Introduction Neutrophils have been involved in sepsis-induced organ damage. Neutrophils could be directly activated by TLR binding ligands including LPS. IRAK-1 is one of many intracellular proteins that are activated upon stimulation of TL receptors. This triggers a series of events that results in the migration of NF-κB into the nucleus and the activation NF-κB-dependent genes. Objectives To identify a single nucleotide polymorphism at position 532 (coding SNP) in volunteers and patients with sepsis. To determine whether IRAK-1 SNP532 results in a decrease in neutrophil NF-κB activation in volunteers and patients with sepsis. To evaluate neutrophil gene expression patterns in IRAK-1 SNP532 and wildtype patients with sepsis. Methods Thirty severe sepsis patients and 34 healthy volunteers were enrolled in this study. Peripheral blood was obtained and neutrophils were isolated by plasma-percoll gradients after dextran sedimentation of erythrocytes. Neutrophils from volunteers were resuspended in RPMI and cultured with or without 100 ng/ml LPS for 60 min. The electrophoretic mobility shift assay technique was used to measure the NF-κB activation. Real-time PCR allelic discrimination assays were developed by the assay-by-design service offered by Applied Biosystems (Foster City, CA, USA). Probe and primer combinations were designed at the single nucleotide polymorphism 532. PCR reactions were performed according to the manufacturer's manual using the Applied Biosystems 7500 Real-Time PCR system. Microarray analysis was used to evaluate the neutrophil gene expression in unstimulated neutrophils and after LPS stimulus. Results The median AUC for NF-κB activation was higher in wildtype genotyped neutrophils as compared with IRAK-1 SNP532 genotyped neutrophils (85.2 vs 100.5, P = 0.05) (Fig. 1). In terms of kinetics pattern, we found some differences on nuclear levels of NF-κB in neutrophils from volunteers cultured with LPS. At 30 min after LPS, the culture nuclear translocation of NK-κB was significantly greater in wildtype genotyped neutrophils than in IRAK-1 SNP532 genotyped neutrophils. Even after 60 min, the NF-κB translocation remained high in wildtype genotyped neutrophils, while in IRAK-1 SNP532 genotyped neutrophils the NF-κB translocation was similar to baseline (Fig. 2). In unstimulated neutrophils from septic patients, the NF-κB translocation was significantly lower in IRAK-1 SNP532 genotyped neutrophils than in wildtype genotyped neutrophils (1.20 vs 2.10, P = 0.05) (Fig. 3). Finally, the expression of some inflammatory related genes (IL-8, IL1β, MIP-2, COX-2, and SOD2) was decreased in IRAK-1 SNP532 genotyped neutrophils. Conclusion IRAK-1 SNP532 genotyped neutrophils from volunteers (after LPS ex vivo challenge) and from septic patients are associated with lower NF-κB activation and lower expression of some IRAK1-related genes. These results demonstrate that IRAK1 Introduction Neutrophils play a major role in sepsis-induced organ dysfunction, especially in the lung. HMGB1 has emerged as a late cytokine...

Objectives To 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 Hemodynamic and metabolic features of a porcine systemic low flow state model

Objectives To 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 Hemodynamic and metabolic features of a porcine systemic low flow state model

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