Profiling gene expression of antimony response genes in Leishmania (Viannia) panamensis and infected macrophages and its relationship with drug susceptibility.

The mechanisms of Leishmania resistance to antimonials have been primarily determined in experimentally derived Leishmania strains. However, their participation in the susceptibility phenotype in field isolates has not been conclusively established. Being an intracellular parasite, the activity of antileishmanials is dependent on internalization of drugs into host cells and effective delivery to the intracellular compartments inhabited by the parasite. In this study we quantified and comparatively analyzed the gene expression of nine molecules involved in mechanisms of xenobiotic detoxification and Leishmania resistance to antimonial drugs in resistant and susceptible laboratory derived and clinical L.(Viannia) panamensis strains(n=19). In addition, we explored the impact of Leishmania susceptibility to antimonials on the expression of macrophage gene products having putative functions in transport, accumulation and metabolism of antimonials. As previously shown for other Leishmania species, a trend of increased abcc3 and lower aqp-1 expression was observed in the laboratory derived Sb-resistant L.(V.) panamensis line. However, this was not found in clinical strains, in which the expression of abca2 was significantly higher in resistant strains as both, promastigotes and intracellular amastigotes. The effect of drug susceptibility on host cell gene expression was evaluated on primary human macrophages from patients with cutaneous leishmaniasis (n=17) infected ex-vivo with the matched L.(V.) panamensis strains isolated at diagnosis, and in THP-1 cells infected with clinical strains (n=6) and laboratory adapted L.(V.) panamensis lines. Four molecules, abcb1 (p-gp), abcb6, aqp-9 and mt2a were differentially modulated by drug resistant and susceptible parasites, and among these, a consistent and significantly increased expression of the xenobiotic scavenging molecule mt2a was observed in macrophages infected with Sb-susceptible L. (V.) panamensis. Our results substantiate that different mechanisms of drug resistance operate in laboratory adapted and clinical Leishmania strains, and provide evidence that parasite-mediated modulation of host cell gene expression of molecules involved in drug transport and metabolism could contribute to the mechanisms of drug resistance and susceptibility in Leishmania.

An Innovative Field-Applicable Molecular Test to Diagnose Cutaneous Leishmania Viannia spp. Infections.

Cutaneous and mucosal leishmaniasis is widely distributed in Central and South America. Leishmania of the Viannia subgenus are the most frequent species infecting humans. L. (V.) braziliensis, L. (V.) panamensis are also responsible for metastatic mucosal leishmaniasis. Conventional or real time PCR is a more sensitive diagnostic test than microscopy, but the cost and requirement for infrastructure and trained personnel makes it impractical in most endemic regions. Primary health systems need a sensitive and specific point of care (POC) diagnostic tool. We developed a novel POC molecular diagnostic test for cutaneous leishmaniasis caused by Leishmania (Viannia) spp. Parasite DNA was amplified using isothermal Recombinase Polymerase Amplification (RPA) with primers and probes that targeted the kinetoplast DNA. The amplification product was detected by naked eye with a lateral flow (LF) immunochromatographic strip. The RPA-LF had an analytical sensitivity equivalent to 0.1 parasites per reaction. The test amplified the principal L. Viannia species from multiple countries: L. (V.) braziliensis (n = 33), L. (V.) guyanensis (n = 17), L. (V.) panamensis (n = 9). The less common L. (V.) lainsoni, L. (V.) shawi, and L. (V.) naiffi were also amplified. No amplification was observed in parasites of the L. (Leishmania) subgenus. In a small number of clinical samples (n = 13) we found 100% agreement between PCR and RPA-LF. The high analytical sensitivity and clinical validation indicate the test could improve the efficiency of diagnosis, especially in chronic lesions with submicroscopic parasite burdens. Field implementation of the RPA-LF test could contribute to management and control of cutaneous and mucosal leishmaniasis.

Parasitological Confirmation and Analysis of Leishmania Diversity in Asymptomatic and Subclinical Infection following Resolution of Cutaneous Leishmaniasis.

