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Closed-loop insulin delivery systems have the potential to address suboptimal glucose control in children and adolescents with type 1 diabetes. We compared safety and efficacy of the Cambridge hybrid closed-loop algorithm with usual care over 6 months in this population.In a multicentre, multinational, parallel randomised controlled trial, participants aged 6-18 years using insulin pump therapy were recruited at seven UK and five US paediatric diabetes centres. Key inclusion criteria were diagnosis of type 1 diabetes for at least 12 months, insulin pump therapy for at least 3 months, and screening HbAOf 147 people initially screened, 133 participants (mean age 13·0 years [SD 2·8]; 57% female, 43% male) were randomly assigned to either the closed-loop group (n=65) or the control group (n=68). Mean baseline HbAThe Cambridge hybrid closed-loop algorithm had an acceptable safety profile, and improved glycaemic control in children and adolescents with type 1 diabetes. To ensure optimal efficacy of the closed-loop system, usage needs to be consistently high, as demonstrated with CamAPS FX.National Institute of Diabetes and Digestive and Kidney Diseases.

Hypoxia is a global and increasingly important stressor in aquatic ecosystems, with major impacts on biodiversity worldwide. Hypoxic waters are often contaminated with a wide range of chemicals but little is known about the interactions between these stressors. We investigated the effects of hypoxia on the responses of zebrafish (Danio rerio) embryos to copper, a widespread aquatic contaminant. We showed that during continuous exposures copper toxicity was reduced by over 2-fold under hypoxia compared to normoxia. When exposures were conducted during 24 h windows, hypoxia reduced copper toxicity during early development and increased its toxicity in hatched larvae. To investigate the role of the hypoxia signaling pathway on the suppression of copper toxicity during early development, we stabilized the hypoxia inducible factor (HIF) pathway under normoxia using a prolyl-4-hydroxylase inhibitor, dimethyloxalylglycine (DMOG) and demonstrated that HIF activation results in a strong reduction in copper toxicity. We also established that the reduction in copper toxicity during early development was independent of copper uptake, while after hatching, copper uptake was increased under hypoxia, corresponding to an increase in copper toxicity. These findings change our understanding of the current and future impacts of worldwide oxygen depletion on fish communities challenged by anthropogenic toxicants.

In pouchitis, the mucosa is infiltrated by activated polymorphonuclear neutrophils capable of producing calprotectin, a stable antimicrobial myelomonocytic protein.The aim is to assess the ability of faecal calprotectin to differentiate between inflamed and noninflamed ileal pouches, and to correlate this with inflammation severity using the newly developed Objective Pouchitis Score.Fifty-four stool samples were collected from patients who had undergone restorative proctocolectomy; 46 from patients with ulcerative colitis and eight from those with familial adenomatous polyposis coli. Faecal calprotectin concentrations were determined by quantitative enzyme-linked immunosorbant assay.Of the ulcerative colitis patients, six were diagnosed with pouchitis and pre-pouch ileitis (median faecal calprotectin: 865 microg/g, with a range of 95-2350 microg/g); 13 had pouchitis alone (145, 33-3350 microg/g) and 27 were uninflamed (56, 4-705 microg/g). Of the familial adenomatous polyposis patients, one had pouchitis and pre-pouch ileitis (305 microg/g), and seven had noninflamed pouches (9, 6-26 microg/g). Stool samples obtained from pouchitis patients had significantly higher calprotectin concentrations compared with those obtained from uninflamed pouches (Mann-Whitney: P0.0001). Faecal calprotectin concentrations correlated closely with the Objective Pouchitis Score, the Pouch Disease Activity Index and endoscopic and histological inflammatory scores (Spearman rank test: P values0.0001). Using a faecal calprotectin threshold ofor=92.5 microg/g to define a positive result, Receiver Operating Characteristic analysis demonstrated a sensitivity of 90% and a specificity of 76.5%.Faecal calprotectin measurement is a useful noninvasive tool in the diagnosis of acutely inflamed ileal pouches and correlates well with the severity of pouchitis.

BACKGROUND: Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTR gene therapy in patients with cystic fibrosis.METHODS: We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Patients (aged ≥12 years) with a forced expiratory volume in 1 s (FEV1) of 50-90% predicted and any combination of CFTR mutations, were randomly assigned, via a computer-based randomisation system, to receive 5 mL of either nebulised pGM169/GL67A gene-liposome complex or 0·9% saline (placebo) every 28 days (plus or minus 5 days) for 1 year. Randomisation was stratified by % predicted FEV1 (FINDINGS: Between June 12, 2012, and June 24, 2013, we randomly assigned 140 patients to receive placebo (n=62) or pGM169/GL67A (n=78), of whom 116 (83%) patients comprised the per-protocol population. We noted a significant, albeit modest, treatment effect in the pGM169/GL67A group versus placebo at 12 months' follow-up (3·7%, 95% CI 0·1-7·3; p=0·046). This outcome was associated with a stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group. We recorded no significant difference in treatment-attributable adverse events between groups.INTERPRETATION: Monthly application of the pGM169/GL67A gene therapy formulation was associated with a significant, albeit modest, benefit in FEV1 compared with placebo at 1 year, indicating a stabilisation of lung function in the treatment group. Further improvements in efficacy and consistency of response to the current formulation are needed before gene therapy is suitable for clinical care; however, our findings should also encourage the rapid introduction of more potent gene transfer vectors into early phase trials.FUNDING: Medical Research Council/National Institute for Health Research Efficacy and Mechanism Evaluation Programme.

