Dispositifs de délivrance de principes actifs pour des applications ophtalmologiques [Drug delivery systems for intraocular applications].

Numerous drug delivery systems (DDSs) can be used as intraocular tools to provide a sustained and calibrated release for a specific drug. Great progress has been made on the design, biocompatibility, bioavailability, and efficacy of DDSs. Although several of them are undergoing clinical trials, a few are already on the market and could be of a routine use in clinical practice. Moreover, miniaturization of the implants makes them less and less traumatic for the eye tissues and some DDSs are now able to target certain cells or tissues specifically. An overview of ocular implants with therapeutic application potentials is provided.

Early effects of intravitreal triamcinolone on macular edema: mechanistic implication.

PURPOSE: To evaluate the early effects of intravitreal triamcinolone acetonide (TA) on cystoid macular edema associated with retinal vein occlusion and diabetic retinopathy. DESIGN: Prospective, interventional, small case series. PARTICIPANTS: Four patients with cystoid macular edema resulting from retinal vein occlusion or diabetic retinopathy of more than 4 months' duration and evaluated as suitable for treatment with intravitreous injection of TA. METHODS: After ophthalmic examination, including visual acuity assessment, intraocular pressure (IOP) measurement, and optical coherence tomography (OCT) analysis, the patients received a single intravitreal injection of 4 mg TA. After the injection, consecutive visual acuity assessment, IOP measurement, and OCT analysis were performed after 1 hour, 6 hours, 1 week, and 2 weeks. MAIN OUTCOME MEASURE: Optical coherence tomography assessment of macular thickness. RESULTS: Macular thickness and edema initially were reduced as early as 1 hour after TA injection. A further continuous decrease was observed during the 2 weeks after treatment. CONCLUSIONS: This rapid effect of intravitreal TA is interpreted to indicate that nongenomic effects on retinal or retinal pigment epithelial cell membranes, or both, may be responsible for this phenomenon. Identifications of these mechanisms may help design alternative, more specific drugs for the treatment of macular edema.

Effects of a short-term overfeeding with fructose or glucose in healthy young males.

Consumption of simple carbohydrates has markedly increased over the past decades, and may be involved in the increased prevalence in metabolic diseases. Whether an increased intake of fructose is specifically related to a dysregulation of glucose and lipid metabolism remains controversial. We therefore compared the effects of hypercaloric diets enriched with fructose (HFrD) or glucose (HGlcD) in healthy men. Eleven subjects were studied in a randomised order after 7 d of the following diets: (1) weight maintenance, control diet; (2) HFrD (3.5 g fructose/kg fat-free mass (ffm) per d, +35 % energy intake); (3) HGlcD (3.5 g glucose/kg ffm per d, +35 % energy intake). Fasting hepatic glucose output (HGO) was measured with 6,6-2H2-glucose. Intrahepatocellular lipids (IHCL) and intramyocellular lipids (IMCL) were measured by 1H magnetic resonance spectroscopy. Both fructose and glucose increased fasting VLDL-TAG (HFrD: +59 %, P < 0.05; HGlcD: +31 %, P = 0.11) and IHCL (HFrD: +52 %, P < 0.05; HGlcD: +58 %, P = 0.06). HGO increased after both diets (HFrD: +5 %, P < 0.05; HGlcD: +5 %, P = 0.05). No change was observed in fasting glycaemia, insulin and alanine aminotransferase concentrations. IMCL increased significantly only after the HGlcD (HFrD: +24 %, NS; HGlcD: +59 %, P < 0.05). IHCL and VLDL-TAG were not different between hypercaloric HFrD and HGlcD, but were increased compared to values observed with a weight maintenance diet. However, glucose led to a higher increase in IMCL than fructose.

PPARbeta regulates vitamin A metabolism-related gene expression in hepatic stellate cells undergoing activation.

Activation of cultured hepatic stellate cells correlated with an enhanced expression of proteins involved in uptake and storage of fatty acids (FA translocase CD36, Acyl-CoA synthetase 2) and retinol (cellular retinol binding protein type I, CRBP-I; lecithin:retinol acyltransferases, LRAT). The increased expression of CRBP-I and LRAT during hepatic stellate cells activation, both involved in retinol esterification, was in contrast with the simultaneous depletion of their typical lipid-vitamin A (vitA) reserves. Since hepatic stellate cells express high levels of peroxisome proliferator activated receptor beta (PPARbeta), which become further induced during transition into the activated phenotype, we investigated the potential role of PPARbeta in the regulation of these changes. Administration of L165041, a PPARbeta-specific agonist, further induced the expression of CD36, B-FABP, CRBP-I, and LRAT, whereas their expression was inhibited by antisense PPARbeta mRNA. PPARbeta-RXR dimers bound to CRBP-I promoter sequences. Our observations suggest that PPARbeta regulates the expression of these genes, and thus could play an important role in vitA storage. In vivo, we observed a striking association between the enhanced expression of PPARbeta and CRBP-I in activated myofibroblast-like hepatic stellate cells and the manifestation of vitA autofluorescent droplets in the fibrotic septa after injury with CCl4 or CCl4 in combination with retinol.

