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Increasing evidence suggests that maternal cholesterol represents an important risk factor for atherosclerotic disease in offspring already during pregnancy, although the underlying mechanisms have not yet been elucidated. Eighteen human fetal aorta samples were collected from the spontaneously aborted fetuses of normal cholesterolemic and hypercholesterolemic mothers. Maternal total cholesterol levels were assessed during hospitalization. DNA methylation profiling of the whole SREBF2 gene CpG island was performed (p value SREBF2 gene shows 4 significant differentially hypermethylated sites in the 5′UTR-CpG island. This finding indicates that more effective long-term primary cardiovascular prevention programs need to be designed for the offspring of mothers with hypercholesterolemia. Further studies should be conducted to clarify the epigenetic mechanisms underlying the association between early atherogenesis and maternal hypercholesterolemia during pregnancy.

Background DNA methylation can play a pathogenic role in the early stages of hyperglycemia linking homeostasis imbalance and vascular damage. Material and methods We investigated DNA methylome by RRBS in CD04+ and CD08+ T cells from healthy subjects (HS) to pre-diabetics (Pre-Diab) and type 2 diabetic (T2D) patients to identify early biomarkers of glucose impairment and vascular damage. Our cross-sectional study enrolled 14 individuals from HS state to increasing hyperglycemia (pilot study, PIRAMIDE trial, NCT03792607). Results Globally, differentially methylated regions (DMRs) were mostly annotated to promoter regions. Hypermethylated DMRs were greater than hypomethylated in CD04+ T cells whereas CD08+ T showed an opposite trend. Moreover, DMRs overlapping between Pre-Diab and T2D patients were mostly hypermethylated in both T cells. Interestingly, SPARC was the most hypomethylated gene in Pre-Diab and its methylation level gradually decreased in T2D patients. Besides, SPARC showed a significant positive correlation with DBP (+0.76), HDL (+0.54), Creatinine (+0.83), LVDd (+0.98), LVSD (+0.98), LAD (+0.98), LVPWd (+0.84), AODd (+0.81), HR (+0.72), Triglycerides (+0.83), LAD (+0.69) and AODd (+0.52) whereas a negative correlation with Cholesterol (−0.52) and LDL (−0.71) in T2D. Conclusion SPARC hypomethylation in CD08+ T cells may be a useful biomarker of vascular complications in Pre-Diab with a possible role for primary prevention warranting further multicenter clinical trials to validate our findings.

Network medicine can advance current medical practice by arising as response to the limitations of a reductionist approach focusing on cardiovascular (CV) diseases as a direct consequence of a single defect. This molecular-bioinformatic approach integrates heterogeneous "omics" data and artificial intelligence to identify a chain of perturbations involving key components of multiple molecular networks that are closely related in the human interactome. The clinical view of the network-based approach is greatly supported by the general law of molecular interconnection governing all biological complex systems. Recent advances in bioinformatics have culminated in numerous quantitative platforms able to identify CV disease modules underlying perturbations of the interactome. This might provide novel insights in CV disease mechanisms as well as putative biomarkers and drug targets. We describe the network-based principles and discuss their application to classifying and treating common CV diseases. We compare the strengths and weaknesses of molecular networks in comparison with the classical current reductionist approach, and remark on the necessity for a two-way approach connecting network medicine with large clinical trials to concretely translate novel insights from bench to bedside.

Background Acute coronary syndrome (ACS) is the most severe clinical manifestation of coronary heart disease and the leading cause of death worldwide. Purpose To perform an epigenome-wide analysis in circulating CD4+ and CD8+ T cells of ACS patients and healthy subjects (HS) enrolled in the DIANA clinical trial (NCT04371809) in order to identify differentially methylated genes (DMGs). Methods Genomic DNA was extracted from CD4+ and CD8+ T cells of all subjects and sequenced by the reduced representation bisulfite sequencing (RRBS) platform. Functional pathway analysis was performed and significant DMGs were selected for gene expression validation by qRT-PCR in ACS patients and HS. GeneMANIA was used to built a prediction gene network. Correlation analyses between molecular data and clinical variables were performed. Results In CD4+ T cells we identified 61 differentially methylated regions (DMRs) associated to 57 annotated genes of which 53% (n=32) were hyper- and 47% (n=29) were hypo-methylated in ACS patients vs HS. In CD8+ T cells we identified 613 DMRs associated to 569 annotated genes of which 28% (n=173) were hyper- and 72% (n=440) were hypo-methylated between two groups. In both cell type of ACS patients, 175 DMRs were associated to 157 annotated genes of which 41% (n=72) were hyper- and 59% (n=103) were hypo-methylated. From functional analysis, we selected the top 5 DMGs in the prevalent pathways with the highest differential of methylation values. Specifically, we considered 6 hub genes: NFATC1, TCF7, PDGFA, PRKCB, PRKCZ and ABCA1 and determined their respective expression levels by q-RT-PCR. We found a significant up-regulation of the selected genes in ACS patients vs HS (P Conlusions This study is the first single-base resolution map of DNA methylome by RRBS in CD4+ and CD8+ T cells, providing specific methylation signatures that could help to clarify the role of aberrant methylation in ACS pathogenesis, and provide the basis for the search of novel epigenetic-sensitive biomarkers in the prevention and early diagnosis of this pathology. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Health;Italian Ministry of Research and University

