RESUMEN
ABSTRACT: Inhibitor development is the most severe complication of hemophilia A (HA) care and is associated with increased morbidity and mortality. This study aimed to use a novel immunoglobulin G epitope mapping method to explore the factor VIII (FVIII)-specific epitope profile in the SIPPET cohort population and to develop an epitope mapping-based inhibitor prediction model. The population consisted of 122 previously untreated patients with severe HA who were followed up for 50 days of exposure to FVIII or 3 years, whichever occurred first. Sampling was performed before FVIII treatment and at the end of the follow-up. The outcome was inhibitor development. The FVIII epitope repertoire was assessed by means of a novel random peptide phage-display assay. A least absolute shrinkage and selection operator (LASSO) regression model and a random forest model were fitted on posttreatment sample data and validated in pretreatment sample data. The predictive performance of these models was assessed by the C-statistic and a calibration plot. We identified 27 775 peptides putatively directed against FVIII, which were used as input for the statistical models. The C-statistic of the LASSO and random forest models were good at 0.78 (95% confidence interval [CI], 0.69-0.86) and 0.80 (95% CI, 0.72-0.89). Model calibration of both models was moderately good. Two statistical models, developed on data from a novel random peptide phage display assay, were used to predict inhibitor development before exposure to exogenous FVIII. These models can be used to set up diagnostic tests that predict the risk of inhibitor development before starting treatment with FVIII.
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Factor VIII , Hemofilia A , Biblioteca de Péptidos , Humanos , Factor VIII/inmunología , Mapeo Epitopo , Masculino , Estudios de Cohortes , Epítopos/inmunologíaRESUMEN
BACKGROUND & AIMS: The PNPLA3 rs738409 C>G (encoding for I148M) variant is a risk locus for the fibrogenic progression of chronic liver diseases, a process driven by hepatic stellate cells (HSCs). We investigated how the PNPLA3 I148M variant affects HSC biology using transcriptomic data and validated findings in 3D-culture models. METHODS: RNA sequencing was performed on 2D-cultured primary human HSCs and liver biopsies of individuals with obesity, genotyped for the PNPLA3 I148M variant. Data were validated in wild-type (WT) or PNPLA3 I148M variant-carrying HSCs cultured on 3D extracellular matrix (ECM) scaffolds from human healthy and cirrhotic livers, with/without TGFB1 or cytosporone B (Csn-B) treatment. RESULTS: Transcriptomic analyses of liver biopsies and HSCs highlighted shared PNPLA3 I148M-driven dysregulated pathways related to mitochondrial function, antioxidant response, ECM remodelling and TGFB1 signalling. Analogous pathways were dysregulated in WT/PNPLA3-I148M HSCs cultured in 3D liver scaffolds. Mitochondrial dysfunction in PNPLA3-I148M cells was linked to respiratory chain complex IV insufficiency. Antioxidant capacity was lower in PNPLA3-I148M HSCs, while reactive oxygen species secretion was increased in PNPLA3-I148M HSCs and higher in bioengineered cirrhotic vs. healthy scaffolds. TGFB1 signalling followed the same trend. In PNPLA3-I148M cells, expression and activation of the endogenous TGFB1 inhibitor NR4A1 were decreased: treatment with the Csn-B agonist increased total NR4A1 in HSCs cultured in healthy but not in cirrhotic 3D scaffolds. NR4A1 regulation by TGFB1/Csn-B was linked to Akt signalling in PNPLA3-WT HSCs and to Erk signalling in PNPLA3-I148M HSCs. CONCLUSION: HSCs carrying the PNPLA3 I148M variant have impaired mitochondrial function, antioxidant responses, and increased TGFB1 signalling, which dampens antifibrotic NR4A1 activity. These features are exacerbated by cirrhotic ECM, highlighting the dual impact of the PNPLA3 I148M variant and the fibrotic microenvironment in progressive chronic liver diseases. IMPACT AND IMPLICATIONS: Hepatic stellate cells (HSCs) play a key role in the fibrogenic process associated with chronic liver disease. The PNPLA3 genetic mutation has been linked with increased risk of fibrogenesis, but its role in HSCs requires further investigation. Here, by using comparative transcriptomics and a novel 3D in vitro model, we demonstrate the impact of the PNPLA3 genetic mutation on primary human HSCs' behaviour, and we show that it affects the cell's mitochondrial function and antioxidant response, as well as the antifibrotic gene NR4A1. Our publicly available transcriptomic data, 3D platform and our findings on NR4A1 could facilitate the discovery of targets to develop more effective treatments for chronic liver diseases.
