Activation of retinoid X receptors protects retinal neurons and pigment epithelial cells from BMAA-induced death.

Exposure to the non-protein amino acid cyanotoxin ß-N-methylamino-L-alanine (BMAA), released by cyanobacteria found in many water reservoirs has been associated with neurodegenerative diseases. We previously demonstrated that BMAA induced cell death in both retina photoreceptors (PHRs) and amacrine neurons by triggering different molecular pathways, as activation of NMDA receptors and formation of carbamate-adducts was only observed in amacrine cell death. We established that activation of Retinoid X Receptors (RXR) protects retinal cells, including retina pigment epithelial (RPE) cells from oxidative stress-induced apoptosis. We now investigated the mechanisms underlying BMAA toxicity in these cells and those involved in RXR protection. BMAA addition to rat retinal neurons during early development in vitro increased reactive oxygen species (ROS) generation and polyADP ribose polymers (PAR) formation, while pre-treatment with serine (Ser) before BMAA addition decreased PHR death. Notably, RXR activation with the HX630 agonist prevented BMAA-induced death in both neuronal types, reducing ROS generation, preserving mitochondrial potential, and decreasing TUNEL-positive cells and PAR formation. This suggests that BMAA promoted PHR death by substituting Ser in polypeptide chains and by inducing polyADP ribose polymerase activation. BMAA induced cell death in ARPE-19 cells, a human epithelial cell line; RXR activation prevented this death, decreasing ROS generation and caspase 3/7 activity. These findings suggest that RXR activation prevents BMAA harmful effects on retinal neurons and RPE cells, supporting this activation as a broad-spectrum strategy for treating retina degenerations.

Sphingosine-1-phosphate and ceramide-1-phosphate promote migration, pro-inflammatory and pro-fibrotic responses in retinal pigment epithelium cells.

Retinal pigment epithelium (RPE) cells, essential for preserving retina homeostasis, also contribute to the development of retina proliferative diseases, through their exacerbated migration, epithelial to mesenchymal transition (EMT) and inflammatory response. Uncovering the mechanisms inducing these changes is crucial for designing effective treatments for these pathologies. Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are bioactive sphingolipids that promote migration and inflammation in several cell types; we recently established that they stimulate the migration of retina Müller glial cells (Simón et al., 2015; Vera et al., 2021). We here analyzed whether S1P and C1P regulate migration, inflammation and EMT in RPE cells. We cultured two human RPE cell lines, ARPE-19 and D407 cells, and supplemented them with either 5 µM S1P or 10 µM C1P, or their vehicles, for 24 h. Analysis of cell migration by the scratch wound assay showed that S1P addition significantly enhanced migration in both cell lines. Pre-treatment with W146 and BML-241, antagonists for S1P receptor 1 (S1P1) and 3 (S1P3), respectively, blocked exogenous S1P-induced migration. Inhibiting sphingosine kinase 1 (SphK1), the enzyme involved in S1P synthesis, significantly reduced cell migration and exogenous S1P only partially restored it. Addition of C1P markedly stimulated cell migration. Whereas inhibiting C1P synthesis did not affect C1P-induced migration, inhibiting S1P synthesis strikingly decreased it; noteworthy, addition of C1P promoted the transcription of SphK1. These results suggest that S1P and C1P stimulate RPE cell migration and their effect requires S1P endogenous synthesis. Both S1P and C1P increase the transcription of pro-inflammatory cytokines IL-6 and IL-8, and of EMT marker α-smooth muscle actin (α-SMA) in ARPE-19 cells. Collectively, our results suggest new roles for S1P and C1P in the regulation of RPE cell migration and inflammation; since the deregulation of sphingolipid metabolism is involved in several proliferative retinopathies, targeting their metabolism might provide new tools for treating these pathologies.

High glucose-induced phospholipase D activity in retinal pigment epithelium cells: New insights into the molecular mechanisms of diabetic retinopathy.

