Laura Iaffaldano

MicroRNA-449a Overexpression, Reduced NOTCH1 Signals and Scarce Goblet Cells Characterize the Small Intestine of Celiac Patients

MiRNAs play a relevant role in regulating gene expression in a variety of physiological and pathological conditions including autoimmune disorders. MiRNAs are also important in the differentiation and function of the mouse intestinal epithelium. Our study was aimed to look for miRNA-based modulation of gene expression in celiac small intestine, and particularly for genes involved in cell intestinal differentiation/proliferation mechanisms. A cohort of 40 children (20 with active CD, 9 on a gluten-free diet (GFD), and 11 controls), were recruited at the Paediatrics Department (University of Naples Federico II). The expression of 365 human miRNAs was quantified by TaqMan low-density arrays. We used bioinformatics to predict putative target genes of miRNAs and to select biological pathways. The presence of NOTCH1, HES1, KLF4, MUC-2, Ki67 and beta-catenin proteins in the small intestine of CD and control children was tested by immunohistochemistry. The expression of about 20% of the miRNAs tested differed between CD and control children. We found that high miR-449a levels targeted and reduced both NOTCH1 and KLF4 in HEK-293 cells. NOTCH1, KLF4 signals and the number of goblet cells were lower in small intestine of children with active CD and in those on a GFD than in controls, whereas more nuclear beta-catenin staining, as a sign of the WNT pathway activation, and more Ki67 staining, as sign of proliferation, were present in crypts from CD patients than in controls. In conclusion we first demonstrate a miRNA mediated gene regulation in small intestine of CD patients. We also highlighted a reduced NOTCH1 pathway in our patients, irrespective of whether the disease was active or not. We suggest that NOTCH pathway could be constitutively altered in the celiac small intestine and could drive the increased proliferation and the decreased differentiation of intestinal cells towards the secretory goblet cell lineage.

miRNA and protein expression profiles of visceral adipose tissue reveal miR-141/YWHAG and miR-520e/RAB11A as two potential miRNA/protein target pairs associated with severe obesity.

Adipose tissues show selective gene expression patterns, to whom microRNAs (miRNAs) may contribute. We evaluated in visceral adipose tissue (VAT) from obese and nonobese females, both miRNA and protein expression profiles, to identify miRNA/protein target pairs associated with obesity (metabolic pathways miRNA-deregulated during obesity). Obese and nonobese females [BMI 42.2 ± 1.6 and 23.7 ± 1.2 kg/m(2) (mean ± SEM), respectively] were enrolled in this study. Notably, most miRNAs were down-expressed in obese tissues, whereas most of the proteins from the investigated spots were up-expressed. Bioinformatics integration of miRNA expression and proteomic data highlighted two potential miRNA/protein target pairs: miR-141/YWHAG (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, gamma polypeptide) and miR-520e/RAB11A (Ras-related protein RAB-11A); the functional interaction between these miRNAs and their target sequences on the corresponding mRNAs was confirmed by luciferase assays. Both RAB11A and YWHAG proteins are involved in glucose homeostasis; YWHAG is also involved in lipid metabolism. Hence, the identified miRNA/protein target pairs are potential players in the obese phenotype.

High Aminopeptidase N/CD13 Levels Characterize Human Amniotic Mesenchymal Stem Cells and Drive Their Increased Adipogenic Potential in Obese Women