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Matriz Extracelular , Células Estrelladas Hepáticas , Lipasa , Cirrosis Hepática , Proteínas de la Membrana , Factor de Crecimiento Transformador beta1 , Humanos , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/genética , Lipasa/genética , Lipasa/metabolismo , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Cirrosis Hepática/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Células Cultivadas , Hígado/patología , Hígado/metabolismo , Transducción de Señal/genética , Obesidad/genética , Obesidad/metabolismo , Masculino , Aciltransferasas , Fosfolipasas A2 Calcio-IndependienteRESUMEN
Fatty liver disease (FLD) caused by metabolic dysfunction is the leading cause of liver disease and the prevalence is rising, especially in women. Although during reproductive age women are protected against FLD, for still unknown and understudied reasons some develop rapidly progressive disease at the menopause. The patatin-like phospholipase domain-containing 3 (PNPLA3) p.I148M variant accounts for the largest fraction of inherited FLD variability. In the present study, we show that there is a specific multiplicative interaction between female sex and PNPLA3 p.I148M in determining FLD in at-risk individuals (steatosis and fibrosis, P < 10-10; advanced fibrosis/hepatocellular carcinoma, P = 0.034) and in the general population (P < 10-7 for alanine transaminase levels). In individuals with obesity, hepatic PNPLA3 expression was higher in women than in men (P = 0.007) and in mice correlated with estrogen levels. In human hepatocytes and liver organoids, PNPLA3 was induced by estrogen receptor-α (ER-α) agonists. By chromatin immunoprecipitation and luciferase assays, we identified and characterized an ER-α-binding site within a PNPLA3 enhancer and demonstrated via CRISPR-Cas9 genome editing that this sequence drives PNPLA3 p.I148M upregulation, leading to lipid droplet accumulation and fibrogenesis in three-dimensional multilineage spheroids with stellate cells. These data suggest that a functional interaction between ER-α and PNPLA3 p.I148M variant contributes to FLD in women.
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Aciltransferasas , Enfermedad del Hígado Graso no Alcohólico , Fosfolipasas A2 Calcio-Independiente , Receptores de Estrógenos , Animales , Femenino , Humanos , Masculino , Ratones , Aciltransferasas/genética , Aciltransferasas/metabolismo , Carcinoma Hepatocelular , Fibrosis , Predisposición Genética a la Enfermedad , Hígado/metabolismo , Neoplasias Hepáticas/patología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Fosfolipasas A2 Calcio-Independiente/genética , Fosfolipasas A2 Calcio-Independiente/metabolismo , Receptores de Estrógenos/metabolismoRESUMEN
Duchenne muscular dystrophy (DMD) is a progressive severe muscle-wasting disease caused by mutations in DMD, encoding dystrophin, that leads to loss of muscle function with cardiac/respiratory failure and premature death. Since dystrophic muscles are sensed by infiltrating inflammatory cells and gut microbial communities can cause immune dysregulation and metabolic syndrome, we sought to investigate whether intestinal bacteria support the muscle immune response in mdx dystrophic murine model. We highlighted a strong correlation between DMD disease features and the relative abundance of Prevotella. Furthermore, the absence of gut microbes through the generation of mdx germ-free animal model, as well as modulation of the microbial community structure by antibiotic treatment, influenced muscle immunity and fibrosis. Intestinal colonization of mdx mice with eubiotic microbiota was sufficient to reduce inflammation and improve muscle pathology and function. This work identifies a potential role for the gut microbiota in the pathogenesis of DMD.
