Antibody response against a Leishmania donovani amastigote-stage-specific protein in patients with visceral leishmaniasis

Shoots of higher plants grow upward in response to gravity. To elucidate the molecular mechanism of this response, we have isolated shoot gravitropism (sgr) mutants in Arabidopsis thaliana. In this report, we describe three novel mutants, sgr4-1, sgr5-1 and sgr6-1 whose inflorescence stems showed abnormal gravitropic responses as previously reported for sgr1, sgr2 and sgr3. These new sgr mutations were recessive and occurred at three independent genetic loci. The sgr4-1 mutant showed severe defect in gravitropism of both inflorescence stem and hypocotyl but were normal in root gravitropism as were sgr1 and sgr2. The sgr5-1 and sgr6-1 mutants showed reduced gravitropism only in inflorescence stems but normal in both hypocotyls and roots as sgr3. These results support the hypothesis that some mechanisms of gravitropism are genetically different in these three organs in A. thaliana. In addition, these mutants showed normal phototropic responses, suggesting that SGR4, SGR5 and SGR6 genes are specifically involved in gravity perception and/or gravity signal transduction for the shoot gravitropic response.     

Leishmania donovani: acquired resistance to visceral leishmaniasis in the golden hamster

Multiple rough endoplasmic cisternae were found in "C" cells of the adult rat, at interphase. They are considered to be normal constituents of "C" cells. Their morphological relation to rough endoplasmic reticulum and their close proximity to mitochondria, Golgi dictyosomes and secretory granules suggest that they may have a role in the secretory activity of this endocrine cells.     

Altered transport properties of pentamidine-resistant Leishmania donovani and L. amazonensis promastigotes

Pentamidine-resistant clones of Leishmania donovani and L. amazonensis promastigotes were developed by increase of the drug pressure in the culture medium and characterized. The resistant clones could grow in 40 and 20 microM pentamidine as determined for L. donovani and L. amazonensis, respectively, with 50% inhibitory concentrations (IC50 values) being 140 and 60 microM, which were 18 and 75 times higher than those recorded for the parental clones, respectively. Biochemical analysis of the clones showed that the acquired pentamidine resistance was specific (no cross-resistance to unrelated drugs and no reversibility with verapamil) and stable in vitro and in vivo. Pentamidine resistance is related to decreased drug uptake and highly increased efflux in both clones of Leishmania spp., accompanied by an alteration in polyamine carriers. Furthermore, a modification of the uptake of pyrimidine nucleosides and several amino acids by these resistant clones indicates alterations in the surface membrane.     

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.     

The importance of TGF-beta in murine visceral leishmaniasis

IFN-gamma is critical for the cure of leishmaniasis in humans and mice. BALB/c mice are genetically susceptible to infection with the visceralizing species of Leishmania, L. chagasi. We have evidence that a soluble factor(s) inhibits IFN-gamma production by cultured liver granuloma cells from BALB/c mice during L. chagasi infection. In contrast, liver granulomas from C3H.HeJ mice, which are genetically resistant to L. chagasi infection, produce abundant IFN-gamma. According to ELISAs and neutralization studies, there was not evidence that the Th2-type cytokines IL-10 or IL-4 contributed to IFN-gamma suppression. However, both Ab neutralization and immunohistochemistry showed that granuloma-derived TGF-beta was, at least in part, responsible for inhibiting IFN-gamma release by CD4+ cells in BALB/c liver granuloma cultures. Consistently, TGF-beta levels were high in liver granulomas from susceptible BALB/c mice but low in resistant C3H mice or in BALB/c mice that were immunized against L. chagasi disease. Administration of recombinant adenovirus expressing TGF-beta (AdV-TGFbeta) but not IL-10 (AdV-IL10) caused genetically resistant C3H mice to become significantly more susceptible to L. chagasi infection. In contrast, either AdV-TGFbeta or AdV-IL10 could abrogate the protective immune response achieved by immunization of BALB/c mice. We conclude that locally secreted TGF-beta inhibits Th1-associated cure of murine visceral leishmaniasis caused by L. chagasi, independently of Th2-type cytokines.     

