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ABSTRACT: Venous thromboembolic (VTE) disease has a high incidence following trauma, but debate remains regarding optimal prophylaxis. Thrombelastography (TEG) has been suggested to be optimal in guiding prophylaxis. Thus, we designed a phase II randomized controlled trial to test the hypothesis that TEG-guided prophylaxis with escalating low-molecular weight heparin (LMWH), followed by antiplatelet therapy would reduce VTE incidence.
Surgical intensive care unit trauma patients (n = 50) were randomized to receive 5,000 IU of LMWH daily (control) or to TEG-guided prophylaxis, up to 5,000 IU twice daily with the addition of aspirin, and were followed up for 5 days. In vitro studies were also conducted in which apheresis platelets were added to blood from healthy volunteers (n = 10).
Control (n = 25) and TEG-guided prophylaxis (n = 25) groups were similar in age, body mass index, Injury Severity Score, and male sex. Fibrinogen levels and platelet counts did not differ, and increased LMWH did not affect clot strength between the control and study groups. The correlation of clot strength (G value) with fibrinogen was stronger on Days 1 and 2 but was superseded by platelet count on Days 3, 4, and 5. There was also a trend in increased platelet contribution to clot strength in patients receiving increased LMWH. In vitro studies demonstrated apheresis platelets significantly increased clot strength (7.19 ± 0.35 to 10.34 ± 0.29), as well as thrombus generation (713.86 ± 12.19 to 814.42 ± 7.97) and fibrin production (274.03 ± 15.82 to 427.95 ± 16.58).
Increased LMWH seemed to increase platelet contribution to clot strength early in the study but failed to affect the overall rise clot strength. Over time, platelet count had the strongest correlation with clot strength, and in vitro studies demonstrated that increased platelet counts increase fibrin production and thrombus generation. In sum, these data suggest an important role for antiplatelet therapy in VTE prophylaxis following trauma, particularly after 48 hours.
Therapeutic study, level III.
The journal of trauma and acute care surgery. 03/2013; 74(3):756-65.
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ABSTRACT: Firm neutrophil (PMN)-endothelial (EC) adhesion is crucial to the PMN-mediated hyperinflammation observed in acute lung injury. Hypertonic saline (HTS) used for resuscitation of hemorrhagic shock has been associated with a decreased incidence of PMN-mediated lung injury/acute respiratory distress syndrome. We hypothesize that physiologically accessible hypertonic incubation (170mM vs. 140mM, osmolarity ranging from 360-300 mOsm/L) inhibits pro-inflammatory activation of human pulmonary microvascular endothelial cells (HMVECs). Pro-inflammatory activation of HMVECs was investigated in response to TNFα including IL-8 release, ICAM-1 surface expression, PMN adhesion, and signaling mechanisms under both isotonic (control) and hypertonic conditions. Hyperosmolarity alone had no effect on either basal IL-8 release or ICAM-1 surface expression, but did lead to concentration-dependent decreases in TNFα-induced IL-8 release, ICAM-1 surface expression, and PMN:HMVEC adhesion. Conversely, HTS activated p38 mitogen-activated protein kinase (MAPK) and enhanced TNFα activation of p38 MAPK. Despite this basal activation, hyperosmolar incubation attenuated TNFα stimulated IL-8 release and ICAM-1 surface expression and subsequent PMN adherence, while p38 MAPK inhibition did not further influence the effects of hyperosmolar conditions on ICAM-1 surface expression. In addition, TNFα induced NF-kB DNA binding, but HTS conditions attenuated this by 31% (p<0.01). In conclusion, HTS reduces PMN:HMVEC adhesion as well as TNFα-induced pro-inflammatory activation of primary HMVECs via attenuation of NF-kB signaling.
