RESUMO
A modern aqueous-ethanolic formulation of the roots of Pelargonium sidoides (Eps 7630), elaborated from the traditional herbal medicine used in areas of southern Africa, is effectively employed for the treatment of ENT and respiratory tract infections in modern phytotherapy. Previous studies have demonstrated antibacterial and immunomodulatory activities. To gain insight into the mode of action at the molecular level, gene expression analyses for the inducible nitric oxide synthase and the cytokines interleukin (IL)-1, IL-12, IL-18, tumour necrosis factor (TNF)-alpha, interferon (IFN)-alpha, and IFN-gamma, were performed using reverse transcription-polymerase chain reaction (RT-PCR). The experiments were carried out in parallel in non-infected and in Leishmania major-infected RAW 264.7 cells and the expression profiles were compared with those mediated by IFN-gamma+LPS. Eps 7630 induced low mRNA levels in non-infected cells, and it considerably up-regulated the transcript expressions in parasitised cells. Interestingly, and in contrast to activation by IFN-gamma+LPS, Eps 7630 also stimulated infected cells to produce IFN-gamma mRNA. A similar expression profile was observed for the methanol-insoluble fraction (MIF) of Eps 7630 and gallic acid, a trace constituent of the extract, while the methanol-soluble fraction and umckalin, an exclusive and representative member of the occurring coumarins, proved to be devoid of any remarkable gene-inducing capabilities. The present results provide not only convincing support for the improvement of immune functions as previously demonstrated in functional bioassays, but also evidence for activation at the transcriptional level and suggest that the underlying inducing principle is located in the MIF.
Assuntos
Citocinas/efeitos dos fármacos , Leishmania major/enzimologia , Leishmania major/imunologia , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Pelargonium , Extratos Vegetais/farmacologia , Animais , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Camundongos , Raízes de PlantasRESUMO
Invasive fungal infections are a major cause of morbidity and mortality in immunodeficient individuals (such as AIDS patients) and in transplant recipients or tumor patients undergoing immunosuppressive chemotherapy. Amphotericin B is one of the oldest, yet most efficient antimycotic agents. However, its usefulness is limited due to dose-dependent side-effects, notably nephrotoxicity. In order to improve its safety margin, new pharmaceutical formulations of amphotericin B have been designed especially to reduce its detrimental effects on the kidneys. Since the 1980s, a wide variety of new amphotericin B formulations have been brought forward for clinical testing, many of which were approved and reached market value in the 1990s. This review describes and discusses the molecular genetics, pharmacological, toxicological, and clinical aspects of amphotericin B itself and many of its innovative formulations.
Assuntos
Anfotericina B , Anti-Infecciosos , Anfotericina B/química , Anfotericina B/metabolismo , Anfotericina B/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Química Farmacêutica , Colesterol/metabolismo , Engenharia Genética , Canais Iônicos/química , Canais Iônicos/metabolismo , Cinética , Modelos MolecularesRESUMO
A series of 27 hydrolyzable tannins and related compounds was tested for antiparasitic effects against both extracellular promastigote and intracellular amastigote Leishmania donovani organisms. In parallel, the compounds were evaluated for their immunomodulatory effects on macrophage functions, including release of nitric oxide (NO), tumour necrosis factor-alpha (TNF-alpha) and interferon (IFN)-like properties using several functional assays. Of the series of polyphenols tested, only gallic acid (54 microM NO) and its methyl ester (32 microM NO) induced murine macrophage-like RAW 264.7 cells to release NO in appreciable amounts (IFN-gamma/LPS 119 microM NO). The in vitro TNF-inducing potential of the polyphenols examined increased in the order of oligomeric ellagitannins (EC(50) > 25 microg/ml) < monomeric ellagitannins, gallotannins (EC(50) 8.5 to > 25 microg/ml) < C-glucosidic ellagitannins, dehydroellagitannins (EC(50) 0.6 - 2.8 microg/ml) at the host cell subtoxic concentration of 50 microg/ml. Furthermore, promastigotes of Leishmania donovani were assayed in the presence of these polyphenols and the results showed that none of the compounds was significantly toxic (EC(50) > 25 microg/ml) to the extracellular forms. In contrast, all polyphenols showed pronounced antileishmanial activities (EC(50) < 0.4 - 12.5 versus 7.9 microg/ml for Pentostam) against intracellular amastigotes of L. donovani residing within RAW cells. Noteworthy, most compounds exhibited low cytotoxicity against the murine host cells (EC(50) >25 microg/ml). Furthermore, some ellagitannins and the majority of dehydroellagitannins induced potent interferon-like activities as reflected by inhibition of the cytopathic effect of encephalomyocarditis virus on fibroblast L929 cells. This is the first report on hydrolyzable tannins as a new class of natural products with leishmanicidal activity including their potential for inducing the release of NO, TNF and IFN-like activity in macrophage-like RAW cells.