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Microbiota , Distrofia Muscular de Duchenne , Animales , Ratones , Distrofina/genética , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Disbiosis , Distrofia Muscular de Duchenne/genética , Sistema Inmunológico/metabolismo , Sistema Inmunológico/patología , Modelos Animales de EnfermedadRESUMEN
BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is the leading cause of liver disorders and has a strong heritable component. The aim of this study was to identify new loci that contribute to severe NAFLD by examining rare variants. METHODS: We performed whole-exome sequencing in individuals with NAFLD and advanced fibrosis or hepatocellular carcinoma (n = 301) and examined the enrichment of likely pathogenic rare variants vs. the general population. This was followed by validation at the gene level. RESULTS: In patients with severe NAFLD, we observed an enrichment of the p.P426L variant (rs143545741 C>T; odds ratio [OR] 5.26, 95% CI 2.1-12.6; p = 0.003) of autophagy-related 7 (ATG7), which we characterized as a loss-of-function, vs. the general population, and an enrichment in rare variants affecting the catalytic domain (OR 13.9; 95% CI 1.9-612; p = 0.002). In the UK Biobank cohort, loss-of-function ATG7 variants increased the risk of cirrhosis and hepatocellular carcinoma (OR 3.30; 95% CI 1.1-7.5 and OR 12.30, 95% CI 2.6-36, respectively; p <0.001 for both). The low-frequency loss-of-function p.V471A variant (rs36117895 T>C) was also associated with severe NAFLD in the clinical cohort (OR 1.7; 95% CI 1.2-2.5; p = 0.003), predisposed to hepatocellular ballooning (p = 0.007) evolving to fibrosis in the Liver biopsy cohort (n = 2,268), and was associated with liver injury in the UK Biobank (aspartate aminotransferase levels, p <0.001), with a larger effect in severely obese individuals in whom it was linked to hepatocellular carcinoma (p = 0.009). ATG7 protein localized to periportal hepatocytes, particularly in the presence of ballooning. In the Liver Transcriptomic cohort (n = 125), ATG7 expression correlated with suppression of the TNFα pathway, which was conversely upregulated in p.V471A carriers. CONCLUSIONS: We identified rare and low-frequency ATG7 loss-of-function variants that promote NAFLD progression by impairing autophagy and facilitating ballooning and inflammation. LAY SUMMARY: We found that rare mutations in a gene called autophagy-related 7 (ATG7) increase the risk of developing severe liver disease in individuals with dysmetabolism. These mutations cause an alteration in protein function and impairment of self-renewal of cellular content, leading to liver damage and inflammation.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Enfermedad del Hígado Graso no Alcohólico , Proteína 7 Relacionada con la Autofagia/genética , Biopsia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Humanos , Inflamación/patología , Hígado/patología , Cirrosis Hepática/complicaciones , Neoplasias Hepáticas/patología , Enfermedad del Hígado Graso no Alcohólico/complicacionesRESUMEN
Inflammation has profound but poorly understood effects on metabolism, especially in the context of obesity and nonalcoholic fatty liver disease (NAFLD). Here, we report that hepatic interferon regulatory factor 3 (IRF3) is a direct transcriptional regulator of glucose homeostasis through induction of Ppp2r1b, a component of serine/threonine phosphatase PP2A, and subsequent suppression of glucose production. Global ablation of IRF3 in mice on a high-fat diet protected against both steatosis and dysglycemia, whereas hepatocyte-specific loss of IRF3 affects only dysglycemia. Integration of the IRF3-dependent transcriptome and cistrome in mouse hepatocytes identifies Ppp2r1b as a direct IRF3 target responsible for mediating its metabolic actions on glucose homeostasis. IRF3-mediated induction of Ppp2r1b amplified PP2A activity, with subsequent dephosphorylation of AMPKα and AKT. Furthermore, suppression of hepatic Irf3 expression with antisense oligonucleotides reversed obesity-induced insulin resistance and restored glucose homeostasis in obese mice. Obese humans with NAFLD displayed enhanced activation of liver IRF3, with reversion after bariatric surgery. Hepatic PPP2R1B expression correlated with HgbA1C and was elevated in obese humans with impaired fasting glucose. We therefore identify the hepatic IRF3-PPP2R1B axis as a causal link between obesity-induced inflammation and dysglycemia and suggest an approach for limiting the metabolic dysfunction accompanying obesity-associated NAFLD.
