Andrea Surrage Calheiros

Cutting Edge: Prostaglandin D2 Enhances Leukotriene C4 Synthesis by Eosinophils during Allergic Inflammation: Synergistic In Vivo Role of Endogenous Eotaxin

In addition to the well-recognized ability of prostaglandin D2 (PGD2) to regulate eosinophil trafficking, we asked whether PGD2 was also able to activate eosinophils and control their leukotriene C4 (LTC4)-synthesizing machinery. PGD2 administration to presensitized mice enhanced in vivo LTC4 production and formation of eosinophil lipid bodies–potential LTC4-synthesizing organelles. Immunolocalization of newly formed LTC4 demonstrated that eosinophil lipid bodies were the sites of LTC4 synthesis during PGD2-induced eosinophilic inflammation. Pretreatment with HQL-79, an inhibitor of PGD synthase, abolished LTC4 synthesis and eosinophil lipid body formation triggered by allergic challenge. Although PGD2 was able to directly activate eosinophils in vitro, in vivo PGD2-induced lipid body-driven LTC4 synthesis within eosinophils was dependent on the synergistic activity of endogenous eotaxin acting via CCR3. Our findings, that PGD2 activated eosinophils and enhanced LTC4 synthesis in vivo in addition to the established PGD2 roles in eosinophil recruitment, heighten the interest in PGD2 as a target for antiallergic therapies.

Eosinophils as a Novel Cell Source of Prostaglandin D2: Autocrine Role in Allergic Inflammation

PGD2 is a key mediator of allergic inflammatory diseases that is mainly synthesized by mast cells, which constitutively express high levels of the terminal enzyme involved in PGD2 synthesis, the hematopoietic PGD synthase (H-PGDS). In this study, we investigated whether eosinophils are also able to synthesize, and therefore, supply biologically active PGD2. PGD2 synthesis was evaluated within human blood eosinophils, in vitro differentiated mouse eosinophils, and eosinophils infiltrating inflammatory site of mouse allergic reaction. Biological function of eosinophil-derived PGD2 was studied by employing inhibitors of synthesis and activity. Constitutive expression of H-PGDS was found within nonstimulated human circulating eosinophils. Acute stimulation of human eosinophils with A23187 (0.1–5 μM) evoked PGD2 synthesis, which was located at the nuclear envelope and was inhibited by pretreatment with HQL-79 (10 μM), a specific H-PGDS inhibitor. Prestimulation of human eosinophils with arachidonic acid (10 μM) or human eotaxin (6 nM) also enhanced HQL-79–sensitive PGD2 synthesis, which, by acting on membrane-expressed specific receptors (D prostanoid receptors 1 and 2), displayed an autocrine/paracrine ability to trigger leukotriene C4 synthesis and lipid body biogenesis, hallmark events of eosinophil activation. In vitro differentiated mouse eosinophils also synthesized paracrine/autocrine active PGD2 in response to arachidonic acid stimulation. In vivo, at late time point of the allergic reaction, infiltrating eosinophils found at the inflammatory site appeared as an auxiliary PGD2-synthesizing cell population. Our findings reveal that eosinophils are indeed able to synthesize and secrete PGD2, hence representing during allergic inflammation an extra cell source of PGD2, which functions as an autocrine signal for eosinophil activation.

Suppressive effect of distinct bradykinin B2 receptor antagonist on allergen-evoked exudation and leukocyte infiltration in sensitized rats

Bradykinin is suggested to play a role in the pathophysiology of several acute and chronic diseases, including allergic disorders such as asthma. In the present study, we have investigated the importance of bradykinin in mediating allergic inflammation in rats. To this end we have tested the effects of the B2 receptor antagonists Hoe 140, FR173657 or FR172357 on the pleural inflammatory response triggered by intrapleural (i.pl.) injection of allergen (ovalbumin, 12 μg cavity−1) in 14 day‐actively sensitized Wistar rats. Analysis of the pleural fluid effluent revealed a sequence of mast cell‐dependent inflammatory events, including early protein exudation and neutrophilia and late pleural eosinophil influx. Local treatment with Hoe 140 (0.1 and 1 μg cavity−1), FR173657 (1 and 10 μg cavity−1) or FR172357 (1 and 10 μg cavity−1) inhibited dose‐dependently allergen‐induced mast cell activation with impairment of pleural plasma leakage, neutrophil accumulation and late eosinophil influx. Moreover, the B2 receptor antagonists also dose‐dependently inhibited the allergic like inflammatory pleurisy triggered by bradykinin (50 μg cavity−1), which is characterized by acute mast cell degranulation, protein leakage and pleural eosinophil infiltration. Taken together, our findings provide substantial evidence to suggest that bradykinin acting on its B2 receptors play a critical role in mediating allergic mast cell‐dependent inflammation in rats, and suggest that B2 receptor antagonists may be useful therapeutically to control allergic dysfunction.

