The Dysfunctional Beliefs and Attitudes about Sleep scale: Dimensions of the European Portuguese DBAS-30 and development of a new short version (DBAS-SF-16).

OBJECTIVES: The Dysfunctional Beliefs and Attitudes about Sleep Scale (DBAS) is the most widely validated instrument for assessing sleep-related cognitions. This study aimed to examine the reliability of the DBAS-30 European Portuguese version, explore its dimensionality, and develop a new short version suitable for differentiating the presence/absence of insomnia. METHODS: From 824 participants aged 18-85 years, the Insomnia Group (IG, n = 355, 261 females and 94 males) and Normal Sleepers Group (NSG, n = 292, 237 females, 54 males and 1 with no response) were constituted. Thirty-one patients with Obstructive Sleep Apnea Syndrome were also recruited. For the DBAS 16-items version, the ability to differentiate dysfunctional beliefs between people with and without insomnia was used as the main criterion for item retention. RESULTS: DBAS-30 PT demonstrated good internal consistency and significantly discriminated IG from NSG. Based on a robust EFA (RDWLS), a three-dimensional structure was determined for IG (Ageing and Hopelessness, Sleep Expectations, and Consequences and Helplessness). DBAS-SF-16 presented as an internally-consistent measure with a reliable two-factor structure (Consequences and Helplessness, Medication and Hopelessness) and showed construct and known groups validity. ROC analysis demonstrated DBAS-SF-16's relevant clinical accuracy, and 4.3 provides the best cut-off score in detecting the level of dysfunctional beliefs associated with clinical insomnia. CONCLUSIONS: A new and meaningful dimensionality of the DBAS-30 was found. DBAS-SF-16 showed to be a reliable, valid, and robust tool for evaluating dysfunctional beliefs about insomnia in clinical and non-clinical populations.

Understanding neuron-glia crosstalk and biological clocks in insomnia.

According to the World Health Organization, about one-third of the population experiences insomnia symptoms, and about 10-15% suffer from chronic insomnia, the most common sleep disorder. Sleeping difficulties associated with insomnia are often linked to chronic sleep deprivation, which has a negative health impact partly due to disruption in the internal synchronisation of biological clocks. These are regulated by clock genes and modulate most biological processes. Most studies addressing circadian rhythm regulation have focused on the role of neurons, yet glial cells also impact circadian rhythms and sleep regulation. Chronic insomnia and sleep loss have been associated with glial cell activation, exacerbated neuroinflammation, oxidative stress, altered neuronal metabolism and synaptic plasticity, accelerated age-related processes and decreased lifespan. It is, therefore, essential to highlight the importance of glia-neuron interplay on sleep/circadian regulation and overall healthy brain function. Hence, in this review, we aim to address the main neurobiological mechanisms involved in neuron-glia crosstalk, with an emphasis on microglia and astrocytes, in both healthy sleep, chronic sleep deprivation and chronic insomnia.

Minicircle-based expression of vascular endothelial growth factor in mesenchymal stromal cells from diverse human tissues.

BACKGROUND: Mesenchymal stromal cells (MSC) have been exploited for the treatment of ischemic diseases given their angiogenic potential. Despite bone marrow (BM) being the most studied tissue source, cells with similar intrinsic properties can be isolated from adipose tissue (AT) and umbilical cord matrix (UCM). The present study aims to compare the angiogenic potential of MSC obtained from BM, AT and UCM that were genetically modified with vascular endothelial growth factor (VEGF)-encoding minicircle (MC) vectors. The overexpression of VEGF combined with the intrinsic properties of MSC could represent a promising strategy towards angiogenic therapies. METHODS: We established a microporation-based protocol to transfect human MSC using VEGF-encoding MC (MC-VEGF). VEGF production levels were measured by an enzyme-linked immunosorbent assay and a quantitative polymerase chain reaction. The in vitro angiogenic potential of transfected cells was quantified using cell tube formation and migration functional studies. RESULTS: MSC isolated from BM, AT or UCM showed similar levels of VEGF secretion after transfection with MC-VEGF. Those values were significantly higher when compared to non-transfected cells, indicating an effective enhancement of VEGF production. Transfected cells displayed higher in vitro angiogenic potential than non-transfected controls, as demonstrated by functional in vitro assays. No significant differences were observed among cells from different sources. CONCLUSIONS: Minicircles can be successfully used to transiently overexpress VEGF in human MSC, regardless of the cell tissue source, representing an important advantage in a clinical context (i.e., angiogenic therapy) because a standard protocol might be applied to MSC of different tissue sources, which can be differentially selected according to the application (e.g., autologous versus allogeneic settings).

