RESUMO
Recombinant factor VIII (FVIII) products represent a life-saving intervention for patients with hemophilia A. However, patients can develop antibodies against FVIII that prevent its function and directly increase morbidity and mortality. The development of anti-FVIII antibodies varies depending on the type of recombinant product used, with previous studies suggesting that second-generation baby hamster kidney (BHK)-derived FVIII products display greater immunogenicity than do third-generation Chinese hamster ovary (CHO)-derived FVIII products. However, the underlying mechanisms responsible for these differences remain incompletely understood. Our results demonstrate that BHK cells express higher levels of the nonhuman carbohydrate α1-3 galactose (αGal) than do CHO cells, suggesting that αGal incorporation onto FVIII may result in anti-αGal antibody recognition that could positively influence the development of anti-FVIII antibodies. Consistent with this, BHK-derived FVIII exhibits increased levels of αGal, which corresponds to increased reactivity with anti-αGal antibodies. Infusion of BHK-derived, but not CHO-derived, FVIII into αGal-knockout mice, which spontaneously generate anti-αGal antibodies, results in significantly higher anti-FVIII antibody formation, suggesting that the increased levels of αGal on BHK-derived FVIII can influence immunogenicity. These results suggest that posttranslational modifications of recombinant FVIII products with nonhuman carbohydrates may influence the development of anti-FVIII antibodies.
Assuntos
Anticorpos , Formação de Anticorpos , Inibidores dos Fatores de Coagulação Sanguínea , Fator VIII , Polissacarídeos , Processamento de Proteína Pós-Traducional/imunologia , Animais , Anticorpos/genética , Anticorpos/imunologia , Inibidores dos Fatores de Coagulação Sanguínea/genética , Inibidores dos Fatores de Coagulação Sanguínea/imunologia , Células CHO , Cricetinae , Cricetulus , Fator VIII/imunologia , Fator VIII/farmacologia , Hemofilia A/genética , Hemofilia A/imunologia , Camundongos , Camundongos Knockout , Polissacarídeos/genética , Polissacarídeos/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologiaRESUMO
Hemostasis encompasses the tightly regulated processes of blood clotting, platelet activation, and vascular repair. After wounding, the hemostatic system engages a plethora of vascular and extravascular receptors that act in concert with blood components to seal off the damage inflicted to the vasculature and the surrounding tissue. The first important component that contributes to hemostasis is the coagulation system, while the second important component starts with platelet activation, which not only contributes to the hemostatic plug, but also accelerates the coagulation system. Eventually, coagulation and platelet activation are switched off by blood-borne inhibitors and proteolytic feedback loops. This review summarizes new concepts of activation of proteases that regulate coagulation and anticoagulation, to give rise to transient thrombin generation and fibrin clot formation. It further speculates on the (patho)physiological roles of intra- and extravascular receptors that operate in response to these proteases. Furthermore, this review provides a new framework for understanding how signaling and adhesive interactions between endothelial cells, leukocytes, and platelets can regulate thrombus formation and modulate the coagulation process. Now that the key molecular players of coagulation and platelet activation have become clear, and their complex interactions with the vessel wall have been mapped out, we can also better speculate on the causes of thrombosis-related angiopathies.
Assuntos
Transtornos da Coagulação Sanguínea/sangue , Inibidores dos Fatores de Coagulação Sanguínea/metabolismo , Fatores de Coagulação Sanguínea/metabolismo , Plaquetas/metabolismo , Hemostasia , Animais , Coagulação Sanguínea , Transtornos da Coagulação Sanguínea/etiologia , Transtornos da Coagulação Sanguínea/genética , Inibidores dos Fatores de Coagulação Sanguínea/genética , Fatores de Coagulação Sanguínea/genética , Comunicação Celular , Retroalimentação Fisiológica , Humanos , Ativação Plaquetária , Transdução de SinaisRESUMO
Anti-drug antibody formation following factor VIII (FVIII) replacement therapy is the most important treatment-related complication in patients with severe haemophilia A. A significant number of these antibodies show neutralising activity against FVIII and are referred to as FVIII inhibitors. Alloimmunity to FVIII, given the absence of endogenous circulating FVIII protein, may be predictable to some extent; however, only 30% of patients develop inhibitors. Genetic and environmental risk factors have been identified, contributing to the likelihood of inhibitor development. Multiple immunological theories have been proposed which in part explain the outcomes of many epidemiological studies. Significant differences exist among replacement therapies, including the source, FVIII sequence, glycosylation, formulation components, impurities and aggregation potential, which significantly complicate interpretation of the results from these studies. In this review, we present recent advances in the understanding of the cellular mechanisms of inhibitor formation and highlight some areas of uncertainty requiring further investigation.
