Major Metacyclic Promastigotes Research Articles

Revisiting Leishmania GP63 host cell targets reveals a limited spectrum of substrates.

Colonization of host phagocytic cells by Leishmania metacyclic promastigotes involves several parasite effectors, including the zinc-dependent metalloprotease GP63. The major mode of action of this virulence factor entails the cleavage/degradation of host cell proteins. Given the potent proteolytic activity of GP63, identification of its substrates requires the adequate preparation of cell lysates to prevent artefactual degradation during cell processing. In the present study, we re-examined the cleavage/degradation of reported GP63 substrates when GP63 activity was efficiently neutralized during the preparation of cell lysates. To this end, we infected bone marrow-derived macrophages with either wild type, Δgp63, and Δgp63+GP63 L. major metacyclic promastigotes for various time points. We prepared cell lysates in the absence or presence of the zinc-metalloprotease inhibitor 1,10-phenanthroline and examined the levels and integrity of ten previously reported host cell GP63 substrates. Inhibition of GP63 activity with 1,10-phenanthroline during the processing of macrophages prevented the cleavage/degradation of several previously described GP63 targets, including PTP-PEST, mTOR, p65RelA, c-Jun, VAMP3, and NLRP3. Conversely, we confirmed that SHP-1, Synaptotagmin XI, VAMP8, and Syntaxin-5 are bona fide GP63 substrates. These results point to the importance of efficiently inhibiting GP63 activity during the preparation of Leishmania-infected host cell lysates. In addition, our results indicate that the role of GP63 in Leishmania pathogenesis must be re-evaluated.

Imaging Leishmania major Antigens in Experimentally Infected Macrophages and Dermal Scrapings from Cutaneous Leishmaniasis Lesions in Tunisia.

Leishmania major cutaneous leishmaniasis (CL) lesions are characterized by an intense process of parasite destruction and antigen processing that could limit microscopic amastigote detection. The aim of our study was to develop a direct immunofluorescence (DIF) assay for in situ visualization of L. major antigens and access its reliability in the routine diagnosis of CL. The developed DIF assay used IgG polyclonal antibodies produced in rabbits by intravenous injections of live L. major metacyclic promastigotes chemically coupled to fluorescein isothiocyanate. Applied to L. major infected RAW macrophages, corresponding macrophage-derived amastigotes and dermal scrapings from CL lesions, the immunofluorescence assay stained specifically Leishmania amastigotes and showed a diffuse Leishmania antigen deposit into cytoplasm of phagocytic cells. Reliability of DIF in CL diagnosis was assessed on 101 methanol-fixed dermal smears from 59 positive and 42 negative CL lesions diagnosed by direct microscopy and/or kDNA real-time PCR. Sensitivity and specificity of DIF was 98.3% and 100%, respectively, being more sensitive than microscopy (p < 0.001) and as sensitive as ITS1-PCR. ITS1-PCR-RFLP allowed Leishmania species identification in 56 out of the 58 DIF-positive smears, identifying 52 L. major, two L. infantum and two L. tropica cases, which indicates antigenic cross-reactivity between Leishmania species.

Evaluation of a New Topical Treatment for the Control of Cutaneous Leishmaniasis.

Leishmania major (L. major) causes cutaneous leishmaniasis in the Old World. The infection mostly induces a localized lesion restricted to the sand fly bite. The costs and the side effects of current treatments render imperative the development of new therapies that are affordable and easy to administrate. Topical treatment would be the ideal option for the treatment of cutaneous leishmaniasis. MF29 is a 3-haloacetamidobenzoate that was shown in vitro to inhibit tubulin assembly in Leishmania. Here, we tested a topical cream formulated with MF29. BALB/c mice were infected in the ear dermis with L. major metacyclic promastigotes and once the lesion appeared, mice were treated with different concentrations of MF29 and compared to the control group treated with the cream used as the vehicle. We observed that topical application of MF29 reduced the progression of the infection while control groups developed an unhealing lesion that became necrotic. The treatment decreased the type 2 immune response. Comparison with SinaAmphoLeish, another topical treatment, revealed that MF29 treatment once a day was sufficient to control lesion development, while application SinaAmphoLeish needed applications twice daily. Collectively, our data suggest that MF-29 topical application could be a promising topical treatment for cutaneous leishmaniasis.