Dimethyloxalylglycine, a small molecule, synergistically increases the homing and angiogenic properties of human mesenchymal stromal cells when cultured as 3D spheroids.

Strategies aiming at increasing the survival and paracrine activity of human mesenchymal stromal cells (MSCs) are of utmost importance to achieve the full therapeutic potential of these cells. Herein, we propose both physical and biochemical strategies to enhance the survival, homing, angiogenic, and immunomodulatory properties of MSCs in vitro. To that purpose, we compared the effect of exposing either 2D monolayer or 3D spheroids of MSCs to (i) hypoxia (2% O2 ) or to (ii) a hypoxic-mimetic small molecule, dimethyloxalylglycine (DMOG), with cells cultured at 21% O2 . 3D-cultured MSC spheroids evidenced higher survival upon exposure to oxidative stress and expressed higher levels of factors involved in tissue repair processes, namely tumor necrosis factor-stimulated gene-6, matrix metalloproteinase-2, and vascular endothelial growth factor. MSCs cultured as 3D spheroids and further exposed to hypoxia or hypoxic-mimetic conditions provided by DMOG synergistically favored the expression of the cell surface marker C-X-C chemokine receptor type-4, involved in homing processes to injured tissues, and adhesion to extracellular matrix components as fibronectin. These results highlight the role of ex vivo preconditioning approaches, presenting a novel strategy that combine biochemical stimuli with 3D spheroid organization of MSCs to maximize their tissue regeneration potential.

The European Portuguese version of the insomnia severity index.

Insomnia is the most prevalent sleep complaint, but remains largely an unidentified public health issue. The Insomnia Severity Index (ISI) is a brief self-report questionnaire to assess insomnia, long-established both in clinical and research settings. The present study aimed to analyse the reliability, validity, and accuracy of the ISI European Portuguese version. After the translation protocol, 1,274 participants (65.54% female), with a mean (SD, range) age of 37.52 (16.82, 18-95) years, completed the ISI. This sample included 250 patients with insomnia from a Sleep Medicine Centre, presenting a diagnosis of insomnia disorder (Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition; International Classification of Sleep Disorders, Third Edition), and 1,024 individuals from the community. A group of 30 patients with obstructive sleep apnea (OSA) was also recruited. Cronbach's α was 0.88 (internal consistency), and corrected item-total correlations ranged from 0.56 to 0.83. An exploratory factor analysis (oblique rotation) revealed a two-factor solution for both clinical and community samples. The ISI total score significantly differentiated insomnia disorder, no insomnia, and OSA subgroups with a large effect size (η 2  = 0.42). The correlation between ISI and Pittsburgh Sleep Quality Index supported concurrent validity (0.82), and discriminant validity was confirmed by a moderate correlation between ISI and Beck Depression Inventory Second Edition (0.32). The area under the curve was 0.88, and the optimal cut-off to detect clinical insomnia was 14 (82.1% sensitivity, 79.5% specificity). In conclusion, the Portuguese version of the ISI is a reliable and valid measure of insomnia in clinical and non-clinical populations. Our present study also contributes to relevant data for the international literature regarding the cut-off score of the scale for the detection of clinical insomnia.

CORRELATION STUDY BETWEEN DRUSEN MORPHOLOGY AND FUNDUS AUTOFLUORESCENCE.

