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Background: Mycobacterium tuberculosis infection is thought to induce oxidative stress. N-acetyl-cysteine (NAC) is widely used in patients with chronic pulmonary diseases including tuberculosis due to its mucolytic and anti-oxidant activities. Here, we tested whether NAC exerts a direct antibiotic activity against mycobacteria. Methods: Oxidative stress status in plasma was compared between pulmonary TB (PTB) patients and those with latent M. tuberculosis infection (LTBI) or healthy uninfected individuals. Lipid peroxidation, DNA oxidation and cell death, as well as accumulation of reactive oxygen species (ROS) were measured in cultures of primary human monocyte-derived macrophages infected with M. tuberculosis and treated or not with NAC. M. tuberculosis, M. avium and M. bovis BCG cultures were also exposed to different doses of NAC with or without medium pH adjustment to control for acidity. The anti-mycobacterial effect of NAC was assessed in M. tuberculosis infected human THP-1 cells and bone marrow-derived macrophages from mice lacking a fully functional NADPH oxidase system. The capacity of NAC to control M. tuberculosis infection was further tested in vivo in a mouse (C57BL/6) model. Results: PTB patients exhibited elevated levels of oxidation products and a reduction of anti-oxidants compared with LTBI cases or uninfected controls. NAC treatment in M. tuberculosis-infected human macrophages resulted in a decrease of oxidative stress and cell death evoked by mycobacteria. Importantly, we observed a dose-dependent reduction in metabolic activity and in vitro growth of NAC treated M. tuberculosis, M. avium and M. bovis BCG. Furthermore, anti-mycobacterial activity in infected macrophages was shown to be independent of the effects of NAC on the host NADPH oxidase system in vitro. Short-term NAC treatment of M. tuberculosis infected mice in vivo resulted in a significant reduction of mycobacterial loads in the lungs. Conclusions: NAC exhibits potent anti-mycobacterial effects and may limit M. tuberculosis infection and disease both through suppression of the host oxidative response and through direct antimicrobial activity. [ABSTRACT FROM AUTHOR]

Background: Mycobacterium tuberculosis infection is known to cause inflammation and lung tissue damage in high-risk populations. Nevertheless, direct associations between mycobacterial loads, systemic inflammation and pulmonary lesions upon treatment initiation have not been fully characterized. In the present exploratory study, we prospectively depict the immune profile, microbial clearance and evolution of radiographic lesions in a pulmonary tuberculosis (PTB) patient cohort before and 60 days after anti-tuberculous treatment (ATT) initiation.Methods: Circulating levels of cytokines (IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α) and C-reactive protein (CRP), as well as values of erythrocyte sedimentation rate (ESR) were measured in cryopreserved serum samples obtained from 73 PTB patients at pre-ATT and day 60 of treatment. Changes of the immune profile over time were compared with mycobacterial loads in sputum and culture conversion at day 60 of ATT. Additional analyses tested associations between improvement of chest radiographic lesions at day 60 and pre-treatment status of inflammation and mycobacterial loads.Results: Within the inflammatory parameters evaluated, values of CRP, IL-2, IL-4, TNF-α and ESR significantly decreased upon treatment initiation. On the converse, IL-10 levels substantially increased at day 60 of ATT, whereas concentrations of IL-6 and IFN-γ remained unchanged. Multidimensional analyses revealed that ESR, IL-2, IL-4 and CRP were the parameters with the highest power to discriminate individuals before and after treatment initiation. We further demonstrated that higher bacterial loads in sputum at pre-ATT were associated with increased systemic inflammation and higher risk for positive M. tuberculosis sputum cultures at day 60 of treatment. Furthermore, we found that pre-ATT mycobacterial loads in sputum and systemic inflammation synergistically associated with the status of radiographic lesions during treatment (Relative risk for chest X-ray improvement: 10.0, 95 % confidence interval: 2.4-40.0, P = 0.002).Conclusions: M. tuberculosis loads in sputum are directly associated to the status of systemic inflammation and potentially impact the immune profile, culture conversion and evolution of lung lesions upon ATT initiation. [ABSTRACT FROM AUTHOR]