In June 2013, a record-breaking female Isurus oxyrinchus (total length 373 cm, mass 600 kg) was captured by rod and reel off Huntington Beach, California, where it was subsequently donated to research and provided a rare opportunity to collect the first data for a female I. oxyrinchus of this size. Counts of vertebral band pairs estimate the shark to have been c. 22 years old, depending upon assumptions of band-pair deposition rates, and the distended uteri and spent ovaries indicated that this shark had recently given birth. The stomach contained a c. 4 year-old female California sea lion Zalophus californianus that confirmed the high trophic position of this large I. oxyrinchus, which was corroborated with the high levels of measured contaminants and tissue isotope analyses.

Coeliac disease is a common condition and its prevalence in UK is now thought to be approximately 1:100. It is being diagnosed and treated more frequently as awareness at the primary care level has increased. Coeliac disease is a complex disorder and is frequently associated with other disease processes. The management of these patients needs to take on a holistic approach, whilst the physician needs to be aware of the rare complications. This article gives an up-to-date review of the literature written on the pathogenesis of coeliac disease. We have attempted to paint a picture from beginning to end, whilst clarifying the grey areas in between. General epidemiological factors are reviewed before looking at genetic risk factors. We assess the sensitivity and specificity of the investigative modalities available for clinical use and comment on optimum management of these patients thereafter. The future of coeliac disease looks promising for patients with several novel therapies on the horizon. Whilst further work is still needed to breed out the toxic epitopes from wheat, novel therapies may come from other areas such as the work aimed at restoring normal tolerance to gluten.

Celiac disease is a chronic enteropathy caused by intolerance to gluten. The true prevalence of this condition is much greater than previously recognized, with increasing numbers of silent cases being diagnosed. Population-based studies, using serologic screening, have indicated that the prevalence of celiac disease in Caucasian populations is .5%-1%. The pattern of incidence is changing, with a greater proportion of cases diagnosed later in adulthood. The pathologic lesion is characterized by a flattened small intestinal mucosa with a lymphocytic infiltrate, crypt hyperplasia, and villous atrophy. Absorptive function may be impaired and patients can experience gastrointestinal symptoms and malabsorption leading to development of anemia, osteoporosis, or other complications. Untreated celiac disease is associated with significant morbidity and increased mortality, largely owing to the development of enteropathy-associated intestinal lymphoma. The pathologic changes and symptoms resolve when gluten is excluded from the diet for a sustained period.

Background. Dysphonia is a known local adverse eAect of inhaled corticosteroids. This symptom was investigated by laryngoscopy and assessment in a voice laboratory. The eAects of changing the treatment of patients with dysphonia, reported whilst using the pMDI, to pMDI plus Nebuhaler 1 or Tubuhaler 1 was also assessed. Methods. Seventy-two patients reporting dysphonia and taking inhaled steroids from a pMDI entered a 12-week, open, parallel group study. Fifty-one completed the study per protocol; 26 in the Nebuhaler group [21 female, mean age 57 years (22‐77)] and 25 in the Turbuhaler 1 group [18 female, mean age 58 years (21‐81)]. A dysphonia diary card was completed weekly. Voice laboratory assessments and laryngoscopy were performed on entry and at 12 weeks. Results. There were no diAerences in voice laboratory data, laryngoscopic evidence of disordered glottic closure and diary data between the two groups at 12 weeks. At study entry laryngoscopic appearances were normal in almost half the patients. Vocal cord bowing was rarely seen. Glottic closure changed in nine patients during the study period, but there was no correlation with voice symptoms. The trend of symptomatic improvement of voice status in the Turbuhaler 1 group did not correlate with voice laboratory assessments and laryngoscopic evidence of disordered glottic closure. After 4 weeks, 40% of patients using Turbuhaler 1 and 8% in the Nebuhaler 1 group scored their voice status as better (P

Ipratropium bromide (IB), typically delivered by pressurized metered dose inhaler (pMDI), is used to treat patients with reversible airways obstruction. Use of the pMDI, unlike the Turbuhaler (TH), demands co-ordination of actuation with inspiration for efficient use. Two studies were carried out to compare the relative efficacy and potency of IB delivered by TH or pMDI. Both studies were of a randomized, double-blind and cross-over design. For the efficacy study, 15 patients received a cumulative dose of 160 microg IB via TH or pMDI as doses of 20, 20, 40 and 80 microg at 45 min intervals on two days. Forced expiratory volume in one second (FEV1) was measured prior to and 40 min after dosing. For the potency study, 33 patients received 10, 20 or 40 microg of IB via TH, 20 microg IB via pMDI, or placebo, on five days. FEV1 was recorded prior to and 15-360 min after dosing. For the efficacy study, there was no difference in FEV1 response to a cumulative dose of IB via pMDI and TH. More than 80% of the maximum effect was seen at the lowest dose (20 microg of IB). Regarding the potency study, the FEV1 response to 20 microg IB administered via pMDI was similar to that of 10 microg via TH; 20 microg via TH was significantly more effective than 20 microg via pMDI (p

