Joseph Cuschieri

Genomic responses in mouse models poorly mimic human inflammatory diseases

A cornerstone of modern biomedical research is the use of mouse models to explore basic pathophysiological mechanisms, evaluate new therapeutic approaches, and make go or no-go decisions to carry new drug candidates forward into clinical trials. Systematic studies evaluating how well murine models mimic human inflammatory diseases are nonexistent. Here, we show that, although acute inflammatory stresses from different etiologies result in highly similar genomic responses in humans, the responses in corresponding mouse models correlate poorly with the human conditions and also, one another. Among genes changed significantly in humans, the murine orthologs are close to random in matching their human counterparts (e.g., R2 between 0.0 and 0.1). In addition to improvements in the current animal model systems, our study supports higher priority for translational medical research to focus on the more complex human conditions rather than relying on mouse models to study human inflammatory diseases.

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.

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.

Effect of chlorhexidine whole-body bathing on hospital-acquired infections among trauma patients.

OBJECTIVE To demonstrate whether daily bathing with cloths impregnated with 2% chlorhexidine gluconate will decrease colonization of resistant bacteria and reduce the rates of health care-associated infections in critically injured patients. DESIGN Retrospective analysis of data collected 6 months before and after institution of a chlorhexidine bathing protocol. SETTING A 12-bed intensive care unit in a level I trauma center. PATIENTS Two hundred eighty-six severely injured patients underwent daily chlorhexidine bathing during the 6-month intervention; 253 patients were bathed without chlorhexidine prior to the intervention. INTERVENTIONS Daily chlorhexidine bathing. MAIN OUTCOMES MEASURES Rates of ventilator-associated pneumonia (VAP), bloodstream infection, and colonization with resistant organisms (methicillin-resistant Staphylococcus aureus [MRSA] or Acinetobacter species). RESULTS Baseline patient and injury characteristics were similar between cohorts. Patients receiving chlorhexidine baths were significantly less likely to acquire a catheter-related bloodstream infection than comparators (2.1 vs 8.4 infections per 1000 catheter-days, P = .01). The incidence of VAP was not affected by chlorhexidine baths (16.9 vs 21.6 infections per 1000 ventilator-days in those with vs those without chlorhexidine baths, respectively, P = .30). However, patients who received chlorhexidine baths were less likely to develop MRSA VAP (1.6 vs 5.7 infections per 1000 ventilator-days, P = .03). The rate of colonization with MRSA (23.3 vs 69.3 per 1000 patient-days, P < .001) and Acinetobacter (1.0 vs 4.6 per 1000 patient-days, P = .36) was significantly lower in the chlorhexidine group than in the comparison group. CONCLUSIONS Daily bathing of trauma patients with cloths impregnated with 2% chlorhexidine gluconate is associated with a decreased rate of colonization by MRSA and Acinetobacter and lower rates of catheter-related bloodstream infection and MRSA VAP.

Hypertonic Resuscitation Modulates the Inflammatory Response in Patients With Traumatic Hemorrhagic Shock

Objective:To determine the effect of resuscitation with hypertonic saline/dextran (HSD) on the innate immune response after injury. Summary of Background Data:Hypovolemic shock causes a whole body ischemia/reperfusion injury, leading to dysregulation of the inflammatory response and multiple organ dysfunction syndrome. Hypertonicity has been shown to modulate the innate immune response in vitro and in animal models of hemorrhagic shock, but the effect on the inflammatory response in humans is largely unknown. Methods:Serial blood samples were drawn (12, 24, 72 hours and 7 days after injury) from patients enrolled in a prospective, randomized, double-blind trial of traumatic hypovolemic shock, HSD (250 mL) versus lactated Ringers solution (LR) as the initial resuscitation fluid. Neutrophil (PMN) CD11b/CD18 expression was assessed via whole blood FACS analysis with and without stimulation (fMLP 5 &mgr;mol/L or PMA 5 &mgr;mol/L). PMN respiratory burst was assessed using the nitro-blue tetrazolium assay. Monocytes stimulated with 100 ng LPS for 18 hours were assessed for cytokine production (TNF-α, IL-1&Bgr;, IL-6, IL-10, IL-12). Results:Sixty-two patients (36 HSD, 26 LR) and 20 healthy volunteers were enrolled. CD11b expression, 12 hours after injury, was increased 1.5-fold in patients resuscitated with LR compared with controls. Those resuscitated with HSD had a significant reduction in CD11b expression 12 hours after injury, compared with LR. There was no difference in respiratory burst early after injury. Monocytes from injured patients expressed lower levels of all cytokines in comparison to normal controls. Patients give HSD showed a trend toward higher levels of IL-1β and IL10 production in response to LPS, 12 hours after injury. Conclusion:HSD resuscitation results in transient inhibition of PMN CD11b expression and partial restoration of the normal monocyte phenotype early after injury.