Shock (Augusta, Ga.) 01/2013; · 2.87 Impact Factor
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ABSTRACT: Thromboelastography (TEG) is emerging as the standard in the management of acute coagulopathies in injured patients. Although TEG is sensitive in detecting abnormalities in clot strength, one shortcoming is differentiating between fibrinogen and platelet contributions to clot integrity. Current American algorithms suggest platelet transfusion, whereas European guidelines suggest fibrinogen concentrates for correcting low clot strength. Therefore, we hypothesized that a TEG-based functional fibrinogen (FF) assay would assess the contribution of fibrinogen and platelets to clot strength and provide insight to transfusion priorities. Blood samples were obtained from trauma patients on arrival to the emergency department or who were admitted to the surgical intensive care unit (n = 68). Citrated kaolin TEG, FF, and von Clauss fibrinogen levels (plasma-based clinical standard) were measured. Correlations were assessed using linear regression models. In vitro studies were also performed with adding fibrinogen concentrates to blood collected from healthy volunteers (n = 10). Functional fibrinogen and citrated kaolin TEG parameters were measured. Functional fibrinogen strongly correlated with von Clauss fibrinogen levels (R = 0.87) and clot strength (R = 0.80). The mean fibrinogen contribution to clot strength was 30%; however, there was a direct linear relationship with fibrinogen level and percent fibrinogen contribution to clot strength (R = 0.83). Traditional TEG parameters associated with fibrinogen activity (α angle and kinetic time) had significantly lower correlations with FF (R = 0.70 and 0.35). Furthermore, platelet count had only a moderate correlation to clot strength (R = 0.51). The addition of fibrinogen concentrate in in vitro studies increased clot strength (MA) (60.44 ± 1.48 to 68.12 ± 1.39) and percent fibrinogen contribution to clot strength (23.8% ± 1.8% to 37.7% ± 2.5%). Functional fibrinogen can be performed rapidly with TEG and correlates well with the standard von Clauss fibrinogen assay. Both fibrinogen and platelet contribution of clot strength can be derived from FF. Moreover, FF had a stronger correlation to clot strength, and increased levels were directly associated with increased percent contribution to clot strength. In vitro studies also demonstrated an increase in FF, clot strength, and percent fibrinogen contribution to clot strength with the addition of fibrinogen concentrate. These data suggest that fibrinogen should be addressed early in trauma patients manifesting acute coagulopathy of trauma.
Shock (Augusta, Ga.) 01/2013; 39(1):45-9. · 2.87 Impact Factor
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Jeffrey N Harr,
Ernest E Moore,
Jeffrey Johnson,
Theresa L Chin,
Max V Wohlauer,
Ronald Maier,
Joseph Cuschieri,
Jason Sperry, Anirban Banerjee,
Christopher C Silliman,
Angela Sauaia
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ABSTRACT: OBJECTIVE:: To determine whether prehospital antiplatelet therapy was associated with reduced incidence of acute lung dysfunction, multiple organ failure, and mortality in blunt trauma patients. DESIGN:: Secondary analysis of a cohort enrolled in the National Institute of General Medical Sciences Trauma Glue Grant database. SETTING:: Multicenter study including nine U.S. level-1 trauma centers. PATIENTS:: A total of 839 severely injured blunt trauma patients at risk for multiple organ failure (age > 45 yr, base deficit > 6 mEq/L or systolic blood pressure < 90 mm Hg, who received a blood transfusion). Severe/isolated head injuries were excluded. MEASUREMENTS AND MAIN RESULTS:: Primary outcomes were lung dysfunction (defined as grades 2-3 by the Denver multiple organ failure score), multiple organ failure (Denver multiple organ failure score >3), and mortality. Patients were documented as on antiplatelet therapy if taking acetylsalicylic acid, clopidogrel, and/or ticlopidine. Fifteen percent were taking antiplatelet therapy prior to injury. Median injury severity score was 30 (interquartile range 22-51), mean age 61 + 0.4 yr and median RBCs volume transfused was 1700 mL (interquartile range 800-3150 mL). Overall, 63% developed lung dysfunction, 19% had multiple organ failure, and 21% died. After adjustment for age, gender, comorbidities, blood products, crystalloid/12 hrs, presence of any head injury, injury severity score, and 12 hrs base deficit > 8 mEq/L, 12 hrs RBC transfusion was associated with a significantly smaller risk of lung dysfunction and multiple organ failure among the group receiving antiplatelet therapy compared with those not receiving it (lung dysfunction p = 0.0116, multiple organ failure p = 0.0291). In addition, antiplatelet therapy had a smaller risk (albeit not significant, p = 0.06) of death for patients receiving RBC compared to those not on antiplatelet therapy after adjustment for confounders, CONCLUSIONS:: Pre-injury antiplatelet therapy is associated with a decreased risk of lung dysfunction, multiple organ failure, and possibly mortality in high-risk blunt trauma patients who received blood transfusions. These findings suggest platelets have a role in organ dysfunction development and have potential therapeutic implications.
