Nanoscale chemical imaging of phagocytosis : a battle for metals between host and microbe

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Combined use of x-ray fluorescence microscopy, phase contrast imaging for high resolution quantitative iron mapping in inflamed cells

X-ray fluorescence microscopy (XRFM) is a powerful technique to detect and localize elements in cells. To derive information useful for biology and medicine, it is essential not only to localize, but also to map quantitatively the element concentration. Here we applied quantitative XRFM to iron in phagocytic cells. Iron, a primary component of living cells, can become toxic when present in excess. In human fluids, free iron is maintained at 10-18 M concentration thanks to iron binding proteins as lactoferrin (Lf). The iron homeostasis, involving the physiological ratio of iron between tissues/secretions and blood, is strictly regulated by ferroportin, the sole protein able to export iron from cells to blood. Inflammatory processes induced by lipopolysaccharide (LPS) or bacterial pathoge inhibit ferroportin synthesis in epithelial and phagocytic cells thus hindering iron export, increasing intracellular iron and bacterial multiplication. In this respect, Lf is emerging as an important regulator of both iron and inflammatory homeostasis. Here we studied phagocytic cells inflamed by bacterial LPS and untreated or treated with milk derived bovine Lf. Quantitative mapping of iron concentration and mass fraction at high spatial resolution is obtained combining X-ray fluorescence microscopy, atomic force microscopy and synchrotron phase contrast imaging

IRM du manganèse (MEMRI) (couplage à l'imagerie chimique par microsonde synchrotron pour optimiser l'imagerie fonctionnelle du transport neuronal)

Résumé Le manganèse (Mn2+) est un élément essentiel du corps humain. Ses propriétés paramagnétiques permettent son utilisation comme agent de contraste pour l'IRM (Mn-MRI ou MEMRI). Analogue du calcium (Ca2+), il pénètre les neurones essentiellement par les canaux calciques. Il est ensuite transporté le long des microtubules jusqu'aux synapses où il est libéré, puis capturé par les autres neurones. Ainsi, il peut rendre compte du transport axonal antérograde et rétrograde. L'approche MEMRI peut ainsi apporter des informations uniques sur la connectivité fonctionnelle cérébrale. Toutefois, deux problèmes limitent l'emploi de ce puissant outil d'imagerie in vivo : (i) A doses élevées, le Mn2+ est toxique pour l'organisme et peut provoquer une atteinte grave du système nerveux central, appelé manganisme. Les niveaux et les mécanismes de toxicité sont mal connus. (ii) Le mode de transport du manganèse dans l'approche MEMRI est mal connu. Afin d'apporter des éléments de réponse à ces deux problèmes, nous avons entrepris une étude couplant IRM et microscopie synchrotron pour mieux comprendre le comportement du Mn2+ in vivo. Nous avons précisé les distributions cellulaire et sub-cellulaire du Mn et d'autres métaux pour un modèle de cellules de type neuronal (lignée de neuroblastome N2A), pour des cultures primaires de neurones hippocampiques, mais aussi au niveau de coupes d'hippocampe de rats. En parallèle, nous avons étudié les effets du Mn sur le métabolisme cérébral par une technique de RMN-HRMAS du proton. Pour compléter ce travail, nous avons mis en œuvre l'imagerie MEMRI chez les souris KO MAP6 présentant un déficit d'une protéine stabilisatrice des microtubules pour évaluer la connectivité fonctionnelle du tract thalamo-cortical. Mots clés Hippocampe, MAP6, manganèse, métabolisme, métal, neurone, MRI, rongeurs, synchrotron.Abstract Manganese (Mn2+) is an essential element for human body. The paramagnetic properties of Mn2+ permit it use as a contrast agent for MRI (Mn-MRI or MEMRI). Analogue of calcium (Ca2+), it enters neurons primarily by calcium channels. It is then transported along microtubules to the synapse where it is released and then captured by other neurons. Thus, it can account for the anterograde and retrograde axonal transport. The MEMRI approach can provide unique information about cerebral functional connectivity. Two problems limit the use of this powerful tool for in vivo imaging: (i) At high doses, Mn2+ is toxic to the body and can cause serious problem of the central nervous system, called manganism. The level and the mechanisms of toxicity are poorly understood. (ii) The mode of manganese transport in the MEMRI approach is unclear. To address these two issues, we undertook a study coupling MRI and synchrotron microscopy to study the Mn 2+ behavior in vivo. We characterized the cellular and subcellular distributions of Mn and other metals in "pseudo neurons" cell line N2A, primary cultures of hippocampal neurons, andin hippocampal slices from rats. In parallel, we studied the effects of Mn on brain metabolism by proton-HRMAS NMR . In parallel, weevaluated MEMRI in MAP6 KO mice which exhibit a deficit in microtubule stabilizing protein, to assess the functional connectivity of the thalamocortical tract. Key words hippocampus, MAP6, manganese, metabolism, metal, neuron, MRI, rodent, synchrotron.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Adsorption and release of BMP-2 on nanocrystalline apatite-coated and uncoated hydroxyapatite/b-tricalcium phosphate porous ceramics

