Wendy S Bril

University of Amsterdam, Amsterdam, North Holland, Netherlands

Are you Wendy S Bril?

Claim your profile

Publications (7)33 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Inhibitory antibodies develop in approximately 25% of patients with severe hemophilia. A following treatment with factorVIII. In E-16KO or E-17KO mice, in which the factor VIII gene has been inactivated by insertion of a neo cassette, inhibitors develop following administration of factor VIII. Here, we describe the generation of transgenic mice expressing human factor VIII-R593C (huFVIII-R593C). Human factor VIII-R593C cDNA under control of a mouse albumin enhancer/promoter was injected into fertilized oocytes. Analysis of transgenic mice revealed that human factor VIII-R593C was expressed in the liver. Transgenic mice were crossed with factor VIII-deficient mice (E-16KO mice). In plasma of E-16KO mice antibodies were detected after five serial intravenous injections of factor VIII, while plasma of huFVIII-R593C/E-16KO mice did not contain detectable levels of antibodies. No antibody secreting cells were observed in either spleen or bone marrow of huFVIII-R593C/E-16KO mice. Also, factor VIII-specific memory B cells were not observed in the spleen of huFVIII-R593C/E-16KO mice. Analysis of T cell responses revealed that splenocytes derived of E-16KO mice secreted IL-10 and IFN-gamma following restimulation with factor VIII in vitro. In contrast, no factor VIII-specific T cell responses were observed in huFVIII-R593C/E-16KO mice. These results indicate that huFVIII-R593C/E-16KO mice are tolerant to intravenously administered factor VIII. It is anticipated that this model may prove useful for studying immune responses in the context of factor VIII gene therapy.
    Thrombosis and Haemostasis 02/2006; 95(2):341-7. · 6.09 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We evaluated inhibitor formation in a group of patients with mild haemophilia A caused by an Arg593 to Cys mutation. A remarkably high cumulative inhibitor incidence of 14% over 22 years was observed. Three of 49 patients developed transient, low-titre inhibitors, which remained below 2.0 BU mL(-1). Four patients with an Arg593 to Cys mutation developed high-titre inhibitors (>5.0 BU mL(-1)). Three of these patients have been described previously. In this study, we characterized inhibitory antibodies in a fourth patient with high-titre inhibitors. Epitope mapping studies revealed that antibodies were predominantly directed to the A2 domain of factor VIII. We addressed the role of human leucocyte antigen (HLA) class II alleles in inhibitor development in patients with an Arg593 to Cys mutation by HLA genotyping. In the group of inhibitor patients raised frequencies of HLA-DRB1*01 and HLA-DQB1*05 were observed that did not reached statistical significance. Our data suggest that inhibitor development in mild haemophilia A patients with an Arg593 to Cys mutation is not linked to HLA class II profile.
    Haemophilia 10/2004; 10(5):509-14. · 3.17 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We report the molecular cloning and characterization of the first leukocyte-associated Ig-like receptor 1 (LAIR-1) homologue in mice that we have named mouse LAIR-1 (mLAIR-1). The mLAIR-1 gene maps to the proximal end of mouse chromosome 7 in a region syntenic with human chromosome 19q13.4 where the leukocyte receptor cluster is located. The protein shares 40% sequence identity with human LAIR-1, has a single Ig-like domain, and contains two immunoreceptor tyrosine-based inhibitory motif-like structures in its cytoplasmic tail. Mouse LAIR-1 is broadly expressed on various immune cells, and cross-linking of the molecule on stably transfected RBL-2H3 and YT.2C2 cells results in strong inhibition of their degranulation and cytotoxic activities, respectively. Upon pervanadate stimulation, the mLAIR-1 cytoplasmic tail becomes phosphorylated, thereby recruiting Src homology region 2-containing tyrosine phosphatase-2. Interestingly, unlike human LAIR-1, Src homology region 2-containing tyrosine phosphatase-1 is not recruited to the mLAIR-1 cytoplasmic tail. Screening human and mouse cell lines for mLAIR-1 and human LAIR-1 binding partners identified several lines expressing putative ligand(s) for both receptors.
