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1.
Am J Respir Crit Care Med ; 207(6): 693-703, 2023 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-36457159

RESUMO

Rationale: Shared symptoms and genetic architecture between coronavirus disease (COVID-19) and lung fibrosis suggest severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may lead to progressive lung damage. Objectives: The UK Interstitial Lung Disease Consortium (UKILD) post-COVID-19 study interim analysis was planned to estimate the prevalence of residual lung abnormalities in people hospitalized with COVID-19 on the basis of risk strata. Methods: The PHOSP-COVID-19 (Post-Hospitalization COVID-19) study was used to capture routine and research follow-up within 240 days from discharge. Thoracic computed tomography linked by PHOSP-COVID-19 identifiers was scored for the percentage of residual lung abnormalities (ground-glass opacities and reticulations). Risk factors in linked computed tomography were estimated with Bayesian binomial regression, and risk strata were generated. Numbers within strata were used to estimate posthospitalization prevalence using Bayesian binomial distributions. Sensitivity analysis was restricted to participants with protocol-driven research follow-up. Measurements and Main Results: The interim cohort comprised 3,700 people. Of 209 subjects with linked computed tomography (median, 119 d; interquartile range, 83-155), 166 people (79.4%) had more than 10% involvement of residual lung abnormalities. Risk factors included abnormal chest X-ray (risk ratio [RR], 1.21; 95% credible interval [CrI], 1.05-1.40), percent predicted DlCO less than 80% (RR, 1.25; 95% CrI, 1.00-1.56), and severe admission requiring ventilation support (RR, 1.27; 95% CrI, 1.07-1.55). In the remaining 3,491 people, moderate to very high risk of residual lung abnormalities was classified at 7.8%, and posthospitalization prevalence was estimated at 8.5% (95% CrI, 7.6-9.5), rising to 11.7% (95% CrI, 10.3-13.1) in the sensitivity analysis. Conclusions: Residual lung abnormalities were estimated in up to 11% of people discharged after COVID-19-related hospitalization. Health services should monitor at-risk individuals to elucidate long-term functional implications.


Assuntos
COVID-19 , Doenças Pulmonares Intersticiais , Humanos , SARS-CoV-2 , COVID-19/epidemiologia , Teorema de Bayes , Pulmão/diagnóstico por imagem , Hospitalização
2.
Development ; 147(21)2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33033118

RESUMO

Mitchell-Riley syndrome (MRS) is caused by recessive mutations in the regulatory factor X6 gene (RFX6) and is characterised by pancreatic hypoplasia and neonatal diabetes. To determine why individuals with MRS specifically lack pancreatic endocrine cells, we micro-CT imaged a 12-week-old foetus homozygous for the nonsense mutation RFX6 c.1129C>T, which revealed loss of the pancreas body and tail. From this foetus, we derived iPSCs and show that differentiation of these cells in vitro proceeds normally until generation of pancreatic endoderm, which is significantly reduced. We additionally generated an RFX6HA reporter allele by gene targeting in wild-type H9 cells to precisely define RFX6 expression and in parallel performed in situ hybridisation for RFX6 in the dorsal pancreatic bud of a Carnegie stage 14 human embryo. Both in vitro and in vivo, we find that RFX6 specifically labels a subset of PDX1-expressing pancreatic endoderm. In summary, RFX6 is essential for efficient differentiation of pancreatic endoderm, and its absence in individuals with MRS specifically impairs formation of endocrine cells of the pancreas head and tail.