Cross-Talk between Macrophage Migration Inhibitory Factor and Eotaxin in Allergic Eosinophil Activation Forms Leukotriene C4–Synthesizing Lipid Bodies

Recent studies have demonstrated an essential and nonredundant role for macrophage migration inhibitory factor (MIF) in asthma pathogenesis. Here we investigate the mechanisms involved in MIF-induced eosinophil activation. By using a model of allergic pulmonary inflammation, we observed that allergen challenge-elicited eosinophil influx, lipid body (also known as lipid droplets) biogenesis, and leukotriene (LT) C₄ synthesis are markedly reduced in Mif(-/-) compared with wild-type mice. Likewise, in vivo administration of MIF induced formation of new lipid bodies within eosinophils recruited to the inflammatory reaction site that corresponded to the intracellular compartment of increased LTC₄ synthesis. MIF-mediated eosinophil activation was at least in part due to a direct effect on eosinophils, because MIF was able to elicit lipid body assembly within human eosinophils in vitro, a phenomenon that was blocked by neutralization of the MIF receptor, CD74. MIF-induced eosinophil lipid body biogenesis, both in vivo and in vitro, was dependent on the cooperation of MIF and eotaxin acting in a positive-feedback loop, because anti-eotaxin and anti-CCR3 antibodies inhibit MIF-elicited lipid body formation, whereas eotaxin-induced lipid body formation is affected by anti-CD74 and MIF expression deficiency. Therefore, allergy-elicited inflammatory MIF acts in concert with eotaxin as a key activator of eosinophils to form LTC₄-synthesizing lipid bodies via cross-talk between CD74 and CCR3. Due to the effect of MIF on eosinophils, strategies that inhibit MIF activity might be of therapeutic value in controlling allergic inflammation.

Curine inhibits eosinophil activation and airway hyper-responsiveness in a mouse model of allergic asthma.

Allergic asthma is a chronic inflammatory airway disease with increasing prevalence around the world. Current asthma therapy includes drugs that usually cause significant side effects, justifying the search for new anti-asthmatic drugs. Curine is a bisbenzylisoquinoline alkaloid that modulates calcium influx in many cell types; however, its anti-allergic and putative toxic effects remain to be elucidated. Our aim was to investigate the effects of curine on eosinophil activation and airway hyper-responsiveness (AHR) and to characterize its potential toxic effects. We used a mouse model of allergic asthma induced by sensitization and challenge with ovalbumin (OVA) to evaluate the anti-allergic effects of oral treatment with curine. The oral administration of curine significantly inhibited eosinophilic inflammation, eosinophil lipid body formation and AHR in animals challenged with OVA compared with animals in the untreated group. The curine treatment also reduced eotaxin and IL-13 production triggered by OVA. Verapamil, a calcium channel antagonist, had similar anti-allergic properties, and curine pre-treatment inhibited the calcium-induced tracheal contractile response ex-vivo, suggesting that the mechanism by which curine exerts its effects is through the inhibition of a calcium-dependent response. A toxicological evaluation showed that orally administered curine did not significantly alter the biochemical, hematological, behavioral and physical parameters measured in the experimental animals compared with saline-treated animals. In conclusion, curine showed anti-allergic activity through mechanisms that involve inhibition of IL-13 and eotaxin and of Ca(++) influx, without inducing evident toxicity and as such, has the potential for the development of anti-asthmatic drugs.

Natural Products as a Source for New Anti-Inflammatory and Analgesic Compounds through the Inhibition of Purinergic P2X Receptors

Natural products have reemerged in traditional medicine as a potential source of new molecules or phytomedicines to help with health disorders. It has been established that members of the P2X subfamily, ATP-gated ion channels, are crucial to the inflammatory process and pain signalization. As such, several preclinical studies have demonstrated that P2X2R, P2X3R, P2X4R and P2X7R are promising pharmacological targets to control inflammatory and pain disorders. Several studies have indicated that natural products could be a good source of the new specific molecules needed for the treatment of diseases linked to inflammation and pain disorders through the regulation of these receptors. Herein, we discuss and give an overview of the applicability of natural products as a source to obtain P2X receptors (P2XR) selective antagonists for use in clinical treatment, which require further investigation.

Curine inhibits mast cell-dependent responses in 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 1h 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 D2 (PGD2) 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 PGD2 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.

