A Lutzomyia longipalpis Salivary Protein Induces Cross-Reactive Antibodies to Pemphigus Autoantigen Desmoglein 1.

Fogo selvagem (FS) is a blistering skin disease caused by pathogenic IgG4 autoantibodies to desmoglein 1 (DSG1). Preclinical FS and leishmaniasis are endemic to certain regions of Brazil and exhibit nonpathogenic anti-DSG1 antibodies. Recurring bites from Lutzomyia longipalpis, the sand fly vector of leishmaniasis, immunize individuals with L. longipalpis salivary antigens LJM17 and LJM11. We measured the antibody responses to LJM17, LJM11, and DSG1 in normal settlers and patients with FS from an endemic focus of FS and nonendemic control populations. We also immunized mice with these antigens and assessed the IgG response. Healthy individuals and patients with FS from endemic areas had significantly higher values of IgG4 anti-LJM17 antibodies than nonendemic controls (P < 0.001 for both). The levels of IgG anti-DSG1 and IgG4 anti-LJM17 and anti-LJM11 antibodies correlated positively in normal settlers and patients with FS. Mice immunized with recombinant LJM17 produced IgG1 antibodies (human IgG4 homolog) that strongly cross-reacted with recombinant DSG1; these IgG1 antibodies were inhibited by LJM17, LJM11, and DSG1 in a dose-dependent manner. However, they did not bind human or mouse epidermis by indirect immunofluorescence. Lastly, we identified short-sequence homologies of surface-exposed residues within the human DSG1 ectodomain and LJM17. Inoculation by LJM17 from L. longipalpis-elicited DSG1-cross-reactive IgG4 antibodies may lead to FS in genetically predisposed individuals.

Chicken eggs as a surveillance tool for malaria and leishmaniasis vector presence.

INTRODUCTION: Malaria and leishmaniases are transmitted by vectors during blood-feeding. Vector-infected animals develop antibodies against the vector's saliva. This study evaluated IgY antibody detection in the chicken eggs exposed to bites from Migonemyia migonei, Lutzomyia longipalpis and Anopheles aquasalis. METHODS: We used ELISA to quantify the antibody levels in the sera and exposed chicken eggs. RESULTS: High IgY levels were observed following immunization; furthermore, higher reactivity was observed in the eggs and species-specific immune response was observed post final immunization. CONCLUSIONS: Chicken eggs can be used as sentinels to surveil vector saliva antibodies.

Lutzomyia longipalpis TGF-ß Has a Role in Leishmania infantum chagasi Survival in the Vector.

Despite the increasing number of studies concerning insect immunity, Lutzomyia longipalpis immune responses in the presence of Leishmania infantum chagasi infection has not been widely investigated. The few available studies analyzed the role of the Toll and IMD pathways involved in response against Leishmania and microbial infections. Nevertheless, effector molecules responsible for controlling sand fly infections have not been identified. In the present study we investigated the role a signal transduction pathway, the Transforming Growth Factor-beta (TGF-ß) pathway, on the interrelation between L. longipalpis and L. i. chagasi. We identified an L. longipalpis homolog belonging to the multifunctional cytokine TGF-ß gene family (LlTGF-ß), which is closely related to the activin/inhibin subfamily and potentially involved in responses to infections. We investigated this gene expression through the insect development and in adult flies infected with L. i. chagasi. Our results showed that LlTGF-ß was expressed in all L. longipalpis developmental stages and was upregulated at the third day post L. i. chagasi infection, when protein levels were also higher as compared to uninfected insects. At this point blood digestion is finished and parasites are in close contact with the insect gut. In addition, we investigated the role of LlTGF-ß on L. longipalpis infection by L. i. chagasi using either gene silencing by RNAi or pathway inactivation by addition of the TGF-ß receptor inhibitor SB431542. The blockage of the LlTGF-ß pathway increased significantly antimicrobial peptides expression and nitric oxide levels in the insect gut, as expected. Both methods led to a decreased L. i. chagasi infection. Our results show that inactivation of the L. longipalpis TGF-ß signal transduction pathway reduce L. i. chagasi survival, therefore suggesting that under natural conditions the parasite benefits from the insect LlTGF-ß pathway, as already seen in Plamodium infection of mosquitoes.

