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1
Shi, Jianguo. "Interaction of human CD23 with IgE and CD21." Thesis, King's College London (University of London), 1997. https://kclpure.kcl.ac.uk/portal/en/theses/interaction-of-human-cd23-with-ige-and-cd21(373685f2-b918-4cae-9404-c10d226ff134).html.
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Van, Zyl Dwain George. "Production of recombinant human CD21 and CD23 : towards a better understanding of their interaction." Thesis, Nelson Mandela Metropolitan University, 2013. http://hdl.handle.net/10948/d10211135.
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The prevalence of allergic diseases has dramatically increased over the last three decades. Presently, it is estimated that 20-30 per cent of the developed world suffers from allergic diseases. The majority of allergic diseases are rooted in the activities of IgE; an immunoglobulin which exerts its effector functions by interacting with a network of proteins. This network includes its low affinity receptor CD23. Cross linking of membrane IgE and CD21 by soluble CD23 results in an increase in IgE synthesis. This marks the interaction between CD23 and CD21 as an attractive therapeutic target. However, details regarding this interaction are inadequate for rational drug design. To obtain a deeper understanding of the CD23-CD21 interaction recombinant human CD21 (SCR1-2 and SCR5-8) and CD23 (16 kD and 25 kDa) were produced. The cloning, expression and purification of recombinant proteins comprised a significant portion of this study. Recombinant CD23 was expressed as inclusion bodies, refolded by rapid dilution and purified by size exclusion chromatography. Conversely, recombinant CD21 was expressed as soluble MBP-fusions and purified with an amylose affinity resin. The interaction between recombinant CD23 and CD21 was analysed by flow cytometry and ELISA experiments. Flow cytometry showed that 16 kDa and 25 kDa CD23 interacted with SCR5-8 to the same extent. Semi-quantitative ELISA experiments showed that both SCR1-2 and SCR5-8 were able to interact with 16 kDa and 25 kDa CD23. This suggests that the binding sites of SCR1-2 and SCR5-8 occur on 16 kDa CD23. Furthermore, since proteins were expressed in E. coli it suggests that the CD23-CD21 interaction does not require glycosylation. Furthermore, considering what is known about the SCR1-2-CD23 interaction from previous NMR studies; i.e. that the C-terminal tail (residues residues 289-298) of CD23 is responsible for binding SCR1-2, indicates that SCR5-8 binds somewhere within the lectin domain of CD23. This indicates that the CD23-CD21 interaction involves C-terminal tail-SCR1-2 and lectin domain-SCR5-8 interactions.
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Karagiannis, Sophia. "The process of endocytosis of CD23." Thesis, King's College London (University of London), 1995. https://kclpure.kcl.ac.uk/portal/en/theses/the-process-of-endocytosis-of-cd23(553202e7-a9c8-444e-b0a5-f7561d1e6297).html.
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Yahya, Mohd Norhakim. "Analysis of the IgE network : inhibition of CD23-mediated IgE upregulation and CD21/C3d interaction." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:bc5ff165-2d2c-4e4f-a0e9-5651cacd2ddf.
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Allergic reactions are mainly mediated by the interactions between the IgE and its ligands, amongst them CD23 and CD21 in what is termed the IgE network. CD23 is involved in upregulating IgE expression by forming a trimolecular complex with CD21 and IgE on the B-cell surface, resulting in the specific activation of IgE-positive B cells. CD21 also interacts with C3d and is a bridge between the innate and the immune system. A crystal structure of the interaction has been solved (Szakonyi et al., 2001) but was controversial because it contradicted previous biochemical analyses. The aims of this thesis were to use various biophysical techniques to study the interactions between the molecules in the IgE network and its possible inhibition. Part 1: Characterisation of a phage display-derived peptide that inhibit IgE binding to CD23 A peptide was previously derived using phage display technology and tested for binding ability to CD23 using SPR and ITC. Subsequent NMR experiments were performed to identify the binding site, followed by characterization of its derivatives. Crystallisation of CD23 with the peptide and soaking with its truncated tripeptide, NWP, were also attempted. Part 2: Characterisation of CD23 and its interaction with its ligands X-ray crystallography was undertaken to solve the structure of derCD23 in complex with a phage display-derived peptide (Part1) followed by crystal soaking with a truncated tripeptide, NWP. However, a reproducible, high-resolution wild type derCD23 structure was determined at 1.9 Å. A comparison of the binding behaviour between the monomeric derCD23 and a trimeric CD23 construct was carried out in order to see the effect of oligomerisation upon IgE binding. Using the known interaction map as well as a crystal structure, the possible interacting residues between CD23 and IgE were examined. The characterisation of the CD23/CD21 interaction was continued from previous efforts in order to confirm that the binding epitope of CD23 for CD21 lies within the C-terminus of CD23. Characterisation of the interactions of CD23/IgE/FcεRI was performed to examine these multimolecular interactions and possible regulatory mechanisms in mast cell degranulation. It was shown that CD23 can form multimeric complexes with IgE-Fc that bind to FcεRI with higher apparent affinity than IgE-Fc alone, which may lead to increases in mast cell degranulation. It was also found that the IgE bound on FcεRI still binds to CD23 although with a lower binding capacity, presumably due allosteric changes. The binding of CD23 with a monoclonal antibody IDEC-152 was also characterised using SPR and NMR spectroscopy. It was proposed that IDEC-152 might interfere with the trimerisation site of CD23 thus reducing its affinity for IgE. A thermofluor assay was developed and optimised for potential screening of compounds that bind to derCD23 using a qPCR machine, which may be useful to screen compounds that bind to CD23 as part of future drug discovery project. Crystallisation of the derCD23/CD21 and IgE/triCD23/CD21 complexes was also attempted as part of ongoing crystallisation projects. Part 3: The interaction between C3d and CD21 The interaction between C3d and CD21 is believed to be a bridge between the innate and adaptive immune response, and is thought to be pivotal in the initiation of autoimmune disease. Following from previous studies on this interaction, further characterisations were performed using NMR and ITC to confirm the involved sites on CD21 (SCR1-2) in binding to C3d. Several potential salt bridges have been identified so far, allowing a high-resolution docked structure of the C3d/CD21 complex.
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Ford, Jill Wallace. "CD23's Role as a Negative Regulator of Allergic Disease: in vivo Effects of Murine CD23 Destabilization and Allelic Mutations." VCU Scholars Compass, 2007. http://hdl.handle.net/10156/2104.
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Ferreira, Lauren. "Cytokine properties of CD23 on human Eosinophilic cells." Thesis, Nelson Mandela Metropolitan University, 2007. http://hdl.handle.net/10948/503.
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CD23, the low affinity IgE receptor, is expressed by various cell types and has numerous functions depending on the form of the protein, its interaction with various ligands and the type of cell involved. CD23 is pivotal in the regulation of IgE, with the soluble form involved in up-regulation, while the membrane bound form is involved in the down-regulation. It is clear why it is believed to be a central molecule in allergic responses, and a therapeutic target for the treatment of allergic disease. In this study a recombinant form of the entire extracellular domain of the protein, exCD23, was produced by PCR cloning and expressed in E. coli. His•Tag™s were introduced onto the C-terminus and N-terminus, respectively, in order to simplify the purification procedure. After renaturation and purification, the recombinant exCD23 bound IgE, indicating its activity. From the IgE binding studies it was established that the position of the tag did not influence the binding. GST•Tagged™ exCD23 was also produced in an attempt to increase the solubility of the recombinant protein, but this proved unsuccessful. Butyrate differentiated EoL-1 cells were treated with the Nterminal His•Tagged™ exCD23, and the protein appeared to suppress the secretion of the constitutively expressed cytokines, especially IL-8 and IFN- , when compared to untreated cells. In addition, treatment of the EoL-1 cells with exCD23 had a significant proliferative effect, but could not induce differentiation of this cell line into mature eosinophilic-like cells.
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Grundy, Gabrielle Jane. "The structure and function of human soluble CD23." Thesis, King's College London (University of London), 2001. https://kclpure.kcl.ac.uk/portal/en/theses/the-structure-and-function-of-human-soluble-cd23(1c2f66b1-b335-4192-892f-bbbf3afde432).html.
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Yuan, Daopeng. "Structural studies of human CD23 and its complexes." Thesis, King's College London (University of London), 2012. https://kclpure.kcl.ac.uk/portal/en/theses/structural-studies-of-human-cd23-and-its-complexes(b8946602-66b9-4b39-a02e-4cda67c1c26d).html.
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IgE plays a central role in the pathogenesis of immediate hypersensitivity reactions through interacting with its receptors, in particular the high affinity receptor FcεRI. The low-affinity IgE receptor, CD23, affects IgE-dependent immune responses by regulating the synthesis of IgE, facilitating allergen presentation to the immune system, and influencing the activation and differentiation of B- and T-cells. Surprisingly, CD23 is different from other Ig receptors and belongs to the C-type (calcium-dependent) lectin family. Calcium binding to CD23 affects IgE binding to CD23, but previous NMR and crystal structures of CD23 gave conflicting results concerning the calcium binding sites. Investigation of calcium binding site(s) in CD23 and its complexes may assist CD23- targeted drug design. DerCD23 (a fragment of human CD23 that consists of the lectin domain and part of the C-terminal tail) and derCD23 mutants designed to remove each of the two potential calcium binding sites, were expressed and purified. The crystal structures of Ca2+-bound wild type derCD23, the complex with the Fcε3-4 sub-fragment of IgE-Fc, and four putative calcium binding site mutants of derCD23 were solved. Binding affinities of derCD23 and its mutants for calcium and for IgE-Fc were measured with ITC and SPR. The results indicate a loop of derCD23 (loop 4) is stabilized upon calcium binding to “site 2” and thereby contributes to the increased binding affinity for IgE. In addition, a residue (D258) in the non-conserved “site 1” is observed to bind IgE directly in the Ca2+-bound derCD23/Fcε3-4 complex. Thus, Ca2+ bound to site 2 in CD23 stabilizes loop 4 for IgE binding, whereas site 1 has evolved to bind IgE directly. Clinical studies of IDEC152 (also known as Lumiliximab), a primatized IgG1, anti- CD23 monoclonal antibody (IDEC Pharmaceuticals, San Diego, CA) show positive clinical effects in patients with allergic asthma and chronic lymphocytic leukemia. The Fab fragment of IDEC152 was prepared by enzymatic digestion from IDEC152 and crystals of the complex of derCD23 with IDEC152-Fab were grown, which diffracted to 2.4 Å resolution.
