A design pilot study proposed as a Canadian, multicenter, open-label, randomized waitlist clinical trial over the duration of six months to determine the success of running a full-scale RCT with the newly designed WAPPS-Hemo calculator in the Canadian Hemophilia community. The WAPPS-Hemo calculator is widely viewed as a beneficial tool to clinicians and patients and has the potential to optimize and tailor the prophylaxis regimen for hemophilia patients. / Thesis / Master of Science (MSc)

Thesis (PhD)--University of South Australia, 2000

No description available.

Hemophilia A is an X-linked recessive bleeding disorder caused by the deficiency of coagulation factor VIII [1]. Current treatment for hemophilia A consists of prophylactic or on demand replacement therapy of either plasma-derived or recombinant FVIII concentrates [2]. Albeit effective, there are several limitations associated with factor concentrates, including high cost that limits its availability for close to 80% of hemophilia patients in developing countries [3-5]. An alternative treatment would thus be desirable. Gene therapy for hemophilia has seen many successes in animal models and represents a more cost-effective alternative to the current treatment modalities [6]. In the current work, I present a gene therapy system for hemophilia that uses mouse fetal myoblasts engineered to secrete FVIII, enclosed in immuno-protective alginate-poly-L-lysine-alginate (APA) microcapsules, as a sustained source of FVIII. In this study, a thorough examination of the encapsulated myoblasts using a novel flow cytometry assay was performed. This method yielded an accurate and precise method for encapsulated cell viability calculation, and also allowed for analysis of several other parameters such as health (cell morphology), cell size and distribution. Flow cytometry was also used to monitor the time-course proliferation profile of encapsulated myoblasts secreting cFVIII, using the division tracking dye CFSE. We found that encapsulated cells display a decreased proliferation rate as well as lower viability than non-encapsulated cells. Implantation of encapsulated G8 myoblasts secreting cFVIII into hemophilia A mice resulted in maximum plasma levels of protein on day 1 ( ~18% of normal canine FVIII levels). Delivery of cFVIII in hemophilic mice also offered protection against blood loss after the mice were subjected to injury (as measured by hematocrit levels); indicating that biologically functional cFVIII continued for at least 7 days post-capsule implantation. Low levels of FVIII antigen returned on day 28 after a transient disappearance on day 14. However, the presence of antigen must be reconciled with appearance of anti-cFVIII antibodies that were detected in the plasma of treated mice at the end of five weeks. The neutralizing nature of these antibodies still needs to be characterized by Bethesda assay Overall, our study demonstrates the feasibility of delivering therapeutic levels of FVIII using encapsulated G8 fetal myoblasts. The presence of functional FVIII protein on day 7, suggests that this treatment was not met by transcriptional repression in vivo, thereby overcoming one of the major obstacles faced by using the transformed C2C12 cell line secreting hFVIII [7] If such levels of FVIII were achieved in humans, it would be sufficient to convert a severe hemophiliac into a mild phenotype. Thus, this gene therapy strategy may be a suitable therapeutic alternative for hemophilia patients. Further work ought to focus on the long-term persistence of FVIII in hemophilia A mice, and also determining the protection following trauma over time to determine if the FVIII remains functional. Other cell lines should be explored for higher expression, reduced immunogenicity and improved viability Still, there is a need to develop human cells expressing high levels of biologically active hFVIII with similar properties to the fetal cells described in this study [8, 9] / Thesis / Master of Applied Science (MASc)

Thesis (Doctoral)--Department of Pediatrics, University Hospital of Malmö, University of Lund. / Added t.p. with thesis statement inserted. Summary in Swedish. Includes bibliographical references.

Thesis (Doctoral)--Department of Pediatrics, University Hospital of Malmö, University of Lund. / Added t.p. with thesis statement inserted. Summary in Swedish. Includes bibliographical references.