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Resistencia a la Insulina , Enfermedad del Hígado Graso no Alcohólico , Animales , Resistencia a la Insulina/fisiología , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Ratones , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/genética , Obesidad/complicaciones , Obesidad/metabolismoRESUMEN
Fatty liver disease (FLD) is a growing health issue with burdening unmet clinical needs. FLD has a genetic component but, despite the common variants already identified, there is still a missing heritability component. Using a candidate gene approach, we identify a locus (rs71519934) at the Pleckstrin and Sec7 domain-containing 3 (PSD3) gene resulting in a leucine to threonine substitution at position 186 of the protein (L186T) that reduces susceptibility to the entire spectrum of FLD in individuals at risk. PSD3 downregulation by short interfering RNA reduces intracellular lipid content in primary human hepatocytes cultured in two and three dimensions, and in human and rodent hepatoma cells. Consistent with this, Psd3 downregulation by antisense oligonucleotides in vivo protects against FLD in mice fed a non-alcoholic steatohepatitis-inducing diet. Thus, translating these results to humans, PSD3 downregulation might be a future therapeutic option for treating FLD.
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Susceptibilidad a Enfermedades , Hígado Graso/etiología , Hígado Graso/metabolismo , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Alelos , Animales , Biomarcadores , Línea Celular , Hígado Graso/patología , Perfilación de la Expresión Génica , Variación Genética , Genotipo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Hígado/patología , Ratones , Polimorfismo de Nucleótido Simple , RNA-Seq , RibonucleasasRESUMEN
A postprandial increase of translation mediated by eukaryotic Initiation Factor 6 (eIF6) occurs in the liver. Its contribution to steatosis and disease is unknown. In this study we address whether eIF6-driven translation contributes to disease progression. eIF6 levels increase throughout the progression from Non-Alcoholic Fatty Liver Disease (NAFLD) to hepatocellular carcinoma. Reduction of eIF6 levels protects the liver from disease progression. eIF6 depletion blunts lipid accumulation, increases fatty acid oxidation (FAO) and reduces oncogenic transformation in vitro. In addition, eIF6 depletion delays the progression from NAFLD to hepatocellular carcinoma, in vivo. Mechanistically, eIF6 depletion reduces the translation of transcription factor C/EBPß, leading to a drop in biomarkers associated with NAFLD progression to hepatocellular carcinoma and preserves mitochondrial respiration due to the maintenance of an alternative mTORC1-eIF4F translational branch that increases the expression of transcription factor YY1. We provide proof-of-concept that in vitro pharmacological inhibition of eIF6 activity recapitulates the protective effects of eIF6 depletion. We hypothesize the existence of a targetable, evolutionarily conserved translation circuit optimized for lipid accumulation and tumor progression.
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Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Factores de Iniciación de Péptidos/genética , Biosíntesis de Proteínas/genética , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Carcinoma Hepatocelular/metabolismo , Línea Celular , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Clofazimina/farmacología , Dieta Alta en Grasa/efectos adversos , Progresión de la Enfermedad , Silenciador del Gen , Humanos , Lipogénesis/efectos de los fármacos , Lipogénesis/genética , Neoplasias Hepáticas/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/etiología , Obesidad/genética , Obesidad/metabolismo , Factores de Iniciación de Péptidos/antagonistas & inhibidores , Factores de Iniciación de Péptidos/metabolismoRESUMEN
BACKGROUND AND AIMS: Farnesoid X receptor (FXR) plays a key role in bile acid and lipid homeostasis. Experimental evidence suggests that it can modulate liver damage related to nonalcoholic fatty liver disease (NAFLD). We examined the impact of the NR1H4 rs35724 G>C, encoding for FXR, on liver damage in a large cohort of patients at risk of steatohepatitis. METHODS: We considered 2,660 consecutive individuals at risk of steatohepatitis with liver histology. The rs35724 G>C polymorphisms were genotyped by TaqMan assays. Gene expression was evaluated by RNASeq in a subset of patients (n = 124). RESULTS: The NR1H4 rs35724 CC genotype, after adjusting for clinic-metabolic and genetic confounders and for enrolling centre, was protective against severity of steatosis (GG vs CC OR 0.77, 95% CI 0.62-0.95; P = .01), steatohepatitis (GG vs CC OR 0.62, 95% CI 0.47-0.83; P = .001) and severity of fibrosis (GG vs CC OR 0.83, 95% CI 0.67-0.98; P = .04). The C allele was associated with higher total circulating cholesterol (P = .01). Patients carrying the NR1H4 rs35724 C allele had significantly higher hepatic mRNA levels of FXR and were associated with higher hepatic FGFR4 and Cyp39A1 that are in turn involved in bile acid synthesis. CONCLUSIONS: Increased hepatic FXR expression due to the NR1H4 rs35724 C allele is linked to higher serum cholesterol but protects against steatosis, steatohepatitis and liver fibrosis. The translational relevance of these results for patient risk stratification and FXR-targeted therapy warrants further investigation.