    The Journal of Immunology 06/2004; 172(9):5535-43. · 5.52 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: After treatment with factor (F) VIII concentrate a significant number of patients with hemophilia A develop inhibitory antibodies that neutralize FVIII. Epitope mapping revealed that antibodies bind to selected regions within the A2, A3, and C2 domains of FVIII. Anti-A2 and anti-A3 antibodies interfere with assembly of FVIIIa with FIXa, whereas anti-C2 antibodies impede the interaction of FVIII with phospholipids. The immunologic mechanisms underlying inhibitor development in hemophilia A have not been fully elucidated. FVIII is recognized by the immune system as a foreign antigenic substance that evokes the T cell-dependent formation of high-affinity antibodies. Clonal analysis of B cell responses in hemophilia A patients has given further insight into the epitope specificity and molecular characteristics of FVIII inhibitors. Costimulatory blockade of FVIII-reactive T cells in a mouse model for hemophilia A has suggested new approaches for treatment of inhibitor patients. In this article, recent studies on the immunobiology of FVIII inhibitors are summarized and discussed with reference to their potential impact on treatment and prevention of immune responses in patients with hemophilia.
    Seminars in Thrombosis and Hemostasis 03/2003; 29(1):61-8. · 4.22 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We characterized anti-factor VIII antibodies in a mild haemophilia A patient with an Arg593-->Cys mutation in the A2 domain, using V gene phage-display technology. All isolated single-chain variable-domain antibody fragments were directed against residues Arg484-Ile508, a binding site for factor VIII inhibitors in the A2 domain. After a further period of replacement therapy, a transient rise in inhibitor titre was observed. These antibodies were directed against the A2 domain. Activation of a pre-existing pool of B cells, which express antibodies against residues Arg484-Ile508, could explain the rapid anamnestic response.
    British Journal of Haematology 11/2002; 119(2):393-6. · 4.94 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Most plasmas from patients with inhibitors contain antibodies that are reactive with the C2 domain of factor VIII. Previously, we have shown that the variable heavy chain (V(H)) regions of antibodies to the C2 domain are encoded by the closely related germline gene segments DP-10, DP-14, and DP-88, which all belong to the V(H)1 gene family. Here, we report on the isolation and characterization of additional anti-C2 antibodies that are derived from V(H) gene segments DP-88 and DP-5. Competition experiments using murine monoclonal antibodies CLB-CAg 117 and ESH4 demonstrated that antibodies derived from DP-5 and DP-88 bound to different sites within the C2 domain. Epitope mapping studies using a series of factor VIII/factor V hybrids revealed that residues 2223 to 2332 of factor VIII are required for binding of the DP-10-, DP-14-, and DP-88-encoded antibodies. In contrast, binding of the DP-5-encoded antibodies required residues in both the amino- and carboxy-terminus of the C2 domain. Inspection of the reactivity of the antibodies with a series of human/porcine hybrids yielded similar data. Binding of antibodies derived from germline gene segments DP-10, DP-14, and DP-88 was unaffected by replacement of residues 2181 to 2243 of human factor VIII for the porcine sequence, whereas binding of the DP-5-encoded antibodies was abrogated by this replacement. Together these data indicate that antibodies assembled from V(H) gene segments DP-5 and the closely related germline gene segments DP-10, DP-14, and DP-88 target 2 distinct antigenic sites in the C2 domain of factor VIII.
