Babies born before 28 weeks’ gestation have lower plasma thyroid hormone concentrations than more mature infants. This may contribute to their risk of poor developmental outcome. Previous studies have suggested that thyroxine supplementation for extremely preterm neonates may be beneficial. The aim of this study was to investigate the effect of administration of supplemental thyroxine to very premature babies on brain size and somatic growth at 36 weeks’ corrected gestational age (CGA).
In this explanatory multicentre double blind randomised placebo controlled trial, 153 infants born below 28 weeks’ gestation were randomised to levothyroxine (LT4) supplementation or placebo until 32 weeks’ CGA. The primary outcome was brain size assessed by the width of the subarachnoid space measured by cranial ultrasound at 36 weeks’ CGA. Lower leg length was measured by knemometry.
Babies in the LT4-supplemented and placebo groups had similar baseline characteristics. There were no significant differences between infants given LT4 (n=78) or placebo (n=75) for width of the subarachnoid space, head circumference at 36 weeks’ CGA, body weight at 36 weeks’ CGA or mortality. Infants who received LT4 had significantly shorter leg lengths at 36 weeks’ CGA although adjusted analysis for baseline length did not find a statistical difference. There was a significant correlation between low FT4 and wider subarachnoid space. No unexpected serious adverse events were noted and incidence of adverse events did not differ between the two groups.
This is the only randomised controlled trial of thyroxine supplementation targeting extremely premature infants. Supplementing all babies below 28 weeks’ gestation with LT4 had no apparent effect on brain size. These results do not support routine supplementation with LT4 for all babies born below 28 weeks’ gestation.
Current Controlled Trials ISRCTN89493983
EUDRACT number: 2005-003-09939
Thyroxine supplementation; RCT; Extreme preterm; Brain; Growth
At the onset of an influenza pandemic, when the severity of a novel strain is still undetermined and there is a threat of introduction into a new environment, e.g., via the deployment of military troops, sensitive screening criteria and conservative isolation practices are generally recommended.
In response to elevated rates of influenza-like illness among U.S. military base camps in Kuwait, U.S. Naval Medical Research Unit No. 3 partnered with local U.S. Army medical units to conduct an A(H1N1) pdm09 outbreak investigation.
Initial clinical data and nasal specimens were collected via the existent passive surveillance system and active surveillance was conducted using a modified version of the World Health Organization/U.S. Centers for Disease Control and Prevention influenza-like illness case definition [fever (T > 100.5˚F/38˚C) in addition to cough and/or sore throat in the previous 72 hours] as the screening criteria. Samples were tested via real-time reverse-transcription PCR and sequenced for comparison to global A(H1N1) pdm09 viruses from the same time period.
The screening criteria used in Kuwait proved insensitive, capturing only 16% of A(H1N1) pdm09-positive individuals. While still not ideal, using cough as the sole screening criteria would have increased sensitivity to 73%.
The results of and lessons learned from this outbreak investigation suggest that pandemic influenza risk management should be a dynamic process (as information becomes available regarding true attack rates and associated mortality, screening and isolation criteria should be re-evaluated and revised as appropriate), and that military operational environments present unique challenges to influenza surveillance.
The purpose of this study was to examine the efficacy of introducing a fish protein hydrolysate (PEP) concurrently with carbohydrate (CHO) and whey protein (PRO) on endurance exercise metabolism and performance.
In a randomised, double blind crossover design, 12 male volunteers completed an initial familiarisation followed by three experimental trials. The trials consisted of a 90 min cycle task corresponding to 50% of predetermined maximum power output, followed by a 5 km time trial (TT). At 15 min intervals during the 90 min cycle task, participants consumed 180 ml of CHO (67 g.hr-1 of maltodextrin), CHO-PRO (53.1 g.hr of CHO, 13.6 g.hr-1 of whey protein) or CHO-PRO-PEP (53.1 g.hr-1 of CHO, 11 g.hr-1 of whey protein and 2.4 g.hr-1of hydrolyzed marine peptides).
Results and conclusions
During the 90 min cycle task, the respiratory exchange ratio (RER) in the CHO-PRO condition was significantly higher than CHO (p < 0.001) and CHO-PRO-PEP (p < 0.001). Additionally, mean heart rate for the CHO condition was significantly lower than that for CHO-PRO (p = 0.021). Time-to-complete the 5 km TT was not significantly different between conditions (m ± SD: 456 ± 16, 456 ± 18 and 455 ± 21 sec for CHO, CHO-PRO and CHO-PRO-PEP respectively, p = 0.98). Although the addition of hydrolyzed marine peptides appeared to influence metabolism during endurance exercise in the current study, it did not provide an ergogenic benefit as assessed by 5 km TT performance.
