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Item A structural study of human complement subcomponent C1s : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University(Massey University, 1981) Morris, Peter JohnThe aim of this project was to determine the carboxyl terminal amino acid sequence of the heavy chain of human complement subcomponent c1s. The proteolytic cleavage of a peptide bond(s), probably at an Arg-Ile bond, of the single chain C1s yields the active serine protease C1s composed of a heavy and light chain. A knowledge of the amino acid sequence preceding the scissile Arg-Ile bond would allow the chemical synthesis of a model peptide substrate for the C1s-activating enzyme C1r. Human C1s was purified to homogeneity by euglobulin precipitation and repeated ion exchange chromatography. Unactivated C1s, which could be activated by incubation with partially purified C1r, was isolated by performing all purification steps in the presence of the serine protease inhibitor phenylmethane sulphonylfluoride and at low temperature. The heavy and light chains of activated C1s were separated by ion exchange chromatography in the presence of denaturant following thorough disulphide bond reduction. Isolation of the carboxyl terminal-derived peptide of the C1s heavy chain by peptide mapping as well as by chemical modification of protein carboxyl groups was unsuccessful largely due to the high molecular weight of the protein substrate. Digestion of c1s by carboxypeptidase B resulted in the very rapid release of arginine in a quantitative yield presumably from the carboxyl terminus of the C1s heavy chain. Affinity chromatography using immobilized anhydrotrypsin was successful in isolating the carboxyl terminal chymotryptic peptide of the C1s heavy chain. Anhydrotrypsin displays a remarkably specific affinity for trypsin product-like peptides possessing a carboxylterminal arginine residue. Attempts to determine the entire amino acid sequence of the isolated peptide were prevented by the difficulty in obtaining sufficient material. However, by determining the N-terminal amino acid sequence and amino acid composition of this peptide as well as by performing further peptide fragmentation by trypsin the following partial primary structure is proposed: Gln-Gln-Lys-Glx-Val-Pro-Glx-Gly- [Thr, Ser, (Leu), Ala] - Lys-Glx-Glx-Asx-Arg.Item A study of circulating neutrophils and exosomes associated with innate immune function in the periparturient grazing dairy cow : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Manawatū, New Zealand(Massey University, 2017) Crookenden, Mallory AnnDairy cows are at greatest risk of infectious and metabolic disease during the periparturient period. This period of three weeks either side of calving is also known as the transition period due to the transition into lactation. This thesis had several aims; one was to characterise innate immune function during the transition period in grazing dairy cows by investigating molecular changes in circulating neutrophils and to assess if common on-farm management strategies (pre-calving feeding level and body condition at calving) were able to influence these molecular changes. Next, metabolic stress on neutrophil function was assessed by establishing a model of cows divergent in metabolic health status. This model was further utilised with the aim to investigate nanoparticles (exosomes), which are regulators of innate immune function and indicators of disease state. To address these aims blood was collected from pasture-fed transition dairy cows. Cellular and molecular methods used included cell and exosome isolation, reverse transcriptase (RT)-quantitative PCR, RNA sequencing, cell culture, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that grazing dairy cows experience a change in innate immune function during the transition period, reflective of reduced functional capacity of the immune system to overcome infectious agents. This altered function was similar to that experienced by housed cows fed a total mixed ration, which adds evidence to support that the dysfunction is a natural part of the transition into lactation at calving. These results also indicated that the functional changes could be influenced by nutrition status, feeding level, and metabolic stress. Analysis of exosomes isolated from the blood of transition cows indicated that these particles carried cargo indicative of metabolic state during the transition period and that they had the ability to alter target cell processes (gene expression, protein expression, and cell proliferation). The conclusions from this thesis increase our understanding of transition cow immune function and how it is influenced by nutrition and cow metabolism. These data are particularly relevant for grazing dairy cows and the findings will contribute to on-farm recommendations and the improvement in animal health and well-being. iiiItem The effect of probiotics on host