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Gastric absorption of oral paracetamol (acetaminophen) may be unreliable perioperatively in the starved and stressed patient. We compared plasma concentrations of parenteral paracetamol given preoperatively and oral paracetamol when given as premedication. Patients scheduled for elective ear; nose and throat surgery or orthopaedic surgery were randomised to receive either oral or intravenous paracetamol as preoperative medication. The oral dose was given 30 minutes before induction of anaesthesia and the intravenous dose given pre-induction. All patients were given a standardised anaesthetic by the same specialist anaesthetist who took blood for paracetamol concentrations 30 minutes after the first dose and then at 30 minute intervals for 240 minutes. Therapeutic concentrations of paracetamol were reached in 96% of patients who had received the drug parenterally, and 67% of patients who had received it orally. Maximum median plasma concentrations were 19 mg.l(-1) (interquartile range 15 to 23 mg.l(-1)) and 13 mg.l(-1) (interquartile range 0 to 18 mg.l(-1)) for the intravenous and oral group respectively. The difference between intravenous and oral groups was less marked after 150 minutes but the intravenous preparation gave higher plasma concentrations throughout the study period. It can be concluded that paracetamol gives more reliable therapeutic plasma concentrations when given intravenously. [ABSTRACT FROM AUTHOR]

Objectives: To assess the validity of an International Classification of Diseases (ICD) code based definition of non-fatal head trauma caused by child abuse (abusive head trauma) for population surveillance in New Zealand., Design: A retrospective cohort study of hospital inpatient records., Setting: A tertiary children's hospital in Auckland, New Zealand., Participants: 1731 children less than 5 years of age who were discharged after a non-fatal head trauma event over a 10-year period from 1 January 2010 to 31 December 2019., Outcome Measures: The outcome of assessment by the hospital's multidisciplinary child protection team (CPT) was compared with the outcome of ICD, Tenth Revision (ICD-10) discharge coding for non-fatal abusive head trauma (AHT). The ICD-10 code definition of AHT was derived from an ICD, Ninth Revision, Clinical Modification definition developed by the Centers for Disease Control, Atlanta, Georgia, which requires both a clinical diagnosis code and a cause-of-injury code., Results: There were 1755 head trauma events with 117 determined as AHT by the CPT. The ICD-10 code definition had a sensitivity of 66.7% (95% CI 57.4 to 75.1) and specificity of 99.8% (95% CI 99.5 to 100). There were only three false positives but 39 false negatives, with 18 of the false negatives coded with X59 (exposure to unspecified factor)., Conclusions: The ICD-10 code broad definition of AHT is a reasonable epidemiological tool for passive surveillance of AHT in New Zealand but it underestimates the incidence. Its performance could be improved by clear documentation of child protection conclusions in clinical notes, clarifying coding practice and removing the exclusion criteria from the definition., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Aim: To assess participation with a structured transition programme for adolescents with diabetes., Methods: Data from a regional cohort aged less than 16 years of age with type 1 (T1) and type 2 diabetes (T2D) in Auckland, New Zealand (2006-2016). Participation was defined as opting into a structured transition programme., Results: Five hundrend and twelve adolescents who were to be transferred to adult care (476 type 1 (T1D) and 36 type 2 (T2D)), overall participation rate of 83%, 86% (408/476) with T1D compared to 47% (17/36) with T2D. Within the cohort of T1D, participation rates for Māori and Pacific were lower (74% and 77%, respectively) than New Zealand Europeans (88%, p = 0.020 and p = 0.039, respectively). Lower socio-economic status was associated with reduced participation (77%) compared to higher socio-economic status (90%, p = 0.002). Of the 476 T1D who participated, 408 (96%) subsequently attended at least one adult service clinic ("capture"). 42% attended an adult clinic within the planned 3 months, 87% at 6 months and retention in adult clinics over 5 years of follow-up was 78%. By contrast, the 68 young people with T1D who did not participate in the structured transition had a capture rate of 78% (p < 0.001) and retention of 63% (p = 0.036)., Conclusions: In adolescents with diabetes, a formal transition from a paediatric service was associated with high rates of adult capture and subsequent retention in adult care over a 5-year follow-up period. Low socio-economic status, Māori or Pacific ethnicity and T2D were associated with reduced participation in the structured transition programme., (© 2022 Diabetes UK.)

μ-Crystallin, encoded by the CRYM gene, binds the thyroid hormones, T 3 and T 4 . Because T 3 and T 4 are potent regulators of metabolism and gene expression, and CRYM levels in human skeletal muscle can vary widely, we investigated the effects of overexpression of Crym . We generated transgenic mice, Crym tg, that expressed Crym protein specifically in skeletal muscle at levels 2.6-147.5 fold higher than in controls. Muscular functions, Ca 2+ transients, contractile force, fatigue, running on treadmills or wheels, were not significantly altered, although T 3 levels in tibialis anterior (TA) muscle were elevated ~190-fold and serum T 4 was decreased 1.2-fold. Serum T 3 and thyroid stimulating hormone (TSH) levels were unaffected. Crym transgenic mice studied in metabolic chambers showed a significant decrease in the respiratory exchange ratio (RER) corresponding to a 13.7% increase in fat utilization as an energy source compared to controls. Female but not male Crym tg mice gained weight more rapidly than controls when fed high fat or high simple carbohydrate diets. Although labeling for myosin heavy chains showed no fiber type differences in TA or soleus muscles, application of machine learning algorithms revealed small but significant morphological differences between Crym tg and control soleus fibers. RNA-seq and gene ontology enrichment analysis showed a significant shift towards genes associated with slower muscle function and its metabolic correlate, β-oxidation. Protein expression showed a similar shift, though with little overlap. Our study shows that μ-crystallin plays an important role in determining substrate utilization in mammalian muscle and that high levels of μ-crystallin are associated with a shift toward greater fat metabolism., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier B.V.)

Mother-Baby Unit research has focussed on maternal psychopathology over the course of an admission. Less is known about the baby's well-being, the shared relationship, or the mother's recovery. In an initial sample of 45 women, we describe discharge and post-discharge outcomes for maternal psychopathology (using maternal report and the Global Assessment of Function, GAF) and the mother-infant relationship (using the Child and Adult Relational Experimental Index, CARE Index). Three months post-discharge, one third of women described themselves as "completely recovered," one third were experiencing significant deterioration and 17% were readmitted to inpatient care. Poorer GAF scores were associated with a clinical diagnosis of comorbid personality disorder, antenatal presence of the index illness, partner illicit substance use, maternal perception of her bond, infant social withdrawal, and child protection concern. Post-discharge, the mother-infant relationship results were concerning. Only 17% were regarded as adequate. Improvement was observed across this period in 56% but relational deterioration occurred for 35%. Maternal and relational outcomes were weakly correlated at discharge (r² = 0.29, p = 0.07) but this was lost post-discharge (r² = 0.03, p = 0.89). The shared relationship and infant mental health should both be targets for intervention; both during MBU admission, and post-discharge., (© 2020 Michigan Association for Infant Mental Health.)

