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FVIII Immunity : early events and tolerance mechanisms to FVIIIQadura, Mohammad Imad 15 August 2008 (has links)
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
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EFFECT OF FVIII CO-ADMINISTRATED WITH IVIG IN IMMUNITY TO FVIII IN HEMOPHILIA A MICEAfraz, Sajjad January 2016 (has links)
Background: Hemophilia A is X-linked recessive congenital bleeding disorder. Exogenous infusion of FVIII is the treatment of choice in hemophilia A patients. However, inhibitor development remains the major problem in management of Hemophilia A. It has been showed that IVIG has immunomodulatory effects and it has been being used in the treatment of several autoimmune and inflammatory disorders. Here, we investigated the effect of co-administration of FVIII with IVIG on the development of inhibitor in naive and previously immunized hemophilia A mice.
Methods: Initially, hemophiliac mice were immunized by weekly intraperitoneal injection of human recombinant FVIII (rFVIII). The mice then were treated, either by rFVIII/IVIG co-injection or rFVIII alone. In the other experimental group, naive hemophiliac mice were treated with rFVIII/IVIG co-injection for four weeks followed by injection of either rFVIII or rFVIII/IVIG. Plasma's anti-FVIII Ab titer was measured using ELISA.
Results: Weekly injection of rFVIII led to the development of anti-FVIII Ab in all previously untreated mice. Treatment of those immunized mice with rFVIII/IVIG co-injection did not reduce the level of pre-existing Ab. On the other hand, naive mice treated with rFVIII/IVIG co-injection showed significantly less Ab titer compared to the mice received rFVIII alone after 4 weeks (mean Ab titre of 1 compared to 39, in rFVIII/IVIG co-injection and rFVIII groups respectively). Although the rFVIII/IVIG-treated mice developed immune response following the injection of rFVIII alone, Ab titer in those that kept receiving rFVIII/IVIG co-injection remained lower compared to other groups during the whole twelve weeks of the experiment.
Conclusions: Co-injection of rFVIII with IVIG decreased the anti-FVIII immune response in previously untreated hemophilia A mice. These findings suggest that IVIG co-administration can be effective in management of hemophilia A patients at risk of inhibitor development. / Thesis / Master of Applied Science (MASc)
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Spécificité épitopique de la réponse immunitaire humorale non-neutralisante et neutralisante chez l'hémophile A / Epitope specificity of non-neutralising and neutralising humoral immune response in haemophilia A patientsLebreton, Aurélien 29 November 2012 (has links)
L'hémophilie A (HA) est une maladie hémorragique héréditaire due au déficit en facteur FVIII (FVIII) de la coagulation. Le traitement préventif de l'HA est basé sur des injections répétées de FVIII. Une réponse immunitaire anti-FVIII peut se développer secondairement au traitement, mettant en jeu des anticorps (Ac) inhibiteurs (neutralisant l'activité procoagulante du FVIII) et/ou des anticorps non-neutralisants (ANN). La cartographie épitopique fine des Ac anti-FVIII permet de mieux connaître les mécanismes physiopathologiques de cette réponse immunitaire. Les travaux de cette thèse comportent deux axes principaux : un premier axe a pour objectif d'identifier des épitopes discontinus au sein des domaines C2 et A2 du FVIII, à l'aide de peptides synthétiques prédits par un algorithme informatique, utilisés ensuite dans des tests d'inhibition basés sur la technologie Luminex. Ces travaux nous ont permis d'identifier 8 peptides mimant des épitopes discontinus répartis à la surface du domaine C2 et 2 peptides correspondant à des épitopes voisins, à la surface du domaine A2. Ces études ont permis de démontrer que la combinaison de la bioinformatique et d'un outil expérimental adapté à la réponse immunitaire anti-FVIII est fructueuse. Le second axe a pour objectif d'étudier la prévalence et la spécificité épitopique des ANN à l'aide d'un test Luminex multiplexé. Cette étude a permis de mettre en évidence une prévalence d'ANN de 18,1% chez 210 hémophiles A sans inhibiteurs provenant d'une cohorte multicentrique rétrospective française. Une forte spécificité épitopique de la réponse immune pour la chaîne lourde du FVIII est observée. Les nouveaux outils que nous avons mis en place permettront d'affiner la cartographie épitopique et le suivi de son évolution chez l'hémophile A avec anticorps anti-FVIII / Haemophilia A (HA) is an inherited bleeding disorder due to factor VIII (FVIII) deficiency. The preventive treatment of HA is based on regular infusions of FVIII. Secondary to the treatment, an immune response often occurs, composed by inhibitory antibodies and by non-neutralising antibodies (NNA). Fine epitope mapping of anti-FVIII antibodies may help for a better understanding of the physiopathology of this immune response. There was two axes in this PhD thesis: the first part is dedicated to the identification of discontinuous epitopes on FVIII C2 and A2 domains, by using synthetic peptides predicted by a bioinformatic tool in inhibition tests based on Luminex technology. Results allowed us to identify 8 peptides mimicking discontinuous epitopes around the C2 domain and 2 peptides mimicking close epitopes on the A2 domain surface. These studies demonstrate that our approach combining bioinformatics with an assay adapted for the anti-FVIII immune response study is fruitful. The second part was dedicated to the evaluation of the prevalence and epitope specificity of NNA, using a multiplexed Luminex assay. A prevalence of 18.1% of NNA was thus found in 210 HA patients without inhibitors from a french multicentric retrospective cohort. An marked epitope specificity againt the heavy chain was noticed. The new tools that we developped will be helpful for refining epitope mapping and for the follow-up of the epitope specificity in HA patients with anti-FVIII Abs.
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Caracterização do padrão de integração do retrovírus pMFG-FVIII-P140K em linhagens celulares humanas produtoras de fator VIII recombinante / Characterization of the integration pattern of pMFG-FVIII-P140K retroviral vector in human cell lines producer of recombinant factor VIIIFreitas, Marcela Cristina Corrêa de 14 March 2011 (has links)
A hemofilia A é uma doença caracterizada pela deficiência do fator VIII (FVIII) de coagulação sanguínea e atinge 1 em 5000 homens. Países desenvolvidos, utilizam como terapia o FVIII recombinante (rFVIII), por apresentar maior segurança e consistir uma fonte ilimitada. Estudos realizados em nosso laboratório, utilizando o vetor retroviral pMFG-FVIII-P140K, originaram as linhagens celulares humanas, HepG2FVIIIdB/P140K (hepática) e Hek293FVIIIdB/P140K (renal), que foram selecionadas pelo mesmo sistema de seleção, o tratamento in vitro com as drogas Benzilguanina e Temozolomide. O presente trabalho teve por objetivo caracterizar e detalhar o padrão de integração do vetor retroviral pMFG-FVIII-P140K nas duas linhagens celulares humanas, e verificar se o perfil de integração está relacionado ao tipo celular específico ou se este sofre influência da estratégia de seleção a qual as células foram submetidas anteriormente. Foi utilizada a técnica de LM-PCR (ligação-mediada por PCR) que permite localizar o sítio de integração do vetor viral, por meio do sequênciamento do produto de PCR obtido após a digestão com enzimas de restrição e ligação de uma sequência adaptadora (LINKER) ao DNA genômico. Após o seqüenciamento as sequências foram submetidas a análises em bancos de dados de genomas (Human BLAT, QuickMap e DAVID/EASE) para caracterização dos respectivos clones. Também foi realizada uma análise da expressão relativa do mRNA relativo ao rFVIII por RT-PCR e da atividade biológica pelo teste de TTPA. Dessa forma foi possível observar que ambas as linhagens modificadas expressam o mRNA relativo ao rFVIII e que as células HepG2 e Hek293 apresentam níveis de secreção da proteína recombinante biologicamente ativa da ordem de 7,9 UI/mL e 2,1 UI/mL, respectivamente. Foram sequenciados um total de 201 clones da HepG2, sendo 123 similares ao genoma humano e dentre estes 73 considerados verdadeiros e 50 ambíguos. Da Hek293 foram sequenciados 221 clones, sendo 179 similares ao genoma humano e dentre estes 64 verdadeiros e 115 ambíguos. Observamos que o vetor retroviral pMFG-FVIII-P140K apresenta um perfil de integração não randômico e diferente entre as células estudadas, uma vez que na linhagem HepG2 houve uma preferência pelos cromossomos 19, 17 e 11, e na Hek293 pelo cromossomo 9. Em relação a regiões genômicas tais como distância de ilhas CpGs e de sítios de ligação de fatores de transcrição não houve diferença no perfil de integração em ambas as linhagens celulares, visto que nas duas células o vetor se inseriu preferencialmente a uma distância de ± 30-60Kb dessas regiões. Observamos também que houve uma integração dentro de genes codificadores de proteínas da ordem de 52% e 44% nas células HepG2 e Hek293, respectivamente, contudo em ambos os casos mais de 90% dessas inserções ocorreram em regiões intrônicas. Houve 