OnPLS-Based Multi-Block Data Integration: A Multivariate Approach to Interrogating Biological Interactions in Asthma.

Integration of multiomics data remains a key challenge in fulfilling the potential of comprehensive systems biology. Multiple-block orthogonal projections to latent structures (OnPLS) is a projection method that simultaneously models multiple data matrices, reducing feature space without relying on a priori biological knowledge. In order to improve the interpretability of OnPLS models, the associated multi-block variable influence on orthogonal projections (MB-VIOP) method is used to identify variables with the highest contribution to the model. This study combined OnPLS and MB-VIOP with interactive visualization methods to interrogate an exemplar multiomics study, using a subset of 22 individuals from an asthma cohort. Joint data structure in six data blocks was assessed: transcriptomics; metabolomics; targeted assays for sphingolipids, oxylipins, and fatty acids; and a clinical block including lung function, immune cell differentials, and cytokines. The model identified seven components, two of which had contributions from all blocks (globally joint structure) and five that had contributions from two to five blocks (locally joint structure). Components 1 and 2 were the most informative, identifying differences between healthy controls and asthmatics and a disease-sex interaction, respectively. The interactions between features selected by MB-VIOP were visualized using chord plots, yielding putative novel insights into asthma disease pathogenesis, the effects of asthma treatment, and biological roles of uncharacterized genes. For example, the gene ATP6 V1G1, which has been implicated in osteoporosis, correlated with metabolites that are dysregulated by inhaled corticoid steroids (ICS), providing insight into the mechanisms underlying bone density loss in asthma patients taking ICS. These results show the potential for OnPLS, combined with MB-VIOP variable selection and interaction visualization techniques, to generate hypotheses from multiomics studies and inform biology.

Multitissue Transcriptomics Delineates the Diversity of Airway T Cell Functions in Asthma.

Asthma arises from the complex interplay of inflammatory pathways in diverse cell types and tissues. We sought to undertake a comprehensive transcriptomic assessment of the epithelium and airway T cells that remain understudied in asthma and investigate interactions between multiple cells and tissues. Epithelial brushings and flow-sorted CD3+ T cells from sputum and BAL were obtained from healthy subjects (n = 19) and patients with asthma (mild, moderate, and severe asthma; n = 46). Gene expression was assessed using Affymetrix HT HG-U133+ PM GeneChips, and results were validated by real-time quantitative PCR. In the epithelium, IL-13 response genes (POSTN, SERPINB2, and CLCA1), mast cell mediators (CPA3 and TPSAB1), inducible nitric oxide synthase, and cystatins (CST1, CST2, and CST4) were upregulated in mild asthma, but, except for cystatins, were suppressed by corticosteroids in moderate asthma. In severe asthma-with predominantly neutrophilic phenotype-several distinct processes were upregulated, including neutrophilia (TCN1 and MMP9), mucins, and oxidative stress responses. The majority of the disease signature was evident in sputum T cells in severe asthma, where 267 genes were differentially regulated compared with health, highlighting compartmentalization of inflammation. This signature included IL-17-inducible chemokines (CXCL1, CXCL2, CXCL3, IL8, and CSF3) and chemoattractants for neutrophils (IL8, CCL3, and LGALS3), T cells, and monocytes. A protein interaction network in severe asthma highlighted signatures of responses to bacterial infections across tissues (CEACAM5, CD14, and TLR2), including Toll-like receptor signaling. In conclusion, the activation of innate immune pathways in the airways suggests that activated T cells may be driving neutrophilic inflammation and steroid-insensitive IL-17 response in severe asthma.

Impaired innate immune gene profiling in airway smooth muscle cells from chronic cough patients.

