Brian G. Harbrecht

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.

Fulminant Clostridium difficile: an underappreciated and increasing cause of death and complications.

ObjectiveTo review the epidemiology and characteristics of patients who died or underwent colectomy secondary to fulminant Clostridium difficile colitis. Summary Background DataIn patients with C. difficile colitis, a progressive, systemic inflammatory state may develop that is unresponsive to medical therapy; it may progress to colectomy or death. MethodsThe authors reviewed 2,334 hospitalized patients with C. difficile colitis from January 1989 to December 2000. Sixty-four patients died or underwent colectomy for pathologically proven C. difficile colitis. ResultsIn 2000, the incidence of C. difficile colitis in hospitalized patients increased from a baseline of 0.68% to 1.2%, and the incidence of patients with C. difficile colitis in whom life-threatening symptoms developed increased from 1.6% to 3.2%. Forty-four patients required a colectomy and 20 others died directly from C. difficile colitis. Twenty-two percent had a prior history of C. difficile colitis. A recent surgical procedure and immunosuppression were common predisposing conditions. Lung transplant patients were 46 times more likely to have C. difficile colitis and eight times more likely to have severe disease. Abdominal computed tomography scan correctly diagnosed all patients, whereas 12.5% of toxin assays and 10% of endoscopies were falsely negative. Patients undergoing colectomy for C. difficile colitis had an overall death rate of 57%. Significant predictors of death after colectomy were preoperative vasopressor requirements and age. ConclusionsC. difficile colitis is a significant and increasing cause of death. Surgical treatment of C. difficile colitis has a high death rate once the fulminant expression of the disease is present.

Blunt splenic injury in adults: Multi-institutional study of the Eastern Association for the Surgery of Trauma

BACKGROUND Nonoperative management of blunt injury to the spleen in adults has been applied with increasing frequency. However, the criteria for nonoperative management are controversial. The purpose of this multi-institutional study was to determine which factors predict successful observation of blunt splenic injury in adults. METHODS A total of 1,488 adults (>15 years of age) with blunt splenic injury from 27 trauma centers in 1997 were studied through the Multi-institutional Trials Committee of the Eastern Association for the Surgery of Trauma. Statistical analysis was performed with analysis of variance and extended chi2 test. Data are expressed as mean +/- SD; a value of p < 0.05 was considered significant. RESULTS A total of 38.5 % of patients went directly to the operating room (group I); 61.5% of patients were admitted with planned nonoperative management. Of the patients admitted with planned observation, 10.8% failed and required laparotomy; 82.1% of patients with an Injury Severity Score (ISS) < 15 and 46.6% of patients with ISS > 15 were successfully observed. Frequency of immediate operation correlated with American Association for the Surgery of Trauma (AAST) grades of splenic injury: I (23.9%), II (22.4%), III (38.1%), IV (73.7%), and V (94.9%) (p < 0.05). Of patients initially managed nonoperatively, the failure rate increased significantly by AAST grade of splenic injury: I (4.8%), II (9.5%), III (19.6%), IV (33.3%), and V (75.0%) (p < 0.05). A total of 60.9% of the patients failed nonoperative management within 24 hours of admission; 8% failed 9 days or later after injury. Laparotomy was ultimately performed in 19.9% of patients with small hemoperitoneum, 49.4% of patients with moderate hemoperitoneum, and 72.6% of patients with large hemoperitoneum. CONCLUSION In this multicenter study, 38.5% of adults with blunt splenic injury went directly to laparotomy. Ultimately, 54.8% of patients were successfully managed nonoperatively; the failure rate of planned observation was 10.8%, with 60.9% of failures occurring in the first 24 hours. Successful nonoperative management was associated with higher blood pressure and hematocrit, and less severe injury based on ISS, Glasgow Coma Scale, grade of splenic injury, and quantity of hemoperitoneum.

Modulation of nitrogen oxide synthesis in vivo: NG-monomethyl-L-arginine inhibits endotoxin-induced nitrate/nitrate biosynthesis while promoting hepatic damage.

