Quality assurance in severe sepsis: an individualised audit/feedback system results in substantial improvements in sepsis care at a large UK teaching hospital

Critical Care, Nov 2013

Mark Simmonds, Esme Blyth, Marc Chikhani, Jaimie Coleman, Vivienne Weston, Tim Hills

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Quality assurance in severe sepsis: an individualised audit/feedback system results in substantial improvements in sepsis care at a large UK teaching hospital

0 P3 Heparin-binding protein improves prediction of severe sepsis in the emergency department Adam Linder 1 Örebro University Hospital , Örebro , Sweden 2 P2 Acute kidney injury decreases long-term survival over a 10-year observation period Adam Linder 3 Cooper University Hospital , Camden, NJ , USA 4 Skåne University Hospital , Lund , Sweden 5 P1 Validation of a novel surveillance paradigm for ventilator-associated events Peter MC Klein Klouwenberg 6 , Bertil Christensson 7 Linköping University Hospital , Linköping , Sweden Critical Care 2013, 17(Suppl 4):P3; doi:10.1186/cc12904 8 Skåne University Hospital , Malmö , Sweden 9 P5 Passive immunotherapy of extended peritonitis as abdominal sepsis prevention Olexandr Butyrsky 10 , Viktor Starosek Department of Surgical Diseases, Crimean State Medical University , Simferopol, Ukraine Critical Care 2013, 17(Suppl 4):P5; doi:10.1186/cc12906 11 Department of Hygiene, Crimean State Medical University , Simferopol, Ukraine Critical Care 2013, 17(Suppl 4):P6; doi:10.1186/cc12907 12 Department of Surgical Diseases, Crimean State Medical University , Simferopol , Ukraine 13 P6 Efficacy of specific immunotherapy of abdominal sepsis Olexandr Butyrsky 14 Hackensack University Medical Center , Hackensack, NJ, USA Critical Care 2013, 17(Suppl 4):P7; doi:10.1186/cc12908 15 Section of Critical Care Medicine, University of Toronto , ON , Canada 16 Laurentian University , Sudbury, ON , Canada 17 Rush University , Chicago, IL , USA 18 Sirtris, A GSK Company , Cambridge, MA, USA Critical Care 2013, 17(Suppl 4):P8; doi:10.1186/cc12909 19 Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam , the Netherlands 20 P8 SRT2379, a small-molecule SIRT1 activator, fails to reduce cytokine release in a human endotoxemia model Maryse A Wiewel 21 Armauer Hansen Research Institute , Addis Ababa , Ethiopia 22 Department of Public Health Officer, Jigjiga University , Jigjiga , Ethiopia 23 Department of Medical Laboratory Science, College of Health Science, Addis Ababa University , Ethiopia 24 P11 Bacteriological profile and antimicrobial sensitivity pattern of blood culture isolates among septicemia-suspected children at Tikur Anbessa Specialized Hospital and Yekatit 12 Hospital , Addis Ababa , Ethiopia Adugna Negussie 25 Tikur Anbessa Specialized Hospital, Department of Pediatrics , Addis Ababa, Ethiopia Critical Care 2013, 17(Suppl 4):P11; doi:10.1186/cc12911 26 Division of Allergy and Clinical Imunology, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia , Jakarta , Indonesia 27 Division of Respirology and Critical Care, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia , Jakarta, Indonesia Critical Care 2013, 17(Suppl 4):P15; doi:10.1186/cc12915 28 Clinical Hospital, Federal University of Uberlândia , Brazil Critical Care 2013, 17(Suppl 4):P16; doi:10.1186/cc12916 29 Biomedical Sciences Institute, Federal University of Uberlândia , Brazil 30 Genetics and Biochemistry Institute, Federal University of Uberlândia , Brazil 31 P16 Is Strongyloides stercoralis a risk factor for sepsis severity? Patrícia Terra Alves 32 P18 Clinical manifestations, etiology and outcome of sepsis in pediatric patients admitted to the ICU Taís C São Pedro 33 , André M Morcillo, Emilio CE Baracat Department of Pediatrics, State University of Campinas, UNICAMP , Campinas, SP, Brazil Critical Care 2013, 17(Suppl 4):P18; doi:10.1186/cc12918 34 School of Medical Sciences, Rio de Janeiro State Federal University , Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 4):P19; doi:10.1186/cc12919 35 State University , Rio de Janeiro , Brazil 36 Central Laboratory, Pedro Ernesto University Hospital, Rio de Janeiro State University , Rio de Janeiro , Brazil 37 Bacteriology, Pedro Ernesto University Hospital, Rio de Janeiro State University , Rio de Janeiro , Brazil 38 School of Medical Sciences, Rio de Janeiro State University , Rio de Janeiro , Brazil 39 Pharmacy, Pedro Ernesto University Hospital, Rio de Janeiro State University , Rio de Janeiro , Brazil 40 Research Department of Emergency Medicine, Beth Israel Deaconess Medical Center , Boston, MA , USA 41 P24 Increasing number of organ dysfunctions is an excellent predictor of in-hospital mortality in emergency department patients with suspected infection: an internal and external prospective validation study Marie K Jessen 42 Research Department of Emergency Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School , Boston, MA, USA Critical Care 2013, 17(Suppl 4):P24; doi:10.1186/cc12924 43 Research Center for Emergency Medicine, Aarhus University Hospital , Aarhus , Denmark 44 P23 Prediction of bacteremia in emergency department patients with suspected infection: an external validation of a clinical decision rule Marie K Jessen 45 Department of Clinical Microbiology, Aalborg University Hospital, Aarhus University Hospital , Aalborg, Denmark Critical Care 2013, 17(Suppl 4):P23; doi:10.1186/cc12923 46 Research Department of Emergency Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School , Boston, MA, USA Critical Care 2013, 17(Suppl 4):P25; doi:10.1186/cc12925 47 P25 Lactate levels in emergency department patients across all causes of physiologic instability Kimie Ødorf 48 Central Laboratory Division, University of Sao Paulo Medical School , Sao Paulo, Brazil Critical Care 2013, 17(Suppl 4):P26; doi:10.1186/cc12926 49 Molecular Biology Branch, Central Laboratory Division, University of Sao Paulo Medical School , Sao Paulo , Brazil 50 Infection Control Unit, Heart Institute (InCor), University of Sao Paulo Medical School , Sao Paulo , Brazil 51 P26 Rapid molecular test (SeptiFast®) reduced time for adjustment of antibiotic treatment in comparison with conventional blood cultures in critically ill sepsis patients: a randomized controlled clinical trial (preliminary results) Cristhieni Rodrigues 52 Department of Mathematics and Statistics, University of Canterbury , Christchurch , New Zealand Critical Care 2013, 17(Suppl 4):P67; doi:10.1186/cc12966 53 Department of Intensive Care, Christchurch Hospital , Christchurch , New Zealand 54 Centre for Bioengineering, Department of Mechanical Engineering, University of Canterbury , Christchurch , New Zealand 55 P67 Hourly and accurate severe sepsis classification using kernel density estimates Jacquelyn D Parente 56 Department of Surgery, Federal University of São Paulo , Brazil Critical Care 2013, 17(Suppl 4):P70; doi:10.1186/cc12969 57 P70 Possible variables related to paradoxical findings between PCR and hemoculture assays in rat experimental sepsis Marcello R da Silva 58 Department of Pediatrics, Lusíada University Center , Lisbon , Portugal 59 Department of Pediatrics, Federal University of São Paulo , Brazil 60 P69 Beneficial effects of vigorous fluid resuscitation therapy varied depending on the time of onset and the sepsis stage in rats: preliminary study Ana MA Liberatore 61 Department of Pediatrics, Federal University of São Paulo , Brazil Critical Care 2013, 17(Suppl 4):P73; doi:10.1186/cc12972 62 Institute of Biology of São Paulo State , São Paulo , Brazil 63 Department of Surgery, Federal University of São Paulo , Brazil 64 P73 Effect of sepsis challenge in chronic inflammation state on mortality and long-term pathological findings in rats Roberto Tussi-Junior 65 Department of Veterinary Medicine, Rakuno Gakuen University , Ebetsu, Hokkaido , Japan Critical Care 2013, 17(Suppl 4):P86; doi:10.1186/cc12985 66 P86 Study of the effect of C1-esterase inhibitor administration to the sepsis pig model Hisashi Imahase 67 Department of Veterinary Science, Rakuno Gakuen University , Ebetsu, Hokkaido , Japan 68 Emergency and Critical Care Center, Saga University Hospital , Saga City , Japan 69 P85 Effect of polymicrobial sepsis on the respiratory mechanism of rats previously exposed to cigarette smoking Glauber C Lima 70 Institute of Biomedical Sciences, Federal University of Rio de Janeiro , Brazil Critical Care 2013, 17(Suppl 4):P90; doi:10.1186/cc12989 71 P90 Synaptic deficits in sepsis: role of glial cells Carolina A Moraes 72 Institute Oswaldo Cruz , FIOCRUZ, Rio de Janeiro , Brazil 73 P88 Epigenetic profile in lipopolysaccharide-stimulated macrophages Ester Correia Sarmento Rios 74 , Thais Martins Lima Salgado, Francisco Garcia Soriano Department of Emergency Medicine, University of São Paulo Medical School , Brazil Critical Care 2013, 17(Suppl 4):P88; doi:10.1186/cc12987 75 P92 Feasibility of gene transfer with nonviral vectors in murine models of sepsis Vanessa B Faiotto 76 , Rodolfo ME Hubert, Devanira S Paixao, Gleice R Souza, Maiara ML Fiusa, Carolina Costa-Lima, Sara TO Saad, Joyce M Annichino-Bizzacchi, Erich V De Paula Faculty of Medical Sciences, University of Campinas , SP, Brazil Critical Care 2013, 17(Suppl 4):P92; doi:10.1186/cc12991 77 Innate Immunology Group, National Veterinary Institute, Technical University of Denmark , Frederiksberg , Denmark 78 Department of Food Science, University of Copenhagen , Frederiksberg , Denmark 79 Department of Veterinary Disease Biology, University of Copenhagen , Frederiksberg , Denmark 80 P95 Gene expression patterns in multiple organs in experimentally induced Staphylococcus aureus sepsis in pigs Helle G Olsen 81 Department of Small Animal Clinical Sciences, University of Copenhagen , Frederiksberg, Denmark Critical Care 2013, 17(Suppl 4):P95; doi:10.1186/cc12994 82 Department of Mathematical Sciences, University of Copenhagen , Denmark 83 P99 Modulation of peroxynitrite improves host response to vasopressin in ovine sepsis Osamu Fujiwara 84 Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of São Paulo , Ribeirão Preto, SP , Brazil 85 P98 Arginine vasopressin V1a agonist attenuates methicillin-resistant Staphylococcus aureus-induced vascular leakage by inhibiting bradykinin Perenlei Enkhbaatar 86 Shriners Hospital for Children , Galveston, TX, USA Critical Care 2013, 17(Suppl 4):P99; doi:10.1186/cc12998 87 Department of Anesthesiology, University of Texas Medical Branch , Galveston, TX , USA 88 P97 Role of NOX2-derived ROS in the development of cognitive impairment after sepsis Joana C D'Avila 89 Evandro Chagas Clinical Research Institute (IPEC), FIOCRUZ and D'Or Institute for Research and Education (IDOR) , Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 4):P97; doi:10.1186/cc12996 90 , Sebastian Rehberg, Osamu Fujiwara, Ernesto Lopez, Donald S Prough Department of Anesthesiology, University of Texas Medical Branch , Galveston, TX, USA Critical Care 2013, 17(Suppl 4):P98; doi:10.1186/cc12997 91 Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo , Ribeirão Preto, Brazil Critical Care 2013, 17(Suppl 4):P100; doi:10.1186/cc12999 92 Department of Morphology, Physiology and Basic Pathology, Dental School of Ribeirão Preto, University of São Paulo , Ribeirão Preto , Brazil 93 P100 Effects of solid dispersion of curcumin in metabolic and immunological alterations during experimental sepsis Letycia Silvano da Silva 94 Veterinary Department at AMIB (Brazilian Intensive Care Association) and Intensivet Veterinary Consulting , Brasília, DF, Brazil Critical Care 2013, 17(Suppl 4):P110; doi:10.1186/cc13009 95 Universidade de Brasília - FAV , Brasília, DF , Brazil 96 Universidade Estadual Paulista 'Julio de Mesquita Filho' , FCAV UNESP, Jaboticabal, SP , Brazil 97 P110 Severe sepsis and septic shock survival in a clinical canine model JGMP Isola P O S T E R P R E S E N T A T I O N S © 2013 various authors, licensee BioMed Central Ltd. All articles published in this supplement are distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. - Background: Reliable surveillance methods are indispensable for benchmarking of healthcare-associated infection rates. The National Healthcare Safety Network (NHSN) recently introduced surveillance of ventilator-associated events (VAE), including ventilator-associated conditions (VAC) [1]. This new algorithm is amenable to automated implementation and strives for more consistent interpretation. We assess the feasibility and reliability of automated implementation. Materials and methods: Retrospective analysis of an ICU cohort with prospective assessment of ventilator-associated pneumonia (VAP) in two academic medical centers (January 2011 to June 2012). The algorithm was electronically implemented as specified by the NHSN using minuteto-minute ventilator data. Two minor modifications were developed to improve stability and comparability with manual surveillance (10th percentile and intermittent ventilation). Concordance was assessed between the algorithms and prospective surveillance. Attributable mortality of VAC was estimated by multivariable competing-risk survival analysis. Results: Two thousand and eighty patients contributed 2,296 episodes of mechanical ventilation (MV). VAC incidence was 10.0/1,000 MV days. Prospective surveillance identified 8 VAP cases/1,000 MV days. The original VAC algorithm detected 32% (38/115) of patients affected by VAP; positive predictive value was 25% (38/152). Using the 10th percentile identified the same number of VAC cases, but only 116 were identical. VAC incidence was 24.9/1,000 MV days with the intermittent ventilation modification. Concordance between the original algorithm and the modified versions was suboptimal. Estimates of attributable mortality varied by implementation: original VAC subdistribution hazard ratio (sdHR) = 4.33, 10th percentile sdHR = 6.26 and intermittent ventilation sdHR = 2.40. Conclusions: Concordance between manual VAP surveillance and the VAE algorithm was poor. Although electronic implementation of the VAE algorithm was feasible, small variations considerably altered the events detected and their effect on mortality. Using the current specifications, comparability across institutions using different electronic or manual implementations remains questionable. Background: We hypothesized that one single episode of acute kidney injury (AKI) reduces long-term survival compared with no acute kidney injury (No AKI) following recovery from critical illness. Materials and methods: A prospective cohort of 2,010 patients admitted to the ICU between 2000 and 2009 at a provincial referral hospital was followed to determine whether AKI influences long-term survival. Results: Of the 1,844 eligible patients, 18.4% had AKI stage 1, 12.1% had stage 2, 26.5% had stage 3, and 43.0% had No AKI, using the KDIGO classification. The mean and median follow-up time was 8.1 and 8.7 years. The 28-day, 1-year, 5-year and 10-year survival rates were 59.6%, 44.9%, 37.4%, and 33.4%, in patients with any AKI (stage 1, stage 2, stage 3), which was significantly worse compared with the critically ill patients with no AKI at any time (P < 0.01). The adjusted 10-year mortality risk associated with AKI was 1.44 (95% CI = 1.2 to 1.7) among 28-day survivors. Patients who had mild AKI (stage 1) had significantly worse survival at 28 days, 1 year, 3 years, 5 years and 10 years compared with No AKI (P < 0.01) (Figure 1A). Patients with sepsis and AKI who survived 28 days had significantly poorer 5-year and 10-year survival compared with nonseptic AKI (P < 0.01) (Figure 1B). Conclusions: Patients with one episode of mild (stage 1) AKI have significantly lower survival rates over 10 years than critically ill patients without AKI. The causes and mechanisms of this association warrant further careful study. Close medical follow-up of these patients may be warranted and mechanistic research required understanding how AKI influences distant events. Figure 1(abstract P2) A: Bar chart showing that patients with AKI of any stage had significantly poorer mean survival rates compared to control patients with no AKI, at 28-days, 90-days, 1-year, 3-years, 5-years and 10-years after enrolment. B: Unadjusted Kaplan-Meier curves showing the10-year survival from ICU admission for patients classified as having any stage of AKI according to the KDIGO classification using serum creatinine. Time is calculated from 28 days after admission (28-day survivors). Mantel-Cox Log Rank showed a significant difference in mortality between the two curves with or without AKI. Background: The early identification of risk of developing severe sepsis in patients with suspected infection remains a difficult challenge. We hypothesized that an elevated plasma level of heparin-binding protein (HBP), a neutrophil-secreted mediator of vascular leakage, would be a predictor of delayed clinical deterioration and progressive organ dysfunction in emergency department (ED) sepsis patients. Materials and methods: A prospective, multicenter study in Sweden and the US was conducted of 763 patients presenting to an ED with a suspected infection and signs of systemic inflammation. Based on recorded clinical and laboratory parameters and final diagnoses, patients were classified into various groups depending on the severity of the infection and inflammatory response. Plasma levels of HBP were measured and compared with levels of other standard sepsis biomarkers including procalcitonin, lactate, WBC, and C-reactive protein. Results: The final diagnoses were severe sepsis with organ failure in 338 patients, nonsevere sepsis without organ failure in 340 patients, and no infection in 85 patients. One-hundred and forty-three patients (19%) presented without signs of severe sepsis, but developed delayed circulatory failure and/or organ dysfunction within 72 hours of enrolment. In this patient group, an elevated HBP level could predict the delayed development of severe sepsis with an AUC value of 0.86. Elevated HBP levels (>30 ng/ml) were found in 80% of the patients and elevated procalcitonin levels (>0.5 ng/ml) were detected in 59%, 10.5 hours (median) before developing severe sepsis. Conclusions: Detection of elevated plasma-HBP levels may help to provide an early risk-stratification of patients with suspected infections in the ED. An elevated HBP level was independently able to predict delayed clinical deterioration to overt shock or severe sepsis with organ failure. Acknowledgements: This project was supported in part by Axis-Shield Diagnostics and the Swedish Government Funds for Clinical Research (ALF), the University Hospital, Lund, Sweden. Clinical trial number: ClinicalTrials.gov NCT01392508 (the IMPRESSED study). Potential conflicts of interests: AL, BC, and PÅ are listed as inventors on a patent filed by Hansa Medical AB. P4 Impact of the Surviving Sepsis Campaign clinical guideline of in sepsis mortality in a public health institution in Brazil Suellen C de Aguiar*, Guilherme F Garcia, Daniela N Ferreira, Francisco C de Souza, Valda MF Mendonça, Vanuza F Ribeiro, Lívia M Ferreira, Flávio D Capanema, Luana C de Carvalho, Marina F de Gomes Comissão Central de Protocolos Clínicos, Fundação Hospitalar do Estado de Minas Gerais, Belo Horizonte, MG, Brazil Critical Care 2013, 17(Suppl 4):P4; doi:10.1186/cc12905 Background: Sepsis is the principal cause of mortality in intensive therapy units (ITUs) around the world [1]. Several international organizations created in 2002 the Surviving Sepsis Campaign (SSC), targeting the reduction of sepsis mortality in 25% during 5 years [2]. The Fundação Hospitalar do Estado de Minas Gerais (FHEMIG), Brazil, was incorporated in this campaign with eight hospitals (four general hospitals, one trauma hospital, one oncologic center, one infectious diseases center, one maternity hospital). The aim of this study is to evaluate the impact of using the SSC sepsis protocol in severe sepsis and sepsis shock lethality in the FHEMIG net hospitals. Materials and methods: This is a retrospective cohort study based on eight ITU public hospitals. The inclusion criteria were patients with severe sepsis and sepsis shock according to the SSC protocol, from January 2010 to December 2012, aged older than 18 years, which had a final outcome of hospital discharge or death. The sepsis lethality was compared annually from 2010 to 2012. Since 2010, the implementation of educative and managerial measures was based on the SSC guidelines: auditing of medical charts; education in sepsis care; issue of booklet and posters about sepsis; inclusion of sepsis information in the medical residence program; and collaboration of hospital directors in monitoring and giving information of the sepsis guideline. The study was approved by the Institutional Ethical and Research Committee. Data were collected and analyzed on EPIINFO software, using ANOVA test for comparisons with precision of 95%. Results: In the period of 3 years, 1,698 severe sepsis and sepsis shock patients were registered and 1,152 (67.84%) died. We verified a reduction of 12% (P = 0.0073) on lethality global. Hospitals 2 and 6 had a significant reduction on lethality, of 35% (P < 0.0001) and 17% (P = 0.0073) respectively (Table 1). Conclusions: The sepsis lethality is still high in this institution (64.1%), compared with the Public Hospitals in Brazil (59.6%) and the world rate (30.8%) [3]. After the adoption of managerial measures based on the SSC protocol, there was a significantly reduction in lethality, but only one hospital reached the target reduction of 25% on lethality. This heterogeneity could be explained by different engagements of the professional board and directory and different patient’s profiles. The sepsis mortality is a major challenge in the world [4], and application of the SSC protocol led to a significant reduction in sepsis lethality. Acknowledgements: The authors would like to acknowledge the assistance of the staff and local protocol team of the participant hospitals: Hospital João XXIII, Hospital Alberto Cavalcanti, Hospital Geral de Barbacena, Hospital Júlia Kubitschek, Hospital Eduardo de Menezes, Maternidade Odete Valadares, Hospital Regional João Penido and Hospital Regional Antônio Dias. References 1. Vincent JL, Rello J, Marshall J, Silva E, Anzueto A, Martin CD, Moreno R, Lipman J, Gomersall C, Sakr Y, et al: International study of the prevalence and outcomes of infection in intensive care units. JAMA 2009, 302:2323-2329. 2. Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM, Sevransky JE, Sprung CL, Douglas IS, Jaeschke R, et al: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit Care Med 2013, 41:580-637. 3. Instituto Latino Americano de Sepse. [http://www.sepsisnet.org]. 