Promastigote Cultures Research Articles

Cell mediated immunity and specific IgG1 and IgG2 antibody response in natural and experimental canine leishmaniosis

In the present study, we have followed up Leishmania infantum infection in dogs: (1) naturally infected; (2) experimentally infected with amastigotes; and (3) experimentally infected with culture promastigotes. The main objective was to evaluate the differences of the humoral and cellular immune responses of each group. Sera from 12 beagle dogs were analysed for total anti-leishmanial antibodies and IgG1 and IgG2 subclasses by enzyme-linked immunosorbent assay (ELISA). Lymphoproliferation to L. infantum antigen was also performed. All naturally infected animals were symptomatic with a marked humoral response. Dogs inoculated with amastigotes were asymptomotic and presented lower antibody titres than naturally infected. Dogs inoculated with culture promastigotes were asymptomotic with no significant humoral response. Strong proliferative responses to Leishmania antigen was observed in dogs inoculated with promastigotes. In our experimental model, IgG1 antibody levels presented a similar pattern in all infected animals, and IgG2 reactivity was high in naturally infected dogs.

Infectivity of promastigotes and amastigotes of Leishmaniainfantum in a canine model for leishmaniosis

Seven dogs experimentally infected with amastigotes or culture promastigotes of Leishmania infantum MON-1 were observed for a period of up to 38 months. The course of infection was monitored by clinical and parasitological examinations, haematological and serum protein analysis, and by anti-leishmania antibody levels. Two of the three amastigote-inoculated dogs developed a symptomatic infection with haematological and protein alterations, and a strong humoral immune response. The third dog was asymptomatic with no haematological or protein alterations and developed a steady humoral response. Four promastigote-inoculated dogs remained asymptomatic throughout the observation period, with only transient antibody responses to leishmanial antigen, and no haematological or protein alterations. The detection of the parasite in biological material obtained at necropsy showed that dogs with no clinical signs or other manifestations of disease may be infected. This indicates that asymptomatic carriers may be present in the canine population, but not identifiable by the usual serological tests, and suggests that epidemiological surveys based on serology may underestimate the prevalence of canine leishmaniosis and the parasite transmission risk.

Early detection of Leishmania promastigotes in dog bone marrow cultures by acridine orange stain

An acridine orange staining technique was evaluated in comparison with other well-known methods for the laboratory diagnosis of leishmaniasis. A higher number of promastigotes was found in Novy-MacNeal-Nicolle (NNN) cultures inoculated with canine bone marrow, when culture samples were stained with acridine orange vital stain, compared with those detected using either Giemsa staining or unstained wet mount examination. Based on our data the acridine orange stain is a useful and timely technique in reflecting the true numbers of microorganisms present in a culture and also enhances the visualization of the parasites. The present results warrant further studies with human samples from suspected leishmaniasis patients.

13C Labelling and electrospray mass spectrometry reveal a de novo route for inositol biosynthesis in Leishmania donovani parasite

In this paper we provide evidence for the presence of a de novo route for myo-inositol biosynthesis in the protozoan parasite Leishmania donovani, by the application of 13C labelling and electrospray ionisation mass spectrometry (ESMS). For this myo-[1-13C]inositol is prepared from D-[6-13C]glucose and biosynthetically incorporated in the parasite promastigote cell culture. Biosynthetic phosphatidylinositol (PI) and its hydrolysis products glycero-PI and inositol are analysed by ESMS and the isotopomeric ratio determined. The incorporation experiments show substantial isotopic dilution, indicating the presence of myo-inositol 1-phosphate synthase (MIP synthase) enzyme in the parasite; this is further confirmed by incorporation of D-[6-13C]glucose in the parasite phosphatidylinositol.

Stage-specific activity of pentavalent antimony against Leishmania donovani axenic amastigotes.