Attempts were made to promote or inhibit nitric oxide (·N=O) synthesis in a murine model of hepatic damage (Corynebacterium parvum followed by lipopolysaccharide; LPS) to determine the role of N=O in the liver injury. Moderate hepatic damage and increases in circulating NO2 ‐/NO3 ‐ levels were detectable after C. parvum alone. Administration of LPS to these mice resulted in severe hepatic damage and acute elevations in circulating nitrogen oxide levels. L‐arg had no influence on the C. parvum or LPS‐induced changes. NG‐monomethyl‐L‐arginine (NMA) had no effect in the absence of LPS, but when given with LPS, a dose‐dependent suppression in plasma NO2 ‐/NO3 ‐ levels and an increase in liver injury were seen. The NMA‐induced changes were partially reversed by the simultaneous administration of L‐arg. These findings suggest a protective role for · N=O in this model.

Inhibition of nitric oxide synthesis during endotoxemia promotes intrahepatic thrombosis and an oxygen radical-mediated hepatic injury.

Corynebacterium parvum‐treated mice produce large amounts of circulating nitrogen oxides and develop a severe liver injury in response to lipopolysaccharide (LPS). Concurrent administration of NG‐monomethyl‐L‐ arginine not only suppresses nitric oxide synthesis in these animals but also profoundly increases the hepatic damage following LPS. In this report, we present evidence that the increased hepatic damage from inhibition of nitric oxide synthesis is mediated in part by superoxide and hydroxyl radicals. The hepatic damage induced by suppressing nitric oxide production during endotoxemia could be reduced by treating mice with superoxide dismutase and deferoxamine, scavengers of superoxide and hydroxyl radicals, respectively. This damage could also be prevented by treating mice with the anticoagulant heparin sodium. The results suggest that nitric oxide synthesis during endotoxemia is important in preventing hepatic damage by reducing oxygen radical‐mediated hepatic injury and preventing intravascular thrombosis.

Multiple cytokines are required to induce hepatocyte nitric oxide production and inhibit total protein synthesis.

The etiology and mechanisms by which severe trauma or sepsis induce hepatic failure are unknown. Previously we showed that Kupffer cells (KC), the fixed macrophages of the liver, induce a profound decrease in hepatocyte (HC) total-protein synthesis when exposed to endotoxin. Furthermore we demonstrated that endotoxin-activated KCs induce these changes in HC protein synthesis through the induction of a novel L-arginine-dependent biochemical pathway within the HC. In this pathway, the guanido nitrogen of L-arginine is converted to the highly reactive molecule nitric oxide (NO.). To identify the KC factors that act as signals for induction of HC NO. biosynthesis, recombinant cytokines were added to HC cultures and HC nitrogen oxide production and protein synthesis levels were determined. We found that no single cytokine, but rather a specific combination of tumor necrosis factor, interleukin-1, interferon-gamma, and endotoxin, were required for maximal induction of HC nitrogen oxide production. This specific combination of cytokines induced a 248.8 +/- 26.0 mumol/L (micromolar) increase in HC nitrogen oxide production and simultaneously inhibited HC total protein synthesis by 36.1% +/- 3.1%. These data demonstrate that multiple cytokines, produced by endotoxin-activated KC, induce the production of NO. within HC, which in turn leads to the inhibition of HC total-protein synthesis.

Fresh frozen plasma is independently associated with a higher risk of multiple organ failure and acute respiratory distress syndrome.

