Increased platelet activation and platelet-inflammasome engagement during chikungunya infection.

Chikungunya fever is a viral disease transmitted by mosquitoes of the genus Aedes. The infection is usually symptomatic and most common symptoms are fever accompanied by joint pain and swelling. In most cases symptoms subside within a week. However, severe prolonged and disabling joint pain, that may persist for several months, even years, are reported. Although the pathogenesis of Chikungunya infection is not fully understood, the evolution to severe disease seems to be associated with the activation of immune mechanisms and the action of inflammatory mediators. Platelets are recognized as inflammatory cells with fundamental activities in the immune response, maintenance of vascular stability and pathogenicity of several inflammatory and infectious diseases. Although the involvement of platelets in the pathogenesis of viral diseases has gained attention in recent years, their activation in Chikungunya has not been explored. The aim of this study was to analyze platelet activation and the possible role of platelets in the amplification of the inflammatory response during Chikungunya infection. We prospectively included 132 patients attended at the Quinta D'Or hospital and 25 healthy volunteers during the 2016 epidemic in Rio de Janeiro, Brazil. We observed increased expression of CD62P on the surface of platelets, as well as increased plasma levels of CD62P and platelet-derived inflammatory mediators indicating that the Chikungunya infection leads to platelet activation. In addition, platelets from chikungunya patients exhibit increased expression of NLRP3, caspase 4, and cleaved IL-1ß, suggestive of platelet-inflammasome engagement during chikungunya infection. In vitro experiments confirmed that the Chikungunya virus directly activates platelets. Moreover, we observed that platelet activation and soluble p-selectin at the onset of symptoms were associated with development of chronic forms of the disease. Collectively, our data suggest platelet involvement in the immune processes and inflammatory amplification triggered by the infection.

Curine Inhibits Macrophage Activation and Neutrophil Recruitment in a Mouse Model of Lipopolysaccharide-Induced Inflammation.

Curine is a bisbenzylisoquinoline alkaloid (BBA) with anti-allergic, analgesic, and anti-inflammatory properties. Previous studies have demonstrated that this alkaloid is orally active at non-toxic doses. However, the mechanisms underlying its anti-inflammatory effects remain to be elucidated. This work aimed to investigate the effects of curine on macrophage activation and neutrophil recruitment. Using a murine model of lipopolysaccharide (LPS)-induced pleurisy, we demonstrated that curine significantly inhibited the recruitment of neutrophils in association with the inhibition of cytokines tumor necrosis factor (TNF-α), interleukin (IL)-1ß, IL-6, monocyte chemotactic protein (CCL2/MCP-1) as well as leukotriene B4 in the pleural lavage of mice. Curine treatment reduced cytokine levels and the expression of iNOS in in vitro cultures of macrophages stimulated with LPS. Treatment with a calcium channel blocker resulted in comparable inhibition of TNF-α and IL-1ß production, as well as iNOS expression by macrophages, suggesting that the anti-inflammatory effects of curine may be related to the inhibition of calcium-dependent mechanisms involved in macrophage activation. In conclusion, curine presented anti-inflammatory effects that are associated with inhibition of macrophage activation and neutrophil recruitment by inhibiting the production of inflammatory cytokines, LTB4 and nitric oxide (NO), and possibly by negatively modulating Ca2+ influx.

Emergence of the East-Central-South-African genotype of Chikungunya virus in Brazil and the city of Rio de Janeiro may have occurred years before surveillance detection.

Brazil, which is hyperendemic for dengue virus (DENV), has had recent Zika (ZIKV) and (CHIKV) Chikungunya virus outbreaks. Since March 2016, CHIKV is the arbovirus infection most frequently diagnosed in Rio de Janeiro. In the analysis of 1835 syndromic patients, screened by real time RT-PCR, 56.4% of the cases were attributed to CHIKV, 29.6% to ZIKV, and 14.1% to DENV-4. Sequence analyses of CHIKV from sixteen samples revealed that the East-Central-South-African (ECSA) genotype of CHIKV has been circulating in Brazil since 2013 [95% bayesian credible interval (BCI): 03/2012-10/2013], almost a year before it was detected by arbovirus surveillance program. Brazilian cases are related to Central African Republic sequences from 1980's. To the best of our knowledge, given the available sequence published here and elsewhere, the ECSA genotype was likely introduced to Rio de Janeiro early on 2014 (02/2014; BCI: 07/2013-08/2014) through a single event, after primary circulation in the Bahia state at the Northestern Brazil in the previous year. The observation that the ECSA genotype of CHIKV was circulating undetected underscores the need for improvements in molecular methods for viral surveillance.

Differential Shedding and Antibody Kinetics of Zika and Chikungunya Viruses, Brazil.

