The relationship between body mass index and mortality is not linear in patients requiring venovenous extracorporeal support.

OBJECTIVE: Morbid obesity may influence candidacy for venovenous extracorporeal membrane oxygenation (VVECMO) support. Indeed, body mass index (BMI) >40 is considered to be a relative contraindication due to increased mortality observed in patients with BMI above this value. There is scant evidence to characterize this relationship beyond speculating about the technical challenges of cannulation and difficulty in optimizing flows. We examined a national cohort to evaluate the influence of BMI on mortality in patients requiring VVECMO for severe acute respiratory syndrome coronavirus 2 infection. METHODS: We performed a retrospective cohort analysis on National COVID Cohort Collaborative data evaluating 1,033,229 patients with BMI ≤60 from 31 US hospital systems diagnosed with severe acute respiratory syndrome virus coronavirus 2 infection from September 2019 to August 2022. We performed univariate and multivariable mixed-effects logistic regression analysis on data pertaining to those who required VVECMO support during their hospitalization. A subgroup risk-adjusted analysis comparing ECMO mortality in patients with BMI 40 to 60 with the 25th, 50th, and 75th BMI percentile was performed. Outcomes of interest included BMI, age, comorbidity score, body surface area, and ventilation days. RESULTS: A total of 774 adult patients required VVECMO. Of these, 542 were men, median age was 47 years, mean adjusted Charlson Comorbidity Index was 1, and median BMI was 33. Overall mortality was 47.8%. There was a nonsignificant overall difference in mortality across hospitals (SD, 0.31; 95% CI, 0-0.57). After mixed multivariable logistic regression analysis, advanced age (P < .0001) and Charlson Comorbidity Index (P = .009) were each associated with increased mortality. Neither gender (P = .14) nor duration on mechanical ventilation (P = .39) was associated with increased mortality. An increase in BMI from 25th to 75th percentile was not associated with a difference in mortality (P = .28). In our multivariable mixed-effects logistic regression analysis, there exists a nonlinear relationship between BMI and mortality. Between BMI of 25 and 32, patients experienced an increase in mortality. However, between BMI of 32 and 37, the adjusted mortality in these patients subsequently decreased. Our subgroup analysis comparing BMIs 40 to 60 with the 25th, 50th, and 75th percentile of BMI found no significant difference in ECMO mortality between BMI values of 40 and 60 with the 25th, 50th, 75th percentile. CONCLUSIONS: Advancing age and higher CCI are each associated with increased risk for mortality in patients requiring VVECMO. A nonlinear relationship exists between mortality and BMI and those between 32 and 37 have lower odds of mortality than those between BMI 25 and 32. This nonlinear pattern suggests a need for further adjudication of the contraindications associated with VVECMO, particularly those based solely on BMI.

Cannulate, extubate, ambulate approach for extracorporeal membrane oxygenation for COVID-19.

OBJECTIVE: We compared outcomes in patients with severe COVID-19 versus non-COVID-19-related acute respiratory distress syndrome (ARDS) managed using a dynamic, goal-driven approach to venovenous extracorporeal membrane oxygenation (ECMO). METHODS: We performed a retrospective, single-center analysis of our institutional ECMO registry using data from 2017 to 2021. We used Kaplan-Meier plots, Cox proportional hazard models, and propensity score analyses to evaluate the association of COVID-19 status (COVID-19-related ARDS vs non-COVID-19 ARDS) and survival to decannulation, discharge, tracheostomy, and extubation. We also conducted subgroup analyses to compare outcomes with the use of extracorporeal cytoreductive techniques (CytoSorb [CytoSorbents Corp] and plasmapheresis). RESULTS: The sample comprised 128 patients, 50 with COVID-19 and 78 with non-COVID-19 ARDS. Advancing age was associated with decreased probability of survival to decannulation (P = .04). Compared with the non-COVID-19 ARDS group, patients with COVID-19 had a greater probability of survival to extubation (P < .01) and comparable survival to discharge (P = .14). CONCLUSIONS: Patients with COVID-19 managed with ECMO had comparable outcomes as patients with non-COVID ARDS. A strategy of early extubation and ambulation might be a safe and effective strategy to improve outcomes and survival, even for patients with severe COVID-19.

Bivalirudin Versus Unfractionated Heparin in Patients With Cardiogenic Shock Requiring Venoarterial Extracorporeal Membrane Oxygenation.

