Study on the Efficacy and Safety of Xueyou Mixture (血友合剂) in Treating Hemophilia

Objective: To observe the effect of Xueyou Mixture (血友合剂, XYM) on blood coagulation factors and its safety in treating hemophilia. Methods: To the randomly selected 65 inpatients of hemophilia, XYM was administered accompanied with intravenous dripping of liver cell growth factor 60-100 mg once a day to protect the liver, with no blood products like concentrated Ⅷ and FⅨ factors or blood plasma given. The treatment lasted for 3 weeks. The short-term efficacy and adverse reactions were observed. The long-term efficacy in patients was observed in a follow-up study of 6-12 months after they were discharged from the hospital but continuously took XYM orally. Results: The short-term markedly effective rate in the patients was 95.38% (62/65). After they were treated for 3 weeks, the level of FⅧ factor activity increased in 56 patients of type A from (3.32 ± 2.21) % to (4.18 ± 2.23) %, and in 9 of type B from (4.92 ± 1.81) % to (5.64 ± 1.96) %. Compared with that before treatment, the difference was significant in both of them (P＜0.01). No obvious adverse reaction was found in the treatment period.The follow-up study showed that in 22 patients of type A, the FⅧ factor activity ratio increased from (3.25±2.11) % to (6.31 ±2.16) %, (8.36±1.05) %, and (16.38±2.71) % in the 2nd, 3rd and 6th month after discharge respectively, all showing significant difference to that before treatment (P＜0.01); and in 4 patients of type B, it increased from (4.15 ± 2.26) % to 7.8% and 11.6% (mean value) in the 2nd and 6th month respectively. Conclusion: XYM could raise the activity of factors Ⅷ and Ⅸ in patients with hemophilia, and the degree of the rise is related with the duration of the therapy, with no obvious adverse reaction, which strikes out a new path and new train of thinking for the treatment of the disease by nonblood preparation.     

Power Doppler sonography in the diagnosis of hemophilic synovitis – a promising tool

Summary. Background: Recurrent hemarthroses in hemophilia results in synovitis and joint arthropathy. Primary prophylaxis when universally instituted at current doses can prevent joint deterioration but is expensive. Alternatively, the selective implementation of prophylaxis would require a more sensitive tool for detecting synovitis than possible with clinical surveillance or plain radiographs. Magnetic resonance imaging (MRI) is such a tool and is utilized for the evaluation of hemophilic joint disease (HJD). However, it is expensive, and requires sedation in younger children precluding its utility for monitoring of synovitis. Ultrasonography (USG) with power Doppler (USG‐PDS) has been utilized to detect and quantitate synovial vascularity in other arthritides and could provide an equally effective but less costly tool for HJD, particularly in children who would not require sedation. Objectives: To determine whether USG‐PDS is comparable to MRI in the evaluation of hemophilic synovitis. Patients: A prospective cohort of 31 subjects including 33 joints (knees, elbows, ankles) underwent dynamic contrast enhanced (DCE)‐MRI and USG‐PDS. Results: USG‐PDS measurements of synovial thickness(r = 0.70, P < 0.0001) and synovial vascularity (r = 0.73, P < 0.0001) correlated strongly with those obtained with DCE‐MRI. A cutoff of PDS intensity of 1.3 decibels (dB) per mm² was found to yield a sensitivity of 100% and a specificity of 94.1% in 17 joints with/without a history of hemarthroses. Pettersson radiographic scores correlated significantly with synovial thickness in adults but not children. Conclusions: Our data suggest that USG‐PDS may be an inexpensive and easily implemented imaging tool for detecting hemophilic synovitis and could be useful in tailoring effective prophylaxis.     

Efficient factor VIII affinity purification using a small synthetic ligand

Summary.  Background:  Hemophilia A is currently treated by infusions of the coagulation factor (F) VIII, of which production and purification remain a challenging task. Current purification procedures using immunoaffinity chromatography are cumbersome, expensive, and suffer from the instability of the applied antibody ligands, which elute along with the product and contaminate it. Recently, FVIII was purified using octapeptide ligands, but their use is limited due to the low resistance to proteases. Objective:  Our goal was to develop and evaluate a novel ligand for FVIII purification, overcoming the drawbacks of current procedures. Methods:  Peptide ligands were screened for binding of ¹²⁵I-plasma-derived-FVIII (pdFVIII) in a microbead assay. A selected ligand-coated Toyopearl resin was then used for pdFVIII purification from cell-conditioned Delbucco's modified Eagle's medium (DMEM) containing fetal bovine serum. The proteolytic stability of ligand was measured by incubating with human serum and proteinase K, and its cytotoxicity towards human OV-MZ-6 cells was assayed. Results:  A high-affinity octapeptidic FVIII ligand was modified into the small, highly stable and non-toxic peptidomimetic ligand L4 by rational and combinatorial design without affecting its affinity for FVIII. Using ligand L4-coated Toyopearl resin, pdFVIII was isolated from cell-conditioned medium with high purity and 89% column retention after elution with a mild buffer containing 0.6  m NaCl at pH 6.8. Conclusions:  Ligand L4 offers a valuable alternative to antibody-based procedures for laboratory and industrial production. Its synthesis by established solid-phase procedures is straightforward and considerably cheaper than the biotechnological production of antibodies, and safety concerns associated with the use of biological material are overcome.     

