Gene expression patterns in multiple organs in experimentally induced Staphylococcus aureus sepsis in pigs

Background: Sepsis- associated encephalopathy (SAE) is an early and common feature of severe infections. Oxidative stress is one of the mechanisms associated with the pathophysiology of SAE. The goal of this study was to investigate the involvement of NADPH oxidase in neuroinflammation and in the long-term cognitive impairment of sepsis survivors. Methods: Sepsis was induced in WT and gp91 phox knockout mice (gp91 phox�/� ) by cecal ligation and puncture (CLP) to induce fecal peritonitis. We measured oxidative stress, Nox2 and Nox4 gene expression and neuroinflammation in the hippocampus at six hours, twenty-four hours and five days post-sepsis. Mice were also treated with apocynin, a NADPH oxidase inhibitor. Behavioral outcomes were evaluated 15 days after sepsis with the inhibitory avoidance test and the Morris water maze in control and apocynin-treated WT mice. Results: Acute oxidative damage to the hippocampus was identified by increased 4-HNE expression in parallel with an increase in Nox2 gene expression after sepsis. Pharmacological inhibition of Nox2 with apocynin completely inhibited hippocampal oxidative stress in septic animals. Pharmacologic inhibition or the absence of Nox2 in gp91 phox�/� mice prevented glial cell activation, one of the central mechanisms associated with SAE. Finally, treatment with apocynin and inhibition of hippocampal oxidative stress in the acute phase of sepsis prevented the development of long-term cognitive impairment. Conclusions: Our results demonstrate that Nox2 is the main source of reactive oxygen species (ROS) involved in the oxidative damage to the hippocampus in SAE and that Nox2-derived ROS are determining factors for cognitive impairments after sepsis. These findings highlight the importance of Nox2-derived ROS as a central mechanism in the development of neuroinflammation associated with SAE.

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. 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%.   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.  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:  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.

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 1,2* , Gebru Mulugeta 1 , Ahmed Bedru 3 , Ibrahim Ali 1,2 , Damte Shimeles 4 , Tsehaynesh Lema 3 , Abraham Aseffa 3 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][2][3][4]. It has been confirmed by culture that is associated with clinical manifestation and systemic response [5][6][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.   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 (R 2 = 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.

Conclusions:
The assay shown here for the quantification of PCT fulfils all requirements for POCT. Within 9 minutes, PCT can be quantified near the patient's bed in whole blood without sample preparation. Acknowledgements: The authors thank Dr A Geerlof (HMGU) for producing recombinant PCT, Dr E Kremmer (HMGu) for producing the mAbs and Dr P Miethe from the Forschungszentrum für Medizintechnik und Biotechnologie (fzmb GmbH) for the delivery of the patient plasma samples. 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.
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.  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 anti-S. 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   [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][3][4][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. Background: Sepsis still represents the leading cause of mortality among children and its etiology changes according to age, immune status and geographic location [1][2][3][4]. Prevention of this disease has key role in reducing morbidity and mortality and includes development and application of vaccines [5][6][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.  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.  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]. 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.   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 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     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  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.

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Severe pneumonia in critically ill cancer patients: clinical outcomes and a comparison between healthcare-associated pneumonia and community-acquired pneumonia 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 TNFα, soluble TNF receptor (STNFR)-1 and STNFR-2, IL-1β, 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-1β suggesting that this cytokine is differently modulated when comparing septic and nonseptic patients with delirium. Background: Sepsis is a complex and multifactorial syndrome, whose incidence, morbidity and mortality have been increasing worldwide. The knowledge of clinical, epidemiological and hemodynamic parameters responsible for its evolution, diagnosis and treatment are still the subject of many studies. Therefore, this study aims to evaluate clinical, laboratory and hemodynamic parameters of morbidity and mortality in patients with severe sepsis and septic shock.  the emergency room (ER). The same intensivist team assists all patients in the ER and during ICU permanence. The principal investigator did not perform any orientation or intervention in the treatment of selected patients. Clinical (age, sex, infection focus, fluid balance, hemodialysis, use of corticosteroids, antibiotic therapy, APACHE II, SOFA), laboratory (blood cell counting, lactate, creatinine, bilirubin, glucose, cortisol, NT-proBNP, C-reactive protein (CRP), procalcitonin (PCT), Troponin I), hemodynamic (blood pressure, heart rate, left ventricular systolic function (echocardiography)) and respiratory parameters (respiratory rate, PaO 2 /FiO 2 ), PEEP and peak inspiratory pressure (PIP)) were analyzed from ICU admission until discharge or death. Echocardiography was performed at 48 hours and on the 10th day after ICU admission. Results: Seventy-two patients (64% male), mean age 52 ± 19 years, were consecutively included, 21% (15/72) with severe sepsis and 79% (57/72) with septic shock. Mortality was 18% (13/72), of these 21% (3/13) for severe sepsis and 79% (10/13) for septic shock. Median APACHE II score was 28 (16 to 37) and SOFA score 6 (5 to 10) ( Table 1). There was positive correlation between mortality with: male gender, APACHE II, SOFA, positive 24-hour fluid balance, hemodialysis indication, corticosteroid use, leukopenia, lactate, NT-proBNP and PCT levels ( Table 2). From univariate analysis, practically the same significant association with mortality was observed (Table 3). In addition, the final multivariate Cox model showed    Background: Rapid response teams (RRTs) represent an intuitively simple concept: when a patient demonstrates signs of imminent clinical deterioration, a team of providers is summoned to the bedside to immediately assess and treat the patient with the goal of preventing ICU transfer, cardiac arrest, or death [1]. Patients whose condition deteriorates acutely while hospitalized often exhibit warning signs (such as abnormal vital signs) in the hours before experiencing adverse clinical outcomes. Sepsis is an illness in which the body has a severe response to bacteria or other germs. This response may be called systemic inflammatory response syndrome (SIRS) [2]. The criteria for calling the RRT are the same as/similar to symptoms of sepsis. We aimed to describe the various criteria for calling the RRT for patients who developed sepsis, initial treatment before transfer to the ICU or step-down unit and outcomes.  was also initiated in the ward. The serum lactate + measured blood culture was 40 (63.5%) and fluid administration was 41 (64.1%) ( Table 1).
Pressing the RRT was in 43 (66.2%) cases by the staff member with significant concern about the patient's condition, 27 (41.5%) cases by changes in systolic blood pressure, and 23 (35.4%) cases due to change in oxygen saturation ( Table 2).