Chronic hyperglycemia, oxidative stress and inflammation are key players in the pathogenesis of diabetic retinopathy (DR). In this work we study the role of phospholipase D (PLD) pathway in an in vitro model of high glucose (HG)-induced damage. To this end, we exposed human retinal pigment epithelium (RPE) cell lines (ARPE-19 and D407) to HG concentrations (16.5 or 33 mM) or to normal glucose concentration (NG, 5.5 mM) for 4, 24 or 72 h. Exposure to HG increased reactive oxygen species levels and caspase-3 cleavage and reduced cell viability after 72 h of incubation. In addition, short term HG exposure (4 h) induced the activation of early events, that involve PLD and ERK1/2 signaling, nuclear factor kappa B (NFκB) nuclear translocation and IκB phosphorylation. The increment in pro-inflammatory interleukins (IL-6 and IL-8) and cyclooxygenase-2 (COX-2) mRNA levels was observed after 24 h of HG exposure. The effect of selective pharmacological PLD1 (VU0359595) and PLD2 (VU0285655-1) inhibitors demonstrated that ERK1/2 and NFκB activation were downstream events of both PLD isoforms. The increment in IL-6 and COX-2 mRNA levels induced by HG was reduced to control levels in cells pre-incubated with both PLD inhibitors. Furthermore, the inhibition of PLD1, PLD2 and MEK/ERK pathway prevented the loss of cell viability and the activation of caspase-3 induced by HG. In conclusion, our findings demonstrate that PLD1 and PLD2 mediate the inflammatory response triggered by HG in RPE cells, pointing to their potential use as a therapeutic target for DR treatment.

Effect of the Essential Oil of Minthostachys verticillata (Griseb.) Epling and Limonene on Biofilm Production in Pathogens Causing Bovine Mastitis.

Bovine mastitis causes large annual economic losses around the world. Different microorganisms are associated with the disease. The capacity of pathogens to adhere to bovine mammary epithelial cells is associated with biofilm production which leads to antibiotic resistance. Research is now leading to search alternative control methods and medicinal plants constitute a natural, safe, effective and inexpensive option. Minthostachys verticillata is an autochthonous medicinal plant of Argentina with multiple ethnobotanical properties. In a previous study, we demonstrated that the essential oil (EO) of this species and limonene, one of its compounds, inhibited the growth of mastitis pathogens. The objective of the present work was to determine the inhibitory effect of the essential oil of M. verticillata and limonene, on biofilm formation and on mature biofilm produced by pathogens isolated from bovine mastitis. Time kill assay and bacterial lysis were also determined. Furthermore, RAPD-PCR assays were performed to determine changes in bacterial DNA after EO and limonene exposition. Bacterial isolates were identified as Escherichia coli (EC3 and EC9), Bacillus pumilus (BP5, BP6, and BP7) and Enterococcus faecium (EF1) by rRNA 16S sequencing and MALDI-TOF MS. All the strains were able to form biofilm. Addition of both lactose and sucrose did not affect biofilm production. MIC values for EO were 3.6 mg/ml for E. faecium; 0.9 mg/ml for E. coli (EC3), 14.5 mg/ml for E. coli (EC9), 1.8 mg/ml for B. pumilus (BP7), 3.63 mg/ml for B. pumilus (BP6) and 29.0 mg/ml for B. pumilus (BP7). MIC values for limonene were 6.6 mg/ml for B. pumilus (BP6) and 105 mg/ml for B. pumilus (BP5). These results demonstrated that EO was more effective than limonene, showing also bactericidal action against E. faecium (minimal inhibitory concentration (MBC) = 29.0 mg/ml). This result was corroborated by time of death assay, observing a cell decrease after at 6 h, and then by bacterial lysis assay. Both EO and limonene affected mature biofilm of isolated strains. The results contribute to the study of EO and limonene which may serve as a therapy against bovine mastitis pathogens inhibiting the development of pathogenic bacteria.

