[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: Postshock mesenteric lymph (PSML) is the mechanistic link between splanchnic ischemia reperfusion (IR) and remote organ injury. We hypothesize that an unbiased inspection of the proteome of PSML will reveal previously unrecognized aberrations in systems biology provoked by hemorrhage-induced mesenteric IR injury in vivo.
Shock was induced in male Sprague-Dawley rats by controlled hemorrhage, and the mesenteric duct was cannulated for lymph collection. Preshock and postshock lymph were collected for differential in-gel electrophoresis (DIGE)-based proteomics. Proteins that increased or decreased in relative concentration > or =1.5-fold were selected for trypsin digestion and analysis by mass spectrometry (MS).
Evidence of tissue injury was detected by an increase in cell/tissue proteins in PSML. Components of coagulation were depleted, whereas products of hemolysis were increased. Haptoglobin was decreased, which supports an early postshock hemolytic process. Interestingly, several protective protease inhibitors were decreased in PSML. The unexpected findings were an increase in alpha-enolase (a key glycolitic enzyme and cell-surface plasminogen binding receptor, +2.4-fold change) and increased major urinary protein (MUP, a sex-specific lipid-binding protein, +17.1-fold change) in PSML.
A proteomic evaluation of PSML revealed evidence of several shock-associated processes: protein release from tissue injury, depletion of coagulation factors and evidence of hemolysis, depletion of protective protease inhibitors, and an increase in abundance of lipid carriers. These results suggest that constitutive changes in the proteome of PSML may provide novel insights into the complex pathophysiology of postshock systems biology.
Surgery 09/2009; 146(2):347-57. · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High-mobility group box 1 (HMGB1) is a late mediator of the systemic inflammation associated with sepsis. Recently, HMGB1 has been shown in animals to be a mediator of hemorrhage-induced organ dysfunction. However, the time course of plasma HMGB1 elevations after trauma in humans remains to be elucidated. Consequently, we hypothesized that mechanical trauma in humans would result in early significant elevations of plasma HMGB1. Trauma patients at risk for multiple organ failure (ISS > or = 15) were identified for inclusion (n = 23), and postinjury plasma samples were assayed for HMGB1 by enzyme-linked immunosorbent assay. Comparison of postinjury HMGB1 levels with markers for patient outcome (age, injury severity score, units of red blood cell (RBC) transfused per first 24 h, and base deficit) was performed. To investigate whether postinjury transfusion contributes to elevations of circulating HMGB1, levels were determined in both leuko-reduced and non-leuko-reduced packed RBCs. Plasma HMGB1 was elevated more than 30-fold above healthy controls within 1 h of injury (median, 57.76 vs. 1.77 ng/mL; P < 0.003), peaked from 2 to 6 h postinjury (median, 526.18 ng/mL; P < 0.01 vs. control), and remained elevated above control through 136 h. No clear relationship was evident between postinjury HMGB1 levels and markers for patient outcome. High-mobility group box 1 levels increase with duration of RBC storage, although concentrations did not account for postinjury plasma levels. Leuko-reduced attenuated HMGB1 levels in packed RBCs by approximately 55% (P < 0.01). Plasma HMGB1 is significantly increased within 1 h of trauma in humans with marked elevations occurring from 2 to 6 h postinjury. These results suggest that, in contrast to sepsis, HMGB1 release is an early event after traumatic injury in humans. Thus, HMGB1 may be integral to the early inflammatory response to trauma and is a potential target for future therapeutics.
[Show abstract][Hide abstract] ABSTRACT: The clinical sequel of ischemia and reperfusion remains a challenge in several clinical areas. Overexpression of heme oxygenase-1 (HO-1), using viral vectors, endotoxemia, and hypoxia, provides protection against ischemia and reperfusion injury. To date, however, no clinically viable therapy exists to safely induce HO-1. We have recently observed that administration of a hemoglobin-based oxygen carrier (HBOC) attenuates postinjury systemic inflammation. We have further demonstrated that an HBOC can induce HO-1 in vitro. We now explore the tissue-specific induction of heme oxygenase-1 after administration of an HBOC.
Rats were infused with doses of HBOC or saline through femoral vein injection (n=5 per group). Animals were sacrificed and organs were flushed. Heart, lung, and brain samples were taken for evaluation of total organ levels of HO-1 induction and for histologic localization of the cellular expression of the HO-1. Heat shock protein 72 levels were also analyzed to determine whether HO-1 induction was a generalized stress response.
Both the heart and lung demonstrated a dose-dependent induction of total organ HO-1. Interestingly, brain tissue did not have any significant amount of HO-1, either at baseline or after HBOC therapy. The cellular localization of HO-1 between organs was also specific, predominantly occurring in the cardiac myocyte and alveolar macrophages. Heat shock protein 72 levels were not significantly changed in any group examined, suggesting the induction of HO-1 is specific.
