Pre-B-cell-colony-enhancing factor is critically involved in thrombin-induced lung endothelial cell barrier dysregulation

Prior genomic and genetic studies identified pre-B-cell colony-enhancing factor (PBEF) as a novel candidate gene and biomarker in acute lung injury (ALI). As increased vascular permeability is a cardinal feature of ALI, we assessed the role of PBEF in in vitro vascular barrier regulation using confluent human pulmonary artery endothelial cell (HPAEC) monolayers. Reductions in PBEF protein expression (>70%) by siRNA significantly attenuated EC barrier dysfunction induced by the potent edemagenic agent, thrombin, reflected by reductions in transendothelial electric resistance (TER, approximately 60% reduction). Furthermore, PBEF siRNA blunted thrombin-mediated increases in Ca(2+) entry, polymerized actin formation, and myosin light chain phosphorylation, events critical to the thrombin-mediated permeability response. Finally, PBEF siRNA also significantly inhibited thrombin-stimulated increase of IL-8 secretion in HPAEC, a chemokine known to induce actin fiber formation and intercellular gap formation of endothelial cells. Taken together, these studies demonstrate that PBEF may be required for complete expression of the thrombin-induced inflammatory response and reveal potentially novel role for PBEF in the regulation of EC Ca(2+)-dependent cytoskeletal rearrangement and endothelial barrier dysfunction. Ongoing studies will continue to address the molecular mechanisms by which PBEF contributes to ALI susceptibility.     

Convergence and amplification of toll-like receptor (TLR) and receptor for advanced glycation end products (RAGE) signaling pathways via high mobility group B1 (HMGB1)

Sustained proinflammatory responses in rheumatoid arthritis, atherosclerosis, and diabetic retinopathy, as well as in cancer, are often associated with increased angiogenesis that contributes to tissue disruption and disease progression. High mobility group B1 (HMGB1) has been recognized as a proinflammatory cytokine and more recently, as a proangiogenic factor. HMGB1 can either be passively released from necrotic cells or actively secreted in response to angiogenic and inflammatory signals. HMGB1 itself may signal through the receptor for advanced glycation end products (RAGE), and via toll-like receptors, TLR2 and TLR4. Activation of these receptors results in the activation of NFκB, which induces the upregulation of leukocyte adhesion molecules and the production of proinflammatory cytokines and angiogenic factors in both hematopoietic and endothelial cells, thereby promoting inflammation. Interestingly, HMGB1 seems to be involved in a positive feedback mechanism, that may help to sustain inflammation and angiogenesis in several pathological conditions, thereby contributing to disease progression. Endothelial cells express HMGB1, as well as the receptors RAGE, TLR2, and TLR4, and in diverse pathologies HMGB1 and its receptors are overexpressed. Furthermore, HMGB1-induced signaling can activate NFκB, which can subsequently induce the expression of HMGB1 receptors. Thus, HMGB1 can mediate amplification of inflammation and angiogenesis through increased secretion of HMGB1 and increased expression of the receptors it can interact with. In this review, we discuss signaling cascades that HMGB1 can induce via TLRs and RAGE, as well as its contribution to pathologies involving endothelial cells.     

高迁移率族蛋白B1与中枢神经系统疾病

高迁移率族蛋白Bl(high mobility group box 1,HMGB1)是高迁移率族蛋白家族成员.HMGB1可与细胞表面特定受体--晚期糖基化终产物受体(receptor for advanced glvcation end products,RAGE)和Toll样受体2(toll like receptor2,TLR2)结合,从而具有广泛的生物学活性.在中枢神经系统,HMGB1参与炎症反应、血脑屏障通透性调节等病理生理学过程,并与缺血性卒中、阿尔茨海默病以及神经胶质瘤等疾病密切相关.     

