Variation in Leishmania chemokine suppression driven by diversification of the GP63 virulence factor.

Leishmaniasis is a neglected tropical disease with diverse outcomes ranging from self-healing lesions, to progressive non-healing lesions, to metastatic spread and destruction of mucous membranes. Although resolution of cutaneous leishmaniasis is a classic example of type-1 immunity leading to self-healing lesions, an excess of type-1 related inflammation can contribute to immunopathology and metastatic spread. Leishmania genetic diversity can contribute to variation in polarization and robustness of the immune response through differences in both pathogen sensing by the host and immune evasion by the parasite. In this study, we observed a difference in parasite chemokine suppression between the Leishmania (L.) subgenus and the Viannia (V.) subgenus, which is associated with severe immune-mediated pathology such as mucocutaneous leishmaniasis. While Leishmania (L.) subgenus parasites utilize the virulence factor and metalloprotease glycoprotein-63 (gp63) to suppress the type-1 associated host chemokine CXCL10, L. (V.) panamensis did not suppress CXCL10. To understand the molecular basis for the inter-species variation in chemokine suppression, we used in silico modeling to identify a putative CXCL10-binding site on GP63. The putative CXCL10 binding site is in a region of gp63 under significant positive selection, and it varies from the L. major wild-type sequence in all gp63 alleles identified in the L. (V.) panamensis reference genome. Mutating wild-type L. (L.) major gp63 to the L. (V.) panamensis sequence at the putative binding site impaired cleavage of CXCL10 but not a non-specific protease substrate. Notably, Viannia clinical isolates confirmed that L. (V.) panamensis primarily encodes non-CXCL10-cleaving gp63 alleles. In contrast, L. (V.) braziliensis has an intermediate level of activity, consistent with this species having more equal proportions of both alleles. Our results demonstrate how parasite genetic diversity can contribute to variation in immune responses to Leishmania spp. infection that may play critical roles in the outcome of infection.

Role of thrombopoiesis in leishmaniasis.

The blood vascular system of mammals is unique in nature; inhabited with a pool of tiny small cell fragments called platelets; attributed with the most important patrolling tasks to check integrity of the entire endothelial landscape. Their production is tightly coupled with hematopoietic system where everything starts from self renewable multipotent hematopoietic stem cells (HSCs) which eventually undergo dual step (megakaryopoiesis-thrombopoiesis) thrombocytes production. Several cytokines tune the fate of every progenitor cells during hematopoiesis through temporal activation of specific transcription factors. Though platelets generated through steady state hematopoiesis are involved in the regulation of vascular homeostasis, these cells can sense pathogens through its innate immune sensors and can mount crucial responses against the invading pathogen. For this, the primary aim of many infections including Leishmania is to induce thrombocytopenia within infected host. But the underlying mechanism of this induced thrombocytopenia in Leishmania infection has not been evaluated. Elucidation of these mechanisms will be fruitful to design new chemotherapeutic strategies.

In Search of Biomarkers for Pathogenesis and Control of Leishmaniasis by Global Analyses of Leishmania-Infected Macrophages.

