A Multi-Species Phenotypic Screening Assay for Leishmaniasis Drug Discovery Shows That Active Compounds Display a High Degree of Species-Specificity.

High genetic and phenotypic variability between Leishmania species and strains within species make the development of broad-spectrum antileishmanial drugs challenging. Thus, screening panels consisting of several diverse Leishmania species can be useful in enabling compound prioritization based on their spectrum of activity. In this study, a robust and reproducible high content assay was developed, and 1280 small molecules were simultaneously screened against clinically relevant cutaneous and visceral species: L. amazonensis, L. braziliensis, and L. donovani. The assay is based on THP-1 macrophages infected with stationary phase promastigotes and posterior evaluation of both compound antileishmanial activity and host cell toxicity. The profile of compound activity was species-specific, and out of 51 active compounds, only 14 presented broad-spectrum activity against the three species, with activities ranging from 52% to 100%. Notably, the compounds CB1954, Clomipramine, Maprotiline, Protriptyline, and ML-9 presented pan-leishmanial activity, with efficacy greater than 70%. The results highlight the reduced number of compound classes with pan-leishmanial activity that might be available from diversity libraries, emphasizing the need to screen active compounds against a panel of species and strains. The assay reported here can be adapted to virtually any Leishmania species without the need for genetic modification of parasites, providing the basis for the discovery of broad spectrum anti-leishmanial agents.

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.

Conventional Versus Natural Alternative Treatments for Leishmaniasis: A Review.

Leishmaniasis is a neglected disease caused by protozoan belonging to the Leishmania genus. There are at least 16 pathogenic species for humans that are able to cause different clinical forms, such as cutaneous or visceral leishmaniasis. In spite of the different species and clinical forms, the treatment is performed with few drug options that, in most cases, are considered outdated. In addition, patients under classical treatment show serious side effects during drug administration, moreover parasites are able to become resistant to medicines. Thus, it is believed and well accepted that is urgent and necessary to develop new therapeutic options to overpass these concerns about conventional therapy of leishmaniasis. The present review will focus on the efficacy, side effects and action mechanism of classic drugs used in the treatment of leishmaniasis, as well as the importance of traditional knowledge for directing a rational search toward the discovery and characterization of new and effective molecules (in vivo assays) from plants to be used against leishmaniasis.

Calophyllum brasiliense Modulates the Immune Response and Promotes Leishmania amazonensis Intracellular Death.

Calophyllum brasiliense is a plant from the Brazilian rain forests and has been used in folk medicine for the treatment of various diseases, including leishmaniasis. This infectious disease depends on the Leishmania sp. and the host immune response. C. brasiliense antileishmanial activity is well known, but the effects on immune response remain to be investigated. This study showed the leishmanicidal and immunomodulatory effects of a 30 µg/mL of hydroalcoholic extract of C. brasiliense in murine macrophages before and after Leishmania (Leishmania) amazonensis infection. The semiquantitative cytokine RNA expression was determined by RT-PCR and the anti-Leishmania activity was measured by infection index (IF). Hydroalcoholic extract of C. brasiliense reduced more than 95% of IF when used before and after Leishmania infection, with 3 and 24 h of treatment (p < 0.05). C. brasiliense inhibited or reduced significantly (p < 0.05) the TNF-α, IL-1ß, IL-18, and IL-10 mRNA expression. The antileishmanial and anti-inflammatory effects showed the potential of C. brasiliense as an alternative therapy for leishmaniasis and it must be investigated.

Melatonin attenuates Leishmania (L.) amazonensis infection by modulating arginine metabolism.

