1) The study examined how an initial trauma-hemorrhage event (the first hit) modulated the response of plasminogen activator inhibitor 1 (PAI-1) in a subsequent polymicrobial sepsis infection (the second hit).
2) PAI-1 levels were measured in the blood, tissues, and organs. An initial rise in circulating PAI-1 levels was seen after trauma-hemorrhage, followed by a delayed decrease in PAI-1 mRNA levels in organs.
3) After sepsis infection, circulating PAI-1 profiles were similar between mice that experienced trauma-hemorrhage and those that did not. However, PAI-1 mRNA declined more in
The study found that viral hepatitis causes downregulation of antioxidant genes like superoxide dismutase 1 (SOD1) in the liver. Mice lacking SOD1 (Sod1-/- mice) exhibited significantly increased liver damage when infected with lymphocytic choriomeningitis virus (LCMV), as shown by greater weight loss, higher liver enzyme levels, and more severe histological lesions, compared to wild-type mice. This liver pathology in Sod1-/- mice was independent of viral load and occurred through a mechanism of increased oxidative stress and hepatocyte apoptosis driven by type I interferon (IFN-I) signaling. Genetic or pharmacological inhibition of IFN-I signaling protected both Sod
Faustman & Davis Nature Drug Disc 2010; 9;482-93Miriam Davis
Tumour necrosis factor (TNF) is a key signaling protein in the immune system that orchestrates communication between immune cells and controls their functions. It is involved in both immune defenses and controlling whether cells live or die. Defects in the TNF signaling pathway have been found in autoreactive T cells associated with various autoimmune diseases like Crohn's disease, multiple sclerosis, and type 1 diabetes. Targeting the TNF receptor 2 (TNFR2) pathway may provide a safer way to selectively induce apoptosis in autoreactive T cells and treat certain autoimmune diseases while avoiding toxicity seen with other therapies.
This document describes an experiment investigating the effects of fenugreek extract on macrophage polarization using a THP-1 cell model. THP-1 macrophages were treated with two concentrations of fenugreek extract and the M1 marker CD86 and M2 marker CD163 were measured using flow cytometry. Preliminary experiments determined that EDTA was the optimal method for detaching adherent THP-1 macrophages without reducing cell viability. The results found that fenugreek upregulated CD86 but downregulated CD163 expression. This suggests fenugreek caused polarization of macrophages toward an M1 phenotype, though further shifted this to an M4 phenotype. The effect on CD163 was possibly due to multiple M2 subclasses making expression harder to measure
1. General anaesthesia can have both direct and indirect effects on the immune system by impacting the innate immune response, adaptive immune response, cytokine production, neutrophil activity, and immunoglobulin levels.
2. Surgery alone increases pro-inflammatory cytokine levels, but anaesthetic agents may increase or decrease specific cytokine production depending on the agent.
3. Perioperative interventions like mechanical ventilation, blood transfusions, chronic pain, and immunosuppressive drugs for transplant patients can further impact the immune response. Precautions are needed for patients with these factors.
Immunosuppression involves reducing the activation of the immune system through medications, surgery, radiation or other means. The document discusses the history of immunosuppression and developments in transplantation including the identification of cortisone and research on the immune system. It outlines current methods of immunosuppression including calcineurin inhibitors, mTOR inhibitors, antimetabolites, corticosteroids, depleting antibodies and fusion proteins. The mechanisms, types and treatment of allograft rejection such as hyperacute, acute and chronic rejection are also summarized.
This document summarizes research examining how the HIV envelope protein gp120 induces apoptosis in lung microvascular endothelial cells. The key findings are:
1) Gp120 upregulates surface expression of the proinflammatory cytokine EMAP II and its receptor CXCR3 on endothelial cells in a rapid and robust manner.
2) This upregulation occurs via gp120 signaling through its CXCR4 receptor and activation of the p38 MAPK pathway.
3) Both EMAP II and CXCR3 are essential for gp120-induced endothelial cell apoptosis.
4) The findings suggest a novel mechanism by which HIV infection causes endothelial cell loss and damage, contributing to lung diseases like emphysema.
The Effects of CD20 inhibitors therapy in comparison to TNF α inhibitors ther...IOSR Journals
Rheumatoid arthritis (RA) is a chronic , systemic , inflammatory disorder that may affect many tissues and organs ,once a diagnosis is made , the maintreatment goals are to control disease activity and slow the rate of joint damage ,in addition to minimizing pain, stiffnes, in flammation and complications. important role of IL-17 in the development of disease and can be used as a marker for monitoring of disease activity . Aim of the Study The aim of the present study to evaluate the effects of CD20 inhibitors therapy in comparison to effects of TNF α inhibitors therapy on IL-17 in patients with active rheumatoid arthritis . Results:- results obtained in the present stydy showd that serum level of IL-17were also decreasesignificantly in patients treated with Rituximab group 3 ( 2.28 ) than those of group 2 Etanercept (anti-TNFα) treated group patients (3.3). Conclution:- the ro
The document discusses the immune system and inflammation. It describes how the immune system protects the host from foreign molecules but can also result in problems like inflammation. Inflammation is characterized by redness, heat, swelling, pain and loss of function. These signs are caused by changes in blood vessels and cells driven by cytokines and other inflammatory mediators released during inflammation. The document also examines the role of various immune cells, cytokines and other mediators in both innate and adaptive immunity as well as the pathogenesis of diseases like arthritis, asthma and cancer. It explores potential therapeutic approaches targeting these mechanisms.
The study found that viral hepatitis causes downregulation of antioxidant genes like superoxide dismutase 1 (SOD1) in the liver. Mice lacking SOD1 (Sod1-/- mice) exhibited significantly increased liver damage when infected with lymphocytic choriomeningitis virus (LCMV), as shown by greater weight loss, higher liver enzyme levels, and more severe histological lesions, compared to wild-type mice. This liver pathology in Sod1-/- mice was independent of viral load and occurred through a mechanism of increased oxidative stress and hepatocyte apoptosis driven by type I interferon (IFN-I) signaling. Genetic or pharmacological inhibition of IFN-I signaling protected both Sod
Faustman & Davis Nature Drug Disc 2010; 9;482-93Miriam Davis
Tumour necrosis factor (TNF) is a key signaling protein in the immune system that orchestrates communication between immune cells and controls their functions. It is involved in both immune defenses and controlling whether cells live or die. Defects in the TNF signaling pathway have been found in autoreactive T cells associated with various autoimmune diseases like Crohn's disease, multiple sclerosis, and type 1 diabetes. Targeting the TNF receptor 2 (TNFR2) pathway may provide a safer way to selectively induce apoptosis in autoreactive T cells and treat certain autoimmune diseases while avoiding toxicity seen with other therapies.
This document describes an experiment investigating the effects of fenugreek extract on macrophage polarization using a THP-1 cell model. THP-1 macrophages were treated with two concentrations of fenugreek extract and the M1 marker CD86 and M2 marker CD163 were measured using flow cytometry. Preliminary experiments determined that EDTA was the optimal method for detaching adherent THP-1 macrophages without reducing cell viability. The results found that fenugreek upregulated CD86 but downregulated CD163 expression. This suggests fenugreek caused polarization of macrophages toward an M1 phenotype, though further shifted this to an M4 phenotype. The effect on CD163 was possibly due to multiple M2 subclasses making expression harder to measure
1. General anaesthesia can have both direct and indirect effects on the immune system by impacting the innate immune response, adaptive immune response, cytokine production, neutrophil activity, and immunoglobulin levels.
2. Surgery alone increases pro-inflammatory cytokine levels, but anaesthetic agents may increase or decrease specific cytokine production depending on the agent.
3. Perioperative interventions like mechanical ventilation, blood transfusions, chronic pain, and immunosuppressive drugs for transplant patients can further impact the immune response. Precautions are needed for patients with these factors.
Immunosuppression involves reducing the activation of the immune system through medications, surgery, radiation or other means. The document discusses the history of immunosuppression and developments in transplantation including the identification of cortisone and research on the immune system. It outlines current methods of immunosuppression including calcineurin inhibitors, mTOR inhibitors, antimetabolites, corticosteroids, depleting antibodies and fusion proteins. The mechanisms, types and treatment of allograft rejection such as hyperacute, acute and chronic rejection are also summarized.
This document summarizes research examining how the HIV envelope protein gp120 induces apoptosis in lung microvascular endothelial cells. The key findings are:
1) Gp120 upregulates surface expression of the proinflammatory cytokine EMAP II and its receptor CXCR3 on endothelial cells in a rapid and robust manner.
2) This upregulation occurs via gp120 signaling through its CXCR4 receptor and activation of the p38 MAPK pathway.
3) Both EMAP II and CXCR3 are essential for gp120-induced endothelial cell apoptosis.
4) The findings suggest a novel mechanism by which HIV infection causes endothelial cell loss and damage, contributing to lung diseases like emphysema.
The Effects of CD20 inhibitors therapy in comparison to TNF α inhibitors ther...IOSR Journals
Rheumatoid arthritis (RA) is a chronic , systemic , inflammatory disorder that may affect many tissues and organs ,once a diagnosis is made , the maintreatment goals are to control disease activity and slow the rate of joint damage ,in addition to minimizing pain, stiffnes, in flammation and complications. important role of IL-17 in the development of disease and can be used as a marker for monitoring of disease activity . Aim of the Study The aim of the present study to evaluate the effects of CD20 inhibitors therapy in comparison to effects of TNF α inhibitors therapy on IL-17 in patients with active rheumatoid arthritis . Results:- results obtained in the present stydy showd that serum level of IL-17were also decreasesignificantly in patients treated with Rituximab group 3 ( 2.28 ) than those of group 2 Etanercept (anti-TNFα) treated group patients (3.3). Conclution:- the ro
The document discusses the immune system and inflammation. It describes how the immune system protects the host from foreign molecules but can also result in problems like inflammation. Inflammation is characterized by redness, heat, swelling, pain and loss of function. These signs are caused by changes in blood vessels and cells driven by cytokines and other inflammatory mediators released during inflammation. The document also examines the role of various immune cells, cytokines and other mediators in both innate and adaptive immunity as well as the pathogenesis of diseases like arthritis, asthma and cancer. It explores potential therapeutic approaches targeting these mechanisms.
Dr aaditya recent advances in immunopharmacology 30 jan10Aaditya Udupa
The document discusses recent advances in immunopharmacology. It covers understanding immunology, modulating the immune response, evaluating immunological agents, and recent markers and parameters. Newer immunomodulatory drugs and their mechanisms of action and adverse drug reactions are also described. These include glucocorticoids, immunophilin ligands like cyclosporine and tacrolimus, proliferation signal inhibitors like sirolimus, mycophenolate mofetil, and thalidomide.
The role of natural products in regulating pyroptosisLucyPi1
This document reviews the role of natural products in regulating pyroptosis. It begins with background on pyroptosis, describing it as a form of inflammatory programmed cell death. It then discusses how pyroptosis is involved in various diseases and its molecular pathways. The document categorizes 14 natural products that have been shown to affect pyroptosis, including flavonoids like dihydromyricetin and terpenoids like andrographolide. These natural products were found to negatively or positively impact pyroptosis pathways by regulating factors like caspase-1, gasdermin D, and cytokines. The review concludes that natural products have potential as sources of new drugs for diseases related to uncontrolled pyroptosis.
The document discusses the systemic inflammatory response that occurs after injury or infection. It describes two phases: an acute pro-inflammatory phase aimed at restoring function and fighting infection, and an anti-inflammatory phase that modulates the pro-inflammatory response to prevent excess and restore homeostasis. It then defines terms related to infection and inflammation and discusses the central nervous system regulation of inflammation through hormonal and neuronal pathways.
This document discusses immunosuppressant drugs, their mechanisms of action, uses, and complications. It describes how immunosuppressants work by inhibiting cytokines, cytokine production, or cytotoxic immune cells. Major drugs covered include calcineurin inhibitors cyclosporine and tacrolimus, mTOR inhibitor sirolimus, corticosteroids, antimetabolites azathioprine and mycophenolate, cytotoxic drugs, and immunosuppressive antibodies. Complications of immunosuppression can include infection, nephrotoxicity, bone marrow toxicity, and increased cancer risk. Each drug profile provides therapeutic uses and adverse effects.
Immunosuppressants are drugs used to modulate the immune system. They have two main objectives: producing desired immunity through immunization, and eliminating unwanted immune reactions. The main classes of immunosuppressants are calcineurin inhibitors like cyclosporine and tacrolimus, antiproliferative drugs, glucocorticoids, and antibodies. Cyclosporine inhibits T cell activation to suppress transplant rejection and autoimmune diseases, while having fewer side effects than other drugs. Immunosuppressants are indicated for organ transplants, infectious diseases, allergies, and autoimmune disorders like rheumatoid arthritis.
Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune diseases characterized by inflammation of small blood vessels. The diseases include granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). AAV is diagnosed based on clinical presentation and presence of ANCAs. Treatment aims to suppress the immune system and minimize organ damage through individualized regimens. Advances in treatment have improved survival but infection risk remains high.