Leishmaniasis is a vector-borne, neglected tropical disease with a worldwide distribution that can present in a variety of clinical forms, depending on the parasite species and host genetic background. The pathogenesis of this disease remains far from being elucidated because the involvement of a complex immune response orchestrated by host cells significantly affects the clinical outcome. Among these cells, macrophages are the main host cells, produce cytokines and chemokines, thereby triggering events that contribute to the mediation of the host immune response and, subsequently, to the establishment of infection or, alternatively, disease control. There has been relatively limited commercial interest in developing new pharmaceutical compounds to treat leishmaniasis. Moreover, advances in the understanding of the underlying biology of Leishmania spp. have not translated into the development of effective new chemotherapeutic compounds. As a result, biomarkers as surrogate disease endpoints present several potential advantages to be used in the identification of targets capable of facilitating therapeutic interventions considered to ameliorate disease outcome. More recently, large-scale genomic and proteomic analyses have allowed the identification and characterization of the pathways involved in the infection process in both parasites and the host, and these analyses have been shown to be more effective than studying individual molecules to elucidate disease pathogenesis. RNA-seq and proteomics are large-scale approaches that characterize genes or proteins in a given cell line, tissue, or organism to provide a global and more integrated view of the myriad biological processes that occur within a cell than focusing on an individual gene or protein. Bioinformatics provides us with the means to computationally analyze and integrate the large volumes of data generated by high-throughput sequencing approaches. The integration of genomic expression and proteomic data offers a rich multi-dimensional analysis, despite the inherent technical and statistical challenges. We propose that these types of global analyses facilitate the identification, among a large number of genes and proteins, those that hold potential as biomarkers. The present review focuses on large-scale studies that have identified and evaluated relevant biomarkers in macrophages in response to Leishmania infection.

Leishmania infection activates host mTOR for its survival by M2 macrophage polarization.

Mammalian target of rapamycin (mTOR) is a central regulator of growth and immunity of host cells. It's involvement in cancer and tuberculosis is well documented but least explored in Leishmania donovani invasion of host cells. Therefore, in the present study, we aimed to investigate the role of mTOR in M2 macrophage polarization for Leishmania survival. We observed that Leishmania infection activated host mTOR pathway characterized by phosphorylation of mTOR, 70S6K and 4-EBP1. Inhibition of mTOR resulted in decreased parasite load and percent infectivity. Moreover, Leishmania infection triggered cell proliferation as was evidenced by increased expression of cyclin A and p-RPS6. mTOR activation during Leishmania infection resulted in reduced expression of M1 macrophage markers (eg, ROS, NO, iNOS, NOX-1, IL-12, IL-1ß and TNF-α), and increased expression of M2 macrophage markers (eg, arginase-1, IL-10, TGF-ß, CD206 and CD163). Furthermore, we observed that in case of Leishmania infection, mTOR inhibition increased the translocation of NF-κB to nucleus and deactivation of STAT-3. Eventually, we observed that inhibition of M2 macrophage polarization reduced Leishmania survival inside macrophages. Therefore, our findings suggest that mTOR plays a crucial role in regulation of M2 macrophage polarization and direct the innate immune homeostasis towards parasite survival inside host.

Human leishmaniasis in Brazil: A general review / Leishmaniose humana no Brasil: uma revisão geral

Summary Leishmaniasis is a disease with ample clinical spectrum and epidemiological diversity and is considered a major public health problem. This article presents an overview of the transmission cycles, host-parasite interactions, clinical, histological and immunological aspects, diagnosis and treatment of various forms of the human disease.

Resumo A leishmaniose representa um complexo de doenças com amplo espectro clínico e diversidade epidemiológica, sendo considerada um grande problema de saúde pública. O presente artigo apresenta uma revisão geral sobre os ciclos de transmissão, as interações parasito-hospedeiro, os aspectos clínicos, histopatológicos e imunológicos, o diagnóstico e o tratamento das diversas formas da doença humana.

Treatment of canine leishmaniasis with marbofloxacin in dogs with renal disease.