NYVAC vector modified by C7L viral gene insertion improves T cell immune responses and effectiveness against leishmaniasis

The NYVAC poxvirus vector is used as vaccine candidate for HIV and other diseases, although there is only limited experimental information on its immunogenicity and effectiveness for use against human pathogens. Here we defined the selective advantage of NYVAC vectors in a mouse model by comparing the immune responses and protection induced by vectors that express the LACK (Leishmania-activated C-kinase antigen), alone or with insertion of the viral host range gene C7L that allows the virus to replicate in human cells. Using DNA prime/virus boost protocols, we show that replication-competent NYVAC-LACK that expresses C7L (NYVAC-LACK-C7L) induced higher-magnitude polyfunctional CD8+ and CD4+ primary adaptive and effector memory T cell responses (IFNγ, TNFα, IL-2, CD107a) to LACK antigen than non-replicating NYVAC-LACK. Compared to NYVAC-LACK, the NYVAC-LACK-C7L-induced CD8+ T cell population also showed higher proliferation when stimulated with LACK antigen. After a challenge by subcutaneous Leishmania major metacyclic promastigotes, NYVAC-LACK-C7L-vaccinated mouse groups showed greater protection than the NYVAC-LACK-vaccinated group. Our results indicate that the type and potency of immune responses induced by LACK-expressing NYVAC vectors is improved by insertion of the C7L gene, and that a replication-competent vector as a vaccine renders greater protection against a human pathogen than a non-replicating vector.

Targeting Leishmania major parasite with peptides derived from a combinatorial phage display library

Cutaneous leishmaniasis (CL) is a global problem caused by intracellular protozoan pathogens of the genus Leishmania for which there are no suitable vaccine or chemotherapy options. Thus, de novo identification of small molecules binding to the Leishmania parasites by direct screening is a promising and appropriate alternative strategy for the development of new drugs. In this study, we used a random linear hexapeptide library fused to the gene III protein of M13 filamentous bacteriophage to select binding peptides to metacyclic promastigotes from a highly virulent strain of Leishmania major (Zymodeme MON-25; MHOM/TN/94/GLC94). After four rounds of stringent selection and amplification, polyclonal and monoclonal phage-peptides directed against L. major metacyclic promastigotes were assessed by ELISA, and the optimal phage-peptides were grown individually and characterized for binding to L. major by monoclonal phage ELISA. The DNA of 42 phage-peptides clones was amplified by PCR, sequenced, and their amino acid sequences deduced. Six different peptide sequences were obtained with frequencies of occurrence ranging from 2.3% to 85.7%. The biological effect of the peptides was assessed in vitro on human monocytes infected with L. major metacyclic promastigotes, and in vivo on susceptible parasite-infected BALB/c mice. The development of cutaneous lesions in the right hind footpads of infected mice after 13 weeks post-infection showed a protection rate of 81.94% with the injected peptide P2. Moreover, Western blots revealed that the P2 peptide interacted with the major surface protease gp63, a protein of 63kDa molecular weight. Moreover, bioinformatics were used to predict the interaction between peptides and the major surface molecule of the L. major. The molecular docking showed that the P2 peptide has the minimum interaction energy and maximum shape complimentarity with the L. major gp63 active site. Our study demonstrated that the P2 peptide occurs at high frequency during the screening procedure, best inhibits L. major growth kinetics in vitro, and reduces cutaneous lesions in BALB/c mice, thus showing great promise in the development of new therapeutic molecules.

