Acute respiratory distress syndrome (ARDS) remains a significant hazard to human health and is clinically challenging because there are no prognostic biomarkers and no effective pharmacotherapy. The lung compartment metabolome may detail the status of the local environment that could be useful in ARDS biomarker discovery and the identification of drug target opportunities. However, neither the utility of bronchoalveolar lavage fluid (BALF) as a biofluid for metabolomics nor the optimal analytical platform for metabolite identification are established. To address this, we undertook a study to compare metabolites in BALF samples from patients with ARDS and healthy controls using a newly developed liquid chromatography (LC)-mass spectroscopy (MS) platform for untargeted metabolomics. Following initial testing of three different high performance liquid chromatography (HPLC) columns, we determined that reversed phase (RP)-LC and hydrophilic interaction chromatography (HILIC), were the most informative chromatographic methods because they yielded the most and highest quality data. Following confirmation of metabolite identification, statistical analysis resulted in 37 differentiating metabolites in the BALF of ARDS compared with health across both analytical platforms. Pathway analysis revealed networks associated with amino acid metabolism, glycolysis and gluconeogenesis, fatty acid biosynthesis, phospholipids and purine metabolism in the ARDS BALF. The complementary analytical platforms of RPLC and HILIC-LC generated informative, insightful metabolomics data of the ARDS lung environment.
biomarkers; critical illness; metabolomics; lung injury; bioinformatics; phospholipids; lactate; xanthine oxidase; hippurate; pharmacotherapy
Pediatric pharmacotherapy is often challenging due to the paucity of available clinical data on the safety and efficacy of medications that are commonly used in children. This quandary is even more prevalent in children with rare diseases. Although extrapolations for dosing and administration are often made from available adult data with similar disease states, this translation becomes even more problematic in rare pediatric diseases. Understanding of disease pathophysiology is typically poor, and few, if any, effective therapies have been studied and identified. One condition that illustrates these issues is plastic bronchitis, a rare, most often pediatric disease that is characterized by the production of obstructive bronchial airway casts. This illness primarily occurs in children with congenital heart disease, often after palliative surgery. Plastic bronchitis is a highly clinically relevant and therapeutically challenging problem with a high mortality rate, and, to date, a generally accepted effective pharmacotherapy regimen has not been identified. Furthermore, the disease is ill defined, which makes timely identification and treatment of children with plastic bronchitis difficult. The pharmacotherapies currently used to manage this disease are largely anecdotal and vary between the use of macrolide antibiotics, mucolytics, bronchodilators, and inhaled fibrinolytics in a myriad of combinations. The purpose of this review is twofold: first, to highlight the dilemma of treating plastic bronchitis, and second, to bring attention to the continuing need for studies of drug therapies used in children so that safe and effective drug regimens can be established, particularly for rare diseases, which often have no treatment options.
congenital heart disease; Fontan procedure; adverse effects; pulmonary disease; fibrinolysis; antiinflammatory agents
We sought to identify key variables in cellular architecture and physiology that might explain observed differences in the passive transport properties of small molecule drugs across different airway epithelial cell types.
Propranolol (PR) was selected as a weakly basic, model compound to compare the transport properties of primary (NHBE) vs. tumor-derived (Calu-3) cells. Differentiated on Transwell™ inserts, the architecture of pure vs. mixed cell co-cultures was studied with confocal microscopy followed by quantitative morphometric analysis. Cellular pharmacokinetic modeling was used to identify parameters that differentially affect PR uptake and transport across these two cell types.
Pure Calu-3 and NHBE cells possessed different structural and functional properties. Nevertheless, mixed Calu-3 and NHBE cell co-cultures differentiated as stable cell monolayers. After measuring the total mass of PR, the fractional areas covered by Calu-3 and NHBE cells allowed deconvoluting the transport properties of each cell type. Based on the apparent thickness of the unstirred, cell surface aqueous layer, local differences in extracellular microenvironment explained the measured variations in passive PR uptake and permeation between Calu-3 and NHBE cells.
