Quantitative Differences In Phagocytosis and Degradation of Pneumocystis By Alveolar Macrophages In Aids and Non-Hiv Patients In Vivo

Broncholaveolar lavage (BAL) specimens (n= 213) from AIDS and non-HIV immunosuppressed patients were investigated for the presence of Pneumocystis carinii infection by fluorescence microscopy of Papanicolaou-stained slides. Alveolar casts, extracellular pneumocysts and phagocytosed cysts and their degradation products in pulmonary alveolar macrophages were identified. the number of phagocytosed pneumocysts within human pulmonary alveolar macrophages was recorded and correlated with the number of extracellular cysts and alveolar casts, in both groups of patients. Both phagocytic and degradation capacity were depressed in AIDS patients. This observation may explain the large number of extracellular organisms found in BAL specimens of AIDS patients compared with non-HIV-positive immunocompromised individuals.     

Influence of Pneumocystis carinii on Nitrite Production by Rat Alveolar Macrophages1

ABSTRACT Nitrite production by rat alveolar macrophages was studied to determine the role of L-arginine oxidation in the interaction between these cells and Pneumocystis carinii. Alveolar macrophages from rats obtained from two different breeders were used: rats from Janvier breeder had latent P. carinii infection, while those from Charles River breeder were bred in a germ-free environment. Pneumocystis carinii increased in vitro nitrite generation by unstimulated alveolar macrophages from Janvier rats only, and this was blocked by N^G-monomethyl-L-arginine. Incubation of cells from Janvier and Charles River rats with lipopolysaccharide and/or interferon-gamma increased nitrite production to a similar extent. Pneumocystis carinii partially decreased nitrite release by activated alveolar macrophages, and this was still inhibited by N^G-monomethyl-L-arginine. In the presence of P. carinii, superoxide dismutase used as a superoxide anion scavenger had no effect on nitrite production by activated cells. These results show that prior exposure to P. carinii leads to nitric oxide production by rat alveolar macrophages. Although the magnitude of this production seems to be moderate, it is of biological significance since cells of P. curinii-naive rats do not generate nitrite whereas those of latently infected rats do.     

In vitro attachment of Pneumocystis carinii from mouse and rat origin

Summary— 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 mouseor 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.     

Analysis of Cellular Response and Gamma Interferon Synthesis in Bronchoalveolar Lavage Fluid and Lung Homogenate of Mice Infected with Pneumocystis carinii

The cellular and cytokine responses in the lungs of mice infected with Pneumocystis carinii were examined on both lung homogenates and bronchoalveolar lavage (BAL) fluids. In the lungs of infected mice, the number of P. carinii cysts rapidly decreased by day 7, then started to increase with a peak on day 14, and thereafter decreased gradually. When the presence of P. carinii was examined at the DNA level by dot blot hybridization, a similar clearance curve was obtained, and the organisms were shown to be completely eliminated on day 28. In the late phase of infection, leukocytes, mainly lymphocytes, increased in number when analyzed on lung homogenates, while no significant increase of inflammatory cells was observed in BAL fluids. An accumulation of both CD4⁺ and CD8⁺ T cells and an increase of activated T cells expressing IL-2Rα were observed in lung homogenates of the infected mice. In addition, a considerable amount of IFN-γ was detected in lung homogenates, but not in BAL fluids. These data indicate that lung homogenates are more suitable than BAL fluids for the analysis of cellular and cytokine responses in the lungs of mice infected with P. carinii. To define the involvement of IFN-γ in host defense against P. carinii, the effect of this cytokine on the killing activity of macrophages against P. carinii was examined in vitro. IFN-γ was found to augment this activity by increasing nitric oxide synthesis of the macrophages. Thus, it is suggested that IFN-γ plays an important role in the protection of mice from P. carinii infection.     

