A doença pneumocócica invasiva é um grande desafio global. Bacteremia está fortemente relacionada às condições clínicas de imunodepressão. Pacientes com câncer raramente são alvo de estudos clínicos. Neste estudo, nosso objetivo primário foi avaliar os fatores prognósticos em pacientes com câncer com bacteremia pneumocócica. Os objetivos secundários foram descrever os episódios de bacteremia e as características fenotípicas das cepas isoladas. O desenho foi uma coorte retrospectiva, que incluiu pacientes com câncer com mais de 18 anos de idade que tiveram bacteremia pneumocócica, de janeiro de 2009 a junho de 2015, no Instituto do Câncer do Estado de São Paulo. Os critérios de infecção foram aqueles delineados pelo NHSN/CDC. Os desfechos avaliados foram mortalidade em 48h e em 10 dias, e sobrevida dos pacientes. As variáveis independentes analisadas foram: idade, sexo, etnia, ECOG, Karnofsky escore, SOFA, diagnóstico do câncer, presença de metástases, quimioterapia, radioterapia, neutropenia, uso prévio de antibióticos, internação hospitalar prévia, internação prévia em UTI, comorbidades (DPOC sistêmica, Hipertensão Arterial, Diabetes Mellitus e Insuficiência Cardíaca), tabagismo, vacinação antipneumocócica prévia, sítio de infecção, infecção polimicrobiana, suscetibilidade antimicrobiana, sorotipo e tratamento. A associação das características com os desfechos foi verificada por meio dos testes qui-quadrado, teste de razão de verossimilhança ou teste exato de Fisher. A análise multivariada foi realizada por regressão logística stepwise, incluindo todas as variáveis com P < 0,10. A sobrevida foi estimada de acordo com cada característica avaliada pela função de Kaplan-Meier, seguida de testes log-rank para comparar a sobrevida entre as categorias das variáveis qualitativas. As Razões de Risco (HR) foram calculadas com os respectivos intervalos de confiança de 95% usando regressão de Cox bivariada. Todas as variáveis que apresentaram nível de significância de 0,10 (p < 0,1) foram testadas no modelo múltiplo, e as variáveis do modelo final foram selecionadas pelo método stepwise com critério de seleção Backward com entrada e saída de 5%. Os testes foram realizados com nível de significância de 5%. Foram detectadas 165 bacteremias pneumocócicas no período do estudo em 161 pacientes oncológicos; destas, 4 episódios foram excluídos por se tratarem de recidivas. Assim, 161 episódios foram incluídos para análise descritiva e estatística. A casuística foi composta em sua maioria por homens (62,7%), com uma média de idade de 61,3 anos. A maioria da casuística foi composta por indivíduos com tumores sólidos (121/75,2%). Entre os tumores sólidos, os mais prevalentes foram os do trato gastrointestinal, seguido dos tumores de cabeça e pescoço e trato respiratório. Entre as neoplasias hematológicas (40/24,8%), as mais prevalentes foram mieloma múltiplo (20/50,0%), seguidos de linfomas não Hodgkin (14/35,0%). A mortalidade em 48h e em 10 dias foi, respectivamente, de 20,5% e 33,5%. Entre 120 cepas testadas, 61 (50,8%) corresponderam aos encontrados na vacina VCV13 e 89 (74,2%) na VPV23. As variáveis associadas a mortalidade em 48h, na análise multivariada, foram: SOFA (OR 1,37, p < 0,001) bacteremia polimicrobiana (OR 3,78, p0,018). Na análise multivariada para mortalidade em 10 dias, os fatores associados foram: febre 48h antes da bacteremia (OR 0,35, p0,026), neutropenia vigente (OR 4,01, p0,011), ECOG ¾ (OR 5,83, p0,001), SOFA (OR 1,41, P < 0,001 - um aumento unitário do SOFA resultou em um aumento de 59% na chance de mortalidade) e uso de quinolonas (OR 0,08, p0,023). Na sobrevida global, as variáveis relacionadas detectadas foram: presença de metástases (HR 1,97, p < 0,001), KARNOFSKY < 90 (HR 1,56, p0,021), SOFA (HR 1,27, p < 0,001), uso de anticorpos no último mês (HR 3,25, p0,026) e bacteremia polimicrobiana (HR 1,69, p0,046). Em conclusão, a bacteremia pneumocócica apresentou alta mortalidade em pacientes oncológicos, com prognóstico relacionado a fatores intrínsecos do hospedeiro e aos episódios de infecção, como o SOFA presente como preditor de mortalidade em 48h, 10 dias e piora da sobrevida global. A presença de bacteremia polimicrobiana associou-se a mortalidade em 48h e piorou a sobrevida. Presença de febre e uso de quinolonas foram fatores de proteção para a mortalidade em 10 dias; a presença de febre provavelmente encurtou o tempo entre o início da sepse e a demanda por serviços de saúde, com antibioticoterapia mais precoce. Não foi possível estimar o efeito protetor da vacinação antipneumocócica devido ao pequeno número de pacientes vacinados / Invasive