Effect of new diamidines against Leishmania donovani infection

The impact of interamidine distance on antileishmanial activity of new aryldiamidines have been evaluated against amastigotes of L. donovani in hamster. Of the 20 compounds tested, only four (2,8-diamidino-9,10-dihydrodibenzoxepin; 2,7-diamidinoxanthone; 2,7-diamidinothioxanthone and 2,7-diamidinoxanthene) showed significant inhibition (more than 80%) of multiplication of amastigotes in spleen. The interamidine distance in the structure appears to have bearing on antileishmanial activity. The observations made are likely to evoke new understanding on the structure activity relationship of diarylamidines.     

Atypical cutaneous histological features of visceral leishmaniasis in acquired immunodeficiency syndrome

OBJECTIVE: A sex hormone imbalance has been reported in patients with hepatocellular carcinoma (HCC). We investigated the serum levels of eight sex hormones in patients with alcohol-related and non-alcohol-related cirrhosis and HCC. METHODS: Luteinizing hormone, follicle-stimulating hormone, estradiol, progesterone, testosterone, androstenedione, dehydroepiandrosterone and sex hormone binding globulin were assayed in 81 patients with cirrhosis (59 men, 22 women) and 97 with HCC and cirrhosis (82 men, 15 women). Hepatitis B or hepatitis C virus infection was present in 58% of patients with cirrhosis and 69% of patients with HCC. Alcohol abuse was the aetiopathogenetic factor in the remaining patients. RESULTS: In men, mean testosterone levels were at the lower limit of the normal range for both patients with HCC and for controls with cirrhosis. Mean estradiol levels were increased both in patients with HCC and in those with cirrhosis, but patients with alcohol-related HCC had higher estradiol levels (P = 0.0002). An index of sex hormone imbalance, the estradiol to testosterone ratio (ETR), was calculated. The ETR was significantly higher in patients with alcohol-related HCC (P = 0.0002). Multiple regression analysis showed that the ETR correlated best with patients' diagnosis (P < 0.05). In women, the ETR was significantly lower in patients with HCC than in controls with cirrhosis. CONCLUSIONS: Men with alcohol-related HCC are characterized by an oestrogen and androgen imbalance and have a higher ETR than patients with other types of liver damage. Since sex hormones modulate hepatocellular proliferation, our data suggest that a sex hormone imbalance plays a role in hepatocarcinogenesis in patients with alcohol-related cirrhosis.     

Leishmania major-like parasite, a pathogenic agent of cutaneous leishmaniasis in Paraguay

Leishmania parasites isolated from two patients with cutaneous leishmaniasis from geographically different localities in Paraguay have been characterized by enzyme electrophoresis (zymodeme) and digestion profiles of kinetoplast DNA with restriction enzymes (schizodeme). Both Paraguayan isolates showed identical zymodeme profiles to each other using 14 enzymes (glutamic pyruvate transaminase, glutamic oxaloacetic transaminase, enolase, fumarate hydratase, glucose phosphate isomerase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, malic enzyme, mannose phosphate isomerase, nucleoside phosphorylase, peptidase-D, 6-phosphogluconate dehydrogenase, phosphoglucomutase, and pyruvate kinase). Although two Paraguayan isolates showed different zymodeme profiles from those of six Leishmania reference strains of Old and New World Leishmania species, they showed identical zymodeme profiles to those of an L. major-like parasite from Ecuador. These observations were confirmed by schizodeme analysis using three restriction endonucleases (Msp I, Hae III, and Taq I). These results indicate that Leishmania parasites isolated in Paraguay are identified as an L. major-like parasite, and it is necessary to consider the existence of L. major-like parasites when classifying Leishmania isolates from the New World.     

Protective effect of Picroliv from Picrorhiza kurroa against Leishmania donovani infections in Mesocricetus auratus

The prevalent drugs for treatment of kala azar viz. sodium stibogluconate (SSG) and pentamidine cause severe toxic side effects and acute immunosuppression in the treated individuals. Picroliv, a standardized mixture of iridoid glycosides, prepared from the alcoholic extract of the root and rhizome of Picrorhiza kurroa has shown strong hepatoprotective activity against several models of hepatotoxicity. Therefore, the present study was undertaken with an objective to study the effects of Picroliv (12.5 mg/kg x 7 days oral) alone and in combination with SSG on parasitemia, lipid peroxidation and hepatic marker enzymes of golden hamsters during Leishmania donovani infection. The results indicated a marked hepatoprotective effect of Picroliv in terms of biochemical markers, and a significant antileishmanial activity implying that it can be utilized as an adjunct to chemotherapy or in combination therapy of kala azar along with sodium stibogluconate, thus enhancing the efficacy of antileishmanials.     

