LeishIF4E-5 Is a Promastigote-Specific Cap-Binding Protein in Leishmania.

Leishmania parasites cycle between sand fly vectors and mammalian hosts, transforming from extracellular promastigotes that reside in the vectors' alimentary canal to obligatory intracellular non-motile amastigotes that are harbored by macrophages of the mammalian hosts. The transition between vector and host exposes them to a broad range of environmental conditions that induces a developmental program of gene expression, with translation regulation playing a key role. The Leishmania genome encodes six paralogs of the cap-binding protein eIF4E. All six isoforms show a relatively low degree of conservation with eIF4Es of other eukaryotes, as well as among themselves. This variability could suggest that they have been assigned discrete roles that could contribute to their survival under the changing environmental conditions. Here, we describe LeishIF4E-5, a LeishIF4E paralog. Despite the low sequence conservation observed between LeishIF4E-5 and other LeishIF4Es, the three aromatic residues in its cap-binding pocket are conserved, in accordance with its cap-binding activity. However, the cap-binding activity of LeishIF4E-5 is restricted to the promastigote life form and not observed in amastigotes. The overexpression of LeishIF4E-5 shows a decline in cell proliferation and an overall reduction in global translation. Immuno-cytochemical analysis shows that LeishIF4E-5 is localized in the cytoplasm, with a non-uniform distribution. Mass spectrometry analysis of proteins that co-purify with LeishIF4E-5 highlighted proteins involved in RNA metabolism, along with two LeishIF4G paralogs, LeishIF4G-1 and LeishIF4G-2. These vary in their conserved eIF4E binding motif, possibly suggesting that they can form different complexes.

Evaluation of Cytopathological Techniques for the Diagnosis of Canine Visceral Leishmaniosis with Lymph Node Samples.

The identification of the parasite in cytological smears of lymph node aspirates is a widely applied technique for the direct diagnosis of Leishmania spp. infection, especially in endemic areas. Although very specific, this method has limited sensitivity, and improving the technique would be highly desirable. This study aimed to evaluate the efficacy of conventional smear cytology (SC), liquid-based cytology (LBC), cell block (CB) stained with haematoxylin and eosin (HE) and immunocytochemistry (ICC), and formalin-fixed paraffin wax-embedded tissue immunohistochemistry (FFPE-IHC) compared with serology and polymerase chain reaction for the diagnosis of canine visceral leishmaniosis (CVL) in lymphoid tissue. The use of a preservative medium and centrifugation for cytological samples reduced the number of unsatisfactory artefacts/background. Moreover, LBC allowed excellent cellular preservation and the application of ancillary techniques, such as CB and ICC. SC was the most accurate morphological diagnostic method (45.0%). CB-ICC alone or associated with SC demonstrated significantly higher sensitivity (70.0% and 72.0%, respectively) when compared with SC alone (34.00%). CB-ICC was found to be more effective in the detection of infected animals with mild clinical signs, similar to FFPE-IHC. The specificity and positive predictive value were similar between all methods. Finally, the detection limit for CB-ICC and SC + CB-ICC was identical (18.46 amastigotes/mm2). Our study suggests that CB-ICC is a promising tool for improvement of the cytopathological diagnosis of CVL and may be applied in routine epidemiological screening.

Site specific microbiome of Leishmania parasite and its cross-talk with immune milieu.

Microbiota consists of commensal, symbiotic and pathogenic microorganisms found in all multicellular organisms. These micro-organisms are found in or on many parts of the body, including the intestinal tract, skin, mouth, and the reproductive tract. This review focuses on interplay of site specific microbiota, vector microbiota along with immune response and severity of Leishmaniasis. Herein, we have reviewed and summarized the counter effect of microbiome post infection with the Leishmania parasite. We have studied skin microbiome along with the gut microbiome of sand-fly which is the vector for transmission of this disease. Our major focus was to understand the skin and gut microbiome during Leishmania infection,their interaction and effect on immunological responses generated during the infection.Moreover, systems biology approach is envisioned to enumerate bacterial species in skin microbiota and Phlebotmus gut microbiota during Leishmania infection.

Chemogenomic Profiling of Antileishmanial Efficacy and Resistance in the Related Kinetoplastid Parasite Trypanosoma brucei.

The arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited and beset by toxicity and emergent resistance. Furthermore, our understanding of drug mode of action and potential routes to resistance is limited. Forward genetic approaches have revolutionized our understanding of drug mode of action in the related kinetoplastid parasite Trypanosoma brucei Therefore, we screened our genome-scale T. brucei RNA interference (RNAi) library against the current antileishmanial drugs sodium stibogluconate (antimonial), paromomycin, miltefosine, and amphotericin B. Identification of T. brucei orthologues of the known Leishmania antimonial and miltefosine plasma membrane transporters effectively validated our approach, while a cohort of 42 novel drug efficacy determinants provides new insights and serves as a resource. Follow-up analyses revealed the antimonial selectivity of the aquaglyceroporin TbAQP3. A lysosomal major facilitator superfamily transporter contributes to paromomycin-aminoglycoside efficacy. The vesicle-associated membrane protein TbVAMP7B and a flippase contribute to amphotericin B and miltefosine action and are potential cross-resistance determinants. Finally, multiple phospholipid-transporting flippases, including the T. brucei orthologue of the Leishmania miltefosine transporter, a putative ß-subunit/CDC50 cofactor, and additional membrane-associated hits, affect amphotericin B efficacy, providing new insights into mechanisms of drug uptake and action. The findings from this orthology-based chemogenomic profiling approach substantially advance our understanding of antileishmanial drug action and potential resistance mechanisms and should facilitate the development of improved therapies as well as surveillance for drug-resistant parasites.

Leishmania amazonensis isolated from human visceral leishmaniasis: histopathological analysis and parasitological burden in different inbred mice.

Leishmania amazonensis is a major etiological agent of human cutaneous leishmaniasis in the Americas; nevertheless there are some reports of this species causing visceral disease in dogs and men. In the present work we have studied a Leishmania strain isolated from a human case of visceral leishmaniasis. We have infected different mouse strains and analyzed the development of the disease, studying the parasite's ability to visceralize and whether this ability is influenced by host genetics. Female BALB/c, C57BL/6, C57BL/10, CBA, DBA/2, and C3H/He mice were subcutaneously infected with 104 L. amazonensis amastigotes. BALB/c, C57BL/6 and C57BL/10 mice were found to be very susceptible to infection, showing lesions that developed to necrosis and ulceration. CBA mice developed a late but severe lesion. DBA/2 mice developed only discrete lesions, while C3H/He mice did not develop any lesions. All mouse strains except C3H/He showed some degree of visceralization, presenting parasites in the spleen, while BALB/c, C57BL/6 and CBA presented parasites also in the liver. Moreover, most of the strains presented high parasite load at the infection site, whereas DBA and C3H/He mice showed low or no parasite load 90 days after infection, respectively. Histopathology corroborates the results, showing that susceptible mice presented an inflammatory reaction with parasites in the skin, lymph nodes and spleen, while strains that are more resistant presented low parasitism and discrete inflammatory reaction. Results indicate that this isolate is extremely virulent, can easily visceralize and that the pathogenesis of leishmaniasis is, at least in part, related to the genetic background of the host.

Estudo da contribuição da autofagia para a susceptibilidade por L. Amazonensis ou resistência por L. Major na infecção de macrófagos murinos da linhagem CBA / Study of the contribution of autophagy to susceptibility by L. Amazonensis or resistance by L. Major in the infection of murine macrophages of the CBA

