Flow cytometry for the evaluation of anti-plasmodial activity of drugs on Plasmodium falciparum gametocytes

<p>Abstract</p> <p>Background</p> <p>The activity of promising anti-malarial drugs against <it>Plasmodium </it>gametocytes is hard to evaluate even in vitro. This is because visual examination of stained smears, which is commonly used, is not totally convenient. In the current study, flow cytometry has been used to study the effect of established anti-malarial drugs against sexual stages obtained from W2 strain of <it>Plasmodium falciparum</it>. Gametocytes were treated for 48 h with different drug concentrations and the gametocytaemia was then determined by flow cytometry and compared with visual estimation by microscopy.</p> <p>Results and conclusions</p> <p>Initially gametocytaemia was evaluated either using light microscopy or flow cytometry. A direct correlation (r²= 0.9986) was obtained. Two distinct peaks were observed on cytometry histograms and were attributed to gametocyte populations. The activities of established anti-malarial compounds were then measured by flow cytometry and the results were equivalent to those obtained using light microscopy. Primaquine and artemisinin had IC₅₀of 17.6 μM and 1.0 μM, respectively.</p> <p>Gametocyte sex was apparently distinguishable by flow cytometry as evaluated after induction of exflagellation by xanthurenic acid. These data form the basis of further studies for developing new methods in drug discovery to decrease malaria transmission.</p

Double-walled carbon nanotubes trigger IL-1β release in human monocytes through Nlrp3 inflammasome activation

Because of their outstanding physical properties, carbon nanotubes (CNTs) are promising new materials in the field of nanotechnology. It is therefore imperative to assess their adverse effects on human health. Monocytes/macrophages that recognize and eliminate the inert particles constitute the main target of CNTs. In this article, we report our finding that double-walled CNTs (DWCNTs) synergize with Tolllike receptor agonists to enhance IL-1β release in human monocytes. We show that DWCNTs–induced IL-1β secretion is exclusively linked to caspase-1 and to Nlrp3 inflammasome activation in human monocytes. We also establish that this activation requires DWCNTs phagocytosis and potassium efflux, but not reactive oxygen specied (ROS) generation. Moreover, inhibition of lysosomal acidification or cathepsin-B activation reduces DWCNT-induced IL-1β secretion, suggesting that Nlrp3 inflammasome activation occurs via lysosomal destabilization. Thus, DWCNTs present a health hazard due to their capacity to activate Nlrp3 inflammasome, recalling the inflammation caused by asbestos and hence demonstrating that they should be used with caution. From the Clinical Editor: This is a very important biosafety/toxicity study regarding double walled carbon nanotubes. The investigators demonstrate that such nanotubes do represent a health hazard due to their capacity to activate Nlrp3 inflammasome, resembling the inflammation caused by asbestos. While further study of this phenomenon is definitely needed, the above findings clearly suggest that special precautions need to be taken when applying these nanoparticles in human disease research

Tumor necrosis factor alpha and adalimumab differentially regulate CD36 expression in human monocytes

In chronic inflammatory diseases, such as rheumatoid arthritis, inflammation acts as an independent cardiovascular risk factor and the use of anti-inflammatory drugs, such as anti-tumor necrosis factor alpha (anti-TNFα), may decrease this risk. The phagocytosis of oxidized low density lipoproteins (LDLs) accumulated in the subendothelium by mononuclear cells influences atherosclerosis and depends on CD36 expression. We investigated the role of TNFα and adalimumab, a human anti-TNFα monoclonal antibody widely used in human pathology, in CD36 expression in human monocytes. Human monocytes were prepared by adherence from whole-blood buffy-coat fractions from healthy donors. CD36 expression was assessed by RT-PCR and flow cytometry, with various TNFα or adalimumab concentrations. Implication of peroxisome proliferator-activated receptor (PPAR)γ in the regulation of CD36 expression was assessed using specific inhibitor or gel shift assays. The impact of redox signaling was investigated using quantification of reactive oxygen species, antioxidant and a NADPH oxidase inhibitor. The F(ab')2 fragment of adalimumab was isolated and its effect was analyzed. TNFα inhibits both CD36 membrane expression and mRNA expression. This inhibition involves a reduction in PPARγ activation. In contrast, adalimumab increases both CD36 membrane expression and mRNA expression. This induction is independent of the Fc portion of adalimumab and involves redox signaling via NADPH oxidase activation. CD36 expression on human monocytes is inhibited by TNFα and independently increased by adalimumab. These data highlight that pro-inflammatory cytokines and their specific neutralization influence the expression of cellular receptors implicated in atherosclerosis. Further studies are needed to investigate the clinical implications of these results in accelerated atherosclerosis observed in rheumatoid arthritis

