Kathleen A. Stringer

Metscape 2 bioinformatics tool for the analysis and visualization of metabolomics and gene expression data

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 metscape-help@umich.edu; akarnovs@umich.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Metabolic consequences of sepsis-induced acute lung injury revealed by plasma 1H-nuclear magnetic resonance quantitative metabolomics and computational analysis

Metabolomics is an emerging component of systems biology that may be a viable strategy for the identification and validation of physiologically relevant biomarkers. Nuclear magnetic resonance (NMR) spectroscopy allows for establishing quantitative data sets for multiple endogenous metabolites without preconception. Sepsis-induced acute lung injury (ALI) is a complex and serious illness associated with high morbidity and mortality for which there is presently no effective pharmacotherapy. The goal of this study was to apply ¹H-NMR based quantitative metabolomics with subsequent computational analysis to begin working towards elucidating the plasma metabolic changes associated with sepsis-induced ALI. To this end, this pilot study generated quantitative data sets that revealed differences between patients with ALI and healthy subjects in the level of the following metabolites: total glutathione, adenosine, phosphatidylserine, and sphingomyelin. Moreover, myoinositol levels were associated with acute physiology scores (APS) (ρ = -0.53, P = 0.05, q = 0.25) and ventilator-free days (ρ = -0.73, P = 0.005, q = 0.01). There was also an association between total glutathione and APS (ρ = 0.56, P = 0.04, q = 0.25). Computational network analysis revealed a distinct metabolic pathway for each metabolite. In summary, this pilot study demonstrated the feasibility of plasma ¹H-NMR quantitative metabolomics because it yielded a physiologically relevant metabolite data set that distinguished sepsis-induced ALI from health. In addition, it justifies the continued study of this approach to determine whether sepsis-induced ALI has a distinct metabolic phenotype and whether there are predictive biomarkers of severity and outcome in these patients.

Electrolysis-induced myocardial dysfunction: A novel method for the study of free radical mediated tissue injury

Oxygen-derived free radicals and other oxidizing species are thought to be involved in inflammation and ischemic tissue injuries. Recently, oxygen-derived free radicals also have been implicated in tissue injury of the myocardium subjected to ischemia/reperfusion. The purpose of this investigation was to determine if electrolysis of a physiological buffer would serve as a source of free radicals, and if these radicals would lead to alterations in myocardial function. Isolated Langendorff-perfused rabbit hearts perfused with buffer subjected to a 20 mA D.C. current for 2 min demonstrated significant increases in coronary perfusion pressure (37 +/- 6 mmHg), left ventricular end diastolic pressure (41 +/- 7 mmHg), and loss in left ventricular developed pressure (35 +/- 5%). The free radical scavengers, superoxide dismutase and a combination of tryptophan plus glycine, were effective in protecting the hearts from the effects of electrolysis. The presence of free radicals was semiquantitated with a radical-luminol chemiluminescent assay. In this assay a variety of radical scavengers and antioxidants were effective (i.e., dimethyl sulfoxide, nitro blue tetrazolium, ascorbate, superoxide dismutase, 1, 3-diphenylisobenzofuran, and glycine, catalase), whereas mannitol and tryptophan were not effective. The data indicate that electrolysis of a physiological buffer produces a milieu containing several reactive oxygen species or free radicals that have the potential to produce alterations in a biological system. This method has the advantage over existing protocols for the generation of radicals in that it is a blood-free and an enzyme-free system.

Hirudins: Antithrombin Anticoagulants

OBJECTIVE: To review the chemistry, pharmacology, available clinical data, and adverse effects of the hirudin anticoagulants. DATA SOURCES: A MEDLINE search and a review of recent scientific abstracts was conducted to identify pertinent literature. STUDY SELECTION: Focus was placed on studies conducted in humans. Because hirudin is still an investigational agent, however, relevant animal data, particularly pharmacokinetic studies and studies of preclinical efficacy, were also selected. DATA EXTRACTION: Data from both human and animal studies were evaluated; emphasis was placed on human trials. DATA SYNTHESIS: Hirudin has demonstrated potent anticoagulant effects. Although hirudin could have a significant impact on the therapeutic management of patients requiring anticoagulant therapy, only a limited number of human studies have been published to date. Trials comparing hirudin and heparin in specific patient populations are still ongoing. CONCLUSIONS: Although still in clinical trials, hirudin is a unique agent that may represent a breakthrough in anticoagulant therapy. The specific role that this agent will play in the management of patients has yet to be determined.

Untargeted LC-MS metabolomics of bronchoalveolar lavage fluid differentiates acute respiratory distress syndrome from health.

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 is 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.

