Expression of green fluorescent protein as a marker for effects of antileishmanial compounds in vitro.

Transgenic Leishmania infantum promastigotes, which constitutively express green fluorescent protein (GFP) in their cytoplasm, were used to monitor the effects of antileishmanial compounds in real time. The GFP-based assay provided a reliable measure of drug-induced inhibitory effects on protein expression, resulting in a dynamic picture of the responses of leishmanial promastigotes to the compounds tested.

Flow cytometry analysis of the effect of allopurinol and the dinitroaniline compound (Chloralin) on the viability and proliferation of Leishmania infantum promastigotes.

BACKGROUND: Leishmaniasis is a major parasitic disease in the tropical regions. However, Leishmania infantum has recently emerged as a very important cause of opportunistic infections for individuals positive for human immunodeficiency virus (HIV). However, there is a lack of in vitro tests for assessing the effect of anti-parasitic drugs on the viability and proliferation of Leishmania infantum. The aim of this study is to assess the efficacy of anti-parasitic drugs like allopurinol and Chloralin on the viability and proliferation of L. infantum promastigotes by utilizing two complementary flow cytometric approaches after exposure of the promastigotes to various concentrations of the drugs. RESULTS: The density of the cultures in the presence and absence of allopurinol was determined by haemocytometer enumeration. The two flow cytometric approaches used to monitor the drug effect were: (i) a quantitative method to measure cell division using 5-,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining and (ii) evaluation of cell viability by dual-staining with the membrane-permeable nuclear stain, SBRY-14 and propidium iodide. It was found that concentrations of allopurinol above 50 microg/ml yielded a clear decrease in the proliferation rate of the promastigotes. However, the viability results showed that about 46.8% of the promastigotes incubated in the presence of 800 microg/ml of allopurinol were still alive after 96 hours. In sharp contrast, more than 90% of promastigotes treated with Chloralin 10 microM (2.7 microg/ml) were dead after 48 hours of treatment. These flow cytometric findings suggest that allopurinol has a leishmaniostatic effect while the dinitroaniline compound (Chloralin) has a leishmaniocidal effect against promastigotes. CONCLUSIONS: The flow cytometric data on proliferation and viability were consistent with results obtained from haemocytometer counts and allowed us to develop a model for assessing in vitro the effects of medicaments like allopurinol and chloralin on L. infantum promastigotes on a cellular level.

Flow cytometric assessment of allopurinol susceptibility in Leishmania infantum promastigote.

BACKGROUND: Leishmaniasis is a major tropical and subtropical parasitic disease. Sodium stibogluconate, N-methyl -D-glucamine antimoniate, amphotericin B, pentamidine, and ketoconazole are drugs used to treat this disease. Some of these drugs cause severe adverse side effects and treatment failures are common. Allopurinol, a purine analog, has been used to treat leishmaniasis, alone or combined with the previously mentioned drugs. Low cost, ease of administration (oral), and lack of toxicity make allopurinol a particularly appealing candidate. METHODS: The effect of allopurinol on Leishmania infantum (MCAN/ES/89/IPZ229/1/89, zymodeme MON1) wild-type promastigotes (wt-p229), and an altered form of these promastigotes (allo-p229) resulting from long term in vitro exposure to allopurinol, was determined by [(3)H]-thymidine incorporation assays and by diverse flow cytometric approaches. RESULTS: Allopurinol arrested the proliferative capacity of wt-p229 promastigotes, reduced the proportion of viable cells, and decreased their total protein content. In contrast, allo-p229 promastigote proliferation was only slightly decelerated and the proportion of viable cells and the protein content were not affected by the allopurinol treatment. CONCLUSIONS: The flow cytometry approach allowed us to demonstrate differences in allopurinol susceptibility of the two promastigote forms, expanding the spectrum of flow cytometry applications in studies of parasite resistance.

Financial analysis of animal trypanosomiasis control using cypermethrin pour-on in Kenya.

The financial impact of use of cypermethrin pour-on (Ectopor(R)) in control of animal trypanosomiosis was determined in a trial undertaken by the Kenya Trypanosomiasis Research Institute (KETRI). This trial started in December 1990 and ended in February 1992. It was undertaken in two adjacent ranches in the coast province of Kenya. The trial site was in an area of high apparent density (AD) of tsetse flies, and at the start of the trial no cattle were kept in this area. Cypermethrin was applied fortnightly to the 1100 steers which were kept in pour-on ranch 'A' while another 100 steers were kept in control ranch 'B' to act as control sentinels. From the main pour-on group, 100 animals were identified as the pour-on sentinels and compared to the control sentinels which received no pour-on.Pour-on application led to a significant decrease in the tsetse AD in the pour-on ranch A to 90% of the initial AD in some areas. The animals treated with pour-on had a significantly higher mean packed-cell volume (PCV). The weekly prevalence of trypanosome infections in animals treated with pour-on was <4% with only one exception when it was <10%. In the control animals, the prevalence ranged between 10 and 50% (with a few exceptions when it was <10%). The incidence of tick-borne diseases was lower in the pour-on animals. The mean monthly weights of the pour-on animals was significantly higher, and at the end of the trial the pour-on animals had a mean weight gain of 136.70+/-16.7kg while the control animals had gained 97.16+/-22.6kg. The financial net return of using cypermethrin pour-on was positive and the financial rate of return of 122.6% indicated that use of the pour-on was highly beneficial despite the high cost of the product.

A comparison of the effects of a benzimidazole and the dinitroanilines against Leishmania infantum.

Leishmania infantum promastigotes and amastigotes were axenically cultured and exposed to the known tubulin binding compounds, the dinitroanilines, trifluralin, benfluralin, pendimethalin, oryzalin and the precursor of the dinitroanilines, chloralin, as well as isomers of chloralin and trifluralin and to the benzimidazole, albendazole. Drug induced inhibition was observed using [3H]thymidine uptake compared with untreated controls. In vitro analysis demonstrated a significant difference in the activity of five of the seven dinitroanilines between both life cycle stages of L. infantum. The amastigotes were 20-times more sensitive to chloralin and its isomer than to the dinitroanilines whereas the promastigotes were similar in sensitivity to the dinitroanilines and to chloralin and its isomer. This interesting finding suggests that the dinitroaniline precursors may have different target sites in the amastigotes to those within the promastigotes. Additionally, both chloralin and its isomer, and to a lesser extent benfluralin, caused a substantial stimulation of thymidine incorporation (up to 50%) at low concentrations. Dose response analysis suggests that the dinitroanilines may have more than one mode of action against L. infantum amastigotes and promastigotes. The inhibitory effects of the dinitroanilines against L. infantum vary from previous findings using the dinitroanilines against other Leishmania spp. The 348 base pair DNA sequence coding for beta-tubulin from amino acid residues 132 to 248 was obtained for L. infantum and used to compare the in vivo efficacy of albendazole with predicted activity based on beta-tubulin sequences of known benzimidazole sensitive protozoa. The use of beta-tubulin sequence as a predictive model of benzimidazole activity is discussed with particular reference to L. infantum.