylogeny of their hosts. This observation suggested that cophylogeny could explain both the current distribution of pathogens in their hosts and the speciation. Thus, molecular, ultrastructural and biological differences among organisms from different mammals strengthen the view of multiple species existing within the genus Pneumocystis. The following species were subsequently described: Pneumocystis jirovecii in humans, Pneumocystis carinii and Pneumocystis wakefieldiae in rats, and Pneumocystis murina in mice. The present work focuses on Pneumocystis oryctolagi sp. nov. from Old-World rabbits. This new species has been described on the basis of both biological and phylogenetic species concepts.

We have compared the searching of the presence of "honeycomb" structures by direct microscopy on wet mount preparations with the direct immunofluorescence (DIF) for the diagnosis of Pneumocystis carinii pneumonia (PCP) in 115 bronchoalveolar (BAL) fluids. The samples belonged to 115 AIDS patients; 87 with presumptive diagnosis of PCP and 28 with presumptive diagnosis other than PCP. The obtained results were coincident in 114 out of 115 studied samples (27 were positive and 87 negative) with both techniques. A higher percentage of positive results (32.18%) among patients with presumptive diagnosis of PCP with respect to those with presumptive diagnosis other than PCP (3.57%) was observed. One BAL fluid was positive only with DIF, showed scarce and isolated P. carinii elements and absence of typical "honeycomb" structures. The searching for "honeycomb" structures by direct microscopy on wet mount preparations could be considered as a cheap and rapid alternative for diagnosis of PCP when other techniques are not available or as screening test for DIF. This method showed a sensitivity close to DIF when it was applied to BAL fluids of AIDS patients with poor clinical condition and it was performed by an experienced microscopist.

A 14-month-old male Yorkshire terrier was presented to the Autonomous University of Barcelona Veterinary Teaching Hospital because of a history of chronic non-productive cough and acute dyspnea. A follow-up radiograph revealed a diffuse, bilaterally interstitial-alveolar lung disease with presence of air bronchograms. The dog died 5 h after admission with severe dyspnea. Histological sections of the necropsy specimens revealed the presence of characteristic Pneumocystis carinii cysts within alveolar spaces. A diagnosis of P. carinii pneumonia (PCP) was made on the basis of these results. To our knowledge, PCP has not been described in a Yorkshire terrier dog.

Electron microscopy (EM) is still an important tool for the investigation of infectious diseases, despite the introduction of powerful new methods, mainly involving the polymerase chain reaction. Particularly in the field of parasitic protozoology associated with AIDS, where many new species of human pathogens have been recognized in tissue biopsies, EM remains an essential 'catch-all' diagnostic method. The resolved ultrastructural details of these newly recognized parasites allows a unique insight into the biology of these organisms. The information produced by EM is different, but complementary, to that provided by alternative methods.

High levels of heterogeneity have been observed among isolates of Pneumocystis carinii derived from different mammalian host species. We report the characterization of P. carinii isolated from a rhesus monkey (Macaca mulatta), which was immunosuppressed as a result of infection with a chimeric simian-human immunodeficiency virus (SHIVsbg). Histopathological examination showed evidence of severe P. carinii pneumonia with a large predominance of trophozoite forms. Alveolitis consisted of typical foamy, honeycomb exudate, with only a few alveolar macrophages. The lung inflammatory response was rather moderate without type-2 pneumocyte hyperplasia or collagenosis. P. carinii organisms were sometimes observed in the bronchiolar lumen. Ultrastructurally, macaque-derived P. carinii was more similar to human- or rabbit-derived parasites than to mouse-derived P. carinii. Molecular studies were carried out on the macaque-derived P. carinii DNA at two genetic loci: the genes encoding the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) and the mitochondrial small subunit ribosomal RNA (mt SSU rRNA). Comparison of the DNA sequences with those from P. carinii isolated from eight other host species demonstrated that the macaque-derived P. carinii was genetically distinct at both loci, and was more closely related to human-derived P. carinii than to P. carinii derived from non-primate sources. We propose that macaque-derived P. carinii be named Pneumocystis carinii f.sp. macacae.

