Evaluation of Exosomal Coding and Non-Coding RNA Signature in Obese Adolescents.

Exosomes may contribute to the pathogenesis of obesity through their action as communication mediators. As we have previously demonstrated, in obese adolescents, some circulating miRNAs modified the C-type natriuretic peptide (CNP) expression and were associated with changes in metabolic functions. At present no data are available on miRNA transport by exosomes in this condition. To verify and compare the presence and the expression of CNP/NPR-B/NPR-C, and some miRNAs (miR-33a-3p/miR-223-5p/miR-142-5p/miRNA-4454/miRNA-181a-5p/miRNA-199-5p), in circulating exosomes obtained from the same cohort of obese (O, n = 22) and normal-weight adolescents (N, n = 22). For the first time, we observed that exosomes carried CNP and its specific receptors only randomly both in O and N, suggesting that exosomes are not important carriers for the CNP system. On the contrary, exosomal miRNAs resulted ubiquitously and differentially expressed in O and N. O showed a significant decrease (p < 0.01) in the expression of all miRNAs except for miR-4454 and miR-142-5p. We have found significant correlations among miRNAs themselves and with some inflammatory/metabolic factors of obesity. These relationships may help in finding new biomarkers, allowing us to recognize, at an early stage, obese children and adolescents at high risk to develop the disease complications in adult life.

Preclinical vascular alterations in obese adolescents detected by Laser-Doppler Flowmetry technique.

BACKGROUND AND AIMS: Childhood obesity promotes adverse changes in cardiovascular structure and function. This study evaluated whether alterations in skin microcirculation were already present in obese adolescents in a pre-clinical phase of cardiovascular disease. METHODS AND RESULTS: After an overnight fasting 22 obese adolescents and 24 normal-weight controls of similar age and gender distribution underwent clinical and blood examination and assessment of microvascular function by using two non-invasive techniques such as Peripheral Artery Tonometry (PAT) and Laser-Doppler Flowmetry (LDF). As compared to normal weight subjects, obese children had higher blood pressure, were significantly more hyper-insulinemic and insulin resistant, showing significantly higher plasma total cholesterol, LDL cholesterol, triglycerides and alanine aminotransferase (ALT). LDF showed lower pre- and post-occlusion forearm skin perfusion (perfusion units/second (PU/sec); median [IQR]) in obese than in normal weight subjects (pre-occlusion: 1633.8 [1023.5] vs. 2281.1 [1344.2]; p = 0.015. Post-occlusion: 4811.3 [4068.9] vs. 7072.8 [7298.8]; p = 0.021), while PAT revealed similar values of reactive hyperemia index (RHI). In entire population, fat mass % (FM%) was an independent determinant of both pre-and post-occlusion skin perfusion. Finally, being obese was associated with a higher risk to have a reduction of both pre- and post-occlusion skin perfusion (OR = 5,82 and 9,27, respectively). CONCLUSION: LDF showed very early, pre-clinical, vascular involvement in obese adolescents, characterized by impaired skin microcirculation, possibly reflecting a more diffuse microvascular dysfunction to other body tissues. Whether changing life style and improving weight may reverse such pre-clinical alterations remains to be established.

Vitamin D status, enterovirus infection, and type 1 diabetes in Italian children/adolescents.

