B cells have a central role in the pathogenesis of collagen-induced arthritis (CIA), an animal model of the autoimmune disease rheumatoid arthritis. In this report, a specific subset of an innate type of B cells, B-1 B cells, have been studied for the involvement in CIA. The B-1 B cells were shown to produce small amounts of collagen-specific antibodies upon stimulation in vitro, suggesting that they play a minor role in the development of CIA. This report also includes how marginal zone B cells, another innate type of B cells with natural collagen-reactivity, can be identified in the medullary sinuses of lymph nodes of collagen-immunized mice, implying involvement in auto antigen trapping.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 5 uppsatser.

Les anticorps monoclonaux (AcM) sont des protéines ayant une reconnaissance antigénique spécifique utilisée pour le développement de réactifs thérapeutiques et diagnostiques. La production commerciale est réalisée en cultivant des cellules hôtes dans des bioréacteurs spécifiques. La densité cellulaire et le métabolisme cellulaire sont des paramètres clés pour le rendement élevé des AcM. Bioréacteur à fibres creuses (HFB), une cartouche contenant des fibres poreuses emballées, est l'un des systèmes de production disponibles dans le commerce. Si la densité de cellules obtenue peut conduire à un rendement élevé, le coût de l'ensemble du dispositif, y compris les pompes et les cartouches très coûteuses, empêche son utilisation de petites unités. Comme une alternative économique, nous avons proposé ici d'étudier le potentiel des modules de dialyse de polysulfone du commerce, classiquement employés dans le traitement de l'insuffisance rénale en phase terminale. Cependant, la membrane de polysulfone native a démontré une adsorption de protéines non spécifique significative préjudiciable à la production d'AcMs. De plus des enjeux normatifs viennent se greffer à ces problématiques scientifiques et technico-économiques, avec le cas des normes (ISO 13485/ AC S99-104/ GMP FDA /BPF…) qui imposent des méthodes de travail normalisées. Ce travail de thèse consiste en la conception d’un bioréacteur jetable, sur la base d’une cartouche de dialyse médicale à fibres creuses. Ce système doit offrir toutes les garanties en termes de production, de facilité d’utilisation, de stérilité, et permettre de concentrer les produits de cytoculture. La méthodologie scientifique a été couplée à une démarche qualité. La gestion de ce projet a été couplé à l’analyse de risques. En effet ce projet a été divisé en ses 5 composantes élémentaires décrite par Ishikawa par la méthode des 5 M. L’analyse de risque a consisté au calcul d’indice de criticité par la méthode AMDEC de chacune de ses familles de risques. Cette approche nous a permis de formaliser deux axes de recherche : i) la mise en œuvre d’une oxygénation efficace du milieu de culture (chapitre 4) et ii) les moyens de limiter le colmatage dans le module à fibres creuses pour obtenir une culture cellulaire conforme aux objectifs (chapitre 5). Le taux d’oxygénation est un facteur à prendre en compte dans un processus de culture cellulaire. L’oxygène peut être supplémenté selon deux modes, le mode passif ou le mode actif [Ozturk et Palsson 1990; Zhang S. et al 1992]. Il existe 2 types de système d’oxygénation : les système dit passif ou les échanges se font a travers une paroi de silicone et un système actif par aération directe dans le milieu de culture. Ce système est de loin l'opération la plus simple pour fournir de l'oxygène. Cependant, lorsque celui-ci est utilisé pour apporter de l’oxygène à des cultures de cellules mammifères, cela peut engendrer des altérations cellulaires. Des agents protecteurs chimiques peuvent être utilisés pour réduire les dommages cellulaires et la formation de mousse [Kamase et Moo-yung 1990; van Der pol L.A et al 1993]. Nos études ont démontré que l'addition d'agents anti-mousse peut entraîner une diminution du coefficient de transfert de masse d’O2 en phase liquide (Kl) [Kamase et Moo-yung 1990]. Nous avons établi l’efficacité de l’utilisation d’un polymère silice/silicone pour éliminer la mousse sur des cultures bactériennes et de cellules mammifères. Afin de limiter ces phénomènes de colmatage, les fibres de polysulfone ont été traitées avec plusieurs tensioactifs (acide pluronique F127, D-limonen et différentes huiles de silicone) qui ont conduit à une diminution significative de l'adsorption protéique. L'effet de ces surfactants sur les performances de filtration et sur la cytotoxicité a été étudié. Certains d'entre eux n'ont pas influencé ces paramètres alors que d'autres ont présenté des effets négatifs. / Monoclonal Antibodies (mAbs) are proteins with specific antigen recognition used for development of both therapeutic and diagnostic reagents. Commercial production is achieved by growing host cells in specific bioreactors. Cell density and cell metabolism are key parameters for high yield of mAbs. Hollow fiber bioreactor (I-IFB), a cartridge containing packed porous fibres, is one of the system for production commercially available. If the cell density achieved can lead to high yield, the cost of the whole device, including pumps and very expensive cartridges prevents its use of small units. As an economical alternative, we proposed here to investigate the potential of commercial polysulfone dialysis modules, classically employed in the treatment of end stage renal failure. However, the native polysulfone membrane demonstrated a significant non-specific protein adsorption detrimental to mAbs production. Moreover normative issues are added to these scientific and techno-economic issues, with the case of standards (ISO 13485 / AC S99-104 / GMP FDA / BPF ...) which impose standard working methods. This thesis consists of the design of a disposable bioreactor, based on a hollow-fiber medical dialysis cartridge. This system must offer all the guarantees in terms of production, ease of use, sterility, and allow to concentrate the cytoculture products. Scientific methodology has been coupled with a quality approach. The management of this project was coupled with the risk analysis. Indeed this project was divided into its 5 elementary components describe by Ishikawa by the 5M method. The risk analysis consisted in the calculation of the criticality index by the AMDEC method of each of its families of risks. This approach allowed us to formalize two research axes: i) the implementation of an effective oxygenation of the culture medium (chapter 4) and ii) the means of limiting the clogging in the hollow fiber module to obtain a culture consistent with the objectives (Chapter 5). The rate of oxygenation is a factor to be taken into account in a cell culture process. Oxygen can be supplemented in two modes, passive mode or active mode [Ozturk and Palsson 1990; Zhang S. et al 19921. There are two types of oxygenation system: the so-called passive system or the exchanges are made through a silicone wall and an active system by direct aeration in the culture medium. This system is by far the simplest operation for providing oxygen. However, when it is used to supply oxygen to mammalian cell cultures, this can cause cellular damage. Chemical protective agents can be used to reduce cell damage and DKamase and Moo-yung 1990 foam formation; van Der pol L.A. et al 1993 Cl. Our studies have shown that the addition of antifoaming agents can lead to a decrease in the liquid phase (K2) mass transfer coefficient of D Kamase and Moo-yung 1990C]. We have established the effectiveness of using a silica / silicone polymer to remove foam on bacterial and mammalian cell cultures. In order to limit these adsorption phenomena, polysulfone fibers were treated with several surfactants (pluronic acid F 127, D-limonen, and different silicone oils) which led to a significant decrease in protein adsorption. The effect of such surfactants on the filtration performances and on cytotoxicity were investigated. Some of the them did not influence these parameters while some presented negative effects. Finally, different cell culture parameters (cells densities, production yield, flow properties, fouling) were studied, as well as the performance of the bioreactor in perfusion continuous mode. The bioreactor was maintained in continuous mode for fifteen days and the production yield per batch was 250 mg of AcMs. The results obtained in this work allowed us to define the next steps to be taken, and are the subject of the Perspectives section.