The standard treatment of human visceral leishmaniasis involves the use of pentavalent antimony (SbV) compounds. In recent years increasing numbers of clinical failures of treatment with SbV have been reported, probably due to the development of parasite resistance to this compound. The mode of action and mechanisms of resistance to SbV have not been fully elucidated. In the present study an axenic amastigote culture was used to study the in vitro responses of Leishmania donovani to SbV. Susceptibility to both sodium stibogluconate and meglumine antimoniate was found to be stage specific. Amastigotes were 73 to 271 times more susceptible to SbV than were promastigotes. As opposed to SbV, trivalent antimony (SbIII) was similarly toxic to both developmental stages. When promastigotes were transformed to amastigotes, susceptibility to meglumine antimoniate developed after 4 to 5 days, upon the completion of differentiation. In contrast, with transformation from amastigotes to promastigotes, resistance to meglumine antimoniate was acquired rapidly, within 24 h, before the completion of differentiation. The culture of promastigotes at an acidic pH (5.5) or at an elevated temperature (37 degrees C) alone did not lead to the appearance of SbV susceptibility, emphasizing the requirement of both these environmental factors for the development of SbV susceptibility. A previously isolated sodium stibogluconate (Pentostam)-resistant L. donovani mutant (Ld1S.20) is also resistant to meglumine antimoniate, indicating cross-resistance to SbV-containing compounds. In contrast, no cross-resistance was found with SbIII, suggesting a mechanism of SbV resistance different from that described in Leishmania tarentolae. These data show that L. donovani susceptibility to SbV is parasite intrinsic, stage specific, and macrophage independent.

In vitro Leishmania major promastigote-induced macrophage migration is modulated by sensory and autonomic neuropeptides.

Recruitment, migration and adherence of macrophages and their interaction with inoculated promastigotes are key steps in the initiation of the inflammatory process in cutaneous leishmaniasis. Parasite- and nervous system-derived factors might be involved in this process. In the present study the chemotactic activities of live, killed and sonicated Leishmania major promastigotes and of the promastigote culture supernatant as well as the L. major surface protease gp63 towards a murine macrophage cell line, Raw 264.7, were investigated, using the Boyden technique. The sensory neuropeptides SOM, CGRP and SP, and the autonomic neuropeptides VIP and NPY, were also investigated for possible modulatory effects on this chemotaxis, using the living promastigotes. Living promastigotes were the most efficient attractants for macrophages compared with other forms of the parasites. Prior incubation of the macrophages with the parasites completely abolished the chemotactic activity. This might indicate that the living promastigote chemotaxis is a receptor-mediated process. On the other hand, paraformaldehyde-killed promastigotes not only failed to induce macrophage chemotaxis but also inhibited it in comparison with the control. The surface protease gp63 tended to inhibit the macrophage chemotactic activity and the sonicate tended to stimulate it compared with controls. The culture supernatant had no effect, indicating that the chemoattractive factors putatively synthesized by the living promastigotes are not released to the surrounding medium. Somatostatin inhibited L. major promastigote-induced macrophage migration at a high concentration, 10(-6) M, while substance P inhibited it at both low concentrations, 10(-10) and 10(-9) M, and a high one, 10(-6) M, the last-mentioned having the greatest inhibitory effect. A stimulatory effect of calcitonin gene-related peptide was found at high concentrations, 10(-5) and 10(-6) M. Vasoactive intestinal peptide stimulated macrophage chemotactic activity at both a high, 10(-5) M, and at a low, 10(-9) M, concentration, the same concentration at which neuropeptide Y exerted its maximum inhibitory effect.

Human and murine immune responses to a novel Leishmania major recombinant protein encoded by members of a multicopy gene family.

Vaccination of BALB/c mice with Leishmania major promastigote culture filtrate proteins plus Corynebacterium parvum confers resistance to infection with L. major. To define immunogenic components of this protein mixture, we used sera from vaccinated mice to screen an L. major amastigote cDNA expression library. One of the immunoreactive clones thus obtained encoded a novel protein of L. major with a molecular mass of 22.1 kDa. The predicted amino acid sequence of this clone exhibited significant homology to eukaryotic thiol-specific-antioxidant (TSA) proteins. Therefore, we have designated this protein L. major TSA protein. Southern blot hybridization analyses indicate that there are multiple copies of the TSA gene in all species of Leishmania analyzed. Northern blot analyses demonstrated that the TSA gene is constitutively expressed in L. major promastigotes and amastigotes. Recombinant TSA protein containing an amino-terminal six-histidine tag was expressed in Escherichia coli with the pET17b system and was purified to homogeneity by affinity chromatography. Immunization of BALB/c mice with recombinant TSA protein resulted in the development of strong cellular immune responses and conferred protective immune responses against infection with L. major when the protein was combined with interleukin 12. In addition, recombinant TSA protein elicited in vitro proliferative responses from peripheral blood mononuclear cells of human leishmaniasis patients and significant TSA protein-specific antibody titers were detected in sera of both cutaneous-leishmaniasis and visceral-leishmaniasis patients. Together, these data suggest that the TSA protein may be useful as a component of a subunit vaccine against leishmaniasis.