P010 Cytokine milieu and nitrite release during early challenge of human PBMC by Leishmania infantum and Leishmania major

Although Th1/Th2 paradigma may account for experimental Leishmaniasis pathogenesis in animals, data on cellular immunity mediators in early phase of human Leishmania infections are still limited and controversial. Therefore the aim of this study was to investigate some Th1 and Th2 cytokines, MCP-1, among the monocyte chemokines, and nitrite release from human PBMC stimulated by Leishmania infantum and Leishmania major metacyclic promastigotes (at a MOI of 2:1 and 0.2:1), the infective form of such parasite, in order to mimic the early phase of the natural infection. After 4 and 24 hours (h) of incubation of PBMC cultures stimulated by live metacyclic Leishmania promastigotes, supernatants were obtained and used for simultaneous determination of cytokine/chemokine levels by a cytokine biochip array on the Evidence Investigator analyser (Randox Laboratories Ltd., Crumlin, UK); as well as for quantitative determination of nitrite by Griess reagent. Results were presented as means ± SEM of three independent experiments each carried out in quadruplicate. Statistic analysis was carried out by Student’s t test. A p value < 0.05 was considered significant. During the very early phase (4 h) of L. major challenge TNF α , IL-1 β , IL-6 levels were significantly (p < 0.05) increased, and a negative correlation was found between such cytokine concentrations and Leishmania burden. On the contrary L. infantum did not cause any TNF α , IL-1 β and IL-6 release. At 24 h promastigote-stimulated TNF α , IL-1 β , IL-6 levels were significantly ( p < 0.05) higher than controls, although each concentration of L. major produced a greater release of these cytokines than the same concentration of L. infantum. Regarding chemokine release an interesting dose-dependent effect was observed for both species and the higher concentration of the promastigotes of L. infantum and L. major stimulated a more elevated release of MCP-1, yet this chemokine levels evaluated at 4 h were not significantly different vs. controls. The nitrite analysis was carried out in order to estimate the release of nitric oxide, whose nitrite is a stable metabolite. At 4 h the nitrite levels were slightly, but significantly ( p < 0.05), elevated in comparison to medium-treated controls following all the concentrations of the two species of Leishmania used. A different scenario was observed at 24 h: the lower parasite burden significantly enhanced the nitrite release after the stimulation with both Leishmania species; on the contrary the higher promastigote concentration of L. major and L. infantum did not cause any significant change of the nitrite level vs. control values. It appears that the larger amount of parasites may modulate both Th1 and Th2 cytokines evaluated and, more importantly, inhibit the release of nitric oxide, the host molecule that ultimately will kill such protozoa. On the other side promastigotes of L. infantum and L. major stimulated a significant and dose-dependent release of MCP-1. Our data may aid to spread a light on differential mechanisms used by Leishmania species in order to evade the host immune system.

Leishmania amazonensis impairs DC function by inhibiting CD40 expression via A2B adenosine receptor activation

Dendritic cells (DCs) play an essential role in the modulation of immune responses and several studies have evaluated the interactions between Leishmania parasites and DCs. While extracellular ATP exhibits proinflammatory properties, adenosine is an important anti-inflammatory mediator. Here we investigated the effects of Leishmania infection on DC responses and the participation of purinergic signalling in this process. Bone marrow-derived dendritic cells (BMDCs) from C57BL/6J mice infected with Leishmania amazonensis, Leishmania braziliensis or Leishmania major metacyclic promastigotes showed decreased major histocompatibility complex (MHC) class II and CD86 expression and increased ectonucleotidase expression as compared with uninfected cells. In addition, L. amazonensis-infected DCs, which had lower CD40 expression, exhibited a decreased ability to induce T-cell proliferation. The presence of MRS1754, a highly selective A(2B) adenosine receptor antagonist at the time of infection increased MHC class II, CD86 and CD40 expression in L. amazonensis-infected DCs and restored the ability of the infected DCs to induce T-cell proliferation. Similar results were obtained through the inhibition of extracellular ATP hydrolysis using suramin. In conclusion, we propose that A(2B) receptor activation may be used by L. amazonensis to inhibit DC function and evade the immune response.