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Ilkow, Veronica Franciszka. "Engineering IgE antibodies and CD23 for therapeutic discovery." Thesis, King's College London (University of London), 2018. https://kclpure.kcl.ac.uk/portal/en/theses/engineering-ige-antibodies-and-cd23-for-therapeutic-discovery(54f73d64-5c16-42c4-9dea-42855873eeb6).html.
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Immunoglobulin E (IgE) is fundamental to the allergic response and the functions of IgE are mediated by its Fc region binding to two receptors, FcεRI and CD23 (FcεRII). The interaction of IgE with other proteins have complicated our investigations of the unique role each receptor plays. To solve this, a small-scale library of IgE-Fc proteins was designed with two key positions, one at each receptor-binding site mutated. The unpredictable allosteric nature of IgE prevents rational engineering approaches, thus the design of a membrane-bound IgE-Fc-GFP-tagged protein allowed for the generation of a membrane-surface display library of stable cell lines. A FACS selection assay identified IgE-Fc proteins with weakened binding to a single IgE-receptor, which serves as a proof-of-principle for this concept. Additional studies into human CD23 and the differences between it and murine CD23 revealed additional levels of regulation for IgE-binding not seen in other species and this is due to its unique properties. Human CD23 is an unusual antibody receptor, being a calcium dependent (C-type) lectin that has lost its carbohydrate binding capability. Ca2+ binds to and increases CD23’s affinity for IgE, and one of two Ca2+ binding sites usually present in C-type lectins is absent in human but present in murine CD23. To understand if the loss of the second Ca2+ binding site has led to a regulatory gain/loss of function in human CD23, a panel of CD23 mutant proteins with increasingly ‘mouse-like’ sequences was generated. The insertion of the second Ca2+ binding site was verified by HSQC-NMR whilst molecular dynamic simulations provided a means of understanding the flexibility of the proteins. It revealed that binding of two Ca2+ ions tethers the soluble CD23 loops into position in the most mouse-like mutant protein, limiting possible conformations for IgE binding. Complementary Biacore experiments indicated that higher calcium binding affinity may have come at a cost of weakened IgE binding, as data in the presence and absence of Ca2+ showed decreased binding affinities of the proteins for human IgE. This regulatory difference between murine and human soluble CD23 could inform the development of CD23/IgE inhibitor therapeutics for the treatment of allergy.
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Bansal, Amolak Singh. "The influence of HLA DR on soluble CD23 secretion and serum soluble CD23 as a marker of immune dysregulation in human disease." Thesis, University of Southampton, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266381.
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ALLIOT, CAROL. "Formes solubles des cd23 et cd25 et hemopathies lymphoides : interet pronostique dans une serie de 40 leucemies lymphoides chroniques." Amiens, 1993. http://www.theses.fr/1993AMIEM041.
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Bund, Dagmar. "hTERT, CD23 und CD229 als Tumorantigene bei der B-CLL." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-145282.
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Jutton, Mark Robert. "Biophysical and biological characterisation of a soluble human CD23 pigment." Thesis, King's College London (University of London), 2007. https://kclpure.kcl.ac.uk/portal/en/theses/biophysical-and-biological-characterisation-of-a-soluble-human-cd23-pigment(4a13a8ad-ea7a-4ba1-877d-ddd3811786a6).html.
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C3-antigen complexes induce B cell proliferation and the synthesis of specific antibodies in the immune response. The mechanism is understood to involve the co-ligation of antigen receptor (lgM) and CD21 on the membrane of antigen-specific B cells. CD23 is the low-affinity IgE receptor and also binds to CD21. It has been shown by NMR spectroscopy that the binding sites for IgE and CD21 on CD23 are distinct and nonoverlapping. CD23 is expressed as a homotrimer on the membrane of B cells and is cleaved by an endogenous metalloprotease to release soluble fragments that contain the f binding sites for both IgE and CD21. It is suggested that these fragments, like the C3antigen complexes, can co-ligate membrane IgE (mlgE) and CD21 on the surface of B cells to up-regulate IgE synthesis. This would enhance allergic responses in vivo. To test this hypothesis, a construct was generated to express a trimeric fragment of soluble human CD23 (LZ-CD23) and provide evidence for the protein's oligomeric state in solution using analytical ultracentrifugation. Surface plasmon resonance studies also showed that LZ-CD23 binds IgE with a high affinity. Confocal microscopy was used to demonstrate that LZ-CD23 caused the time-dependent re-distribution of fluorescentlylabelled mlgE and CD21 on human tonsillar B cells, stimulated with anti-CD40 and IL-4 to induce heavy-chain switching to 19B. As early as 15 minutes following incubation with LZ-CD23, rnIgE and CD21 were seen to co-cap in large aggregated clusters on the B cell surface. Additionally, the specificity of LZ-CD23 towards these markers was demonstrated as LZ-CD23 had no effect on the surface distribution of CD38. LZ-CD23 was shown to be biologically active by enhancing the secretion of IgE from B cells. Moreover, evidence is presented that CD23 exerts its IgE-potentiating action by specifically targeting cells that are pre-committed to 19B. The mechanism thus appears to resemble that of the C3-antigen complex in the immune response, except that it is isotype rather than antigen-specific. These results shed light on the role of CD23 in IgE homeostasis and suggest a novel means of targeting CD23 for intervention in the allergic response.
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Reljic, Rajko. "The interaction of CD23 and CR2 and its functional consequences." Thesis, King's College London (University of London), 1996. https://kclpure.kcl.ac.uk/portal/en/theses/the-interaction-of-cd23-and-cr2-and-its-functional-consequences(01e4a5aa-37fc-4892-8fa8-458ac1183001).html.
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Ewart, Marie-Ann. "Analysis of transcriptional regulatory elements of the human CD23 gene." Thesis, University of Glasgow, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343975.
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Daniels, Brodie Belinda. "Molecular and cellular analysis of the interaction between soluble CD23 and CD11/CD18 integrins." Thesis, Nelson Mandela Metropolitan University, 2010. http://hdl.handle.net/10948/1217.
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The low affinity IgE receptor, CD23, is expressed by a wide variety of cells and cleaved from its original 45 kDa size to several smaller soluble CD23 proteins. Soluble CD23 function depends on the form of the protein and its interaction with various ligands. CD23 is believed to play an important role in regulating allergic responses and in inflammation, amongst others. β2 integrins are important in a variety of cell-adhesion reactions during immune-inflammatory mechanisms and the binding of their natural ligands generates outside-in cellular signalling, leading to cell activation. Although the binding of CD23 to β2 integrins contributes to this signalling in monocytes, the interaction site for CD23 is unknown. This study focused on the interaction of three soluble CD23 proteins with the β2 integrins CD11b/CD18 and CD11c/CD18. Differentiated HL60, THP1 and U937 monocytic cells were used to demonstrate the binding of three recombinant CD23 constructs (corresponding to 16, 25 and 33 kDa human soluble CD23) to upregulated CD11b/CD18 and CD11c/CD18. This binding was partially blocked by an antibody specific for the CD11b/CD18 αI domain, demonstrating that αI domains are involved in binding to CD23. Recombinant αI domain proteins of CD11b and CD11c were demonstrated to bind CD23 using ELISA and in surface plasmon resonance spectroscopy. The dissociation constants for CD23-CD11b/CD18 and CD23-CD11c/CD18 are comparable to other integrin ligands. This study has shown that CD23 interacts directly with the αI domains of β2 integrins and that the interaction surface likely spans the lectin domain as well as either the stalk and/or C-terminal tail of CD23. This study also looked at the effect that soluble CD23 proteins had on monocyte biology. It appears that iv sCD23 proteins have little effect on the phagocytic or chemotactic ability of monocytes, while an increase in oxidative burst was shown with the 16 kDa and 25 kDa CD23 proteins. Signalling pathways for the production of reactive oxygen species were investigated and it appears that the CD23 proteins signal mainly through the phosphoinositide-3 kinase pathway, although the mitogen activated protein kinase and Src kinase pathways may also play a role. These data suggest that sCD23 proteins induce outside-in signalling of β2 integrins and are able to change the activation state of CD11b/CD11c by stimulating oxidative burst. This needs to be further investigated by determining how the three sCD23 proteins are binding the CD11 proteins and investigating further leukocyte function and inflammatory responses by the cells.
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Pereira, Melanie Claire. "The molecular analysis of the interation surface between sCD23 and the B2-integrins, CD11b & CD11c." Thesis, Nelson Mandela Metropolitan University, 2012. http://hdl.handle.net/10948/d1014734.
