Frankfurt (Main), Univ., Diss., 2001.

Universiẗat, Diss., 1996--Konstanz.

Thesis (Ph. D.)--University of Iowa, 2007. / Supervisor: Elizabeth A. Chrischilles. Includes bibliographical references (leaves 272-282).

Universiẗat, Diss., 2004--Düsseldorf.

Guillain Barré Syndrome in humans is characterised by ascending paralysis. It is often associated with preceding infections two to four weeks prior to nadir and is fatal in five percent of cases. Antibodies specific to several nerve components are frequently associated with clinical symptoms in GBS. These antibodies were found to be specific to various gangliosides and ganglioside complexes. It was also found that antibody reactivity to gangliosides is affected by membrane components. The most prevalent (20-30%) immunoglobulin in GBS is anti-GM1 (20-30%), which also binds to the LPS of the PEN O:19 Campylobacter jejuni serotype. This is the most common infectious agent associated with GBS and emphasizes the importance of infection and anti-ganglioside antibodies in disease development. Intravenous infusion of pooled immunoglobulin from healthy donors, also called intravenous immunoglobulin (IVIg), halves the severity of disease manifestation. The action mechanism of IVIg in curing GBS is not clear, but intravenous immunoglobulin was shown to neutralize anti-ganglioside binding activity and its pathogenic effects. It was further found that anti-idiotypic antibodies in IVIg inhibit anti-ganglioside antibody activity. Treatment with IVIg is not equally effective in all GBS cases, which might be due to the inability of IVIg to neutralize anti-ganglioside antibodies in all patients adequately. Therefore, the treatment of GBS with IVIg needs to be better understood in order to improve its use as a cure for GBS. This study confirmed previous findings that the interaction of patient serum anti-GM1 antibodies and ganglioside auto-antigens is greatly impaired by components in healthy serum. Bound anti-GM1 antibodies could be displaced by (presumably) anti-idiotypic antibodies from healthy donor serum. This study found that the displacement potential between donor sera differs. Anti-GM1 antibody displacement was found to be dependent on the character of both anti-GM1 and anti-idiotypic antibody. This demonstrated the feasibility of improving the efficiency of treatment by IVIg by sourcing it from only those sera that test best for displacing auto-antibodies from their ganglioside antigens in ELISA. IVIg selection may therefore greatly benefit from the use of recombinant phage display antibodies to distinguish between the various types of GBS for treatment. To develop a method to characterize anti-ganglioside antibodies sensitively, an evanescent field biosensor was employed in which gangliosides were presented in a liposome environment. This provided a more physiological way of antibody antigen recognition. The optimized method determined the ganglioside binding specificity of purified IgG from a GBS patient, and mouse monoclonal anti-GM1 and anti-GD1a antibodies accurately. The results compared well with those from ELISA. The results obtained with purified IgG were far better than that obtained with whole serum analysis. This could be due to non-specific binding or the presence of inhibiting anti-idiotypic antibodies in patient sera. The biosensor method for antibody detection in GBS may allow the detection of anti-idiotypic antibodies in patients in future, because it requires no prior labelling of antibodies. Anti-idiotypic interaction may be detected by displacement of Ab1 from antigen, or by capturing Ab2 on Ab1 immobilized on the biosensor surface. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2008. / Biochemistry / MSc / unrestricted

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.

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.

archives@tulane.edu / Effective adaptive immune responses depend on the presentation to CD4+ T cells antigen peptides bound to major histocompatibility complex class II proteins. The structure of an antigen strongly influences its processing within the endolysosome and potentially controls the identity and abundance of peptides that are presented to T cells. The dissertation presented here sought to expand our understanding of how antigen structure and stability influence adaptive immune responses for two model antigens. Pseudomonas exotoxin A domain III (PE-III) functions as an ADP-ribosyltransferase with significant cellular toxicity and has been incorporated into a recombinant immunotoxin for the treatment of cancer. The bacterial component of the PE-III immunotoxin is highly immunogenic and generates neutralizing antibodies that render subsequent treatments ineffective. A group of six single-amino-acid substitutions in PE-III that were predicted to disrupt CD4+ T-cell epitopes have been shown to reduce antibody responses in mice. Here we demonstrate that only one of the substitutions, R494A, exhibits reduced folding stability and proteolytic resistance through the removal of a hydrogen bond. This destabilization significantly reduces its antibody immunogenicity while generating CD4+ T-cell epitopes that are indistinguishable from those of wildtype PE-III. PE-III specific B cells isolated from R494A-immunized animals contained fewer somatic mutations, which are associated with affinity maturation, and exhibited a weaker germinal-center gene signature, compared to B cells from wildtype-immunized animals. Chicken ovalbumin (cOVA) has been studied for decades primarily due to the robust genetic and molecular resources that are available for experimental investigations. cOVA is a member of the serpin superfamily of proteins that function as protease inhibitors, although cOVA does not exhibit this activity. As a serpin, cOVA possess a protease-sensitive reactive center loop that lies adjacent to the OT-II epitope. We took advantage of the previously described single-substitution-variant, OVA R339T, which can undergo the dramatic structural transition observed in serpins to study how changes in loop size and protein stability influences CD4+ T-cell priming in vivo. We observed that OVA R339T loop-insertion increases overall stability and protease resistance and significantly shortens the reactive center loop. This results in reduced CD4+ T-cell priming of the OT-II epitope in SJL mice. These findings have implications for the design of more effective vaccines for the treatment of infectious diseases and cancer as well as the development of more robust CD4+ T-cell epitope prediction tools. / 1 / Daniel Moss

No description available.

Bacillus anthracis is a facultative intracellular bacterial pathogen that can cause cutaneous, gastrointestinal or respiratory disease in many vertebrates, including humans. Commercially available anthrax vaccines for immunization of humans are of limited duration and do not protect against the respiratory form of the disease. Brucella abortus is a facultative intracellular bacterium that causes chronic infection in animals and humans. As with other intracellular pathogens, cell mediated immune responses (CMI) are crucial in affording protection against brucellosis. B. abortus strain RB51 has been shown to be useful in eliciting protective cell mediated immunity and humoral responses against Brucella in cattle and other animal species. Since the protective antigen (PA) of B. anthracis is known to induce protective antibodies, it was decided that the objective of this research was to test whether the gene encoding PA could be expressed in Brucella producing a bivalent vaccine to protect against both brucellosis and anthrax. The pag gene was transcriptionally fused to promoters of genes encoding superoxide dismutase or heat shock protein groE, subcloned into a broad host range plasmid (pBBR1MCS) and shown to express in E. coli by immunoblotting using antiserum specific for PA. The immunoblot results revealed that E. coli produced a PA protein of the expected size. In addition, the culture medium was shown to contain the same PA protein using immunoblotting. These results show that E. coli is capable of expressing B. anthracis PA in both the cellular and extracellular forms. The pBB/PA plasmid was used to transform B. abortus RB51 and CmR clones screened for the expression of PA by immunoblotting. Twenty clones of strain RB51/pBBSOD were show to express a 30kDa PA protein. Three clones of strain RB51/pBBGroE-PA were shown to express a 63-83kDa protein as detected by antiserum specific for PA. Using the A/J mouse, an immunocompromised vertebrate model, immunization and challenge studies were performed. Preliminary results demonstrate that the bivalent vaccine is capable of producing protection against a live challenge with B. abortus and some protection against live non-disease producing spores of B. anthracis. / Master of Science