This study evaluated differences in efficacy and safety outcomes with bivalirudin compared with unfractionated heparin (UFH) in patients with cardiogenic shock requiring venoarterial extracorporeal membrane oxygenation (VA ECMO). We performed a retrospective study at an academic medical center that included patients greater than 18 years of age supported with VA ECMO due to cardiogenic shock from January 2009 to February 2021. The primary endpoint was ECMO-associated thrombotic events normalized to duration of ECMO support. Secondary safety endpoints included major bleeding (per ELSO criteria) and blood product administration. Overall, 143 patients were included in our analysis with 54 having received bivalirudin and 89 having received UFH. Median duration of ECMO support was 92 (interquartile range, 56-172) hours. ECMO-associated thrombotic events per ECMO day were significantly less among those that received bivalirudin ( P < 0.001). In adjusted regression, bivalirudin was independently associated with an increased time to thrombosis when compared with UFH (Exp[B] -3.8; 95% confidence interval, 1.7-8.8; P = 0.002). Patients receiving bivalirudin experienced less major bleeding events ( P = 0.02) with less total red blood cell and fresh frozen plasma administration ( P = 0.04 and P = 0.03, respectively). Bivalirudin is a safe and efficacious alternative to UFH in patients requiring VA ECMO for cardiogenic shock.

Oxygenated right ventricular assist device with a percutaneous dual-lumen cannula as a bridge to lung transplantation.

Background: Oxygenated right ventricular assist device (oxyRVAD) placement has become more streamlined with the introduction of the dual-lumen pulmonary artery cannula. Peripherally cannulated oxyRVAD may provide oxygenation support with right heart support as an alternative to venoarterial extracorporeal membrane oxygenation (ECMO) as a bridge to lung transplantation. Methods: A single-institution, retrospective analysis was performed on patients placed on oxyRVAD with a dual-lumen pulmonary artery cannula with the intention of bridging to lung transplantation in 2019. Results: Four patients with idiopathic pulmonary fibrosis were placed on oxyRVAD as a bridge to transplantation. Two patients were extubated and ambulated while waiting for a lung offer, and two patients required conversion to venoarteriovenous ECMO (VAV ECMO) from oxyRVAD. The median waiting time for extracorporeal life support (ECLS) was 42 h. All patients underwent double lung transplantation. Two patients stayed on oxyRVAD, and one patient was placed on venovenous ECMO (VV ECMO) after transplantation. Primary graft dysfunction score at 72 h after transplantation was grade 1 in three patients and grade 3 in one patient. Conclusions: Peripherally cannulated oxyRVAD with percutaneous dual-lumen venous cannula could be an ambulatory bridge for lung transplantation. It is unknown whether oxyRVAD is feasible as a long-term bridge to lung transplantation.

Comparison of Anticoagulation Strategies in Patients Requiring Venovenous Extracorporeal Membrane Oxygenation: Heparin Versus Bivalirudin.

OBJECTIVES: Extracorporeal membrane oxygenation is a life-sustaining therapy for severe respiratory failure. Extracorporeal membrane oxygenation circuits require systemic anticoagulation that creates a delicate balance between circuit-related thrombosis and bleeding-related complications. Although unfractionated heparin is most widely used anticoagulant, alternative agents such as bivalirudin have been used. We sought to compare extracorporeal membrane oxygenation circuit thrombosis and bleeding-related outcomes in respiratory failure patients receiving either unfractionated heparin or bivalirudin for anticoagulation on venovenous extracorporeal membrane oxygenation support. DESIGN: Retrospective cohort study. SETTING: Single-center, cardiothoracic ICU. PATIENTS: Consecutive patients requiring venovenous extracorporeal membrane oxygenation who were maintained on anticoagulation between 2013 and 2020. INTERNVENTIONS: IV bivalirudin or IV unfractionated heparin. MEASUREMENTS AND MAIN RESULTS: Primary outcomes were the presence of extracorporeal membrane oxygenation in-circuit-related thrombotic complications and volume of blood products administered during extracorporeal membrane oxygenation duration. One hundred sixty-two patients receiving unfractionated heparin were compared with 133 patients receiving bivalirudin for anticoagulation on venovenous extracorporeal membrane oxygenation. In patients receiving bivalirudin, there was an overall decrease in the number of extracorporeal membrane oxygenation circuit thrombotic complications (p < 0.005) and a significant increase in time to circuit thrombosis (p = 0.007). Multivariable Cox regression found that heparin was associated with a significant increase in risk of clots (Exp[B] = 2.31, p = 0.001). Patients who received bivalirudin received significantly less volume of packed RBCs, fresh frozen plasma, and platelet transfusion (p < 0.001 for each). There was a significant decrease in the number major bleeding events in patients receiving bivalirudin, 40.7% versus 11.7%, p < 0.001. CONCLUSIONS: Patients receiving bivalirudin for systemic anticoagulation on venovenous extracorporeal membrane oxygenation experienced a decrease in the number of extracorporeal membrane oxygenation circuit-related thrombotic events as well as a significant decrease in volume of blood products administered.