Maternal obesity is associated to increased fetal risk of obesity and other metabolic diseases. Human amniotic mesenchymal stem cells (hA-MSCs) have not been characterized in obese women. The aim of this study was to isolate and compare hA-MSC immunophenotypes from obese (Ob-) and normal weight control (Co-) women, to identify alterations possibly predisposing the fetus to obesity. We enrolled 16 Ob- and 7 Co-women at delivery (mean/SEM prepregnancy body mass index: 40.3/1.8 and 22.4/1.0 kg/m2, respectively), and 32 not pregnant women. hA-MSCs were phenotyped by flow cytometry; several maternal and newborn clinical and biochemical parameters were also measured. The expression of membrane antigen CD13 was higher on Ob-hA-MSCs than on Co-hA-MSCs (P = 0.005). Also, serum levels of CD13 at delivery were higher in Ob- versus Co-pregnant women and correlated with CD13 antigen expression on Ob-hA-MSCs (r2 = 0.84, P < 0.0001). Adipogenesis induction experiments revealed that Ob-hA-MSCs had a higher adipogenic potential than Co-hA-MSCs as witnessed by higher peroxisome proliferator-activated receptor gamma and aP2 mRNA levels (P = 0.05 and P = 0.05, respectively), at postinduction day 14 associated with increased CD13 mRNA levels from baseline to day 4 postinduction (P < 0.05). Adipogenesis was similar in the two sets of hA-MSCs after CD13 silencing, whereas it was increased in Co-hA-MSCs after CD13 overexpression. CD13 expression was high also in Ob-h-MSCs from umbilical cords or visceral adipose tissue of not pregnant women. In conclusion, antigen CD13, by influencing the adipogenic potential of hA-MSCs, could be an in utero risk factor for obesity. Our data strengthen the hypothesis that high levels of serum and MSC CD13 are obesity markers.

Proteome analysis of human amniotic mesenchymal stem cells (hA-MSCs) reveals impaired antioxidant ability, cytoskeleton and metabolic functionality in maternal obesity

Maternal obesity increases the risk of obesity and/or obesity-related diseases in the offspring of animal models. The aim of this study was to identify metabolic dysfunctions that could represent an enhanced risk for human obesity or obesity-related diseases in newborn or in adult life, similar to what occurs in animal models. To this aim, we studied the proteome of 12 obese (Ob-) and 6 non-obese (Co-) human amniotic mesenchymal stem cells (hA-MSCs) obtained from women at delivery by cesarean section (pre-pregnancy body mass index [mean ± SD]: 42.7 ± 7.7 and 21.3 ± 3.3 kg/m2, respectively). The proteome, investigated by two-dimensional fluorescence difference gel electrophoresis/mass spectrometry, revealed 62 differently expressed proteins in Ob- vs Co-hA-MSCs (P < 0.05), nine of which were confirmed by western blotting. Bioinformatics analysis showed that these 62 proteins are involved in several statistically significant pathways (P < 0.05), including the stress response, cytoskeleton and metabolic pathways. Oxidative stress was shown to be an early triggering factor of tissue fat accumulation and obesity-related disorders in the offspring of obese animal models. Our finding of a reduced stress response in Ob-hA-MSCs suggests that a similar mechanism could occur also in humans. Long-term follow-up studies of newborns of obese mothers are required to verify this hypothesis.

Changes in the MicroRNA Profile Observed in the Subcutaneous Adipose Tissue of Obese Patients after Laparoscopic Adjustable Gastric Banding

Background. Laparoscopic adjustable gastric banding (LAGB) results in significant lasting weight loss and improved metabolism in obese patients. To evaluate whether epigenetic factors could concur to these benefits, we investigated the subcutaneous adipose tissue (SAT) microRNA (miRNA) profile before (T0) and three years (T1) after LAGB in three morbidly obese women. Case Reports. SAT miRNA profiling, evaluated by TaqMan Array, showed four downexpressed (miR-519d, miR-299-5p, miR-212, and miR-671-3p) and two upexpressed (miR-370 and miR-487a) miRNAs at T1 versus T0. Bioinformatics predicted that these miRNAs regulate genes belonging to pathways associated with the cytoskeleton, inflammation, and metabolism. Western blot analysis showed that PPAR-alpha, which is the target gene of miR-519d, increased after LAGB, thereby suggesting an improvement in SAT lipid metabolism. Accordingly, the number and diameter of adipocytes were significantly higher and lower, respectively, at T1 versus T0. Bioinformatics predicted that the decreased levels of miR-212, miR-299-5p, and miR-671-3p at T1 concur in reducing SAT inflammation. Conclusion. We show that the miRNA profile changes after LAGB. This finding, although obtained in only three cases, suggests that this epigenetic mechanism, by regulating the expression of genes involved in inflammation and lipid metabolism, could concur to improve SAT functionality in postoperative obese patients.

