Sandflies (Diptera: Psychodidae) associated with opossum nests at urban sites in southeastern Brazil: a risk factor for urban and periurban zoonotic Leishmania transmission?

Sandflies associated with opossum nests are reported for the first time in the yards of residences located in the urban area of the municipality of Monte Mor, situated in the metropolitan region of Campinas, state of São Paulo, Brazil. Eleven specimens of Evandromyia cortelezzii and one of Evandromyia lenti were captured in two Didelphis albiventris nests. Ev. cortelezzii is considered a secondary vector species for the transmission of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum in the Neotropics. This association may contribute to the introduction, establishment and maintenance of urban and periurban zoonotic transmission outbreaks of Leishmania and should therefore be investigated further.

A comparison of molecular markers to detect Lutzomyia longipalpis naturally infected with Leishmania (Leishmania) infantum

The aim of the present study was to detect natural infection by Leishmania (Leishmania) infantum in Lutzomyia longipalpis captured in Barcarena, state of Pará, Brazil, through the use of three primer sets. With this approach, it is unnecessary to previously dissect the sandfly specimens. DNA of 280 Lu. longipalpis female specimens were extracted from the whole insects. PCR primers for kinetoplast minicircle DNA (kDNA), the mini-exon gene and the small subunit ribosomal RNA (SSU-rRNA) gene of Leishmania were used, generating fragments of 400 bp, 780 bp and 603 bp, respectively. Infection by the parasite was found with the kDNA primer in 8.6% of the cases, with the mini-exon gene primer in 7.1% of the cases and with the SSU-rRNA gene primer in 5.3% of the cases. These data show the importance of polymerase chain reaction as a tool for investigating the molecular epidemiology of visceral leishmaniasis by estimating the risk of disease transmission in endemic areas, with the kDNA primer representing the most reliable marker for the parasite.

Cross-presenting dendritic cells are required for control of Leishmania major infection.

Leishmania major infection induces self-healing cutaneous lesions in C57BL/6 mice. Both IL-12 and IFN-γ are essential for the control of infection. We infected Jun dimerization protein p21SNFT (Batf3(-/-) ) mice (C57BL/6 background) that lack the major IL-12 producing and cross-presenting CD8α(+) and CD103(+) DC subsets. Batf3(-/-) mice displayed enhanced susceptibility with larger lesions and higher parasite burden. Additionally, cells from draining lymph nodes of infected Batf3(-/-) mice secreted less IFN-γ, but more Th2- and Th17-type cytokines, mirrored by increased serum IgE and Leishmania-specific immunoglobulin 1 (Th2 indicating). Importantly, CD8α(+) DCs isolated from lymph nodes of L. major-infected mice induced significantly more IFN-γ secretion by L. major-stimulated immune T cells than CD103(+) DCs. We next developed CD11c-diptheria toxin receptor: Batf3(-/-) mixed bone marrow chimeras to determine when the DCs are important for the control of infection. Mice depleted of Batf-3-dependent DCs from day 17 or wild-type mice depleted of cross-presenting DCs from 17-19 days after infection maintained significantly larger lesions similar to mice whose Batf-3-dependent DCs were depleted from the onset of infection. Thus, we have identified a crucial role for Batf-3-dependent DCs in protection against L. major.

Leishmania major metacaspase can replace yeast metacaspase in programmed cell death and has arginine-specific cysteine peptidase activity.