Acute inflammatory responses induced by bacteria or fungi block nocturnal melatonin synthesis by rodent pineal glands. Here, we show Leishmania infection does not impair daily melatonin rhythm in hamsters. Remarkably, the attenuated parasite burden and lesion progression in hamsters infected at nighttime was impaired by blockage of melatonin receptors with luzindole, whereas melatonin treatment during the light phase attenuated Leishmania infection. In vitro studies corroborated in vivo observations. Melatonin treatment reduced macrophage expression of Cat-2b, Cat1, and ArgI, genes involved in arginine uptake and polyamine synthesis. Indeed, melatonin reduced macrophage arginine uptake by 40%. Putrescine supplementation reverted the attenuation of infectivity by melatonin indicating that its effect was due to the arrest of parasite replication. This study shows that the Leishmania/host interaction varies in a circadian manner according to nocturnal melatonin pineal synthesis. Our results provide new data regarding Leishmania infectiveness and show new approaches for applying agonists of melatonin receptors in Leishmaniasis therapy.

New heterocyclic hybrids of pyrazole and its bioisosteres: design, synthesis and biological evaluation as dual acting antimalarial-antileishmanial agents.

A new series of pyrazole derivatives were synthesized by hybridization with five-membered heterocyclic moieties such as thiazoles, thiazolidinones, 1,3,4-thiadiazoles and pyrazolines. The compounds were evaluated for their in vivo antimalarial activity against Plasmodium berghei infected mice and the most active derivatives were further examined for their in vitro antimalarial activity against chloroquine resistant (RKL9) strain of Plasmodium falciparum. Compounds 2c, 2d, 4b, 4c, 4d, 5a, 6c, 8c and 9b had more than 90% parasite suppression activity of that found with the antimalarial reference standard drug, chloroquine phosphate and had lower IC50 values than chloroquine. Compounds 4b and 9b were the most active derivatives, and their activities were 5-fold higher than chloroquine. All the newly synthesized compounds were evaluated for their in vitro antileishmanial activity against Leishmania aethiopica promastigotes and amastigote. The results showed that compounds 2c, 2d, 3d, 4b, 4c, 4d and 5a had lower or similar IC50 values than the reference standard drugs, amphotericin B and miltefosine. Compound 3d had the highest antileishmanial activity. Collectively, compounds 2c, 2d, 4b, 4c, 4d and 5a exhibited dual activity against malaria and leishmaniasis and were safe and well tolerated by the experimental animals orally up to 300 mg/kg and parenterally up to 100 mg/kg.

Avaliação do papel do ferro e de proteínas envolvidas em seu metabolismo na infecção in vitro de macrófagos por Leishmania amazonenses ou Leishmania major / Iron paper and evaluation of proteins involved in the metabolism of macrophages in vitro infection by Leishmania amazonenses or Leishmania major

A leishmaniose é uma antropozoonose causada por protozoários do gênero Leishmania e é considerada uma das principais doenças negligenciadas. Modelos experimentais são amplamente utilizados para uma melhor compreensão da doença e dos mecanismos relacionados à resistência e susceptibilidade à infecção. Macrófagos de camundongos CBA controlam a infecção por Leishmania major ao passo que são permissivos a Leishmania amazonensis. Além disso, estudos baseados em abordagem proteômica demonstraram padrões distintos de expressão proteica em macrófagos derivados de medula óssea (BMMΦ) infectados por essas espécies de Leishmania. Dentre as proteínas diferentemente expressas, foram identificadas proteínas envolvidas no metabolismo de ferro moduladas positivamente em macrófagos infectados por L. amazonensis. Adicionalmente, embora ainda existam controvérsias, diversos estudos têm abordado a participação do elemento ferro na interação parasito-hospedeiro e no estabelecimento das infecções por tripanossomatídeos, incluindo Leishmania. Assim, para melhor compreender os mecanismos envolvidos nessa doença, o presente estudo buscou explorar o modelo comparativo de resistência e suscetibilidade do camundongo CBA para determinar o papel do ferro na infecção por Leishmania. Nossa hipótese é que a expressão de proteínas envolvidas no metabolismo de ferro é modulada diferentemente em macrófagos de camundongos CBA infectados por L. amazonensis, em comparação à L. major, favorecendo a sobrevivência intracelular do parasito. Nosso objetivo foi avaliar a expressão de proteínas que participam do metabolismo de ferro, como receptor de transferrina (Tf), CD71, e heme oxigenasse-1, HO-1, e determinar o efeito da modulação da disponibilidade de ferro na infecção por Leishmania. Observamos maior expressão de HO-1 em BMMΦ infectados por L. amazonensis (18,34 ± SD ng/mL), quando comparados a BMMΦ infectados por L. major (7,07 ± SD ng/mL), utilizando ELISA. Maior expressão de CD71 também foi observada na infecção por L. amazonensis (MFI 2.103) em comparação à infecção por L. major (MFI 472), utilizando FACS, além de uma maior ligação e captação de HoloTf (Tf carregada com ferro). Embora tenha sido observado que essas proteínas encontram-se diferentemente expressas em BMMΦ infectados por essas duas espécies de Leishmania, não foram observadas diferenças significativas na concentração intracelular do ferro. Em seguida, ensaios funcionais a partir da modulação da disponibilidade intracelular de ferro foram realizados com o objetivo de avaliar seu papel no desfecho da infecção por Leishmania. Os resultados mostraram que a depleção de ferro reduz em 90% o percentual de BMMΦ infectados por L. amazonensis e 70% dos infectados por L. major...

