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1.
Commun Med (Lond) ; 4(1): 37, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38443590

RESUMO

BACKGROUND: Lung cancer is associated with the greatest cancer mortality as it typically presents with incurable distributed disease. Biomarkers relevant to risk assessment for the detection of lung cancer continue to be a challenge because they are often not detectable during the asymptomatic curable stage of the disease. A solution to population-scale testing for lung cancer will require a combination of performance, scalability, cost-effectiveness, and simplicity. METHODS: One solution is to measure the activity of serum available enzymes that contribute to the transformation process rather than counting biomarkers. Protease enzymes modify the environment during tumor growth and present an attractive target for detection. An activity based sensor platform sensitive to active protease enzymes is presented. A panel of 18 sensors was used to measure 750 sera samples from participants at increased risk for lung cancer with or without the disease. RESULTS: A machine learning approach is applied to generate algorithms that detect 90% of cancer patients overall with a specificity of 82% including 90% sensitivity in Stage I when disease intervention is most effective and detection more challenging. CONCLUSION: This approach is promising as a scalable, clinically useful platform to help detect patients who have lung cancer using a simple blood sample. The performance and cost profile is being pursued in studies as a platform for population wide screening.


Lung cancer is responsible for more deaths worldwide than all other cancers. It is often detected with the appearance of symptoms when treatment is limited and outcomes for the patient are much worse. While imaging chest scans can detect disease, they are poorly used even in the United States where it is an approved screening method. When cancer is present, protease enzymes are responsible for making space and modifying the lung tissue for the growing tumor. This report describes a panel of 18 sensors that release a fluorescent signal when these enzymes are present in a blood sample. The signal acts like a fingerprint of activity that can be used to identify people with lung cancer. This sensor platform can detect patients with curable lung cancer and could provide a platform for screening very large populations of at-risk individuals.

2.
JCI Insight ; 4(5)2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30702443

RESUMO

Molecular profiling of prostate cancer with liquid biopsies, such as circulating tumor cells (CTCs) and cell-free nucleic acid analysis, yields informative yet distinct data sets. Additional insights may be gained by simultaneously interrogating multiple liquid biopsy components to construct a more comprehensive molecular disease profile. We conducted an initial proof-of-principle study aimed at piloting this multiparametric approach. Peripheral blood samples from men with metastatic castrate-resistant prostate cancer were analyzed simultaneously for CTC enumeration, single-cell copy number variations, CTC DNA and matched cell-free DNA mutations, and plasma cell-free RNA levels of androgen receptor (AR) and AR splice variant (ARV7). In addition, liquid biopsies were compared with matched tumor profiles when available, and a second liquid biopsy was drawn and analyzed at disease progression in a subset of patients. In this manner, multiparametric liquid biopsy profiles were successfully generated for each patient and time point, demonstrating the feasibility of this approach and highlighting shared as well as unique cancer-relevant alterations. With further refinement and validation in large cohorts, multiparametric liquid biopsies can optimally integrate disparate but clinically informative data sets and maximize their utility for molecularly directed, real-time patient management.


Assuntos
Biópsia Líquida/métodos , Neoplasias da Próstata/sangue , Neoplasias da Próstata/patologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Ácidos Nucleicos Livres/sangue , Variações do Número de Cópias de DNA , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Células Neoplásicas Circulantes , Neoplasias da Próstata/genética , Receptores Androgênicos/sangue , Receptores Androgênicos/genética
3.
Breast Cancer Res Treat ; 163(2): 219-230, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28271309

RESUMO

PURPOSE: The molecular characterization of circulating tumor cells (CTCs) is critical to identify the key drivers of cancer metastasis and devising therapeutic approaches, particularly for inflammatory breast cancer (IBC) which is usually diagnosed at advance stages and progresses rapidly. METHODS: Genomic alterations in tumor tissue samples were studied using Foundation One™. Single CTCs were isolated using CellSearch followed by single-cell isolation by DEPArray™. Samples with 20 or more CTCs were chosen to isolate single CTCs using the DEPArray™. RESULTS: Genomic alterations were studied in primary tumor or metastatic sites from 32 IBC patients. Genes with high-frequency mutations were as follows: TP53 (69%), RB1 (16%), PIK3CA (13%), and also ErbB2 (3%). At least once during treatment, CTCs were detected in 26 patients with metastatic IBC, in two patients with locally advanced IBC, and four patients had no detectable CTCs. Per 7.5 mL of blood, fifteen patients (47%) had ≥20 CTCs and six of them were chosen at random to isolate single CTCs. These cells were tested for the presence of TP53, RB1, PIK3CA, and/or ErbB2 mutations previously found in matching tissue biopsies. The isolated CTCs showed the same mutations as primary or metastatic tumor samples. Intra-patient CTC heterogeneity was found by the presence of different CTC subclones, with some CTCs harboring different combinations of mutated and wild-type genes. CONCLUSIONS: Our results indicate that CTCs could represent a non-invasive source of cancer cells from which to determine genetic markers as the disease progresses and identify potential therapeutic targets in IBC patients.