Mixed cell co-cultures can be used to compare the local effects of the extracellular microenvironment on drug uptake and transport across two epithelial cell types.
cellular pharmacokinetics; Calu-3 cells; local drug absorption; inhaled drug delivery; computational modeling
Rationale: Plastic bronchitis (PB) is a rare and deadly condition that is characterized by the formation of airway casts. It most frequently occurs in children with underlying congenital heart disease that has been surgically palliated by the Fontan procedure. The Fontan circulation results in above-normal central venous pressure, and it has been hypothesized that the formation of airway casts is due to lymph leak. Knowledge of plastic bronchitis pathogenesis is poor and stems mostly from published case reports.
Objectives: To garner information about cast pathogenesis by characterizing inflammatory cell phenotypes in existing formalin-preserved, paraffin-embedded samples and generating protein and cytokine–chemokine profiles of airway cast homogenates.
Methods: We used immunofluorescence confocal microscopy, state-of-the-science proteomics, and a cytokine array assay to immunophenotype cellular content and to generate protein and cytokine profiles of plastic bronchitis airway casts, respectively.
Measurements and Main Results: Neutrophils, eosinophils, macrophages, and B lymphocytes were identified in cast samples; there were notably fewer T lymphocytes. Fibrin(ogen) was an abundant protein in the cast proteome. Histone H4 was also abundant, and immunofluorescence microscopy demonstrated it to be mostly extracellular. The cytokine profile of plastic bronchitis casts was proinflammatory.
Conclusions: Plastic bronchitis airway casts from children with Fontan physiology are composed of fibrin and are cellular and inflammatory in nature, providing evidence that their formation cannot be explained simply by lymph leak into the airways. Consequences of cellular necrosis including extracellular histones and the apparent low number of T cells indicate that a derangement in inflammation resolution likely contributes to cast formation.
inflammation; proteomics; fibrin; histones; cytokines
Plastic bronchitis (PB) is a poorly understood disease that can complicate any underlying pulmonary disease. However, it appears to most often occur in patients with surgically palliated congenital heart disease, particularly after the Fontan procedure. Few data exist about the prevalence and etiology of PB in this population. In an effort to establish data about prevalence, we conducted a retrospective study of an existing Fontan surgery database (n = 654) comprised of data, including sex, age at date of surgery, alive/dead status, New York Heart Association classification at last follow-up, right-ventricular end-diastolic pressure and pulmonary artery pressure before Fontan surgery, and the presence of a Fontan fenestration. An initial medical record review of 173 patients in the database who were followed at the University of Michigan identified seven patients with PB resulting in an estimated prevalence of 4 %. Subsequently, 14 % of 211 surveyed patients reported that they presently expectorate mucus or fibrin plugs (casts). Demographic and clinical variables did not differ between patients with or without possible PB. Collectively, these findings suggest that Fontan patients presently with PB may range from 4 to 14 %, indicating potential under-diagnosis of the disease. There were no remarkable physical or hemodynamic indicators that differentiated patients with or without possible PB. These data also highlight the need for more elaborate, prospective studies to improve our understanding of PB pathogenesis so that more definitive diagnostic criteria for this devastating disease can be established and its prevalence more accurately determined.
Congenital heart disease; Hypoplastic left heart syndrome; Fontan procedure adverse effects
We hypothesized that radiation induced thoracic toxicity (RITT) of the lung, esophagus and pericardium share a similar mechanism, and aimed to examine whether genetic variation of transforming growth factor-beta1 (TGFβ1), tissue plasminogen activator (tPA) and angiotensin converting enzyme (ACE), are associated with RITT in patients with non-small cell lung cancer (NSCLC).
Material and methods
Patients with stage I-III NSCLC were enrolled and received radiotherapy (RT). Blood samples were obtained pre-RT and at 4–5 weeks during RT and plasma TGF-β1 was measured using an enzyme-linked immunosorbent assay. The DNA samples extracted from blood pre-RT were analyzed for the following frequent genetic variations: TGFβ1 509C/T, tPA −7351 C/T, and ACE I/D. RITT score was defined as the sum of radiation induced toxicity grades in esophagus, lung and pericardium.
