Beckwith–Wiedemann syndrome prenatal diagnosis by methylation analysis in chorionic villi

Beckwith–Wiedemann syndrome (BWS) is an imprinting disorder that can be prenatally suspected or diagnosed based on established clinical guidelines. Molecular confirmation is commonly performed on amniocytes. The possibility to use fresh (CVF) and cultured (CVC) chorionic villi has never been investigated. To verify whether CVF and CVC are reliable sources of DNA to study fetal methylation, we used pyrosequencing to test the methylation level of a number of differentially methylated regions (DMRs) at several imprinted loci (ICR1, ICR2, H19, PWS/AS-ICR, GNASXL, GNAS1A, ZAC/PLAGL1, and MEST) and at non-imprinted MGMT and RASSF1A promoters. We analyzed these regions in 19 healthy pregnancies and highlighted stable methylation levels between CVF and CVC at ICR1, ICR2, GNASXL, PWS/AS-ICR, and MEST. Conversely, the methylation levels at H19 promoter, GNAS1A and ZAC/PLAGL1 were different in CVC compared to fresh CV. We also investigated ICR1 and ICR2 methylation level of CVF/CVC of 2 BWS-suspected fetuses (P1 and P2). P1 showed ICR2 hypomethylation, P2 showed normal methylation at both ICR1 and ICR2. Our findings, although limited to one case of BWS fetus with an imprinting defect, can suggest that ICR1 and ICR2, but not H19, could be reliable targets for prenatal BWS diagnosis by methylation test in CVF and CVC. In addition, PWS/AS-ICR, GNASXL, and MEST, but not GNAS1A and ZAC/PLAGL1, are steadily hemimethylated in CV from healthy pregnancies, independently from culture. Thus, prenatal investigation of genomic imprinting in CV needs to be validated in a locus-specific manner.     

Differential uPAR recruitment in caveolar‐lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis

Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid‐rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar‐lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro‐angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid‐supported mobile bilayer lipid membranes with raft‐like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR‐GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1‐enriched biomimetic membranes, were validated by identifying a pro‐angiogenic activity of GM1‐enriched EPCs, based on GM1‐dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti‐angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar‐raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar‐raft partitioning of uPAR, as opposed to control and GM3‐challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis.     

Polyinosinic-polycytidylic acid limits tumor outgrowth in a mouse model of metastatic lung cancer

Polyinosinic-polycytidylic acid (poly I:C), a TLR3 ligand, is currently being tested in human clinical trials as an adjuvant to anti-cancer vaccines and in combination with other therapies. However, little is known about its activity in established pulmonary metastasis. The aim of our study was to elucidate the effect of poly I:C (1, 10, or 100 μg/mouse) in a mouse model of B16-F10-induced metastatic lung cancer. Lung tumor growth was arrested after a single administration of poly I:C. This was associated with higher influx of mature dendritic cells (DCs), which drove toward a Th1-like, Th17-like, and cytotoxic immune environment. The interference with IFN type I receptor signaling by means of a specific mAb reversed poly I:C-mediated tumor regression due to lower presence of myeloid DCs, cytotoxic DCs (CD11c(+)CD8(+)), NKT cells, CD8(+) T cells, and Th1-like cytokines. Moreover, the adoptive transfer of poly I:C-activated bone marrow-derived DCs into tumor-bearing mice resulted in activities similar to those of the systemic administration of poly I:C on lung tumor burden. In conclusion, our data prove that poly I:C has potential anti-tumor activity in a mouse model of established pulmonary metastasis. The activation of DCs and the production of IFN type I are responsible for an effective T cytotoxic immune response against metastatic lung cancer progression after poly I:C treatment.     