4. Sales Júnior JAL, David CM, Hatum R, Souza PCSP, Japiassú A, Pinheiro CTS, Friedman G, Silva OB, Dias MD, Koterba E, et al: Sepse Brasil: estudo epidemiológico da sepse em Unidades de Terapia Intensiva brasileiras. Rev Bras Ter Intensiva 2006, 18:9-17. Background: The outcome of extended peritonitis is determined by many factors including antimicrobial defense. Microbial invasion, surgery, and intensive therapy cause secondary immunity deficiency associated with septic complication incidence and post-surgery lethality. The great importance in initialization and supporting these processes belongs to Escherichia coli endotoxin that participates in digestive tract immunity and general immunoresistance. Table 1(abstract P4) Severe sepsis and sepsis shock death in the eight FHEMIG hospitals, from 2010 to 2012 Table 1(abstract P5) Indexes of anti-endotoxin immunity in extended peritonitis Before treatment, opt.un. 5th day of treatment, opt.un. p1, evidence between donors and day of admission; p2, evidence between day of admission and the 5th day; p3, evidence between patients of different immunity level. High immunity level patients (group I, n = 5) Low immunity level patients (group II, n = 27) 0.276 ± 0.004 (p1 > 0.05) 0.210 ± 0.03 (p1 < 0.01) 0.121 ± 0.01 (p1 < 0.01) 0.084 ± 0.007 (p1 < 0.001, p3 < 0.001) 0.202 ± 0.02 (p1 < 0.05, p3 > 0.05) 0.069 ± 0.008 (p1 < 0.01, p3 < 0.001) Table 2(abstract P5) Dynamics of anti-LPS-antibodies in low immunity level patients with peritonitis after sandoglobulin H injection Before injection, opt.un. After injection, opt.un. Materials and methods: Thirty-two patients ages 15 to 86 (male:female = 24:8) treated for extended peritonitis were investigated. Blood was sampled after admission and in 5 days to determine anti-lipopolysaccharide antibodies of different classes (anti-LPS-IgA, anti-LPS-IgG, anti-LPS-IgM, respectively) by hard-phase immunoenzyme analysis. The control group included 10 healthy donors (opt.un.): anti-LPS-IgA - 0.348 ± 0.053, anti-LPSIgM - 0.162 ± 0.01, anti-LPS-IgG - 0.333 ± 0.051. Results: Patients with high levels of anti-endotoxin immunity were 15.6% (n = 5) (Table 1); after surgery they had rapid recovery, normalization of peristalsis and laboratory parameters by the 5th day. Patients of low immunity level were 84.4% (n = 27); they had a long complicated recovery period. In group I for standard treatment within 5 days one noticed evident shifts of all parameters that witnesses its sufficiency. In group II the parameters are not increased evidently, which testifies to necessity of additional immunocorrection. Low immunity level patients were introduced to 3 ml sandoglobulin H on the 5th day after surgery that was associated with a sharp increase of anti-LPS antibody titer (Table 2). Growth of anti-LPS antibody titer was associated with positive dynamics of the post-surgery period. Conclusions: The majority of peritonitis patients have decreased competent anti-LPS antibodies, which determines the severity of the postsurgery period. Low immunity level patients need passive nonspecific immunotherapy that stimulates protective functions, blocks mechanisms of inflammation progress, and prevents abdominal sepsis. Background: According to the 2004 WHO Annual Report, abdominal sepsis (AS) is one of the most dangerous diseases of the 21st century. But the issue of its treatment including immunotherapy is very far from being completely solved. Our aim was the demonstration of specific immunotherapy efficacy in AS. Materials and methods: We investigated activity of immunogenesis (production of specific antibodies) in AS in 50 patients under impact of hyperimmune plasma infusion obtained from the donors who recently 0.452 ± 0.02 (p2 < 0.001) 0.286 ± 0.04 (p2 < 0.05) 0.184 ± 0.02 (p2 < 0.01) 0.154 ± 0.015 (p2 < 0.01) 0.213 ± 0.01 (p2 > 0.05) 0.083 ± 0.007 (p2 > 0.05) Table 1(abstract P6) Dynamics of specific antibody titer in AS patients after hyperimmune plasma introduction Before introducing, After introducing, units/l units/l Anti-E. coli (n = 10) Anti-P. aeruginosa (n = 10) Anti-S. aureus (n = 10) Anti-bacteroids (n = 10) Anti-peptococci (n = 10) *Parameters change evidently (P < 0.05). endured acute inflammatory abdominal diseases. The control group was made up of 10 healthy people. Results: Our investigations demonstrated that the donors’ titer of specific antibodies is evidently more according to indexes of anti-Escherichia coli, anti-Pseudomonas aeruginosa, anti-Staphylococcus aureus, anti-bacteroids, anti-peptococci immunity. Introducing hyperimmune plasma obtained from such donors evidently increases specific antibody titer in AS patients (Table 1). Our results demonstrated that the titer of specific antibodies to AS main agents in the dead patients was evidently lower in comparison with the control group and in recovered people. This trend is observed 7 to 10 days after surgery. For example, at admission the titer of anti-S. aureus antibodies in dead patients was 11% lower in comparison with the control group, and the titer of anti-E. coli antibodies 19% lower in comparison with control group and 32% lower in comparison with recovered patients; 7 to 10 days after surgery in dead patients in comparison with recovered patients, the titer of anti-S. aureus antibodies was lower by 22%, and the titer of anti-E. coli antibodies by 47%. Conclusions: Introducing hyperimmune plasma (specific immunotherapy) increases titer of specific antibodies, and increased concentration of specific antibodies improves forecast of survival in AS. P7 Characteristics and outcomes of patients with culture negative septic shock compared with patients with culture positive septic shock: a retrospective cohort study Shravan Kethireddy1*, Amanda Bengier2, H Lester Kirchner2, R Bruce Light3, Yazdan Mirzanejad4, Dennis Maki5, Yaseen Arabi6, Steven Lapinsky7, David Simon8, Aseem Kumar9, Joseph E Parrillo10, Anand Kumar3, the Cooperative Antimicrobial Therapy of Septic Shock (CATSS) Database Group 1Section of Critical Care Medicine and Infectious Diseases, Geisinger Medical Center, Danville, PA, USA; 2Clinical Innovation and Biostatistics, Division of Internal Medicine, Geisinger Medical Center, Danville, PA, USA; 3Section of Critical Care, Section of Infectious Diseases, University of Manitoba, Winnipeg, MB, Canada; 4Surrey Memorial Hospital, Surrey, BC, Canada; 5University of Wisconsin Hospital and Clinics, Madison, WI, USA; 6King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia; Background: Previous studies have identified that nearly 30% of patients with severe sepsis and septic shock lack a definitive microbial etiology. The characteristics and outcomes of culture negative septic shock are not well defined despite large epidemiologic studies on septic shock Materials and methods: Retrospective nested cohort study of 2,651 patients with culture-negative septic shock and 6,019 culture-positive septic shock patients derived from a trinational, 8,760-patient database of patients with septic shock between 1989 and 2008. Results: In total, 30.6% of cases of septic shock cases were identified as culture-negative within the database. Patients with culture-negative septic shock (CNSS) experienced similar ICU mortality as did those with culturepositive septic shock (CPSS) (41.7% vs. 40.5% P = 0.276) and identical overall hospital mortality (51.9% vs. 51.9% P = 0.976). Severity of illness was similar between CNSS and CPSS (median APACHE II 25 (IQR 6 to 54) vs. 25 (IQR 4 to 70) respectively). Initial and 6-hour lactate levels were also similar among CNSS and CPSS patients (mean 4.4 vs. 4.1, P = 0.237 and mean 4.0 vs. 4.1, P = 0.221 respectively). Interestingly CNSS patients were significantly more likely to be hypothermic than CPSS patients (temperature <36°C 18.9% vs. 15.3%, P < 0.0001). CNSS patients presented significantly more often from the community (63.3% vs. 58.0%, P < 0.0001), where patients with CPSS presented more often with nosocomial infections (36.7% vs. 42.0%, P < 0.0001). Gastrointestinal and respiratory tract infections were the predominant sources of infection in both groups. However, CNSS patients with respiratory tract infections experienced lower mortality than their CPSS counterparts (49.6% vs. 56.3%, P = 0.008) but similar mortality rates with gastrointestinal infections (61.0% vs. 58.2%, P = 0.289) (Tables 1 and 2). Table 1(abstract P7) Comparison of variables of culture-positive and culture-negative septic shock Table 1(abstract P7): Comparison of variables of culture-positive and culture-negative septic shock (Continued) Days to extubation Lactate - 6 hours Lactate - 24 hours Similar to our previous findings, we identified by the second hour after onset of persistent/recurrent hypotension that the in-hospital mortality rate was significantly increased relative to receiving therapy within the first hour (odds ratio, 1.62; 95% CI, 1.21 to 2.15; P < 0.001) in the CPSS group. Following increasing delays in the administration of appropriate antimicrobial therapy over the first 6 hours after the onset of hypotension, patients in both groups experienced nearly congruent, significant increases in hospital mortality; at 6 hours the CNSS group (odds ratio, 2.87; 95% CI, ICU LOS (median, IQR) 15-day survival (n, %) Hospital survival (n, %) APACHE (mean, SD) ICU LOS (median, IQR) 15-day survival (n, %) Hospital survival (n, %) APACHE (mean, SD) 1.72 to 4.78; P < 0.0001) and the CPSS group (odds ratio, 3.44; 95% CI, 2.17 to 5.48; P < 0.0001) (Figure 1). Survival differences between these time intervals are not significantly different in patients with CNSS and CPSS. Conclusions: Patients with CNSS behave similarly to CPSS patients in nearly all respects. As with bacterial septic shock, early appropriate antimicrobial therapy appears to improve mortality. Earlier recognition of infection is the most obvious effective strategy to improve hospital survival. Optimal duration of therapy is not well defined among patients with CNSS. In addition to early, appropriate antimicrobial therapy, use of de-escalation strategies such as serial procalcitonin levels may be useful to determine the length of empiric broad-spectrum antimicrobial use in this population. Background: SRT2379 is a selective small-molecule activator of the NAD+-dependent deacetylase, Sirtuin 1 (SIRT1), which has broad antiinflammatory effects in cell cultures and rodents. The aim of the current Figure 1(abstract P7) Odds ratio of death by antibiotic delay in culture-positive and culture-negative septic shock. study (EUDRACT # 2011-002266-20) was to determine the effect of SRT2379 on the inflammatory responses in normal healthy male subjects after exposure to LPS. Materials and methods: This single-blind, placebo-controlled study consisted of four treatment arms (n = 8 per arm): (1) oral SRT2379 50 mg; (2) oral SRT2379 250 mg; (3) oral SRT2379 1000 mg; and (4) placebo. All subjects received a single dose of study drug on day 1 followed by intravenous LPS 4 hours later. Laboratory parameters of inflammation along with assessment of clinical signs, safety assessments, and pharmacokinetic measurements were recorded at baseline and after LPS administration. Results: SRT2379 was well tolerated. Adverse events were similar across all treatment groups and were predominantly as expected with LPS administration. Pharmacokinetic exposures increased in a dose-dependent manner. SRT2379 did not significantly impact cytokine release as compared with placebo: TNFa (183.52, 177.57, 123.84 vs. 195.30 pg/ml for groups 1, 2, 3 vs. group 4, respectively, P > 0.05), IL-6 (195.25, 237.51, 180.26 vs. 250.08 pg/ml, respectively, P > 0.05), IL-17 (3.88, 2.59, 6.42 vs. 8.09 pg/ml, respectively, P > 0.05), IL-8 (126.11, 105.25, 110.56 vs. 108.77 pg/ml, respectively, P > 0.05), and IL-10 (12.61, 13.03, 40.40 vs. 11.90 pg/ml, respectively, P > 0.05). SRT2379 also had no impact on vital signs, leukocyte counts, or coagulation activation markers compared with placebo. Conclusions: Although SRT2379 suppresses inflammatory markers in preclinical experiments, we were unable to demonstrate a similar impact in this human model of endotoxemia. This may be due to potency or exposure issues, with the compound. SRT2379 terminated for further clinical development. More promising candidates are being identified for future clinical exploration. Pavlo Melnychenko*, Alexander Potapov, Andrey Babanin Background: One of the perspective directions for the improvement of surgical patients’ treatment results is a multimodal approach in perioperative management including wide administration of regional anesthesia, early enteral feeding and modification of infusion therapy. The goal of this study is the assessment of the multimodal approach effect on antiendotoxin immunity and systemic inflammation after major abdominal surgery. Materials and methods: Open nonrandomized research. In the control group (n = 52), perioperative management was carried out with perioperative starvation, total intravenous anesthesia and analgesia on the basis of opiates. In the multimodal approach group (n = 40) we used a thoracic epidural analgesia in combination with early enteral feeding and preoperative infusion of HES 130/0.42 of 15 ml/kg body weight. In vein blood tests we analyzed C-reactive protein (CRP) and antibodies to lipopolysaccharide of Escherichia coli by IgM (anti-LPS-IgM) class. Data are submitted in the form of the median and 95% CI. The Mann-Whitney U criterion is used for comparisons between groups. Results: The median maintenance of CRP was 135.7 mkg/ml (95% CI = 153.5 to 249.5) in the control group for 3 days after operation but in the multimodal approach group was significantly lower - 89.0 mkg/ml (95% CI = 56.9 to 212.4; P < 0.05). The median anti-LPS-IgM level was 0.087 MU (95% CI, 0.084 to 0.226) in the control group in the same time but in the multimodal approach group was significantly higher - 0.181 MU (95% CI, 0.153 to 0.241; P < 0.001). The obtained data can mean that the expressed system inflammatory reaction has negative impact on the postoperative period. Reduced antiendotoxin immunity increases terms of hospitalization as an independent factor. This also increases the number of complications and lethality in surgery. Conclusions: The multimodal approach that includes thoracic epidural analgesia, early enteral feeding and preoperative infusion of HES 130/0.42 after volume abdominal operations prevents exhaustion of antiendotoxin immunity and system inflammatory reaction. Reference 1. Bennet-Guerrero E, Michael HP, Robin GB, et al: Decreased endotoxin immunity is associated with greater mortality and/or prolonged hospitalization after surgery. Anesthesiology 2001, 94:992-998. Background: Septicemia is a systemic disease caused by the spread of microorganisms and their toxins in the blood. These bloodstream infections are a major cause of morbidity and mortality in children in developing country [1-4]. It has been confirmed by culture that is associated with clinical manifestation and systemic response [5-7]. It is crucial to continuously monitor any change in the local patterns of infection and susceptibility to various antibiotics. The aim of this study was to determine the bacteriological profile and antimicrobial sensitivity patterns among children suspected of having septicemia. Materials and methods: A cross-sectional study involved about 201 pediatric patients (≤12 years) was conducted from October 2011 to February 2012 at Tikur Anbessa Specialized Hospital and Yekatit 12 Hospital’s pediatric units after the proposal of this study was approved by National Ethics Review Committee. Standard procedure was followed for blood sample collection. Samples were incubated in the BACTEC 9050 System, followed by isolate identifications based on standard microbiological procedures and testing for their susceptibility to antimicrobial agents using the disc diffusion method. Data were analyzed using the SPSS version 19 software package. Results: Out of 201 study subjects, 110 (54.7%) were male. The majority (147, 73.1%) of them were neonates (≤28 days). The mean length of hospitalization was 11.24 days. Out of the 201 tested blood samples, blood cultures were positive in 56 (27.9%) cases (Figure 1). Gram-negative and Gram-positive bacteria constituted 51.8% and 46.4%, respectively. The most frequent pathogen found was Staphylococcus aureus (23.2%), followed by Serratia marcescens (21.4%), CoNS (19.6%), Klebsiella spp. (16%), Salmonella spp. (5.4%) and Enterobacter cloacae (3.6%) (Figure 2). The majority of bacterial isolates showed high resistance to ampicillin, penicillin, co-trimoxazole, gentamicin and tetracycline. Ciprofloxacin and nalidixic acid were the most effective antimicrobial agents for Gramnegative bacteria, while vancomycin and clindamycin for Gram-positive bacteria (Table 1). Deaths occurred in 25 (12.4%) children, out of which 13 (23.2%) had bacteremia. Conclusions: The present study revealed that both Gram-positive and Gram-negative bacteria were responsible for bloodstream infections and the majority of the isolates were multidrug resistant. S. aureus and S. marcescens were the most common isolated bacteria from blood cultures. The alarmingly higher percentages of multidrug-resistant isolates urge us to take infection prevention measures and to conduct other large studies for appropriate empiric antibiotic choice. Acknowledgements: The authors would like to acknowledge the technical support provided by the members of the Departments of Microbiology and Pediatrics of Tikur Anbessa Specialized and Yekatit 12 Hospitals. They also thank Mr Yoseph Kenea for his excellence statistic support. This work was supported by AHRI/ALERT and AAU. References 1. Archibald LK, McDonald LC, Nwanyanwu O, Kazembe P, Dobbie H, Tokars J, et al: A hospital-based prevalence survey of bloodstream infections in febrile patients in Malawi: implications for diagnosis and therapy. J Infect Dis 2000, 181:1414-1420. 2. Ogunleye VO, Ogunleye AO, Ajuwape ATP, Olawole OM, Adetosoye AI: Childhood septicaemia due to salmonella species in Ibadan, Nigeria. Afr J Biomed Res 2005, 8:131-134. Figure 1(abstract P11) Distribution of 56 blood culture isolates by age interval and gender. Figure 2(abstract P11) Distribution of blood culture isolates in children with suspected of having sepsis. Table 1(abstract P11) Antimicrobial resistance pattern of bacteria isolated from blood culture Resistance proportion of bacterial isolates (R%) ND, not done; SXT, sulphamethaxozol/trimethoprim. aGram-negative bacteria (Acinetobacter baumanii, Escherichia coli, and Pseudomonas leutole). bGram-positive bacteria (Enterococcus spp. and Streptococcus. spp.). P13 Development of a new monoclonal antibody-based point-of-care testing assay for the quantification of procalcitonin in whole blood for a rapid sepsis diagnostic Martin Rieger*, Daniela Rascher, Anton Hartmann Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Department of Environmental Science, Research Unit Microbe-Plant Interactions, Neuherberg, Germany Critical Care 2013, 17(Suppl 4):P13; doi:10.1186/cc12913 Background: After recent studies of the BMBF (SepNet), sepsis causes about 150 deaths per day in Germany, making it the third leading cause of death in Germany. In acute sepsis, rapid diagnosis and rapid medication is crucial. Both as a reliable parameter for diagnosis of sepsis and for guiding the antibiotic therapy, procalcitonin (PCT) is a very sensitive available biomarker [1] and is recommended in the current guidelines [2] to be quantified under sepsis suspicion. Although there are a couple of commercially available fast analytical devices for the quantification of PCT, none of these devices completely fulfill all requirements for a point-of-care testing (POCT) device which are: bedside testing; no sample preparation (whole blood testing); simple handling with ready-to-use and single-use cartridges; and short turnaround time between analysis and medical treatment in the clinical necessary concentration range. Whereas most devices fulfill the latter requirements they are still too big for bedside testing or cannot handle whole blood. Materials and methods: Based on newly developed monoclonal antibodies (mAbs) [3], a fast and sensitive immunoassay for the quantification of PCT in whole blood was developed and transferred to a commercially developed (not available on market) POCT device (respons®IQ) from pes diagnosesysteme GmbH. Results: With the new developed mAbs the achieved limit of detection for PCT in plasma and whole blood is 0.04 ng/ml and 0.05 ng/ml respectively, which is within the clinical necessary range (<0.05 ng/ml). The now established assay shows high reproducibility within 9 minutes, independent of different plasma samples due to the selection of suitable additive compounds. In a first set of leftover patient samples, the PCT-POCT assay showed good correlation (R2 = 0.988, n = 14, m = 2) with the state-of-the-art technology Kryptor (BRAHMs) (D Rascher, M Rieger, HMGU, AMP, unpublished data). Moreover, in cooperation with Dr A Geerlof (HMGU), human recombinant PCT (hrPCT) was produced in two biological and clinical relevant forms (amino acids 1 to 116 and 3 to 116) in high amounts and high purity (A Geerlof, D Rascher, M Rieger, unpublished data). This hrPCT will replace expensive (5 k$/mg) and batch-to-batch varying commercial available hrPCTs as standard reference material. Jessamine C Sareno*, Jacinto Blas V Mantaring Background: Pentoxifylline, a xanthine derivative, has raised new interest in neonatal research due to its immunomodulatory functions and its potential role in reducing mortality from sepsis. Two small studies on a per-protocol analysis have shown promising results. This larger trial on an intention-to-treat basis will determine whether the use of pentoxifylline as an adjunctive therapy for sepsis in preterm neonates (≤36 weeks) weighing <1,500 g will truly result in a reduction in the all-cause mortality. Materials and methods: Preterm infants ≤1,500 g with suspected infection admitted to the NICU of a large tertiary, training, government hospital were eligible for inclusion in the study. After informed consent, they were randomized to receive either pentoxifylline at a dose of 6 mg/kg/hour or placebo. Patients with major congenital malformations, congenital infections and severe hemorrhage were excluded from the study. Pentoxifylline was administered as a 6 ml infusion for 6 hours for 6 days. The control group received normal saline in the same manner as the pentoxifylline infusion. Patients, parents and physicians (outcome assessors) were blinded to the treatment assignments. The primary outcome was analyzed on an intention to treat basis. The primary outcome measured in the study is the occurrence of all-cause mortality between the two groups. Secondary outcomes measured include mortality from sepsis, adverse drug reactions and length of hospital stay. Results: A total of 312 neonates are included in this interim analysis: 156 in the pentoxifylline group and 156 in the control group. Baseline characteristics were comparable between the two groups. In this analysis, there is no difference in the occurrence of death among patients in the pentoxifylline group versus the placebo group (RR: 1.08 (0.83, 1.41)). There is no statistical difference in the risk of death from septic shock (RR: 1.03 (0.67, 1.59), P = 1.0). There was also no significant difference in the length of hospital stay in the two groups (36 days in treatment group vs. 35 days in control group, P = 0.910). No significant adverse drug reactions were noted with pentoxifylline use. Conclusions: Pentoxifylline as an adjunct therapy for sepsis did not show a decrease in the all-cause mortality. There is also no difference in the occurrence of death from sepsis and length of hospital stay. No adverse drug reactions were noted with pentoxifylline. Acknowledgements: The authors thank the neonatology fellows of the Philippine General Hospital and Ms Carmi Pitajen, RN, research assistant. P15 Effect of semi-quantitative procalcitonin assay on the adequacy of empirical antibiotics and mortality in septic patients Dana Dharaniyadewi1*, Khie Chen Lie2, Nanang Sukmana3, C Martin Rumende4 1Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia; 2Division of Tropical Medicine and Infectious Diseases, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Background: Sepsis is a serious clinical condition with a considerable morbidity and mortality. Procalcitonin (PCT) is a good biomarker for early diagnosis and infection monitoring. A semi-quantitative PCT assay can be performed at the bedside and has good diagnostic value [1,2]. The present study aimed to investigate the effect of a semi-quantitative PCT test on the empirical antibiotic initiation time, the appropriateness of empirical antibiotics and mortality in septic patients. Materials and methods: The study design was a randomized diagnostic trial, which was also a pragmatic trial. Septic patients more than 18 years old with and without signs of organ hypoperfusion or dysfunction who were admitted to Cipto Mangunkusomo Hospital emergency department in the internal medicine unit were eligible. Subjects were randomly assigned to either a semi-quantitative PCT-examined group (study group) or a control group. Semi-quantitative PCT test results will be informed to the physicians taking care of the patients. The primary outcome was 14-day mortality. Secondary outcomes were the time of initiation and appropriateness of empirical antibiotics. A Tropical Infection Consultant will assess the appropriateness of empirical antibiotics based on Pedoman Umum Penggunaan Antibiotik Departemen Kesehatan Republik Indonesia. Results: Two hundred and five patients met the inclusion criteria. Ninetyfive of 100 subjects from the study group and 102 of 105 subjects from the control group were included in the analysis (Figure 1). Both groups have equal baseline characteristics (Table 1). The mortality risk was lower in the study group (RR 0.53; 95% CI 0.36 to 0.77). The study group had greater probability to have a first dose of empirical antibiotic in less than 6 hours compared with the control group (RR 2.48; 95% CI 1.88 to 3.26). No effect was seen in appropriateness of empirical antibiotics between groups (RR 0.99; 95% CI 0.92 to 1.08) (Table 2). Conclusions: Semi-quantitative PCT examination affects the empirical antibiotic initiation time and mortality in septic patients, but not the appropriateness of empirical antibiotics. Figure 1(abstract P15) Enrollment of patients and completion of the study. Table 1(abstract P15) Baseline characteristics of the patients Semi-quantitative PCT-examined group, n (%) Control group, n (%) ≤60 years Source of infection ≥2 sources Severe sepsis and septic shock 2.48 (1.88 to 3.26) 0.99 (0.92 to 1.08) 0.53 (0.36 to 0.77) Table 2(abstract P15) Effect of semi-quantitative procalcitonin assay on adequacy of empirical antibiotics and mortality in septic patients Semi-quantitative PCT assay, n (%) Empirical antibiotic initiation time Appropriateness of empirical antibiotics ≤6 hours Background: Sepsis is a complex disease with an initial proinflammatory profile triggered by an infection process, which is typically followed by a compensatory anti-inflammatory response, leading to immunosuppression. There are few cases in literature relating sepsis with opportunistic infections, such as strongyloidiasis, which may lead to severe clinical consequences due to hyperinfection. Human strongyloidiasis is a neglected tropical disease of major worldwide distribution, affecting millions of people. Despite of the fact that infection with Strongyloides stercoralis is usually self-limited and with low morbidity in immunocompetent individuals, it may become lethal in cases of immunosuppression, such as AIDS, corticosteroid treatment and transplantation. Our aim in this work was to investigate the presence of S. stercoralis antigens and anti-parasitic IgG in sepsis patients in a highly endemic area of strongyloidiasis. Materials and methods: Serum samples from 27 individuals with strongyloidiasis and 27 healthy subjects were used as positive and negative controls, respectively, according to their parasitological analyses. Additionally, 27 sepsis patients were also investigated. We have used ELISA tests to detect S. stercoralis antigens and IgG antiS. stercoralis in all three groups. The cutoff value was determined by the ROC curves obtained by Prism 5.0 software. Results: IgG anti-S. stercoralis was detected in six patients; five under septic shock and one with sepsis. Among them, four were positive for the parasite antigen-antibody immune complex; three under septic shock and one with sepsis, demonstrating that 15% of sepsis patients were infected by the parasite, which may have significantly contributed with the hyperinfection presented by septic-shock patients (10%). Conclusions: There are only two reports of an association between S. stercoralis infection and immunosuppression, which led to lethal sepsis cases. However, our preliminary analysis through antigen-antibody immune complex demonstrated that this parasitic infection might be more common in sepsis than expected. The correct diagnosis of the causal infection in sepsis may support the correct therapeutic choice, which is fundamental to avoid the continuous spread of specific pathogen/parasite triggers that will eventually lead to hyperinfection, and consequently to severe sepsis. Acknowledgements: The authors would like to thank the patients and their families for the direct collaboration in this work, the medical staff from the ICU of the university hospital for providing the biological samples and the clinical parameters, and financial support by CNPq, CAPES, and FAPEMIG. P17 Procalcitonin, presepsin, pro-adrenomedullin, fibrin degradation products, and lactate in early diagnosis and prognosis of septic patients newly admitted to the intermediate care unit from the emergency department Filippo Mearelli1*, Nicola Fiotti1, Nicola Altamura1, Irene Paoli1, Chiara Casarsa1, Maurizio Ruscio2, Gianni Biolo1 1Unit of Clinica Medica Generale e Terapia Medica, Department of Medical Surgical and Health Sciences, University of Trieste, Italy; 2Laboratory Medicine Ospedale Sant’ Antonio, San Daniele Del Friuli, Italy Critical Care 