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1.
Cell Syst ; 12(8): 780-794.e7, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34139154

RESUMO

COVID-19 is highly variable in its clinical presentation, ranging from asymptomatic infection to severe organ damage and death. We characterized the time-dependent progression of the disease in 139 COVID-19 inpatients by measuring 86 accredited diagnostic parameters, such as blood cell counts and enzyme activities, as well as untargeted plasma proteomes at 687 sampling points. We report an initial spike in a systemic inflammatory response, which is gradually alleviated and followed by a protein signature indicative of tissue repair, metabolic reconstitution, and immunomodulation. We identify prognostic marker signatures for devising risk-adapted treatment strategies and use machine learning to classify therapeutic needs. We show that the machine learning models based on the proteome are transferable to an independent cohort. Our study presents a map linking routinely used clinical diagnostic parameters to plasma proteomes and their dynamics in an infectious disease.


Assuntos
Biomarcadores/análise , COVID-19/patologia , Progressão da Doença , Proteoma/fisiologia , Fatores Etários , Contagem de Células Sanguíneas , Gasometria , Ativação Enzimática , Humanos , Inflamação/patologia , Aprendizado de Máquina , Prognóstico , Proteômica , SARS-CoV-2/imunologia
2.
Cell Syst ; 11(1): 11-24.e4, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32619549

RESUMO

The COVID-19 pandemic is an unprecedented global challenge, and point-of-care diagnostic classifiers are urgently required. Here, we present a platform for ultra-high-throughput serum and plasma proteomics that builds on ISO13485 standardization to facilitate simple implementation in regulated clinical laboratories. Our low-cost workflow handles up to 180 samples per day, enables high precision quantification, and reduces batch effects for large-scale and longitudinal studies. We use our platform on samples collected from a cohort of early hospitalized cases of the SARS-CoV-2 pandemic and identify 27 potential biomarkers that are differentially expressed depending on the WHO severity grade of COVID-19. They include complement factors, the coagulation system, inflammation modulators, and pro-inflammatory factors upstream and downstream of interleukin 6. All protocols and software for implementing our approach are freely available. In total, this work supports the development of routine proteomic assays to aid clinical decision making and generate hypotheses about potential COVID-19 therapeutic targets.


Assuntos
Proteínas Sanguíneas/metabolismo , Infecções por Coronavirus/sangue , Pneumonia Viral/sangue , Proteômica/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/isolamento & purificação , Biomarcadores/sangue , Proteínas Sanguíneas/análise , COVID-19 , Infecções por Coronavirus/classificação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias/classificação , Pneumonia Viral/classificação , Pneumonia Viral/patologia , Pneumonia Viral/virologia , SARS-CoV-2 , Adulto Jovem
3.
Eur Neurol ; 59(3-4): 143-7, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18057901

RESUMO

BACKGROUND: Family and twin studies suggest predisposing genetic factors in stroke. Lacunar infarcts represent a homogeneous phenotype, which is a prerequisite for genetic analyses. Applying an affected sib -pair analysis, we prospectively assessed the prevalence of microangiopathic brain lesions (MBL) and associated risk factors among siblings of patients with lacunar stroke. METHODS: Index patients fulfilled clinical criteria of a lacunar stroke in combination with a corresponding MBL on CT or MRI. Siblings were characterized as affected if MBL demonstrated on MRI. The prevalence of MBL was compared with population prevalence data extracted from other studies. RESULTS: From 784 patients screened, 81 index patients with lacunar stroke and 97 siblings were recruited, of which 42% were identified as affected. Compared with data from unselected historical controls and stratified by age groups, prevalence was between 2 and 5 times higher. CONCLUSIONS: Our results indicate that genetic stroke studies are feasible even in subtypes of ischemic stroke. The high prevalence of MBL among siblings of patients with lacunar infarct might suggest a familial aggregation. However, due to the small sample size these results need to be interpreted with caution and require confirmation by planned genetic analyses.