Background—Electronic diary cards have advantages over paper diaries for daily collection of data on lung function and symptoms in patients with respiratory disorders. The suitability of a pen-based electronic diary (Apple MessagePad) for this purpose was assessed in a clinical trial setting.Methods—Two studies were carried out in patients with chronic obstructive airways disease: 1. An open randomized two-period crossover study comparing electronic and paper diaries in 22 clinic outpatients aged 18–70. Data were collected for four weeks on each type of diary, and 2. An open study in 37 patients in general practice aged 13–80. Data were collected for four weeks on electronic diaries only.Results—In Study 1, 59% of patients preferred the electronic diary and 18% preferred paper. Both paper and electronic diaries were found to be easy to use. There were fewer problematic data from the electronic diaries (0.24% of data points) compared with paper (5.6%), resulting in improved data reliability. There were mor...

BACKGROUND: Clinical trials in cystic fibrosis (CF) have been hindered by the paucity of well characterised and clinically relevant outcome measures. AIM: To evaluate a range of conventional and novel biomarkers of CF lung disease in a multicentre setting as a contributing study in selecting outcome assays for a clinical trial of CFTR gene therapy. METHODS: A multicentre observational study of adult and paediatric patients with CF (>10 years) treated for a physician-defined exacerbation of CF pulmonary symptoms. Measurements were performed at commencement and immediately after a course of intravenous antibiotics. Disease activity was assessed using 46 assays across five key domains: symptoms, lung physiology, structural changes on CT, pulmonary and systemic inflammatory markers. RESULTS: Statistically significant improvements were seen in forced expiratory volume in 1 s (p

Sustainable (aerobic) swimming performance in yellowfin tuna (Thunnus albacares) is examined through a model estimating red muscle oxygen demand and the cardiovascular system's capacity for oxygen delivery. At maximum oxygen delivery (25°C), red muscle oxygen consumption (VO2RM) is estimated to be 0.197 ml O2/g/min, which is higher than previous estimates of maximum VO2RM. However, due to the high costs of swimming at high speeds, maximum sustained velocity at this rate would only be 3.7 fork lengths/s, which is lower than previous estimates for yellowfin. Hypoxia and reduced ambient temperature that may be encountered by yellowfin in the course of their natural movement patterns will decrease oxygen delivery capacity through effects on the cardiovascular system, limiting maximum VO2RM. Moderate swimming velocities could, however, be maintained at an oxygen tension of 75 mmHg, and at an ambient temperature of 15°C.

Tunas are pelagic, continuous swimmers, with numerous specializations for achieving a high aerobic scope. Tunas must maintain a high rate of energy turnover, and therefore require elevated levels of aerobic performance in multiple physiological functions simultaneously. Based on a model of oxygen demand and delivery to the swimming musculature, the yellowfin's total oxygen consumption at the predicted maximum sustainable (aerobic) swimming velocity is well below estimates of its maximum oxygen consumption. This suggests that the high aerobic scope of tunas may be a specialization that permits continuous swimming in addition to supplying oxygen to other metabolic functions. Estimates of the metabolic costs of oxygen-debt repayment, growth, and specific dynamic action have been combined with this model of aerobic swimming performance to evaluate the total energy budget in relation to the aerobic scope of the yellowfin tuna. Repayment of the oxygen debt incurred during burst swimming is potentially a large component of tuna respiratory metabolism and the relatively high aerobic capacity of tuna white muscle may be a specialization for rapid lactate clearance.

The metabolic rates (V(dot)O2) of three tropical tunas [yellowfin tuna (Thunnus albacares), kawakawa (Euthynnus affinis) and skipjack (Katsuwonus pelamis)] were estimated using a large water-tunnel respirometer. Experiments lasting up to 31 h were used to determine the effects of velocity (U) on tuna V(dot)O2 over a range of U (17-150 cm s-1) and temperatures (18shy;30deg;C). Replicate tests were carried out on several fish. The swimming V(dot)O2 of yellowfin is temperature-dependent (Q10=1.67, determined over intervals of 3shy;5deg;C). For yellowfin and skipjack, it was also possible to partition metabolic costs between maintenance and locomotion. The standard metabolic rate (SV(dot)O2) was estimated by extrapolation of the U/V(dot)O2 function to U=0. Comparisons of SV(dot)O2 for different size groups of yellowfin show that the mass-specific scaling exponent for V(dot)O2 is -0.40. The SV(dot)O2 of tuna is comparable to values determined previously by stasis respirometry and is approximately three times higher than that of salmonids. Further comparisons with salmonids show that the slope of the U/V(dot)O2 function is less for tunas, which demonstrate a greater swimming efficiency.

