Impaired Mitochondrial Morphology and Functionality in Lonp1wt/- Mice

LONP1 is a nuclear-encoded mitochondrial protease crucial for organelle homeostasis; mutations ofLONP1have been associated with Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies (CODAS) syndrome. To clarify the role of LONP1 in vivo, we generated a mouse model in whichLonp1was ablated. The homozygousLonp(-/-)mouse was not vital, while the heterozygousLonp1(wt/-)showed similar growth rate, weight, length, life-span and histologic features as wild type. Conversely, ultrastructural analysis of heterozygous enterocytes evidenced profound morphological alterations of mitochondria, which appeared increased in number, swollen and larger, with a lower complexity. Embryonic fibroblasts (MEFs) fromLonp1(wt/-)mice showed a reduced expression ofLonp1andTfam, whose expression is regulated by LONP1. Mitochondrial DNA was also reduced, and mitochondria were swollen and larger, albeit at a lesser extent than enterocytes, with a perinuclear distribution. From the functional point of view, mitochondria from heterozygous MEF showed a lower oxygen consumption rate in basal conditions, either in the presence of glucose or galactose, and a reduced expression of mitochondrial complexes than wild type. In conclusion, the presence of one functional copy of theLonp1gene leads to impairment of mitochondrial ultrastructure and functions in vivo

Evidence for mitochondrial Lonp1 expression in the nucleus

The coordinated communication between the mitochondria and nucleus is essential for cellular activities. Nonetheless, the pathways involved in this crosstalk are scarcely understood. The protease Lonp1 was previously believed to be exclusively located in the mitochondria, with an important role in mitochondrial morphology, mtDNA maintenance, and cellular metabolism, in both normal and neoplastic cells. However, we recently detected Lonp1 in the nuclear, where as much as 22% of all cellular Lonp1 can be found. Nuclear localization is detectable under all conditions, but the amount is dependent on a response to heat shock (HS). Lonp1 in the nucleus interacts with heat shock factor 1 (HSF1) and modulates the HS response. These findings reveal a novel extramitochondrial function for Lonp1 in response to stress

Prognostic immune markers identifying patients with severe COVID-19 who respond to tocilizumab

Introduction: A growing number of evidences suggest that the combination of hyperinflammation, dysregulated T and B cell response and cytokine storm play a major role in the immunopathogenesis of severe COVID-19. IL-6 is one of the main pro-inflammatory cytokines and its levels are increased during SARS-CoV-2 infection. Several observational and randomized studies demonstrated that tocilizumab, an IL-6R blocker, improves survival in critically ill patients both in infectious disease and intensive care units. However, despite transforming the treatment options for COVID-19, IL-6R inhibition is still ineffective in a fraction of patients. Methods: In the present study, we investigated the impact of two doses of tocilizumab in patients with severe COVID-19 who responded or not to the treatment by analyzing a panel of cytokines, chemokines and other soluble factors, along with the composition of peripheral immune cells, paying a particular attention to T and B lymphocytes. Results: We observed that, in comparison with non-responders, those who responded to tocilizumab had different levels of several cytokines and different T and B cells proportions before starting therapy. Moreover, in these patients, tocilizumab was further able to modify the landscape of the aforementioned soluble molecules and cellular markers. Conclusions: We found that tocilizumab has pleiotropic effects and that clinical response to this drug remain heterogenous. Our data suggest that it is possible to identify patients who will respond to treatment and that the administration of tocilizumab is able to restore the immune balance through the re-establishment of different cell populations affected by SARS-COV-2 infection, highlighting the importance of temporal examination of the pathological features from the diagnosis

Immunosenescence and vaccine efficacy revealed by immunometabolic analysis of SARS-CoV-2-specific cells in multiple sclerosis patients