Treatment of canine leishmaniasis (CanL) represents a challenge. Due to the high prevalence of renal disease associated to CanL, it is important to find an effective drug that does not damage the kidneys. Marbofloxacin has been shown to be effective and well tolerated in non-azotemic dogs with leishmaniasis. To evaluate the safety and efficacy of marbofloxacin in dogs with leishmaniasis and decreased renal function, 28 dogs suffering from leishmaniasis and chronic kidney disease (CKD) were treated with oral marbofloxacin at 2 mg/Kg/day for 28 days. During treatment dogs were assessed by performing weekly physical exams, measuring blood pressure and evaluating blood and urine parameters. Lymph node aspirations were also obtained at days 0 and 28. The global clinical score decreased significantly, from 6.2±3.4 to 4.7±3.1 (p = 0.0001), after treatment. Marbofloxacin also decreased parasitic load in 72% of the dogs. No significant differences in plasma creatinine, urine specific gravity, urinary concentrations of cystatin C, ferritin and urinary protein loss were detected during treatment. A transient but significant decrease in blood pressure was detected up to day 14 (from 180.1±36.6 to 166.0±32.7 mmHg; p = 0.016). Moreover, dogs showed a significant increase in plasma albumin concentration (from 15.0±5.2 to 16.6±3.9 g/L; p = 0.014) and a significant decrease in globulin concentration (from 59.0±18.1 to 54.1±18.0 g/L; p = 0.005). The results demonstrate that, in addition to being effective for treatment of CanL, marbofloxacin is a very safe drug in dogs with CKD and leishmaniasis.

Cardiac manifestations of parasitic diseases.

The heart may be affected directly or indirectly by a variety of protozoa and helminths. This involvement may manifest in different ways, but the syndromes resulting from impairment of the myocardium and pericardium are the most frequent. The myocardium may be invaded by parasites that trigger local inflammatory response with subsequent myocarditis or cardiomyopathy, as occurs in Chagas disease, African trypanosomiasis, toxoplasmosis, trichinellosis and infection with free-living amoebae. In amoebiasis and echinococcosis, the pericardium is the structure most frequently involved with consequent pericardial effusion, acute pericarditis, cardiac tamponade or constrictive pericarditis. Chronic hypereosinophilia due to helminth infections, especially filarial infections, has been associated with the development of tropical endomyocardial fibrosis, a severe form of restrictive cardiomyopathy. Schistosomiasis-associated lung vasculature involvement may cause pulmonary hypertension (PH) and cor pulmonale Tropical pulmonary eosinophilia, which is characterised by progressive interstitial fibrosis and restrictive lung disease, may lead to PH and its consequences may occur in the course of filarial infections. Intracardiac rupture of an Echinococcus cyst can cause membrane or secondary cysts embolisation to the lungs or organs supplied by the systemic circulation. Although unusual causes of cardiac disease outside the endemic areas, heart involvement by parasites should be considered in the differential diagnosis especially of myocardial and/or pericardial diseases of unknown aetiology in both immunocompetent and immunocompromised individuals. In this review, we updated and summarised the current knowledge on the major heart diseases caused by protozoan and metazoan parasites, which either involve the heart directly or otherwise influence the heart adversely.

Laryngeal leishmaniasis in a patient taking inhaled corticosteroids.

We present a case of a man in his late 60s, who had spent 3-4 months of the year in rural Spain, presenting with intermittent hoarseness of voice. He had a background of asthma and bronchiectasis, and was taking inhaled corticosteroids. His dysphonia was initially managed as bronchiectasis with little improvement. Bronchoscopy revealed a cystic lesion on his left vocal fold, and tissue biopsy revealed Leishmania amastigotes. This confirmed a diagnosis of laryngeal leishmaniasis. We propose that this is likely secondary to his inhaled corticosteroid therapy. The infection was treated with a 30-day course of miltefosine, and at most recent follow-up the patient was deemed free from leishmanial infection.

Iron acquisition in Leishmania and its crucial role in infection.

Iron is an essential cofactor for many basic metabolic pathways in pathogenic microbes and their hosts. It is also dangerous as it can catalyse the production of reactive free radicals. This dual character makes the host can either limit iron availability to invading microbes or exploit iron to induce toxicity to pathogens. Successful pathogens, including Leishmania species, must possess mechanisms to circumvent host's iron limitation and iron-induced toxicity in order to survive. In this review, we discuss the regulation of iron metabolism in the setting of infection and delineate the iron acquisition strategies used by Leishmania parasites and their subversions to host iron metabolism to overcome host's iron-related defences.