Assuntos
Antiparasitários/farmacologia , Leishmania donovani/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Taninos/farmacologia , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Animais , Linhagem Celular , Macrófagos/metabolismo , Macrófagos/parasitologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Extratos Vegetais/farmacologia , Estereoisomerismo , Taninos/química , Taninos/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A series of naturally occurring aurones (e. g., Rubiaceae, Cyperaceae) was synthesized and tested for the ability to inhibit erythrocytic stages of Plasmodium falciparum strains in vitro. Some of the these compounds exhibited antiplasmodial activity in the micromol range, determined as fifty percent-inhibitory concentrations (IC(50)). Drug activity was not associated with cytotoxicity for the mammalian tumor cell lines KB and SKMel (IC(50) > 3.0 microM). The most active compound was 4,6,4'-triacetyl-3',5'-dimethoxy-2-aurone with IC(50) values of 0.007 microM and 0.18 microM for the P. falciparum strains K1 and NF54, respectively. Interestingly, the multiple drug-resistant P. falciparum strain K1 was more sensitive to tested aurones than the drug-susceptible strain NF54.
Assuntos
Antimaláricos/farmacologia , Antiprotozoários/farmacologia , Benzofuranos/farmacologia , Preparações de Plantas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Animais , Antimaláricos/química , Antiprotozoários/química , Benzofuranos/química , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Humanos , Técnicas In Vitro , Concentração Inibidora 50 , Magnoliopsida , Malária Falciparum/tratamento farmacológico , Estrutura Molecular , Fitoterapia , Especificidade da Espécie , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
A series of 17 proanthocyanidins and structurally related compounds was tested for activity against Leishmania donovani amastigotes and promastigotes in vitro. Most of the polyphenols significantly inhibited the intracellular survival of L. donovani amastigotes (EC50 0.8-10.6 nM) when compared with the antileishmanial drug Pentostam (EC50 10.6 nM), but all were inactive against the extracellular form (EC50 7.8 to >86 nM). Noteworthy is that all compounds exhibited only moderate or no cytotoxicity against the murine host cells (EC50 7.8 to >56 nM; >25 microg/ml). These polyphenols were further evaluated for immunomodulatory effects on macrophage functions, including release of nitric oxide (NO), tumor necrosis factor-alpha (TNF) and interferon (IFN)-like properties using several functional assays. The results showed that all compounds induced murine RAW 264.7 cells only moderately to release NO (7-26 microM) relative to the reference stimulus IFN-gamma/LPS (119 microM). The TNF-inducing potential of the polyphenols producing 50% lysis in murine L929 cells ranged from absent to 138 U/ml at the host cell subtoxic concentration of 50 microg/ml. The highest TNF-inducing activity was associated with those flavan-3-ols with galloyl groups (98-127 U/ml). For proanthocyanidins, it appeared that an increase in the flavanyl chain length did not enhance the induction of TNF-release (32-86 U/ml and below detection limits for oligomers and polymers, respectively). With interferon-like activities, phylloflavan and a prodelphinidin polymer showed appreciable cytoprotective effects, as reflected by the inhibition of the cytopathic effect of encephalomyocarditis virus on L929 fibroblast cells (38 and 36 U/ml, respectively). All remaining compounds displayed only negligible or moderate protective effects at subtoxic concentrations up to 25 microg/ml (<5 to 12 U/ml). These results indicate that proanthocyanidins and related compounds have favorable antileishmanial activity in vitro and might be considered as beneficial immunological response modifiers provided there are no bioavailability problems.
