Evaluation of cellular immunological responses in mono- and polymorphic clinical forms of post-kala-azar dermal leishmaniasis in India.

Post-kala-azar dermal leishmaniasis (PKDL) is a chronic dermal complication that occurs usually after recovery from visceral leishmaniasis (VL). The disease manifests into macular, papular and/or nodular clinical types with mono- or polymorphic presentations. Here, we investigated differences in immunological response between these two distinct clinical forms in Indian PKDL patients. Peripheral blood mononuclear cells of PKDL and naive individuals were exposed in vitro to total soluble Leishmania antigen (TSLA). The proliferation index was evaluated using an enzyme-linked immunosorbent assay (ELISA)-based lymphoproliferative assay. Cytokines and granzyme B levels were determined by cytometric bead array. Parasite load in tissue biopsy samples of PKDL was quantified by quantitative polymerase chain reaction (qPCR). The proportion of different lymphoid subsets in peripheral blood and the activated T cell population were estimated using flow cytometry. The study demonstrated heightened cellular immune responses in the polymorphic PKDL group compared to the naive group. The polymorphic group showed significantly higher lymphoproliferation, increased cytokines and granzyme B levels upon TSLA stimulation, and a raised proportion of circulating natural killer (NK) T cells against naive controls. Furthermore, the polymorphic group showed a significantly elevated proportion of activated CD4(+) and CD8(+) T cells upon in-vitro TSLA stimulation. Thus, the polymorphic variants showed pronounced cellular immunity while the monomorphic form demonstrated a comparatively lower cellular response. Additionally, the elevated level of both activated CD4(+) and CD8(+) T cells, coupled with high granzyme B secretion upon in-vitro TSLA stimulation, indicated the role of cytotoxic cells in resistance to L. donovani infection in polymorphic PKDL.

Effective humoral and cellular immunoprotective responses in Li ESAp-MDP vaccinated protected dogs.

Cell-mediated and humoral immunity were explored in LiESAp-MDP vaccinated protected dogs versus susceptible placebo dogs 2 months and 8 months post-vaccination. As previously described, a strong and long-lasting cell-mediated immunity, critical for protection against Leishmania infantum was exclusively revealed in vaccinated dogs as confirmed by a positive response to the intradermal inoculation of leishmanin and by a significant higher anti-leishmanial activity of canine monocytes-derived macrophages. Moreover, our results support the view that cooperation of humoral antibody with cell-mediated immunity might be important in developing protective immunity in LiESAp-MDP vaccinated dogs. Anti-LiESAp serum samples were found functionally active in vitro, promoting (i) early killing of pretreated promastigotes and amastigotes, (ii) strong inhibitory effect on the in vitro growth of both parasitic developmental stages of L. infantum and (iii) most importantly, a significant inhibition of pretreated promastigotes in vitro infectivity in canine macrophages. However, anti-LiESAp antibody response was not implicated in the promastigotes-amastigotes differentiation process. In these experiments, we have added additional support to the concept that antibodies to Leishmania may be important in developing protective immunity.

Spirolactone iridoids might be responsible for the antileishmanial activity of a Peruvian traditional remedy made with Himatanthus sucuuba (Apocynaceae).

Extracts of seven medicinal plants used specifically against cutaneous leishmaniasis in the Madre de Dios region of Peru were evaluated in vitro against promastigote and axenic amastigote forms of Leishmania amazonensis. One of them showed interesting leishmanicidal activities (IC(50)=5 microg/ml in amastigotes). Bio-guided isolation of the stem bark's ethanol extract of Himatanthus sucuuba (Spruce ex Müll. Arg.) Woodson (Apocynaceae) afforded the spirolactone iridoids isoplumericin and plumericin. The latter showed a reduction of macrophage infection similar to that of the reference drug Amphotericin B (IC(50)=0.9 and 1 microM, respectively). These findings validate the traditional use of Himatanthus sucuuba in the treatment of cutaneous leishmaniasis (Uta) in Peru.

Lymphocytes of dogs immunised with purified excreted-secreted antigens of Leishmania infantum co-incubated with Leishmania infected macrophages produce IFN gamma resulting in nitric oxide-mediated amastigote apoptosis.