Assuntos
Neoplasias Inflamatórias Mamárias/sangue , Adulto , Idoso , Sequência de Bases , Classe I de Fosfatidilinositol 3-Quinases/genética , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Humanos , Neoplasias Inflamatórias Mamárias/genética , Neoplasias Inflamatórias Mamárias/mortalidade , Neoplasias Inflamatórias Mamárias/patologia , Pessoa de Meia-Idade , Células Neoplásicas Circulantes , Receptor ErbB-2/genética , Proteínas de Ligação a Retinoblastoma/genética , Deleção de Sequência , Análise de Célula Única , Análise de Sobrevida , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genética
4.
Oncotarget ; 7(18): 26724-38, 2016 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-27049831

RESUMO

Targeted cancer therapeutics are promised to have a major impact on cancer treatment and survival. Successful application of these novel treatments requires a molecular definition of a patient's disease typically achieved through the use of tissue biopsies. Alternatively, allowing longitudinal monitoring, biomarkers derived from blood, isolated either from circulating tumor cell derived DNA (ctcDNA) or circulating cell-free tumor DNA (ccfDNA) may be evaluated. In order to use blood derived templates for mutational profiling in clinical decisions, it is essential to understand the different template qualities and how they compare to biopsy derived template DNA as both blood-based templates are rare and distinct from the gold-standard. Using a next generation re-sequencing strategy, concordance of the mutational spectrum was evaluated in 32 patient-matched ctcDNA and ccfDNA templates with comparison to tissue biopsy derived DNA template. Different CTC antibody capture systems for DNA isolation from patient blood samples were also compared. Significant overlap was observed between ctcDNA, ccfDNA and tissue derived templates. Interestingly, if the results of ctcDNA and ccfDNA template sequencing were combined, productive samples showed similar detection frequency (56% vs 58%), were temporally flexible, and were complementary both to each other and the gold standard. These observations justify the use of a multiple template approach to the liquid biopsy, where germline, ctcDNA, and ccfDNA templates are employed for clinical diagnostic purposes and open a path to comprehensive blood derived biomarker access.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/genética , DNA Tumoral Circulante/sangue , Análise Mutacional de DNA/métodos , Células Neoplásicas Circulantes , Adulto , Idoso , Neoplasias da Mama/sangue , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade
5.
PLoS One ; 9(1): e86717, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24489774

RESUMO

BACKGROUND: Contemporary cancer diagnostics are becoming increasing reliant upon sophisticated new molecular methods for analyzing genetic information. Limiting the scope of these new technologies is the lack of adequate solid tumor tissue samples. Patients may present with tumors that are not accessible to biopsy or adequate for longitudinal monitoring. One attractive alternate source is cancer cells in the peripheral blood. These rare circulating tumor cells (CTC) require enrichment and isolation before molecular analysis can be performed. Current CTC platforms lack either the throughput or reliability to use in a clinical setting or they provide CTC samples at purities that restrict molecular access by limiting the molecular tools available. METHODOLOGY/PRINCIPAL FINDINGS: Recent advances in magetophoresis and microfluidics have been employed to produce an automated platform called LiquidBiopsy®. This platform uses high throughput sheath flow microfluidics for the positive selection of CTC populations. Furthermore the platform quantitatively isolates cells useful for molecular methods such as detection of mutations. CTC recovery was characterized and validated with an accuracy (<20% error) and a precision (CV<25%) down to at least 9 CTC/ml. Using anti-EpCAM antibodies as the capture agent, the platform recovers 78% of MCF7 cells within the linear range. Non specific recovery of background cells is independent of target cell density and averages 55 cells/mL. 10% purity can be achieved with as low as 6 CTCs/mL and better than 1% purity can be achieved with 1 CTC/mL. CONCLUSIONS/SIGNIFICANCE: The LiquidBiopsy platform is an automated validated platform that provides high throughput molecular access to the CTC population. It can be validated and integrated into the lab flow enabling CTC enumeration as well as recovery of consistently high purity samples for molecular analysis such as quantitative PCR and Next Generation Sequencing. This tool opens the way for clinically relevant genetic profiling of CTCs.