pneumococcal disease is a major global challenge. Bacteremia is strongly related to the clinical conditions of immunodepression. Patients with cancer are rarely the target of clinical trials. In this study, our primary objective was to evaluate the prognostic factors in patients with pneumococcal bacteremia. The secondary objectives were to describe the episodes of bacteremia and the phenotypic characteristics of the isolated strains. The design was a retrospective cohort that included cancer patients over 18 years of age who had pneumococcal bacteremia from January 2009 to June 2015 at the \"Instituto do Câncer do Estado de São Paulo\". The criteria for infection were those outlined by the NHSN / CDC. The outcomes evaluated were mortality in 48h and 10 days and overall survival. The independent variables analyzed were: age, sex, ethnicity, ECOG, Karnofsky score, SOFA, diagnosis of cancer, presence of metastasis, chemotherapy, radiotherapy, neutropenia, previous use of antibiotics, previous hospital admission, previous ICU admission, comorbidities (systemic COPD, Hypertension, Diabetes mellitus and Congestive Heart Failure), smoking, previous antipneumococcal vaccination, site of infection, polymicrobial infection, antimicrobial susceptibility, serotype and treatment. The association of the characteristics with the outcomes was verified using chi-square tests, likelihood ratio test or Fisher\'s exact test. Multivariate analysis was performed by stepwise logistic regression, including all variables with P < 0.10. Survival was estimated according to each characteristic assessed by the Kaplan-Meier function, followed by log-rank tests to compare survival between categories of qualitative variables. The Hazard Ratios (HR) were calculated with the respective 95% confidence intervals using bivariate Cox regression. All variables that presented a significance level of 0.10 (p < 0.1) were tested in the multiple model, and the variables of the final model were selected by stepwise method with Backward selection criterion with input and output of 5%. The tests were performed with a significance level of 5%. We detected 165 pneumococcal bacteremias during the study period in 161 cancer patients; of these, 4 episodes were excluded because they were recurrences. Thus, 161 episodes were included for descriptive and statistical analysis. Our study consisted mostly of men (62.7%), with a mean age of 61.3 years. The majority of cases were individuals with solid tumors (121 / 75.2%). Among the solid tumors, the most prevalent tumors were those of the gastrointestinal tract, followed by tumors of the head and neck and respiratory tract. Among the hematological malignancies (40/24.8%), the most prevalent were multiple myeloma (20 / 50.0%), followed by non-Hodgkin\'s lymphomas (14 / 35.0%). Mortality in 48h and 10 days was, respectively, 20.5% and 33.5%. Among 120 strains tested, 61 (50.8%) corresponded to those found in the VCV13 vaccine and 89 (74.2%) in the VPV23. The variables associated with 48-h mortality in the multivariate analysis were: SOFA (OR 1.37, p < 0.001) and polymicrobial bacteremia (OR 3.78, p 0.018). In the multivariate analysis for 10-day mortality, the associated factors were: fever 48h before bacteremia (OR 0.35, p 0.026), current neutropenia (OR 4.01, p 0.011), ECOG ¾ (OR 5.83 , p 0.001), SOFA (OR 1.41, P < 0.001 - a unit increase of SOFA resulted in a 59% increase in chance of mortality) and use of quinolones (OR 0.08, p0.023). In the overall survival, the related variables detected were: presence of metastases (HR 1.97, p < 0.001), KARNOFSKY < 90 (HR 1.56, p0.021), SOFA (HR 1.27, p < 0.001), use of antibodies in the last month (HR 3.25, p0.026) and polymicrobial bacteremia (HR 1.69, p0.046). In conclusion, pneumococcal bacteremia presented high mortality in cancer patients, with a prognosis related to intrinsic host factors and infection episodes, such as SOFA present as a predictor of mortality in 48h, 10 days and worsening of overall survival. The presence of polymicrobial bacteremia was associated with 48-h mortality and worsened survival. Presence of fever and use of quinolones were protective factors for mortality in 10 days; the presence of fever probably shortened the time between the onset of sepsis and the demand for health services, with earlier antibiotic therapy. It was not possible to estimate the protective effect of anti-pneumococcal vaccination due to the small number of patients vaccinated