Assuntos
Autoanticorpos , Inibidores dos Fatores de Coagulação Sanguínea , Fator VIII , Autoanticorpos/genética , Autoanticorpos/imunologia , Inibidores dos Fatores de Coagulação Sanguínea/genética , Inibidores dos Fatores de Coagulação Sanguínea/imunologia , Fator VIII/genética , Fator VIII/imunologia , Fator VIII/uso terapêutico , Hemofilia A/tratamento farmacológico , Hemofilia A/genética , Hemofilia A/imunologia , Hemofilia A/patologia , Humanos , Masculino , Fatores de RiscoRESUMO
BACKGROUND/OBJECTIVE: Hemophilia A is a genetic disorder through which patients suffer from recurrent bleeding. This can be caused by a defect in human plasma coagulation factor VIII. High incidence of FVIII inhibitors in some severe hemophilia A patients after FVIII therapy is a considerable complication. Determination of good predictive factors can improve the safety of this treatment. HLA-II have been shown as a predictive element for inhibitor development. The goal of this study is to determine the association between HLA-DRB1*15:03, HLA-DRB1*11 and HLA-DRB1*01:01 alleles and FVIII inhibitors in severe hemophilia A patients in Iran. MATERIALS/METHODS: HLA-DRB1 genotyping was performed using Multiplex sequences Specific Primers (PCR-SSP) in two groups of severe hemophilia A patients comprising 51 and 50 individuals with and without FVIII inhibitors respectively. The levels of inhibitor were determined through Nijmegen-modified Bethesda assay. HLA-DRB1 allele frequencies were compared between groups by using multiple logistic regression models. RESULTS: HLA-DRB1*01:01 allele frequency was significantly higher in patients without inhibitor ORadj: 2.7 (95%CI: 1.08, 6.97; P = 0.034). There wasn't any statistically significant difference in HLA-DRB1*11 allele frequency between groups ORadj: 0.7 (95%CI: 0.27, 1.82; P = 0.47). There was no connection between HLA-DRB1*15:03 and inhibitor development ORadj: 0.94 (95%CI: 0.38, 2.35; P = 0.94). CONCLUSION: An association between HLA-DRB1*01:01 and paucity of FVIII inhibitor showed that this allele has probably a protective effect in severe hemophilia A patients in Iran. Determination of the predictive and protective alleles are beneficial in pre-treatment activities and decrease the risk of unsuccessful therapy with FVIII in each population.
Assuntos
Alelos , Inibidores dos Fatores de Coagulação Sanguínea/genética , Frequência do Gene , Cadeias HLA-DRB1/genética , Hemofilia A/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Fator VIII/genética , Feminino , Humanos , Lactente , Irã (Geográfico) , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
A major problem of hemophilia A (HA) treatment is the development of factor VIII (FVIII) inhibitor, which usually occurs shortly after initiating replacement therapy. Several studies showed the correlation between inhibitor development and polymorphisms in inflammatory and immune response genes of HA patients; however, literature data are not available to prove this association in Iranian population. The aim of this study was to investigate a possible association between FVIII inhibitor formation and the polymorphisms of 16 inflammatory and immune response genes in Iranian severe HA patients (FVIII activity < 1%). This case-control study was performed on 55 patients with severe HA inhibitors and 45 samples without inhibitors from Iranian Comprehensive Hemophilia Care center. After extraction of whole genomic DNA from blood samples and design of primers for 16 genes, the genotyping was performed by Tetra primer ARMS PCR, and the validation of single nucleotide polymorphisms was determined by DNA sequencing. The data indicated that there was a significant association between inhibitor development, and F13A1 (TT), DOCK2 (CC& CT), and MAPK9 (TT) genotypes. Moreover, a considerably increased inhibitor risk carrying T, C, and T allele for F13A1, DOCK2, and MAPK9 genes was observed in patients with inhibitors, respectively. In contrast, there was no statistically significant difference between the genotypic and allelic frequencies for other genes in patients with inhibitors compared to patients without inhibitors. These results demonstrate that only polymorphisms in F13A1, DOCK2, and MAPK9 genes are associated with the risk of developing FVIII inhibitors in Iranian HA patients.