At the time of the clinical onset of type 1 diabetes (T1D), we investigated 82 pediatric cases in parallel with 117 non-diabetic controls matched by age, geographic area, and time of collection. The occurrence of an enteroviral infection was evaluated in peripheral blood using a sensitive method capable of detecting virtually all human enterovirus (EV) types. While non-diabetic controls were consistently EV-negative, 65% of T1D cases carried EVs in blood. The vitamin D status was assessed by measuring the concentration of 25-hydroxyvitamin D [25(OH)D] in serum. Levels of 25(OH)D were interpreted as deficiency (≤50 nmol/L), insufficiency (52.5-72.5 nmol/L), and sufficiency (75-250 nmol/L). In T1D cases, the median serum concentration of 25(OH)D was 54.4 ± 27.3 nmol/L vs 74.1 ± 28.5 nmol/L in controls (P = .0001). Diabetic children/adolescents showed deficient levels of vitamin D 25(OH)D (ie, 72.5 nmol/L) in 48.8% cases vs 17.9% in non-diabetic controls (P = .0001). Unexpectedly, the median vitamin D concentration was significantly reduced in virus-positive vs virus-negative diabetics (48.2 ± 22.5 vs 61.8 ± 31.2 nmol/L; P = .015), with deficient levels in 58.5% vs 31.0%, respectively. Thus, at the time of clinical onset, EV-positive cases had reduced vitamin D levels compared with EV-negative cases. This could indicate either that the virus-negative children/adolescents had been hit by a non-infectious T1D-triggering event, or that children/adolescents with proper levels of vitamin D had been able to rapidly clear the virus. Thus, it would be important to assess whether adequate vitamin D supplementation before or during the prediabetic phase of T1D may counteract the diabetogenic potential of infectious pathogens.

Mid-regional-pro-adrenomedullin plasma levels are increased in obese adolescents.

PURPOSE: Recently, adrenomedullin (ADM) was defined as a new member of the adipokine family. ADM secreted by adipocytes, through its vasodilator and antioxidant actions, might be protective against metabolic syndrome-associated cardiovascular complications. The aim of the study was to assess plasma mid-regional (MR)-proADM levels in obese adolescents compared to normal-weight subjects and its relation with BMI, body composition and metabolic indices. METHODS: Plasma MR-proADM was measured in 32 healthy adolescents [BMI z-score (mean ± SEM) = 0.6 ± 0.09 and 0.8 ± 0.07 in females and males, respectively] and in 51 age-matched obese adolescents [BMI z-score (mean ± SEM) = 2.8 ± 0.12 and 2.9 ± 0.08 in female and males, respectively] by a time-resolved amplified cryptate emission technology assay. RESULTS: Plasma MR-proADM levels resulted significantly higher in obese than in normal-weight adolescents (MR-proADM: 0.33 ± 0.1 vs 0.40 ± 0.1 nmol/L, p < 0.0001). Using univariate analysis, we observed that MR-proADM correlated significantly with BMI z-score (p < 0.0001), fat mass (p < 0.0001), circulating insulin (p < 0.004), HOMA-IR (p < 0.005), total cholesterol (p < 0.03) and LDL-cholesterol (p < 0.05). Including MR-proADM as response variable and its significant correlates into a multiple regression analysis, we observed that fat mass (p = 0.014) and BMI z-score (p = 0.036) were independent determinants of circulating MR-proADM. CONCLUSIONS: Our study shows for the first time that obese adolescents have higher circulating levels of MR-proADM compared with normal-weight, appropriate controls suggesting its important involvement in obese patients.

An Italian MODY family with proband and son carrying variants in GCK and HFN1A: is it a true case of digenic MODY?

Maturity Onset Diabetes of the Young (MODY) is a monogenic autosomal dominant disorder affecting 1-5 % of all patients with diabetes mellitus. In Caucasians, GCK and HNF1A mutations are the most common cause of MODY. Here, we report two family members carrying a genetic variant of both GCK and HNF1A gene and their nine year clinical follow-up. Our report urges physicians to be cautious when variants in two genes are found in a single patient and suggests that collaboration with MODY genetics experts is necessary for correct diagnosis and treatment.

Circulating and Exosomal microRNA-33 in Childhood Obesity.