Conclusions:
The criteria for calling the RRT can support the prompt identification of patients who have sepsis and prevent disease progression. Furthermore, the treatment may also be performed in the ward and may result in a reduction in mortality. Results: Twenty-six publications found and gave rise to two categories. First, the identification of clinical signs and CPR maneuver by the nursing staff and the nurse in the PCR are essential for successful resuscitation: the authors agree that the service systematized-based SAV protocol is essential for there to be success in CPR. Recognition theoretical and practical skills of the staff are among the most important determinants of the success rates of RC [1]. Thus, it is necessary that health professionals, especially nursing staff, be aware of the clinical signs of PCR. Furthermore, the residence time of the professional nursing staff in the ICU causes them to gain more experience, making it easier to identify clinical signs and cardiac rhythms [2,3]. Second, the impact of continuing education on quality of nursing care in a PCR: the proper training of the nursing staff, especially those that operate in the ICU, is vital for emergency treatment PCR. Identifying the theoretical and practical knowledge of staff about the PCR and PCR is an important prerequisite for planning a training service [2]. The nurse as team leader and organizer of the ICU is the right professional to establish measures to be taken at the time of the PCR. The nurse has a responsibility to properly distribute the measures to be implemented at the time of service of the PCR, identifying it early and minimizing damage [4]. Conclusion: Continuing education has significant impact in improving the level of knowledge of nursing professionals, leading to survival of patients in a hospitalized ICU, as it ensures the identification of the signs and symptoms of CRP in patients in the ICU. Background: Increased prevalence of vitamin D deficiency (VDD) in sepsis and its association with sepsis severity has been documented in adults [1][2][3]. However, data on the pediatric population are scarce. This study aims at assessing the prevalence of VDD (25-hydroxyvitamin D (25(OH)D) level <20 ng/ml) among children with sepsis in developing nations and its implication on sepsis severity. Materials and methods: A prospective observational study conducted between January and December 2012. During the study period all consecutive PICU admissions between the ages of 1 and 12 years were screened for sepsis at the time of admission to the ICU. Out of 613 PICU admissions, 124 patients satisfying the criteria for sepsis [4] were randomly enrolled and followed up throughout the hospital stay. Patients with an immunosuppressed state or receipt of vitamin D within the 3 months prior to hospital admission were excluded. A control group comprising of 40 healthy children was also included for comparison with the general population. The serum 25(OH)D level was measured in all patients with sepsis within 24 hours of admission to the PICU. Severity of sepsis was assessed using the Pediatric Risk of Mortality III (PRISM III) score and the daily Sequential Organ Function Assessment (SOFA) score. Results: Patients with sepsis had low 25(OH)D levels compared with healthy controls (P = 0.04). Median 25(OH)D level among patients was 19.7 ng/ml (interquartile range (IQR): 12.5, 31.2) and median 25(OH)D level among controls was 30.4 ng/ml (IQR: 22.1, 38). Prevalence of VDD was high among patients 51% (95% confidence interval (CI), 42 to 59) compared with the VDD of 17% (95% CI, 8 to 32) in healthy controls (P < 0.001) ( Table 1). No significant correlation was found between vitamin D level and PRISM III score or daily SOFA score. Out of 19 deaths, 17 (90%) deaths occurred in patients with vitamin D deficiency and insufficiency (odds ratio 3.09, 95% CI: 0.6 to 20.7). However, the difference in mortality was not statistically significant (P = 0.58). Factors such as septic shock, multiorgan Staff member has significant concern about the patient's condition

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Critical Care 2013, Volume 17 Suppl 4 http://ccforum.com/supplements/17/S4 dysfunction syndrome (MODS), duration of mechanical ventilation, blood culture positivity, hypocalcemia and length of PICU stay were not modified by the presence of VDD (Table 2). Conclusions: We found a high prevalence of VDD among children with sepsis when compared with healthy children but VDD was not associated with the severity of sepsis or its outcome. Background: Renal transplantation is the treatment of choice for endstage renal disease as it is cost-effective, and improves survival and quality of life as compared with maintenance dialysis [1,2]. However, the need for immunosuppression increases the hazard of septic complications [3]. Sepsis is one of the leading causes of death among renal transplant recipients and little is known about its characteristics in this population [4,5]. The aim of this study was to evaluate the factors associated with mortality in renal transplant patients admitted to the ICU with severe sepsis and septic shock.

