Insulin is a major autoantigen in type 1 diabetes, targeted by both CD8 and CD4 T-cells. We studied an insulin-reactive T-cell receptor (TCR) alpha-chain transgenic non-obese diabetic (NOD) mouse on a TCRC and proinsulin2 (PI2)-deficient background, designated as A22Cα -/-PI2 -/-NOD mice. These mice develop a low incidence of autoimmune diabetes. To test the role of gut microbiota on diabetes development in this model system, we treated the A22Cα -/-PI2 -/-NOD mice with enrofloxacin, a broad-spectrum antibiotic. The treatment led to male mice developing accelerated diabetes. We found that enrofloxacin increased the frequency of the insulin-reactive CD8+ T-cells and activated the cells in the Peyer's patches (PP) and pancreatic lymph nodes (PLNs), together with induction of immunological effects on the antigen-presenting cell populations. The composition of gut microbiota differed between the enrofloxacin-treated and untreated mice and also between the enrofloxacintreated mice that developed diabetes, compared with those that remained normoglycemic.Our results provide evidence that the composition of the gut microbiota is important for determining the expansion and activation of insulin-reactive CD8+ T-cells. Materials and methodsMice NOD/Caj mice were originally obtained from Yale University. G9Cα -/-NOD, G9Cα -/-PI2 -/-NOD and A22Cα -/-PI2 -/-NOD have all been previously described and are summarized in Supplementary Table 1 (34-36). All mice used in the current study were male, from litters divided between treatment groups (Fig.1A). Mice from several breeder pairs were mixed and housed in microisolators or scantainers with food and water ad libitum, with 12-hour light and dark cycles in the specific pathogen-free facility at Cardiff University. All procedures were performed in accordance with UK Home Office approved protocols. Preparation and administration of enrofloxacin-treated waterEnrofloxacin (Bayer) was added to autoclaved, filtered water at a final concentration of 0.4mg/ml (diluted 1:250), prepared freshly every week. Untreated mice received the same autoclaved, filtered water. Mice were treated from 3-weeks of age (at weaning) continuously until 10-weeks of age, unless otherwise stated. Diabetes IncidenceMice were monitored weekly for glycosuria (Bayer Diastix) from 5-weeks of age until termination. Diabetes was diagnosed after 2 consecutive positive glycosuria tests, confirmed by a blood glucose concentration >13.9mmol/L (>250mg/dl). Statistical analysis was performed using the log-rank test.
Light and smell have both been shown to induce beneficial changes to human psychophysiology. Bright light therapy has been shown to have a positive impact on anxiety and depression and smell has also been shown to have positive effects on mood, stress, anxiety and depression. We developed a method for the delivery of integrated light and smell stimulation to try to optimise positive psychophysiological benefit. We tested its effectiveness on a physiological measure, EEG frontal alpha asymmetry (FA) and a psychological paradigm, the POMS test, both of which have been used as a measure of emotional state and mood. Light, pleasant smell, combined light+smell and a no stimulus control were delivered for 90s while the frontal alpha asymmetry (FA) was monitored. Smell and light+smell caused significant reductions in negative FA during stimulation. Exposure to a longer 15 min nonadaptive light+smell stimulus protocol reduced negative FA and decreased negative affect (POMS). The effects were greater in the negative FA group. Both the physiological (EEG) and psychometric (POMS) data indicate that integrated light and smell stimulation can reduce negative affect and reduce a marker for anxiety/ depression. This light+smell sensory stimulation protocol could offer a safe treatment for depression/anxiety.
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