Impaired vitreous composition and retinal pigment epithelium function in the FoxG1::LRP2 myopic mice.

High myopia (HM) is one of the main causes of visual impairment and blindness all over the world and an unsolved medical problem. Persons with HM are predisposed to other eye pathologies such as retinal detachment, myopic retinopathy or glaucomatous optic neuropathy, complications that may at least partly result from the extensive liquefaction of the myopic vitreous gel. To identify the involvement of the liquid vitreous in the pathogenesis of HM we here analyzed the vitreous of the recently described highly myopic low density lipoprotein receptor-related protein 2 (Lrp2)-deficient eyes. Whereas the gel-like fraction was not apparently modified, the volume of the liquid vitreous fraction (LVF) was much higher in the myopic eyes. Biochemical and proteome analysis of the LVF revealed several modifications including a marked decrease of potassium, sodium and chloride, of proteins involved in ocular tissue homeostasis and repair as well as of ADP-ribosylation factor 4 (ARF4), a protein possibly involved in LRP2 trafficking. A small number of proteins, mainly comprising known LRP2 ligands or proteins of the inflammatory response, were over expressed in the mutants. Moreover the morphology of the LRP2-deficient retinal pigment epithelium (RPE) cells was affected and the expression of ARF4 as well as of proteins involved in degradative endocytosis was strongly reduced. Our results support the idea that impairment of the RPE structure and most likely endocytic function may contribute to the vitreal modifications and pathogenesis of HM.

Brain proteomic modifications associated to protective effect of grape extract in a murine model of obesity.

In recent years, the obesity epidemic has developed into a major health crisis worldwide. With current treatments limited to expensive, high-risk surgery and minimally efficacious pharmacotherapy, new therapeutic options are urgently needed to fight against this alarming trend. Though brain dysfunction has been studied linked to high fat diet (HFD) and grape seed and skin extract (GSSE) correction, the proteomic modifications linking the two effects on brain lipotoxicity are not well understood. To this end rats were exposed for 8 weeks to HFD treatment, to GSSE (500mg/kg BW) and to binary mixture of HFD and GSSE to gain insight into the potential pathways altered with metabolic disease and the protection afforded by GSSE. Significant modifications of brain proteins were detected using mass spectrometry-based differential proteomics. These proteins were mainly related to oxidative stress, glycolysis and calcium signaling. Additionally, proteins involved in the cytoskeleton were also affected by HFD treatment. Interestingly, whether up- or down regulated protein abundances, GSSE corrected most of the disturbances of HFD treatment. These findings provide impetus for future therapeutic investigation on GSSE against other metabolic disorders.

Overexpression of MHC class I in muscle of lymphocyte-deficient mice causes a severe myopathy with induction of the unfolded protein response.

Muscle fibers do not normally express major histocompatibility complex class I (MHC-I) molecules, and their reexpression is a hallmark of inflammatory myopathies. It has been shown in mice that overexpression of MHC-I induces a poorly inflammatory myositis accompanied by the unfolded protein response (UPR), but it is unclear whether it is attributable to T-cell-mediated MHC-I-dependent immune responses or to MHC-I forced expression per se. Indeed, besides presenting antigenic peptides to CD8(+) T cells, MHC-I may also possibly exert nonimmunologic, yet poorly understood pathogenic effects. Thus, we investigated the pathogenicity of MHC-I expression in muscle independently of its immune functions. HT transgenic mice that conditionally overexpress H-2K(b) in muscle were bred to an immunodeficient Rag2(-/-) background. The muscle proteome was analyzed by label-free high-resolution protein quantitation and Western blot. Despite the absence of adaptive immunity, HT Rag2(-/-) mice developed a very severe myopathy associated with the cytoplasmic accumulation of H-2K(b) molecules. The UPR was manifest by up-regulation of characteristic proteins. In humans, we found that HLA class I molecules not only were expressed at the sarcolemma but also could accumulate intracellularly in some patients with inclusion body myositis. Accordingly, the UPR was triggered as a function of the degree of HLA accumulation in myofibers. Hence, reexpression of MHC-I in normally negative myofibers exerts pathogenic effects independently of its immune function.

