RESUMEN
While KRAS is the most frequently mutated oncogene in non-small cell lung cancer (NSCLC), KRAS-mutant tumors have long been considered difficult to treat and thus, an unmet need still remains. Partly due to the lack of targeted treatments, comprehensive real-world description of NSCLC patients with KRAS mutation is still largely missing in Finland. In this study, all adult patients diagnosed with locally advanced and unresectable or metastatic NSCLC from 1 January 2018 to 31 August 2020 at the Hospital District of Helsinki and Uusimaa were first identified in this retrospective registry-based real-world study. The final cohort included only patients tested with next generation sequencing (NGS) and was stratified by the KRAS mutation status. A total of 383 patients with locally advanced and unresectable or metastatic NSCLC and with NGS testing performed were identified. Patients with KRAS mutation (KRAS G12C n = 35, other KRAS n = 74) were younger than patients without KRAS mutations, were all previous or current smokers, and had more often metastatic disease at diagnosis. Also, these patients had poorer survival, with higher age, Charlson comorbidity index (CCI) being 5 or above, and KRAS G12C being the most significant risk factors associated with poorer survival. This suggests that the patients with KRAS mutation have a more aggressive disease and/or tumors with KRAS mutation are more difficult to treat, at least without effective targeted therapies.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Mutación , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Carcinoma de Pulmón de Células no Pequeñas/genética , Finlandia , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias Pulmonares/genética , Femenino , Masculino , Persona de Mediana Edad , Anciano , Estudios Retrospectivos , Adulto , Anciano de 80 o más Años , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Familial cardiomyopathy in pediatric stages is a poorly understood presentation of heart disease in children that is attributed to pathogenic mutations. Through exome sequencing, we report a homozygous variant in tropomodulin 1 (TMOD1; c.565C>T, p.R189W) in three individuals from two unrelated families with childhood-onset dilated and restrictive cardiomyopathy. To decipher the mechanism of pathogenicity of the R189W mutation in TMOD1, we utilized a wide array of methods, including protein analyses, biochemistry and cultured cardiomyocytes. Structural modeling revealed potential defects in the local folding of TMOD1R189W and its affinity for actin. Cardiomyocytes expressing GFP-TMOD1R189W demonstrated longer thin filaments than GFP-TMOD1wt-expressing cells, resulting in compromised filament length regulation. Furthermore, TMOD1R189W showed weakened activity in capping actin filament pointed ends, providing direct evidence for the variant's effect on actin filament length regulation. Our data indicate that the p.R189W variant in TMOD1 has altered biochemical properties and reveals a unique mechanism for childhood-onset cardiomyopathy.
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Citoesqueleto de Actina , Cardiomiopatías , Niño , Humanos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Miocitos Cardíacos/metabolismo , Mutación , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Tropomodulina/genética , Tropomodulina/química , Tropomodulina/metabolismoRESUMEN
BACKGROUND: We aimed to quantify ductular reaction (DR) in biliary atresia using a neural network in relation to underlying pathophysiology and prognosis. METHODS: Image-processing neural network model was applied to 259 cytokeratin-7-stained native liver biopsies of patients with biliary atresia and 43 controls. The model quantified total proportional DR (DR%) composed of portal biliary epithelium (BE%) and parenchymal intermediate hepatocytes (PIH%). The results were related to clinical data, Sirius Red-quantified liver fibrosis, serum biomarkers, and bile acids. RESULTS: In total, 2 biliary atresia biopsies were obtained preoperatively, 116 at Kasai portoenterostomy (KPE) and 141 during post-KPE follow-up. DR% (8.3% vs. 5.9%, p=0.045) and PIH% (1.3% vs. 0.6%, p=0.004) were increased at KPE in patients remaining cholestatic postoperatively. After KPE, patients with subsequent liver transplantation or death showed an increase in DR% (7.9%-9.9%, p = 0.04) and PIH% (1.6%-2.4%, p = 0.009), whereas patients with native liver survival (NLS) showed decreasing BE% (5.5%-3.0%, p = 0.03) and persistently low PIH% (0.9% vs. 1.3%, p = 0.11). In Cox regression, high DR predicted inferior NLS both at KPE [DR% (HR = 1.05, p = 0.01), BE% (HR = 1.05, p = 0.03), and PIH% (HR = 1.13, p = 0.005)] and during follow-up [DR% (HR = 1.08, p<0.0001), BE% (HR = 1.58, p = 0.001), and PIH% (HR = 1.04, p = 0.008)]. DR% correlated with Sirius red-quantified liver fibrosis at KPE (R = 0.47, p<0.0001) and follow-up (R = 0.27, p = 0.004). A close association between DR% and serum bile acids was observed at follow-up (R = 0.61, p<0.001). Liver fibrosis was not prognostic for NLS at KPE (HR = 1.00, p = 0.96) or follow-up (HR = 1.01, p = 0.29). CONCLUSIONS: DR predicted NLS in different disease stages before transplantation while associating with serum bile acids after KPE.