Simultaneous gene expression profiling in human macrophages infected with Leishmania major parasites using SAGE

BackgroundLeishmania (L) are intracellular protozoan parasites that are able to survive and replicate within the harsh and potentially hostile phagolysosomal environment of mammalian mononuclear phagocytes. A complex interplay then takes place between the macrophage (MΦ) striving to eliminate the pathogen and the parasite struggling for its own survival.To investigate this host-parasite conflict at the transcriptional level, in the context of monocyte-derived human MΦs (MDM) infection by L. major metacyclic promastigotes, the quantitative technique of serial analysis of gene expression (SAGE) was used.ResultsAfter extracting mRNA from resting human MΦs, Leishmania-infected human MΦs and L. major parasites, three SAGE libraries were constructed and sequenced generating up to 28,173; 57,514 and 33,906 tags respectively (corresponding to 12,946; 23,442 and 9,530 unique tags). Using computational data analysis and direct comparison to 357,888 publicly available experimental human tags, the parasite and the host cell transcriptomes were then simultaneously characterized from the mixed cellular extract, confidently discriminating host from parasite transcripts. This procedure led us to reliably assign 3,814 tags to MΦs' and 3,666 tags to L. major parasites transcripts. We focused on these, showing significant changes in their expression that are likely to be relevant to the pathogenesis of parasite infection: (i) human MΦs genes, belonging to key immune response proteins (e.g., IFNγ pathway, S100 and chemokine families) and (ii) a group of Leishmania genes showing a preferential expression at the parasite's intra-cellular developing stage.ConclusionDual SAGE transcriptome analysis provided a useful, powerful and accurate approach to discriminating genes of human or parasitic origin in Leishmania-infected human MΦs. The findings presented in this work suggest that the Leishmania parasite modulates key transcripts in human MΦs that may be beneficial for its establishment and survival. Furthermore, these results provide an overview of gene expression at two developmental stages of the parasite, namely metacyclic promastigotes and intracellular amastigotes and indicate a broad difference between their transcriptomic profiles. Finally, our reported set of expressed genes will be useful in future rounds of data mining and gene annotation.

Leishmania major: Effects of proteophosphoglycan on reactive oxygen species, IL-12, IFN-γ and IL-10 production in healthy individuals

Protozoan parasites of the genus Leishmania secrete a range of proteophosphoglycans (PPG) known to be important for successful colonization of Leishmania in the sandfly and for virulence in the mammalian host. PPGs are a large family of extensively glycosylated proteins with some unusual and unique features. In this study we purified PPG from culture supernatant of Leishmania major metacyclic promastigotes. In discontinuous SDS–PAGE, PPG could not enter the resolving gel but after mild acid hydrolysis several bands resolved. Agarose gel electrophoresis and immunoblot analysis using monoclonal antibody (WIC 79.3) indicated that the PPG preparation consisted of heterogeneous molecules. Compositional analysis showed that the PPG preparation contained 67% glycan, 28% protein and 5% phosphate. Additionally, the effect of PPG on reactive oxygen species (ROS) production and induction of IL-10, IL-12 and IFN-γ secretion by human peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals was investigated. The water-soluble secreted form of PPG at a concentration of 1μg glycan/ml seems to be a potent inducer of ROS and IL-10 and to a lesser extent of IFN-γ and IL-12. Cytokines and ROS production was decreased in a dose-dependent manner as the concentration of PPG was increased to 100μg glycan/ml.

The prevention of the growth of Leishmania major progeny in BALB/c iron-loaded mice: a process coupled to increased oxidative burst, the amplitude and duration of which depend on initial parasite developmental stage and dose

BALB/c mice were given or not iron around the time of intradermal parasite inoculation, in their ears, of either 10 6 stationary-phase (designated “high-dose model”) or 10 3 Leishmania major metacyclic promastigotes (designated “low-dose model”). Iron-loaded mice in the high-dose model displayed delayed and limited pathogenic processes, whereas in the low-dose model, the mice remained ear lesion-free over 12 months post-parasite inoculation. These phenotypes were coupled to an increased leukocyte oxidative burst displayed mainly by neutrophils: it was early and transient in the high-dose model, whereas it was sustained in the low-dose model. In the latter model, injection of an antioxidant (diphenyleneiodonium chloride) at week 2 post- L. major inoculation resulted in a significant decrease in oxidative burst and reversed the protective status. The increased and sustained oxidative burst displayed by the neutrophils, the sustained presence of IL-12 (p40/p70)-positive leukocytes in the ear dermis, the low number of inflammatory leukocytes in the ear dermis and their concomitant high number in the draining lymph node are three related features that likely contribute to the shaping of the protective status, the onset and dynamic maintenance of which are antioxidant sensitive.