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Enfermedad del Hígado Graso no Alcohólico , Receptores Citoplasmáticos y Nucleares/genética , Ácidos y Sales Biliares , Humanos , Hígado/patología , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos , Esteroide HidroxilasasRESUMEN
OBJECTIVE: Neddylation is a druggable and reversible ubiquitin-like post-translational modification upregulated in many diseases, including liver fibrosis, hepatocellular carcinoma, and more recently, non-alcoholic fatty liver disease (NAFLD). Herein, we propose to address the effects of neddylation inhibition and the underlying mechanisms in pre-clinical models of NAFLD. METHODS: Hepatic neddylation measured by immunohistochemical analysis and NEDD8 serum levels measured by ELISA assay were evaluated in NAFLD clinical and pre-clinical samples. The effects of neddylation inhibition by using a pharmacological small inhibitor, MLN4924, or molecular approaches were assessed in isolated mouse hepatocytes and pre-clinical mouse models of diet-induced NAFLD, male adult C57BL/6 mice, and the AlfpCre transgenic mice infected with AAV-DIO-shNedd8. RESULTS: Neddylation inhibition reduced lipid accumulation in oleic acid-stimulated mouse primary hepatocytes and ameliorated liver steatosis, preventing lipid peroxidation and inflammation in the mouse models of diet-induced NAFLD. Under these conditions, increased Deptor levels and the concomitant repression of mTOR signaling were associated with augmented fatty acid oxidation and reduced lipid content. Moreover, Deptor silencing in isolated mouse hepatocytes abolished the anti-steatotic effects mediated by neddylation inhibition. Finally, serum NEDD8 levels correlated with hepatic neddylation during the disease progression in the clinical and pre-clinical models CONCLUSIONS: Overall, the upregulation of Deptor, driven by neddylation inhibition, is proposed as a novel effective target and therapeutic approach to tackle NAFLD.
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Ácidos Grasos/metabolismo , Hepatocitos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Adolescente , Adulto , Anciano , Animales , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Transducción de Señal , Adulto JovenRESUMEN
We herein characterize the immunopathological features of two Italian COVID-19 patients who underwent bilateral lung transplantation (bLTx). Removed lungs underwent histopathological evaluation. Gene expression profiling (GEP) for immune-related signatures was performed on lung specimens and SARS-CoV-2-stimulated peripheral blood mononuclear cells (PBMCs). Cytokine levels were measured on lungs, bronchoalveolar lavage fluids and in culture supernatants. Pathological assessment showed extensive lung damage with the pattern of proliferative to fibrotic phases, with diffuse alveolar damage mimicking usual interstitial pneumonia (UIP). Lungs' GEP revealed overexpression of pathogen recognition receptors, effector cytokines and chemokines, immune activation receptors and of the inflammasome components. Multiplex cytokine analysis confirmed a proinflammatory state, with high levels of monocyte/macrophage chemotactic and activating factors and of IL-6 and TNF-α. A similar profile was observed in SARS-CoV-2-stimulated PBMCs collected 7 days after transplant. The pattern of tissue damage observed in the lungs suggests that this may represent the output of protracted disease, resembling a diffuse UIP-like picture. The molecular immune profiling supports the paradigm of a persistent proinflammatory state and sustained humoral immunity, conditions that are maintained despite the iatrogenic immunosuppression.