Reduced Expression of Lipophosphoglycan (LPG) and Kinetoplastid Membrane Protein (KMP)-11 in Leishmania donovani Promastigotes in Axenic Culture

The expression of surface lipophosphoglycan (LPG) and the lipophosphoglycan-associated kinetoplastid membrane protein (KMP)-11 was studied in the strain AG83 of Leishmania donovani in axenic culture. The expression of LPG and KMP-11 decreased along with parasite virulence as a function of the time of the subculture.

Leishmania amazonensis:Long-Term Cultivation of Axenic Amastigotes Is Associated to Metacyclogenesis of Promastigotes

Cysne-Finkelstein, L., Temporal, R. M., Aguiar Alves, F., and Leon, L. L. 1998.Leishmania amazonensis:Long-term cultivation of axenic amastigotes is associated to metacyclogenesis of promastigotes.Experimental Parasitology89,58–62. AxenicLeishmania amazonensiscultures were obtained (with 2 years of follow-up) in Schneider's medium at pH 5.5 and a temperature of 32°C. Differentiation of promastigotes into amastigotes is directly associated with the metacyclogenesis rate of the original promastigotes. Promastigote cultures containing different percentages of metacyclic forms, as evaluated by the complement lysis test, resulted in axenic amastigote cultures containing a variable amount of undifferentiated forms. We observed that a culture originally containing a large amount of metacyclic forms (73%) underwent a complete differentiation process, resulting in a 100% amastigote culture with no promastigote forms. All other cultures (with a decreasing amount of metacyclic forms: 50% and fewer) failed to differentiate completely, resulting in cultures with increasing percentages of promastigote contamination. The amastigote/promastigote rate in these cultures has been maintained at the same level thus far. The axenicLeishmania amazonensisamastigotes were cryopreserved, thawed as such, and successfully cultivated under appropriately defined growth conditions.

Leishmania sp.:Growth and Survival Are Impaired by Ion Channel Blockers

In the present work we examined the effect of ion transport blockers on the growth and viability ofLeishmania sp.and on the infection of macrophages by the parasite. 4-aminopyridine and glibenclamide block voltage-dependent and K+ATP channels, respectively; amiloride is used to detect Na+channels and Na+/H+antiporters; and anthracene-9-carboxylic acid affects chloride channels. The EC50for promastigote cultures of three strains of theLeishmaniasubgenus, namely,Leishmania (Leishmania)NR,Leishmania (Leishmania) amazonensisLTB0016, andLeishmania (Leishmania) major,at their stationary phase of growth, were, respectively, 39, 46, and 464 μMfor 4-aminopyridine; 7, 0.8, and 10 μMfor glibenclamide and 66, 170, and 10 μMfor anthracene-9-carboxylic acid. The amiloride EC50for NR was 264 μMand 10 μMforL. (L.) major,but was never reached for LTB0016. Higher concentrations of the drugs impaired the exponential growth ofLeishmaniapromastigotes. These results suggest the susceptibility ofLeishmania sp.to blockers associated with K+and Cl−and to Na+or Na+/H+transport systems. Blockade of such systems might have impaired the survival of the parasites as promastigotes. In addition, it affected the persistence of parasites in host cells. Although the infection of the macrophage cell line J774 and peritoneal-exudate macrophages was not significantly decreased by concentrations of the drugs around the promastigotes' EC50, the survival of intracellular parasites decreased significantly in the presence of these drugs without affecting the viability of the macrophages. Some blockers consistently gave small EC50and significantly decreased the infection process as well as the survival of intracellular parasites. Thus, elucidation of their mechanism of action inLeishmaniais relevant, since they could represent a potential subject for the development of leishmanicidal drugs.

