Abstract 1211
The development of neutralizing anti-Factor VIII (FVIII) antibodies is the major complication of the treatment of patients with hemophilia A (HA). Several mechanisms of inhibition have ...been described: steric hindrance, immune complex formation and catalytic antibodies. Anti-FVIII catalytic antibodies act like enzymes and lead to the hydrolysis of FVIII. Lacroix-Desmazes et al. had shown the presence of catalytic antibodies in the plasma of patients with severe HA who had developed inhibitors (13/24) (Lacroix-Desmazes et al, NEJM, 2002). Previous studies on catalytic antibodies reported results for patients with severe HA at one time point, exclusively. Thus, we proposed to extend the analysis of catalytic antibodies to patients with a mild or moderate HA and to follow over their lifetime patients who develop inhibitor.
We studied plasma samples from 33 patients with HA. Sixteen were patients with severe HA, including 8 patients with inhibitor (Inh+) and 8 patients without inhibitor (Inh-), and 17 were mild or moderate HA patients (7 Inh+ and 10 Inh-). Among Inh+ patients, 6 were treated on-demand (3 severe and 3 moderate HA patients) and 9 were submitted to an immune tolerance induction (ITI) protocol (4 severe and 5 mild or moderate patients with HA). A therapeutic preparation of pooled normal IgG (IVIg) from healthy donors was used as a source of normal IgG. We also used plasma from 13 male healthy donors. As described previously 2, IgG were purified from plasma by affinity-chromatography on protein G followed by a size-exclusion chromatography in presence of urea. Then, catalytic activity was evaluated by the hydrolysis of FVIII after incubation with purified IgG (Lacroix-Desmazes et al, Nature, 1999). Inhibitor titer is measured by modified Bethesda test.
Mean FVIII-hydrolyzing rates were determined for healthy donors, HA patients and IVIg, used as control. Catalytic activity of HA patients IgG was significantly higher than those of healthy donors or IVIg (p<0.01 and p<0.001, respectively). Sixty four per cent of patients with HA had catalytic antibodies, regardless the phenotype nor inhibitor presence. In addition, prevalence of FVIII-hydrolyzing antibodies for Inh+ and Inh- HA patients was 94% and 47%, respectively. However, the mean FVIII-hydrolysis rate was comparable for both groups (0.23 ± 0.06 mmol/min/mol). Surprisingly, we showed that the mean catalytic activity of mild or moderate HA patients were significantly lower than those of severe HA patients (0.17 ± 0.05 versus 0.31 ± 0.07). Interestingly, we were able to study the evolution of both catalytic and inhibitory activities for patients who developed inhibitor. We observed 2 profiles: in the first case, FVIII-hydrolysis rate and inhibitor titer followed the same trend, but in the second case, these two parameters showed a dissociated evolution. These results were independent of the type of treatment (on-demand or ITI).
For the first time, we studied catalytic activity for patients with mild or moderate HA. In comparison with patients with severe HA, catalytic activity is much lower for patients with mild or moderate HA. In addition, most of patients with severe HA had FVIII-hydrolytic antibodies (88 %), in contrast with previous studies (50%) (Lacroix-Desmazes et al, NEJM, 2002). In the same way, we showed the presence of catalytic antibodies in patients without inhibitor. Moreover, we studied the evolution of catalytic activity and inhibitor titer over the time for patients with inhibitor and showed that catalytic activity did not necessarily follow the same trend as inhibitory activity. The results suggested that catalytic antibodies could not act like neutralizing antibodies.
No relevant conflicts of interest to declare.
Abstract 38
The occurrence of inhibitory anti-factor VIII (FVIII) antibodies is the major complication of replacement therapy in patients with hemophilia A. Heme oxygenase-1 (HO-1) is a stress ...inducible enzyme with anti-inflammatory activity. Induction of HO-1 in hemophilic mice reduces the immunogenicity of therapeutic FVIII. Interestingly, polymorphisms in the promoter of the HO-1-encoding gene (HMOX1) modulate the expression of HO-1.
We investigated the relationship between polymorphisms in the promoter of HMOX1 in severe hemophilia A patients and the development of FVIII inhibitors.
We analyzed 362 patients with severe hemophilia A involving 99 patients with FVIII inhibitors and 263 patients who did not develop inhibitor within the first 150 cumulative exposure days to therapeutic FVIII. Direct sequencing and DNA fragment analysis were used to study the variable (GT)n polymorphism and single nucleotide polymorphisms located at −1135 and −413 in the promoter of HMOX1. We assessed associations between the individual allele frequencies and genotypes, and the development of inhibitors.
