Influence of Chronic Obstructive Pulmonary Disease and Moderate-To-Severe Sleep Apnoea in Overnight Cardiac Autonomic Modulation: Time, Frequency and Non-Linear Analyses.

Chronic obstructive pulmonary disease (COPD) is one of the most prevalent lung diseases worldwide. COPD patients show major dysfunction in cardiac autonomic modulation due to sustained hypoxaemia, which has been significantly related to higher risk of cardiovascular disease. Obstructive sleep apnoea syndrome (OSAS) is a frequent comorbidity in COPD patients. It has been found that patients suffering from both COPD and OSAS simultaneously, the so-called overlap syndrome, have notably higher morbidity and mortality. Heart rate variability (HRV) has demonstrated to be useful to assess changes in autonomic functioning in different clinical conditions. However, there is still little scientific evidence on the magnitude of changes in cardiovascular dynamics elicited by the combined effect of both respiratory diseases, particularly during sleep, when apnoeic events occur. In this regard, we hypothesised that a non-linear analysis is able to provide further insight into long-term dynamics of overnight cardiovascular modulation. Accordingly, this study is aimed at assessing the usefulness of sample entropy (SampEn) to distinguish changes in overnight pulse rate variability (PRV) recordings among three patient groups while sleeping: COPD, moderate-to-severe OSAS, and overlap syndrome. In order to achieve this goal, a population composed of 297 patients were studied: 22 with COPD alone, 213 showing moderate-to-severe OSAS, and 62 with COPD and moderate-to-severe OSAS simultaneously (COPD+OSAS). Cardiovascular dynamics were analysed using pulse rate (PR) recordings from unattended pulse oximetry carried out at patients' home. Conventional time- and frequency- domain analyses were performed to characterise sympathetic and parasympathetic activation of the nervous system, while SampEn was applied to quantify long-term changes in irregularity. Our analyses revealed that overnight PRV recordings from COPD+OSAS patients were significantly more irregular (higher SampEn) than those from patients with COPD alone (0.267 [0.210-0.407] vs. 0.212 [0.151-0.267]; p < 0.05) due to recurrent apnoeic events during the night. Similarly, COPD + OSAS patients also showed significantly higher irregularity in PRV during the night than subjects with OSAS alone (0.267 [0.210-0.407] vs. 0.241 [0.189-0.325]; p = 0.05), which suggests that the cumulative effect of both diseases increases disorganization of pulse rate while sleeping. On the other hand, no statistical significant differences were found between COPD and COPD + OSAS patients when traditional frequency bands (LF and HF) were analysed. We conclude that SampEn is able to properly quantify changes in overnight cardiovascular dynamics of patients with overlap syndrome, which could be useful to assess cardiovascular impairment in COPD patients due to the presence of concomitant OSAS.

Cell-TypeAnalyzer: A flexible Fiji/ImageJ plugin to classify cells according to user-defined criteria.

Fluorescence microscopy techniques have experienced a substantial increase in the visualization and analysis of many biological processes in life science. We describe a semiautomated and versatile tool called Cell-TypeAnalyzer to avoid the time-consuming and biased manual classification of cells according to cell types. It consists of an open-source plugin for Fiji or ImageJ to detect and classify cells in 2D images. Our workflow consists of (a) image preprocessing actions, data spatial calibration, and region of interest for analysis; (b) segmentation to isolate cells from background (optionally including user-defined preprocessing steps helping the identification of cells); (c) extraction of features from each cell; (d) filters to select relevant cells; (e) definition of specific criteria to be included in the different cell types; (f) cell classification; and (g) flexible analysis of the results. Our software provides a modular and flexible strategy to perform cell classification through a wizard-like graphical user interface in which the user is intuitively guided through each step of the analysis. This procedure may be applied in batch mode to multiple microscopy files. Once the analysis is set up, it can be automatically and efficiently performed on many images. The plugin does not require any programming skill and can analyze cells in many different acquisition setups.

Pivotal role of TLR4 receptors in alcohol-induced neuroinflammation and brain damage.