Factor VIII (FVIII) is an essential blood-clotting protein and mutations in the FVIII gene are the cause of hemophilia A, a severe inherited bleeding disorder. FVIII synthesis has been observed in discreet endothelial sub-populations including liver sinusoidal endothelial cells and in selected microvascular beds. The mechanistic basis for this differential expression is unknown. Differences in shear stress are believed to play an important role in determining endothelial heterogeneity. In this study, we have evaluated the effect of various shear stress conditions on FVIII expression in blood outgrowth endothelial progenitor cells (BOECs) with an in vitro flow system. Under static conditions, BOECs do not express FVIII. In contrast, after exposure to laminar shear stress for 48 hrs, a significant increase in FVIII expression was documented by qRT-PCR, regardless of the magnitude of shear stress studied (1, 5, 15 and 30 dynes/cm2). To determine the effect of prolonged shear stress, laminar flow was applied over 120 hrs and FVIII mRNA levels returned to static levels. Induction of gene expression by laminar shear stress followed by repression after longer durations is common to other pro-coagulant genes induced by non-laminar or oscillatory flow (eg. tissue factor). BOECs exposed to 15 dyne/cm2 of shear stress, oscillating every 0.5 sec for 120 hrs, had FVIII mRNA levels 4.7-fold that of cells in static conditions. This was significantly higher than FVIII expression in BOECs exposed to 15 dyne/cm2 of laminar shear stress for the same duration. Expression of KLF2, a transcription factor that suppresses endothelial pro-coagulant gene expression under laminar shear stress, was significantly reduced in BOECs exposed to oscillatory as opposed to laminar shear stress. Finally, in BOECs exposed to oscillatory shear stress, FVIII protein was synthesized and co-localizes with its carrier protein VWF in Weibel-Palade bodies. These studies show that shear stress is a significant regulator of FVIII expression in BOECs, that FVIII expression is inversely correlated with that of KLF2, and that FVIII protein co-localizes with VWF in these cells. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-03-04 17:00:27.994

Among the complications of current treatments for hemophilia A, the development of anti-FVIII antibodies including “FVIII inhibitors” remains the major clinical problem in treating hemophiliacs. Factor VIII inhibitors work through neutralizing the coagulation cofactor activity of the infused FVIII and preventing the restoration of normal hemostasis. This thesis explains the influence of genetic background on the generation of FVIII inhibitors, introduces a new pre-clinical approach that reduces the immunological response towards FVIII and predicts the in vivo behavior of recombinant and plasma-derived FVIII products in hemophilic patients. First, we studied the influence of the genetic background on the formation of FVIII antibodies by treating hemophilia A Balb/c and C57BL/6 mice with repetitive FVIII infusions. We observed that the C57BL/6 mice developed higher FVIII antibody titers than the Balb/c mice. Our results suggest that differences in the cytokine immune responses due to FVIII in Balb/c and C57BL/6 mice are responsible for the different FVIII antibody titers in each of these strains. Second, we investigated the use of FVIII-pulsed immature dendritic cells in inducing immune tolerance against FVIII prior to the FVIII treatment. We showed that in vivo, FVIII does not induce the activation and proliferation of hemophilic T cells. Furthermore, infusing FVIII-pulsed immature dendritic cells into hemophilic mice resulted in a long-term reduction in immune reactivity towards FVIII. Also, we have proposed methods on how to improve the tolerogenic abilities of dendritic cells. Our results indicate that the immature dendritic cells induced the formation of T regulatory cells and that these T regulatory cells were responsible for the observed reduction in immune reactivity. Finally, we were able to identify the mechanisms behind the immune system activation in mice treated with either recombinant or plasma-derived FVIII products. We showed that plasma-derived FVIII results in reduced FVIII antibody titer formation in hemophilic mice. Our results demonstrate that the differences in antibody formation in hemophilic mice treated with either recombinant or plasma- derived FVIII products are due to the distinct cytokine micro-environment induced by each product. This thesis contributes to the current knowledge on FVIII immunology and the in vivo behavior of FVIII in hemophilic mice. The results generated from this thesis can be used to modify the available FVIII treatments in order to minimize the immunological complications of FVIII and improve the quality of life of hemophilic patients. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2008-08-14 18:22:51.56

Thesis (M.Med.Sc.) -- University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 2000. / Bibliography: leaves 115-125.

Thesis (Ph. D.)--University of Washington, 1990. / Vita. Includes bibliographical references (leaves [274]-288).