The Negative Clinical Consequences Due to the Lack of the Elaboration of a Sc...asclepiuspdfs
Until a few years ago, the immune system was considered as responsible for the only defense against microbial infections and other external agents. On the contrary, the immune cells have been proven to be linked not only through cell-cell contact but also by releasing proteins capable of influencing the immune-inflammatory response, the so-called cytokines or interleukins. Moreover, the cytokines have appeared to play not only immune activities but also metabolic and systemic effects influencing the overall biological systems, including the nervous, the endocrine, and the cardiovascular systems, by representing the main endogenous molecules responsible for the maintenance of the unity of the biological life. Therefore, only the systematic clinical consideration of cytokine effects may allow the generation of real future holistic medicine.
vascular insulin and IGF-1 in diabetic woundsSaeed Aghdam
This document summarizes a study examining the role of vascular endothelial insulin/IGF-1 signaling in skin wound healing. The study found that deleting the insulin receptor and IGF-1 receptor specifically in endothelial cells of mice (DKOIVE mice) did not significantly impact skin vessel homeostasis under normal conditions. However, when the skin was wounded, DKOIVE mice showed strongly reduced neovascularization during wound healing compared to controls, accompanied by reduced granulation tissue formation. This indicates that endothelial insulin/IGF signaling is essential for neovascularization during wound healing and implies its reduction directly contributes to impaired healing associated with diabetes.
Novel immunotherapies for rheumatoid arthritisNJayabalan
This document discusses novel immunotherapies for rheumatoid arthritis that have been approved by the FDA. It provides details on 6 categories of immunotherapies: B cell depletion therapy, T cell costimulation inhibition, anti-IL-6 therapy, anti-TNFα therapy, Janus kinase inhibitors, and biosimilars. For each category, it describes example drugs, their mechanisms of action, effects, and potential side effects and risks. Overall, the document suggests that improved understanding of RA immunopathogenesis has led to more personalized, effective, and cost-efficient treatment options.
Cytokines are proteins involved in cell signaling and communication that are important in immune responses and inflammation. They are produced by immune cells and modulate processes like immune cell development, activation of lymphocytes and phagocytes, and the inflammatory response. Cytokines play roles in diseases like cancer, rheumatoid arthritis, sepsis, and tuberculosis by influencing processes such as immune cell recruitment and activation, tissue damage, and granuloma formation. Their levels can provide information about disease activity and progression in some conditions.
This document discusses rheumatoid arthritis (RA) and the evolution of drugs used to treat it. RA is a chronic inflammatory disease that causes joint damage and disability. While the cause is unknown, it involves an immune system response leading to inflammation. Treatment has progressed from nonsteroidal anti-inflammatory drugs (NSAIDs) and disease-modifying antirheumatic drugs (DMARDs) like methotrexate, to biological DMARDs that target specific cytokines and cells involved in the immune response, such as tumor necrosis factor (TNF) inhibitors and interleukin-6 (IL-6) inhibitors. Larger clinical trials were required to develop these targeted biologic therapies compared to earlier drugs. Animal models of collagen
This presentation discusses immunosuppressant drugs, which inhibit the immune system. These drugs are used for organ transplantation to prevent rejection and for autoimmune diseases. The presentation classifies immunosuppressants and describes the mechanisms of several major drug classes, including calcineurin inhibitors, antiproliferatives, antibodies, and glucocorticoids. Key mechanisms covered include inhibition of T-cell signaling, blocking of DNA synthesis and cell proliferation, and disruption of T-cell receptor function. The goal of the presentation is to explain how different immunosuppressant drugs work at the molecular level.
The document discusses various types of immunosuppressive drugs used in clinical practice including corticosteroids, cytostatic drugs, immunsuppressive agents like cyclosporine A and tacrolimus, cytokine and cytokine receptor antibodies, and leukopheresis. These drugs act through different mechanisms such as inhibiting T cell activation, cell proliferation, cytokine production and signaling. The document also covers the indications for immunosuppression including inflammation, allergic diseases, autoimmune diseases, and transplantation. It describes the mechanisms, examples and side effects of the main classes of immunosuppressive medications.
This study developed a new mouse model that lacks all isoforms of the Trim24 gene. Mice with complete loss of Trim24 (Trim24-/-) spontaneously developed hepatic lipid accumulation, inflammation, fibrosis, and hepatocellular carcinoma without a high-fat diet or other induction methods. Trim24-/- mice had decreased expression of genes involved in oxidation, lipid metabolism, and increased expression of genes related to endoplasmic reticulum stress and cell cycle pathways. By 6 months of age, Trim24-/- mice exhibited macroscopic white liver lesions composed of steatosis and fibrosis that progressed to hepatocellular carcinoma, recapitulating features of non-alcoholic fatty liver disease in humans
1) Treatment with the TNF-α antagonist etanercept was associated with improvements in asthma-related quality of life scores, lung function, and exacerbations in patients with severe corticosteroid-refractory asthma in an open-label clinical trial.
2) A double-blind placebo-controlled trial found that 10 weeks of etanercept treatment was associated with increased bronchial hyperresponsiveness and improved asthma control in patients with refractory asthma but not mild-moderate asthma.
3) Intravenous infusion of the TNF-α antagonist infliximab was associated with decreased exacerbations and improved lung function in a double-blind placebo-controlled trial of patients with moderate asthma uncontrolled by inhaled
Silencing c-Myc translation as a therapeutic strategy through targeting PI3Kd...Mark Lipstein
This document summarizes a study examining the combination of a novel PI3Kδ inhibitor, TGR-1202, with the proteasome inhibitor carfilzomib for treating hematological malignancies. The study found that TGR-1202 synergizes strongly with carfilzomib in lymphoma, leukemia, and myeloma cell lines and primary cells by silencing c-Myc translation. This synergistic effect is driven by TGR-1202's unexpected additional activity of inhibiting CK1ε, which contributes to repressing phosphorylation of 4E-BP1 and lowering c-Myc protein levels. The results suggest that TGR-1202, as a dual PI3Kδ/CK1ε inhibitor, may have
This document provides an overview of immunosuppressant drugs. It begins with an introduction to immunity and the types of immunity. It then discusses antigens and antibodies, as well as the action of antigens and antibodies. The document covers non-specific defenses and introduces immunosuppressant drugs. It classifies immunosuppressants and discusses their mechanisms and side effects. The document focuses on calcineurin inhibitors like cyclosporine and tacrolimus, mTOR inhibitors like sirolimus, and antiproliferative drugs. It provides details on the mechanisms, uses, and side effects of these major classes of immunosuppressant drugs.
This document presents information about autoimmune diseases from MD. Zahirul Islam. It defines autoimmune disease as a condition where the body produces antibodies that attack its own tissues, leading to damage. It provides examples of common autoimmune diseases like lupus, rheumatoid arthritis, and diabetes. The document notes there are two main types - systemic disorders involving non-specific autoantibodies and local disorders targeting specific tissues. It discusses the most common organs and tissues affected by autoimmune diseases and describes methods of diagnosis and treatment options for some conditions.
The document describes the four life cycle stages of a butterfly: egg, larva/caterpillar, pupa/chrysalis, and adult. The female attaches eggs to leaves which hatch into caterpillars that feed and grow, then form a protective pupa casing while transforming inside, and finally emerge as adult butterflies that migrate or find new habitats.
El documento presenta la información personal de Viviana Patricia Cepeda Quenguan, de 19 años, quien se graduó de la institución educativa San Juan y estudió técnico en sistemas en el Servicio Nacional de Aprendizaje (SENA). Además, describe la misión y visión del SENA, así como algunos de sus símbolos como el himno, escudo y bandera. Finalmente, explica brevemente los diferentes tipos de vinculación laboral que puede tener un aprendiz del SENA, como contratos de aprendizaje, pasantías y proyect
Dr aaditya recent advances in immunopharmacology 30 jan10Aaditya Udupa
The document discusses recent advances in immunopharmacology. It covers understanding immunology, modulating the immune response, evaluating immunological agents, and recent markers and parameters. Newer immunomodulatory drugs and their mechanisms of action and adverse drug reactions are also described. These include glucocorticoids, immunophilin ligands like cyclosporine and tacrolimus, proliferation signal inhibitors like sirolimus, mycophenolate mofetil, and thalidomide.
The role of natural products in regulating pyroptosisLucyPi1
This document reviews the role of natural products in regulating pyroptosis. It begins with background on pyroptosis, describing it as a form of inflammatory programmed cell death. It then discusses how pyroptosis is involved in various diseases and its molecular pathways. The document categorizes 14 natural products that have been shown to affect pyroptosis, including flavonoids like dihydromyricetin and terpenoids like andrographolide. These natural products were found to negatively or positively impact pyroptosis pathways by regulating factors like caspase-1, gasdermin D, and cytokines. The review concludes that natural products have potential as sources of new drugs for diseases related to uncontrolled pyroptosis.
The document discusses the systemic inflammatory response that occurs after injury or infection. It describes two phases: an acute pro-inflammatory phase aimed at restoring function and fighting infection, and an anti-inflammatory phase that modulates the pro-inflammatory response to prevent excess and restore homeostasis. It then defines terms related to infection and inflammation and discusses the central nervous system regulation of inflammation through hormonal and neuronal pathways.
This document discusses immunosuppressant drugs, their mechanisms of action, uses, and complications. It describes how immunosuppressants work by inhibiting cytokines, cytokine production, or cytotoxic immune cells. Major drugs covered include calcineurin inhibitors cyclosporine and tacrolimus, mTOR inhibitor sirolimus, corticosteroids, antimetabolites azathioprine and mycophenolate, cytotoxic drugs, and immunosuppressive antibodies. Complications of immunosuppression can include infection, nephrotoxicity, bone marrow toxicity, and increased cancer risk. Each drug profile provides therapeutic uses and adverse effects.
Immunosuppressants are drugs used to modulate the immune system. They have two main objectives: producing desired immunity through immunization, and eliminating unwanted immune reactions. The main classes of immunosuppressants are calcineurin inhibitors like cyclosporine and tacrolimus, antiproliferative drugs, glucocorticoids, and antibodies. Cyclosporine inhibits T cell activation to suppress transplant rejection and autoimmune diseases, while having fewer side effects than other drugs. Immunosuppressants are indicated for organ transplants, infectious diseases, allergies, and autoimmune disorders like rheumatoid arthritis.
Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune diseases characterized by inflammation of small blood vessels. The diseases include granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). AAV is diagnosed based on clinical presentation and presence of ANCAs. Treatment aims to suppress the immune system and minimize organ damage through individualized regimens. Advances in treatment have improved survival but infection risk remains high.
The Negative Clinical Consequences Due to the Lack of the Elaboration of a Sc...asclepiuspdfs
Until a few years ago, the immune system was considered as responsible for the only defense against microbial infections and other external agents. On the contrary, the immune cells have been proven to be linked not only through cell-cell contact but also by releasing proteins capable of influencing the immune-inflammatory response, the so-called cytokines or interleukins. Moreover, the cytokines have appeared to play not only immune activities but also metabolic and systemic effects influencing the overall biological systems, including the nervous, the endocrine, and the cardiovascular systems, by representing the main endogenous molecules responsible for the maintenance of the unity of the biological life. Therefore, only the systematic clinical consideration of cytokine effects may allow the generation of real future holistic medicine.
vascular insulin and IGF-1 in diabetic woundsSaeed Aghdam
This document summarizes a study examining the role of vascular endothelial insulin/IGF-1 signaling in skin wound healing. The study found that deleting the insulin receptor and IGF-1 receptor specifically in endothelial cells of mice (DKOIVE mice) did not significantly impact skin vessel homeostasis under normal conditions. However, when the skin was wounded, DKOIVE mice showed strongly reduced neovascularization during wound healing compared to controls, accompanied by reduced granulation tissue formation. This indicates that endothelial insulin/IGF signaling is essential for neovascularization during wound healing and implies its reduction directly contributes to impaired healing associated with diabetes.
Novel immunotherapies for rheumatoid arthritisNJayabalan
This document discusses novel immunotherapies for rheumatoid arthritis that have been approved by the FDA. It provides details on 6 categories of immunotherapies: B cell depletion therapy, T cell costimulation inhibition, anti-IL-6 therapy, anti-TNFα therapy, Janus kinase inhibitors, and biosimilars. For each category, it describes example drugs, their mechanisms of action, effects, and potential side effects and risks. Overall, the document suggests that improved understanding of RA immunopathogenesis has led to more personalized, effective, and cost-efficient treatment options.
Cytokines are proteins involved in cell signaling and communication that are important in immune responses and inflammation. They are produced by immune cells and modulate processes like immune cell development, activation of lymphocytes and phagocytes, and the inflammatory response. Cytokines play roles in diseases like cancer, rheumatoid arthritis, sepsis, and tuberculosis by influencing processes such as immune cell recruitment and activation, tissue damage, and granuloma formation. Their levels can provide information about disease activity and progression in some conditions.
