Edson F. Assis

Cytokine profiles as markers of disease severity in sepsis: a multiplex analysis

IntroductionThe current shortage of accurate and readily available, validated biomarkers of disease severity in sepsis is an important limitation when attempting to stratify patients into homogeneous groups, in order to study pathogenesis or develop therapeutic interventions. The aim of the present study was to determine the cytokine profile in plasma of patients with severe sepsis by using a multiplex system for simultaneous detection of 17 cytokines.MethodsThis was a prospective cohort study conducted in four tertiary hospitals. A total of 60 patients with a recent diagnosis of severe sepsis were included. Plasma samples were collected for measurement of cytokine concentrations. A multiplex analysis was performed to evaluate levels of 17 cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, interferon-γ, granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor, monocyte chemoattractant protein [MCP]-1, macrophage inflammatory protein-1 and tumour necrosis factor-α). Cytokine concentrations were related to the presence of severe sepsis or septic shock, the severity and evolution of organ failure, and early and late mortality.ResultsConcentrations of IL-1β, IL-6, IL-7, IL-8, IL-10, IL-13, interferon-γ, MCP-1 and tumour necrosis factor-α were significantly higher in septic shock patients than in those with severe sepsis. Cytokine concentrations were associated with severity and evolution of organ dysfunction. With regard to the severity of organ dysfunction on day 1, IL-8 and MCP-1 exhibited the best correlation with Sequential Organ Failure Assessment score. In addition, IL-6, IL-8 and G-CSF concentrations during the first 24 hours were predictive of worsening organ dysfunction or failure of organ dysfunction to improve on day three. In terms of predicting mortality, the cytokines IL-1β, IL-4, IL-6, IL-8, MCP-1 and G-CSF had good accuracy for predicting early mortality (< 48 hours), and IL-8 and MCP-1 had the best accuracy for predicting mortality at 28 days. In multivariate analysis, only MCP-1 was independently associated with prognosis.ConclusionIn this exploratory analysis we demonstrated that use of a multiple cytokine assay platform allowed identification of distinct cytokine profiles associated with sepsis severity, evolution of organ failure and death.

Multiplex cytokine profile from dengue patients: MIP-1beta and IFN-gamma as predictive factors for severity

BackgroundDengue virus pathogenesis is not yet fully understood and the identification of patients at high risk for developing severe disease forms is still a great challenge in dengue patient care. During the present study, we evaluated prospectively the potential of cytokines present in plasma from patients with dengue in stratifying disease severity.MethodsSeventeen-cytokine multiplex fluorescent microbead immunoassay was used for the simultaneous detection in 59 dengue patients. GLM models using bimodal or Gaussian family were determined in order to associate cytokines with clinical manifestations and laboratory diagnosis.ResultsIL-1β, IFN-γ, IL-4, IL-6, IL-13, IL-7 and GM-CSF were significantly increased in patients with severe clinical manifestations (severe dengue) when compared to mild disease forms (mild dengue). In contrast, increased MIP-1β levels were observed in patients with mild dengue. MIP-1β was also associated with CD56+NK cell circulating rates. IL-1β, IL-8, TNF-α and MCP-1 were associated with marked thrombocytopenia. Increased MCP-1 and GM-CSF levels correlated with hypotension. Moreover, MIP-1β and IFN-γ were independently associated with both dengue severity and disease outcome.ConclusionOur data demonstrated that the use of a multiple cytokine assay platform was suitable for identifying distinct cytokine profiles associated with the dengue clinical manifestations and severity. MIP-β is indicated for the first time as a good prognostic marker in contrast to IFN-γ that was associated with disease severity.

