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J M Saint-Remy

Publications and source records attributed to J M Saint-Remy.

At least 19 recordsLinked to original sources
PubMed2001

Structure of a factor VIII C2 domain-immunoglobulin G4kappa Fab complex: identification of an inhibitory antibody epitope on the surface of factor VIII.

The development of an immune response to infused factor VIII is a complication affecting many patients with hemophilia A. Inhibitor antibodies bind to antigenic determinants on the factor VIII molecule and block its procoagulant activity. A patient-derived inhibitory immunoglobulin G4kappa antibody (BO2C11) produced by an immortalized memory B-lymphocyte cell line interferes with the binding of factor VIII to phospholipid surfaces and to von Willebrand factor. The structure of a Fab fragment derived from this antibody complexed with the factor VIII C2 domain was determined at 2.0 A resolution. The Fab interacts with solvent-exposed basic and hydrophobic side chains that form a membrane-association surface of factor VIII. This atomic resolution structure suggests a variety of amino acid substitutions in the C2 domain of factor VIII that might prevent the binding of anti-C2 inhibitor antibodies without significantly compromising the procoagulant functions of factor VIII.

Antibodies, Monoclonal↗
PubMed2001

Antigenicity of putative phospholipid membrane-binding residues in factor VIII.

Most inhibitory antibodies to human factor VIII (fVIII) bind to epitopes in the A2, ap-A3, or C2 domains. The anticoagulant action of antibodies to the C2 domain is due to inhibition of binding of fVIII to phospholipid. The x-ray structure of the human fVIII C2 domain shows a putative hydrophobic, 3-prong, phospholipid membrane-binding site consisting of Met2199/Phe2200, Val2223, and Leu2251/Leu2252. Additionally, Lys2227, near Val2223, is part of a ring of positively charged residues that may contribute to electrostatic interaction of fVIII with negatively charged phosphatidylserine. In this study, 8 active mutants of human fVIII (Met2199Ile, Leu2252Phe, Phe2200Leu, Val2223Ala, Lys2227Glu, Met2199Ile/Phe2200Leu, Val2223Ala/Lys2227Glu, and Met2199Ile/Phe2200Leu/Val2223Ala/Lys2227Glu), which were constructed on the basis of differences between human, porcine, murine, and canine fVIII at proposed phospholipid binding sites, were expressed. The antigenicity of the mutants toward 5 C2-specific polyclonal human antibodies was measured by using the Bethesda assay. A human monoclonal anti-C2 antibody, BO2C11, and a murine C2-specific monoclonal antibody, NMC VIII-5, were also included in the analysis. In comparison with wild-type, B-domainless fVIII, the Met2199Ile, Phe2200Leu, and Leu2252 single mutants had lower antigenicity toward most of the inhibitors. In contrast, the Val2223Ala and Lys2227Glu mutants usually showed increased antigenicity. These results suggest that C2 inhibitors frequently target the Met2199/Phe2200 and Leu2251/Leu2252 beta-hairpins and are consistent with the hypothesis that these residues participate in binding to phospholipid membranes. In contrast, Val2223 and Lys2227 may oppose antibody binding sterically or through stabilization of a low-affinity membrane-binding conformation of the C2 domain.

Amino Acid Sequence↗
PubMed2000

A novel cause of mild/moderate hemophilia A: mutations scattered in the factor VIII C1 domain reduce factor VIII binding to von Willebrand factor.

