Function of TRADD in tumor necrosis factor receptor 1 signaling and in TRIF-dependent inflammatory responses.

Tumor necrosis factor receptor 1 (TNFR1) and Toll-like receptors (TLRs) regulate immune and inflammatory responses. Here we show that the TNFR1-associated death domain protein (TRADD) is critical in TNFR1, TLR3 and TLR4 signaling. TRADD deficiency abrogated TNF-induced apoptosis, prevented recruitment of the ubiquitin ligase TRAF2 and ubiquitination of the adaptor RIP1 in the TNFR1 signaling complex, and considerably inhibited but did not completely abolish activation of the transcription factor NF-kappaB and mitogen-activated protein kinases 'downstream' of TNFR1. TRIF-dependent cytokine production induced by the synthetic double-stranded RNA poly(I:C) and lipopolysaccharide was lower in TRADD-deficient mice than in wild-type mice. Moreover, TRADD deficiency inhibited poly(I:C)-mediated RIP1 ubiquitination and activation of NF-kappaB and mitogen-activated protein kinase signaling in fibroblasts but not in bone marrow macrophages. Thus, TRADD is an essential component of TNFR1 signaling and has a critical but apparently cell type-specific function in TRIF-dependent TLR responses.

Intrinsically photosensitive retinal ganglion cells.

The recent discovery of melanopsin-expressing retinal ganglion cells that mediate the pupil light reflex has provided new insights into how the pupil responds to different properties of light. These ganglion cells are unique in their ability to transduce light into electrical energy. There are parallels between the electrophysiologic behavior of these cells in primates and the clinical pupil response to chromatic stimuli. Under photopic conditions, a red light stimulus produces a pupil constriction mediated predominantly by cone input via trans-synaptic activation of melanopsin-expressing retinal ganglion cells, whereas a blue light stimulus at high intensity produces a steady-state pupil constriction mediated primarily by direct intrinsic photoactivation of the melanopsin-expressing ganglion cells. Preliminary data in humans suggest that under photopic conditions, cones primarily drive the transient phase of the pupil light reflex, whereas intrinsic activation of the melanopsin-expressing ganglion cells contributes heavily to sustained pupil constriction. The use of chromatic light stimuli to elicit transient and sustained pupil light reflexes may become a clinical pupil test that allows differentiation between disorders affecting photoreceptors and those affecting retinal ganglion cells.

IROme, a New High-Throughput Molecular Tool for the Diagnosis of Inherited Retinal Dystrophies.

The molecular diagnosis of retinal dystrophies is difficult because of the very important number of genes implicated and is rarely helped by genotype-phenotype correlations. This prompted us to develop IROme, a custom designed in solution-based targeted exon capture assay (SeqCap EZ Choice library, Roche NimbleGen) for 60 retinitis pigmentosa-linked genes and three candidate genes (942 exons). Pyrosequencing was performed on a Roche 454 GS Junior benchtop high-throughput sequencing platform. In total, 23 patients affected by retinitis pigmentosa were analyzed. Per patient, 39.6 Mb were generated, and 1111 sequence variants were detected on average, at a median coverage of 17-fold. After data filtering and sequence variant prioritization, disease-causing mutations were identified in ABCA4, CNGB1, GUCY2D, PROM1, PRPF8, PRPF31, PRPH2, RHO, RP2, and TULP1 for twelve patients (55%), ten mutations having never been reported previously. Potential mutations were identified in 5 additional patients, and in only 6 patients no molecular diagnosis could be established (26%). In conclusion, targeted exon capture and next-generation sequencing are a valuable and efficient approach to identify disease-causing sequence variants in retinal dystrophies.

Anti-vascular endothelial growth factor acts on retinal microglia/macrophage activation in a rat model of ocular inflammation.

