Ninety-nine patients had been diagnosed with a FVIII inhibitor. disorder.1 The reasons for such immunogenicity of FVIII concentrates, as compared to other therapeutic proteins, remain unclear. Different risk factors have been associated with the appearance of FVIII inhibitors, including the type of mutations responsible for hemophilia A, the HLA haplotypes and polymorphisms in and genes.2C5 Inflammatory events in act or occurring at the time of therapeutic FVIII administration have also been proposed as potential risk factors. Thus, repeated joint bleeds create a chronic inflammation favoring the local recruitment and activation of antigen-presenting cells and immune effectors.3,6 Likewise, surgery, which, in conjuncture with intensive FVIII treatment, has been proposed as a risk factor for inhibitor development,7 induces acute inflammation. Besides, the very administration of therapeutic FVIII has been controversially proposed to bring about inflammatory signals by virtue of the capacity of FVIII to induce a burst of thrombin Rabbit polyclonal to LRRC15 generation, that in turn triggers proteinase-activated receptors.8,9 Heme oxygenase (HO) is an essential enzyme for Tacrolimus monohydrate the catabolism of heme and has been shown to have potent anti-oxidant, cytoprotective, immunosuppressive and anti-inflammatory properties via the production of bile pigments, carbon monoxide (CO) and the induction of ferritin.10,11 Two isoforms of HO have been identified:12,13 HO-2 is produced constitutively, whereas HO-1 is inducible. Thus, HO-1 is normally undetectable in resting cells, but may be induced as a result of inflammation or oxidative stress, by various stimuli, such as pro-oxidative compounds, pro-inflammatory cytokines, toxins or toll-like receptor ligands.10,12,14 In animal models, the pharmacological induction of HO-1 ameliorates chronic and severe inflammation,15,16 has beneficial effects in various autoimmune conditions17,18 and improves graft survival.16,19 HO-1 was demonstrated to participate in the resolution of physiological inflammation and in wound healing.14,20 Accordingly, congenital defects in HO-1 expression are associated with systemic inflammation in both mice and humans.20 Recently, Tacrolimus monohydrate we demonstrated that the induction of HO-1 before the administration of FVIII to FVIII-deficient mice protects against the anti-FVIII immune response.21 The protective effect of HO-1 induction was reverted by tin-mesoporphyrin, an inhibitor of HO-1, and was reproduced by the administration of the end-degradation products of heme by HO-1, i.e., CO and bilirubin. The human HO-1-encoding gene (than HUVEC from healthy donors with 32 GT repeats following stimulation with H2O2.26 Polymorphisms in the promoter of the gene that result in greater inducibility of the enzyme have been associated with positive outcomes in a number of human pathologies characterized by cellular/tissue damage and inflammation.22 We hypothesized that polymorphisms in the promoter may confer different genetic predispositions to the induction of the immune response against exogenous FVIII among patients with hemophilia A by differentially influencing the capacity to modulate the inflammatory status of the patients. To test our hypothesis, we analyzed polymorphisms in the gene promoter of a large international cohort of patients with severe hemophilia A, and correlated the polymorphisms present with the development of FVIII inhibitors. Methods Study population Our study included 362 patients Tacrolimus monohydrate with severe hemophilia A from different hemophilia centers in France (Caen, Kremlin-Bictre, Paris, Rennes) and Germany (Bonn). Ninety-nine patients had been diagnosed with a FVIII inhibitor. The 263 inhibitor-negative patients matched with inhibitor-positive patients for mutation type except for missense mutations (Table 1). The selection criterion was severe hemophilia A (FVIII:C<1%). Inhibitor historical peak titers were documented for 75 of the 99 inhibitor-positive patients: 26 patients had a historical peak titer <5 Bethesda units (BU)/mL (mean 2.6; range 1.0C4.8) and 49 patients had a historical peak titer 5 BU/mL Tacrolimus monohydrate (mean 1259; range 5 C 50000). Patients who had never developed an inhibitor after 150 cumulative exposure days (CED) or more were defined as inhibitor-negative patients. Approval for these studies was obtained from the Caen University institutional review board. Written informed consent was provided by each patient according to the Declaration of Helsinki. Table 1. Characteristics of the studied population. Open in a separate window Factor VIII activity Tacrolimus monohydrate and factor VIII inhibitory titers FVIII activity was assessed using standard techniques. The original Bethesda method and the Nijmegen modification of the Bethesda assay were used to test for the presence or absence of FVIII-specific inhibitors in patients with hemophilia A.27 Genotyping of the variable (GT)n polymorphism in the promoter of the HO-1 gene Genomic DNA was obtained from blood, anticoagulated with ethylene-diamine-tetra-acetic acid (EDTA), using commercial DNA isolation kits and.