Comparison of Hydromorphone versus Fentanyl-based Sedation in Extracorporeal Membrane Oxygenation: A Propensity-Matched Analysis.

INTRODUCTION: Data comparing sedatives in patients receiving extracorporeal membrane oxygenation (ECMO) are sparse. However, it is known that the ECMO circuit alters the pharmacokinetic properties of medications via drug sequestration of lipophilic agents and increased volume of distribution. OBJECTIVES: This study evaluated the difference in days alive without delirium or coma and the sedative requirements in patients receiving fentanyl versus hydromorphone in ECMO patients. METHODS: This single-center retrospective observational study evaluated adults receiving ECMO for more than 48 hours and continuous infusion of either fentanyl or hydromorphone for at least 6 hours. Of 148 patients evaluated, 88 received fentanyl and 60 received hydromorphone continuous infusion sedation. Outcomes included delirium-free and coma-free (DFCF) days, narcotic use, and sedative use. MAIN RESULTS: There was an increase in the number of DFCF days in the hydromorphone group at day 7 (p=0.07) and day 14 (p=0.08) and a significant reduction in daily fentanyl equivalent exposure. Propensity score matching yielded 54 matched pairs. An 11.1% increase was observed in the proportion of ECMO days alive without delirium or coma in the hydromorphone group at 7 days (53.2% vs 42.1%, p=0.006). Patients in the hydromorphone group received significantly fewer narcotics with a median of 555 µg (interquartile range [IQR] 287-905 µg) of fentanyl equivalents per day compared with 2291 µg (IQR 1053-4023 µg) in the fentanyl group (p<0.005). CONCLUSION: The use of hydromorphone-based sedation in ECMO patients resulted in more days alive without delirium or coma while significantly reducing narcotic requirements.

Challenges in the development and implementation of a healthcare system based extracorporeal cardiopulmonary resuscitation (ECPR) program for the treatment of out of hospital cardiac arrest.

INTRODUCTION: Extracorporeal cardiopulmonary resuscitation (ECPR) can treat cardiac arrest refractory to conventional therapies. Many institutions are interested in developing their own ECPR program. However, there may be challenges in logistics and implementation. AIMS: The aim of our protocol was to demonstrate that an ECPR team was feasible within our healthcare system and that the identification of UPMC Presbyterian as a receiving center allowed for successful treatment within 30 min from EMS dispatch. METHODS: We developed out of hospital cardiac arrest (OHCA) ECPR protocols for Emergency Medical Services (EMS), EMS communications, and our in-hospital ECPR team. Inclusion criteria indentified patients with a potentially reversible arrest etiology and high probability of recoverable brain injury using a simple checklist: witnessed collapse, layperson CPR, initial shockable rhythm, and age 18-60 years. We trained local EMS crews to screen patients and reviewed the criteria with a Medic Command Physician prior to transport to our hospital. RESULTS: From October 2015 to March 31st 2018, EMS treated 1165 EMS OHCA cases, transported 664 (57%) to a local hospital, and transported 120 (10%) to our institution. Of these, five (4.1%) patients underwent ECPR. Among excluded cases, 64 (53%) had nonshockable rhythms, 48 (40%) were unwitnessed arrests, 50 (42%) were over age 60 and the remaining 20 (17%) had no documented reasons for exclusion. For ECPR cases, median pre-hospital CPR duration was 26 [IQR 25-40] min. Four patients (80%) received mechanical CPR. Interval from arrest to arrival on scene was 5 [IQR 4-6] min and interval from radio call to activation of ECPR was 13 [IQR 7-21] min. Interval from EMS dispatch to departure from scene was 20 [IQR 19-21] min. Time from EMS dispatch to initiation of ECPR was 63 [IQR 59-69] min. CONCLUSIONS: ECPR is an infrequent occurrence in EMS practice. Most apparently eligible patients did not get ECPR, highlighting the need for ongoing programmatic development, provider education, and qualitative work exploring barriers to implementation.

