RESUMO
Dendritic cells (DCs) transfected with mRNA encoding human telomerase reverse transcriptase (hTERT) have been shown to represent potent inducers of CTLs and antitumor immunity. However, it has become widely accepted that not only CTLs but also CD4(+) T helper cells are critical to the generation, as well as to the maintenance, of potent antitumor responses in vivo. In this study, we sought to determine whether human DCs transfected with mRNA encoding a chimeric hTERT/lysosome-associated membrane protein (LAMP-1) protein, carrying the endosomal/lysosomal sorting signal of the LAMP-1, are capable of stimulating concomitant hTERT-specific CD8(+) and CD4(+) T-cell responses in vitro. We show that processing of hTERT/LAMP-1 transcripts leads to enhanced stimulation of hTERT-specific CD4(+) T cells and does not negatively affect intracellular generation and subsequent presentation of MHC class I epitopes, hence, generating a CTL response. These findings provide a preclinical rationale of using DCs transfected with the chimeric hTERT/LAMP-1 RNA in vaccine trials to facilitate generation of antigen-specific CD4(+) T-cell responses that may be required to stimulate and maintain an optimal CD8(+) CTL response in vivo.
Assuntos
Antígenos CD/genética , Antígenos CD/imunologia , Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer/genética , Células Dendríticas/fisiologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Telomerase/genética , Telomerase/imunologia , Vacinas Anticâncer/imunologia , Proteínas de Ligação a DNA , Células Dendríticas/imunologia , Epitopos de Linfócito T/imunologia , Humanos , Ativação Linfocitária , Proteínas de Membrana Lisossomal , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Citotóxicos/imunologia , Telomerase/biossíntese , Transfecção , Células Tumorais CultivadasRESUMO
MicroRNAs (miRNAs) have emerged as biomarkers of metabolic status, etiological factors in complex disease, and promising drug targets. Recent reports suggest that miRNAs are critical regulators of pathways underlying the pathophysiology of type 2 diabetes. In this study, we demonstrate by deep sequencing and real-time quantitative PCR that hepatic levels of Foxa2 mRNA and miR-29 are elevated in a mouse model of diet-induced insulin resistance. We also show that Foxa2 and miR-29 are significantly upregulated in the livers of Zucker diabetic fatty (fa/fa) rats and that the levels of both returned to normal upon treatment with the insulin-sensitizing agent pioglitazone. We present evidence that miR-29 expression in human hepatoma cells is controlled in part by FOXA2, which is known to play a critical role in hepatic energy homeostasis. Moreover, we demonstrate that miR-29 fine-tunes FOXA2-mediated activation of key lipid metabolism genes, including PPARGC1A, HMGCS2, and ABHD5. These results suggest that miR-29 is an important regulatory factor in normal metabolism and may represent a novel therapeutic target in type 2 diabetes and related metabolic syndromes.
Assuntos
Resistência à Insulina/genética , MicroRNAs/fisiologia , Animais , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Feminino , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/fisiologia , Humanos , Camundongos , Modelos Animais , Pioglitazona , Ratos , Ratos Zucker , Tiazolidinedionas/uso terapêutico , Regulação para CimaRESUMO
Prostate-specific membrane antigen (PSMA) is a prototypical differentiation antigen expressed on normal and neoplastic prostate epithelial cells, and on the neovasculature of many solid tumors. Monoclonal antibodies specific for PSMA are in development as therapeutic agents. Methodologies to actively immunize against PSMA may be limited by immunologic ignorance and/or tolerance that restrict the response to self-antigens. Our studies have previously shown that xenogeneic immunization with DNA vaccines encoding melanosomal differentiation antigens induces immunity in a mouse melanoma model. Here we apply this approach to PSMA to establish proof of principle in a mouse model. Immunization with xenogeneic human PSMA protein or DNA induced antibodies to both human and mouse PSMA in mice. Monoclonal antibodies specific for mouse PSMA were generated to analyze antibody isotypes and specificity for native and denatured PSMA at the clonal level. Most antibodies recognized denatured PSMA, but C57BL/6 mice immunized with xenogeneic PSMA DNA followed by a final boost with xenogeneic PSMA protein yielded autoantibodies that reacted with native folded mouse PSMA. Monoclonal antibodies were used to confirm the expression of PSMA protein in normal mouse kidney. These results establish the basis for clinical trials to test PSMA DNA vaccines in patients with solid tumors that either express PSMA directly or that depend on normal endothelial cells expressing PSMA for their continued growth.