Browsing by Issue Date, starting with "2023-08-21"
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- Targeting lysosomal proteases for a host-directed therapy for tuberculosisPublication . Pires, DavidTuberculosis is a disease caused by the bacteria Mycobacterium tuberculosis (Mtb) which latently infects one-quarter of the human population and is the leading cause of death by an infectious agent. Contributing to the challenge of this old disease is the lack of (1) an effective vaccine, (2) reliable biomarkers for latent infection, (3) the limited number of effective antimicrobial drugs, (4) a prolonged therapeutical regime, and (5) the evolution of multi-drug resistant strains. This increasingly foments the need for novel therapies that target the bacterial niche or improve the host response, alone and in combination with the current conventional therapy. We have been probing the intracellular niche of Mtb, the macrophage, for how these bacteria survive and replicate inside them while impairing their bactericidal response. In that search, we found that Mtb induces a decrease in the expression of a group of proteolytic enzymes, the cathepsins, that participate in key cellular processes regulating homeostasis, cell death, inflammation, antigen presentation, and microbial killing. Regarding Mtb infection, this downregulation results in improved bacterial survival and replication inside macrophages as well as poor lymphocyte priming by the infected cells. To address this problem, we have been exploring the different pathways by which cathepsin activity is regulated in our cells. So far, we found three levels of cathepsins regulation that can be manipulated to our advantage: One is by targeting miRNAs to restore cathepsins gene expression; another by targeting cystatins, the natural inhibitors of cathepsins, to restore their activity; and finally, by using saquinavir, a repurposed inhibitor of the HIV protease that unexpectedly improves the activity of some human cathepsins. Together, these strategies were shown to improve the intracellular killing of Mtb by macrophages, as well as enhance the ability of these cells to prime CD4+ T-lymphocytes and induce their proliferation and IFNγ secretion. Our approach suggests a potential host-targeted strategy that can be developed as a complementary therapy to current antibiotics. Human monocytes were isolated from buffy-coats of healthy human donors provided by the National Blood Institute (Instituto Português do Sangue e da Transplantação, IP, Lisbon, Portugal).
- Mass spectrometry‑based identification of peptides presented by major histocompatibility complex in macrophagesPublication . Mateus, Hugo; Pinheiro, Ricardo; Santos, Hugo M.; Bettencourt, Paulo J. G.Immunopeptidomics is a field of research that has progressed in thelast years due to advances in sophisticated analytical techniquesbased on mass spectrometry and bioinformatics. The ability to identifymolecules to the extent of a single ion led to a step forward inimmunopeptidomics. Mass spectrometry enables the identificationof thousands of peptide sequences in a single sample, thus providinglarge-scale reliable information. The immunopeptidome is the entire group of peptides presented by the major histocompatibility complexClass-I (MHC-I), at the surface of all nucleated cells and Class II, at thesurface of professional antigen presenting cells. The MHC-bound peptidesare recognized by T cells and constitute the immunological synapse,leading to the initiation of the adaptive immune response. Underpathological conditions, peptides originating from the proteolysis ofpathogen proteins are presented to the cells of the host immune systemvia MHC. Thus, the identification of pathogen peptides throughimmunopeptidomics is an unbiased method for understanding thegeneration of adaptive immune responses against pathogens.Here we describe the establishment of a new mass spectrometrybasedimmunopeptidomics platform for peptide identification inphysiological and pathological conditions. Using the macrophage cellline with THP-1, with a known HLA-type, we were able to identify atotal of 2913 unique MHC-I bound peptides. The peptide length distribution,NetMHCpan-4.1 rank affinity, and best match HLA bindingallele for each peptide will be presented.Finally, identifying MHC-I and MHC-II peptides under physiologicaland pathological conditions could uncover the most relevant peptidesable to stimulate the right type of T-cell response for vaccine designand development.