Polymorphism of the Thymidylate Synthase Gene of Pneumocystis carinii from Different Host Species

Pneumocystis carinii is an opportunistic agent found in the lung of various mammals which often causes severe pneumonia in immunocompromised humans, especially in AIDS patients. In the past several years significant additions have been made to the collection of knowledge we have concerning the genetic diversity of P. carinii. These additions provide new understanding of Pneumocystis transmission and the effect of possible reservoirs of Pneumocystis in the various species. In this study, a 400-bp fragment of the thymidylate synthase (TS) gene of P. carinii has been amplified by PCR from 43 parasite isolates obtained from 4 mammalian host species: rat, mouse, rabbit and human. A probe selected from the TS gene sequence of rat-derived P. carinii was hybridized with the amplified products from rat- and mouse-derived P. carinii, but not with rabbit or human P. carinii DNA. Restriction profiles were performed on amplified fragments from all isolates, and the 4 nucleotide sequences of the TS gene fragment amplifed from rat, mouse, rabbit and human P. carinii were determined. Differences were detected in the gene fragment in P. carinii isolates from the 4 host species; however no difference was revealed in P. carinii isolates within a single host species, whatever the host strain or its geographic origin. Thus, the sequence differences of the P. carinii TS gene appeared as host-species specific. A specific probe which recognized all human P. carinii isolates was defined.     

Molecular Genetic Distinction of Pneumocystis carinii from Rats and Humans

Pneumocystis carinii from rats and from humans were compared with respect to electrophoretic karyotype, presence of DNA sequences known to be repeated in rat-derived P. carinii, overall DNA sequence homology, and the sequences at two genetic loci. The organisms from each host species were different in each respect. Neither of two repeated DNAs from rat-derived P. carinii was found in the genome of human-derived organisms, and total DNA from rat-derived P. carinii failed to hybridize to human-derived P. carinii DNA. The sequences of the α-tubulin genes from the two P. carinii were strikingly different and the base composition of the α-tubulin gene from rat-derived P. carinii was rich in adenine and thymine, while the base composition of this gene from human-derived P. carinii was rich in guanine and cytosine. The sequence from the 18S rRNA gene of human-derived P. carinii was twice as divergent from that of rat-derived P. carinii as the sequence from the corresponding region of Candida albicans was from that of Candida tropicalis. These data show that rats and humans can harbor distinct types of P. carinii that are sufficiently different to suggest that P. carinii from the two hosts could be different species.     

Transcription Factor Genes from Rat Pneumocystis carinii

Genes encoding the TFIID TATA-box binding protein (TBP) from two probable species of rat Pneumocystis carinii (prototype and variant) were sequenced. The two P. carinii TBP gene sequences were 91% identical to each other, and 65-77% identical to TBP genes from other species. A cDNA from one of the two P. carinii TBP genes was sequenced, which showed that four small introns resided in identical positions within the TBP genes from the prototype and variant rat P. carinii. Conservation of the 180 amino acids that constitute the conserved core of TBP was 97% between the P. carinii TBP, which were 95% and 97% identical to conserved core sequences of TBP from Saccharomyces cerevisiae and Schizosaccharomyces pombe respectively.     

Localization of surfactant protein A (SP-A) in alveolar macrophage subpopulations of normal and fibrotic rat lung

