Anthropometric, hormonal and biochemical differences in lean and obese women before and after menopause

The menopausal status is associated with an increased risk of metabolic and cardiovascular diseases. Since the post-menopausal modifications have not been clearly investigated in obese women, we evaluated the influences of menopausal status on anthropometric, hormonal and biochemical characteristics in selected groups of normal-weight and obese women. We studied 92 female outpatients: 24 normal-weight pre-menopausal (Pre-NW) [body mass index (BMI) 23.6 +/- 0.48, age 44.8 +/- 0.68], 24 normal-weight post-menopausal (Post-NW) (BMI 23.7 +/- 0.44, age 55.5 +/- 0.69), 24 obese pre-menopausal (pre-OB) (BMI 32.3 +/- 0.45, age 44.6 +/- 0.75), 20 obese post-menopausal women (Post-OB) (BMI 32.9 +/- 0.57, age 55.2 +/- 0.82). All the subjects were non smokers and free from hypertension, diabetes or impaired glucose tolerance (IGT). Anthropometric parameters, body composition, 17 beta-estradiol, LH, FSH, androstenedione, SHBG, testosterone and leptin were determined. Free androgen index (FAI) and insulin resistance index (HOMA) were calculated. In comparison with Pre-OB, Post-OB had higher values of waist circumferences (p < 0.02), while Post-NW showed no difference. Total and LDL-cholesterol were high in Post-NW women, whereas in the obese subjects they were already elevated in the premenopausal period. SHBG levels declined and FAI increased in Post-OB in comparison with Pre-OB. SHBG levels showed an inverse correlation with BMI, waist and waist-to-hip ratio (WHR), while FAI positively correlated with waist values. Serum leptin levels were higher in Post-OB than in Pre-OB, whereas they were similar in normal-weight women. The rise of leptin levels may be related to the greater abdominal fat deposition. In addition, menopausal status of uncomplicated obese women is associated with a greater abdominal fat deposition and with higher values of free androgen index, which may be considered as factors of cardiovascular risk.     

A dystroglycan mutation (p.Cys667Phe) associated to muscle‐eye‐brain disease with multicystic leucodystrophy results in ER‐retention of the mutant protein

Dystroglycan (DG) is a cell adhesion complex composed by two subunits, the highly glycosylated α‐DG and the transmembrane β‐DG. In skeletal muscle, DG is involved in dystroglycanopathies, a group of heterogeneous muscular dystrophies characterized by a reduced glycosylation of α‐DG. The genes mutated in secondary dystroglycanopathies are involved in the synthesis of O‐mannosyl glycans and in the O‐mannosylation pathway of α‐DG. Mutations in the DG gene (DAG1), causing primary dystroglycanopathies, destabilize the α‐DG core protein influencing its binding to modifying enzymes. Recently, a homozygous mutation (p.Cys699Phe) hitting the β‐DG ectodomain has been identified in a patient affected by muscle‐eye‐brain disease with multicystic leucodystrophy, suggesting that other mechanisms than hypoglycosylation of α‐DG could be implicated in dystroglycanopathies. Herein, we have characterized the DG murine mutant counterpart by transfection in cellular systems and high‐resolution microscopy. We observed that the mutation alters the DG processing leading to retention of its uncleaved precursor in the endoplasmic reticulum. Accordingly, small‐angle X‐ray scattering data, corroborated by biochemical and biophysical experiments, revealed that the mutation provokes an alteration in the β‐DG ectodomain overall folding, resulting in disulfide‐associated oligomerization. Our data provide the first evidence of a novel intracellular mechanism, featuring an anomalous endoplasmic reticulum‐retention, underlying dystroglycanopathy.     

Therapeutic and clinical aspects of portal vein thrombosis in patients with cirrhosis

Portal vein thrombosis(PVT) is a frequent complication in cirrhosis, particularly in advanced stages of the disease. As for general venous thromboembolism, risk factors for PVT are slow blood flow, vessel wall damage and hypercoagulability, all features of advanced cirrhosis. Actually, the old dogma of a hemorrhagic tendency in cirrhosis has been challenged by new laboratory tools and the clinical evidence that venous thrombosis also occurs in cirrhosis. The impaired hepatic synthesis of both pro- and anticoagulants leads to a rebalanced hemostasis, more liable to be tipped towards thrombosis or even bleeding. Conventional anticoagulant drugs(low molecular weight heparin or vitamin K antagonists) may be used in cirrhosis patients with PVT, particularly in those eligible for liver transplantation, to prevent thrombosis progression thus permitting/facilitating liver transplant. However, several doubts exist on the level of anticoagulation achieved as estimated by coagulation tests, on the efficacy of treatment monitoring and on the correct timing for discontinuation in non-transplant candidates, while in transplant candidates there is expert consensus on continuing anticoagulation until transplantation. The recent introduction of direct acting oral anticoagulant drugs(DOACs) in other clinical settings generates much interest on their possible application in patients with cirrhosis and PVT. However, DOACs were not evaluated yet in patients with liver disease and cannot be recommended for the present time.     