Cooperating pre-T-cell receptor and TCF-1-dependent signals ensure thymocyte survival.

Intrathymic T-cell maturation critically depends on the selective expansion of thymocytes expressing a functionally rearranged T-cell receptor (TCR) beta chain. In addition, TCR-independent signals also contribute to normal T-cell development. It is unclear whether and how signals from the 2 types of pathways are integrated. Here, we show that T-cell factor-1 (TCF-1), a nuclear effector of the canonical wingless/int (wnt)/catenin signaling pathway, ensures the survival of proliferating, pre-TCR(+) thymocytes. The survival of pre-TCR(+) thymocytes requires the presence of the N-terminal catenin-binding domain in TCF-1. This domain can bind the transcriptional coactivator beta-catenin and may also bind gamma-catenin (plakoglobin). However, in the absence of gamma-catenin, T-cell development is normal, supporting a role for beta-catenin. Signaling competent beta-catenin is present prior to and thus arises independently from pre-TCR signaling and does not substantially increase on pre-TCR signaling. In contrast, pre-TCR signaling significantly induces TCF-1 expression. This coincides with the activation of a wnt/catenin/TCF reporter transgene in vivo. Collectively, these data suggest that efficient TCF-dependent transcription requires that pre-TCR signaling induces TCF-1 expression, whereas wnt signals may provide the coactivator such as beta-catenin. The 2 pathways thus have to cooperate to ensure thymocyte survival at the pre-TCR stage.

Transgenic reexpression of GLUT1 or GLUT2 in pancreatic beta cells rescues GLUT2-null mice from early death and restores normal glucose-stimulated insulin secretion.

GLUT2-null mice are hyperglycemic, hypoinsulinemic, hyperglucagonemic, and glycosuric and die within the first 3 weeks of life. Their endocrine pancreas shows a loss of first phase glucose-stimulated insulin secretion (GSIS) and inverse alpha to beta cell ratio. Here we show that reexpression by transgenesis of either GLUT1 or GLUT2 in the pancreatic beta cells of these mice allowed mouse survival and breeding. The rescued mice had normal-fed glycemia but fasted hypoglycemia, glycosuria, and an elevated glucagon to insulin ratio. Glucose tolerance was, however, normal. In vivo, insulin secretion assessed following hyperglycemic clamps was normal. In vitro, islet perifusion studies revealed that first phase of insulin secretion was restored as well by GLUT1 or GLUT2, and this was accompanied by normalization of the glucose utilization rate. The ratio of pancreatic insulin to glucagon and volume densities of alpha to beta cells were, however, not corrected. These data demonstrate that 1) reexpression of GLUT1 or GLUT2 in beta cells is sufficient to rescue GLUT2-null mice from lethality, 2) GLUT1 as well as GLUT2 can restore normal GSIS, 3) restoration of GSIS does not correct the abnormal composition of the endocrine pancreas. Thus, normal GSIS does not depend on transporter affinity but on the rate of uptake at stimulatory glucose concentrations.

Polyene macrolide antifungal drugs trigger interleukin-1β secretion by activating the NLRP3 inflammasome.

The use of antimycotic drugs in fungal infections is based on the concept that they suppress fungal growth by a direct killing effect. However, amphotericin and nystatin have been reported to also trigger interleukin-1β (IL-1β) secretion in monocytes but the molecular mechanism is unknown. Here we report that only the polyene macrolides amphotericin B, nystatin, and natamycin but none of the tested azole antimycotic drugs induce significant IL-1β secretion in-vitro in dendritic cells isolated from C57BL/6 mouse bone marrow. IL-1β release depended on Toll-like receptor-mediated induction of pro-IL-1β as well as the NLRP3 inflammasome, its adaptor ASC, and caspase-1 for enzymatic cleavage of pro-IL-1β into its mature form. All three drugs induced potassium efflux from the cells as a known mechanism for NLRP3 activation but the P2X7 receptor was not required for this process. Natamycin-induced IL-1β secretion also involved phagocytosis, as cathepsin activation as described for crystal-induced IL-1β release. Together, the polyene macrolides amphotericin B, nystatin, and natamycin trigger IL-1β secretion by causing potassium efflux from which activates the NLRP3-ASC-caspase-1. We conclude that beyond their effects on fungal growth, these antifungal drugs directly activate the host's innate immunity.