Disease-modifying therapies (DMT) administered to patients with multiple sclerosis (MS) can influence immune responses to SARS-CoV-2 and vaccine efficacy. However, data on the detailed phenotypic, functional and metabolic characteristics of antigen (Ag)-specific cells following the third dose of mRNA vaccine remain scarce. Here, using flow cytometry and 45-parameter mass cytometry, we broadly investigate the phenotype, function and the single-cell metabolic profile of SARS-CoV-2-specific T and B cells up to 8 months after the third dose of mRNA vaccine in a cohort of 94 patients with MS treated with different DMT, including cladribine, dimethyl fumarate, fingolimod, interferon, natalizumab, teriflunomide, rituximab or ocrelizumab. Almost all patients display functional immune response to SARS-CoV-2. Different metabolic profiles characterize antigen-specific-T and -B cell response in fingolimod- and natalizumab-treated patients, whose immune response differs from all the other MS treatments.Disease-modifying therapies (DMT) administered to patients with multiple sclerosis (MS) can influence immune responses to SARS-CoV-2 and vaccine efficacy. However, data on the detailed phenotypic, functional and metabolic characteristics of antigen (Ag)-specific cells following the third dose of mRNA vaccine remain scarce. Here, using flow cytometry and 45-parameter mass cytometry, we broadly investigate the phenotype, function and the single-cell metabolic profile of SARS-CoV-2-specific T and B cells up to 8 months after the third dose of mRNA vaccine in a cohort of 94 patients with MS treated with different DMT, including cladribine, dimethyl fumarate, fingolimod, interferon, natalizumab, teriflunomide, rituximab or ocrelizumab. Almost all patients display functional immune response to SARS-CoV-2. Different metabolic profiles characterize antigen-specific-T and -B cell response in fingolimod- and natalizumab-treated patients, whose immune response differs from all the other MS treatments

Detailed characterization of SARS-CoV-2-specific T and B cells after infection or heterologous vaccination

: The formation of a robust long-term antigen (Ag)-specific memory, both humoral and cell-mediated, is created following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or vaccination. Here, by using polychromatic flow cytometry and complex data analyses, we deeply investigated the magnitude, phenotype, and functionality of SARS-CoV-2-specific immune memory in two groups of healthy subjects after heterologous vaccination compared to a group of subjects who recovered from SARS-CoV-2 infection. We find that coronavirus disease 2019 (COVID-19) recovered patients show different long-term immunological profiles compared to those of donors who had been vaccinated with three doses. Vaccinated individuals display a skewed T helper (Th)1 Ag-specific T cell polarization and a higher percentage of Ag-specific and activated memory B cells expressing immunoglobulin (Ig)G compared to those of patients who recovered from severe COVID-19. Different polyfunctional properties characterize the two groups: recovered individuals show higher percentages of CD4+ T cells producing one or two cytokines simultaneously, while the vaccinated are distinguished by highly polyfunctional populations able to release four molecules, namely, CD107a, interferon (IFN)-γ, tumor necrosis factor (TNF), and interleukin (IL)-2. These data suggest that functional and phenotypic properties of SARS-CoV-2 adaptive immunity differ in recovered COVID-19 individuals and vaccinated ones

Human astrocytes and microglia show augmented ingestion of synapses in Alzheimer's disease via MFG-E8

Synapse loss correlates with cognitive decline in Alzheimer's disease (AD). Data from mouse models suggests microglia are important for synapse degeneration, but direct human evidence for any glial involvement in synapse removal in human AD remains to be established. Here we observe astrocytes and microglia from human brains contain greater amounts of synaptic protein in AD compared with non-disease controls, and that proximity to amyloid-β plaques and the APOE4 risk gene exacerbate this effect. In culture, mouse and human astrocytes and primary mouse and human microglia phagocytose AD patient-derived synapses more than synapses from controls. Inhibiting interactions of MFG-E8 rescues the elevated engulfment of AD synapses by astrocytes and microglia without affecting control synapse uptake. Thus, AD promotes increased synapse ingestion by human glial cells at least in part via an MFG-E8 opsonophagocytic mechanism with potential for targeted therapeutic manipulation.</p

Adjuvant capecitabine in triple negative breast cancer patients with residual disease after neoadjuvant treatment: real-world evidence from CaRe, a multicentric, observational study

