Modern dietary habits are linked to high exposure to Advanced Glycation End products (AGEs) mainly due to the dramatic increase in the consumption of highly processed foods in recent years. Body levels of these compounds vary with food intake and are almost interconnected with age and health status, formally embodying indicators of oxidative stress and inflammation in adults. However, the relationship between AGEs and health issues has not been definitively understood in children, and several pediatric investigations have produced conflicting evidence. Besides, despite extensive research, there are no universally accepted analytical techniques for measuring AGE levels in the human body, with several approaches available, each with its advantages and disadvantages. This pilot study aimed to investigate the association between urinary AGEs, measured using spectrofluorimetry-based assays, and circulating microRNAs (c-miRNAs) in a subsample (n = 22) of Italian children participating in the I.Family Study. Anthropometric measurements, biochemical markers, and miRNA profiles were assessed. The first indication of a relationship between urinary AGEs and c-miRNAs in the context of obesity was found. Specifically, four miRNAs, hsa-miR-10b-5p, hsa-miR-501-5p, hsa-miR-874-3p, and hsa-miR-2355-5p were significantly associated with levels of urinary AGEs. The association between AGEs, obesity, inflammation markers, and specific miRNAs highlights the complex interplay between these factors and their potential impact on cellular and tissue homeostasis. The discovery of altered c-miRNAs profiling has the potential to offer innovative methods for assessing early changes in the body's AGE pool and allow recognition of an increased risk of disease susceptibility, routinely undetected until metabolic complications are identified.

miRNAs are small noncoding RNAs that control gene expression at the posttranscriptional level. It has been recognized that dysregulation of miRNAs reflects the state and function of cells and tissues, contributing to their dysfunction. The identification of hundreds of extracellular miRNAs in biological fluids has underscored their potential in the field of biomarker research. In addition, the therapeutic potential of miRNAs is receiving increasing attention in numerous conditions. On the other hand, many operational issues, including stability, delivery systems, and bioavailability, have yet to be resolved. In this dynamic field, biopharmaceutical companies are increasingly engaged, and ongoing clinical trials point to anti-miR and miR-mimic molecules as novel classes of molecules for upcoming therapeutic applications. This reprint aims to provide a broad overview of current knowledge on several outstanding issues and new opportunities offered by miRNAs in the treatment of diseases and as early diagnostic tools for next-generation medicine.

miRNAs are small noncoding RNAs that control gene expression at the posttranscriptional level. It has been recognised that miRNA dysregulation reflects the state and function of cells and tissues, contributing to their dysfunction. The identification of hundreds of extracellular miRNAs in biological fluids has underscored their potential in the field of biomarker research. In addition, the therapeutic potential of miRNAs is receiving increasing attention in numerous conditions. On the other hand, many operative problems including stability, delivery systems, and bioavailability, still need to be solved. In this dynamic field, biopharmaceutical companies are increasingly engaged, and ongoing clinical trials point to anti-miR and miR-mimic molecules as an innovative class of molecules for upcoming therapeutic applications.

Tritordeum is a cereal species that resulted from the hybridization between durum wheat (T. durum) and wild barley (H. chilense). It is considered a natural crop since obtained using traditional breeding techniques. In this study, Tritordeum was explored as a candidate for low-toxic cereal species.

Abstract: miRNAs are small noncoding RNAs that control gene expression at the posttranscriptional level. It has been recognised that miRNA dysregulation reflects the state and function of cells and tissues, contributing to their dysfunction. The identification of hundreds of extracellular miRNAs in biological fluids has underscored their potential in the field of biomarker research. In addition, the therapeutic potential of miRNAs is receiving increasing attention in numerous conditions. On the other hand, many operative problems including stability, delivery systems, and bioavailability, still need to be solved. In this dynamic field, biopharmaceutical companies are increasingly engaged, and ongoing clinical trials point to anti-miR and miR-mimic molecules as an innovative class of molecules for upcoming therapeutic applications. This article aims to provide a comprehensive overview of current knowledge on several pending issues and new opportunities offered by miRNAs in the treatment of diseases and as early diagnostic tools in next-generation medicine.

