Grant E. O'Keefe

A genomic storm in critically injured humans

Critical injury in humans induces a genomic storm with simultaneous changes in expression of innate and adaptive immunity genes.

Is hypothermia in the victim of major trauma protective or harmful? A randomized, prospective study.

OBJECTIVE The purpose of this randomized, prospective clinical trial was to determine whether hypothermia during resuscitation is protective or harmful to critically injured trauma patients. SUMMARY BACKGROUND DATA Hypothermia has both protective and harmful clinical effects. Retrospective studies show higher mortality in patients with hypothermia; however, hypothermia is more common in more severely injured patients, which makes it difficult to determine whether hypothermia contributes to mortality independently of injury severity. There are no randomized, prospective treatment studies to assess hypothermias impact as an independent variable. METHODS Fifty-seven hypothermic (T < or = 34.5 C), critically injured patients requiring a pulmonary artery catheter were randomized to a rapid rewarming protocol using continuous arteriovenous rewarming (CAVR) or to a standard rewarming (SR) control group. The primary outcome of interest was first 24-hour blood product and fluid resuscitation requirements. Other comparative analyses included coagulation assays, hemodynamic and oxygen transport measurements, length of stay, and mortality. RESULTS The two groups were well matched for demographic and injury severity characteristics. CAVR rewarmed significantly faster than did SR (p < 0.01), producing two groups with different amounts of hypothermia exposure. The patients who underwent CAVR required less fluid during resuscitation to the same hemodynamic goals (24,702 mL vs. 32,540 mL, p = 0.05) and were significantly more likely to rewarm (p = 0.002). Only 2 (7%) of 29 patients who underwent CAVR failed to warm to 36 C and both died, whereas 12 (43%) of 28 patients who underwent SR failed to reach 36 C, and all 12 died. Patients who underwent CAVR had significantly less early mortality (p = 0.047). CONCLUSION Hypothermia increases fluid requirements and independently increases acute mortality after major trauma.

Quantitative proteome analysis of human plasma following in vivo lypopolysaccharide administration using 16O/18O labeling and the accurate mass and time tag approach

Identification of novel diagnostic or therapeutic biomarkers from human blood plasma would benefit significantly from quantitative measurements of the proteome constituents over a range of physiological conditions. Herein we describe an initial demonstration of proteome-wide quantitative analysis of human plasma. The approach utilizes postdigestion trypsin-catalyzed 16O/18O peptide labeling, two-dimensional LC-FTICR mass spectrometry, and the accurate mass and time (AMT) tag strategy to identify and quantify peptides/proteins from complex samples. A peptide accurate mass and LC elution time AMT tag data base was initially generated using MS/MS following extensive multidimensional LC separations to provide the basis for subsequent peptide identifications. The AMT tag data base contains >8,000 putative identified peptides, providing 938 confident plasma protein identifications. The quantitative approach was applied without depletion of high abundance proteins for comparative analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Accurate quantification of changes in protein abundance was demonstrated by both 1:1 labeling of control plasma and the comparison between the plasma samples following LPS administration. A total of 429 distinct plasma proteins were quantified from the comparative analyses, and the protein abundances for 25 proteins, including several known inflammatory response mediators, were observed to change significantly following LPS administration.

High Dynamic Range Characterization of the Trauma Patient Plasma Proteome

Although human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with two-dimensional LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this “divide-and-conquer” strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3654 different proteins with 1494 proteins identified by multiple peptides. Numerous low abundance proteins were identified, exemplified by 78 “classic” cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally a total of 2910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients that provides a foundation for future high throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.

Effect of Immediate Enteral Feeding on Trauma Patients with an Open Abdomen: Protection from Nosocomial Infections

BACKGROUND Damage-control laparotomy has become increasingly common after operative resuscitation of severe hemorrhagic shock after injury. Despite increased use, uncertainty exists about the safety and timing of enteral nutrition. The purpose of this study was to determine the safety and effect of immediate enteral nutrition. STUDY DESIGN Data were obtained from a multicenter prospective cohort study evaluating clinical outcomes in adults with hemorrhagic shock after injury and were limited to patients with an open abdomen and no hollow viscus injury. The immediate enteral nutrition cohort was defined as initiation of enteral feeds within 36 hours after acute resuscitation completion. Multivariate stepwise logistic regression was used to evaluate factors associated with immediate enteral nutrition. RESULTS One hundred subjects met inclusion criteria; 32 immediate enteral nutrition and 68 nonimmediate enteral nutrition. Nearly all patients underwent fascial closure (93.8% immediate enteral nutrition versus 94.1% nonimmediate enteral nutrition), with an average closure day of 6.47 +/- 0.83 with immediate enteral nutrition and 8.55 +/- 0.85 with nonimmediate enteral nutrition (p = 0.129). No significant difference in multiorgan dyfunction syndrome, length of ventilator days, ICU days, hospital days, or mortality was seen between groups. The rate of pneumonia was significantly different: 14 (43.8%) in immediate enteral nutrition and 49 (72.1%) in nonimmediate enteral nutrition (p = 0.008). Immediate enteral nutrition remained independently associated with a reduction in pneumonia within our stepwise regression (odds ratio = 0.32; 95% CI, 0.13 to 0.79). CONCLUSIONS Immediate enteral nutrition after damage control appears safe, with no effect on abdominal closure rate. In addition, the reduction in pneumonia associated with immediate enteral nutrition suggests a tangible benefit. Immediate enteral nutrition should be considered in patients with open abdomens after severe trauma.

