RESUMO
Rare mutations in IRF6 and GRHL3 cause Van der Woude syndrome, an autosomal dominant orofacial clefting disorder. Common variants in IRF6 and GRHL3 also contribute risk for isolated orofacial clefting. Similarly, variants within genes that encode receptor tyrosine kinase (RTK) signaling components, including members of the FGF pathway, EPHA3 and SPRY2, also contribute risk for isolated orofacial clefting. In the mouse, loss of Irf6 or perturbation of Fgf signaling leads to abnormal oral epithelial adhesions and cleft palate. Oral adhesions can result from a disruption of periderm formation. Here, we find that IRF6 and SPRY4 signaling interact in periderm function. We crossed Irf6 heterozygous ( Irf6+/-) mice with transgenic mice that express Spry4 in the basal epithelial layer ( TgKRT14::Spry4). While embryos with either of these mutations can have abnormal oral adhesions, using a new quantitative assay, we observed a nonadditive effect of abnormal oral epithelial adhesions in the most severely affected double mutant embryos ( Irf6+/-;TgKRT14::Spry4). At the molecular level, the sites of abnormal oral adhesions maintained periderm-like cells that express keratin 6, but we observed abnormal expression of GRHL3. Together, these data suggest that Irf6 and RTK signaling interact in regulating periderm differentiation and function, as well as provide a rationale to screen for epistatic interactions between variants in IRF6 and RTK signaling pathway genes in human orofacial clefting populations.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Fatores Reguladores de Interferon/genética , Proteínas do Tecido Nervoso/genética , Aderências Teciduais/genética , Anormalidades Múltiplas/embriologia , Anormalidades Múltiplas/genética , Animais , Fenda Labial/embriologia , Fissura Palatina/embriologia , Cistos/embriologia , Cistos/genética , Modelos Animais de Doenças , Anormalidades Maxilomandibulares/embriologia , Anormalidades Maxilomandibulares/genética , Lábio/anormalidades , Lábio/embriologia , Camundongos , Camundongos Transgênicos , Anormalidades da Boca/embriologia , Anormalidades da Boca/genética , Mutação , Fenótipo , Transdução de Sinais , Aderências Teciduais/embriologiaRESUMO
The signaling lymphocytic activation molecule-associated adaptor Ewing's sarcoma's-activated transcript 2 (EAT-2) is primarily expressed in dendritic cells, macrophages and natural killer cells. Including EAT-2 in a vaccination regimen enhanced innate and adaptive immune responses toward pathogen-derived antigens, even in the face of pre-existing vaccine immunity. Herein, we investigate whether co-vaccinations with two recombinant Ad5 (rAd5) vectors, one expressing the carcinoembryonic antigen (CEA) and one expressing EAT-2, can induce more potent CEA-specific cytotoxic T lymphocyte (CTL) and antitumor activity in the therapeutic CEA-expressing MC-38 tumor model. Our results suggest that inclusion of EAT-2 significantly alters the kinetics of Th1-biasing proinflammatory cytokine and chemokine responses, and enhances anti-CEA-specific CTL responses. As a result, rAd5-EAT2-augmented rAd5-CEA vaccinations are more efficient in eliminating CEA-expressing target cells as measured by an in vivo CTL assay. Administration of rAd5-EAT2 vaccines also reduced the rate of growth of MC-38 tumor growth in vivo. Also, an increase in MC-38 tumor cell apoptosis (as measured by hematoxylin and eosin staining, active caspase-3 and granzyme B levels within the tumors) was observed. These data provide evidence that more efficient, CEA-specific effector T cells are generated by rAd5 vaccines expressing CEA, when augmented by rAd5 vaccines expressing EAT-2, and this regimen may be a promising approach for cancer immunotherapy in general.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Vacinas Anticâncer/farmacologia , Antígeno Carcinoembrionário/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenoviridae/genética , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Antígeno Carcinoembrionário/biossíntese , Antígeno Carcinoembrionário/genética , Vetores Genéticos/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Linfócitos T Citotóxicos/imunologiaRESUMO
Alternative human and non-human Ad serotype vectors are currently studied for gene therapy and/or vaccine applications to capitalize upon their likely ability to avoid pre-existing immunity to HAd5. However, relatively little attention has been given to the nature and scope of innate immune responses generated by alternative Ad serotypes. In this study, we characterized several innate immune responses after intravenous administration of wild-type Ad serotypes HAd31, HAd3, HAd5, HAd37, SAd23 and HAd41, representing groups A-F, respectively. Notably, biodistribution studies revealed significant differences between the serotypes, with high levels of HAd3 genomes found in the liver and lung, and HAd37 genomes found in the spleen after systemic administration. Relative to similar treatments with other Ad serotypes, HAd3 and SAd23 induced altered innate immune responses, illustrated by induction of higher levels of cellular gene transcription in several tissues, and higher plasma levels of cytokines and chemokines. We also investigated whether complement interactions have a role in HAd3- and SAd23-induced responses. We confirmed complement dependent gene transcription, plasma cytokine/chemokine responses, and liver toxicities incurred after administration of HAd3 and SAd23. This study highlights the potential benefits and/or limitations to the proposed use of alternative Ad serotypes for gene therapy or vaccine applications.