BACKGROUND: The contribution of individuals with subclinical infection to the transmission and endemicity of cutaneous leishmaniasis (CL) is unknown. Immunological evidence of exposure to Leishmania in residents of endemic areas has been the basis for defining the human population with asymptomatic infection. However, parasitological confirmation of subclinical infection is lacking. METHODS: We investigated the presence and viability of Leishmania in blood and non-invasive mucosal tissue samples from individuals with immunological evidence of subclinical infection in endemic areas for CL caused by Leishmania (Viannia) in Colombia. Detection of Leishmania kDNA was conducted by PCR-Southern Blot, and parasite viability was confirmed by amplification of parasite 7SLRNA gene transcripts. A molecular tool for genetic diversity analysis of parasite populations causing persistent subclinical infection based on PCR amplification and sequence analysis of an 82bp region between kDNA conserved blocks 1 and 2 was developed. PRINCIPAL FINDINGS: Persistent Leishmania infection was demonstrated in 40% (46 of 114) of leishmanin skin test (LST) positive individuals without active disease; parasite viability was established in 59% of these (27 of 46; 24% of total). Parasite burden quantified from circulating blood monocytes, nasal, conjunctival or tonsil mucosal swab samples was comparable, and ranged between 0.2 to 22 parasites per reaction. kDNA sequences were obtained from samples from 2 individuals with asymptomatic infection and from 26 with history of CL, allowing genetic distance analysis that revealed diversity among sequences and clustering within the L. (Viannia) subgenus. CONCLUSIONS: Our results provide parasitological confirmation of persistent infection among residents of endemic areas of L. (Viannia) transmission who have experienced asymptomatic infection or recovered from CL, revealing a reservoir of infection that potentially contributes to the endemicity and transmission of disease. kDNA genotyping establishes proof-of-principle of the feasibility of genetic diversity analysis in previously inaccessible and unexplored parasite populations in subclinically infected individuals.

Human macrophage response to L. (Viannia) panamensis: microarray evidence for an early inflammatory response.

BACKGROUND: Previous findings indicate that susceptibility to Leishmania (Viannia) panamensis infection of monocyte-derived macrophages from patients and asymptomatically infected individuals were associated with the adaptive immune response and clinical outcome. METHODOLOGY/PRINCIPAL FINDINGS: To understand the basis for this difference we examined differential gene expression of human monocyte-derived macrophages following exposure to L. (V.) panamensis. Gene activation profiles were determined using macrophages from healthy volunteers cultured with or without stationary phase promastigotes of L. (V.) panamensis. Significant changes in expression (>1.5-fold change; p<0.05; up- or down-regulated) were identified at 0.5, 4 and 24 hours. mRNA abundance profiles varied over time, with the highest level of activation occurring at earlier time points (0.5 and 4 hrs). In contrast to observations for other Leishmania species, most significantly changed mRNAs were up- rather than down-regulated, especially at early time points. Up-regulated transcripts over the first 24 hours belonged to pathways involving eicosanoid metabolism, oxidative stress, activation of PKC through G protein coupled receptors, or mechanism of gene regulation by peroxisome proliferators via PPARα. Additionally, a marked activation of Toll-receptor mediated pathways was observed. Comparison with published microarray data from macrophages infected with L. (Leishmania) chagasi indicate differences in the regulation of genes involved in signaling, motility and the immune response. CONCLUSIONS: Results show that the early (0.5 to 24 hours) human monocyte-derived macrophage response to L. (Viannia) panamensis is not quiescent, in contrast to published reports examining later response times (48-96 hours). Early macrophage responses are important for the developing cellular response at the site of infection. The kinetics and the mRNA abundance profiles induced by L. (Viannia) panamensis illustrate the dynamics of these interactions and the distinct biologic responses to different Leishmania species from the outset of infection within their primary host cell.

Discovery of factors linked to antimony resistance in Leishmania panamensis through differential proteome analysis.