This study demonstrates that a clinically accessible product, HBOC, can specifically and selectively induce the expression of the protective enzyme HO-1 in vivo. These findings begin to characterize which organ systems may benefit by preischemic treatments with HBOC and further expand potential clinical applications of HBOCs.
Journal of the American College of Surgeons 05/2009; 208(4):592-8. · 4.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion death. We hypothesize that TRALI requires 2 events: (1) the clinical condition of the patient and (2) the infusion of antibodies against MHC class I antigens or the plasma from stored blood. A 2-event rat model was developed with saline (NS) or endotoxin (LPS) as the first event and the infusion of plasma from packed red blood cells (PRBCs) or antibodies (OX18 and OX27) against MHC class I antigens as the second event. ALI was determined by Evans blue dye leak from the plasma to the bronchoalveolar lavage fluid (BALF), protein and CINC-1 concentrations in the BALF, and the lung histology. NS-treated rats did not evidence ALI with any second events, and LPS did not cause ALI. LPS-treated animals demonstrated ALI in response to plasma from stored PRBCs, both prestorage leukoreduced and unmodified, and to OX18 and OX27, all in a concentration-dependent fashion. ALI was neutrophil (PMN) dependent, and OX18/OX27 localized to the PMN surface in vivo and primed the oxidase of rat PMNs. We conclude that TRALI is the result of 2 events with the second events consisting of the plasma from stored blood and antibodies that prime PMNs.
[Show abstract][Hide abstract] ABSTRACT: High-mobility group box 1 (HMGB1) is a late mediator of the systemic inflammation associated with sepsis. Recently, HMGB1 has been shown in animals to be a mediator of hemorrhage-induced organ dysfunction. However, the time course of plasma HMGB1 elevations following trauma in humans remains to be elucidated. Consequently, we hypothesized that mechanical trauma in humans would result in early, significant elevations of plasma HMGB1. METHODS:: Trauma patients at risk for MOF (ISS >/= 15) were identified for inclusion (n=23) and post-injury plasma samples were assayed for HMGB1 by ELISA. Comparison of post-injury HMGB1 levels with markers for patient outcome (age, injury severity score, units of red blood cell (RBC) transfused/1ST 24 hours, and base deficit) was performed. To investigate whether post-injury transfusion contributes to elevations of circulating HMGB1, levels were determined in both leukoreduced (LR) and non-leukoreduced (NLR) packed RBCs. RESULTS:: Plasma HMGB1 was elevated >30 fold above healthy controls within one hour of injury (median 57.76ng/ml vs 1.77ng/ml, p<0.003), peaked from 2-6 hours post-injury (median 526.18ng/ml, p<0.01 vs control), and remained elevated above control through 136 hours. No clear relationship was evident between post-injury HMGB1 levels and markers for patient outcome. HMGB1 levels increase with duration of RBC storage, though concentrations did not account for post-injury plasma levels. LR attenuated HMGB1 levels in packed RBCs by approximately 55% (p<0.01). CONCLUSION:: Plasma HMGB1 is significantly increased within one hour of trauma in humans with marked elevations occurring from 2-6 hours post-injury. These results suggest that, in contrast to sepsis, HMGB1 release is an early event following traumatic injury in humans. Thus, HMGB1 may be integral to the early inflammatory response to trauma and is a potential target for future therapeutics.
[Show abstract][Hide abstract] ABSTRACT: The inflammatory response after an insult may provoke further tissue damage, and the macrophage is central in this pathophysiology. Induction of heme oxygenase-1 (HO-1) attenuates postshock organ dysfunction, although the mechanism remains unclear. We hypothesized that HO-1 induction modifies the cytokine profile of LPS-stimulated macrophages. Heme oxygenase-1 was induced in murine and human macrophages with varying concentrations of a hemoglobin-based oxygen carrier (HBOC). Heme oxygenase-1 expression was analyzed by Western blotting of whole cell lysates. Macrophages were pretreated with HBOC for 4 h, then media with LPS were added for up to 24 h. The specific HO-1 inhibitor zinc protoporphyrin (ZnPP) was used to inhibit the effects of HO-1. Supernatants were analyzed for IL-6, IL-10, TNF-alpha, and monocyte chemotactic protein 1 (MCP-1) by enzyme-linked immunosorbent assay. Incubation of cells with HBOC produced a dose-dependent expression of HO-1. Heme oxygenase-1 expression decreased LPS-stimulated secretion of MCP-1, IL-6, IL-10, and TNF-alpha at both 4 and 24 h in murine and human macrophages. The addition of ZnPP to inhibit HO-1 partially restored MCP-1 and IL-6 secretion in murine macrophages. Furthermore, immunofluorescent microscopy revealed HBOC-induced HO-1 inhibited LPS-stimulated nuclear translocation of the p65 subunit of nuclear factor-kappaB. In summary, HBOC incubation of macrophages induced HO-1 expression, which reduced LPS-mediated cytokine release, and that MCP-1 and IL-6 secretion could be partially restored with ZnPP. These data encourage continued investigation into the role of HO-1 in protecting against posttraumatic organ dysfunction and the clinical potential of HBOC for HO-1 induction.