The colocalization of surfactant protein A (SP-A) and the alveolar macrophage markers ED1 and RM-1, as well as various lectins of the N-acetyl-galactosamine group [Maclura pomifera lectin (MPA), Dolichos biflorus lectin (DBA), soybean agglutinin (SBA)] and of the mannose group [Canavalia ensiformis lectin (ConA), Galanthus nivalis lectin (GNA)] was studied in normal and fibrotic rat lung tissues. In normal tissue, SP-A was located preferentially in the alveolar macrophage subpopulation lacking specific binding sites for lectins of the N-acetylgalactosamine group (DBA and SBA), although 50% of MPA-binding macrophages contained SP-A. The ED1-positive cells were SP-A-negative, whereas SP-A uptake could be detected among the RM-1 immunoreactive as well as the ConA and GNA binding macrophages. In fibrotic lung tissue, however, a small number of .DBA and SBA binding macrophages contained SP-A and the percentage of GNA and ConA binding alveolar macrophages exhibiting SP-A immunoreactivity was reduced. Additionally, the number of ED1⁺/SP-A⁺ macrophages was found to be increased. Immunoelectron microscopy revealed accumulation of SP-A in the extracellular space. The differing SP-A content in different alveolar macrophage subpopulations suggests a more complex mechanism of uptake and degradation of surfactant proteins in normal and pathological conditions, which cannot simply be explained by the glycoconjugate pattern on the surface of alveolar macrophages.     

Artificial Infections of Pneumocystis carinii in the SCID Mouse and their Use in the In Vivo Evaluation of Antipneumocystis Drugs

A model for the in vivo evaluation of antipneumocystis drugs has been developed in SCID mice infected intratracheally with cryopreserved mouse-derived Pneumocystis carinii. The development of a highly reproducible fatal P. carinii pneumonia occured within 10 weeks (mean survival time ± SEM = 72.2 ± 1.2 days). Continuous administration of dexamethasone (2 mg/liter in the drinking water) exacerbated the rate of onset of severe P. carinii pneumonia (mean survival time ± SEM = 63 ± 1.3 days) in SCID mice. The number of cysts per g of lung homogenate (homogenate counts) were maximal with an inoculum of 20,000 cysts at 6 weeks post infection. Homogenate counts correlated with infection scores (graded assessments of immunofluorescent cysts on lung impression smears) suggesting that infection scoring accurately and rapidly reflects the severity of P. carinii pneumonia in SCID mice. These studies led to the development of a drug screening protocol in which Pneumocystis-free female SCID mice (20–25 g) were started on dexamethasone 7 days prior to IT inoculation with a single dose of 20,000 cysts. Drugs were evaluated either for: a) prophylaxis (continuously from day 1 post infection) or b) treatment (from day 21 post infection) until day 42 post infection, when all mice were killed and infection scores determined. Co-trimoxazole (at 250 mg sulfamethoxazole + 50 mg trimethoprim/kg/day) given in the drinking water was found to be highly effective in both the prophylaxis and treatment of mouse P. carinii pneumonia. Co-trimoxazole remained very effective in the prophylaxis P. carinii pneumonia in the SCID mouse at 125 mg sulfamethoxazole + 25 mg trimethoprim/kg/day p.o. and showed some enhancement of efficacy over sulfamethoxazole alone at 125 mg/kg/day p.o., suggesting limited synergy between sulfamethoxazole and trimethoprim. The results presented provide confirmation of the usefulness and predictability of the model.     

Pneumocystis carinii: Genetic Diversity and Cell Biology

As an important opportunistic pulmonary pathogen, Pneumocystis carinii has been the focus of extensive research over the decades. The use of laboratory animal models has permitted a detailed understanding of the host–parasite interaction but an understanding of the basic biology of P. carinii has lagged due in large part to the inability of the organism to grow well in culture and to the lack of a tractable genetic system. Molecular techniques have demonstrated extensive heterogeneity among P. carinii organisms isolated from different host species. Characterization of the genes and genomes of the Pneumocystis family has supported the notion that the family comprises different species rather than strains within the genus Pneumocystis and contributed to the understanding of the pathophysiology of infection. Many of the technical obstacles in the study of the organisms have been overcome in the past decade and the pace of research into the basic biology of the organism has accelerated. Biochemical pathways have been inferred from the presence of key enzyme activities or gene sequences, and attempts to dissect cellular pathways have been initiated. The Pneumocystis genome project promises to be a rich source of information with regard to the functional activity of the organism and the presence of specific biochemical pathways. These advances in our understanding of the biology of this organism should provide for future studies leading to the control of this opportunistic pathogen.     