Critical care medicine 12/2012; · 6.37 Impact Factor
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ABSTRACT: Initiating prehospital resuscitation with plasma in patients with trauma-associated hemorrhagic shock will result in more rapid and durable clot formation and, thus, the need for fewer packed cell infusions, less frequent use of cryoprecipitate, and more ventilator-free hospital days compared with those of patients randomized to standard crystalloid field resuscitation.
Surgery 11/2012; · 3.10 Impact Factor
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ABSTRACT: While minimizing hyperglycemia in critically injured patients improves outcomes, it is debatable whether postinjury glucose control should aim for conventional glucose control levels (≤180 mg/dL) or tight glucose control levels (81-108 mg/dL). We queried our 17-year prospective database of patients at risk for postinjury multiple organ failure to examine the association between glucose levels and adverse outcomes.
Acutely injured patients admitted to a Level I trauma center intensive care unit from 1992 to 2008 who were more than 15 years of age, had Injury Severity Scores >15, and who survived >48 hours were eligible for the study. Multiple logistic regression was used to determine the independent association of glucose control with adverse outcomes (death, ventilator-free days, intensive care unit-free days, and major infections), adjusted for Injury Severity Score, age, and red blood cell transfusion in the first 12 hours.
Overall, 2,231 patients were eligible, of whom 153 (6.9%) died. The mean age was 37.8 ± 0.4 years, and the median Injury Severity Score was 27 (interquartile range, 21-35). The majority (77%) of these patients maintained mean glucose within conventional glucose control levels and only 10% achieved mean glucose levels within tight glucose control levels. Nonsurvivors required greater doses of insulin to control glucose levels and had greater mean insulin to glucose ratios (t test; P = .025). After adjusting for confounders, mean glucose remained significantly associated with the studied adverse outcomes. Age significantly modified all these associations with older patients seeming to benefit more from tight glucose control levels than their younger counterparts.
Age is an effect modifier of the association between glucose levels and adverse outcomes. Future studies including larger samples of elderly trauma patients are needed to determine the ideal levels for glucose control in this growing population.
Surgery 09/2012; 152(3):315-21. · 3.10 Impact Factor
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ABSTRACT: Growing evidence suggests platelets are essential in posttraumatic, acute lung injury (ALI). Halogenated ethers interfere with the formation of platelet-granulocyte aggregates. The potential benefit of halogenated ethers has not been investigated in models of trauma/hemorrhagic shock (T/HS). Therefore, we hypothesized that isoflurane decreases T/HS-mediated ALI through platelet inhibition.
Sprague-Dawley rats (n = 47) were anesthetized by either pentobarbital or inhaled isoflurane and placed into (1) control, (2) trauma (laparotomy) sham shock, (3) T/HS (mean arterial pressure, 30 mmHg × 45 min), (4) pretreatment with an ADP receptor antagonist, or (5) T/HS with isoflurane initiated during resuscitation groups. ALI was determined by protein and pulmonary immunofluorescence bronchoalveolar lavage (BAL) fluid. Platelet Mapping specifically evaluated thrombin-independent inhibition of the ADP and AA pathways of platelet activation.
Pretreatment with isoflurane abrogated ALI as measured by both BAL fluid protein and pulmonary immunofluorescence (P < .001). Platelet Mapping revealed specific inhibition of the platelet ADP-pathway with isoflurane (P < .001). Pretreatment with an ADP receptor antagonist decreased ALI to sham levels, confirming that specific platelet ADP inhibition decreases ALI. Isoflurane initiated during resuscitation also decreased ALI (P < .001).
Isoflurane attenuates ALI through an antiplatelet mechanism, in part, through inhibition of the platelet ADP pathway. Isoflurane given postinjury also protects against ALI, and highlights the potential applications of this therapy in various clinical scenarios of ischemia/reperfusion.
Surgery 08/2012; 152(2):270-6. · 3.10 Impact Factor
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Max Wohlauer,
Ernest E Moore,
Christopher C Silliman,
Miguel Fragoso,
Fabia Gamboni,
Jeffrey Harr,
Frank Accurso,
Frank Wright,
James Haenel,
David Fullerton, Anirban Banerjee
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ABSTRACT: We hypothesized that aerosolized inhaled hypertonic saline given at the onset of resuscitation will decrease acute lung injury following hemorrhagic shock, by inhibiting the release of epithelial derived proinflammatory mediators.
