title: Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent.
abstract: BACKGROUND
  Deoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown.
  
  
  RESULTS
  In the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG). Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte numbers decreased. The secretion of tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) from peripheral mononuclear blood cells ex vivo increased after treatments with DNA, while IL-10 secretion decreased. Horses treated with DNA had significantly higher myeloid cell numbers and chemokine (C-X-C motif) ligand (CXCL)-10 expression in skin samples at the intradermal injection sites compared to horses treated with transfection reagent only, suggesting an inflammatory response to DNA treatment. In horses treated with expressing DNA, however, local CXCL-10 expression was highest and immunohistochemistry revealed more intradermal IL-12-positive cells when compared to the other treatment groups. In contrast to non-grey horses, grey horses showed fewer effects of DNA treatments on blood lymphocyte counts, TNFα secretion and myeloid cell infiltration in the dermis.
  
  
  CONCLUSIONS
  Treatment with complexed linear DNA constructs induced an inflammatory response independent of the coding sequence and of CG motif content. Expressing IL-12/IL-18 DNA locally induces expression of the downstream mediator CXCL-10. The grey horses included appeared to display an attenuated immune response to DNA treatment, although grey horses bearing melanoma responded to this treatment with moderate tumour remission in a preceding study. Whether the different immunological reactivity compared to other horses may contributes to the melanoma susceptibility of grey horses remains to be elucidated.
authors.affiliation: University of Veterinary Medicine Hannover, Clinic for Horses, Buenteweg 9, 30559, Hannover, Germany. Christiane.Schnabel@tiho-hannover.de.
authors.forename: Christiane L
authors.initials: CL
authors.lastname: Schnabel
authors.affiliation: University of Veterinary Medicine Hannover, Clinic for Horses, Buenteweg 9, 30559, Hannover, Germany. Patrick.Steinig@tiho-hannover.de.
authors.forename: P
authors.initials: P
authors.lastname: Steinig
authors.affiliation: University of Veterinary Medicine Hannover, Immunology Unit, Bischofsholer Damm 15, 30173, Hannover, Germany. Mirja.Koy@tiho-hannover.de.
authors.forename: M
authors.initials: M
authors.lastname: Koy
authors.affiliation: University of Veterinary Medicine Hannover, Immunology Unit, Bischofsholer Damm 15, 30173, Hannover, Germany. Hans-Joachim.Schuberth@tiho-hannover.de.
authors.forename: H-J
authors.initials: HJ
authors.lastname: Schuberth
authors.affiliation: Mologen AG, Fabeckstrasse 30, 14195, Berlin, Germany. Juhls@mologen.com.|Foundation Institute Molecular Biology and Bioinformatics, Freie Universitaet Berlin, Berlin, Germany. Juhls@mologen.com.
authors.forename: C
authors.initials: C
authors.lastname: Juhls
authors.affiliation: Mologen AG, Fabeckstrasse 30, 14195, Berlin, Germany. Oswald@mologen.com.|Foundation Institute Molecular Biology and Bioinformatics, Freie Universitaet Berlin, Berlin, Germany. Oswald@mologen.com.
authors.forename: D
authors.initials: D
authors.lastname: Oswald
authors.affiliation: Foundation Institute Molecular Biology and Bioinformatics, Freie Universitaet Berlin, Berlin, Germany. burghardt.wittig@fu-berlin.de.
authors.forename: B
authors.initials: B
authors.lastname: Wittig
authors.affiliation: University of Veterinary Medicine Hannover, Small Animal Clinic, Buenteweg 9, 30559, Hannover, Germany. Saskia.Willenbrock@tiho-hannover.de.
authors.forename: S
authors.initials: S
authors.lastname: Willenbrock
authors.affiliation: University of Veterinary Medicine Hannover, Small Animal Clinic, Buenteweg 9, 30559, Hannover, Germany. hugo.murua.escobar@med.uni-rostock.de.|Division of Medicine, Clinic III, Haematology, Oncology and Palliative Medicine, University of Rostock, 18057, Rostock, Germany. hugo.murua.escobar@med.uni-rostock.de.
authors.forename: H
authors.initials: H
authors.lastname: Murua Escobar
authors.affiliation: University of Veterinary Medicine Hannover, Institute of Anatomy, Bischofsholer Damm 15, 30173, Hannover, Germany. Christiane.Pfarrer@tiho-hannover.de.
authors.forename: C
authors.initials: C
authors.lastname: Pfarrer
authors.affiliation: Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell Universit, 240 Farrier Rd, Ithaca, NY, 14853, USA. bw73@cornell.edu.
authors.forename: B
authors.initials: B
authors.lastname: Wagner
authors.affiliation: pj statistics, Niedstrasse 16, 12159, Berlin, Germany. peter.jaehnig@pj-statistics.com.
authors.forename: P
authors.initials: P
authors.lastname: Jaehnig
authors.affiliation: Department of Veterinary Medicine, Clinical Sciences, Clinical Pathology and Clinical Pathophysiology, Justus-Liebig-Universitaet, Frankfurter Strasse 126, 35392, Giessen, Germany. Andreas.Moritz@vetmed.uni-giessen.de.
authors.forename: A
authors.initials: A
authors.lastname: Moritz
authors.affiliation: University of Veterinary Medicine Hannover, Clinic for Horses, Buenteweg 9, 30559, Hannover, Germany. Karsten.Feige@tiho-hannover.de.
authors.forename: K
authors.initials: K
authors.lastname: Feige
authors.affiliation: University of Veterinary Medicine Hannover, Clinic for Horses, Buenteweg 9, 30559, Hannover, Germany. Jessika.Cavalleri@tiho-hannover.de.
authors.forename: J-M V
authors.initials: JM
authors.lastname: Cavalleri
mesh_terms: D000818:Animals; D019496:Cancer Vaccines / Q000008:administration & dosage / Q000276:immunology*; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000276:immunology; D006734:Horse Diseases / Q000276:immunology / Q000517:prevention & control*; D006736:Horses; D007271:Injections, Intradermal; D007273:Injections, Intramuscular; D008297:Male; D008545:Melanoma / Q000517:prevention & control / Q000662:veterinary*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000685:Serum Amyloid A Protein / Q000378:metabolism; D019444:Vaccines, DNA / Q000276:immunology
chemical_list: D019496:Cancer Vaccines; D016207:Cytokines; D012333:RNA, Messenger; D000685:Serum Amyloid A Protein; D019444:Vaccines, DNA