Background: Concurrent malaria and dengue infection is frequently diagnosed in endemic countries, but its immunopathology remains largely unknown. In the present study, a large panel of cytokines/chemokines and clinical laboratory markers were measured in patients with Plasmodium vivax and dengue co-infection as well as in individuals with malaria or dengue mono-infections in order to identify biosignatures of each clinical condition. Methods: Individuals from the Brazilian Amazon were recruited between 2009 and 2013 and classified in three groups: vivax malaria (n = 52), dengue (n = 30) and vivax malaria and dengue co-infection (n = 30). P. vivax malaria was diagnosed by thick blood smear and confirmed by PCR; dengue cases were detected by IgM ELISA or NS1 protein. The plasma levels of cytokines and chemokines were determined by multiplex assay. Results: Individuals with malaria and dengue co-infection displayed lower levels of platelets and haemoglobin than those with malaria or dengue mono-infections (p = 0.0047 and p = 0.0001, respectively). The group of individuals coinfected exhibited the highest median concentrations of IFN-γ, IL-6, CCL4 than the mono-infected groups. Network analyses of plasma cytokines/chemokines revealed that malaria and dengue co-infection exhibits a distinct immune profile with critical roles for TNF, IL-6 and IFN-γ. Further, parasitaemia levels displayed positive significant interactions with IL-6, CCL4 and IL-10 in the group of patients co-infected with malaria and dengue. No differences were observed in distribution of dengue virus serotypes and Plasmodium parasitaemia levels between the groups. Conclusions: The findings described here identify unique patterns of circulating immunological markers in cases of malaria and dengue co-infection and provide insights on the immunopathology of this co-morbid condition. [ABSTRACT FROM AUTHOR]

Background Eicosanoids and sand fly saliva have a critical role in the Leishmania infection. Here, we evaluated the effect of Lutzomyia longipalpis salivary gland sonicate (SGS) on neutrophil and monocyte recruitment and activation of eicosanoid production in a murine model of inflammation. Methods C57BL/6 mice were inoculated intraperitonealy with Lutzomyia longipalpis SGS or Leishmania infantum or both, followed by analyses of cell recruitment, parasite load and eicosanoid production. Results Intraperitoneal injection of Lutzomyia longipalpis SGS together with Leishmania infantum induced an early increased parasite viability in monocytes and neutrophils. L. longipalpis SGS increased prostaglandin E2 (PGE2), but reduced leukotriene B4 (LTB4) production ex vivo in peritoneal leukocytes. In addition, the pharmacological inhibition of cyclooxygenase 2 (COX-2) with NS-398 decreased parasite viability inside macrophages during Leishmania infection in the presence of L. longipalpis SGS arguing that PGE2 production is associated with diminished parasite killing. Conclusions These findings indicate that L. longipalpis SGS is a critical factor driving immune evasion of Leishmania through modulation of PGE2/ LTB4 axis, which may represent an important mechanism on establishment of the infection. [ABSTRACT FROM AUTHOR]