Materials and methods:
We conducted a single-institution retrospective observational cohort study in consecutive renal transplant patients admitted to the ICU with severe sepsis or septic shock in a public high-volume kidney transplant center from 1 June 2010 and 31 December 2011. We registered demographic data, transplant characteristics and sepsis management to identify predictive factors of ICU, hospital and 1-year mortality.
Results: A total of 190 patients were enrolled. The mean age was 51 ± 13 years, 115 (60.5%) were male, 122 (64.2%) were deceased donors, median APACHE was 20 (16 to 23) and median admission SOFA was 5 (4 to 8). The most common source of infection was respiratory (59.5%) followed by urinary tract (16.8%). Tachypnea, tachycardia, fever, hypothermia, leukocytosis and leukopenia were present in 74.7%, 67.9%, 24.2%, 6.3%, 26.3% and 16.3% of the patients. The most prevalent dysfunction was respiratory (68.4%) followed by cardiovascular (41.1%) and renal (40.5%). The median time between transplantation and the septic event was 2.1 (0.6 to 7.8) years. The duration of organ dysfunction before the diagnosis of sepsis was 2.5 (1.1 to 5.2) hours. The median length of ICU and hospital stay was 6 (3 to 13) and 20 (12 to 35) days, respectively. Hospital and 1-year mortalities were 38.4% and 42.6%, respectively. In the multivariate analysis, male gender, the variation in the SOFA score after the first 24 hours, the need for mechanical ventilation, the presence of hematologic dysfunction, being admitted from the wards and AKI stage 3 were predictors of hospital mortality.
Conclusions: In the present study, independent factors associated with mortality were related to features of sepsis severity and not to factors associated with transplantation. Another interesting finding was the low frequency of signs of systemic inflammatory response.    [2]. Results: Severe sepsis was found in 1,018 (52%), cryptic shock in 162 (8%), vasoplegic shock in 549 (28%) and dysoxic shock in 219 (12%) patients. Mean age was 60 years, 47% were male and the majority was admitted form the emergency department (47%). The lung was the principal source of infection, followed by the urinary tract and abdominal. Overall, the four groups presented significant differences in APACHE II and SOFA scores (P < 0.001 for both), dysoxic shock being the most severe group. In post-hoc analysis, patients in the severe sepsis group presented similar SOFA score to patients in the cryptic shock group (P = 0.20). Overall, 28-day crude survival was different between groups (P < 0.001), being higher for the severe sepsis group (69%, P < 0.001 vs. other), similar between cryptic and vasoplegic shock (53%, P = 0.39) and lower for dysoxic shock (38%, P < 0.001 vs. other). In an adjusted analysis considering age, APACHE II and SOFA, the 28-day survival remained different between groups (P < 0.001) and the hazard ratio for the dysoxic shock group was the highest: Conclusions: The present study has some limitations because of being a retrospective observational study. However, the mortality was quite low in the group of patients included in this study. Moreover, after IVIG treatment values of WBC, CRP and procalcitonin were improved. The median value of serum IgG before treatment was within the normal range, but after treatment was also significantly improved. There is a possibility that severe septic patients require additional IgG regardless of its normal concentrations in their blood.   .7)). The main reason for ICU admission was respiratory failure (70%). Nonsurvivors needed mechanical ventilation (88% vs. 48%, P < 0.01) and vasopressors (71% vs. 41%, P = 0.05) more frequently. Neurological dysfunction was more common in nonsurvivors (79% vs. 41%, P = 0.01, odds ratio 5.2 (95% CI 1.5 to 18.2)). After multivariate analysis, neurological dysfunction was associated with hospital mortality, while HAART in the first 30 days of hospitalization was a protective associated factor. Conclusions: Disseminated TB was the most common presentation in HIV/AIDS critically ill patients. Nonsurvivors were more prone to multiple organ dysfunction syndrome, and neurological dysfunction was associated with hospital mortality. The administration of HAART within 30 days of hospitalization was associated with survival. Background: Sepsis is a systemic inflammation caused by severe infection.

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It is a life-threatening condition, progresses rapidly, and affects multiple system functions. An evidence-based medical sepsis bundles model has been used for sepsis care in clinical practice. All patients who have at least two signs or symptoms of systemic inflammatory response syndrome (SIRS) secondary to an infectious process are considered septic. Sepsis is the leading cause of death in ICUs and a major cause of late hospital mortality rate, exceeding the acute coronary syndromes and neoplasms. Mortality in Brazil reaches 60%, while the world average is around 30%, overcoming countries such as India and Argentina. The early recognition and treatment of these patients are key to reducing mortality. The aim of this study is to evaluate the implementation of the protocol of sepsis in a university general hospital in Porto Alegre. Materials and methods: Retrospective evaluation of protocols for sepsis in emergency in 2012.
Results: A total of 200 patients were enrolled in the protocol during the study period. The average age was 35 years (SD ± 16.5), 51% of patients were male, the most frequent focus was respiratory 61%, and the second urinary with 14%. Clinical criteria for inclusion in the protocol that most prevailed were: axillary temperature and heart rate, with more than 95%. Altered axillary temperature was present in 98% of the sample. Of these cases, 86.5% (n = 173) of patients were discharged within 24 hours. Twentyseven patients met criteria for hospitalization, 22% required the ICU. Around 75% (n = 20) of inpatients had no blood cultures collected before starting antibiotics. Only 7% mortality (n = 2). Conclusions: The criteria for inclusion in the protocol are quite sensitive and the number of visits per month in the emergency exceeds 10,000. A total of 200 patients enrolled to the sepsis care protocol in a year, over 80% of these being discharged within 24 hours, suggests a low adherence to institutional protocol, especially in patients with septic shock, which is reinforced by the very low mortality compared with literature data. The evaluation of these data was essential to bring the knowledge that adherence to the protocol is still very low in our institution.
Background: A 69-year-old woman underwent elective surgical repair of an abdominal aortic aneurysm. Intraoperative lesions were intestinal and splenic, requiring performing segmental bowel resection and splenectomy. By hemodynamic instability the patient was maintained on mechanical ventilation in norepinephrine and was transferred to the ICU. After 3 days she had fever, tachycardia, hypotension and anuria, with output fetid and purulent secretion by the tracheal tube. Chest X-ray showed opacity in the right lung; cultures were collected and cefepime initiated empirically for treatment of ventilator-associated pneumonia. Acinetobacter baumannii was isolated sensitive only to polymyxin-E in the sample of tracheal secretions. An exchange of antimicrobial therapy was made, but the patient developed refractory shock and died. Materials and methods: We report the case of a patient with septic shock. Background: Elevated serum arterial lactate levels are often associated with an imbalance between oxygen demand and delivery, which has a strong correlation with poorer outcomes in critically ill patients [1,2]. This study aims to evaluate serum arterial lactate as a predictor of mortality in critical patients admitted with severe sepsis and septic shock. Materials and methods: Retrospective cohort study conducted in the ICU of Hospital Anchieta, Brasília, DF, Brazil, during 3 years. For the first analysis, patients were divided into two groups: group with arterial lactate >2 mmol/l and group with low arterial lactate ≤2 mmol/l at the time of admission. For a second analysis, patients were divided into two groups: group with arterial lactate >3.3 mmol/l and group with arterial lactate ≤3.3 mmol/l at the time of admission.