Cell-specific expression and pathway analyses reveal alterations in trauma-related human T cell and monocyte pathways.

Monitoring genome-wide, cell-specific responses to human disease, although challenging, holds great promise for the future of medicine. Patients with injuries severe enough to develop multiple organ dysfunction syndrome have multiple immune derangements, including T cell apoptosis and anergy combined with depressed monocyte antigen presentation. Genome-wide expression analysis of highly enriched circulating leukocyte subpopulations, combined with cell-specific pathway analyses, offers an opportunity to discover leukocyte regulatory networks in critically injured patients. Severe injury induced significant changes in T cell (5,693 genes), monocyte (2,801 genes), and total leukocyte (3,437 genes) transcriptomes, with only 911 of these genes common to all three cell populations (12%). T cell-specific pathway analyses identified increased gene expression of several inhibitory receptors (PD-1, CD152, NRP-1, and Lag3) and concomitant decreases in stimulatory receptors (CD28, CD4, and IL-2Rα). Functional analysis of T cells and monocytes confirmed reduced T cell proliferation and increased cell surface expression of negative signaling receptors paired with decreased monocyte costimulation ligands. Thus, genome-wide expression from highly enriched cell populations combined with knowledge-based pathway analyses leads to the identification of regulatory networks differentially expressed in injured patients. Importantly, application of cell separation, genome-wide expression, and cell-specific pathway analyses can be used to discover pathway alterations in human disease.

ANGPT2 Genetic Variant Is Associated with Trauma-associated Acute Lung Injury and Altered Plasma Angiopoietin-2 Isoform Ratio

RATIONALE Acute lung injury (ALI) acts as a complex genetic trait, yet its genetic risk factors remain incompletely understood. Large-scale genotyping has not previously been reported for ALI. OBJECTIVES To identify ALI risk variants after major trauma using a large-scale candidate gene approach. METHODS We performed a two-stage genetic association study. We derived findings in an African American cohort (n = 222) using a cardiopulmonary disease-centric 50K single nucleotide polymorphism (SNP) array. Genotype and haplotype distributions were compared between subjects with ALI and without ALI, with adjustment for clinical factors. Top performing SNPs (P < 10(-4)) were tested in a multicenter European American trauma-associated ALI case-control population (n = 600 ALI; n = 2,266 population-based control subjects) for replication. The ALI-associated genomic region was sequenced, analyzed for in silico prediction of function, and plasma was assayed by ELISA and immunoblot. MEASUREMENTS AND MAIN RESULTS Five SNPs demonstrated a significant association with ALI after adjustment for covariates in Stage I. Two SNPs in ANGPT2 (rs1868554 and rs2442598) replicated their significant association with ALI in Stage II. rs1868554 was robust to multiple comparison correction: odds ratio 1.22 (1.06-1.40), P = 0.0047. Resequencing identified predicted novel splice sites in linkage disequilibrium with rs1868554, and immunoblots showed higher proportion of variant angiopoietin-2 (ANG2) isoform associated with rs1868554T (0.81 vs. 0.48; P = 0.038). CONCLUSIONS An ANGPT2 region is associated with both ALI and variation in plasma angiopoietin-2 isoforms. Characterization of the variant isoform and its genetic regulation may yield important insights about ALI pathogenesis and susceptibility.