mucosal immune responses : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Nutritional Science at Massey University, Palmerston North, New Zealand(Massey University, 1998) Wong, Mei ChingLactic acid bacteria (LAB) are a group of Gram-positive anaerobic bacteria that convert carbohydrates and complex sugars into lactic acid as the end product through fermentation. Several species of LAB have been used as probiotics. Probiotics are mono- or mixed cultures of live microorganisms which, when orally administered to animals or man, benefit them by improving the balance of the indigenous microflora. Lactic acid bacteria are claimed to have several beneficial effects; one of them being stimulation of the immune system. Many studies have demonstrated the immunostimulatory effects of LAB and various mechanisms have been suggested as to how LAB stimulate the immune system. These include the ability of LAB to translocate to Peyer's Patches (PP) and other gut-associated lymphoid tissues (GALT) for immunological processing by immunocompetent cells and production of cytokines. There were three aims in our present studies. The first was to determine the effect of dose of an immunoenhancing probiotic strain L. rhamnosus on the mucosal and serum immune responses of mice to oral antigens cholera toxin (CT) and ovalbumin (OV). The second aim was to examine the effect of viability of L. rhamnosus on these responses. Various mucosal immune parameters were measured in these studies. Results indicate that the immunostimulatory effects of L. rhamnosus were dose-dependent and that the 1 x 109 cfu dose was the most appropriate dose for L. rhamnosus for its immunostimulatory effects. Viability also affects the immunostimulatory effects of L. rhamnosus as shown by the higher efficacy of viable L rhamnosus than non-viable L. rhamnosus in stimulation of several aspects of the mucosal immune system. In some other immune parameters, non-viable L. rhamnosus was found to be the same as, or more effective than the viable bacteria. These findings were significant in that they provide additional evidence of the dose- and viability-dependency of different LAB. This information will help those involved in the development of probiotic products to consider these factors when formulating their products so that the concentration of live LAB can be adjusted to ensure the product can convey the maximum beneficial effect to the consumer. The third aim of our studies was to examine the role played by the immune system in protecting against enteric infection. It was found that L rhamnosus increased the resistance of mice to S. typhimurium infection as demonstrated by the lower numbers of bacteria found in the liver and spleen, and a maintenance of liveweight. This was also accompanied by increased mucosal and systemic immune responses to S. typhimurium. This result suggests that the immune system may play an important role in mediating the protection against enteric infection. Various other mechanisms have also been postulated by which LAB protect against enteric infection, for example, production of antibacterial substances, competition for adherence to the gut wall and for nutrients. However, the precise role and relative importance of these mechanisms in mediating protection against enteric infection is unknown.Item Study of an exported protein of Mycobacterium avium subspecies paratuberculosis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand(Massey University, 2004) Copland, Susan MareeJohne's disease is a chronic, progressive enteric disease of ruminants caused by infection with Mycobacterium avium subspecies paratuberculosis (M. ptb) from the MAIS complex (M. avium, M. ptb, M. intracellulare and M. scrofulaceum). The lack of specific and sensitive diagnostic tests often leads to M. ptb infected animals being diagnosed with bovine tuberculosis, a member of the MTB complex (M. tb, M. bovis, M. bovis BCG, M africanum, M. microti and M. canetti). Secreted proteins from pathogenic mycobacteria have been found to be important for the development of protective immunity, namely a cell mediated immune response (CMI). The development of reliable differential diagnostic tests will require the use of species-specific secreted protein antigens and the CMI response. Due to the taxonomic distance between the MAIS and MTB complexes our hypothesis was that the M. ptb genome may encode for secreted proteins that are absent from members of the MTB complex. If such proteins can stimulate an immune response they may be suitable for use as antigens in a differential diagnostic test for Johne's disease. To this end, the secreted protein library clone pJEMIl-M ptb281 was examined and its insert found to contain the 5' region of the hypothetical M.ptb281 ORF fused in frame with phoA. The entire ORF was determined using M. avium and M. ptb database sequences then cloned into E. coli and mycobacterial expression systems. These systems incorporate 