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Atresia Biliar , Aprendizaje Profundo , Humanos , Atresia Biliar/diagnóstico , Atresia Biliar/cirugía , Pronóstico , Portoenterostomía Hepática/métodos , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/cirugía , Cirrosis Hepática/complicaciones , Ácidos y Sales BiliaresRESUMEN
OBJECTIVES: It has been suggested that after partial tonsillectomy, referred here as tonsillotomy (TT), the remaining tonsillar tissue might be altered, leading to scarring or chronic tonsillitis. The objectives were to compare the histology of regrown tonsillar tissues with native tonsils and to assess the incidence of reoperations and predictive factors for tonsillar regrowth after TT. METHODS: Tonsillar tissues of 1) children that underwent TT and later requiring resurgery and 2) children operated on for the first time with TT were prospectively analysed. To assess the incidence of resurgery because of tonsillar regrowth and predictive factors for tonsillar regrowth, the data covering TTs and tonsillectomies performed in 2009-2020 were retrospectively retrieved. RESULTS: Altogether 11 children formed the regrowth group, with the control group consisting of 19 children. In the histological analysis of tonsillar tissues, neutrophil infiltration in the epithelium and crypts and severe lymphoplasmacytic infiltration in the epithelium were significantly more frequent in samples of the regrowth group relative to the control group. The number of germinal centres was greater in the regrowth group. In the retrospective analysis of 3141 children, the incidence of resurgery after primary TT was 1.9%. Logistic regression analysis showed that age was the only significant predisposing factor for resurgery. CONCLUSION: Inflammatory cells were present more often in regrown tonsillar tissues but there was no evidence of severe chronic inflammation or scarring in the regrown tonsils. The risk of resurgery after TT was low. Young age predisposed to tonsillar regrowth, no other risk factors were found.
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Tonsilectomía , Tonsilitis , Niño , Cicatriz/cirugía , Humanos , Tonsila Palatina/patología , Estudios Retrospectivos , Factores de Riesgo , Tonsilectomía/efectos adversos , Tonsilitis/patologíaRESUMEN
BACKGROUND Subclinical graft inflammation and fibrosis after pediatric liver transplantation (LT) are common. Biomarkers are needed that precede and are associated with these changes and graft outcome. MATERIAL AND METHODS We evaluated immunohistochemical expression of 6 biomarkers [alpha-smooth muscle actin (alpha-SMA), collagen I, decorin, vimentin, P-selectin glycoprotein ligand-1 (PSGL-1), and CD34] in biopsies taken intraoperatively at LT (baseline) (n=29) and at 11.3 years after LT (first follow-up) (n=51). Liver biochemistry and graft histology were assessed at the first follow-up and at final assessment (19.6 years after LT) (n=48). Second follow-up biopsies for histology were available from 24 patients. The immunostainings were correlated with liver histology, biochemistry, and outcome at these time-points. RESULTS Baseline levels of the biomarkers were unrelated to presence of fibrosis at follow-up. Increased alpha-SMA, collagen I levels, decorin, and vimentin were associated with simultaneous fibrosis at the first follow-up (p=0.001-0.027). Increased SMA, collagen I, decorin, vimentin, PSGL-1, and CD34 expression at first follow-up were associated with simultaneous portal inflammation (p=0.001-0.025). alpha-SMA, decorin, and vimentin expression were increased in patients without fibrosis at the first follow-up but who developed fibrosis in second follow-up (p=0.014 p=0.024 and p=0.024). Significant fibrosis (F2) and markedly increased alpha-SMA, collagen I, decorin, and vimentin levels at first follow-up were associated with suboptimal liver status at the final assessment (p=0.002-0.042). CONCLUSIONS The expression of the biomarkers at LT was unrelated to later development of graft fibrosis. a-SMA, decorin, and vimentin were associated with later graft fibrosis and suboptimal liver status.
