Matthew Leighton

Multiple epiphyseal dysplasia mutations in MATN3 cause misfolding of the A-domain and prevent secretion of mutant matrilin-3

Multiple epiphyseal dysplasia (MED) is a relatively common skeletal dysplasia that can present in childhood with a variable phenotype of short stature and pain and stiffness in the large joints, and often progresses to early‐onset osteoarthritis in adulthood. Mutations in the matrilin‐3 gene (MATN3) have recently been shown to underlie some forms of autosomal dominant MED. To date all MED mutations in matrilin‐3 cluster in the single A‐domain, suggesting that they may disrupt the structure and/or function of this important domain. To determine the effects of MATN3 mutations on the structure and function of matrilin‐3 we expressed both normal and mutant matrilin‐3 in mammalian cells. Wild‐type (wt) matrilin‐3 was efficiently secreted into conditioned medium, whereas mutant matrilin‐3 was retained and accumulated within the cell. Furthermore, when the mutant A‐domains were examined individually, they existed primarily in an unfolded conformation. Co‐immunoprecipitation experiments demonstrated that the mutant A‐domains were specifically associated with ERp72, a chaperone protein known to be involved in mediating disulfide bond formation. Light microscopy of cartilage from an MED patient with a MATN3 mutation showed the presence of intracellular material within the chondrocytes, whilst the overall matrix appeared normal. On electron micrographs, the inclusions noted at the light microscopy level appeared to be dilated cisternae of rough endoplasmic reticulum and immunohistochemical analysis confirmed that the retained protein was matrilin‐3. In summary, the data presented in this paper suggest that MED caused by MATN3 mutations is the result of an intracellular retention of the mutant protein. Hum Mutat 26(6), 557–565, 2005.

A mechanistic multicentre, parallel group, randomised placebo-controlled trial of mesalazine for the treatment of IBS with diarrhoea (IBS-D)

Introduction Immune activation has been reported in the mucosa of IBS patients with diarrhoea (IBS-D), and some small studies have suggested that mesalazine may reduce symptoms. We performed a double-blind, randomised placebo-controlled trial of 2 g mesalazine twice daily versus placebo for 3 months in patients with Rome III criteria IBS-D. Primary outcome was daily average stool frequency during weeks 11–12; secondary outcomes were abdominal pain, stool consistency, urgency and satisfactory relief of IBS symptoms. Methods Participants were randomised after a 2-week baseline stool diary. All participants completed a 12-week stool diary and at the end of each week recorded the presence of ‘satisfactory relief of IBS symptoms’. Results 136 patients with IBS-D (82 women, 54 men) were randomised, 10 patients withdrew from each group. Analysis by intention to treat showed the daily average stool frequency during weeks 11 and 12 were mean (SD), 2.8 (1.2) in mesalazine and 2.7 (1.9) in the placebo group with no significant group difference, (95% CI) 0.1 (−0.33 to 0.53), p=0.66. Mesalazine did not improve abdominal pain, stool consistency nor percentage with satisfactory relief compared with placebo during the last two-weeks follow-up. Conclusions This study does not support any clinically meaningful benefit or harm of mesalazine compared with placebo in unselected patients with IBS-D. More precise subtyping based on underlying disease mechanisms is needed to allow more effective targeting of treatment in IBS. Trial registration number NCT01316718.

A mechanistic multi-centre, parallel group, randomised placebo controlled trial of Mesalazine for treatment of irritable bowel syndrome with diarrhoea (IBS-D)