Viral adaptation to host immune responses occurs in chronic hepatitis B virus (HBV) infection, and adaptation is greatest in HBV e antigen-negative disease

Hepatitis B virus (HBV)-specific T-cell responses are important in the natural history of HBV infection. The number of known HBV-specific T-cell epitopes is limited, and it is not clear whether viral evolution occurs in chronic HBV infection. We aimed to identify novel HBV T-cell epitopes by examining the relationship between HBV sequence variation and the human leukocyte antigen (HLA) type in a large prospective clinic-based cohort of Asian patients with chronic HBV infection recruited in Australia and China (n = 119). High-resolution 4-digit HLA class I and II typing and full-length HBV sequencing were undertaken for treatment-naïve individuals (52% with genotype B, 48% with genotype C, 63% HBV e antigen [HBeAg] positive). Statistically significant associations between HLA types and HBV sequence variation were identified (n = 49) at 41 sites in the HBV genome. Using prediction programs, we determined scores for binding between peptides containing these polymorphisms and associated HLA types. Among the regions that could be tested, HLA binding was predicted for 14/18 (78%). We identified several HLA-associated polymorphisms involving likely known anchor residues that resulted in altered predicted binding scores. Some HLA-associated polymorphisms fell within known T-cell epitopes with matching HLA restriction. Enhanced viral adaptation (defined as the presence of the relevant HLA and the escaped amino acid) was independently associated with HBeAg-negative disease (P = 0.003). Thus, HBV appears to be under immune pressure in chronic HBV infection, particularly in HBeAg-negative disease.     

The interaction between CD44 on tumour cells and hyaluronan under physiologic flow conditions: implications for metastasis formation

The adhesion of tumour cells to the endothelial cells of blood vessels of the microcirculation represents a crucial step in haematogenous metastasis formation. Similar to leukocyte extravasation, selectins mediate initial tumour cell rolling on endothelium. An additional mechanism of leukocyte adhesion to endothelial cells is mediated by hyaluronan (HA). However, data on the interaction of tumour cells with hyaluronan under shear stress are lacking. The expression of the hyaluronan binding protein CD44 on tumour cell surfaces was evaluated using flow cytometry. The adhesion of tumour cells to HA with regard to adhesive events and rolling velocity was determined in flow assays in the human small cell lung cancer (SCLC) cell lines SW2, H69, H82, OH1 and OH3, the colon carcinoma cell line HT29 and the melanoma cell line MeWo. Hyaluronan deposition in human and mouse lung blood vessels was histochemically determined. MeWo adhered best to HA followed by HT29. SCLC cell lines showed the lowest CD44 expression on the cell surface and lowest number of adhesive events. While hyaluronan was deposited in patches in the microvasculature of the alveolar septum in the human lung, it was only present in the periarterial space in the mouse lung. Certain tumour entities bind to HA under physiological shear stresses so that HA can be considered a further ligand for cell extravasation in haematogenous metastasis. As hyaluronan is deposited within the pulmonary microvasculature, it may well serve as a ligand for its binding partner CD44, which is expressed by many tumour cells.     

Association of MAOA and COMT gene polymorphisms with palatable food intake in children

Several studies have implicated dopamine (DA) in appetite regulation. The enzymes catechol-o-methyltransferase (COMT) and monoamine oxidase A (MAOA) control DA availability and their genes have well-characterized functional variants. In this study, we examined three polymorphisms in these genes, T941G and MAOAu-VNTR in the MAOA gene and Val158Met in the COMT gene, to investigate how heritable variations in enzymes that determine DA levels might influence food intake and nutritional status. This investigation was a cross-sectional examination of 354 Brazilian children of three to four years old. Polymorphisms were analyzed by PCR-based methods. Means of dietary and anthropometric data were compared among genotypes by one-way analyses of variance or Kruskal Wallis tests. The MAOAu-VNTR and COMT Val158Met polymorphisms were associated with the amount of palatable food intake in boys. Presence of the MAOAu-VNTR*long allele was associated with higher intake of lipid-dense foods (LDF) when compared with the *short allele (P=.009); the amount of sugar-dense foods (SDF) intake was also higher in males carriers of the MAOAu-VNTR *long allele than in carriers of the *short allele (P=.034). In the girls' sample, MAOAu-VNTR polymorphism was not associated with food intake and nutritional status. Carriers of the COMT Val158Met*Val allele presented higher intake of LDF when compared with Met/Met homozygotes (P=.008). This study provides the first indication that genetic variants of enzymes that control DA availability might be involved in determination of the amount of palatable food intake in children.     

