Bone marrow metabolism is affected by body weight and response to exercise training varies according to anatomical location.

AIM: High body weight is a protective factor against osteoporosis, but obesity also suppresses bone metabolism and whole-body insulin sensitivity. However, the impact of body weight and regular training on bone marrow (BM) glucose metabolism is unclear. We studied the effects of regular exercise training on bone and BM metabolism in monozygotic twin pairs discordant for body weight. METHODS: We recruited 12 monozygotic twin pairs (mean ± SD age 40.4 ± 4.5 years; body mass index 32.9 ± 7.6, mean difference between co-twins 7.6 kg/m2 ; eight female pairs). Ten pairs completed the 6-month long training intervention. We measured lumbar vertebral and femoral BM insulin-stimulated glucose uptake (GU) using 18 F-FDG positron emission tomography, lumbar spine bone mineral density and bone turnover markers. RESULTS: At baseline, heavier co-twins had higher lumbar vertebral BM GU (p < .001) and lower bone turnover markers (all p < .01) compared with leaner co-twins but there was no significant difference in femoral BM GU, or bone mineral density. Training improved whole-body insulin sensitivity, aerobic capacity (both p < .05) and femoral BM GU (p = .008). The training response in lumbar vertebral BM GU was different between the groups (time × group, p = .02), as GU tended to decrease in heavier co-twins (p = .06) while there was no change in leaner co-twins. CONCLUSIONS: In this study, regular exercise training increases femoral BM GU regardless of weight and genetics. Interestingly, lumbar vertebral BM GU is higher in participants with higher body weight, and training counteracts this effect in heavier co-twins even without reduction in weight. These data suggest that BM metabolism is altered by physical activity.

Pediatric Short Bowel Syndrome: Real-World Evidence on Incidence and Hospital Resource Use From a Finnish Data Lake.

OBJECTIVES: Little is known about the epidemiology and healthcare burden of pediatric intestinal failure (IF). We aimed to assess the incidence, prevalence, healthcare resource utilization (HCRU), and related costs of pediatric short bowel syndrome (SBS) using follow-up data from the largest hospital district in Finland. METHODS: This retrospective registry study utilized electronic healthcare data covering all pediatric patients with SBS-IF born between 2010 and 2019 at the Hospital District of Helsinki and Uusimaa in Finland. Patients were followed from birth until the end of 2020 and compared to control patients, all from the same hospital system. RESULTS: In total, 38 patients with SBS-IF and 1:5 matched controls were included, with median follow-up time of almost 6 years from birth. Over half of the patients were born early preterm (gestational age ≤30 weeks). The incidence of pediatric SBS-IF was 24 per 100,000 live births. The HCRU was higher compared to controls and most of the inpatient days incurred during the first year of the SBS-IF patients' life. The average hospital-based HCRU costs were €221,000 for the first year and €57,000 for whole follow-up annually. The costs were higher for the early preterm patients and accumulated mainly from inpatient days. CONCLUSIONS: SBS-IF is a rare disease with a relatively low number of patients treated at each hospital district. The burden on the hospital system, as well as the patient's family, is especially high at the onset as the newborns with SBS-IF spend a significant part of their first year of life in the hospital.

Corneal nerve and nerve conduction abnormalities in children with type 1 diabetes.

OBJECTIVE: In vivo corneal confocal microscopy (CCM) is a novel, rapid, and non-invasive technique that identifies early small fiber damage and can predict the progression and development of clinical neuropathy in adults with type 1 diabetes. However, its usefulness in children is not well established. This study compared corneal confocal microscopy with neuropathic symptoms, signs, and objective measures of neuropathy for the diagnosis of diabetic neuropathy in children with type 1 diabetes. RESEARCH DESIGN AND METHODS: A total of 83 children with type 1 diabetes and 83 healthy participants of similar age underwent assessment of neuropathy symptoms, signs, nerve conduction studies, quantitative sensory and autonomic function testing, and in vivo CCM. RESULTS: Only of 3/83 (4%) children with type 1 diabetes had subclinical neuropathy. However, corneal nerve fiber density (p = 0.001), branch density (p = 0.006), fiber length (p = 0.002), tibial motor nerve amplitude and conduction velocity, and sural sensory nerve amplitude and conduction velocity (all p < 0.004) were lower in participants with type 1 diabetes than in the controls. Vibration, cooling, and warm perception thresholds and deep breathing heart rate variability were not found to be different (all p > 0.05) between children with type 1 diabetes and healthy controls. Multivariate regression analysis identified a possible association between body mass index and decreased corneal nerves. CONCLUSIONS: Decreased corneal nerves and abnormal nerve conduction were found in children with type 1 diabetes. CCM may allow rapid objective detection of subclinical diabetic neuropathy in children and adolescents with type 1 diabetes.