Continuous axenic culture of Pneumocystis carinii has been achieved. A culture vessel is used that allows for frequent medium exchange without disturbance of organisms that grow attached to a collagen-coated porous membrane. The growth medium is based on Minimal Essential Medium with Earle's salt supplemented with S-adenosyl-L-methionine, putrescine, ferric pyrophosphate, N-acetyl glucosamine, putrescine, p-aminobenzoic acid, L-cysteine and L-glutamine, and horse serum. Incubation is in room air at 31 degrees C. The pH of the medium begins at 8.8 and rises to approximately 9 as the cells grow. Doubling times calculated from growth curves obtained from cultures inoculated at moderate densities ranged from 35 to 65 hours. With a low-density inoculum, the doubling time is reduced to 19 hours. The morphology of cultured organisms in stained smears and in transmission electron micrographs is that of P. carinii, and P. carinii-specific mAbs label the cultured material. Cultured organisms are infective for immunosuppressed rats and can be stored frozen and used to reinitiate culture.

The pneumocystis carinii (PNC) is a challenge, because its taxonomy has not been completely explained. PNC seems to be a chimera with fungal and parasitic characteristics. The objective of the present study is to analyze the different stages observed in PNC using transmission electron microscopy in order to correlate them with bibliographic reports regarding a possible taxonomic classification. The trophic stage was the form most frequently encountered in alveolar spaces and their organisms varying in size and shape from 6 to 20 microns. Cyst were round or oval, with diameters ranging between 4 and 12 microns, showing four intracystic bodies were individually limited by a membrane. Connections between the membrane of intracystic bodies and junctions between them and the inner cell membrane of the cyst wall were also observed. Our ultrastructural findings appear to be in agreement with several reports which considered PNC among the fungi, most likely related them to the non-gemating Ascomycetes.

Ultrastructural studies indicate that the attachment of Pneumocystis carinii to alveolar epithelial cells is a central feature during development of this pneumonia. P. carinii attaches preferentially to type I alveolar epithelial cells, and rarely binds to type II cells. Extensive in vitro investigations support roles for both fibronectin and vitronectin, which bind to the organism, and mediate P. carinii attachment to respiratory epithelial cells through corresponding integrin receptors. Additional studies indicate that P. carinii cytoskeletal rearrangement occurs after initial binding of the organism to lung cells. Further, the attachment of P. carinii to host epithelial cells is believed to induce ultrastructural changes in these cells and to result in enhanced epithelial cell permeability. Further studies will be required to determine the mechanisms and consequences of P. carinii attachment to host epithelial cells in the intact host.

Because of their constant exposure to normal rodent definitive hosts, least weasels (Mustela nivalis) were trapped in southern Finland in late fall 1994 and examined for lung parasites. Histological examination showed that 46% of the weasels (n = 46) were infected with adiaspores identified as Chrysosporium sp. Granulomas surrounding the adiaspores consisted of mostly unorganized layers of mononuclear cells. The adiaspores from least weasels were much smaller than those reported from their prey animals. Infection with Pneumocystis carinii also was found in two weasels.

Pneumocystis is a eukaryotic unicellular microorganism with marked fungal affinities. All known life cycle stages of this parasite were observed in the lung of mammals. The cystic forms of this microorganism may be observed microscopically by using stains with affinity for the components of their relatively thick cell wall. However, about 100 years ago they were observed for the first time thanks to panoptic stains which do not stain their cell wall. Methanol-Giemsa technique as well as Giemsa-like rapid stainings are often used to reveal vegetative or cystic forms of this parasite on air dried smears of clinical or experimental samples. For many years, hypotheses on its life cycle, which remains unknown, were based on transmission electron microscopy (TEM) studies. However, only for the last years progresses in the quality of fixation for TEM led to a better understanding of the Pneumocystis cell structure. In this chapter, strategies to reveal Pneumocystis organisms in clinical or experimental specimens by using light microscopy, as well as techniques allowing a good preparation of parasitic samples for TEM, are given and shortly discussed.

