Regional Center for Multiple Sclerosis e and Clinical Neurobiology

Orbassano, Italy

Regional Center for Multiple Sclerosis e and Clinical Neurobiology

Orbassano, Italy

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van Beers M.M.C.,University Utrecht | van Beers M.M.C.,Leiden University | Sauerborn M.,University Utrecht | Gilli F.,Regional Center for Multiple Sclerosis e and Clinical Neurobiology | And 4 more authors.
Journal of Immunological Methods | Year: 2010

To date, the therapeutic efficacy of recombinant human proteins is limited by their potential to break B cell tolerance in patients. The formation of neutralising antibodies (NABs) directed against recombinant human interferon beta (rhIFNβ) is associated with a decrease in the therapeutic effect of the protein. For this reason, there is a need to study factors that can cause the immunogenicity of rhIFNβ. Transgenic C57Bl/6 mice that are immune tolerant for human interferon beta (hIFNβ) have been employed in a mouse model for assessing the breaking of immune tolerance by rhIFNβ. In this study, we used the original C57Bl/6 mouse model as well as the hybrid offspring from crossings of transgenic C57Bl/6 mice with wildtype FVB/N mice to study the immunogenicity of three commercial rhIFNβ products, Rebif®, Avonex® and Betaferon®. As determined by ELISA, wildtype C57Bl/6 mice failed to form binding antibodies (BABs) against Rebif® and Avonex® formulated with human serum albumin. Because not all interferon beta products induce antibodies in wildtype C57Bl/6 mice, the transgenic C57Bl/6 mice cannot be used to study the breaking of tolerance by these products. However, the crossing of transgenic C57Bl/6 mice with FVB/N mice resulted in wildtype hybrid offspring in which all products were immunogenic and transgenic hybrid offspring that showed immune tolerance for hIFNβ. Thus, these C57Bl/6 × FVB/N hybrid transgenic mice can be used to study the breaking of immune tolerance for all rhIFNβ products. Of the three products, only Betaferon® was able to break immune tolerance in the transgenic hybrids. With an MxA gene expression inhibition assay, NABs were detected in Betaferon® treated wildtype hybrid mice, but not in transgenic hybrid mice, indicating a distinct immune mechanism in wildtype and transgenic mice. A pegylated rhIFNβ-1a variant, PEG-rhIFNβ-1a, induced antibodies in wildtype hybrid mice, but did not break the immune tolerance of transgenic hybrid mice. This suggests that pegylation did not affect the potential of rhIFNβ-1a to break B cell tolerance. © 2009 Elsevier B.V. All rights reserved.


Van Beers M.M.C.,University Utrecht | Van Beers M.M.C.,Leiden University | Sauerborn M.,University Utrecht | Gilli F.,Regional Center for Multiple Sclerosis e and Clinical Neurobiology | And 3 more authors.
Pharmaceutical Research | Year: 2010

Purpose: To study the influence of protein aggregation on the immunogenicity of recombinant human interferon beta (rhIFNβ) in wild-type mice and transgenic, immune-tolerant mice, and to evaluate the induction of immunological memory. Methods: RhIFNβ-1b and three rhIFNβ-1a preparations with different aggregate levels were injected intraperitoneally in mice 15× during 3 weeks, and the mice were rechallenged with rhIFNβ-1a. The formation of binding (BABs) and neutralizing antibodies (NABs) was monitored. Results: Bulk rhIFNβ-1a contained large, mainly non-covalent aggregates and stressed rhIFNβ-1a mainly covalent, homogeneous (ca. 100 nm) aggregates. Reformulated rhIFNβ-1a was essentially aggregate-free. All products induced BABs and NABs in wild-type mice. Immunogenicity in the transgenic mice was product dependent. RhIFNβ-1b showed the highest and reformulated rhIFNβ-1a the lowest immunogenicity. In contrast with wild-type mice, transgenic mice did not show NABs, nor did they respond to the rechallenge. Conclusions: The immunogenicity of the products in transgenic mice, unlike in wild-type mice, varied. In the transgenic mice, neither NABs nor immunological memory developed. The immunogenicity of rhIFNβ in a model reflecting the human immune system depends on the presence and the characteristics of aggregates. © 2010 The Author(s).


Gilli F.,Regional Center for Multiple Sclerosis e and Clinical Neurobiology | Lindberg R.L.P.,University of Basel | Valentino P.,Regional Center for Multiple Sclerosis e and Clinical Neurobiology | Marnetto F.,Regional Center for Multiple Sclerosis e and Clinical Neurobiology | And 8 more authors.
PLoS ONE | Year: 2010

Background: Pregnancy is associated with reduced activity of multiple sclerosis (MS). However, the biological mechanisms underlying this pregnancy-related decrease in disease activity are poorly understood. Methodology: We conducted a genome-wide transcription analysis in peripheral blood mononuclear cells (PBMCs) from 12 women (7 MS patients and 5 healthy controls) followed during their pregnancy. Samples were obtained before, during (i.e. at the third, sixth, and ninth month of gestation) and after pregnancy. A validation of the expression profiles has been conducted by using the same samples and an independent group of 25 MS patients and 11 healthy controls. Finally, considering the total group of 32 MS patients, we compared expression profiles of patients relapsing during pregnancy (n = 6) with those of relapse-free patients (n = 26). Principal Findings: Results showed an altered expression of 347 transcripts in non-pregnant MS patients with respect to non-pregnant healthy controls. Complementary changes in expression, occurring during pregnancy, reverted the previous imbalance particularly for seven inflammation-related transcripts, i.e. SOCS2, TNFAIP3, NR4A2, CXCR4, POLR2J, FAM49B, and STAG3L1. Longitudinal analysis showed that the overall deregulation of gene expression reverted to "normal" already within the third month of gestation, while in the post-partum gene expressions rebounded to pre-pregnancy levels. Six (18.7%) of the 32 MS patients had a relapse during pregnancy, mostly in the first trimester. The latter showed delayed expression profiles when compared to relapse-free patients: in these patients expression imbalance was reverted later in the pregnancy, i.e. at sixth month. Conclusions: Specific changes in expression during pregnancy were associated with a decrease in disease activity assessed by occurrence of relapses during pregnancy. Findings might help in understanding the pathogenesis of MS and may provide basis for the development of novel therapeutic strategies. © 2010 Gilli et al.

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