In late years, a substantial advance has been made in the study of the role of microRNAs (miRNAs) in the pathogenesis of diseases. New evidence shows that dysregulation of miRNAs represents an etiologic factor of a variety of disorders, including cancer. Besides, miRNAs have also emerged as fundamental regulators of metabolic processes taking part in maintaining energy balance and metabolic homeostasis. Dysregulation of miRNAs, by directly affecting the status and functions of adipose tissue, pancreas, liver, and muscle, contributes to metabolic abnormalities being fully implicated in body fat accumulation, obesity, and obesity-related diseases. The discovery of circulating miRNAs has highlighted their potential as endocrine signalling molecules and disease indicators. Nevertheless, the study of the involvement of miRNAs in metabolic dysfunctions is still a young field of research and information on their role is nearly limited to date. As well, the contemporary rising in childhood obesity rates creates a need for tools that quantify metabolic changes in obese children and adolescents for the early detection or prevention of comorbidities. This chapter aims to provide current insights into the role of miRNAs as biological markers focusing on their link to obesity and obesity-related morbid conditions.

Immuno-laser capture microdissection (Immuno-LCM) has been used to analyze cell-specific gene expression profiles. However, the usefulness of such a technique is frequently limited by RNA degradation. We, therefore, developed a rapid protocol of LCM on mirror sections, which allows for preserving RNA integrity. With such a procedure, we investigated cell-type-specific gene expression of ?? intraepithelial lymphocytes (IELs) in untreated celiac disease (CD). An increase in TGF-? mRNA expression levels was observed in ?? + IELs compared to intestinal enterocytes (IEs), whereas anti-inflammatory IL-10 mRNA production from ?? + IELs was lower compared to IEs. In untreated CD patients, the production of anti-inflammatory cytokines by ?? + IELs is suggestive of a regulatory function, thus playing a critical role in limiting inflammation. This work underscores the importance of LCM on mirror sections as a valuable tool to perform cell-type-specific molecular analysis in tissue.

Background: The promising emergence of circulating miRNAs as stable, reproducible, and consistent among individuals has opened a promising research avenue for non-invasive biomarker detection. A solid connection between circulating miRNAs and glycaemic as well as metabolic homeostasis has been established, demonstrating that levels of specific miRNAs vary under different physio-pathological conditions. Objective: In this study, we explored the expression of candidate miRNAs, in relation to biomarkers of insulin sensitivity (insulin levels, HOMA index) in a subgroup (n=58) of subjects participating in the European I.Family Study, a project aimed at assessing the determinants of eating behavior in children and adolescents and related health outcomes (Trial registration: ISRCTN62310987). The sample included children/adolescents with overweight/obesity, as overweight/obesity is a known risk factor for impaired glucose homeostasis and metabolic disorders. Besides, dysregulation in insulin signaling is among the typical and earliest metabolic signs predisposing to the development of type 2 diabetes. Of note, no one of the study-subject suffered from metabolic syndrome or TD2, no alterations in blood levels of glucose and HbA1c were recognized. Results: The differential expression of miR-191-3p and miR-375 was statistically confirmed to be associated with early changes in insulin level and HOMA index in both girls and boys. The analyses were adjusted for confounding factors, including puberty since puberty is characteristically associated with a decrease in insulin sensitivity. Covariates also included the country of origin, age, BMI z-score, total energy intake, energy from fats, energy from carbohydrates, energy from proteins, and the highest educational level of parents since several studies indicated that parental influences have a marked effect on nutritional habits, dietary intakes, and food preferences of young children. To gain a mechanistic understanding of how the miRNAs could be associated with glycaemic impairment, molecular interactions of confirmed miRNAs were also predicted by bioinformatics. Conclusion: In recent years, numerous efforts have been made to identify early, reliable and predictive biomarkers of altered insulin sensitivity; nevertheless, risk-screening methods are nearly limited. Identification of circulating miRNAs associated with insulin impairment may offer novel approaches to assessing early variations in insulin sensitivity to timely detect insulin impairment and prediabetes. Furthermore, the results provide new evidence about the underlying molecular mechanisms connected to early changes in glycaemic homeostasis.