76 NSCLC patients receiving definitive RT were enrolled. Patients with TGFß1 509CC had higher mean grade of esophagitis (1.4±0.2 vs. 0.8±0.2, p=0.019) and RITT score (2.6±0.3 vs. 1.6±0.3, p=0.009) than T allele carriers. Although no significant relationship was observed between RITT and the tPA or ACE variants individually, patients with any high risk alleles (tPA CC or ACE D or TGFß1 509CC) had significantly higher grade of developing combined RITT (p<0.001). Patients with TGFß1 509CC had greater increase of plasma TGF ß1 levels at 4-5 weeks during-RT than T allele carriers (CC 1.2±0.2 vs. T 0.7±0.1, p=0.047).
This exploratory study demonstrated that sensitivity of radiation toxicity may be determined by genomic factors associated with TGFβ1 and genes involved in TGFβ1 pathway.
single nucleotide polymorphism; toxicity; radiotherapy; non-small cell lung cancer
We discovered that serious issues could arise that may complicate interpretation of metabolomic data when identical samples are analyzed at more than one NMR facility, or using slightly different NMR parameters on the same instrument. This is important because cross-center validation metabolomics studies are essential for the reliable application of metabolomics to clinical biomarker discovery. To test the reproducibility of quantified metabolite data at multiple sites, technical replicates of urine samples were assayed by 1D-1H-NMR at the University of Alberta and the University of Michigan. Urine samples were obtained from healthy controls under a standard operating procedure for collection and processing. Subsequent analysis using standard statistical techniques revealed that quantitative data across sites can be achieved, but also that previously unrecognized NMR parameter differences can dramatically and widely perturb results. We present here a confirmed validation of NMR analysis at two sites, and report the range and magnitude that common NMR parameters involved in solvent suppression can have on quantitated metabolomics data. Specifically, saturation power levels greatly influenced peak height intensities in a frequency-dependent manner for a number of metabolites, which markedly impacted the quantification of metabolites. We also investigated other NMR parameters to determine their effects on further quantitative accuracy and precision. Collectively, these findings highlight the importance of and need for consistent use of NMR parameter settings within and across centers in order to generate reliable, reproducible quantified NMR metabolomics data.
Local or systemic inflammation can result in acute lung injury (ALI), and is associated with capillary leakage, reduced lung compliance, and hypoxemia. Curcumin, a plant-derived polyphenolic compound, exhibits potent anti-inflammatory properties, but its poor solubility and limited oral bioavailability reduce its therapeutic potential. A novel curcumin formulation (CDC) was developed by complexing the compound with hydroxypropyl-γ-cyclodextrin (CD). This results in greatly enhanced water solubility and stability that facilitate direct pulmonary delivery. In vitro studies demonstrated that CDC increased curcumin’s association with and transport across Calu-3 human airway epithelial cell monolayers, compared with uncomplexed curcumin solubilized using DMSO or ethanol. Importantly, Calu-3 cell monolayer integrity was preserved after CDC exposure, whereas it was disrupted by equivalent uncomplexed curcumin solutions. We then tested whether direct delivery of CDC to the lung would reduce severity of ALI in a murine model. Fluorescence microscopic examination revealed an association of curcumin with cells throughout the lung. The administration of CDC after LPS attenuated multiple markers of inflammation and injury, including pulmonary edema and neutrophils in bronchoalveolar lavage fluid and lung tissue. CDC also reduced oxidant stress in the lungs and activation of the proinflammatory transcription factor NF-κB. These results demonstrate the efficacy of CDC in a murine model of lung inflammation and injury, and support the feasibility of developing a lung-targeted, curcumin-based therapy for the treatment of patients with ALI.
cyclodextrin; LPS; turmeric; Calu-3; oxidative stress; TEER
Chronic exposure to some well-absorbed but slowly eliminated xenobiotics can lead to their bioaccumulation in living organisms. Here, we studied the bioaccumulation and distribution of clofazimine, a riminophenazine antibiotic used to treat mycobacterial infection. Using mice as a model organism, we performed a multiscale, quantitative analysis to reveal the sites of clofazimine bioaccumulation during chronic, long-term exposure. Remarkably, between 3 and 8 weeks of dietary administration, clofazimine massively redistributed from adipose tissue to liver and spleen. During this time, clofazimine concentration in fat and serum significantly decreased, while the mass of clofazimine in spleen and liver increased by >10-fold. These changes were paralleled by the accumulation of clofazimine in the resident macrophages of the lymphatic organs, with as much as 90% of the clofazimine mass in spleen sequestered in intracellular crystal-like drug inclusions (CLDIs). The amount of clofazimine associated with CLDIs of liver and spleen macrophages disproportionately increased and ultimately accounted for a major fraction of the total clofazimine in the host. After treatment was discontinued, clofazimine was retained in spleen while its concentrations decreased in blood and other organs. Immunologically, clofazimine bioaccumulation induced a local, monocyte-specific upregulation of various chemokines and receptors. However, interleukin-1 receptor antagonist was also upregulated, and the acute-phase response pathways and oxidant capacity decreased or remained unchanged, marking a concomitant activation of an anti-inflammatory response. These experiments indicate an inducible, immune system-dependent, xenobiotic sequestration response affecting the atypical pharmacokinetics of a small molecule chemotherapeutic agent.