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COVID-19/cirugía , Quimiocinas/metabolismo , Citocinas/metabolismo , Leucocitos Mononucleares/metabolismo , Trasplante de Pulmón , Pulmón/patología , Síndrome de Dificultad Respiratoria/cirugía , Adolescente , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/virología , COVID-19/sangre , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/inmunología , Genotipo , Humanos , Inflamasomas/metabolismo , Interleucina-6/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/virología , Ganglios Linfáticos/patología , Ganglios Linfáticos/virología , Masculino , Persona de Mediana Edad , Plasma/virología , Polimorfismo de Nucleótido Simple , Síndrome de Dificultad Respiratoria/inmunología , Síndrome de Dificultad Respiratoria/patología , Síndrome de Dificultad Respiratoria/virología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
SARS-CoV-2 virus infection is responsible for coronavirus disease (COVID-19), which is characterised by a hyperinflammatory response that plays a major role in determining the respiratory and immune-mediated complications of this condition. While isolating peripheral blood mononuclear cells (PBMCs) from whole blood of COVID-19 patients by density gradient centrifugation, we noticed some changes in the floating properties and in the sedimentation of the cells on density medium. Investigating this further, we found that in early phase COVID-19 patients, characterised by reduced circulating lymphocytes and monocytes, the PBMC fraction contained surprisingly high levels of neutrophils. Furthermore, the neutrophil population exhibited alterations in the cell size and in the internal complexity, consistent with the presence of low density neutrophils (LDNs) and immature forms, which may explain the shift seen in the floating abilities and that may be predictive of the severity of the disease. The percentage of this subset of neutrophils found in the PBMC band was rather spread (35.4 ± 27.2%, with a median 28.8% and IQR 11.6-56.1, Welch's t-test early phase COVID-19 versus blood donor healthy controls P < 0.0001). Results confirm the presence of an increased number of LDNs in patients with early stage COVID-19, which correlates with disease severity and may be recovered by centrifugation on a density gradient together with PBMCs.
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COVID-19/sangre , Separación Celular , Leucocitos Mononucleares/metabolismo , SARS-CoV-2/metabolismo , Adulto , COVID-19/patología , Centrifugación por Gradiente de Densidad , Femenino , Humanos , Leucocitos Mononucleares/patología , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: The Milan metropolitan area in Northern Italy was among the most severely hit by the SARS-CoV-2 outbreak. The aim of this study was to examine the seroprevalence trends of SARS-CoV-2 in healthy asymptomatic adults, and the risk factors and laboratory correlates of positive tests. MATERIALS AND METHODS: We conducted a cross-sectional study in a random sample of blood donors, who were asymptomatic at the time of evaluation, at the beginning of the first phase (February 24th to April 8th 2020; n=789). Presence of IgM/IgG antibodies against the SARS-CoV-2-Nucleocapsid protein was assessed by a lateral flow immunoassay. RESULTS: The test had a 100/98.3 sensitivity/specificity (n=32/120 positive/negative controls, respectively), and the IgG test was validated in a subset by an independent ELISA against the Spike protein (n=34, p<0.001). At the start of the outbreak, the overall adjusted seroprevalence of SARS-CoV-2 was 2.7% (95% CI: 0.3-6%; p<0.0001 vs 120 historical controls). During the study period, characterised by a gradual implementation of social distancing measures, there was a progressive increase in the adjusted seroprevalence to 5.2% (95% CI: 2.4-9.0; 4.5%, 95% CI: 0.9-9.2% according to a Bayesian estimate) due to a rise in IgG reactivity to 5% (95% CI: 2.8-8.2; p=0.004 for trend), but there was no increase in IgM+ (p=not significant). At multivariate logistic regression analysis, IgG reactivity was more frequent in younger individuals (p=0.043), while IgM reactivity was more frequent in individuals aged >45 years (p=0.002). DISCUSSION: SARS-CoV-2 infection was already circulating in Milan at the start of the outbreak. The pattern of IgM/IgG reactivity was influenced by age: IgM was more frequently detected in participants aged >45 years. By the end of April, 2.4-9.0% of healthy adults had evidence of seroconversion.
