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
Sepsis, the body's response to infection, is associated with extremely high mortality rates. Why a protective mechanism turns into a deadly clinical picture is a matter of debate, and goes largely unexplained. In previous work we demonstrated that platelet-derived microparticles (MP) can induce endothelial and vascular smooth muscle cell apoptosis in septic patients through NADPH oxidasedependent superoxide release [1]. In this work we sought to create a model for ex vivo generation of septic-like MP and to identify the pathways responsible for MP free radical release and effects. Septic shock is a condition related to the generation of high amounts of thrombin, TNFα and nitrogen reactive species. Human platelets exposed to the NO donors diethylamine-NONOate (0.5 mM) and nitroprusside (2 mM) for 20 minutes generated MP similar to those found in the blood of septic shock patients. Flow cytometry and western blot analysis of those MP, like their septic counterparts, revealed exposure of the tetraspanin markers CD9, CD63, and CD81, but little phosphatidylserine. Such a membrane exposure, associated with their size, characterizes them as exosomes. Furthermore, we identified the Nox2 and p22phox NADPH oxidase subunits and the inducible isoform of NO synthase (NOS), but not the NOS I and III isoforms. On the other hand, platelets exposed to thrombin or TNFα released particles with clearly distinct characteristics, such as high phosphatidylserine and low tetraspanin. Like the septic MP, the MP obtained by NO exposure generated the superoxide radical and NO, as disclosed by lucigenin (5 µM) and coelenterazine (5 µM) chemiluminescence and by 4,5-diaminofluorescein (10 mM) and 2′,7′-dichlorofluorescein (10 mM) fluorescence. As expected, NOS inhibitors or NADPH oxidase inhibitors significantly reduced signals. In addition, endothelial cells exposed to this type of MP underwent apoptotic death, while control MP had negligible effects. NADPH oxidase as well as NOS inhibition significantly reduced apoptosis rates. Concomitant generation of NO and superoxide suggests biological effects of the highly reactive radical peroxynitrite. In fact, the peroxynitrite scavenger urate (1 mM) showed an additive effect on fluorescent signal inhibition, as well as on endothelial apoptosis rate reduction. We thus propose that platelet-derived exosomes may be another class of actors in the complex play known as 'vascular redox signaling'. In this sense, an exosome-based approach can provide novel tools for further understanding and even treating vascular dysfunction related to sepsis. Introduction The intestinal hypothesis of sepsis has been attributed to bacterial translocation (BT), and the aggravation of sepsis is related to the increased vascular permeability state that potentates the BT index. In this study we examined the BT index during sepsis with or without mesenteric lymph exclusion. Materials and methods Wistar rats (±200 g) were submitted to the BT process (E. coli R6 10 ml of 10 10 CFU/ml) and nonlethal sepsis (E. cloacae 89 2 ml ...
plays a critical role in the inflammatory response and, potentially, a polymorphism in IRAK1 may alter the immune response impacting clinical outcome. P2Gene expression and intracellular NF-κ κB activation after HMGB1 and LPS stimuli in neutrophils from septic patients E Silva, 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...
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...
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...
Introduction Many authors have written about the need to treat patients closer to their beds, in order to observe them more as distinct people. The FAST HUG mnemonic, which consists of a checklist, was suggested as an idea to be employed everyday, by professionals dealing with patients who are critically ill. Minding these questions and motivated by an idea of follow patients' treatment closer, we have put into practice the instrument developed by Jean-Louis Vincent, evaluating the seven most important procedures in critically ill patients, and performed the FAST HUG. This checklist consists of seven items to be evaluated: Feeding, Analgesia, Sedation, Thromboembolic prophylaxis, Head-of-bed elevation, stress Ulcer prevention, and Glucose control. Knowing that the pressure ulcer is one of the challenges faced by ICU nurses, related to patients' need to stay at rest, to be under rigorous control or more complex therapy, it was decided to create the eighth item on the checklist: S, for skin. It stands for skin treatment, with the techniques used in the unit (Braden Scale), monitoring and evaluating closer skin integrity, and allowing nurses to calculate the scoring average of the Braden Scale, and greater incidence of ulcer in interned patients. Objective To expose the shortcomings found during the FAST HUG application, and to show results obtained with the eighth item of the FAST HUG mnemonic: S-Skin. Methods A descriptive study, based on institutional data, was carried out in the adult ICU of a private hospital. It was performed from 2 to 27 June 2008, except on weekends. Three hundred and twenty-three patients were involved. The checklist was carried out during the afternoons by the head nurse, or the assistant nurse of the unit. In order to do this job, a spreadsheet was elaborated to control data, updated every week. This spreadsheet provided graphics for a more objective control of the results obtained. The idea was exposed to the team, during a training program, and so we started the activities. Results and discussion For 20 days of the checklist, 323 patients were evaluated for the eight items. The real shortcomings most frequently found were related to thromboembolic prophylaxis (85%) and glucose control (90%). These shortcomings were immediately evaluated and, depending on this analysis, this item would go on or not, according to the patient's clinical situation. The shortcomings found were tracked just as they were detected, and their cause would be discussed in a multidisciplinary group, and a solution was found. If the item was not observed, it would be 1. Vincent JL: Give your patient a fast hug (at least) once a day.
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