Marine peptide; Hydrolyzed protein; Exercise metabolism
A lack of age-appropriate formulations can make it difficult to administer medicines to children. A manipulation of the dosage form may be required to achieve the required dose. This study aimed to describe medicines that are manipulated to achieve the required dose in paediatric practice.
A structured, undisguised observational study and postal survey. The observational study investigated drug manipulations occurring in clinical practice across three sites. The questionnaire, administered to a sample of paediatric nurses throughout the UK, surveyed manipulations conducted and nurses’ experiences and views.
The observational study identified 310 manipulations, of which 62% involved tablets, 21% were intravenous drugs and 10% were sachets. Of the 54 observed manipulations 40 involved tablets with 65% of the tablets being cut and 30% dispersed to obtain a smaller dose. 188 manipulations were reported by questionnaire respondents, of these 46% involved tablets, 12% were intravenous drugs, and 12% were nebuliser solutions. Manipulations were predominantly, but not exclusively, identified in specialist clinical areas with more highly dependent patients. Questionnaire respondents were concerned about the accuracy of the dose achieved following manipulations and the lack of practice guidance.
Manipulations to achieve the required dose occur throughout paediatric in-patient settings. The impact of manipulations on the efficacy of the drugs, the accuracy of the dose and any adverse effects on patients is not known. There is a need to develop evidence-based guidance for manipulations of medicines in children.
Drug manipulation; Survey; Dosage forms; Children's medicines
During construction of several gene deletion mutants in Lactococcus lactis MG1363 which involved a high-temperature (37.5°C) incubation step, additional spontaneous mutations were observed which resulted in stable heat resistance and in some cases salt-hypersensitive phenotypes. Whole-genome sequencing of one strain which was both heat resistant and salt hypersensitive, followed by PCR and sequencing of four other mutants which shared these phenotypes, revealed independent mutations in llmg_1816 in all cases. This gene encodes a membrane-bound stress signaling protein of the GdpP family, members of which exhibit cyclic dimeric AMP (c-di-AMP)-specific phosphodiesterase activity. Mutations were predicted to lead to single amino acid substitutions or protein truncations. An independent llmg_1816 mutant (Δ1816), created using a suicide vector, also displayed heat resistance and salt hypersensitivity phenotypes which could be restored to wild-type levels following plasmid excision. L. lactis Δ1816 also displayed improved growth in response to sublethal concentrations of penicillin G. High-temperature incubation of a wild-type industrial L. lactis strain also resulted in spontaneous mutation of llmg_1816 and heat-resistant and salt-hypersensitive phenotypes, suggesting that this is not a strain-specific phenomenon and that it is independent of a plasmid integration event. Acidification of milk by the llmg_1816-altered strain was inhibited by lower salt concentrations than the parent strain. This study demonstrates that spontaneous mutations can occur during high-temperature growth of L. lactis and that inactivation of llmg_1816 leads to temperature resistance and salt hypersensitivity.
Spores of thermophilic Geobacillus species are a common contaminant of milk powder worldwide due to their ability to form biofilms within processing plants. Genotyping methods can provide information regarding the source and monitoring of contamination. A new genotyping method was developed based on multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) in conjunction with high-resolution melt analysis (MLV-HRMA) and compared to the currently used method, randomized amplified polymorphic DNA PCR (RAPD-PCR). Four VNTR loci were identified and used to genotype 46 Geobacillus isolates obtained from retailed powder and samples from 2 different milk powder processing plants. These 46 isolates were differentiated into 16 different groups using MLV-HRMA (D = 0.89). In contrast, only 13 RAPD-PCR genotypes were identified among the 46 isolates (D = 0.79). This new method was then used to analyze 35 isolates obtained from powders with high spore counts (>104 spores · g−1) from a single processing plant together with 27 historical isolates obtained from powder samples processed in the same region of Australia 17 years ago. Results showed that three genotypes can coexist in a single processing run, while the same genotypes observed 17 years ago are present today. While certain genotypes could be responsible for powders with high spore counts, there was no correlation to specific genotypes being present in powder plants and retailed samples. In conclusion, the MLV-HRMA method is useful for genotyping Geobacillus spp. to provide insight into the prevalence and persistence of certain genotypes within milk powder processing plants.
To obtain reliable information about the incidence of adverse drug reactions, and identify potential areas where intervention may reduce the burden of ill-health.
Prospective observational study.
A large tertiary children’s hospital providing general and specialty care in the UK.
All acute paediatric admissions over a one year period.