Aims Immune endothelial inflammation, underlie coronary heart disease (CHD) related phenotypes, could provide new insight into the pathobiology of the disease. We investigated DNA methylation level of the unique CpG island of HLA-G gene in CHD patients and evaluated the correlation with cardiac computed tomography angiography (CCTA) features. Methods Thirty-two patients that underwent CCTA for suspected CHD were enrolled for this study. Obstructive CHD group included fourteen patients, in which there was a stenosis greater than or equal to 50% in one or more of the major coronary arteries detected; whereas subjects with Calcium (Ca) Score=0, uninjured coronaries and with no obstructive CHD were considered as control subjects (Ctrls) (n=18). For both groups, DNA methylation profile of the whole 5'UTR-CpG island of HLA-G was measured. The plasma soluble HLA-G (sHLA-G) levels were detected in all subjects by specific ELISA assay. Statistical analysis was performed using R software. Results For the first time, our study reported that 1) a significant hypomethylation characterized three specific fragments (B, C and F) of the 5'UTR-CpG island (p=0.05) of HLA-G gene in CHD patients compared to Ctrl group; 2) hypomethylation level of one specific fragment positively correlated with coronary Ca score, a relevant parameter of CCTA (p Conclusions Our results showed that reduced levels of circulating HLA-G molecules could derive from epigenetic marks inducing hypomethylation of specific regions into 5'UTR-CpG island of HLA-G gene in CHD patients with obstructive coronary stenosis vs non critical stenosis group. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Italian Minister of Health

Hyperglycaemia can strongly alter the epigenetic signatures in many types of human vascular cells providing persistent perturbations of protein–protein interactions both in micro- and macro-domains. The establishment of these epigenetic changes may precede cardiovascular (CV) complications and help us to predict vascular lesions in diabetic patients. Importantly, these epigenetic marks may be transmitted across several generations (transgenerational effect) and increase the individual risk of disease. Aberrant DNA methylation and imbalance of histone modifications, mainly acetylation and methylation of H3, represent key determinants of vascular lesions and, thus, putative useful biomarkers for prevention and diagnosis of CV risk in diabetics. Moreover, a differential expression of some micro-RNAs (miRNAs), mainly miR-126, may be a useful prognostic biomarker for atherosclerosis development in asymptomatic subjects. Recently, also environmental-induced chemical perturbations in mRNA (epitranscriptome), mainly the N6-methyladenosine, have been associated with obesity and diabetes. Importantly, reversal of epigenetic changes by modulation of lifestyle and use of metformin, statins, fenofibrate, and apabetalone may offer useful therapeutic options to prevent or delay CV events in diabetics increasing the opportunity for personalized therapy. Network medicine is a promising molecular-bioinformatic approach to identify the signalling pathways underlying the pathogenesis of CV lesions in diabetic patients. Moreover, machine learning tools combined with tomography are advancing the individualized assessment of CV risk in these patients. We remark the need for combining epigenetics and advanced bioinformatic platforms to improve the prediction of vascular lesions in diabetics increasing the opportunity for CV precision medicine.

