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1.
J Invest Dermatol ; 133(2): 489-98, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22992806

RESUMO

Toxic epidermal necrolysis (TEN) is a severe immune-mediated adverse cutaneous drug eruption characterized by rapid and extensive epithelial cell death in the epidermis and mucosae. The molecular events leading to this often fatal condition are only partially understood, but evidence suggests a dual mechanism implicating a "drug"-specific immune response on one side and the onset of target cell death by proapoptotic molecules including FasL on the other side. Herein, we describe a potential molecular bridge between these two events that involves inducible nitric oxide synthase (iNOS), which is highly upregulated in the skin of TEN patients. We show that activated T cells secrete high amounts of tumor necrosis factor-α (TNF-α) and IFN-γ, and that both cytokines lead to increased expression and activity of keratinocyte iNOS. A similar observation has been made with drug-specific T lymphocytes from a TEN patient exposed to the culprit drug. The resulting increase in nitric oxide significantly upregulates keratinocyte FasL expression, resulting in Fas- and caspase-8-mediated keratinocyte cell death. Taken together, our data suggest that T-lymphocyte activation by drugs in TEN patients may indirectly lead to FasL-mediated keratinocyte apoptosis, via a molecular bridge involving TNF-α, IFN-γ, and iNOS.


Assuntos
Apoptose/imunologia , Interferon gama/imunologia , Óxido Nítrico Sintase Tipo II/imunologia , Síndrome de Stevens-Johnson/imunologia , Fator de Necrose Tumoral alfa/imunologia , Receptor fas/imunologia , Caspase 8/imunologia , Caspase 8/metabolismo , Linhagem Celular , Sinergismo Farmacológico , Proteína Ligante Fas/genética , Proteína Ligante Fas/imunologia , Proteína Ligante Fas/metabolismo , Prepúcio do Pênis/citologia , Humanos , Interferon gama/metabolismo , Interferon gama/farmacologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Masculino , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/imunologia , Cultura Primária de Células , RNA Mensageiro/metabolismo , Síndrome de Stevens-Johnson/metabolismo , Síndrome de Stevens-Johnson/patologia , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Receptor fas/metabolismo
2.
J Immunol ; 180(9): 5826-32, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18424701

RESUMO

Efficient priming of adaptive immunity depends on danger signals provided by innate immune pathways. As an example, inflammasome-mediated activation of caspase-1 and IL-1beta is crucial for the development of reactive T cells targeting sensitizers like dinitrofluorobenzene (DNFB). Surprisingly, DNFB and dinitrothiocyanobenzene provide cross-reactive Ags yet drive opposing, sensitizing vs tolerizing, T cell responses. In this study, we show that, in mice, inflammasome-signaling levels can be modulated to turn dinitrothiocyanobenzene into a sensitizer and DNFB into a tolerizer, and that it correlates with the IL-6 and IL-12 secretion levels, affecting Th1, Th17, and regulatory T cell development. Hence, our data provide the first evidence that the inflammasome can define the type of adaptive immune response elicited by an Ag, and hint at new strategies to modulate T cell responses in vivo.


Assuntos
Dinitrobenzenos/farmacologia , Dinitrofluorbenzeno/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Animais , Antígenos/imunologia , Caspase 1/imunologia , Reações Cruzadas/efeitos dos fármacos , Reações Cruzadas/imunologia , Humanos , Tolerância Imunológica/imunologia , Imunidade Inata/imunologia , Inflamação/imunologia , Interleucina-1beta/imunologia , Interleucina-2/imunologia , Interleucina-6/imunologia , Camundongos , Transdução de Sinais/imunologia
3.
Blood ; 111(9): 4780-7, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18314443

RESUMO

Because of the low proliferative potential of tumor cells in patients with Sézary syndrome (SzS), their accumulation has been suggested to be due to defective regulation of apoptosis. We analyzed the sensitivity to soluble Fas-ligand (FasL) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), 2 members of the TNF superfamily in peripheral blood leukocytes (PBL) from patients with SzS. Compared with healthy donors, CD4(+) cells from patients with SzS were completely resistant to FasL in 9 of 16 cases. Of these 9 FasL-resistant cases, 4 revealed a loss in Fas (CD95) expression, whereas the remaining 5 exhibited normal or enhanced Fas expression. In the latter 5 cases, the apoptosis inhibitor cFLIP was overexpressed in CD4(+)/CD26(-) tumor cells compared with CD4(+)/CD26(-) cells from Fas-expressing FasL-sensitive patients and healthy donors. Furthermore, resistance to TRAIL and tumor cell-restricted loss of TRAIL-receptor 2 were observed in 16 of 16 SzS PBLs. It is noteworthy that resistance to FasL could be overcome by the use of a hexameric FasL or upon exposure of SzS cells to interferon-alpha (IFN-alpha) or IFN-gamma, the latter by an increase of Fas expression. Our data on primary SzS lymphocytes reveal frequent resistance to apoptosis induced by FasL and TRAIL, which may contribute to their accumulation in patients with SzS and be relevant at a therapeutic level.