- Usability testing of the implant disease risk assessment IDRA, a tool for preventing peri-implant disease: a protocol designPublication . Bornes, Rita; Montero, Javier; Correia, André; Rosa, Nuno; Ferreira, Ana
- Implementation of a pre‑Good Laboratory Practice management system for academic researchPublication . Pinheiro, Ricardo; Abreu, Cloé; Bettencourt, Paulo J. G.The implementation of quality control procedures, at an academiclaboratory, relies on a system that flows information to scientists, staff,and students in a clear and accountable manner.The organization and implementation of new methodologies, in a newlaboratory, implies the definition of a work culture and structure frominception to completion. Establishing and maintaining a new workphilosophy is demanding and requires constant and close supervisionof all laboratory actions. Particularly, when the methods are innovativeand require a significant change of work culture from users.By establishing a system that standardizes common laboratory protocolsto facilitate training while simultaneously tracking progress, wesuccessfully implemented a pre-Good Laboratory Practices (pre-GLP)facility at the Faculty of Medicine of the Universidade Católica Portuguesa(FM).The pre-GLP system is an adaption of the system adopted by theJenner Institute, University of Oxford. Briefly, the new users aretrained on Standard Operations Procedures (SOP), provided by acompetent user. Once training is successfully completed, the user isapproved and qualified as competent user. All training actions arerecorded in the researcher’s internal record. The internal records areinternally verified by the laboratory manager, and laboratory director,and externally audited.The SOPs are regularly updated and improved to reflect any significantupdates on procedures, equipment, and reagents. UpdatedSOP´s are reassessed and follow the pipeline of approval. Implementationof this laboratory management system is a step forwardin quality assurance and standardization of methodologies towardsgood laboratorial practices, increased health, and safety, and qualitydata production.Finally, the implementation of this quality assurance method at theFM, provides an additional layer of health and safety protection forusers, simultaneously assuring reproducibility and reliability of protocolsacross the campus.
- Development of a new mRNA vaccine platform for tuberculosisPublication . Matarazzo, Laura; Taina‑González, Laura; Pinheiro, Ricardo; Pires, David; de la Fuente, María; Bettencourt, Paulo J. G.Background Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), is the frst cause of death by an infectious disease worldwide, killed 1.6 million people in 2021. Bacillus Calmette-Guerin (BCG) is the only approved vaccine for TB to date. However, while BCG is efective in preventing severe forms in children, its efcacy in adults is inconsistent and it does not prevent transmission, highlighting the need for new vaccine development [1]. The recent success of COVID-19 vaccines raised the interest for mRNA-based vaccines, as they are efective, safe and easy to produce. This project aims to develop a new mRNA vaccine platform for TB, based on mRNA coding for antigenic peptides from BCG and M.tb identifed by immunopeptidomics [2], and formulated with a patented technology of lipid nanoemulsions (NE) (WO2019138139A1), adapted for efcient intracellular delivery of mRNA [3]. Materials and methods We tested diferent prototypes of NE-mRNA formulations, coding for EGFP, in vitro. Human alveolar basal epithelial cells (A549), human monocytic cells (THP-1), and primary human monocyte-derived macrophages, were transfected with NE-mRNA formulations. Transfection efciency was assessed by measuring the percentage of transfected cells, and the intensity of GFP fuorescence. The cytotoxicity of the formulations was evaluated using AlamarBlue, and by 7-AAD viability staining. Results In vitro preliminary data using EGFP-mRNA-NE formulations indicate that NE formulations can efciently deliver mRNA and induce expression of the encoded protein in diferent cell types, with low cytotoxicity. Conclusions The NE technology presented here is safe, stable, and can efciently deliver mRNA to various cell types. Selected NE formulations will be used as a carrier for a new vaccine candidate against TB, based on mRNA encoding relevant antigenic peptides. These will be tested in mice for safety, immunogenicity, efcacy and dose optimization in order to generate an efective and sustained humoral and cellular immune response against TB. The mRNA vaccines are rapid and relatively simple to produce. The vaccine platform described here could be adapted to develop vaccines against other infectious diseases, particularly to quickly respond to emerging pathogens.
