Small Extracellular Vesicles and Oxidative Pathophysiological Mechanisms in Retinal Degenerative Diseases.

This review focuses on the role of small extracellular vesicles in the pathophysiological mechanisms of retinal degenerative diseases. Many of these mechanisms are related to or modulated by the oxidative burden of retinal cells. It has been recently demonstrated that cellular communication in the retina involves extracellular vesicles and that their rate of release and cargo features might be affected by the cellular environment, and in some instances, they might also be mediated by autophagy. The fate of these vesicles is diverse: they could end up in circulation being used as markers, or target neighbor cells modulating gene and protein expression, or eventually, in angiogenesis. Neovascularization in the retina promotes vision loss in diseases such as diabetic retinopathy and age-related macular degeneration. The importance of micro RNAs, either as small extracellular vesicles' cargo or free circulating, in the regulation of retinal angiogenesis is also discussed.

JNK signalling is necessary for a Wnt- and stem cell-dependent regeneration programme.

Regeneration involves the integration of new and old tissues in the context of an adult life history. It is clear that the core conserved signalling pathways that orchestrate development also play central roles in regeneration, and further study of conserved signalling pathways is required. Here we have studied the role of the conserved JNK signalling cascade during planarian regeneration. Abrogation of JNK signalling by RNAi or pharmacological inhibition blocks posterior regeneration and animals fail to express posterior markers. While the early injury-induced expression of polarity markers is unaffected, the later stem cell-dependent phase of posterior Wnt expression is not established. This defect can be rescued by overactivation of the Hh or Wnt signalling pathway to promote posterior Wnt activity. Together, our data suggest that JNK signalling is required to establish stem cell-dependent Wnt expression after posterior injury. Given that Jun is known to be required in vertebrates for the expression of Wnt and Wnt target genes, we propose that this interaction may be conserved and is an instructive part of planarian posterior regeneration.

PBX/extradenticle is required to re-establish axial structures and polarity during planarian regeneration.

Recent advances in a number of systems suggest many genes involved in orchestrating regeneration are redeployed from similar processes in development, with others being novel to the regeneration process in particular lineages. Of particular importance will be understanding the architecture of regenerative genetic regulatory networks and whether they are conserved across broad phylogenetic distances. Here, we describe the role of the conserved TALE class protein PBX/Extradenticle in planarians, a representative member of the Lophotrocozoa. PBX/Extradenticle proteins play central roles in both embryonic and post-embryonic developmental patterning in both vertebrates and insects, and we demonstrate a broad requirement during planarian regeneration. We observe that Smed-pbx has pleiotropic functions during regeneration, with a primary role in patterning the anterior-posterior (AP) axis and AP polarity. Smed-pbx is required for expression of polarity determinants notum and wnt1 and for correct patterning of the structures polarized along the AP axis, such as the brain, pharynx and gut. Overall, our data suggest that Smed-pbx functions as a central integrator of positional information to drive patterning of regeneration along the body axis.

Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation.

Planarian adult stem cells (pASCs) or neoblasts represent an ideal system to study the evolution of stem cells and pluripotency as they underpin an unrivaled capacity for regeneration. We wish to understand the control of differentiation and pluripotency in pASCs and to understand how conserved, convergent or divergent these mechanisms are across the Bilateria. Here we show the planarian methyl-CpG Binding Domain 2/3 (mbd2/3) gene is required for pASC differentiation during regeneration and tissue homeostasis. The genome does not have detectable levels of 5-methylcytosine (5(m)C) and we find no role for a potential DNA methylase. We conclude that MBD proteins may have had an ancient role in broadly controlling animal stem cell pluripotency, but that DNA methylation is not involved in planarian stem cell differentiation.

Association of carbohydrate 125 antigen with sepsis mortality in critical patients. / Asociación del antígeno carbohidrato 125 con la mortalidad por sepsis en pacientes críticos.

