Chao Hung Lee

Clinical correlation of variations in the internal transcribed spacer regions of rRNA genes in Pneumocystis carinii f.sp. hominis.

ObjectivesTo analyse the importance of sequence variations in the internal transcribed spacer (ITS) regions 1 and 2 of the nuclear rRNA operon in AIDS patients with Pneumocystis carinii pneumonia (PCP). Design and methodsITS 1 and 2 genotypes were determined in 162 bronchoalveolar lavage samples from 130 patients participating in a prospective cohort study of PCP. ResultsA total of 49 different ITS genotypes were detected. ITS genotype was not associated with the clinical severity or outcome of PCP. In 37 of 162 (23%) samples infection with two or more genotypes was observed. A genotype switch was detected in six of 10 patients (60%) with recurrent episodes of PCP. However, genotype changes were also seen in 10 of 19 patients (53%) who had repeated bronchoscopies within the same episode of PCP. The same ITS type was observed twice in 13 (46%) of the 28 patients with repeat bronchoscopies during single or recurrent episodes of pneumonia, but in only 14 of 81 (17%) randomly selected pairs (P < 0.01). ConclusionAlthough the detection of ITS genotypes is not a random event, changes in genotype can be detected in a single episode of disease, with 23% of PCP patients being infected with more than one P. carinii genotype, thus complicating the use of this locus as a genetic marker to separate new infection from the reactivation of latent infection. ITS genotypes are not associated with the clinical severity of PCP.

Toll-Like Receptor 2 Mediates Alveolar Macrophage Response to Pneumocystis murina

ABSTRACT The innate immune response to Pneumocystis infection is not well understood. In this study, normal C57BL/6 mouse alveolar macrophages were found to respond to Pneumocystis murina organisms through Toll-like receptor 2 (TLR2), leading to the nuclear translocation of NF-κB and the production of proinflammatory cytokine tumor necrosis factor alpha (TNF-α) and chemokine macrophage inflammatory protein 2 (MIP-2). P. murina stimulation of normal alveolar macrophages from C57BL/6 mice resulted in increased TLR2 transcription but not increased TLR4 transcription. In gain-of-function studies with HEK293 cells expressing TLR2 or TLR4, only TLR2 was found to stimulate an NF-κB response to P. murina. TNF-α and MIP-2 production in response to P. murina by mouse alveolar macrophages was inhibited by a monoclonal antibody that specifically blocked the ligand-binding ability of TLR2. Alveolar macrophages from TLR2 knockout (TLR2−/−) mice showed little increase in TNF-α and MIP-2 mRNA levels upon P. murina stimulation. An in vivo study showed that TLR2−/− mice challenged with P. murina had reduced cytokine responses. These results indicate that TLR2 plays a major role in the innate immune response to P. murina.

Correlation of Organism Burden and Alveolar Macrophage Counts during Infection with Pneumocystis carinii and Recovery

ABSTRACT 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.

Effect of Transcription Factor GATA-2 on Phagocytic Activity of Alveolar Macrophages from Pneumocystis carinii-Infected Hosts

ABSTRACT Alveolar macrophages from Pneumocystis carinii-infected hosts are defective in phagocytosis (W. Chen, J. W. Mills, and A. G. Harmsen, Int. J. Exp. Pathol. 73:709-720, 1992; H. Koziel et al., J. Clin. Investig. 102:1332-1344, 1998). Experiments were performed to determine whether this defect is specific for P. carinii organisms. The results showed that these macrophages were unable to phagocytose both P. carinii organisms and fluorescein isothiocyanate (FITC)-conjugated latex beads, indicating that alveolar macrophages from P. carinii-infected hosts have a general defect in phagocytosis. To determine whether this defect correlates with the recently discovered down-regulation of the GATA-2 transcription factor gene during P. carinii infection, alveolar macrophages from dexamethasone-suppressed or healthy rats were treated with anti-GATA-2 oligonucleotides and then assayed for phagocytosis. Aliquots of the alveolar macrophages were also treated with the sense oligonucleotides as the control. Cells treated with the antisense oligonucleotides were found to have a 46% reduction in phagocytosis of P. carinii organisms and a 65% reduction in phagocytosis of FITC-latex beads compared to those treated with the sense oligonucleotides. To determine whether the defect in phagocytosis in alveolar macrophages from P. carinii-infected hosts can be corrected by overexpression of GATA-2, a plasmid containing the rat GATA-2 gene in the sense orientation driven by the cytomegalovirus (CMV) promoter was introduced into alveolar macrophages from P. carinii-infected rats. Aliquots of the same cells transfected with a plasmid containing GATA-2 in the antisense orientation relative to the CMV promoter served as the control. Alveolar macrophages treated with the sense GATA-2 expression construct were found to increase their phagocytic activity by 66% in phagocytosis of P. carinii organisms and by 280% in phagocytosis of FITC-latex beads compared to those that received the antisense GATA-2 construct. The results of this study indicate that GATA-2 plays an important role in the regulation of phagocytosis in alveolar macrophages during P. carinii infection.

