DPD epitope-specific glutamic acid decarboxylase (GAD)65 autoantibodies in children with Type 1 diabetes.

AIM: To study whether DPD epitope-specific glutamate decarboxylase autoantibodies are found more frequently in children with milder forms of Type 1 diabetes. METHODS: We prospectively evaluated 75 children with new-onset autoimmune Type 1 diabetes, in whom we collected demographic, anthropometric and clinical data and measured islet autoantibodies. Glutamate decarboxylase 65 autoantibody-positive samples were analysed for epitope specificities using recombinant Fab against the DPD-defined epitope of glutamate decarboxylase 65. RESULTS: After adjustment for age, positive DPD epitope recognition was significantly associated with higher C-peptide levels at onset (P = 0.02, r2 =0.21, n = 35), and high DPD recognition in the highest quartile tended to be associated with HbA1c ≤ 53 mmol/mol (7%) at the last follow-up [mean (sd) follow-up 1.3 (0.4) years; P = 0.07; for the model, P = 0.044, n = 30)]. Age- and sex-adjusted BMI percentile was significantly correlated with recognition of the DPD-defined epitope (P < 0.03, r2 =0.14, n = 34), but this correlation was driven by the older age group (age ≥ 10 years; P = 0.016, r2 =0.27, n = 21) and was not significant in younger children (P = 0.93, n = 13). There were no independent associations with sex, race/ethnicity, diabetic ketoacidosis, HbA1c , HLA DR3-DQ2/DR4-DQ8 or autoantibody number. CONCLUSIONS: Our findings suggest that recognition of the DPD-defined glutamate decarboxylase 65 autoantibody epitope at Type 1 diabetes onset is directly associated with ß-cell function, BMI and age, which supports the hypothesis that immunological factors contribute to the clinical heterogeneity of Type 1 diabetes. Larger studies relating epitope-specific glutamate decarboxylase 65 autoantibody to clinical phenotype in children with Type 1 diabetes are warranted.

Decreased bone mineralization in children with Noonan syndrome: another consequence of dysregulated RAS MAPKinase pathway?

INTRODUCTION: Noonan syndrome (NS) is a disorder of RAS- mitogen activated protein kinase (MAPK) pathway with clinical features of skeletal dysplasia. This pathway is essential for regulation of cell differentiation and growth including bone homeostasis. Currently, limited information exists regarding bone mineralization in NS. MATERIALS AND METHODS: Using dual-energy X-ray absorptiometry (DXA), bone mineralization was evaluated in 12 subjects (mean age 8.7 years) with clinical features of NS. All subjects underwent genetic testing which showed mutations in PTPN11 gene (N=8) and SOS1 gene (N=1). In a subgroup of subjects with low bone mass, indices of calcium-phosphate metabolism and bone turnover were obtained. RESULTS: 50% of subjects had low bone mass as measured by DXA. Z-scores for bone mineral content (BMC) were calculated based on age, gender, height, and ethnicity. Mean BMC z-score was marginally decreased at -0.89 {95% CI -2.01 to 0.23; p=0.1}. Mean total body bone mineral density (BMD) z-score was significantly reduced at -1.87 {95% CI -2.73 to -1.0; p=0.001}. Mean height percentile was close to - 2 SD for this cohort, thus total body BMD z-scores were recalculated, adjusting for height age. Adjusted mean total body BMD z-score was less reduced but still significant at -0.82 {95% CI -1.39 to -0.25; p=0.009}. Biochemical evaluation for bone turnover was unremarkable except serum IGF-I and IGF-BP3 levels which were low-normal for age. DISCUSSION: Children with NS have a significantly lower total body BMD compared to age, gender, ethnicity and height matched controls. In addition, total BMC appears to trend lower in children with NS compared to controls. We conclude that the metabolic bone disease present resulted from a subtle variation in the interplay of osteoclast and osteoblast activity, without clear abnormalities being defined in the metabolism of either. Clinical significance of this finding needs to be validated by larger longitudinal studies. Also, histomorphometric analysis of bone tissue from NS patients and mouse model of NS may further elucidate the relationship between the RAS-MAPK pathway and skeletal homeostasis.

A randomized controlled trial to evaluate the effects of high-dose versus low-dose of arginine therapy on hepatic function tests in argininosuccinic aciduria.

