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1.
J Transl Med ; 14(1): 181, 2016 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-27320496

RESUMO

BACKGROUND: Exosomes are nano-sized extracellular vesicles participating in cell-to-cell communication both in health and disease. However, the knowledge about the functions and molecular composition of exosomes in the upper airways is limited. The aim of the current study was therefore to determine whether nasal exosomes can influence inflammatory cells and to establish the proteome of nasal lavage fluid-derived exosomes in healthy subjects, as well as its alterations in individuals with chronic airway inflammatory diseases [asthma and chronic rhinosinusitis (CRS)]. METHODS: Nasal lavage fluid was collected from 14 healthy subjects, 15 subjects with asthma and 13 subjects with asthma/CRS. Exosomes were isolated with differential centrifugation and the proteome was analysed by LC-MS/MS with the application of two exclusion lists as well as using quantitative proteomics. Ingenuity Pathways Analysis and GO Term finder was used to predict the functions associated with the exosomal proteome and a migration assay was used to analyse the effect on immune cells by nasal exosomes. RESULTS: Firstly, we demonstrate that nasal exosomes can induce migration of several immune cells, such as monocytes, neutrophils and NK cells in vitro. Secondly, a mass spectrometry approach, with the application of exclusion lists, was utilised to generate a comprehensive protein inventory of the exosomes from healthy subjects. The use of exclusion lists resulted in the identification of ~15 % additional proteins, and increased the confidence in ~20 % of identified proteins. In total, 604 proteins were identified in nasal exosomes and the nasal exosomal proteome showed strong associations with immune-related functions, such as immune cell trafficking. Thirdly, a quantitative proteomics approach was used to determine alterations in the exosome proteome as a result of airway inflammatory disease. Serum-associated proteins and mucins were more abundant in the exosomes from subjects with respiratory diseases compared to healthy controls while proteins with antimicrobial functions and barrier-related proteins had decreased expression. CONCLUSIONS: Nasal exosomes were shown to induce the migration of innate immune cells, which may be important as the airway epithelium is the first line of defence against pathogens and allergens. The decreased expression in barrier and antimicrobial exosomal proteins in subjects with airway diseases, could possibly contribute to an increased susceptibility to infections, which have important clinical implications in disease progression.


Assuntos
Exossomos/metabolismo , Inflamação/imunologia , Inflamação/patologia , Leucócitos/patologia , Mucosa Nasal/metabolismo , Sistema Respiratório/imunologia , Sistema Respiratório/patologia , Western Blotting , Movimento Celular , Cromatografia Líquida , Doença Crônica , Feminino , Citometria de Fluxo , Humanos , Masculino , Mucinas/metabolismo , Lavagem Nasal , Transporte Proteico , Proteoma/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem
2.
J Pathol ; 229(5): 719-28, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23335350

RESUMO

Current or recent enteroviral infections show an association with type 1 diabetes. However, evidence for this has mainly been generated using a particular mouse monoclonal antibody (clone 5-D8/1) which binds the viral capsid protein VP1. Difficulty in confirming these findings using other independent methods has led to the concern that this might be artefactual. To address this, we examined the potential cross-reactivity of clone 5-D8/1 with normal islet proteins. Western blotting, two-dimensional gel electrophoresis, and mass spectrometry were used to identify human islet proteins bound by the clone 5-D8/1. We found a distinct reactivity with two mitochondrial proteins, creatine kinase B-type and ATP synthase beta subunit. Immunohistochemistry using the clone 5-D8/1 revealed a granular cytoplasmic staining pattern in mitochondria-rich cells, ie hepatocytes, ductal epithelial cells, vascular endothelial cells, skeletal muscle cells, and the neoplastic salivary gland oncocytoma cells, whereas connective tissue and infiltrating immune cells were negative. Staining on islets of Langerhans from subjects with recent-onset type 1 diabetes, but not on isolated human islets infected in vitro with enteroviruses, could be blocked after mixing the clone 5-D8/1 with the mitochondrial proteins. Collectively, our data show that the clone 5-D8/1 detects two human mitochondrial enzymes in addition to enteroviral VP1. The notion that the previously reported VP1 positivity in islets of recent-onset type 1 diabetes patients could reflect cross-reactivity to native islet proteins and not the presence of EV is supported by difficulties in demonstrating EV infection by independent techniques such as PCR or in situ hybridization. These findings call for revisiting the presence of enteroviruses in pancreatic islets of patients with type 1 diabetes.


