RESUMO
Ciliopathies are associated with wide spectrum of structural birth defects (SBDs), indicating important roles for cilia in development. Here, we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140, an intraflagellar transport (IFT) protein regulating ciliogenesis. Ift140-deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula (TEF), randomized heart looping, congenital heart defects (CHDs), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAGGCre-ER deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD were not observed with 4 Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest-mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathies.
Assuntos
Ciliopatias , Cardiopatias Congênitas , Animais , Camundongos , Cílios/metabolismo , Cardiopatias Congênitas/genética , Desenvolvimento Embrionário , Proteínas de Transporte/metabolismo , Crânio , Ciliopatias/genética , Ciliopatias/metabolismo , Ciliopatias/patologiaRESUMO
Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice.
Assuntos
Cílios , Ciliopatias , Humanos , Animais , Camundongos , Cílios/genética , Cílios/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas/genética , Aminoácidos/metabolismo , Mamíferos/metabolismo , Proteínas do Citoesqueleto/genéticaRESUMO
Primary cilia (PC) are important signaling hubs, and we here explored their role in colonic pathology. In the colon, PC are mostly present on fibroblasts, and exposure of mice to either chemically induced colitis-associated colon carcinogenesis (CAC) or dextran sodium sulfate (DSS)-induced acute colitis decreases PC numbers. We generated conditional knockout mice with reduced numbers of PC on colonic fibroblasts. These mice show increased susceptibility to CAC, as well as DSS-induced colitis. Secretome and immunohistochemical analyses of DSS-treated mice display an elevated production of the proinflammatory cytokine IL-6 in PC-deficient colons. An inflammatory environment diminishes PC presence in primary fibroblast cultures, which is triggered by IL-6 as identified by RNA-seq analysis together with blocking experiments. These findings suggest an activation loop between IL-6 production and PC loss. An analysis of PC presence on biopsies of patients with ulcerative colitis or colorectal cancer (CRC) reveals decreased numbers of PC on colonic fibroblasts in pathological compared with surrounding normal tissue. Taken together, we provide evidence that a decrease in colonic PC numbers promotes colitis and CRC.
Assuntos
Cílios , Interleucina-6 , Camundongos , Animais , Interleucina-6/genéticaRESUMO
The interactions between viruses and actin cytoskeleton have been widely studied. We showed that rotaviruses remodel microfilaments in intestinal cells and demonstrated that this was due to the VP4 spike protein. Microfilaments mainly occur in the apical domain of infected polarized enterocytes and favor the polarized apical exit of viral progeny. The present work aims at the identification of molecular determinants of actin-VP4 interactions. We used various deletion mutants of VP4 that were transfected into Cos-7 cells and analyzed interactions by immunofluorescence confocal microscopy. It has been established that the C-terminal part of VP4 is embedded within viral particles when rotavirus assembles. The use of specific monoclonal antibodies demonstrated that VP4 is expressed in different forms in infected cells: classically as spike on the outer layer of virus particles, but also as free soluble protein in the cytosol. The C terminus of free VP4 was identified as interacting with actin microfilaments. The VP4 actin binding domain is unable to promote microfilament remodeling by itself; the coiled-coil domain is also required in this process. This actin-binding domain was shown to dominate a previously identified peroxisomal targeting signal, located in the three last amino acids of VP4. The newly identified actin-binding domain is highly conserved in rotavirus strains from species A, B, and C, suggesting that actin binding and remodeling is a general strategy for rotavirus exit. This provides a novel mechanism of protein-protein interactions, not involving cell signaling pathways, to facilitate rotavirus exit.IMPORTANCE Rotaviruses are causal agents of acute infantile viral diarrhea. In intestinal cells, in vitro as well as in vivo, virus assembly and exit do not imply cell lysis but rely on an active process in which the cytoskeleton plays a major role. We describe here a novel molecular mechanism by which the rotavirus spike protein VP4 drives actin remodeling. This relies on the fact that VP4 occurs in different forms. Besides its structural function within the virion, a large proportion of VP4 is expressed as free protein. Here, we show that free VP4 possesses a functional actin-binding domain. This domain, in coordination with a coiled-coil domain, promotes actin cytoskeleton remodeling, thereby providing the capacity to destabilize the cell membrane and allow efficient rotavirus exit.