Animal study.
Animal-care facility procedure room in a medical center.
Adult male Sprague-Dawley rats.
Rats underwent hemorrhagic shock followed by 2 hrs of resuscitation and 1 hr of observation. In the study group, nebulized hypertonic saline was delivered at the end of the shock period and after 1 hr and 2 hrs of resuscitation.
Shock provoked acute lung injury, which was attenuated with inhaled hypertonic saline (1.56 ± 0.2 mg protein/mL vs. 0.95 ± 0.3 mg protein/mL bronchoalveolar lavage fluid, shock vs. shock + hypertonic saline, p < .01). Nebulized hypertonic saline reduced inflammation (cytokine-induced neutrophil chemoattractant-1 accumulation in bronchoalveolar lavage fluid 5999 ± 1267 pg/mL vs. 3342 ± 859 pg/mL, shock vs. shock + hypertonic saline, p = .006). Additionally, nebulized hypertonic saline inhibited matrix -metalloproteinase-13 accumulation in the bronchoalveolar lavage fluid (1513 ± 337 pg/mL bronchoalveolar lavage fluid vs. 230 ± 19 pg/mL, shock vs. shock + hypertonic saline, p = .009) and pretreatment with a matrix metalloproteinase-13 inhibitor was sufficient to attenuate postshock acute lung injury (1.42 ± 0.09 mg/mL vs. 0.77 ± 0.23 mg/mL bronchoalveolar lavage protein, shock vs. shock + matrix metalloproteinase-13 inhibitor CL-82198, p = .002).
Inhaled hypertonic saline attenuates postshock acute lung injury by exerting an anti-inflammatory effect on the pulmonary epithelium, suggesting a new clinical strategy to treat acute lung injury/acute respiratory distress syndrome.
Critical care medicine 06/2012; 40(9):2647-53. · 6.37 Impact Factor
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ABSTRACT: The cellular and biochemical mechanisms leading to acute lung injury (ALI) and subsequent multiple organ failure are only partially understood. To study the potential role of eicosanoids, particularly leukotrienes, as possible mediators of ALI, we used a murine experimental model of ALI induced by hemorrhagic shock after blood removal via cardiac puncture. Neutrophil sequestration, as shown by immunofluorescence and protein leakage into the alveolar space were measured as markers of injury. We used liquid chromatography coupled to tandem mass spectrometry to unequivocally identify several eicosanoids in the bronchoalveolar lavage fluid of experimental animals. MK886, a specific inhibitor of the 5-lipoxygenase (5-LO) pathway, and transgenic mice deficient in 5-LO were used to determine the role of this enzymatic pathway in this model. Leukotriene B4 and leukotriene C4 were consistently elevated in shock-treated mice compared with sham-treated mice. MK886 attenuated neutrophil infiltration and protein extravasation induced by hemorrhagic shock. 5-Lipoxygenase-deficient mice showed reduced neutrophil infiltration and protein extravasation after shock treatment, indicating greatly reduced lung injury. These results support the hypothesis that 5-LO, most likely through the generation of leukotrienes, plays an important role in the pathogenesis of ALI induced by hemorrhagic shock in mice. This pathway could represent a new target for pharmacological intervention to reduce lung damage following severe primary injury.
Shock (Augusta, Ga.) 03/2012; 37(6):599-604. · 2.87 Impact Factor
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ABSTRACT: Despite improved resuscitation strategies, acute kidney injury (AKI) remains an important cause of morbidity and high resource use among severely injured patients. Thus, we conducted a comprehensive evaluation of the epidemiology and outcomes of early AKI among severely injured patients as well as its impact on the development of postinjury multiple organ failure (MOF).
We queried our 17-year database of high-risk postinjury patients (Injury Severity Score >15, age >15 years, survival >48 hours, and no isolated head injury). MOF and AKI (creatinine >1.8 mg/dL) were defined by the Denver MOF score. Patients with documented preexisting renal, hepatic, cardiac, or pulmonary disease (120, 5%) were excluded, leaving 2157 for analysis.