Aim: To review indications and use of angiotensin-converting enzyme-inhibitor (ACEI) therapy for the treatment of persistent microalbuminuria (MA) and/or hypertension (HTN) in adolescents with type 1 diabetes mellitus (T1DM)., Methods: Retrospective chart review of adolescent patients with T1DM seen within the paediatric diabetes service in Auckland, New Zealand, from 2006 to 2016. MA, HTN, patient demographic characteristics and ACEI prescribing and monitoring indices were examined., Results: Five hundred adolescents with T1DM were included. There were 26 patients (5%) with MA and/or HTN. MA alone was present in 16, HTN alone in 3 and both HTN and MA in 7. The 5-year MA/HTN-free rate was 98%, and the 10-year MA/HTN-free rate was 93%. Longer disease duration and earlier diagnosis were predictors of MA/HTN. There was no significant difference in standard clinical indices between study patients and others. ACEI was prescribed for 17 of 26 patients for either HTN or MA. Within 6 weeks of ACEI commencement, less than half of the subjects had repeat serum creatinine and MA screens and no record of repeat blood pressure measurement. Despite this, all patients had 3-monthly reviews within outpatient clinics where adjustments of ACEI doses were made., Conclusion: In our regional adolescent population with T1DM, there were low rates of both MA and/or HTN. In those who required treatment with ACEI, clinical monitoring post-commencement of therapy was inconsistent. Local consensus guidelines for the management of persistent MA in children and adolescents with diabetes mellitus were developed in response to this study., (© 2017 Paediatrics and Child Health Division (The Royal Australasian College of Physicians).)

Background: Infectious diseases are the leading cause of hospital admissions in young children. Hospitalisation with an infectious disease is a recurrent event for some children. Our objective was to describe risk factors for infectious disease readmission following hospital admission with an infectious disease in the first two years of life., Methods: We performed a national cohort study of New Zealand children, born 2005-2009, with an infectious disease admission before age 24 months. Children readmitted with an infectious disease within 12 months of the first infectious disease admission were identified. Every infectious disease admission was categorised as a respiratory, enteric, skin and soft tissue, urinary or other infection. Independent associations of demographic and child health factors with infectious disease readmission were determined using multiple variable logistic regression., Results: From 2005 to 2011, there were 69,902 infectious disease admissions for 46,657 children less than two years old. Of these 46,657 children, 10,205 (22%) had at least one infectious disease readmission within 12 months of their first admission. The first infectious disease admission was respiratory (54%), enteric (15%), skin or soft tissue (7%), urinary (4%) or other (20%). Risk of infectious disease readmission was increased if the first infectious disease admission was respiratory (OR = 1.87, 95% CI 1.78-1.95) but not if it was in any other infectious disease category. Risk factors for respiratory infectious disease readmission were male gender, Pacific or Māori ethnicity, greater household deprivation, presence of a complex chronic condition, or a first respiratory infectious disease admission during autumn or of ≥3 days duration. Fewer factors (younger age, male gender, presence of a complex chronic condition) were associated with enteric infection readmission. The presence of a complex chronic condition was the only factor associated with urinary tract infection readmission and none of the factors were associated with skin or soft tissue infection readmission., Conclusions: In children less than two years old, infectious disease readmission risk is increased if the first infectious disease admission is a respiratory infectious disease but not if it is another infectious disease category. Risk factors for respiratory infectious disease readmission are different from those for other infectious disease readmissions.

Background: Bioequivalence between Tacrolimus Prograf® and generic tacrolimus formulations has been demonstrated in adult populations, however clinical experience and safety data regarding generic tacrolimus in pediatric transplant recipients is limited. This study aimed to evaluate conversion from Tacrolimus Prograf® to Sandoz® in pediatric renal transplant recipients nationwide. The primary outcome was a change in mean trough tacrolimus concentration. Additionally, changes in tacrolimus intra-patient coefficient of variation (CoV), allograft function, requirement for dose adjustments, and episodes of biopsy-proven rejection were evaluated., Methods: Retrospective cohort study in 37 pediatric renal transplant recipients who switched to Tacrolimus Sandoz®. Each patient had three pre-conversion tacrolimus trough and creatinine concentrations within the 4 months prior and three post-conversion concentrations on day 3, 10, and the next subsequent level. Mean pre- and post-conversion tacrolimus trough concentrations and glomerular filtration rate (eGFR) were calculated. Tacrolimus concentration, CoV, and creatinine differences were compared by paired t test., Results: Thirty-seven patients (41% females, age 3-18 years) were included. Average intra-patient difference in trough tacrolimus concentration was 0.05μg/l (95% CI -0.37 to 0.47). Average intra-patient difference in eGFR was -1.20 ml/min/1.73 2 (95% CI -3.53 to 1.13). Three patients had acute rejection during 12 months post-conversion compared to none during 12 months pre-conversion., Conclusions: Pediatric renal transplant recipients can be converted from Tacrolimus Prograf® to Sandoz® with negligible change in trough concentration, dose adjustments, or immediate allograft function. Of concern was the number of acute rejection episodes, however non-adherence contributed to at least one episode and this difference was determined clinically and statistically not significant.

Aim: To investigate the incidence and severity of anti-N-methyl-d-aspartate (anti-NMDA) receptor encephalitis in children from New Zealand., Method: A retrospective case series was undertaken of all children (≤18y) diagnosed with anti-NMDA receptor encephalitis from January 2008 to October 2015., Results: Sixteen patients were identified with anti-NMDA receptor antibodies in the cerebrospinal fluid, three of whom had an associated teratoma. Fifteen children had Māori and/or Pacific Island ancestry. The incidence of anti-NMDA receptor encephalitis in Māori children was 3.4 per million children per year (95% confidence interval [CI] 1.4-7.0) and the incidence in Pacific children was 10.0 per million children per year (95% CI 4.3-19.8) compared with 0.2 per million children per year (95% CI 0.0-1.0) in children without Māori or Pacific Island ancestry. Sixty-seven per cent of children had a good outcome (modified Rankin Score ≤2) at 2 years' follow-up. This compares unfavourably with other cohorts despite a shorter median time to first-line immunotherapy (13d; range 4-89) and a higher proportion of children being treated with second-line therapy (50%)., Interpretation: Māori and Pacific Island children have a higher incidence of anti-NMDA receptor encephalitis and possibly a more severe phenotype. These data suggest a genetic predisposition to anti-NMDA receptor encephalitis in these populations., (© 2017 Mac Keith Press.)