Assuntos
Actinas/química , Actinas/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Rotavirus/metabolismo , Animais , Sítios de Ligação , Células COS , Proteínas do Capsídeo/genética , Linhagem Celular , Chlorocebus aethiops , Microscopia Confocal , Microscopia de Fluorescência , Mutação , Ligação Proteica , Domínios Proteicos , Rotavirus/química , Rotavirus/genéticaRESUMO
Primary cilia (PC) are antenna-like organelles that protrude from most mammalian cells. They are essential for the regulation of several signaling pathways such as Hedgehog and WNT It is therefore not surprising that a dysfunction of PC is frequently associated with pathologies. Originally, PC were found to be involved in a variety of diseases commonly referred to as ciliopathies including cystic kidney diseases. Evidence is accumulating that PC play also an important role in cancer formation and regulation, which is the focus of this review.
Assuntos
Cílios/metabolismo , Cílios/patologia , Neoplasias/patologia , Animais , Feminino , Proteínas Hedgehog/metabolismo , Humanos , Masculino , Neoplasias/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Via de Sinalização WntRESUMO
Hair follicle morphogenesis requires precisely controlled reciprocal communications, including hedgehog (Hh) signaling. Activation of the Hh signaling pathway relies on the primary cilium. Disrupting ciliogenesis results in hair follicle morphogenesis defects due to attenuated Hh signaling; however, the loss of cilia makes it impossible to determine whether hair follicle phenotypes in these cilia mutants are caused by the loss of cilia, disruption of Hh signaling, or a combination of these events. In this study, we characterized the function of Ift27, which encodes a subunit of intraflagellar transport (IFT) complex B. Hair follicle morphogenesis of Ift27-null mice was severely impaired, reminiscent of phenotypes observed in cilia and Hh mutants. Furthermore, the Hh signaling pathway was attenuated in Ift27 mutants, which was in association with abnormal ciliary trafficking of SMO and GLI2, and impaired processing of Gli transcription factors; however, formation of the ciliary axoneme was unaffected. The ciliary localization of IFT25 (HSPB11), the binding partner of IFT27, was disrupted in Ift27 mutant cells, and Ift25-null mice displayed hair follicle phenotypes similar to those of Ift27 mutants. These data suggest that Ift27 and Ift25 operate in a genetically and functionally dependent manner during hair follicle morphogenesis. This study suggests that the molecular trafficking machineries underlying ciliogenesis and Hh signaling can be segregated, thereby providing important insights into new avenues of inhibiting Hh signaling, which might be adopted in the development of targeted therapies for Hh-dependent cancers, such as basal cell carcinoma.
Assuntos
Folículo Piloso/embriologia , Proteínas Hedgehog/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Organogênese/genética , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Transporte Biológico/genética , Diferenciação Celular , Células Cultivadas , Cílios/fisiologia , Folículo Piloso/metabolismo , Proteínas Hedgehog/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Queratinócitos/citologia , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Knockout , Transporte Proteico/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Transplante de Pele , Receptor Smoothened , Proteína Gli2 com Dedos de Zinco , Proteínas rab de Ligação ao GTP/biossíntese , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Oral-facial-digital syndromes (OFDS) gather rare genetic disorders characterised by facial, oral and digital abnormalities associated with a wide range of additional features (polycystic kidney disease, cerebral malformations and several others) to delineate a growing list of OFDS subtypes. The most frequent, OFD type I, is caused by a heterozygous mutation in the OFD1 gene encoding a centrosomal protein. The wide clinical heterogeneity of OFDS suggests the involvement of other ciliary genes. For 15 years, we have aimed to identify the molecular bases of OFDS. This effort has been greatly helped by the recent development of whole-exome sequencing (WES). Here, we present all our published and unpublished results for WES in 24 cases with OFDS. We identified causal variants in five new genes (C2CD3, TMEM107, INTU, KIAA0753 and IFT57) and related the clinical spectrum of four genes in other ciliopathies (C5orf42, TMEM138, TMEM231 and WDPCP) to OFDS. Mutations were also detected in two genes previously implicated in OFDS. Functional studies revealed the involvement of centriole elongation, transition zone and intraflagellar transport defects in OFDS, thus characterising three ciliary protein modules: the complex KIAA0753-FOPNL-OFD1, a regulator of centriole elongation; the Meckel-Gruber syndrome module, a major component of the transition zone; and the CPLANE complex necessary for IFT-A assembly. OFDS now appear to be a distinct subgroup of ciliopathies with wide heterogeneity, which makes the initial classification obsolete. A clinical classification restricted to the three frequent/well-delineated subtypes could be proposed, and for patients who do not fit one of these three main subtypes, a further classification could be based on the genotype.