OBJECTIVE: To compare the effects of combinatorial therapy with low-dose arginine and a nitrogen scavenging agent (sodium phenylbutyrate) vs. monotherapy with high-dose arginine on liver function tests in patients with argininosuccinic aciduria (ASA). STUDY DESIGN: Twelve patients with ASA were enrolled in a double-blind, placebo-controlled, cross-over study design. Subjects were randomized to receive either a low-dose of arginine therapy (100 mg · kg(-1) · d(-1)) combined with sodium phenylbutyrate (500 mg · kg(-1) · d(-1)) (LDA arm) or a high-dose of arginine alone (500 mg · kg(-1) · d(-1)) (HDA arm) for one week. At the end of one week of therapy, liver function tests were assessed and metabolite fluxes were measured using a multi-tracer stable isotope protocol. RESULTS: Plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), and measures of synthetic functions of the liver were the primary outcomes. Subjects had significantly increased levels of argininosuccinate (P<0.03) and AST levels (P<0.01) after treatment with high-dose arginine. In the subset of subjects with elevated AST or ALT, treatment with high-dose of arginine was associated with further increases in plasma levels of both aminotransferases. Whereas subjects had increased arginine and citrulline flux with high-dose arginine therapy, the glutamine flux was not different between the two treatment arms. The synthetic liver functions as assessed by prothrombin time, INR, and coagulation factor levels were not different between the HDA and LDA arms. CONCLUSIONS: Administering higher doses of arginine in subjects with ASA results in increases in AST and ALT levels, especially in the subset of patients with elevated baseline aminotransferases. Hence, low-dose arginine sufficient to normalize arginine levels in plasma combined with nitrogen scavenging therapy should be considered as a therapeutic option for treatment of ASA in patients with elevations of hepatic aminotransferases.

Beta cell function and BMI in ethnically diverse children with newly diagnosed autoimmune type 1 diabetes.

OBJECTIVE: To examine the relationship between BMI and beta-cell function at diagnosis of autoimmune type 1 diabetes (T1D) in a large group of ethnically diverse children. METHODS: Cross-sectional analysis of 524 children (60.8% White, 19.5% Hispanic, 14.5% African-American, 5.2% other non-Hispanic; mean age = 9.8 yr [SD = 2.5]) with newly diagnosed autoimmune T1D. RESULTS: As much as 22.2% of children were overweight or obese. Median random serum C-peptide was 0.40 ng/mL (25th-75th percentiles = 0.3-0.8), with median glycemia of 366 mg/dL (25th-75th percentiles = 271-505). Median C-peptide was 0.3, 0.5, 0.7, and 0.85 ng/mL, respectively, in underweight, normal weight, overweight, and obese children (p < 0.0001, Kruskal-Wallis). In the final model (p < 0.0001), the odds of having preserved C-peptide (≥0.6 ng/mL) were increased by 2.4-fold (95% CI = 1.2-4.9, p < 0.015) and 4.1-fold (1.9-8.5, p < 0.0001), respectively, in overweight and obese compared to lean children; 1.3-fold per each year of age; 2.5-fold in girls compared to boys; 4-fold in children who presented without, compared to with, diabetes ketoacidosis (DKA); and decreased by 21% for each point increase in HbA1c. Tanner stage, race/ethnicity, glycemia, and number of anti-islet antibodies expressed were not independently associated with preserved C-peptide. The association between BMI and C-peptide levels was significant in children with and without preserved C-peptide. Excluding patients who presented with DKA and/or using BMI obtained 5 wk after diagnosis did not alter the results. CONCLUSION: Obese and overweight children compared to lean children have greater beta-cell function at the onset of autoimmune T1D. Prospective studies on the relationships among BMI, beta-cell function, and progression to clinical T1D are warranted.

Spironolactone in pulmonary arterial hypertension: results of a cross-over study.