Molecular characterization of the heat-inducible LmSTI1 protein of Leishmania major

We have recently isolated a cDNA encoding the Leishmania major homologue of the yeast stress-inducible protein STI1. Southern blot analyses indicate that this protein is encoded by a single copy gene in L. major and that this gene is highly conserved throughout the Leishmania genus. The STI1 gene is constitutively expressed in both L. major promastigotes and amastigotes however, STI1 transcript levels can be upregulated in promastigotes by a shift in culture temperature from 26 to 37°C. Upregulation of transcript was detectable within 5′ of heat shock and continued to increase for a further 8 h before returning to constitutive levels. In addition, biosynthetic incorporation of [ 35S]methionine followed by immunoprecipitation revealed an increase in the level of nascent STI1 protein synthesized when promastigote cultures were shifted from 26 to 37°C. The L. major STI1 protein and the heat shock proteins Hsp83 and Hsp70 form a salt-sensitive complex in L. major promastigotes as evidenced by co-immunoprecipitation using an antiserum specific for L. major STI1. Furthermore, this complex can be reconstituted in vitro by adding recombinant STI1 containing an amino-terminal histidine tag to promastigote lysate and subsequent purification using metal chelate affinity chromatography.

Alpha-difluoromethylornithine-resistant cell lines obtained after one-step selection of Leishmania mexicana promastigote cultures.

Proliferation of Leishmania mexicana promastigotes in synthetic medium can be blocked by the depletion of intracellular polyamine pools induced by the presence of D,L-alpha-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase (ODC). Here we report that DFMO-resistant cell lines growing normally at DFMO levels of 10 mM have been obtained from non-proliferating cultures after a single-step selection in the presence of high concentrations of the drug. The DFMO-resistant promastigotes underwent a morphological transformation into an 'amastigote-like' form after incubation for several hours at gradually increasing temperatures up to 35 degrees C. The uptake of DFMO was not significantly altered in the drug-resistant cell lines but in both cases (promastigote and 'amastigote-like' forms) the ODC specific activity was increased approx. 15-fold over the normal enzymic levels found in the wild-type Leishmania. The enzyme affinities for its substrate and for DFMO gave very similar values in the drug-resistant promastigotes and the wild-type parasites. In contrast, ODC from the 'amastigote-like' Leishmania showed a higher affinity for ornithine and a decreased capacity for the binding of DFMO. An 80-fold amplification of the ODC gene and a corresponding increase in its transcripts have been detected in both DFMO-resistant Leishmania cell lines. The drug-resistant phenotypes with their characteristic morphologies, the increased levels of ODC activity and the amplification of the ODC gene have been stable for at least 6 months in the absence of selective pressure.

Activity of liposomal amphotericin B against experimental cutaneous leishmaniasis.

The polyene antibiotic amphotericin B is currently a second-line treatment for visceral leishmaniasis (VL) and mucocutaneous leishmaniasis. Lipid-amphotericin B formulations with lower toxicity than the parent drug that were developed for the treatment of systemic mycoses have proved to be an effective treatment for VL, especially AmBisome, a small unilamellar negatively charged liposome. In vitro, free amphotericin B was three to six times more active than the liposomal formulation AmBisome against both Leishmania major promastigotes in culture and amastigotes in murine macrophages. In a BALB/c L. major model of cutaneous infection, liposomal amphotericin B administered once a day on six alternate days by the intravenous route produced a dose-response effect between 6.25 and 50 mg/kg. Liposomal amphotericin B administered subcutaneously close to a lesion had no significant activity. Free drug was ineffective at nontoxic doses. The results suggest that liposomal amphotericin B may be useful in the treatment of cutaneous leishmaniasis.