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.

Hypertonic Preconditioning Inhibits Macrophage Responsiveness to Endotoxin

Hypertonic saline has been shown to modulate cell shape and the response of components of the innate immune response. However, the effect of hypertonic saline on the macrophage remains unknown. We hypothesized that hypertonic preconditioning would impair subsequent inflammatory mediator signaling through a reduction in stress fiber polymerization and mitogen-activated protein kinase activity after LPS stimulation. Rabbit alveolar macrophages were stimulated with 100 ng/ml of LPS. Selected cells were preconditioned with 40–100 mM of NaCl, mannitol, or urea for 4 h and returned to isotonic medium before LPS stimulation. Cellular protein was harvested and subjected to Western blot analysis for the dually phosphorylated active forms of p38 and extracellular signal-related kinase (ERK) 1/2. TNF production was determined by an L929 bioassay, and stress fiber polymerization was evaluated by confocal microscopy. Preconditioning of macrophages with NaCl or mannitol resulted in dose-dependent reduction in ERK 1/2 phosphorylation with no effect on p38 phosphorylation. Urea preconditioning had no effect on either mitogen-activated protein kinase. A dose-dependent attenuation of TNF production was seen with NaCl and mannitol preconditioning (p < 0.05), but not with urea. NaCl and mannitol preconditioning resulted in failure of LPS-induced stress fiber polymerization, whereas urea did not. Extracellular hypertonic conditions (i.e., NaCl and mannitol) have an immunomodulatory effect on macrophages, demonstrated through failure of optimal stress fiber polymerization, ERK 1/2 activity, and TNF production. Intracellular hypertonic conditions (i.e., urea) had no significant effect. Hypertonic saline or mannitol resuscitation, therefore, may help protect against multiple-organ dysfunction syndrome as a result of this reduced proinflammatory responsiveness.

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)

Mitogen-activated protein kinase (MAPK).