Early (day 2) AKI was evident in 2.13% of the patients and associated with a 78% MOF incidence and 27% mortality. Both rates were higher than those associated with early heart, lung, or liver failure.
Early AKI is a harbinger of adverse outcome postinjury, outperforming hepatic, cardiac, or pulmonary dysfunction as a predictor of MOF and death. Prevention of early AKI and a better understanding of organ crosstalk may help reduce AKI-associated morbidity, mortality, and obligatory costs of this complication.
I, prognostic study.
The journal of trauma and acute care surgery. 02/2012; 72(2):373-8; discussion 379-80.
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ABSTRACT: Predicting refractory coagulopathy early in resuscitation of injured patients may decrease a leading cause of preventable death. We hypothesized that clot strength (G) measured by point-of-care rapid thrombelastography (r-TEG) on arrival in the emergency department can predict massive transfusion (MT) and coagulation-related mortality (MT-death).
Trauma alerts/activations from May 2008 to September 2010 were reviewed. The variables included the following: age, sex, injury severity score (ISS), systolic blood pressure (SBP), base deficit (BD), traditional coagulation tests (international normalized ratio ([INR], partial thromboplastin time [PTT]), TEG-derived G, and blood products transfused within the first 6 hours. Independent predictors of 2 outcomes (MT [≥10 packed red blood cells units/6 h] and MT-related death) were identified using logistic regression. The individual predictive values of BD, INR, PTT, and G were assessed comparing the areas under the receiver operating characteristic curves (AUC ROC), while adjusting for age, ISS, and SBP.
Among the 80 study patients, 48% required MT, and 21% died of MT-related complications. INR, ISS, and G were independent predictors of MT, whereas age, ISS, SBP, and G were independently associated with MT-death. The predictive power for outcome MT did not differ among INR (adjusted AUC ROC = 0.92), PTT (AUC ROC = 0.90, P = .41), or G (AUC ROC = 0.89, P = .39). For outcome MT-death, G had the greatest adjusted AUC ROC (0.93) compared with the AUC ROC for BD (0.87, P = .05), INR (0.88, P = .11), and PTT (0.89; P = .19).
These data suggest that the point-of-care TEG parameter clot strength (G) provides consistent, independent prediction of MT and MT-death early in the resuscitation of injured patients.
Surgery 09/2011; 151(1):48-54. · 3.10 Impact Factor
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ABSTRACT: Plasma is vital for the resuscitation of injured patients and to restore necessary procoagulants, especially Factors (F)II, FV, FVII, FX, and FXIII; however, female plasma has been implicated in the majority of transfusion-related acute lung injury (TRALI) cases and male-only plasma transfusion regimens have significantly decreased the incidence of TRALI. Little is known about the human plasma proteome, and no comparisons have been made between male and female plasma; therefore, we hypothesize that there are significant differences between plasma from male and female donors.
Five units of fresh-frozen plasma each were collected from nulliparous female donors and male donors, and the proteome was analyzed by depleting the 14 most common proteins by immunoaffinity columns followed by protein separation by one dimension gel electrophoresis, tryptic digestion of the proteins, analysis of the peptides by liquid chromatography-tandem mass spectrometry, and identification employing human protein sequence databases.
Female plasma versus male plasma contained pregnancy zone protein (419- to 580-fold), FV (twofold), α(1)-antitrypsin (twofold), β(2) -microglobulin (twofold), and Complement Factors H and C4B (1.5- to 2-fold) at significantly higher concentrations than males and males contained significant increases in Fc-binding protein (twofold), protein Z-dependent protease inhibitor (twofold), phosphatidylinositol glycan-specific phospholipase (fourfold), protein S-100 (threefold), and transgelin-2 (14-fold) versus females (p < 0.005). The increases in FV, α(1)-antitrypsin, and β(2)-microglobulin were confirmed by an activity assay or immunoblots.
We conclude that there are proteomic differences between male and female plasma, which could be exploited to improve clinical outcomes in transfused patients.
Transfusion 08/2011; 52(2):417-24. · 3.22 Impact Factor
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ABSTRACT: Although melanoma can elicit robust tumor antigen-specific immune responses, advanced melanoma is associated with immune tolerance. We have previously described several mechanisms of melanoma-induced immunosuppression, including the skewing of the immune response towards a Th2 cytokine profile and the induction of regulatory T cells. Since dendritic cells (DCs) are potentially important players that can direct other cells of the immune system towards a cytotoxic, humoral, or regulatory phenotype, we hypothesized that melanoma-produced factors directly affect the maturation and function of DCs, influencing the nature and magnitude of the resulting immune response.