Toll-like receptors play an important role in the innate immune response, although emerging evidence indicates their role in brain injury and neurodegeneration. Alcohol abuse induces brain damage and can sometimes lead to neurodegeneration. We recently found that ethanol can promote TLR4 signaling in glial cells by triggering the induction of inflammatory mediators and causing cell death, suggesting that the TLR4 response could be an important mechanism of ethanol-induced neuroinflammation. This study aims to establish the potential role of TLR4 in both ethanol-induced glial activation and brain damage. Here we report that TLR4 is critical for ethanol-induced inflammatory signaling in glial cells since the knockdown of TLR4, by using both small interfering RNA or cells from TLR4-deficient mice, abolished the activation of microtubule-associated protein kinase and nuclear factor-kappaB pathways and the production of inflammatory mediators by astrocytes. Our results demonstrate, for the first time, that whereas chronic ethanol intake upregulates the immunoreactive levels of CD11b (microglial marker) and glial fibrillary acidic protein (astrocyte marker), and also increases caspase-3 activity and inducible nitric oxide synthase, COX-2, and cytokine levels [interleukin (IL)-1beta, tumor necrosis factor-alpha, IL-6] in the cerebral cortex of female wild-type mice, TLR4 deficiency protects against ethanol-induced glial activation, induction of inflammatory mediators, and apoptosis. Our findings support the critical role of the TLR4 response in the neuroinflammation, brain injury, and possibly in the neurodegeneration induced by chronic ethanol intake.

A practical approach to FRET-based PNA fluorescence in situ hybridization.

Given the demand for improved methods for detecting and characterizing RNA variants in situ, we developed a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction as FRET efficiency measure. The FRET-based PNA fluorescence in situ hybridization (FP-FISH) method offers a conceptually new approach for characterizing at the subcellular level not only splice variant isoform structure, location, and dynamics but also potentially a wide variety of close range RNA-RNA interactions. In this paper, we explain the FP-FISH technique workflow for reliable and reproducible results.

A FRET-based assay for characterization of alternative splicing events using peptide nucleic acid fluorescence in situ hybridization.

We describe a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction by FRET measure. As a proof of concept we analyzed two alternative splicing events originating from lymphocyte antigen 6 (LY6) complex, locus G5B (LY6G5B) pre-mRNA. These are characterized by the removal of the first intron (Fully Spliced Isoform, FSI) or by retention of such intron (Intron-Retained Isoform, IRI). The use of PNA probe pairs labeled with donor (Cy3) and acceptor (Cy5) fluorophores, suitable to FRET, flanking FSI and IRI specific splice junctions specifically detected both mRNA isoforms in HeLa cells. We have observed that the method works efficiently with probes 5-11 nt apart. The data supports that this FRET-based PNA fluorescence in situ hybridization (FP-FISH) method offers a conceptually new approach for characterizing at the subcellular level not only splice variant isoform structure, location and dynamics but also potentially a wide variety of close range RNA-RNA interactions.

Ethanol mimics ligand-mediated activation and endocytosis of IL-1RI/TLR4 receptors via lipid rafts caveolae in astroglial cells.

We have recently reported that ethanol-induced inflammatory processes in the brain and glial cells are mediated via the activation of interleukin-1 beta receptor type I (IL-1RI)/toll-like receptor type 4 (TLR4) signalling. The mechanism(s) by which ethanol activates these receptors in astroglial cells remains unknown. Recently, plasma membrane microdomains, lipid rafts, have been identified as platforms for receptor signalling and, in astrocytes, rafts/caveolae constitute an important integrators of signal events and trafficking. Here we show that stimulation of astrocytes with IL-1beta, lipopolysaccharide or ethanol (10 and 50 mM), triggers the translocation of IL-1RI and/or TLR4 into lipid rafts caveolae-enriched fractions, promoting the recruitment of signalling molecules (phospho-IL-1R-associated kinase and phospho-extracellular regulated-kinase) into these microdomains. With confocal microscopy, we further demonstrate that IL-1RI is internalized by caveolar endocytosis via enlarged caveosomes organelles upon IL-1beta or ethanol treatment, which sorted their IL-1RI cargo into the endoplasmic reticulum-Golgi compartment and into the nucleus of astrocytes. In short, our findings demonstrate that rafts/caveolae are critical for IL-1RI and TLR4 signalling in astrocytes, and reveal a novel mechanism by which ethanol, by interacting with lipid rafts caveolae, promotes IL-1RI and TLR4 receptors recruitment, triggering their endocytosis via caveosomes and downstream signalling stimulation. These results suggest that TLRs receptors are important targets of ethanol-induced inflammatory damage in the brain.

Assessment of automated analysis of portable oximetry as a screening test for moderate-to-severe sleep apnea in patients with chronic obstructive pulmonary disease.