Statins decrease neuroinflammation and prevent cognitive impairment after cerebral malaria

Cerebral malaria (CM) is the most severe manifestation of Plasmodium falciparum infection in children and non-immune adults. Previous work has documented a persistent cognitive impairment in children who survive an episode of CM that is mimicked in animal models of the disease. Potential therapeutic interventions for this complication have not been investigated, and are urgently needed. HMG-CoA reductase inhibitors (statins) are widely prescribed for cardiovascular diseases. In addition to their effects on the inhibition of cholesterol synthesis, statins have pleiotropic immunomodulatory activities. Here we tested if statins would prevent cognitive impairment in a murine model of cerebral malaria. Six days after infection with Plasmodium berghei ANKA (PbA) mice displayed clear signs of CM and were treated with chloroquine, or chloroquine and lovastatin. Intravital examination of pial vessels of infected animals demonstrated a decrease in functional capillary density and an increase in rolling and adhesion of leukocytes to inflamed endothelium that were reversed by treatment with lovastatin. In addition, oedema, ICAM-1, and CD11b mRNA levels were reduced in lovastatin-treated PbA-infected mice brains. Moreover, HMOX-1 mRNA levels are enhanced in lovastatin-treated healthy and infected brains. Oxidative stress and key inflammatory chemokines and cytokines were reduced to non-infected control levels in animals treated with lovastatin. Fifteen days post-infection cognitive dysfunction was detected by a battery of cognition tests in animals rescued from CM by chloroquine treatment. In contrast, it was absent in animals treated with lovastatin and chloroquine. The outcome was similar in experimental bacterial sepsis, suggesting that statins have neuroprotective effects in severe infectious syndromes in addition to CM. Statin treatment prevents neuroinflammation and blood brain barrier dysfunction in experimental CM and related conditions that are associated with cognitive sequelae, and may be a valuable adjuvant therapeutic agent for prevention of cognitive impairment in patients surviving an episode of CM.

Platelet Activation and Apoptosis Modulate Monocyte Inflammatory Responses in Dengue

Dengue is the most prevalent human arbovirus disease in the world. Dengue infection has a large spectrum of clinical manifestations, from self-limited febrile illness to severe syndromes accompanied by bleeding and shock. Thrombocytopenia and vascular leak with altered cytokine profiles in plasma are features of severe dengue. Although monocytes have been recognized as important sources of cytokines in dengue, the contributions of platelet–monocyte interactions to inflammatory responses in dengue have not been addressed. Patients with dengue were investigated for platelet–monocyte aggregate formation. Platelet-induced cytokine responses by monocytes and underlying mechanisms were also investigated in vitro. We observed increased levels of platelet–monocyte aggregates in blood samples from patients with dengue, especially patients with thrombocytopenia and increased vascular permeability. Moreover, the exposure of monocytes from healthy volunteers to platelets from patients with dengue induced the secretion of the cytokines IL-1β, IL-8, IL-10 and MCP-1, whereas exposure to platelets from healthy volunteers only induced the secretion of MCP-1. In addition to the well-established modulation of monocyte cytokine responses by activated platelets through P-selectin binding, we found that interaction of monocytes with apoptotic platelets mediate IL-10 secretion through phosphatidylserine recognition in platelet–monocyte aggregates. Moreover, IL-10 secretion required platelet–monocyte contact but not phagocytosis. Together, our results demonstrate that activated and apoptotic platelets aggregate with monocytes during dengue infection and signal specific cytokine responses that may contribute to the pathogenesis of dengue.

Lipid bodies in oxidized LDL-induced foam cells are leukotriene-synthesizing organelles: a MCP-1/CCL2 regulated phenomenon

Lipid-laden foam macrophages are emerging as key players in early atherogenesis. Even though cytoplasmic lipid bodies (lipid droplets) are now recognized as organelles with cell functions beyond lipid storage, the mechanisms controlling lipid body biogenesis within macrophages and their additional functions in atherosclerosis are not completely elucidated. Here we studied oxLDL-elicited macrophage machinery involved in lipid body biogenesis as well as lipid body roles in leukotriene (LT) synthesis. Both in vivo and in vitro, oxLDL (but not native LDL) induced rapid assembly of cytoplasmic lipid bodies-bearing ADRP within mice macrophages. Such oxLDL-elicited foamy-like phenotype was a pertussis toxin-sensitive process that depended on a paracrine activity of endogenous MCP-1/CCL2 and activation of ERK. Pretreatment with neutralizing anti-MCP-1/CCL2 inhibited macrophage ADRP protein expression induced by oxLDL. By directly immuno-localizing leukotrienes at their sites of synthesis, we showed that oxLDL-induced newly formed lipid bodies function as active sites of LTB(4) and LTC(4) synthesis, since oxLDL-induced lipid bodies within foam macrophages compartmentalized the enzyme 5-lipoxygenase and five lipoxygenase-activating protein (FLAP) as well as newly formed LTB(4) and LTC(4). Consistent with MCP-1/CCL-2 role in ox-LDL-induced lipid body biogenesis, in CCR2 deficient mice both ox-LDL-induced lipid body assembly and LT release were reduced as compared to wild type mice. In conclusion, oxLDL-driven foam cells are enriched with leukotriene-synthesizing lipid bodies--specialized organelles whose biogenic process is mediated by MCP-1/CCL2-triggered CCR2 activation and ERK-dependent downstream signaling--that may amplify inflammatory mediator production in atherosclerosis.