PURPOSE: To evaluate whether anti-vascular endothelial growth factor (VEGF) neutralizing antibodies injected in the vitreous of rat eyes influence retinal microglia and macrophage activation. To dissociate the effect of anti-VEGF on microglia and macrophages subsequent to its antiangiogenic effect, we chose a model of acute intraocular inflammation. METHODS: Lewis rats were challenged with systemic lipopolysaccharide (LPS) injection and concomitantly received 5 µl of rat anti-VEGF-neutralizing antibody (1.5 mg/ml) in the vitreous. Rat immunoglobulin G (IgG) isotype was used as the control. The effect of anti-VEGF was evaluated at 24 and 48 h clinically (uveitis scores), biologically (cytokine multiplex analysis in ocular media), and histologically (inflammatory cell counts on eye sections). Microglia and macrophages were immunodetected with ionized calcium-binding adaptor molecule 1 (IBA1) staining and counted based on their differential shapes (round amoeboid or ramified dendritiform) on sections and flatmounted retinas using confocal imaging and automatic quantification. Activation of microglia was also evaluated with inducible nitric oxide synthase (iNOS) and IBA1 coimmunostaining. Coimmunolocalization of VEGF receptor 1 and 2 (VEGF-R1 and R2) with IBA1 was performed on eye sections with or without anti-VEGF treatment. RESULTS: Neutralizing rat anti-VEGF antibodies significantly decreased ocular VEGF levels but did not decrease the endotoxin-induced uveitis (EIU) clinical score or the number of infiltrating cells and cytokines in ocular media (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α, and monocyte chemoattractant protein [MCP]-1). Eyes treated with anti-VEGF showed a significantly decreased number of activated microglia and macrophages in the retina and the choroid and decreased iNOS-positive microglia. IBA1-positive cells expressed VEGF-R1 and R2 in the inflamed retina. CONCLUSIONS: Microglia and macrophages expressed VEGF receptors, and intravitreous anti-VEGF influenced the microglia and macrophage activation state. Taking into account that anti-VEGF drugs are repeatedly injected in the vitreous of patients with retinal diseases, part of their effects could result from unsuspected modulation of the microglia activation state. This should be further studied in other ocular pathogenic conditions and human pathology.

Alteration in neuromuscular function after a 5 km running time trial

The aim of this study was to characterize the effect of a 5 km running time trial on the neuromuscular properties of the plantar flexors. Eleven well-trained triathletes performed a series of neuromuscular tests before and immediately after the run on a 200 m indoor track. Muscle activation (twitch interpolation) and normalized EMG activity were assessed during maximal voluntary contraction (MVC) of plantar flexors. Maximal soleus H-reflexes and M-waves were evoked at rest (i.e. H (MAX) and M (MAX), respectively) and during MVC (i.e. H (SUP) and M (SUP), respectively). MVC significantly declined (-27%; P < 0.001) after the run, due to decrease in muscle activation (-8%; P < 0.05) and M (MAX)-normalized EMG activity (-13%; P < 0.05). Significant reductions in M-wave amplitudes (M (MAX): -13% and M (SUP): -16%; P < 0.05) as well as H (MAX)/M (MAX) (-37%; P < 0.01) and H (SUP)/M (SUP) (-25%; P < 0.05) ratios occurred with fatigue. Following exercise, the single twitch was characterized by lower peak torque (-16%; P < 0.001) as well as shorter contraction (-19%; P < 0.001) and half-relaxation (-24%; P < 0.001) times. In conclusion, the reduction in plantar flexors strength induced by a 5 km running time trial is caused by peripheral adjustments, which are attributable to a failure of the neuromuscular transmission and excitation-contraction coupling. Fatigue also decreased the magnitude of efferent motor outflow from spinal motor neurons to the plantar flexors and part of this suboptimal neural drive is the result of an inhibition of soleus motoneuron pool reflex excitability.

Insulin and NPY pathways and the control of GnRH function and puberty onset.

Energy balance exerts a critical influence on reproductive function. Leptin and insulin are among the metabolic factors signaling the nutritional status of an individual to the hypothalamus, and their role in the overall modulation of the activity of GnRH neurons is increasingly recognized. As such, they participate to a more generalized phenomenon: the signaling of peripheral metabolic changes to the central nervous system. The physiological importance that the interactions occurring between peripheral metabolic factors and the central nervous system bear for the control of food intake is increasingly recognized. The central mechanisms implicated are the focus of attention of very many research groups worldwide. We review here the experimental data that suggest that similar mechanisms are at play for the metabolic control of the neuroendocrine reproductive function. It is appearing that metabolic signals are integrated at the levels of first-order neurons equipped with the proper receptors, ant that these neurons send their signals towards hypothalamic GnRH neurons which constitute the integrative element of this network.