The Use of ECMO for the Treatment of Refractory Cardiac Arrest or Postarrest Cardiogenic Shock Following In-Hospital Cardiac Arrest: A 10-Year Experience.

OBJECTIVES: Extracorporeal membrane oxygenation (ECMO) has been increasingly used in the treatment of refractory cardiac arrest (extracorporeal cardiopulmonary resuscitation [ECPR]) and postarrest cardiogenic shock (PACS). Our primary objective was to determine the 1-year survival of patients who were treated with ECMO for PACS or in ECPR. METHODS: We conducted a retrospective analysis of hospitalized patients in a tertiary care facility who underwent treatment with ECMO for ECPR or PACS. Between January 2004 and December 2013, patients were prospectively entered into an institutional registry. One-year follow-up was assessed by electronic medical record or social security death index if clinical follow-up was unavailable. RESULTS: Fifty-one patients were treated with ECMO during the study period. The mean age was 54.0 ± 10.9 years; the majority of patients were men (80.4%). The most common etiology of arrest was acute myocardial infarction (51.0%). Overall, 13 (25.4%) patients survived for at least 1 year. Preexisting coronary artery disease, hypertension, and hyperlipidemia were associated with reduced likelihood of survival. We observed a significant improvement in 1-year mortality in patients treated for PACS when compared to ECPR, 46.7% versus 16.7%, respectively. CONCLUSION: The use of ECMO for treatment of refractory cardiac arrest or cardiogenic shock may be a suitable treatment in a very select cohort of patients. Our results support a significantly higher 1-year survival in patients with PACS compared to refractory cardiac arrest.

Extracorporeal membrane oxygenation support in acute coronary syndromes complicated by cardiogenic shock.

BACKGROUND: Acute coronary syndrome (ACS) complicated by shock is associated with high mortality despite the use of percutaneous support devices. Extracorporeal membrane oxygenation (ECMO) offers cardiopulmonary support but its safety and efficacy in the ACS setting is still under investigation. METHODS: We reviewed the clinical characteristics and course of 18 consecutive patients who received femoral veno-arterial ECMO in the cardiac catheterization lab for severe shock due to ACS at our center between 2007 and 2013. RESULTS: The average age was 59.9 years, 72.2% male. Of the 18 patients, 83% had a ST-segment elevation myocardial infarction, of which 55% had a left main or left anterior descending artery occlusion. Thirteen patients received stents, three were referred for coronary artery bypass grafting alone, and two received balloon angioplasty. All patients received aspirin, a thienopyridine (either clopidogrel or ticagrelor), and heparin. Five patients received a glycoprotein IIb/IIIa inhibitor during the catheterization. The average length of ECMO was 3.2 ± 2.5 days, length of stay was 23.4 days, and 67% survived to discharge. Seventeen of eighteen patients (94%) required at least one blood transfusion and use of blood products was significantly higher in the group receiving glycoprotein IIb/IIIa inhibitors [19 U of packed red blood cells (PRBC) vs. 8.2 U (P = 0.003)]. CONCLUSIONS: In patients with severe shock or refractory ventricular arrhythmias due to ACS, VA-ECMO likely offers an alternative form of biventricular support albeit with significant resource utilization and morbidity. A better understanding of how to manage patients with ACS requiring VA-ECMO support including the associated morbidities such as bleeding is necessary.

