Pneumocystis carinii causes severe pneumonia in immunocompromised hosts. The binding of P. carinii to alveolar epithelial cells and extracellular matrix constituents such as fibronectin and vitronectin is a central feature of infection, which initiates proliferation of the organism. Herein, we demonstrate that P. carinii binding to lung cells specifically alters the gene expression of the organism, regulating fungal growth. Subtractive hybridization was performed to isolate P. carinii genes expressed following binding to mammalian extracellular matrix constituents. P. carinii STE20 (PCSTE20), a gene participating in mating and pseudohyphal growth of other fungi, was identified following adherence to the extracellular matrix constituents fibronectin, vitronectin, collagen, and lung epithelial cells. The expression of PCSTE20 and a related P. carinii mitogen-activated protein kinase (MAPK) kinase gene, also implicated in signaling of mating, were both specifically upregulated by binding to matrix protein. The expression of general cyclin-dependent kinases and other MAPKs not involved in mating pathways were not altered by organism binding. PCSTE20 expression was also strongly enhanced following organism attachment to A549 lung epithelial cells. When expressed in a Saccharomyces cerevisiae ste20Δ mutant, PCSTE20 suppressed defects in both mating and pseudohyphal growth. These findings are consistent with the observed proliferation and filopodial extension of Pneumocystis organisms adherent to the epithelium in the lungs of immunocompromised hosts. PCSTE20 expression appears to represent a significant component in the regulation of the life cycle of this intractable opportunistic pathogen.
Pneumocystis carinii attaches to alveolar epithelial cells during the development of pneumonia. Adhesive proteins found within the alveolar space have been proposed to mediate P. carinii adherence to lung cells. Vitronectin (Vn), a 75-kDa glycoprotein present in the lower respiratory tract, has substantial cell-adhesive properties and might participate in the host-parasite interaction during P. carinii pneumonia. To address whether Vn binds to P. carinii, we studied the interaction of radiolabeled Vn with purified P. carinii organisms. Vn bound to P. carinii, occupying an estimated 5.47 x 10(5) binding sites per organism, with an affinity constant, Kd, of 4.24 x 10(-7) M. Interestingly, the interaction of Vn with P. carinii was not mediated through the Arg-Gly-Asp cell-adhesive domain of Vn. Addition of Arg-Gly-Asp-Ser (RGDS) peptides did not inhibit binding. In contrast, Vn binding to P. carinii was substantially inhibited by the addition of heparin or by digesting the organisms with heparitinase, suggesting that P. carinii may interact with the glycosaminoglycan-binding domain of Vn. To determine whether Vn might enhance P. carinii attachment to lung epithelial cells, we studied the binding of 51Cr-labeled P. carinii to cultured A549 lung cells. Addition of Vn resulted in significantly increased binding of P. carinii to A549 cells, whereas a neutralizing anti-Vn serum substantially reduced the binding of P. carinii to A549 cells. These data suggest that Vn binds to P. carinii and that Vn might provide an additional means by which P. carinii attaches to respiratory epithelial cells.
Pneumocystis carinii causes pneumonia in immunocompromised patients with a high morbidity and mortality rate, but the interaction between this organism and the host cell is not well understood. The purpose of this research was to study the response of host cells to P. carinii infection on a molecular level.
The technique of mRNA differential display was used to detect genes whose expression may be affected by P. carinii infection. The nucleotide sequence of one differentially displayed DNA fragment was found to be identical to that of the rat mitochondrial ATPase 6 gene, which is a subunit of the F0F1-ATP synthase complex. A four-fold increase in expression of this gene was verified by Northern blot analysis of total RNA extracted from P. carinii-infected rat lung versus that from mock-infected rat lung. Localization of the cells containing ATPase 6 mRNA was accomplished by in situ hybridization. In sections of non-infected rat lung, these cells were found lining the distal parts of the respiratory tree and in apical areas of the alveoli. Histological location of these cells suggested that they were Clara cells and type II pneumocytes. This hypothesis was confirmed by co-localizing the mRNAs for ATPase 6 and surfactant protein B (SP-B) to the same cells by two-color fluorescent in situ hybridization.
The ATPase 6 gene is over expressed during P. carinii infection, and type II pneumocytes and Clara cells are the cell types responsible for this over-expression.