Background: In triple negative breast cancer patients treated with neoadjuvant chemotherapy, residual disease at surgery is the most relevant unfavorable prognostic factor. Current guidelines consider the use of adjuvant capecitabine, based on the results of the randomized CREATE-X study, carried out in Asian patients and including a small subset of triple negative tumors. Thus far, evidence on Caucasian patients is limited, and no real-world data are available. Methods: We carried out a multicenter, observational study, involving 44 oncologic centres. Triple negative breast cancer patients with residual disease, treated with adjuvant capecitabine from January 2017 through June 2021, were recruited. We primarily focused on treatment tolerability, with toxicity being reported as potential cause of treatment discontinuation. Secondarily, we assessed effectiveness in the overall study population and in a subset having a minimum follow-up of 2 years. Results: Overall, 270 patients were retrospectively identified. The 50.4% of the patients had residual node positive disease, 7.8% and 81.9% had large or G3 residual tumor, respectively, and 80.4% a Ki-67 >20%. Toxicity-related treatment discontinuation was observed only in 10.4% of the patients. In the whole population, at a median follow-up of 15 months, 2-year disease-free survival was 62%, 2 and 3-year overall survival 84.0% and 76.2%, respectively. In 129 patients with a median follow-up of 25 months, 2-year disease-free survival was 43.4%, 2 and 3-year overall survival 78.0% and 70.8%, respectively. Six or more cycles of capecitabine were associated with more favourable outcomes compared with less than six cycles. Conclusion: The CaRe study shows an unexpectedly good tolerance of adjuvant capecitabine in a real-world setting, although effectiveness appears to be lower than that observed in the CREATE-X study. Methodological differences between the two studies impose significant limits to comparability concerning effectiveness, and strongly invite further research

Influenza vaccination for immunocompromised patients: systematic review and meta-analysis from a public health policy perspective.

Immunocompromised patients are vulnerable to severe or complicated influenza infection. Vaccination is widely recommended for this group. This systematic review and meta-analysis assesses influenza vaccination for immunocompromised patients in terms of preventing influenza-like illness and laboratory confirmed influenza, serological response and adverse events