Assuntos
Diferenciação Celular , Diabetes Mellitus/genética , Diabetes Mellitus/patologia , Endoderma/embriologia , Doenças da Vesícula Biliar/genética , Doenças da Vesícula Biliar/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Atresia Intestinal/genética , Atresia Intestinal/patologia , Mutação/genética , Pâncreas/embriologia , Fatores de Transcrição de Fator Regulador X/genética , Alelos , Sequência de Bases , Diferenciação Celular/genética , Cromatina/metabolismo , Consanguinidade , Diabetes Mellitus/diagnóstico por imagem , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Família , Feminino , Doenças da Vesícula Biliar/diagnóstico por imagem , Genoma Humano , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Atresia Intestinal/diagnóstico por imagem , Masculino , Linhagem , Transcrição Gênica , Transcriptoma/genética , Microtomografia por Raio-X
3.
Diabetes Obes Metab ; 25(12): 3529-3537, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37646197

RESUMO

BACKGROUND: Donor hyperglycaemia following brain death has been attributed to reversible insulin resistance. However, our islet and pancreas transplant data suggest that other mechanisms may be predominant. We aimed to determine the relationships between donor insulin use and markers of beta-cell death and beta-cell function in pancreas donors after brain death. METHODS: In pancreas donors after brain death, we compared clinical and biochemical data in 'insulin-treated' and 'not insulin-treated donors' (IT vs. not-IT). We measured plasma glucose, C-peptide and levels of circulating unmethylated insulin gene promoter cell-free DNA (INS-cfDNA) and microRNA-375 (miR-375), as measures of beta-cell death. Relationships between markers of beta-cell death and islet isolation outcomes and post-transplant function were also evaluated. RESULTS: Of 92 pancreas donors, 40 (43%) required insulin. Glycaemic control and beta-cell function were significantly poorer in IT donors versus not-IT donors [median (IQR) peak glucose: 8 (7-11) vs. 6 (6-8) mmol/L, p = .016; C-peptide: 3280 (3159-3386) vs. 3195 (2868-3386) pmol/L, p = .046]. IT donors had significantly higher levels of INS-cfDNA [35 (18-52) vs. 30 (8-51) copies/ml, p = .035] and miR-375 [1.050 (0.19-1.95) vs. 0.73 (0.32-1.10) copies/nl, p = .05]. Circulating donor miR-375 was highly predictive of recipient islet graft failure at 3 months [adjusted receiver operator curve (SE) = 0.813 (0.149)]. CONCLUSIONS: In pancreas donors, hyperglycaemia requiring IT is strongly associated with beta-cell death. This provides an explanation for the relationship of donor IT with post-transplant beta-cell dysfunction in transplant recipients.


Assuntos
Ácidos Nucleicos Livres , Hiperglicemia , Transplante das Ilhotas Pancreáticas , MicroRNAs , Humanos , Peptídeo C , Morte Encefálica , Insulina/genética , Doadores de Tecidos , Morte Celular
4.
Genome Res ; 29(5): 723-736, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962179

RESUMO

Esophageal adenocarcinoma (EAC) is one of the most frequent causes of cancer death, and yet compared to other common cancers, we know relatively little about the molecular composition of this tumor type. To further our understanding of this cancer, we have used open chromatin profiling to decipher the transcriptional regulatory networks that are operational in EAC. We have uncovered a transcription factor network that is usually found in primitive intestinal cells during embryonic development, centered on HNF4A and GATA6. These transcription factors work together to control the EAC transcriptome. We show that this network is activated in Barrett's esophagus, the putative precursor state to EAC, thereby providing novel molecular evidence in support of stepwise malignant transition. Furthermore, we show that HNF4A alone is sufficient to drive chromatin opening and activation of a Barrett's-like chromatin signature when expressed in normal human epithelial cells. Collectively, these data provide a new way to categorize EAC at a genome scale and implicate HNF4A activation as a potential pivotal event in its malignant transition from healthy cells.


Assuntos
Adenocarcinoma/genética , Esôfago de Barrett/genética , Neoplasias Esofágicas/genética , Fator de Transcrição GATA6/metabolismo , Redes Reguladoras de Genes/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Adenocarcinoma/metabolismo , Biomarcadores Tumorais/genética , Progressão da Doença , Neoplasias Esofágicas/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Transcriptoma
5.
Proc Natl Acad Sci U S A ; 116(44): 22294-22299, 2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31611378

