Proefschrift Universiteit van Amsterdam. / Met lit. opg. - Met samenvatting in het Nederlands.

The development of inhibitory antibodies to the factor VIII (FVIII) protein is the greatest complication in the management of hemophilia A patients. These antibodies, which form in approximately 25% of patients, neutralize the procoagulant activity of FVIII. There are limited treatment options to manage FVIII “inhibitors”, and this significantly increases morbidity within the hemophilia population. Therefore, understanding the immunobiology of FVIII, and developing safe, efficacious therapies to induce immunological tolerance to FVIII is a clinical priority. In 2010, there is no therapy available to prevent the formation of FVIII inhibitors in boys with hemophilia. Therefore, we evaluated the efficacy of anti-CD3 to induce tolerance to FVIII in a prophylactic setting. Low-dose anti-CD3 significantly increased the level CD4+CD25+ T regulatory cells, and prevented formation of inhibitors in >80% in hemophilia A mice. Depleting CD4+CD25+ cells in vivo completely abrogated tolerance. Furthermore, cytokine production by splenocytes from tolerant mice were shifted toward a Th1 response. Anti-CD3 therefore represents one of the most efficacious pre-clinical therapies for FVIII tolerance induction. Surgery is widely regarded in the hemophilia community as a trigger for inducing de novo inhibitor formation. There is, however, only conflicting clinical evidence, and no basic science data to lend support to this clinical hypothesis. Therefore, we developed a novel surgical procedure in hemophilia A mice to study the influence of surgery on FVIII immunogenicity. We found that surgery induced a systemic proinflammatory response (upregulated plasma IL-1 and IL-6), but surprisingly the immunogenicity of FVIII was not enhanced when infused at the perioperative time. These results are significant, however, because they suggest that surgery is not as important for de novo inhibitor formation as previously thought. Finally, it is unknown whether central tolerance to FVIII shapes the peripheral T cell repertoire. Therefore, we studied the murine thymus for evidence of FVIII expression. Whole thymus expressed FVIII mRNA but not protein. FVIII mRNA expression in the thymus was due, at least in part, by the thymic epithelium (CD45-/loEpCAM+). In FVIII-/-AIRE+/-, the immunogenicity of FVIII appeared to be unaltered. This study is the first to investigate a possible role for central tolerance to FVIII. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-09-23 10:22:07.39

Proefschrift Universiteit van Amsterdam. / Met lit. opg. - Met samenvatting in het Nederlands.

No description available.

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

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

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

Thesis (MTech(Medical Technology))--Cape Technikon, 1997 / Cryoprecipitates are used as the raw material for the preparation of Factor VIII (FVIIIl) for replacement therapy for haemophiliacs. Routinely, cryoprecipitate only recovers 50% of the Factor VIII in the plasma. The purpose of this study, production of cryoprecipitate, was to investigate those variables which play a key role in determining the yield of Factor VIII present in cryoprecipitate. Cryoprecipitate production involves a wide range of variables which could effect the final outcome of the product. These vary from the donor blood group, time of donation, exercise levels of the donor, to a time delay prior to processing, temperature storage conditions, to the method utilised for plasma freezing and thawing. The objective was to explore which combination of variables in the procedure would lead to a process which would optimize the preparation of cryoprecipitate in a routine environment, to yield the highest levels of Factor VIII. Frequently in scientific investigations, particularly when a practical approach has to be adopted, questions arise in which the effects of a number of different variables in a process, require evaluation. Such questions can usually be most economically investigated, by arranging the analysis according to an ordered plan in which all the factors are viewed in a regular way. Provided the plan has been correctly chosen, it is possible to determine not only the effect of each individual variable, but also the way in which each effect depends on the other factor, by means of an interaction. This makes it possible to obtain a more complete picture of what is happening, than would have been obtained by varying each of the variables one at a time while keeping the others constant. Designs of this sort lend themselves well to statistical analysis, and provide their own estimates of experimental error. This type of statistical analysis called, 2K Fractional Factorial Experimental Design, forms the basis of this study in which 14 key variables in the production process of cryoprecipitate were defined as possible areas in which Factor VIII levels in the cryoprecipitate are effected. Key variables have been identified on an individual basis in previous studies (Burka et al., 1975), however this blended approach to optimise the key variables within the production environment, and define further combinations which could be incorporated into the production, has never been attempted. The statistical design used in the study was compiled by the Institute for Biostatistics of the Medical Research Council (MRC). Units of blood were collected and processed, from blood donors under the stipulated criteria, corresponding to the study design.

Factor VIII is a protein which has therapeutic applications for the treatment of Hemophilia A. Its deficiency, either qualitative or quantitative, results in Hemophilia A, a disorder affecting approximately 1 in 10,000 males. Currently, FVIII replacement therapy uses FVIII derived from plasma or cell culture. The current cost of this therapy is in excess of $150,000 per patient per year. Thus, alternative sources that are more economical are attractive. The present work focuses upon the characterization of recombinant FVIII (rFVIII) made in the milk of transgenic pigs. Two dimensional western analysis of rFVIII obtained from pig whey showed a range of FVIII species having different isoelectric points (pI) consistent with diverse glycosylation patterns. The pI of these diverse FVIII populations were accurately predicted using theoretical calculations based upon primary protein structure as variable biantennary glycosylation patterns having 0, 1, or 2 sialic acid groups present. Kinetic limitations in the adsorption of rFVIII to anion exchange media due to the nature of the complex milk environment were observed. rFVIII was purified quantitatively using batch equilibration of whey with DEAE Sepharose. This material showed proteolytic processing that was very similar to FVIII obtained from human plasma. Based upon these results, it was postulated that a dissociation of the light (A3C1C2) and heavy (A1A2B) chain due to a lack of vWF may be responsible for the low FVIII activity. / Master of Science

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 142-167).