Knowledge and practice of harm-reduction behaviours for alcohol and other illicit substance use in adolescents with type 1 diabetes.

OBJECTIVE: To survey adolescents with type 1 diabetes mellitus (T1DM) about their knowledge and application of harm-reduction recommendations when they engage in alcohol and other illicit substance use. METHODS: Cross-sectional survey and chart review of adolescents with T1DM aged 13 to 18 years. RESULTS: One hundred and ninety patients were approached and 164 were included in the analysis. Mean age was 15.6 years (standard deviation [SD]=1.5). Fifty-one per cent were male. Of those who reported consuming alcohol, 95% knew that they should have a friend or parent check their blood glucose in the middle of the night after drinking but only 62% reported actually doing this in practice. Similarly, 98% reported knowing that they should wear a medic alert identification but only 79% reported actually doing this. Of those who reported consuming cannabis, 14% reported forgetting to check blood glucose and 14% reported forgetting insulin when using cannabis. From the chart review, a significantly lower proportion of adolescents reported substance use during their clinic visits (alcohol 26%, tobacco 19%, illicit substance 25%) compared to the self report in the survey (alcohol 55%, tobacco 30%, illicit substance 32%). CONCLUSIONS: Adolescents' knowledge of harm-reduction practices for the use of alcohol and other illicit substances is not always put to practice. Motivating adolescents to use their knowledge in practice is an important area to improve in diabetes self-management. Those who reported engaging in substance use in the survey had not always reported use during interactions with health care providers. This emphasizes the need for unbiased, universal education of all adolescents in the clinic.

Prevalence of alcohol, tobacco, cannabis and other illicit substance use in a population of Canadian adolescents with type 1 diabetes compared to a general adolescent population.

BACKGROUND: Youth with chronic conditions may engage in risky behaviour to the same, if not higher, degree as their healthy peers. OBJECTIVES: To determine the prevalence of alcohol, tobacco, cannabis and illicit substance use in adolescents with type 1 diabetes (T1DM) compared to a general adolescent population. METHODS: Cross-sectional survey of adolescents with T1DM (13 to 18 years). A published contemporary Canadian youth survey on use of alcohol, tobacco and illicit drugs was used as data representative of the general adolescent population. Outcome measures between the T1DM and general group were compared using Chi-square and Fisher's exact test where appropriate. RESULTS: One hundred and sixty-four adolescents with T1DM (mean 15.6 years [SD 1.5]; 51.3% male) were participated. The proportions of adolescents with T1DM who have tried substances were: alcohol 51.8%, tobacco 27.4%, cannabis 22.6% and other illicit substances 7.3%. Compared to the general population (n=3469), there were no significant differences in the proportion of adolescents that reported ever consuming alcohol, tobacco or cannabis. Reported illicit substance use was significantly lower in adolescents with T1DM compared to general population (7.3% versus 36.0%, P<0.0001). CONCLUSIONS: Proportions reporting having ever consumed alcohol, tobacco or cannabis were not significantly different between the two groups. However, the proportion of adolescents with T1DM who reported ever consuming an illicit substance was different from the comparison group. It is important to explore risky behaviours with adolescents with T1DM and focus on prevention and education during routine clinic visits.

The readiness and motivation interview for families (RMI-Family) managing pediatric obesity: study protocol.

