Principal components analysis, using the reported properties of 250 brewing strains of Saccharomyces cerevisiae held at the National Collection of Yeast Cultures, differentiated five groups of strains, each group consisting of two sub-groups. An antiserum was prepared against one strain from each group: these were then used to carry out micro-immunoelectrophoresis of 43 strains, chosen to be representative of the groups. 19 antigens were recognised. Multi-variate analysis methods (principal components and cluster analysis) were then used to determine whether any associations could be observed between antigenic structure and the brewing properties of the strains. Associations were demonstrated between the antigens designated 6 and 7 and the brewing property of head formation, between antigens 4A, 5 and 10 and deposit formation, between antigen 8 and rate of attenuation and between antigens 5 and 7 and clarification with finings. The validity of these findings was confirmed by antigenic analysis of variant strains differing in brewing properties from the parent strain: in these variant strains a change in a particular brewing property was accompanied by corresponding changes in antigenic structure. It became apparent that variation and mutation were more prevalent in brewing yeast than had been realised; the incidence and effects of mutation to respiratory deficiency (RD) were examined as a particular case of this. In the production of brewers wort on laboratory, pilot and production scale formaldehyde was added to the mash. During subsequent fermentations it was shown that in the case of ale yeast A (AYA) the rate of production of RD mutants from the parent strain was dependent on the initial formaldehyde concentration; formaldehyde added at the beginning of the mashing programme caused the greatest degree of mutation. After mashing, levels of free formaldehyde were low (0.1-0.5 ppm), suggesting the formation of a mutagenic reaction product: this was apparently a heat stable dialysable nitrogen compound of less than 10,000 molecular weight. RD mutants were more sedimentary than the parent and some had faster fermentation times. A further ale yeast (AYB) and a lager yeast (LYA) were examined: LYA showed a high mutation rate even in the absence of formaldehyde.

olecular parameters controlling antigenicity* that is immunogenialty and antigenic specificity, are reviewed together with acme aspects of humoral immunity and tolerance, and characteristics and functions of cells involved in these immunological phenomena* Antigenicity of native monomer, heat-denatured monomer and heatpolymerised bovine serum albumin (BSA) have been compared. Both aonooeric antigens were similar in the three aspects of antigenicity examined, namely their humoral iraaunogenicity, tolerogenicity and serological specificity. The antigenicity of polymerised BSA differed from that of monomers in both its imaunogenlcity and serological behaviour. Polymerised BSA Induced stronger primary and secondary humoral responses of anti-native specificity than did the monomers in both mice and rabbits. The apparent immunogenic (priming) threshold dose of polymerised BSA was approximately ten fold lower than that of monomer in CM mice. Partial humoral tolerance (low-gone) was induced by all three BSA antigens in CBA mice. The threshold tolerogenic dose was essentially the same (lpg) for monomer and polymerized BSA. The hyporespemsiveness induced by polymerised BSA was both leas pronounced and more transient than that due to monomer. Serological activity of the BSA antigens was examined by a primary binding assay of Parr. Distinct differences between monomelic and polymerized BSA were observed with respect to the slope of their binding curves, association and dissociation rates of their binding to antibody, and mutual cross-reactivity in an inhibition assay. The results of the serological studies could be attributed to either a new specificity or, store comprehensively, to an enhanced capacity of polymerised BSA to establish avid bonds with antibody* The polymerised B3A was removed from circulation of CBA mice more rapidly than were the momwers* However, a large proportion of polymerised BSA was released in a little degraded form from peritoneal exudate cells cultured in vitro after the uptake* A possible relationship between the serological and immunogenic properties of the BSA antigens, as well as the significance of their handling by phagocytic cells, were discussed.

