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1.
Mol Cell Biol ; 26(6): 2399-407, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16508014

RESUMO

The mitogen-activated protein kinase (MAPK) p38/MAPK-activated protein kinase 2 (MK2) signaling pathway plays an important role in the posttranscriptional regulation of tumor necrosis factor (TNF), which is dependent on the adenine/uridine-rich element (ARE) in the 3' untranslated region of TNF mRNA. After lipopolysaccharide (LPS) stimulation, MK2-deficient macrophages show a 90% reduction in TNF production compared to the wild type. Tristetraprolin (TTP), a protein induced by LPS, binds ARE and destabilizes TNF mRNA. Accordingly, macrophages lacking TTP produce large amounts of TNF. Here, we generated MK2/TTP double knockout mice and show that, after LPS stimulation, bone marrow-derived macrophages produce TNF mRNA and protein levels comparable to those of TTP knockout cells, indicating that in the regulation of TNF biosynthesis TTP is genetically downstream of MK2. In addition, we show that MK2 is essential for the stabilization of TTP mRNA, and phosphorylation by MK2 leads to increased TTP protein stability but reduced ARE affinity. These data suggest that MK2 inhibits the mRNA destabilizing activity of TTP and, in parallel, codegradation of TTP together, with the target mRNA resulting in increased cellular levels of TTP.


Assuntos
Proteínas Quinases/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Tristetraprolina/metabolismo , Fator de Necrose Tumoral alfa/genética , Regiões 3' não Traduzidas , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Adenina/metabolismo , Animais , Células Cultivadas , Regulação para Baixo , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Mutantes , Fosforilação , Biossíntese de Proteínas , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Tristetraprolina/genética , Fator de Necrose Tumoral alfa/metabolismo , Uridina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
2.
Respir Res ; 8: 47, 2007 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-17610738

RESUMO

BACKGROUND: Although the contribution of alveolar type II epithelial cell (AEC II) activities in various aspects of respiratory immune regulation has become increasingly appreciated, our understanding of the contribution of AEC II transcriptosome in immunopathologic lung injury remains poorly understood. We have previously established a mouse model for chronic T cell-mediated pulmonary inflammation in which influenza hemagglutinin (HA) is expressed as a transgene in AEC II, in mice expressing a transgenic T cell receptor specific for a class II-restricted epitope of HA. Pulmonary inflammation in these mice occurs as a result of CD4+ T cell recognition of alveolar antigen. This model was utilized to assess the profile of inflammatory mediators expressed by alveolar epithelial target cells triggered by antigen-specific recognition in CD4+ T cell-mediated lung inflammation. METHODS: We established a method that allows the flow cytometric negative selection and isolation of primary AEC II of high viability and purity. Genome wide transcriptional profiling was performed on mRNA isolated from AEC II isolated from healthy mice and from mice with acute and chronic CD4+ T cell-mediated pulmonary inflammation. RESULTS: T cell-mediated inflammation was associated with expression of a broad array of cytokine and chemokine genes by AEC II cell, indicating a potential contribution of epithelial-derived chemoattractants to the inflammatory cell parenchymal infiltration. Morphologically, there was an increase in the size of activated epithelial cells, and on the molecular level, comparative transcriptome analyses of AEC II from inflamed versus normal lungs provide a detailed characterization of the specific inflammatory genes expressed in AEC II induced in the context of CD4+ T cell-mediated pneumonitis. CONCLUSION: An important contribution of AEC II gene expression to the orchestration and regulation of interstitial pneumonitis is suggested by the panoply of inflammatory genes expressed by this cell population, and this may provide insight into the molecular pathogenesis of pulmonary inflammatory states. CD4+ T cell recognition of antigen presented by AEC II cells appears to be a potent trigger for activation of the alveolar cell inflammatory transcriptosome.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Imunofenotipagem , Pulmão/patologia , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/imunologia , Mucosa Respiratória/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Regulação da Expressão Gênica/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Alvéolos Pulmonares/metabolismo , Mucosa Respiratória/metabolismo
3.
FEMS Immunol Med Microbiol ; 43(3): 373-84, 2005 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-15708311