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Infecciones Asintomáticas/epidemiología , Donantes de Sangre/estadística & datos numéricos , COVID-19/epidemiología , Pandemias , SARS-CoV-2/inmunología , Adulto , Factores de Edad , Anticuerpos Antivirales/sangre , Teorema de Bayes , COVID-19/inmunología , Prueba Serológica para COVID-19/métodos , Intervalos de Confianza , Estudios Transversales , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Italia/epidemiología , Masculino , Persona de Mediana Edad , Análisis de Regresión , Factores de Riesgo , Seroconversión , Estudios Seroepidemiológicos , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a common prelude to cirrhosis and hepatocellular carcinoma. The genetic rs641738 C>T variant in the lysophosphatidylinositol acyltransferase 1 (LPIAT1)/membrane bound O-acyltransferase domain-containing 7, which incorporates arachidonic acid into phosphatidylinositol (PI), is associated with the entire spectrum of NAFLD. In this study, we investigated the mechanism underlying this association in mice and cultured human hepatocytes. DESIGN: We generated the hepatocyte-specific Lpiat1 knockout mice to investigate the function of Lpiat1 in vivo. We also depleted LPIAT1 in cultured human hepatic cells using CRISPR-Cas9 systems or siRNA. The effect of LPIAT1-depletion on liver fibrosis was examined in mice fed high fat diet and in liver spheroids. Lipid species were measured using liquid chromatography-electrospray ionisation mass spectrometry. Lipid metabolism was analysed using radiolabeled glycerol or fatty acids. RESULTS: The hepatocyte-specific Lpiat1 knockout mice developed hepatic steatosis spontaneously, and hepatic fibrosis on high fat diet feeding. Depletion of LPIAT1 in cultured hepatic cells and in spheroids caused triglyceride accumulation and collagen deposition. The increase in hepatocyte fat content was due to a higher triglyceride synthesis fueled by a non-canonical pathway. Indeed, reduction in the PI acyl chain remodelling caused a high PI turnover, by stimulating at the same time PI synthesis and breakdown. The degradation of PI was mediated by a phospholipase C, which produces diacylglycerol, a precursor of triglyceride. CONCLUSION: We found a novel pathway fueling triglyceride synthesis in hepatocytes, by a direct metabolic flow of PI into triglycerides. Our findings provide an insight into the pathogenesis and therapeutics of NAFLD.
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Aciltransferasas/genética , Proteínas de la Membrana/genética , Enfermedad del Hígado Graso no Alcohólico/etiología , Fosfatidilinositoles/metabolismo , Triglicéridos/metabolismo , Animales , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Metabolismo de los Lípidos , Ratones , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patologíaRESUMEN
BACKGROUND & AIMS: Fatty liver disease (FLD) is a growing epidemic that is expected to be the leading cause of end-stage liver disease within the next decade. Both environmental and genetic factors contribute to the susceptibility of FLD. Several genetic variants contributing to FLD have been identified in exome-wide association studies. However, there is still a missing hereditability indicating that other genetic variants are yet to be discovered. METHODS: To find genes involved in FLD, we first examined the association of missense and nonsense variants with alanine aminotransferase at an exome-wide level in 425,671 participants from the UK Biobank. We then validated genetic variants with liver fat content in 8930 participants in whom liver fat measurement was available, and replicated 2 genetic variants in 3 independent cohorts comprising 2621 individuals with available liver biopsy. RESULTS: We identified 190 genetic variants independently associated with alanine aminotransferase after correcting for multiple testing with Bonferroni method. The majority of these variants were not previously associated with this trait. Among those associated, there was a striking enrichment of genetic variants influencing lipid metabolism. We identified the variants rs2792751 in GPAM/GPAT1, the gene encoding glycerol-3-phosphate acyltransferase, mitochondrial, and rs429358 in APOE, the gene encoding apolipoprotein E, as robustly associated with liver fat content and liver disease after adjusting for multiple testing. Both genes affect lipid metabolism in the liver. CONCLUSIONS: We identified 2 novel genetic variants in GPAM and APOE that are robustly associated with steatosis and liver damage. These findings may help to better elucidate the genetic susceptibility to FLD onset and progression.