INTRODUCTION: The increased synthesis of CA125 in mesothelial cells is connected with pathophysiological processes, also present in sepsis, that link inflammation with systemic congestion. We propose to evaluate serum levels of this biomarker in patients with sepsis and to study its association with the severity and evolution of the disease. METHODS: Longitudinal retrospective observational study, which included 126 patients admitted to an Intensive Care Unit with sepsis criteria. The main variables analyzed were: CA125 values for 7 days, the variation of its levels according to the source of infection (abdominal, pulmonary, nephrourinary and others), sepsis, septic shock, APACHE-II score and mortality. RESULTS: CA125 levels remained elevated throughout the study period. The abdominal focus presented higher mean levels of CA125 (62±55.5U/mL; P=.001) and were higher in non-survivors (77.2U/mL; interquartile range 35.9-118.5; P=.0273). CA125 levels>35U/mL throughout the weal had an independently effect on the evolution (relative risk [RR] 3.1; 95% confidence interval [CI] 1.6-6.2; P=.001) and the elevated mean value of CA125 was also associated with mortality (RR 1.004; 95% CI 1.001-1.005; P=.0001). CONCLUSIONS: Septic patients presented high levels of CA125 on the study days, being higher in abdominal infections. In our study, serial determination of CA125 is a prognostic marker of mortality independent of age, origin of infection or severity.

Chronic alcohol feeding induces biochemical, histological, and functional alterations in rat retina.

AIMS: Ethanol consumption originates a wide spectrum of disorders, including alteration of visual function. Oxidative stress is included among the mechanisms by which alcohol predisposes nervous tissue to injury. Retina, which is the neurosensorial eye tissue, is particularly sensitive to oxidative stress. METHODS: In this study we analyze the effect of long-term alcohol consumption on oxidative stress parameters of the rat retina, and its correlation to retinal function, as well as to the expression of the antiapoptotic protein Bcl-2. We also study the protective effect of ebselen, a synthetic selenoorganic antioxidant. RESULTS: Herein we show that ethanol has a toxic effect on rat retina associated with oxidative stress. Decreases in retina glutathione concentration and increases in malondialdehyde content in whole eye homogenate significantly correlate with ERG b-wave decrease and Bcl-2 overexpression. We also show how ebselen is able to prevent all the alterations observed. CONCLUSION: Chronic ethanol consumption induces oxidative stress in rat retina associated with an impairment of ERG and Bcl-2 overexpression, suggesting a role for glial cells. All these alterations in the rat allow the proposal of an alcoholic retinopathy in this species.

Hepatic and renal metallothionein concentrations in Commerson's dolphins (Cephalorhynchus commersonii) from Tierra del Fuego, South Atlantic Ocean.

The Commerson's dolphin is the most common endemic odontocete of subantarctic waters of Tierra del Fuego, Argentina incidentally caught in fishing nets. The species is classified as "Data Deficient" by the IUCN. Metallothioneins (MTs) are considered as suitable biomarkers for health and environmental monitoring. The aims of the study were to assess MT concentrations in the liver and kidney of bycaught specimens. Moreover, correlations with Zn, Se, Cd, Ag and Hg, and the molar ratios of MT:metals were estimated to evaluate if there is an indication of their respective protective role against metal toxicity in tissues. Hepatic and renal MT concentrations were similar, ranging from 11.6 to 29.1nmol·g(-1) WW, and Kidney/Liver ratios ranging from 0.73 to 1.93 corresponded to normal ranges. Results suggest that MTs are related to physiological ranges for the species. This information constitutes the first MT report on Commerson's dolphins and possibly considered as baseline for species' conservation.

Role of oxygen and nitrogen species in experimental uveitis: anti-inflammatory activity of the synthetic antioxidant ebselen.

