Inhibitor Formation Research Articles

Patients with Severe Factor XI Deficiency Who Have An Inhibitor or IgA Deficiency Can Undergo Uneventful Major Surgery by a Single Infusion of Low Dose Recombinant Factor VIIa and Use of Tranexamic Acid.

Inhibitors to factor XI (FXI) can develop following exposure to plasma products in 33% of patients with severe FXI deficiency who are homozygous for the type II nonsense mutation prevalent in Jews (Salomon et al. Blood 2003; 101:4783). Hemostasis in five such patients was previously achieved during surgery by infusion of recombinant factor VIIa (rFVIIa) and tranexamic acid, but two patients experienced arterial thrombosis following surgery (Schulman et al. Hemophilia 2006; 12:223). Conceivably, the rather high dose of rFVIIa used was responsible for the thrombotic events in these patients. In a previous in vitro study, we showed that low concentrations of rFVIIa can correct thrombin generation in plasma of patients with severe FXI deficiency and an inhibitor to FXI (Livnat et al. J Thromb Hemost 2006; 4:192). In the present study we addressed the question whether low doses of rFVIIa and use of tranexamic would be hemostatically effective and less thrombogenic during and after major surgery in patients with severe FXI deficiency in whom plasma derivatives could not be used. Three patients (two with FXI inhibitor and one with severe IgA deficiency) underwent major surgery managed by a single low dose infusion of rFVIIa and administration of tranexamic acid 1 G q.i.d for 7 days (Table). In patient 1 the infusion was given immediately prior to surgery and in patients 2 and 3 at the end of surgery. Hemostasis was achieved in all patients and no thrombosis occurred.No.Sex/ageFXI (U/dL)FXI inhibitor (B.U)OperationSingle dose rVIIa (uG/Kg)Adverse events1M/62< 0.015.0Laparoscopic cholecystectomy30None2M/61< 0.015.0Transuretheral prostatectomy16None3M/630.020*Parathyroidectomy15None* IgA deficiencyThese observations suggest that a single infusion of low dose rFVIIa and use of tranexamic acid can be a safe modality of treatment in patients with severe FXI deficiency who cannot receive blood products. Furthermore, the protocol outlined here may be used to avoid inhibitor formation in patients with extremely severe FXI deficiency who undergo surgery.

Mutations M184V and Y115F in HIV-1 Reverse Transcriptase Discriminate against “Nucleotide-competing Reverse Transcriptase Inhibitors”

Indolopyridones are potent inhibitors of reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1). Although the structure of these compounds differs from established nucleoside analogue RT inhibitors (NRTIs), previous studies suggest that the prototype compound INDOPY-1 may bind in close proximity to the polymerase active site. NRTI-associated mutations that are clustered around the active site confer decreased, e.g. M184V and Y115F, or increased, e.g. K65R, susceptibility to INDOPY-1. Here we have studied the underlying biochemical mechanism. RT enzymes containing the isolated mutations M184V and Y115F cause 2-3-fold increases in IC(50) values, while the combination of the two mutations causes a >15-fold increase. K65R can partially counteract these effects. Binding studies revealed that the M184V change reduces the affinity to INDOPY-1, while Y115F facilitates binding of the natural nucleotide substrate and the combined effects enhance the ability of the enzyme to discriminate against the inhibitor. Studies with other strategic mutations at residues Phe-61 and Ala-62, as well as the use of chemically modified templates shed further light on the putative binding site of the inhibitor and ternary complex formation. An abasic site residue at position n, i.e. opposite the 3'-end of the primer, prevents binding of INDOPY-1, while an abasic site at the adjacent position n+1 has no effect. Collectively, our findings provide strong evidence to suggest that INDOPY-1 can compete with natural deoxynucleoside triphosphates (dNTPs). We therefore propose to refer to members of this class of compounds as "nucleotide-competing RT inhibitors" (NcRTIs).

