ABSTRACT
Sepsis remains a major cause of morbidity and mortality worldwide, with increased burden in low- and middle-resource settings. The role of the inflammatory response in the pathogenesis of the syndrome has supported the modern concept of sepsis. Nevertheless, a definition of sepsis and the criteria for its recognition is a continuous process, which reflects the growing knowledge of its mechanisms and the success and failure of diagnostic and therapeutic interventions. Here we review the evolving concepts of sepsis, from the "systemic inflammatory response syndrome triggered by infection" (Sepsis-1) to "a severe, potentially fatal, organic dysfunction caused by an inadequate or dysregulated host response to infection" (Sepsis-3). We focused in the pathophysiology behind the concept and the criteria for recognition and diagnosis of sepsis. A major challenge in evaluating the host response in sepsis is to characterize what is protective and what is harmful, and we discuss that, at least in part, the apparent dysregulated host response may be an effort to adapt to a hostile environment. The new criteria for recognition and diagnosis of sepsis were derived from robust databases, restricted, however, to developed countries. Since then, the criteria have been supported in different clinical settings and in different economic and epidemiological contexts, but still raise discussion regarding their use for the identification versus the prognostication of the septic patient. Clinicians should not be restricted to definition criteria when evaluating patients with infection and should wisely use the broad array of information obtained by rigorous clinical observation.
Subject(s)
Sepsis/immunology , Sepsis/physiopathology , Humans , Lactic Acid/blood , Medical Illustration , Organ Dysfunction Scores , Sepsis/diagnosis , Sepsis/metabolismABSTRACT
Sepsis remains a major cause of morbidity and mortality worldwide, with increased burden in low- and middle-resource settings. The role of the inflammatory response in the pathogenesis of the syndrome has supported the modern concept of sepsis. Nevertheless, a definition of sepsis and the criteria for its recognition is a continuous process, which reflects the growing knowledge of its mechanisms and the success and failure of diagnostic and therapeutic interventions. Here we review the evolving concepts of sepsis, from the "systemic inflammatory response syndrome triggered by infection" (Sepsis-1) to "a severe, potentially fatal, organic dysfunction caused by an inadequate or dysregulated host response to infection" (Sepsis-3). We focused in the pathophysiology behind the concept and the criteria for recognition and diagnosis of sepsis. A major challenge in evaluating the host response in sepsis is to characterize what is protective and what is harmful, and we discuss that, at least in part, the apparent dysregulated host response may be an effort to adapt to a hostile environment. The new criteria for recognition and diagnosis of sepsis were derived from robust databases, restricted, however, to developed countries. Since then, the criteria have been supported in different clinical settings and in different economic and epidemiological contexts, but still raise discussion regarding their use for the identification versus the prognostication of the septic patient. Clinicians should not be restricted to definition criteria when evaluating patients with infection and should wisely use the broad array of information obtained by rigorous clinical observation.
Subject(s)
Humans , Sepsis/physiopathology , Sepsis/immunology , Sepsis/diagnosis , Sepsis/metabolism , Lactic Acid/blood , Organ Dysfunction Scores , Medical IllustrationABSTRACT
BACKGROUND: Ataxia-telangiectasia (AT) is a well-known primary immunodeficiency with recurrent sinopulmonary infections and variable abnormalities in both the humoral and cellular immune system. Dysfunctions in immunoglobulin production, reduced number of B cells, and B-cell receptor excision circles copies have been reported. We aimed to understand the immunological mechanisms involving the humoral compartment in AT patients by analysing peripheral blood B cells subsets, B-T lymphocyte cooperation through the expression of CD40 and CD40 ligand (CD40L), and cytokines involved in class-switch recombination production. METHODS: We compared the proportion of B-cell subsets, the expression of CD40/CD40L, and the plasma levels of IL-6 and IFN-γ of 18 AT patients and 15 healthy age-sex-matched controls using flow cytometry. RESULTS: We found that some steps in peripheral B cell development were altered in AT with a pronounced reduction of cell-surface CD40 expression. The proportions of transitional and naïve-mature B cells were reduced, whereas CD21-low, natural effector memory, IgM-only memory, and IgG atypical memory B cells were present in a higher proportion. CONCLUSIONS: These findings revealed a disturbed B-cell homeostasis with unconventional maturation of B lymphocyte memory cells, which can explain the consequent impairment of humoral immunity.
