Leishmania RNA virus-1 is similarly detected among metastatic and non-metastatic phenotypes in a prospective cohort of American Tegumentary Leishmaniasis.

American Tegumentary Leishmaniasis (ATL) is an endemic and neglected disease of South America. Here, mucosal leishmaniasis (ML) disproportionately affects up to 20% of subjects with current or previous localised cutaneous leishmaniasis (LCL). Preclinical and clinical reports have implicated the Leishmania RNA virus-1 (LRV1) as a possible determinant of progression to ML and other severe manifestations such as extensive cutaneous and mucosal disease and treatment failure and relapse. However, these associations were not consistently found in other observational studies and are exclusively based on cross-sectional designs. In the present study, 56 subjects with confirmed ATL were assessed and followed out for 24-months post-treatment. Lesion biopsy specimens were processed for molecular detection and quantification of Leishmania parasites, species identification, and LRV1 detection. Among individuals presenting LRV1 positive lesions, 40% harboured metastatic phenotypes; comparatively 58.1% of patients with LRV1 negative lesions harboured metastatic phenotypes (p = 0.299). We found treatment failure (p = 0.575) and frequency of severe metastatic phenotypes (p = 0.667) to be similarly independent of the LRV1. Parasite loads did not differ according to the LRV1 status (p = 0.330), nor did Leishmanin skin induration size (p = 0.907) or histopathologic patterns (p = 0.780). This study did not find clinical, parasitological, or immunological evidence supporting the hypothesis that LRV1 is a significant determinant of the pathobiology of ATL.

Metabolomic Reprogramming of C57BL/6-Macrophages during Early Infection with L. amazonensis.

Leishmania survival inside macrophages depends on factors that lead to the immune response evasion during the infection. In this context, the metabolic scenario of the host cell-parasite relationship can be crucial to understanding how this parasite can survive inside host cells due to the host's metabolic pathways reprogramming. In this work, we aimed to analyze metabolic networks of bone marrow-derived macrophages from C57BL/6 mice infected with Leishmania amazonensis wild type (La-WT) or arginase knocked out (La-arg-), using the untargeted Capillary Electrophoresis-Mass Spectrometry (CE-MS) approach to assess metabolomic profile. Macrophages showed specific changes in metabolite abundance upon Leishmania infection, as well as in the absence of parasite-arginase. The absence of L. amazonensis-arginase promoted the regulation of both host and parasite urea cycle, glycine and serine metabolism, ammonia recycling, metabolism of arginine, proline, aspartate, glutamate, spermidine, spermine, methylhistidine, and glutathione metabolism. The increased L-arginine, L-citrulline, L-glutamine, oxidized glutathione, S-adenosylmethionine, N-acetylspermidine, trypanothione disulfide, and trypanothione levels were observed in La-WT-infected C57BL/6-macrophage compared to uninfected. The absence of parasite arginase increased L-arginine, argininic acid, and citrulline levels and reduced ornithine, putrescine, S-adenosylmethionine, glutamic acid, proline, N-glutamyl-alanine, glutamyl-arginine, trypanothione disulfide, and trypanothione when compared to La-WT infected macrophage. Moreover, the absence of parasite arginase leads to an increase in NO production levels and a higher infectivity rate at 4 h of infection. The data presented here show a host-dependent regulation of metabolomic profiles of C57BL/6 macrophages compared to the previously observed BALB/c macrophages infected with L. amazonensis, an important fact due to the dual and contrasting macrophage phenotypes of those mice. In addition, the Leishmania-arginase showed interference with the urea cycle, glycine, and glutathione metabolism during host-pathogen interactions.

Innate immune response: ally or enemy in cutaneous leishmaniasis?

Cutaneous leishmaniasis (CL) is an infectious and neglected disease caused by parasites of the genus Leishmania, which produces a wide spectrum of cutaneous manifestations. CL research has shown that the innate immune activity of cells such as neutrophils, natural killers, macrophages, dendritic cells and the complement system are capable of controlling this infection. However, Leishmania can also modulate the immune activity of these cells to promote its own survival and proliferation at the intracellular level. This review discusses the role of the innate immune response in the control and spread of this infection.