INTRODUÇÃO: Macrófagos de camundongos CBA controlam a infecção por Leishmania major, no entanto, são permissivos à infecção por Leishmania amazonensis. Os estudos conduzidos, até o momento, sobre o papel desempenhado pela autofagia na infecção por Leishmania levaram a dados controversos. OBJETIVO: No presente trabalho, avaliamos se a resposta autofágica de macrófagos infectados pode ser responsável pela diferença no curso da infecção por essas duas espécies de Leishmania. MATERIAL e MÉTODOS e RESULTADOS: Inicialmente, demonstramos por qPCR e por análise de dados de microarranjos públicos que um número maior de genes relacionados à autofagia é modulado positivamente em células infectadas por L. amazonensis em comparação às infectadas L. major. Ingenuity Pathway Analysis (IPA) demonstrou modulação oposta dos genes relacionados à autofagia entre os macrófagos infectados com L. amazonensis daqueles infectados com L. major. Após 24 h de infecção, a relação LC3-II/Act é aumentada tanto em macrófagos infectados por L. amazonensis quanto nos infectados por L. major em comparação com controles não infectados, mas menos do que em células tratadas com cloroquina. Embora, os vacúolos parasitóforos induzidos por L. major tenham apresentado maior positividade para o marcador degradativo, DQBSA, o recrutamento de LC3 foi maior nos vacúolos parasitóforos induzidos por L. amazonensis. Interessantemente, tanto a indução farmacológica quanto a fisiológica da autofagia aumentaram a viabilidade intracelular de L. amazonensis e L. major, enquanto a inibição da autofagia não teve efeito sobre a viabilidade intracelular desses parasitas. Também demonstramos que a indução da autofagia reduziu a produção de NO por macrófagos infectados por L. amazonensis ou L. major, mas não alterou a atividade da arginase, A análise de componentes principais e agrupamento hierárquico de clusters discriminaram completamente os macrófagos infectados por L. major de células infectadas por L. amazonensis de acordo com a intensidade da infecção e características autofágicas dos vacúolos induzidos por essas duas cepas. CONCLUSÃO: Em conclusão, a infecção por L. amazonensis ou L. major, apesar de ativar similarmente o fluxo autofágico em macrófagos infectados e os parasitos terem sua viabilidade favorecida pela indução da autofagia, promove expressão diferenciada de genes relacionados à autofagia e interação distinta dos vacúolos parasitóforos com compartimentos autofágicos. Essas diferenças são capazes de separar completamente os macrófagos infectados por L. amazonensis daqueles por L. major

INTRODUCTION: CBA mouse macrophages (MΦ) control Leishmania major infection yet are permissive to Leishmania amazonensis. The role played by autophagy in Leishmania infection needs further investigation. OBJECTIVE: Thus, we assessed whether activation of autophagic pathway may account for differences in the response of infected MΦ to these two parasite strains. MATERIAL and METHODS and RESULTS: First, we demonstrated by qPCR and by analysis of publicly available microarray data that a greater number of autophagy-related genes (Atg) are positively modulated in cells infected by L. amazonensis compared to those infected by L. major. Ingenuity Pathway Analyses (IPA) demonstrated opposite modulation in genes in L. amazonensisand L. major-infected MΦ. After 24 h of infection, the autophagic flux measured by LC3-II/Act ratio was similarly increased in either L. amazonensis- or L. majorinfected MΦ compared to uninfected cells. Although L. major-induced parasitophorous vacuoles exhibited greater positivity for the degradative marker, DQ-BSA, LC3 recruitment was increased in L. amazonensis-induced parasitophorous vacuoles. Interestingly, autophagy induction enhanced intracellular L. amazonensis and L. major viability, although autophagy inhibition caused no effect on infection profile. We also demonstrated that autophagy induction reduced NO production by Leishmania-infected MΦ, yet did not alter arginase activity. Moreover, principal component analysis completely discriminated L. major-infected MΦ from L. amazonensis-infected cells regarding infection intensity and autophagic features of parasite-induced PV. CONCLUSION: In conclusion, infection by L. amazonensis or L. major, although similarly activates the autophagic flux in infected MΦ and the parasites have their viability favored by autophagy induction, these Leishmania species cause differentiated expression of Atg and distinct interaction of their parasitophorous vacuoles with autophagic vacuoles. These differences are capable to discriminate MΦ infected by L. amazonensis from those infected by L. major

Terapias alternativas e combinadas para o tratamento da Leishmaniose Cutânea Americana: uma abordagem experimental in vitro e in vivo