2013, 17(Suppl 4):P17; doi:10.1186/cc12917 Background: More than 50% of all septic patients admitted to intensive care departments derive from intermediate care units (INCU). Biomarkers represent the most promising tool for early diagnosis of sepsis; but their accuracy in INCU has been largely disregarded [1]. Moreover, given the complexity of the septic pathophysiology, a panel of biomarkers could be more effective than a single one. For this reason we tested acute phase protein, cell surface, vasotonous related, coagulation system, and tissue hypoxia markers in early ruling in/out of sepsis in patients suffering from systemic inflammatory response syndrome (SIRS) [2-5]. Materials and methods: This prospective observational study included all SIRS [5] patients newly admitted to a medical ward from February to May 2012. Cases were diagnosed as sepsis or non-infective SIRS by clinical examination, cultures of the biological fluid, and imaging during a 7-day follow-up. Investigators were blinded to biomarker results. Survivors at 7 and 30 days were also assessed. Samples for procalcitonin (PCT), presepsin (sCD14-ST), pro-adrenomedullin (PRO-ADM), fibrin degradation products (FDP) and lactate were collected within 4 hours of admission. Their role in predicting diagnosis and survival, alone or in combination, have been investigated by receiver operating characteristic (ROC) curve, Youden index, relative risk and binary logistic regression. Results: Among the 60 sepsis patients (microbiological and clinical sepsis), the most common sites of infection were the lung (67%), urinary tract (17%), abdomen (5%), and skin (8%). The sepsis group had significantly higher levels of PCT, sCD14-ST and FDP than the noninfective SIRS group. The area under the ROC was 0.80, 0.78, and 0.67 for FDP, PCT, and sCD14-ST respectively. Main results are reported in Table 1: the combination of FDP and PCT detected correctly 10 more cases, leaving misdiagnosed only nine out of 80 patients. ROC curves are reported in Figure 1. sCD14-ST (cutoff 1.317 ng/ml, OR 12.2 (95% CI 2.6 to 55.5) P = 0.0002) and lactate (cutoff 20 mg/dl OR 11.9 (95% CI 2.23 to 62.5) P = 0.001) were comparable in predicting 7-day survival. Mortality at 30 days was significantly higher in patients with PRO-ADM level ≥4.09 nmol/l (OR 26 (95% CI 4.8 to 143) P = 0.000002). The Kaplan-Meier curves for PRO-ADM are reported in Figure 2. Table 1(abstract P17) 180 + 0.1 ng/ml Background: Sepsis still represents the leading cause of mortality among children and its etiology changes according to age, immune status and geographic location [1-4]. Prevention of this disease has key role in reducing morbidity and mortality and includes development and application of vaccines [5-7]. In 2010, pneumococcal and meningococcal C vaccines were introduced in the basic immunization schedule in Brazil. The application of these may already be influencing the etiologic profile of sepsis in childhood [7]. The evaluation of this profile, as well as the clinical manifestations and course of sepsis in the post vaccine, becomes essential for better clinical decision and effective therapeutic approach in hospitalized patients. The objective was to determine clinical manifestations, etiology and outcome of sepsis in patients admitted to a pediatric ICU. Materials and methods: A retrospective cohort study, by collecting data from medical records of patients diagnosed with sepsis admitted to the pediatric ICU of Hospital Municipal Dr. Mario Gatti, Campinas, SP, from January 2011 to December 2012. The variables studied were: age, sex, vaccination schedule, etiologic agent identified in cultures and clinical outcome. Results: Eighty-seven patients were included in the study (56 male, 31 female) with a mean hospital stay of 8.16 days, vasoactive drug use of 2.82 days and 5.33 days of mechanical ventilation. In total, 57/87 cultures were negative. Among the positive, the majority (21/30) was collected less than 48 hours after admission and the most frequent etiologies were: Gram-negative bacteria (10), Staphylococcus aureus (7) and Neisseria meningitidis (4). Two cultures were positive for Streptococus pyogenes and only one for Streptococcus pneumoniae. Twenty-four (16.1%) patients died. Figure 1(abstract P17) ROC curves of PCT, FDP, sCD-14ST, and the combination of FDP + PCT. Solid thick line, PCT; dashed line, FDP; dotted line, PCT+FDP; dot-dash line, sCD-14ST; thin solid line, reference line. Mortality was higher in patients with incomplete immunization (P = 0.047). Among the cases with meningococcal etiology, 3/4 were not vaccinated. Conclusions: The clinical group of patients diagnosed with sepsis showed short time of hospitalization, use of vasoactive drugs and mechanical ventilation. Mortality was high and higher in the group of patients with incomplete immunization. Among the causative agents, it P19 Difficulties in implementation of the project ‘HUPE against sepsis’: speaking of people who watch Sérgio da Cunha1*, Mário Castro Álvares Perez1, Elisabete Novello Ferreira2, Luana Ferreira de Almeida2, Eliane Passos Pereira Assumpção2, Paulo Vieira Damasco3, Jorge da Silva Motta4, Rogério Marques de Souza5, Viviane Silva e Silva2, Elizabeth de Andrade Marques6, Vagner Ismerim Lobão7, Irene de Souza e Silva8, Ana Alice de A Triani8, Jessica Bernardes Almeida Borges da Silva2, Julio Cesar Delgado Correal9, Catherine Valdez10, Jessica Oliveira10 1Department of Clinical Medicine, College of Medical Ciences, Rio de Janeiro State University, Rio de Janeiro, Brazil; 2Nursing Department, Pedro Ernesto University Hospital, Rio de Janeiro State University, Rio de Janeiro, Brazil; 3Department of Internal Medicine, College of Medical Ciences, Rio de Janeiro State University, Rio de Janeiro, Brazil; 4Medical Coordination, Pedro Ernesto University Hospital, Rio de Janeiro State University, Rio de Janeiro, Brazil; 5Nursing Coordination, Pedro Ernesto University Hospital, Rio de Janeiro Figure 2(abstract P17) Thirty-day survival curve (Kaplan-Meier) according to PRO-ADM levels. Introduction: The project ‘HUPE against sepsis’ seeks to emphasize the importance of early recognition of sepsis, in order to accelerate the implementation of measures associated with decreased mortality for severe sepsis. It is therefore important that the professionals involved in healthcare are attentive to quick detection of signs and symptoms associated with the condition. The objective was to identify the difficulties for the implementation of the protocol advocated by the Surviving Sepsis Campaign and adopted by the project ‘HUPE against sepsis’. Materials and methods: The study was conducted in clinical medical and surgical wards, DIP, general duty, cardiac and ICUs of the Pedro Ernesto University Hospital (HUPE), totaling 11 inpatient units. In January 2013, a questionnaire was applied to doctors, nurses and nurse technicians, including effective, contractors and residents. This instrument contained closed questions, professional profile and was related to the topic in question. Results: Fifty-one professionals participated in the study: 22 (43%) medical staff and 29 (57%) nurses. Of these, 12 were medical residents, and eight were nursing residents, all in the first year (approximately 78% of workers investigated). Most physicians (55%), 38% of nurses and 40% of nurses claimed to have greatest difficulty administering the first dose of antibiotics within up to 1 hour after the diagnosis. About 45% of doctors and 31% of nurses also reported difficulty in the distribution of materials to acquire the sepsis kit (which contains materials for deep venous puncture, invasive hemodynamic monitoring and collecting blood cultures). Physicians (41%) and nurses (40%) still reported as a problem going to the pharmacy to get the first dose of the antibiotic. Other limiting factors were also appointed: obtaining the vesical catheterization of delay (for hourly diuresis control); rapid identification of severe sepsis; printed data record of the protocol; samples of blood culture for aerobic; and peripheral venous access puncture. Conclusion: The difficulties pointed out by the professionals investigated are common and include factors that prevent the correct and early implementation of the protocol, be they of institutional and/or professional responsibility. Seeking solutions to the problems raised allows a targeting of future actions to be developed, among them the constant updating and training of professionals involved in assistance for the inpatients investigated. This allows, also, the search for better institutional infrastructure appropriate to meeting the demands of the patient with severe sepsis. Background: Neutrophils as a part of nonspecific immunity factors play a crucial role in antimicrobial resistance. Reactive oxygen species (ROS) are an important compound of the neutrophils’ microbicidal action. Analysis of neutrophils’ ROS production could provide valuable data on a phagocyte link of immunity [1]. A chemiluminescent (CL) assay being highly sensitive allows evaluating oxidative output of the cells in dynamics. Many studies on neutrophil CL in humans with different diseases have been published [2,3]. However, the results often vary between authors because of the lack of standardized method of CL analysis. So we have developed a methodology of neutrophils’ CL analysis according to the principles of evidence-based medicine. Materials and methods: One hundred and twenty healthy donors and 17 ICU patients with second-third-degree burns participated in this study. We held an assay on the 1st, 8th and 15th day after injury and later; 37 observations in total. To dilute blood samples we used Hank’s balanced salt saline (HBSS) with glucose, pH 7.4. Luminol (Sigma-Aldrich) was dissolved in double-distilled water at 1 mM. N-formyl-methionyl-leucylphenylalanine (FMLP; Sigma-Aldrich) and 4-phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich) were diluted in dimethyl sulfoxide (MP Biomedicals, LLC) to make stock solutions that were dissolved in HBSS on the day of experiment. CL was evaluated by means of a chemiluminometer Lum-12 (Department of Biophysics, Moscow State University) [4]. Results: We substantiate an optimal experiment design in the context of obtaining the highest intensity of analytic signal and reproducible findings. Thus we have developed a method for evaluation of a neutrophil function, based on a step-by-step stimulation of the cells by PMA and FMLP. Using our approach, we investigated the distributions of CL characteristics for the population of 80 healthy donors. We obtained reproducible kinetic profiles with intensive flash and absent glow phase of emission in all of the samples. Profiles of ICU patients’ samples showed high intensity of both flash and glow phase of emission (Figure 1). Insufficient glow phase indicated subsequent development of severe septic complications. Figure 1(abstract P20) Kinetics of CL response in ICU patient and donor. Conclusions: As a result we suggest a reliable and replicable method for the evaluation of a neutrophil function. Investigation of the glow phase of the emission is promising to forecast risks of septic complications; we constructed a range of values of adjusted CL glow amplitude at different neutrophil counts that indicates a low probability of SIRS and septic complications that could be useful for correction of intensive treatment tactics. Acknowledgements: The author would like to express deepest appreciation to all those who provided the possibility to perform this research. The author wishes to thank Prof. YA Vladimirov and the team of Department of Biophysics at Moscow State University (Russia) and Dr MA Godkov for assistance and guidance with this study and for submitting of equipment and reagents. Also the author would like to thank Dr EN Kobzeva and Prof. Dr SV Smirnov for the opportunity to work with blood donors and ICU patients. Furthermore, the author would also like to acknowledge with much appreciation Dr VV Kulabukhov and the staff of the Department of Burn Resuscitation at Vishnevsky Institute of Surgery (Moscow, Russia) for their suggestions and encouragement. References 1. Edwards SW: Biochemistry and Physiology of the Neutrophil Cambridge University Press 1994. 2. Vladimirov YA, Proscurnina EV: Free radicals and cell chemiluminescence. Biochemistry 2009, 74:1545-1566. 3. Dahlgren C, Karlsson A, Bylund J: Measurement of respiratory burst products generated by professional phagocytes. Methods Mol Biol 2007, 412:349-363. 4. Obraztsov IV: An evaluation of neutrophil function: a new approach to the chemiluminescent analysis [abstract]. Immunology 2012, 137(s1):199. Background: Bacteremia is a common clinical condition with an incidence of approximately 140 to 160 per 100,000 person-years. Since sepsis is a time-critical diagnosis, identification of emergency department (ED) patients at risk of bacteremia is therefore a priority. The study objective was to validate a previously published clinical decision rule for predicting a positive blood culture in ED patients with suspected infection based on minor criteria, major criteria and a total score [1]. Table 1(abstract P23) Decision rule Suspected endocarditis (3 points) Temperature >39.4°C (103.0°F) (3 points) Indwelling vascular catheter (2 points) A blood culture is indicated by the rule if at least one major criterion or two minor criteria are present. Otherwise, cultures may be omitted. Points used to calculate the total score. Materials and methods: This was a retrospective matched cohort study, set in a large urban academic tertiary ED at Aarhus University Hospital, Aarhus, Denmark with approximately 56,000 patient visits annually. Adult ED patients with blood cultures obtained from 1 January through 31 December 2011. ED patients with blood culture-confirmed bacteremia were matched 1:3 to patients with negative cultures. The outcome was true bacteremia. Features of the clinical history, co-morbid illnesses, physical observations and laboratory tests were used to evaluate the performance of the clinical decision rule including calculation of the total score (Table 1). We report operating characteristics and the summary c-statistic for the decision rule. Results: Among 1,526 patients, 105 (6.9%) patients were classified with true bacteremia. The sensitivity of the prediction rule was 94% (95% confidence interval (CI) 88 to 98%) and specificity 48% (95% CI 42 to 53%). Positive and negative predictive values were 37% (95% CI 32 to 44%) and 96% (95% CI 92 to 99%), respectively. The area under the receiver-operating characteristics curve was 0.83 ± 0.02 standard error (Figure 1). Conclusions: The clinical decision rule performed well in our ED setting and is likely to be a useful supplement to clinical judgment. Acknowledgements: The CONSIDER Sepsis Network is a collaboration of clinical researchers with an interest in sepsis at Aarhus University Hospital, Aarhus, Denmark. Reference 1. Shapiro, et al: Who needs a blood culture? A prospectively derived and validated prediction rule. J Emerg Med 2008, 35:255-264. Background: Conscious assessment for organ dysfunction in infected patients is not uniformly performed since the prognostic performance of organ dysfunction has not been validated. We hypothesize that the number of organ dysfunctions is a prognostic marker in emergency department (ED) patients with suspected infection and that an increasing number of organ dysfunctions correlates with in-hospital mortality. Materials and methods: A prospective observational study of adult (18+ years) ED patients with suspected infection presenting to one of two urban, academic medical center EDs. The inclusion criterion was clinically suspected infection at ED presentation. At Beth Israel Deaconess Medical Center (BIDMC), Boston, USA, consecutive patients were enrolled over a 1-year period (internal validation set) and at Aarhus University Hospital (AUH), Aarhus, Denmark, a case-control study was performed (external validation set). Laboratory and clinical data were collected at enrollment to assess organ dysfunction. Primary outcome was in-hospital mortality. Minor criteria (1 point each) Temperature 38.3 to 39.3°C Hypotension (systolic blood pressure <90 mmHg) White blood cell count >18,000 cells/mm3 Bands >5% (in our setting, immature cells >0.5%) Platelets <150,000 cells/mm3 Creatinine >2.0 mg/dl (177 µl/l) Figure 1(abstract P23) Receiver operating characteristics curve (ROC) for external validation of the bacteremia prediction rule, calculated using the total score. Figure 1(abstract P24) Table 1(abstract P24) Effect of number of organ dysfunctions on in-hospital mortality adjusted for age and Charlson Comorbidity score Number of organ dysfunctions Internal validation set in-hospital mortality External validation set in-hospital mortality Data presented as OR (95% CI). 9.3 (4.8 to 18.1) 18.0 (8.8 to 36.9) 50.5 (22.0 to 115.8) 39.0 (8.9 to 170.7) 3.1 (0.9 to 10.4) 7.3 (2.1 to 24.7) 33.6 (8.56 to 127.3) 45.0 (8.56 to 236.2) 285.9 (16.9 to 483.2) Figure 2(abstract P24) Logistic regression was performed to determine the independent mortality odds. Results: Four thousand, nine hundred and fifty-two patients were enrolled at BIDMC and 483 patients at AUH. Overall mortality rates were 4% and 11% with mean ages of 58 ± 21 and 69 ± 16 years, respectively. The mortality rate increased with increasing number of organ dysfunctions: BIDMC: 0 organ dysfunctions, 0.6% mortality; 1 dysfunction, 3.3%; 2 dysfunctions, 7.8%; 3 dysfunctions, 15.9%; and ≥4 dysfunctions, 34.3%; and AUH: 2.2%, 6.7%, 17%, 41%, and 57% mortality (Figure 1). The number of organ dysfunctions remained an independent predictor after adjustment for age and Charlson Index (Table 1). The AUCs for the models were 0.82 and 0.87, respectively (Figure 2). The effect of specific types of organ dysfunction on mortality was largest for respiratory dysfunction (OR 3.57 (95% CI 2.5 to 5.1)) in the internal and for Table 2(abstract P24) Effect of organ dysfunctions on mortality adjusted for age and Charlson Index Cardiovascular 3.2 (0.9 to 11.5) Data presented as OR (95% CI). 29.0 (7.1 to 116.9) 18.5 (7.3 to 46.8) 6.7 (2.9 to 11.2) 8.9 (4.7 to 17.1) 5.4 (2.7 to 10.9) 4.5 (1.1 to 18.2) hematologic dysfunction (OR 33.57 (8.56 to 127.3)) in the external validation set (Table 2). Conclusions: Using readily available criteria in the ED to assess the number of organ dysfunctions is a reliable tool in predicting in-hospital mortality in both validation sets and could assist in risk prognostication and aid with earlier, targeted therapy. Background: Physiologic instability (PI) is a common, critical problem in the emergency department (ED) [1,2], and can have different underlying causes. The ability to determine the underlying cause of instability is paramount for early treatment and risk stratification [3]. Lactate has been shown to have prognostic value in some categories of unstable patients [4,5]. The objective of this study was to investigate how serum lactate concentrations differ across categories of PI and the association of lactate concentrations with clinical deterioration for each category. Materials and methods: A prospective observational study of adult patients with PI at a university ED. PI was defined as lactate ≥4 mmol/l, or >5 minutes of heart rate (HR) ≥130, or respiratory rate (RR) ≥24, or shock index ≥1, or systolic blood pressure ≤90 mmHg. We excluded patients with no lactate measurements, isolated atrial tachycardia, seizure, intoxication, psychiatric agitation, or tachycardia due to pain. A physician retrospectively Figure 1(abstract P25) Lactate levels across groups of physiological instability. Figure 2(abstract P25) Levels of lactate across groups of physiologic instability stratified by deterioration. no/yes = deterioration present or not. (*) Significant differences between groups (p < 0.05). categorized PI cause. Categories were defined as septic, cardiogenic, hemorrhagic, hypovolemic, or other. The primary outcome was deterioration, defined as: acute renal failure (elevated creatinine to ≥2× baseline levels), intubation, vasopressors, or in-hospital mortality. Results: We identified 1,156 patients with PI and excluded 324. Of the remaining, 304 did not have lactate measurements, leaving 528 for the analysis: 302 septic, 46 cardiogenic, 29 hemorrhagic, 57 hypovolemic, and 94 with another cause of instability. The differences in lactate levels between groups were not statistically significant (Figure 1). The lactate levels were statistically different between patients who deteriorated when compared with patients who did not deteriorate in the sepsis group (3.05 mmol/l vs. 1.91 mmol/l, P < 0.0001) and the other group (2.89 mmol/l vs. 1.94 mmol/l, P = 0.002). No statistically significant differences were demonstrated for the cardiogenic, the hemorrhagic or the hypovolemic groups (Figure 2). Conclusions: Lactate levels were not significantly different between the five groups with PI. However, in patients in the sepsis or other group, elevated lactate predicted deterioration. This was not demonstrated for the other causes of PI. This study suggests that in unstable patients lactate has the same likelihood of elevation between different causes of instability, but it may not have the same prognostic value for deterioration across underlying causes. Acknowledgements: CONSIDER Sepsis Network is a collaboration of clinical researchers with an interest in sepsis at Aarhus University Hospital, Aarhus, Denmark. References 1. Jones AE, Aborn LS, Kline JA: Severity of emergency department hypotension predicts adverse hospital outcome. Shock 2004, 22:410-414. 2. Jones AE, Stiell IG, Nesbitt LP, Spaite DW, Hasan N, Watts BA, Kline JA: Nontraumatic out-of-hospital hypotension predicts inhospital mortality. Ann Emerg Med 2004, 43:106-113. 3. Sebat F, Musthafa Aa, Johnson D, Kramer Aa, Shoffner D, Eliason M, Henry K, Spurlock B: Effect of a rapid response system for patients in shock on time to treatment and mortality during 5 years. Crit Care Med 2007, 35:2568-2575. 4. Shapiro NI, Howell MD, Talmor D, Nathanson LA, Wolfe RE, Weiss JW: Serum lactate as a predictor of mortality in emergency department patients with infection. 2005, 45:524-528. 5. Vermeulen RP, Hoekstra M, Nijsten MW, van der Horst IC, van Pelt LJ, Jessurun Ga, Jaarsma T, Zijlstra F, van den Heuvel AF: Clinical correlates of arterial lactate levels in patients with ST-segment elevation Background: Sepsis is the main cause of death in ICUs all over the world. Early detection of the pathogen is essential for appropriate antimicrobial treatment. Materials and methods: To evaluate the reduction in time of antimicrobial adjustment therapy in patients with sepsis comparing a rapid molecular test (SeptiFast®) with conventional blood cultures, a randomized controlled clinical trial was conducted between October 2012 and May 2013 in a cardiology hospital. Adult patients staying more than 48 hours in hospital with clinical suspicion of sepsis were included in the study. Blood samples were collected for cultures (BacT/ALERT®) and Septifast® test immediately prior to initiation of antibiotic therapy. Patients were allocated into two groups. In the Intervention Group (GI), Septifast® results were communicated to the medical researcher and antimicrobials were adjusted. In the Control Group (GII), Septifast® results were not informed and therapy adjustment was based on the blood culture. Registered in Clinical trials.gov (NCT 01450358). Results: Forty-six patients were included, 17 in GI and 29 in GII. Key data are shown in Table 1. In GI therapy adjustment was done in 580 minutes compared with 3,007 minutes in GII (P = 0.004). Conclusions: The rapid molecular test (SeptiFast®) reduced the time for adjustment of antibiotic treatment in comparison with conventional blood cultures in critically ill sepsis patients. Table 1(abstract P26) Distribution of characteristics in the two groups Intervention group (n= 17) Control group (n= 29) Hospital stay (mean, days) Ejection fraction <40% Receiving antibiotics prior to study Patients with adjustment therapy based on SeptiFast® Patients with adjustment therapy based on blood culture Pathogens detected in SeptiFast® Pathogens detected in blood culture P. aeruginosa (2), K. pneumonia/oxytoca (1), E. aerogenes/cloacae (1), S. marcescens (1), A. baumanii (1) P27 Comparison between inflammatory biomarkers procalcitonin, IL-6 and C-reactive protein for infection diagnosis and fever evolution in neutropenic patients, submitted to hematopoietic stem cell transplantation Karin Schmidt Rodrigues Massaro1*, Rodrigo Macedo2, Maria Aparecida Shikanai-Yasuda3, Silvia Figueiredo Costa3 1Department of the School of Medicine of Universidade de São Paulo, Brazil; 2Fanem Ltd, Brazil, São Paulo, Brazil; 3Infectious and Parasitology Diseases, Department of the School of Medicine of Universidade de São Paulo, Brazil Critical Care 2013, 17(Suppl 4):P27; doi:10.1186/cc12927 Background: Biomarkers were assessed during neutropenic fever in hematopoietic stem cell transplantation (HSCT). The objective was to assess serum values of C-reactive protein (CRP), procalcitonin (PCT) and IL-6 to identify infection in HSCT and risk factors for death. Materials and methods: Prospective study with 296 patients submitted to autologous or allogeneic HSCT. PCT, CRP and IL-6 dosed at the following moments: afebrile neutropenia, fever, 24 hours upon fever, 72 hours upon fever and long-lasting fever. Patients were classified into groups (I, afebrile; II, fever of unknown origin; and III, clinically or microbiologically proven fever). ROC curves, sensitivity, specificity, and multivariate analysis were used to evaluate factors associated with death. Results: One hundred and ninety patients had fever. Mean and median values of IL-6 at fever onset in group I with regard to group II (P = 0.013) presented significantly higher values. Levels of CRP in group I differed significantly from those found in group III (P < 0.05). Groups differed in levels of IL-6 and CRP at fever onset. Group II presented IL-6 and CRP concentrations significantly lower than group III. Cutoff values of PCT: fever onset, 24 hours upon fever, 72 hours of fever, and long-standing fever were: 0.32; 0.47; 0.46 and 0.35 µg/l. At fever onset, sensitivity was 52.3 and specificity 52.6 for infection diagnosis. Best cutoff values of CRP for fever onset, 24 hours upon fever, 72 hours upon fever and longstanding fever were: 79, 120, 108 and 72 mg/l. At