Pneumocystis carinii: inhibition of lung cell growth mediated by parasite attachment.

Pneumocystis carinii pneumonia is a significant cause of mortality in immunocompromised patients. Current concepts suggest that attachment of P. carinii to alveolar epithelium is required for development of pneumonia. We examined the mechanism of P. carinii adherence to cultured A549 cells, a permanent cell line derived from human alveolar epithelium. P. carinii adherence was quantified by measuring attachment of 51Cr-labeled P. carinii to cultured A549 cells. After 8 h of incubation, 37.4 +/- 4.2% of P. carinii were adherent to A549 cells. In the presence of agents known to impair cytoskeletal function, including 10(-5) M cytochalasin B, 10(-5) M colchicine, and 10(-5) M trimethylcolchicinic acid (TMCA), adherence was decreased from 57.4 +/- 4.2% to 9.3 +/- 3.4%, 12.5 +/- 3.6%, and 21.5 +/- 3.6%, respectively (P less than 0.01, all comparisons). Secondly, we examined the effect of P. carinii on the function of A549 cells. P. carinii resulted in significant impairment of A549 cell growth, indicating P. carinii adversely affected the function of target lung cells. A P. carinii:A549 cell ratio of 50:1 resulted in 43.5 +/- 2.9% inhibition of A549 cell growth (P less than 0.001). Additionally, TMCA, which significantly prevented attachment of P. carinii, reversed the impairment of A549 cell growth. These data demonstrate that P. carinii attachment to cultured lung cells can be quantified, is dependent on intact cytoskeletal function and is necessary for impairment of lung cell replication.

The role of alveolar macrophages in Pneumocystis carinii degradation and clearance from the lung.

Although studies indicate that alveolar macrophages participate in host defense against Pneumocystis carinii, their role in organism degradation and clearance from the lung has not yet been established. We, therefore, quantified the uptake and degradation of 35S-labeled P. carinii by cultured macrophages, demonstrating significant degradation of P. carinii over 6 h. We further evaluated the role of macrophages in elimination of P. carinii from the living host. Rats received either intratracheal PBS, liposomal PBS (L-PBS), or liposomal dichloromethylene diphosphonate (L-Cl2MDP), a preparation which leads to selective depletion of macrophages. Over 72 h, L-Cl2MDP-treated animals had loss of > 85% of their alveolar macrophages. In contrast, L-PBS-treated rats had cellular differentials identical to rats receiving PBS. Macrophage-depleted rats and controls were next inoculated with P. carinii and organism clearance was determined after 24 h. P. carinii elimination was evaluated with both cyst counts and an ELISA directed against glycoprotein A (gpA), the major antigen of P. carinii. Both assays indicated that macrophage-depleted rats had substantial inpairment of P. carinii clearance compared to L-PBS- or PBS-treated rats. These data provide the first direct evidence that macrophages mediate elimination of P. carinii from the living host.

Pneumocystis carinii inhibits cyclin-dependent kinase activity in lung epithelial cells.

Pneumocystis carinii remains an important cause of pneumonia in patients with AIDS. Attachment of the organism to epithelial cells is a central event in establishing infection, impairing the growth potential of lung epithelial cells and thereby slowing repair. In light of investigations documenting a central role for cyclin-dependent kinases in controlling the cell cycle, we addressed the hypothesis that P. carinii inhibits epithelial cell growth by interfering with host epithelial cyclin-dependent kinase (cdk) activity. We observed that P. carinii significantly impaired growth of cultured mink lung epithelial cells, with effects observed after 48-72 h of treatment. However, the kinase activity associated with p34cdc2 or p33cdk2 was maximally inhibited as early as 24 h after P. carinii exposure. The inhibitory effect on cyclin-dependent kinase activity was mediated by the trophozoite form of P. carinii, in that highly purified trophozoites exerted marked inhibition of p34cdc2 activity. Growth impairment was similarly preceded by P. carinii-induced alteration in the state of epithelial cell p34cdc2 phosphorylation, with no change in p34cdc2 or p33cdk2 protein levels. These data strongly suggest that the antiproliferative activity of P. carinii on respiratory epithelium is mediated in part through modulation of the host cell cycle machinery.

