Efficacy of the vaccination of C57BL/6 mice against infection with different species of Leishmania

Antigens were isolated from lysates of promastigotes of Leishmania infantum by electro-elution from polyacrylamide gels. Antigens with respective molecular weights for F2 = 94-67 kd; F5 = 30-20 kd and F6 below 20 kd, were injected intravenously in C 57 BL/6 mice. The immune sera were studied by indirect immunofluorescence; an in vivo test showed their inhibitory effect on the life cycle of several Leishmania species from the Old and the New world. Furthermore, mice immunized with F2, F5 or F6 were protected against an infection by Leishmania major. These results demonstrate that vaccination is efficient in mice differing genetically for either susceptibility (BALB/c) or partial resistance (C 57 BL/6) to Leishmania infections. Recently, we reported that a single monoclonal antibody raised against Leishmania infantum can prevent the development of Leishmania major and Leishmania mexicana amazonensis. Indeed, BALB/c mice injected subcutaneously with promastigotes pre-treated with this monoclonal antibody, did not present any cutaneous lesions over a period of 3 months. Using a mouse in vivo model--intraperitoneal injection of TG 180 mouse sarcoma cells along with monoclonal antibody pre-treated promastigotes--such an antibody-mediated inhibition was also observed against Old and New World Leishmania species. Protective monoclonal antibodies recognized by immunoblotting technique three antigenic fractions (40, 70 and 113 kd) common to several Leishmania species. Antigenic preparations from Leishmania infantum extracts were isolated by electro-elution from polyacrylamide gels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leishmania parasites (Kinetoplastida: Trypanosomatidae) reversibly inhibit visceral muscle contractions in hemimetabolous and holometabolous insects

Female sand flies can acquire protozoan parasites in the genus Leishmania when feeding on an infected vertebrate host. The parasites complete a complex growth cycle in the sand fly gut until they are transmitted by bite to another host. Recently, a myoinhibitory peptide was isolated from Leishmania major promastigotes. This peptide caused significant gut distension and reversible, dose-dependent inhibition of spontaneous hindgut contractions in the enzootic sand fly vector, Phlebotomus papatasi. The current study further characterizes myoinhibitory activity in L. major and other kinetoplastid parasites, using the P. papatasi hindgut and other insect organ preparations. Myoinhibitory activity was greatest in cultured promastigotes and in culture medium in late log-phase and early stationary-phase, coinciding with development of infective Leishmania morphotypes in the sand fly midgut. L. major promastigote lysates inhibited spontaneous contractions of visceral muscle preparations from hemimetabolous (Blattaria and Hemiptera) and holometabolous (Diptera) insects. Inhibition of visceral muscle contractions in three insect orders indicates a conserved mode of action. Myoinhibitory activity was detected also in Leishmania braziliensis braziliensis, a Sudanese strain of Leishmania donovani, and the kinetoplastid parasite Leptomonas seymouri. Protozoan-induced myoinhibition mimics the effect of insect myotropins. Inhibiting host gut contractions protects Leishmania parasites from being excreted after blood meal and peritrophic matrix digestion, allowing development and transmission of infective forms.     

Evidence of transferrin binding sites on the surface of Leishmania promastigotes.

A glycoprotein of 78,000 molecular mass (78 kDa), associated with the membrane of Leishmania infantum promastigotes, was identified and immunopurified by monoclonal antibody (mAb) LD9 produced against isolated membrane preparations. mAb LD9 was subsequently found to bind to human transferrin, also of 78 kDa. Binding of LD9 to transferrin was completely abolished when the mAb was preabsorbed by Leishmania membranes, thereby indicating that the 78-kDa Leishmania membrane-associated glycoprotein and transferrin have common antigenic epitope(s). The 78-kDa Leishmania membrane-associated protein was released in soluble nonaggregated form by mild treatment with acetic acid saline. Anti-transferrin polyclonal antibodies, recognized both the membrane-associated and the soluble form of the 78-kDa glycoprotein. The 78-kDa soluble form was characterized further as an iron-containing protein. The above data combined with iron uptake by promastigotes as demonstrated by the Prussian blue reaction indicate that the 78-kDa Leishmania membrane-associated glycoprotein is transferrin. The binding of 125I-human transferrin to Leishmania-purified membrane preparations was then investigated. The results indicate the presence of a high affinity saturable binding site (Kd = 2.2 10(-8) M) that is specific for transferrin. We suggest that the 78-kDa glycoprotein recognized by mAb LD9 is transferrin that binds to the surface of Leishmania promastigotes via a transferrin receptor.     