This document discusses rheumatoid arthritis (RA) and the evolution of drugs used to treat it. RA is a chronic inflammatory disease that causes joint damage and disability. While the cause is unknown, it involves an immune system response leading to inflammation. Treatment has progressed from nonsteroidal anti-inflammatory drugs (NSAIDs) and disease-modifying antirheumatic drugs (DMARDs) like methotrexate, to biological DMARDs that target specific cytokines and cells involved in the immune response, such as tumor necrosis factor (TNF) inhibitors and interleukin-6 (IL-6) inhibitors. Larger clinical trials were required to develop these targeted biologic therapies compared to earlier drugs. Animal models of collagen
This presentation discusses immunosuppressant drugs, which inhibit the immune system. These drugs are used for organ transplantation to prevent rejection and for autoimmune diseases. The presentation classifies immunosuppressants and describes the mechanisms of several major drug classes, including calcineurin inhibitors, antiproliferatives, antibodies, and glucocorticoids. Key mechanisms covered include inhibition of T-cell signaling, blocking of DNA synthesis and cell proliferation, and disruption of T-cell receptor function. The goal of the presentation is to explain how different immunosuppressant drugs work at the molecular level.
The document discusses various types of immunosuppressive drugs used in clinical practice including corticosteroids, cytostatic drugs, immunsuppressive agents like cyclosporine A and tacrolimus, cytokine and cytokine receptor antibodies, and leukopheresis. These drugs act through different mechanisms such as inhibiting T cell activation, cell proliferation, cytokine production and signaling. The document also covers the indications for immunosuppression including inflammation, allergic diseases, autoimmune diseases, and transplantation. It describes the mechanisms, examples and side effects of the main classes of immunosuppressive medications.
This study developed a new mouse model that lacks all isoforms of the Trim24 gene. Mice with complete loss of Trim24 (Trim24-/-) spontaneously developed hepatic lipid accumulation, inflammation, fibrosis, and hepatocellular carcinoma without a high-fat diet or other induction methods. Trim24-/- mice had decreased expression of genes involved in oxidation, lipid metabolism, and increased expression of genes related to endoplasmic reticulum stress and cell cycle pathways. By 6 months of age, Trim24-/- mice exhibited macroscopic white liver lesions composed of steatosis and fibrosis that progressed to hepatocellular carcinoma, recapitulating features of non-alcoholic fatty liver disease in humans
1) Treatment with the TNF-α antagonist etanercept was associated with improvements in asthma-related quality of life scores, lung function, and exacerbations in patients with severe corticosteroid-refractory asthma in an open-label clinical trial.
2) A double-blind placebo-controlled trial found that 10 weeks of etanercept treatment was associated with increased bronchial hyperresponsiveness and improved asthma control in patients with refractory asthma but not mild-moderate asthma.
3) Intravenous infusion of the TNF-α antagonist infliximab was associated with decreased exacerbations and improved lung function in a double-blind placebo-controlled trial of patients with moderate asthma uncontrolled by inhaled
Silencing c-Myc translation as a therapeutic strategy through targeting PI3Kd...Mark Lipstein
This document summarizes a study examining the combination of a novel PI3Kδ inhibitor, TGR-1202, with the proteasome inhibitor carfilzomib for treating hematological malignancies. The study found that TGR-1202 synergizes strongly with carfilzomib in lymphoma, leukemia, and myeloma cell lines and primary cells by silencing c-Myc translation. This synergistic effect is driven by TGR-1202's unexpected additional activity of inhibiting CK1ε, which contributes to repressing phosphorylation of 4E-BP1 and lowering c-Myc protein levels. The results suggest that TGR-1202, as a dual PI3Kδ/CK1ε inhibitor, may have
This document provides an overview of immunosuppressant drugs. It begins with an introduction to immunity and the types of immunity. It then discusses antigens and antibodies, as well as the action of antigens and antibodies. The document covers non-specific defenses and introduces immunosuppressant drugs. It classifies immunosuppressants and discusses their mechanisms and side effects. The document focuses on calcineurin inhibitors like cyclosporine and tacrolimus, mTOR inhibitors like sirolimus, and antiproliferative drugs. It provides details on the mechanisms, uses, and side effects of these major classes of immunosuppressant drugs.
This document presents information about autoimmune diseases from MD. Zahirul Islam. It defines autoimmune disease as a condition where the body produces antibodies that attack its own tissues, leading to damage. It provides examples of common autoimmune diseases like lupus, rheumatoid arthritis, and diabetes. The document notes there are two main types - systemic disorders involving non-specific autoantibodies and local disorders targeting specific tissues. It discusses the most common organs and tissues affected by autoimmune diseases and describes methods of diagnosis and treatment options for some conditions.
The document describes the four life cycle stages of a butterfly: egg, larva/caterpillar, pupa/chrysalis, and adult. The female attaches eggs to leaves which hatch into caterpillars that feed and grow, then form a protective pupa casing while transforming inside, and finally emerge as adult butterflies that migrate or find new habitats.
El documento presenta la información personal de Viviana Patricia Cepeda Quenguan, de 19 años, quien se graduó de la institución educativa San Juan y estudió técnico en sistemas en el Servicio Nacional de Aprendizaje (SENA). Además, describe la misión y visión del SENA, así como algunos de sus símbolos como el himno, escudo y bandera. Finalmente, explica brevemente los diferentes tipos de vinculación laboral que puede tener un aprendiz del SENA, como contratos de aprendizaje, pasantías y proyect
Este certificado reconoce la participación de Juan Arevalo Gutierrez en calidad de en el Primer Congreso Internacional de Ingeniería de Sistemas e Informática (CIS 2014) organizado por la Facultad de Ingeniería y la Escuela Académico Profesional de Ingeniería de Sistemas e Informática de la Universidad Cesar Vallejo del 10 al 12 de enero de 2014.
El documento describe tres tipos de herramientas de búsqueda de información en Internet: metabuscadores, motores de búsqueda y directorios temáticos. Los metabuscadores como Metacrawler permiten realizar una única búsqueda que consulta múltiples motores de búsqueda. Motores como AltaVista ofrecen búsquedas simples y avanzadas para encontrar páginas web. Finalmente, los directorios temáticos como InfoMine organizan la información de forma jerárquica por temas para una navegación más sencilla.
Este documento presenta una guía de práctica sobre Windows 8. Explica cómo usar funciones como Aero Snaps para ver varias ventanas a la vez, personalizar el escritorio, organizar iconos, tomar capturas de pantalla, usar notas rápidas, cambiar las vistas del explorador, seleccionar archivos y carpetas, y abrir múltiples ventanas usando atajos de teclado. También incluye ejercicios prácticos sobre cómo crear carpetas y archivos de texto en WordPad y eliminar elementos seleccionados.
This document contains three invitation for tenders for construction projects. The first is for construction of a new medical college building in Thiruvananthapuram, Kerala with an estimated cost of Rs. 4.94 crore. The second is for construction of two limited height subway crossings in West Bengal with a value of Rs. 2.83 crore. The third is for construction of a school building in Jampui, Tripura with an estimated cost of Rs. 3.78 crore.
Bakal Nyesal Seumur Hidup Kalau Kamu Belum Pernah ke 10 Tempat Ini!Caroline Winata
Pemerintah mengumumkan paket stimulus ekonomi baru untuk menyelamatkan bisnis dan pekerjaan. Paket ini memberi insentif pajak dan bantuan tunai untuk UMKM. Tujuannya menjaga stabilitas ekonomi selama pandemi.
Detlef Braun has over 30 years of experience in banking with a focus on payments, cash management, and liquidity management. He has worked for SmartStream Technologies, IBM, and several German banks. Most recently, he was a Pre-Sales consultant for SmartStream, supporting sales across Europe and globally. He has deep expertise in TARGET2, Basel regulations, and other major payment systems.
Клуб анонимных аналитиков, Инфографер, Москва, 19 октябряMaxim Gorchakov
Разбор текущих трендов в годовых отчетах крупных российских компаний. Представлено на собрании Клуба анонимных аналитиков в Москве 19 октября 2016 года
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1) The document describes four studies related to sepsis and the immune response.
2) The first study finds that autophagy plays a protective role in organ dysfunction in a mouse sepsis model, with autophagy induced in several organs over 24 hours after cecal ligation and puncture.
3) The second study finds that TNFα decreases heart rate, contractile force, and speeds of contraction and relaxation in an isolated perfused rat heart, and that these effects are partially reversible.
Morphological and functional state of immune organs in rats with experimental...QUESTJOURNAL
This study examined the morphological and functional changes in the immune organs (thymus and spleen) of rats with experimentally induced type 1 diabetes mellitus (DM-1). Rats with DM-1 showed initial pathological changes in these organs, including increased medulla in the thymus and increased white pulp in the spleen, indicating early inflammatory and degenerative processes. Treatment with the phytopreparation BNO 10.30 was found to help restore cell structure in the thymus and spleen by stimulating immune function. The results demonstrate the importance of early detection of immune organ changes in patients with type 1 diabetes.
The document discusses a study that found propagermanium, an organic germanium compound, reduces atherosclerosis in mice by inhibiting macrophage infiltration. Propagermanium inhibits the MCP-1/CCR2 pathway, which plays an important role in macrophage recruitment during plaque formation. The study showed propagermanium treated mice had significantly smaller atherosclerotic lesions and fewer macrophages in plaques than untreated mice. By suppressing macrophage infiltration through the MCP-1/CCR2 pathway, propagermanium may help prevent atherosclerosis and be a potential drug for treating vulnerable plaque.
The document discusses a study that found propagermanium, an organic germanium compound, reduces atherosclerosis in mice by inhibiting macrophage infiltration. Propagermanium inhibits the MCP-1/CCR2 pathway, which plays an important role in macrophage recruitment during plaque formation. The study showed propagermanium treated mice had significantly smaller atherosclerotic lesions and fewer macrophages in plaques than untreated mice. By suppressing macrophage infiltration through the MCP-1 receptor, propagermanium may prevent atherosclerosis and be a potential drug for treating vulnerable plaque.
The document discusses a study that found propagermanium, an organic germanium compound, reduces atherosclerosis in mice by inhibiting macrophage infiltration. Propagermanium inhibits the MCP-1/CCR2 pathway, which plays an important role in macrophage recruitment during plaque formation. The study showed propagermanium treated mice had significantly smaller atherosclerotic lesions and fewer macrophages in plaques than untreated mice. By suppressing macrophage infiltration through the MCP-1 receptor, propagermanium may prevent atherosclerosis and be a potential drug for treating vulnerable plaque.
Deletion of TLR4 Ameliorates Inflammation Response and Apoptosis in Septic Ca...semualkaira
Septic cardiomyopathy (SCM) is featured by
severe myocardial dysfunction and remains one of the lethal complications in clinical sepsis. Toll-like receptor 4 (TLR4) signaling
is known as a classical innate pathway in heart diseases, whereas
the precise underlying mechanism of TLR4 in SCM remains elusive. This study was designed to examine the specific role of TLR4
in SCM with a focus on inflammation and apoptosis.
Deletion of TLR4 Ameliorates Inflammation Response and Apoptosis in Septic Ca...semualkaira
Septic cardiomyopathy (SCM) is featured by
severe myocardial dysfunction and remains one of the lethal complications in clinical sepsis. Toll-like receptor 4 (TLR4) signaling
is known as a classical innate pathway in heart diseases, whereas
the precise underlying mechanism of TLR4 in SCM remains elusive. This study was designed to examine the specific role of TLR4
in SCM with a focus on inflammation and apoptosis.
This document summarizes a lecture on acute rheumatic fever (ARF). It begins with definitions of ARF as an acute inflammatory disease affecting connective tissue and primarily the cardiovascular system, developing after a streptococcal infection in predisposed individuals. It then covers the epidemiology, etiology, pathogenesis, clinical manifestations including carditis, arthritis, chorea and others, diagnostic criteria, classification, treatment and prevention of ARF. The lecture traces the history of understanding and research on ARF and rheumatic heart disease. It emphasizes the role of streptococcal infection in causing ARF and discusses virulence factors and the immune response involved in its pathogenesis.
EVIDENCE OF POSTTRAUMATIC COAGULOPATHY IN CASE OF THE SEVERE COMBINED THORACI...pijans
Early coagulopathy associated with trauma – result of exaggerate activity during the initiation phase of
coagulation. The aim of this study was to determine the diagnostic value of the coagulopathy markers for
metabolic monitoring of the severe combined thoracic trauma and it’s possibly to help in outcome
prediction. This retrospective study was performed on 73 male polytrauma patients from 20 to 68 years
old. The prothrombin time, fibrinogen concentration and β-Naphthol test were estimated on 1-2-d, 3-4-th
and 5-6-th days after trauma. The results suggest that hypocoagulation occurs early in equal extent for
survivals and non-survivals. Coagulation abnormalities are the result of vital functions disturbances rather
than direct tissue injury.
This document provides a review of recent developments in the understanding of disseminated intravascular coagulation (DIC). It discusses the pathophysiology of DIC, including the mechanisms of increased thrombin generation, impaired anticoagulant pathways, impaired fibrinolysis, and activation of inflammatory pathways. It also addresses the diagnosis of DIC through laboratory tests and scoring systems. Finally, it reviews current standard and experimental therapies for DIC, including replacement therapy, anticoagulants, restoration of anticoagulant pathways, and other agents. The treatment of the underlying condition that triggered DIC is also emphasized.
- The document examines the role of plasminogen activator inhibitor 1 (PAI-1) in the recruitment of mast cells (MCs) to glioma tumors.
