Ricardo J. Gonzalez

Plasma from aged stored red blood cells delays neutrophil apoptosis and primes for cytotoxicity: abrogation by poststorage washing but not prestorage leukoreduction.

BACKGROUND Blood transfusion-particularly that of older stored red blood cells (RBCs)--is an independent risk factor for postinjury multiple organ failure. Immunomodulatory effects of RBC transfusion include neutrophil (PMN) priming for cytotoxicity, an effect exacerbated by longer RBC storage times. We have found that delayed PMN apoptosis in trauma patients is provoked by transfusion, independent of injury severity. We hypothesized that aged stored RBCs delay PMN apoptosis, but that prestorage leukodepletion or poststorage washing could abrogate the effect. METHODS Healthy volunteers each donated 1 unit of blood. One half was leukodepleted, and RBC units were processed in the usual fashion and stored at 4 degrees C. Aliquots were removed on days 1, 14, 21, and 42 and the plasma fraction isolated. Selected aliquots were washed with normal saline before plasma isolation. PMNs harvested from healthy controls were incubated (5% CO2, 37 degrees C) with unmodified, leukoreduced, or washed RBC plasma (20% plasma/80% RPMI 1640), and apoptosis assessed by morphology after 24 hours. Apoptotic index (apoptotic PMNs/total PMNs) was compared. PMN priming for superoxide release was also assessed after plasma exposure. RESULTS PMN apoptosis was delayed by RBCs stored for 21 or 42 days. Prestorage leukodepletion did not alter the effect. However, washing 42-day-old RBCs abrogated the effect. PMN priming for superoxide was provoked by stored packed RBCs in an identical pattern to delayed apoptosis. CONCLUSION Plasma from stored RBCs-even if leukoreduced-delays apoptosis and primes PMNs. The effect becomes evident at 21 days and worsens through product outdate (42 days), but may be prevented by poststorage washing. Inflammatory agents contaminating stored blood likely mediate the effect. Modification of transfusion practices (e.g., giving fresher or washed RBCs or blood substitutes) may attenuate adverse immunomodulatory effects of transfusion in trauma patients.

Transfusion of the injured patient: Proceed with caution

Transfusion of the injured patient with packed red blood cells (PRBCs) is a dynamic process requiring vigilance during the acute resuscitative and recovery phases postinjury. Although adverse events have been reported in 2% to 10% of injured patients, the advent of new detection techniques for viral pathogens has markedly decreased the risk of infectious transmission. However, transfusions are strongly associated with immunosuppression in the host, which may occur days after the initial injury and may lead to bacterial infections. Conversely, early transfusion of stored PRBCs, >6 units in the first 12 h postinjury, contributes to an early state of hyperinflammation that is a strong, independent predictor of multiple organ failure (MOF) in those patients with intermediate injury severity scores. The roles of prestorage leukoreduction are also reviewed with respect to the promotion of both immunosuppression and hyperinflammation. We further summarize studies with hemoglobin substitutes, whose use may obviate many of the untoward events of transfusion and promise to lead to better outcomes for injured patients.

Influence of Obesity on Cancer-Related Outcomes After Pancreatectomy to Treat Pancreatic Adenocarcinoma

OBJECTIVE To examine the influence of obesity, as measured by body mass index (BMI) (calculated as weight in kilograms divided by height in meters squared), on clinicopathologic factors and survival after pancreatectomy to treat adenocarcinoma. DESIGN Retrospective review and statistical analysis using prospectively collected data. SETTING Referral center with a dedicated multidisciplinary pancreas cancer program. PATIENTS Two hundred eighty-five consecutive patients with data available for BMI calculation who underwent potentially curative pancreas resection to treat adenocarcinoma from January 1, 1999, to October 31, 2006. MAIN OUTCOME MEASURE Influence of BMI and other known prognostic variables on the incidence of lymph node metastasis and disease-free and overall survival. RESULTS We identified a subset of obese patients (BMI >35) who were at 12-fold risk of lymph node metastasis compared with nonobese patients (BMI < or =35). The estimated disease-free and overall survival rates were decreased in the obese patients, and the risk of cancer recurrence and death after pancreatectomy was nearly twice that in nonobese patients. CONCLUSIONS Obese patients with a BMI of more than 35 are more likely to have node-positive pancreatic cancer and decreased survival after surgical resection. Data suggest that the negative influence of BMI of more than 35 on cancer-related end points is unrelated to the potential complexity of performing major oncologic surgery in obese patients.

Post-hemorrhagic shock mesenteric lymph activates human pulmonary microvascular endothelium for in vitro neutrophil-mediated injury: the role of intercellular adhesion molecule-1.