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Both CD23 and the β2 integrins (also known as CD11/CD18) have very important immunological functions, especially during the allergic response where the binding of CD23 to β2 integrins contributes to various types of signalling in monocytes which can result in drastic sensitivities experienced by some allergic individuals. CD23, also known as the low affinity receptor for immunoglobulin E or (FcεRII), is a type II transmembrane glycoprotein which is synthesized by haematopoietic cells and has biological activity in both membrane-bound and freely soluble forms. It acts via a number of receptors, including the β2 integrins. β2 integrins are specifically found on leukocytes and they play important roles in cell–cell or cell–matrix adhesion via their ability to bind multiple ligands. These molecules occur as heterodimers consisting of an alpha (α) and beta (β) subunit. The α-subunits of β2 integrins contain an approximately 200-amino-acid inserted domain or I-domain which is implicated in ligand binding function. There are four different types of β2 integrins, namely CD11a, CD11b, CD11c and CD11d, all dimers with the common beta subunit, CD18. CD23 and CD11/18 are natural ligands of each other; however the interaction site for CD23 is unknown. It is postulated that the integrin recognizes a tripeptide motif in a small disulfide-bonded loop at the N-terminus of the lectin head region of CD23, which is focussed around Arg172, Lys173 and Cys174 (RKC). This study thus focused on the interaction between the I-domain of CD11 (b and c) and a recombinant 25kDa construct of sCD23. In order to understand the characteristics of ligand binding between the relevant proteins of interest, alanine substitutions on the RKC motif of CD23 were made via site-directed mutagenesis. Consequently, a recombinant form of the I-domain of CD11 (b and c) as well as a wild type (containing the RKC motif) and mutant form (containing an AAC motif) of sCD23 were expressed and purified. The CD11 recombinant proteins were purified via affinity chromatography and the CD23 recombinant proteins via gel filtration chromatography. In addition, synthetic (CD23 derived) peptides, one containing the RKC sequence and the other the AAC sequence, were designed and custom synthesized. The synthetic peptides as well as the recombinant CD23 proteins were then analyzed for their interaction with the CD11 I-domain via ELISA. Subsequent ELISA analyses showed that the native sCD23 and the RKC peptide were able to bind to the integrin α I-domain whereas the mutant sCD23 and the corresponding synthetic AAC peptide failed to bind. This interaction was also analysed via flow cytometry using differentiated U937 cells, yielding similar results. ELISA analyses for the sCD23-CD11b I-domain interaction showed a Kd of 0.36 ± 0.14 μM whereas the RKC-CD11b I-domain interaction yielded a Kd of 1.75 ± 0.58 μM. Similarly, the sCD23-CD11c I-domain interaction yielded a Kd of 0.39 ± 0.09 μM and 1.53 ± 0.72 μM for the RKC-CD11c I-domain interaction. Peptide inhibitory analysis, analysed via ELISA and flow cytometry, reinforced the fact that the RKC motif on sCD23 is a prerequisite for ligand binding of the CD11b/c I-domain.
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Caven, Timothy Hays. "IGE PRODUCTION REGULATION VIA CD23 STALK ENGAGEMENT AND CELL CYCLE STIMULATION." VCU Scholars Compass, 2006. http://hdl.handle.net/10156/1643.
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Potter, Sarah Jane. "Molecular analysis of human CD23 (Fc[epsilon]RII) protein isoform function." Thesis, University of Glasgow, 2001. http://theses.gla.ac.uk/2107/.
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In the research described in this thesis, the trafficking of each CD23 isoform was studied in detail in both wild-type and mutant CD23 proteins, using an identical cell system and the same method of CD23 ligation for both isoforms. Confocal microscopic analysis demonstrated intracellular sorting differences to exist between the two isoforms, with CD23a utilising the endocytic pathway, and CD23b following both the endocytic and phagocytic pathways in a B-cell line. Site-directed mutagenesis was used to investigate a number of potentially key residues present in the unique N-terminal tail of each isoform. The serine groups at positions 7 and 5 in the CD23a and b isoforms, respectively, and the NNP tri-peptide motif in the b isoform were found to be necessary for accurate trafficking of these proteins. The discovery that the CD23 isoforms utilise different trafficking pathways corroborates the hypothesis that CD23a and CD23b may have functionally different roles. Current models of human CD23 signalling link the CD23a isoforms to a cAMP-generating pathway and the CD23b isoform to stimulation of inositol-1,4,5-trisphosphate production and calcium mobilisation. The ability of CD23 to transmit a signal within various cells and the fact it has a very short cytoplasmic tail, of only 23 amino acids and bereft of catalytic motifs, strongly suggests that CD23 may associate with other molecules involved in signal transduction. The divergence in the signalling pathways associated with each CD23 isoform has been attributed to the unique amino acids at the N-terminal cytoplasmic tails, as the remainder of the proteins are identical. However, there is no definitive evidence to link directly CD23 to any of these individual signalling pathways. The research presented in this thesis utilises the yeast two-hybrid assay to investigate binding partners for the N-terminal tail of CD23. Filamin A was identified as a potential interacting protein, and was found to interact with both the CD23a and CD23b isoforms. It is hypothesised that CD23 interacts with filamin A, which functions as an adaptor protein, to enable downstream responses to CD23 through connections to the appropriate signalling pathways in an isoform-specific manner.
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Cooper, Ali. "The role of human CD23 in lgE homeostasis & allergic disease." Thesis, King's College London (University of London), 2013. https://kclpure.kcl.ac.uk/portal/en/theses/the-role-of-human-cd23-in-lge-homeostasis--allergic-disease(9cf99ae7-3243-441e-b146-4a63c01127ec).html.
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CD23, the low affinity receptor for IgE on B cells, exists in membrane and soluble forms. CD23 also binds CD21 with a distinct binding site to IgE. Soluble CD23 (sCD23) fragments are released from trimeric membrane CD23 (mCD23) by the endogenous metalloprotease, ADAM10. It has been suggested that trimeric sCD23 fragments can co-ligate membrane IgE (mIgE) and membrane CD21 (mCD21) on the surface of human B cells, in a similar way to C3d-antigen complexes and mIgM, to upregulate IgE synthesis and provoke allergic responses. To test this hypothesis, purified tonsil B cells were stimulated with IL-4 and anti-CD40 to induce class switching to IgE in vitro. mCD23 was up-regulated and sCD23 accumulated in the medium prior to IgE synthesis. IL-10 and IL-21 were shown to enhance IgE synthesis by increasing cell division and plasma cell differentiation. siRNA inhibition of CD23 synthesis or inhibition of mCD23 cleavage by an ADAM10 inhibitor, GI254023X, were shown to suppress IgE synthesis. Addition of a recombinant trimeric sCD23, triCD23, enhanced IgE synthesis. This occurred even when endogenous mCD23 was protected from cleavage by GI254023X, indicating that IgE synthesis is positively controlled by sCD23. triCD23 was shown to bind to cells coexpressing mIgE and mCD21 and caused capping of these proteins on the B cell membrane. triCD23-mediated up-regulation of IgE secretion and capping of mCD21 was blocked in the presence of an anti-CD21 monoclonal antibody. Up-regulation of IgE secretion by sCD23 occurred after class switch recombination and the effects were isotype-specific. Together, these results suggest that mIgE and mCD21 co-operate in the sCD23-mediated positive regulation of IgE synthesis by IgEcommitted B cells. These results have improved our understanding of the regulation of IgE in human B cells and provide evidence for sCD23 as a potential therapeutic target in allergy and asthma.
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Sprong, Kaitlin. "Analysis of the interaction between recombinant human Beta2 integrin I-domains and CD23." Thesis, Nelson Mandela Metropolitan University, 2014. http://hdl.handle.net/10948/d1021078.
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In order to further elucidate the interaction between CD23 and β2 integrins (CD11b/CD18) the following objectives were established: Expression and purification of CD11b I-domain as a GST-fusion protein using Escherichia coli; Cloning, synthesis and expression of CD18 I-Like domain.CD11b I-domain has previously been expressed as a GST-fusion protein (Daniels, 2010) and consequently led to comparable expression of CD18 I-like domain as a GST-fusion protein; Preparation of two site-directed mutants of CD18 I-Like domain in order to study the function of the serine residue involved in the S116P mutation. The serine was mutated to proline, as in LAD patients, as well as alanine, a non-polar alternative, in order to contrast and compare binding characteristics. Expression, refolding and purification of sCD23, and a double mutatedsCD23 (RKΔAA) from E. coli; This was performed according to the method described by Daniels et al. (2005); Investigation of the CD23-CD11b I-like domain interaction through surface plasmon resonance spectroscopy.
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Bertho, Jean-Marc. "Caracterisation des precurseurs lymphocytaires t humains : role du cd23 dans leur differenciation." Paris 6, 1991. http://www.theses.fr/1991PA066032.
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La nature des precurseurs de la lignee lymphocytaire t humaine reste difficile a etablir. Nous avons pu isoler trois populations differentes de cellules pre-t, l'une d'origine medullaire, les deux autres d'origine thymique, sur la base de l'expression de l'antigene cd7, qui est l'un des marqueurs les plus precoces de la lignee lymphocytaire t. Ces cellules sont capables de se differencier in vitro pour donner des cellules t matures. L'etude de leurs caracteristiques phenotypiques et fonctionnelles a permis de proposer un schema des premieres etapes de la differenciation lymphocytaire t. La differenciation in vitro de ces precurseurs est induite par le fragment soluble de cd23, en presence d'il1. D'autres activites biologiques ont pu etre attribuees a cette molecule. Nous avons ainsi montre que le cd23 induit la differenciation et la proliferation des precurseurs myeloides, et la proliferation des cellules t cd4+ medullaires. De par ses activites biologiques variees, le cd23 acquiere une importance grandissante dans la regulation de l'hematopoiese et de la reponse allergique
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FOURCADE, CHRISTINE. "Expression et role du cd23 au niveau des stroma hematopoietiques chez l'homme." Paris 11, 1993. http://www.theses.fr/1993PA112209.
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Sur la base de l'expression de l'antigene cd7 qui est l'un des marqueurs les plus precoces de la lignee lymphocytaire t, nous avons pu isoler des cellules cd7+ cd2. Ces cellules sont capables de se differencier in vitro en lymphocytes t matures en presence d'il1 et de cd23 et de proliferer en reponse a l'il3. La signification physiologique de ces donnees a ete confirmee par la mise en evidence de ces facteurs in vivo sur coupe de thymus au niveau des cellules epitheliales thymiques. Le cd23 est egalement exprime par les monocytes/macrophages du stroma medullaire ou il intervient dans l'autorenouvellement des precurseurs hematopoietiques et la proliferation des cellules t cd4+ medullaires. Le cd23 et l'il3 sont egalement secretes par les keratinocytes de la peau ou ils pourraient etre des facteurs potentiels de differenciation extra thymique. L'ensemble de ces resultats souligne le polymorphisme fonctionnel du cd23 et le role majeur des stroma hematopoietiques dans l'hematopoiese chez l'homme
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ROUSSELET, GERMAIN. "La proteine cd23 dans les carcinomes nasopharynges : etude biologique et approche clinique." Paris 6, 1992. http://www.theses.fr/1992PA066317.
