Targeting chalcone binding sites in living Leishmania using a reversible fluorogenic benzochalcone probe.

Chalcones (1,3-diphenyl-2-propen-1-ones) either natural or synthetic have a plethora of biological properties including antileishmanial activities, but their development as drugs is hampered by their largely unknown mechanisms of action. We demonstrate herein that our previously described benzochalcone fluorogenic probe (HAB) could be imaged by fluorescence microscopy in live Leishmania amazonensis promastigotes where it targeted the parasite acidocalcisomes, lysosomes and the mitochondrion. As in the live zebrafish model, HAB formed yellow-emitting fluorescent complexes when associated with biological targets in Leishmania. Further, we used HAB as a reversible probe to study the binding of a portfolio of diverse chalcones and analogues in live promastigotes, using a combination of competitive flow cytometry analysis and cell microscopy. This pharmacological evaluation suggested that the binding of HAB in promastigotes was representative of chalcone pharmacology in Leishmania, with certain exogenous chalcones exhibiting competitive inhibition (ca. 20-30%) towards HAB whereas non-chalconic inhibitors showed weak capacity (ca. 3-5%) to block the probe intracellular binding. However, this methodology was restricted by the strong toxicity of several competing chalcones at high concentration, in conjunction with the limited sensitivity of the HAB fluorophore. This advocates for further optimization of this undirect target detection strategy using pharmacophore-derived reversible fluorescent probes.

Neutralization of Apis mellifera bee venom activities by suramin.

In this work we evaluated the ability of suramin, a polysulfonated naphthylurea derivative, to antagonize the cytotoxic and enzymatic effects of the crude venom of Apis mellifera. Suramin was efficient to decrease the lethality in a dose-dependent way. The hemoconcentration caused by lethal dose injection of bee venom was abolished by suramin (30 µg/g). The edematogenic activity of the venom (0.3 µg/g) was antagonized by suramin (10 µg/g) in all treatment protocols. The changes in the vascular permeability caused by A. mellifera (1 µg/g) venom were inhibited by suramin (30 µg/g) in the pre- and posttreatment as well as when the venom was preincubated with suramin. In addition, suramin also inhibited cultured endothelial cell lesion, as well as in vitro myotoxicity, evaluated in mouse extensor digitorum longus muscle, which was inhibited by suramin (10 and 25 µM), decreasing the rate of CK release, showing that suramin protected the sarcolemma against damage induced by components of bee venom (2.5 µg/mL). Moreover, suramin inhibited the in vivo myotoxicity induced by i.m. injection of A. mellifera venom in mice (0.5 µg/g). The analysis of the area under the plasma CK vs. time curve showed that preincubation, pre- and posttreatment with suramin (30 µg/g) inhibited bee venom myotoxic activity in mice by about 89%, 45% and 40%, respectively. Suramin markedly inhibited the PLA2 activity in a concentration-dependent way (1-30 µM). Being suramin a polyanion molecule, the effects observed may be due to the interaction of its charges with the polycation components present in A. mellifera bee venom.

Endothelial P2X7 receptors' expression is reduced by schistosomiasis.

Endothelial cells control vascular tone, permeability and leukocyte transmigration and are modulated by pro-inflammatory mediators. Schistosomiasis is an intravascular disease associated with inflammation, therefore altering endothelial cells' phenotype. Purinergic P2X7 receptors (P2X7R) play an important role in inflammation; however, the impact of the disease upon endothelial P2X7R function or expression has not been explored. Using ethidium bromide uptake to investigate P2X7R function, we observed that the effects of ATP (3 mM) and the P2X7R agonist 3'-O-(4-benzoyl)-ATP (BzATP) were smaller in mesenteric endothelial cells from the Schistosoma mansoni-infected group than in the control group. In the control group, BzATP induced endothelial nitric oxide production, which was blocked by the P2X7R antagonists KN-62 and A740003. However, in the infected group, we observed a reduced effect of BzATP and no effect of both P2X7R antagonists, suggesting a downregulation of endothelial P2X7R in schistosomiasis. We observed similar results in both infected and P2X7R(-/-) groups, which were also comparable to data obtained with KN-62- or A740004-treated control cells. Data from Western blot and immunocytochemistry assays confirmed the reduced expression of P2X7R in the infected group. In conclusion, our data show a downregulation of P2X7R in schistosomiasis infection, which likely limits the infection-related endothelial damage.