Mineralocorticoid effects in the kidney: correlation between alphaENaC, GILZ, and Sgk-1 mRNA expression and urinary excretion of Na+ and K+.

Aldosterone exerts its effects through interactions with two types of binding sites, the mineralocorticoid (MR) and the glucocorticoid (GR) receptors. Although both receptors are known to be involved in the anti-natriuretic response to aldosterone, the mechanisms of signal transduction leading to modulation of electrolyte transport are not yet fully understood. This study measured the Na(+) and K(+) urinary excretion and the mRNA levels of three known aldosterone-induced transcripts, the serum and glucocorticoid-induced kinase (Sgk-1), the alpha subunit of the epithelial Na(+) channel (alphaENaC), and the glucocorticoid-induced-leucine-zipper protein (GILZ) in the whole kidney and in isolated cortical collecting tubules of adrenalectomized rats treated with low doses of aldosterone and/or dexamethasone. The resulting plasma concentrations of both steroids were close to 1 nmol/L. Aldosterone, given with or without dexamethasone, induced anti-natriuresis and kaliuresis, whereas dexamethasone alone did not. GILZ and alphaENaC transcripts were higher after treatment with either or both hormones, whereas the mRNA abundance of Sgk-1 was increased in the cortical collecting tubule by aldosterone but not by dexamethasone. We conclude the increased expression of Sgk-1 in the cortical collecting tubules is a primary event in the early antinatriuretic and kaliuretic responses to physiologic concentrations of aldosterone. Induction of alphaENaC and/or GILZ mRNAs may play a permissive role in the enhancement of the early and/or late responses; these effects may be necessary for a full response but do not by themselves promote early changes in urinary Na(+) and K(+) excretion.

The endocrine disruptor monoethyl-hexyl-phthalate is a selective peroxisome proliferator-activated receptor gamma modulator that promotes adipogenesis.

The ability of pollutants to affect human health is a major concern, justified by the wide demonstration that reproductive functions are altered by endocrine disrupting chemicals. The definition of endocrine disruption is today extended to broader endocrine regulations, and includes activation of metabolic sensors, such as the peroxisome proliferator-activated receptors (PPARs). Toxicology approaches have demonstrated that phthalate plasticizers can directly influence PPAR activity. What is now missing is a detailed molecular understanding of the fundamental basis of endocrine disrupting chemical interference with PPAR signaling. We thus performed structural and functional analyses that demonstrate how monoethyl-hexyl-phthalate (MEHP) directly activates PPARgamma and promotes adipogenesis, albeit to a lower extent than the full agonist rosiglitazone. Importantly, we demonstrate that MEHP induces a selective activation of different PPARgamma target genes. Chromatin immunoprecipitation and fluorescence microscopy in living cells reveal that this selective activity correlates with the recruitment of a specific subset of PPARgamma coregulators that includes Med1 and PGC-1alpha, but not p300 and SRC-1. These results highlight some key mechanisms in metabolic disruption but are also instrumental in the context of selective PPAR modulation, a promising field for new therapeutic development based on PPAR modulation.

Differential effects of GABAB receptor subtypes, {gamma}-hydroxybutyric Acid, and Baclofen on EEG activity and sleep regulation.

The role of GABA(B) receptors in sleep is still poorly understood. GHB (γ-hydroxybutyric acid) targets these receptors and is the only drug approved to treat the sleep disorder narcolepsy. GABA(B) receptors are obligate dimers comprised of the GABA(B2) subunit and either one of the two GABA(B1) subunit isoforms, GABA(B1a) and GABA(B1b). To better understand the role of GABA(B) receptors in sleep regulation, we performed electroencephalogram (EEG) recordings in mice devoid of functional GABA(B) receptors (1(-/-) and 2(-/-)) or lacking one of the subunit 1 isoforms (1a(-/-) and 1b(-/-)). The distribution of sleep over the day was profoundly altered in 1(-/-) and 2(-/-) mice, suggesting a role for GABA(B) receptors in the circadian organization of sleep. Several other sleep and EEG phenotypes pointed to a more prominent role for GABA(B1a) compared with the GABA(B1b) isoform. Moreover, we found that GABA(B1a) protects against the spontaneous seizure activity observed in 1(-/-) and 2(-/-) mice. We also evaluated the effects of the GHB-prodrug GBL (γ-butyrolactone) and of baclofen (BAC), a high-affinity GABA(B) receptor agonist. Both drugs induced a state distinct from physiological sleep that was not observed in 1(-/-) and 2(-/-) mice. Subsequent sleep was not affected by GBL whereas BAC was followed by a delayed hypersomnia even in 1(-/-) and 2(-/-) mice. The differential effects of GBL and BAC might be attributed to differences in GABA(B)-receptor affinity. These results also indicate that all GBL effects are mediated through GABA(B) receptors, although these receptors do not seem to be involved in mediating the BAC-induced hypersomnia.