High-mobility group box 1 represents a potential marker of disease activity and novel therapeutic target in systemic lupus erythematosus

High-mobility group box 1 (HMGB1) protein is a nuclear DNA-binding protein, which functions as an alarmin when released from cells. Recent studies implicate extracellular HMGB1 in the pathogenesis of systemic lupus erythematosus (SLE), a prototypical autoimmune disease characterized by the formation of multiple autoantibodies, especially those directed against nucleosomes and double-stranded (ds)DNA. Elevated concentrations of HMGB1 are observed in sera as well as in skin lesions of patients with lupus. Of importance, serum HMGB1 and anti-HMGB1 autoantibody levels correlate with disease activity. In the blood of patients with SLE, HMGB1 is complexed with nucleosomes, at least partially. Moreover, HMGB1-nucleosome complexes from apoptotic cells activate antigen-presenting cells. Injection of HMGB1-nucleosome complexes into nonautoimmune mice results in the formation of autoantibodies against dsDNA and histones in a Toll-like receptor (TLR) 2-dependent manner. Additionally, HMGB1, as a part of DNA-anti-DNA immune complexes, can interact with receptor for advanced glycation end products (RAGE) on the surface of plasmacytoid dendritic cells and B cells leading to TLR9-dependent interferon (IFN)α release and activation of autoreactive B cells, respectively. HMGB1 attached to neutrophil extracellular traps may contribute to IFNα production by facilitating the recognition of self-nucleic acids. Furthermore, HMGB1, complexed with DNA and pathogenic anti-DNA autoantibodies, activates its receptors, TLR2, TLR4 and RAGE, and may thereby be involved in anti-DNA autoantibody-induced kidney damage in lupus nephritis. Collectively, these findings suggest that HMGB1 is a potential marker of disease activity and, because of its probable involvement in the pathogenesis, a novel therapeutic target in SLE.     

Sepsis-induced lung injury and the effects of ibuprofen pretreatment. Analysis of early alveolar events via repetitive bronchoalveolar lavage.

Current knowledge of alveolar pathophysiology during early sepsis-induced acute lung injury (ALI) and the role of resident alveolar macrophages (AM) in mediating alveolar inflammatory events during sepsis is limited. Further, the effects of ibuprofen pretreatment upon alveolar pathophysiology and AM function during early sepsis-induced ALI is unclear. Utilizing repetitive bronchoalveolar lavage (BAL) in a porcine model of sepsis-induced ALI, we studied changes in alveolar cellular constituents, BAL protein content and molecular composition, and AM superoxide anion (O2-.) generation during early sepsis. The neutrophil percentage of recovered alveolar cells (17 +/- 8%, t = 300 min versus 2 +/- 1%, t = 0; p = 0.06) and the bronchoalveolar lavage total protein content (493 +/- 110 micrograms/ml, t = 300 min versus 109 +/- 18 micrograms/ml, t = 0; p less than 0.05) increased in septic animals. Increases in BAL fluid total protein were primarily due to low-molecular-weight plasma protein, indicating relative preservation of alveolar-capillary membrane size selectivity. Alveolar macrophages harvested following 300 min of sepsis generated significantly less O2-. following phorbol myristate acetate (PMA) stimulation compared to AM harvested at baseline. Ibuprofen pretreatment of septic animals completely blocked leakage of plasma proteins into the alveoli and attenuated neutrophil migration but did not prevent downregulation of AM O2-. generation. Increased alveolar-capillary membrane permeability, neutrophil migration into the alveoli, and downregulation of AM oxidant generation occur within hours of the onset of sepsis. Ibuprofen pretreatment significantly attenuates early sepsis-induced ALI without altering sepsis-induced AM dysfunction.     

miR-126-5p expression in the plasma of patients with sepsis-induced acute lung injury and its correlation with inflammation and immune function

Objective

This work was implemented to elucidate the miR-126-5p expression in the plasma of patients with sepsis-induced acute lung injury (ALI) and its correlation with inflammation and immune function.

Methods

The peripheral blood of patients with sepsis-induced ALI was obtained, and the levels of inflammatory factors (interleukin-6 [IL-6], C-reactive protein [CRP], and procalcitonin [PCT]) were determined. Meanwhile, T lymphocyte subsets (CD3+, CD4+, and CD8+), and immunoglobulins (IgA, IgM, and IgG) were tested. miR-126-5p and TRAF6 mRNA expression in plasma was assessed. Receiver operating characteristic (ROC) curve was performed to assess the diagnostic accuracy of miR-126-5p in sepsis without ALI and sepsis with ALI. Correlation between miR-126-5p expression and clinical indicators was analyzed. The targets of miR-126-5p were predicted using the bioinformatics method, and the direct targets were verified through investigations.

