Elvira M. Saraiva

Evidence that the High Incidence of Treatment Failures in Indian Kala-Azar Is Due to the Emergence of Antimony-Resistant Strains of Leishmania donovani

The possibility that the high frequency of treatment failures in Indian kala-azar might be due to infection with antimony-resistant strains of Leishmania donovani has not been experimentally addressed. L. donovani isolates were obtained from splenic aspiration smears of 24 patients in Bihar, India, who either did not respond (15) or did respond (9) to 1 or more full courses of treatment with sodium antimony gluconate (SAG). A strong correlation (P<.001) between clinical response and SAG sensitivity in vitro was observed only when strains were assayed as intracellular amastigotes: responsive isolates ED50=2.4+/-2.6, ED90=6.4+/-7.8 microgram SAG/mL; unresponsive isolates ED50=7.4+/-3.7 microgram SAG/mL, ED90=29.1+/-11.1 SAG/mL. No correlation with clinical response was found by use of extracellular promastigotes (ED50=48+/-22 vs. 52+/-29 microgram/mL). The emergence of antimony-resistant L. donovani strains appears to be a cause of treatment failures in India.

Antileishmanial Activity of an Indole Alkaloid from Peschiera australis

ABSTRACT In this study, we show the leishmanicidal effects of a chloroform fraction (CLF) and a purified indole alkaloid obtained from crude stem extract of Peschiera australis againstLeishmania amazonensis, a causative agent of cutaneous leishmaniasis in the New World. In a bioassay-guided chemical fractionation, the leishmanicidal activity in CLF completely and irreversibly inhibited promastigote growth. This fraction was also active against amastigotes in infected murine macrophages. Chemical analysis of CLF identified an iboga-type indole alkaloid coronaridine as one of its major compounds. Coronaridine showed potent antileishmanial activity, inhibiting promastigote and amastigote growth. Promastigotes and amastigotes treated with CLF or coronaridine showed pronounced alterations in their mitochondria as assessed by transmission electron microscopy.

ETosis: A Microbicidal Mechanism beyond Cell Death.

Netosis is a recently described type of neutrophil death occurring with the release to the extracellular milieu of a lattice composed of DNA associated with histones and granular and cytoplasmic proteins. These webs, initially named neutrophil extracellular traps (NETs), ensnare and kill microorganisms. Similarly, other cell types, such as eosinophils, mast cells, and macrophages, can also dye by this mechanism; thus, it was renamed as ETosis, meaning death with release of extracellular traps (ETs). Here, we review the mechanism of NETosis/etosis, emphasizing its role in diseases caused by protozoan parasites, fungi, and viruses.

The comparative fine structure and surface glycoconjugate expression of three life stages of Leishmania major

The cellular ultrastructure and surface glycoconjugate expression of three life stages of Leishmania major were compared. Noninfective logarithmic phase promastigotes (LP) are immature cells bearing a thin cell coat, short flagellum, small and empty flagellar pocket, and a loose cytoplasm filled with profiles of ER and large Golgi complex. LP also contain subpopulations of maturing cells containing less ER and Golgi and synthesizing cytoplasmic granules of different size, number, and electron-density. Infective or metacyclic promastigotes (MP) are fully differentiated nondividing forms with a thickened, prominent cell coat, long flagellum, distended flagellar pocket filled with secretory material, and few cytoplasmic organelles other than abundant electron-dense granules. Tissue amastigotes also contain electron-dense cytoplasmic granules, their flagellar pockets are also enlarged and contain secretory material, but they lack a discernable cell coat. Immunogold labeling of GP63 on the cell surface was extensive only on amastigotes. Promastigote GP63 appeared to be masked by the presence of a densely packed lipophosphoglycan (LPG) coat which was extensively labeled on the entire surface of MP and LP. An elongated, developmentally modified form of LPG was abundantly labeled only on MP. LPG was poorly labeled on amastigotes, arguing that the promastigote cell coat is a stage-specific structure which is lost during intracellular transformation.

Presentation of the Leishmania antigen LACK by infected macrophages is dependent upon the virulence of the phagocytosed parasites.