[Show abstract][Hide abstract] ABSTRACT: The earliest response of esophageal mucosa to gastric reflux is the development of oxidative damage and inflammation. These processes contribute to the development of metaplasia known as Barrett's esophagus, as well as the progression to malignancy. Secretory phospholipase A(2) is a mediator of inflammation with levels that are increased in Barrett's metaplasia and carcinoma when compared with levels in normal samples. Our goal is to determine the role of secretory phospholipase A(2) in the development of reflux-associated changes in the esophageal mucosa.
Secretory phospholipase A(2)-deficient mice (C57BL/6, n = 5) and mice known to express high levels of secretory phospholipase A(2) (BALB/c, n = 5) underwent side-to-side surgical anastomosis of the first portion of the duodenum and gastroesophageal junction, allowing exposure of esophageal mucosa to duodenal and gastric contents duodeno-gastroesophageal anastomosis. Control animals (n = 5) of each strain underwent laparotomy with esophagotomy and repair. Tissue was frozen in embedding medium. Hematoxylin and eosin staining and Ki67 and secretory phospholipase A(2) immunohistochemistry were used to evaluate esophageal tissue and its response to duodeno-gastroesophageal anastomosis.
Immunofluorescent staining confirmed the absence of secretory phospholipase A(2) in C57BL/6 mice and its presence in BALB/c mice. Hematoxylin and eosin staining demonstrated significant thickening of the esophageal mucosa in response to gastroesophageal reflux in the presence of secretory phospholipase A(2). Mice known to express high levels of secretory phospholipase A(2) also demonstrated increased numbers of proliferating cells. Secretory phospholipase A(2)-deficient mice were immune to the early changes induced by mixed reflux.
The presence of secretory phospholipase A(2) appears necessary for early histologic changes produced by exposure of the esophagus to gastroduodenal contents. This enzyme is identified as a promising target for evaluation of mechanisms of carcinogenesis and chemoprevention of esophageal carcinoma.
The Journal of thoracic and cardiovascular surgery 07/2008; 135(6):1220-7. · 3.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mesenteric lymph is the mechanistic link between splanchnic hypoperfusion and acute lung injury (ALI), but the culprit mediator(s) remains elusive. Previous work has shown that administration of a phospholipase A(2) (PLA(2)) inhibitor attenuated postshock ALI and also identified a non-ionic lipid within the postshock mesenteric lymph (PSML) responsible for polymorphonuclear neutrophil (PMN) priming. Consequently, we hypothesized that gut-derived leukotriene B(4) (LTB(4)) is a key mediator in the pathogenesis of ALI. Trauma/hemorrhagic shock (T/HS) was induced in male Sprague-Dawley rats and the mesenteric duct cannulated for lymph collection/diversion. PSML, arachidonic acid (AA), and a LTB(4) receptor antagonist were added to PMNs in vitro. LC/MS/MS was employed to identify bioactive lipids in PSML and the lungs. T/HS increased AA in PSML and increased LTB(4) and PMNs in the lung. Lymph diversion decreased lung LTB(4) by 75% and PMNs by 40%. PSML stimulated PMN priming (11.56 +/- 1.25 vs. 3.95 +/- 0.29 nmol O(2)(-)/min; 3.75 x 10(5) cells/ml; P < 0.01) that was attenuated by LTB(4) receptor blockade (2.64 +/- 0.58; P < 0.01). AA stimulated PMNs to produce LTB(4), and AA-induced PMN priming was attenuated by LTB(4) receptor antagonism. Collectively, these data indicate that splanchnic ischemia/reperfusion activates gut PLA(2)-mediated release of AA into the lymph where it is delivered to the lungs, provoking LTB(4) production and subsequent PMN-mediated lung injury.