Introduction Immune activation has been reported in the mucosa of IBS patients with diarrhoea (IBS-D), and some small studies have suggested that mesalazine may reduce symptoms. We performed a double-blind, randomised placebo-controlled trial of 2 g mesalazine twice daily versus placebo for 3 months in patients with Rome III criteria IBS-D. Primary outcome was daily average stool frequency during weeks 11–12; secondary outcomes were abdominal pain, stool consistency, urgency and satisfactory relief of IBS symptoms. Methods Participants were randomised after a 2-week baseline stool diary. All participants completed a 12-week stool diary and at the end of each week recorded the presence of ‘satisfactory relief of IBS symptoms’. Results 136 patients with IBS-D (82 women, 54 men) were randomised, 10 patients withdrew from each group. Analysis by intention to treat showed the daily average stool frequency during weeks 11 and 12 were mean (SD), 2.8 (1.2) in mesalazine and 2.7 (1.9) in the placebo group with no significant group difference, (95% CI) 0.1 (−0.33 to 0.53), p=0.66. Mesalazine did not improve abdominal pain, stool consistency nor percentage with satisfactory relief compared with placebo during the last two-weeks follow-up. Conclusions This study does not support any clinically meaningful benefit or harm of mesalazine compared with placebo in unselected patients with IBS-D. More precise subtyping based on underlying disease mechanisms is needed to allow more effective targeting of treatment in IBS. Trial registration number NCT01316718.

An NMR and enzyme study of the carbon metabolism of Neisseria meningitidis

The pathogenic neisseriae are fastidious bacteria that are only able to grow on a restricted range of carbon sources. The genome sequence of Neisseria meningitidis strain MC58 predicts the presence of a complete citric acid cycle (CAC), but there have been no detailed biochemical studies of carbon metabolism in this important pathogen. In this study, both NMR and conventional enzyme assays were used to investigate the central metabolic pathways of a serogroup B strain (K454). (13)C-NMR labelling patterns of amino acids from hydrolysed cell proteins after growth with either 2- or 3-[(13)C]pyruvate were consistent with the operation of a complete oxidative CAC. Enzyme assays showed that cell-free extracts contained all the CAC enzymes predicted from the genome sequence, including a membrane-bound malate:quinone oxidoreductase which is present in place of the conventional NAD-linked cytoplasmic malate dehydrogenase. (1)H-NMR studies showed that growth on glucose, lactate and, especially, pyruvate, resulted in the excretion of significant amounts of acetate into the culture supernatant. This occurred via the phosphotransacetylase (PTA)-acetate kinase (ACK) pathway. Extremely high specific activities of PTA (7-14 micromol min(-1) mg(-1)) were detected in cell-free extracts, although ACK activities were much lower (46-298 nmol min(-1) mg(-1)). Expression of PTA and ACK activities was not co-ordinately regulated during growth on combinations of carbon sources. This may be related to the presence of two ackA paralogues in N. meningitidis which are, unusually, unlinked to the pta gene.

A novel form of chondrocyte stress is triggered by a COMP mutation causing pseudoachondroplasia

Pseudoachondroplasia (PSACH) results from mutations in cartilage oligomeric matrix protein (COMP) and the p.D469del mutation within the type III repeats of COMP accounts for approximately 30% of PSACH. To determine disease mechanisms of PSACH in vivo, we introduced the Comp D469del mutation into the mouse genome. Mutant animals were normal at birth but grew slower than their wild‐type littermates and developed short‐limb dwarfism. In the growth plates of mutant mice chondrocyte columns were reduced in number and poorly organized, while mutant COMP was retained within the endoplasmic reticulum (ER) of cells. Chondrocyte proliferation was reduced and apoptosis was both increased and spatially dysregulated. Previous studies on COMP mutations have shown mutant COMP is co‐localized with chaperone proteins, and we have reported an unfolded protein response (UPR) in mouse models of PSACH‐MED (multiple epiphyseal dysplasia) harboring mutations in Comp (T585M) and Matn3, Comp etc (V194D). However, we found no evidence of UPR in this mouse model of PSACH. In contrast, microarray analysis identified expression changes in groups of genes implicated in oxidative stress, cell cycle regulation, and apoptosis, which is consistent with the chondrocyte pathology. Overall, these data suggest that a novel form of chondrocyte stress triggered by the expression of mutant COMP is central to the pathogenesis of PSACH. Hum Mutat 33:218–231, 2012.