Macrophage microbicidal mechanisms in vivo: reactive nitrogen versus oxygen intermediates in the killing of intracellular visceral Leishmania donovani

To determine the relative contributions of respiratory burst-derived reactive oxygen intermediates (ROI) versus reactive nitrogen intermediates (RNI) to macrophage-mediated intracellular host defense, mice genetically deficient in these mechanisms were challenged with Leishmania donovani, a protozoan that selectively parasitizes visceral tissue macrophages. During the early stage of liver infection at wk 2, both respiratory burst-deficient gp91(phox)-/- (X-linked chronic granulomatous disease [X-CGD]) mice and inducible nitric oxide synthase (iNOS) knockout (KO) mice displayed comparably increased susceptibility. Thereafter, infection was unrestrained in mice lacking iNOS but was fully controlled in X-CGD mice. Mononuclear cell influx into infected liver foci in X-CGD and iNOS KO mice was also overtly impaired at wk 2. However, granuloma assembly in parasitized tissue eventually developed in both hosts but with divergent effects: mature granulomas were functionally active (leishmanicidal) in X-CGD mice but inert in iNOS-deficient animals. These results suggest that (a) ROI and RNI probably act together in the early stage of intracellular infection to regulate both tissue recruitment of mononuclear inflammatory cells and the initial extent of microbial replication, (b) RNI alone are necessary and sufficient for eventual control of visceral infection, and (c) although mature granulomas have traditionally been associated with control of such infections, these structures fail to limit intracellular parasite replication in the absence of iNOS.     

The activities of four anticancer alkyllysophospholipids against Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei

The in-vitro activities of four anticancer alkyllysophospholipids, ET-18-OCH3 (edelfosine), hexadecylphosphocholine (miltefosine), ilmofosine and SRI 62-834, were determined with ED50 values for Leishmania donovani and Trypanosoma cruzi amastigotes between 0.2 and 5.0 microM and for Trypanosoma brucei trypomastigotes between 7.0 and 50.8 microM. In BALB/c mice miltefosine and ilmofosine were the most active compounds with ED50 values of 2.9 and 14.5 mg/kg x 5 doses against L. donovani and a suppressive effect on T. cruzi infections at 30 mg/kg x 5 doses.     

Genetic analysis of purine metabolism in Leishmania donovani

To dissect the contributions of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenine phosphoribosyltransferase (APRT), and adenosine kinase (AK) to purine salvage in Leishmania donovani, null mutants genetically deficient in HGPRT and/or APRT were generated by targeted gene replacement in wild type cells and preexisting mutant strains lacking either APRT or AK activity. These knockouts were obtained either by double targeted gene replacement or by single gene replacement followed by negative selection for loss-of-heterozygosity. Genotypes were confirmed by Southern blotting and the resultant phenotypes evaluated by enzymatic assay, resistance to cytotoxic drugs, ability to incorporate radiolabeled purine bases, and growth on various purine sources. All mutant strains could propagate in defined growth medium containing any single purine source and could metabolize exogenous [3H]hypoxanthine to the nucleotide level. The surprising ability of mutant L. donovani lacking HGPRT, APRT, and/or AK to incorporate and grow in hypoxanthine could be attributed to the ability of the parasite xanthine phosphoribosyltransferase enzyme to salvage hypoxanthine. These genetic studies indicate that HGPRT, APRT, and AK, individually or in any combination, are not essential for the survival and growth of the promastigote stage of L. donovani and intimate an important, if not crucial, role for xanthine phosphoribosyltransferase in purine salvage.     

Leishmania pifanoi amastigote antigen P-4: epitopes involved in T-cell responsiveness in human cutaneous leishmaniasis

