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PRIMVAC is a VAR2CSA-derived placental malaria vaccine candidate aiming to prevent serious clinical outcomes of Plasmodium falciparum infection during pregnancy. We assessed the safety and immunogenicity of PRIMVAC adjuvanted with Alhydrogel or glucopyranosyl lipid adjuvant in stable emulsion (GLA-SE) in French and Burkinabe women who were not pregnant., Bundesministerium für Bildung und Forschung, through Kreditanstalt für Wiederaufbau, Germany; Inserm, and Institut National de Transfusion Sanguine, France; Irish Aid, Department of Foreign Affairs and Trade, Ireland.

Recently, a new class of lipid-lowering agents has been described that upregulate LDL receptor (LDLr) activity. These agents are proposed to activate sterol-regulated gene expression through binding to the sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP). Here, we show that the steroidal LDLr upregulator, GW707, induces accumulation of lysosomal free cholesterol and inhibits LDL-stimulated cholesterol esterification, similar to that observed in U18666A-treated cells and in Niemann-Pick type C1 (NPC1) mutants. Moreover, we demonstrate that induction of the NPC-like phenotype by GW707 is independent of SCAP function. We find that treatment with GW707 does not increase SREBP-dependent gene expression above that observed in lipoprotein-starved cells. Rather, we show that the apparent increase in SREBP-dependent activity in GW707-treated cells is attributable to a failure to appropriately suppress sterol-regulated gene expression, as has been shown previously for U18666A-treated cells and NPC mutant fibroblasts. We further demonstrate that cells treated with either GW707 or U18666A fail to appropriately generate 27-hydroxycholesterol in response to LDL cholesterol. Taken together, these findings support a mechanism in which GW707 exerts its hypolipidemic effects through disruption of late endosomal/lysosomal sterol trafficking and subsequent stimulation of LDLr activity.

The physiological processing of the beta-amyloid precursor protein (betaAPP) by a protease called alpha-secretase gives rise to APP alpha, a C-terminally truncated fragment of betaAPP with known neurotrophic and cytoprotective properties. Several lines of evidence indicate that protein kinase C (PKC)-mediated events regulate this physiological pathway. We show here that the proteasome multicatalytic complex modulates the phorbol 12,13-dibutyrate-stimulated APP alpha secretion at several levels in human kidney 293 (HK293) cells. Two blocking agents of the proteasome, namely, Z-IE(Ot-Bu)A-leucinal and lactacystin, elicit a dual effect on PKC-regulated APP alpha secretion by metabolically labeled HK293 cells. Thus, short periods of preincubation (2-5 h) of the cells with the inhibitors trigger a drastic potentiation of APP alpha recovery, whereas long-term treatment of the cells (15-20 h) with the blocking agents leads to an overall decrease in the secretion of APP alpha. Such a dual effect was not observed on constitutive APP alpha secretion and intracellular formation generated by HK293 cells, which both only increase upon inhibitor treatments. Similar effects on the constitutive and PKC-regulated APP alpha secretion were observed with PC12 cells. Altogether, these data suggest distinct mechanisms underlying basal and PKC-regulated APP alpha production, indicating that this multicatalytic complex appears as a key contributor of the alpha-secretase pathway.

La production exacerbee de peptide amyloide contribue vraisemblablement a l’etiologie de la maladie d’Alzheimer. En effet, ce peptide s’accumule, s’agrege et conduit aux depots proteiques -les plaques seniles - qui envahissent le cortex des patients atteints de maladie d’Alzheimer. Une des approches therapeutiques theoriques visant a ralentir ou a bloquer l’apparition des plaques seniles consiste donc a cibler les enzymes qui sont responsables de la formation du peptide amyloide. Celles-ci sont regroupees sous le terme generique de secretases. La liberation du peptide amyloide fait intervenir de maniere sequentielle la β-secretase puis la y-secretase. Une autre activite enzymatique, l’α-secretase, a un role oppose et protecteur puisqu’elle « detruit » le peptide amyloide. Cet article presente les voies metaboliques de la maturation physiopathologique de la (βAPP et les avancees recentes de la recherche concernant la nature des secretases impliquees dans la maladie d’Alzheimer.