Results

miR-126-5p expression in plasma of patients with sepsis-induced ALI was reduced than that of patients with sepsis without ALI. miR-126-5p expression was negatively correlated with IL-6, CRP, and PCT but positively correlated with IgA, IgM, and IgG as well as CD3+, CD4+, and CD8+ in patients with sepsis-induced ALI. ROC curve suggested that miR-126-5p (AUC: 0.777; 95%CI: 0.689–0.866) could distinguish patients with sepsis with ALI from patients with sepsis without ALI. TRAF6 expression in patients with sepsis-induced ALI was higher than that in patients with sepsis without ALI. TRAF6 was a target gene of miR-126-5p,

Conclusion

This research highlights that miR-126-5p is reduced in the plasma of patients with sepsis-induced ALI, and miR-126-5p relates to systemic inflammation and immune function indicators.     

High-mobility group box 1 activates integrin-dependent homing of endothelial progenitor cells

Endothelial progenitor cells (EPCs) are recruited to ischemic regions and improve neovascularization. Integrins contribute to EPC homing. High-mobility group box 1 (HMGB1) is a nuclear protein that is released extracellularly on cell necrosis and tissue damage, eliciting a proinflammatory response and stimulating tissue repair. In the present study, we investigated the effects of HMGB1 on EPC homing. EPCs express the HMGB1 receptors RAGE (receptor for advanced glycation end products) and TLR2 (Toll-like receptor 2). EPC migration was stimulated by HMGB1 in a RAGE-dependent manner. In addition, the HMGB1-induced migration of EPCs on fibronectin and fibrinogen was significantly inhibited by antibodies against beta1 and beta2 integrins, respectively. Short-term prestimulation of EPCs with HMGB1 also increased EPC adhesion to endothelial cell monolayers, and this effect was blocked by antibodies to beta2 integrins or RAGE. HMGB1 increased EPC adhesion to the immobilized integrin ligands intercellular adhesion molecule-1 and fibronectin in a RAGE-dependent manner. Strikingly, HMGB1 rapidly increased integrin affinity and induced integrin polarization. Using intravital microscopy in a tumor model of neovascularization, prestimulation of EPCs with HMGB1 enhanced the initial in vivo adhesion of EPCs to microvessels and the recruitment of EPCs in the tumor tissue. In addition, prestimulation of EPCs with HMGB1 increased the homing of EPCs to ischemic muscles. In conclusion, these data represent a link between HMGB1 and integrin functions of EPCs and demonstrate that HMGB1 stimulates EPC homing to ischemic tissues. These results may provide a platform for the development of novel therapeutic approaches to improve EPC homing.     

Insulin reduces LPS-induced lethality and lung injury in rats

Insulin is a main glucose homeostatic hormone in the body. Previous reports showed that insulin also exerted anti-inflammatory actions and attenuated systemic inflammatory response. Here, we observed the effects and the underlying mechanisms of insulin on lipopolysaccharide (LPS)-induced acute lung injury (ALI). As revealed by survival study, insulin reduced mortality of rats and prolonged their survival time. Meanwhile, insulin significantly reduced the levels of inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and high mobility group box 1 (HMGB1) in bronchoalveolar lavage fluid (BALF). Besides, insulin markedly inhibited the expression of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB). Taken together, these data provided information that insulin attenuated LPS-induced ALI may attribute partly to the inhibition of the production of cytokines, and the expression of TLR2, TLR4 and NF-κB.     