Factor 8 (F8) gene mutation profile of Turkish hemophilia A patients with inhibitors

Factor VIII (FVIII) replacement therapy is ineffective in hemophilia A patients who develop alloantibodies (inhibitors) against FVIII. The type of factor 8 (F8) gene mutation, genes in the major histocompatibility complex loci, and also polymorphisms in IL-10 and tumor necrosis factor-alpha are the major predisposing factors for inhibitor formation. The present study was initiated to reveal the F8 gene mutation profile of 30 severely affected high-responder patients with inhibitor levels of more than 5 Bethesda U (BU)/ml and four low-responder patients with inhibitors less than 5 BU/ml. Southern blot and PCR analysis were performed to detect intron 22 and intron 1 inversions, respectively. Point mutations were screened by DNA sequence analysis of all coding regions, intron/exon boundaries, promoter and 3' UTR regions of the F8 gene. The prevalent mutation was the intron 22 inversion among the high-responder patients followed by large deletions, small deletions, and nonsense mutations. Only one missense and one splicing error mutation was seen. Among the low-responder patients, three single nucleotide deletions and one intron 22 inversion were found. All mutation types detected were in agreement with the severe hemophilia A phenotype, most likely leading to a deficiency of and predisposition to the development of alloantibodies against FVIII. It is seen that Turkish hemophilia A patients with major molecular defects have a higher possibility of developing inhibitors.

Factor VIII genotype and inhibitor development in patients with haemophilia A: highest risk in patients with splice site mutations

The appearance of inhibitory antibodies against factor VIII (FVIII) is the most severe and costly complication of replacement therapy in patients with haemophilia A (HA). To determine the relationship between FVIII genotype and inhibitor development, baseline FVIII activity, genotype and inhibitor development were reviewed in 1104 patients with HA. In patients with severe HA, splicing errors present the highest frequency of inhibitors, ahead of inversion of intron 1 and of intron 22, nonsense mutations and large deletions. The lowest inhibitor frequency in severe HA is found in patients with missense mutations and small deletions/insertions. Subanalyses indicate that nonsense mutations and small deletions/insertions leading to a frameshift in the light chain are associated with a significant higher risk of inhibitor formation than similar mutations occurring in the heavy chain (27% vs. 14%). These mutation types also have a higher frequency of inhibitors when occurring in exons 23-26, where a second FVIII transcript originates, compared with similar mutations in exons 1-22 (28% vs. 17%). These results suggest that complete absence of FVIII because of null mutations, including splice site mutations, or the absence of a second transcript result in an increased risk of inhibitor development.

Volatile Components of Camellia sinensis Inhibit Growth and Biofilm Formation of Oral Strepto

This study aimed to evaluate the efficacy of semi fermented and non fermented Camellia sinensis extracts (Black and Green tea) and comparison between them against Streptococcus mutans ATCC 25175, S. mitis ATCC 9811 and S. sanguis ATCC 10556 that are responsible for dental caries and bacteremias following dental manipulations. Minimum inhibitory concentrations of both tea extracts were assessed by Well diffusion and Broth dilution methods and examination of cell adherence (Biofilm inhibitory concentrations) was observed on glass slides under phase contrast microscope and colony counts from glass beads. Concentration of 1 mg mL(-1) of semi fermented tea extract was completely biofilm inhibitor but biofilm formation by these bacteria was seen 7 days after treatment with 1 mg mL(-1) of non fermented Camellia sinensis on glass beads and BIC for oral streptococci treated with this extract was 1.5, 2.5 mg mL(-1) of semi fermented and 3 mg mL(-1) of non fermented extracts had bactericidal effect on these bacteria. Semi fermented and non fermented Camellia sinensis extracts were able to prevent growth of oral streptococci. Therefore dental caries significantly reduce and the efficiency of semi fermented tea was higher due to rich content of volatile components rather than non fermented extracts.

Introduction to Session 2: Enzyme Catalysis and Engineering

The role of enzymes in biomass-to-fuels is rapidly expanding from a narrow focus on the hydrolysis of cellulose remaining after pretreatment to a more comprehensive approach, where increasing attention is focused on noncellulase activities, including hemicellulases, lignin-modifying, and accessory enzymes. This transition is being driven from several directions. Foremost is the realization that pretreatment may be best viewed as an activation or preconditioning step in the hydrolysis of biomass. Pretreatment conditions severe enough to produce highly accessible biomass for subsequent enzymatic conversion may be saddled with the significant problems of high cost, yield losses, and inhibitor formation. Mild severities may significantly reduce these problems; however, lower severity pretreatment results in higher recalcitrance, and often higher chemical complexity compared to more severely pretreated biomass.