A leishmaniose é uma doença tropical causada por protozoários do gêneroLeishmania que afeta 12 milhões de pessoas em 98 países. Seu tratamentoconta com um restrito arsenal terapêutico e exige a administração defármacos tóxicos por longos períodos. Na busca por novas terapias, oreposicionamento de fármacos e a associação terapêutica têm sidoaplicados com sucesso para doenças negligenciadas. O presente estudoteve como objetivo a avaliação in vitro, ex vivo e in vivo do potencial antiLeishmania(Leishmania) amazonensis dos fármacos amitriptilina, econazol,sertralina e triclosan, bem como o estudo de associações terapêuticas invitro e/ou ex vivo e mecanismo de ação in vitro dos fármacos amitriptilina etriclosan. Os resultados demonstraram que todos os fármacos estudados apresentaram atividade contra formas promastigotas e amastigotasintracelulares de L. (L.) amazonensis, com valores de Concentração Efetiva50% que variam de 1,50 a 51,48 µM. Os resultados obtidos a partir das associações entre os fármacos estudados e fármacos padrões foram classificados como aditivos ou indiferentes. Por meio da investigação domecanismo de ação leishmanicida, foi possível concluir que a mitocôndria é uma organela alvo do fármaco amitriptilina, enquanto que o fármaco triclosaninduz danos à membrana plasmática parasitária. Quando tratados comeconazol por via oral (10 mg/kg/dia por 28 dias consecutivos) ou triclosanpor via tópica (creme 1% por 14 dias consecutivos), houve uma redução de75 a 89% da carga parasitária dos camundongos infectados com L. (L.) amazonensis. Os resultados obtidos contribuem para a investigação de novas alternativas para o tratamento da leishmaniose cutânea e sugerem que novos estudos utilizando associação ou coadministração dessesfármacos com fármacos padrões podem ser promissores em modelosanimais.

Leishmaniasis is a tropical disease caused by protozoa of the genusLeishmania that affects 12 million people in 98 countries. There is a limitedtherapeutic arsenal and the treatment requires the administration of toxicdrugs for long periods. In the search for new therapies, the drug repositioningand therapeutic association have been successfully applied to neglecteddiseases. The aim of the present study was to evaluate in vitro, ex vivo andin vivo anti-Leishmania (Leishmania) amazonensis potential of the drugsamitriptyline, econazole, sertraline and triclosan, as well as the study of invitro and / or ex vivo therapeutic associations and mechanism of action of thedrugs amitriptyline and triclosan. The results showed that all studied drugshave activity against L. (L.) amazonensis promastigotes and intracellularamastigotes, with 50% Effective Concentration values ranging from 1.50 to51.48 μM. The results obtained from the combination between the studieddrugs and standard drugs were classified as additives or indifferent. Throughthe investigation of the leishmanicial mechanism of action, it was possible toconclude that the mitochondria is a target organelle of the drug amitriptyline,whereas the drug triclosan induces damage to the parasitic plasmamembrane. When treated with oral econazole (10 mg/kg/day for 28consecutive days) or triclosan topically (1% cream for 14 consecutive days),there was a 75 - 89% reduction in the parasite load of the mice infected withL. (L.) amazonensis. The results obtained contribute to the investigation ofnew alternatives for the treatment of cutaneous leishmaniasis and suggestthat new studies using association or coadministration of these drugs withstandard drugs may be promising in animal models.

Molecular typing reveals the co-existence of two transmission cycles of American cutaneous leishmaniasis in the Andean Region of Venezuela with Lutzomyia migonei as the vector

BACKGROUND The transmission routes for American cutaneous leishmaniasis (ACL) are in flux, so studies examining its transmission in humans, mammalian hosts, and sand fly vectors are urgently needed. OBJECTIVES The aim of this work was understand the epidemiological cycles of Leishmania spp., which causes ACL in the Andean Region of Venezuela, by identifying the Leishmania and the sand fly species involved in human and dog infections. METHODS Thirty-one biopsies from patients in Mérida and Táchira states with suspected ACL were studied by both parasitological tests (cultures and hamster inoculation) and a molecular test [Internal transcribed spacer 1 (ITS1) nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)]. We also conducted a survey to detect Leishmania infection in dogs (Immunifluorescence antibody test and ITS1 nested PCR-RFLP) and sand flies (ITS1 nested PCR-RFLP) from El Carrizal, a highly endemic focus of ACL in Venezuela. FINDINGS Three different Leishmania species were identified in the clinical samples from humans (Leishmania braziliensis, L. guyanensis, and L. mexicana) and dogs (L. guyanensis and L. mexicana). The predominant sand fly species found were those from the Verrucarum group (infected with L. mexicana) and Lutzomyia migonei (infected with L. guyanensis and L. mexicana). MAIN CONCLUSIONS We show that Lu. migonei may be the putative vector in two ACL epidemiological cycles, involving L. guyanensis and L. mexicana. We also report for the first time the presence of L. guyanensis in domestic animals.