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration.

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.

Transcriptional repression of peroxisome proliferator-activated receptor beta/delta in murine keratinocytes by CCAAT/enhancer-binding proteins.

The roles of peroxisome proliferator-activated receptors (PPARs) and CCAAT/enhancer-binding proteins (C/EBPs) in keratinocyte and sebocyte differentiation suggest that both families of transcription factors closely interact in the skin. Initial characterization of the mouse PPARbeta promoter revealed an AP-1 site that is crucial for the regulation of PPARbeta expression in response to inflammatory cytokines in the skin. We now present evidence for a novel regulatory mechanism of the expression of the PPARbeta gene by which two members of the C/EBP family of transcription factors inhibit its basal promoter activity in mouse keratinocytes. We first demonstrate that C/EBPalpha and C/EBPbeta, but not C/EBPdelta, inhibit the expression of PPARbeta through the recruitment of a transcriptional repressor complex containing HDAC-1 to a specific C/EBP binding site on the PPARbeta promoter. Consistent with this repression, the expression patterns of PPARbeta and C/EBPs are mutually exclusive in keratinocytes of the interfollicular epidermis and hair follicles in mouse developing skin. This work reveals the importance of the regulatory interplay between PPARbeta and C/EBP transcription factors in the control of proliferation and differentiation in this organ. Such insights are crucial for the understanding of the molecular control regulating the balance between proliferation and differentiation in many cell types including keratinocytes.

Effects of salbutamol on the contractile properties of human skeletal muscle before and after fatigue.

AIM: The study examined the effects of an oral acute administration of the beta2-agonist salbutamol (Sal) (6 mg) vs. placebo on muscle strength and fatigability in 12 non-asthmatic recreational male athletes in a randomized double-blind protocol. METHODS: Contractile properties of the right quadriceps muscle were measured during electrical stimulations, i.e. twitch, 1-s pulse trains at 20 (P(20) ) and 80 Hz (P(80) ) and during maximal voluntary isometric contraction (MVIC) before (PRE) and after (POST) a fatigue-producing protocol set by an electromyostimulation (30 contractions, frequency: 75 Hz, on-off ratio: 6.25-20s). In addition, the level of muscle voluntary activation was measured. RESULTS: In PRE and POST conditions, the peak torque (PT) of twitch, P(80) and MVIC were not modified by the treatment. The PT in POST P(20) was slightly, although not significantly, less affected by fatigue in Sal compared with placebo condition. Moreover, twitch half-relaxation time at PRE was smaller under Sal than under placebo (P < 0.05). No significant changes in the degree of voluntary activation were observed with Sal treatment in PRE or POST condition. CONCLUSION: Although these findings did not exclude completely an effect of Sal on peripheral factors of human skeletal muscle, oral acute administration of the beta2-agonist Sal seems to be without any relevant ergogenic effect on muscle contractility and fatigability in non-asthmatic recreational male athletes.

Calmodulin kinase IV: expression and function during rat brain development.

The expression of calmodulin kinase IV (CaMKIV) can be induced by the thyroid hormone T3 in a time- and concentration-dependent manner at a very early stage of brain differentiation using a fetal rat telencephalon primary cell culture system which can grow and differentiate under chemically defined conditions (Krebs et al. (1996) J. Biol. Chem. 271, 11055-11058). After the induction of CaMKIV by T3 we examined the influence of prolonged absence of T3 from the culture medium on the expression of CaMKIV. We could demonstrate that after the T3-dependent induction of CaMKIV, omission of the hormone, even for 8 days, from the medium did not downregulate the expression of CaMKIV indicating that different regulatory mechanisms became important for the expression of the enzyme. We further showed that CaMKIV could be involved in the Ca(2+) -dependent expression of the immediate early gene c-fos, probably via phosphorylation of the transcription factor CREB. Convergence of signal transduction pathways on this transcription factor by using different protein kinases may explain the importance of CREB for the regulation of different cellular processes.