Very long chain acyl-CoA dehydrogenase deficiency (VLCADD, OMIM #201475) has been increasingly diagnosed since the advent of expanded newborn screening (NBS). Elevated levels of tetradecenoyl-L-carnitine (C14:1) in newborn screening blood spot samples are particularly common in New Zealand, however this has not translated into increased VLCADD clinical presentations. A high proportion of screen-positive cases in NZ are of Maori or Pacific ethnicity and positive for the c.1226C > T (p.Thr409Met) ACADVL gene variant. We performed a retrospective, blinded, case-control study of 255 cases, born between 2006 and 2013, with elevated NBS C14:1 levels between 0.9 and 2.4 μmol/L, below the NZ C14:1 notification cut-off of 2.5 μmol/L. Coded healthcare records were audited for cases and age- and ethnicity- matched controls. The clinical records of those with possible VLCADD-related symptoms were reviewed. The follow-up period was 6 months to 7 years. Two of 247 cases (0.8 %) had possible VLCADD-like symptoms while four of 247 controls (2 %) had VLCADD-like symptoms (p = 0.81). Maori were overrepresented (68 % of the cohort vs 15 % of population). Targeted analysis of the c.1226 locus revealed the local increase in screening C14:1 levels is associated with the c.1226C > T variant (97/152 alleles tested), found predominantly in Maori and Pacific people. There was no increase in clinically significant childhood disease, irrespective of ethnicity. The study suggests that children with elevated C14:1, between 0.9-2.4 μmol/L, on NBS are at very low risk of clinically significant childhood disease. A minimally interventional approach to managing these patients is indicated, at least in the New Zealand population.

Background: Anemia is a major complication for patients on chronic dialysis. Erythropoietin is effective if iron is available, however unnecessary iron supplementation results in iron overload. Reticulocyte hemoglobin equivalent (Ret-He) may be useful for assessing iron status., Methods: A national retrospective cohort study including all children on chronic dialysis in New Zealand between 2007 and 2013, pairing Ret-He with demographic information, anemia indices, and markers of iron status., Results: In 606 observations, we found a modest relationship between Ret-He and transferrin saturation (TSAT) (r = 0.34, p < 0.001) and a poor correlation between Ret-He and ferritin (r = 0.09, p = 0.04). There was a negative correlation between ferritin and hemoglobin (r = -0.14, p = 0.002), a weak positive correlation between TSAT and hemoglobin (r = 0.12, p = 0.007), and a modest positive correlation between Ret-He and hemoglobin (r = 0.22, p < 0.001). The diagnostic performance of Ret-He to detect absolute iron deficiency (cut-off value 28.9 pg, sensitivity 90 %, specificity 75 %, AUC 0.87) was good., Conclusions: Ret-He is a more relevant marker of iron status than ferritin and TSAT. This supports prospectively testing Ret-He to distinguish between iron deficiency and suboptimal erythropoietin dosing as competing causes for anemia. Ferritin is an unhelpful biomarker of iron deficiency in this setting.

Background: Intravenous cannulation is a painful procedure that can provoke anxiety and stress. Injecting local anaesthetic can provide analgesia at the time of cannulation, but it is a painful procedure. Topical anaesthetic creams take between 30 and 90 minutes to produce an effect. A quicker acting analgesic allows more timely investigation and treatment. Vapocoolants have been used in this setting, but studies have reported mixed results., Objectives: To determine effects of vapocoolants on pain associated with intravenous cannulation in adults and children. To explore variables that might affect the performance of vapocoolants, including time required for application, distance from the skin when applied and time to cannulation. To look at adverse effects associated with the use of vapocoolants., Search Methods: We searched the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, Latin American Caribbean Health Sciences Literature (LILACS), the Cumulative Index to Nursing and Allied Health Literature (CINAHL), the Institute for Scientific Information (ISI) Web of Science and the http://clinicaltrials.gov/, http://www.controlled-trials.com/ and http://www.trialscentral.org/ databases to 1 May 2015. We applied no language restrictions. We also scanned the reference lists of included papers., Selection Criteria: We included all blinded and unblinded randomized controlled trials (RTCs) comparing any vapocoolant with placebo or control to reduce pain during intravenous cannulation in adults and children., Data Collection and Analysis: Three review authors independently assessed trial quality and extracted data, contacted study authors for additional information and assessed included studies for risk of bias. We collected and analysed data for the primary outcome of pain during cannulation, and for the secondary outcomes of pain associated with application of the vapocoolant, first attempt success rate of intravenous cannulation, adverse events and participant satisfaction. We performed subgroup analyses for the primary outcome to examine differences based on age of participant, type of vapocoolant used, application time of vapocoolant and clinical situation (emergency vs elective). We used random-effects model meta-analysis in RevMan 5.3 and assessed heterogeneity between trial results by examining forest plots and calculating the I(2) statistic., Main Results: We found nine suitable studies of 1070 participants and included them in the qualitative analyses. We included eight studies of 848 participants in the meta-analysis for the primary outcome (pain during intravenous cannulation). Use of vapocoolants resulted in a reduction in pain scores as measured by a linear 100 mm visual analogue scale (VAS 100) compared with controls (difference between means -12.5 mm, 95% confidence interval (CI) -18.7 to -6.4 mm; moderate-quality evidence). We could not include in the meta-analysis one study, which showed no effects of the intervention.Use of vapocoolants resulted in increased pain scores at the time of application as measured by a VAS 100 compared with controls (difference between means 6.3 mm, 95% CI 2.2 to 10.3 mm; four studies, 461 participants; high-quality evidence) and led to no difference in first attempt success compared with controls (risk ratio (RR) 1.00, 95% CI 0.94 to 1.06; six studies, 812 participants; moderate-quality evidence). We documented eight minor adverse events reported in 279 vapocoolant participants (risk difference (RD) 0.03, 95% CI 0 to 0.05; five studies, 551 participants; low quality-evidence).The overall risk of bias of individual studies ranged from low to high, with high risk of bias for performance and detection bias in four studies. Sensitivity analysis showed that exclusion of studies at high or unclear risk of bias did not materially alter the results of this review., Authors' Conclusions: Moderate-quality evidence indicates that use of a vapocoolant immediately before intravenous cannulation reduces pain during the procedure. Use of vapocoolant does not increase the difficulty of cannulation nor cause serious adverse effects but is associated with mild discomfort during application.