Background Endothelial cells arise from endothelial progenitor cells (EPCs), which reside in bone marrow vascular niches, and are classified according to specific functional differences. Oxidative stress and inflammation lead to endothelial dysfunction that is a critical event in the initiation and progression of atherosclerotic plaques. Patients with chronic renal failure frequently show endothelial dysfunction and are at a greatly increased risk of developing atherosclerosis. Owing to their ability to partially restore vascular damage, both the number and functional changes in EPCs may be considered as useful prognostic biomarkers of cardiovascular events in hemodialysis (HD) patients. Purpose This study investigated EPC features and several biomarkers of systemic vascular inflammation and carotid atherogenesis in hemodialysis (HD) patients. Methods We studied 104 HD patients (males=55 and females 49, mean age: 51±12 years) and 40 healthy controls (males=20/females=20; mean age: 52±11 years). Isolated EPCs were cultured in the fibronectin-covered culture dishes and counted. EPC markers were studied by flow cytometry (FACS) by using anti-CD34, anti-CD133 and anti-vascular endothelial growth factor receptor 2 (VEGFR-2) antibodies. Serum levels of intercellular cell adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), IL-6, TNF-α, and asymmetric dimethyl-arginine (ADMA) were measured by ELISA method. Carotid intima-media thickness (CIMT) and ratio (CIMR) were also evaluated. Results In our experimental conditions, EPC number was decreased in HD patients when compared to controls (p Conclusions Our study shows that EPC number was decreased in HD patients and it was associated with systemic inflammation. TNF-α could activate inhibitory actions on EPC in HD patients. A significant relationship was present between ICAM/VCAM and carotid atherosclerosis, while this was not evident with EPC number. These pathogenic mechanisms can contribute to the high incidence of cardiovascular diseases in HD patients. However, further larger studies should investigate this working hypothesis.

Aim Hyperglycemia status induces endothelial dysfunction, although the underlying pathogenic mechanisms are not fully understood. There are several studies connecting sugar/sweetened beverages to the cardiovascular disease. Currently, many sweeteners have been extensively introduced into lifestyle to normalize blood glucose levels without altering the sweet taste. However, there is growing concern for their impact on metabolic health. Methods Human endothelial cells were treated with Glucose, Fructose, Aspartame, Rebaudioside A, Stevioside, or Steviol. Morphological characteristics, in vitro angiogenesis and array gene expression were analyzed. Results High-glucose and fructose concentrations significantly decreased cell features such as angiogenic capability. Interestingly, non-caloric sweeteners did not significantly modified all cell characteristics and they did not compromised cell angiogenic ability. Array gene expression analysis revealed that the chemokine fractalkine (CX3CL1) and the enzyme transferase (HPRT1) were always significantly upregulated and downregulated respectively, after glucose and fructose treatments (P > .05), whereas they were non-differentially expressed with all the other sweeteners. Interestingly, both genes are considered as cardiovascular disease risk biomarkers. Specifically, upregulation of CX3CL1/CX3CR1 occurs in the human placenta and serum levels of the ligand are associated with markers of insulin resistance in GDM. Conclusions Differently from glucose and fructose, steviol glycosides do not damage endothelial cells. Prospective preclinical studies and clinical trials are warranted to confirm the long-term safety of such compounds.

AIMS:Immune endothelial inflammation, underlying coronary heart disease (CHD) related phenotypes, could provide new insight into the pathobiology of the disease. We investigated DNA methylation level of the unique CpG island of HLA-G gene in CHD patients and evaluated the correlation with cardiac computed tomography angiography (CCTA) features. METHODS:Thirty-two patients that underwent CCTA for suspected CHD were enrolled for this study. Obstructive CHD group included fourteen patients, in which there was a stenosis greater than or equal to 50% in one or more of the major coronary arteries detected; whereas subjects with Calcium (Ca) Score = 0, uninjured coronaries and with no obstructive CHD (no critical stenosis, NCS) were considered as control subjects (n = 18). For both groups, DNA methylation profile of the whole 5'UTR-CpG island of HLA-G was measured. The plasma soluble HLA-G (sHLA-G) levels were detected in all subjects by specific ELISA assay. Statistical analysis was performed using R software. RESULTS:For the first time, our study reported that 1) a significant hypomethylation characterized three specific fragments (B, C and F) of the 5'UTR-CpG island (p = 0.05) of HLA-G gene in CHD patients compared to control group; 2) the hypomethylation level of one specific fragment of 161bp (+616/+777) positively correlated with coronary Ca score, a relevant parameter of CCTA (p