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Le carcinome indifferencie du nasopharynx constitue un probleme de sante publique, et un modele unique de cancerogenese multi-factorielle. Sa repartition geographique endemique suggere l'existence de facteurs environnementaux et/ou genetiques decisifs; le virus d'epstein-barr est toujours present dans les cellules tumorales et joue certainement un role dans la cancerogenese; les interactions entre les lymphocytes infiltrant la tumeur et les cellules malignes sont un element majeur de la biologie tumorale. La proteine cd23 ayant ete impliquee dans les processus d'immortalisation des lymphocytes b par le virus d'epstein-barr, nous avons entrepris son etude dans ce nouveau modele. Nos travaux demontrent que cd23 peut etre exprime par des cellules epitheliales de la tumeur, et regule positivement ou negativement par des cytokines produites par des lymphocytes t actives (interleukine 4, interferon gamma). Ils permettent de completer le schema des interactions paracrines au sein de la tumeur. De plus, cd23 apparait comme un marqueur pronostique en terme de survie et de rechutes loco-regionales, pour des patients atteints d'une tumeur de stade eleve. Son dosage dans le serum pourrait donc permettre de mieux adapter les protocoles therapeutiques et le suivi des patients. Nous suggerons que cd23 pourrait etre le marqueur d'une sous-population de tumeurs primaires, capables de developper des interactions particulieres avec leur micro-environnement, et presentant une plus grande agressivite loco-regionale
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Gu, Baijun. "TWO PATHWAYS OF SHEDDING OF L-SELECTIN AND CD23 FROM HUMAN B-LYMPHOCYTES." University of Sydney, 2000. http://hdl.handle.net/2123/821.
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Lymphocytes from patients with B-chronic lymphocytic leukemia (B-CLL) express large numbers of P2X7 receptors for extracellular adenosine triphosphate (ATP). Activation of P2X7 receptors induces multiple downstream effects, of which the best documented is the opening of an ionic channel that is selective for divalent cations. Another effect of ATP is to induce the shedding of L-selectin (CD62L), a molecule which is involved in the adhesive interactions of lymphocytes on endothelial cells. High levels of soluble L-selectin and CD23 are found in the serum of patients with B-CLL, although the mechanisms involved in their production are poorly characterized. Because extracellular ATP causes shedding of L-selectin, we studied the effect of ATP on shedding of CD23, an adhesion molecule expressed on the surface of B-CLL lymphocytes. ATP induced the shedding of CD23 at an initial rate of 12% of that for L-selectin, while the EC50 of ATP (35 uM) and BzATP (10 uM) was identical for shedding of both molecules. Inactivation of the P2X7 receptor by pre-incubation with OxATP, an irreversible inhibitor of P2X7 purinoceptor, abolished ATP-induced shedding of both molecules. Moreover, KN-62, the most potent inhibitor for the P2X7 receptor inhibited ATP-induced shedding of both CD23 and L-selectin with the same IC50 (12 nM). Ro 31-9790, a membrane permeant zinc chelator which inhibits the phorbol-ester stimulated shedding of L-selectin also inhibited shedding of CD23 from B-CLL lymphocytes, but the IC50 was different for the two shed molecules (25 versus 1 ug/ml respectively). Although L-selectin was completely shed by incubation of cells with phorbol-ester no CD23 was lost under these conditions. Also, Ca2+ inhibits ATP-induced CD23 shedding but not L-selectin shedding. Since soluble CD23 and L-selectin are found in the serum of normal subjects and B-CLL patients, the expression of these two adhesion molecules on lymphocytes before and after transendothelial migration was studied in an in vitro model of this process. In normal and B-CLL subjects, 71�b5% of L-selectin from both T and B cells and 90% of CD23 from B cells was lost following transmigration, while the expression of a range of other adhesion molecules such as VLA-4, ICAM-1, LFA-1 and CD44 was unchanged. Lymphocytes incubated with OxATP retained their capacity for transendothelial migration and showed the same loss of L-selectin as control leukaemic lymphocytes. Ro 31-9790, which can protect ATP-induced both L-selectin and CD23 shedding, had no effect on inhibiting L-selectin and CD23 lost during transmigration. These data show the presence of a second pathway for the downregulation of L-selectin and CD23 from the lymphocyte surface. Data in vivo from 'knock-out' mice show that L-selectin is essential for the emigration of lymphocytes through high endothelial venules into lymph nodes. The migration of normal and B-CLL lymphocytes across confluent human umbilical vein endothelial monolayers was studied in an in vitro model of this process. Lymphocytes treated with ATP or BzATP showed 56�b25% or 67�b16% loss of L-selectin on the surface and 36�b24% or 64�b19% decrease of transmigration, respectively, while OxATP, which does not alter the L-selectin level, had no effect on lymphocyte transmigration. Further experiments examined this correlation between L-selectin expression and lymphocyte transendothelial migration in this model system. A quantitative assay for cell surface L-selectin showed that expression of L-selectin was lower on B-CLL lymphocytes (8,880�b5,700 molecules/cell) than on normal lymphocytes (29,500�b7,500 molecules/cell, p less than 0.001). Also the rate of transmigration of B-CLL lymphocytes (1.5�b0.9 migrated cells/HUVEC) was lower than normal peripheral lymphocytes (2.4�b0.9 migrated cells/HUVEC, p=0.04). Incubation of lymphocytes in complete medium for 24 hrs increased the expression of L-selectin on B-CLL lymphocytes by 1.5 to 2 fold while the normal lymphocyte L-selectin remained at the initial level. This upregulation of B-CLL L-selectin correlated with a 2 fold increased rate of transendothelial migration. A correlation was found between L-selectin expression on lymphocytes and their ability for transendothelial migration (r^2=0.6). This study shows that the adhesion molecules L-selectin and CD23 can be lost from lymphocytes by two different physiological pathways. One is by P2X7 receptor activation by extracellular ATP while the second is activated by transendothelial migration of these cells. A second finding is that B-CLL lymphocytes have lower level of L-selectin expression and an impaired ability for transendothelial migration compared with normal peripheral blood lymphocytes. Do these results explain the high serum levels of soluble L-selectin and CD23 observed in B-CLL? Although B-CLL lymphocytes do not recirculate as rapidly as normal peripheral blood lymphocytes, the greatly increased number of leukaemic cells in B-CLL ensures that much more soluble L-selectin and CD23 is generated during the recirculation of these cells through the body.
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Wright, Tracey Jane. "Characterisation of CD23 cleavage by endogenous and exogenous proteases using neo-epitope antibodies." Thesis, University of Leicester, 2000. http://hdl.handle.net/2381/29820.
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CD23 is the low affinity IgE receptor. It is a type II integral transmembrane glycoprotein that can be shed from the cell surface forming soluble products of approximately 37, 33, 29, 25 and 16kDa. It appears that membrane and soluble CD23 have opposing regulatory functions and that inhibition of CD23 shedding may have potential to alleviate both allergic and inflammatory diseases. This has focused attention on the endogenous protease(s) responsible for CD23 shedding, leading to the demonstration that both the 37 and 33kDA sCD23 fragments are cleaved from the cell surface by a metalloprotease. In order to characterise the cleavage events within CD23, anti-neoepitope antibodies specific for the newly created amino and carboxy termini of the two predominant cleavage sties within CD23 (producing the 37 and 25kDa soluble CD23 products) were raised. Characterisation of these antibodies demonstrated that the proteolytic cleavage events responsible for creating the 37 and 25kDa sCD23 fragments are independent of each other. Furthermore, two different proteases were shown to be responsible for cleaving these two fragments. The work described in this thesis confirms previous reports that 37kDA sCD23 is cleaved by a metalloprotease, however cleavage of the 25kDa fragment was not inhibited by metalloprotease inhibitors. The production of the two different sized sCD23 molecules by different proteases has important implications for targeting the proteolytic cleavage events to alleviate symptoms of allergic and inflammatory diseases. This emphasises the importance of defining the biological functions of mCD23 and each of the sCD23 molecules.
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Rückert, René. "Bakterielles Superantigen verstärkt die Atemwegsinflammation und bronchiale Atemwegsreagibilität in einem Mausmodell der allergischen Sensibilisierung." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2000. http://dx.doi.org/10.18452/14539.
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Asthma Bronchiale (AB) ist eine chronisch- obstruktive, teilweise reversible Entzündung der Atemwege, deren klinisches Korellat die bronchiale Hyperreagibilität (BHR) ist. Es lassen sich aufgrund ethiologischer Faktoren extrinsiches und intrinsisches AB unterscheiden, wobei ersteres auf einer allergischen Sensibilisierung und letzteres auf irritativen oder infektbedingten entzündlichen Prozessen beruht. In der vorliegenden Arbeit wurde der Einfluß von bakteriellem Superantigen auf die Entzündungsreaktion und die bronchiale Hyperreagibilität untersucht. Stapylococcal enterotoxin B (SEB) wurde hierbei als Modellsubstanz in einem Mausmodell eingesetzt, da SEB produzierende Staphylokokken im Nasenrachenraum von Asthmatikern nachgewiesen werden konnten. Nasale Applikation von SEB induzierte in C57BL/6 Mäusen eine Entzündungsreaktion mit Influx von Lymphozyen und eosinophilen Granulozyten sowie gesteigerte Produktion von IL-4, IL-5 und TNF-alpha in der Lunge, welches in der Histologie und Bronchiallavage nachgewiesen wurde. Desweiteren führt SEB allergenunabhängig zur Ausbildung von BHR. SEB Applikation in einem Mausmodell der allergischen Sensibilisierung (gegen Ovalbumin in C57BL/6 Mäusen) verstärkt die allergische Entzündung in der Lunge und die BHR. CD23 (Low-Affinity IgE Rezeptor) Knock out Tiere zeigen nach allergischer Sensibilisierung und SEB Behandlung keinen Anstieg der TNF-alpha Produktion und keine Hyperreagibilität. Aus diesen Ergebnisse läßt sich schlußfolgern: I. Bakterielles Superantigen induziert das Vollbild des intrinsischen AB im Tiermodell. II. Bakterielles Superantigen kann das extrinsische, allergische AB verstärken. III. Der CD23 Rezeptor ist essentiell für die TNF-alpha Produktion und die Induktion von BHR. Diese Resultate sollten in klinischen Studien am Patienten überprüft werden, da aufgrund der hier vorliegenden Daten zu erwarten ist, daß Antibiotikatherapie, und damit Elimination superantigenproduzierender Bakterien im Nasenrachenraum, die klinische Symptomatik des AB reduzieren kann.<br>Asthma bronchiale (AB) is an obstructive, partially reversible chronic inflammatatory disease of the small airways, which clinical correlate is represented by airway hyperreactivity. Based on etiological factors, AB can be divided in extrinsic and intrinsic AB, where the first depends on an allergic sensitization and the latter on airway irritation by environmental factors or airway inflammation due to viral or bacterial infection. In this thesis, the role of bacterial superantigens in airway inflammation and -hyperreactivity is analyzed. Staphylococcal enterotoxin B (SEB) was used as a prototypic substance, since SEB producing Staphylo-coccal aureus can be found in the nose and pharynx of asthmatic patients. Nasal application of SEB in C57BL/6 mice resulted in airway inflammation characterized by an influx of lymphocytes and eosinophil granulocytes and increased production of IL-4, IL-5, and TNF-alpha, which was analyzed by histology and bronchiolalveolar lavage. Furthermore, SEB induced independent of aller-gens airway hyperreactivity. SEB application in a mouse-model of allergic sensitization (to ovalbumin in C57BL/6 mice) boosts the allergen-induced allergic inflammation and airway hyperreactivity. CD23 (low-affinity IgE receptor) knock out mice showed no increased TNF-alpha production and no air-way hyperreactivity after allergic sensitization and SEB treatment. These results demonstrate: I. Bacterial superantigen can induce intrinsic AB in a mouse model. II. Bacte-rial superantigen can significantly boost the allergic, extrinsic AB. III. The CD23 receptor is essential for TNF-alpha production and for the induction of airway hyperreactivity. Based on these findings, clinical surveys should be performed, since one could expect, that eradication of nasal bacterial carriage and therefore local superantigen sezernation should improve the AB- symptoms in affected patients.
