Biomedical subjects

D Scandella

Publications and source records attributed to D Scandella.

At least 19 recordsLinked to original sources

PubMed2001

Long-term induction of immune tolerance after blockade of CD40-CD40L interaction in a mouse model of hemophilia A.

A factor VIII-deficient knockout mouse was used as a model for severe hemophilia A to characterize the immune response to recombinant human factor VIII (fVIII) and to study new approaches for induction of immune tolerance to fVIII. Mice initially received periodic injections of fVIII in doses similar to those used for the treatment of human hemophilia A. To induce immune tolerance, a hamster monoclonal antibody specific for murine CD40 ligand (CD40L or CD154) was injected with fVIII. Control mice received fVIII alone or fVIII and hamster immunoglobulin G. After treatment, humoral and cellular immune responses were evaluated. Ninety-five percent of anti-CD40L-treated mice had lower titers of anti-fVIII antibody (less than 1 microg/mL) compared with fVIII-injected control mice (mean, 18 microg/mL). To determine whether anti-CD40L treatment induces long-term immune tolerance, mice were rechallenged 3 times with fVIII alone. At 150 days after treatment, 12 of 22 anti-CD40L-treated mice remained tolerant to fVIII (anti-fVIII antibody titers less than 1 microg/mL). However, tolerant mice immunized with tetanus toxoid (TT) developed high anti-TT antibody, demonstrating that tolerance is fVIII specific. T cells from tolerant mice showed impaired proliferative responses after stimulation with fVIII in vitro and lack of production of the cytokines interleukin-2 (IL-2), IL-4, interferon gamma, and IL-10. These results demonstrate that long-term immune tolerance to fVIII was effectively induced after early blockade of CD40-CD40L interaction. In addition, the lack of tolerance in this model was associated with the expression of a Th2 phenotype.

Animals↗

PubMed2001

Detection of all anti-factor VIII antibodies in haemophilia A patients by the Bethesda assay and a more sensitive immunoprecipitation assay.

Plasmas from 40 haemophilia A patients enrolled in a study by the paediatric group of the German Society on Thrombosis and Hemostasis were tested by the Bethesda assay for inhibitor antibodies and by a more sensitive immunoprecipitation assay (IP) for all antifactor VIII antibodies. Of the 26 severe, 11 moderate and three mild haemophiliacs, 18, two, and none, respectively, had positive Bethesda titres after several factor VIII infusions. In 275 plasmas with Bethesda titres of 0, 0.6--1.0, > 1--5, and > 5--655, the IP responses were 0-238, 0--61, 0--786, and 43--6141, respectively, and a reliable positive IP titre was > 4.2. The overlapping ranges of IP titres indicated large differences in the ratio of inhibitory to noninhibitory antibodies in individual plasmas. In five of seven patients with Bethesda titres of 0.6--1, the IP titres were < 4.2, suggesting a lack of precision of Bethesda titres < or = 1. Detection of the primary immune response was found in only three patients by IP assay before a positive Bethesda assay. This precludes early, reliable testing of which patients will be immunologically responsive. In four patients undergoing immune tolerance therapy, antifactor VIII antibodies were still detectable by the IP assay in the absence of a Bethesda titre, which indicates that antibodies were completely eradicated in none of the patients. Our results show that the use of both the Bethesda and IP assays can provide more accurate detection of antifactor VIII antibodies in all patients.

Antibodies↗

PubMed2001

Low factor VIII recovery in haemophilia A patients without inhibitor titre is not due to the presence of anti-factor VIII antibodies undetectable by the Bethesda assay.