Subject(s)
Ataxia Telangiectasia/immunology , B-Lymphocyte Subsets/immunology , Adolescent , Adult , CD40 Antigens/biosynthesis , Child , Child, Preschool , Female , Humans , Immunophenotyping , Male , Young AdultABSTRACT
Inflammasome signalling induces the processing and secretion of interleukin (IL)-1ß and IL-18 which, coupled with pyroptosis, activate further the inflammatory response. In the present study we evaluated the expression of genes involved in inflammasome signalling pathways in septic patients, their interaction networks and the predicted functions modulated in survivors and non-survivors. Twenty-seven patients with sepsis secondary to community-acquired pneumonia admitted to intensive care units from three general hospitals in São Paulo were included into the study. We performed a polymerase chain reaction (PCR) array encompassing 35 genes related to the nucleotide-binding oligomerization domain and leucine-rich repeat-containing (NLR)-inflammasome in peripheral blood mononuclear cells obtained at admission and after 7 days of follow-up. Eleven healthy volunteers were used as the reference group. Increased NLRC4 and NLRP3 and decreased nucleotide-binding oligomerization domain (NOD1), and NLRP1 expression was observed in septic patients compared to healthy individuals; the IL-1ß and IL-18 expression levels were also high in the patients. The gene expression changes followed the same patterns in surviving and non-surviving patients, with higher magnitudes observed in non-survivors. Functional analyses revealed, however, that activation and inhibition intensity for representing functions were different in survivors and non-survivors, as for production of reactive oxygen species, synthesis of nitric oxide and for the control of bacterial infections. Our results showed that the genes involved in the activation of the NLR-inflammasome cascades were altered substantially in septic patients, with a higher number of altered genes and a higher intensity in the disturbance of gene expression found among patients dying of sepsis.
Subject(s)
Community-Acquired Infections/complications , Inflammasomes/genetics , Sepsis/genetics , Transcriptome , Adult , Aged , Aged, 80 and over , Brazil , Case-Control Studies , Cell Line , Female , Gene Expression , Humans , Interleukin-18/genetics , Interleukin-18/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Prospective Studies , Pyroptosis , Reactive Oxygen Species/metabolism , Signal Transduction , Toll-Like Receptors/genetics , Toll-Like Receptors/metabolismABSTRACT
Recognition of pathogens is performed by specific receptors in cells of the innate immune system, which may undergo modulation during the continuum of clinical manifestations of sepsis. Monocytes and neutrophils play a key role in host defense by sensing and destroying microorganisms. This study aimed to evaluate the expression of CD14 receptors on monocytes; CD66b and CXCR2 receptors on neutrophils; and TLR2, TLR4, TLR5, TLR9, and CD11b receptors on both cell types of septic patients. Seventy-seven septic patients (SP) and 40 healthy volunteers (HV) were included in the study, and blood samples were collected on day zero (D0) and after 7 days of therapy (D7). Evaluation of the cellular receptors was carried out by flow cytometry. Expression of CD14 on monocytes and of CD11b and CXCR2 on neutrophils from SP was lower than that from HV. Conversely, expression of TLR5 on monocytes and neutrophils was higher in SP compared with HV. Expression of TLR2 on the surface of neutrophils and that of TLR5 on monocytes and neutrophils of SP was lower at D7 than at D0. In addition, SP who survived showed reduced expression of TLR2 and TLR4 on the surface of neutrophils at D7 compared to D0. Expression of CXCR2 for surviving patients was higher at follow-up compared to baseline. We conclude that expression of recognition and cell signaling receptors is differentially regulated between SP and HV depending on the receptor being evaluated.
Subject(s)
Adult , Aged , Child, Preschool , Female , Humans , Male , Middle Aged , Chemokines/blood , Integrins/blood , Monocytes/chemistry , Neutrophils/chemistry , Sepsis/immunology , Toll-Like Receptors/blood , Anti-Bacterial Agents/therapeutic use , Antigens, CD/blood , /blood , /blood , Cell Adhesion Molecules/blood , Flow Cytometry , GPI-Linked Proteins/blood , Hospital Mortality , Immunophenotyping , Intensive Care Units , /blood , Statistics, Nonparametric , Sepsis/therapy , Treatment Outcome , Toll-Like Receptor 9/blood , /blood , /blood , /bloodABSTRACT
Recognition of pathogens is performed by specific receptors in cells of the innate immune system, which may undergo modulation during the continuum of clinical manifestations of sepsis. Monocytes and neutrophils play a key role in host defense by sensing and destroying microorganisms. This study aimed to evaluate the expression of CD14 receptors on monocytes; CD66b and CXCR2 receptors on neutrophils; and TLR2, TLR4, TLR5, TLR9, and CD11b receptors on both cell types of septic patients. Seventy-seven septic patients (SP) and 40 healthy volunteers (HV) were included in the study, and blood samples were collected on day zero (D0) and after 7 days of therapy (D7). Evaluation of the cellular receptors was carried out by flow cytometry. Expression of CD14 on monocytes and of CD11b and CXCR2 on neutrophils from SP was lower than that from HV. Conversely, expression of TLR5 on monocytes and neutrophils was higher in SP compared with HV. Expression of TLR2 on the surface of neutrophils and that of TLR5 on monocytes and neutrophils of SP was lower at D7 than at D0. In addition, SP who survived showed reduced expression of TLR2 and TLR4 on the surface of neutrophils at D7 compared to D0. Expression of CXCR2 for surviving patients was higher at follow-up compared to baseline. We conclude that expression of recognition and cell signaling receptors is differentially regulated between SP and HV depending on the receptor being evaluated.