Pneumocystis carinii (PC) is a fungus present in the lungs of many mammal species. Even though studies of the genome, the isoenzymes, and the antigens have proved some host-species-linked heterogeneity, the existence of distinct Pneumocystis species or subspecies has still not been accepted. Comparative studies of the ultrastructural morphology of pneumocysts derived from several host species may support evidence of host-species-linked heterogeneity. We have compared the ultrastructural morphology of pneumocysts derived from mice, rats, and rabbits. The density of membrane-limited electron-dense cytoplasmic granules was found to be higher in mouse-derived pneumocysts than in rabbit-derived pneumocysts, and furthermore the average diameter of the granules from mouse pneumocysts was larger than that of granules from rabbit-derived pneumocysts. The average diameter of the filopodia of mouse-derived pneumocysts was smaller than that of filopodia from rat-derived pneumocysts, which was smaller than that of filopodia from rabbit-derived pneumocysts. Globular electron-dense bulbous dilatations at the tip of the filopodia were described for the first time and they were only found on filopodia of mouse-derived pneumocysts. These distinct host-species-linked morphological differences of pneumocysts from mouse, rat, and rabbit may support previous biochemical data indicating the existence of different Pneumocystis species or subspecies.

Even for routine purposes, standard staining of Pneumocystis- or Leishmania-containing materials, e.g., with Giemsa or Diff-Quik, is often unsatisfactory due to poor contrast and to staining of irrelevant structures. In comparison, the bisbenzimide dye Hoechst 33258, a DNA-binding fluorochrome, allows a more precise analysis of such materials. Bisbenzimide stained all stages of these fungal or protozoal organisms with brilliant contrast against a uniformly dark background. The level of background luminescence and staining of detritus or non-DNA structures was very low. Organisms were stained both outside of and within phagocytic cells with equal intensity. Counting of individual microorganisms, e.g., in macrophages heavily parasitized with Leishmania or in Pneumocystis-infected bronchoalveolar lavage, was simplified and more precise. Air-dried cell suspensions, cytocentrifuge preparations, impression smears, or cryocut micrographs showed the advantages of bisbenzimide staining over Diff-Quik. Staining with bisbenzimide could be a valuable auxiliary technique for the analysis of material infected with a variety of microorganisms.

The aim of this study was to detect the surface antigens in different stages of experimental induced Pneumocystis carinii in Sprague-Dawley rats. Immunohistochemical staining with monoclonal (900, 902 and 904) and polyclonal (SP-D) antibodies demonstrated that the P. carinii organisms were mostly in the alveolar lumina. The binding sites of the monoclonal (900, 902 and 904) and polyclonal (SP-D) antibodies developed against P. carinii were examined at the ultrastructural level by using a post-embedding immunogold labeling. The gold particles were observed evenly on the surface of precyst and cyst stages of the P. carinii. In the trophozoite stage, scattered gold particles were seen on the pellicles and tubular expansions. The monoclonal antibodies reacted mainly with pellicles of P. carinii, whereas SP-D labeled pellicles, intracystic bodies, cytoplasms of alveolar macrophages, free floating surfactant material in the alveolar spaces, and adjacent type II epithelial cells. In the immunogold labeling, basically no significant differences were found in the precyst, cyst, and ruptured cyst stages. These results indicate that the gold particles were observed adhering to every stage of P. carinii, mostly concentrated on the pellicles, and more concentrated in the precyst or cyst stage than trophozoite stage which may be due to an increase in antigen accumulation during development from the trophozoite to the cyst.

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. araneus did not differ from organisms of rat origin.