BACKGROUND: The coexistence of obstructive sleep apnea syndrome (OSAS) and chronic obstructive pulmonary disease (COPD) leads to increased morbidity and mortality. The development of home-based screening tests is essential to expedite diagnosis. Nevertheless, there is still very limited evidence on the effectiveness of portable monitoring to diagnose OSAS in patients with pulmonary comorbidities. OBJECTIVE: To assess the influence of suffering from COPD in the performance of an oximetry-based screening test for moderate-to-severe OSAS, both in the hospital and at home. METHODS: A total of 407 patients showing moderate-to-high clinical suspicion of OSAS were involved in the study. All subjects underwent (i) supervised portable oximetry simultaneously to in-hospital polysomnography (PSG) and (ii) unsupervised portable oximetry at home. A regression-based multilayer perceptron (MLP) artificial neural network (ANN) was trained to estimate the apnea-hypopnea index (AHI) from portable oximetry recordings. Two independent validation datasets were analyzed: COPD versus non-COPD. RESULTS: The portable oximetry-based MLP ANN reached similar intra-class correlation coefficient (ICC) values between the estimated AHI and the actual AHI for the non-COPD and the COPD groups either in the hospital (non-COPD: 0.937, 0.909-0.956 CI95%; COPD: 0.936, 0.899-0.960 CI95%) and at home (non-COPD: 0.731, 0.631-0.808 CI95%; COPD: 0.788, 0.678-0.864 CI95%). Regarding the area under the receiver operating characteristics curve (AUC), no statistically significant differences (p >0.01) between COPD and non-COPD groups were found in both settings, particularly for severe OSAS (AHI ≥30 events/h): 0.97 (0.92-0.99 CI95%) non-COPD vs. 0.98 (0.92-1.0 CI95%) COPD in the hospital, and 0.87 (0.79-0.92 CI95%) non-COPD vs. 0.86 (0.75-0.93 CI95%) COPD at home. CONCLUSION: The agreement and the diagnostic performance of the estimated AHI from automated analysis of portable oximetry were similar regardless of the presence of COPD both in-lab and at-home. Particularly, portable oximetry could be used as an abbreviated screening test for moderate-to-severe OSAS in patients with COPD.

Chronic ethanol treatment enhances inflammatory mediators and cell death in the brain and in astrocytes.

Inflammatory processes and cytokine expression have been implicated in the pathogenesis of several neurodegenerative disorders. Chronic ethanol intake induces brain damage, although the mechanisms involved in this effect are not well understood. We tested the hypothesis that activation of glial cells by ethanol would induce stimulation of signaling pathways and inflammatory mediators in brain, and would cause neurotoxicity. We used cerebral cortex from control and chronic ethanol-fed rats, which received ethanol-liquid diet for 5 months and cultured of astrocytes exposed to 75 mM ethanol for 7 days. Our results demonstrate that chronic ethanol treatment up-regulates iNOS, COX-2 and IL-1beta in rat cerebral cortex and in cultured astrocytes. Under both experimental conditions, up-regulation of these inflammatory mediators and IL-1RI concomitantly occurs with the stimulation of IRAK and MAP kinases, including ERK1/2, p-38 and JNK, which trigger the downstream activation of oxidant-sensitive transcription factors NF-KB and AP-1. These effects were associated with an increased in both caspase-3 and apoptosis in ethanol-fed rats and in astrocytes exposed to ethanol. In conclusion, chronic ethanol treatment stimulates glial cells, up-regulating the production and the expression of inflammatory mediators in the brain, and activating signalling pathways and transcription factors involved in inflammatory damage and cell death.

Ethanol-induced iNOS and COX-2 expression in cultured astrocytes via NF-kappa B.

The CNS is particularly susceptible to the effects of alcohol and toxicity. Astrocytes are immunoactive cells, and the activation of these cells is associated with several neurodegenerative disorders. By using cultured cortical astrocytes, we show that a short ethanol treatment (100 mM) is able to up-regulate both cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, and that these effects are regulated via nuclear factor kappa B (NF-kappa B) as revealed by the inhibition of NF-kappa B activation with pyrrolidine dithiocarbamate (PDTC) or BAY 11-7082. These results suggest that ethanol is able to induce inflammatory mediators in astrocytes through the NF-kappa B activation.

PreTCR and TCRγδ signal initiation in thymocyte progenitors does not require domains implicated in receptor oligomerization.