Peripheral blood fibrocytes: new information to explain the dynamics of Leishmania infection

Fibrocytes are important for understanding the progression of many diseases because they are present in areas where pathogenic lesions are generated. However, the morphology of fibrocytes and their interactions with parasites are poorly understood. In this study, we examined the morphology of peripheral blood fibrocytes and their interactions with Leishmania (L.) amazonensis . Through ultrastructural analysis, we describe the details of fibrocyte morphology and how fibrocytes rapidly internalise Leishmania promastigotes. The parasites differentiated into amastigotes after 2 h in phagolysosomes and the infection was completely resolved after 72 h. Early in the infection, we found increased nitric oxide production and large lysosomes with electron-dense material. These factors may regulate the proliferation and death of the parasites. Because fibrocytes are present at the infection site and are directly involved in developing cutaneous leishmaniasis, they are targets for effective, non-toxic cell-based therapies that control and treat leishmaniasis.

Diseño, síntesis y evaluación biológica de un grupo de pirazinoisoquinolinas y quinolinas con posible actividad esquistosomicida y leishmanicida respectivamente / Design, synthesis and biological evaluation of a group of pyrazinoisoquinolines and quinolines with possible schistosomicidal and leishmanicidal activity respectively

La esquistosomiasis y la leishmaniasis son dos parasitosis con una alta incidencia en el mundo y con la menor cantidad de medicamentos disponibles para sus tratamientos. Para la esquistosomiasis, el praziquantel (PZQ) es la única droga que existe en los actuales momentos contra la enfermedad, mientras que, en el caso de la leishmaniasis, los antimoniales pentavalentes, empleados como drogas de primera línea, son altamente tóxicos o presentan problemas de resistencia. Por ello, esta tesis describe el diseño, la síntesis y los estudios de actividad biológica de un grupo de pirazinoisoquinolinas y quinolinas sustituidas con posible actividad esquistosomicida y leishmanicida respectivamente. Luego de ensayar diversas vías, se logró la síntesis del PZQ y compuestos relacionados (quince compuestos) mediante una secuencia de cinco pasos, con rendimientos entre el 20% y el 60%. El PZQ se obtuvo en un 33%, con un exceso del enantiómero levo, mostrando ser más activo que el PZQ comercial (mezcla racémica). Los compuestos obtenidos, evaluados en cepas de S. mansoni, no mostraron ser más activos que el PZQ, a las dos concentraciones evaluadas. También, se cuantificó la relación entre la estructura química y la actividad biológica (QSAR) de derivados de PZQ reportados en la literatura. En cuanto a los compuestos con posible actividad leishmanicida, también se ensayaron varios métodos de síntesis hasta lograr obtener veintidós quinolínas de los tipos 2-metil, 2-propil, 4-metil-2-propil y 2-alquildiamino con rendimientos entre un 10 y un 70%. Los compuestos evaluados que mostraron una actividad prometedora en promastigotes de L. mexicanafueron la 2-metilquinolina y 6,7-metilendioxi-2-propil-quinolina. En cuanto a los estudios QSAR, no fue posible encontrar una ecuación representativa que relacionara la actividad biológica con la estructura química.

Schistosomiasis and leishmaniasis are two parasitic diseaseswell spread in the world, and at the same time both have very few medications for their treatment. For schistosomiasis, praziquantel (PZQ) is the drug of choice for its treatment, while in the case of leishmaniasis, the pentavalent antimonials used as first line drugs are highly toxic or present resistance problems. This work describes the design, synthesis and biological activity studies of a group of pirazinoisoquinolines and substituted quinolines with a possible schistosomicidal and leishmanicidal activities, respectively. After several intents, it was possible to synthesize PZQ and some related compounds (fifteen) through a sequence of five steps, with yields between 20 and 60%. PZQ was obtained with a yield of 33%, with a levoenantiomeric excess, and showed a better activity than the commercial compound. The related compounds obtained, evaluated against S. mansonistrains did not have an activity comparable to that of PZQ, at the concentrations evaluated. Quantitative structure­activity relationships (QSAR)studieswere also performed with PZQ derivatives reported in the literature.Several ways of synthesis were also probed for the possible leishmanicidal compounds proposed, until it was possible to obtain twenty two quinoline derivatives (of the type 2-methyl-, 2-propyl-, 4-methyl-2-propyl-and 2-alquildiamino-), with yields between10 and 70%. Of the compounds evaluated, two showed promising activity against L. mexicana promastigotes, 2-iimethylquinoline and 6,7-methylendioxi-2-propyl-quinoline. QSAR studies with these compounds did not yield a representative model for the set.