Wolbachia introduction into Lutzomyia longipalpis (Diptera: Psychodidae) cell lines and its effects on immune-related gene expression and interaction with Leishmania infantum.

BACKGROUND: The leishmaniases are important neglected diseases caused by Leishmania spp. which are transmitted by sand flies, Lutzomyia longipalpis being the main vector of visceral leishmaniasis in the Americas. The methodologies for leishmaniasis control are not efficient, causing 1.5 million reported cases annually worldwide, therefore showing the need for development of novel strategies and interventions to control transmission of the disease. The bacterium Wolbachia pipientis is being used to control viruses transmitted by mosquitoes, such as dengue and Zika, and its introduction in disease vectors has been effective against parasites such as Plasmodium. Here we show the first successful establishment of Wolbachia into two different embryonic cell lines from L. longipalpis, LL-5 and Lulo, and analysed its effects on the sand fly innate immune system, followed by in vitro Leishmania infantum interaction. RESULTS: Our results show that LL-5 cells respond to wMel and wMelPop-CLA strains within the first 72 h post-infection, through the expression of antimicrobial peptides and inducible nitric oxide synthase resulting in a decrease of Wolbachia detection in the early stages of infection. In subsequent passages, the wMel strain was not able to infect any of the sand fly cell lines while the wMelPop-CLA strain was able to stably infect Lulo cells and LL-5 at lower levels. In Wolbachia stably infected cells, the expression of immune-related genes involved with downregulation of the IMD, Toll and Jak-Stat innate immune pathways was significantly decreased, in comparison with the uninfected control, suggesting immune activation upon Wolbachia transinfection. Furthermore, Wolbachia transinfection did not promote a negative effect on parasite load in those cells. CONCLUSIONS: Initial strong immune responses of LL5 cells might explain the inefficiency of stable infections in these cells while we found that Lulo cells are more permissive to infection with Wolbachia causing an effect on the cell immune system, but not against in vitro L. infantum interaction. This establishes Lulo cells as a good system for the adaptation of Wolbachia in L. longipalpis.

Chicken eggs as a surveillance tool for malaria and leishmaniasis vector presence

Abstract INTRODUCTION: Malaria and leishmaniases are transmitted by vectors during blood-feeding. Vector-infected animals develop antibodies against the vector's saliva. This study evaluated IgY antibody detection in the chicken eggs exposed to bites from Migonemyia migonei, Lutzomyia longipalpis and Anopheles aquasalis. METHODS: We used ELISA to quantify the antibody levels in the sera and exposed chicken eggs. RESULTS: High IgY levels were observed following immunization; furthermore, higher reactivity was observed in the eggs and species-specific immune response was observed post final immunization. CONCLUSIONS: Chicken eggs can be used as sentinels to surveil vector saliva antibodies.

Immunization of Experimental Dogs With Salivary Proteins From Lutzomyia longipalpis, Using DNA and Recombinant Canarypox Virus Induces Immune Responses Consistent With Protection Against Leishmania infantum.