- It finds that neutralizing PAI-1 attenuates the infiltration of MCs into glioma tumors. It also finds that MCs express the PAI-1 receptor LRP1, and blocking LRP1 also attenuates MC migration.
- Activation of the potential PAI-1/LRP1 axis in MCs by purified PAI-1 promotes increased phosphorylation of STAT3 and subsequent MC exocytosis. This indicates the PAI-1/LRP1 axis influences MC recruitment in glioma tumors.
This document outlines the history and mechanisms of the indoleamine-2,3-dioxygenase (IDO) pathway and its role in cancer immunotherapy. It discusses key findings such as:
1) Munn's 1999 discovery that IDO suppresses T-cell mediated rejection of tumors and fetal allografts by depleting tryptophan.
2) Mechanistic studies showing IDO induces T-cell arrest and inhibits proliferation through tryptophan depletion and kynurenine production, leading to Treg differentiation and CTL inhibition.
3) Preclinical studies combining IDO inhibitors with chemotherapy or vaccines, showing enhanced anti-tumor effects.
4) Ongoing clinical
Clinical and Laboratory Prognostic Factors in Malignant form of Mediterranean...inventionjournals
Mediterranean spotted fever (MSF) caused by Rickettsia conorii has become a significant health risk for suffering people and international travelers. In the past, overlooked as a serious disease, at present it is known that MSF was wrongly considered a benign condition. In this report, we present a set of clinical features and laboratory parameters in 55 patients (19 fatalities and 36 survivors) with malignant forms of the disease. The purpose of the study was to outline the prognostic factors of the fatal outcome in patients with malignant MSF. Based on our data, the main prediction factors for mortality in malignant MSF patients were: advanced age, delayed hospital admission, severe concomitant diseases, and failure to start or to complete appropriate antibiotic treatment. Laboratory prognostic factors in fatalities were: leukocytosis with a marked shift to the left; extremely high serum urea and creatinine levels; low levels of fibrinogen and prolongation of thrombin time. The most frequently involved organ systems of malignant cases were: central nervous system 100%, liver 92.72%, kidneys 60%, lungs 58.18%, myocardium 30.9%, and gastrointestinal tract 23.63%. The conducted histopathological investigations revealed lethal complications: encephalitis, brain edema, acute respiratory distress syndrome, non-cardiogenic lung swelling, acute myocarditis, gastrointestinal bleeding, hemorrhagicnecrotizing pancreatitis and acute renal failure
Clinical and Laboratory Prognostic Factors in Malignant form of Mediterranean...inventionjournals
Mediterranean spotted fever (MSF) caused by Rickettsia conorii has become a significant health risk for suffering people and international travelers. In the past, overlooked as a serious disease, at present it is known that MSF was wrongly considered a benign condition. In this report, we present a set of clinical features and laboratory parameters in 55 patients (19 fatalities and 36 survivors) with malignant forms of the disease. The purpose of the study was to outline the prognostic factors of the fatal outcome in patients with malignant MSF. Based on our data, the main prediction factors for mortality in malignant MSF patients were: advanced age, delayed hospital admission, severe concomitant diseases, and failure to start or to complete appropriate antibiotic treatment. Laboratory prognostic factors in fatalities were: leukocytosis with a marked shift to the left; extremely high serum urea and creatinine levels; low levels of fibrinogen and prolongation of thrombin time. The most frequently involved organ systems of malignant cases were: central nervous system 100%, liver 92.72%, kidneys 60%, lungs 58.18%, myocardium 30.9%, and gastrointestinal tract 23.63%. The conducted histopathological investigations revealed lethal complications: encephalitis, brain edema, acute respiratory distress syndrome, non-cardiogenic lung swelling, acute myocarditis, gastrointestinal bleeding, hemorrhagicnecrotizing pancreatitis and acute renal failure.
Clinical and Laboratory Prognostic Factors in Malignant form of Mediterranean...inventionjournals
The document summarizes a study of 55 patients with malignant forms of Mediterranean spotted fever (MSF). The study aimed to identify clinical and laboratory prognostic factors for fatal outcomes in these patients. 19 patients died, while 36 survived. Key findings were that advanced age, delayed hospital admission of over 6 days, presence of pre-existing conditions like diabetes or hypertension, and failure to receive full antibiotic treatment were associated with higher mortality. Laboratory factors predicting death included higher white blood cell and kidney function test levels, as well as lower fibrinogen and prolonged blood clotting times. The most commonly affected organ systems in severe cases were the central nervous system, liver, kidneys, lungs and heart.
This study assessed the safety, tolerability, and immunogenicity of a polyvalent WT1 peptide vaccine in patients with acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS). Sixteen patients received at least one vaccination of four WT1 peptides designed to stimulate both CD4 and CD8 T-cell responses. Vaccinations were well-tolerated with no discontinuations due to toxicity. Two AML patients experienced relapse-free survival over 1 year, exceeding the duration of their first remission, suggesting potential clinical benefit from the vaccine. The vaccine engaged protective immune responses against WT1, warranting further clinical trials.
This document discusses cell and tissue damage from various causes and their effects. It begins by explaining prefixes, suffixes, and roots used in medical terminology. It then defines pathology and discusses basic terminology like disease, etiology, pathogenesis, diagnosis, and prognosis. Various types of cellular adaptation and degeneration are described, including atrophy, hypertrophy, hyperplasia, metaplasia, and dysplasia. Necrosis, or lethal cell injury, is defined and the stages are outlined. Various causes of cell injury are provided like oxygen deprivation, chemicals, infections, immunological reactions, genetics, nutrition, physical agents, and aging.
This document discusses cell and tissue damage from various causes and their effects. It begins by explaining prefixes, suffixes, and roots used in medical terminology. It then defines pathology and discusses basic terminology like disease, etiology, pathogenesis, diagnosis, and prognosis. It explains cellular adaptation, degeneration, and death. Cells can adapt through atrophy, hypertrophy, hyperplasia, metaplasia, and dysplasia. Injuries can cause non-lethal cell degeneration or lethal necrosis. Necrosis is irreversible cell death marked by biochemical and morphological changes. Various causes of cell injury are discussed like oxygen deprivation, chemicals, infections, immunological reactions, genetics, nutrition, physical agents, and aging.
The document discusses various topics related to sepsis including:
1) Definitions of terms like SIRS, sepsis, septic shock, and their associated signs and symptoms.
2) The inflammatory cascades that occur in response to infection and lead to organ dysfunction.
3) How sepsis affects different organs in the body like the brain, heart, lungs, kidneys, and liver.
4) Two theories for the origin of sepsis - the gut origin theory and lung origin theory.
5) The role of activated protein C in reducing thrombin production and inflammation.
T.W. presents with signs and symptoms consistent with rheumatoid arthritis (RA), including morning stiffness lasting hours, fatigue, joint pain, and swelling in the hands and feet. Examination finds symmetrical swelling and tenderness in the small joints of the hands and feet. Laboratory tests show elevated ESR and CRP, positive RF and anti-CCP, and mild anemia. Radiographs show soft tissue swelling, joint space narrowing, and bone erosions in the hands. These findings meet criteria for a diagnosis of RA and help assess disease activity and response to treatment over time.
1. A Non-Lethal Traumatic/Hemorrhagic Insult Strongly
Modulates the Compartment-Specific PAI-1 Response in
the Subsequent Polymicrobial Sepsis
Pierre Raeven, Alma Salibasic, Susanne Drechsler, Katrin Maria Weixelbaumer, Mohammad Jafarmadar,
Martijn van Griensven¤
, Soheyl Bahrami, Marcin Filip Osuchowski*
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology in the Trauma Research Center of Allgemeine Unfallversicherungsanstalt, Vienna, Austria
Abstract
Introduction: Plasminogen activator inhibitor 1 (PAI-1) is a key factor in trauma- and sepsis-induced coagulopathy. We
examined how trauma-hemorrhage (TH) modulates PAI-1 responses in subsequent cecal ligation and puncture (CLP)-
induced sepsis, and the association of PAI-1 with septic outcomes.
Methods: Mice underwent TH and CLP 48 h later in three separate experiments. In experiment 1, mice were sacrificed pre-
and post-CLP to characterize the trajectory of PAI-1 in plasma (protein) and tissues (mRNA). Post-CLP dynamics in TH-CLP
(this study) and CLP-Only mice (prior study) were then compared for modulatory effects of TH. In experiment 2, to relate
PAI-1 changes to outcome, mice underwent TH-CLP and were sampled daily and followed for 14 days to compare non-
survivors (DEAD) and survivors (SUR). In experiment 3, plasma and tissue PAI-1 expression were compared between mice
predicted to die (P-DIE) and to live (P-LIVE).
Results: In experiment 1, an early post-TH rise of circulating PAI-1 was contrasted by a delayed (post-TH) decrease of PAI-1
mRNA in organs. In the post-CLP phase, profiles of circulating PAI-1 were similar between TH-CLP and CLP-Only mice.
Conversely, PAI-1 mRNA declined in the liver and heart of TH-CLP mice versus CLP-Only. In experiment 2, there were no
DEAD/SUR differences in circulating PAI-1 prior to CLP. Post-CLP, circulating PAI-1 in DEAD was 2–4-fold higher than in SUR.
PAI-1 increase heralded septic deaths up to 48 h prior but DEAD/SUR thrombomodulin (endothelial injury marker) levels
were identical. In experiment 3, levels of circulating PAI-1 and its hepatic gene expression were higher in P-DIE versus P-LIVE
mice and those increases closely correlated with liver dysfunction.
Conclusions: Trauma modulated septic PAI-1 responses in a compartment-specific fashion. Only post-CLP increases in
circulating PAI-1 predicted septic outcomes. In posttraumatic sepsis, pre-lethal release of PAI-1 was mostly of hepatic origin
and was independent of endothelial injury.
Citation: Raeven P, Salibasic A, Drechsler S, Weixelbaumer KM, Jafarmadar M, et al. (2013) A Non-Lethal Traumatic/Hemorrhagic Insult Strongly Modulates the
Compartment-Specific PAI-1 Response in the Subsequent Polymicrobial Sepsis. PLoS ONE 8(2): e55467. doi:10.1371/journal.pone.0055467
Editor: Charles C. Caldwell, University of Cincinnati, United States of America
Received August 20, 2012; Accepted December 23, 2012; Published February 8, 2013
Copyright: ß 2013 Raeven et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was funded by WWTF Grant LS07-065 (awarded to MFO and SB). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Marcin.Osuchowski@trauma.lbg.ac.at
¤ Current address: Experimental Trauma Surgery, Clinic for Trauma Surgery, Klinikum Rechts der Isar, Technical University Munich, Munich, Germany
Introduction
Trauma [1] and sepsis [2,3] are among the leading causes of
death worldwide and up to 10% of sepsis cases are preceded by
trauma [4]. In trauma patients without brain injury, acute and
early deaths are typically due to hemorrhage, whereas sepsis is the
third most frequent cause of late death (in 31% of cases), after
multiple organ failure (40%) and acute lung injury (26%) [5,6]. An
increased severity of traumatic injury increases the risk of
developing sepsis by 6 to 16-fold [7].
Both trauma and sepsis trigger the innate immune response,
activate the endothelium and non-specific host defense mechan-
isms, and affect blood clotting homeostasis [8–10]. Although their
mechanisms and evolution can be diverse (reviewed in [11] and
[12]), trauma and sepsis alike cause a deregulation of the
coagulation/fibrinolysis balance leading to coagulopathy of
different severity [10,13–15]. When present, coagulopathy in-
creases mortality by approximately 4-fold in trauma [13] and by
approximately 2-fold in sepsis [16]. A single traumatic challenge,
depending on its severity, may independently affect the host
response and facilitate development of sepsis. It has been
demonstrated that rats were more sensitive to endotoxin following
surgical trauma (i.e. implantation of intravascular catheters 1 to 7
days earlier) [17]. Depending on the mediator(s) studied, the more
recent reports demonstrate that an initial traumatic and/or
hemorrhagic injury may cause either ‘‘priming’’ or ‘‘desensitiza-
tion’’ of the immune system [18,19].
PLOS ONE | www.plosone.org 1 February 2013 | Volume 8 | Issue 2 | e55467
2. Plasminogen activator inhibitor type 1 (PAI-1), a member of the
serine protease inhibitor family, is an antagonist of both tissue- and
urokinase-plasminogen activators and is deemed to be the key
inhibitor of fibrinolysis [20]. PAI-1 is expressed in various cell
types, predominantly in endothelial cells and hepatocytes [20,21].
Apart from its role in coagulopathy, PAI-1 has been also shown to
be a relevant factor in basic defense processes such as leukocyte
activation [22] and clearance of apoptotic neutrophils [23], in
tissue remodeling, development of cancer, atherosclerosis and the
metabolic syndrome (reviewed in [21]). PAI-1 can be found in an
active form which spontaneously converts into a latent conforma-
tion (half life of less than 2 hours), or bound to vitronectin, its main
stabilizing protein [21].