We measured the vertical and horizontal movements of striped marlin (Tetrapturus audax) off the leeward coast of the Island of Hawaii between 20 November and 18 December 1992 while simultaneously gathering data on water temperature and oceanic currents. Fish movements were monitored by ultrasonic depth-sensitive transmitters, depth-temperature profiles by an expendable bathythermograph system, and oceanic current patterns by an acoustic Doppler current profiler. Like Indo-Pacific blue marlin (Makaira mazara), striped marlin near Hawaii spend >85% of their time in the mixed layer (i.e., above 90 m depth). The maximum depth for striped marlin appears to be limited by water temperatures 8 C° colder than the mixed layer, rather than by an absolute lower temperature. We also found that the horizontal displacements of some striped marlin can be strongly influenced by currents.

Activin A is a homodimer of inhibin βA subunits, and was first isolated from gonadal fluids on the basis of its ability to stimulate FSH secretion by rat pituitary cells in vitro. The βA subunits of activin and their mRNAs have been found in many cell types, in several species and at different stages of development, suggesting that activin A has a wide range of diverse biological roles. Apart from the modulation of gonadotroph function, in-vitro studies have demonstrated inhibitory effects of activin A on GH synthesis, GH secretion and possibly somatotroph proliferation. We have therefore investigated the potential role of activin A in the pathophysiological regulation of GH secretion by human somatotrophinoma cells using in-vitro techniques. Cell cultures were established by enzyme dispersion of adenoma tissue obtained from six patients with acromegaly, and treated for 72 h with 0·01–10 nmol recombinant human activin A/1 followed by a 2-h stimulation test with 10 nmol GH-releasing factor (GRF)/l. Medium was collected at 24, 48 and 72 h, as well as after GRF treatment, and GH concentrations were measured by immunoradiometric assay. Basal GH secretion from the cells of two tumours was significantly stimulated 12–63% above control values during treatment with 0·01–10 nmol activin A/1, whereas the peptide had no effect on GH release from cells of the remainder of the tumours. GRF significantly stimulated GH release from the cells of two different adenomas, and pretreatment with 0·01–1 nmol activin A/1 partially but significantly blocked GRF-stimulated GH release from the cells of one of these. These data demonstrate that activin A stimulates basal GH secretion from the cells of some, but not all, human somatotrophinomas in vitro. Pretreatment with the peptide may also partially block GRF-stimulated GH release from GRF-responsive somatotrophinoma cells. The importance of these actions in the pathophysiological regulation of human somatotrophinomas remains to be determined. Journal of Endocrinology (1993) 137, 329–334

Restorative procto-colectomy with ileal pouch anal anastomosis has become the most common elective surgical procedure for patients with ulcerative colitis and is becoming popular in those with familial adenomatous polyposis coli. The procedure itself is primarily carried out in specialist surgical centres but an increasing number are being performed and followed up in district general hospitals. These patients are now filtering through general surgical and gastroenterology clinics and are frequently seen in primary care. Pouchitis, an inflammatory condition of the ileal pouch, has become the third most important form of inflammatory bowel disease. As research develops in this area, other complications are being found. The aim of this review is to provide an up-to-date, evidence-based approach to the clinical management of these patients.

Serum concentrations of LH and FSH and their response to the separate administration of GnRH (100 micrograms i.v.) and TRH (200 micrograms i.v.) have been studied preoperatively in 12 patients with a clinically functionless pituitary adenoma, of whom nine (3F: 6M) were found to secrete gonadotrophins in vitro. In three patients with a gonadotrophin-secreting adenoma (GSA) the pulsatile release of LH and FSH was also assessed preoperatively. An elevated serum FSH was recorded in six of nine patients with a GSA, and was subnormal in one, whilst an elevated LH was recorded in only two and was subnormal in six. A doubling of LH occurred in only four of the nine patients after GnRH and in three of six after TRH. None of the three patients with a non-GSA was shown to have an aberrant response to GnRH or TRH. In patients with a GSA, pulsatile release of LH and FSH was usually asynchronous and neither hormone demonstrated any regular harmonic pattern. These data show that in patients with a GSA the serum FSH level is usually elevated but this is not invariable, and the LH may well be low. Pathological responses of LH are frequently found following the administration of either GnRH or TRH and these stimulation tests should be performed separately in patients presenting with a clinically 'non-functioning' pituitary tumour to assist in the preoperative diagnosis. The absence of normal LH and FSH pulsing also appears to be a feature of GS adenomas, and suggests that tumorous gonadotrophin secretion is not under physiological control by hypothalamic GnRH.