In under a century, melioidosis, the infection due to Burkholderia pseudomallei, has emerged from Whitmore’s series of glanders-like infections amongst the morphia addicts in Burma to a major cause of mortality in northeastern Thailand and northern Australia. Also endemic in other parts of south-east Asia, melioidosis may have varied presentations ranging from severe, overwhelming infection to chronic, low grade disease. Observational evidence had suggested that granulocyte colony stimulating factor (G-CSF), a naturally occurring substance produced by the body in response to infection, may have been useful in reducing the high mortality associated with the more severe forms of this infection. Other observations linked the occurrence of this disease to various environmental factors, such as contamination of drinking water and the annual rainfall. This thesis explores and attempts to quantify these associations. There are three parts to this thesis. In the first part, I reviewed the epidemiology and management of patients with melioidosis. The use of G-CSF and meropenem was associated with a fall in mortality, although other factors may have at least partially contributed to this effect. In the second part, I progressed towards a clinical trial of G-CSF. There was no other evidence supporting the use of G-CSF in severe sepsis and ethical issues precluded a trial in Darwin. There was not evidence from laboratory models of G-CSF action in melioidosis to support the use of G-CSF in patients, although there remained some doubt regarding the applicability of such models to human disease. I examined clinical methods to identify patients at high risk of death from melioidosis. A simple scoring system based on clinical and laboratory parameters was developed and externally validated. However, clinical definitions of severe sepsis appeared to be better predictors of mortality. A clinical trial based on clinical definitions was commenced in Thailand. In the final part, I explored the question of whether different strains or B. pseudomallei or different environmental conditions caused different patterns of infection. There was no evidence that strain types of this bacterium determine the pattern or severity of disease, but weather conditions appeared to influence the distribution of disease in northern Australia.

Doctor of Philosophy / Department of Industrial & Manufacturing Systems Engineering / David H. Ben-Arieh / Chih-Hang Wu / Patients who suffer from sepsis or septic shock are of great concern in the healthcare system. Recent data indicate that more than 900,000 severe sepsis or septic shock cases developed in the United States with mortality rates between 20% and 80%. In the United States alone, almost $17 billion is spent each year for the treatment of patients with sepsis. Clinical trials of treatments for sepsis have been extensively studied in the last 30 years, but there is no general agreement of the effectiveness of the proposed treatments for sepsis. Therefore, it is necessary to find accurate and effective tools that can help physicians predict the progression of disease in a patient-specific way, and then provide physicians recommendation on the treatment of sepsis to lower risk for patients dying from sepsis. The goal of this research is to develop a risk assessment tool and a risk management tool for sepsis. In order to achieve this goal, two system dynamic mathematical models (SDMMs) are initially developed to predict dynamic patterns of sepsis progression in innate immunity and adaptive immunity. The two SDMMs are able to identify key indicators and key processes of inflammatory responses to an infection, and a sepsis progression. Second, an integrated-mathematical-multi-agent-based model (IMMABM) is developed to capture the stochastic nature embedded in the development of inflammatory responses to a sepsis. Unlike existing agent-based models, this agent-based model is enhanced by incorporating developed SDMMs and extensive experimental data. With the risk assessment tools, a Markov decision process (MDP) is proposed, as a risk management tool, to apply to clinical decision-makings on sepsis. With extensive computational studies, the major contributions of this research are to firstly develop risk assessment tools to identify the risk of sepsis development during the immune system responding to an infection, and secondly propose a decision-making framework to manage the risk of infected individuals dying from sepsis. The methodology and modeling framework used in this dissertation can be expanded to other disease situations and treatment applications, and have a broad impact to the research area related to computational modeling, biology, medical decision-making, and industrial engineering.