Molecular characterization of infectious pancreatic necrosis virus strains isolated from the three types of salmonids farmed in Chile.

BACKGROUND: The infectious pancreatic necrosis virus (IPNV) causes significant economic losses in Chilean salmon farming. For effective sanitary management, the IPNV strains present in Chile need to be fully studied, characterized, and constantly updated at the molecular level. METHODS: In this study, 36 Chilean IPNV isolates collected over 6 years (2006-2011) from Salmo salar, Oncorhynchus mykiss, and Oncorhynchus kisutch were genotypically characterized. Salmonid samples were obtained from freshwater, estuary, and seawater sources from central, southern, and the extreme-south of Chile (35° to 53°S). RESULTS: Sequence analysis of the VP2 gene classified 10 IPNV isolates as genogroup 1 and 26 as genogroup 5. Analyses indicated a preferential, but not obligate, relationship between genogroup 5 isolates and S. salar infection. Fifteen genogroup 5 and nine genogroup 1 isolates presented VP2 gene residues associated with high virulence (i.e. Thr, Ala, and Thr at positions 217, 221, and 247, respectively). Four genogroup 5 isolates presented an oddly long VP5 deduced amino acid sequence (29.6 kDa). Analysis of the VP2 amino acid motifs associated with clinical and subclinical infections identified the clinical fingerprint in only genogroup 5 isolates; in contrast, the genogroup 1 isolates presented sequences predominantly associated with the subclinical fingerprint. Predictive analysis of VP5 showed an absence of transmembrane domains and plasma membrane tropism signals. WebLogo analysis of the VP5 BH domains revealed high identities with the marine birnavirus Y-6 and Japanese IPNV strain E1-S. Sequence analysis for putative 25 kDa proteins, coded by the ORF between VP2 and VP4, exhibited three putative nuclear localization sequences and signals of mitochondrial tropism in two isolates. CONCLUSIONS: This study provides important advances in updating the characterizations of IPNV strains present in Chile. The results from this study will help in identifying epidemiological links and generating specific biotechnological tools for controlling IPNV outbreaks in Chilean salmon farming.

Insulin-related signaling pathways elicited by light in photoreceptor nuclei from bovine retina.

Retina light stimulation triggers phototransduction events as well as different signaling mechanisms in outer segments (sensorial portion) of photoreceptor cells. We have recently reported a novel light-dependent activation of diacylglycerol kinase (DAGK) and protein kinase C (PKC) at the nuclear level of photoreceptor cells. The aim of the present study was to analyze whether ex-vivo light exposure of bovine retinas also modulates insulin-related signaling pathways in nuclei from photoreceptor cells. To this end, a nuclear fraction enriched in small nuclei from photoreceptor cells (PNF) was obtained using a modified nuclear isolation protocol. In PNF obtained from bovine retinas exposed to light or darkness, the presence of insulin receptor (IR) and phosphorylated insulin receptor (pIR), the activation of Akt, p38 and extracellular signal-regulated kinase (ERK1/2) and the local action of insulin on lipid kinases were studied. Immunofluorescence (IF) and Western blot (WB) studies revealed the presence of IR in photoreceptor nuclei. In PNF a light-dependent increase in IR total content was observed. The presence of activated IR (pIR) was also observed in PNF by WB, being its content higher in PNF from light than in to darkness. Light exposure also produced a significant increase in the content of p-Akt (3 fold) and p-p38 (60%) without changes in total Akt and p38. In addition, an increase in the content of total ERK1/2 (2 fold) was found without changes in p-ERK/total ERK ratio, indicating that light induces translocation of p-ERK to the nucleus. Polyphosphoinositide kinase and diacylglycerol kinase (DAGK) activities were measured in isolated nuclei from light-activated or darkness-adapted retinas through the formation of polyphosphoinositides (PPIs) and phosphatidic acid (PA) using nuclear lipid substrates and [γ-(32)P]ATP as radioactive substrate. A light-dependent increase in PPIs and PA formation was detected when isolated nuclei were exposed to 0.8 µM insulin plus 0.2 mM vanadate. WB studies revealed that retina's exposure to insulin under light condition increased nuclear IR content. In addition, PNF exposure to insulin increased ERK1/2 phosphorylation with no changes in total ERK1/2. Our results demonstrate the presence and the functional state of IR in the nucleus from photoreceptor cells. They also show that molecular signaling components linked to tyrosine kinase receptors and MAPK pathways, such as Akt and ERK1/2, respectively, are present in photoreceptor nuclei and are regulated by insulin and light.