Any medication taken in the two weeks prior to admission.
Occurrence of adverse drug reaction.
240/8345 admissions in 178/6821 patients admitted acutely to a paediatric hospital were thought to be related to an adverse drug reaction, giving an estimated incidence of 2.9% (95% CI 2.5, 3.3), with the reaction directly causing, or contributing to the cause, of admission in 97.1% of cases. No deaths were attributable to an adverse drug reaction. 22.1% (95% CI 17%, 28%) of the reactions were either definitely or possibly avoidable. Prescriptions originating in the community accounted for 44/249 (17.7%) of adverse drug reactions, the remainder originating from hospital. 120/249 (48.2%) reactions resulted from treatment for malignancies. The drugs most commonly implicated in causing admissions were cytotoxic agents, corticosteroids, non-steroidal anti-inflammatory drugs, vaccines and immunosuppressants. The most common reactions were neutropenia, immunosuppression and thrombocytopenia.
Adverse drug reactions in children are an important public health problem. Most of those serious enough to require hospital admission are due to hospital-based prescribing, of which just over a fifth may be avoidable. Strategies to reduce the burden of ill-health from adverse drug reactions causing admission are needed.
There is little research on parents' experiences of suspected adverse drug reactions in their children and hence little evidence to guide clinicians when communicating with families about problems associated with medicines.
To identify any unmet information and communication needs described by parents whose child had a suspected adverse drug reaction.
Semi-structured qualitative interviews with parents of 44 children who had a suspected adverse drug reaction identified on hospital admission, during in-patient treatment or reported by parents using the Yellow Card Scheme (the UK system for collecting spontaneous reports of adverse drug reactions). Interviews were conducted face-to-face or by telephone; most interviews were audiorecorded and transcribed. Analysis was informed by the principles of the constant comparative method.
Many parents described being dissatisfied with how clinicians communicated about adverse drug reactions and unclear about the implications for their child's future use of medicines. A few parents felt that clinicians had abandoned their child and reported refusing the use of further medicines because they feared a repeated adverse drug reaction. The accounts of parents of children with cancer were different. They emphasised their confidence in clinicians' management of adverse drug reactions and described how clinicians prospectively explained the risks associated with medicines. Parents linked symptoms to medicines in ways that resembled the established reasoning that clinicians use to evaluate the possibility that a medicine has caused an adverse drug reaction.
Clinicians' communication about adverse drug reactions was poor from the perspective of parents, indicating that improvements are needed. The accounts of parents of children with cancer indicate that prospective explanation about adverse drug reactions at the time of prescription can be effective. Convergence between parents and clinicians in their reasoning for linking children's symptoms to medicines could be a starting point for improved communication.
To describe the use of ciprofloxacin and fluconazole for the treatment of sepsis in European neonatal intensive care units (NICUs) in order to better orient research aimed at acquiring essential knowledge in this critical area.
The survey consisted of an online questionnaire for all participating NICUs on treatment schemes employed, rationales behind drug choices and interest in participation in research involving the two drugs.
A total of 189 level II and III NICUs participated in the survey, representing 25 countries, with Italy, UK and France providing the greatest number of centres (54 % of total). Ciprofloxacin is used in 25 % of NICUs that responded, although the indications for administering it vary between centres and the dosage ranges vary considerably, with 25 % of NICUs giving ≤10 mg/kg/day and another 25 % giving ≥21 mg/kg/day. Factors given as affecting the decision to use ciprofloxacin are uncertainty about its safety and pharmacokinetics and level of penetration in the cerebrospinal fluid. Among the 70 % of responding units that use fluconazole to treat fungal infection, 45 % administer 6 mg/kg unit doses while 33 % administer 12 mg/kg; 41 % of NICUs use a 24-h interval between administrations while 20 % wait 72 h. Among the responding NICUs, 57 % were willing to participate in a project on ciprofloxacin and 59 % would consider participating in a randomized controlled trial evaluating fluconazole versus micafungin.
Great variability in therapies exists within and between countries. Numerous centres are interested in participating in research on these drugs, highlighting the need for further knowledge on sepsis treatment and European centres’ interest in off-patent medicine research.
Data collection; Intensive care units, neonatal; Sepsis; Ciprofloxacin; Fluconazole
Information on the neonatal exposure to excipients is limited. Our aim was to describe the extent of excipient intake by Estonian neonates; to classify the excipients according to potential neonatal toxicity and thereby to measure the extent of exposure of neonates to potentially harmful excipients.
