Interferon-beta antibodies have a higher affinity in patients with neutralizing antibodies compared to patients with non-neutralizing antibodies.

In this study, we investigated the affinity, determined by a relative affinity assay, using increasing concentrations of sodium-isothiocyanate to disrupt the antigen antibody binding of neutralizing and non-neutralizing antibodies against interferon-beta (IFNbeta)-1a and -1b in 73 serum samples of MS patients treated with IFNbeta-1a or -1b. Relative affinity values were significantly higher in NAB-positive compared to NAB-negative samples and in samples of IFNbeta-1a-treated patients compared to IFNbeta-1b. A significant positive correlation between relative affinity values and therapy duration indicates affinity maturation as another qualitative factor in IFNbeta neutralization.

A comparative study of the relative bioavailability of different interferon beta preparations.

BACKGROUND: Three different recombinant interferon beta (IFNbeta) preparations are currently available for the treatment of MS, two IFNbeta-1a products (Rebif and Avonex) and one IFNbeta-1b product (Betaferon). These products differ with respect to the recommended dose, the dosage regimen, and the injection route. This study compared the relative biologic activity of these three products in vitro and in vivo. METHODS: Blood samples were collected from 237 patients with MS (170 on IFNbeta therapy and 67 control subjects receiving no therapy). Samples with neutralizing antibodies were excluded. Levels of the antiviral protein MxA and soluble vascular cell adhesion molecule-1 (sVCAM) in the four groups were measured by ELISA. In the in vitro investigation, untreated blood was incubated for 24 hours with increasing concentrations of the three IFNs from a dose of 1 IU/mL to 1000 IU/mL, after which levels of MxA were measured. RESULTS: A difference between the groups was observed in vitro, with a significant change from baseline in MxA levels being observed at 10 IU for Betaferon compared with 100 IU for Rebif and Avonex. However, this might be due to the different methods used for the determination of IU by the manufacturers. At the single-dose level there were no significant differences between IFNbeta preparations. In vivo, there were significantly different levels of MxA in the four groups. In the Betaferon group, the median value for MxA was 2.29 ng/105 peripheral blood leukocytes (PBL), compared with 1.00 ng/105 PBL for Rebif, 0.57 ng/105 PBL for Avonex, and 0.14 ng/105 PBL for the control group. Some significant differences between the groups were also apparent with respect to levels of sVCAM, which were higher with Betaferon than with Rebif. CONCLUSION: IFNbeta-1b induces higher levels of the above markers of IFNbeta bioactivity than either of the two IFNbeta-1a preparations. Moreover, there is a less striking difference between the two IFNbeta-1a preparations in favor of subcutaneous IFNbeta-1a.

A critical comparison of frequently used methods for the analysis of tumor necrosis factor-alpha expression by human immune cells.

A variety of methods have been developed for the measurement of tumor necrosis factor (TNF)-alpha synthesis by immune cells. Here we have compared the results of the most common used methods, including in vitro stimulation of whole blood or peripheral blood mononuclear cell (PBMC) cultures with phytohaemagglutinin (PHA) or lipopolysaccharide (LPS) and RT-PCR analysis of TNF-alpha transcription in unstimulated PBMC. When we used EDTA treated blood samples we observed a significant correlation between the PHA and LPS stimulated TNF-alpha responses in whole blood or PBMC cultures. In contrast, TNF-alpha concentrations obtained from PHA and LPS stimulated whole blood cultures from citrate-treated blood did not show a correlation. We also found that the PHA stimulated TNF-alpha response was significantly higher in PBMC than in whole blood cultures, whereas the highest LPS stimulated TNF-alpha response was observed in citrate-treated blood. Moreover, the TNF-alpha response in both, citrate and EDTA treated whole blood cultures was significantly higher after LPS than after PHA stimulation. In contrast, in PBMC cultures the PHA stimulated TNF-alpha response was significantly higher than the LPS stimulated response. The results of RT-PCR analysis revealed a significant correlation with the PHA stimulated TNF-alpha response, both in whole blood assays and in PBMC cultures. In addition our results demonstrate that these different methods can only be compared when the influence of external factors such as the immediate processing of blood samples or the use of an appropriate anticoagulant and stimulant is considered.

Differing immunogenic potentials of interferon beta preparations in multiple sclerosis patients.

Interferon beta (IFNbeta) is a first-line therapy for multiple sclerosis (MS). However, some patients experience a decline in efficacy with continued therapy due to the development of anti-IFNbeta neutralizing antibodies (NAb). We investigated the frequency of NAb cross-sectionally in 846 MS patients who were receiving IFNbeta-1b, IFNbeta-1a im, or IFNbeta-1a sc. The frequency of NAb in patients receiving IFNbeta-1a im was lower (5%) than in patients treated with any other form of IFNbeta (22-35%) (P < 0.0001). Binding antibodies (BAb) were measured in 808 patients. The frequency differed significantly between treatment groups, ranging from 45% (IFNbeta-1a im) to 88% (IFNbeta-1b). The proportion of NAb-positive patients within the BAb-positive group differed significantly among treatment groups, ranging between 12% (IFNbeta-1a im) and 51% (IFNbeta-1a sc). The median NAb titer from all IFNbeta-1a-treated patients was higher than from IFNbeta-1b-treated patients (446 versus 171 NU/ mL, P = 0.04). Among NAb-positive patients, the frequency of NAb titers > 100 NU/mL was 71% for IFNbeta-1a compared with 58% for IFNbeta-1b (P = 0.04). Except for conflicting data regarding IFNbeta-1a sc, the results are generally consistent with the literature and together with the differing proportion of NAb-positive patients within the BAb-positive group, provide further insight into the immunogenicity of the IFNbeta preparations.

Correlation between the IgG index, oligoclonal bands in CSF, and the diagnosis of demyelinating diseases.

Intrathecal immunoglobulin synthesis can be assessed by different approaches. It can be measured quantitatively by the IgG index or qualitatively by isoelectric focusing (IEF) to detect oligoclonal bands (OCB). In this study we investigated if there is a correlation between the frequency of OCB and the IgG index and if the IgG index predicts the diagnosis of a demyelinating CNS disease (DMD). We found a positive correlation between the IgG index and the frequency of OCB as well as the probability of DMD. We conclude that quantitative assessment of CSF-IgG can be useful because it is easier and quicker to perform than IEF but cannot replace IEF in general because this is the most sensitive method to detect abnormal IgG in CSF.