BACKGROUND Splanchnic hypoperfusion is believed to be central in the pathogenesis of hemorrhagic shock-induced acute respiratory distress syndrome and multiple organ failure. Our previous work focused on the portal circulation as the conduit for gut-derived mediators of acute respiratory distress syndrome. Our current focus is the proinflammatory effects of postshock mesenteric lymph. We hypothesize that postshock lymph induces neutrophil (PMN)-mediated endothelial cell damage in an intercellular adhesion molecule-1 (ICAM-1)-dependent fashion, and devised a two-insult model to test this hypothesis. METHODS Rats (n > or = 5) underwent hemorrhagic shock (mean arterial pressure, 40 mm Hg for 30 minutes) and resuscitation (shed blood plus two times crystalloid) with lymph collection. Human pulmonary microvascular endothelial cells (HMVECs) were divided into three groups and grown to near confluence. Group 1 was incubated for 6 hours in 1% preshock or postshock lymph and ICAM-1 was measured by flow cytometry. Group 2 consisted of coculture of HMVECs and PMNs after endothelial cell activation to determine whether postshock lymph would stimulate PMN adherence. Group 3 was incubated under identical conditions, but PMNs were added for 30 minutes, and then activated with 4.5 micromol/L lysophosphatidylcholine (lyso-PC) for 1 hour to ascertain cytotoxicity. HMVEC density was measured using microscopy and recorded as HMVECs per millimeter squared. ICAM-1-blocking antibody and isotype control were used to assess the effects of ICAM-1 on PMN cytotoxicity. A buffer control was used for comparison using analysis of variance with Tukeys correction. RESULTS Postshock lymph activated HMVECs for increased surface expression of ICAM-1 and stimulated PMNs to adhere to endothelial cell monolayers. Activation of PMNs with lyso-PC in the presence of postshock lymph resulted in marked HMVEC death. The addition of an ICAM-1-blocking antibody abrogated this effect. Neither postshock lymph alone (758 +/- 35 HMVECs/mm(2)), nor postshock lymph in the presence of quiescent PMNs alone (734 +/- 28 HMVECs/mm(2)), nor lymph plus lyso-PC (834 +/- 21 HMVECs/mm(2)) provoked endothelial cell damage. CONCLUSION Postshock mesenteric lymph activates endothelial cells for increased ICAM-1 expression and PMN adherence. Furthermore, postshock lymph acts as an inciting event in a two-event in vitro model of PMN-mediated endothelial cell injury. These findings further substantiate the key mechanistic role of mesenteric lymph in hemorrhagic shock-induced acute lung injury and suggest that ICAM-1 expression is pivotal in the two-event model of multiple organ failure.

Hypertonic saline inhibits neutrophil (PMN) priming via attenuation of p38 MAPK signaling.

Priming of the neutrophil cytotoxic response is central to the pathogenesis of early postinjury multiple organ failure (MOF). Platelet-activating factor (PAF) has been implicated as a key inflammatory mediator in postinjury neutrophil priming and requires p38 MAPK signaling to produce its biologic effects. Hypertonic saline (HTS) resuscitation decreases the postinjury inflammatory response following shock in animals and decreases receptor-mediated neutrophil (PMN) cytotoxic functions in vitro. We hypothesized that HTS attenuates PAF priming of the PMN cytotoxic response by interfering with PAF-mediated p38 MAPK signal transduction. Isolated PMNs were preincubated in isotonic buffer or HTS (Na+ = 180 mM), then primed with PAF. Neutrophil CD11b/CD18 expression was measured by flow cytometry. Receptor-dependent (fMLP), N-formyl-methionyl-leucyl-phenylalanine, fMLP) and receptor-independent (PMA) O2- production was measured by reduction of cytochrome c in resting and PAF primed PMNs. Total p38 MAPK protein PAF-mediated p38 MAPK activation was assessed by western blot of PMN lysates. Clinically relevant levels of HTS attenuated PAF-mediated beta2-integrin expression. While HTS attenuated receptor-dependent (fMLP and PAF/fMLP) O2- production, receptor-independent (PMA) O2- production was unaffected. Conversely, HTS attenuated PAF priming of PMA-mediated O2- production. PAF and HTS did not alter total cellular p38 MAPK content. Clinically relevant levels of HTS alone did not activate p38 MAPK but inhibited PAF mediated p38 MAPK activation. HTS attenuates PAF priming of the PMN cytotoxic response by altering intracellular signal transduction. Therefore, HTS resuscitation may attenuate postinjury PMN priming and ultimately the risk of developing MOF.

The lipid fraction of post-hemorrhagic shock mesenteric lymph (PHSML) inhibits neutrophil apoptosis and enhances cytotoxic potential.