Effect of phospholipase A2 inhibitors during infection caused by Leishmania (Leishmania) amazonensis

Lipid metabolites play an important role in parasite differentiation and virulence. Studies have revealed that Leishmania sp. uses prostaglandins to evade innate barriers, thus enabling the parasites to survive inside immune cells. Despite the role of the enzyme Phospholipase A2 (PLA2) in prostaglandins production, few studies have investigated the role of parasite PLA2 during the interaction between L. (L.) amazonensis and the host (in vitro and in vivo) immune cells. Methods: In the present work, the leishmanicidal effect of PLA2 inhibitors, methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL) and aristolochic acid (AA) were investigated in vitro (promastigote and intracellular amastigote forms of L. (L.) amazonensis) and during in vivo infection using BALB/c mice. Results: The aforementioned inhibitors were deleterious to promastigote and amastigote forms of the L. (L.) amazonensis and were non-toxic to peritoneal macrophages from BALB/c mice. L. (L.) amazonensis-infected BALB/c mice treated with the inhibitor BEL presented decreased lesion size and skin parasitism; however, BEL treatment induced hepatotoxicity in BALB/c mice. Conclusions: Results presented herein suggested that PLA2 inhibitors altered L. (L.) amazonensis viability. In spite of liver toxicity, treatment with BEL was the most selective compound in vitro, as well in vivo, resulting in lower skin parasitism in the infected mice. These findings corroborate the role of PLA2 in parasite virulence and maintenance in vertebrate hosts, and suggest that molecules structurally related to BEL should be considered when planning compounds against Leishmania sp.(AU)

Combining epidemiology with basic biology of sand flies, parasites, and hosts to inform leishmaniasis transmission dynamics and control.

Quantitation of the nonlinear heterogeneities in Leishmania parasites, sand fly vectors, and mammalian host relationships provides insights to better understand leishmanial transmission epidemiology towards improving its control. The parasite manipulates the sand fly via production of promastigote secretory gel (PSG), leading to the "blocked sand fly" phenotype, persistent feeding attempts, and feeding on multiple hosts. PSG is injected into the mammalian host with the parasite and promotes the establishment of infection. Animal models demonstrate that sand flies with the highest parasite loads and percent metacyclic promastigotes transmit more parasites with greater frequency, resulting in higher load infections that are more likely to be both symptomatic and efficient reservoirs. The existence of mammalian and sand fly "super-spreaders" provides a biological basis for the spatial and temporal clustering of clinical leishmanial disease. Sand fly blood-feeding behavior will determine the efficacies of indoor residual spraying, topical insecticides, and bed nets. Interventions need to have sufficient coverage to include transmission hot spots, especially in the absence of field tools to assess infectiousness. Interventions that reduce sand fly densities in the absence of elimination could have negative consequences, for example, by interfering with partial immunity conferred by exposure to sand fly saliva. A deeper understanding of both sand fly and host biology and behavior is essential to ensuring effectiveness of vector interventions.

Interaction of KMP-11 with Phospholipid Membranes and Its Implications in Leishmaniasis: Effects of Single Tryptophan Mutations and Cholesterol.

KMP-11 is a small protein that is believed to control the overall bilayer pressure of the Leishmania parasite. Recent results have suggested that membrane binding and the presence of cholesterol affect the efficacy of Leishmanial infection, in which KMP-11 plays an important role. Nevertheless, there exists no systematic study of membrane interaction with KMP-11 either in the absence or presence of cholesterol. In this article, we investigated the interaction between KMP-11 and phospholipid membranes using an unsaturated (PC 18:1; 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)) and saturated (PC 12:0; 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)) lipid as membrane mimics. Additionally, we studied the effect of cholesterol on the protein-membrane interaction. Steady-state as well as time-resolved fluorescence spectroscopy, isothermal titration calorimetry (ITC), and ζ-potential measurements were used for the determination of the binding constants for the wild-type (WT) and single-site tryptophan mutants. Single-site tryptophan mutants were designed to make sure that the tryptophan residues sample different surface exposures in different mutants. In the absence of cholesterol, the membrane-binding affinities of the partially exposed and buried tryptophan mutants (Y5W and Y48W, respectively) were found to be greater than those of the WT protein. In the presence of cholesterol, the binding constants of the WT and Y48W mutant were found to decrease with an increase in cholesterol concentration. This was in contrast to that in the Y5W and F77W mutants, in which the binding constants increased on adding cholesterol. The present study highlights the interplay among the conformational architecture of a protein, its interaction with the membrane, and membrane composition in modulating the survival of a Leishmania parasite inside host macrophages.