Leishmaniasis is an anthropozoonosis caused by the protozoan parasite Leishmania and is considered one of the main neglected diseases. Animal models are widely used to better understand the disease and the mechanisms involved in resistance and susceptibility to infection. CBA mouse macrophages control the infection by L. major, while are permissive to L. amazonensis. Proteomic studies showed different protein profiles in bone marrow macrophages (BMMΦ) infected these species of Leishmania. We also observed that proteins involved in iron metabolism were positively modulated in L. amazonensis-infected macrophages. In addition, although literature review showed controverse data, several studies have addressed the role iron plays in host-parasite interaction and the establishment of trypanosomatids infections, including Leishmania. To better understand the mechanisms of the disease, this study sought to evaluate in a comparative model of resistance and susceptibility, using CBA macrophages, the role iron plays in Leishmania infection. Our hypothesis is that the expression of proteins involved in iron metabolism is differently modulated in CBA mice macrophages infected with L. amazonensis in comparison to L. major, favoring the intracellular survival of the parasite. Our goal was to evaluate the expression of proteins involved in iron metabolism of CBA mice macrophages, such as transferrin receptor (Tf), CD71, and heme oxygenase 1 (HO-1) and determine the effect of the modulation of intracellular iron in Leishmania infection. Using ELISA, we confirmed a higher expression of HO-1 in L. amazonensis- (18.34 ng/mL) compared to L. major-infected CBA macrophages (7.07 ng/mL). Using FACS analysis, CD71 showed to be higher expressed in L. amazonensis- (MFI 2.103) than in L. major-infected macrophages (MFI 472), in addition to higher binding and take up of HoloTf in these cells. Although it has been observed that proteins involved in iron metabolism were differently expressed in BMMΦ infected with these Leishmania species, no significant differences were observed in intracellular iron concentration. To further evaluate the role iron plays in the outcome of Leishmania infection, we modulated iron availability to Leishmania-infected cells using iron chelates or iron supplements. The results show that iron depletion reduces in 90% L. amazonensis infection and in 70% L. major infection. In addition, iron supplementation increased the percentage of L. amazonensis-infected cells from 69.64 to 82.79% and parasite load from 2,996 to 4,001 Leishmania/cell, as well as in the intracellular viability of both Leishmania species. In sum, these data indicate that although there is a positive modulation...

High content analysis of primary macrophages hosting proliferating Leishmania amastigotes: application to anti-leishmanial drug discovery.