Metacyclic Leishmania promastigotes are transmitted by sand flies that inject parasites and saliva into the host's skin. Previous studies have demonstrated that DNA plasmids encoding Lutzomyia longipalpis salivary proteins LJM17 and LJL143, when used to immunize dogs, resulted in a systemic and local Th1 cell-mediated immunity that interfered in parasite survival in vitro. Here we evaluated the ability of these same salivary antigens to induce anti-Leishmania immunity and to confer protection by immunizing dogs using a novel vaccination strategy more suitable for use in the field. The strategy consisted of a single dose of plasmid followed by two doses of recombinant Canarypoxvirus (rCanarypoxvirus) expressing L. longipalpis salivary proteins (LJM17 or LJL143). Thirty days after the final immunization, dogs were intradermally challenged with 107Leishmania infantum promastigotes in the presence of L. longipalpis saliva. We followed the experimentally infected dogs for 10 months to characterize clinical, parasitological, and immunological parameters. Upon vaccination, all immunized dogs presented strong and specific humoral responses with increased serum concentrations of IFN-γ, TNF, IL-7, and IL-15. The serum of dogs immunized with LJM17 also exhibited high levels of IL-2, IL-6, and IL-18. L. infantum infection was established in all experimental groups as evidenced by the presence of anti-Leishmania IgG, and by parasite detection in the spleen and skin. Dogs immunized with LJM17-based vaccines presented higher circulating levels of IFN-γ, IL-2, IL-6, IL-7, IL-15, IL-18, TNF, CXCL10, and GM-CSF post-infection when compared with controls. Results demonstrated that relevant Leishmania-specific immune responses were induced following vaccination of dogs with L. longipalpis salivary antigen LJM17 administered in a single priming dose of plasmid DNA, followed by two booster doses of recombinant Canarypox vector. Importantly, a significant increase in pro-inflammatory cytokines and chemokines known to be relevant for protection against leishmaniasis was evidenced after challenging LJM17-vaccinated dogs as compared to controls. Although similar results were observed following immunization with LJL143, the pro-inflammatory response observed after immunization was attenuated following infection. Collectively, these data suggest that the LJM17-based vaccine induced an immune profile consistent with the expected protective immunity against canine leishmaniosis. These results clearly support the need for further evaluation of the LJM17 antigen, using a heterologous prime-boost vaccination strategy against canine visceral leishmaniosis (CVL).

Leishmania, microbiota and sand fly immunity.

In this review, we explore the state-of-the-art of sand fly relationships with microbiota, viruses and Leishmania, with particular emphasis on the vector immune responses. Insect-borne diseases are a major public health problem in the world. Phlebotomine sand flies are proven vectors of several aetiological agents including viruses, bacteria and the trypanosomatid Leishmania, which are responsible for diseases such as viral encephalitis, bartonellosis and leishmaniasis, respectively. All metazoans in nature coexist intimately with a community of commensal microorganisms known as microbiota. The microbiota has a fundamental role in the induction, maturation and function of the host immune system, which can modulate host protection from pathogens and infectious diseases. We briefly review viruses of public health importance present in sand flies and revisit studies done on bacterial and fungal gut contents of these vectors. We bring this information into the context of sand fly development and immune responses. We highlight the immunity mechanisms that the insect utilizes to survive the potential threats involved in these interactions and discuss the recently discovered complex interactions among microbiota, sand fly, Leishmania and virus. Additionally, some of the alternative control strategies that could benefit from the current knowledge are considered.

The small non-coding RNA response to virus infection in the Leishmania vector Lutzomyia longipalpis.

Sandflies are well known vectors for Leishmania but also transmit a number of arthropod-borne viruses (arboviruses). Few studies have addressed the interaction between sandflies and arboviruses. RNA interference (RNAi) mechanisms utilize small non-coding RNAs to regulate different aspects of host-pathogen interactions. The small interfering RNA (siRNA) pathway is a broad antiviral mechanism in insects. In addition, at least in mosquitoes, another RNAi mechanism mediated by PIWI interacting RNAs (piRNAs) is activated by viral infection. Finally, endogenous microRNAs (miRNA) may also regulate host immune responses. Here, we analyzed the small non-coding RNA response to Vesicular stomatitis virus (VSV) infection in the sandfly Lutzoymia longipalpis. We detected abundant production of virus-derived siRNAs after VSV infection in adult sandflies. However, there was no production of virus-derived piRNAs and only mild changes in the expression of vector miRNAs in response to infection. We also observed abundant production of virus-derived siRNAs against two other viruses in Lutzomyia Lulo cells. Together, our results suggest that the siRNA but not the piRNA pathway mediates an antiviral response in sandflies. In agreement with this hypothesis, pre-treatment of cells with dsRNA against VSV was able to inhibit viral replication while knock-down of the central siRNA component, Argonaute-2, led to increased virus levels. Our work begins to elucidate the role of RNAi mechanisms in the interaction between L. longipalpis and viruses and should also open the way for studies with other sandfly-borne pathogens.