The rate of treatment failure to antileishmanial chemotherapy in Latin America is up to 64%. Parasite drug resistance contributes to an unknown proportion of treatment failures. Identification of clinically relevant molecular mechanisms responsible for parasite drug resistance is critical to the conservation of available drugs and to the discovery of novel targets to reverse the resistant phenotype. We conducted comparative proteomic-based analysis of Leishmania (Viannia) panamensis lines selected in vitro for resistance to trivalent antimony (Sb(III)) to identify factors associated with antimony resistance. Using 2-dimensional gel electrophoresis, two distinct sub-proteomes (soluble in NP-40/urea and Triton X-114, respectively) of promastigotes of WT and Sb(III)-resistant lines were generated. Overall, 9 differentially expressed putative Sb-resistance factors were detected and identified by mass spectrometry. These constituted two major groups: (a) proteins involved in general stress responses and (b) proteins with highly specific metabolic and transport functions, potentially directly contributing to the Sb-resistance mechanism. Notably, the sulfur amino acid-metabolizing enzymes S-adenosylmethionine synthetase (SAMS) and S-adenosylhomocysteine hydrolase (SAHH) were over-expressed in Sb(III)-resistant lines and Sb(III)-resistant clinical isolates. These enzymes play a central role in the upstream synthesis of precursors of trypanothione, a key molecule involved in Sb-resistance in Leishmania parasites, and suggest involvement of epigenetic regulation in response to drug exposure. These data re-enforce the importance of thiol metabolism in Leishmania Sb resistance, reveal previously unrecognized steps in the mechanism(s) of Sb tolerance, and suggest a cross-talk between drug resistance, metabolism and virulence.

Leishmania (Viannia) infection in the domestic dog in Chaparral, Colombia.

Peridomestic transmission of American cutaneous leishmaniasis is increasingly reported and dogs may be a reservoir of Leishmania (Viannia) in this setting. We investigated the prevalence of infection in dogs in Chaparral County, Colombia, the focus of an epidemic of human cutaneous leishmaniasis caused by Leishmania (Viannia) guyanensis. Two (0.72%) of 279 dogs had lesions typical of cutaneous leishmaniasis that were biopsy positive by kinetoplast DNA polymerase chain reaction-Southern blotting. Seroprevalence was 2.2% (6 of 279) by enzyme-linked immunosorbent assay. Buffy coat and ear skin biopsy specimens were positive by polymerase chain reaction-Southern blotting in 7.3% (10 of 137) and 11.4% (12 of 105) of dogs, respectively. Overall 20% of dogs (21 of 105) showed positive results for one or more tests. Amplification and sequencing of the Leishmania 7SL RNA gene identified L. guyanensis in one dog and L. braziliensis in two dogs. No association was identified between the risk factors evaluated and canine infection. Dogs may contribute to transmission but their role in this focus appears to be limited.

Murine model of chronic L. (Viannia) panamensis infection: role of IL-13 in disease.

Leishmania (Viannia) organisms are the most prevalent etiologic agents of human cutaneous leishmaniasis in the Americas. Nevertheless, our knowledge of the immunological mechanisms exploited by L. (Viannia) organisms remains limited and the mechanisms underlying disease are not well understood. Here, we report the development of a BALB/c mouse model of L. (V.) panamensis infection that is able to reproduce chronic disease, with persistent infection and clinically evident lesions for over 1 year. The immune response of the mouse resembles that found for L. (V.) panamensis-infected patients with chronic and recurrent lesions, presenting a mixed Th1/Th2 response with the presence of TNF-α, IFN-γ, IL-10 and IL-13. Using immunodeficient mice, the critical role for IL-13 and/or IL-4Rα in determining susceptibility to chronic infection was evident. With the induction of healing in the immunodeficient mice, increases in IFN-γ and IL-17 were found, concomitant with parasite control and elimination. Specifically, increases in CD4(+) (but not CD8(+)) T cells producing IFN-γ were observed. These results suggest that IL-13 represents an important target for disease control of L. (V.) panamensis infection. This murine model should be useful to further understand the pathology associated with chronic disease and to develop methods for the treatment and prevention of leishmaniasis caused by L. (Viannia) parasites.

Antimony resistance and trypanothione in experimentally selected and clinical strains of Leishmania panamensis.