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Cirrosis Hepática , Trasplante de Hígado , Adolescente , Biomarcadores , Biopsia , Niño , Preescolar , Femenino , Fibrosis , Humanos , Lactante , Hígado/patología , Cirrosis Hepática/etiología , Cirrosis Hepática/patología , Trasplante de Hígado/efectos adversos , MasculinoRESUMEN
Amyloid precursor protein (APP) is ubiquitously expressed. Studies in neuronal cells have implicated APP or its fragments as negative regulators of cholesterol metabolism. In the current study, APP acted, via its α-cleavage, as a positive regulator of sterol regulatory element-binding protein-2 (SREBP2) signaling in human astrocytic cells (U251MG), hepatic cells (HepG2), and primary fibroblasts, leading to an approximate 30% increase in SRE-dependent gene expression and, consequently, enhanced cholesterol biosynthesis and LDL receptor levels. This effect was mediated via the secretory ectodomain APPsα. The ß-cleaved ectodomain, in turn, repressed SRE-dependent gene expression by up to â¼ 30%. This resulted in decreased cholesterol synthesis and LDL receptor content, establishing a physiological feedback loop in cholesterol-loaded cells, where APP undergoes preferential ß-cleavage. Patients with familial Alzheimer's disease had decreased circulating lathosterol, reflecting hepatic cholesterol synthesis, and their fibroblasts had reduced LDL receptor content, which was alleviated by decreasing ß-cleavage. These results show that APP regulates cholesterol metabolism in cells relevant for whole-body cholesterol balance and reveal that APP α- and ß-cleavages produce opposing paracrine regulators of SREBP2 signaling.
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Precursor de Proteína beta-Amiloide/metabolismo , Colesterol/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Western Blotting , Línea Celular , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Células Hep G2 , Humanos , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/fisiología , Reacción en Cadena de la Polimerasa , ARN Interferente Pequeño , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMEN
Hydroxysteroid (17beta) dehydrogenase 7 (HSD17B7) has been shown to catalyze the conversion of both estrone to estradiol (17-ketosteroid reductase activity) and zymosterone to zymosterol (3-ketosteroid reductase activity involved in cholesterol biosynthesis) in vitro. To define the metabolic role of the enzyme in vivo, we generated knockout mice deficient in the enzyme activity (HSD17B7KO). The data showed that the lack of HSD17B7 results in a blockage in the de novo cholesterol biosynthesis in mouse embryos in vivo, and HSD17BKO embryos die at embryonic day (E) 10.5. Analysis of neural structures revealed a defect in the development of hemispheres of the front brain with an increased apoptosis in the neuronal tissues. Morphological defects in the cardiovascular system were also observed from E9.5 onward. Mesodermal, endodermal, and hematopoietic cells were all detected by the histological analysis of the visceral yolk sac, whereas no organized vessels were observed in the knockout yolk sac. Immunohistological staining for platelet endothelial cell adhesion molecule-1 indicated that the complexity of the vasculature also was reduced in the HSD17B7KO embryos, particularly in the head capillary plexus and branchial arches. At E8.5-9.5, the heart development and the looping of the heart appeared to be normal in the HSD17B7KO embryos. However, at E10.5 the heart was dilated, and the thickness of the cardiac muscle and pericardium in the HSD17B7KO embryos was markedly reduced, and immunohistochemical staining for GATA-4 revealed that HSD17B7KO embryos had a reduced number of myocardial cells. The septum of the atrium was also defected in the knockout mice.