Assuntos
Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Ligante Fas/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/análise , Síndrome de Sézary/patologia , Neoplasias Cutâneas/patologia , Linfócitos T/patologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/análise , Estudos de Casos e Controles , Regulação da Expressão Gênica , Humanos , Receptores de Morte Celular , Síndrome de Sézary/imunologia , Neoplasias Cutâneas/imunologia
4.
J Gene Med ; 7(9): 1178-86, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15880606

RESUMO

BACKGROUND: Genetically modified keratinocytes generate transplantable self-renewing epithelia suitable for delivery of therapeutic polypeptides. However, the variety of viral vectors and experimental conditions currently used make fragmented or contradictory the information on the transduction efficiency of the human primary keratinocytes. To compare the suitability of the most currently used viral vectors for efficient gene transfer to human keratinocytes, we have performed a comparative study using a panel of recombinant constructs. METHODS: For each vector, the transduction efficiency and the persistence of the transgene expression were quantified by fluorescence microscopy and flow cytometry analysis of the infected cells. RESULTS: We show that: (1) canine and human adenoviral vectors achieve a highly efficient but transient transduction of both primary and immortalized keratinocytes; (2) the adenovirus-associated virus (AAV) vectors transduce immortalized keratinocytes, albeit with a short-lived gene expression (<4 days), but fail to infect primary keratinocytes; and (3) under appropriate conditions, the oncoretroviral and lentiviral vectors can permanently transduce up to 100% of primary keratinocytes, but the highly clonogenic keratinocytes are more efficiently targeted by lentiviral vectors. CONCLUSIONS: Therefore, AAV vectors are unsuitable to transduce primary keratinocytes, while human and canine adenoviral vectors appears to be appropriate to achieve short-term delivery of therapeutic products. Recombinant retroviruses provide sustained expression of the transgene, but the lentiviral vectors are the most suitable for ex vivo gene therapy because of their ability to transduce clonogenic primary keratinocytes.


Assuntos
Adenovirus Caninos/genética , Adenovírus Humanos/genética , Terapia Genética , Vetores Genéticos/uso terapêutico , Queratinócitos/transplante , Transdução Genética , Animais , Células Cultivadas , Cães , Humanos , Lentivirus/genética , Vírus da Leucemia Murina , Camundongos , Plasmídeos , Recombinação Genética
5.
Exp Cell Res ; 293(2): 219-28, 2004 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-14729459

RESUMO

Adipocyte differentiation involves dramatic cell shape alterations that are accompanied by changes in the expression of cytoskeletal and extracellular matrix (ECM) proteins. Aortic carboxypeptidase-like protein (ACLP) is a secreted protein associated with the extracellular matrix whose expression is induced during smooth muscle (SM) differentiation. We analyzed the expression of ACLP gene during adipocyte differentiation of 3T3-F442A, 3T3-L1, and Ob1771 preadipocytes. Our results show that ACLP mRNA and protein are expressed in growing cells and after commitment. Thereafter, their expression levels decrease, as opposed to that of aP2 and PPARgamma2. Consistent with these observations, ACLP mRNA is expressed in the stromal-vascular fraction of adipose tissue but not in the adipocyte fraction. Overexpression of ACLP in 3T3-F442A preadipocytes inhibits adipocyte differentiation at both morphological and molecular level. However, ACLP overexpression promotes transdifferentiation of preadipocytes into smooth muscle-like cells, which express specific markers such as SM22alpha, SM alpha-actin, SM-MHC, and caldesmon. These findings demonstrate that overexpression of a single extracellular matrix protein is sufficient to induce transdifferentiation and that ACLP may modulate the commitment of mesodermal cells into different lineages depending upon its pattern of expression.


Assuntos
Adipócitos/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Células-Tronco Mesenquimais/metabolismo , Músculo Liso/metabolismo , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Proteínas/metabolismo , Actinas/metabolismo , Adipócitos/citologia , Animais , Biomarcadores , Proteínas de Ligação a Calmodulina/metabolismo , Carboxipeptidases , Proteínas de Transporte/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Mesenquimais/citologia , Camundongos , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Músculo Liso/citologia , Cadeias Pesadas de Miosina/metabolismo , Células NIH 3T3 , Proteínas/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Regulação para Cima/genética
6.
J Invest Dermatol ; 121(6): 1336-43, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14675179

RESUMO

Genetic mutations in alpha6beta4 integrin cause junctional epidermolysis bullosa with pyloric atresia, a genodermatosis characterized by blistering of the skin and pyloric occlusion. The lethal form of junctional epidermolysis bullosa with pyloric atresia has been mainly associated with the presence of premature termination codons in the mRNA encoding either the alpha6 or beta4 subunit causing rapid decay of the mutated transcript and absence of alpha6beta4 integrin. In this study, we disclose the genetic background of lethal junctional epidermolysis bullosa with pyloric atresia in a patient presenting absent expression of alpha6 integrin despite normal steady-state level of the alpha6beta4 mRNA. Screening for mutation in the alpha6 gene detected a homozygous base pair substitution (286 C-to-T), which results in the substitution of a serine with a leucine residue (S47L). The amino acid substitution S47L localizes in the first beta-strand of the seven-bladed beta-propeller structure of the extracellular head of alpha6 integrin, and triggers a rapid proteolysis of the aberrant polypeptides involving the lysosomal degradation pathway. This study provides new insight into the pathogenic effect of a mis-sense mutation affecting a functional domain of a protein, and identifies a critical peptide sequence of the beta-propeller domain conserved among the alpha integrin cell receptors.


Assuntos
Epidermólise Bolhosa/genética , Epidermólise Bolhosa/metabolismo , Integrina alfa6/genética , Integrina alfa6/metabolismo , Mutação de Sentido Incorreto , Piloro/anormalidades , Sequência de Aminoácidos , Células Cultivadas , DNA Complementar , Células Epidérmicas , Epidermólise Bolhosa/patologia , Evolução Fatal , Feminino , Expressão Gênica , Humanos , Recém-Nascido , Integrina alfa6/química , Integrina beta4/genética , Queratinócitos/patologia , Queratinócitos/fisiologia , Ligantes , Masculino , Dados de Sequência Molecular , Linhagem , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Índice de Gravidade de Doença
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