Impact of chlorhexidine digluconate and temperature on curli production in Escherichia coli-consequence on its adhesion ability.

Chlorhexidine-Digluconate (CHX-Dg) is a biocide widely used as disinfectant or antiseptic in clinical and domestic fields. It is often found in the formulation of solutions to treat superficial wounds. Nevertheless, few studies have focused on its effects on Escherichia coli while this bacterium is commonly involved in mixed infections. Therefore, the impact of CHX-Dg and temperature on E. coli was investigated; particularly the curli production. In accordance with bibliographic data, the curli production decreased when the temperature of the culture was shift from 30 °C to 37 °C. The bacterial adhesion to abiotic surfaces was also reduced. Surprisingly, the curli production at 37 °C was maintained in presence of antiseptic and the bacterial adhesion was improved at a very low concentration (1 µg ml-1) of CHX-Dg. Complementary investigations with a cpxR mutant demonstrated that the CpxA/R-TCS (Two-Component System) is involved in the temperature-dependent control of the curli expression. Indeed, the curli production was not altered by the growth temperature in the mutant. Otherwise, no relationship between CHX-Dg and the Cpx-TCS was shown. A subsequent proteomic investigation revealed the alteration of the production of 44 periplasmic and outer membrane proteins in presence of CHX-Dg. These proteins are involved in the transport of small molecules, the envelope integrity, the stress response as well as the protein folding.

Major remodeling of brain microvessels during neonatal period in the mouse: A proteomic and transcriptomic study.

Preterm infants born before 29 gestation weeks incur major risk of subependymal/intracerebral/intraventricular hemorrhage. In mice, neonate brain endothelial cells are more prone than adult cells to secrete proteases under glutamate challenge, and invalidation of the Serpine 1 gene is accompanied by high brain hemorrhage risk up to five days after birth. We hypothesized that the structural and functional states of microvessels might account for age-dependent vulnerability in mice up to five days after birth and might represent a pertinent paradigm to approach the hemorrhage risk window observed in extreme preterms. Mass spectrometry proteome analyses of forebrain microvessels at days 5, 10 and in adult mice revealed 899 proteins and 36 enriched pathways. Microarray transcriptomic study identified 5873 genes undergoing at least two-fold change between ages and 93 enriched pathways. Both approaches pointed towards extracellular matrix, cell adhesion and junction pathways, indicating delayed microvascular strengthening after P5. Furthermore, glutamate receptors, proteases and their inhibitors exhibited convergent evolutions towards excitatory aminoacid sensitivity and low proteolytic control likely accounting for vascular vulnerability in P5 mice. Thus, age vascular specificities must be considered in future therapeutic interventions in preterms. Data are available on ProteomeXchange (identifier PXD001718) and NCBI Gene-Expression-Omnibus repository (identification GSE67870).

Identification of 7 Proteins in Sera of RA Patients with Potential to Predict ETA/MTX Treatment Response.

OBJECTIVE: The recent growth of innovating biologics has opened fascinating avenues for the management of patients. In rheumatoid arthritis, many biologics are currently available, the choice of which being mostly determined empirically. Importantly, a given biologic may not be active in a fraction of patients and may even provoke side effects. Here, we conducted a comparative proteomics study in attempt to identify a predictive theranostic signature of non-response in patients with rheumatoid arthritis treated by etanercept/methotrexate combination. METHODS: A serum sample was collected prior to treatment exposure from a cohort of 22 patients with active RA. A proteomic "label free" approach was then designed to quantitate protein biomarkers using mass spectrometry. To verify these results, a relative quantification followed by an absolute quantification of interesting protein candidates were performed on a second cohort. The criterion of judgment was the response to etanercept/methotrexate combination according to the EULAR criteria assessed at 6 months of treatment. RESULTS: These investigations led to the identification of a set of 12 biomarkers with capacity to predict treatment response. A targeted quantitative analysis allowed to confirm the potential of 7 proteins from the latter combination on a new cohort of 16 patients. Two highly discriminating proteins, PROS and CO7, were further evaluated by ELISA on this second cohort. By combining the concentration threshold of each protein associated to a right classification (responders vs non-responders), the sensitivity and specificity reached 88.9 % and 100 %, respectively. CONCLUSION: Prior to methotrexate/etanercept treatment, abundance of several sera proteins, notably PROS and CO7, were associated to response status of RA patients 6 month after treatment initiation.

