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Studies on the Clinical Features、Immunologic Characteristics and FⅧ Binding Epitope in Bullous Pemphigoid Complicating with Acquired FⅧ Inhibitor

Author: ZuoWenLi
Tutor: ZhangGuangSen
School: Central South University
Course: Internal Medicine
Keywords: bullous pemphigoid clinical feature acquired FVⅢinhibitors IgG subclass FVⅢbinding epitope
CLC: R55
Type: PhD thesis
Year: 2006
Downloads: 56
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Research background: Acquired factorⅧ(FⅧ) inhibitor is an uncommon hemorrhagic disorder, which caused by the development of antibodies directed against the coagulation FⅧ. The incidence of acquired FⅧinhibitor has been estimated to be 0.2~1.0 case per 1 million persons per year. Acquired FⅧinhibitor is more frequent in the elderly and affects both sexes equally. Patients with FⅧantibodies hemorrhage into the skin、muscles、gastrointestinal and urological system、prolonged postpartum bleeding and excessive bleeding following trauma or surgery, whereas hemarthroses are unusual. The hemorrhage in acquired FⅧinhibitor is serious or life threatening, patients with anti-FⅧantibodies become resistant to substitution therapy, the mortality rate is as high as 16%~22%. The disease is commonly associated with other underlying medical conditions such as pregnancy and postpartum status、immunological disorders et al. However, in approximately 50% of cases, FⅧautoantibodies occure in patients lacking relevant concomitant diseases. Acquired FⅧinhibitor is rarely found to be associated with bullous pemphigoid. In the literature, only 8 cases with acquired FⅧinhibitor have been described and limited in clinical analysis, however, there is nothing literature similar to our investigation in China. Most FⅧinhibitors were IgG, IgA or IgM were rarely to be reported. The IgG subclass of anti-FⅧantibody have been characterized as being predominantly polyclonal, a prevalence of the IgG4 subclass (although IgG4 constitutes only 4% of serum IgG) often combined with at least one other heavy chain subclass (primarily IgG1). Previous studies have shown that the major inhibitor epitopes were located in the A2、A3、C2 FⅧdomains, majority bound to the 44kD and or 72KD thrombin cleaved fragment. We observed and analyzed the clinical features and laboratory examination and identified the diagnosis of a patient, who suffered from bullous pemphigoid with acquired FⅧinhibitor. Treated the patient with plasmapheresis therapies, the therapeutic efficacy was estimated by observing the change of symptoms、FⅧ:C activity and FⅧinhibitor titre; By FⅧ:C neutralizing test in vitro and "temporary" animal hemophilia model was set up, we not only proved FⅧinhibitor in patient’s plasma was able to inhibit FⅧ:C activity obviously, but also strengthened the clinical diagnosis; we used immuno-nephelometric methods, combined with the immunoblotting assay, and the distribution table of IgG subclass was obtained; By solid-phase binding assay of FⅧand FⅧinhibitor, combined with western blot, the binding epitope at which FⅧinhibitor binded with FⅧwas recognized, thus we elucidated the immune characteristics of FⅧinhibitor and molecular mechanism for FⅧinhibitor pathogeny partly; and provided new data for the clinical features recognizing, laboratory monitoring and reasonable treatment. PartⅠThe clinical features and diagnosis of an acquired FⅧinhibitor in a patient with bullous pemphigoidObjective: To analyze the clinical and laboratory features of an acquired FⅧinhibitor in a patient with bullous pemphigoid, determine the diagnosis of acquired FⅧinhibitor, and to improve the understanding of acquired FⅧinhibitor. Methods:①Collected and analyzed the patient’s case history in detail;②Laboratory tests about hemorrhagic disorder such as activated partial thromboplastin time (APTT)、Prothrombin time (PT)、Thromboplastin generation test(TGT) were performed; The plasma FⅧ:C activity was determined based on One-stage assay; the titre of FⅧinhibitor was evaluated by Bethesda Unit(BU); ) The therapeutic efficacy of immunosuppressant combined with plasmapheresis was estimated by observe the change of clinical phenotype、FⅧ:C activity and FⅧinhibitor titre. Results:①Patient’s clinical manifestation was consistent with the phenotype of FⅧdeficiency, who presented with multiple episodes of bleeding after she was diagnosed with bullous pemphigoid. There was no family or prior bleeding history, so we concluded that the bleeding was acquired;②APTT was 80.5s (normal: 25~35s) and the abnormal APTT could not be corrected by normal plasma; PT