Lufaxin, a Novel Factor Xa Inhibitor From the Salivary Gland of the Sand Fly Lutzomyia longipalpis Blocks Protease-Activated Receptor 2 Activation and Inhibits Inflammation and Thrombosis In Vivo

Objective-Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. Methods and Results-Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant approximate to 3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl3-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme. Conclusion-Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events. (Arterioscler Thromb Vasc Biol. 2012;32:2185-2196.)

Dual effect of Lutzomyia longipalpis saliva on Leishmania braziliensis infection is mediated by distinct saliva-induced cellular recruitment into BALB/c mice ear

Abstract Background Leishmania parasites are transmitted to their vertebrate hosts by infected Phlebotomine sand flies during the blood meal of the flies. Sand fly saliva is known to enhance Leishmania spp. infection, while pre-exposure to saliva protects mice against parasitic infections. In this study, we investigated the initial inflammatory leucocyte composition induced by one or three inocula of salivary gland extract (SGE) from Lutzomyia longipalpis in the presence or absence of Leishmania braziliensis. Results We demonstrated that inoculating SGE once (SGE-1X) or three times (SGE-3X), which represented a co-inoculation or a pre-exposure to saliva, respectively, resulted in different cellular infiltrate profiles. Whereas SGE-1X led to the recruitment of all leucocytes subtypes including CD4+ T cells, CD4+CD25+ T cells, dendritic cells, macrophages and neutrophils, the immune cell profile in the SGE-3X group differed dramatically, as CD4+ T cells, CD4+CD25+ T cells, dendritic cells, macrophages and neutrophils were decreased and CD8+ T cells were increased. The SGE-1X group did not show differences in the ear lesion size; however, the SGE-1X group harbored a higher number of parasites. On the other hand, the SGE-3X group demonstrated a protective effect against parasitic disease, as the parasite burden was lower even in the earlier stages of the infection, a period in which the SGE-1X group presented with larger and more severe lesions. These effects were also reflected in the cytokine profiles of both groups. Whereas the SGE-1X group presented with a substantial increase in IL-10 production, the SGE-3X group showed an increase in IFN-γ production in the draining lymph nodes. Analysis of the inflammatory cell populations present within the ear lesions, the SGE-1X group showed an increase in CD4+FOXP3+ cells, whereas the CD4+FOXP3+ population was reduced in the SGE-3X group. Moreover, CD4+ T cells and CD8+ T cells producing IFN-γ were highly detected in the ears of the SGE-3X mice prior to infection. In addition, upon treatment of SGE-3X mice with anti-IFN-γ monoclonal antibody, we observed a decrease in the protective effect of SGE-3X against L. braziliensis infection. Conclusions These results indicate that different inocula of Lutzomyia longipalpis salivary gland extract can markedly modify the cellular immune response, which is reflected in the pattern of susceptibility or resistance to Leishmania braziliensis infection.

Toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly Lutzomyia longipalpis

The saliva of blood-sucking arthropods contains powerful pharmacologically active substances and may be a vaccine target against some vector-borne diseases. Subtractive cloning combined with biochemical approaches was used to discover activities in the salivary glands of the hematophagous fly Lutzomyia longipalpis. Sequences of nine full-length cDNA clones were obtained, five of which are possibly associated with blood-meal acquisition, each having cDNA similarity to: (i) the bed bug Cimex lectularius apyrase, (ii) a 5′-nucleotidase/phosphodiesterase, (iii) a hyaluronidase, (iv) a protein containing a carbohydrate-recognition domain (CRD), and (v) a RGD-containing peptide with no significant matches to known proteins in the blast databases. Following these findings, we observed that the salivary apyrase activity of L. longipalpis is indeed similar to that of Cimex apyrase in its metal requirements. The predicted isoelectric point of the putative apyrase matches the value found for Lutzomyia salivary apyrase. A 5′-nucleotidase, as well as hyaluronidase activity, was found in the salivary glands, and the CRD-containing cDNA matches the N-terminal sequence of the HPLC-purified salivary anticlotting protein. A cDNA similar to α-amylase was discovered and salivary enzymatic activity demonstrated for the first time in a blood-sucking arthropod. Full-length clones were also found coding for three proteins of unknown function matching, respectively, the N-terminal sequence of an abundant salivary protein, having similarity to the CAP superfamily of proteins and the Drosophila yellow protein. Finally, two partial sequences are reported that match possible housekeeping genes. Subtractive cloning will considerably enhance efforts to unravel the salivary pharmacopeia of blood-sucking arthropods.