20% de integrações em sítios frágeis do genoma na linhagem HepG2 e 17% na Hek293. Em suma este trabalho mostrou a integração do vetor retroviral pMFG-FVIII-P140K específica para as duas linhagens em estudo. Além disso, pudemos observar que em ambas as linhagens celulares, HepG2FVIIIdB/P140K e Hek293FVIIIdB/P140K, existem regiões gênicas dentro dos cromossomos na qual o vetor exibe uma preferência. O perfil de integração caracterizado aqui difere de outros trabalhos da literatura que utilizaram retrovírus derivados de MLV. Isso nos leva a crer que o padrão de inserção descrito pode estar relacionado ao fato de que essas células foram tratadas anteriormente com drogas quimioterapêuticas para seleção de um clone celular com maior nível de produção de rFVIII, levando consequentemente a seleção de um perfil de integração semelhante entre as linhagens celulares, mesmo estas tendo origens teciduais diferentes. / Hemophilia A is a disease characterized by deficiency of blood clotting factor VIII (FVIII) and affects 1 in 5000 men. Developed countries use as therapy for Hemophilia A recombinant FVIII (rFVIII) because rFVIII concentrates have proven efficacy and safety and this therapy consist of an unlimited supply.. Studies in our laboratory using the retroviral vector pMFG-FVIII-P140K, originate the human recombinant cell lines, HepG2FVIIIdB/P140K (liver origen) and Hek293FVIIIdB/P140K (renal origen) which were selected with high doses of Benzyladenine and Temozolomide. This study aimed to characterize the integration pattern of retroviral vector pMFG-FVIII-P140K in these human cell lines, and verify if the profile of integration is related to specific cell type or it was influenced by the selection strategy which cells have been previously submitted. We used the technique of LM-PCR (ligation-mediated PCR) that allows to locate the site of viral vector integration by the sequencing of PCR products. The sequences were obtained after genomic DNA digestion with restriction enzymes and subsequent connection of an adapter (LINKER) to 5or 3 of the DNA fragment. The sequences were analyzed in genomic databases (Human BLAT, and Quickmap DAVID / EASE) for characterization of the respective clones. It was also performed an analysis of relative expression of rFVIII mRNA by RT-PCR and the biological activity was measured by APTT test. Thus it was observed that both cell lines express the mRNA relative to rFVIII and HepG2 and HEK293 cells have levels biologically active recombinant protein approximately 7.9 IU / mL and 2.1 IU / mL, respectively . We sequenced a total of 201 clones of HepG2FVIIIdB/P140K, 123 of these sequences match to the human genome and, among those 73 were considered true and 50 were ambiguous. In Hek293FVIIIdB/P140K 221 clones were sequenced, these total, 179 were similar to the human genome. 64 were true and 115 were ambiguous. We note that the retroviral vector pMFG-FVIII-P140K presents a profile of integration different and non-random between the studied cells lines, in HepG2FVIIIdB/P140K was observed a preference of integration for chromosomes 19, 17 and 11, and in Hek293FVIIIdB/P140K for the chromosome 9. In genomic regions such as CpG islands and transcription factors binding sites (TFBS), there was no difference in the profile of integration in both cell lines. In the two cell lines the vector is inserted preferably at a distance of ± 30 - 60Kb of these regions. We also observed that there was 52% of integration within genes encoding proteins (RefSeq genes) in HepG2FVIIIdB/P140K and 44% in Hek293FVIIIdB/P140K cells. In both cases more than 90% of the insertions occurred in intronic regions. There were 20% of integrations at fragile sites in the genome of HepG2 cell line and 17% in Hek293. In summary this study showed the integration profile of retroviral vector pMFG -FVIII-P140K in two different cell lines that produces FVIIIr protein are very similar. Furthermore, we observed that in both cell lines, HepG2FVIIIdB/P140K and Hek293FVIIIdB/P140K, there are genetic regions within the chromosomes in which the vector displays a preference. The integration profile featured described here differs in CpGs island and TFBS from other studies in the literature that used retroviruses derived from MLV. This leads us to believe that the integration pattern described here may be related to the fact that these cells were previously treated with chemotherapeutic drugs for selecting a cell clone with the highest level of production of rFVIII, consequently leading to selection of a similar profile integration between cell lines, even those having different tissue origins.
