Sheltered in Stromal Tissue Cells, Trypanosoma cruzi Orchestrates Inflammatory Neovascularization via Activation of the Mast Cell Chymase Pathway

Microangiopathy may worsen the clinical outcome of Chagas disease. Given the obstacles to investigating the dynamics of inflammation and angiogenesis in heart tissues parasitized by Trypanosoma cruzi, here we used intravital microscopy (IVM) to investigate microcirculatory alterations in the hamster cheek pouch (HCP) infected by green fluorescent protein-expressing T. cruzi (GFP-T. cruzi). IVM performed 3 days post-infection (3 dpi) consistently showed increased baseline levels of plasma extravasation. Illustrating the reciprocal benefits that microvascular leakage brings to the host-parasite relationship, these findings suggest that intracellular amastigotes, acting from inside out, stimulate angiogenesis while enhancing the delivery of plasma-borne nutrients and prosurvival factors to the infection foci. Using a computer-based analysis of images (3 dpi), we found that proangiogenic indexes were positively correlated with transcriptional levels of proinflammatory cytokines (pro-IL1β and IFN-γ). Intracellular GFP-parasites were targeted by delaying for 24 h the oral administration of the trypanocidal drug benznidazole. A classification algorithm showed that benznidazole (>24 h) blunted angiogenesis (7 dpi) in the HCP. Unbiased proteomics (3 dpi) combined to pharmacological targeting of chymase with two inhibitors (chymostatin and TY-51469) linked T. cruzi-induced neovascularization (7 dpi) to the proangiogenic activity of chymase, a serine protease stored in secretory granules from mast cells

Ecotin-Like ISP of L. major Promastigotes Fine-Tunes Macrophage Phagocytosis by Limiting the Pericellular Release of Bradykinin from Surface-Bound Kininogens: A Survival Strategy Based on the Silencing of Proinflammatory G-Protein Coupled Kinin B-2 and B-1 Receptors

Inhibitors of serine peptidases (ISPs) expressed by Leishmania major enhance intracellular parasitism in macrophages by targeting neutrophil elastase (NE), a serine protease that couples phagocytosis to the prooxidative TLR4/PKR pathway. Here we investigated the functional interplay between ISP-expressing L. major and the kallikrein-kinin system (KKS). Enzymatic assays showed that NE inhibitor or recombinant ISP-2 inhibited KKS activation in human plasma activated by dextran sulfate. Intravital microscopy in the hamster cheek pouch showed that topically applied L. major promastigotes (WT and Delta isp2/3 mutants) potently induced plasma leakage through the activation of bradykinin B-2 receptors (B2R). Next, using mAbs against kininogen domains, we showed that these BK-precursor proteins are sequestered by L. major promastigotes, being expressed at higher % in the Delta isp2/3 mutant population. Strikingly, analysis of the role of kinin pathway in the phagocytic uptake of L. major revealed that antagonists of B2R or B1R reversed the upregulated uptake of Delta isp2/3mutants without inhibiting macrophage internalization of WT L. major. Collectively, our results suggest that L. major ISP-2 fine-tunes macrophage phagocytosis by inhibiting the pericellular release of proinflammatory kinins from surface bound kininogens. Ongoing studies should clarify whether L. major ISP-2 subverts TLR4/PKR-dependent prooxidative responses of macrophages by preventing activation of G-protein coupled B2R/B1R

Estimation of genetic parameters for postweaning growth traits in crossbred Angus x Zebu cattle

The objective of this study was to verify the effect of individual heterozygosity on weights at 365 (W365) and 550 (W550) days of age, and to estimate the heritability of these traits and the breeding values of animals of different breed compositions involving Angus and Zebu breeds in order to evaluate the possible use of these traits as selection criteria in crossbreeding and genetic improvement programs. Records from 5,629 crossbred Angus x Zebu animals born between 1995 and 2004 were used. Univariate and bivariate analyses of the traits were performed using an animal model and Bayesian inference. The effects included in the model were contemporary group, individual heterozygosis, age of animal at recording, and random direct genetic effect of the animal and residual. The size of the chain requested, as well as the burn-in period and thinning interval, was sufficient to obtain convergence of the posterior estimates of the genetic parameters. The heritability estimates obtained in univariate and bivariate analysis were 0.38 and 0.30 for W365 and 0.32 and 0.30 for W550, respectively. The genetic correlation between traits was 0.98. Comparison of the rank of the 10 best sires based on the breeding values for W365 and W550 showed a small difference between the traits studied. The effect of heterozygosity has importance for crossings and for obtaining higher postweaning weights, indicating that the use of animals with maximum individual heterozygosity is advantageous. W365 and W550 were found to be appropriate selection criteria and should be included in breeding programs of crossbred Angus x Zebu cattle. </div

Sheltered in Stromal Tissue Cells, <i>Trypanosoma cruzi</i> Orchestrates Inflammatory Neovascularization via Activation of the Mast Cell Chymase Pathway