The Diagnosis of Pneumocystis Carinii Pneumonia By Cytologic Evaluation of Papanicolaou and Leishman-Stained Bronchoalveolar Specimens In Patients With the Acquired Immunodeficiency Syndrome

The presence of foamy alveolar casts or flocculent material in Papanicolaou and Leishman-stained smears of bronchoalveolar lavage (BAL) fluid is said to be indicative of infection with Pneumocystis carinii. We have investigated the sensitivity and specificity of this method of diagnosing pneumocystis pneumonia in patients with the acquired immunodeficiency syndrome (AIDS). Patients (n= 114) with diffuse lung infiltrates were submitted to fibreoptic broncoscopy and BAL. Seventy of them were patients with AIDS. the other 44 individuals were not infected by the human immunodeficiency virus (HIV). Pneumocystis carinii organisms were identified on Grocott's methenamine silver (GMS)-stained BAL smears in 30 patients with AIDS. Flocculent material was present in the Papanicolaou and Leishman-stained smears from all of these cases. Conversely, P. carinii were not seen on GMS-stained smears in the remaining 84 individuals with or without AIDS. No flocculent material was observed in Papanicolaou or Leishman-stained smears in these 84 patients. We concluded that the presence of flocculent material in Papanicolaou or Leishman-stained smears of BAL fluid is indicative of P. carinii pneumonia in patients with AIDS. La présence de cylindres alvéolaires spumeux ou de matériel floculé dans les étalements de liquide de lavage bronchoalvéolaire (LBA) colorés selon Papanicolaou ou Leishman est considérée comme symptomatique d'une infection par Pneumocystis carinii. Nous avons étudié la sensibilité et la spécificité de cette méthode de diagnostic de l'infection par Pneumocystis carinii chez des patients atteints de syndrome de déficience immunitaire acquise (SIDA). Cent quatorze malades avec des infiltrats pulmonaires diffus ont subi une fibroscopie bronchique et un lavage broncho-alvéolaire. Soixante dix d'entre eux edtaient atteints de SIDA, 44 n'étaient pas infectés par le Virus de l'Immunodéficience Humaine (VIH). Le Pneumocystis carinii a été identifiié par la coloration de Grocott chez 30 patients atteints de SIDA. Chez ces patients, la présence d'un matériel floculé est constante sur les étalements colorés au Papanicolaou et au Leishman. A l'inverse, Pneumocystis carinii n'a pas été retrouvé chez les 84 autres malades, atteints ou non du SIDA et les étalements de LBA ne contenaient pas de matériel floculé. En conclusion, la présence de matériel floculé dans les étalements de LBA colorés selon Papanicolaou ou Leishmanest associée à une pneumpathie àPneumocystis carinii chez les patients atteints de SIDA. Sensitivität und Spezifität des Nachweises schaumiger oder flockiger Alveolarausgüsse bei Pneumocystis carinii wurden in 114 Fällen diffuser Lungeninfiltrate untersucht. 70 Patienten waren an AIDS erkrankt, 44 weitere waren HIV-negative. In 30 der AIDS-Fälle wurde P. carinii mit der Grocott'schen Färbung nachgewiesen. Die typischen Eiweißniederschläge waren in all diesen Fällen nachweisbar. Umgekehrt ergab die Grocottfärbung in 84 Fällen mit oder ohne AIDS ein negatives Ergebnis. In all diesen Fällen war kein Eiweißniederschlag nachweisbar. Daraus ergibt sich, daß die Eiweißniederschläge in Präparaten, die nach Papanicolaou oder Leishman gefärbt wurden, kennziechned sind für die P. carinii Pneumonie.     