Differences in gene expression between Pneumocystis carinii-infected and noninfected rats were examined. Total RNA was isolated from homogenized rat lungs and then subjected to differential display with combinations of oligo(dT) and various arbitrary PCR primers. Approximately 50 differentially expressed bands were observed. Several of these DNA bands were isolated, reamplified, and cloned. The cloned DNA fragments were used as probes to perform Northern hybridization on RNA from P. carinii-infected and noninfected rat lungs. One clone was found to react with a 3-kb mRNA from noninfected but not from P. carinii-infected rat lung, suggesting that the gene represented by this clone was down-regulated during P. carinii infection. The nucleotide sequence of this clone was determined and found to be 97% homologous to the mouse GATA-2 transcription factor. In situ hybridization using RNA probes derived from this clone revealed that alveolar macrophages, resident lung monocytes, and bronchial epithelial cells express the GATA-2 gene in the lung.
Changes in the number of alveolar macrophages were correlated with organism burden during Pneumocystis carinii infection. The lungs of healthy, dexamethasone-treated, and dexamethasone-treated and P. carinii-infected rats were lavaged with phosphate-buffered saline. Counting of alveolar macrophages in the lavage fluids revealed that P. carinii infection caused a 58% decrease in the number of alveolar macrophages and that higher P. carinii organism burdens caused a more rapid decrease in alveolar macrophage number. As a control, healthy rats were challenged with the same number of organisms as that normally used to generate P. carinii infections in dexamethasone-treated rats. Thirteen days after challenge, these rats had a profound (54%) increase in alveolar macrophage number in response to the challenge, while the number of alveolar macrophages in immunosuppressed and P. carinii-infected rats had decreased significantly by this time point. These experiments created the first animal model to mimic human pneumocystis pneumonia in alveolar macrophage number alterations. Reduction of P. carinii organism numbers by treatment of rats with trimethoprim and sulfamethoxazole brought a slow rebound in alveolar macrophage number, while recovery from P. carinii infection by cessation of immunosuppression brought a rapid rebound in alveolar macrophage number. These results suggest that both the immune state of the host and P. carinii burden affect alveolar macrophage number.
Pneumocystis carinii surface glycoprotein A (gpA) exhibits host species-specific phenotypic and genotypic variation. Despite this heterogeneity, the gpAs of P. carinii isolated from different host species appear to be homologous molecules sharing certain biochemical and antigenic characteristics. Using two degenerate oligodeoxyribonucleotide primers corresponding to conserved cysteine regions from ferret and rat P. carinii gpAs, a PCR product of approximately 300 bp was amplified from ferret, rat, and SCID mouse P. carinii-infected lung genomic DNA. Northern (RNA) hybridization revealed a transcript of 3,450 nucleotides in P. carinii-infected SCID mouse lung mRNA, which is similar in size to the transcripts for ferret and rat P. carinii gpAs. Nucleotide sequence analysis of SCID mouse P. carinii gpA subclones derived from the PCR products identified two isoforms, which were 89% identical to each other in the amplified region and 73 and 54% identical to the rat- and ferret-derived P. carinii gpA genes, respectively. Comparison of the deduced amino acid sequences of mouse, ferret, and rat P. carinii gpAs revealed striking similarity in residues adjacent to and including the conserved cysteines. Furthermore, the spacing of two proline residues is invariant, and a potential N-linked glycosylation site is found at a similar position in all of the gpAs. Despite the heterogeneity observed in P. carinii gpAs, the conservation of cysteine residues and adjacent sequences implies similar secondary structure and, most likely, similar function for the gpAs of P. carinii isolated from different host species.
