Brigitte Arbeille

Abnormal activation of autophagy-induced crinophagy in Paneth cells from patients with Crohn's disease.

Autophagy-related 16 like-1 (ATG16L-1), immunity-related GTPase-M (IRGM), and nucleotide-binding oligomerization domain-containing 2 (NOD2) regulate autophagy, and variants in these genes have been associated with predisposition to Crohns disease (CD). However, little is known about the role of autophagy in CD. Intestinal biopsies from untreated pediatric patients with CD, celiac disease, or ulcerative colitis were analyzed by immunohistochemistry and electron microscopy. We observed that autophagy was specifically activated in Paneth cells from patients with CD, independently of mucosal inflammation or disease-associated variants of ATG16L1 or IRGM. In these cells, activation of autophagy was associated with a significant decrease in number of secretory granules and features of crinophagy. These observations might account for the disorganization of secretory granules previously reported in Paneth cells from patients with CD.

Evidence for an α-Granular Pool of the Cytoskeletal Protein α-Actinin in Human Platelets That Redistributes With the Adhesive Glycoprotein Thrombospondin-1 During the Exocytotic Process

Abstract In a previous study, we have demonstrated that the platelet adhesive glycoprotein thrombospondin-1 (TSP-1) interacts specifically with the cytoskeletal protein α-actinin in a solid-phase binding assay. Stored in the α-granules of platelets, TSP-1 is secreted during cell activation and binds to the plasma membrane promoting the platelet macroaggregate formation. However, the molecular mechanism by which TSP-1 reaches and binds to the platelet surface is to date unelucidated. α-Actinin is an actin-binding and actinin–cross-linking protein that is present in most cells and may act as a link between the bundles of F-actin and the plasma membrane. In this study, we have investigated a possible interaction of α-actinin with TSP-1 in platelets by examining their respective subcellular location during the platelet activation process. By indirect immunofluorescence, α-actinin was found to display a granular staining in resting platelets similar to that of TSP-1. Performing postembedding immunogold labelin...

Ultrasound and microbubble-assisted gene delivery in Achilles tendons: Long lasting gene expression and restoration of fibromodulin KO phenotype

The aim of this study is to deliver genes in Achilles tendons using ultrasound and microbubbles. The rationale is to combine ultrasound-assisted delivery and the stimulation of protein expression induced by US. We found that mice tendons injected with 10 μg of plasmid encoding luciferase gene in the presence of 5×10⁵ BR14 microbubbles, exposed to US at 1 MHz, 200 kPa, 40% duty cycle for 10 min were efficiently transfected without toxicity. The rate of luciferase expression was 100-fold higher than that obtained when plasmid alone was injected. Remarkably, the luciferase transgene was stably expressed for up to 108 days. DNA extracted from these sonoporated tendons was efficient in transforming competent E. coli bacteria, indicating that persistent intact pDNA was responsible for this long lasting gene expression. We used this approach to restore expression of the fibromodulin gene in fibromodulin KO mice. A significant fibromodulin expression was detected by quantitative PCR one week post-injection. Interestingly, ultrastructural analysis of these tendons revealed that collagen fibrils diameter distribution and circularity were similar to that of wild type mice. Our results suggest that this gene delivery method is promising for clinical applications aimed at modulating healing or restoring a degenerative tendon while offering great promise for gene therapy due its safety compared to viral methods.

Very-High-Frequency Ultrasound Corneal Imaging as a New Tool for Early Diagnosis of Ocular Surface Toxicity in Rabbits Treated with a Preserved Glaucoma Drug

