Antileishmanial activities associated with plants used in the Malian traditional medicine.

Sixty-four extracts issued from twenty-one plants used in the Malian traditional medicine--several of them as antiparasitic drugs--were assayed for their antileishmanial effects against both extracellular and intracellular forms of Leishmania major. Seven extracts from six different plants--Sarcocephalus latifolius, Zanthoxylum zanthoxyloides, Entada africana, Bobgunnia madagascarensis, Pseudocedrela kotschyi and Psorospermum guineense--were found to be significantly active against the intracellular form of the parasite.

Antileishmanial and antifungal acridone derivatives from the roots of Thamnosma rhodesica.

Eight furanocoumarins, one coumarin and four acridone derivatives have been identified in the roots of Thamnosma rhodesica (Rutaceae). Rhodesiacridone, one of these acridone derivatives, is reported here for the first time. Its structure was elucidated by spectrometric methods including ESI-HR, EI, DCI mass spectrometry, 1H, 13C and 2D NMR experiments. This novel compound showed activities against the intracellular form of a human pathogen, the protozoan parasite Leishmania major. Two known acridone related compounds, gravacridonediol and 1-hydroxy-10-methylacridone, exhibited activities against the intracellular form of the same parasite and the fungus Cladosporium cucumerinum, respectively.

A new function of the Fas-FasL pathway in macrophage activation.

Upon infection with the protozoan parasite Leishmania major, susceptible BALB/c mice develop unhealing lesions associated with the maturation of CD4(+)Th2 cells secreting IL-4. In contrast, resistant C57BL/6 mice heal their lesions, because of expansion and secretion of IFN-gamma of CD4(+) Th1 cells. The Fas-FasL pathway, although not involved in Th cell differentiation, was reported to be necessary for complete resolution of lesions. We investigate here the role of IFN-gamma and IL-4 on Fas-FasL nonapoptotic signaling events leading to the modulation of macrophage activation. We show that addition of FasL and IFN-gamma to BMMø led to their increased activation, as reflected by enhanced secretion of TNF, IL-6, NO, and the induction of their microbicidal activity, resulting in the killing of intracellular L. major. In contrast, the presence of IL-4 decreased the synergy of IFN-gamma/FasL significantly on macrophage activation and the killing of intracellular L. major. These results show that FasL synergizes with IFN-gamma to activate macrophages and that the tight regulation by IFN-gamma and/or IL-4 of the nonapoptotic signaling events triggered by the Fas-FasL pathway affects significantly the activation of macrophages to a microbicidal state and may thus contribute to the pathogenesis of L. major infection.

Culture and functional studies of mouse macrophages on native-like fibrillar type I collagen.

Freshly isolated, starch-elicited mouse peritoneal macrophages (Mø) attached very efficiently to type I collagen in vitro, if collagen molecules were arranged in ordered supra-molecular assemblies corresponding to the precursor native fibrils. After 6-20h of incubation, the collagen-bound cells were observed to secrete fibronectin, which presumably enhanced cell-collagen interaction associated with cellular differentiation. Mø attachment to collagen could be temporarily inhibited by addition of the linear tri-peptide Arg-Gly-Asp (RGD) to the culture media. This inhibition was much more pronounced when using the cyclic RGD-containing peptide cGRGDSPA. Similarly, cells could be easily detached from the fibrillar collagen layers within 20 min at 37 degrees C by RGDS, GRGDS or cGRGDSPA but not by the glutamate-containing RGES peptide. Using antibodies to known collagen receptors, attachment of Mø to type I collagen fibers was best inhibited by antibodies directed against the alpha2 and beta1 integrin subunits. The presence of these integrins on Mø was confirmed by immunofluorescence. Binding of the alpha2beta1 integrin on collagen was divalent cation-dependent and was supported by magnesium but not by calcium. Cells recovered by RGD-mediated detachment from collagen were highly phagocytic and synthesized DNA when exposed to growth factors. These cells could be activated for cytotoxicity by treatment with interferon-gamma and lipopolysaccharide. Comparative in vitro assays performed on macrophages cultured on plastic and on collagen allowed the detection of NO production by activated macrophages followed by spontaneous deactivation for cells cultivated on collagen. These findings suggest that Mø can recognize native collagen of type I through functional interactions with their specific triple helix-binding integrin receptors indicating that integrins other than those directed to fibronectin may also occupy active focal points on the cell at the initial phase of attachment.

Impairment of Brucella growth in human macrophagic cells that produce nitric oxide.

In mice, nitric oxide (NO) production by inducible NO synthase (iNOS), is a component of the control of Brucella infection. In humans, the involvement of iNOS in infection is still a matter of debate. Based on in vitro experiments, it was recently postulated that in humans, Brucella infection tends to become chronic because NO cannot exert its deleterious effect. In fact, conditions allowing NO production by human macrophages in culture are poorly defined, rendering the in vitro study of NO function difficult. Using DFGiNOS U937 macrophagic cells engineered to produce NO and U937 cells activated by ligation of IgE receptors, we showed that the intracellular development of Brucella was impaired in human macrophages, which produced NO. Although Brucella-infected human macrophagic phagocytes did not release NO in commonly used models of infection, the machinery required to produce NO was expressed in these cells and could be triggered by cell membrane receptors present on the infected cells. Therefore, the lack of NO production in isolated human macrophages infected by Brucella under in vitro conditions did not exclude a possible involvement of NO in the control of human brucellosis.

