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    Application of flow cytometry for enumerating individual bacterial cultures from a mixed culture system : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 2014) Horváth, Kylie
    Cultured dairy products are often made with more than one microbial culture. Yoghurt requires the cultivation of several bacterial species for its production and the level of each is important for different reasons. Differential plate count methods to enumerate the separate species in yoghurt are not ideal because many of the bacteria used have similar growth profiles and plate counts take several days to produce a result. A fast specific method for enumerating each culture would be beneficial because quick results would enable tighter control of processing or experimental conditions and the ability to track individual species amongst a background of similar bacteria. Flow cytometry combined with fluorescent in-situ hybridisation (FLOW-FISH) was investigated as a potential solution and successful enumeration was achieved within 1 day for a yoghurt microorganism, Streptococcus thermophilus (ST55), grown in M17 medium. This method may be improved to increase the signal-to-noise ratio and to reduce the assay time. The chemical propidium monoazide enabled a closer match to plate counts for flow cytometry results using a total viable count assay and may be useful combined with the FLOW-FISH assay for removing non-viable or viable, but non-culturable, cells from the results. An enzyme and/or detergent pre-treatment may achieve successful FLOW-FISH enumeration of cells grown in reconstituted skim milk – a similar matrix to yoghurt.
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    Non-invasive assessment of airway inflammation in asthma : a thesis by publications presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Public Health, Massey University, Wellington, New Zealand
    (Massey University, 2013) Brooks, Collin Richard
    Inflammation is a central feature in current definitions of asthma. Despite this, airway inflammation remains infrequently assessed in either population-based studies or clinical practice. In this thesis, conventional and novel non-invasive methods (based on exhaled nitric oxide (FENO) measurement and sputum induction) were used to assess airway inflammation and examine the presence, characteristics and stability of inflammatory asthma phenotypes in a general population sample, which included very young and very old individuals. It was shown that FENO measurement could be easily and cost-effectively conducted, and that flow cytometric analysis of sputum leukocyte populations is a feasible alternative to conventional manual cell counts. In particular, flow cytometric analysis was shown to be well suited to the detection of rare cell populations, and provided data suggesting that airway invariant natural killer T cells may not be a key player in asthma pathophysiology and that basophils may be a useful indicator of allergic airway inflammation in asthma. When examining inflammatory asthma phenotypes, it was shown that less than 50% of asthmatics (both children and adults) had evidence of eosinophilic inflammation, although in one small study, altered treatment resulted in phenotype changes in more than 50% of asthmatics studied. Neutrophilic airway inflammation was rare, and was statistically significantly associated with age. Approximately half of all the asthmatics studied had no detectable evidence of airway inflammation at the time of assessment. In conclusion, the methods developed and validated for the non-invasive assessment of airway inflammation allow more detailed investigations of asthma aetiology in populationbased studies. However, a single assessment of airway inflammation may not be adequate for valid identification of inflammatory asthma phenotypes. The results of the studies described in this thesis suggest that 50% of asthmatics may have eosinophilic airway inflammation, with the remainder having no airway inflammation. Further investigations of noninflammatory mechanisms are therefore warranted, as a better understanding of the mechanisms and the associated environmental exposures involved may guide the development of more effective therapies and control measures for this common phenotype.
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    Antigen-specific cytotoxic T lymphocytes target airway CD103+ and CD11b+ dendritic cells to suppress allergic inflammation
    (Nature Publishing Group, 2016-01) Daniels NJ; Hyde E; Ghosh S; Seo K; Price KM; Hoshino K; Kaisho T; Okada T; Ronchese F
    Allergic airway inflammation is driven by the recognition of inhaled allergen by T helper type 2 (Th2) cells in the airway and lung. Allergen-specific cytotoxic T lymphocytes (CTLs) can strongly reduce airway inflammation, however, the mechanism of their inhibitory activity is not fully defined. We used mouse models to show that allergen-specific CTLs reduced early cytokine production by Th2 cells in lung, and their subsequent accumulation and production of interleukin (IL)-4 and IL-13. In addition, treatment with specific CTLs also increased the proportion of caspase+ dendritic cells (DCs) in mediastinal lymph node (MLN), and decreased the numbers of CD103+ and CD11b+ DCs in the lung. This decrease required expression of the cytotoxic mediator perforin in CTLs and of the appropriate MHC-antigen ligand on DCs, suggesting that direct CTL-DC contact was necessary. Lastly, lung imaging experiments revealed that in airway-challenged mice XCR1-GFP+ DCs, corresponding to the CD103+ DC subset, and XCR1-GFP− CD11c+ cells, which include CD11b+ DCs and alveolar macrophages, both clustered in the areas surrounding the small airways and were closely associated with allergen-specific CTLs. Thus, allergen-specific CTLs reduce allergic airway inflammation by depleting CD103+ and CD11b+ DC populations in the lung, and may constitute a mechanism through which allergic immune responses are regulated.