The role of nitric oxide (NO) in the anti-leishmanial activity has been confirmed both in vitro and in vivo. Recently, we demonstrated that NO-mediated apoptosis-like amastigote death pathway is an important and highly regulated mechanism used for the clearance of Leishmania within infected murine macrophages stimulated to produce NO endogenously. To further characterize these important effector mechanisms in dog, a natural host-reservoir of L. infantum/L. chagasi, we have developed an ex vivo infection model of canine macrophages. Exposure of L. infantum-infected macrophages to autologous peripheral lymphocytes derived from dogs immunised with purified excreted-secreted antigens of L. infantum promastigotes (LiESAp) formulated with muramyl dipeptide (MDP) as adjuvant resulted in a significant leishmanicidal effect due to interferon (IFN)-gamma dependent macrophage activation. Concomitant accumulation of NO(3)(-)/NO(2)(-) in supernatants of co-cultured cells and in situ staining of parasites with terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and YOPRO-1 showed that NO-mediated apoptosis of intracellular L. infantum amastigotes is occurring in canine macrophages as previously observed in mouse models. Monitoring these parameters in dogs after immunisation and before experimental challenge can represent a useful and easy way to rapidly evaluate vaccine candidates against canine visceral leishmaniasis.

Cloning of a Leishmania major gene encoding for an antigen with extensive homology to ribosomal protein S3a.

Following purification by affinity chromatography, a Leishmania major S-hexylglutathione- binding protein of molecular mass 66kDa was isolated. The immune serum against the parasite 66kDa polypeptide when used to screen a L. major cDNA library could identify clones encoding for the human v-fos transformation effector homologue, namely ribosomal protein S3a, and thus was named LmS3a-related protein (LmS3arp). A 1027bp cDNA fragment was found to contain the entire parasite gene encoding for a highly basic protein of 30kDa calculated molecular mass sharing homology to various ribosomal S3a proteins from different species. Using computer methods for a multiple alignment and sequence motif search, we found that LmS3arp shares a sequence homology to class theta glutathione S-transferase mainly in a segment containing critical residues involved in glutathione binding. These new findings are discussed in the light of recent published data showing multiple function(s) of the ribosomal proteins S3a.

Identification of a major 72 kilodalton surface antigen in twelve isolates of Leishmania braziliensis braziliensis.

The study of the surface antigens of Leishmania braziliensis braziliensis revealed a great homogeneity among ten strains isolated from Bolivia and two reference strains from Brazil and Belize. A 72 kDa major protein, present in all L. b. braziliensis strains, was recognized by both cutaneous and mucocutaneous human sera, but was not recognized by Kala-azar and chagasic sera. No cross-reactive antigens were found among strains of Leishmania braziliensis guyanensis, Leishmania braziliensis panamensis, Leishmania mexicana amazonensis and Leishmania donovani chagasi testing these strains with hamster and human anti-L. b. braziliensis sera. Moreover, these strains possessed major antigens with molecular weights different from those of L. b. braziliensis strains. A microheterogeneity of L. b. braziliensis surface antigens was detected for the high molecular weight antigens and seemed to be related to the isoenzymic microheterogeneity.

Leishmania spp: completely defined medium without serum and macromolecules (CDM/LP) for the continuous in vitro cultivation of infective promastigote forms.

The elimination of serum or of serum-derived macromolecules that supplant the fetal calf serum requirement from Leishmania culture media could decrease costs and improve the feasibility of large-scale production of well-defined parasite material. We report a completely defined medium, without serum-derived protein and/or macromolecules as a serum substitute, of common, available, and inexpensive constituents that can be used in place of serum-supplemented media for the continuous in vitro cultivation of promastigote forms of various Leishmania species. Typical promastigote morphology was observed in Giemsa-stained smears, regardless of the strain analyzed. Electrophoretic analysis showed that the proteinase patterns of aserically grown promastigote forms were similar to those obtained in serum-supplemented RPMI 1640 medium for all Leishmania studied. Similar antigenic profiles were recognized in immunoblots by sera from hosts with visceral or cutaneous leishmaniasis after growing promastigotes in the two different culture media. For parasites causing both cutaneous and visceral leishmaniasis, the absence of serum and macromolecules in the culture medium did not markedly change their in vitro infectivity for resident mouse macrophages and their virulence in animals compared with parasites cultivated in nondefined medium. Serum-free technology will be increasingly important in providing stability and reproducibility as research using promastigote moves closer to therapeutic applications.