Our results demonstrate a higher frequency of alleles with large (GT)n repeat (n≥30, L, associated with a lesser expression of HO-1) in inhibitor-positive patients odds ratio (OR) 2.31; 95% CI 1.46–3.66, p<0.001. Six genotypes (L/L, L/M, L/S, M/M, M/S and S/S) of (GT)n repeats were identified (S: n<21; M: 21≤n<30). The genotype group including L alleles (L/L, L/M and L/S) was statistically more frequent among inhibitor-positive than inhibitor-negative patients, as compared to the other genotypes (33.3% vs 17.1%) OR 2.21, 95% CI 1.30–3.76, p<0.01.
To our knowledge, this is the first association between HMOX1 promoter polymorphism and development of anti-drug antibodies. Modulating the endogenous anti-inflammatory machinery of hemophilia A patients appears as a plausible therapeutic option for reducing the risk of inhibitor development.
No relevant conflicts of interest to declare.
Inhibitors of Factor VIII in Hemophilia Lacroix-Desmazes, Sebastien; Dimitrov, Jordan D; Repesse, Yohann
The New England journal of medicine,
07/2009, Letnik:
361, Številka:
3
Journal Article
Recenzirano
To the Editor:
Viel et al. (April 16 issue)
1
propose that treatment of hemophilia A with factor VIII that is matched for polymorphic variants may reduce the risk of the development of factor VIII ...inhibitors. Their study included patients with different types of causative mutations and disease severity. We believe that stratification of patients according to the presence or absence of factor VIII protein, whether it is functional or not, may be more suitable than evaluation of mismatched factor VIII as a risk factor for the development of inhibitors.
Mismatched replacement therapy appears to be a risk factor in patients . . .
The role of Von Willebrand Factor (VWF) as a chaperone molecule for procoagulant factor VIII (FVIII) has been extensively documented. Under physiological conditions, VWF binds to FVIII after its ...release in the circulation. VWF protects FVIII from proteolysis by lipid−bound proteases, stabilizes FVIII and further regulates its elimination by lipoprotein receptors. We investigated whether VWF might modulate FVIII endocytosis by professionnal antigen presenting cells (APCs). Immature dendritic cells (DCs), utilised as model of APCs, were generated from circulating monocytes of healthy blood donors and incubated with FITC−labeled FVIII (FVIII−FITC). T cell assays were performed by co−culturing the human FVIII−specific CD4+ T cell clone, D9E9, with immature DCs from MHC−matched donors in presence of unconjugated FVIII and increasing concentrations of VWF (1:1 to 1:130). Preincubation of FVIII−FITC with VWF at 1 to 130−fold molar excess resulted in a dose−dependent inhibition of FVIII endocytosis (12 ± 2% to 94 ± 18%, respectively). Human serum albumin used in equivalent molar ratios did not prevent FVIII internalization. In contrast, the protective effect of VWF on FVIII uptake was significantly restored by co−incubation of FVIII and VWF in the presence of F(ab')2 fragments of BO2C11, an anti−FVIII IgG, and Ac418, an anti−VWF IgG, that both disrupt the interaction between FVIII and VWF. In addition, blocking DC entry of FVIII by VWF resulted in a dose−dependent reduction of the activation of D9E9 (up to 75%). Interestingly, D9E9 activation by DCs loaded with a synthetic FVIII−derived peptide, I2144–T2161, was not altered in the presence of equivalent concentrations of VWF, indicating that VWF does not have a direct inhibitory effect on T cell activation. The administration of exogenous FVIII to patients with hemophilia A, results in up to 30% of the cases, in the developement of FVIII−specific antibodies that inhibit FVIII procoagulant activity. In vivo experimental evidences and clinical observations have documented that the presence of VWF in FVIII therapeutic concentrates may be associated with a lower incidence of FVIII inhibitors. Here, we demonstrate that VWF may reduce the immunogenicity of FVIII by preventing its internalization by professionnal APCs, and reducing the subsequent presentation of FVIII−derived peptides to T lymphocytes.
