Augmenter of liver regeneration (ALR) is a novel biomarker of hepatocellular stress/inflammation: in vitro, in vivo and in silico studies.

The liver is a central organ involved in inflammatory processes, including the elaboration of acute-phase proteins. Augmenter of liver regeneration (ALR) protein, expressed and secreted by hepatocytes, promotes liver regeneration and maintains viability of hepatocytes. ALR also stimulates secretion of inflammatory cytokines (tumor necrosis factor [TNF]-α and interleukin [IL]-6) and nitric oxide from Kupffer cells. We hypothesized that ALR may be involved in modulating inflammation induced by various stimuli. We found that hepatic ALR levels are elevated at 24 h, before or about the same time as an increase in the mRNA expression of TNF-α and IL-6, after portacaval shunt surgery in rats. Serum ALR also increased, but significantly only on d 4 when pathological changes in the liver become apparent. In rats, serum ALR was elevated after intraperitoneal administration of lipopolysaccharide alone and in a model of gram-negative sepsis. Serum ALR increased before alanine aminotransferase (ALT) in endotoxemia and in the same general time frame as TNF-α and IL-6 in the bacterial sepsis model. Furthermore, mathematical prediction of tissue damage correlated strongly with alterations in serum ALR in a mouse model of hemorrhagic shock. In vitro, monomethyl sulfonate, TNF-α, actinomycin D and lipopolysaccharide all caused increased release of ALR from rat hepatocytes, which preceded the loss of cell viability and/or inhibition of DNA synthesis. ALR may thus serve as a potential diagnostic marker of hepatocellular stress and/or acute inflammatory conditions.

Hemoadsorption reprograms inflammation in experimental gram-negative septic peritonitis: insights from in vivo and in silico studies.

UNLABELLED: Improper compartmentalization of the inflammatory response leads to systemic inflammation in sepsis. Hemoadsorption (HA) is an emerging approach to modulate sepsis-induced inflammation. We sought to define the effects of HA on inflammatory compartmentalization in Escherichia coli-induced fibrin peritonitis in rats. HYPOTHESIS: HA both reprograms and recompartmentalizes inflammation in sepsis. Sprague Dawley male rats were subjected to E. coli peritonitis and, after 24 h, were randomized to HA or sham treatment (sepsis alone). Venous blood samples collected at 0, 1, 3 and 6 h (that is, 24-30 h of total experimental sepsis), and peritoneal samples collected at 0 and 6 h, were assayed for 14 cytokines along with NO(2)(-/)NO(3)(-). Bacterial counts were assessed in the peritoneal fluid at 0 and 6 h. Plasma tumor necrosis factor (TNF)-α, interleukin (IL)-6, CXCL-1, and CCL2 were significantly reduced in HA versus sham. Principal component analysis (PCA) suggested that inflammation in sham was driven by IL-6 and TNF-α, whereas HA-associated inflammation was driven primarily by TNF-α, CXCL-1, IL-10 and CCL2. Whereas -peritoneal bacterial counts, plasma aspartate transaminase levels and peritoneal IL-5, IL-6, IL-18, interferon (IFN)-γ and NO(2)(-)/NO(3)(-) were significantly lower, both CXCL-1 and CCL2 as well as the peritoneal-to-plasma ratios of TNF-α, CXCL-1 and CCL2 were significantly higher in HA versus sham, suggesting that HA-induced inflammatory recompartmentalization leads to the different inflammatory drivers discerned in part by PCA. In conclusion, this study demonstrates the utility of combined in vivo/in silico methods and suggests that HA exerts differential effects on mediator gradients between local and systemic compartments that ultimately benefit the host.

Transcriptomic response of murine liver to severe injury and hemorrhagic shock: a dual-platform microarray analysis.