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Caracterização do padrão de integração do retrovírus pMFG-FVIII-P140K em linhagens celulares humanas produtoras de fator VIII recombinante / Characterization of the integration pattern of pMFG-FVIII-P140K retroviral vector in human cell lines producer of recombinant factor VIIIMarcela Cristina Corrêa de Freitas 14 March 2011 (has links)
A hemofilia A é uma doença caracterizada pela deficiência do fator VIII (FVIII) de coagulação sanguínea e atinge 1 em 5000 homens. Países desenvolvidos, utilizam como terapia o FVIII recombinante (rFVIII), por apresentar maior segurança e consistir uma fonte ilimitada. Estudos realizados em nosso laboratório, utilizando o vetor retroviral pMFG-FVIII-P140K, originaram as linhagens celulares humanas, HepG2FVIIIdB/P140K (hepática) e Hek293FVIIIdB/P140K (renal), que foram selecionadas pelo mesmo sistema de seleção, o tratamento in vitro com as drogas Benzilguanina e Temozolomide. O presente trabalho teve por objetivo caracterizar e detalhar o padrão de integração do vetor retroviral pMFG-FVIII-P140K nas duas linhagens celulares humanas, e verificar se o perfil de integração está relacionado ao tipo celular específico ou se este sofre influência da estratégia de seleção a qual as células foram submetidas anteriormente. Foi utilizada a técnica de LM-PCR (ligação-mediada por PCR) que permite localizar o sítio de integração do vetor viral, por meio do sequênciamento do produto de PCR obtido após a digestão com enzimas de restrição e ligação de uma sequência adaptadora (LINKER) ao DNA genômico. Após o seqüenciamento as sequências foram submetidas a análises em bancos de dados de genomas (Human BLAT, QuickMap e DAVID/EASE) para caracterização dos respectivos clones. Também foi realizada uma análise da expressão relativa do mRNA relativo ao rFVIII por RT-PCR e da atividade biológica pelo teste de TTPA. Dessa forma foi possível observar que ambas as linhagens modificadas expressam o mRNA relativo ao rFVIII e que as células HepG2 e Hek293 apresentam níveis de secreção da proteína recombinante biologicamente ativa da ordem de 7,9 UI/mL e 2,1 UI/mL, respectivamente. Foram sequenciados um total de 201 clones da HepG2, sendo 123 similares ao genoma humano e dentre estes 73 considerados verdadeiros e 50 ambíguos. Da Hek293 foram sequenciados 221 clones, sendo 179 similares ao genoma humano e dentre estes 64 verdadeiros e 115 ambíguos. Observamos que o vetor retroviral pMFG-FVIII-P140K apresenta um perfil de integração não randômico e diferente entre as células estudadas, uma vez que na linhagem HepG2 houve uma preferência pelos cromossomos 19, 17 e 11, e na Hek293 pelo cromossomo 9. Em relação a regiões genômicas tais como distância de ilhas CpGs e de sítios de ligação de fatores de transcrição não houve diferença no perfil de integração em ambas as linhagens celulares, visto que nas duas células o vetor se inseriu preferencialmente a uma distância de ± 30-60Kb dessas regiões. Observamos também que houve uma integração dentro de genes codificadores de proteínas da ordem de 52% e 44% nas células HepG2 e Hek293, respectivamente, contudo em ambos os casos mais de 90% dessas inserções ocorreram em regiões intrônicas. Houve 20% de integrações em sítios frágeis do genoma na linhagem HepG2 e 17% na Hek293. Em suma este trabalho mostrou a integração do vetor retroviral pMFG-FVIII-P140K específica para as duas linhagens em estudo. Além disso, pudemos observar que em ambas as linhagens celulares, HepG2FVIIIdB/P140K e Hek293FVIIIdB/P140K, existem regiões gênicas dentro dos cromossomos na qual o vetor exibe uma preferência. O perfil de integração caracterizado aqui difere de outros trabalhos da literatura que utilizaram retrovírus derivados de MLV. Isso nos leva a crer que o padrão de inserção descrito pode estar relacionado ao fato de que essas células foram tratadas anteriormente com drogas quimioterapêuticas para