The β-amyloid precursor protein (βAPP) undergoes a physiological cleavage triggered by one or several proteolytic activities referred to as α-secretases, leading to the secretion of sAPPα. Several lines of evidence indicate that the α-secretase cleavage is a highly regulated process. Thus, besides constitutive production of sAPPα, several studies have reported on protein kinase C-regulated sAPPα secretion. Studies aimed at identifying α-secretase(s) candidates suggest the involvement of enzymes belonging to the pro-hormone convertases and disintegrin families. The delineation of respective contributions of proteolytic activities in constitutive and regulated sAPPα secretion is rendered difficult by the fact that the overall regulated response always includes the basal constitutive counterpart that cannot be selectively abolished. Here we report on the fact that the furin-deficient LoVo cells are devoid of regulated PKC-dependent sAPPα secretion and therefore represent an interesting model to study exclusively the constitutive sAPPα secretion. We show here, by a pharmacological approach using selective inhibitors, that pro-hormone convertases and proteases of the ADAM (disintegrin metalloproteases) family participate in the production/secretion of sAPPαs in LoVo cells. Transfection analysis allowed us to further establish that the pro-hormone convertase 7 and ADAM10 but not ADAM17 (TACE, tumour necrosis factor α-converting enzyme) likely contribute to constitutive sAPPα secretion by LoVo cells.

Presenilins 1 and 2 are two homologous proteins which, when mutated, appear responsible for most of the early-onset familial forms of Alzheimer's disease. Among various functional aspects, presenilins appear to behave as chaperoning partners of a series of proteins including the beta-amyloid precursor protein. Recently, presenilins were shown to interact with Rab11, a GTPase involved in intracellular transport. This suggested that Rab11-presenilin interaction could influence the routing of betaAPP and thereby modulate its maturation. In this context, we examined whether overexpression of Rab11 or its constitutively active mutant Rab11Q70L could affect betaAPP maturation in human HEK293 cells. We show here that the overexpression of both Rab11-related proteins does not modify the recovery of secreted sAPPalpha or Abeta in HEK293 cells expressing wild-type betaAPP or betaAPP harboring the Swedish double mutation. These data indicate that Rab11 does not influence betaAPP processing in HEK293 cells. However, it does not preclude the possibility for Rab11 to modulate other presenilin-mediated functions in human cells.

Several lines of evidence suggest that most of the early-onset forms of familial Alzheimer’s disease (FAD) are due to inherited mutations borne by a chromosome 14–encoded protein, presenilin 1 (PS1). This is likely related to an increased production of amyloid β-peptide (A β)42, one of the main components of the extracellular deposits called senile plaques that invade human cortical areas during the disease. We set up stably transfected HEK293 cells overexpressing wild-type (wt) and various FAD-linked mutated PS1. By Western blot analysis, we examined the influence of specific proteasome inhibitors on PS1-like immunoreactivities. Furthermore, by means of metabolic labeling and immunoprecipitation with A β40 and A β42-directed specific antibodies, we assessed the effect of the inhibitors on the production of A βs by wt and mutated PS1-expressing cells transiently transfected with βAPP751. We show that two distinct proteasome inhibitors, Z-IE(Ot-Bu)A-Leucinal and lactacystin, increase in a time- and dose-dependent manner the immunoreactivities of both wt and mutated PS1. Furthermore, we demonstrate that PS1 is polyubiquitinated in these cells. Other inhibitors, ineffective on the proteasome, fail to protect wt and mutated PS1-like immunoreactivities. We also establish that the FAD-linked mutations of PS1 trigger a selective increased formation of Aβ42 as reflected by higher Aβ42 over total Aβ ratios when compared with wtPS1-expressing cells. Interestingly, this augmentation was further amplified by proteasome inhibitors in cells expressing mutated but not wtPS1. Altogether, our data indicate that PS1 undergoes polyubiquitination in HEK293 cells and that the proteasome contributes to the degradation of wt and FAD-linked PS1, thereby directly influencing the Aβ production in human cells.