Surfactant protein A (SP-A), a member of the collectin family, selectively binds to Pneumocystis carinii and mediates interactions between pathogen and host alveolar macrophages in vitro. To test the hypothesis that mice lacking SP-A have delayed clearance of Pneumocystis organisms and enhanced lung injury, wild-type C57BL/6 (WT) and SP-A-deficient mice (SP-A−/−) with or without selective CD4+-T-cell depletion were intratracheally inoculated with Pneumocystis organisms. Four weeks later, CD4-depleted SP-A-deficient mice had developed a more severe Pneumocystis infection than CD4-depleted WT (P. carinii pneumonia [PCP] scores of 3 versus 2, respectively). Whereas all non-CD4-depleted WT mice were free of PCP, intact SP-A−/− mice also had evidence of increased organism burden. Pneumocystis infection in SP-A-deficient mice was associated histologically with enhanced peribronchial and/or perivascular cellularity (score of 4 versus 2, SP-A−/− versus C57BL/6 mice, respectively) and a corresponding increase in bronchoalveolar lavage (BAL) cell counts. Increases in SP-D content, gamma interferon, interleukin-4, interleukin-5, and tumor necrosis factor alpha in BAL fluid occurred but were attenuated in PCP-infected SP-A−/− mice compared to WT mice. There were increases in total BAL NO levels in both infected groups, but nitrite levels were higher in SP-A−/− mice, indicating a reduction in production of higher oxides of nitrogen that was also reflected in lower levels of 3-nitrotyrosine staining in the SP-A−/− group. We conclude that despite increases in inflammatory cells, SP-A-deficient mice infected with P. carinii exhibit an enhanced susceptibility to the organism and attenuated production of proinflammatory cytokines and reactive oxygen-nitrogen species. These data support the concept that SP-A is a local effector molecule in the lung host defense against P. carinii in vivo.
An enzyme-linked immunosorbent assay and a Western blot analysis were developed to study the antibody response to Pneumocystis carinii in serum and bronchoalveolar lavage fluid from 27 human immunodeficiency virus 27 (HIV)-infected patients with P. carinii pneumonia (Pcp), 32 patients without Pcp, and 51 HIV-negative controls. Urea was used for the correct dilution of epithelial lining fluid, and albumin was used to evaluate transudation from plasma for the assessment of local production of antibodies to P. carinii. By contrast with those of immunoglobulin G (IgG), IgA responses to P. carinii were increased in serum from HIV-positive patients compared to negative controls. Local production of antibodies to P. carinii, especially IgA, was decreased in patients with Pcp. In a study of 10 patients of each group, IgG and IgA responses to gp116 from P. carinii were lower in patients with Pcp than in other groups. These results suggest that, in addition to alveolar macrophages, local antibodies may play a role in host defense against P. carinii.
Pneumocystis carinii obtained from infected rats and patients was cultured in the A549 cell line, a presumptive alveolar type 2 cell line derived from a human lung carcinoma. Standard criteria were established for organism sampling, quantitation, and growth. The trophozoite form of P. carinii was a more sensitive indicator of growth than was the cyst. Rat P. carinii increased 10-fold in primary culture and could be serially passed three additional times to new cultures; success in growing human P. carinii was limited and appeared to be related to the quality of the specimen received for culture. Growth pattern experiments suggested that close interaction of P. carinii with the cell monolayer is an important step in the life cycle of the organism. Thus, the A549 culture system should be useful for in vitro studies of the immunobiology of P. carinii.
The opportunistic pathogen Pneumocystis carinii causes
pneumonia (P. carinii pneumonia, or PCP) in
immunocompromised individuals such as AIDS patients. Rat-derived
P. carinii carinii organisms have distinct sterols which
are not synthesized by mammals and not found in other microbes
infecting mammalian lungs. The dominant sterol present in the organism
is cholesterol (which is believed to be scavenged from the host), but
other sterols in P. carinii carinii have an alkyl group at
C-24 of the sterol side chain (C28 and C29
24-alkylsterols) and a double bond at C-7 of the nucleus. Recently,
pneumocysterol (C32), which is essentially lanosterol with
a C-24 ethylidene group, was detected in lipids extracted from a
formalin-fixed human P. carinii-infected lung, and its
structures were elucidated by gas-liquid chromatography, mass
spectrometry, and nuclear magnetic resonance spectrometry in
conjunction with analyses of chemically synthesized authentic
standards. The sterol composition of isolated P. carinii
hominis organisms has yet to be reported. If P.
carinii from animal models is to be used for identifying
potential drug targets and for developing chemotherapeutic approaches
to clear human infections, it is important to determine whether the
24-alkylsterols of organisms found in rats are also present in
organisms in humans. In the present study, sterol analyses of P.