The human protozoan parasite Leishmania major has been shown to exhibit several morphological and biochemical features characteristic of a cell death program when differentiating into infectious stages and under a variety of stress conditions. Although some caspase-like peptidase activity has been reported in dying parasites, no caspase gene is present in the genome. However, a single metacaspase gene is present in L. major whose encoded protein harbors the predicted secondary structure and the catalytic dyad histidine/cysteine described for caspases and other metacaspases identified in plants and yeast. The Saccharomyces cerevisiae metacaspase YCA1 has been implicated in the death of aging cells, cells defective in some biological functions, and cells exposed to different environmental stresses. In this study, we describe the functional heterologous complementation of a S. cerevisiae yca1 null mutant with the L. major metacaspase (LmjMCA) in cell death induced by oxidative stress. We show that LmjMCA is involved in yeast cell death, similar to YCA1, and that this function depends on its catalytic activity. LmjMCA was found to be auto-processed as occurs for caspases, however LmjMCA did not exhibit any activity with caspase substrates. In contrast and similarly to Arabidopsis thaliana metacaspases, LmjMCA was active towards substrates with arginine in the P1 position, with the activity being abolished following H147A and C202A catalytic site mutations. These results suggest that metacaspases are members of a family of peptidases with a role in cell death conserved in evolution notwithstanding possible differences in their catalytic activity.

The prominent role of neutrophils during the initial phase of infection by Leishmania parasites.

Neutrophils are rapidly and massively recruited to the site of Leishmania inoculation, where they phagocytose the parasites, some of which are able to survive within these first host cells. Neutrophils can thus provide a transient safe shelter for the parasites, prior to their entry into macrophages where they will replicate. In addition, neutrophils release and synthesize rapidly several factors including cytokines and chemokines. The mechanism involved in their rapid recruitment to the site of parasite inoculation, as well as the putative consequences of their massive presence on the microenvironment of the focus of infection will be discussed in the context of the development of the Leishmania-specific immune response.

An essential role for the Leishmania major metacaspase in cell cycle progression.

Metacaspases (MCAs) are distant orthologues of caspases and have been proposed to play a role in programmed cell death in yeast and plants, but little is known about their function in parasitic protozoa. The MCA gene of Leishmania major (LmjMCA) is expressed in actively replicating amastigotes and procyclic promastigotes, but at a lower level in metacyclic promastigotes. LmjMCA has a punctate distribution throughout the cell in interphase cells, but becomes concentrated in the kinetoplast (mitochondrial DNA) at the time of the organelle's segregation. LmjMCA also translocates to the nucleus during mitosis, where it associates with the mitotic spindle. Overexpression of LmjMCA in promastigotes leads to a severe growth retardation and changes in ploidy, due to defects in kinetoplast segregation and nuclear division and an impairment of cytokinesis. LmjMCA null mutants could not be generated and following genetic manipulation to express LmjMCA from an episome, the only mutants that were viable were those expressing LmjMCA at physiological levels. Together these data suggest that in L. major active LmjMCA is essential for the correct segregation of the nucleus and kinetoplast, functions that could be independent of programmed cell death, and that the amount of LmjMCA is crucial. The absence of MCAs from mammals makes the enzyme a potential drug target against protozoan parasites.

Leishmania (Viannia) naiffi: rare enough to be neglected?

In the Brazilian Amazon, American tegumentary leishmaniasis (ATL) is endemic and presents a wide spectrum of clinical manifestations due, in part, to the circulation of at least seven Leishmaniaspecies. Few reports of Leishmania (Viannia) naiffiinfection suggest that its occurrence is uncommon and the reported cases present a benign clinical course and a good response to treatment. This study aimed to strengthen the clinical and epidemiological importance of L. (V.) naiffiin the Amazon Region (Manaus, state of Amazonas) and to report therapeutic failure in patients infected with this species. Thirty Leishmania spp samples isolated from cutaneous lesions were characterised by multilocus enzyme electrophoresis. As expected, the most common species was Leishmania (V.) guyanensis (20 cases). However, a relevant number ofL. (V.) naiffi patients (8 cases) was observed, thus demonstrating that this species is not uncommon in the region. No patient infected withL. (V.) naiffievolved to spontaneous cure until the start of treatment, which indicated that this species may not have a self-limiting nature. In addition, two of the patients experienced a poor response to antimonial or pentamidine therapy. Thus, either ATL cases due to L. (V.) naifficannot be as uncommon as previously thought or this species is currently expanding in this region.

Polymerase chain reaction detection of LeishmaniaDNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detectLeishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmaniagenus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.

