ABSTRACT Background Triatoma infestans (Hemiptera: Reduviidae) is a hematophagous insect and the main vector of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae). In the present study, the authors investigated whether a serine protease activity from the saliva of T. infestans has a role in vasomotor modulation, and in the insect-blood feeding by cleaving and activating protease-activated receptors (PARs). Methods T. infestans saliva was chromatographed as previously reported for purification of triapsin, a serine protease. The cleavage activity of triapsin on PAR peptides was investigated based on FRET technology. Mass spectrometry was used to analyze the sites of PAR-2 peptide cleaved by triapsin. NO measurements were performed using the DAN assay (2,3-diaminonapthalene). The vasorelaxant activity of triapsin was measured in vessels with or without functional endothelium pre-contracted with phenylephrine (3 µM). Intravital microscopy was used to assess the effect of triapsin on mouse skin microcirculation. Results Triapsin was able to induce hydrolysis of PAR peptides and showed a higher preference for cleavage of the PAR-2 peptide. Analysis by mass spectrometry confirmed a single cleavage site, which corresponds to the activation site of the PAR-2 receptor. Triapsin induced dose-dependent NO release in cultured human umbilical vein endothelial cells (HUVECs), reaching a maximum effect at 17.58 nM. Triapsin purified by gel-filtration chromatography (10-16 to 10-9 M) was applied cumulatively to mouse mesenteric artery rings and showed a potent endothelium-dependent vasodilator effect (EC30 = 10-12 M). Nitric oxide seems to be partially responsible for this vasodilator effect because L-NAME (L-NG-nitroarginine methyl ester 300 µM), a nitric oxide synthetase inhibitor, did not abrogate the vasodilation activated by triapsin. Anti-PAR-2 antibody completely inhibited vasodilation observed in the presence of triapsin activity. Triapsin activity also induced an increase in the mouse ear venular diameter. Conclusion Data from this study suggest a plausible association between triapsin activity mediated PAR-2 activation and vasodilation caused by T. infestans saliva.

In the present paper, we developed a primary culture of Rhodnius prolixus salivary gland and main salivary canal cells. Cells remained viable in culture for 30 days. Three types of cells were indentified in the salivary gland cultures, with binuclear cells being the most abundant. The supernatants of salivary cultures contained mainly 16-24 kDa proteins and presented anticoagulant and apyrase activities. Secretion vesicles were observed budding from the cellular monolayer of the main salivary canal cells. These results indicate that R. prolixus salivary proteins may be produced in vitro and suggest that the main salivary canal may have a possible secretory role.

A presença de algumas macromoléculas no sangue esta associada a fenômenos de hiperviscosidade sangüínea e hiperagregação eritrocitária, quadro freqüente em algumas patologias (Diabetes, Hipertensão Arterial,etc). Este estudo investiga a agregação eritrocitária e sedimentação em amostras de sangue suíno, humano e bovino utilizando duas técnicas experimentais. O sangue humano e o de suínos agregam naturalmente formando roleaux enquanto o sangue bovino não agrega. A microestrutura foi investigada por microscopia óptica em amostras de sangue bovino (hematócrito 40%), suíno (hematócrito 1%, 5%, 10%, 30% e 40%) e humano (hematócrito 40%) e amostras de sangue bovino (hematócrito 40%) com adição de fibrinogênio nas concentrações de 5g/l, 10g/l, 20g/l, 25g/l e 30g/l. Os ensaios no Leitor de Microplacas foram realizados para se determinar o comportamento da agregação eritrocitária e sedimentação do sangue em função do hematócrito com e sem adição de fibrinogênio (para o sangue bovino). A microscopia óptica de luz transmitida permite a caracterização das microestruturas do sangue sob diferentes condições experimentais. Com o Leitor de Microplacas é possível distinguir-se três regiões no processo: a- Região I onde ocorre a agregação eritrocitária; b-Região II onde ocorre à predominância da etapa de sedimentação dos eritrócitos; e c- Região III onde ocorre um aumento lento da absorbância ocasionado também pela sedimentação dos eritrócitos. Associando a microscopia óptica com o Leitor de Microplacas é possível criar parâmetros para quantificar o processo de agregação e sedimentação do sangue, ferramentas úteis no diagnóstico e evolução de patologias que afligem tanto seres humanos como animais.

The presence of some macromolecules in the blood is associated with blood hiperviscosity and hyperaggregation. We have studied erythrocyte aggregation and blood sedimentation on samples of swine, human and bovine blood. Bovine blood does not present erythrocyte aggregation while human and swine blood naturally aggregates at low shear rates and at stasis. The action of different bovine fibrinogen concentrations on the microstructure of bovine blood was analyzed and compared with swine and human blood. The phenomena were investigated using optical microscopy (to analyze blood microstructures) and Microplate Reader to measure absorbance (at a fixed wavelength, l=655nm) versus time(s). Experiments were performed using samples of bovine blood (hematocrit 40%), swine blood (H= 1%, 5%, 10%, 30% and 40%) and human blood (H= 40%); and samples of bovine blood (H= 40%) with the addition of bovine fibrinogen at different concentrations (5g/l, 10g/l, 20g/l, 25g/l and 30g/l). Using the Microplate Reader it was possible to distinguish three regions in the process: a-The region I where the erythrocyte aggregation occurs; b-region II where the sedimentation process is predominant and c-Region III where a very slow increase indicates that the persistence of sedimentation process. The association of the optical microscopy with the Microplate Reader technique makes possible the determination of quantitative parameters for the process of aggregation and sedimentation. These parameters will facilitate the diagnosis and a more accurate analysis of the evolution of pathologies that afflict both humans as animals.

The aim of this study was to obtain experimental evidence that phlebotomine saliva is actually ingested during the carbohydrate ingestion phase (before and after blood digestion). The ingestion of carbohydrate was simulated as it occurs in the field by offering the insects balls of cotton soaked in sucrose, sucrose crystals or orange juice cells. The results obtained here showed that ingestion occurred under each condition investigated, as indicated by the presence of apyrase, an enzyme used as a marker to detect saliva in the insect gut and/or carbohydrate sources. Saliva ingestion by phlebotomine during the carbohydrate ingestion phase is important to explain how it could promote starch digestion and to trigger Leishmania promastigotes to follow a differentiation pathway as proposed previously by some authors.