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.     

Destruction of Leishmania mexicana amazonensis promastigotes by normal human serum

Fresh normal human serum was observed to have a lethal effect on Leishmania mexicana amazonensis promastigotes obtained from laboratory-bred Lutzomyia longipalpis or on promastigotes grown in liquid culture medium, inoculated with the same isolates. Heat inactivation abolished the Leishmania lytic activity from the sera. Resistance of culture promastigotes to lysis by normal human serum was investigated in three isolates of L. m. amazonensis. Development of resistance (up to 7%) was found in only one isolate, obtained from the bone marrow in a human case of visceral leishmaniasis.     

Tissue eosinophilia and Leishmania mexicana mexicana eosinophil interactions in murine cutaneous leishmaniasis

Summary Outbred albino mice were infected subcutaneously with 10⁶ amastigotes of Leishmania mexicana mexicana and the subsequent lesions were evaluated by light and electron microscopy at various intervals after infection. The animals developed persistent nodules and a spectrum of lesions of variable size which was correlated with the host's ability to control the parasite in the tissue. During the acute phase of the disease the histopathological results showed an accumulation of granulocytes, some mononuclear phagocytes and a predominance of eosinophils as compared to other cell types. In this early acute phase, eosinophils were found in the tissue together with normal and degranulating mast cells. In the granulomatous inflammatory reaction of the chronic phases, there was infiltration of granulocytes parallel to parasite multiplication and the formation of parasitized vacuolated macrophages. The number of eosinophils was consistently greater than neutrophils, regardless of lesion type or number of parasites present in the tissue. During the acute reaction, the granulocytes apparently destroyed many parasites; however, there was an unvaryingly low level of phagocytosis of amastigotes during the chronic stages by both eosinophils and neutrophils. Neutrophils seemed to be more effective than eosinophils in the killing of ingested parasites. A close association between eosinophils and parasitized macrophages was seen in the chronic lesions; thus, eosinophils might contribute to parasite destruction through co-operation with macrophages.     

Leishmania mexicana: destruction of isolated amastigotes by amino acid esters

Amino acid esters can destroy intracellular as well as isolated amastigotes of Leishmania mexicana amazonensis. In the present study we examined, using a tetrazolium reduction assay, the toxicity of the esters for amastigotes isolated from mouse lesions. Parasite killing by the "prototype" compound L-leucine methyl ester at 1 mM concentration and at pH 7.3 took place within 15-30 min. Time-lapse cinematographic observations showed that the amastigotes rounded up and became less phase-dense before they rapidly broke down. Ammonium chloride, ethylamine or monensin, known to raise the intracellular pH, reduced the sensitivity of the amastigotes to L-Leu-OMe. This finding suggests that an acidified compartment is involved in the destruction of the parasites. The leishmanicidal activity of a series of L-amino acid esters was also investigated. The ED50 (concentration for half maximal effect) for methyl esters was (in mM): Leu (0.62), Trp (0.96), Met (1.13), Glu (2.0), Phe (2.5), and Tyr (3.8). In contrast, the methyl esters of Ile, Val, Ala, beta Ala, Gly, Ser, His, and Pro were either inactive or weakly active at 15 mM. Benzyl esters were more active than their methyl homologs: the ED50 of the benzyl esters of Leu, Val, Ile, Gly, Ala, beta Ala, and Pro were, respectively, 0.07, 0.20, 0.22, 0.88, 1.5, 2.3, and 6.7 mM. Ranks of leishmanicidal activity may reflect differences in the rates of ester uptake and trapping by the amastigotes, in the specificity of the relevant hydrolytic enzyme(s), in the accumulation and metabolic fate of the released amino acids, or in the toxicity of the amino acid or alcohol released within the amastigotes.     

Host-parasite relationship of Leishmania mexicana mexicana and Lutzomyia abonnenci (Diptera: Psychodidae)

The life cycle of Leishmania mexicana mexicana in the gut of the sand fly, Lutzomyia abonnenci, was studied by light and electron microscopy. Development was suprapylarian with initial establishment of parasites in the bloodmeal (posterior midgut), and anterior migration of parasites to the cardia/stomodeal valve region beginning at 2.5 days post-infection. Flagellates were first observed in the esophagus at 3.5 days, in the posterior armature region of the pharynx at 5 days, and in the anterior pharynx at 7 days; but they were not detected in the cibarium or proboscis. Infection of the pylorus region of the hindgut and of the Malpighian tubules was also commonly observed. Three different morphological forms of L. m. mexicana developed in the gut: nectomonad promastigotes, short promastigotes, and paramastigotes. Nectomonads occurred primarily in the abdominal midgut after bloodmeal digestion, where they were oriented in longitudinal masses in the lumen, or interdigitated with epithelial microvilli via the flagellum. Short promastigotes found in the cardia/stomodeal valve region are described for the first time. These forms were smaller than nectomonads, showed an amplification of the kinetoplast, apposition of kinetoplast and nucleus, and were embedded in a gel-like matrix. To maintain position in the cardia, parasites commonly inserted the flagellum deep into microvilli or cytoplasm of the epithelium; adherence to the cuticular intima of the stomodeal valve was by flagellar modification and formation of hemidesmosome plaques. Paramastigotes occurred in the esophagus, were sometimes degenerated in appearance, and were attached via flagellar hemidesmosomes. Paramastigotes observed in the lumen of the pharynx were commonly degenerated and were not attached to the intima. L. m. mexicana was able to colonize the various gut habitats of Lu. abonnenci by a number of adaptations; this sand fly appears to be a suitable biological host for the parasite.     