Background: DDX39B (BAT1) encodes an RNA helicase known to regulate expression of TNF and IL-6. Elevated levels of these two cytokines are associated with increased severity of clinical malaria. The aim of this study was to investigate the relationship between single nucleotide polymorphisms (SNPs) in the DDX39B, TNF and IL6 genes and the clinical outcomes of patients with Plasmodium vivax malaria. Methods: Cross-sectional investigations were carried out in two regions of the Brazilian Amazon where several studies on the pathogenesis of vivax malaria had been performed. Individuals were categorized according to infection status as well as clinical presentation into the following groups: uninfected, asymptomatic infection, mild infection, or complicated infection. Polymorphisms were identified using PCR restriction fragment-length polymorphism analysis and the restriction enzymes NlaIII or NcoI. The plasma levels of cytokines were determined using ELISA. Results: The G allele of DDX39B-22C > G was associated with absent or decreased manifestations of malaria and the C allele was a risk factor for disease complications. Study participants heterozygous for TNF-308 (GA) and DDX39B-348 (CT) had higher TNF levels than wild-type participants. Haplotypes that included DDX39B (−22C > G and -348C > T) and TNF polymorphisms were not directly associated with mild or complicated malaria infections; however, haplotypes AGC, ACC, GGT, AGT and ACT were associated with increased TNF levels. Participants with genotype combinations GC/CC/ GG/GG and GG/CT/GG/GG (DDX39B-22/DDX39B-348/TNF-308/IL6-176) had decreased and increased risk of mild malaria, respectively, compared with asymptomatic and uninfected participants. GC/CC/GG/GG was linked to decreased TNF and IL-6 levels. Conclusions: This is the first study to describe patients with DDX39B and IL6 SNPs who had vivax malaria. These findings support the postulation that a set of mutations in immune-related genes is associated with inflammatory mediators and the clinical outcomes of patients with malaria. [ABSTRACT FROM AUTHOR]

Background: Plasmodium vivax malaria clinical outcomes are a consequence of the interaction of multiple parasite, environmental and host factors. The host molecular and genetic determinants driving susceptibility to disease severity in this infection are largely unknown. Here, a network analysis of large-scale data from a significant number of individuals with different clinical presentations of P. vivax malaria was performed in an attempt to identify patterns of association between various candidate biomarkers and the clinical outcomes. Methods: A retrospective analysis of 530 individuals from the Brazilian Amazon, including P. vivax-infected individuals who developed different clinical outcomes (148 asymptomatic malaria, 187 symptomatic malaria, 13 severe non-lethal malaria, and six severe lethal malaria) as well as 176 non-infected controls, was performed. Plasma levels of liver transaminases, bilirubins, creatinine, fibrinogen, C-reactive protein, superoxide dismutase (SOD)-1, haem oxygenase (HO)-1 and a panel composed by multiple cytokines and chemokines were measured and compared between the different clinical groups using network analysis Results: Non-infected individuals displayed several statistically significant interactions in the networks, including associations between the levels of IL-10 and IL-4 with the chemokine CXCL9. Individuals with asymptomatic malaria displayed multiple significant interactions involving IL-4. Subjects with mild or severe non-lethal malaria displayed substantial loss of interactions in the networks and TNF had significant associations more frequently with other parameters. Cases of lethal P. vivax malaria infection were associated with significant interactions between TNF ALT, HO-1 and SOD-1. Conclusions: The findings imply that clinical immunity to P. vivax malaria is associated with multiple significant interactions in the network, mostly involving IL-4, while lethality is linked to a systematic reduction of complexity of these interactions and to an increase in connections between markers linked to haemolysis-induced damage. [ABSTRACT FROM AUTHOR]

Background: Accurate malaria diagnosis is mandatory for the treatment and management of severe cases. Moreover, individuals with asymptomatic malaria are not usually screened by health care facilities, which further complicates disease control efforts. The present study compared the performances of a malaria rapid diagnosis test (RDT), the thick blood smear method and nested PCR for the diagnosis of symptomatic malaria in the Brazilian Amazon. In addition, an innovative computational approach was tested for the diagnosis of asymptomatic malaria. Methods: The study was divided in two parts. For the first part, passive case detection was performed in 311 individuals with malaria-related symptoms from a recently urbanized community in the Brazilian Amazon. A crosssectional investigation compared the diagnostic performance of the RDT Optimal-IT, nested PCR and light microscopy. The second part of the study involved active case detection of asymptomatic malaria in 380 individuals from riverine communities in Rondônia, Brazil. The performances of microscopy, nested PCR and an expert computational system based on artificial neural networks (MalDANN) using epidemiological data were compared. Results: Nested PCR was shown to be the gold standard for diagnosis of both symptomatic and asymptomatic malaria because it detected the major number of cases and presented the maximum specificity. Surprisingly, the RDT was superior to microscopy in the diagnosis of cases with low parasitaemia. Nevertheless, RDT could not discriminate the Plasmodium species in 12 cases of mixed infections (Plasmodium vivax + Plasmodium falciparum). Moreover, the microscopy presented low performance in the detection of asymptomatic cases (61.25% of correct diagnoses). The MalDANN system using epidemiological data was worse that the light microscopy (56% of correct diagnoses). However, when information regarding plasma levels of interleukin-10 and interferon-gamma were inputted, the MalDANN performance sensibly increased (80% correct diagnoses). Conclusions: An RDT for malaria diagnosis may find a promising use in the Brazilian Amazon integrating a rational diagnostic approach. Despite the low performance of the MalDANN test using solely epidemiological data, an approach based on neural networks may be feasible in cases where simpler methods for discriminating individuals below and above threshold cytokine levels are available. [ABSTRACT FROM AUTHOR]