RESUMO

Androgen biosynthesis in the human fetus proceeds through the adrenal sex steroid precursor dehydroepiandrosterone, which is converted to testosterone in the gonads, followed by further activation to 5α-dihydrotestosterone in genital skin, thereby facilitating male external genital differentiation. Congenital adrenal hyperplasia due to P450 oxidoreductase deficiency results in disrupted dehydroepiandrosterone biosynthesis, explaining undervirilization in affected boys. However, many affected girls are born virilized, despite low circulating androgens. We hypothesized that this is due to a prenatally active, alternative androgen biosynthesis pathway from 17α-hydroxyprogesterone to 5α-dihydrotestosterone, which bypasses dehydroepiandrosterone and testosterone, with increased activity in congenital adrenal hyperplasia variants associated with 17α-hydroxyprogesterone accumulation. Here we employ explant cultures of human fetal organs (adrenals, gonads, genital skin) from the major period of sexual differentiation and show that alternative pathway androgen biosynthesis is active in the fetus, as assessed by liquid chromatography-tandem mass spectrometry. We found androgen receptor expression in male and female genital skin using immunohistochemistry and demonstrated that both 5α-dihydrotestosterone and adrenal explant culture supernatant induce nuclear translocation of the androgen receptor in female genital skin primary cultures. Analyzing urinary steroid excretion by gas chromatography-mass spectrometry, we show that neonates with P450 oxidoreductase deficiency produce androgens through the alternative androgen pathway during the first weeks of life. We provide quantitative in vitro evidence that the corresponding P450 oxidoreductase mutations predominantly support alternative pathway androgen biosynthesis. These results indicate a key role of alternative pathway androgen biosynthesis in the prenatal virilization of girls affected by congenital adrenal hyperplasia due to P450 oxidoreductase deficiency.


Assuntos
17-alfa-Hidroxiprogesterona/metabolismo , Androgênios/biossíntese , Fenótipo de Síndrome de Antley-Bixler/genética , Feto/metabolismo , Receptores Androgênicos/genética , Virilismo/metabolismo , Glândulas Suprarrenais/embriologia , Glândulas Suprarrenais/metabolismo , Androgênios/genética , Células Cultivadas , Feminino , Feto/embriologia , Genitália/embriologia , Genitália/metabolismo , Gônadas/embriologia , Gônadas/metabolismo , Humanos , Masculino , Receptores Androgênicos/metabolismo , Diferenciação Sexual , Virilismo/genética
6.
Diabetologia ; 64(6): 1375-1384, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33665687

RESUMO

AIMS/HYPOTHESIS: Approximately 50% of organ donors develop hyperglycaemia in intensive care, which is managed with insulin therapy. We aimed to determine the relationships between donor insulin use (DIU) and graft failure in pancreas transplantation. METHODS: UK Transplant Registry organ donor data were linked with national data from the UK solid pancreas transplant programme. All pancreas transplants performed between 2004 and 2016 with complete follow-up data were included. Logistic regression models determined associations between DIU and causes of graft failure within 3 months. Area under the receiver operating characteristic curve (aROC) and net reclassification improvement (NRI) assessed the added value of DIU as a predictor of graft failure. RESULTS: In 2168 pancreas transplant recipients, 1112 (51%) donors were insulin-treated. DIU was associated with a higher risk of graft loss from isolated islet failure: OR (95% CI), 1.79 (1.05, 3.07), p = 0.03, and this relationship was duration/dose dependent. DIU was also associated with a higher risk of graft loss from anastomotic leak (2.72 [1.07, 6.92], p = 0.04) and a lower risk of graft loss from thrombosis (0.62 [0.39, 0.96], p = 0.03), although duration/dose-dependent relationships were only identified in pancreas transplant alone/pancreas after kidney transplant recipients with grafts failing due to thrombosis (0.86 [0.74, 0.99], p = 0.03). The relationships between donor insulin characteristics and isolated islet failure remained significant after adjusting for potential confounders: DIU 1.75 (1.02, 2.99), p = 0.04; duration 1.08 (1.01, 1.16), p = 0.03. In multivariable analyses, donor insulin characteristics remained significant predictors of lower risk of graft thrombosis in pancreas transplant alone/pancreas after kidney transplant recipients: DIU, 0.34 (0.13, 0.90), p = 0.03; insulin duration/dose, 0.02 (0.001, 0.85), p = 0.04. When data on insulin were added to models predicting isolated islet failure, a significant improvement in discrimination and risk reclassification was observed in all models: no DIU aROC 0.56; DIU aROC 0.57, p = 0.86; NRI 0.28, p < 0.00001; insulin duration aROC 0.60, p = 0.47; NRI 0.35, p < 0.00001. CONCLUSIONS/INTERPRETATION: DIU predicts graft survival in pancreas transplant recipients. This assessment could help improve donor selection and thereby improve patient and graft outcomes.