Subject(s)
Chemokines/blood , Integrins/blood , Monocytes/chemistry , Neutrophils/chemistry , Sepsis/immunology , Toll-Like Receptors/blood , Adult , Aged , Anti-Bacterial Agents/therapeutic use , Antigens, CD/blood , CD11b Antigen/blood , Cell Adhesion Molecules/blood , Child, Preschool , Female , Flow Cytometry , GPI-Linked Proteins/blood , Hospital Mortality , Humans , Immunophenotyping , Intensive Care Units , Lipopolysaccharide Receptors/blood , Male , Middle Aged , Receptors, Interleukin-8B/blood , Sepsis/therapy , Statistics, Nonparametric , Toll-Like Receptor 2/blood , Toll-Like Receptor 4/blood , Toll-Like Receptor 5/blood , Toll-Like Receptor 9/blood , Treatment OutcomeABSTRACT
Lipopolysaccharide (LPS) activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R) and smooth (S) forms signal through Toll-like receptor 4 (TLR4), but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS) and nitric oxide (NO) generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30 percent increase followed by a 40 percent decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50 percent reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.
Subject(s)
Adult , Female , Humans , Male , Lipopolysaccharides/pharmacology , Monocytes/drug effects , Neutrophil Activation/drug effects , Neutrophils/drug effects , Nitric Oxide/metabolism , Reactive Oxygen Species/metabolism , Receptors, Cell Surface/metabolism , /immunology , /metabolism , Flow Cytometry , Monocytes/immunology , Monocytes/metabolism , Neutrophils/immunology , Neutrophils/metabolism , Nitric Oxide/biosynthesis , Salmonella , /immunology , /metabolism , Up-Regulation/drug effects , Up-Regulation/immunologyABSTRACT
Tolerance to lipopolysaccharide (LPS) occurs when animals or cells exposed to LPS become hyporesponsive to a subsequent challenge with LPS. This mechanism is believed to be involved in the down-regulation of cellular responses observed in septic patients. The aim of this investigation was to evaluate LPS-induced monocyte tolerance of healthy volunteers using whole blood. The detection of intracellular IL-6, bacterial phagocytosis and reactive oxygen species (ROS) was determined by flow cytometry, using anti-IL-6-PE, heat-killed Staphylococcus aureus stained with propidium iodide and 2',7'-dichlorofluorescein diacetate, respectively. Monocytes were gated in whole blood by combining FSC and SSC parameters and CD14-positive staining. The exposure to increasing LPS concentrations resulted in lower intracellular concentration of IL-6 in monocytes after challenge. A similar effect was observed with challenge with MALP-2 (a Toll-like receptor (TLR)2/6 agonist) and killed Pseudomonas aeruginosa and S. aureus, but not with flagellin (a TLR5 agonist). LPS conditioning with 15 ng/mL resulted in a 40 percent reduction of IL-6 in monocytes. In contrast, phagocytosis of P. aeruginosa and S. aureus and induced ROS generation were preserved or increased in tolerant cells. The phenomenon of tolerance involves a complex regulation in which the production of IL-6 was diminished, whereas the bacterial phagocytosis and production of ROS was preserved. Decreased production of proinflammatory cytokines and preserved or increased production of ROS may be an adaptation to control the deleterious effects of inflammation while preserving antimicrobial activity.