Η σήψη αποτελεί μια από τις σημαντικότερες αιτίες νοσηλείας και θνησιμότητας στον ανεπτυγμένο κόσμο, όπου σχεδόν τα δύο - τρίτα του πληθυσμού υποφέρουν από παχυσαρκία. Σαν αποτέλεσμα, η συνύπαρξη των δύο αυτών καταστάσεων έχει γίνει όλο και συχνότερη στην κλινική πράξη και ένας συνεχώς αυξανόμενος αριθμός κλινικών μελετών προσπαθεί να προσεγγίσει την πιθανή επίδραση της παχυσαρκίας στην νοσηρότητα και θνησιμότητα των ασθενών με σήψη, με έως τώρα αντιφατικά αποτελέσματα. Σκοπός της παρούσας μελέτης είναι να διερευνήσει τον τρόπο με τον οποίο η παχυσαρκία επηρεάζει την ανοσιακή απάντηση των σηπτικών ασθενών, εκτιμώντας τον αριθμό και την κατάσταση ενεργοποίησης των μακροφάγων του λιπώδους ιστού, τα επίπεδα του TNFα στον ορό και στον λιπώδη ιστό και δείκτες οξειδωτικού stress στο πλάσμα. Ασθενείς και Μέθοδοι: Μελετήθηκαν 106 ασθενείς, οι οποίοι χωρίστηκαν σε τέσσερις ομάδες (Ελέγχου n=26, Παχυσαρκίας n=27, Σήψης n=27, Σήψης & Παχυσαρκίας n=26). Ο αριθμός των μακροφάγων στο υποδόριο και ενδοκοιλιακό λίπος και οι υπότυποί τους (M1 και M2) αναγνωρίστηκαν με ανοσοϊστοχημική τεχνική υπό μικροσκόπηση. Τα επίπεδα του TNFα mRNA στο υποδόριο και ενδοκοιλιακό λίπος μετρήθηκαν με real-time reverse transcription-PCR. Στον ορό τα επίπεδα του TNFα μετρήθηκαν με sandwich enzyme-linked immunosorbent assay (ELISA). Το οξειδωτικό stress στο πλάσμα εκτιμήθηκε χρησιμοποιώντας επιλεγμένους βιοδείκτες [TBARS (thiobarbituric acid-reactive substances), Πρωτεϊνικά Καρβονύλια, TAC (total antioxidant capacity)]. Αποτελέσματα: Παρατηρήθηκε ότι η σήψη αυξάνει τον ολικό αριθμό και τον Μ2 υπότυπο των μακροφάγων στο ενδοκοιλιακό λίπος, ενώ η παχυσαρκία δεν φάνηκε να επηρεάζει τη συγκέντρωση των μακροφάγων στο λίπος. Η παχυσαρκία βρέθηκε ότι αυξάνει τα επίπεδα του TNFα mRNA (P<0.05) στο ενδοκοιλιακό λίπος καθώς επίσης και τα επίπεδα των TBARS (P<0.001) και Πρωτεϊνικών Καρβονυλίων (P<0.001) στο πλάσμα των σηπτικών ασθενών. Τα επίπεδα της TAC στο πλάσμα βρέθηκε ότι μειώνονται και τα επίπεδα TNFα στον ορό ότι αυξάνονται με τη σήψη, ενώ δεν επηρεάζονταν από την παχυσαρκία. Συμπεράσματα: Η παχυσαρκία σχετίζεται με αυξημένη παραγωγή TNFα στον λιπώδη ιστό και αύξηση του οξειδωτικού stress, προάγοντας την προ-φλεγμονώδη απάντηση στους σηπτικούς ασθενείς. / Sepsis is one of the most important causes of mortality in the developed world, where almost two thirds of the population suffer from obesity. Therefore, the coexistence of both conditions has become frequent in clinical practice and a growing number of clinical studies attempts to examine the potential effect of obesity on sepsis with controversial results up to now. The present study investigates how obesity influences the immune response of septic patients, by assessing the number and activation state of adipose tissue macrophages, serum and adipose tissue tumor necrosis factor-alpha (TNFα) levels and plasma oxidative stress markers. Subjects/methods: The study included 106 patients, divided into four groups (control n = 26, obesity n = 27, sepsis n = 27 and sepsis and obesity n = 26). The number of macrophages in subcutaneous and visceral adipose tissue (SAT and VAT) and their subtypes (M1 and M2) were defined with immunohistochemical staining techniques under light microscopy. TNFα mRNA levels were determined in SAT and VAT using real-time reverse transcription-PCR. Serum levels of TNFα were determined with sandwich enzyme-linked immunosorbent assay. Plasma oxidative stress was evaluated using selective biomarkers [thiobarbituric acid-reactive substances (TBARS), protein carbonyls and total antioxidant capacity (TAC)]. Results: Sepsis increased the total number of macrophages and their M2 subtype in VAT, whereas obesity did not seem to affect the concentration of macrophages in fat. Obesity increased TNFα mRNA levels (P < 0.05) in VAT as well as the plasma TBARS (P < 0.001) and protein carbonyls (P < 0.001) in septic patients. The plasma TAC levels were decreased and the serum TNFα levels were increased in sepsis although they were not influenced by obesity. Conclusions: Obesity is associated with elevated TNFα adipose tissue production and increased oxidative stress biomarkers, promoting the proinflammatory response in septic patients.

