Gene expression profiling of whole blood: A comparative assessment of RNA-stabilizing collection methods.

Peripheral Blood gene expression is widely used in the discovery of biomarkers and development of therapeutics. Recently, a spate of commercial blood collection and preservation systems have been introduced with proprietary variations that may differentially impact the transcriptomic profiles. Comparative analysis of these collection platforms will help optimize protocols to detect, identify, and reproducibly validate true biological variance among subjects. In the current study, we tested two recently introduced whole blood collection methods, RNAgard® and PAXgene® RNA, in addition to the traditional method of peripheral blood mononuclear cells (PBMCs) separated from whole blood and preserved in Trizol reagent. Study results revealed striking differences in the transcriptomic profiles from the three different methods that imply ex vivo changes in gene expression occurred during the blood collection, preservation, and mRNA extraction processes. When comparing the ability of the three preservation methods to accurately capture individuals' expression differences, RNAgard® outperformed PAXgene® RNA, and both showed better individual separation of transcriptomic profiles than PBMCs. Hence, our study recommends using a single blood collection platform, and strongly cautions against combining methods during the course of a defined study.

Assessment of Coagulation Homeostasis in Blunt, Penetrating, and Thermal Trauma: Guidance for a Multicenter Systems Biology Approach.

INTRODUCTION: Provisioning care for traumatically injured patients makes conducting research very proximal to injury difficult. These studies also inherently have regulatory barriers to overcome. Here we outline a protocol for acute-phase enrollment of traumatically injured patients into a prospective observational clinical trial with precise and comprehensive sample acquisition in support of a systems biology approach to a research study. METHODS: Experts in trauma, burn, blood coagulation, computational biology, and integrative systems biology developed a prospective study that would capture the natural history of coagulation pathology after traumatic injury. Blood was sampled at admission and serial time points throughout hospitalization. Concurrently, demographic and outcomes data were recorded and on-site point-of-care testing was implemented. Protocols were harmonized across sites and sampling protocols validated through demonstration of feasibility and sample quality assurance testing. A novel data integration platform was developed to store, visualize, and enable large-scale analysis of empirical and clinical data. Regulatory considerations were also addressed in protocol development. RESULTS: A comprehensive Manual of Operations (MOO) was developed and implemented at 3 clinical sites. After regulatory approval, the MOO was followed to collect 5,348 longitudinal samples from 1,547 patients. All samples were collected, processed, and stored per the MOO. Assay results and clinical data were entered into the novel data management platform for analyses. CONCLUSION: We used an iterative, interdisciplinary process to develop a systematic and robust protocol for comprehensive assessment of coagulation in traumatically injured patients. This MOO can be a template for future studies in the acute setting.

Modeling the effects of a Staphylococcal Enterotoxin B (SEB) on the apoptosis pathway.

BACKGROUND: The lack of detailed understanding of the mechanism of action of many biowarfare agents poses an immediate challenge to biodefense efforts. Many potential bioweapons have been shown to affect the cellular pathways controlling apoptosis 1234. For example, pathogen-produced exotoxins such as Staphylococcal Enterotoxin B (SEB) and Anthrax Lethal Factor (LF) have been shown to disrupt the Fas-mediated apoptotic pathway 24. To evaluate how these agents affect these pathways it is first necessary to understand the dynamics of a normally functioning apoptosis network. This can then serve as a baseline against which a pathogen perturbed system can be compared. Such comparisons can expose both the proteins most susceptible to alteration by the agent as well as the most critical reaction rates to better instill control on a biological network. RESULTS: We explore this through the modeling and simulation of the Fas-mediated apoptotic pathway under normal and SEB influenced conditions. We stimulated human Jurkat cells with an anti-Fas antibody in the presence and absence of SEB and determined the relative levels of seven proteins involved in the core pathway at five time points following exposure. These levels were used to impute relative rate constants and build a quantitative model consisting of a series of ordinary differential equations (ODEs) that simulate the network under both normal and pathogen-influenced conditions. Experimental results show that cells exposed to SEB exhibit an increase in the rate of executioner caspase expression (and subsequently apoptosis) of 1 hour 43 minutes (+/- 14 minutes), as compared to cells undergoing normal cell death. CONCLUSION: Our model accurately reflects these results and reveals intervention points that can be altered to restore SEB-influenced system dynamics back to levels within the range of normal conditions.

Feasibility of assessment of regulatory lipids in breath condensate as potential presymptomatic harbingers of pulmonary pathobiology.

Regulatory lipids from the airway surface readily form aerosols that can be recovered non-invasively by cooling expired breath to form breath condensate (BC). Regulatory lipids have been detected previously utilizing enzyme-linked-immunosorbent serologic assay (ELISA). Here we test the feasibility of assessment of regulatory lipids in BC by mass spectrometry so presently unknown lipid regulatory components can be detected without addition of specific antibodies as in the ELISA procedure. Baseline regulatory lipids were detected in >pg/mL BC in control animals or human lung tissue culture cells. In nearly every case animals exposed to toxins or infectious bacteria showed increases in the BC regulatory components. Lipids were recovered from BC by solid phase extraction. Phosphatidylcholine (PC) based lipids were detected as the progenitor (parent) ions of isomers that fragmented in producing product positive ions at m/z 184 (of phosphocholine) in tandem MS using capillary HPLC and electrospray ionization. BC eicosanoids such as prostaglandins, thromboxane, and isoprostanes require capillary gas chromatography for separation and detection that necessitates methoximation, pentafluorobenzyl (PFB) ester formation, and trimethyl silylation of hydroxyls prior to gas chromatography/ion trap tandem mass spectrometry of negative ions after chemical ionization (NICI). Tetradeuterated internal standards were utilized for quantitation with the GC/NICI/MS. Changes in concentrations of lipids and eicosanoids were observed in piglets, and rats exposed to aerosolized 100 mug/kg lipopolysaccharide (LPS), or 50 mug/kg and 150 mug/kg aerosolized Staphylococcal enterotoxin B (SEB) in BC as well as in human THP-1 cell culture cell supernatants and bronchoalveolar lavage (BAL) samples in rats. Responses of the molecular species of phosphatidylcholines (PCs), platelet activating factors (PAFs) and specific eicosanoids correlated to the toxin and bacterial infections suggesting that patterns of differential responses could be detected with further experimentation. Initial targets included prostaglandins (PGE(2), PGF(2alpha)), thromboxane (TXB2), and prostacyclin (as 6-Keto PGF(1alpha)) that show differential responses to inflammation, the leukotriene (LTB4) and PGD2 for allergic responses, isoprostanes (8-iso-PGF(2alpha)) for free radical oxidative stress responses, and HETEs for differential lipoxygenase activities. PAFs and lysoPAFs have been shown to increase with inflammation and in the feasibility experiments reported here. Preliminary studies show pulmonary responses of piglets to intrathecal exposure of toxicants (LPS and SEB) or infections with Actinobacillus pleuropneumoniae induce increased levels of lipids and two eicosanoids with the suggestion that differential patterns might be detected with expanded testing. Preliminary experience indicates numerous other eicosanoids were available for assay in BC. This suggests an important potential application of BC to observe a wide array of factors to establish comprehensive profiles for physiological and pathophysiological states. Ultimately this technique could be used as a non-invasive possibly presymptomatic assessment of pulmonary pathobiology.