PURPOSE: To correlate drusen morphology and outer retinal status with autofluorescence (AF) imaging in patients with intermediate age-related macular degeneration. METHODS: Drusen type and morphology were analyzed using color fundus photography and spectral-domain optic coherence tomography, whereas fundus AF was used for drusen AF evaluation. Additional structural changes on spectral-domain optic coherence tomography, such as disruption of external limiting membrane, ellipsoid zone, and retinal pigment epithelium/Bruch membrane complex, as well as the presence of choroidal hypertransmission at correspondent locations were also evaluated and correlated with fundus AF findings. Spearman's correlation coefficient was used to analyze the correlation between spectral-domain optic coherence tomography morphological characteristics of drusen and AF appearance of the corresponding drusen. Strength of correlation was calculated (r), and a P value < 0.05 was considered statistically significant. RESULTS: Two hundred and twenty-eight drusen from 53 eyes of 53 patients were analyzed, 130 soft drusen (57.02%) and 98 cuticular drusen (42.98%). Sixty percent of the drusen were isoautofluorescent (n = 136), 35% hyperautofluorescent (n = 80), and 5% hypoautofluorescent (n = 12). We found positive correlation between drusen AF and hyperreflective foci (r = 0.4). Outer retinal layers morphology (external limiting membrane and ellipsoid zone status and hypertransmission) also correlates with autofluorescent findings (r = 0.3). CONCLUSION: Multimodal imaging reveals a broad spectrum of ultrastructural changes, which may reflect different stages in the evolution of drusen. Our results suggest that drusen morphological characteristics and autofluorescent findings are correlated but other factors or cofactors may be involved. The described correlations will help us understand new progression biomarkers of nonexudative age-related macular degeneration.

Modulation of the in vitro angiogenic potential of human mesenchymal stromal cells from different tissue sources.

Mesenchymal stromal cells (MSCs) have been widely exploited for the treatment of several conditions due to their intrinsic regenerative and immunomodulatory properties. MSC have demonstrated to be particularly relevant for the treatment of ischemic diseases, where MSC-based therapies can stimulate angiogenesis and induce tissue regeneration. Regardless of the condition targeted, recent analyses of MSC-based clinical trials have demonstrated limited benefits indicating a need to improve the efficacy of this cell product. Preconditioning MSC ex vivo through microenvironment modulation was found to improve MSC survival rate and thus prolong their therapeutic effect. This workstudy aims at enhancing the in vitro angiogenic capacity of a potential MSC-based medicinal product by comparing different sources of MSC and culture conditions. MSC from three different sources (bone marrow [BM], adipose tissue [AT], and umbilical cord matrix [UCM]) were cultured with xenogeneic-/serum-free culture medium under static conditions and their angiogenic potential was studied. Results indicated a higher in vitro angiogenic capacity of UCM MSC, compared with cells derived from BM and AT. Physicochemical preconditioning of UCM MSC through a microcarrier-based culture platform and low oxygen concentration (2% O2 , compared with atmospheric air) increased the in vitro angiogenic potential of the cultured cells. Envisaging the clinical manufacturing of an allogeneic, off-the-shelf MSC-based product, preconditioned UCM MSC maintain the angiogenic gene expression profile upon cryopreservation and delivery processes in the conditions of our study. These results are expected to contribute to the development of MSC-based therapies in the context of angiogenesis.

Engineering of Human Mesenchymal Stem/Stromal Cells with Vascular Endothelial Growth Factor-Encoding Minicircles for Angiogenic Ex Vivo Gene Therapy.

Peripheral artery disease (PAD) is a debilitating and prevalent condition characterized by blockage of the arteries, leading to limb amputation in more severe cases. Mesenchymal stem/stromal cells (MSC) are known to have intrinsic regenerative properties that can be potentiated by the introduction of pro-angiogenic genes such as the vascular endothelial growth factor (VEGF). Herein, the use of human bone marrow MSC transiently transfected with minicircles encoding for VEGF is proposed as an ex vivo gene therapy strategy to enhance angiogenesis in PAD patients. The VEGF gene was cloned in minicircle and conventional plasmid vectors and used to transfect bone marrow-derived MSC ex vivo. VEGF expression was evaluated both by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The number of VEGF transcripts following MSC transfection with minicircles increased 130-fold relative to the expression in non-transfected MSC, whereas for the plasmid (pVAX1)-based transfection, the increase was 50-fold. Compared to the VEGF basal levels secreted by MSC (11.1 ± 3.4 pg/1,000 cells/day), significantly higher values were detected by enzyme-linked immunosorbent assay after both minicircle and pVAX1 transfection (644.8 ± 82.5 and 508.3 ± 164.0 pg/1,000 cells/day, respectively). The VEGF overexpression improved the angiogenic potential of MSC in vitro, as confirmed by endothelial cell tube formation and cell migration assays, without affecting the expansion potential ex vivo, as well as multilineage differentiation capacity or immunophenotype of MSC. Although preclinical in vivo studies are required, these results suggest that minicircle-mediated VEGF gene delivery, combined with the unique properties of human MSC, could represent a promising ex vivo gene therapy approach for an improved angiogenesis in the context of PAD.