carinii hominis organisms isolated from cryopreserved human
P. carinii-infected lungs and from bronchoalveolar lavage
fluid were performed. Several of the same distinct sterols (e.g.,
fungisterol and methylcholest-7-ene-3β-ol) previously identified in
P. carinii carinii were also present in organisms isolated
from human specimens. Pneumocysterol was detected in only some of the
Pneumocystis carinii pneumonia (PcP) is a clinically important infection of immunocompromised patients. Although the interaction of Pneumocystis with the alveolar epithelium has been well documented, very little information regarding the epithelial response to Pneumocystis is currently available. In order to study Pneumocystis-epithelium interactions, a murine cell line derived specifically from an alveolar epithelial cell (AEC) was utilized. The coculture of murine AECs with mouse Pneumocystis induced a dose- and time-dependent release of the CXC chemokine MIP-2. Importantly, the specific removal of Pneumocystis from the preparation, or the pretreatment of AECs with sulfasalazine, a potent and specific inhibitor of NF-κB, nearly completely abrogated the chemokine response to Pneumocystis. Since the murine MIP-2 promoter contains consensus κB binding sequences, the ability of Pneumocystis to stimulate NF-κB signaling in AECs was examined. Pneumocystis stimulation of an AEC line stably transfected with a κB-dependent reporter construct triggered the NF-κB signaling pathway and reporter production. These data were confirmed in gel shift assays, providing direct evidence that Pneumocystis induced the nuclear translocation of the p50/p65 heterodimeric form of NF-κB. Maximal NF-κB activation was dependent upon direct contact with viable Pneumocystis organisms. These data demonstrate that Pneumocystis activates NF-κB signaling in AECs and establish a reporter cell line for studying NF-κB activation in AECs. Given the global regulatory functions of the NF-κB family, these findings suggest that Pneumocystis directly alters AEC gene expression in a manner that promotes pulmonary immune and inflammatory responses.
Pneumocystis carinii pneumonia was produced in two groups of rats by the administration of corticosteroids, a low-protein (8%) diet, and tetracycline in the drinking water. A third group not on corticosteroids or a low-protein diet served as controls. Members of the first group were sacrificed weekly for 8 weeks, and lungs were examined. A highly significant correlation was found between the histopathological assessment of the intensity of P. carinii infection and the number of cysts counted in enzyme-digested lungs. P. carinii progressively filled alveoli, and cyst counts increased from less than or equal to 10(4) to 10(9) cysts/g of lung at peak intensity of infection at 7 to 8 weeks. The second group of rats was placed on a regular diet and tapering doses of corticosteroids after week 4, and they were sacrificed at varying intervals for up to 21 weeks. P. carinii was not cleared from the lungs until after week 13 (more than 6 weeks after discontinuation of all steroids). Histologically, there was an increased prominence of alveolar macrophages and the progressive development of interstitial mononuclear cell infiltrate and fibrosis. Thus, P. carinii grows grows slowly in vivo and interacts with specific host cells. The resulting changes may be important in the pathogenesis of the infection and in the clearance of the organism from the lung after immunocompetence has been restored.
While CD8+ cells have been shown to contribute to lung injury during Pneumocystis carinii pneumonia (PCP), there are conflicting reports concerning the ability of CD8+ cells to kill P. carinii. To address these two issues, we studied the effect of the presence of CD8+ cells in two mouse models of PCP. In the reconstituted SCID mouse model, depletion of CD8+ cells in addition to CD4+ cells after reconstitution did not result in increased numbers of P. carinii cysts compared to the numbers of cysts in mice with only CD4+ cells depleted. This result was observed regardless of whether the mice were reconstituted with naïve or P. carinii-sensitized lymphocytes. In contrast, reconstitution with sensitized lymphocytes resulted in more rapid onset of lung injury that was dependent on the presence of CD8+ cells. The course of organism replication over a 6-week period was also examined in the CD4+-T-cell-depleted and CD4+- and CD8+-T-cell-depleted mouse model of PCP. Again, the organism burdens were identical at all times regardless of whether CD8+ cells were present. Thus, in the absence of CD4+ T cells, CD8+ T cells are a key contributor to the inflammatory lung injury associated with PCP. However, we were unable to demonstrate an in vivo effect of these cells on the course of P. carinii infection.
