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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.

The aim of this study was to characterize both by flow cytometry analysis and immunohistochemistry cervix uteri cells of nulliparous women screened for cervical intraepithelial neoplasia (CIN) in comparison to a group without CIN by using mesenchymal stem cell-like and hematopoietic lineage markers. A significant expression for CD29, CD38, HLA-I, and HLA-II was correlated positively to the CIN degree and it was more relevant in patients positive for human papilloma virus (HPV). Thus, identification and detailed characterization of pluripotent resident in uteri cells could be a promising therapeutic target.

The risk of coronary heart disease (CHD) clinical manifestations and patient management is estimated according to risk scores accounting multifactorial risk factors, thus failing to cover the individual cardiovascular risk. Technological improvements in the field of medical imaging, in particular, in cardiac computed tomography angiography and cardiac magnetic resonance protocols, laid the development of radiogenomics. Radiogenomics aims to integrate a huge number of imaging features and molecular profiles to identify optimal radiomic/biomarker signatures. In addition, supervised and unsupervised artificial intelligence algorithms have the potential to combine different layers of data (imaging parameters and features, clinical variables and biomarkers) and elaborate complex and specific CHD risk models allowing more accurate diagnosis and reliable prognosis prediction. Literature from the past 5 years was systematically collected from PubMed and Scopus databases, and 60 studies were selected. We speculated the applicability of radiogenomics and artificial intelligence through the application of machine learning algorithms to identify CHD and characterize atherosclerotic lesions and myocardial abnormalities. Radiomic features extracted by cardiac computed tomography angiography and cardiac magnetic resonance showed good diagnostic accuracy for the identification of coronary plaques and myocardium structure; on the other hand, few studies exploited radiogenomics integration, thus suggesting further research efforts in this field. Cardiac computed tomography angiography resulted the most used noninvasive imaging modality for artificial intelligence applications. Several studies provided high performance for CHD diagnosis, classification, and prognostic assessment even though several efforts are still needed to validate and standardize algorithms for CHD patient routine according to good medical practice.

Rationale About 50% of hospitalized coronavirus disease 2019 (COVID-19) patients with diabetes mellitus (DM) developed myocardial damage. The mechanisms of direct SARS-CoV-2 cardiomyocyte infection include viral invasion via ACE2-Spike glycoprotein-binding. In DM patients, the impact of glycation of ACE2 on cardiomyocyte invasion by SARS-CoV-2 can be of high importance. Objective To evaluate the presence of SARS-CoV-2 in cardiomyocytes from heart autopsy of DM cases compared to Non-DM; to investigate the role of DM in SARS-COV-2 entry in cardiomyocytes. Methods and results We evaluated consecutive autopsy cases, deceased for COVID-19, from Italy between Apr 30, 2020 and Jan 18, 2021. We evaluated SARS-CoV-2 in cardiomyocytes, expression of ACE2 (total and glycosylated form), and transmembrane protease serine protease-2 (TMPRSS2) protein. In order to study the role of diabetes on cardiomyocyte alterations, independently of COVID-19, we investigated ACE2, glycosylated ACE2, and TMPRSS2 proteins in cardiomyocytes from DM and Non-DM explanted-hearts. Finally, to investigate the effects of DM on ACE2 protein modification, an in vitro glycation study of recombinant human ACE2 (hACE2) was performed to evaluate the effects on binding to SARS-CoV-2 Spike protein. The authors included cardiac tissue from 97 autopsies. DM was diagnosed in 37 patients (38%). Fourth-seven out of 97 autopsies (48%) had SARS-CoV-2 RNA in cardiomyocytes. Thirty out of 37 DM autopsy cases (81%) and 17 out of 60 Non-DM autopsy cases (28%) had SARS-CoV-2 RNA in cardiomyocytes. Total ACE2, glycosylated ACE2, and TMPRSS2 protein expressions were higher in cardiomyocytes from autopsied and explanted hearts of DM than Non-DM. In vitro exposure of monomeric hACE2 to 120 mM glucose for 12 days led to non-enzymatic glycation of four lysine residues in the neck domain affecting the protein oligomerization. Conclusions The upregulation of ACE2 expression (total and glycosylated forms) in DM cardiomyocytes, along with non-enzymatic glycation, could increase the susceptibility to COVID-19 infection in DM patients by favouring the cellular entry of SARS-CoV2.

Background Glycemic control is a strong predictor of long-term cardiovascular risk in patients with diabetes mellitus, and poor glycemic control influences long-term risk of cardiovascular disease even decades after optimal medical management. This phenomenon, termed glycemic memory, has been proposed to occur due to stable programs of cardiac and endothelial cell gene expression. This transcriptional remodeling has been shown to occur in the vascular endothelium through a yet undefined mechanism of cellular reprogramming. Methods In the current study, we quantified genome-wide DNA methylation of cultured human endothelial aortic cells (HAECs) via reduced-representation bisulfite sequencing (RRBS) following exposure to diabetic (250 mg/dL), pre-diabetic (125 mg/dL), or euglycemic (100 mg/dL) glucose concentrations for 72 h (n = 2). Results We discovered glucose-dependent methylation of genomic regions (DMRs) encompassing 2199 genes, with a disproportionate number found among genes associated with angiogenesis and nitric oxide (NO) signaling-related pathways. Multi-omics analysis revealed differential methylation and gene expression of VEGF (↑5.6% DMR, ↑3.6-fold expression), and NOS3 (↓20.3% DMR, ↓1.6-fold expression), nodal regulators of angiogenesis and NO signaling, respectively. Conclusion In the current exploratory study, we examine glucose-dependent and dose-responsive alterations in endothelial DNA methylation to examine a putative epigenetic mechanism underlying diabetic vasculopathy. Specifically, we uncover the disproportionate glucose-dependent methylation and gene expression of VEGF and NO signaling cascades, a physiologic imbalance known to cause endothelial dysfunction in diabetes. We therefore hypothesize that epigenetic mechanisms encode a glycemic memory within endothelial cells.