Identification of key mechanisms controlling gene expression in Leishmania infected macrophages using genome-wide promoter analysis.

The present study describes the in silico prediction of the regulatory network of Leishmania infected human macrophages. The construction of the gene regulatory network requires the identification of Transcription Factor Binding Sites (TFBSs) in the regulatory regions (promoters, enhancers) of genes that are regulated upon Leishmania infection. The promoters of human, mouse, rat, dog and chimpanzee genes were first identified in the whole genomes using available experimental data on full length cDNA sequences or deep CAGE tag data (DBTSS, FANTOM3, FANTOM4), mRNA models (ENSEMBL), or using hand annotated data (EPD, TRANSFAC). A phylogenetic footprinting analysis and a MATCH analysis of the promoter sequences were then performed to predict TFBS. Then, an SQL database that integrates all results of promoter analysis as well as other genome annotation information obtained from ENSEMBL, TRANSFAC, TRED (Transcription Regulatory Element Database), ORegAnno and the ENCODE project, was established. Finally publicly available expression data from human Leishmania infected macrophages were analyzed using the genome-wide information on predicted TFBS with the computer system ExPlain™. The gene regulatory network was constructed and activated signal transduction pathways were revealed. The Irak1 pathway was identified as a key pathway regulating gene expression changes in Leishmania infected macrophages.

Leishmaniasis / Leishmaniasis

La leishmaniasis se considera una enferemedad zoonótica que puede afectar al hombre al entrar en contacto con el ciclo de transmisión del parásito, convirtiéndose en una antropozoonosis. En la actualidad, es una de las seis endemias consideradas prioridad en el mundo. Las zonas de mayor prevalencia de leishmaniasis en paraguay, corresponden a los nuevos asentamientos poblacionales, especialmente de los departamentos de canindeyú y San Pedro, en ñareas rurales boscosas de tierra poco explotadas producto del avance de la frontera agrícola. El objetivo de este trabajo es brindar información actualizada y completa de la epidemiología, etiología, ciclo biológico, reservorios, histopatología, diagnostico, clínica y tratamiento. Con el propósito de que sirva al profesional de la salud como material de lectura acerca estas zoonosis tan freceuntes en nuestro país, de manera que se logre diagnosticar precozmente al paciente, y así sentar base al tratamiento adecuado.

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.

Leishmania GP63 alters host signaling through cleavage-activated protein tyrosine phosphatases.

With more than 12 million people affected worldwide, 2 million new cases occurring per year, and the rapid emergence of drug resistance and treatment failure, leishmaniasis is an infectious disease for which research on drug and vaccine development, host-pathogen, and vector-parasite interactions are current international priorities. Upon Leishmania-macrophage interaction, activation of the protein tyrosine phosphatase (PTP) SHP-1 rapidly leads to the down-regulation of Janus kinase and mitogen-activated protein kinase signaling, resulting in the attenuation of host innate inflammatory responses and of various microbicidal macrophage functions. We report that, in addition to SHP-1, the PTPs PTP1B and TCPTP are activated and posttranslationally modified in infected macrophages, and we identify an essential role for PTP1B in the in vivo progression of Leishmania infection. The mechanism underlying PTP modulation involves the proteolytic activity of the Leishmania surface protease GP63. Access of GP63 to macrophage PTP1B, TCPTP, and SHP-1 is mediated in part by a lipid raft-dependent mechanism, resulting in PTP cleavage and stimulation of phosphatase activity. Collectively, our data present a mechanism of cleavage-dependent activation of macrophage PTPs by an obligate intracellular pathogen and show that internalization of GP63, a key Leishmania virulence factor, into host macrophages is a strategy the parasite uses to interact and survive within its host.