<p>Nitric oxide (NO) is an endogenous gaseous signaling molecule produced by three NO synthase isoforms (NOS1, 2, 3) and important in host defense. The induction of NOS2 during bacterial sepsis is critical for pathogen clearance but its sustained activation has long been associated with increased mortality secondary to multiple organ dysfunction syndrome (MODS). High levels of NO produced by NOS2 incite intrinsic cellular dysfunction, in part by damaging macromolecules through nitration and/or nitrosylation. These include mitochondrial DNA (mtDNA) and enzymes of key mitochondrial pathways required for maintenance of normal O2 utilization and energy homeostasis. However, animal studies and clinical trials inhibiting NOS2 have demonstrated pronounced organ dysfunction and increased mortality in response to live bacterial infections, confirming that NOS2 confers pro-survival benefits. Of particular interest here, the constitutive NOS1 and NOS3 have been linked to the up-regulation of nuclear genes involved in mitochondrial biogenesis but no comparable role has been described for NOS2. <italic> Therefore, I hypothesized that NOS2 is indispensible for host protection but must be tightly regulated to ensure NO levels are high enough to activate mitochondrial and other pro-survival genes, but below the threshold for cellular damage.</italic></p><p>This hypothesis was explored with two major Aims. The <italic>first Aim</italic> was to define the role of NOS2 in the activation of mitochondrial biogenesis in the heart of <italic>E. coli</italic>-treated mice. The <italic>second</italic> was to investigate the ability of NOS2 to be transcriptionally regulated by an enzyme previously shown to induce mitochondrial biogenesis, heme oxygenase-1 (HO-1). This hypothesis was tested using an <italic>in vivo</italic> model of sublethal heat-killed <italic>E. coli</italic> (<italic>HkEC</italic>) peritonitis in C57B/L6 (Wt), NOS2-/-, and TLR4-/- mice. Additionally, <italic>in vitro</italic> systems of mouse AML-12 or Hepa 1-6 cells pretreated with HO-1 activators or <italic>Hmox1</italic> shRNA prior to inflammatory challenge with lipopolysaccharide (LPS) +/- tumor necrosis factor-&alpha; (TNF-&alpha;). For the first Aim, Wt, NOS2-/-, and TLR4-/- mice were treated with (<italic>HkEC</italic> and cardiac tissue analyzed for mitochondrial function, expression of nuclear and mitochondrial proteins needed for mitochondrial biogenesis, and histological expression of NOS2 and TLR4 relative to changes in mitochondrial mass. For the second Aim, Wt mice were pretreated with hemin or carbon monoxide (CO) to activate HO-1 prior to <italic>HkEC</italic>-peritonitis. Liver tissue in these animals was evaluated at four hours for HO-1 induction, <italic>Nos2</italic> mRNA expression, cytokine profiles, and nuclear factor (NF)-&kappa;B activation. Liver cell lines were pretreated with hemin, CO-releasing molecule (CORM), or bilirubin one hour before LPS exposure and the <italic>Nos2</italic> transcriptional response evaluated at two and 24 hours. The MTT assay was used to confirm that <italic>in vitro</italic> treatments were not lethal. </p><p>These studies demonstrated that <italic>HkEC</italic> induced mtDNA damage in the heart that was repaired in Wt mice but not in NOS2-deficient mice. In KO mice, sustained mtDNA damage was associated with the reduced expression of nuclear (NRF-1, PGC-1&alpha;) and mitochondrial (Tfam, Pol-&gamma;) proteins needed for mitochondrial biogenesis. The findings thus supported that NOS2 is required for mitochondrial biogenesis in the heart during Gram-negative challenge. Evaluation of the relationship between HO-1 and NOS2 in murine liver was more complex; HO-1 activation in <italic>HkEC</italic>-treated Wt mice attenuated 4-hour <italic>Nos2</italic> gene transcription. In liver cell lines, hemin, CORM, and bilirubin were unable to suppress <italic>Nos2</italic> expression at the time of maximal induction (2 hours). <italic>Nos2</italic> was, however, suppressed by 24 hours, suggesting that the regulatory impact of HO-1 induction was not engaged early enough to reduce <italic>Nos2</italic> transcription at 2 hours. It is concluded that NOS2 induction in bacterial sepsis optimizes the expression of the mitochondrial biogenesis transcriptional program, which subsequently can also be regulated by HO-1/CO in murine liver. This provides a potential new mechanism by which immune suppression and mitochondrial repair can occur in tandem during the acute inflammatory response.</p> / Dissertation