A novel hypothesis: up-regulation of HO-1 by activation of PPARγ inhibits HMGB1-RAGE signaling pathway and ameliorates the development of ALI/ARDS

Suppression of inflammation in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) by activation of peroxisome proliferator-activated receptor (PPAR)-γ has been well demonstrated in animal model studies. However, the molecular mechanisms underlying this effect remain largely unknown. The induction of heme oxygenase-1 (HO-1) exerts antioxidant, anti-apoptotic, and immunomodulatory functions in various situations. Recent studies have indicated that activation of PPARγ induces expression of HO-1, suggesting that HO-1 is a downstream target of PPARγ. Meanwhile, study has shown that activation of PPARγ ameliorates inflammatory response of cells by inhibiting high mobility group box 1 (HMGB1) release. In pulmonary system, binding of HMGB1 to its receptor for advanced glycation end-products (RAGE) triggers the production of pro-inflammatory cytokines, chemokines, adhesion molecules and reactive oxygen species, promoting the development of ALI/ARDS. Based on the recent findings that induction of HO-1 protects tissues and cells from extracellular stress by reducing HMGB1 production, we propose the hypothesis that HO-1 may mediate the protective effects of PPARγ on inhibition of HMGB1-RAGE signaling pathway to attenuate the development of ALI/ARDS.KEYWORDS : Peroxisome proliferator-activated receptor (PPAR)-γ, heme oxygenase-1 (HO-1), high mobility group box 1 (HMGB1), receptor for advanced glycation end-products (RAGE), acute lung injury/acute respiratory distress syndrome (ALI/ARDS)     

Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction

Thrombin-induced barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contraction, and gap formation. Phosphorylation of regulatory myosin light chains (MLC) is a key mechanism of endothelial cell (EC) contraction and barrier dysfunction, which is triggered by Ca(2+)/calmodulin-dependent MLC kinase (MLCK) and Rho-associated kinase (Rho-kinase). The role of MLCK in EC barrier regulation has been previously described; however, Rho-mediated pathway in thrombin-induced pulmonary EC dysfunction is not yet precisely characterized. Here, we demonstrate that thrombin-induced decreases in transendothelial electrical resistance (TER) indicating EC barrier dysfunction are universal for human and bovine pulmonary endothelium, and involve membrane translocation and direct activation of small GTPase Rho and its downstream target Rho-kinase. Transient Rho membrane translocation coincided with translocation of upstream Rho activator, guanosine nucleotide exchange factor p115-RhoGEF. Rho mediated activation of downstream target, Rho-kinase induced phosphorylation of the EC MLC phosphatase (MYPT1) at Thr(686) and Thr(850), resulting in MYPT1 inactivation, accumulation of diphospho-MLC, actin remodeling, and cell contraction. The specific Rho-kinase inhibitor, Y27632, abolished MYPT1 phosphorylation, MLC phosphorylation, significantly attenuated stress fiber formation and thrombin-induced TER decrease. Furthermore, expression of dominant-negative Rho and Rho-kinase abolished thrombin-induced stress fiber formation and MLC phosphorylation. Our data, which provide comprehensive analysis of Rho-mediated signal transduction in pulmonary EC, demonstrate involvement of guanosine nucleotide exchange factor, p115-RhoGEF, in thrombin-mediated Rho regulation, and suggest Rho, Rho-kinase, and MYPT1 as potential pharmacological and gene therapy targets critical for prevention of thrombin-induced EC barrier disruption and pulmonary edema associated with acute lung injury.     

氯胺酮对急性肺损伤大鼠HMGB1及TLR4表达的影响

目的研究氯胺酮对急性肺损伤大鼠肺组织中高迁移率族蛋白(HMGB1)及Toll样受体4(TLR4)表达的影响。方法创建大鼠脓毒症致急性肺损伤模型,随机分为对照组、脓毒症组、氯胺酮组。观察建模后大鼠的一般状态。于6h、12h、24h颈椎脱臼法处死大鼠,取肺组织,HE染色显微镜下观察大鼠肺组织结构改变。RT-PCR法检测大鼠肺组织HMGB1及TLR4表达的变化。结果对照组大鼠健康正常,肺组织结构清晰;脓毒症组大鼠30min后逐渐出现肺损伤症状,肺组织结构破坏;氯胺酮组大鼠肺损伤症状轻微,组织破坏程度轻。脓毒症组与氯胺酮组大鼠肺组织HMGB1、TLR4的表达均高于对照组;随着建模时间延长,脓毒症组及氯胺酮组大鼠肺组织HMGB1、TLR4表达均逐渐升高,且氯胺酮组两者的表达均低于脓毒症组。结论氯胺酮能够减轻脓毒症所致的急性大鼠肺损伤,这可能与其降低了肺组织的HMGB1、TLR4表达相关。     