Leishmaniose visceral americana: avaliação dos parâmetros da capacidade vetorial de Lutzomyia longipalpis em área urbana do muncípio de Panorama, São Paulo, Brasil

Introdução: A leishmaniose visceral (LV) é um importante problema de saúde pública no Brasil, com cerca 3000 mil casos notificados anualmente. Nos últimos anos, a LV tem ampliado sua distribuição em vários estados do país, associada principalmente aos processos socioambientais, antrópicos e migratórios. A LV é causada pela infecção com Leishmania infantum chagasi, transmitida, principalmente, por Lutzomyia longipalpis (Diptera: Psychodidae). Este flebotomíneo apresenta ampla distribuição nas Américas, todavia, evidências sugerem que se constitui em um complexo de espécies crípticas. A dinâmica de transmissão da LV é modulada por fatores ecológicos locais que influenciam a interação entre populações do patógeno, do vetor e dos hospedeiros vertebrados. Portanto, o estudo das variáveis associadas a esta interação pode contribuir para elucidar aspectos dos elos epidemiológicos e contribuir para a tomada de decisões em saúde pública. Objetivo: Avaliar parâmetros relacionados à capacidade vetorial da população de Lu. longipalpis presente em área urbana do município de Panorama, estado de São Paulo. Métodos: Foram realizadas capturas mensais durante 48 meses para avaliar a distribuição espaço-temporal de Lu. longipalpis e investigar a circulação de Le. i. chagasi. Também foram realizados os seguintes experimentos com o vetor: captura-marcação-soltura-recaptura para estimar a sobrevida da população e a duração do seu ciclo gonotrófico, a atratividade dos hospedeiros mais frequentes em áreas urbanas, a proporção de repasto em cão, infecção experimental e competência vetorial. Resultados: Observou-se que no município de Panorama, Lu. longipalpis apresentou as frequências mais elevadas na estação chuvosa (entre outubro e março), maior densidade em áreas com presença de vegetação e criação de animais domésticos, locais aonde também foi demonstrada a circulação natural de espécimes de Lu. longipalpis infectados com Le. i. chagasi. Além disto, foi corroborado que a população de Lu. longipalpis apresentou hábito hematofágico eclético, altas taxas de sobrevivência e que foi competente para transmitir o agente da LV. Nos experimentos de laboratório foi evidenciada a heterogeneidade na infecção de fêmeas de Lu. longipalpis desafiadas a se alimentarem em cães comprovadamente infectados por L. i. chagasi e o rápido desenvolvimento do parasita neste vetor natural. Conclusões. As observações do presente estudo corroboram a capacidade vetora de Lu. longipalpis para transmitir a Le. i. chagasi e ressaltam a importância da espécie na transmissão do agente etiológico da LV. Ações de manejo ambiental, educação e promoção à saúde são recomendadas às autoridades municipais para diminuir o risco potencial de infecção na população humana e canina, considerando-se o elevado potencial vetor de Lu. longipalpis e a presença de condições que favorecem a interação dos componentes da tríade epidemiológica da LV.

Abundance of Lutzomyia longipalpis in urban households as risk factor of transmission of visceral leishmaniasis

Urban occurrence of human and canine visceral leishmaniasis (VL) is linked to households with characteristics conducive to the presence of sand flies. This study proposes an ad hoc classification of households according to the environmental characteristics of receptivity to phlebotominae and an entomological study to validate the proposal. Here we describe the phlebotominae population found in intra- and peridomiciliary environments and analyse the spatiotemporal distribution of the VL vector Lutzomyia longipalpis of households receptive to VL. In the region, 153 households were classified into levels of receptivity to VL followed by entomological surveys in 40 of those properties. Kruskal-Wallis verified the relationship between the households’ classification and sand fly abundance and Kernel analysis evaluated L. longipalpis spatial distribution: of the 740 sand flies were captured, 91% were L. longipalpis; 82% were found peridomiciliary whilst the remaining 18% were found intradomiciliary. No statistically significant association was found between sandflies and households levels. L. longipalpis counts were concentrated in areas of high vulnerability and some specific households were responsible for the persistence of the infestation. L. longipalpis prevails over other sand fly species for urban VL transmission. The entomological study may help target the surveillance and vector control strategies to domiciles initiating and/or maintaining VL outbreaks.

Should reproductively isolated populations of Lutzomyia longipalpis sensu lato receive taxonomically valid names?

A group of 18 research workers involved in different aspects of the biology of Lutzomyia longipalpis discussed whether or not it is important to give taxonomically valid names to populations that have been defined by biological, biochemical and molecular methods to be reproductively isolated. The type material of this medically important species has been lost and because of this it was recommended that a colony should be established from insects captured in the region of the type area and that their description should serve as the basis for future descriptions. It was pointed out that there is a lack of uniformity in the naming of closely related American sand flies and that some of the differences between populations of Lu. longipalpis are greater than those between accepted species. The majority of the participants agreed that the populations that have been defined in the literature as sibling species should be named.

