Basic And Clinical Research On Hereditary Coagulation Factor Deficienc

Background:The development of anti-factor Ⅷ(FⅧ)antibodies,termed as inhibitors,is the most challenging therapeutic complication of hemophilia A(HA).Immune tolerance induction is the only widely accepted method for eradicating inhibitors and inducing FⅧimmune tolerance,but it is difficult to administer,incompletely effective and expensive.Human umbilical cord-derived mesenchymal stromal cells(hUC-MSCs)exhibit low immunogenicity and high immunosuppressive property,and have been used to treat various human diseases.Objective:This study aims to clarify whether hUC-MSCs can prevent or eliminate inhibitors in HA mice,and to explore the mechanism.Methods:HA mice were intravenously infused with repeated doses of recombinant human factor Ⅷ(rhFⅧ)to establish inhibitors,with or without simultaneous hUC-MSCs infusion.Inhibitor titers were measured by Bethesda assay and were reported as Bethesda units per milliliter(BU/ml).Flow cytometry analysis was used to analyze the proportion of T follicular helper cells(TFHs)and regulatory T cells(Tregs)in CD4 T cells in mice spleen.Mice plasma total immunoglobulin G(IgG)levels were measured by enzymelinked immunosorbent assay.Results:For mice with pre-existing FⅧ inhibitors,hUC-MSCs could not eradicate or accelerate the eradication of inhibitors and could not inhibit the increase of inhibitor titers when inhibitor-positive mice were restimulated by rhFⅧ.For na(?)ve HA mice,simultaneous infusion of hUC-MSCs with rhFⅧ prevented inhibitor development.After six weeks of treatment,mice treated with hUC-MSC and rhFⅧ,compared to mice treated with rhFⅧ alone,had significantly lower incidence of inhibitors[15.8%(3/19)VS 76.5%(13/17)(P=0.001)]and lower mean(± standard error of mean,SEM)inhibitor titers[13.7±13.5 BU/ml VS 124.8± 43.8 BU/ml(P=0.000)].Mice treated with hUC-MSC and rhFⅧ and with hUC-MSC alone,compared to mice treated with rhFⅧ alone or with PBS,had higher mean(± SEM)percentage of TFHs[hUC-MSC and rhFⅧ(1.0 ± 0.3%)VS rhFⅧ alone(0.3 ± 0.1%):P=1.000,hUC-MSC and rhFⅧ VS PBS(0.2±0.1%):P=1.000.hUC-MSC alone(2.3 ± 0.5%)VS rhFⅧ alone:P=0.040,hUC-MSC alone VS PBS:P=0.027]and lower mean(± SEM)percentage of Tregs[hUC-MSC and rhFⅧ(8.9± 0.4%)VS rhFⅧ alone(12.0 ± 0.4%):P=0.000,hUC-MSC and rhFⅧ VS PBS(11.2± 0.4%):P=0.004,hUC-MSC alone(8.8 ± 0.4%)VS rhFⅧ alone:P=0.000,hUC-MSC alone VS PBS:P=0.009]in CD4 T cells in the spleen,and had significantly higher plasma total IgG levels[hUC-MSC and rhFⅧ(12.9 ± 4.7 mg/ml)VS rhFⅧ alone(1.6±0.3 mg/ml):P=0.000,hUC-MSC and rhFⅧ VS PBS(1.4 ± 0.2 mg/ml):P=0.000,hUC-MSC alone(3.9 ± 0.4 mg/ml)VS rhFⅧ alone:P=0.001,hUC-MSC alone VS PBS:P=0.001].Mouse umbilical cord-derived mesenchymal stromal cells(mUC-MSCs)could not prevent HA mice inhibitor development,and could not elevate mice plasma total IgG levels.But mUC-MSCs could alter the proportion of TFHs and Tregs in CD4 T cells in the spleen.Mice treated with mUC-MSC and rhFⅧ and with mUC-MSC alone,compared to mice treated with rhFⅧ alone or with PBS,had higher mean(± SEM)percentage of TFHs[mUC-MSC and rhFⅧ(2.5 ± 0.2%)VS rhFⅧ alone(0.7 ± 0.1%):P=0.000,mUCMSC and rhFⅧ VS PBS(0.6±0.1%):P=0.000,mUC-MSC alone(2.3±0.2%)VS rhFⅧ alone:P=0.001,mUC-MSC alone VS PBS:P=0.000]and lower mean(± SEM)percentage of Tregs[mUC-MSC and rhFⅧ(10.1±0.3%)VS rhFⅧ alone(12.4 ±0.4%):P=0.008,mUC-MSC and rhFⅧ VS PBS(12.6± 0.6%):P=0.007,mUC-MSC alone(10.7 ± 0.5%)VS rhFⅧ alone:P=0.071,mUC-MSC alone VS PBS:P=0.060]in CD4 T cells in the spleen.Conclusion:hUC-MSCs could not eradicate or accelerate the eradication of established inhibitors in HA mice and could not inhibit the increase of inhibitor titers in inhibitorpositive mice when they were restimulated by rhFⅧ.Simultaneous hUC-MSCs infusion with rhFⅧ prevented inhibitor development in HA mice through immune competition.In HA mice with non-inflammatory internal environment,hUC-MSCs were predominantly immunogenic,not immunosuppressive.Background:Congenital factor Ⅶ(FⅦ)deficiency is a rare bleeding disorder characterized by a wide molecular and clinical heterogeneity.In China,case reports or case series on patients with FⅦ deficiency only came from a limited number of provinces.Objective:In this study,we aimed to investigate the clinical phenotype and F7 genotype of the registered patients with FⅦ deficiency from throughout China and to show their current status of management.Methods:Data of 193 patients with FⅦ deficiency were retrospectively analyzed.Data recorded on each patient included:demographics,patient characteristics,clinical history and bleeding manifestations,gene variants and treatment strategy.Results:The most frequent bleeding symptoms were epistaxis(86,44.6%),cutaneous(75,38.9%),oral cavity bleeding(78,40.4%)and menorrhagia(39,44.3%of females in reproductive age).Fatal central nervous system bleeding and disabling joint bleeding occurred in 3 patients each.The majority of patients(173/193,89.6%)had FⅦ activity(FⅦ:C)≤10%and the proportion of symptomatic patients in this group(138/173,79.8%)was significantly higher than that in the groups with FⅦ:C>10-25%(5/12,41.7%)and>25-50%(3/8,37.5%)(Chi2=13.641,P=0.001).Major bleeds occurred only in patients with FⅦ:C≤10%.In total,fifty-five patients underwent genotype analysis:most variants were missense(62.5%)and most patients had homozygous/compound heterozygous(85.4%)variants.Prothrombin complex concentrates(72.4%)were the most frequently used on-demand replacement therapy.Regular prophylaxis was applied in only five patients,all with severe recurrent hemarthrosis.Prophylaxis before delivery decreased the risk of postpartum bleeding in women(Chi2=69.243,P=0.000).Conclusion:Most patients experienced only minor bleeds.However,life-or limbthreatening hemorrhage also occurred in severe patients with FⅦ:C≤10%.Most F7 variants were missense and most patients were homozygous/compound heterozygous.Prophylactic treatment before delivery could decrease the risk of postpartum bleeding in women with FⅦ deficiency.This study provides useful information on the clinical phenotype,F7 genotype and current status of FⅦ-deficiency patients management,and may promote further exploration of FⅦ deficiency in the future,as well as promotion of care of this population in China.