Daniel Parzy

Integrated Profiling of Basal and Luminal Breast Cancers

Basal and luminal are two molecular subtypes of breast cancer with opposite histoclinical features. We report a combined, high-resolution analysis of genome copy number and gene expression in primary basal and luminal breast cancers. First, we identified and compared genomic alterations in 45 basal and 48 luminal tumors by using 244K oligonucleotide array comparative genomic hybridization (aCGH). We found various genome gains and losses and rare high-level gene amplifications that may provide therapeutic targets. We show that gain of 10p is a new alteration in basal breast cancer and that a subregion of the 8p12 amplification is specific of luminal tumors. Rare high-level amplifications contained BCL2L2, CCNE, EGFR, FGFR2, IGF1R, NOTCH2, and PIK3CA. Potential gene breaks involved ETV6 and FLT3. Second, we analyzed both aCGH and gene expression profiles for 42 basal and 32 luminal breast cancers. The results support the existence of specific oncogenic pathways in basal and luminal breast cancers, involving several potential oncogenes and tumor suppressor genes (TSG). In basal tumors, 73 candidate oncogenes were identified in chromosome regions 1q21-23, 10p14, and 12p13 and 28 candidate TSG in regions 4q32-34 and 5q11-23. In luminal breast cancers, 33 potential oncogenes were identified in 1q21-23, 8p12-q21, 11q13, and 16p12-13 and 61 candidate TSG in 16q12-13, 16q22-24, and 17p13. HORMAD1 (P = 6.5 x 10(-5)) and ZNF703 (P = 7 x 10(-4)) were the most significant basal and luminal potential oncogenes, respectively. Finally, among 10p candidate oncogenes associated with basal subtype, we validated CDC123/C10orf7 protein as a basal marker.

Concentration and purification by magnetic separation of the erythrocytic stages of all human Plasmodium species

BackgroundParasite concentration methods facilitate molecular, biochemical and immunological research on the erythrocytic stages of Plasmodium. In this paper, an adaptation of magnetic MACS® columns for the purification of human Plasmodium species is presented. This method was useful for the concentration/purification of either schizonts or gametocytes.Results and conclusionsThe magnetic removal of non-parasitized red blood cells (in vivo and in vitro) using magnetic columns (MACS) was evaluated. This easy-to-use technique enriched schizonts and gametocytes from Plasmodium falciparum in vitro cultures with a very high degree of purity. In addition, all haemozoin-containing stages (schizonts and/or gametocytes) from the peripheral blood of infected patients could be concentrated using this method. This method is particularly useful for the concentration of non-falciparum species, which do not grow in culture and are otherwise difficult to obtain in large amounts.

In Vitro Activities of Antibiotics against Plasmodium falciparum Are Inhibited by Iron

ABSTRACT The in vitro activities of cyclines (tetracycline, doxycycline, minocycline, oxytetracycline, and rolitetracycline), macrolides (erythromycin, spiramycin, roxithromycin, and lincomycin), quinolones (norfloxacin and ofloxacin), rifampin, thiamphenicol, tobramycin, metronidazole, vancomycin, phosphomycin, and cephalosporins (cephalexin, cefaclor, cefamandole, cefuroxime, ceftriazone, cefotaxime, and cefoxitin) were evaluated onPlasmodium falciparum clones, using an isotopic, micro-drug susceptibility test. Only tetracyclines, macrolides, quinolones, and rifampin demonstrated in vitro activity againstP. falciparum, which increased after a prolonged exposure (96 or 144 h). In the presence of iron (FeCl3), only the activities of tetracyclines and norfloxacin were decreased. Their in vitro activity against intraerythrocytic stages of multidrug-resistant P. falciparum and their efficacy in vivo favor the use of antibiotics as antimalarial drugs. However, due to their slow antimalarial action and to the fact that they act better after prolonged contact, they probably need to be administered in conjunction with a rapidly acting antimalarial drug, such as a short course of chloroquine or quinine.

New ferrocenic pyrrolo[1,2-a]quinoxaline derivatives: synthesis, and in vitro antimalarial activity.

