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1.
Mil Med ; 186(Suppl 1): 129-136, 2021 01 25.
Article in English | MEDLINE | ID: mdl-33499487

ABSTRACT

INTRODUCTION: Bacterial sepsis is a life-threatening disease and a significant clinical problem caused by host responses to a microbial infection. Sepsis is a leading cause of death worldwide and, importantly, a significant cause of morbidity and mortality in combat settings, placing a considerable burden on military personnel and military health budgets. The current method of treating sepsis is restricted to pathogen identification, which can be prolonged, and antibiotic administration, which is, initially, often suboptimal. The clinical trials that have been performed to evaluate bacterial separation as a sepsis therapy have been unsuccessful, and new approaches are needed to address this unmet clinical need. MATERIALS AND METHODS: An inertial-based, scalable spiral microfluidic device has been created to overcome these previous deficiencies through successful separation of infection-causing pathogens from the bloodstream, serving as a proof of principle for future adaptations. Fluorescent imaging of fluorescent microspheres mimicking the sizes of bacteria cells and blood cells as well as fluorescently stained Acinetobacter baumannii were used to visualize flow within the spiral. The particles were imaged when flowing at a constant volumetric rate of 0.2 mL min-1 through the device. The same device was functionalized with colistin and exposed to flowing A. baumannii at 0.2 mL h-1. RESULTS: Fluorescent imaging within the channel under a constant volumetric flow rate demonstrated that smaller, bacteria-sized microspheres accumulated along the inner wall of the channel, whereas larger blood cell-sized microspheres accumulated within the center of the channel. Additionally, fluorescently stained A. baumannii displayed accumulation along the channel walls in agreement with calculated performance. Nearly 106 colony-forming units of A. baumannii were extracted with 100% capture efficiency from flowing phosphate-buffered saline at 0.2 mL h-1 in this device; this is at least one order of magnitude more bacteria than present in the blood of a human at the onset of sepsis. CONCLUSIONS: This type of bacterial separation device potentially provides an ideal approach for treating soldiers in combat settings. It eliminates the need for immediate pathogen identification and determination of antimicrobial susceptibility, making it suitable for rapid use within low-resource environments. The overall simplicity and durability of this design also supports its broad translational potential to improve military mortality rates and overall patient outcomes.


Subject(s)
Blood-Borne Pathogens , Acinetobacter baumannii , Anti-Bacterial Agents , Colistin , Humans , Microbial Sensitivity Tests
2.
J Immunol ; 199(9): 3074-3085, 2017 11 01.
Article in English | MEDLINE | ID: mdl-28947540

ABSTRACT

Histological chorioamnionitis (HCA) is an intrauterine inflammatory condition that increases the risk for preterm birth, death, and disability because of persistent systemic and localized inflammation. The immunological mechanisms sustaining this response in the preterm newborn remain unclear. We sought to determine the consequences of HCA exposure on the fetal CD4+ T lymphocyte exometabolome. We cultured naive CD4+ T lymphocytes from HCA-positive and -negative preterm infants matched for gestational age, sex, race, prenatal steroid exposure, and delivery mode. We collected conditioned media samples before and after a 6-h in vitro activation of naive CD4+ T lymphocytes with soluble staphylococcal enterotoxin B and anti-CD28. We analyzed samples by ultraperformance liquid chromatography ion mobility-mass spectrometry. We determined the impact of HCA on the CD4+ T lymphocyte exometabolome and identified potential biomarker metabolites by multivariate statistical analyses. We discovered that: 1) CD4+ T lymphocytes exposed to HCA exhibit divergent exometabolomic profiles in both naive and activated states; 2) ∼30% of detected metabolites differentially expressed in response to activation were unique to HCA-positive CD4+ T lymphocytes; 3) metabolic pathways associated with glutathione detoxification and tryptophan degradation were altered in HCA-positive CD4+ T lymphocytes; and 4) flow cytometry and cytokine analyses suggested a bias toward a TH1-biased immune response in HCA-positive samples. HCA exposure primes the neonatal adaptive immune processes by inducing changes to the exometabolomic profile of fetal CD4+ T lymphocytes. These exometabolomic changes may link HCA exposure to TH1 polarization of the neonatal adaptive immune response.