Assuntos
Infarto Encefálico , Angiografia Cerebral/métodos , Irmãos , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/genética , Idoso , Infarto Encefálico/epidemiologia , Infarto Encefálico/genética , Infarto Encefálico/patologia , Eletroencefalografia , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Prevalência , Estudos Prospectivos , Fatores de Risco , Acidente Vascular Cerebral/epidemiologia , Tomografia Computadorizada por Raios X
4.
Cancer Res ; 66(2): 649-52, 2006 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-16423992

RESUMO

The immune system can recognize antigenic peptides derived from tumors by their presentation on MHC class I complexes to CTLs. Immunoproteasomes (i20S) can substantially enhance the MHC class I peptide repertoire, making down-regulation of i20S an important strategy of tumor cells in manipulating immune surveillance. Here, we report that human cancer cells express the nonfunctional immunosubunit-variant LMP7E1, in addition to, or instead of LMP7E2, in response to IFN-gamma. This preferential expression of LMP7E1 and the consequent down-regulation of LMP7E2 results in i20S deficiency. The molecular explanation for this phenomenon is the incapacity of LMP7E1 to interact efficiently with the proteasome maturation protein, which regularly recruits LMP7E2 into nascent i20S precursor complexes. In contrast to previous reports, i20S formation in these cancer cells cannot be restored by IFN-gamma treatment. However, expression of LMP7E2 in these cells restores the i20S-deficient phenotype. Thus, our data describe a novel mechanism that contributes to the process of oncogenesis.


Assuntos
Transformação Celular Neoplásica/imunologia , Complexos Multienzimáticos/biossíntese , Antígenos de Neoplasias , Células CACO-2 , Regulação para Baixo , Regulação da Expressão Gênica , Células HeLa , Humanos , Interferon gama/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Melanoma/patologia , Fenótipo , Complexo de Endopeptidases do Proteassoma , Isoformas de Proteínas , Neoplasias Cutâneas/patologia , Linfócitos T Citotóxicos/imunologia
5.
Proc Natl Acad Sci U S A ; 102(26): 9241-6, 2005 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-15944226

RESUMO

Peptide generation by the proteasome is rate-limiting in MHC class I-restricted antigen presentation in response to IFN-gamma. IFN-gamma-induced de novo formation of immunoproteasomes, therefore, essentially supports the rapid adjustment of the mammalian immune system. Here, we report that the molecular interplay between the proteasome maturation protein (POMP) and the proteasomal beta5i subunit low molecular weight protein 7 (LMP7) has a key position in this immune adaptive program. IFN-gamma-induced coincident biosynthesis of POMP and LMP7 and their direct interaction essentially accelerate immunoproteasome biogenesis compared with constitutive 20S proteasome assembly. The dynamics of this process is determined by rapid LMP7 activation and the immediate LMP7-dependent degradation of POMP. Silencing of POMP expression impairs recruitment of both beta5 subunits into the proteasome complex, resulting in decreased proteasome activity, reduced MHC class I surface expression, and induction of apoptosis. Furthermore, our data reveal that immunoproteasomes exhibit a considerably shortened half-life, compared with constitutive proteasomes. In consequence, our studies demonstrate that the cytokine-induced rapid immune adaptation of the proteasome system is a tightly regulated and transient response allowing cells to return rapidly to a normal situation once immunoproteasome function is no longer required.


Assuntos
Interferon gama/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Apoptose , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Clonagem Molecular , Citocinas/metabolismo , Inativação Gênica , Genes MHC Classe I/genética , Células HeLa , Humanos , Sistema Imunitário , Imunoprecipitação , Cinética , Complexo Principal de Histocompatibilidade , Chaperonas Moleculares/metabolismo , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Regulação para Cima
6.
Int J Radiat Oncol Biol Phys ; 61(5): 1482-92, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15817354

