Evaluation of in vitro and in vivo Efficacy of a Novel Amphotericin B-Loaded Nanostructured Lipid Carrier in the Treatment of Leishmania braziliensis Infection.

BACKGROUND: Leishmaniasis is a neglected disease, and the current therapeutic arsenal for its treatment is seriously limited by high cost and toxicity. Nanostructured lipid carriers (NLCs) represent a promising approach due to high drug loading capacity, controlled drug release profiles and superior stability. Here, we explore the efficacy of a unique pH-sensitive amphotericin B-loaded NLC (AmB-NLC) in Leishmania braziliensis infection in vitro and in vivo. METHODS AND RESULTS: AmB-NLC was assessed by dynamic light scattering and atomic force microscopy assays. The carrier showed a spherical shape with a nanometric size of 242.0 ± 18.3 nm. Zeta potential was suggestive of high carrier stability (-42.5 ± 1.5 mV), and the NLC showed ~99% drug encapsulation efficiency (EE%). In biological assays, AmB-NLC presented a similar IC50 as free AmB and conventional AmB deoxycholate (AmB-D) (11.7 ± 1.73; 5.3 ± 0.55 and 13 ± 0.57 ng/mL, respectively), while also presenting higher selectivity index and lower toxicity to host cells, with no observed production of nitric oxide or TNF-α by in vitro assay. Confocal microscopy revealed the rapid uptake of AmB-NLC by infected macrophages after 1h, which, in association with more rapid disruption of AmB-NLC at acidic pH levels, may directly affect intracellular parasites. Leishmanicidal effects were evaluated in vivo in BALB/c mice infected in the ear dermis with L. braziliensis and treated with a pentavalent antimonial (Sb5+), liposomal AmB (AmB-L) or AmB-NLC. After 6 weeks of infection, AmB-NLC treatment resulted in smaller ear lesion size in all treated mice, indicating the efficacy of the novel formulation. CONCLUSION: Here, we preliminarily demonstrate the effectiveness of an innovative and cost-effective AmB-NLC formulation in promoting the killing of intracellular L. braziliensis. This novel carrier system could be a promising alternative for the future treatment of cutaneous leishmaniasis.

Ageing dangerously; homing of senescent CD8 T cells in cutaneous Leishmaniasis.

Both CD8+ T cells and NK cells contribute to the immune response against the protozoan Leishmania parasite. Both are able to generate IFN-γ and both display cytotoxic features. These features may enable them to not only contribute to parasite clearance but also to cause immune-mediated pathology. This pathology is evident, for example, in the Leismania-induced skin lesions found in patients with cutaneous leismaniasis (CL). Here we highlight new data demonstrating that CD8+ T cells and NK cells in CL display a highly cytotoxic senescent phenotype, and that the senescent T cells play a major role in mediating skin pathology. This is the first demonstration that senescent CD8 T cells contribute to immunopathology in vivo.

Compartmentalized cytotoxic immune response leads to distinct pathogenic roles of natural killer and senescent CD8+ T cells in human cutaneous leishmaniasis.

Cytotoxic activity mediated by CD8+ T cells is the main signature of the immunopathogenesis of cutaneous leishmaniasis (CL). Here, we performed a broad evaluation of natural killer (NK) cell phenotypic and functional features during cutaneous leishmaniasis. We demonstrate for the first time that CL patients present the accumulation of circulating NK cells with multiple features of replicative senescence including low proliferative capacity and shorter telomeres, elevated expression of CD57, KLRG1 but diminished CD27 stimulatory receptor expression. Moreover, they exhibited higher cytotoxic and inflammatory potential than age-matched controls. The accumulation of circulating senescent NK cells (CD56dim  CD57bright ) correlated positively with skin lesion size in the same patients, suggesting that they, like circulating senescent CD8+ T cells, may contribute to the immunopathology of CL. However, this senescent population had lower cutaneous lymphocyte antigen expression and so had diminished skin-homing potential compared with total or senescent CD8+ T cells. This was confirmed in CL skin lesions where we found a predominance of CD8+ T cells (both senescent and non-senescent) that correlated with the severity of the disease. Although there was also a correlation between the proportions of senescent NK cells (CD56+  CD57+ ) in the skin and lesion size, this was less evident. Collectively our results demonstrate first-hand that senescent cytotoxic cells may mediate skin pathology during human cutaneous leishmaniasis. However, as senescent cytotoxic CD8+ T cells predominate in the skin lesions, they may have a greater role than NK cells in mediating the non-specific skin damage in CL.