fever onset, sensitivity was 55.4 and specificity was 55.1. Best cutoff values of IL-6 for fever onset, 24 hours upon fever, 72 hours upon fever and long-standing fever were: 34, 32, 16 and 9 pg/ml. At fever onset, sensitivity and specificity were: 59.8 and 59.7. In the autologous’ group, IL-6 presents significant values at initial moments. Independent risk factors identified in the multivariate analysis were: related donor, unrelated donor, Gram-negative infection, DHL ≥390 (UI/l), urea ≥25 (mg/dl) and CRP ≥120 (mg/l). Conclusions: IL-6 and CRP are associated with the early diagnosis of clinically or microbiologically confirmed infection in post-HSCT febrile neutropenia. The association of the three biomarkers did not present any advantage, nor did it improve diagnostic accuracy. IL-6 was the only biomarker significantly associated at an early stage with infection when assessed only in patients submitted to autologous HSCT. The independent variables associated with death were: allogeneic transplantation, Gramnegative infection, DHL ≥390 UI/l at fever onset and urea ≥25 mg/dl at fever onset and CRP ≥120 mg/l. P28 Severe pneumonia in critically ill cancer patients: clinical outcomes and a comparison between healthcare-associated pneumonia and community-acquired pneumonia Ligia SCF Rabello1*, Luciano CP Azevedo2, Ivens Augusto Oliveira de Souza2, Viviane Bogado Leite Torres1, Maíra M Rosolem3, Jose Roberto Lapa e Silva11, Marcio Soares1,4,5, Jorge IF Salluh1,4,5 1Postgraduate Program of Internal Medicine, Universidade Federal do Rio de Janeiro, Brazil; 2Hospital Sirio-Libanes, São Paulo, Brazil; 3Hospital Barra Dor, Rio de Janeiro, Brazil; 4D’Or Institute for Research and Education, Rio de Janeiro, Brazil; 5Postgraduate Program, Instituto Nacional de Câncer, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 4):P28; doi:10.1186/cc12928 Background: Pneumonia is the most frequent source of infection in cancer patients and accounts for 50% of all cases of septic shock. Frequently cancer patients attend a hospital for several treatments and an event of pneumonia in this scenario is called healthcare-associated pneumonia (HCAP) by current ATS/IDSA guidelines. The aims of this study were to describe a population of cancer patients with severe pneumonia (not acquired in the hospital setting) who required ICU admission; identify predictors of hospital mortality; and classify the study population based on ATS CAP/HCAP definitions providing a comparison of clinical data, microbiologic variables and outcomes between the two groups. Materials and methods: A prospective cohort study was performed from 2002 to 2011 at Instituto Nacional de Cancer and Hospital Sirio-Libanes, Brazil. Adult patients (>18 years) with a definite diagnosis of cancer and presenting with pneumonia (not acquired in the hospital setting) were evaluated at ICU admission. Demographic, clinical and laboratory data were collected during the first day of ICU including the CURB-65, the SAPS II, the SOFA score, comorbidities, Performance Status and cancerrelated and treatment-related data. Results: A total of 268 patients were admitted to the ICU with pneumonia and classified as CAP (n = 109/40.7%) and HCAP (n = 159/59.3%). There were 187 (69.8%) patients with solid tumors and 81 (30.2%) patients with hematological malignancies. One hundred and sixty-seven (62.3%) patients had septic shock at ICU admission. ICU and hospital mortality rates were 45.5% and 67.9%. When we compared CAP and HCAP populations, we observed similar characteristics and outcomes in both groups. As expected, higher severity of illness, organ failures, need for life-sustaining therapies and failure of NIV were associated with increased mortality. In a multivariate analysis, mechanical ventilation in the ICU (OR 2.52 (1.19 to 5.32)), dialysis in the ICU (OR 3.86 (1.23 to 12.10)) and higher severity of illness (SAPS2 per point OR 1.03 (1.01 to 1.05)) were associated with increased hospital mortality whereas successful noninvasive ventilation was associated with lower mortality (OR 0.32 (0.13 to 0.77)). The model showed good discrimination (AROC 0.832). Conclusions: We believe that cancer patients are a distinct group of patients with pneumonia regardless of HCAP or CAP classification. They have specific characteristics and predictors of outcome, and treatment should be based on their clinical characteristics and local microbiologic profiles. Background: Delirium is a common occurrence in critically ill patients and is associated with an increase in morbidity and mortality [1]. Some evidence suggests that septic patients with delirium may differ from a general critically ill population. In a subgroup analysis of the MENDS study, a benefit of dexmedetomidine sedation over lorazepam was only evident in septic patients [2]. The aim of our study was investigate the relationship between systemic inflammation and the development of delirium in septic and nonseptic critically ill patients. Materials and methods: We performed a cohort study in a 20-bed mixed ICU that included consecutive patients admitted for more than 24 hours. Delirium was diagnosed using the Confusion Assessment Method for the Intensive Care Unit (CAM-ICU). Coma was defined as a Richmond Agitation Sedation Scale (RASS) score of -4 or -5. Blood samples were collected within 12 hours of enrollment for determination of TNFa, soluble TNF receptor (STNFR)-1 and STNFR-2, IL-1b, IL-6, IL-10 and adiponectin. Results: Seventy-eight patients were included in the study: 26 nonseptic/ nondelirium (control), 13 nonseptic/delirium (delirium), 21 septic/ nondelirium (septic) and 18 septic/delirium (sepsis-associated delirium (SAD)). From all analyzed biomarkers only STNFR1, STNFR2 and adiponectin were independently associated with delirium occurrence, but none of these biomarkers had a significant interaction with sepsis. In contrast, there was significantly interaction between sepsis and IL-1b suggesting that this cytokine is differently modulated when comparing septic and nonseptic patients with delirium. P65 Burden of mortality related to sepsis in Brazil from 2002 to 2011 Luciano CP Azevedo1,2,3*, Leandro U Taniguchi1,2, Guilherme PP Schettino1, Cristiana M Toscano1, Ana L Bierrenbach1 1Instituto Sírio-Libanês de Ensino e Pesquisa, São Paulo, Brazil; 2State University of São Paulo, Brazil; 3Federal University of São Paulo, Brazil Critical Care 2013, 17(Suppl 4):P65; doi:10.1186/cc12964 Background: Sepsis represents a substantial healthcare burden. Limited epidemiologic information about the demography of sepsis mortality or about its temporal changes is available. Few population-based sources of data have been used to investigate the burden of sepsis-associated mortality on a national level. We investigated the epidemiology of sepsis deaths in Brazil from 2002 to 2011 using secondary data from the Brazilian Mortality Information System (Sistema de Informações de Mortalidade (SIM)). Materials and methods: A retrospective descriptive study using data reported to the Brazilian SIM for the years 2002 to 2011. SIM is an electronic, case-based mortality registry that derives its information almost entirely from death certificates. Sepsis-associated deaths from 2002 to 2011 were identified based on International Classification of Diseases 10th Revision codes listed on the underlying and on the contributing causes-ofdeath. Population-based sepsis-associated mortality rates and trends were estimated. In addition, age, gender, ethnicity, and outcome variables were assessed. Considering the cases of sepsis identified during the study period, annual population-based mortality rates were calculated using as denominators population estimates provided by the Brazilian Institute of Geography and Statistics with the 2010 census age-stratified population as the standard. Trends of mortality rates over time were explored with the chi-square test for trend. Rate changes were considered significant when P < 0.05. Results: The total number of deaths recorded in SIM increased over the decade. In 2002 there were 982,294 deaths reported and in 2010 this number was 1,133,761. The number of sepsis deaths increased from 95,972 (9.8%) to 186,712 (16.5%). The average age of sepsis-associated deaths progressively increased from 60.2 years in 2002 to 2003 to 67.1 years in 2010 to 2011. During the same period the average age of all deaths increased from 57.8 years to 62.7 years. White individuals were more frequent (60.4%), as compared with mixed race (24.4%) and blacks (6.6%). A substantial part of sepsis deaths occurred in the hospital (94.8%). The age-adjusted rate of sepsis-associated mortality increased from 69.5 deaths per 100,000 to 97.8 deaths per 100,000 from 2002 to 2010 (P < 0.001). Conclusions: Between 2002 and 2011, the contribution of sepsis to allcause mortality increased significantly in Brazil. Moreover, age-adjusted mortality by sepsis also augmented in the last decade. These numbers confirm the importance of sepsis as a significant healthcare issue in Brazil as well as the need for adequate strategies of early recognition and treatment. Joel Passos*, Paulo Pires, Sérgio Curvelo, Decio Netto, Bruna Passos, Background: Critical patients requiring prolonged intensive care are more at risk of being colonized by germs acquired in an ICU and present infections. The factors that contribute to the high rate of infection and mortality in ICUs are possibly associated with the severity of the underlying disease, invasive proceedings, the long period of hospitalization and use of antibiotics, especially the expanded spectrum, so that there are multidrug-resistant bacteria, which complicates therapy. Approximately 5% of patients admitted to ICUs will acquire a nosocomial infection, resulting in increased length of hospitalization, around 5 to 10 days, and will be considered a consequence of healthcare in 30% of cases. Diagnostic or therapeutic interventions provide breakdown of the mechanical barrier of the skin and mucus assigned to invasive, skin lesions caused by devitalization, trauma or by removing the skin secondary to burns or debridement. In addition to the mechanical factors that disrupt the natural barriers of defense, there are others that are inherent in clinical conditions of patients and promote the acquisition of infections in the hospital environment; the immune ability is compromised because the natural defense mechanisms are altered by the very nature base or as a result of therapeutic interventions. The rate of infection is high among intensive care patients, especially respiratory infections. Pseudomonas aeruginosa was the prevalent bacteria in our ICU. That is why the prevalence of infection acquired in the ICU is high and suggests that preventive measures are important to reduce the occurrence of infection in critical patients. Materials and methods: Retrospective study, analyzing culture results for 1 year in a ICU with 10 beds in the northern of state of Rio de Janeiro. We considered cultures of urine, blood, cerebrospinal fluid, tracheal aspirate, nasal swabs and catheter tip, and detected the most prevalent microorganisms in our ICU. Results: We analyzed 453 cultures, 178 (39.29%) were positive for some germ, 240 (52.98%) were negative and 35 (7.72%) had impaired analysis. Among the cultures were performed 152 blood cultures, 38 (25%) positive and 114 (75%) negative, 96 urine cultures, 36 (52.17%) positive and 60 (47.83) negative, 31 samples of tracheal secretions, 20 (64.51%) positive and 11 negative (35.49%), 141 nasal swabs, 71 (50.35%) positive and 70 negative (49.65%), and 27 cultures from the catheter tip, six (22.22%) positive and 21 (77.78%) negative. Among the positive blood cultures assayed as being prevalent was 31.57% with P. aeruginosa, the second Staphylococcus aureus and Proteus mirabilis with the same number of specimens, 15.78%. Among the 36 positive urine cultures, Candida albicans was the prevalent with 22.22%, second place was 13.88% Escherichia coli and P. mirabilis was third with 11.11%. The cultures were tracheal P. aeruginosa as the most prevalent in half of the cases (50%), and secondly C. albicans and Acinetobacter baumannii at 10%. Among the cultures of nasal swab taken on admission of patients, the prevalent germ was P. aeruginosa with 26.76%, in second place with 12.67% was P. mirabilis and third with the same number of cases were A. baumannii and Serratia marcescens, 11.26%. Among the catheter tip cultures, P. aeruginosa was prevalent with 40%, and P. mirabilis second with 20%. There no was positive cerebrospinal fluid culture in the period. Conclusions: This study contributes to the knowledge of local resistance rates, which is one of the basic steps for the establishment of individualized strategies regarding the use of antimicrobials. Acknowledgements: We are thankful to the Director, Laboratory and Committee of the Hospital Infection Control from Hospital Unimed Costa do Sol, whose contributions have made this study possible. Background: Sepsis score classifications increase conditionally with concurrent systemic inflammatory response syndrome (SIRS) score, Sequential Organ Failure Assessment score, and clinical intervention. However, hierarchical criteria fail to accurately classify sepsis when related physiological manifestations are resolved, while the underlying infection remains. Materials and methods: To enable hour-to-hour sepsis classification, we examined the diagnostic performance of a continuous sepsis score. We identified 36 adult patients in the Christchurch Hospital ICU with sepsis from a patient database. A severe sepsis biomarker was developed from model-based insulin sensitivity, temperature, heart rate, respiratory rate, blood pressures, and SIRS score. Sepsis and nonsepsis patient-hours were categorized by the ACCP/SCCM guidelines, where each category was scored independently, rather than hierarchically. Kernel density estimates were used to classify severe sepsis (including septic shock) of 1,690 hours over 6,550 total hours. Optimal diagnostic performance from the receiver operating characteristic (ROC) curve was determined for in-sample, out-ofsample, and overall estimates. Results: The severe sepsis biomarker achieved 86% sensitivity (81 to 94%), 85% specificity (80 to 95%), 0.93 (0.88 to 0.99) area under the ROC curve, 8.2 (4.0 to 19.0) positive likelihood ratio, 0.17 (0.06 to 0.23) negative likelihood ratio, 68% (58 to 87%) positive predictive value, 94% (92 to 98%) negative predictive value, and a diagnostic odds ratio of 116 (17 to 308) at an optimal probability cutoff value of 0.25. Conclusions: This clinical biomarker can thus be readily assessed at the bedside to yield a non-invasive and continuous estimate of the probability of severe sepsis. The results show high accuracy as a potential severe sepsis diagnostic and monitoring response to sepsis interventions in real time. P68 Potential anti-inflammatory role of 2-chloroadenosine treatment during acute lung inflammation in BALB/c mice suffering from Klebsiella pneumoniae B5055-induced acute lung infection Vijay Kumar*, Kusum Harjai, Sanjay Chhibber Department of Microbiology, Panjab University, Chandigarh, India Critical Care 2013, 17(Suppl 4):P68; doi:10.1186/cc12967 Background: Acute lung inflammation (ALI) is a life-threatening pathology and can develop during the course of several clinical conditions such as pneumonia, acid aspiration or sepsis. Adenosine plays a significant role in controlling acute inflammation via binding to A2A receptors on inflammatory cells; that is, neutrophils or macrophages. The present study was designed to evaluate the anti-inflammatory and immunomodulatory effects of 2-chloroadenosine (2-CADO), alone or in combination with amoxicillin/clavulanic acid (AMC), in Klebsiella pneumoniae B5055-induced acute lung infection in mice. Materials and methods: Acute lung infection in mice was induced by directly instilling the selected dose (104 colony-forming units/ml) of bacteria intranasally. Histopathological examination of the lungs was performed to reveal neutrophil infiltration into the lung alveoli. In addition to the major proinflammatory cytokines TNFa and IL-1a, levels of the anti-inflammatory cytokine IL-10 were also determined by ELISA. Results: Intranasal instillation of bacteria caused profound neutrophil infiltration into the lung alveoli as well as a significant increase in the levels of proinflammatory mediators (that is, TNFa and IL-1a). However, intravenous administration of 2-CADO 10 μg/kg/day, alone or in combination with an antibiotic (that is, AMC 20 μg/ml/day i.p. 1 day after establishment of infection), significantly decreased neutrophil infiltration into the lung alveoli. A significant decrease in TNFa and IL-1a along with elevation of IL-10 levels in the lung homogenate of mice with acute lung infection was observed upon treatment with 2CADO alone, with no significant decrease in bacterial counts. Moreover in combination with AMC, 2-CADO exhibited its immunomodulatory action in acute lung infection and prevented ALI observed during acute bacterial pulmonary infection, whilst an antibacterial action was exhibited by AMC. Conclusions: 2-CADO proved a potent immunomodulatory agent during acute Gram-negative bacteria-induced ALI and exhibited its antiinflammatory and immunomodulatory potential even in the presence of antibiotics. Thus, it has a potential to be used as an adjunct immunomodulatory agent during acute inflammatory conditions like ALI or sepsis. Background: Fluid replacement has been a usually recommended maneuver in sepsis; however, growing clinical controversies in the management of critically ill patients with severe sepsis have questioned its benefit. Herein, we evaluated the effect of a rapid hyperhydration (HH) therapy in varying stages of sepsis. Materials and methods: Wistar-EPM rats, weighing 200 to 250 g, were submitted to two sepsis models: S8 group, submitted to 2 ml Escherichia coli 108 CFU/ml intravenous (i.v.) inoculation, LD60, or S9 group, with E. coli 109 CFU/ml inoculation, LD80. Both groups were treated with HH (30 ml/kg of Ringer lactate i.v., in 20 minutes) in the early (E30 minute) and late (L6 hour) phases of sepsis. The mortality was followed up to 30 days (n = 6/group) and the splanchnic microcirculation was monitored by sidestream dark field imaging (SDF) video microscopy at 6-hour and 24-hour periods (n = 3/group/period). Results: The HH at the E30 minute phase of S8 improved the survival rate from 40% to 90%, and L6 hour phase HH promoted an 80% survival rate. Besides, the survival rate in S9 (LD80), with E30 minute HH, improved the survival rate from 20% to 50%. However, it was less effective as compared with the E6H phase HH, which resulted in an expressive survival rate (from 20% to 70%). These intriguing results suggested that there is an interdependent and time-dependent pathophysiology feature within the host response based on sepsis severity stage and a rapid high-volume reposition. The SDF analysis in control sepsis groups (S8 and S9), without fluid therapy, showed a broadly distributed microcirculation dysfunction in the liver lobules and kidney tubules at 6 hours after sepsis challenge, and such findings were similar between groups, but after 24 hours the survivors showed an improved microcirculation hemodynamic pattern and it was more evident in the S8 group. The survivals of the S8 E30 minute treated group showed less injury at 6 hours and 24 hours as compared with nontreated groups and S8 L6 hour treated animals. In S9 treated groups, both showed a partial repair at 24 hours post sepsis. Conclusions: The hyperfluid therapy given rapidly in both early and late phases in sepsis and severe sepsis states showed that its beneficial effect was more or less effective dependent on the phase and sepsis intensity; however, the more prominent survival rates were seen at the early phase of sepsis (S8) and at the later phase of severe sepsis (S9). The underlying pathophysiology evolved in these paradoxical conditions needs to be better elucidated. Acknowledgements: Grant number 2012/20841-7, São Paulo Research Foundation (FAPESP). Background: A positive blood culture (BC) is considered the gold standard method for the sepsis diagnosis, although its sensibility is low (10 to 30%) which demands a better diagnostic tool to limit broadspectrum antibiotic use in the majority of patients without culture-based sepsis diagnosis. Besides, after microbial invasion, they can remain live, dead or fragmented in the bloodstream, thus limiting BC efficiency. Herein we evaluated the PCR diagnostic efficacy under live, dead and bacterial DNA contents in the bloodstream. Materials and methods: Wistar rats were distributed in three groups (n = 20/group) based on live, dead and DNA inoculations. The LPS+DNA group (1 mg/kg LPS injection plus 4 hours later DNA injection, n = 10) was designed for DNA detection under an induced inflammatory state. Live, Dead and extracted DNA forms of Pseudomonas aeruginosa (ATCC 27853) relative to 2 ml of 107 colony-forming units/ml were injected into the circulation. Blood samples were collected after 20 minutes and 6 hours (n = 10/group/period), and were submitted to nested PCR assay using general and specific primers. BC was performed with 200 μl and 3,000 μl only in the Live group. Results: In the Live group, at 20 minutes the sensibility was 100% by both BC and PCR and at 6 hours the sensitivity was 60% (with 200 μl) and 90% (with 3,000 μl) in BC, and 80% in PCR sampled with 50 μl blood volume. In the Dead group, the PCR sensitivity was 90% at 20 minutes and 50% at 6 hours. In the DNA group, the sensitivity remained at 50% independent of time. The inflamed condition did not change PCR sensitivity. Overall data showed that in both techniques the sensitivity dropped with time. In the BC assay the positivity was dependent on sampled blood volume, and in the PCR it was related to live or dead condition. These findings suggest that the live bacteria remain for a short period of time in the bloodstream while DNA can last for longer periods. Conclusions: Considering that PCR is performed with 40× less blood compared with a habitual BC, PCR can be an assay of choice when BC is negative and in conjunction in a live bacteria circulating condition. Besides, the PCR assay with specific primers can be a useful method for sepsis diagnosis in specific bacterial surge events in the ICU, thus improving antibiotic usage potentials. Mayara Andrade Ferrari*, Marcelo Eduardo Batalhão, Evelin Capellari Cárnio Background: Prior exposure to infection, particularly during the neonatal phase, contributes to individual differences in susceptibility to disease during adult life. Animal neonates undergoing lipopolysaccharide administration (LPS) react differently to the front endotoxemia in adulthood. Ghrelin, a peptide hormone originally found in the stomach, has effects on the modulation of the inflammatory response. Specific receptors are found for ghrelin on neutrophils, macrophages and lymphocytes and their activation by ghrelin inhibits the production of several inflammatory cytokines, including nitric oxide (NO). Therefore, our objective is to evaluate the role of ghrelin in the attenuation of fever during endotoxemia in adulthood induced by neonatal exposure to LPS. Materials and methods: The study was conducted using rats in the pregnancy period. After the birth of pups (day 0 of the experiment) we selected only male rats. All animals were weaned at 21 days and at 14 days of age received neonatal administration of LPS 100 μg/kg intraperitoneally (i.p.). Subsequently they were separate in cages until they reached 8 to 12 weeks of age for the experiment (by endotoxemia in adult administration of 10 mg/kg LPS i.p.). To determine the body temperature, the animals were anesthetized and a capsule inserted into the peritoneal cavity biotelemetry. Body temperature was measured for a period of 6 hours after induction of endotoxemia. To verify the effect of ghrelin and ghrelin antagonist on body temperature during endotoxemia, ghrelin was administered 0.1 mg/kg ghrelin antagonist or 50 nmol/kg i.p. concomitant administration of LPS. After decapitation, blood samples were collected and centrifuged to separate the plasma. The plasma was stored at -70°C for subsequent determination of NO. Results: In our preliminary data we observed no significant difference in fever-induced endotoxemia in animals subjected to LPS administration in the neonatal period, when compared with their respective controls. Conclusions: These data do not corroborate the findings of the literature and we believe it is due to the fact that the animals used until now have had prior exposure to pathogens. So in our next experiments we will use experimental animals that are specific pathogen free. P72 Interruption of the intestinal immune route to the systemic circulation associated with early hyperhydration minimized splanchnic microcirculation damage and improved sepsis survival Fernando M Dulcini1*, Ana MA Liberatore2, Bianca C Zychar1, Ivan HJ Koh1 1Department of Surgery, Federal University of São Paulo, Brazil; 2Department of Pediatrics, Federal University of São Paulo, Brazil Critical Care 2013, 17(Suppl 4):P72; doi:10.1186/cc12971 Background: Considering that the communication of the intestinal immunity with the systemic bloodstream can be a relevant adjuvant factor in the amplification of the host systemic inflammatory response and subsequent multiple organ dysfunction in sepsis, we aimed to evaluate the effect of the obstruction of the mesenteric lymph duct (OMLD) associated with massive fluid therapy in the early phase of sepsis and severe sepsis models. Materials and methods: Adult Wistar-EPM rats were submitted to 108 (S8) or 109 (S9) CFU/ml Escherichia coli inoculum intravenously (i.v.) (DL80 within 26 hours), and were treated with hyperhydration (HH) with or without previous OMLD (n = 5/group). Control group were naïve animals (N) and animals submitted to HH or sepsis only. The mortality of groups was followed up to 30 days after experiments and microcirculation monitoring was observed at 6 hours post sepsis induction by videomicroscopy (sidestream darkfield imaging (SDF)). Results: The effect of OMLD + HH reduced significantly the sepsis mortality rate: S8 (60% to 14.5%) and S9 (80% to 60%). Besides, the liver and kidney microcirculatory features were better preserved as compared with untreated sepsis groups under video-microscopy (SDF) monitoring. (Figure 