Introduction Cardiac surgery with cardiopulmonary bypass (CPB) is a recognized trigger of systemic inflammatory response, usually related to postoperative acute lung injury (ALI).As an attempt to dampen inflammatory response, steroids have been perioperatively administered to patients.Macrophage migration inhibitory factor (MIF), a regulator of the endotoxin receptor, is implicated in the pathogenesis of ALI.We have previously detected peak circulating levels of MIF, 6 hours post CPB.Experimental data have shown that steroids may induce MIF secretion by mononuclear cells.This study aims to correlate levels of MIF assayed 6 hours post CPB to the intensity of postoperative pulmonary dysfunction, analysing the impact of perioperative steroid administration. MethodsWe included patients submitted to cardiac surgery with CPB, electively started in the morning, performed by the same team under a standard technique except for the addition of methylprednisolone (15 mg/kg) to the CPB priming solution for patients from group MP (n = 37), but not for the remaining patients -group NS (n = 37).MIF circulating levels were assayed at the anesthesia induction, 3, 6, and 24 hours after CPB.A standard weaning protocol with fast track strategy was adopted, and indicators of organ dysfunction and therapeutic intervention were registered during the first 72 hours postoperative.Results Levels of MIF assayed 6 hours post CPB correlated directly to the postoperative duration of mechanical ventilation (P = 0.014, rho = 0.282) and inversely to PaO 2 /FiO 2 ratio (P = 0.0021, rho = -0.265).No difference in MIF levels was noted between the groups.The duration of mechanical ventilation was higher (P = 0.005) in the group MP (7.92 ± 6.0 hours), compared with the group NS (4.92 ± 3.6 hours). ConclusionCirculating levels of MIF assayed 6 hours post CPB are correlated to postoperative pulmonary performance.Immunosuppressive doses of methylprednisolone did not affect circulating levels of MIF and may be related to prolonged mechanical ventilation.
Introduction Neutrophils play a major role in sepsis-induced organ dysfunction, especially in the lung.HMGB1 has emerged as a late cytokine and is implicated in the perpetuation of inflammatory stimulus and organ dysfunction development as well.There are limited data about neutrophil response patterns to HMGB1 in septic patients, and whether those patterns could be different from those following LPS exposure.Objectives To evaluate the differences of gene expression and activation of NF-κB, Akt, and p38MAPK in blood neutrophils from septic patients exposed to HMGB1 and LPS; and to compare response patterns between blood neutrophils from patients and healthy volunteers.Methods Twenty-two sepsis-induced acute lung injury patients and 34 healthy volunteers were enrolled in this study.The primary clinical variables collected were the 28-day survival and the presence of shock at ICU admission.Peripheral blood was obtained and neutrophils were isolated by plasma-percoll gradients after dextran sedimentation of erythrocytes.Neutrophils were resuspended in RPMI and cultured with or without 1000 ng/ml rHMGB1 or with or without 100 ng/ml LPS for 15, 30, and 60 min.The electrophoretic mobility shift assay technique was used to measure the NF-κB translocation, while western blot analysis was used to determine Akt phosphorylation and an ELISA was used to determine p38MAPK phosphorylation.Microarray analysis was used to evaluate the neutrophil gene expression in unstimulated neutrophils and after either HMGB1 stimulus or LPS stimulus.P < 0.05 was considered significant.Results Although with some similarities, HMGB1 and LPS induced distinct patterns of gene expression in peripheral blood neutrophils from septic patients.A Venn diagram (Fig. 1) displays genes upregulated greater than twofold that are both common and unique after both stimuli.Using functional ontology, the genes upregulated by both HMGB1 and LPS primarily consisted of cytokines, chemokines, coagulation-related proteins, phosphatases, and transcriptional regulators factors.Importantly, while HMGB1 induced an HMGB1-related gene downregulation, LPS did not induce any changes in HMGB1 gene expression in these patients.Regarding intracellular activation, both HMGB1 and LPS increased translocation of NF-κB and the phosphorylation of Akt and p38MAPK in neutrophils from septic patients.However, there were some differences in terms of the degree and kinetics of activation between neutrophils cultured with LPS and HMGB1 (Fig. 2).There are no important differences in terms of intracellular activation when we compared neutrophils from septic patients with those from volunteers.Finally, neither NF-κB translocation nor kinase phosphorylation was associated with sepsis severity.However, the majority of genes in unstimulated neutrophils and after HMGB1 had a higher expression in mild patients.In contrast, CCL20, CCRL2, CIAS1, PTGER, PTX3, and MAP3K8 had a higher expression in severe patients only after LPS stimulus. ConclusionAlthough with some similarities, HMGB1 and LPS induced distinct pattern of gene expression in neutrophils from septic patients.Both stimuli were able to increase intracellular activation and this activation was similar to that found in neutrophils from volunteers, showing that even after sepsis stimulus the neutrophil keeps its ability to respond to a second hit.