Circulating antibodies directed against tryptophan-like epitopes in sera of patients with human African trypanosomiasis.

Human African trypanosomiasis is often associated with an intense proliferation of B lymphocytes, leading to polyclonal antibody synthesis. Using a modified enzyme-linked immunosorbent assay method, we have found highly significant levels of circulating anti-conjugated tryptophan-like epitope antibodies in sera of patients with sleeping sickness. These antibodies were immunoglobulins (Ig) of the M isotype. There was no correlation between immunologic binding and the Ig levels found in sera of patients with human African trypanosomiasis. Higher antibody levels in stage II of the disease than in stage I may be related to damage to the central nervous system. The specificity of this immunologic binding was evaluated by 1) comparison with that obtained with other related conjugates and 2) serum titration. Anti-conjugated tryptophan-like epitope antibodies were not found in other neurologic diseases tested. Their involvement in this pathology remains unknown.

Correlation of high serum levels of tumor necrosis factor-alpha with disease severity in human African trypanosomiasis.

The levels of tumor necrosis factor-alpha (TNF-alpha) in sera from Trypanosoma brucei gambiense-infected patients from the endemic region of Boko Songho (Bouenza focus in Congo) were measured. An increase was observed in sera from patients (geometric mean = 53.75 pg/ml, n = 69) compared with control subjects from the same endemic area (6.72 pg/ml, n = 31). The patients were classified as being in the early (blood lymphatic) stage and late (meningo-encephalitic) stage of disease according to the presence of parasites and cells in cerebrospinal fluid (CSF). An increase in TNF-alpha was noted in late stage patients (68.42 pg/ml, n = 28) compared with early stage patients (43.68 pg/ml, n = 41). Those patients with fever, asthenia, and edema and those with neurologic signs had higher levels of TNF-alpha (89.36 pg/ml, n = 26) than others (38.07 pg/ml, n = 43). No differences in TNF-alpha levels were seen when trypanosomes were detected in one location (blood, lymph nodes, or CSF) or two or three locations. These data show that the levels of TNF-alpha in serum of T. b. gambiense-infected patients were correlated with disease severity (presence of signs of inflammation or presence of major neurologic signs) and indicate that TNF-alpha could be involved in some aspects of human African trypanosomiasis physiopathology.

Different monoclonal antibodies against the component 5 specific for Trypanosoma cruzi.

Three murine monoclonal antibodies (I-35/67, II-190/30, III-160/18) produced by immunization against total epimastigote extract or component 5-enriched fractions of Trypanosoma cruzi (Tehuantepec strain) have been demonstrated to recognize the component 5 specific for T. cruzi. By immunofluorescence studies, one of these monoclonal antibodies (I-35/67) was shown to bind to the epimastigote cell surface, and the two others (II-190/30 III-160/18) were preferentially directed against internal subcellular organelles. Immunoprecipitation using these monoclonal antibodies followed by SDS-PAGE analysis, has led to identification of four molecules with apparent molecular weights of 72 Kd, 51 Kd, 43 Kd, and 24 Kd in T. cruzi epimastigotes. Potential uses of these monoclonal antibodies in serological tests and immunochemical analysis of target antigens are discussed.

Specific and sensitive immunological diagnosis of Chagas' disease by competitive antibody enzyme immunoassay using a Trypanosoma cruzi-specific monoclonal antibody.

Coexistence of Chagas' disease with leishmaniasis and T. rangeli infection in endemic areas and cross-reactivity between corresponding etiological agents can confuse the immunodiagnosis of Chagas' disease. A discriminative serological test could therefore represent a major advance in specific immunodiagnosis. A competitive antibody enzyme immunoassay against a component 5-enriched preparation, using a T. cruzi species-specific monoclonal antibody has allowed development of a specific serodiagnosis of Chagas' disease with high sensitivity (96.6% in undetermined and chronic phases of infection). This test can differentiate Chagas' disease from other cross-reacting parasitic diseases in areas where concomitant infections are unknown or suspected.