The renin-angiotensin-aldosterone system is implicated in the pathophysiology of pulmonary arterial hypertension. We undertook this study to determine the effects of spironolactone, a mineralocorticoid receptor blocker, on collagen metabolism in pulmonary arterial hypertension patients. After obtaining institutional review board approval and informed consent, 42 pulmonary arterial hypertension patients were prospectively enrolled and 35 patients completed the 16-week randomized double-blinded crossover clinical trial. Subjects received 50 mg spironolactone or placebo and at the end of week 8, treatment arm was switched. Circulating levels of collagen biomarkers, brain natriuretic peptide, and aldosterone levels were measured, and six-minute walk distance, liver function tests, and echocardiogram data were collected at weeks 0, 8, and 16. Mean age was 45 ± 15 years and 87% were females. At baseline, brain natriuretic peptide and aldosterone levels were 74 ± 95 pg/ml and 7 ± 8 pg/ml, respectively. There was no change in the levels of amino-terminal propeptide of procollagen type III (PIIINP), MMP-9, TIMP-1, and MMP-9/TIMP-1 ratio at weeks 8 and 16 compared to baseline values in placebo arm and treatment arm. The baseline six-min walk distance was 436 ± 115 meters at baseline and no change in walk distance was noted at weeks 8 and 16 (P = 0.372). None of the patients developed hyperkalemia or liver function test abnormalities at weeks 8 and 16 requiring discontinuation of study drug. Our study showed no change in collagen metabolite levels in pulmonary arterial hypertension patients treated with spironolactone. Spironolactone was safe and well tolerated by pulmonary arterial hypertension patients with no increased hyperkalemia or liver function test abnormalities.

Generalized metabolic bone disease in Neurofibromatosis type I.

Skeletal abnormalities are a recognized component of Neurofibromatosis type I (NF1) but a generalized metabolic bone defect in NF1 has not been fully characterized thus far. The purpose of this study was to characterize at the densitometric, biochemical and pathological level the bone involvement in NF1 patients. Using dual energy X-ray absorptiometry (DXA) we analyzed bone status in 73 unselected NF1 subjects, 26 males and 47 females, mainly children and adolescents (mean age: 16.6 years). In a subgroup of subjects with low bone mass, we measured indices of calcium-phosphate metabolism, bone turnover, and bone density before and after vitamin D and calcium treatment. We found statistically significant and generalized reduction in bone mass with the mean lumbar bone mineral density (BMD) z-score being -1.38+/-1.05 (CI 95% -1.62 to -1.13), and whole body bone mineral content (BMC) z-score -0.61+/-1.19 (CI 95% -0.94 to -0.29), both significantly reduced compared to normal controls (p<.001). PTH was moderately elevated and after 4 months of supplemental therapy with calcium and vitamin D, it decreased to the normal range. However, BMD z-scores did not significantly improve after 2 years of follow-up. Histological analysis of bone samples from NF1 patients revealed substantial alteration of bone microarchitecture due mainly to reduced trabecular bone. Our observations are consistent with a generalized bone metabolic defect due to loss of the function of neurofibromin. Early identification of patients with osteoporosis may permit more timely and aggressive treatments to prevent the likely substantial morbidity associated with increased fracture risk later in life.

Worsening renal function in children hospitalized with decompensated heart failure: evidence for a pediatric cardiorenal syndrome?

OBJECTIVES: The purpose of this study was to determine the incidence of renal insufficiency in children hospitalized with acute decompensated heart failure and whether worsening renal function is associated with adverse cardiovascular outcome. DESIGN: Prospective observational cohort study. SETTING: Single-center children's hospital. PATIENTS: All pediatric patients from birth to age 21 yrs admitted to our institution with acute decompensated heart failure from October 2003 to October 2005. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Acute decompensated heart failure was defined as new-onset or acute exacerbation of heart failure signs or symptoms requiring hospitalization and inpatient treatment. We required that heart failure be attributable to ventricular dysfunction only. Worsening renal function was defined as an increase in serum creatinine by > or = 0.3 mg/dL during hospitalization. Sixty-three patients (35 male, 28 female) comprised 73 patient hospitalizations. Median age at admission was 10 yrs (range 0.1-20.3 yrs). Median serum creatinine at admission was 0.6 mg/dL (range 0.2-3.5 mg/dL), and median creatinine clearance was 103 mL/min/1.73 m2 (range 22-431 mL/min/1.73 m2). Serum creatinine increased during 60 of 73 (82%) patient hospitalizations (median increase 0.2 mg/dL, range 0.1-2.7 mg/dL), and worsening renal function occurred in 35 of 73 (48%) patient hospitalizations. Clinical variables associated with worsening renal function included admission serum creatinine (p = .009) and blood urea nitrogen (p = .04) and, during hospitalization, continuous infusions of dopamine (p = .028) or nesiritide (p = .007). Worsening renal function was independently associated with the combined end point of in-hospital death or need for mechanical circulatory support (adjusted odds ratio 10.2; 95% confidence interval 1.7-61.2, p = .011). Worsening renal function was also associated with longer observed length of stay (33 +/- 30 days vs. 18 +/- 25 days, p < .03). CONCLUSIONS: These data suggest that an important cardiorenal interaction occurs in children hospitalized for acute decompensated heart failure. Renal function commonly worsens in such patients and is associated with prolonged hospitalization and in-hospital death or the need for mechanical circulatory assistance.

