Revealing and Harnessing Tumour-Associated Microglia/Macrophage Heterogeneity in Glioblastoma

Cancer heterogeneity and progression are subject to complex interactions between neoplastic cells and their microenvironment, including the immune system. Although glioblastomas (GBMs) are classified as ‘cold tumours’ with very little lymphocyte infiltration, they can contain up to 30–40% of tumour-associated macrophages, reported to contribute to a supportive microenvironment that facilitates tumour proliferation, survival and migration. In GBM, tumour-associated macrophages comprise either resident parenchymal microglia, perivascular macrophages or peripheral monocyte-derived cells. They are recruited by GBMs and in turn release growth factors and cytokines that affect the tumour. Notably, tumour-associated microglia/macrophages (TAMs) acquire different expression programs, which shape the tumour microenvironment and contribute to GBM molecular subtyping. Further, emerging evidence highlights that TAM programs may adapt to specific tumour features and landscapes. Here, we review key evidence describing TAM transcriptional and functional heterogeneity in GBM. We propose that unravelling the intricate complexity and diversity of the myeloid compartment as well as understanding how different TAM subsets may affect tumour progression will possibly pave the way to new immune therapeutic avenues for GBM patients.publishedVersio

Elucidating tumour-associated microglia/macrophage diversity along glioblastoma progression and under ACOD1 deficiency

In glioblastoma (GBM), tumour-associated microglia/macrophages (TAMs) represent the major cell type of the stromal compartment and contribute to tumour immune escape mechanisms. Thus, targeting TAMs is emerging as a promising strategy for immunotherapy. However, TAM heterogeneity and metabolic adaptation along GBM progression represent critical features for the design of effective TAM-targeted therapies. Here, we comprehensively study the cellular and molecular changes of TAMs in the GL261 GBM mouse model, combining single-cell RNA-sequencing with flow cytometry and immunohistological analyses along GBM progression and in the absence of Acod1 (also known as Irg1), a key gene involved in the metabolic reprogramming of macrophages towards an anti-inflammatory phenotype. Similarly to patients, we identify distinct TAM profiles, mainly based on their ontogeny, that reiterate the idea that microglia- and macrophage-like cells show key transcriptional differences and dynamically adapt along GBM stages. Notably, we uncover decreased antigen-presenting cell features and immune reactivity in TAMs along tumour progression that are instead enhanced in Acod1-deficient mice. Overall, our results provide insight into TAM heterogeneity and highlight a novel role for Acod1 in TAM adaptation during GBM progression.publishedVersio

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

TUMOUR-ASSOCIATED MICROGLIA/MACROPHAGE HETEROGENEITY IN GLIOBLASTOMA

Glioblastoma (GBM) is the most common and aggressive primary brain tumour in adults, characterized by high degrees of both inter- and intra-tumour heterogeneity. GBM cells secrete numerous factors promoting the recruitment and infiltration of cellular players to the local tumour microenvironment. Tumour-associated microglia/macrophages (TAMs) represent the major cell type of the stromal compartment in GBM playing important roles along tumour development. Along GBM progression, these cells are supposed to be geared towards a tumour-supportive phenotype, therefore TAMs are pursued as key targets for the development of novel strategies aimed at re-educating them towards anti-tumour phenotypes. However, it is yet unclear how these immune suppressive properties are acquired and whether TAM subsets may phenotypically and functionally differently contribute to tumour development. Hence, the main goal of the present PhD project was to elucidate TAM diversity under defined temporal and spatial settings in GBM. Taking advantage of the GBM GL261 syngeneic and patient-derived orthotopic xenograft mouse models, we comprehensively studied the cellular and transcriptional heterogeneity of TAMs by combining single-cell RNA-sequencing, multicolour flow cytometry, immunohistological and functional analyses. We demonstrated that, as observed in patients, the myeloid compartment is the most affected and heterogeneous stromal compartment, with microglia and macrophage-like cells acquiring key transcriptional differences and rapidly adapting along GBM progression. Specifically, we uncovered that TAM transcriptional programmes converge over time, suggesting a context-dependent symbiosis mechanism characterized by decreased antigen-presenting cell signatures at late tumour stages. In the absence of Acod1/Irg1, a key gene involved in the metabolic reprogramming of macrophages towards an anti-inflammatory phenotype, we detected higher TAM diversity in the TME displaying increased immunogenicity and correlating with increased lymphocytic recruitment to the tumour site. Additionally, we uncovered that TAMs exhibit niche-specific functional adaptations in the tumour microenvironment, with microglia in the invasive landscapes displaying higher immune reactive profiles when compared to the corresponding cells in the angiogenic tumour phenotypes. Taken together, our data provide insights into the spatial and molecular heterogeneity of TAMs dynamically adapting along tumour progression or across specific tumour sites and revealing potential reactive anti-tumorigenic cell subsets that may be harnessed for therapeutic intervention in GBM