Subject(s)
Adult , Female , Humans , Male , Lipopeptides/pharmacology , Lipopolysaccharides/pharmacology , Monocytes/immunology , Pseudomonas aeruginosa/immunology , Reactive Oxygen Species/metabolism , Staphylococcus aureus/immunology , /immunology , Monocytes/drug effects , Monocytes/metabolism , Phagocytosis/immunology , Pseudomonas aeruginosa/metabolism , Reactive Oxygen Species/immunology , Staphylococcus aureus/metabolism , Toll-Like Receptors/antagonists & inhibitorsABSTRACT
Lipopolysaccharide (LPS) activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R) and smooth (S) forms signal through Toll-like receptor 4 (TLR4), but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS) and nitric oxide (NO) generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.
Subject(s)
Lipopolysaccharides/pharmacology , Monocytes/drug effects , Neutrophil Activation/drug effects , Neutrophils/drug effects , Nitric Oxide/metabolism , Reactive Oxygen Species/metabolism , Receptors, Cell Surface/metabolism , Adult , Female , Flow Cytometry , Humans , Lipopolysaccharide Receptors/immunology , Lipopolysaccharide Receptors/metabolism , Male , Monocytes/immunology , Monocytes/metabolism , Neutrophils/immunology , Neutrophils/metabolism , Nitric Oxide/biosynthesis , Salmonella , Toll-Like Receptor 4/immunology , Toll-Like Receptor 4/metabolism , Up-Regulation/drug effects , Up-Regulation/immunologyABSTRACT
Tolerance to lipopolysaccharide (LPS) occurs when animals or cells exposed to LPS become hyporesponsive to a subsequent challenge with LPS. This mechanism is believed to be involved in the down-regulation of cellular responses observed in septic patients. The aim of this investigation was to evaluate LPS-induced monocyte tolerance of healthy volunteers using whole blood. The detection of intracellular IL-6, bacterial phagocytosis and reactive oxygen species (ROS) was determined by flow cytometry, using anti-IL-6-PE, heat-killed Staphylococcus aureus stained with propidium iodide and 2',7'-dichlorofluorescein diacetate, respectively. Monocytes were gated in whole blood by combining FSC and SSC parameters and CD14-positive staining. The exposure to increasing LPS concentrations resulted in lower intracellular concentration of IL-6 in monocytes after challenge. A similar effect was observed with challenge with MALP-2 (a Toll-like receptor (TLR)2/6 agonist) and killed Pseudomonas aeruginosa and S. aureus, but not with flagellin (a TLR5 agonist). LPS conditioning with 15 ng/mL resulted in a 40% reduction of IL-6 in monocytes. In contrast, phagocytosis of P. aeruginosa and S. aureus and induced ROS generation were preserved or increased in tolerant cells. The phenomenon of tolerance involves a complex regulation in which the production of IL-6 was diminished, whereas the bacterial phagocytosis and production of ROS was preserved. Decreased production of proinflammatory cytokines and preserved or increased production of ROS may be an adaptation to control the deleterious effects of inflammation while preserving antimicrobial activity.
Subject(s)
Lipopeptides/pharmacology , Lipopolysaccharides/pharmacology , Monocytes/immunology , Pseudomonas aeruginosa/immunology , Reactive Oxygen Species/metabolism , Staphylococcus aureus/immunology , Adult , Female , Humans , Interleukin-6/immunology , Male , Monocytes/drug effects , Monocytes/metabolism , Phagocytosis/immunology , Pseudomonas aeruginosa/metabolism , Reactive Oxygen Species/immunology , Staphylococcus aureus/metabolism , Toll-Like Receptors/antagonists & inhibitorsABSTRACT
We evaluated the recovery of cardiovascular function after transient cardiogenic shock. Cardiac tamponade was performed for 1 h and post-shock data were collected in 5 domestic large white female pigs (43 +/- 5 kg) for 6 h. The control group (N = 5) was observed for 6 h after 1 h of resting. During 1 h of cardiac tamponade, experimental animals evolved a low perfusion status with a higher lactate level (8.0 +/- 2.2 vs 1.9 +/- 0.9 mEq/L), lower standard base excess (-7.3 +/- 3.3 vs 2.0 +/- 0.9 mEq/L), lower urinary output (0.9 +/- 0.9 vs 3.0 +/- 1.4 mL x kg(-1) x h(-1)), lower mixed venous saturation, higher ileum partial pressure of CO2-end tidal CO2 (EtCO2) gap and a lower cardiac index than the control group. Throughout the 6-h recovery phase after cardiac tamponade, tamponade animals developed significant tachycardia with preserved cardiac index, resulting in a lower left ventricular stroke work, suggesting possible myocardial dysfunction. Vascular dysfunction was present with persistent systemic hypotension as well as persistent pulmonary hypertension. In contrast, oliguria, hyperlactatemia and metabolic acidosis were corrected by the 6th hour. The inflammatory characteristics were an elevated core temperature and increased plasma levels of interleukin-6 in the tamponade group compared to the control group. We conclude that cardiovascular recovery after a transient and severe low flow systemic state was incomplete. Vascular dysfunction persisted up to 6 h after release of tamponade. These inflammatory characteristics may also indicate that inflammatory activation is a possible pathway involved in the pathogenesis of cardiogenic shock.