A prospective cohort study that recorded all medicines prescribed to patients aged below 28 days admitted to Tartu University Hospital from 01.02-01.08 2008 and to Tallinn Children’s Hospital from 01.02- 01.08 2009 was conducted. Excipients were identified from Summaries of Product Characteristics and classified according to toxicity following a literature review.
1961 prescriptions comprising 107 medicines were written for 348/490 neonates admitted. A total of 123 excipients were found in 1620 (83%) prescriptions and 93 (87%) medicines. 47 (38%) of these excipients were classified as potentially or known to be harmful to neonates. Most neonates (97%) received at least one medicine (median number 2) with potentially or known to be harmful excipient. Parabens were the most commonly used known to be harmful excipients and sodium metabisulphite the most commonly used potentially harmful excipient, received by 343 (99%) and 297 (85%) of treated neonates, respectively.
Hospitalised neonates in Estonia are commonly receiving a wide range of excipients with their medication. Quantitative information about excipients should be made available to pharmacists and neonatologists helping them to take into account excipient issues when selecting medicines and to monitor for adverse effects if administration of medicines containing excipients is unavoidable.
Harmful excipient; Neonate
Premature infants are frequently exposed to aminoglycoside antibiotics. Novel urinary biomarkers may provide a non-invasive means for the early identification of aminoglycoside-related proximal tubule renal toxicity, to enable adjustment of treatment and identification of infants at risk of long-term renal impairment. In this proof-of-concept study, urine samples were collected from 41 premature neonates (≤32 weeks gestation) at least once per week, and daily during courses of gentamicin, and for 3 days afterwards. Significant increases were observed in the three urinary biomarkers measured (Kidney Injury Molecule-1 (KIM-1), Neutrophil Gelatinase-associated Lipocalin (NGAL), and N-acetyl-β-D-glucosaminidase (NAG)) during treatment with multiple courses of gentamicin. When adjusted for potential confounders, the treatment effect of gentamicin remained significant only for KIM-1 (mean difference from not treated, 1.35 ng/mg urinary creatinine; 95% CI 0.05–2.65). Our study shows that (a) it is possible to collect serial urine samples from premature neonates, and that (b) proximal tubule specific urinary biomarkers can act as indicators of aminoglycoside-associated nephrotoxicity in this age group. Further studies to investigate the clinical utility of novel urinary biomarkers in comparison to serum creatinine need to be undertaken.
To develop and test a new adverse drug reaction (ADR) causality assessment tool (CAT).
A comparison between seven assessors of a new CAT, formulated by an expert focus group, compared with the Naranjo CAT in 80 cases from a prospective observational study and 37 published ADR case reports (819 causality assessments in total).
Main Outcome Measures
Utilisation of causality categories, measure of disagreements, inter-rater reliability (IRR).
The Liverpool ADR CAT, using 40 cases from an observational study, showed causality categories of 1 unlikely, 62 possible, 92 probable and 125 definite (1, 62, 92, 125) and ‘moderate’ IRR (kappa 0.48), compared to Naranjo (0, 100, 172, 8) with ‘moderate’ IRR (kappa 0.45). In a further 40 cases, the Liverpool tool (0, 66, 81, 133) showed ‘good’ IRR (kappa 0.6) while Naranjo (1, 90, 185, 4) remained ‘moderate’.
The Liverpool tool assigns the full range of causality categories and shows good IRR. Further assessment by different investigators in different settings is needed to fully assess the utility of this tool.
AIM: To investigate the patient characteristics, relationship between the Logistic EuroSCORE (LES) and the observed outcomes in octogenarians who underwent surgical aortic valve replacement (AVR).
METHODS: Two hundred and seventy three octogenarians underwent AVR between 1996 and 2008 at Bristol Royal Infirmary. Demographics, acute outcomes, length of hospital stay and mortality were obtained. The LES was calculated to characterize the predicted operative risk. Two groups were defined: LES ≥ 15 (n = 80) and LES < 15 (n = 193).
RESULTS: In patients with LES ≥ 15, 30 d mortality was 14% (95% CI: 7%-23%) compared with 4% (95% CI: 2%-8%) in the LES < 15 group (P < 0.007). Despite the increase in number of operations from 1996 to 2008, the average LES did not change. Only 5% of patients had prior bypass surgery. The LES identified a low risk quartile of patients with a very low mortality (4%, n = 8, P < 0.007) at 30 d. The overall surgical results for octogenarians were excellent. The low risk group had an excellent outcome and the high risk group had a poor outcome after surgical AVR.
CONCLUSION: It may be better treated with transcatheter aortic valve implantation.
