title: Endocytosis of DNA-Hsp65 alters the pH of the late endosome/lysosome and interferes with antigen presentation.
abstract: BACKGROUND
  Experimental models using DNA vaccine has shown that this vaccine is efficient in generating humoral and cellular immune responses to a wide variety of DNA-derived antigens. Despite the progress in DNA vaccine development, the intracellular transport and fate of naked plasmid DNA in eukaryotic cells is poorly understood, and need to be clarified in order to facilitate the development of novel vectors and vaccine strategies.
  
  
  RESULTS
  Using confocal microscopy, we have demonstrated for the first time that after plasmid DNA uptake an inhibition of the acidification of the lysosomal compartment occurs. This lack of acidification impaired antigen presentation to CD4 T cells, but did not alter the recruitment of MyD88. The recruitment of Rab 5 and Lamp I were also altered since we were not able to co-localize plasmid DNA with Rab 5 and Lamp I in early endosomes and late endosomes/lysosomes, respectively. Furthermore, we observed that the DNA capture process in macrophages was by clathrin-mediated endocytosis. In addition, we observed that plasmid DNA remains in vesicles until it is in a juxtanuclear location, suggesting that the plasmid does not escape into the cytoplasmic compartment.
  
  
  CONCLUSIONS
  Taken together our data suggests a novel mechanism involved in the intracellular trafficking of plasmid DNA, and opens new possibilities for the use of lower doses of plasmid DNA to regulate the immune response.
authors: Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, São Paulo, Brazil.
authors: Ana Paula F
authors: AP
authors: Trombone
authors: Célio L
authors: CL
authors: Silva
authors: Karla M
authors: KM
authors: Lima
authors: Constance
authors: C
authors: Oliver
authors: Maria Célia
authors: MC
authors: Jamur
authors: Alan R
authors: AR
authors: Prescott
authors: Arlete A M
authors: AA
authors: Coelho-Castelo
mesh_terms: D000818:Animals; D017951:Antigen Presentation / Q000276:immunology / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics; D001692:Biological Transport; D002460:Cell Line; D018834:Chaperonin 60; D018833:Chaperonins / Q000235:genetics; D002966:Clathrin / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism*; D006433:Hemocyanins / Q000276:immunology / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D010957:Plasmids / Q000235:genetics; D051217:Toll-Like Receptor 9 / Q000235:genetics / Q000502:physiology; D014168:Transferrin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; D002966:Clathrin; D018076:DNA, Complementary; C495370:Tlr9 protein, mouse; D051217:Toll-Like Receptor 9; D014168:Transferrin; C074119:heat-shock protein 65, Mycobacterium; D006433:Hemocyanins; D018833:Chaperonins; C032808:keyhole-limpet hemocyanin