Materials and methods:
The present study is a retrospective cohort conducted over a 3-year period in the ICU of Hospital Anchieta, Brasília, Brazil. Patients were divided into two groups: severe sepsis without shock septic (SW) and severe sepsis with septic shock (SS). The patients coming from other ICUs or transferred to other ICUs were excluded.
Conclusions: Patients admitted with septic shock had higher mortality than patients admitted with severe sepsis without septic shock, but there was no difference between the groups with respect to length of stay in the ICU.

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Prognostic factors in the first hour post admission for intra-hospital mortality in patients with septic shock in an ICU Background: Severe sepsis and septic shock are common and are associated with substantial mortality and substantial consumption of healthcare resources [1]. Although the incidence of septic shock has steadily increased during the past several decades, the associated mortality rates have remained constant or have decreased only slightly [2]. Our study aimed to identify the prognostic factors during the first hour after admission for intra-hospital mortality in patients with septic shock in a general ICU. Background: Sequential Organ Failure Assessment (SOFA) is a largely used score in the evaluation of organ dysfunction/failure in septic patients [1]. Repeated scoring can also assess patient condition and disease development [2]. The present study aims to describe the association between SOFA score and the organ dysfunction components of this score with mortality in septic patients admitted to an ICU. Background: The platelet count is an established index in the evaluation of severity in patients with sepsis, and therefore is a component of the SOFA score [1]. Furthermore, the alterations in leukocyte count are also used in the definition of SIRS. The present study aims to evaluate the accuracy of the platelet/leukocyte ratio (P/L) as a predictor of mortality in septic patients. Materials and methods: Retrospective cohort study conducted on patients admitted to the ICU of Hospital Anchieta, Brasília, DF, Brazil, during 3 years. The patients with sepsis were divided according to P/L as follows: P/L ≥8 group (HPL) or P/L <8 (LPL). The primary outcome was  , and length of stay in the ICU was 9 ± 11 days. Mortality at 4 days was 10.8% (n = 21) and at 28 days was 12.3% (n = 24). The groups P/L <8 and P/L ≥8 did not present differences regarding age (59 ± 20 vs. 65 ± 22, P = 0.07) and APACHE II (22 ± 9 vs. 20 ± 9, P = 0.19). The LPL group had higher SAPS3 (68 ± 18 vs. 59 ± 13, P = 0.00). The LPL was significantly associated with mortality in 4 days (18% vs. 7%, P = 0.02) and 28 days (19% vs. 9%, P = 0.03 Background: The increase of the older population, with higher incidence of co-morbidities and age-related decline in organic functions, suggests a need for better understanding the peculiarities of the process leading to adequate critical care in this group of patients [1,2]. This study aims to evaluate morbimortality of sepsis on older and nonolder patients and its impact on their outcomes. Materials and methods: Retrospective cohort study conducted in the ICU of Hospital Santa Luzia, Brasilia, DF, Brazil, during 6 months. Patients diagnosed with sepsis were divided into two groups: older, defined as age ≥65 years, and nonolder, with age <65, for the analysis of the outcomes. Results: A total of 130 patients with sepsis were enrolled, 11.5% (n = 15) with septic shock. Mean age was 64 ± 22 years, ICU length of stay 9.6 ± 13.2 days, SAPS3 50 ± 13. ICU mortality in 4 days was 6.9% (n = 13), in 28 days was 9.2% (n = 12) and hospital morality was 17.7% (n = 23). Seventy patients were older (60.8%). The older patients had higher SAPS3 (56 ± 12 vs. 40 ± 9, P = 0.00), Charlson Comorbidity Index (CCI) (2.4 ± 1.9 vs. 1.0 ± 1.7, P = 0.00), and incidence of Glasgow Coma Scale <15 (19% vs. 5.9%, P = 0.03). There was no difference between the groups regarding the incidence of septic shock (12.7% vs. 9.8%, P = 0.62), need for dialysis ( Results: A total of 76 patients were enrolled, 10.5% (n = 8) with septic shock. Age was 70 ± 18 years, SAPS3: 52.9 ± 13.9, APACHE II: 15.5 ± 8.8. The ICU length of stay was 9 ± 10 days. ICU mortality was 21% (n = 16). The most prevalent sites of infections were respiratory (57.9%, n = 44), followed by urinary (25%, n = 19) and cutaneous (6.6%, n = 5). The incidence of tachycardia was the only parameter higher in the NSG (37.5% vs. 9.1%, P = 0.00). There was no difference regarding the incidence of fever or hypothermia ( Background: Measures to ensure an appropriate early treatment for critically ill patients result in significant decreases in mortality [1,2]. This study aims to evaluate the impact of the elapsed time between ICU request and actual admission of patients with SIRS/sepsis on ICU mortality and length of stay. Materials and methods: Retrospective cohort study conducted in the ICU of Hospital Santa Luzia, Brasilia, DF, during 3 months. Patients being consecutively admitted to the ICU with diagnostic of SIRS/sepsis were divided into two groups: those with elapsed time between ICU request and admission less than 6 hours (short waiting period group (SWP)) or over 6 hours (long waiting period group (LWP)). Results: A total of 70 patients were enrolled (46% of admissions), 14 patients with SIRS, 27 with sepsis, 13 with severe sepsis and 17 with septic shock. For the entire cohort, the mean age was 61 ± 22 years, APACHE II was 12 ± 7.7, ICU length of stay was 15 ± 22.8 days, and 39 were male (54,9%). Thirty-five patients belonged to the LWP (50%). LWP patients had higher mortality (50% vs. 19.6%, P = 0.04), and longer ICU length of stay (13.6 ± 18.5 vs. 23.5 ± 40.7 days, P = 0.04). Relative risk for death in the LWP was 2.83 (95% CI: 1.28 to 6.28). Conclusions: The elapsed time between ICU request and actual admission of patients with SIRS/sepsis over 6 hours resulted in increased ICU mortality and ICU length of stay for this group of patients. Background: Advances in the treatment of burns have reduced mortality rates and improved quality of life of victims. However, the most frequent complication is infection [1]. Thermal injury over 20% of the body surface area may lead to conditions similar to SIRS, as in septic shock. Beyond the extent of body surface area burned, which causes structural changes in skin coverage, other factors lead to infectious complications in burned patients: immunosuppression resulting from thermal injury, the possibility of gastrointestinal bacterial translocation, prolonged hospitalization, the use of devices and surgical procedures related to the burned areas [2,3]. C-reactive protein (CRP) is a known marker of infection and sepsis in patients admitted to the ICU. Materials and methods: CRP was measured in a cohort of 18 critically ill mechanically ventilated victims of a fire disaster in the city of Santa Maria, Brazil, on 27 January 2013, admitted to the ICU of the Hospital de Clínicas de Porto Alegre. The patients were divided into groups according to CPR levels, group 1 (CPR ≤190 mg/l) and group 2 (CRP >190 mg/l), and the Mann-Whitney test was performed to compare groups according to mortality, length of ICU and hospital stay, presence of sepsis and SOFA score on days 1, 3 and 7.