Trauma-hemorrhagic shock (HS/T) is a complex process that elicits numerous molecular pathways. We hypothesized that a dual-platform microarray analysis of the liver, an organ that integrates immunology and metabolism, would reveal key pathways engaged following HS/T. C57BL/6 mice were divided into five groups (n = 4/group), anesthetized, and surgically treated to simulate a time course and trauma severity model: 1) nonmanipulated animals, 2) minor trauma, 3) 1.5 h of hemorrhagic shock and severe trauma (HS/T), 4) 1.5 h HS/T followed by 1 h resuscitation (HS/T+1.0R), 5) 1.5 h HS/T followed by 4.5 h resuscitation (HS/T+4.5R). Liver RNA was hybridized to CodeLink and Affymetrix mouse whole genome microarray chips. Common genes with a cross-platform correlation >0.6 (2,353 genes in total) were clustered using k-means clustering, and clusters were analyzed using Ingenuity Pathways Analysis. Genes involved in the stress response and immunoregulation were upregulated early and remained upregulated throughout the course of the experiment. Genes involved in cell death and inflammatory pathways were upregulated in a linear fashion with elapsed time and in severe injury compared with minor trauma. Three of the six clusters contained genes involved in metabolic function; these were downregulated with elapsed time. Transcripts involved in amino acid metabolism as well as signaling pathways associated with glucocorticoid receptors, IL-6, IL-10, and the acute phase response were elevated in a severity-dependent manner. This is the first study to examine the postinjury response using dual-platform microarray analysis, revealing responses that may enable novel therapies or diagnostics.

The role of initial trauma in the host's response to injury and hemorrhage: insights from a correlation of mathematical simulations and hepatic transcriptomic analysis.

Trauma and hemorrhagic shock (HS) elicit severe physiological disturbances that predispose the victims to subsequent organ dysfunction and death. The general lack of effective therapeutic options for these patients is mainly due to the complex interplay of interacting inflammatory and physiological elements working at multiple levels. Systems biology has emerged as a new paradigm that allows the study of large portions of physiological networks simultaneously. Seeking a better understanding of the interplay among known inflammatory pathways, we constructed a mathematical model encompassing the dynamics of the acute inflammatory response that incorporates the intertwined effects of inflammation and global tissue damage. The model was calibrated using data from C57Bl/6 mice subjected to endotoxemia, sham operation (i.e., surgical trauma induced by cannulation [ST]) or ST + HS+ resuscitation (ST-HS-R). An in silico simulation, made at whole-organism level, suggested that similar pathways of different magnitudes were operant as the degree of total body damage increased. We sought to validate this hypothesis by subjecting mice to HS and comparing the models predictions to circulating markers of inflammation and tissue injury as well as the global transcriptomic response of the liver. C57Bl/6 mice were subjected to ST or ST-HS (without resuscitation). Liver gene expression was assessed using an Affymetrix DNA microarray (GeneChip Mouse Expression Set 430A, Affymetrix, Santa Clara, CA), which contains 22,621 probe sets and effectively interrogates 12,341 mouse genes. The microarray data sets were subjected to hierarchical clustering and pathway analysis. In agreement with model predictions, circulating levels of inflammation/tissue injury markers and the microarray analysis both demonstrated that ST alone accounts for a substantial proportion of the observed phenotypic and genetic/molecular changes versus untreated animals. The addition of HS further increased the magnitude of gene expression, but relatively few additional genes were recruited. Mathematical simulations and DNA microarrays, both systems biology tools, may provide valuable insight into the complex global physiological interactions that occur in response to trauma and hemorrhagic shock.

In silico models of acute inflammation in animals.

Trauma and hemorrhagic shock elicit an acute inflammatory response, predisposing patients to sepsis, organ dysfunction, and death. Few approved therapies exist for these acute inflammatory states, mainly due to the complex interplay of interacting inflammatory and physiological elements working at multiple levels. Various animal models have been used to simulate these phenomena, but these models often do not replicate the clinical setting of multiple overlapping insults. Mathematical modeling of complex systems is an approach for understanding the interplay among biological interactions. We constructed a mathematical model using ordinary differential equations that encompass the dynamics of cells and cytokines of the acute inflammatory response, as well as global tissue dysfunction. The model was calibrated in C57Bl/6 mice subjected to (1) various doses of lipopolysaccharide (LPS) alone, (2) surgical trauma, and (3) surgery + hemorrhagic shock. We tested the model's predictive ability in scenarios on which it had not been trained, namely, (1) surgery +/- hemorrhagic shock + LPS given at times after the beginning of surgical instrumentation, and (2) surgery + hemorrhagic shock + bilateral femoral fracture. Software was created that facilitated fitting of the mathematical model to experimental data, as well as for simulation of experiments with various inflammatory challenges and associated variations (gene knockouts, inhibition of specific cytokines, etc.). Using this software, the C57Bl/6-specific model was recalibrated for inflammatory analyte data in CD14-/- mice and was used to elucidate altered features of inflammation in these animals. In other experiments, rats were subjected to surgical trauma +/- LPS or to bacterial infection via fibrin clots impregnated with various inocula of Escherichia coli. Mathematical modeling may provide insights into the complex dynamics of acute inflammation in a manner that can be tested in vivo using many fewer animals than has been possible previously.