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_a 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_a 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_max of the reaction; factor VIII:C does not affect the K_m 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_a. 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_a.

Call number: LD2668 .T4 1984 H35 / Master of Science

<p>The most common primary malignant brain tumor, glioblastoma, is uniformly fatal. Current therapy provides only incremental benefits in survival and is often incapacitating owing to limits defined by nonspecific toxicity. By contrast, immunotherapy offers a particularly promising approach, and has the theoretical potential to target and eliminate malignant cells with unprecedented specificity. The goal of this dissertation is to apply recombinant technologies to develop a new immune-based therapy for patients with malignant glioma. This work will span the design, production, and preclinical testing of a novel bispecific antibody designed to redirect T cells against a tumor-specific mutant of the epidermal growth factor receptor, EGFRvIII.</p><p>Chapters 1 and 2 will provide an overview of broad topics in antitumor immunotherapy and immune biology, with special focus on concepts as they relate to tumors of the central nervous system. In addition, the history and current state of bispecific antibodies, particularly those of the bispecific T-cell engager (BiTE) subclass, as well as their potential role in the treatment of malignant disease, will be considered in detail. Data presented in Chapter 3 will describe our approach to generating novel bispecific tandem single-chain antibody reagents, while experiments in Chapter 4 will demonstrate the capacity of one of these molecules, an EGFRvIII-specific BiTE, to achieve antitumor efficacy both <italic>in vitro</italic> and <italic>in vivo</italic> using murine models of glioma. Addressing a major barrier to the translation of immune therapies for cancer, chapter 5 will establish a potential role for BiTEs in overcoming cell-mediated immune suppression associated with malignant disease. Lastly, Chapter 6 and 7 will report on emerging areas of study, including the use of syngeneic, transgenic murine systems, and strategies by which BiTEs may be propelled rapidly into early phase clinical trials. </p><p>In summary, separating BiTEs from other available immunotherapeutic approaches, our work in this field suggests that BiTEs are (1) highly-specific molecules that greatly reduce the risk of toxicity, (2) have the ability to penetrate the blood-brain barrier and accumulate in intracerebral tumors, and (3) may potentially overcome multiple mechanisms of immunosuppression present in patients with glioblastoma. Together, these studies have the potential to improve the clinical management of patients with glioblastoma through the generation of a novel therapeutic.</p> / Dissertation