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Keith, Brooks. "IgE Enhances B Cell-Derived Exosomal Induced T Cell Proliferation." VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/2909.
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For many years it has been known that the injection of antigen bound to an antibody leads to more than a 1000-fold increase in antigen specific antibody response. This observation holds true for IgE, which is dependent upon CD23 expression, as this enhancement is not present in mice deficient in CD23. It also has been shown that when mice are injected with IgE-antigen complexes also display an increase in antigen specific T cell proliferation. While there are published studies that demonstrate a role for B cell derived exosomes in the activation and proliferation of T cells, none have focused upon the potential role of CD23 as a molecular basis for this phenomenon, at least in the context of allergy and asthma. This thesis provides direct evidence that B cell-derived exosomes possess co-stimulatory molecules, including CD80 and CD86, which act in concert with CD23 to induce T cell proliferation, at least in vitro. This is due to, or enhanced by, the exosomal transfer of the antigen or peptide to T cells. Importantly, the antigen transfer is dependent upon the availability of IgE and the expression of CD23.
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BOURGET, ISABELLE. "Role de la molecule cd20 dans l'activation des lymphocytes b : regulation de l'expression du recepteur pour l'antigene et de la molecule cd23 (fc epsilon rii)." Nice, 1994. http://www.theses.fr/1994NICE4724.
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La molecule cd20 est une phosphoproteine transmembranaire de 35/37 kda exprimee a la surface des cellules b depuis un stade precoce de leur maturation et jusqu'au stade plasmocyte. Nos resultats montrent que les anticorps diriges contre la molecule cd20 reduisent considerablement le niveau d'expression du recepteur pour l'antigene (migm) a la surface des cellules b. De plus, ils inhibent les effets inducteurs de l'interleukine-4 (il-4) sur l'expression de cette molecule. Dans ces conditions, on observe une diminution de la capacite des cellules b a etre activees par ce recepteur. Les anticorps cd20 n'agissent pas au niveau de la transcription du gene codant pour les igm. De plus, ils n'affectent ni leur biosynthese, ni leur maturation, ni leur internalisation. Par contre, ils empechent l'insertion a la membrane des migm neosynthetisees en les deroutant vers un compartiment intracellulaire ou elles sont degradees. Les anticorps cd20 reduisent egalement l'expression basale du recepteur de basse affinite pour les ige (fc epsilon rii/cd23) a la surface des cellules b et sa surexpression induite par l'il-4. En fait, les anticorps cd20 stimulent le clivage proteolytique extracellulaire de la molecule cd23 membranaire et la liberation de fragments solubles. Ces resultats prennent de l'importance puisque cette molecule, sous sa forme membranaire ou soluble, est connue pour reguler la proliferation et la maturation des cellules lymphoides et myeloides. Elle est egalement impliquee dans la production d'ige et la liberation de mediateurs de l'inflammation. En controlant l'expression du recepteur pour l'antigene et de cd23 a la surface des lymphocytes b, la molecule cd20 pourrait donc jouer un role regulateur sur differents aspects de la reponse immune
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Mathews, Joel. "ADAM10 exacerbation of allergic disease is potentially explained by its role in CD23 exosomal sorting." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/2372.
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CD23, the natural negative regulator of IgE, has been shown to be involved in asthma progression through its regulation of IgE. To investigate if its sheddase, ADAM10, is also involved in asthma progression, three mouse models were utilized; an IgE/mast cell dependent model, an IgE dependent, mast cell independent model and a mast cell and IgE independent model. Experimental asthma was then induced in mice which were selectively deficient for ADAM10 in B cells (ADAM10-/-) and compared to WT controls. The ADAM-/- mice had decreased signs of asthma, including eosinophilia, AHR and IgE synthesis in the IgE dependent model compared to LM controls, while with the IgE independent model there was no significant difference. Thus, CD23Tg and ADAM10-/- B cell mice have reduced IgE dependent lung inflammation in mouse models compared to WT controls. As a follow up, ADAM10 was inhibited in WT mice by intranasal administration of an ADAM10 inhibitor, compared to carrier (DMSO) treated mice. As with ADAM10-/- mice, inhibition of ADAM10 was only able to control IgE dependent models. These results thus show that ADAM10 is a possible target in controlling IgE dependent allergic disease, possibly as blocking ADAM10 would cause an increase in CD23 membrane expression. To better understand how ADAM10 cleaves CD23 we first sought to confirm previous studies that CD23 is internalized, with the hypothesis that shedding takes place intracellularly, rather than at the cell surface as previously assumed. Indeed, ADAM10 is more highly expressed intracellularly than at the cell surface. At 37 ºC, crosslinking CD23, especially with the anti-stalk mAb 19G5, resulted in extensive CD23 internalization. In addition, the expected increase in soluble CD23 (sCD23) production when 19G5 was added was blocked by the addition of NH4Cl. NH4Cl is known to block the progression of the endosomal pathway. These findings thus confirmed our hypothesis that cleavage of CD23 requires internalization and progression through the endosomal pathway before it is released into the extracellular space. We further demonstrated that ADAM10 is not only involved in cleaving CD23, but also in sorting CD23 into exosomes, as B cells lacking ADAM10 do not incorporate CD23 into exosomes. In addition, we found that exosomes secreted from the cell contain full length CD23, thus showing that they could bind IgE/antigen complex and be involved in the known CD23 dependent enhancement of antigen presentation by the injection of IgE/antigen complexes compared to antigen alone. These results also show that the change in ADAM10 expression specifically in a B cell could be involved in enhancement of IgE dependent inflammation. To determine what signals change ADAM10 expression, ADAM10 promoter studies were initiated. We found that both IL-21 and anti-CD40 increased ADAM10 promoter activity, while IL-4 and IL-13 had no effect. Overall our data show that increasing ADAM10 activity and expression leads to increased inflammation and IgE and is a possible target in controlling IgE dependent diseases.
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Santamaria, Kathleen. "Etude de l’hétérogénéité des centrocytes humains à travers l’expression du CD23 : différenciation en plasmablastes et expression d’une signature minimale transcriptionnelle au niveau cellule-unique comportant DEC2." Thesis, Rennes 1, 2020. http://www.theses.fr/2020REN1B038.
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La différenciation terminale des lymphocytes B à travers le centre germinatif conduit à la production de cellules sécrétrices d’anticorps : les plasmocytes (PC) à longue durée de vie et de haute affinité pour l’antigène. Cette réaction implique la formation d’une microstructure anatomique qui comprend une zone sombre : lieu d’intenses proliférations des centroblastes et de maturation d’affinité de leur BCR, et une zone claire dans laquelle ont lieu les commutations de classes isotypiques du BCR et la sélection des centrocytes (CC). Ce processus est finement contrôlé par les lymphocytes T folliculaires auxiliaires (Tfh) notamment à travers la production de molécules comme l’IL-4, le CD40L et l’IL-21. Ce travail de thèse s'intéresse à l'expression du CD23 à la surface des CC lors de leur métamorphose en PC. J'ai d’abord montré que l'expression de ce récepteur de faible affinité aux IgE est régulée par les signaux Tfh : CD40L et IL-4. De plus j'ai mis en évidence que les CC humains exposés aux signaux Tfh mais négatifs pour le CD23 sont capables de se différencier en PC. Dans ce cadre, la signature transcriptionnelle de ces progéniteurs de PC a été étudiée à l'échelle de la cellule unique et a permis d’identifier un gène jusqu’alors jamais décrit dans les PC qui code pour le facteur de transcription DEC2 et dont la fonction reste à déterminer. J'ai par ailleurs étudié l'expression du CD23 dans le lymphome folliculaire et mis en évidence qu’il existe des différences entre les populations tumorales positives et négatives pour le CD23, suggérant que les cellules CD23neg ont un avantage de survie et une capacité de différenciation supérieure en culture que les cellules CD23pos<br>Terminal B cell differentiation through the germinal center leads to the production of antibody-secreting cells: plasma cells (PC) with a long lifespan and high affinity for the antigen. This reaction involves the formation of an anatomical microstructure that includes a dark zone: site of intense proliferation and affinity maturation of the BCR of the centroblasts, and a light zone in which the class switch recombination of the BCR and centrocyte (CC) selection take place. This differentiation is finely controlled by follicular helper T cells (Tfh) that produce molecules such as IL-4, CD40L and IL-21. This phD work focus on the CD23 expression on the surface of CC during their metamorphosis into PC. Firstly, I showed that the expression of the low affinity IgE receptor is regulated by Tfh derived IL-4 and CD40L. Moreover, I showed that CC exposed to Tfh signals and which do not express the CD23 were the ones that have the ability to differentiate into PC. In this context, I identified at the single cell level a transcriptional signature expressed specifically by these cells which contains a gene never described in PC, encoding the transcription factor DEC2 whose function remains to be determined. In addition, I studied CD23 expression in follicular lymphoma and showed the existence of differences between these two populations, suggesting a preferential survival and differentiation of CD23neg cells compared to CD23pos cells
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OUAAZ, FATEH. "Fonctions du cd23 soluble et membranaire dans la lignee myelomonocytaire. Etude des signaux intracellulaires." Paris 6, 1994. http://www.theses.fr/1994PA066419.