A prospective surveillance study of factor VIII inhibitor development in the Canadian haemophilia A population following the switch to a recombinant factor VIII product formulated with sucrose

The introduction of new factor concentrates has, at times, resulted in an increase in inhibitor development; hence large systematic surveys of inhibitor development are necessary whenever new products are introduced. This study presents the results of a surveillance study conducted by the Inhibitor Subcommittee of the Association of Hemophilia Clinic Directors of Canada that evaluated inhibitor development in patients with haemophilia A following the switch to a second generation recombinant FVIII product (rFVIII-FS; Kogenate((R)) Bayer). Four hundred and sixty haemophilia A paediatric and adults patients from 17 Canadian Comprehensive Hemophilia Care Centers were enrolled in the study. Of these, 274 patients had evaluable data. Blood samples collected at baseline (prior to the switch to rFVIII-FS), and at 12 and 24 months following conversion were tested for inhibitors by the Nijmegen-modified Bethesda assay. Four subjects had positive inhibitor titres at baseline, with values ranging from 3.3 to 160 BU. Of the 274 patients who had baseline samples collected, 225 had postswitch samples collected at 12 months and 189 subjects had samples collected at 24 months. Only patients with positive baseline inhibitor titres (n = 4) had positive inhibitor titres at either the 12- or 24-month postswitch time points; therefore no de novo inhibitors developed over the 2-year evaluation period in this patient population. The results of this surveillance study suggest that the altered formulation of this recombinant FVIII concentrate was not associated with an increased incidence of inhibitor formation.

Safety and efficacy of sucrose-formulated full-length recombinant factor VIII: Experience in the standard clinical setting

The safety of full-length sucrose-formulated recombinant factor VIII (rFVIII-FS; Kogenate FS) for up to 24 months of use was evaluated in a postmarketing observational study in Europe. Long-term safety and efficacy data were available for 212 patients with severe haemophilia A, including 13 previously untreated patients (PUPs) and 12 patients with 1-19 exposure days (EDs). Patients accumulated a mean (+/- SD) of 187 (121) EDs to rFVIII-FS and received a total of 39,627 infusions, mainly for prophylaxis and for the treatment of 4,283 spontaneous or trauma-related bleeds during an average observation time of 710 (136) days. Of these bleeding episodes, 85.4% were successfully treated with one or two infusions of rFVIII-FS. Haemostasis was also evaluated during 46 minor to major surgical procedures, and the response to infusion was "excellent" or "good" in all cases. FVIII inhibitor formation was observed in six patients (two de novo; four persistent or recurrent). The de novo cases represent 8.0% (2 of 25) of patients who reported 0-19 previous EDs at study entry. Four of the five patients who reported possible drug-related adverse effects developed inhibitors. The results of this observational study demonstrate the efficacy and safety of rFVIII-FS during normal clinical use in the treatment of patients with severe haemophilia A. Furthermore, these findings are consistent with those of previous phase III clinical studies with rFVIII-FS, particularly with regard to its efficacy and low incidence of inhibitor formation.

An observational study of sucrose-formulated recombinant factor VIII for Japanese patients with haemophilia A

The safety and efficacy of sucrose-formulated recombinant factor VIII (rFVIII-FS; Kogenate FS) under usual clinical practice were evaluated for 12 months in an observational, postmarketing surveillance study conducted at 214 treatment centres throughout Japan. The study included 631 patients with haemophilia A, 80% of whom had severe or moderately-severe disease (< or = 2% FVIII:C). Most patients (n = 477; 75.6%) had >100 prior exposure days (EDs), but the study also included 62 (9.8%) patients with <20 EDs who were at high risk for inhibitor development. A total of 71,240 infusions were administered during the observation (mean, 113 +/- 108 per patient). Physicians rated efficacy and tolerability of rFVIII-FS as "very good" or "good" in >99% of patients. FVIII inhibitors were observed in seven patients (5 de novo; 1 persistent/fluctuating; 1 recurrent). The overall de novo inhibitor incidence was 0.8% (5/631; or 5/599 among the subgroup of patients with negative baseline titre and no known inhibitor history). De novo cases represented 3.2% (2/62) of patients with <20 EDs at enrollment (2/57 in the no inhibitor subgroup) and 0.2% (1/477) of patients pretreated with >100 EDs (1/452 in the no inhibitor subgroup) at enrollment. The results of this large observational study demonstrate that rFVIII-FS is both safe and efficacious as used in the usual clinical setting for the treatment of Japanese patients with mild to severe haemophilia A. This study supports the efficacy of rFVIII-FS with an incidence of inhibitor formation no greater than in a comparable European study or previous phase III clinical studies.