Is the reactive oxygen species-dependent-NF-κB activation observed in iron-loaded BALB/c mice a key process preventing growth of Leishmania major progeny and tissue-damage?

Systemic iron delivery to BALB/c mice, at time points surrounding the inoculation of 1000 Leishmania major metacyclic promastigotes intradermally in the ear results in the complete absence of onset and further development of ear lesion. In these iron-protected mice, the L. major intracellular progeny remains very low in both the ear and the draining lymph node. The iron-induced protective status is associated with a diphenyleneiodonium-sensitive sustained increased oxidative burst. We showed that iron-loaded mice developed no lesions at the site of the primary inoculation and were also resistant to reinoculation at a distant site (intradermal re-inoculation of 1000 metacyclic promastigotes in the contra-lateral ear). Interestingly, in the lymph node cell population recovered from iron-loaded mice at weeks 8 and 12 after the second parasite inoculation, and whatever the protective status studied – primary or resistant to re-inoculation – three potentially related features were observed: (i) NF-κB activation, (ii) enhanced TCR-mediated T lymphocyte proliferation, and (iii) high number of IFN-γ-positive CD4 +T cells. These results show a putative role of an iron-induced reactive oxygen species-dependent activation of NF-κB in the development of protective immunity against L. major.

Design and Development of Thermolytic DNA Oligonucleotide Prodrugs

Deoxyribonucleoside phosphoramidites functionalized with the thermolytic 2-(N-formyl-N-methyl)aminoethyl group for phosphorus protection (1a-d) have been prepared and employed in the solid-phase synthesis of CpG ODN fma1555. Given that this modified oligonucleotide can be converted to the immunomodulatory CpG ODN 1555 under neutral conditions at 37 degrees C, its biologic activity was demonstrated in vivo by studies showing that intraperitoneal administration of CpG ODN fma1555 in mice resulted in the activation of cytokine-secreting splenocytes. Furthermore, administration of CpG ODN fma1555 to mice that were challenged intradermally in the ear with live L. major metacyclic promastigotes, reduced the severity of Leishmania skin lesions over time to an extent similar to that obtained with CpG ODN 1555. In another infectious model experiment, CpG ODN fma1555 protected newborn mice from death (65% survival) when administered 3 days before infection with the aggressive Tacaribe (TCRV) virus. A comparable immunoprotection was obtained by treatment of TCRV-infected mice with CpG ODN 1555 administered on the same day of infection (45% survival). However, when TCRV-infected mice were treated with CpG ODN fma1555 on the day of infection, they died as a consequence of the relatively slow conversion of the oligonucleotide prodrug to the bioactive CpG ODN 1555. Co-administration of both CpG ODN 1555 and CpG ODN fma1555 to mice 3 days prior to TCRV infection or on the day of infection provided protection from death (45-65% survival) and thus widened the immunoprotection window against TCRV-infection.

The persistence, dissemination, and visceralization tendency of Leishmania major in Syrian hamsters