seleção de um clone celular com maior nível de produção de rFVIII, levando consequentemente a seleção de um perfil de integração semelhante entre as linhagens celulares, mesmo estas tendo origens teciduais diferentes. / Hemophilia A is a disease characterized by deficiency of blood clotting factor VIII (FVIII) and affects 1 in 5000 men. Developed countries use as therapy for Hemophilia A recombinant FVIII (rFVIII) because rFVIII concentrates have proven efficacy and safety and this therapy consist of an unlimited supply.. Studies in our laboratory using the retroviral vector pMFG-FVIII-P140K, originate the human recombinant cell lines, HepG2FVIIIdB/P140K (liver origen) and Hek293FVIIIdB/P140K (renal origen) which were selected with high doses of Benzyladenine and Temozolomide. This study aimed to characterize the integration pattern of retroviral vector pMFG-FVIII-P140K in these human cell lines, and verify if the profile of integration is related to specific cell type or it was influenced by the selection strategy which cells have been previously submitted. We used the technique of LM-PCR (ligation-mediated PCR) that allows to locate the site of viral vector integration by the sequencing of PCR products. The sequences were obtained after genomic DNA digestion with restriction enzymes and subsequent connection of an adapter (LINKER) to 5or 3 of the DNA fragment. The sequences were analyzed in genomic databases (Human BLAT, and Quickmap DAVID / EASE) for characterization of the respective clones. It was also performed an analysis of relative expression of rFVIII mRNA by RT-PCR and the biological activity was measured by APTT test. Thus it was observed that both cell lines express the mRNA relative to rFVIII and HepG2 and HEK293 cells have levels biologically active recombinant protein approximately 7.9 IU / mL and 2.1 IU / mL, respectively . We sequenced a total of 201 clones of HepG2FVIIIdB/P140K, 123 of these sequences match to the human genome and, among those 73 were considered true and 50 were ambiguous. In Hek293FVIIIdB/P140K 221 clones were sequenced, these total, 179 were similar to the human genome. 64 were true and 115 were ambiguous. We note that the retroviral vector pMFG-FVIII-P140K presents a profile of integration different and non-random between the studied cells lines, in HepG2FVIIIdB/P140K was observed a preference of integration for chromosomes 19, 17 and 11, and in Hek293FVIIIdB/P140K for the chromosome 9. In genomic regions such as CpG islands and transcription factors binding sites (TFBS), there was no difference in the profile of integration in both cell lines. In the two cell lines the vector is inserted preferably at a distance of ± 30 - 60Kb of these regions. We also observed that there was 52% of integration within genes encoding proteins (RefSeq genes) in HepG2FVIIIdB/P140K and 44% in Hek293FVIIIdB/P140K cells. In both cases more than 90% of the insertions occurred in intronic regions. There were 20% of integrations at fragile sites in the genome of HepG2 cell line and 17% in Hek293. In summary this study showed the integration profile of retroviral vector pMFG -FVIII-P140K in two different cell lines that produces FVIIIr protein are very similar. Furthermore, we observed that in both cell lines, HepG2FVIIIdB/P140K and Hek293FVIIIdB/P140K, there are genetic regions within the chromosomes in which the vector displays a preference. The integration profile featured described here differs in CpGs island and TFBS from other studies in the literature that used retroviruses derived from MLV. This leads us to believe that the integration pattern described here may be related to the fact that these cells were previously treated with chemotherapeutic drugs for selecting a cell clone with the highest level of production of rFVIII, consequently leading to selection of a similar profile integration between cell lines, even those having different tissue origins.