Chronic cough is associated with airway inflammation and remodelling. Abnormal airway smooth muscle cell (ASMC) function may underlie mechanisms of chronic cough. Our objective was to examine the transcriptome and focused secretome of ASMCs from chronic cough patients and healthy non-cough volunteers. ASMC gene expression profiling was performed at baseline and/or after stimulation with polyinosinic:polycytidylic acid (poly(I:C)) to mimic viral infection. Supernatants were collected for multiplex analysis. Our results showed no significant differentially expressed genes (DEGs, false discovery rate (FDR) <0.05) between chronic cough and healthy non-cough ASMCs at baseline. Poly(I:C) stimulation resulted in 212 DEGs (>1.5 fold-change, FDR <0.05) in ASMCs from chronic cough patients compared with 1674 DEGs in healthy non-cough volunteers. The top up-regulated genes included chemokine (C-X-C motif) ligand (CXCL) 11 (CXCL11), CXCL10, chemokine (C-C motif) ligand (CCL) 5 (CCL5) and interferon-induced protein 44 like (IFI44L) corresponding with inflammation and innate immune response pathways. ASMCs from cough subjects had enhanced activation of viral response pathways in response to poly(I:C) compared with healthy non-cough subjects, reduced activation of pathways involved in chronic inflammation and equivalent activation of neuroregulatory genes. The poly(I:C)-induced release of inflammatory mediators, including CXCL8, interleukin (IL)-6 and CXCL1, from ASMCs from cough patients was significantly impaired compared with healthy non-cough subjects. Addition of fluticasone propionate (FP) to poly(I:C)-treated ASMCs resulted in greater gene expression changes in healthy non-cough ASMCs. FP had a differential effect on poly(I:C)-induced mediator release between chronic cough and healthy non-cough volunteers. In conclusion, altered innate immune and inflammatory gene profiles within ASMCs, rather than infiltrating cells or nerves, may drive the cough response following respiratory viral infection.

Validated and longitudinally stable asthma phenotypes based on cluster analysis of the ADEPT study.

BACKGROUND: Asthma is a disease of varying severity and differing disease mechanisms. To date, studies aimed at stratifying asthma into clinically useful phenotypes have produced a number of phenotypes that have yet to be assessed for stability and to be validated in independent cohorts. The aim of this study was to define and validate, for the first time ever, clinically driven asthma phenotypes using two independent, severe asthma cohorts: ADEPT and U-BIOPRED. METHODS: Fuzzy partition-around-medoid clustering was performed on pre-specified data from the ADEPT participants (n = 156) and independently on data from a subset of U-BIOPRED asthma participants (n = 82) for whom the same variables were available. Models for cluster classification probabilities were derived and applied to the 12-month longitudinal ADEPT data and to a larger subset of the U-BIOPRED asthma dataset (n = 397). High and low type-2 inflammation phenotypes were defined as high or low Th2 activity, indicated by endobronchial biopsies gene expression changes downstream of IL-4 or IL-13. RESULTS: Four phenotypes were identified in the ADEPT (training) cohort, with distinct clinical and biomarker profiles. Phenotype 1 was "mild, good lung function, early onset", with a low-inflammatory, predominantly Type-2, phenotype. Phenotype 2 had a "moderate, hyper-responsive, eosinophilic" phenotype, with moderate asthma control, mild airflow obstruction and predominant Type-2 inflammation. Phenotype 3 had a "mixed severity, predominantly fixed obstructive, non-eosinophilic and neutrophilic" phenotype, with moderate asthma control and low Type-2 inflammation. Phenotype 4 had a "severe uncontrolled, severe reversible obstruction, mixed granulocytic" phenotype, with moderate Type-2 inflammation. These phenotypes had good longitudinal stability in the ADEPT cohort. They were reproduced and demonstrated high classification probability in two subsets of the U-BIOPRED asthma cohort. CONCLUSIONS: Focusing on the biology of the four clinical independently-validated easy-to-assess ADEPT asthma phenotypes will help understanding the unmet need and will aid in developing tailored therapies. TRIAL REGISTRATION: NCT01274507 (ADEPT), registered October 28, 2010 and NCT01982162 (U-BIOPRED), registered October 30, 2013.

Paradox and privilege: A 55-year follow-up of the mortality of Yale College graduates.

OBJECTIVE: Two hypotheses were tested: 1. People from privileged backgrounds had better survival than those from less privileged backgrounds. 2. The advantages of privilege were vitiated by fraternity membership. METHODS: A 55-year retrospective cohort study of survival since 1960 of 945 graduates of Yale College followed to 2015. RESULTS: The survival of graduates of private secondary schools (the privileged group) did not differ from that of public school graduates. However, graduates of private secondary schools who had not joined a fraternity in college had significantly better survival than private school graduates who had joined fraternities and than public school graduates, whether fraternity members or not. CONCLUSIONS: The benefits of a privileged background in respect of survival were undermined by fraternity membership. It is suggested that both self-selection and substance mis-use may have contributed to the survival difference.