- Diagnosis and risk prevention of Covid 19 in homeless people in LisbonPublication . Figueiredo, Amélia Simões; Resende, Ana; Ferrito, Cândida; Deodato, Sérgio; Neves-Amado, João; Manso, Dina; Almeida, António; Feliciano, Amélia; Rosa, Nuno; Barros, MarleneBackground The study is part of a broader research project – Public Bathouse Nursing - dedicated to the study of vulnerable populations that use the Public Bathouse in the city of Lisbon [1]. When SARS-CoV-2 pandemic situation started, structures for homeless people, at that time counted as 3029, were reinforced in Lisbon city. These people do not manage health autonomously, thus requiring defnition of strategies for testing and early diagnosis, to promote isolation and quick endorsementto Portuguese National Health Service. The project was approved by the Health Ethics Committee of the Regional Health Administration of Lisbon and Tagus Valley (Opinion 2776/CES/2021). Materials and methods Tracking SARS-CoV-2 lab tests were performed to the people from Temporary Housing Centers, Social Emergency Housing Centers and to those who, being homeless, were identifed by street technical teams. Our objectives were the detection of SARS-CoV-2 in the homeless people using the Public Bathhouse of Alcântara and of 10 structures of Planning and Intervention Centers in homelessness, such as street teams, temporary emergency shelters and cafeterias, promoting early detection and adequate endorsement of positive cases. The process of collection of spittle was performed between November 29th,2021and February 7th, 2022. From the initial 455 samples collected, 59 were annulled, due to poor quality and quantity. An error of 3,6% was considered, with a confdence level of 95,0%, thus making viable samples n= 396. Results Those 396 tracked people revealed an incidence rate of the disease of 2%, while, during the same period, the whole country rounded 18%. A study carried out in the United States of America did not fnd statistically signifcant relevance between the values of the incidence of the disease COVID-19 among homeless and non-homeless [2]. On the other hand, a Danish study revealed that the homeless population seems not to have been afected by COVID-19 in the frst wave [3]. The positive cases of COVID-19 who were lodged temporarily in Social Emergency Housing Centers, were isolated there, upon indication of Regional and Local Authorities, that were properly informed. Positive cases of homelessness people identifed and tracked on the streets, were endorsed to the Rear Support Structure, to guarantee appropriate isolation and dignifed convalescence. Conclusions The conclusion was that the incidence rate of COVID-19 in homeless people is below general population in Portugal.
- Liposomal delivery of repurposed antiviral drug saquinavir to macrophages as a host-directed therapy for tuberculosisPublication . Mandal, Manoj; Pires, David; Pinho, Jacinta; Catalão, Maria João; Almeida, António José; Azevedo-Pereira, José Miguel; Gaspar, Maria Manuela; Anes, ElsaMycobacterium tuberculosis (Mtb) latently infects approximately a quarter of the world’s population and 10 % of these will develop the disease tuberculosis. Mtb infects macrophages, manipulating the proteolytic mechanisms, particularly, by decreasing the expression and activity of lysosomal cathepsins. Consequently, Mtb survives and even replicates inside macrophages concomitant with poor priming of the adaptive immune response. Our group found that the protease inhibitor used in antiretroviral therapy for HIV infection, saquinavir (SQV), restores and further improves the overall activity of cathepsins in Mtb-infected macrophages and more specifcally, that of cathepsin S [1]. In this study, we tested the incorporation of SQV in liposomes to establish an improved delivery method for SQV to human monocyte-derived macrophages. Using fuorophore-tagged liposomes we demonstrated the effciency of SQV-loaded liposome internalization by human macrophages. Additionally, using a general fuorescent substrate of human cathepsins we could observe improved proteolytic activity in treated macrophages. When applying this treatment to Mtb-infected macrophages these effects resulted in better control of the infection. Furthermore, liposomal delivery of SQV reduced the cytotoxicity of the treatment and allowed the usage of higher concentrations without impacting cell viability. By using this strategy, we overcame the cathepsin activity blockade that is induced by the pathogen [2]. The results further demonstrate the effcacy of SQV-loaded liposomes to help control infections by Mtb clinical strains susceptible or resistant to the current antibiotic therapy. Our results suggest the use of liposomal delivery of SQV as a potential complementary therapy against Mtb infection. Human monocytes were isolated from buffy-coats of healthy human donors provided by the National Blood Institute (Instituto Português do Sangue e da Transplantação, IP, Lisbon, Portugal).