Avaliação da tendência temporal e da prevalência de leishmaniose visceral canina e humana dentre os anos de 2010 a 2019 em municípios de Minas Gerais ­ Brasil / Evaluation of the temporal trend and prevalence of canine and human visceral leishmaniosis between 2010 and 2019 in municipalities of Minas Gerais - Brazil / Evaluación de la tendencia temporal y de la prevalencia de leishmaniosis visceral canina y humana entre los años 2010 a 2019 en los municipios de Minas Gerais ­ Brasil

Leishmanioses são causadas por protozoários do gênero Leishmania, parasitos que infectam grande número de mamíferos, incluindo o homem. A Leishmaniose Visceral (LV) é a forma mais severa da doença e invariavelmente leva ao óbito, se não diagnosticada e tratada precocemente. O objetivo deste estudo foi realizar uma análise de natureza documental, descritiva e analítica, de abordagem quantitativa das informações contidas no banco de dados Fundação Ezequiel Dias/Gerenciador de Ambientes Laboratoriais (FUNED/GAL), da Secretaria Estadual de Saúde de Minas Gerais. Os dados epidemiológicos que compõe este estudo são registros de casos positivos e negativos da cidade de Patos de Minas-MG e 18 municípios das mesorregiões do Triângulo Mineiro e Alto Paranaíba, Noroeste de Minas e Norte de Minas. No período compreendido entre janeiro de 2010 e junho de 2019 foram notificados um total de 1170 indivíduos suspeitos de Leishmaniose Visceral Canina e/ou Humana. 304 (25,98%) indivíduos receberam o resultado positivo, enquanto 866 foram negativos, e em alguns casos, inconclusivos. Os dados obtidos no estudo revelaram a tendência temporal crescente e alta prevalência da doença, mostrando que a doença está em expansão na região estudada onde o cão é o principal reservatório doméstico da doença, permanecendo como principal elo de ligação entre o protozoário e o hospedeiro humano.(AU)

Leishmaniasis are caused by protozoa of the genus Leishmania, parasites that infect a large number of mammals, including humans. Visceral Leishmaniasis (VL) is the most severe form of the disease and invariably leads to death if not diagnosed and treated early. The objective of this study was to carry out a documentary, descriptive and analytical analysis, with a quantitative approach to the information contained in the Fundação Ezequiel Dias/Manager of Laboratory Environments (FUNED/GAL) database from the Minas Gerais State Health Secretariat. The epidemiological data that make up this study are records of positive and negative cases in the city of Patos de Minas - MG and of 18 municipalities in the mesoregions of the Triângulo Mineiro and Alto Paranaíba, Northwest of Minas and North of Minas. In the period between January 2010 and June 2019, a total of 1170 individuals suspected of Canine and/or Human Visceral Leishmaniasis were notified. A total of 304 (25.98%) individuals received a positive result, while 866 were considered negative, and in some cases, inconclusive. The data obtained in the study revealed the growing temporal trend and high prevalence of the disease, showing that the disease is expanding in the studied region where the dog is presented as the main domestic reservoir of the disease, remaining as the main link between the protozoan and the human host.(AU)