AIMS: To investigate the effectiveness of digoxigenin incorporated double stranded DNA probes produced by the polymerase chain reaction (PCR), for the detection of Pneumocystis carinii, using in situ hybridisation (ISH). METHODS: Formalin fixed, paraffin wax embedded sections of 26 human lung samples from 11 patients with P carinii pneumonia (PCP), and 15 with various types of fungal and viral pneumonia, were obtained during necropsy or transbronchial lung biopsy. Three additional PCP induced rat lung samples were also tested. PCR probes were prepared using the digoxigenin labelling mixture, and they were amplified from the DNA of a PCP induced rat lung after administration of dexamethasone, on the basis that 5S ribosomal RNA sequences are identical in human and rat P carinii. ISH was performed using this probe, and visualised using the digoxigenin nucleic acid detection kit. An immunohistochemical study using anti-human Pneumocystis monoclonal antibody was also carried out in parallel. RESULTS: ISH positively stained eight (of eight) lung necropsy specimens from patients with PCP, three (of three) transbronchial lung biopsy specimens from patients with PCP, and none of the three PCP induced rat lung specimens. In contrast, none of the specimens from patients with pneumonia caused by Aspergillus sp (n = 5), Candida sp (n = 4), Cryptococcus sp (n = 2), mucormycosis (n = 2), or cytomegalovirus (n = 2) were positive on ISH or immunohistochemistry. CONCLUSIONS: Using a digoxigenin labelled PCR probe for the entire 5S rRNA sequence in conjunction with conventional staining, ISH is highly reactive and specific for the diagnosis of PCP.
Pneumocystis carinii is an opportunistic fungal pathogen that causes life-threatening pneumonia in immunocompromised individuals. Infants appear to be particularly susceptible to Pneumocystis pulmonary infections. We have previously demonstrated that there is approximately a 3-week delay in the clearance of Pneumocystis organisms from pup mouse lungs compared to that in adults. We have further shown that there is approximately a 1-week delay in alveolar macrophage activation in pups versus adult mice. Alveolar macrophages are the primary effector cells responsible for the killing and clearance of Pneumocystis, suggesting that pup alveolar macrophages may be involved in the delayed clearance of this organism. Alveolar macrophages cultured in vitro with Pneumocystis alone demonstrate little to no activation, as indicated by a lack of cytokine production. However, when cultured with lipopolysaccharide (LPS) or zymosan, cytokine production was markedly increased, suggesting that pup alveolar macrophages are specifically unresponsive to Pneumocystis organisms rather than being intrinsically unable to become activated. Furthermore, pup mice treated with aerosolized, heat-killed Escherichia coli in vivo were able to clear Pneumocystis more efficiently than were control mice. Together, these data suggest that while pup alveolar macrophages are unresponsive to P. carinii f. sp. muris organisms, they are capable of activation by heat-killed E. coli in vivo, as well as LPS and zymosan in vitro. The lack of response of pup mice to P. carinii f. sp. muris may reflect protective mechanisms specific to the developing pup lung, but ultimately it results in insufficient clearance of Pneumocystis organisms.
Host defense mechanisms against Pneumocystis carinii are not fully understood. Previous work in the murine model has shown that host defense against infection is critically dependent upon host CD4+ T cells. The recently described Th17 immune response is predominantly a function of effector CD4+ T cells stimulated by interleukin-23 (IL-23), but whether these cells are required for defense against P. carinii infection is unknown. We tested the hypothesis that P. carinii stimulates the early release of IL-23, leading to increases in IL-17 production and lung effector CD4+ T-cell population that mediate clearance of infection. In vitro, stimulation of alveolar macrophages with P. carinii induced IL-23, and IL-23p19 mRNA was expressed in lungs of mice infected with this pathogen. To address the role of IL-23 in resistance to P. carinii, IL-23p19−/− and wild-type control C57BL/6 mice were infected and their fungal burdens and cytokine/chemokine responses were compared. IL-23p19−/− mice displayed transient but impaired clearance of infection, which was most apparent 2 weeks after inoculation. In confirmatory studies, the administration of either anti-IL-23p19 or anti-IL-17 neutralizing antibody to wild-type mice infected with P. carinii also caused increases in fungal burdens. IL-17 and the lymphocyte chemokines IP-10, MIG, MIP-1α, MIP-1β, and RANTES were decreased in the lungs of infected IL-23p19−/− mice in comparison to their levels in the lungs of wild-type mice. In IL-23p19−/− mice infected with P. carinii, there were fewer effector CD4+ T cells in the lung tissue. Collectively, these studies indicate that the IL-23-IL-17 axis participates in host defense against P. carinii.
