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Find video protocols related to scientific articles indexed in Pubmed.
Pneumocystis.
Cold Spring Harb Perspect Med
PUBLISHED: 11-05-2014
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Since its initial misidentification as a trypanosome some 100 years ago, Pneumocystis has remained recalcitrant to study. Although we have learned much, we still do not have definitive answers to such basic questions as, where is the reservoir of infection, how does Pneumocystis reproduce, what is the mechanism of infection, and are there true species of Pneumocystis? The goal of this review is to provide the reader the most up to date information available about the biology of Pneumocystis and the disease it produces.
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Pneumonia. Treatment and diagnosis.
Ann Am Thorac Soc
PUBLISHED: 08-23-2014
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Pneumonia remains a leading cause of morbidity and mortality despite advances in treatment and therapy. The "Pneumonia: Treatment and Diagnosis" session of the Pittsburgh International Lung Conference examined topics related to improving care of patients with pneumonia. These topics included the process and quality of care for community-acquired pneumonia (CAP), diagnosis and treatment of emerging fungal pathogens, an overview of the strengths and weaknesses of different diagnostic modalities, and an example of how basic science is exploring immunomodulatory strategies for pneumonia treatment. Systematic health care provider and institutional improvements can decrease mortality rates in CAP, particularly in patients with increasingly complex comorbidities. Aspects of current guidelines for the diagnosis and treatment of fungal pneumonia were reviewed through a series of case presentations. Proper treatment of pneumonia hinges on correct pathogen identification but is complicated by the variety of diagnostic assays with variable specificity, sensitivity, and interpretation. In addressing this topic, Dr. Patrick Murray, Ph.D., discussed a range of diagnostic tests for a variety of pathogens and guidelines for their use. In addition to the current state of CAP treatment, Bill (Beibei) Chen, M.D., Ph.D., presented a new potential therapeutic agent called forsythin, an immunomodulatory compound derived from a plant used in traditional Chinese medicine. These topics, ranging from institution-sized policy to interactions at the molecular scale, paint a broad perspective of the efforts against pneumonia.
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Pneumocystis jirovecii Rtt109, a novel drug target for Pneumocystis pneumonia in immunosuppressed humans.
Antimicrob. Agents Chemother.
PUBLISHED: 04-14-2014
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Pneumocystis pneumonia (PcP) is a significant cause of morbidity and mortality in immunocompromised patients. In humans, PcP is caused by the opportunistic fungal species Pneumocystis jirovecii. Progress in Pneumocystis research has been hampered by a lack of viable in vitro culture methods, which limits laboratory access to human-derived organisms for drug testing. Consequently, most basic drug discovery research for P. jirovecii is performed using related surrogate organisms such as Pneumocystis carinii, which is derived from immunosuppressed rodents. While these studies provide useful insights, important questions arise about interspecies variations and the relative utility of identified anti-Pneumocystis agents against human P. jirovecii. Our recent work has identified the histone acetyltransferase (HAT) Rtt109 in P. carinii (i.e., PcRtt109) as a potential therapeutic target for PcP, since Rtt109 HATs are widely conserved in fungi but are absent in humans. To further address the potential utility of this target in human disease, we now demonstrate the presence of a functional Rtt109 orthologue in the clinically relevant fungal pathogen P. jirovecii (i.e., PjRtt109). In a fashion similar to that of Pcrtt109, Pjrtt109 restores H3K56 acetylation and genotoxic resistance in rtt109-null yeast. Recombinant PjRtt109 is an active HAT in vitro, with activity comparable to that of PcRtt109 and yeast Rtt109. PjRtt109 HAT activity is also enhanced by the histone chaperone Asf1 in vitro. PjRtt109 and PcRtt109 showed similar low micromolar sensitivities to two reported small-molecule HAT inhibitors in vitro. Together, these results demonstrate that PjRtt109 is a functional Rtt109 HAT, and they support the development of anti-Pneumocystis agents directed at Rtt109-catalyzed histone acetylation as a novel therapeutic target for human PcP.
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Correlation of regional emphysema and lung cancer: a lung tissue research consortium-based study.
J Thorac Oncol
PUBLISHED: 03-26-2014
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Chronic obstructive pulmonary disease and lung cancer are linked because both airflow obstruction and emphysema, on computer tomography, are independent risk factors for lung cancer. However, the local risk of malignancy relative to development of regional emphysema has not yet been defined. Specifically, it is not known if primary lung cancers are associated with regions of worse emphysema within individual patients.
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AIDS-related mycoses: the way forward.
Trends Microbiol.
PUBLISHED: 03-04-2014
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The contribution of fungal infections to the morbidity and mortality of HIV-infected individuals is largely unrecognized. A recent meeting highlighted several priorities that need to be urgently addressed, including improved epidemiological surveillance, increased availability of existing diagnostics and drugs, more training in the field of medical mycology, and better funding for research and provision of treatment, particularly in developing countries.
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Reduced GM1 ganglioside in CFTR-deficient human airway cells results in decreased ?1-integrin signaling and delayed wound repair.
Am. J. Physiol., Cell Physiol.
