Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps

Neutrophils are short-lived leukocytes that die by apoptosis, necrosis, and NETosis. Upon death by NETosis, neutrophils release fibrous traps of DNA, histones, and granule proteins named neutrophil extracellular traps (NETs), which can kill bacteria and fungi. Inoculation of the protozoan Leishmania into the mammalian skin causes local inflammation with neutrophil recruitment. Here, we investigated the release of NETs by human neutrophils upon their interaction with Leishmania parasites and NETs'' ability to kill this protozoan. The NET constituents DNA, elastase, and histones were detected in traps associated to promastigotes by immunofluorescence. Electron microscopy revealed that Leishmania was ensnared by NETs released by neutrophils. Moreover, Leishmania and its surface lipophosphoglycan induced NET release by neutrophils in a parasite number- and dose-dependent manner. Disruption of NETs by DNase treatment during Leishmania–neutrophil interaction increased parasite survival, evidencing NETs'' leishmanicidal effect. Leishmania killing was also elicited by NET-rich supernatants from phorbol 12-myristate 13-acetate-activated neutrophils. Immunoneutralization of histone during Leishmania–neutrophil interaction partially reverted Leishmania killing, and purified histone killed the parasites. Meshes composed of DNA and elastase were evidenced in biopsies of human cutaneous leishmaniasis. NET is an innate response that might contribute to diminish parasite burden in the Leishmania inoculation site.     

Immune serum from both susceptible and resistant strains of mice increases phagocytosis of Leishmania mexicana amazonensis by macrophages

Immune sera obtained from either BALB/c mice (susceptible) at 7 weeks, or C57BL/6 mice (resistant), at 7 weeks after infection with L. m. amazonensis, were effective in increasing internalization of homologous promastigotes into starch-induced peritoneal macrophages (from both mouse strains). Both the internalization enhancing effect and the levels of anti-leishmanial antibody (ELISA) were removed from sera by absorption with heat-killed promastigotes. Sera at 1/200 dilution obtained from either mouse strain at 2 weeks after infection did not enhance parasite internalization into macrophages. The factors leading to susceptibility or resistance during leishmaniasis do not appear to be related to differences in antibody-mediated opsonic activity.     

Activity of ketoconazole derivatives against Leishmania mexicana amazonensis within mouse peritoneal macrophages

Imidazoles such as ketoconazole have proven antileishmanial activity, both in vitro and in vivo. New derivatives of ketoconazole have been synthesized in order to improve the therapeutic index and antileishmanial activity as assessed by mouse peritoneal macrophages infected with Leishmania mexicana amazonesis. Amino-acid derivatives of ketoconazole are at least 10 times more effective than ketoconazole in vitro, and the best effect is observed using the phenylalanyl-ketoconazole. Fatty acid derivatives, such as oleoyl-ketoconazole, also possess a greater therapeutic activity but to a lesser extent than amino-acid derivatives. Moreover, oleoyl-ketoconazole showed a remarkable property in terms of effective dose. Our results demonstrate the potential antileishmanial efficacy of some ketoconazole derivatives, and suggest that phenylalanyl-ketoconazole should be considered for experimental evaluation in animal models.     

Granulocyte-macrophage and macrophage colony-stimulating factors activate intramacrophage killing of Leishmania mexicana amazonensis

Leishmania organisms are important pathogens, causing diseases worldwide. Standard therapies are often toxic and are not always effective. The effect of recombinant human granulocyte-macrophage and macrophage colony-stimulating factors (GM-CSF and M-CSF) on intramacrophage survival of Leishmania mexicana amazonensis (Lma) were compared with those of interferon-gamma (IFN-gamma). Macrophages previously infected with Lma were treated with or without GM-CSF and M-CSF. Compared with no cytokine treatment, treatment with GM-CSF (0.1-100 ng/ml) or M-CSF (1:3.5 X 10(6) - 1:3.5 X 10(3) dilutions) caused a significant dose-dependent reduction in intracellular parasites, 427 +/- 20 (mean +/- SE) Lma/100 macrophages. GM-CSF or M-CSF in combination with IFN-gamma resulted in more effective inhibition of intracellular parasites. Thus, the cytostatic activity appears to require interaction between cytokines, macrophages, and amastigotes. These cytokines are potential therapeutic agents for visceral leishmaniasis.     