高迁移率族蛋白B1与脑缺血

高迁移率族蛋白B1(high mobility group box 1,HMGB1)是高迁移率族蛋白家族成员之一.细胞外HMGB1和细胞表面受体--晚期糖基化终产物受体(receptor for advanced glycation endproduct,RAGE)、Toll样受体2(toll like receptor 2,TLR2)和Toll样受体4(toll like receptor 4,ILR4)结合,与其他促炎介质协同作用,促进炎症级联反应.脑缺血后,过度表达的HMGB1参与了炎症反应、神经元凋亡等病理生理学过程.     

Activated protein C inhibits high mobility group box 1 signaling in endothelial cells

A pathogenic role for high-mobility group box 1 (HMGB1) protein has been postulated in severe sepsis. Activated protein C (APC) is the only drug approved by the Food and Drug Administration for severe sepsis; however, its effect on HMGB1 signaling has never been investigated. Here, we monitored the effect of APC on the lipopolysaccharide-mediated release of HMGB1 and the HMGB1-mediated modulation of proinflammatory responses in HUVECs. APC potently inhibited the release of HMGB1 and down-regulated the adhesion of the monocytic cell line, THP-1, to HMGB1-activated endothelial cells. HMGB1 up-regulated proinflammatory responses by interacting with 3 pathogen-related pattern recognition receptors: TLR2 and TLR4 and the receptor for advanced glycation end products. APC not only inhibited HMGB1 release but also down-regulated the cell surface expression of all 3 HMGB1 receptors in endothelial cells. The protective effects of APC were mediated through endothelial cell protein C receptor (EPCR) and protease-activated receptor 1 (PAR-1). Interestingly, a thrombin derivative containing the Gla-domain of APC recapitulated all protective effects of APC with a 20- to 50-fold higher efficacy. These results suggest that the EPCR- and PAR-1-dependent protective effects of APC in severe sepsis may partially be mediated through the inhibition of HMGB1 signaling and that the chimeric thrombin mutant has potential therapeutic utility for severe sepsis.     

High mobility group box 1 induces a negative inotropic effect on the left ventricle in an isolated rat heart model of septic shock: a pilot study.

BACKGROUND: Sepsis can be exacerbated by an inappropriate immune response and the severe impact of this disease on the cardiovascular system is well documented. High mobility group box 1 (HMGB1) protein is an important mediator in the pathogenesis of sepsis and its role in cardiovascular system dysfunction was investigated in an lipopolysaccharide (LPS)-induced rat model of sepsis. METHODS AND RESULTS: Twelve hours after intravenous bolus injections of LPS (5 mg/kg), rats were killed and heart samples were harvested. Immunoblot analysis was performed to assess expression levels of HMGB1 in cardiac myocytes. Left ventricular developed pressure (LVDP) served as a measure of systolic function. LPS administration was associated with an increase in the expression of HMGB1 in cardiac myocytes and a decrease in cardiac function. Hearts from the LPS-treated rats were also perfused with recombinant HMGB1 and cardiac function measured. The dose-dependent effects observed with elevated HMGB1 included decreased LVDP, decreased left ventricular (LV) + dP/dt(max), decreased absolute value of LV- dP/dt(min), and increased LV end-diastolic pressure. CONCLUSIONS: HMGB1 stimulation produces a negative inotropic effect during septic shock, suggesting an important role for this molecule in cardiovascular system dysfunction during sepsis.     