The ability of the gliadin fraction of wheat gluten to exacerbate coeliac disease is well documented. We investigated the possible toxicity of high molecular weight glutenin subunits (HMW-GS) in coeliac disease in vitro using gluten-sensitive T cells, and in vivo with challenge studies in patient volunteers.A mixture of four HMW-GS was chemically separated from wheat flour and checked for purity by HPLC, SDS-PAGE and ELISA. T-cell lines, grown up from small intestinal biopsies from coeliac patients (n=17), were tested for their reactivity to HMW-GS. Adults with coeliac disease and who were on a gluten-free diet (n=3) underwent in-vivo challenges with HMW-GS. Duodenal biopsies, taken prior to the challenge and at intervals up to 6 h afterwards, were assessed for morphology, intra-epithelial lymphocyte count, and interleukin 15 (IL-15) expression, by immunohistochemistry.T-cell lines from 11 of 17 patients were stimulated by HMW-GS. There was a significant change in small intestinal morphology 4 h after commencing infusions with HMW-GS in all three subjects. For example villus height to crypt depth ratios were reduced in the three patients from 3.0+/-0.5 to 1.29+/-0.2, 2.53+/-0.7 to 0.81+/-0.6 and 3.0+/-0.7 to 1.85+/-0.3, P0.0001 in all cases. There was increased expression of IL-15 in the small intestine from 2 h after the HMW-GS challenges.Mixed HMW-GS stimulate T-cell lines from some coeliac patients and exacerbate coeliac disease in vivo, inducing expression, within 2 h, of IL-15. This suggests an innate immune response to these proteins.

This paper describes the procedure and outcome of an audit exercise designed to identify the equipment of choice from among the nasogastric (NG) tubes available on the UK market. In order to assess some of these in a systematic way, a working party of dietitians and nurses was established. Standards expected from a NG tube were set and agreed. These were then used to evaluate seven tubes to see which reached the agreed standard. This was not an exhaustive survey of all available tubes on the market, but a selection of those commonly available. Among the seven tubes investigated, the Corslo®Merck tube was found to be the NG tube of choice in Oxfordshire. Future reassessments will be required as new NG tubes become available.

Coeliac disease is a chronic enteropathy caused by intolerance to gluten proteins. The true prevalence of this condition is greater than previously thought, with increasing numbers of 'silent' cases being diagnosed. Untreated coeliac disease is associated with significant morbidity and increased mortality. There have been a number of advances in our understanding of the pathogenesis of coeliac disease, in particular the mechanisms whereby gluten epitopes are processed, become modified by tissue transglutaminase (tTG) and then interact with HLA restricted T cells. An improved understanding of the immune response to gluten is likely to lead to the development of novel strategies for the treatment of coeliac disease.

Advances in animal tag technologies now offer a significant method for gathering oceanographic information throughout the world's oceans. By employing marine organisms as oceanic samplers (MOOS), vast amounts of in situ data (e.g. temperature, productivity and salinity in relation to depth and location) can be archived and transmitted via satellite or obtained directly from the tag if the tag is recovered. The beauty of this technique is that no on-board navigation system nor external propulsion supply is required. The animal performs the work necessary to transport the equipment and chooses what transect to run. The gathered data, guided by the animal's point of view, reveal secrets of the animal's life and pinpoint environmental features most important to its lifestyle. Depending on the species tagged, the animal may perform a number of tasks of great interest to oceanographers e.g. gathering data from remote regions like the poles, performing repeated deep dives, targeting specific food sources or oceanographic features and traversing massive expanses of ocean. The breadth of animal species with which this technology has been successfully deployed is growing steadily. The list includes bluefin and yellowfin tuna, white sharks, thresher sharks, whale sharks, ocean sunfish, marine turtles, seals, sea lions, and seabirds. It is hoped that the wealth of tag data already being collected worldwide can serve both the needs of the biologic and oceanographic communities alike.

Background/Objectives: Environmentally friendly hydrofluoroalkane (HFA) pressurised metered-dose inhalers are currently being marketed to replace chlorofluorocarbon (CFC)-driven devices. It is uncertain whether these new formulations with different properties are acceptable to patients. Similarly, switching a patient to a dry powder inhaler (DPI) carries the risk of non-acceptance. Methods: One hundred patients with obstructive airway disease on regular CFC aerosol inhaler medication underwent a standardised, structured interview. During the interview patients were asked to use a new HFA aerosol inhaler and three DPIs in random order. Patients’ notions were recorded. Results: Most patients (96) agreed to change from their CFC to the HFA inhaler, of those, only 12 did so with some reservation. Properties (taste, user-friendliness, design) of the HFA inhaler were rated favourably. DPIs represented an acceptable alternative to aerosol inhalers. In fact, 57 patients preferred a DPI over the HFA inhaler. Not all powder devices were equally acceptable. Replacing the CFC inhaler with patients’ preferred alternative devices resulted in a more than 3-fold increase in costs. Conclusion: Concerns about the acceptability of reformulated CFC-free aerosol inhalers are ill founded. However, if given the choice, many patients prefer a DPI over the HFA inhaler. The transition offers an opportunity to review patients’ current treatment and the proficiency of their inhaling technique. Moving to CFC-free inhalers will have revenue implications.