Canine leishmaniasis: Genome-wide analysis and antibody response to Lutzomyia longipalpis saliva.

The anti-inflammatory properties of sand fly saliva favor the establishment of the Leishmania infantum infection. In contrast, an antibody response against Lutzomyia longipalpis saliva is often associated with a protective cell-mediated response against canine visceral leishmaniasis. Genetic studies may demonstrate to what extent the ability to secrete anti-saliva antibodies depends on genetic or environmental factors. However, the genetic basis of canine antibody response against sand fly saliva has not been assessed. The aim of this study was to identify chromosomal regions associated with the anti-Lu. longipalpis salivary IgG response in 189 dogs resident in endemic areas in order to provide information for prophylactic strategies. Dogs were classified into five groups based on serological and parasitological diagnosis and clinical evaluation. Anti-salivary gland homogenate (SGH) IgG levels were assessed by Enzyme-Linked Immunosorbent Assay (ELISA). Genomic DNA was isolated from blood samples and genotyped using a SNP chip with 173,662 single nucleotide polymorphism (SNP) markers. The following linear regression model was fitted: IgG level = mean + origin + sex + age + use of a repellent collar, and the residuals were assumed as pseudo-phenotypes for the association test between phenotypes and genotypes (GWA). A component of variance model that takes into account polygenic and sample structure effects (EMMAX) was employed for GWA. Phenotypic findings indicated that anti-SGH IgG levels remained higher in exposed and subclinically infected dogs than in severely diseased dogs even in regression model residuals. Five associated markers were identified on chromosomes 2, 20 and 31. The mapped genes included CD180 (RP105) and MITF related to the rapid activation of B lymphocytes and differentiation into antibody-secreting plasma cells. The findings pointed to chromosomal segments useful for functional confirmation studies and a search for adjuvant molecules of the anti-saliva response.

Antibody response to sand fly saliva is a marker of transmission intensity but not disease progression in dogs naturally infected with Leishmania infantum.

BACKGROUND: Antibody responses to sand fly saliva have been suggested to be a useful marker of exposure to sand fly bites and Leishmania infection and a potential tool to monitor the effectiveness of entomological interventions. Exposure to sand fly bites before infection has also been suggested to modulate the severity of the infection. Here, we test these hypotheses by quantifying the anti-saliva IgG response in a cohort study of dogs exposed to natural infection with Leishmania infantum in Brazil. METHODS: IgG responses to crude salivary antigens of the sand fly Lutzomyia longipalpis were measured by ELISA in longitudinal serum samples from 47 previously unexposed sentinel dogs and 11 initially uninfected resident dogs for up to 2 years. Antibody responses were compared to the intensity of transmission, assessed by variation in the incidence of infection between seasons and between dogs. Antibody responses before patent infection were then compared with the severity of infection, assessed using tissue parasite loads and clinical symptoms. RESULTS: Previously unexposed dogs acquired anti-saliva antibody responses within 2 months, and the rate of acquisition increased with the intensity of seasonal transmission. Over the following 2 years, antibody responses varied with seasonal transmission and sand fly numbers, declining rapidly in periods of low transmission. Antibody responses varied greatly between dogs and correlated with the intensity of transmission experienced by individual dogs, measured by the number of days in the field before patent infection. After infection, anti-saliva antibody responses were positively correlated with anti-parasite antibody responses. However, there was no evidence that the degree of exposure to sand fly bites before infection affected the severity of the infection. CONCLUSIONS: Anti-saliva antibody responses are a marker of current transmission intensity in dogs exposed to natural infection with Leishmania infantum, but are not associated with the outcome of infection.