Assuntos
Separação Celular/métodos , Células Neoplásicas Circulantes/metabolismo , Anticorpos/química , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Automação Laboratorial , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Contagem de Células , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial , Expressão Gênica , Humanos , Imãs , Técnicas Analíticas Microfluídicas , Células Neoplásicas Circulantes/patologia , Reologia
7.
Cell Res ; 21(7): 1068-79, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21467996

RESUMO

Monocytes are mobilized to sites of infection via interaction between the chemokine MCP-1 and its receptor, CCR2, at which point they differentiate into macrophages that mediate potent antimicrobial effects. In this study, we investigated the mechanisms by which monocytes are mobilized in response to systemic challenge with the intracellular bacterium Francisella tularensis. We found that mice deficient in MyD88, interferon-γ (IFNγ)R or CCR2 all had defects in the expansion of splenic monocyte populations upon F. tularensis challenge, and in control of F. tularensis infection. Interestingly, MyD88-deficient mice were defective in production of IFNγ, and IFNγR-deficient mice exhibited defective production of MCP-1, the ligand for CCR2. Transplantation of IFNγR-deficient bone marrow (BM) into wild-type mice further suggested that mobilization of monocytes in response to F. tularensis challenge required IFNγR expression on BM-derived cells. These studies define a critical host defense circuit wherein MyD88-dependent IFNγ production signals via IFNγR expressed on BM-derived cells, resulting in MCP-1 production and activation of CCR2-dependent mobilization of monocytes in the innate immune response to systemic F. tularensis challenge.


Assuntos
Francisella tularensis/fisiologia , Interações Hospedeiro-Patógeno , Camundongos/microbiologia , Monócitos/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Receptores CCR2/imunologia , Receptores de Interferon/imunologia , Animais , Quimiocina CCL2/imunologia , Deleção de Genes , Expressão Gênica , Imunidade Inata , Camundongos/imunologia , Monócitos/citologia , Monócitos/microbiologia , Fator 88 de Diferenciação Mieloide/genética , Receptores CCR2/genética , Receptores de Interferon/genética , Transdução de Sinais , Baço/citologia , Tularemia/imunologia , Receptor de Interferon gama
8.
Sci Signal ; 3(123): ra41, 2010 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-20501937

RESUMO

Canonical and noncanonical nuclear factor kappaB (NF-kappaB) signaling are the two basic pathways responsible for the release of NF-kappaB dimers from their inhibitors. Enhanced NF-kappaB signaling leads to inflammatory and proliferative diseases; thus, inhibitory pathways that limit its activity are critical. Whereas multiple negative feedback mechanisms control canonical NF-kappaB signaling, none has been identified for the noncanonical pathway. Here, we describe a mechanism of negative feedback control of noncanonical NF-kappaB signaling that attenuated the stabilization of NF-kappaB-inducing kinase (NIK), the central regulatory kinase of the noncanonical pathway, induced by B cell-activating factor receptor (BAFF-R) and lymphotoxin beta receptor (LTbetaR). Inhibitor of kappaB (IkappaB) kinase alpha (IKKalpha) was previously thought to lie downstream of NIK in the noncanonical NF-kappaB pathway; we showed that phosphorylation of NIK by IKKalpha destabilized NIK. In the absence of IKKalpha-mediated negative feedback, the abundance of NIK increased after receptor ligation. A form of NIK with mutations in the IKKalpha-targeted serine residues was more stable than wild-type NIK and resulted in increased noncanonical NF-kappaB signaling. Thus, in addition to the regulation of the basal abundance of NIK in unstimulated cells by a complex containing tumor necrosis factor receptor-associated factor (TRAF) and cellular inhibitor of apoptosis (cIAP) proteins, IKKalpha-dependent destabilization of NIK prevents the uncontrolled activity of the noncanonical NF-kappaB pathway after receptor ligation.