Social Media Methods for Studying Rare Diseases

For pediatric rare diseases, the number of patients available to support traditional research methods is often inadequate. However, patients who have similar diseases cluster “virtually” online via social media. This study aimed to (1) determine whether patients who have the rare diseases Fontan-associated protein losing enteropathy (PLE) and plastic bronchitis (PB) would participate in online research, and (2) explore response patterns to examine social media’s role in participation compared with other referral modalities. A novel, internet-based survey querying details of potential pathogenesis, course, and treatment of PLE and PB was created. The study was available online via web and Facebook portals for 1 year. Apart from 2 study-initiated posts on patient-run Facebook pages at the study initiation, all recruitment was driven by study respondents only. Response patterns and referral sources were tracked. A total of 671 respondents with a Fontan palliation completed a valid survey, including 76 who had PLE and 46 who had PB. Responses over time demonstrated periodic, marked increases as new online populations of Fontan patients were reached. Of the responses, 574 (86%) were from the United States and 97 (14%) were international. The leading referral sources were Facebook, internet forums, and traditional websites. Overall, social media outlets referred 84% of all responses, making it the dominant modality for recruiting the largest reported contemporary cohort of Fontan patients and patients who have PLE and PB. The methodology and response patterns from this study can be used to design research applications for other rare diseases.

Utility of magnetic resonance imaging and nuclear magnetic resonance-based metabolomics for quantification of inflammatory lung injury

Magnetic resonance imaging (MRI) and metabolic nuclear magnetic resonance (NMR) spectroscopy are clinically available but have had little application in the quantification of experimental lung injury. There is a growing and unfulfilled need for predictive animal models that can improve our understanding of disease pathogenesis and therapeutic intervention. Integration of MRI and NMR could extend the application of experimental data into the clinical setting. This study investigated the ability of MRI and metabolic NMR to detect and quantify inflammation-mediated lung injury. Pulmonary inflammation was induced in male B6C3F1 mice by intratracheal administration of IL-1beta and TNF-alpha under isoflurane anesthesia. Mice underwent MRI at 2, 4, 6, and 24 h after dosing. At 6 and 24 h lungs were harvested for metabolic NMR analysis. Data acquired from IL-1beta+TNF-alpha-treated animals were compared with saline-treated control mice. The hyperintense-to-total lung volume (HTLV) ratio derived from MRI was higher in IL-1beta+TNF-alpha-treated mice compared with control at 2, 4, and 6 h but returned to control levels by 24 h. The ability of MRI to detect pulmonary inflammation was confirmed by the association between HTLV ratio and histological and pathological end points. Principal component analysis of NMR-detectable metabolites also showed a temporal pattern for which energy metabolism-based biomarkers were identified. These data demonstrate that both MRI and metabolic NMR have utility in the detection and quantification of inflammation-mediated lung injury. Integration of these clinically available techniques into experimental models of lung injury could improve the translation of basic science knowledge and information to the clinic.

Multiscale Distribution and Bioaccumulation Analysis of Clofazimine Reveals a Massive Immune System-Mediated Xenobiotic Sequestration Response

ABSTRACT 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.

Emerging Role of Anticoagulants and Fibrinolytics in the Treatment of Acute Respiratory Distress Syndrome

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.

Fontan-Associated Protein-Losing Enteropathy and Plastic Bronchitis

OBJECTIVE To characterize the medical history, disease progression, and treatment of current-era patients with the rare diseases Fontan-associated protein-losing enteropathy (PLE) and plastic bronchitis. STUDY DESIGN A novel survey that queried demographics, medical details, and treatment information was piloted and placed online via a Facebook portal, allowing social media to power the study. Participation regardless of PLE or plastic bronchitis diagnosis was allowed. Case control analyses compared patients with PLE and plastic bronchitis with uncomplicated control patients receiving the Fontan procedure. RESULTS The survey was completed by 671 subjects, including 76 with PLE, 46 with plastic bronchitis, and 7 with both. Median PLE diagnosis was 2.5 years post-Fontan. Hospitalization for PLE occurred in 71% with 41% hospitalized ≥ 3 times. Therapy varied significantly. Patients with PLE more commonly had hypoplastic left ventricle (62% vs 44% control; OR 2.81, 95% CI 1.43-5.53), chylothorax (66% vs 41%; OR 2.96, CI 1.65-5.31), and cardiothoracic surgery in addition to staged palliation (17% vs 5%; OR 4.27, CI 1.63-11.20). Median plastic bronchitis diagnosis was 2 years post-Fontan. Hospitalization for plastic bronchitis occurred in 91% with 61% hospitalized ≥ 3 times. Therapy was very diverse. Patients with plastic bronchitis more commonly had chylothorax at any surgery (72% vs 51%; OR 2.47, CI 1.20-5.08) and seasonal allergies (52% vs 36%; OR 1.98, CI 1.01-3.89). CONCLUSIONS Patient-specific factors are associated with diagnoses of PLE or plastic bronchitis. Treatment strategies are diverse without clear patterns. These results provide a foundation upon which to design future therapeutic studies and identify a clear need for forming consensus approaches to treatment.