The participation of trypanothione in clinical and experimental antimony (Sb) resistance in Leishmania panamensis was examined by using specific inhibitors. Buthionine sulfoximine (BSO) significantly reversed the resistance to trivalent Sb (Sb(III)) of promastigotes of experimentally derived Sb-resistant lines, supporting the participation of a trypanothione-mediated mechanism of resistance. In contrast, promastigotes of strains isolated at the time of clinical treatment failure and resistant to pentavalent Sb (Sb(V)) as intracellular amastigotes were not cross resistant to Sb(III), and BSO had little or no effect on susceptibility. Difluoromethylornithine did not alter the Sb(III) susceptibilities of experimentally selected lines or clinical strains. The mechanisms of acquired resistance emerging in clinical settings may differ from those selected by in vitro exposure to Sb.

Etiologic agent of an epidemic of cutaneous leishmaniasis in Tolima, Colombia.

American cutaneous leishmaniasis (ACL) has been characterized as a zoonotic disease. However, peridomestic and domestic transmission have been recorded in at least nine countries in Central and South America. The present study was undertaken to identify the etiologic agent of a peridomestic epidemic of ACL in the Department of Tolima, Colombia. Leishmania isolates were obtained during the diagnosis of 56 patients with ACL who consulted the local leishmaniasis control program in three municipalities in Tolima. Species were identified using monoclonal antibodies and isoenzyme electrophoresis. A total of 53 (94.6%) of 56 isolates were identified as Leishmania (Viannia) guyanensis. Three isolates (5.4%) were identified as L. (V.) panamensis. Leishmania (V.) guyanensis is the probable etiologic agent of the largest epidemic of cutaneous leishmaniasis recorded in Colombia. This species has not previously been reported outside the Amazon and southeastern regions of Colombia, and has not been described in the peridomestic setting or linked with an epidemic.

Treponema pallidum elicits innate and adaptive cellular immune responses in skin and blood during secondary syphilis: a flow-cytometric analysis.

BACKGROUND: Syphilis is caused by the spirochetal pathogen Treponema pallidum. The local and systemic cellular immune responses elicited by the bacterium have not been well studied in humans. METHODS: We used multiparameter flow cytometry to characterize leukocyte immunophenotypes in skin and peripheral blood from 23 patients with secondary syphilis and 5 healthy control subjects recruited in Cali, Colombia. Dermal leukocytes were obtained from fluid aspirated from epidermal suction blisters raised over secondary syphilis skin lesions. RESULTS: Compared with peripheral blood (PB), blister fluids (BFs) were enriched for CD4(+) and CD8(+) T cells, activated monocytes/macrophages, and CD11c(+) monocytoid and CD11c(-) plasmacytoid dendritic cells (mDCs and pDCs, respectively). Nearly all mDCs in BFs expressed the human immunodeficiency virus (HIV) coreceptors CCR5 and DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and high levels of human leukocyte antigen (HLA)-DR. Dermal pDCs expressed both HIV coreceptors without increases in HLA-DR intensity. Compared with normal blood, circulating mDCs in patients with syphilis expressed higher levels of both CCR5 and DC-SIGN, whereas circulating pDCs in patients expressed only higher levels of DC-SIGN. Most dermal T cells were CCR5(+) and displayed a memory (CD27(+)/CD45RO(+)) or memory/effector (CD27(-)/CD45RO(+)) immunophenotype. A corresponding shift toward memory and memory/effector immunophenotype was clearly discernible among circulating CD4(+) T cells. Compared with PB from control subjects, a larger percentage of CD4(+) T cells in PB from patients with syphilis expressed the activation markers CD69 and CD38. CONCLUSIONS: During secondary syphilis, T. pallidum simultaneously elicits local and systemic innate and adaptive immune responses that may set the stage for the bidirectional transmission of HIV.

Pharmacokinetics of antimony in children treated for leishmaniasis with meglumine antimoniate.