BACKGROUND: Experts recommend that clinicians assess motivational factors before initiating care for pediatric obesity. Currently, there are no well-established clinical tools available for assessing motivation in youth with obesity or their families. This represents an important gap in knowledge since motivation-related information may shed light on which patients might fail to complete treatment programs. Our study was designed to evaluate the measurement properties and utility of the Readiness and Motivational Interview for Families (RMI-Family), a structured interview that utilizes a motivational interviewing approach to (i) assess motivational factors in youth and their parents, and (ii) examine the degree to which motivation and motivation-related concordance between youth and parents are related to making changes to lifestyle habits for managing obesity in youth. METHODS: From 2016 to 2020, this prospective study will include youth with obesity (body mass index [BMI] ≥97th percentile; 13-17 years old; n = 250) and their parents (n = 250). The study will be conducted at two primary-level, multidisciplinary obesity management clinics based at children's hospitals in Alberta, Canada. Participants will be recruited and enrolled after referral to these clinics, but prior to initiating clinical care. Each youth and their parent will complete the RMI-Family (~1.5 h) at baseline, and 6- and 12-months post-baseline. Individual (i.e., youth or parent) and family-level (i.e., across youth and parent) responses to interview questions will be scored, as will aspects of interview administration (e.g., fidelity to motivational interviewing tenets). The RMI-Family will also be examined for test-retest reliability. Youth data collected at each time point will include demography, anthropometry, lifestyle habits, psychosocial functioning, and health services utilization. Cross-sectional and longitudinal associations between individual and family-level interview scores on the RMI-Family and these clinical measures will be examined. DISCUSSION: As a measurement tool drawing on family-centered care and motivational interviewing, the RMI-Family was designed to increase understanding of the role of motivational factors in pediatric obesity management, allowing healthcare providers and policymakers to manage pediatric obesity more effectively and efficiently. Findings will help to create an innovative, tailored model of health care delivery that uses resources judiciously and is designed to best meet families' needs.

Motor unit number estimations are smaller in children with type 1 diabetes mellitus: A case-cohort study.

INTRODUCTION: We studied the potential for motor unit number estimation (MUNE) to detect subclinical changes in motor unit numbers in children with type 1 diabetes mellitus (DM). METHODS: Blinded observers performed clinical assessment, electrophysiology, and multipoint MUNE of the extensor digitorum brevis muscle in children with DM for ≥ 5 years and age-matched healthy controls. RESULTS: For 51 DM subjects, the disease duration was 9.1 ± 2.6 years. Subjects with DM and healthy controls (n=21) had similar demographics. There were no clinical symptoms or signs of peripheral neuropathy in any subject, nor differences in standard electrophysiology between cohorts. Estimated motor unit numbers were decreased significantly in children with DM (224 ± 87 vs. 274 ± 101, P=0.036). CONCLUSION: Despite the absence of clinical or standard electrophysiological differences from normal control subjects, MUNE can detect a small significant difference in children with DM, suggesting that motor unit loss begins early and subclinically in the disease.

A Randomized Trial of Closed-Loop Insulin Delivery Postpartum in Type 1 Diabetes.

OBJECTIVE: This study aimed to evaluate the efficacy of closed-loop insulin delivery postpartum. RESEARCH DESIGN AND METHODS: In this open-label, randomized controlled trial, postpartum individuals with type 1 diabetes were randomized to hybrid closed-loop insulin delivery with the MiniMed 670G/770G system in automode or sensor-augmented pump therapy in the first 12-weeks postpartum followed by a continuation phase with closed-loop insulin delivery for all until 24 weeks postpartum. RESULTS: Eighteen participants (mean ± SD age 32 ± 3.5 years, diabetes duration 22 ± 7.3 years, and early pregnancy HbA1c 52 ± 6.8 mmol/mol [6.9 ± 0.9%]) completed 24 weeks of postpartum follow-up. In the randomized phase, percent time in range 70-180 mg/dL (3.9-10 mmol/L) did not differ between groups (79.2 ± 8.7% vs. 78.2 ± 6.0%; P = 0.41). Participants randomized to closed-loop insulin delivery spent less time <70 mg/dL (3.9 mmol/L) and <54 mg/dL (3.0 mmol/L) (1.7 ± 0.8% vs. 5.5 ± 3.3% [P < 0.001] and 0.3 ± 0.2% vs. 1.1 ± 0.9% [P = 0.008]). Time >180 mg/dL (10 mmol/L) was not different between groups (18.7 ± 8.8% vs. 15.9 ± 7.7%; P = 0.21). In the continuation phase, those initially randomized to sensor-augmented pump therapy had less time <70 mg/dL after initiation of closed-loop insulin delivery (5.5 ± 3.3% vs. 3.3 ± 2.2%; P = 0.039). The closed-loop group maintained similar glycemic metrics in both study phases. There were no episodes of diabetic ketoacidosis or severe hypoglycemia in the randomized or continuation phase in either group. CONCLUSIONS: Women randomized to closed-loop insulin delivery postpartum had less hypoglycemia than those randomized to sensor-augmented pump therapy. There were no safety concerns. These findings are reassuring for use of closed-loop insulin delivery postpartum because of its potential to reduce hypoglycemia.