Assuntos
Antocianinas/farmacologia , Antiprotozoários/farmacologia , Leishmania donovani/efeitos dos fármacos , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Proantocianidinas , Fator de Necrose Tumoral alfa/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Flavonoides/farmacologia , Interferons/biossíntese , Interferons/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II , Relação Estrutura-AtividadeRESUMO
The antileishmanial and immunomodulatory potencies of a series of 28 polyphenols were evaluated in terms of extra- and intracellular leishmanicidal activity and macrophage activation for release of nitric oxide (NO), tumour necrosis factor (TNF) and interferon (IFN)-like properties. For this, several functional bioassays were employed including an in vitro model for leishmaniasis in which murine bone marrow-derived macrophages (BMMphi) were infected with the obligate intracellular parasite Leishmania donovani, an extracellular Leishmania proliferation assay, a fibroblast-lysis assay (TNF-activity), and a biochemical assay for NO. Except for gallic acid, its methyl ester, shikimic acid and catechin (EC50 25.8-67.9 nM) all polyphenols tested significantly inhibited the intracellular survival of L. donovani amastigotes (EC50 0.4-13.9 nM) when compared with the clinically used agent, sodium stibogluconate (EC50 10.6 nM). In contrast, none of the samples proved to be directly toxic for the extracellular promastigote form of the parasite. Noteworthy, the phenolic samples showed only moderate or no cytotoxicity against the murine host cells (EC50 10 to >144 nM). Although NO is an important effector molecule in macrophage microbicidal activity, the inducing potential of the test compounds for its release was found to be very moderate ranging from 7-54 microM (IFN-gamma/LPS 119 microM). On the other hand, inhibition of NO production had no apparent effect on intracellular leishmanicidal activity of polyphenols. Their in vitro TNF-inducing potential producing 50% lysis in murine L929 cells increased in the order of simple phenols and flavanols (34-48 U/ml) < A-type proanthocyanidins (53-80 U/ml) < B-type proanthocyanidins (64-200 U/ml) < hydrolyzable tannins (287-350 U/ml) at the host cell subtoxic concentration of 50 microg/ml. Furthermore, gallic acid and some hydrolyzable tannins showed appreciable IFN-like activities (14-23 U/ml) as reflected by inhibition of the cytopathic effect of encephalomyocarditis virus on fibroblast L 929 cells. The results provide a rational basis for the recorded anti-infectious efficacy of traditionally used herbal medicines containing tannins in vivo, in the light of both only moderate direct antimicrobial activities of distinct polyphenols in vitro and the limited knowledge on their uptake in humans.
Assuntos
Flavonoides , Leishmania donovani/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Fenóis/farmacologia , Polímeros/farmacologia , Taninos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células da Medula Óssea/citologia , Interferon gama/farmacologia , Células L , Ativação de Macrófagos , Macrófagos/fisiologia , Camundongos , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Polifenóis , Proteínas Recombinantes , Relação Estrutura-Atividade , ômega-N-Metilarginina/farmacologiaRESUMO
Extracts and isolated constituents (coumarins and phenols) of Pelargonium sidoides DC, a plant species used in folk medicine by the Southern African native population, were evaluated for their effects on nonspecific immune functions. Although this herbal medicine is also successfully employed in modern phytotherapy in Europe to cure infectious diseases of the respiratory tract, the scientific basis of its remedial effects is still unclear. Thus, functional bioassays including an in vitro model for intracellular infection with Leishmania parasites, an extracellular Leishmania growth assay, a fibroblast-virus protection assay (IFN activity), a fibroblast-lysis assay (TNF activity) and a biochemical assay for inorganic nitric oxides (iNO) were employed. None of the test samples revealed significant activity against extracellular, promastigote Leishmania donovani, the causative agent of human visceral leishmaniasis. In contrast, apart from the coumarin samples, all the Pelargonium extracts (EC(50) <0.1-3.3 microg/mL), gallic acid (EC(50) 4.4 microg/mL) and its methyl ester (EC(50) 12.5 microg/mL) significantly reduced the intracellular survival of L. donovani amastigotes within murine macrophages. These data indicate that the samples acted indirectly on Leishmania parasites, possibly by activating leishmanicidal macrophage functions. Macrophage activation was confirmed by detection of tumour necrosis factor (TNF-alpha) and inorganic nitric oxides (iNO) in supernatants of sample-treated macrophage cultures. Synthesis of iNO is a well-known effector mechanism of macrophages against microorganisms such as Leishmania. Interestingly, blocking iNO-synthase with L-NMMA had no substantial effect on sample-induced intracellular Leishmania kill. From bioassay-guided fractionation, gallic acid and its methyl ester present in large amounts in P. sidoides and in its active extracts, were identified as the prominent immunomodulatory principle for this herbal medicine. The results, when taken together with recent reported antibacterial activity, provide a rational basis for both the traditional and the present utilization of P. sidoides in the claimed conditions.