BACKGROUND: In some settings, the response to pentavalent antimonial therapy for leishmaniasis may be lower in children than in adults. We hypothesized that there are age-dependent pharmacokinetic differences of potential clinical relevance. METHODS: We compared the pharmacokinetics of antimony (Sb) in adults and 2 groups of children 3-6 years old who had cutaneous leishmaniasis treated with intramuscular meglumine antimoniate. Adults (n=9) and the first group of children (n=9) received 20 mg Sb/kg/day for 20 days; the second group of children (n=6) received 20 mg Sb/kg for 19 days and 30 mg Sb/kg on day 20. Drug exposure was assessed by the area under the 24-h time-concentration curve (AUC(0-24)) in plasma. RESULTS: Children (vs. adults) who received 20 mg/kg had a 42% lower AUC(0-24) (mean +/- SE, 111+/-7 vs. 190+/-10 mg x h/L, compared with adults; P<.001), a 16% lower peak concentration (32.7+/-0.9 vs. 38.8+/-2.1 mg/L; P=.04), and a 75% higher weight-adjusted clearance (0.185+/-0.013 vs. 0.106+/-0.006 L/h/kg; P<.001). The 30 mg/kg dose in children increased the AUC(0-24) to 164+/-10 mg x h/L and the peak concentration to 43.8+/-2.3 mg/L. CONCLUSIONS: Drug exposure is significantly lower in children than in adults treated with the same weight-adjusted regimen of meglumine antimoniate, which primarily stems from a higher antimony clearance rate.

Resistance to oxidative stress is associated with metastasis in mucocutaneous leishmaniasis.

Mucocutaneous leishmaniasis (MCL) in South and Central America is characterized by the dissemination (metastasis) of Leishmania Viannia subgenus parasites from a cutaneous lesion to nasopharyngeal tissues. Little is known about the pathogenesis of MCL, especially with regard to the virulence of the parasites and the process of metastatic dissemination. We previously examined the functional relationship between cytoplasmic peroxiredoxin and metastatic phenotype using highly, infrequently, and nonmetastatic clones isolated from an L. (V.) guyanensis strain previously shown to be highly metastatic in golden hamsters. Distinct forms of cytoplasmic peroxiredoxin were identified and found to be associated with the metastatic phenotype. We report here that peroxidase activity in the presence of hydrogen peroxide and infectivity differs between metastatic and nonmetastatic L. (V.) guyanensis clones. After hydrogen peroxide treatment or heat shock, peroxiredoxin was detected preferentially as dimers in metastatic L. (V.) guyanensis clones and in L. (V.) panamensis strains from patients with MCL, compared with nonmetastatic parasites. These data provide evidence that resistance to the first microbicidal response of the host cell by Leishmania promastigotes is linked to peroxiredoxin conformation and may be relevant to intracellular survival and persistence, which are prerequisites for the development of metastatic disease.

Resistance to antimony and treatment failure in human Leishmania (Viannia) infection.

BACKGROUND: Failure of antimonial therapy has been increasingly reported in anthroponotic visceral leishmaniasis and in cutaneous disease. The role of drug resistance in treatment failure has been difficult to ascertain because therapeutic response is multifactorial, and the efficacy of antimonial drugs depends on an effective immune response. In this study, we sought to determine whether standard treatment selects for resistant organisms and whether drug resistance contributes to treatment failure. METHODS: We evaluated the susceptibility to antimony of 19 strains isolated before treatment with meglumine antimoniate and 21 strains isolated at treatment failure from 20 patients. The 50% effective dose (ED50) of antimony in the form of additive-free meglumine antimoniate was determined for intracellular amastigotes in human promonocytic U-937 cells. RESULTS: Before treatment, 16% of strains (3/19) showed primary resistance (ED50 of >128 microg Sb/mL), whereas 84% (16/19) were susceptible (ED50 of <20 microg Sb/mL). However, 88% of susceptible strains (14/16) had ED90 values of >128 microg Sb/mL. At treatment failure, 40% of strains (8/20) were resistant. Secondary resistance was documented in 4 patients. CONCLUSIONS: Primary and secondary resistance to antimony can contribute to treatment failure in American cutaneous leishmaniasis. Selection for resistance to antimony occurs during standard treatment with antimonial drugs, and primary resistance to antimony supports the plausibility of anthroponotic transmission.

Comparative protein profiling identifies elongation factor-1beta and tryparedoxin peroxidase as factors associated with metastasis in Leishmania guyanensis.