Leishmaniosis son causadas por protozoos del género Leishmania, parásitos que infectan a un gran número de mamíferos, incluyendo el hombre. La Leishmaniosis Visceral (LV) es la forma más grave de la enfermedad e invariablemente conduce a la muerte, si no se la diagnostica y la trata a tiempo. El objetivo de ese estudio fue realizar un análisis de naturaleza documental, descriptivo y analítico, de enfoque cuantitativo de las informaciones contenidas en la base de datos Fundação Ezequiel Dias/Gerente de Ambientes de Laboratorio (FUNED /GAL), de la Secretaría de Salud del Estado de Minas Gerais. Los datos epidemiológicos que conforman este estudio son registros de casos positivos y negativos en la ciudad de Patos de Minas-MG y 18 municipios de las mesorregiones del Triângulo Mineiro y Alto Paranaíba, Noroeste de Minas y Norte de Minas. En el período comprendido entre enero de 2010 y junio de 2019, se notificó a un total de 1170 personas sospechosas de Leishmaniosis Visceral Canina y/o Humana. 304 (25,98%) individuos recibieron un resultado positivo, mientras que 866 fueron negativos y, en algunos casos, no concluyentes. Los datos obtenidos en el estudio revelaron la tendencia creciente temporal y la alta prevalencia de la enfermedad, mostrando que la enfermedad se está expandiendo en la región estudiada donde el perro es el principal reservorio doméstico de la enfermedad, permaneciendo como principal conexión entre el protozoo y el hospedero humano.(AU)

Diagnostic usefulness of immunohistochemical evaluation of CD1a antigen and polyclonal anti-leishmania antibodies in cutaneous leishmaniasis.

BACKGROUND: Different immunohistochemical markers to detect amastigotes in cutaneous leishmaniasis have been proposed with variable diagnostic usefulness. OBJECTIVES: To evaluate the diagnostic usefulness of immunohistochemical amastigotes identification by specific polyclonal anti-Leishmania antibodies and CD1a expression (clone EP3622) in a series of PCR confirmed cutaneous leishmaniasis. MATERIALS AND METHODS: Thirty-three skin samples corresponding to PCR confirmed cutaneous leishmaniasis were included in the study. All samples were stained with Hematoxylin-eosin and Giemsa. Moreover, immunohistochemical studies with anti-CD1a and anti-Leishmania antibodies were performed. The patients clinical features and the observed histopathological features were also recorded. RESULTS: From the selected 33 biopsies, Leishmania spp. amastigotes were detected in 48.4% of cases with conventional Hematoxylin-eosin stain and in 57.5% of cases by Giemsa staining. In 31/33 cases, anti-CD1a allowed us to identify parasitic structures, and in 33/33 cases amastigotes were detected with anti-Leishmania antibodies. Concordance between both techniques, anti-CD1a and anti-Leishmania, was 94% [CI 95%: (79,8%-99,3%)] ; p value <0.05. The sensitivity of anti-CD1a in comparison with the PCR was 94%, with a positive predictive value of 100%. Two cases of low parasitic index were negative for CD1a immunostaining. In cases with high parasitic index, anti-CD1a stained amastigotes in superficial and deep dermis. Only a few cases were originally diagnosed with the available histological techniques, needing PCR for Leishmania spp. CONCLUSIONS: Anti-CD1a antibody seems to be a useful technique to identify amastigotes when PCR and anti-Leishmania antibodies are not available. The sensitivity to detect amastigotes is increased when the CD1a immunostaining is added to the classical Haematoxylin - eosin and Giemsa staining.

Regulation of macrophage subsets and cytokine production in leishmaniasis.

Macrophages are host cells for parasites of the genus Leishmania where they multiply inside parasitophorous vacuoles. Paradoxically, macrophages are also the cells responsible for killing or controlling parasite growth, if appropriately activated. In this review, we will cover the patterns of macrophage activation and the mechanisms used by the parasite to circumvent being killed. We will highlight the impacts of the vector bite on macrophage activation. Finally, we will discuss the ontogeny of macrophages that are infected by Leishmania spp.

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice.