Circulating biomarkers available in clinical practice do not allow to stratify patients with coronary heart disease (CHD) prior the onset of a clinically relevant event. We evaluated the methylation status of specific genomic segments and gene expression in peripheral blood of patients undergoing Cardiac Computed Tomography (CCT) for CHD (n = 95). We choose to investigate cholesterol metabolism. Methylation and gene expression of low density lipoprotein receptor (LDLR), sterol regulatory element-binding factor 2 (SREBF2) and ATP-binding cassette transporter 1 (ABCA1) were evaluated by qRT-PCR. Calcium score (CACS), stenosis degree, total plaque volume (TPV), calcified plaque volume (CPV), non-calcified plaque volume (NCPV) and plaque burden (PB) were assessed in all CHD patients (n = 65). The percentage of methylation at the specific analyzed segment of LDLR promoter was higher in CHD patients vs healthy subjects (HS) (n = 30) (p = 0.001). LDLR, SREBF2 and ABCA1 mRNAs were up-regulated in CHD patients vs HS (p = 0.02; p = 0.019; p = 0.008). SREBF2 was overexpressed in patients with coronary stenosis 50% vs subjects with stenosis

Serological assays are still considered the most useful tests in the diagnosis of syphilis. Since no single serological assay is able to provide a satisfactory result, in our laboratory we have evaluated the usefulness of a commercially-available immunoblot to diagnose syphilis infection among blood donors. From October 2012 to June 2013, 4572 blood donors were screened for syphilis with an automated chemiluminescent microparticle immunoassay (CMIA). To confirm the presence of treponemal antibodies, CMIA-reactive sera were tested by standard Treponema pallidum haemagglutination assay (TPHA). In addition, an alternative confirmatory test – the immunoblot INNO-LIA assay was introduced in our laboratory. Since two additional positives among CMIA-reactive-TPHA-negative samples were found, we concluded that the INNO-LIA immunoblot allowed a better detection of syphilis compared to TPHA. A confirmatory strategy based on the use of two treponemal assays could meet the screening requirements for blood donors as well as in our centre.

Background Patients with end-stage liver disease (ESLD) have a compromised nutritional status because of the liver crucial role in regulating metabolic homeostasis and energy balance. Data sources A systematic review of literature based on extensive relevant articles published from 2001 to 2017 in English in PubMed database was performed by searching keywords such as liver disease, non-alcoholic liver disease, alcoholic liver disease, malnutrition, epigenetics, gut microbiota, and probiotics. Results Liver transplantation would be one eligible therapy for ESLD patients, even if, the clinical outcome is negatively influenced by malnutrition and/or infections. The malnutrition is a condition of nutrient imbalance with a high incidence in ESLD patients. An accurate evaluation of nutritional status could be fundamental for reducing complications and prolonging the survival of ESLD patients including those undergoing liver transplantation. In addition, the interaction among nutrients, diet and genes via epigenetics has emerged as a potential target to reduce the morbidity and mortality in ESLD patients. The malnutrition induces changes in gut microbiota causing dysbiosis with a probable translocation of bacteria and/or pathogen-derived factors from the intestine to the liver. Gut microbiota contribute to the progression of chronic liver diseases as well as hepatocellular carcinoma. The administration of probiotics modulating gut microbiota could improve all chronic liver diseases. Conclusions This review provides an update on malnutrition status linked to epigenetics and the potential benefit of some probiotics on the management of ESLD patients. In support of this view and to reveal the constant and growing interest in this field, some clinical trials are reported.

From the 1980s, extracorporeal photochemotherapy (ECP) has been shown to be effective in a variety of pathological conditions such as erythrodermic cutaneous T-cell lymphoma, autoimmune diseases, solid organ allograft rejection and graft versus host disease. To date, ECP represents a non-aggressive immune modulatory therapy with a low spectrum of toxicity. ECP reduces the alloreactivity promoting the immune tolerance to self. At the same time, it allows the maintenance of immune response integrity of both naive and memory T-cells. However, the molecular mechanisms of action by which ECP exerts its therapeutic activity are still under investigation. Here, we review molecular mechanisms and clinical applications involved in ECP. The outcome of ECP is difficult due to the lack of reliable predictor factors for the selection of patients and their adequate follow-up. Since the study of such predictors is important, we also describe some biological markers that enable us to investigate the clinical management of the patients considered for the use of ECP.