Pneumocystis carinii remains an important and potentially fatal cause of opportunistic pneumonia. Animal studies reveal that substantial quantities of surfactant protein D (SP-D) accumulate in the airspaces during P. carinii pneumonia and are particularly abundant in aggregates of organisms. Due to the multimeric structure of SP-D, we hypothesized that SP-D mediates aggregation of the organism. From previous clinical studies it is known that aggregated organisms are conspicuous in sections of lung tissue and bronchoalveolar lavage (BAL) fluids of humans with active P. carinii pneumonia. Herein, we observe that SP-D levels increased at least fourfold in BAL fluids of patients with P. carinii pneumonia. Next, a spectrophotometric sedimentation assay was developed to assess the aggregation of P. carinii in vitro by SP-D. P. carinii organisms were first stripped with glutathione to remove bound SP-D and subsequently incubated in the presence of SP-D and 2 mM calcium. P. carinii incubated with natural SP-D (10 μg/ml) containing dodecamers and higher-order forms exhibited aggregation and enhanced sedimentation compared to that of glutathione-stripped P. carinii. Aggregation was also enhanced by the concentrated supernatant of rat BAL fluid, and this effect was abolished by the selective removal of SP-D from the lavage fluid. P. carinii aggregation was reduced by maltose, mannose, and EDTA, consistent with the role of the SP-D C-type lectin domain (CRD) in the aggregation event. Comparisons of different molecular forms of SP-D showed that dodecamers—but not trimeric subunits—mediate optimal aggregation of P. carinii. Aggregation of P. carinii by SP-D was shown to be responsible for the impaired phagocytosis of the organisms by alveolar macrophages. Thus, SP-D-mediated aggregation of P. carinii may represent one means by which the organism avoids elimination by the host.
Although a clear relationship between αβ T-cell receptor-positive (αβ-TCR+) CD4+ T cells and susceptibility to Pneumocystis carinii infection exists, the role of other T-cell subsets is less clearly defined. Previous studies have shown that γδ-TCR+ T cells infiltrate into the lung during P. carinii pneumonia. Therefore, the present study examined the role of γδ-TCR+ T cells in host defense against P. carinii pneumonia. C57BL/6 (control) and B6.129P2-Tcrdtm1Mom (γδ-TCR+ T-cell-deficient) mice were inoculated intratracheally with P. carinii. At specific time points, mice were sacrificed and analyzed for P. carinii burden, T-cell subsets, and cytokine levels in lung tissue. Analysis of P. carinii burden showed a more rapid and complete resolution of infection in γδ-TCR+ T-cell-deficient mice than in C57BL/6 controls. This augmented resolution was associated with elevated gamma interferon (IFN-γ) levels in bronchoalveolar lavage fluid predominantly produced by CD8+ T cells, as well as an increased recruitment of CD8+ T cells in general. In separate experiments, neutralization of IFN-γ or depletion of CD8+ T cells early during infection abolished the augmented resolution previously observed in γδ-TCR+ T-cell-deficient mice. These results show that the presence of γδ-TCR+ T cells modulates host susceptibility to P. carinii pneumonia through interactions with pulmonary CD8+ T cells and tissue production of IFN-γ.
Pneumocystis carinii has been shown to cause extra-alveolar infections in humans, but the lack of a reproducible animal model has hindered the elucidation of mechanisms of P. carinii dissemination. In the present study, PCR and the immunosuppressed rat model were used to gain further insight into the dissemination of P. carinii organisms in extrapulmonary (EP) tissues. Primer sequences specific to major surface glycoprotein (MSG) and dihydrofolate reductase (DHFR) were used to detect P. carinii in lungs and EP tissues. Sprague-Dawley rats were grouped into two classes: one group included rats that had primary episodes of pneumocystosis and the other group included rats that had undergone treatment for P. carinii infection and that had second episodes of pneumocystosis. PCR analysis with MSG primers with tissues obtained from both groups of rats showed the presence of P. carinii DNA in adrenal tissue, bone marrow, blood, and heart, kidney, liver, lymph node, spleen, and thyroid tissues. Reverse transcriptase-PCR (RT-PCR) analysis was carried out with DHFR primers with lung, spleen, heart, kidney, and liver tissues from both groups of rats. Only those tissues that showed a positive PCR result and hybridization signal for the MSG gene were used for the RT-PCR experiments. RT-PCR analysis showed that the P. carinii DHFR gene is actively transcribed in these tissues, thereby indicating the presence of viable P. carinii organisms in EP tissues. Our observations suggest that P. carinii dissemination is influenced by factors other than P. carinii chemotherapy and that heavy organism load and destruction of lung tissue may contribute to the dissemination of P. carinii. The study provides an animal model that can be used for further investigations of the causes of EP pneumocystosis.