Hydroxyethyl starches (HES) are the most routinely used synthetic colloids during fluid resuscitation and have reported effects on coagulation. The overall goal of the investigation in this thesis was to evaluate the effects of tetrastarch administration on hemodynamic, laboratory, and hemostatic variables in healthy dogs and dogs with systemic inflammation. The objectives were to compare hemodynamic and laboratory variables in dogs receiving an isotonic crystalloid (0.9% NaCl) or tetrastarch during health and after induction of systemic inflammation; to compare the hemostatic effects of an isotonic crystalloid (0.9% NaCl) and synthetic colloid (tetrastarch) in healthy dogs and dogs with induced systemic inflammation; to compare two different protocols for TEG® activation and to determine the correlation between TEG® variables and traditional coagulation test results. Sixteen adult purpose-bred Beagles were randomized into one of two groups receiving fluid resuscitation with either 40 mL/kg IV isotonic crystalloid (0.9% NaCl) or synthetic colloid (tetrastarch) after administration of lipopolysaccharide (LPS; 5 μg/kg, IV) or an equal volume of placebo (0.9% NaCl, IV). Blood samples, for analysis, were collected at 0, 1, 2, 4, and 24 hours from the time of fluid resuscitation. After a 14-day washout period, the study was repeated such that dogs received the opposite treatment (LPS or placebo) and the same resuscitation fluid. Resuscitation with equal volumes of 0.9% NaCl and tetrastarch caused similar changes in hemodynamic and laboratory variables in dogs with LPS-induced systemic inflammation; however, larger increases in HR and blood pressure were seen within the first 2 hours following tetrastarch administration compared to 0.9% NaCl. Tetrastarch administration increased COP in all dogs, despite a decrease in TS. Tetrastarch bolus administration to dogs with LPS-induced systemic inflammation also resulted in a transient hypocoagulability characterized by a prolonged PTT, decreased clot formation speed and clot strength, and acquired type 1 von Willebrand disease. Considering the limited additional benefit of tetrastarch administration on hemodynamic variables demonstrated, as well as the transient adverse hemostatic effects of tetrastarch administration, the increased cost associated with the use of tetrastarch likely negates its use as a first line treatment during fluid resuscitation in dogs. / Pet Trust Fund

Sepsis claims over 215,000 lives in the US annually. Inducible glucocorticoids (iGC) is produced during sepsis. However, the precise effects of iGC in sepsis remain unclear due to a lack of appropriate animal models. Glucocorticoid (GC) insufficiency is associated with a marked increase in mortality and occurs in 60% of severe septic patients. Yet the conclusion of GC therapy on septic patients is still controversial. Scavenger receptor class B type I (SR-BI) in the adrenal mediates the selective uptake of cholesteryl ester from lipoproteins for GC synthesis. SR-BI-/- mice completely lack iGC during sepsis and are highly susceptible to septic death, which presents SR-BI-/- mice as a GC insufficient model. However, SR-BI-/- mice display multiple defects contributing to septic death, making it difficult to study iGC by using these mice. Therefore, we utilized adrenal-specific SR-BI-/- mice (ADR-T SR-BI-/-) generated by adrenal transplantation. As expected, the ADR-T SR-BI-/- mice failed to generate iGC under cecal ligation and puncture (CLP)-induced sepsis and showed a significantly higher mortality than the control mice, demonstrating that iGC is essential for preventing septic death. High blood urea nitrogen (BUN) was observed in the ADR-T SR-BI-/- mice but not in the control mice in CLP, indicating that iGC protects kidney injury in sepsis. Plasma IL-6 was remarkably higher in the ADR-T SR-BI-/- mice than the control mice, demonstrating an anti-inflammatory effect of iGC in sepsis. The ADR-T SR-BI-/- mice also displayed significantly lower phagocytic activity of monocytes and neutrophils in the blood and lower activation of T cells in the spleen compared to the control mice in CLP, suggesting that iGC is immunomodulatory in sepsis. Low-dose GC supplementation significantly improved the survival of SR-BI-/- mice in CLP, but did not increase the survival rate of SR-BI+/+ mice in CLP, indicating that GC supplementation improves the survival specifically in mice with adrenal insufficiency. Overall, we revealed that iGC is essential for sepsis survival. iGC prevents kidney damage, modulates inflammatory responses and exerts immunomodulatory functions in sepsis. GC supplementation specifically improves survival of individuals with adrenal insufficiency in sepsis.