Impaired left ventricular function as a predictive factor for mid-term survival in octogenarians after primary coronary artery bypass surgery.

BACKGROUND: The impact of preoperative impaired left ventricular ejection fraction (EF) in octogenarians following coronary bypass surgery on short-term survival was evaluated in this study. METHODS: A total of 147 octogenarians (mean age 82.1 ± 1.9 years) with coronary artery diseases underwent elective coronary artery bypass graft between January 2000 and December 2009. Patients were stratified into: Group I (n = 59) with EF >50%, Group II (n = 59) with 50% > EF >30% and in Group III (n = 29) with 30% > EF. RESULTS: There was no difference among the three groups regarding incidence of COPD, renal failure, congestive heart failure, diabetes, and preoperative cerebrovascular events. Postoperative atrial fibrillation was the sole independent predictive factor for in-hospital mortality (odds ratio (OR), 18.1); this was 8.5% in Group I, 15.3% in Group II and 10.3% in Group III. Independent predictive factors for mortality during follow up were: decrease of EF during follow-up for more that 5% (OR, 5.2), usage of left internal mammary artery as free graft (OR, 18.1), and EF in follow-up lower than 40% (OR, 4.8). CONCLUSIONS: The results herein suggest acceptable in-hospital as well short-term mortality in octogenarians with impaired EF following coronary artery bypass grafting (CABG) and are comparable to recent literature where the mortality of younger patients was up to 15% and short-term mortality up to 40%, respectively. Accordingly, we can also state that in an octogenarian cohort with impaired EF, CABG is a viable treatment with acceptable mortality.

Dysfonction endothéliale et risque cardiovasculaire : l'exercice protège la fonction endothéliale et prévient la maladie cardiovasculaire [Endothelial dysfunction and cardiovascular risk : xercise protects endothelial function and prevents cardiovascular disease]

Objectifs : Décrire les caractéristiques de la dysfonction endothéliale associée au risque cardiovasculaire et expliciter les mécanismes biologiques par lesquels l'exercice stimule et/ou restaure la fonction endothéliale. Actualités : La fonction endothéliale, via les effets vasculoprotecteurs du monoxyde d'azote (NO), préserve la santé cardiovasculaire. Le dysfonctionnement endothélial est un facteur prédictif de la survenue des événements cardiovasculaires. L'endothélium est donc un organe cible préventif et thérapeutique prioritaire pour diminuer le risque cardiovasculaire. Perspectives : Les études épidémiologiques mettent en évidence les bienfaits de l'exercice régulier sur la fonction endothéliale, via une action endothéliale directe. L'approche expérimentale permet aujourd'hui de mieux cerner les mécanismes biologiques protecteurs de l'exercice. L'exercice, via l'élévation des forces de cisaillement, protège et/ou normalise la fonction endothéliale en augmentant la biodisponibilité en NO soit par une stimulation de la production de NO et/ou, soit par une augmentation des défenses antioxydantes et/ou une atténuation des enzymes prooxydantes. Conclusion : La connaissance des mécanismes biologiques protecteurs de l'exercice doit permettre d'encourager la pratique d'un exercice régulier par tous pour prévenir et réduire la mortalité cardiovasculaire.

Gain-of-function mutations in PDR1, a regulator of antifungal drug resistance in Candida glabrata, control adherence to host cells.

