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Liquid-chromatography tandem mass-spectrometry (LC-MS/MS) was developed in parallel to Immunoassays (IAs) and today is proposed as the “gold standard” for steroid assays. Leydig cells of men with Klinefelter syndrome (KS) are able to respond to human chorionic gonadotropin (hCG) stimulation, even if testosterone (T) production was impaired. The aim was to evaluate how results obtained by IAs and LC-MS/MS can differently impact on the outcome of a clinical research on gonadal steroidogenesis after hCG stimulation. A longitudinal, prospective, case-control clinical trial. (clinicaltrial.gov NCT02788136) was carried out, enrolling KS men and healthy age-matched controls, stimulated by hCG administration. Serum steroids were evaluated at baseline and for 5 days after intramuscular injection of 5000 IU hCG using both IAs and LC-MS/MS. 13 KS patients (36 ± 9 years) not receiving Treplacement therapy and 14 controls (32 ± 8 years) were enrolled. T, progesterone, cortisol, 17-hydroxy-progesterone (17OHP) and androstenedione, were significantly higher using IAs than LC-MS/MS. IAs and LC-MS/MS showed direct correlation for all five steroids, although the constant overestimation detected by IAs. Either methodology found the same 17OHP and Tincreasing profile after hCG stimulation, with equal areas under the curves (AUCs). Although a linearity between IA and LC-MS/MS is demonstrated, LC-MS/MS is more sensitive and accurate, whereas IA shows a constant overestimation of sex steroid levels. This result suggests the need of reference intervals built on the specific assay. This fundamental difference between these two methodologies opens a deep reconsideration of what is needed to improve the accuracy of steroid hormone assays.

Quality of life is negatively affected in children with food allergy. Oral immunotherapy is an approach to food allergy that leads to patient desensitization by administering gradually increasing amounts of a given food allergen. The aim of this pilot study is to evaluate how oral immunotherapy affects quality of life in children allergic to cow milk proteins. Thirty children (aged 3–12 years) with cow milk allergy were recruited. Their parents were provided with a validated disease specific quality of life questionnaire (the food allergy quality of life questionnaire – parent form, FAQLQ-PF) before and again 2 months after completing an oral immunotherapy protocol with cow milk. A significant improvement in all the investigated domains - emotional impact, food anxiety and social and dietary limitations - was found. The separate analysis of the different age groups demonstrated that the emotional impact and the food-related anxiety improved in children older than 4, while the social domains improved in each age group. In this pilot experience, oral immunotherapy significantly improves quality of life in children with cow milk allergy. The improvement seems particularly evident in children over 4 years old, who are most likely to benefit from the oral immunotherapy approach. Further placebo-controlled studies are needed to confirm these preliminary results.

Abstract: Esophageal candidiasis (EC) is a common and serious complication in patients infected with the human immunodeficiency virus (HIV). Micafungin has been shown to have dose-related efficacy and to be well tolerated in patients with HIV and EC. This analysis of data from a randomized, double-blind study examined pharmacokinetic parameters of micafungin (dosed at 50, 100, and 150 mg/day) and its metabolites in a subset of patients with HIV and EC. Micafungin exhibited linear, predictable pharmacokinetics, similar to the previous observations in healthy control subjects. Micafungin peak plasma concentration and exposure were increased with dose, while half-life and clearance remained consistent with increasing dose. Plasma concentrations of the metabolites M-1, M-2, and M-5 remained low throughout the study (24 h exposure ≤14% relative to micafungin at end of therapy for each). No differences in micafungin pharmacokinetic parameters were observed according to the sex or race of the patients. The high systemic exposures associated with micafungin 100 and 150 mg/day relative to micafungin 50 mg/day were found to directly correlate with endoscopic clearance. These data provide evidence that the pharmacokinetics of micafungin underlie the dose-related efficacy in patients with HIV and EC.