Following our search for antimalarial compounds, novel series of ferrocenic pyrrolo[1,2-a]quinoxaline derivatives 1-2 were synthesized from various substituted nitroanilines and tested for in vitro activity upon the erythrocytic development of Plasmodiumfalciparum strains with different chloroquine-resistance status. The pyrrolo[1,2-a]quinoxalines 1 were prepared in 6-8 steps through a regioselective palladium-catalyzed monoamination by coupling 4-chloropyrrolo[1,2-a]quinoxalines with 1,3-bis(aminopropyl)piperazine or -methylamine using Xantphos as the ligand. The ferrocenic bispyrrolo[1,2-a]quinoxalines 2 were prepared by reductive amination of previously described bispyrrolo[1,2-a]quinoxalines 9 with ferrocene-carboxaldehyde, by treatment with NaHB(OAc)(3). The best results were observed with ferrocenic pyrrolo[1,2-a]quinoxalines linked by a bis(3-aminopropyl)piperazine. Moreover, it was observed that a methoxy group on the pyrrolo[1,2-a]quinoxaline nucleus and no substitution on the terminal N-ferrocenylmethylamine function enhanced the pharmacological activity. Selected compounds 1b, 1f-h, 1l and 2a were tested for their ability to inhibit beta-haematin formation, the synthetic equivalent of hemozoin, by using the HPIA (heme polymerization inhibitory activity) assay. Of the tested compounds, only 2a showed a beta-haematin formation inhibition, but no inhibition of haem polymerization was observed with the other selected ferrocenic monopyrrolo[1,2-a]quinoxaline derivatives 1b, 1f-h and 1l, as the IC(50) values were superior to 10 equivalents.

cAMP-dependent protein kinase from Plasmodium falciparum: an update.

One of the most important public health problems in the world today is the emergence and dissemination of drug-resistant malaria parasites. Plasmodium falciparum is the causative agent of the most lethal form of human malaria. New anti-malarial strategies are urgently required, and their design and development require the identification of potential therapeutic targets. However, the molecular mechanisms controlling the life cycle of the malaria parasite are still poorly understood. The published genome sequence of P. falciparum and previous studies have revealed that several homologues of eukaryotic signalling proteins, such as protein kinases, are relatively conserved. Protein kinases are now widely recognized as important drug targets in protozoan parasites. Cyclic AMP-dependent protein kinase (PKA) is implicated in numerous processes in mammalian cells, and the regulatory mechanisms of the cAMP pathway have been characterized. P. falciparum cAMP-dependent protein kinase plays an important role in the parasites life cycle and thus represents an attractive target for the development of anti-malarial drugs. In this review, we focus on the P. falciparum cAMP/PKA pathway to provide new insights and an improved understanding of this signalling cascade.

Intramuscular Bioavailability and Clinical Efficacy of Artesunate in Gabonese Children with Severe Malaria

ABSTRACT Artesunate (ARS) is a water-soluble artemisinin derivative that is a potential alternative to quinine for the treatment of severe childhood malaria. We studied the pharmacokinetics and bioavailability of ARS given by the intramuscular (i.m.) route in an open crossover study design. Fourteen children were randomized to receive intravenous (i.v.) ARS in a loading dose (2.4 mg/kg of body weight) followed 12 h later by an i.m. dose (1.2 mg/kg) (group I), and 14 children were randomized to receive i.m. ARS (2.4 mg/kg) followed by an i.v. dose of ARS (1.2 mg/kg) (group II). We carried out a two-compartment analysis of ARS and dihydroartemisinin (DHA; the principal antimalarial metabolite) levels in 21 children (groups I and II combined). Absorption of i.m. ARS was rapid, with the maximum concentration of DHA in serum being achieved in less than 1 h in most children (median time to the maximum concentration of drug in serum, 35.1 min; range, 10.8 to 71.9 min). The absolute bioavailability of DHA was a median of 86.4% (range, 11.4 to 462.1%), the median steady-state volume of distribution was 1.3 liters/kg (range, 0.5 to 7.9 liters/kg), and the median clearance was 0.028 liters/kg/min (range, 0.001 to 1.58 liters/kg/min). There were no major adverse events attributable to ARS. Parasite clearance kinetics were comparable between the two treatment groups. These results support the use of i.m. ARS in children with severe malaria.

Immunization with Recombinant Duffy Binding-Like-γ3 Induces Pan-Reactive and Adhesion-Blocking Antibodies against Placental Chondroitin Sulfate A-Binding Plasmodium falciparum Parasites

Maternal malaria is associated with the sequestration, in the placenta, of Plasmodium falciparum-infected erythrocytes onto chondroitin sulfate A (CSA), via the duffy binding-like (DBL)-gamma3 domain of the P. falciparum erythrocyte membrane protein 1 (PfEMP1(CSA)) (DBL-gamma3(CSA)). The production of antibodies against CSA-binding infected erythrocytes (IEs(CSA)) is correlated with resistance to maternal malaria in multiparous women. We produced recombinant DBL-gamma3(CSA) (rDBL-gamma3(CSA)) in insect cells, corresponding to 2 variant DBL-gamma3(CSA) subtypes that mediate binding to CSA in laboratory lines and placental isolates. Both recombinant cysteine-rich DBL-gamma3(CSA) domains blocked IEs(CSA) binding to CSA. Immunization of mice, with the rDBL-gamma3(CSA)-FCR3 and rDBL-gamma3(CSA)-3D7 domains, resulted in the generation of antibodies recognizing homologous and heterologous rDBL-gamma3(CSA), a finding indicating conserved epitopes inducing a pan-reactive immune response. Mouse monoclonal antibodies (MAbs) against both recombinant proteins were pan-reactive with various IEs(CSA). One MAb efficiently inhibited and reversed IE(CSA) cytoadhesion to endothelial cells in vitro. Thus, DBL-gamma3(CSA) is the target of inhibitory and pan-reactive antibodies. Saimiri sciureus monkeys immunized with FCR3-rDBL-gamma3(CSA) developed pan-reactive and inhibitory antibodies, a finding suggesting that the development of a vaccine to prevent maternal malaria is feasible.