Background: During the past decade, obesity has become a global epidemic with interconnected health, social, and economic concerns. Cumulative evidence proposes that over-nutrition-induced obesity leads to an upturn in the innate immune system and local inflammatory process in adipose tissue. The resulting persistent chronic low-grade inflammation designates a hallmark of obesity that leads to and perpetuates the metabolic alterations and insulin resistance in target organs. Of note, inflammation and its supporting mechanisms are considered closely related to the progression of numerous diseases. Several actors have been involved in governing the inflammatory response, including epigenetic players. Among these, miRNAs are emerging as crucial regulators of immune cell development, immune responses, autoimmunity, and inflammation. Objective: In this study, we aimed at recognizing the involvement of candidate miRNAs in relation to inflammation-associated biomarkers in a subsample of European children with overweight and obesity participating in the I.Family Study (www.ifamilystudy.eu). The study subjects included healthy individuals (n=79) with increased adiposity since this condition contributes to the early occurrence of chronic low-grade inflammation and the intertwisted metabolic changes. We focused on the acute phase reagent CRP and several cytokines selected as conceivable inflammation biomarkers. Associations of miRNAs expression with CRP and the different interleukins were performed using linear regression analyses adjusting for covariates including country of origin, age, BMI z-score, puberty status. Results: We found that the chronic low-grade CRP elevation exhibited a highly significant association with miR-26b-3p and miR-576-5p in the boys' subgroup. Furthermore, the association of CRP with miR-10b-5p and miR-31-5p was highly significant in girls. We also found major sex-related associations of candidate miRNAs with cytokines: except for IL-6, significant associations of miR-10b-5p and miR-26b-3p with TNF-?, IL1-Ra, IL-8, and IL-15 levels were found exclusively in boys. Conclusion: The findings of this exploratory study propose major differences in the relationship of circulating miRNAs and inflammatory biomarkers across sexes pointing to a conceivable role of miRNAs among the candidate epigenetic mechanisms related to the low-grade inflammation process in childhood obesity, calling for more attention in this largely underexplored area.

Increasing data suggest that overnutrition-induced obesity may trigger an inflammatory process in adipose tissue and upturn in the innate immune system. Numerous players have been involved in governing the inflammatory response, including epigenetics. Among epigenetic players, miRNAs are emerging as crucial regulators of immune cell development, immune responses, autoimmunity, and inflammation. In this study, we aimed at identifying the involvement of candidate miRNAs in relation to inflammation-associated biomarkers in a subsample of European children with overweight and obesity participating in the I.Family study. The study sample included individuals with increased adiposity since this condition contributes to the early occurrence of chronic low-grade inflammation. We focused on the acute-phase reagent C-reactive protein (CRP) as the primary outcome and selected cytokines as plausible biomarkers of inflammation. We found that chronic low-grade CRP elevation shows a highly significant association with miR-26b-3p and hsa-miR-576-5p in boys. Furthermore, the association of CRP with hsa-miR-10b-5p and hsa-miR-31-5p is highly significant in girls. We also observed major sex-related associations of candidate miRNAs with selected cytokines. Except for IL-6, a significant association of hsa-miR-26b-3p and hsa-miR-576-5p with TNF-, IL1-Ra, IL-8, and IL-15 levels was found exclusively in boys. The findings of this exploratory study suggest sex differences in the association of circulating miRNAs with inflammatory response biomarkers, and indicate a possible role of miRNAs among the candidate epigenetic mechanisms related to the process of low-grade inflammation in childhood obesity.

Background In recent years, the exciting emergence of circulating miRNAs as stable, reproducible, and consistent among individuals has opened a promising research opportunity for the detection of non-invasive biomarkers. A firm connection has been established between circulating miRNAs and glycaemic as well as metabolic homeostasis, showing that levels of specific miRNAs vary under different physio-pathological conditions. Objective In this pilot study, we investigated the expression of candidate miRNAs, hsa-miR-191-3p and hsa-miR-375, in relation to biomarkers associated with insulin sensitivity in a subgroup (n=58) of subjects participating to the European I.Family Study, a project aimed to assess the determinants of eating behaviour in children and adolescents and related health outcomes. The sample included overweight/obese children/adolescents since overweight/obesity is a known risk factor for impaired glucose homeostasis and metabolic disorders. Biological targets of candidate miRNAs were also explored in silico. Results We observed a significant association of the two miRNAs and early changes in glycaemic homeostasis, independent of covariates including country of origin, age, BMI z-score, puberty status, highest educational level of parents, total energy intake, energy from fats, energy from carbohydrates, and energy from proteins. Conclusion Identification of circulating miRNAs associated with insulin impairment may offer novel approaches of assessing early variations in insulin sensitivity and provide evidence about the molecular mechanisms connected to early changes in glycaemic homeostasis.