Aim: To evaluate very-high-frequency (VHF) ultrasound imaging as a new method to detect and quantify early corneal epithelium changes induced by chronic exposure to a benzalkonium-chloride-containing antiglaucoma drug. Methods: Timolol preserved with 0.01% benzalkonium chloride solution was applied b.i.d. in 1 eye of 10 rabbits for 56 days. Unpreserved timolol solution was used as control. Ocular surface changes were assessed weekly combining clinical examinations, in vivo 60-MHz ultrasound imaging and ex vivo histological analysis. Results: VHF ultrasound imaging allowed quantitative measurement of corneal epithelium thickness and qualitative imaging of toxic epithelial damage. It revealed significantly decreased epithelial thickness in vivo as early as the 21st day of treatment (40.75 ± 1.72 µm at D0 vs. 39 ± 2 at D21, vs. 31.9 ± 2.98 at D56; p = 0.017 and p = 0.005, respectively). The first clinical changes appeared from the 42nd day of treatment (conjunctival redness, conjunctival staining and corneal staining; D56 compared to D0: p = 0.005, 0.01 and 0.004, respectively) and then correlated with VHF ultrasound data. Epithelial thickness measured with VHF ultrasound was correlated with histological epithelial pachymetry (p < 0.001) and with the corneal damage score assessed with scanning electron microscopy (p = 0.038). Conclusion: VHF ultrasound imaging provided an early in vivo diagnosis of corneal epithelium pathology induced by chronic exposure to a preserved glaucoma drug, before the first clinical evidence of ocular toxicity. It could be a new reproducible method to detect the toxicity of glaucoma medication so that therapy can then be adapted.

Ferric Iron Reduction by Fermentative Strain BS2 Isolated From an Iron-Rich Anoxic Environment (Lake Pavin, France)

A facultatively Fe-reducing strain BS2 related to Clostridium saccarobutylicum was isolated from the anoxic zone of iron-rich Lake Pavin. A comparative study was performed on the growth, fermentative profile and heat production of BS2 on glucose in presence and absence of Fe(III). Fe(III) availability led to an optimization of glucose uptake and higher maintenance of strain BS2 in the growth media. Given the prevailing conditions (e.g., scarcity of metabolizable organic matter) in the anoxic zone of Lake Pavin, Fe(III) reduction by this facultative Fe(III) reducer may confer ecological benefits to strain BS2. Moreover, in presence of Fe(III), there was a modification of the fermentative balance that may influence the terminal-accepting processes in this layer.

Effect of transitory glucose deprivation on mitochondrial structure and functions in cultured cerebellar granule neurons.

We found that 60-min glucose deprivation leads to progressive decrease in the mitochondrial membrane potential and increase in [Ca(2+)](i) in cultured cerebellar granule neurons. The latter effect was fully reversible, returning to the basal level 60 min after restoration of normal glucose level in the incubation medium, whereas mitochondrial membrane potential remained at 10.0+/-1.8% below the initial value. Electron microscopy indicated that glucose deprivation induced appearance of mitochondria with local lightening of the matrix and destruction of cristae. This mitochondrial conformation was preserved during the restoration phase after glucose level in the cultivation medium returned to the normal level. Neuronal death within a 24-h period after 60-min glucose deprivation was relatively small, being 14.0+/-4.4%.

The pig as a model for investigating the role of neutrophil serine proteases in human inflammatory lung diseases

The serine proteases released by activated polymorphonuclear neutrophils [NSPs (neutrophil serine proteases)] contribute to a variety of inflammatory lung diseases, including CF (cystic fibrosis). They are therefore key targets for the development of efficient inhibitors. Although rodent models have contributed to our understanding of several diseases, we have previously shown that they are not appropriate for testing anti-NSP therapeutic strategies [Kalupov, Brillard-Bourdet, Dade, Serrano, Wartelle, Guyot, Juliano, Moreau, Belaaouaj and Gauthier (2009) J. Biol. Chem. 284, 34084–34091). Thus NSPs must be characterized in an animal model that is much more likely to predict how therapies will act in humans in order to develop protease inhibitors as drugs. The recently developed CFTR−/− (CFTR is CF transmembrane conductance regulator) pig model is a promising alternative to the mouse model of CF [Rogers, Stoltz, Meyerholz, Ostedgaard, Rokhlina, Taft, Rogan, Pezzulo, Karp, Itani et al. (2008) Science 321, 1837–1841]. We have isolated blood neutrophils from healthy pigs and determined their responses to the bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus, and the biochemical properties of their NSPs. We used confocal microscopy and antibodies directed against their human homologues to show that the three NSPs (elastase, protease 3 and cathepsin G) are enzymatically active and present on the surface of triggered neutrophils and NETs (neutrophil extracellular traps). All of the porcine NSPs are effectively inhibited by human NSP inhibitors. We conclude that there is a close functional resemblance between porcine and human NSPs. The pig is therefore a suitable animal model for testing new NSP inhibitors as anti-inflammatory agents in neutrophil-associated diseases such as CF.