6x histidine (His6) affinity tags into recombinant proteins allowing them to be semi-purified by Ni-NTA affinity chromatography. Semi-purified recombinant proteins tested positive by western blot analysis to highly specific anti-His6-tag antibodies. Amino acid sequencing to confirm the identity of these recombinant proteins and screening for their ability to stimulate an immune response were prevented by time constraints. Homologs to M. ptb281 were absent from M. tb, M. bovis and M. bovis BCG but present in the MAIS complex, making this protein unsuitable for use as an antigen to differentiate between MAIS complex species in a diagnostic test. M. ptb281 homologs found in the genomes of two members of the Acetomycetes order corresponded to hypothetical proteins predicted by computer software programs trained to identify genes, which may indicate that the hypothetical M. ptb281 ORF may encode a functional protein.Item A genetic approach to identify Mycobacterium bovis exported protein antigens : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Biology, Massey University(Massey University, 1997) Borich, Suzanne MarieA novel approach, combining phoA-fusion technology with T cell screening of a recombinant cosmid library, was used to detect Mycobacterium bovis exported T cell antigens. An M. bovis BCG library of phoA-fusions was constructed in Escherichia coli and Mycobacterium smegmatis using the plasmid vector pJEM11. The M. bovis BCG DNA inserts from ten PhoA+ clones were partially sequenced and used to search databases for similarities to known genes. These revealed similarities to a family of genes coding for high temperature-requirement serine proteases and a Mycobacterium leprae putative exported lipoprotein gene (pel). The DNA inserts from PhoA+ clones were used to probe an M. bovis cosmid library expressed in M. smegmatis 10 identify cosmids containing the full-length genes coding for these exported proteins. Culture filtrates (CFs) prepared from selected M. smegmatis recombinants (cosmids) were assayed for their ability to induce proliferation and IFN-γ-production from peripheral blood mononuclear cells (PBMCs) taken from M. bovis BCG-immunised and non-immunised control cattle. Culture filtrates from two recombinant M. smegmatis (cosmids 44 and 56) induced significant IFN-γ-production and proliferation by PBMCs from immunised animals. An exported protein gene, identified using the phoA-fusion technology, was subcloned from cosmid 56 and its sequence determined and analysed. Database searches using the deduced amino acid sequence of this gene revealed similarities to an M. leprae putative exported lipoprotein (Pel) and a family of MalE maltose-binding proteins. The M. bovis pel gene was shown to be expressed by recombinant M. smegmatis. Preliminary evidence from this study indicates that the M. bovis Pel protein is recognised by antigen-specific lymphocytes from M. bovis BCG-immunised animals. The PBMCs taken from M. bovis challenged and M. bovis BCG vaccinated / challenged cattle also recognised CF from recombinant M. smegmatis expressing the pel gene in in vitro immunoassays. The combined strategy of using phoA-gene fusions and T cell screening of CFs from a recombinant M. bovis cosmid library proved a sensitive and rapid method for the detection of potential M. bovis T cell antigens.Item Identification and characterisation of an exported immunogenic protein of Mycobacterium avium subspecies paratuberculosis : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand(Massey University, 2002) Dupont, ChristineExported proteins of mycobacteria are available to interact with the immune system at an early stage of infection and are potent inducers of immune responses. Potentially exported proteins of Mycobacterium avium subspecies paratuberculosis were identified using alkaline phosphatase gene fusion technology. A library of partial gene fusions from a New Zealand clinical isolate of M. a. paratuberculosis was constructed in the shuttle vector pJEM11 and expressed in the surrogate hosts E. coli and M. smegmatis. The DNA inserts from a portion of the resulting clones expressing alkaline phosphatase-positive fusion proteins were partially sequenced to identify the proteins. Eleven proteins not previously described for M. a. paratuberculosis were identified as containing signal sequences for export. One of these, a putative lipoprotein named P22 was selected for further study. The full nucleic acid sequence of the p22 gene was determined and the open reading frame was cloned into die mycobacterial expression vector pMIP12. This enabled P22 to be produced as a polyhistidine-tagged protein in M. smegmatis and facilitated purification by chromatography. N-terminal sequencing of the recombinant protein confirmed cleavage of an N-terminal signal sequence. Native P22 was detected in culture supernatants and cell sonicates of M. a. paratuberculosis strain 316F using rabbit antibody raised