Background: The main challenge in type 2 diabetes (T2D) is to detect the regulators of pathogenic events during early stages of disease, as well as prevention and progression follow-up of cardiovascular (CV) complications. DNA methylation and micro-RNAs (miRNAs) are major components of the epigenome, which are involved in the diabetic interactome. This study protocol may contribute to advance our knowledge on molecular basis underlying T2D and its CV complications, as well as provide putative useful prognostic biomarkers.Methods: The perturbation of interactome through micro-RNA and methylome analysis in diabetes endophenotypes: the PIRAMIDE pathogenic clinical study protocol is a cross-sectional research program planned to combine big data and network-based analysis aimed to investigate whether DNA methylation and miRNAs may act as simultaneous regulators of the interactome in T2D patients. Clinical datasets will be aggregate to large-scale DNA methylation, mRNA-Seq, and miRNA-Seq analysis performed both on purified CD4+ and CD8+ T cells isolated from 35 T2D patients and 35 sex and age-matched controls. DNA methylome data will be used as input for the weighted human DNA methylation PPI network (WMPN) algorithm. RNA sequencing data will be used as input data for the TargetScan algorithm. The primary endpoint will be to integrate both DNA methylation and miRNA networks to potentially capture which genes are simultaneously modulate by interactions between epigenetic changes. Then, statistical analysis will be performed to correlate these molecular modifications with development of T2D-related CV complications.Conclusions: PIRAMIDE pathogenic clinical study protocol will test the hypothesis that simultaneous interactions between DNA methylation and miRNAs may hit T2D-associated candidate genes and predict the development of T2D-related CV complications.Trial Registration: The ongoing PIRAMIDE pathogenic clinical study protocol has been registered on NIH website (NCT03792607).

Intravenous immunoglobulin (IVIG) products are known to have beneficial immunomodulatory effects on several inflammatory and autoimmune disorders. These effects could be attributed to a different inhibitory action on complement factors, but other mechanisms could be implicated, e.g., immunocomplexes development and/or anti-idiotypic antibodies. Positive results on the reduction of anti-Human Leukocyte Antigens (HLA) antibodies in highly sensitized patients have also been found. The present study focuses on the effect of IVIG on the reduction of Panel Reactive Antibody level and crossmatch positivity in sensitized patients awaiting kidney transplantation.The study was performed adapting an in vitro assay on sensitized patients' sera in waiting list for kidney transplantation. Sera of twelve highly sensitized patients were evaluated for the cytotoxicity inhibition after 10% IVIG treatment.A reduction of anti- HLA antibody levels was observed in 75% (9/12) of treated patients in vitro, while 25% (3/12) resulted unresponsiveness. Particularly, our data showed a significantly higher Panel Reactive Antibody reduction for T lymphocytes (p0.010) than B lymphocytes (p0.032).In this study, we have used an in vitro assay to investigate susceptibility to desensitization with IVIG treatment of sensitized patient sera. These findings reveal that the variable effect of IVIG on reducing Panel Reactive Antibody in our immunized patients could be attributed to a different inhibitory action on complement, likely due to the type and the titre of anti-HLA antibodies.

In this chapter we will describe the use of the tubular network assay as a useful tool in the evaluation of cancer neoangiogenesis (as reported by Arnaoutova et al. [Angiogenesis 12:267–274, 2009]). Angiogenesis is involved not only in pathological conditions including cancer biology and non-neoplastic diseases, but also many biological processes including reproduction, development and repair. A detailed working protocol applicable to the study of any type of endothelial cells is provided below and in addition, details on data interpretation analysis and troubleshooting.

The growing improvements of computed tomography have made this technique more and more available for cardiac evaluation. Coronary artery anomalies (CAAs) are often incidental findings in subjects with suspected coronary artery disease (CAD) undergoing coronary angiography or computed tomography coronary angiography (CTCA). In some cases, CAAs can be clinically relevant so their identification could change radically patient management and treatment. We report the case of a 68-year-old male patient with known CAD and associated anomalous origination of the left coronary artery from the opposite sinus.