Parasites of the Leishmania Viannia subgenus are major causative agents of mucocutaneous leishmaniasis (MCL), a disease characterised by parasite dissemination (metastasis) from the original cutaneous lesion to form debilitating secondary lesions in the nasopharyngeal mucosa. We employed a protein profiling approach to identify potential metastasis factors in laboratory clones of L. (V.) guyanensis with stable phenotypes ranging from highly metastatic (M+) through infrequently metastatic (M+/M-) to non-metastatic (M-). Comparison of the soluble proteomes of promastigotes by two-dimensional electrophoresis revealed two abundant protein spots specifically associated with M+ and M+/M- clones (Met2 and Met3) and two others exclusively expressed in M- parasites (Met1 and Met4). The association between clinical disease phenotype and differential expression of Met1-Met4 was less clear in L. Viannia strains from mucosal (M+) or cutaneous (M-) lesions of patients. Identification of Met1-Met4 by biological mass spectrometry (LC-ES-MS/MS) and bioinformatics revealed that M+ and M- clones express distinct acidic and neutral isoforms of both elongation factor-1 subunit beta (EF-1beta) and cytosolic tryparedoxin peroxidase (TXNPx). This interchange of isoforms may relate to the mechanisms by which the activities of EF-1beta and TXNPx are modulated, and/or differential post-translational modification of the gene product(s). The multiple metabolic functions of EF-1 and TXNPx support the plausibility of their participation in parasite survival and persistence and thereby, metastatic disease. Both polypeptides are active in resistance to chemical and oxidant stress, providing a basis for further elucidation of the importance of antioxidant defence in the pathogenesis underlying MCL.

Advances in leishmaniasis.

Governed by parasite and host factors and immunoinflammatory responses, the clinical spectrum of leishmaniasis encompasses subclinical (inapparent), localised (skin lesions), and disseminated infection (cutaneous, mucosal, or visceral). Symptomatic disease is subacute or chronic and diverse in presentation and outcome. Clinical characteristics vary further by endemic region. Despite T-cell-dependent immune responses, which produce asymptomatic and self-healing infection, or appropriate treatment, intracellular infection is probably life-long since targeted cells (tissue macrophages) allow residual parasites to persist. There is an epidemic of cutaneous leishmaniasis in Afghanistan and Pakistan and of visceral infection in India and Sudan. Diagnosis relies on visualising parasites in tissue or serology; culture and detection of parasite DNA are useful in the laboratory. Pentavalent antimony is the conventional treatment; however, resistance of visceral infection in India has spawned new treatment approaches--amphotericin B and its lipid formulations, injectable paromomycin, and oral miltefosine. Despite tangible advances in diagnosis, treatment, and basic scientific research, leishmaniasis is embedded in poverty and neglected. Current obstacles to realistic prevention and proper management include inadequate vector (sandfly) control, no vaccine, and insufficient access to or impetus for developing affordable new drugs.

Amplification of human DNA by primers targeted to Leishmania kinetoplast DNA and post-genome considerations in the detection of parasites by a polymerase chain reaction.

We evaluated the Leishmania Viannia-specific primers B1-B2 to detect Leishmania in normal skin and peripheral blood monocytes of patients with active cutaneous leishmaniasis. Southern blotting and sequencing of polymerase chain reaction (PCR) products confirmed the specificity of kinetoplast DNA (kDNA) amplification from tissue fluid from healthy skin, whereas the PCR with monocytes also amplified a human sequence of a size similar (718 basepairs) to the expected kDNA product (750 basepairs), resulting in false-positive results. Although B1 was not homologous to any human DNA sequence, B2 showed homology to a human chromosome 2 intergenic region (AC010878) at positions 35,881-36,599, which are spaced 718 nucleotides apart. Amplification of the human artifact from monocyte DNA was confirmed using the primer B2 alone. Examination of other primers reported for the PCR of kDNA from various species of Leishmania showed that six of seven were homologous to human DNA sequences. These findings underscore the importance of exploiting sequencing, bioinformatics, and DNA probes to refine molecular amplification techniques and to validate the performance of primers when used for new applications.

Role of the ABC transporter MRPA (PGPA) in antimony resistance in Leishmania infantum axenic and intracellular amastigotes.