Estudo dos flebotomíneos (Diptera: Psychodidae) na localidade de Casa Branca, município de Brumadinho, área de transmissão para leishmanioses no Estado de Minas Gerais

As leishmanioses são doenças endêmicas em vários países do mundo, incluindo o Brasil e são causadas por parasitas do gênero Leishmania. O conhecimento sobre os vetores dessas doenças, os flebotomíneos, pode auxiliar no delineamento das medidas de controle da doença em determinados locais. O objetivo deste trabalho foi estudar a fauna de flebotomíneos, verificar a presença de DNA de Leishmanianas fêmeas capturadas e verificar a alimentação sanguínea nas fêmeas ingurgitadas coletadas na localidade de Casa Branca, pertencente ao município de Brumadinho, Minas Gerais. Durante o período de maio de 2013 a julho de 2014 foram realizadas coletas bimensais totalizando oito coletas sistematizadas de flebotomíneos utilizando 18 armadilhas luminosas expostas no peridomicílio de nove casas, selecionadas por apresentarem casos de leishmanioses humana ou canina. A fauna de flebotomíneos foi composta por 23 espécies, com um total de 16.771 flebotomíneos capturados, sendo a espécie Nyssomyia whitmani a mais abundante na área de estudo, seguida por Lutzomyialongipalpis e Migonemyiamigonei. As fêmeas não alimentadas pertencentes às coletas dos meses de maio/2013, setembro/2013, janeiro/2014 e maio/2014 foram dissecadas e colocadas em pools de no máximo 20 indivíduos onde foi feita a extração de DNA e posterior pesquisa de DNA de Leishmania por meio da técnica de PCR dirigida ao alvo ITS1.

As espécies de Leishmania e outros tripanossomatídeos detectados nos pools das fêmeas de flebotomíneos neste estudo foram identificados através da técnica de sequenciamento genético do produto amplificado. Foi possível detectar DNA de Leishmania em nove pools: Lu. longipalpis (1), Ny. whitmani (6) e Psychodopygus lloydi (2).Outros tripanossomatídeos foram detectados em 10 pools: Crithidia sp.em 1 pool de Ps. lloydi, Endotrypanum sp. em 6 pools de Ny. whitmani e Herpetomonas sp. nas espécies Ny. whitmani (3 pools) e no complexo cortelezzii (1 pool). As fêmeas alimentadas foram dissecadas e o DNA foi extraído para posterior identificação de fontes alimentares através da PCR dirigida ao gene do citocromo b com a confirmação pelo sequenciamento genético. Foram detectadas as fontes alimentares Canis familiaris, Gallus gallus, Homo sapiens e Rattus rattus, e a espécie Ny. whitmani foi a mais abundante capturada alimentada no peridomicílio. Os resultados da pesquisa de DNA de Leishmania chamam atenção para a presença das espécies de importância médica como Lu. longipalpis e Ny. whitmani presentes na área de estudo detectadas com Leishmania, reforçando seus papéis na epidemiologia das leishmanioses. Os outros tripanossomatídeos detectados nesse estudo mostram que pela PCR-ITS1 foi possível detectar outras espécies da família Trypanosomatidae além de Leishmania, evidenciando que essas espécies estão presentes na área de estudo. Os resultados do estudo das fontes alimentares mostraram que os flebotomíneos estão adaptados ao peridomicílio se alimentando em animais comuns a este ambiente

Effects of specific antisera targeting peritrophic matrix-associated proteins in the sand fly vector Phlebotomus papatasi.

In many hematophagous insects, the peritrophic matrix (PM) is formed soon after a blood meal (PBM) to compartmentalize the food bolus. The PM is an important component of vector competence, functioning as a barrier to the development of many pathogens including parasites of the genus Leishmania transmitted by sand flies. PM morphology and permeability are associated with the proteins that are part of the PM scaffolding, including several peritrophins, and chitin fibers. Here, we assessed the effects of specific antisera targeting proteins thought to be an integral part of the PM scaffolding and its process of maturation and degradation. Phlebotomus papatasi sand flies were fed with red blood cells reconstituted with antisera targeting the chitinase PpChit1, and the peritrophin PpPer2. Sand fly midguts were dissected at different time points and processed for light microscopy (LM), confocal and transmission electron (TEM) microscopies (24, 42-46, 48 and 72h PBM), scanning electron (SEM) (48h PBM) and atomic force (AFM) (30h PBM) microscopies. TEM and WGA-FITC staining indicate PM degradation was significantly delayed following feeding of flies on anti-PpChit1. AFM analysis at 30h PBM point to an increase in roughness' amplitude of the PM of flies that fed on either anti-PpChit1 or anti-PpPer2. Collective, our data suggest that antibodies targeting PM-associated proteins affects the kinetics of PM maturation, delaying its degradation and disruption and are potential targets on transmission-blocking vaccines strategies.

