Characterising developmental and activity-dependent myelination in neocortical brain slice cultures

Studying the process of myelination during early brain development is an essential part of understanding several key topics, such as memory, learning, demyelinating diseases, traumatic brain injury, and brain plasticity. In this study, a novel method of culturing murine brain slices is presented and characterised which provides a stable, three-dimensional, in vitro model for studying oligodendrocyte maturation in the neocortex. The slices were from transgenic PLP-dsRED mice and were stained using a fluorescent anti-MBP (Myelin basic protein) marker to visualise oligodendrocyte morphology and track the progress of myelination. Oligodendrocyte differentiation and myelination were examined qualitatively where images were ranked on a scale of 1-4 based on differentiation and myelination levels. It was found that the optimal time window for making viable slice cultures was at postnatal days 3-4, as this gave better oligodendrocyte maturation than slices taken later, at postnatal day 7. To appraise this method’s success as a technique for testing the effects of drug treatments on oligodendrocytes, slices were treated with factors expected to modulate myelination. Both TTX (Tetrodotoxin), which has been shown to block oligodendrocyte proliferation and myelination, and BDNF (Brain-derived neurotrophic factor), which enhances the maturation and myelination of oligodendrocytes, were used in this study. Using the fluorescent imaging technique and ranking system, it was concluded that TTX had a negative impact on the level of differentiation and myelination in the neocortex, whereas BDNF had no effect. These findings were followed by analysing the expression of key oligodendrocyte genes. qPCR was used to determine how TTX and BDNF treatments influenced the expression of oligodendrocyte lineage maturation markers PDGFRα (Platelet-derived growth factor receptor A), Enpp6 (Ectonucleotide pyrophosphatase/phosphodiesterase 6), or MOG (Myelin oligodendrocyte glycoprotein). In agreement with the imaging data, BDNF showed no significant effect on oligodendrocyte genes. In contrast, TTX treatment reduced the expression of MOG in the brain slices, indicating that this neurotoxin reduced the number of oligodendrocyte lineage cells that reached the final myelinating stage of maturity. These results are consistent with findings that oligodendrocyte differentiation is stimulated by axon activity, and that blocking that with TTX reduces myelination. Moreover, they confirm that the neocortical slice culture system is a suitable platform for ex vivo studies of oligodendrocyte and myelin plasticity and should prove useful for further research to increase the depth of knowledge of myelin development, and for testing potential drug treatments for oligodendrocyte-related disorders

Diabetic neuropathy: a mechanism of TRPV1 sensitisation and the treatment with Vascular Endothelial Growth Factor-A165b (VEGF-A165b)

Diabetic neuropathy affects up to 50% of diabetic patients and commonly presents as neuropathic pain. Streptozotocin (STZ) injected type 1 diabetic rats exhibit thermal hypersensitivity and this is caused by the sensitization of transient receptor potential vanilloid 1 (TRPV1) on DRG neurons.Thermal hypersensitivity is ameliorated in STZ diabetic rats with the systemic treatment of vascular endothelial growth factor-A165b (VEGF-A165b). This thesis investigated the role of the activation of the receptor for advanced glycated end products (RAGE) on the sensitization of TRPV1 on DRG neurons and determined the effects of VEGF-A165b on this mechanism. Capsaicin-evoked TRPV1 activity was measured in DRG neurons from adult STZ diabetic rats to determine a sensitization effect and these effects were modeled in vitro by exposing DRG neuronal cultures from naïve rats to hyperglycemic conditions. STZ diabetic rats had sensitized agonist-evoked TRPV1 activity, increased RAGE expression on the DRG neurons, and increased expression of high mobility group box-1 (HMGB1, a RAGE agonist) protein around nociceptor terminals. In vitro it was determined that hyperglycemic conditions sensitize capsaicin-evoked TRPV1 activity and this effect was mediated by the activation of RAGE, indicating that RAGE activation in diabetes is likely to cause TRPV1 sensitization. It was determined that HMGB1 binds to RAGE on DRG neurons and this binding results in the sensitization of TRPV1 activity and this sensitization event was a PKC mediated effect. PKC is able to phosphorylate serine residues on the intracellular domain of TRPV1. This phosphorylation causes increased agonist-evoked TRPV1 activity. Here it was demonstrated that there was increased TRPV1 phosphorylation at serine 800, a PKC dependent phosphorylation site, on DRG neurons in hyperglycemia and this event is likely to contribute towards the TRPV1 sensitization effect induced by HMGB1-RAGE binding. In vivo VEGF-A165b did not alter the expression of HMGB1 around nociceptor terminals but it did reduce the expression of RAGE on DRG neurons. In vitro VEGF-A165b blocked TRPV1 sensitization in DRG neurons exposed to hyperglycemic conditions, blocked the PKC mediated phosphorylation event, and blocked the HMGB1 mediated sensitization of TRPV1. VEGF-A165b may, therefore, be preventing this sensitization effect by blocking by the PKC activation that occurs downstream of RAGE activation. These results demonstrate a novel mechanism of neuronal TRPV1 sensitization in vitro involving the activation of RAGE on the DRG neurons. This mechanism may contribute to the sensitization of nociceptors in diabetes and consequently the development of neuropathic pain. VEGF-A165b blocks this mechanism indicating that 1) VEGF-A165b has direct actions on the DRG neurons in preventing them from hyperglycemia mediated damage and 2) VEGF-A165b may be exerting its analgesic effects in STZ diabetic rats through this mechanism