Modeling and Hemofiltration Treatment of Acute Inflammation.

The body responds to endotoxins by triggering the acute inflammatory response system to eliminate the threat posed by gram-negative bacteria (endotoxin) and restore health. However, an uncontrolled inflammatory response can lead to tissue damage, organ failure, and ultimately death; this is clinically known as sepsis. Mathematical models of acute inflammatory disease have the potential to guide treatment decisions in critically ill patients. In this work, an 8-state (8-D) differential equation model of the acute inflammatory response system to endotoxin challenge was developed. Endotoxin challenges at 3 and 12 mg/kg were administered to rats, and dynamic cytokine data for interleukin (IL)-6, tumor necrosis factor (TNF), and IL-10 were obtained and used to calibrate the model. Evaluation of competing model structures was performed by analyzing model predictions at 3, 6, and 12 mg/kg endotoxin challenges with respect to experimental data from rats. Subsequently, a model predictive control (MPC) algorithm was synthesized to control a hemoadsorption (HA) device, a blood purification treatment for acute inflammation. A particle filter (PF) algorithm was implemented to estimate the full state vector of the endotoxemic rat based on time series cytokine measurements. Treatment simulations show that: (i) the apparent primary mechanism of HA efficacy is white blood cell (WBC) capture, with cytokine capture a secondary benefit; and (ii) differential filtering of cytokines and WBC does not provide substantial improvement in treatment outcomes vs. existing HA devices.

The acute inflammatory response in diverse shock states.

A poorly controlled acute inflammatory response can lead to organ dysfunction and death. Severe systemic inflammation can be induced and perpetuated by diverse insults such as the administration of toxic bacterial products (e.g., endotoxin), traumatic injury, and hemorrhage. Here, we probe whether these varied shock states can be explained by a universal inflammatory system that is initiated through different means and, once initiated, follows a course specified by the cellular and molecular mechanisms of the immune and endocrine systems. To examine this question, we developed a mathematical model incorporating major elements of the acute inflammatory response in C57Bl/6 mice, using input from experimental data. We found that a single model with different initiators including the autonomic system could describe the response to various insults. This model was able to predict a dose range of endotoxin at which mice would die despite having been calibrated only in nonlethal inflammatory paradigms. These results show that the complex biology of inflammation can be modeled and supports the hypothesis that shock states induced by a range of physiologic challenges could arise from a universal response that is differently initiated and modulated.

Effects of volume resuscitation on splanchnic perfusion in canine model of severe sepsis induced by live Escherichia coli infusion.

INTRODUCTION: We conducted the present study to investigate whether early large-volume crystalloid infusion can restore gut mucosal blood flow and mesenteric oxygen metabolism in severe sepsis. METHODS: Anesthetized and mechanically ventilated male mongrel dogs were challenged with intravenous injection of live Escherichia coli (6 x 10(9) colony-forming units/ml per kg over 15 min). After 90 min they were randomly assigned to one of two groups - control (no fluids; n = 13) or lactated Ringer's solution (32 ml/kg per hour; n = 14) - and followed for 60 min. Cardiac index, mesenteric blood flow, mean arterial pressure, systemic and mesenteric oxygen-derived variables, blood lactate and gastric carbon dioxide tension (PCO2; by gas tonometry) were assessed throughout the study. RESULTS: E. coli infusion significantly decreased arterial pressure, cardiac index, mesenteric blood flow, and systemic and mesenteric oxygen delivery, and increased arterial and portal lactate, intramucosal PCO2, PCO2 gap (the difference between gastric mucosal and arterial PCO2), and systemic and mesenteric oxygen extraction ratio in both groups. The Ringer's solution group had significantly higher cardiac index and systemic oxygen delivery, and lower oxygen extraction ratio and PCO2 gap at 165 min as compared with control animals. However, infusion of lactated Ringer's solution was unable to restore the PCO2 gap. There were no significant differences between groups in mesenteric oxygen delivery, oxygen extraction ratio, or portal lactate at the end of study. CONCLUSION: Significant disturbances occur in the systemic and mesenteric beds during bacteremic severe sepsis. Although large-volume infusion of lactated Ringer's solution restored systemic hemodynamic parameters, it was unable to correct gut mucosal PCO2 gap.