Severe combined immunodeficient (SCID) mice lack functional lymphocytes and therefore develop Pneumocystis carinii pneumonia. However, when infected SCID mice are immunologically reconstituted with congenic spleen cells, a protective inflammatory cascade is initiated. Proinflammatory cytokines are produced, and lymphocytes and macrophages are recruited specifically to alveolar sites of infection. Importantly, uninfected regions of the lung remain free from inflammatory involvement, suggesting that there are specific mechanisms that limit inflammation in the infected lung. Therefore, to determine whether chemokines are involved in targeting the P. carinii-driven inflammatory response, steady-state mRNA levels of several chemokines were measured in the lungs of both reconstituted and nonreconstituted P. carinii-infected SCID mice. Despite significant organism burdens in the lungs of 8- and 10-week-old SCID mice, there was no evidence of elevated chemokine gene expression, which is consistent with the lack of an inflammatory response in these animals. However, when 8-week-old infected SCID mice were immunologically reconstituted, signs of focal pulmonary inflammation were observed, and levels of RANTES, MCP-1, lymphotactin, MIP-1α, MIP-1β, and MIP-2 mRNAs were all significantly elevated. Chemokine mRNA abundance was elevated at day 10 postreconstitution (PR), was maximal at day 12 PR, and returned to baseline by day 22 PR. In situ hybridization demonstrated that during the peak of inflammation, RANTES gene expression was localized to sites of inflammatory cell infiltration and P. carinii infection. Thus, these observations indicate that chemokines play a role in the focal targeting of inflammatory cell recruitment to sites of P. carinii infection after the passive transfer of lymphocytes to the host.
During Pneumocystis carinii pneumonia (PCP) in mice, the degree of pulmonary inflammation correlates directly with the severity of lung function deficits. Therefore, studies were undertaken to determine whether the host inflammatory response contributes to PCP-related respiratory impairment, at least in part, by disrupting the pulmonary surfactant system. Protein and phospholipid content and surfactant activity were measured in the lavage fluid of infected mice in either the absence or presence of an inflammatory response. At 9 weeks postinfection with P. carinii, nonreconstituted SCID mice exhibited no signs of pulmonary inflammation, respiratory impairment, or surfactant dysfunction. Lavage fluid obtained from these mice had protein/phospholipid (Pr/PL) ratios (64% ± 4.7%) and minimum surface tension values (4.0 ± 0.9 mN/m) similar to those of P. carinii-free control mice. However, when infected SCID mice were immunologically reconstituted, an intense inflammatory response ensued. Pr/PL ratios (218% ± 42%) and minimum surface tension values (27.2 ± 2.7 mN/m) of the lavage fluid were significantly elevated compared to those of the lavage fluid from infected, nonreconstituted mice (P < 0.05). To examine the specific role of CD8+ T-cell-mediated inflammation in surfactant dysfunction during PCP, mice with defined T-cell populations were studied. P. carinii-infected, CD4+-depleted mice had elevated lavage fluid Pr/PL ratios (126% ± 20%) and elevated minimum surface tension values (16.3 ± 1.0 mN/m) compared to normal mice (P < 0.05). However, when infected mice were additionally depleted of CD8+ cells, Pr/PL ratios were normal and surfactant activity was improved. These findings demonstrate that the surfactant pathology associated with PCP is related to the inflammatory process rather than being a direct effect of P. carinii. Moreover, CD8+ lymphocytes are involved in the mechanism leading to surfactant dysfunction.