Subject(s)
Cardiac Tamponade/physiopathology , Hypotension/physiopathology , Shock, Cardiogenic/physiopathology , Systemic Inflammatory Response Syndrome/physiopathology , Animals , Cardiac Tamponade/blood , Female , Hypotension/etiology , Recovery of Function , Shock, Cardiogenic/blood , Swine , Systemic Inflammatory Response Syndrome/etiology , Time FactorsABSTRACT
We evaluated the recovery of cardiovascular function after transient cardiogenic shock. Cardiac tamponade was performed for 1 h and post-shock data were collected in 5 domestic large white female pigs (43 ± 5 kg) for 6 h. The control group (N = 5) was observed for 6 h after 1 h of resting. During 1 h of cardiac tamponade, experimental animals evolved a low perfusion status with a higher lactate level (8.0 ± 2.2 vs 1.9 ± 0.9 mEq/L), lower standard base excess (-7.3 ± 3.3 vs 2.0 ± 0.9 mEq/L), lower urinary output (0.9 ± 0.9 vs 3.0 ± 1.4 mL·kg-1·h-1), lower mixed venous saturation, higher ileum partial pressure of CO2-end tidal CO2 (EtCO2) gap and a lower cardiac index than the control group. Throughout the 6-h recovery phase after cardiac tamponade, tamponade animals developed significant tachycardia with preserved cardiac index, resulting in a lower left ventricular stroke work, suggesting possible myocardial dysfunction. Vascular dysfunction was present with persistent systemic hypotension as well as persistent pulmonary hypertension. In contrast, oliguria, hyperlactatemia and metabolic acidosis were corrected by the 6th hour. The inflammatory characteristics were an elevated core temperature and increased plasma levels of interleukin-6 in the tamponade group compared to the control group. We conclude that cardiovascular recovery after a transient and severe low flow systemic state was incomplete. Vascular dysfunction persisted up to 6 h after release of tamponade. These inflammatory characteristics may also indicate that inflammatory activation is a possible pathway involved in the pathogenesis of cardiogenic shock.
Subject(s)
Animals , Female , Cardiac Tamponade/physiopathology , Hypotension/physiopathology , Shock, Cardiogenic/physiopathology , Systemic Inflammatory Response Syndrome/physiopathology , Cardiac Tamponade/blood , Hypotension/etiology , Recovery of Function , Swine , Shock, Cardiogenic/blood , Systemic Inflammatory Response Syndrome/etiology , Time FactorsABSTRACT
Experimental models of sepsis-induced pulmonary alterations are important for the study of pathogenesis and for potential intervention therapies. The objective of the present study was to characterize lung dysfunction (low PaO2 and high PaCO2, and increased cellular infiltration, protein extravasation, and malondialdehyde (MDA) production assessed in bronchoalveolar lavage) in a sepsis model consisting of intraperitoneal (ip) injection of Escherichia coli and the protective effects of pentoxifylline (PTX). Male Wistar rats (weighing between 270 and 350 g) were injected ip with 10(7) or 10(9) CFU/100 g body weight or saline and samples were collected 2, 6, 12, and 24 h later (N = 5 each group). PaO2, PaCO2 and pH were measured in blood, and cellular influx, protein extravasation and MDA concentration were measured in bronchoalveolar lavage. In a second set of experiments either PTX or saline was administered 1 h prior to E. coli ip injection (N = 5 each group) and the animals were observed for 6 h. Injection of 10(7) or 10(9) CFU/100 g body weight of E. coli induced acidosis, hypoxemia, and hypercapnia. An increased (P < 0.05) cell influx was observed in bronchoalveolar lavage, with a predominance of neutrophils. Total protein and MDA concentrations were also higher (P < 0.05) in the septic groups compared to control. A higher tumor necrosis factor-alpha (P < 0.05) concentration was also found in these animals. Changes in all parameters were more pronounced with the higher bacterial inoculum. PTX administered prior to sepsis reduced (P < 0.05) most functional alterations. These data show that an E. coli ip inoculum is a good model for the induction of lung dysfunction in sepsis, and suitable for studies of therapeutic interventions.