Scope: Several studies reported a role of amylase/trypsin-inhibitors (ATIs) of common wheat species in promoting immune reactions. Here, we investigated in celiac disease (CD), the immunogenic properties of ATIs from diploid compared to common hexaploid wheats after an in vitro proteolytic hydrolysis. Methods and Results: ATIs purified from two lines of diploid Triticum monococcum (TM), Monlis and Norberto-ID331, and from Triticum aestivum (TA), Sagittario, were digested with pepsin-chymotrypsin (PC) enzymes and analyzed using a proteomic approach, and subsequently their immune stimulatory properties were investigated on jejunal biopsies and T-cell lines from CD patients. No significant expression of IL-8 and TNF-? were detected on biopsies cultured with ATIs from TM in comparison with ATIs from TA. No significant IFN-? production was observed in intestinal gliadin- raised T-cells in response to ATIs from both TM and TA wheats. Proteomic results revealed that both TM ATIs showed reduced stability to proteolytic enzymes compared to TA ones. Conclusion: TM ATIs are substantially different from those of TA, showing a reduced ability to trigger the innate immunity in CD and a higher susceptibility to enzymatic hydrolysis.

Background and Aim. Although the expansion of TCR?D+ intraepithelial lymphocytes(IELs) is a hallmark of Celiac Disease (CD), to date little is known about the mechanismunderlying their expansion and their role in CD pathogenesis. Different evidence pointstoward an immunoregulatory role for?D+ IEL. We have shown that IL-15, highly expressedin CD intestinal mucosa, impairs the functions of Foxp3+ Treg cells. Different approacheshave been used to study the pattern of cytokines produced by?D+ IELs in CD, which haveled sometimes to contradictory results. It is essential to analyse specific cell types to identifyand define biologically important processes in CD pathogenesis. The development in thelast decade of laser capture microdissection (LCM) has allowed this goal to be achieved.The aim of this study was to investigate the regulatory function of TCR?D+ IELs, isolatedby LCM, and to understand if their role could be impaired by IL-15.Methods.Frozensection of jejunum was obtained from 10 untreated CD patients. Using LCM in combinationwith immunohistochemistry, the?D+ IELs and intestinal epithelial cells were isolated andtotal RNA was extracted. The relative expression of TGF-?, IL-10 and IL-15 was evaluatedby quantitative reverse transcriptase real-time PCR (qRT-PCR).Results were expressed asthe ratio of each cytokine cDNA concentration relative to the concentration of the GAPDHhousekeeping gene cDNA in the same sample. Statistical analyses were performed inGraphPad Prism and data were reported as normalized mean expression ± standard error. Results. An increased gene expression level of TGF-?and IL-10 (p<0.05) was observed in?D+ IELs from untreated CD patients compared to the intestinal epithelial cells which,otherwise, have been characterized by an increased gene expression level of IL-15 (p<0.05).Conclusion.In the epithelium compartment of untreated CD jejunal mucosa we found thatenterocytes and?D+ T cells produce respectively IL-15 and anti-inflammatory cytokinesTGF-?and IL-10. Taken toghether our results suggest that in CD the?D+ IELs have regulatoryfunction, which is not affected by IL-15, produced by enterocytes.

In recent years, significant progress has been made on the role of regulatory microRNAs (miRNAs) in the development of diseases. miRNAs have emerged as key regulators of metabolic processes playing critical roles in maintaining organismal energy balance and metabolic homeostasis. Dysregulation of miRNAs may contribute to metabolic abnormalities since they play pivotal roles in body fat accumulation, obesity, and obesity-related disease by directly affecting status and functions of adipose tissue, pancreas, liver, and muscle. The discovery of circulating miRNAs highlighted their potential as either endocrine signalling molecules or disease indicators. However, the study of miRNAs involvement in metabolic diseases is still a young research field and information concerning their roles remains nearly limited nowadays. This chapter aims to provide recent findings concerning the contribution of miRNAs to obesity and obesity-related morbid conditions.