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Clinical performance of a point-of-care assay for measurement of presepsin in patients with bacteremia Yasuo Fukui 1* , Yoshikazu Okamura 2 Background: Severe sepsis is the main cause of death in the ICU. Relatives are at risk for post-traumatic stress disorder (PTSD) or anxiety and depression [1]. The objective is to assess whether the ICU experience may predict these psychological symptoms of relatives at 90 days after the patient's death or discharge. Materials and methods: Prospective observational study in four ICUs of one university hospital, including all patients with severe sepsis and endof-life-decisions. At 90 days, the main relative was interviewed with the Impact of Event Scale (to measure PTSD), the Hospital Anxiety and Depression Scale and self-developed items on satisfaction with the ICU experience, including medical care and communication in general as well as specifically in the end-of-life context, and decision-making. Three multiple linear regression models were calculated to predict anxiety, depression and post-traumatic stress each.
Results: Eighty-four relatives were included. They were mostly female (74%), spouse (42%) or child (42%), median age was 57 years. Seventyseven percent acted as proxies. After 90 days, 51% relatives were at risk for PTSD, 48% for anxiety and 33% for depression. Overall satisfaction with the ICU experience was high. Relatives' satisfaction with medical care and communication in general predicted lower anxiety (P = 0.025).
Conclusions: Relatives of patients with sepsis have a high psychological burden. Improving communication between ICU staff and relatives may reduce their symptoms of anxiety.

