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Abstract 2030 Sickle cell disease (SCD) is associated with a high rate of thromboembolic complications, particularly in patients with advanced vasculopathy. Recent functional studies have revealed pathological platelet activation associated with intravascular hemolysis, the severity of pulmonary hypertension (PAH), and impaired NO-signaling in platelets mediated by NO scavenging by plasma hemoglobin (Villagra J et al, Blood 2007; Hu W et al, Blood 2010, prepublished online). In addition, our previous amplified platelet transcriptome studies in SCD patients have demonstrated dysregulated arginine and polyamine metabolism, potentially contributing to this dysfunction at the transcriptional level (Raghavachari N et al, Circulation 2007). New RNA technologies for purification of total RNA including small RNAs now allow for more refined study of transcriptional events in platelets from individual patients. We have undertaken the present study to demonstrate and validate the presence and differential expression of microRNAs (miRNAs) in platelets from patients with SCD compared with African American control subjects. We hypothesize that posttranscriptional regulation of the platelet function by miRNAs is an important contributor to SCD pathogenesis associated with pathological platelet activation. To date, there have been no studies undertaken to study the role of platelet miRNAs in SCD. Platelet rich plasma samples from 43 subjects were obtained from University of Pittsburgh Medical Center (UPMC) and National Heart, Lung, Blood Institute (NHLBI). Out of these there were 27 SCD patients and 17 were sex-, race- matched healthy controls. Out of the SCD patients, 18 were known to have PAH and 14 were on hydroxyurea (HU) at the time of study enrollment. Total platelet RNA including miRNAs were extracted using the miRNeasy Minikit (Qiagen, Valencia, CA) with yield in the range of 110–1875 ng from 16 ml of whole blood per donor. We hybridized these samples on a miRNA microarray platform (Agilent version 3) containing 866 human miRNAs. The array data was normalized using quantile normalization and were analyzed with GeneSpring GX 11 software. Differential expression profiles between sickle cell disease patients and controls were determined separately for the NHLBI and UPMC cohort to detect a greater than 2 fold difference change in miRNA expression at p-value of 0.05. Our final expression list included only common miRNAs identified in both cohorts, allowing for an additional level of validation. Final validation was performed by RT-PCR of a subset of the differentially expressed miRNAs. Our analysis identified 42 significantly differentially expressed miRNAs in platelets when comparing the SCD with the control groups, at a fold-change > 2 and p-value Disclosures: No relevant conflicts of interest to declare.

Background: Chronic obstructive pulmonary disease (COPD) is characterized by both airway remodeling and parenchymal destruction. The identification of unique biomarker patterns associated with airway dominant versus parenchymal dominant patterns would support the existence of unique phenotypes representing independent biologic processes. A cross-sectional study was performed to examine the association of serum biomarkers with radiographic airway and parenchymal phenotypes of COPD. Methodology/Principal Findings: Serum from 234 subjects enrolled in a CT screening cohort was analyzed for 33 cytokines and growth factors using a multiplex protein array. The association of serum markers with forced expiratory volume in one second percent predicted (FEV1%) and quantitative CT measurements of airway thickening and emphysema was assessed with and without stratification for current smoking status. Significant associations were found with several serum inflammatory proteins and measurements of FEV1%, airway thickening, and parenchymal emphysema independent of smoking status. The association of select analytes with airway thickening and emphysema was independent of FEV1%. Furthermore, the relationship between other inflammatory markers and measurements of physiologic obstruction or airway thickening was dependent on current smoking status. Conclusions/Significance: Airway and parenchymal phenotypes of COPD are associated with unique systemic serum biomarker profiles. Serum biomarker patterns may provide a more precise classification of the COPD syndrome, provide insights into disease pathogenesis and identify targets for novel patient-specific biological therapies.

