Insights on COVID-19 Vaccination in Portugal: A Qualitative Study among Health Professionals and Teachers.

BACKGROUND: Vaccination against COVID-19 has had a major impact over the course of the pandemic, leading to a reduced number of hospitalizations and deaths. However, the mass vaccination process has been accompanied by skepticism and hesitancy since its beginning. As health professionals and teachers are important public health actors who can strongly intervene to reduce vaccination hesitancy among their patients and students, respectively, this study aimed to assess their main perceptions towards COVID-19 vaccination. METHODS: Two focus group sessions, one with health professionals and the other with teachers, were conducted according to the COREQ checklist. Qualitative data were analyzed through theoretical thematic analysis. RESULTS: In general, none of the groups showed vaccine hesitancy, although both groups had concerns regarding the safety and efficacy of the vaccines. The main concerns of health professionals were mostly related to the long-term impact of the COVID-19 pandemic, while teachers were more worried about the lack of access to reliable information about the COVID-19 vaccination. CONCLUSIONS: It is plausible to conclude that it is imperative to provide clear and accurate information for the population in order to avoid vaccination hesitancy.

Perspectives and experiences of Covid-19: Two Irish studies of families in disadvantaged communities.

The Covid-19 pandemic has been recognised to affect families' socio-emotional well-being. Collecting the views of families in diverse socio-economic contexts can contribute to understanding their specific needs and resources in relation to the Covid-19 pandemic. The overarching objective of the current research was to explore the views and experiences of families in relation to the Covid-19 pandemic, who were living in the Republic of Ireland, including in areas designated as disadvantaged. In Study 1, the objective was to explore changes, difficulties, and concerns experienced by parents of children up to six years old during the pandemic, and related associations with socio-demographic characteristics. Data were collected from 168 parents/carers via an online questionnaire, and examined using conceptual content analysis. The most frequently identified experiences related to restrictions, social isolation, negative impacts on parents' emotional and psychological well-being, negative impacts on children's emotional well-being and development, concerns with physical health, uncertainty about the future, and positive changes regarding family time and activities. Associations were found with parents' age and work situation, and family's income and composition. In Study 2, the objective was to explore the views of children, parents, and service providers about the impact of the Covid-19 pandemic on families' life, and relevant supports. Data were collected from 50 children aged between eight and 17 years old, 17 parents, and 20 service providers through focus group discussions, and examined using thematic analysis. The participants reported experiences related to challenges with online education, uncertainty regarding children's education, food poverty, and children's socio-emotional health. The findings of both studies reinforced the importance of implementing measures to promote parents' and children's socio-emotional well-being, combat educational inequalities, and ensure economic and employment security.

TARGETing Transcriptional Regulation in the Medicinal Plant Catharanthus roseus.

Transcriptional regulation is a central piece of the highly valuable monoterpenoid indole alkaloid pathway of C. roseus , and the ultimate tool for its understanding and manipulation. Here, we describe the adaptation of the TARGET methodology to identify specific and genome-wide leaf targets of C. roseus candidate transcription factors (TFs).

The Impact of Lifestyle on Prostate Cancer: A Road to the Discovery of New Biomarkers.

Prostate cancer (PCa) is one of the most common cancers among men, and its incidence has been rising through the years. Several risk factors have been associated with this disease and unhealthy lifestyles and inflammation were appointed as major contributors for PCa development, progression, and severity. Despite the advantages associated with the currently used diagnostic tools [prostate-specific antigen(PSA) serum levels and digital rectal examination (DRE)], the development of effective approaches for PCa diagnosis is still necessary. Finding lifestyle-associated proteins that may predict the development of PCa seems to be a promising strategy to improve PCa diagnosis. In this context, several biomarkers have been identified, including circulating biomarkers (CRP, insulin, C-peptide, TNFα-R2, adiponectin, IL-6, total PSA, free PSA, and p2PSA), urine biomarkers (PCA3, guanidine, phenylacetylglycine, and glycine), proteins expressed in exosomes (afamin, vitamin D-binding protein, and filamin A), and miRNAs expressed in prostate tissue (miRNA-21, miRNA-101, and miRNA-182). In conclusion, exploring the impact of lifestyle and inflammation on PCa development and progression may open doors to the identification of new biomarkers. The discovery of new PCa diagnostic biomarkers should contribute to reduce overdiagnosis and overtreatment.

The Effect of Nutrition on Aging-A Systematic Review Focusing on Aging-Related Biomarkers.