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Quality assurance in severe sepsis: an individualised audit Background: Severe sepsis has a high mortality and high healthcare costs. Rapid recognition and treatment can save lives but requires a coordinated response [1]. Hospital-wide audits in 2005 and 2010 showed significant deficiencies when compared with international guidelines, with 35% of cases receiving antibiotics in <1 hour and only 25% receiving basic pre-ICU interventions in a timely manner. By time-lining our response to severe sepsis, we identified system and process failures [2]. Some system improvements (for example, providing first-line antibiotics in acute areas) were straightforward to tackle, but sepsis care remained reliant on individual clinician response. Equally, whilst dissemination of organisation-level audit data raised the profile of sepsis, it appeared that individual clinicians did not view it as 'their problem'. It is recognised that individualised feedback can improve care, as pride and the competitive nature of healthcare workers drives improvement. This is especially true when adherence to recommended practice is low [3]. We tried to change behaviour by creating a rapid response audit/feedback mechanism that informed clinicians of their own response to the severely septic patient, from which they could learn and improve. Materials and methods: Patients admitted to any critical care unit (58 beds, four units, two sites) with a primary admission diagnosis of infection were screened for severe sepsis. The pre-ICU care of patients who met the criteria was then audited against the Surviving Sepsis Guidelines [1]. Time zero was defined as when the criteria for severe sepsis were first met. Information on timings of key interventions (such as doctor review and request for critical care escalation) was also gathered. An individualised traffic-light report was then generated and emailed to the patient's consultant and other stakeholders such as junior doctors or nurses involved in the patient's care (Figure 1). We aimed to report cases back within 7 days of arrival to ensure the patient story was fresh in the clinician's mind. A cumulative report is generated monthly to track organisation-wide performance.
Results: Since November 2011 we have provided feedback on over 300 severe sepsis cases. Antibiotic administration in <1 hour has risen from 35% to 75% (Figure 2), and pre-ICU bundle compliance has risen from 25% to 70% (Figure 3)    Critical Care 2013, Volume 17 Suppl 4 http://ccforum.com/supplements/17/S4 2013 were enrolled, Discrimination and calibration of the ISS, NISS, SIRS score (the first 3 days post trauma), ISS/LD 50ISS , NISS/LD 50NISS , ISS´SIRS score, NISS´SIRS score, ISS/LD 50ISS´S IRS score, NISS/LD 50NISS´S IRS score, ISS+SIRS score, NISS+SIRS score, ISS/LD 50ISS +SIRS score, and NISS/LD 50NISS +SIRS score in predicting post-trauma sepsis were compared using receiver operator characteristic (ROC) curves and Hosmer-Lemeshow statistics. Results: The LD 50 values of ISS for age 16 to 44, 45 to 64, and ≥65 were 55, 49, and 33, respectively (for males: 54, 50, and 31; for females: 60, 47, and 39). The LD 50 values of NISS for age 16 to 44, 45 to 64, and ≥65 were 62, 56 and 43, respectively (for males: 62, 56, and 42; for females: 65, 53, and 48). The predicting capability of ISS/LD 50ISS +SIRS score and NISS/LD 50NISS +SIRS score were equivalent (area under the ROC curve = 0.932 versus 0.932) and both showed better discrimination than others in predicting post-trauma sepsis. For ISS/LD 50ISS +SIRS score, the cutoff value of ROC curve was 2.2128, with a positive predictive value of 65.86%, a negative predictive value of 95.95%, a sensitivity of 87.13%, a specificity of 87.11%, a positive likelihood ratio of 6.76, a negative likelihood ratio of 0.15, a Youden index of 0.7424, and an accuracy of 87.11%. For NISS/ LD 50NISS +SIRS score, the cutoff value of ROC curve was 2.3208, with a positive predictive value of 66.87%, a negative predictive value of 95.98%, a sensitivity of 87.13%, a specificity of 87.68%, a positive likelihood ratio of 7.07, a negative likelihood ratio of 0.15, a Youden index of 0.7481, and an accuracy of 87.56%. Conclusions: This study calculates for the first time the LD 50 values of ISS or NISS from Chinese trauma patients. The novel and simple formulae ISS/ LD 50ISS +SIRS score and NISS/LD 50NISS +SIRS score are then set up to predict the incidence of sepsis following traumatic injury, which perform better at predicting capability than ISS, NISS, SIRS score and other formulae including LD 50 values of ISS or NISS. Background: Sepsis is a major challenge in medicine, its high incidence, mortality and high costs making this syndrome the leading cause of mortality in ICUs, and is considered a health problem in a worldwide extension that affects millions of people and results in high morbidity and mortality. It is believed there are 18 million annually reported cases, and of every four people diagnosed one is victimized by sepsis [1][2][3]. The aim of this study was to identify the main aspects in the treatment of sepsis in the last 10 years. Materials and methods: A quantitative, descriptive and cross-sectional, literature study, concerning the main aspects in the treatment of sepsis. A semi-structured instrument developed by the authors was used to collect data to categorize the studies obtained. After collection, an electronic spreadsheet was generated, and data were analyzed using descriptive statistics. Results: Ten studies with a central theme focused on the treatment of sepsis were used. Seventy percent of these studies were between the years 2008 and 2011. Fifty percent of the articles mentioned that the early approach of the infectious agent is very important for successful treatment, while 60% reported that the control of the infectious focus is one of the main alternatives. Fifty percent of the studies also reported an infusion of antibiotics in accordance with the infectious focus as essential to the treatment of sepsis, and 80% reported the use of activated protein C as an indicator for diagnosis septic patients. It is observed that most studies seek early detection of the infection and early antibiotic administration, which reinforces the need for optimization of processes for the bundle of the first hour of the sepsis protocol proposed by the Latin American Institute of Sepsis (ILAS). Conclusions: The present study therefore concludes that for effective treatment of sepsis an early approach right after diagnosis of the disease is indispensable. Likewise, the treatment of sepsis primarily seeks to control the infectious focus using specific antibiotics. Also, the use of activated protein C may be a good alternative in the diagnosis of this pathology and a good indicator for controlling this disease.