Replacement therapy in severe hemophilia A leads to factor VIII (FVIII) inhibitors in 30% of patients. Factor VIII gene (F8) mutation type, a family history of inhibitors, ethnicity and intensity of ...treatment are established risk factors, and were included in two published prediction tools based on regression models. Recently investigated immune regulatory genes could also play a part in immunogenicity. Our objective is to identify bio-clinical and genetic markers for FVIII inhibitor development, taking into account potential genetic high order interactions. The study population consisted of 593 and 79 patients with hemophilia A from centers in Bonn and Frankfurt respectively. Data was collected in the European ABIRISK tranSMART database. A subset of 125 severely affected patients from Bonn with reliable information on first treatment was selected as eligible for risk stratification using a hybrid tree-based regression model (GPLTR). In the eligible subset, 58 (46%) patients developed FVIII inhibitors. Among them, 49 (84%) were "high risk" F8 mutation type. 19 (33%) had a family history of inhibitors. The GPLTR model, taking into account F8 mutation risk, family history of inhibitors and product type, distinguishes two groups of patients: a high-risk group for immunogenicity, including patients with positive HLA-DRB1*15 and genotype G/A and A/A for IL-10 rs1800896, and a low-risk group of patients with negative HLA-DRB1*15 / HLA-DQB1*02 and T/T or G/T for CD86 rs2681401. We show associations between genetic factors and the occurrence of FVIII inhibitor development in severe hemophilia A patients taking into account for high-order interactions using a generalized partially linear tree-based approach.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Etomidate and ketamine are used during induction of anesthesia in high-risk patients. However, their effects on action potential (AP) variables and ischemia/reperfusion-induced arrhythmias and ...conduction blocks are unknown.
Guinea pig right ventricular muscle strips were mounted in a 5-mL double chamber bath with the strips separated into two zones by an impermeable latex membrane. One-half (normal zone) was exposed to normal perfusate while the other half (altered zone) was exposed to hypoxia, hyperkalemia, acidosis, and lack of glucose. AP variables were recorded continuously in the normal and altered zones. Spontaneous arrhythmias and conduction blocks were noted. Etomidate (10(-7), 10(-6), and 10(-5) M) and ketamine (10(-6), 10(-5), and 10(-4) M) were superfused into the bath throughout the experiment and the electrophysiologic effects compared with the control group.
We found that under control conditions, etomidate and ketamine did not modify resting membrane potential, maximal upstroke velocity, AP amplitude, or AP duration at 90% of repolarization (APD90). Ketamine (10(-4) M), but not weaker concentrations and none of the concentration of etomidate, reversed the ischemia-induced shortening of APD90 and APD dispersion. Etomidate and ketamine did not modify the occurrence of conduction block during simulated ischemia. In contrast, ketamine (25% at 10(-6) M, 13% at 10(-5) M, and 13% at 10(-4) M vs 90% in the control group, P < 0.05) but not etomidate (38% at 10(-7) M, 63% at 10(-6) M, and 63% at 10(-5) M vs 90% in the control group, NS) decreased the incidence of reperfusion-induced spontaneous arrhythmias.
In guinea pig myocardium, our data suggest that ketamine, in clinically relevant concentrations, decreases ischemia-induced AP shortening and spontaneous reperfusion-induced ventricular arrhythmias. Further study is required to precisely determine the effect of etomidate on reperfusion-induced arrhythmias.
Summary
Background: As the publication of the sequence of the factor VIII gene (FVIII) in 1984, a large number of mutations that cause hemophilia A (HA) have been identified. Thanks to the advances ...in the detection of mutations, it is now possible to identify a putative FVIII sequence alteration in the vast majority of patients with HA.Objectives: Our main objective was to report on the spectrum of FVIII mutations and their distribution throughout the gene in 120 patients with HA.Methods: Screening of FVIII mutations was performed using direct sequencing. Newly described missense mutations were further studied by molecular modeling.Results: A total of 47 different HA causative FVIII mutations have been identified, 26 of which are described for the first time. These novel mutations include 14 missense and six nonsense mutations, two small deletions, one large deletion and three splice‐site mutations. We further investigated the development of FVIII‐specific inhibitors in all patients with HA. We found that four novel mutations (Ser882X, Tyr1786Ser, Ala2218Thr and a splice‐site defect in intron 22) were associated with inhibitor development.Conclusion: These data extend our insight into the mechanisms by which novel amino acid substitutions may lead to HA, and how HA patient genotypes influence the risk of FVIII inhibitor development.
We report the case of a 64-year-old Jarvik 2000 recipient with a high risk of bleeding (anticoagulation treatment and acquired von Willebrand disease), who presented with intractable gastrointestinal ...hemorrhage due to severe gastric angiodysplasia. He was successfully treated with long-acting octreotide.
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