ObjectivesTo characterize an experimental model of pulmonary embolism by studying hemodynamics, lung mechanics and histopathologic derangements caused by pulmonary microembolism in pigs.To identify lung alterations after embolism that may be similar to those evidenced in pulmonary inflammatory conditions.Materials and methods Ten Large White pigs (weight 35-42 kg) were instrumented with arterial and pulmonary catheters, and pulmonary embolism was induced in five pigs by injection of polystyrene microspheres (diameter ~300 µM), in order to obtain a pulmonary mean arterial pressure of twice the baseline value.Five other animals injected with saline served as controls.Hemodynamic and respiratory data were collected and pressure x volume curves of the respiratory system were performed by a quasi-static low flow method.Animals were followed for 12 hours, and after death lung fragments were dissected and sent to pathology.Results Pulmonary embolism induced a significant reduction in stroke volume (71 ± 18 ml/min/bpm pre vs 36 ± 9 ml/min/bpm post, P < 0.05), an increase in pulmonary mean arterial pressure (27 ± 4 mmHg pre vs 39 ± 6 mmHg post, P < 0.05) and pulmonary vascular resistance (193 ± 122 mmHg/l/min pre vs 451 ± 149 mmHg/l/min post, P < 0.05).Respiratory dysfunction was evidenced by significant reductions in the PaO 2 /FiO 2 ratio (480 ± 50 pre vs 159 ± 55 post, P < 0.05), the dynamic lung compliance (27 ± 6 ml/cmH 2 O pre vs 19 ± 5 ml/cmH 2 O post, P < 0.05), the increase in dead space ventilation (20 ± 4 pre vs 47 ± 20 post, P < 0.05) and, the shift of pressure x volume curves to the right, with reduction in pulmonary hysteresis.Pathology depicted inflammatory neutrophil infiltrates, alveolar edema, collapse and hemorrhagic infarctions.Conclusion This model of embolism is associated with cardiovascular dysfunction, as well as respiratory injury characterized by a decrease in oxygenation, lung compliance and hysteresis.Pathology findings were similar to those verified in inflammatory pulmonary injury conditions.This model may be useful to study pathophysiology, as well as pharmacologic and ventilatory interventions useful to treat pulmonary embolism. P6
Introduction Neutrophils play a major role in sepsis-induced organ dysfunction, especially in the lung.HMGB1 has emerged as a late cytokine and is implicated in the perpetuation of inflammatory stimulus and organ dysfunction development as well.There are limited data about neutrophil response patterns to HMGB1 in septic patients, and whether those patterns could be different from those following LPS exposure.Objectives To evaluate the differences of gene expression and activation of NF-κB, Akt, and p38MAPK in blood neutrophils from septic patients exposed to HMGB1 and LPS; and to compare response patterns between blood neutrophils from patients and healthy volunteers.Methods Twenty-two sepsis-induced acute lung injury patients and 34 healthy volunteers were enrolled in this study.The primary clinical variables collected were the 28-day survival and the presence of shock at ICU admission.Peripheral blood was obtained and neutrophils were isolated by plasma-percoll gradients after dextran sedimentation of erythrocytes.Neutrophils were resuspended in RPMI and cultured with or without 1000 ng/ml rHMGB1 or with or without 100 ng/ml LPS for 15, 30, and 60 min.The electrophoretic mobility shift assay technique was used to measure the NF-κB translocation, while western blot analysis was used to determine Akt phosphorylation and an ELISA was used to determine p38MAPK phosphorylation.Microarray analysis was used to evaluate the neutrophil gene expression in unstimulated neutrophils and after either HMGB1 stimulus or LPS stimulus.P < 0.05 was considered significant.Results Although with some similarities, HMGB1 and LPS induced distinct patterns of gene expression in peripheral blood neutrophils from septic patients.A Venn diagram (Fig. 1) displays genes upregulated greater than twofold that are both common and unique after both