Opposite effects of ANP receptors in attenuation of LPS-induced endothelial permeability and lung injury

Atrial natriuretic peptide (ANP) has been recently identified as a modulator of acute lung injury (ALI) induced by pro-inflammatory agonists. While previous studies tested effects of exogenous ANP administration, the role of endogenous ANP in the course of ALI remains unexplored. This study examined regulation of ANP and its receptors NPR-A, NPR-B and NPR-C by LPS and involvement of ANP receptors in the modulation of LPS-induced lung injury. Primary cultures of human pulmonary endothelial cells (EC) were used in the in vitro tests. Expression of ANP and its receptors was determined by quantitative RT-PCR analysis. Agonist-induced cytoskeletal remodeling was evaluated by immunofluorescence staining, and EC barrier function was characterized by measurements of transendothelial electrical resistance. In the murine model of ALI, LPS-induced lung injury was assessed by measurements of protein concentration and cell count in bronchoalveolar lavage fluid (BAL). LPS stimulation significantly increased mRNA expression levels of ANP and NPR-A in pulmonary EC. Pharmacological inhibition of NPR-A augmented LPS-induced EC permeability and blocked barrier protective effects of exogenous ANP on LPS-induced intercellular gap formation. In contrast, pharmacological inhibition of ANP clearance receptor NPR-C significantly attenuated LPS-induced barrier disruptive effects. Administration of NPR-A inhibitor in vivo exacerbated LPS-induced lung injury, whereas inhibition of NPR-C suppressed LPS-induced increases in BAL cell count and protein content. These results demonstrate for the first time opposite effects of NPR-A and NPR-C in the modulation of ALI and suggest a compensatory protective mechanism of endogenous ANP in the maintenance of lung vascular permeability in ALI.     

Phosphorylated ERM Mediates Lipopolysaccharide Induced Pulmonary Microvascular Endothelial Cells Permeability Through Negatively Regulating Rac1 Activity

IntroductionThe endotoxin lipopolysaccharide (LPS)-induced pulmonary endothelial barrier disruption is a key pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the molecular mechanisms underlying LPS-impaired permeability of pulmonary microvascular endothelial cells (PMVECs) are not fully understood.MethodsRat PMVECs were isolated and monolayered cultured, then challenged with different doses of LPS (0.1 mg/L, 1 mg/L, and 10 mg/L). Trans-endothelial electrical resistance (TER) was utilized to measure the integrity of the endothelial barrier. Ras-related C3 botulinum toxin substrate 1 (Rac1) activity and the phosphorylation of Ezrin/Radixin/Moesin proteins (ERM) were assessed by pulldown assay and Western Blotting. Small interfering RNA (siRNA) inhibition of Rac1 and Moesin were applied to evaluate the effect of PMVEs permeability and related pathway.ResultsLPS induced dose and time-dependent decreases in TER and increase in ERM threonine phosphorylation, while inactivated Rac1 activity in PMVEC. siRNA study demonstrated that both Rac1 and Moesin were involved in the mediation of the LPS-induced hyperpermeability in PMVECs monolayers, and Rac1 and Moesin could regulate each other.ConclusionPhosphorylated ERM mediates LPS induced PMVECs permeability through negatively regulating Rac1 activity.     

Platelet Toll-like receptor expression and activation induced by lipopolysaccharide and sepsis

Platelets and Toll-like receptor (TLR) signalling play a role in the immune response during sepsis. Although preclinical knowledge about the role of platelet TLR signalling is increasing, data during human sepsis are less abundant. Moreover, controversy remains about the effect of the TLR4 agonist lipopolysaccharide (LPS) on platelet activation. We therefore assessed platelet TLR expression during human and murine sepsis. Moreover, we investigated the effect of TLR4 signalling on platelet activation and TLR expression. Platelets from healthy controls stimulated with LPS did not show classical platelet activation (P-selectin, CD63 and phosphatidylserine expression), potentiation of subthreshold agonist stimulation nor platelet-leukocyte complex formation. LPS stimulation however did increase maximal mitochondrial respiration in a TLR4-dependent manner. Platelet stimulation with LPS did not alter TLR expression. Platelet stimulation with thrombin receptor activating peptide increased TLR5 and TLR9, but not TLR2 or TLR4 expression. Platelets from patients with sepsis and mice with experimental sepsis showed platelet activation, but unaltered TLR expression. These results indicate that sepsis-induced platelet activation is not associated with altered platelet TLR expression and, although platelets are responsive to LPS, stimulation of platelet TLR4 does not result in classical platelet activation.     

