Evaluation of bioavailability of three types of IFNβ in multiple sclerosis patients by a new quantitative-competitive-PCR method for MxA quantification

• Published in: Journal of immunological methods Vol. 256; no. 1; pp. 141 - 152
• Main Authors: Bertolotto, Antonio, Gilli, Francesca, Sala, Arianna, Audano, Luisa, Castello, Anna, Magliola, Umberto, Melis, Fabio, Giordana, Maria Teresa
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01-10-2001; Elsevier
• Subjects: b-Interferon; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; human myxovirus; IFNα therapy; IFNβ; Microbiology; Multiple sclerosis; MxA mRNA; MxA protein; Quantitative-competitive PCR; Techniques used in virology; Virology; co-GAPDH; NABs; PBS; Multiple sclerosis; RT; IFNβ; MxA mRNA; MS; IFNα therapy; PBMC; Et-Br; IFN; co-MxA; Quantitative-competitive PCR; MxA; qc-PCR; GAPDH; Human; Nervous system diseases; Multiple; Alpha interferon; Competitive reaction; Bioavailability; Method; Protein; Inflammatory disease; Polymerase chain reaction; Blood cell; Messenger RNA; Treatment; Central nervous system disease; Beta interferon; Intracellular
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(01)00434-3

Intracellular expression of human myxovirus protein A (MxA) is exclusively induced by type I IFNs (IFNα,β,ω) or by some viruses and it is strongly increased under IFN treatment.
We set up an internally controlled quantitative-competitive polymerase chain reaction (qc-PCR) that quantifies MxA mRNA expressed in human peripheral blood mononuclear cells (PBMC).
Our qc-PCR is accurate because the mean ratio of copy number estimated by qc-PCR to that quantified spectrophotometrically is 1.08±0.03, moreover it is repeatable with high sensitivity (1 fg MxA/pg GAPDH).
MxA mRNA was tested in 47 Relapsing–Remitting Multiple Sclerosis (RR-MS) untreated patients and in 48 patients treated with one of the 3 IFNβ licensed for MS (24 with Rebif, 14 with Avonex and 10 with Betaferon). All the 48 treated patients were negative to IFNβ neutralising antibodies (NABs) as tested in our laboratory using a cytopatic assay (CPE).
MxA mRNA levels were detectable in all untreated patients (mean 24±18 fg MxA/pg GAPDH) and significantly higher levels were found in all the treated patients 12 h after IFNβ administration (mean 499±325 fg MxA/pg GAPDH); furthermore, the three types of IFNβ showed comparable bioavailability.
Our data indicate that the bioavailability of the three available types of IFNβ can be evaluated by MxA qc-PCR.