Additional data on Trypanosoma cruzi isozymic strains encountered in Bolivian domestic transmission cycles.

We have collected in Bolivia 212 stocks of Trypanosoma cruzi from domestic transmission cycles and have assayed for nine enzyme systems (11 gene loci). Only a few different isozyme profiles exist, without recombination between them, a situation also encountered in previous Bolivian samples. The 212 stocks, combined with 207 stocks previously studied, have been analysed to uncover any spatial patterns. The frequency of heterozygous strains (2 and 2a) decreases westwards and with increasing altitude. Given that longitude and altitude are correlated with each other, it is not possible to decide which of these two geographic variables is the relevant one, or if both are. These associations might be due to climatic factors. Studies by other authors have shown, however, that heterozygous strains are rare or absent in the Amazon Basin, which is at low altitude.

Serodiagnosis of sleeping sickness in the Republic of the Congo: comparison of indirect immunofluorescent antibody test and card agglutination test.

The card agglutination test for trypanosomiasis (CATT) was evaluated and compared to the classical immunofluorescent antibody test (IFAT) in the immunological diagnosis of Gambian trypanosomiasis. Tests were performed on serum and whole blood. Cross-reactions were found in the CATT with sera from patients suffering from parasitic infections other than sleeping sickness, but could be largely overcome by selecting 1/10 as the specific threshold dilution. At 1/40 dilution no false positive result was observed in the IFAT. At the specific threshold dilution, the sensitivity of IFAT was 94.7%, compared with 91.6% for the CATT. On whole blood, a more convenient sample in the field, IFAT specificity (100%) was greater than that of the CATT (94.3%), as was its sensitivity (92% compared with 82.5%). In view of its simplicity and rapidity of execution, the CATT is an efficient serological test to detect new foci. When greater sensitivity is required, IFAT should be preferred to CATT.

Serum lipid and lipoprotein abnormalities in human African trypanosomiasis.

We have studied the serum lipoprotein system in human African trypanosomiasis (Trypanosoma brucei gambiense infection). The study was carried out on 74 Congolense patients suffering from sleeping sickness and 34 Congolense control subjects living in the endemic region of Boko Songho. We have determined the serum concentrations of lipids (triglycerides, cholesterol, phospholipids) and apolipoproteins (apolipoprotein A-I and B), and the separation of serum lipoproteins by electrophoresis. For the patients infected with T. b. gambiense, in comparison with control subjects, the results have shown (i) a significant increase in triglyceride concentration and a decrease in cholesterol concentration; (ii) a significant rise in apolipoprotein B concentration and a significant reduction in apolipoprotein A-I concentration; and (iii) an increase in low density lipoproteins and a decrease in high density lipoproteins. We conclude, therefore, that human African trypanosomiasis is associated with marked alterations in the composition and levels of host lipoproteins.

Efficacy of second line drugs on antimonyl-resistant amastigotes of Leishmania infantum.

In a previous paper we have demonstrated that the induction, by direct drug pressure, of a resistance to Sb(III) antimony at physiological concentration in the amastigote stage of the parasite, led to a high cross-resistance to Sb(V) species in the form of Glucantime. In this paper, further chemoresistant clones were characterized. Axenic amastigotes of Leishmania infantum were adapted to survive in culture medium containing 4, 20, 30 and 120 microg/ml of potassium antimonyl tartrate Sb(II). These mutants were 12, 28, 35 and 44-fold more resistant to Sb(III) than the parental wild-type clone. They were able to resist at concentrations of Glucantime Sb(V) as high as 160 microg/ml when growing in THP-1 cells. We have investigated the efficacy of second line drugs in clinical use (pentamidine and amphotericin B) on the antimony-resistant mutants. Amphotericin B was toxic for both wild-type and chemoresistant mutants at concentrations ranging from 0.05 to 0.15 microM. Pentamidine which is extensively used when the first course of antimonial pentavalent compounds is unsuccessful, was more toxic for all the chemoresistant organisms than for the wild-type clone. In the same way, chemoresistant amastigotes growing within THP-1 cells were more susceptible to pentamidine than the wild-type clone. Our results showed that the resistance of the mutants was restricted to the antimony containing drugs and did not led to a cross-resistance against the other clinically relevant drugs. These results confirmed that these two drugs (pentamidine and amphotericin B) are good candidates to treat pentavalent antimonial unresponsiveness.