    Blood 05/2002; 99(8):2828-34. · 9.06 Impact Factor
  • Source
    Wendy Simone Bril
    [Show abstract] [Hide abstract]
    ABSTRACT: The X-linked bleeding disorder haemophilia A is due to a deficiency or functional defect of coagulation factor VIII. The bleeding tendency can be corrected by administration of factor VIII concentrates. A serious complication of factor VIII replacement therapy is the development of anti-factor VIII antibodies (inhibitors) that neutralize factor VIII activity. In recent years, the epitope-specifities and the inhibitory mechanisms of factor VIII inhibitors have gained increasing interest. The generation of factor VIII knock-out mice has opened the possibility of studying the immunobiology of inhibitor formation in murine models of haemophilia A. In spite of these recent developments however, the immunological mechanisms underlying the anti-factor VIII immune response have remained poorly understood so far. Most of our current knowledge is based on studies on inhibitor formation in the severe form of haemophilia. However, inhibitors also occur in patients with mild haemophilia A, in particular after a period of extensive factor VIII replacement therapy. These patients differ from severe haemophiliacs in that they have low levels of circulating factor VIII activity (5-25% of normal). The presence of endogenous factor VIII may have major impact on the immune response to exogenous factor VIII during replacement therapy. The studies presented in this thesis were performed to obtain a better understanding of the immunobiology of inhibitor development in mild haemophilia A. In the introduction (chapter 1), recent studies on the immunobiology of factor VIII inhibitors in haemophilia A patients are summarized and discussed. We have characterized the anti-factor VIII antibodies in patients with mild haemophilia A employing phage display technology. In chapter 2, anti-C2 antibodies were isolated and characterized from the repertoire of a mild haemophilia A patient. Our results provide evidence for the presence of two classes of anti-C2 antibodies that recognize distinct antigenic sites in factor VIII. The characteristics of the anti-C2 antibodies were further analysed in chapter 3, and compared to the epitopes of previously described murine monoclonal antibodies. The first class of anti-C2 antibodies bind to the epitope defined by monoclonal antibody ESH4. The second class of antibodies bind to the epitope defined by monoclonal antibody CLB-CAg 117. Antibodies belonging to this second class of antibodies were also isolated from a different patient with mild haemophilia A (chapter 4). The VH gene segment usage of the antibodies directed at the epitope defined by CLB-CAg 117 is less restricted compared to the first class of anti-C2 antibodies. Based on the long CDR3 region, we argue that this second class of antibodies originates from a pool of polyreactive human antibodies. In chapter 5, we describe the inhibitor development of a patient with mild haemophilia A caused by an Arg593 to Cys mutation. We have isolated and characterized anti-A2 antibodies using phage display and we have performed epitope-mapping studies of anti-factor VIII antibodies in plasma using immunoprecipitation analysis. The data presented in chapter 5 provide a possible explanation for anamnestic responses observed in patients with a history of inhibitor development. We propose that activation of a quiescent pool of memory B cells underlies the rise in inhibitor titer observed in haemophilia A patients with a history of inhibitor development. Chapter 6 describes the epitope specificities of anti-factor VIII antibodies in another patient from our cohort of mild haemophilia A patients with the Arg593 to Cys mutation. Results from this chapter and previous studies show that high responder patients with the Arg593 to Cys substitution develop inhibitory antibodies predominantly directed at the A2 domain of factor VIII. This suggests that inhibitor formation proceeds via a common mechanism in these patients. The role of HLA class II alleles in inhibitor formation was investigated by HLA genotyping of 42 patients with the Arg593 to Cys mutation. Our data suggest a weak association between inhibitor development and HLA class II alleles in mild haemophilia A patients with the Arg593 to Cys mutation. In Chapter 7, we present the characteristics of a mouse transgenic for human factor VIII with the Arg593 to Cys mutation (hufVIII-R593C mouse). The anti-factor VIII immune response was analysed in transgenic hufVIII-R593C mice crossed with factor VIII-deficient mice (exon 16 knock out, or E-16 KO mice). Serial intravenous injections of human factor VIII do not evoke an immune response in hufVIII-R593C/E-16 KO mice. The introduction of the human factor VIII–R593C transgene renders the mice tolerant to human factor VIII. However, when hufVIII-R593C/E-16 KO mice were subcutaneously injected with factor VIII in the presence of an adjuvant, loss of tolerance to factor VIII was observed. The results of chapter 7 demonstrate that hufVIII-R593C/E-16 KO mice provide a valuable model for studies directed at the mechanisms underlying inhibitor development in haemophilia A. Finally, chapter 8 provides a general overview that discusses the implications of our findings.