Aortic valve replacement; Transcatheter aortic valve implantation; Logistic EuroSCORE; Coronary artery bypass grafting
Cytokines are secreted from macrophages and other cells of the immune system in response to pathogens. Additionally, in autoinflammatory diseases cytokine secretion occurs in the absence of pathogenic stimuli. In the case of TRAPS [TNFR (tumour necrosis factor receptor)-associated periodic syndrome], inflammatory episodes result from mutations in the TNFRSF1A gene that encodes TNFR1. This work remains controversial, however, with at least three distinct separate mechanisms of receptor dysfunction having been proposed. Central to these hypotheses are the NF-κB (nuclear factor κB) and MAPK (mitogen-activated protein kinase) families of transcriptional activators that are able to up-regulate expression of a number of genes, including pro-inflammatory cytokines. The present review examines each proposed mechanism of TNFR1 dysfunction, and addresses how these processes might ultimately impact upon cytokine secretion and disease pathophysiology.
mitogen-activated protein kinase (MAPK); nuclear factor κB (NF-κB); periodic syndrome; tumour necrosis factor (TNF); tumour necrosis factor receptor-associated periodic syndrome (TRAPS); ADAM, a disintegrin and metalloproteinase; AP, activator protein; APR, acute phase response; ATF6, activating transcription factor 6; CRD, cysteine-rich domain; CREBH, cAMP-responsive-element-binding protein H; CRP, C-reactive protein; ER, endoplasmic reticulum; IFN, interferon; IRF, IFN-regulatory factor; JNK, c-Jun N-terminal kinase; IL, interleukin; JAK/STAT, Janus kinase/signal transducer and activator of transcription; mAb, monoclonal antibody; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor κB; PBMC, peripheral blood mononuclear cell; ROS, reactive oxygen species; SAP, serum amyloid P; TNF, tumour necrosis factor; TACE, TNFα-converting enzyme; TNFR, TNF receptor; sTNFR1, soluble TNFR1; TRAPS, TNFR-associated periodic syndrome; UPR, unfolded protein response
The consumption of fresh fruit & vegetable in concentrate form (FVC) have recently become an alternative approach to combating excessive renal acid loads often associated with Western Diets. Additionally, these FVC's have been purported to induce metabolic alkalosis, which perhaps may enhance the blood buffering capacity of an individual. Therefore, the aim of this preliminary study was to profile the acid-base response after ingestion of an acute dose of fruit and vegetable extract (Energised Greens™ (EG), Nottingham, UK) and compare it to a standard, low dose (0.1 g·kg-1) of sodium bicarbonate (NaHCO3).
As part of a randomized, cross over design participants consumed 750 mL of water with either 9 g of EG (manufacturer recommendations), 0.1 g·kg-1 of NaHCO3 or a placebo (plain flour) in opaque encapsulated pills following an overnight fast. Capillary samples were obtained and analyzed every 15 min for a period of 120 min following ingestion. Significant interactions (p < 0.01), main effects for condition (p < 0.001) and time (p < 0.001) were evident for all acid-base variables (pH, HCO3-, BE). Interactions indicated significant elevation in blood alkalosis for only the NaHCO3 condition when compared to both placebo and EG from 15 to 120 minutes.
Despite previous findings of elevated blood pH following acute mineral supplementation, manufacturer recommended doses of EG do not induce any significant changes in acid-base regulation in resting males.
Infants born <29 weeks gestation are at high risk of neurocognitive disability. Early postnatal growth failure, particularly head growth, is an important and potentially reversible risk factor for impaired neurodevelopmental outcome. Inadequate nutrition is a major factor in this postnatal growth failure, optimal protein and calorie (macronutrient) intakes are rarely achieved, especially in the first week. Infants <29 weeks are dependent on parenteral nutrition for the bulk of their nutrient needs for the first 2-3 weeks of life to allow gut adaptation to milk digestion. The prescription, formulation and administration of neonatal parenteral nutrition is critical to achieving optimal protein and calorie intake but has received little scientific evaluation. Current neonatal parenteral nutrition regimens often rely on individualised prescription to manage the labile, unpredictable biochemical and metabolic control characteristic of the early neonatal period. Individualised prescription frequently fails to translate into optimal macronutrient delivery. We have previously shown that a standardised, concentrated neonatal parenteral nutrition regimen can optimise macronutrient intake.