Background: MicroRNAs (miRNAs) are short, non-coding RNA regulators of protein coding genes. miRNAs play a very important role in diverse biological processes and various diseases. Many algorithms are able to predict miRNA genes and their targets, but their transcription regulation is still under investigation. It is generally believed that intragenic miRNAs (located in introns or exons of protein coding genes) are co-transcribed with their host genes and most intergenic miRNAs transcribed from their own RNA polymerase II (Pol II) promoter. However, the length of the primary transcripts and promoter organization is currently unknown. Methodology: We performed Pol II chromatin immunoprecipitation (ChIP)-chip using a custom array surrounding regions of known miRNA genes. To identify the true core transcription start sites of the miRNA genes we developed a new tool (CPPP). We showed that miRNA genes can be transcribed from promoters located several kilobases away and that their promoters share the same general features as those of protein coding genes. Finally, we found evidence that as many as 26% of the intragenic miRNAs may be transcribed from their own unique promoters. Conclusion: miRNA promoters have similar features to those of protein coding genes, but miRNA transcript organization is more complex.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disease associated with substantial morbidity and mortality. The objective of this study was to determine whether there is a peripheral blood protein signature in IPF and whether components of this signature may serve as biomarkers for disease presence and progression.We analyzed the concentrations of 49 proteins in the plasma of 74 patients with IPF and in the plasma of 53 control individuals. We identified a combinatorial signature of five proteins-MMP7, MMP1, MMP8, IGFBP1, and TNFRSF1A-that was sufficient to distinguish patients from controls with a sensitivity of 98.6% (95% confidence interval [CI] 92.7%-100%) and specificity of 98.1% (95% CI 89.9%-100%). Increases in MMP1 and MMP7 were also observed in lung tissue and bronchoalveolar lavage fluid obtained from IPF patients. MMP7 and MMP1 plasma concentrations were not increased in patients with chronic obstructive pulmonary disease or sarcoidosis and distinguished IPF compared to subacute/chronic hypersensitivity pneumonitis, a disease that may mimic IPF, with a sensitivity of 96.3% (95% CI 81.0%-100%) and specificity of 87.2% (95% CI 72.6%-95.7%). We verified our results in an independent validation cohort composed of patients with IPF, familial pulmonary fibrosis, subclinical interstitial lung disease (ILD), as well as with control individuals. MMP7 and MMP1 concentrations were significantly higher in IPF patients compared to controls in this cohort. Furthermore, MMP7 concentrations were elevated in patients with subclinical ILD and negatively correlated with percent predicted forced vital capacity (FVC%) and percent predicted carbon monoxide diffusing capacity (DLCO%).Our experiments provide the first evidence for a peripheral blood protein signature in IPF to our knowledge. The two main components of this signature, MMP7 and MMP1, are overexpressed in the lung microenvironment and distinguish IPF from other chronic lung diseases. Additionally, increased MMP7 concentration may be indicative of asymptomatic ILD and reflect disease progression.

To accomplish this, STAT3 phosphorylation, as well as DNAbinding activity of STAT3 in response to cigarette smoke expo-sure, were assessed by immunoblot andelectrophoretic mobilityshift assay, respectively. In addition, normal human bronchialepithelial cells wereexposed tocigarette smokeextract with andwithout transfection with siRNA specifically targeting STAT3(SMARTpool; Dharmacon, Lafayette, CO). Cigarette smokeexposure resulted in STAT3 phosphorylation in a time- andconcentration-dependent manner. Cigarette smoke also increasedSTAT3-DNA binding activity. Introduction of STAT3 siRNA sig-nificantly suppressed STAT3 expression and caused cell deathin response to cigarette smoke exposure. These results suggestthat STAT3 is activated by cigarette smoke and that the STAT3signal pathway plays an important role in modulating survivalor death in human bronchial epithelial cells exposed to cigarettesmoke.

Patients and their families can be our greatest teachers. Such was the case with the initiation of the Warm Autopsy Program at the Dorothy P. & Richard P. Simmons Center for Interstitial Lung Disease at the University of Pittsburgh. This brief essay describes events that led to our beginning the program, which we believe uniquely benefits researchers whose work focuses on the quest to further understand idiopathic pulmonary fibrosis (IPF). We outline the lessons that we have learned in the hope that our experience may be useful for other centers that are considering a warm autopsy program for patients and their families. A detailed description of the regulatory and ethical issues related to warm autopsy is beyond the scope of this essay and has been previously reviewed [ 1]. The idea for the Warm Autopsy Program was planted as one of our support group meetings was ending. One of the patients approached our clinical nurse specialist (KOL) privately, and said that he was “going to die soon” and wanted to donate his lungs to our program for research. He then said, “I don't want others who get this disease to suffer like me.” We had been holding a support group for patients with IPF for about two years, and this patient and his wife attended frequently. He was a retired firefighter who was unable to be seen in our clinical program because his insurance would not cover any medical expenses at our institution. His wife learned about our support group on the Internet and they actively participated. He had also been a trumpet player in a band and said that his participation in the band had decreased over the course of his illness. It was obvious that it was important to him to be able to donate his lungs to assist in finding a cure for this disease.

Gene expression patterns from peripheral blood cells may be useful as biomarkers for monitoring MS progression and response to therapy, argue Kaminski and Achiron.

Streptomycin sulphate is an effective aminoglycoside with limited nephrotoxicity. We report a case of renal electrolyte wasting that mimicked Bartter's syndrome and was induced by streptomycin. A 40 year old woman presented with a four month history of fever, enlarged liver, and a macular rash. Results of physical examination, bone marrow and lymph node biopsies, computed tomography, and …

Feature selection is used extensively in biomedical research for biomarker identification and patient classification, both of which are essential steps in developing personalized medicine strategies. However, the structured nature of the biological datasets and high correlation of variables frequently yield multiple equally optimal signatures, thus making traditional feature selection methods unstable. Features selected based on one cohort of patients, may not work as well in another cohort. In addition, biologically important features may be missed due to selection of other co-clustered features We propose a new method, Tree-guided Recursive Cluster Selection (T-ReCS), for efficient selection of grouped features. T-ReCS significantly improves predictive stability while maintains the same level of accuracy. T-ReCS does not require an a priori knowledge of the clusters like group-lasso and also can handle "orphan" features (not belonging to a cluster). T-ReCS can be used with categorical or survival target variables. Tested on simulated and real expression data from breast cancer and lung diseases and survival data, T-ReCS selected stable cluster features without significant loss in classification accuracy.