Assuntos
Alelos , Inibidores dos Fatores de Coagulação Sanguínea/genética , Frequência do Gene , Fatores de Troca do Nucleotídeo Guanina/genética , Hemofilia A/genética , Proteína Quinase 9 Ativada por Mitógeno/genética , Polimorfismo Genético , Adulto , Fator VIII/genética , Proteínas Ativadoras de GTPase , Humanos , Irã (Geográfico) , MasculinoRESUMO
Factor VIII (FVIII)-neutralizing antibodies ("inhibitors") are a serious problem in hemophilia A (HA). The aim of this study was to characterize HLA-restricted T-cell responses from a severe HA subject with a persistent inhibitor and from 2 previously studied mild HA inhibitor subjects. Major histocompatibility complex II tetramers corresponding to both of the severe HA subject's HLA-DRA-DRB1 alleles were loaded with peptides spanning FVIII-A2, C1, and C2 domains. Interestingly, only 1 epitope was identified, in peptide FVIII2194-2213, and it was identical to the HLA-DRA*01-DRB1*01:01-restricted epitope recognized by the mild HA subjects. Multiple T-cell clones and polyclonal lines having different avidities for the peptide-loaded tetramer were isolated from all subjects. Only high- and medium-avidity T cells proliferated and secreted cytokines when stimulated with FVIII2194-2213 T-cell receptor ß (TCRB) gene sequencing of 15 T-cell clones from the severe HA subject revealed that all high-avidity clones expressed the same TCRB gene. High-throughput immunosequencing of high-, medium-, and low-avidity cells sorted from a severe HA polyclonal line revealed that 94% of the high-avidity cells expressed the same TCRB gene as the high-avidity clones. TCRB sequencing of clones and lines from the mild HA subjects also identified a limited TCRB gene repertoire. These results suggest a limited number of epitopes in FVIII drive inhibitor responses and that the T-cell repertoires of FVIII-responsive T cells can be quite narrow. The limited diversity of both epitopes and TCRB gene usage suggests that targeting of specific epitopes and/or T-cell clones may be a promising approach to achieve tolerance to FVIII.
Assuntos
Epitopos de Linfócito T , Fator VIII , Hemofilia A , Receptores de Antígenos de Linfócitos T alfa-beta , Linfócitos T/imunologia , Adolescente , Autoanticorpos/genética , Autoanticorpos/imunologia , Inibidores dos Fatores de Coagulação Sanguínea/genética , Inibidores dos Fatores de Coagulação Sanguínea/imunologia , Criança , Pré-Escolar , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Fator VIII/genética , Fator VIII/imunologia , Antígenos HLA/genética , Antígenos HLA/imunologia , Hemofilia A/genética , Hemofilia A/imunologia , Humanos , Masculino , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologiaRESUMO
Inhibitor development is the most serious complication in patients with hemophilia. We investigated association of HLA genotypes with inhibitor development in Korean patients with severe hemophilia A (HA). HLA genotyping was done in 100 patients with severe HA including 27 patients with inhibitors. The allele frequencies between inhibitor-positive and inhibitor-negative patients were compared. HLA class I alleles were not associated with the inhibitor status. In HLA class II, DRB1*15 [n = 100, odds ratio (OR) 0.217, P = 0.028] and DPB1*05:01 [OR 0.461, P = 0.026] were negatively associated with inhibitor development. In a subgroup of patients with intron 22 inversion, C*07:02 was positively associated with inhibitor development [n = 30, OR 5.500, P = 0.043]. In the subgroup of patients without intron 22 inversion, the negative association between DPB1*05:01 and inhibitor development was reinforced [n = 70, OR 0.327, P = 0.010], and positive association of DRB1*13:02 and DPB1*04:01 with inhibitor development was identified [OR 3.059, P = 0.037 for both]. Previously reported risk alleles were not consistently associated with inhibitor risk in our series. This study demonstrated the profile of HLA alleles associated with inhibitor risk in Korean patients with severe HA was different from that in patients of other ethnicities, which needs to be considered in risk assessment and management.
Assuntos
Inibidores dos Fatores de Coagulação Sanguínea/sangue , Genótipo , Antígenos HLA-C/genética , Cadeias HLA-DRB1/genética , Hemofilia A/sangue , Hemofilia A/genética , Adolescente , Adulto , Idoso , Povo Asiático , Inibidores dos Fatores de Coagulação Sanguínea/genética , Criança , Pré-Escolar , Feminino , Hemofilia A/epidemiologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , República da Coreia/epidemiologia , Fatores de RiscoRESUMO
The two plasma inhibitors, protein Z-dependent protease inhibitor (ZPI) and tissue factor pathway inhibitor (TFPI), effectively inhibit the activity of activated factor X (FXa); however, neither inhibitor exhibits any reactivity with the homologous protease activated factor IX (FIXa). In this study, we investigated the molecular basis for the lack of reactivity of FIXa with these plasma inhibitors and discovered that unique structural features within residues of the 39-loop are responsible for restricting the inhibitor specificity of FIXa. This loop in FXa is highly acidic and contains three Glu residues at positions 36, 37, and 39. On the other hand, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. We discovered that replacing residues of the 39-loop (residues 31-41) of FIXa with corresponding residues of FXa renders the FIXa chimera susceptible to inactivation by both ZPI and TFPI. Thus, the inactivation rate of the FIXa chimera by ZPI in the presence of protein Z (PZ), negatively charged membrane vesicles, and calcium ions approached the same diffusion-limited rate (>10(7) m(-1) s(-1)) that has been observed for the PZ-dependent inhibition of FXa by ZPI. Interestingly, sequence alignments indicated that, similar to FXa, residue 36 is a Glu in both mouse and bovine FIXa and that both proteases are also susceptible to inhibition by the PZ-ZPI complex. These results suggest that structural features within residues of the 39-loop contribute to the resistance of FIXa to inhibition by plasma inhibitors ZPI and TFPI.