Evaluation of serological assays based on a novel excreted antigen preparation for the diagnosis of Cutaneous Leishmaniasis in Panama

The objective of the present study was to determine the efficacy of prototype diagnostic serological assays for American Cutaneous Leishmaniasis (ACL) in Panama. As such, we prospectively sampled 100 cutaneous leishmaniasis case-patients and tested their sera in two serological assays based upon novel soluble antigen preparations made from propagating the parasites in a protein-free, serum free media. Using serum and a Leishmania mexicana antigen preparation to sensitize plates, the assay correctly identified 89% of the case-patients. While using serum with an antigen preparation from Leishmania braziliensis, the assay correctly identified 71% of the patients. Concerning both test formats, performance was near equal in true positive and presumptive positive subsets demonstrating the improved sensitivity of these assays over reference methods of choice. Since the incidence of leishmaniasis in Panama has increased dramatically in the past 10 years, these assays may be useful in clinical and epidemiological studies and control programs.     

Functional expression of a multidrug P-glycoprotein transporter of Leishmania

P-glycoprotein (Pgp) transporters play an important role in multidrug resistance in eukaryotic cells and in protozoan parasites such as Leishmania. To search for new reversal agents of the Leishmania tropica Pgp, we developed a screening assay using the Baculovirus-insect cell expression system. We demonstrated a MgATP-dependent, vanadate-sensitive transport of Hoechst 33342 in membrane preparations of Sf9 insect cells expressing Pgp. We have found that dihydro-beta-agarofuran sesquiterpenes from Maytenus cuzcoina inhibited Hoechst 33342 transport that correlates with their reversal effect in a multidrug-resistant L. tropica line overexpressing Pgp. The results suggest that Sf9 cell membrane Hoechst 33342 transport system represents an efficient tool for examining the interactions of Leishmania Pgp with pharmacological agents.     

Axenic promastigote forms of Leishmania (Viannia) lainsoni as an alternative source for Leishmania antigen production

The present study demonstrates that axenic cultures of Leishmania (Viannia) lainsoni produce larger cell masses in NNN-LIT medium, as well as higher amounts of total proteins in cell extracts, than Leishmania (Leishmania) amazonensis. Antigenicity of L. (V.) lainsoni whole promastigotes is similar to that of L. (L.) amazonensis, as demonstrated by an indirect immunofluorescence diagnostic test using sera from human patients and dogs infected with visceral leishmaniasis. Infectivity of the L. (V.) lainsoni strain used in the present work was demonstrated by the detection by transmission-electron microscopy of tissue amastigotes in skin lesion samples from an experimentally infected hamster. Incubation of lesion fragments in NNN-LIT medium allowed us to obtain promastigote forms, which could be cultivated successfully in vitro. lsoenzyme analysis of such promastigotes confirmed the parasite strain as L. (V.) lainsoni, as compared to other Leishmania reference strains. Our data indicate that L. (V.) lainsoni is a useful alternative source for antigen production as well for use in assays that depend on large cell volumes of Leishmania spp. parasites.     

The expression of biologically active human p53 in Leishmania cells: a novel eukaryotic system to produce recombinant proteins.

We have investigated the use of Leishmania cells as a novel eukaryotic expression system for the production of recombinant protein. These cells are easy to maintain, requiring no CO2 incubator or shaker, and can be grown in standard tissue culture media. Leishmania cells can be readily transfected with plasmid DNA by electroporation and transformants selected with antibiotic resistance. Recent studies have shown that it is possible to express foreign genes in Leishmania for the purpose of understanding the biology of this protozoan cell. In the present study we report the use of this system as a means of producing a biologically functional human p53 protein. The conformation of the p53 protein is critical for its ability to bind specific DNA sequences. It is demonstrated that Leishmania-synthesized human p53 is phosphorylated and can bind specifically to its enhancer DNA sequence. These data demonstrate that Leishmania may represent a simple eukaryotic expression system for the production of biologically active recombinant proteins.     

Respiratory chain components of Leishmania tropica promastigotes.

Crude preparations of kinetoplast vesicles were used to investigate the respiratory chain components in Leishmania tropica promastigotes. In difference spectra from enzymically and chemically reduced preparations, cytochrome b was the predominant component. By utilizing special assays designed to minimize the influence of cytochrome b on difference spectra, cytochromes a, a3 and c555 were demonstrated. Difference spectra from chemically reduced preparations indicated that pyridine nucleotides (NADH) and flavoproteins were also part of the respiratory chain. The presence of these components as well as their response to respiratory inhibitors and ascorbate provide evidence for the presence of a typical trypanosomatid respiratory chain in L. tropica promastigotes.     