It is commonly recognized that sleep is essential for children's health, and that insufficient sleep duration is associated with negative health outcomes. In humans, sleep duration and quality are influenced by genetic, environmental and social factors. Epigenetic mechanisms, likewise, regulate circadian rhythms and sleep patterns. In the present study, we aimed to identify circulating microRNAs associated with sleep duration in a subsample of normal-weight European children/adolescents (n=111) participating in the I.Family Study. Study subjects were divided into two groups based upon the self-reported sleep duration, according to the recommended amount of sleep for pediatric populations. Sleep needs for children <13 years were at least 9 hours per day, and for children >13 were at least 8 hours per day. There were group differences (short sleepers versus normal sleepers) in circulating levels of miR-26b-3p [mean (I.C.)] = 2.0 (1.3 - 2.7) versus 2.3 (1.9 - 2.7), p-value = 0.05 and miR-485-5p [mean (I.C.)] = 0.6 (0.3 - 0.9) versus 0.9 (0.7 - 1.0), p-value < 0.001 adjusting for country of origin, age, sex, pubertal status, screen time, and highest educational level of parents. Our findings show for the first time that the sleep duration reflects the profile of specific circulating microRNAs in school-aged children and adolescents. It is conceivable that epigenetic modifications, mainly related to circadian rhythm control, may be modulated or interfere with sleep duration.

Background and aims: Laser capture microdissection (LCM) is a powerful tool for the isolation of specific tissue compartments. We aimed to investigate the mucosal immune response that takes place in different intestinal compartments of IBD patients, dissected by LCM, analyzing cytokines expression profile and endoplasmic reticulum (ER) stress markers. Methods: Frozen sections of gut were obtained from patients with Crohn's disease (CD), ulcerative colitis (UC) and from controls. Using LCM, surface epithelium (SE) and lamina propria (LP) compartments were isolated and total RNA extracted. The relative expression of Th1, Th17 and Treg cytokines was evaluated by quantitative reverse transcriptase real-time PCR (qRT-PCR), in addition to the assessment of mRNA splicing of the transcription factor X-box binding protein-1 (XBP1). Human neutrophil elastase (HNE) and the transcription factor forkhead box P3 (Foxp3) were also analyzed by immunohistochemistry. Results: The increased expression of IL-17 was observed in both intestinal compartments of IBD patients when compared to controls. IFN- ?, TNF-?, IL-10, HNE and Foxp3 were overexpressed in the LP compartment of both IBD patients as compared to controls. An upregulation of IFN-? and an infiltration of HNE+ cells was found in the SE of patients with UC. Splicing of XBP1 mRNA was recognized in both intestinal compartments of IBD patients when compared to controls. Conclusions: In IBD patients, both intestinal compartments are involved in Th17 response, whereas, LP compartment plays a prominent role in Th1 and Treg immune responses. Nevertheless, high level of IFN- ? was found in the SE of UC patients, suggesting that this compartment is involved in the Th1 immune response. Our data also suggested that ER stress signalling is active in both LP and SE compartment of IBD patients, thus advocating that ER stress and immunity are intertwined.

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Background: Nearly ten years ago, the WHO reported the increasing prevalence of obesity worldwide as a challenge for public health due the associated adverse consequences. Omic studies demonstrated that microRNA (miRNA) changes in tissues correlate with several diseases, including obesity. Other studies suggested a remarkable stability of miRNA also in blood, emphasing their potential as theranostic agents. Aim: This study investigated the profiles of circulating miRNAs in plasma samples of normal weight (n=159) and overweight/obese (n=149) children participating to the I.Family study, an EC funded study finalized to investigate the etiology of overweight, obesity and related disorders in children of eight European countries (www.ifamilystudy.eu). Differences in miRNA expression patterns with respect to anthropometric and biochemical variables were explored. Results: A high degree of variability in levels of circulating miRNAs was recognised among children from different countries. Several miRNAs differentially expressed in overweight/low grade obesity children were characterized (miR-551a and miR-501-5p up-regulated; miR-10b-5p, miR-191-3p, miR-215-5p and miR-874-3p down-regulated). ROC curves were constructed for confirmed miRNAs. Single miRNAs exhibited low AUC values with the highest values for miR-874-3p and miR-501-5p which in combination provided an interesting value (AUC=0.755). Pearson's analysis confirmed that miR-10b-5p, miR-215-5p, miR-501-5p, miR-551a, and miR-874-3p correlated with BMI z-score. Molecular interactions of obesity-associated miRNAs were also predicted. Computational analysis indicated that miRNAs act as key regulators of metabolism, playing pivotal roles in early stages of obesity by affecting multiple candidate genes. Conclusions: Although causal pathways cannot be definitely inferred it is conceivable that circulating miRNAs may be new biomarkers of early childhood obesity.