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to visceral lesions. Currently, 12 million people are estimated to be infected with Leishmania worldwide and over 1 billion people live at the risk of infection. Leishmania amazonensis is endemic in Central and South America and usually leads to the cutaneous form of the disease, which can be directly visualized in an animal model. Therefore, L. amazonensis strains are good models for cutaneous leishmaniasis studies because they are also easily cultivated in vitro. C57BL/6 mice mimic the L. amazonensis-driven disease progression observed in humans and are considered one of the best mice strains model for cutaneous leishmaniasis. In the vertebrate host, these parasites inhabit macrophages despite the defense mechanisms of these cells. Several studies use in vitro macrophage infection assays to evaluate the parasite infectivity under different conditions. However, the in vitro approach is limited to an isolated cell system that disregards the organism's response. Here, we compile an in vivo murine infection method that provides a systemic physiological overview of the host-parasite interaction. The detailed protocol for the in vivo infection of C57BL/6 mice with L. amazonensis comprises parasite differentiation into infective amastigotes, mice footpad cutaneous inoculation, lesion development, and parasite load determination. We propose this well-established method as the most adequate method for physiological studies of the host immune and metabolic responses to cutaneous leishmaniasis.

The First Non-LRV RNA Virus in Leishmania.

In this work, we describe the first Leishmania-infecting leishbunyavirus-the first virus other than Leishmania RNA virus (LRV) found in trypanosomatid parasites. Its host is Leishmaniamartiniquensis, a human pathogen causing infections with a wide range of manifestations from asymptomatic to severe visceral disease. This virus (LmarLBV1) possesses many characteristic features of leishbunyaviruses, such as tripartite organization of its RNA genome, with ORFs encoding RNA-dependent RNA polymerase, surface glycoprotein, and nucleoprotein on L, M, and S segments, respectively. Our phylogenetic analyses suggest that LmarLBV1 originated from leishbunyaviruses of monoxenous trypanosomatids and, probably, is a result of genomic re-assortment. The LmarLBV1 facilitates parasites' infectivity in vitro in primary murine macrophages model. The discovery of a virus in L.martiniquensis poses the question of whether it influences pathogenicity of this parasite in vivo, similarly to the LRV in other Leishmania species.

LAX28 is required for the stable assembly of the inner dynein arm f complex, and the tether and tether head complex in Leishmania flagella.

Motile eukaryotic flagella beat through coordinated activity of dynein motor proteins; however, the mechanisms of dynein coordination and regulation are incompletely understood. The inner dynein arm (IDA) f complex (also known as the I1 complex), and the tether and tether head (T/TH) complex are thought to be key regulators of dynein action but, unlike the IDA f complex, T/TH proteins remain poorly characterised. Here, we characterised T/TH-associated proteins in the protist Leishmania mexicana Proteome analysis of axonemes from null mutants for the CFAP44 T/TH protein showed that they lacked the IDA f protein IC140 and a novel 28-kDa axonemal protein, LAX28. Sequence analysis identified similarities between LAX28 and the uncharacterised human sperm tail protein TEX47, both sharing features with sensory BLUF-domain-containing proteins. Leishmania lacking LAX28, CFAP44 or IC140 retained some motility, albeit with reduced swimming speed and directionality and a propensity for flagellar curling. Expression of tagged proteins in different null mutant backgrounds showed that the axonemal localisation of LAX28 requires CFAP44 and IC140, and the axonemal localisations of CFAP44 and IC140 both depend on LAX28. These data demonstrate a role for LAX28 in motility and show mutual dependencies of IDA f and T/TH-associated proteins for axonemal assembly in Leishmania.

Mutations of key substrate binding residues of leishmanial peptidase T alter its functional and structural dynamics.

BACKGROUND: M20 aminopeptidases, such as Peptidase T (PepT), are implicated in the hydrolysis of oligopeptides during the terminal stages of protein degradation pathway to maintain turnover. Therefore, specific inhibition of PepT bores well for the development of novel next-generation antileishmanials. This work describes the metal dependence, substrate preferences and inhibition of PepT, and demonstrates in detail the role of its two conserved substrate binding residues. METHODS: PepT was purified and characterized using a scheme of peptide substrates and peptidomimetic inhibitors. Residues T364 and N378 were mutated and characterized with an array of biochemical, biophysical and structural biology methods. RESULTS: PepT sequence carries conserved motifs typical of M20 peptidases and our work on its biochemistry shows that this cytosolic enzyme carries broad substrate specificity with best cleavage preference for peptides carrying alanine at the P1 position. Peptidomimetics amastatin and actinonin occupied S1 pocket by competing with the substrate for binding to active site and inhibited PepT potently, while arphamenine A and bestatin were less effective inhibitors. We further show that the mutation of conserved substrate binding residues (T364 and N378) to alanine affects structure, reduces substrate binding and alters the amidolytic activity of this dimeric enzyme. CONCLUSIONS: PepT preferentially hydrolyzes oligopeptides carrying alanine at P1 position and is potently inhibited by peptidomimetics. Reduced substrate binding after mutations was a key factor involved in amidolytic digressions. GENERAL SIGNIFICANCE: This study provides insights for further exploration of the druggability of PepT and highlights prospective applications of this enzyme along with its mutazyme T364A/N378A.