Factors affecting the offer of pulmonary rehabilitation to patients with chronic obstructive pulmonary disease by primary care professionals : a qualitative study

Aim: To explore health professionals&rsquo; experiences of barriers and facilitators to referring patients for pulmonary rehabilitation in a primary care setting. Background: Pulmonary rehabilitation involves a multidisciplinary teamwork approach to improvingthe quality of life for people with chronic obstructive pulmonary disease. This study aimed to find out about health care professionals&rsquo; experiences when referring patients. Reports suggest that a health care professional&rsquo;s attitude towards a treatment affects the willingness of patients to accept advice. Methods: Five focus group interviews were undertaken with 21 health professionals from North Midlands, UK. Data were analysed using a thematic analysis drawing on the techniques of grounded theory.Findings: Chronic disease management has been delegated to Practice Nurses in many cases leaving some nurses feeling unsupported and some General Practitioners feeling deskilled. Problems with communication, a lack of adequate and timely local service provision, a difficult referral process, time pressures and lack of information were barriers to health care professionals making an offer of pulmonary rehabilitation. An explanatory model is proposed to describe how addressing barriers to referral may improve health care professionals views about pulmonary rehabilitation and therefore may mean that they present it in a more positive manner.<br /

Sensory neuronal sensitisation occurs through HMGB-1/RAGE and TRPV1 in high glucose conditions

Many potential causes for painful diabetic neuropathy have been proposed including actions of cytokines and growth factors. High mobility group protein B1 (HMGB1) is a RAGE agonist, increased in diabetes, that contributes to pain by modulating peripheral inflammatory responses. HMGB1 enhances nociceptive behaviour in naïve animals through an unknown mechanism. We tested the hypothesis that HMGB1 causes pain through direct neuronal activation of RAGE and alteration of nociceptive neuronal responsiveness. HMGB1 and RAGE expression were increased in skin and primary sensory (DRG) neurons of diabetic rats at times when pain behaviour was enhanced. Agonist-evoked TRPV1-mediated calcium responses increased in cultured DRG neurons from diabetic rats and in neurons from naïve rats exposed to high glucose concentrations. HMGB1-mediated increases in TRPV1-evoked calcium responses in DRG neurons were RAGE and PKC-dependent, and this was blocked by co-administration of the growth factor splice variant, VEGF-A165b. Pain behaviour and DRG RAGE expression increases were blocked by VEGF-A 165 b treatment of diabetic rats in vivo. HMGB-1-RAGE activation sensitizes DRG neurons in vitro. VEGF-A165b blocks HMGB-1/RAGE DRG activation, which may contribute to its analgesic properties in vivo

New models of care: a liaison psychiatry service for medically unexplained symptoms and frequent attenders in primary care