Asunto(s)
1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , Alanina Transaminasa/sangre , Apolipoproteínas E/genética , Variación Genética , Enfermedad del Hígado Graso no Alcohólico/genética , Biomarcadores/sangre , Europa (Continente) , Exoma , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/diagnóstico , Fenotipo , Reproducibilidad de los Resultados , Medición de Riesgo , Factores de Riesgo , TranscriptomaRESUMEN
Malignant pleural mesothelioma (MPM) is often associated with a poor prognosis and options for the treatment of this disease are few. To date, the important role of the immune microenvironment in modifying the disease natural history is well established. The programmed cell death pathway (PD-1/PD-L1) limits the T lymphocyte activation in peripheral tissues when an inflammatory response occurs, and controls the tumour immune escape. PD-L1 is broadly expressed in several malignant tumours and associated with poor clinical outcomes. Thus, the aim of our study is to investigate the potential role of PD-L1 expression in MPM prognosis. Biopsy samples from 198 patients diagnosed with MPM were examined by immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR) to evaluate PD-L1 protein and gene expression. For PD-L1 protein expression we consider at least 5% membranous staining as positive. Gene expression levels were calculated with ΔΔCt method. Positive expression of PD-L1 by IHC was correlated with worse overall survival (OS; p=0.0225) in MPM patients. PD-L1 positive status was correlated with worse OS in the subgroup of patients with ECOG score <2 (p=0.0004, n=129) and these data were confirmed by multivariate analysis. No significant correlation was found between PD-L1 gene expression and OS. Our results show that PD-L1 evaluated by IHC assay may be a prognostic biomarker for MPM patients with good performance status.
Asunto(s)
Antígeno B7-H1/metabolismo , Mesotelioma Maligno/diagnóstico , Neoplasias Pleurales/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Antígeno B7-H1/genética , Biomarcadores/metabolismo , Estudios de Cohortes , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Italia , Masculino , Mesotelioma Maligno/metabolismo , Mesotelioma Maligno/patología , Persona de Mediana Edad , Neoplasias Pleurales/metabolismo , Neoplasias Pleurales/patología , Pronóstico , Estudios RetrospectivosRESUMEN
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) risk stratification in individuals with dysmetabolism is a major unmet need. Genetic predisposition contributes to non-alcoholic fatty liver disease (NAFLD). We aimed to exploit robust polygenic risk scores (PRS) that can be evaluated in the clinic to gain insight into the causal relationship between NAFLD and HCC, and to improve HCC risk stratification. METHODS: We examined at-risk individuals (NAFLD cohort, n = 2,566; 226 with HCC; and a replication cohort of 427 German patients with NAFLD) and the general population (UK Biobank [UKBB] cohort, n = 364,048; 202 with HCC). Variants in PNPLA3-TM6SF2-GCKR-MBOAT7 were combined in a hepatic fat PRS (PRS-HFC), and then adjusted for HSD17B13 (PRS-5). RESULTS: In the NAFLD cohort, the adjusted impact of genetic risk variants on HCC was proportional to the predisposition to fatty liver (p = 0.002) with some heterogeneity in the effect. PRS predicted HCC more robustly than single variants (p <10-13). The association between PRS and HCC was mainly mediated through severe fibrosis, but was independent of fibrosis in clinically relevant subgroups, and was also observed in those without severe fibrosis (p <0.05). In the UKBB cohort, PRS predicted HCC independently of classical risk factors and cirrhosis (p <10-7). In the NAFLD cohort, we identified high PRS cut-offs (≥0.532/0.495 for PRS-HFC/PRS-5) that in the UKBB cohort detected HCC with ~90% specificity but limited sensitivity; PRS predicted HCC both in individuals with (p <10-5) and without cirrhosis (p <0.05). CONCLUSIONS: Our results are consistent with a causal relationship between hepatic fat and HCC. PRS improved the accuracy of HCC detection and may help stratify HCC risk in individuals with dysmetabolism, including those without severe liver fibrosis. Further studies are needed to validate our findings. LAY SUMMARY: By analyzing variations in genes that contribute to fatty liver disease, we developed two risk scores to help predict liver cancer in individuals with obesity-related metabolic complications. These risk scores can be easily tested in the clinic. We showed that the risk scores helped to identify the risk of liver cancer both in high-risk individuals and in the general population.