Leishmania amazonensis resistance in murine macrophages: Analysis of possible mechanisms.

Leishmaniasis encompass a group of infectious parasitic diseases occurring in 97 endemic countries where over one billion people live in areas at risk of infection. It is in the World Health Organization list of neglected diseases and it is considered a serious public health problem, with more than 20,000 deaths a year and high morbidity. Infection by protozoa from the genus Leishmania can cause several forms of the disease, which may vary from a self-healing ulcer to fatal visceral infection. Leishmania species, as well as host immune response and genetics can modulate the course of the disease. Leishmania sp are obligatory intracellular parasites that have macrophages as their main host cell. Depending on the activation phenotype, these cells may have distinct roles in disease development, acting in parasite control or proliferation. Therefore, the purpose of this work was to analyze Leishmania amazonensis infection in primary macrophage cells obtained from mice with two distinct genetic backgrounds, ie. different susceptibility to the infection; evaluating the cause for that difference. After infection, peritoneal macrophages from the resistant C3H/He strain presented lower parasite load when compared to susceptible BALB/c macrophages. The same was also true when cells received a Th2 stimulus after infection, but the difference was abrogated under Th1 stimulus. Nitric oxide production and arginase activity was different between the strains under Th1 or Th2 stimulus, respectively, but iNOS inhibition was unable to suppress C3H/He resistance. Hydrogen peroxide production was also higher in C3H/He than BALB/c under Th1 stimulus, but it could not account for differences in susceptibility. These results led us to conclude that, although they have an important role in parasite control, neither NO nor H2O2 production can explain C3H/He resistance to infection. Other studies are needed to uncover different mechanisms of resistance/susceptibility to L. amazonensis.

LeishIF4E1 Deletion Affects the Promastigote Proteome, Morphology, and Infectivity.

Leishmania parasites cycle between sand-fly vectors and mammalian hosts, adapting to changing environmental conditions by driving a stage-specific program of gene expression, which is tightly regulated by translation processes. Leishmania encodes six eIF4E orthologs (LeishIF4Es) and five eIF4G candidates, forming different cap-binding complexes with potentially varying functions. Most LeishIF4E paralogs display temperature sensitivity in their cap-binding activity, except for LeishIF4E1, which maintains its cap-binding activity under all conditions. We used the CRISPR-Cas9 system to successfully generate a null mutant of LeishIF4E1 and examine how its elimination affected parasite physiology. Although the LeishIF4E1-/- null mutant was viable, its growth was impaired, in line with a reduction in global translation. As a result of the mutation, the null LeishIF4E1-/- mutant had a defective morphology, as the cells were round and unable to grow a normal flagellum. This was further emphasized when the LeishIF4E1-/- cells failed to develop the promastigote morphology once they shifted from conditions that generate axenic amastigotes (33°C, pH 5.5) back to neutral pH and 25°C, and they maintained their short flagellum and circular structure. Finally, the LeishIF4E1-/- null mutant displayed difficulty in infecting cultured macrophages. The morphological changes and reduced infectivity of the mutant may be related to differences in the proteomic profile of LeishIF4E1-/- cells from that of controls. All defects monitored in the LeishIF4E1-/- null mutant were reversed in the add-back strain, in which expression of LeishIF4E1 was reconstituted, establishing a strong link between the cellular defects and the absence of LeishIF4E1 expression.IMPORTANCELeishmania parasites are the causative agents of a broad spectrum of diseases. The parasites migrate between sand-fly vectors and mammalian hosts, adapting to changing environments by driving a regulated program of gene expression, with translation regulation playing a key role. The leishmanias encode six different paralogs of eIF4E, the cap-binding translation initiation factor. Since these vary in function, expression profile, and assemblage, it is assumed that each is assigned a specific role throughout the life cycle. Using the CRISPR-Cas9 system for Leishmania, we generated a null mutant of LeishIF4E1, eliminating both alleles. Although the mutant cells were viable, their morphology was altered and their ability to synthesize the flagellum was impaired. Elimination of LeishIF4E1 affected their protein expression profile and decreased their ability to infect cultured macrophages. Restoring LeishIF4E1 expression restored the affected features. This study highlights the importance of LeishIF4E1 in diverse cellular events during the life cycle of Leishmania.