BACKGROUND Blood transfusion is known to be an independent risk factor for mortality, multiple organ failure (MOF), acute respiratory distress syndrome (ARDS), and nosocomial infection after injury. Less is known about the independent risks associated with plasma-rich transfusion components including fresh frozen plasma (FFP), platelets (PLTS), and cryoprecipitate (CRYO) after injury. We hypothesized that plasma-rich transfusion components would be independently associated with a lower risk of mortality but result in a greater risk of morbid complications. METHODS Data were obtained from a multicenter prospective cohort study evaluating clinical outcomes in bluntly injured adults with hemorrhagic shock. All patients required blood transfusion for enrollment. Patients with isolated traumatic brain injury and those not surviving beyond 48 hours were excluded. Cox proportional hazard regression models were used to estimate the outcome risks (per unit) associated with plasma-rich transfusion requirements during the initial 24 hours after injury after controlling for important confounders. RESULTS For the entire study population (n = 1,175), 65%, 41%, and 28% of patients received FFP, PLTS and CRYO, respectively. There was no association with plasma-rich transfusion components and mortality or nosocomial infection. For every unit given, FFP was independently associated with a 2.1% and 2.5% increased risk of MOF and ARDS, respectively. CRYO was associated with a 4.4% decreased risk of MOF (per unit), and PLTS were not associated with any of the outcomes examined. When early deaths (within 48 hours) were included in the model, FFP was associated with a 2.9% decreased risk of mortality per unit transfused. CONCLUSIONS In patients who survive their initial injury, FFP was independently associated with a greater risk of developing MOF and ARDS, whereas CRYO was associated with a lower risk of MOF. Further investigation into the mechanisms by which these plasma-rich component transfusions are associated with these effects are required.

Endogenous nitric oxide inhibits the synthesis of cyclooxygenase products and interleukin-6 by rat Kupffer cells

Macrophage production of nitric oxide (N=O) leads to considerable alterations of vital metabolic pathways in various target cells. The present study tested whether N=O synthesis by Kupffer cells (KCs), the resident macrophages of the liver, interferes with the secretory function of these cells. As in other macrophage‐ type cells, the combination of lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) was a potent stimulus of N=O synthesis by KC. Treatment with LPS and IFN‐γ also induced significant production of prostaglandin E2 (PGE2), thromboxane B2 (TBX2), tumor necrosis factor a (TNF‐ α), interleukin‐1 (IL‐1), and IL‐6. Inhibition of N=O synthesis by the L‐arginine analogue NG‐monomethyl‐L‐ar‐ ginine (NMA) resulted in a further increase of PGEs, TXBs, and EL‐6 but not BL‐l and TNF‐α production, indicating specific inhibitory effects of endogenous N=O synthesis on the secretory activity of KCs. PGE2 production was most sensitive to the suppressive effect of N=O and increased 24 h after stimulation with LPS and IFN‐γ from 16.3 ± 4.9 ng/106 KCs without NMA to 94.3 ± 17.9 ng/106 KCs with NMA. This effect of NMA was reversed by a 10‐fold increase of the L‐arginine concentration. No recovery of PGE2 production was seen when N=O synthesis was blocked after 24 h. NMA treatment increased cyclooxygenase activity more than threefold, suggesting that N=O inhibits PGE2 and TXB2 production through diminished PGH2 availability. N=O synthesis did not significantly affect total protein synthesis or viability of the KCs. These results show that N=O influences the production of specific inflammatory mediators by KCs./

Nitric oxide synthesis serves to reduce hepatic damage during acute murine endotoxemia.

Background and MethodsNitric oxide synthesis occurs both in vitro and in vivo in response to inflammatory stimuli and can have profound effects on the local cellular environment. Hepatocytes, Kupfler cells, and endothelial cells produce nitric oxide in vitro, but the in vivo role of this reactive mediator in the liver is unknown. We assessed the role of nitric oxide synthesis during endotoxemia in mice by inhibiting its synthesis with NG-monomethyl-L-arginine after lipopolysaccharide injection and by determining the effects of this inhibition on hepatic damage. ResultsInjection of lipopolysaccharide in mice increased plasma nitrite and nitrate concentrations, the stable end products of nitric oxide metabolism, and caused mild hepatic damage as measured by increased circulating hepatocellular enzyme levels. NG-monomethyl-L-arginine decreased plasma nitrite and nitrate values, but increased the lipopolysaccharide-induced hepatic injury. NG-monomethyl-L-arginine caused no hepatic damage when given without lipopolysaccharide. The extent of hepatic damage with NG-monomethyl-L-arginine was proportional to the dose of lipopolysaccharide used and could be reduced with concurrent administration of L-arginine but not D-arginine. ConclusionsNitric oxide synthesis provides a protective function against lipopolysaccharide-induced liver injury that increases in importance as the degree of endotoxemia increases. The production of nitric oxide is, therefore, an important part of the livers response to a systemic inflammatory stimulus.

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.