Extracorporeal membrane oxygenation for advanced refractory shock in acute and chronic cardiomyopathy.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) has been used to obtain rapid resuscitation and stabilization in advanced refractory cardiogenic shock (CS), but clear strategies to optimize outcomes and minimize futile support have not been established. METHODS: We retrospectively reviewed our experience with ECMO in patients with advanced refractory CS, after an acute myocardial infarct (AMI) compared with patients receiving ECMO after an acute decompensating chronic cardiomyopathy (CCM). RESULTS: Between January 2003 and February 2009, 33 patients required ECMO support for advanced refractory CS secondary to AMI (AMI-CS) and 9 patients were supported by ECMO in the presence of an acutely decompensated CCM (CCM-CS). Survival at 30 days, 1 and 2 years for patients with AMI-CS, was 64%, 48%, and 48% compared with 56%, 11%, and 11% at the same time points for those with CCM-CS (p = 0.05). In the AMI-CS group, 14 of 33 (42%) patients were weaned directly from ECMO after revascularization; 15 of 33 (45%) patients were bridged to ventricular assist device (VAD) support and subsequently either underwent heart transplantation (n = 6), were successfully weaned from VAD (n = 2) or died while on VAD support (n = 7). In the CCM-CS group, 7 patients were bridged to VAD support (77%), with 1 patient surviving after VAD weaning. CONCLUSIONS: Extracorporeal membrane oxygenation in advanced refractory AMI-CS is associated with acceptable outcomes in a well-selected population. The ECMO in patients with an acute decompensation of a chronic CM should be carefully considered, to avoid futile support.

Extracorporeal membrane oxygenation as a bridge to lung transplant: midterm outcomes.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) is used occasionally as a bridge to lung transplantation. The impact on mid-term survival is unknown. We analyzed outcomes after lung transplant over a 19-year period in patients who received ECMO support. METHODS: From March 1991 to October 2010, 1,305 lung transplants were performed at our institution. Seventeen patients (1.3%) were supported with ECMO before lung transplant. Diagnoses included retransplantation (n = 6), pulmonary fibrosis (n = 6), cystic fibrosis (n = 4), and chronic obstructive pulmonary disease (n = 1). Fifteen patients underwent double lung transplant, one patient had single left lung transplant and one patient had a heart-lung transplant. Venovenous and venoarterial ECMO were implanted in eight and nine cases, respectively. Median duration of support was 3.2 days (range, 1 to 49 days). Mean patient follow-up was 2.3 years. RESULTS: Thirty-day, 1-year, and 3-year survivals were 81%, 74%, and 65%, respectively, for the supported patients and 93%, 78%, and 62% in the control group (p = 0.56). Two-year survival was not affected by ECMO type, with survival of five out of nine patients supported by venoarterial ECMO vs seven out of eight patients supported by venovenous ECMO (p = 0.17). At 1- year follow-up, allograft function for the ECMO-supported patients did not differ from the control group (forced expiratory volume in one second, 2.35 L vs 2.09 L, p = 0.39) (forced vital capacity, 3.06 L vs 2.71 L, p = 0.34). CONCLUSIONS: Extracorporeal membrane oxygenation as a bridge to lung transplantation is associated with higher perioperative mortality but acceptable mid-term survival in carefully selected patients. Late allograft function did not differ in patients who received ECMO support before lung transplant from those who did not receive ECMO.

Isolation of Aspergillus in three 2009 H1N1 influenza patients.

OBJECTIVES: To describe the association of Aspergillus with influenza. DESIGN/SETTING/SAMPLE: Three case reports of ICU patients with influenza complicated by the isolation of Aspergillus species are described and a review of the literature on the topic was performed. CONCLUSIONS: Severe influenza cases can be complicated by Aspergillus infection.

Initial experience with single cannulation for venovenous extracorporeal oxygenation in adults.

PURPOSE: Historically, venovenous extracorporeal membrane oxygenation has required dual cannulation. A single-venous cannulation strategy may facilitate implantation and patient mobilization. Here we present our early experience with a single cannulation technique. DESCRIPTION: Review of venovenous extracorporeal membrane oxygenation support using internal jugular vein insertion of the Avalon elite bicaval dual lumen catheter (Avalon Laboratories, Rancho Dominguez, CA) in 11 consecutive patients with severe respiratory failure. EVALUATION: Adequate oxygenation was obtained in all patients: 115 mm Hg PaO(2) (median), 53 to 401 mm Hg (range). Median time of support was 78 hours (range, 3 to 267 hours). No mortality was directly related to the cannulation strategy. There were three nonfatal cannulation-related events. Two patients had proximal cannula displacement requiring repositioning. One patient suffered an acute thrombosis of the cannula. CONCLUSIONS: Our series supports single-venous cannulation in venovenous extracorporeal membrane oxygenation as a promising technique. It may be an excellent alternative to current cannulation strategies in patients requiring prolonged support and specifically for those considered for a bridge-to-lung transplantation.

The role of RAGE in the pathogenesis of intestinal barrier dysfunction after hemorrhagic shock.