Clinical and Pathological Aspects of Canine Cutaneous Leishmaniasis: A Meta-analysis.

PURPOSE: In the present study, we described the most prevalent clinical symptoms, the most affected organs, and the macro and microscopic lesions associated with cutaneous leishmaniasis. METHODS: Two independent researchers performed an extensive systematic review of the literature in four stages (identification, screening, eligibility, and inclusion) to identify studies published between January 2002 and November 2018 from the following electronic databases: Web of Science, PubMed, SciELO, Science Direct, and Google Scholar. Meta-analysis was conducted in "Metaprop" package of R 3.4.2 software. RESULTS: The electronic search yielded 3896 results, out of which 155 were further analyzed based on the full-text. Data extracted from 16 articles were included in the meta-analysis, representing a total of 430 leishmaniasis cases. Only 43% of all animals were identified to exhibit the clinical and cutaneous changes characteristic of leishmaniasis based on the observation that skin lesions were the most prevalent clinical sign and were present in 86% of all cases. Other less prevalent symptoms included weight loss, cachexia, apathy and lymph node enlargement. Histopathological analysis showed that the skin was the most affected organ, affecting 64% of cases, followed by lymph nodes (12%), spleen (8%) and liver (7%). CONCLUSIONS: Therefore, our current findings suggest that cutaneous leishmaniasis could lead to visceral disease. Notably, our findings indicated no clinical manifestation patterns in cutaneous leishmaniasis, since the same host species may present different clinical conditions.

Intracellular iron availability modulates the requirement for Leishmania Iron Regulator 1 (LIR1) during macrophage infections.

The Leishmania plasma membrane transporter Leishmania Iron Regulator 1 (LIR1) facilitates iron export and is required for parasite virulence. By modulating macrophage iron content, we investigated the host site where LIR1 regulates Leishmania amazonensis infectivity. In bone marrow-derived macrophages, LIR1 null mutants demonstrated a paradoxical increase in virulence during infections in heme-depleted media, while wild-type growth was inhibited under the same conditions. Loading the endocytic pathway of macrophages with cationized ferritin prior to infection reversed the effect of heme depletion on both strains. Thus, LIR1 contributes to Leishmania virulence by protecting the parasites from toxicity resulting from iron accumulation inside parasitophorous vacuoles.

Caffeic acid has antipromastigote activity by apoptosis-like process; and anti-amastigote by TNF-α/ROS/NO production and decreased of iron availability.