To test this hypothesis, immature myeloid-derived DCs (mdDCs) were derived with cytokines from CD14+ peripheral blood mononuclear cells (PBMCs) and exposed to 20% melanoma-conditioned media (MCM). After 2 d, the expression of maturation markers and the function of these mdDCs, measured by cytokine production, the amount of endocytosis, expression of the inhibitory molecule indoleamine 2,3-dioxygenase (IDO), and the ability to stimulate T cells were determined.
We found that incubation with MCM did not inhibit the expression of maturation markers or IDO, the production of cytokines, the amount of antigen uptake, or the ability to induce T cell proliferation in mixed-lymphocyte reactions by mdDC.
These results suggest that the immunosuppressive effects of melanoma-produced factors are independent of directly measurable changes in mdDC function or maturation in vitro.
Journal of Surgical Research 08/2011; 176(1):301-8. · 2.25 Impact Factor
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ABSTRACT: The return of heparinized shed blood (SB) in trauma/hemorrhagic shock (T/HS) models remains controversial because of potential anti-inflammatory properties. Although ubiquitous as an anticoagulant, heparin is ineffective on cellular coagulation as an antithrombotic agent. Therefore, we hypothesized that returning heparinized SB would paradoxically enhance acute lung injury (ALI) after T/HS because of the infusion of activated platelets. Sprague-Dawley rats, anesthetized with pentobarbital, underwent laparotomy and hemorrhage-induced shock (MAP of 30 mmHg × 45 min). Animals were resuscitated with a combination of normal saline and returned SB. Shed blood was collected in either 80 U/kg of heparin, 800 U/kg of heparin, or citrate or diluted 1:8 with normal saline. An additional group of animals were pretreated with a platelet P2Y12 receptor antagonist (clopidogrel) before T/HS. Bronchoalveolar lavage, lung myeloperoxidase assays, pulmonary immunofluorescence, and blood smears were conducted. Bronchoalveolar lavage protein increased in animals resuscitated with heparinized SB (T/HS + 80 U/kg Hep 1.62 ± 0.29, T/HS + 800 U/kg Hep 1.30 ± 0.15 vs. T/SS 0.51 ± 0.16 and T/HS Citrate 0.7 ± 0.09) (P < 0.0001). Blood smears and platelet function assays revealed platelet aggregates and increased platelet activation. Animals pretreated with a platelet P2Y12 receptor antagonist were protected from postinjury ALI (P < 0.0001). Animals with return of SB had increased pulmonary polymorphonuclear leukocyte sequestration (P < 0.0001). Pulmonary immunofluorescence demonstrated microthrombi only in the T/HS group receiving heparinized SB (P < 0.0001). The return of heparinized SB functions as a "second hit" to enhance ALI, with activated platelets propagating microthrombi and pulmonary polymorphonuclear leukocyte recruitment.
Shock (Augusta, Ga.) 08/2011; 36(6):595-603. · 2.87 Impact Factor
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ABSTRACT: Arachidonic acid (AA, and its leukotriene derivatives, e.g., LTB(4)) is an inflammatory mediator in post-shock mesenteric lymph that appears to act as an agonist on G-protein coupled receptors (GPCRs). These mediators prime neutrophils (PMNs) for an increased production of superoxide, implicated in the development of acute lung injury (ALI). Hypertonic saline (HTS) has also been shown to have immunomodulatory effects such as attenuation of PMN priming by precluding appropriate clathrin-mediated endocytosis of activated GPCRs, thereby potentially attenuating ALI. We hypothesize that HTS inhibits priming of the PMN oxidase by these lipid mediators.
After PMNs were isolated from healthy donors, incubation was done in either isotonic buffer (control) or HTS (180 mmol/L) for 5 min at 37°C. The PMNs were then primed for 10 min with AA [5 μM] or 5 min with LTB(4) [1 μM] and the oxidase was activated with 200 ng/mL of phorbol 12-myristate 13-acetate (PMA), a non-GPCR activator, and superoxide anion generation was measured via reduction of cytochrome c.