We have previously demonstrated that murine macrophages (Mϕ) infected with Leishmania promastigotes, in contrast to Mϕ infected with the amastigote stage of these parasites, are able to present the Leishmania antigen LACK (Leishmania homologue of receptors for activated C kinase) to specific, I‐Ad‐restricted T cell hybrids and to the T cell clone 9.1‐2. These T cells react with the LACK (158 – 173) peptide, which is immunodominant in BALB/c mice. Here, we show that the level of stimulation of the LACK‐specific T cell hybridoma OD12 by promastigote‐infected Mϕ is clearly dependent upon the differentiation state of the internalized parasites. Thus, shortly after infection with log‐phase or stationary‐phase promastigotes of L. major or of L. amazonensis, Mϕ strongly activated OD12. The activity was transient and rapidly lost. However, under the same conditions, activation of OD12 by Mϕ infected with metacyclic promastigotes of L. major or of L. amazonensis was barely detectable. At the extreme, Mϕ infected with amastigotes were incapable to stimulate OD12. Thus, the presentation of LACK by infected Mϕ correlates with the degree of virulence of the phagocytosed parasites, the less virulent being the best for the generation/expression of LACK (158 – 173)‐I‐Ad complexes. While the intracellular killing of the parasites appears to be an important condition for the presentation of LACK, it is not the only requisite. The partial or total destruction of intracellular L. amazonensis amastigotes does not allow the presentation of LACK to OD12. A preferential interaction of LACK (158 – 173) with recycling rather than newly synthesized MHC class II molecules does not explain the transient presentation of LACK by Mϕ infected with log‐phase or stationary‐phase promastigotes because brefeldin A strongly inhibited the presentation of LACK to OD12. Taken together, these results suggest that virulent stages of Leishmania, namely metacyclics and amastigotes, have evolved strategies to avoid or minimize their recognition by CD4+ T lymphocytes.

The role of the lipophosphoglycan of Leishmania in vector competence

The surface lipophosphoglycans (LPG) of Leishmania promastigotes express stage- and species-specific polymorphisms that are defined by variations in the type and number of phosphorylated oligosaccharide repeats. We have studied how these polymorphic structures control the development of transmissible infections in the sandfly vector as well as the species-specificity of vectorial competence. Procyclic promastigotes displayed an inherent capacity to bind to midgut epithelial cells of a competent vector. This capacity was lost during their transformation of metacyclic promastigotes, permitting the selective release and anterior migration of infective-stage parasites for subsequent transmission by bite. Midgut attachment and release were found to be controlled by developmental modifications in terminally exposed saccharides on LPG, which, depending on the species of Leishmania, involved either substitution or capping of terminal side-chain sugars, loss of terminal side-chain sugars, substitution or loss of neutral capping sugars. The stage-specific terminal sugars involved in midgut adhesion are, in some cases, also species-specific, and the extent to which these differences affect midgut attachment, forcefully predicted vectorial competence.

Characterization of neutrophil extracellular traps in cats naturally infected with feline leukemia virus

Feline leukemia virus (FeLV), a common, naturally occurring gammaretrovirus in domestic cats, is associated with degenerative diseases of the haematopoietic system, immunodeficiency and neoplasia. FeLV infection causes an important suppression of neutrophil function, leading to opportunistic infections. Recently, a new microbicidal mechanism named NETosis was described in human, bovine and fish neutrophils, as well as in chicken heterophils. The purpose of the present study was to characterize NETosis in feline neutrophils, as well as to evaluate neutrophil function in FeLV naturally infected symptomatic and asymptomatic cats through the phagocytosis process, release of neutrophil extracellular traps (NETs) and myeloperoxidase (MPO) activity. The results showed that feline neutrophils stimulated with protozoa parasites released structures comprising DNA and histones, which were characterized as NETs by immunofluorescence. Quantification of NETs after neutrophil stimulation showed a significant increase in NET release by neutrophils from FeLV(-) and FeLV(+) asymptomatic cats compared with FeLV(+) symptomatic cats. Moreover, the number of released NETs and MPO activity in unstimulated neutrophils of FeLV(+) symptomatic cats were higher than those in unstimulated neutrophils from FeLV(-) and FeLV(+) asymptomatic cats. This study reports, for the first time, NET release by feline neutrophils, along with the fact that NET induction may be modulated by a viral infection. The results indicate that the NET mechanism appears to be overactivated in FeLV(+) cats and that this feature could be considered a marker of disease progression in FeLV infection.