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Le cd23 est le recepteur de basse affinite pour l'ige et possede sous sa forme soluble ou membranaire diverses activites biologiques. En synergie avec l'interleukine-1, le cd23 soluble est capable d'induire la differenciation in vitro des precurseurs lymphoides t et myeloides de la moelle osseuse humaine. Nous avons montre dans ce travail deux nouvelles activites cytokine du cd23 soluble: a) la regulation de la proliferation des precurseurs myeloides leucemiques et b) l'augmentation des reponses inflammatoires des monocytes humains. L'analyse des signaux intracellulaires et du recepteur du cd23 soluble est en cours. Nous avons montre l'expression in vivo du cd23 par les macrophages du stroma medullaire ce qui illustre la relevance physiologique de l'action de cette molecule dans l'hematopoiese humaine. La forme membranaire du cd23 apparait comme etant le recepteur fonctionnel aux ige a la surface des monocytes humains normaux, des eosinophiles et des keratinocytes. J'ai pu montre que la stimulation du cd23 membranaire peut constituer une voie de differenciation terminale vers le macrophage des precurseurs myelomonocytaires leucemiques. Grace a cette etude, j'ai mis en evidence une cascade d'evenements moleculaires decoulant de la stimulation du cd23 membranaire et impliques dans la differenciation terminale des cellules leucemiques. Nous avons montre d'abord une generation de nucleotides cycliques et du monoxyde d'azote, ensuite l'activation du facteur transcriptionnel nfkb et enfin la modulation de l'expression de divers proto-oncogenes comme le c-fms. L'ensemble de ce travail montre le role du recepteur/cytokine cd23 dans la proliferation et la differenciation des cellules hematopoietiques de la lignee myelomonocytaire ainsi que les reponses inflammatoires
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Yoshikawa, Tsutomu. "Characterization of novel FcεRII/CD23 isoforms lacking the transmembrane segment in human cell lines". Kyoto University, 2002. http://hdl.handle.net/2433/149338.
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Bund, Dagmar [Verfasser], and Michael [Akademischer Betreuer] Hallek. "hTERT, CD23 und CD229 als Tumorantigene bei der B-CLL / Dagmar Bund. Betreuer: Michael Hallek." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2012. http://d-nb.info/1024243443/34.
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Housden, Jonathan E. M. "Lys 352 in human IgE is a major effector determinant residue in IgE-CD23 interaction." Thesis, University of Sheffield, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.443882.
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Kao, Wen-Pin. "Protein engineering and characterization of a stable trimeric form of CD23 and a fluorescent IgE biosensor." Thesis, King's College London (University of London), 2013. https://kclpure.kcl.ac.uk/portal/en/theses/protein-engineering-and-characterization-of-a-stable-trimeric-form-of-cd23-and-a-fluorescent-ige-biosensor(4b861bc3-fa5b-4590-9665-9507bcfcdbf5).html.
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Immunoglobulin E (IgE) plays a critical role in allergic diseases such as asthma and atopic dermatitis. Cross-linking of IgE bound to its high affinity receptor, FceRI, by allergen on mast cells and basophils results in the release of inflammatory mediators and induces allergic reactions. IgE can also form a complex with its low affinity receptor, CD23, and CD21 on the B cell surface to regulate IgE synthesis. Hence, there are two possible strategies in the therapy of IgE-mediated disease: blocking IgE binding to FceRI and inhibiting IgE production. Crystal log raphic and FRET studies with a fluorescently labelled IgE-Fc biosensor revealed conformational change in IgE when it binds to FceRI. It also implied that an IgE-Fc biosensor is not only a useful tool to study the binding of IgE and its receptor, but also can be used to screen drug candidates for IgE-mediated therapy. To improve the reliability of the IgE-Fc biosensor, we characterized the fluorescent molecules used to construct the biosensor and generated a biosensor by other methods. Results suggested that a biosensor made by biotinylated IgE-Fc and monovalent streptavidin, both labelled with fluorescent dyes, is a useful tool. Therapies to block IgE binding to FceRI are clearly efficacious but still have limitations. Hence, a strategy to block IgE synthesis remains an extremely important goal of current research. Since CD23 was discovered in 1975, it has been proposed to play a critical role in IgE synthesis. Soluble CD23 is released by proteolytic cleavage from membrane-bound CD23 and interacts with membrane-bound IgE and CD21 to increase IgE production. Membrane-bound CD23 can also be ligated by IgE-allergen complexes to reduce IgE production. To test this model, we produced trimeric CD23, triCD23, which was designed by adding an isoleucine-rich a-helical coiled-coil motif to its N-terminal region and characterised this protein by various biophysical methods. Biophysical characterization suggested that triCD23 has a well folded lectin head domain and forms a trimer. Surface Plasmon Resonance results indicated that triCD23 binds to IgE-Fc fragments with two different affinities, 2.7x10"6 and 1.5x10"8M. Biological functional measurements showed that it binds to IgE on human B cells and increases IgE production.
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Brignone, Chrystelle. "Régulation du clivage du récepteur de basse affinité pour les IgE (FceRII/CD23) : implication dans l'inflammation." Nice, 2001. http://www.theses.fr/2001NICE5667.
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Siddorn, Philip David. "Efficient numerical modelling of wave-structure interaction." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:de36bd2f-cd23-4f11-b67f-9d8cd48ecd3c.
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Offshore structures are required to survive in extreme wave environments. Historically, the design of these offshore structures and vessels has relied on wave-tank experiments and linear theory. Today, with advances in computing power, it is becoming feasible to supplement these methods of analysis with fully nonlinear numerical simulation. This thesis is concerned with the development of an efficient method to perform this numerical modelling, in the context of potential flow theory. The interaction of a steep ocean wave with a floating body involves a moving free surface and a wide range of length scales. Attempts to reduce the size of the simulation domain cause problems with wave reflection from the domain edge and with the accurate creation of incident waves. A method of controlling the wave field around a structure is presented. The ability to effectively damp an outgoing wave in a very short distance is demonstrated. Steep incident waves are generated without the requirement for the wave to evolve over a large time or distance before interaction with the body. This enables a general wave-structure interaction problem to be modelled in a small tank, and behave as if it were surrounded by a large expanse of water. The suitability of the boundary element method for performing this modelling is analysed. Potential improvements are presented with respect to accuracy, robustness, and computational complexity. Evidence of third order diffraction is found for an FPSO model.
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Munoz, Olivier. "Régulation du clivage du récepteur de basse affinité pour les IgE (Fc[epsilon]RII/CD23 : importance de l'oligomérisation." Nice, 2000. http://www.theses.fr/2000NICE5417.
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CD23 est le récepteur de basse affinité pour les IgE exprimé à la surface des cellules hématopoi͏̈étiques, dont les lymphocytes B ou les monocytes. La partie extracellulaire la plus distale de ce récepteur présente certaines homologies avec les lectines de type C, et contient les sites d'interaction pour différents ligands (IgE, CD21, CD11b /CD11c, CD47/VnR) qui rendent compte des propriétés biologiques de CD23. Le domaine lectine est connecté à la partie transmembranaire par une région en hélice α présentant un motif leucine-zipper, responsable de l'oligomérisation de la molécule. Cette région contient en outre des sites de clivage protéolytique libérant des formes solubles dont certaines gardent la capacité à former des oligomères. Toutes conservent le domaine lectine et interagissent à des degrés divers avec les ligands précédents. Le mécanisme protéolytique responsable de ce clivage reste toujours à déterminer. CD23 est impliqué dans d'importantes fonctions du système immunitaire, dont la production d'IgE et l'inflammation. Les formes membranaires ou solubles régulent ses fonctions souvent de manière opposée. Ainsi CD23 joue un rôle non négligeable dans la pathogénèse de l'allergie ou certaines maladies inflammatoires chroniques, dont la polyarthrite rhumatoi͏̈de. Nos résultats montrent que la désorganisation de cette structure augmente fortement le clivage spontané de CD23, ainsi que la sensibilité du récepteur vis-à-vis des protéases exogènes telles que l'élastase. A l'inverse, les IgE stabilisent une forme oligomérique, peu sensible au clivage. D'autre part, ce travail met en exergue la prédisposition de CD23 à être clivé par les protéases de l'inflammation et l'activité pro-inflammatoire des fragments solubles alors libérés. L'ensemble de ces données atteste d'un rôle central de la conformation de CD23 dans sa protéolyse.
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Good, Samantha. "An investigation into the interaction of truncated recombinant IgE-Fc fragments with Fc&RI and CD23." Thesis, University of Sheffield, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274954.
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Becherel, Pierre-André. "Role du cd23 et de la no-synthetase dans l'activation des keratinocytes humains : implications en immunophysiopathologie et pharmacologie." Paris 6, 1999. http://www.theses.fr/1999PA066044.