Effects of Artemetin on Nitric Oxide Release and Protection against Peroxidative Injuries in Porcine Coronary Artery Endothelial Cells

Artemetin is one of the main components of Achillea millefolium L. and Artemisia absinthium, which have long been used for the treatment of various diseases. To date, however, available information about protective effects of their extracts on the cardiovascular system is scarce. Therefore, we planned to analyze the effects of artemetin on nitric oxide (NO) release and the protection exerted against oxidation in porcine aortic endothelial (PAE) cells. In PAE, we examined the modulation of NO release caused by artemetin and the involvement of muscarinic receptors, β2‐adrenoreceptors, estrogenic receptors (ER), protein‐kinase A, phospholipase‐C, endothelial‐NO‐synthase (eNOS), Akt, extracellular‐signal‐regulated kinases 1/2 (ERK1/2) and p38 mitogen activated protein kinase (p38 MAPK). Moreover, in cells treated with hydrogen peroxide, the effects of artemetin were examined on cell survival, glutathione (GSH) levels, apoptosis, mitochondrial membrane potential and transition pore opening. Artemetin increased eNOS‐dependent NO production by the involvement of muscarinic receptors, β2‐adrenoreceptors, ER and all the aforementioned kinases. Furthermore, artemetin improved cell viability in PAE that were subjected to peroxidation by counteracting GSH depletion and apoptosis and through the modulation of mitochondrial function. In conclusion, artemetin protected endothelial function by acting as antioxidant and antiapoptotic agent and through the activation of ERK1/2 and Akt. Copyright © 2015 John Wiley & Sons, Ltd.     

Unbiased optical mapping of telomere-integrated endogenous human herpesvirus 6

Next-generation sequencing technologies allowed sequencing of thousands of genomes. However, there are genomic regions that remain difficult to characterize, including telomeres, centromeres, and other low-complexity regions, as well as transposable elements and endogenous viruses. Human herpesvirus 6A and 6B (HHV-6A and HHV-6B) are closely related viruses that infect most humans and can integrate their genomes into the telomeres of infected cells. Integration also occurs in germ cells, meaning that the virus can be inherited and result in individuals harboring the virus in every cell of their body. The integrated virus can reactivate and cause disease in humans. While it is well established that the virus resides in the telomere region, the integration locus is poorly defined due to the low sequence complexity (TTAGGG)n of telomeres that cannot be easily resolved through sequencing. We therefore employed genome imaging of the integrated HHV-6A and HHV-6B genomes using whole-genome optical site mapping technology. Using this technology, we identified which chromosome arm harbors the virus genome and obtained a high-resolution map of the integration loci of multiple patients. Surprisingly, this revealed long telomere sequences at the virus−subtelomere junction that were previously missed using PCR-based approaches. Contrary to what was previously thought, our technique revealed that the telomere lengths of chromosomes harboring the integrated virus genome were comparable to the other chromosomes. Taken together, our data shed light on the genetic structure of the HHV-6A and HHV-6B integration locus, demonstrating the utility of optical mapping for the analysis of genomic regions that are difficult to sequence.