I monitored the persistence, dissemination, and the possible visceralization tendency of Leishmania major, the causative parasite of cutaneous leishmaniasis in North Africa and the Middle East in Syrian hamsters ( Mesocricetus auratus). Hamsters were inoculated subcutaneously in the hind footpad, with 1 × 10 6 L. major metacyclic promastigotes and were sacrificed at months 1, 3, 6 and 10 post-infection (pi). Skin lesions, blood, spleens, livers and kidneys were screened by both Giemsa-stained smears and a polymerase chain reaction (PCR) for detection of L. major amastigotes. A few weeks pi, 61.7% of the inoculated hamsters developed a cutaneous lesion only at the inoculation site, while 38.3% of them developed non-self-healed lesions at sites distant from the inoculation site. PCR identified all the positive stained smears as well as false-negative ones, indicating that PCR was more sensitive than stained smears. The results confirmed the dissemination and persistence of L. major amastigotes in all tissues examined, except the kidneys, for a period extending to 10 months, only in those hamsters suffering from disseminated cutaneous lesions. Parasite DNA was detected in the bloods starting from the first month pi and starting from month 3 pi in the spleens and livers. Some, but not all, the animals with disseminated infections proved to be positive for parasite DNA in their organs. Persistence of the L. major amastigotes in the tissues differed from those of other species causing visceral diseases. These findings demonstrate a possible visceralization tendency for L. major previously recorded for L. tropica and L. mexicana.

Thermolytic CpG-containing DNA oligonucleotides as potential immunotherapeutic prodrugs.

A CpG-containing DNA oligonucleotide functionalized with the 2-(N-formyl-N-methyl)aminoethyl thiophosphate protecting group (CpG ODN fma1555) was prepared from phosphoramidites 1a–d using solid-phase techniques. The oligonucleotide behaved as a prodrug by virtue of its conversion to the well-studied immunomodulatory CpG ODN 1555 through thermolytic cleavage of the 2-(N-formyl-N-methyl)aminoethyl thiophosphate protecting group. Such a conversion occurred at 37°C with a half-time of 73 h. The immunostimulatory properties of CpG ODN fma1555 were evaluated in two in vivo assays, one of which consisted of mice challenged in the ear with live Leishmania major metacyclic promastigotes. Local intradermal administration of CpG ODN fma1555 was as effective as that of CpG ODN 1555 in reducing the size of Leishmania lesions over time. In a different infectious model, CpG ODN 1555 prevented the death of Tacaribe-infected mice (43% survival) when administered between day 0 and 3 post infection. Administration of CpG ODN fma1555 three days before infection resulted in improved immunoprotection (60–70% survival). Moreover, co-administration of CpG ODN fma1555 and CpG ODN 1555 in this model increased the window for therapeutic treatment against Tacaribe virus infection, and thus supports the use of thermolytic oligonucleotides as prodrugs in the effective treatment of infectious diseases.

Behavior of Leishmania major metacyclic promastigotes during the course of infection and immune response development in resistant versus susceptible hosts

Little is known on the epitopes derived from metacyclic promastigotes of Leishmania that are important on the regulation or destruction of the parasite, as targets of immune attack in the vertebrate host. In this study we investigated an alternative method to obtain metacyclic promasigotes of Leishmania major, as evaluated by the course of infection and delayed-type hipersensitivity (DTH) in resistant versus susceptible inbred mice. Non-infective (procyclic) promastigotes of L. major recently transformed from tissue amastigotes were attached to a negatively charged glass-wool column, whereas metacyclic promastigotes were not bound to columns and could be easily recovered. Optimal chromatography conditions were validated through statistical analyses. Parasite average yield from glass wool columns and promastigote viability were estimated by light microscopy. Metacyclic promastigotes yielded 43.5% to 57.5%. Different patterns of cutaneous lesions were obtained in BALB/c (susceptible) and C57BL/6 (resistant) mice, the former with highly infective lesions induced by metacyclic promastigotes. DTH responses proved to be higher in groups of C57BL/6 mice which were infected with metacyclic promastigotes. These results indicate that the new method could be integrated with the investigation of metacyclogenesis of Leishmania in vivo.

The levels and patterns of cytokines produced by CD4 T lymphocytes of BALB/c mice infected with Leishmania major by inoculation into the ear dermis depend on the infectiousness and size of the inoculum.