stimuli.Using functional ontology, the genes upregulated by both HMGB1 and LPS primarily consisted of cytokines, chemokines, coagulation-related proteins, phosphatases, and transcriptional regulators factors.Importantly, while HMGB1 induced an HMGB1-related gene downregulation, LPS did not induce any changes in HMGB1 gene expression in these patients.Regarding intracellular activation, both HMGB1 and LPS increased translocation of NF-κB and the phosphorylation of Akt and p38MAPK in neutrophils from septic patients.However, there were some differences in terms of the degree and kinetics of activation between neutrophils cultured with LPS and HMGB1 (Fig. 2).There are no important differences in terms of intracellular activation when we compared neutrophils from septic patients with those from volunteers.Finally, neither NF-κB translocation nor kinase phosphorylation was associated with sepsis severity.However, the majority of genes in unstimulated neutrophils and after HMGB1 had a higher expression in mild patients.In contrast, CCL20, CCRL2, CIAS1, PTGER, PTX3, and MAP3K8 had a higher expression in severe patients only after LPS stimulus. ConclusionAlthough with some similarities, HMGB1 and LPS induced distinct pattern of gene expression in neutrophils from septic patients.Both stimuli were able to increase intracellular activation and this activation was similar to that found in neutrophils from volunteers, showing that even after sepsis stimulus the neutrophil keeps its ability to respond to a second hit.
Introduction Neutrophils play a major role in sepsis-induced organ dysfunction, especially in the lung.HMGB1 has emerged as a late cytokine and is implicated in the perpetuation of inflammatory stimulus and organ dysfunction development as well.There are limited data about neutrophil response patterns to HMGB1 in septic patients, and whether those patterns could be different from those following LPS exposure.Objectives To evaluate the differences of gene expression and activation of NF-κB, Akt, and p38MAPK in blood neutrophils from septic patients exposed to HMGB1 and LPS; and to compare response patterns between blood neutrophils from patients and healthy volunteers.Methods Twenty-two sepsis-induced acute lung injury patients and 34 healthy volunteers were enrolled in this study.The primary clinical variables collected were the 28-day survival and the presence of shock at ICU admission.Peripheral blood was obtained and neutrophils were isolated by plasma-percoll gradients after dextran sedimentation of erythrocytes.Neutrophils were resuspended in RPMI and cultured with or without 1000 ng/ml rHMGB1 or with or without 100 ng/ml LPS for 15, 30, and 60 min.The electrophoretic mobility shift assay technique was used to measure the NF-κB translocation, while western blot analysis was used to determine Akt phosphorylation and an ELISA was used to determine p38MAPK phosphorylation.Microarray analysis was used to evaluate the neutrophil gene expression in unstimulated neutrophils and after either HMGB1 stimulus or LPS stimulus.P < 0.05 was considered significant.Results Although with some similarities, HMGB1 and LPS induced distinct patterns of gene expression in peripheral blood neutrophils from septic patients.A Venn diagram (Fig. 1) displays genes upregulated greater than twofold that are both common and unique after both stimuli.Using functional ontology, the genes upregulated by both HMGB1 and LPS primarily consisted of cytokines, chemokines, coagulation-related proteins, phosphatases, and transcriptional regulators factors.Importantly, while HMGB1 induced an HMGB1-related gene downregulation, LPS did not induce any changes in HMGB1 gene expression in these patients.Regarding intracellular activation, both HMGB1 and LPS increased translocation of NF-κB and the phosphorylation of Akt and p38MAPK in neutrophils from septic patients.However, there were some differences in terms of the degree and kinetics of activation between neutrophils cultured with LPS and HMGB1 (Fig. 2).There