Although most CF patients express CFTR protein (albeit mutant) and should therefore not recognise the wild-type CFTR protein as foreign, there is an inherent risk of activation of T-cells against the recombinant wild-type protein after gene therapy. In addition, we have previously shown that approximately 10% of CF and non-CF subjects carry self-reactive CFTR-specific T-cells ( Calcedo et al , Hum Gene Ther Clin Dev 2013 ). The reason for this is unknown and it is also unclear whether being positive for self-reactive T-cells affects disease severity or increases the risk of further T-cell activation after gene therapy. As part of the UKCFGTC Phase I/IIa Pilot study [in which patients received a single dose (5, 10 or 20 mls) of the non-viral formulation pGM169/GL67A] peripheral blood mononuclear cells (PBMC) were collected prior to dosing and approximately 4 weeks after nebulisation of 5 ml (n = 2), 10 ml (n = 6) or 20 ml (n = 17) of pGM169/GL67A. IFN-g ELISPOT to detect CFTR-specific T-cells in PBMC was performed. CFTR-specific T-cells were detectable in one patient pre- and post-dosing. In the remaining 18 patients we did not detect CFTR-specific T-cells. In addition we quantified anti-DNA antibodies (anti-nuclear and anti-cytoplasmic) in blood samples taken pre- and approximately 4 weeks post-dosing (n = 7 (5 ml), n = 10 (10 ml) and n = 17 (20 ml). We did not observe any evidence for induction of anti-DNA antibodies after a single dose of pGM169/GL67A. The UKCFGTC has now completed a Phase IIb multi-dose clinical trial in May 2014 (ClinicalTrials.gov identification number – NCT01621867). CF patients received 12 monthly doses of pGM169/GL67A (115 completed nine or more doses), or placebo by aerosol. PBMC were collected on two occasions prior to dose 1 to establish baseline levels for CFTR-specific T-cells, approximately 4 weeks after Dose 4 or 5, and 2 to 4 weeks after Dose 12 and the ELISPOT was performed. In addition anti-DNA antibodies were quantified. The Phase IIb trial will be unblinded in Summer 2014 to allow data analysis and all data will be presented at the conference. Funded by the NIHR/EME Programme and the Cystic Fibrosis Trust.

We have used the polymorphic chloroplast (cp) and nuclear simple sequence repeats (SSRs) to analyse levels of cytoplasmic and nuclear diversity in the gene pool of the European cultivated potato (Solanum tuberosum ssp. tuberosum). Primers designed from the complete chloroplast sequence of tobacco (Nicotiana tabacum) were used to amplify polymorphic products in a range of potato cultivars. Combining the data from seven polymorphic cpSSR loci gave 26 haplotypes, one of which (haplotype A) accounted for 151 out of the 178 individuals studied and corresponded to the T-type cytoplasm previously identified in cultivated potatoes using chloroplast restriction fragment length polymorphism analysis. Phylogenetic and diversity analyses of the relationships between cpSSR haplotypes confirmed much higher levels of cytoplasmic diversity outwith the T-type group. Diversity levels at eight nuclear SSR loci, however, were not significantly different between cytoplasmic groups, suggesting a severe maternal bottleneck in the evolution of the modern cultivated potato. These results highlight the importance in quantifying levels of cytoplasmic as well as nuclear diversity and confirm the need for a change in breeding practices to increase levels of non-T-type cytoplasm in the cultivated gene pool, thus helping reduce problems associated with pollen sterility. This may be facilitated by germplasm analysis using cpSSRs, which will allow efficient selection of diverse cytoplasm donors.

The structure-activity relationships of some novel coronary dilator derivatives of palmitoyl carnitine in the rat isolated perfused heart are described. It has been shown previously that esterification of palmitoyl carnitine changes the activity of the compound from a coronary constrictor to a coronary dilator. In this study, it was found that the ester group is not a necessary requirement for coronary dilator activity, but only the absence of the negatively charged carboxylic acid group of palmitoyl carnitine, as compounds containing an ethyl group in place of the ester group were also active coronary dilators. Furthermore, substituting the methyl groups attached to the nitrogen atom of the molecule profoundly altered coronary dilator activity. A quaternary ammonium group was a necessary requirement for potent coronary dilator activity.

AIM: To investigate all patients referred to our center with non-responsive celiac disease (NRCD), to establish a cause for their continued symptoms. METHODS: We assessed all patients referred to our center with non-responsive celiac disease over an 18-mo period. These individuals were investigated to establish the eitiology of their continued symptoms. The patients were first seen in clinic where a thorough history and examination were performed with routine blood work including tissue transglutaminase antibody measurement. They were also referred to a specialist gastroenterology dietician to try to identift any lapses in the diet and sources of hidden gluten ingestion. A repeat small intestinal biopsy was also performed and compared to biopsies from the referring hospital where possible. Colonoscopy, lactulose hydrogen breath testing, pancreolauryl testing and computed tomography scan of the abdomen were undertaken if the symptoms persisted. Their clinical progress was followed over a minimum of 2 years. RESULTS: One hundred and twelve consecutive patients were referred with NRCD. Twelve were found not to have celiac disease (CD). Of the remaining 100 patients, 45% were not adequately adhering to a strict gluten-free diet, with 24 (53%) found to be inadvertently ingesting gluten, and 21 (47%) admitting non-compliance. Microscopic colitis was diagnosed in 12% and small bowel bacterial overgrowth in 9%. Refractory CD was diagnosed in 9%. Three of these were diagnosed with intestinal lymphoma. After 2 years, 78 patients remained well, eight had continuing symptoms, and four had died. CONCLUSION: In individuals with NRCD, a remediable cause can be found in 90%: with continued gluten ingestion as the leading cause. We propose an algorithm for investigation.

