Leishmania donovani infection in heterozygous and recombinant H-2 haplotype mice

On a B10 (Lshs) genetic background the development of acquired T-cell-mediated immunity to Leishmania donovani infection in mice is under H-2-linked genetic control. Three phenotypic patterns of recovery were previously observed: "early cure" (H-2s, H-2r), "cure" (H-2b) and "noncure" (H-2d, H-2q, H-2f), with cure behaving as a recessive trait in H-2b/H-2d mice. In this study the long-term response to L. donovani is followed over 130 days of infection in eight recombinant haplotype strains and in six further heterozygous haplotype combinations. Noncure in B10.HTG mice, which carry d alleles for loci at the K end and b alleles for loci at the D end of H-2, confirms that H-2-linked genetic control of the acquired response to L. donovani infection is located in the K end. The complex pattern of dominance relationships observed in the additional heterozygous haplotypes studied, the variable phenotypic response of H-2k mice and of recombinant haplotype strains carrying IEk in common, and the differential early curing activity observed in heterozygotes involving the s but not the r early cure haplotype and in recombinant haplotype mice carrying s alleles to the left of IE suggest, however, that more than one subregion (IE and presumably IA) are involved. Results are interpreted in the light of immunoregulatory T-cell populations previously demonstrated in noncure, cure, and early cure strains.     

Leishmania major and Leishmania donovani: effect of LPG-containing and LPG-deficient strains on monocyte chemotaxis and chemiluminescence.

Lipophosphoglycan (LPG) is a major glycolipid present on the membrane of Leishmania promastigotes and amastigotes. We have previously shown that preincubation of peripheral blood monocytes with purified LPG inhibits IL-1 production, chemotactic locomotion, and luminol-dependent chemiluminescence (LDCL). In the present study we tested the effect of LPG present on live parasites on monocyte activity. For this purpose, we used two mutant strains deficient in LPG and two LPG-containing strains. One pair was Leishmania major and the other Leishmania donovani. Monocytes in suspension were infected with the different parasite strains and tested for chemotactic locomotion and LDCL at different times between 1 and 72 hr after infection. In parallel, the percentage of infected monocytes was measured in stained cytospin preparations. The results obtained showed that at 1 hr of incubation only the LPG-containing strains inhibited chemotaxis, while the mutant strains showed a normal response. From 4 hr of incubation onwards, the mutant strains also inhibited monocyte chemotactic locomotion. LDCL was only slightly inhibited by the LPG-containing strains after 1 hr, because of a high level of spontaneous stimulation, probably due to phagocytosis. At 24 and 72 hr all strains inhibited LDCL. These results suggest that LPG is responsible for early inhibition of macrophage activity, but that other factors are responsible for inhibition at later stages of in vitro infection. The model described here might represent a useful tool to further analyze the mechanisms involved in immune evasion of Leishmania parasites.     

Antileishmanial effect of free and encapsulated sinefungin against Leishmania donovani infections in BALB/c mice

Sinefungin, a C-nucleoside antibiotic, displays a high antiparasitic action. The effect of free and microencapsulated sinefungin was compared on BALB/c mice infected with Leishmania donovani; the encapsulated form proved more effective by one order of magnitude.     

Comparison of the A2 gene locus in Leishmania donovani and Leishmania major and its control over cutaneous infection

In Old World Leishmania infections, Leishmania donovani is responsible for fatal visceral leishmaniasis, and L. major is responsible for non-fatal cutaneous leishmaniasis in humans. The genetic differences between these species which govern the pathology or site of infection are not known. We have therefore carried out detailed analysis of the A2 loci in L. major and L. donovani because A2 is expressed in L. donovani but not L. major, and A2 is required for survival in visceral organs by L. donovani. We demonstrate that although L. major contains A2 gene regulatory sequences, the multiple repeats that exist in L. donovani A2 protein coding regions are absent in L. major, and the remaining corresponding A2 sequences appear to represent non-expressed pseudogenes. It was possible to restore amastigote-specific A2 expression to L. major, confirming that A2 regulatory sequences remain functional in L. major. Although L. major is a cutaneous parasite in rodents and humans, restoring A2 expression to L. major inhibited its ability to establish a cutaneous infection in susceptible BALB/c or resistant C57BL6 mice, a phenotype typical of L. donovani. There was no detectable cellular immune response against L. major after cutaneous infection with A2-expressing L. major, suggesting that the lack of growth was not attributable to acquired host resistance but to an A2-mediated suppression of parasite survival in skin macrophages. These observations argue that the lack of A2 expression in L. major contributed to its divergence from L. donovani with respect to the pathology of infection.     