was 12.0s (normal, 11~14s); TGT showed FⅧdeficiency, and the FⅧactivity was < 1.5%, FⅧinhibitor titer in patient’s plasma was 147.8 BU;③Patient’s hematoma slowly resolved and skin ecchymoses disappeared, the FⅧ:C activity went up to 13.2% and FⅧinhibitor titer dropped to 28 BU/ml after treated with plasmapheresis and cyclophosphamide. Conclusion: We determined the clinical and laboratory diagnosis of the patient suffered from bullous pemphigoid with acquired FⅧinhibitor; APTT and PT were the preliminary screening tests of hemorrhagic disease, further APTT correct test、TGT、the detection of FⅧ:C activity and FⅧinhibitor titre can determine the diagnosis; Immunosuppressant and plasmapheresis are effective in treating acquired FⅧinhibitor.PartⅡInhibitory effect of FⅧinhibitor on FⅧ:C in normal pooled plasma and rabbit modleObjective: To purify the IgG from patient plasma or normal pooled plasma; To certify the effects of FⅧinhibitor on FⅧ:C activity in normal pooled plasma or rabbits in vivo. Methods: IgG purification was finished by protein A-agrose column chromatograph and identified by SDS-polyacrylamide gel ectrophoresis (SDS-PAGE); Purified patient IgG was added into normal pooled plasma with different dose (Oμg, 6.25μg, 12.5μg, 25μg, 50μg, 75μg), then APTT was assayed for discovering FⅧinhibitor effect on FⅧin vitro, equal amounts of normal human IgG was added into normal pooled plasma as control; Pateint’s plasma was infused into rabbits by intravenous injection and blood sample were collected at different time-point (including pre-infusion 30′, post-infusion 30′、60′、90′、120′separately). Then rabbit plasma APTT values were evaluated, equal volume of normal plasma was infused into rabbits as control. Results:①we got the purified IgG, the molecular weight of purified IgG are 55KD (heavy chain) and 28KD light chain);②The patient’s IgG was able to prolong normal human plasma APTT significantly with a dose-dependent manner, while normal IgG had no effect on normal human plasma APTT;③Animal experiments showed that the patient’s plasma markedly prolonged rabbits plasma APTT in a time-dependent fashion; while normal human plasma did not interfere with rabbits APTT. Conclusion: The FⅧinhibitor in the patient with bullous pemphigoid is IgG; The FⅧinhibitor has significant inhibitory effect on FⅧ:C in vivo or vitro, which strengthened the clinical diagnosis of acquired FⅧinhibitor.PartⅢIgG subclass assay and FⅧbinding epitope identification of acquired FⅧinhibitorObjective: To determine the IgG subclass concentration of patient; To identify FⅧinhibitor IgG subclass and FⅧbinding epitope, and to elucidate the molecular mechanism for FⅧinhibitor pathogeny. Methods:①The determination of IgG subclass concentration by immuno-nephelometric method;②Western blot analysis was used, combined with optical density(OD)scanning for protein band, we determined the relative concentration of patient’s IgG subclass;③The FⅧof thrombin cleaved was run on 8% SDS-PAGE, then separated FⅧfragments were electrophoretically transferred on PVDF membranes, the membranes were incubated with patient’s plasma overnight (solid-phase binding assay of FⅧand FⅧinhibitor), combined with Western blot, the IgG subclass and the binding epitope of FⅧInhibitor was identified; The membranes were incubated with normal human plasma as control. Results:①Immuno-nephelometric method displayed the concentration of IgG4 in patient’s plasma or purified IgG was significantly higher than that in normal plasma or purified IgG, the patient’s IgG1 subclass (both plasma and IgG) was also somewhat higher than that in normal. However, there are not obviously difference in IgG2 and IgG3 between patient and normal (both plasma and IgG);②Western blot analysis combined with OD scanning displayed the patient’s inhibitors were IgG4 predominant, followed by IgG1;③Solid-phase binding method followed by Western blot showed that reactivity of the 44KD FⅧfragment with monoclonal anti-IgG4 and IgG1, but lacked the reactivity with monoclonal anti-IgG2 and IgG3 when incubated PVDF membrane with patient plasma; However, there is not evidence for the binding when incubated PVDF membrane with normal plasma(IgG1、IgG2、IgG3 or IgG4). Conclusion: We definited the IgG subclass of FⅧinhibitor in Chinese patient with bullous pemphigoid, which was IgG4 predominant and IgG1 was secondary; the binding epitope for FⅧinhibitor was identified as FⅧ44KD fragment, the immunologic characteristics and molecular mechanism for acquired FⅧinhibitor pathogeny were elucidated partly.in our case.

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