Dendritic Cells and Leishmania Infection: Adding Layers of Complexity to a Complex Disease.

Leishmaniasis is a group of neglected diseases whose clinical manifestations depend on factors from the host and the pathogen. It is an important public health problem worldwide caused by the protozoan parasite from the Leishmania genus. Cutaneous Leishmaniasis (CL) is the most frequent form of this disease transmitted by the bite of an infected sandfly into the host skin. The parasites can be uptook and/or recognized by macrophages, neutrophils, and/or dendritic cells (DCs). Initially, DCs were described to play a protective role in activating the immune response against Leishmania parasites. However, several reports showed a dichotomic role of DCs in modulating the host immune response to susceptibility or resistance in CL. In this review, we discuss (1) the interactions between DCs and parasites from different species of Leishmania and (2) the crosstalk of DCs and other cells during CL infection. The complexity of these interactions profoundly affects the adaptive immune response and, consequently, the disease outcome, especially from Leishmania species of the New World.

Infecção por leishmania modula a ativação de Beta-1 integrinas e altera a cinética do espalhamento celular de monócitos sobre fibronectina/Leishmania infection modulates the activation of Beta-1 integrins and alters the kinetics of monocyte cell spread on fibronectin / Leishmania infection modulates the activation of beta-1 integrins and alters the kinetics of monocyte cell spread on fibronectin / Leishmania infection modulates the activation of beta-1 integrins and alters the kinetics of monocyte cell spread on fibronectin

A leishmaniose é considerada como um grupo de doenças, causadas por parasitos do gênero Leishmania. O gênero inclui mais de 20 espécies de parasitos que são transmitidas ao homem pela picada da fêmea, infectada, dos insetos da família dos flebotomíneos e as diferentes espécies estão associadas a diferentes manifestações clínicas da doença. Uma característica marcante na infecção é a presença de macrófagos infectados no sítio de inoculação do parasito e em órgãos internos do hospedeiro. Estudos prévios, demonstraram que a infecção de fagócitos mononucleares com Leishmania amazonensis, L. infantum ou L. braziliensis promove uma diminuição na adesão celular ao tecido conjuntivo inflamado da pele. Esta diminuição da adesão é decorrente, principalmente, de mecanismos envolvidos na regulação da molécula VLA-4, uma integrina da família beta 1 que se liga à VCAM-1...

Leishmaniasis is considered a group of diseases, caused by parasites of the genus Leishmania. The genus includes more than 20 species of parasites that are transmitted to humans by the bite of vector female insect, of the phlebotomine family, and the different species are associated with different clinical manifestations of the disease. A striking feature of infection is the presence of infected macrophages at the site of inoculation of the parasite and internal organs of the host. Previous studies have demonstrated that infection of mononuclear phagocytes with Leishmania amazonensis, L. infantum or L. braziliensis promotes a decrease in cellular adhesion to the inflamed connective tissue of the skin. This decrease in adhesion results mainly from mechanisms involved in the regulation of the VLA-4 molecule, a beta 1 integrin that binds to VCAM-1...

Alterações histopatológicas em cães com leishmaniose visceral naturalmente infectados do município de Jequié-BA(Brasil) / Histopathological changes in dogs with visceral leishmaniasis naturally infected in the city of Jequié-Ba (Brazil)

INTRODUÇÃO: Leishmaniose visceral é uma zoonose grave que acomete uma gama de animais. O cão é considerado o principal reservatório urbano da Leishmania infantum e pode ser clinicamente assintomático ou sintomático na infecção. Poucos estudos abordam os aspectos clínicos e histopatológicos na leishmaniose visceral, sobretudo estudos sistemáticos, que fazem descrição detalhada dos achados de histopatologia e façam a correlação desses aspectos na doença. A hipótese da pesquisa é que cães naturalmente infectados com L. infantum apresentam alterações histopatológicas distintas que se relacionam com as manifestações clínicas, tendo significados diagnóstico e prognóstico. OBJETIVO: O objetivo do trabalho foi a descrição sistemática das alterações histopatológicas encontradas nos diversos órgãos de cães com leishmaniose visceral naturalmente infectados, correlacionando com diferentes manifestações clínicas. MÉTODOS: Para este estudo foram coletados amostras e dados provenientes de 58 cães...