B-type natriuretic peptide predicts adverse cardiovascular events in pediatric outpatients with chronic left ventricular systolic dysfunction.

BACKGROUND: Plasma B-type natriuretic peptide (BNP) levels are elevated in adults with heart failure and correlate with functional classification and prognosis. The range and predictive power of BNP concentrations in children with chronic heart failure, however, are not known. METHODS AND RESULTS: Whole blood BNP concentrations were measured in 53 consecutive patients with chronic left ventricular (LV) systolic dysfunction (biventricular hearts, ejection fraction < 50%, > 3 months since diagnosis). Children who had been hospitalized within 3 months before potential enrollment and those < 2 months or > 21 years of age were excluded. BNP concentrations were measured with the Triage assay (Biosite Diagnostics, Inc, San Diego, Calif). Echocardiographers and clinicians were blinded to BNP levels. An adverse cardiovascular event was defined as cardiac death, cardiac-related hospitalization, or listing for cardiac transplantation. The median age of patients with LV dysfunction was 9.3 years (interquartile range [IQR], 2.7 to 15.1 years). BNP levels were elevated in children with LV dysfunction compared with healthy controls (median, 78 pg/mL [IQR, 22 to 551 pg/mL] versus median, 7 pg/mL [IQR, 5 to 11 pg/mL]; P < 0.0001). Whole blood BNP concentrations were increased in patients who had a 90-day adverse cardiovascular event compared with those who did not (median, 735 pg/mL [IQR, 685 to 1510 pg/mL] versus median, 37 pg/mL [IQR, 14 to 92 pg/mL]; P < 0.001). Patients with a BNP concentration > or = 300 pg/mL were at increased risk of death, hospitalization, or listing for cardiac transplantation (adjusted hazard ratio, 63.6; P < 0.0001). CONCLUSIONS: BNP concentrations are elevated in children with chronic LV systolic dysfunction and predict the 90-day composite end point of death, hospitalization, or listing for cardiac transplantation.

Efficacy of a low-cost bubble CPAP system in treatment of respiratory distress in a neonatal ward in Malawi.

BACKGROUND: Respiratory failure is a leading cause of neonatal mortality in the developing world. Bubble continuous positive airway pressure (bCPAP) is a safe, effective intervention for infants with respiratory distress and is widely used in developed countries. Because of its high cost, bCPAP is not widely utilized in low-resource settings. We evaluated the performance of a new bCPAP system to treat severe respiratory distress in a low resource setting, comparing it to nasal oxygen therapy, the current standard of care. METHODS: We conducted a non-randomized convenience sample study to test the efficacy of a low-cost bCPAP system treating newborns with severe respiratory distress in the neonatal ward of Queen Elizabeth Central Hospital, in Blantyre, Malawi. Neonates weighing >1,000 g and presenting with severe respiratory distress who fulfilled inclusion criteria received nasal bCPAP if a device was available; if not, they received standard care. Clinical assessments were made during treatment and outcomes compared for the two groups. FINDINGS: 87 neonates (62 bCPAP, 25 controls) were recruited. Survival rate for neonates receiving bCPAP was 71.0% (44/62) compared with 44.0% (11/25) for controls. 65.5% (19/29) of very low birth weight neonates receiving bCPAP survived to discharge compared to 15.4% (1/13) of controls. 64.6% (31/48) of neonates with respiratory distress syndrome (RDS) receiving bCPAP survived to discharge, compared to 23.5% (4/17) of controls. 61.5% (16/26) of neonates with sepsis receiving bCPAP survived to discharge, while none of the seven neonates with sepsis in the control group survived. INTERPRETATION: Use of a low-cost bCPAP system to treat neonatal respiratory distress resulted in 27% absolute improvement in survival. The beneficial effect was greater for neonates with very low birth weight, RDS, or sepsis. Implementing appropriate bCPAP devices could reduce neonatal mortality in developing countries.