are no important differences in terms of intracellular activation when we compared neutrophils from septic patients with those from volunteers.Finally, neither NF-κB translocation nor kinase phosphorylation was associated with sepsis severity.However, the majority of genes in unstimulated neutrophils and after HMGB1 had a higher expression in mild patients.In contrast, CCL20, CCRL2, CIAS1, PTGER, PTX3, and MAP3K8 had a higher expression in severe patients only after LPS stimulus. ConclusionAlthough with some similarities, HMGB1 and LPS induced distinct pattern of gene expression in neutrophils from septic patients.Both stimuli were able to increase intracellular activation and this activation was similar to that found in neutrophils from volunteers, showing that even after sepsis stimulus the neutrophil keeps its ability to respond to a second hit.
Although enteropathogenic Escherichia coli (EPEC) are well-recognized diarrheal agents, their ability to translocate and cause extraintestinal alterations is not known. We investigated whether a typical EPEC (tEPEC) and an atypical EPEC (aEPEC) strain translocate and cause microcirculation injury under conditions of intestinal bacterial overgrowth. Bacterial translocation (BT) was induced in female Wistar-EPM rats (200-250 g) by oroduodenal catheterization and inoculation of 10 mL 10(10) colony forming unit (CFU)/mL, with the bacteria being confined between the duodenum and ileum with ligatures. After 2 h, mesenteric lymph nodes (MLN), liver and spleen were cultured for translocated bacteria and BT-related microcirculation changes were monitored in mesenteric and abdominal organs by intravital microscopy and laser Doppler flow, respectively. tEPEC (N = 11) and aEPEC (N = 11) were recovered from MLN (100%), spleen (36.4 and 45.5%), and liver (45.5 and 72.7%) of the animals, respectively. Recovery of the positive control E. coli R-6 (N = 6) was 100% for all compartments. Bacteria were not recovered from extraintestinal sites of controls inoculated with non-pathogenic E. coli strains HB101 (N = 6) and HS (N = 10), or saline. Mesenteric microcirculation injuries were detected with both EPEC strains, but only aEPEC was similar to E. coli R-6 with regard to systemic tissue hypoperfusion. In conclusion, overgrowth of certain aEPEC strains may lead to BT and impairment of the microcirculation in systemic organs.
Sepsis is the result from a complex bacterial-host interaction, which is an often-fatal response when host protective molecular mechanisms designed to fight invading bacteria surpass the beneficial intensity to the point of causing injury to the host. Increasing evidences have implicated the bacterial translocation (BT) as the main source for the induction of sepsis, although the beneficial effect of BT process has been related to the development of the intestinal immune response by physiological interaction between bacteria and host. In this article, we examined evolving concepts concerning to BT and discussed about its potential role in the promotion of microcirculation injury, moreover, its possible participation in the sepsis induction. According to our data obtained from in-vivo BT animal-model, both bacterial overgrowth and bacterial pathogenic determinants seem to be major predisposing factors for the induction of BT. Besides, translocation of luminal bacteria through the lymphatic via elicits the activation of the GALT inflammatory response contributing to microcirculation injuries, and the haematological via of BT was responsible to the systemic bacterial spread. On other hand, the combination of BT process to the pre-existing host systemic infection played a crucial role in the worsening of the clinical outcome. In our understanding, studies concerning to intestinal immune response and the pathophysiology of bacterial-host interaction, under normal and disease conditions, seems to be the key elements to the development of therapeutic approaches towards sepsis.
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