Purpose: To describe self-reported psychosocial wellbeing of adolescent childhood cancer survivors (CCS) compared with a control group of their peers., Methods: In this case-control study, 170 CCS aged 12-18 years completed an internet survey. The survey was a modified version of the Youth'07 Health and Wellbeing Survey of Secondary School Students in New Zealand. The control group (historical comparison) were the 9107 Youth'07 survey participants. Psychosocial wellbeing was assessed by measures of a) wellbeing (WHO-5), b) anxiety (MASC-10), c) depression (RADS2-SF) and d) emotional and behavioural difficulties (SDQ)., Results: The majority of CCS scored within the normal range across all four measures: wellbeing (89%), anxiety (93%), depression (94%) and emotional and behavioural difficulties (82%), leaving a small but important minority of CCS reporting significant clinical issues. Compared to their peers, adolescent CCS were no more likely to have an abnormal score for any of the psychosocial measures, and less likely to report abnormal psychosocial wellbeing (OR = 0.44, p = 0.0003) and prosocial behaviour problems (OR = 0.53, p = 0.009). Survivors of central nervous system tumours, older age, older age at diagnosis, and lower socioeconomic status were associated with some psychosocial difficulty., Conclusions: Following a diagnosis of childhood cancer, intensive therapy, and the subsequent risk of adverse health outcomes, one might expect CCS to be doing less well than their peers in terms of psychosocial wellbeing. The findings of this study, however, show that CCS are doing as well, and in some respects better, than their peers., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: New Zealand accepts 750 refugees annually who enter via the Mangere Refugee Resettlement Centre., Aims: To evaluate the health needs of refugee children less than 5 years of age., Methods: Retrospective audit on the outcomes of health screening and referrals in children less than 5 years of age at the Mangere Refugee Resettlement Centre between 2007 and 2011., Results: Of the 343 children, the most common infectious diseases were latent tuberculosis (15%) and parasitic infections (15%). In those older than 1 year old who had rubella and measles serology information, immunity was found in 50% and 59%, respectively. Hepatitis B immunity was found in 68%. Complete vaccination certificates were available for 66% on arrival to New Zealand. Vaccinations were administered to 73% while at the Mangere Refugee Resettlement Centre. Iron deficiency and vitamin D deficiency were the main noninfectious diseases found and were present in 33% and 12%, respectively. The total requiring referral for further medical assessment or support was 58% with 19% requiring referral to more than one service., Conclusions: Screening identified health needs in otherwise asymptomatic newly arriving refugee children. A proportion of children required access to multiple specialized medical services and may benefit from a comprehensive pediatric service.

Aim: A nationwide 24-month study was conducted (2007-2009), via the New Zealand Paediatric Surveillance Unit to define epidemiology and clinical features of acute poststreptococcal glomerulonephritis (APSGN) in children hospitalised with the illness., Methods: Paediatricians (n = 215) were requested to report new hospitalised cases fulfilling a case definition of definite (haematuria with low C3 and high streptococcal titres or biopsy proven APSGN) or probable (haematuria with low C3 or high streptococcal titres)., Results: A total of 176 cases were identified (definite: n = 138, probable: n = 38) with 63% residing in the Auckland metropolitan region. Sixty-seven percent were in the most deprived quintile. Annual incidence (0-14 years) was 9.7/100,000 (Pacific 45.5, Maori 15.7, European/other 2.6 and Asian 2.1/100,000). Annual incidence was highest in the South Auckland Metropolitan region (31/100,000), Central Auckland 14.9, West/North Auckland metropolitan region 5.9 and for the remainder of New Zealand 5.5/100,000. Age-specific incidence was highest in age 5-9 years (15.1/100,000). Reduced serum complement C3, gross haematuria, hypertension, impairment of renal function and heavy proteinuria were present in 93%, 87%, 72%, 67% and 44% of patients, respectively. Severe hypertension was closely associated with either symptoms of an acute encephalopathy or congestive heart failure., Conclusions: New Zealand children carry a significant disease burden of hospitalised APSGN with socio-economically deprived; Pacific and Maori children are being over-represented. Significant short-term complications were observed in hospitalised children with APSGN. Persistently very low rates in European/other suggest a preventable disease., (© 2013 The Authors. Journal of Paediatrics and Child Health © 2013 Paediatrics and Child Health Division (Royal Australasian College of Physicians).)

Aim: School-based interventions to tackle the rise in childhood overweight and obesity remain inconclusive and are often limited in their application to diverse populations. To inform and measure the effect of the implementation of a primary school-based longitudinal randomised controlled nutrition and activity intervention, Project Energize, baseline measures of body size and blood pressure were required., Methods: This cross-sectional study stratified by age, sex, ethnicity, rurality and school socio-economic-status (school-SES) measured body mass index (BMI), percentage body fat (%BF), waist and resting blood pressure from 2752 5- and 10-year-old children (62% European, 31% Māori) representative of the Waikato region of New Zealand., Result: Waikato children have a high prevalence of overweight and obesity that is linked with hypertension. Cardiovascular risk factors including raised blood pressure and hypertension, waist and arm circumference and percentage body fat (%BF) were more prevalent in 10-year-olds, lower school-SES and to some extent, urban living. In European children, BMI and waist circumference were similarly predictive of %BF, but for Māori children, waist circumference predicted %BF better than BMI., Conclusions: A variety of stratified, baseline measurements is important when designing school-based interventions. In particular, waist circumference measures may be a more accurate predictor of %BF than BMI when determining measurement protocols that consider different ethnic groups and environments among children. The effect of targeted improvements of the school physical activity and nutrition environment on the rate of increase of weight, fatness and blood pressure in children should be examined., (© 2012 The Authors. Journal of Paediatrics and Child Health © 2012 Paediatrics and Child Health Division (Royal Australasian College of Physicians).)

Background: Abnormalities on magnetic resonance imaging scans are common both before and after surgery for congenital heart disease in early infancy. The aim of this study was to prospectively investigate the nature, timing, and consequences of brain injury on magnetic resonance imaging in a cohort of young infants undergoing surgery for congenital heart disease both with and without cardiopulmonary bypass., Methods and Results: A total of 153 infants undergoing surgery for congenital heart disease at <8 weeks of age underwent serial magnetic resonance imaging scans before and after surgery and at 3 months of age, as well as neurodevelopmental assessment at 2 years of age. White matter injury (WMI) was the commonest type of injury both before and after surgery. It occurred in 20% of infants before surgery and was associated with a less mature brain. New WMI after surgery was present in 44% of infants and at similar rates after surgery with or without cardiopulmonary bypass. The most important association was diagnostic group (P<0.001). In infants having arch reconstruction, the use and duration of circulatory arrest were significantly associated with new WMI. New WMI was also associated with the duration of cardiopulmonary bypass, postoperative lactate level, brain maturity, and WMI before surgery. Brain immaturity but not brain injury was associated with impaired neurodevelopment at 2 years of age., Conclusions: New WMI is common after surgery for congenital heart disease and occurs at the same rate in infants undergoing surgery with and without cardiopulmonary bypass. New WMI is associated with diagnostic group and, in infants undergoing arch surgery, the use of circulatory arrest.