PALB2 gene is mutated in about 1–2 % of familial breast cancer as well as in 3–4 % of familial pancreatic cancer cases. Few studies have reported mutations in Italian patients with breast or pancreatic cancer. We evaluate the occurrence of PALB2 mutations in Italian patients affected with hereditary breast and ovarian cancers and define the pathological significance of the putative allelic variants. We recruited 98 patients (F = 93, M = 5) affected with breast and/or ovarian cancer, negative for mutations in BRCA1 and BRCA2 (BRCAX). Genomic DNA was isolated from peripheral blood lymphocytes, PALB2 coding regions and adjacent intronic were sequenced; in silico predictions were carried out using prediction programs. Mutational analysis of PALB2 gene revealed the novel mutation c.1919C>A (p.S640X) in a 29 years old woman with breast cancer. The c.1919C>A (p.S640X) mutation causes the lack of C-terminus region inducing alteration of MORF4L1–PALB2 association and the lack of interaction of PALB2 with RAD51 and BRCA2. In addition, we identified two novel PALB2 variants, c.3047T>C (p.F1016S) and c.*146A>G. In silico analysis conducted for c.*146A>G indicates that this variant does not affect the splicing while c.3047T>C (p.F1016S) was predicted as damaging in three classifier algorithms. The proband carrier of c.3047T>C (p.F1016S) showed two breast cancer cases, two ovarian cancer cases and one pancreatic cancer in mother’s family. c.3047T>C (p.F1016S) and c.*146A>G should be considered PALB2 UVs even though the genotype–phenotype correlation for these variants remains still unclear. Our findings indicate that the presence of PALB2 mutation should be routinely investigated in hereditary breast and ovarian cancers families since it could be of clinical relevance for clinical management.

Background: The use of cardiac resynchronization therapy (CRT) and implantable cardioverter- defibrillator (ICD) for advanced heart failure (HF) is increasing. Renal dysfunction is a common condition in HF which is associated with a worse survival. The study aims at identifying in patients with advanced HF treated with CRT the effect of baseline glomerular filtration rate (GFR), GFR improvement and left ventricular ejection fraction (LVEF) change, after 6-months of CRT implant, on survival. Methods: The study population consisted of 375 advanced HF patients who received a CRT between 1999 and 2009, of these 277 received also an ICD implant. Clinical characteristics (New York Heart Association [NYHA] functional class, ischemic vs. non-ischemic etiology, atrial fibrillation, diabetes, hypertension, LVEF, QRS duration and GFR were recorded. The use of common used drugs was evaluated. Cox proportional hazards analysis was calculated in order to evaluate variables associated to mortality. Results: During a median follow-up of 43.0 months, 93 (24.8%) patients died. Patients deceased during the study had at baseline higher NYHA class and lower LVEF and GFR. In Cox regression analysis, GFR predicts long-term mortality (hazard ratio [HR] 0.983; 95% confidence interval [CI] 0.969–0.998; p = 0.023) independently from the effect of others covariates. In addition, a positive GFR improvement 6 months after CRT implant is significantly associated with a lower hazard of mortality (for each 10 mL/min of GFR improvement HR 0.86; 95% CI 0.75–0.99; p = 0.038). Conclusions: GFR is a significant predictor of mortality in advanced HF patients who received CRT. A GFR improvement 6 months after CRT implant is significantly associated with a lower hazard of mortality.

Automated chemiluminescent immunoassays (CLIAs) are useful for the detection of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus 1/2 antigen/antibodies (HIV 1/2 Ag/Ab) in blood donor screening. Eight hundred and forty serum samples were tested for hepatitis B surface antigen (HBsAg), HCV antibodies (anti-HCV), and HIV1/2 Ag/Ab in parallel using 2 different CLIAs (Abbott Architect i2000SR and Roche Cobas e411). The concordance between the 2 systems was high (Cohen's kappa 0.97 for HBsAg, 0.77 for anti-HCV, 0.92 for HIV1/2 Ag/Ab) and the specificity and the positive predictive value were comparable. Among the 12 discrepant results, 11 were false-positive and 1 (reactive by Architect) was true-positive for anti-HCV. Positivity for HBV DNA, HCV RNA, and HIV RNA was recorded in 90.9%, 38.9%, and 100% of true-positive samples, respectively. This study represents the first stringent comparison between Architect i2000SR and Cobas e411 in blood donors. We observed a good correlation and high agreement among HBV, HCV, and HIV with the 2 automated systems.

Background Bartonella henselae is the etiologic agent of cat-scratch disease. B. henselae infections are responsible for a widening spectrum of human diseases, although often symptomless, ranging from self-limited to life-threatening and show different courses and organ involvement due to the balance between host and pathogen. The role of the host immune response to B. henselae is critical in preventing progression to systemic disease. Indeed in immunocompromised patients, such as solid organ transplant patients, B. henselae results in severe disseminated disease and pathologic vasoproliferation. The purpose of this study was to determine the seroprevalence of B. henselae in patients awaiting heart transplant compared to healthy individuals enrolled in the Regional Reference Laboratory of Transplant Immunology of Second University of Naples. Methods Serum samples of 38 patients awaiting heart transplant in comparison to 50 healthy donors were examined using immunfluorescence assay. Results We found a B. henselae significant antibody positivity rate of 21% in patients awaiting heart transplant ( p = 0.002). There was a positive rate of 8% ( p > 0.05) for immunoglobulin (Ig)M and a significant value of 13% ( p = 0.02) for IgG, whereas controls were negative both for IgM and IgG antibodies against B. henselae . The differences in comorbidity between cases and controls were statistically different (1.41 ± 0.96 vs 0.42 ± 0.32; p = 0.001). Conclusions Although this study was conducted in a small number of patients, we suggest that the identification of these bacteria should be included as a routine screening analysis in pretransplant patients.