Microangiopathy may worsen the clinical outcome of Chagas disease. Given the obstacles to investigating the dynamics of inflammation and angiogenesis in heart tissues parasitized by Trypanosoma cruzi, here we used intravital microscopy (IVM) to investigate microcirculatory alterations in the hamster cheek pouch (HCP) infected by green fluorescent protein-expressing T. cruzi (GFP-T. cruzi). IVM performed 3 days post-infection (3 dpi) consistently showed increased baseline levels of plasma extravasation. Illustrating the reciprocal benefits that microvascular leakage brings to the host-parasite relationship, these findings suggest that intracellular amastigotes, acting from inside out, stimulate angiogenesis while enhancing the delivery of plasma-borne nutrients and prosurvival factors to the infection foci. Using a computer-based analysis of images (3 dpi), we found that proangiogenic indexes were positively correlated with transcriptional levels of proinflammatory cytokines (pro-IL1β and IFN-γ). Intracellular GFP-parasites were targeted by delaying for 24 h the oral administration of the trypanocidal drug benznidazole. A classification algorithm showed that benznidazole (>24 h) blunted angiogenesis (7 dpi) in the HCP. Unbiased proteomics (3 dpi) combined to pharmacological targeting of chymase with two inhibitors (chymostatin and TY-51469) linked T. cruzi-induced neovascularization (7 dpi) to the proangiogenic activity of chymase, a serine protease stored in secretory granules from mast cells

Contact System Activation in Plasma from Dengue Patients Might Harness Endothelial Virus Replication through the Signaling of Bradykinin Receptors

Since exacerbated inflammation and microvascular leakage are hallmarks of dengue virus (DENV) infection, here we interrogated whether systemic activation of the contact/kallikrein-kinin system (KKS) might hamper endothelial function. In vitro assays showed that dextran sulfate, a potent contact activator, failed to generate appreciable levels of activated plasma kallikrein (PKa) in the large majority of samples from a dengue cohort (n = 70), irrespective of severity of clinical symptoms. Impaired formation of PKa in dengue-plasmas correlated with the presence of cleaved Factor XII and high molecular weight kininogen (HK), suggesting that the prothrombogenic contact system is frequently triggered during the course of infection. Using two pathogenic arboviruses, DENV or Zika virus (ZIKV), we then asked whether exogenous BK could influence the outcome of infection of human brain microvascular endothelial cells (HBMECs). Unlike the unresponsive phenotype of Zika-infected HBMECs, we found that BK, acting via B2R, vigorously stimulated DENV-2 replication by reverting nitric oxide-driven apoptosis of endothelial cells. Using the mouse model of cerebral dengue infection, we next demonstrated that B2R targeting by icatibant decreased viral load in brain tissues. In summary, our study suggests that contact/KKS activation followed by BK-induced enhancement of DENV replication in the endothelium may underlie microvascular pathology in dengue

Contact System Activation in Plasma from Dengue Patients Might Harness Endothelial Virus Replication through the Signaling of Bradykinin Receptors

Since exacerbated inflammation and microvascular leakage are hallmarks of dengue virus (DENV) infection, here we interrogated whether systemic activation of the contact/kallikrein-kinin system (KKS) might hamper endothelial function. In vitro assays showed that dextran sulfate, a potent contact activator, failed to generate appreciable levels of activated plasma kallikrein (PKa) in the large majority of samples from a dengue cohort (n = 70), irrespective of severity of clinical symptoms. Impaired formation of PKa in dengue-plasmas correlated with the presence of cleaved Factor XII and high molecular weight kininogen (HK), suggesting that the prothrombogenic contact system is frequently triggered during the course of infection. Using two pathogenic arboviruses, DENV or Zika virus (ZIKV), we then asked whether exogenous BK could influence the outcome of infection of human brain microvascular endothelial cells (HBMECs). Unlike the unresponsive phenotype of Zika-infected HBMECs, we found that BK, acting via B2R, vigorously stimulated DENV-2 replication by reverting nitric oxide-driven apoptosis of endothelial cells. Using the mouse model of cerebral dengue infection, we next demonstrated that B2R targeting by icatibant decreased viral load in brain tissues. In summary, our study suggests that contact/KKS activation followed by BK-induced enhancement of DENV replication in the endothelium may underlie microvascular pathology in dengue

Synthesis and Biological Evaluation of Novel 6-Hydroxy-benzo[d][1,3]oxathiol-2-one Schiff Bases as Potential Anticancer Agents

With the aim of discovering new anticancer agents, we have designed and synthesized novel 6-hydroxy-benzo[d][1,3]oxathiol-2-one Schiff bases. The synthesis started with the selective nitration at 5-position of 6-hydroxybenzo[d][1,3]oxathiol-2-one (1) leading to the nitro derivative 2. The nitro group of 2 was reduced to give the amino intermediate 3. Schiff bases 4a–r were obtained from coupling reactions between 3 and various benzaldehydes and heteroaromatic aldehydes. All the new compounds were fully identified and characterized by NMR (1H and 13C) and specifically for 4q by X-ray crystallography. The in vitro cytotoxicity of the compounds was evaluated against cancer cell lines (ACP-03, SKMEL-19 and HCT-116) by using MTT assay. Schiff bases 4b and 4o exhibited promising cytotoxicity against ACP-03 and SKMEL-19, respectively, with IC50 values lower than 5 μM. This class of compounds can be considered as a good starting point for the development of new lead molecules in the fight against cancer