BACKGROUND: MicroRNA-33 may control a wide range of different metabolic functions. METHODS: This study aims to assess the miR-33a circulating profile in normal-weight (N = 20) and obese (O = 30) adolescents and to correlate its expression levels to their metabolic parameters. In a subset of subjects, we compared circulating miR-33a with exosomal miR-33a. RESULTS: Metabolic parameters were altered in O, with initial hyperinsulinemia. Circulating miR-33a was significantly higher in O than in N (p = 0.0002). Significant correlations between miR-33a and auxological and metabolic indices (Insulin p = 0.01; Cholesterol p = 0.01; LDL p = 0.01; HbA1c p = 0.01) were found. Splitting our population (O + N) into two groups, according to the median value of mRNA expression miR-33a levels (0.701), irrespective of the presence or absence of obesity, we observed that those having a higher expression of miR-33a were more frequently obese (87.5% vs. 12.5%; p < 0.0001) and had significantly increased values of auxological and metabolic parameters. Exosomes extracted from plasma of N and O carried miR-33a, and its expression was lower in O (p = 0.026). No correlations with metabolic parameters were observed. CONCLUSION: While exosome miR-33a does not provide any advantage, circulating miR-33a can provide important indications in an initial phase of metabolic dysfunction, stratifying obese adolescents at higher cardiometabolic risk.

Ultrasound and Clinical Alterations in the Foot of Children with Obesity and Diabetes.

BACKGROUND: Alterations in plantar soft tissues are often reported in adults with diabetes, whereas data on children are conflicting. Also, the extent of foot damage caused by excess body fat in children has not been fully characterized yet. This study aimed to address the relationship between body mass and structural changes of the foot in children and adolescents with and without diabetes. METHODS: In a case-control study, 43 participants (age 13 ± 2.6 years) were recruited, 29 (67%) with type 1 diabetes (T1D) and 14 (33%) controls. Anthropometric parameters [body mass index (BMI), waist circumference (WC), and waist-to-height ratio (WHtR)], foot posture index-6 (FPI-6) for static foot posture, and navicular drop test (NDT) for medial longitudinal arch height (MLA) were measured in all participants. The thickness of the midfoot plantar fascia (MPF) and medial midfoot fat pad (MMFP) were quantified using ultrasound. RESULTS: No differences in clinical and ultrasonographical parameters were observed between the study groups. MMFP thickness was correlated with MPF thickness (p = 0.027). MMFP and MPF thicknesses were positively associated with BMI (p < 0.001 and p = 0.013, respectively), WC (p < 0.001 and p = 0.013), and WHtR (p < 0.001 and p = 0.026). The NDT measured on the right and left foot correlated with WHtR (p = 0.038 and p = 0.009, respectively), but not with WC and BMI. CONCLUSIONS: Children with T1D show structural alterations of plantar soft tissues which seem related to body mass increase rather than diabetes pathology. Ultrasound is a valuable tool to assess early structural changes of the foot in young people with an elevated BMI.

Expression of miRNAs in Pre-Schoolers with Autism Spectrum Disorders Compared with Typically Developing Peers and Its Effects after Probiotic Supplementation.

Alteration of the microbiota-gut-brain axis has been recently recognized as a possible contributor to the physiopathology of autism spectrum disorder (ASD). In this context, microRNA (miRNAs) dysfunction, implicated both in several neuropathological conditions including ASD and in different gastrointestinal disorders (GIDs), could represent an important modulating factor. In this contextual framework, we studied the transcriptional profile of specific circulating miRNAs associated with both ASD (miR-197-5p, miR-424-5p, miR-500a-5p, miR-664a-5p) and GID (miR-21-5p, miR-320a-5p, miR-31-5p, miR-223-5p) in a group of pre-schoolers with ASD and in typically developing (TD) peers. In the ASD group, we also assessed the same miRNAs after a 6-month supplementation with probiotics and their correlation with plasma levels of zonulin and lactoferrin. At baseline, the expression of miRNAs involved in ASD were significantly reduced in ASD pre-schoolers vs. TD controls. Regarding the miRNAs involved in GID, the expression levels of miR-320-5p, miR-31-5p, and miR-223-5p were significantly higher in ASD than in TD subjects, whereas miR-21-5p showed significantly reduced expression in the ASD group vs. TD group. Supplementation with probiotics did not significantly change the expression of miRNAs in the ASD population. We found a significative negative correlation between zonulin and miR-197-5p and miR-21-5p at baseline, as well as between lactoferrin and miR-223-5p after 6 months of probiotic supplementation. Our study confirms the presence of an altered profile of the miRNAs investigated in ASD versus TD peers that was not modified by supplementation with probiotics.

