title: New and cost effective cell-based assay for Dialyzed Leukocyte Extract (DLE)-induced Jurkat cells proliferation under azathioprine treatment.
abstract: The human Dialyzed Leukocyte Extract (DLE) is a heterogeneous mix of oligopeptides of <10kDa, extracted from leukocytes of healthy donors. There is significant clinical evidence of improvement using DLE during treatment of allergies, cancer,immunodeficiencies, and in mycotic and viral infections. Nevertheless, the DLE exact nature and mechanism of action have been elusive for more than 50 years. DLE biological activity testing is necessary in DLE production and quality control. Both in vitro and in vivo assays exist: E-rosette test, induction of delayed type hypersensitivity in mice, leukocyte migration and IFN-γ secretion. The animal-origin materials and in vivo assays convey a considerable logistic, ethic and economic burden, meanwhile the available in vitro assays have been reported with limited reproducibility and sometimes contradictory results. Here we are reporting a new DLE biological activity cell-based assay. The A20 and Jurkat cell lines were treated with (+Aza) or without (-Aza) azathioprine, DLE (+DLE) or both (+Aza/+DLE). After 72h, the cell proliferation was analyzed by the MTT or BrdU incorporation assays. In +Aza/+DLE treated cells, we observed a significant higher proliferation, when compared with +Aza/-DLE. In the absence of Aza, cells did not present any proliferation difference between -DLE or +DLE treatments. Both assays, MTT and BrdU showed similar results, being the MTT test more cost effective and we select it for validation as DLE biological assay using Jurkat cells only. We tested three different lyophilized DLE batches and we found consistent results with acceptable assay reproducibility and linearity. The DLE capacity for rescuing Jurkat cell proliferation during +Aza treatment was consistent using different liquid and lyophilized DLE batches, presenting also consistent chromatographic profiles. Finally, DLE treatment in Jurkat cells did not result into significant IL-2 of IFN-γ secretion, and known lymphocyte proliferative drugs failed to rescue Jurkat cells viability in presence of +Aza, as +DLE treatment did in our MTT assay. In conclusion, our new cell-based MTT assay has excellent DLE biological activity consistency, robustness and is cost effective, presenting important advantages over previous DLE activity in vitro and in vivo assays.
authors.affiliation: R&D Department, Imuna Pharm a. s., Jarková 17, 082 22 Šarišské Michaľany, Slovak Republic. Electronic address: miguel.lopez.cardoso@gmail.com.
authors.forename: F M
authors.initials: FM
authors.lastname: Cardoso
authors.affiliation: R&D Department, Imuna Pharm a. s., Jarková 17, 082 22 Šarišské Michaľany, Slovak Republic.
authors.forename: M
authors.initials: M
authors.lastname: Tomkova
authors.affiliation: R&D Department, Imuna Pharm a. s., Jarková 17, 082 22 Šarišské Michaľany, Slovak Republic.
authors.forename: D
authors.initials: D
authors.lastname: Petrovajova
authors.affiliation: R&D Department, Imuna Pharm a. s., Jarková 17, 082 22 Šarišské Michaľany, Slovak Republic.
authors.forename: M
authors.initials: M
authors.lastname: Bubanova
authors.affiliation: R&D Department, Imuna Pharm a. s., Jarková 17, 082 22 Šarišské Michaľany, Slovak Republic.
authors.forename: O
authors.initials: O
authors.lastname: Ragac
authors.affiliation: R&D Department, Imuna Pharm a. s., Jarková 17, 082 22 Šarišské Michaľany, Slovak Republic; Olomouc University Social Health Institute (OUSHI), Univerzitní 22, 771 11 Olomouc, Czech Republic.
authors.forename: T
authors.initials: T
authors.lastname: Hornakova
mesh_terms: D000818:Animals; D001379:Azathioprine / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D003362:Cost-Benefit Analysis / Q000379:methods; D006801:Humans; D019169:Jurkat Cells / Q000187:drug effects* / Q000502:physiology*; D007962:Leukocytes / Q000187:drug effects* / Q000502:physiology*; D051379:Mice; D015203:Reproducibility of Results; D014165:Transfer Factor / Q000494:pharmacology*
chemical_list: D014165:Transfer Factor; D001379:Azathioprine
keywords: Azathioprine; DLE; Jurkat; MTT assay