SummaryReal-Time PCR was used to determine whether the levels of mRNAs coding for anti-oxidant enzymes varied in apple peel during post-harvest cold storage, and if these were correlated with the occurrence of apple scald symptoms. Levels of the scald-related compounds, αfarnesene and conjugated trienes (CT; particularly, CT281 and CT258), were determined in fruit peel of the scald-resistant apple ‘Belfort’ and the scald-susceptible apple ‘Granny Smith’ during storage. αFarnesene levels varied, irrespective of the incidence of scald, whereas the levels of CTs were correlated with the degree of scald. On the other hand, the level of H2O2in ‘Granny Smith’ apple peel increased gradually, but significantly, from harvest during storage, whereas this value did not vary in ‘Belfort’ peel. In both apple cultivars, the levels of mRNA for Mn superoxide dismutase (MnSOD) decreased with storage time, whereas the levels of ascorbate peroxidase (APX) mRNA did not vary. In ‘Granny Smith’ apples, mRNA levels for catalase (CAT) increased dramatically (7.8-fold) late in the storage period. Monodehydro-ascorbate reductase (MDHAR) and glutathione reductase (GR) mRNA levels also increased greatly (12- and 18.8-fold, respectively) early in storage. Conversely, in ‘Belfort’ apples the transcript levels of these genes did not change during storage. These results indicate that scaldresistance in ‘Belfort’ apples was not directly related to higher levels of mRNAs for anti-oxidant enzymes. However, the higher levels of H2O2in ‘Granny Smith’apples was positively correlated with the higher levels of mRNAs for the anti-oxidant enzymes CAT, GR and MDHAR, suggesting direct regulation by the level of H2O2on transcription of these anti-oxidant enzyme genes.

Exhaled nitric oxide (ENO) has been proposed as a marker of airway inflammation in asthma and could be useful to evaluate the response to anti-inflammatory treatment. We investigated the effect of budesonide and nedocromil sodium on ENO levels and lung function in asthmatic children. Twenty stable steroid-naı̈ve asthmatic children were randomized in a single blind, cross-over study to receive inhaled budesonide (group A) or nedocromil sodium (group B) for 6 weeks. ENO was measured with a chemiluminescence analyser at baseline and at the end of each treatment period. Repeated-measures ANOVA was carried out. In asthmatic baseline ENO levels [mean 32·5 ppb, 95% confidence interval (CI) 26·4 to 38·7] were significantly higher compared to reference values (8·7 ppb, 95% CI 8·1 to 9·2,P<0·001). There were no treatment-order effect, no carry-over effect and in both groups the response pattern was the same: budesonide significantly lowered ENO levels from 41·0 ppb to 22·8 ppb in group A (mean,P<0·001) and from 22·6 ppb to 13·0 ppb in group B, (mean, P<0·05), while nedocromil did not reduce ENO values (from 24·4 ppb to 22·6 ppb in group B and from 22·8 ppb to 38·0 ppb in group A, mean, P=NS and P<0·01 respectively). After budesonide treatment ENO values of asthmatics were still significantly higher than in healthy children. The baseline values of FEV1and FEF25–75were normal in both groups and no significant changes were observed during the study. In conclusion, our study shows that budesonide, but not nedocromil sodium, significantly reduces ENO levels in stable asthmatic children even in absence of changes in the lung function.

BACKGROUND: Methylxanthines are often administered to preterm infants for the treatment of apnoea. AIMS: To study the effects of theophylline on energy metabolism, physical activity, and lung mechanics in preterm infants. METHODS: Indirect calorimetry was performed for six hours before and after administration of a bolus of theophylline (5 mg/kg) in 18 preterm infants while physical activity was recorded with a video camera. Lung mechanics measurements were performed at baseline and 12 and 24 hours after theophylline treatment. RESULTS: Theophylline increased mean (SEM) energy expenditure by 15 (5) kJ/kg/day and augmented carbohydrate utilisation from 6.8 to 8.0 g/kg/day, but fat oxidation was unchanged. After theophylline treatment, preterm infants had faster respiration, lower transcutaneous CO2, and improved static respiratory compliance without increased physical activity. CONCLUSIONS: A bolus of 5 mg/kg theophylline increased energy expenditure independently of physical activity, increased carbohydrate utilisation, and improved respiratory compliance. The increased energy expenditure could be detrimental to the growth of the preterm infant.