Quantitation of Absolute Pneumocystis carinii Nuclear DNA Content. Trophic and Cystic Forms Isolated from Infected Rat Lungs are Haploid Organisms

The Pneumocystis carinii carinii DNA content in nuclei of trophic forms and cysts (spore cases) containing 2, 4, or 8 intracystic bodies, were compared using quantitative fluorescence image analysis. The nuclear DNA content was found to be lower than the theoretical limits of Feulgen cytophotometry. Several fluorescent DNA dyes provide brighter staining, but these techniques suffer from nonspecific binding to other cellular components, such as RNA. It was demonstrated that the thick glycocalyx surfaces of trophic forms and the cyst walls of P. carinii organisms, as well as the cell wall of S. cerevisiae, bound all fluorescent dyes tested to varying degrees. Hence in this study, measurements were performed on cells in which the outer surfaces of organisms were first removed with lyticase. Two stains that appeared most specific for DNA, DB181 and 4′,6-diamidino-2-phenylindole (DAPI), were used for quantitations; lower deviations of fluorescence intensities were observed with DB181. Haploid wild type Saccharomyces cerevisiae and cdc-28 temperature-sensitive mutant cells, accumulated at the restrictive temperature (37° C), were used as quantitative internal standards for estimating the absolute nuclear DNA content of P. carinii. Haploid wild type and mutant nuclei stained with DAPI had the same relative fluorescence intensities. The P. carinii nuclear DNA content of trophic forms and individual intracystic bodies (spores), regardless of life cycle stage, were not different. The mean values obtained were 6.9 and 6.7 fg DNA/nucleus with DB181 and DAPI, respectively (approximately 9.26 and 8.99 Mbp nucleotides, respectively). Since these would include 2C (G-2 phase) and S-phase nuclei, a 1C population of nuclei was selected by histogram distributions of DB181-stained nuclei. Almost all nuclei analyzed in all life cycle stages fell within this population. The 1C mean of 6.55 fg DNA/nucleus (median, 6.62 fg DNA/nucleus) was estimated as representing 8.79 Mbp nucleotides, assuming only A-T binding of the dye and taking into account the G+C content of S. cerevisiae and P. carinii. A 4C (G-2-phase diploid nuclei) population was not detected in histograms of DB181- or DAPI-stained nuclei. The P. carinii nuclear DNA content values obtained in this study were similar to those independently obtained by calculating the total DNA in the organism's chromosomes resolved by electrophoretic techniques. Together, the data on total chromosome numbers and the estimated DNA content of those chromosomes, with our quantitation of nuclear DNA content of different life-cycle stages demonstrate that P. carinii carinii isolated from infected rat lungs are haploid organisms.     

The Challenge of Pneumocystis carinii Culture1

ABSTRACT. Published and unpublished data on the cultivation of P. carinii were reviewed by a panel of investigators convened by the National Institutes of Health. Although several cell culture systems allow propagation of P. carinii for a limited time with modest rates of replication, these have not proved adequate for isolation of P. carinii in sufficient quantity to explore important basic biological investigation. Attempts at cell-free culture have yielded only transient proliferation. Because much of the unsuccessful work on cultivation of the organism has been unpublished, the panel agreed that these data may be useful to other investigators in designing experimental strategies for cultivation. Therefore, the purpose of this report is to make available this information to researchers, lest others unknowingly repeat unsuccessful methods. It is hoped that by documenting the history and the complexities of Pneumocystis culture, renewed interest and efforts will be directed toward this fundamental scientific challenge.     