Measurements of factor VIII (FVIII) recovery in previously untreated patients with haemophilia A were done as part of the clinical trial of safety and efficacy of the recombinant FVIII, Recombinate. In 22 of 72 assessable patients, positive inhibitor titres > or = 0.6 Bethesda units mL-1 were detected by the Bethesda assay in one or more plasma samples, and the remaining 50 patients were negative at all timepoints. Of the latter group, 16 individuals without inhibitors unexpectedly had both normal (111) and low (52) recoveries during the study. We investigated the possibility that other antibodies not detectable in the Bethesda assay were responsible for the low recovery, by using a highly sensitive immunopreciptation (IP) assay for detection of all antiFVIII antibodies. Eight of the 16 patients with low and normal recoveries did indeed have antibodies detected by the IP assay, and the remaining eight were negative. Four antibody-positive individuals had insignificantly low titres, and the other four had modest to high titres. In the latter group, antibodies were found with similar frequencies and titre in plasmas from patients with low or normal recovery. Low recovery in haemophilia A patients without inhibitor titres must therefore be attributed to factors other than antiFVIII.

Antibodies↗

PubMed2000

Antibodies to the FVIII light chain that neutralize FVIII procoagulant activity are present in plasma of nonresponder patients with severe hemophilia A and in normal polyclonal human IgG.

We have analyzed the properties of anti-factor VIII (FVIII) immunoglobulin (Ig) G recovered by affinity chromatography on FVIII-Sepharose from the IgG fraction of the plasma of healthy individuals and nonresponder patients with hemophilia A. Affinity-purified anti-FVIII antibodies were found to neutralize FVIII activity and to bind to FVIII with an affinity similar to that of anti-FVIII IgG that had been affinity-purified from the plasma of inhibitor-positive hemophilia patients and of patients with anti-FVIII autoimmune disease. The antibodies also exhibited patterns of reactivity with thrombin-digested FVIII similar to those of FVIII inhibitors and preferentially recognized epitopes located in the light chain of FVIII. These observations suggest that FVIII inhibitors occurring in hemophilia A and in patients with anti-FVIII autoimmune disease originate from the expansion of preexisting natural anti-FVIII clones that exhibit FVIII-neutralizing properties.

Antibodies↗

PubMed2000

Reduction of the antigenicity of factor VIII toward complex inhibitory antibody plasmas using multiply-substituted hybrid human/porcine factor VIII molecules.

Factor VIII (fVIII) circulates as a heavy chain/light chain (A1-A2-B/ap-A3-C1-C2) heterodimer. The 41-residue light chain activation peptide, ap, is cleaved from fVIII during proteolytic activation by thrombin or factor Xa. We constructed 7 active recombinant hybrid B-domainless human/porcine fVIII molecules that contained combinations of porcine sequence replacements within the A2, ap-A3, and C2 domains. The cross-reactivity of 23 high-titer inhibitory antibodies between human fVIII and the hybrids was inversely related to the degree of porcine substitution. In all plasmas, the substitution of all 3 regions yielded cross-reactivities that were not significantly different from those of porcine fVIII. To differentiate between inhibitor binding to the ap region and the A3 domain, we constructed 2 additional hybrids that contained porcine A2 and C2 domain substitutions and either porcine A3 or porcine ap substitutions. The porcine ap segment was less antigenic than the human ap segment in several plasmas that had activity against the ap-A3 region. This indicates that some inhibitor plasmas contain antibodies directed against the fVIII ap segment in addition to A2, A3, and C2 domain epitopes identified in previous studies. Substitution of porcine sequences within the A2, A3, C2, and ap regions of human fVIII is necessary and sufficient to achieve a maximal reduction in antigenicity relative to porcine fVIII with respect to most inhibitory antibody plasmas. (Blood. 2000;95:564-568)

Animals↗

PubMed2000

An alloantibody recognizing the FVIII A1 domain in a patient with CRM reduced haemophilia A due to deletion of a large portion of the A1 domain DNA sequence.