PUBLISHED: 02-05-2014
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Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function reduces chloride secretion and increases sodium uptake, but it is not clear why CFTR mutation also results in progressive lung inflammation and infection. We previously demonstrated that CFTR-silenced airway cells migrate more slowly during wound repair than CFTR-expressing controls. In addition, CFTR-deficient cells and mouse models have been reported to have altered sphingolipid levels. Here, we investigated the hypothesis that reduced migration in CFTR-deficient airway epithelial cells results from altered sphingolipid composition. We used cell lines derived from a human airway epithelial cell line (Calu-3) stably transfected with CFTR short hairpin RNA (CFTR-silenced) or nontargeting short hairpin RNA (controls). Cell migration was measured by electric cell substrate impedance sensing (ECIS). Lipid analyses, addition of exogenous glycosphingolipids, and immunoblotting were performed. We found that levels of the glycosphingolipid, GM1 ganglioside, were ~60% lower in CFTR-silenced cells than in controls. CFTR-silenced cells exhibited reduced levels of activated ?1-integrin, phosphorylated tyrosine 576 of focal adhesion kinase (pFAK), and phosphorylation of Crk-associated substrate (pCAS). Addition of GM1 (but not GM3) ganglioside to CFTR-silenced cells restored activated ?1-integrin, pFAK, and pCAS to near control levels and partially restored (~40%) cell migration. Our results suggest that decreased GM1 in CFTR-silenced cells depresses ?1-integrin signaling, which contributes to the delayed wound repair observed in these cells. These findings have implications for the pathology of cystic fibrosis, where altered sphingolipid levels in airway epithelial cells could result in a diminished capacity for wound repair after injury.
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Idiopathic pulmonary fibrosis: evolving concepts.
Mayo Clin. Proc.
PUBLISHED: 02-03-2014
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Idiopathic pulmonary fibrosis (IPF) occurs predominantly in middle-aged and older adults and accounts for 20% to 30% of interstitial lung diseases. It is usually progressive, resulting in respiratory failure and death. Diagnostic criteria for IPF have evolved over the years, and IPF is currently defined as a disease characterized by the histopathologic pattern of usual interstitial pneumonia occurring in the absence of an identifiable cause of lung injury. Understanding of the pathogenesis of IPF has shifted away from chronic inflammation and toward dysregulated fibroproliferative repair in response to alveolar epithelial injury. Idiopathic pulmonary fibrosis is likely a heterogeneous disorder caused by various interactions between genetic components and environmental exposures. High-resolution computed tomography can be diagnostic in the presence of typical findings such as bilateral reticular opacities associated with traction bronchiectasis/bronchiolectasis in a predominantly basal and subpleural distribution, along with subpleural honeycombing. In other circumstances, a surgical lung biopsy may be needed. The clinical course of IPF can be unpredictable and may be punctuated by acute deteriorations (acute exacerbation). Although progress continues in unraveling the mechanisms of IPF, effective therapy has remained elusive. Thus, clinicians and patients need to reach informed decisions regarding management options including lung transplant. The findings in this review were based on a literature search of PubMed using the search terms idiopathic pulmonary fibrosis and usual interstitial pneumonia, limited to human studies in the English language published from January 1, 2000, through December 31, 2013, and supplemented by key references published before the year 2000.
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Identification of a cell-of-origin for fibroblasts comprising the fibrotic reticulum in idiopathic pulmonary fibrosis.
Am. J. Pathol.
PUBLISHED: 01-02-2014
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Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the middle aged and elderly with a prevalence of one million persons worldwide. The fibrosis spreads from affected alveoli into contiguous alveoli, creating a reticular network that leads to death by asphyxiation. Lung fibroblasts from patients with IPF have phenotypic hallmarks, distinguishing them from their normal counterparts: pathologically activated Akt signaling axis, increased collagen and ?-smooth muscle actin expression, distinct gene expression profile, and ability to form fibrotic lesions in model organisms. Despite the centrality of these fibroblasts in disease pathogenesis, their origin remains uncertain. Here, we report the identification of cells in the lungs of patients with IPF with the properties of mesenchymal progenitors. In contrast to progenitors isolated from nonfibrotic lungs, IPF mesenchymal progenitor cells produce daughter cells manifesting the full spectrum of IPF hallmarks, including the ability to form fibrotic lesions in zebrafish embryos and mouse lungs, and a transcriptional profile reflecting these properties. Morphological analysis of IPF lung tissue revealed that mesenchymal progenitor cells and cells with the characteristics of their progeny comprised the fibrotic reticulum. These data establish that the lungs of patients with IPF contain pathological mesenchymal progenitor cells that are cells of origin for fibrosis-mediating fibroblasts. These fibrogenic mesenchymal progenitors and their progeny represent an unexplored target for novel therapies to interdict fibrosis.
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Pneumocystis pneumonia in patients treated with rituximab.
Chest
PUBLISHED: 10-01-2013
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Pneumocystis pneumonia (PcP) is an opportunistic fungal infection. Although T-cell immunity is classically related to Pneumocystis defense, recent data support roles for B lymphocytes in the development of PcP in animals, and we have observed several cases of PcP in patients receiving rituximab. These observations prompted a systematic review of our experience to define the spectrum of clinical presentations in which PcP has occurred in the setting of rituximab therapy.
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The Pneumocystis Ace2 transcription factor regulates cell wall-remodeling genes and organism virulence.
J. Biol. Chem.
PUBLISHED: 06-25-2013
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Pneumocystis carinii (Pc) ?-glucans are major components of the organism cell wall; yet, the regulation of Pc cell wall genesis and remodeling is not well understood. Ace2 transcription factors, which are present in many fungi, regulate glucanases and other enzymes needed for cell wall remodeling. The cloning and heterologous expression of PcAce2 in ace2? Saccharomyces cerevisiae demonstrated that PcAce2 can restore the defective glucanase and endochitinase gene expression of the mutant as well as regulate cell wall ?-glucan biosynthetic genes. Furthermore, when a reconstructed yeast system was used, PcAce2 activated the transcription of the Pneumocystis gsc1 ?-glucan synthetase, confirming the activity of a Pc transcription factor on a native Pneumocystis promoter and gene for the first time. We further observed that Pneumocystis binding to host extracellular matrix proteins and lung epithelial cells induced the phosphorylation (activation) of the PcAce2 transcription factor. Finally, we present a novel method that confirms the role of PcAce2 in modulating organism virulence using ace2? Candida glabrata infection in neutropenic mice. Together, these results indicate that the adherence of Pc to lung matrix proteins and epithelial cells leads to the activation of the Ace2 transcription factor, which regulates cell wall degradation and biosynthesis genes that are required for cell wall remodeling.