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ENHANCEMENT OF hFVIII ACTIVITY THROUGH LC MODIFICATIONS FOR GENE THERAPY OF HEMOPHILIA AFirrman, Jenni Ann January 2015 (has links)
Gene therapy for Hemophilia A (HA) using the recombinant Adeno-associated virus (rAAV) offers an alternative to classic treatment, which consists of FVIII protein infusions. However, due to limitations associated with rAAV and the FVIII protein itself, the end result is a transgene expression below therapeutic limits. One approach to improving the therapeutic value of rAAV gene therapy for HA is to engineer a more active FVIII protein through genetic modifications. Preliminary testing revealed that canine FVIII Light Chain (kLC) enhances coagulation activity, and that it would be possible to improve FVIII activity through modifications of the light chain. Through the process of engineering, evaluation, and negative selection of kLC, a final construct was engineered. The hLC-K12 is a human Light Chain (hLC) construct containing 12 amino acid changes that work together to enhance coagulation activity. A comparison of the FVIII clotting activity to the amount of protein produced determined that hLC-K12 produced a 3.28 fold increase in specific activity over hLC in vitro. Similar in vitro results were observed when hLC-k12 was tested with the X5 heavy chain (X5HC), a heavy chain that has been genetically modified to enhance production. CD4KO/HA mice were injected with a rAAV vector carrying the hLC-K12 gene in conjugation with a rAAV vector carrying the X5HC gene. Replacing the hLC vector with the hLC-K12 vector produced an average 7.43 fold increase in FVIII clotting activity. An ELISA assay revealed no significant difference between productions of the heavy or light chains at any time point. By comparing the clotting activity to the amount of protein produced, it was determined that the increase in coagulation activity was due to an increase in specific activity. In fact, replacing the hLC vector with the hLC-K12 vector resulted in an average 5.8 fold increase in FVIII specific activity. The K12 modifications were evaluated using a single chain FVIII conformation. In vitro, the addition of the K12 mutations to the human heavy chain, hHCK12BDD, resulted in a 4.3 fold increase in clotting activity, but no increase in protein production. There was however, a 3.3 fold increase in specific activity of the protein. Adding the K12 mutations to the X5 heavy chain, X5K12BDD, in vitro, resulted in a 2.7 fold increase in clotting activity and a 1.42 fold increase in specific activity of the protein. Single chain rAAV vectors were packaged and delivered to CD4KO/HA mice. Compared to mice injected with hFVIIIBDD, the hHCK12BDD produced an average 4.6 fold increase in clotting activity. An ELISA revealed no significant difference in production between these two groups. However, mice injected with hHCK12BDD produced FVIII with an average of 4.13 fold increase in specific activity. Similarly, when compared to mice injected with X5FVIIIBDD, the X5K12BDD produced an average 2.14 fold increase in clotting activity. An ELISA assay demonstrated no significant increase in protein production between these two groups. However, when compared to X5BDD, mice injected with the X5K12BDD vector produced FVIII with an average 1.98 fold increase in specific activity. Results demonstrate that the K12 light chain modifications are able to enhance clotting activity of hFVIII both in vitro and in vivo, using either a dual chain or single chain delivery method. In order to determine the mechanism of enhancement, hFVIIIBDD and hHCK12BDD protein was partially purified and tested for activity. Results demonstrated that the hHCK12BDD protein produced a specific activity of 39,153.69 Units/mg, which is a 6.28 fold increase over hFVIIIBDD specific activity, which was 6,237.92 Units/mg. Measurement of conversion from FX to FXa revealed that the hHCK12BDD protein generated a higher amount of FXa at a quicker rate. In conclusion, these results provide evidence that the K12 modifications enhance specific activity through an increase in FXa generation. / Microbiology and Immunology
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Inovação no processo de fracionamento de plasma humano através do uso de cromatografia líquida de pseudoafinidade. / Innovation in human plasma fractionation process using liquid pseudo-affinity chromatography.Feliciano, Gabriel Pinna 27 October 2016 (has links)
Neste trabalho mostramos que o método de purificação empregando a coluna ANX Sepharose FF, alternando eluições com CaCl2 (pseudoafinidade) e NaCl, permite obter concentrados do complexo protrombínico e do fator VIII em uma etapa cromatográfica. Testamos as colunas ANX e Q Sepharose FF, monolito QA e membrana Sartobind Q e os tampões citrato, MES e Bis-Tris. Empregando a ANX Sepharose FF e o tampão citrato as proteínas do complexo protrombínico eluiram com CaCl2 25 mM e o FVIII com NaCl 500mM. A recuperação da atividade de FVIII foi de cerca de 60% e o fator de purificação de 220 vezes. Na fração contendo as proteínas do complexo protrombínico foram identificadas por espectrometria de massas a presença de fibrinogênio, proteína C4 do complemento e C4b Binding Protein. A análise das frações mostra que a presença destas proteínas não é devido à variação da concentração de CaCl2, possivelmente se deve à difusão lenta destas através da resina. Mais de 99% das proteínas do plasma não são adsorvidas na coluna e podem ser purificadas em etapas posteriores. / In this study we showed that the purification method employing the anion exchange column ANX Sepharose FF, and alternating CaCl2 (pseudoaffinity) and NaCl elutions, allows us to obtain concentrates of prothrombin complex proteins and factor VIII in a single chromatographic step. We tested the ANX, Q Sepharose FF and QA monolithic column and Sartobind Q membrane and the citrate, MES and Bis-Tris buffers. Using ANX Sepharose FF and the citrate buffer the prothrombin complex proteins eluted with 25 mM CaCl2 and FVIII with 500 mM NaCl. FVIII activity recovery was approximately 60% with a purification factor of 220. In the fraction containing the prothrombin complex proteins presence of fibrinogen, complement C4 and C4bBinding Protein were identified by mass spectrometry. Analysis of the fractions showed that their presence is not due to the variation of concentration of CaCl2, but possibly due to their slow diffusion through the resin. More than 99% of the plasma proteins are not adsorbed in the column and can be purified in following chromatographic steps.