Studies in trauma and sepsis alone imply that PAI-1 plays
a critical role in coagulopathy occurring after either of the above
conditions [15,24,25]. Circulating levels of PAI-1 were lower
immediately after trauma [11,26], but higher at later time points
post-injury [26,27]. In sepsis, plasma levels of non-survivors were
.2-fold higher compared to survivors [25,28,29] and patients with
homozygosity for the 4G allele (PAI-1 4G/5G promoter poly-
morphism) were 2 to 3-fold more susceptible for development of
septic shock and multiple organ dysfunction syndrome MODS,
and had a 2-fold higher risk of dying of MODS compared to
patients with different genotypes [30,31]. Clinical studies demon-
strated that sepsis alone induces a rapid increase of PAI-1 in the
blood (37–39), while we and others have shown that the
magnitude of the post-septic PAI-1 response is highly compart-
ment-specific [25,32–34]. Despite its key role in critical diseases,
the evolution of PAI-1 in post-traumatic sepsis is virtually
unknown. The only, to our knowledge, relevant experimental
inflammatory 2-hit study reported a 2-fold increase of PAI-1
mRNA expression in the rat lung (other tissues/organs were not
examined) after an intra-tracheal endotoxin administration
following hemorrhage (compared to endotoxin after sham
treatment) [35].
In the present study, we aimed to characterize the evolution of
PAI-1 over the course of post-traumatic sepsis and to establish if
and/or how the initial trauma-hemorrhage hit modulates the PAI-
1 protein and gene expression responses in the subsequent
polymicrobial sepsis. Specifically, we focused on PAI-1 gene
expression and presence of the protein in different compartments
(i.e. in the blood, major organs and vascular endothelium) and its
association with early septic outcomes, endothelial injury and risk
of death.
Materials and Methods
Animals
Four week old (n = 42, weight 20–25 g, experiment 1) and
three-month old (n = 72, 30 g, experiment 2 and 3), female CD-1
outbred mice (Harlan, Italy) were used. Mice were kept in groups
of five animals per Type-III cage on a 12 h light –dark cycle with
temperature maintained between 22–24uC and provided with
a standard rodent diet and water ad libitum throughout all
experiments (see the Ethics Statement for approvals/ethical details).
Cages were enriched with houses, wood wool for nesting as well as
wooden boards, tunnels and small blocks for gnawing (Abedd Lab
& Vet Service, Austria) to facilitate natural behaviour prior to and
throughout the experiments.
Ethics Statement
All animal procedures were approved by the Viennese (Austria)
legislative committee (Animal Use Proposal Permission no:
000794/2009/13) and conducted according to National Institute
of Health guidelines.
Due to the severe nature of the TH-CLP model additionally
complicated by the two age groups, a particular focus was put on
monitoring of animals in the study to minimize suffering within the
borders of the experimental design. All mice enrolled in the study
were kept in the small in-house animal facility of our institute to
enable optimal monitoring: the overall health status was checked
by trained professionals (i.e. DVMs and/or MDs) at least three
times per day during non-moribund disease period and every 2–
3 h whenever mice were in an acutely severe state (defined by, e.g.
decreased activity, progressing hypothermia, rapid weight gain;
complete symptom list in [36]). To further enhance monitoring
capacity and ensure maximal data reproducibility, all TH-CLP
experiments were conducted in small sets of animals.
Given that in experiment 2 the objective of our study was to
compare levels of circulating mediators between non-surviving and
surviving mice, the time point of septic deaths was the most critical
endpoint. This precluded us to follow typical humane endpoints as
they are inadequate and too imprecise in critical illness models
such as sepsis, shock, trauma and burns [36]: sacrificing mice using
the classical (and accepted) humane endpoints (e.g. temperature,
weight loss) in any of those severe disease experiments may be
premature and produce type I and/or II errors. For example, in
the CLP model, a weight gain (but not its loss) is more predictive of
impending death, while hypothermia of 30uC has a positive
predictive value for death (within 24 h) of only 56% [36].
Therefore, as suggested by Nemzek et al. [36] we followed
a more accurate, empirically established set of guidelines that
offered, although a very narrow, yet a feasible window of
opportunity for induction of death. This was possible due to the
frequent monitoring and did not hinder the experimental setup of
this study. Specifically, mice were killed only at the end of each
experiment or upon signs of imminent death (i.e. inability to
maintain upright position/ataxia/tremor and prolonged/deep
hypothermia and/or agonal breathing) by using deep inhalation
anesthesia (isoflurane, ForaneH, Abott, Austria) followed by an
overdose of barbiturate (thiopental, Thiopental SandozH, Austria).
Study Design
The study consisted of three experimental parts displayed in
Figure 1. In experiment 1, young female mice suffering from mild
sepsis (as defined in the two-hit model subsection below) were
sacrificed to obtain organ/tissue and blood samples for charac-
terization of PAI-1 dynamics in different compartments (Fig. 1A).
Four week old females were used to exclude possible estrogen-
related variations and to reliably compare data from this study
with the data from our previous study [25].
In experiment 2 (Fig. 1B), septic mice were never sacrificed but
subjected to CLP of medium-severity, blood was sampled daily
(see the Blood sampling subsection below) and followed until day 14
post-CLP. At the end of the study, mice were retrospectively
divided into surviving (SUR) and dying (DEAD) subgroups. The
1100 ng/ml PAI-1 cut-off used for retrospective stratification of
mice into DEAD and SUR was selected from the pooled values of
circulating PAI-1 measured between 6 72 h post-CLP (this study).
In experiment 2 and 3 (below), three month old mice were chosen
as they appropriately recapitulate an adolescent population of
trauma victims and are more suitable for daily monitoring (e.g.
larger volume of daily blood samples).
In experiment 3, mice were again subjected to TH-CLP (CLP of
medium-severity) and were stratified into either predicted to die
(P-DIE) or predicted to live (P-LIVE) and sacrificed in pairs (1:1
ratio) at 48 h post-CLP (Fig. 1C). Stratification was based on the
Preceding Trauma Changes the Septic PAI-1 Response
PLOS ONE | www.plosone.org 2 February 2013 | Volume 8 | Issue 2 | e55467
3. post-CLP changes in body temperature (BT) as CLP mice dying in
the acute phase of sepsis typically develop medium-to-severe
hypothermia [37,38]. Specifically, TH-CLP mice displaying
BT,28uC were identified as P-DIE, while mice with BT.36uC
were identified as P-LIVE. The predictive accuracy of BT
stratification (Table S1) was established based on a large database
that included BT measurements from experiment 2 mice and TH-
CLP mice (all females) from another study [39]. BT-based
stratification has a number of advantages: 1) P-DIE mice are fully
symptomatic, thus are closer to death compared to IL-6-based
prediction [40], 2) the BT separation is highly reliable (Table S1),
and 3) maximal comparison consistency can be obtained by
selecting any specific post-CLP day for sacrifice. In experiment 3,
the 48 h was selected as an optimal time-point – a total of nine P-
DIE/P-LIVE pairs were identified and sacrificed for comparative
analysis.
Two-hit Model
We used a post-traumatic sepsis model originally described by
van Griensven et al. [41] with additional modifications for
improved clinical relevance as detailed elsewhere [42]. All surgical
procedures were performed in the morning, between 8–10 am.
Trauma/hemorrhage – the first hit. Briefly, mice were
anesthetized (induction 3%, maintenance 1.5% isoflorane) and
subjected to a non-comminuted, unilateral midshaft femur fracture
with local tissue damage immediately followed by hemorrhage
(TH, 248 h time-point). The femur fracture was produced by
a brief compression with custom-designed blunt pliers. The
Figure 1. Schematic view of the experimental design of the two-hit models. A) 3 week-old mice were subjected to trauma and hemorrhage
(TH) at 248 h followed by mild (23G) cecal ligation and puncture (CLP) sepsis at 0 h. The experimental period was divided into a pre-and post-CLP
phase. At 248 , 242 h, 236 h, 224 h, 0 h, 6 h, 12 h and 24 h, mice (n = 6 per time point) were sacrificed (indicated by black crosses) and blood and
organs were collected. B) 3 month-old mice (n = 42) were subjected to TH (248 h) followed by medium-severity (17G) CLP-induced sepsis (0 h). The
experimental period was divided into a pre-and post-CLP phase. From the TH time point (248 h) on, 20 ml of blood was collected daily, and
additionally at 6 h, until 96 h (day 6 post-TH/day 4 post-CLP). Animals were observed until death or day 14 post-CLP, whichever occurred first. C) 3
month-old mice (n = 30) were subjected to TH (248 h) followed by CLP sepsis of medium-severity (17G) at 0 h. Pair of mice with a body temperature
(BT) ,28uC (predicted to die) and BT .36uC (predicted to live) were identified at the 48 h time-point and sacrificed.
doi:10.1371/journal.pone.0055467.g001
Preceding Trauma Changes the Septic PAI-1 Response
PLOS ONE | www.plosone.org 3 February 2013 | Volume 8 | Issue 2 | e55467
4. effectiveness and reproducibility of injury was confirmed by X-ray.
Hemorrhage was performed via retro-orbital puncture to produce
a 40% blood loss of the total blood volume (calculated as 6% of the
total body weight; individually in each mouse). Overall, the TH
step was typically sub-lethal with only incidental deaths (0–5%
mortality). All pre-CLP deaths in our model occurred within the
first 2 h of TH – no late TH mortality was ever observed [42].
Post-TH, mice were subcutaneously resuscitated with 0.9% saline
with four times the volume of shed blood: 1 mL containing
analgesia (0.05 mg/kg buprenorphine, BuprenovetH) was given
immediately after hemorrhage (simulating the phase of restricted
resuscitation), while the remaining volume was administered 1 h
post-TH (simulating the phase of unrestricted resuscitation).
Polymicrobial sepsis – the second hit. Mice were sub-
jected to CLP (0 h time-point) at 48 h post-TH. We followed the
original CLP protocol by Wichterman et al. [43] with relevant
modifications [42]. Given that two different levels of CLP severity
were employed in our study, we utilized two different needle
gauges for puncturing the cecum: a 23-gauge (23G) needle was
used in the first experiment (Fig. 1A), while a 17G needle in
experiments 2 and 3 (Fig. 1B and C). As previously established,
CLP with 23G needle ensures 100% survival within the first 24
post-surgery (and minimal long-term mortality), while 17G needle
produces approx. 40% mortality at day 5 post-CLP (day 7 post-
TH) [42]. Beginning at 0 h until the sacrifice time-point
(experiment 1 and 3) or day 5 post-CLP (experiment 2), septic
mice were resuscitated twice daily with 1 ml of saline including
analgesia (0.05 mg/kg buprenorphine, BuprenovetH). Additional-
ly, at 2 h after-CLP, each mouse was resuscitated by a single
subcutaneous injection of 1 ml Ringer solution containing
antibiotics (25 mg/kg imipenem/cilastatin, ZienamH, MSD,
Austria) to emulate the clinical anti-microbial therapy [44].
We employed a subcutaneous instead of peritoneal route of
administration given that the latter is more stressful to mice and it
occasionally may result in inadvertent punctures of internal
organs. Additionally, in CLP, intraperitoneal application is
a potential confounder as progressing peritonitis may differentially
modulate effects of antibiotic treatment among individuals. For
pain management, all mice were administered metamizol (6 mg/
kg, NovalginH, Sanofi, Austria) via the drinking water from day 5
post-CLP until the end of each experiment. For all surgical
procedures, mice were anesthetized with isoflurane (ForaneH,
Abbott, Austria). Given that post-CLP responses have been well
described by us and others [25,32–34,45], sham surgeries were not
performed to reduce the total number of mice in the study and to
comply with the 3R tenet. All experiments were performed in
small sets of animals at the time and the results of all separate
experiments were combined in the final analysis.
Blood Sampling
In experiment 1, 50 ml of blood was collected via facial vein
puncture [46] immediately prior to sacrifice and at indicated time
points (Fig. 1A). In experiment 2, mice were not sacrificed and
20 ml of blood was collected repetitively from each animal every
24 h (including an additional sample at 6 h post-CLP) [46] until
day 5 (Fig. 1B). In experiment 3, 50 ml of blood was collected
immediately prior to sacrifice via facial puncture (Fig. 1C). All
blood samples were drawn into a pipette rinsed with ethylenedia-
minetetraacetic acid (EDTA) and then immediately diluted 1:10 in
phosphate-buffered saline containing 2% EDTA. After centrifu-
gation (10006g, 5 min, 22uC) supernatant (1:10 diluted plasma)
was removed and stored at –80uC until analysis.
Complete Blood Count
The remaining cell pellets after centrifugation and removal of
plasma were resuspended to the original 1:10 diluted blood
volume with CD3700 diluent (Abbot, Austria) containing EDTA.
Complete blood counts (CBC) with differential were then
performed on the CD3700 counter (Abbott, Austria) optimized
for mouse blood.
Organ and Metabolic Function Parameters
Alanine transaminase (ALT), aspartate transaminase (AST),
blood urea nitrogen (urea), glucose, and lactate dehydrogenase
(LDH) were analyzed in plasma samples using the Cobas c111
analyzer (Roche, Switzerland).