Multiomics and digital monitoring during lifestyle changes reveal independent dimensions of human biology and health.

We explored opportunities for personalized and predictive health care by collecting serial clinical measurements, health surveys, genomics, proteomics, autoantibodies, metabolomics, and gut microbiome data from 96 individuals who participated in a data-driven health coaching program over a 16-month period with continuous digital monitoring of activity and sleep. We generated a resource of >20,000 biological samples from this study and a compendium of >53 million primary data points for 558,032 distinct features. Multiomics factor analysis revealed distinct and independent molecular factors linked to obesity, diabetes, liver function, cardiovascular disease, inflammation, immunity, exercise, diet, and hormonal effects. For example, ethinyl estradiol, a common oral contraceptive, produced characteristic molecular and physiological effects, including increased levels of inflammation and impact on thyroid, cortisol levels, and pulse, that were distinct from other sources of variability observed in our study. In total, this work illustrates the value of combining deep molecular and digital monitoring of human health. A record of this paper's transparent peer review process is included in the supplemental information.

Exploring Knowledge and Safety Practices for Driving in Youth With Type 1 Diabetes.

OBJECTIVES: The aim of this work was to survey how well adolescents with type 1 diabetes mellitus (T1D) of legal driving age understand the risks associated with driving with diabetes and report adherence to recommendations. METHODS: This study was a cross-sectional survey of 191 adolescents, 14 to 18 years of age, with T1D. RESULTS: The average (standard deviation [SD]) glycated hemoglobin of respondents with a learner's or a driver's license was 9.0% (SD, 1.9%) and 8.9% (SD, 1.9%), respectively (p=0.91). The proportions of adolescents with a learner's or a driver's license who almost always or always checked their blood glucose prior to driving was 69% and 41%, respectively (p=0.01). Eighteen percent of adolescents with a learner's license and 41% with a driver's license experienced hypoglycemia while driving. The average number of weekly hypoglycemic events in each group was 2.0 (SD, 1.4) and 2.3 (SD, 2.0), respectively. There was a higher reported frequency of weekly mild hypoglycemic events between drivers who reported pulling over at least once while driving due to symptoms of hypoglycemia (3.25±2.38) and those who reported never having pulled over for hypoglycemia (1.87±1.31) (p=0.012). Respondents with a learner's license reported higher adherence to guidelines than those with a full license. CONCLUSIONS: Clinical education needs to reinforce adherence to recommendations, particularly checking blood glucose or wearing a continuous glucose monitor prior to driving, for all adolescents of driving age. The frequency of mild hypoglycemic events per week is associated with self-reported hypoglycemic events while driving.

A Practical Approach for the Verification and Determination of Site- and Trimester-Specific Reference Intervals for Thyroid Function Tests in Pregnancy.