The mechanisms responsible for the low factor VIII (fVIII) activity in the plasma of patients with mild/moderate hemophilia A are poorly understood. In such patients, we have identified a series of fVIII mutations (Ile2098Ser, Ser2119Tyr, Asn2129Ser, Arg2150His, and Pro2153Gln) clustered in the C1 domain and associated with reduced binding of fVIII to von Willebrand factor (vWf). For each patient plasma, the specific activity of mutated fVIII was close to that of normal fVIII. Scatchard analysis showed that the affinity for vWf of recombinant Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII mutants was reduced 8-fold, 80-fold, and 3-fold, respectively, when compared with normal fVIII. Given the importance of vWf for the stability of fVIII in plasma, these findings suggested that the reduction of fVIII binding to vWf resulting from the above-mentioned mutations could contribute to patients' low fVIII plasma levels. We, therefore, analyzed the effect of vWf on fVIII production by Chinese hamster ovary (CHO) cells transfected with expression vectors for recombinant B domain-deleted normal, Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII. These 3 mutations impaired the vWf-dependent accumulation of functional fVIII in culture medium. Analysis of fVIII production by transiently transfected CHO cells indicated that, in addition to the impaired stabilization by vWf, the secretion of functional Ile2098Ser and Arg2150His fVIII was reduced about 2-fold and 6-fold, respectively, by comparison to Ser2119Tyr and normal fVIII. These findings indicate that C1-domain mutations resulting in reduced fVIII binding to vWf are an important cause of mild/moderate hemophilia A.

Animals↗
PubMed2000

A human antibody directed to the factor VIII C1 domain inhibits factor VIII cofactor activity and binding to von Willebrand factor.

The occurrence of factor VIII (fVIII) inhibitory antibodies is a rare complication of fVIII substitution therapy in mild/moderate hemophilia A patients. fVIII mutations in certain regions such as the C1 domain are, however, more frequently associated with inhibitor, for reasons which remain unclear. To determine whether inhibitors could map to the mutation site, we analyzed at the clonal level the immune response of such a patient with an inhibitor to wild-type but not self-fVIII and an Arg2150His substitution in the C1 domain. Immortalization of the patient B lymphocytes provided a cell line producing an anti-fVIII IgG4kappa antibody, LE2E9, that inhibited fVIII cofactor activity, following type 2 kinetics and prevented fVIII binding to von Willebrand factor. Epitope mapping with recombinant fVIII fragments indicated that LE2E9 recognized the fVIII C1 domain, but not the Arg2150His-substituted C1 domain. Accordingly, LE2E9 did not inhibit Arg2150His fVIII activity. These observations identify C1 as a novel target for fVIII inhibitors and demonstrate that Arg2150His substitution alters a B-cell epitope in the C1 domain, which may contribute to the higher inhibitor incidence in patients carrying such substitution. (Blood. 2000; 95:156-163)

Amino Acid Sequence↗
PubMed2000

Major T cell epitope-containing peptides can elicit strong antibody responses.

Peptides containing major T cell epitopes have the capacity to induce T cell anergy and have therefore been proposed for the treatment of allergic and autoimmune diseases. Such peptides should not be immunogenic, i. e. should not contain a B cell recognition site. We have evaluated in BALB/c mice the therapeutic potential of a 15-mer peptide (p21 - 35) derived from Der p2, a major allergen of the house dust mite Dermatophagoides pteronyssinus, which contains a dominant T cell epitope but is not recognized by antibodies to Der p2. Unexpectedly, p21 - 35 elicited strong immune responses, suggesting the presence of a cryptic B cell epitope. Similar results were obtained with mice of three additional MHC haplotypes. A core sequence of four amino acids (Ile-Ile-His-Arg) corresponding to residues 28 - 31 was shared by the B and T cell epitopes. Critical residues for B cell recognition were Arg31 and Lys33, while Ile28 was essential for T cell recognition. A Lys33Ala mutant of p21 - 35 still activated T cells but had much reduced immunogenic properties, making it a suitable alternative peptide for T cell anergy induction. Careful investigation of the immunogenic potential of peptides used to induce T cell anergy should be carried out prior to their clinical application.

Allergens↗
PubMed2000

Factor VIII Inhibitors. Natural autoantibodies and anti-idiotypes.