Measurement of Circulating PAI-1 and Thrombomodulin
The detection of antigens in plasma samples was based on
standard enzyme-linked immunosorbent assays (ELISA) using
commercially available mouse kits for total PAI-1 (Innovative
Research, USA) and soluble thrombomodulin (sTM, Uscn Life
Science Inc., P.R. China) according to the manufacturer’s
instructions. Compatibility of PAI-1 and sTM antigen assays with
the EDTA-preserved blood (versus citrate-preserved) was pre-
viously validated [25]. To determine general PAI-1 dynamics in
the systemic circulation, we assessed total (including the active,
latent and bound forms) circulating PAI-1 rather than active PAI-1
alone.
Gene Expression Analysis
At sacrifice (experiment 1), parts of the liver, kidney, lungs, heart
and left cranial vena cava (LCVC) were collected, immediately
snap-frozen in liquid nitrogen and stored at 280uC. In experiment
3, parts of the liver and LCVC were harvested and stored at
280uC. LCVC collections were pooled (n = 3–6 animals/group/
time point for experiments 1 and 3) due to very small tissue
volumes obtained from a single animal. Total RNA was extracted
using the QIAzol Lysis Reagent/RNeasy Mini kit combination
(Qiagen, Germany). RNA integrity was confirmed by agarose gel
electrophoresis. Of the total RNA, 2 mg reverse transcribed into
using anchored oligo-dT18-primers (Invitrogen, USA) and AMV
Reverse Transcriptase (Finnzymes, Finland). Gene expression of
murine PAI-1 and hypoxanthine-guanine phosphoribosyltransfer-
ase (HPRT) was analyzed by real-time polymerase chain reaction
(PCR) on a CFX96 real-time cycler (Bio-Rad, Austria) using
KAPA SYBR Green Universal 2X Mastermix (KAPA Biosystems,
USA). Following primers were used: HPRT sense 59-
GCAAGTCTTTCAGTCCTGTCC-39 and antisense 59-
GCAGCGTTTCTGAGCCAT-39, PAI-1 sense 59-TCCTCCA-
CAGCCTTTGTCAT-39 and antisense 59-ACATCTC-
CACTTTCGTCCCA-39. PCR protocol consisted of 3 min at
95uC for initial Taq-polymerase activation, followed by 40 cycles
each including a denaturation step of 10 s at 95uC, an annealing
step of 30 s at 60uC, and a 10 s extension step at 72uC with plate
read. PAI-1 expression relative to HPRT (and normalized to 0 h
time point) were calculated using the 22DCt
method [47].
Immunohistochemistry
At sacrifice (experiment 1, 248 h, 242 h and 24 h time points
only), parts of the liver and lungs were collected, immediately
snap-frozen in liquid nitrogen and stored at 280uC until further
processing by immunohistochemistry (IHC). The tissue samples
were embedded in OCT compound (Tissue-Tek, USA) and 5 mm-
thick cryostat sections were cut. The sections were fixed in ice cold
acetone and subsequently subjected to IHC staining. Endogenous
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5. peroxidase blocking was performed by treating the cryostat
sections with 0.3% H2O2 in 80% methanol for 20 min. The
sections were then washed with TRIS-buffered saline and
incubated with anti-PAI-1 primary antibody (1:500, Abcam,
UK) for 1 h at room temperature. After another buffer rinse, all
sections were incubated with an anti-rabbit polymer (Dako,
Denmark) for 30 minutes at room temperature. After washing,
staining was developed by using a peroxidase substrate kit
(ImmPACTTM
NovaREDTM
, Vector Laboratories, USA). The
slides were then counterstained with hematoxylin, dehydrated and
mounted permanently using an automatic glass coverslipper
(CTM 6, Microm, Germany). Immunohistochemical controls
were performed by replacing the primary antibody with antibody
diluent (Zytomed Systems, Germany). Images were generated
using the Olympus XC10 camera and OlyVIA dotSlide 2.4
software (Olympus, Austria).
Statistical Analysis
Normality was tested in all recorded parameters and non-
Gaussian data were log-transformed prior to further analyses.
Normally distributed data were tested by the analysis of variance
followed by the Tukey’s multiple comparison test, whereas skewed
data were analyzed by the Mann–Whitney U-test. Data in tables
that show comparisons of parameters collected in the TH-CLP
(this study) and CLP-Only [25] mice, are presented as mean 6
standard deviation. Data in graphs are presented on the original
scale as box plot diagrams. Statistical assessment of LCVC
expression in experiment 1 and 2 was not possible due to sample
pooling.
All survival curves were plotted using the Kaplan-Meier
method, and differences were analyzed by Log-rank (Mantel-
Cox) test. The retrospective comparison of PAI-1 trajectory
between SUR and DEAD (3:1 ratio) data sets was performed at
each time point separately using the unpaired t-test (normally
distributed data) and Mann-Whitney test (skewed data). To
characterize plasma PAI-1 using the time point of death as the
reference point (regardless of the day of death), three consecutive
pre-lethal PAI-1 values from each DEAD mouse were retrospec-
tively tallied, displayed in an inverted fashion (i.e. 72 h, 48 h and
24 h prior to death) and matched against PAI-1 values from
(randomly selected) SUR (alive at day 14) animals recorded on the
same post-CLP day (1:1 DEAD/SUR ratio).
Predictive accuracy of BT and circulating PAI-1 for post-TH-
CLP outcomes was evaluated by the Receiver Operating
Characteristics (ROC) curve and was expressed by the area under
the curve (AUC). The predictive accuracy of the ROC-AUC was
defined as: 0.9–1 = excellent, 0.8–0.9 = good, 0.7–0.8 = fair, 0.6–
0.7 = poor and ,0.6 = not useful. Correlations between selected
parameters were tested by non-parametric analysis and expressed
by Spearman’s rank correlation coefficient (r). All tests were
carried out using Prism 5 (GraphPad, USA). The level of
significance was determined at p,0.05.
Results
Trauma/Hemorrhage does not Alter the Dynamics of
Circulating PAI-1 after the Subsequent CLP Hit
First, we portrayed the dynamics of circulating PAI-1 in the
TH-CLP period (Fig. 2). At 248 h (baseline, immediately prior to
TH), the total PAI-1 plasma concentration was 5.4 ng/ml
(Fig. 2A). TH induced an elevation of plasma PAI-1 by 6-fold
(32 ng/ml) at 242 h (6 h post-TH), followed by a decrease to the
baseline level at 224 h (5.7 ng/ml). CLP (2nd
hit) provoked
a gradual increase of circulating total PAI-1 from 6 h onward (32-
and a 52-fold rise at 6 and 12 h) with the peak value (590 ng/ml)
at 24 h (Fig. 2B). Pre-and post-CLP trajectories of selected
hematological parameters (CBC) are shown in Table S2.
Second, we compared the evolution of total PAI-1 release into
the blood after post-traumatic sepsis versus sepsis alone (Table 1).
Baseline PAI-1 levels in mice subjected to TH-CLP (this study;
measured at 248 h) and CLP-Only (previous study [25]) were
identical (5.4 ng/ml). In TH-CLP mice, PAI-1 concentration at
0 h (immediately prior to CLP) was 50% higher compared to
CLP-Only mice at 0 h (i.e. baseline) (Table 1). However, in both
groups, PAI-1 measured post-CLP displayed an identical trajec-
tory: its circulating concentrations rose gradually and were similar
at all of the time points assessed.
Figure 2. Circulating PAI-1 response to TH-CLP. 3 week-old mice
were subjected to trauma and hemorrhage (TH) at 248 h followed by
mild (23G) cecal ligation and puncture (CLP) sepsis at 0 h. At 248 h,
242 h, 236 h, 224 h, 0 h, 6 h, 12 h and 24 h, mice (n = 6 per time
point) were sacrificed and blood was collected and analyzed for total
plasminogen activator inhibitor type 1 (PAI-1) plasma concentration.
Figure shows box plot diagrams, with whiskers indicating minimum and
maximum and dots representing values outside of 1.5 inter-quartile
range. *P,0.05 versus 248 h, 1 P,0.05 versus 6 h.
doi:10.1371/journal.pone.0055467.g002
Table 1. Circulating PAI-1 during the post-CLP phase in post-
traumatic sepsis versus sepsis alone.
Plasma 0 h 6 h 12 h 24 h
TH-CLP 11.862.8* 168.56121.9 278.56126.0 590.06432.8
CLP-Only1
5.462.1 97.1635.7 422.36323.6 11946759.9
fold difference 2.2 1.7 0.7 0.5
P-value 0.008 0.096 0.427 0.934
Three week-old mice were subjected to trauma and hemorrhage (TH) followed
by polymicrobial cecal ligation and puncture (CLP) sepsis or CLP alone (CLP-
Only). At 0, 6, 12 and 24 h post-CLP, mice (n = 6 mice per time point) were
sacrificed and blood was collected and analyzed for total plasminogen activator
inhibitor type 1 (PAI-1) in plasma. Data as mean 6 SD (TH-CLP and CLP-Only
rows) representing 5–6 mice per time point. Fold PAI-1 difference between TH-
CLP and CLP-Only.
1
Values obtained from a separate study (22).
*P,0.05 versus CLP-Only.
doi:10.1371/journal.pone.0055467.t001
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6. Trauma/Hemorrhage Modulates the PAI-1 Gene
Expression in the Post-CLP Phase
In the next step, we characterized the trajectory of PAI-1
mRNA over the TH-CLP duration in various organs (Fig. 3). TH
alone induced a strong PAI-1 mRNA decrease in the liver (by 93%
at 0 h, Fig. 3A) and heart (by 83% at 224 h and 0 h, Fig. 3B). In
the kidney and lungs, PAI-1 mRNA transcription was not affected
by TH (Fig. 3 C–D). A statistically insignificant increasing trend
Figure 3. Organ-specific PAI-1 gene expression after TH-CLP. 3 week-old mice were subjected to trauma and hemorrhage (TH) at 248 h
followed by mild (23G) cecal ligation and puncture (CLP) sepsis at 0 h. At 248 h, 242 h, 236 h, 224 h, 0 h, 6 h, 12 h and 24 h, mice (n = 6 per time
point) were sacrificed and organs were collected and analyzed for expression of plasminogen activator inhibitor type 1 mRNA in the A) liver, B) heart,
C) kidney, D) lung and E) left cranial vena cava (LCVC). Panels A-D show box plot diagrams representing 5–6 mice per time point, with whiskers
indicating minimum and maximum and dots representing values outside of 1.5 inter-quartile range. In panel E, each LCVC dot represents a pooled
collection of samples (n = 5–6 mice per time point). *P,0.05 versus 248 h; 1P,0.05 versus 0 h.
doi:10.1371/journal.pone.0055467.g003
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7. (20-fold peak rise at 224 h) in PAI-1 mRNA was observed in the
LCVC (Fig. 3E).
2nd
hit CLP induced an immediate (from 6 h onward, lasting up
to 24 h) increase of PAI-1 mRNA the liver (.600-fold vs. 0 h;
Fig. 3A) and a statistically insignificant rise (.20-fold vs. 0 h;
Fig. 3E) in the LCVC. A milder increase of PAI-1 mRNA was
recorded in the heart (3 to 7-fold; Fig. 3B) and kidney (Fig. 3C). In
contrast, CLP did not consistently up regulate PAI-1 mRNA in the
lungs, although PAI-1 mRNA level at 12 h was 4-fold (p,0.05)
higher compared to baseline (Fig. 3D). Pre-and post-CLP
trajectories of circulating ALT and AST as well as urea, glucose
and LDH are shown in Figure S1.
Similar to the comparison in Table 1, we next compared the
trajectory of PAI-1 mRNA fluctuations between post-traumatic
sepsis and sepsis alone (i.e. the post-CLP phases in both groups,
Table 2). At 0 h (i.e. immediately before CLP), PAI-1 gene
expression in TH-CLP animals was lower in the liver (by 293%),
kidney (281%) and heart (283%), but not in the lung compared
to CLP-Only mice. In the post-CLP phase, PAI-1 mRNA level in
TH-CLP mice was significantly lower in the liver (283% at 24 h)
and the heart (278% at 12 h), but not in the kidney and lung
compared to CLP-Only animals. PAI-1 mRNA in the LCVC
appeared to increase faster after TH-CLP than CLP-Only but this
comparison was not statistically verified due to sample pooling.
To corroborate the above changes, we additionally examined
the expression of PAI-1 protein in the liver and lungs by IHC
(Fig. 4). At baseline, there was no evident expression of PAI-1.
Whereas both TH (1st
hit) and CLP (2nd
hit) increased PAI-1
protein expression in the liver (at 242 h and 24 h), only post-
traumatic CLP (but not TH alone) triggered a moderate
expression of PAI-1 protein in the lungs.
Increase of Circulating PAI-1 in Post-traumatic Sepsis is
Associated with Worse Outcome
In the second part of the study, we investigated the relationship
between PAI-1 in plasma and TH-CLP outcome. Mice subjected
to TH-CLP were never sacrificed but sampled daily (until day 5
post-CLP). At the end of the study, all animals were retrospectively
divided into either dying (DEAD) or surviving (SUR) and
compared (see Materials and Methods for details).
TH-CLP mortality reached 60% by day 14 post-CLP (Fig. 5A).