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17-Hidroxiesteroide Deshidrogenasas/metabolismo , Diferenciación Celular/genética , Colesterol/biosíntesis , Corazón/embriología , Placa Neural/embriología , 17-Hidroxiesteroide Deshidrogenasas/genética , Animales , Apoptosis/genética , Vasos Sanguíneos/embriología , Vasos Sanguíneos/enzimología , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Ratones , Ratones Noqueados , Miocardio/enzimología , Placa Neural/enzimología , Saco Vitelino/irrigación sanguínea , Saco Vitelino/embriologíaAsunto(s)
Encéfalo/metabolismo , Colesterol/metabolismo , Genética , Animales , Encéfalo/citología , Encéfalo/fisiología , RatonesRESUMEN
RATIONALE: The synthetic sphingosine analog FTY720 is undergoing clinical trials as an immunomodulatory compound, acting primarily via sphingosine 1-phosphate receptor activation. Sphingolipid and cholesterol homeostasis are closely connected but whether FTY720 affects atherogenesis in humans is not known. OBJECTIVE: We examined the effects of FTY720 on the processing of scavenged lipoprotein cholesterol in human primary monocyte-derived macrophages. METHODS AND RESULTS: FTY720 did not affect cholesterol uptake but inhibited its delivery to the endoplasmic reticulum, reducing cellular free cholesterol cytotoxicity. This was accompanied by increased levels of Niemann-Pick C1 protein (NPC1) and ATP-binding cassette transporter (ABC)A1 proteins and increased efflux of endosomal cholesterol to apolipoprotein A-I. These effects were not dependent on sphingosine 1-phosphate receptor activation. Instead, FTY720 stimulated the production of 27-hydroxycholesterol, an endogenous ligand of the liver X receptor, leading to liver X receptor-induced upregulation of ABCA1. Fluorescently labeled FTY720 was targeted to late endosomes, and the FTY720-induced upregulation of ABCA1 was NPC1-dependent, but the endosomal exit of FTY720 itself was not. CONCLUSIONS: We conclude that FTY720 decreases cholesterol toxicity in primary human macrophages by reducing the delivery of scavenged lipoprotein cholesterol to the endoplasmic reticulum and facilitating its release to physiological extracellular acceptors. Furthermore, FTY720 stimulates 27-hydroxycholesterol production, providing an explanation for the atheroprotective effects and identifying a novel mechanism by which FTY720 modulates signaling.
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Aterosclerosis/prevención & control , Colesterol/metabolismo , Hidroxicolesteroles/metabolismo , Macrófagos/efectos de los fármacos , Glicoles de Propileno/farmacología , Esfingosina/análogos & derivados , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Apolipoproteína A-I/metabolismo , Aterosclerosis/metabolismo , Transporte Biológico , Proteínas Portadoras/metabolismo , Técnicas de Cultivo de Célula , Muerte Celular , Supervivencia Celular , Células Cultivadas , Ésteres del Colesterol/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Clorhidrato de Fingolimod , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lipoproteínas LDL/metabolismo , Receptores X del Hígado , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteína Niemann-Pick C1 , Receptores Nucleares Huérfanos/agonistas , Receptores Nucleares Huérfanos/metabolismo , Receptores Depuradores/efectos de los fármacos , Receptores Depuradores/metabolismo , Transducción de Señal/efectos de los fármacos , Esfingosina/farmacología , Factores de TiempoRESUMEN
Cathepsin D (CTSD) deficiencies are fatal neurological diseases that in human infants and in sheep are characterized by extreme loss of neurons and myelin. To date, similar morphological evidence for myelin disruption in CTSD knockout mice has not been reported. Here, we show that CTSD deficiency leads to pronounced myelin changes in the murine brain: myelin-related proteolipid protein and myelin basic protein were both markedly reduced at postnatal day 24, and the amount of lipids characteristically high in myelin (e.g. plasmalogen-derived alkenyl chains and glycosphingolipid-derived 20- and 24-carbon acyl chains) were significantly lowered compared with controls. These changes were accompanied by ultrastructural alterations of myelin, including significant thinning of myelin sheaths. Furthermore, in CTSD knockout brains there was a pronounced accumulation of cholesteryl esters and abnormal levels of proteins related to cholesterol transport, with an increased content of apolipoprotein E and a reduced content of ATP-binding cassette transporter A1. These results provide evidence for dysmyelination and altered trafficking of cholesterol in brains of CTSD knockout mice, and warrant further studies on the role of lipid metabolism in the pathogenesis of CTSD deficiencies.