Motivation: Metabolomics is a rapidly evolving field that holds promise to provide insights into genotype–phenotype relationships in cancers, diabetes and other complex diseases. One of the major informatics challenges is providing tools that link metabolite data with other types of high-throughput molecular data (e.g. transcriptomics, proteomics), and incorporate prior knowledge of pathways and molecular interactions.
Results: We describe a new, substantially redesigned version of our tool Metscape that allows users to enter experimental data for metabolites, genes and pathways and display them in the context of relevant metabolic networks. Metscape 2 uses an internal relational database that integrates data from KEGG and EHMN databases. The new version of the tool allows users to identify enriched pathways from expression profiling data, build and analyze the networks of genes and metabolites, and visualize changes in the gene/metabolite data. We demonstrate the applications of Metscape to annotate molecular pathways for human and mouse metabolites implicated in the pathogenesis of sepsis-induced acute lung injury, for the analysis of gene expression and metabolite data from pancreatic ductal adenocarcinoma, and for identification of the candidate metabolites involved in cancer and inflammation.
Availability: Metscape is part of the National Institutes of Health-supported National Center for Integrative Biomedical Informatics (NCIBI) suite of tools, freely available at http://metscape.ncibi.org. It can be downloaded from http://cytoscape.org or installed via Cytoscape plugin manager.
Contact: email@example.com; firstname.lastname@example.org
Supplementary information: Supplementary data are available at Bioinformatics online.
Plastic bronchitis (PB) is a rare disease that often occurs in patients with congenital heart disease (CHD) who have undergone staged single ventricle palliation. It is characterized by the formation of rubbery “casts” in the airways. PB treatment frequently includes inhaled tPA. However, the efficacy of tPA to reduce cast burden is unknown. This is further complicated by our lack of knowledge of cast composition. We obtained spontaneously expectorated PB casts from children (n=4) with CHD and one adult patient with idiopathic PB. Pathological assessment was made from paraffin-preserved samples. Casts were treated with phosphate-buffered saline (PBS) or tPA. Cast response to tPA was assessed by changes in cast weight and the production of fibrin D-dimer. Independent of dose, tPA reduced cast weight compared with PBS-treatment (p=0.001) and increased D-dimer levels. Histological staining showed that PB casts from all patients were comprised of fibrin and contained notable numbers of lymphocytes. Cast composition did not change over time. Collectively, these data support that in our PB patients, casts are comprised of fibrin and are responsive to tPA treatment. This makes inhaled tPA a potentially viable option for symptomatic relief of PB while we work to unravel the complexity of PB pathogenesis.
congential heart disease; Fontan physiology; pulmonary drug delivery
Metabolomics, a science of systems biology, is the global assessment of endogenous metabolites within a biologic system and represents a “snapshot” reading of gene function, enzyme activity, and the physiological landscape. Metabolite detection, either individual or grouped as a metabolomic profile, is usually performed in cells, tissues, or biofluids by either nuclear magnetic resonance spectroscopy or mass spectrometry followed by sophisticated multivariate data analysis. Because loss of metabolic homeostasis is common in critical illness, the metabolome could have many applications, including biomarker and drug target identification. Metabolomics could also significantly advance our understanding of the complex pathophysiology of acute illnesses, such as sepsis and acute lung injury/acute respiratory distress syndrome. Despite this potential, the clinical community is largely unfamiliar with the field of metabolomics, including the methodologies involved, technical challenges, and, most importantly, clinical uses. Although there is evidence of successful preclinical applications, the clinical usefulness and application of metabolomics in critical illness is just beginning to emerge, the advancement of which hinges on linking metabolite data to known and validated clinically relevant indices. In addition, other important aspects, such as patient selection, sample collection, and processing, as well as the needed multivariate data analysis, have to be taken into consideration before this innovative approach to biomarker discovery can become a reliable tool in the intensive care unit. The purpose of this review is to begin to familiarize clinicians with the field of metabolomics and its application for biomarker discovery in critical illnesses such as sepsis.