Risk Factors for Respiratory Failure Following Operative Stabilization of Thoracic and Lumbar Spine Fractures

BACKGROUND Respiratory failure is a serious complication that can adversely affect the hospital course and survival of multiply injured patients. Some studies have suggested that delayed surgical stabilization of spine fractures may increase the incidence of respiratory complications. However, the authors of these studies analyzed small sets of patients and did not assess the independent effects of multiple risk factors. METHODS A retrospective cohort study was conducted at a regional level-I trauma center to identify risk factors for respiratory failure in patients with surgically treated thoracic and lumbar spine fractures. Demographic, diagnostic, and procedural variables were identified. The incidence of respiratory failure was determined in an adult respiratory distress syndrome registry maintained concurrently at the same institution. Univariate and multivariate analyses were used to determine independent risk factors for respiratory failure. An algorithm was formulated to predict respiratory failure. RESULTS Respiratory failure developed in 140 of the 1032 patients in the study cohort. Patients with respiratory failure were older; had a higher mean Injury Severity Score (ISS) and Charlson Comorbidity Index Score; had greater incidences of pneumothorax, pulmonary contusion, and thoracic level injury; had a lower mean Glasgow Coma Score (GCS); were more likely to have had a posterior surgical approach; and had a longer mean time from admission to surgical stabilization than the patients without respiratory failure (p < 0.05). Multivariate analysis identified five independent risk factors for respiratory failure: an age of more than thirty-five years, an ISS of > 25 points, a GCS of < or = 12 points, blunt chest injury, and surgical stabilization performed more than two days after admission. An algorithm was created to determine, on the basis of the number of preoperative predictors present, the relative risk of respiratory failure when surgery was delayed for more than two days. CONCLUSIONS Independent risk factors for respiratory failure were identified in an analysis of a large cohort of patients who had undergone operative stabilization of thoracic and lumbar spine fractures. Early operative stabilization of these fractures, the only risk factor that can be controlled by the physician, may decrease the risk of respiratory failure in multiply injured patients.

The changing pattern and implications of multiple organ failure after blunt injury with hemorrhagic shock

Objectives:To describe the incidence of postinjury multiple organ failure and its relationship to nosocomial infection and mortality in trauma centers using evidence-based standard operating procedures. Design:Prospective cohort study wherein standard operating procedures were developed and implemented to optimize postinjury care. Setting:Seven U.S. level I trauma centers. Patients:Severely injured patients (older than age 16 yrs) with a blunt mechanism, systolic hypotension (<90 mm Hg), and/or base deficit (≥6 mEq/L), need for blood transfusion within the first 12 hrs, and an abbreviated injury score ≥2 excluding brain injury were eligible for inclusion. Measurements and Main Results:One thousand two patients were enrolled and 916 met inclusion criteria. Daily markers of organ dysfunction were prospectively recorded for all patients while receiving intensive care. Overall, 29% of patients had multiple organ failure develop. Development of multiple organ failure was early (median time, 2 days), short-lived, and predicted an increased incidence of nosocomial infection, whereas persistence of multiple organ failure predicted mortality. However, surprisingly, nosocomial infection did not increase subsequent multiple organ failure and there was no evidence of a “second-hit”-induced late-onset multiple organ failure. Conclusions:Multiple organ failure remains common after severe injury. Contrary to current paradigms, the onset is only early, and not bimodal, nor is it associated with a “second-hit”-induced late onset. Multiple organ failure is associated with subsequent nosocomial infection and increased mortality. Standard operating procedure-driven interventions may be associated with a decrease in late multiple organ failure and morbidity. (Crit Care Med 2012; 40:–1135)

Innate Immunity Genes Influence the Severity of Acute Appendicitis

Objective:Using acute appendicitis as a model, we tested the hypothesis that polymorphisms in genes involved in host defense can be associated with the severity of local infection-inflammation in humans. Summary Background Data:Innate immunity is the bodys front-line system for antimicrobial host defense. Local inflammation is a major innate immune mechanism for containing and destroying microbes, but it may also contribute to tissue injury. Methods:We studied 134 patients with acute appendicitis treated at an urban hospital. We looked for associations between the severity of appendicitis (uncomplicated vs. perforated or gangrenous), plasma and peritoneal cytokine concentrations, and single nucleotide polymorphisms in genes involved in recognizing bacterial molecules [CD14 (−159 C→T); TLR4 (896 A→G)] and in mounting an inflammatory response [IL-6 (−174 G→C), TNF-&agr; (−308 G→A), IL-1&bgr; (−31 C → T)]. Results:Ninety-one patients (68%) had uncomplicated appendicitis and 43 (32%) had complicated disease. The SNPs in the CD14, TLR4, IL-1&bgr;, and TNF-&agr; genes were not associated with the severity of appendicitis. A strong association was found between C-allele carriage at −174 in the IL-6 gene and decreased risk of complicated disease (adjusted odds ratio = 0.24, 95% CI = 0.07–0.76). Lower plasma and peritoneal fluid IL-6 concentrations in the IL-6 −174 C-carriers than in the GG homozygotes suggest that this polymorphism contributes to decreased IL-6 production in vivo. Conclusions:Polymorphism in the IL-6 gene was associated with the severity of appendicitis, even after adjustment for duration of symptoms. The risk for developing appendiceal perforation or gangrene may be determined, in part, by variation in the IL-6 gene.