In vivo and in vitro phagocytosis of Leishmania (Leishmania) amazonensis promastigotes by B‐1 cells

Leishmaniasis is caused by Leishmania parasites that infect several cell types. The promastigote stage of Leishmania is internalized by phagocytic cells and transformed into the obligate intracellular amastigote form. B‐1 cells are a subpopulation of B cells that are able to differentiate in vitro and in vivo into mononuclear phagocyte‐like cells with phagocytic properties. B‐1 cells use several receptors for phagocytosis, such as the mannose receptor and third complement receptor. Leishmania binds to the same receptors on macrophages. In this study, we demonstrated that phagocytes derived from B‐1 cells (B‐1 CDP) were able to internalize promastigotes of L. (L.) amazonensis in vitro. The internalized promastigotes differentiated into amastigotes. Our results showed that the phagocytic index was higher in B‐1 CDP compared to peritoneal macrophages and bone marrow‐derived macrophages. The in vivo phagocytic ability of B‐1 cells was also demonstrated. Parasites were detected inside purified B‐1 cells after intraperitoneal infection with L. (L.) amazonensis promastigotes. Intraperitoneal stimulation with the parasites led to an increase in both IL‐10 and TNF‐α. These results highlight the importance of studying B‐1 CDP cells as phagocytic cells that can participate and contribute to immunity to parasites.     

Low serum levels of dehydroepiandrosterone and cortisol in human diffuse cutaneous leishmaniasis by Leishmania mexicana

Low levels of dehydroepiandrosterone (DHEA) and cortisol hormones produced by the suprarenal cortex have been associated with diseases involving chronic inflammation, low interferon (IFN)-gamma, and high interleukin (IL)-6. Diffuse cutaneous leishmaniasis (DL), a long-lasting intracellular parasitic infectious disease, can spread unknown levels of DHEA and cortisol. Serum concentrations of both were measured in 5 patients with DL, in 15 patients with localized lesions produced by Leishmania (LL), and in 20 healthy volunteers. Leishmania mexicana mexicana was identified as the causal agent in patients with DL and LL. Hormone levels were lower in DL compared with controls and LL. Furthermore, we detected a lower percentage of IFN-gamma-positive cells with higher levels of IL-6 and higher titers of anti-Leishmania antibodies in patients with DL, whereas patients with LL were similar to controls. These data suggest that patients with DL may be good candidates for DHEA and cortisol supplementation.     

Amine production by Leishmania mexicana

Growing promastigotes of Leishmania mexicana mexicana excreted large amounts of urea and ammonia into the culture medium. Both promastigotes and amastigotes in short-term, high-density cultures also produced urea and ammonia; the excretion rate was higher in promastigotes. Putrescine was excreted by growing promastigotes but spermine and spermidine excretion apparently did not occur. Both promastigote and amastigote cell extracts contained putrescine and spermidine. Both polyamines, but especially putrescine, were present at higher concentrations in promastigotes than amastigotes. Trace amounts of spermine were found in the promastigote extracts but none was detected in the amastigotes.     

Isolation of Leishmania mexicana amazonensis from the bone marrow in a case of American visceral leishmaniasis

The first documented human case of visceral leishmaniasis caused by L. mexicana amazonensis is reported. Leishmania were isolated from bone marrow aspirate material from a typical visceral leishmaniasis patient. Further characterization by isoenzyme electrophoresis and by a panel of species- and subspecies-specific monoclonal antibodies established its classification as L. m. amazonensis.     

Combined effect of the essential oil from Chenopodium ambrosioides and antileishmanial drugs on promastigotes of Leishmania amazonensis

To date, there are no vaccines against Leishmania, and chemotherapy remains the mainstay for the control of leishmaniasis. The drugs of choice used for leishmaniasis therapy are significantly toxic, expensive and with a growing frequency of refractory infections. Because of these limitations, a combination therapy is the better hope. This work demonstrates that the essential oil from Chenopodium ambrosioides shows a synergic activity after incubation in conjunction with pentamidine against promastigotes of Leishmania amazonensis. However, an indifferent effect has been found for combinations of meglumine antimoniate or amphotericin B and the essential oil.     

Different patterns of disease in two inbred mouse strains infected with a clone of Leishmania mexicana amazonensis

We have infected BALB/c and C57BL/6 mice with a cloned Leishmania mexicana amazonensis population, obtained from the "Maria" strain. Progression of infection and histopathological examination confirmed the extreme susceptibility of BALB/c mice and the resistant pattern of the C57 BL/6. Anti-Leishmania antibody titers were higher in BALB/c than in C57BL/6 mice through the period of infection. Tests of delayed type hypersensitivity reaction with Leishmania antigens were positive in both strains in the beginning of the infection, but were negative later on in BALB/c mice. Our results are similar to those obtained with mixed parasite populations, and rule out the possibility of selection among different parasite subpopulations as responsible for the divergent course of the disease exhibited by these two strains of mice.     

