Repositório Científico do Instituto Nacional de Saúde
Entradas recentes
Statistical analysis of longitudinal RBC omics data
Publication . Silva, Carolina; Antunes, Marília; Penque, Deborah
Red blood cells (RBCs) are emerging as important modulators of the immune system. Despite evidence that alterations in RBC functionality are associated with disease severity in COVID-19 patients, there is no information regarding the impact of RBC activity on the immune response to COVID-19 vaccination. This work aims to establish an adequate methodology for the statistical analysis of longitudinal RBC metabolomics data collected during COVID-19 vaccination (n=22, 5 time points) to identify metabolites with significant changes throughout the immunization process.
For the pre-treatment of the metabolomics data set, different pre-treatment methodologies comprised of imputation and normalization steps were compared to investigate which algorithm and application order was more adequate. Testing of these methods showed that normalization followed by kNN imputation using cosine distance was highlighted as the best-performing pre-treatment strategy. Following its application, generalized estimating equations (GEEs) created from the normalized data led to the identification of 30 metabolites with significant changes in concentration between different time points of COVID-19 vaccination.
Significant RBC metabolites were linked to major metabolic pathways in the cells, such as the metabolism of amino acids and purines, and the transport of small molecules through the cellular membrane. Some of these metabolites were discovered to have relevant functions in the development of an effective immune response against infections, like COVID-19. The connections between these metabolites and the defense mechanisms commonly used by cells to fight viral infections offer a strong clue for the immune functions that those metabolites may have in the human body, suggesting that the RBC metabolism could play a significant part in the generation of an immune response to COVID-19
vaccination.
Further work is in progress to integrate and correlate proteomic data retrieved from the same longitudinal experiment for a comprehensive depiction of the RBC function in the COVID-19 vaccine-induced immunization process.
Assessing the pro inflammatory effects of bisphenol compounds using exposure relevant in vitro co culture models
Publication . Pereira, Gonçalo Alexandre Candeia; Jordan, Peter; Rodrigues, Cecília
Inflammation has reached epidemic proportions in industrialized countries, mainly due to unhealthy habits, poor diet, environmental pollution and other factors not yet understood. If uncontrolled or prolonged, inflammation can become chronic and contribute to the development of a number of human diseases, including autoimmune diseases, intestinal diseases and, in the worst cases, tumorigenesis and tumor progression. Exposure to endocrine disrupting chemicals (EDCs) is one environmental factor contributing to inflammation, and recent studies have brought the bisphenol (BP) group of EDCs into the scientific spotlight. They have been strongly linked to various pathologies, including chronic inflammation, and their effect on human gut health is a hot topic in the scientific community. With this in mind, the aim of this work was proposed to analyze the effects of four bisphenols, BPA, BPS-MAE, BPAP and BPP, on intestinal barrier stress and associated pro-inflammatory effects. To achieve this, a co-culture system was optimized and established, consisting of an improved protocol of polarized Caco-2 epithelial cells seeded on PET insert filters in an apical compartment, together with THP-1 derived macrophages in a basolateral compartment. Subsequently, the effects of BPs exposure on barrier integrity, cellular stress and pro-inflammatory cytokine were tested in a wide range of concentrations (from 100 μM to 0.1 μM). Experimentally, we found that the model was capable of delivering BP-specific data on potential health effects. In terms of transepithelial resistance and epithelial stress, we were able to identify some clear trends that need to be consolidated with more independent experimental replicates. In particular, BPA was the least potent inducer of cellular stress responses and changes in epithelial polarization, whereas the BP analogues tested proved to be more disruptive than BPA, with BPP appearing to be the most potentially hazardous, followed by BPAP and then BPS-MAE. To access the inflammation-modulating effects of these compounds, we tested macrophages, either directly or as co-cultured cells, for expression of the pro-inflammatory marker IL-1β using a semiquantitative RT-PCR approach. An important optimization was their priming with IFN-γ to increase the sensitivity of the model and allow for more physiological relevance. Our observations showed that, once again, the BP analogues induced greater effects compared to BPA. BPP appeared to be the more potent inducer of inflammation, followed by BPS-MAE. Both showed elevated levels of the IL-1β marker at all concentrations tested. BPAP and BPA produced more attenuated effects, although significant at higher concentrations. In conclusion, this work has provided us with landmark results on these BPA analogues and their effects on gut health, adding new insights into the 'new generation' of emerging BPs and their potential adverse health effects.
Establishment of a Human iPSC Line from Mucolipidosis Type II That Expresses the Key Markers of the Disease
Publication . Moutinho, Maria Eduarda; Gonçalves, Mariana; Duarte, Ana J.; Encarnação, Marisa; Coutinho, Maria Francisca; Matos, Liliana; Santos, Juliana I.; Ribeiro, Diogo; Amaral, Olga; Gaspar, Paulo; Alves, Sandra; Moreira, Luciana V.