Assuntos
Cuidados Críticos , Sobrevivência de Enxerto , Hiperglicemia/tratamento farmacológico , Insulina/uso terapêutico , Transplante de Pâncreas , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Sistema de Registros , Adulto Jovem
7.
Diabetes Obes Metab ; 23(1): 49-57, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32893472

RESUMO

AIMS: The relationship between peri-transplant glycaemic control and outcomes following pancreas transplantation is unknown. We aimed to relate peri-transplant glycaemic control to pancreas graft survival and to develop a framework for defining early graft dysfunction. METHODS: Peri-transplant glycaemic control profiles over the first 5 days postoperatively were determined by an area under the curve [AUC; average daily glucose level (mmol/L) × time (days)] and the coefficient of variation of mean daily glucose levels. Peri-transplant hyperglycaemia was defined as an AUC ≥35 mmol/day/L (daily mean blood glucose ≥7 mmol/L). Risks of graft failure associated with glycaemic control and variability and peri-transplant hyperglycaemia were determined using covariate-adjusted Cox regression. RESULTS: We collected 7606 glucose readings over 5 days postoperatively from 123 pancreas transplant recipients. Glucose AUC was a significant predictor of graft failure during 3.6 years of follow-up (unadjusted HR [95% confidence interval] 1.17 [1.06-1.30], P = .002). Death censored non-technical graft failure occurred in eight (10%) recipients with peri-transplant normoglycaemia, and eight (25%) recipients with peri-transplant hyperglycaemia such that hyperglycaemia predicted a 3-fold higher risk of graft failure [HR (95% confidence interval): 3.0 (1.1-8.0); P = .028]. CONCLUSION: Peri-transplant hyperglycaemia is strongly associated with graft loss and could be a valuable tool guiding individualized graft monitoring and treatment. The 5-day peri-transplant glucose AUC provides a robust and responsive framework for comparing graft function.


Assuntos
Transplante de Pâncreas , Glicemia , Controle Glicêmico , Sobrevivência de Enxerto , Humanos , Pâncreas
8.
BMC Infect Dis ; 21(1): 700, 2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294037

RESUMO

BACKGROUND: Predicting hospital length of stay (LoS) for patients with COVID-19 infection is essential to ensure that adequate bed capacity can be provided without unnecessarily restricting care for patients with other conditions. Here, we demonstrate the utility of three complementary methods for predicting LoS using UK national- and hospital-level data. METHOD: On a national scale, relevant patients were identified from the COVID-19 Hospitalisation in England Surveillance System (CHESS) reports. An Accelerated Failure Time (AFT) survival model and a truncation corrected method (TC), both with underlying Weibull distributions, were fitted to the data to estimate LoS from hospital admission date to an outcome (death or discharge) and from hospital admission date to Intensive Care Unit (ICU) admission date. In a second approach we fit a multi-state (MS) survival model to data directly from the Manchester University NHS Foundation Trust (MFT). We develop a planning tool that uses LoS estimates from these models to predict bed occupancy. RESULTS: All methods produced similar overall estimates of LoS for overall hospital stay, given a patient is not admitted to ICU (8.4, 9.1 and 8.0 days for AFT, TC and MS, respectively). Estimates differ more significantly between the local and national level when considering ICU. National estimates for ICU LoS from AFT and TC were 12.4 and 13.4 days, whereas in local data the MS method produced estimates of 18.9 days. CONCLUSIONS: Given the complexity and partiality of different data sources and the rapidly evolving nature of the COVID-19 pandemic, it is most appropriate to use multiple analysis methods on multiple datasets. The AFT method accounts for censored cases, but does not allow for simultaneous consideration of different outcomes. The TC method does not include censored cases, instead correcting for truncation in the data, but does consider these different outcomes. The MS method can model complex pathways to different outcomes whilst accounting for censoring, but cannot handle non-random case missingness. Overall, we conclude that data-driven modelling approaches of LoS using these methods is useful in epidemic planning and management, and should be considered for widespread adoption throughout healthcare systems internationally where similar data resources exist.


