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Attachment and Detachment of Circulating Tumor Cells in an Antibody-Functionalized MicrosystemCheung, Siu Lun January 2009 (has links)
The attachment and detachment of circulating tumor cells in a functionalized microchannel under hydrodynamic loading have been studied. For the cell attachment experiments, EpCAM antibodies are immobilized on the microchannel surface to capture either PC3N prostate or MDA-MB-231 breast cancer cells from homogeneous cell suspensions. Using the same protocol, N-Cadherin antibodies are immoblilzed and used to study the detachment of target cancer cells captured in the microchannels.A critical flow rate Qc has been identified to characterize the kinetics of cell capture in a functionalized microchannel. Approaching one limit, when the receptor-ligand interaction dominates, more than 90% of moving cells can be captured and a sharp peak is observed in the spatial distribution of the captured cells. Approaching another limit, when hydrodynamic loading dominates, almost all cells cannot be captured in the channel. Between these two limits, there is a transition region in which both capture efficiency and cell distribution are sensitive to the flow parameters. Proper characteristic time and length scales have been identified to describe the cell spatial distribution using a log-normal statistical model. The kinetic details of cell capture are determined by the competition between the flow rate and the ligand-receptor association/dissociation rates.Additionally, the attachment dynamics of circulating tumor cells in a bio-functionalized microchannel under hydrodynamic loading has been explored. The target cells initially role along the microchannel with fluctuating velocity prior to firm adhesion. When a successful bond is established, the cancer cells require a certain length to come to a complete stop; this stopping length is found to depend linearly on the applied hydrodynamic flow rate. The force balance in the vertical cross stream direction is dominated by the gravitational force; as a result, all cells loaded into a microchannel intimately contact the functionalized channel bottom surface within a short time. The streamwise horizontal motion of the cells on the surface is dominated by the balance between the shear flow hydrodynamic loading and the receptor-ligand binding interaction. A linear spring element is incorporated in the physical model to represent the dynamics of a cancer cell captured by immobilized antibodies. Featuring a mobility matrix, a proposed theoretical model is utilized to estimate the binding and hydrodynamic forces acting on the cell in a microchannel. Inserting certain fitting parameters, the time evolution of a stopping cell is successfully predicted by a simplified exponential function.The mechanical response of a captured cancer cell to a hydrodynamic flow field is investigated and, in particular, the effect of flow acceleration is examined. The observed cell deformation is dramatic under low acceleration, but is negligible under high acceleration. Consequently, the detachment of captured cells depends on both flow rate and flow acceleration. The flow rate required for cell detachment is a random variable that can be described by a log-normal distribution. Two flow acceleration limits have been identified for proper scaling of the flow rate required to detach captured cells. A time constant on the order of 1min for the mechanical response of a captured cell has been identified for scaling the flow acceleration. Based on these acceleration limits and the time constant, an exponential-like empirical model is proposed to predict the flow rate required for cell detachment as a function of flow acceleration.
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VHH Antibody Fragments Against Internalin B, a Virulence Factor of Listeria monocytogenes: Reagents for Biosensor DevelopmentGene, Robert 04 October 2012 (has links)
The food processing industry requires alternative methods for detecting the foodborne pathogen Listeria monocytogenes that are cheaper and faster than the current methods. Conventional antibodies and their fragments have been used as biorecognition elements in sensors before, but their use is hindered by high production cost and relative instability. These issues are resolved by VHH fragments, derived from the heavy chain-only antibodies found in Camelidae. VHHs are inexpensive to produce, and are more resistant to environmental stressors. This work describes the isolation of phage-displayed VHHs that recognize recombinant Internalin B, a virulence factor characteristic of L. monocytogenes. Clone R303 was chosen for further characterization, and shown to bind full-length Internalin B. Furthermore, immobilized R303 was shown to capture L. monocytogenes cells. This panel of VHHs, particularly R303, can be utilized by colleagues within the Sentinel Bioactive Paper Network to make a viable biosensor for L. monocytogenes. / Sentinel Bioactive Paper Network
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Tolerance to neonatal porcine islet xenografts induced by a combination of monoclonal antibodiesArefanian, Hossein Unknown Date
No description available.