This study was aimed at examining the role of oxygen and nitrogen reactive species in a model of experimental uveitis upon intravitreal injection of bacterial endotoxin to albino New Zealand rabbits. The inflammatory response was evaluated in terms of: (i) the integrity of the blood aqueous barrier (protein and cell content in samples of aqueous humor), (ii) histopathological changes of the eyes, (iii) clinical evaluation (with a score index based on clinical symptoms), and (iv) the concentration of malondialdehyde (MDA), in aqueous humor, as a marker of oxidative stress. Betamethasone was used as reference treatment, superoxide dismutase as quencher of superoxide anion, L-N(G)-nitro-L-arginine-methyl-esther (L-NAME) and chlorpromazine as nitric oxide synthase inhibitors, and ebselen, a glutathione peroxidase mimic, as peroxynitrite reductant. All the substances were injected subconjunctivally to the rabbits immediately after the intravitreal endotoxin injection. Ebselen was the only treatment able to decrease MDA concentration to control values, exerting an effect similar to that elicited by L-NAME on the rest of the parameters tested. The data presented render ebselen a notable choice for the treatment of uveitis, with implications for clinical trials.

Serum malondialdehyde correlates with therapeutic efficiency of high activity antiretroviral therapies (HAART) in HIV-1 infected children.

Serum malondialdehyde (MDA) levels are increased in human immunodeficiency virus (HIV)-infected children, as it happens also in infected adult individuals. Introduction of high activity antiretroviral therapy (HAART) has promoted an intense decline in morbidity and mortality of these patients. Here we present data on the effect of HAART on serum MDA of HIV+ children and compare them with levels prior to HAART. MDA levels reflect, as other markers do, the HAART-induced clinical improvement and probably also the pro-oxidant/antioxidant side effects of the different drugs used. The results herein allow the proposal of including serum MDA levels as an additional parameter for the clinical management of HIV+ children.

FACS analysis of the planarian stem cell compartment as a tool to understand regenerative mechanisms.

Planarians provide a relatively simple model system in which to study stem cell dynamics and regenerative phenomena. As with other systems understanding the dynamics of stem cell and stem cell progeny is crucial in order to get at the molecular mechanisms orchestrating stem cell biology. Planarians have an abundant adult stem cell population that can be observed using Fluorescence-Activated Cell Sorting (FACS). This approach allows different subpopulations of stem cells and their progeny to be monitored and sorted for downstream studies in response to different regenerative scenarios, drug treatments, or RNAi knockdown of genes required for regenerative events.

Combining classical and molecular approaches elaborates on the complexity of mechanisms underpinning anterior regeneration.

The current model of planarian anterior regeneration evokes the establishment of low levels of Wnt signalling at anterior wounds, promoting anterior polarity and subsequent elaboration of anterior fate through the action of the TALE class homeodomain PREP. The classical observation that decapitations positioned anteriorly will regenerate heads more rapidly than posteriorly positioned decapitations was among the first to lead to the proposal of gradients along an anteroposterior (AP) axis in a developmental context. An explicit understanding of this phenomenon is not included in the current model of anterior regeneration. This raises the question what the underlying molecular and cellular basis of this temporal gradient is, whether it can be explained by current models and whether understanding the gradient will shed light on regenerative events. Differences in anterior regeneration rate are established very early after amputation and this gradient is dependent on the activity of Hedgehog (Hh) signalling. Animals induced to produce two tails by either Smed-APC-1(RNAi) or Smed-ptc(RNAi) lose anterior fate but form previously described ectopic anterior brain structures. Later these animals form peri-pharyngeal brain structures, which in Smed-ptc(RNAi) grow out of the body establishing a new A/P axis. Combining double amputation and hydroxyurea treatment with RNAi experiments indicates that early ectopic brain structures are formed by uncommitted stem cells that have progressed through S-phase of the cell cycle at the time of amputation. Our results elaborate on the current simplistic model of both AP axis and brain regeneration. We find evidence of a gradient of hedgehog signalling that promotes posterior fate and temporarily inhibits anterior regeneration. Our data supports a model for anterior brain regeneration with distinct early and later phases of regeneration. Together these insights start to delineate the interplay between discrete existing, new, and then later homeostatic signals in AP axis regeneration.