Surfactant protein D interacts with Pneumocystis carinii and mediates organism adherence to alveolar macrophages.

Pneumocystis carinii interacts with glycoproteins present in the lower respiratory tract through its mannose-rich surface antigen complex termed gpA. Surfactant protein D (SP-D) is a recently described component of the airspace lining material that possesses a calcium-dependent lectin domain capable of interacting with glycoconjugates present on microorganisms and leukocytes. Accordingly, we evaluated the extent and localization of SP-D in the lower respiratory tract during Pneumocystis pneumonia in an immunosuppressed rat model and examined its role in modulating interaction of P. carinii with macrophages. We report that SP-D is a major component of the alveolar exudates that typify P. carinii pneumonia and is present bound to the surface of P. carinii organisms in vivo. We further demonstrate that SP-D binds to P. carinii through saccharide-mediated interactions with gpA present on the surface of the organism. Lastly, we show that SP-D augments binding of P. carinii to alveolar macrophages, but does not significantly enhance macrophage phagocytosis of the organism. The interaction of SP-D with gpA represents an additional important component of the host-parasite relationship during P. carinii pneumonia.

Vitronectin interacts with Candida albicans and augments organism attachment to the NR8383 macrophage cell line.

Candida albicans is an increasingly important cause of mucocutaneous and bloodstream infections. The potential role of circulating adhesive glycoproteins such as vitronectin (Vn) in host defense against C. albicans is currently unknown. Accordingly, we investigated the binding of plasma-derived Vn with C. albicans strain 36082. Vn specifically bound to C. albicans in a concentration-dependent fashion. Higher affinity binding sites numbered 9.8 x 10(4) sites per organism, with a dissociation constant, Kd of 3.5 x 10(-7) M. Vn binding with C. albicans was significantly inhibited by heparin, suggesting interaction of the organism with Vn's glycosaminoglycan-binding region. To further determine which molecule(s) on the fungus interacted with Vn, C. albicans components were extracted, separated by SDS and blotted with radiolabeled Vn. These studies revealed that Vn binds to a 30 kDa molecule on C. albicans. Finally, we investigated the role of Vn in promoting interaction of C. albicans with phagocytic cells. Incubation of C. albicans in the presence of Vn significantly increased binding of the organism to cultured NR8383 macrophages compared to incubations performed in the absence of Vn. These data demonstrate that C. albicans interacts with the heparin-binding domain Vn and further suggest that Vn augments organism uptake by phagocytic cells.

Fungal beta-glucans modulate macrophage release of tumor necrosis factor-alpha in response to bacterial lipopolysaccharide.

Tumor necrosis factor-alpha (TNF alpha) is a potent cytokine believed to participate in the development of endotoxin-induced shock and the adult respiratory distress syndrome. Treatment of animals with beta-glucan prior to bacterial challenge reduces TNF alpha release and prevents death. We therefore hypothesized that beta-glucan might regulate TNF alpha secretion from macrophages in response to lipopolysaccharide (LPS). Rat alveolar macrophages were cultured in the presence of beta-glucan alone and the TNF alpha secretion quantified using an L929 cytotoxicity assay. Concentrations of beta-glucan less than 500 micrograms/ml were found to stimulate TNF alpha release from macrophages. However, concentrations of beta-glucan greater than 500 micrograms/ml resulted in suppression of the TNF alpha activity released. This reduction in TNF alpha release was not mediated by a toxic effect of beta-glucan, as large concentrations of beta-glucan had no effect on macrophage viability. We further observed that the incubation of macrophages with large concentrations of beta-glucan (500 micrograms/ml) also inhibited the secretion of TNF alpha induced by bacterial LPS. Furthermore, interferon-gamma (IFN gamma), a potent activator of TNF alpha expression, failed to overcome the inhibition of TNF alpha caused by beta-glucan. These data suggest an immunomodulatory role for beta-glucan which may explain both the TNF alpha-stimulating and -inhibiting effects of fungal beta-glucans during infection.