Dysfunctional neutrophil (PMN) apoptosis facilitates hyperinflammatory tissue injury. Previous work has demonstrated that post-hemorrhagic shock mesenteric lymph (PHSML) provokes PMN-mediated acute lung injury in animal models, but the mechanism remains unclear. We have documented that the lipid fraction of PHSML is responsible for PMN priming of the respiratory burst. In this study, we hypothesized that PHSML lipids delay PMN apoptosis and thereby further enhance PMN cytotoxic potential. Mesenteric lymph was collected from rats (n = 5) before (control), during non-lethal hemorrhagic shock (MAP 40 mmHg, 30 min), and during resuscitation (shed blood + 2x crystalloid). Human PMNs were incubated with control, PHSML, PHSML lipid extracts, and heat-treated PHSML (60 degrees C, 30 min.) at 1-10% (v:v) in RPMI 1640 for 24 h. Apoptosis was assessed using acridine orange/ethidium bromide staining and fluorescence microscopy. Priming of the respiratory burst was evaluated by incubating PMNs with (a) control PHSML or (b) PHSML lipid extracts for 24 h and by activating with fMLP (1 micromol/L). PHSML and PHSML lipid extracts (5-10%) inhibited PMN apoptosis. Heat denaturing the PHSML (to eliminate cytokines and complement) had no effect on the inhibition of PMN apoptosis. Similarly, incubation with polymixin B at a concentration that binds endotoxin had no effect. Both the PHSML and PHSML lipids (5%) following 24-h incubation primed the fMLP-activated oxidase. At physiologic concentrations, both PHSML and the lipid fraction of PHSML delay PMN apoptosis and prime the NADPH oxidase. These data further implicate the lipid components of mesenteric lymph as central in the pathogenesis of hemorrhagic shock induced PMN-mediated acute lung injury.

Mesenteric lymph is responsible for post-hemorrhagic shock systemic neutrophil priming

BACKGROUND Hemorrhagic shock-induced splanchnic hypoperfusion has been implicated as a priming event in the two event model of multiple organ failure (MOF). We have previously shown that early postinjury neutrophil (PMN) priming identifies the injured patient at risk for MOF. Recent in vitro studies have demonstrated that postshock mesenteric lymph primes isolated human neutrophils. We hypothesize that lymphatic diversion before hemorrhagic shock abrogates systemic PMN priming and subsequent lung injury. METHODS Sprague-Dawley rats (n >or= 5 per group) underwent hemorrhagic shock (MAP 40 mm Hg x 30 min) and resuscitation (shed blood + 2x crystalloid) with and without mesenteric lymphatic duct diversion. Sham animals underwent anesthesia and laparotomy. Whole blood was taken 2 hours after resuscitation, heparinized, and incubated for 5 min at 37 degrees C. Surface expression of CD11b (a marker for PMN priming) was determined by flow-cytometry compared with isotype controls. In addition, lung myeloperoxidase (MPO) was measured for PMN sequestration, and Evans blue lung leak was assessed in the bronchoalveolar lavage fluid in sham, and shock +/- lymph diversion animals. RESULTS Hemorrhagic shock resulted in increased surface expression of PMN CD11b relative to sham (23.8 +/- 6.7 vs. 9.9 +/- 0.6). Mesenteric lymphatic diversion before hemorrhagic shock abrogated this effect (8.0 +/- 2.6). Lung PMN accumulation, as assessed by MPO, was greater in the lungs of nondiverted (113 +/- 14 MPO/mg lung) versus sham (55 +/- 4 MPO/mg lung, p < 0.05); lymph diversion reduced lung PMNs to control levels (71 +/- 6.5 MPO/mg lung, p < 0.05). Evans blue lung leak was 1.6 times sham in the hemorrhagic shock group; this was returned to sham levels after lymph diversion (p < 0.05). CONCLUSION Post-hemorrhagic shock mesenteric lymph primes circulating PMNs, promotes lung PMN accumulation, and provokes acute lung injury. Lymphatic diversion abrogates these pathologic events. These observations further implicate the central role of mesenteric lymph in hemorrhagic shock-induced lung injury. Characterizing the PMN priming agents could provide insight into the pathogenesis of postinjury MOF and ultimately new therapeutic strategies.

Hypertonic saline activation of p38 MAPK primes the PMN respiratory burst.

Investigation of hypertonic saline (HTS) modulation of neutrophils (PMN) cytotoxic responses has generated seemingly contradictory results. Clinically relevant levels of HTS attenuate receptor-mediated p38 MAPK signaling, whereas higher levels activate p38 MAPK. Concurrently, HTS exerts a dose-dependent attenuation of the PMN respiratory burst, most notably at concentrations where p38 MAPK is activated. We hypothesized that HTS-mediated p38 MAPK activation augments the PMN respiratory burst on return to normotonicity. We found that although clinically relevant levels of HTS (Na+ > or = 200 mM) did not activate p38 MAPK, higher concentrations (Na+ > or = 300 mM) resulted in activation comparable with that after PAF stimulation. Transient stimulation with high levels of HTS primed the PMN respiratory burst in response to fMLP and PMA. This effect was attenuated by pretreatment with SB 203580, a p38 MAPK specific inhibitor. We conclude that severe osmotic shock primes the respiratory burst via p38 MAPK signaling, further supporting the role of this signaling cascade in PMN priming.