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Other HIV-associated pneumonias.
Clin. Chest Med.
PUBLISHED: 04-08-2013
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The incidence, mortality, and epidemiology of human immunodeficiency virus (HIV)-associated pulmonary infections have changed as a result of effective antiretroviral and prophylaxis antimicrobial therapy. The clinical presentation, radiographic abnormalities, and treatment of pneumonia from various uncommon pathogens in patients with AIDS can be different from those in immunocompetent patients. Advances in invasive and noninvasive testing and molecular biological techniques have improved the diagnosis and prognosis of pulmonary infections in patients infected with HIV. This review focuses on pulmonary infections from nontuberculosis mycobacteria, cytomegalovirus, fungi (aspergillosis, cryptococcosis, endemic fungi), and parasites (toxoplasmosis), and uncommon bacterial pneumonia (nocardiosis, rhodococcosis) in these patients.
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Characterization of the Pneumocystis carinii histone acetyltransferase chaperone proteins PcAsf1 and PcVps75.
Infect. Immun.
PUBLISHED: 04-08-2013
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Rtt109 is a lysine acetyltransferase that acetylates histone H3 at lysine 56 (H3K56) in fungi. This acetylation event is important for proper DNA replication and repair to occur. Efficient Rtt109 acetyltransferase activity also requires a histone chaperone, vacuolar protein sorting 75 (Vps75), as well as the major chaperone of the H3-H4 dimer, anti-silencing factor 1 (Asf1). Little is known about the role of these proteins in the opportunistic fungal pathogen Pneumocystis carinii. To investigate the functions of Asf1 and Vps75 in Pneumocystis carinii, we cloned and characterized both of these genes. Here, we demonstrate that both genes, P. carinii asf1 (Pcasf1) and Pcvps75, function in a fashion analogous to their Saccharomyces cerevisiae counterparts. We demonstrate that both P. carinii Asf1 (PcAsf1) and PcVps75 can bind histones. Furthermore, when Pcasf1 is expressed heterologously in S. cerevisiae asf1? cells, PcAsf1 can restore full H3 lysine acetylation. We further demonstrated that the Pcasf1 cDNA expressed in asf1? S. cerevisiae cells can restore growth to wild-type levels in the presence of genotoxic agents that block DNA replication. Lastly, we observed that purified PcAsf1 and PcVps75 proteins enhance the ability of PcRtt109 to acetylate histone H3-H4 tetramers. Together, our results indicate that the functions of the Rtt109-Asf1-Vps75 complex in the acetylation of histone H3 lysine 56 and in DNA damage response are present in P. carinii DNA and cell cycle progression.
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Diffuse pulmonary lymphangiomatosis.
Can. Respir. J.
PUBLISHED: 03-05-2013
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Diffuse pulmonary lymphangiomatosis (DPL) is a rare disease characterized by infiltration of the lung, pleura and mediastinum with thin-walled lymphangiomas. DPL can result in mass effect from infiltrative disease, restrictive and obstructive pulmonary physiology, chylous effusions and respiratory failure. The present article discusses clinical, radiographic and pathological features, and treatment options for DPL.
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Genetic variants associated with the risk of chronic obstructive pulmonary disease with and without lung cancer.
Cancer Prev Res (Phila)
PUBLISHED: 11-01-2011
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Chronic obstructive pulmonary disease (COPD) is a strong risk factor for lung cancer. Published studies about variations of genes encoding glutathione metabolism, DNA repair, and inflammatory response pathways in susceptibility to COPD were inconclusive. We evaluated 470 single-nucleotide polymorphisms (SNP) from 56 genes of these three pathways in 620 cases and 893 controls to identify susceptibility markers for COPD risk, using existing resources. We assessed SNP- and gene-level effects adjusting for sex, age, and smoking status. Differential genetic effects on disease risk with and without lung cancer were also assessed; cumulative risk models were established. Twenty-one SNPs were found to be significantly associated with risk of COPD (P < 0.01); gene-based analyses confirmed two genes (GCLC and GSS) and identified three additional genes (GSTO2, ERCC1, and RRM1). Carrying 12 high-risk alleles may increase risk by 2.7-fold; eight SNPs altered COPD risk without lung cancer by 3.1-fold and 4 SNPs altered the risk with lung cancer by 2.3-fold. Our findings indicate that multiple genetic variations in the three selected pathways contribute to COPD risk through GCLC, GSS, GSTO2, ERCC1, and RRM1 genes. Functional studies are needed to elucidate the mechanisms of these genes in the development of COPD, lung cancer, or both.
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Guidelines for the naming of genes, gene products, and mutants in the opportunistic protists.
J. Eukaryot. Microbiol.
PUBLISHED: 08-25-2011
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The opportunistic protists encompass a wide diversity of organisms including Pneumocystis, Toxoplasma, cryptosporidia, microsporidia, and related genera. Recent advances in the molecular biology and cellular biochemistry of these organisms have led to the identification of an ever growing numbers of key genes and their cognate proteins. Until now, these molecules have not been designated using any consistent nomenclature system, leading to considerable confusion. The participants of the 11th International Workshop on Opportunistic Protists met on August 3, 2010 to reach consensus of a nomenclature system for genes, gene products, and mutants in the opportunistic protists. The following summary reports the consensus agreement to move toward a unified nomenclature system for these organisms. The system is adapted from that used for Saccharomyces cerevisiae.