Protein misfolding and aggregation in neurodegenerative disorders

Dissertação de mestrado em Investigação Biomédica, apresentada à Faculdade de Medicina da Universidade de CoimbraProtein misfolding and aggregation is a common hallmark in neurodegenerative disorders, including Parkinson’s disease (PD) and Huntington Disease (HD). The misfolding and aggregation of specific proteins contributes to neuronal degeneration in somewhat specific areas of the brain. However, the mechanisms by which these proteins lead to degeneration remains poorly understood. The main goals of this study was to elucidate and identify the molecular determinants of the subcellular localization of alpha-synuclein (α-syn) in PD and also to study the effect of N-terminal phosphorylation in the aggregation and toxicity of mutant huntingtin (Htt) in HD using Drosophila melanogaster as model. Previous results from our lab have shown that the mutant form of α-syn protein (α-syn-EGFP A30P) fails to localize the photoreceptor’s synapses. In this study we used RNAi knockdown screen to identify gene modulators of α-syn-EGFP A30P localization by performing head cryossections of adult flies using GMR-Gal4 driver in order to express the protein α-syn (WT and mutant form) in the Drosophila eye. We validated that α-syn-EGFP A30P has a mislocalized distribution throughout the photoreceptors cytoplasm. Using specific genetic knockdown for spaghetti squash, tomosyn isoform C and synaptotagmin 4, we saw an increase of α-syn-EGFP A30P localization at the synapse and these specific interactors may be possible modulators of this localization. Phosphorylation pathways have been shown to modulate the toxicity of mutant Htt in vitro by affecting its oligomerization and aggregation dynamics. We used a Drosophila model where the amino acid residues T3, S13 and S16 of Httex1 97Q were mutated to aspartate (T3D, S13D and S16D - phosphomimics) or alanine (T3A, S13A and S16A - phosphoresistant). We did functional assays to evaluate the effect of N-terminal phosphorylation on motor abilities and survival rate and we also performed dissections of larval eye-imaginal discs and adult brains in order to characterize the pattern of aggregation of these mutants. Moreover, we studied the effect on Httex1 protein aggregation of particular phosphatases that may dephosphorylate NT17 using RNAi gene knockdown experiments in dopaminergic neurons, using TH-Gal4 driver. From our results it was possible to confirm that single phosphorylation on NT17 domain modulates Htt aggregation and neurotoxicity in Drosophila since phosphomimic mutants showed an improvement in motor function with the exception of the T3D mutant. Phosphomimic mutants also exhibited increased life span compared to the phosphoresistant mutants. Interestingly, phosphomimic mutants showed bigger aggregates than phosphoresistant mutants in two different cell types analyzed, the dopaminergic neurons of the adult brain and the photoreceptor cells of the larval eye-imaginal discs. We also test the effect of genetic knockdown for specific protein phosphatases on dopaminergic neurons and a significant reduction of mutant Htt aggregation was observed when PP1α-96A and PP1-87B protein phosphatases where downregulated. These findings suggest the mislocalization of α-syn-EGFP A30P may due to its interaction with other proteins in the cell and impairs its traffic to the synapse. In HD, phosphorylation has an important role in modulating aggregation and toxicity of Httex1 and specific phosphatases may improve the outcomes

A Molecular and Epidemiological Investigation of a Large SARS-CoV-2 Outbreak in a Long-Term Care Facility in Luxembourg, 2021

In spring 2021, a long-term care facility (LTCF) of 154 residents in Luxembourg experienced a large severe, acute respiratory-syndrome coronavirus 2 (SARS-CoV-2) outbreak a few days after a vaccination campaign. We conducted an outbreak investigation and a serosurvey two months after the outbreak, compared attack rates (AR) among residents and staff, and calculated hospitalization and case-fatality rates (CFR). Whole genome sequencing (WGS) was performed to detect variants in available samples and results were compared to genomes published on GISAID. Eighty-four (55%) residents and forty-five (26%) staff members tested positive for SARS-CoV-2; eighteen (21%) residents and one (2.2%) staff member were hospitalized, and twenty-three (CFR: 27%) residents died. Twenty-seven (21% of cases) experienced a reinfection. Sequencing identified seventy-seven cases (97% of sequenced cases) with B.1.1.420 and two cases among staff with B.1.351. The outbreak strain B.1.1.420 formed a separate cluster from cases from other European countries. Convalescent and vaccinated residents had higher anti-SARS-CoV-2 IgG antibody concentrations than vaccinated residents without infection (98% vs. 52%, respectively, with >120 RU/mL, p < 0.001). We documented an extensive outbreak of SARS-CoV-2 in an LTCF due to the presence of a specific variant leading to high CFR. Infection in vaccinated residents increased antibody responses. A single vaccine dose was insufficient to mitigate the outbreak