BACKGROUND: Leishmaniasis is a disease caused by protozoan parasites of the Leishmania genus whose current treatment has high cost, highly toxic, and difficult administration, which makes it very important to find alternative natural compounds of high efficiency and low cost. PURPOSE: This study assessed the in vitro effect of caffeic acid (CA) on promastigotes and L. amazonensis-infected macrophages. METHODS: Evaluation of the in vitro leishmanicidal activity of CA against promastigotes and L. amazonensis infected peritoneal macrophages, as well its microbicide mechanisms. RESULTS: CA 12.5-100 µg/ml were able to inhibit promastigotes proliferation at all tested periods. The IC50, 12.5 µg/ml, also altered promastigote cell morphology and cell volume accompanied by loss of mitochondrial integrity, increase in reactive oxygen species (ROS) production, phosphatidylserine exposure, and loss of plasma membrane integrity - characterizing the apoptosis-like process. Moreover, CA reduced the percentage of infected macrophages and the number of amastigotes per macrophages increasing TNF-α, ROS, NO and reducing IL-10 levels as well as iron availability. CONCLUSION: CA showed in vitro antipromastigote and antiamostigote by increasing oxidant and inflammatory response important to eliminate the parasite.

Leishmania Lipophosphoglycan Triggers Caspase-11 and the Non-canonical Activation of the NLRP3 Inflammasome.

Activation of the NLRP3 inflammasome by Leishmania parasites is critical for the outcome of leishmaniasis, a disease that affects millions of people worldwide. We investigate the mechanisms involved in NLRP3 activation and demonstrate that caspase-11 (CASP11) is activated in response to infection by Leishmania species and triggers the non-canonical activation of NLRP3. This process accounts for host resistance to infection in macrophages and in vivo. We identify the parasite membrane glycoconjugate lipophosphoglycan (LPG) as the molecule involved in CASP11 activation. Cytosolic delivery of LPG in macrophages triggers CASP11 activation, and infections performed with Lpg1-/- parasites reduce CASP11/NLRP3 activation. Unlike bacterial LPS, purified LPG does not activate mouse CASP11 (or human Casp4) in vitro, suggesting the participation of additional molecules for LPG-mediated CASP11 activation. Our data identify a parasite molecule involved in CASP11 activation, thereby establishing the mechanisms underlying inflammasome activation in response to Leishmania species.

Alterations induced in the ileum of mice upon inoculation with different species of Leishmania: a preliminary study

Abstract INTRODUCTION: Leishmania species cause skin, mucosal, and disseminated lesions. We studied the effects of three Leishmania species on ileal morphology in mice. METHODS: BALB/c mice were intraperitoneally inoculated with Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) major (4 animals/group). After 72h, the ilea were collected and histologically processed. RESULTS: Following inoculation, the goblet cell and intraepithelial lymphocyte populations increased, while Paneth cell number and crypt width decreased. In addition, enterocyte size, villi height, and mucosa, submucosa, and muscular tunic thickness increased. CONCLUSIONS: Leishmania modified the quantity of cells in and morphology of mice ilea.

The role of heparan sulfate in host macrophage infection by Leishmania species.

The leishmaniases are a group of neglected tropical diseases caused by parasites from the Leishmania genus. More than 20 Leishmania species are responsible for human disease, causing a broad spectrum of symptoms ranging from cutaneous lesions to a fatal visceral infection. There is no single safe and effective approach to treat these diseases and resistance to current anti-leishmanial drugs is emerging. New drug targets need to be identified and validated to generate novel treatments. Host heparan sulfates (HSs) are abundant, heterogeneous polysaccharides displayed on proteoglycans that bind various ligands, including cell surface proteins expressed on Leishmania promastigote and amastigote parasites. The fine chemical structure of HS is formed by a plethora of specific enzymes during biosynthesis, with various positions (N-, 2-O-, 6-O- and 3-O-) on the carbon sugar backbone modified with sulfate groups. Post-biosynthesis mechanisms can further modify the sulfation pattern or size of the polysaccharide, altering ligand affinity to moderate biological functions. Chemically modified heparins used to mimic the heterogeneous nature of HS influence the affinity of different Leishmania species, demonstrating the importance of specific HS chemical sequences in parasite interaction. However, the endogenous structures of host HSs that might interact with Leishmania parasites during host invasion have not been elucidated, nor has the role of HSs in host-parasite biology. Decoding the structure of HSs on target host cells will increase understanding of HS/parasite interactions in leishmaniasis, potentiating identification of new opportunities for the development of novel treatments.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites.

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite's ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/ΔLmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.