Population Structure of Rat-Derived Pneumocystis carinii in Danish Wild Rats

The rat model of Pneumocystis carinii pneumonia is frequently used to study human P. carinii infection, but there are many differences between the rat and human infections. We studied naturally acquired P. carinii in wild rats to examine the relevance of the rat model for human infection. P. carinii DNA was detected in 47 of 51 wild rats and in 10 of 12 nonimmunosuppressed laboratory rats. Evidence for three novel formae speciales of rat-derived P. carinii was found, and these were provisionally named Pneumocystis carinii f. sp. rattus-secundi, Pneumocystis carinii f. sp. rattus-tertii, and Pneumocystis carinii f. sp. rattus-quarti. Our data suggest that low-level carriage of P. carinii in wild rats and nonimmunosuppressed laboratory rats is common and that wild rats are frequently coinfected with more than one forma specialis of P. carinii. We also examined the diversity in the internally transcribed spacer (ITS) regions of the nuclear rRNA operon of Pneumocystis carinii f. sp. carinii by using samples from wild rats and laboratory rats and spore trap samples. We report a lack of variation in the ITS1 and ITS2 regions that is consistent with an evolutionary bottleneck in the P. carinii f. sp. carinii population. This study shows that human- and rat-derived P. carinii organisms are very different, not only in genetic composition but also in population structure and natural history.     

Antigenic properties of recombinant glycosylated and nonglycosylatedPneumocystis carinii glycoprotein A polypeptides expressed in baculovirus-infected insect cells

Since a continuous culture system is not yet available for the opportunistic fungal pathogenPneumocystis carinii, obtaining suitable amounts of purifiedP. carinii antigens free of mammalian-host lung contaminants is difficult. Hence, production of recombinant antigen possessing epitopes found in nativeP. carinii antigens is critical for immunological studies. We utilized the baculovirus expression vector system (BEVS) in insect cells to determine whether B-cell epitopes present in the protein core of a nativeP. carinii surface glycoprotein were conserved in the recombinant polypeptide, and to investigate its glycosylation by insect cells. B-cell epitopes were retained, but the insect cells appeared to hyperglycosylate the recombinant protein.     

Specificity in structure-based drug design: Identification of a novel,selective inhibitor of Pneumocystis carinii dihydrofolate reductase

Specificity is an important aspect of structure-based drug design. Distinguishing between related targets in different organisms is often the key to therapeutic success. Pneumocystis carinii is a fungal opportunist which causes a crippling pneumonia in immunocompromised individuals. We report the identification of novel inhibitors of P. cariniidihydrofolate reductase (DHFR) that are selective versus inhibition of human DHFR using computational molecular docking techniques. The Fine Chemicals Directory, a database of commercially available compounds, was screened with the DOCK program suite to produce a list of potential P. carinii DHFR inhibitors. We then used a postdocking refinement directed at discerning subtle structural and chemical features that might reflect species specificity. Of 40 compounds predicted to exhibit anti-PneumocystisDHFR activity, each of novel chemical framework, 13 (33%) show IC₅₀ values better than 150 μM in an enzyme assay. These inhibitors were further assayed against human DHFR: 10 of the 13 (77%) bind preferentially to the fungal enzyme. The most potent compound identified is a 7 μM inhibitor of P. carinii DHFR with 25-fold selectivity. The ability of molecular docking methods to locate selective inhibitors reinforces our view of structure-based drug discovery as a valuable strategy, not only for identifying lead compounds, but also for addressing receptor specificity. Proteins 29:59–67, 1997. © 1997 Wiley-Liss, Inc.     

A 55 kDa antigen of Pneumocystis carinii: analysis of the cellular immune response and characterization of the gene

Rat-derived Pneumocystis carinii contains a major antigen complex of 45–55 kDa. The fusion protein of a cDNA encoding the 3′ portion of the 55 kDa antigen, which had previously been shown to be recognized by serum antibodies of exposed subjects, was investigated for its ability to stimulate a cellular immune response. Rats exposed to P. carinii via the environment exhibited a vigorous proliferative response to the antigen whereas unexposed rats did not. The full-length cDNA for a 55 kDa antigen was cloned and found to contain a 1245 bp open reading frame capable of encoding a 414-amino-acid peptide. The gene encoding this protein contained a single 39 bp intron and transcribed a 1.45 kb RNA message. The cloning and characterization of the 55 kDa antigen gene will allow production of the specific immunological reagents necessary to characterize this molecule and study its role in the biology and pathogenesis of P. carinii.