Insights into the epidemiological link between biting flies and pemphigus foliaceus in southeastern Brazil.

BACKGROUND: Black fly and sandfly bites are related to the endemicity of pemphigus foliaceus (PF); however, an immune reaction against the salivary proteins from these flies still requires confirmation in the case of PF patients living in southeastern Brazil. PURPOSE: To georeference the distribution of Simuliidae (Diptera: Simuliidae) and Phlebotominae (Diptera: Psychodidae) and of PF cases in the northeastern region of São Paulo State, and to assess the humoral immune response against salivary gland extracts (SGEs) from biting flies in PF patients, relatives, and neighbours. METHODS: PF patients' medical information recorded between 1965 and 2014 were obtained from the database of the University Hospital. Data on the distribution of fly species were collected from scientific reports and epidemiological databases. Spatial maps relating the distribution of biting flies with PF cases were plotted. Serum IgG antibodies against the SGEs from Simulium nigrimanum, Nyssomyia neivai, and Aedes aegypti (as control) were determined by ELISA. RESULTS: Two hundred and eighty-five PF cases were distributed in 60 municipalities with a prevalence of 57.5 per million inhabitants, revealing well-defined geographical clusters. S. nigrimanum and N. neivai specimens were registered in eight (13.3%) and 26 (43.3%) of these municipalities, respectively. PF patients, and their relatives presented higher levels of IgG against the SGEs of S. nigrimanum and N. neivai (P<0.001 for both), but not against the SGE from A. aegypti (P=0.115 and P=0.552, respectively), as compared to controls. IgG against the SGEs from S. nigrimanum and N. neivai but not against the SGE from A. aegypti correlated with levels of anti-Desmoglein 1 in PF patients (r=0.3848, P=0.039; and r=0.416, P=0.022, respectively). CONCLUSION: An epidemiological link between biting flies and PF in southeastern Brazil is proposed, implying a possible role of the salivary proteins from these flies in PF etiopathogenesis.

Seroconversion to Lutzomyia intermedia LinB-13 as a biomarker for developing cutaneous leishmaniasis.

Sand flies inject saliva while feeding in the vertebrate host and anti-saliva antibodies can be used as biomarkers of exposure to Leishmania vectors. We expressed recombinant salivary proteins from Lutzomyia intermedia, a vector of Leishmania braziliensis, and evaluated the seroreactivity in exposed individuals in search for exposure markers. We found a strong correlation among positive serology to recombinant proteins LinB-13, 26, 15, 21 and to salivary proteins: rLinB-13 was the top performing molecule; IgG4 was the most predominant antibody subclass and antibodies to rLinB-13 did not cross react with Lu. longipalpis salivary proteins. By evaluating a cohort of contacts of CL patients, we confirmed that rLinB-13, an antigen 5-related protein, is a marker of exposure to Lu. intermedia with high degree of accuracy. In a 5-year follow up, we determined that individuals who developed CL presented higher anti-rLinB13 IgG responses, before the appearance of clinical symptoms. They also presented a lower frequency of cellular responses to the parasite (DTH). Our results show that seroconversion to a salivary molecule, rLinB-13, is a marker of risk for CL development caused by Leishmania braziliensis. This highlight the possibility of developing tools based on vector molecules to manage the disease in endemic areas.

Immunity to Lutzomyia whitmani Saliva Protects against Experimental Leishmania braziliensis Infection.