Mucolipidosis type II (ML II) is a rare and fatal disease of acid hydrolase trafficking. It is caused by pathogenic variants in the GNPTAB gene, leading to the absence of GlcNAc-1-phosphotransferase activity, an enzyme that catalyzes the first step in the formation of the mannose 6-phosphate (M6P) tag, essential for the trafficking of most lysosomal hydrolases. Without M6P, these do not reach the lysosome, which accumulates undegraded substrates. The lack of samples and adequate disease models limits the investigation into the pathophysiological mechanisms of the disease and potential therapies. Here, we report the generation and characterization of an ML II induced pluripotent stem cell (iPSC) line carrying the most frequent ML II pathogenic variant [NM_024312.5(GNPTAB):c.3503_3504del (p.Leu1168fs)]. Skin fibroblasts were successfully reprogrammed into iPSCs that express pluripotency markers, maintain a normal karyotype, and can differentiate into the three germ layers. Furthermore, ML II iPSCs showed a phenotype comparable to that of the somatic cells that originated them in terms of key ML II hallmarks: lower enzymatic activity of M6P-dependent hydrolases inside the cells but higher in conditioned media, and no differences in an M6P-independent hydrolase and accumulation of free cholesterol. Thus, ML II iPSCs constitute a novel model for ML II disease, with the inherent iPSC potential to become a valuable model for future studies on the pathogenic mechanisms and testing potential therapeutic approaches.
Systemic lupus erythematosus and the gut microbiome: To look forward is to look within – A systematic review and narrative synthesis
Publication . Oliveira, Daniel Guimarães; Machado, Alexandra; Castro, Pedro Lacerda; Karakikla-Mitsakou, Zoe; Vasconcelos, Carlos
Background: Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease shaped by complex interactions involving genetic and environmental factors. Among these, the gut microbiome is emerging as potentially modulating immune responses and influencing disease susceptibility, progression, and activity.
Objectives: To synthesize current evidence on gut microbiome changes in adult SLE patients, framed along the clinical pathway – from diagnosis to treatment – to help bridge bench and bedside for microbiome-informed SLE care and research.
Methods: A systematic search identified primary research studies examining gut microbiota in adult SLE patients. Studies were reviewed in a stepwise manner by independent investigators. Findings were synthesized narratively, emphasizing human data.
Results: SLE patients exhibit gut microbiome dysbiosis, with reduced microbial richness and altered bacterial taxa. A lower Firmicutes/Bacteroidetes ratio is frequently observed. Enrichment of specific taxa, such as Enterococcus, Lactobacillus, and Ruminococcus gnavus, is reported. Dysbiosis correlates with increased gut permeability,
immune activation, and autoreactivity. Clinical associations include disease activity, flares, nephritis, and other manifestations. SLE treatments, such as hydroxychloroquine and corticosteroids, influence the microbiome.
Emerging interventions such as dietary modulation and fecal microbiota transplantation show promise in early studies. However, considerable heterogeneity exists across studies in terms of patient characteristics, methodology, and taxa-level findings.
Conclusions: The gut microbiome has multifaceted associations with SLE pathogenesis, disease activity, and therapeutic response. Translation will require standardized methods, functional validation, longitudinal followup, and clinical integration. While uncertainties remain, the gut microbiome is increasingly relevant, and clinicians caring for patients with SLE should be aware of its emerging implications.
Toward harmonizing protein data in food composition databases: evaluating perspectives, methods and implications
Publication . Pferdmenges, Larissa E.; Colombani, Paolo C.; Carlsen, Monica Hauger; Pajari, Anne-Maria; Poulsen, Anders; Dias, Maria da Graça; Moller, Anders; Lisciani, Silvia; Wust, Matthias; Bonsmann, Stefan; Schweiggert-Weisz, Ute
Protein content in foods has historically been estimated by multiplying measured nitrogen content with a universal nitrogen-to-protein conversion factor (NCF) of 6.25. Despite scientific consensus that this approach leads to systematic overestimations due to variations in amino acid composition and non-protein nitrogen (NPN) content, no universally accepted revision has been implemented. This review critically examines diverse perspectives on protein quantification and their implications for Food Composition Databases (FCDBs). A structured definition of protein for FCDBs is proposed, including amino acid residues, free amino acids and small peptides, while explicitly excluding NPN and prosthetic groups. Furthermore, analytical methods and NCF calculations are evaluated in order to provide more accurate assessments of protein content across a range of food matrices. The review highlights the importance of selecting food-specific NCFs to reduce overestimations, ensuring both scientific accuracy and practical feasibility. By addressing methodological shortcomings and proposing a refined protein quantification framework, this work aims to facilitate the transition toward more precise and harmonized protein values in FCDBs, benefiting nutritional research, dietary guidelines, and food labeling regulations.