This paper describes the rate of violent episodes at a youth psychiatric unit, identifies significant clinical and demographic differences between service users who had admissions with violent episodes and those who did not, and describes characteristics of violent incidents, including antecedents, consequences, victim type, and severity of violence. A retrospective file audit over a 2-year period reviewed 303 admissions. Characteristics of violent incidents (n = 242) and service users (violent/non-violent) were recorded. Of 263 service users, 21.7% exhibited violent behaviour. Significant differences between admissions with and without violent episodes were found in terms of ethnicity, legal status, length of admission, and diagnosis. Staff were the most frequent victims and less severe incidents were most common. The most frequent antecedents to violence were positive symptoms of psychosis, hostility, and agitation, while the most common consequences were seclusion, physical restraint and 'as-required' medication. This study has identified that violent incidents are a common and significant issue. The findings might help staff in reviewing current management approaches. Future areas of study have been identified., (© 2012 The Authors. International Journal of Mental Health Nursing © 2012 Australian College of Mental Health Nurses Inc.)

Background: The incidence of type 2 diabetes mellitus (T2DM) is increasing in adolescents in most western countries. The time-course of glycemic control and impact of early treatment remain poorly understood., Objectives: To determine the change in incidence of T2DM, and the time-course of glycemic control in a regional pediatric cohort with T2DM., Methods: Retrospective analysis of prospectively collected data on 52 patients with T2DM from a population-based treatment referral cohort from 1 January 1995 to 31 December 2007., Results: The annual incidence of new cases of T2DM in children <15 yr increased fivefold in the Auckland region of New Zealand from 1995 [0.5/100,000; 95% confidence interval (CI) 0.0–2.2] to 2007 (2.5/100,000; 95% CI 1.0–5.5). The average annual incidence per 100,000 over the entire period was 1.3 (95% CI 1.0–1.8) overall, 0.1 (0.0–0.4) in Europeans, and 3.4 in both Maori (2.0–5.3) and Pacifica (2.2–5.0). Fifty-seven percent of children were symptomatic at presentation. Fifty-eight percent of patients were treated with insulin from diagnosis, most of whom were symptomatic (p = 0.003). Follow-up data were available for 48 patients with a mean of 2.4 yr. Although insulin therapy was associated with a greater fall in HbA1c values in the first 12 months of treatment (to a nadir of 7.1 vs. 8.1%, p < 0.05), there was a rapid deterioration after 12 months, and subsequent mean HbA1c values were >9% in both groups. Therapy did not affect body mass index standard deviation score (BMI SDS)., Conclusions: The incidence of T2DM in childhood or adolescence increased markedly over a 13-yr period in the Auckland region. Long-

We describe improved methods for large format, two-dimensional gel electrophoresis (2DE) that improve protein solubility and recovery, minimize proteolysis, and reduce the loss of resolution due to contaminants and manipulations of the gels, and thus enhance quantitative analysis of protein spots. Key modifications are: (i) the use of 7 M urea and 2 M thiourea, instead of 9 M urea, in sample preparation and in the tops of the gel tubes; (ii) standardized deionization of all solutions containing urea with a mixed bed ion exchange resin and removal of urea from the electrode solutions; and (iii) use of a new gel tank and cooling device that eliminate the need to run two separating gels in the SDS dimension. These changes make 2DE analysis more reproducible and sensitive, with minimal artifacts. Application of this method to the soluble fraction of muscle tissues reliably resolves ~1800 protein spots in adult human skeletal muscle and over 2800 spots in myotubes., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: We aimed to evaluate the incidence of type 1 diabetes mellitus in children <15 years of age (yr) in the Auckland region (New Zealand) over 20 years (1990-2009)., Methods: We performed a retrospective review of all patients <15 yr diagnosed with type 1 diabetes, from an unselected complete regional cohort., Results: There were 884 new cases of type 1 diabetes, and age at diagnosis rose from 7.6 yr in 1990/1 to 8.9 yr in 2008/9 (r(2) = 0.31, p = 0.009). There was a progressive increase in type 1 diabetes incidence among children <15 yr (p<0.0001), reaching 22.5 per 100,000 in 2009. However, the rise in incidence did not occur evenly among age groups, being 2.5-fold higher in older children (10-14 yr) than in the youngest group (0-4 yr). The incidence of new cases of type 1 diabetes was highest in New Zealand Europeans throughout the study period in all age groups (p<0.0001), but the rate of increase was similar in New Zealand Europeans and Non-Europeans. Type 1 diabetes incidence and average annual increase were similar in both sexes. There was no change in BMI SDS shortly after diagnosis, and no association between BMI SDS and age at diagnosis., Conclusions: There has been a steady increase in type 1 diabetes incidence among children <15 yr in Auckland over 20 years. Contrary to other studies, age at diagnosis has increased and the greatest rise in incidence occurred in children 10-14 yr. There was little change in BMI SDS in this population, providing no support for the 'accelerator hypothesis'.

Electroporation (EP) is used to transfect skeletal muscle fibers in vivo, but its effects on the structure and function of skeletal muscle tissue have not yet been documented in detail. We studied the changes in contractile function and histology after EP and the influence of the individual steps involved to determine the mechanism of recovery, the extent of myofiber damage, and the efficiency of expression of a green fluorescent protein (GFP) transgene in the tibialis anterior (TA) muscle of adult male C57Bl/6J mice. Immediately after EP, contractile torque decreased by ∼80% from pre-EP levels. Within 3 h, torque recovered to ∼50% but stayed low until day 3. Functional recovery progressed slowly and was complete at day 28. In muscles that were depleted of satellite cells by X-irradiation, torque remained low after day 3, suggesting that myogenesis is necessary for complete recovery. In unirradiated muscle, myogenic activity after EP was confirmed by an increase in fibers with central nuclei or developmental myosin. Damage after EP was confirmed by the presence of necrotic myofibers infiltrated by CD68+ macrophages, which persisted in electroporated muscle for 42 days. Expression of GFP was detected at day 3 after EP and peaked on day 7, with ∼25% of fibers transfected. The number of fibers expressing green fluorescent protein (GFP), the distribution of GFP+ fibers, and the intensity of fluorescence in GFP+ fibers were highly variable. After intramuscular injection alone, or application of the electroporating current without injection, torque decreased by ∼20% and ∼70%, respectively, but secondary damage at D3 and later was minimal. We conclude that EP of murine TA muscles produces variable and modest levels of transgene expression, causes myofiber damage due to the interaction of intramuscular injection with the permeabilizing current, and that full recovery requires myogenesis.