Background: Despite clinical descriptions of severe vivax malaria cases having been reported, data regarding immunological and inflammatory patterns are scarce. In this report, the inflammatory and immunological status of both mild and severe vivax malaria cases are compared in order to explore immunopathological events in this disease. Methods and Results: Active and passive malaria case detections were performed during 2007 in Buritis, Rondônia, in the Brazilian Amazon. A total of 219 participants enrolled the study. Study individuals were classified according to the presence of Plasmodium vivax infection within four groups: non-infected (n = 90), asymptomatic (n = 60), mild (n = 50) and severe vivax infection (n = 19). A diagnosis of malaria was made by microscopy and molecular assays. Since at present no clear criteria define severe vivax malaria, this study adapted the consensual criteria from falciparum malaria. Patients with severe P. vivax infection were younger, had lived for shorter time in the endemic area, and recalled having experienced less previous malaria episodes than individuals with no malaria infection and with mild or asymptomatic infection. Strong linear trends were identified regarding increasing plasma levels of C reactive protein (CRP), serum creatinine, bilirubins and the graduation of disease severity. Plasma levels of tumour necrosis factor (TNF), interferon-gamma(IFN-gamma) and also IFN-gamma/interleukin-10 ratios were increased and exhibited a linear trend with gradual augmentation of disease severity. Both laboratory parameters of organ dysfunction and inflammatory cytokines were reduced during anti-parasite therapy in those patients with severe disease. Conclusion: Different clinical presentations of vivax malaria infection present strong association with activation of pro-inflammatory responses and cytokine imbalance. These findings are of utmost importance to improve current knowledge about physiopathological concepts of this serious widespread disease. [ABSTRACT FROM AUTHOR]

Background: Eicosanoids and sand fly saliva have a critical role in the Leishmania infection. Here, we evaluated the effect of Lutzomyia longipalpis salivary gland sonicate (SGS) on neutrophil and monocyte recruitment and activation of eicosanoid production in a murine model of inflammation., Methods: C57BL/6 mice were inoculated intraperitonealy with Lutzomyia longipalpis SGS or Leishmania infantum or both, followed by analyses of cell recruitment, parasite load and eicosanoid production., Results: Intraperitoneal injection of Lutzomyia longipalpis SGS together with Leishmania infantum induced an early increased parasite viability in monocytes and neutrophils. L. longipalpis SGS increased prostaglandin E2 (PGE2), but reduced leukotriene B4 (LTB4) production ex vivo in peritoneal leukocytes. In addition, the pharmacological inhibition of cyclooxygenase 2 (COX-2) with NS-398 decreased parasite viability inside macrophages during Leishmania infection in the presence of L. longipalpis SGS arguing that PGE2 production is associated with diminished parasite killing., Conclusions: These findings indicate that L. longipalpis SGS is a critical factor driving immune evasion of Leishmania through modulation of PGE2/LTB4 axis, which may represent an important mechanism on establishment of the infection.