Leishmania major and L. donovani: effects on proteolytic enzymes of Phlebotomus papatasi (Diptera, Psychodidae)

Phlebotomus papatasi is susceptible to Leishmania major which it transmits in nature, but is resistant to L. donovani. The present study compares the effect of L. major and L. donovani on the proteolytic activity of P. papatasi gut enzymes. The experiments measured digestion of C14-labeled globin by gut homogenates of flies. Homogenates were prepared from flies fed on serum only (controls) or from flies fed serum containing promastigotes or their dried culture overlayer. In other experiments, the promastigotes or dried culture overlayers were added in vitro to the gut homogenate of control flies. Proteolytic activity of gut homogenate from flies infected with L. major was about one-third less than that of controls, while that from flies infected with L. donovani was one-third greater. Ingestion of L. major dried culture overlayer had an effect on flies similar to that of the promastigotes, while L. donovani dried culture overlayer produced no significant effect. When added to gut homogenate in vitro, promastigotes of both species promoted proteolysis as did dried culture overlayer of L. major. Dried culture overlayer of L. donovani, however, had an opposite effect. It is suggested that the observed reduction in proteolytic activity caused by L. major infection may result from inhibition of enzyme production.     

Expression of the E3L gene of vaccinia virus in transgenic mice decreases host resistance to vaccinia virus and Leishmania major infections

The E3L gene of vaccinia virus (VACV) encodes the E3 protein that in cultured cells inhibits the activation of interferon (IFN)-induced proteins, double-stranded RNA-dependent protein kinase (PKR), 2′-5′-oligoadenylate synthetase/RNase L (2-5A system) and adenosine deaminase (ADAR-1), thus helping the virus to evade host responses. Here, we have characterized the in vivo E3 functions in a murine inducible cell culture system (E3L-TetOFF) and in transgenic mice (TgE3L). Inducible E3 expression in cultured cells conferred on cells resistance to the antiviral action of IFN against different viruses, while expression of the E3L gene in TgE3L mice triggered enhanced sensitivity of the animals to pathogens. Virus infection monitored in TgE3L mice by different inoculation routes (intraperitoneal and tail scarification) showed that transgenic mice became more susceptible to VACV infection than control mice. TgE3L mice were also more susceptible to Leishmania major infection, leading to an increase in parasitemia compared to control mice. The enhanced sensitivity of TgE3L mice to VACV and L. major infections occurred together with alterations in the host immune system, as revealed by decreased T-cell responses to viral antigens in the spleen and lymph nodes and by differences in the levels of specific innate cell populations. These results demonstrate that expression of the E3L gene in transgenic mice partly reverses the resistance of the host to viral and parasitic infections and that these effects are associated with immune alterations.     

Sandfly midgut lectin: effect of galactosamine on Leishmania major infections

Galactosamine, which has been shown in vitro to specifically inhibit sandfly midgut lectin activity, was fed to Phlebotomus duboscqi females with blood containing promastigotes of Leishmania major . Non-inhibitory sugar, galactose, was added in controls. For two strains of L. major (LV 561 and Neal-P), galactosamine substantially enhanced the establishment of infection in the sandfly posterior midgut and significantly increased parasite loads after defaecation, but did not affect anterior migration of Leishmania . On day 3 post-infection, most infections in galactosamine-fed sandfly groups (92% of LV 561 and 100% of Neal-P) were found in the ectoperitrophic space of the posterior midgut, whereas most infections in the galactose-fed groups of sandflies (85% in LV 561 and 96% in Neal-P) were restricted to the peritrophic sac. On day 9, however, the proportion of infections colonizing the stomodeal valve was similar in both dietary groups of sandflies for both strains of L. major . The addition of galactosamine prevented the decrease of parasite loads which occurred in controls between days 3 and 6 post-infection. On days 6 and 9, heavy infections were observed almost exclusively in galactosamine-fed females. Differences between groups were more pronounced for the Neal-P strain, which normally developed poorly in sandflies. Morphology of L. major LV 561 was not affected by galactosamine supplement: the lengths of parasite body and flagellum were similar in both sandfly groups. Two hypotheses are considered for the role of sandfly midgut lectin in Leishmania development in the vector midgut. One proposes that sandfly lectin kills Leishmania promastigotes, the other assumes that lectin blocks LPG-mediated binding of promastigotes to sandfly midgut microvilli.     