Assuntos
Anticorpos/imunologia , Proteínas do Capsídeo/imunologia , Creatina Quinase/imunologia , Diabetes Mellitus Tipo 1/imunologia , Infecções por Enterovirus/imunologia , Enterovirus/imunologia , Ilhotas Pancreáticas/imunologia , ATPases Mitocondriais Próton-Translocadoras/imunologia , Artefatos , Western Blotting , Reações Cruzadas , Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 1/virologia , Eletroforese em Gel Bidimensional , Infecções por Enterovirus/complicações , Infecções por Enterovirus/virologia , Humanos , Imuno-Histoquímica , Ilhotas Pancreáticas/enzimologia , Ilhotas Pancreáticas/virologia , Proteômica/métodos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Técnicas de Cultura de Tecidos
3.
Hepatol Commun ; 6(10): 2689-2701, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35833455

RESUMO

In nonalcoholic fatty liver disease (NAFLD) the patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 variant is a contributor. In mice, the Pnpla3 148M variant accumulates on lipid droplets and probably leads to sequestration of a lipase cofactor leading to impaired mobilization of triglycerides. To advance our understanding of the localization and abundance of PNPLA3 protein in humans, we used liver biopsies from patients with NAFLD to investigate the link to NAFLD and the PNPLA3 148M genotype. We experimentally qualified an antibody against human PNPLA3. Hepatic PNPLA3 protein fractional area and localization were determined by immunohistochemistry in biopsies from a well-characterized NAFLD cohort of 67 patients. Potential differences in hepatic PNPLA3 protein levels among patients related to degree of steatosis, lobular inflammation, ballooning, and fibrosis, and PNPLA3 I148M gene variants were assessed. Immunohistochemistry staining in biopsies from patients with NAFLD showed that hepatic PNPLA3 protein was predominantly localized to the membranes of small and large lipid droplets in hepatocytes. PNPLA3 protein levels correlated strongly with steatosis grade (p = 0.000027) and were also significantly higher in patients with lobular inflammation (p = 0.009), ballooning (p = 0.022), and significant fibrosis (stage 2-4, p = 0.014). In addition, PNPLA3 levels were higher in PNPLA3 rs738409 148M (CG, GG) risk allele carriers compared to 148I (CC) nonrisk allele carriers (p = 0.0029). Conclusion: PNPLA3 protein levels were associated with increased hepatic lipid content and disease severity in patients with NAFLD and were higher in PNPLA3 rs738409 (148M) risk allele carriers. Our hypothesis that increased hepatic levels of PNPLA3 may be part of the pathophysiological mechanism of NAFLD is supported.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Aciltransferases , Alelos , Animais , Fibrose , Humanos , Inflamação/genética , Lipase/genética , Proteínas de Membrana/genética , Hepatopatia Gordurosa não Alcoólica/genética , Fosfolipases/genética , Fosfolipases A2 Independentes de Cálcio/genética , Triglicerídeos
5.
J Alzheimers Dis ; 16(2): 389-97, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19221428

RESUMO

Amyloid-beta(Abeta) aggregation is a major hallmark of Alzheimer's disease (AD). Previous studies have suggested that only unbound Abeta can take part in the aggregation process. Therefore, endogenous Abeta-binding proteins may have an important role in preventing AD. Here, we analyzed cerebrospinal fluid (CSF) samples from 35 subjects with AD, 18 subjects with frontotemporal dementia (FTD) and 29 non-demented controls to test if reduced Abeta-binding capacity in CSF is a specific feature of AD. A panel of known Abeta-binding CSF proteins, including beta-trace/prostaglandin D2 synthase (beta-trace), transthyretin (TTR), cystatin C (CysC) and alpha(1)-antitrypsin (AAT), were quantified and related to diagnosis and CSF levels of Abeta(1-38), Abeta(1-40) and Abeta(1-42). AD patients displayed a mild reduction in the CSF levels of beta-trace (p=0.020), CysC (p=0.017), AAT (p=0.019) and TTR (p=0.012) compared with controls. While the reductions in AAT and TTR were AD-specific, the levels of beta-trace and CysC were also reduced in FTD. As expected, CSF Abeta(1-42) was reduced in AD compared with controls (p=0.00005) and with FTD patients (p=0.015). Positive correlations between Abeta(1-42) and beta-trace, CysC and TTR, respectively, were seen only in the AD group, suggesting that deficient Abeta-binding capacity in CSF may contribute to the amyloidogenic process in AD.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Pré-Albumina/líquido cefalorraquidiano , alfa 1-Antitripsina/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Cistatina C/líquido cefalorraquidiano , Demência/líquido cefalorraquidiano , Eletroquímica , Feminino , Humanos , Oxirredutases Intramoleculares/líquido cefalorraquidiano , Modelos Lineares , Lipocalinas/líquido cefalorraquidiano , Masculino , Nefelometria e Turbidimetria/métodos , Fragmentos de Peptídeos/líquido cefalorraquidiano , Estatísticas não Paramétricas
6.
PLoS One ; 13(4): e0196601, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29702679