Photopheresis (ECP) is a new type of photochemotherapy, used for the treatment of oncological and autoimmune diseases. Lymphocytes are drawn from the patients by leukapheresis, treated with 8-methoxypsoralen (8-MOP) and ultraviolet light A (UVA) in an extracorporeal system and then reinfused. Skin exposure to 8-MOP and UVA (PUVA) has been shown to relieve cutaneous symptoms of graft-versus-host disease (GVHD) in bone marrow transplant (BMT) recipients. ECP, which is similar in some ways to PUVA, has been used in this study to treat four paediatric patients who developed chronic GVHD following BMT and in whom GVHD had failed to respond to conventional immunosuppressive therapy. Following ECP, skin lesions cleared almost completely and pulmonary function tests improved in two of three patients with cutaneous and lung involvement. Serum bilirubin and transaminases gradually normalized, and γGT decreased considerably in the remaining patient who had a severe cholestatic hepatopathy. The Karnofsky performance score increased to 90% in the three patients with positive responses to ECP and remained unchanged (40%) in the patient who did not respond. Immunosuppressive therapy was reduced in three patients and eventually discontinued in two. No significant side-effects were observed during the treatment. Our results suggest that ECP is a non-aggressive treatment that may benefit patients with chronic GVHD who do not respond to standard immunosuppressive therapy.

Twenty-three obese children, aged 9 to 14 years, ranging in percentage overweight from 26% to 83% (median 51.6%±16.3%), and 37 normal-weight children, matched for sex, age and height, performed a maximal exercise test on a treadmill. Cardiorespiratory performance was assessed by determination of the ventilatory anaerobic threshold (VAT) expressed in ml O2/min per kg and as a percent of maximal oxygen uptake (% VO2max). VAT and VO2max related to body weight were significantly lower (P<0.01) in the obese than in the normal-weight children. VAT % VO2max was similar in the two groups. A significant correlation was found between VAT and VO2max both in the obese (r=0.85) and in the control groups (r=0.79). The habitual level of physical activity was lower in the obese subjects compared to the control subjects (P<0.001). In conclusion our study shows that physical fitness of overweight children is quantitatively lowered and that it can be assessed by VAT. VAT does not require a maximal test and is particularly useful in the ergometric study of subjects with exercise intolerance.

Summary: The Bio-Rad HPLC method for the assay of urinary free catecholamines was employed during a period of 13 months, on samples of 720 subjects which were all normal but one with a pheochromocytoma diagnosis. The non-parametric reference limits were in accordance with those of other authors. Preliminary clean-up was carried out both in a standard urine and in the sample urine by means of a disposable micro-cation column prefilled with Bio-Rex 70 and ammonium acetate buffer which retain the weakly basic amines. Next epinephrine (E), norepinephrine (NE), dopamine (DA) and an internal standard (IS, N-methildopamine) were removed using ammonium pentaborate, a highly specific eluant. The final eluate was separated by HPLC on a Bio-Rad cation-exchange column, using 5% methanol in 0.06 M phosphate buffer (pH 5.2) as the mobile phase. Lyphochek normal and abnormal urine controls were elaborated with Lyphline Quality Control Program based on Westgard’s rules.Many amines may be retained on a carboxylic acid resin. However, very few compounds possess a cis-diol structure. In the absence of drug therapy, only three compounds are commonly seen in HPLC chromatograms in addition to E, NE, DA and IS. Two unidentified compounds elute before E. Alpha-methyldopa (Aldomet TM) is a common anti-hypertensive drug which forms two metabolites closely related to biogenic amines.Alpha-methylnorepinephrine elutes between E and NE. Alpha-methyldopamine coelutes with dopamine.The easy and quick method, giving good reproducibility and excellent clinic agreement, is suitable to pheochromocytoma differential diagnosis in clinical chemestry laboratories.

