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Studies of immunomodulatory effects of soluble factors derived from plasma using the effect of factor concentrates on stimulated leucocytes in vitro - as a model /Hodge, Gregory Lionel Unknown Date (has links)
Thesis (PhD)--University of South Australia, 2000
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Factor VIII inhibitors in haemophilia A /Ling, Min. January 2000 (has links) (PDF)
Thesis (M.Med.Sc.) -- University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 2000. / Bibliography: leaves 115-125.
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Optimization of production variables governing yield and stability of factor VIII in cryoprecipitateCollette, Carol Joan January 1997 (has links)
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.
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The role of factor VIII in blood coagulationNeal, G. G. January 1982 (has links)
Factor VIII, a component of the intrinsic pathway of blood coagulation, has yet to be purified to homogeneity. It appears that, in vivo, the factor VIII coagulant protein is closely associated with one or more other proteins (factor VHI-related antigen and platelet aggregating factor). The material normally isolated from bovine plasma as 'factor VIII' possesses all three activities and is therefore either a mixture or a complex of the various proteins. In the present study, bovine factor VIII:C was purified approximately fivethousand- fold by a combination of ion-exchange chromatography and fractional precipitation. The factor VIII coagulant activity can be separated from the other activities of the 'factor VIII complex' but the procedures involved are not suitable for preparative use as the factor VIII:C which is obtained is unstable. During coagulation, factor VIII:C is required during the activation of factor X. Studies with purified bovine clotting factors indicate that factor IX<sub>a</sub> is the enzyme responsible for the cleavage of factor X, in a calcium-dependent reaction which is stimulated by phospholipid. Factor VIII:C further accelerates the rate at which factor X<sub>a</sub> is generated. Preliminary investigations of the kinetic parameters of the reaction indicate that the stimulation by factor VIII:C occurs through a marked increase in the V<sub>max</sub> of the reaction; factor VIII:C does not affect the K<sub>m</sub> for factor X. The coagulant activity of factor VIII is enhanced by exposure to thrombin, but the 'activated' factor VIII:C which is produced is not itself capable of activating factor X in the absence of factor IX<sub>a</sub>. Thus, the 'activation' of factor VIII:C, in contrast to the activation of, for example, factors IX and X, does not appear to result in the formation of an enzyme. That is, factor VIII:C is a non-enzymic, high molecular weight cofactor for factor IX<sub>a</sub>.
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Interaction of recombinant factor VIII and the nonionic surfactant Tween 80 at interfacesJoshi, Omkar 05 December 2005 (has links)
The role of the nonionic surfactant Tween 80 on the behavior of the
therapeutic recombinant protein Factor VIII (rFVIII) was investigated at solid/liquid and
air/water interfaces. In order to provide a model system to compare results obtained for
the complicated rFVIII-Tween system, a well-characterized globular protein lysozyme
was used. The experimental scheme involved the introduction of the protein and Tween to
the adsorption substrate in different manners, either lysozyme Tween together or in
sequence as lysozyme followed by Tween or vice versa. It was observed that the addition
of Tween together with lysozyme reduced the amounts adsorbed at hydrophobic surfaces,
while no such reduction was observed on hydrophilic surfaces. A high Tween
concentration was required to effect the removal of the lysozyme molecules from the
hydrophobic surface and Tween was not effective in removing lysozyme from the
hydrophilic surface at any concentration. These results suggest that the Tween-surface
interaction is important in determining lysozyme adsorption. Similar observations were
made for the rFVIII-Tween system at hydrophobic and hydrophilic silica interfaces. In
this case, the presence of interfacial and solution Tween together resulted in complete
prevention of rFVIII adsorption. Electrostatic forces were observed to be play an
important role in rFVIII adsorption. The rFVIII-Tween interactions at solid interfaces
were also evaluated using intrinsic fluorescence and biological activity measurements.
Results obtained with respect rFVIII adsorbed mass, and structure or biological activity
change upon adsorption, were evaluated in parallel. This parallel evaluation suggested that
rFVIII adsorption on hydrophilic, negatively charged surfaces is likely to be highly
ordered and oriented in a manner that retains the solvent accessibility of the active sites in
rFVIII. On the other hand, rFVIII may adsorb to hydrophobic surfaces in different
orientations, with a likelihood of surface induced unfolding. rFVIII-Tween interaction at
the air/water interface was investigated separately. Surface tension data recorded for
rFVIII-Tween mixtures suggested that Tween dominated the air/water interface as the
Tween concentration was increased. Reduced interface-induced unfolding was observed at
high Tween concentrations. These results were also thought to contribute to the reduction
in rFVIII aggregation typically observed as a result of exposure to the air/water interface. / Graduation date: 2006
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The adsorption of human recombinant factor VIII in the presence of the nonionic triblock surfactant Pluronic® F-68 at the air-water interface /Alkhatib, Aveen K. January 1900 (has links)
Thesis (M.S.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 42-44). Also available on the World Wide Web.