Nuclear glutaredoxin 3 is critical for protection against oxidative stress-induced cell death.

Mammalian glutaredoxin 3 (Grx3) has been shown to be critical in maintaining redox homeostasis and regulating cell survival pathways in cancer cells. However, the regulation of Grx3 is not fully understood. In the present study, we investigate the subcellular localization of Grx3 under normal growth and oxidative stress conditions. Both fluorescence imaging of Grx3-RFP fusion and Western blot analysis of cellular fractionation indicate that Grx3 is predominantly localized in the cytoplasm under normal growth conditions, whereas under oxidizing conditions, Grx3 is translocated into and accumulated in the nucleus. Grx3 nuclear accumulation was reversible in a redox-dependent fashion. Further analysis indicates that neither the N-terminal Trx-like domain nor the two catalytic cysteine residues in the active CGFS motif of Grx3 are involved in its nuclear translocation. Decreased levels of Grx3 render cells susceptible to cellular oxidative stress, whereas overexpression of nuclear-targeted Grx3 is sufficient to suppress cells' sensitivity to oxidant treatments and reduce reactive oxygen species production. These findings provide novel insights into the regulation of Grx3, which is crucial for cell survival against environmental insults.

Energy requirements during pregnancy based on total energy expenditure and energy deposition.

BACKGROUND: Energy requirements during pregnancy remain controversial because of uncertainties regarding maternal fat deposition and reductions in physical activity. OBJECTIVE: This study was designed to estimate the energy requirements of healthy underweight, normal-weight, and overweight pregnant women and to explore energetic adaptations to pregnancy. DESIGN: The energy requirements of 63 women [17 with a low body mass index (BMI; in kg/m(2)), 34 with a normal BMI, and 12 with a high BMI] were estimated at 0, 9, 22, and 36 wk of pregnancy and at 27 wk postpartum. Basal metabolic rate (BMR) was measured by calorimetry, total energy expenditure (TEE) by doubly labeled water, and activity energy expenditure (AEE) as TEE - BMR. Energy deposition was calculated from changes in body protein and fat. Energy requirements equaled the sum of TEE and energy deposition. RESULTS: BMR increased gradually throughout pregnancy at a mean (+/-SD) rate of 10.7 +/- 5.4 kcal/gestational week, whereas TEE increased by 5.2 +/- 12.8 kcal/gestational week, which indicated a slight decrease in AEE. Energy costs of pregnancy depended on BMI group. Although total protein deposition did not differ significantly by BMI group (mean for the 3 groups: 611 g protein), FM deposition did (5.3, 4.6, and 8.4 kg FM in the low-, normal-, and high-BMI groups; P = 0.02). Thus, energy costs differed significantly by BMI group (P = 0.02). In the normal-BMI group, energy requirements increased negligibly in the first trimester, by 350 kcal/d in the second trimester, and by 500 kcal/d in the third trimester. CONCLUSION: Extra energy intake is required by healthy pregnant women to support adequate gestational weight gain and increases in BMR, which are not totally offset by reductions in AEE.

Early clinical markers for the development of bronchopulmonary dysplasia: soluble E-Selectin and ICAM-1.

OBJECTIVE: To test the hypothesis that in infants born at

Platelet-monocyte complex formation: effect of blocking PSGL-1 alone, and in combination with alphaIIbbeta3 and alphaMbeta2, in coronary stenting.