The localization and biochemical nature of antigens found in the electron-lucent layer (ELL) of Pneumocystis carinii cysts using polyclonal rabbit antibodies are described. These antigens, specific for the cystic stages of the parasite, were shared by organisms from different hosts, suggesting that they represent functionally important components of the cyst cell wall. The binding sites were situated on an interwoven net of fibrils in the ELL produced by mild to strong proteolysis. Degradation of this residue by glucanase and chitinase confirms that this layer contains branched glucan and chitin. In contrast, the prompt susceptibility of the polysaccharide-rich ELL to proteolysis reveals that proteins are also relevant in building up the cyst-wall glucan skeleton. It is therefore concluded that the formation of the Pneumocystis cyst wall shows differences to the typical fungal cell-wall architecture. The taxonomical debate regarding this unique protist is ongoing, and consideration of these immunological and morphological findings may be useful for the study of the biology and phylogeny of Pneumocystis.

Cyclic lipodepsipeptide compounds of the echinocandin class exhibit broad-spectrum antifungal activity and have been shown to be effective in the treatment of Pneumocystis carinii pneumonia in laboratory animal models. Previous studies have led investigators to propose that these compounds, active against fungal cell walls, are selectively active against the cyst forms of P. carinii. We demonstrate that a semisynthetic, water-soluble echinocandin analog, LY307853, is effective in reducing the number of all life cycle forms of P. carinii and is more effective in mice immunosuppressed with monoclonal antibody to L3T4+ cells than in mice immunosuppressed with dexamethasone. Treatment of P. carinii isolates with LY307853 in a short-term in vitro culture model resulted in cytoarchitectural alterations suggesting that this echinocandin may interfere with the export of surface glycoprotein and the formation of the tubular elements normally found on the surfaces of trophic forms. The cytoarchitectural changes in trophic forms treated in vitro with LY307853 were also observed in trophic forms in the lung tissue of rats treated with a closely related echinocandin analog, LY303366.

The purpose of this review is to assist mycologists in having a better understanding of Pneumocystis carinii and the disease that it causes. Now considered to be a fungus, P. carinii is unusual in its life cycle and relationship with the host. P. carinii pneumonia (PCP) pathogenesis, immunology and host defence mechanisms are examined, as well as epidemiological and control strategies. Most pneumocystosis pathophysiological changes result from the parasite's attachment and proliferation in the lungs, resulting in a filling of the alveoli with masses of the micro-organism. Pathological changes include an increase in alveolar capillary membrane permeability and injury to the alveolar epithelium, which may be mediated by the release of degradative enzymes from the pathogen. A host response takes place by hypertrophy, and hyperplasia involving type II epithelial alveolar cells. P carinii interacts with pulmonary surfactants by binding to the hydrophilic proteins A and D, and by modifying their phospholipid composition. Alveolar macrophages and CD4+ T cells play a key role in the host's defence against Pneumocystis. The epidemiology of PCP remains poorly understood. Airborne transmission has been established, but the actual infective form and its source remains unknown. Studies concerning P. carinii genetic diversity have shown that the parasite polymorphism is related, at least partially, to the host species. A strong host-species specificity in P. carinii has been found. From an epidemiological perspective, there appears to be no animal reservoir for the agent of human PCP. Thus, this disease should not be considered to be zoonotic. Although a significant decrease in the incidence of pneumocystosis has been obtained when employing chemoprophylaxis, anti-P. carinii drugs are not completely successful, often inducing deleterious side-effects. For these reasons, new prophylactic and therapeutic strategies need to be developed. One approach could be based on the anti-P. carinii effect of yeast killer toxins and antibiotic anti-idiotypic antibodies.

Pneumocystis carinii-infected immunosuppressed ferrets and rats were searched for P. carinii cysts and trophozoites in extrapulmonary organs, including the heart, liver, stomach, kidney, and spleen. Foci of P. carinii were found in the liver or kidney of 4 (10%) of 40 ferrets but in no extrapulmonary sites of 30 rats with P. carinii pneumonitis. Attempts to identify P. carinii from immunosuppressed rat or ferret blood or propagate the organism intraorbitally in immunosuppressed rats were unsuccessful. Pneumocystis carinii was identified from aspirated stomach contents of ferrets but in none of the rats infected with P. carinii pneumonitis.