1). Conclusions: These preliminary findings showed that both HH and OMLD have a potential therapeutic application in sepsis by minimizing the splanchnic organ’s microcirculation dysfunction. Acknowledgements: Grant number 2011/204014, São Paulo Research Foundation (FAPESP). Background: Recent studies from our laboratory showed that animals subjected to 50% shortening of the small intestine developed bacterial translocation unleashed chronically. Bacterial translocation has shown the effect of exacerbation of systemic inflammatory response by crosstalk between intestinal and systemic immune response. In this sense, the aim of this study was to evaluate whether a septic challenge in the state of chronic inflammation resulting from the shortening of the small bowel can modify the mortality outcome and trigger organ alterations in the long term. Materials and methods: Wistar-EPM rats were submitted to 50% small intestine shortening (IS group, n = 20) or sham intestinal anastomosis (IA group, n = 20), and after 4 months were submitted to sepsis challenge with 2 ml 108CFU/ml Escherichia coli i.v. The mortality was observed up to 30 days and the survivors of both groups were killed after 6 months for histological analysis. The other 10 animals were killed after 4 months of intestinal shortening in order to determine the histological pattern related to the bowel shortening effect. Results: The mortality rate after sepsis was 80% in the IS group and 35% in the IA group. The bowel shortening without sepsis challenge showed hepatic mild steatosis with inflammation similar to acute hepatitis, vascular congestion and focal necrosis. The distal ileum showed shortening and broadening of villus, focal cryptic necrosis and mild macrophages and eosinophil infiltration in the lamina propria. In the IS group was seen a generalized steatosis and vascular congestion in the liver; alveolar atelectasis, BALT hyperplasia, a large number of macrophages, mast cells, foam cells, lymphocytes, eosinophil and plasmocyte infiltration and alveolar edema, plus vascular congestion and sclerosis in the lung; villus apical necrosis, intense inflammatory cell infiltration and vascular congestion in the lamina propria of the ileum; and the kidney with tubular nephrosis, tubular obstruction, vascular congestion with interstitium hemorrhage and tubular hyaline material deposition. In the IA group was seen moderate liver steatosis, intestinal lamina propria cellular infiltrations, glomerulonephritis, kidney tubular edema, parenchymal hemorrhage and Figure 1(abstract P72) Splanchnic organ’s microcirculation following 6 hours after sepsis or HH procedures by SDF monitoring: (a) HH; (b) S8 + OMLD + HH; (c) S9 + OMLD + HH. Bowman capsule thickness. However, the alterations were less compared with the IS group. Conclusions: The chronic inflammatory state, in combination with sepsis, might be an important aggravating factor related to sepsis P74 Cholecystokinin inhibits inducible nitric oxide synthase expression in lipopolysaccharide-stimulated macrophages Evelin C Carnio1*, Luiz GS Branco2, Rafael S Saia3 1Escola de Enfermagem de Ribeirão Preto - USP, Brazil; 2Faculdade de Odontologia de Ribeirão Preto - USP, Brazil; 3Faculdade de Medicina de Ribeirão Preto - USP, Brazil Critical Care 2013, 17(Suppl 4):P74; doi:10.1186/cc12973 Background: Cholecystokinin (CCK) receptors are expressed in macrophages and are upregulated by inflammatory stimulus. In vitro and in vivo studies have demonstrated the ability of CCK to decrease the production of various proinflammatory cytokines. This study investigates the role of CCK on iNOS expression in lipopolysaccharide (LPS)-activated peritoneal macrophages, as well as the intracellular signaling pathways involved in affecting iNOS synthesis. Materials and methods: Experimental procedures were approved by the Comitê de Ética em Experimentação Animal - FMRP (protocol number 152/ 2009). Thioglicollate-elicited macrophages were obtained by peritoneal lavage and cultured in RPMI 1640 medium, 10% fetal bovine serum and antibiotics. Nuclear p65, cAMP and iNOS levels were determined using ELISA kits, CCK receptors and I Ba expression by western blot and nitrite by the Griess method. Data were compared by one-way ANOVA and significant differences obtained using the Tukey multiple variances post hoc test. Results: CCK reduced NO production attenuating iNOS mRNA expression (15.49 ± 10.80 vs. 113.16 ± 0.23 AU; P < 0.05) and protein formation. Furthermore, CCK inhibited the NF- B pathway reducing I Ba degradation and minor p65-dependent translocation to the nucleus (543.78 ± 84.57 vs. 90.42 ± 9.13%, P < 0.05). Moreover, CCK restored the intracellular cAMP content activating the cAMP-protein kinase A (PKA) pathway, which resulted in a negative modulatory role on iNOS expression and nitrite production. In peritoneal macrophages, the CCK-1R expression was predominant and upregulated by LPS (0.61 ± 0.08 vs. 0.30 ± 0.09 AU; P < 0.05). The pharmacological studies confirmed that CCK-1R subtype is the major receptor responsible for the biological effects of CCK. Conclusions: These data suggest an anti-inflammatory role for the peptide CCK in modulating iNOS-derived NO synthesis, possibly controlling the macrophage hyper-activation through NF- B, cAMP-PKA and CCK-1R pathways. Acknowledgements: Fapesp and CNPq. P75 CD11b and TLR4 in human neutrophil priming by endotoxins from Escherichia coli Isabella Prokhorenko*, Dimitry Kabanov, Svetlana Zubova, Sergay Grachev Institute of Basic Biological Problems, Pushchino, Moscow Region, Russia Critical Care 2013, 17(Suppl 4):P75; doi:10.1186/cc12974 Background: The interaction of endotoxins (lipopolysaccharides (LPS)) from Gram-negative bacteria with peripheral blood mononuclear cells leads to the assembly of a receptor cluster composed from mCD14, CD11b/CD18, TLR4, CD16A and CD36 [1,2]. It is well known that the main signal transducing receptor complex is TLR4/MD-2 while mCD14 is involved in the recognition of S or R endotoxin’s glycoforms [3,4]. A growing body of evidence indicates that the CD11b/CD18 receptor plays the significant role in the endotoxin signaling machinery because it can influence TLR4mediated cell activation [5]. So, using mAbs, we carried out experiments to elucidate the influence of CD11b inhibition on neutrophil priming by endotoxins for N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced respiratory burst. Materials and methods: Human neutrophils were isolated from heparinized blood of healthy volunteers by standard procedure and incubated with or without anti-TLR4 mAbs (HTA125, IgG2a) or anti-CD11b mAbs (clone 44, IgG1) or isotypic immunoglobulin controls, respectively, for 30 minutes before stimulation with S-LPS or Re-LPS from Escherichia coli O55: B5 or JM103, respectively. The cells (2 × 105), 2% of autologous serum, glucose and luminol in Ca2+-PBS buffer (pH 7.3), were placed in the chemiluminometer’s chambers (37°C) and primed by S-LPS or Re-LPS (100 ng/ml) for 30 minutes (37°C). Reactive oxygen species (ROS) production was triggered by addition of fMLP (1 µM). The chemiluminescence reaction was monitored continuously for 7 minutes. Total ROS production by control Introduction: Attention has been paid in recent years to studies showing immune cell death mechanisms during the course of sepsis in response to proinflammatory and anti-inflammatory mediators that are involved in its pathophysiology. Taurine (Tau) is an abundant amino acid in polymorphonuclear leucocytes that reacts with hypochlorous acid to form taurine chloramine (TauCl) under inflammatory conditions. In this context, we investigated potential interactions between lymphocytes and TauCl in rats submitted to cecal ligation and perforation (CLP), analyzing cell viability and cytokine secretion profile (TNFa, IFNg, IL-6, IL-17A, IL-23 and IL-10). Materials and methods: Adult male rats were divided in two groups: sham and CLP that were killed 24 or 120 hours after sepsis induction to isolate lymphocytes from the blood and spleen. Lymphocytes (>95.0% purity determined by differentiation with Giemsa staining) were cultured Figure 1(abstract P75) for 24 hours at a concentration of 1 × 106 cells/ml and activated by 2 mg/ml concanavalin A. After 24 hours, Tau and TauCl were added at concentrations of 0.1, 0.2, 0.3, 0.4 and 0.5 mM for 1 hour. After this time, cells were incubated with MTT (500 μg/ml) for 3 hours to evaluate cell viability and supernatants were used to determine cytokine concentrations. Results: Tau-treated cells exhibited better viability than those treated with TauCl, in both time and organs. TauCl, in a time and dosedependent ratio, decreased cytokines secretion when compared with untreated cells. See Figures 1 to 7. Conclusion: These findings show a possible impairment in lymphocyte function promoted by TauCl, correlated with immunosuppression and cell death characteristic of the late stages of sepsis. Figure 1(abstract P76) Cell viability by MTT assay. Viability of lymphocytes treated with Tau and TauCl in different molar concentrations. Rats were submitted to CLP or Sham, and 24 or 120 hours after the surgery their blood and spleens were collected, the lymphocytes were isolated, cultured and the cell viability was measured by MTT assay. (A) blood, 24 hours; (B) blood, 120 hours; (C) spleen, 24 hours; (D) spleen, 120 hours. *P < 0.05, compared with sham group (Tau-treated); #P < 0.05, compared with sham group (TauCl-treated), n = 5. Figure 2(abstract P76) Cytokine secretion. Effect of TauCl on production of proinflammatory mediator IL-17A by Th17 lymphocytes. Activated lymphocytes (1 × 106 cells/ml) were preincubated with TauCl (0.1 or 0.5 mM) for 1 hour. After this, supernatants were collected and IL-17A was measured by ELISA. (A) blood; (B) spleen. Results are expressed as means ± SD. *Compared with sham control 24 hours; #compared with Clp control 24 hours; &compared with sham control 120 hours; $compared with Clp control 120 hours, all with P < 0.05 significant (n = 5). Figure 3(abstract P76) Effect of TauCl on production of proinflammatory mediator IL-23 by Th17 lymphocytes. Activated lymphocytes (1 × 106 cells/ml) were preincubated with TauCl (0.1 or 0.5 mM) for 1 hour. After this, supernatants were collected and IL-23 was measured by ELISA. (A) blood; (B) spleen. Results are expressed as means ± SD. *Compared with sham control 24 hours; #compared with Clp control 24 hours; &compared with sham control 120 hours; $compared with Clp control 120 hours, all with P < 0.05 significant (n = 5). Figure 4(abstract P76) Effect of TauCl on production of proinflammatory mediator IFNg by Th1 lymphocytes. Activated lymphocytes (1 × 106 cells/ml) were preincubated with TauCl (0.1 or 0.5 mM) for 1 hour. After this, supernatants were collected and IFNg was measured by ELISA. (A) blood; (B) spleen. Results are expressed as means ± SD. *Compared with sham control 24 hours; #compared with Clp control 24 hours; &compared with sham control 120 hours; $compared with Clp control 120 hours, all with P < 0.05 significant (n = 5). Figure 5(abstract P76) Effect of TauCl on production of proinflammatory mediator TNFa by Th1 lymphocytes. Activated lymphocytes (1 × 106 cells/ml) were preincubated with TauCl (0.1 or 0.5 mM) for 1 hour. After this, supernatants were collected and TNFa was measured by ELISA. (A) blood; (B) spleen. Results are expressed as means ± SD. *Compared with sham control 24 hours; #compared with Clp control 24 hours; &compared with sham control 120 hours; $compared with Clp control 120 hours, all with P < 0.05 significant (n = 5). Figure 6(abstract P76) Effect of TauCl on production of proinflammatory mediator IL-6 by Th2 lymphocytes. Activated lymphocytes (1 × 106 cells/ml) were preincubated with TauCl (0.1 or 0.5 mM) for 1 hour. After this, supernatants were collected and IL-6 was measured by ELISA. (A) blood; (B) spleen. Results are expressed as means ± SD. *Compared with sham control 24 hours; #compared with Clp control 24 hours; &compared with sham control 120 hours; $compared with Clp control 120 hours, all with P < 0.05 significant (n = 5). Background: Sepsis is a clinical condition resulting from the excessive inflammatory response of the host against an infectious agent and is associated with high morbidity and mortality in patients in ICUs. In sepsis the brain can be targeted, associated with mental damage and decline, impaired attention, disorientation, delirium and coma. It has been seen that the permeability of the blood-brain barrier (BBB) is associated with septic encephalopathy, allowing cell infiltration and increased oxidative stress. Accordingly, such events can be potentiated through the involvement of molecules that when activated perpetuate the inflammatory response and the breaking of the BBB, and it is possible to postulate that the CD40 molecule may be involved by being under increased expression in microglia in inflammatory events occurring systemically. The aim of this study therefore is to evaluate the role of CD40 in the breakdown of the BBB, cell infiltration and oxidative damage in the brain of rats with sepsis. Materials and methods: Male Wistar rats were subjected to cecal ligation and puncture (CLP) to induce sepsis. The animals (n = 10) were Figure 7(abstract P76) Effect of TauCl on production of anti-inflammatory mediator IL-10 by Th2 lymphocytes. Activated lymphocytes (1 × 106 cells/ml) were preincubated with TauCl (0.1 or 0.5 mM) for 1 hour. After this, supernatants were collected and IL-10 was measured by ELISA. (A) blood; (B) spleen. Results are expressed as means ± SD. *Compared with sham control 24 hours; #compared with Clp control 24 hours; &compared with sham control 120 hours; $compared with Clp control 120 hours, all with P < 0.05 significant (n = 5). divided into sham, CLP, CLP + 1 ng, CLP + 10 ng and CLP + 100 ng antiCD40 antibody administered intracerebroventricularly. The rats were killed at 24 hours for assessment of oxidative damage in lipids (TBARS), damage to proteins by protein carbonylation, nitrite/nitrate concentration (NO), myeloperoxidase (MPO) and breakdown of the BBB. The other group was subjected to CLP and after 24 hours they were killed and the hippocampus removed to analyse expression of CD40 and CD40L by western blotting. Data were evaluated by ANOVA and post-hoc Tukey test with significance P < 0.05. Results: Our results show that in the most effective dose of 100 ng/kg anti-CD40 showed a decrease in the breakdown of the BBB, MPO, nitrite/ nitrate concentration and TBARS. A dose of 1 ng/kg was effective only in the reduction of nitrite/nitrate concentration and 10 ng/kg was not effective in TBARS and carbonyl. Western blotting analysis showed increased expression of CD40 and CD40L in CLP animals when compared with sham. Conclusions: Modulation of the levels of CD40 may represent a potential therapeutic target in sepsis. Acknowledgements: CAPES, CNPq, UNESC and UNISUL. P78 Endotoxin induces conversion of endothelial cells into activated fibroblasts César Echeverría1*, Ignacio Montorfano1, Daniela Sarmiento1, Alvaro Becerra1, Claudio Cabello-Verrugio1, Felipe Simon1,2 1Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago, Chile; 2Millennium Institute on Immunology and Immunotherapy, Santiago, Chile Critical Care 2013, 17(Suppl 4):P78; doi:10.1186/cc12977 Background: Endothelial dysfunction is a key step in endotoxemiaderived sepsis syndrome pathogenesis. It is well accepted that the bacterial endotoxin lipopolysaccharide (LPS) induces endothelial cell (EC) dysfunction through immune system overactivation [1-3]. However, LPS can also affect ECs in the absence of participation by immune cells [4-6]. Although interactions between LPS and ECs evoke endothelial death, a significant portion of ECs are resistant to LPS challenge [6-8]. However, the mechanism that confers endothelial resistance to LPS is not known. Considering that LPS-resistant ECs exhibit a fibroblast-like morphology, suggesting that these ECs enter in a fibrotic program in response to LPS, our aim was to investigate whether LPS induces endothelial fibrosis and explore the underlying mechanism. Materials and methods: We used two different models: primary ECs, and intact blood vessels (IBV). Both preparations were freshly obtained from umbilical cord veins from normal pregnancies, after patients’ informed consent. The investigation conforms with the principles outlined in the Declaration of Helsinki. The Commission of Bioethics and Biosafety of Universidad Andres Bello approved all experimental protocols. Once the preparation was established they were cultured with or without LPS as a Sandra Crestani*, Jose Eduardo da Silva Santos, Jamil Assreuy Background: Calcium activity is essential to vascular smooth muscle contraction. Although it is well established that arteries from rats in septic shock present hyporesponsiveness to vasoconstrictor drugs, the role of calcium mobilization in this contractile dysfunction is far less investigated. We hypothesized that during septic shock calcium dynamics is changed and may have a role in the vascular dysfunction in sepsis. Materials and methods: Female Wistar rats (3 months old) were anesthetized by oxygen-isoflurane (3%) inhalation and subjected to cecal ligature and puncture surgery (CLP). Immediately after and every 12 hours rats received physiological saline solution (PBS 30 ml/kg, subcutaneously) and tramadol (5 mg/kg, subcutaneously). After 6 hours (CLP-6) or 24 hours (CLP-24) rats were killed, the aorta was harvested and cut in rings, the endothelium was removed and rings were mounted in baths. Rings were exposed to KCl 120 mM and phenylephrine (PE 1 µM). Aorta rings were kept in a modified depolarizing Krebs solution nominally Ca2+ free and contracted by CaCl2 (1 to 100 mM). The same protocol were repeated in presence of thapsigargin (3 µM), DTNB (100 µM) or PTIO (100 µM). Different vessels were exposed to single concentrations of PE (1 μM) or caffeine (20 mM) in Ca2+-free solution, in the presence or absence of thapsigargin. Results: Maximal contraction (Emax) induced by KCl or PE was reduced, especially in the CLP-24 group. Similarly, CaCl2-induced contraction was reduced (60%) in the CLP-24 group. Thapsigargin (sarcoplasmatic calcium reuptake blocker) and DTNB (sulphydryl oxidation) restored the contraction elicited by CaCl2 in septic rings, but without effect in control rings. PE-induced contraction in calcium-free solution was significantly reduced in CLP-24 rings (Emax 1.6 ± 0.4 g control vs. 0.3 ± 0.1 g CLP-24 rings). Thapsigargin did not change the hyporesponsiveness to PE but PTIO (nitric oxide scavenger) restored it partially. Caffeine-induced contraction in Ca2+-free solution was reduced in CLP-24 rings (0.2 ± 0.06 g control vs. 0.03 ± 0.01 g in CLP-24). Thapsigargin or PTIO restored the contraction induced by caffeine. Conclusions: These data suggest that in septic shock septic calcium mobilization is strongly impaired. Although preliminary, our results suggest that calcium channel nitrosylation and calcium reuptake may be reasons for the vascular hyporesponsiveness of septic shock. Acknowledgements: Financial support: CNPq. FINEP. FAPESC and CAPES. Background: Hypotension and cardiac dysfunction are frequently found in severe sepsis and septic shock. Vasoactive and inotropic drugs are largely used to reverse hypotension, but its effects on heart function have been scarcely investigated [1]. We thus evaluated the influence of both norepinephrine and dobutamine on the cardiovascular function of rats subjected to cecal ligation and puncture (CLP). Materials and methods: The measurement of the cardiac function was performed in male Wistar rats (3 to 4 months old), kept under isofluraneinduced anesthesia (1 to 3%), using a pressure-volume catheter, which was inserted into the left ventricle through the carotid artery. Blood samples were collected from all animals for hematological analyses. The experiments were conducted at 24 and 48 hours after CLP. For this, the cecum was ligated with a ratio of 50% and perforated with a needle (18 G, four holes; mortality rate ~50% after 48 hours), followed by four subcutaneous injections (12/12 hours) of sterile saline (30 ml/kg) and tramadol (5 mg/kg), for fluid resuscitation and analgesia, respectively. Data were recorded at baseline and after single bolus administration of norepinephrine (1, 3 and 10 nmol/kg, i.v.) or dobutamine (3, 10 and 30 nmol/kg, i.v.). The results obtained in CLP groups were compared with control (CT) animals, which did not undergo any manipulation. Results: Both CLP 24 and 48 hour groups presented thrombocytopenia (~40% reduction), lymphopenia, hypoglycemia and leukopenia (P < 0.05), a clear indication of severe sepsis. However, only CLP 48 hour animals displayed refractory hypotension (MAP = 59 mmHg, vs. 78 mmHg in CT; P < 0.05) in spite of volume resuscitation. The highest doses of norepinephrine and dobutamine increased the MAP to 133.8 ± 8.1 and 97.8 ± 3.1 mmHg in CT, and to 120.6 ± 6.7 and 77.3 ± 4.4 mmHg in CLP 48 hour animals, respectively. The heart rate was significantly increased by norepinephrine and dobutamine in control, but not in CLP 48 hour animals. In addition, the basal values of both dP/dtmax and dP/dtmin, as well as after 1 nmol/kg dobutamine, were reduced in CLP 48 hour animals. Conclusions: Using a pressure-volume catheter in a closed-chest approach we demonstrated that, in spite of the ability to increase blood pressure, the chronotropic effects of norepinephrine and dobutamine are reduced at 48 hours after CLP in rats subjected to CLP. In addition, all doses of norepinephrine, but only by the highest doses of dobutamine, improved systolic and diastolic function in these animals. Acknowledgements: CNPq and FAPESC (2012000367 and 2012000078). Reference 1. Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM, Sevransky JE, Sprung CL, Douglas IS, Jaeschke R, Osborn TM, Nunnally ME, Townsend SR, Reinhart K, Kleinpell RM, Angus DC, Deutschman CS, Machado FR, Rubenfeld GD, Webb S, Beale RJ, Vincent JL, Moreno R: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012. Intensive Care Med 2013, 39:165-228. P81 Increased sympathetic tone contributes to cardiovascular dysfunction in sepsis Ana Maria Favero1*, Regina Sordi1, Geisson Nardi2, Jamil Assreuy1 1Department of Pharmacology, Universidade Federal de Santa Catarina, Florianópolis, Brazil; 2Department of Biological Sciences and Health, Universidade do Oeste de Santa Catarina, Joaçaba, Brazil Critical Care 2013, 17(Suppl 4):P81; doi:10.1186/cc12980 Background: The cardiovascular dysfunction of sepsis/septic shock is characterized by hypotension, tachycardia/bradycardia, endothelial dysfunction and hyporesponsiveness to vasoconstrictors. Hypotension and low tissue perfusion trigger an increase in sympathetic tone probably as an attempt to restore blood pressure to normal levels. The persistently higher sympathetic stimulation may lead to the exhaustion of the capacity of vascular response and thus create a vicious circle contributing to vascular hyporesponsiveness and higher adrenergic stimulation. In addition, in septic shock patients, increased arterial levels of norepinephrine (NE) were significantly associated with mortality. The aim of this work was to evaluate the vascular response to an adrenergic agonist during severe sepsis and the effects of the early inhibition of sympathetic tone in sepsisinduced cardiovascular dysfunction. Materials and methods: Sepsis was induced by cecal ligation and puncture (CLP) surgery in female Wistar rats. Septic animals and controls (CT) were treated with the ganglionic blockers pentolinium (PENT; 5 mg/kg, s.c.) or hexamethonium (HEX, 15 mg/kg, s.c.) or vehicle (saline) 1 hour after surgery. The vascular response to the administration of NE was assessed 6 hours or 24 hours after CLP surgery. The survival rate was also evaluated. All procedures were approved by our Institutional Ethics Committee (PP00631/ CEUA-UFSC) and are in accordance with NIH Animal Care Guidelines. Results: Six hours after CLP surgery, septic animals were hypotensive. Treatment with hexamethonium or pentolinium prevented the development of hypotension (control 84.8 ± 2.6; CLP 60.7 ± 4.5*; CLP + HEX 72.7 ± 3.1; CLP + PENT 78.1 ± 2.7 mmHg; *P < 0.05 compared with control group). However, 24 hours after surgery, the ganglionic blockers failed to prevent hypotension (control 88.5 ± 1.7; CLP 62.7 ± 1.7*; CLP + HEX 68.8 ± 2.7*; CLP + PENT 70.9 ± 3.4* mmHg; *P < 0.05 compared with control group). The vascular hyporesponsiveness to NE observed both 6 hours and 24 hours after CLP was completely blocked by the early treatment with both ganglionic blockers (NE 10 nmol/kg, expressed as increase in blood pressure compared with baseline: control 54.2 ± 4.5; CLP 6 hours 21.9 ± 3.1*; CLP 6 hours + HEX 52.6 ± 7.0; CLP 6 hours + PENT 54.1 ± 4.9; CLP 24 hours 31.1 ± 5.6*; CLP 24 hours + HEX 74.6 ± 3.0; CLP 24 hours + PENT 64.4 ± 7.8 mmHg; *P < 0.05 compared with control group). The early ganglionic blockade with PENT decreased the mortality observed after 96 hours. Conclusions: Our data indicate that increased sympathetic tone in sepsis contributes, at least in part, to the development of hypotension, hyporesponsiveness to vasoactive agents and mortality. Blockade of increased sympathetic tone thus may be considered as an adjuvant therapy for the treatment of septic cardiovascular dysfunction. Acknowledgements: Financial support: CAPES, CNPq, FAPESC and FINEP. P82 Vascular smooth muscle cell activation depends on NOS-1-derived NO and consequent peroxynitrite generation Karin Scheschowitsch1*, Regina de Sordi1, João Alfredo de Moraes2, Christina Barja-Fidalgo2, Jamil Assreuy1 1Department of Pharmacology, UFSC, Florianópolis, SC, Brazil; 2Department of Pharmacology, UERJ, Rio de Janeiro, RJ, Brazil Critical Care 2013, 17(Suppl 4):P82; doi:10.1186/cc12981 Background: Low levels of nitric oxide (NO) play a key role in vascular tonus maintenance. Previous results from our laboratory show that hypotension and mortality during sepsis are prevented by the early administration of NOS-1 inhibitors. The aim of this study was thus to investigate the role of NOS-1 and NOS-3-derived NO and of other reactive oxygen species (ROS) in smooth muscle cell activation. Materials and methods: Smooth muscle cell line of rat aorta (A7r5) was used. Control cells and NOS-1 or NOS-3 silenced cells (siNOS-1 and siNOS-3, respectively) were stimulated with LPS 1 µg/ml and IFN 200 U/ml (LPS/IFN). NO and ROS