Trypanosomatid and fungal glycolipids and sphingolipids as infectivity factors and potential targets for development of new therapeutic strategies

Several (glyco)(sphingo)lipids from different human pathogens have been characterized, and frequently many of these molecules are participating in host-pathogen interaction. In Leishmania (Leishmania) amazonensis, for example, amastigotes present on their surface glycosphingolipids (GSLs) with the structure Galbeta1-3Galalpha, which is recognized by 30 kDa receptor of macrophages. Furthermore, other Leishmania species, such as Leishmania (Leishmania) major and Leishmania (Viannia) braziliensis present glycosylinositolphospholipids (GIPLs) which are involved in Leishmania-macrophage interaction. It is worth to mention that these antigens are not expressed in mammalian cells. Leishmania promastigotes also present inositol phosphorylceramide (IPC), a unique sphingolipid characteristic of fungi and plants. It was observed that IPC synthesis is essential for parasite division, since Aureobasidin A, an inhibitor of IPC synthase, inhibited significantly promastigote and amastigote growths. Recently, it was also demonstrated that GIPLs, IPC and sterols are preferentially present in the parasite membrane microdomains resistant to Triton X-100 at 4 degrees C. The disruption of these microdomains by incubating parasites with methyl-beta-cyclodextrin inhibited significantly macrophage infectivity by Leishmania. Other pathogens, such as fungi, also present unique glycolipids which may have an important role for the fungal development and/or disease establishment. Taking together these results, this review will discuss different biological roles for (glyco)(sphingo)lipids of different pathogens.     

Antigenic extracts of Leishmania braziliensis and Leishmania amazonensis associated with saponin partially protects BALB/c mice against Leishmania chagasi infection by suppressing IL-10 and IL-4 production

This study evaluated two vaccine candidates for their effectiveness in protecting BALB/c mice against Leishmania chagasi infection. These immunogenic preparations were composed of Leishmania amazonensis or Leishmania braziliensis antigenic extracts in association with saponin adjuvant. Mice were given three subcutaneous doses of one of these vaccine candidates weekly for three weeks and four weeks later challenged with promastigotes of L. chagasi by intravenous injection. We observed that both vaccine candidates induced a significant reduction in the parasite load of the liver, while the L. amazonensis antigenic extract also stimulated a reduction in spleen parasite load. This protection was associated with a suppression of both interleukin (IL)-10 and IL-4 cytokines by spleen cells in response to L. chagasi antigen. No change was detected in the production of IFN-γ. Our data show that these immunogenic preparations reduce the type 2 immune response leading to the control of parasite replication.     

The heat shock proteins,Hsp70 and Hsp83, of Leishmania infantum are mitogens for mouse B cells

Extending earlier studies, this report demonstrates that Leishmania infantum heat shock proteins (Hsps), Hsp70 and Hsp83, expressed as recombinant proteins fused to the Escherichia coil maltose-binding protein (MBP), are potent mitogens for murine splenocytes. The response was not due to lipopolysaccharide (LPS) because the stimulatory activity of Hsp preparations was sensitive to boiling and trypsin treatments, whereas the corresponding activity of LPS was resistant to both treatments. It was found that in vitro incubation of spleen cells with the Leishmania Hsps leads to the expansion of CD220-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. In fact, splenocytes from B cell-deficient mice did not proliferate in response to the Leishmania Hsps. In contrast, spleen cells from athymic nude mice were significantly stimulated by these recombinant proteins as an indication that the MBP-Hsp70 and MBP-Hsp83 recombinant proteins behave as T cell-independent mitogens of B cells. Furthermore, both proteins were able to induce proliferation on B cell populations purified from BALB/c spleen.     

Studies on the Attachment of Leishmania Flagella to Sand Fly Midgut Epithelium

An in vitro assay was developed to study the recognition mechanism for attachment of Leishmania flagella to sand fly midgut epithelium. Frozen sections of sand fly guts were incubated with Hagella preparations, and probed with a flagella-specific monoclonal antibody. Tissue-specific adhesion of flagella to midgut epithelium was demonstrated by indirect immunofluorescence. None of the 13 sugars, screened to test for possible lectin-mediation, appeared to significantly inhibit the adhesion of flagella to gut sections. Similarly no inhibition was achieved by incubating flagella with pep 63 which inhibits the promastigote-macrophage recognition mechanism. Significant inhibition was attained by incubating flagella preparations with a monoclonal antibody which binds to a flagellar membrane-component. The possible relevance of the described mechanism for the biology of Leishmania in their sand fly hosts, is discussed.     