Synergism Between Platelet-Activating Factor-Like Phospholipids and Peroxisome Proliferator-Activated Receptor γ Agonists Generated During Low Density Lipoprotein Oxidation That Induces Lipid Body Formation in Leukocytes

Oxidized low density lipoprotein (LDL) has an important proinflammatory role in atherogenesis. In this study, we investigated the ability of oxidized LDL (oxLDL) and its phospholipid components to induce lipid body formation in leukocytes. Incubation of mouse peritoneal macrophages with oxidized, but not with native LDL led to lipid body formation within 1 h. This was blocked by platelet-activating factor (PAF) receptor antagonists or by preincubation of oxLDL with rPAF acetylhydrolase. HPLC fractions of phospholipids purified from oxLDL induced calcium flux in neutrophils as well as lipid body formation in macrophages. Injection of the bioactive phospholipid fractions or butanoyl and butenoyl PAF, a phospholipid previously shown to be present in oxLDL, into the pleural cavity of mice induced lipid body formation in leukocytes recovered after 3 h. The 5-lipoxygenase and cyclooxygenase-2 colocalized within lipid bodies formed after stimulation with oxLDL, bioactive phospholipid fractions, or butanoyl and butenoyl PAF. Lipid body formation was inhibited by 5-lipoxygenase antagonists, but not by cyclooxygenase-2 inhibitors. Azelaoyl-phosphatidylcholine, a peroxisome proliferator-activated receptor-γ agonist in oxLDL phospholipid fractions, induced formation of lipid bodies at late time points (6 h) and synergized with suboptimal concentrations of oxLDL. We conclude that lipid body formation is an important proinflammatory effect of oxLDL and that PAF-like phospholipids and peroxisome proliferator-activated receptor-γ agonists generated during LDL oxidation are important mediators in this phenomenon.

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.

Effects of bone marrow-derived mononuclear cells from healthy or acute respiratory distress syndrome donors on recipient lung-injured mice.

Objective:The advantage of using autologous bone marrow–derived mononuclear cells to treat acute respiratory distress syndrome patients is to prevent immunological rejection. However, bone marrow–derived mononuclear cells may be altered by different acute respiratory distress syndrome etiologies, resulting in questionable efficacy and thus limited clinical application. We aimed to investigate the effects of bone marrow–derived mononuclear cells obtained from healthy and acute respiratory distress syndrome donors on pulmonary and extrapulmonary acute respiratory distress syndrome. Design:Prospective, randomized, controlled experimental study. Setting:University research laboratory. Subjects:Two hundred and twenty-five C57BL/6 mice. Interventions:Acute respiratory distress syndrome was induced by Escherichia coli lipopolysaccharide intratracheally (ARDSp) or intraperitoneally (ARDSexp). Control mice (Healthy) received saline solution intratracheally (Cp) or intraperitoneally (Cexp). After 24 hours, whole bone marrow cells were analyzed in vitro: 1) colony-forming unit–fibroblasts and 2) hematopoietic stem cells, neutrophils, T helper lymphocytes, B lymphocytes, and nonhematopoietic precursors. After cell characterization, all groups received saline or bone marrow–derived mononuclear cells (2 × 106), obtained from Cp, Cexp, ARDSp, and ARDSexp donor mice, IV, on day 1. Measurements and Main Results:On day 1, in ARDSp, different patterns of colony formation were found, with nonstromal cells (mainly neutrophils) predominating over fibroblastoid colonies. In ARDSexp, irregular colony-forming unit–fibroblasts morphology with dispersed proliferating colonies and a greater number of hematopoietic stem cells were observed. In ARDSp, colony-forming unit–fibroblasts count was higher but not measurable in ARDSexp. In ARDSp, monocytes and T lymphocytes were increased and hematopoietic precursor cells reduced, with no significant changes in ARDSexp. On day 7, bone marrow–derived mononuclear cells improved survival and attenuated changes in lung mechanics, alveolar collapse, inflammation, pulmonary fibrosis, and apoptosis in the lung and distal organs, regardless of donor type. Conclusions:Bone marrow–derived mononuclear cells from ARDSp and ARDSexp donors showed different characteristics but were as effective as cells obtained from healthy donors in reducing inflammation and remodeling, suggesting the utility of autologous transplant of bone marrow–derived mononuclear cells in the clinical setting.