Inosine analogs. Their metabolism in mouse L cells and in Leishmania donovani

The growth of Leishmania donovani promastigotes and mouse L cells is differentially inhibited by several inosine analogs with modifications in the imidazole ring. The protozoal and mammalian cells also demonstrate differential metabolism of these analogs. 7-Deazainosine, 7-thia-7,9-dideazainosine, and formycin B were converted to their respective ATP analogs by both cell types. 8-Azainosine was converted to a GTP analog by mouse L cells; L. donovani did not metabolize this nucleoside. 9-Deazainosine and allopurinol riboside were metabolized only to their respective IMP analogs by L cells. L. donovani metabolized 9-deazainosine and allopurinol riboside to their ATP analogs and also metabolized 9-deazainosine to its GTP analog. All nucleosides studied were resistant to cleavage by either organism. From metabolism studies in the presence of a specific enzyme inhibitor, it was deduced that allopurinol riboside, formycin B, and 9-deazainosine were phosphorylated by at least two different routes in the mouse L cells. The metabolism of formycin B was inhibited 65% by the adenosine kinase inhibitor, 5-iodotubercidin, whereas the metabolism of allopurinol riboside (14% inhibition) and 9-deazainosine (0% inhibition) was only slightly affected by this inhibitor. The metabolism of allopurinol riboside and 9-deazainosine by L. donovani was not affected by 5-iodotubercidin. In contrast to the results of L cells, the metabolism of formycin B by L. donovani was also not affected by 5-iodotubercidin. The abilities of mouse L cells and L. donovani to metabolize these inosine analogs to the corresponding nucleotide analogs of ATP or GTP may be considered to be an activating step and correlates well with the respective cytotoxic effects of these compounds.     

An integrated pipeline for the development of novel panels of mapped microsatellite markers for Leishmania donovani complex, Leishmania braziliensis and Leishmania major

A panel of microsatellites mapped to the Leishmania genome might make it possible to find associations between specific loci and phenotypic traits. To identify such loci, a Perl programme was written that scans the sequence of a genome and writes all loci containing microsatellites to a MySQL database. The programme was applied to the sequences of the L. braziliensis, L. infantum and L. major genomes. The database is publicly available over the internet: http://www.genomics.liv.ac.uk/tryps/resources.html 'Microsatellite Locus Extractor', and allows the selection of mapped microsatellites that meet user-defined criteria from a specified region of the selected genome. The website also incorporates a primer design pipeline that will design primers to amplify the selected loci. Using this pipeline 12 out of 17 primer sets designed against the L. infantum genome generated polymorphic PCR products. A tailed primer protocol was used to label all microsatellite primers with a single set of labelled primers. To avoid the culture of parasites prior to genotyping, sets of nested PCR primers were developed to amplify parasite DNA eluted from microscope slides. The limit of detection was approximately 1.6 parasite equivalents. However, only 6/56 DNA from slides stored at ambient temperature for over 6 months gave positive PCR results.     

Leishmania major proteophosphoglycan is expressed by amastigotes and has an immunomodulatory effect on macrophage function

Proteophosphoglycan (PPG) is a newly described mucin-like glycoprotein found on the surface of Leishmania major promastigotes and secreted in the culture supernatant. We show here that antigenically similar PPGs are present in several Leishmania species. PPG could also be detected on the surface of amastigotes and in small, parasite-free vesicles in infected macrophages. Because of the similarity of its carbohydrate chains to lipophosphoglycan, a parasite receptor for host macrophages, PPG was tested for binding to macrophages. PPG bound to macrophages and was internalized in a time-dependent manner. PPG inhibited the production of tumor necrosis factor-alpha and synergized with interferon-gamma to stimulate the production of nitric oxide by macrophages. PPG may contribute to the binding of Leishmania to host cells and may play a role in modulating the biology of the infected macrophage at the early stage of infection.