Hyperosmolarity abrogates neutrophil cytotoxicity provoked by post-shock mesenteric lymph.

Hypertonic saline (HTS) resuscitation inhibits acute lung injury in animal models of shock, but some argue this may simply represent more efficient fluid resuscitation. Inflammatory mediators within mesenteric lymph have been identified as a link between splanchnic hypoperfusion and acute respiratory distress syndrome (ARDS). We hypothesize that HTS resuscitation abrogates post-shock lymph-mediated neutrophil (PMN) priming and PMN-mediated human endothelial cell cytotoxicity. Mesenteric lymph was collected from rats (n = 5) before (control), during non-lethal hemorrhagic shock, defined as a mean arterial pressure (MAP) of 40 mmHg for 30 min, and after resuscitation (shed blood + 2 × lactated Ringers (LR) versus 7.5% NaCl, 4 cc/kg, over 5 min). Isolated human PMNs were primed with physiologic concentrations (5% v:v) of lymph either from animals resuscitated with LR or HTS and activated with either PMA or fMLP. In a separate set of experiments, human PMNs were primed with LR lymph after incubation with HTS (180 mM NaCl). The maximal rate of superoxide production was measured by reduction of cytochrome C. In addition, the effect of HTS pretreatment on PMN adherence to human pulmonary microvascular endothelial cells (HMVEC) and PMN-mediated cytotoxicity was determined after lymph-mediated PMN priming. PHSML primed isolated PMNs above buffer controls and pre-shock lymph in a normotonic environment; HTS resuscitation abrogated this effect. HTS preincubation of isolated PMNs inhibited PHSML-induced PMN priming, adherence to HMVECs, and PMN-mediated HMVEC cytotoxicity. Hypertonic resuscitation (HTS) abrogates PHSML priming of the PMN and PMN-mediated HMVEC cytotoxicity. Furthermore, incubation of PMNs in clinically relevant HTS (180 mM NaCl) prevents PHSML PMN priming and PMN:HMVEC interactions. These studies suggest inhibition of PMN signal transduction is a mechanism whereby HTS resuscitation abrogates acute lung injury.

Post-hemorrhagic shock mesenteric lymph lipids prime neutrophils for enhanced cytotoxicity via phospholipase A2

Hemorrhagic shock induced mesenteric hypoperfusion has long been implicated as a key event in the pathogenesis of the adult respiratory distress syndrome (ARDS) and multiple organ failure (MOF). Previous work links post-hemorrhagic shock mesenteric lymph (PHSML) lipids and neutrophil (PMN) priming in the pathogenesis of ARDS. We hypothesize that gut phospholipase A2 (PLA2) liberates proinflammatory lipids following hemorrhagic shock, which are responsible for enhanced PMN cytotoxicity. Mesenteric lymph was collected from rats (n > or = 5) before hemorrhagic shock, during hemorrhagic shock (MAP 40 mm Hg x 30 min), and after resuscitation (shed blood + 2x lactated Ringers). PMNs were incubated with physiologic concentrations (1-5%, v:v) of (a) buffer control, (b) sham (c) pre-shock lymph, (c) PHSML, (d) PHSML lipid extracts, (e) heat-denatured PSHML, and (f) PHSML harvested after i.v. pretreatment with a known PLA2 inhibitor (quinacrine, 10 mg/kg). PMNs were activated with fMLP (1 micromol), and the maximal rate of superoxide production measured by reduction of cytochrome c. Gut morphology was assessed histologically using hematoxalin and eosin (HE) staining. PHSML and PHSML lipid extracts (5%, v:v) primed for enhanced superoxide production compared to buffer controls (2.5-fold and 3.6-fold), sham (2.5-fold) and pre-shock lymph (2.0-fold). Lymph collected after systemic PLA2 inhibition, in contrast, abrogated the PMN priming response. Gut mucosal morphology, at end-resuscitation, was intact on HE staining both with and without PLA2 inhibition. Heat denaturing the PHSML (eliminating cytokines and complement), on the other hand, did not reduce PMN priming. Physiologic concentrations of PHSML lipids prime the PMN respiratory burst. Lymph priming is diminished with systemic PLA2 inhibition, implicating gut PLA2 as a source of proinflammatory lipids that may be central in the pathogenesis of hemorrhagic shock induced ARDS/MOF.