We propose a single centre, randomised controlled exploratory trial of two standardised, concentrated neonatal parenteral nutrition regimens comparing a standard macronutrient content (maximum protein 2.8 g/kg/day; lipid 2.8 g/kg/day, dextrose 10%) with a higher macronutrient content (maximum protein 3.8 g/kg/day; lipid 3.8 g/kg/day, dextrose 12%) over the first 28 days of life. 150 infants 24-28 completed weeks gestation and birthweight <1200 g will be recruited. The primary outcome will be head growth velocity in the first 28 days of life. Secondary outcomes will include a) auxological data between birth and 36 weeks corrected gestational age b) actual macronutrient intake in first 28 days c) biomarkers of biochemical and metabolic tolerance d) infection biomarkers and other intravascular line complications e) incidence of major complications of prematurity including mortality f) neurodevelopmental outcome at 2 years corrected gestational age
Current controlled trials: ISRCTN76597892; EudraCT Number: 2008-008899-14
The vulnerability of newborn babies' skin creates the potential for a number of skin problems. Despite this, there remains a dearth of good quality evidence to inform practice. Published studies comparing water with a skin-cleansing product have not provided adequate data to inform an adequately powered trial. Nor have they distinguished between babies with and without a predisposition to atopic eczema. We conducted a pilot study as a prequel to designing an optimum trial to investigate whether bathing with a specific cleansing product is superior to bathing with water alone. The aims were to produce baseline data which would inform decisions for the main trial design (i.e. population, primary outcome, sample size calculation) and to optimize the robustness of trial processes within the study setting.
100 healthy, full term neonates aged <24 hours were randomly assigned to bathing with water and cotton wool (W) or with a cleaning product (CP). A minimum of bathing 3 times per week was advocated. Groups were stratified according to family history of atopic eczema. Transepidermal water loss (TEWL), stratum corneum hydration and skin surface pH were measured within 24 hours of birth and at 4 and 8 weeks post birth. Measurements were taken on the thigh, forearm and abdomen. Women also completed questionnaires and diaries to record bathing practices and medical treatments.
Forty nine babies were randomized to cleansing product, 51 to water. The 95% confidence intervals (CI) for the average TEWL measurement at each time point were: whole sample at baseline: 10.8 g/m2/h to 11.7 g/m2/h; CP group 4 weeks: 10.9 g/m2/h to 13.3 g/m2/h; 8 weeks: 11.4 g/m2/h to 12.9 g/m2/h; W group 4 weeks:10.9 g/m2/h to 12.2 g/m2/h; 8 weeks: 11.4 g/m2/h to 12.9 g/m2/h.
This pilot study provided valuable baseline data and important information on trial processes. The decision to proceed with a superiority trial, for example, was inconsistent with our data; therefore a non-inferiority trial is recommended.
Phosphoinositide 3-kinases (PI3Ks) are critical regulators of pancreatic β cell mass and survival, whereas their involvement in insulin secretion is more controversial. Furthermore, of the different PI3Ks, the class II isoforms were detected in β cells, although their role is still not well understood. Here we show that down-regulation of the class II PI3K isoform PI3K-C2α specifically impairs insulin granule exocytosis in rat insulinoma cells without affecting insulin content, the number of insulin granules at the plasma membrane, or the expression levels of key proteins involved in insulin secretion. Proteolysis of synaptosomal-associated protein of 25 kDa, a process involved in insulin granule exocytosis, is impaired in cells lacking PI3K-C2α. Finally, our data suggest that the mRNA for PI3K-C2α may be down-regulated in islets of Langerhans from type 2 diabetic compared with non-diabetic individuals. Our results reveal a critical role for PI3K-C2α in β cells and suggest that down-regulation of PI3K-C2α may be a feature of type 2 diabetes.
Diabetes; Exocytosis; Insulin Secretion; Phosphatidylinositol 3-Kinase; Signal Transduction
Ischaemic preconditioning results in a reduction in ischaemic‐reperfusion injury to the heart. This beneficial effect is seen both with direct local preconditioning of the myocardium and with remote preconditioning of easily accessible distant non‐vital limb tissue. Ischaemic postconditioning with a comparable sequence of brief periods of local ischaemia, when applied immediately after the ischaemic insult, confers benefits similar to preconditioning.
To test the hypothesis that limb ischaemia induces remote postconditioning and hence reduces experimental myocardial infarct size in a validated swine model of acute myocardial infarction.
Acute myocardial infarction was induced in 24 pigs with 90 min balloon inflations of the left anterior descending coronary artery. Remote ischaemic postconditioning was induced in 12 of the pigs by four 5 min cycles of blood pressure cuff inflation applied to the lower limb immediately after the balloon deflation. Infarct size was assessed by measuring 72 h creatinine kinase release, MRI scan and immunohistochemical analysis.
Area under the curve of creatinine kinase release was significantly reduced in the postconditioning group compared with the control group with a 26% reduction in the infarct size (p<0.05). This was confirmed by MRI scanning and immunohistochemical analysis that revealed a 22% (p<0.05) and a 47.52% (p<0.01) relative reduction in the infarct size, respectively.
