The effect of thiamine deficiency on inflammation, oxidative stress and cellular migration in an experimental model of sepsis.

BACKGROUND: Sepsis is a prevalent condition in critically ill patients and may be associated with thiamine deficiency (TD). The aim of this study was to evaluate the effect of TD on inflammation, oxidative stress and cellular recruitment in a sepsis model. METHODS: The experimental sepsis model, cecal ligation and puncture (CLP), was utilized on mice in comparison with a sham procedure. The following four groups were compared against each other: SHAM with AIN93G complete chow, SHAM with thiamine deficient (TD) chow, CLP with AIN93G complete chow, and CLP with TD chow. Thiamine pyrophosphate (TPP) blood concentrations were determined, and blood and peritoneal fluid were evaluated for differences in TNF-alpha, IL-1, IL-6, KC and MCP-1/CCL2 levels. In addition, the levels of 4-HNE adducts in liver proteins were evaluated by Western Blot. RESULTS: The mean TPP blood concentration from the mice fed with the complete chow was 303.3 ± 42.6 nmol/L, and TD occurred within 10 days. TNF-α and MCP-1 concentrations in the peritoneal fluid were significantly greater in the CLP with TD chow group when compared with the other groups. The blood IL-1ß level, however, was lower in the CLP with TD chow group. Liver 4-HNE levels were highest in the TD chow groups. Blood mononuclear cell numbers, as well as peritoneal total leukocyte, mononuclear cell and neutrophil numbers were greater in the CLP with TD chow group. Peritoneal bacterial colony forming units (CFU) were significantly lower in the CLP with TD chow group. CONCLUSION: TD was associated with greater bacterial clearance, oxidative stress and inflammatory response changes.

IL-6 and IL-8 in cerebrospinal fluid from patients with aseptic meningitis and bacterial meningitis: their potential role as a marker for differential diagnosis.

Cytokines are molecules that act as mediators of immune response; cerebral spinal fluid (CSF) IL-6 is found in all meningeal inflammatory diseases, but IL-8 is associated with acute bacterial meningitis (ABM). A case control study was done to ascertain the discriminatory power of these cytokines in differentiating ABM from aseptic meningitis (AM); IL-6 and IL-8 CSF concentrations were tested through ELISA in samples collected from patients who underwent investigation for meningitis. Sixty patients, 18 with AM, nine with bacteriologic confirmed ABM and 33 controls, assisted in 2005 (MA and controls) and 2007 (ABM) were included. Differently from controls, IL-6 concentrations were increased both in MA and ABM patients (p < 0.05). CSF IL-8 levels were higher in ABM than in AM and controls (p < 0.05). Discriminatory power in ABM as assessed by the area under receiver operator (ROC) curve was 0.951 for IL-8, using a cut-off of 1.685 ng/dL (100% of sensitivity and 94% of specificity). The CSF concentration of both IL-6 and IL-8 are increased in the presence of meningeal inflammation, IL-8 could be an important tool to differentiate ABM from AM.

IL-6 and IL-8 in cerebrospinal fluid from patients with aseptic meningitis and bacterial meningitis: their potential role as a marker for differential diagnosis

Cytokines are molecules that act as mediators of immune response; cerebral spinal fluid (CSF) IL-6 is found in all meningeal inflammatory diseases, but IL-8 is associated with acute bacterial meningitis (ABM). A case control study was done to ascertain the discriminatory power of these cytokines in differentiating ABM from aseptic meningitis (AM); IL-6 and IL-8 CSF concentrations were tested through ELISA in samples collected from patients who underwent investigation for meningitis. Sixty patients, 18 with AM, nine with bacteriologic confirmed ABM and 33 controls, assisted in 2005 (MA and controls) and 2007 (ABM) were included. Differently from controls, IL-6 concentrations were increased both in MA and ABM patients (p < 0.05). CSF IL-8 levels were higher in ABM than in AM and controls (p < 0.05). Discriminatory power in ABM as assessed by the area under receiver operator (ROC) curve was 0.951 for IL-8, using a cut-off of 1.685 ng/dL (100 percent of sensitivity and 94 percent of specificity). The CSF concentration of both IL-6 and IL-8 are increased in the presence of meningeal inflammation, IL-8 could be an important tool to differentiate ABM from AM.

Potencial terapêutico da PAF acetilhidrolase (PAF-AH) no controle da sepse / Therapeutical potential of the PAF acetilhidrolase (PAF-AH) in the control of sepse