We report the development of a FVIII inhibitor in a patient with severe, cross reacting material reduced (CRM(R)) haemophilia A. The level of Factor VIII antigen (FVIII:Ag) measured by ELISA using anti-C2 monoclonal and alloantibodies was 1.9 U/dl. This baseline FVIII:Ag level was increased to 8.3 U/dl after administration of DDAVP. The anti-FVIII inhibitor titer was 2.9 Bethesda U/ml. DNA analysis showed a large deletion of the FVIII gene from exon 4 to 7, corresponding to amino acid residues 111-317 included within the A1 domain. The size of the gene deletion was approximately 28 kb. 5' and 3' breakpoints were identified by sequencing in intron 3 and intron 7, respectively. FVIII mRNA was detected in the patient's peripheral lymphocytes and the deletion spanning exon 4 to 7 was confirmed at the RNA level. Immunoprecipitation experiments using 125I labeled A1, A2 and light chain demonstrated that the inhibitor reacted only with the 54 kDa A1 domain. The inhibitor activity was more than 95% neutralized by A1 domain polypeptide. Our findings suggest a close relationship between the inhibitor epitope and the specific gene deletion with regard to the pathogenesis of the inhibitor in this patient.

Adult↗

PubMed2000

Characterization of antibodies to factor VIII in hemophilia A patients treated by immune tolerance therapy.

The treatment of hemophilia A patients has improved in this decade by production of recombinant factor VIII (FVIII) in mammalian cells, which has significantly reduced contamination by infectious agents. A remaining serious problem in 25-30% of patients with severe hemophilia A is the appearance of antibodies that inactivate FVIII. The present therapy for this condition is frequent treatment with high doses of FVIII to induce tolerance, which is defined as a negative Bethesda assay. Serial plasma samples from 50 evaluable patients in a large study of 72 previously untreated patients were tested to determine whether tolerized patients have actually lost all their anti-FVIII by using a 10 fold more sensitive immunoprecipitation (IP) method of measuring all anti-FVIII antibodies. Six of the 22 patients with inhibitors were given tolerance therapy, and 3 of them were only partially tolerized as determined by IP assay. Seven patients with 1-11 BU/mL lost their inhibitor spontaneously, while 5 non-inhibitor patients with low level immune responses similarly became antibody negative. In a smaller study, a tolerized patient with 0 BU/mL had remaining non-inhibitory antibody levels high enough to reduce the FVIII half-life significantly. Plasmas from 2 patients who were not tolerized, were tested by the IP assay for the A2 and/or C2 domain specificity of the anti-FVIII over time. The antibodies detected were directed against both the heavy and light chains of FVIII, and they increased and decreased at the same rate before and during tolerance therapy.

Antibody Specificity↗

PubMed1999

Role of factor VIII C2 domain in factor VIII binding to factor Xa.

Factor VIII (FVIII) is activated by proteolytic cleavages with thrombin and factor Xa (FXa) in the intrinsic blood coagulation pathway. The anti-C2 monoclonal antibody ESH8, which recognizes residues 2248-2285 and does not inhibit FVIII binding to von Willebrand factor or phospholipid, inhibited FVIII activation by FXa in a clotting assay. Furthermore, analysis by SDS-polyacrylamide gel electrophoresis showed that ESH8 inhibited FXa cleavage in the presence or absence of phospholipid. The light chain (LCh) fragments (both 80 and 72 kDa) and the recombinant C2 domain dose-dependently bound to immobilized anhydro-FXa, a catalytically inactive derivative of FXa in which dehydroalanine replaces the active-site serine. The affinity (K(d)) values for the 80- and 72-kDa LCh fragments and the C2 domain were 55, 51, and 560 nM, respectively. The heavy chain of FVIII did not bind to anhydro-FXa. Similarly, competitive assays using overlapping synthetic peptides corresponding to ESH8 epitopes (residues 2248-2285) demonstrated that a peptide designated EP-2 (residues 2253-2270; TSMYVKEFLISSSQDGHQ) inhibited the binding of the C2 domain or the 72-kDa LCh to anhydro-FXa by more than 95 and 84%, respectively. Our results provide the first evidence for a direct role of the C2 domain in the association between FVIII and FXa.