The role of dentritic cells in leishmania infection : use of novel DC lines for the development of monoclonal antibodies

Les cellules dendritiques (DCs) sont des cellules multifonctionnelles qui font le lien entre le sytème immunitaire inné et adaptatif chez les mammifères. Il existe plusieurs sous-types de DCs basés sur leurs fonctions et l'endroit où elles se situent dans le corps. Dans le cadre de cette thèse, nous avons étudié le rôle de ces cellules face à une infection parasitaire. La Leishmania est un parasite causant une maladie appelée Leishmaniose, maladie endémique de l'Afrique, de l'Asie et de certaines régions de l'Amérique du Sud. Certaines espèces causent des lésions cutanées, alors que d'autres causent des lésions dans les muqueuses ou dans les organes internes. Le système immunitaire répond en générant une réponse inflammatoire qui élimine l'infection. Lors d'une réponse non-inflammatoire (de type cytokines, chemokines), cela va amener à une persistance du parasite sur le long terme. Les DC s'activant en présence du parasite dans la peau, vont le transporter vers un ganglion. A cet endroit, se trouvent différents sous-types de DC qui ont la particularité de présenter l'antigène (spécifique à la Leishmaniose) aux lymphocytes T, ce qui va alors amener à une réponse immunitaire puissante contre le parasite. Nous avons comparé différentes espèces de Leishmaniose dans leur façon d'activer les DC et différents modèles de souris ont été utilisé dans ce but-là. Les souris du type C57BL/6 sont connues pour être résistantes à L. major et sensibles à L. mexicana, alors qu'au contraire, les souris Balb/c sont connues pour être sensibles à ces deux espèces. En utilisant des parasites fluorescents transgéniques, nous avons comparé ces deux espèces de parasites (L. major et L. mexicana) en recherchant quelles cellules elles sont capables d'infecter in-vivo dans un modèle murin. Le rôle général des DC dans une infection à L. major a déjà été décrit. Dans notre étude, nous avons étudié le besoin en DC CD8a+ dans les ganglions afin d'engendrer une réponse face à une infection à L. major. Les souris qui n'ont pas ce sous-type de DC sont beaucoup plus sensibles à l'infection : elles ont des marqueurs inflammatoires plus bas et des lésions plus grandes. Nous avons également remarqué que les DC CD8a+ jouent un rôle crucial dans une phase plus avancée de l'infection. Dans notre laboratoire, nous avons la chance d'avoir une source illimitée de DCs de sous-type CD8a+ provenant d'une souris génétiquement modifiée par nos soin. Grâce à cela, nous avons utilisé ces cellules CD8a+ pour immuniser des rats afin de produire des anticorps monoclonaux ayant des propriétés spécifiques comme l'identification de protéines uniques présentes à la surface des DC et qui ensuite, modulent une réponse immunitaire in-vivo. Nous sommes actuellement en phase de caractérisation de plus de 750 hybridomes générés dans notre laboratoire. - Les cellules dendritiques (DCs) constituent le lien entre le système inné et adaptatif de la réponse immunitaire, car elles sont capables de présenter l'antigène, de donner la co- stimulation et de relâcher des cytokines et chimokines. Au cours de cette thèse, nous avons exploré différentes familles de DC lors d'infections parasitaires, telles que la Leishmaniose, parasite intracellulaire qui infecte les mammifères. La plupart des lésions cutanées résistantes sont caractérisées par une réponse pro-inflammatoire générée par l'IL-12. A l'inverse, pour la forme non résistante, la réponse est générée par l'IL-4 et l'IL-10, dans les modèles murins vulnérables. L'infection avec Lmajor a été caractérisée chez la souris C57BL/6 (Thl) et chez la souris Balb/c (Th2). Chez la souris C57BL/6 la lésion guérit, alors que chez la souris Balb/c, la lésion est au contraire non-cicatrisante. Nous avons comparé l'activation causée dans l'ensemble des DC par différentes espéces de Leishmania, et plus spécifiquement dans les DC CD8a+ présentes dans les ganglions lymphatiques et leur rôle dans la vulnérabilité à L. major. Ces cellules sont spécialisées dans la présentation croisée d'antigènes exogènes par le CMH-I et le haut taux de production d'IL-12 après activation. En utilisant des DC dérivées de moelle osseuse, nous avons constaté que L. guyanensis V+ (transportant un retrovirus) était le plus efficace pour l'activation des DC in-vitro comparé à L. major, L. mexicana et L. guyanensis (V-). Toutefois, in-vivo, les souris infectées avec L. major ont vu la taille de leur ganglions lymphatiques drainants augmentée, 3-6 semaines après l'infection dans les deux