BACKGROUND: Binding of platelet P-selectin to P-selectin glycoprotein ligand 1 (PSGL-1) is an initial event in the interactions between platelets and monocytes. Platelet-monocyte complexes (PMCs) have been implicated in several vascular disease processes, including acute coronary syndromes (ACS) and complications after percutaneous coronary intervention (PCI). We investigated the effect of ex vivo blockade of PSGL-1, alone and in combination with blockade of the alphaMbeta(2) (Mac-1) and alpha(IIb)beta(3) (GP IIb/IIIa) integrins, on PMC formation. METHODS AND RESULTS: Dual-label flow cytometry was used to detect PMCs in the blood of 10 volunteers and 10 patients undergoing PCI who received intravenous GP IIb/IIIa antagonists. PSGL-1 blockade, both prior to and after platelet stimulation, markedly reduced the formation of PMCs. Concomitant ex vivo blockade of the alphaMbeta(2) and alpha(IIb)beta(3) integrins did not result in further decreases of PMCs compared to PSGL-1 blockade alone. Antagonism of PSGL-1 also led to near elimination of leukocyte-platelet interactions under flowing conditions. CONCLUSION: Blockade of PSGL-1 alone is sufficient to inhibit and reverse the formation of PMCs following platelet stimulation. Concurrent antagonism of PSGL-1 and the alpha(IIb)beta(3) and alphaMbeta(2) integrins was not more effective than inhibition of PSGL-1 alone. These results suggest that platelet-monocyte complex formation is mostly dependent on PSGL-1.

Impact of cardiac growth on Doppler tissue imaging velocities: a study in healthy children.

BACKGROUND: Doppler tissue imaging (DTI) is a useful modality to quantitatively assess regional myocardial function. Studies attempting to establish reference values for DTI velocities in healthy children have been limited by small sample sizes and limited age distribution. In addition, the clinical effect of cardiac growth and other demographic and echocardiographic parameters on DTI velocities during childhood has not been adequately evaluated. METHODS: Pulsed wave DTI velocities were obtained in 325 healthy children at the lateral mitral annulus, interventricular septum, and lateral tricuspid annulus during early diastole, late diastole, and ventricular systole and were compared with demographic and echocardiographic study variables. RESULTS: In healthy children, parameters of cardiac growth, most notably left ventricular end-diastolic dimension, have the most significant correlation with the majority of DTI velocities. Age was also significantly correlated with most DTI velocities whereas sex, heart rate, and other echocardiographic parameters demonstrated minimal or no correlation. CONCLUSIONS: This study establishes reference values for DTI velocities and demonstrates the important clinical effects of cardiac growth and age on DTI velocities in neonates and children.

Effects of age, feeding regimen, and glucocorticoids on catecholamine and cortisol excretion in preterm infants.

BACKGROUND: The sympathoadrenal system is important in maintaining normal physiologic functioning in infants and increased output also can reflect stress. We sought to determine the effects of age, feeding regimen, and glucocorticoids on catecholamine and cortisol excretion in preterm infants and to assess whether a particular strategy of feeding enhanced sympathoadrenal development or was stressful. METHODS: Preterm infants (26-30 wk gestation; n = 171) were assigned randomly to begin trophic feedings from day 4 through 14 (trophic group) or to start feedings at day 15 (standard group) with feedings administered either by bolus every 3 hours (bolus) or continuously over 24 hours (continuous). At 10, 28, 40, 50, and 60 days of age, urine was collected continuously for 6 hours for measurement of catecholamines (norepinephrine, epinephrine, dopamine), cortisol, and creatinine. Data were available for 98 infants. RESULTS: Norepinephrine excretion increased with postnatal age. The increase with age was significantly greater in the trophic group compared with that in the standard group. Epinephrine excretion did not change with age, and there were no differences between trophic and standard groups. Dopamine excretion increased with age but was similar between trophic and standard groups (borderline significantly greater in the trophic group). Cortisol excretion increased with age and also was similar between trophic and standard groups. There was no effect on catecholamine or cortisol excretion of bolus vs continuous feedings, antenatal or postnatal corticosteroids, gestational age at birth, age at which full feedings were attained, or use of human milk compared with preterm formula. CONCLUSIONS: The greatest determinant of catecholamine and cortisol excretion is postnatal age. Feeding method, type of feeding, and glucocorticoid administration in the amounts customarily used have little significant effect on catecholamine or cortisol excretion. The apparent link between early feeding and norepinephrine (and possibly dopamine) excretion warrants further investigation.

Evaluation of alternative methods to prepare porcine adipocytes for measurement with an electronic particle number and size determination apparatus.