Polyamine-mediated apoptosis of alveolar macrophages during Pneumocystis pneumonia

The number of alveolar macrophages is decreased during Pneumocystis pneumonia (Pcp), partly because of activation of apoptosis in these cells. This apoptosis occurs in both rat and mouse models of Pcp. Bronchoalveolar lavage (BAL) fluids from Pneumocystis-infected animals were found to contain high levels of polyamines, including spermidine, N1-acetylspermine, and N1-acetylspermidine. These BAL fluids and exogenous polyamines were able to induce apoptosis in alveolar macrophages. Apoptosis of alveolar macrophages during infection, after incubation with BAL fluids from Pneumocystis-infected animals, or after incubation with polyamines was marked by an increase in intracellular reactive oxygen species, activation of caspases-3 and -9, DNA fragmentation, and leakage of mitochondrial cytochrome c into the cytoplasm. When polyamines were depleted from the BAL fluids of infected animals, the ability of these BAL fluids to induce apoptosis was lost. Interestingly, the apoptosis inducing activity of the polyamine-depleted BAL fluids was restored when polyamines were added back. The results of this study suggested that Pneumocystis infection results in accumulation of high levels of polyamines in the lung. These polyamines activate apoptosis of alveolar macrophages, perhaps because of the ROS that are produced during polyamine metabolism.

Pneumocystis jiroveci in Portuguese immunocompromised patients: association of specific ITS genotypes with treatment failure, bad clinical outcome and childhood

We analyzed the genetic variation among isolates of Pneumocystis jiroveci from Portuguese immunocompromised patients with PCP at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon and at the dihydropteroate synthase (DHPS) gene. Pulmonary secretions from 42 patients with PCP corresponding to 43 episodes were studied. Demographic, immunological, and clinical data were obtained from all patients. By combining the two regions ITS1 and ITS2, we found 17 different ITS types of P. jiroveci, two of them were new types (Pb and Pe). The four most prevalent ITS types were Eg (23.3%), Eb and Ne (11.6% each), and Bi (9.3%). A single type was detected in 95.3% of the samples and 4.7% had mixed infections with three different ITS types. DHPS mutants were present in 17 (46%), and the wildtype was present in 20 (54%) of 37 isolates. No association was found between ITS and DHPS types and between DHPS types and therapy or response to anti-PCP treatment. Type Ne presented an association with negative response to anti-PCP treatment (P<0.001) and with death before 120 days after PCP diagnosis (P=0.025). Type Eb was significantly more common in children than in adults (P=0.001). Our data suggest an association of specific ITS genotypes with treatment failure, bad clinical outcome and childhood.

Effect of Bronchoalveolar Lavage Fluid from Pneumocystis carinii- Infected Hosts on Phagocytic Activity of Alveolar Macrophages