Requirements of defined cultivation conditions for standard growth of Leishmania promastigotes in vitro.

The growth characteristics of L. chagasi (MHOM/BR/79/LI01) and L. braziliensis (MHOM/BR/72/1670), the causative agents of visceral and muco-cutaneous leishmaniases, respectively, were compared. Inoculum size clearly influences the growth course of both Leishmania species, whatever the culture medium used (serum-supplemented media: GLSH or RPMI, and a chemically defined medium: LITR9). Cultures initiated with low concentrations failed to promote cell growth, while typical growth curves were obtained when higher promastigote inocula were used. For all the species tested, the higher the initial density of flagellates in the medium, the shorter were the periods covered by the latent and particularly by the logarithmic growth phases. In contrast, using constant inocula, variations in the volume of the incubation medium did not change the time-course of the different culture phases of either Leishmania species, provided that the ratio of incubation medium to total flask volume was comparable. Only cell division time significantly increased with the culture volume. We also determined whether or not the growth characteristics of the promastigotes of L. chagasi or L. braziliensis could be generalized to other members of the genus. Our results show that, whatever the culture medium used, L. infantum behaves in the same way as does L. chagasi, whereas L. panamensis, L. guyanensis, L. mexicana and L. amazonensis display growth patterns similar to that of L. braziliensis.

Experimental studies on the evolution of antimony-resistant phenotype during the in vitro life cycle of Leishmania infantum: implications for the spread of chemoresistance in endemic areas.

Pentavalent antimonial unresponsiveness is an emerging problem in endemic areas and information on factors which could modulate the transmission of drug-resistant phenotypes and parasites during life cycle are warranted. Using axenic amastigotes resistant to potassium antimonyl tartrate (Sb(III)) we investigated the modulation of antimonyl resistance during the in vitro life cycle. We assessed: (i) the stability of the drug-resistant phenotype during the in vitro life cycle; (ii) the transmission of drug-resistant clones when mixed with a wild-type clone at different susceptible/chemoresistant ratios (50/50,90/10,10/90) after one or two in vitro life cycles. We demonstrate that: (i) mutants which were 12,28,35 and 44 fold more resistant to Sb(III)-antimonial than their parental wild-type, were Glucantime Sb(V)-resistant when growing in THP-1 cells; (ii) the drug-resistant phenotype was partially retained during long-term in vitro culture (3 months) in drug free medium; (iii) the antimonyl-resistant phenotype was retained after one or more in vitro life cycles. However, when drug-resistant parasites were mixed with susceptible, mutants could not be detected in the resulting population, after one or two in vitro life cycles, whatever the initial wild-type/chemoresistant ratio. These results could be explained by the lower capacity of drug-resistant amastigotes to undergo the amastigote-promastigote differentiation process, leading probably to their sequential elimination during life cycle. Taken together, these observations demonstrate that different factors could modulate the transmission of Leishmania drug resistance during the parasite's life cycle.

Secreted antigens of the amastigote and promastigote forms of Leishmania infantum inducing a humoral response in humans and dogs.

To study the antigens secreted by promastigote and amastigote forms of Leishmania infantum which are able to induce a humoral response in human patients and dogs, we have carried out immunoprecipitation assays with different supernatants of in vitro cultured parasites, metabolically labelled with [35S]methionine, using serum samples from human patients and dogs. In addition, some metabolic labelling experiments were performed daily during the in vitro culture parasite's life cycle to follow the time course excretion-secretion of parasitic antigens. The results demonstrated that the two different hosts developed an antibody response against secreted antigens of both stages of Leishmania infantum. Nevertheless, the humoral response directed against the excreted-secreted antigens of the promastigote forms was qualitatively and quantitatively different when we compare the human and the dog immune responses. On the other hand, when the excreted-secreted antigens of the amastigote forms are immunoprecipitated with either human or canine immune serum, the humoral response is similar. In addition, the time course study showed that excretion-secretion of antigens was qualitatively and quantitatively modulated during the parasitic in vitro life cycle.