RESUMO
A method for the genetic modification of dendritic cells (DC) was previously established based on the in vitro differentiation of embryonic stem (ES) cells to DC (ES-DC). The unavailability of human ES cells genetically identical to the patients will be a problem in the future clinical application of this technology. This study attempted to establish a strategy to overcome this issue. The TAP1 or beta(2)-microglobulin (beta(2)m) gene was disrupted in 129 (H-2(b))-derived ES cells and then expression vectors for the H-2K(d) or beta(2)m-linked form of K(d) (beta2m-K(d)) were introduced, thus resulting in two types of genetically engineered ES-DC, TAP1(-/-)/K(d) ES-DC and beta(2)m(-/-)/beta(2)m-K(d) ES-DC. As intended, both of the transfectant ES-DC expressed K(d) but not the intrinsic H-2(b) haplotype-derived MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) and TAP1(-/-)/K(d) ES-DC were not recognized by pre-activated H-2(b)-reactive CTL and did not prime H-2(b) reactive CTL in vitro or in vivo. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC and TAP1(-/-)/K(d) ES-DC had a survival advantage in comparison to beta(2)m(+/-)/beta(2)m-K(d) ES-DC and TAP1(+/+)/K(d) ES-DC, when transferred into BALB/c mice. K(d)-restricted RSV-M2-derived peptide-loaded ES-DC could prime the epitope-specific CTL upon injection into the BALB/c mice, irrespective of the cell surface expression of intrinsic H-2(b) haplotype-encoded MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC were significantly more efficient in eliciting immunity against RSV M2 protein-expressing tumor cells than beta(2)m(+/-)/beta(2)m-K(d) ES-DC. The modification of the beta(2)m or TAP gene may therefore be an effective strategy to resolve the problem of HLA class I allele mismatch between human ES or induced pluripotent stem cells and the recipients to be treated.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Células Dendríticas/imunologia , Células-Tronco Embrionárias/imunologia , Epitopos de Linfócito T/imunologia , Antígenos H-2/genética , Ativação Linfocitária/imunologia , Linfócitos T Citotóxicos/imunologia , Microglobulina beta-2/genética , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adenocarcinoma/prevenção & controle , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/prevenção & controle , Citotoxicidade Imunológica/genética , Células Dendríticas/metabolismo , Células Dendríticas/transplante , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/transplante , Feminino , Antígenos H-2/biossíntese , Antígenos H-2/metabolismo , Haplótipos/imunologia , Teste de Histocompatibilidade , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Ligação Proteica/genética , Ligação Proteica/imunologia , Linfócitos T Citotóxicos/metabolismo , Microglobulina beta-2/deficiênciaRESUMO
Rheumatoid arthritis (RA), which afflicts about 1% of the world population, is a chronic systemic inflammatory disease of unknown aetiology that primarily affects the synovial membranes of multiple joints. Although CD4(+) T cells seem to be the prime mediators of RA, it remains unclear how arthritogenic CD4(+) T cells are generated and activated. Given that highly self-reactive T-cell clones are deleted during normal T-cell development in the thymus, abnormality in T-cell selection has been suspected as one cause of autoimmune disease. Here we show that a spontaneous point mutation of the gene encoding an SH2 domain of ZAP-70, a key signal transduction molecule in T cells, causes chronic autoimmune arthritis in mice that resembles human RA in many aspects. Altered signal transduction from T-cell antigen receptor through the aberrant ZAP-70 changes the thresholds of T cells to thymic selection, leading to the positive selection of otherwise negatively selected autoimmune T cells. Thymic production of arthritogenic T cells due to a genetically determined selection shift of the T-cell repertoire towards high self-reactivity might also be crucial to the development of disease in a subset of patients with RA.
Assuntos
Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Modelos Animais de Doenças , Mutação de Sentido Incorreto/genética , Proteínas Tirosina Quinases/genética , Linfócitos T/imunologia , Timo/patologia , Sequência de Aminoácidos , Animais , Apoptose , Artrite Reumatoide/enzimologia , Artrite Reumatoide/patologia , Sequência de Bases , Doença Crônica , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Tirosina Quinases/química , Transdução de Sinais , Linfócitos T/patologia , Timo/imunologia , Proteína-Tirosina Quinase ZAP-70 , Domínios de Homologia de srcAssuntos
Adenocarcinoma/secundário , Neoplasias do Ânus/patologia , Doença de Paget Extramamária/patologia , Pele/patologia , Adenocarcinoma/química , Idoso de 80 Anos ou mais , Neoplasias do Ânus/química , Biomarcadores Tumorais/análise , Biópsia , Humanos , Imuno-Histoquímica , Masculino , Invasividade Neoplásica , Doença de Paget Extramamária/química , Pele/químicaRESUMO
Atopic dermatitis (AD) is one of the most common inflammatory diseases of the skin and is usually associated with a family history of atopic diathesis. It has been well established that many environmental or psychological factors aggravate AD. However, it is not clear whether psychological stress by itself can trigger AD. We examined the effect of psychological stress on the onset of AD, using an animal model, the NC/Nga mouse. The animals were exposed to the water avoidance stress (WAS) test to induce psychological stress. Additionally, we examined how corticotropin-releasing factor (CRF) affected the development of AD induced by psychological stress. Under specific pathogen-free (SPF) conditions, NC/Nga mice did not develop AD-like skin lesions. In contrast, NC/Nga mice exposed to psychological stress developed AD-like skin lesions along with elevated levels of serum immunoglobulin E even when kept under SPF conditions. The AD-like skin lesions induced by WAS were completely blocked by pretreating the animals with CRF. Our data indicate that a psychological factor is capable of eliciting AD-like skin lesions in NC/Nga mice. It is possible that the inhibitory effect of CRF may be mediated by the functional modification of various cells that have CRF receptors.