BACKGROUND: Population-, assay-, and trimester-specific reference intervals for thyroid function tests are necessary to assess thyroid status accurately and manage thyroid disease throughout pregnancy. This study's objective was to verify if the manufacturer's recommended trimester-specific reference intervals for thyroid tests and the American Thyroid Association's recommended total thyroxine (TT4) pregnancy reference intervals were verifiable and appropriate for use in the authors' multicultural population. METHODS: Blood samples were obtained from the following sources: stored frozen surplus blood from women undergoing routine aneuploidy screening (first- and second-trimester samples, n = 274), women participating in an observational cohort study (second- and third-trimester samples, n = 135), and blood collected from women presenting for assessment to the labor and delivery ward (third-trimester samples, n = 35). Exclusions included thyroid medication or disease and positive thyroid peroxidase antibodies (anti-TPO). Samples were analyzed for thyrotropin (TSH), free T4 (fT4), free triiodothyronine (fT3), TT4, and anti-TPO using the Roche Cobas 8000 Modular e602 electrochemiluminescence immunoassay. RESULTS: Nine percent of the aneuploidy screening samples were excluded prior to thyroid testing due to maternal use of thyroid medication. Six percent of analyzed samples were excluded: 5.9% with positive anti-TPO and one with a TSH >10 mIU/L. The manufacturer's recommended trimester-specific reference intervals for TSH were not verified by described standardized methods. Therefore, 95th percentile reference intervals were determined using a minimum number of samples. Reference intervals for TSH and fT4 were as follows: 9-12 weeks, 0.18-2.99 mIU/L and 11-19.2 pmol/L; second trimester, 0.11-3.98 mIU/L and 10.5-18.2 pmol/L; and third trimester, 0.48-4.71 mIU/L and 9.0-16.1 pmol/L, respectively. The TT4 reference interval after 19 weeks' gestation was 77-186 nmol/L. CONCLUSIONS: This study provides a simple approach to verify or establish trimester-specific thyroid function reference intervals in local populations. The TT4 reference interval was lower than the interval proposed by the American Thyroid Association, suggesting the need for further study of TT4 in pregnancy and reliance on locally established fT4 reference intervals after 19 weeks, especially when there are no equivalent reference intervals for TT4.

Metabolic consequences of discretionary fortified beverage consumption containing excessive vitamin B levels in adolescents.

Over the past decade, there has been a substantial increase in the number of beverage products containing added vitamins and minerals. Often viewed as a healthier choice by consumers, the metabolic impacts of excessive vitamin consumption are relatively unknown, especially in children. The aim of this study was to examine the effects of a widely available, vitamin fortified beverage (5h Energy Decaffeinated) on insulin sensitivity, metabolic hormones and serum metabolomic responses in adolescents. Twenty adolescents (13-19y, 10M/10F) completed two randomized trials, consuming either coloured water as placebo (PL) or a vitamin fortified, sugar free beverage (FB, 1.5ml/kg) 40min prior to a modified oral glucose tolerance test (OGTT, 1.75g/kg glucose). Samples were collected at baseline and at 30, 45, 60, 90 and 120min during the OGTT. No differences in blood glucose response were observed between the treatments. However, compared to PL, postprandial plasma C-peptide and insulin excursion was significantly greater with FB, resulting in a 28% decline in the insulin sensitivity index. This was accompanied by elevated GLP-1, glucagon and PYY responses with FB compared to PL. Serum metabolomics (1H-NMR) analysis also revealed perturbations to vitamin B-linked one carbon metabolism flux with FB consumption that became more pronounced over time. These included a transient reduction in homocysteine flux accompanied by increases in betaine, vitamin B6, vitamin B12, choline, folate and taurine. Although these impacts are likely short-lived, results show that beverages fortified with excessive amounts of vitamins are not metabolically inert, but likely result in greater insulin secretion, differential gut hormone secretion and elevated one-carbon flux to process the excessive vitamin loads.

Effect of Prebiotic on Microbiota, Intestinal Permeability, and Glycemic Control in Children With Type 1 Diabetes.

CONTEXT: Patients with type 1 diabetes (T1D) have lower microbiota diversity and distinct gut microbial profiles that have been linked to changes in intestinal permeability. Prebiotics are nondigestible carbohydrates that alter gut microbiota and could potentially improve glycemic control and reduce intestinal permeability and thereby insulin sensitivity. OBJECTIVE: To determine the effect of prebiotics on glycemic control, gut microbiota, and intestinal permeability in children with T1D. DESIGN: A randomized, placebo-controlled trial in children 8 to 17 years of age with T1D using placebo or prebiotic oligofructose-enriched inulin for 12 weeks. Baseline, 3-month, and 6-month assessments included HbA1c, C-peptide, gut microbiota, intestinal permeability, frequency of diabetic ketoacidosis (DKA), and severe hypoglycemia. RESULTS: Forty-three subjects were randomized and 38 completed the study. The groups were similar at baseline: prebiotic (N = 17), age 12.5 years (SD of 2.8), HbA1c 8.02% (SD of 0.82); placebo (N = 21), age 12.0 years (SD of 2.6), HbA1c 8.08% (SD of 0.91). No significant differences were found in the frequency of DKA or severe hypoglycemia. At 3-months, C-peptide was significantly higher (P = 0.029) in the group who received prebiotics, which was accompanied by a modest improvement in intestinal permeability (P = 0.076). There was a significant increase in the relative abundance of Bifidobacterium within the prebiotic group at 3 months that was no longer present after the 3-month washout. The placebo group had significantly higher relative abundance of Streptococcus, Roseburia inulinivorans, Terrisporobacter, and Faecalitalea compared with the prebiotic group at 3 months. CONCLUSION: Prebiotics are a potentially novel, inexpensive, low-risk treatment addition for T1D that may improve glycemic control. Further larger-scale trials are needed.