RESUMO

Beta cell dysfunction accompanies and drives the progression of type 2 diabetes mellitus (T2D), but there are few clinical biomarkers available to assess islet cell stress in humans. Secretagogin, a protein enriched in pancreatic islets, demonstrates protective effects on beta cell function in animals. However, its potential as a circulating biomarker released from human beta cells and islets has not been studied. In this study primary human islets, beta cells and plasma samples were used to explore secretion and expression of secretagogin in relation to the T2D pathology. Secretagogin was abundantly and specifically expressed and secreted from human islets. Furthermore, T2D patients had an elevated plasma level of secretagogin compared with matched healthy controls, which was confirmed in plasma of diabetic mice transplanted with human islets. Additionally, the plasma secretagogin level of the human cohort had an inverse correlation to clinical assessments of beta cell function. To explore the mechanism of secretagogin release in vitro, human beta cells (EndoC-ßH1) were exposed to elevated glucose or cellular stress-inducing agents. Secretagogin was not released in parallel with glucose stimulated insulin release, but was markedly elevated in response to endoplasmic reticulum stressors and cytokines. These findings indicate that secretagogin is a potential novel biomarker, reflecting stress and islet cell dysfunction in T2D patients.


Assuntos
Diabetes Mellitus Tipo 2/sangue , Ilhotas Pancreáticas/metabolismo , Secretagoginas/sangue , Adulto , Idoso , Animais , Biomarcadores/sangue , Núcleo Celular/metabolismo , Estudos de Coortes , Citocinas/metabolismo , Citoplasma/metabolismo , Diabetes Mellitus Experimental/sangue , Retículo Endoplasmático/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Glucagon/metabolismo , Glucose/farmacologia , Teste de Tolerância a Glucose , Humanos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/fisiopatologia , Transplante das Ilhotas Pancreáticas , Masculino , Camundongos , Pessoa de Meia-Idade
7.
J Proteome Res ; 7(5): 2114-20, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18351740

RESUMO

The neurodegenerative disorder Alzheimer's disease (AD) is the most common cause of dementia in the elderly. The presence of neurofibrillary tangles, consisting of hyperphosphorylated tau protein, is one of the major neuropathologic characteristics of the disease, making this protein an attractive biomarker for AD and a possible target for therapy. Here, we describe an optimized immunoprecipitation mass spectrometry method that enables, for the first time, detailed characterization of tau in human cerebrospinal fluid. The identities of putative tau fragments were confirmed using nanoflow liquid chromatography and tandem mass spectrometry. Nineteen tryptic fragments of tau were detected, of which 16 are found in all tau isoforms while 3 represented unique tau isoforms. These results pave the way for clinical CSF studies on the tauopathies.


Assuntos
Imunoprecipitação/métodos , Espectrometria de Massas/métodos , Isoformas de Proteínas/líquido cefalorraquidiano , Isoformas de Proteínas/química , Proteínas tau/líquido cefalorraquidiano , Proteínas tau/química , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Química Encefálica , Cromatografia Líquida/métodos , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/líquido cefalorraquidiano , Fragmentos de Peptídeos/química , Isoformas de Proteínas/genética , Proteínas tau/genética
8.
Ann Neurol ; 62(2): 193-6; discussion 205, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16900522

RESUMO

OBJECTIVE: A recent study using surface-enhanced laser desorption/ionization time-of-flight analysis of cerebrospinal fluid identified a 12.5 kDa truncated isoform of cystatin C (CysC) as a specific biomarker for multiple sclerosis (MS). METHODS: Surface-enhanced laser desorption/ionization time-of-flight analysis of cerebrospinal fluid samples from 43 MS patients and 46 healthy control subjects. RESULTS: Full-length CysC (13.4 kDa) concentration was similar in MS and control samples. The 12.5 kDa CysC protein was produced from full-length CysC by N-terminal cleavage during storage at -20 degrees C. INTERPRETATION: The 12.5 kDa CysC isoform is a storage-related artifact and is not useful as a diagnostic marker for MS.


Assuntos
Cistatinas/líquido cefalorraquidiano , Esclerose Múltipla/líquido cefalorraquidiano , Sequência de Aminoácidos , Artefatos , Biomarcadores/líquido cefalorraquidiano , Cistatina C , Cistatinas/química , Cistatinas/genética , Armazenamento de Medicamentos , Congelamento , Humanos , Peso Molecular , Concentração Osmolar , Fragmentos de Peptídeos/líquido cefalorraquidiano , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Isoformas de Proteínas/líquido cefalorraquidiano , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo
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