Sterol composition and biosynthesis in Trypanosoma cruzi amastigotes.

A detailed analysis of the endogenous sterols present in the clinically relevant intracellular (amastigote) stages of Trypanosoma cruzi, is presented. The parasites were grown in cultured Vero cells in the absence or presence of different sterol biosynthesis inhibitors, including the C14alpha demethylase inhibitor ketoconazole and two inhibitors of delta24(25)-sterol methyl transferase, 20 piperidin-2-yl-5alpha-pregnan-3beta-20-R-diol (22,26-azasterol) and 24-(R,S),25-epiminolanosterol. Amastigotes were isolated and purified from their host cells and neutral lipids were extracted, separated and analyzed by chromatographic and mass spectrometric methods. Control (untreated) amastigotes contained as main endogenous sterols 24-methyl-cholesta-7-en-3beta-ol (ergosta-7-en-3beta-ol) and its 24-ethyl analog, plus smaller amounts of their precursor, ergosta-7,24(28)dien-3beta-ol; these cells also contained cholesterol (up to 80% by weight of total sterols), probably derived from host cells. Amastigotes that proliferated in the presence of 10 nM ketoconazole (minimal inhibitory concentration, MIC) for 24 h had a sharply reduced content of endogenous 4-desmethyl sterols with a concomitant accumulation of 24-methyl-dihydrolanosterol and 24-methylene-dihydrolanosterol. On the other hand, amastigotes incubated during the same period of time with the two inhibitors of 24(25)-SMT at their respective MICs (100-300 nM) accumulated large amounts of C27 sterols whose structure suggested, in the case of 22,26-azasterol, that delta14 sterol reductase was also inhibited. Ketoconazole produced a dose-dependent reduction in the incorporation of [2-(14)C]-acetate into the parasite's endogenous C4-desmethyl sterols with an IC50 of 50 nM, indistinguishable from the value reported previously for the extracellular epimastigote form. Taken together, the results showed that amastigotes have a simpler sterol biosynthetic pathway than that previously described for epimastigotes, lacking both delta5 and delta22 reductases. They also suggest that the 100-fold higher potency of antifungal azoles as antiproliferative agents against amastigotes, when compared with epimastigotes, is most probably due to a smaller pool of endogenous sterols in the intracellular parasites.

Antiproliferative effects and mechanism of action of SCH 56592 against Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies.

We have investigated the antiproliferative effects of SCH 56592, a new experimental triazole, against Trypanosoma (Schizotrypanum) cruzi, the etiological agent of Chagas' disease in Latin America. SCH 56592 blocked the proliferation of the epimastigote form of the parasite in vitro at 30 nM, a concentration 30- to 100-fold lower than that required with the reference compounds ketoconazole and itraconazole. At that concentration all the parasite's endogenous sterols (ergosterol, 24-ethyl-cholesta-5,7,22-trien-3 beta-ol, and its 22-dihydro analogs), were replaced by methylated sterols (lanosterol and 24-methylene-dihydrolanosterol), as revealed by high-resolution gas chromatography coupled with mass spectrometry. This indicated that the primary mechanism of action of the drug was inhibition of the parasite's sterol C-14 alpha demethylase. Against the clinically relevant intracellular amastigote form, grown in cultured Vero cells at 37 degrees C, the MIC of SCH 56592 was 0.3 nM, again 33- to 100-fold lower than that of ketoconazole or itraconazole. In a murine model of acute Chagas' disease, SCH 56592 given at > or = 10 mg/kg of body weight/day for a total of 43 doses allowed 85 to 100% survival and 90 to 100% cure of the surviving animals, as verified by parasitological, serological, and PCR-based tests, while ketoconazole given at 30 mg/kg day allowed 60% survival but only 20% cure. In a murine model of chronic Chagas' disease, SCH 56592 was again more effective than ketoconazole, providing 75 to 85% protection from death, with 60 to 75% parasitological cures of the surviving animals, while no parasitological cures were observed with ketoconazole. The results indicate that SCH 56592 is the most powerful sterol biosynthesis inhibitor ever tested against T. cruzi and may be useful in the treatment of human Chagas' disease.

Cure of short- and long-term experimental Chagas' disease using D0870.