Subject(s)
Chorioamnionitis/immunology , Chorioamnionitis/metabolism , Th1 Cells/immunology , Th1 Cells/metabolism , Biomarkers/metabolism , Chorioamnionitis/pathology , Enterotoxins/pharmacology , Female , Humans , Infant, Newborn , Infant, Premature , Male , Pregnancy , Th1 Cells/pathology
3.
PLoS One ; 10(2): e0117685, 2015.
Article in English | MEDLINE | ID: mdl-25723555

ABSTRACT

To address the challenges of tracking the multitude of signaling molecules and metabolites that is the basis of biological complexity, we describe a strategy to expand the analytical techniques for dynamic systems biology. Using microfluidics, online desalting, and mass spectrometry technologies, we constructed and validated a platform well suited for sampling the cellular microenvironment with high temporal resolution. Our platform achieves success in: automated cellular stimulation and microenvironment control; reduced non-specific adsorption to polydimethylsiloxane due to surface passivation; real-time online sample collection; near real-time sample preparation for salt removal; and real-time online mass spectrometry. When compared against the benchmark of "in-culture" experiments combined with ultraperformance liquid chromatography-electrospray ionization-ion mobility-mass spectrometry (UPLC-ESI-IM-MS), our platform alleviates the volume challenge issues caused by dilution of autocrine and paracrine signaling and dramatically reduces sample preparation and data collection time, while reducing undesirable external influence from various manual methods of manipulating cells and media (e.g., cell centrifugation). To validate this system biologically, we focused on cellular responses of Jurkat T cells to microenvironmental stimuli. Application of these stimuli, in conjunction with the cell's metabolic processes, results in changes in consumption of nutrients and secretion of biomolecules (collectively, the exometabolome), which enable communication with other cells or tissues and elimination of waste. Naïve and experienced T-cell metabolism of cocaine is used as an exemplary system to confirm the platform's capability, highlight its potential for metabolite discovery applications, and explore immunological memory of T-cell drug exposure. Our platform proved capable of detecting metabolomic variations between naïve and experienced Jurkat T cells and highlights the dynamics of the exometabolome over time. Upregulation of the cocaine metabolite, benzoylecgonine, was noted in experienced T cells, indicating potential cellular memory of cocaine exposure. These metabolomics distinctions were absent from the analogous, traditional "in-culture" UPLC-ESI-IM-MS experiment, further demonstrating this platform's capabilities.


Subject(s)
Mass Spectrometry/methods , Metabolome , Metabolomics/methods , Microfluidics/methods , Cocaine/metabolism , Extracellular Space/metabolism , Humans , Jurkat Cells , Solid Phase Extraction/methods , Spectrometry, Mass, Electrospray Ionization/methods
4.
Exp Biol Med (Maywood) ; 239(11): 1433-42, 2014 Nov.
Article in English | MEDLINE | ID: mdl-24903164

ABSTRACT

The human body is a complex assembly of physiological systems designed to manage the multidirectional transport of both information and nutrients. An intricate interplay between the nervous, circulatory, and secretory systems is therefore necessary to sustain life, allow delivery of nutrients and therapeutic drugs, and eliminate metabolic waste products and toxins. These systems also provide vulnerable routes for modification by substances of abuse. Addictive substances are, by definition, neurologically active, but as they and their metabolites are spread throughout the body via the nervous, circulatory, respiratory and digestive systems, there is abundant opportunity for interaction with numerous cell and tissue types. Cocaine is one such substance that exerts a broad physiological effect. While a great deal of the research concerning addiction has addressed the neurological effects of cocaine use, only a few studies have been aimed at delineating the role that cocaine plays in various body systems. In this paper, we probe the current research regarding cocaine and the immune system, and map a systems-level view to outline a broader perspective of the biological response to cocaine. Specifically, our overview of the neurological and immunomodulatory effects of the drug will allow a broader perspective of the biological response to cocaine. The focus of this review is on the connection between the nervous and immune systems and the role this connection plays in the long-term complications of cocaine use. By describing the multiplicity of these connections, we hope to inspire detailed investigations into the immunological interplay in cocaine addiction.