Assuntos
Inibidores dos Fatores de Coagulação Sanguínea/metabolismo , Fator IXa/metabolismo , Lipoproteínas/metabolismo , Serpinas/metabolismo , Animais , Inibidores dos Fatores de Coagulação Sanguínea/química , Inibidores dos Fatores de Coagulação Sanguínea/genética , Bovinos , Fator IXa/química , Fator IXa/genética , Fator Xa/química , Fator Xa/genética , Fator Xa/metabolismo , Humanos , Lipoproteínas/química , Lipoproteínas/genética , Camundongos , Estrutura Secundária de Proteína , Alinhamento de Sequência , Serpinas/química , Serpinas/genéticaRESUMO
BACKGROUND: Black patients with hemophilia A (factor VIII deficiency) are twice as likely as white patients to produce inhibitors against factor VIII proteins given as replacement therapy. There are six wild-type factor VIII proteins, designated H1 through H6, but only two (H1 and H2) match the recombinant factor VIII products used clinically. H1 and H2 are found in all racial groups and are the only factor VIII proteins found in the white population to date. H3, H4, and H5 have been found only in blacks. We hypothesized that mismatched factor VIII transfusions contribute to the high incidence of inhibitors among black patients. METHODS: We sequenced the factor VIII gene (F8) in black patients with hemophilia A to identify causative mutations and the background haplotypes on which they reside. Results from previous Bethesda assays and information on the baseline severity of hemophilia, age at enrollment, and biologic relationships among study patients were obtained from review of the patients' medical charts. We used multivariable logistic regression to control for these potential confounders while testing for associations between F8 haplotype and the development of inhibitors. RESULTS: Of the 78 black patients with hemophilia enrolled, 24% had an H3 or H4 background haplotype. The prevalence of inhibitors was higher among patients with either of these haplotypes than among patients with haplotype H1 or H2 (odds ratio, 3.6; 95% confidence interval, 1.1 to 12.3; P=0.04), despite a similar spectrum of hemophilic mutations and degree of severity of illness in these two subgroups. CONCLUSIONS: These preliminary results suggest that mismatched factor VIII replacement therapy may be a risk factor for the development of anti-factor VIII alloantibodies.
Assuntos
População Negra/genética , Inibidores dos Fatores de Coagulação Sanguínea/imunologia , Fator VIII/genética , Fator VIII/imunologia , Hemofilia A/etnologia , Hemofilia A/imunologia , Adolescente , Adulto , Sequência de Aminoácidos , Anticorpos , Inibidores dos Fatores de Coagulação Sanguínea/genética , Criança , Pré-Escolar , Fator VIII/uso terapêutico , Haplótipos , Hemofilia A/genética , Hemofilia A/terapia , Humanos , Isoanticorpos , Masculino , Mutação , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Adulto JovemRESUMO
Development of inhibitors to factor VIII, a serious complication of replacement therapy in haemophilia A patients, leads to increased bleeding, morbidity and mortality. There is no data on the risk factors for inhibitor development in Indian patients with severe haemophilia A. Our aim was to study the role of immune regulatory gene polymorphisms in the development of inhibitors. Fourteen immune regulatory gene polymorphisms (IL1ß, IL4, IL10, TNFA and CTLA4) were analysed in 120 patients with severe haemophilia A, i.e. 50 inhibitor positive patients, and 70 inhibitor negative control patients, by PCR-RFLP, DNA sequencing and allele-specific PCRs. The IL10 promoter 'GCC' haplotypes overall (P: 0.002, OR: 3.452, 95% CI: 1.607-7.416), and 'GCC/ATA' (P: 0.011, OR: 3.492, 95% CI: 1.402-8.696) haplotype, associated with high and intermediate IL10 production, respectively, were significantly higher in inhibitor positive patients, whereas the 'non-GCC' haplotypes overall (P: 0.002,OR: 0.290, 95% CI 0.135-0.622) and 'ATA/ATA' haplotype (P: 0.025, OR: 0.278, 95% CI: 0.096-0.802), associated with low IL10 synthesis, were significantly higher among inhibitor negative patients. The TNFA rs1799724 C/T heterozygote prevalence was significantly higher in the inhibitor positive group (P: 0.021, OR: 3.190, 95% CI: 1.273-7.990), whereas the other polymorphisms showed no statistically significant association with the presence of inhibitors. Different immune regulatory gene polymorphisms play a significant role as possible risk factors for the development of inhibitors in severe haemophilia A patients.