Diving for food: a switch of foraging strategy of dusky dolphins in Argentina

During winter, dusky dolphins (Lagenorhynchus obscurus) were observed in coordinated diving apparently in a feeding activity, contrasting with the surface feeding observed during summer. The aim of this work consisted in analyzing the diving activity as an alternative foraging strategy in Argentine dusky dolphins, based on sequential analysis. The study area was Golfo Nuevo, located in Northern Patagonia, Argentina. Random transects were surveyed by a research boat from 2001 to 2007. During behavioral sampling, group members were observed continuously and the predominant activity was recorded at 2-min intervals. Six predominant activities were identified. Each 2-min interval was classified according to the activity at the previous interval (preceding activity), the activity at the interval (following activity), and the season (cold or warm). Z scores were calculated and then used to construct sequence diagrams. An association between diving and milling behavior was observed. This could be another foraging tactics different to the surface foraging sequences and this could be related with the distribution or abundance of preys.     

Lysosomal vitamin E accumulation in Niemann-Pick type C disease

Niemann-Pick C disease (NPC) is a neuro-visceral lysosomal storage disorder mainly caused by genetic defects in the NPC1 gene. As a result of loss of NPC1 function large quantities of free cholesterol and other lipids accumulate within late endosomes and lysosomes. In NPC livers and brains, the buildup of lipids correlates with oxidative damage; however the molecular mechanisms that trigger it remain unknown. Here we study potential alterations in vitamin E (α-tocopherol, α-TOH), the most potent endogenous antioxidant, in liver tissue and neurons from NPC1 mice. We found increased levels of α-TOH in NPC cells. We observed accumulation and entrapment of α-TOH in NPC neurons, mainly in the late endocytic pathway. Accordingly, α-TOH levels were increased in cerebellum of NPC1 mice. Also, we found decreased mRNA levels of the α-TOH transporter, α-Tocopherol Transfer Protein (α-TTP), in the cerebellum of NPC1 mice. Finally, by subcellular fractionation studies we detected a significant increase in the hepatic α-TOH content in purified lysosomes from NPC1 mice. In conclusion, these results suggest that NPC cells cannot transport vitamin E correctly leading to α-TOH buildup in the endosomal/lysosomal system. This may result in a decreased bioavailability and impaired antioxidant function of vitamin E in NPC, contributing to the disease pathogenesis.     

Cardiovascular autonomic imbalance and baroreflex dysfunction in the apolipoprotein E-deficient mouse

Genetically engineered mouse models and advances in molecular biotechnology have given extensive aid to experimental studies of cardiovascular mechanisms and dysfunction in pathological states such as atherosclerosis. Among the available animal models that have been developed to study atherosclerosis, the apolipoprotein E-deficient (apoE(-/-)) mouse is the most ideal genetically modified animal presently available. The apoE(-/-)mouse develops spontaneous severe hypercholesterolemia in a short-time and subsequently develops atherosclerotic lesions similar to those found in humans. Since its creation two decades ago, the apoE(-/-)mouse has greatly contributed to the understanding of atherosclerosis, but the consequences of hypercholesterolemia and atherosclerosis for the autonomic control of cardiovascular function in this mouse model have not been reviewed. In this article, we provide an overview of abnormalities of the parasympathetic and sympathetic nervous systems controlling heart rate and blood pressure and emphasize the dysfunction of the baroreflex control of cardiovascular function and how this dysfunction is influenced by nitric oxide, reactive oxygen species, aging and an atherogenic diet in the apoE(-/-)mouse.