Chagas' disease, a protozoan infection by the kinetoplastid Trypanosoma cruzi, constitutes a major public health problem in Latin America. With the use of mouse models of both short- and long-term forms of the disease, the efficacy of D0870, a bis-triazole derivative, was tested. D0870 was able to prevent death and induced parasitological cure in 70 to 90 percent of animals, in both the short- and long-term disease. In contrast, currently used drugs such as nifurtimox or ketoconazole prolonged survival but did not induce significant curing effects. D0870 may be useful in the treatment of human long-term Chagas' disease, a condition that is currently incurable.

Lupus eritematoso sistémico: relación de factores hormonales con evolución clínica / Lupus erythematosus systemic: relations of hormonals factors with clinic evolution

OBJETIVOS: Nos propusimos comprobar si los factores hormonales tienen influencia en la actividad y evolución del Lupus Eritematoso Sistémico (LES), determinando los niveles séricos de estradiol, testosterona, FSH y de la concentración de receptores estrogénicos en células monoclonales de sangre periférico(CMSP). METODOLOGIA: Se seleccionaron 22 pacientes con diagnóstico de LES basados en los criterios de la ARA 1982, del sexo femenino culminando sólo 10 de ellos, la investigación. Se realizó registro clínico, inmunoserológico, niveles séricos de estradiol, testosterona, FSH y concentración de receptores estrogénicos en CMSP tanto para los pacientes como para los controles. El análisis estadístico se realizó mediante la "t" de Student y el índice de correlación lineal de Pearson. RESULTADOS: En el 80 por ciento de los pacientes se observó Anyti-Adn elevados. Factores del Complemento disminuidos en el 90 por ciento y complejos inmunes circulantes elevados en la totalidad de los casos. La creatinina sérica fue normal contrastando con el hallazgo de nefritis lúpica en el 100 por ciento. Los estrógenos séricos elevados significativamente (p<0,01), testosterona disminuida (p<0,01), FSH sin diferencia estadística en ambos grupos (p<0,5). No hubo correlación entre el estradiol, testosterona en relación a la concentración de receptores estrogénicos en CMSP e igualmente no se constató correlación entre la concentración de receptores estrogénicos y los índices de actividad de la enfermedad. CONCLUSION: La determinación de los factores del coplemento viene a ser índices precisos de actividad, superiores a los anticuerpos Anti-Adn bicatenario. En todo paciente lúpico, existe nefropatía. Proponemos que las pacientes con LES, tienen a poseer estradiol y por otra parte, la testosterona disminuida en relación a la población normal. En cuanto a la concentración de receptores de estrógeno en CMSP, no se encontró diferencia entre los grupos. No pareciera relacionarse los niveles elevados de estradiol con la actividad de la enfermedad, ni existir correlación entre la actividad estrogénica y la lúpica.

Inhibition of phosphatidylcholine biosynthesis and cell proliferation in Trypanosoma cruzi by ajoene, an antiplatelet compound isolated from garlic.

Ajoene [(E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide], a potent antiplatelet compound derived from garlic, inhibits the proliferation of both epimastigotes and amastigotes of Trypanosoma cruzi, the causative agent of Chagas' disease. The growth of the epimastigote form was immediately arrested by 80 microM ajoene, while 100 microM induced cell lysis in 24 hr. In the amastigote form proliferating inside VERO cells, 40 microM ajoene was sufficient to eradicate the parasite from the host cells in 96 hr. Growth inhibition of the epimastigotes was accompanied by a gross alteration of the phospholipid composition of the treated cells in which phosphatidylcholine (PC), the major phospholipid class present in control cells, dropped to the least abundant phospholipid in cells treated with 60 microM ajoene for 96 hr, while its immediate precursor, phosphatidylethanolamine (PE), became the predominant species; this was correlated with a marked drop in the incorporation of [14C-U]acetate in PC and a corresponding increase in PE. Concomitant with the change in the phospholipid headgroup composition of the cells, the fatty acids esterified to this lipid fraction underwent a dramatic alteration due to the increase in the content of saturated fatty acids and a marked reduction in the content of linoleic (18:2) acid, which is the predominant fatty acid in control cells. We also found that ajoene inhibited the de novo synthesis of neutral lipids and, in particular, of sterols in the epimastigotes, but the resultant changes in the sterol composition were not sufficient to explain the antiproliferative effects of the drug. Electron-microscopy showed a concentration-dependent alteration of intracellular membranous structures, particularly the mitochondrion and endoplasmatic reticulum. The results suggest that one important factor associated with the antiproliferative effects of ajoene against T. cruzi is its specific alteration of the phospholipid composition of these cells.