Assuntos
Inibidores dos Fatores de Coagulação Sanguínea/genética , Inibidores dos Fatores de Coagulação Sanguínea/imunologia , Fator VIII/antagonistas & inibidores , Fator VIII/imunologia , Hemofilia A/genética , Hemofilia A/imunologia , Polimorfismo Genético , Adolescente , Adulto , Inibidores dos Fatores de Coagulação Sanguínea/sangue , Antígeno CTLA-4/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , Predisposição Genética para Doença , Haplótipos , Hemofilia A/sangue , Humanos , Índia , Interleucina-10/genética , Interleucina-1beta/genética , Interleucina-4/genética , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Fatores de Risco , Fator de Necrose Tumoral alfa/genética , Adulto JovemRESUMO
The mechanisms of Ebola (EBOV) pathogenesis are only partially understood, but the dysregulation of normal host immune responses (including destruction of lymphocytes, increases in circulating cytokine levels, and development of coagulation abnormalities) is thought to play a major role. Accumulating evidence suggests that much of the observed pathology is not the direct result of virus-induced structural damage but rather is due to the release of soluble immune mediators from EBOV-infected cells. It is therefore essential to understand how the candidate therapeutic may be interrupting the disease process and/or targeting the infectious agent. To identify genetic signatures that are correlates of protection, we used a DNA microarray-based approach to compare the host genome-wide responses of EBOV-infected nonhuman primates (NHPs) responding to candidate therapeutics. We observed that, although the overall circulating immune response was similar in the presence and absence of coagulation inhibitors, surviving NHPs clustered together. Noticeable differences in coagulation-associated genes appeared to correlate with survival, which revealed a subset of distinctly differentially expressed genes, including chemokine ligand 8 (CCL8/MCP-2), that may provide possible targets for early-stage diagnostics or future therapeutics. These analyses will assist us in understanding the pathogenic mechanisms of EBOV infection and in identifying improved therapeutic strategies.
Assuntos
Ebolavirus/imunologia , Predisposição Genética para Doença , Genoma , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/terapia , Transcrição Gênica , Animais , Coagulação Sanguínea/genética , Inibidores dos Fatores de Coagulação Sanguínea/genética , Inibidores dos Fatores de Coagulação Sanguínea/metabolismo , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Doença pelo Vírus Ebola/genética , Ativação Linfocitária/genética , Macaca mulatta , Família Multigênica , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Thrombophilia screening is controversial. In a retrospective family cohort, where probands had thrombosis and a thrombophilic defect, 2479 relatives were tested for thrombophilia. In antithrombin-, protein C-, and protein S-deficient relatives, annual incidences of venous thrombosis were 1.77% (95% CI, 1.14-2.60), 1.52% (95% CI, 1.06-2.11), and 1.90% (95% CI, 1.32-2.64), respectively, at a median age of 29 years and a positive family history of more than 20% symptomatic relatives. In relatives with factor V (FV) Leiden, prothrombin 20210G>A, or high FVIII levels, these were 0.49% (95% CI, 0.39-0.60), 0.34% (95% CI, 0.22-0.49), and 0.49% (95% CI, 0.41-0.51), respectively. High FIX, FXI, and TAFI, and hyperhomocysteinemia were not independent risk factors. Annual incidence of major bleeding in antithrombin-, protein C-, or protein S-deficient relatives on anticoagulants was 0.29% (95% CI, 0.03-1.04). Cumulative recurrence rates in relatives with antithrombin, protein C, or protein S deficiency were 19% at 2 years, 40% at 5 years, and 55% at 10 years. In relatives with FV Leiden, prothrombin 20210G>A, or high levels FVIII, these were 7%, 11%, and 25%, respectively. Considering its clinical implications, thrombophilia testing should address hereditary deficiencies of antithrombin, protein C, and protein S in patients with first venous thrombosis at young age and/or a strong family history of venous thrombosis.