Both AA [5 μM] and LTB(4) [1 μM] significantly primed the PMA activated respiratory burst (P < 0.05, ANOVA, Newman-Keuls, n = 4). HTS inhibited both AA and LTB(4) priming of the respiratory burst.
These data indicate that HTS reduces the cytotoxicity of PMNs stimulated by these lipid mediators in vitro and further support the immunomodulatory effects of HTS.
Journal of Surgical Research 07/2011; 174(1):24-8. · 2.25 Impact Factor
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ABSTRACT: Substantial investigation has implicated mesenteric lymph as the mechanistic link between gut ischemia/reperfusion (I/R) and distant organ injury. Specifically, lymph diversion prevents acute lung injury (ALI) in vitro, and bioactive lipids and proteins isolated from postshock mesenteric lymph (PSML) maintain bioactivity in vitro. However, Koch's postulates remain to be satisfied via direct cross-transfusion into a naïve animal. We therefore hypothesized that real time cross-transfusion of postshock mesenteric lymph provokes acute lung injury.
One set of Sprague-Dawley rats (lymph donors) was anesthetized, with the mesenteric lymph ducts cannulated and exteriorized to drain freely into a siliconates plastic cup; concurrently, a second group of rats ( lymph recipients) was anesthetized, with a cannula inserted into the animal's right internal jugular vein. Blood was removed from the donor rats to induce hemorrhagic shock (MAP of 35 mmHg × 45 min). The recipient rats were positioned 10 cm below the plastic cup, which emptied into the jugular vein cannula. Thus, mesenteric lymph from the shocked donor rat was delivered to the recipient rat at the rate generated during shock and the subsequent 3 h of resuscitation.
Neutrophil (PMN) accumulation in the lungs was substantially elevated in the postshock lymph cross-transfusion group compared to both sham lymph cross-transfusion and instrumented control (MPO: 9.42 ± 1.55 versus 2.81 ± 0.82 U/mg lung tissue in postshock versus sham lymph cross-transfusion, n = 6 in each group, P = 0.02). Additionally, cross-transfusion of PSML induced oxidative stress in the lung (0.21 ± 0.03 versus 0.10 ± 0.01 micromoles MDA per mg lung tissue in lymph cross-transfusion versus instrumented control, n = 6 in each group, P = 0.046). Furthermore, transfusion of PSML provoked lung injury (BAL protein 0.77 ± 0.18 versus 0.15 ± 0.02 mg/mL protein in BALF, postshock versus sham lymph cross-transfusion, n = 6 in each group, P = 0.004).
Cross-transfusion of PSML into a naïve animal leads to PMN accumulation and provokes ALI. These data provide evidence that postshock agents released into mesenteric lymph are capable of provoking distant organ injury.
Journal of Surgical Research 04/2011; 170(2):314-8. · 2.25 Impact Factor
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ABSTRACT: Acute coagulopathy of trauma (ACOT) has been described as a very early hypocoagulable state, but the mechanism remains controversial. One proposed mechanism is tissue hypoperfusion leading to protein C activation, with subsequent inhibition of Factors V and VIII. Variability in trauma has impeded the use of clinical data towards the elucidation of the mechanisms of ACOT, but thrombelastography (TEG) may provide insight by assessing hemostatic function from initial thrombin activation to fibrinolysis. We hypothesized that in a controlled animal model of trauma/hemorrhagic shock, clotting factor dysfunction is the predominant mechanism in early ACOT.
Rats anesthetized by inhaled isoflurane (n = 6) underwent laparotomy, and hemorrhage was induced to maintain a MAP of 35 mm Hg for 30 min. Rats were then resuscitated with twice their shed blood volume in normal saline. TEG was performed at baseline, shock, and post-resuscitation periods. No heparin was given. Statistical analysis was performed by ANOVA with post-hoc Fisher's test.
Coagulation factor function was significantly impaired in the early stages of trauma/hemorrhagic shock. TEG R and SP-values were significantly increased from baseline to shock (P < 0.001) and from shock to post-resuscitation periods (P < 0.05). Delta (R-SP), a measure of thrombin generation, showed a significant increase (P < 0.05) from baseline to shock. No significant changes were found in K, Angle, MA, and LY30 values.