Efficacy and safety of recombinant factor VIII products in patients with hemophilia A

The introduction of recombinant factor VIII (rFVIII) clotting factor concentrates nearly 20 years ago represented a significant advance in the treatment of hemophilia A. The major advantage of rFVIII products compared with plasma-derived FVIII products is related to product safety, with rFVIII products virtually eliminating bloodborne pathogen transmission. The most challenging aspect of hemophilia A management today is the development of FVIII inhibitors; previously untreated patients are at the highest risk for inhibitor formation. Presented in this article are results of clinical trials in previously treated and untreated patients and postmarketing surveillance studies for the four commercially available rFVIII products (Recombinate, ReFacto, Kogenate FS/Kogenate Bayer and Advate). Recombinant FVIII therapies are highly efficacious when used ondemand and prophylactically, and they have excellent safety profiles; there have been no reports of viral- or prion-based disease transmission associated with rFVIII administration. The incidence rate of inhibitors in previously untreated patients ranges from 15% to approximately 30%. Because rFVIII concentrates have proven efficacy and safety profiles, a number of hemophilia treatment groups recommend rFVIII products as first-line therapy in the management of hemophilia A.

Increased frequency of the CTLA‐4 49 A/G polymorphism in patients with acquired haemophilia A compared to healthy controls

Acquired haemophilia (AH) is an autoimmune disorder characterized by autoantibodies against endogenous factor VIII (FVIII). Half of the patients present with an underlying disease known to cause the FVIII autoantibodies whereas in the other half the disease is of idiopathic nature. Recently, it has been shown that variants of the polymorphic cytotoxic T lymphocyte antigen-4 (CTLA-4) gene are associated with autoimmune diseases and also represent a risk factor for inhibitor formation in inherited haemophilia A. In the present study, we investigated whether CTLA-4 variants also play a role in the pathogenesis of AH. Therefore, we analyzed three single nucleotide polymorphisms (SNPs) of the CTLA-4 gene (-318 C/T, +49 A/G and CT60 A/G) in 57 AH patients and 98 controls. The CTLA-4 + 49 G allele occurred with a significantly higher frequency in patients with AH compared with controls [odds ratio (OR) = 2.17, 95% confidence interval (CI): 1.36-3.48, P = 0.001]. This effect was mainly caused by a higher frequency of the 49 G allele in female patients (OR = 5.1, 95% CI: 1.76-15.02, P = 0.002), whereas in males the frequencies were not significantly different (OR = 1.4, P = 0.5). A higher frequency of the G allele was also observed in the subcohort with AH and underlying autoimmune disease (OR = 3.1, P = 0.04). Our observations of a higher frequency of the CTLA-4 + 49 A/G SNP in AH patients are in concordance with findings in other autoimmune disorders. In conclusion, on the background of the CTLA-4 gene polymorphism, further genetic and/or environmental factors might contribute to and finally trigger the clinical manifestation of AH.

Immunogenicity of Factor VIII: Is Clotting the “Danger” Signal?.

It is well established that mice lacking factor VIII (E16 or fVIII−/−) respond to intravenous (i.v.) or intraperitoneal (i.p.) injections of therapeutic doses of human fVIII in PBS. This leads to both T-cell priming for cytokine production and inhibitor formation against fVIII with specificity similar to that of hemophilia A patients (Pratt et al., 2004). The lack of tolerance to murine fVIII may be important in the ability of fVIII−/− mice to respond to human fVIII. Nonetheless, the ability to respond effectively in the absence of extrinsic adjuvant begs the question of what is the “danger signal” required for immune responsiveness to fVIII. Even more surprising is the fact that mice with normal hemostasis respond to therapeutic doses of human fVIII given intravenously (i.v.), a normally tolerogenic route (Lin, Soukhareva, and Scott, ASH 2004). We proposed to investigate whether fVIII has any intrinsic adjuvanticity. Preliminary experiments failed to support the notion that fVIII was a polyclonal B cell activator or could stimulate up regulation of MHC class II or co-stimulatory molecules in vitro or in vivo. We hypothesized that the immunogenicity of fVIII is due to its ability to initiate clotting and that this cascade leads to local co-stimulatory events. To test this, fVIII−/− (C57Bl/6 background) or normal Balb/c mice were injected with either native (N) fVIII or heat inactivated (HI) fVIII (56o, 30′). This heat inactivation method was verified to destroy biologic activity of fVIII in chromogenic assays. After five injections, the antibody response elicited by NfVIII was three- to fourfold higher than that elicited by HIfVIII. ELISA assays with monoclonal antibodies against different fVIII domains (a gift of Dr. Pete Lollar) indicated a partial loss of binding to conformational B cell epitopes in HIfVIII. T cell proliferation to both preparations of fVIII demonstrated conservation of T cell epitopes. Importantly, the T cell response to fVIII was dramatically reduced in the group immunized with HIfVIII. These results suggest that the ability of fVIII to initiate a localized clotting event plays a critical role in the immunogenicity of fVIII.(Supported by NIH grants R01 HL061883 and T32 HL07698).