Fifteen very low birth weight children, 9 appropriate for gestational age (AGA, mean birth weight 1302 ± 164 g) and 6 small for gestational age children (SGA, mean birth weight 1263±117 g), were studied at the age of 7–12 years, and compared to a group of 26 healthy, age-, sex-, and height-matched children born at term. None of the VLBW children had developed chronic bronchopulmonary disease. Pulmonary function tests and progressive exercise tests on a treadmill were performed. Forced vital capacity, forced expiratory volume at 1 s and forced expiratory flow between 25% and 75% of vital capacity were normal for all subjects. No differences were found in maximum oxygen consumption, anaerobic threshold and maximal heart rate between the AGA and SGA children and the respective controls. Both in the AGA and SGA subgroups, the pre-exercise oxygen uptake results were comparable to those of the controls. In the SGA subgroup the energy cost of running was significantly higher with respect to the controls, while no difference was found between the AGA and the control children. In conclusion, children with birth weight less than 1501 g have normal values of aerobic fitness. In SGA children the efficiency of running is slightly reduced.

Background: While surfactant protein composition of bronchoalveolar lavage (BAL) has been described in lung diseases of adults and premature infants, scanty data are available on surfactant protein composition beyond the neonatal period.Objective: To measure SP-A and SP-B in BALs of children with chronic lung disease and to compare them with controls.Methods: We recruited 12 children undergoing bronchoscopy for chronic lung disease (CLD group, median age 2,5 months, IQR 1,3-6,6) and 8 children who had bronchoscopy for laringotracheomalacia assessment (median age 15,7 months, IQR 11,7-19,8). BALs were collected and SP-B and SP-A amounts were quantified by ELISA using a rabbit antiserum directed against SP-B and SP-A, respectively. Data were compared by Mann-Whitney test, and a p< 0.05 regarded as statistically significant.Results: SP-A levels were significantly lower in CLD children compared to controls (median 1,49% of phospholipid amount (PL), IQR 0,48-6,00 versus 11,50% PL, IQR 5,04-15,05, p=0,003, in the CLD group and in controls, respectively). SP-B was measured in all 12 CLD patients and in 2 controls (analysis in progress). SP-B levels tended to be lower in the CLD group (median 0,56% PL, IQR 0,29-4,27) compared to controls (mean 2,96% PL, the two values being 1,80 and 4,12% PL).One girl in the CLD group had non detectable SP-A and SP-B levels and she was diagnosed to have ABCA3 mutation.Conclusions: SP-A levels appear to be lower in chronic lung disease. Preliminary data suggest a similar pattern for SP-B, but this needs to be confirmed by the ongoing analysis.

A recent report demonstrated that the values of dynamic compliance measured in mechanical breaths are lower than those evaluated in spontaneous ones (Mammel Ped Pulmonol B:222, 1990). To evaluate the influence of mechanical and spontaneous ventllation on respiratory function values we measured and compared static compliance of the respiratory system (Crs) in these two different states, performing 44 studies in 33 stable intubated infants (age 3 days±1 SD, weight 2000 g±400 SD). All infants had acute or chronic lung disease, no sedat ion was given. We used the single breath occlusion technique and a computerized system (SensorMedics 2600); compliance measurements (atleast 7 breaths) were performed during mechanical (CrsV) and spontaneous (CrsS) breaths (after a brief disconnection of the infant from the ventilator). In both situations the exalation is externa) to the ventilator circuit. The mean values of CrsV resulted 0.46±0.06 ml/cmH20/kg (mean coefficient of variation cv; 9.35±3-8%, ranee 3.3-16%> and there was no significant difference (p=NS paired t-test) from the values of CrsS (0.43±0.07 ml/cmH20/kg, cv: 9.85±3.5%, range 4-17%). Assuming that the lung is on the same part of the pressure-volume curve, we conclude that measuring static compliance by the single breath occlusion technique is not necessary to obtain measurements performed both during mechanical and spontaneous breaths as suggested for dynamic compliance.