Early corneal nerve fibre damage and increased Langerhans cell density in children with type 1 diabetes mellitus.

Corneal confocal microscopy (CCM) has been used to identify corneal nerve damage and increased Langerhans cell (LC) density in adults with Type 1 diabetes mellitus (T1DM). The purpose of this study was to evaluate whether corneal confocal microscopy can identify early corneal nerve damage and change in LC density in children and adolescents with T1DM. 64 participants with T1DM (age-14.6 ± 2.5 years, duration of diabetes-9.1 ± 2.7 years, HbA1c-75.66 ± 2.53 mmol/mol [9.1 ± 1.8%]) and 48 age-matched healthy control subjects underwent CCM. Sub-basal corneal nerve morphology and the density of mature and immature LCs was quantified. Corneal nerve fibre length and branch density were lower, whilst fibre density and tortuosity did not differ and both immature and mature LC density was significantly higher in T1DM compared to control subjects. There was no association between HbA1c and duration of diabetes with nerve fibre parameters or LC's density. Children and adolescents with T1DM demonstrate early immune activation and nerve degeneration.

The first cysteine-rich domain of the receptor GFRalpha1 stabilizes the binding of GDNF.

The GDNF (glial cell line-derived neurotrophic factor)-binding receptor GFRalpha1 (GDNF family receptor alpha1) is attached to the membrane by a GPI (glycosylphosphatidylinositol) anchor and consists of three cysteine-rich domains. The region corresponding to the second and third domains has been shown previously to participate in ligand binding, and to interact with the transmembrane tyrosine kinase receptor RET. No function has so far been found for the N-terminal, first domain (D1). Here we show that the GPI-anchored full-length receptor binds 125I-GDNF two times more tightly than does a GPI-anchored truncated receptor lacking D1. Scintillation proximity assays with purified receptor proteins also show that the GDNF-binding capacity of the soluble full-length GFRalpha1 is two times higher than the GDNF-binding capacity of the soluble D1-truncated GFRalpha1. As RET stabilizes the binding of GDNF equally well to the full-length and truncated receptors, D1 seems not to be involved in the interaction between GFRalpha1 and RET. Moreover, soluble full-length GFRalpha1 mediates GDNF-promoted neurite outgrowth in PC6-3 cells more efficiently than the soluble truncated GFRalpha1 protein. At low concentrations, the soluble fulllength receptor mediates the phosphorylation of RET more efficiently than the soluble truncated receptor. However, when the receptors are overexpressed on the cell surface as GPI-anchored proteins, or added to the growth medium at high concentrations as soluble proteins, full-length and truncated GFRalpha1 are indistinguishable in GDNF-dependent RET-phosphorylation assays. High levels of the receptors can thus mask a slightly impaired function in the phosphorylation assay. Based on assays with both GPI-anchored and soluble receptors, we therefore conclude that D1 contributes to the optimal function of GFRalpha1 by stabilizing the interaction between GFRalpha1 and GDNF.

Family Intervention for Obese/Overweight Children Using Portion Control Strategy (FOCUS) for Weight Control: A Randomized Controlled Trial.

Nutritional counseling for children with obesity is an important component of management. This randomized controlled trial was conducted to compare change in body mass index (BMI) z score after 6 months. Children 8 to 16 years with a BMI greater than the 85th percentile were randomized to standard of care nutrition counseling versus intervention with standard nutrition counseling including portion control tool training for the family. Measures were completed at baseline, 3 months, and 6 months. Fifty-one children were randomized to control and 48 to intervention. Mean age was 11 years (SD = 2.2). Mean BMI z score was 2.7 (SD = 0.4). Forty-five percent were male (n = 45). Follow-up at 6 months was 73.7% (73/99). Although no differences were seen between the groups, there was a significant decrease in BMI z score between baseline and 6 months within each group.

