Jean-Claude Ameisen

Ligands of the Peripheral Benzodiazepine Receptor Are Potent Inhibitors of Plasmodium falciparum and Toxoplasma gondii In Vitro

ABSTRACT The increase in resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands the development of new antimalarial agents. In this quest, we have found that ligands to the peripheral benzodiazepine receptor such as flurazepam, an agonist of the benzodiazepine family, and PK11195, an antagonist derived from isoquinoline, were active against Plasmodium falciparum. These two compounds effectively and rapidly inhibited parasite growth in vitro, irrespective of parasite resistance to chloroquine and mefloquine. Treatment with both drugs induced a sharp and consistent decline in parasitemia, a complete inhibition of parasite replication, and the destruction of parasites within the host red blood cells. Using electron microscopy, we showed that dramatic morphological changes, involving swollen endoplasmic reticulum and the reduction of hemozoin, were consistent with parasite death. The potent activities of flurazepam and PK11195 were also evaluated for antagonist or synergistic effects with currently used antimalarial drugs such as chloroquine and mefloquine. Moreover, flurazepam was found to be active against Toxoplasma gondii, another member of the phylum Apicomplexa. Taken together, our results indicated that benzodiazepines could be considered promising candidates in the treatment of both malaria and toxoplasmosis.

Inhibition of multicellular development switches cell death of Dictyostelium discoideum towards mammalian-like unicellular apoptosis

The multicellular development of the single celled eukaryote Dictyostelium discoideum is induced by starvation and consists of initial aggregation of the isolated amoebae, followed by their differentiation into viable spores and dead stalk cells. These stalk cells retain their structural integrity inside a stalk tube that support the spores in the fruiting body. Terminal differentiation into stalk cells has been shown to share several features with programmed cell death (Cornillon et al. (1994), J. Cell Sci. 107, 2691-2704). Here we report that, in the absence of aggregation and differentiation, D. discoideum can undergo another form of programmed cell death that closely resembles apoptosis of most mammalian cells, involves loss of mitochondrial transmembrane potential, phosphatidylserine surface exposure, and engulfment of dying cells by neighboring D. discoideum cells. This death has been studied by various techniques (light microscopy and scanning or transmission electron microscopy, flow cytometry, DNA electrophoresis), in two different conditions inhibiting D. discoideum multicellular development. The first one, corresponding to an induced unicellular cell death, was obtained by starving the cells in a conditioned cell-free buffer, prepared by previous starvation of another D. discoideum cell population in potassium phosphate buffer (pH 6.8). The second one, corresponding to death of D. discoideum after axenic growth in suspension, was obtained by keeping stationary cells in their culture medium. In both cases of these unicellular-specific cell deaths, microscopy revealed morphological features known as hallmarks of apoptosis for higher eukaryotic cells and apoptosis was further corroborated by flow cytometry. The occurrence in D. discoideum of programmed cell death with two different phenotypes, depending on its multicellular or unicellular status, is further discussed.

Molecular and cellular mechanisms of erythrocyte programmed cell death: impact on blood transfusion.

