Activation of cellular functions in macrophages by venom secretory Asp-49 and Lys-49 phospholipases A(2).

The in vitro effects of myotoxin III (MT-III), an Asp-49 catalytically-active phospholipase A(2), and myotoxin II (MT-II), a catalytically-inactive Lys-49 variant, isolated from Bothrops asper snake venom, on phagocytosis and production of hydrogen peroxide (H(2)O(2)) by thioglycollate-elicited macrophages were investigated. MT-II and MT-III were cytotoxic to mouse peritoneal macrophages at concentrations higher than 25 microg/ml. At non-cytotoxic concentrations, MT-II stimulated Fcgamma, complement, mannose and beta-glucan receptors-mediated phagocytosis, whereas MT-III stimulated only the mannose and beta-glucan receptors-mediated phagocytosis. Moreover, both myotoxins induced the release of H(2)O(2) by thioglycollate-elicited macrophages, MT-III being the most potent stimulator. MT-II induced the release of H(2)O(2) only at a concentration of 3.2 microg/ml (130% increment) while MT-III induced this effect at all concentrations tested (0.5-2.5 microg/ml; average of 206% increment). It is concluded that, at non-cytotoxic concentrations, MT-II and MT-III activate defense mechanisms in macrophages up regulating phagocytosis, mainly via mannose and beta-glucan receptors, and the respiratory burst.     

Inflammation induced by Bothrops asper venom: release of proinflammatory cytokines and eicosanoids, and role of adhesion molecules in leukocyte infiltration.

Bothrops asper venom (BaV) causes systemic and local effects characterized by an acute inflammatory reaction with accumulation of leukocytes and release of endogenous mediators. In this study, the effects of BaV on the release of the cytokines IL-1, IL-6 and TNF-alpha and the eicosanoids LTB4 and TXA2 in the peritoneal cavity of mice were analyzed. We also investigated the participation of beta2 integrin chain, l-selectin, LFA-1, ICAM-1 and PECAM-1 adhesion molecules in the BaV-induced leukocyte accumulation. Levels of proinflammatory cytokines IL-6 and TNF-alpha, as well as eicosanoids LTB4 and TXA2 were significantly increased after BaV injection (250 microg/kg), whereas no increment in IL-1 was observed. Anti-mouse l-selectin, LFA-1, ICAM-1, PECAM-1 and beta2 integrin chain monoclonal antibodies resulted in a reduction of neutrophil accumulation induced by BaV injection compared with isotype-matched control injected animals. These data suggest that BaV is able to induce the activation of leukocytes and endothelium to express adhesion molecules involved in the recruitment of neutrophils into the inflammed site. Furthermore, these results showed that BaV induces the release of cytokines and eicosanoids in the local of the venom injection; these inflammatory mediators may be important for the initiation and amplification of the inflammatory reaction characteristic from Bothrops sp envenomation.     

Endoplasmic Stress Affects the Coinfection of Leishmania Amazonensis and the Phlebovirus (Bunyaviridae) Icoaraci

Viral coinfections can modulate the severity of parasitic diseases, such as human cutaneous leishmaniasis. Leishmania parasites infect thousands of people worldwide and cause from single cutaneous self-healing lesions to massive mucosal destructive lesions. The transmission to vertebrates requires the bite of Phlebotomine sandflies, which can also transmit Phlebovirus. We have demonstrated that Leishmania infection requires and triggers the Endoplasmic stress (ER stress) response in infected macrophages. In the present paper, we tested the hypothesis that ER stress is increased and required for the aggravation of Leishmania infection due to coinfection with Phlebovirus. We demonstrated that Phlebovirus Icoaraci induces the ER stress program in macrophages mediated by the branches IRE/XBP1 and PERK/ATF4. The coinfection with L. amazonensis potentiates and sustains the ER stress, and the inhibition of IRE1α or PERK results in poor viral replication and decreased parasite load in macrophages. Importantly, we observed an increase in viral replication during the coinfection with Leishmania. Our results demonstrated the role of ER stress branches IRE1/XBP1 and PERK/ATF4 in the synergic effect on the Leishmania increased load during Phlebovirus coinfection and suggests that Leishmania infection can also increase the replication of Phlebovirus in macrophages.     

Clinicopathological prognostic factors for gallbladder carcinoma: a retrospective study