INTRODUCTION: Visceral leishmaniasis is a serious zoonosis that affects a range of animals. The dog is considered the main urban reservoir of Leishmania infantum and may be clinically asymptomatic or symptomatic in infection. Few studies address the clinical and histopathological aspects in visceral leishmaniasis, especially systematic studies, which are detailed description of histopathology findings and making the correlation of these aspects of the disease. The hypothesis of the research is that dogs naturally infected with L. infantum have distinct histopathologic changes that relate to the clinical manifestations, diagnosis and prognosis. OBJECTIVE: The objective was the systematic description of histopathology found in the various organs of dogs with visceral leishmaniasis naturally infected, correlated with different clinical manifestations. METHODS: For this study were collected samples and data from 58 dogs...

Triagem fitoquímica, atividade antioxidante e leishmanicida do extrato hidroetanólico 70% (v/v) e das frações obtidas de (Annona crassiflora Mart. ) / Phytochemical screening, antioxidant and leishmanicidal activity of the hydroethanolic extract 70% (v / v) obtained from fractions (Annona crassiflora Mart. )

A fitoterapia é uma prática sociocultural presente ao longo dos anos e utilizada para tratar enfermidades que acometem o ser humano. Nesse contexto, a Annona crassiflora Mart. destaca-se por ser uma das espécies utilizadas na fitoterapia pouco estudada química e biologicamente. Conhecida popularmente como marolo denota, segundo a literatura científica, uma constituição química composta por ácidos fenólicos, alcaloides, flavonoides, taninos, terpenoides e acetogeninas. Esses ativos são responsáveis pelo grande potencial farmacológico da espécie onde se destacam as atividades antimicrobianas, antioxidantes, citotóxicas, leishmanicidas e hipoglicêmicas. Considerando o exposto, este estudo propôs investigar do ponto de vista químico e biológico a espécie Annona crassiflora. Para esse fim, foi obtido o extrato hidroetanólico 70% (v/v) e frações hidroetanólicas das folhas de marolo. Com essas amostras realizou-se uma triagem fitoquímica que permitiu a detecção de compostos como: alcaloides, flavonoides, taninos, terpenos, ácidos fenólicos e catequinas. Avaliou-se, também, a atividade antioxidante pelo método sequestrante do radical DPPH, e os valores das frações hidroetanólicas revelaram-se mais significativos comparados ao extrato hidroetanólico. A atividade leishmanicida foi executada utilizando placas de 96 poços e os resultados mostraram que o extrato e as frações apresentaram-se inativos contra as formas promastigotas de Leishmania amazonensis.(AU)

Phytotherapy is a sociocultural practice present over the years and used to treat diseases that affect humans. In this context, Annona crassiflora Mart. is notable for being one of the species used in phytotherapy, seldom studied chemically or biologically. Popularly known as "marolo", its chemical constitution is composed of phenolic acids, alkaloids, flavonoids, tannins, terpenoids and acetogenins, according to scientific literature. The presence of these actives is responsible for the great pharmacological potential of this species, where the antimicrobial, antioxidant, cytotoxic, leishmanicidal and hypoglycemic activities stand out. Considering this, the present study proposes to investigate the Annona crassiflora species from the chemical and biological point of view. For this purpose, we obtained hydroethanolic extract 70% (v / v) and fractions from the marolo leaves. With these samples, a phytochemical screening was carried out, which allowed us to detect compounds such as: alkaloids, flavonoids, tannins, terpenes, phenolic acids and catechins. We also assessed the antioxidant activity using the DPPH radical capturing method, where the values ​​of the hydroethanolic fractions were more significant compared to its extract. The leishmanicidal activity was performed using 96-well plates and the results show that both the extract and the fractions were inactive against the promastigote forms of Leishmania amazonensis.(AU)