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IMMUNE CELLS EXPRESS ELEVATED COAGULATION FACTOR VIII IN PROTHROMBOTIC PONATINIB-TREATED MICEZENG, PENG 23 May 2022 (has links)
No description available.
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Etude du répertoire épitopique et isotypique des anticorps anti-facteur VIII chez les patients atteints d'hémophilie A / Analysis of epitopic and isotypic profile of anti-FVIII antibodies in haemophilia A patientsLapalud, Priscilla 20 September 2012 (has links)
Le facteur VIII (FVIII) joue un rôle essentiel dans la coagulation sanguine. Lorsque le FVIII fait génétiquement défaut, une pathologie hémorragique grave survient: l'hémophilie A (HA) congénitale. La complication majeure de la prise en charge de ces patients est l'apparition d'allo-anticorps (alloAcs) dirigés contre le FVIII thérapeutique administré. Dès lors, la seule thérapeutique efficace est l'induction de tolérance immune (ITI) qui vise à les éradiquer. Cependant, ce traitement échoue dans 30% des cas, sans qu'aucun facteur ne permette actuellement de prédire l'échec de ce traitement contraignant et coûteux. des facteurs immunologiques prédictifs de l'efficacité de l'ITI ont été recherchés chez 25 patients par analyse du répertoire épitopique et isotypique des Acs anti-FVIII à l'aide de la technologie x-MAP. Des biomarqueurs individuels (Acs anti-A2 et -A1 du FVIII), et des combinaisons originales ont été identifiés (0,841 < AUC < 0,946). Des manifestations hémorragiques peuvent apparaitre chez des patients non hémophiles, dues à des autoAcs anti-FVIII (HA acquise). Dans certains cas, les autoAcs se développent au moment du postpartum. peu de données sont disponibles sur cette réponse immune. Dans une seconde étude portant sur 73 cas, nous avons découvert un profil immunologique (autoAcs anti-A1) différenciant les HA du postpartum et les autres HA acquises. Les profils d'IgG anti-FVIII que nous avons établis s'avèrent prometteurs pour prédire l'efficacité de l'ITI et engendrer une cartographie précise de la réponse autoimmune chez les patients atteints d'HA acquise. / Factor VIII (FVIII) plays a critical role in blood coagulation. When FVIII s genetically defective, a serious hemorrhagic disease occurs: congenital hemophilia A (HA). The main complication of the management of these patients is the appearance of alloantibodies (alloAbs) directed against administred therapeutic FVIII. therefore, the only effective treatment is the immune tolerance induction (ITI), which aims to eradicate these alloAbs. However, this treatment fails in up to 30% of cases, without any factor currently able to predict the failure of this constraining and expensive treatment. Immunological factors predictive to the efficacy of ITI were investigated in 25 patients by analysis of epitopic and isotypic IgG profile of anti-FVIII Abs using x-MAP technology. Individual biomarkers (anti-FVIII A1 and -A2 Abs), and original combinations were identified (0,841 < AUC < 0,946). Hemorrhagic manifestations can occur in non-hemophiliac patients, due to anti-FVIII autoAbs (acquired HA). In some patients, the autoAbs appear in postpartum period but few data are available on the immune response due to the rarity of the disease. In a second study of 73 cases, we found a different immunological profile between patients with postpartum HA and the other acuired HA patients. IgG profiles of anti-FVIII we have established are promising for predicting the effectiveness of ITI and generate an accurate mapping of autoimmune response in patients with acquired HA.
