title: New strategy for testing efficacy of immunotherapeutic compounds for diabetes in vitro.
abstract: The valuable role of immunotherapy in treating autoimmune diseases is increasingly recognized by those involved in the research and clinical application of new biopharmaceuticals products. However, many aspects related to the mechanisms of immune-modulated therapies remain to be elucidated in order to explore fully the emerging opportunities. The non-obese diabetic NOD mouse develops insulin-dependent diabetes mellitus spontaneously as a consequence of an autoimmune process in the presence of pathogenic CD4(+) T cells that typically exhibit Th17 cell phenotypes. The change of a Th17 phenotype into a pattern of regulatory T cells (Treg) is extremely important in controlling autoimmune diseases. Heat shock proteins (HSPs) are stress-induced proteins with immunoregulatory properties. In the current study, the capacity of Hsp65 and Hsp70 mycobacterial HSPs and a constructed DNA encoded Hsp65 (DNAhsp65) to transform the pattern of the immune response from Th17 into Treg cells has been studied in vitro using co-cultures of antigen presenting cells (APCs) and T cells in NOD mice.
  
  
  
  Cells harvested from NOD mice and cultured for 48 h (without immunoregulatory compounds) presented with Th1/Th17 patterns and secretions of IL-6, IFN-γ, IL-10 and IL-17 cytokines. The cultured cells from the non-diabetic BALB/C mice exhibited a Th1 pattern and the production of IL 6 and IFN-γ secretions. An up-regulation was observed in the supernatants from the co-cultures of NOD cells that were stimulated with DNAhsp65, Hsp65 or Hsp70 through increased levels of IL-10 secretion and the suppression of IL-6, IFN-γ and IL-17 production. In addition, immunoregulation was demonstrated through IL-17 suppression in the co-culture stimulated by the specific insulin antigen. Moreover, an increase of immunoregulatory compounds were observed in the co-culture through the expression of CD11b(+)CD86(+) activation markers on APCs, as well as the frequency of Treg cells expressing CD4(+)CD3(+) and CD4(+)CD25(hi).
  
  
  
  The in vitro observation of Th17 cells differentiating into Tregs in NOD mice could raise the hypothesis that the immune regulatory activity of HSPs could be an efficient strategy for diabetes prevention and treatment.
authors.affiliation: Department of Pediatrics, Ribeirão Preto Medical School, University of São Paulo (USP), Av. Bandeirantes, 3900, 7 Floor, 14049-900, Ribeirão Preto, SP, Brazil. gecilmara@gmail.com.
authors.forename: Gecilmara Salviato
authors.initials: GS
authors.lastname: Pileggi
authors.affiliation: Department of Pediatrics, Ribeirão Preto Medical School, University of São Paulo (USP), Av. Bandeirantes, 3900, 7 Floor, 14049-900, Ribeirão Preto, SP, Brazil.
authors.forename: Aline Dayana
authors.initials: AD
authors.lastname: Clemencio
authors.affiliation: Department of Biochemistry and Immunology, Ribeirão Preto Medical School, USP, Ribeirão Preto, SP, Brazil.
authors.forename: Thiago
authors.initials: T
authors.lastname: Malardo
authors.affiliation: Department of Pediatrics, Ribeirão Preto Medical School, University of São Paulo (USP), Av. Bandeirantes, 3900, 7 Floor, 14049-900, Ribeirão Preto, SP, Brazil.
authors.forename: Sonir R
authors.initials: SR
authors.lastname: Antonini
authors.affiliation: Department of Biochemistry and Immunology, Ribeirão Preto Medical School, USP, Ribeirão Preto, SP, Brazil.
authors.forename: Vania Luiza Deperon
authors.initials: VL
authors.lastname: Bonato
authors.affiliation: Department of Biochemistry and Immunology, Ribeirão Preto Medical School, USP, Ribeirão Preto, SP, Brazil.
authors.forename: Wendy Martin
authors.initials: WM
authors.lastname: Rios
authors.affiliation: Department of Biochemistry and Immunology, Ribeirão Preto Medical School, USP, Ribeirão Preto, SP, Brazil.
authors.forename: Celio L
authors.initials: CL
authors.lastname: Silva
mesh_terms: D000818:Animals; D000938:Antigen-Presenting Cells / Q000187:drug effects* / Q000276:immunology* / Q000473:pathology; D001681:Biological Assay / Q000379:methods; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D003920:Diabetes Mellitus / Q000188:drug therapy* / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005260:Female; D007004:Hypoglycemic Agents / Q000008:administration & dosage; D007155:Immunologic Factors / Q000008:administration & dosage; D051379:Mice; D008807:Mice, Inbred BALB C; D016688:Mice, Inbred NOD; D017063:Outcome Assessment, Health Care / Q000379:methods; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects* / Q000276:immunology* / Q000473:pathology
chemical_list: D007004:Hypoglycemic Agents; D007155:Immunologic Factors
keywords: Diabetes; Heat-shock proteins; Immunotherapy; NOD mice; Regulatory T cells; Th17 cells