Leishmania infantum: Infection of macrophages in vitro with promastigotes

Leishmania infantum promastigotes in axenic culture exhibit limited infectivity for mouse peritoneal macrophages (Mϕ) in vitro using standard culture conditions (37°C; 95% air/5% CO 2) compared to Leishmania donovani promastigotes which induce notable infections. The infectivity of logarithmic (log) and stationary (stat) phase promastigotes of L. infantum was enhanced by the addition of fresh homologous serum, but no amastigotes were observed after 4 days. Prolonged infections, including transformation and survival of intracellular amastigotes in BALB c mouse and hamster resident peritoneal Mϕ and Mϕ cell line J774.G8 were obtained by incubating Mϕ for 48 h at 26°C prior to standard culture. Enhanced infectivity was observed in a number of L. infantum strains subject to this transient thermal change.

Chronic lupoid leishmaniasis. Evaluation by polymerase chain reaction.

The cutaneous lesions in chronic lupoid leishmaniasis resemble those of lupus vulgaris, both clinically and histologically. The differential diagnosis is difficult and may depend on the detection of a few Leishmania amastigotes in the histologic sections, the growth of the promastigotes in cultures, or the identification of amastigotes by other techniques. Polymerase chain reaction was used to detect Leishmania amastigote DNA in tissue samples obtained from 65 patients with chronic lupoid leismaniasis, and the results were confirmed by Southern blot analysis. The histologic findings of a predominantly epithelioid cell granuloma surrounded by lymphocytic infiltrate in chronic lupoid leishmaniasis are very similar to those observed in lupus vulgaris. Extensive histologic examination of the sections in this series revealed occasional macrophages containing a few amastigotes in only 12 cases. Cultures in NNN medium yielded Leishmania promastigotes in 20 cases. Polymerase chain reaction studies using a Leishmania-specific primer identified Leishmania DNA in 30 of 63 cases, and those using a Mycobacterium tuberculosis primer were found to be negative for mycobacteria in 47 cases tested, including 11 cases with a positive tuberculin skin reaction. The histologic findings in chronic lupoid leishmaniasis resemble those of lupus vulgaris. Polymerase chain reaction studies were useful in identifying amastigotes in 30 (47.6%) of 63 cases. This study confirms the presence of DNA molecules of Leishmania amastigotes in samples of formalin-fixed, paraffin-embedded granulomatous tissue obtained from patients with chronic lupoid leishmaniasis.

A simple method for maintaining, detecting and recovering virulent Leishmania donovani in hamsters

The ability of hamsters ( Mesocricetus auratus) to retain amastigotes of Leishmania donovani at cutaneous sites was examined. Following intradermal inoculation of L. donovani stationary phase culture promastigotes in fore and hind footpads, nasal area and belly skin, cultures of aspirates taken fortnightly from these sites showed that amastigotes can survive in the skin for up to 10 months without visceralizing. Hairless cutaneous sites were better at retaining L. donovani amastigotes than the hairy belly skin. L. donovani promastigotes cultivated from aspirates of sites of inoculation were highly virulent. The skin is suggested as one of the sites where viscerotropic L. donovani can remain cryptic for a long time before the infection either visceralizes or is aborted. Skins of hamsters when inoculated intradermally can serve as an easy site for maintaining, detecting and recovering virulent L. donovani without killing the hamster.

Leishmania braziliensis: isolation of carbohydrate-containing antigen and possibility of its use in the immunodiagnosis of American cutaneous leishmaniasis.

Leishmania braziliensis is a causative agent of American Cutaneous Leishmaniasis (ACL). The 034-JCG strain, isolated from a patient from the northern region of Paraná State, Brazil, was cultivated in Blood Agar Base medium, lyophilized and submitted to phenol-water extraction. The extract was treated with RNase I. The carbohydrate containing-antigen (Ag-CHO) was immunogenic to rabbits and showed at least a fraction with some negative charge at pH 8.2. This antigen showed cross-reactivity with the phenol-water extract of the growth medium used for the culture of promastigotes and with the surface antigens of promastigotes. Its composition is: 24.3% of total sugars, from which 11.2% of galactose, 7.5% of mannose and 5.6% of ribose. Protein content was 5.4% and phosphate 18.5%. The antigenic activity was maintained after: repeated freezing-thawing; lyophilization; heating at 100 degrees C for 30 minutes; treatment with RNase, trichloroacetic acid and sodium metaperiodate. The precipitin line obtained is Periodic Acid Schiff positive. The application of the Ag-CHO in counterimmunoelectrophoresis reaction for the immunodiagnosis of ACL showed 60% sensitivity, and no cross-reaction with the five sera of Chagas' disease patients tested. The use of this antigen in a more sensitive technique, with more samples of sera, may improve these results.