U.S. Inhibitor Pilot Project: Participant Characteristics and Infusion Log Adherence.

Inhibitors are now considered to be the most important complication of clotting factor replacement therapy in hemophilia. In January 2005, the Universal Data Collection (UDC) project, which includes data on over 14,000 patients with hemophilia A or B, was amended to include prospective data collection on inhibitors. We report interim results of a pilot project involving 9 selected Hemophilia Treatment Centers in the U.S. Dedicated data managers collected risk factor and product exposure data from enrolled patients on a monthly basis. Tracked infusion log submissions were used to estimate patient adherence to infusion log completion. A blood specimen is collected at baseline, annually, at product switch, or for clinical indication and is tested centrally for inhibitor using the Nijmegen modification method. Results are reported and any above a threshold value are followed up with repeat testing and clinical correlation. As of August 2007, 514 male patients with hemophilia, ages 2–84 years (mean 23 yrs SD 17) had been enrolled. Of these, 415 (81%) had FVIII deficiency (64% severe, 20% moderate, 16% mild) and 99 (19%) had FIX deficiency (35%, 43%, and 22%, respectively). 85% of the patients were caucasian, 8% black, and 4% Hispanic. A previous history of inhibitor was present at enrollment in 71 (14%). Prophylactic treatment was used by 37% and 80% received product by home infusion. Historical exposure to product collected at enrollment was as follows: 14% had 0–20 exposure days (ED), 12% had 21–100 ED, 7% had 101–150 ED, and 64% > 150 ED. During follow-up, 6 patients have had inhibitor titer measurements over 0.4 BU. One of these was known to have an active inhibitor at enrollment. Confirmatory testing of remaining cases is underway. Mean adherence rate for patient infusion log completion in the 9 HTC's was 45% (range 7% to 74%). Overall adherence increased with time on study from about 40% at one month to nearly 60% for those enrolled at least 10 months. In summary, enrolled participants represent a wide range of characteristics including age, race, disease severity and number of factor exposures. Interim findings indicate that prospective collection of factor infusion data from hemophilia patients necessary to appropriately monitor inhibitor formation, is feasible but labor intensive and requires patient adherence to completion and submission of accurate infusion logs. Because inhibitor formation is rare and the potential risk factors are many, large scale studies with long-term follow-up will be needed to fully assess risk factors for inhibitors.

Co-Delivery of Sleeping Beauty Transposons Encoding FVIII and Indoleamine 2,3-Dioxygenase Attenuate Inhibitor Formation Resulting in Long-Term FVIII Expression.

Gene therapy in adult hemophilia A mice is known to generate a robust immune response against the newly produced FVIII protein. This phenomenon has been extensively reported for both viral and non-viral vectors. T cell activation promotes the proliferation of B cells that produce antibodies against FVIII (inhibitors) leading to a loss of circulating FVIII protein and activity. Approaches to attenuate this immune response have included the use of cytotoxic chemotherapeutic agents (such as cyclophosphamide) or the use of antibodies or soluble fusion proteins that prevent T cell activation (such as the fusion protein between the cytotoxic T lymphocyte-associated antigen 4 and the immunoglobulin heavy chain, CTLA-4-Ig). Recently, the mechanism by which CTLA-4-Ig exerts its effect has been elucidated and is thought to involve tryptophan catabolism by upregulation of the enzyme indoleamine 2,3-dioxygenase (IDO). Catabolites of tryptophan degradation, such as kynurenine, have been shown to block T cell proliferation and promote T cell apoptosis. Here we report that hydrodynamic co-administration of Sleeping Beauty transposons encoding both FVIII and IDO are able to attenuate, but not fully block, inhibitor formation. With this approach, long term expression of FVIII at therapeutic levels (∼10% of normal) can be achieved in adult animals without the need for additional immune suppression. Only in animals receiving FVIII and IDO together can we detect FVIII protein by western blot at 24 weeks. Serum kynurenine levels are slightly elevated after gene delivery in animals receiving the IDO gene, but fall to within normal levels by 24 weeks. These results suggest that modulation of the levels of tryptophan catabolites in vivo can influence the formation of FVIII inhibitors.

Epitope Identification and Generation of an Antigen-Specific T-Cell Clone in a Mild Hemophilia A Inhibitor Subject with Missense Genotype R593C.