Background: There is minimal literature available on the long-term outcome of pediatric non-cystic fibrosis (CF) bronchiectasis., Aim: To document 5-year outcomes of children with chest computerized tomography (CT) scan diagnosed bronchiectasis from a tertiary New Zealand (NZ) respiratory clinic., Methods: Review of a clinical database identified 91 children. Demographics, clinical data, lung function, chest X-ray (CXR), sputum, presumed etiology, admission data, and the NZ deprivation index (NZDep) were collected. Univariate and multivariate regression were used to correlate clinical findings with lung function data and CXR scores using the Brasfield Scoring System., Results: Of the 91 children, 53 (59%) were Pacific Island, 22 (24%) Maori, 14 (15%) European, and 2 (2%) Other. The median follow-up period was 6.7 years (range 5.0-15.3 years) and median age at diagnosis was 7.3 years (range 11 months-16 years). Lung function data (n = 64) showed a mean decline of -1.6% predicted/year. In 30 children lung function declined (mean FEV(1) -4.4% predicted/year, range 1-17%), remained stable in 13 and improved in 21 children (mean FEV(1) of +3% predicted/year, range 1-15%). Reduced lung function was associated with male gender, chronic Haemophilus influenzae infection, longevity of disease, and Maori and Pacific Island ethnicity. There was a significant correlation with FEV(1) and CXR score at beginning (n = 47, r = 0.45, P = 0.001) and end (n = 26, r = 0.59, P = 0.002) of the follow-up period. The only variable consistently related to CXR score was chronic Haemophilus influenzae infection occurring in 27 (30%) (r(2)  = 0.52, P = <0.0001). Only four children were chronically infected with Pseudomonas species. Six children died., Conclusion: In our experience despite management in a tertiary multidisciplinary bronchiectasis clinic, progression of lung disease continues in a group of children and young adults., (Copyright © 2011 Wiley-Liss, Inc.)

Background: Type 1 diabetes mellitus (T1DM) may lead to severe long-term health consequences. In a longitudinal study, we aimed to identify factors present at diagnosis and 6 months later that were associated with glycosylated haemoglobin (HbA(1c)) levels at 24 months after T1DM diagnosis, so that diabetic children at risk of poor glycaemic control may be identified., Methods: 229 children <15 years of age diagnosed with T1DM in the Auckland region were studied. Data collected at diagnosis were: age, sex, weight, height, ethnicity, family living arrangement, socio-economic status (SES), T1DM antibody titre, venous pH and bicarbonate. At 6 and 24 months after diagnosis we collected data on weight, height, HbA(1c) level, and insulin dose., Results: Factors at diagnosis that were associated with higher HbA(1c) levels at 6 months: female sex (p<0.05), lower SES (p<0.01), non-European ethnicity (p<0.01) and younger age (p<0.05). At 24 months, higher HbA(1c) was associated with lower SES (p<0.001), Pacific Island ethnicity (p<0.001), not living with both biological parents (p<0.05), and greater BMI SDS (p<0.05). A regression equation to predict HbA(1c) at 24 months was consequently developed., Conclusions: Deterioration in glycaemic control shortly after diagnosis in diabetic children is particularly marked in Pacific Island children and in those not living with both biological parents. Clinicians need to be aware of factors associated with poor glycaemic control beyond the remission phase, so that more effective measures can be implemented shortly after diagnosis to prevent deterioration in diabetes control.

We studied the response of dysferlin-null and control skeletal muscle to large- and small-strain injuries to the ankle dorsiflexors in mice. We measured contractile torque and counted fibers retaining 10-kDa fluorescein dextran, necrotic fibers, macrophages, and fibers with central nuclei and expressing developmental myosin heavy chain to assess contractile function, membrane resealing, necrosis, inflammation, and myogenesis. We also studied recovery after blunting myogenesis with X-irradiation. We report that dysferlin-null myofibers retain 10-kDa dextran for 3 days after large-strain injury but are lost thereafter, following necrosis and inflammation. Recovery of dysferlin-null muscle requires myogenesis, which delays the return of contractile function compared with controls, which recover from large-strain injury by repairing damaged myofibers without significant inflammation, necrosis, or myogenesis. Recovery of control and dysferlin-null muscles from small-strain injury involved inflammation and necrosis followed by myogenesis, all of which were more pronounced in the dysferlin-null muscles, which recovered more slowly. Both control and dysferlin-null muscles also retained 10-kDa dextran for 3 days after small-strain injury. We conclude that dysferlin-null myofibers can survive contraction-induced injury for at least 3 days but are subsequently eliminated by necrosis and inflammation. Myogenesis to replace lost fibers does not appear to be significantly compromised in dysferlin-null mice.

Background: This study was conducted to identify patient-related, surgical, and anesthetic factors that would help predict adverse events and allow for better planning of perioperative care in children with myotonic dystrophy., Methods: This is a retrospective chart review from a large tertiary pediatric hospital. Data were collected on demographics, disease severity, surgical procedure, and anesthetic technique. Perioperative adverse events were recorded., Results: Records on 27 patients having 78 anesthetics over a 17.5-year period were reviewed. The overall frequency of postoperative respiratory complications was 10%. Significant risk factors were high muscular impairment rating scale (MIRS) grade (P = 0.007), at least 2300 cytosine, thymine, guanine (CTG) repeats on the protein kinase gene of chromosome 19q (P = 0.009), a longer duration of surgery (RR = 14.0 for surgery lasting at least 1 h; P = 0.002), perioperative morphine use (RR = 7.7, 95% CI 2.2-12.8; P = 0.005), intubation (P = 0.02), and the use of muscle relaxant without reversal (RR = 15.5, P = 0.0002). Using a multivariate risk model, only MIRS grade and the use of muscle relaxant without reversal were shown to be significant independent risk factors (RR = 24.9, P < 0.0001)., Conclusions: The MIRS is a statistically significant and clinically useful tool for predicting high perioperative risk. Patients with a high MIRS grade should therefore be considered for postoperative intensive care. The use of muscle relaxant without reversal was also shown to be a significant risk factor. Patients who require morphine infusions postoperatively might also be most safely managed in a high dependency unit.

Objective: It is difficult to know when children hospitalized with pertussis can be safely discharged. We sought to identify clinical features of children hospitalized with pertussis that are associated with readmission., Methods: A case series of 207 children hospitalized with pertussis was studied. The 33 children readmitted with pertussis were compared with the 174 who did not require readmission., Results: Demographic characteristics and immunization status of the children with pertussis requiring readmission did not differ from the children who were not readmitted. Median duration of initial hospital stay was 4 days for both groups (P=.11). The children who were readmitted had more cyanotic episodes per day (0.8 vs 0.0 episodes, P=.03) and on greater proportion of hospital days (0.5 vs 0.1, P=.01). On the last day of admission, the children subsequently readmitted had more coughing episodes (4 vs 0, P < .001), and a larger proportion had a cyanotic episode (30% vs 10%, P=.003). The risk of readmission was increased in children who had > or =1 cyanotic episode per day (relative risk [RR]=2.5, 95% confidence interval [95% CI] 1.3-4.6); cyanosis on > or =50% of days (RR=2.6, 95% CI 1.4-4.8);> or =2 coughing paroxysms on the last hospital day (RR=2.4, 95% CI 1.3-4.4); or any cyanosis on the last day (RR=2.9, 95% CI 1.5-5.2)., Conclusions: Paroxysmal cough and cyanosis are clinical signs that can be used in children hospitalized with pertussis to help decide when to discharge them from hospital.