Lutzomyia longipalpis (Diptera, Psychodidae) em Cuesta Basáltica, na bacia hidrográfica do Rio Corumbataí, Região Centro-leste do Estado de São Paulo

A incidência das leishmanioses tegumentar e visceral americanas, em especial esta última (LVA), em hospedeiros caninos e humanos, encontra-se em crescente processo de expansão no Estado de São Paulo. Para a vigilância epidemiológica dessas endemias, torna-se fundamental o conhecimento da distribuição e da ecologia das diferentes espécies da fauna flebotomínea vetoras. Assim, a divulgação de novos encontros de seus vetores, sobretudo da Lutzomyia longipalpis, o principal vetor da LVA, é fundamental para apontar novas áreas de risco para a transmissão dessas doenças. Neste estudo, capturas de flebotomíneos foram realizadas em ambiente domiciliar, peridomiciliar e de mata, em diferentes localidades rurais dos municípios de Ipeúna e Itirapina, entre outubro de 2001 e fevereiro de 2004. Foram utilizadas armadilhas luminosas automáticas do tipo CDC, das 18h às 8h, em 14 noites, resultando 420 horas de exposição. Foram capturados 177 flebotomíneos pertencentes a doze espécies. A espécie mais abundante, Nyssomyia neivai, apontada como a principal vetora de LTA no Estado, contribuiu com 85,4% dos espécimes capturados em Ipeúna. O encontro de Lutzomyia longipalpis em uma caverna em Itirapina, aponta para o risco de estabelecimento da LVA na área e a necessidade de mais estudos locais sobre sua ecologia, sobretudo em relação à ocupação de ambientes antrópicos.

Sôbre o encontro de Lutzomyia longipalpis (Lutz & Neiva, 1912) no Estado de São Paulo, Brasil

Relata-se o encontro de Lutzomyia longipalpis na localidade denominada Bairro do Arado, município de Salto de Pirapora, Estado de São Paulo, Brasil. Trata-se do primeiro registro concreto da presença dessa espécie em território paulista. Sugere-se a necessidade de investigar o significado epidemiológico dêsse achado.

Novos encontros de flebotomíneos no Estado de São Paulo, Brasil, com especial referência à Lutzomyia longipalpis

Relata-se o encontro de várias espécies de Phleboteminae, no Estado de São Paulo, Brasil, até agora raramente ou mesmo não assinaladas nessa região. São registrados novos achados de Lutzomyia longipalpis e fornecidos alguns dados sobre as circunstâncias desses encontros.

Primeiro encontro de Lutzomyia longipalpis (Lutz & Neiva, 1912) na área urbana de Campo Grande, MS, Brasil

Relata-se a ocorrência, pela primeira vez, do vetor da leishmaniose visceral, Lutzomyia longipalpis, na área urbana de Campo Grande, Mato Grosso do Sul. Discute-se a importância deste encontro na transmissão da doença nessa área.

Lutzomyia longipalpis behavior and control at an urban visceral leishmaniasis focus in Argentina

During the earlier stages of visceral leishmaniasis transmission in Posadas City, Misiones, both the night activity and attraction to humans of Lutzomyia longipalpis were assessed, in order to provide preliminary recommendations. The impact of peridomestic deltamethrin spraying performed by local officials was also evaluated. Although Lu. longipalpis were found in traps located over a dog the entire night, 90% of the females were captured from 20.30h to 1.30h, and only landed on a human when he was at a distance of 1.5 m from the dog. Peridomestic spraying of deltamethrin (25 mg/m²) reduced the sand fly capture up to seven days post-intervention without dispersion in the border of the sprayed areas. These results support the recommendations about time-space focus of the protection measures: first half of the night, in the backyard, with pets and domestic animals kept at least 5 m from humans. The deltamethrin as it was used did not seem very effective in this scenario; neither did the eventual use of bed nets, at least in adults, due to the place/hour of sand fly higher activity. This study strengthens the need for a multidisciplinary approach to develop prevention strategies based both on biological and anthropological studies.

Tentativa de transmissão da Leishmania donovani pela picada do Lutzomyia longipalpis entre cães

Os Autores apresentam dados sôbre tentativas de transmissão experimental da Leishmania donovani pela picada de Lutzomyia longipalpis entre cães. Dois cães jovens sadios foram picados respectivamente por dois e sete flebótomos ricamente infectados e não adquiriram leishmaniose.

Sobre a ocorrência da Lutzomyia longipalpis (Lutz & Neiva, 1912) na Ilha Grande, Estado do Rio de Janeiro, Brasil

Os autores descrevem peta primeira vez a presença de Lutzomyia longipalpis na Praia Vermelha, praia localizada na Ilha Grande, litoral do Estado do Rio de Janeiro e acentuam a importância epidemiológica do achado.

Nota sobre a presença da Lutzomyia longipalpis (Lutz & Neiva, 1912) na Ilha Grande, Estado do Rio de Janeiro, Brasil

Foi relatado o encontro pela primeira vez, de Lutzomyia longipalpis na Praia Vermelha, município de Angra dos Reis, Rio de Janeiro, e chamada atenção do fato em relação à possível conotação em Leishmania Visceral no futuro, tal qual, ocorreu em Bangu e Jacarepaguá no Rio de Janeiro.