Assuntos
Retroalimentação Fisiológica/fisiologia , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Receptor do Fator Ativador de Células B/metabolismo , Western Blotting , Linhagem Celular , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Quinase I-kappa B/genética , Imunoprecipitação , Proteínas Inibidoras de Apoptose/metabolismo , Receptor beta de Linfotoxina/metabolismo , Camundongos , Mutação , NF-kappa B/genética , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Serina/genética , Serina/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Transfecção , Quinase Induzida por NF-kappaB
9.
Cancer Res ; 70(7): 2595-603, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-20233880

RESUMO

Inflammation has increasingly been recognized as a critical component influencing tumor growth. Recent reports have revealed conflicting evidence for the role of Toll-like receptors (TLR) in modulating tumorigenesis. In our study, we implicate TLR3 in mediating immune surveillance with increased growth of implanted transgenic adenocarcinoma of the mouse prostate (TRAMP) tumors in TLR3(-/-) compared with TLR3(+/+) mice. Activation of TLR3 by polyinosinic-polycytidylic acid (polyI:C) leads to induction of multiple inflammatory pathways, including NF-kappaB, mitogen-activated protein kinases, and interferon (IFN) regulatory factors. We explored the potential of TLR3 stimulation in prostate cancer immunotherapy and showed that treatment with polyI:C can strongly suppress both s.c. implanted TRAMP tumors in syngenic mice as well as orthotopic prostate cancers in TRAMP C57Bl6 x FvB F1 Tg(+/-) transgenic mice. Treated tumors remained well differentiated to moderately differentiated with increased infiltration of T lymphocytes and natural killer (NK) cells compared with poorly differentiated adenocarcinoma observed in untreated tumors. Like TLR3(-/-) mice, IFN-alpha receptor 1 (IFNAR1)(-/-) mice exhibited reduced tumor surveillance and impaired tumor suppression following polyI:C treatment. We observed that type I IFN-dependent induction of cytokines was responsible for NK activation, with depletion of NK cells leading to increased tumor growth as well as expansion of CD4(+)CD25(+)Foxp3(+) T regulatory lymphocytes. Our study therefore delineates the importance of IFNAR-dependent functions in TLR3-mediated tumor suppression and supports the use of TLR3 agonists for prostate cancer immune-based therapies.


Assuntos
Interferon Tipo I/imunologia , Neoplasias da Próstata/imunologia , Receptor 3 Toll-Like/imunologia , Animais , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/imunologia , Linhagem Celular Tumoral , Tolerância Imunológica , Indutores de Interferon/farmacologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Células Matadoras Naturais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Poli I-C/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Receptor de Interferon alfa e beta/imunologia , Linfócitos T Reguladores/imunologia
10.
PLoS One ; 4(6): e5810, 2009 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-19503839

RESUMO

Antimicrobial effector mechanisms are central to the function of the innate immune response in host defense against microbial pathogens. In humans, activation of Toll-like receptor 2/1 (TLR2/1) on monocytes induces a vitamin D dependent antimicrobial activity against intracellular mycobacteria. Here, we report that TLR activation of monocytes triggers induction of the defensin beta 4 gene (DEFB4), requiring convergence of the IL-1beta and vitamin D receptor (VDR) pathways. TLR2/1 activation triggered IL-1beta activity, involving the upregulation of both IL-1beta and IL-1 receptor, and downregulation of the IL-1 receptor antagonist. TLR2/1L induction of IL-1beta was required for upregulation of DEFB4, but not cathelicidin, whereas VDR activation was required for expression of both antimicrobial genes. The differential requirements for induction of DEFB4 and cathelicidin were reflected by differences in their respective promoter regions; the DEFB4 promoter had one vitamin D response element (VDRE) and two NF-kappaB sites, whereas the cathelicidin promoter had three VDREs and no NF-kappaB sites. Transfection of NF-kappaB into primary monocytes synergized with 1,25D3 in the induction of DEFB4 expression. Knockdown of either DEFB4 or cathelicidin in primary monocytes resulted in the loss of TLR2/1-mediated antimicrobial activity against intracellular mycobacteria. Therefore, these data identify a novel mechanism of host defense requiring the induction of IL-1beta in synergy with vitamin D activation, for the TLR-induced antimicrobial pathway against an intracellular pathogen.