In experimental murine cutaneous leishmaniasis, the purified Leishmania pifanoi amastigote protein P-4 has been shown to induce significant protection against infection. Further, recent studies examining the response of peripheral blood mononuclear cells (PBMC) from Leishmania braziliensis-infected human patients have demonstrated that the P-4 protein selectively elicits a significant TH1-like response. Because a TH1-like response is associated with cure, epitope studies were conducted to further evaluate the human response to P-4. PBMC from confirmed cutaneous leishmaniasis patients infected with L. braziliensis in Rio de Janeiro, Brazil, an area where the disease is endemic, were examined for T-cell proliferation and/or cytokine production in response to whole-parasite homogenate, isolated P-4 protein, and/or P-4 peptides. Twenty of the 22 patients (91%) examined responded to the native P-4 protein by proliferation and/or gamma interferon (IFN-gamma) production. According to the proliferation data, PBMC from 14 patients (64%) were found to respond to the intact P-4 protein (stimulation index of >/=2.5). Fifty-seven percent of the P-4-responsive patients studied responded to at least one of the P-4 peptides; 11 individual peptides were found to elicit a proliferative response. Of 17 patients examined for cytokine production, no PBMC produced detectable interleukin-4 in response to P-4 protein or peptides. However, PBMC from 14 patients (82%) produced significant levels of IFN-gamma (>/=20 pg/ml) in response to native P-4 protein. Nineteen of the 23 peptides were found to elicit an IFN-gamma response from at least two patients. These data indicate that multiple epitopes spanning the entire P-4 molecule are responsible for the TH1-like immune response observed, indicating that the intact P-4 amastigote molecule, rather than selected peptides, may prove to be the most useful for leishmaniasis vaccine development.     

Interleukin-7 enhances antimicrobial activity against Leishmania major in murine macrophages

Recently, it has been shown that interleukin-7 (IL-7) is able to induce secretion of cytokines and tumoricidal activity by human monocytes. This study shows that treatment of murine macrophages infected with Leishmania major with IL-7 without any other stimulus reduced the percentage of infected cells, as well as the parasite burden per cell, in a dose-dependent manner to a limited degree (45% reduction of the number of amastigotes per 100 macrophages). Simultaneous treatment of macrophages with gamma interferon and IL-7 led to nearly complete (> 99%) elimination of amastigotes. Addition of anti-tumor necrosis factor alpha or N omega-monomethyl-L-arginine acetate reversed the leishmanicidal effects of IL-7, and production of nitric oxide was induced in the presence of IL-7.     

A case of visceral leishmaniasis in Austria

We report a patient aged 41 years with fever of unknown origin. Notable aspects of his travel history were a trip to the Philippines and a sailing trip around Sicily. The patient presented with fever up to 40 degrees C since 4 weeks, weakness, headache, hepatosplenomegaly and night sweat. No specific cause could be found. Based on clinical findings tuberculosis was suspected and empirical tuberculostatic treatment was started. However, during the following 6 weeks the patient's condition deteriorated. A bone marrow biopsy performed to exclude a haematological malignancy revealed Leishmania sp. in macrophages. This histological diagnosis was confirmed retrospectively by re-examination of a previously performed liver biopsy and by an increased anti-leishmania serum antibody titer of 1:1280. The patient was treated with sodium stibogluconate (pentostam, 850 mg) for 30 days and recovered slowly.     

Survival strategies of Leishmania donovani in mammalian host macrophages

Microbes have evolved a variety of strategies to survive inside their host cells. Some have the molecular machinery to survive in the hostile environment of phagolysosomes; others escape the phagosome to the more cozy environment of the cell cytoplasm; others inhibit the phagosome fusion with hydrolase-enriched endocytic organelles. This is the case for the promastigote form of the protozoan parasite Leishmania donovani which resides in a phagosome displaying poor fusogenic properties towards endosomes and lysosomes. Recent results indicate that the lipophosphoglycan (LPG), the major cell surface molecule of Leishmania, is involved in the inhibition of phagosome maturation. Further studies in our laboratories are addressing the molecular mechanisms of action of LPG to modulate phagosome fusion properties and its effect on the biogenesis of phagolysosomes.     

Inducible expression of suicide genes in Leishmania donovani amastigotes

This study tests the feasibility of using the A2 gene regulatory system to create a Leishmania cell line in which attenuation is developmentally regulated when the parasite differentiates from promastigotes to amastigotes. The Leishmania donovani- inducible A2 gene regulatory system was used to differentially express in amastigotes two potential suicide genes: a truncated version of the L. donovani 3'-nucleotidase/nuclease expressed in the cytoplasm and the herpes simplex virus thymidine kinase gene. These genes were inserted between A2 noncoding regulatory sequences for up-regulation of expression in amastigotes. The accumulation of toxic products affected L. donovani cell replication and viability both in vitro and in vivo. The inducible expression of toxic gene products represents a valuable tool for the development of safe and effective vaccines.