Thoracic Diseases Research Unit

Rochester, MN, United States

Thoracic Diseases Research Unit

Rochester, MN, United States
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Villegas L.R.,Thoracic Diseases Research Unit | Kottom T.J.,Thoracic Diseases Research Unit | Limper A.H.,Thoracic Diseases Research Unit | Limper A.H.,Mayo Medical School
Medical Microbiology and Immunology | Year: 2012

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 β-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 TNFα 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. © Springer-Verlag 2012.

Kottom T.J.,Thoracic Diseases Research Unit | Limper A.H.,Thoracic Diseases Research Unit
Journal of Biological Chemistry | Year: 2013

Background: Ace2 transcription factors regulate fungal cell wall remodeling. Results: Pneumocystis carinii Ace2 (PcAce2) is activated by lung matrix proteins and participates in fungal virulence. Conclusion: PcAce2 represents a key component of life cycle regulation that is induced by Pneumocystis contact with host substrates. Significance: This is the first example demonstrating a contact-induced activation of the Ace2 transcription factor. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Kottom T.J.,Thoracic Diseases Research Unit | Han J.,Mayo Medical School | Zhang Z.,Mayo Medical School | Limper A.H.,Thoracic Diseases Research Unit
American Journal of Respiratory Cell and Molecular Biology | Year: 2011

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 panspecific 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 rtt109Δ S. cerevisiae cells restored H3-K56 acetylation and the sensitivity toward DNA-damaging agents of rtt109Δ 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.

Limper A.H.,Thoracic Diseases Research Unit | Limper A.H.,Foundation Medicine
Proceedings of the American Thoracic Society | Year: 2010

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.

Belete H.A.,Thoracic Diseases Research Unit | Hubmayr R.D.,Thoracic Diseases Research Unit | Wang S.,Thoracic Diseases Research Unit | Singh R.-D.,Thoracic Diseases Research Unit
PLoS ONE | Year: 2011

Cell wounding is an important driver of the innate immune response of ventilator-injured lungs. We had previously shown that the majority of wounded alveolus resident cells repair and survive deformation induced insults. This is important insofar as wounded and repaired cells may contribute to injurious deformation responses commonly referred to as biotrauma. The central hypothesis of this communication states that extracellular adenosine-5′ triphosphate (ATP) promotes the repair of wounded alveolus resident cells by a P2Y2-Receptor dependent mechanism. Using primary type 1 alveolar epithelial rat cell models subjected to micropuncture injury and/or deforming stress we show that 1) stretch causes a dose dependent increase in cell injury and ATP media concentrations; 2) enzymatic depletion of extracellular ATP reduces the probability of stretch induced wound repair; 3) enriching extracellular ATP concentrations facilitates wound repair; 4) purinergic effects on cell repair are mediated by ATP and not by one of its metabolites; and 5) ATP mediated cell salvage depends at least in part on P2Y2-R activation. While rescuing cells from wounding induced death may seem appealing, it is possible that survivors of membrane wounding become governors of a sustained pro-inflammatory state and thereby perpetuate and worsen organ function in the early stages of lung injury syndromes. Means to uncouple P2Y2-R mediated cytoprotection from P2Y2-R mediated inflammation and to test the preclinical efficacy of such an undertaking deserve to be explored. © 2011 Belete et al.

Plataki M.,Thoracic Diseases Research Unit | Hubmayr R.D.,Thoracic Diseases Research Unit
Current Opinion in Critical Care | Year: 2011

Purpose of review: Despite the well recognized role of mechanical ventilation in lung injury, appropriate surrogate markers to guide titration of ventilator settings remain elusive. One would like to strike a balance between protecting aerated units from overdistension while recruiting unstable units, thereby reducing tissue damage associated with their cyclic recruitment and derecruitment. To do so requires some estimate of the topographical distribution of parenchymal stress and strain. Recent findings: Recent studies have highlighted the importance of chest wall recoil and its effect on pleural pressure (Ppl) in determining lung stress. Although esophageal pressure (Pes) has traditionally been used to measure the average Ppl in normal upright patients, in recumbent acute lung injury/acute respiratory distress syndrome patients Pes-based estimates of Ppl are subject to untestable assumptions. Nevertheless, Pes measurements in recumbent patients with injured lungs strongly suggest that Ppl over dependent parts of the lung can exceed airway pressure by substantial amounts. Moreover, results of a pilot study in which Pes was used to titrate positive end-expiratory pressure (PEEP) suggest clinical benefit. Summary: Notwithstanding its theoretical limitations, esophageal manometry has shown promise in PEEP titration and deserves further evaluation in a larger trial on patients with injured lungs. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Villegas L.R.,Thoracic Diseases Research Unit | Kottom T.J.,Thoracic Diseases Research Unit | Limper A.H.,Thoracic Diseases Research Unit | Limper A.H.,Mayo Medical School
American Journal of Respiratory Cell and Molecular Biology | Year: 2010

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, wehave identified a P.carinii β-1,3-endoglucanasegene (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 β-glucans is a fundamental process during completion of the Pneumocystis life cycle.