Journal of Applied Physiology 05/2008; 104(4):1161-6. · 3.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gelsolin is a plasma protein that functions to depolymerize actin filaments preventing capillary plug formation following tissue injury. It also functions to mediate the inflammatory response by binding proinflammatory lipids such as lysophosphatidic acid, sphingosine-1-phosphate and phosphoinositides. Clinically, reduced gelsolin concentrations have been associated with increased mortality in critically ill, trauma, and burn patients. We have previously shown that following hemorrhagic shock with splanchnic hypoperfusion, mesenteric lymph contains lipid components that cause neutrophil and EC activation and that protein concentrations are severely diluted due to resuscitation. We hypothesized that lipid binding proteins such as gelsolin may be depleted after trauma/hemorrhagic shock leading to increased lipid bioactivity.
Shock was induced in SD rats by controlled hemorrhage and the mesenteric duct cannulated for lymph collection. Resuscitation was performed by infusing 2x SB volume in NS over 30 min, followed by 1/2 SB volume over 30 min, then 2x SB volume in NS over 60 min. Pre and post-shock lymph was loaded at equal protein concentrations on 2D-gels, followed by trypsin digestion and identification with mass spectrometry (MS-MS). Proteomics data were confirmed with Western blotting then quantitated by densitometry. Analysis of variance was used evaluate statistical data.
Gelsolin decreased in mesenteric lymph following hemorrhagic shock.
Gelsolin is found at high levels (comparable to plasma) in mesenteric lymph. Following hemorrhagic shock, gelsolin levels decrease significantly, possibly due to consumption by the actin scavenging system. The magnitude of this change in concentration could release lipid bioactivity and predispose the lung and other organs to capillary injury.
Journal of Surgical Research 12/2007; 143(1):130-5. · 2.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Posthemorrhagic shock mesenteric lymph (PSML) has been shown to activate pulmonary endothelial cells and cause lung injury. Although multiple mediators may be involved, most of these effects are mediated by nuclear factor-kappa B (NF-kappaB) activation. Degradation of the inhibitor of kappa B (IkappaB) is a key regulatory step in the activation of NF-kappaB. We therefore hypothesized that PSML would cause IkappaB degradation with subsequent NF-kappaB phosphorylation and nuclear translocation.
Mesenteric lymph was collected from male rats before shock and each hour after shock for up to 3 h (n = 5). Buffer (control), buffer + 10% (v/v) lymph, or buffer + tumor necrosis factor (10 ng/mL) were incubated with human pulmonary endothelial cells for 30 min and then lysed. Immunoblots of lysates were probed for IkappaB and phospho-p65. Immunohistochemistry was performed on cells grown on glass slides and then treated as above with the third PSML sample. Cells were fixed and then probed for p65. Statistical analysis was performed with Student's t-test and analysis of variance with significance was set at P < 0.05.
Western blots of cell lysates for IkappaB demonstrated a steady decrease in total IkappaB with each lymph sample. Phosphorylation of NF-kappaB , p65 component, steadily increased with each PSML sample, with a maximum reached during the third PSML sample, which also significantly increased translocation of NF-kappaB to the nucleus.
Postshock mesenteric lymph bioactivity is mediated by pathways which involved IkappaB degradation. These pathways offer novel off targets for clinical intervention to prevent the distal organ injury caused by postinjury hemorrhagic shock.
Journal of Surgical Research 11/2007; 143(1):136-40. · 2.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Kupffer cells (liver macrophages) are a key initiator of inflammation following hepatic insults such as infection, ischemia/reperfusion, and rejection. Heme oxygenase 1 (HO-1) is protective against inflammatory injury. A hemoglobin-based oxygen carrier (HBOC) has been shown to prevent organ inflammation from hemorrhagic shock as well as induce HO-1 at the cellular level. Therefore, we hypothesize that HBOC can induce Kupffer cell HO-1 production.
Mice administered 20% blood volume HBOC or saline intravenously were sacrificed at 0, 12, 24, 48 hours (n = 4-6/group). Hepatic protein underwent Western blotting for HO-1 and heat shock protein 72. Hepatic frozen sections underwent immunofluorescent staining for HO-1/CD68.
Following HBOC injection, hepatic HO-1 fold change peaked at 12 hours (7.3 +/- 0.8) (p < .01), remained increased at 24 hours (4.7 +/- 0.4) (p < .01), and returned to baseline by 48 hours. HSP72 expression was unaffected in all groups. Twleve-hour liver section immunostaining confirmed significant induction of HO-1 by HBOC. Double staining for HO-1 and CD68 identified Kupffer cells as the majority of cells expressing HO-1.
HBOC induces hepatic HO-1 expression in Kupffer cells without heat shock protein response. These data provide the basis for further investigation into a clinical therapy to induce Kupffer cell HO-1 expression with the goal of attenuating the hepatic immunoresponse to various insults.
Surgery 08/2007; 142(2):289-94. · 3.37 Impact Factor