The impact of arginase activity on virulence factors of Leishmania amazonensis.

The outcome of Leishmania infection depends on the parasite species and the host immune response. Virulence factors have been extensively studied over the years in an effort to find efficient vaccines and/or treatments for Leishmania infection. Arginase activity in Leishmania has been described as an essential player for the polyamines pathway, impacting parasite replication and infectivity. Considering previous studies showing that the absence of arginase activity leads to low infectivity of Leishmania amazonensis, we reanalyzed transcriptomic data comparing both promastigotes and axenic amastigotes from L. amazonensis wild type (La-WT) and L. amazonensis arginase knockout (La-arg-) backgrounds. The analysis produced a new compilation of modulated transcripts that indicated the role of arginase not only in the polyamines pathway but also in the modulation of virulence factors involved in parasite recognition, growth and differentiation.

Comparative Proteomic Analysis of Murine Cutaneous Lesions Induced by Leishmania amazonensis or Leishmania major.

Cutaneous leishmaniasisis is the most common clinical form of leishmaniasis and one of the most relevant neglected diseases. It is known that the progress of the disease is species specific and the host's immune response plays an important role in its outcome. However, the pathways that lead to parasite clearance or survival remain unknown. In this work, skin tissue from mice experimentally infected with L. amazonensis, one of the causative agents of cutaneous leishmaniasis in the Amazon region, L. major, another causative agent of cutaneous leishmaniasis in Africa, the Middle East, China, and India, or lipopolysaccharides from Escherichia coli as an inflammation model were investigated using label-free proteomics to unveil Leishmania-specific protein alterations. Proteomics is a powerful tool to investigate host-pathogen relationships to address biological questions. In this work, proteins from mice skin biopsies were identified and quantified using nano-LC coupled with tandem mass spectrometry analyses. Integrated Proteomics Pipeline was used for peptide/protein identification and quantification. Western blot was used for validation of protein quantification by mass spectrometry, and protein pathways were predicted using Ingenuity Pathway Analysis. In this proteomics study, several proteins were pointed out as hypothetical targets to guide future studies on Leishmania-specific modulation of proteins in the host. We identified hundreds of exclusively modulated proteins after Leishmania spp. infection and 17 proteins that were differentially modulated in the host after L. amazonensis or L. major infection.

In vitro effect of Bothrops leucurus lectin (BLL) against Leishmania amazonensis and Leishmania braziliensis infection.

Leishmania amazonensis and Leishmania braziliensis are the main causative agents of American Tegumentary Leishmaniasis (ATL) in Brazil. As intracellular parasites, the infection by Leishmania species is dependent on the host immune response and the immunotherapy could be promissory for the development of new strategies to combat ATL. In this work we investigated the leishmanicidal potential of a galactose-binding lectin from the snake venom of Bothrops leucurus (BLL) during the infection with L. amazonensis and L. braziliensis. BLL inhibited the promastigote growth and viability of both species in a mechanism dependent on galactose and calcium. The treatment with BLL also decreases the survival of intracellular parasites for both species and induced profound ultrastructural changes on amastigotes without apparent damage to the host cells. The analysis of the cytokine profile revealed that BLL induced an increase in the proinflammatory cytokines IL-6 and TNF-α by infected macrophages in both species, but differed in relation to IL-1ß and IL-10 response. Future works using in vitro and in vivo models are necessary to support the use of these lectins as biotechnological tool in immunological studies.

Susceptibility of dendritic cells from individuals with schistosomiasis to infection by Leishmania braziliensis.