Rapid antigen tests for dengue virus serotypes and Zika virus in patient serum

A low-cost, equipment-free rapid antigen test distinguishes dengue virus serotypes and Zika virus in patient sera without detectable cross-reactivity. Distinguishing dengue from Zika More than mere summer pests, mosquitoes can transmit viruses, such as dengue and Zika. Diagnosing infections of these related flaviviruses can be difficult because of cross-reactivity in diagnostic tests. Bosch et al. developed monoclonal antibodies to detect viral nonstructural 1 (NS1) protein antigens specific to dengue and Zika. Incorporating the antibodies into an immunochromatography format yielded a rapid diagnostic assay that produces a visual readout in the presence of NS1. The assay identified the four dengue serotypes and Zika viral infections without cross-reaction when testing human serum samples from endemic areas in Central and South America and India. This approach could be useful for developing rapid diagnostics for other emerging pathogens. The recent Zika virus (ZIKV) outbreak demonstrates that cost-effective clinical diagnostics are urgently needed to detect and distinguish viral infections to improve patient care. Unlike dengue virus (DENV), ZIKV infections during pregnancy correlate with severe birth defects, including microcephaly and neurological disorders. Because ZIKV and DENV are related flaviviruses, their homologous proteins and nucleic acids can cause cross-reactions and false-positive results in molecular, antigenic, and serologic diagnostics. We report the characterization of monoclonal antibody pairs that have been translated into rapid immunochromatography tests to specifically detect the viral nonstructural 1 (NS1) protein antigen and distinguish the four DENV serotypes (DENV1–4) and ZIKV without cross-reaction. To complement visual test analysis and remove user subjectivity in reading test results, we used image processing and data analysis for data capture and test result quantification. Using a 30-μl serum sample, the sensitivity and specificity values of the DENV1–4 tests and the pan-DENV test, which detects all four dengue serotypes, ranged from 0.76 to 1.00. Sensitivity/specificity for the ZIKV rapid test was 0.81/0.86, respectively, using a 150-μl serum input. Serum ZIKV NS1 protein concentrations were about 10-fold lower than corresponding DENV NS1 concentrations in infected patients; moreover, ZIKV NS1 protein was not detected in polymerase chain reaction–positive patient urine samples. Our rapid immunochromatography approach and reagents have immediate application in differential clinical diagnosis of acute ZIKV and DENV cases, and the platform can be applied toward developing rapid antigen diagnostics for emerging viruses.

Curine, an Alkaloid Isolated from Chondrodendron platyphyllum Inhibits Prostaglandin E2 in Experimental Models of Inflammation and Pain

Curine is a bisbenzylisoquinoline alkaloid that is isolated from Chondrodendron platyphyllum, a plant that is used to treat malaria, inflammation, and pain. Recent reports have demonstrated the antiallergic effects of curine at nontoxic doses. However, its anti-inflammatory and analgesic properties remain to be elucidated. This study investigated the anti-inflammatory and analgesic effects of curine in mice. We analyzed the effects of an oral treatment with curine in the formation of paw edema, vascular permeability, abdominal contortion, licking behavior, and hyperalgesia using different inflammatory stimuli. Curine significantly inhibited the formation of paw edema by decreasing vascular permeability, inhibited the acetic acid-induced writhing response, inhibited the licking behavior during inflammation but not during the neurogenic phase of the formalin test, and inhibited carrageenan-induced hyperalgesia. Finally, curine inhibited prostaglandin E2 production in vitro without affecting cyclooxygenase-2 expression. The effects of curine treatment were similar to the effects of indomethacin, but were different from the effects of morphine treatment, suggesting that the analgesic effects of curine do not result from the direct inhibition of neuronal activation but instead depend on anti-inflammatory mechanisms that, at least in part, result from the inhibition of prostaglandin E2 production. In conclusion, curine presents anti-inflammatory and analgesic effects at nontoxic doses and has the potential for use in anti-inflammatory drug development.

Time course of pulmonary burden in mice exposed to residual oil fly ash

Residual oil fly ash (ROFA) is a common pollutant in areas where oil is burned. This particulate matter (PM) with a broad distribution of particle diameters can be inhaled by human beings and putatively damage their respiratory system. Although some studies deal with cultured cells, animals, and even epidemiological issues, so far a comprehensive analysis of respiratory outcomes as a function of the time elapsed after exposure to a low dose of ROFA is wanted. Thus, we aimed to investigate the time course of mechanical, histological, and inflammatory lung changes, as well as neutrophils in the blood, in mice exposed to ROFA until 5 days after exposure. BALB/c mice (25 ± 5 g) were randomly divided into 7 groups and intranasally instilled with either 10 μL of sterile saline solution (0.9% NaCl, CTRL) or ROFA (0.2 μg in 10 μL of saline solution). Pulmonary mechanics, histology (normal and collapsed alveoli, mononuclear and polymorphonuclear cells, and ultrastructure), neutrophils (in blood and bronchoalveolar lavage fluid) were determined at 6 h in CTRL and at 6, 24, 48, 72, 96, and 120 h after ROFA exposure. ROFA contained metal elements, especially iron, polycyclic aromatic hydrocarbons (PAHs), and organochlorines. Lung resistive pressure augmented early (6 h) in the course of lung injury and other mechanical, histological and inflammatory parameters increased at 24 h, returning to control values at 120 h. Blood neutrophilia was present only at 24 and 48 h after exposure. Swelling of endothelial cells with adherent neutrophils was detected after ROFA instillation. No neutrophils were present in the lavage fluid. In conclusion, the exposure to ROFA, even in low doses, induced early changes in pulmonary mechanics, lung histology and accumulation of neutrophils in blood of mice that lasted for 4 days and disappeared spontaneously.