The intrapulmonary life cycle of Pneumocystis carinii has been proposed to include asexual reproduction of haploid trophic forms and sexual reproduction to form cysts or ascospores containing 8 intracystic bodies or spores. This study presents the ultrastructural morphology of trophic forms and developing ascospores. Nuclear-associated organelles (NAO) were observed in these various forms and in a binucleated trophozoite. The unique configuration of the nuclei and NAO in this binucleated trophic form were similar to ultrastructural features observed in conjugation of Saccharomyces cerevisiae. NAOs in developing ascospores were present in pairs and were associated with multiple nuclei. In more mature ascospores, NAO were associated with individual spores. The intermediate forms described in this study support an asexual and sexual reproductive process in P. carinii.

Neutrophils from human immunodeficiency virus (HIV)-negative blood donors, asymptomatic HIV-positive patients, AIDS patients with previous Pneumocystis carinii pneumonia (PCP), and AIDS patients without previous PCP were compared for their ability to activate the respiratory burst, measured as luminol-amplified chemiluminescence. P. carinii, Staphylococcus aureus, phorbol-12-myristate-13-acetate, and FMLP were used to stimulate the neutrophils. When stimulated with P. carinii, neutrophils from PCP patients had a significantly lower response than the other groups, whereas no difference was found when S aureus was used. A somewhat but not significantly lower response to P. carinii was also seen in non-PCP patients compared with HIV-negative donors. Priming of the neutrophils with recombinant granulocyte colony-stimulating factor (G-CSF) or recombinant human granulocyte-macrophage (GM)-CSF corrected this defect. A similar effect of these cytokines was seen on phagocytosis, whereas the chemiluminescence in unprimed cells did not correlate with phagocytosis.

The ultrastructural immunolocalization of target antigens recognized by two monoclonal antibodies against Pneumocystis carinii was investigated using both human- and rat-derived organism. Labelling was seen using both cryo electron microscopy and postembedding in Epon after fixation in glutaraldehyde. The antibodies showed different host species- and developmental stage specificity. One of the antibodies reacted with the outer membrane of all developmental stages of human-derived but not rat-derived organisms, whereas the other reacted only with the cyst form. The distribution of the latter was similar to that reported for methenamine silver, a widely used cytochemical stain for the parasite, and reacted with both human- and rat-derived organisms. The antibodies described appear to be useful markers in studies on the differentiation of Pneumocystis carinii.

Aim: To prepare a monoclonal antibody (EB9) against the trophozoite form of Pneumocystis carinii and to test its efficacy for detecting infection with this organism., Method: The sensitivity and specificity of the EB9 antibody were assessed by comparing it with other conventional stains (Papanicolaou, Giemsa and Grocott) and 3F6 antibody in 33 bronchoalveolar lavage specimens from HIV positive patients suspected of having P carinii pneumonia., Results: P carinii infection was detected in 15 of 33 patients. In 14 cases the organism was detected by two or more of the staining methods used; however, EB9 failed to detect infection in two cases which were positive by other staining techniques. In one case P carinii infection was detected by EB9 only., Conclusion: The results of this study suggest that P carinii infection in the lung may occur in two forms: the cyst form and the trophozoite form, which may explain the observed variation in response to treatment.

A new class of promising antipneumocystis agents, cyclic lipopeptide pneumocandin analogs, has been shown to effectively prevent Pneumocystis carinii cyst development in murine models. These compounds are believed to inhibit the biosynthesis of beta-1,3-glucan, a major constituent of the cell walls of various pathogenic fungi. However, all evidence of the presence of this polymer in P. carinii cysts is based on indirect methods. To address this, highly specific rabbit polyclonal antiserum was raised against a laminariheptaose-human transferrin hapten conjugate. This antiserum was used to demonstrate the presence of beta-1,3-glucan in alkaline extracts of P. carinii-infected rat lung tissue and to quantitate the degree of infection in this tissue as laminarin equivalents. The antiserum was also used to localize beta-1,3-glucan in P. carinii-infected rat lung tissue at the transmission electron microscopic level by immunogold labeling. High concentrations of beta-1,3-glucan were present in the electron-lucent layer of the P. carinii cyst wall, but beta-1,3-glucan was absent from intracystic bodies and trophozoites. Ultrastructural evaluation of lung tissue from P. carinii-infected rats treated with the pneumocandin analog L-733,560 demonstrated that the few cysts which remained are deformed, lack the translucent layer of the cyst wall, and contain minimal amounts of beta-1,3-glucan.