- Adaptation and implementation of the European matrix for teaching spiritual care to nursing studentsPublication . Sitefane, Sara; Afonso, Ana; Rabiais, Isabel; Caldeira, SílviaBackground The World Health Organization’s concept of health currently comprises eight dimensions: emotional, spiritual, intellectual, physical, environmental, fnancial, occupational, and social. Including the spiritual dimension [1] represents an essential milestone in recognizing its positive impact on health, well-being, and quality of life. In this sense, nursing students’ acquisition and development of spiritual care skills are required, particularly in undergraduate nursing degrees. Also, the evidence demonstrates the positive relationship between spiritual education and spiritual competencies, emphasizing the need for spiritual education as an integral and regular part of the undergraduate nursing curriculum. Regardless of this evidence, the educational strategies for improving and developing undergraduate nursing students’ skills and competencies are scarce and should be urgently considered as nurses and midwives still report feeling unprepared for providing spiritual care. Recently, the EPICC project (Nurses’ and Midwives’ Competence in Providing Spiritual Care through Innovative Education and Compassionate Care)[2] has been implemented as a turning point in nursing education for spiritual care and spirituality, through a systematic, consensual, and efective response, by involving multiple partners and experts from diferent European countries. Portugal has been a participant in that Erasmus-funded project. Materials and methods This Ph.D. project concerns the translation, adaptation, and implementation of the EPICC matrix. First, a translation and cultural adaptation process will be conducted according to the core project guidelines [3]. Then the matrix for education and assessing spiritual care competencies will be implemented in a pilot study in a Portuguese nursing school involving undergraduate nursing students. Results The core project guidelines represent V stage of the cross-cultural adaptation process. The preliminary results point to the beginning of stage III with the back translation of the synthesized written version of the EPICC Spiritual Care Education Standard and EPICC Spiritual Care Competency Self-assessment tool. Stage I (initial translation with written reports of each – T1 and T2) and Stage II (synthesis of the translations to version T12) of the translation and cultural adaptation of the EPICC matrix are already concluded. Conclusions This innovative project could help improve Portuguese schools’ nursing curricula from an evidence-based perspective.
- Teaching spiritual care in Portuguese nursing schoolsPublication . Afonso, Ana; Sitefane, Sara; Rabiais, Isabel; Caldeira, SílviaSpiritual care is an essential dimension of holistic care. Organizations, such as the International Council of Nurses, mention the importance of spirituality for health and the urgency of nurses providing spiritual care. At the same time, studies show that spirituality brings benefts at the level of coping strategies both in crisis or struggling experiences, greater gratitude, facilitating forgiveness and meaning of life. However, spirituality and spiritual care seem to have been neglected and, among other factors, the lack of training in the nursing degree has been described as critical. The undergraduate nursing degree in Portugal should ensure scientifc, technical, human, and cultural training for of providing and managing general nursing care. As so, the undergraduate degree should provide the conditions to learn about spiritual care based on a holistic approach to patients, families, and communities. The school curriculum and respective syllabuses are not random, and the choice of diferent curricular units results from refexive and intentional processes in each institution. Materials and methods An exploratory study was conducted to map the explicit reference of spiritual care in the undergraduate nursing degree in all nursing schools in Portugal. Data were collected in September 2022 by searching for the spiritual* research term in the designation of the undergraduate nursing degree curricular units as displayed on the websites of the higher education institutions (HEI). Results Of the 36 HEI, none had curricular units entitled with spirituality or spiritual care. Conclusions Although it cannot be inferred that spiritual care is not addressed in the undergraduate nursing degree, as the curriculum is a form of social visibility of a discipline and a profession, these data should lead us to refect on the (in)visibility that is given to the spiritual dimension. So further studies are needed to disclose and understand how students are prepared to attend to patients in a holistic paradigm that includes attention to the spiritual dimension of health.