The aim of the work carried out in this thesis was to investigate the effects of GM-CSF and IL-3 after gene-gun vaccination. Initial studies focused on the histological effects of GM-CSF and IL-3 in ovine skin. GM-CSF induced a linear increase in IL-1β transcripts from 1-24 hours concomitant with pronounced neutrophilic infiltration and micro-abscess formation by 24 hours. Maximal GM-CSF and IL-3 mRNA expression was evident at approx. 4 hours, whereas IL-1β and TNF-α transcripts remained highly elevated up to 96 hours. Infiltration of DC, co-ordinately with a rise in MHC class II DRα expression in perivascular regions, was observed 72-96 hours after administration of IL-3, whereas infiltates of B cells and DC were observed at 24 and 72 hours, respectively. Subsequent work characterised DC draining skin before and after GM-CSF administration by means of cannulation of the pseudoafferent lymphatics. Two sub-populations of afferent lymph DC (ALDC) were isolated based on differential expression of SIRPα as described in both cattle and rats, Ovine SIRPα⁺ ALDC constitutively express high levels of IL-1β, IL-18 and IL-10 mRNA. Conversely, SIRPα^- ALDC does not express IL-10 but express high levels of IL-12p40. SIRPα⁺ ALDC may contain Langerhans’ cells since both ATPase and langerin were detected. Both subpopulations express Toll-like receptor 3 (TLR3) and TLR9, whereas SIRPα⁺ ALDC uniquely express TLR4 (+/- CD14). Vaccination with pGM-CSF resulted in increased cytokine production by ALDC subpopulations and upregulation of MHC class IIα, CD1b, CD40, CD86 and CD11c. These studies have extended the knowledge of GM-CSF and IL-3 as adjuvants and highlight the use of the cannulation model to further decipher the immune mechanisms of adjuvants. This work has demonstrated that GM-CSF not only recruits DC into skin but also activates DC, possibly as a result of increased expression of “danger signals” including IL-1β and TNF-α.

In this study the diversity of mast cell proteases and some of the factors regulating mast cell growth and protease expression were examined in rodents. Five proteases were isolated from mouse small intestinal mucosa and their substrate specificities defined. The isolated proteases were all of mast cell origin and were chymotrypsin-like in their substrate specificities. The proteases were all identified as variants of mouse mast cell protease-1 which differed only in their carbohydrate moieties. Despite the fact that these enzymes shared a common core polypeptide they all differed significantly in the rate at which they hydrolysed synthetic substrates and in the rates at which they were inhibited by α<SUB>1</SUB>-proteinase inhibitor. A related, but distinct protease was isolated from peritoneal cavity mast cells of mice. This enzyme, also a chymase, had N-terminal sequence identity with mouse mast cell protease-4. This enzyme was not inhibited by α<SUB>1</SUB>-proteinase inhibitor. Factors which regulate mast cell survival, growth, proliferation and protease expression were examined in the rat. Stem cell factor (SCF) or cytokine-rich lymph node-conditioned medium (LNCM) was administered to normal rats by intraperitoneal injection and the effects on the connective tissue mast cells (CTMC) of the peritoneal cavity were monitored. LNCM did not cause in increase in CTMC numbers but it did stimulate a switch in protease expression from rat mast cell protease 1 (RMCP I) alone to dual expression of both RMCP I and II. SCF alone caused a significant increase in CTMC numbers coupled with a decrease in RMCP I content, and an increase in RMCP II content/cell. Treatment with both LNCM and SCF together caused an even greater increase in both CTMC numbers and RMCP II expression than with SCF alone. Daily intravenous injection for SCF for 14 days into both normal and <I>Nippostrongylus brasiliensis-</I>infected rats resulted in a five-fold increase in CTMC numbers with a concomitant decrease in RMCP I content. There was no significant expression of RMCP II in the CTMC of these animals. These results show that SCF and the cytokines in LNCM play an important role in the regulation of mast cell populations and their expression of proteases.