IL RUOLO DELL’IMMUNITA INNATA NEL COVID-19 GRAVE

La complessa risposta immunitaria causata dall'infezione da SARS-CoV-2 può portare alla malattia chiamata COVID-19. L'immunità innata è la prima linea di difesa contro le infezioni e, come precedentemente descritto dal nostro gruppo, una sua eccessiva attivazione può causare uno stato infiammatorio persistente che contribuisce all'immunopatogenesi della forma grave di COVID-19. Pertanto, il mio progetto si è focalizzato sull'identificazione dei principali meccanismi fisiopatologici innescati dai componenti dell'immunità innata, tra cui molecole solubili e cellule come monociti (MC) e neutrofili (NP), che possono contribuire all'eterogeneità della malattia. Sono state studiate due coorti di pazienti con polmonite da COVID-19. La prima comprendeva 28 pazienti e 27 soggetti sani (HD) ed è stata studiata per caratterizzare il fenotipo, il metabolismo e le funzioni dei MC circolanti. La seconda coorte includeva 88 pazienti e 59 HD ed è stata studiata per individuare i meccanismi alla base della risposta neutrofilica generata durante l'infezione da SARS-CoV-2. I risultati ottenuti hanno dimostrato che i MC nei pazienti COVID-19 gravi rispetto ai HD, sono caratterizzati da una profonda alterazione metabolica. Vi è una maggiore percentuale di MC che presentano mitocondri depolarizzati e un aumento della massa mitocondriale, insieme a profonde alterazioni ultrastrutturali. I MC disfunzionali, inoltre, esprimono bassi livelli di HLA-DR e hanno una limitata capacità di fare burst ossidativo rispetto ai MC di HD, ma mantengono la capacità di produrre citochine. Inoltre, abbiamo rilevato a livello periferico, un’importante ridistribuzione delle sottopopolazioni monocitarie, con un aumento della classe intermedia pro-infiammatoria, e una riduzione di quella non classica. Tutte le sottoclassi presentano un’overespressione dei checkpoint inibitori PD-1 e PD-L1 riconducibile al fenomeno di exhaustion. Infine, la concentrazione plasmatica di alcuni fattori coinvolti nella regolazione e migrazione dei MC è maggiore nei pazienti COVID-19, come quella di GM-CSF, che associata ad un aumento significativo di MC immaturi circolanti, suggerisce la presenza di mielopoiesi di emergenza. Analizzando la seconda coorte dello studio, abbiamo dimostrato la presenza di un’alterazione funzionale e metabolica nei granulociti circolanti dei pazienti COVID-19 gravi. La percentuale di NP immaturi aumenta e i NP degranulano di più. A livello bioenergetico, i NP dei pazienti gravi rispetto ai HD hanno una ridotta capacità respiratoria di riserva e di burst ossidativo, ma un’aumentata capacità glicolitica, che sostiene la formazione delle trappole extracellulari (NET). Inoltre, abbiamo osservato la formazione di grandi depositi intracellulari di glicogeno nei neutrofili dei pazienti rispetto ai HD e un aumento dell’espressione genica dell’enzima glicogeno fosforilasi L (PYGL). La glicogenolisi, quindi, può essere utilizzata dai neutrofili come via energetica per la formazione di NET. La quantificazione dei livelli plasmatici di citochine e chemochine nei pazienti con COVID-19 grave ha rivelato la presenza di un pattern di attivazione correlabile alla gravità della malattia. In conclusione, i dati suggeriscono che l'infezione da SARS-CoV-2 può influenzare in maniera importante l’immunità innata. I diversi profili fenotipico e metabolico di MC e NP, nella malattia, possono diventare target efficaci per lo sviluppo di nuove terapie. L’overespressione dei checkpoint inibitori sui MC rappresenta un possibile bersaglio per l'immunoterapia nella lotta contro il cancro e l'aumento dell’attività glicolitica e glicogenolitica nei NP apre la strada a nuove strategie per controllare le malattie infiammatorie.SARS-CoV-2 infection triggers a complex immune response that can cause the onset of COVID-19, and that starts by the activation of innate immunity, the first line of defense. As previously described by our group, a hyperactivation of innate immune cells leads to a strong inflammatory status, whose persistence contributes to the immunopathogenesis of severe COVID-19. Therefore, my studies aimed at the identification of pivotal pathophysiological mechanisms that are triggered by main soluble molecules and cells belonging to innate immunity, like monocytes (MC) and neutrophils (NP), and their contribution to the heterogeneity of COVID-19. Two cohorts of patients with COVID-19 pneumonia were investigated. The first comprised 28 patients and 27 healthy donors (HD) and was used to characterize metabolism, phenotype, and functions of circulating MC. The second included 88 patients and 59 HD and was studied to dissect mechanisms underlying the neutrophilic response during SARS-CoV-2 infection. We found that MC from severe COVID-19 patients were metabolically impaired. The percentage of MC with depolarized mitochondria (mt) was increased as well as their mt mass and mt ultrastructure was profoundly altered compared to healthy MC. In COVID-19 patients dysfunctional MC expressed low levels of HLA-DR and showed a reduced capacity to perform the oxidative burst, but they were still able to produce cytokines. A relevant redistribution of MC subsets was also detected, with an expansion of intermediate MC, which have pro-inflammatory function, and a reduction of nonclassical MC. In all subsets inhibitory checkpoints PD-1 and PD-L1 were overexpressed, probably contributing to immune exhaustion. Finally, plasma concentration of several mediators involved in MC regulation and migration was higher in COVID-19 patients such as GM-CSF, suggesting the presence of emergency myelopoiesis in severe patients. In fact, immature MC were significantly increased in peripheral blood from COVID-19 patients. Regarding NP, formal evidence of their functional status in severe COVID-19 patients was missing. We found that circulating granulocytes were altered in severe COVID-19 patients, with a higher proportion of immature NP and more degranulated NP. The analysis of NP bioenergetic profile revealed a decreased spare respiratory capacity and a defective respiratory burst in NP from COVID-19 patients versus HD. However, glycolysis and glycolytic capacity were strongly increased which sustain neutrophils extracellular traps (NET) formation. Other than glycolysis, other metabolic routes have been recently associated with NP’ differentiation and function. We discovered large intracytoplasmic deposits of glycogen in NP from COVID-19 patients compared to HD and increased levels of intracellular glycogen and mRNA levels of glycogen phosphorylase L (PYGL), which catalyses glycogenolysis. Indeed, NP can use glycogen for NET formation. The analysis of cytokine and chemokine profile in patients with severe COVID-19 revealed that they are profoundly altered delineating an activation pattern which correlates with the severity of the disease of COVID-19. In conclusion, data suggested that infection with SARS-CoV-2 can heavily affect the innate compartment of immune system. MC and NP have a remodelled phenotype and metabolism that can prove effective targets for innovative therapies. The upregulation of inhibitory checkpoints on MC represents an interesting point for immunotherapy against cancer, and increased glycolysis and glycogenolysis crucial for NET formation in NP suggest possible novel strategies to control inflammatory diseases