The production of cytokines by CD4 lymph node T lymphocytes derived from BALB/c mice recently infected in the ear dermis with high (10(6) parasites) or low (10(3) parasites) doses of Leishmania major metacyclic promastigotes (MP) was examined over a 3-week period following inoculation. Results were compared with those obtained when mice were injected with less infectious parasite populations, namely, stationary-phase or log-phase promastigotes (LP). Cells were purified 16 h and 3, 8, and 19 days after inoculation, and the amounts of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) released in response to LACK (Leishmania homolog of receptors for activated C kinase) or total L. major antigens were assessed. We found that LACK-reactive T cells from mice inoculated with a high dose of parasites first produced IFN-gamma and later on IL-4; the level of IFN-gamma produced early by these cells was dependent upon the stage of the promastigotes inoculated, the highest level being reached with cells recovered from mice inoculated with the least infectious parasites, LP; sequential production of IFN-gamma and then of IL-4 also characterized L. major antigen-reactive CD4 T cells, suggesting that the early production of IFN-gamma does not impede the subsequent rise of IL-4 and finally the expansion of the parasites; after low-dose inoculation of MP, cutaneous lesions developed with kinetics similar to that of lesions induced after inoculation of 10(6) LP, but in this case CD4 T lymphocytes did not release IFN-gamma or IL-4 in the presence of LACK and neither cytokine was produced in response to L. major antigens before the onset of lesion signs. These results suggest the existence of a discreet phase in terms of CD4 T-cell reactivity for at least the first 8 days following inoculation, a time period during which parasites are able to grow moderately. In conclusion, the levels and profiles of cytokines produced by Leishmania-specific CD4 T lymphocytes clearly depend on both the stage of differentiation and number of parasites used for inoculation.

Immunological and pathological evaluation of rhesus macaques infected with Leishmania major

Cutaneous leishmaniasis, a parasitic infection causing ulcerating skin lesions, is an important disease worldwide and urgently requires a vaccine. Animal models that closely mimic human disease are essential for designing preventive vaccines against Leishmania major. We have evaluated both biologic and immunologic parameters of cutaneous L. major infection in nonhuman primates. Naı̈ve rhesus macaques or monkeys previously exposed to L. major were infected with varying doses of L. major metacyclic promastigotes, and lesion size was assessed over a 10-week period. Monkeys previously infected with L. major had much smaller lesions that resolved faster compared with those of naı̈ve monkeys in response to the two higher doses of infection. Moreover, eight of nine naı̈ve monkeys had parasites detected in their lesions during the course of the infection. In addition, the cellular infiltrate within the lesions was qualitatively and quantitatively different in naı̈ve versus previously infected monkeys. Finally, an ELIspot assay determined that the magnitude and kinetics of responses differed between previously infected and naı̈ve monkeys. Index descriptors and abbreviations: Leishmania major; L. amazonensis; L. brasiliensis; parasitic infection; Th1 cells; cellular immunity; vaccines; CpG ODN, cytosine phosphate guanosine oligodeoxynucleotides; PBMC, peripheral blood mononuclear cells; PVI, perivascular inflammation; DI, dermal inflammation; SLA, soluble Leishmania antigen

Leishmania major reaches distant cutaneous sites where it persists transiently while persisting durably in the primary dermal site and its draining lymph node: a study with laboratory mice.

So far, studies of Leishmania persistence in mice have used injections of parasites administered either intravenously in the tail vein or subcutaneously in the footpad. These routes poorly reflect the natural conditions when the sandfly delivers metacyclic promastigotes intradermally. In this study B10D2 and BALB/c mice were inoculated within the ear dermis with 10(4) Leishmania major metacyclic promastigotes. The parasite load was monitored by quantitative PCR in different tissues from the dermal inoculation site to distant tissues. The two sites of multiplication and persistence of parasites were the site of L. major inoculation and the draining lymph node (DLN), with a different pattern in the two mouse inbred lines. These two organs were the only sites harboring parasites 12 months postinoculation, with the DLN of BALB/c mice harboring around 10(7) parasites, a stable load from months 3 to 12. In these two sites, 8 and 12 months after inoculation, interleukin 4 (IL-4), gamma interferon, and inducible nitric oxide synthase transcripts parallel the parasite load while IL-10 transcript levels remain high. In addition, at early time points until month 3, parasite DNA was also detected in distant tissues such as the contralateral noninoculated ear or the tail skin, indicating that blood was at least transiently disseminating the parasites. In contrast, L. major DNA in liver, spleen, and femoral bone marrow remained sporadic in mice of both lines. This study is discussed within the framework of Leishmania transmission from the vertebrate host to the sandfly vector, a complex process still poorly understood.