Foxg1-Cre Mediated Lrp2 Inactivation in the Developing Mouse Neural Retina, Ciliary and Retinal Pigment Epithelia Models Congenital High Myopia.

Myopia is a common ocular disorder generally due to increased axial length of the eye-globe. Its extreme form high myopia (HM) is a multifactorial disease leading to retinal and scleral damage, visual impairment or loss and is an important health issue. Mutations in the endocytic receptor LRP2 gene result in Donnai-Barrow (DBS) and Stickler syndromes, both characterized by HM. To clearly establish the link between Lrp2 and congenital HM we inactivated Lrp2 in the mouse forebrain including the neural retina and the retinal and ciliary pigment epithelia. High resolution in vivo MRI imaging and ophthalmological analyses showed that the adult Lrp2-deficient eyes were 40% longer than the control ones mainly due to an excessive elongation of the vitreal chamber. They had an apparently normal intraocular pressure and developed chorioretinal atrophy and posterior scleral staphyloma features reminiscent of human myopic retinopathy. Immunomorphological and ultrastructural analyses showed that increased eye lengthening was first observed by post-natal day 5 (P5) and that it was accompanied by a rapid decrease of the bipolar, photoreceptor and retinal ganglion cells, and eventually the optic nerve axons. It was followed by scleral thinning and collagen fiber disorganization, essentially in the posterior pole. We conclude that the function of LRP2 in the ocular tissues is necessary for normal eye growth and that the Lrp2-deficient eyes provide a unique tool to further study human HM.

Identification of S100A9 as biomarker of responsiveness to the methotrexate/etanercept combination in rheumatoid arthritis using a proteomic approach.

OBJECTIVES: One way to optimize the drug prescription in rheumatoid arthritis (RA) is to identify predictive biomarkers of drug responsiveness. Here, we investigated the potential "theranostic" value of proteins of the S100 family by monitoring levels of both S100A8 and S100A9 in blood samples from RA patients. DESIGN: For proteomic analysis, peripheral blood mononuclear cells (PBMC) and serum samples were collected in patients prior to initiation of the methotrexate/etanercept (MTX/ETA) combination. Firstly, relative mass spectrometry (MS) quantification focusing on S100A8 and S100A9 proteins was carried out from PBMCs samples to identify potential biomarkers. The same approach was also performed from serum samples from responder (R) and non responder (NR) patients. Finally, to confirm these results, an absolute quantification of S100A8, S100A9 proteins and calprotectin (heterodimer of S100A8/S100A9) was carried out on the serum samples using ELISA. RESULTS: MS analyses revealed that both S100A8 and S100A9 proteins were significantly accumulated in PBMC from responders. In contrast to PBMC, only the S100A9 protein was significantly overexpressed in the serum of R patients. Absolute quantification by ELISA confirmed this result and pointed out a similar expression level of S100A8 protein and calprotectin in sera from both R and NR groups. Thus, the S100A9 protein revealed to be predictive of MTX/ETA responsiveness, contrarily to parameters of inflammation and auto-antibodies which did not allow significant discrimination. CONCLUSION: This is the first report of an overexpression of S100A9 protein in both PBMCs and serum of patients with subsequent response to the MTX/ETA combination. This protein thus represents an interesting biomarker candidate of therapeutic response in RA.