Coinfection with leishmaniasis and schistosomiasis has been associated with increased time to healing of cutaneous lesions of leishmaniasis. The objective of this study was to evaluate the effect of Leishmania braziliensis infection on co-cultures of monocyte-derived dendritic cells (MoDCs) with autologous lymphocytes from patients with schistosomiasis and patients with cutaneous leishmaniasis. MoDCs were differentiated from peripheral blood monocytes, isolated by magnetic beads, infected with L. braziliensis, and co-cultured with autologous lymphocytes. Expression of HLA-DR, CD1a, CD83, CD80, CD86, CD40, and the IL-10 receptor (IL-10R) on MoDCs as well as CD28, CD40L, CD25, and CTLA-4 on lymphocytes were evaluated by flow cytometry. The production of the cytokines IL-10, TNF, IL-12p40, and IFN-γ were evaluated by sandwich ELISA of the culture supernatant. The infectivity evaluation was performed by light microscopy after concentration of cells by cytospin and Giemsa staining. It was observed that the frequency of MoDCs expressing CD83, CD80, and CD86 as well as the MFI of HLA-DR were smaller in the group of patients with schistosomiasis compared to the group of patients with leishmaniasis. On the other hand, the frequency of IL-10R on MoDCs was higher in patients with schistosomiasis than in patients with leishmaniasis. CD4+ and CD8+ T lymphocytes from patients with schistosomiasis presented a lower frequency of CD28 and a higher frequency of CTLA-4 compared to lymphocytes from patients with leishmaniasis. Levels of IL-10 were higher in the supernatants of co-cultures from individuals with schistosomiasis compared to those with leishmaniasis. However, levels of TNF, IL-12p40, and IFN-γ were lower in the group of individuals with schistosomiasis. Regarding the frequency of MoDCs infected by L. braziliensis after 72h in culture, it was observed that higher frequencies of cells from patients with schistosomiasis were infected compared to cells from patients with leishmaniasis. It was concluded that MoDCs from patients with schistosomiasis are more likely to be infected by L. braziliensis, possibly due to a lower degree of activation and a regulatory profile.

Knockdown of Host Antioxidant Defense Genes Enhances the Effect of Glucantime on Intracellular Leishmania braziliensis in Human Macrophages.

Leishmaniasis is a neglected tropical disease that affects millions of people worldwide and represents a major public health problem. Information on protein expression patterns and functional roles within the context of Leishmania-infected human monocyte-derived macrophages (MDMs) under drug treatment conditions is essential for understanding the role of these cells in leishmaniasis treatment. We analyzed functional changes in the expression of human MDM genes and proteins during in vitro infection by Leishmania braziliensis and treatment with Glucantime (SbV), using quantitative PCR (qPCR) arrays, Western blotting, confocal microscopy, and small interfering RNA (siRNA) human gene inhibition assays. Comparison of the results from gene transcription and protein expression analyses revealed that glutathione S-transferase π1 (GSTP1), glutamate-cysteine ligase modifier subunit (GCLM), glutathione reductase (GSR), glutathione synthetase (GSS), thioredoxin (TRX), and ATP-binding cassette, subfamily B, member 5 (ABCB5), were strongly upregulated at both the mRNA and protein levels in human MDMs that were infected and treated, compared to the control group. Subcellular localization studies showed a primarily phagolysosomal location for the ABCB5 transporter, indicating that this protein may be involved in the transport of SbV By inducing a decrease in L. braziliensis intracellular survival in THP-1 macrophages, siRNA silencing of GSTP1, GSS, and ABCB5 resulted in an increased leishmanicidal effect of SbV exposure in vitro Our results suggest that human MDMs infected with L. braziliensis and treated with SbV express increased levels of genes participating in antioxidant defense, whereas our functional analyses provide evidence for the involvement of human MDMs in drug detoxification. Therefore, we conclude that GSS, GSTP1, and ABCB5 proteins represent potential targets for enhancing the leishmanicidal activity of Glucantime.

Oral Pentoxifylline Associated with Pentavalent Antimony: A Randomized Trial for Cutaneous Leishmaniasis.