Assuntos
Face/anormalidades , Síndromes Orofaciodigitais/genética , Anormalidades Múltiplas/genética , Transtornos da Motilidade Ciliar/genética , Encefalocele/genética , Feminino , Heterozigoto , Humanos , Masculino , Mutação/genética , Doenças Renais Policísticas/genética , Proteínas/genética , Retinose PigmentarRESUMO
Recent studies identified a previously uncharacterized gene C5ORF42 (JBTS17) as a major cause of Joubert syndrome (JBTS), a ciliopathy associated with cerebellar abnormalities and other birth defects. Here we report the first Jbts17 mutant mouse model, Heart Under Glass (Hug), recovered from a forward genetic screen. Exome sequencing identified Hug as a S235P missense mutation in the mouse homolog of JBTS17 (2410089e03rik). Hug mutants exhibit multiple birth defects typical of ciliopathies, including skeletal dysplasia, polydactyly, craniofacial anomalies, kidney cysts and eye defects. Some Hug mutants exhibit congenital heart defects ranging from mild pulmonary stenosis to severe pulmonary atresia. Immunostaining showed JBTS17 is localized in the cilia transition zone. Fibroblasts from Hug mutant mice and a JBTS patient with a JBTS17 mutation showed ciliogenesis defects. Significantly, Hug mutant fibroblasts showed loss of not only JBTS17, but also NPHP1 and CEP290 from the cilia transition zone. Hug mutants exhibited reduced ciliation in the cerebellum. This was associated with reduction in cerebellar foliation. Using a fibroblast wound-healing assay, we showed Hug mutant cells cannot establish cell polarity required for directional cell migration. However, stereocilia patterning was grossly normal in the cochlea, indicating planar cell polarity is not markedly affected. Overall, we showed the JBTS pathophysiology is replicated in the Hug mutant mice harboring a Jbts17 mutation. Our findings demonstrate JBTS17 is a cilia transition zone component that acts upstream of other Joubert syndrome associated transition zone proteins NPHP1 and CEP290, indicating its importance in the pathogenesis of Joubert syndrome.
Assuntos
Doenças Cerebelares/genética , Cerebelo/anormalidades , Proteínas de Membrana/genética , Retina/anormalidades , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Polaridade Celular , Células Cultivadas , Doenças Cerebelares/patologia , Cerebelo/patologia , Cílios , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Anormalidades do Olho/genética , Anormalidades do Olho/patologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Doenças Renais Císticas/genética , Doenças Renais Císticas/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Gravidez , Transporte Proteico/genética , Retina/patologiaRESUMO
Short chain fatty acids (SCFAs) are primarily produced in the caecum and proximal colon via the bacterial fermentation of undigested carbohydrates that have avoided digestion in the small intestine. Increasing evidence supports the critical role that SCFAs play in health and homeostasis. Microbial SCFAs, namely butyric acid, serve as a principal energy source for colonocytes, and their production is essential for gut integrity. A direct link between SCFAs and some human pathological conditions, such as inflammatory bowel disease, irritable bowel syndrome, diarrhea, and cancer, has been proposed. The direct measurement of SCFAs in feces provides a non-invasive approach to demonstrating connections between SCFAs, microbiota, and metabolic diseases to estimate their potential applicability as meaningful biomarkers of intestinal health. This study aimed to adapt a robust analytical method (liquid-liquid extraction, followed by isobutyl chloroformate derivatization and GC-MS analysis), with comparable performances to methods from the literature, and to use this tool to tackle the question of pre-analytical conditions, namely stool processing. We focused on the methodology of managing stool samples before the analysis (fresh stool or dilution in either ethanol/methanol, lyophilized stool, or RNAlater®), as this is a significant issue to consider for standardizing results between clinical laboratories. The objective was to standardize methods for future applications as diagnostic tools. In this paper, we propose a validated GC-MS method for SCFA quantification in stool samples, including pre- and post-analytical comparison studies that could be easily used for clinical laboratory purposes. Our results show that using lyophilization as a stool-processing method would be the best method to achieve this goal.