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Epidemiology of sepsis in a university hospital in Rio de Janeiro Sergio da Cunha 1* , Mario CA Perez 1 , Elisabete N Ferreira 2 , Julio CD Correal 2 , Viviane S Silva 2 , Eliane PP Assumpção 2 , Luana F Almeida 2 , Catherine C Valdez 3 , Jéssica R Oliveira 3  Background: Severe sepsis and septic shock are challenges in critical medicine care and there are few epidemiologic studies in public university hospitals in Brazil. Materials and methods: A prospective study was performed to determine the epidemiology of the sepsis in hospitalized patients in our institution (600-bed tertiary teaching urban hospital) from September 2012 to May 2013. The criteria for sepsis definition were obtained from the 2013 guidelines [1]. Clinical and epidemiological data were collected from patients' records. A univariate, bivariate and multivariate analyses were performed. Results: In total, we analyzed 103 patients with severe sepsis and septic shock during the period of study. The frequency of male gender was 55.8% and a median age of 62 years was observed in the patients. The median acute physiology and chronic health disease classification system II (APACHE II) score estimated was 21.1 and a community origin of sepsis was present in 53.4% of them with a mortality rate of 61.3%. Yet in 57 (64.0%) patients with healthcare-associated sepsis, the mortality rate was 63.1% and the risk of death was higher for this group (odds ratio (OR) = 5.54; 95% confidence interval (CI) = 2.19 to 14.0; P < 0.05). In the entire group, 53.4% had septic shock and 60.1% entered the vasopressor protocol. In relation to the source of infection, the top three were: pulmonary (51.4%), abdominal (14.5%) and urinary (12.6%). We observed the greatest risk of death in the group with pulmonary infection (OR = 3.08; 95% CI = 1.1 to 8.5; P = 0.03). The prevalence of positive blood cultures was 32.1% and 23 microorganisms were identified, these being 65.2% Gram-negative bacilli (Klebsiella pneumoniae (21.7%), Escherichia coli (17.3%)), 21.7% Gram-positive cocci and 13.6% fungi. Lethality in sepsis episodes was associated independently with the delay in starting antibiotic therapy (more than 6 hours: OR = 2.94; 95% CI = 1.05 to 8.02; P = 0.04), inappropriate plasmatic volume expansion use (less than 20 ml/kg: OR = 2.84; 95% CI = 1.07 to 7.5; P = 0.03) and pulmonary source of sepsis (OR = 3.08; 95% CI = 1.1 to 8.5; P = 0.03). The use of corticosteroids seemed to increase the mortality rate, but in the multivariate analysis this association failed to reach statistical significance (OR = 2.2; 95% CI = 0.08 to 6.5, P = 0. 1). Conclusions: Enterobacteriaceae and pulmonary sepsis were the main factors responsible for triggering sepsis. Fast and aggressive fluid therapy and early adequate antibiotics are mandatory to change the lethality in severe sepsis and septic shock. Further studies evaluating the effect of therapy with corticosteroids should be assessed.  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 (10 4 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 TNFα and IL-1α, 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, TNFα and IL-1α). 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 TNFα and IL-1α along with elevation of IL-10 levels in the lung homogenate of mice with acute lung infection was observed upon treatment with 2-CADO 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 10 8 CFU/ml intravenous (i.v.) inoculation, LD 60 , or S9 group, with E. coli 10 9 CFU/ml inoculation, LD 80 . 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 (LD 80 ), 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.
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 10 7 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. 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. 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 10 8 (S8) or 10 9 (S9) CFU/ml Escherichia coli inoculum intravenously (i.v.) (DL 80 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.  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 10 8 CFU/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 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 morbidity and mortality by promoting an increasing organ dysfunction in the long term.  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 IBα 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 IBα 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. 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, IgG 2a ) or anti-CD11b mAbs (clone 44, IgG 1 ) 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 × 10 5 ), 2% of autologous serum, glucose and luminol in Ca 2+ -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 and LPS primed neutrophils during the first 50 seconds after fMLP addition is presented in Figure 1.
Results: Re-LPS revealed the most neutrophil priming activity in comparison with S-LPS ( Figure 1A), which is in accordance with our previous work [6]. Actually, mAbs against TLR4 as well as against CD11b did not inhibit neutrophil priming by E. coli endotoxins. Moreover, the incubation of cells with anti-TLR4 or anti-CD11b mAbs followed by endotoxin priming increased fMLP-induced ROS production ( Figure 1A). However, the differences between priming effectiveness of S-LPS and Re-LPS, which had been seen in endotoxin primed cells, were leveled by prior cell exposure to anti-CD11b mAbs. Neutrophils exposed to anti-TLR4 mAbs retained their ability to distinguish between S-LPS or Re-LPS being primed, respectively ( Figure 1A). Incubation with isotypic IgG 2a decreased fMLP-induced ROS production from unprimed neutrophils ( Figure 1B) that was not observed in the case of IgG 1 . These results demonstrate that Fc regions of isotypic immunoglobulins and therefore of mAbs used in our study are not silent parts of these molecules regarding neutrophil surface receptors and their intracellular signaling pathways. Finally, the incubation of cells with isotypic immunoglobulins and then with Re-LPS did not abrogate neutrophil priming for subsequently fMLP-triggered ROS production.
Conclusions: The inhibition of human neutrophil CD11b by specific mAbs (clone 44) did not abolish LPS-dependent neutrophil priming for fMLP-induced respiratory burst, but eliminated the capacity of these cells to distinguish between S-LPS or Re-LPS glycoforms. Unlike the effect of anti-CD11b mAbs, neutrophil exposition to anti-TLR4 mAbs (HTA125) did not inhibit neutrophil priming and capacity of these cells to distinguish endotoxin's glycoforms.

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Taurine chloramine decreases cell viability and cytokine production in blood and spleen lymphocytes from septic rats submitted to sepsis 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 (TNFα, IFNγ, 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 Critical Care 2013, Volume 17 Suppl 4 http://ccforum.com/supplements/17/S4 for 24 hours at a concentration of 1 × 10 6 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.