Aims and method: This paper describes the process of setting up and the early results from a new liaison psychiatry service in primary care for people identified as frequent general practice attenders with long-term conditions or medically unexplained symptoms. Using a rapid evidence synthesis, we identified existing service models, mechanisms to identify and refer patients, and outcomes for the service. Considering this evidence, with local contingencies we defined options and resources. We agreed a model to set up a service in three diverse general practices. An evaluation explored the feasibility of the service and of collecting data for clinical, service and economic outcomes. Results: High levels of patient and staff satisfaction, and reductions in the utilisation of primary and secondary healthcare, with associated cost savings are reported. Clinical implications: A multidisciplinary liaison psychiatry service integrated in primary care is feasible and may be evaluated using routinely collected data

Physical Activity Characteristics across GOLD Quadrants Depend on the Questionnaire Used

BACKGROUND:The GOLD multidimensional classification of COPD severity combines the exacerbation risk with the symptom experience, for which 3 different questionnaires are permitted. This study investigated differences in physical activity (PA) in the different GOLD quadrants and patient's distribution in relation to the questionnaire used. METHODS:136 COPD patients (58±21% FEV1 predicted, 34F/102M) completed COPD assessment test (CAT), clinical COPD questionnaire (CCQ) and modified Medical Research Council (mMRC) questionnaire. Exacerbation history, spirometry and 6MWD were collected. PA was objectively measured for 2 periods of 1 week, 6 months apart, in 5 European centres; to minimise seasonal and clinical variation the average of these two periods was used for analysis. RESULTS:GOLD quadrants C+D had reduced PA compared with A+B (3824 [2976] vs. 5508 [4671] steps.d-1, p<0.0001). The choice of questionnaire yielded different patient distributions (agreement mMRC-CAT κ = 0.57; CCQ-mMRC κ = 0.71; CCQ-CAT κ = 0.72) with different clinical characteristics. PA was notably lower in patients with an mMRC score ≥2 (3430 [2537] vs. 5443 [3776] steps.d-1, p <0.001) in both the low and high risk quadrants. CONCLUSIONS:Using different questionnaires changes the patient distribution and results in different clinical characteristics. Therefore, standardization of the questionnaire used for classification is critical to allow comparison of different studies using this as an entry criterion. CLINICAL TRIAL REGISTRATION:ClinicalTrials.gov NCT01388218

Use of low-dose oral theophylline as an adjunct to inhaled corticosteroids in preventing exacerbations of chronic obstructive pulmonary disease: study protocol for a randomised controlled trial.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with high morbidity, mortality, and health-care costs. An incomplete response to the anti-inflammatory effects of inhaled corticosteroids is present in COPD. Preclinical work indicates that 'low dose' theophylline improves steroid responsiveness. The Theophylline With Inhaled Corticosteroids (TWICS) trial investigates whether the addition of 'low dose' theophylline to inhaled corticosteroids has clinical and cost-effective benefits in COPD. METHOD/DESIGN: TWICS is a randomised double-blind placebo-controlled trial conducted in primary and secondary care sites in the UK. The inclusion criteria are the following: an established predominant respiratory diagnosis of COPD (post-bronchodilator forced expiratory volume in first second/forced vital capacity [FEV1/FVC] of less than 0.7), age of at least 40 years, smoking history of at least 10 pack-years, current inhaled corticosteroid use, and history of at least two exacerbations requiring treatment with antibiotics or oral corticosteroids in the previous year. A computerised randomisation system will stratify 1424 participants by region and recruitment setting (primary and secondary) and then randomly assign with equal probability to intervention or control arms. Participants will receive either 'low dose' theophylline (Uniphyllin MR 200 mg tablets) or placebo for 52 weeks. Dosing is based on pharmacokinetic modelling to achieve a steady-state serum theophylline of 1-5 mg/l. A dose of theophylline MR 200 mg once daily (or placebo once daily) will be taken by participants who do not smoke or participants who smoke but have an ideal body weight (IBW) of not more than 60 kg. A dose of theophylline MR 200 mg twice daily (or placebo twice daily) will be taken by participants who smoke and have an IBW of more than 60 kg. Participants will be reviewed at recruitment and after 6 and 12 months. The primary outcome is the total number of participant-reported COPD exacerbations requiring oral corticosteroids or antibiotics during the 52-week treatment period. DISCUSSION: The demonstration that 'low dose' theophylline increases the efficacy of inhaled corticosteroids in COPD by reducing the incidence of exacerbations is relevant not only to patients and clinicians but also to health-care providers, both in the UK and globally. TRIAL REGISTRATION: Current Controlled Trials ISRCTN27066620 was registered on Sept. 19, 2013, and the first subject was randomly assigned on Feb. 6, 2014

The DANTE trial protocol: a randomised phase III trial to evaluate the Duration of ANti-PD-1 monoclonal antibody Treatment in patients with metastatic mElanoma.