The 400 MHz 1H NMR spectrum of captopril in a variety of solvents is analysed and compared with those of epicaptopril and its disulphide analogue. A method for detecting isomeric and oxidative impurities by examination of a 1H NMR spectrum of captopril in DMSO-d6 is proposed. 13C NMR and MS data enable differentiation of captopril from its disulphide analogue but not from its diastereoisomer epicaptopril.

The vasodilator action of the isopropyl ester of palmitoyl carnitine (P1Pi) has been examined in perfused rat hearts and mesenteric vessels. The coronary vasodilator effect P1Pi was not significantly inhibited by flurbiprofen (10 microM), BW755C (10 microM), glibenclamide (10 microM) or the bradykinin B2 receptor antagonist D-Arg0[Hyp3,Thi5,8,D-Phe7]bradykinin (1 microM), indicating that the action of P1Pi is not mediated via arachidonic acid metabolites, ATP-dependent K+ channels or bradykinin B2 receptors. L-NG-Nitro arginine (100 microM) did not inhibit the vasodilator action of P1Pi whilst superoxide dismutase (20 and 50 U.ml-1) attenuated its vasodilator action. Methylene blue (10 microM) caused inhibition in three out of four hearts, while haemoglobin (1 microM) caused an irreversible inhibition of the action of P1Pi which was associated with a depression of myocardial contractility. In air-damaged mesenteric vascular beds the vasodilator action of P1Pi was not attenuated, whilst that of acetylcholine was abolished. In K(+)-depolarised mesenteric vascular beds the constrictor action of Ca2+ was attenuated by P1Pi. Therefore the vasodilator effect of P1Pi appears to be the result of a direct effect on smooth muscle.

Introduction and Objectives The “Run-In” study is an on-going longitudinal, observational study of CF patients assessing outcome measures for a future gene therapy trial. Spirometry is performed at each visit and volumes are converted to % predicted values according to published reference equations; historically these were separate for adults and children. Here, we describe the issues arising from this approach, and highlight the benefit of using a reference source which bridges the transition from child to adulthood. Methods CF subjects (=10 years; FEV 1 =40% predicted) were recruited from three sites in London and Edinburgh. Visits were undertaken during periods of stability every 3–6 months; data presented here are from the first four visits. Spirometry was performed on an Easyone spirometer. Volumes were converted to % predicted values according to Rosenthal ( 1 raw data were subsequently re-analysed using Stanojevic reference equations, which span all age ranges. Comparisons were made using paired t-tests. Results 191 patients attended visit 1 (mean age 22.7 years, 55% male; 91 patients 1 % predicted values were significantly higher than the Stanojevic values: mean differences 2.8 (95% CI 1.9 to 3.7) for children with Rosenthal equations (p 1 % over visits 1–4 was significantly greater with Rosenthal/Quanjer references than with Stanojevic (p=0.001) largely due to an artefactual drop when switching from Rosenthal to Quanjer values. As an example, a female patient aged 17.8 years at visit 1 had a drop in absolute FEV 1 % predicted between visits 1 and 2 of 11% when Rosenthal/Quanjer were used but only 3% with Stanojevic reference values. Conclusions Our results highlight issues raised when separate adult and paediatric spirometry reference ranges are used in longitudinal study. The UK CF Gene Therapy Consortium has adopted the Stanojevic reference source for all spirometry analysis in its ongoing Clinical Programme.

Large prolactin-secreting pituitary adenomas with suprasellar extension are often complicated by visual field defects, for which surgical decompression is the accepted treatment. However, surgical management of large prolactinomas is often not curative. This report describes a group of six unselected male patients who presented with visual field defects and reduced visual acuity due to large pituitary tumours with suprasellar extension. All six patients also had loss of libido and/or impotence. A rapid serum prolactin estimation enabled the diagnosis of prolactinoma to be made, and CT revealed a large pituitary adenoma with suprasellar extension. The patients were treated with bromocriptine, in doses increasing from 2.5 to 20 mg daily, as the sole therapy. Symptoms were relieved and serum prolactin levels were restored to normal or near normal; visual field defects resolved and visual acuity recovered in all patients. A repeat CT showed evidence of tumour shrinkage especially of the suprasellar extension, in all the patients. A diagnosis of prolactinoma should always be considered in a patient with a large pituitary tumour. The clinical history and a rapid prolactin assay will confirm the diagnosis. Treatment with bromocriptine leads to rapid improvement in perimetry and visual acuity as well as tumour shrinkage, obviating the need for pituitary surgery.