Natural antibodies to factor VIII are present in the normal antibody repertoire as other self-reactive antibodies to soluble proteins. The question as to whether they represent just a chance occurrence linked to the huge diversification of the antibody repertoire or whether these antibodies have an actual physiological relevance is not entirely settled. Evidence is in favor of a role in the maintenance of immune homeostasis, however, namely self-reactive antibodies are required to maintain the capacity of the immune system to distinguish self from nonself. Anti-factor VIII antibodies pose an interesting case in point because they exhibit the capacity to inhibit the function of factor VIII. Such a property is neutralized at least in part by the production of corresponding anti-idiotypic antibodies. Normal homeostasis can therefore be viewed as a network of interacting molecules, idiotypes, and anti-idiotypes; disruption of this equilibrium leads to the development of autoimmunity. A question that remains open for the time being is whether this network of interactions can be modulated in a defined way for the treatment of autoimmune reactions. This would mean either passive administration of anti-idiotypic antibodies or active immunization with idiotypes. The former has proved to be efficient, and the latter has still to be demonstrated. Further, and probably most importantly, is the question of the possible application of the idiotypic network concept to the treatment of hemophilia patients producing inhibitors. This essentially requires that an analysis of the anti-factor VIII immune response be carried out at the clonal level. Such work is ongoing in our laboratory.

Animals↗
PubMed2000

Natural antibodies to factor VIII.

Anti-factor VIII antibodies represent a unique model to study the relationship between natural autoreactivity (natural antibodies to factor VIII of healthy individuals), disease-associated autoimmunity ("spontaneous" factor VIII inhibitors of patients with anti-factor VIII autoimmune disease) and antigen-driven immune responses (immune inhibitors in multitransfused patients with hemophilia A) to a single human protein antigen. Although natural and disease-associated anti-factor VIII antibodies are not readily distinguished based on the comparison of their isotypic distribution and epitope mapping, available studies of cross-reacting idiotypes suggest that factor VIII inhibitors in patient's plasma encompass two populations of anti-factor VIII antibodies. Some antibodies result from the clonal expansion of B lymphocytes that exist before treatment with factor VIII and secrete anti-factor VIII antibodies with properties similar to those of natural anti-factor VIII antibodies present in healthy individuals; other inhibitors are produced by B cell clones that have undergone affinity maturation and hypermutation of the V regions of the antibodies they produce. The implications for the treatment of patients with anti-factor VIII inhibitors are discussed.

Antibodies, Anti-Idiotypic↗
PubMed2000

Humanized severe combined immunodeficient mice as a potential model for the study of tolerance to factor VIII.

A Severe Combined Immunodeficient (SCID) mouse model has been established to evaluate experimental conditions leading to the production of factor VIII (FVIII) autoantibodies. To this end, we humanized 10 groups of 7 mice with peripheral blood mononuclear cells of 10 unrelated healthy blood donors (15 x 10(6) cells/mouse). Mice were injected with saline or immunized i.p. with 50 IU of a plasma derived human FVIII 24 h after reconstitution. Further immunization was made with 25 IU of FVIII every fortnight during 6 weeks and animals were sacrificed after 8 weeks. All reconstituted mice showed a spontaneous production of anti-FVIII antibodies in the absence of immunization with the corresponding antigen. However, no differences were observed regarding the quantity or the quality of these antibodies produced in the immunized or the saline group, indicating that tolerance to FVIII had been transferred with cell reconstitution. Affinity purified FVIII specific antibodies were capable of inhibiting FVIII activity and preventing the binding of FVIII to phospholipids in a dose-dependent manner. Immunoprecipitation experiments showed that the antibodies recognized only the C1 and C2 light chain domains. Since antibodies of interest can be found in the SCID mouse model and, moreover, since they are qualitatively comparable with the source donor's antibodies, this model provides a tool to study the regulation of tolerance against self antigens in normal subjects and in acquired haemophilia patients.

Animals↗
PubMed2000

Hemophilia factor VIII therapy. B- and T-cell tolerance: from basic concepts to clinical practice.