Whether thymocytes adopt an αß or a γδ T cell fate in the thymus is determined at the ß selection checkpoint by the relatively weak or strong signals that are delivered by either the pre-T cell receptor (preTCR) or the γδ TCR, respectively. Signal initiation at the ß selection checkpoint is thought to be independent of ligand engagement of these receptors. Some reports have suggested that receptor oligomerization, which is thought to be mediated by either the immunoglobulin (Ig)-like domain of the preTCRα (pTα) chain or the variable domain of TCRδ, is a unifying mechanism that initiates signaling in early CD4(-)CD8(-) double-negative (DN) thymocyte progenitors. Here, we demonstrate that the extracellular regions of pTα and TCRδ that are implicated in mediating receptor oligomerization were not required for signal initiation from the preTCR or TCRγδ. Indeed, a truncated TCRγδ that lacked all of its extracellular Ig-like domains still formed a signaling-competent TCR that drove cells through the ß selection checkpoint. These observations suggest that signal initiation in DN thymocytes is simply a consequence of the surface-pairing of TCR chains, with signal strength being a function of the abundances of surface TCRs. Thus, processes that regulate the surface abundances of TCR complexes in DN cells, such as oligomerization-induced endocytosis, would be predicted to have a major influence in determining whether cells adopt an αß versus γδ T cell fate.

Intermittent ethanol exposure induces inflammatory brain damage and causes long-term behavioural alterations in adolescent rats.

Adolescent brain development seems to be important for the maturation of brain structures and behaviour. Intermittent binge ethanol drinking is common among adolescents, and this type of drinking can induce brain damage. Because we have demonstrated that chronic ethanol treatment induces inflammatory processes in the brain, we investigate whether intermittent ethanol intoxication enhances cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in adolescent rats, and whether these mediators induce brain damage and cause permanent cognitive dysfunctions. Adolescent rats were exposed to ethanol (3.0 g/kg) for two consecutive days at 48-h intervals over 14 days. Levels of COX-2, iNOS and cell death were assessed in the neocortex, hippocampus and cerebellum 24 h after the final ethanol administration. The following day or 20 days after the final injection (adult stage), animals were tested for different behavioural tests (conditional discrimination learning, rotarod, object recognition, beam-walking performance) to assess cognitive and motor functions. Our results show that intermittent ethanol intoxication upregulates COX-2 and iNOS levels, and increases cell death in the neocortex, hippocampus and cerebellum. Furthermore, animals treated with ethanol during adolescence exhibited behavioural deficits that were evident at the end of ethanol treatments and at the adult stage. Administration of indomethacin, a COX-2 inhibitor, abolishes the induction of COX-2 and iNOS expression and cell death, preventing ethanol-induced behavioural deficits. These findings indicate that binge pattern exposure to ethanol during adolescence induces brain damage by inflammatory processes and causes long-lasting neurobehavioural consequences. Accordingly, administering indomethacin protects against ethanol-induced brain damage and prevents detrimental ethanol effects on cognitive and motor processes.

RhoE participates in the stimulation of the inflammatory response induced by ethanol in astrocytes.

Astroglial cells are involved in the neuropathogenesis of several inflammatory diseases of the brain, where the activation of inflammatory mediators and cytokines plays an important role. We have previously demonstrated that ethanol up-regulates inflammatory mediators in both brain and astroglial cells. Since Rho GTPases are involved in inflammatory responses of astrocytes where loss of stress fibers takes place and RhoE/Rnd3 disorganizes the actin cytoskeleton, the aim of the present study was to investigate the implication of this protein in the stimulation of inflammatory signaling induced by ethanol. Our findings show that RhoE expression induces a decrease in both RhoA and Rac. In addition, RhoE not only induces actin cytoskeleton disorganization but it also stimulates both the IRAK/ERK/NF-kappaB pathway and the COX-2 expression associated with the inflammatory response in these cells. Our results also show that ethanol exposure induces RhoE signaling in astrocytes. Preincubation of astrocytes with GF109203X, an inhibitor of PKCs, reduces the RhoE levels and abolishes the ethanol-induced activation of IRAK, NF-kappaB and the COX-2 expression. Furthermore, RhoE overexpression restores ethanol responses in astrocytes treated with the PKCs inhibitor. Altogether, our findings suggest that this small GTPase is involved in the stimulation of the inflammatory signaling induced by ethanol in astrocytes. These findings provide new insights into the molecular mechanism involved in the inflammatory responses in astrocytes.