Minimally-invasive Technique for Injection into Rat Optic Nerve.

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Pathogenic aquaporin-4 reactive T cells are sufficient to induce mouse model of neuromyelitis optica.

INTRODUCTION: Neuromyelitis Optica (NMO) is an autoimmune disease primarily targeting the spinal cord and optic nerve leading to paralysis and blindness. The discovery of an antibody against the astrocytic water channel, aquaporin-4 (AQP4), in the majority of patients, has led to the presumption that the antibody was necessary for disease pathogenesis. The potential role of T cells in the central nervous system, however, has not been thoroughly examined. RESULTS: We generated an anti-AQP4 antibody seronegative model of NMO using pathogenic AQP4-reactive T cells in mice by immunizing AQP4 null mice with peptides corresponding to the second extracellular loop of AQP4, loop C. When polarized to a Th17 phenotype and transferred to wild-type mice, these cells caused tail and limb weakness. Histology showed demyelination and T cell infiltration in the spinal cord, optic nerve and brain. Animals receiving cells re-stimulated in culture with non-specific proteins resulted in no behavioral disease, indicating that specific targeting of AQP4 is essential for this phenotype. CONCLUSIONS: In summary, we show that AQP4-reactive T cells are sufficient to trigger an NMO-like disease in mice, independent of antibodies, indicating that pathogenic AQP4-reactive T cells may play a similar role in humans.

Lactobacillus reuteri CRL1098 soluble factors modulate tumor necrosis factor alpha production in peripheral blood mononuclear cells: involvement of lipid rafts.

The aim of the present study was to evaluate the capacity of Lactobacillus reuteri CRL1098 soluble factors (Lr-S) to modulate TNF-α production in peripheral blood mononuclear cells (PBMC) and to study lipid rafts participation in this response. PBMC treated with Lr-S showed a reduced production of TNF-α. In addition, Lr-S treatment activated ERK and p38 MAPK pathways in PBMC. Lipid rafts participation in the reduced production of TNF-α by PBMC induced by Lr-S was verified by lipid rafts disruption with methyl-ß-cyclodextrin and the reduction of the Src-tyrosine kinase Lck localization in rafts. Moreover, PBMC pre-treatment with Lck inhibitors blocked the effect of Lr-S on TNF-α production suggesting that activation and mobilization of Lck from lipid rafts would be involved in the modulatory effect of L. reuteri CRL1098. A secreted peptide of 5785 Da would be responsible of the modulatory effect of CRL1098 strain. This study demonstrated for the first time the lipid rafts participation in a response induced by a beneficial bacterium. Also, these results open new possibilities for investigating the molecular mechanisms involved in the interaction of probiotic bacterial extracellular compounds with immune cells.

Three-dimensional imaging of the mouse neurovasculature with magnetic resonance microscopy.