Assuntos
Fibroblastos/efeitos dos fármacos , Leishmania donovani/efeitos dos fármacos , Plantas Medicinais , Rosales , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Animais , Macrófagos/efeitos dos fármacos , Medicinas Tradicionais Africanas , Camundongos , Fitoterapia , Extratos Vegetais/farmacologia , Raízes de PlantasRESUMO
A variety of chalcones have been shown to exhibit activity against Leishmania parasites. In contrast to synthetic or semisynthetic chalcones, only a few plant-derived compounds have been investigated. To provide a scientific rational for the antiprotozoal potency of plants used in ethnomedicine and containing chalcones, and in the search for new antiprotozoal drugs, we have carried out a primary screening for in vitro leishmanicidal activity of 20 chalcones isolated from plants. The compounds were tested against extracellular promastigotes of Leishmania donovani, L. infantum, L. enrietii and L. major, and against intracellular amastigote L. donovani residing within murine macrophages. Against the extracellular Leishmania (L. donovani), most compounds were active with EC(50) values between 0.07 and 2.01 microg/mL. Some of these chalcones, 2',4'-dihydroxy-4-methoxychalcone, 2'-hydroxy-3,4-dimethoxychalcone and 2-hydroxy-4,4'-dimethoxychalcone also significantly inhibited the intracellular survival of L. donovani parasites with EC(50) values between 0.39 and 0.41 microg/mL. When tested against murine bone marrow-derived macrophages as a mammalian host cell control, all compounds with antileishmanial activities also proved to be cytotoxic to varying extents (EC(50) 0.19-2.06 microg/mL). Correlations between molecular structures and antileishmanial activity are discussed in detail. Specific compounds are illustrated with emphasis on their mode of action and potential for the development of selective antiprotozoal agents.
Assuntos
Antiprotozoários/farmacologia , Chalcona/análogos & derivados , Chalcona/farmacologia , Leishmania/efeitos dos fármacos , Plantas Medicinais , Animais , Macrófagos/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Relação Estrutura-AtividadeRESUMO
Aphidicolin and a series of semisynthetic aphidicolan derivatives have been identified in in vitro tests as novel drugs with antiparasitic potential. All compounds have been tested against extracellular promastigotes of Leishmania donovani, L. infantum, L. enriettii, and L. major and against intracellular amastigotes of L. donovani in murine macrophages. The compounds showed antileishmanial activity at concentrations in the microgram range (50% effective concentration [EC(50)] = 0.02 to 1.83 microg/ml). The most active derivative (aphidicolin-17-glycinate hydrochloride) had EC(50)s of 0. 2 microg/ml against extracellular and 0.02 microg/ml against intracellular L. donovani parasites. To validate the pharmacological potential of tested drugs, pharmacological safety was determined by testing all compounds against two neoplastic cell lines (squamous carcinoma [KB] and melanoma [SK-Mel]) and against murine bone marrow-derived macrophages as host cells. With minor exceptions only for macrophages, tested aphidicolans did not show significant cytotoxicity (EC(50) > 25.0 microg/ml). Structure-activity relationships of these aphidicolan derivatives are discussed.
Assuntos
Antiprotozoários/farmacologia , Afidicolina/análogos & derivados , Afidicolina/farmacologia , Leishmania/efeitos dos fármacos , Animais , Células da Medula Óssea/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura , Humanos , Camundongos , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
A series of monomeric and dimeric naphthoquinones with potential for treatment of Leishmania infections was identified in vitro using both a direct cytotoxicity assay against extracellular promastigotes of Leishmania donovani, L. infantum, L. enriettii, and L. major and a test against intracellular amastigote L. donovani residing within murine macrophages. Several naphthoquinones proved to be active at concentrations in the microgram range (EC(50) 0.9-17.0 microg/ml). When tested against a panel of human cancer cell lines (KB, SKMel, A549, MDA) and murine bone marrow culture-derived macrophages (BMMPhi) as mammalian host cell controls, compounds with anti-Leishmania-activity showed moderate (EC(50)>25 microg/ml) to pronounced (EC(50)<10 microg/ml) toxic effects.
Assuntos
Antiprotozoários/farmacologia , Leishmania/efeitos dos fármacos , Naftoquinonas/farmacologia , Animais , Humanos , Relação Estrutura-AtividadeRESUMO
A series of monomeric and dimeric naphthoquinones with potential for treatment of Leishmania infections was identified in vitro using both a direct cytotoxicity assay against extracellular promastigotes of Leishmania donovani, Leishmania infanturn, Leishmania enriettii, and Leishmania major and a test against intracellular amastigote L. donovani residing within murine macrophages. Several naphthoquinones proved to be active at concentrations in the microgram range (EC(50) 0.9-17.0 microg/ml). When tested against a panel of human cancer cell lines (KB, SKMel, A549, MDA) and murine bone marrow culture-derived macrophages (BMMPhi) as mammalian host cell controls, compounds with anti-Leishmania-activity showed moderate (EC(50)>25 microg/ml) to pronounced (EC(50)<10 microg/ml) toxic effects.
Assuntos
Leishmania/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Naftoquinonas/farmacologia , Animais , Humanos , Leishmania donovani/efeitos dos fármacos , Leishmania enriettii/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Leishmania major/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/parasitologiaRESUMO
Whole spleen cell cultures from SCID mice release high levels of IFN-gamma when exposed to heat-killed Listeria monocytogenes (HKL). This microbe-induced and T cell-independent response depends on both macrophages (MPhi) and NK cells: HKL-stimulated MPhi release TNF-alpha and IL-12, which together activate NK cells for IFN-gamma release. We show here that this cytokine-mediated activation cascade can be modulated by a mAb against the MPhi surface glycoprotein F4/80. HKL-induced IL-12, TNF-alpha, and IFN-gamma in SCID whole spleen cell cultures was inhibited by coincubation with anti-F4/80 mAb whereas IL-1 and IL-10 were enhanced. Both effects were apparent at mRNA and protein release levels. Whereas inhibitory activities were F4/80 Ag specific, stimulatory effects were Fc dependent and nonspecific. Furthermore, cytokine inhibition by anti-F4/80 was only apparent when MPhi and NK cells were present simultaneously and in close vicinity, indicating that direct cell-to-cell contact is a prerequisite. These data suggest a novel pathway for microbe-induced MPhi/NK cell interaction involving direct cell-to-cell signaling and give the first evidence for a functional role of the MPhi surface glycoprotein F4/80.
Assuntos
Adjuvantes Imunológicos/farmacologia , Anticorpos Monoclonais/farmacologia , Antígenos de Diferenciação/imunologia , Células Matadoras Naturais/imunologia , Listeria monocytogenes/imunologia , Macrófagos/imunologia , Adjuvantes Imunológicos/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Comunicação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Inibição de Contato/imunologia , Citocinas/biossíntese , Citocinas/genética , Citocinas/metabolismo , Epitopos/fisiologia , Feminino , Temperatura Alta , Imunidade Inata/imunologia , Fragmentos Fab das Imunoglobulinas/fisiologia , Imunossupressores/metabolismo , Imunossupressores/farmacologia , Interferon gama/antagonistas & inibidores , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/microbiologia , Cinética , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos SCID , RNA Mensageiro/biossíntese , Baço/citologia , Baço/imunologia , Baço/metabolismoRESUMO
A series of aurones with drug-potential for Leishmania infections was identified in vitro using both a direct cytotoxicity assay against extracellular promastigotes of Leishmania donovani, L. infantum, L. enriettii, and L. major, and a test against intracellular amastigote forms of L. donovani residing within murine macrophages. The most active aurone (6-hydroxy-2-[phenylmethylene]-3(2H)-benzofuranone) had an EC50 of 0.45 microgram/ml in the extra-, and an EC50 of 1.40 micrograms/ml in the intracellular assay. Other aurones were active between 0.06-12.50 micrograms/ml and 0.04-7.81 micrograms/ml, respectively. When tested against murine bone marrow-derived macrophages as a mammalian host cell control, the compounds showed only moderate cytotoxicity (EC50 2.32 to > 25.0 micrograms/ml). This is the first report on aurones as a new class of natural products with leishmanicidal activity.
Assuntos
Antiprotozoários/farmacologia , Benzofuranos/farmacologia , Compostos de Benzilideno/farmacologia , Leishmania/efeitos dos fármacos , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/parasitologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Even for routine purposes, standard staining of Pneumocystis- or Leishmania-containing materials, e.g., with Giemsa or Diff-Quik, is often unsatisfactory due to poor contrast and to staining of irrelevant structures. In comparison, the bisbenzimide dye Hoechst 33258, a DNA-binding fluorochrome, allows a more precise analysis of such materials. Bisbenzimide stained all stages of these fungal or protozoal organisms with brilliant contrast against a uniformly dark background. The level of background luminescence and staining of detritus or non-DNA structures was very low. Organisms were stained both outside of and within phagocytic cells with equal intensity. Counting of individual microorganisms, e.g., in macrophages heavily parasitized with Leishmania or in Pneumocystis-infected bronchoalveolar lavage, was simplified and more precise. Air-dried cell suspensions, cytocentrifuge preparations, impression smears, or cryocut micrographs showed the advantages of bisbenzimide staining over Diff-Quik. Staining with bisbenzimide could be a valuable auxiliary technique for the analysis of material infected with a variety of microorganisms.
Assuntos
Bisbenzimidazol , Corantes Fluorescentes , Leishmania enriettii/isolamento & purificação , Pneumocystis/isolamento & purificação , Animais , Líquido da Lavagem Broncoalveolar/microbiologia , Núcleo Celular/ultraestrutura , Feminino , Leishmania enriettii/ultraestrutura , Leishmaniose/diagnóstico , Leishmaniose/parasitologia , Pulmão/microbiologia , Macrófagos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Monócitos/parasitologia , Pneumocystis/ultraestrutura , Pneumonia por Pneumocystis/diagnóstico , Pneumonia por Pneumocystis/microbiologia , Ratos , Ratos WistarRESUMO
The regulatory role of soluble cytokines in innate cellular immune responses induced by Pneumocystis carinii was assessed in vitro in direct comparison to induction by Listeria monocytogenes. This report shows that P. carinii organisms, as well as L. monocytogenes, stimulated in whole spleen cell cultures of SCID mice the release of IFN-gamma, TNF-alpha/beta, IL-10, IL-12, and iNO. This response was independent of functional T cells. Both macrophages (M phi) and natural killer (NK) cells were necessary for either microorganism to induce release of these cytokines. Cocultures of purified M phi--including alveolar M phi--and purified NK cells indicated that no other cell population was necessarily involved. Microbial induction of NK cell-derived IFN-gamma has been reported to be mediated by the combined effects of TNF-alpha and IL-12 released by M phi upon adequate microbial stimulation. Interestingly, only L. monocytogenes, but not P. carinii organisms could directly induce detectable amounts of TNF-alpha/beta, IL-12, or iNO in purified M phi cultures. In dose-response experiments, release of IFN-gamma, TNF-alpha/beta, and iNO was reduced at high relative concentrations of either microorganism. This high-dose suppression was at least partially controlled by M phi-produced IL-10. Our data show that, P. carinii potently induces activating and inhibitory innate cellular immune response mechanisms and indicate that the initial step of macrophage-mediated NK cell activation might also involve other pathways than those described to date.
Assuntos
Pneumocystis/imunologia , Animais , Feminino , Imunidade Celular , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-12/biossíntese , Células Matadoras Naturais/imunologia , Listeria monocytogenes/imunologia , Listeria monocytogenes/metabolismo , Linfotoxina-alfa/biossíntese , Macrófagos Alveolares/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Óxido Nítrico/síntese química , Pneumocystis/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
This is the first report on aurones as a new class of natural products with leishmanicidal activity. A series of aurones with drug-potential for Leishmania infections was identified in vitro using both a direct cytotoxicity test against extracellular promastigotes of Leishmania donovani, L. infantum, L. enriettii, and L. major, and a test against intracellular amastigote L. donovani residing within murine macrophages. The compounds proved to be active at concentrations in the microgram range between 0.4 and 5.0 microg/ml. When tested against murine bone marrow-derived macrophages as a mammalian host cell control, all compounds showed only moderate cytotoxicity (EC50 2.32-25.0 microg/ml).
Assuntos
Antiprotozoários/farmacologia , Benzofuranos/farmacologia , Leishmania/efeitos dos fármacos , Animais , Antiprotozoários/química , Benzofuranos/química , Células Cultivadas , Humanos , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Células Tumorais CultivadasRESUMO
The course of cutaneous leishmaniasis was examined in mice from two genetically closely related strains, C57BL/10ScCr (Cr) and C57BL/10ScSn (Sn). Sn mice are able to heal Leishmania major infections, while Cr mice are unable to heal. The cutaneous lesions of the Cr mice progressed continuously and the increase in lesion size was paralleled by an unrestricted growth of the parasites in vivo. Cr mice, in contrast to their Sn counterparts, are highly resistant to all effects of lipopolysaccharide (LPS). The nonhealing L. major infection in Cr mice is in sharp contrast to the course of infection in another endotoxin-nonresponder mouse strain, C3H/HeJ, which heal infections with L. major. Cr mice exhibit, in addition to the defective LPS responsiveness, an impaired interferon-gamma (IFN-gamma) response after infection with a variety of microorganisms. The insufficient activation of parasitized macrophages to kill intracellular L. major could be due to the inability of splenocytes from infected Cr mice to secrete IFN-gamma upon restimulation with L. major. IFN-gamma is essential for the efficient activation of parasitized macrophages to kill intracellular L. major by producing nitric oxide (NO). Although bone marrow-derived Cr macrophage do not produce NO in response to LPS, both Sn and Cr macrophages release NO upon stimulation with IFN-gamma and tumor necrosis factor, indicating that they are responsive to activation by these cytokines.
Assuntos
Leishmania major , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/imunologia , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL/genética , Animais , Feminino , Técnicas In Vitro , Interferon gama/biossíntese , Leishmania major/imunologia , Ativação de Macrófagos , Masculino , Camundongos , Óxido Nítrico/biossíntese , Fenótipo , Especificidade da Espécie , Fator de Necrose Tumoral alfa/biossínteseRESUMO
The well-established methods of generating stably transfected cell lines, and the detection of nanomolar amounts of an enzyme in a fast and reproducible assay, were utilised to establish non-radiometric cytotoxicity assays. In these assay systems, the detection of released enzymes was used to quantitate the leakage of intracellular proteins after membrane disintegration. Target cell lines were transfected with a luciferase reporter gene under the control of a strong eucaryotic promoter. Release of the intracellular expressed enzyme into the culture supernatant occurred after membrane perforation and was measured as an indicator of cellular death. The quantitation of released enzyme was a reliable indicator of cell death initiated either by complement-mediated killing, or by cell-mediated cytotoxicity. This system was initially established with P815 mastocytoma cells as an example of a target cell line. Transfection with the firefly luciferase gene provided an intracellular enzyme absent in mammalian cells. In a parallel approach, P815 and BW5147 target cells were transfected with bacterial beta-galactosidase to provide a similar cytotoxicity system. This enzyme, however, has a considerably longer half life in tissue culture medium than luciferase. In a direct comparison between the standard 51Cr release and beta-galactosidase release, the enzyme release showed a much higher signal-to-noise ratio, i.e., low background and high induced release if spontaneous release and detergent induced maximal lysis were measured. Since a wide range of human and murine cell lines can be stably transfected and several reporter genes are available, the system should provide an alternative for conventional cytotoxicity assays. The detection of released enzymes by colorimetric or luminometric methods makes this cytotoxicity assay independent of radionuclides. The sensitivity of luminometric enzyme detection systems should also permit the measurement of apoptotic processes and might make in vivo studies of cellular death using transgenic animals feasible.