Multiple sclerosis is a complex disease and one for which the aetiology remains largely unanswered. Anti-dsDNA antibodies have been found intrathecally and bordering lesions in multiple sclerosis patients and in view of their known pathogenity in lupus nephritis the aim of this project was to further investigate their role in multiple sclerosis. Using the acute experimental allergic encephalomyelitis (EAE) model in the Lewis rat, the inflammatory phase of disease was profiled using immunohistological and ELISA methods and was related to clinical sign severity. The parameters of interest were central nervous system deposits of IgM, IgG, B cells and C3 and anti-DNA antibodies in sera, cerebrospinal fluid and in situ. In situ evaluation of anti-dsDNA antibodies was also performed in tissue taken from Biozzi (AH) mice (relapsing/remitting EAE model) and from a multiple sclerosis patient. Inflammatory deposits specifically at sites of perivascular cuffing were found to increase with increasing clinical sign severity. At the time clinical signs had plateaued in the Lewis rat, intrathecal anti-dsDNA antibodies were at their highest level and anti-ssDNA antibodies at their lowest. The latter possibly due to their involvement in the 'clearing-up' process following tissue damage. Using novel DNA probes fluorescence suggestive of the presence of anti-dsDNA iii antibodies was seen in both animal and human tissue. Within human tissue the antibodies appeared to accumulate around active lesions and within vessels, raising the question of these antibodies having differing location dependent functions. EAE models have the potential to investigate these findings further and to evaluate new therapies.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Immunochemical reagents were characterized under carefully controlled laboratory conditions using conventional high performance liquid chromatography instrumentation. The stationary phase was prepared by attaching antigen molecules to an insoluble support through a covalent linkage. Experiments were carried out by introducing antibody molecules into the mobile phase and monitoring their interaction with the stationary phase. Monoclonal antibodies were employed because of their more homogeneous properties compared to polyclonal antisera. Radioisotopes were employed to study low level adsorption on the stationary phase. Recovery experiments were carried out in which it was possible to account for all of the material introduced into the mobile phase. Antibodies were purified over a preparative scale antigen affinity column following labeling to insure high immunoreactivity. Studied under normally dissociating conditions, irreversible adsorption of picomole amounts of protein on the antigen stationary phase was greater than on other ligand modified stationary phases. This accumulation decreased with repeated use of the affinity column. The present study provides a framework for evaluation of other immunoaffinity systems and demonstrates that reproducible recovery of immunologically active material in high yield is possible. Monoclonal antibodies labeled with fluorescein were different from unlabeled molecules in binding and physical characteristics. Computer simulations were used to describe binding behavior. Although fluorescein labels improve detection sensitivity over native protein absorbance, their use in this case decreased binding affinity significantly. Heterogeneity of affinity purified fluorescein labeled and unlabeled monoclonal antibodies was examined with two dimensional gel electrophoresis. In addition to increased charge heterogeneity in the labeled antibody fragments, both light and heavy chains possessed more negative character. These results agree with each other. Fluorescein contains a carboxylic acid group, and modification of antibody light chains may interfere with binding affinity. The number and location of labels covalently attached to antibodies must be carefully controlled to obtain maximum detection sensitivity and preserve immunoreactivity.

To determine the antigenic relatedness of the different developmental stages of Cryptosporidium parvum, monoclonal IgG3 antibody (mAb), Cmg-3, was produced by immunizing mice with partially purified merozoites. The monoclonal Cmg-3 reacted with a 3.5 kDa antigen of sporozoites in western blots and appeared to react with cell surface antigens of air-dried merozoites and sporozoites using immunofluorescence (IF). Additional mAbs, C6B6 (IgG1) and C4A1 (IgM), which react with a 20 kDa and multiple sporozoite antigens, respectively, were employed for immunoelectron microscopic studies with Cmg-3. These mAbs showed similar (surface/cytoplasmic) immunoelectron microscopic colloidal gold labeling patterns with all C. parvum life cycle stages. The three mAbs were also examined for potential modulation of cryptosporidial infections in vivo by daily oral mAb administration to oocyst-inoculated neonatal mice. Monoclonal-treated neonatal mice were sacrificed four and eight days post infection (pi). Differences in infection rates were observed among the treatment groups (p < .05). Suckling mice treated daily with orally administered mixtures of mAbs (ascitic fluids) showed significantly reduced parasite loads compared to control mice at four and eight days pi, while suckling mice receiving mAb Cmg-3 alone showed significant differences only at four days pi. Passive transfer of immunity using hyperimmune bovine colostrum was performed to determine the therapeutic and prophylactic efficacy of daily oral administration of anti-C. parvum antibody on the manifestation of cryptosporidial disease in neonatal mice as a model for treating cryptosporidiosis in immunocompromised patients. Hyperimmune colostrum was found to provide therapeutic and prophylactic efficacy against cryptosporidiosis in neonatal mice. Significantly fewer (p < 0.05) stages of C. parvum were found in mice that received hyperimmune skim colostrum (HSC) or hyperimmune original colostrum (HC) than in those treated with control colostrum (CC) or saline. Using IF, antigen-specific IgG in HSC and HC to C. parvum was 35 times greater than that of CC. There was no significant difference between groups treated with HSC or HC (p < .05), which suggests that the immunoglobulins, other biologically active factors such as cytokines, or both, might be active factors of immunity against cryptosporidiosis.