The transmission of Pneumocystis carinii from person to person was studied by detecting P. carinii-specific DNA in prospectively obtained noninvasive deep-nasal-swab samples from a child with a documented P. carinii pneumonia (PCP), his mother, two contact health care workers, and 30 hospital staff members who did not enter the patient's room (controls). Nested-DNA amplification was done by using oligonucleotide primers designed for the gene encoding the mitochondrial large subunit rRNA of rat P. carinii (P. carinii f. sp. carinii) that amplifies all forms of P. carinii and internal primers specific for human P. carinii (f. sp. hominis). P. carinii f. sp. hominis DNA was detected in samples from the patient and all of his contacts versus none of the 30 hospital staff members. The results, as previously shown in murine models of P. carinii pneumonia, document that person-to-person transmission of P. carinii is possible. This observation suggests that immunocompromised patients not on PCP prophylaxis should not enter the room of a patient with PCP, and it also raises the question as to whether healthy contacts can transmit the disease to immunocompromised patients at risk.
The prevalence of Pneumocystis carinii pneumonia (PCP) in humans caused by more than a single genotype has been reported to range from 10 to 67%, depending on the method used for detection (3, 19). Most coinfections were associated with primary rather than recurrent disease. To better understand the factors influencing the development of coinfections, the time periods between inoculations and the genotype of the infecting organisms were evaluated in the chronically immunosuppressed-inoculated rat model of PCP. P. carinii f. sp. carinii infecting rats differentiated by karyotypic profiles exhibit the same low level of genetic divergence manifested by organisms infecting humans. P. carinii f. sp. carinii karyotype forms 1, 2, and 6 were inoculated into immunosuppressed rats, individually and in dual combinations, spaced 0, 10, and 20 days apart. Infections comprised of both organism forms resulted from admixtures inoculated at the same time. In contrast, coinfections did not develop in most rats, where a 10- or 20-day gap was inserted between inoculations; only the first organism form inoculated was detected by pulsed-field gel electrophoresis in the resultant infection. Organism burdens were reduced with combinations of forms 1 and 2 spaced 20 days apart but not in rats inoculated with forms 1 and 6. A role for the host response in the elimination of the second population and in reduction of the organism burden was suggested by the lack of direct killing of forms 1 and 2 in an in vitro ATP assay, by reduction of the burden by autoclaved organisms, and by the specific reactions of forms 1 and 2 but not forms 1 and 6. These studies showed that the time between inoculations was critical in establishing coinfections and P. carinii f. sp. carinii karyotype profiles were associated with differences in biological responses. This model provides a useful method for the study of P. carinii coinfections and their transmission in humans.
A repetitive genomic DNA clone (B12-2) that specifically hybridizes to Pneumocystis carinii DNA has been identified. No cross-hybridization to genomic DNA prepared from bacteria, other fungi, protozoa, or mammals was observed. Clone B12-2 is multiply represented in the P. carinii genome. By direct hybridization to DNA prepared from the lungs of immunosuppressed rats, the probe can detect the equivalent of fewer than 1,000 P. carinii organisms. A hybridization assay employing clone B12-2 has been developed to quantitate organism load in the rat model for P. carinii. Application of the assay to track the accumulation of organisms during the immunosuppression regimen as well as to monitor the efficacy of two drug therapies used clinically for the treatment of P. carinii pneumonia is described here. The clone B12-2 hybridization assay for the determination of P. carinii organism load possesses several advantageous features and thus should serve to complement conventional staining and immunohistochemical methods.
The objective of this study was to type, analyze, and compare Pneumocystis carinii hominis strains obtained from different samples during a given or recurrent episodes of P. carinii pneumonia (PCP) for epidemiologic purposes. We studied 36 bronchoalveolar lavage (BAL) or induced sputum (IS) samples from 16 human immunodeficiency virus-infected patients with one or several episodes of PCP. PCR amplification and direct sequencing were performed on the two internal transcribed spacers (ITS1 and ITS2) of P. carinii hominis rRNA genes by using DNA extracted from BAL or IS samples, and the sequences were compared to the mitochondrial large-subunit (mt LSU) gene sequence determined in a previous study in our laboratory. The studies of the mt LSU and ITS sequences showed that some patients (n = 10) were infected with the same strains of P. carinii hominis during a given episode of PCP. In one patient infected with strains with identical sequences in several episodes, the recurrence could have been due to reactivation of organisms not eliminated by treatment during the first episode or to de novo infection by an identical strain. In five patients infected with strains with different sequences in each episode, recurrence was due to de novo infection. Sequence analysis of these two P. carinii hominis gene regions showed that de novo infection can occur in AIDS patients with recurrent PCP.