AbstractCutaneous leishmaniasis (CL) by Leishmania braziliensis is associated with decreasing cure rates in Brazil. Standard treatment with pentavalent antimony (Sbv) cures only 50-60% of the cases. The immunopathogenesis of CL ulcer is associated with high interferon-γ and tumor necrosis factor (TNF) production. Pentoxifylline, a TNF inhibitor, has been successfully used in association with Sbv in mucosal and cutaneous leishmaniasis. This randomized, double-blind, and placebo-controlled trial aimed to evaluate the efficacy and safety of oral pentoxifylline plus Sbv versus placebo plus Sbv in patients with CL in Bahia, Brazil. A total of 164 patients were randomized in two groups to receive the combination or the monotherapy. Cure rate 6 months after treatment was 45% in the pentoxifylline group and 43% in the control group. There was also no difference between the groups regarding the healing time (99.7 ± 66.2 days and 98.1 ± 72.7 days, respectively). Adverse events were more common in the pentoxifylline group (37.8%), versus 23% in the placebo group. This trial shows that Sbv combined therapy with pentoxifylline is not more effective than Sbv monotherapy in the treatment of CL caused by L. braziliensis.

Analysis of expression of FLI1 and MMP1 in American cutaneous leishmaniasis caused by Leishmania braziliensis infection.

FLI1 (Friend leukemia virus integration 1) and IL6 (interleukin 6; IL-6) are associated with Leishmania braziliensis susceptibility. Cutaneous lesions show exaggerated matrix metalloproteinase 1 (MMP1). In other skin diseases, FLI1 promoter methylation reduces FLI1 expression, and low FLI1 down-regulates MMP1. IL-6 increases FLI1 expression. We hypothesized that epigenetic regulation of FLI1 in cutaneous leishmaniasis, together with IL-6, might determine MMP1 expression. While generally low (<10%), percent FLI1 promoter methylation was lower (P=0.001) in lesion biopsies than normal skin. Contrary to expectation, a strong positive correlation occurred between FLI1 methylation and gene expression in lesions (r=0.98, P=0.0005) and in IL-6-treated L. braziliensis-infected macrophages (r=0.99, P=0.0004). In silico analysis of the FLI1 promoter revealed co-occurring active H3K27ac and repressive DNA methylation marks to enhance gene expression. FLI1 expression was enhanced between 3 and 24hour post infection in untreated (P=0.0002) and IL-6-treated (P=0.028) macrophages. MMP1 was enhanced in lesion biopsies (P=0.0002), induced (P=0.007) in infected macrophages, but strongly inhibited by IL-6. No correlations occurred between FLI1 and MMP1 expression in lesions or infected macrophages (with/without IL-6). We conclude that MMP1 is regulated by factors other than FLI1, and that the influence of IL-6 on MMP1 was independent of its effect on FLI1.

Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response.

Many Leishmania (Viannia) parasites harbor the double-stranded RNA virus Leishmania RNA virus 1 (LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in both Leishmania braziliensis and Leishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lacks cis- or trans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For both L. braziliensis and L. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Toll-like receptor 3-dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence of L. braziliensis, the Leishmania species responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of other Leishmania viruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi-viral relationships in evolution: one of balance rather than elimination.

Exploring the unbinding of Leishmania (L.) amazonensis CPB derived-epitopes from H2 MHC class I proteins.

New strategies to control Leishmania disease demand an extensive knowledge about several aspects of infection including the understanding of its molecular events. In murine models, cysteine proteinase B from Leishmania amazonensis promotes regulation of immune response, and fragments from its C-terminus extension (cyspep) can play a decisive role in the host-parasite interaction. The interaction between cyspep-derived peptides and major histocompatibility complex (MHC) proteins is a crucial factor in Leishmania infections. Seven cyspep-derived peptides, previously identified as capable of interacting with H-2 (murine) MHC class I proteins, were studied in this work. We established a protocol to simulate the unbinding of these peptides from the cleft of H-2 receptors. From the simulations, we estimated the corresponding free energy of dissociation (ΔGd ) and described the molecular events that occur during the exit of peptides from the cleft. To test the reliability of this method, we first applied it to a calibration set of four crystallographic MHC/peptide complexes. Next, we explored the unbinding of the seven complexes mentioned above. Results were consistent with ΔGd values obtained from surface plasmon resonance (SPR) experiments. We also identified some of the primary interactions between peptides and H-2 receptors, and we detected three regions of influence for the interaction. This pattern was systematically observed for the peptides and helped determine a minimum distance for the real interaction between peptides and H-2 proteins occurring at ∼ 25 Å.

Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes.

Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole.

CK2 Secreted by Leishmania braziliensis Mediates Macrophage Association Invasion: A Comparative Study between Virulent and Avirulent Promastigotes.

CK2 is a protein kinase distributed in different compartments of Leishmania braziliensis: an externally oriented ecto-CK2, an intracellular CK2, and a secreted CK2. This latter form is constitutively secreted from the parasite (CsCK2), but such secretion may be highly enhanced by the association of specific molecules, including enzyme substrates, which lead to a higher enzymatic activity, called inductively secreted CK2 (IsCK2). Here, we examined the influence of secreted CK2 (sCK2) activity on the infectivity of a virulent L. braziliensis strain. The virulent strain presented 121-fold higher total CK2 activity than those found in an avirulent strain. The use of specific CK2 inhibitors (TBB, DRB, or heparin) inhibited virulent parasite growth, whereas no effect was observed in the avirulent parasites. When these inhibitors were added to the interaction assays between the virulent L. braziliensis strain and macrophages, association index was drastically inhibited. Polyamines enhanced sCK2 activity and increased the association index between parasites and macrophages. Finally, sCK2 and the supernatant of the virulent strain increased the association index between the avirulent strain and macrophages, which was inhibited by TBB. Thus, the kinase enzyme CK2 seems to be important to invasion mechanisms of L. braziliensis.

Protective and pathological functions of CD8+ T cells in Leishmania braziliensis infection.

Cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is characterized by a strong Th1 response that leads to skin lesion development. In areas where L. braziliensis transmission is endemic, up to 15% of healthy subjects have tested positive for delayed-type hypersensitivity to soluble leishmania antigen (SLA) and are considered to have subclinical (SC) infection. SC subjects produce less gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) than do CL patients, but they are able to control the infection. The aim of this study was to characterized the role of CD8(+) T cells in SC infection and in CL. Peripheral blood mononuclear cells (PBMC) were stimulated with SLA to determine the frequencies of CD4(+) IFN-γ(+) and CD8(+) IFN-γ(+) T cells. Monocytes from PBMC were infected with L. braziliensis and cocultured with CD8(+) T cells, and the frequencies of infected monocytes and levels of cytotoxicity markers, target cell apoptosis, and granzyme B were determined. The frequency of CD8(+) IFN-γ(+) cells after SLA stimulation was higher for SC individuals than for CL patients. The frequency of infected monocytes in SC cells was lower than that in CL cells. CL CD8(+) T cells induced more apoptosis of infected monocytes than did SC CD8(+) T cells. Granzyme B production in CD8(+) T cells was higher in CL than in SC cells. While the use of a granzyme B inhibitor decreased the number of apoptotic cells in the CL group, the use of z-VAD-FMK had no effect on the frequency of these cells. These results suggest that CL CD8(+) T cells are more cytotoxic and may be involved in pathology.

Estudo da interação entre neutrófilos, células dendríticas humanas e L. braziliensis / Study of the interaction between neutrophils, human dendritic cells and L. braziliensis

Os neutrófilos são essenciais para a resposta imune inata contra uma variedade de patógenos. Eles são capazes de modular a resposta imune através da produção de citocinas e quimiocinas, degranulação e a sua interação direta com outras células no local da infecção, tais como as células dendríticas. A interação entre as células do sistema imune inato é essencial para direcionar a resposta imune adaptativa, a qual é responsável pela eliminação de microrganismos e manutenção de memória imunológica. Objetivo: Este estudo avaliou a interação de neutrófilos humanos com Leishmania braziliensis, através da análise da expressão de moléculas de superfície, liberação de enzimas presentes nos grânulos e produção de espécies reativas de oxigênio (ROS). Também foi avaliada a interação entre neutrófilos humanos infectados e células dendríticas, a fim de se observar o efeito desta interação na indução da ativação das células dendríticas. Metodologia: Os neutrófilos foram purificados a partir do sangue periférico de doadores saudáveis e as células dendríticas foram geradas in vitro. Os neutrófilos foram infectados ou não com L. braziliensis e co-cultivados com as células dendríticas. Em seguida, os sobrenadantes e as células foram coletadas para avaliar a liberação de enzimas, tais como mieloperoxidase (MPO) e metaloproteinase 9 (MMP-9). O fenótipo e a função dos neutrófilos foram analisados através da expressão de Mac-1 (CD18 e CD11b), CD16, CD62-L e produção de ROS...