Recent advances in the understanding of the mechanisms by which tolerance develops in both B- and T- cells have led to a re-appraisal of possible methods which could be applicable in clinical settings such as hemophilia patients with inhibitors. Thus, it is now clear that a number of mechanisms operate in such a way as to actively induce anergy of B- or T- cells, or even to specifically delete cells in the periphery. The characteristics inherent to the B- and T- cell repertoires and peripheral pools play a major role in the efficiency and maintenance of tolerance induction. The purpose of this manuscript is to review the mechanisms of tolerance induction briefly so as to provide a framework for the understanding of current and future attempts to achieve Factor VIII unresponsiveness in hemophilia A patients.

B-Lymphocytes↗
PubMed1999

Antifactor VIII antibody inhibiting allogeneic but not autologous factor VIII in patients with mild hemophilia A.

Two unrelated patients with the same Arg2150His mutation in the factor VIII (FVIII) C1 domain, a residual FVIII activity of 0.09 IU/mL, and inhibitor titres of 300 and 6 Bethesda Units, respectively, were studied. Further analysis of patient LE, with the highest inhibitor titer, showed that (1) plasma or polyclonal IgG antibodies prepared from LE plasma inhibited the activity of allogeneic (wild-type) but not of self FVIII; (2) the presence of von Willebrand factor (vWF) increased by over 10-fold the inhibitory activity on wild-type FVIII; (3) the kinetics of FVIII inhibition followed a type II pattern, but in contrast to previously described type II inhibitors, LE IgG was potentiated by the presence of vWF instead of being in competition with it; (4) polyclonal LE IgG recognized the FVIII light chain in enzyme-linked immunosorbent assay and the recombinant A3-C1 domains in an immunoprecipitation assay, indicating that at least part of LE antibodies reacted with the FVIII domain encompassing the mutation site; and (5) LE IgG inhibited FVIII activity by decreasing the rate of FVIIIa release from vWF, but LE IgG recognized an epitope distinct from ESH8, a murine monoclonal antibody exhibiting the same property. We conclude that the present inhibitors are unique in that they clearly distinguish wild-type from self, mutated FVIII. The inhibition of wild-type FVIII by LE antibody is enhanced by vWF and is associated with an antibody-dependent reduced rate of FVIIIa release from vWF.

Amino Acid Substitution↗
PubMed1999

Anti-idiotypic antibodies: from regulation to therapy of factor VIII inhibitors.

Evidence has recently accumulated showing that anti-idiotypic antibodies specific to anti-FVIII antibodies are present in the plasma of healthy individuals and of haemophilia A patients with or without inhibitors, where they can neutralise the FVIII inhibitory activity. Additionally, patients successfully desensitised towards FVIII have an increased production of anti-idiotypic antibodies with no significant reduction in anti-FVIII antibodies. We review here possible strategies for modulating the anti-FVIII immune response by idiotypic interactions.

Antibodies, Anti-Idiotypic↗
PubMed1999

Some factor VIII (FVIII) inhibitors recognise a FVIII epitope(s) that is present only on FVIII-vWF complexes.

A mild haemophilia A patient (LE) with an Arg2150His mutation in the C1 domain of the factor VIII (FVIII) light chain was shown to have anti-FVIII antibodies inhibiting wild type but not self FVIII. Polyclonal anti-FVIII antibodies of this patient were purified by affinity adsorption using recombinant FVIII (rFVIII) and/or plasma-derived FVIII-von Willebrand factor (vWF) complexes. A distinct population of antibodies was obtained that bound to FVIII-vWF complexes but not to rFVIII, indicating that an epitope was created by the association of FVIII to vWF. Such antibodies belonged to the IgG2 isotype, but the FVIII epitopes to which they bind could not be mapped with precision due to vWF dependency. Depletion experiments showed that anti-FVIII antibodies recognising FVIII-vWF complex also distinguished wildtype from mutated self FVIII, indicating that the Arg2150His mutation alters the B cell epitope formed by the association of FVIII to vWF. To determine whether the Arg2150His substitution also alters the formation of the FVIII-vWF complex, the interaction between mutated or normal FVIII with vWF was evaluated in plasma. The dissociation rate of mutated FVIII from vWF was found to be significantly increased. The presence of an Arg2150His mutation therefore results in the disappearance of a FVIII B cell epitope generated by the association of FVIII with vWF. Patients carrying such an Arg2150His mutation and receiving infusion of wild-type FVIII may therefore be at risk of developing inhibitors to allogeneic FVIII only.

