Evaluation of bioavailability of three types of IFNbeta in multiple sclerosis patients by a new quantitative-competitive-PCR method for MxA quantification.
Intracellular expression of human myxovirus protein A (MxA) is exclusively induced by type I IFNs (IFNalpha,beta,omega) 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 IFNbeta licensed for MS (24 with Rebif, 14 with Avonex and 10 with Betaferon). All the 48 treated patients were negative to IFNbeta neutralising antibodies (NABs) as tested in our laboratory using a cytopathic 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 IFNbeta administration (mean 499+/-325 fg MxA/pg GAPDH); furthermore, the three types of IFNbeta showed comparable bioavailability. Our data indicate that the bioavailability of the three available types of IFNbeta can be evaluated by MxA qc-PCR.
Bertolotto
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Centro Sclerosi Multipla and Laboratorio di Neurobiologia Clinica, Divisione Universitaria di Neurologia, Azienda Ospedaliera S. Luigi, Università di Torino, Regione Gonzole 10, 10043 Orbassano, Italy. NSGLB@TIN.IT
Gilli
F
F
Sala
A
A
Audano
L
L
Castello
A
A
Magliola
U
U
Melis
F
F
Giordana
M T
MT
D019204:GTP-Binding Proteins; D006801:Humans; D016899:Interferon-beta / Q000494:pharmacology*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D009103:Multiple Sclerosis / Q000188:drug therapy* / Q000276:immunology; D064547:Myxovirus Resistance Proteins; D016133:Polymerase Chain Reaction / Q000379:methods*; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
C579071:MX1 protein, human; D064547:Myxovirus Resistance Proteins; D011506:Proteins; D012333:RNA, Messenger; D016899:Interferon-beta; D019204:GTP-Binding Proteins