Screening and Identification of Putative Long Non-Coding RNA in Childhood Obesity: Evaluation of Their Transcriptional Levels.

Background and Methods: Long non-coding RNAs (LncRNAs) and microRNAs are involved in the pathogenesis of obesity, a multifactorial disease that is characterized by inflammation, cardiometabolic complications, and increased cancer risk among other co-morbidities. The up/down regulation of LncRNAs and microRNAs may play an important role in this condition to identify new diagnostic/prognostic markers. The aim of the study was to identify circulating inflammatory LncRNAs in obese adolescents (n = 54) and to evaluate whether their expression behaved differently compared to normal-weight adolescents (n = 26). To have a more complete insight, the expression of some circulating miRNAs that are linked to obesity (miR-33a, miR-223, miR-142, miR-199a, miR-181a, and miR-4454) were also analyzed. Results: LncRNAs and miRNAs were extracted simultaneously from plasma samples and amplified by Real-Time PCR. Among the 86 LncRNAs that were analyzed with custom pre-designed plates, only four (RP11-347E10.1, RP11-10K16.1, LINC00657, and SNHG12) were amplified in both normal-weight and obese adolescents and only SNHG12 showed significantly lower expression compared to the normal-weight adolescents (p = 0.026). Circulating miRNAs showed a tendency to increase in obese subjects, except for miR-181a expression. LncRNAs and miRNAs correlated with some clinical and metabolic parameters. Conclusions: Our results suggest the importance of these new biomarkers to better understand the molecular mechanisms of childhood obesity and its metabolic disorder.

Peripheral Arterial Tonometry (EndoPAT)-measured Endothelial Dysfunction in Migraine with Aura children.

BACKGROUND: The association between Migraine with Aura (MA) and vascular disease has been previously reported. We investigated whether pre-clinical vascular alterations, such as Endothelial Dysfunction, are already present in children and adolescents with Migraine with Aura. METHODS: We retrospectively enrolled 27 patients having Migraine with Aura, aged9 -18 years, and 31 age matched healthy control subjects to evaluateEndothelial Function by Peripheral Arterial Tonometry. This technique measures finger pulse wave amplitude, before and during reactive hyperaemia, and calculates the Augmentation Index (AI) and the Reactive Hyperaemia Index (RHI). We also set-up an Aura Severity Scale to assess disease severity and its relationship with AI and RHI alterations. RESULTS: Also if the case-control study resulted only partially as significant, we found there is an inversely proportional relationship between the severity of the migraine measured with Aura Severity Scale and the values of the endoscore (a significantly reduced levels of AI (p-value <0,03) and a marginal reduction of RHI levels (p-value <0,07). CONCLUSION: Further studies should explore the impact of pre-clinical vascular alterations in children and adolescents with Migraine with Aura.

Cardiometabolic risk in childhood cancer survivors.

The Italian Cancer Registry Association has estimated that for the five-year period 2016-2020, in line with the previous five years, approximately 7000 neoplasms have been diagnosed among children and 4000 among adolescents. Leukemias, brain tumors and lymphomas together account for more than two-thirds of all pediatric cancers. Fortunately, the five-years survival rate has progressively improved reaching 80% thanks to the continuing improvement of therapeutic protocols but most of these cancer survivors will have at least one chronic health condition by 40 years of age. Long-term complications concern various organs and systems and have a multifactorial etiopathogenesis. Obesity, diabetes, and metabolic syndrome represent chronic diseases that affect life expectancy. Cardiovascular risk partly linked to therapies and genetic susceptibility and partly linked to the presence of obesity, diabetes and metabolic syndrome predispose childhood cancer survivors to heart failure, coronary artery disease, valvular disease, arrhythmia. Hence the cardio- metabolic risk of childhood cancer survivors can have a significant impact on their lives, families, and on society at-large. Therefore, it is very important to know the risk factors that predispose to the development of cardio-metabolic pathologies in childhood cancer survivors, the possible primary and secondary prevention strategies, the methods of surveillance and the therapeutic approaches.

Circulating microRNAs associated with C-type natriuretic peptide in childhood obesity.