to P22. Investigation of the presence of genes similar to p22 in other mycobacterial species, revealed p22 was present in Mycobacterium avium subspecies avium and similar genes existed in M. intracellularae (88.5% identity) and M. scrofulaceum (87.7% identity). Database searches showed P22 belonged to the LppX/LprAFG family of mycobacterial lipoproteins also found in M. leprae and in members of the M. tuberculosis complex. P22 shared less than 75% identity to these proteins. Recombinant P22 was able to elicit significantly increased interferon-gamma secretion in blood from a group of eight sheep vaccinated with a live, attenuated strain of M. a. paratuberculosis (strain 316F) compared to a group of five unvaccinated sheep. Antibody to P22 was detected by Western blot analysis in 10 out of 11 vaccinated sheep, in two out of two clinically affected cows and in 11 out of 13 subclinically infected cows.Item The effect of bovine colostrum supplementation on levels of secretory immunoglobulin-A (S-IgA) in saliva of elite atheletes, non-exercising controls and non-exercising older adults : a project [i.e. thesis] completed as fulfilment of the requirements of a doctoral thesis in Clinical Nutrition, Massey University, Albany Campus, New Zealand(Massey University, 2007) Crooks, ChristineSecretory immunoglobulin-A (S-IgA) in saliva may reflect levels of immune defence at other mucosal sites. Reduced levels of salivary S-IgA have been associated with an increased risk for upper respiratory symptoms (URS) in athletes. Previously, the consumption of a nutrition supplement, bovine colostrum (BC) by distance runners, was shown to significantly increase levels of salivary S-IgA compared to baseline; however the mechanism was not known. The immunomodulatory effect of BC is investigated further in these current studies. Twenty-five swimmers (12 males [M], 13 females [F], age 14-23 years) training at an elite level, 28 lightly-exercising students (9M, 19F, age 18-27 years), and 45 healthy older adults (20M, 20F, age 65-76 years), consumed a supplement of either BC or placebo for ten weeks. Saliva samples were collected at baseline, weekly for four weeks during supplementation and post-supplementation. Blood samples were collected at baseline, monthly during supplementation and post-supplementation. No significant changes were seen in levels of S-IgA in either BC or placebo groups within any of the cohorts. There was a trend towards a significant difference in URS reportage between BC and placebo groups in the swimmers cohort, but not in the students or older adults. There was also a trend towards a difference in the number of swimmers reporting URS. Fewer numbers of swimmers consuming BC reported URS compared the placebo (P=0.062) after consuming BC for four weeks compared to those consuming the placebo. Post-exercise plasma cortisol results were significantly reduced in the BC subgroup compared to the placebo (P=0.004). These results do not support the findings of previous intervention studies investigating the immunomodulatory effect of BC in athletes. However the reduced reportage of URS, among swimmers consuming the BC supplement, suggested there was some benefit to their health. A possible explanation is that BC has impacted on non-infectious causes of URS. Growth factors present in BC may enhance intestinal repair which could be advantageous to athletes recovering from bouts of prolonged intensive exercise. The effect of gastrointestinal disturbances on local and systemic immunity may be minimised which benefits immune protection. However an inconsistent effect of BC supplementation on immune protection in athletes means further research is still required. In these studies there was no benefit to immune protection in the student or older adult cohort. Further investigation into the safety of BC for all population groups is still required.Item A search for genetic factors influencing immune responses to a killed Mycobacterium avium subspecies paratuberculosis vaccine in Australian fine-wool merino sheep : thesis in fulfilment of the degree of Doctor of Philosophy in Animal Science, Institute of Veterinary, Animal and Biomedical Sciences, College of Sciences, Massey University(Massey University, 2007) Dukkipati, Venkata Sayoji RaoVSR Dukkipati (2007). A search for genetic factors influencing immune responses to Mycobacterium avium subspecies paratuberculosis. Doctoral thesis, Massey University, Palmerston North, New Zealand. A study was conducted to identify associations between genetic markers and immune responses in Australian fine-wool Merino sheep to a killed Mycobacterium avium subspecies paratuberculosis (Map) vaccine (GudairTM). Blood samples and immune response data (antibody and interferon gamma, IFN-gamma results) were obtained from 934 sheep from a longterm Map vaccination trial undertaken on three independent properties in New South Wales, Australia. Blood samples were genotyped for