Biomimetic nanocrystalline apatites: Emerging perspectives in cancer diagnosis and treatment

Nanocrystalline calcium phosphate apatites constitute the mineral part of hard tissues, and the synthesis of biomimetic analogs is now wellmastered at the labscale. Recent advances in the fine physicochemical characterization of these phases enable one to envision original applications in the medical field along with a better understanding of the underlying chemistry and related pharmacological features. In this contribution, we specifically focused on applications of biomimetic apatites in the field of cancer diagnosis or treatment. We first report on the production and first biological evaluations (cytotoxicity, proinflammatory potential, internalization by ZR751 breast cancer cells) of individualized luminescent nanoparticles based on Eudoped apatites, eventually associated with folic acid, for medical imaging purposes. We then detail, in a first approach, the preparation of tridimensional constructs associating nanocrystalline apatite aqueous gels and drugloaded pectin microspheres. Sustained releases of a fluorescein analog (erythrosin) used as model molecule were obtained over 7 days, in comparison with the ceramic or microsphere reference compounds. Such systems could constitute original bonefilling materials for in situ delivery of anticancer drug

PPARγ ligands switched high fat diet-induced macrophage M2b polarization toward M2a thereby improving intestinal Candida elimination.

International audienceObesity is associated with a chronic low-grade inflammation that predisposes to insulin resistance and the development of type 2 diabetes. In this metabolic context, gastrointestinal (GI) candidiasis is common. We recently demonstrated that the PPARγ ligand rosiglitazone promotes the clearance of Candida albicans through the activation of alternative M2 macrophage polarization. Here, we evaluated the impact of high fat diet (HFD)-induced obesity and the effect of rosiglitazone (PPARγ ligand) or WY14643 (PPARα ligand) both on the phenotypic M1/M2 polarization of peritoneal and cecal tissue macrophages and on the outcome of GI candidiasis. We demonstrated that the peritoneal macrophages and the cell types present in the cecal tissue from HF fed mice present a M2b polarization (TNF-α(high), IL-10(high), MR, Dectin-1). Interestingly, rosiglitazone induces a phenotypic M2b-to-M2a (TNF-α(low), IL-10(low), MR(high), Dectin-1(high)) switch of peritoneal macrophages and of the cells present in the cecal tissue. The incapacity of WY14643 to switch this polarization toward M2a state, strongly suggests the specific involvement of PPARγ in this mechanism. We showed that in insulin resistant mice, M2b polarization of macrophages present on the site of infection is associated with an increased susceptibility to GI candidiasis, whereas M2a polarization after rosiglitazone treatment favours the GI fungal elimination independently of reduced blood glucose. In conclusion, our data demonstrate a dual benefit of PPARγ ligands because they promote mucosal defence mechanisms against GI candidiasis through M2a macrophage polarization while regulating blood glucose level

Biomimetic Apatite-Based Functional Nanoparticles as Promising Newcomers in Nanomedicine: Overview of 10 Years of Initiatory Research

Biomimetic calcium phosphate apatites, analogous to bone mineral, may now be produced synthetically. Their intrinsic biocompatibility and the nanometer dimensions of their constitutive crystals not only allow one to envision applications in bone tissue regeneration, but also in other medical fields such as nanomedicine, and in particular in view of cell diagnosis. In this mini-review, we look back at 10 years of our dedicated research, and summarize the main advances made in terms of preparation, physical-chemical characterizations and biological evaluations of colloidal formulations of biomimetic apatite-based nanoparticles, which we illustrate here with the angle of cancer diagnosis. The confirmed exceptional biocompatibility of these engineered nanoparticles, associated to the possibility to confer them luminescence properties by way of controlled lanthanide doping, and their capacity to be internalized by cells, including with cancer cell addressing abilities (shown here as a proof of concept), underline that biomimetic apatite-based colloidal nanoparticles are particularly promising for nanomedicine applications, for example related to diseased cells diagnosis. Multidisciplinary research on these functional nanoparticles, initiated as described here, has now generated emulation in the scientific community where the concept of apatite nanoparticles for nanomedicine is being, gratifyingly, appropriated