Pneumocystis carinii pneumonia in patients without acquired immunodeficiency syndrome: associated illness and prior corticosteroid therapy.

OBJECTIVE: To determine the clinical spectrum of immunosuppressive conditions and systemic corticosteroid therapy associated with the development of Pneumocystis carinii pneumonia in a consecutive series of patients without acquired immunodeficiency syndrome (AIDS). DESIGN: We retrospectively analyzed a consecutive series of 116 patients without AIDS who were assessed at Mayo Medical Center for a first episode of P. carinii pneumonia between 1985 and 1991. METHODS: Medical records were examined to determine underlying immunosuppressive disorders, premorbid corticosteroid dosage and duration of therapy, associated infections, and subsequent respiratory failure and in-hospital mortality. RESULTS: Conditions associated with a first episode of P. carinii pneumonia were hematologic malignant disorders (30.2%), organ transplantation (25.0%), inflammatory disorders (22.4%), solid tumors (12.9%), and miscellaneous conditions (9.5%). Regardless of the associated underlying disease, corticosteroids had been administered systemically in 105 patients (90.5%) within 1 month before the diagnosis of P. carinii pneumonia. The median daily corticosteroid dose was equivalent to 30 mg of prednisone; however, 25% of patients had received as little as 16 mg of prednisone daily. The median duration of corticosteroid therapy was 12 weeks before the development of pneumonia; however, P. carinii pneumonia developed after 8 weeks or less of corticosteroid therapy in 25% of these patients. Respiratory failure occurred in 43%, and in-hospital mortality was 34% for patients with P. carinii pneumonia in conditions other than AIDS. CONCLUSION: Although these results do not suggest that premorbid administration of corticosteroids is the only factor that contributes to the development of P. carinii pneumonia in these patients, they show that, in this large consecutive series, systemic corticosteroid therapy, even in moderate doses, was administered to most patients during the month preceding the onset of P. carinii pneumonia. Consideration should be given to instituting P. carinii prophylaxis (when not contra-indicated) in patients for whom prolonged systemic corticosteroid therapy is prescribed.

Cardiopulmonary metastatic lesions of osteosarcoma and associated cerebral infarction.

Osteogenic sarcoma frequently disseminates by hematogenous routes. A 32-year-old patient underwent evaluation for an acute cerebral infarction. Cardiac auscultation disclosed an abnormal diastolic sound. Echocardiographic examination revealed a large left atrial mass, which was found at thoracotomy to be metastatic osteogenic sarcoma. Cerebral computed tomographic scans at the time of initial examination and 3 months later demonstrated new cerebral lesions consistent with metastatic growths. The abrupt cerebral infarction, other clinical findings, and results of diagnostic studies strongly suggested that the acute cerebrovascular event was the result of metastatic cerebral embolization from the tumor material found in the thorax. Cerebral infarction is an unusual and catastrophic complication of thoracic metastatic lesions of osteogenic sarcoma.

Diagnosing ventilator-associated pneumonia: the role of bronchoscopy.