BACKGROUND: Immunotherapy is revolutionising the treatment of patients diagnosed with melanoma and other cancers. The first immune checkpoint inhibitor, ipilimumab (targeting cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4)), showed a survival advantage over standard chemotherapy. Subsequently the anti-programmed cell death protein 1 (PD-1) antibodies, nivolumab and pembrolizumab were shown to be more effective than ipilimumab. Ipilimumab combined with nivolumab gives an incremental gain in overall survival compared with nivolumab alone but increases the risk of severe, potentially life-threatening toxicities. In contrast to ipilimumab monotherapy, anti-PD-1 antibodies are licensed to be continued until disease progression. Follow-up of patients recruited to the first trials evaluating 2 years of pembrolizumab showed that three-quarters of responding patients continue responding after stopping treatment. Suggestive of early response, we hypothesised that continuing anti-PD-1 treatment beyond 1 year in progression-free patients may be unnecessary and so designed the DANTE trial. METHODS: DANTE is a multicentre, randomised, phase III, non-inferiority trial to evaluate the duration of anti-PD-1 therapy in patients with metastatic (unresectable stage III and stage IV) melanoma. It uses a two-stage recruitment strategy, registering patients before they complete 1 year of first-line anti-PD-1 +/- CTLA-4 therapy and randomising eligible patients who have received 12 months of treatment and are progression-free at 1 year. At randomisation, 1208 patients are assigned (1:1) to either 1) continue anti-PD-1 treatment until disease progression/ unacceptable toxicity/ for at least 2 years in the absence of disease progression/ unacceptable toxicity or 2) to stop treatment. Randomisation stratifies for baseline prognostic factors. The primary outcome is progression-free survival at 3, 6, 9 and 12 months and then, 6-monthly for up to 4-years. Secondary outcomes collected at all timepoints include overall survival, response-rate and duration and safety, with quality of life and cost-effectiveness outcomes collected 3-monthly for up to 18-months. Sub-studies include a qualitative analysis of patient acceptance of randomisation and sample collection to inform future translational studies into response/ toxicity biomarkers. DISCUSSION: DANTE is a unique prospective trial investigating the optimal duration of anti-PD-1 therapy in metastatic melanoma patients. Outcomes will inform future use of these high burden drugs. TRIAL REGISTRATION: ISRCTN15837212 , 31 July 2018

Breathlessness is associated with urinary incontinence in men: A community-based study

<p>Abstract</p> <p>Background</p> <p>Urinary incontinence (UI) is a distressing problem for older people. To investigate the relationship between UI and respiratory symptoms among middle-aged and older men, a community-based study was conducted in Japan.</p> <p>Methods</p> <p>A convenience sample of 668 community-dwelling men aged 40 years or above was recruited from middle and southern Japan. The International Consultation on Incontinence Questionnaire-Short Form, the Medical Research Council's dyspnoea scale and the Australian Lung Foundation's Feeling Short of Breath scale, were administered by face-to-face interviews to ascertain their UI status and respiratory symptoms.</p> <p>Results</p> <p>The overall prevalence of UI was 7.6%, with urge-type leakage (59%) being most common among the 51 incontinent men. The presence of respiratory symptoms was significantly higher among incontinent men than those without the condition, especially for breathlessness (45% versus 30%, <it>p </it>= 0.025). The odds of UI for breathlessness was 2.11 (95% confidence interval 1.10-4.06) after accounting for age, body mass index, smoking and alcohol drinking status of each individual.</p> <p>Conclusions</p> <p>The findings suggested a significant association between UI and breathlessness in middle-aged and older men.</p