Large multi-centre pilot randomized controlled trial testing a low-cost, tailored, self-help smoking cessation text message intervention for pregnant smokers (MiQuit).

Abstract Aims To estimate the effectiveness of pregnancy smoking cessation support delivered by short message service (SMS) text message and key parameters needed to plan a definitive trial. Design Multi‐centre, parallel‐group, single‐blinded, individual randomized controlled trial. Setting Sixteen antenatal clinics in England. Participants Four hundred and seven participants were randomized to the intervention (n = 203) or usual care (n = 204). Eligible women were < 25 weeks gestation, smoked at least one daily cigarette (> 5 pre‐pregnancy), were able to receive and understand English SMS texts and were not already using text‐based cessation support. Intervention All participants received a smoking cessation leaflet; intervention participants also received a 12‐week programme of individually tailored, automated, interactive, self‐help smoking cessation text messages (MiQuit). Outcome measurements Seven smoking outcomes, including validated continuous abstinence from 4 weeks post‐randomization until 36 weeks gestation, design parameters for a future trial and cost‐per‐quitter. Findings Using the validated, continuous abstinence outcome, 5.4% (11 of 203) of MiQuit participants were abstinent versus 2.0% (four of 204) of usual care participants [odds ratio (OR) = 2.7, 95% confidence interval (CI) = 0.93–9.35]. The Bayes factor for this outcome was 2.23. Completeness of follow‐up at 36 weeks gestation was similar in both groups; provision of self‐report smoking data was 64% (MiQuit) and 65% (usual care) and abstinence validation rates were 56% (MiQuit) and 61% (usual care). The incremental cost‐per‐quitter was £133.53 (95% CI = –£395.78 to 843.62). Conclusions There was some evidence, although not conclusive, that a text‐messaging programme may increase cessation rates in pregnant smokers when provided alongside routine NHS cessation care.

Efficacy and mode of action of mesalazine in the treatment of diarrhoea-predominant irritable bowel syndrome (IBS-D): study protocol for a randomised controlled trial

BackgroundIrritable bowel syndrome (IBS) is reported by one in ten of the population accounting for up to 40% of new referrals to gastroenterology outpatients. Patients characteristically have abdominal discomfort and disturbed bowel habit. Diarrhoea-predominant IBS is characterised by frequent loose stools with associated urgency and abdominal cramps. Current symptomatic treatments can reduce bowel frequency but often fail to reduce discomfort.Mesalazine is an anti-inflammatory drug used to treat patients with inflammatory bowel disease. There is one pilot study suggesting it may be beneficial to patients who have diarrhoea-predominant IBS but these findings need to be confirmed in a larger trial. The current study aims to test the effectiveness of mesalazine to reduce symptoms in diarrhoea-predominant IBS patients. The study will also investigate the mode of action of the drug, especially its impact on mast cell activation.Methods/designThis is a multicentre randomised, double-blind, placebo-controlled trial using a parallel group design. At least 108 participants with diarrhoea-predominant IBS will be recruited through at least six hospitals. The intervention is a 12-week course of 2g mesalazine granules taken up to twice a day. The comparator is a blinded placebo granule formulation.Outcome measures include stool diaries, symptom questionnaires, stool and blood samples together with rectal mucosal biopsies. The daily stool diary will record stool frequency and form, urgency, bloating, abdominal pain and a global satisfaction with control of IBS scored each week. The questionnaires will assess bowel symptoms, while the samples and biopsies will be used to analyse underlying mechanisms of any response.Primary outcome will be the average stool frequency during weeks 11 and 12 of the treatment period and will be compared between treatment arms using an analysis of covariance in the form of a general linear model incorporating baseline characteristics that are thought a priori to strongly predict outcome. The primary efficacy parameter will be the difference in mean frequency between treatment arms.DiscussionThis report describes a randomised controlled trial that will provide evidence of any benefit of treating diarrhoea-predominant IBS patients with mesalazine. The results will be available toward the end of 2013.Trial registrationISRCTN76612274