Septic shock is a major cause of deaths resulting from uncontrolled inflammation and circulatory failure. Recent studies suggest that the vascular isoform of ATP-sensitive K+ (KATP) channels is an important contributor to septic susceptibility. To understand the molecular mechanisms for channel regulation during sepsis, we performed studies in isolated endothelium-denuded mesenteric rings. Lipopolysaccharides (LPS) induced vascular relaxation and hyporeactivity to phenylephrine. The LPS-treated aortic smooth muscle cells displayed hyperpolarization and augmentation of KATP channel activity. Both were due to an up-regulation of Kir6.1 and SUR2B surface expression. The up-regulation relied on transcriptional and translational mechanisms, in which nuclear factor-¦ÊB (NF-¦ÊB) and Protein kinase A (PKA) played a critical role. Oxidative stress occurs during sepsis and may act as another regulatory mechanism affecting KATP channel activity and vascular contractility. We found that micromolar concentrations of H2O2 impaired the pinacidil-induced vasodilation. The effect attributed to the suppression of KATP channel activity, which can be fully produced by reactivity oxidants. Unlike the Kir6.1/SUR2B channel, the Kir6.2/SUR2B channel was insensitive to 1mM H2O2, indicating that the modulation sites are located in Kir6.1. Site-directed mutational analysis showed that three cysteine residues located in N-terminus and the core region of Kir6.1 were likely to mediate the redox-dependent channel modulation. Arginine vasopressin (AVP) is a vasoconstrictor that is successfully applied to manage sepsis. However, the downstream target of AVP is uncertain. Our studies show that AVP-induced vasoconstriction depended on V1a receptor, Protein kinase C (PKC) and KATP channel. Additionally, AVP decreased Kir6.1/SUR2B channel activity through V1a receptor. The inhibitory effect was caused by a suppression of the channel open state probability. The channel inhibition was mediated by phosphorylation of the channel protein by PKC. The widespread involvement of the vascular KATP channel in vascular responses to endotoxemia strongly suggests that the temporospatial control of channel activity may constitute an important intervention to vascular tone, blood pressure and organ-tissue perfusion in septic shock. Such a control appears feasible by targeting several modulatory mechanisms of intracellular signaling, Kir6.1/SUR2B expression, redox state and channel protein phosphorylation as demonstrated in this dissertation.

The purpose of this research study was to improve the nursing care of intravenous catheters by providing evidence on the effects of prolonged duration of intravenous administration set use. Intravenous therapy is a vital part of modern health care. However, its invasive nature can result in infection, with high associated morbidity and mortality. The highest infection rates are displayed in intensive care patients with central venous catheters. The duration of intravenous administration set use may have an impact on infection rates,however the current practice usage and the optimum duration of use is unknown. Previous studies of central venous catheters have reported equal infection rates with 1 to 4 days of administration set use; however few patients have been evaluated with administration sets used beyond this time. Previous research has been limited by the inadequacy of available definitions for Catheter-Related Infection. A prospective, randomised, controlled clinical trial was performed to assess the infection risk of using administration sets for prolonged periods. In the developmental phase prior to the clinical trial; definitions of Catheter-Related Bloodstream Infection (CRBSI) were developed; a nursing practice survey was undertaken to establish the current duration of administration set use; and laboratory experiments were executed to assess the impact of prolonged use on administration set physical integrity and performance. Central venous catheters were randomised to have their administration sets used for 4 days (n = 203) or 7 days (n = 201). Percutaneous central venous catheters were enrolled into the study from two adult intensive care units at a metropolitan, tertiary-referral, teaching hospital. Catheters were multiple-lumen, chlorhexidine-gluconate and silver-sulphadiazine coated lines, both inserted and removed in the intensive care unit. Catheters were cultured for microbial colonisation on removal using the Maki roll-plate technique. Patients were assessed for CRBSI using the developed definitions consisting of categories: definite, probable (type I and II), possible and absent. Prior to the clinical trial, a practice survey questionnaire was administered, and laboratory experimentation was performed. Normality of distribution for continuous variables was assessed using the Kolmogorov- Smirnov statistic. The distribution between groups of variables considered risk factors for Catheter-Related Infection were tested to assess for bias using Chi-square and T-test. Logistic regression modelling was performed to analyse the influence of potentially confounding variables. The incidence of catheter colonisation and CRBSI was tested between groups using Kaplan-Meier survival curve with Log-rank test. Paired T-tests were performed to test for difference in programmed and delivered volumes of administration sets. A general linear model (ANOVA)± a Scheffe post hoc test to isolate difference was fitted to the standardised values of delivered volumes to determine the effects of day of measurement and volume delivery rate on the accuracy of volume delivery. There were 10 colonised tips in the intervention group and 19 in the control group. This difference was not statistically significant (Kaplan Meier survival analysis, Log Rank = 0.87, df = 1, p = 0.35). There were 3 cases of CRBSI per group and the difference in survival from CRBSI was not statistically significant (Kaplan Meier with Log Rank test, p = 0.86). The pre-clinical trial phases of the research programme established that current clinical practice was 3 to 7-day use of administration sets; that administration sets were physically intact and delivered clinically accurate volumes after 7 days of use; and developed useful definitions of CRBSI. Prolonged intravenous administration set use of 7 days was found to have no significant impact on patient infection indicators or physical performance of the sets. This finding is congruent with previous research and trends in current clinical practice. In conclusion, the research findings support the use of intravenous administration sets for 7 days.