Assuntos
Anti-Infecciosos/farmacologia , Regulação da Expressão Gênica , Interleucina-1beta/metabolismo , Receptores de Calcitriol/metabolismo , Receptor 2 Toll-Like/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Humanos , Modelos Biológicos , Monócitos/metabolismo , Monócitos/microbiologia , Mycobacterium tuberculosis/metabolismo , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Células-Tronco , Receptor 1 Toll-Like/metabolismo , beta-Defensinas/genética , Catelicidinas
11.
PLoS Pathog ; 5(4): e1000379, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19360122

RESUMO

Here we investigated the role of the Nod/Rip2 pathway in host responses to Chlamydophila pneumoniae-induced pneumonia in mice. Rip2(-/-) mice infected with C. pneumoniae exhibited impaired iNOS expression and NO production, and delayed neutrophil recruitment to the lungs. Levels of IL-6 and IFN-gamma levels as well as KC and MIP-2 levels in bronchoalveolar lavage fluid (BALF) were significantly decreased in Rip2(-/-) mice compared to wild-type (WT) mice at day 3. Rip2(-/-) mice showed significant delay in bacterial clearance from the lungs and developed more severe and chronic lung inflammation that continued even on day 35 and led to increased mortality, whereas WT mice cleared the bacterial load, recovered from acute pneumonia, and survived. Both Nod1(-/-) and Nod2(-/-) mice also showed delayed bacterial clearance, suggesting that C. pneumoniae is recognized by both of these intracellular receptors. Bone marrow chimera experiments demonstrated that Rip2 in BM-derived cells rather than non-hematopoietic stromal cells played a key role in host responses in the lungs and clearance of C. pneumoniae. Furthermore, adoptive transfer of WT macrophages intratracheally was able to rescue the bacterial clearance defect in Rip2(-/-) mice. These results demonstrate that in addition to the TLR/MyD88 pathway, the Nod/Rip2 signaling pathway also plays a significant role in intracellular recognition, innate immune host responses, and ultimately has a decisive impact on clearance of C. pneumoniae from the lungs and survival of the infectious challenge.


Assuntos
Infecções por Chlamydophila/imunologia , Imunidade Inata , Proteínas Adaptadoras de Sinalização NOD/imunologia , Pneumonia Bacteriana/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Quimiocinas/imunologia , Quimiocinas/metabolismo , Infecções por Chlamydophila/metabolismo , Infecções por Chlamydophila/patologia , Chlamydophila pneumoniae/imunologia , Chlamydophila pneumoniae/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos/imunologia , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Proteínas Adaptadoras de Sinalização NOD/metabolismo , Pneumonia Bacteriana/metabolismo , Pneumonia Bacteriana/patologia , RNA Mensageiro/análise , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia
12.
Nat Immunol ; 9(12): 1371-8, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18997794

RESUMO

Recent studies suggest that nuclear factor kappaB-inducing kinase (NIK) is suppressed through constitutive proteasome-mediated degradation regulated by TRAF2, TRAF3 and cIAP1 or cIAP2. Here we demonstrated that the degradation of NIK occurs upon assembly of a regulatory complex through TRAF3 recruitment of NIK and TRAF2 recruitment of cIAP1 and cIAP2. In contrast to TRAF2 and TRAF3, cIAP1 and cIAP2 seem to play redundant roles in the degradation of NIK, as inhibition of both cIAPs was required for noncanonical NF-kappaB activation and increased survival and proliferation of primary B lymphocytes. Furthermore, the lethality of TRAF3 deficiency in mice could be rescued by a single NIK gene, highlighting the importance of tightly regulated NIK.


Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Proteínas Inibidoras de Apoptose/imunologia , NF-kappa B/imunologia , Fator 2 Associado a Receptor de TNF/imunologia , Fator 3 Associado a Receptor de TNF/imunologia , Animais , Linfócitos B/citologia , Sobrevivência Celular , Células Cultivadas , Ativação Enzimática/imunologia , Immunoblotting , Imunoprecipitação , Proteínas Inibidoras de Apoptose/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Interferente Pequeno , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Transfecção
13.
J Exp Med ; 203(11): 2413-8, 2006 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-17015635

RESUMO

Proper activation of nuclear factor (NF)-kappaB transcription factors is critical in regulating fundamental biological processes such as cell survival and proliferation, as well as in inflammatory and immune responses. Recently, the NF-kappaB signaling pathways have been categorized into the canonical pathway, which results in the nuclear translocation of NF-kappaB complexes containing p50, and the noncanonical pathway, which involves the induced processing of p100 to p52 and the formation of NF-kappaB complexes containing p52 (Bonizzi, G., and M. Karin. 2004. Trends Immunol. 25:280-288). We demonstrate that loss of tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) results in constitutive noncanonical NF-kappaB activity. Importantly, TRAF3-/- B cells show ligand-independent up-regulation of intracellular adhesion molecule 1 and protection from spontaneous apoptosis during in vitro culture. In addition, we demonstrate that loss of TRAF3 results in profound accumulation of NF-kappaB-inducing kinase in TRAF3-/- cells. Finally, we show that the early postnatal lethality observed in TRAF3-deficient mice is rescued by compound loss of the noncanonical NF-kappaB p100 gene. Thus, these genetic data clearly demonstrate that TRAF3 is a critical negative modulator of the noncanonical NF-kappaB pathway and that constitutive activation of the noncanonical NF-kappaB pathway causes the lethal phenotype of TRAF3-deficient mice.


Assuntos
Subunidade p52 de NF-kappa B/deficiência , Subunidade p52 de NF-kappa B/genética , Fator 3 Associado a Receptor de TNF/deficiência , Fator 3 Associado a Receptor de TNF/genética , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Regulação para Baixo , Genes Letais , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subunidade p52 de NF-kappa B/antagonistas & inibidores , Subunidade p52 de NF-kappa B/fisiologia , Processamento de Proteína Pós-Traducional
14.
J Immunol ; 174(3): 1602-7, 2005 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-15661922

RESUMO

Type I IFNs are well established antiviral cytokines that have also been shown to be induced by bacteria. However, the signaling mechanisms regulating the activation of these cytokines during bacterial infections remain poorly defined. We show that although Gram-negative bacteria can activate the type I IFN pathway through TLR4, the intracellular Gram-positive bacterium Listeria monocytogenes (LM) can do so independently of TLR4 and TLR2. Furthermore, experiments using genetic mutants and chemical inhibitors suggest that LM-induced type I IFN activation occurs by an intracellular pathway involving the serine-threonine kinase TNFR-associated NF-kappaB kinase (TANK)-binding kinase 1 (TBK1). Interestingly, receptor-interacting protein 2, a component of the recently discovered nucleotide-binding oligomerization domain-dependent intracellular detection pathway, was not involved. Taken together, our data describe a novel signal transduction pathway involving TBK1 that is used by LM to activate type I IFNs. Additionally, we provide evidence that both the LM- and TLR-dependent pathways converge at TBK1 to activate type I IFNs, highlighting the central role of this molecule in modulating type I IFNs in host defense and disease.


Assuntos
Interferon Tipo I/biossíntese , Listeria monocytogenes/imunologia , Ativação Linfocitária/imunologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptores de Superfície Celular/fisiologia , Transdução de Sinais/imunologia , Animais , Células da Medula Óssea/enzimologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/microbiologia , Células Cultivadas , Citosol/enzimologia , Citosol/imunologia , Citosol/microbiologia , Interferon Tipo I/deficiência , Interferon Tipo I/genética , Interferon Tipo I/fisiologia , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Transdução de Sinais/genética , Receptor 2 Toll-Like , Receptor 4 Toll-Like
15.
Nat Immunol ; 5(9): 891-8, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15311277