Neutrophils are essential in the innate immune response against a variety of pathogens. They are able to modulate immune response by cytokine and chemokine production, release of granules and their direct interaction with other cells at the infection site. Dendritic cells are recruited in response to cytokines and chemokines produced by neutrophils. The interaction between cells of the innate immune system is essential for targeting the adaptive immune response, which is responsible for eliminating microorganisms and the maintenance of immunological memory. Objective: Evaluate the interaction of human neutrophils with Leishmania braziliensis, through the analysis of surface molecule expression, release of granules enzymes and production of reactive oxygen species (ROS). We also evaluated the interaction between human infected neutrophils and dendritic cells, in order to observe the effect of this interaction on dendritic cells. Methodology: Neutrophils were purified from peripheral blood of healthy donors and dendritic cells were generated in vitro. Neutrophils were infected or not with L. braziliensis and cocultured with DC. Afterwards, supernatants and cells were harvested to evaluate the release of granules enzymes, such as myeloperoxidase (MPO) and metalloproteinase 9 (MMP-9). Neutrophils phenotype and function were analyzed by the expression of Mac-1 (CD18 and CD11b), CD16 and ROS production...

Native rodent species are unlikely sources of infection for Leishmania (Viannia) braziliensis along the Transoceanic Highway in Madre de Dios, Peru.

An estimated 2.3 million disability-adjusted life years are lost globally from leishmaniasis. In Peru's Amazon region, the department of Madre de Dios (MDD) rises above the rest of the country in terms of the annual incidence rates of human leishmaniasis. Leishmania (Viannia) braziliensis is the species most frequently responsible for the form of disease that results in tissue destruction of the nose and mouth. However, essentially nothing is known regarding the reservoirs of this vector-borne, zoonotic parasite in MDD. Wild rodents have been suspected, or proven, to be reservoirs of several Leishmania spp. in various ecosystems and countries. Additionally, people who live or work in forested terrain, especially those who are not regionally local and whose immune systems are thus naïve to the parasite, are at most risk for contracting L. (V.) braziliensis. Hence, the objective of this study was to collect tissues from wild rodents captured at several study sites along the Amazonian segment of the newly constructed Transoceanic Highway and to use molecular laboratory techniques to analyze samples for the presence of Leishmania parasites. Liver tissues were tested via polymerase chain reaction from a total of 217 rodents; bone marrow and skin biopsies (ear and tail) were also tested from a subset of these same animals. The most numerous rodent species captured and tested were Oligoryzomys microtis (40.7%), Hylaeamys perenensis (15.7%), and Proechimys spp. (12%). All samples were negative for Leishmania, implying that although incidental infections may occur, these abundant rodent species are unlikely to serve as primary reservoirs of L. (V.) braziliensis along the Transoceanic Highway in MDD. Therefore, although these rodent species may persist and even thrive in moderately altered landscapes, we did not find any evidence to suggest they pose a risk for L. (V.) braziliensis transmission to human inhabitants in this highly prevalent region.

The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of human tegumentary leishmaniasis.