An exploratory pathways analysis of temporal changes induced by spinal cord injury in the rat bladder wall: insights on remodeling and inflammation.

BACKGROUND: Spinal cord injuries (SCI) can lead to severe bladder pathologies associated with inflammation, fibrosis, and increased susceptibility to urinary tract infections. We sought to characterize the complex pathways of remodeling, inflammation, and infection in the urinary bladder at the level of the transcriptome in a rat model of SCI, using pathways analysis bioinformatics. METHODOLOGY/PRINCIPAL FINDINGS: Experimental data were obtained from the study of Nagatomi et al. (Biochem Biophys Res Commun 334: 1159). In this study, bladders from rats subjected to surgical SCI were obtained at 3, 7 or 25 days post-surgery, and Affymetrix GeneChip Rat Genome U34A arrays were used for cRNA hybridizations. In the present study, Ingenuity Pathways Analysis (Ingenuity Systems, www.ingenuity.com) of differentially expressed genes was performed. Analysis of focus genes in networks, functional analysis, and canonical pathway analysis reinforced our previous findings related to the presence of up-regulated genes involved in tissue remodeling, such as lysyl oxidase, tropoelastin, TGF-beta1, and IGF-1. This analysis also highlighted a central role for inflammation and infection, evidenced by networks containing genes such as CD74, S100A9, and THY1. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that tissue remodeling, infection, inflammation, and tissue damage/dysfunction all play a role in the urinary bladder, in the complex response to SCI.

In silico and in vivo approach to elucidate the inflammatory complexity of CD14-deficient mice.

The inflammatory phenotype of genetically modified mice is complex, and the role of Gram-negative lipopolysaccharide (LPS) in acute inflammation induced by surgical cannulation trauma, alone or in combination with hemorrhage and resuscitation ("hemorrhagic shock"), is both complex and controversial. We sought to determine if a mathematical model of acute inflammation could elucidate both the phenotype of CD14-deficient (CD14(-/-)) mice--following LPS, cannulation, or hemorrhagic shock--and the role of LPS in trauma/hemorrhage-induced inflammation. A mathematical model of inflammation initially calibrated in wild-type (C57Bl/6) mice subjected to LPS, cannulation, and hemorrhagic shock was recalibrated in CD14(-/-) mice subjected to the same insults, yielding an ensemble of models that suggested specific differences at the cellular and molecular levels (for example, 43-fold lower activation of leukocytes by LPS). The CD14(-/-)-specific model ensemble could account for complex changes in inflammatory analytes in these mice following LPS treatment. Model prediction of similar organ damage in CD14(-/-) and wild-type mice subjected to cannulation alone or with hemorrhagic shock was verified in vivo (similar ALT levels). These studies suggest that LPS-CD14 responses do not cause inflammation in surgical trauma/hemorrhagic shock and demonstrate a novel use of combined in silico and in vivo methods to elucidate the complex inflammatory phenotype of genetically modified animals.

The role of hepatic type 1 plasminogen activator inhibitor (PAI-1) during murine hemorrhagic shock.

Hemorrhagic shock (HS) followed by resuscitation (HS-R) is characterized by profound physiological changes. Even if the patient survives the initial blood loss, these poorly understood changes can lead to morbidity. One of the tissues most often affected is liver. We sought to recognize specific hepatic changes induced by this stressor to identify targets for therapeutic intervention. Gene array analyses using mouse liver mRNAs were used to identify candidate genes that contribute to hepatic damage. To verify the role of one of the genes identified using the arrays, mice were subjected to HS-R, and multiple parameters were analyzed. A profound increase in plasminogen activator inhibitor type 1 (PAI-1) mRNA was observed using hepatic mRNAs from C57Bl/6 mice after HS, both with and without resuscitation. Constitutive loss of PAI-1 resulted in notable tissue preservation and lower (P < .05) alanine aminotransferase (ALT) levels. Fibrin degradation products (FDPs) and interleukins 6 and 10 (IL-6 and IL-10) were unaffected by loss of PAI-1; however, enhanced urokinase activity, an elevation of active hepatocyte growth factor (HGF), an increase in unprocessed transforming growth factor-beta1 (TGF-beta1), and retention of ERK phosphorylation after HS-R were associated with improved hepatic function. In conclusion, PAI-1 protein is a negative effector of hepatic damage after HS-R through its influence on classic regulators of hepatic growth, as opposed to its role in fibrinolysis.