An immunosuppressed rat model was established by injecting cortisone acetate 25 mg/rat twice a week for 4 weeks and 12.5 mg/rat for another 2 weeks subcutaneously. A development of Pneumocystis carinii pneumonia (PCP) was found at the end of the 6th week in all rats. These rats were injected intramuscularly with artemether at 100 mg/kg once a day for 5 consecutive days. All rats were necropsied at the end of the 8th week. The lung impression smears were stained with Gomori's stain, and Pneumocystis carinii cysts were counted. The ultrastructural changes of trophozoite, precyst and cyst were investigated by transmission electron microscopy on the 7th day after treatment.

The proposed life cycle of the opportunistic organism Pneumocystis carinii (PC) includes the formation of a thick-walled cyst containing haploid progeny. The progeny cells mature within the cyst and are somehow released, leaving a collapsed empty cyst behind. Pneumocystis carinii cysts containing 8 intracystic bodies (ICB) as well as empty collapsed cysts have been commonly observed. In this study a single pore in the cyst walls of PC was observed by transmission electron microscopy in infected rat-lung preparations. Presence of a single pore in the cyst wall of an empty but noncollapsed cyst and in collapsed, empty cysts suggests that pore formation may be a mode of excystation for ICB of P. carinii.

Iron plays a critical role in host-parasite interactions, and iron chelators have been demonstrated to serve as effective adjunct therapeutic agents against malaria. The effects of the parenteral iron chelator deferoxamine (DFO) on the growth of rat-derived Pneumocystis carinii were studied in a human fibroblast cell culture model and in two in vivo models of experimental infection. In addition, the effects of the investigational oral iron chelator CP20 and its 3-hydroxypyridin-4-one analogs CP51, CP94, and CP96 on the growth of P. carinii in vitro were assessed. DFO suppressed the growth of P. carinii in vitro in a dose-dependent manner, and daily injections of DFO markedly reduced the intensity of P. carinii infection in both mice and rats. Cell cultures treated with iron chelators that are administered orally to humans also showed substantial P. carinii growth inhibition. Reduction of P. carinii numbers after iron chelator therapy correlated with alterations in P. carinii morphology, as viewed by transmission electron microscopy. Since the use of current anti-P. carinii drugs is limited by toxicity or incomplete efficacy, or both, the role of iron chelation as adjunctive anti-P. carinii chemotherapy merits additional investigation.

To investigate the morphology of Pneumocystis carinii in vivo, we studied the ultrastructure of P carinii in bronchoalveolar lavage fluid (BALF) obtained from four non-AIDS patients with P carinii pneumonia (PCP), and observed the morphologic changes after treatment in one patient. Every form recognized in the normal life cycle of P carinii, ie, thick-walled cysts, crescent-shaped cysts, and trophozoites, was detected in the pretreatment BALF specimens. In the posttreatment BALF, P carinii revealed marked ultrastructural changes such as the elongation of thick-walled cysts, the degeneration of intracystic bodies, and the disappearance of trophozoites. Massive debris derived from P carinii was still found in the BALF even after the treatment. Examination of P carinii in BALF, including ultrastructural observations, might help us to evaluate the disease status and the effect of treatment in patients with PCP.

Typical Pneumocystis carinii pneumonia was found in a male thoroughbred foal (46 days old) suffering from diarrhea and hypogammaglobulinemia after birth. The characteristics of the organisms were demonstrated by Grocott methenamine silver staining, and scanning and transmission electron microscopy. The present focal had no histological lesions suggesting immunodeficiency. However, he could not get adequate colostrum from his dam, because she died of hemorrhagic shock due to immuno-mediated thrombocytopenia at delivery. This condition was considered to have predisposed the foal to the pneumonia.