RESUMO

Interferon (IFN) activates the signal transducer and activator of transcription (STAT) pathway to regulate immune responses. The protein inhibitor of activated STAT (PIAS) family has been suggested to negatively regulate STAT signaling. To understand the physiological function of PIAS1, we generated Pias1(-/-) mice. Using PIAS1-deficient cells, we show that PIAS1 selectively regulates a subset of IFN-gamma- or IFN-beta-inducible genes by interfering with the recruitment of STAT1 to the gene promoter. The antiviral activity of IFN-gamma or IFN-beta was consistently enhanced by Pias1 disruption. Pias1(-/-) mice showed increased protection against pathogenic infection. Our data indicate that PIAS1 is a physiologically important negative regulator of STAT1 and suggest that PIAS1 is critical for the IFN-gamma- or IFN-beta-mediated innate immune responses.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata , Interferons/fisiologia , Proteínas/imunologia , Animais , Células Cultivadas , Proteínas de Ligação a DNA/imunologia , Infecções por Herpesviridae/imunologia , Humanos , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas/imunologia , Proteínas Inibidoras de STAT Ativados , Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções por Rhabdoviridae/imunologia , Rhadinovirus/imunologia , Fator de Transcrição STAT1 , Transativadores/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia
16.
Cytokine Growth Factor Rev ; 14(3-4): 193-209, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12787559

RESUMO

Members of the TNF receptor superfamily play pivotal roles in numerous biological events in metazoan organisms. Ligand-mediated trimerization by corresponding homo- or heterotrimeric ligands, the TNF family ligands, causes recruitment of several intracellular adaptors, which activate multiple signal transduction pathways. While recruitment of death domain (DD) containing adaptors such as Fas associated death domain (FADD) and TNFR associated DD (TRADD) can lead to the activation of a signal transduction pathway that induces apoptosis, recruitment of TRAF family proteins can lead to the activation of transcription factors such as, NF-kappaB and JNK thereby promoting cell survival and differentiation as well as immune and inflammatory responses. Individual TNF receptors are expressed in different cell types and have a range of affinities for various intracellular adaptors, which provide tremendous signaling and biological specificities. In addition, numerous signaling modulators are involved in regulating activities of signal transduction pathways downstream of receptors in this superfamily. Most of the TNF receptor superfamily members as well as many of their signaling mediators, have been uncovered in the last two decades. However, much remains unknown about how individual signal transduction pathways are regulated upon activation by any particular TNF receptor, under physiological conditions.


Assuntos
Fator de Necrose Tumoral alfa/fisiologia , Animais , Apoptose , Sobrevivência Celular , Humanos , Inflamação/etiologia , Ligantes , Modelos Biológicos , NF-kappa B/fisiologia , Proteínas/química , Proteínas/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Transdução de Sinais , Fator 1 Associado a Receptor de TNF
17.
Nature ; 416(6877): 190-4, 2002 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-11894097

RESUMO

Host defences to microorganisms rely on a coordinated interplay between the innate and adaptive responses of immunity. Infection with intracellular bacteria triggers an immediate innate response requiring macrophages, neutrophils and natural killer cells, whereas subsequent activation of an adaptive response through development of T-helper subtype 1 cells (TH1) proceeds during persistent infection. To understand the physiological role of receptor-interacting protein 2 (Rip2), also known as RICK and CARDIAK, we generated mice with a targeted disruption of the gene coding for Rip2. Here we show that Rip2-deficient mice exhibit a profoundly decreased ability to defend against infection by the intracellular pathogen Listeria monocytogenes. Rip2-deficient macrophages infected with L. monocytogenes or treated with lipopolysaccharide (LPS) have decreased activation of NF-kappaB, whereas dominant negative Rip2 inhibited NF-kappaB activation mediated by Toll-like receptor 4 and Nod1. In vivo, Rip2-deficient mice were resistant to the lethal effects of LPS-induced endotoxic shock. Furthermore, Rip2 deficiency results in impaired interferon-gamma production in both TH1 and natural killer cells, attributed in part to defective interleukin-12-induced Stat4 activation. Our data reflect requirements for Rip2 in multiple pathways regulating immune and inflammatory responses.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Drosophila , Imunidade Inata/imunologia , Listeriose/imunologia , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/imunologia , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Deleção de Genes , Humanos , Switching de Imunoglobulina , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-12/imunologia , Interleucina-18/imunologia , Células Matadoras Naturais/imunologia , Lipopolissacarídeos/imunologia , Listeria monocytogenes/imunologia , Listeria monocytogenes/fisiologia , Listeriose/metabolismo , Listeriose/patologia , Ativação Linfocitária , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1 , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores , Receptores de Superfície Celular/metabolismo , Choque Séptico/induzido quimicamente , Choque Séptico/imunologia , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo , Receptor 4 Toll-Like , Receptores Toll-Like
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