RESUMO
OBJECTIVE, DESIGN, AND METHODS: Although 17-hydroxyprogesterone (17OHP) has historically been the steroid assayed in the diagnosis of congenital adrenal 21-hydroxylase deficiency (CAH-21D), its C11-hydroxylated metabolite, 21-deoxycortisol (21DF), which is strictly of adrenal origin, is assayed in parallel in this pathology. This steroid (21DF) is oxidized by 11beta-hydroxysteroid dehydrogenase type 2 into 21-deoxycortisone (21DE). In the context of CAH-21D confirmation testing, confounding factors (such as intensive care unit admission, stress, prematurity, early sampling, and variations of sex development) can interfere with the interpretation of the gold-standard biomarkers (17OHP and 21DF). Since its tissue concentrations are especially high in the placenta, we hypothesized that 21DE quantification in the neonatal periods could be an interesting biomarker in addition to 17OHP and 21DF. To verify this hypothesis, we developed a new mass spectrometry-based assay for 21DE in serum and applied it to newborns screened for CAH-21D. RESULTS: In newborns with CAH-21D, the mean serum levels of 21DE reached 17.56â ng/mL (ranging from 8.58â ng/mL to 23.20â ng/mL), and the mean 21DE:21DF ratio was 4.99. In contrast, in newborns without CAH-21D, the 21DE serum levels were low and not statistically different from the analytical 21DE limit of quantification (0.01â ng/mL). CONCLUSION: Basal serum 21DE appears to be a novel sensitive and specific biomarker of CAH-21D in newborns.
Assuntos
Hiperplasia Suprarrenal Congênita , Biomarcadores , Cortodoxona , Humanos , Hiperplasia Suprarrenal Congênita/diagnóstico , Hiperplasia Suprarrenal Congênita/sangue , Recém-Nascido , Feminino , Cortodoxona/sangue , Biomarcadores/sangue , Masculino , 17-alfa-Hidroxiprogesterona/sangue , Triagem Neonatal/métodos , Sensibilidade e EspecificidadeRESUMO
Ciliopathies are associated with wide spectrum of structural birth defects (SBD), indicating important roles for cilia in development. Here we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140 , an intraflagellar transport protein regulating ciliogenesis. Ift140 deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula, randomized heart looping, congenital heart defects (CHD), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAG-Cre deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD was not observed with four Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathy.
RESUMO
Motile and non-motile cilia are critical to mammalian development and health. Assembly of these organelles depends on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). A series of human and mouse IFT74 variants were studied to understand the function of this IFT subunit. Humans missing exon 2, which codes for the first 40 residues, presented an unusual combination of ciliary chondrodysplasia and mucociliary clearance disorders while individuals carrying biallelic splice site variants developed a lethal skeletal chondrodysplasia. In mice, variants thought to remove all Ift74 function, completely block ciliary assembly and result in midgestational lethality. A mouse allele that removes the first 40 amino acids, analogous to the human exon 2 deletion, results in a motile cilia phenotype with mild skeletal abnormalities. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia could account for the motile cilia phenotype observed in human and mice.
RESUMO
Tryptophan, an essential amino acid, and its metabolites are involved in many physiological processes including neuronal functions, immune system, and gut homeostasis. Alterations to tryptophan metabolism are associated with various pathologies such as neurologic, psychiatric disorders, inflammatory bowel diseases (IBD), metabolic disorders, and cancer. It is consequently critical to develop a reliable, quantitative method for the analysis of tryptophan and its downstream metabolites from the kynurenine, serotonin, and indoles pathways. An LC-MS/MS method was designed for the analysis of tryptophan and 20 of its metabolites, without derivatization and performed in a single run. This method was validated for both serum and stool. The comparisons between serum and plasma, collected with several differing anticoagulants, showed significant differences only for serotonin. References values were established in sera and stools from healthy donors. For stool samples, as a proof of concept, the developed method was applied to a healthy control group and an IBD patient group. Results showed significant differences in the concentrations of tryptophan, xanthurenic acid, kynurenic acid, indole-3-lactic acid, and picolinic acid. This method allowed an extensive analysis of the three tryptophan metabolic pathways in two compartments. Beyond the application to IBD patients, the clinical use of this method is wide-ranging and may be applied to other pathological conditions involving tryptophan metabolism, such as neurological, psychiatric, or auto-inflammatory pathologies.