Amino Acid Sequence↗

PubMed1999

Human inhibitor antibodies specific for the factor VIII A2 domain disrupt the interaction between the subunit and factor IXa.

Factor VIIIa, a heterotrimer of the A1, A2, and A3-C1-C2 subunits, increases the catalytic efficiency for factor IXa-catalyzed activation of factor X. A significant fraction of naturally occurring, anti-factor VIII inhibitor antibodies reacts with the A2 domain. Utilizing the capacity for isolated A2 subunit to stimulate factor IXa activity, we show that a panel of these inhibitors block this activity. Inhibition of activity parallels the antibody potency as measured in the Bethesda assay. These antibodies also block the A2-dependent increases in fluorescence anisotropy of fluorescein-Phe-Phe-Arg factor IXa. Similar to the IgG fractions, a peptide representing the sequence of the inhibitor epitope (A2 residues 484-509) blocked the A2-dependent stimulation of factor IXa. These results indicate that antibodies possessing this specificity directly inhibit the interaction of A2 subunit with factor IXa, thus abrogating the contribution of this subunit to cofactor activity. Furthermore, these results also suggest that factor VIII residues 484-509 contribute to a factor IXa-interactive site.

Antibodies, Monoclonal↗

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

Identification of a factor VIII peptide, residues 2315-2330, which neutralizes human factor VIII C2 inhibitor alloantibodies: requirement of Cys2326 and Glu2327 for maximum effect.

Factor VIII (FVIII) inhibitor alloantibodies react with combinations of the A2, C2 and A3-C1 domains of the FVIII molecule. Some inhibitors block binding of FVIII to both von Willebrand factor (VWF) and phospholipid, and recognize a C2 domain epitope which overlaps both binding sites. In order to determine the essential binding regions for alloantibodies inhibitory for FVIII activity, we have performed inhibitor neutralization assays and competitive inhibition assays using 10 overlapping synthetic peptides spanning the carboxy-terminal region of the C2 domain (residues 2288-2332). We found one peptide (2315-2330, L9) which neutralized the anti-FVIII activity of four out of five different C2 alloantibodies by 50%, 39%, 47% and 57%, respectively. Neutralization of these alloantibodies by recombinant C2 domain (residues 2173-2332) was 68%, 50%, 59%, 86% and >95%, respectively. The inhibitor which was not neutralized by L9 peptide and reacted by immunoblotting with peptide 2218-2307, did not prevent binding of FVIII to VWF and only partially inhibited binding of FVIII to phosphatidylserine. Mutants of the L9 peptide were prepared in which each residue from 2315-2330 was sequentially substituted by glycine. Inhibitor neutralization experiments using these peptides demonstrated that Arg2320 and Cys2326 or Glu2327 are important for the effect of L9 peptide, since their substitution by glycine reduced its neutralizing effect by 60% to >90%, suggesting that they are crucial for formation of the one of the C2 inhibitor epitopes.

Amino Acid Sequence↗

PubMed1999

Epitope specificity and inactivation mechanisms of factor VIII inhibitor antibodies.

The domain specificity of anti-factor VIII (FVIII) inhibitor antibodies was determined in assays using FVIII domains generated by thrombin cleavage or expressed as recombinant polypeptides to neutralise the inhibitor. The results revealed the existence of three major types of inhibitors, and various combinations of these antibodies were found in haemophilic and autoantibody patients. Anti-A2 domain inhibitors prevent normal function of the FVIII/factor IXa (FIXa)/phospholipid complex in an unknown manner. Binding of FVIII to phospholipid and to von Willebrand factor is blocked by anti-C2 domain antibodies, and the binding of FVIII to FIXa is prevented by anti-A3 domain antibodies. A rare type of inhibitor prevents release of activated FVIII from von Willebrand factor (vWf), and another probably interferes with FVIII binding to factor X (FX) because it shares the epitope of a monoclonal antibody with this property.

Binding Sites↗

PubMed1999

Inhibitors in German hemophilia A patients treated with a double virus inactivated factor VIII concentrate bind to the C2 domain of FVIII light chain.