Antibodies↗
PubMed1998

Mechanism and kinetics of factor VIII inactivation: study with an IgG4 monoclonal antibody derived from a hemophilia A patient with inhibitor.

The development of an immune response towards factor VIII (fVIII) remains a major complication for hemophilia A patients receiving fVIII infusions. The design of a specific therapy to restore unresponsiveness to fVIII has been hampered by the diversity of the anti-fVIII antibody. Molecular analysis of the specific immune response is therefore required. To this end, we have characterized an fVIII-specific human IgG4kappa monoclonal antibody (BO2C11) produced by a cell line derived from the memory B-cell repertoire of a hemophilia A patient with inhibitor. BO2C11 recognizes the C2 domain of fVIII and inhibits its binding to both von Willebrand factor (vWF) and phospholipids. It completely inhibits the procoagulant activity of native and activated fVIII, with a specific activity of approximately 7,000 Bethesda units/mg. vWF reduces the rate of fVIII inactivation by BO2C11. The antibody-fVIII association rate constant (kass approximately 7.4 x 10(5) M-1 s-1) is eightfold lower than that for vWF-fVIII association, whereas its dissociation rate constant (kdiss < or = 1 x 10(-5) s-1) is 100-fold lower than that for the vWF-fVIII complex, which suggests that BO2C11 almost irreversibly neutralizes fVIII after its dissociation from vWF. BO2C11 is the first human monoclonal anti-fVIII IgG antibody that has been isolated and allows the study of fVIII inactivation at the molecular level.

Antibodies, Monoclonal↗
PubMed1998

Factor VIII immunogenicity.

The immunogenicity of factor VIII depends on the interaction of multiple parametres including host susceptibility and characteristics of the factor VIII preparations. We briefly review here the basic mechanisms by which tolerance to self is established, maintained, and possibly broken in the context of haemophilia A, with special emphasis on the severity of the disease. Reference is also made to the situation observed in healthy individuals and patients with autoantibodies to factor VIII.

Antibody Formation↗
PubMed1997

Epitope mapping: a new method for biological evaluation and immunotoxicology.

The extraordinary capacity of the immune system to recognise and distinguish between molecules that are almost identical can be exploited to generate reagents useful for the identification of specific determinants on drugs, blood-derived products, hormones, receptors or micro-organisms. This manuscript provides a rationale for the use of epitope mapping and a brief outline of its multiple applications.

Animals↗
PubMed1997

Heating lyophilised factor VIII does not alter its recognition by specific antibodies.

BACKGROUND AND OBJECTIVES: Alterations of factor VIII (FVIII) during preparation procedures can potentially affect its immunogenicity. One method evaluating such alterations could be by determining the reactivity of FVIII with specific antibodies. MATERIALS AND METHODS: Since heat treatment is currently used to reduce the risk of viral transmission, we evaluated the immunoreactivity of plasma-derived FVIII before and after heating at different temperatures and for different periods. Freeze-dried FVIII was used for these experiments as part of the validation procedure of a novel FVIII preparation. RESULTS: Heating FVIII for up to 72 h at 80 degrees C does not alter its reactivity with specific rabbit antibodies or mouse monoclonal antibodies, although some loss of FVIII activity occurred after 72 h. After heating for 2 h at 100 degrees C, a procedure that reduced FVIII activity by about 50%, there were still no significant effects on FVIII reactivity with monoclonal antibodies. CONCLUSIONS: Freeze-dried preparations of plasma-derived FVIII seem to be resistant to heat-induced structural denaturation.

Animals↗