Evolution of lesion formation, parasitic load, immune response, and reservoir potential in C57BL/6 mice following high- and low-dose challenge with Leishmania major.

A model of cutaneous leishmaniasis using 10(2) Leishmania major metacyclic promastigotes inoculated into the footpads of genetically resistant C57BL/6 mice was studied in order to more accurately reproduce the evolution of lesion formation and the kinetics of parasite growth and immune response as they might occur in naturally exposed reservoirs and in human hosts. In contrast to the more conventional experimental model employing 10(6) metacyclic promastigotes, in which the rapid development of footpad lesions was associated with an increasing number of amastigotes in the site, the low-dose model revealed a remarkably "silent" phase of parasite growth, lasting approximately 6 weeks, during which peak parasitic loads were established in the absence of any overt pathology. Footpad swelling was observed after 6 weeks, coincident with the onset of parasite clearance and with production of high levels of interleukin-12 (IL-12) and gamma interferon (IFN-gamma) in draining lymph nodes. Low-dose challenge of IL-12- and IFN-gamma-depleted or -deficient mice provided strong evidence that the induction or expression of cellular immunity is essentially absent during the first 6 to 8 weeks of intracellular growth, since the concentration of amastigotes in the site was not enhanced compared to that for wild-type animals during this time. By monitoring the ability of infected mice to transmit parasites to vector sand flies, it was observed that following low-dose challenge, footpads without apparent lesions provided an efficient source of parasites for exposed flies and that the low-dose challenge actually extended the duration of parasite transmissibility during the course of infection.

Characterization of the Elongating α-d-Mannosyl Phosphate Transferase from Three Species of Leishmania Using Synthetic Acceptor Substrate Analogues

Leishmania express lipophosphoglycans and proteophosphoglycans that contain Galbeta1-4Manalpha1-P phosphosaccharide repeat structures assembled by the sequential addition of Manalpha1-P and betaGal. The synthetic acceptor substrate Galbeta1-4Manalpha1-P-decenyl and a series of analogues were used to probe Leishmania alpha-D-mannosyl phosphate transferase activity. We show that the activity detected with Galbeta1-4Manalpha1-P-decenyl is the elongating alpha-D-mannosyl phosphate transferase associated with lipophosphoglycan biosynthesis (eMPT(LPG)). Differences in the apparent K(m) values for the donor and acceptor substrates were found using L. major, L. mexicana, and L. donovani promastigote membranes, but total activity correlated with the number of lipophosphoglycan repeats. Further comparisons showed that lesion-derived L. mexicana amastigotes, that do not express lipophosphoglycan, lack eMPT(LPG) and that nondividing L. major metacyclic promastigotes contain 5-fold less eMPT(LPG) activity than dividing procyclic promastigotes. The fine specificity of promastigote eMPT(LPG) activity was determined using 24 synthetic analogues of Galbeta1-4Manalpha1-P-decenyl. The three species gave similar results: the negative charge of the phosphodiester and the C-6 hydroxyl of the alphaMan residue are essential for substrate recognition, the latter most likely acting as a hydrogen bond acceptor. The C-6' hydroxyl of the betaGal residue is required for substrate recognition as well as for catalysis. The rate of Manalpha1-P transfer declines with increasing acceptor substrate chain length. The presence of a monosaccharide substituent at the C-3 position of the terminal betaGal residue abrogates Man-P transfer, showing that chain elongation must precede side chain modification during lipophosphoglycan biosynthesis. In contrast, substitution of the penultimate phosphosaccharide repeat does not abrogate transfer but is slightly stimulatory in L. mexicana and inhibitory in L. major.