Knowledge of the three-dimensional (3D) architecture of blood vessels in the brain is crucial because the progression of various neuropathologies ranging from Alzheimer's disease to brain tumors involves anomalous blood vessels. The challenges in obtaining such data from patients, in conjunction with development of mouse models of neuropathology, have made the murine brain indispensable for investigating disease induced neurovascular changes. Here we describe a novel method for "whole brain" 3D mapping of murine neurovasculature using magnetic resonance microscopy (µMRI). This approach preserves the vascular and white matter tract architecture, and can be combined with complementary MRI contrast mechanisms such as diffusion tensor imaging (DTI) to examine the interplay between the vasculature and white matter reorganization that often characterizes neuropathologies. Following validation with micro computed tomography (µCT) and optical microscopy, we demonstrate the utility of this method by: (i) combined 3D imaging of angiogenesis and white matter reorganization in both, invasive and non-invasive brain tumor models; (ii) characterizing the morphological heterogeneity of the vascular phenotype in the murine brain; and (iii) conducting "multi-scale" imaging of brain tumor angiogenesis, wherein we directly compared in vivo MRI blood volume measurements with ex vivo vasculature data.

Rins endo: um novo sistema para irrigação dos canais radiculares / Rins endo: a new system for irrigation of the root canals

A completa limpeza do sistema de canais radiculares, por meio da instrumentação e irrigação, é um fator importante no sucesso na terapia endodôntica. Além de novos instrumentos, novos dispositivos foram desenvolvidos para irrigação dos canais radiculares, destacando, entre eles, o sistema Rins Endo com ativação hidrodinâmica baseado na tecnologia de pressão-sucção. A finalidade deste trabalho é descrever os componentes desse novo sistema e a técnica para sua utilização.

In the attempt to reach the complete cleanness of the root canals system and to increase the success in the endodontics therapy, new devices had been developed to irrigation of root canals, amongst them, the Rins Endo system, with hydrodynamic activation based in the pressure-suction technology. This system is a alternative to the endodontics during the biomechanical preparation. The objective of this work was to describe the components of this new system and the technique for its use.

FLT-3 expression and function on microglia in multiple sclerosis.

Inflammatory cell infiltration and resident microglial activation within the central nervous system (CNS) are pathological events in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). While MS therapies target the peripheral immune system, no treatment is currently known to also modulate microglia. FMS-like tyrosine-3 (FLT-3) is expressed on hematopoietic and dendritic cells. We reported that FLT-3 inhibition ameliorates early actively induced EAE by predominantly modulating dendritic cell function as compared to microglia. We demonstrate in this report that FLT-3 is expressed in perivascular cuffs, brain parenchyma and in non-lesioned gray and white matter within MS brain but not in these regions within control brain. Furthermore, we demonstrate that FLT-3 is expressed on two populations of cells within MS brain; one which expresses the dendritic cell marker CD209, and the other which does not, suggesting that FLT-3 within MS brain is expressed on infiltrating dendritic cells and a non-dendritic cell such as microglia. Additionally, we report that FLT-3 inhibition in murine microglia blocks, in a dose-dependent manner, IFN-γ-induced expression of MHC class II and CD86, and LPS-induced secretion of IL-6. These data suggest that FLT-3 is involved in microglial cells' capacity to respond to environmental cues to function as antigen presenting cells and mediate CNS inflammation. Furthermore these data suggest that FLT-3 may be a therapeutic target on microglia to mitigate CNS inflammation.

Interleukin-17 in transverse myelitis and multiple sclerosis.

CSF IL-6 is elevated in transverse myelitis (TM) and predicts disability. Since IL-17 regulates cytokines (TNFalpha, IL-1beta and IL-6) known to stimulate IL-6 production by astrocytes, we sought to determine whether IL-17 was increased in TM and MS compared to healthy controls (HC) and other neurologic diseases (OND). IL-17 and IL-6 levels were measured in stimulated peripheral blood mononuclear cell (PBMC) supernatants from HC, MS, TM and OND. IL-17 was increased in TM compared to HC, MS, and OND (mean pg/ml+/-standard error; HC: 36.1+/-11.7, MS: 89.4+/-23.3, TM: 302.6+/-152.5, OND: 41.2+/-13.0, p=0.01). IL-6 was increased in TM relative to MS and HC (HC: 2624 pg/ml+/-641, MS: 6129+/-982, TM: 12,536+/-2657, OND: 6920+/-1801, p<0.002). MS patients with early disease (<2 years) also had increased levels of IL-17 (p<0.04) and IL-6 (p<0.05). Cytokine neutralization experiments demonstrated that IL-6 was the main inducer of astrocyte IL-6 production. We conclude that IL-17 and IL-6 production from PBMC in TM and early MS are increased and induce astrocyte IL-6 production through IL-6.