To test whether an early raise in PAI-I was associated with an
increased mortality, survival curves were re-plotted to divide mice
based on their circulating PAI-1 (cut-off set at 1100 ng/ml)
measured at 24 h post-CLP (Fig. 5B). TH-CLP animals with
plasma PAI-1 level $1100 ng/ml showed 22% higher mortality
compared to their counterparts with PAI-1,1100 ng/ml (n = 24;
p,0.05).
Circulating PAI-1 Predicts Death after but not Prior to the
CLP Hit
Next, we compared the time course of circulating PAI-1
between SUR and DEAD mice (Fig. 6A). In the pre-CLP phase,
plasma PAI-1 increase between DEAD/SUR mice was identical.
In the post-CLP phase, however, plasma PAI-1 trajectories
gradually separated over time. Compared to SUR, PAI-1 in
DEAD was insignificantly elevated at 6 and 24 h (by 50 and 70%),
approx. 2-fold higher at 48 h, and 4-fold higher at 72 h. This
observation was corroborated by ROC analysis: the predictive
accuracy for early death was poor at 6 h and 24 h (AUC = 0.57
and 0.65), good (AUC = 0.81) at 48 h and excellent (AUC = 0.9) at
72 h time point (Table S3).
Given that the exact time of the onset of sepsis is frequently
unknown, we additionally assessed the exact time line of a feasible
outcome-dependent separation. DEAD and SUR values were re-
plotted in an inverted fashion using the day of death (days 1–5
post-CLP) as the reference point (Fig. 6B, see Materials and
Methods). Prediction of post-CLP deaths was not possible earlier
than 48 h prior to the event: while circulating PAI-1 levels in
DEAD and SUR mice overlapped at 72 h before death, PAI-1 in
DEAD (vs. SUR) gradually increased to reach a 2-fold and 3-fold
difference at 48 h and 24 h prior to death, respectively.
Pre-lethal Release of PAI-1 during Post-traumatic Sepsis is
Independent of Endothelial Injury
To establish whether the above DEAD vs. SUR separation was
reflected by the degree of endothelial injury, we studied the time
course of sTM in the same set of mice (Fig. 7). The trajectory of
sTM was identical between SUR and DEAD at all time points.
Both groups had equal baseline values and TH induced an
equivalent increase of sTM at 242 h (to approx. 100 ng/ml) that
remained unchanged until 0 h (2nd
hit CLP). In the post-CLP
Table 2. Organ/tissue PAI-1 mRNA expression during the
post-CLP in posttraumatic sepsis versus sepsis alone.
0 6 12 24
Liver TH-CLP 0.0760.04* 46.53623.92 55.63648.01* 41.35626.43*
CLP-Only1
1.0061.31 96.17684.28 127.06100.90 246.76262.20
fold
difference
0.07 0.67 0.44 0.17
P-value 0.0047 0.8149 0.1471 0.0103
Heart TH-CLP 0.1760.08* 0.4960.21 0.4860.13* 1.1160.81
CLP-Only1
1.0060.61 1.1960.73 2.1761.81 0.9261.17
fold
difference
0.17 0.42 0.22 1.21
P-value 0.0043 0.1255 0.0168 0.2729
Kidney TH-CLP 0.1860.09* 4.5263.08 5.1362.62 3.0162.35
CLP-Only1
1.0061.27 2.4763.36 2.2362.20 5.00611.08
fold
difference
0.19 1.83 2.3 0.6
P-value 0.035 0.065 0.0559 0.5362
Lung TH-CLP 1.2260.63 2.4961.09 4.0461.05 5.7263.75
CLP-Only1
1.0061.06 2.3461.08 2.7861.66 3.6663.68
fold
difference
1.21 1.07 1.45 1.56
P-value 0.3095 0.9626 0.1807 0.2254
LCVC TH-CLP 8.28 166.57 334.69 329.32
CLP-Only1
1.00 4.30 14.29 140.90
fold
difference
8.28 38.74 23.42 2.34
P-value N/A N/A N/A N/A
3 week-old mice were subjected to trauma and hemorrhage (TH) followed by
polymicrobial cecal ligation and puncture (CLP) sepsis or CLP alone (CLP-Only).
At 0, 6, 12 and 24 h post-CLP, mice (n = 6 mice per time point) were sacrificed
and organs were collected and analyzed for total plasminogen activator
inhibitor type 1 (PAI-1) mRNA expression. Data as mean 6 SD (TH-CLP and CLP-
Only rows) representing 5–6 mice per time point. Fold PAI-1 difference between
TH-CLP and CLP-Only.
1
Values obtained from a separate study (22).
*P,0.05 versus CLP-Only. N/A, not applicable.
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8. phase, concentration of sTM was 5 to 7-fold higher than baseline
but it was equal between SUR and DEAD at all time points.
Pre-lethal Release of PAI-1 during Post-traumatic Sepsis
Correlates with Exacerbated Hepatic PAI-1 Gene
Expression and Liver Dysfunction
Finally, we aimed to establish the main source of pre-lethal PAI-
1 release in post-traumatic sepsis. To achieve that, we employed
a prediction of outcome based on changes in body temperature
occurring during early CLP phase. This approach ensured high
predictive accuracy (see Table S1 and Study Design) and at the
same time, it allowed comparative organ analysis in mice with
opposite sepsis severity.
Pairs of P-DIE and P-LIVE mice were identified and the
concentration of circulating PAI-1 (and ALT, AST) and PAI-1
gene expression in the liver and LCVC were measured (Fig. 8).
Compared to P-LIVE animals, a 2-fold increase of circulating
PAI-1 in P-DIE mice (Fig. 8A) was accompanied by a 7-fold
increase of PAI-1 mRNA in the liver (Fig. 8B). Notably, the above
effect was paralleled by deterioration of liver function: compared
to P-LIVE, there was an additional 2-fold increase in both ALT
and AST in P-DIE mice (Fig. 8C and D). A strong correlation in
outcome-dependent changes was recorded between circulating
PAI-1 and all above-mentioned parameters (Table 3). In the
LCVC, the level of PAI-1 mRNA expression was similar between
P-DIE and P-LIVE mice (Fig. 8E).
Discussion
A traumatic challenge may independently affect the evolution of
the host response and regulation of important mediators during
the development of a subsequent sepsis. In the present study, we
investigated how initial trauma-hemorrhage influences the PAI-1
response in a following sepsis in mice, focusing on its expression in
different compartments and its correlation with outcome.
An insight into the systemic compartment is clinically most
important due to the potential diagnostic and/or predictive utility
of measured parameters. The combination of the two current
experiments (Fig. 1) with the data from our recently published
CLP-Only study [25] allowed us to: 1) follow concentration of
PAI-1 in the blood over the seven-day period (from the TH onset),
2) provide a comprehensive outcome-dependent characterization
of early and delayed fluctuations of PAI-1 as mice transitioned
from the trauma/hemorrhage to post-traumatic sepsis, and finally
3) establish the modulatory importance of the TH hit in post-
traumatic CLP sepsis.
TH induced a marked increase of plasma PAI-1 (and
a simultaneous decrease of hemoglobin, platelets and red blood
cell count) that was followed by a complete recovery of PAI-1 (and
of platelet counts) by the time of sepsis onset (Fig. 2A, Table S2).
Thus, in the experimental context, this model [39,41,42,48,49]
allows enough time for the TH-induced coagulation-fibrinolysis
imbalance to be largely restored prior to the CLP hit, while, as we
have recently demonstrated, the immuno-inflammatory system in
TH mice remains highly activated at 0 h [39]. The above
dynamics closely emulate the clinical scenario as the SIRS/
inflammatory response in trauma patients can remain elevated for
days [50], especially in patients developing subsequent secondary
complications [51,52], while the circulating PAI-1 subsides
relatively quickly [27]. Moreover, two studies demonstrated that
an acute blood loss in the volume comparable to the one shed in
the current study (i.e. ,40% of the total) leads to a period of severe
hypotension as observed in patients suffering from hemorrhagic
traumatic shock [53,54]. Hence, this model provides an ideal
milieu for investigating modulatory effects of the (TH - induced)
isolated immuno-inflammatory component upon desired sequelae
of secondary sepsis.
Regarding the PAI-1 gene/protein dynamics, this two-hit study
reveals two novel findings. First, we demonstrate that the increase
of circulating PAI-1 after TH hit did not evidently influence its
subsequent dynamic after sepsis (Table 1). In general, both the
post-CLP trajectory and the magnitude of the observed PAI-1
increase remained similar between the TH-CLP and CLP-Only
groups. Although we did not measure it earlier than 6 h post-TH,
the transient PAI-1 increase (at 6 and 12 hours after TH) is also
reported in patients [27] and it likely reflects a compensatory
rebound after the steep decrease of PAI-1 immediately after
trauma [26]. In contrast, the increase of plasma PAI-1 during
sepsis is expected to be a direct counter-regulatory response
against induction of plasminogen activators and successive plasmin
production. The post-CLP increase in PAI-1 expression is also
triggered by systemic release of TNFa and IL-1b [12].
Figure 4. Immunohistochemical staining of PAI-1 in the liver and lung post-TH and TH-CLP. 3 week-old mice were subjected to trauma
and hemorrhage (TH) at 248 h followed by mild (23G) cecal ligation and puncture (CLP) sepsis at 0 h. At 248 h, 242 h and 24 h, mice were
sacrificed and organs were collected and stained for plasminogen activator inhibitor type 1 (PAI-1) in the liver and lung. Representative images for 3–
6 animals per time point per tissue are shown. NC, negative control for PAI-1 (incubated without primary antibody). Original magnification x100.
doi:10.1371/journal.pone.0055467.g004
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9. Second, we show a differential (TH-dependent) priming of the
PAI-1 gene response in various organs and an evident mismatch
between the mRNA expression and circulating PAI-1 protein. The
liver has been identified as one of the most prominent sources of
PAI-1 after both hemorrhage [55,56] and CLP-induced sepsis
[25,33]. After TH alone, we observed an early trend to increasing
hepatic PAI-1 gene expression (Fig. 3A) that was similar to the
early hepatic PAI-1 mRNA increase reported by Yamashita et al.
in hemorrhagic rats [55]. The delayed drop of PAI-1 mRNA in
the liver and heart at 0 h (i.e. 48 h after TH) is currently
unsubstantiated as this finding has not been scrutinized by others.
In the acute phase of post-traumatic sepsis, we recorded a distinct
upregulation of PAI-1 gene expression in the liver (also a strong
trend to increases in the LCVC). Yet, the mRNA upregulation
after two hits was much lower when compared to the hepatic PAI-
1 expression recorded in CLP-Only mice (Table 2). Interestingly,
while this elevation was remarkably constant across all time-points
in TH-CLP mice, it increased additionally in CLP-Only animals.
Regarding other organs, both heart and kidney remained
quiescent whereas only a delayed, low-grade increase of PAI-1
mRNA was observed in the lung. In contrast to the aforemen-
tioned findings of Fan et al. (30), the increase of PAI-1 mRNA in
the lung was not additionally modulated by TH (vs. CLP-Only,
Table 2). This disparity can be explained by the difference in the
magnitude/localization of the inflammatory response – a systemic
one after CLP (this study) vs. an isolated response after a local
Figure 5. Increased plasma PAI-1 at 24 h post-TH-CLP is
associated with lower 14-day survival. 3 month-old mice (n = 42)
were subjected to trauma and hemorrhage (TH) at 248 h followed by
mild (23G) cecal ligation and puncture (CLP) sepsis at 0 h. From the TH
time point on, 20 ml of blood was collected daily and additionally at 6 h,
until 96 h (day 6 post-TH/day 4 post-CLP). Animals were observed until
death or day 14 post-CLP, whichever occurred first. A) 14-day survival.
B) Survival of mice based on plasma PAI-1 at 24 h post-CLP below or
above a subjective threshold. Digits on the graph indicate the number
of deaths at the indicated time point. *p,0.05 versus ,1100 ng/ml.
CLP time point indicated by arrow.
doi:10.1371/journal.pone.0055467.g005
Figure 6. Plasma PAI-1 is increased in dying versus surviving
mice. 3 month-old mice (n = 42) were subjected to trauma and
hemorrhage (TH) at 248 h followed by mild (23G) cecal ligation and
puncture (CLP) sepsis at 0 h. From the TH time point on, 20 ml of blood
was collected daily and additionally at 6 h, until 96 h (day 6 post-TH/
day 4 post-CLP. Animals were observed until death or day 14 post-CLP,
whichever occurred first. The experimental period was divided into
a pre-and post-CLP (red and black dots, respectively) phase. A) Time
course of total circulating plasminogen activator inhibitor type 1 (PAI-1)
of dying (DEAD, filled dots) and surviving (SUR, empty dots) mice. For
SUR, n = 17 at each time point. For DEAD: n = 24 at 248 h, 224 h, 0 h
and 6 h; n = 23 at 24 h; n = 15 at 48 h; n = 6 at 72 h. B) Circulating levels
of PAI-1 of DIE (n = 24 at each time point) and time-matched levels of
SUR (n = 24 at each time point) at the indicated time points prior to
death. *p,0.05 versus SUR. CLP time point indicated by arrow.
doi:10.1371/journal.pone.0055467.g006
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10. (intratracheal) endotoxin administration (30). In addition, we have
recently shown that acute CLP does not produce a significant lung
injury in female CD-1 (ICR) mice [57]. Overall, the current data
convincingly implicate liver as the chief source of PAI-1 pro-
duction and release in post-traumatic sepsis.