The receptor for advanced glycation end products (RAGE) has been implicated in the pathogenesis of numerous conditions associated with excessive inflammation. To determine whether RAGE-dependent signaling is important in the development of intestinal barrier dysfunction after hemorrhagic shock and resuscitation (HS/R), C57Bl/6, rage(-/-), or congenic rage(+/+) mice were subjected to HS/R (mean arterial pressure of 25 mmHg for 3 h) or a sham procedure. Twenty-four hours later, bacterial translocation to mesenteric lymph nodes and ileal mucosal permeability to FITC-labeled dextran were assessed. Additionally, samples of ileum were obtained for immunofluorescence microscopy, and plasma was collected for measuring IL-6 and IL-10 levels. HS/R in C57Bl/6 mice was associated with increased bacterial translocation, ileal mucosal hyperpermeability, and high circulating levels of IL-6. All of these effects were prevented when C57Bl/6 mice were treated with recombinant human soluble RAGE (sRAGE; the extracellular ligand-binding domain of RAGE). HS/R induced bacterial translocation, ileal mucosal hyperpermeability, and high plasma IL-6 levels in rage(+/+) but not rage(-/-) mice. Circulating IL-10 levels were higher in rage(-/-) compared with rage(+/+) mice. These results suggest that activation of RAGE-dependent signaling is a key factor leading to gut mucosal barrier dysfunction after HS/R.

HMGB1 is secreted by immunostimulated enterocytes and contributes to cytomix-induced hyperpermeability of Caco-2 monolayers.

High-mobility group box 1 (HMGB1), a cytokine-like proinflammatory protein, is secreted by activated macrophages and released by necrotic cells. We hypothesized that immunostimulated enterocytes might be another source for this mediator. Accordingly, Caco-2 cells or primary mouse intestinal epithelial cells (IECs) were incubated with "cytomix" (a mixture of TNF, IL-1beta, and IFN-gamma) for various periods. HMGB1 in cell culture supernatants was detected by Western blot analysis and visualized in Caco-2 cells with the use of fluorescence confocal and immunotransmission electron microscopy. Caco-2 cells growing on filters in diffusion chambers were stimulated with cytomix for 48 h in the absence or presence of anti-HMGB1 antibody, and permeability to fluorescein isothiocyanate-dextran (average molecular mass, 4 kDa; FD4) was assessed. Cytomix-stimulated Caco-2 cells secreted HMGB1 into the apical but not the basolateral compartments of diffusion chambers. Although undetectable at 6 and 12 h after the start of incubation with cytomix, HMGB1 was present in supernatants after 24 h of incubation. HMGB1 secretion by Caco-2 monolayers also was induced when the cells were exposed to FSL-1, a Toll-like receptor (Tlr)-2 agonist, or flagellin, a Tlr5 agonist, but not lipopolysaccharide, a Tlr4 agonist. Cytomix also induced HMGB1 secretion by primary IECs. Cytoplasmic HMGB1 is localized within vesicles in Caco-2 cells and is secreted, at least in part, associated with exosomes. Incubating Caco-2 cells with cytomix increased FD4 permeation, but this effect was significantly decreased in the presence of anti-HMGB1 antibody. Collectively, these data support the view that HMGB1 is secreted by immunostimulated enterocytes. This process may exacerbate inflammation-induced epithelial hyperpermeability via an autocrine feedback loop.

The ethyl pyruvate analogues, diethyl oxaloproprionate, 2-acetamidoacrylate, and methyl-2-acetamidoacrylate, exhibit anti-inflammatory properties in vivo and/or in vitro.