Studies of the association of rat-origin Pneumocystis carinii with culture cells were performed both to learn more about the role of cells in P. carinii culture and to evaluate additional cell lines in an effort to improve culture methods. Proliferation of trophozoites of P. carinii from rat lung in cultures with six lung cell lines was demonstrated by light microscopic evaluations of both Giemsa-stained and immune-specific-stained culture samples. Scanning electron microscopy and transmission electron microscopy were used to study the organism's interaction with culture cells and demonstrated a close association of P. carinii with cells in cell lines that supported growth. Proliferation with the MVILU line was suboptimal and there was less organism interaction with these cells than with other cell lines that allowed proliferation. Two cell lines evaluated, Chinese Hamster ovary CHOKI and CHOLEKI, did not allow proliferation and had no association of P. carinii with cells. Scanning and transmission electron micrographs demonstrated the close association of organisms with rat fetal lung (RFL), human embryonic lung (HEL), human diploid lung (HFL), and feline embryonic lung (AKD) culture cells. It appears that the association of rat-origin P. carinii with cells is essential for parasite proliferation in short-term culture.

Pneumocystis carinii organisms were isolated from viral antibody-negative rats that had been infected by intratracheal intubation of organism preparations tested negative for common bacteria and fungi. Infection scores of lungs from infected animals at the time of parasite isolation was > 5 (100-1,000 organisms/oil immersion field). Electron microscopy of heavily infected lungs revealed that the pathogens adhered to Type I pneumocytes and to each other, resulting in obstructions up to several cell layers thick, which extended into the alveolar lumen. Protocols for purifying the organisms were developed to optimize separation from each other and from host cells, and to optimize preparation purity, recovery efficiency, and organism viability. The study tested mucolytic agents, sieving, various centrifugation speeds, lysis of host cells by osmotic shock and filtration through membranes of different pore diameter. Final preparations contained no intact host cells as determined by light microscopy. Only minor amounts (< 5%) of host debris were detected by electron microscopy. Most organisms and their pellicles were ultrastructurally intact but no longer adhered to one another. The final preparation was characterized biochemically by quantitation of the specific lung surfactant marker surfactant protein A, which indicated > 99.5% purity. The total non-P. carinii protein in the final preparation (< 6%, depending on the level of infection) was estimated by the protein content of pelletable material resulting from processing uninfected lungs in an identical manner. Elimination of free cholesterol and phospholipids from host lung tissue was monitored during the purification process. Exogenous stigmasterol, added as an extracellular marker, decreased during the purification process and was undetectable in the final organism preparation. Yields of 10(8)-10(9) organisms/rat were routinely obtained. Viability, assessed by the calcein acetoxymethyl ester-propidium iodide assay, was 80-95%.

Primaquine and other 8-aminoquinolines are effective against Pneumocystis carinii in culture and animal models but the way(s) in which they affect P. carinii are not known. This study used transmission electron microscopy to observe early effects of 8-aminoquinolines on P. carinii grown with human embryonic lung fibroblasts in microcarrier suspension culture. The 8-aminoquinolines evaluated were primaquine and Walter Reed Army Institute for Research (WR) compounds WR6026, WR238605 and WR242511. Samples of P. carinii were taken at 0, 3, 6, 12, 24 and 48 hours from culture flasks containing selected concentrations of the drugs. Time matched samples from a parallel culture without drug served as controls. All the 8-aminoquinolines produced similar morphologic alterations of the internal structure of P. carinii. Initially, dilatation of the nuclear envelopes and membranous arrays arising from the reticular system were observed. Later, more organisms displayed large arrays of smooth membranous material often presenting a concentric membranous pattern. Subsequently, cellular organization was lost resulting in necrosis. At concentrations tested WR242511 appeared to be the most effective, producing alterations in many trophozoites after 6 hours of exposure; WR6026 appeared to be the least effective with some organisms unaffected after 48 hours. The changes observed are consistent with damage to the reticular system of P. carinii, which might be caused by oxidation by the 8-aminoquinolines or their metabolites.