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The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibodyLiu, Bo January 2014 (has links)
The glycosylation pattern of a chimeric heavy chain antibody (EG2) produced from CHO cells was affected by the glucose concentration (0-25 mM) of cultures established at high density (>106 cells/ml) over 24 h. The resulting proportion of non-glycosylated Mab was directly correlated to the exposure time of cells to media depleted of glucose. Deprivation of glucose for the full 24 h resulted in a 52% non-glycosylated Mab fraction. Analysis of steady state levels of intracellular lipid-linked oligosaccharides (LLOs) showed that under glucose limitation there was a reduction in the amount of full length LLO (Glc3Man9GlcNac2), with a concomitant increase in the smaller mannosyl-glycans (Man2-5GlcNAc2). Glycan microheterogeneity was quantified by galactosylation and sialylation indices (GI and SI), which showed a direct correlation to the cell specific glucose uptake. These findings are important in relation to the low substrate that may occur in fed-batch cultures for Mab production.
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In vitro cellular studies on the human immune response to Plasmodium falciparum malariaBrown, James January 1983 (has links)
This thesis reports the results of a large number of experiments which were designed to elucidate the mechanisms whereby Gambian children, suffering from acute Plasmodium falciparum malaria may eventually control their infections. These experiments were carried out in vitro and success or failure of the various test systems was judged by their effect on parasite multiplication. Early in the course of these investiqations it was demonstrated that mononuclear cells from these children could cooperate with antibodies present in their serum to bring about a marked reduction in parasite growth. The efficiency of this antibody-dependent cellular cytotoxicity (ADCC) mechanism was related to levels of parasitaemia in the children, being greater in convalescent children than in those with acute malaria. Attempts were now made to identify the effector cells in this ADCC. Purified T and B cells were ineffective and although purified adherent cells (A) had an effect, it was much less than that mediated by the undepleted mononuclear cell population. Adherent cells were, however, fully effective in ADCC if they were exposed to the supernatant from T cells non-specifically activated by PHA. Thus cell cooperation leading to activation appears to play an important role in this system. Finally, experiments were set up to determine whether activated mononuclear cells could exert an inhibitory effect on parasite multiplication which was independent of anti-malarial antibody. It was shown that depression of parasite growth could be achieved by mononuclear cells, either from the children or from Europeans, if these cells were exposed to supernatants of previously stimulated mononuclear cells. These findings can be assembled to provide a tentative model of the development of protective responses in vivo. Perhaps following phagocytosis of parasite antigens and their presentation on the cell surface, T cells become activated: they may cooperate with B cells to produce parasite specific antibodies; they may also activate other mononuclear cells (non T, non B) to become effector cells. These cells, either alone, or perhaps more efficiently in cooperation with antibody, are able to kill parasites by the release of toxic factors, and the infection is brought under control. Finally, large amounts of specific antibodies of appropriate isotypes are synthesized. Acting as opsonins or by activating complement, they may serve to destroy remaining parasites. Their continued presence, by preventing merozoite penetration, may provide at least a temporary defense against reinfection. It is assumed that Gambian adults who have suffered repeated malaria infections and are now immune are defended by their possession of circulating IgG antibodies and B memory cells of all appropriate specificities.
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Tolerance to neonatal porcine islet xenografts induced by a combination of monoclonal antibodiesArefanian, Hossein 11 1900 (has links)
Islet transplantation is a more physiological way to treat type 1 diabetes. However, shortage of donor tissue and chronic administration of immune suppressive drugs has limited the widespread application of this therapy for all patients with type 1 diabetes, particularly children suffering from this disease. Xenogeneic islet transplantation particularly neonatal porcine islets (NPI) holds promise for clinical transplantation because of the potentially unlimited supply of islets. New evidence suggests that monoclonal antibodies (mAbs) specific for immune cell surface molecules could be employed in the prevention of islet graft rejection as well as induction of immunological tolerance to the transplanted grafts without the need for continuous administration of harmful immune suppressive drugs. It was shown by our group that short-term administrations of a combination of anti-LFA-1 and anti-CD154 mAbs which targets both adhesion and costimulatory pathways of T cell activation, is highly effective in preventing NPI xenograft rejection. In this thesis, we determined whether short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs could induce tolerance to NPI xenografts. Our data show that this combination of mAbs can induce dominant, species and tissue specific tolerance to NPI xenografts which is mediated by regulatory T cells in non-autoimmune prone B6 mice. We also found that T cell subsets such as CD4+ and CD8+ T cells as well as antigen presenting cells (APC) play an important role in the induction and maintenance of tolerance to NPI xenografts. In addition we found that PD-1/PDL interaction is important for induction and maintenance of tolerance to NPI xenografts. Finally, we found that this combined mAb therapy was effective in preventing NPI xenografts rejection in autoimmune prone NOD mice when it was combined with anti-CD4 mAb. It is may hope that the research presented in this thesis will provide insight into the nature of the immune responses to xenogeneic islet transplantation in humans and aid in the development of effective, tolerance inducing therapies, so that patients with T1DM will once again know a life free from their disease. / Experimental Surgery