Studies on the attachment of Leishmania flagella to sand fly midgut epithelium

An in vitro assay was developed to study the recognition mechanism for attachment of Leishmania flagella to sand fly midgut epithelium. Frozen sections of sand fly guts were incubated with flagella preparations, and probed with a flagella-specific monoclonal antibody. Tissue-specific adhesion of flagella to midgut epithelium was demonstrated by indirect immunofluorescence. None of the 13 sugars, screened to test for possible lectin-mediation, appeared to significantly inhibit the adhesion of flagella to gut sections. Similarly no inhibition was achieved by incubating flagella with pep 63 which inhibits the promastigote-macrophage recognition mechanism. Significant inhibition was attained by incubating flagella preparations with a monoclonal antibody which binds to a flagellar membrane-component. The possible relevance of the described mechanism for the biology of Leishmania in their sand fly hosts, is discussed.     

The dendritic cell receptor DC-SIGN discriminates among species and life cycle forms of Leishmania

Infection of dendritic cells by the human protozoal parasite Leishmania is part of its survival strategy. The dendritic cell receptors for Leishmania have not been established and might differ in their interactions among Leishmania species and infective stages. We present evidence that the surface C-type lectin DC-SIGN (CD 209) is a receptor for promastigote and amastigote infective stages from both visceral (Leishmania infantum) and New World cutaneous (Leishmania pifanoi) Leishmania species, but not for Leishmania major metacyclic promastigotes, an Old World species causing cutaneous leishmaniasis. Leishmania binding to DC-SIGN was found to be independent of lipophosphoglycan, the major glycoconjugate of the promastigote plasma membrane. Our findings emphasize the relevance of DC-SIGN in Leishmania-dendritic cell interactions, an essential link between innate and Leishmania-specific adaptive immune responses, and suggest that DC-SIGN might be a therapeutic target for both visceral and cutaneous leishmaniasis     

Minicircle variable region probes for characterization of Leishmania (Viannia) species.

The minicircle molecules present in the kinetoplast DNA (kDNA) network constitute a particularly useful molecular tool because they are a multicopy target and present a variable region that differs among minicircle classes in the same network. Using the polymerase chain reaction (PCR) and a set of primers directed outwardly from the minicircle conserved region, it is possible to prepare molecular probes representing the pool of variable regions from the different minicircle classes in the kDNA. In order to examine the specificity of the minicircle variable region as hybridization probes in Leishmania (Viannia) species, such fragments were amplified from reference strains and from a panel of isolates representing the zymodeme diversity of Leishmania (Viannia) in Colombia. The size of the amplified products was conserved in Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, and Leishmania (Viannia) panamensis (650 bp) and diverged in Leishmania (Viannia) equatorensis and Leishmania (Viannia) colombiensis (850 bp). The amplified products were further hybridized to variable region pools of Leishmania braziliensis, Leishmania panamensis, Leishmania guyanensis, and Leishmania equatorensis reference strains. The results obtained from the hybridization experiments support this approach as a means of defining relationships among strains. Hybridization allowed homologies to be perceived, whereas restriction fragment length analysis of the amplified products yielded strain-specific profiles. Apparently, L. (V.) equatorensis and L. (V.) colombiensis minicircle variable regions have no or only low homology with those of other Leishmania (Viannia) species, showing the divergence of those species within the subgenus.     

Occurrence of co-infection by Leishmania (Leishmania) chagasi and Trypanosoma (Trypanozoon) evansi in a dog in the state of Mato Grosso do Sul, Brazil

A natural case of co-infection by Leishmania and Trypanosoma is reported in a dog (Canis familiaris) in south- western state of Mato Grosso do Sul, Brazil. Both amastigote and trypomastigote forms were observed after Giemsa staining of cytological preparations of the dog's bone marrow aspirate. No parasite was detected using medium culture inoculation of the sample. DNA obtained from the bone marrow aspirate sample and from the blood buffy coat was submitted to polymerase chain reaction (PCR) with a set of rDNA-based primers S4/S12. The nucleotide sequence of the PCR product was identical to that of Trypanosoma (Trypanozoon) evansi. The S4/S12 PCR was then used as template in a nested-PCR using a specific Leishmania set S17/S18 as primers, to explain the amastigote forms. The nucleotide sequence of the new PCR product was identical to that of Leishmania (Leishmania) chagasi. This case, as far as we know, is the first report of a dog co-infected with these parasites, suggesting that besides L. (L.) chagasi, the natural transmission of T. (T.) evansi occurs in the area under study.     