Circulating microRNAs (miRNAs) are potential biomarkers of metabolic disease implicated in the pathogenesis of obesity and at present, no data are available on a possible contribution of C-type natriuretic peptides (CNP)-linked miRNAs to childhood obesity. Our aims were to 1) perform an in silico-analysis to identify miRNAs targeting CNP gene; 2) recognize CNP-linked miRNAs associated with obesity; 3) characterize their circulating profiling in normal-weight (N) and obese adolescents (O). A clinical examination was performed in 25 N and 52 O adolescents. CNP plasma levels were detected by immunometric assay while miRNA expression was carried out on peripheral blood using Real-Time PCR. Plasma CNP resulted significantly lower in O than in N (5.58 ±â€¯0.62 vs.14.78 ±â€¯1.35 pg/mL, p < 0.0001). In silico-analysis disclosed several specific circulating CNP-linked miRNAs among which miR-33a-3p, miR-223-5p and miR-142-5p also associated with obesity. MiR-199-5p and miR-4454, known to be associated with obesity but not with CNP, were also studied. miR-223-5p and miR-33a-3p resulted significantly (p = 0.05) higher in O (0.97 ±â€¯0.1; 0.85 ±â€¯0.1, respectively) than in N (0.66 ±â€¯0.11; 0.51 ±â€¯0.08, respectively). Plasma CNP correlated inversely with miR-33a-3p (p = 0.036), miR-223-5p (p = 0.004), miR-199-5p (p = 0.003) and miR-4454 (p < 0.0001). Significantly positive correlations were observed between miR-33a-3p and miR-223-5p (p = 0.002) and between miR-199-5p and miR-4454 (p = 0.0001). Applying a multiple linear regression model, miR-142-5p, miR-199a-5p, miR-223-5p, miR33a-3p, diastolic blood pressure (DBP) and age were independent determinants of CNP. Our results underline the concept that expanding our knowledge on the behaviour of circulating miRNA profile may have a promising role for early identification of obese children at increased risk of cardiometabolic alterations.

C-type natriuretic peptide plasma levels and whole blood mRNA expression show different trends in adolescents with different degree of endothelial dysfunction.

C-type natriuretic peptide (CNP) is an endogenous adipogenesis regulator whose plasma levels in childhood are known, while no data are available on its expression. Our aim was to evaluate both CNP plasma levels and CNP system expression in whole blood obtained from normal-weight (N, n = 24) and obese (O, n = 16) adolescents (age:13.5 ± 0.4 years). Endothelial function was assessed measuring reactive hyperemia index (RHI). CNP plasma levels, evaluated with specific RIA, resulted significantly lower in O than in N (6.1 ± 0.8 vs.15.2 ± 1.3 pg/mL; p < 0.0001), while CNP/NPR-B/NPR-C mRNA, measured by Real-Time PCR, resulted similar in N (4.1 ± 1.7; 5.0 ± 1.6; 2.2 ± 0.9) and in O (4.3 ± 1.6; 3.5 ± 1.1; 2.3 ± 0.8). RHI was significantly lower in O than in N (1.4 ± 0.08 vs.2.1 ± 0.04, p < 0.0001). Dividing all subjects according to the RHI median value, irrespective of the presence or absence of obesity (Group 1 > 1.9, n = 23, Group 2 < 1.9, n = 17), CNP plasma concentrations resulted significantly (p = 0.014) higher in Group 1 (14.6 ± 1.6) than in Group 2 (7.5 ± 1.0), showing a significant correlation with RHI (p = 0.0026), while CNP mRNA expression was, surprisingly, higher in Group 2 (7.0 ± 2.3) than in Group 1 (1.8 ± 0.4; p = 0.02). NPR-B mRNA resulted similar in both Groups (4.3 ± 1.6; 4.7 ± 1.3) and NPR-C significantly higher in Group 2 (p = 0.02). Our data suggest different trends between CNP plasma levels and expression, assessed for the first time in whole blood, that could reflect changes occurring both at CNP transcriptional level in activated leukocytes due to inflammation, and at circulating levels, due to CNP paracrine/autocrine activities. This could represent an interesting area for new therapies able to modulate endothelial dysfunction.