Characterization of a polymorphic family of integral membrane proteins in promastigotes of different Leishmania species

Antibodies raised against a Leishmania major recombinant promastigote surface antigen 2(PSA-2) fragment recognized three major polypeptides of approximate M r 96 000, 80 000 and 50 000 in promastigotes of three Israeli isolates of L. major including the cloned line LRC-L137-V121, but detected a different array of polypeptides in other L. major isolates. The pattern was different both in number of polypeptides detected and their molecular weight. The antibodies to L. major PSA-2 also recognized polypeptides in L. tropica, L. donovani and very weakly in L. mexicana promastigotes and in Crithidia lucilliae. The number and size of the polypeptides was different in each species. In addition to the membrane-bound PSA-2 polypeptides we identified water-soluble forms of PSA-2 released in promastigote culture supermatants. Peptide maps of the various L. major PSA-2 membrane polypeptides showed they were different from each other. N-terminal amino acid sequence of the three polypeptides expressed by L. major PSA-2 gene and shown that it produces a M r35 000 polypeptide recognized by polymorphic family. Because of the extensive sequence similarity between the PSA-2 genes it has been difficult to assign protein products to individual genes. As a first step towards solving this problem, we have transfected into L. mexicana a genomic clone of a L. major PSA-2 gene and shown that it produces a M r 35 000 polypeptide recognized by monoclonal and polyclonal antibodies to L. major PSA-2.

Determination of Pentamidine in Leishmania Infantum Promastigotes by Ion-Paired Liquid Chromatography

Abstract Pentamidine is an aromatic diaraidine, which have recently been shown to be non-competitive inhibitors of putrescine uptake in parasitic protozoa. In order to understand the mechanism involved in Pentamidine uptake by parasitic protozoa cells, a HPLC analytical method for determination of pentamidine in Leishmania infantum promastigote cultures was developed. The influence on the capacity factor of various characteristic parameters in the mobile phase such as pH, percentage of methanol and temperature of column is studied, taking into account that pentamidine can form ion-pairs with sulphonic compounds, the influence of the length of the lateral chain and concentration of various compounds of this type are also studied. The results obtained in this study allow us to know the best chomatographic conditions for the determination of pentamidine in Leishmania infantum promastigotes and they can also be used for the optimization of analytical methods in order to determine pentamidine in other biologic...

Distribution of lipophosphoglycan-associated epitopes in differentLeishmania species and in African trypanosomes

Monoclonal antibody (mAb) CA7AE binds specifically to the phosphorylated Gal-beta 1,4-Man disaccharide repeat epitope of Leishmania donovani lipophosphoglycan (LPG). This mAb detected the repeat epitope in most but not all of a wide variety of Leishmania species and strains examined. MAb CA7AE also bound to both glycoprotein and carbohydrate antigens in medium from L. donovani promastigote cultures. Specifically, mAb CA7AE bound the delipidated form of LPG, the phosphoglycan, and a glycoprotein both of which are released into the medium by the parasite indicating that both share a specific phosphorylated carbohydrate epitope. The epitope was detected in sera from L. donovani-infected (kala-azar positive) patients when mAb CA7AE was used in an antigen-capture enzyme-linked immunosorbent assay (ELISA). MAb L157 is specific for a protein that is found associated with L. donovani LPG, the lipophosphoglycan-associated protein (LPGAP). This mAb bound to molecules in all 19 strains (representing 9 species) of Leishmania promastigotes and to molecules in 2 species of Trypanosoma procyclic culture forms. This wide distribution of the LPGAP epitope implies that it may have a conserved function, for example, in the biochemistry or arrangement of parasite surface molecules. In addition, since the LPGAP is involved in the stimulation of T lymphocyte proliferation, its wide distribution amongst different Leishmania species suggests that it may be an ideal molecule for testing as a vaccine for leishmaniasis.