Purpose: The purposes of this study are to determine the incidence of infectious opportunistic choroiditis in patients with the acquired immune deficiency syndrome (AIDS), to study the association of these choroidal infections with systemic dissemination, and to investigate the life expectancy and cause of death in patients with infectious opportunistic choroiditis., Methods: A total of 470 eyes of 235 consecutive autopsies of patients with AIDS were examined by histopathologic methods. The clinical charts and autopsy reports of these patients were subsequently reviewed for presence of systemic dissemination of various infectious agents., Results: Of the 235 patients, 18 were found to have infectious choroiditis. The etiologic agents found were: Cryptococcus neoformans, Pneumocystis carinii, Mycobacterium tuberculosis, Histoplasma capsulatum, Candida, Aspergillus fumigatus, Toxoplasma gondii, and Mycobacterium avium-intracellulare. In 15 of these 18 patients, the cause of death was considered to be due to systemic dissemination of the organism causing the choroiditis. Only 4 of the 18 cases of infectious choroiditis were diagnosed during life, and the survival time of these patients after diagnosis was only 25 days. Five of the 18 patients also were found to have cytomegalovirus (CMV) infection of the retina., Conclusion: Multifocal choroiditis due to endogenous infectious emboli in patients with AIDS reflects systemic dissemination and localization of infectious agents predominantly in the choriocapillaris. Early diagnosis and treatment are imperative and may be life-saving.

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.

Pneumocystis carinii infected rat lungs were postfixed with a mixture of OsO4 and K4Fe(CN)6. A marked improvement in staining of cell membranes, endoplasmic reticulum, nuclear membranes and glycogen was observed. These improvements were seen in both the trophic and cystic forms of the organisms. The addition of K4Fe(CN)6 did not improve the staining of cell walls, microtubules or ribosomes. Trophozoites were seen attached to both type 1 and type 2 pneumocytes by filopodia and/or intercalation of the cell body of P. carinii with the host lung cells. It is expected that the improvement in ultrastructural detail will allow better understanding of the ultrastructure of P. carinii and provide insights into the modes of action of various antimicrobial compounds on this organism.

Immunosuppression suspected to be associated with retrovirus infection was diagnosed in an 18-month-old female llama. The llama had a 6-month history of weight loss, intermittent lameness, and infections that were nonresponsive to treatment. Serial CBC indicated persistent nonregenerative anemia and leukopenia characterized by absolute neutropenia and lymphopenia. Functional hypoplasia of myeloid and erythroid cell lines was detected in serial bone marrow biopsy specimens. Notable pathologic findings included inadequate hematopoiesis, generalized lymphoid hypoplasia and plasma cell depletion, and pulmonary alveolar histiocytosis. Pneumocystis carinii cysts and viral particles of the size and morphologic features consistent with the retrovirus family were observed in lung sections examined by transmission electron microscopy. Antemortem macrophage and postmortem lymph node cultures were positive for reverse transcriptase activity.

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.

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.

It has been hypothesized that coinfection with mycobacteria occurs in patients with Pneumocystis carinii choroiditis, but cases demonstrating ocular infection by both organisms have not been reported. This study reports the case of a patient with P. carinii choroiditis who was treated with intravenous trimethoprim and sulfamethoxazole, followed by intravenous trimethoprim and dapsone. The choroidal lesions failed to resolve despite 6 weeks of treatment, and the patient died from massive pulmonary infection caused by P. carinii, Mycobacterium avium-intracellulare, and cytomegalovirus infections. Ocular histologic and electron microscopic examinations revealed choroidal infection by both P. carinii and M. avium-intracellulare. Serum levels of sulfamethoxazole were below the recommended therapeutic range for treating P. carinii infection during the first week of therapy, but adequate drug levels were subsequently obtained. Failure of choroidal lesions of P. carinii to resolve in some cases may suggest insufficient antimicrobial levels in the blood or raise the possibility of coexistent M. avium-intracellulare or other opportunistic infection.

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.