Assuntos
COVID-19/terapia , Unidades de Terapia Intensiva/estatística & dados numéricos , Tempo de Internação/estatística & dados numéricos , Idoso , COVID-19/epidemiologia , Análise de Dados , Inglaterra/epidemiologia , Feminino , Número de Leitos em Hospital , Planejamento Hospitalar/métodos , Humanos , Masculino , Pessoa de Meia-Idade
9.
Diabetes Obes Metab ; 22(10): 1874-1879, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32452110

RESUMO

Insulin is routinely used to manage hyperglycaemia in organ donors and during the peri-transplant period in islet transplant recipients. However, it is unknown whether donor insulin use (DIU) predicts beta-cell dysfunction after islet transplantation. We reviewed data from the UK Transplant Registry and the UK Islet Transplant Consortium; all first-time transplants during 2008-2016 were included. Linear regression models determined associations between DIU, median and coefficient of variation (CV) peri-transplant glucose levels and 3-month islet graft function. In 91 islet cell transplant recipients, DIU was associated with lower islet function assessed by BETA-2 scores (ß [SE] -3.5 [1.5], P = .02), higher 3-month post-transplant HbA1c levels (5.4 [2.6] mmol/mol, P = .04) and lower fasting C-peptide levels (-107.9 [46.1] pmol/l, P = .02). Glucose at 10 512 time points was recorded during the first 5 days peri-transplant: the median (IQR) daily glucose level was 7.9 (7.0-8.9) mmol/L and glucose CV was 28% (21%-35%). Neither median glucose levels nor glucose CV predicted outcomes post-transplantation. Data on DIU predicts beta-cell dysfunction 3 months after islet transplantation and could help improve donor selection and transplant outcomes.


Assuntos
Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Transplante das Ilhotas Pancreáticas , Glicemia , Peptídeo C , Glucose , Humanos , Insulina , Doadores de Tecidos
10.
Development ; 142(18): 3126-37, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26395141

RESUMO

A wealth of data and comprehensive reviews exist on pancreas development in mammals, primarily mice, and other vertebrates. By contrast, human pancreatic development has been less comprehensively reviewed. Here, we draw together those studies conducted directly in human embryonic and fetal tissue to provide an overview of what is known about human pancreatic development. We discuss the relevance of this work to manufacturing insulin-secreting ß-cells from pluripotent stem cells and to different aspects of diabetes, especially permanent neonatal diabetes, and its underlying causes.


Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células Secretoras de Insulina/citologia , Morfogênese/fisiologia , Pâncreas/embriologia , Pâncreas/crescimento & desenvolvimento , Células-Tronco Pluripotentes/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Especificidade da Espécie
11.
Hepatology ; 65(2): 710-721, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27775817