Despite the increasing life expectancy, an individual's later years tends to be accompanied by a decrease in the quality of life. Though biological changes that occur through the natural process of aging cannot be controlled, the risk factors associated with lifestyle can. Thus, the main goal of this systematic review was to evaluate how nutrition can modulate aging. For this purpose, thirty-six studies were selected on (i) the efficiency of nutrition's effect on aging, (ii) the evaluation of biomarkers that promote healthy aging, and (iii) how to increase longevity through nutrition, and their quality was assessed. The results showed that choosing low carbohydrate diets or diets rich in vegetables, fruits, nuts, cereals, fish, and unsaturated fats, containing antioxidants, potassium, and omega-3 decreased cardiovascular diseases and obesity risk, protected the brain from aging, reduced the risk of telomere shortening, and promoted an overall healthier life. With this study, the conclusion is that since the biological processes of aging cannot be controlled, changing one's nutritional patterns is crucial to prevent the emergence and development of diseases, boost longevity, and, mostly, to enhance one's quality of life and promote healthy aging.

l-α-aminoadipate causes astrocyte pathology with negative impact on mouse hippocampal synaptic plasticity and memory.

Increasing evidence shows that astrocytes, by releasing and uptaking neuroactive molecules, regulate synaptic plasticity, considered the neurophysiological basis of memory. This study investigated the impact of l-α-aminoadipate (l-AA) on astrocytes which sense and respond to stimuli at the synaptic level and modulate hippocampal long-term potentiation (LTP) and memory. l-AA selectivity toward astrocytes was proposed in the early 70's and further tested in different systems. Although it has been used for impairing the astrocytic function, its effects appear to be variable in different brain regions. To test the effects of l-AA in the hippocampus of male C57Bl/6 mice we performed two different treatments (ex vivo and in vivo) and took advantage of other compounds that were reported to affect astrocytes. l-AA superfusion did not affect the basal synaptic transmission but decreased LTP magnitude. Likewise, trifluoroacetate and dihydrokainate decreased LTP magnitude and occluded the effect of l-AA on synaptic plasticity, confirming l-AA selectivity. l-AA superfusion altered astrocyte morphology, increasing the length and complexity of their processes. In vivo, l-AA intracerebroventricular injection not only reduced the astrocytic markers but also LTP magnitude and impaired hippocampal-dependent memory in mice. Interestingly, d-serine administration recovered hippocampal LTP reduction triggered by l-AA (2 h exposure in hippocampal slices), whereas in mice injected with l-AA, the superfusion of d-serine did not fully rescue LTP magnitude. Overall, these data show that both l-AA treatments affect astrocytes differently, astrocytic activation or loss, with similar negative outcomes on hippocampal LTP, implying that opposite astrocytic adaptive alterations are equally detrimental for synaptic plasticity.

In-Depth Analysis of the Impact of Different Serum-Free Media on the Production of Clinical Grade Dendritic Cells for Cancer Immunotherapy.

Dendritic cell (DC)-based antitumor vaccines have proven to be a safe approach, but often fail to generate robust results between trials. Translation to the clinic has been hindered in part by the lack of standard operation procedures for vaccines production, namely the definition of optimal culture conditions during ex-vivo DC differentiation. Here we sought to compare the ability of three clinical grade serum-free media, DendriMACS, AIM-V, and X-VIVO 15, alongside with fetal bovine serum-supplemented Roswell Park Memorial Institute Medium (RPMI), to support the differentiation of monocyte-derived DCs (Mo-DCs). Under these different culture conditions, phenotype, cell metabolomic profiles, response to maturation stimuli, cytokines production, allogenic T cell stimulatory capacity, as well as priming of antigen-specific CD8+ T cells and activation of autologous natural killer (NK) cells were analyzed. Immature Mo-DCs differentiated in AIM-V or X-VIVO 15 presented lower levels of CD1c, CD1a, and higher expression of CD11c, when compared to cells obtained with DendriMACS. Upon stimulation, only AIM-V or X-VIVO 15 DCs acquired a full mature phenotype, which supports their enhanced capacity to polarize T helper cell type 1 subset, to prime antigen-specific CD8+ T cells and to activate NK cells. CD8+ T cells and NK cells resulting from co-culture with AIM-V or X-VIVO 15 DCs also showed superior cytolytic activity. 1H nuclear magnetic resonance-based metabolomic analysis revealed that superior DC immunostimulatory capacities correlate with an enhanced catabolism of amino acids and glucose. Overall, our data highlight the impact of critically defining the culture medium used in the production of DCs for clinical application in cancer immunotherapy. Moreover, the manipulation of metabolic state during differentiation could be envisaged as a strategy to enhance desired cell characteristics.