Insult-dependent effect of bone marrow cell therapy on inflammatory response in a murine model of extrapulmonary acute respiratory distress syndrome

IntroductionAdministration of bone marrow-derived cells produces beneficial effects in experimental extrapulmonary acute respiratory distress syndrome (ARDS). However, there are controversies regarding the effects of timing of cell administration and initial insult severity on inflammatory response. We evaluated the effects of bone marrow-derived mononuclear cells (BMDMC) in two models of extrapulmonary ARDS once lung morphofunctional changes had already been installed.MethodsBALB/c mice received lipopolysaccharide (LPS) intraperitoneally (5 mg/kg in 0.5 ml saline) or underwent cecal ligation and puncture (CLP). Control mice received saline intraperitoneally (0.5 ml) or underwent sham surgery. At 24 hours, groups were further randomized to receive saline or BMDMC (2 × 106) intravenously. Lung mechanics, histology, and humoral and cellular parameters of lung inflammation and remodeling were analyzed 1, 3 and 7 days after ARDS induction.ResultsBMDMC therapy led to improved survival in the CLP group, reduced lung elastance, alveolar collapse, tissue and bronchoalveolar lavage fluid cellularity, collagen fiber content, and interleukin-1β and increased chemokine (keratinocyte-derived chemokine and monocyte chemotactic protein-1) expression in lung tissue regardless of the experimental ARDS model. Intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression in lung tissue increased after cell therapy depending on the insult (LPS or CLP).ConclusionsBMDMC therapy at day 1 successfully reduced lung inflammation and remodeling, thus contributing to improvement of lung mechanics in both extrapulmonary ARDS models. Nevertheless, the different inflammatory responses induced by LPS and CLP resulted in distinct effects of BMDMC therapy. These data may be useful in the clinical setting, as they suggest that the type of initial insult plays a key role in the outcome of treatment.

Oleanolic acid improves pulmonary morphofunctional parameters in experimental sepsis by modulating oxidative and apoptotic processes

We compared the effects of oleanolic acid (OA) vs. dexamethasone on lung mechanics and histology, inflammation, and apoptosis in lung and distal organs in experimental sepsis. Seventy-eight BALB/c mice were randomly divided into two groups. Sepsis was induced by cecal ligation and puncture, while the control group underwent sham surgery. 1h after surgery, all animals were further randomized to receive saline (SAL), OA and dexamethasone (DEXA) intraperitoneally. Both OA and DEXA improved lung mechanics and histology, which were associated with fewer lung neutrophils and less cell apoptosis in lung, liver, and kidney than SAL. However, only animals in the DEXA group had lower levels of interleukin (IL)-6 and KC (murine analog of IL-8) in bronchoalveolar lavage fluid than SAL animals. Conversely, OA was associated with lower inducible nitric oxide synthase expression and higher superoxide dismutase than DEXA. In the experimental sepsis model employed herein, OA and DEXA reduced lung damage and distal organ apoptosis through distinct anti-inflammatory mechanisms.