Global response of Plasmodium falciparum to hyperoxia: a combined transcriptomic and proteomic approach.

BackgroundOver its life cycle, the Plasmodium falciparum parasite is exposed to different environmental conditions, particularly to variations in O2 pressure. For example, the parasite circulates in human venous blood at 5% O2 pressure and in arterial blood, particularly in the lungs, at 13% O2 pressure. Moreover, the parasite is exposed to 21% O2 levels in the salivary glands of mosquitoes.MethodsTo study the metabolic adaptation of P. falciparum to different oxygen pressures during the intraerythrocytic cycle, a combined approach using transcriptomic and proteomic techniques was undertaken.ResultsEven though hyperoxia lengthens the parasitic cycle, significant transcriptional changes were detected in hyperoxic conditions in the late-ring stage. Using PS 6.0™ software (Ariadne Genomics) for microarray analysis, this study demonstrate up-expression of genes involved in antioxidant systems and down-expression of genes involved in the digestive vacuole metabolism and the glycolysis in favour of mitochondrial respiration. Proteomic analysis revealed increased levels of heat shock proteins, and decreased levels of glycolytic enzymes. Some of this regulation reflected post-transcriptional modifications during the hyperoxia response.ConclusionsThese results seem to indicate that hyperoxia activates antioxidant defence systems in parasites to preserve the integrity of its cellular structures. Moreover, environmental constraints seem to induce an energetic metabolism adaptation of P. falciparum. This study provides a better understanding of the adaptive capabilities of P. falciparum to environmental changes and may lead to the development of novel therapeutic targets.

Influence of oxygen on asexual blood cycle and susceptibility of Plasmodium falciparum to chloroquine: requirement of a standardized in vitro assay

ObjectiveThe main objective of this study was to assess the influence of gas mixtures on in vitro Plasmodium falciparum growth and 50% inhibitory concentration (IC50) for chloroquine.MethodsThe study was performed between February 2004 and December 2005. 136 Plasmodium falciparum isolates were used to evaluate gas mixtures effect on IC50 for chloroquine by isotopic microtest. The oxygen effect on asexual blood cycle of 3D7 and W2 clones was determined by thin blood smears examination and tritiated hypoxanthine uptake.ResultsFrom 5% O2 to 21% O2 conditions, no parasiticide effect of O2 concentration was observed in vitro on the clones 3D7 and W2. A parasitostatic effect was observed during the exposure of mature trophozoïtes and schizonts at 21% O2 with an increase in the length of schizogony. The chloroquine IC50 at 10% O2 were significantly higher than those at 21% O2, means of 173.5 nM and 121.5 nM respectively (p < 0.0001). In particular of interest, among the 63 isolates that were in vitro resistant to chloroquine (IC50 > 100 nM) at 10% O2, 17 were sensitive to chloroquine (IC50 < 100 nM) at 21% O2.ConclusionBased on these results, laboratories should use the same gas mixture to realize isotopic microtest. Further studies on comparison of isotopic and non-isotopic assays are needed to establish a standardized in vitro assay protocol to survey malaria drug resistance.

Culture and Antibiotic Susceptibility of Bartonella quintana in Human Erythrocytes

ABSTRACT Bartonella quintana, the agent of trench fever, has recently been implicated in various diseases, in particular, bacteremia and endocarditis in homeless people. The host cell of Bartonella spp. is believed to be the erythrocyte, and in the present study we demonstrate that B. quintana can be cultured in vitro in human erythrocytes. The bacteria were found to be intraerythrocytic by laser confocal microscopy with Bartonella species-specific monoclonal antibodies. Infections with B. quintana decreased the life span of erythrocytes in culture from 8.6 to 4.8 days. In the culture system we found that most of the antibiotics that we tested (doxycycline, fluoroquinolone compounds, and beta-lactams) were not bactericidal. Gentamicin was bactericidal at 4 μg/ml, as was rifampin, but to a lesser extent. At this concentration, gentamicin has been shown to enter erythrocytes slowly and to reach a peak level of 0.26 μg/ml after 24 h. At 0.26 μg/ml, however, we found that gentamicin was not able to kill extracellular B. quintana, even after 96 h of incubation. We hypothesize that erythrocytes may be a reservoir for B. quintana and that the bactericidal activity of gentamicin that we observed occurs mainly when the bacteria emerge from the erythrocytes and are found extracellularly. It would appear that gentamicin should be administered for at least 5 days to cure patients infected with B. quintana.