[PCR-RFLP and RAPD for typing neotropical Leishmania]. / PCR-RFLP y RAPD para la tipificación de Leishmania neotropical.

INTRODUCTION: The analysis of the PCR-restriction fragment length polymorphism and random amplified polymorphic DNA have been useful tools for Leishmania identification. OBJECTIVES: Molecular procedures were demonstrated for identification and typing of reference strains of New World Leishmania and their applicability was validated for clinical samples. MATERIALS AND METHODS: DNA was extracted from 16 reference strains of Latin American Leishmania as well as from clinical samples of leishmaniasis patients. A sequence coding for cysteine proteinase B was amplified by PCR and subjected to restriction fragment length polymorphism analysis. The enzyme used was Taq1. For eight of the reference strains, the random amplified polymorphic desoxyribonucleic acid technique (RAPD) was applied. Band patterns for Leishmania species differentiation were established each each method. The sample size of the clinical sample was of 5. RESULTS: PCR products of the cysteine proteinase B gene were obtained for L. braziliensis, L. peruviana, L. panamensis and L. guyanensis. For the other species, L. mexicana, L. amazonensis, L. garnhami, L. lainsoni, L. chagasi, L. naiffi, no amplification occurred. The patterns of restriction fragments revealed band patterns in common for L. peruviana, L. guyanensis and L. panamensis, whereas L. braziliensis had a distinctive pattern. When human samples were examined, amplification occurred for all cases, and the profiles corresponded to the common profile of L. peruviana, L. guyanensis and L. panamensis. The RAPD technique demonstrated reproducible and distinctive patterns for each of the 8 reference strains, L. mexicana, L. amazonensis, L. garnhami, L. lainsoni, L. chagasi, L. naiffi, making possible to differentiate all them. The advantages and limitations of each procedure are discussed. CONCLUSIONS: The combination of RFP and RAPD methodologies provide useful tools to identify medical important species of Leishmania by recognizing DNA sequences characteristic of each species.

Immunization with H1, HASPB1 and MML Leishmania proteins in a vaccine trial against experimental canine leishmaniasis.

The protective capabilities of three Leishmania recombinant proteins - histone 1 (H1) and hydrophilic acylated surface protein B1 (HASPB1) immunized singly, or together as a protein cocktail vaccine with Montanide, and the polyprotein MML immunized with MPL-SE adjuvant - were assessed in beagle dogs. Clinical examination of the dogs was carried out periodically under blinded conditions and the condition of the dogs defined as asymptomatic or symptomatic. At the end of the trial, we were able to confirm that following infection with L. infantum promastigotes, five out of eight dogs immunized with H1 Montanide, and four out of eight dogs immunized with either the combination of HASPB1 with Montanide or the combination of H1+HASPB1 with Montanidetrade mark, remained free of clinical signs, compared with two out of seven dogs immunized with the polyprotein MML and adjuvant MPL-SE, and two out of eight dogs in the control group. The results demonstrate that HASPB1 and H1 antigens in combination with Montanide were able to induce partial protection against canine leishmaniasis, even under extreme experimental challenge conditions.

Surface determinants of Leishmania parasites and their role in infectivity in the mammalian host.