RESUMO

PURPOSE: To study in detail the temporal and spatial release of the pro-inflammatory cytokines tumor necrosis factor alpha, interleukin (IL)-1alpha, and IL-6 in the lung tissue of C57BL/6 mice after thoracic irradiation with 12 Gy. METHODS AND MATERIALS: C57BL/6J mice were exposed to either sham irradiation or a single fraction of 12 Gy delivered to the thorax. Treated and sham-irradiated control mice were killed at 0.5 h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 1 week, 2 weeks, 4 weeks, 8 weeks, 16 weeks, and 24 weeks post-irradiation (p.i.). Real-time multiplex reverse transcriptase polymerase chain reaction was established to evaluate the relative messenger RNA (mRNA) expression of TNF-alpha, IL-1alpha, and IL-6 in the lung tissue of the mice (compared with nonirradiated lung tissue). Immunohistochemical detection methods (alkaline phosphatase anti-alkaline phosphatase, avidin-biotin-complex [ABC]) and automated image analysis were used to quantify the protein expression of TNF-alpha, IL-1alpha, and IL-6 in the lung tissue (percentage of the positively stained area). RESULTS: Radiation-induced release of the pro-inflammatory cytokines TNF-alpha, IL-1alpha, and IL-6 in the lung tissue was detectable within the first hours after thoracic irradiation. We observed statistically significant up-regulations for TNF-alpha at 1 h p.i. on mRNA (4.99 +/- 1.60) and at 6 h p.i. on protein level (7.23% +/- 1.67%), for IL-1alpha at 6 h p.i. on mRNA (11.03 +/- 0.77) and at 12 h p.i. on protein level (27.58% +/- 11.06%), for IL-6 at 6 h p.i. on mRNA (6.0 +/- 3.76) and at 12 h p.i. on protein level (7.12% +/- 1.93%). With immunohistochemistry, we could clearly demonstrate that the bronchiolar epithelium is the most prominent source of these inflammatory cytokines in the first hours after lung irradiation. During the stage of acute pneumonitis, the bronchiolar epithelium, as well as inflammatory cells in the lung interstitium, produced high amounts of TNF-alpha (with the maximal value at 4 weeks p.i.: 9.47% +/- 1.78%), IL-1alpha (with the peak value at 8 weeks p.i.: 14.76% +/- 7.77%), and IL-6 (with the peak value at 8 weeks p.i.: 4.28% +/- 1.33%). CONCLUSIONS: In the present study we have clearly demonstrated the immediate expression of the pro-inflammatory cytokines TNF-alpha, IL-1alpha, and IL-6 in the bronchiolar epithelium in the first hours after lung irradiation. A second, long-lasting release of these cytokines by the bronchiolar and alveolar epithelium, as well as by inflammatory cells, was observed at the onset of acute pneumonitis. Therefore, we postulate that lung irradiation causes immediate epithelial reaction, with the bronchiolar epithelium becoming a significant source of pro-inflammatory cytokines capable of promoting inflammation through recruitment and activation of inflammatory cells.


Assuntos
Brônquios/metabolismo , Brônquios/efeitos da radiação , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Pulmão/efeitos da radiação , Fator de Necrose Tumoral alfa/metabolismo , Animais , Epitélio/metabolismo , Epitélio/efeitos da radiação , Feminino , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Doses de Radiação
7.
Strahlenther Onkol ; 181(3): 197-204, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15756525

RESUMO

BACKGROUND AND PURPOSE: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ZD1839 (Iressa) reduces survival and augments radiation response of certain tumor cells. The aim of this study was to identify cellular events that are associated with the modulation of radiosensitivity by ZD1839. MATERIAL AND METHODS: Three tumor cell lines (A549, H596, FaDu) were exposed to ionizing radiation, treatment with ZD1839, and combined treatment. Clonogenic cell survival was determined by colony assays, EGFR and transforming growth factor-(TGF-)alpha expression by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), cell cycle distribution and apoptosis by flow cytometry. RESULTS: In A549 and H596 cells ZD1839 had little effect on clonogenic growth, but survival curves revealed a radiosensitizing effect of 5 microM ZD1839 on A549 cells. Both cell lines expressed moderate amounts of EGFR mRNA and very low levels of TGF-alpha mRNA. FaDu cells expressed relatively high amounts of EGFR and TGF-alpha transcripts and showed marked inhibition of clonogenic growth, reduction of S-phase cells, and induction of apoptosis after treatment with 1 microM ZD1839 and combined treatment. Surprisingly, the subpopulation of FaDu cells surviving ZD1839 pretreatment was more radioresistant. Exposure to ZD1839 caused a decrease in EGFR mRNA expression in A549 cells, no change in H596, and even an increase in FaDu cells. CONCLUSION: The sensitivity to ZD1839 correlated with the EGFR expression level, an inhibition of cell proliferation, and induction of apoptosis in the cell lines analyzed. A radiosensitizing effect of ZD1839 was associated with downregulation of EGFR mRNA expression.


Assuntos
Receptores ErbB/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Tolerância a Radiação/fisiologia , Adenocarcinoma , Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Linhagem Celular Tumoral , Gefitinibe , Humanos , Neoplasias Pulmonares , Tolerância a Radiação/efeitos dos fármacos
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