OBJECTIVE: To discuss the two diagnostic procedures used most frequently to obtain uncontaminated lower airway secretions during bronchoscopy. DESIGN: This article reviews the contributing risk factors of ventilator-associated pneumonia (VAP) and the recent studies that have assessed the usefulness of the protected specimen brush (PSB) and bronchoalveolar lavage (BAL) in the nonimmunocompromised host. RESULTS: A prompt, accurate diagnosis of VAP, including specific identification of the bacterial pathogen, remains a common challenge in the intensive-care unit. Standard clinical criteria are of suboptimal specificity for making decisions, including selecting antibiotic therapy. Bronchoscopic techniques of lung secretion sampling can be used in the intensive-care unit in an effort to overcome the effects of oropharyngeal contamination. The PSB and BAL, used appropriately, can help intensive-care clinicians formulate specific antimicrobial therapy. Evaluation of intracellular bacteria obtained by BAL has been reported to be useful in guiding empiric antibiotic therapy while the final results of cultures obtained with the PSB are pending. Prior antibiotic therapy, however, may confound the interpretation and clinical utility of results. CONCLUSION: Currently, for a patient taking antibiotic therapy, no reliable technique nor quantitative culture threshold exists to help in diagnosing suspected VAP or in guiding antibiotic therapy. If the clinical situation allows, antibiotic therapy should be discontinued for 48 hours; then, the PSB, BAL, protected BAL, or endobronchial aspiration should be used. These contemporary modalities, however, necessitate further clinical trials before widespread use is warranted.

Constrictive pericarditis and pleuropulmonary disease linked to ergot dopamine agonist therapy (cabergoline) for Parkinson's disease.

Cabergoline is one of several ergoline dopamine agonist medications used in the treatment of Parkinson's disease (PD). We diagnosed constrictive pericarditis (CP) in a patient with PD receiving cabergoline therapy (10 mg daily), who had symptoms and signs of congestive heart failure (CHF). In the absence of previous reported cases of this condition linked to ergoline drugs, cabergoline was not initially identified as the cause. Shortly thereafter, however, the patient developed of a severe pleuropulmonary inflammatory-fibrotic syndrome, a recognized complication of ergoline medications, thus suggesting a common pathogenesis due to cabergoline therapy. To our knowledge, this is the first case in the English literature, although we speculate that CP may be more common than reported among patients with PD who are treated with an ergoline drug (cabergoline, bromocriptine, pergolide, or lisuride). The diagnosis of CP is difficult and requires a high level of suspicion; symptoms may masquerade as CHF due to common mechanisms such as coronary artery disease. In patients with PD who are taking not only cabergoline but also one of the other ergoline drugs, CP should be suspected if symptoms of CHF develop.

Fibronectin. A versatile matrix protein with roles in thoracic development, repair and infection.

Fibronectin, a dimeric cell-adhesive extracellular matrix glycoprotein, is secreted by mesenchymal cells and assembled into insoluble matrices which have important biological functions in embryologic development as well as in tissue response to injury. Fibronectin interacts with numerous cell types including mesenchymal cells and inflammatory cells which bear appropriate fibronectin receptors. In vitro, fibronectin serves as an adhesive substrate and promotes cell proliferation and cytodifferentiation. During development, fibronectin-rich matrices are deposited in specific location and regulate the directional migration of embryonic cells. In particular, fibronectin matrices appear to be of critical importance to normal cardiopulmonary development. Following embryologic development, the tissue expression of fibronectin is greatly reduced, but increases markedly following tissue injury, where newly expressed fibronectin matrices appear critical to tissue repair. Recent evidence has documented increased expression of fibronectin in numerous pulmonary conditions including the adult respiratory distress syndrome (ARDS), bronchiolitis obliterans organizing pneumonia (BOOP) and idiopathic pulmonary fibrosis (IPF). Additionally, fibronectin also interacts with a large number of microorganisms and therefore also is potentially important in microbial adherence to airway epithelium and subsequent infections of the respiratory system.

Laboratory diagnosis of Pneumocystis carinii infections by PCR directed to genes encoding for mitochondrial 5S and 28S ribosomal RNA.