BACKGROUND/OBJECTIVES: Human leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a biologically sound High Content Analysis assay, based on the use of homogeneous populations of primary mouse macrophages hosting Leishmania amazonensis amastigotes. In contrast to classical promastigote-based screens, our assay more closely mimics the environment where intracellular amastigotes are growing within acidic parasitophorous vacuoles of their host cells. This multi-parametric assay provides quantitative data that accurately monitors the parasitic load of amastigotes-hosting macrophage cultures for the discovery of leishmanicidal compounds, but also their potential toxic effect on host macrophages. We validated our approach by using a small set of compounds of leishmanicidal drugs and recently published chemical entities. Based on their intramacrophagic leishmanicidal activity and their toxicity against host cells, compounds were classified as irrelevant or relevant for entering the next step in the drug discovery pipeline. CONCLUSIONS/SIGNIFICANCE: Our assay represents a new screening platform that overcomes several limitations in anti-leishmanial drug discovery. First, the ability to detect toxicity on primary macrophages allows for discovery of compounds able to cross the membranes of macrophage, vacuole and amastigote, thereby accelerating the hit to lead development process for compounds selectively targeting intracellular parasites. Second, our assay allows discovery of anti-leishmanials that interfere with biological functions of the macrophage required for parasite development and growth, such as organelle trafficking/acidification or production of microbicidal effectors. These data thus validate a novel phenotypic screening assay using virulent Leishmania amastigotes growing inside primary macrophage to identify new chemical entities with bona fide drug potential.

Efficacy of miltefosine treatment in Leishmania amazonensis-infected BALB/c mice.

Leishmaniasis is one of the most serious worldwide diseases caused by protozoan parasites of the Leishmania genus, affecting millions of people around the world. All currently available treatments present severe toxic side effects, require long-term compliance, cause serious side effects and are uncomfortable for patients. Leishmania amazonensis, a species endemic to Brazil, causes severe localised or diffuse skin lesions in humans. Owing to the unsatisfactory nature of the currently available chemotherapies, new approaches have been assessed for improved therapeutic intervention strategies against leishmaniasis. Miltefosine is an alkylphospholipid analogue that exhibits potent activity against the different clinical manifestations of leishmaniasis. Thus, the aim of this study was to investigate the long-term efficacy of miltefosine in BALB/c mice infected with L. amazonensis owing to the lack of a profound study demonstrating its dose-dependent and long-term effects. It was observed that animals treated with 20-50 mg/kg/day of miltefosine exhibited a significant dose-dependent reduction in lesion size; furthermore, in mice receiving higher doses, lesions disappeared after the end of treatment. To confirm a possible parasitological cure, mice up to 250 days after the end of treatment were analysed. No lesions or presence of parasite DNA were found in mice treated with 30, 40 and 50 mg/kg/day of miltefosine. In summary, these results show that miltefosine may be used to treat cutaneous leishmaniasis caused by L. amazonensis, alone or as combination therapy.

Structure/antileishmanial activity relationship study of naphthoquinones and dependency of the mode of action on the substitution patterns.

A series of naphthoquinones was tested for activity against both extracellular promastigote and intracellular amastigote Leishmania major GFP in vitro. In parallel, the compounds were evaluated for cytotoxic effects against bone marrow-derived macrophages (BMM Φ) as a mammalian host cell control. Most of the compounds noticeably inhibited the growth of extracellular parasites (IC (50) 0.5 to 6 µM) and the intracellular survival of L. major GFP amastigotes (IC (50) 1 to 7 µM) when compared with the antileishmanial drug amphotericin B (IC (50) of 2.5 and 0.2 µM, respectively). In general, antiprotozoal activity and host cell cytotoxicity seemed to increase in parallel. Conspicuously, the cytotoxic effect was less pronounced on infected host cells when compared with that on noninfected cells. Concerning structure/activity relationships for the tested naphthoquinones, some interesting structural features emerged from this study. Introduction of a methyl or methoxyl group at C-2 of the parent 1,4-naphthoquinone slightly increased the antileishmanial activity against clinically relevant amastigotes, while the presence of a hydroxyl function in this position dramatically reduced the effectiveness. In contrast, hydroxylation at C-5 and dihydroxy substitution at C-5 and C-8 significantly enhanced the antiprotozoal activity. Similarly, the presence of a side chain hydroxyl group PERI to a carbonyl function as represented in the series of shikonin/alkannin derivatives increased the activity when compared with substituted analogs. Within the series of naphthoquinones tested, the dimeric mixture of vaforhizin and isovaforhizin showed the highest activity IN VITRO against the clinically relevant intracellular amastigote with an IC (50) of 1.1 µM. With IC (50) values mostly in the range of 1-3 µM, the shikonin/alkannin derivatives proved to be similarly considerably leishmanicidal. None of the compounds tested was capable to induce NO production known to play a crucial role in the host resistance against intracellular pathogens, excluding activation of microbicidal mechanisms in macrophages. The mode of action apparently depended on the substitution pattern, associated with the electrophilicity of the naphthoquinone or the efficiency of redox cycling. Conspicuously, members oxygenated in the quinone ring proved to be leishmanicidal when coincubated with glutathione, while the majority of the remaining compounds lost activity.