ABSTRACT Alveolar macrophages from Pneumocystis carinii-infected rats are defective in phagocytosis. To investigate whether this defect is due to a certain factor present in P. carinii-infected lungs, alveolar macrophages from uninfected rats were incubated with bronchoalveolar lavage (BAL) fluid samples from P. carinii-infected rats. Alveolar macrophages treated with these BAL fluid samples became defective in phagocytosis but remained normal when treated with BAL fluid samples from noninfected or Toxoplasma gondii-infected rats. The suppressive activity of the BAL fluid samples from P. carinii-infected rats on phagocytosis was retained when the BAL fluid samples were passed through a filter with a pore size of 0.45 μm but was lost when the BAL fluid samples were digested with proteases such as trypsin, pepsin, papain, or endopeptidase Gly-C. Lipid fractions of these BAL fluid samples had no suppressive activity on phagocytosis. The suppressive activity of these BAL fluid samples was also lost when they were incubated with concanavalin A-agarose beads, suggesting that the inhibitor is a glycoprotein. The inhibitor was estimated to be larger than 100,000 Da by exclusion filtration. After binding to the concanavalin A-agarose beads, the inhibitor in BAL fluid samples and P. carinii lysate could be eluted with 200 mM methylmannose. Treatment of both the crude BAL fluid samples and P. carinii lysate and the 200 mM methylmannose eluate with antibody against the major surface glycoprotein of P. carinii eliminated their suppressive activity. These results suggest that the factor capable of suppressing the phagocytic activity of alveolar macrophages is P. carinii major surface glycoprotein or one or more of its derivatives.

(1–3)-Beta-D-glucan in association with lactate dehydrogenase as biomarkers of Pneumocystis pneumonia (PcP) in HIV-infected patients

Pneumocystis pneumonia (PcP) is a major HIV-related illness caused by Pneumocystis jirovecii. Definitive diagnosis of PcP requires microscopic detection of P. jirovecii in pulmonary specimens. The objective of this study was to evaluate the usefulness of two serum markers in the diagnosis of PcP. Serum levels of (1–3)-beta-d-glucan (BG) and lactate dehydrogenase (LDH) were investigated in 100 HIV-positive adult patients and 50 healthy blood donors. PcP cases were confirmed using indirect immunofluorescence with monoclonal anti-Pneumocystis antibodies and nested-PCR to amplify the large subunit mitochondrial rRNA gene of P. jirovecii in pulmonary specimens. BG and LDH levels in serum were measured using quantitative microplate-based assays. BG and LDH positive sera were statistically associated with PcP cases (Pu2009≤u20090.001). Sensitivity, specificity, positive/negative predictive values (PPV/NPV), and positive/negative likelihood ratios (PLR/NLR) were 91.3xa0%, 61.3xa0%, 85.1xa0%, 79.2xa0%, 2.359, and 0.142, respectively, for the BG kit assay, and 91.3xa0%, 35.5xa0%, 75.9xa0%, 64.7xa0%, 1.415 and 0.245, respectively, for the LDH test. Serologic markers levels combined with the clinical diagnostic criteria for PcP were evaluated for their usefulness in diagnosis of PcP. The most promising cutoff levels for diagnosis of PcP were determined to be 400xa0pg/ml of BG and 350 U/l of LDH, which combined with clinical data presented 92.8xa0% sensitivity, 83.9xa0% specificity, 92.8xa0% PPV, 83.9xa0% NPV, 5.764 PLR and 0.086 NLR (Pu2009<u20090.001). This study confirmed that BG is a reliable indicator for detecting P. jirovecii infection. The combination between BG/LDH levels and clinical data is a promising alternative approach for PcP diagnosis.

Down-regulation of GATA-2 transcription during Pneumocystis carinii infection.

ABSTRACT 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.

Effects of Decreased Calmodulin Protein on the Survival Mechanisms of Alveolar Macrophages during Pneumocystis Pneumonia

ABSTRACT Pneumocystis infection causes increased intracellular levels of reactive oxygen species (ROS) and the subsequent apoptosis of alveolar macrophages (Amø). Assessments of key prosurvival molecules in Amø and bronchoalveolar lavage fluids from infected rats and mice showed low levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and reduced activation of phosphoinositide-3 kinase (PI-3K). Ubiquitous calcium-sensing protein calmodulin protein and mRNA levels were also reduced in Amø during Pneumocystis pneumonia (Pcp). Calmodulin has been implicated in control of GM-CSF production and PI-3K activation in other immune cell types. Experiments to determine the control of GM-CSF and PI-3K by calmodulin in Amø showed that GM-CSF expression and PI-3K activation could not be induced when calmodulin was inhibited. Calmodulin inhibition also led to increased levels of ROS and apoptosis in cells exposed to bronchoalveolar lavage fluids from infected animals. Supplementation of Amø with exogenous calmodulin increased survival signaling via GM-CSF and PI-3K and reduced ROS and apoptosis. These data support the hypotheses that calmodulin levels at least partially control survival signaling in Amø and that restoration of GM-CSF or PI-3K signaling will improve host response to the organism.