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

Sequence Analysis of the UCP1 Gene in a Severe Obese Population from Southern Italy

Brown adipose tissue, where Uncoupling Protein 1 (UCP1) activity uncouples mitochondrial respiration, is an important site of facultative energy expenditure. This tissue may normally function to prevent obesity. Our aim was to investigate by sequence analysis the presence of UCP1 gene variations that may be associated with obesity. We studied 100 severe obese adults (BMI > 40 kg/m2) and 100 normal-weight control subjects (BMI range = 19–24.9 kg/m2). We identified 7 variations in the promoter region, 4 in the intronic region and 4 in the exonic region. Globally, 72% of obese patients bore UCP1 polymorphisms. Among UCP1 variants, g.IVS4−208T>G SNP was associated with obesity (OR: 1.77; 95% CI = 1.26–2.50; P = .001). Further, obese patients bearing the g.−451C>T (CT+TT) or the g.940G>A (GA+AA) genotypes showed a higher BMI than not polymorphic obese patients (P = .008 and P = .043, resp.). In conclusion, UCP1 SNPs could represent “thrifty” factors that promote energy storage in prone subjects

Celiac disease-associated Neisseria flavescens decreases mitochondrial respiration in CaCo-2 epithelial cells: Impact of Lactobacillus paracasei CBA L74 on bacterial-induced cellular imbalance

: We previously identified a Neisseria flavescens strain in the duodenum of celiac disease (CD) patients that induced immune inflammation in ex vivo duodenal mucosal explants and in CaCo-2 cells. We also found that vesicular trafficking was delayed after the CD-immunogenic P31-43 gliadin peptide-entered CaCo-2 cells and that Lactobacillus paracasei CBA L74 (L. paracasei-CBA) supernatant reduced peptide entry. In this study, we evaluated if metabolism and trafficking was altered in CD-N. flavescens-infected CaCo-2 cells and if any alteration could be mitigated by pretreating cells with L. paracasei-CBA supernatant, despite the presence of P31-43. We measured CaCo-2 bioenergetics by an extracellular flux analyser, N. flavescens and P31-43 intracellular trafficking by immunofluorescence, cellular stress by TBARS assay, and ATP by bioluminescence. We found that CD-N. flavescens colocalised more than control N. flavescens with early endocytic vesicles and more escaped autophagy thereby surviving longer in infected cells. P31-43 increased colocalisation of N. flavescens with early vesicles. Mitochondrial respiration was lower (P < .05) in CD-N. flavescens-infected cells versus not-treated CaCo-2 cells, whereas pretreatment with L. paracasei-CBA reduced CD-N. flavescens viability and improved cell bioenergetics and trafficking. In conclusion, CD-N. flavescens induces metabolic imbalance in CaCo-2 cells, and the L. paracasei-CBA probiotic could be used to correct CD-associated dysbiosis

Age-Related Reference Intervals of the Main Biochemical and Hematological Parameters in C57BL/6J, 129SV/EV and C3H/HeJ Mouse Strains

BACKGROUND: Although the mouse is the animal model most widely used to study the pathogenesis and treatment of human diseases, reference values for biochemical parameters are scanty or lacking for the most frequently used strains. We therefore evaluated these parameters in the C57BL/6J, 129SV/EV and C3H/HeJ mice. METHODOLOGY/PRINCIPAL FINDINGS: We measured by dry chemistry 26 analytes relative to electrolyte balance, lipoprotein metabolism, and muscle/heart, liver, kidney and pancreas functions, and by automated blood counter 5 hematological parameters in 30 animals (15 male and 15 female) of each mouse strain at three age ranges: 1-2 months, 3-8 months and 9-12 months. Whole blood was collected from the retro-orbital sinus. We used quality control procedures to investigate analytical imprecision and inaccuracy. Reference values were calculated by non parametric methods (median and 2.5(th) and 97.5(th) percentiles). The Mann-Whitney and Kruskal-Wallis tests were used for between-group comparisons. Median levels of GLU, LDH, Chol and BUN were higher, and LPS, AST, ALP and CHE were lower in males than in females (p range: 0.05-0.001). Inter-strain differences were observed for: (1) GLU, t-Bil, K+, Ca++, PO(4)- (p<0.05) and for TAG, Chol, AST, Fe++ (p<0.001) in 4-8 month-old animals; (2) for CK, Crea, Mg++, Na++, K+, Cl- (p<0.05) and BUN (p<0.001) in 2- and in 10-12 month-old mice; and (3) for WBC, RBC, HGB, HCT and PLT (p<0.05) during the 1 year life span. CONCLUSION/SIGNIFICANCE: Our results indicate that metabolic variations in C57BL/6J, 129SV/EV and C3H/HeJ mice after therapeutic intervention should be evaluated against gender- and age-dependent reference intervals

Bariatric surgery improves blood metabolic profile and miRNA based regulation of gene expression in subcutaneous adipose tissue from morbid obese patients

Obesity is a multifactorial disorder influenced by the interaction of genetic, behavioral and environmental factors, control of appetite and energy expenditure, and the availability of high-calorie food. Adipocyte and adipose tissue dysfunctions are the primary defects in obesity and may link obesity to such disorders as increased insulin resistance, type 2 diabetes, fatty liver disease, hypertension, dyslipidemia, atherosclerosis and cancer. Bariatric procedures have been shown to be more effective in the management of morbid obesity than lifestyle interventions and pharmacotherapy. The aim of this study was to investigate if the significant and sustained weight loss after laparoscopic adjustable gastric banding (LAGB) resulted in an improvement in the metabolism of obese subjects in terms of serum biochemical parameters and phenotypic characteristics (cell size and number) of subcutaneous adipose tissue (SAT). Moreover, we investigated if miRNA based regulation of gene expression could be involved in the mechanisms underlying the weight loss. We evaluated 20 severely obese subjects before LAGB (T0, mean body mass index [BMI] 44.9 kg/m2) and after the loss of >30% excess weight (T1, mean BMI 31.5 kg/m2). We also evaluated 10 normal weight subjects. We collected SAT and serum samples from all subjects. Conventional biochemical parameters were measured by routine laboratory procedures, and leptin and adiponectin by Luminex xMAP technology. Five-micron sections were prepared from all paraffin-embedded SAT blocks. Slides were then stained with hematoxylin & eosin. Macrophagic infiltration were evaluated by CD68 immunohistochemical analysis. Levels of insulin, homeostasis model assessment-insulin resistance, triglycerides and liver markers as well as the leptin/adiponectin ratio were significantly lower at T1 vs T0 (p<0.05). The number of SAT adipocytes was greater and their size smaller at T1 than at T0 (p<0.05). Moreover, the morphological characteristics of SAT adipocytes at T1 did not differ from those of control adipocytes (p=0.89). Weight loss induced by bariatric surgery resulted in a significant reduction in the inflammation level, as measured by CD68 score. We found that 4% of miRNAs was differently expressed in T1 vs T0, of these 3% was up-expressed and 1% was down-expressed. Bioinformatic analysis of deregulated miRNAs showed several target genes which were involved in relevant pathways among which: pathways in cancer, regulation of endocytosis, MAPK signaling, TGF-beta signaling. LAGB induces an improvement in the obese metabolic status, which could result in a decreased risk of obese-associated diseases. Moreover, the normalization of adipocyte features at T1 vs T0 suggests a regression of SAT inflammation. Furthermore, our data support that the improvement of the metabolic status induced by bariatric surgery in our obese patients could be obtained also by miRNA-based regulation of gene expression