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Testing different purification methods for sample preparation in recombinant FVIII pharmaceutical production / Utvärdering av olika reningsmetoder för provberedning av rekombinant FVIIIJohansson, Malin January 2024 (has links)
Hemofili A är en X-länkad ärftlig sjukdom som påverkar blodkoagulationen och beror på avsaknaden eller bristen på proteinet faktor VIII (fVIII). Nuwiq® är en rekombinant fVIII B- domän borttagen produkt från Octapharma AB som behandlar hemofili A och produceras med en cellinje av mänskliga njurceller från embryon. FVIII är 170 kDa som hel-längds protein, men klyvs till 90 kDa och 80 kDa kedjor innan de släpps ut i kroppen för cirkulation. Enligt reglementen måste 170 kDa kedjan vara under en specifik gräns under odlings processen för den farmaceutiska produkten och den relative distributionen av de tre olika isoformerna behövs mätas. Idag upparbetas proverna och analyseras med en droppkolon och reverse-phase high performance liquid chromatography (HPLC). Droppkolonen är en tidskrävande process och målet med detta projekt var att hitta en metod som kan förkorta tiden för detta analyssteg. I denna studie ska möjligheterna utvärderas för att använda Western Blot metod och att använda ett ÄKTA pure system för att rena proverna innan de analyseras med HPLC. Resultatet från Western Blot visa liknades resultat som HPLC analysen gör och en linjäritets, noggrannhets och precisions studie utfördes. Linjäritets studien resulterade i att provkoncentrationen bestämdes till 2 IU/mL. Noggrannhets testerna visade att det var liknande resultat men med mindre skillnader mellan de två metoderna och viss olikhet är förväntad då HPLC analyserar med avseende på hydrofobisitet och Western Blot använder antikroppar och kemiluminiscens. En mer omfattande precisions studie behövs innan några beslut kan tas angående en ändring av analysmetod. Western Blot visade lovande resultat och skulle förkorta metodtiden med ungefär 1-2 dagar. De första ÄKTA reningarna med odlingsprover visade inget protein i eluatet, men med en högre protein mängd pålagd på kolonen från ett prov med mindre cell rester hade eluatet en bra mängd protein i sig. Analysen av eluatet med Western Blot visade goda resultat. Fortsatta tester behövs göras med ÄKTA reningen för att metoden ska kunna betrakta det som en möjlig metod att använda på laboratoriet för att analysera faktor VIII protein. / Hemophilia A is a X-linked hereditary disease that effects the coagulation of the blood and is due to the absence or deficiency of the protein factor VIII (FVIII). Nuwiq® is a recombinant FVIII B-domain deleted product from Octapharma AB that treats hemophilia A and are produced with human embryonic kidney cell lines. The fVIII protein is 170 kDa full length but are cleaved into a 90 kDa chain and an 80 kDa chain before release into circulation in the body. Due to regulations the 170 kDa chain needs to be below a certain limit during the cultivation process of the pharmaceutical product and the relative distribution between the three isoforms needs to be measured. Today the sample is prepared and analysed through a drop column and reverse-phase high performance liquid chromatography (HPLC). The drop column is a time-consuming process and the aim for this project was to find a method that can reduce the time for this analysis step. This study investigated the possibilities to use Western Blot as a method and to use an ÄKTA pure system for the preparation of the samples and then analyse with HPLC. Results from Western Blot showed similar results as the HPLC analysis and a linearity, accuracy and precision study were performed. The linearity study showed promising results for a sample concentration of 2 IU/mL to be used. Accuracy testing showed similar but slightly different results between the two methods and some differences are expected since the HPLC analyses on hydrophobicity and Western Blot uses antibodies and chemiluminescence. There would need to be a more extensive precision study before any decision could be made about changing the analysis method. Western Blot showed promising results and would shorten the method time with around 1-2 days. The first ÄKTA purification runs with cultivation samples did not show any protein levels in the eluate, but with a higher protein amount put on the column from a sample with less cell debris the eluate had good amount of protein in it. The analysis of the eluate with Western Blot showed good results. Further testing of the ÄKTA purific tion is needed to consider it an option to use at the laboratory for analysis of factor VIII protein.
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