Peripheral blood proteins predict mortality in idiopathic pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of unknown etiology with a variable and unpredictable course. OBJECTIVES: The aim of this study was to identify and validate plasma proteins that are predictive of outcome in IPF. METHODS: Plasma samples were available for 241 patients with IPF (140 derivation and 101 validation). In the derivation cohort, concentrations of 92 proteins were analyzed using a multiplex bead-based immunoassay and concentrations of matrix metalloproteinase (MMP)-7, MMP-1, and surfactant protein D were assessed by ELISA. In the validation cohort concentrations of intercellular adhesion molecule (ICAM)-1, IL-8, and vascular cell adhesion molecule (VCAM)-1 were assessed by bead-based multiplex assay, and S100A12 and MMP-7 by ELISA. Associations of biomarkers with mortality, transplant-free survival, and disease progression were tested in the derivation and validation cohorts using nonparametric methods of survival analysis and the Cox proportional hazards model, and an integrated risk prediction score was derived and tested. MEASUREMENTS AND MAIN RESULTS: High concentrations of MMP-7, ICAM-1, IL-8, VCAM-1, and S100A12 predicted poor overall survival, poor transplant-free survival, and poor progression-free survival in the derivation cohort. In the independent validation cohort high concentrations of all five were predictive of poor transplant-free survival; MMP-7, ICAM-1, and IL-8 of overall survival; and ICAM-1 of poor progression-free survival. The personal clinical and molecular mortality prediction index derived in the derivation cohort was highly predictive of mortality in the validation cohort. CONCLUSIONS: Our results suggest that plasma proteins should be evaluated as a tool for prognosis determination in prioritization of patients for lung transplantation and stratification in drug studies.

Reprint of: Comparison of affinity tags for protein purification.

Affinity tags are highly efficient tools for purifying proteins from crude extracts. To facilitate the selection of affinity tags for purification projects, we have compared the efficiency of eight elutable affinity tags to purify proteins from Escherichia coli, yeast, Drosophila, and HeLa extracts. Our results show that the HIS, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG, HPC (heavy chain of protein C) peptide tags, and the GST and MBP protein fusion tag systems differ substantially in purity, yield, and cost. We find that the HIS tag provides good yields of tagged protein from inexpensive, high capacity resins but with only moderate purity from E. coli extracts and relatively poor purification from yeast, Drosophila, and HeLa extracts. The CBP tag produced moderate purity protein from E. coli, yeast, and Drosophila extracts, but better purity from HeLa extracts. Epitope-based tags such as FLAG and HPC produced the highest purity protein for all extracts but require expensive, low capacity resin. Our results suggest that the Strep II tag may provide an acceptable compromise of excellent purification with good yields at a moderate cost.

Persistent Tn polyagglutination syndrome during febrile neutropenia: a case report and review of the literature.

INTRODUCTION: Tn polyagglutination syndrome is a rare disorder that has been reported on only a few occasions in the literature, and, to the best of our knowledge, never before in the context of febrile neutropenia. CASE PRESENTATION: We report the case of a 26-year-old Caucasian woman who presented to our emergency department complaining of a persistent fever over the previous three days. She had a history of long-standing refractory pancytopenia with multi-lineage dysplasia and severe neutropenia, but she had rarely experienced infection. The results of a physical examination and multiple laboratory tests were unremarkable. While investigating the possible causes of the refractory, long-standing pancytopenia, the possibility of a polyagglutinable state was suggested. Blood samples were sent to the laboratory for an analysis of mixed-field seed lectin agglutination assay. A serum lectin panel confirmed the final diagnosis of Tn-activation. CONCLUSIONS: We should include Tn-activation in our differential whenever we encounter cases of refractory long-standing idiopathic cytopenias and inconclusive bone marrow results displaying multi-lineage dysplasia. Novel genetic techniques have recently revealed the interesting pathophysiology of this phenomenon. The recognition and inclusion of Tn polyagglutination syndrome in our differential diagnoses has important clinical implications, given its main associated features, such as severe thrombocytopenia and neutropenia, which are usually linked to a benign clinical course and prognosis. Increased awareness of the polyagglutinable disorders will potentially decrease the need for invasive and costly medical interventions and also raises the need for monitoring of this specific sub-set of patients. In addition, the study of the expression and implications of Tn, and other similar antigens, offers a fascinating perspective for the study of its role in the diagnosis, prognosis and immunotherapy of solid tumors and hematological malignancies. The infrequency with which Tn polyagglutination syndrome is encountered, its clinical features and its pathophysiology make it a formidable diagnostic challenge.

Beyond informed consent: educating the patient.