Assuntos
Hormônio Liberador da Corticotropina/farmacologia , Dermatite Atópica/etiologia , Estresse Psicológico/complicações , Animais , Anticorpos Bloqueadores/farmacologia , Corticosterona/sangue , Citocinas/biossíntese , Citocinas/sangue , Defecação/efeitos dos fármacos , Dermatite Atópica/patologia , Dermatite Atópica/fisiopatologia , Imunoglobulina E/biossíntese , Imunoglobulina E/genética , Imuno-Histoquímica , Linfonodos/metabolismo , Masculino , Camundongos , Prurido/etiologia , Prurido/psicologia , Estresse Psicológico/fisiopatologia , Substância P/antagonistas & inibidores , ÁguaRESUMO
We describe a case of a 7-year-old boy diagnosed as xeroderma pigmentosum complementation group A (XPA). Severe photosensitivity developed at 5 months after birth, and at a visit to our hospital at the age of five years, multiple brownish freckles were present on the face. XPA complementing (XPAC) gene compensated the ability of DNA repair after UV-irradiation of the fibroblasts. PCR-RFLP and DNA sequencing analyses revealed compound heterozygosity for a splicing mutation (IV3 -1G => C) at the splicing acceptor site of intron 3 and a nonsense mutation (Arg228 => stop) in exon 6. The former mutation can be detected by a restriction enzyme Alw NI and the latter detected by Hph I. Neither obvious neurological symptoms nor malignant skin tumors were noted. This genotype is associated with milder clinical symptoms than homozygosity for the IV3 -1G => C mutation.
Assuntos
Predisposição Genética para Doença , Homozigoto , Mutação Puntual , Raios Ultravioleta/efeitos adversos , Xeroderma Pigmentoso/diagnóstico , Xeroderma Pigmentoso/genética , Sequência de Bases , Criança , Análise Mutacional de DNA , Fibroblastos/efeitos da radiação , Teste de Complementação Genética , Humanos , Masculino , Dados de Sequência Molecular , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , PrognósticoRESUMO
We report two cases of Werner's syndrome (WS). First, a 42-year-old Japanese man was referred on suspicion of systemic sclerosis (SSc) because of scleroderma-like skin atrophy and foot ulcers. Second, a 51-year-old woman with malignant fibrous histiocytoma was referred on suspicion of premature aging syndrome. Because both patients had many typical manifestations compatible with WS, we made a clinical diagnosis of WS. Genetic analyses revealed a homozygous mutation, an A deletion at nucleotide 3677 of WS gene (WRN) in the first case and a homozygous mutation, a G to C substitution at one base upstream of exon 26 of WRN in the second case. Both mutations were consistent with those previously reported in Japanese WS patients.
Assuntos
DNA Helicases/genética , Síndrome de Werner/diagnóstico , Síndrome de Werner/genética , Adulto , Primers do DNA , Diagnóstico Diferencial , Exodesoxirribonucleases , Feminino , Úlcera do Pé , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Reação em Cadeia da Polimerase , RecQ Helicases , Úlcera Cutânea , Helicase da Síndrome de WernerRESUMO
Adult murine epidermis contains members of the epithelial gammadelta T-cell family called dendritic epidermal T cells (DETCs). Their development and maturation have been the subjects of investigations, but the details are still unclear. T-cell receptor (TCR) zeta-chain-associated protein-70 (ZAP-70), one of the protein tyrosine kinases required for TCR signaling, plays a pivotal role in the development of alphabeta T cells. In mice lacking ZAP-70, thymic development of alphabeta T cells was completely arrested at the immature CD4(+)CD8(+) TCR(low) stage. Here, we examined whether or not the development and maturation of DETCs were altered in ZAP-70-deficient mice. Immunohistochemical analyses of epidermal sheets revealed that the number of DETCs was reduced and their characteristic dendrites were lost or markedly shortened in ZAP-70(-/-) mice. In flow cytometric analyses, the expression levels of gammadelta TCR and Thy-1.2 on the ZAP-70(-/-) DETCs were lower than those on ZAP-70(+/-) DETCs. The expression of a very early activation antigen, CD69, was not detected on ZAP-70(-/-) DETCs, whereas CD69 was expressed on ZAP-70(+/-) DETCs. Furthermore, ZAP-70(-/-) DETCs showed markedly reduced proliferation and no IL-2 gene expression in response to anti-CD3epsilon or concanavalin A stimulation. These results suggest that ZAP-70 is essential for TCR signaling which induces the proliferation and the final maturation of DETCs in the epidermis.