Mast Cell Coupling to the Kallikrein–Kinin System Fuels Intracardiac Parasitism and Worsens Heart Pathology in Experimental Chagas Disease

During the course of Chagas disease, infectious forms of Trypanosoma cruzi are occasionally liberated from parasitized heart cells. Studies performed with tissue culture trypomastigotes (TCTs, Dm28c strain) demonstrated that these parasites evoke neutrophil/CXCR2-dependent microvascular leakage by activating innate sentinel cells via toll-like receptor 2 (TLR2). Upon plasma extravasation, proteolytically derived kinins and C5a stimulate immunoprotective Th1 responses via cross-talk between bradykinin B2 receptors (B2Rs) and C5aR. Awareness that TCTs invade cardiovascular cells in vitro via interdependent activation of B2R and endothelin receptors [endothelin A receptor (ETAR)/endothelin B receptor (ETBR)] led us to hypothesize that T. cruzi might reciprocally benefit from the formation of infection-associated edema via activation of kallikrein–kinin system (KKS). Using intravital microscopy, here we first examined the functional interplay between mast cells (MCs) and the KKS by topically exposing the hamster cheek pouch (HCP) tissues to dextran sulfate (DXS), a potent “contact” activator of the KKS. Surprisingly, although DXS was inert for at least 30 min, a subtle MC-driven leakage resulted in factor XII (FXII)-dependent activation of the KKS, which then amplified inflammation via generation of bradykinin (BK). Guided by this mechanistic insight, we next exposed TCTs to “leaky” HCP—forged by low dose histamine application—and found that the proinflammatory phenotype of TCTs was boosted by BK generated via the MC/KKS pathway. Measurements of footpad edema in MC-deficient mice linked TCT-evoked inflammation to MC degranulation (upstream) and FXII-mediated generation of BK (downstream). We then inoculated TCTs intracardiacally in mice and found a striking decrease of parasite DNA (quantitative polymerase chain reaction; 3 d.p.i.) in the heart of MC-deficient mutant mice. Moreover, the intracardiac parasite load was significantly reduced in WT mice pretreated with (i) cromoglycate (MC stabilizer) (ii) infestin-4, a specific inhibitor of FXIIa (iii) HOE-140 (specific antagonist of B2R), and (iv) bosentan, a non-selective antagonist of ETAR/ETBR. Notably, histopathology of heart tissues from mice pretreated with these G protein-coupled receptors blockers revealed that myocarditis and heart fibrosis (30 d.p.i.) was markedly and redundantly attenuated. Collectively, our study suggests that inflammatory edema propagated via activation of the MC/KKS pathway fuels intracardiac parasitism by generating infection-stimulatory peptides (BK and endothelins) in the edematous heart tissues

A neuro-fuzzy system to support in the diagnostic of epileptic events and non-epileptic events using different fuzzy arithmetical operations Um sistema neuro-difuso para auxiliar no diagnóstico de eventos epilépticos e eventos não epilépticos utilizando diferentes operações aritméticas difusas

OBJECTIVE: To investigate different fuzzy arithmetical operations to support in the diagnostic of epileptic events and non epileptic events. METHOD: A neuro-fuzzy system was developed using the NEFCLASS (NEuro Fuzzy CLASSIfication) architecture and an artificial neural network with backpropagation learning algorithm (ANNB). RESULTS: The study was composed by 244 patients with a bigger frequency of the feminine sex. The number of right decisions at the test phase, obtained by the NEFCLASS and ANNB was 83.60% and 90.16%, respectively. The best sensibility result was attained by NEFCLASS (84.90%); the best specificity result were attained by ANNB with 95.65%. CONCLUSION: The proposed neuro-fuzzy system combined the artificial neural network capabilities in the pattern classifications together with the fuzzy logic qualitative approach, leading to a bigger rate of system success.<br>OBJETIVO: Investigar diferentes operações aritméticas difusas para auxíliar no diagnóstico de eventos epilépticos e eventos não-epilépticos. MÉTODO: Um sistema neuro-difuso foi desenvolvido utilizando a arquitetura NEFCLASS (NEuro Fuzzy CLASSIfication) e uma rede neural artificial com o algoritmo de aprendizagem backpropagation (RNAB). RESULTADOS: A amostra estudada foi de 244 pacientes com maior freqüência no sexo feminino. O número de decisões corretas na fase de teste, obtidas através do NEFCLASS e RNAB foi de 83,60% e 90,16%, respectivamente. O melhor resultado de sensibilidade foi obtido com o NEFCLASS (84,90%); o melhor resultado de especificidade foi obtido com a RNAB (95,65%). CONCLUSÃO: O sistema neuro-difuso proposto combinou a capacidade das redes neurais artificiais na classificação de padrões juntamente com a abordagem qualitativa da logica difusa, levando a maior taxa de acertos do sistema