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Pneumocystis carinii interactions with lung epithelial cells and matrix proteins induce expression and activity of the PcSte20 kinase with subsequent phosphorylation of the downstream cell wall biosynthesis kinase PcCbk1.
Infect. Immun.
PUBLISHED: 07-18-2011
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Eukaryotic cell proliferation and phenotype are highly regulated by contact-dependent mechanisms. We have previously shown that the binding and interaction of the opportunistic fungal pathogen Pneumocystis carinii to lung epithelial cells and extracellular matrix proteins induces mRNA expression of both the mitogen-activated protein (MAP) kinase P. carinii Ste20 (PcSte20) and the cell wall-remodeling enzyme PcCbk1 (16). Herein, we report that in addition to PcSte20 mRNA expression being upregulated, Pneumocystis PcSte20 kinase activity is increased upon interacting with these same lung targets. This activity is also significantly suppressed by Clostridium difficile toxin B, a pan-specific inhibitor of small GTPases, demonstrating the potential role of a Cdc42-like molecule in this signaling cascade. We further observed that the PcSte20 kinase physically interacts with a specific region of the P. carinii cell wall biosynthesis kinase, PcCbk1, a downstream kinase important for mating projection formation and cell wall remodeling. This direct binding was mapped to a specific region of the PcCbk1 protein. We also demonstrated that PcSte20 obtained from whole P. carinii lysates has the ability to phosphorylate PcCbk1 after the organism interacts with lung epithelial cells and extracellular matrix components. These observations provide new insights into P. carinii signaling induced by interactions of this important opportunistic fungal pathogen with lung epithelial cells and matrix.
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Substrate analysis of the Pneumocystis carinii protein kinases PcCbk1 and PcSte20 using yeast proteome microarrays provides a novel method for Pneumocystis signalling biology.
Yeast
PUBLISHED: 07-14-2011
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Pneumocystis carinii (Pc) undergoes morphological transitions between cysts and trophic forms. We have previously described two Pc serine/threonine kinases, termed PcCbk1 and PcSte20, with PcSte20 belonging to a family of kinases involved in yeast mating, while PcCbk1 is a member of a group of protein kinases involved in regulation of cell cycle, shape, and proliferation. As Pc remains genetically intractable, knowledge on specific substrates phosphorylated by these kinases remains limited. Utilizing the phylogenetic relatedness of Pc to Saccharomyces cerevisiae, we interrogated a yeast proteome microarray containing >4000 purified protein based peptides, leading to the identification of 18 potential PcCbk1 and 15 PcSte20 substrates (Z-score?>?3.0). A number of these potential protein substrates are involved in bud site selection, polarized growth, and response to mating ? factor and pseudohyphal and invasive growth. Full-length open reading frames suggested by the PcCbk1 and PcSte20 protoarrays were amplified and expressed. These five proteins were used as substrates for PcCbk1 or PcSte20, with each being highly phosphorylated by the respective kinase. Finally, to demonstrate the utility of this method to identify novel PcCbk1 and PcSte20 substrates, we analysed DNA sequence data from the partially complete Pc genome database and detected partial sequence information of potential PcCbk1 kinase substrates PcPxl1 and PcInt1. We additionally identified the potential PcSte20 kinase substrate PcBdf2. Full-length Pc substrates were cloned and expressed in yeast, and shown to be phosphorylated by the respective Pc kinases. In conclusion, the yeast protein microarray represents a novel crossover technique for identifying unique potential Pc kinase substrates.
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An official American Thoracic Society statement: Treatment of fungal infections in adult pulmonary and critical care patients.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 01-04-2011
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With increasing numbers of immune-compromised patients with malignancy, hematologic disease, and HIV, as well as those receiving immunosupressive drug regimens for the management of organ transplantation or autoimmune inflammatory conditions, the incidence of fungal infections has dramatically increased over recent years. Definitive diagnosis of pulmonary fungal infections has also been substantially assisted by the development of newer diagnostic methods and techniques, including the use of antigen detection, polymerase chain reaction, serologies, computed tomography and positron emission tomography scans, bronchoscopy, mediastinoscopy, and video-assisted thorascopic biopsy. At the same time, the introduction of new treatment modalities has significantly broadened options available to physicians who treat these conditions. While traditionally antifungal therapy was limited to the use of amphotericin B, flucytosine, and a handful of clinically available azole agents, current pharmacologic treatment options include potent new azole compounds with extended antifungal activity, lipid forms of amphotericin B, and newer antifungal drugs, including the echinocandins. In view of the changing treatment of pulmonary fungal infections, the American Thoracic Society convened a working group of experts in fungal infections to develop a concise clinical statement of current therapeutic options for those fungal infections of particular relevance to pulmonary and critical care practice. This document focuses on three primary areas of concern: the endemic mycoses, including histoplasmosis, sporotrichosis, blastomycosis, and coccidioidomycosis; fungal infections of special concern for immune-compromised and critically ill patients, including cryptococcosis, aspergillosis, candidiasis, and Pneumocystis pneumonia; and rare and emerging fungal infections.
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Update on the diagnosis and treatment of Pneumocystis pneumonia.
Ther Adv Respir Dis
PUBLISHED: 08-24-2010
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Pneumocystis is an opportunistic fungal pathogen that causes an often-lethal pneumonia in immunocompromised hosts. Although the organism was discovered in the early 1900s, the first cases of Pneumocystis pneumonia in humans were initially recognized in Central Europe after the Second World War in premature and malnourished infants. This unusual lung infection was known as plasma cellular interstitial pneumonitis of the newborn, and was characterized by severe respiratory distress and cyanosis with little or no fever and no pathognomic physical signs. At that time, only anecdotal cases were reported in adults and usually these patients had a baseline malignancy that led to a malnourished state. In the 1960-1970s additional cases were described in adults and children with hematological malignancies, but Pneumocystis pneumonia was still considered a rare disease. However, in the 1980s, with the onset of the HIV epidemic, Pneumocystis prevalence increased dramatically and became widely recognized as an opportunistic infection that caused potentially life-treating pneumonia in patients with impaired immunity. During this time period, prophylaxis against this organism was more generally instituted in high-risk patients. In the 1990s, with widespread use of prophylaxis and the initiation of highly active antiretroviral therapy (HAART) in the treatment of HIV-infected patients, the number of cases in this specific population decreased. However, Pneumocystis pneumonia still remains an important cause of severe pneumonia in patients with HIV infection and is still considered a principal AIDS-defining illness. Despite the decreased number of cases among HIV-infected patients over the past decade, Pneumocystis pneumonia continues to be a serious problem in immunodeficient patients with other immunosuppressive conditions. This is mostly due to increased use of immunosuppressive medications to treat patients with autoimmune diseases, following bone marrow and solid organ transplantation, and in patients with hematological and solid malignancies. Patients with hematologic disorders and solid organ and hematopoietic stem cell transplantation are currently the most vulnerable groups at risk for developing this infection. However, any patient with an impaired immunity, such as those receiving moderate doses of oral steroids for greater than 4 weeks or those receiving other immunosuppressive medications are at also at significant risk.
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Pneumocystis carinii expresses an active Rtt109 histone acetyltransferase.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 07-23-2010
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Species in the genus Pneumocystis can cause severe pneumonia in immune-compromised hosts. The identification of specific targets present in Pneumocystis species, but lacking in mammalian hosts, is paramount to developing new means to treat this infection. One such potential protein is Rtt109, which is a type of histone acetyltransferase (HAT) required for DNA replication in fungi, but not found in mammals. Sequence orthologues of Rtt109 are present in other fungi, but are absent in mammals, making it a potential pan-specific target against medically relevant fungi. Accordingly, we sought to identify the presence of an Rtt109 in P. carinii. A Pneumocystis carinii (Pc) Rtt109 165-bp partial sequence was initially identified from the incomplete P. carinii genome database. Subsequently, a full-length, 1,128-bp cDNA with homology to Saccharomyces cerevisiae Rtt109 (39% Basic Local Alignment Search Tool (BLASTP)) was cloned and characterized. Sequence analysis of PcRtt109 indicated that the P. carinii molecule contains the putative catalytic aspartate present in yeast. We further demonstrated that the PcRtt109 expressed in rtt109? S. cerevisiae cells restored H3-K56 acetylation and the sensitivity toward DNA-damaging agents of rtt109? mutant cells. Purified PcRtt109 had the ability to acetylate lysine-56 of histone H3, similar to the ability of Schizosaccharomyces pombe Rtt109 protein. The site-directed mutagenesis of PcRtt109 D84A, a potential regulatory site in the Rtt109 HAT family, abolished H3 acetylation, whereas a DD218/219AA mutation that compromised the activity of ScRtt109 had little effect, demonstrating similarities and differences in Pneumocystis PcRtt109 compared with yeast Saccharomyces cerevisiae Rtt109. These results indicate that P. carinii contains an Rtt109 HAT molecule, and represent the complete identification and characterization of a HAT molecule from this important opportunistic fungal pathogen.
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Pneumocystis cell wall beta-glucan stimulates calcium-dependent signaling of IL-8 secretion by human airway epithelial cells.
Respir. Res.
PUBLISHED: 07-13-2010
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Respiratory failure secondary to alveolar inflammation during Pneumocystis pneumonia is a major cause of death in immunocompromised patients. Neutrophil infiltration in the lung of patients with Pneumocystis infection predicts severity of the infection and death. Several previous studies indicate that airway epithelial cells release the neutrophil chemoattractant proteins, MIP-2 (rodents) and IL-8 (humans), in response to Pneumocystis and purified Pneumocystis cell wall beta-glucans (PCBG) through the NF-kappaB-dependent pathway. However, little is known about the molecular mechanisms that are involved in the activation of airway epithelium cells by PCBG resulting in the secretion of IL-8.
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The changing spectrum of fungal infections in pulmonary and critical care practice: clinical approach to diagnosis.
Proc Am Thorac Soc
PUBLISHED: 05-14-2010
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Fungal lung infections are being diagnosed with increasing frequency. This is related to the increased numbers of immune-compromised and other susceptible patient groups. This article will focus on the evolving epidemiology of fungal lung infections and clinical manifestations that should prompt the clinician to consider the possibility of fungal lung infection. In addition, current approaches for the diagnosis of these infection are also reviewed. Heightened awareness of fungal lung infection, and appropriate use of the available diagnostic modalities, will permit appropriate treatment of these important clinical infections in immune-compromised individuals.
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Cigarette smoke promotes dendritic cell accumulation in COPD; a Lung Tissue Research Consortium study.
Respir. Res.
PUBLISHED: 04-26-2010
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Abnormal immune responses are believed to be highly relevant in the pathogenesis of chronic obstructive pulmonary disease (COPD). Dendritic cells provide a critical checkpoint for immunity by their capacity to both induce and suppress immunity. Although evident that cigarette smoke, the primary cause of COPD, significantly influences dendritic cell functions, little is known about the roles of dendritic cells in the pathogenesis of COPD.