Alkylacylglycerolipid domain of GPI molecules of Leishmania is responsible for inhibition of PKC-mediated c-fos expression

Glycosylphosphatidylinositols (GPIs) are the most abundant molecules present in the membranes of the parasitic protozoa Leishmania responsible for multiple forms of leishmaniasis. Among the prominent biological activities displayed by the major Leishmania GPIs [lipophosphoglycan (LPG) and glycoinositolphospholipids (GIPLs)] is the inhibition of macrophage functions such as the protein kinase C (PKC)-dependent signaling pathway. The bioactivity of Leishmania GPIs is in contrast to Trypanosoma brucei and Plasmodium falciparum GPIs, which activate the macrophage functions. To address the question as to which structural domain of Leishmania GPIs is responsible for dramatic down-regulation of PKC-dependent transient c-fos expression, the chemically synthesized defined alkylacylglycerolipids domain of corresponding GPIs, and LPG and GIPLs isolated from Leishmania donovani, were evaluated for inhibition of PKC and c-fos expression in macrophages. The results presented here demonstrate that the unusual lipid domain of Leishmania GPIs is primarily responsible for inhibition of PKC-dependent transient c-fos expression.     

The immunomodulatory effect of plant lectins: a review with emphasis on ArtinM properties

Advances in the glycobiology and immunology fields have provided many insights into the role of carbohydrate-protein interactions in the immune system. We aim to present a comprehensive review of the effects that some plant lectins exert as immunomodulatory agents, showing that they are able to positively modify the immune response to certain pathological conditions, such as cancer and infections. The present review comprises four main themes: (1) an overview of plant lectins that exert immunomodulatory effects and the mechanisms accounting for these activities; (2) general characteristics of the immunomodulatory lectin ArtinM from the seeds of Artocarpus heterophyllus; (3) activation of innate immunity cells by ArtinM and consequent induction of Th1 immunity; (4) resistance conferred by ArtinM administration in infections with intracellular pathogens, such as Leishmania (Leishmania) major, Leishmania (Leishmania) amazonensis, and Paracoccidioides brasiliensis. We believe that this review will be a valuable resource for more studies in this relatively neglected area of research, which has the potential to reveal carbohydrate targets for novel prophylactic and therapeutic strategies.     

Haemoflagellates: commercially available liquid media for rapid cultivation.

The successful cultivation of a variety of haemoflagellates in three different liquid media is reported. These media include medium 199, Grace's insect tissue-culture medium and Schneider's drosophila medium, each in combination with 30% (v/v) foetal calf serum. These media were used to cultivate Old and New World species of visceral and cutaneous human Leishmania, as well as Leishmania species isolated from sandflies, rodents, and reptiles. Four strains of Trypanosoma cruzi, an isolate of T. R-angeli and and an isolate of T. lewisi have also been cultivated in these media. One or more of these media have been used to cultivate 121 strains of haemoflagellates, including at least 14 different species (11 Leishmania and 3 Trypanosoma) and many geographic isolates or strains. The Leishmania include L. braziliensis, L. peruviana, L. mexicana, L. tropica, L. donovani, L. chagasi, L. enriettii, L. hertigi, L. hoogstraali, L. adleri, and L. agamae. Using the Schneider's based medium, we have obtained primary isolates of both cutaneous and visceral Leishmania of man and of experimentally infected laboratory rodents and canines. Freeze-dried preparations of the Schneider's based medium that were reconsituted with distilled water after 24 months of storage at ambient temperature have proven to be suitable cultivation media. This feature makes the media valuable field tools. The various species of human Leishmania cultivated in these media have in our experience demonstrated no differences in growth rate, viability after liquid nitrogen preservation, or infectivity for laboratory animals and tissue-culture cells compared with promastigotes derived from blood-agar cultivation.     

Cellulase Activity of Leishmania major in the Sandfly Vector and in Culture

ABSTRACT. The secretion of cellulose-degrading enzymes by Leishmania promastigotes in culture and in the sandfly vector was demonstrated. Two types of activity of cellulase enzyme-complexes were measured: endoglucanases, which randomly cleave cellulose chains and cellobioydrolases, which remove cellobiose from the nonreducing end of the molecule. The assays demonstrated that enzymes with these activities were secreted into the culture medium by Leishmania major, L. donovani , and L. braziliensis . These activities were also found in cultures of Sauroleishmania agamae, Leptomonas seymouri, Herpetomonas muscarum, Crithidia fasciculata and Trypanosoma brucei brucei that had a relatively low endoglucanase activity. Both endoglucanase and cellobiohydrolase activities were found in the gut of L. major-infected Phlebotomus papatasi , while gut preparations of uninfected sandflies had only cellobiohydrolase activity. The similar growth of L. major parasites in medium supplemented with either cellulose or glucose suggests these parasites can utilize cellulose.