In tegumentary leishmaniasis caused by Leishmania braziliensis, there is evidence that increased production of IFN-γ, TNF-α and absence of IL-10 is associated with strong inflammatory reaction and with tissue destruction and development of the lesions observed in cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). We evaluate the role of regulatory cytokines and cytokine antagonists in the downregulation of immune response in L. braziliensis infection. Peripheral blood mononuclear cells from CL and ML were stimulated with soluble Leishmania antigen in the presence or absence of regulatory cytokines (IL-10, IL-27 and TGF-ß) or antagonists of cytokines (α-TNF-α and α-IFN-γ). Cytokines production (IL-10, IL-17, TNF-α and IFN-γ) was measured by ELISA. IL-10 and TGF-ß downmodulate TNF-α and IL-17 production, whereas IL-27 had no effect in the production of TNF-α, IFN-γ and IL-17 in these patients. Neutralization of TNF-α decreased IFN-γ level and the neutralization of IFN-γ decreased TNF-α level and increased IL-10 production. This study demonstrate that IL-10 and TGF-ß are cytokines that appear to be more involved in modulation of immune response in CL and ML patients. IL-10 might have a protective role, since the neutralization of IFN-γ decreases the production of TNF-α in an IL-10-dependent manner.

Avaliação da capacidade protetora de antígenos recombinantes contra a Leishmaniose Tegumentar / Protective capacity of recombinant antigens against tegumentary leishmaniasis

A leishmaniose é uma doença de escala global, que afeta 12 milhões de pessoas e pode causar um espectro de doenças que vai desde a forma cutânea localizada, que tende para a cura espontânea, até a forma visceral que é fatal. Apesar da gravidade da doença, até o momento não existe uma vacina efetiva para prevenir a leishmaniose. Dentre os antígenos promissores para o desenvolvimento de uma vacina, destacam-se as proteínas ribossomais (S4, S6, L3 e L5) e a KMP-11, uma proteína de superfície presente nos membros da família tripanosomatidae. Nosso estudo consistiu em avaliar os efeitos da imunização com estes antígenos frente ao desafio com L. major e com L. braziliensis, empregando modelos experimentais de infecção. Primeiramente, avaliamos a capacidade protetora dos antígenos ribossomais frente à infecção por L. major. Dos quatro antígenos avaliados, apenas L3 ou L5 foram capazes de prevenir o desenvolvimento da lesão e de diminuir a carga parasitária. A vacinação de camundongos com estes antígenos, na presença de CpG, induziu um perfil de resposta Th1, com elevada produção de IFN-γ, baixa produção de IL-10 e presença de anticorpos IgG2a. Em seguida, avaliamos a capacidade protetora dos antígenos L3 e L5...

Leishmaniasis is a global disease affecting 12 million people and can cause diseases that range from self-healing localized cutaneous leishmaniasis to fatal visceral leishmaniasis. Despite the severity of the disease, there is no effective vaccine to prevent leishmaniasis. Among the promising antigens for the development of a vaccine, stand out the ribosomal proteins (S4, S6, L3, and L5) and KMP-11, a surface protein, widely found in the members of family Trypanosomatidae. Our study evaluated the effects of immunization with these antigens upon challenge with L. major and L. braziliensis, employing the experimental models of infection. First, we evaluated the protective ability of ribosomal antigens to infection by L. major. Among the four antigens examined only L3 or L5...

Golden hamster (Mesocricetus auratus) as an experimental model for Leishmania (Viannia) braziliensis infection.

The lack of an adequate model for Leishmania (Viannia) braziliensis infection is a limiting factor for studying American tegumentary leishmaniasis (ATL). The golden hamster (Mesocricetus auratus) is a promising model because besides being highly susceptible to dermotropic Leishmania infection, the lesions are very similar to cutaneous leishmaniasis (CL) in humans. However, different Leishmania isolates or species and/or protocols have resulted in different outcomes, whereas no study has evaluated the reproducibility of L. braziliensis infection in this model. The natural history of L. braziliensis infection in 34 hamsters was evaluated by using a single parasite isolate in 8 independent experiments under similar experimental conditions. Clinical, histological and immunological analyses were performed. The hamsters presented skin ulcers similar to those observed in ATL. The intra-experiment lesion increment tended to show an intermediary variance. Histological analysis of infected skins showed granulomatous reaction, scarce amastigotes, and Schaumann's bodies. Blood lymphocytes proliferated in response to leishmanial antigens. The severity of the infection was positively correlated to spleen weight, and the titres of anti-Leishmania IgG antibodies. Our findings indicate that the hamster is an appropriate model for immunopathogenesis studies of CL caused by L. braziliensis, supporting its use in clinical, vaccine and chemotherapy experimental protocols.