Assuntos
Trombofilia/diagnóstico , Trombose Venosa/epidemiologia , Trombose Venosa/etiologia , Adulto , Inibidores dos Fatores de Coagulação Sanguínea/genética , Fatores de Coagulação Sanguínea/genética , Transtornos de Proteínas de Coagulação/genética , Estudos de Coortes , Saúde da Família , Predisposição Genética para Doença , Humanos , Incidência , Países Baixos/epidemiologia , Estudos Retrospectivos , Risco , Trombofilia/epidemiologia , Trombofilia/genética , Trombose Venosa/sangueRESUMO
Continuous expression of activated factor VII (FVIIa) via gene transfer is a potential therapeutic approach for hemophilia patients with or without inhibitory antibodies to human factor VIII (FVIII) or IX (FIX). Here, we investigate whether gene transfer of an engineered canine FVIIa (cFVIIa) transgene can affect hemostasis in a canine model of hemophilia, a good predictor of efficacy of hemophilia treatments. Purified recombinant cFVIIa exhibited 12-fold higher tissue factor-dependent activity than purified recombinant zymogen cFVII. Subsequently, we generated a serotype 8 recombinant adeno-associated viral vector expressing cFVIIa from a liver-specific promoter. Vector delivery via the portal vein in hemophilia A and B dogs was well tolerated, and long-term expression of cFVIIa resulted in a shortening of the prothrombin time, partial correction of the whole blood clotting time and thromboelastography parameters, and a complete absence of spontaneous bleeding episodes. No evidence of hepatotoxicity, thrombotic complications, or inhibitory immune response was found. These data provide the first evidence for in vivo efficacy and safety of continuously expressed FVIIa as a FVIII/FIX-bypassing agent in a large animal model of hemophilia, avoiding the risk of inhibitor formation associated with bolus FVIII or FIX infusion.
Assuntos
Dependovirus , Expressão Gênica , Terapia Genética , Hemofilia A/terapia , Animais , Inibidores dos Fatores de Coagulação Sanguínea/genética , Inibidores dos Fatores de Coagulação Sanguínea/metabolismo , Modelos Animais de Doenças , Cães , Fator VIIa , Hemofilia A/genética , Hemofilia A/metabolismo , Hemorragia/genética , Hemorragia/metabolismo , Hemorragia/terapia , Humanos , Fígado/metabolismo , Especificidade de Órgãos , Regiões Promotoras Genéticas , Tempo de Protrombina , TromboelastografiaRESUMO
Several genes that modify risk of factor VIII (FVIII) inhibitors in haemophilia A patients have been identified. Aside from the underlying mutations that cause haemophilia A, inhibitor risk appears to be modified by polymorphisms in various cytokines and immunomodulators including IL10, TNFα and CTLA4. HLA haplotypes have not been strong determinants of inhibitor risk. We sought to confirm previous observations on FVIII inhibitor risk-modifying genes and to test new candidate genes encoding various otherTH1/TH2 cytokines. We also sought to determine whether normal FVIII gene polymorphisms affect inhibitor risk in caucasians. We studied 915 caucasian, severe haemophilia A patients (282 inhibitor cases and 633 non-inhibitor controls). Genes were analysed using 368 tagging single nucleotide polymorphisms starting 20 kb 5' and ending 10 kb 3' of each gene's coding sequence; four other polymorphisms (factor V Leiden & prothrombin 20210 polymorphisms and two in HFE) were also evaluated. Haplotypes that increased inhibitor risk were found in IL10 (OR = 1.33, P = 0.04), IL12 (OR = 1.31, P = 0.04) and IL1α (OR = 2.16, P = 0.034). Protective haplotypes were seen in IL2 (OR = .69, P = 0.008) and IL1ß (OR = 0.75, P = 0.02). One rare haplotype in the FVIII gene increased the risk of inhibitor development by nearly fourfold (OR = 3.8, P = 0.004). We replicate previous findings for IL10; identify new associations with IL1, IL2 and IL12; and identify a rare FVIII haplotype in caucasians that is associated with increased inhibitor risk.
Assuntos
Inibidores dos Fatores de Coagulação Sanguínea/genética , Haplótipos/genética , Hemofilia A/genética , Hemofilia A/imunologia , Interleucinas/genética , Adolescente , Adulto , Estudos de Casos e Controles , Genótipo , Humanos , Polimorfismo Genético/genética , Adulto JovemRESUMO
UNLABELLED: The development of inhibitors in haemophilia B is one of the most important complications of replacement therapy, affecting mortality and morbidity. Inhibitor development is based on complex immunological factors, and to date, only little is known about its underlying mechanisms. Here, we present first results of the haemophilia B group of our Inhibitor-Immunology study. PATIENTS, METHODS: So far we have analysed 15 patients with haemophilia B. Four of them developed a high titre inhibitor; the remaining 11 had no inhibitor. We evaluated 9 SNPs in 8 genes (CD40, CTLA-4 , IL-1ß, IL-10, TLR2 , TLR4, TLR9, TNF-α). We compared the distribution of these alleles between inhibitor and non-inhibitor haemophilia B patients and between haemophilia B patients and a normal male control population. HLA typing was performed in all patients. Results, discussion: There appears to be a trend towards a skewed distribution of TLR 9, IL-10 and CTLA4 alleles in haemophilia B patients. Due to the limited number these differences are, however, not statistically significant. The t-test of all patients with inhibitor versus without inhibitor was significant for HLA-A*03 and DPB1*0401 and borderline for DRB1*0201.