Increased endothelial cell-leukocyte interaction in murine schistosomiasis: possible priming of endothelial cells by the disease.

BACKGROUND AND AIMS: Schistosomiasis is an intravascular parasitic disease associated with inflammation. Endothelial cells control leukocyte transmigration and vascular permeability being modulated by pro-inflammatory mediators. Recent data have shown that endothelial cells primed in vivo in the course of a disease keep the information in culture. Herein, we evaluated the impact of schistosomiasis on endothelial cell-regulated events in vivo and in vitro. METHODOLOGY AND PRINCIPAL FINDINGS: The experimental groups consisted of Schistosoma mansoni-infected and age-matched control mice. In vivo infection caused a marked influx of leukocytes and an increased protein leakage in the peritoneal cavity, characterizing an inflamed vascular and cellular profile. In vitro leukocyte-mesenteric endothelial cell adhesion was higher in cultured cells from infected mice as compared to controls, either in the basal condition or after treatment with the pro-inflammatory cytokine tumor necrosis factor (TNF). Nitric oxide (NO) donation reduced leukocyte adhesion to endothelial cells from control and infected groups; however, in the later group the effect was more pronounced, probably due to a reduced NO production. Inhibition of control endothelial NO synthase (eNOS) increased leukocyte adhesion to a level similar to the one observed in the infected group. Besides, the adhesion of control leukocytes to endothelial cells from infected animals is similar to the result of infected animals, confirming that schistosomiasis alters endothelial cells function. Furthermore, NO production as well as the expression of eNOS were reduced in cultured endothelial cells from infected animals. On the other hand, the expression of its repressor protein, namely caveolin-1, was similar in both control and infected groups. CONCLUSION/SIGNIFICANCE: Schistosomiasis increases vascular permeability and endothelial cell-leukocyte interaction in vivo and in vitro. These effects are partially explained by a reduced eNOS expression. In addition, our data show that the disease primes endothelial cells in vivo, which keep the acquired phenotype in culture.

Schistosomiasis differentially affects vasoconstrictor responses: up-regulation of 5-HT receptor-mediated aorta contraction.

Schistosomiasis, classified by the World Health Organization as a neglected tropical disease, is an intravascular parasitic disease associated to a chronic inflammatory state. Evidence implicating inflammation in vascular dysfunction continues to mount, which, broadly defined, reflects a failure in the control of intracellular Ca2+ and consequently, vascular contraction. Therefore, we measured aorta contraction induced by 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1), two important regulators of vascular contraction. Isometric aortic contractions were determined in control and Schistosoma mansoni-infected mice. In the infected animals, 5-HT induced a 50% higher contraction in relation to controls and we also observed an increased contraction in response to Ca2+ mobilisation from sarcoplasmic reticulum. Nevertheless, Rho kinase inhibition reduced the contraction in response to 5-HT equally in both groups, discarding an increase of the contractile machinery sensitivity to Ca2+. Furthermore, no alteration was observed for contractions induced by ET-1 in both groups. Our data suggest that an immune-vascular interaction occurs in schistosomiasis, altering vascular contraction outside the mesenteric portal system. More importantly, it affects distinct intracellular signalling involved in aorta contraction, in this case increasing 5-HT receptor signalling.

Schistosomiasis differentially affects vasoconstrictor responses: up-regulation of 5-HT receptor-mediated aorta contraction

Schistosomiasis, classified by the World Health Organization as a neglected tropical disease, is an intravascular parasitic disease associated to a chronic inflammatory state. Evidence implicating inflammation in vascular dysfunction continues to mount, which, broadly defined, reflects a failure in the control of intracellular Ca2+ and consequently, vascular contraction. Therefore, we measured aorta contraction induced by 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1), two important regulators of vascular contraction. Isometric aortic contractions were determined in control and Schistosoma mansoni-infected mice. In the infected animals, 5-HT induced a 50 percent higher contraction in relation to controls and we also observed an increased contraction in response to Ca2+ mobilisation from sarcoplasmic reticulum. Nevertheless, Rho kinase inhibition reduced the contraction in response to 5-HT equally in both groups, discarding an increase of the contractile machinery sensitivity to Ca2+. Furthermore, no alteration was observed for contractions induced by ET-1 in both groups. Our data suggest that an immune-vascular interaction occurs in schistosomiasis, altering vascular contraction outside the mesenteric portal system. More importantly, it affects distinct intracellular signalling involved in aorta contraction, in this case increasing 5-HT receptor signalling.