The association of bone morphogenetic proteins (BMPs) with calcium phosphate bioceramics is known to confer them osteoinductive properties. The aim of this study was to evaluate the surface properties, especially regarding recombinant human BMP-2 (rhBMP-2) adsorption and release, of commercial sintered biphasic calcium phosphate ceramics after coating with biomimetic nanocrystalline apatite. The raw and coated ceramics exhibited similar macroporous structures but different nanometer-sized pores contents. Both types of ceramics showed Langmuir-type adsorption isotherms of rhBMP-2. The coating noticeably increased the rate of adsorption and the total amount of growth factor taken up, but the maximum coverage per surface area unit as well as the affinity constant appeared lower for coated ceramics compared with raw ceramic surfaces. The limited advantage gained by coating the ceramics can be assigned to a lower accessibility of the surface adsorption sites compared with the raw ceramics. The quantity of rhBMP-2 spontaneously released in cell culture medium during the first weeks was lower for coated samples than for uncoated ceramics and represented a minor fraction of the total adsorbed amount. In conclusion, the nanocrystalline apatite coating was found to favor the adsorption of rhBMP-2 while providing a mean to fine tune the release of the growth factor

Manganese Cytotoxicity Assay on Hippocampal Neuronal Cell Culture

Compared to an in vivo experiment, neuronal cell cultures are immediately accessibleto observation and manipulation. In this protocol, we describe a technique to evaluate thecytotoxicity of a metal, manganese (Mn2+), on hippocampal neuronal cell cultures. Interestingly, this protocol is easily adaptable to any type of primary culture (e.g., cortical neurons) and any type of toxic compound (e.g., chemical product).Fil: Daoust, Alexia. Inserm; Francia. Universite Joseph Fourier; FranciaFil: Saoudi, Yasmina. Inserm; Francia. Universite Joseph Fourier; FranciaFil: Brocard, Jacques. Inserm; Francia. Universite Joseph Fourier; FranciaFil: Collomb, Nora. Inserm; Francia. Universite Joseph Fourier; FranciaFil: Batandier, Cecile. Laboratoire de Bioénergétique Fondamentale et Appliquée; FranciaFil: Bisbal, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Inserm; Francia. Universite Joseph Fourier; FranciaFil: Salome, Murielle. European Synchrotron Radiation Facility; FranciaFil: Andrieux, Annie. Inserm; Francia. Universite Joseph Fourier; FranciaFil: Bohic, Sylvain. Inserm; Francia. Universite Joseph Fourier; Francia. European Synchrotron Radiation Facility; FranciaFil: Barbier, Emmanuel. Inserm; Francia. Universite Joseph Fourier; Franci

Calcium carbonate-calcium phosphate mixed cement compositions for bone reconstruction

The feasibility of making calcium carbonate-calcium phosphate (CaCO3-CaP) mixed cements, comprising at least 40 % (w/w) CaCO3 in the dry powder ingredients, has been demonstrated. Several original cement compositions were obtained by mixing metastable crystalline calcium carbonate phases with metastable amorphous or crystalline calcium phosphate powders in aqueous medium. The cements set within at most 1 hour at 37°C in atmosphere saturated with water. The hardened cement is microporous and exhibits weak compressive strength. The setting reaction appeared to be essentially related to the formation of a highly carbonated nanocrystalline apatite phase by reaction of the mestastable CaP phase with part or almost all of the metastable CaCO3 phase. The recrystallization of metastable CaP varieties led to a final cement consisting of a highly carbonated poorly crystalline apatite (PCA) analogous to bone mineral associated with various amounts of vaterite and/or aragonite. The presence of controlled amounts of CaCO3 with a higher solubility than the apatite formed in the well-developed calcium phosphate cements might be of interest to increase resorption rates in biomedical cement and favor its replacement by bone tissue. Cytotoxicity testing revealed excellent cytocompatibility of CaCO3-CaP mixed cement compositions

Loss of bhlha9 Impairs Thermotaxis and Formalin-Evoked Pain in a Sexually Dimorphic Manner

International audienceC-LTMRs are known to convey affective aspects of touch and to modulate injury-induced pain in humans and mice. However, a role for these neurons in temperature sensation has been suggested, but not fully demonstrated. Here, we report that deletion of C-low-threshold mechanoreceptor (C-LTMR)-expressed bhlha9 causes impaired thermotaxis behavior and exacerbated formalin-evoked pain in male, but not female, mice. Positive modulators of GABAA receptors failed to relieve inflammatory formalin pain and failed to decrease the frequency of spontaneous excitatory post-synaptic currents (sEPSCs) selectively in bhlha9 knockout (KO) males. This could be explained by a drastic change in the GABA content of lamina II inner inhibitory interneurons contacting C-LTMR central terminals. Finally, C-LTMR-specific deep RNA sequencing revealed more genes differentially expressed in male than in female bhlha9 KO C-LTMRs. Our data consolidate the role of C-LTMRs in modulation of formalin pain and provide in vivo evidence of their role in the discriminative aspects of temperature sensation

Sub-ppm level high energy resolution fluorescence detected X-ray absorption spectroscopy of selenium in articular cartilage

The speciation of highly-diluted elements by X-ray absorption spectroscopy in a diverse range of materials is extremely challenging, especially in biological matrices such as articular cartilage. Here we show that using a high energy resolution fluorescence detected X-ray absorption spectroscopy (HERFD-XAS) technique coupled to an array of crystal analyzers, selenium speciation down to 400 ppb (μg kg-1) within articular cartilage can be demonstrated. This is a major advance in the speciation of highly-diluted elements through X-ray absorption spectroscopy and opens new possibilities to study the metabolic role of selenium and other elements in biological samples