The ileal Peyer's patch (IPP) of lambs is a region of intense lymphopoiesis and B cell development. Monoclonal antibodies against ovine lymphoctye antigens have been used to characterise the IPP lymphocyte. Three murine monoclonal antibodies against ovine IgM, IgG1 and Ig light chain were produced and are described fully. IgM and MHC class II antigens are expressed on the vast majority of IPP cells whilst cells bearing other serum Ig isotypes and T cell antigens are rare. A novel Ig molecule appears to be coexpressed with IgM, it is proposed that this is the ovine equivalent of IgD. IPP cells can be induced to proliferate and differentiate when cultured with lipo-polysaccharide (LPS) and interleukin 2 (IL2). Proliferation is inhibited by rabbit anti-sheep Ig antibodies. Using an ELISA for Ig, it has been possible to quantitate Ig synthesis and secretion. Mean cellular Ig increases greater than 25-fold during differentiation. High-rate secretion begins 4 days after initiation of culture and is virtually complete by day 7. As IPP B cells differentiate to IgM secretion, membrane Ig is rapidly lost so that by day 6, only 15% of cells express Ig on their surfaces. Changes in MHC class II antigens were also studied. Surface expression of MHC class II molecules doubled by 24 hours and slowly declined to resting levels as differentiation proceeded. A large increase in cytoplasmic MHC class II content was noted on day 3. The reasons for this increase are discussed. Kinetic studies suggest that IL2 responsiveness is acquired approximately 20 hours after activation by LPS. The concentration required to give half maximal Ig secretion is 125 pM indicating that the interaction between IL2 and its receptor is one of high affinity. During differentiation, the cells enlarge and show an increase in the cytoplasmic:nuclear ratio. The formation of extensive rough endoplasmic reticulum and additional mitochondria is indicative of the functional changes occurring. This is the first description of a sheep B cell differentiation assay. It is proposed that this system is a suitable model on which to base further studies into the molecular biology of sheep Ig genes, Ig isotype switching and lymphokines and their receptors.

The aim of the present study was to develop enzyme linked immunosorbent assays (ELISAs) for detection of specific antibodies against <i>Babesia bovis</i> and <i>B.bigemina</i> for use in epidemiological studies of bovine babesiosis in Brazil. These tests were developed using purified proteins of each parasite, which were identified as species-specific in a comprehensive immunochemical characterisation of diffrent stocks of <i>Babesia</i> parasites. The review of the literature on bovine babesiosis covers the geographical distribution, transmission and life cycle, and <i>in vitro</i> cultivation of both species, as well as immunology, diagnosis, epidemiology and control of the disease and its current situation in Brazil. The thesis first describes a series of studies on the <i>in vitro</i> culture of <i>B.bovis</i>, which include the evaluation of the effect of different sera on <i>in vitro</i> growth, the incorporation of feeder cells (bovine aortic endothelial and mouse peritoneal cells) into cultures, cloning of one isolate by <i>in vitro</i> limiting dilutions, initiation of cultures from low parasitaemia blood, and several attempts to establish African isolates of <i>B.bigemina in vitro</i>. Methods for concentration of <i>Babesia</i> infected red blood cells and free parasites were then examined. These included the use of differential hypotonic lysis, density gradient centrifugation, induction of free merozoites into culture supernatant and techniques for fractionation of exoantigens present in culture supernatant, namely high performance liquid chromatography. Somatic components and exoantigens of different stocks of parasites were characterised immunochemically by Western immuno-blotting, and immunoprecipitation of <SUP>35</SUP>S-methionine labelled proteins by acrylamide gel electrophoresis, using a wide variety of sera which included calf sera experimentally raised against different stocks of each parasite, sera collected in the field in Brazil and a panel of monoclonal antibodies previously produced against <i>B.bovis</i> and characterised by IFAT, Western immuno-blotting and ELISA in the present study.