Remote ischaemic postconditioning is a simple technique to reduce infarct size without the hazards and logistics of multiple coronary artery balloon inflations. This type of conditioning promises clear clinical potential.
Idiopathic recurrent miscarriage is defined as 3 consecutive pregnancy losses with no contributing features found on investigations. At present there are no treatments of proven efficacy for idiopathic recurrent miscarriage. Uterine natural killer (uNK) cells, the most predominant leucocyte in the endometrium are adjacent to foetal trophoblast cells and thought to be involved in implantation. The exact mechanisms of how uNK cells affect implantation are not clear but are probably through the regulation of angiogenesis. Multiple studies have shown an association between high density of uterine natural killer cells and recurrent miscarriage. We have shown that prednisolone reduces the number of uNK cells in the endometrium. The question remains as to whether reducing the number of uNK cells improves pregnancy outcome.
We propose a randomised, double-blind, placebo controlled trial of prednisolone with a pilot phase to assess feasibility of recruitment, integrity of trial procedures, and to generate data to base future power calculations. The primary aim is to investigate whether prednisolone therapy during the first trimester of pregnancy is able to improve live birth rates in patients with idiopathic recurrent miscarriage and raised uNK cells in the endometrium. Secondary outcomes include conception rate, karyotype of miscarriage, miscarriages (first and second trimester), stillbirths, pregnancy complications, gestational age at delivery, congenital abnormality and side effects of steroids. The trial has 2 stages: i) screening of non-pregnant women and ii) randomisation of the pregnant cohort. All patients who fit the inclusion criteria (<40 years old, ≥3 consecutive miscarriages with no cause found and no contraindications to prednisolone therapy) will be asked to consent to an endometrial biopsy in the mid-luteal phase to assess their levels of uNK cells. Women with high levels of uNK cells (≥5%), will be randomised to either prednisolone or placebo when a pregnancy is confirmed. Follow-up includes 2 weekly ultrasound scans in the first trimester, an anomaly scan at 20 weeks gestation, growth scans at 28 and 34 weeks gestation and a postnatal follow-up at 6 weeks.
Current Controlled Trials ISRCTN28090716
Lactobacillus reuteri BR11 possesses a novel mechanism of oxidative defense involving an abundant cystine ABC transporter encoded by the cyuABC gene cluster. Large amounts of thiols, including H2S, are secreted upon cystine uptake by the CyuC transporter. A cystathionine γ-lyase (cgl) gene is cotranscribed with the cyu genes in several L. reuteri strains and was hypothesized to participate in cystine-mediated oxidative defense by producing reducing equivalents. This hypothesis was tested with L. reuteri BR11 by constructing a cgl mutant (PNG901) and comparing it to a similarly constructed cyuC mutant (PNG902). Although Cgl was required for H2S production from cystine, it was not crucial for oxidative defense in de Mann-Rogosa-Sharpe medium, in contrast to CyuC, whose inactivation resulted in lag-phase arrest in aerated cultures. The importance of Cgl in oxidative defense was seen only in the presence of hemin, which poses severe oxidative stress. The growth defects in aerated cultures of both mutants were alleviated by supplementation with cysteine (and cystine in the cgl mutant) but not methionine, with the cyuC mutant showing a much higher concentration requirement. We conclude that L. reuteri BR11 requires a high concentration of exogenous cysteine/cystine to grow optimally under aerobic conditions. This requirement is fulfilled by the abundant CyuC transporter, which has probably arisen due to the broad substrate specificity of Cgl, resulting in a futile pathway which degrades cystine taken up by the CyuC transporter to H2S. Cgl plays a secondary role in oxidative defense by its well-documented function of cysteine biosynthesis.
The dosing regimen and indications for many medicines in current use in neonatology are not well defined. There is a need to prioritise research in this area, but currently there is little information about which drugs are used in UK neonatal units and the research needs in this area as perceived by UK neonatologists.
The Neonatal Clinical Studies Group (CSG) of the Medicines for Children Research Network (MCRN) undertook a 2 week prospective scoping survey study to establish which medicines are used in UK neonatal units; how many babies are receiving them; and what clinicians (and other health professionals) believe are important issues for future research.