Destruction of normal human eosinophils by Entamoeba histolytica

We evaluated the in-vitro interaction of normal human eosinophil leucocytes and a virulent strain of Entamoeba histolytica (HM1-IMSS) in the presence of immune serum. At a 10:1 (eosinophil:amoeba) ratio a significant time-dependent destruction of eosinophils was found from the first hour onward, and a similar, albeit weaker, cytopathic effect was found in the 200:1 ratio mixtures, with some delay in the microscopic evidence of such effect. Results were unaffected by serum factors, and amoebae emerged virtually unharmed throughout these experiments, again regardless of the presence of serum factors. These results indicate that, as with neutrophil leucocytes, virulent E. histolytica is capable of destroying normal human eosinophils in vitro.     

The effect of parasitization by Leishmania mexicana mexicana on macrophage function in vitro

Macrophages infected with amastigotes of Leishmania mexicana mexicana as compared to normal macrophages show decreased migration both randomly and through a 5 microns pore in response to a known chemotaxin, an increased ability to pinocytose and an increased bactericidal ability. Unless very heavily parasitized their ability to phagocytose is unaltered. Parasitized macrophages are unaltered in their ability to secrete extracellularly lysosomal enzymes, prostaglandins and lysozyme in response to known stimuli, or to kill target cells in an antibody dependent cell mediated cytotoxicity assay.     

The interaction of Leishmania donovani promastigotes and human fibroblasts in vitro

Leishmania donovani promastigotes derived from infected hamster spleens, in either log phase or stationary phase growth, associated with human foreskin fibroblasts in vitro and assumed the morphological characteristics of amastigotes. This apparent conversion was noted within hours at 26 degrees C, 32 degrees C or 37 degrees C; in the continued presence of promastigotes, increasing numbers of amastigote-like forms were seen for 2 weeks at 26 degrees C or 32 degrees C. At 37 degrees C amastigote-like forms declined sharply after 6 days. Multiplication of amastigote-like forms was not observed at any temperature, this was also true of freshly isolated amastigotes from hamster spleens which associated with fibroblasts but did not multiply. Approximately 0.1% of promastigotes appeared to convert per day. Amastigote-like forms were seen within fibroblasts by transmission electron microscopy, surrounded by a closely applied host membrane. Scanning electron microscopy showed promastigotes with their flagellae under or within fibroblasts, but phagocytosis was not observed. These experiments suggest that the conditions required for promastigote-to-amastigote conversion may be different than those required for amastigote multiplication, and the mammalian core body temperature may not be required for promastigote conversion.     

Leishmania mexicana complex: human infections in the Republic of Panama

Parasites of the genus Leishmania responsible for human cutaneous leishmaniasis in the New World form 2 major taxonomic divisions: the Leishmania braziliensis and the L. mexicana complexes. We report the isolation and characterization of the L. mexicana complex among humans in the Republic of Panama. Characterization was based on parasite morphology, pathogenesis in infected golden hamsters, cellulose acetate isoenzyme electrophoretic mobilities, and membrane-specific monoclonal antibodies using the radioimmune binding assay technique.     

Attachment of plasma membrane vesicles of human macrophages to Leishmania tropica promastigotes

Intracellular parasitism of host macrophages is the pathologic hallmark of leishmaniasis. Since the organisms are found almost exclusively in this type of cell, the possibility that specific macrophage plasma-membrane determinants mediate the attachment to promastigotes of Leishmania tropica was investigated. Plasma membrane vesicles were prepared from human monocytes/macrophages and erythrocytes, radioiodinated, and incubated with L tropica promastigotes, erythrocytes, or Sephadex beads. Macrophage plasma-membrane vesicles bound to promastigotes to a significant extent but did not bind to intact erythrocytes or to an inert particle. In contrast, erythrocyte membrane vesicles did not bind to promastigotes. The binding of macrophage plasma-membrane vesicles to promastigotes demonstrated the characteristics of a receptor-ligand interaction in terms of specificity, saturability, competitive inhibition, and temperature independence. These results suggest the presence of one or more intrinsic binding sites on the macrophage plasma membrane to which promastigotes can attach. If this is the case, therapeutic intervention by strategies that inhibit attachment of this obligate intracellular parasite to its target host cell may be possible.