production was assessed with fluorescent probes. NOS content was evaluated by western blot and NOS-2 activity was indirectly measured by Griess reaction. Further, control cells were treated for 30 minutes with a NO scavenger (c-PTIO), a NOS inhibitor (7-NI) or a NADPH oxidase inhibitor (DPI) before stimulation. Immunofluorescence was used to evaluate protein nitration and NF- B nuclear translocation. To confirm the role of peroxynitrite in cell activation, control cells were stimulated with a sub-effective amount of LPS/IFN together with a NO donor and a superoxide anion generator and treated with a NOS-2 inhibitor 4 hours after stimulation. Griess reaction was performed 48 hours after. Statistical comparisons were performed by two-way ANOVA followed by the Bonferroni test. Results: A7r5 control cells stimulated with LPS/IFN presented a rapid increase in intracellular NO and ROS content. These increases were prevented by c-PTIO, 7-NI and DPI, as well as in siNOS-1 and siNOS-3 cells. NOS-2 was only expressed after cell stimulation. Control cells incubated with c-PTIO or 7-NI and stimulated with LPS/IFN presented a diminished NOS-2 expression and activity. Only in siNOS-1 cells was NOS2 expression and activity also reduced. Nuclear translocation of NF- B and positive nitrotyrosine reaction were reduced in c-PTIO or 7-NI treated groups. Sub-effective concentrations of LPS/IFN did not induce significant nitrite production. However, when sub-effective LPS/IFN was associated with the production of low concentrations of peroxynitrite, nitrite accumulation was as high as in cells stimulated with activating concentrations of LPS/IFN. Conclusions: We show for the first time the importance of NOS-1-derived NO and peroxynitrite for smooth muscle cell activation. Cell stimulation with LPS/IFN causes an early NOS-1-derived NO pulse and a ROS pulse that forms peroxynitrite. The interplay between these species seems to be key events for NF- B nuclear translocation and NOS-2 expression. Acknowledgements: CNPQ, CAPES and FAPESC. P83 Estradiol cypionate modulates immunological response during sepsis Luiz E da Silva*, Angelita M Stabile, Marcel E Batalhão, Evelin C Cárnio College of Nursing at Ribeirão Preto, São Paulo, Brazil Critical Care 2013, 17(Suppl 4):P83; doi:10.1186/cc12982 Background: Sepsis and its common complication septic shock are generally induced by the action of lipopolysaccharide (LPS) and characterized by peripheral arteriolar vasodilatation that results in hypotension and inadequate tissue perfusion. During sepsis, secretion occurs of large amounts of inflammatory mediators such as nitric oxide (NO), interleukin 1 (IL-1) and TNFa that will modulate the inflammatory response. One significant finding in clinics is that men and women respond differently to sepsis, with better prognosis related to women [1]. Materials and methods: Male and female (ovariectomized and sham surgery) rats were injected intraperitoneally (i.p.) for three consecutive days with ECP 40 µg/kg or vehicle. On the third day, after ECP injection, rats receive i.p. injection of 10 mg/kg bacterial LPS or saline solution. Plasma was collected 2, 4 and 6 hours after LPS for NO and cytokine measurements. Results: Administration of LPS increased the NO plasma concentration in males and females (2, 4 and 6 hours). ECP pretreatment decreased the NO concentration in sham females at 4 and 6 hours; conversely, it increased nitrate levels in ovariectomized and in males at 4 and 6 hours. IL-1 plasma concentration was increased in the three groups after LPS administration at 2 and 4 hours and in Sham at 6 hours; ECP pretreatment decreased IL-1 plasma concentration in all groups at 2 hours. LPS administration also increased TNFa plasma concentration at 2, 4 and 6 hours in the three groups; ECP pretreatment inhibited the increase of TNFa at 2 hours in three groups. Conclusions: Our results indicate that estradiol may have proinflammatory or anti-inflammatory actions depending on the gender and the mediator evaluated; this balance in mediator secretion may be protective and explain in part the better outcomes of woman during sepsis. Acknowledgements: FAPESP. Reference 1. Martin GS, Mannino DM, Eaton S, Moss M: The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 2003, 348:1546-1554. Patrícia de Oliveira Benedet*, Gustavo Campos Ramos, Ana Maria Favero, Background: The profound decrease in vasomotor tone accompanied by hyporesponsiveness to vasoconstrictors is an important contributor to morbidity and mortality in septic shock. Overproduction of nitric oxide (NO) has been shown to play a relevant role in septic shock vascular dysfunction. One of the mechanisms whereby NO exerts some of its effects is the reaction with thiol groups of cysteine residues in a process called S-nitrosylation, producing S-nitrosothiols. The aim of the present study is to show that modification in S-nitrosylation has an important impact in sepsis-induced vascular dysfunction and mortality. Materials and methods: Wistar female rats were anesthetized and submitted to cecal ligation and puncture (CLP) for induction of sepsis. Thirty minutes before and 4 hours after surgery, animals received 5,5’dithio-bis-(2-nitrobenzoic acid) (DTNB), an oxidizing agent of sulfhydryl groups or vehicle. Eight hours after CLP the rats were prepared for invasive blood pressure measurements and vascular reactivity to phenylephrine was assessed. The effect of DTNB on survival was also evaluated. All of the procedures were approved by the institutional Animal Ethics Committee (protocol number PP00790/CEUA/UFSC). Results: Eight hours after sepsis induction, rats displayed a pronounced hyporesponsiveness to phenylephrine (10 nmol/kg; 21.3 ± 1.1 mmHg CLP group compared with 42.3 ± 0.8 mmHg in control group; P < 0.05, n = 6). When DTNB was injected 30 minutes before and 4 hours after CLP surgery, the response to phenylephrine was completely normalized (10 nmol/kg; 46.2 ± 2.2 mmHg; P < 0.05, n = 6). DTNB also reduced the mortality of septic rats by 40%. Conclusions: Our results suggest that NO overproduction during septic shock may cause nitrosylation of critical proteins important for alphaadrenergic contractile response. Oxidation of protein sulfhydryls by DTNB prevents nytrosylation and restores the response to phenylephrine in septic animals. Another important finding is that DTNB restored the alpha-adrenergic response even after sepsis is installed. Understanding the role of S-nitrosylation may help to develop strategies to prevent or reverse the vascular dysfunction of sepsis. Acknowledgements: Financial support: CNPq, CAPES, FAPESC and FINEP. Background: The objective was to evaluate the profile of respiratory mechanism of septic female rats previously submitted to exposure of cigarette smoking. Materials and methods: Initially, female rats (230 to 300 g) were randomly divided into a control group (NS) kept with no manipulation and a cigarette smoking-induced respiratory disorders group (S). A rat model used to induce respiratory disorders was established by exposure to cigarette smoking (8 units/15 minutes) daily for 6 weeks. Twenty-four hours after the last cigarette smoking exposure session, each group underwent cecal ligation and puncture procedures to induce polymicrobial sepsis (CLP group) or only underwent a laparotomy (sham group), resulting in the following four experimental groups: Sham-NS (n = 11), Sham-S (n = 11), CLP-NS (n = 6) and CLP-S (n = 9). The profile of respiratory mechanism was evaluated by forced oscillation measurements using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc.) at 24 hours CLP or Sham procedures. The respiratory system parameters evaluated were calculated in flexiWare7 software. All experimental procedures used in our study were approved by the Institutional Animal Ethics Committee (nº 11221971-3/47). Results: Among the experimental groups, no significant difference in airway resistance was verified, while prior exposure to cigarette smoking decreased the tissue resistance of sham-operated rats (0.77 ± 0.03 vs. 0.55 ± 0.01 cmH2O.second/ml, Sham-NF and Sham-F, respectively) as well as inhibiting the increase in tissue resistance induced by sepsis (1.11 ± 0.11 vs. 0.76 ± 0.03 cmH2O.second/ml, CLP-NS and CLP-S, respectively). The prior exposure to cigarette smoking did not alter the lung compliance of sham-operated rats, but it blocked the CLP-induced reduction of lung compliance (0.82 ± 0.04, 0.21 ± 0.11 and 0.57 ± 0.07 cmH2O.second/ml, Sham-NS, CLP-NS and CLP-S, respectively). Similarly, cigarette smoking blocked the CLP-induced decrease of inspiratory capacity (7.85 ± 0.25, 4.96 ± 1.49 and 7.00 ± 0.41 cmH2O.second/ml, Sham-NS, CLP-NS and CLP-S, respectively) but did not alter the inspiratory capacity from sham-operated rats (8.68 ± 0.2 cmH2O.second/ml, Sham-S) compared with Sham-NS. Conclusions: In contrast to sham-operated rats, cigarette smoking inhibited changes in the resistance, compliance and inspiratory capacity of the respiratory system of CLP-operated rats. Background: New therapy is required that improves the prognosis of patients suffering from severe sepsis or septic shock. C1-esterase inhibitor (C1-Inh) was introduced in clinical medicine for patients with hereditary angioedema. Some studies show that C1-Inh administration may also have a beneficial effect in other clinical conditions such as sepsis [1,2]. We examined the effect of C1-Inh administration to the sepsis pig model. Materials and methods: The experiments were performed divided into two groups: the treatment group and the control group. We administered LPS (40 μg/kg) to pigs of about 10 kg over 30 minutes. At the same time, we administered C1-Inh in the control group (500 U, n = 3; 1,000 U, n = 3), and saline in the control group (n = 3). We examined the effect of C1-Inh for the outcome of the two groups, physiological indicators such as heart rates (HR) and mean arterial pressure (MAP), and autopsy results such as pleural effusion and ascites. Results: The outcome of the two groups was that 5/6 in the treatment group and 2/3 in the control group survived at 240 minutes from the end of LPS administration. HR (/minute) at 180 minutes from the end of LPS administration was 157.5 ± 12.3 in the treatment group and 205.3 ± 42.6 in the control group, and MAP (mmHg) at the same time was 60.0 ± 8.2 in the treatment group and 58.3 ± 5.6 in the control group. As for the autopsy results, pleural effusion (ml) was 13.28 ± 3.13 in the treatment group and 9.87 ± 4.33 in the control group, and ascites (ml) was 165.8 ± 32.99 in the treatment group and 210.0 ± 60.8 in the control group. Seeing each individual, the individual showing a large effect of C1-inh was observed. Conclusions: C1-Inh tended to stabilize the hemodynamics of the sepsis pig model, but was not able to reduce significantly the amount of pleural effusion and ascites. Acknowledgements: This is a collaborative study of Emergency and Critical Care Center, Saga University Hospital and the Department of Veterinary Medicine, Rakuno Gakuen University. References 1. Caliezi C, Wuillemin WA, Zeerleder S, Redondo M, Eisele B, Hack CE: C1 esterase inhibitor: an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema. Pharmacol Rev 2000, 52:91-112. 2. Igonin AA, Protsenko DN, Galstyan GM, Vlasenko AV, Khachatryan NN, Nekhaev IV, Shlyapnikov SA, Lazareva NB, Herscu P: C1-esterase inhibitor infusion increases survival rates for patients with sepsis. Crit Care Med 2012, 40:770-777. P87 Brain markers of neurodegeneration in sepsis survivor rats Larissa de Souza Constantino1*, Cristiane Damiani Tomasi1, Matheus Pasquali2, Samantha Pereira Miguel3, João Paulo Almeida dos Santos2, Francieli Vuolo1, Clarissa Martinelo Comim1, Fabrícia Petronilho3, João Quevedo1, Daniel Pens Gelain2, José Cláudio Fonseca Moreira2, Felipe Dal-Pizzol1 1University of the Extreme-South Catarinense, Criciúma, Brazil; 2Federal University of the Rio Grande do Sul (UFRGS), Porto Alegre, Brazil; 3University of Southern Santa Catarina, Tubarão, Brazil Critical Care 2013, 17(Suppl 4):P87; doi:10.1186/cc12986 Background: Several preclinical and clinical reports indicate a significant role for systemic inflammation in chronic neurodegenerative diseases [1], with commitment of different brain regions. Several studies have demonstrated hippocampal atrophy, EEG changes [2], profound glial activation, the generation of nitric oxide and changes in expression of mediator apoptosis [3]. The release of these mediators and oxidative stress occur mainly in acute phase inflammation in sepsis survivor rats and are associated with long-term cognitive impairment [4]. These cognitive deficits have been associated with decreased quality of life and increased long-term morbidity. Some of these alterations resembled the pathophysiological mechanisms of neurodegenerative diseases. For this reason, we analyzed parameters related to neurodegeneration in rats that survived sepsis, and their relation to cognitive dysfunction. Materials and methods: Wistar rats were subjected to sepsis by cecal ligation and puncture and 30 days after surgery the hippocampus and prefrontal cortex were isolated just after cognitive evaluation by the inhibitory avoidance test. The immunocontent of b-amyloid peptide (Ab), receptor for advanced glycation endproducts (RAGE) and synaptophysin were analyzed by western blot. Results: Ab was increased in septic animals in the hippocampus, but not in the prefrontal cortex. RAGE was upregulated in both structures after sepsis, and the immunocontent of synaptophysin was decreased only in the prefrontal, and inversely correlated to Ab levels. Prefrontal levels of synaptophysin correlated with performance in the inhibitory avoidance. Background: Sepsis remains a clinical challenge for the ICUs. However, it is known that the tolerance mechanism using low doses of lipopolysaccharide (LPS) reduces the expression of proinflammatory genes and involves epigenetic regulation. The chromatin openness is regulated by histone acetyltransferases (HATs) and these enzymes could be modulated by nitric oxide (NO) interaction. In the present work, we demonstrate the pathway of tolerance to LPS from HAT activity and level of histone openness to production of cytokines as well as the influence of NO inhibition. Materials and methods: THP1 differentiated into macrophages (with 2.5 nM PMA treatment) were cultivated in RPMI medium (Control group), submitted to tolerance (500 ng/ml LPS 24 hours before challenge with 1,000 ng/ml LPS - Tolerant group) and challenge (1,000 ng/ml LPS - D group) during 24 hours. NO production was inhibited by addition of 100 μM LNAME. The HAT activity and cytokine production (IL-6) were measured with biochemistry kits. Histone acetylated H3 and H4 were analyzed by western blotting. Results: Tolerance reduced HAT activity compared with the group directly challenged (P < 0.05). Acetylated H4 was maintained at basal levels in the tolerant group and increased in the D group (P < 0.05). However, the tolerance increases the acetylation of histone H3 in a NO-dependent response. Similarly, the IL-6 release was reduced by induction of tolerance (P < 0.05 vs. D group). However, this effect was abolished by inhibition of NO production. Conclusions: The induction of tolerance diminishes HAT activity and cytokine production. The tolerance triggers a complex epigenetic modulation dependent of NO. Acknowledgements: FAPESP 09/15530-0. P89 Histone acetyltransferase and DNA methyltransferase expression in response to LPS stimulation Ester Correia Sarmento Rios*, Francisco Garcia Soriano Departamento de Clínica Médica da Universidade de São Paulo, SP, Brazil Critical Care 2013, 17(Suppl 4):P89; doi:10.1186/cc12988 Background: Tolerance is a defense strategy capable of reducing the proinflammatory impact of infection. Tolerance capacity differs among the different tissues. It is known that epigenetic regulation is cell type specific. The cell machinery regulates the expression and activity of the enzymes that regulate chromatin openness. Understanding the epigenetic mechanism activated by different doses of LPS is important to define new approaches for the treatment of systemic infections. The objective of this work was to study the LPS-induced epigenetic response, analyzing the expression of histone acetyltransferases (HAT) and DNA methyltransferases (DNMTs). Materials and methods: THP-1 human promonocytes were cultivated in RPMI (C group), submitted to different doses of LPS (T group - tolerance with 500 ng/ml during 24 hours and challenge with 1 µg/ml during 24 hours; D group - 1 µg/ml during 24 hours). The inhibition of nitric oxide production was performed with LNAME (100 µM). Male Balb C mice (8 weeks old) were divided into two groups: C group - without manipulation; D group - received 5 mg/kg LPS. The spleens were collected 48 hours after. The HAT, DNMTs, lysine acetylated and histone H3 acetylated amounts were determinate by western blot. The results represent three similar experiments. The statistical analysis was performed by ANOVA. Research protocol number 0950/09 was approved by the ethics committee. Results: The challenge with LPS reduced the expression of DNMT1 in THP1 cells. However, the tolerance increased the amount of this enzyme (25%). Challenge with LPS reduces DNTM3a production (50%) in mice spleen. The expression of HAT was reduced (50%) in the T group and this event was NO dependent. The LPS addition to THP1 culture decreases the production of acetylated lysine (P < 0.05) in a dose-dependent way (68% and 33% with 0, 1 and 5 µg/ml LPS respectively). Low doses of LPS reduce the acetylation of histone H3 (30% and 60% with 500 ng and 1 µg/ml LPS respectively). Conclusions: Different concentrations of LPS are required for selective regulation of subsequent LPS-stimulated epigenetic mechanisms. Acknowledgements: FAPESP 09/15530-0. Background: Recent clinical studies have shown that sepsis survivors can develop long-term cognitive impairment. The cellular and molecular mechanisms involved in these events are not yet completely understood. In this study, we investigate the synaptic deficits in sepsis and the involvement of glial cells in this process. Materials and methods: Using a clinically relevant model of sepsis (cecal ligation and puncture), we observed a decrease of recognition memory 9 days after sepsis. At the same time, by colocalization between presynaptic and post-synaptic protein, synaptophysin and PSD-95, we observed a reduction of structural synapses in the hippocampus and cerebral cortex of septic mice. To define the molecular mechanisms accountable for synaptic loss in sepsis, we used an in vitro approach treating neuronal cultures with conditioned medium from astrocyte (ACM) and microglial (MCM) cultures stimulated with LPS. Results: We observed that the MCM reduced the synapse number and the ACM increased the number of synapses. The analysis of conditioned medium composition showed that MCM had increased levels of IL-1b while the ACM had increased levels of TGF-b1, as compared with medium from the non-LPS-stimulated cultures. The increased levels of IL-1b, from microglial activated with LPS, accompanied by an increase of TGF-b1, from LPS-activated astrocytes, suggests an anti-synaptic activity in IL-1b and pro-synaptic actions in TGF-b1. Inhibition assays with the addition of soluble IL-1b receptor (IL-1 Ra) prevented the MCM-induced synapsis loss. To understand whether the loss in synapse density would have functional outcomes we performed patch clamp experiments in neurons treated with microglia conditioned medium (MCM) and MCM of LPS-stimulated cultures. Patch-clamp recordings in the MCM-treated neurons showed a reduction in postsynaptic current frequency, while an increase in current amplitudes suggests a functional synaptic deficit. Conclusions: These findings show, for the first time, a correlation between synaptic deficits and memory dysfunction, suggesting a possible mechanism for cognitive impairment after sepsis as well as a glial-derived molecule mediating synapse reduction. P91 Association of the immature platelet fraction with the diagnosis and severity of sepsis: an observational study Melina V Rodrigues*, Bruna D Andreguetto, Thiago M Santos, Maria de Fátima P Gilberti, Joyce M Annichino-Bizzacchi, Desanka Dragosavac, Marco A Carvalho-Filho, Erich V De Paula Faculty of Medical Sciences, University of Campinas, SP, Brazil Critical Care 2013, 17(Suppl 4):P91; doi:10.1186/cc12990 Background: An ideal sepsis biomarker should be able to segregate infected patients from other causes of SIRS, and also to allow some kind of risk stratification. Furthermore, it should be capable of identifying subgroups of patients with specific sepsis complications, enabling targetspecific preventive and therapeutic measures. Finally, access to this biomarker should not depend on complex and high-cost equipments and reagents, allowing access to more patients. New hematologic automated analyzers used for evaluation of the complete blood count provide a series of advanced analytical parameters that permit more detailed evaluations of circulating blood cells. Parameters such as the immature reticulocyte fraction (IRF) and immature platelet fraction (IPF) identify early signs of hematopoietic recovery, and have been studied in several inflammatory conditions. Recently, a study performed in critically ill patients suggested that the IPF could be a more accurate biomarker of sepsis development than C-reactive protein (CRP) and procalcitonin. The aim of this study was to evaluate whether IPF and IRF levels presented any association with clinical and laboratory parameters of sepsis severity. Materials and methods: During 30 days the IPF and IRF were obtained using an automated hematologic analyzer (Sysmex XE5000) within 24 hours from admission for consecutive patients with sepsis. Results: In total, 23 patients with sepsis were enrolled in the study, of which 12 (52%) presented severe sepsis or septic shock. The median APACHE II and SOFA scores at admission were 15 (6 to 37) and 6 (1 to 17). Median IPF and IRF levels at admission were 4% (1.1 to 11.0%) and 14% (1.6 to 47.1%) respectively, and were significantly higher than in a population of healthy individuals (IPF = 2.1% and IRF = 2.9%; both P < 0.001). As opposed to the CRP, both IPF and IRF were significantly correlated with the SOFA at admission (Rs = 0.52 and 0.45; P = 0.01 and 0.02 respectively). However, when patients were stratified by the median SOFA score at admission, only the IPF was significantly higher in patients with SOFA ≥6 (IPF = 6.2% vs. 2.9%; P = 0.01). Similar results were observed when patients were stratified by the presence of severe sepsis. The IPF presented a significant correlation with the platelet count (Rs = -0.71; P < 0.001), but with not with PT, aPTT and D-dimer. Conclusions: In patients with sepsis, both IPF and IRF were higher than in healthy individuals, and the IPF was associated with increased sepsis severity. Larger studies are warranted to define and validate the precise role of the IPF as a sepsis biomarker. Background: Although several target-specific therapies for sepsis failed to translate into clinical benefits during the last decades, the increasing knowledge about sepsis pathogenesis continues to reveal new therapeutic targets that could be explored in the future. One of the challenges of previous target-specific treatments for sepsis was the short half-life of agents, some in the range of minutes. Gene transfer strategies can overcome this limitation, by providing a platform for longer expression of secreted therapeutic proteins. On the other hand, the transient nature of sepsis precludes the use of gene transfer strategies leading to long-term expression such as viral vectors. In this context, the use of nonviral vectors emerges as an attractive strategy for the treatment of sepsis, provided that sufficient expression of any therapeutic gene can be obtained. Materials and methods: Two gene constructs were used to evaluate the feasibility of gene transfer in the endotoxemia model: a lacZ expression plasmid driven by the CMV promoter, and a coagulation factor IX expression plasmid with the hAAT liver-specific promoter. The latter was used as a reporter gene for secreted proteins. C57Bl/6 mice were challenged with LPS and gene transfer was performed 6 hours thereafter, so as to mimic the timepoint when sepsis treatments would be initiated. Fifty micrograms of plasmid were injected into the tail vein using hydrodynamic transfection. A less aggressive protocol, which could in principle be translatable to the clinical setting, was also tested. Gene expression was evaluated 72 hours after gene transfer by a blinded investigator. Results: Factor IX activity levels (FIX:C) were significantly lower in nontransfected LPS-challenged mice (n = 12) compared with nontransfected controls (n = 14), suggesting that endotoxemia decreases baseline FIX:C levels. Higher FIX:C levels (twofold higher than controls) were observed in control mice submitted to hydrodynamic transfection (n = 5), as expected. When gene transfer was evaluated in the context of sepsis, LPS-challenged mice (n = 9) presented 1.7-fold higher FIX:C levels than control mice (n = 12) (P < 0.01). Moreover, mice that were exposed to a less aggressive intravenous transfection protocol (n = 8) presented FIX:C levels that were 1.4-fold higher than controls (P = 0.04). Liver-expression of b-galactosidase also demonstrated the feasibility of gene transfer in LPS-challenged mice. Conclusions: Our results suggest that the cellular and molecular events of sepsis reproduced in the endotoxemia model could facilitate gene transfer, thus offering a unique opportunity for gene therapy with nonviral vectors, without the need for traumatic gene transfer protocols that would be required in other pathological conditions. P93 Effect of IL-1 receptor antagonist on the cerebrospinal fluid nitric oxide concentrations during experimental polymicrobial sepsis in rats Fazal Wahab1*, Lucas F Tazinafo1, Marcelo Eduardo Batalhão2, Evelin Capellari Carnio2, Maria Jose Alves da Rocha1 1Department of Morphology, Physiology and Basic Pathology, FORP, University of São Paulo Campus de Ribeirão Preto, São Paulo, Brazil; 2Department of General and Specialized Nursing, EERP, University of São Paulo Campus de Ribeirão Preto, São Paulo, Brazil Critical Care 2013, 17(Suppl 4):P93; doi:10.1186/cc12992 Background: Recently, we observed that blocking the IL-1-IL-1r signaling pathway by central administration of IL-1ra (an IL-1 receptor antagonist) can result in increased AVP secretion and survival rate in the late phase of sepsis [1]. The mechanism of this effect of IL-1ra on AVP concentration and survival rate remains elusive. Many studies have implicated excessive production of nitric oxide (NO) as one of the important factors responsible for decreased AVP secretion during the late phase of sepsis [2]. Currently, the effect of IL-1ra on the central NO production and release during sepsis is not known. Materials and methods: In this study, we checked the effect of IL-1ra on sepsis-induced increased release of NO in cerebrospinal fluid (CSF). Sepsis was induced by cecal ligation and puncture (CLP). IL-1ra (9 nmol/animal) and vehicle (PBS: 2 μl/animal) were injected intracerebroventricularly to separate groups of CLP (n = 8/group) and control (n = 8/group) animals. CSF and blood samples were collected from different groups of rats (n = 6 to 8/group) after 1, 2, 4, 6 and 24 hours. The NO concentration in CSF was determined by chemiluminescence assay. Specific ELISA was used for AVP analysis. All experiments were carried out according to an institutional ethic committee-approved protocol (CEUA protocol number 12.1.1205.53.0). Results: NO levels were significantly (P < 0.05 to 0.005) increased in postCLP 6-hour and 24-hour as compared with control, post-CLP 1-hour, 2-hour, and 4-hour animals. IL-1ra administration did not significantly alter the NO concentration in CSF after 1, 2, 4 and 6 hours as compared with vehicle treatment in CLP animals as well as in control. In contrast, after 24 hours NO levels were significantly (P < 0.02) lowered in IL-1ratreated animals (22.36 ± 2.07 μM) as compared with vehicle-treated animals (31.97 ± 2.88 μM). The AVP concentration in IL-1ra-treated rats was significantly higher in IL-1ra-treated animals in comparison with vehicle treatment. Moreover, the survival rate of IL-1ra-treated rats was >80% while that of vehicle-treated rats was 47%. Conclusions: Our results have demonstrated that blocking the IL-1-IL-1r signaling pathway by central administration of IL-1ra increases AVP secretion in the late phase of sepsis, which may be beneficial for survival. We believe that one of the mechanisms for this effect of IL-1ra is through reduction of NO concentration in CSF of the septic rats. Acknowledgements: This research work was funded by FAPESP. FW is supported by a postdoctoral fellowship of FAPESP. References 1. Wahab F, Tazinafo LF, Rocha MJA: Study of the effect of IL-1 receptor antagonist on vasopressin secretion during experimental polymicrobial sepsis in rats. Covian Symposium. University of Sao Paulo, Brazil 1913. 