Aim: Most children are continent of urine by the time they are in primary school. Balanced micturition and paradoxically dysfunctional voiding (DV) are acquired behaviour. Children need easy access to toilets at school, to comply with timed voiding which is part of the treatment for DV. This study investigated children's access to primary school toilets in Auckland., Methods: A questionnaire was sent to 97 primary schools listed on the New Zealand ministry of education (MoE) website for the Auckland region. Information regarding the school decile rating, population, rota, toilet facilities and toilet policy was requested. Six randomly selected schools were visited to verify the facilities available there., Results: Eighty-four percent of the schools in our sample complied with the prescribed statutory minimum for both boys and girls toilets. There was a median ratio of 11 children per facility. The median duration to use a facility during the first recess was 2 min., Conclusion: In general, the toilet facilities and utilization ratios in primary schools in the Auckland region appear to provide a healthy environment for urination. Schools must be encouraged to draft and adhere to a toilet policy to ensure a uniform toilet environment.

Aims/hypothesis: This study was performed to evaluate the influence of ethnicity and socioeconomic status (SES) on metabolic control in a population-based cohort of children with type 1 diabetes mellitus, and to evaluate whether any relationship between ethnicity and HbA(1c) is mediated by SES., Methods: We performed a retrospective review of all patients under age 16 years with type 1 diabetes (n = 555) from 1995 to 2005 in the greater Auckland region, New Zealand. Diabetes care variables and HbA(1c) values were collected prospectively, during clinic visits., Results: The mean population HbA(1c) was 8.3 +/- 1.3%. Maori and Pacific patients had poorer metabolic control than their European counterparts (9.1% and 9.3% vs 8.1%, p < 0.001) and higher rates of moderate to severe hypoglycaemia (31.1 and 24.8 vs 14.9 events/100 patient-years, p = 0.03). In multiple linear regression analysis, both ethnicity and SES were independently associated with HbA(1c) (p < 0.001). Other factors associated with higher HbA(1c) level were longer duration of diabetes, higher insulin dose, lower BMI z score and less frequent blood glucose monitoring (p < 0.001)., Conclusions/interpretation: Both ethnicity and SES independently influenced metabolic control in a large, unselected population of children with type 1 diabetes. Irrespective of SES, Maori and Pacific youth with type 1 diabetes were at greater risk of both moderate to severe hypoglycaemia and long-term complications associated with poor metabolic control.

Objective: Previous studies have shown that circulating concentrations of TSH, free T4, and free T3 are genetically regulated, but the genes responsible remain largely unknown. The aim of this study was to identify genetic loci associated with these parameters., Design: We performed a multipoint, nonparametric genome-wide linkage scan of 613 female dizygotic twin pairs. All subjects were euthyroid (TSH 0.4-4.0 mU/liter) with negative thyroid peroxidase antibodies and no history of thyroid disease. The genome scan comprised 737 microsatellite markers supplemented with dinucleotide markers. Data were analyzed using residualized thyroid hormone data after adjustment for age, smoking, and body mass index., Results: Multipoint linkage analysis gave linkage peaks for free T4 on chromosome 14q13 and 18q21 [logarithm of odds (LOD) 2.4-3.2]; TSH on chromosomes 2q36, 4q32, and 9q34 (LOD 2.1-3.2); and free T3 on chromosomes 7q36, 8q22, and 18q21 (LOD 2.0-2.3)., Conclusions: This study has identified eight genomic locations with linkage of LOD of 2.0 or greater. These results should enable targeted positional candidate and positional cloning studies to advance our understanding of genetic control of the pituitary-thyroid axis.

Intermediate filaments, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. We examined the contractile properties and morphology of fast-twitch skeletal muscle from mice lacking keratin 19. Tibialis anterior muscles of keratin-19-null mice showed a small but significant decrease in mean fiber diameter and in the specific force of tetanic contraction, as well as increased plasma creatine kinase levels. Costameres at the sarcolemma of keratin-19-null muscle, visualized with antibodies against spectrin or dystrophin, were disrupted and the sarcolemma was separated from adjacent myofibrils by a large gap in which mitochondria accumulated. The costameric dystrophin-dystroglycan complex, which co-purified with gamma-actin, keratin 8 and keratin 19 from striated muscles of wild-type mice, co-purified with gamma-actin but not keratin 8 in the mutant. Our results suggest that keratin 19 in fast-twitch skeletal muscle helps organize costameres and links them to the contractile apparatus, and that the absence of keratin 19 disrupts these structures, resulting in loss of contractile force, altered distribution of mitochondria and mild myopathy. This is the first demonstration of a mammalian phenotype associated with a genetic perturbation of keratin 19.

To identify proteins expressed abnormally in facioscapulohumeral muscular dystrophy (FSHD), we extracted soluble proteins from deltoid muscle biopsies from unaffected control and FSHD patients and analyzed them using two-dimensional electrophoresis, mass spectrometry and immunoblotting. Muscles from patients with FSHD showed large increases over controls in a single soluble, 34 kDa protein (pI=5.08) identified by mass spectrometry and immunoblotting as mu-crystallin (CRYM). Soluble fractions of biopsies of several other myopathies and muscular dystrophies showed no appreciable increases in mu-crystallin. Mu-crystallin has thyroid hormone and NADPH binding activity and so may influence differentiation and oxidative stress responses, reported to be altered in FSHD. It is also linked to retinal and inner ear defects, common in FSHD, suggesting that its up-regulation may play a specific and important role in pathogenesis of FSHD.

Obesity is a multifactorial disorder with a complex phenotype. It is a significant risk factor for diabetes and hypertension. We assessed obesity-related traits in a large cohort of twins and performed a genome-wide linkage scan and positional candidate analysis to identify genes that play a role in regulating fat mass and distribution in women. Dizygous female twin pairs from 1,094 pedigrees were studied (mean age 47.0+/-11.5 years (range 18-79 years)). Nonparametric multipoint linkage analyses showed linkage for central fat mass to 12q24 (141 cM) with LOD 2.2 and body mass index to 8q11 (67 cM) with LOD 1.3, supporting previously established linkage data. Novel areas of suggestive linkage were for total fat percentage at 6q12 (LOD 2.4) and for total lean mass at 2q37 (LOD 2.4). Data from follow-up fine mapping in an expanded cohort of 1243 twin pairs reinforced the linkage for central fat mass to 12q24 (LOD 2.6; 143 cM) and narrowed the -1 LOD support interval to 22 cM. In all, 45 single-nucleotide polymorphisms (SNPs) from 26 positional candidate genes within the 12q24 interval were then tested for association in a cohort of 1102 twins. Single-point Monks-Kaplan analysis provided evidence of association between central fat mass and SNPs in two genes - PLA2G1B (P = 0.0067) and P2RX4 (P = 0.017). These data provide replication and refinement of the 12q24 obesity locus and suggest that genes involved in phospholipase and purinoreceptor pathways may regulate fat accumulation and distribution.