Considerable advances in heart transplantation outcome have been achieved through the improvement of donor-recipient selection, better organ preservation, lower rates of perioperative mortality and the use of innovative immunosuppressive protocols. Nevertheless, long-term survival is still influenced by late complications. We support the introduction of HLA matching as an additional criterion in the heart allocation. Indeed, allosensitization is an important factor affecting heart transplantation and the presence of anti-HLA antibodies causes an increased risk of antibody-mediated rejection and graft failure. On the other hand, the rate of heart-immunized patients awaiting transplantation is steadily increasing due to the limited availability of organs and an increased use of ventricular assist devices. Significant benefits may result from virtual crossmatch approach that prevents transplantation in the presence of unacceptable donor antigens. A combination of both virtual crossmatch and a tailored desensitization therapy could be a good compromise for a favorable outcome in highly sensitized patients. Here, we discuss the unresolved issue on the clinical immunology of heart transplantation.

Summary Cell-based therapies, as potential approach to cure peripheral artery disease (PAD), have been clinically investigated after promising results in preclinical models. The so far published studies are very heterogeneous, as different cell sources, cell types, amounts of administered cells and delivering strategies have been used. Overall, cell therapies for PAD bring about a general improvement of patient's clinical condition, even though conclusions cannot be established due to the small size and non-randomized design of these trials. In this context, non-invasive imaging techniques, aimed to monitor angiogenesis and neovascularization after cell therapy, will help the follow-up of clinical studies. However, still much work is needed to establish advanced imaging procedure to overcome the limitation of the current techniques and to accumulate more data in large populations of patients. Here, we report the main imaging techniques employed to evaluate the outcome of the different cell-based therapies in PAD. Moreover, we focus on both published and ongoing clinical trials utilizing cell therapy in PAD.

CXCL12, also known as SDF-1, is the single natural ligand for chemokine receptors CXCR4 and CXCR7. CXCL12 has angiogenic properties in normal endothelial tissue and is involved in the outgrowth and metastasis of CXCR4 expressing tumors. Recent investigations have indicated that CXCL12 levels increase after chemo- and anti- VEGF therapy, favouring recurrences. The blockade of CXCL12/CXCR4 axis has emerged as a potential additional or alternative target for neo-adjuvant treatments. We have reviewed recent patent applications between 2008 and 2011 in tumor angiogenesis and the most clinical data supporting the potential use of anti-CXCR4 agents in this field. Among these, AMD3100, also known as Plerixaform (Mozobil® by Genzyme), is approved for stem cell mobilisation in patients with leukaemia, while BKT140 (Emory University), POL6326 (Polyphor Ag) and TG-0054 (ChemoCentryx) are currently in clinical trials in combination with chemotherapy for multiple myeloma and leukaemia. The aptamer Nox-A12 (Noxxon) is in trials for chronic lymphatic leukaemia treatment. MSX-122 (Metastatix) is in Phase I trials for solid tumor treatment, while CXCR7-specific inhibitor CCX2066 (ChemoCentryx) is still in preclinical studies. We have also considered other strategies, such RNA interference and miRNA, which could be tested for solid tumor adjuvant therapy.

Circulating endothelial progenitor cells (EPCs) play a significant role in regeneration of damaged blood vessels. Levels and functional activities of EPCs are noticeable altered by risk factors for coronary heart disease (CHD) and compounds that can prevent or ameliorate EPC dysfunction are currently of special clinical interest. Here, we evaluate the effects of red wine (RW) on EPCs in C57BL/6J mice subjected to physical exercise. FACS computed counting showed a significant increase of EPC number (P