The development of inhibitory anti-FVIII antibodies is a major clinical problem in hemophilia A. While less common in mild/moderately severe patients, the relative risk of inhibitor formation is elevated in patients with missense mutations in the FVIII A2 domain, especially those with an R593C genotype. In this study, T-cell responses to 14 FVIII A2 domain peptides with predicted DRB1*1101 MHC binding motifs were investigated using MHC class II tetramer reagents. CD4+ T cells were isolated from a hemophilic subject (DRB1*1101, 1302) with FVIII missense genotype R593C and stimulated with pooled peptides. This subject had developed a longstanding low titer inhibitor after receiving multiple FVIII infusions. Staining with fluorescent, peptide-loaded tetramers revealed that the hemophilic subject, but not an HLA-matched healthy control, had a DRB1*1101-restricted response to peptide A2589–608, which contained the wild-type R593 sequence. MHC class II tetramers with bound A2589–608 were used to sort antigen-specific T cells and then generate a T-cell clone recognizing this high avidity DRB1*1101-restricted epitope. FVIII residues 594–602 (FLPNPAGVQ) comprise a predicted high-affinity binding motif. Peptide binding assays confirmed that A2589–608 bound to recombinant, monomeric DRB1*1101 protein with high affinity. A peptide with the corresponding hemophilic sequence (A2589–608, 593C) bound with affinity that was threefold lower, but in a range that should be sufficient for presentation to T cells. The other 12 A2 peptides had a wide spectrum of binding affinities for the DRB1*1101 protein. Interestingly, prediction algorithms suggest that the peptides containing residue 593 occupy the MHC Class II peptide-binding groove in a register that places this residue just outside the p1 anchor position. Thus we anticipate that residue 593 modulates T-cell recognition of peptide A2589–608. This provides a potential explanation for inhibitor development in mild hemophilia A patients with a FVIII R593C genotype who are HLA-DRB1*1101. Immunogenicity of this FVIII region with respect to other HLA types is under investigation.

Re-Establishment and Characterization of an Extinct Line of Sheep with a Spontaneous Bleeding Disorder That Closely Recapitulates Human Hemophilia A.

Hemophilia A, or Factor VIII (FVIII) deficiency, is the most common severe hereditary coagulation disorder, affecting 1 in 5000 male live births. Animal models in dog, mouse, and rabbit have been developed and used to study FVIII function and to evaluate new methods of treatment and prevention of inhibitor formation. Unfortunately, for unknown reasons, results obtained using these models didn't always result in successful therapies when applied to humans. For new treatments to be safely and successfully translated from surrogate models to clinical trials, it is critical to develop an animal model that simultaneously and accurately parallels normal human physiology while mimicking human hemophilia's physiopathological process. Due to its striking physiological and anatomical similarities to humans, sheep are considered an ideal model to study a vast array of pathologies. The aim of these studies was to re-establish, study, and characterize an extinct line of sheep with a spontaneous bleeding disorder that closely recapitulated human hemophilia A (ThrombHaemost 68:618, 1992). Thus, we used frozen semen from an affected male to generate hemophilia A carriers. We obtained 20 females that when compared to pooled control sheep plasma, exhibited slightly increased PTT levels (38.1±1.1; N=30s), normal PT and platelet number, and slightly decreased FVIII:C (70±3%). Levels of Fibrinogen, FIX, vWF activity and vWF:ag were also normal. A second round of reproductive manipulations using the carriers' oocytes and the affected semen produced 23 more animals, 16 of which were obligate carriers with a similar phenotype. The other 8 animals exhibited prolonged bleeding from the umbilical cord that promptly stopped upon administration of purified human FVIII concentrate using recommended dosing. Due to the unfeasibility of clamping the umbilical cord, therapy with human FVIII was continued each 12 hours until the umbilical cord dropped off. Blood collected prior to the administration of FVIII showed that these animals had almost non-existent levels of FVIIIc, and an extremely prolonged PTT (91.5±2.9, N=30.3) with normal levels of platelets, fibrinogen, FVII, FIX, and vWF. 2 of the animals died shortly after birth due to extensive hematomas related to lambing trauma. The other 6 animals, now 5 months old (maturity 6–9 months), developed clinical symptomatology closely mimicking that of human patients with severe hemophilia A. Each of these animals had between 2–6 episodes of severe bleeding including hemarthroses of the elbow, shoulder, hip, and knee, multiple muscle hematomas, including 1 hematoma of the tongue and 1 episode of mild hematuria. All of the bleeding episodes resolved upon administration of 1–2 treatments with human FVIII. Animals have thus far received between 964-4546U of human FVIII. Of interest is that low-titer inhibitors (1.3; 2.6; 3 BU) were detected in 3 of the animals showing that the nature of the mutation present in these sheep renders them prone to inhibitor development. Characterization of the mutation is currently underway. We hope that this large animal model will contribute to a better understanding of hemophilia and the development of novel treatments that can directly translate to human patients, such as stem cell transplantation and gene therapy-based approaches.