2. Corrêa PB, Pancoto JA, de Oliveira-Pelegrin GR, Cárnio EC, Rocha MJ: Participation of iNOS-derived NO in hypothalamic activation and vasopressin release during polymicrobial sepsis. J Neuroimmunol 2007, 183:17-25. Background: Since its discovery by Kojima and colleagues in 1999 [1], the hormone ghrelin has been studied in different contexts, since this peptide has the ability to promote hormonal, vascular and immune changes. His well-established functions are the release of growth hormone, by a mechanism distinct from the growth hormone release factor, and stimulation of hunger, by activating hippothalamic neurons, leading to release of neuropeptide Y, thus promoting orexigenic effects [2]. Because of its ability to release hormones, including vasopressin [3], and by possessing immunomodulatory properties, ghrelin has been studied in different contexts of inflammatory states, as present in endotoxemia and sepsis [4]. The infusion of lipopolysaccharide (LPS) is capable of generating an inflammatory state, with augmenting of TNFa, IL-1b and nitric oxide, which in turn leads to cardiac depression and systemic vasodilation and hypotension [5]. Due to its properties to modulate the inflammatory response, in a way of diminishing the levels of TNFa, IL-1b and nitric oxide, which are augmented in the endotoxemic state, as well the ability to augment the plasma levels of vasopressin, ghrelin emerges as a potential neuro-immunomodulator in hypotension caused by endotoxemia. We speculate that ghrelin, mediating the inflammatory response and by augmenting vasopressin blood levels, could attenuate the hypotension caused by endotoxin. Materials and methods: Male Wistar rats (250 to 300 g) had their jugular vein and/or their right cerebral ventricle cannulated for drug administration, and the femoral artery cannulated for mean arterial pressure (MAP) and heart rate (HR) records, respectively. All experimental procedures were approved by the Comitê de Ética em Experimentação Animal-campus de Ribeirão Preto (protocol number 12.1.1441.53.5). The endotoxemia model was induced by endovenous injection of lipopolysaccharide (LPS; 1.5 mg/kg). Data were compared using two-way analyses of variance and significant differences were obtained using the Bonferroni post test. Results: LPS administration leads to a drop in MAP in the first 2 hours, followed by a partial recovery of the MAP, and then a second drop in MAP, with a peak in 6 hours. The HR was augmented in this group. Systemic administration of ghrelin alone, through a bolus followed by subcutaneous implantation of an osmotic pump, did not alter the response, in comparison with the saline-treated group. The icv administration of ghrelin, however, diminished the HR in some intervals, although did not present a difference in MAP, in comparison with the saline-treated group. The administration of ghrelin, centrally and peripherally, when given at the same time as the LPS bolus, attenuated the first drop in MAP and completely restored the second drop present in the LPS group. Conclusions: Ghrelin is capable of attenuating the hypotension caused by endotoxin, and we speculate that the improvement is due to Background: Animal research in sepsis needs analytical tools that can capture and exploit the complexity of the condition. To summarise the disease progression in a porcine model of severe Staphylococcus aureus sepsis, we used principal component analysis (PCA) as a multivariate approach to identify early dynamic expression patterns of 34 selected genes in the liver, lung, and spleen tissue. Materials and methods: We combined data from two related experimental studies in pigs haematogenously infected with a porcine pathogenic strain of S. aureus [1,2]. Seventeen infected pigs were euthanised at the following time points post infection (p.i.): 6 hours (n = 3), 12 hours (n = 3), 24 hours (n = 3), 30 hours (n = 1), 36 hours (n = 2), and 48 hours (n = 5). Five healthy controls were managed in parallel. Gene expression of 34 genes related to acute inflammation and haemostasis was measured in the liver, lung, and spleen by quantitative real-time PCR. The data matrix of 22 samples and 102 (34 × 3) variables were log-transformed, scaled to unit variance, and subjected to PCA. Results: Three (PC1 to PC3) distinct dynamic response patterns were identified. PC1: hepatic positive and negative acute-phase genes were the main influencers of a protracted pattern induced between 12 and 48 hours of infection, which explained 23% of the total variation in the dataset (Figure 1A, C). PC2: an acute pattern distinguished infected pigs from controls already after 6 hours and peaked around 12 hours p.i. After 30 to 48 hours, pigs had either reverted back to basal levels (n = 7) or below basal levels (n = 2) (Figure 1A). This pattern explained 14% of the total variation and was influenced by a systemic (nonorganspecific) mixture of proinflammatory, anti-inflammatory and haemostatic genes (Figure 1C). The two pigs with low PC2 levels had suffered from overt disseminated intravascular coagulation when euthanised [3], and this outcome was clearly reflected by PC2. PC3: a per-acute pattern, influenced mainly by pulmonary proinflammatory genes (explaining 11% of the total variation), was induced in infected pigs at 6 hours p.i., while at later time points most pigs had moved towards basal levels (Figure 1B, D). Conclusions: Multivariate analysis (PCA) identified three temporally distinct patterns in gene expression data from the liver, lung, and spleen tissue: pulmonary inflammation was rapidly induced, followed by transient induction of a generalised inflammatory and haemostatic response, and initiation of the hepatic acute-phase response. Figure 1(abstract P95) Pigs (scores) and genes (loadings) in PC1-PC2 (A, C) and PC1-PC3 space (B, D). (A) Increased PC1 scores are seen in infected pigs from 12 hours until the end of the study (48 hours), with a peak around 24 hours. PC2 scores are increased after 6 hours of infection, with a peak at 12 hours. Thereafter, scores decline towards basal levels, or below. (B) PC3 scores are increased mainly in infected pigs at 6 hours p.i. (C) Genes with largest influence on the PC1 axis are mainly hepatic positive and negative acute phase genes (for example, SAA, ITIH4, TTR, TRF). Genes with largest influence on the PC2 axis are not specifically related to a single tissue. (D) The main influencers of the PC3 axis are inflammatory genes measured in lung tissue. Gene symbols are used according to the National Center for Biotechnology Information (NCBI). Background: Previous studies demonstrated the presence of microparticles (exosomes) in plasma of septic patients. These are cell-derived vesicles containing specific collections of proteins, lipids and genetic material that participate in the intercellular communication, changing the function and physiology of their target cells. The role of exosomes in sepsis, however, remains deeply unexplored. This study aimed to investigate the composition of microRNAs and messenger RNAs related to inflammatory response in circulating microparticles of septic shock patients. Materials and methods: Fourteen patients had 30 ml blood collected in the first 48 hours of sepsis and 7 days after for those who survived. Five healthy volunteers served as controls. Exosomes were isolated from plasma by filtration (0.22 μM) and ultracentrifugation. Thirty nanograms of the total RNA were reversely transcribed and the expression profile of 754 human miRNAs and 91 mRNAs from immune response was evaluated by real-time quantitative PCR using the Taqman Low Density Array (Applied Biosystems). The raw data were processed in Expression Suite v1.0.1 software and analyzed in StatMiner v3.0 software considering the global expression level for normalization. The fold-change was calculated based on the estimated mean difference (2(-ΔCT)). Results: Different miRNA expression was observed in the exosomes from septic patients in comparison with healthy donors. In the first 48 hours of septic shock, three miRNAs were differentially expressed: miR-1290 (2.78fold, P = 0.02), miR-1298 (4.02-fold, P = 0.03) and miR-146a (-2.51-fold, P = 0.02). In the recovery phase of sepsis, five miRNAs were differently expressed as compared with controls: miR-1260 (2.29-fold, P = 0.02), miR1274A (2.83-fold, P = 0.02), miR-1274B (3.31-fold, P = 0.02), miR-192 (1.83fold, P = 0.02) and miR-604 (-6.41-fold, P = 0.02). The miRNA expression profiles in different stages of sepsis were similar. Moreover, exosomes from patients after 1 week of sepsis carry less CCL5 mRNA than in the beginning of the disease (-2.49-fold, P = 0.02). Conclusions: Exosomes from septic shock patients carry different miRNA expression profiles at different stages of the disease, as compared with healthy individuals. CCL5 mRNA is less expressed in the recovery phase of sepsis. The composition of these vesicles may help to understand the underlying mechanisms involved in their role in the pathogenesis of sepsis. Acknowledgements: Financial support from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). Background: Septic encephalopathy (SE) is a frequent complication in severe sepsis. Here we have explored the role of NADPH oxidase in different aspects of SE pathophysiology. We investigated the involvement of NADPH oxidase in neuroinflammation and in the long-term cognitive impairment of sepsis survivors. Materials and methods: Our approach included pharmacological inhibition of NADPH oxidase activity with apocynin and the use of genetically deficient (knockout) mice for gp91phox (gp91phox-/-), the catalytic subunit of Nox2. Sepsis was induced by cecal ligation and puncture and fecal peritonitis. We measured the hippocampal oxidative stress, Nox2 and Nox4 gene expression and neuroinflammation in WT and gp91phox-/mice at 6 hours, 24 hours and 5 days post sepsis. Behavioral outcomes were evaluated 15 days after sepsis with the inhibitory avoidance and the Morris water maze tests. Results: The data show progressive oxidative damage to the hippocampus, identified by increased 4-hydroxynonenal expression, associated with an increase in Nox2 gene expression in the first days after sepsis. Pharmacological inhibition of Nox2 with apocynin completely inhibits hippocampal oxidative damage in septic animals as well as the development of long-term cognitive impairment in the survivors. Pharmacologic inhibition or the absence of Nox2 in gp91phox-/- mice prevents glial cells activation, one of the central mechanisms associated with SE and other neurodegenerative diseases. Conclusions: We identified Nox2 activation as a necessary step for glial cell activation in SE. Our data indicate that Nox2 is as a major source of oxidative stress in the brain and consequently has a central role in the development of cognitive impairments observed in sepsis survivors. Background: Previously, we have shown that methicillin-resistant Staphylococcus aureus (MRSA) sepsis was associated with more severely pronounced vascular leakage compared with Pseudomonas aeruginosa sepsis. We have also demonstrated that the arginine vasopressin V1a receptor (V1aR) agonist significantly attenuated the severity of MRSAinduced vascular leakage [1]. The goal of the present study was to explore mechanistic aspects of V1aR agonist’s action. Materials and methods: Twelve adult female sheep were operatively prepared for chronic study. After 5 days of recovery, tracheostomy was performed under anesthesia and injury was given. The injury consisted of insufflation of cooled cotton smoke (48 breaths) and instillation of 2.5 × 106 CFU MRSA into the lungs by bronchoscope under maintenance isoflurane anesthesia. Following the injury, sheep were awakened, placed on mechanical ventilation and randomly allocated into two groups: control group, saline treated, n = 6; and POV group, treated with intravenous V1aR agonist, Phe2-Orn8-Vasotocin (POV) (Ferring Research Institute, Inc., San Diego, CA, USA), n = 6. The titration of POV was started when mean arterial blood pressure (MAP) dropped by 10 mmHg from the baseline with the initial dose of 30 pmol/minute, which was further adjusted to maintain MAP close to baseline. All sheep were resuscitated with lactated Ringer’s solution with initial rate of 2 ml/kg/hour that was further adjusted according to hematocrit. The experiment lasted 24 hours. Plasma levels of nitric oxide (NO; Grease reaction), asymmetric dymethylarginine (ADMA; mass spectrometry) and bradykinin (mass spectrometry) were determined at 0 hours and every 3 hours after the injury. Results: MRSA-induced plasma levels of NO (nitrite/nitrate) as well as cumulative body fluid were significantly inhibited by V1aR agonist. The treatment with POV also attenuated the MRSA-induced hypotension. The plasma levels of ADMA were higher in the treated group compared with the control at 24 hours after the injury (0.93 ± 0.14 in control, n = 3 vs. 1.23 ± 0.08 in POV, n = 6). In addition, the treatment with POV significantly inhibited the MRSA-induced bradykinin increases at 3 hours after the injury (1.14 ± 0.4 in control vs. 0.52 ± 0.001 in POV, P < 0.05). Conclusions: Arginine vasopressin V1aR agonist attenuates the severity of MRSA-induced vascular leakage by inhibiting potent permeability factor bradykinin and excessive NO. The V1aR agonist may modulate NO production by promoting the release of endogenous NO synthase inhibitor ADMA. Reference 1. Rehberg S, et al: Selective V(1a) agonism attenuates vascular dysfunction and fluid accumulation in ovine severe sepsis. Am J Physiol Heart Circ Physiol 2012, 303:H1245-H1254. Background: The standard therapy for sepsis is becoming less effective due to increasing microorganism resistance to antibiotics and cardiovascular collapse refractory to fluid resuscitation and vasopressors. In this study, we demonstrate a critical role of peroxynitrite in vascular hyporesponsiveness to vasopressin (VP) in methicillin-resistant Staphylococcus aureus (MRSA)induced ovine sepsis. Materials and methods: Sheep were instrumented with Swan Ganz (common jugular vein), femoral artery, and left atrium catheters to monitor hemodynamics for 24 hours. Sepsis was induced by instillation of live MRSA (2.5 to 3.5 × 1011 CFU) into the lungs by bronchoscope under anesthesia. Sheep were then awakened, placed on a ventilator, and fluid resuscitated. Urine output was measured via a Foley catheter. Groups: MRSA, received MRSA, n = 4; MRSA + peroxynitrite decomposition catalyst (PDC), received MRSA and were treated with PDC starting 6 hours post injury (0.1 mg/kg bolus followed by 0.02 mg/kg/hour), n = 4; MRSA + VP, received MRSA and were titrated with VP when mean arterial pressure fell by 10 mmHg, n = 4; and MRSA + VP + PDC, received MRSA, treated with VP and PDC, n = 4. Results: MRSA induced severe hypotension refractory to aggressive fluid and AVP. PDC and AVP alone partially attenuated the severe hypotension. When combined they more effectively reversed the hypotension. Inhibition of peroxynitrite formation by PDC also markedly reduced AVP requirement. In addition, the in vitro effects of AVP (5 nM) on isolated arterial ring tone were abolished with co-incubation with peroxynitrite (50 μM). Conclusions: Peroxynitrite modulation may be a novel treatment option for management of sepsis-induced cardiovascular collapse refractory to vasopressors. These findings are especially provocative since peroxynitrite is the product of excessive nitric oxide regardless of which NOS isoform is involved and the major debate of whether the use of NOS inhibitors in management of sepsis is beneficial still remains. Background: Studies suggest that curcumin, found in the tropical plant Curcuma longa, has anti-inflammatory and antioxidant properties and can act in sepsis, decreasing the release of proinflammatory mediators and free radicals. In the search to increase curcumin’s bioavailability a fitotecnologic process was developed that generated a solid dispersion of curcumin named DS17. This dispersion is water soluble and seems to increase the curcumin absorption by the gastrointestinal tract. The aim of our study was to assess the biological activity of the solid dispersion of curcumin (DS17) in immunological and metabolic alterations observed in a model of sepsis in rats induced by CLP. Materials and methods: Male Wistar rats (250 to 300 g) were divided into two groups: polymicrobial sepsis model by cecal ligation and puncture (CLP) and sham operation (OF). The animals were pretreated with DS17 (100 mg/kg) orally for 7 days prior to CLP and treated 2 hours after surgery. The animals were used to analyze curcumin absorption through HPLC, plasma glucose, cytokines, nitric oxide (NO) and HSP70. Another group had the survival rate assessed for 48 hours. Results: Our results showed that curcumin is present in the plasma at 4 and 6 hours but absent 24 hours following the DS17 administration. The dispersion decreased IL-6 in plasma and peritoneal fluid at 6 and 24 hours, and IL-1b 6 hours after sepsis stimulus. Moreover, we observed an increase in the hematocrit and a decrease in plasma glucose in the same animals. Paradoxically, plasma IL-10 and serum HSP70 decreased in 24 hours while plasma NO increased in the same period. These changes were not sufficient to increase significantly the survival although we observed a biological improvement of 20% 24 hours following CLP. Conclusions: Our results suggest that despite a significant decrease in proinflammatory cytokines (IL-1b and IL-6), treatment with curcumin solid dispersion produced no beneficial biological effect in septic animals. Further studies are necessary to better clarify the suggested antioxidant and anti-inflammatory effect of curcumin. P101 Effects of PPARg in dendritic cells during severe sepsis and sepsisinduced immunosuppression Raphael Molinaro1*, Roberta Navarro-Xavier1, Papp Attila2, Adriana Vieira-de-Abreu1, Adriana Ribeiro Silva1, Hugo Caire Castro-Faria-Neto1, Claudia Farias Benjamim3, Laszlo Nagy2, Patrícia Torres Bozza1 1Immunopharmacology Laboratory, IOC/FIOCRUZ, RJ, Brazil; 2Nuclear Hormone Receptors Laboratory, Debrecen, Hungary; 3Inflammation Laboratory, UFRJ, Brazil Critical Care 2013, 17(Suppl 4):P101; doi:10.1186/cc13000 Background: Sepsis is a systemic inflammatory response syndrome against infection, which can develop in sepsis-associated immunosuppression. Actually, several inflammatory dysfunctions have been described in dendritic cells (DCs) that could be responsible for impairing the immune response towards the secondary infection. PPARg is a lipid-activated nuclear receptor, which participates in inflammation, lipid metabolism and cellular differentiation. Previous studies have shown the role of PPARg in acute sepsis besides its effects in sepsis-induced immunosuppression still being unclear. Our aims were to evaluate the phenotypic changes in DCs in lungs from post-septic mice and to assess the effects of PPARg on DC functions. Materials and methods: Mice were subjected to cecum ligation and puncture (CLP) or Sham and, 6 hours after, all groups were treated with antibiotics. Fourteen post-septic and Sham mice were infected with BCG and 24 hours after challenge the lungs were collected, minced and digested to investigate the cytokine production, gene expression and phenotype analysis. To evaluate the effects of PPARg, post-septic derived BMDC were pretreated with PPARg agonist (rosiglitazone) before BCG infection. After 24 hours, lipid droplet formation, phagocytosis, cytokines and oxide nitric production were analyzed. Results: Post-septic mice were susceptible against Mycobacterium bovis, BCG and exhibited higher cellular infiltration. Lungs from post-septic mice showed increased IL-10 level and COX2, CCR2 and IL-1b expression. When post-septic and Sham mice were infected with BCG, we observed higher increased COX2, CCR2 and IL-1b expression in lungs from postseptic mice as compared with lungs from Sham mice but the IL-10 level was reduced. In addition, lungs from post-septic mice showed higher Ly6G cells compared with lungs from Sham mice. Infected BMDC exhibited an immature profile (lower expression of CD80 and CD40) and a positive shift to anti-inflammatory cytokine production (increased IL-10 and reduced TNFa, CCL2 and IL-1b levels). PPARg flanked mice in CD11c cells were more susceptible to severe sepsis. Activation of PPARg in infected BMDC from post-septic mice reduced lipid droplet formation, phagocytosis and oxide nitric production but not cytokine production when compared with infected BMDC from Sham mice. Conclusions: After severe sepsis, phenotypic changes modulate DC functions and may contribute to sepsis-induced immunosuppression. The understanding of PPARg could be important for development of new therapy in sepsis-associated immunosuppression and long-term inflammatory diseases. Raphael Molinaro1*, Papp Attila2, Adriana Ribeiro Silva1, Hugo Caire Castro-Faria-Neto1, Claudia Farias Benjamim3, Laszlo Nagy2, Patrícia Torres Bozza1 Background: Sepsis is a systemic inflammatory response syndrome against infection, which can develop in sepsis-associated immunosuppression. Actually, several inflammatory dysfunctions have been described in dendritic cells (DCs), which could be responsible for impairing the immune response towards the secondary infection, although how these stable modifications maintain is still unknown. Our hypothesis is that DCs from post-septic mice have chromatin alteration and differential microRNA expression. Materials and methods: To investigate the global gene expression, postseptic and Sham-derived BMDC were infected or not with BCG for 24 hours. Total RNA were collected and the gene expression profile was assessed by Affymetrix GeneChip technology. The gene expression profiles were classified by Gene Ontology (GEO). Also, the microRNA analysis was obtained from Affymetrix microarray. To investigate the chromatin modifications, post-septic and Sham BMDC were performed to Chip-Seq analysis. Results: Supervised analysis identified a set of 2,755 genes that distinguished very accurately between post-septic BMDC and Sham BMDC. The gene expression signature showed 1,805 stimulated genes and 950 inhibited genes in post-septic BMDC compared with Sham BMDC. The gene expression signature of post-septic BMDC provided a molecular and functional profile based in GEO. It is noteworthy that post-septic BMDC were mostly found in the downregulated genes to encode proteins involved in the biological pathways of the inflammatory process (IL-1a, IL-12, CD28, TLR2, Hmgb1, CCL2), lipid metabolism (FABP4, Elovl2, PTGS1, PPARδ) and histone modifications (ACAT3, CBx2, Oip5, Hist2hX). When post-septic and Sham BMDC were infected with BCG, downregulated gene sets were classified in 130 significant GEO terms (mainly involved in inflammatory and lipid metabolism process) while surprisingly upregulated gene sets were classified in 10 significant GEO terms (nine inflammatory processes of 10 terms). In microRNA expression, we observed higher microRNA expression in post-septic compared with Sham BMDC. When BMDC were infected with BCG, post-septic BMDC exhibited higher numbers of microRNA compared with Sham BMDC. Furthermore, we assessed the presence of H3K27ac and H3K4me3 in inflammatory (IL-10, TNFa, IL-6 and TGF-b) and lipid metabolism genes (ABCA1, PLIN2, CD36 and FABP4). Both H3K4me3 and H3K27ac on PLIN2, CD36 and FABP4 gene bodies were reduced and the presence of H3K4me3/H3K27ac was increased on TNFa and TGF-b gene bodies. Conclusions: These results demonstrate the global gene expression signature, higher microRNA expression and H3K4me3/H3K27ac profile on chromatin structure in post-septic BMDC. The present study suggests epigenetic changes may play a role in transcriptional regulation in postseptic DCs. P103 Vasopressin secretion in sepsis-surviving animals following dehydration Lucas Favaretto Tazinafo*, Tatiana Tocchini Felippotti, Maria José Alves da Rocha Department of Morphology, Physiology and Basic Pathology, Faculty of Dentistry of Ribeirão Preto - USP, Ribeirão Preto, Brazil Critical Care 2013, 17(Suppl 4):P103; doi:10.1186/cc13002 Background: Vasopressin (AVP) plasma levels increase in the early phase of sepsis but remain at basal levels in the late phase of sepsis [1]. It is also known that one-half of septic patients do not properly respond to an osmotic challenge, one of the strongest stimuli for AVP secretion [2]. However, whether these AVP secretion changes persist in sepsis survivors is not known. This study investigated the possible alterations in plasma AVP levels in sepsis-surviving animals. Materials and methods: Male Wistar rats were separated into two groups: sepsis induced by cecal ligation and puncture (CLP), or sham animals. They received saline solution (50 mg/ml; s.c) immediately and 12 hours after CLP, and also ceftriaxone (30 mg/kg; s.c.) and clyndamicin (25 mg/kg; s.c.) after every 6 hours for 3 days. Sham animals received the volume of saline corresponding to antibiotic administration. After 10 days, the animals were dehydrated or left as control. After 2 days, the animals were decapitated, and the serum and plasma collected for sodium, hematocrit and hormone determination. The posterior pituitary glands were removed for hormone stock analysis. Results: Sepsis-surviving animals presented a higher serum sodium even without the osmotic stimulus (147.8 ± 0.97 SEM vs. 151.4 ± 0.6 SEM mmol/l CLP; P < 0.001). Following dehydration, as expected, there was an increase of serum sodium in CLP animals (151.4 ± 0.6 SEM vs. 155.71 ± 0.47 SEM mmol/l; P < 0.001) and sham animals (147.8 ± 0.97 SEM vs. 154 ± 0.26 SEM mmol/l dehydrated; P < 0.001) with difference between the groups (154 ± 0.26 SEM vs. 155.71 ± 0.47 SEM mmol/l CLP; P < 0.041). Hematocrit also increased in both CLP (42.63 ± 1.58 SEM vs. 50.17 ± 1.67% SEM dehydrated; P = 0.002) and sham (mean: 41.8 ± 1.43 SEM vs. 49.5 ± 1.0% SEM; P = 0.003) groups but without difference between the groups. The animals responded with an increase in the AVP plasma levels (6.12 ± 0.68 SEM vs. 6.16 ± 0.94 SEM pg/ml CLP, P > 0.05), and a decrease in AVP neurohypophysis stocks (4.0 ± 1.02 SEM vs. 1.91 ± 0.67 SEM ng/μg CLP; P = 0.107), with no difference between the groups. Conclusions: The results suggest that sepsis-surviving animals do not present alterations in secretion of AVP in relation to volemia. However, serum sodium results suggest that AVP secretion is impaired in sepsissurviving animals. Acknowledgements: Fazal Wahab, Nilton Nascimento dos Santos Junior, Nadir Martins Fernandes, José Antunes Rodrigues and Milene M. Lopes. References 1. Correa PB, Pancoto JA, De Oliveira-Pelegrin GR, Carnio EC, Rocha MJ: Participation of iNOS-derived NO in hypothalamic activation and vasopressin release during polymicrobial sepsis. J Neuroimmunol 2007, 183:17-25. 