Candida glabrata is an emerging opportunistic pathogen that is known to develop resistance to azole drugs due to increased drug efflux. The mechanism consists of CgPDR1-mediated upregulation of ATP-binding cassette transporters. A range of gain-of-function (GOF) mutations in CgPDR1 have been found to lead not only to azole resistance but also to enhanced virulence. This implicates CgPDR1 in the regulation of the interaction of C. glabrata with the host. To identify specific CgPDR1-regulated steps of the host-pathogen interaction, we investigated in this work the interaction of selected CgPDR1 GOF mutants with murine bone marrow-derived macrophages and human acute monocytic leukemia cell line (THP-1)-derived macrophages, as well as different epithelial cell lines. GOF mutations in CgPDR1 did not influence survival and replication within macrophages following phagocytosis but led to decreased adherence to and uptake by macrophages. This may allow evasion from the host's innate cellular immune response. The interaction with epithelial cells revealed an opposite trend, suggesting that GOF mutations in CgPDR1 may favor epithelial colonization of the host by C. glabrata through increased adherence to epithelial cell layers. These data reveal that GOF mutations in CgPDR1 modulate the interaction with host cells in ways that may contribute to increased virulence.

Placental growth factor contributes to micro-vascular abnormalization and blood-retinal barrier breakdown in diabetic retinopathy.

OBJECTIVE: There are controversies regarding the pro-angiogenic activity of placental growth factor (PGF) in diabetic retinopathy (DR). For a better understanding of its role on the retina, we have evaluated the effect of a sustained PGF over-expression in rat ocular media, using ciliary muscle electrotransfer (ET) of a plasmid encoding rat PGF-1 (pVAX2-rPGF-1). MATERIALS AND METHODS: pVAX2-rPGF-1 ET in the ciliary muscle (200 V/cm) was achieved in non diabetic and diabetic rat eyes. Control eyes received saline or naked plasmid ET. Clinical follow up was carried out over three months using slit lamp examination and fluorescein angiography. After the control of rPGF-1 expression, PGF-induced effects on retinal vasculature and on the blood-external barrier were evaluated respectively by lectin and occludin staining on flat-mounts. Ocular structures were visualized through histological analysis. RESULTS: After fifteen days of rPGF-1 over-expression in normal eyes, tortuous and dilated capillaries were observed. At one month, microaneurysms and moderate vascular sprouts were detected in mid retinal periphery in vivo and on retinal flat-mounts. At later stages, retinal pigmented epithelial cells demonstrated morphological abnormalities and junction ruptures. In diabetic retinas, PGF expression rose between 2 and 5 months, and, one month after ET, rPGF-1 over-expression induced glial activation and proliferation. CONCLUSION: This is the first demonstration that sustained intraocular PGF production induces vascular and retinal changes similar to those observed in the early stages of diabetic retinopathy. PGF and its receptor Flt-1 may therefore be looked upon as a potential regulatory target at this stage of the disease.

Light-induced retinal degeneration correlates with changes in iron metabolism gene expression, ferritin level, and aging.

PURPOSE: Retinal degeneration is associated with iron accumulation in several rodent models in which iron-regulating proteins are impaired. Oxidative stress is catalyzed by unbound iron. METHODS: The role of the heavy chain of ferritin, which sequesters iron, in regulating the thickness of the photoreceptor nuclear layer in the 4- and 16-month-old wild-type H ferritin (HFt(+/+)) and heterozygous H ferritin (HFt(+/-)) mice was investigated, before and 12 days after exposure to 13,000-lux light for 24 hours. The regulation of gene expression of the various proteins involved in iron homeostasis, such as transferrin, transferrin receptor, hephaestin, ferroportin, iron regulatory proteins 1 and 2, hepcidin, ceruloplasmin, and heme-oxygenase 1, was analyzed by quantitative (q)RT-PCR during exposure (2, 12, and 24 hours) and 24 hours after 1 day of exposure in the 4-month-old HFt(+/+) and HFt(+/-) mouse retinas. RESULTS: Retinal degeneration in the 4-month-old HFt(+/-) mice was more extensive than in the HFt(+/+) mice. Yet, it was more extensive in both of the 16-month-old mouse groups, revealing the combined effect of age and excessive light. Injury caused by excessive light modified the temporal gene expression of iron-regulating proteins similarly in the HFt(+/-) and HFt(+/+) mice. CONCLUSIONS: Loss of one allele of H ferritin appears to increase light-induced degeneration. This study highlighted that oxidative stress related to light-induced injury is associated with major changes in gene expression of iron metabolism proteins.