U.S. INHIBITOR PILOT PROJECT: STUDY DESIGN AND METHODS VALIDATION

ISTH07A1_P-T-118: Contact View [P-T-118] U.S. INHIBITOR PILOT PROJECT: STUDY DESIGN AND METHODS VALIDATION J.M. Soucie, C.H. Miller, M. Creary, W.C. Hooper, T.C. Abshire, D.B. Brettler, P. Bockenstedt, J. DiPaola, G. Massey, A. Neff, A. Shapiro, M. Tarantino, B.M. Wicklund, D. DiMichele, U.D.C. Working Group. Div of Blood Disorders, NCBDDD, Centers for Disease Control and Prevention; Dept. of Pediatrics, Emory University, Atlanta, GA; Dept. of Medicine, UMass Memorial Medical Center, Worcester, MA; Dept. of Internal Medicine, University of Michigan, Ann Arbor, MI; Dept. of Pediatrics, Univ. of Iowa Hospitals amp] Clinics, Iowa City, IA; Dept. of Pediatrics, Virginia Commonwealth Univ., Richmond, VA; Dept. of Medicine, Vanderbilt Univ., Nashville, TN; Hematology, Indiana Hemophilia and Thrombosis Center, Indianapolis, IN; Hematology, Comprehensive Bleeding Disorders Center, Peoria, IL; Dept. of Pediatrics, Children's Mercy Hospital, Kansas City, MO; Dept. of Pediatrics, Weill Medical College of Cornell Univ., New York, NY; Hemophilia Treatment Centers, Various Institutions, U.S., United States Introduction: The Universal Data Collection (UDC) Program of the Division of Blood Disorders at the Centers for Disease Control and Prevention (CDC) has initiated a pilot project at 9 U.S. Hemophilia Treatment Centers to perform post market surveillance for inhibitors. Goals are to prospectively collect risk factor and product exposure data, to provide centralized inhibitor monitoring, and to correlate inhibitor formation with genotype. Methods: Patients provide infusion logs with data on product brand, dose, and reason infused. A blood specimen is collected at baseline, annually, at product switch, or for clinical indication. Plasma and cells for DNA are shipped on cold packs to CDC, where inhibitor testing is done by the Nijmegen modification of the Bethesda assay, using commercially prepared buffered normal pooled plasma (BNPP) and deficient plasma from a hemophilic donor. A positive control with a known inhibitor titer is run with each assay. Specimens are screened for inhibitor at 3 parts patient plasma to 1 part BNPP. Positive specimens are retested in dilutions. Results are reported in Nijmegen-Bethesda units (NBU). The intron 22 inversion of the FVIII gene is detected using a modified version of longrange PCR. Utilizing the ABI 3730 Automated DNA sequencer, FVIII and FIX genes are sequenced in both directions, including both the 5' and 3' untranslated regions. Results: In the first year, 438 patients (80% hemophilia A) were enrolled. Comparison of 50 cold pack specimens to frozen specimens shipped on dry ice showed a correlation coefficient of 0.998. 406 inhibitor tests have been performed. The effects of infused FVIII or FIX and heat treatment of specimens are being investigated. More than 200 gene analyses have been done. Conclusions: Centralized monitoring of inhibitors is feasible and promotes quality control. Concurrent mutation analysis will add to the data on risk factors. International coordination of such data collection will produce a powerful tool for surveillance. Soucie JM, Miller CH, Creary M, Hooper WC, Abshire TC, Brettler DB, Bockenstedt P, DiPaola J, Massey G, Neff A, Shapiro A, Tarantino M, Wicklund BM, DiMichele D, U.D.C. Working Group. U.S. INHIBITOR PILOT PROJECT: STUDY DESIGN AND METHODS VALIDATION. J Thromb Haemost 2007; 5 Supplement 2: P-T-118 Date: Tuesday, July 10, 2007 file:////ddn-452/d/working/Rajan/Wileyml-3/2014/January/16-Jan/Tuesday/P-T-118.htm (1 of 2) [1/16/2014 8:20:24 PM] Abstract ISTH07A1_P-T-118: Contact ViewISTH07A1_P-T-118: Contact View Session Info: Poster: Haemophilia A and B Room: Exhibition Area file:////ddn-452/d/working/Rajan/Wileyml-3/2014/January/16-Jan/Tuesday/P-T-118.htm (2 of 2) [1/16/2014 8:20:24 PM]