sepsis;; acute lung injury;; pneumonia;; trauma
Modeling the local absorption and retention patterns of membrane-permeant small molecules in a cellular context could facilitate development of site-directed chemical agents for bioimaging or therapeutic applications. Here, we present an integrative approach to this problem, combining in silico computational models, in vitro cell based assays and in vivo biodistribution studies. To target small molecule probes to the epithelial cells of the upper airways, a multiscale computational model of the lung was first used as a screening tool, in silico. Following virtual screening, cell monolayers differentiated on microfabricated pore arrays and multilayer cultures of primary human bronchial epithelial cells differentiated in an air-liquid interface were used to test the local absorption and intracellular retention patterns of selected probes, in vitro. Lastly, experiments involving visualization of bioimaging probe distribution in the lungs after local and systemic administration were used to test the relevance of computational models and cell-based assays, in vivo. The results of in vivo experiments were consistent with the results of in silico simulations, indicating that mitochondrial accumulation of membrane permeant, hydrophilic cations can be used to maximize local exposure and retention, specifically in the upper airways after intratracheal administration.
We have developed an integrative, cell-based modeling approach to facilitate the design and discovery of chemical agents directed to specific sites of action within a living organism. Here, a computational, multiscale transport model of the lung was adapted to enable virtual screening of small molecules targeting the epithelial cells of the upper airways. In turn, the transport behaviors of selected candidate probes were evaluated to establish their degree of retention at a site of absorption, using computational simulations as well as two in vitro cell-based assay systems. Lastly, bioimaging experiments were performed to examine candidate molecules' distribution in the lungs of mice after local and systemic administration. Based on computational simulations, the higher mitochondrial density per unit absorption surface area is the key parameter determining the higher retention of small molecule hydrophilic cations in the upper airways, relative to lipophilic weak bases, specifically after intratracheal administration.
Disruption of fibrinolytic homeostasis participates in the pathogenesis of severe lung diseases like acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF) and plastic bronchitis. We have developed a pulmonary formulation of tissue plasminogen activator (pf-tPA) that withstands nebulization and reaches the lower airways.
Since treatment of ARDS, IPF and plastic bronchitis will require repeated administration of pf-tPA, the purpose of this study was to determine the safety of prolonged, repeated administration of pf-mouse tPA (pf-mtPA) to the lungs of healthy mice.
Male and female B6C3F1 mice received one of two intratracheal (IT) doses of either nebulized pf-mtPA or sterile saline twice daily for 28 days. Weekly blood samples were collected to estimate hematocrit. Following the dosing period, animals were sacrificed for gross necropsy, the acquisition of bronchoalveolar lavage fluid (BALF), and histological assessment of the lungs and other major organs.
The low dose of pf-mtPA was well-tolerated by both female and male mice. However, female and male mice that received the high dose experienced a 16% and 8% incidence, respectively, of fatal pulmonary hemorrhage. Although male mice had a lower incidence of bleeding, these events occurred at lower mean (±S.E.) doses (1.06±0.02 mg/kg/d) of pf-mtPA compared with females (1.48±0.03 mg/kg/d, p < 0.001). In addition, male mice had higher BALF mtPA concentrations. Bleeding occurred six and 12 days in male and female mice, respectively, after the initiation of dosing suggesting that mtPA accumulated in the lungs.