In seroconversion panels obtained from patients from Brazil, diagnostic testing for Zika virus infection was improved by combining multiple antibody isotypes, techniques, and antigens, but sensitivity remained suboptimal. In contrast, chikungunya virus diagnostic testing was unambiguous. Recurrent recent arbovirus infections suggested by serologic data and unspecific symptoms highlight the need for exhaustive virologic testing.

New Insights in Purinergic Therapy: Novel Antagonists for Uridine 5'-Triphosphate-Activated P2Y Receptors from Brazilian Flora.

P2Y2 and P2Y4 receptors are physiologically activated by uridine 5'-triphosphate (UTP) and are widely expressed in many cell types in humans. P2Y2 plays an important role in inflammation and proliferation of tumor cells, which could be attenuated with the use of antagonists. However, little is known about the physiological functions related to P2Y4, due to the lack of selective ligands for these receptors. This can be solved through the search for novel compounds with antagonistic activity. The aim of this study was to discover new potential antagonist candidates for P2Y2 and P2Y4 receptors from natural products. We applied a calcium measurement methodology to identify new antagonist candidates for these receptors. First, we established optimal conditions for the calcium assay using J774.G8, a murine macrophage cell line, which expresses functional P2Y2 and P2Y4 receptors and then, we performed the screening of plant extracts at a cutoff concentration of 50 µg/mL. ATP and ionomycin, known intracellular calcium inductors, were used to stimulate cells. The calculated EC50 were 11 µM and 103 nM, respectively. These cells also responded to the UTP stimulation with an EC50 of 1.021 µM. Screening assays were performed and a total of 100 extracts from Brazilian plants were tested. Joannesia princeps Vell. (stem) and Peixotoa A. Juss (flower and leaf) extracts stood out due to their ability to inhibit UTP-induced responses without causing cytotoxicity, and presented an IC50 of 32.32, 14.99, and 12.98 µg/mL, respectively. Collectively, our results point to the discovery of potential antagonist candidates from Brazilian flora for UTP-activated receptors.

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

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.

Photobiomodulation in the Prevention of Tooth Sensitivity Caused by In-Office Dental Bleaching. A Randomized Placebo Preliminary Study.

OBJECTIVE: Analyze the effect of photobiomodulation in the prevention of tooth sensitivity after in-office dental bleaching. BACKGROUND DATA: Tooth sensitivity is a common clinical consequence of dental bleaching. Therapies for prevention of sensitivity have been investigated in literature. MATERIALS AND METHODS: This study was developed as a randomized, placebo blind clinical trial. Fifty patients were selected (n = 10) and randomly divided into five groups: (1) control, (2) placebo, (3) laser before bleaching, (4) laser after bleaching, and (5) laser before and after bleaching. Irradiation was performed perpendicularly, in contact, on each tooth during 10 sec per point in two points. The first point was positioned in the middle of the tooth crown and the second in the periapical region. Photobiomodulation was applied using the following parameters: 780 nm, 40 mW, 10 J/cm2, 0.4 J per point. Pain was analyzed before, immediately after, and seven subsequent days after bleaching. Patients were instructed to report pain using the scale: 0 = no tooth sensitivity, 1 = gentle sensitivity, 2 = moderate sensitivity, 3 = severe sensitivity. RESULTS: There were no statistical differences between groups at any time (p > 0.05). More studies, with others parameters and different methods of tooth sensitivity analysis, should be performed to complement the results found. CONCLUSIONS: Within the limitation of the present study, the laser parameters of photobiomodulation tested in the present study were not efficient in preventing tooth sensitivity after in-office bleaching.

An Improved Method for P2X7R Antagonist Screening.

ATP physiologically activates the P2X7 receptor (P2X7R), a member of the P2X ionotropic receptor family. When activated by high concentrations of ATP (i.e., at inflammation sites), this receptor is capable of forming a pore that allows molecules of up to 900 Da to pass through. This receptor is upregulated in several diseases, particularly leukemia, rheumatoid arthritis and Alzheimer's disease. A selective antagonist of this receptor could be useful in the treatment of P2X7R activation-related diseases. In the present study, we have evaluated several parameters using in vitro protocols to validate a high-throughput screening (HTS) method to identify P2X7R antagonists. We generated dose-response curves to determine the EC50 value of the known agonist ATP and the ICs50 values for the known antagonists Brilliant Blue G (BBG) and oxidized ATP (OATP). The values obtained were consistent with those found in the literature (0.7 ± 0.07 mM, 1.3-2.6 µM and 173-285 µM for ATP, BBG and OATP, respectively) [corrected].The Z-factor, an important statistical tool that can be used to validate the robustness and suitability of an HTS assay, was 0.635 for PI uptake and 0.867 for LY uptake. No inter-operator variation was observed, and the results obtained using our improved method were reproducible. Our data indicate that our assay is suitable for the selective and reliable evaluation of P2X7 activity in multiwell plates using spectrophotometry-based methodology. This method might improve the high-throughput screening of conventional chemical or natural product libraries for possible candidate P2X7R antagonist or agonist.

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.

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.

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.

