The aetiology of Crohn's disease (CD) involves disorders in host genetic factors and intestinal microbiota. Adherent-invasive Escherichia coli (AIEC) are receiving increased attention because in studies of mucosa-associated microbiota, they are more prevalent in CD patients than in healthy subjects. AIEC are associated both with ileal and colonic disease phenotypes. In this study, we reported a protease called Vat-AIEC from AIEC that favours the mucosa colonization. The deletion of the Vat-AIEC-encoding gene resulted in an adhesion-impaired phenotype in vitro and affected the colonization of bacteria in contact with intestinal epithelial cells in a murine intestinal loop model, and also their gut colonization in vivo. Furthermore, unlike LF82Δvat-AIEC, wild-type AIEC reference strain LF82 was able to penetrate a mucus column extensively and promoted the degradation of mucins and a decrease in mucus viscosity. Vat-AIEC transcription was stimulated by several chemical conditions found in the ileum environment. Finally, the screening of E. coli strains isolated from CD patients revealed a preferential vat-AIEC association with AIEC strains belonging to the B2 phylogroup. Overall, this study revealed a new component of AIEC virulence that might favour their implantation in the gut of CD patients.
In different animal models, photoreceptor degeneration was correlated to an abnormal increase in cGMP concentration. The cGMP-induced photoreceptor toxicity was demonstrated by applying the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine on retinal explants. To assess the role of cGMP-gated channels in this cGMP toxicity, the Ca(2+) channel blockers verapamil and L- and D-diltiazem, which block cGMP-gated channels with different efficacies, were applied to in vitro animal models of photoreceptor degeneration. These models included: (i) adult rat retinal explants incubated with zaprinast, a more specific inhibitor of the rod phosphodiesterase than 3-isobutyl-1-methylxanthine and (ii) rd mouse retinal explants. Photoreceptor apoptosis was assessed by terminal dUTP nick end labelling and caspase 3 activation. Effects of the blockers on the synaptic rod Ca(2+) channels were measured by patch-clamp recording. In the zaprinast-induced photoreceptor degeneration model, both diltiazem isomers rescued photoreceptors whereas verapamil had no influence. Their neuroprotective efficacy was correlated to their inhibition of cGMP-gated channels (l-diltiazem>d-diltiazem>verapamil=0). In contrast, all three Ca(2+) channel blockers suppressed rod Ca(2+) channel currents similarly. This suppression of the currents by the diltiazem isomers was very weak (16.5%) at the neuroprotective concentration (10 microm). In rd retinal explants, both diltiazem isomers also slowed down rod degeneration in contrast to verapamil. L-diltiazem exhibited this effect at concentrations ranging from 1 to 20 microm. This study further supports the photoreceptor neuroprotection by diltiazem particularly in the rd mouse retina, whereas the absence of neuroprotection by verapamil further suggests the role of cGMP-gated channel activation in the induction of photoreceptor degeneration.
. Voltage-gated channels and calcium homeostasis in mammalian rod photoreceptors. J Neurophysiol 93: 1468 -1475, 2005. First published October 13, 2004; doi:10.1152/jn.00874.2004. Recent reports on rod photoreceptor neuroprotection by Ca 2ϩ channel blockers have pointed out the need to assess the effect of these blockers on mammalian rods. However, in mammals, rod electrophysiological characterization has been hampered by the small size of these photoreceptors, which were instead extensively studied in nonmammalian vertebrates. To further characterize ionic conductances and to assess the pharmacology of Ca 2ϩ channels in mammalian rods, freshly dissociated pig rod photoreceptors were recorded with the whole cell patch-clamp technique. Rod cells expressed 1) a hyperpolarization-activated inward-rectifying conductance (I h ) sensitive to external Cs ϩ ; 2) a sustained outward K ϩ current (I K ) sensitive to tetraethylammonium; 3) a sustained voltage-gated Ca 2ϩ current (I Ca ) sensitive to benzothiazepine (diltiazem) and phenylalkylamine (verapamil) derivatives; 4) a Ca 2ϩ -activated Cl Ϫ current (I Cl(Ca) ); and 5) a plasma membrane Ca 2ϩ -ATPase. The Ca 2ϩ current showed a range of activation from positive potentials to -60 mV with a maximum between -30 and -20 mV. In contrast to other L-type Ca 2ϩ channels, rod Ca 2ϩ channels were blocked at similar and relatively high concentrations by the diltiazem isomers and verapamil. The biphasic dose-response for D-diltiazem confirmed the low sensitivity of Ca 2ϩ channels for the molecule. The ATPase, which was localized at the axon terminal, was found to contribute to Ca 2ϩ extrusion. These results suggest that the electrophysiological features of rod photoreceptors had been preserved during evolution from nonmammalian vertebrates to mammals. This work indicates further that mammalian rods express nonclassic L-type Ca 2ϩ channels, showing a low sensitivity to the diltiazem isomers used in neuroprotective studies.
Among retinal macular diseases, the juvenile recessive Stargardt disease and the age-related degenerative disease arise from carbonyl and oxidative stresses (COS). Both stresses originate from an accumulation of all-trans-retinal (atRAL) and are involved in bisretinoid formation by condensation of atRAL with phosphatidylethanolamine (carbonyl stress) in the photoreceptor and its transformation into lipofuscin bisretinoids (oxidative stress) in the retinal pigment epithelium (RPE). As atRAL and bisretinoid accumulation contribute to RPE and photoreceptor cell death, our goal is to select powerful chemical inhibitors of COS. Here, we describe that phloroglucinol, a natural phenolic compound having anti-COS properties, protects both rat RPE and mouse photoreceptor primary cultures from atRAL-induced cell death and reduces hydrogen peroxide (H 2 O 2 )-induced damage in RPE in a dose-dependent manner. Mechanistic analyses demonstrate that the protective effect encompasses decrease in atRAL-induced intracellular reactive oxygen species and free atRAL levels. Moreover, we show that phloroglucinol reacts with atRAL to form a chromene adduct which prevents bisretinoid A2E synthesis in vitro. Taken together, these data show that the protective effect of phloroglucinol correlates with its ability to trap atRAL and to prevent its further transformation into deleterious bisretinoids. Phloroglucinol might be a good basis to develop efficient therapeutic derivatives in the treatment of retinal macular diseases.
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Our study shows that EGCG pretreatment can protect primary rat RPE cells from H(2)O(2)-induced death. This suggests potential effect of EGCG in the prevention of retinal diseases associated with H(2)O(2)-induced oxidative stress.
In vertebrates, visual perception occurs in the rod and cone photoreceptor outer segments (POS) through visual pigments, G protein-coupled receptors (rhodopsin and cone opsins) consisting of the apoprotein opsin and a prosthetic group, the vitamin A-derived chromophore 11-cis-retinal (11cRAL). 1 The light isomerizes 11cRAL to trans configuration (atRAL) and subsequently elicits the conformational transition within the opsin proteins, followed by the activation of the phototransduction cascade. After photoisomerization of
These results are consistent with previous studies suggesting that Ca(2+) channel activation contributes to retinal cell death following either glutamate excitotoxicity or retinal ischemia. Under both conditions, the L-type Ca(2+) channel blocker, diltiazem, can limit cell death. These results extend the potential application of diltiazem in retinal neuroprotection to retinal pathologies involving glutamate excitotoxicity and ischemia.
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