The Cause of Death of a Child in the 18th Century Solved by Bone Microbiome Typing Using Laser Microdissection and Next Generation Sequencing

The history of medicine abounds in cases of mysterious deaths, especially by infectious diseases, which were probably unresolved because of the lack of knowledge and of appropriate technology. The aim of this study was to exploit contemporary technologies to try to identify the cause of death of a young boy who died from a putative “infection” at the end of the 18th century, and for whom an extraordinarily well-preserved minute bone fragment was available. After confirming the nature of the sample, we used laser microdissection to select the most “informative” area to be examined. Tissue genotyping indicated male gender, thereby confirming the notary’s report. 16S ribosomal RNA sequencing showed that Proteobacteria and Actinobacteria were more abundant than Firmicutes and Bacteroidetes, and that Pseudomonas was the most abundant bacterial genus in the Pseudomonadaceae family. These data suggest that the patient most likely died from Pseudomonas osteomyelitis. This case is an example of how new technological approaches, like laser microdissection and next-generation sequencing, can resolve ancient cases of uncertain etiopathology. Lastly, medical samples may contain a wealth of information that may not be accessible until more sophisticated technology becomes available. Therefore, one may envisage the possibility of systematically storing medical samples for evaluation by future generations.

Oropharyngeal microbiome evaluation highlights Neisseria abundance in active celiac patients

We previously profiled duodenal microbiome in active (a-), gluten-free diet (GFD) celiac disease (CD) patients and controls finding higher levels of the Proteobacterium Neisseria flavescens in a-CD patients than in the other two groups. Here, we investigate the oropharyngeal microbiome in CD patients and controls to evaluate whether this niche share microbial composition with the duodenum. We characterized by 16S rRNA gene sequencing the oropharyngeal microbiome in 14 a-CD, 22 GFD patients and 20 controls. Bacteroidetes, Proteobacteria and Firmicutes differed significantly between the three groups. In particular, Proteobacteria abounded in a-CD and Neisseria species mostly accounted for this abundance (p < 0.001), whereas Bacteroidetes were more present in control and GFD microbiomes. Culture-based oropharyngeal microbiota analysis confirmed the greater abundance of Proteobacteria and of Neisseria species in a-CD. Microbial functions prediction indicated a greater metabolic potential for degradation of aminoacids, lipids and ketone bodies in a-CD microbiome than in control and GFD microbiomes, in which polysaccharide metabolism predominated. Our results suggest a continuum of a-CD microbial composition from mouth to duodenum. We may speculate that microbiome characterization in the oropharynx, which is a less invasive sampling than the duodenum, could contribute to investigate the role of dysbiosis in CD pathogenesis.

3q29 microduplication in a small family with complex metabolic phenotype from Southern Italy

aAndrea Vitale and Giuseppe Labruna contributed equally to this work. *Corresponding authors: Pasqualina Buono, Ceinge Biotecnologie Avanzate, Via G. Salvatore 486, 80145, Naples, Italy, Phone: 00390813737892, E-mail: buono@uniparthenope.it; Dipartimento di Scienze Motorie e del Benessere, Università “Parthenope”, Naples, Italy; and IRCCS SDN, Naples, Italy; and Barbara Lombardo, Ceinge Biotecnologie Avanzate, Via G. Salvatore 486, 80145, Naples, Italy, Phone: 00390813737917, E-mail: lombardo@ceinge.unina.it; and Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, Naples, Italy Andrea Vitale, Annamaria Mancini and Andreina Alfieri: Dipartimento di Scienze Motorie e del Benessere, Università “Parthenope”, Naples, Italy; and Ceinge Biotecnologie Avanzate, Naples, Italy Giuseppe Labruna: IRCCS SDN, Naples, Italy Laura Iaffaldano and Carmela Nardelli: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, Naples, Italy Fabrizio Pasanisi: Dipartimento di Medicina Clinica e Chirurgia, Università Federico II, Naples, Italy Lucio Pastore: Ceinge Biotecnologie Avanzate, Naples, Italy; and Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, Naples, Italy Letter to the Editor