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Imatinib treatment for idiopathic pulmonary fibrosis: Randomized placebo-controlled trial results.
Am. J. Respir. Crit. Care Med.
PUBLISHED: 12-10-2009
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Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with no known efficacious therapy. Imatinib is a tyrosine kinase inhibitor with potential efficacy to treat fibrotic lung disease.
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Clinical significance of radiologic characterizations in COPD.
COPD
PUBLISHED: 11-27-2009
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COPD is a heterogeneous disorder with clinical assessment becoming increasingly multidimensional. We hypothesized HRCT phenotype would strongly influence clinical outcomes including health status, exacerbation frequency, and BODE. COPD subjects were characterized via the SF-12, SGRQ, MMRC, physiologic testing, and standardized volumetric chest HRCT. Visual semi-quantitative estimation of bronchial wall thickness (VBT) and automated quantification of emphysema percent and bronchial wall thickness were generated. Multivariate modeling compared emphysema severity and airway abnormality with clinical outcome measures. Poisson models were used to analyze exacerbation frequency. SGRQ and SF-12 physical component scores were influenced by FEV(1)% predicted, emphysema percent, and VBT. VBT scores > 2 (scale 0-48) were associated with increased exacerbation frequency (p = 0.009) in the preceding year adjusting for age, gender, emphysema percent, smoking history and FEV(1)% predicted, although this effect was attenuated by age. Emphysema percent correlated with total BODE score in unadjusted (r = 0.73; p < 0.0001) and adjusted (p < 0.0001) analyses and with BODE individual components. HRCT provides unique COPD phenotyping information. Radiographic quantification of emphysema and bronchial thickness are independently associated with SGRQ and physical component score of the SF-12. Bronchial thickness but not emphysema is associated with exacerbation frequency, whereas emphysema is a stronger predictor of BODE and its systemic components MMRC, 6MWT, and BMI. Future research should clarify whether CT parameters complement BODE score in influencing survival.
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Characterization of the PcCdc42 small G protein from Pneumocystis carinii, which interacts with the PcSte20 life cycle regulatory kinase.
Am. J. Physiol. Lung Cell Mol. Physiol.
PUBLISHED: 11-13-2009
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Pneumocystis carinii (Pc) causes severe pneumonia in immunocompromised hosts. The binding of Pc trophic forms to alveolar epithelial cells is a central feature of infection, inducing the expression and activation of PcSte20, a gene participating in mating, proliferation, and pseudohyphal growth. In related fungi, Ste20 proteins are generally activated by immediate upstream small G proteins of the Cdc42-like family. PcCdc42 has not been previously described in Pneumocystis. To address the potential role of such a G protein in Pneumocystis, PcCdc42 was cloned from a Pc cDNA library. Using the full-length 576-bp PcCdc42 cDNA sequence, a CHEF blot of genomic DNA yielded a single band, providing evidence that this gene is present as a single copy within the genome. The total length of PcCdc42 cDNA was 576 bp with an estimated molecular mass of approximately 38 kDa. BLASTP analysis demonstrated greater than 80% homology with other fungal Cdc42p proteins. Northern analysis indicated equal mRNA expression in both cystic and trophic life forms. Heterologous expression of PcCdc42 in Saccharomyces cerevisiae (Sc) demonstrated that PcCdc42p was able to restore growth in an ScCdc42Delta yeast strain. Additional assays with purified PcCdc42 protein demonstrated GTP binding and intrinsic GTPase activity, which was partially but significantly suppressed by Clostridium difficile toxin B, characteristic of Cdc42 GTPases. Furthermore, PcCdc42 protein was also shown to bind to the downstream PCSte20 kinase partner in the presence (but not the absence) of GTP. These data indicate that Pc possesses a Cdc42 gene expressing an active G protein, which binds the downstream regulatory kinase PcSte20, important in Pc life cycle regulation.
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Characterization of PCEng2, a {beta}-1,3-endoglucanase homolog in Pneumocystis carinii with activity in cell wall regulation.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 09-25-2009
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Pneumocystis jirovecii pneumonia is an opportunistic fungal infection that causes severe respiratory impairment in immunocompromised patients. The viability of Pneumocystis organisms is dependent on the cyst cell wall, a structural feature that is regulated by essential cell wall-associated enzymes. The formation of the glucan-rich cystic wall has been previously characterized, but glucan degradation in the organism-specifically, degradation during trophic excystment-is not yet fully understood. Most studies of basic Pneumocystis biology have been conducted in Pneumocystis carinii or Pneumocystis murina, the varieties of this genus that infect rats and mice, respectively. Furthermore, all known treatments for P. jirovecii were initially discovered through studies of P. carinii. Accordingly, in this study, we have identified a P. carinii beta-1,3-endoglucanase gene (PCEng2) that is demonstrated to play a significant role in cell wall regulation. The cDNA sequence contained a 2.2-kb open reading frame with conserved amino acid domains homologous to similar fungal glycosyl hydrolases (GH family 81). The gene transcript showed up-regulation in cystic isolates, and the expressed protein was detected within both cyst and trophic forms. Complementation assays in Eng2-deleted Saccharomyces cerevisiae strains showed restoration of the cell wall separation defect during proliferation, demonstrating the importance of PCEng2 protein. during fungal growth. These findings suggest that regulation of cyst cell wall beta-glucans is a fundamental process during completion of the Pneumocystis life cycle.
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Gender influences Health-Related Quality of Life in IPF.
Respir Med
PUBLISHED: 08-07-2009
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HRQL in IPF patients is impaired. Data from other respiratory diseases led us to hypothesize that significant gender differences in HRQL in IPF also exist.
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Characterization of a novel ADAM protease expressed by Pneumocystis carinii.
