ABSTRACT
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.
Subject(s)
GTP-Binding Proteins , Interferon-beta/pharmacology , Multiple Sclerosis/drug therapy , Polymerase Chain Reaction/methods , Proteins/genetics , Humans , Leukocytes, Mononuclear/immunology , Multiple Sclerosis/immunology , Myxovirus Resistance Proteins , Protein Biosynthesis , RNA, Messenger/biosynthesis , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and SpecificityABSTRACT
Quantification of tumor necrosis factor-alpha (TNF-alpha) mRNA in peripheral blood mononuclear cells (PBMC) could provide information about disease activity in multiple sclerosis (MS); however, specific competitive methods must be utilized. A competitor cDNA, having the same sequence of the target TNF-alpha cDNA, a part from an internal 49-bp deletion, was generated and used to set-up a quantitative polymerase chain reaction (PCR) to quantify mRNA of TNF-alpha. Competitor and target were co-amplified using the same primers. The rates of generation of competitor and target TNF-alpha conformed closely to the prediction of the mathematical model, and a high level of accuracy and reproducibility was achieved. The method was applied to quantify TNF-alpha mRNA in PBMC of normal subjects and multiple sclerosis (MS) patients both during clinical relapses and remissions. A statistically significant higher level of TNF-alpha mRNA was detected during relapses than during remissions. High levels of TNF-alpha mRNA were found in 44% of relapses and 12% of samples during remissions, suggesting that TNF-alpha mRNA synthesis is abnormal in MS.
Subject(s)
Leukocytes, Mononuclear/metabolism , Multiple Sclerosis/metabolism , Polymerase Chain Reaction/methods , RNA, Messenger/biosynthesis , Tumor Necrosis Factor-alpha/genetics , Adult , Binding, Competitive , Case-Control Studies , Female , Humans , Logistic Models , Male , Middle Aged , Recurrence , Reproducibility of ResultsABSTRACT
Eight relapsing-remitting multiple sclerosis (MS) patients were tested for the level of transforming growth factor beta1 (TGFbeta1) mRNA in peripheral blood mononuclear cells every 15 days for 6 months. Disease activity was evaluated every 4 weeks by magnetic resonance imaging (MRI) and neurological examination. An inverse correlation was found between the level of TGFbeta1 mRNA and MRI disease activity. The level of TGFbeta1 mRNA predicted the presence of disease activity in the scans performed 2-4 weeks later with high sensitivity (88%) and specificity (87.5%) suggesting that TGFbeta1 mRNA quantification could be an indicator of disease activity in MS.