The platelet receptor for type III collagen (TIIICBP) is present in platelet membrane lipid microdomains (rafts)

Platelet interactions with collagen are orchestrated by the presence or the migration of platelet receptor(s) for collagen into lipid rafts, which are specialized lipid microdomains from the platelet plasma membrane enriched in signalling proteins. Electron microscopy shows that in resting platelets, TIIICBP, a receptor specific for type III collagen, is present on the platelet membrane and associated with the open canalicular system, and redistributes to the platelet membrane upon platelet activation. After platelet lysis by 1% Triton X-100 and the separation of lipid rafts on a discontinuous sucrose gradient, TIIICBP is recovered in lipid raft-containing fractions and Triton X-100 insoluble fractions enriched in cytoskeleton proteins. Platelet aggregation, induced by type III collagen, was inhibited after disruption of the lipid rafts by cholesterol depletion, whereas platelet adhesion under static conditions did not require lipid raft integrity. These results indicate that TIIICBP, a platelet receptor involved in platelet interaction with type III collagen, is localized within platelet lipid rafts where it could interact with other platelet receptors for collagen (GP VI and α2β1 integrin) for efficient platelet activation.

Different Role of Platelet Glycoprotein GP Ia/IIa in Platelet Contact and Activation Induced by Type I and Type III Collagens

The role of glycoprotein Ia/IIa was studied during platelet contact and aggregation induced by type I and type III collagen. The anti-glycoprotein Ia/IIa (6F1) antibody inhibited type I collagen-induced aggregation but did not inhibit the first contact between platelets and collagen. In contrast, it was without effect either on type III collagen-induced contact or platelet interaction with the subendothelium in a static assay. Platelet aggregation induced by type III collagen was only slightly slowed down by 6F1 but pp72 spleen tyrosine kinase phosphorylation was not modified even at concentrations of 6F1 that completely blocked platelet activation induced by type I collagen. Our results indicate that glycoprotein Ia/IIa is not a primary binding site for type I or type III collagen on the platelet membrane. This receptor is more specifically involved in type I collagen-induced platelet spreading and aggregation.

Quantitative analysis of immunogold labellings of collagen types I, III, IV and VI in healthy and pathological human corneas

Abstract• Background: We studied the distribution of collagen types I, III, IV and VI in one healthy human cornea and in seven pathological human corneas, in which the disorders were three cases of pseudophakic bullous keratopathy (two severe, one moderate) and one case each of stage IV keratoconus, chronic ulcer, vascularized cornea and disciform keratitis. • Methods: Transmission electron microscopy examinations were performed on post-embedding immunogold-labelled sections. The staining was evaluated by gold particle count in the different tissues. The presence or absence of a given antigen was determined by statistical analysis, using a d-value test. • Results: Our results on healthy corneal tissues corroborate the data available from previous studies, except for collagen type VI, which we found to be absent in Bowmans layer. In pathological corneas with a collagenous layer posterior to Descemets membrane, collagen types I, III and especially IV were detected in this collagenous layer. Collagen types I, III and VI were detected in the anterior healed stroma of other pathological corneas, except for the keratoconus cornea, in which intense collagen III staining was observed. • Conclusion: The presence of collagen types I and III in the posterior collagenous layer of our pseudophakic bullous keratopathy corneas suggests that this layer corresponds to scar tissue secreted by stimulated endothelial cells.