Domains of p85cdc10 required for function of the fission yeast DSC-1 factor.

p85cdc10 is a component of the S.pombe DSC-1 complex, which is thought to mediate periodic transcription of genes in late G1. In order to understand the role of p85cdc10 in the function of this complex, we have analysed which domains of p85cdc10 are required for biological activity and the formation of a stable DSC-1 complex in vitro, both in cdc10 temperature sensitive and null backgrounds. No DSC-1 activity is found in the absence of p85cdc10 and the activity of the complex is reduced or absent in all cdc10ts mutants tested. Full biological activity and rescue of a cdc10::ura4+ null allele requires the N-terminal domain, the cdc10/SWI6 repeats and the helical C-terminal region. In the absence of p85cdc10, both the C-terminal and cdc10/SWI6 repeat domains are required for DSC-1 activity in vitro. In a cdc10ts background, rescue of DSC-1 activity and complementation of mutants, requires only expression of the C-terminal domain, though the presence of the cdc10/SWI6 motifs enhances its activity. The N-terminal domain, alone, or in combination with the cdc10/SWI6 motifs, does not have biological activity, and does not restore DSC-1 activity. We conclude that both the C-terminal domain of p85cdc10 is critical for formation of the DSC-1 complex and that the cdc10/SWI6 motifs also play a role, perhaps by stabilizing the complex. Our data also suggest that the S.pombe DSC-1 complex contains more than one molecule of p85cdc10.

The rationale of retinal endovascular fibrinolysis in the treatment of retinal vein occlusion : from experimental data to clinical application.

PURPOSE: : We describe a retinal endovascular fibrinolysis technique to directly reperfuse experimentally occluded retinal veins using a simple micropipette. METHODS: : Retinal vein occlusion was photochemically induced in 12 eyes of 12 minipigs: after intravenous injection of 10% fluorescein (1-mL bolus), the targeted retinal vein segment was exposed to thrombin (50 units) and to Argon laser (100-200 mW) through a pars plana approach. A beveled micropipette with a 30-μm-diameter sharp edge was used for micropuncture of the occluded vein and endovascular microinjection of tissue plasminogen activator (50 μg/mL) in 11 eyes. In one control eye, balanced salt solution was injected. The lesion site was examined histologically. RESULTS: : Retinal vein occlusion was achieved in all cases. Endovascular microinjection of tissue plasminogen activator or balanced salt solution led to reperfusion of the occluded retinal vein in all cases. Indicative of successful reperfusion were the following: continuous endovascular flow, unaffected collateral circulation, no optic disk ischemia, and no venous wall bleeding. However, balanced salt solution injection was accompanied by thrombus formation at the punctured site, whereas no thrombus was observed with tissue plasminogen activator injection. CONCLUSION: : Retinal endovascular fibrinolysis constitutes an efficient method of micropuncture and reperfusion of an experimentally occluded retinal vein. Thrombus formation at the punctured site can be prevented by injection of tissue plasminogen activator.

Estimation de la fonction rénale par l'equation MDRD: intérêt et limites pour l'adaptation des doses de médicaments [Renal function estimation by MDRD equation: interest and limitations for drug dosing].

The MDRD (Modification of diet in renal disease) equation enables glomerular filtration rate (GFR) estimation from serum creatinine only. Thus, the laboratory can report an estimated GFR (eGFR) with each serum creatinine assessment, increasing therefore the recognition of renal failure. Predictive performance of MDRD equation is better for GFR < 60 ml/min/1,73 m2. A normal or near-normal renal function is often underestimated by this equation. Overall, MDRD provides more reliable estimations of renal function than the Cockcroft-Gault (C-G) formula, but both lack precision. MDRD is not superior to C-G for drug dosing. Being adjusted to 1,73 m2, MDRD eGFR has to be back adjusted to the patient's body surface area for drug dosing. Besides, C-G has the advantage of a greater simplicity and a longer use.