Both clinical and experimental studies have shown that high
levels of circulating PAI-1 early after the sepsis onset are associated
with worse long-term outcome [25,28,58]. Pralong et al. found that
71% of the patients with PAI-1 levels of more than 550 ng/ml at
time of entry to the study died within 7 days (acute septic deaths)
compared to only 6% of patients with PAI-1 levels lower than
550 ng/ml [58]. We now report an identical relationship in post-
traumatic sepsis, albeit at a different PAI-1 cut-off. Out of all mice
with PAI-1 level higher than or equal to 1100 ng/ml (measured at
24 h post-CLP), 72% died within 7 days, compared to 50% of
mice with PAI-1 below 1100 ng/ml (Fig. 5B). The outcome
discrepancy between the two low-risk groups is largely due to the
relatively dense accumulation of deaths within the first 5 days post-
CLP in our 2-hit model compared to a more protracted mortality
in the clinical study [58]. In patients dying of sepsis, plasma PAI-1
typically rises over time and remains high until the time of death,
whereas PAI-1 in survivors peaks early and recovers to normal
levels within approx. one week [24,28]. We observed a similar
trajectory; at 48 h and 72 h after the 2nd
hit (but not earlier)
circulating PAI-1 in dying septic mice continued to rise, whereas it
remained stable after the initial post-CLP increase in all survivors
(Fig. 5A). Disappointingly, the trauma-induced fluctuations of
PAI-1 during the post-TH phase (but pre-CLP), did not
discriminate post-traumatic sepsis outcomes prior to its onset. In
the context of a prospective risk-assessment in any patient suffering
from post-traumatic sepsis, the impending outcome is a much
more relevant reference point than the time of the onset of sepsis
as the latter event may be easily overlooked. After re-plotting the
data using the day of death as the reference time point (Fig. 6B), it
became apparent that PAI-1 concentration in dying mice was 2 to
3-fold higher than in matched survivors, yet not earlier than 48 h
prior to death. Although this time-window is identical to the
effective time-span in predictive capacity of IL-6 (and other
cytokines) for septic deaths [39,45,59], a relatively loose clustering
of individual, outcome-based PAI-1 values disqualifies it as
a potential stand-alone predictor of outcome in sepsis syndromes.
Yet, when viewed collectively, the above findings invite a much
more important speculation: i.e. that a detectable pre-lethal shift in
the systemic coagulation/fibrinolytic (as well as inflammatory)
responses occurs in a relatively advanced stage of sepsis (and
irrespective of its chronological scale). In other words, given that
septic deaths are not typically heralded immediately after the onset
of the disease, it is suggestive that interventions undertaken even in
late sepsis may effectively save lives – provided that they are
appropriately fine-tuned.
Given that the vascular bed constitutes a rich source of PAI-1,
the last investigative element of experiment 2 aimed to assess the
relationship between the pre-lethal increase of circulating PAI-1
and the level of endothelial injury. Soluble thrombomodulin is
regarded as an accurate marker of endothelial injury and both
trauma and sepsis trigger its rapid release into the bloodstream
[60,61]. In our study, sTM increased immediately after TH and
remained strongly elevated until day 3 post-CLP. This is indicative
of a lasting and relatively high-degree of endothelial injury with
the first hit serving as the main injurious trigger (given that 2nd hit
did not further aggravate the magnitude of the TH-induced
damage). Surprisingly, the strong outcome-dependent separation
of circulating PAI-1 (Fig. 5) was not reflected by fluctuation of
sTM: its trajectories in dying and surviving TH-CLP mice were
nearly superimposable (Fig. 6). Given a relatively short half-life
(,2 h) of PAI-1 and sTM in circulation [20,61], the above data
strongly imply that in post-traumatic sepsis, the pre-lethal PAI-1
release is independent of the level of endothelial injury.
Out of all investigated organs in our two-hit model, liver
demonstrated the most robust and consistent expression of the
PAI-1 gene. Therefore, in the final step of our study, we sought to
characterize the outcome-dependent transcription of hepatic PAI-
1 and correlate it with the release of circulating PAI-1 protein.
Given that analyses of outcome-based gene expression in tissues
were not possible in experiment 2, we devised an alternative
protocol to enable accurate comparison of lethal versus non-lethal
PAI-1 responses (see Study Design). Specifically, we relied on
Figure 7. Plasma thrombomodulin level during TH-CLP devel-
ops irrespective of outcome. 3 month-old mice (n = 42) were
subjected to trauma and hemorrhage (TH) at 248 h followed by mild
(23G) cecal ligation and puncture (CLP) sepsis at 0 h. The experimental
period was divided into a pre- and post-CLP (red and black dots,
respectively) phase. From the TH time point (248 h) on, 20 ml of blood
was collected daily, and additionally at 6 h, until 96 h (day 6 post-TH/
day 4 post-CLP). Animals were observed until death or day 14 post-CLP,
whichever occurred first. For SUR, n = 17 for each time point. For DEAD:
n = 24 at 248 h 224 h, 0 h and 6 h; n = 23 at 24 h; n = 15 at 48 h; n = 6
at 72 h. Figure depicts the time course of circulating thrombomodulin
of dying (DEAD, black-filled dots) and surviving (SUR, white-filled dots)
mice. CLP time point indicated by arrow.
doi:10.1371/journal.pone.0055467.g007
Table 3. Correlation of circulating PAI-1 with its hepatic gene
expression and liver function.
Variable 1 Variable 2 Spearman-r 95% CI P-value N
PAI-1
plasma
PAI-1 mRNA
liver
0.86 0.63 to 0.95 ,0.01 18
PAI-1
plasma
ALT 0.66 0.27 to 0.87 ,0.01 18
PAI-1
plasma
AST 0.66 0.26 to 0.87 ,0.01 18
PAI-1, plasminogen activator inhibitor type 1; mRNA, messenger ribonucleic
acid; ALT, alanine transaminase; AST, aspartate transaminase; CI, confidence
interval; N, number of values.
3 month-old mice were subjected to trauma and hemorrhage (248 h) followed
by CLP sepsis of medium-severity (17G) at 0 h. In the post-CLP phase, mice with
body temperature (BT) ,28uC and littermates with BT .36uC were identified
(1:1 ratio) and instantly sacrificed. PAI-1 mRNA expression in the liver and
plasma concentrations of total PAI-1, ALT and AST, and were correlated
independent of BT and expressed by Spearman’s rank correlation coefficient (r).
doi:10.1371/journal.pone.0055467.t003
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11. changes in body temperature to stratify TH-CLP mice into those
predicted to die (P-DIE; hypothermic) and those predicted to live
(P-LIVE; normothermic). The comparison revealed a strong
outcome-dependent activation of PAI-1 transcription in the liver
(but not in LCVC). Notably, circulating PAI-1 tightly correlated
with its hepatic gene expression (Table 3) and pre-lethal increases
in both parameters demonstrated a strong positive correlation with
the level of liver dysfunction (by ALT and AST). While this does
not establish a clear causal relationship, Lagoa et al., suggested that
hepatic expression of PAI-1 might directly influence liver function
or vice versa. Using a rat model of hemorrhagic shock, they showed
that generalized liver dysfunction as well as localized damage of
hepatic endothelium were absent in PAI-1 knockout mice but this
tissue preservation was gone after administration of stable PAI-1
[56]. Interestingly, they also observed a post-resuscitation shift of
the immunohistochemical PAI-1 protein signal from hepatocytes
into the endothelium-rich hepatic sinusoids. It remains to be
verified whether similar effects can be triggered by systemic
infections. However, the combined results of our study convinc-
ingly imply that liver (either parenchymal cells and/or hepatic
Figure 8. Release of PAI-1 into circulation is of hepatic origin and associated with liver dysfunction. 3 month-old mice (n = 30) were
subjected to trauma and hemorrhage (TH) at 248 h followed by mild (23G) cecal ligation and puncture (CLP) sepsis at 0 h. Body temperature (BT)
was measured in all mice in the post-CLP phase. Mice with body temperature (BT) ,28uC (n = 9) were identified as predicted to die (P-DIE), while
littermates with BT .36uC (n = 9) were identified as predicted to live (P-LIVE). Identified mice were instantly sacrificed (all at 48 h post-CLP) for
collection of blood, liver and left cranial vena cava (LCVC). A) total plasminogen activator inhibitor type 1 (PAI-1) in plasma B) PAI-1 mRNA expression
in the liver, C) plasma alanine transaminase (ALT), D) plasma aspartate transaminase (AST), E) PAI-1 mRNA expression in the left cranial vena cava
(LCVC). Data as box plot diagrams representing 9 mice per column, with whiskers indicating minimum and maximum. In E, each column represents
a total of 9 mice (3 pooled collections of LCVC samples, each collection comprising 3 mice). *P,0.05 versus ,28uC.
doi:10.1371/journal.pone.0055467.g008
Preceding Trauma Changes the Septic PAI-1 Response
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12. endothelium) is the key contributor behind the robust pre-lethal
release of circulating PAI-1 in post-traumatic sepsis.
In conclusion, we postulate that initial blunt trauma coupled
with sublethal hemorrhage does not alter the concentration of
PAI-1 in the plasma during subsequent sepsis, yet they strongly
modulate PAI-1 gene expression in selected organs. Moreover,
plasma PAI-1 in post-traumatic sepsis is chiefly secreted in the liver
and its synthesis and release is higher in dying subjects than in
survivors. Interestingly, the latter phenomenon is irrespective of
the level of damage to vascular endothelium. Finally, since the
TH-CLP model closely mimics the evolution of PAI-1 observed in
trauma and/or septic patients it may serve as a relevant platform
for pre-clinical testing of potential PAI-1-modulating therapeutics.
Supporting Information
Figure S1 Organ function response to TH-CLP. 3 week-
old mice were subjected to trauma and hemorrhage (TH) at-48 h
followed by mild (23G) polymicrobial cecal ligation and puncture
(CLP) sepsis at 0 h. At 248 h, 242 h, 236 h, 224 h, 0 h, 6 h,
12 h and 24 h, mice (6 per time point) were sacrificed and blood
was collected and analyzed for A) alanine transaminase (ALT), B)
aspartate transaminase (AST), C) blood urea nitrogen (urea), D)
glucose, and E) lactate dehydrogenase (LDH) plasma concentra-
tions. Figure shows box plot diagrams, with whiskers indicating
minimum and maximum and dots representing values outside of
1.5 inter-quartile range. *P,0.05 versus 248 h, 1P,0.05 versus
0 h.
(TIF)
Table S1 Prediction of TH-CLP outcomes based on
body temperature. BT, body temperature; AUC, area under
the curve; CI, confidence interval, NPV, negative predictive value
for outcome; PPV, positive predictive value for outcome. 3 month-
old female mice (n = 57) were subjected to trauma and
hemorrhage (TH, 248 h) followed by cecal ligation and puncture
(CLP) sepsis of medium-severity (17G) at 0 h. Body temperature
was recorded in all mice in the post-CLP phase (days 1–5 post-
CLP). Table displays predictive accuracy (within next 48 h) for BT
measurements taken at 48 h post-CLP only.
(DOC)
Table S2 Hematological response to TH-CLP. 3 week-old
mice were subjected to trauma and hemorrhage (248 h) followed
by mild (23G) polymicrobial cecal ligation and puncture (CLP)
sepsis at 0 h. At 248 h, 242 h, 236 h, 224 h, 0 h, 6 h, 12 h and
24 h, mice (6 per time point) were sacrificed and blood was
collected and analyzed. RBC, red blood cell count; PLT, platelet
count; Hb, hemoglobin; WBC, white blood cell count; NEU,
neutrophil granulocyte, LYM, lymphocyte count. a
P,0.05 versus
248 h, b
P,0.05 versus 242 h, c
P,0.05 versus 236 h, d
P,0.05
versus 224 h, e
P,0.05 versus 0 h, f
P,0.05 versus 6 h, g
P,0.05
versus 24 h, 05 versus all other time points. Data as mean 6 SD; n
$5 per time point.
(DOC)
Table S3 Predictive accuracy of plasma PAI-1 for death.
AUC, area under the curve; ROC, receiver operating character-
istic; CI, confidence interval. *P,0.05. Plasma PAI-1 levels
obtained were evaluated by the ROC curve to determine the
predictive accuracy for the outcome as expressed by the AUC.
The predictive accuracy of the ROC-AUC was defined as: 0.9–
1 = excellent, 0.8–0.9 = good, 0.7–0.8 = fair, 0.6–0.7 = poor and
,0.6 = not useful.
(DOC)
Acknowledgments
We gratefully acknowledge the help of A. Meinl with IHC, and the expert
technical assistance of C. Kober and A. Khadem. We thank J. Ferguson for
proofreading the manuscript.
Author Contributions
Conceived and designed the experiments: PR MVG SB MFO. Performed
the experiments: PR AS SD KMW. Analyzed the data: PR AS MJ MFO.
Contributed reagents/materials/analysis tools: PR MJ. Wrote the paper:
PR MFO.
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