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CD40 expression in the hippocampus and its role in blood-brain barrier permeability, neutrophil infiltration and oxidative stress: implication for brain damage associated with sepsis in rats Monique Michels 1* , Lucinéia G Danielski 1 , Andriele A da Silva Vieira 1 , Luiz Carlos Vieira 1     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   divided into sham, CLP, CLP + 1 ng, CLP + 10 ng and CLP + 100 ng anti-CD40 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. 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][2][3]. However, LPS can also affect ECs in the absence of participation by immune cells [4][5][6]. Although interactions between LPS and ECs evoke endothelial death, a significant portion of ECs are resistant to LPS challenge [6][7][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 model of endotoxemia. ECs were exposed to 20 mg/ml LPS for 72 hours, while IBV was challenged to 20 mg/ml LPS on the inside for 48 hours. Results: ECs exposed to LPS showed a fibroblast-like morphologic change. In addition, LPS-treated ECs showed an upregulation of both fibroblastspecific protein expression such as fibroblast specific protein-1 and αsmooth muscle actin, and extracellular matrix proteins secretion including fibronectin and collagen type III. In concordance, ECs exposed to endotoxin showed a severe downregulation of endothelial markers such as vascular endothelial cadherin and the platelet endothelial cell adhesion molecule-1 (CD31). Similar results were obtained in the endothelial monolayer from IBV perfused with LPS in which abundant fibrosis was observed. Furthermore, we demonstrate that LPS-induced EC fibrosis is dependent on the endotoxin receptor toll-like receptor-4. In addition, the participation of NAD(P)H oxidase activity and ROS generation was demonstrated using specific blockers. Finally, we demonstrated by means of small interfering technology and a pharmacological inhibitor that LPS-induced EC fibrosis is dependent on the activin like kinase-5 kinase activity, suggesting that tumor growth factor beta is involved in this fibrotic process. Conclusions: We conclude that LPS is able and sufficient to promote endothelial fibrosis. It is noteworthy that LPS-induced endothelial fibrosis perpetuates endothelial dysfunction as a maladaptive process rather than a survival mechanism for protection against LPS. These findings are useful in improving current treatment against endotoxemia-derived sepsis syndrome and other inflammatory diseases. 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 Ca 2+ free and contracted by CaCl 2 (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 Ca 2+ -free solution, in the presence or absence of thapsigargin. Results: Maximal contraction (E max ) induced by KCl or PE was reduced, especially in the CLP-24 group. Similarly, CaCl 2 -induced contraction was reduced (60%) in the CLP-24 group. Thapsigargin (sarcoplasmatic calcium reuptake blocker) and DTNB (sulphydryl oxidation) restored the contraction elicited by CaCl 2 in septic rings, but without effect in control rings. PE-induced contraction in calcium-free solution was significantly reduced in CLP-24 rings (E max 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 Ca 2+ -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/dt max and dP/dt min , 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. 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. 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 NOS-2 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. 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 TNFα 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 TNFα plasma concentration at 2, 4 and 6 hours in the three groups; ECP pretreatment inhibited the increase of TNFα 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. 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).
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.

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Effect of polymicrobial sepsis on the respiratory mechanism of rats previously exposed to cigarette smoking 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. 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.  [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 β-amyloid peptide (Aβ), receptor for advanced glycation endproducts (RAGE) and synaptophysin were analyzed by western blot. Results: Aβ 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 Aβ levels. Prefrontal levels of synaptophysin correlated with performance in the inhibitory avoidance.
Conclusions: The brain from sepsis survivor animals presented several markers of neurodegeneration, and inhibitory avoidance test performance seems to be dependent on the levels at some of these markers.

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Epigenetic 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. 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: 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. 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.

P92
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 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 TNFα, IL-1β 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 TNFα, IL-1β 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 modulation of cytokines, nitric oxide and augmented vasopressin blood levels.
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  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.  [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 × 10 6 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.

P98
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). 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-1β 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-1β 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. 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. PPARγ is a lipid-activated nuclear receptor, which participates in inflammation, lipid metabolism and cellular differentiation. Previous studies have shown the role of PPARγ 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 PPARγ 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 PPARγ, post-septic derived BMDC were pretreated with PPARγ 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-1β expression. When post-septic and Sham mice were infected with BCG, we observed higher increased COX2, CCR2 and IL-1β 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 TNFα, CCL2 and IL-1β levels). PPARγ flanked mice in CD11c cells were more susceptible to severe sepsis. Activation of PPARγ 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 PPARγ could be important for development of new therapy in sepsis-associated immunosuppression and long-term inflammatory diseases.

P102
Epigenetic changes in dendritic cells in sepsis-associated 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-1α, 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 Critical Care 2013, Volume 17 Suppl 4 http://ccforum.com/supplements/17/S4 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, TNFα, IL-6 and TGF-β) 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 TNFα and TGF-β 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. 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.
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-1β 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. 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.

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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 β-amyloid protein in the brain of CCR2 -/naive mice, we observed the increased in β-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. 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.

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Atorvastatin and simvastatin protects cognitive impairment in an animal model of sepsis 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.

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Dasatinib has a dual effect on sepsis Cassiano F Gonçalves-deAlbuquerque * , Alessandra 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.

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Peroxisome proliferator-activated receptor agonist rosiglitazone improves host defense against Pseudomonas aeruginosa in a murine model of pneumonia 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 PPARγ, 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 PPARγ 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 PPARγ activation may improve the host defense against bacteria. Acknowledgements: Financial support: FIOCRUZ, CNPq and FAPERJ.