BackgroundGallbladder carcinoma (GBC) is an uncommon neoplasm with poor long-term survival. Worldwide the incidence rates vary according to geographic area. The multifactorial aetiology and the rarity of the disease limits the studies to improve outcomes in patients, since the treatment remains mostly surgical. The aim of this study was to identify clinicopathological prognostic factors for survival in patients with GBC submitted to surgery in our institution—a tertiary centre in Portugal. Also, to assess the expression of possible biomarkers (HER2, CD44 and ALDH1) in GBC, as well as the frequency of microsatellite instability (MSI) tumours.MethodsClinicopathological characteristics of 41 consecutive patients that underwent surgical resection for GBC (2008–2019) at our hospital were retrospectively reviewed. Clinicopathological factors were assessed and an immunohistochemical (IHC) analysis was done. Microsatellite stability (MSS) was considered if there was maintenance of nuclear expression of MLH1, MSH2, MSH6 and PMS2. Human epidermal growth factor receptor 2 (HER2) expression was evaluated according to the rules applied for gastric cancer and expression of CD44 and ALH1 was evaluated in order to detect cancer stem cells (CSC). Survival analysis was conducted using Kaplan-Meier and Cox regression was used to find prognostic factors.ResultsIncidence of GBC in our cohort of patients was 0.45%, most commonly affecting females. Median overall survival (OS) was 23 months with a 39.6% 5-year survival rate. Stage > II [hazard ratios (HR) =8.58; P=0.007], lymphovascular invasion (LVI) (HR =4.06; P=0.045) and hepatic resection (HR =0.288; P=0.034) independently influenced survival. HER2 positivity and high expression of CD44 or ADLH1 did not show significant influence in survival (P=0.649, P=0.868 and P=0.914, respectively), although HER2 and ALDH1 positive patients showed a tendency to a shorter OS, compared to negative patients. We found no relation between these biomarkers expression and disease stage. All analysed samples had MSS.ConclusionsGBC patients with a worse prognosis can be identified. The overexpression of HER2 could select patients for targeted therapy and prompt tissue sampling in unresectable patients.     

Anti‐TNF‐α Activity of Brazilian Medicinal Plants and Compounds from Ouratea semiserrata

Several plant species are used in Brazil to treat inflammatory diseases and associated conditions. TNF‐α plays a pivotal role on inflammation, and several plant extracts have been assayed against this target, both in vitro and in vivo. The effect of 11 Brazilian medicinal plants on TNF‐α release by LPS‐activated THP‐1 cells was evaluated. The plant materials were percolated with different solvents to afford 15 crude extracts, whose effect on TNF‐α release was determined by ELISA. Among the evaluated extracts, only Jacaranda caroba (Bignoniaceae) presented strong toxicity to THP‐1 cells. Considering the 14 non‐toxic extracts, TNF‐α release was significantly reduced by seven of them (inhibition > 80%), originating from six plants, namely Cuphea carthagenensis (Lythraceae), Echinodorus grandiflorus (Alismataceae), Mansoa hirsuta (Bignoniaceae), Ouratea semiserrata (Ochnaceae), Ouratea spectabilis and Remijia ferruginea (Rubiaceae). The ethanol extract from O. semiserrata leaves was fractionated over Sephadex LH‐20 and RP‐HPLC to give three compounds previously reported for the species, along with agathisflavone and epicatechin, here described for the first time in the plant. Epicatechin and lanceoloside A elicited significant inhibition of TNF‐α release, indicating that they may account for the effect produced by O. semiserrata crude extract. Copyright © 2015 John Wiley & Sons, Ltd.     

Inhibitory effects of Zataria multiflora essential oil and its main components on nitric oxide and hydrogen peroxide production in glucose-stimulated human monocyte

The inhibitory effects of Zataria multiflora essential oil on nitric oxide (NO) and hydrogen peroxide (H₂O₂) production were examined in human monocytes cultured in the presence of 20 mM glucose. Z. multiflora essential oil was extracted by water-distillation and then analyzed by GC–MS. Carvacrol (29.2%), thymol (25.4%), p-cymene (11.2%), linalool (9.6%) and γ-terpinene (8%) were the main components detected in the essential oil. Cells cultured in the presence of 20 mM glucose showed an increase in NO and H₂O₂ production as well as NO synthase (NOS) and NADH oxidase (NOX) activities compared to cells cultured in the presence of 5 mM glucose. Pretreatment with Z. multiflora essential oil, carvacrol and thymol reduced NO and H₂O₂ production as well as NOS and NOX activities in those cells cultured in the presence of 20 mM glucose. However, p-cymene, linalool and γ-terpinene did not show any such activities. Accordingly, it was concluded that Z. multiflora can reduce oxidative stress and can be used in the therapy of oxidative damage accompanying hyperglycemia and some inflammatory conditions.     

Potential therapeutic effect of Allium cepa L. and quercetin in a murine model of Blomia tropicalis induced asthma

Background

Asthma is an inflammatory condition characterized by airway hyperresponsiveness and chronic inflammation. The resolution of inflammation is an essential process to treat this condition. In this study we investigated the effect of Allium cepa L. extract (AcE) and quercetin (Qt) on cytokine and on smooth muscle contraction in vitro and its therapeutic potential in a murine model of asthma.

Methods

AcE was obtained by maceration of Allium cepa L. and it was standardized in terms of quercetin concentration using high performance liquid chromatography (HPLC). In vitro, using AcE 10, 100 or 1000 μg/ml or Qt 3.5, 7.5, 15 μg/ml, we measured the concentration of cytokines in spleen cell culture supernatants, and the ability to relax tracheal smooth muscle from A/J mice. In vivo, Blomia tropicalis (BT)-sensitized A/J mice were treated with AcE 100, 1000 mg/kg or 30 mg/kg Qt. We measured cell influx in bronchoalveolar lavage (BAL), eosinophil peroxidase (EPO) in lungs, serum levels of Bt-specific IgE, cytokines levels in BAL, and lung histology.

Results

We observed a reduction in the production of inflammatory cytokines, a relaxation of tracheal rings, and a reduction in total number of cells in BAL and EPO in lungs by treatment with AcE or Qt.

Conclusion

AcE and Qt have potential as antiasthmatic drugs, as they possess both immunomodulatory and bronchodilatory properties.