In the Large(myd) mouse, dystroglycan is incompletely glycosylated and thus cannot bind its extracellular ligands, causing a muscular dystrophy that is usually lethal in early adulthood. We show that the Large(myd) mutation alters the composition and organization of the sarcolemma of fast-twitch skeletal muscle fibers in young adult mice. Costameres at the sarcolemma of the tibialis anterior muscle of Large(myd) mice contain reduced levels of several membrane cytoskeletal proteins, including dystrophin and beta-spectrin. In the quadriceps, longitudinally oriented costameric structures tend to become thickened and branched. More strikingly, proteins of the dystrophin complex present between costameres in controls are absent from Large(myd) muscles. We propose that the absence of the dystrophin complex from these regions destabilizes the sarcolemma of the Large(myd) mouse and thereby contributes to the severity of its muscular dystrophy. Thus, the positioning of sarcolemmal proteins may have a profound effect on the health of skeletal muscle.

Low bone mineral density (BMD) is a major risk factor for osteoporotic fracture. Studies of BMD in families and twins have shown that this trait is under strong genetic control. To identify regions of the genome that contain quantitative trait loci (QTL) for BMD, we performed independent genomewide screens, using two complementary study designs. We analyzed unselected nonidentical twin pairs (1,094 pedigrees) and highly selected, extremely discordant or concordant (EDAC) sib pairs (254 pedigrees). Nonparametric multipoint linkage (NPL) analyses were undertaken for lumbar spine and total-hip BMD in both cohorts and for whole-body BMD in the unselected twin pairs. The maximum evidence of linkage in the unselected twins (spine BMD, LOD 2.7) and the EDAC pedigrees (spine BMD, LOD 2.1) was observed at chromosome 3p21 (76 cM and 69 cM, respectively). These combined data indicate the presence, in this region, of a gene that regulates BMD. Furthermore, evidence of linkage in the twin cohort (whole-body BMD; LOD 2.4) at chromosome 1p36 (17 cM) supports previous findings of suggestive linkage to BMD in the region. Weaker evidence of linkage (LOD 1.0-2.3) in either cohort, but not both, indicates the locality of additional QTLs. These studies validate the use, in linkage analysis, of large cohorts of unselected twins phenotyped for multiple traits, and they highlight the importance of conducting genome scans in replicate populations as a prelude to positional cloning and gene discovery.

The CD3 gene region on chromosome 11q23 has been implicated in susceptibility to type 1 (insulin-dependent) diabetes mellitus. Using semi-automated fluorescence-based technology, we have undertaken association and linkage analysis of a dinucleotide microsatellite in the CD3 delta (CD3D) gene. We have also performed a large case-control analysis of a restriction fragment length polymorphism (RFLP) in the CD3 epsilon (CD3E) gene, 26 kb from CD3D. We found no evidence for the previously reported association between the 8 kb allele of the RFLP and disease in a UK dataset of 403 diabetic patients and 446 nondiabetic controls. Furthermore, the use of the transmission/disequilibrium test (TDT) showed no evidence of linkage or association to type 1 diabetes at either marker locus. We conclude that the CD3 gene region does not contribute significantly to IDDM susceptibility. We have successfully applied semi-automated, fluorescence-based technology to undertake association analysis on the CD3D microsatellite. Moreover, by analysing 94 other dinucleotide repeat markers, we conclude that fluorescence-based methodology can generally be applied to large-scale, semi-automated association studies with most microsatellite markers.

The role of human chromosome 2 in type 1 diabetes was evaluated by analysing linkage and linkage disequilibrium at 21 microsatellite marker loci, using 348 affected sibpair families and 107 simplex families. The microsatellite D2S152 was linked to, and associated with, disease in families from three different populations. Our evidence localizes a new diabetes susceptibility gene, IDDM7, to within two centiMorgans of D2S152. This places it in a region of chromosome 2q that shows conserved synteny with the region of mouse chromosome 1 containing the murine type 1 diabetes gene, Idd5. These results demonstrate the utility of polymorphic microsatellites for linkage disequilibrium mapping of genes for complex diseases.

We have searched the human genome for genes that predispose to type 1 (insulin-dependent) diabetes mellitus using semi-automated fluorescence-based technology and linkage analysis. In addition to IDDM1 (in the major histocompatibility complex on chromosome 6p21) and IDDM2 (in the insulin gene region on chromosome 11p15), eighteen different chromosome regions showed some positive evidence of linkage to disease. Linkages to chromosomes 11q (IDDM4) and 6q (IDDM5) were confirmed by replication, and chromosome 18 may encode a fifth disease locus. There are probably no genes with large effects aside from IDDM1. Therefore polygenic inheritance is indicated, with a major locus at the major histocompatibility complex.

To facilitate large-scale genetic mapping of the human genome, we have developed chromosome-specific sets of microsatellite marker loci suitable for use with a fluorescence-based automated DNA fragment analyser. We present 254 dinucleotide repeat marker loci (80% from the Généthon genetic linkage map) arranged into 39 sets, covering all 22 autosomes and the X chromosome. The average distance between adjacent markers is 13 centiMorgans, and less than 4% of the genome lies more than 20 cM from the nearest marker. Each set of microsatellites consists of up to nine marker loci, with allele size ranges that do not overlap. We selected marker loci on the basis of their reliability in the polymerase chain reaction, polymorphism content, map position and the accuracy with which alleles can be scored automatically by the Genotyper program.

The sensitivity of the 5-lipoxygenase to inhibition by 5,8,11,14-eicosatetraynoic acid (ETYA) is species- and/or tissue-dependent. Guinea pig peritoneal polymorphonuclear leukocytes prelabeled with [3H]arachidonic acid and stimulated with ionophore A23187 formed 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), as well as several dihydroxy fatty acids, including 5(S),12(R)-dihydroxy-6,8,10-(cis/trans/trans)-14-(cis)-eicosatetraenoic acid. ETYA (40 microM) did not inhibit, but, rather, increased the incorporation of 3H label into 5-HETE. In contrast, ETYA markedly inhibited the formation of radiolabeled dihydroxy acid metabolites by the A23187-stimulated cells. Assay of products from polymorphonuclear leukocytes incubated with exogenous arachidonic acid plus A23187, by reverse phase high performance liquid chromatography combined with ultraviolet absorption, showed a concentration-dependent inhibition of the formation of dihydroxy acid metabolite by ETYA (1-50 microM) and an increase in 5-HETE levels (maximum of 2- to 3-fold). The latter finding was verified by stable isotope dilution assay with deuterated 5-HETE as the internal standard. Another lipoxygenase inhibitor, nordihydroguaiaretic acid, potently inhibited the formation of both 5-HETE and dihydroxy acids, with an IC50 of 2 microM. The data suggest that ETYA can inhibit the enzymatic step whereby 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid is converted to leukotriene A4 in guinea pig polymorphonuclear leukocytes.

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.