Cooperation between Apoptotic and Viable Metacyclics Enhances the Pathogenesis of Leishmaniasis

Mimicking mammalian apoptotic cells by exposing phosphatidylserine (PS) is a strategy used by virus and parasitic protozoa to escape host protective inflammatory responses. With Leishmania amazonensis (La), apoptotic mimicry is a prerogative of the intramacrophagic amastigote form of the parasite and is modulated by the host. Now we show that differently from what happens with amastigotes, promastigotes exposing PS are non-viable, non-infective cells, undergoing apoptotic death. As part of the normal metacyclogenic process occurring in axenic cultures and in the gut of sand fly vectors, a sub-population of metacyclic promastigotes exposes PS. Apoptotic death of the purified PS-positive (PSPOS) sub-population was confirmed by TUNEL staining and DNA laddering. Transmission electron microscopy revealed morphological alterations in PSPOS metacyclics such as DNA condensation, cytoplasm degradation and mitochondrion and kinetoplast destruction, both in in vitro cultures and in sand fly guts. TUNELPOS promastigotes were detected only in the anterior midgut to foregut boundary of infected sand flies. Interestingly, caspase inhibitors modulated parasite death and PS exposure, when added to parasite cultures in a specific time window. Efficient in vitro macrophage infections and in vivo lesions only occur when PSPOS and PS-negative (PSNEG) parasites were simultaneously added to the cell culture or inoculated in the mammalian host. The viable PSNEG promastigote was the infective form, as shown by following the fate of fluorescently labeled parasites, while the PSPOS apoptotic sub-population inhibited host macrophage inflammatory response. PS exposure and macrophage inhibition by a subpopulation of promastigotes is a different mechanism than the one previously described with amastigotes, where the entire population exposes PS. Both mechanisms co-exist and play a role in the transmission and development of the disease in case of infection by La. Since both processes confer selective advantages to the infective microorganism they justify the occurrence of apoptotic features in a unicellular pathogen.

Changes in lipophosphoglycan and gene expression associated with the development of Leishmania major in Phlebotomus papatasi.

Stage-specific molecular and morphogenic markers were used to follow the kinetics of appearance, number, and position of metacyclic promastigotes developing during the course of L. major infection in a natural vector, Phlebotomus papatasi. Expression of surface lipophosphoglycan (LPG) on transformed promastigotes was delayed until the appearance of nectomonad forms on day 3, and continued to be abundantly expressed by all promastigotes thereafter. An epitope associate with arabinose substitution of LPG side-chain oligosaccharides, identified by its differential expression by metacyclics in vitro, was detected on the surface of a low proportion of midgut promastigotes beginning on day 5, and on up to 60% of promastigotes on days 10 and 15. In contrast 100% of the parasites egested from the mouthparts during forced feeding of 15 day infected flies stained strongly for this epitope. At each time-point, the surface expression of the modified LPG was restricted to morphologically distinguished metacyclic forms. Ultrastructural study of the metacyclic surface revealed an approximate 2-fold increase in the thickness of the surface coat compared to nectomonad forms, suggesting elongation of LPG as occurs during metacyclogenesis in vitro. A metacyclic-associated transcript (MAT-1), another marker identified by its differential expression in vitro, also showed selective expression by promastigotes in the fly, and was used in in situ hybridization studies to demonstrate the positioning of metacyclics in the anterior gut.

Classical ROS-dependent and early/rapid ROS-independent release of Neutrophil Extracellular Traps triggered by Leishmania parasites

Neutrophil extracellular traps (NETs) extruded from neutrophils upon activation are composed of chromatin associated with cytosolic and granular proteins, which ensnare and kill microorganisms. This microbicidal mechanism named classical netosis has been shown to dependent on reactive oxygen species (ROS) generation by NADPH oxidase and also chromatin decondensation dependent upon the enzymes (PAD4), neutrophil elastase (NE) and myeloperoxidase (MPO). NET release also occurs through an early/rapid ROS-independent mechanism, named early/rapid vital netosis. Here we analyze the role of ROS, NE, MPO and PAD4 in the netosis stimulated by Leishmania amazonensis promastigotes in human neutrophils. We demonstrate that promastigotes induce a classical netosis, dependent on the cellular redox imbalance, as well as by a chloroamidine sensitive and elastase activity mechanism. Additionally, Leishmania also induces the early/rapid NET release occurring only 10 minutes after neutrophil-parasite interaction. We demonstrate here, that this early/rapid mechanism is dependent on elastase activity, but independent of ROS generation and chloroamidine. A better understanding of both mechanisms of NET release, and the NETs effects on the host immune system modulation, could support the development of new potential therapeutic strategies for leishmaniasis.