Establishment of permanent cell lines purified from human mesothelioma: morphological aspects, new marker expression and karyotypic analysis.

This study reports the establishment of three major subtypes of human mesothelioma cells in tissue culture, i.e. the epithelioid, sarcomatoid and biphasic forms, and compares their phenotypic and biological characteristics. Primary cells isolated from biopsies or pleural exudates were subcultured for over 50 passages. We evaluated immunoreactivity using various mesothelial markers related to histological patterns of these cell lines. For epithelioid cells, calretinin and cytokeratin were found to be useful and easily interpretable markers as for control mesothelial cells. The biphasic form was only partially positive and the sarcomatoid type negative. Vimentin was expressed by all cell lines. BerEP4, a specific marker for adenocarcinoma, was negative. Interestingly, while the macrophage marker CD14 was negative, immunoreactivity for a mature macrophage marker (CD68) was expressed by all cell types, suggesting that this marker might constitute an additional tool useful in the differential diagnosis of mesothelioma. At the ultrastructural level, a cell surface rich in microvilli confirmed their mesothelial origin. PCR analysis revealed that none of the cell lines contained SV40 DNA. Karyotypic analyses showed more complex abnormalities in the epithelioid subtype than in the sarcomatoid form. These cell lines may be useful in the study of cellular, molecular and genetic aspects of the disease.

Both the Fas ligand and inducible nitric oxide synthase are needed for control of parasite replication within lesions in mice infected with Leishmania major whereas the contribution of tumor necrosis factor is minimal.

Following infection with the protozoan parasite Leishmania major, C57BL/6 mice develop a small lesion that heals spontaneously. Resistance to infection is associated with the development of CD4(+) Th1 cells producing gamma interferon (IFN-gamma) and tumor necrosis factor (TNF), which synergize in activating macrophages to their microbicidal state. We show here that C57BL/6 mice lacking both TNF and Fas ligand (FasL) (gld TNF(-/-) mice) infected with L. major neither resolved their lesions nor controlled Leishmania replication despite the development of a strong Th1 response. Comparable inducible nitric oxide synthase (iNOS) activities were detected in lesions of TNF(-/-), gld TNF(-/-), and gld mice, but only gld and gld TNF(-/-) mice failed to control parasite replication. Parasite numbers were high in gld mice and even more elevated in gld TNF(-/-) mice, suggesting that, in addition to iNOS, the Fas/FasL pathway is required for successful control of parasite replication and that TNF contributes only a small part to this process. Furthermore, FasL was shown to synergize with IFN-gamma for the induction of leishmanicidal activity within macrophages infected with L. major in vitro. Interestingly, TNF(-/-) mice maintained large lesion size throughout infection, despite being able to largely control parasite numbers. Thus, IFN-gamma, FasL, and iNOS appear to be essential for the complete control of parasite replication, while the contribution of TNF is more important in controlling inflammation at the site of parasite inoculation.

Novel peptide inhibitors of Leishmania gp63 based on the cleavage site of MARCKS (myristoylated alanine-rich C kinase substrate)-related protein.

The zinc metalloprotease gp63 (leishmanolysin; promastigote surface protease) is expressed at high density at the surface of Leishmania promastigotes. Efficient non-toxic inhibitors of gp63 do not exist, and its precise role in parasite physiology remains unknown. MARCKS (myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in various cells, including macrophages. We reported previously that MRP is an excellent substrate for gp63. A major cleavage site was identified within the MRP effector domain (ED), a highly basic 24-amino-acid sequence, and the synthetic ED peptide (MRP(ED)) was shown to inhibit MRP hydrolysis. In the present study, MRP cleavage was used as an assay to measure the capacity of various MRP or MARCKS ED peptides to block gp63 activity. On a molar basis, MRP(ED) inhibited gp63 to a greater extent than two previously described gp63 inhibitors, o -phenanthroline and benzyloxycarbonyl-Tyr-Leu-NHOH. MARCKS(ED) analogues containing modifications in the gp63 consensus cleavage site showed significant differences in inhibitory capacity. As phosphorylation of ED serine residues prevented gp63-mediated MRP degradation, we synthesized a pseudophosphorylated peptide in which serine residues were substituted by aspartate (3DMRP(ED)). 3DMRP(ED) was a highly effective inhibitor of both soluble and parasite-associated gp63. Finally, MRP ED peptides were synthesized together with an N-terminal HIV-1 Tat transduction domain (TD) to obtain cell-permeant peptide constructs. Such peptides retained gp63 inhibitory activity and efficiently entered both macrophages and parasites in a Tat TD-dependent manner. These studies may provide the basis for developing potent cell-permeant inhibitors of gp63.