Assuntos
Doenças Inflamatórias Intestinais , Triptofano , Humanos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Serotonina/metabolismo , CinureninaRESUMO
Neonatal screening for congenital adrenal hyperplasia (CAH) faces many specific challenges. It must be done using a performant analytical approach that combines sensitivity and specificity to capture the potential causes of mortality during the first week of life, such as salt wasting and glucocorticoid deficiency. Here, we confirm that maternal inhaled corticosteroid intake during pregnancy is a possible cause of missed CAH diagnosis. Thanks to liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis, we were able to quantify endogenous steroid metabolites and also detect the presence of exogenous steroids in the dried blood spot of a newborn. Adding LC-MS/MS analysis as second-tier test, especially one that includes both 17-hydroxyprogesterone and 21-deoxycortisol measurements, would probably improve CAH diagnosis. In familial neonatal screening one could also look for maternal corticosteroid therapies that are hidden to prevent false-negative tests.
RESUMO
In children with premature pubarche (PP), late onset 21-hydroxylase deficiency (21-OHD), also known as non-classical congenital adrenal hyperplasia (NCCAH), can be routinely ruled out by an adrenocorticotropic hormone (ACTH) test. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a quantitative assay of the circulating steroidome can be obtained from a single blood sample. We hypothesized that, by applying multivariate machine learning (ML) models to basal steroid profiles and clinical parameters of 97 patients, we could distinguish children with PP from those with NCCAH, without the need for ACTH testing. Every child presenting with PP at the Trousseau Pediatric Endocrinology Unit between 2016 and 2018 had a basal and stimulated steroidome. Patients with central precocious puberty were excluded. The first set of patients (year 1, training set, n = 58), including 8 children with NCCAH verified by ACTH test and genetic analysis, was used to train the model. Subsequently, a validation set of an additional set of patients (year 2, n = 39 with 5 NCCAH) was obtained to validate our model. We designed a score based on an ML approach (orthogonal partial least squares discriminant analysis). A metabolic footprint was assigned for each patient using clinical data, bone age, and adrenal steroid levels recorded by LC-MS/MS. Supervised multivariate analysis of the training set (year 1) and validation set (year 2) was used to validate our score. Based on selected variables, the prediction score was accurate (100%) at differentiating premature pubarche from late onset 21-OHD patients. The most significant variables were 21-deoxycorticosterone, 17-hydroxyprogesterone, and 21-deoxycortisol steroids. We proposed a new test that has excellent sensitivity and specificity for the diagnosis of NCCAH, due to an ML approach.
Assuntos
Hiperplasia Suprarrenal Congênita , Puberdade Precoce , Hiperplasia Suprarrenal Congênita/genética , Hormônio Adrenocorticotrópico , Algoritmos , Criança , Cromatografia Líquida , Feminino , Cabelo , Humanos , Aprendizado de Máquina , Masculino , Puberdade Precoce/diagnóstico , Puberdade Precoce/genética , Esteroides , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Human-induced pluripotent stem cell-derived hepatocytes (iHeps) have been shown to have considerable potential in liver diseases, toxicity, and pharmacological studies. However, there is a growing need to obtain iHeps that are truly similar to primary adult hepatocytes in terms of morphological features and functions. We generated such human iHeps, self-assembled as organoids (iHep-Orgs). METHODS: iPSC-derived hepatoblasts were self-assembled into spheroids and differentiated into mature hepatocytes modulating final step of differentiation. RESULTS: In about four weeks of culture, the albumin secretion levels and the complete disappearance of α-fetoprotein from iHep-Orgs suggested the acquisition of a greater degree of maturation than those previously reported. The expression of apical transporters and bile acid secretion evidenced the acquisition of complex hepatocyte polarity as well as the development of a functional and well-defined bile canalicular network confirmed by computational analysis. Activities recorded for CYP450, UGT1A1, and alcohol dehydrogenase, response to hormonal stimulation, and glucose metabolism were also remarkable. Finally, iHep-Orgs displayed a considerable ability to detoxify pathological concentrations of lactate and ammonia. CONCLUSIONS: With features similar to those of primary adult hepatocytes, the iHep-Orgs thus produced could be considered as a valuable tool for the development and optimization of preclinical and clinical applications.