Hemostatic effect of recombinant factor VIIa, NN1731 and recombinant factor VIII on needle-induced joint bleeding in hemophilia A mice

Summary.  Background:  Hemophilia A is the most common serious bleeding disorder, and the hallmark of this disease is joint bleeding episodes. These result in hemophilic synovitis, an inflammatory and proliferative condition of the joint, which progresses into a chronic degenerative arthritis, hemophilic arthropathy. Methods:  In this paper, we describe the effect of recombinant factor VIIa (rFVIIa), and an analogue NN1731 as well as rFVIII on needle-induced bleeding in hemophilia A mice. Conclusions:  Here we show a reducing effect of rFVIIa and NN1731 on bleeding induced in hemophilic mice, and we show that preventive treatment with rFVIII normalizes bleeding.     

Molecular mechanisms underlying hemophilia A phenotype in seven females

Summary.  Background:  Hemophilia A (HA) in females is a rare observation. Here we describe various genetic mechanisms that result in phenotypic expression of HA in seven females. Methods:  The F8 gene was examined in all patients and relatives by direct sequencing. Multiplex ligation-dependent probe amplification (MLPA) was performed for large deletion screening. X chromosome inactivation was studied by PCR analysis of a polymorphic CAG repeat in the first exon of the human androgen receptor (HUMARA) gene. Results:  In two females sequencing of the F8 gene revealed homozygous missense mutations (Arg593Cys and Tyr1680Phe) as a consequence of consanguineous marriage. The third case was due to compound heterozygosity comprising the missense mutation Leu412Phe inherited from the carrier mother, together with a de novo large deletion spanning exon 9–22, probably originating from the germ cells of the healthy father. Three further cases shared a common mechanism representing heterozygous mutations in the F8 gene (Arg1781His, Arg327His, small deletion in exon 10) combined with non-random inactivation of the X chromosome. The final case describes a coincidental inheritance of HA and Coffin–Lowry syndrome in the same family. The HA phenotype results from a heterozygous small deletion affecting the F8 gene (c.6872 del CT leading to Thr2272 fs ) and a complete inactivation of the maternal X chromosome, which segregates with Coffin–Lowry syndrome in the two brothers of the proposita. Conclusions:  In conclusion, molecular genetic analysis represents an essentially valuable tool in elucidating the nature of the molecular mechanisms underlying the HA phenotype in females.     

Validation of a composite score for clinical severity of hemophilia

Summary.  Introduction:  Evaluation of modulators of the phenotypic expression of hemophilia may benefit from a scoring system that reflects several aspects of the clinical severity instead of only one dimension. Methods:  We describe here how we constructed a composite Hemophilia Severity Score (HSS) and performed validation. The items in the HSS are annual incidence of joint bleeds, World Federation of Hemophilia Orthopedic joint score, and annual factor consumption. The latter two were adjusted for age at start of prophylaxis and body weight. Data for 100 adolescent or adult patients with hemophilia A or B in the mild, moderate or severe form without inhibitors were collected for the 1990–1999 period. We evaluated the reliability (multidimension property, test–retest) and validity (content, convergent, discriminant and known groups) of the score. Results:  The HSS ranged from 0 to 0.94 and was higher in severe hemophilia A than severe hemophilia B (median 0.50 and 0.24; P  = 0.031). The validation indicated that the HSS is reliable and reflective of the clinical severity of hemophilia. The presence of factor V G1691A or prothrombin G20210A polymorphisms was found in 13 patients. The clinical severity, measured as the HSS or each of the three components, appeared to be modified by prothrombin G20210A but not by FV G1691A. Conclusion:  The HSS is a well-defined tool that provides a comprehensive representation of the clinical severity of hemophilia in adults. It would be useful in larger studies on the assessment of modulators of the phenotypic expression of hemophilia.     

用 Ion Torrent 半导体测序技术对血友病 B 家系 F9 基因行突变分析及产前诊断

目的用Ion Torrent半导体测序技术对11个血友病B(HB)家系的F9基因进行序列分析,寻找致病突变,并为遗传咨询和产前诊断提供依据。方法采集11个HB家系先证者及女性成员的外周血样本,抽提基因组DNA,利用Ion Torrent半导体测序技术检测F9基因致病突变,对发现的致病突变进行Sanger测序验证。进一步对7例高危家系孕妇进行羊膜腔穿刺,采集胎儿羊水样本,并进行产前诊断。结果 13例HB患者均存在F9基因致病性突变,共发现10种突变,其中1种为新的突变类型。对8例胎儿进行产前诊断,其中1例为半合子突变,2例为杂合突变,5例不携带母源致病突变。结论用Ion Torrent半导体测序技术检测F9基因的突变谱,对于HB家系携带者的产前诊断具有一定的临床意义。     

Increased clearance of von Willebrand factor antigen post‐DDAVP in Type 1 von Willebrand disease: is it a potential pathogenic process?