Subject(s)
Lung Diseases/drug therapy , Pentoxifylline/therapeutic use , Phosphodiesterase Inhibitors/therapeutic use , Pulmonary Gas Exchange/drug effects , Sepsis/drug therapy , Acute Disease , Animals , Disease Models, Animal , Escherichia coli Infections/complications , Escherichia coli Infections/drug therapy , Inflammation/drug therapy , Inflammation Mediators/blood , Male , Malondialdehyde/blood , Rats , Rats, Wistar , Sepsis/microbiologyABSTRACT
Experimental models of sepsis-induced pulmonary alterations are important for the study of pathogenesis and for potential intervention therapies. The objective of the present study was to characterize lung dysfunction (low PaO2 and high PaCO2, and increased cellular infiltration, protein extravasation, and malondialdehyde (MDA) production assessed in bronchoalveolar lavage) in a sepsis model consisting of intraperitoneal (ip) injection of Escherichia coli and the protective effects of pentoxifylline (PTX). Male Wistar rats (weighing between 270 and 350 g) were injected ip with 10(7) or 10(9) CFU/100 g body weight or saline and samples were collected 2, 6, 12, and 24 h later (N = 5 each group). PaO2, PaCO2 and pH were measured in blood, and cellular influx, protein extravasation and MDA concentration were measured in bronchoalveolar lavage. In a second set of experiments either PTX or saline was administered 1 h prior to E. coli ip injection (N = 5 each group) and the animals were observed for 6 h. Injection of 10(7) or 10(9) CFU/100 g body weight of E. coli induced acidosis, hypoxemia, and hypercapnia. An increased (P < 0.05) cell influx was observed in bronchoalveolar lavage, with a predominance of neutrophils. Total protein and MDA concentrations were also higher (P < 0.05) in the septic groups compared to control. A higher tumor necrosis factor-alpha (P < 0.05) concentration was also found in these animals. Changes in all parameters were more pronounced with the higher bacterial inoculum. PTX administered prior to sepsis reduced (P < 0.05) most functional alterations. These data show that an E. coli ip inoculum is a good model for the induction of lung dysfunction in sepsis, and suitable for studies of therapeutic interventions.
Subject(s)
Animals , Male , Rats , Lung Diseases/drug therapy , Pentoxifylline/therapeutic use , Phosphodiesterase Inhibitors/therapeutic use , Pulmonary Gas Exchange/drug effects , Sepsis/drug therapy , Acute Disease , Disease Models, Animal , Escherichia coli Infections/complications , Escherichia coli Infections/drug therapy , Inflammation Mediators/blood , Inflammation/drug therapy , Malondialdehyde/blood , Rats, Wistar , Sepsis/microbiologyABSTRACT
Glycolipoprotein (GLP) from pathogenic serovars of Leptospira has been implicated in the pathogenesis of leptospirosis by its presence in tissues of experimental animals with leptospirosis, the inhibition of the Na,K-ATPase pump activity, and induced production of cytokines. The aims of the present study were to investigate the induction of IL-6 by GLP in peripheral blood mononuclear cells (PBMC) and to demonstrate monocyte stimulation at the cellular level in whole blood from healthy volunteers. PBMC were stimulated with increasing concentrations (5 to 2500 ng/ml) of GLP extracted from the pathogenic L. interrogans serovar Copenhageni, lipopolysaccharide (positive control) or medium (negative control), and supernatants were collected after 6, 20/24, and 48 h, and kept at -80 degrees C until use. Whole blood was diluted 1:1 in RPMI medium and cultivated for 6 h, with medium, GLP and lipopolysaccharide as described above. Monensin was added after the first hour of culture. Supernatant cytokine levels from PBMC were measured by ELISA and intracellular IL-6 was detected in monocytes in whole blood cultures by flow-cytometry. Monocytes were identified in whole blood on the basis of forward versus side scatter parameters and positive reactions with CD45 and CD14 antibodies. GLP ( > or = 50 ng/ml)-induced IL-6 levels in supernatants were detected after 6-h incubation, reaching a peak after 20/24 h. The percentage of monocytes staining for IL-6 increased with increasing GLP concentration. Thus, our findings show a GLP-induced cellular activation by demonstrating the ability of GLP to induce IL-6 and the occurrence of monocyte activation in whole blood at the cellular level.