RESUMO

Current preclinical drug testing does not predict some forms of adverse drug reactions in humans. Efforts at improving predictability of drug-induced tissue injury in humans include using stem cell technology to generate human cells for screening for adverse effects of drugs in humans. The advent of induced pluripotent stem cells means that it may ultimately be possible to develop personalized toxicology to determine interindividual susceptibility to adverse drug reactions. However, the complexity of idiosyncratic drug-induced liver injury means that no current single-cell model, whether of primary liver tissue origin, from liver cell lines, or derived from stem cells, adequately emulates what is believed to occur during human drug-induced liver injury. Nevertheless, a single-cell model of a human hepatocyte which emulates key features of a hepatocyte is likely to be valuable in assessing potential chemical risk; furthermore, understanding how to generate a relevant hepatocyte will also be critical to efforts to build complex multicellular models of the liver. Currently, hepatocyte-like cells differentiated from stem cells still fall short of recapitulating the full mature hepatocellular phenotype. Therefore, we convened a number of experts from the areas of preclinical and clinical hepatotoxicity and safety assessment, from industry, academia, and regulatory bodies, to specifically explore the application of stem cells in hepatotoxicity safety assessment and to make recommendations for the way forward. In this short review, we particularly discuss the importance of benchmarking stem cell-derived hepatocyte-like cells to their terminally differentiated human counterparts using defined phenotyping, to make sure the cells are relevant and comparable between labs, and outline why this process is essential before the cells are introduced into chemical safety assessment. (Hepatology 2017;65:710-721).


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Hepatócitos/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Testes de Toxicidade , Células Cultivadas/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes/metabolismo , Valor Preditivo dos Testes , Sensibilidade e Especificidade
14.
Am J Hum Genet ; 92(2): 259-64, 2013 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-23313374

RESUMO

Urofacial syndrome (UFS) (or Ochoa syndrome) is an autosomal-recessive disease characterized by congenital urinary bladder dysfunction, associated with a significant risk of kidney failure, and an abnormal facial expression upon smiling, laughing, and crying. We report that a subset of UFS-affected individuals have biallelic mutations in LRIG2, encoding leucine-rich repeats and immunoglobulin-like domains 2, a protein implicated in neural cell signaling and tumorigenesis. Importantly, we have demonstrated that rare variants in LRIG2 might be relevant to nonsyndromic bladder disease. We have previously shown that UFS is also caused by mutations in HPSE2, encoding heparanase-2. LRIG2 and heparanase-2 were immunodetected in nerve fascicles growing between muscle bundles within the human fetal bladder, directly implicating both molecules in neural development in the lower urinary tract.


Assuntos
Glicoproteínas de Membrana/genética , Mutação/genética , Doenças Urológicas/genética , Sequência de Bases , Criança , Pré-Escolar , Análise Mutacional de DNA , Fácies , Família , Feminino , Humanos , Imuno-Histoquímica , Lactente , Masculino , Dados de Sequência Molecular , Linhagem , Bexiga Urinária/patologia , Bexiga Urinaria Neurogênica/genética , Doenças Urológicas/fisiopatologia
15.
Am J Hum Genet ; 92(4): 605-13, 2013 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-23541340

RESUMO

Perrault syndrome is a genetically and clinically heterogeneous autosomal-recessive condition characterized by sensorineural hearing loss and ovarian failure. By a combination of linkage analysis, homozygosity mapping, and exome sequencing in three families, we identified mutations in CLPP as the likely cause of this phenotype. In each family, affected individuals were homozygous for a different pathogenic CLPP allele: c.433A>C (p.Thr145Pro), c.440G>C (p.Cys147Ser), or an experimentally demonstrated splice-donor-site mutation, c.270+4A>G. CLPP, a component of a mitochondrial ATP-dependent proteolytic complex, is a highly conserved endopeptidase encoded by CLPP and forms an element of the evolutionarily ancient mitochondrial unfolded-protein response (UPR(mt)) stress signaling pathway. Crystal-structure modeling suggests that both substitutions would alter the structure of the CLPP barrel chamber that captures unfolded proteins and exposes them to proteolysis. Together with the previous identification of mutations in HARS2, encoding mitochondrial histidyl-tRNA synthetase, mutations in CLPP expose dysfunction of mitochondrial protein homeostasis as a cause of Perrault syndrome.