Subject(s)
Cocaine-Related Disorders/physiopathology , Immune System/drug effects , Immune System/physiopathology , Nervous System/drug effects , Nervous System/physiopathology , Systems Biology , Humans
5.
Anal Chem ; 86(13): 6563-71, 2014 Jul 01.
Article in English | MEDLINE | ID: mdl-24856386

ABSTRACT

A metabolic system is composed of inherently interconnected metabolic precursors, intermediates, and products. The analysis of untargeted metabolomics data has conventionally been performed through the use of comparative statistics or multivariate statistical analysis-based approaches; however, each falls short in representing the related nature of metabolic perturbations. Herein, we describe a complementary method for the analysis of large metabolite inventories using a data-driven approach based upon a self-organizing map algorithm. This workflow allows for the unsupervised clustering, and subsequent prioritization of, correlated features through Gestalt comparisons of metabolic heat maps. We describe this methodology in detail, including a comparison to conventional metabolomics approaches, and demonstrate the application of this method to the analysis of the metabolic repercussions of prolonged cocaine exposure in rat sera profiles.


Subject(s)
Algorithms , Cocaine-Related Disorders/metabolism , Mass Spectrometry/methods , Metabolome , Metabolomics/methods , Animals , Cluster Analysis , Cocaine-Related Disorders/blood , Multivariate Analysis , Phenotype , Rats , Workflow
6.
IEEE Trans Biomed Eng ; 60(3): 682-90, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23380852

ABSTRACT

The sophistication and success of recently reported microfabricated organs-on-chips and human organ constructs have made it possible to design scaled and interconnected organ systems that may significantly augment the current drug development pipeline and lead to advances in systems biology. Physiologically realistic live microHuman (µHu) and milliHuman (mHu) systems operating for weeks to months present exciting and important engineering challenges such as determining the appropriate size for each organ to ensure appropriate relative organ functional activity, achieving appropriate cell density, providing the requisite universal perfusion media, sensing the breadth of physiological responses, and maintaining stable control of the entire system, while maintaining fluid scaling that consists of ~5 mL for the mHu and ~5 µL for the µHu. We believe that successful mHu and µHu systems for drug development and systems biology will require low-volume microdevices that support chemical signaling, microfabricated pumps, valves and microformulators, automated optical microscopy, electrochemical sensors for rapid metabolic assessment, ion mobility-mass spectrometry for real-time molecular analysis, advanced bioinformatics, and machine learning algorithms for automated model inference and integrated electronic control. Toward this goal, we are building functional prototype components and are working toward top-down system integration.


Subject(s)
Artificial Organs , Biomedical Engineering , Lab-On-A-Chip Devices , Models, Biological , Biomedical Engineering/instrumentation , Biomedical Engineering/methods , Humans , Systems Biology/instrumentation
7.
Anal Chem ; 84(20): 8467-74, 2012 Oct 16.
Article in English | MEDLINE | ID: mdl-22967262

ABSTRACT

Current desalination techniques for mass spectrometry-based protocols are problematic for performing temporal response studies where increased temporal resolution requires small samples and faster sampling frequencies, which greatly increases the number of samples and sample preparation time. These challenges are pertinent to cellular dynamics experiments, where it is important to sample the biological system frequently and with as little sample waste as possible. To address these needs, we present a dual-column online solid phase extraction (SPE) approach capable of preconcentrating and preparing a constantly perfusing sample stream, with minimal to no sample loss. This strategy is evaluated for use in microfluidic bioreactor studies specifically aimed at characterizing suitable sample flow rates, temporal resolving power, and analyte concentrations. In this work, we demonstrate that this strategy may be used for flow rates as low as 500 nL/min, with temporal resolving power on the order of 3 min, with analyte loadings ranging from femtomoles to picomoles for metabolites. Under these conditions, recoveries of ca. 80% are obtained even at femtomole loadings.


Subject(s)
Salts/isolation & purification , Solid Phase Extraction/instrumentation , Bioreactors , Equipment Design , Mass Spectrometry/instrumentation , Microfluidic Analytical Techniques/instrumentation
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