Leishmania are intracellular protozoan parasites that reside primarily in host mononuclear phagocytes. Infection of host macrophages is initiated by infective promastigote stages and perpetuated by an obligate intracellular amastigote stage. Studies undertaken over the last decade have shown that the composition of the complex surface glycocalyx of these stages (comprising lipophosphoglycan, GPI-anchored glycoproteins, proteophosphoglycans and free GPI glycolipids) changes dramatically as promastigotes differentiate into amastigotes. Marked stage-specific changes also occur in the expression of other plasma membrane components, including type-1, polytopic and peripheral membrane proteins, reflecting the distinct microbicidal responses and nutritional environments encountered by these stages. More recently, a number of Leishmania mutants lacking single or multiple surface components have been generated. While some of these mutants are less virulent than wild type parasites, many of these mutants exhibit only mild or no loss of virulence. These studies suggest that, 1) the major surface glycocalyx components of the promastigote stage (i.e. LPG, GPI-anchored proteins) only have a transient or minor role in macrophage invasion, 2) that there is considerable functional redundancy in the surface glycocalyx and/or loss of some components can be compensated for by the acquisition of equivalent host glycolipids, 3) the expression of specific nutrient transporters is essential for life in the macrophage and 4) the role(s) of some surface components differ markedly in different Leishmania species. These mutants will be useful for identifying other surface or intracellular components that are required for virulence in macrophages.

Evaluation of adenosine 5'-diphosphate (ADP)- and collagen-induced platelet aggregation in canine leishmaniasis.

Leishmania-infected dogs, which represent an important reservoir of infection in many parts of the world, frequently suffer from haematological disorders, including thrombocytopenia. In this study, the ability of platelets from healthy (control) dogs (n = 11) and from dogs with naturally acquired clinical leishmaniasis (n = 24) to aggregate in the presence of two different agonists (adenosine 5'-diphosphate [ADP] and collagen) was assayed. Haematological parameters examined consisted of the platelet count, prothrombin time, activated partial thromboplastin time (aPTT), fibrinogen concentration and D-dimer concentration. In dogs with leishmaniasis, a significant decrease in ADP- and collagen-induced platelet aggregation was observed. Compared with platelets from the control dogs, those from leishmania-infected dogs showed a higher sensitivity to collagen, as demonstrated by a reduction in platelet aggregation of up to 20.4%, and a significant (P < 0.0001) difference for all the doses tested. With ADP the reduction was up to 10.4%, the difference reaching a significant level of P < 0.0001 only at the maximum dose used. The nature of this response, which was not accompanied by any clinical signs of bleeding other than an increase in aPTT, emphasizes the role of platelets in the parasite-host cell interaction.

Both the Fas ligand and inducible nitric oxide synthase are needed for control of parasite replication within lesions in mice infected with Leishmania major whereas the contribution of tumor necrosis factor is minimal.

Following infection with the protozoan parasite Leishmania major, C57BL/6 mice develop a small lesion that heals spontaneously. Resistance to infection is associated with the development of CD4(+) Th1 cells producing gamma interferon (IFN-gamma) and tumor necrosis factor (TNF), which synergize in activating macrophages to their microbicidal state. We show here that C57BL/6 mice lacking both TNF and Fas ligand (FasL) (gld TNF(-/-) mice) infected with L. major neither resolved their lesions nor controlled Leishmania replication despite the development of a strong Th1 response. Comparable inducible nitric oxide synthase (iNOS) activities were detected in lesions of TNF(-/-), gld TNF(-/-), and gld mice, but only gld and gld TNF(-/-) mice failed to control parasite replication. Parasite numbers were high in gld mice and even more elevated in gld TNF(-/-) mice, suggesting that, in addition to iNOS, the Fas/FasL pathway is required for successful control of parasite replication and that TNF contributes only a small part to this process. Furthermore, FasL was shown to synergize with IFN-gamma for the induction of leishmanicidal activity within macrophages infected with L. major in vitro. Interestingly, TNF(-/-) mice maintained large lesion size throughout infection, despite being able to largely control parasite numbers. Thus, IFN-gamma, FasL, and iNOS appear to be essential for the complete control of parasite replication, while the contribution of TNF is more important in controlling inflammation at the site of parasite inoculation.