49 out of 116 units responded to at least one element of the survey (42%). 37 units reported the number of neonates who received medicines over a 2 week period. A total of 3924 medicine-patient pairs were reported with 119 different medicines. 70% of medicine-patient pairs involved medicines that were missing either a license or dose for either term or preterm neonates. 4.3% of medicine-patient pairs involved medicines that were missing both license and dose for any neonate. The most common therapeutic gap in need of additional research identified by UK neonatologists was chronic lung disease (21 responding units), followed by patent ductus arteriosus and vitamin supplements (11 responding units for both)
The research agenda for neonatal medicines can be informed by knowledge of current medicine use and the collective views of the neonatal community.
Lactococcus lactis is a gram-positive bacterium that is widely used in the food industry and is therefore desirable as a candidate for the production and secretion of recombinant proteins. Previously, we generated a L. lactis strain that expressed and secreted the antimicrobial cell wall-lytic enzyme lysostaphin. To identify lactococcal gene products that affect the production of lysostaphin, we isolated and characterized mutants generated by random transposon mutagenesis that had altered lysostaphin activity. Out of 35,000 mutants screened, only one with no lysostaphin activity was identified, and it was found to contain an insertion in the lysostaphin expression cassette. Ten mutants with higher lysostaphin activity contained insertions in only four different genes, which encode an uncharacterized putative transmembrane protein (llmg_0609) (three mutants), an enzyme catalyzing the first step in peptidoglycan biosynthesis (murA2) (five mutants), a putative regulator of peptidoglycan modification (trmA) (one mutant), and an uncharacterized enzyme possibly involved in ubiquinone biosynthesis (llmg_2148) (one mutant). These mutants were found to secrete larger amounts of lysostaphin than the control strain (MG1363[lss]), and the greatest increase in secretion was 9.8- to 16.1-fold, for the llmg_0609 mutants. The lysostaphin-oversecreting llmg_0609, murA2, and trmA mutants were also found to secrete larger amounts of another cell wall-lytic enzyme (the Listeria monocytogenes bacteriophage endolysin Ply511) than the control strain, indicating that the phenotype is not limited to lysostaphin.
Newly synthesized secretory granule content proteins are delivered via the Golgi complex for storage within mature granules, whereas constitutive secretory proteins are not stored. Most soluble proteins traveling anterograde through the trans-Golgi network are not excluded from entering immature secretory granules, whether or not they have granule-targeting signals. However, the ‘sorting-for-entry’ hypothesis suggests that soluble lumenal proteins lacking signals enter transport intermediates for the constitutive secretory pathway. We aimed to investigate how these constitutive secretory proteins are sorted. In a pancreatic β-cell line, we stably expressed two lumenal proteins whose normal sorting information has been deleted: alkaline phosphatase, truncated to eliminate its glycosylphosphatidylinositol membrane anchor (SEAP); and Cab45361, a Golgi lumenal resident, truncated to eliminate its intracellular retention (Cab308Myc). Both truncated proteins are efficiently secreted, but whereas SEAP enters secretory granules, Cab308Myc behaves as a true constitutive marker excluded from granules. Interestingly, upon permeabilization of organelle membranes with saponin, SEAP is extracted as a soluble protein whereas Cab308Myc remains associated with the membrane. These are among the first data to support a model in which association with the lumenal aspect of Golgi and/or post-Golgi membranes can serve as a means for selective sorting of constitutive secretory proteins.
Trans-Golgi network; Granule maturation; Constitutive; like secretory pathway
Infants born at extreme prematurity are at high risk of developmental disability. A major risk factor for disability is having a low level of thyroid hormone described as hypothyroxinaemia, which is recognised to be a frequent phenomenon in these infants. Derangements of critical thyroid function during the sensitive window in prematurity when early development occurs, may have a range of long term effects for brain development. Further research in preterm infants using neuroimaging techniques will increase our understanding of the specificity of the effects of hypothyroxinaemia on the developing foetal brain. This is an explanatory double blinded randomised controlled trial which is aimed to assess the effect of thyroid hormone supplementation on brain size, key brain structures, extent of myelination, white matter integrity and vessel morphology, somatic growth and the hypothalamic-pituitary-adrenal axis.
The study is a multi-centred double blinded randomised controlled trial of thyroid hormone supplementation in babies born below 28 weeks' gestation. All infants will receive either levothyroxine or placebo until 32 weeks corrected gestational age. The primary outcomes will be width of the sub-arachnoid space measured using cranial ultrasound and head circumference at 36 weeks corrected gestational age. The secondary outcomes will be thyroid hormone concentrations, the hypothalamic pituitary axis status and auxological data between birth and expected date of delivery; thyroid gland volume, brain size, volumes of key brain structures, extent of myelination and brain vessel morphology at expected date of delivery and markers of morbidity which include duration of mechanical ventilation and/or oxygen requirement and chronic lung disease.
Current Controlled Trials ISRCTN89493983