Chiral cholinergic ligands with actions characteristic of nicotine rather than muscarine (e.g., effects blocked by hexamethonium but not by atropine) have already been encountered in preceding sections of Chapter 8. These include α-methylacetylcholine and muscarone, and a more general account of such agents is now presented. Interpretations of their stereostructure-activity relationships is made difficult by evidence that nicotinic agents often function indirectly by promoting the presynaptic release of ACh,(1–3) a feature of the action of certain muscarinic agents as well.(4) Thus nicotine itself has been classified as having indirect cholinergic action at guinea-pig ileum sites.(1) Tissue incubated with hemicholinium (which inhibits the synthesis of ACh(5) for 15 min) showed a 70–100% reduction in its response to the spasmogenic action of nicotine.

Although 5-hydroxytryptamine (1; 5-HT, serotonin) was detected in the mammalian CNS more than 40 years ago,(1) advances in the understanding of central serotonergic mechanisms lapsed behind those of other neurotransmitters, due chiefly to the relative lack of compounds with selectivity for 5-HT receptors. Hence stereochemical study of chiral 5-HT ligands has been limited, with the notable exception of investigations of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) described later. Interest in 5-HT receptors and their ligands has risen dramatically during the past decade and several valuable reviews on the topic are available.(2–7) The detection and subtype classification of 5-HT receptors rests heavily on the results of binding experiments, briefly outlined here. From work with [3H]5-HT, Peroutka and Snyder(8) proposed the existence of two major populations of central 5-HT binding sites, namely, 5-H1 sites labeled with high affinity by the natural ligand and 5-HT2 sites labeled (front cortex especially) by [3H]spiperidone. The ability of the DA-receptor ligand spiperidone to label 5-HT sites was originally reported by Leysen et al. (9) The further classification of 5-HT1 receptors followed observation that a portion of the binding of [3H]5-HT to brain receptor membranes was displaceable by low concentrations of spiperidone—such sites were subsequently termed 5-HT1A while sites insensitive to spiperidone were termed 5-HT1B.

It is assumed that most readers of this book will already be familiar with stereochemical principles and will have knowledge of pharmacological concepts and methods. Nevertheless, a summary of basic principles of these kinds is held to form an appropriate and useful starting point for the main topic of the book. The purpose of this summary is to serve as an “aide-memoire,” to update prior knowledge, and to direct attention to stereochemical aspects which recur throughout the main text. Material presented relates, on the one hand, to the molecular geometry of organic structures and, on the other, to pharmacological methodology.

Dopamine (DA) 1 is an achiral molecule and thus presents a case akin to those of acetylcholine and histamine discussed in later chapters in which a pair of pharmacophores are separated by a bimethylene unit. Its receptors, nevertheless, display marked degrees of stereoselectivity toward chiral dopaminergic ligands of both agonist and antagonist character as reviewed by Cannon(1) and a Danish group,(2) respectively, and briefly by Baindur and Neumeyer.(3) The dopamine system has much in common with that of adrenergic agents, including features of in vivo synthesis, metabolism, uptake and release, and interaction with adenylate cyclase.

We are conducting a large, longitudinal study to assess outcome measures and identify optimal patients for a multidose trial of CF gene therapy. Two imaging modalities are being employed: radioisotope deposition scans and high resolution CT. Subjects have undergone these scans on a single occasion, whilst clinically stable. The purpose was: Deposition scan —to determine which patients would be most optimal for topical drug delivery and CT —to assess the suitability of various parameters as efficacy measures. Following inhalation of 99m Tc-labelled human serum albumin, planar gamma camera images and SPECT were used to assess 3-D deposition. Images were scored both digitally and visually (I- no defects; II- patchy deposition; III- patchy deposition with large defects; IV- grossly abnormal). HRCT scans were scored by two radiologists on a lobar basis for the following: bronchiectasis (extent/severity), airway wall thickening, mucus plugging and gas trapping. 147 deposition scan were available; digital indices (DI) ranged from 34 (best) to 150 (severely abnormal). Visual scores correlated well with DI (R2 0.63; p 1 % (p 1 of 43.9(4.3)%, significantly lower than groups I-III (p

The measurement of cocaine and its major metabolites has been achieved by an HPLC method that compensates for their different solubilities and detection properties. Although ecgonine methyl ester is a major metabolite it is generally not measured by HPLC because it is poorly detectable by UV, and its water solubility makes recovery from urine difficult. Using modified solid-phase extraction procedures recoveries of 85% for ecgonine methyl ester, 97% for cocaine, 106% for benzoylecgonine and 80% for ethylcocaine have been obtained from urine. Increased chromatographic retention and detection sensitivity has been obtained by formation of the t-butyldimethylsilyl derivative of ecgonine methyl ester which was found to be stable in the HPLC mobile phase for at least 1 week. Alkylation of norcocaine and benzoylecgonine has improved their detection sensitivity and also chromatographic resolution. All calibrations were linear over the range 200-1000 ng ml-1 in urine with correlation coefficients > 0.99.