Targeted Magnetic Intra-Lysosomal Hyperthermia produces lysosomal reactive oxygen species and causes Caspase-1 dependent cell death

Therapeutic strategies using drugs which cause Lysosomal Cell Death have been proposed for eradication of resistant cancer cells. In this context, nanotherapy based on Magnetic Intra-Lysosomal Hyperthermia (MILH) generated by magnetic nanoparticles (MNPs) that are grafted with ligands of receptors overexpressed in tumors appears to be a very promising therapeutic option. However, mechanisms whereby MILH induces cell death are still elusive. Herein, using Gastrin-grafted MNPs specifically delivered to lysosomes of tumor cells from different cancers, we provide evidences that MILH causes cell death through a non-apoptotic signaling pathway. The mechanism of cell death involves a local temperature elevation at the nanoparticle periphery which enhances the production of reactive oxygen species through the lysosomal Fenton reaction. Subsequently, MILH induces lipid peroxidation, lysosomal membrane permeabilization and leakage of lysosomal enzymes into the cytosol, including Cathepsin-B which activates Caspase-1 but not apoptotic Caspase-3. These data highlight the clear potential of MILH for the eradication of tumors overexpressing receptors

Targeting a G-Protein-Coupled Receptor Overexpressed in Endocrine Tumors by Magnetic Nanoparticles To Induce Cell Death

Nanotherapy using targeted magnetic nanoparticles grafted with peptidic ligands of receptors overexpressed in cancers is a promising therapeutic strategy. However, nanoconjugation of peptides can dramatically affect their properties with respect to receptor recognition, mechanism of internalization, intracellular trafficking, and fate. Furthermore, investigations are needed to better understand the mechanism whereby application of an alternating magnetic field to cells containing targeted nanoparticles induces cell death. Here, we designed a nanoplatform (termed MG-IONP-DY647) composed of an iron oxide nanocrystal decorated with a ligand of a G-protein coupled receptor, the cholecystokinin-2 receptor (CCK2R) that is overexpressed in several malignant cancers. MG-IONP-DY647 did not stimulate inflammasome of Raw 264.7 macrophages. They recognized cells expressing CCK2R with a high specificity, subsequently internalized via a mechanism involving recruitment of β-arrestins, clathrin-coated pits, and dynamin and were directed to lysosomes. Binding and internalization of MG-IONP-DY647 were dependent on the density of the ligand at the nanoparticle surface and were slowed down relative to free ligand. Trafficking of CCK2R internalized with the nanoparticles was slightly modified relative to CCK2R internalized in response to free ligand. Application of an alternating magnetic field to cells containing MG-IONP-DY647 induced apoptosis and cell death through a lysosomal death pathway, demonstrating that cell death is triggered even though nanoparticles of low thermal power are internalized in minute amounts by the cells. Together with pioneer findings using iron oxide nanoparticles targeting tumoral cells expressing epidermal growth factor receptor, these data represent a solid basis for future studies aiming at establishing the proof-of-concept of nanotherapy of cancers using ligand-grafted magnetic nanoparticles specifically internalized via cell surface receptors

PPARγ Controls Dectin-1 Expression Required for Host Antifungal Defense against Candida albicans

We recently showed that IL-13 or peroxisome proliferator activated receptor γ (PPARγ) ligands attenuate Candida albicans colonization of the gastrointestinal tract. Here, using a macrophage-specific Dectin-1 deficient mice model, we demonstrate that Dectin-1 is essential to control fungal gastrointestinal infection by PPARγ ligands. We also show that the phagocytosis of yeast and the release of reactive oxygen intermediates in response to Candida albicans challenge are impaired in macrophages from Dectin-1 deficient mice treated with PPARγ ligands or IL-13. Although the Mannose Receptor is not sufficient to trigger antifungal functions during the alternative activation of macrophages, our data establish the involvement of the Mannose Receptor in the initial recognition of non-opsonized Candida albicans by macrophages. We also demonstrate for the first time that the modulation of Dectin-1 expression by IL-13 involves the PPARγ signaling pathway. These findings are consistent with a crucial role for PPARγ in the alternative activation of macrophages by Th2 cytokines. Altogether these data suggest that PPARγ ligands may be of therapeutic value in esophageal and gastrointestinal candidiasis in patients severely immunocompromised or with metabolic diseases in whom the prevalence of candidiasis is considerable