Rituximab for the treatment of childhood chronic idiopathic thrombocytopenic purpura and hemophilia with inhibitors

Rituximab, a monoclonal chimeric antibody to the CD20 antigen, is an effective treatment for non-Hodgkin lymphomas. Moreover, rituximab has also shown efficacy in various autoimmune disorders. In this review, we will focus on the use of rituximab in childhood disorders of hemostasis associated with inhibitor formation. Although the results presented suggest that rituximab can be useful in the treatment of this subset of pediatric patients, most of the data come from isolated case reports or descriptions of small, uncontrolled series. Therefore, large, prospective, and randomized trials are needed to confirm the positive, preliminary results.

High expression reduces an antibody response after neonatal gene therapy with B domain-deleted human factor VIII in mice.

Gene therapy could prevent bleeding in patients with hemophilia A, but might induce antibodies that block factor VIII (FVIII) function. To test the efficacy of gene therapy in the newborn period for preventing a response to human FVIII (hFVIII) because of immaturity of the immune system. Varying doses of a retroviral vector (RV) expressing a B domain-deleted hFVIII cDNA were injected i.v. into newborn hemophilia A C57BL/6 or normal C3H mice. Mice were evaluated for hFVIII expression, hemostasis, and development of anti-hFVIII antibodies with inhibitory activity. Injection of a high RV dose [10(10) transducing units (TU) kg(-1)] into newborn hemophilia A or C3H mice resulted in 61% and 13% of normal hFVIII antigen in plasma, respectively; most mice did not produce anti-hFVIII antibodies, and hemophilia A mice did not bleed. Furthermore, most mice with >20 ng mL(-1) of hFVIII in plasma (10% normal, 1 x 10(-10) m) were tolerant to hFVIII, as an antibody response was markedly reduced after challenge with hFVIII with or without adjuvant. However, most RV-treated animals with lower antigen levels developed antibodies before or after challenge. Thus, initiation of a gene therapy trial with low RV doses might increase inhibitor formation. Furthermore, frequent hFVIII infusions in newborns with hemophilia A might reduce inhibitor formation. Finally, difficulties in achieving tolerance after gene therapy for hemophilia A as compared to hemophilia B may relate to lower expression of FVIII than FIX, as high antigen levels are most effective at inducing tolerance.

Efficacy, safety and tolerability of recombinant factor VIII (REFACTO®) in patients with haemophilia A: interim data from a postmarketing surveillance study in Germany and Austria

An open-label, multicentre, postmarketing surveillance study conducted in Germany and Austria with recombinant factor VIII (REFACTO) has enrolled 217 patients (mean age 26.3 years) from 38 haemophilia centres during the first 4.8 years. Most patients (188/217; 86.6%) had severe to moderately severe haemophilia A, of whom 153 completed sufficient diary information for the main efficacy analysis. These 153 patients experienced a median of 6.6 (interquartile range 1.4-18.6) bleeding episodes per year. Patients treated with prophylaxis experienced a median of 4.4 (1.1-9.3) bleeds per year, while patients treated on-demand experienced a median of 22.8 (11.3-29.0) bleeds per year. Overall, most physicians (41/43 [95.3%]) were 'very satisfied' or 'satisfied' with the efficacy of REFACTO in the treatment of bleeding episodes. A total of 137 non-serious adverse events have been reported in 52/217 patients (24.0%) to date. In addition, 129 serious adverse events in 87 patients (40%) were reported, including 41 cases of 'less than expected therapeutic effect' (LETE). Of these, 39 LETE cases were reported in one centre; however, patients in this centre experienced considerably fewer bleeding episodes per year than patients outside this centre. Overall, six patients (2.8%) have developed de novo inhibitors, three of which were considered high titre. Four of these patients were at high risk (0-50 exposure days [ED]) of inhibitor formation, one was at intermediate risk (51-100 ED) and one was at low risk (>100 ED). These results emphasize the benefit of postmarketing surveillance and, overall, this study confirms the efficacy, safety and tolerability of REFACTO in the treatment of patients with haemophilia A.