BACKGROUND: Previous works showed that immunization with saliva from Lutzomyia intermedia, a vector of Leishmania braziliensis, does not protect against experimental infection. However, L. braziliensis is also transmitted by Lutzomyia whitmani, a sand fly species closely related to Lu. intermedia. Herein we describe the immune response following immunization with Lu. whitmani saliva and the outcome of this response after L. braziliensis infection. METHODS AND FINDINGS: BALB/c mice immunized with Lu. whitmani saliva developed robust humoral and cellular immune responses, the latter characterized by an intense cellular infiltrate and production of IFN-γ and IL-10, by both CD4+ and CD8+ cells. Mice immunized as above and challenged with L. braziliensis plus Lu. whitmani saliva displayed significantly smaller lesions and parasite load at the challenge site. This protection was associated with a higher (p<0.05) IFN-γ production in response to SLA stimulation. Long-term persisting immunity was also detected in mice immunized with Lu. whitmani saliva. Furthermore, individuals residing in an endemic area for cutaneous leishmaniasis (CL) presented antibody responses to Lu. whitmani saliva. However CL patients, with active lesions, displayed a lower humoral response to Lu. whitmani saliva compared to individuals with subclinical Leishmania infection. CONCLUSION: Pre-exposure to Lu. whitmani saliva induces protection against L. braziliensis in a murine model. We also show that Lu. whitmani salivary proteins are immunogenic in naturally exposed individuals. Our results reinforce the importance of investigating the immunomodulatory effect of saliva from different species of closely related sand flies.

Antissaliva Antibodies of Lutzomyia Longipalpis in area of Visceral Leishmaniasis.

The aim of the present study was to assess the presence of antissaliva antibodies of Lutzomyia longipalpis in human hosts living in area of visceral leishmaniasis, located in the Center-West region of Brazil. The presence of antissaliva antibodies of L. longipalpis exhibited a strong correlation with the protection and development of antibodies against Leishmania sp. Of the 492 children studied, elevated antissaliva antibodies of L. longipalpis were detected in 38.4% of the participants. There was a higher percentage of positivity (64.7%) among children who exhibited anti-Leishmania sp. antibodies and among those who were positive in the delayed hypersensitivity test (34.8%).

The sandfly Lutzomyia longipalpis LL5 embryonic cell line has active Toll and Imd pathways and shows immune responses to bacteria, yeast and Leishmania.

BACKGROUND: Lutzomyia longipalpis is the main vector of visceral leishmaniasis in Latin America. Sandfly immune responses are poorly understood. In previous work we showed that these vector insects respond to bacterial infections by modulating a defensin gene expression and activate the Imd pathway in response to Leishmania infection. Aspects of innate immune pathways in insects (including mosquito vectors of human diseases) have been revealed by studying insect cell lines, and we have previously demonstrated antiviral responses in the L. longipalpis embryonic cell line LL5. METHODS: The expression patterns of antimicrobial peptides (AMPs) and transcription factors were evaluated after silencing the repressors of the Toll pathway (cactus) and Imd pathway (caspar). AMPs and transcription factor expression patterns were also evaluated after challenge with heat-killed bacteria, heat-killed yeast, or live Leishmania. RESULTS: These studies showed that LL5 cells have active Toll and Imd pathways, since they displayed an increased expression of AMP genes following silencing of the repressors cactus and caspar, respectively. These pathways were also activated by challenges with bacteria, yeast and Leishmania infantum chagasi. CONCLUSIONS: We demonstrated that L. longipalpis LL5 embryonic cells respond to immune stimuli and are therefore a good model to study the immunological pathways of this important vector of leishmaniasis.

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.

Atração de vetores de leishmaniose tegumentar americana por compostos orgânicos voláteis presentes na pele humana / Attraction of American tegumentary leishmaniasis vectors by volatile organic compounds present on human skin