Clotting factor derangement leading to impaired thrombin generation is the principle etiology of ACOT in this model and not the dynamics of clot formation, fibrin cross-linking, clot strength/platelet function, or fibrinolysis.
Journal of Surgical Research 04/2011; 170(2):319-24. · 2.25 Impact Factor
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ABSTRACT: Experiments show that upon traumatic injury the composition of mesenteric lymph changes such that it initiates an immune response that can ultimately result in multiple organ dysfunction syndrome (MODS). To identify candidate protein mediators of this process we carried out a quantitative proteomic study on mesenteric lymph from a well characterized rat shock model. We analyzed three animals using analytical 2D differential gel electrophoresis. Intra-animal variation for the majority of protein spots was minor. Functional clustering of proteins revealed changes arising from several global classes that give novel insight into fundamental mechanisms of MODS. Mass spectrometry based proteomic analysis of proteins in mesenteric lymph can effectively be used to identify candidate mediators and loss of protective agents in shock models.
Clinical Proteomics 01/2011; 8(1):1.
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ABSTRACT: Extensive animal work has established mesenteric lymph as the mechanistic link between gut ischemia/reperfusion and distant organ injury. Our trauma and transplant services provide a unique opportunity to assess the relevance of our animal data to human mesenteric lymph under conditions that simulate those used in the laboratory. Mesenteric lymph was collected from 11 patients with lymphatic injuries, during semielective spine reconstruction or immediately before organ donation. The lymph was tested for its ability to activate human neutrophils in vitro and was analyzed by label-free proteomic analysis. Human mesenteric lymph primed human polymorphonuclear neutrophils in a pattern similar to that observed in previous rodent, swine, and primate studies. A total of 477 proteins were identified from the 11 subjects' lymph samples with greater than 99% confidence. In addition to classic serum proteins, markers of hemolysis, extracellular matrix components, and general tissue damage were identified. Both tissue injury and shock correlate strongly with production of bioactive lymph. Products of red blood cell hemolysis correlate strongly with human lymph bioactivity, and immunoglobulins have a negative correlation with the proinflammatory lymph. These human data corroborate the current body of research implicating postshock mesenteric lymph in the development of systemic inflammation and multiple organ failure. Further studies will be required to determine if the proteins identified participate in the pathogenesis of multiple organ failure and if they can be used as diagnostic markers.
Shock (Augusta, Ga.) 12/2010; 35(4):331-8. · 2.87 Impact Factor
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ABSTRACT: Lyso-PCs (lysophosphatidylcholines) are a mixture of lipids that accumulate during storage of cellular blood components, have been implicated in TRALI (transfusion-related acute lung injury) and directly affect the physiology of neutrophils [PMNs (polymorphonuclear leucocytes)]. Because the G2A receptor, expressed on PMNs, has been reported to recognize lyso-PCs, we hypothesize that lyso-PC activation of G2A causes the increases in cytosolic Ca²(+) via release of G(α) and G(βγ) subunits, kinase activation, and the recruitment of clathrin, β-arrestin-1 and GRK6 (G-protein receptor kinase 6) to G2A for signal transduction. PMNs were isolated by standard techniques, primed with lyso-PCs for 5-180 s, and lysed for Western blot analysis, immunoprecipitation or subcellular fractionation, or fixed and smeared on to slides for digital microscopy. The results demonstrated that lyso-PCs cause rapid activation of the G2A receptor through S-phosphorylation and internalization resulting in G(αi)₋₁ and G(αq/)₁₁ release leading to increases in cytosolic Ca²(+), which was inhibited by an antibody to G2A or intracellular neutralization of these subunits. Lyso-PCs also caused the release of the G(βγ) subunit which demonstrated a physical interaction (FRET+) with activated Hck (haemopoietic cell kinase; Tyr⁴¹¹). Moreover, G2A recruited clathrin, β-arrestin-1 and GRK6: clathrin is important for signal transduction, GRK6 for receptor de-sensitization, and β-arrestin-1 both propagates and terminates signals. We conclude that lyso-PC activation of G2A caused release of G(αi)₋₁, G(αq/)₁₁ and G(βγ), resulting in cytosolic Ca²(+) flux, Hck activation, and recruitment of clathrin, β-arrestin-1 and GRK6.
Biochemical Journal 11/2010; 432(1):35-45. · 4.90 Impact Factor