Experimental investigations with mammalian adipose tissue require a determination of adipocyte number as a basis for expression of metabolic and growth data. Determination of cell size is also important in adipose tissue because the fivefold or greater variation in adipocyte diameter in most growing and adult mammals precludes simple determination of cell number to interpret the biological observations. There are two approaches to determine adipocyte size and number: microscopic methods and electronic particle counter methods. Microscopic methods use embedded sections, frozen sections, or isolated cells, whereas electronic particle number and size instrumental methods use adipocytes released from fixed tissue fragments or adipocytes fixed after isolation. The advantage of the electronic approach is that it evaluates thousands of particles, although the standard fixative, osmium, is quite toxic. Consequently, we evaluated a number of alternative fixation methods to prepare isolated porcine adipocytes for number and size determination by electronic instrumentation. Fixation in 3, 4, or 5% glutaraldehyde or in 4% formaldehyde were not acceptable procedures for porcine adipocytes. The 4% glutaraldehyde fixation procedure was acceptable for isolated rat adipocytes (Stewart and Kaplan, 1993); porcine adipocytes seem to be much more susceptible to breakage using these procedures than rat adipocytes. We also added urea or Triton X-100 to glutaraldehyde- and osmium-fixed cells to decrease clumping and adhesion of individual cells; none of these additions was beneficial. Ability to store samples would improve the logistics for these time-consuming analyses. Samples of osmium-fixed adipocytes were stored in osmium, in .9% NaCl (saline) after removal of osmium, in 8 M urea after osmium removal with saline, or in .01% Triton X-100 after osmium removal with saline. Storage in urea or Triton was inappropriate because of irreversible clumping of individual cells. Storage in osmium was acceptable for at least 30 d. and storage in saline was marginally acceptable. The variability of the size determination process for osmium-fixed adipocytes was evaluated.

Disparity in the availability of injectable epinephrine in a large, diverse US school district.

BACKGROUND: Food allergies affect 2.5% of the US population. Results of studies show that minorities have the highest prevalence of food allergies. The Houston Independent School District (HISD) has an urban, socioeconomically diverse population and the role of ethnicity and socioeconomic status (SES) with availability of epinephrine has not been explored in this population. OBJECTIVE: This study sought to understand the association of SES and the presence of epinephrine in urban schools. METHODS: A 6-item questionnaire about food allergy characteristics was sent by e-mail to one nurse per elementary school in the HISD to identify the number, characteristics and treatment of food allergic reactions, and the presence of epinephrine injectors. The reactions and presence of injectors were assessed for the previous school year. Schools were categorized by socioeconomic variables as "low" or "non-low" based on National School Lunch Program student participation. Poisson, logistic, and linear regression analyses were used for group comparisons. RESULTS: One or more children with food allergies were reported in 97% of responding schools, but only 43% of schools reported having epinephrine injectors. A larger number of injectors in schools were associated with students of higher SES (r(2) = 0.701; P < .001). There were 6 times more injectors in non-low SES schools than in low SES schools (P < .03). Low SES and limited English proficiency were associated with decreased epinephrine injectors in schools. CONCLUSION: In the HISD, epinephrine injectors were more likely to be found in non-low SES schools versus low SES schools. Because minority students are disproportionately highly represented in low SES schools, there appears to be a disparity in the availability of injectable epinephrine for minority students in HISD schools.

Evaluating the association of autonomic drug use to the development and severity of retinopathy of prematurity.

PURPOSE: To explore the association of autonomic agents with the development and severity of retinopathy of prematurity (ROP). METHODS: The medical records of all preterm infants screened for ROP were retrospective reviewed. The association between development and severity of ROP and the use and dose(s) of autonomic agents was analyzed, after adjustment for the covariates gestational age, weight, development of septicemia, intraventricular hemorrhage, and respiratory distress syndrome. RESULTS: A total of 350 infants were screened. Caffeine was used in 338 infants; dopamine in 98 infants. There was a significant association between the use of dopamine and development of ROP (P < 0.001; relative risk [RR] = 1.6 [95% CI, 1.23-2.06]) and the need for ROP treatment (P = 0.001; RR = 4.63 [95% CI, 1.82-11.79]). The number of dopamine doses was significantly associated with the development of any ROP (P < 0.001; RR = 1.07 [95% CI, 1.03-1.1]), the severity of ROP (P < 0.001; RR = 1.09 [95% CI, 1.05-1.14]), and the need for treatment (P < 0.001; RR = 1.09 [95% CI, 1.05-1.14]). The total dose of caffeine was significantly associated with the development of any ROP (P = 0.003; RR = 1.03 [95% CI, 1.01-1.05]) and the need for treatment (P = 0.006, RR = 1.073 [95% CI; 1.021-1.13]). CONCLUSIONS: Although a causal relationship was not identified, the use of the autonomic agents caffeine and dopamine was associated with the development and severity of retinopathy of prematurity in this cohort.