Up-regulation of low-density lipoprotein receptor (LDLr) is a key mechanism to control elevated plasma LDL-cholesterol levels. In the present paper, we compare the ability of four distinct pharmacological drugs to up-regulate LDLr expression in human hepatocytes. HepG2 cells were treated with the steroidal analog GW707, the oxidosqualene cyclase inhibitor U18666A, the 3β-hydroxysterol Δ 7 -reductase inhibitor AY-9944 and the vacuolar-type ATPase inhibitor bafilomycin A1. We found that the four compounds induced sequestration of free cholesterol in the endosomal/lysosomal compartment leading to a positive filipin staining pattern and a complete inhibition of cholesteryl ester synthesis. As a consequence of the sequestration of cholesterol, the expression and the activity of LDLr were strongly induced resulting from a transcriptional effect which was measured by a reporter gene assay. These effects were fully abolished when an exogenous water soluble cholesterol analog was added to the cells. These findings have led to the identification of a common mechanism to up-regulate LDLr expression in human hepatocytes and may represent an interesting alternative approach to identify new hypolipidemic drugs.

We showed previously that PrPc undergoes constitutive and phorbol ester-regulated cleavage inside the 106–126 toxic domain of the protein, leading to the production of a fragment referred to as N1. Here we show by a pharmacological approach thato-phenanthroline, a general zinc-metalloprotease inhibitors, as well as BB3103 and TAPI, the inhibitors of metalloenzymes ADAM10 (A disintegrinand metalloprotease); and TACE,tumor necrosis factorα-converting enzyme; ADAM17), respectively, drastically reduce N1 formation. We set up stable human embryonic kidney 293 transfectants overexpressing human ADAM10 and TACE, and we demonstrate that ADAM10 contributes to constitutive N1 production whereas TACE mainly participates in regulated N1 formation. Furthermore, constitutive N1 secretion is drastically reduced in fibroblasts deficient for ADAM10 whereas phorbol 12,13-dibutyrate-regulated N1 production is fully abolished in TACE-deficient cells. Altogether, our data demonstrate for the first time that disintegrins could participate in the catabolism of glycosyl phosphoinositide-anchored proteins such as PrPc. Second, our study identifies ADAM10 and ADAM17 as the protease candidates responsible for normal cleavage of PrPc. Therefore, these disintegrins could be seen as putative cellular targets of a therapeutic strategy aimed at increasing normal PrPcbreakdown and thereby depleting cells of the putative 106–126 “toxic” domain of PrPc.

Human neutrophils in whole blood become bipolar in shape after exposure to chemokinetic stimuli. In normal blood, the proportion of non-spherical neutrophils was 1.2 +/- 0.07% (n = 101). After incubation of blood samples with corticotropin-releasing hormone (CRF, 1 to 20 microM) 36 of 101 subjects exhibited aor = 10% bipolar-shape ellipsoid response. This ellipsoid response was more frequent in female than in male subjects (32/75 vs. 4/26, p0.01). Female Caucasian subjects were more sensitive to CRF than female East Asian subjects (25/48 vs. 2/15, p0.01). Age was not a factor in sensitivity to CRF. In young female East Asian subjects (23 +/- 0.4 years, n = 8) that did not manifest the ellipsoid response to CRF, formyl-Met-Leu-Phe (fMLP), a chemotactic peptide, 10(-9) M increased non-spherical neutrophils to 31 +/- 0.8%. In these individuals, the fMLP response was inhibited in a dose-dependent manner by CRF. The pharmacological profile of the stimulatory and fMLP-inhibitory actions of CRF on neutrophil shape was consistent with that of a CRF1-receptor mediated response. Expression of mRNA for the CRF1-receptor was detected in hematopoietic cell lines (e.g., HL-60) using a reverse transcriptase polymerase chain-reaction method. The bipolar-shape response of human neutrophils to CRF has the potential to be a useful indicator of the functional state of this hormone-receptor system in inflammation.