Assuntos
Proteases Dependentes de ATP/genética , Endopeptidase Clp/genética , Exoma/genética , Genes Recessivos , Disgenesia Gonadal 46 XX/etiologia , Perda Auditiva Neurossensorial/etiologia , Mitocôndrias/enzimologia , Mutação/genética , Proteases Dependentes de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Adolescente , Adulto , Feminino , Homozigoto , Humanos , Hibridização In Situ , Masculino , Mitocôndrias/genética , Linhagem , Fenótipo , Adulto Jovem
16.
J Biol Chem ; 289(13): 8931-46, 2014 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-24488492

RESUMO

Glucocorticoids (GC) regulate cell fate and immune function. We identified the metastasis-promoting methyltransferase, metastasis-related methyltransferase 1 (WBSCR22/Merm1) as a novel glucocorticoid receptor (GR) regulator relevant to human disease. Merm1 binds the GR co-activator GRIP1 but not GR. Loss of Merm1 impaired both GR transactivation and transrepression by reducing GR recruitment to its binding sites. This was accompanied by loss of GR-dependent H3K4Me3 at a well characterized promoter. Inflammation promotes GC resistance, in part through the actions of TNFα and IFNγ. These cytokines suppressed Merm1 protein expression by driving ubiquitination of two conserved lysine residues. Restoration of Merm1 expression rescued GR transactivation. Cytokine suppression of Merm1 and of GR function was also seen in human lung explants. In addition, striking loss of Merm1 protein was observed in both inflammatory and neoplastic human lung pathologies. In conclusion, Merm1 is a novel regulator of chromatin structure affecting GR recruitment and function, contributing to loss of GC sensitivity in inflammation, with suppressed expression in pulmonary disease.


Assuntos
Neoplasias Pulmonares/metabolismo , Metiltransferases/metabolismo , Receptores de Glucocorticoides/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Brônquios/patologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Interferon gama/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Lisina/metabolismo , Metilação/efeitos dos fármacos , Metiltransferases/química , Ligação Proteica , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/genética , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitinação/efeitos dos fármacos
17.
J Hepatol ; 62(3): 581-9, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25457200

RESUMO

BACKGROUND & AIMS: Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. METHODS: Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. RESULTS: HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. CONCLUSIONS: HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes.


Assuntos
Células-Tronco Fetais/citologia , Células-Tronco Fetais/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Adulto , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Metaboloma , Modelos Biológicos , Fenótipo , Proteoma/metabolismo
18.
Am J Hum Genet ; 89(5): 668-74, 2011 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-22077972

RESUMO

Urinary bladder malformations associated with bladder outlet obstruction are a frequent cause of progressive renal failure in children. We here describe a muscarinic acetylcholine receptor M3 (CHRM3) (1q41-q44) homozygous frameshift mutation in familial congenital bladder malformation associated with a prune-belly-like syndrome, defining an isolated gene defect underlying this sometimes devastating disease. CHRM3 encodes the M3 muscarinic acetylcholine receptor, which we show is present in developing renal epithelia and bladder muscle. These observations may imply that M3 has a role beyond its known contribution to detrusor contractions. This Mendelian disease caused by a muscarinic acetylcholine receptor mutation strikingly phenocopies Chrm3 null mutant mice.


Assuntos
Erros Inatos do Metabolismo/genética , Síndrome do Abdome em Ameixa Seca/genética , Receptor Muscarínico M3 , Bexiga Urinária , Animais , Sequência de Bases , Consanguinidade , Feminino , Mutação da Fase de Leitura/genética , Humanos , Mutação INDEL/genética , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Modelos Moleculares , Síndrome do Abdome em Ameixa Seca/patologia , Receptor Muscarínico M3/deficiência , Receptor Muscarínico M3/genética , Homologia de Sequência do Ácido Nucleico , Fatores Sexuais , Bexiga Urinária/embriologia , Bexiga Urinária/patologia , Obstrução do Colo da Bexiga Urinária/genética , Obstrução do Colo da Bexiga Urinária/patologia
19.
Hepatology ; 58(2): 799-809, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23526496