The informed consent doctrine was conceived as a basis for allowing patients to meaningfully participate in the decision-making process. It has evolved into a formal, legal document that reflects a desire by physicians and surgeons to have patients execute "waivers of liability." In the process it has lost its educational value by shifting the emphasis to obtaining a "preoperative release" from an exchange of information upon which a patient can make important decisions about their healthcare choices. This is unfortunate because, in the process, both patients and physicians have suffered. Patients have become alienated from the informed consent process and, paradoxically, physicians and surgeons may have created more liability exposure through this alienation. We propose that by returning to an educational model, the patients will develop a greater sense of control, become more compliant, and potentially experience improved healthcare outcomes. There may also develop an alliance between the patient and the physician or surgeon, such that the seeds of an antagonistic or litigious relationship will not be planted before treatment begins. Liability reduction, therefore, may more likely arise from the educational model.

Levels of mRNA coding for alpha-, beta-, and gamma-synuclein in the brains of newborn, juvenile, and adult rats.

Synucleins are proteins known for their malfunction in a group of illnesses called synucleopathies, which includes Alzheimer's and Parkinson's disease. To learn more about the role of synucleins in the CNS, we have studied levels of message coding for alpha-, beta-, and gamma-synuclein using quantitative RT-PCR. Levels of synuclein mRNAs were studied in the cerebral cortex (left and right, anterior and posterior), hippocampus, striatum, and cerebellum, obtained from 5-d-old (newborn), 1-mo (juvenile)-, and 6-, and 9-mo (adult)-old rats. The mRNA levels for all synucleins varied significantly among structures. The rank order of mRNA levels in different structures was cortex = hippocampus > striatum > cerebellum for alpha-synuclein; cortex > hippocampus = cerebellum > striatum for beta-synuclein; and hippocampus = striatum > cortex = cerebellum for gamma-synuclein. There was significant effect of age for mRNA levels for all synucleins. The dynamics of these changes were different depending on type of synuclein and brain structure. Levels of mRNA for alpha-synuclein were significantly reduced with age in all structures except hippocampus. For beta- and gamma-synuclein, levels increased significantly only in the cerebral cortex and only from 5 d to 1 mo of age. In contrast, gamma-synuclein levels in the cerebellum were very high at 5 d and significantly reduced at 1 mo of age. The revealed pattern and dynamics of changes in the levels of mRNA coding for synucleins would support the conclusion for an important role of these molecules during development and the aging process.

Nanoliter high throughput quantitative PCR.

Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols.

Integrated expressional analysis: application to the drug discovery process.

Microarray technology enables high-throughput testing of gene expression to investigate various neuroscience related questions. This in turn creates a demand for scalable methods to confirm microarray results and the opportunity to use this information to discover and test novel pathways and therapeutic applications. Discovery of new central nervous system (CNS) treatments requires a comprehensive understanding of multiple aspects including the biology of a target, the pathophysiology of a disease/disorder, and the selection of successful lead compounds as well as efficient biomarker and drug disposition strategies such as absorption (how a drug is absorbed), distribution (how a drug spreads through an organism), metabolism (chemical conversion of a drug, if any, and into which substances), and elimination (how is a drug eliminated) (ADME). Understanding of the toxicity is also of paramount importance. These approaches, in turn, require novel high-content integrative assay technologies that provide thorough information about changes in cell biology. To increase efficiency of profiling, characterization, and validation, we established a new screening strategy that combines high-content image-based testing on Array Scan (Cellomics) with a confocal system and the multiplexed TaqMan RT-PCR method for quantitative mRNA expression analysis. This approach could serve as an interface between high-throughput microarray testing and specific application of markers discovered in the course of a microarray experiment. Markers could pinpoint activation or inhibition of a molecular pathway related, for instance, to neuronal viability. We demonstrate the successful testing of the same cell population in an image-based translocational assay followed by poly(A) mRNA capture and multiplexed single tube RT-PCR. In addition, Ciphergen ProteinChip analysis can be performed on the supernatant, thus allowing significant complementarity in the data output and interpretation by also including the capture and initial analysis of proteins in the integrative approach presented. We have determined various conditions including the number of cells, RT and PCR optimization, which are necessary for successful detection and consequent assay integration. We also show the successful convergence of various different approaches and multiplexing of different targets within a single real-time PCR tube. This novel integrative technological approach has utility for CNS drug discovery, target and biomarker identification, selection and characterization as well as for the study of toxicity- and adverse event-associated molecular mechanisms.