A membrane-permeant peptide that inhibits MLC kinase restores barrier function in in vitro models of intestinal disease

BACKGROUND & AIMS: Maintenance of the mucosal barrier is a critical function of intestinal epithelia. Myosin regulatory light chain (MLC) phosphorylation is a common intermediate in the pathophysiologic regulation of this barrier. The aim of this study was to determine whether a membrane permeant inhibitor of MLC kinase (PIK) could inhibit intracellular MLC kinase and regulate paracellular permeability. METHODS: Recombinant MLC and Caco-2 MLC kinase were used for kinase assays. T84 and Caco-2 monolayers were treated with enteropathogenic Escherichia coli (EPEC) or tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma to induce barrier dysfunction. RESULTS: PIK inhibited MLC kinase in vitro and was able to cross cell membranes and concentrate at the perijunctional actomyosin ring. Consistent with these properties, apical addition of PIK reduced intracellular MLC phosphorylation by 22% +/- 2%, increased transepithelial resistance (TER) by 50% +/- 1%, and decreased paracellular mannitol flux rates by 5.2 +/- 0.2-fold. EPEC infection induced TER decreases of 37% +/- 6% that were limited to 16% +/- 5% by PIK. TNF-alpha and IFN-gamma induced TER decreases of 22% +/- 3% that were associated with a 172% +/- 1% increase in MLC phosphorylation. Subsequent PIK addition caused MLC phosphorylation to decrease by 25% +/- 4% while TER increased to 97% +/- 6% of control. CONCLUSIONS: PIK can prevent TER defects induced by EPEC and reverse MLC phosphorylation increases and TER decreases induced by TNF-alpha and IFN-gamma. The data also suggest that TNF-alpha and IFN-gamma regulate TER, at least in part, via the perijunctional cytoskeleton. Thus, PIK may be the prototype for a new class of targeted therapeutic agents that can restore barrier function in intestinal disease states.     

Pulmonary vs nonpulmonary sepsis and mortality in acute lung injury

BACKGROUND: Acute lung injury (ALI) is a frequent complication of sepsis. It is unclear if a pulmonary vs nonpulmonary source of sepsis affects mortality in patients with sepsis-induced ALI. METHODS: Two hundred eighty-eight consecutive patients with sepsis-induced ALI from 14 ICUs at four hospitals in Baltimore, MD were prospectively classified as having a pulmonary vs nonpulmonary source of sepsis. Multiple logistic regression was conducted to evaluate the independent association of a pulmonary vs nonpulmonary source of sepsis with inpatient mortality. RESULTS: In an unadjusted analysis, in-hospital mortality was lower for pulmonary vs nonpulmonary source of sepsis (42% vs 66%, p < 0.0001). Patients with pulmonary sepsis had lower acute physiology and chronic health evaluation (APACHE) II and sequential organ failure assessment (SOFA) scores, shorter ICU stays prior to the development of ALI, and higher lung injury scores. In the adjusted analysis, several factors were predictive of mortality: age (odds ratio [OR], 1.03; 95% confidence interval [CI], 1.01 to 1.06), Charlson comorbidity index (OR, 1.15; 95% CI, 1.02 to 1.30), ICU length of stay prior to ALI diagnosis (OR, 1.19; 95% CI, 1.01 to 1.39), APACHE II score (OR, 1.07; 95% CI, 1.03 to 1.12), lung injury score (OR, 1.64; 95% CI, 1.11 to 2.43), SOFA score (OR, 1.15; 95% CI, 1.06 to 1.26), and cumulative fluid balance in the first 7 days after ALI diagnosis (OR, 1.06; 95% CI, 1.03 to 1.10). A pulmonary vs nonpulmonary source of sepsis was not independently associated with mortality (OR, 0.72; 95% CI, 0.38 to 1.35). CONCLUSIONS: Although lower mortality was observed for ALI patients with a pulmonary vs nonpulmonary source of sepsis, this finding is likely due to a lower severity of illness in those with pulmonary sepsis. Pulmonary vs nonpulmonary source of sepsis was not independently predictive of mortality for patients with ALI.