Assuntos
Inibidores dos Fatores de Coagulação Sanguínea/sangue , Inibidores dos Fatores de Coagulação Sanguínea/genética , Genes MHC da Classe II/genética , Predisposição Genética para Doença/genética , Hemofilia B/sangue , Hemofilia B/genética , Polimorfismo de Nucleotídeo Único/genética , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Adulto JovemRESUMO
Inhibitors of factor VIII (FVIII) remain the most challenging complication of FVIII protein replacement therapy in hemophilia A (HA). Understanding the mechanisms that guide FVIII-specific B cell development could help identify therapeutic targets. The B cell-activating factor (BAFF) cytokine family is a key regulator of B cell differentiation in normal homeostasis and immune disorders. Thus, we used patient samples and mouse models to investigate the potential role of BAFF in modulating FVIII inhibitors. BAFF levels were elevated in pediatric and adult HA inhibitor patients and decreased to levels similar to those of noninhibitor controls after successful immune tolerance induction (ITI). Moreover, elevations in BAFF levels were seen in patients who failed to achieve FVIII tolerance with anti-CD20 antibody-mediated B cell depletion. In naive HA mice, prophylactic anti-BAFF antibody therapy prior to FVIII immunization prevented inhibitor formation and this tolerance was maintained despite FVIII exposure after immune reconstitution. In preimmunized HA mice, combination therapy with anti-CD20 and anti-BAFF antibodies dramatically reduced FVIII inhibitors via inhibition of FVIII-specific plasma cells. Our data suggest that BAFF may regulate the generation and maintenance of FVIII inhibitors and/or anti-FVIII B cells. Finally, anti-CD20/anti-BAFF combination therapy may be clinically useful for ITI.
Assuntos
Fator Ativador de Células B/imunologia , Inibidores dos Fatores de Coagulação Sanguínea/imunologia , Fator VIII/imunologia , Hemofilia A/imunologia , Adolescente , Adulto , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Fator Ativador de Células B/genética , Inibidores dos Fatores de Coagulação Sanguínea/genética , Criança , Pré-Escolar , Fator VIII/antagonistas & inibidores , Fator VIII/genética , Fator VIII/uso terapêutico , Feminino , Células HEK293 , Hemofilia A/tratamento farmacológico , Hemofilia A/genética , Humanos , Tolerância Imunológica/efeitos dos fármacos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Pessoa de Meia-IdadeRESUMO
The formation of antibodies against factor VIII or factor IX that inhibit replacement therapy is currently the most serious treatment-related complication faced by patients with haemophilia. This review highlights non-modifiable and modifiable risk factors that determine the development of these antibodies. The non-modifiable risk factors include patient genotype for haemophilia, immunogenotype, ethnicity and positive family history. Age, intensity of treatment and the type of clotting factor administered are identified as modifiable risk factors. These risk factors are likely to be identified more accurately in forthcoming prospective randomized controlled trials and current patient registries. Through a more complete picture of a patient's overall risk profile, individually tailored treatment schedules might be developed that could minimize the incidence of inhibitor formation and thus maximize therapeutic benefit.