INTRODUÇÃO. A Leishmaniose Tegumentar Americana (LTA) é a forma de leishmaniose com maior incidência em humanos, e no Brasil a maioria dos casos está concentrada nas regiões Norte e Nordeste. O protozoário Leishmania braziliensis é considerado o principal agente etiológico com ocorrência no país e tem como principais vetores os flebotomíneos Lutzomyia (Nyssomyia) intermedia e Lutzomyia (Nyssomyia) whitmani. Sabe-se que, de modo geral, o olfato é o principal sentido de orientação dos insetos e, assim, foi assumido neste trabalho que esses vetores são atraídos por compostos orgânicos voláteis (COVs) presentes nos odores da pele humana. OBJETIVO. O objetivo geral do projeto foi identificar os COVs presentes na pele humana e avaliar os seus efeitos na atração de vetores de LTA, com a finalidade de desenvolver e aprimorar alternativas para o controle dos mesmos, de modo que estas possam ser baseadas em COVs que mimetizem os odores da pele humana e, portanto, sejam efetivas na captura de flebotomíneos antropofílicos...

INTRODUCTION. American Tegumentary Leishmaniasis (ATL) is the most frequent form of leishmaniasis among humans. In Brazil most of the cases occurs in the North and in the Northeast of the country. Leishmania braziliensis is the main ethilogic agent and its most common vectors are the phlebotomine sand flies Lutzomyia (Nyssomyia) intermedia and Lutzomyia (Nyssomyia) whitmani. It is known that the olfact is the best developed sense of orientation among insects and thus it was assumed that these vectors are attracted by volatile organic compounds (VOCs) from human skin odors. AIM. The aim of this project was to identify the VOCs from human skin and evaluate their effect on the attraction of ATL vectors, aiming to develop and enhance alternatives of its control, in a way that it can be based on VOCs that mimic human skin odors, which can be effective for anthropophilic phlebotomine capture...

Papel da saliva de flebotomos como marcador de exposição e seu efeito na proteção contra Leishmania braziliensis / Role of saliva from sandflies as an exposure marker and its effect on protection against Leishmania braziliensis

A Leishmaniose Tegumentar (LT) é uma importante patologia causada pelo protozoário Leishmania braziliensis, o principal agente etiológico nas Américas. Esse parasita é transmitido aos hospedeiros através durante a alimentação sanguínea do vetor. Comumente encontrados em área endêmica de LT, os flebótomos Lutzomyia intermedia e Lutzomyia whitmani, foram incriminados como vetores da L. braziliensis. Apesar dos amplos estudos acerca da saliva dos vetores e do papel dos flebótomos na transmissão, muito pouco se sabe sobre o envolvimento de L. whitmani na LT. OBJETIVO. Este trabalho visou avaliar o papel de L. whitmani em etapas críticas da transmissão da L. braziliensis, como a avaliação do vetor na área endêmica, a exposição de animais no peridomicílio humano ao flebótomo e o papel da saliva do vetor no estabelecimento da L. braziliensis em infecção experimental murina. MATERIAIS E MÉTODOS. Para a obtenção dos dados, foram realizados testes sorológicos como ELISA e Western blot no monitoramento das galinhas da área endêmica e nos camundongos mantidos em laboratório. Também foram realizadas coletas e identificação dos flebótomos da área endêmica e ensaios de estimulação celular para análise da resposta celular dos camundongos. RESULTADOS. Os resultados mostraram que galinhas no peridomicílio humano produziram anticorpos anti-saliva de L. whitmani, apresentando reconhecimento específico à saliva desta espécie. Também foi observado que a imunização de camundongos com saliva de L. whitmani é capaz de induzir proteção contra a infecção por L. braziliensis em camundongos imunizados. CONCLUSÃO. Estudos envolvendo a participação de importantes vetores em área endêmica são fundamentais para o entendimento da dinâmica da cadeia epidemiológica e futuros estudos envolvendo o desenvolvimento de vacinas eficazes contra a doença.