Plataki M.,Thoracic Diseases Research Unit | Lee Y.D.,Eulji University | Rasmussen D.L.,Thoracic Diseases Research Unit | Hubmayr R.D.,Thoracic Diseases Research Unit
American Journal of Respiratory and Critical Care Medicine | Year: 2011

Wounded alveolus resident cells are identified in human and experimental acute respiratory distress syndrome models. Poloxamer 188 (P188) is an amphiphilic macromolecule shown to have plasma membrane-sealing properties in various cell types. Objectives: To investigate whether P188 (1) protects alveolus resident cells from necrosis and (2) is associated with reduced ventilator-induced lung injury in live rats, isolated perfused rat lungs, and scratch and stretch-wounded alveolar epithelial cells. Methods: Seventy-four live rats and 18 isolated perfused rat lungs were ventilated with injurious or protective strategies while infused with P188 or control solution. Alveolar epithelial cell monolayers were subjected to scratch or stretch wounding in the presence or absence of P188. Measurements and Main Results: P188 was associated with fewer mortally wounded alveolar cells in live rats and isolated perfused lungs. In vitro, P188 reduced the number of injured and necrotic cells, suggesting that P188 promotes cell repair and renders plasma membranes more resilient to deforming stress. The enhanced cell survival was accompanied by improvement in conventional measures of lung injury (peak airway pressure, wet-to-dry weight ratio) only in the ex vivo-perfused lung preparation and not in the live animal model. Conclusions: P188 facilitates plasma membrane repair in alveolus resident cells, but has no salutary effects on lung mechanics or vascular barrier properties in live animals. This discordance may have pathophysiological significance for the interdependence of different injury mechanisms and therapeutic implications regarding the benefits of prolonging the life of stress-activated cells.

Hussein O.,Thoracic Diseases Research Unit | Walters B.,Thoracic Diseases Research Unit | Stroetz R.,Thoracic Diseases Research Unit | Valencia P.,Thoracic Diseases Research Unit | And 2 more authors.
American Journal of Physiology - Lung Cellular and Molecular Physiology | Year: 2013

Mechanical ventilation may cause harm by straining lungs at a time they are particularly prone to injury from deforming stress. The objective of this study was to define the relative contributions of alveolar overdistension and cyclic recruitment and "collapse" of unstable lung units to membrane wounding of alveolar epithelial cells. We measured the interactive effects of tidal volume (VT), transpulmonary pressure (PTP), and of airspace liquid on the number of alveolar epithelial cells with plasma membrane wounds in ex vivo mechanically ventilated rat lungs. Plasma membrane integrity was assessed by propidium iodide (PI) exclusion in confocal images of subpleural alveoli. Cyclic inflations of normal lungs from zero end-expiratory pressure to 40 cmH2O produced VT values of 56.9 ± 3.1 ml/kg and were associated with 0.12 ± 0.12 PI-positive cells/alveolus. A preceding tracheal instillation of normal saline (3 ml) reduced VT to 49.1 ± 6 ml/kg but was associated with a significantly greater number of wounded alveolar epithelial cells (0.52 ± 0.16 cells/alveolus; P < 0.01). Mechanical ventilation of completely saline-filled lungs with saline (VT = 52 ml/kg) to pressures between 10 and 15 cmH2O was associated with the least number of wounded epithelial cells (0.02 ± 0.02 cells/ alveolus; P < 0.01). In mechanically ventilated, partially saline-filled lungs, the number of wounded cells increased substantially with VT, but, once VT was accounted for, wounding was independent of maximal PTP. We found that interfacial stress associated with the generation and destruction of liquid bridges in airspaces is the primary biophysical cell injury mechanism in mechanically ventilated lungs. © 2013 the American Physiological Society.

Suki B.,Boston University | Hubmayr R.,Thoracic Diseases Research Unit
Current Opinion in Critical Care | Year: 2014

PURPOSE OF REVIEW: The adult respiratory distress syndrome (ARDS) is a common cause of respiratory failure with substantial impact on public health. Patients with ARDS generally require mechanical ventilation, which risks further lung damage. Recent improvements in ARDS outcomes have been attributed to reductions in deforming stress associated with lung protective mechanical ventilation modes and settings. The following review details the mechanics of the lung parenchyma at different spatial scales and the response of its resident cells to deforming stress in order to provide the biologic underpinnings of lung protective care. RECENT FINDINGS: Although lung injury is typically viewed through the lens of altered barrier properties and mechanical ventilation-Associated immune responses, in this review, we call attention to the importance of heterogeneity and the physical failure of the load bearing cell and tissue elements in the pathogenesis of ARDS. Specifically, we introduce a simple elastic network model to better understand the deformations of lung regions, intra-Acinar alveoli and cells within a single alveolus, and consider the role of regional distension and interfacial stress-related injury for various ventilation modes. SUMMARY: Heterogeneity of stiffness and intercellular and intracellular stress failure are fundamental components of ARDS and their development also depends on the ventilation mode. © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.

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