RESUMO

UNLABELLED: Failure to predict hepatotoxic drugs in preclinical testing makes it imperative to develop better liver models with a stable phenotype in culture. Stem cell-derived models offer promise, with differentiated hepatocyte-like cells currently considered to be "fetal-like" in their maturity. However, this judgment is based on limited biomarkers or transcripts and lacks the required proteomic datasets that directly compare fetal and adult hepatocytes. Here, we quantitatively compare the proteomes of human fetal liver, adult hepatocytes, and the HepG2 cell line. In addition, we investigate the proteome changes in human fetal and adult hepatocytes when cultured in a new air-liquid interface format compared to conventional submerged extracellular matrix sandwich culture. From albumin and urea secretion, and luciferase-based cytochrome P450 activity, adult hepatocytes were viable in either culture model over 2 weeks. The function of fetal cells was better maintained in the air-liquid interface system. Strikingly, the proteome was qualitatively similar across all samples but hierarchical clustering showed that each sample type had a distinct quantitative profile. HepG2 cells more closely resembled fetal than adult hepatocytes. Furthermore, clustering showed that primary adult hepatocytes cultured at the air-liquid interface retained a proteome that more closely mimicked their fresh counterparts than conventional culture, which acquired myofibroblast features. Principal component analysis extended these findings and identified a simple set of proteins, including cytochrome P450 2A6, glutathione S transferase P, and alcohol dehydrogenases as specialized indicators of hepatocyte differentiation. CONCLUSION: Our quantitative datasets are the first that directly compare multiple human liver cells, define a model for enhanced maintenance of the hepatocyte proteome in culture, and provide a new protein "toolkit" for determining human hepatocyte maturity in cultured cells.


Assuntos
Diferenciação Celular/genética , Hepatócitos/metabolismo , Hepatócitos/patologia , Proteômica/métodos , Álcool Desidrogenase/metabolismo , Células Cultivadas , Sistema Enzimático do Citocromo P-450/metabolismo , Glutationa Transferase/metabolismo , Células Hep G2 , Humanos , Fígado/citologia , Fígado/embriologia , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia
20.
Hepatology ; 56(3): 1108-16, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22488688

RESUMO

UNLABELLED: Osteopontin (OPN) is an important component of the extracellular matrix (ECM), which promotes liver fibrosis and has been described as a biomarker for its severity. Previously, we have demonstrated that Sex-determining region Y-box 9 (SOX9) is ectopically expressed during activation of hepatic stellate cells (HSC) when it is responsible for the production of type 1 collagen, which causes scar formation in liver fibrosis. Here, we demonstrate that SOX9 regulates OPN. During normal development and in the mature liver, SOX9 and OPN are coexpressed in the biliary duct. In rodent and human models of fibrosis, both proteins were increased and colocalized to fibrotic regions in vivo and in culture-activated HSCs. SOX9 bound a conserved upstream region of the OPN gene, and abrogation of Sox9 in HSCs significantly decreased OPN production. Hedgehog (Hh) signaling has previously been shown to regulate OPN expression directly by glioblastoma (GLI) 1. Our data indicate that in models of liver fibrosis, Hh signaling more likely acts through SOX9 to modulate OPN. In contrast to Gli2 and Gli3, Gli1 is sparse in HSCs and is not increased upon activation. Furthermore, reduction of GLI2, but not GLI3, decreased the expression of both SOX9 and OPN, whereas overexpressing SOX9 or constitutively active GLI2 could rescue the antagonistic effects of cyclopamine on OPN expression. CONCLUSION: These data reinforce SOX9, downstream of Hh signaling, as a core factor mediating the expression of ECM components involved in liver fibrosis. Understanding the role and regulation of SOX9 during liver fibrosis will provide insight into its potential modulation as an antifibrotic therapy or as a means of identifying potential ECM targets, similar to OPN, as biomarkers of fibrosis.


Assuntos
Cirrose Hepática/diagnóstico , Cirrose Hepática/etiologia , Osteopontina/fisiologia , Fatores de Transcrição SOX9/fisiologia , Animais , Progressão da Doença , Humanos , Masculino , Osteopontina/biossíntese , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição SOX9/biossíntese
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