This study established a safe dose range and demonstrated the feasibility of prolonged, repeated dosing of pf-tPA. High doses (≥ 1mg/kg/d) were associated with pulmonary hemorrhage that may be due, in part, to accumulation of drug in the lungs.
pulmonary drug delivery; therapeutic protein; plastic bronchitis; experimental lung injury; fibrinolysis; lung clearance; gender differences
Tissue plasminogen activator (tPA) has both fibrinolytic and anti-inflammatory activity. These properties may be useful in treating inflammatory lung diseases, such as acute respiratory distress syndrome (ARDS).We have previously demonstrated the feasibility of targeted pulmonary delivery of tPA. As part of our research to develop a clinically viable pulmonary formulation of tPA, we assessed the tolerability and incidence of haemorrhage associated with the administration of a pulmonary formulation of mouse tPA (pf-mtPA).Intratracheal doses of nebulized pf-mtPA or sterile saline were administered with increasing frequency to male and female B6C3F1 mice. After dosing, the mice entered a recovery period, after which they were killed and their lungs were lavaged and harvested. Post-mortem gross necropsy was performed and all major organs were assessed histologically for haemorrhage. The bronchoalveolar lavage fluid was assessed for markers of lung injury.Mouse tPA that was formulated to mimic a previously characterized human pf-tPA was well tolerated when given intratracheally with increasing dosing frequency. The administration of pf-mtPA did not result in any detectable haemorrhagic-related events or signs of lung injury.The results of the present longitudinal study demonstrate that a maximally feasible dose of pf-mtPA (3 mg/kg) can be given frequently over a short period of time (12 h) without haemorrhagic complications. Although these data were generated in a healthy mouse model, they provide support for the continued evaluation of pf-tPA for the treatment of pulmonary diseases, such as ARDS.
B6C3F1 mice; haemorrhage; nebulization; pulmonary drug delivery; tissue plasminogen activator
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high mortality rates despite therapeutic advances. The pathogenesis of ALI and ARDS is similar to that of sepsis, as these disease states involve uncontrolled host defense responses that lead to inflammation, endothelial damage, enhanced coagulation, diminished fibrinolysis, and fibroproliferation. Recent studies of anticoagulants have shown positive outcomes in patients with severe sepsis. In addition, emerging evidence suggests that the use of anticoagulants, such as tissue factor pathway inhibitor, antithrombin, thrombomodulin, heparin, activated protein C, and fibrinolytics (plasminogen activators and particularly tissue plasminogen activator), may be useful in the treatment of ALI and ARDS. Data from experimental models of sepsis, ALI, and ARDS indicate that some of these agents improve lung function and oxygenation. Although clinical data are less convincing than these findings, results from clinical trials may influence the design of future studies.
acute respiratory distress syndrome; ARDS; acute lung injury; ALI; inflammation; coagulation; fibrinolysis; tissue factor pathway inhibitor; activated protein C; thrombomodulin; antithrombin; heparin; plasminogen activators; t-PA; u-PA
This study was conducted to assess the feasibility of a pulmonary formulation of tissue plasminogen activator (tPA) for nebulization into the airway by measuring protein stability, biologic activity, particle size, and estimating human lung distribution.
Formulations were derived by varying the surfactant and protein concentrations. Protein stability and recovery of each nebulized tPA formulation were assessed by ultraviolet spectroscopy. Formulations that met protein stability feasibility criteria were assessed for biologic and fibrinolytic activities. Biologic activity was determined by their ability to inhibit superoxide anion production by human neutrophils. Fibrinolytic activity was assessed by the cleavage of plasminogen to plasmin. Aerodynamic properties were assessed using a cascade impactor, and an estimation of human airway deposition was made via a human lung replica.
Twenty-seven tPA formulations were initially assessed, 15 of which met protein stability criteria. Subsequently, three of these formulations maintained biologic and fibrinolytic activities. These formulations exhibited particle sizes of 2.4–3.1 μm, and had respirable doses ≥65%. A formulation of 1 mg mL−1 tPA and 0.1% Tween 80 exhibited a 45% deposition in the lower airways of a human lung replica.
A suitable pulmonary tPA formulation was identified that, following nebulization, maintained protein stability as well as biologic and fibrinolytic activities, and resulted in an optimal respirable dose and human airway deposition. This formulation may be applicable in the treatment of lung diseases, such as acute respiratory distress syndrome by permitting targeted pulmonary delivery of a therapeutic protein to the lungs.
acute respiratory distress syndrome; inflammation; nebulization; pulmonary delivery; tissue plasminogen activator