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Delivery of CRISPR/Cas9 RNAs into Blood Cells of Zebrafish: Potential for Genome Editing in Somatic CellsSchneider, Sara Jane 08 1900 (has links)
Factor VIII is a clotting factor found on the intrinsic side of the coagulation cascade. A mutation in the factor VIII gene causes the disease Hemophilia A, for which there is no cure. The most common treatment is administration of recombinant factor VIII. However, this can cause an immune response that renders the treatment ineffective in certain hemophilia patients. For this reason a new treatment, or cure, needs to be developed. Gene editing is one solution to correcting the factor VIII mutation. CRISPR/Cas9 mediated gene editing introduces a double stranded break in the genomic DNA. Where this break occurs repair mechanisms cause insertions and deletions, or if a template oligonucleotide can be provided point mutations could be introduced or corrected. However, to accomplish this goal for editing factor VIII mutations, a way to deliver the components of CRISPR/Cas9 into somatic cells is needed. In this study, I confirmed that the CRISPR/Cas9 system was able to create a mutation in the factor VIII gene in zebrafish. I also showed that the components of CRISPR/Cas9 could be piggybacked by vivo morpholino into a variety of blood cells. This study also confirmed that the vivo morpholino did not interfere with the gRNA binding to the DNA, or Cas9 protein inducing the double stranded break.
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Desenvolvimento de processo cromatográfico para purificação de fator VIII humano. Emprego de anticorpos contra fragmentos específicos da proteína na avaliação da pureza e estabilidade durante as etapas de purificação. / Process development for human factor VIII purification by chromatography, the use of specific antibodies against fragments of the protein for evaluation of purity and stability during purification processes.Jinzenji, Daniela 31 October 2008 (has links)
O fator VIII de coagulação (FVIII), recombinante ou purificado de plasma, é o biofármaco necessário para o tratamento da hemofília A, a doença hemorrágica mais freqüente em humanos. O método tradicional para a purificação de FVIII parte de crioprecipitado de plasma e precipitação alcoólica. No Instituto Butantan, foi proposto um método alternativo, utilizando somente cromatografia para esta purificação. Este projeto teve por objetivo comparar dois métodos cromatográficos de purificação do FVIII: 1 - gel filtração direta do plasma e 2 - pré-purificação de FVIII do plasma por cromatografia de troca aniônica, seguida de gel filtração. A purificação foi analisada por dosagens de atividade específica de FVIII e presença de outras proteínas da cascata de coagulação nas frações de cromatografia. Foram realizadas clonagem de fragmentos gênicos de FVIII e expressão de fragmentos protéicos para imunização de animais. Os soros com anticorpos policlonais anti-FVIII foram usados em ensaios de \"western blot\" para detectar as cadeias de FVIII ou degradação. / Coagulation factor VIII (FVIII), recombinant or purified from plasma, is the biopharmaceutical used for treatment of haemophilia A, the most frequent human hemorrhagic disorder. The traditional method used for purification of FVIII starts from plasma cryoprecipitate and alcoholic precipitation. The Instituto Butantan proposed an alternative methodology using only chromatography for FVIII purification. The main objective of this project was to compare two chromatographic methods for FVIII purification: 1 - direct plasma gel filtration and 2 - pre-purification of FVIII by anion exchange chromatography, followed by gel filtration. The purification process was analyzed by determination of FVIII specific activity and detection of other coagulation factors co eluting in chromatographic fractions. Fragments of FVIII gene were cloned and protein fragments were expressed for animal immunization. Sera with polyclonal antibodies anti-FVIII were used in western blots assays to detect FVIII chains or its degradation.
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Desenvolvimento de processo cromatográfico para purificação de fator VIII humano. Emprego de anticorpos contra fragmentos específicos da proteína na avaliação da pureza e estabilidade durante as etapas de purificação. / Process development for human factor VIII purification by chromatography, the use of specific antibodies against fragments of the protein for evaluation of purity and stability during purification processes.Daniela Jinzenji 31 October 2008 (has links)
O fator VIII de coagulação (FVIII), recombinante ou purificado de plasma, é o biofármaco necessário para o tratamento da hemofília A, a doença hemorrágica mais freqüente em humanos. O método tradicional para a purificação de FVIII parte de crioprecipitado de plasma e precipitação alcoólica. No Instituto Butantan, foi proposto um método alternativo, utilizando somente cromatografia para esta purificação. Este projeto teve por objetivo comparar dois métodos cromatográficos de purificação do FVIII: 1 - gel filtração direta do plasma e 2 - pré-purificação de FVIII do plasma por cromatografia de troca aniônica, seguida de gel filtração. A purificação foi analisada por dosagens de atividade específica de FVIII e presença de outras proteínas da cascata de coagulação nas frações de cromatografia. Foram realizadas clonagem de fragmentos gênicos de FVIII e expressão de fragmentos protéicos para imunização de animais. Os soros com anticorpos policlonais anti-FVIII foram usados em ensaios de \"western blot\" para detectar as cadeias de FVIII ou degradação. / Coagulation factor VIII (FVIII), recombinant or purified from plasma, is the biopharmaceutical used for treatment of haemophilia A, the most frequent human hemorrhagic disorder. The traditional method used for purification of FVIII starts from plasma cryoprecipitate and alcoholic precipitation. The Instituto Butantan proposed an alternative methodology using only chromatography for FVIII purification. The main objective of this project was to compare two chromatographic methods for FVIII purification: 1 - direct plasma gel filtration and 2 - pre-purification of FVIII by anion exchange chromatography, followed by gel filtration. The purification process was analyzed by determination of FVIII specific activity and detection of other coagulation factors co eluting in chromatographic fractions. Fragments of FVIII gene were cloned and protein fragments were expressed for animal immunization. Sera with polyclonal antibodies anti-FVIII were used in western blots assays to detect FVIII chains or its degradation.
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