In this study the expression of MHC class II molecules on T cells from various immunological compartments of the sheep was examined. Monoclonal antibodies (mAbs) were characterized which react specifically with the homologues of human DRα and DQα molecules. The expression of these antigens was established on B cells and T cell subsets derived from peripheral blood, efferent lymph and afferent lymph. B cells from each of the lymphocyte sources expressed both DRα and DQα antigens. Variable expression on T cells was found. Each of the T cell subsets expressed both DR and DQ molecules at much higher levels in afferent lymph. DR expression on peripheral blood lymphocytes and efferent lymphocytes was higher than DQ on each of the T cell subsets. Afferent lymphocytes represent memory type cells and class II DR expression on these T cells may mark activation status. Expression of the class II subtypes was also examined on <I>in vivo</I> activated T cells and correlated with expression of activation markers. Increased expression of class II molecules was coincident with the increased expression of activation markers in each of the T cell subsets with the exception of γδ cells. The lack of correlation on γδ T cells may be a reflection on the type of antigen used. Levels of DQ expression on CD4 positive cells after activation showed a more dramatic increase than levels of DR expression. Cytokine profiles of concanavalin A (Con A) activated T cells (DR positive) were examined and compared with that of DR negative T cells. The data reveal that IL-6 mRNA production correlated with DR expression. IL-6 was induced after activation of the total T cell population and was not induced in the DR negative T cells. No detectable differences in IL-4, Il-10 and γIFN mRNA profiles were observed between the total T cell population and the DR negative T cells.

The aim of this study was to quantify immune cell populations within the cow corpus luteum (CL) throughout the oestrous cycle in order to investigate whether these cells could be involved in controlling luteal function, particularly around the time of luteolysis. Six CL were collected from each of four stages of the oestrous cycle and were identified on the basis of their gross appearance, for preliminary immunohistochemical studies. Immune cell populations and MHCII expression varied throughout the oestrous cycle. In particular, the number of macrophages, T-lymphocytes (CD5+, CD4+) and MHCII expression was significantly higher in late stage CL (after luteolysis) compared to all other stages. To study in more detail the cellular events associated with luteolysis, the oestrous cycles of 19 cows were synchronised. CL were collected between days 16 and 20 of the following oestrous cycle. A significant increase in the number of T-lymphocytes (CD5+, CD8+) was detected in CL collected from day 16 onwards, compared to days 13-14. This increase occurred prior to functional luteolysis. Artificially-induced luteolysis was then assessed as a potential model for further studies around luteolysis. CL collected 6, 12, and 24 hours after luteolysis induced using a single injection of 25mg PGF<SUB>2α</SUB> had undergone dramatic structural regression which bore little resemblance to events during normal luteolysis and so this model was rejected. The role of endogeoous PGF<SUB>2α</SUB> in inducing the influx of T-lymphocytes was then investigated. Production of PGF<SUB>2α</SUB> was inhibited in 12 cows between days 15 and 18 of the oestrous cycle and artificially replaced in six of the cows for 24 hours before collection of the CL on day 18. The number of macrophages was significantly lower in all animals in which PGF<SUB>2α</SUB> was inhibited compared to control animals but T-lymphocyte numbers were not significantly altered.

Several biochemical characteristics of elafin, such as low molecular weight, cationicity, heavy disulphide bonding and tissue distribution of mucosal sites, suggested that it may possess such-endotoxin properties similar to the defensins and other cationic antimicrobial peptides. Here we have demonstrated that elafin binds directly to LPS of both smooth-form and rough-form type. This bindings was shown to occur within the conserved lipid. A portion of the LPS molecule, and binding of elafin to LPS inhibited subsequent interaction of LPS with LPS-binding protein (LBP). Moreover, both terminal domains of elafin were shown to bind LPS and preclude this interaction. Furthermore, elafin inhibited TNF-α secretion in serum-free milieu. These findings suggest that extracellular elafin may play divergent roles in the innate immune response to LPS depending on the site of infection, for example serum-rich bloodstream or serum-deficient mucosal sites. A replication-deficient adenovirus vector encoding human elafin cDNA (Ad-elafin) was used to extend these studies to demonstrate that elafin may also act intracellularly to dampen macrophage responses to LPS. This LPS-modulatory activity prompted us to study the antimicrobial properties of elafin. Ad-elafin infection of primary murine tracheal epithelial cells <i>ex vivo </i>conferred antimicrobial activity against <i>staphylococcus aureus, </i>but compromised the inherent ability of cells to kill <i>Pseudomonas aeruginosa. </i> In summary, elafin may play a role in innate immunity by modulating host responses to Gram-negative bacterial LPS. Elafin could function to enhance immune responses in sites of local inflammation, such as the airways, but to down-regulate potentially deleterious responses to LPS in the circulation.

No description available.