Infect. Immun.
PUBLISHED: 05-18-2009
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Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts. Recent evidence has suggested that unidentified proteases are involved in Pneumocystis life cycle regulation. Proteolytically active ADAM (named for "a disintegrin and metalloprotease") family molecules have been identified in some fungal organisms, such as Aspergillus fumigatus and Schizosaccharomyces pombe, and some have been shown to participate in life cycle regulation. Accordingly, we sought to characterize ADAM-like molecules in the fungal opportunistic pathogen, Pneumocystis carinii (PcADAM). After an in silico search of the P. carinii genomic sequencing project identified a 329-bp partial sequence with homology to known ADAM proteins, the full-length PcADAM sequence was obtained by PCR extension cloning, yielding a final coding sequence of 1,650 bp. Sequence analysis detected the presence of a typical ADAM catalytic active site (HEXXHXXGXXHD). Expression of PcADAM over the Pneumocystis life cycle was analyzed by Northern blot. Southern and contour-clamped homogenous electronic field blot analysis demonstrated its presence in the P. carinii genome. Expression of PcADAM was observed to be increased in Pneumocystis cysts compared to trophic forms. The full-length gene was subsequently cloned and heterologously expressed in Saccharomyces cerevisiae. Purified PcADAMp protein was proteolytically active in casein zymography, requiring divalent zinc. Furthermore, native PcADAMp extracted directly from freshly isolated Pneumocystis organisms also exhibited protease activity. This is the first report of protease activity attributable to a specific, characterized protein in the clinically important opportunistic fungal pathogen Pneumocystis.
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Pneumocystis pneumonia: current concepts in pathogenesis, diagnosis, and treatment.
Clin. Chest Med.
PUBLISHED: 04-21-2009
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Pneumocystis pneumonia (PCP) is an infection of the lungs caused by the opportunistic fungal genus Pneumocystis. In humans, PCP is a serious and potentially life-threatening infection occurring in immunocompromised individuals, particularly those who have AIDS, or following immune suppression from malignancy, organ transplantation, or therapies for inflammatory diseases. Several recent studies have contributed to understanding of the biology and pathogenesis of the organism yielding new diagnostic approaches and therapeutic targets. Although trimethoprim-sulfamethoxazole remains the mainstay of prophylaxis and treatment, ongoing concerns for emerging Pneumocystis resistance supports the continuing investigation for novel therapeutic agents.
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The Pneumocystis meiotic PCRan1p kinase exhibits unique temperature-regulated activity.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 03-13-2009
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Pneumocystis organisms are opportunistic fungal pathogens that cause significant pneumonia in immune-compromised hosts. Recent evidence has suggested that Pneumocystis carinii exists as separate mating types, and expresses and regulates proteins that govern meiosis and progression of the life cycle. This study was undertaken to investigate the activity of three life cycle-regulatory proteins in Pneumocystis, including two proteins essential in mating signaling, and a putative meiotic regulator, to determine the conditions under which they are most active. This study used V5/HIS-tagged PCRan1p, PCSte20p, and PCCbk1, purified from Saccharomyces cerevisiae strain, INVSC, as well as an in vitro Escherichia coli protein expression system to determine the optimal expression conditions of each protein in the presence of varying pH, temperature, and metal ions. These studies demonstrate an atypical enzymatic activity in PCRan1p, whereby the kinase was most active in the environmental conditions between 10 and 25 degrees C, compared with a dramatic reduction in activity above 30 degrees C, temperatures typically found within mammalian hosts. Circular dichroism and fluorescence spectroscopy suggest that PCRan1p becomes partially unfolded at 25 degrees C, leading to its most active conformation, whereas continued unfolding as temperature increases results in strongly suppressed activity. These studies suggest that, in vivo, while under conditions within the mammalian lung (typically 37 degrees C), PCRan1p kinase activity is largely suppressed, allowing better conditions for the activation of meiosis, whereas in ex vivo environments, PCRan1p kinase activity increases to arrest progression of the life cycle until conditions become more favorable.
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Monitoring of nonsteroidal immunosuppressive drugs in patients with lung disease and lung transplant recipients: American College of Chest Physicians evidence-based clinical practice guidelines.
Chest
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Immunosuppressive pharmacologic agents prescribed to patients with diffuse interstitial and inflammatory lung disease and lung transplant recipients are associated with potential risks for adverse reactions. Strategies for minimizing such risks include administering these drugs according to established, safe protocols; monitoring to detect manifestations of toxicity; and patient education. Hence, an evidence-based guideline for physicians can improve safety and optimize the likelihood of a successful outcome. To maximize the likelihood that these agents will be used safely, the American College of Chest Physicians established a committee to examine the clinical evidence for the administration and monitoring of immunosuppressive drugs (with the exception of corticosteroids) to identify associated toxicities associated with each drug and appropriate protocols for monitoring these agents.
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Detection of (1, 3)-?-D-glucan in bronchoalveolar lavage and serum samples collected from immunocompromised hosts.