Assuntos
Inibidores dos Fatores de Coagulação Sanguínea , Hemofilia A/imunologia , Fatores Etários , Inibidores dos Fatores de Coagulação Sanguínea/genética , Citocinas/imunologia , Fator IX/uso terapêutico , Fator VIII/uso terapêutico , Genótipo , Hemofilia A/tratamento farmacológico , Hemofilia A/etnologia , Hemofilia A/genética , Humanos , Lactente , Recém-Nascido , Fatores de RiscoRESUMO
Inhibitor development, because of its impact on patients' morbidity and quality of life, is presently the most serious complication of haemophilia A treatment. The identification of several genetic and non-genetic risk factors may be used for the stratification of inhibitor risk and the definition of prevention strategies, particularly for patients with a high-risk genetic profile. The most extensively studied genetic factor is the type of F8 mutation, i.e. large deletions, nonsense mutations and inversions, which are associated with a higher risk of inhibitor development. This is the basis for the increased risk in patients with inhibitor family history; however, concordance family studies showed that factors other than F8 mutations are involved. An emerging role is investigated for polymorphisms of immune-regulatory genes that may increase (IL-10 and TNF-alpha) or reduce (CTLA-4) inhibitor risk and whose heterogeneous ethnic distribution may correlate to the higher inhibitor risk in non-caucasian patients. A role for FVIII haplotypes, particularly in black haemophiliacs, has been recently proposed. Recent studies report an increased inhibitor risk for initial intensive treatments (surgery or severe bleeds requiring high-dose and/or prolonged treatment, presence of danger signals), whereas regular prophylaxis (absence of danger signals) exerts a protective effect. A clinical score including the type of F8 mutation, family history of inhibitors and intensive treatment has been recently validated for predicting inhibitor risk. Because of the lack of useful data regarding the role of different types of FVIII concentrates, the stratification of risk in patients starting replacement treatment together with the careful evaluation of indications, doses and duration of treatment at first exposures and further efforts for overcoming barriers to early implementation of prophylaxis are encouraged, particularly for patients with a predictable high inhibitor risk.
Assuntos
Inibidores dos Fatores de Coagulação Sanguínea/genética , Fator VIII/genética , Fator VIII/administração & dosagem , Fator VIII/uso terapêutico , Predisposição Genética para Doença , Genótipo , Hemofilia A/tratamento farmacológico , Hemofilia A/genética , Humanos , Mutação , Fatores de RiscoRESUMO
The prevalence of inhibitors in Chinese haemophiliacs has not yet been reported. The aim of this study was to identify the prevalence of factor VIII (FVIII) inhibitors among haemophiliacs who are treated only with plasma-derived FVIII (pdFVIII), cryoprecipitate or fresh frozen plasma (FFP), and tried to explore the relationship between the generation of inhibitors and particular FVIII deficiency genotypes. Clinical information and blood samples of 1435 patients with haemophilia A (HA) were collected by six haemophilia centres in China. The Nijmegen modification of the Bethesda assay was used to detect inhibitors. Multiplex PCR, long-range PCR and direct sequencing were performed for genotyping. The overall prevalence of inhibitors in Chinese HA patients was 3.9% and the prevalence of severe haemophiliacs was 4.3%; 18 of the 56 patients with inhibitors had high titres. A total of 38 different mutations were identified in the 55 patients with inhibitors, including 15 intron 22 and 3 intron 1 inversions, seven large deletions, 14 small deletion/insertions, seven nonsense mutations, one splice site mutations and eight missense mutations. Of 38 mutations, 28 were novel. Patients with large deletions and nonsense mutations were prone to have high titre inhibitors, with incidence rates of 57.1% (4/7) and 42.9% (3/7), respectively. In conclusion, the prevalence of inhibitors in Chinese HA patients is much lower than that reported for other ethnic groups and the large deletion and nonsense mutations are high risk factors for high titre inhibitor development.
Assuntos
Povo Asiático/genética , Inibidores dos Fatores de Coagulação Sanguínea/análise , Inibidores dos Fatores de Coagulação Sanguínea/genética , Fator VIII/antagonistas & inibidores , Hemofilia A/genética , Hemofilia A/imunologia , Mutação , China/etnologia , Análise Mutacional de DNA , Genótipo , Hemofilia A/epidemiologia , Humanos , PrevalênciaRESUMO
One of the most severe and important complication in the treatment of patients with haemophilia A is the formation of neutralizing antibodies (FVIII inhibitors) that inhibit the clotting activity of substituted FVIII. Both genetic and environmental factors influence the susceptibility of patients to develop inhibitors. The objective of this study was to evaluate whether polymorphisms in different genes involved in the regulation of the immune system may confer susceptibility to inhibitor development in patients with HA. We analysed the distribution of polymorphisms in the CTLA4, PTPN22, IL10, TNFalpha, FOXP3 and IRF5 genes that have been reported to be associated with a number of autoimmune disease. In addition, we evaluated the distribution of IL10 haplotypes in haemophilic patients and healthy controls to assess whether specific polymorphisms in IL10 gene were associated to the risk of inhibitor development. We focused on a cohort of Italian unrelated haemophilic patients with and without a history of inhibitors. Genotyping was carried out with standard methods including RFLP, real time PCR and direct DNA sequencing. Our data show that, considering single nucleotide variations, genotype frequencies in patients with inhibitors were not significantly different from those observed in patients without inhibitors, suggesting a lack of association between these polymorphisms and the development of inhibitors. Moreover, no relationship was found between specific combinations of IL10 alleles and the antibody production. Previous contradictory association studies may depend on the different genetic background of the population examined. Further studies may contribute to a clearer understanding of this process.