The Spectrum of Vitamin D Deficiency: Description of a Family.

Background Vitamin D deficiency represents a global health problem, affecting children and adolescents worldwide. Objects To confirm that vitamin D deficiency can present as a spectrum of clinical pictures. Methods We diagnosed nutritional rickets in a 10-month-old infant of Senegal origin with several risk factors for vitamin D deficiency. As many of these factors affected also his cohabitant relatives, we evaluate infant's family members (mother and 4 brothers) looking for other vitamin D deficiency-related comorbidities. Results 3 brothers had asymptomatic vitamin D deficiency and 2 of them (9.8 and 13.4 years-old) showed secondary hyperparathyroidism. The fourth brother (11.3 years-old) had nutritional rickets. Their mother was affected by osteomalacia. None of them received vitamin D supplementation. Conclusion Vitamin D deficiency may present as a spectrum of clinical pictures, representing a continuum ranging from asymptomatic/subtle conditions to overt rickets/osteomalacia. Immigrant families are at high risk for vitamin D deficiency at every age. If a case of symptomatic vitamin D deficiency is recognized, then the evaluation of the all family members is recommended, as they can have the same and/or other risk factors for vitamin D deficiency.

Skin advanced glycation end-products evaluation in infants according to the type of feeding and mother's smoking habits.

OBJECTIVES: This study was conducted to assess whether formula-fed infants had increased skin advanced glycation end-products compared with breastfed ones. We also evaluated the effect of maternal smoke during pregnancy and lactation on infant skin advanced glycation end-products accumulation. METHODS: Advanced glycation end-product-linked skin autofluorescence was measured in 101 infants. RESULTS: In infants born from non-smoking mothers, advanced glycation end-products were higher in formula-fed subjects than in breastfed subjects (0.80 (0.65-0.90) vs 1.00 (0.85-1.05), p < 0.001). Advanced glycation end-products in breastfed infants from smoking mothers were higher than in those from non-smoking mothers (0.80 (0.65-0.90) vs 1.00 (0.90-1.17), p = 0.009). CONCLUSION: Formula-fed infants had increased amounts of advanced glycation end-products compared with the breastfed ones, confirming that breast milk represents the best food for infants. Breastfed infants from mothers smoking during pregnancy and lactation had increased skin advanced glycation end-products, suggesting that smoke-related advanced glycation end-products transfer throughout breast milk. Moreover, advanced glycation end-products may already increase during gestation, possibly affecting fetal development. Thus, we reinforced that smoking must be stopped during pregnancy and lactation.

C-type natriuretic peptide is closely associated to obesity in Caucasian adolescents.

CNP is a natural regulator of adipogenesis playing a role in the development of obesity in childhood. Aim of the study was to evaluate CNP plasma levels in normal-weight (N), overweight (OW) and obese adolescents (O). Eighty two subjects (age:12.8±2.4, years) without cardiac dysfunction were enrolled and CNP plasma levels were measured by RIA. NT-proBNP, MR-proANP, AGEs, reactive hyperemia index (RHI) and standard clinical chemistry parameters were also measured. O and OW adolescents had higher values of BMI and fat mass than N. CNP levels were significantly lower in OW:4.79[3.29-21.15] and O:3.81[1.55-13.4] than in N:13.21[7.6-37.8]; p<0.0001N vs O, p=0.0003N vs OW). LogCNP values correlated significantly and inversely with BMI z-score, FM%, TF% and circulating levels of CRP, insulin, total cholesterol, LDL, and triglycerides, in addition to an inverse relationship with skin AGEs and a direct correlation with RHI. LogCNP was also inversely associated with LogNT-proBNP and LogMR-proANP values. Using ROC analysis the risk of obesity resulted significantly (p≪0.0001) associated with CNP values (AUC=0.9724). These results suggest that CNP may play a more important role than BNP and ANP related peptides, as risk marker of obesity, in addition to its involvement in adipogenesis and endothelial dysfunction.