BACKGROUND: Sepsis is a clinical syndrome characterised by the systemic response to infection. It is a common problem in modern intensive care units and is associated with significant morbidity and mortality. Though the underlying cause of death is often multifactorial, refractory hypotension and cardiovascular collapse are frequently observed in the terminal phases of the condition. The aetiology of these cardiovascular abnormalities is complex but appears to be mediated by a circulating factor(s). The impact of sepsis upon left ventricular systolic function has been studied extensively. This may be because it is more readily assessed than diastolic function. Despite being increasingly appreciated as a contributor to morbidity and mortality in other clinical settings, there are scant data regarding the evaluation of left ventricular diastolic function in sepsis. Review of the haemodynamic monitoring literature reveals that many conventional measures of left ventricular filling, intravascular volume status and fluid responsiveness are influenced by ventricular diastolic (dys)function, such that interpretation can be challenging in critical care settings. In addition, many available techniques, such as pulmonary artery catheterisation, are invasive and potentially associated with risk to the patient. More robust and less invasive measures of left ventricular diastolic function and filling that can be applied within the intensive care unit (ICU) must be developed. The use of cardiac biomarkers, such as B-type natriuretic peptide (BNP), might represent a novel approach to evaluating left ventricular diastolic function and filling. BNP is released by the myocardium in response to wall stretch/tension. It has demonstrated value in the emergency department diagnosis of heart failure but interpretation of plasma BNP concentrations in critical care remains problematic. At least in part, this appears to relate to the significant number of potential confounders in patients with critical illness. Associations between BNP concentration and diastolic function have not previously been evaluated in severe sepsis and septic shock. The overall aim of this thesis is to investigate the usefulness of plasma BNP concentration in the evaluation of left ventricular diastolic function (including ventricular filling) in severe sepsis and septic shock. DIASTOLIC (DYS)FUNCTION IN SEPSIS: Review of the literature reveals that sepsis is associated with a spectrum of diastolic dysfunction. Characterisation of diastolic function in sepsis is challenging. In this regard, tissue Doppler imaging (TDI), offers promise. TDI is an echocardiographic technique that measures myocardial velocities, which are low frequency, high-amplitude signals filtered from conventional Doppler imaging. TDI has gained acceptance amongst cardiologists in the evaluation of diastolic function, particularly as a measure of ventricular relaxation and ventricular filling pressure; however, there are scant data regarding its use in critical care. We analysed echocardiographs from a large heterogeneous cohort of consecutive ICU patients (n=94) who had TDI as part of their clinically requested echocardiography. As well as supporting the feasibility of TDI in critically ill and mechanically ventilated patients, we demonstrated a wide range of TDI variables and a high prevalence of diastolic dysfunction using this modality. RODENT MODELS OF SEPSIS: We also sought to adapt, refine and evaluate rodent models of sepsis. Such models would allow control for a multitude of potential confounders commonly encountered in clinical sepsis. Two commonly employed rodent models of sepsis include caecal ligation and perforation (CLP) and endotoxin infusion. Comparison between CLP, sham and control groups demonstrated no difference in TDI or BNP. The observed changes in echocardiographic diastolic variables did not reflect those expected in sepsis and may be best explained by increases in heart rate rather than diastolic dysfunction per se. Endotoxaemia was associated with changes consistent with impaired myocardial relaxation (TDI) and reversible myocardial injury (histopathology), as expected in sepsis. BNP did not change significantly from baseline. This might be explained by the potential influence of fluid management upon BNP secretion. CLINICAL RESEARCH: The prediction of fluid responsiveness potentially prevents ineffective, excessive or deleterious intravenous fluid administration. Prospective evaluation of plasma BNP concentration in patients with septic shock found that it was not a predictor of a fluid responsive state. Furthermore, elevated BNP did not rule out a favourable response and therefore does not contraindicate a fluid challenge. Both impaired diastolic dysfunction, especially E/e’, and elevated BNP, have been associated with excess mortality in a range of cardiovascular diseases. These have not previously been compared in septic shock. In a cohort of patients with septic shock, E/e’ was a stronger predictor of mortality than cardiac biomarkers, including BNP. Fluid balance was an independent predictor of BNP in septic shock. OVERALL CONCLUSION: BNP appears not to be clinically useful in the evaluation of ventricular filling or diastolic function in sepsis. The association with fluid balance is a new finding and should be evaluated in a wider range of critically ill patients. In contrast to BNP, TDI appears to be a promising bedside tool in the evaluation of diastolic function and should be further evaluated in critical care.