Human herpesvirus 6A and 6B (HHV-6A and HHV-6B) are closely related virus species that infect humans (1). HHV-6B infects almost all humans within the first years of life and causes the febrile illness exanthema subitum (2). Infection with HHV-6A is thought to occur later in life, but the epidemiology of the virus is poorly characterized. Like all herpesviruses, HHV-6A and HHV-6B establish latency upon primary infection, allowing the virus to persist in the host for life. In contrast to other human herpesviruses, HHV-6A and HHV-6B integrate their genomes into the telomere region of host chromosomes of latently infected cells (3–6). The viruses can also heritably integrate into germ cells, resulting in individuals and their offspring that harbor the virus in every nucleated cell (3, 7–11). This phenomenon is referred to as inherited chromosomally integrated HHV-6 (iciHHV-6). Importantly, HHV-6 reactivation from the integrated state is associated with various diseases, including graft-versus-host disease, encephalitis, and heart disease (12–18). More recently, a study indicated that telomeres carrying integrated HHV-6 are shorter and more unstable (19), which, in turn, could influence aging and/or diseases of individuals with iciHHV-6.It remains unknown whether certain integration events or iciHHV-6 genomes at specific loci are responsible for, or contribute directly to, disease in humans. Although we can identify individuals that harbor iciHHV-6A and iciHHV-6B through qPCR (20), a major challenge in studying iciHHV-6 is that we cannot readily examine the integration site with high resolution. Integration loci of HHV-6A and HHV-6B were first detected by fluorescent in situ hybridization (FISH) (3), and the junction has since been sequenced using a PCR-based approach for three different integrations. Sanger sequencing of these PCR fragments indicated that the right direct repeat (DR-R) is fused to the subtelomeres with only a very short stretch of telomere sequences between the virus and host chromosome (4, 5, 19, 21).However, FISH is a challenging and laborious technique, offering very low spatial resolution that does not allow us to directly observe the junction. Although PCR amplification and Sanger sequencing does offer sequence information, this approach requires previous knowledge of the chromosomal location of the virus genome (19, 22), is prone to amplification errors, and has not succeeded in determining structures of junctions. Next-generation sequencing (NGS) using various platforms has not succeeded in sequencing the junction, as either the coverage or the read length was not sufficient to resolve the complex and repetitive nature of the telomere region harboring the virus genome.We therefore set out to develop an approach to study the virus−host junction because understanding its composition is key to understanding the effect of iciHHV-6 on human biology. Using a whole-genome optical mapping technology (23, 24), we were able to generate unbiased, high-resolution maps of HHV-6A and HHV-6B integration sites, which allowed the identification of the chromosomes harboring the integrated HHV-6A and HHV-6B genome in four different iciHHV-6 patient-derived cell lines. We also know, from our recent work on the evolutionary history of the virus, that most known integrations are derived from very few ancestral genome invasion events (25). This means that, for every sample we determine the chromosomal location for, this can be extrapolated to many orthologs without the expense and effort of further experimental work. Our results conclusively determine the orientation of the virus genome, as well as the length of the telomeric virus−chromosome junction and the distal telomeres on the end of the virus genome. Furthermore, the ability to determine the chromosomal location of the virus will be crucial to decipher the role of the integrated virus genome in human diseases.     

Immunohistochemical expression of p16(INK4a) is predictive of HR-HPV infection in cervical low-grade lesions.

The p16(INK4a) is a cyclin-dependent kinase inhibitor that decelerates the cell cycle by inactivating the cyclin-dependent kinases involved in the phosphorylation of the retinoblastoma protein (RB). Expression of E6 and E7 oncogenes of high-risk (HR) human papillomavirus (HPV), affecting the RB-p16 pathway, leads to p16 upregulation. Although it is widely reported that p16 is overexpressed in a high percentage of preneoplastic lesions and in almost all carcinomas of the uterine cervix, protein upregulation and its correlation with HPV infection in low-grade lesions is still being debated. In this study, we investigated in parallel, p16 expression and HPV infection in 100 cervical biopsies (17 normal tissues, 54 CIN1, 10 CIN2, 11 CIN3, eight invasive squamous cancers). Results obtained demonstrated that none of the 17 normal cervical tissues, evaluated by immunohistochemistry, presented p16 positivity whereas, starting from CIN1 (31%) to CIN2 (90%), CIN3 (100%) and carcinomas (100%), a constant and significant increase of protein overexpression (P<0.0001) was observed. In addition, p16 overexpression consistently showed elevated sensitivity (84%) and specificity (98%) in detecting HR-HPV infection with a high positive predictive value (97%) and negative predictive value (86%). Of interest, 93% of the p16-positive CIN1 were also HR-HPV infected. Our findings confirmed that p16 overexpression is associated to high-grade precancerous lesions and cervical carcinomas, and further demonstrated that immunohistochemical evaluation of p16 may be a useful biomarker in identifying HR-HPV-infected low-grade lesions.     

Prolongation of skin allograft survival in rats by the transplantation of microencapsulated xenogeneic neonatal porcine Sertoli cells

Skin rejection remains a major hurdle in skin reconstructive transplantation surgery. In fact, 85% of the grafted patients experience at least one episode of acute skin rejection in the first year. It has been observed that Sertoli cells (SC), when co-transplanted with allo- or xenogeneic cell/tissues, can induce graft acceptance in the absence of systemic immunosuppression. A method aimed at significantly prolonging skin allografts in rats transplanted with barium alginate-based microencapsulated xenogeneic porcine SC (SC-MCs) is described. Results demonstrated that intraperitoneal (IP) transplantation of SC-MCs with high cellular viability and function can significantly prolong allogeneic skin grafts when compared to transplantation controls receiving only empty alginate capsules (E-MCs). Lymphocytic infiltration at the skin graft site was not observed in 80% of the SC-MCs transplanted rats and these recipient animals showed a significant increased expression of T regulatory (Tregs) cells when compared to E-MCs transplantation controls. The findings of this report further substantiate the positive therapeutic effects of SC on transplantation technology mediated by Sertoli cell-induced alterations of the host's immune system and indicate new perspectives and new strategies for successful skin tissue allografts.