Vaccine candidates for leishmaniasis: a review.

Leishmaniasis is a diverse group of clinical syndromes caused by protozoan parasites of the genus Leishmania. The clinical manifestation of the disease varies from self-limiting cutaneous lesions to progressive visceral disease. It is estimated that 350 million people are at risk in 88 countries, with a global incidence of 1-1.5 million cases of cutaneous and 500,000 cases of visceral leishmaniasis. The key control measures mainly rely on early case detection and chemotherapy which has been hampered by the toxicity of drugs, side-effects and by the emergence of drug resistance in parasites. Control of reservoir host and vector is difficult due to operational difficulties and frequent relapses in the host. Therefore, the development of effective and affordable vaccine against leishmaniasis is highly desirable. Although considerable progress has been made over the last decade in understanding immune mechanisms underlying potential candidate antigens, including killed, live attenuated parasites, crude parasites, pure or recombinant Leishmania proteins or DNA encoding leishmanial proteins, as well as immunomodulators from sand fly saliva, very few candidate vaccines have progressed beyond the experimental stage. As such there is no vaccine against any form of human leishmaniasis. In recent years, however, much interest has been stimulated towards vaccination against leishmaniasis focused mainly on cutaneous leishmaniasis with fewer attempts against visceral leishmaniasis.

Current approaches to discover marine antileishmanial natural products.

Leishmaniasis is a neglected infectious disease caused by kinetoplastid protozoans. An urgent need for novel chemotherapeutics exists. The current approaches to discover new antileishmanial compounds present many drawbacks, including high-cost and time-consuming bioassays. Thus, advances in leishmaniasis treatment are limited, and the development of screening assays is hindered. The combination of multidisciplinary approaches using standardised methods and synchronous projects could be an alternative to develop novel drugs for leishmaniasis treatment. In this review, we discuss the current status of leishmaniasis occurrence and treatment. In addition, we address the advantages and limitations of in vitro leishmaniasis bioassays and discuss the findings of drug discovery research using natural products. Finally, we comprehensively review the marine natural products that are active against Leishmania spp., including their natural sources and bioactivity profile.

Cutaneous and mucocutaneous leishmaniasis: emerging therapies and progress in disease management.

IMPORTANCE OF THE FIELD: Cutaneous leishmaniasis (CL) is a major tropical skin disease. Its incidence continues to increase, and disease control and management are challenging. Available therapies remain inadequate and are associated with low efficacy, toxicity, difficulties with administration, or are expensive. AREAS COVERED IN THIS REVIEW: This article describes progress in the therapeutics of CL since 2006. Clinical trials have provided further evidence for the use of alternative systemic agents to first-line antimonials, an enhanced topical paromomycin preparation, the efficacy of thermotherapy, photodynamic therapy as an emerging physical therapy, and the role of immunotherapy and immunomodulators as adjuncts to chemotherapy. In addition, in vitro studies have demonstrated the anti-leishmanial effects of several drugs, which might represent potential future therapeutic agents for CL. WHAT THE READER WILL GAIN: An overview of the magnitude and complexity of this heterogenous disease, and an update on recent advances in therapeutics and future directions for new drug development. TAKE HOME MESSAGE: Drug therapy for CL must be tailored according to infective species, endemic region, and host responses; a range of different therapies and modalities is therefore required. The impetus for new drug development must continue, combination therapies need to be evaluated, and robust and comparative trials of existing agents are required to adequately assess their efficacy and tolerability.