eight microsatellite markers that included four (DYMS1, OLADRW, OLADRB and SMHCC1) from the Ovar-Mhc region, two each from the SLC11A1 (OVINRA1 and OVINRA2) and IFN-gamma (o(IFN)gamma and OarKP6) gene regions. Vaccination with GudairTM induced strong antibody and IFN-gamma responses as early as two weeks post-vaccination. Between-property differences in magnitude and trend of immune responses, concomitant with season of vaccination and magnitude of natural infection prevalent in individual flocks, were evident. Immune responses in controls on all the three properties remained consistently low, except for slightly elevated IFN-gamma levels at a few time points in controls of properties 2 and 3, concomitant with exposure to natural infection. There were only 2 alleles and 3 genotypes for marker o(IFN)gamma but other loci exhibited extensive polymorphisms, the most occurring at OLADRW which had 42 alleles and 137 genotypes. Heterozygosities varied between 33% (OVINRA2) and 87% (SMHCC1), while polymorphic information contents ranged from 0.31 (o(IFN)gamma) to 0.88 (OLADRW). Genotypes at loci DYMS1, OLADRB, SMHCC1, OVINRA1 and o(IFN)gamma were in Hardy- Weinberg equilibrium (HWE), while those at OarKP6 were in HWE only when rare alleles (<1.0% frequency) were pooled with the closest size class. Departure from HWE, resulting from possible preferential amplification of alleles in heterozygotes, was evident at OLADRW and OVINRA2. Associations between immune responses and genetic polymorphisms at the marker loci were examined by analysing both genotypic and allelic affects. The study revealed several genotypes/alleles at different marker loci to be significantly associated with antibody and IFN-gamma responses to vaccination with GudairTM. However, the majority of those effects were inconsistent across the three properties. Based on significance and consistency in effects across the three properties, five genotypes (two at DYMS1 and one each at OLADRB, SMHCC1 and OVINRA1) and three alleles (one each at DYMS1, OLADRB and o(IFN)gamma) were considered either ‘probable’ or ‘most likely’ to be associated with low IFN-gamma responses, while a genotype at o(IFN)gamma was considered ‘most likely’ to influence high IFN-gamma responses. An allele at OarKP6 was considered ‘probable’ to be associated with low antibody responses to vaccination. Considering the significance of IFN-gamma responses in protection against Map, it is likely that the identified genotype/alleles influencing IFN-gamma responses to vaccination would also influence immune responses to natural Map infections. However, further studies need to be conducted to determine the role of these marker genotypes/alleles in protection against paratuberculosis under natural infection conditions. Key words: paratuberculosis, OJD, Johne’s disease, sheep, immune response, genetic markers, gene polymorphisms, MHC, SLC11A1, IFN-gammaItem Siblings, asthma, rhinoconjunctivitis and eczema: a worldwide perspective from the International Study of Asthma and Allergies in Childhood.(WILEY-BLACKWELL, 2015-01) Strachan DP; Aït-Khaled N; Foliaki S; Mallol J; Odhiambo J; Pearce N; Williams HC; ISAAC Phase Three Study GroupBACKGROUND: Associations of larger families with lower prevalences of hay fever, eczema and objective markers of allergic sensitization have been found fairly consistently in affluent countries, but little is known about these relationships in less affluent countries. METHODS: Questionnaire data for 210,200 children aged 6-7 years from 31 countries, and 337,226 children aged 13-14 years from 52 countries, were collected by Phase Three of the International Study of Asthma and Allergies in Childhood (ISAAC). Associations of disease symptoms and labels of asthma, rhinoconjunctivitis and eczema were analysed by numbers of total, older and younger siblings, using mixed (multi-level) logistic regression models to adjust for individual covariates and at the centre level for region, language and national affluence. RESULTS: In both age groups, inverse trends (P < 0.0001) were observed for reported 'hay fever ever' and 'eczema ever' with increasing numbers of total siblings, and more specifically older siblings. These inverse associations were significantly (P < 0.005) stronger in more affluent countries. In contrast, symptoms of severe asthma and severe eczema were positively associated (P < 0.0001) with total sibship size in both age groups. These associations with disease severity were largely independent of position within the sibship and national GNI per capita. CONCLUSIONS: These global findings on sibship size and childhood asthma, rhinoconjunctivitis and eczema suggest at least two distinct trends. Inverse associations with older siblings (observations which prompted the 'hygiene hypothesis' for allergic disease) are mainly a phenomenon of more affluent countries, whereas greater severity of symptoms in larger families is globally more widespread.