At a molecular level, the immune state is controlled through an exhaustive array of interconnected and tightly controlled series of intracellular signaling pathways (1, 2). This is achieved, in part, by activating or deactivating regulatory proteins through protein phosphorylation. The highly conserved mitogen-activated protein kinase (MAPK) family is one of the major kinase families that regulate cells by transducing extracellular signals into cellular responses (3). Three important members of this family are the extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Each is similarly activated by dual phosphorylation on adjacent threonine and tyrosine residues. The classic MAPK cascade consists of three sequential intracellular protein kinase activation steps and is initiated when the first member, MAPK kinase kinase (MAPKKK), is activated (Fig. 1). MAPKKK is a protein kinase that phosphorylates and activates MAPK kinase (MAPKK), and this activation is followed by activation of the specific MAPK. MAPKs are proline-directed protein kinases, meaning that they phosphorylate serine or threonine residues that are neighbors to proline. In this manner, MAPKs activate numerous protein kinases, nuclear proteins, and transcription factors, leading to downstream signal transduction. Activation of the MAPK cascade is rapid and enables cells to respond to environmental changes in a regulated fashion. Erk. ERK was the first identified of all the MAPK family members. ERK is made up of two isoforms, ERK1 and ERK2, and is commonly referred to as ERK 1/2. ERK is primarily involved in proliferation, transformation, and differentiation. ERK activation is initiated by the phosphorylation and activation of Raf (MAPKKK), followed by the phosphorylation and activation of MEK 1/2 (MAPKK) that leads to the activation of ERK 1/2 (MAPK) (4). Raf is a highly conserved kinase that is activated by the G-protein, Ras. Ras is activated through interaction with Grb2SOS complex, where SOS catalyzes the formation of Ras-guanosine triphosphate (GTP) complex. This GTP-bound complex binds to Raf and activates it in a calciumdependent manner (5). This cascade is activated by a number of mitogens, including epidermal growth factor, platelet-derived growth factor, thromboxane A2, angiotensin II, transforming growth factor , and insulin (6). Additionally, ERK is activated in response to endotoxin, oxidative stress, platelet activating factor (PAF), and adherence by innate immune system cells such as monocytes and macrophages. Activation of ERK by oxidative stress, PAF, and adherence is important in monocyte/macrophage proinflammatory reprogramming, characteristic of sepsis, by modulating the activation status of the regulatory kinase calcium/calmodulin-dependent protein kinase II (CaMK II), leading to dysregulated inflammatory cytokine production (7). p38. p38 is an important MAPK member that is activated in response to physiologic stress, endotoxin, osmotic stress, and ultraviolet exposure (8). Five isoforms of the p38 group have been identified: p38 (SAPK2), p38 , p38 2, p38 (SAPK3), and p38 (9). Expression of these isoforms varies among tissues. p38 is highly expressed in leukocytes and bone marrow, p38 is expressed in heart and brain, and p38 is expressed predominately in skeletal muscles. Similar to ERK, p38 is activated by a series of upstream activators. The two major upstream activators are MKK3 and MKK6. MKK6 is poorly expressed in leukocytes, whereas MKK3 is highly expressed in leukocytes. More proximally, there are multiple diverse kinases that act as MAPKKK for this pathway, including TAK1 and AK-1. This diverse array of proximal kinases allows for activation by a wide range of stimuli. Major substrates for p38 include MAP kinase–activated protein kinase-2 (MAPKAP-K2) and MAPKAP-K3. MAPKAPK2/3 are serine protein kinases known to subsequently activate heat shock protein-27 (HSP-27) (10). HSP-27 is an actin binding protein that helps to repair the actin cytoskeleton and inhibit apoptosis, and it may have some anti-inflammatory properties. p38 also phosphorylates and activates the transcription factors ATF-2 and SP1. ATF-2 is critical for the upregulation of a number of different inflammatory proteins, whereas SP1 appears to be critical for the transcription of the antiinflammatory cytokine IL-10 (11). Inflammatory stimuli, such as endotoxin, tumor necrosis factor (TNF), PAF, interleukin (IL)-1, and ischemia-reperfusion, induce p38 activation in innate immune cells (9). Although p38 activation is essential for expression of the inflammatory cytokines or chemokines, such as TNF, IL-1, IL-6, and IL-8, within immune cells, unregulated prolonged activation of p38 appears to be critical for the immunosuppressive phenotype typical of late sepsis through the liberation of IL-10 and expansion of the immunosuppressive Th-2 cytokine phenotype within the T cell population (12). JNK. JNK was originally identified as 54-kDa stress-activated protein kinase (SAPK). JNK is activated in response to many of the stimuli that activate p38, such as lipopolysaccharide, TNF, IL-1, osmotic stress, and ultraviolet radiation (9). The JNK protein-kinases are encoded by three genes: JNK1, JNK2, and JNK3. JNK1/2 are ubiquitously expressed, whereas JNK3 appears to be expressed only in brain, heart, and testis. The imFrom the Department of Surgery, University of Washington, Seattle, WA. The authors have no financial interests to disclose. Copyright

The role of repeat angiography in the management of pelvic fractures

BACKGROUND Angiographic embolization has emerged as the treatment modality of choice for bleeding pelvic fractures. The purpose of this study is to identify potential indicators for ongoing pelvic hemorrhage despite initial therapeutic or non-diagnostic angiography. METHODS The trauma registry of a Level I trauma center was used to identify patients with pelvic fractures between January 2000 and June 2002. Records were reviewed for demographics, severity of injury, hemodynamic status, initial and subsequent base deficit, blood and fluid requirements, length of stay, and mortality. Statistical analysis was performed using Students t test, and univariate and multivariate analysis, significance was assigned to p < or = 0.05. RESULTS During the study period, 678 patients had pelvic fractures. Angiography was performed in 31 (4.6%) of these patients. Arterial hemorrhage was diagnosed initially on 16 (51.6%) patients requiring embolization. Three (18.8%) of these embolized patients required repeat angiography and embolization due to ongoing pelvic hemorrhage. Of the initial 15 patients with negative angiograms, five (33.3%) had repeat angiograms due to continued hypotension and acidosis. Four (80.0%) of these five patients were found to have arterial hemorrhage requiring embolization. Of the seven (22.6%) patients requiring repeat angiography for control of ongoing pelvic hemorrhage, three independent factors were predictive: continued or recurrent hypotension (SBP < 90), absence of intra-abdominal injury, and persistent base deficit of 10 for greater than 6 hours. The presence of all three independent predictors was associated with a 97% probability of pelvic bleeding (p = 0.001). CONCLUSION Angiographic embolization is highly effective in controlling arterial bleeding associated with pelvic fractures. However, repeat angiography should be performed in patients with pelvic fractures with ongoing evidence of hemorrhage demonstrated by persistent base deficit and hypotension once other potential sources of bleeding have been excluded.