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Encéfalo/metabolismo , Catepsina D/deficiencia , Ésteres del Colesterol/metabolismo , Enfermedades Desmielinizantes/metabolismo , Vaina de Mielina/metabolismo , Animales , Transporte Biológico Activo/genética , Encéfalo/patología , Catepsina D/genética , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Vaina de Mielina/genética , Vaina de Mielina/patologíaRESUMEN
Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disorder of children, characterized by selective death of neocortical neurons. To understand early disease mechanisms in INCL, we have studied Ppt1(Deltaex4) knock-out mouse neurons in culture and acute brain slices. Global transcript profiling showed deregulation of key neuronal functions in knock-out mice including cholesterol metabolism, neuronal maturation, and calcium homeostasis. Cholesterol metabolism showed major changes; sterol biosynthesis was enhanced and steady-state amounts of sterols were altered at the cellular level. Changes were also present in early maturation of Ppt1(Deltaex4) neurons indicated by increased proliferative capacity of neuronal stem cells. Knock-out neurons presented unaltered electrophysiological properties suggesting uncompromised synaptic function in young animals. However, knock-out neurons exhibited more efficient recovery from glutamate-induced calcium transients, possibly indicating neuroprotective activation. This study established that the neuronal deregulation in INCL is linked to neuronal maturation, lipid metabolism and calcium homeostasis.
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Calcio/metabolismo , Colesterol/metabolismo , Homeostasis , Neuronas/metabolismo , Tioléster Hidrolasas/deficiencia , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Hipocampo/metabolismo , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Lipofuscinosis Ceroideas Neuronales/fisiopatología , Neuronas/citología , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Células Madre/citología , Sinapsis/metabolismo , Sinapsis/patología , Tioléster Hidrolasas/genéticaRESUMEN
MLN64 is a late endosomal cholesterol-binding membrane protein of an unknown function. Here, we show that MLN64 depletion results in the dispersion of late endocytic organelles to the cell periphery similarly as upon pharmacological actin disruption. The dispersed organelles in MLN64 knockdown cells exhibited decreased association with actin and the Arp2/3 complex subunit p34-Arc. MLN64 depletion was accompanied by impaired fusion of late endocytic organelles and delayed cargo degradation. MLN64 overexpression increased the number of actin and p34-Arc-positive patches on late endosomes, enhanced the fusion of late endocytic organelles in an actin-dependent manner, and stimulated the deposition of sterol in late endosomes harboring the protein. Overexpression of wild-type MLN64 was capable of rescuing the endosome dispersion in MLN64-depleted cells, whereas mutants of MLN64 defective in cholesterol binding were not, suggesting a functional connection between MLN64-mediated sterol transfer and actin-dependent late endosome dynamics. We propose that local sterol enrichment by MLN64 in the late endosomal membranes facilitates their association with actin, thereby governing actin-dependent fusion and degradative activity of late endocytic organelles.
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Actinas/metabolismo , Proteínas Portadoras/metabolismo , Endocitosis , Proteínas de la Membrana/metabolismo , Orgánulos/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular , Chlorocebus aethiops , Factor de Crecimiento Epidérmico/farmacología , Regulación de la Expresión Génica , Humanos , Proteínas de la Membrana/genética , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Orgánulos/efectos de los fármacos , Orgánulos/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factores de TiempoRESUMEN
Astrocytes secrete cholesterol in lipoprotein particles. Here we show that primary murine embryonic astrocytes secrete endogenously synthesized cholesterol but also the cholesterol precursors desmosterol and lathosterol. In astrocyte membranes, desmosterol and cholesterol were the predominant sterols. Astrocytes derived from Niemann-Pick type C lipidosis (NPC1-/-) mice displayed late endosomal cholesterol deposits, but the secretion of biosynthetic sterols from the cells was not inhibited. Both wild-type and NPC1-/- astrocytes secreted the NPC2 protein. Size-exclusion chromatography combined with electron microscopy showed that the majority of sterols were secreted separately from NPC2 in heterogeneous spherical particles with an average diameter of 20 nm. These data suggest that NPC2 and the majority of sterols secreted from astrocytes are not released together and that the secretion of neither sterols nor NPC2 requires NPC1 function. In addition, the findings reveal a complexity of sterol species in astrocytes and bring up the possibility that some of the effects assigned to astrocyte cholesterol may be attributed to its penultimate precursors.