N-acetyl-L: -cysteine reduces the parasitism of BALB/c mice infected with Leishmania amazonensis.

Leishmania amazonensis infection leads to progressive diseases in a majority of inbred strains of mice. Glutathione (GSH) participates in a large number of cellular phenomena and seems to be essential for several immune functions, including host defense during leishmaniasis. In this study, we evaluated the effects of N-acetyl-L: -cysteine (NAC), as GSH supplement, on the course of L. amazonensis infection in susceptible BALB/c mice. The treatment with NAC (200 mg/kg daily) was effective in raising GSH levels in both lymph node and spleen cells. Although this treatment did not change the footpad swelling development in L. amazonensis-infected mice, it caused a significant decrease in the number of parasites recovered from the footpad lesion and draining popliteal lymph node. Our data suggest that intracellular Leishmania killing in vivo was improved by the augment of GSH levels through NAC administration.

Leishmania amazonensis trypanothione reductase: evaluation of the effect of glutathione analogs on parasite growth, infectivity and enzyme activity.

Trypanothione reductase (TR) is a major enzyme in trypanosomatids. Its substrate, trypanothione is a molecule containing a tripeptide (L-glutamic acid-cysteine-glycine) coupled to a polyamine, spermidine. This redox system (TR/Trypanothione) is vital for parasite survival within the host cell and has been described as a good target for chemotherapy anti-Leishmania. The use of tripeptides analogs of glutathione would result in a decrease in trypanothione synthesis and as a consequence in TR activity. In this work, besides the enzyme potential inhibition, it also evaluated the influence of those analogs on parasite growth and on its infective capacity. The results showed a significant effect on parasite growth and infectivity and in addition TR activity was highly inhibited. These results are very promising, suggesting a potential use of those analogs as therapeutic drugs against experimental diseases caused by trypanosomatids.

IL-5-Induced Eosinophils Suppress the Growth of Leishmania amazonensis In Vivo and Kill Promastigotes In Vitro in Response to Either IL-4 or IFN-gamma.

In IL-5 transgenic mice (C3H/HeN-TgN(IL-5)-Imeg), in which 50% of peripheral blood leukocytes are eosinophils, the development of infection by Leishmania amazonensis was clearly suppressed. To determine mechanistically how this protozoan parasite is killed, we performed in vitro killing experiments. Either IL-4 or IFN-gamma effectively stimulated eosinophils to kill Leishmania amazonensis promastigotes, and most of the killing was inhibited by catalase but not by the NO inhibitor L-N5-(1-iminoethyl)-ornithine, suggesting that hydrogen peroxide is responsible for the killing of L. amazonensis by eosinophils. There was no significant degranulation of eosinophils in the culture, because eosinophil peroxidase was not detected in culture supernatants when L. amazonensis promastigotes were killed by activated eosinophils. Such resistance was also observed in BALB/c mice, which are highly susceptible to L. amazonensis. Expression plasmids for IL-4, IL-5, and IFN-gamma were transferred into muscle by electroporation in vivo starting 1 week before infection. Expression plasmid for IL-5 was most effective in slowing the development of infection among three expression plasmids. Expression plasmid for IL-4 was slightly effective and that for IFN-gamma had no effect on the progress of disease. These results suggest that IL-5 gene transfer into muscle by electroporation is useful as a supplementary protection method against L. amazonensis infection.

Leishmania (L.) amazonensis-induced inhibition of nitric oxide synthesis in host macrophages.

Inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production was demonstrated in J774-G8 macrophages infected with Leishmania (L.) amazonensis promastigotes. The downmodulation of NO production observed in infected and LPS-stimulated J774-G8 cells correlated with a reduction in inducible nitric oxide synthase (iNOS) activity. Reduction in iNOS activity was not paralleled by decreased iNOS mRNA expression, suggesting that the parasite affects post-transcriptional events of NO synthesis. Supplementation with L-arginine or tetrahydrobiopterin did not increase NO production, suggesting that inhibition is not due to an insufficiency of substrate or co-factor. Treatment with anti-IL-10, anti-IL-4 or anti-TGF-beta neutralizing antibodies also failed to increase NO production, indicating that these cytokines are not involved in the observed parasite-induced inhibition of NO synthesis. However, treatment of the cultures with IFN-gamma resulted in a marked increase in NO production by infected LPS-stimulated cells. These results show that although L.(L.) amazonensis infection inhibits iNOS activity and NO production by J774-G8 cells, activation by IFN-gamma is capable of overriding the suppression of NO synthesis.

Recent developments in leishmaniasis.

PURPOSE OF REVIEW: The leishmaniases, caused by protozoan parasites of the genus Leishmania, are a significant health problem in many regions of the world. This review highlights the recent advances in the study of leishmaniasis related to parasite biology, disease pathogenesis, clinical evaluation and treatment, and prevention. RECENT FINDINGS: Genetic heterogeneity and clonal diversity is common among Leishmania strains. Gene knockout, overexpression, and re-introduction studies have identified a number of genes that play a role in parasite virulence. Surprisingly, the importance of the surface lipophosphoglycan in parasite virulence appears to differ among Leishmania spp. Studies in experimental animal models have further defined the roles of CD4 and CD8 T cells, IL-4, IL-10, and IL-12 in the control, maintenance, or progression of disease. The effect of Leishmania on dendritic cells and macrophage effector function has also been an important area of investigation. A number of new vaccine candidates have been identified through experimental animal studies. Clinical studies of leishmaniasis have focused on the host determinants of disease (most notably HIV co-infection), serological and DNA-based diagnostic assays, and treatment. Antimony-resistant cases of cutaneous and visceral leishmaniasis have become more common; liposomal amphotericin and oral miltefosine are promising alternative therapies. SUMMARY: Significant advances have been made in the areas of pathogenesis, host defence, and treatment of leishmaniasis. A number of new vaccine candidates and potential targets of drug therapy have been identified, but progress from preclinical studies to clinical trials has been slow. Translational research, built upon the solid foundation of existing and ongoing basic investigation, is a high priority.

Leishmaniasis cutánea en la provincia de Toledo. Estudio de 43 pacientes / Cutaneous leishmaniasis in the Toledo province. A study of 43 patients

Se estudiaron 43 pacientes diagnosticados de leishmaniasis cutánea (botón de Oriente) que acudieron a la consulta de dermatología del Hospital Virgen de la Salud entre enero de 1990 y diciembre de 1997. Recogimos los siguientes datos clínicos: sexo, edad, número de lesiones, tiempo de evolución, forma clínica, tamaño y localización. El diagnóstico fue por biopsia cutánea o hallazgo del microorganismo en frotis de la lesión. Encontramos una alta proporción de mujeres (70 %) con dos picos de frecuencia en la edad de los pacientes, uno en menores de 14 años (30 %) y el otro entre 61 y 75 años (30 %).La lesión era única en casi todos los pacientes (91 %). El tiempo de evolución de las lesiones presentaba dos picos de máxima frecuencia (6 y 12 meses). La localización era fundamentalmente en áreas fotoexpuestas: 75 % en cara y 19 % en extremidades. La forma clínica era polimorfa. La forma papulonodular eritematosa con costra en superficie fue la más observada, con un tamaño entre 0,3 y 5 cm. La infiltración intralesional de antimoniales pentavalentes fue el tratamiento más empleado, con resultado satisfactorio. Discutimos los resultados de este trabajo y los comparamos con otras revisiones publicadas (AU)