Moderate wine intake is associated with a reduced risk of morbidity and mortality from cardiovascular disease. Atherosclerosis is enhanced in arterial segments exposed to disturbed flow. Perturbed shear stress increases also the endothelial expression of oxidation-sensitive responsive genes (such as ELK-1 and p-JUN). This study evaluates the effects of chronic consumption of red wine on perturbed shear stress-induced atherogenesis. Results indicated that chronic treatment with red wine significantly attenuated the activation of redox-sensitive genes (ELK-1 and p-JUN) and increased endothelial nitric oxide synthase (eNOS) expression (which was decreased by perturbed shear stress) in cultured human coronary endothelial cells (EC) and in atherosclerosis-prone areas of hypercholesterolemic mice. Oral administration of red wine to hypercholesterolemic mice reduced significantly the progression of atherosclerosis. Moreover, short-term supplementation with red wine to C57BL/6J mice significantly increased upregulation of aortic eNOS and SIRT1 expression induced by physical training. These findings establish that administration of low doses of red wine can attenuate the proatherogenic effects induced by perturbed shear stress in vitro and in vivo. This evidence may have implications for the prevention of atherosclerotic lesion progression and its clinical manifestations.

Despite advances in imaging, understanding the underlying pathways, and clinical translation of animal models of disease there remains an urgent need for therapies that reduce brain damage after stroke and promote functional recovery in patients. Blocking oxidant radicals, reducing matrix metalloproteinase-induced neuronal damage, and use of stem cell therapy have been proposed and tested individually in prior studies. Here we provide a comprehensive integrative management approach to reducing damage and promoting recovery by combining biological therapies targeting these areas. In a rat model of transient cerebral ischemia (middle cerebral artery occlusion) gene delivery vectors were used to overexpress tissue inhibitor of matrix metalloproteinase 1 and 2 (TIMP1 and TIMP2) 3 days before ischemia. After occlusion, autologous bone marrow cells alone or in combination with agents to improve NO bioavailability were administered intraarterially. When infarct size, BrdU incorporation, and motor function recovery were determined in the treatment groups the largest beneficial effect was seen in rats receiving the triple combined therapy, surpassing effects of single or double therapies. Our study highlights the utility of combined drug, gene, and cell therapy in the treatment of stroke. © 2007 by The National Academy of Sciences of the USA.

Recent evidence indicates that bone-marrow cells (BMCs) can contribute to the healing process of the injured cardiovascular system via the chemokine receptor CXCR4/SDF-1, thymosin beta(4) and integrin alpha(4)beta(1) molecular pathways. During tissue ischemia overwhelming numbers of detrimental oxygen radicals are generated, and therefore treatment with antioxidants and L-arginine, the precursor of nitric oxide (NO), could induce beneficial effects beyond those achieved by BMC transplantation alone. Recent studies have reported that BMCs have enhanced neovascularization capacity in cotreatment with alpha-tocopherol (vitamin E), ascorbic acid (vitamin C) and L-arginine. Moreover, BMC therapy can be combined with gene therapy. Clinical trials employing BMCs in the treatment of cardiovascular diseases have been completed with mixed or positive results, and several trials are ongoing. Here, we discuss the clinical potential of BMC transplantation alone and in combined therapy that aims to restore organ vascularization and function. We also consider the mechanisms of mobilization, differentiation and incorporation of BMCs.

Background and purpose: It is well established that human vascular lesions begin very early stage in life with the development of fatty streak lesions in the arteries of fetuses. Although several studies have demonstrated that maternal cholesterol may exert detrimental effects on the number and degree of vascular lesions in fetuses, however, how cholesterol affects in utero programming is still unclear. Epigenetics migh promote pathogenic mechanisms by which gene-environment interactions could occurr. The aim of this study was to verify whether maternal hypercholesterolemia during pregnancy affects epigenetic signature in fetal aortic tissues. Methods: Human fetal aortas (n=42) were analyzed by immunohistochemistry and dichotomized in their respective high and low maternal cholesterol during pregnancy. Immunohistochemistry in fetal arteries was performed with the following antibodies: Anti-trimethyl-Histone H3, (Lys4, Lys9, Lys27), and Anti-CD68 antibody. Methyl DNA immunoprecipitation was performed by MeDIP kit (DIAGENODE). Results: The data indicated a strong signal >75% of positive cells with histone trimethyl lys 27 antibody present in 27% of the biggest fetal aortic lesion area (>120mm2/section) (p>0.039). Molecular analysis of specific cholesterol regulatory circuit gene expression revealed hypermethylation in promoter and 3' UTR of SREBP2 and LDL receptor gene in aortas from hypercholesterolemic mothers compared to normocholesterolemic group (p