Unknown,Transcriptomics,Genomics,Proteomics

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Oxidative Stress and Reduced Responsiveness of Challenged Circulating Leukocytes Following Pulmonary Instillation of Metal-Rich Particulate Matter in Rats

ABSTRACT: Exposure to welding fume consists of a mixed exposure of metal-rich particulate matter with gases (ozone, carbon monoxide) and/or vapors (VOCs). Data suggests that welders are immune compromised. Given the inability of pulmonary leukocytes to properly respond to a secondary infection, the question arose concerning how far upstream the dysfunction persisted. Our aim was to evaluate circulating leukocyte functionality after welding fume exposure. The results showed that welding fume exposure has rapid effects on the circulating leukocyte population as identified by relative mRNA expression changes. Instillation of MMA-SS reduced inflammatory protein production of circulating leukocytes when challenged with the secondary stimulus LPS. The effects were not related to transcription, but were observed in conjunction with oxidative stress. Rats were intratracheally instilled (ITI) with phosphate buffered saline or 2 mg of welding fume collected from a manual metal arc-stainles steel weld. Rats were sacrificed 4 and 24 h post-exposure and whole blood was collected. Whole blood was used for cellular differential counts, RNA isolation with subsequent microarray and Ingenuity Pathway Analysis, and secondary stimulation with lipopolysaccharide utilizing TruCulture technology from Myriad/Rules Based Medicine. Also, mononuclear cells were isolated 24 h post-exposure to measure oxidative stress by flow cytometry and confocal microscopy. Whole blood collected 4 and 24 h post-exposure was challenged (a paired non-stimulant (null) and stimulant (LPS) tube) for 24 h using Truculture technology from Myriad/ RBM. After the challenge, cells were recovered using RNA later for subsequent RNA isolation and microarray.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Patti Zeidler-Erdely

PROVIDER: E-GEOD-58197 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Diagnosis of acute infection in the critically ill remains a challenge. Changes in physiologic parameters and existing molecular diagnostics are not specific and microbiologic confirmation of infection can take days. As cellular first responders, preclinical studies indicate that circulating leukocytes are activated in response to bacterial infection, manifesting infection-specific transcriptional programs. We hypothesized that circulating leukocyte transcriptional profiles can be used to diagnose infection and monitor response to therapy in the critically ill. Experiment Overall Design: A translational research approach was employed. Fifteen mice underwent intratracheal (i.t.) injections of live P. aeruginosa, P. aeruginosa endotoxin, live S. pneumoniae, or normal saline. At 24 hours after injury, GeneChip microarray analysis of circulating buffy coat RNA identified 219 genes that distinguished between the pulmonary insults and differences in 7-day mortality. Similarly, buffy coat microarray expression profiles were generated from 20 mechanically ventilated patients every two days for up to three weeks; 11 of these patients developed pneumonia. Principal components analysis of the expression levels of the human orthologs for the mouse genes resulted in gene expression trajectories that describe the cohort of 11 patients as they develop, are treated for, and convalesce from ventilator-associated pneumonia (VAP). When the VAP microarray profiles were analyzed independently, 85 genes were identified with consistent changes in abundance during the seven days bracketing the diagnosis of VAP. The estimated prediction accuracy in this small patient cohort was 91%. The abundance of select transcripts was validated using real-time PCR. Principal components analysis of the average microarray-measured abundances of these genes defined a general trajectory (riboleukogram) for the onset of acute infection in this patient population. Individual riboleukograms demonstrated that the onset and course of infection are specific for each individual, but as patients recover from infection the riboleukograms converge. The gene ontologies of the co-expression networks indicate that leukocyte chemokines and granule maturation are especially informative for pneumonia diagnostics.

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Oxidative Stress and Reduced Responsiveness of Challenged Circulating Leukocytes Following Pulmonary Instillation of Metal-Rich Particulate Matter in Rats

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