Relapse of new world diffuse cutaneous leishmaniasis caused by Leishmania (Leishmania) mexicana after miltefosine treatment

A 35-year-old man with a 19-year history of slowly evolving diffuse cutaneous leishmaniasis was treated with oral miltefosine, 50 mg three times a day. The patient responded after four months of miltefosine treatment with clearance of all nodular lesions and plaques from the entire body surface and had negative slit-skin smears and cultures for Leishmania. However, two months after stopping miltefosine, skin lesions reappeared and parasites were observed in samples. The relapsed lesions did not respond to an additional two-month course of miltefosine. No laboratory or clinical adverse events to miltefosine were observed. Parasites from skin lesions were cultured and identified as Leishmania (Leishmania) mexicana by isoenzyme electrophoresis.     

Accidental Leishmania mexicana infection in an immunosuppressed laboratory technician

A 30 year-old female laboratory technician under immunosuppressive treatment because of systemic lupus erythematosus (SLE) developed cutaneous leishmaniasis 8 months after accidental percutaneous inoculation of amastigote culture forms of Leishmania mexicana . Leishmania -specific PCR and restriction analysis patterns were identical for both the laboratory strain and the clinical specimen. The lesion was resistant to local paromomycin and oral ketoconazole, but responded to local application of meglumine antimonate. No signs of dissemination or visceralization occurred during the 5-month period of observation. However, a future recurrence cannot be excluded since a persistent infection even after clinical cure has always to be considered in leishmaniasis. Patients under immunosuppressive therapy are possibly at risk of clinical relapse or disseminating infection although there is no experience with regard to leishmaniasis mexicana . Serious infection may require interferon gamma as part of the treatment which may contribute to deterioration of concomitant diseases like SLE. In any case, the exposure of immunodeficient laboratory workers to Leishmania spp . should be avoided.     

Genetic characterization of glucose transporter function in Leishmania mexicana

Both insect and mammalian life cycle stages of Leishmania mexicana take up glucose and express all three isoforms encoded by the LmGT glucose transporter gene family. To evaluate glucose transporter function in intact parasites, a null mutant line has been created by targeted disruption of the LmGT locus that encompasses the LmGT1, LmGT2, and LmGT3 genes. This deltalmgt null mutant exhibited no detectable glucose transport activity. The growth rate of the deltalmgt knockout in the promastigote stage was reduced to a rate comparable with that of WT cells grown in the absence of glucose. deltalmgt cells also exhibited dramatically reduced infectivity to macrophages, demonstrating that expression of LmGT isoforms is essential for viability of amastigotes. Furthermore, WT L. mexicana were not able to grow as axenic culture form amastigotes if glucose was withdrawn from the medium, implying that glucose is an essential nutrient in this life cycle stage. Expression of either LmGT2 or LmGT3, but not of LmGT1, in deltalmgt null mutants significantly restored growth as promastigotes, but only LmGT3 expression substantially rescued amastigote growth in macrophages. Subcellular localization of the three isoforms was investigated in deltalmgt cells expressing individual LmGT isoforms. Using anti-LmGT antiserum and GFP-tagged LmGT fusion proteins, LmGT2 and LmGT3 were localized to the cell body, whereas LmGT1 was localized specifically to the flagellum. These results establish that each glucose transporter isoform has distinct biological functions in the parasite.     

Preliminary study towards a novel experimental model to study localized cutaneous leishmaniasis caused by Leishmania (Leishmania) mexicana

There is not an experimental model of localized cutaneous leishmaniasis (LCL) caused by Leishmania (Leishmania) mexicana. The aim of the present study was to characterize the clinical and histological features of Peromyscus yucatanicus experimentally infected with L. (L.) mexicana. A total of 54 P. yucatanicus (groups of 18) were inoculated with 1x10(6) promastigotes of L. (L.) mexicana in the base of the tail. They were euthanized at three and six months post experimental infection. The control group was inoculated with RPMI-1640. The predominant clinical sign observed was a single ulcerated lesion in 27.77% (5/18) and in 11.11% (2/18) P. yucatanicus at three and six months respectively. The histological pattern described as chronic granulomatous inflammation with or without necrosis was found in 7/7 (100%) biopsies of euthanized P. yucatanicus at three (n = 5) and six (n = 2) months, respectively. These results resembled clinical and histological features caused by L. (L.) mexicana in humans, and support the possibility to employ P. yucatanicus as a novel experimental model to study LCL caused by this parasite.     

Leishmania mexicana and Leishmania major: attenuation of wild-type parasites and vaccination with the attenuated lines

A method for attenuation of Leishmania species by culturing in vitro under gentamicin pressure has been used successfully with Leishmania mexicana, L. major, L. infantum, and L. donovani. The attenuated lines invaded but were unable to survive within bone marrow-derived macrophages in vitro, whereas wild-type parasites survived and multiplied. The attenuated lines of L. mexicana and L. major both failed to induce cutaneous lesions in the majority of BALB/c mice over a minimum 12-week observation period after subcutaneous injection of stationary phase parasites. The attenuated line of L. mexicana retained its properties in gentamicin-free medium over 40 subcultures. The attenuated lines of L. mexicana and L. major both induced significant protection in mice against challenge with wild-type 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.     

17Beta-estradiol increases Leishmania mexicana killing in macrophages from DBA/2 mice by enhancing production of nitric oxide but not pro-inflammatory cytokines

We have previously shown that female DBA/2 mice are significantly more resistant to Leishmania mexicana compared with males. Here, we have analyzed the effect of 17beta-estradiol (E(2)) on function and cytokine production in male and female DBA/2 macrophages in vitro. We show that E(2) increases NO production and parasite killing in L. mexicana-infected male and female DBA/2 macrophages without increasing production of pro-inflammatory cytokines. These data indicate that E(2) may enhance leishmanicidal activity in macrophages by directly regulating production of NO.