No description available.

No description available.

No description available.

No description available.

No description available.

The production of antibodies to a veterinary drug requires conjugation of the low molecular weight compound to a larger molecular weight substance (carrier protein) to enable immune recognition. The resulting immunogen is usually mixed with an adjuvant to create a prepnration thnt will elicit a strong and lasting inullune response to the complex when administered to the host animal. Fretmd's adjuvant has been previously used but can produce adverse side effects in the host. A replacement adjuvant for Fretllld's was sought: Montanide ISA 50V was found to be suitable for the production of antibodies to three veterinary clmg haptens without the adverse effects associated with Freund·s. Antibodies were required for the development of inummoassays for the coccidiostats diclazuril and . robenidine. TIle lack of a suitable fimctional group for coupling to the protein made immtlllogen preparation difficult for both these compounds. lvlimics possessing part of the relevant structures and more open to chemical manipulation were used as haptens to prepare inummogens and subsequently specific antibodies for diclazuril and robenidine were obtained. Polyclonal antisera to tlle antibiotic chloramphenicol were successfully raised in three different species. All of the antisera were affected to a similar e;-,.1ent when sanlple matrix was introduced to the assays. It was found that a heterologous format of both the ELISA and biosensor assay increased tlle crossreactivity of donkey and goat antibodies but not emile!. A camel antibody was separated into its three IgG subclasses. The conventional four chain molecule displayed affinity to chloramphenicol and the carrier protein \\rule the two heavy chain IgGs were only able to bind the carrier. TIle use of a suitable adjunult. antigen mimics and appropriate chemical manipulation techniques can enable the researcher to generate specific and sensitive antibodies to a range of small molecular weight compounds.

Activation of purified murine CD4⁺ T cells enhanced expression of the Notch target gene <i>hes1</i> and induced differential expression of Notch receptors and ligands compared to unstimulated cells. Surface staining for Notch 1 revealed that unstimulated cells expressed this receptor at the membrane. Activation of T cells induced capping of Notch1, which was found to co-localise with CD4. Capping of surface Notch and co-localisation with CD4 upon T cell activation was reminiscent of immunological synapse formation, suggesting that Notch may interact directly with T cell receptor signalling. Notch signalling was attenuated in CD4⁺T cells using a γ-secretase inhibitor, allowing assessment of Notch function in determining effector function. Notch signal inhibition in the context of anti-CD3-Ab stimulation, resulted in inhibition of TNFα, IFNγ, IL-4 and IL-10 secretion. Blockade of Notch signalling where cells were stimulated with anti-CD3/28-Ab did not down-regulate TNFα, IFNγ or IL-4 secretion, but IL-10 was inhibited. Notch signalling may thus be important as a co-stimulator when CD28 signalling is limiting. Naturally occurring CD25⁺ CD4⁺ T regulatory cells and <i>in vitro </i>generated TrI cells were examined for expression of Notch pathway components to ascertain if Notch may be involved in mediating regulatory function. Both populations were found to express higher levels of the Notch ligand Delta 1 than non-regulatory CD4⁺ T cells. Inhibition of Notch signalling did not affect the ability of CD25⁺ CD4⁺ T cells to regulate proliferation of CD4⁺ CD25⁺ T cells, but did reduce secretion of IL-10. These findings indicate that Notch receptors and ligands are expressed by CD4⁺ T cells. Notch may also be a component of the immune synapse and potentially modulates CD4⁺ T cell effector function through regulation of cytokine secretion, particularly IL-10.

No description available.

No description available.

Human CD55 is a 70kDa protein found on the surface of serum-exposed cells: It has long been recognised as a regulator of the complement system, protecting self-cells by accelerating the decay of the C3 and C5 convertases. Much more recently CD55 has also been identified as a co-stimulator of T cells. I have used a wide range of biophysical techniques to study CD55 and its interaction with two human ligands, Bb and CD97. The interaction of CD55 with Bb has been investigated in the hope of contributing to the understanding of how CD55 accelerates decay of the alternative pathway C3 convertase (C3bBb). Surface plasmon resonance experiments have confirmed that CD55 binds to the vWF-A domain of Bb in the presence of Mg2 + and the KD of this interaction has been measured as 2.2JlM. Electron paramagnetic resonance has been used to obtain experimentally-derived distance restraints in order to model the complex between CD55 and the Bb vWF-A domain. CD55 has previously been shown to co-regulate T cell activity via its interaction with CD97, the archetypal member of the EGF-TM7 family of proteins. Using X-ray crystallography, I have determined the structure of EMR2, a very close homologue of CD97. Nuclear magnetic resonance-based chemical shift mapping of the CD55EMR2 interaction has allowed me to generate a model for the CD55-CD97 complex. The structure of this complex reveals that the T cell and complement regulatory activities of CD55 occur on opposite faces of the molecule. This suggests that CD55 is capable of promoting an adaptive immune response while simultaneously protecting self-cells from the innate immune system.