Assuntos
Células-Tronco Pluripotentes Induzidas , Hepatopatias , Adulto , Diferenciação Celular , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Hepatopatias/metabolismo , Organoides/metabolismoRESUMO
The local immune-inflammatory response elicited by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is still poorly described, as well as the extent to which its characteristics may be associated with the outcome of critical Coronavirus disease 2019 (COVID-19). In this prospective monocenter study, all consecutive COVID-19 critically ill patients admitted from February to December 2020 and explored by fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) were included. Biological assays, including digital ELISA cytokine profiling and targeted eicosanoid metabolomic analysis, were performed on paired blood and BAL fluid (BALF). Clinical outcome was assessed through the World Health Organization 10-point Clinical Progression Scale (WHO-CPS) at the 28th day (D28) following the admission to intensive care unit. A D28-WHO-CPS value higher than 5 defined a poor outcome. Seventy-six patients were included, 45 (59%) had a poor day-28 outcome. As compared to their counterparts, patients with D28-WHO-CPS > 5 exhibited a neutrophil-predominant bronchoalveolar phenotype, with a higher BALF neutrophil/lymphocyte ratio, a blunted local type I interferon response, a decompartimentalized immune-inflammatory response illustrated by lower BALF/blood ratio of concentrations of IL-6 (1.68 [0.30-4.41] vs. 9.53 [2.56-19.1]; p = 0.001), IL-10, IL-5, IL-22 and IFN-γ, and a biological profile of vascular endothelial injury illustrated by a higher blood concentration of VEGF and higher blood and/or BALF concentrations of several vasoactive eicosanoids. In critically ill COVID-19 patients, we identified bronchoalveolar and blood immune-inflammatory biomarker signature associated with poor 28-day outcome.
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COVID-19 , Biomarcadores , Líquido da Lavagem Broncoalveolar , Estado Terminal , Humanos , Estudos Prospectivos , SARS-CoV-2RESUMO
Drug-induced cholestasis is mostly intrahepatic and characterized by alterations of bile canaliculi dynamics and morphology as well as accumulation of bile acids (BAs) in hepatocytes. However, little information exists on first changes in BA content and profile induced by cholestatic drugs in human liver. In this study, we aimed to analyze the effects of a large set of cholestatic and noncholestatic drugs in presence of physiological serum concentrations and 60-fold higher levels of 9 main BAs on cellular accumulation of BAs using HepaRG hepatocytes. BAs were measured in cell layers (cells + bile canaliculi) and culture media using high-pressure liquid chromatography coupled with tandem mass spectrometry after 24 h-treatment. Comparable changes in total and individual BA levels were observed in cell layers and media from control and noncholestatic drug-treated cultures: unconjugated BAs were actively amidated and lithocholic acid (LCA) was entirely sulfated. In contrast, cellular accumulation of LCA and in addition, of the 2 other hydrophobic BAs, chenodeoxycholic acid and deoxycholic acid, was evidenced only with cholestatic compounds in presence of BA mixtures at normal and 60-fold serum levels, respectively, suggesting that LCA was the first BA to accumulate. Cellular accumulation of hydrophobic BAs was associated with inhibition of their amidation and for LCA, its sulfation. In conclusion, these results demonstrated that cellular accumulation of unconjugated hydrophobic BAs can be caused by various cholestatic drugs in human hepatocytes and suggest that their cellular detection, especially that of LCA, could represent a new strategy for evaluation of cholestatic potential of drugs and other chemicals.
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Ácidos e Sais Biliares/metabolismo , Colestase/induzido quimicamente , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Biomarcadores/metabolismo , Colestase/metabolismo , Avaliação Pré-Clínica de Medicamentos , Hepatócitos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fígado/metabolismo , Preparações Farmacêuticas/química , Valor Preditivo dos TestesRESUMO
The vertebrate hedgehog pathway is organized in primary cilia, and hedgehog components relocate into or out of cilia during signaling. Defects in intraflagellar transport (IFT) typically disrupt ciliary assembly and attenuate hedgehog signaling. Determining whether IFT drives the movement of hedgehog components is difficult due to the requirement of IFT for building cilia. Unlike most IFT proteins, IFT27 is dispensable for cilia formation but affects hedgehog signaling similarly to other IFTs, allowing us to examine its role in the dynamics of signaling. Activating signaling at points along the pathway in Ift27 mutant cells showed that IFT is extensively involved in the pathway. Similar analysis of Bbs mutant cells showed that BBS proteins participate at many levels of signaling but are not needed to concentrate Gli transcription factors at the ciliary tip. Our analysis showed that smoothened delivery to cilia does not require IFT27, but the role of other IFTs is not known. Using a rapamycin-induced dimerization system to sequester IFT-B proteins at the mitochondria in cells with fully formed cilia did not affect the delivery of Smo to cilia, suggesting that this membrane protein may not require IFT-B for delivery.