RESUMO

The development of novel approaches that allow accurate targeting of therapeutics to the intestinal mucosa is a major task in the research on intestinal inflammation. For the first time, a live genetically modified bacterial strain has been approved by Dutch authorities as a therapeutic agent for experimental therapy of intestinal bowel disease (IBD) in humans. Genetically modified probiotics can very well be used as carriers for localized antigen delivery into the intestine. Therapeutic safety, however, of such a carrier organism, is crucial, especially when a specific probiotic strain has to be used under diseased conditions. In this study, we tested the potential of Escherichia coli NISSLE 1917 to serve as a safe carrier for targeted delivery of recombinant proteins to the intestinal mucosa. In a well-defined and very sensitive immunological system, we demonstrate that intestinal recombinant E. coli NISSLE 1917 has no effect on migration, clonal expansion and activation status of specific CD4+ T cells, neither in healthy mice nor in animals with acute colitis. Furthermore, recombinant E. coli NISSLE 1917 has no effect on the induction or breakdown of peripheral T-cell tolerance in an autoimmune environment. The excellent colonization properties of E. coli NISSLE 1917 render this strain an ideal candidate as carrier organism for gut-focused in situ synthesis of therapeutic molecules.


Assuntos
Escherichia coli/genética , Escherichia coli/imunologia , Imunidade nas Mucosas , Mucosa Intestinal/imunologia , Probióticos , Animais , Autoimunidade , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Colite/imunologia , Colite/patologia , Colite/terapia , DNA Bacteriano/genética , Portadores de Fármacos , Hemaglutininas/administração & dosagem , Hemaglutininas/genética , Hemaglutininas/imunologia , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Segurança , Tolerância a Antígenos Próprios
4.
J Immunol ; 179(12): 8059-68, 2007 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-18056346

RESUMO

The concept of immune regulation/suppression has been well-established and, besides thymus-derived CD4+CD25+ regulatory T (TR) cells, it became clear that a variety of additional peripherally induced TR cells play vital roles in protection from many harmful immune responses including intestinal inflammation. In the present study, we have analyzed in vivo-induced Ag-specific CD4+ TR cells with respect to their molecular and functional phenotype. By comparative genomics we could show that these Ag-specific TR cells induced by chronic Ag stimulation in vivo clearly differ in their genetic program from naturally occurring thymus-derived CD4+CD25+ TR cells. This distinct population of induced TR cells express neither CD25 nor the TR-associated transcription factor Foxp3. Strikingly, CD25 is not even up-regulated upon stimulation. Despite the lack in Foxp3 expression, these in vivo-induced CD25- TR cells are able to interfere with an Ag-specific CD8+ T cell-mediated intestinal inflammation without significant increase in CD25 and Foxp3 expression. Thus, our results demonstrate that in vivo-induced Ag-specific TR cells represent a distinct population of Foxp3-CD25- TR cells with regulatory capacity both in vitro and in vivo.


Assuntos
Antígenos Virais/imunologia , Fatores de Transcrição Forkhead/análise , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Subunidade alfa de Receptor de Interleucina-2/análise , Ativação Linfocitária/genética , Linfócitos T Reguladores/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Fatores de Transcrição Forkhead/genética , Perfilação da Expressão Gênica , Genômica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Subunidade alfa de Receptor de Interleucina-2/genética , Camundongos , Camundongos Transgênicos
5.
Cell ; 129(5): 891-902, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17540170

RESUMO

In causing disease, pathogens outmaneuver host defenses through a dedicated arsenal of virulence determinants that specifically bind or modify individual host molecules. This dedication limits the intruder to a defined range of hosts. Newly emerging diseases mostly involve existing pathogens whose arsenal has been altered to allow them to infect previously inaccessible hosts. We have emulated this chance occurrence by extending the host range accessible to the human pathogen Listeria monocytogenes by the intestinal route to include the mouse. Analyzing the recognition complex of the listerial invasion protein InlA and its human receptor E-cadherin, we postulated and verified amino acid substitutions in InlA to increase its affinity for E-cadherin. Two single substitutions increase binding affinity by four orders of magnitude and extend binding specificity to include formerly incompatible murine E-cadherin. By rationally adapting a single protein, we thus create a versatile murine model of human listeriosis.