Bone marrow cell response after injury and during early stage of regeneration is independent of the tissue-of-injury in 2 injury models.

Selectively recruiting bone marrow (BM)-derived stem and progenitor cells to injury sites is a promising therapeutic approach. The coordinated action of soluble factors is thought to trigger the mobilization of stem cells from the BM and recruit them to lesions to contribute to tissue regeneration. Nevertheless, the temporal response profile of the major cellular players and soluble factors involved in priming the BM and recruiting BM-derived cells to promote regeneration is unknown. We show that injury alters the BM cellular composition, introducing population-specific fluctuations during tissue regeneration. We demonstrate that injury causes an immediate, transient response of mesenchymal stromal cells and endothelial cells followed by a nonoverlapping increase in hematopoietic stem and progenitor cells. Moreover, BM reaction is identical whether the injury is inflicted on skin and muscle or also involves a bone defect, but these 2 injury paradigms trigger distinct systemic cytokine responses. Together, our results indicate that the BM response to injury in the early stages of regeneration is independent of the tissue-of-injury based on the 2 models used, but the injured tissue dictates the systemic cytokine response.-Leitão, L., Alves, C. J., Alencastre, I. S., Sousa, D. M., Neto, E., Conceição, F., Leitão, C., Aguiar, P., Almeida-Porada, G., Lamghari, M. Bone marrow cell response after injury and during early stage of regeneration is independent of the tissue-of-injury in 2 injury models.

Utility of new-generation pacemakers in sleep apnea screening.

OBJECTIVE: Patients with cardiac pacemakers present a high prevalence of undiagnosed sleep apnea syndrome (SAS). New-generation pacemakers have algorithms that identify sleep respiratory events. Our aim was to evaluate their accuracy in the diagnosis of SAS. METHODS: We performed a prospective study that included patients with new-generation pacemakers (Reply 200 pacemakers). All patients underwent a polysomnography (PSG). On the same night, the respiratory disturbance index of the PSG (RDI-PSG) and of the pacemaker (RDI-PM) were recorded. The agreement between methods was assessed using the kappa coefficient, Bland and Altman statistics and receiver operating characteristic (ROC) curves. RESULTS: Sixty patients were recruited but the RDI-PM for the PSG night was not available in six patients. PSG diagnosed SAS in 74% of patients (20% severe, 19% moderate, 35% mild). Besides snoring (63%), most patients had no SAS symptoms. There was a strong positive correlation between RDI-PSG and RDI-PM (r = 0.522, p < 0.001), but the level of agreement between methods regarding SA diagnosis/severity was poor (k = 0.167). ROC curves identified a RDI-PM of 10 events/h as the optimal cut-off point for diagnosing SAS (area under the curve (AUC): 0.81, sensitivity: 80%, specificity: 79%, positive predictive value: 91%, negative predictive value: 58%). The best cut-off for identifying moderate/severe SAS was at 13 events/h (AUC: 0.86, sensitivity: 100%, specificity: 70%, positive predictive value: 68%, negative predictive value: 100%). CONCLUSIONS: SAS prevalence in patients with pacemakers is high (74%). Most are asymptomatic, which could delay the diagnosis. Patients with clinical indication for a pacemaker may benefit from a device with sleep apnea monitoring.

Thymic crosstalk restrains the pool of cortical thymic epithelial cells with progenitor properties.

Cortical (cTEC) and medullary (mTEC) thymic epithelial cells establish key microenvironments for T-cell differentiation and arise from thymic epithelial cell progenitors (TEP). However, the nature of TEPs and the mechanism controlling their stemness in the postnatal thymus remain poorly defined. Using TEC clonogenic assays as a surrogate to survey TEP activity, we found that a fraction of cTECs generates specialized clonal-derived colonies, which contain cells with sustained colony-forming capacity (ClonoTECs). These ClonoTECs are EpCAM+MHCII-Foxn1lo cells that lack traits of mature cTECs or mTECs but co-express stem-cell markers, including CD24 and Sca-1. Supportive of their progenitor identity, ClonoTECs reintegrate within native thymic microenvironments and generate cTECs or mTECs in vivo. Strikingly, the frequency of cTECs with the potential to generate ClonoTECs wanes between the postnatal and young adult immunocompetent thymus, but it is sustained in alymphoid Rag2-/-Il2rg-/- counterparts. Conversely, transplantation of wild-type bone marrow hematopoietic progenitors into Rag2-/-Il2rg-/- mice and consequent restoration of thymocyte-mediated TEC differentiation diminishes the frequency of colony-forming units within cTECs. Our findings provide evidence that the cortical epithelium contains a reservoir of epithelial progenitors whose abundance is dynamically controlled by continual interactions with developing thymocytes across lifespan.