Subject(s)
Glycoproteins/pharmacology , Interleukin-6/biosynthesis , Leptospira interrogans/immunology , Monocytes/immunology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Interleukin-6/immunology , Leukocyte Common Antigens/immunology , Lipopolysaccharide Receptors/immunology , Monocytes/microbiologyABSTRACT
Glycolipoprotein (GLP) from pathogenic serovars of Leptospira has been implicated in the pathogenesis of leptospirosis by its presence in tissues of experimental animals with leptospirosis, the inhibition of the Na,K-ATPase pump activity, and induced production of cytokines. The aims of the present study were to investigate the induction of IL-6 by GLP in peripheral blood mononuclear cells (PBMC) and to demonstrate monocyte stimulation at the cellular level in whole blood from healthy volunteers. PBMC were stimulated with increasing concentrations (5 to 2500 ng/ml) of GLP extracted from the pathogenic L. interrogans serovar Copenhageni, lipopolysaccharide (positive control) or medium (negative control), and supernatants were collected after 6, 20/24, and 48 h, and kept at -80°C until use. Whole blood was diluted 1:1 in RPMI medium and cultivated for 6 h, with medium, GLP and lipopolysaccharide as described above. Monensin was added after the first hour of culture. Supernatant cytokine levels from PBMC were measured by ELISA and intracellular IL-6 was detected in monocytes in whole blood cultures by flow-cytometry. Monocytes were identified in whole blood on the basis of forward versus side scatter parameters and positive reactions with CD45 and CD14 antibodies. GLP ( > or = 50 ng/ml)-induced IL-6 levels in supernatants were detected after 6-h incubation, reaching a peak after 20/24 h. The percentage of monocytes staining for IL-6 increased with increasing GLP concentration. Thus, our findings show a GLP-induced cellular activation by demonstrating the ability of GLP to induce IL-6 and the occurrence of monocyte activation in whole blood at the cellular level.
Subject(s)
Humans , Glycoproteins/pharmacology , /biosynthesis , Leptospira interrogans/immunology , Monocytes/immunology , /immunology , /immunology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , /immunology , Monocytes/microbiologyABSTRACT
In this report we evaluated CD4(+) T, CD8(+) T and natural killer (NK) cell counts, the levels of naive/memory subsets within the CD4(+) T lymphocyte population, expression of CD38 on T lymphocytes, and CD4(+) and CD8(+) T cell cytokine production in two girls with hyper-IgM (HIM) syndrome. Both girls developed recurrent infections early in infancy, presenting a wide spectrum of clinical manifestations, with a strikingly different disease severity between them. CD4(+) T cell counts were low in both children (patient 1: 214 cells/mm(3) and patient 2: 392 cells/mm(3)), and the CD4/CD8 T cell ratio was 0.4 for patient 1, the patient with the more severe disease, and 1.4 for patient 2. NK cell numbers were low in patient 1 (60 cells/mm(3)) and borderline (286 cells/mm(3)) with regard to normal levels in patient 2. An imbalance of naive and memory/effector cell subsets was found in both girls, with the percentage of CD45RA(+) 27(+) (naive) CD4(+) T lymphocytes being 5.8 and 12.4 for patients 1 and 2, respectively. Expression of CD38 on the surface of T lymphocytes was low in patient 1. Detection of intracellular interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha in CD4(+) and CD8(+) T lymphocytes upon PMA-Io stimulus was preserved in both children. In conclusion, we found low numbers of CD4(+) T lymphocytes and a dramatic redistribution of naive and memory/effector CD4(+) T lymphocytes in two girls with non-X-linked HIM syndrome. Furthermore, we found low expression of CD38 on T lymphocytes and low numbers of NK cells in the patient with the more severe disease, indicating a possible role for these cells in the pathogenesis of this immunodeficiency.