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 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.

Eosinophils as a novel cell source of prostaglandin D2: autocrine role in allergic inflammation.

PGD(2) 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 PGD(2) synthesis, the hematopoietic PGD synthase (H-PGDS). In this study, we investigated whether eosinophils are also able to synthesize, and therefore, supply biologically active PGD(2). PGD(2) 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 PGD(2) 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 PGD(2) 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 PGD(2) synthesis, which, by acting on membrane-expressed specific receptors (D prostanoid receptors 1 and 2), displayed an autocrine/paracrine ability to trigger leukotriene C(4) synthesis and lipid body biogenesis, hallmark events of eosinophil activation. In vitro differentiated mouse eosinophils also synthesized paracrine/autocrine active PGD(2) 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 PGD(2)-synthesizing cell population. Our findings reveal that eosinophils are indeed able to synthesize and secrete PGD(2), hence representing during allergic inflammation an extra cell source of PGD(2), which functions as an autocrine signal for eosinophil activation.

Chemical profile and antinociceptive efficacy of Rheedia longifolia leaf extract.

Different species of the family Clusiaceae, including Rheedia longifolia, are used in folk medicine to treat inflammatory diseases. This family is largely distributed in tropical and subtropical areas of Brazil, but their chemical and pharmacological properties have been the subject of a few studies. In previous studies, we found that the aqueous extract from R. longifolia leaves presented important anti-inflammatory and analgesic activity. We investigated the chemical profile of R. longifolia and characterized the pharmacological effect of different chemically identified fractions in pharmacological models of neurogenic and inflammatory nociception. The pharmacological tests showed that oral treatment with aqueous crude extract and fractions of methanol extract of R. longifolia leaf induced a significant antinociceptive effect using von Frey filaments. In addition, the most polar fractions presented antinociceptive activity in a neurogenic model of nociception (capsaicin model). The chromatographic analysis indicated the presence of bisflavonoids in the fractions obtained from the methanol extract. These results suggest that bisflavonoids found in methanol-extracted fractions are involved in the inhibition of inflammatory and neurogenic nociception. It is important that the R. longifolia aqueous extract treatment inhibited ulcer formation induced by indomethacin, suggesting an anti-ulcerogenic activity closely associated with its analgesic effect.

Effect of Rheedia longifolia leaf extract and fractions on the P2X7 receptor in vitro: novel antagonists?

Recently, the P2X(7) receptor has been reported to be associated with chronic inflammatory and neuropathic pain. Because Rheedia longifolia extract has analgesic and anti-inflammatory activity, we evaluated the in vitro inhibitory potential of methanol extract and fractions from its leaves on the P2X(7) purinergic receptor. The activity of P2X(7) was studied with a dye uptake assay and with the whole-cell patch clamp technique in mouse peritoneal macrophages treated with methanol extract of R. longifolia leaves and fractions. The dye uptake was evaluated by flow cytometry and fluorescence microscopy. The R. longifolia extract and some fractions showed an inhibitory effect on the P2X(7) purinergic receptor in a dose-dependent manner. The ethyl acetate fraction exhibited the most potent inhibitory effects. The methanol extract and the butanol fraction showed the same inhibitory effects, despite their lower potency compared with the other fractions. The R. longifolia extract and some of its fractions may be anti-inflammatory because of their inhibitory effect on the P2X(7) receptor. Further investigation is needed to determine the pattern of inhibition and selectivity. Chromatographic analysis indicated the presence of bisflavonoids in the methanol extract fractions. A member of this chemical family is the most probable active compound responsible for the P2X(7) inhibitory effects present in the R. Longifolia extract and fractions.

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.

Antinociceptive activity of aqueous extract of Bowdichia virgilioides in mice.

Bowdichia virgilioides Kunth (Family Fabaceae) is a plant that is distributed widely in the tropical and subtropical regions of the world. In the northeast region of Brazil, where B. virgilioides is called "sucupira-preta," the stem bark is used in folk medicine to treatment of inflammatory and painful diseases. This study aimed to evaluate the antinociceptive activity of the aqueous extract of the dried stem bark of B. virgilioides. The aqueous extract of B. virgilioides in doses of 50, 100, 200, and 400 mg/kg was administered orally 1 hour prior to pain induction. Only the doses of 200 and 400 mg/kg produced an inhibition by 61% and 74%, respectively, in the number of abdominal writhings induced by acetic acid. This antinociceptive effect was not reversed by pretreatment with naloxone, indicating that the effect is not associated with the activation of opioid receptors. In the formalin test, using the two highest doses, the extract had no effect in the first phase but produced an analgesic effect on the second phase with the inhibition of licking time (P < .001). In the hot plate test, no effect was seen at the dose of 400 mg/kg p.o. Our findings show that B. virgilioides contains pharmacologically active constituents that possess antinociceptive activity justifying its popular therapeutic use in treating conditions associated with the painful conditions.

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.