Antimonial compounds are the mainstay for the treatment of infections with the protozoan parasite Leishmania. We present our studies on Leishmania infantum amastigote parasites selected for resistance to potassium antimonyl tartrate [Sb(III)]. Inside macrophages, the Sb(III)-selected cells are cross-resistant to sodium stibogluconate (Pentostam), the main drug used against Leishmania. Putative alterations in the level of expression of more than 40 genes were compared between susceptible and resistant axenic amastigotes using customized DNA microarrays. The expression of three genes coding for the ABC transporter MRPA (PGPA), S-adenosylhomocysteine hydrolase, and folylpolyglutamate synthase was found to be consistently increased. The levels of cysteine were found to be increased in the mutant. Transfection of the MRPA gene was shown to confer sodium stibogluconate resistance in intracellular parasites. This MRPA-mediated resistance could be reverted by using the glutathione biosynthesis-specific inhibitor buthionine sulfoximine. These results highlight for the first time the role of MRPA in antimony resistance in the amastigote stage of the parasite and suggest a strategy for reversing resistance.

Selective action of fluoroquinolones against intracellular amastigotes of Leishmania (Viannia) panamensis in vitro.

We have demonstrated that fluoroquinolones, a class of antibacterial agents that act through inhibition of type II DNA topoisomerases, exert selective action against intracellular amastigotes of Leishmania (Viannia) panamensis at concentrations that are achievable in vivo. Drug cytotoxicity assays employing the luciferase reporter gene revealed that intracellular amastigotes were 6.6- to 25.9-fold more sensitive than human macrophages (P < 0.05) to second-generation fluoroquinolones in vitro. The most selective agents (enoxacin and ciprofloxacin) exhibited 2 orders of magnitude greater potency against parasites (50% effective dose [ED50] = 54.9-83.4 microM) than host cells (ED50 = 1,425-1,740 microM). Linear regression analysis of ED50 data confirmed a complete lack of correlation (r = 0.001) between the relative drug sensitivities of parasites and host cells. A potential relationship between the structures of fluoroquinolones and their relative leishmanicidal activities was observed. The key substituents of the basic pyridone beta-carboxylic acid nucleus accounting for enhanced antiparasite potency and selectivity appear to be a nitrogen at position 8 of the bicyclic nucleus (enoxacin), a cyclopropyl substituent at the R1 site (ciprofloxacin), and linkage of the R1 and X8 groups by a CH3CHO bridge to form a tricyclic compound (ofloxacin). These findings support the potential of fluoroquinolones and derivatives as novel antileishmanials and encourage their clinical evaluation.

Inhibition of Leishmania donovani promastigote DNA topoisomerase I and human monocyte DNA topoisomerases I and II by antimonial drugs and classical antitopoisomerase agents.

We have compared the inhibitor sensitivities of DNA topoisomerase I (TOPI) from Leishmania donovani promastigotes and TOPs I and II of human monocytes using pentavalent and trivalent antimonials (SbV, SbIII) and classical TOP inhibitors. Bis-benzimidazoles (Hoechst-33258 and -33342) were potent inhibitors of both parasite and human TOPI, but Hoechst-33342 was markedly less cytotoxic to promastigotes than to monocytes in vitro. Leishmania donovani was also considerably less sensitive than monocytes to camptothecin, both at enzyme and cellular levels. Sodium stibogluconate (SSG) was the only antimonial to inhibit TOPI, exhibiting a significant (P < 0.05) 3-fold greater potency against the L. donovani enzyme but showed low cytotoxicities against intact promastigotes. The SbV meglumine antimoniate failed to inhibit TOPI and showed negligible cytotoxicities, whereas SbIII drugs were lethal to parasites and monocytes yet poor inhibitors of TOPI. Monocyte TOPII was inhibited by bis-benzimidazoles and insensitive to antimonials and camptothecin. The disparity between the high leishmanicidal activity and low anti-TOPI potency of SbIII indicates that in vivo targeting of L. donovani TOPI by the reductive pathway of antimonial activation is improbable. Nevertheless, the potent direct inhibition of TOPI by SSG and the differential interactions of camptothecin with L. donovani and human TOPI support the possibility of developing parasite-specific derivatives.