Evidências sugerem que uma regulação deficiente da resposta inflamatória do indivíduo aos produtos microbianos é central para a mortalidade de pacientes com SIRS ou sepse. Estratégias para bloquear essa resposta inflamatória são investigadas frequentemente como novos alvos terapêuticos na sepse. Mediadores lipídicos parecem estar envolvidos nesta síndrome, e dentre estes o PAF exerce um papel crítico. Recentemente, a enzima PAF-acetilhidrolase (PAF-AH), que degrada o PAF, foi clonada e seus efeitos antiinflamatórios foram demonstrados em modelos experimentais. Neste trabalho, tivemos como objetivo central investigar o potencial terapêutico da PAF-AH em modelos de sepse. Inicialmente, medimos a atividade da PAF-AH no plasma desses animais submetidos aos modelos de administração intraperitoneal de LPS, da bactéria N. meningitidis ou laparotomia seguida de ligadura e punção cecal (CLP), como também no plasma de pacientes com sepse ou choque séptico. Observamos uma diminuição da atividade da PAF-AH nos animais que foram submetidos aos modelos de sepse, após 24h do estímulo inicial. Além disso, no plasma de pacientes com choque séptico também observamos uma atividade enzimática reduzida quando comparamos com voluntários sadios. Avaliando a curva de sobrevida de animais submetidos aos modelos CLP ou endotoxemia letal, observamos um aumento significativo na sobrevida de animais (40%) tratados com a rPAF-AH, como também uma proteção em animais trangênicos para a enzima, submetidos ao CLP.

Observamos um aumento nos níveis de mediadores antiinflamatórios como a IL-10 e MCP-1/CCL2 e decréscimo de mediadores pró-inflamatórios como TNF, IL-6 e MIF. Animais geneticamente deficientes do receptor do MCP-1/CCL2 tratados com a rPAF-AH não foram protegidos da sepse induzida pelo modelo CLP, sugerindo que o papel protetor da PAF-AH depende da liberação de MCP-1/CCL2. Em adição, animais geneticamente deficientes de MCP-1/CCL2 foram mais susceptíveis a mortalidade induzida pelo CLP, apresentando níveis aumentados de MIF e diminuídos da citocina IL-10. A associação da rPAF-AH com o antibiótico Imipenem se mostrou de grande valia, visto que 60% dos animais sobreviveram, quando comparada com o tratamento isolado com a PAF-AH (40%) ou mesmo com o antibiótico. Quando contamos o número de unidades formadoras de colônias no grupo tratado com a rPAF-AH observamos um decréscimo no número de bactérias, que se correlacionou com aumento nos níveis de NO. Concluímos que alterações observadas na atividade endógena da PAF-AH contribui na fisiopatogia da sepse e que a administração exógena da enzima reduz a resposta inflamatória e a mortalidade de animais submetidos a modelos experimentais clinicamente relevantes de sepse.

Calcitonin gene-related peptide inhibits local acute inflammation and protects mice against lethal endotoxemia.

Calcitonin gene-related peptide (CGRP), a potent vasodilatory peptide present in central and peripheral neurons, is released at inflammatory sites and inhibits several macrophage, dendritic cell, and lymphocyte functions. In the present study, we investigated the role of CGRP in models of local and systemic acute inflammation and on macrophage activation induced by lipopolysaccharide (LPS). Intraperitoneal pretreatment with synthetic CGRP reduces in approximately 50% the number of neutrophils in the blood and into the peritoneal cavity 4 h after LPS injection. CGRP failed to inhibit neutrophil recruitment induced by the direct chemoattractant platelet-activating factor, whereas it significantly inhibited LPS-induced KC generation, suggesting that the effect of CGRP on neutrophil recruitment is indirect, acting on chemokine production by resident cells. Pretreatment of mice with 1 mug of CGRP protects against a lethal dose of LPS. The CGRP-induced protection is receptor mediated because it is completely reverted by the CGRP receptor antagonist, CGRP 8-37. The protective effect of CGRP correlates with an inhibition of TNF-alpha and an induction of IL-6 and IL-10 in mice sera 90 min after LPS challenge. Finally, CGRP significantly inhibits LPS-induced TNF-alpha released from mouse peritoneal macrophages. These results suggest that activation of the CGRP receptor on macrophages during acute inflammation could be part of the negative feedback mechanism controlling the extension of acute inflammatory responses.

Morphological and biochemical characterization of macrophages activated by carrageenan and lipopolysaccharide in vivo.

Macrophages are able to recognize, internalize and destroy a large number of pathogens, thus restricting the infection until adaptive immunity is initiated. In this work our aim was to analyze the surface charge of cells activated by carrageenan (CAR) and lipopolysaccharide (LPS) through light and electron microscopy approaches as well as the release of inflammatory mediators in vitro. The ultrastuctural analysis and the light microscopy data showed that in vivo administration of CAR represents a potent inflammatory stimulation for macrophages leading to a high degree of spreading, an increase in their size, in the number of the intracellular vacuoles and membrane projections as compared to the macrophages collected from untreated animals as well as mice submitted to LPS. Our data demonstrated that CAR stimulated-macrophages displayed a remarkable increase in nitric oxide production and PGE2 release as compared to the cells collected from non-stimulated and stimulated mice with LPS in vivo. On the other hand, non-stimulated macrophages as well as macrophages stimulated by LPS produce almost the same quantities of TNF-alpha, while in vivo stimulation by CAR leads to a 30-40% increase of cytokine release in vitro compared to the other groups. In conclusion, our morphological and biochemical data clearly showed that in vivo stimulation with CAR induces a potent inflammatory response in macrophages representing an interesting model to analyze inflammatory responses.