espèces de souris (les C57BL/6 résistantes et les Balb/c sensibles). En utilisant un parasite fluorescent transgénique, nous avons trouvé que les souris C57BL/6 sensibles à Lmexicana ont un nombre plus important de cellules Β infectées et un plus petit nombre de DC dérivées des monocytes inflammatoires, comparé au souris infectées avec L. major. Les conséquences de ces observations sont encore à l'étude. Des souris déficientes en CD8ct+DC et CD103+ sont plus sensibles à L. major que les souris WT: leurs lésions sont plus grandes et la charge parasitaire est plus importante. Nous avons généré une chimère de moelles osseuse CD11-DTR et Batf3-/- en mélangeant les moelles de ces deux souris, afin de déterminer le temps après infection où le manque de DC's CD8a+ contribue le plus à l'augmentation de la vulnérabilité chez la souris KO. Ces souris produisent plus d'IgG1 et IgE, font une réponse Th2 plus forte et Thl moins forte. Nous avons constaté que les souris déficientes en DC CD8a+ au début de la réponse immunitaire adaptive (trois semaines après injection) maintiennent un haut taux de lésions de grande taille, semblable à celui des souris chez qui les cellules ont été déplétées avant l'injection. Cela indique que les DC CD8a+ sont nécessaires pour l'efficacité de l'immunité dans la phase chronique de l'infection à L. major. Parallèlement à cela, nous avons aussi commencé une génération d'anticorps monoclonaux dirigés contre les DC CD8a+ activés en utilisant des souches établies dans notre laboratoire. En partant d'une librairie de 763 hybridomes, nous avons identifié plusieurs clones dignes d'intérêt avec une capacité fonctionnelle à moduler la prolifération et la sécrétion de cytokines des cellules T, ainsi que les molécules de co-stimulation présentes à la surface des DC activées elle-même. - Dendritic cells (DCs) are the bridge between the innate and the adaptive arms of the immune systems. They are professional antigen presentation cells and have important cytokine/chemokine release functions. In this dissertation we have focussed on the study of the different subsets of DCs in parasitic infection immunity. Leishmania are intra-cellular parasites of many different species that infect mammals. Most cutaneous lesions that are self- healing are characterized with a pro-inflammatory response with IL-12 while high levels of cytokines such as IL-4 and IL-10 characterized in susceptible mouse models. In mice L. major infection has been well characterized in C57BL/6 mice (Thl) that form healing lesions while Balb/c mice (Th2) form non-healing lesions. This thesis is focussed on comparing DC activation at large by different strains of Leishmania and more specifically, dLN resident CD8a+ DCs and their role in L. major susceptibility. This subset is specialized in cross- presentation of exogenous antigens in the MHC-I pathway and produce high levels of EL-12. Using bone marrow derived DCs we found that L. guyanensis V+ (carrying a retro-virus) was the most efficient at activating DCs in-vitro. In-vivo however L. major infected mice had the largest dLNs 3-6 weeks after infection in both genetically resistant C57BL/6 and susceptible Balb/c mice. Using transgenic fluorescent parasites, we found that C57BL/6 mice which are susceptible to L. mexicana had more number of infected Β cells and fewer number of infected inflammatory monocyte derived DCs in contrast to L. major infection. Using mice deficient in CD8a+ DCs, we found that these mice were more susceptible to L. major than their WT counterparts. They made larger lesions, had higher parasite burdens, higher levels of Th2 indicating immunolgloblins as measured by higher serie IgE levels and lower CD4+ IFNy+ cells. A mixed bone marrow chimera system of CDllc-DTR and Batf3~'~ was generated to determine the time point at which the lack of CD8a+ DCs most contributes to the increased susceptibility in KO mice. We found that mice depleted of CD8a+ DCs at the advent of the adaptive response (3 weeks after infection) maintained the significantly higher lesion size similar to mice whose cells were depleted from the onset of infection. This indicates that CD8a+ DCs are required for effective immunity in the chronic phase of L. major infection. We also began the generation of a valuable tool of monoclonal antibodies against activated CD8a+ DCs using our in-house DC line. From a library of 763 hybridomas we have identified several interesting clones with a functional ability to modulate Τ cell proliferation and cytokine secretion as well as down-modulating co-stimulatory molecules on activated DC cells themselves.

Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection

In Brazil, human and canine visceral leishmaniasis (CVL) caused byLeishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL.

Transforming growth factor beta 1 production by CD4+ CD25+ regulatory T cells in peripheral blood mononuclear cells from healthy subjects stimulated with Leishmania guyanensis.

Transforming growth factor beta (TGF-beta) has been shown to be a central immunomodulator used by leishmaniae to escape effective mechanisms of protection in human and murine infections with these parasites. However, all the information is derived from studies of established infection, while little is known about TGF-beta production in response to Leishmania stimulation in healthy subjects. In this study, TGF-beta1 production was demonstrated in peripheral blood mononuclear cells from healthy subjects never exposed to leishmaniae in response to live Leishmania guyanensis, and the TGF-beta1-producing cells were described as a distinct subpopulation of CD4(+) CD25(+) regulatory T cells. The suppressive properties of CD4(+) CD25(+) T cells were demonstrated in vitro by their inhibition of production of interleukin 2 (IL-2) and IL-10 by CD4(+) CD25(-) T cells in the presence of either anti-CD3 or L. guyanensis. Although neutralization of TGF-beta1 did not reverse the suppressive activity of CD4(+) CD25(+) T cells activated by anti-CD3, it reversed the suppressive activity of CD4(+) CD25(+) T cells activated by L. guyanensis. Altogether our data demonstrated that TGF-beta1 is involved in the suppressive activity of L. guyanensis-stimulated CD4(+) CD25(+) T cells from healthy controls.

Genetically resistant mice lacking interleukin-12 are susceptible to infection with Leishmania major and mount a polarized Th2 cell response.

Mice with homologous disruption of the gene coding for either the p35 subunit or the p40 subunit of interleukin-12 (IL-12) and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in resistance to infection and the differentiation of functional CD4+ T cell subsets in vivo. Wild-type 129/Sv/Ev mice are resistant to infection with L. major showing only small lesions which resolve spontaneously within a few weeks and develop a type 1 CD4+ T cell response. In contrast, mice lacking bioactive IL-12 (IL-12p35-/- and IL-12p40-/-) developed large, progressing lesions. Whereas resistant mice were able to mount a delayed-type hypersensitivity (DTH) response to Leishmania antigen, susceptible BALB/c mice as well as IL-12-deficient 129/Sv/Ev mice did not show any DTH reaction. To characterize the functional phenotype of CD4+ T cells triggered in infected wild-type mice and IL-12-deficient mice, the expression of mRNA for interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in purified CD4+ lymph node cells was analyzed. Wild-type 129/Sv/Ev mice showed high levels of mRNA for IFN-gamma and low levels of mRNA for IL-4 which is indicative of a Th1 response. In contrast, IL-12- deficient mice and susceptible BALB/c mice developed a strong Th2 response with high levels of IL-4 mRNA and low levels of IFN-gamma mRNA in CD4+ T cells. Similarly, lymph node cells from infected wild-type 129 mice produced predominantly IFN-gamma in response to stimulation with Leishmania antigen in vitro whereas lymph node cells from IL-12-deficient mice and susceptible BALB/c mice produced preferentially IL-4. Taken together, these results confirm in vivo the importance of IL-12 in induction of Th1 responses and protective immunity against L. major.