Twenty-five year experience with balloon aortic valvuloplasty for congenital aortic stenosis.

Balloon aortic valvuloplasty (BAV) is the primary therapy for congenital aortic stenosis (AS). Few reports describe long-term outcomes. In this study, a retrospective single-institution review was performed of patients who underwent BAV for congenital AS. The following end points were evaluated: moderate or severe aortic insufficiency (AI) by echocardiography, aortic valve replacement, repeat BAV, surgical aortic valvotomy, and transplantation or death. From 1985 to 2009, 272 patients who underwent BAV at ages 1 day to 30.5 years were followed for 5.8 ± 6.7 years. Transplantation or death occurred in 24 patients (9%) and was associated with depressed baseline left ventricular shortening fraction (LVSF) (p = 0.04). Aortic valve replacement occurred in 42 patients (15%) at a median of 3.5 years (interquartile range 75 days to 5.9 years) after BAV and was associated with post-BAV gradient ≥25 mm Hg (p = 0.02), the presence of post-BAV AI (p = 0.03), and below-average baseline LVSF (p = 0.04). AI was found in 83 patients (31%) at a median of 4.8 years (interquartile range 1.4 to 8.7) and was inversely related to post-BAV gradient ≥25 mm Hg (p <0.04). AI was associated with depressed baseline LVSF (p = 0.02). Repeat valvuloplasty (balloon or surgical) occurred in 37 patients (15%) at a median of 0.51 years (interquartile range 0.10 to 5.15) and was associated with neonatal BAV (p <0.01), post-BAV gradient ≥25 mm Hg (p = 0.03), and depressed baseline LVSF (p = 0.05). In conclusion, BAV confers long-term benefits to most patients with congenital AS. Neonates, patients with post-BAV gradients ≥25 mm Hg, and patients with lower baseline LVSF experienced worse outcomes.

Presence or absence of a known diabetic ketoacidosis precipitant defines distinct syndromes of "A-ß+" ketosis-prone diabetes based on long-term ß-cell function, human leukocyte antigen class II alleles, and sex predilection.

Ketosis-prone diabetes (KPD) is heterogeneous. Longitudinal follow-up revealed that patients with "A-ß+" KPD (absent autoantibodies and preserved ß-cell function) segregated into 2 subgroups with distinct evolution of ß-cell function and glycemic control. Generalized linear analysis demonstrated that the variable that most significantly differentiated them was presence of a clinically evident precipitating event for the index diabetic ketoacidosis (DKA). Hence, we performed a comprehensive analysis of A-ß+ KPD patients presenting with "provoked" compared with "unprovoked" DKA. Clinical, biochemical, and ß-cell functional characteristics were compared between provoked and unprovoked A-ß+ KPD patients followed prospectively for 1 to 8 years. Human leukocyte antigen class II allele frequencies were compared between these 2 groups and population controls. Unprovoked A-ß+ KPD patients (n = 83) had greater body mass index, male preponderance, higher frequency of women with oligo-/anovulation, more frequent African American ethnicity, and less frequent family history of diabetes than provoked A-ß+ KPD patients (n = 64). The provoked group had higher frequencies of the human leukocyte antigen class II type 1 diabetes mellitus susceptibility alleles DQB1*0302 (than the unprovoked group or population controls) and DRB1*04 (than the unprovoked group), whereas the unprovoked group had a higher frequency of the protective allele DQB1*0602. ß-Cell secretory reserve and glycemic control improved progressively in the unprovoked group but declined in the provoked group. The differences persisted in comparisons restricted to patients with new-onset diabetes. "Unprovoked" A-ß+ KPD is a distinct syndrome characterized by reversible ß-cell dysfunction with male predominance and increased frequency of DQB1*0602, whereas "provoked" A-ß+ KPD is characterized by progressive loss of ß-cell reserve and increased frequency of DQB1*0302 and DRB1*04. Unprovoked DKA predicts long-term ß-cell functional reserve, insulin independence, and glycemic control in KPD.