Human red blood cells (RBCs) have a definite life span of 120 days at the end of which they are captured and then endocytized by macrophages. Every day, 360 billion RBCs are phagocytized, i.e. 5 million per second. This fascinating phenomenon of programmed cell death (PCD) raises the following questions: i) what signals the death sentence of RBCs; and ii) what are the physiological mechanisms for sequestration of the effete RBCs from the blood stream with such precision? In other words, the question is: “By what specific membrane signal(s) do the reticulo-endothelial cells distinguish between the truly senescent RBCs and others?” [For review, see ref.11. In addition, even if it is generally asserted that erythrocytes cannot undergo apoptosis because they lack nucleus and mitochondria, we have hypothetized that erythrophagocytosis by macrophages could be regarded as an apoptotic-like mechanism, on the basis of a series of well-known characteristics of senescent RBCs which are characteristics of apoptosis: i) specific modifications of erythrocyte membrane such as progressive release of microvesicles leading to a decrease of cell size and to an increase of density; ii) alteration of erythrocyte cytoskeleton, of spectrin in particular, leading to membrane budding and to the so-called echinocyte forms (crenated forms) and then to the spheroechinocyte form with spicules and to the smooth sphere form which are both specifically phagocytized; iii) enzymatic desialylation demasking terminal D-galactose residues and inducing the capture of erythrocytes by a Dgalactolectin of macrophagic membrane; iv) progressive appearance in the cell outer leaflet of phosphatidylserine that serves as a signal for triggering the recognition and phagocytosis of senescent erythrocytes by macrophages. With a view to verifying this hypothesis, we have undertaken experiments mostly founded on flow cytometry and using two types of senescent erythrocytes: the ones isolated from heparinized blood by ultracentrihgation in a self-forming Percoll gradient (physiological senescence), the others obtained by incubation with Ca?’ alone or in presence of ionophore A23 187 (induced PCD). A third population was constituted of leukodepleted and non-leukodepleted RBCs stored in the blood bank. Morphological changes and cell injury were assessed by flow cytometry using forward (cell size) and side-angle (cell density) scatters (FSC versus SSC). This is a simple, rapid and sensitive method we previously described [2] that discriminates and quantifies viable and senescent erythrocytes. Desialylation of RBCs was measured by flow cytometric analysis of the binding of FITC-labeled lectins specific for sialic acids (WGA. SNA and MAA) and O-galactose (RCA’”) residues. The in vitro phagocytosis of RBCs was measured by using a rapid, sensitive and reproducible flow cytofluorimetric procedure we previously described [3] and which is based on the use of RBCs labeled with the fluorescent probe PKH-26. The procedure involves the following steps: i) incubation of PKH-26-labeled erythrocytes with murine macrophages. ii) removal of unbound red blood cells, iii) lysis of membrane-bound RBCs, and iv) measurement of extent of phagocytosis by direct flow-cytometric analysis of intact macrophages. Dot-plot analysis of red PKH-2blabeled RBC cytofluorescence (ordinate) against the green macrophage autofluorescence (abscissa) clearly defines two regions: those of non-

A suppressive effect of the adenovirus 5 protein E1B 55K on apoptosis induced by IL-3 deprivation and γ-irradiation

The murine IL‐3‐dependent myeloid cell line 32D undergoes a rapid death when deprived of interleukin‐3 (IL‐3), a process that is suppressed or delayed by the constitutive expression of Bcl‐2 or the Bcl‐2‐related Bcl‐xL survival protein. The adenovirus type 5 E1B region encodes an E1B 55K protein, that has been reported to bind and inactivate the p53 protein that plays an important role in the induction of apoptosis. In order to explore the potential effect of the E1B 55K protein on IL‐3 deprival‐induced cell death, we have established 32D cell lines overexpressing the adenovirus E1B 55K protein and compared its ability to modulate the cell death with that of the human Bcl‐2 protein. We observed that E1B 55K, as Bcl‐2, delays the cell death caused by either IL‐3‐deprivation or DNA damage induced by γ‐irradiation. Cell‐cycle analysis after IL‐3 deprivation indicated that surviving Bcl‐2 transfectants accumulate predominantly in the G0/G1 phase of the cell cycle, while E1B 55K transfectants survive in both G0/G1 and the S and G2/M phases of the cell cycle. zVAD‐fmk, a broad caspase inhibitor, prevented chromatin condensation and fragmentation, but not cell death, suggesting that IL‐3 deprivation induces a cell death program in which the caspases are dispensable. In contrast, both E1B 55K and Bcl‐2 allowed cell survival and prevented the typical features of programmed cell death, such as phosphatidyl‐serine exposure, loss of mitochondrial membrane potential, and chromatin condensation and fragmentation. Our findings indicate that the adenovirus 5 E1B 55K protein has the capability to act as a survival factor, and suggest that E1B 55K exerts its effect upstream of the activation of effector caspases, by preventing the loss of mitochondrial membrane potential induced by IL‐3 deprivation.