To reduce the risk of transmission of hepatitis A virus, an Octapharma produced factor VIII (fVIII) concentrate treated with solvent detergent (FVIII-SD) was further pasteurized after purification. This product, Octavi SDPlus (FVIII-SDP), was marketed in Europe in 1993 to 1995. Inhibitors appeared from September to October, 1995, in 12 of 109 previously treated German hemophilia A patients. A study of similarly treated Belgian patients, who also developed inhibitors, had shown antibodies to the fVIII light chain (domains A3-C1-C2) only. In the present study, the epitope specificity of 8 German inhibitor plasmas was also found to be restricted to the light chain. In radioimmunoprecipitation assays to localize the light chain epitope(s), antibody binding to heavy chain (domains A1-A2-B) was 11-148 fold lower than to the C2 domain, and binding to recombinant A3-C1 was barely detectable. These results were supported by >95% neutralization of a high responder inhibitor titer by the C2 domain.

Adolescent↗

PubMed1998

Residues Glu2181-Val2243 contain a major determinant of the inhibitory epitope in the C2 domain of human factor VIII.

The human blood coagulation factor VIII C2 domain (Ser2173-Tyr2332) contains an epitope recognized by most polyclonal inhibitory anti-factor VIII alloantibodies and autoantibodies. We took advantage of the differential reactivity of inhibitory antibodies with human and porcine factor VIII and mapped a major determinant of the C2 epitope by using a series of active recombinant hybrid human/porcine factor VIII molecules. A series of five C2-specific human antibodies and a murine anti-factor VIII monoclonal antibody, NMC-VIII/5, inhibited a hybrid containing a substitution of porcine sequence for Glu2181-Val2243 significantly less than human factor VIII. In contrast, four of the five patient antibodies and NMC-VIII/5 inhibited a hybrid containing a substitution of porcine sequence for Thr2253-Tyr2332 equally well as human factor VIII. Thus, a major factor VIII inhibitor epitope determinant is bounded by Glu2181-Val2243 at the NH2-terminal end of the C2 domain. Because C2 inhibitors block the binding of factor VIII to phospholipid and von Willebrand factor, for which binding sites have been localized to Thr2303-Tyr2332, these results imply that the segment bounded by Glu2181-Val2243 also is involved in these macromolecular interactions.

Amino Acid Sequence↗

PubMed1998

Activation of factor VIII by thrombin increases its affinity for binding to synthetic phospholipid membranes and activated platelets.

Membrane-bound thrombin-activated factor VIII (fVIIIa) functions as a cofactor for factor IXa in the factor Xase complex. We found that binding of heterotrimeric fVIIIa (A1.A2.A3-C1-C2) to synthetic vesicles with a physiologic content of 4% phosphatidylserine (PS), 76% phosphatidylcholine, and 20% phosphatidylethanolamine occurs with a 10-fold higher affinity than that of factor VIII (fVIII). The increased affinity of fVIIIa for PS-containing membranes resulted from the reduced rate of fVIIIa dissociation from the vesicles compared with that of fVIII. Similar affinities of A3-C1-C2, A1.A2. A3-C1-C2, and A3-C1-C2.heavy chain for interaction with PS-containing membranes demonstrate that removal of the light chain (LCh) acidic region by thrombin is responsible for these increased affinities of fVIIIa and its derivatives. Similar kinetic parameters of fVIII and its LCh and C2 domain for binding to PS-containing membranes and to activated platelets indicated that the C2 domain is entirely responsible for the interaction of fVIII with membranes. We conclude that the increased fVIIIa affinity for PS-containing membranes is a result of conformational change(s) within the C2 domain upon removal of the acidic region of the LCh. This conclusion is based on the finding that binding of the monoclonal antibody ESH8 to the C2 domain, which is known to prevent this conformational transition, resulted in fVIIIa binding to PS/phosphatidylcholine/phosphatidylethanolamine vesicles (4/76/20) with a lower affinity similar to that of fVIII. In addition, stabilization of the low affinity binding conformation of the C2 domain of fVIIIa by this antibody led to an inhibition of the fVIIIa activity in the factor X activation complex.