INTRODUÇÃO. A Leishmaniose Tegumentar (LT) é uma importante patologia causada pelo protozoário Leishmania braziliensis, o principal agente etiológico nas Américas. Esse parasita é transmitido aos hospedeiros através durante a alimentação sanguínea do vetor. Comumente encontrados em área endêmica de LT, os flebótomos Lutzomyia intermedia e Lutzomyia whitmani, foram incriminados como vetores da L. braziliensis. Apesar dos amplos estudos acerca da saliva dos vetores e do papel dos flebótomos na transmissão, muito pouco se sabe sobre o envolvimento de L. whitmani na LT. OBJETIVO. Este trabalho visou avaliar o papel de L. whitmani em etapas críticas da transmissão da L. braziliensis, como a avaliação do vetor na área endêmica, a exposição de animais no peridomicílio humano ao flebótomo e o papel da saliva do vetor no estabelecimento da L. braziliensis em infecção experimental murina. MATERIAIS E MÉTODOS. Para a obtenção dos dados, foram realizados testes sorológicos como ELISA e Western blot no monitoramento das galinhas da área endêmica e nos camundongos mantidos em laboratório. Também foram realizadas coletas e identificação dos flebótomos da área endêmica e ensaios de estimulação celular para análise da resposta celular dos camundongos. RESULTADOS. Os resultados mostraram que galinhas no peridomicílio humano produziram anticorpos anti-saliva de L. whitmani, apresentando reconhecimento específico à saliva desta espécie. Também foi observado que a imunização de camundongos com saliva de L. whitmani é capaz de induzir proteção contra a infecção por L. braziliensis em camundongos imunizados. CONCLUSÃO. Estudos envolvendo a participação de importantes vetores em área endêmica são fundamentais para o entendimento da dinâmica da cadeia epidemiológica e futuros estudos envolvendo o desenvolvimento de vacinas eficazes contra a doença.

ß-Adrenergic blockade protects BALB/c mice against infection with a small inoculum of Leishmania mexicana mexicana (LV4).

In order to test the influence of the sympathetic nervous system on Leishmania mexicana infection, groups of female BALB/c mice were treated (i.p.) with the non-selective ß-adrenergic receptor (ß-AR) antagonist (S)-propranolol (5mg/kg thrice a day), the ß2-AR agonist clenbuterol (1mg/kg once a day) or the α2-AR antagonist yohimbine (2mg/kg twice a day) during 5days. During the second day of treatments, mice were inoculated in the footpad with 1×10(6) or 1×10(3) metacyclic promastigotes of L. mexicana mexicana (LV4). The lesion size was measured weekly, and parasite burden on week 12. In mice treated with (S)-propranolol, the percentage of splenic T lymphocytes producing IFN-γ after antigen challenge was determined by flow cytometry. In mice infected with 1×10(6) parasites, only (S)-propranolol caused a reduction of footpad swelling (p<0.05, weeks 11-12), without effects on parasite burden, or in the percentage of IFN-γ-immunopositive CD4(+) or CD8(+) T lymphocytes. In mice infected with 1×10(3) parasites, the effects of treatments vs. control group were as follows: (a) inhibition of footpad swelling by (S)-propranolol (p<0.01, weeks 3-12), clenbuterol (p<0.05, weeks 7-10), and yohimbine (p<0.01, week 7); (b) a decrease of the parasite burden by (S)-propranolol (p<0.01) and yohimbine (p<0.05); (c) in control mice the percentage of CD4(+) T-cells producing IFN-γ was 6.2±0.5%, while in those treated with (S)-propranolol it increased to 8.7±0.6% (p<0.01); (d) in control mice the percentage of CD8(+) T-cells producing IFN-γ was 3.1±0.4%, while in those treated with (S)-propranolol it increased to 10.4±0.2% (p<0.01). These results indicate that the blockade of ß-ARs during infection of BALB/c mice with an inoculum of L. mexicana mexicana similar to that delivered by the bite of a sand fly produces a Th1 bias in the immune response, favoring an increment of T lymphocytes secreting IFN-γ, which correlated with a reduced parasite burden (p<0.05, Spearman's test). We suggest that ß-AR antagonists could be of therapeutic value, either as treatment or as adjuvant of vaccines for L. mexicana.