Molecular characterization of CD44+/CD24-/Ck+/CD45- cells in benign and malignant breast lesions.

Breast cancer epithelial cells with the CD44+/CD24-/low phenotype possess tumor-initiating cells and epithelial-mesenchymal transition (EMT) capacity. Massive parallel sequencing can be an interesting approach to deepen the molecular characterization of these cells. We characterized CD44+/CD24-/cytokeratin(Ck)+/CD45- cells isolated through flow cytometry from 43 biopsy and 6 mastectomy samples harboring different benign and malignant breast lesions. The Ion Torrent Ampliseq Cancer Hotspot panel v2 (CHPv2) was used for the identification of somatic mutations in the DNA extracted from isolated CD44+/CD24-/Ck+/CD45- cells. E-Cadherin and vimentin immunohistochemistry was performed on sections from the corresponding formalin-fixed, paraffin-embedded (FFPE) blocks. The percentage of CD44+/CD24-/Ck+/CD45- cells increased significantly from non-malignant to malignant lesions and in association with a significant increase in the expression of vimentin. Non-malignant lesions harbored only a single-nucleotide polymorphism (SNP). Mutations in the tumor suppressor p53 (TP53), NOTCH homolog 1 (NOTCH1), phosphatase and tensin homolog (PTEN), and v-akt murine thymoma viral oncogene homolog 1 (AKT1) genes were found in isolated CD44+/CD24-/Ck+/CD45- cells from ductal carcinomas in situ (DCIS). Additional mutations in the colony-stimulating factor 1 receptor (CSF1R), ret proto-oncogene (RET), and TP53 genes were also identified in invasive ductal carcinomas (IDCs). The use of massive parallel sequencing technology for this type of application revealed to be extremely effective even when using small amounts of DNA extracted from a low number of cells. Additional studies are now required using larger cohorts to design an appropriate mutational profile for this phenotype.

Predominant role of interferon-γ in the host protective effect of CD8(+) T cells against Neospora caninum infection.

It is well established that CD8(+) T cells play an important role in protective immunity against protozoan infections. However, their role in the course of Neospora caninum infection has not been fully elucidated. Here we report that CD8-deficient mice infected with N. caninum presented higher parasitic loads in the brain and lungs and lower spleen and brain immunity-related GTPases than their wild-type counterparts. Moreover, adoptive transfer of splenic CD8(+) T cells sorted from N. caninum-primed immunosufficient C57BL/10 ScSn mice prolonged the survival of infected IL-12-unresponsive C57BL/10 ScCr recipients. In both C57BL/6 and C57BL/10 ScSn mice CD8(+) T cells are activated and produce interferon-γ (IFN-γ) upon challenged with N. caninum. The host protective role of IFN-γ produced by CD8(+) T cells was confirmed in N. caninum-infected RAG2-deficient mice reconstituted with CD8(+) T cells obtained from either IFN-γ-deficient or wild-type donors. Mice receiving IFN-γ-expressing CD8(+) T cells presented lower parasitic burdens than counterparts having IFN-γ-deficient CD8(+) T cells. Moreover, we observed that N. caninum-infected perforin-deficient mice presented parasitic burdens similar to those of infected wild-type controls. Altogether these results demonstrate that production of IFN-γ is a predominant protective mechanism conferred by CD8(+) T cells in the course of neosporosis.

Lymphocyte gene expression signatures from patients and mouse models of hereditary hemochromatosis reveal a function of HFE as a negative regulator of CD8+ T-lymphocyte activation and differentiation in vivo.