Comparison of affinity tags for protein purification.

Affinity tags are highly efficient tools for purifying proteins from crude extracts. To facilitate the selection of affinity tags for purification projects, we have compared the efficiency of eight elutable affinity tags to purify proteins from Escherichia coli, yeast, Drosophila, and HeLa extracts. Our results show that the HIS, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG, HPC (heavy chain of protein C) peptide tags, and the GST and MBP protein fusion tag systems differ substantially in purity, yield, and cost. We find that the HIS tag provides good yields of tagged protein from inexpensive, high capacity resins but with only moderate purity from E. coli extracts and relatively poor purification from yeast, Drosophila, and HeLa extracts. The CBP tag produced moderate purity protein from E. coli, yeast, and Drosophila extracts, but better purity from HeLa extracts. Epitope-based tags such as FLAG and HPC produced the highest purity protein for all extracts but require expensive, low capacity resin. Our results suggest that the Strep II tag may provide an acceptable compromise of excellent purification with good yields at a moderate cost.

Functional informatics: convergence and integration of automation and bioinformatics.

The biopharmaceutical industry is currently being presented with opportunities to improve research and business efficiency via automation and the integration of various systems. In the examples discussed, industrial high-throughput screening systems are integrated with functional tools and bioinformatics to facilitate target and biomarker identification and validation. These integrative functional approaches generate value-added opportunities by leveraging available automation and information technologies into new applications that are broadly applicable to different types of projects, and by improving the overall research and development and business efficiency via the integration of various systems.

Peripheral blood stem cell mobilization. A role for CXC chemokines.

Chemokines induce rapid hematopoietic stem and progenitor cell mobilization and synergize with hematopoietic cytokines in mobilizing stem and progenitor cells. These proteins alone and in combination offer new paradigms for autologous and allogeneic peripheral blood stem cell transplantation (PBSCT). The mechanisms responsible for hematopoietic stem cell (HSC) mobilization either with growth factors or chemokines are largely unknown, but a better understanding of these mechanisms will permit the development of novel, more rapid and efficacious regimens. Studies presented herein indicate that the CXCR2 chemokine receptor that interacts with selective chemokine ligands, particularly GRObeta/CXCL2 and GRObeta-T, may be the dominant receptor mediating hematopoietic cell mobilization, and that polymorphonuclear neutrophils may be the primary CXCR2 expressing target cell for stem and progenitor cell mobilization.

Inhibition of hematopoietic progenitor cell growth by Tyr-MIF, an endogenous opiate modulator, and its degradation products.

There is increasing evidence that neuronal factors can affect hematopoietic cell proliferation. Endogenous opioids with specificity for several opioid receptor classes were tested for their ability to inhibit murine and human hematopoietic progenitor cell proliferation. Tyr-MIF, an opioid tetrapeptide (H-Tyr-Pro-Leu-Gly-NH2), demonstrated a dose-dependent inhibition of colony formation at concentrations < 10 uM, inhibiting M-CSF and G-CSF-responsive progenitor cells equally. Tyr-MIF did not inhibit the number of colonies responsive to recombinant interleukin 3 (rmIL-3) or recombinant murine granulocyte-macrophage colony stimulating factor (rmGM-CSF), but significantly reduced colony size of GM-CSF responsive colonies. Colony formation by human low density and CD34+ marrow cells in response to G-CSF was also inhibited by Tyr-MIF and was more sensitive to inhibition than murine progenitor cells. Colony formation by single CD34+ cells was also inhibited by Tyr-MIF, indicating an effect directly on progenitor cells. Incubation of marrow cells in liquid culture and removal of Tyr-MIF prior to quantitating progenitor cell proliferation demonstrated that opioid-induced inhibition was reversible. The inhibitory effect of Tyr-MIF was not blocked by naloxone, a mu receptor specific antagonist, or diminished in mu opioid receptor deficient mice. HPLC analysis of cell-free culture medium containing Tyr-MIF showed no presence of the parent peptide after 24 h while progenitor cell inhibitory activity was retained. Analysis of potential degradation products of Tyr-MIF indicated that only H-Gly-NH9 or H-Gly-NH2 containing peptides inhibited colony forming unit (CFU) proliferation. These results indicate that Tyr-MIF is a reversible inhibitor of mature hematopoietic progenitor cell proliferation, and that this effect is most likely mediated by the degradation product H-Gly-NH2. Potential applications including protection of myeloid cells after cytosuppresive therapy are discussed.