Mycopathologia
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The incidence of invasive fungal infections (IFI) has increased in recent years, especially among immunocompromised hosts (ICH). In 2003, the Fungitell(®) assay received FDA clearance for the presumptive diagnosis of IFI using serum and detects (1-3)-?-D-glucan, which is a major cell wall component of certain fungi (e.g., Candida, Aspergillus, and Pneumocystis). The goal of the current study was to assess the performance of the assay on bronchoalveolar lavage (BAL) fluid and serum to identify IFI in ICH. Patients were classified as having proven, probable, possible, or no IFI according to the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) guidelines. Among 109 patients for whom the results of Fungitell were compared to the EORTC/MSG criteria, Fungitell showed a low positive predictive value for the identification of IFI from both BAL (20.0%) and serum (26.7%). However, the negative predictive value of Fungitell was significantly higher for both sample types (BAL, 83.0%; serum, 84.8%). Interestingly, the results of Fungitell were positive in BAL and serum in 7/8 (87.5%) patients diagnosed with Pneumocystis pneumonia (PcP) by real-time, non-nested PCR. These data indicate that the Fungitell assay has a low positive predictive value for the diagnosis of IFI in ICH, regardless of the specimen type that is tested. However, testing of serum samples by Fungitell may permit a rapid and noninvasive initial screening approach in patients with presumed PcP.
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Relationship between lung function impairment and health-related quality of life in COPD and interstitial lung disease.
Chest
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Health-related quality-of-life (HRQL) measures have been correlated with lung function in patients with COPD and interstitial lung disease (ILD). However, different pathophysiologic mechanisms may influence how these distinct diseases affect HRQL, resulting in differing HRQL by pulmonary diagnosis among patients with similar severity of ventilatory impairment.
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Drug-associated acute lung injury: a population-based cohort study.
Chest
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A number of drugs have been reported as risk factors for acute lung injury (ALI) and ARDS. However, evidence is largely limited to case reports, and there is a paucity of data on the incidence and outcome of drug-associated ALI (DALI).
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Chitinases in Pneumocystis carinii pneumonia.
Med. Microbiol. Immunol.
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Pneumocystis pneumonia remains an important complication of immune suppression. The cell wall of Pneumocystis has been demonstrated to potently stimulate host inflammatory responses, with most studies focusing on ?-glucan components of the Pneumocystis cell wall. In the current study, we have elaborated the potential role of chitins and chitinases in Pneumocystis pneumonia. We demonstrated differential host mammalian chitinase expression during Pneumocystis pneumonia. We further characterized a chitin synthase gene in Pneumocystis carinii termed Pcchs5, a gene with considerable homolog to the fungal chitin biosynthesis protein Chs5. We also observed the impact of chitinase digestion on Pneumocystis-induced host inflammatory responses by measuring TNF? release and mammalian chitinase expression by cultured lung epithelial and macrophage cells stimulated with Pneumocystis cell wall isolates in the presence and absence of exogenous chitinase digestion. These findings provide evidence supporting a chitin biosynthetic pathway in Pneumocystis organisms and that chitinases modulate inflammatory responses in lung cells. We further demonstrate lung expression of chitinase molecules during Pneumocystis pneumonia.
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Glycosphingolipids mediate pneumocystis cell wall ?-glucan activation of the IL-23/IL-17 axis in human dendritic cells.
Am. J. Respir. Cell Mol. Biol.
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Pneumocystis species are opportunistic fungal organisms that cause severe pneumonia in immune-compromised hosts, with resultant high morbidity and mortality. Recent work indicates that IL-17 responses are important components of host defense against fungal pathogens. In the present study, we demonstrate that cell-surface ?-glucan components of Pneumocystis (PCBG) stimulate human dendritic cells (DCs) to secrete IL-23 and IL-6. These cytokines are well established to stimulate a T helper-17 (Th17) phenotype. Accordingly, we further observe that PCBG-stimulated human DCs interact with lymphocytes to drive the secretion of IL-17 and IL-22, both Th17-produced cytokines. The activation of DCs was shown to involve the dectin-1 receptor with a downstream activation of the Syk kinase and subsequent translocation of both the canonical and noncanonical components of the NF-?B transcription factor family. Finally, we demonstrate that glycosphingolipid-rich microdomains of the plasma membrane participate in the activation of DCs by PCBG through the accumulation of lactosylceramide at the cell surface during stimulation with PCBG. These data strongly support the idea that the ?-glucan surface components of Pneumocystis drive the activation of the IL-23/IL-17 axis during this infection, through a glycosphingolipid-initiated mechanism.
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Primary alveolar epithelial cell surface membrane microdomain function is required for Pneumocystis ?-glucan-induced inflammatory responses.
Innate Immun
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Intense lung inflammation characterizes respiratory failure associated with Pneumocystis pneumonia. Our laboratory has previously demonstrated that alveolar epithelial cells (AECs) elaborate inflammatory cytokines and chemokines in response to the Pneumocystis carinii cell wall constituent ?-(1?3)-glucan (PCBG), and that these responses require lactosylceramide, a prominent glycosphingolipid constituent of certain cell membrane microdomains. The relevance of membrane microdomains, also termed plasma membrane lipid rafts, in cell signaling and macromolecule handling has been increasingly recognized in many biologic systems, but their role in P. carinii-induced inflammation is unknown. To investigate the mechanisms of microdomain-dependent P. carinii-induced inflammation, we challenged primary rat AECs with PCBG with or without pre-incubation with inhibitors of microdomain function. Glycosphingolipid and cholesterol rich microdomain inhibition resulted in significant attenuation of P. carinii-induced expression of TNF-? and the rodent C-X-C chemokine MIP-2, as well as their known inflammatory secondary signaling pathways. We have previously shown that protein kinase C (PKC) is activated by PCBG challenge and herein show that PKC localizes to AEC microdomains. We also demonstrate by conventional microscopy, fluorescence microscopy, confocal microscopy and spectrophotofluorimetry that AECs internalize fluorescently-labeled PCBG by microdomain-mediated mechanisms, and that anti-microdomain pretreatments prevent internalization. Taken together, these data suggest an important role for AEC microdomain function in PCBG-induced inflammatory responses. This offers a potential novel target for therapeutics for a condition that continues to exert unacceptable morbidity and mortality among immunocompromised populations.
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