The Reliability and Reproducibility of Corneal Confocal Microscopy in Children.

PURPOSE: To assess the image and patient level interrater agreement and repeatability within 1 month for corneal nerve fiber length (CNFL) measured using in vivo corneal confocal microscopy (IVCCM) in children. METHODS: Seventy-one subjects (mean [SD] age 14.3 [2.6] years, range 8-18 years; 44 with type 1 diabetes and 27 controls; 36 males and 35 females) were included. 547 images (∼6 images per subject) were analyzed manually by two independent and masked observers. One-month repeat visit images were analyzed by a single masked observer in 21 patients. Automated image analysis was then performed using a specialized computerized software (ACCMetrics). RESULTS: For CNFL, the ICC (95% CI) were 0.94 (0.93-0.95) for image-level, 0.86 (0.78-0.91) for patient-level, and 0.88 (0.72-0.95) for the 1-month repeat assessment, and the Bland-Altman plots showed minimal bias between observers. Although there was excellent agreement between manual and automated analysis according to an ICC 0.89 (0.82-0.93), the Bland-Altman plot showed a consistent bias with manual measurements providing higher readings. CONCLUSIONS: In vivo corneal confocal microscopy image analysis shows good reproducibility with excellent intraindividual and interindividual variability in pediatric subjects. Since the image-level reproducibility is stronger than the patient-level reproducibility, refinement of the method for image selection will likely further increase the robustness of this novel, rapid, and noninvasive approach to detect early neuropathy in children with diabetes. Further studies on the use of IVCCM to identify early subclinical neuropathy in children are indicated.

PSPN/GFRalpha4 has a significantly weaker capacity than GDNF/GFRalpha1 to recruit RET to rafts, but promotes neuronal survival and neurite outgrowth.

Previously, it was shown that the recruitment of RET into lipid rafts by glial cell line-derived neurotrophic factor (GDNF)/GFRalpha1 is crucial for efficient signal transduction. Here, we show that the mouse GFRalpha4 is a functional, N-glycosylated, glycosylphosphatidylinositol (GPI)-anchored protein, which mediates persephin (PSPN)-induced phosphorylation of RET, but has an almost undetectable capacity to recruit RET into the 0.1% Triton X-100 insoluble membrane fraction. In spite of this, PSPN/mGFRalpha4 promotes neurite outgrowth in PC6-3 cells and survival of cerebellar granule neurons. As we show that also human PSPN/GFRalpha4 is unable to recruit RET into lipid rafts, we propose that the mammalian GFRalpha4 in this respect differs from GFRalpha1.

The structure of the glial cell line-derived neurotrophic factor-coreceptor complex: insights into RET signaling and heparin binding.

Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor alpha1 (GFR alpha 1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF(2).GFR alpha 1(2) complex structure at 2.35 A resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF(2).GFR alpha 1(2) complex and the previously published artemin(2).GFR alpha 3(2) complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFR alpha interface between the two structures, in particular ones that buttress the key conserved Arg(GFR alpha)-Glu(ligand)-Arg(GFR alpha) interaction. Second, the flexible GDNF ligand "finger" loops fit differently into the GFR alphas, which are rigid. Third, and we believe most importantly, the quaternary structure of the two tetramers is dissimilar, because the angle between the two GDNF monomers is different. This suggests that the RET-RET interaction differs in different ligand(2)-co-receptor(2)-RET(2) heterohexamer complexes. Consistent with this, we showed that GDNF(2).GFR alpha1(2) and artemin(2).GFR alpha 3(2) signal differently in a mitogen-activated protein kinase assay. Furthermore, we have shown by mutagenesis and enzyme-linked immunosorbent assays of RET phosphorylation that RET probably interacts with GFR alpha 1 residues Arg-190, Lys-194, Arg-197, Gln-198, Lys-202, Arg-257, Arg-259, Glu-323, and Asp-324 upon both domains 2 and 3. Interestingly, in our structure, sucrose octasulfate also binds to the Arg(190)-Lys(202) region in GFR alpha 1 domain 2. This may explain how GDNF.GFR alpha 1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET.