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Despite the fast development of vaccines, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still circulates through variants of concern (VoC) and escape the humoral immune response. SARS-CoV-2 has provoked over 200,000 deaths/months since its emergence and only a few antiviral drugs showed clinical benefit up to this moment. Thus, chemical structures endowed with anti-SARS-CoV-2 activity are important for continuous antiviral development and natural products represent a fruitful source of substances with biological activity. In the present study, agathisflavone (AGT), a biflavonoid from Anacardium occidentale was investigated as a candidate anti-SARS-CoV-2 compound. In silico and enzymatic analysis indicated that AGT may target mainly the viral main protease (M

The disease leprosy is caused by Mycobacterium leprae. The disease displays a spectrum of clinical manifestations relating to the stage of the infection and the pathogen-specific immune response. The most frequent M. leprae-specific hypersensitivity reactions are erythema nodosum leprosum (ENL) and type-1 (reversal) reaction (T1R). Omega-3 and omega-6 fatty acid-derived lipid mediators are involved in the regulation of these M. leprae-specific inflammatory and immune responses. Studies on lipid mediators showed their presence during different manifestations of leprosy-before and after multidrug therapy (MDT) and during T1R. This review aims to compare the lipid mediators at different stages of the disease. This review also presents new data on the significance of lipid mediators (cysteinyl leukotrienes and leukotriene B4, prostaglandin E2 and D2, lipoxin A4 and resolvin D1) on ENL.

The immune system is a key component of tumorigenesis, with the latter promoting the development of cancer, its progression and metastasis. In fact, abundant infiltration of tumor-associated macrophages (TAM), which are M2-like macrophages, has been associated with a poor outcome in most types of cancers. Here, we show that lactate produced by murine melanoma B16F10 cells induces an M2-like profile in cultured macrophages. Further, we demonstrate that clotrimazole (CTZ), an off-target anti-tumor drug, abolishes lactate effects on the activation of macrophages and induces the expression of M1-like markers. We show that clotrimazole has cytotoxic effects on tumor cells by negatively modulating PI3K, which inhibits glycolytic metabolism and leads to a diminishing lactate production by these cells. These effects are more pronounced in cancer cells exposed to conditioned media of M2-polarized macrophages. Moreover, clotrimazole inhibits tumor growth in a murine model of implanted melanoma, reduces lactate content in a tumor microenvironment and decreases vascular endothelial growth factor expression. Finally, clotrimazole drastically diminishes TAM infiltration in the tumors, thereby inducing M1 polarization. Collectively, these findings identify a new antitumor mechanism of clotrimazole by modulating the tumor microenvironment (TME), particularly the activation and viability of TAM.

Zika virus (ZIKV), an emerging Flavivirus, was recently associated with severe neurological complications and congenital diseases. Therefore, development of antiviral agents capable of inhibiting ZIKV replication is urgent. Chloroquine is a molecule with a confirmed safety history for use with pregnant women, and has been found to exhibit anti-ZIKV activity at concentrations around 10 μM. This suggests that modifications to the chloroquine structure could be promising for obtaining more effective anti-ZIKV agents. Here, we report the ability of a series of N-(2-(arylmethylimino)ethyl)-7-chloroquinolin-4-amine derivatives to inhibit ZIKV replication in vitro. We have found that the quinoline derivative, N-(2-((5-nitrofuran-2-yl)methylimino)ethyl)-7-chloroquinolin-4-amine, 40, was the most potent compound within this series, reducing ZIKV replication by 72% at 10 μM. Compound 40 exhibits an EC50 value of 0.8 ± 0.07 μM, compared to that of chloroquine of 12 ± 3.2 μM. Good activities were also obtained for other compounds, including those with aryl groups = phenyl, 4-fluorophenyl, 4-nitrophenyl, 2,6-dimethoxyphenyl, 3-pyridinyl and 5-nitrothien-2-yl. Syntheses of these quinoline derivatives have been obtained both by thermal and ultrasonic means. The ultrasonic method produced comparable yields to the thermal (reflux) method in very much shorter times 30-180 s compared to 30-180 min reactions times. These results indicate that this group of compounds is a good follow-up point for the potential discovery of new drugs against the Zika disease.

Zika virus (ZIKV), an arthropod-born Flavivirus, has been associated with a wide range of neurological diseases in adults, foetuses and neonates. Since no vaccine is available, repurposing of antiviral drugs currently in medical use is necessary. Mefloquine has confirmed anti-ZIKV activity. We used medicinal chemistry-driven approaches to synthesize and evaluate the ability of a series of new 2,8-bis(trifluoromethyl)quinoline derivatives to inhibit ZIKV replication in vitro, in order to improve the potency of mefloquine. We found that quinoline derivatives 3a and 4 were the most potent compounds within this series, both with mean EC50 values of 0.8 μM, which represents a potency 5 times that of mefloquine. These results indicate that new 2,8-bis(trifluoromethyl)quinoline chemical structures may be promising for the development of novel anti-ZIKV drugs.

Mansonic schistosomiasis is a disease caused by the trematode Schistosoma mansoni, endemic to tropical countries. S. mansoni infection induces the formation of granulomas and potent polarization of Th2-type immune response. There is great interest in understanding the mechanisms used by this parasite that causes a modulation of the immune system. Recent studies from our group demonstrated that lipids of S. mansoni, including lysophosphatidylcholine (LPC) have immunomodulatory activity. In the present study, our aim was to investigate the role of lipids derived from S. mansoni in the activation and polarization of macrophages and to characterize the mechanisms involved in this process. Peritoneal macrophages obtained from wild type C57BL/6mice or bone marrow derived macrophages were stimulated in vitro with lipids extracted from adult worms of S. mansoni. We demonstrated that total schistosomal-derived lipids as well as purified LPC induced alternatively activated macrophages/M2 profile observed by increased expression of arginase-1, mannose receptor, Chi3l3, TGFβ and production of IL-10 and PGE2 24h after stimulation. The involvement of the nuclear receptor PPARγ in macrophage response against LPC was investigated. Through Western blot and immunofluorescence confocal microscopy we demonstrated that schistosomal-derived LPC induces increased expression of PPARγ in macrophages. The LPC-induced increased expression of arginase-1 were significantly inhibited by the PPAR-γ antagonist GW9662. Together, these results demonstrate an immunomodulatory role of schistosomal-derived LPC in activating macrophages to a profile of the type M2 through PPARγ-dependent mechanisms, indicating a novel pathway for macrophage polarization triggered by parasite-derived LPC with potential implications to disease pathogenesis.

Sepsis is a complex life-threatening syndrome caused by dysregulated inflammatory and metabolic host response to infection. Alterations in lipid metabolism and an increased number of lipid droplets (LDs) are observed during sepsis; however LD involvement in maladaptive tissue tolerance that culminates in organ dysfunction during sepsis is poorly understood. Here we show that increased accumulation of LDs is associated with peroxidized lipid production, liver dysfunction and sepsis severity. Through the use of antioxidants or CRISPR/Cas9 deletion of the NADPH oxidase subunit p22phox we demonstrate that ROS induced LD biogenesis and oxidatively altered LD content. Moreover, the LDs associated with mitochondria and peridroplet mitochondria in cells from the septic mice showed increased ultrastructural damage. Strikingly, dampening LD accumulation by DGAT1 inhibition decreased the production of inflammatory mediators, reduced lipid peroxidation while improving tissue function. Altogether, we demonstrate that LDs contribute to sepsis-associated organ dysfunction by disrupting tissue tolerance through the amplification of lipid peroxidation, suggesting that LD and changes in lipid metabolism in sepsis may be a target for therapy.

Toll like receptors (TLRs) are involved in the modulation of diverse host genes expression through a complex network of signalling events that allow for an appropriate response to a microbial pathogen. In the present work we used TLR6KO mice in order to study the role of TLR6 in the immune discrimination of lipids from two Babesia bovis strains, attenuated R1A (LA) and virulent S2P (LV), and the consequent macrophage activation. We demonstrated that TLR6 is required for lipid body induction in murine peritoneal macrophages by both LA and LV. Interestingly, as regards IL-10 and COX-2/PGE2 pathway induction by LA and LV, we observed differences in the biological effects produced by these lipid extracts. Our results indicate a role of TLR6 in the down-modulation of these immunoregulators only in the case of LA, whereas this receptor was not implicated in pro-inflammatory TNFα, IL-6 and KC release induced by LA. Remarkably, LV did not exert the down-modulatory effect observed for LA, supporting the notion that LA and LV possess different lipid composition that could correlate with the polar pathogenic effect of both B. bovis strains. Fil: Gimenez, Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Belaunzarán, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Magalhães, K. G.. Universidade do Brasília; Brasil Fil: Poncini, Carolina Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Lammel, Estela María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: González, Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Bozza, P. T.. Fundación Oswaldo Cruz; Brasil Fil: Durante de Isola, Elvira Luisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina

Hepatic Stellate Cells (HSCs) play a crucial role in pathogenesis of liver inflammation and fibrosis. During chronic liver injury, HSCs lose vitamin A and transform into myofibroblastic cells. In schistosomal granulomas, these activated HSCs are called GR-HSCs. Schistosomal-triggered hepatic fibrogenesis has TGF-β as the most potent fibrogenic stimulus, that also controls gene expression of the angiogenic molecule VEGF in HSCs. COX-dependent production of prostaglandins (PGs) also play role in angiogenic processes. Besides angiogenic roles, prostanoids control immunomodulation of Schistosoma mansoni infection. Specifically, schistosoma-derived PGD2 has emerged as a key parasite regulator of immune defense evasion, while no role is still established to host PGD2. Therefore, the aim of this work is to investigate the ability of GR-HSCs to synthesize COX-derived PGD2 and a potential role of this prostanoid in VEGF production by GR-HSCs in vitro. Here, we confirmed that GR-HSCs express COX-2, which displayed perinuclear localization. While unstimulated GR-HSCs produce basal levels of PGD2, TGF-β stimulation besides increasing COX2- mRNA levels, enhanced synthesis/secretion of PGD2 in GR-HSCs supernatant. Moreover, GR-HSCs-derived PGD2 mediate VEGF production by TGF-β-stimulated GR-HSCs, since the pre-treatment with HQL-79, an inhibitor of hematopoietic PGD synthase inhibited both PGD2 synthesis and VEGF secretion by TGF-β-stimulated GR-HSCs. All together, our findings show an autocrine/paracrine activity of GR-HSCs-derived PGD2 on TGF-β-induced VEGF production by GR-HSCs, unveiling a role for PGD2 as important regulator of HSCs activation in hepatic granulomas from schistosome infected mice.

Ethnopharmacological relevance Curine is a bisbenzylisoquinoline alkaloid and the major constituent isolated from Chondrodendron platyphyllum , a plant that is used to treat inflammatory diseases in Brazilian folk medicine. This study investigates the effectiveness of curine on mast cell-dependent responses in mice. Materials and methods To induce mast cell-dependent responses, Swiss mice were subcutaneously sensitized with ovalbumin (OVA—12 μg/mouse) and Al(OH) 3 in a 0.9% NaCl solution. Fifteen days later, the animals were challenged with OVA through different pathways. Alternatively, the animals were injected with compound 48/80 or histamine, and several parameters, including anaphylaxis, itching, edema and inflammatory mediator production, were analyzed. Promethazine, cromoglycate, and verapamil were used as control drugs, and all of the treatments were performed 1 h before the challenges. Results Curine pre-treatment significantly inhibited the scratching behavior and the paw edema induced by either compound 48/80 or OVA, and this protective effect was comparable in magnitude with those associated with treatment with either cromoglycate or verapamil. In contrast, curine was a weak inhibitor of histamine-induced paw edema, which was completely inhibited by promethazine. Curine and verapamil significantly inhibited pleural protein extravasations and prostaglandin D 2 (PGD 2 ) and cysteinyl leukotrienes (CysLTs) production following allergen-induced pleurisy. Furthermore, like verapamil, curine inhibited the anaphylactic shock caused by either compound 48/80 or an allergen. In in vitro settings, these treatments also inhibited degranulation as well as PGD 2 and CysLT production through IgE-dependent activation of the mast cell lineage RBL-2H3. Conclusion Curine significantly inhibited immediate allergic reactions through mechanisms more related to mast cell stabilization and activation inhibition than interference with the pro-inflammatory effects of mast cell products. These findings are in line with the hypothesis that the alkaloid curine may be beneficial for the treatment of allergic disorders.

Sepsis is a severe disorder characterized by systemic inflammatory responses in the presence of an infection and may progress to multiple organ dysfunction and death. Alterations in cerebral microcirculation fulfill a crucial role in the pathogenesis of severe sepsis, and include a decrease in capillary density and disturbances in leukocyte movement along capillaries. Nevertheless, the mechanisms involved in sepsis-associated cerebral microcirculatory alterations have so far not been defined. We investigated the effect of the peroxisome proliferator-activated receptor gamma (PPARγ) selective agonist rosiglitazone on leukocyte/endothelial cell interaction and functional capillary density in the brain in the cecal ligation and puncture (CLP) model of sepsis. Anti-inflammatory effects of rosiglitazone on the cerebral microcirculation were marked. Functional capillary density increased and leukocyte rolling and adhesion were decreased in animals submitted to CLP and treated with rosiglitazone. Our data provide evidence for involvement of PPARγ activation in leukocyte-endothelium interactions and alterations in capillary density. Improved cerebral perfusion in animals treated with rosiglitazone, suggests that PPARγ activation is protective against cerebral microvascular dysfunction in sepsis.

Introduction Coagulation abnormalities are frequent in patients with severe infections. However, the predictive value of d -dimer and of the presence of associated coagulation derangements in severe community-acquired pneumonia (CAP) remains to be thoroughly evaluated. The aim of this study was to investigate the predictive value of coagulation parameters in patients with severe CAP admitted to the intensive care unit. Methods d -Dimer, antithrombin, International Society of Thrombosis and Hemostasis score, clinical variables, Sequential Organ Failure Assessment (SOFA), The Acute Physiology and Chronic Health Evaluation II (APACHE II) and the CURB-65 score were measured in the first 24 hours. Results are shown as median (25%-75% interquartile range). The main outcome measure was hospital mortality. Results Ninety patients with severe CAP admitted to the intensive care unit were evaluated. Overall hospital mortality was 15.5%. d -Dimer levels in nonsurvivors were higher than those in survivors. In the univariate analysis, d -dimer, SOFA, and APACHE II scores were predictors of death. The discriminative ability of d -dimer (area under receiver operating curve = 0.75 [95% confidence interval, 0.64-0.83]; best cutoff for d -dimer was 1798 ng/mL) for in-hospital mortality was comparable with APACHE II and SOFA and better than C-reactive protein. Moreover, the addition of d -dimer to APACHE II or SOFA score increased the discriminative ability of both scores (area under the receiver operating curve = 0.82 [0.72-0.89] and 0.84 [0.75-0.91], respectively). Conclusions d -Dimer levels are good predictors of outcome in severe CAP and may augment the predictive ability of scoring systems as APACHE II and SOFA.

Introduction Our aim was to evaluate the impact of corticosteroids on clinical course and outcomes of patients with severe community-acquired pneumonia (CAP) requiring invasive mechanical ventilation. Methods This was a cohort study of patients with severe CAP from 2 intensive care units in tertiary hospitals in Brazil and Portugal. Results A total of 111 patients were included (median age, 69 years; 56% men; 34% hospital mortality). Corticosteroids were prescribed in 61 (55%) patients. Main indications for their use were bronchospasm (52.5%) and septic shock (36%). Mortality rate of patients treated with and without corticosteroids was comparable (29.5% vs 32%, P = .837). No significant differences were observed on clinical course from day 1 to day 7 as assessed by the Sequential Organ Failure Assessment score (P = .95). Furthermore, C-reactive protein declined similarly in both groups (P = .147). In a multivariate analysis, mortality was associated with older age and higher Acute Physiology and Chronic Health Evaluation II score. Conclusions In patients with severe CAP requiring invasive mechanical ventilation, adjunctive therapy with corticosteroids did not influence intensive care unit and hospital mortality. In addition, no changes were observed on weaning from vasopressors, on recovery from organ failure/dysfunction as assessed by the Sequential Organ Failure Assessment score, as well as on C-reactive protein course.

Hepatic stellate cells (HSCs) have a critical role in liver physiology, and in the pathogenesis of liver inflammation and fibrosis. Here, we investigated the interplay between leukotrienes (LT) and TGF-β in the activation mechanisms of HSCs from schistosomal granulomas (GR-HSCs). First, we demonstrated that GR-HSCs express 5-lipoxygenase (5-LO), as detected by immunolocalization in whole cells and confirmed in cell lysates through western blotting and by mRNA expression through RT-PCR. Moreover, mRNA expression of 5-LO activating protein (FLAP) and LTC(4)-synthase was also documented, indicating that GR-HSCs have the molecular machinery required for LT synthesis. Morphological analysis of osmium and Oil-Red O-stained HSC revealed large numbers of small lipid droplets (also known as lipid bodies). We observed co-localization of lipid droplet protein marker (ADRP) and 5-LO by immunofluorescence microscopy. We demonstrated that GR-HSCs were able to spontaneously release cysteinyl-LTs (CysLTs), but not LTB(4,) into culture supernatants. CysLT production was highly enhanced after TGF-β-stimulation. Moreover, the 5-LO inhibitor zileuton and 5-LO gene deletion were able to inhibit the TGF-β-stimulated proliferation of GR-HSCs, suggesting a role for LTs in HSC activation. Here, we extend the immunoregulatory function of HSC by demonstrating that HSC from liver granulomas of schistosome-infected mouse are able to release Cys-LTs in a TGF-β-regulated manner, potentially impacting pathogenesis and liver fibrosis in schistosomiasis.

Babesia bovis is an intraerythrocytic apicomplexan protozoa of cattle that causes an acute infection with parasite persistence. Babesiosis limitation depends on macrophages, essential effector cells of the host innate defense, which generate inflammatory cytokines and nitric oxide. Herein, we report quantitative differences in the lipid composition of merozoites from two B. bovis strains with polar behaviour: attenuated R1A and virulent S2P. Accordingly, we observed a distinct inflammatory response induced by the total lipids of R1A (L(A)) and S2P (L(V)) in murine peritoneal macrophages. L(A) and particularly its fractions phosphatidic acid and phosphatidylserine+phosphatidylinositol (PS+PI), produced a strong activation of these cells with lipid body formation, cyclooxygenase-2 expression and pro-inflammatory TNFalpha, IL-6 and KC secretion. Although L(V) did not activate these cells, the corresponding PS+PI fraction induced TNFalpha, IL-6 and KC release. Therefore, these facts might be suggesting the presence of an inhibitor in L(V). Furthermore, the employment of wild type and toll like receptor 2 knockout (TLR2KO) mice allowed us to demonstrate that macrophage activation by the stimulating lipid fractions was mediated through TLR2. Interestingly, only L(A) activated the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Inhibitory studies employing UO126, indicated that the ERK pathway was required for TNFalpha, IL-6 and KC release. In conclusion, the absence of inflammatory response observed with the lipids of S2P virulent strain could constitute an evasion mechanism of the innate immune response enabling parasite establishment in the host.

Lipid body accumulation within leukocytes is a common feature in both clinical and experimental infectious, neoplasic and other inflammatory conditions. Here, we will review the contemporary evidence related to the biogenesis and structure of leukocyte lipid bodies (also known as lipid droplets) as inflammatory organelles. Studies of leukocyte lipid bodies are providing functional, ultrastructural and protein compositional evidences that lipid bodies are not solely storage depots of neutral lipid. Over the past years substantial progresses have been made to demonstrate that lipid body biogenesis is a highly regulated process, that culminate in the compartmentalization of a specific set of proteins and lipids, that place leukocyte lipid bodies as inducible cytoplasmic organelles with roles in cell signaling and activation, regulation of lipid metabolism, membrane trafficking and control of the synthesis and secretion of inflammatory mediators. Pertinent to the roles of lipid bodies in inflammation and cell signaling, enzymes involved in eicosanoid synthesis are localized at lipid bodies and lipid bodies are sites for eicosanoid generation. Collectively, lipid bodies in leukocytes are emerging as critical regulators of different inflammatory diseases, key markers of leukocyte activation and attractive targets for novel anti-inflammatory therapies.

Cytoplasmic lipid bodies (also known as lipid droplets) are intracellular deposits of arachidonic acid (AA), which can be metabolized for eicosanoid generation. PGE2 is a major AA metabolite produced by epithelial cells and can modulate restoration of epithelium homeostasis after injury. We studied lipid body biogenesis and their role in AA metabolic pathway in an epithelial cell line derived from normal rat intestinal epithelium, IEC-6 cells. Lipid bodies were virtually absent in confluent IEC-6 cells. Stimulation of confluent IEC-6 cells with unsaturated fatty acids, including AA or oleic acid (OA), induced rapid lipid body assembly that was independent on its metabolism to PGE(2), but dependent on G-coupled receptor-driven signaling through p38, PKC, and PI3 K. Newly formed lipid bodies compartmentalized cytosolic phospholipase (cPL)A(2)-alpha, while facilitated AA mobilization and synthesis of PGE(2) within epithelial cells. Thus, both lipid body-related events, including highly regulated biogenesis and functional assembly of cPLA (2)-alpha-driven enhanced AA mobilization and PGE(2)production, may have key roles in epithelial cell-driven inflammatory functions, and may represent relevant therapeutic targets of epithelial pathologies.

Background High cortisol levels are frequent in patients with severe infections. However, the predictive value of total cortisol and of the presence of critical illness-related corticosteroid insufficiency (CIRCI) in severe community-acquired pneumonia (CAP) remains to be thoroughly evaluated. The aim of this study was to investigate the predictive value of adrenal response in patients with severe CAP admitted to the ICU. Methods Baseline and postcorticotropin cortisol levels C-reactive protein (CRP), d-dimer, clinical variables, sequential organ failure assessment (SOFA), APACHE (acute physiology and chronic health evaluation) II, and CURB-65 (confusion, urea nitrogen, respiratory rate, BP, age ≥ 65 years) scores were measured in the first 24 h. Results are shown as median (interquartile range [IQR]). The major outcome measure was hospital mortality. Results Seventy-two patients with severe CAP admitted to the ICU were evaluated. Baseline cortisol levels were 18.1 μg/dL (IQR, 14.4 to 26.7 μg/dL), and the difference between baseline and postcorticotropin cortisol after 250 μg of corticotropin was 19 μg/dL (IQR, 12.8 to 27 μg/dL). Baseline cortisol levels presented positive correlations with scores of disease severity, including CURB-65, APACHE II, and SOFA (p Conclusions Baseline cortisol levels are better predictors of severity and outcome in severe CAP than postcorticotropin cortisol or routinely measured laboratory parameters or scores as APACHE II, SOFA, and CURB-65.

International audience; This report addressed the question whether ExoU stimulation of airway epithelial cells may contribute to the inflammatory response detected in the course of Pseudomonas aeruginosa respiratory infections. Infection with PA103 P. aeruginosa elicited a potent release of IL-6 and IL-8, as well as of arachidonic acid (AA) and PGE(2) that was reduced by the bacterial treatment with MAFP, a cPLA(2) inhibitor. Airway cells from the BEAS-2B line and in primary culture were shown to be enriched in lipid bodies (LBs), that are cytoplasmic domains implicated in AA transformation into eicosanoids. However, cells infected with PA103 and with a mutant deficient in exoU but complemented with a functional gene exhibited reduced contents of LBs, and this reduction was inhibited by MAFP. FACS analysis showed that the decrease in the LB content correlated with the presence of intracellular PGE(2). Also, in PA103-infected cells, PGE(2) was immunolocalized in LBs, suggesting that the reduction in the cell content of the organelles was due to consumption of their glycerolipids, resulting in local synthesis of the prostanoid. In conclusion, we showed the ExoU ability to induce airway epithelial cells to overproduce PGE(2) and we speculate that LB may represent intracellular loci involved in ExoU-induced eicosanoid synthesis.

Leptin is an adipocyte-derived hormone/cytokine that links nutritional status with neuroendocrine and immune functions. Lipid bodies (lipid droplets) are emerging as dynamic organelles with roles in lipid metabolism and inflammation. Here we investigated the roles of leptin in signaling pathways involved in cytoplasmic lipid body biogenesis and leukotriene B(4) synthesis in macrophages. Our results demonstrated that leptin directly activated macrophages and induced the formation of adipose differentiation-related protein-enriched lipid bodies. Newly formed lipid bodies were sites of 5-lipoxygenase localization and correlated with an enhanced capacity of leukotriene B(4) production. We demonstrated that leptin-induced macrophage activation was dependent on phosphatidylinositol 3-kinase (PI3K) activity, since the lipid body formation was inhibited by LY294002 and was absent in the PI3K knock-out mice. Leptin induces phosphorylation of p70(S6K) and 4EBP1 key downstream signaling intermediates of the mammalian target of rapamycin (mTOR) pathway in a rapamycin-sensitive mechanism. The mTOR inhibitor, rapamycin, inhibited leptin-induced lipid body formation, both in vivo and in vitro. In addition, rapamycin inhibited leptin-induced adipose differentiation-related protein accumulation in macrophages and lipid body-dependent leukotriene synthesis, demonstrating a key role for mTOR in lipid body biogenesis and function. Our results establish PI3K/mTOR as an important signaling pathway for leptin-induced cytoplasmic lipid body biogenesis and adipose differentiation-related protein accumulation. Furthermore, we demonstrate a previously unrecognized link between intracellular (mTOR) and systemic (leptin) nutrient sensors in macrophage lipid metabolism. Leptin-induced increased formation of cytoplasmic lipid bodies and enhanced inflammatory mediator production in macrophages may have implications for obesity-related cardiovascular diseases.

Lipid bodies (LB), lipid-rich inclusions abundantly present in cells engaged in inflammation, are specialized intracellular domains involved in generating inflammatory mediators, the eicosanoids. Since the acute Trypanosoma cruzi infection triggers a potent inflammatory reaction characterized by a great increase of peripheral blood monocyte (PBM) and macrophage numbers, we investigated the LB occurrence in these cells. The experimental rat infection by T. cruzi (Y strain) induced significant increase of the LB numbers in peritoneal macrophages at day 6 and 12, accompanied by significant enhancement of Prostaglandin E(2) (PGE(2)) production, as measured by EIA. At day 12, ultrastructural analysis of the heart, a target organ of the disease, showed numerous macrophages with LB prominently increased in number (mean of 8.3 per section view, range of 1-25) compared to controls (mean of 2.6 per section view, range of 0-3) and size. PBM from all groups rarely showed LB. Our results demonstrate, for the first time, that T. cruzi infection in rats elicits important LB formation in inflammatory macrophages but not in PBM. The increase in LB numbers during infection positively correlates with increased generation of PGE(2), suggesting that LB may have a role in the heightened eicosanoid production observed during T. cruzi infection.

Adult male rats were administered intrapleural (ipl) or intraperitoneal (i.p.) injection of lipopolysaccharide (LPS), 24 h after being submitted to forced swim test (FST). Four hours after LPS challenge, an increase in pleural and peritoneal leukocyte and neutrophil counts in animals not exposed to FST was noted. FST induced a marked increase in pleural, but not in peritoneal, neutrophil leukocytosis. The results suggest that lowered mood may increase inflammatory response to LPS.

Eosinophils are capable of generating eicosanoid derivatives of arachidonic acid by means of cyclooxygenase and the 5- and 15-lipoxygenase (LO) pathways. Moreover, eosinophils, because of their expression of leukotriene (LT) C 4 synthase, are a major source of 5-LO-derived cysteinyl LTs, which are potent paracrine mediators of bronchial obstruction and inflammation pertinent to asthma. The regulation of eicosanoid formation within eosinophils involves activation of key enzymes at specific intracellular sites. Calcium ionophore-elicited translocation of 5-LO to the membranes of the nuclear envelope is associated with LTC 4 formation. In addition, lipid bodies, the formation of which is initiated by specific receptor-mediated signaling pathways, are sites of cyclooxygenase- and LO-pathway eicosanoid formation. Newly formed LTC 4 can be immunolocalized at perinuclear membranes in ionophore-activated eosinophils and at lipid bodies in CCR3 agonist (eg, eotaxin) chemokine-stimulated eosinophils. The local generation of eicosanoids at distinct sites within eosinophils may be important for the roles of these eicosanoids, both as paracrine mediators pertinent to inflammation and as intracrine signal-transducing mediators that help regulate cellular responses of eosinophils. (J Allergy Clin Immunol 2002;109:393-400.)

Leukocyte lipid bodies, abundant in cells associated with inflammation, can be induced to form in response to stimuli that include cis -unsaturated, but not saturated, fatty acids. Arachidonyl trifluoromethyl ketone (AACOCF 3 ), a non-esterifiable arachidonate analog and an inhibitor of cytosolic phospholipase A 2 enzymes (PLA 2 ), dose-dependently (0–20 μM) stimulated neutrophil lipid body formation, but this stimulation was not attributable to PLA 2 inhibition. Palmitoyl trifluoromethyl ketone, also a PLA 2 inhibitor, failed to stimulate lipid body formation, like palmitic acid itself, and did not inhibit stimulated lipid body formation. Moreover, aspirin, indomethacin and ibuprofen, which inhibit cis -unsaturated fatty acid-induced lipid body formation, inhibited AACOCF 3 -induced lipid body formation. Lipid body induction with AACOCF 3 reflected its structural basis as a cis -unsaturated fatty acid analog. These results indicate that cytosolic PLA 2 enzymes are not active in lipid body induction and cis -fatty acid stimulation of lipid body formation does not require esterification of cis -fatty acids into glycerolipids.

The role of catecholamines in regulating pleural neutrophilia evoked by intrathoracic (i.t.) injection of lipopolysaccharide (LPS) was investigated in Wistar rats by means of surgical adrenalectomy, depletion of catecholamine stores or adrenoceptor blockade. Treatment of animals with a single dose of LPS evoked a dramatic increase in the number of pleural neutrophils concomitant with an increase in the number of these cells in blood at 4 h. Although blood neutrophilia was drastically reduced when catecholamine stores were depleted with intraperitoneal (i.p.) injection of reserpine, pleural neutrophilia was not modified. However, the i.t. injection of reserpine reduced the increase in pleural neutrophils after LPS stimulation. Adrenalectomy failed to inhibit the increase in neutrophil counts in the blood or pleural cavity after LPS challenge. Pretreatment with intravenous (i.v.) injection of prazosin, an alpha(1)/alpha(2B) antagonist, reduced LPS-induced blood but not pleural neutrophilia. On the other hand, although pleural neutrophilia was not affected by systemic pretreatment with the alpha(2)-adrenoceptor antagonist, yohimbine, the local treatment (i. t. injection) with this antagonist markedly reduced the increase in pleural neutrophil counts observed after stimulation by LPS. In contrast, pleural neutrophilia induced by i.t injection of formyl-methionyl-leucyl-phenylalanine (fMLP) was not modified by local treatment with yohimbine. Taken together, our results suggest that catecholamines, through activation of alpha(1) and alpha(2)-adrenoceptors, play a role in the regulation of blood and pleural neutrophilia observed during the inflammatory response evoked by LPS in the pleural cavity.

Purpose: Recently, new arbovirus such as Zika virus (ZIKV) and Chikungunya virus (CHIKV) were introduced in Brazil. Despite the clinical relevance, to date there is no syndromic surveillance system focused in arbovirosis in Brazil. Here, we present the initial results of active syndromic surveillance system for arbovirus infections in Rio de Janeiro, Brazil. Methods & Materials: This is a hospital-based prospective multicenter transversal study. Inclusion criteria were: Presence of fever AND/OR exanthema, with maximal duration of 7 days; PLUS: 2 or more nonspecific symptoms OR a clinical suspicion of arbovirus infection. Patients who met inclusion criteria had blood and urine tested for ZIKV, CHIKV and DENV by RT-PCR. Clinical data were collected using REDCap standardized form. Results: From March 14th to May 31th of 2016 (epidemiological weeks 11 to 22) 269 patients were included in the study. Patients were predominantly female (57.7%), with mean age of 41.9 years old. One hundred ninety patients (71.1%) were PCR-positive for at least one of the arbovirus tested, 4 patients were infected simultaneously by 2 arbovirus (3 ZIKV/CHIKV and 1 ZIKV/DENV4 (co-infection); 79 patients (29.4%) were PCR-negative. Among PCR-positive samples, 146 (54.3%) were confirmed for CHIKV, 36 (13.4%) were confirmed for ZIKV and 8 (3.0%) were confirmed for DENV (1 DENV3 and 7 DENV4). Over time, dengue cases were distributed between EW 11 to 13, most ZIKA cases (63.9%) between EW 12 to 15 and CHIKV cases were almost evenly distributed along the time study interval. Median time (days) between symptoms onset and PCR confirming diagnosis were 2.0 (IQR 2.0 – 4.25), 2.0 (IQR 1.5 – 3.0) and 4.5 (IQR 3.0 – 6.5), for ZIKV, CHIKV and DENV, respectively. Fever and arthralgia were the most frequent symptoms among CHIKV (90% and 89%) and ZIKV cases (78% and 75%); fever (88%) and headache (88%) were the most frequent symptoms among DENV. Conclusion: Co-circulation of dengue, Zika and CHIK has been observed Rio de Janeiro with direct impact on surveillance, diagnostic and management of the cases. Establishing an active syndromic surveillance system focused in arboviruses is paramount for a fast and precise public health response to these new arbovirus infections.

Intrathoracic injection of endotoxin lipopolysaccharide, LPS into rats induced a dose-dependent increase in the number of eosinophils recovered from the pleural cavity. The pleural eosinophil accumulation peaked within 24-48 h, and returned to basal levels within 120 h. This phenomenon was accompanied by mononuclear cell infiltration, and preceded by massive neutrophil accumulation. Pretreatment with indomethacin, BW 755C (a dual cyclo/lipoxygenase inhibitor), BW A4C (a specific lipoxygenase inhibitor) or the platelet activating factor (PAF) antagonists WEB 2086 and PCA 4248 failed to inhibit the endotoxin-induced pleural eosinophilia, whilst dexamethasone (5-10 micrograms/cavity) or cycloheximide (14-28 micrograms/cavity) abolished this phenomenon. Transfer of the cell-free pleural washing from LPS-treated donor rats to normal recipient rats led to a two-fold increase in the eosinophil counts. Treatment of donors, but not recipients, with cycloheximide or dexamethasone inhibited the eosinophil accumulation induced by the pleural washings, indicating that the generation of the eosinophilotactic activity, but not its effects, depends on protein synthesis. This eosinophilotactic activity was maintained after lyophilization and heating (100 degrees C for 30 min), but was destroyed by trypsin. This substance has a molecular weight ranging between 10 and 50 kDa. The available data suggest that the late eosinophil accumulation induced by LPS is independent of arachidonic acid metabolites and PAF, and probably depends on a newly generated heat-stable soluble protein.

The mechanism of rat thrombocytopenia induced by i.v. injections of platelet-activating factor (PAF-acether) was investigated. Platelet counts performed after diluting the blood samples in 1% formalin in saline showed that PAF-acether (6 μg/kg i.v.) induced a significant thrombocytopenia in rats, which peaked within 1 h, followed by a drastic increase of platelet counts at 4 h and a return to basal levels at 24 h. At this time, it was not possible to induce thrombocytopenia with a second challenge with PAF-acether, indicating a clear state of desensitization which disappeared within five days after the first injection of PAF-acether. The pretreatment with the specific PAF-acether receptor antagonists, BN 52021 (2.5–15 mg/kg), 48740 RP (6–25 mg/kg) and WEB 2086 (0.25–1 mg/kg) blocked the thrombocytopenia dose dependently. The lipoxygenase inhibitor nordihydroguaiaretic acid, at 25–100 mg/kg, was also effective against the thrombocytopenia induced by PAF-acether, reinforcing the potential involvement of arachidonic acid derivatives in this process. Adrenal hormones may modulate this process, since adrenalectomized animals responded to PAF-acether with exacerbated thrombocytopenia. No reduction in the platelet counts was noted when the blood was diluted in formalin-free saline, indicating that unstable aggregates were formed in vivo, which tended to resolve in vitro. Our results suggest that the thrombocytopenia induced in rats by PAF results from a reversible process of intravascular platelet aggregation, probably following the secretion of platelet-activating substances released by a first-hit blood cell.