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Les keratinocytes epidermiques (ke) humains normaux sont exposes en permanence a des stimuli inflammatoires varies et a l'action de facteurs solubles secretes par les cellules du derme (lymphocytes, mastocytes, monocytes/macrophages, fibroblastes), notamment au cours des phenomenes allergiques et des processus infectieux ou anti-tumoraux. Apres stimulation prealable par de l'il-4, les keratinocytes apparaissent a ce jour comme les seules cellules non hematopoietiques a pouvoir exprimer le cd23 (recepteur de faible affinite pour les ige, fcrii) a leur surface. Ce cd23 est fonctionnel, puisque nous avons montre que sa ligation par le couple ige/anti-ige ou par un anticorps monoclonal specifique induit une liberation dose-dependante d'il-6 et de tnf- par ces cellules. La ligation du cd23 membranaire sur les ke s'accompagne egalement d'une accumulation intracellulaire de nucleotides cycliques et de l'activation d'une no-synthase, comme en temoigne la secretion de nitrites par ces cellules. En outre, cette voie no-dependante apparait necessaire a la production de cytokines par les keratinocytes, puisqu'un antagoniste de la no-synthase inhibe toute secretion par ces cellules. Les keratinocytes humains peuvent ainsi participer directement aux reponses immunes mediees par les ige par leur capacite a exprimer un cd23 fonctionnel a leur surface apres stimulation par l'il-4 et a pouvoir lier les ige produites au cours des reactions allergiques. De plus, cette voie de l'inos est inhibee dans les keratinocytes par des modulateurs pharmacologiques comme les retinoides ou des immunomodulateurs comme l'il-10. Enfin, cette induction du cd23 et de l'inos semble etre fonctionnelle puisqu'on les retrouve dans les ke au cours de l'urticaire aigue, affection liee a la fois a la presence d'il-4 et d'ige.
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Nazari, Naser. "An investigation into the interaction of truncated recombinant human CD23 fragments with IgE and their relevance in allergic disease." Thesis, University of Sheffield, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.425190.
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Morel, Franck. "Etude de la sensibilité au CD23 soluble de lymphocites T et B humains dans certaines situations normales ou pathologiques." Poitiers, 1994. http://www.theses.fr/1994POIT2314.
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La mise en evidence et l'etude par notre groupe d'un facteur de differenciation et d'activation lymphocytaire t (ptda) a revele ses analogies avec le scd23 de 25 kda. Le scd23 induit la differenciation des prothymocytes, de precurseurs cd34#+, de cellules b et augmente la synthese des ige. Nous avons demontre l'identite du scd23 et de la ptda, presente dans les surnageants de lymphocytes b normaux ou ebv-transformes. Le scd23 induit, en presence d'il1, une augmentation du taux de clonage des lymphocytes t cd4#+ stimules. Le scd23 ne modifie pas l'expression des antigenes cd2, 3, 4, 7, il2rs et hla-dr, ni la synthese d'il2, 4, 6, et 10. Par contre, il stimule la synthese d'ifng. Nous montrons que la preincubation des lymphocytes t cd4#+ en presence de scd23 induit une inhibition de la synthese des ige par les lymphocytes b stimules par l'il4, en accord avec le role inhibiteur de l'ifng sur cette synthese. Si le cd21 parait etre le recepteur du cd23 membranaire et soluble de 29kda, il ne semble pas etre celui de la forme de 25kda. Apres avoir emis l'hypothese que le cd38 puisse etre un recepteur pour le scd23, nous montrons que le cd38, implique comme le scd23 dans l'activation des lymphocytes t et l'inhibition de l'apoptose de cellule b, n'est pas a lui seul le recepteur du scd23 de 25kda, mais nous n'excluons pas sa participation dans un recepteur multimerique. Par ailleurs, les taux seriques eleves de scd23 dans les llc nous ont amenes a nous interroger sur son implication dans cette pathologie. Nous montrons que le scd23 augmente comme les ifn, la proliferation des cellules b de llc induite par l'il2. Le scd23 induit egalement la proliferation de cellules cd34#+ derivees de la transformation aigue d'une lmc, mais non de cellules tumorales plus engagees dans la differenciation t ou b. Le scd23 stimule donc la proliferation et/ou la differenciation de precurseurs hematopoietiques normaux ou leucemiques determines. Enfin, une augmentation de scd23 serique est detectee dans l'arterite temporale, caracterisee par une inflammation chronique. Ces taux eleves sont reduits sous corticotherapie, comme ceux de crp et d'il6. Le role potentiel du scd23 dans la formation des infiltrats leucocytaires est discute. Le cd23 tant membranaire que soluble apparait donc implique dans de nombreuses etapes de la reponse immune. L'etude des mecanismes d'action mis en jeu devrait permettre de mieux apprehender le role complexe de cette molecule "mi-recepteur-mi cytokine
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Montagnac, Guillaume. "Etude du rôle du récepteur de faible affinité aux IgE, CD23, dans le transport transépithélial de complexes IgE/allergènes." Paris 5, 2005. http://www.theses.fr/2005PA05N11S.
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Le récepteur de faible affinité aux IgE (CD) est impliqué dans le transport transépithélial de complexes IgE / allergène. Au niveau intestinal, ce transport est à l'origine du déclenchement allergique. Mon travail de thèse a visé à étudier le rôle du CD23 dans ce processus. Nous avons établi que les cellules épithéliales intestinales murines expriment la forme CD23b ainsi qu'une nouvelle forme épissée, bDELTA5, dont l'expression est induite par la sensibilisation in vivo. La régulation de l'internalisation du CD23 murin est complexe, faisant intervenir des domaines intra et extracellulaires. Nous avons ensuite déterminé que la forme CD23b peut prendre en charge la transcytose de complexes IgE/allergène tandis que bDELTA5 peut transporter des IgE libres. Les études que nous avons menées en parallèle chez l'homme suggèrent que le CD23 y joue un rôle similaire bien que les modalités régulant l'endocytose et la transcytose soient différentes<br>The low affinity receptor for IgE (CD23) has been implicated in the transepithelial transport of IgE/allergen complexes. This transport is known to induce allergic reactions in sensitized animals. The purpose of my thesis was to characterize the precise role of CD23 in this process. We first establish that mouse intestinal epithelial cells express CD23b. A new CD23b derived splice form, bDELTAS, is also expressed upon sensitization. We found that that endocytosis regulation of CD23 is complex involving both extra and intracellular domains of the receptor. We then showed that classical CD23b transports IgE/allergen complexes while bDELTA5 mediates free IgE transcytosis. Studies in human revealed that CD23 plays a similar role in this species but that endocytosis and transcytosis regulation are here very different
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Gibb, David. "ADAM10 is a critical regulator of B cell development, antibody production, and myeloid-derived suppressor cell expansion: Effects of B cell-specific ADAM10 deletion and overexpression in vivo." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/2269.
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Proteolytic processing of transmembrane receptors and ligands can have dramatic effects on cell signaling and subsequent cellular responses. Previous studies demonstrated that a disintegrin and metalloproteinase 10 (ADAM10) may cleave numerous B cell-expressed receptors, including the low affinity IgE receptor (CD23). However, lethality of ADAM10-deficient embryos has limited examination of these cleavage events in lymphocytes. To investigate their role in B cell development and function, we generated B cell-specific ADAM10 knockout mice. Intriguingly, deletion prevented development of the entire marginal zone B cell (MZB) lineage. Further analysis revealed that ADAM10 is required for S2 cleavage of the Notch2 receptor and initiation of Notch2 signaling, which is required for MZB development. Additionally, cleavage of CD23 was dramatically impaired in ADAM10-deficient B cells. This finding and results of ex vivo cleavage assays demonstrated that ADAM10 is the principal in vivo sheddase of CD23. Previous studies have demonstrated that Notch signaling and CD23 cleavage regulate antibody production. Accordingly, deletion of ADAM10 profoundly inhibited germinal center formation, and T-dependent and T-independent antibody responses to immunization, implicating ADAM10 as a novel regulator of adaptive immunity. Additionally, to determine the role of ADAM10 activity in hematopoiesis, we generated transgenic mice (A10Tg) that overexpress the protease on lymphoid and myeloid progenitors. Surprisingly, this markedly suppressed B2 cell development and promoted dramatic expansion of myeloid-derived suppressor cells (MDSCs) via a cell intrinsic mechanism. A10Tg MDSCs inhibited T cell proliferation and adoptive immunotherapy of B16 melanoma, resulting in exacerbated metastatic progression that was prevented by MDSC depletion. Thus, A10Tg mice represent a novel model for the examination of MDSC development and MDSC-mediated immune suppression in a tumor-free environment. Finally, hematopoietic stem cell cultures revealed that ADAM10 overexpression directs myeloid development by dysregulating Notch signaling via uncoupling the highly regulated proteolysis of Notch receptors. Collectively, these findings demonstrate that ADAM10 is a critical regulator of Notch signaling, B cell development, and MDSC expansion. Moreover, they have important implications for the treatment of numerous CD23 and Notch mediated pathologies, ranging from allergy to cancer.
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Rambert, Jérôme. "Recherche de nouvelles approches thérapeutiques anti-inflammatoires : inhibition des fonctions macrophagiques." Bordeaux 2, 2003. http://www.theses.fr/2003BOR21084.
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Les macrpophages occupent un rôle central dans les réactions inflammatooires. Une des voies d'activation des macophages passe par la glycoprotéine de surface CD23. Le CD23 est un récepteur exprimé par de nombreuses cellules hématopoietiques et épithéliales. Quand il interagit avec ses ligands, le CD23 active les macrophages pour produire divers médiateurs dont les cytokines pro-inflammatoires et le monoxyde d'azote. Une augmentation du niveau de CD23 a été rapportée dans de nombreuses maladies inflammatoires dont la polyarthrite rhumatoide. Dans ce travail, nous avons tenté de bloquer la réaction inflammatoire, soit en bloquant spécifiquement le CD23 par des peptides, soit en cherchant des molécules à pouvoir anti-inflammatoire où la quercétine s'est avérée être un excellent candidat. Les perspectives thérapeutiques ouvertes par ce travail sont très encourageantes et nous incitent à développer ces nouvelles approches thérapeutiques pour soigner les pathologies inflammatoires chroniques<br>The macrophages have a central place in the inflammatory reactions. One of the macrophage activation way is the CD23 glycoprotein. CD23 is a receptor expressed by variety of hematopoietic cells and tissular epithelial cells. Interaction of CD23 with his ligands activates macrophages to produce various pro-inflammatory cytokines and nitric oxide. Increased levels of CD23 have been reported in various inflammatory diseases including rheumatoid arthritis. In this work, we have tried to block the inflammatory reaction by peptides which bind CD23 and block the activation of this receptor, or with new anti-inflammatory drugs where quercetin is a potential applicant for anti-inflammatory therapy. The therapeutic perspectives open by this work are exciting and incite us to develop this new therapeutic approaches to treat the chronic inflammatory diseases
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PAUL-EUGENE, DUGAS NATHALIE. "Regulation de l'expression et de la fonction du recepteur de basse affinite pour les ige (fcer2b/cd23) des monocytes/macrophages humains." Paris 7, 1993. http://www.theses.fr/1993PA077193.