Subject(s)
ADP-ribosyl Cyclase/analysis , Antigens, CD/analysis , Cytokines/immunology , Immunoglobulin M/immunology , Immunologic Deficiency Syndromes/immunology , T-Lymphocyte Subsets/immunology , ADP-ribosyl Cyclase 1 , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Child, Preschool , Cytokines/analysis , Female , Flow Cytometry , Humans , Immunologic Memory , Immunophenotyping/methods , Infant , Interferon-gamma/analysis , Intracellular Fluid/chemistry , Killer Cells, Natural/immunology , Lymphocyte Count , Membrane Glycoproteins , Tumor Necrosis Factor-alpha/analysisABSTRACT
Evaluation of HIV-induced IL-2 production by peripheral blood mononuclear cells (PBMC) and HIV-specific T helper and cytotoxic T lymphocyte (CTL) responses in health care workers (HCW) occupationally exposed to HIV reveals a high rate of response to HIV among non-seroconverters. IL-10 is also known to interfere with HIV infection in vitro. To evaluate the induction of IL-10 by HIV antigens in HCW occupationally exposed to HIV, 18 HCW with percutaneous injury were enrolled in this study, 9 of them exposed to HIV-contaminated blood, and 9 exposed to HIV-negative blood. PBMC were incubated on plates coated with HIV-1 antigens, and IL-10 was measured in supernatants by ELISA. Five of nine HCW exposed to HIV-contaminated blood presented HIV-induced IL-10. Two of nine HCW exposed to HIV-negative source patients also had detectable levels of HIV-induced IL-10, one of them in the sample obtained on the day of accidental exposure. There was a relationship between the type of device involved in injury and IL-10 production. Individuals exposed to hollow needles or scalpels presented HIV-induced IL-10, whereas those exposed to solid needles and to digital puncture did not, suggesting a relationship between infectious load and IL-10. Although occupational exposure to HIV leads to a low rate of seroconversion, these individuals can develop an antigen-specific immune response characterized in our study by induction of IL-10 in PBMC in vitro.
Subject(s)
HIV Antigens/immunology , HIV-1/immunology , Health Personnel , Interleukin-10/biosynthesis , Leukocytes, Mononuclear/metabolism , Occupational Exposure , Accidents, Occupational , HIV Infections/immunology , HIV Infections/transmission , HIV Seronegativity/immunology , HIV Seropositivity/immunology , Humans , Infectious Disease Transmission, Patient-to-Professional , Interleukin-10/analysis , Leukocytes, Mononuclear/chemistry , Needlestick Injuries/immunologyABSTRACT
Evaluation of HIV-induced IL-2 production by peripheral blood mononuclear cells (PBMC) and HIV-specific T helper and cytotoxic T lymphocyte (CTL) responses in health care workers (HCW) occupationally exposed to HIV reveals a high rate of response to HIV among non-seroconverters. IL-10 is also known to interfere with HIV infection in vitro. To evaluate the induction of IL-10 by HIV antigens in HCW occupationally exposed to HIV, 18 HCW with percutaneous injury were enrolled in this study, 9 of them exposed to HIV-contaminated blood, and 9 exposed to HIV-negative blood. PBMC were incubated on plates coated with HIV-1 antigens, and IL-10 was measured in supernatants by ELISA. Five of nine HCW exposed to HIV-contaminated blood presented HIV-induced IL-10. Two of nine HCW exposed to HIV-negative source patients also had detectable levels of HIV-induced IL-10, one of them in the sample obtained on the day of accidental exposure. There was a relationship between the type of device involved in injury and IL-10 production. Individuals exposed to hollow needles or scalpels presented HIV-induced IL-10, whereas those exposed to solid needles and to digital puncture did not, suggesting a relationship between infectious load and IL-10. Although occupational exposure to HIV leads to a low rate of seroconversion, these individuals can develop an antigen-specific immune response characterized in our study by induction of IL-10 in PBMC in vitro
Subject(s)
Humans , Accidents, Occupational , Health Personnel , HIV-1 , Interleukin-10 , Leukocytes, Mononuclear , Occupational Exposure , HIV Infections , HIV Seronegativity , HIV Seropositivity , Infectious Disease Transmission, Patient-to-Professional , Interleukin-10 , Leukocytes, Mononuclear , Needlestick InjuriesABSTRACT
The outcome of patients with sepsis arises from multiple factors affecting both the host and the invading microorganisms. Age, presence of underlying disease, source of infection and some specific etiological agents have been related to prognosis. Appropriateness of antimicrobial therapy, considering the in vitro susceptibility tests for the infecting bacteria, has been strongly associated with the outcome. Therefore even after the cascade of sepsis has been triggered, the control of bacteria growth is still fundamental for the outcome of the infection. This is a major distinction point from experimental studies in which whole killed bacteria and their products are used as model of sepsis. However, even within the setting of adequate antimicrobial use, patients still die of sepsis. Thus, strategies focusing on further therapy targets are an important area of interest for basic and clinical research. Although such adjunctive sepsis therapy has failed to achieve consistent better survival rates so far, nevertheless, it improved our understanding of the pathophysiological events seen in sepsis that the possibility that a new and effective treatment may arise has been warmly considered. In this paper we aim to review some aspects of the pathogenesis of sepsis, focusing on recent advances and on possible targets for adjunctive therapy. Published clinical trials and experimental data supporting such trials are commented on.