Strain-dependent variation in Mycobacterium bovis BCG-induced human T-cell activation and gamma interferon production in vitro.

Three commonly used Mycobacterium bovis BCG vaccine strains elicited different magnitudes of T-cell activation and gamma interferon production in vitro in healthy BCG-vaccinated individuals. Glaxo 1077 exhibited the greatest stimulatory capacity, followed by Pasteur 1173 and then Danish 1331. These differences may affect in vitro stimulation and vaccination-induced immunogenicity.

Involvement of TLR4/type I IL-1 receptor signaling in the induction of inflammatory mediators and cell death induced by ethanol in cultured astrocytes.

Activated astroglial cells are implicated in neuropathogenesis of many infectious and inflammatory diseases of the brain. A number of inflammatory mediators and cytokines have been proposed to play a key role in glial cell-related brain damage. Cytokine production seems to be initiated by signaling through TLR4/type I IL-1R (IL-1RI) in response to their ligands, LPS and IL-1beta, playing vital roles in innate host defense against infections, inflammation, injury, and stress. We have shown that glial cells are stimulated by ethanol, up-regulating cytokines and inflammatory mediators associated with TLR4 and IL-1RI signaling pathways in brain, suggesting that ethanol may contribute to brain damage via inflammation. We explore the possibility that ethanol, in the absence of LPS or IL-1beta, triggers signaling pathways and inflammatory mediators through TLR4 and/or IL-1RI activation in astrocytes. We show in this study that ethanol, at physiologically relevant concentrations, is capable of inducing rapid phosphorylation within 10 min of IL-1R-associated kinase, ERK1/2, stress-activated protein kinase/JNK, and p38 MAPK in astrocytes. Then an activation of NF-kappaB and AP-1 occurs after 30 min of ethanol treatment along with an up-regulation of inducible NO synthase and cyclooxygenase-2 expression. Finally, we note an increase in cell death after 3 h of treatment. Furthermore, by using either anti-TLR4- or anti-IL-1RI-neutralizing Abs, before and during ethanol treatment, we inhibit ethanol-induced signaling events, including NF-kappaB and AP-1 activation, inducible NO synthase, and cyclooxygenase-2 up-regulation and astrocyte death. In summary, these findings indicate that both TLR4 and IL-1RI activation occur upon ethanol treatment, and suggest that signaling through these receptors mediates ethanol-induced inflammatory events in astrocytes and brain.

Chronic ethanol consumption enhances interleukin-1-mediated signal transduction in rat liver and in cultured hepatocytes.

BACKGROUND: Interleukin-1 (IL-1) is a central mediator of the inflammatory process. Increased serum levels of IL-1 have been reported in alcoholics with liver damage, but it remains unknown whether chronic ethanol intake, in the presence or absence of lipopolysaccharide (LPS), activates IL-1 release and signaling in the hepatocyte. METHODS: IL-1beta and IL-10 release, expression of their receptors (IL-1RI and IL-10R), and the IL-1RI signal transduction response were evaluated in livers and cultured hepatocytes from ethanol-fed or pair-fed rats exposed in vivo or in vitro to LPS, ethanol, or both. RESULTS: Chronic ethanol intake increased both the serum levels of IL-1beta and IL-10 and the expression of IL-1RI, but not of IL-10R, in the liver microsomal fraction. In vivo LPS administration potentiated the ethanol-induced release of plasma cytokines. It is interesting to note that ethanol, either given in a single dose or chronically fed, stimulated IL-1beta and IL-10 release from cultured hepatocytes. Stimulation of hepatocytes with IL-1beta caused a higher activation of IL-1-associated kinase, extracellular receptor-activated kinases 1 and 2, and nuclear factor-kappaB (NF-kappaB) in hepatocytes from alcohol-fed animals than from controls. Furthermore, in the absence of any stimulation, hepatocytes from alcohol-fed animals showed an activation of both kinases, as well as an increase in NF-kappaB binding. Our results suggest the participation of the extracellular signal-regulated kinase (ERK)1/2 pathway in ethanol-induced NF-kappaB activation, because treatment with PD-98059, an ERK1/2 inhibitor, partially suppressed IL-1beta-induced NF-kappaB expression. CONCLUSIONS: Chronic ethanol intake potentiates the action of the proinflammatory cytokine IL-1beta, enhancing the release and signaling response of IL-1beta in the hepatocyte, which in conjunction with other cytokines or LPS may exacerbate the inflammatory damage associated with alcoholic liver disease.