Ethyl pyruvate (EP) is a simple aliphatic ester derived from the endogenous metabolite, pyruvic acid. EP has been shown to decrease the expression of various pro-inflammatory mediators, including nitric oxide (NO*), tumor necrosis factor (TNF), cyclooxygenase-2, and interleukin (IL)-6, in a variety of in vitro and in vivo model systems. In an effort to better understand the chemical features that might explain the anti-inflammatory properties of EP, we screened 15 commercially available compounds for cytoprotective or anti-inflammatory effects using two in vitro assay systems: TNF and NO* production by lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage-like cells and changes in the permeability of Caco-2 human enterocyte-like monolayers stimulated with a cocktail of pro-inflammatory cytokines called cytomix (1000U/ml IFN-gamma plus 10ng/ml TNF-alpha plus 1ng/ml IL-1beta). Two compounds, namely diethyl oxaloproprionate (DEOP) and 2-acetamidoacrylate (2AA), demonstrated consistent anti-inflammatory or cytoprotective pharmacological properties in this screening process. Treatment of mice with either of these compounds ameliorated LPS-induced ileal mucosal hyperpermeability to the fluorescent probe, fluorescein isothiocyanate-labeled dextran (average molecular mass 4kDa), and bacterial translocation to mesenteric lymph nodes. Treatment with either of these compounds also improved survival in mice challenged with a lethal dose of LPS. Finally, in a study that compared 2AA to its methyl ester, we showed that methyl-2-acetamidoacrylate is at least 100-fold more potent than the parent carboxylate as an inhibitor of LPS-induced NO* production by RAW 264.7 cells. Collectively, these data are consistent with the view that anti-inflammatory activity is demonstrable for a number of compounds that either incorporate an olefinic linkage conjugated to a carbonyl moiety or are capable of undergoing tautomeric rearrangement to form such a structure. Moreover, our findings suggest that esters with these general characteristics, perhaps because of their greater lipophilicity or electrophilicity, are more potent anti-inflammatory agents than are the parent carboxylates.

Ethyl pyruvate provides durable protection against inflammation-induced intestinal epithelial barrier dysfunction.

Ethyl pyruvate (EP) has been shown to be an effective anti-inflammatory agent. Herein, we sought to test the following hypotheses: 1) the pharmacological effects of EP persist after cells have been exposed to the compound in vitro, even if the cultures are washed to minimize the amount of EP that is retained in the media; 2) the pharmacological effects of EP persist in vivo, even after waiting a prolonged period (i.e., 6 h) after the last dose of the compound; and 3) the in vivo pharmacological effects of EP are distinct from those of the closely related compound, sodium pyruvate. Incubation of Caco-2 human enterocyte-like monolayers with cytomix, a mixture of interleukin-1beta, interferon-gamma, and tumor necrosis factor, increased permeability to the fluorescent macromolecule, FITC-labeled Dextran (mol wt 4,000 Da). Co-incubation of the cells with 5 mM EP ameliorated cytomix-induced hyperpermeability and induction of iNOS mRNA expression. EP was associated with similar pharmacological effects when cells were pre-incubated with the compound for 24 h prior and then washed extensively prior to adding the cytokine cocktail. Injecting C57Bl/6 mice with lipopolysaccharide (LPS) resulted in gut barrier dysfunction and hepatocellular injury. Although equivalent doses of both EP and sodium pyruvate ameliorated these phenomena, EP was more efficacious than pyruvate. Pretreatment with EP ameliorated the deleterious effects of LPS, even when the duration between the last dose of EP and the endotoxic challenge was 6 h. We conclude that EP provides durable protection against some of the deleterious effects of LPS or pro-inflammatory cytokines.

NAD+ ameliorates inflammation-induced epithelial barrier dysfunction in cultured enterocytes and mouse ileal mucosa.

In the course of other experiments, we serendipitously observed that extracellular nicotinamide adenine dinucleotide (NAD+) ameliorated the development of epithelial hyperpermeability when monolayers of Caco-2 enterocyte-like cells were incubated with cytomix, a mixture containing interferon-gamma, interleukin-1beta, and tumor necrosis factor-alpha. We sought to characterize the effects of NAD+ on inflammation-induced epithelial barrier dysfunction using Caco-2 monolayers that were exposed to cytomix in the absence or presence of NAD+ or other purine-containing molecules. Paracellular barrier function measured as the apical-to-basolateral passage of fluorescein isothiocyanate-conjugated dextran (mol. wt. approximately 4000) was preserved in a concentration-dependent manner when immunostimulated Caco-2 cells were exposed to extracellular NAD+. Incubation with NAD+ prevented cytomix-induced derangements in the expression and localization of the tight junction proteins occludin and zonula occludens-1 in Caco-2 cells. Treatment of cytomix-stimulated cells with NAD+ also blocked nuclear factor-kappaB (NF-kappaB) activation, inducible nitric-oxide synthase induction, and increased production of nitric oxide (NO.). Ileal mucosal permeability to fluorescein isothiocyanate-dextran mol. wt. approximately 4000 was increased in mice 18 h after lipopolysaccharide (endotoxin) injection, but treatment of endotoxemic mice with NAD+ ameliorated the development of gut mucosal hyperpermeability. Thus, extracellular NAD+ seems to ameliorate inflammation-induced intestinal epithelial barrier dysfunction by inhibiting NF-kappaB activation and increased NO. production.