Assuntos
Proteínas de Bactérias/genética , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Engenharia de Proteínas , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Células CACO-2 , Caderinas/metabolismo , Feminino , Humanos , Listeriose/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Placenta , Gravidez , Ligação Proteica , Organismos Livres de Patógenos Específicos , Fatores de Virulência
6.
Gastroenterology ; 131(2): 510-24, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16890605

RESUMO

BACKGROUND & AIMS: Few data exist regarding mechanisms of mucosal CD8+ T-cell reactivity to epithelial-specific antigen. To dissect the immunologic mechanisms underlying CD8+ T-cell dysregulation, reactivity to a self-antigen expressed in intestinal epithelium of mice bearing a major histocompatibility complex class I-restricted T-cell receptor specific for this antigen was studied. In addition, antigen-specific regulatory CD4+ T cells induced in vivo were tested to control these autoreactive CD8+ T cells. METHODS: Transgenic VILLIN-HA mice were mated with CL4-TCR transgenic mice. Alternatively, adoptive transfer of CL4-TCR transgenic CD8+ T cells into VILLIN-HA transgenic mice was performed to mimic spontaneous encounter of neoantigen. Mucosal CD8+ T cells were characterized under different conditions of tolerance, immunopathology, and active immunosuppression. RESULTS: Transgenic CD8+ T cells from VILLIN-HA x CL4-TCR transgenic mice preferentially migrated and expanded in mucosal lymphoid tissues. Although transgenic CD8+ T cells showed signs of T-cell activation, they failed to cause tissue damage. This was accompanied by the induction/expansion of CD4+ and CD8+, Foxp3-expressing T cells. In contrast, adoptive transfer of naive transgenic CD8+ T cells from CL4-TCR transgenic mice into VILLIN-HA transgenic mice induced severe intestinal inflammation with poor clinical course of disease. Transgenic CD8+ T cells secreted vigorous amounts of proinflammatory cytokines like interferon gamma/tumor necrosis factor alpha. Strikingly, this acute wasting disease was significantly ameliorated by cotransfer of antigen-specific regulatory CD4+ T cells. CONCLUSIONS: Epithelial-specific antigen expression is sufficient to trigger severe antigen-specific CD8+ T-cell-mediated intestinal inflammation; this might be controlled by antigen-specific regulatory T cells under physiological conditions.


Assuntos
Autoimunidade , Linfócitos T CD8-Positivos/imunologia , Enterite/imunologia , Mucosa Intestinal/patologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Movimento Celular , Progressão da Doença , Enterite/patologia , Citometria de Fluxo , Mucosa Intestinal/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Fenótipo
7.
Xenotransplantation ; 13(4): 345-56, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16768728

RESUMO

BACKGROUND: Microinjection of foreign DNA into pronuclei of zygotes has been the method of choice for the production of transgenic domestic animals. Following microinjection the transgene is randomly integrated into the host genome which can be associated with insertional mutagenesis and unwanted pathological side effects. METHODS: Here, we evaluated the health status of pigs transgenic for the human regulator of complement activation (RCA) CD59 and conducted a complete pathomorphological examination on 19 RCA transgenic pigs at 1 to 32 months of age from nine transgenic lines. Nine wild-type animals served as controls. Expression levels of human complement regulator CD59 (hCD59) mRNA were measured by RT-PCR and distribution of hCD59 protein was determined by immunohistochemistry. RESULTS: Albeit variable transgene expression levels, no specific pathomorphologic phenotype associated with the presence of the transgene in all analyzed pig lines could be detected. CONCLUSIONS: Transgenic expression of this human RCA gene construct is not correlated with a specific pathological phenotype in pigs. This is crucial for the application of the technology and the use of transgenic pigs for biomedical and agricultural applications.


Assuntos
Animais Geneticamente Modificados/genética , Antígenos CD59/genética , Sus scrofa/genética , Animais , Animais Geneticamente Modificados/metabolismo , Feminino , Humanos , Masculino , Especificidade de Órgãos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sus scrofa/metabolismo , Análise Serial de Tecidos , Transgenes
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