Activation of phosphoinositide-3 kinase/Akt pathway by FeSO4 in rat cerebral cortex synaptic endings.

The aim of this work was to study the involvement of the phosphoinositide-3-kinase (PI3K)/Akt pathway in synaptic endings incubated under oxidative stress conditions. Synaptosomes purified from rat cerebral cortex were exposed to FeSO4 (50 microM) for different periods of time. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenate (LDH) leakage were significantly affected after 5 min of incubation in the presence of FeSO4, with respect to control conditions. In whole synaptosomes incubated in the presence of [gamma-(32)P]ATP, phosphoinositide (PPI) labeling was increased after 5 min of Fe2+ exposure. This effect was prevented by the specific PI3K inhibitor LY294002. Anti-p85 immunoprecipitates (IPs) obtained from synaptosomes preincubated with Fe2+ (5 min) showed a PI3K activity two-fold higher than the activity recovered under control conditions. Additionally, Akt activation was temporally coincident with PI3K activation. LY294002 was not able to prevent the LDH leakage and diminution of MTT reduction induced by Fe2+. Our results demonstrate that free iron provokes the early activation of PI3K/Akt pathway, but this activation is not sufficient for protecting synaptic endings from oxidative damage.

Prevalence of intestinal metaplasia in the distal oesophagus, oesophagogastric junction and gastric cardia in symptomatic patients in north-east Italy: a prospective, descriptive survey. The Italian Ulcer Study Group "GISU".

BACKGROUND: Incidence of adenocarcinoma of distal oesophagus and gastric cardia, probably arising from areas of intestinal metaplasia, has been increasing rapidly. AIMS: To define prevalence of intestinal metaplasia of distal oesophagus, oesophagogastric junction and gastric cardia and to evaluate potential associated factors, by means of a prospective multicentre study including University and teaching hospitals, and primary and tertiary care centres. PATIENTS: Each of 24 institutions involved in study enrolled 10 consecutive patients undergoing first-time routine endoscopy for dyspeptic symptoms. METHODS: Patients answered symptom questionnaires and underwent gastroscopy Three biopsies were taken from distal oesophagus, oesophago-gastric junction and gastric cardia, and were stained with haematoxylin and eosin. Specimens were also evaluated for Helicobacter pylori infection. RESULTS: A total of 240 patients (124 male, 116 female; median age 56 years, range 20-90) were enrolled in study. Intestinal metaplasia affected distal oesophagus in 5, oesophago-gastric junction in 19 and gastric cardia in 10 patients. Low-grade dysplasia was found at distal oesophagus and/or oesophago-gastric junction of 3/24 patients with intestinal metaplasia vs 2/216 without intestinal metaplasia (p<0.05). A significant association was found between symptoms and presence of intestinal metaplasia, regardless of location, and between Helicobacter pylori infection and intestinal metaplasia at oesophago-gastric junction. CONCLUSIONS: Intestinal metaplasia of distal oesophagus, oesophagogastric-junction and gastric cardia is found in a significant proportion of symptomatic patients undergoing gastroscopy and is associated with dysplasia in many cases. Although prevalence of dysplasia seems to decrease when specialized columnar epithelium is found in short segment, or even focally in oesophago-gastric junction, these small foci of intestinal metaplastic cells may represent source of most adenocarcinomas of cardia.