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Le recepteur de basse affinite pour les ige (cd23) est une molecule qui sous sa forme membranaire ou sous sa forme soluble est impliquee dans un grand nombre de pathologies parmi lesquelles on trouve l'allergie et les maladies parasitaires. L'etude des mecanismes regulant son expression et sa fonction permet d'evaluer son role fonctionnel dans ces pathologies et de definir, eventuellement de nouvelles approches therapeutiques. L'expression du cd23 a la surface des monocytes est induite par l'il-4. Cette expression est augmentee en presence d'agents regulant le metabolisme des nucleotides cycliques tels que les agonistes #2-adrenergiques, et cette interaction fonctionnelle semble etre dependante du metabolisme de la l-arginine (no-synthase). De plus, la stimulation de ce recepteur par des complexes immuns a ige ou par des anticorps anti-cd23 induit la production de mediateurs inflammatoires. Ce processus est en partie associe au metabolisme de la l-arginine puisqu'inhibe en presence de lnmma. De meme, ce metabolisme de la l-arginine est implique dans la regulation de la production d'ige par les cellules mononucleees humaines normales. L'implication du metabolisme du monoxyde d'azote dans la regulation des fonctions dependantes des ige explique en partie du moins les interactions fonctionnelles entre l'il-4 et les agents regulant le metabolisme des nucleotides cycliques
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Bader, Talisa [Verfasser], Manuel [Akademischer Betreuer] Weber, and Falk [Gutachter] Wehrhan. "Anzahl der Tumor und Lymphknoten infiltrierenden NK- Zellen (CD56, CD57) und dendritischen Zellen (CD21, CD23) bei kleinen, primären oralen Plattenepithelkarzinomen (OSCC) und deren Assoziation mit unterschiedlichen klinischen Verläufen / Talisa Bader ; Gutachter: Falk Wehrhan ; Betreuer: Manuel Weber." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2021. http://d-nb.info/1241827265/34.
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DI, BERARDINO WILMA. "Implication du cytosquelette dans l'expression et le clivage du recepteur de basse affinite pour les ige (cd23) dans les lymphocytes b humains." Aix-Marseille 2, 1996. http://www.theses.fr/1996AIX22008.
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La molecule cd20, specifiquement exprimee a la surface des lymphocytes b, intervient dans l'activation et la differenciation de ces cellules. Nos resultats montrent que les anticorps monoclonaux diriges contre la proteine cd20 diminuent l'expression du recepteur de basse affinite pour les ige (cd23) induite par l'il-4 sur des lymphocytes b humains ainsi que son expression constitutive sur des cellules b transformees par le virus epstein-barr. Cet effet resulte d'une stimulation du clivage proteolytique extracellulaire de cd23. De plus, les anticorps diriges contre cd40, cmh classe i et ii produisent les memes effets. Nous avons montre qu'une correlation etroite existe entre le clivage de cd23 et l'agregation cellulaire mettant en jeu une molecule d'adhesion encore inconnue. A la fois le clivage et l'agregation font suite a l'activation des tyrosine kinases pp125#f#a#k et pp59#f#y#n et a la phosphorylation sur tyrosine des proteines associees a l'actine vinculine, paxilline et taline. Nos resultats mettent en evidence le role des proteines cd20, cd40, et les molecules du cmh dans le controle de l'equilibre entre les formes membranaire et soluble de cd23 dont les fonctions physiologiques sont differentes
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ROTIROTI, MARIA CATERINA. "Characterization of Chimeric Antigen Receptors (CARs) as a potential tool for the treatment of Acute Myeloid Leukemia (AML)." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2019. http://hdl.handle.net/10281/241327.
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La Leucemia Mieloide Acuta (LMA) è ancora associata ad alti tassi di ricaduta in seguito al trattamento con le terapie convenzionali che comprendono la chemioterapia e il trapianto di cellule staminali ematopoietiche. Da qui la necessità di identificare nuove strategie terapeutiche. L’immunoterapia con linfociti T ingegnerizzati per esprimere recettori chimerici artificiali (CARs) in grado di riconoscere specifici antigeni tumorali ha mostrato risultati sorprendenti in particolare nel contesto leucemie linfoblastiche di tipo B, suscitando un vivo interesse nell'estendere questo approccio anche ad altre neoplasie ematologiche come la LMA. Tra le molecole di superficie identificate nell’ambito della LMA, il CD33 e CD123 (la subunità α del recettore dell’IL-3), essendo ampiamente espressi sia dai blasti leucemici che dalle cellule staminali leucemiche, rappresentano dei buoni antigeni bersaglio per lo sviluppo di terapie CAR mediate.
Il mio progetto di dottorato è stato dunque incentrato sulla caratterizzazione di cellule killer indotte da citochine (CIK) geneticamente modificate attraverso il sistema non virale “Sleeping Beauty” per esprimere CARs specifici per il CD123 e il CD33 quale strategia terapeutica per il trattamento della LMA.
Nel contesto del targeting dell’antigene CD123, abbiamo focalizzato la nostra attenzione sull’ effetto di diverse variabili coinvolte nel CAR design, e note per la loro capacità di modulare i profili funzionali delle cellule CAR T, come l'affinità di legame e l'espressione del CAR in relazione alla densità dell'antigene bersaglio. Infatti, mentre l'effetto "off-target" associato al targeting del CD33 è per lo più limitato al compartimento ematologico, il targeting del CD123 richiede un livello più alto di cautela, a causa del potenziale riconoscimento da parte del CD123.CAR del tessuto endoteliale che esprime il CD123 a bassi livelli.
Utilizzando il nostro modello in vitro siamo stati in grado di definire sia soglie di antigene "litiche" che di "attivazione" dimostrando che, mentre l'attività citotossica precoce non è influenzata né dall'espressione del CAR né dalla modifica dell’affinità del CAR, l’ espressione del CAR rappresenta la variabile principale che incide sulle funzioni effettrici. Complessivamente, la completa caratterizzazione di tutte queste variabili ha fornito ulteriori informazioni per la corretta progettazione di un CAR anti-CD123 per il trattamento della LMA che possa garantire un corretto equilibrio tra i profili di efficacia e sicurezza.
In parallelo, una corretta valutazione preclinica di queste nuove terapie richiede anche un'accurata valutazione dell'efficacia, in particolare considerando le complessità intrinseca alla LMA, come l'eterogeneità e il microambiente mieloide immunosoppressivo. Pertanto, per quanto riguarda il targeting del CD33, abbiamo studiato i profili di efficacia di cellule CD33.CAR CIK da sole e in combinazione con agenti chemioterapici convenzionali, sfruttando un modello di xenotrapianto abbinato a chemioterapia per testare le cellule CD33.CAR-CIK nei confronti di cellule di LMA resistenti / residue alla chemioterapia. Abbiamo scoperto che le cellule CD33.CAR CIK da sole mostrano una potente attività anti-leucemica in vitro e in vivo, riducendo in modo significativo lo sviluppo della LMA quando somministrate come approccio di trattamento precoce, e ritardando la progressione di LMA in topi con una malattia conclamata. Inoltre, dati preliminari hanno mostrato che le cellule CD33.CAR-CIK sono ancora in grado di eliminare le cellule di LMA nei topi con recidive dopo la chemioterapia.<br>Acute Myeloid Leukemia (AML) is still associated with high relapse rates when treated with conventional chemotherapeutic and hematopoietic transplantation regimens. Thus, new treatment options are urgently needed. Immunotherapy adopting T cells engineered to express tumor-directed Chimeric Antigen Receptors (CARs) has shown striking results particularly in the context of B-cell malignancies, sparking a keen interest in extending this approach also to other hematological malignancies such as AML.
Among the surface molecules identified, the CD33 and CD123 (IL-3 receptor α subunit) molecules have emerged as the main validated targets in AML and, being broadly expressed on both AML blasts and leukemic stem cells (LSCs), represent suitable antigens to be targeted with CAR-T cells.
My PhD project has been focused on the characterization of non-viral Sleeping-Beauty engineered Cytokine-Induced Killer (CIK) cells with both anti-CD123 and CD33 CARs as a potential tool for the treatment of AML.
In the context of CD123 targeting, we focused our attention on dissecting the effect of several variables involved in the CAR design, known to modulate CAR T-cell functional profiles, such as CAR binding affinity and expression in relation to the target antigen density. Indeed, while the “on target-off tumor” effect associated to the CD33 targeting is mostly limited to the hematological compartment, the CD123 targeting demands a higher level of caution, due to the potential recognition of low CD123-positive endothelial tissue.
By using our model we were able to define both “lytic” and “activation” antigen thresholds showing that, while the early cytotoxic activity is not affected either by CAR expression or by CAR affinity tuning, the CAR expression represents the main variable impacting on later effector functions. Overall, the full dissection of all these variables offers additional knowledge for the proper design of a suitable anti-CD123 CAR for the treatment of AML which can grant a proper balance between efficacy and safety profiles.
In parallel, a proper preclinical assessment of novel therapies also demands for accurate efficacy evaluation, particularly considering the AML disease complexity, such as the heterogeneity and the immunosuppressive myeloid microenvironment. Thus, regarding the CD33 targeting, we investigated the efficacy profiles of CD33.CAR CIK cells alone and in combination with conventional chemotherapeutic agents by exploiting a xenograft chemotherapy model to examine the CD33.CAR-CIK cell contribute in the elimination of the chemotherapy resistant/residual AML cells. We found that CD33.CAR CIK cells alone exhibited a potent anti-leukemic activity in vitro and in vivo, significantly reducing AML development when administered as an early treatment approach and delaying the progression of established disease in mice. Moreover, preliminary data showed that CD33.CAR-CIK cells were still capable to target chemotherapy resistant/residual AML cells in mice experiencing disease recurrence after chemotherapy.