PCR with 5S mitochondrial ribosomal RNA (5S) target is a sensitive and specific assay for the detection of Pneumocystis carinii in clinical specimens from the respiratory tract. We developed an oligonucleotide probe directed to a 200 bp amplicon generated by fungal-specific universal primers that anneals with sequences specific for P. carinii in the 28S ribosomal RNA gene (28S). Of 50 archived bronchoalveolar lavage 1(BAL) specimens, 46 of 50 samples (92% agreement) gave the same result (23 positive, 23 negative) by PCR directed to the 5S and 28S assays. Results of calcofluor white staining of BAL smears on slides indicated agreement with the molecular results in 43 of 46 (93.5%) assays. PCR detection of P. carinii by amplification of 28S ribosomal gene target by fungal-specific primers and an organism-specific probe provides an alternate genomic target for the laboratory diagnosis of this organism.

Mechanisms of defence in the lung: lessons from Pneumocystis carinii pneumonia.

Pneumocystis carinii continues to represent an important complication of individuals with compromised immunity. P. carinii interacts with immune and non-immune cells in the lung and mediates lung injury through a variety of mechanisms. CD4+ T lymphocytes are the cornerstone in defence against P. carinii. Recent studies indicate that alveolar macrophages provide essential functions that significantly enhance clearance of P. carinii infection. P. carinii also attaches to alveolar epithelial cells, causing inhibition of epithelial growth and replication. In addition to cellular interactions, P. carinii organisms bind to a variety of host adhesive proteins present in the lower respiratory tract. Binding of these proteins to P. carinii modulates host cell recognition and immune responses to the parasite. During the course of P. carinii pneumonia, several inflammatory mediators are produced in the lung. Although necessary for control of infection, exuberant inflammatory responses also predispose the host to the development of acute lung injury. Thus, host defences against P. carinii depend on complex interactions between immune and non-immune cells as well as several mediators that facilitate host recognition and eventual elimination of infection. Understanding these complex processes may enable development of novel therapeutic approaches for management of this important infection.

Steady-state effects of vitronectin and fibronectin on the binding, uptake, and degradation of Pneumocystis carinii in rat alveolar macrophages.

Pneumocystis carinii pneumonia remains a serious complication of immunodeficiency. Vitronectin (VN) and fibronectin (FN) accumulate in the lung during P. carinii infection and bind to the organism, thereby enhancing macrophage release of TNF alpha. It is not known whether VN and FN also regulate uptake and degradation of P. carinii by macrophage when present in concentrations similar to those in the lung during pneumonia. To address this, macrophages were cultured with 35S-radiolabeled P. carinii and organism binding, phagocytosis, and degradation determined in media alone (control), or in the presence of VN or FN (100 micrograms/ml each). Soluble VN and FN, in concentrations similar to those in the host, did not significantly affect binding uptake or degradation of P. carinii by alveolar macrophages. Thus, although VN and FN enhance macrophage activation during P. carinii pneumonia, phagocytosis of the organism is not increased by these host glycoproteins under steady-state conditions.

Candida albicans induces the release of inflammatory mediators from human peripheral blood monocytes.

Candida albicans (C. albicans) is a major nosocomial pathogen. We examined arachidonic acid (AA) and cytokine production by monocytes stimulated with C. albicans. [14C]-AA labeled monocytes released 8.9 +/- 2.3% of the incorporated AA following stimulation with live C. albicans (C. albicans: monocyte of 16:1) (P = 0.0002). Prior studies indicate that soluble alpha-mannans and beta-glucans antagonize mannose and beta-glucan receptors, respectively. Preincubation of monocytes with alpha-mannan (100 micrograms/ml) caused 45.8 +/- 5.7% inhibition of [14C]-AA release, whereas beta-glucan (100 micrograms/ml) yielded 43.7 +/- 6.0% inhibition (P < 0.05 for each compared to control). Additionally, monocytes stimulated with C. albicans also released interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8). However, alpha-mannan or beta-glucan failed to inhibit IL-1 beta release. These data indicate that C. albicans induces monocytes to release AA and inflammatory cytokines. Furthermore, AA, but not cytokine liberation, is partially mediated by alpha-mannan and beta-glucan components of the fungus.