Summary.  The mechanism of von Willebrand factor (VWF) clearance is not fully understood. The factors that affect VWF clearance, and the normal in vivo mechanism of clearance, may be relevant to the pathogenesis of Type 1 von Willebrand disease (VWD), in which there is a partial deficiency of VWF. In order to investigate the clearance of VWF in Type 1 VWD, the current study assessed the half-life of VWF antigen ( t _½ VWF:Ag) in Type 1 VWD patients and individuals with mild hemophilia A following the administration of 1-deamino-8- d -arginine vasopressin (DDAVP; desmopressin). To date 20 individuals have been assessed, 13 with Type 1 VWD and seven with mild hemophilia A. The median t _½ VWF:Ag in the Type 1 VWD and mild hemophilia A groups were 4.6 h and 9.5 h, respectively. The difference between the t _½ VWF:Ag for the two groups was significant, P  < 0.02. Analysis of the data showed a correlation between the t _½ VWF:Ag and the baseline VWF:Ag level prior to administration of DDAVP: lower baseline VWF:Ag levels were associated with a shorter t _½ VWF:Ag. These data suggest that increased clearance of VWF may be the pathogenic mechanism in some cases of Type 1 VWD.     

T-cell responses over time in a mild hemophilia A inhibitor subject: epitope identification and transient immunogenicity of the corresponding self-peptide

BACKGROUND: Antibodies that neutralize factor (F) VIII activity, clinically referred to as 'inhibitors', complicate the treatment of hemophilia A patients; current tolerance and bypass strategies are extremely costly and sometimes ineffective. The development of inhibitors requires T-cell help. OBJECTIVES: We characterized T-cell responses of a subject with mild hemophilia A with missense genotype A2201P for one year following his initial inhibitor response, with the goals of defining the primary epitope(s) and its (their) MHC Class II restriction. We investigated the possible involvement of regulatory T cells in modulating immune responses. PATIENTS/METHODS: The subject developed high-titer FVIII-neutralizing antibodies (250 BU mL(-1)) that declined over time to 8 BU ml(-1). His clotting activity was initially impaired (3%) but returned to baseline (8-10%) within four weeks. MHC Class II tetramers were used to analyze his CD4 T cells, which were stimulated with peptides spanning the C2 domain. Responses of total and CD25-depleted CD4 cells to sequences containing A2201 (native), P2201 (hemophilic), and other predicted T-cell epitopes were evaluated. RESULTS AND CONCLUSIONS: An HLA-DRA-DRB1*0101 restricted T-cell epitope containing the wild-type A2201 sequence was identified. Interestingly, peptides containing A2201 were recognized by CD4 T cells at all time points, whereas a P2201 peptide was recognized only near the initial peak response. The responsiveness of CD25-depleted CD4 cells to an A2201 peptide was enhanced 11 and 19 weeks following inhibitor detection, suggesting the possible involvement of CD4+CD25+ regulatory T cells in modulating immune responses. Patient-derived T-cell clones proliferated in response to C2 protein and to peptides containing A2201 but not P2201.     

Reduction of the immune response to factor VIII mediated through tolerogenic factor VIII presentation by immature dendritic cells

Summary. Background: The development of neutralizing antibodies to factor FVIII (FVIII) represents the most serious complication in the treatment of hemophilia A. Objective: We have explored the potential of using immature dendritic cells (iDCs) to present FVIII in a tolerogenic manner to T cells. Methods: The iDCs were isolated from hemophilic murine bone marrow and pulsed with canine cFVIII (cFVIII‐iDCs) in the presence or absence of the NFκB pathway blocking compound Andrographolide (Andro‐cFVIII‐iDCs). Three weekly intravenous infusions of one million cFVIII pulsed‐iDCs were administered to a group of five hemophilic Balb/c mice. Anti‐FVIII antibody levels were monitored by functional Bethesda assay after four weekly intravenous challenges with 2 IU of cFVIII. Results: We have shown that cFVIII in the presence or absence of Andro is efficiently taken up by iDCs and that this process does not result in the maturation of DCs or the activation of co‐cultured T cells. Following repeated infusion of the cFVIII‐iDCs and Andro‐cFVIII‐iDCs into hemophilic mice, which were subsequently challenged with cFVIII, long‐term reductions of FVIII inhibitors of 25% and 40%, respectively, were documented. Studies of cytokine release and T‐cell phenotypes indicate that the mechanisms responsible for reducing immunologic responsiveness to cFVIII appear to involve an expansion of Foxp3 T regulatory cells in the case of cFVIII‐iDC infusion and the elaboration of the immunosuppressive cytokines IL‐10 and TGF‐β following andrographolide‐treated cFVIII‐iDCs. Conclusions: This study shows that tolerogenic presentation of cFVIII to the immune system can significantly reduce immunogenicity of the protein.