Binding Sites↗

PubMed1998

Some human inhibitor antibodies interfere with factor VIII binding to factor IX.

Factor VIII (fVIII) functions as a cofactor of factor IXa in the intrinsic pathway of blood coagulation. Its absence or abnormality causes the bleeding disorder hemophilia A. About 23% of hemophiliacs who receive therapeutic fVIII infusions develop antibodies that inhibit its activity. We previously showed by inhibitor neutralization assays that the fVIII A2 and C2 domain polypeptides contain common inhibitor epitopes. Often hemophilic inhibitor plasmas were partially neutralized by C2 and more completely neutralized by fVIII light chain (A3-C1-C2), suggesting the presence of an additional major inhibitor epitope(s) within the A3-C1 domains. In immunoprecipitation assays, 17 of 18 inhibitor IgGs bound to recombinant 35S-A3-C1. Amino acids 1811-1818 of the A3 domain comprise a binding site for factors IX and IXa. Three inhibitor IgGs prevented binding of factor IXa to fVIII light chain, and the binding of each IgG to light chain was competed by A3 peptide 1804-1819. The generation of factor Xa by the fVIIIa/fIXa complex in a chromogenic assay was prevented by these inhibitors. Therefore, we propose that another important mechanism of fVIII inactivation by human inhibitors is the prevention of fVIIIa/fIXa association.

Antibodies, Blocking↗

PubMed1998

The natural history of the immune response to exogenous factor VIII in severe haemophilia A.

The development of inhibitory antibodies to factor VIII (fVIII) in severe haemophilia A patients is a serious therapeutic complication. Using a highly sensitive immunoprecipitation (IP) assay which measures all anti-fVIII antibodies, we have tested severe haemophilic plasmas from two clinical studies. Inhibitor titres in the range of 0.4 to 1 Bethesda units/ml (BU/ml) could not be verified by IP as being due to an immune response to fVIII in 35% of plasmas tested. Low fVIII recoveries were likewise correlated with the presence of antibodies in 29% of plasmas tested. However, 16% of plasmas without inhibitor titres had immune responses as measured by IP. The rapidity of antibody appearance did not allow their effective detection by IP before development of inhibitor titres. These results suggest that the IP assay can provide a valuable confirmation of anti-fVIII antibody production when the Bethesda assay is low or negative and where clinical observations suggest their presence, but they cannot be used reliably to detect early immune responses.

Antibody Formation↗

PubMed1998

Dominant C2 domain epitope specificity of inhibitor antibodies elicited by a heat pasteurized product, factor VIII CPS-P, in previously treated hemophilia A patients without inhibitors.

From June, 1990, to November, 1991, in The Netherlands and Belgium, 16 previously treated severe hemophilia A patients (PTP) developed inhibitors after exposure to factor VIII CPS-P, a new heat pasteurized product. A previously untreated patient (PUP) also developed an inhibitor to CPS-P. In inhibitor neutralization assays with recombinant fVIII C2 and A2 domain polypeptides, plasmas from 14 PTPs were > or = 79% neutralized by C2 and < 10% by A2, but the PUP plasma was partially neutralized by C2 (48%) and A2 (28%). Immunoprecipitation assays of the PTP and PUP plasmas with the fVIII heavy chain and with recombinant C2 and A3-C1 polypeptides confirmed that the C2 dominant immune response to CPS-P was found only in the PTPs. Competition of the binding of 2 inhibitors to 125I-CPS-P by unlabeled CPS-P and another plasma fVIII was similar, demonstrating that the antibody response was not directed to epitopes only present in CPS-P. We propose that the immunogenicity of the CPS-P C2 domain was altered by heat pasteurization.

Adolescent↗