RESUMO
Interleukin-10-deficient mice develop colitis and colorectal cancer similar to the inflammatory bowel disease associated cancer in humans. The aim of this study was to identify possible mutations of oncogenes and tumour suppressor genes involved in tumorigenesis in Interleukin-10 (IL-10)-deficient mice. Twenty colon carcinomas from IL-10-deficient mice were screened for mutations in the K-ras and p53 genes by 'cold' single-strand-conformation polymorphism. Immunohistochemical staining was performed to detect mutations in the proteins P53, APC and MSH2, and the transforming growth factor beta type II receptor. Microsatellite instability was analysed at eight chromosomal loci and plasma levels of transforming growth factor beta1 (TGF-beta1) were also measured. At 9 weeks, 14% of the animals developed colorectal cancer, and at 10-31 weeks the incidence of carcinoma was 65%. No mutations were detected in the analysed oncogene and tumour suppressor genes. Plasma TGF-beta1 levels in IL-10-deficient mice 10-31 weeks old were higher than in wild-type littermates e.g. 45.7 +/- 4.6 ng/ml versus 19.8 +/- 4.5 ng/ml (P<0.01). No alterations in K-ras, p53, APC: and Msh2 genes suggests that other genes are involved in the development of these tumours. Elevated TGF-beta1 plasma levels correspond to the high incidence of dysplasia and cancer. Normal expression of the TGF-beta II receptors hints at genetic alterations in other members of the TGF-beta receptor signal transduction pathway.
Assuntos
Proteínas de Ligação a DNA , Doenças Inflamatórias Intestinais/genética , Interleucina-10/genética , Mutação , Neoplasias/genética , Proteína da Polipose Adenomatosa do Colo , Animais , Carcinoma/genética , Neoplasias do Colo/genética , Neoplasias Colorretais/genética , Proteínas do Citoesqueleto/genética , Genes p53/genética , Genes ras/genética , Humanos , Imuno-Histoquímica , Intestino Grosso/patologia , Intestino Delgado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Repetições de Microssatélites , Proteína 2 Homóloga a MutS , Polimorfismo Conformacional de Fita Simples , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Análise de Sequência de DNA , Transdução de Sinais , Fator de Crescimento Transformador beta/sangue , Fator de Crescimento Transformador beta1RESUMO
Inflammatory bowel disease (IBD) comprises the two disorders ulcerative colitis (UC) and Crohn's disease (CD). Although the etiology is still unclear, initiation and aggravation of the inflammatory processes seem to be due to a massive local mucosal immune response. An increased number of greatly activated macrophages seems to contribute to the onset of IBD by expressing upregulated costimulatory molecules (e.g., CD80/CD86) and a cytokine profile favouring a type I proinflammatory response. The release of interleukin 2 (IL-2) and Interferon-gamma (IFN-gamma) by naive T lymphocytes predominantly stimulates cytotoxic T lymphocytes, macrophages, and natural killer (NK) cells and increases the antigen-presenting potential of all these cell types. Opposite this proinflammatory immune reaction a compensatory type II antiinflammatory response has been suggested in the inflamed mucosa, involving mainly interleukin 4 and interleukin 10. Both cytokines are able to down-regulate inflammatory mediators including tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 and favor a humoral immune response. The main goal of this clinical trial is the local liposome-mediated gene transfer of these two antiinflammatory cytokines, interleukin 4 and interleukin 10, in patients with severe IBD of the rectum. This local administration of antiinflammatory cytokines will avoid toxic systemic side effects, prevents blocking of the beneficial effects of proinflammatory cytokines, e.g., TNF-alpha in other tissue compartments and increases the local concentration of interleukin 4 and interleukin 10 over a prolonged period of time. The combined effects of IL-4 and IL-10 have been shown to shift the Th1/Th2 cell activation in favor of a Th2 immune response which seems to be essential for fighting against the inflammation and ultimative healing.
Assuntos
Terapia Genética , Doenças Inflamatórias Intestinais/terapia , Interleucina-10/genética , Interleucina-4/genética , Doenças Retais/terapia , Ensaios Clínicos como Assunto , Técnicas de Transferência de Genes , Terapia Genética/efeitos adversos , Humanos , Doenças Inflamatórias Intestinais/imunologia , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Lipossomos , Seleção de Pacientes , Doenças Retais/imunologia , Células Th1/metabolismo , Células Th2/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Systemic anti-cytokine therapies have been unsuccessful in preventing mortality from gram-negative bacteremia in humans partly because of the failure to neutralize pro-inflammatory cytokines at sites of exaggerated production. In an attempt to deliver anti-inflammatory cytokines to organs directly, gene transfer was employed. Thirty-six BALB/c mice were injected intraperitoneally with cationic liposomes containing plasmids encoding the human interleukin-4 (hIL-4) or IL-13 gene. Both, hIL-4 and hIL-13 mRNA were detected by reverse transcription-polymerase chain reaction analysis in the liver and the spleen of the animals. Fourty-eight hours after the in vivo gene transfer, these 36 mice and 18 mock-transfected mice, were challenged with a lethal dose of E. coli lipopolysaccharide with D-galactosamine (D-GalN). Gene transfer with hIL-4 reduced the serum tumor necrosis factor (TNF)-alpha production in response to endotoxin/D-GalN by 80% from 113.1 pg/ml in mock-transfected animals to 22.2 pg/ml (p < 0.05); human IL-13 gene transfer reduced serum TNF-alpha levels by 90% (113.1 pg/ml to 11.6 pg/ml; p < 0.05). Survival was improved from 20% to over 83% in both treatment groups (p < 0.001). Our data demonstrate a potent in vivo anti-inflammatory action of both IL-4 and IL-13. In addition, the immune functions of peritoneal macrophages are significantly ameliorated in both treatment groups, with IL-13 demonstrating better macrophage immune modulation than IL-4 (p < 0.05).