Proinflammatory cytokines increase the rate of glycolysis and adenosine-5'-triphosphate turnover in cultured rat enterocytes.

OBJECTIVE: Measurements of steady-state adenosine-5'-triphosphate (ATP) levels in tissue samples from patients or experimental animals with sepsis or endotoxemia provide little information about the rate of ATP production and consumption in these conditions. Accordingly, we sought to use an in vitro "reductionist" model of sepsis to test the hypothesis that proinflammatory cytokines modulate ATP turnover rate. DESIGN: In vitro "reductionist" model of sepsis. SETTING: University laboratory. SUBJECTS: Cultured rat enterocyte-like cells. INTERVENTIONS: IEC-6 nontransformed rat enterocytes were studied under control conditions or following incubation for 24 or 48 hrs with cytomix, a mixture of tumor necrosis factor-alpha (10 ng/mL), interleukin-1beta (1 ng/mL), and interferon-gamma (1000 units/mL). To measure ATP turnover rate, ATP synthesis was acutely blocked by adding to the cells a mixture of 2-deoxyglucose (10 mM), potassium cyanide (8 mM), and antimycin A (1 microM). ATP content was measured at baseline (before metabolic inhibition) and 0.5, 1, 2, 5, and 10 mins later. Log-linear ATP decay curves were generated and the kinetics of ATP utilization thereby calculated. MEASUREMENTS AND MAIN RESULTS: ATP consumption rate was higher in cytomix-stimulated compared with control cells (3.11 +/- 1.39 vs. 1.25 +/- 0.66 nmol/min, respectively; p <.01). Similarly, the half-time for ATP disappearance was shorter in cytomix-stimulated compared with control cells (2.63 +/- 1.00 vs. 6.21 +/- 3.49; p <.05). In contrast to these findings, the rate of ATP disappearance was similar in cytokine-naïve and immunostimulated IEC-6 cells when protein and nucleic acid synthesis were inhibited by adding 50 microg/mL cycloheximide and 5 microg/mL actinomycin D to cultures for 4 hrs. The rates of glucose consumption and lactate production were significantly greater in cytomix-stimulated compared with controls cells. CONCLUSIONS: Incubation of IEC-6 cells with cytomix significantly increased ATP turnover. Increased ATP turnover rate was supported by increases in the rate of anaerobic glycolysis. These findings support the view that proinflammatory mediators impose a metabolic demand on visceral cells. In sepsis, cells may be more susceptible to dysfunction on the basis of diminished oxygen delivery and/or mitochondrial dysfunction.

Ethyl pyruvate ameliorates intestinal epithelial barrier dysfunction in endotoxemic mice and immunostimulated caco-2 enterocytic monolayers.

Ethyl pyruvate (EP) solution ameliorates ileal mucosal hyperpermeability and decreases the expression of several proinflammatory genes in ileal and/or colonic mucosa when it is used instead of Ringer's lactate solution (RLS) to resuscitate mice from hemorrhagic shock. To test the hypothesis that EP can ameliorate gut barrier dysfunction induced by other forms of inflammation, we incubated Caco-2 monolayers for 24 to 48 h with cytomix (a mixture of interferon-gamma, tumor necrosis factor-alpha, and interleukin-1beta) in the presence or absence of graded concentrations of EP or sodium pyruvate. Cytomix increased the permeability of Caco-2 monolayers to fluorescein isothiocyanate-labeled dextran (FD4; average molecular mass 4 kDa), but this effect was inhibited by adding 0.1 to 10 mM EP (but not similar concentrations of sodium pyruvate) to the culture medium. EP inhibited several other cytomix-induced phenomena, including nuclear factor-kappaB activation, inducible nitric oxide synthase mRNA expression, and nitric oxide production. Cytomix altered the expression and localization of the tight junctional proteins, ZO-1 and occludin, but this effect was prevented by EP. Delayed treatment with EP solution instead of RLS ameliorated ileal mucosal hyperpermeability to FD4 and bacterial translocation to mesenteric lymph nodes in mice challenged with lipopolysaccharide (LPS). These data support the view that EP ameliorates cytokine- and/or LPS-induced derangements in intestinal epithelial barrier function.