Abnormally low CD8+ T-lymphocyte numbers is characteristic of some patients with hereditary hemochromatosis (HH), a MHC-linked disorder of iron overload. Both environmental and genetic components are known to influence CD8+ T-lymphocyte homeostasis but the role of the HH associated protein HFE is still insufficiently understood. Genome-wide expression profiling was performed in peripheral blood CD8+ T lymphocytes from HH patients selected according to CD8+ T-lymphocyte numbers and from Hfe-/- mice maintained either under normal or high iron diet conditions. In addition, T-lymphocyte apoptosis and cell cycle progression were analyzed by flow cytometry in HH patients. HH patients with low CD8+ T-lymphocyte numbers show a differential expression of genes related to lymphocyte differentiation and maturation namely CCR7, LEF1, ACTN1, NAA50, P2RY8 and FOSL2, whose expression correlates with the relative proportions of naïve, central and effector memory subsets. In addition, expression levels of LEF1 and P2RY8 in memory cells as well as the proportions of CD8+ T cells in G2/M cell cycle phase are significantly different in HH patients compared to controls. Hfe-/- mice do not show alterations in CD8+ T-lymphocyte numbers but differential gene response patterns. We found an increased expression of S100a8 and S100a9 that is most pronounced in high iron diet conditions. Similarly, CD8+ T lymphocytes from HH patients display higher S100a9 expression both at the mRNA and protein level. Altogether, our results support a role for HFE as a negative regulator of CD8+ T-lymphocyte activation. While the activation markers S100a8 and S100a9 are strongly increased in CD8+ T cells from both, Hfe-/- mice and HH patients, a differential profile of genes related to differentiation/maturation of CD8+ T memory cells is evident in HH patients only. This supports the notion that HFE contributes, at least in part, to the generation of low peripheral blood CD8+ T lymphocytes in HH.

Identification of human T-cell receptors with optimal affinity to cancer antigens using antigen-negative humanized mice.

Identifying T-cell receptors (TCRs) that bind tumor-associated antigens (TAAs) with optimal affinity is a key bottleneck in the development of adoptive T-cell therapy of cancer. TAAs are unmutated self proteins, and T cells bearing high-affinity TCRs specific for such antigens are commonly deleted in the thymus. To identify optimal-affinity TCRs, we generated antigen-negative humanized mice with a diverse human TCR repertoire restricted to the human leukocyte antigen (HLA) A*02:01 (ref. 3). These mice were immunized with human TAAs, for which they are not tolerant, allowing induction of CD8⁺ T cells with optimal-affinity TCRs. We isolate TCRs specific for the cancer/testis (CT) antigen MAGE-A1 (ref. 4) and show that two of them have an anti-tumor effect in vivo. By comparison, human-derived TCRs have lower affinity and do not mediate substantial therapeutic effects. We also identify optimal-affinity TCRs specific for the CT antigen NY-ESO. Our humanized mouse model provides a useful tool for the generation of optimal-affinity TCRs for T-cell therapy.

Transgenic mice with a diverse human T cell antigen receptor repertoire.

Because of tolerance mechanisms, it has been hard to identify the T cell receptors (TCRs) of high-avidity T cells against self (for example, tumor) antigens. TCRs that are specific for foreign human antigens from the nontolerant T cell repertoire can be identified in mice. Moreover, if mice are constructed to express the human TCR repertoire, they can be used to analyze the unskewed repertoire against human self antigens. Here we generated transgenic mice with the entire human TCRalphabeta gene loci (1.1 and 0.7 Mb), whose T cells express a diverse human TCR repertoire that compensates for mouse TCR deficiency. A human major histocompatibility class I transgene increases the generation of CD8+ T cells with human compared to mouse TCRs. Functional CD8+ T cells against several human tumor antigens were induced, and those against the Melan-A melanoma antigen used similar TCRs to those that have been detected in T cell clones from individuals with autoimmune vitiligo or melanoma. These mice will allow researchers to identify pathogenic and therapeutic human TCRs.

The role of TCR specificity and clonal competition during reconstruction of the peripheral T cell pool.

Survival of peripheral CD8(+) T cells requires TCR interactions with peptide-MHC complexes (p-MHC). In the adult mouse, in the presence of homeostatic mechanisms that strictly control T cell numbers, it is likely that diverse T cell clones may compete for shared patterns of p-MHC. In the present study, we investigate whether the recognition of p-MHC overlaps between different T cell populations and what role does this process plays in the establishment of the peripheral T cell pools. Using an experimental strategy that follows the fate of adoptively transferred polyclonal T cells into RAG(0/0) or different TCR transgenic RAG(0/0) hosts, we demonstrate that T cells bearing different TCR specificities share identical TCR-specific requirements for survival and lymphopenia driven proliferation (LDP). This interclonal competition applies to both naive and activated/memory T cells and is partially determined by the clone size of the established/resident T cells. However, clonal competition with activated/memory resident T cells impacts differently on the fate of newly produced bone-marrow-derived T cells or adoptively transferred peripheral T cells. Overall, our findings indicate that p-MHC define multiple diverse resource niches that can be shared by T cells from different compartments.