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Study of the antigenicity of P. yoelii parasitized erythrocyte ghost antigens and their role in protectionTerrientes S., Zilka I January 1990 (has links)
Thesis (Ph. D.)--University of Hawaii at Manoa, 1990. / Includes bibliographical references (leaves 134-152) / Microfiche. / xvi, 152 leaves, bound ill. 29 cm
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Computational analyses of biological sequences -applications to antibody-based proteomics and gene family characterizationLindskog, Mats January 2005 (has links)
Following the completion of the human genome sequence, post-genomic efforts have shifted the focus towards the analysis of the encoded proteome. Several different systematic proteomics approaches have emerged, for instance, antibody-based proteomics initiatives, where antibodies are used to functionally explore the human proteome. One such effort is HPR (the Swedish Human Proteome Resource), where affinity-purified polyclonal antibodies are generated and subsequently used for protein expression and localization studies in normal and diseased tissues. The antibodies are directed towards protein fragments, PrESTs (Protein Epitope Signature Tags), which are selected based on criteria favourable in subsequent laboratory procedures. This thesis describes the development of novel software (Bishop) to facilitate the selection of proper protein fragments, as well as ensuring a high-throughput processing of selected target proteins. The majority of proteins were successfully processed by this approach, however, the design strategy resulted in a number ofnfall-outs. These proteins comprised alternative splice variants, as well as proteins exhibiting high sequence similarities to other human proteins. Alternative strategies were developed for processing of these proteins. The strategy for handling of alternative splice variants included the development of additional software and was validated by comparing the immunohistochemical staining patterns obtained with antibodies generated towards the same target protein. Processing of high sequence similarity proteins was enabled by assembling human proteins into clusters according to their pairwise sequence identities. Each cluster was represented by a single PrEST located in the region of the highest sequence similarity among all cluster members, thereby representing the entire cluster. This strategy was validated by identification of all proteins within a cluster using antibodies directed to such cluster specific PrESTs using Western blot analysis. In addition, the PrEST design success rates for more than 4,000 genes were evaluated. Several genomes other than human have been finished, currently more than 300 genomes are fully sequenced. Following the release of the tree model organism black cottonwood (Populus trichocarpa), a bioinformatic analysis identified unknown cellulose synthases (CesAs), and revealed a total of 18 CesA family members. These genes are thought to have arisen from several rounds of genome duplication. This number is significantly higher than previous studies performed in other plant genomes, which comprise only ten CesA family members in those genomes. Moreover, identification of corresponding orthologous ESTs belonging to the closely related hybrid aspen (P. tremula x tremuloides) for two pairs of CesAs suggest that they are actively transcribed. This indicates that a number of paralogs have preserved their functionalities following extensive genome duplication events in the tree’s evolutionary history. / QC 20101021
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Small animal models of Gal-mediated and xenograft rejectionGock, Hilton Unknown Date (has links) (PDF)
Xenotransplantation is the final frontier of using vascularised organs or cellular grafts to treat end-organ disease and offers a potential solution to the worldwide shortage of human tissue available for transplantation. The main immunological barrier to xenografting from pig-to-primate is the antigen, Galactose-α1,3-Galactose (Gal) which is found in all species except humans and other higher primates. Even with the major advancement of deleting Gal from the potential pig donor species with the aid of cloning technology, complete elimination may be elusive as alternative genes yet to be fully characterised, may still produce Gal at low levels. Thus, the human immune response against Gal may continue to be a barrier to successful xenotransplantation. The aim of this project was to develop small animal models of the important components of xenograft rejection that largely relate to the anti-Gal immune response. These include models of hyperacute, acute vascular and chronic xenograft-like rejection that in turn, provide new insights in the immune mechanisms of the rejection processes. The role of antibody and both innate and cognate cellular immunity are explored. Both vascularised heart grafts and non-vascularised skin graft models are examined as rejection of solid organs may differ from cellular transplantation. The project also provides a platform for future studies in testing genetic and pharmacotherapeutic strategies to overcome the rejection processes uncovered.
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Leucocyte Populations of Barramundi, Lates Calcarifer, and their Interactions with the Bacterial Pathogen Streptococcus IniaeTumbol, Reiny Antonetha Unknown Date (has links)
No description available.
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