179. Helper-Dependent Adenovirus-Mediated Gene Transfer of an LDL Receptor/Transferrin Chimeric Protein Reduces Aortic Atherosclerosis in LDL Receptor-Deficient Mice

Familial hypercholesterolemia (FH) is a well-characterized genetic hyperlipidemia due in most of the cases to mutations in the LDL receptor (LDLR) gene; FH is characterized by elevated concentration of plasma LDL cholesterol (LDL-C) with consequent deposition of LDL-C in tendons, skin and arteries. Statins can lower cholesterol levels but are not effective in all patients whose prognosis is still quite poor. In the past, we have developed safe and effective gene-therapy strategies for hepatocytes transduction and consequent expression of anti-atherogenic proteins using PEGylated helper-dependent adenoviral (HD-Ad) vectors. We have recently devised a therapeutic strategy for reducing LDL using a secreted protein that can potentially be expressed in non-hepatic tissues used as bioreactors. At this aim, we developed an HD-Ad vector for the expression of the soluble form of the extracellular portion of the human LDLR fused with transferrin (LDLR/Tf). We evaluated the efficacy of LDLR/Tf in cellular models such as CHOldla7 in which we restored the cell ability to uptake of labeled LDL; subsequently, we administered intravenously 1X10E11 vp/kg of the HD-Ad vector expressing LDLR/Tf in LDLR-deficient mice and demonstrated the efficacy of the above-mentioned vector in reducing total and LDL cholesterol levels; in addition, expression of LDLR/Tf significantly reduced aortic atherosclerotic lesions in treated LDLR-deficient mice compared to controls 1.78±0.48 vs. 5.38±0.54 sq.mm.). We therefore demonstrated the efficacy of serum secretion of LDLR/Tf in reducing aortic atherosclerosis in FH mice. These results will allow the evaluation of HD-Ad vector-mediated expression of LDLR/Tf in non-hepatic tissues using alternative routes of administration in order to develop safer gene transfer protocol more compatible with clinical applications.

A specific immunophenotype and an increased adipogenic potential characterized human amniotic mesenchymal stem cells (hA-MSCs) isolated from obese pregnant women at delivery

Maternal obesity is associated to increased fetal risk of obesity and other metabolic diseases. Human amniotic mesenchymal stem cells (hA-MSC) have not been characterized in obese women. The aim of this study was to isolate and compare hA-MSC immunophenotypes from obese (Ob-) and normal weight control (Co-) women to identify alterations possibly predisposing the foetus to obesity. We enrolled 16 Ob- and 7 Co-women at delivery (mean/SEM pre-pregnancy BMI: 40.3/1.8 kg/m2 and 22.4/1.0 kg/m2, respectively). hA-MSCs were phenotyped by flow cytometry; several maternal and newborn clinical and biochemical parameters were also measured. The expression of membrane antigen CD13 was higher on Ob-hA-MSCs than on Co-hA-MSCs (P=0.0043). Also serum levels of CD13 at delivery were higher in Ob- versus Co-pregnant women and correlated with CD13 antigen expression on Ob-hA-MSCs (r2=0.84, P<0.0001). Adipogenesis induction experiments revealed that Ob-hA-MSCs had a higher adipogenic potential than Co-hA-MSCs as witnessed by higher PPARg and aP2 mRNA levels (P=0.02 and P=0.03, respectively) at post-induction day 14 associated with an increase of CD13 mRNA from baseline to day 4 post-induction (P<0.05). Adipogenesis was similar in the two sets of hA-MSCs after CD13 silencing, whereas it was increased in Co-hA-MSCs after CD13 overexpression. Pretreatment of Co- and Ob-hA-MSCs with IFN-g ng/mL) for 24 h induced upregulation of CD13 expression. In conclusion, antigen CD13, by influencing the adipogenic potential of hA-MSCs could be an in-utero risk factor for obesity. Our data strengthen the hypothesis that high levels of serum and MSC CD13 are obesity markers.