2. Siami S, Bailly-Salin J, Polito A, Porcher R, Blanchard A, Haymann JP, Laborde K, Maxime V, Boucly C, Carlier R, Annane D, Sharshar T: Osmoregulation of vasopressin secretion is altered in the postacute phase of septic shock. Crit Care Med 2010, 38:1962-1969. P104 Evaluation of inflammatory parameters and cognitive impairment in a murine model of Pseudomonas aeruginosa pneumosepsis Flora Magno*, Danielle O Nascimento, Pedro CB Alexandre, Patrícia A Reis, Patrícia T Bozza, Hugo C Castro-Faria-Neto, Fernando A Bozza Immunopharmacology Laboratory, IOC/FIOCRUZ, RJ, Brazil Critical Care 2013, 17(Suppl 4):P104; doi:10.1186/cc13003 Background: Sepsis is a severe medical condition characterized by systemic inflammatory response secondary to infection, which frequently progresses to multiple organ dysfunction and death. It is currently the leading cause of death in ICUs worldwide. The most frequent source of infection in sepsis is the lung with a high lethality rate. Pseudomonas aeruginosa is one of the most common pathogens found in sepsis patients. Cognitive impairment is a significant consequence of sepsis reported among survivors. The encephalopathy associated with systemic inflammation is not well understood so the development of new clinical relevant models to help understand this sequelae is important. In this study we aimed to evaluate acute inflammatory markers and establish a long-term consequence in a murine model of pneumosepsis. Materials and methods: C57/BL6 mice were submitted to intratracheal instillation of 105 colony-forming units of P. aeruginosa. Six hours later the bronchoalveolar lavage fluid was collected for cell migration, protein (BCA method) and cytokine (ELISA) analysis. Caudal vein blood samples were collected for cell counting. Another group of animals had their lungs perfused for myeloperoxidase quantification and histological analysis. Evan’s blue dye method was used for the assessment of lung permeability. The survival rate of animals submitted to P. aeruginosa instillation was observed daily during 7 days. This group of animals received a single dose of antibiotic meropenem (30 mg/kg), 6 hours after pneumonia induction. Cognitive damage was evaluated through the freezing test. Results: Our results showed that P. aeruginosa infection caused an expressive recruitment of leukocytes, mainly neutrophils to the lung. Myeloperoxidase, a marker for neutrophil migration, was significantly increased in the lungs of animals instilled with P. aeruginosa. The animals instilled with P. aeruginosa also showed a significant increase in IL-6, KC and protein levels. Histological analysis showed an intense cell infiltrate in the lung tissue and the survival rate was extensively lower in P. aeruginosa infected mice. Additionally, the animals submitted to pneumosepsis had a loss of aversive memory 13 days after pneumonia induction and this loss remained 50 days later. Conclusions: Our study demonstrates the acute inflammatory response to P. aeruginosa lung infection and indicates that possibly this pneumonia model can cause irreversible cognitive impairment. Our results reveal a possible experimental model for the study of encephalopathy associated with systemic inflammation. Acknowledgements: Financial support: CNPq, FAPERJ and FIOCRUZ. P105 Role of inflammatory caspases in a murine two-hit model of sepsis: analysis of immunosuppression and cognitive impairment Mariana Gisely Amarante Teixeira da Cunha1*, Danielle Bastos de Albuquerque1, Daiane Chaves1, Silvio Caetano Alves Júnior1, Flora Magno1, Patrícia Torres Bozza1, Fernando Augusto Bozza2, Hugo Caire Castro-Faria-Neto1, Rachel Novaes Gomes1,2 1Laboratório de Imunofarmacologia, Fiocruz, RJ, Brazil; 2Instituto de Pesquisa Evandro Chagas (IPEC), Fiocruz, RJ, Brazil Critical Care 2013, 17(Suppl 4):P105; doi:10.1186/cc13004 Background: Morbidities associated with severe sepsis are serious problems for surviving patients, such as cognitive impairment and immunosuppression. The immunosuppression predisposes the patients to a second infection, which generally is fatal. Several studies have been made to understand the mediators involved with this immunosuppression associated with sepsis. Some data from the literature show that caspase-1 promotes activation of cytokines, such as IL-1b, and actions are inhibited by caspase-12. This study proposes to analyze the role of inflammatory caspases in immunosuppression and cognitive damage associated with a two-hit model of sepsis. Materials and methods: We submitted Swiss animals to the model of two hits of infection. The first hit was the CLP model and the second hit was intratracheal instillation of Pseudomonas aeruginosa. We analyze the mortality rate and the inflammatory profile of the animals submitted to the CLP model and the two-hit sepsis model. The cognition of the animals was tested by the passive avoidance test 15 and 21 days after the CLP and 21 days until 96 days after the two-hit sepsis model. Results: First we characterize the model and we observed a 30% survival rate of the CLP group in comparison with a 100% survival rate in the SHAM group. The high mortality of the CLP group was associated with hypoglycemia in the first 72 hours after the infection, increased neutrophil accumulation in the peritoneal cavity 6 and 24 hours after the CLP and an increase of inflammatory cytokines 6 hours after the CLP, such as CCL2, IL-1b and IL-10. The CLP group had a cognitive impairment 15 days after the CLP, but the memory was recovered 21 days after the infection. The CLP group was more susceptible to P. aeruginosa infection 21 days after the CLP, when we compare with the SHAM group. The CLP + P. aeruginosa group had a low count of neutrophils in BAL when compared with the SHAM + P. aeruginosa group. We observed a decrease in caspase-1 expression and an increased expression of caspase-12 in the lungs of the CLP + P. aeruginosa group. When we look to cognition, both the SHAM + P. aeruginosa and CLP + P. aeruginosa groups had cognitive impairment 21 days after the infection, and the cognitive impairment remained until 96 days in the SHAM + P. aeruginosa group after the infection, but the CLP + P. aeruginosa recovered the memory 96 days after the infection. Conclusions: Our preliminary results suggest that the immunosuppression associated with the CLP model (first hit) led to more susceptibility for survivor animals, which succumbed to a pneumonia model (second hit). We observed the involvement of inflammatory caspases in this immunosuppression phenomenon with a decrease of caspase-1 and an increase in caspase-12 expression. When we observed the cognitive function, we observed that the animals submitted to CLP had a cognitive impairment 15 days after the infection and the infection with P. aeruginosa induced a cognitive impairment until 96 days in both in groups. However, further studies should be made to confirm these results. P106 Involvement of CC-chemokine receptor 2 in sepsis: focus on cognitive impairment Mariana Gisely Amarante Teixeira da Cunha1*, Rachel Novaes Gomes1,2, Daniele Bastos de Albuquerque1, Daiane Chaves1, Silvio Caetano Alves Júnior1, Patrícia Reis1, Rosália Mendez Otero3, Patrícia Torres Bozza1, Fernando Augusto Bozza2, Hugo Caire Castro-Faria-Neto1 1Laboratório Imunofarmacologia, Fiocruz-IOC, Rio de Janeiro, Brazil; 2FiocruzIPEC, Rio de Janeiro, Brazil; 3Lab. Neurobiologia Cellular e Molecular - CCS, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 4):P106; doi:10.1186/cc13005 Background: Sepsis is a major disease entity with important clinical implications. Critical illness survivors present long-term cognitive impairment, including problems with memory and learning. Chemokines are important to the recruitment of leukocytes to infectious tissue, but a few studies described the role of the CC-chemokine receptor 2 (CCL2) in the cognitive process. In this study, we analyze the involvement of CCR2 in the physiopathology of sepsis, especially in development of cognitive dysfunction. Materials and methods: The CCR2-deficient mice (CCR2-/-) were submitted to a CLP model and we analyzed the survival rate, the severity score of the animals during 144 hours and 15 days after the CLP, and we analyzed the memory of the animals. To analyze the contextual memory, the mice were submitted to the open field method and the water maze procedure. To evaluate the aversive memory, the passive avoidance test was performed. Results: First, we observed that the CLP group had cognitive impairment, but the CCR2-/- group submitted to CLP had more severe cognitive impairment in comparison with the WT-CLP group. Interesting, the CCR2-/- Sham group presented cognitive impairment, suggesting that CCR2 is important to the physiological process of cognition. We then submitted CCR2-/- naive mice to water maze and passive avoidance tests. We found that CCR2-/- naive mice have an impairment of aversive and contextual memory. The cognitive impairment was associated with a decrease of BDNF expression in the hippocampus. When we analyze the expression of b-amyloid protein in the brain of CCR2-/- naive mice, we observed the increased in b-amyloid protein expression in the cortex and hippocampus of these animals, accompanied by increased cell proliferation in the dentate girus, and increased caspase-3 and caspase-12 expression in the hippocampus and cortex. We did not observe a difference in the numbers of neurons in the brain from CCR2-/- naïve mice, as well the numbers of microglial cells. But, surprisingly, there was an increase of astrocytes in the hippocampus of CCR2-/- mice. Conclusions: CCR2 is involved with the physiology of cognition, with the important role arising in the amyloid accumulation in the brain and induction of the caspase-3 pathway. P107 Evaluation of compound NXY-059 on cognitive impairment caused by sepsis Nathália S Oliveira1*, Monica F Pereira1, Mariana GAT Cunha1, Silvio CA Júnior1, Rachel N Gomes1, Patrícia A Reis1, Robert Floyd2, Hugo CCF Neto1 1Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, RJ, Brazil; 2Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Critical Care 2013, 17(Suppl 4):P107; doi:10.1186/cc13006 Background: Nitrones are a class of molecules whose main effect on biological systems is their antioxidant action. Some studies showed a neuroprotective effect in ischemia models and neurodegenerative diseases. Those diseases presented an inflammatory profile that leads the production of reactive oxygen species. This characteristic can generate brain injuries, which can affect areas related to memory consolidation. Sepsis is a pathology that forms an inflammatory response, which causes encephalopathy creating cognitive impairment. Therefore, the present study has the aim to evaluate the effect of the compound NXY-059 on the cognitive impairment caused by encephalopathy like sepsis. Materials and methods: For the assays, mice Swiss Webster male (22 to 28 g, n = 15 per group) were submitted to the CLP method and treated with antibiotics (10 mg/kg, i.p.) for three consecutive days (6, 24 and 48 hours) and with NXY-059 (50 mg/kg, i.p.) for five consecutive days (6, 24, 48, 72 and 96 hours after the surgery). At 24 and 48 hours, a gravity score was made to determine the level of sepsis and the percent of survival was assessed until 144 hours. After 4 hours fast, the glucose levels were also measured 24 and 48 hours after CLP performance. The cognitive impairment was evaluated through the open field method on the 15th (training) and 16th (test) day after the surgery. Results: Our results show that treatment with NXY-059 did not offer a protective effect on mortality and the animals developed moderated sepsis according to the gravity score at 24 hours (4 to 6). At 48 hours, the animals recovered for slight sepsis (2 to 3). The glucose levels were slightly restored at 48 hours for the animals treated with the compound. In the cognitive impairment analysis, we observed a as reduction (P < 0.05) in the numbers of crossing and rearings for the animals treated with NXY-059 when compared with animals treated with vehicle (saline). Conclusions: According to these results, we can suggest that treatment with NXY-059 offered protection against cognitive impairment generated by sepsis. P108 Atorvastatin and simvastatin protects cognitive impairment in an animal model of sepsis Pedro CB Alexandre1*, Patricia A Reis1, Joana D’Ávila1, Flora M de J Oliveira1, Fabricio A Pamplona2, Luciana D Siqueira1, Hugo CC Faria Neto1, Fernando A Bozza3 1Laboratorio de Imunofarmacologia, IOC/FIOCRUZ, Rio de Janeiro, Brazil; 2Instituto D’Or de Pesquisa e Ensina - IDOR, Rio de Janeiro, Brazil; 3Instituto de Pesquisa Clinica Evandro Chagas - IPEC/FIOCRUZ, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 4):P108; doi:10.1186/cc13007 Background: Recently it was shown that a significant proportion of sepsis survivors can develop a transitory or permanent cognitive impairment. Statins have the ability to block the cascade of cholesterol formation by acting on HMG-CoA reductase, reducing the synthesis of endogenous cholesterol. Recently it has been observed that statins have anti-inflammatory properties preventing brain dysfunction in malaria models, reducing the production of brain cytokines, oxidative stress and alterations in the blood-brain barrier. The aim of the present study was to evaluate the ability of statins to reduce neuroinflammation and protect septic animals from neurocognitive damage. Materials and methods: Feces were extracted (5 mg/g b.w.) from the large intestine of SW mice and diluted in saline, centrifuged and the supernatant collected and injected into the animals (n = 5 to 8/group). Control animals received 0.5 ml saline. Animals were treated at 6, 24 and 48 hours after sepsis induction with imipenem (30 mg/kg b.w., 0.2 ml s.c.) and 1.0 ml saline (s.c.). Statins (Ator and Sinv) were administrated 1 hour before and 6, 24 and 48 hours after the infection (20 mg/kg b.w., p.o.). Mortality was observed for 96 hours and a score of severity evaluated. The inflammatory profile and oxidative damage was determined at 6 and 24 hours. In addition, mice brains were evaluated for microglial activation and BBB dysfunction. After 15 days we analyzed the cognitive damage using the inhibitory avoidance task and Morris water maze. Results: No significant difference in survival was observed comparing septic animals treated with antibiotics plus atorvastatin or simvastatin (56%; 53%) with septic animals with only antibiotics (37%). We observed lower levels of proinflammatory cytokines (IL-1, IL-6) and chemokines (KC and MCP-1) when comparing statin-treated animals and nontreated. We also observed a decreased in the oxidative damage in brains 6 hours after sepsis in the treated groups. Finally, statin treatment was able to protect septic animals from cognitive damage including avoidance and spatial memory, both affected in untreated infected mice. Conclusions: We can conclude that statins protected septic animals from cognitive damage, reducing neuroinflammation, and adjuvant therapies with statins can be interesting targets for future clinical trials focused on the prevention of long-term cognitive decline in sepsis. P109 Dasatinib has a dual effect on sepsis Cassiano F Gonçalves-deAlbuquerque*, Alessandra F Silveira, Cristina L Nagae, Carlos André M Silva, Mirian Priscila L Lima, Raysa Captivo, Larissa Camisão, Caroline L Hildebrandt, Patrícia T Bozza, Adriana R Silva, Hugo C Castro-Faria-Neto Laboratório de Imunofarmacologia, IOC/Fiocruz, Rio de Janeiro, Brazil Critical Care 2013, 17(Suppl 4):P109; doi:10.1186/cc13008 Background: Sepsis occurs as a result of a systemic inflammatory response to an infection. In this context, homeostasis of biological systems depends on regulatory mechanisms to modulate the amplitude of the immune response to stimuli, such as infection, preventing damage resulting from this imbalance of immune response. The exacerbated immune response can cause serious tissue or systemic damage, as occurs in autoimmune and chronic inflammatory diseases. The main aim of our study is to investigate the effect of dasatinib in polymicrobial sepsis. Materials and methods: Swiss mice were subjected to cecal ligation and puncture and treated with dasatinib 1, 5 and 10 mg/kg 30 minutes before and 6 and 24 hours after the surgery. Survival rate and clinical signs were assayed; cell accumulation, bacterial load were measured in peritoneal lavage and inflammatory mediators were measured in plasma. Results: Animals receiving dasatinib 5 and 10 mg/kg showed the worst clinical score and an increased mortality rate. Animals receiving dasatinib 1 mg/kg showed an increase in survival, a decrease in clinical score, in cell migration, in colony-forming units and cytokine production. Conclusions: Dasatinib has a dual effect in polymicrobial sepsis, where higher doses had deleterious effects but lower doses had beneficial effects, probably because lower doses may downregulate the immune response, avoiding extensive tissue damage. Acknowledgements: Financial support: Fiocruz, CNPq, Faperj, Vichem Chemie and TARKINAID. Background: Sepsis is a major cause of death in veterinary medicine, as in the human field, but there are no survival data described for this syndrome in the veterinary clinical field. This aspect challenges experimental medicine, may alter the baseline data to be applied in the human setting and could explain in part why most results obtained from laboratory research are not completely useful in the human clinical field. The purpose of this prospective observational study was to investigate the 24-hour and 30-day survival from severe sepsis and septic shock in canine septic patients that were approached with the Surviving Sepsis Campaign (SSC) bundles. Materials and methods: Nineteen client-owned puppies with naturally acquired parvovirus haemorrhagic gastroenteritis were classified as severe sepsis and septic shock patients and received medical care according to the guidelines proposed by the SSC. Subsequently, the 24-hour and 30-day survival was evaluated for each case. The results were statistically analysed by Fisher’s exact test at a significance level of 5%. Results: Fifteen patients (78.9%) were admitted to the emergency department and classified as severe sepsis subjects. The mortality rate in the severe sepsis group was 33.33% (five animals), of which four animals died in the first 24 hours of admission and the other on the following day. Four dogs (21.1%) were classified as septic shock patients. The mortality rate in the septic shock group was 100%, of which two animals died in the first 24 hours of admission and two on the day after (Table 1). Conclusions: The observation of clinical outcomes in this clinical canine sepsis model showed that the majority of deaths in both severe sepsis and septic shock occur within the first 24 hours. However, after 30 days there is a significant difference between both groups, showing no survival in septic shock animals. Therefore, this preliminary study suggests a new veterinary database to be applied for future human research. P111 Peroxisome proliferator-activated receptor agonist rosiglitazone improves host defense against Pseudomonas aeruginosa in a murine model of pneumonia Crisitina Lyra Nagae*, Cassiano Felippe Gonçalves-de-Albuquerque, Alessandra Silveira Ferreira, Raysa Captivo, Larissa Camisão, Carlos André Mandarino Silva, Mirian Priscila Lins de Lima, Caroline Loureiro Hildebrandt, Patricia Torres Bozza, Hugo Caire de Castro-Faria-Neto, Adriana Ribeiro Silva Laboratório de Imunofarmacologia, IOC/FIOCRUZ, Rio de Janeiro, RJ, Brazil Critical Care 2013, 17(Suppl 4):P111; doi:10.1186/cc13010 Table 1(abstract P110) Severe sepsis and septic shock animals classified as nonsurvivors and survivors 24 hours and 30 days after admission Nonsurvivors 24 hours Survivors 24 hours Nonsurvivors 30 days Survivors 30 days Background: Pseudomonas aeruginosa is a Gram-negative bacterium regarded as an opportunistic pathogen. It infects immunocompromised patients, and is the second leading cause of nosocomial diseases. This bacterium has numerous virulence factors, adapts quickly to new environments, and requires a few nutrients to survive. All of these mechanisms will generate a host response. The fastest immune response is neutrophil recruitment, followed by phagocytosis and degranulation. There is another mechanism to fight bacteria called NET formation, which is the formation of a neutrophil extracellular network. NET is formed through a process called NETosis where the release of the cell nuclear material can hold and destroy pathogens. The nuclear receptor peroxisome proliferatoractivated receptor PPARg, besides lipid and glucose metabolism, is involved in the inflammatory response modulation, being considered a potential target for the study of new therapies for inflammatory and infectious diseases. We therefore aim to investigate the involvement of PPARg in lung injury caused by P. aeruginosa using an agonist of this receptor, rosiglitazone. Materials and methods: For this purpose, Swiss mice were instilled intratracheally with bacteria and treated with rosiglitazone 5 hours after the operation. We analysed clinical signs using 10 physical parameters, cellularity and DNA measurement to assess NET formation. Results: We found that the animals stimulated with Pseudomonas showed an increase in inflammatory parameters, while the animals treated with rosiglitazone showed improvement in clinical signs and increased NET formation. Conclusions: We can conclude that rosiglitazone has an antiinflammatory role during lung infection, suggesting that PPARg activation may improve the host defense against bacteria. Acknowledgements: Financial support: FIOCRUZ, CNPq and FAPERJ. 1. Leifsson PS , Iburg T , Jensen HE , Agerholm JS , Kjelgaard-Hansen M , Wiinberg B , Heegaard PMH , Astrup LB , Olsson AE , Skov MG , et al: Intravenous inoculation of Staphylococcus aureus in pigs induces severe sepsis as indicated by increased hypercoagulability and hepatic dysfunction . FEMS Microbiol Lett 2010 , 309 : 208 - 216 . 2. Soerensen KE , Nielsen OL , Birck MM , Soerensen DB , Leifsson PS , Jensen HE , Aalbaek B , Kristensen AT , Wiinberg B , Kjelgaard-Hansen M , et al: The use of sequential organ failure assessment parameters in an awake porcine model of severe Staphylococcus aureus sepsis . APMIS 2012 , 120 : 909 - 921 . 3. Soerensen KE , Olsen HG , Skovgaard K , Wiinberg B , Nielsen OL , Leifsson PS , Jensen HE , Kristensen AT , Iburg TM : Disseminated intravascular coagulation in a novel porcine model of severe Staphylococcus aureus sepsis fulfills human clinical criteria . J Comp Pathol 2013 in press. P96 Microparticles from septic shock patients contain microRNA and messenger RNA: new players in the pathogenesis of sepsis? Luciano CP Azevedo1 , 2 , 3 *, Juliana M Real1 , João E Bezerra2 , Flavia R Machado3 , Reinaldo Salomao3 1Instituto Sírio-Libanês de Ensino e Pesquisa , São Paulo , Brazil; 2State University of São Paulo, Brazil; 3Federal University of São Paulo, Brazil Critical Care 2013 , 17 (Suppl 4):P96; doi:10.1186/cc12995

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Mark Simmonds, Esme Blyth, Marc Chikhani, Jaimie Coleman, Vivienne Weston, Tim Hills. Quality assurance in severe sepsis: an individualised audit/feedback system results in substantial improvements in sepsis care at a large UK teaching hospital, Critical Care, 2013, P61,