Purpose Human leukocyte antigen (HLA)-G is a non-classic major histocompatibility complex HLA class I molecule. HLA-G may have tolerogenic properties which are linked to epigenetic-sensitive pathways. There is a correlation of sHLA-G levels and graft acceptance in transplantation studies. There are previous data on correlation of sHLA-G with graft rejection as well as with viral infections such as hepatitis C virus (HCV) in kidney transplanted patients. Here, we report the sHLA-G expression in patients on the waiting list for kidney transplantation, with and without anti-HCV compared to a control group. Methods Serum of 67 patients on the waiting list for kidney transplantation (n = 43 with anti-HCV and n = 24 without anti-HCV) was analyzed. Among these patients, n = 39 were on the waiting list for the first transplantation, while n = 28 were patients who returned in the list. The control group included n = 23 blood donors with anti-HCV (n = 13) and without anti-HCV (n = 10). Results The expression of sHLA-G was significantly lower in the control group (39.6 ± 34.1 U/ml) compared to both - patients on the waiting list for the first transplantation (62.5 ± 42.4 U/ml, p=0.031) and patients who returned in the list (76.7 ± 53.9 U/ml, p=0.006). No significant differences were observed in all anti-HCV positive groups. A positive linear correlation between sHLA-G and TNF-α, and patient age was observed. Conclusions Serum sHLA-G values were significantly increased in both - patients on the waiting list for the first transplantation and patients who returned in the list, as compared to control group. Our findings confirm the key tolerogenic role of sHLA-G levels as epigenetic-related marker for measuring the state of kidney allograft acceptance.

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

This study sought to determine the possible detrimental effects of several low- or non-caloric sweeteners on endothelial progenitor cells (EPCs), inflammation and behavioural changes in mice. C57BL/6 male mice received low and high dose of natural and artificial sweeteners for 4 weeks. EPCs, physical and biochemical variables, inflammation and behavioural changes were evaluated. A significant reduction of about 25% of EPCs was found when mice received a moderate amount of all sweeteners (p p p p

Genome-wide association studies have revealed several genes predisposing to autoimmunity, however, concordance rates in monozygotic twins are significantly below 50% for several autoimmune diseases. The limited presence of a strong genetic association only in some patients supports that other non-genetic mechanisms are active in these pathologies. Epigenetic modifications such as DNA methylation, histone modification, and microRNA signaling regulate gene expression and are sensitive to external stimuli and they might be as bridging between genetic and environmental factors. Some evidence has highlighted the involvement of epigenetic alterations in the pathogenesis of various autoimmune diseases giving rise to great expectations among clinicians and researchers. The direct role of these alterations in the initiation/progression of autoimmune diseases is still unclear. The knowledge in depth of these pathogenic and epigenetic mechanisms will increase the possibility of the control and/or prevention of autoimmune diseases through the use of drugs that target epigenetic pathways. Moreover, we could use epigenetic-related biomarkers to follow this complicated framework (for example H3K4me3 and miRNA-155 are among those proposed biomarkers). This article reviews current understanding of the epigenetic involvement in the field of autoimmune diseases especially in systemic lupus erythematosus, rheumatoid arthritis, sclerosis multiple and type 1 diabetes.

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.

Sensitized candidates for heart transplant usually end up on a long waiting list and have an increased risk of rejection, graft loss, and incidence of cardiac allograft vasculopathy. An increasing number of studies have demonstrated the negative effect of preformed and posttransplant antibodies on graft survival. Thus, in sensitized patients, the combination of new, appropriate, desensitization protocols, and monitoring of posttransplant development of donor-specific antibodies may improve short-term and long-term outcomes. Introduction of more-sensitive and more-specific techniques for antibody detection provides a valid tool for assessing the degree of pretransplant HLA histocompatibility, and, therefore, predicting the results of crossmatch in sensitized patients, which are difficult to transplant. Currently, there are no accurate and standard methods to determine the functional characteristics of antibodies detected by solid-phase assay and, therefore, to predict their clinical relevance. Therefore, the future of heart transplantation requires a better understanding of tissue typing techniques and the effect of anti-HLA antibodies on clinical outcome to prevent discrimination against sensitized patients at the time of organ allocation.

The CD146 cell membrane adhesion molecule is highly expressed on the cell surface of several tumours. The level of its expression has been found to correlate directly with tumour progression and metastatic potential, thus establishing CD146 as an important candidate of tumour growth and metastasis. In order to characterize its expression in human osteosarcoma (OS) cell lines, we have examined the CD146 expression at protein and RNA levels in both normal and tumour osteoblast-like cell lines by several methods. Our results indicate that CD146 protein is expressed at low levels in normal osteoblast cells whereas it is highly expressed in all OS cell lines analysed, (SaOS, MG-63, U-2OS). Moreover, CD146 overexpression was partially reduced in shYY1 cells, where the Yin Yang 1 transcription factor, also found over-expressed in human OS cells, has been silenced.

Increasing evidence indicates that mammalian SIRT1 mediates calorie restriction and influences lifespan regulating a number of biological molecules such as FoxO1. SIRT1 controls the angiogenic activity of endothelial cells via deacetylation of FoxO1. Endothelial dysfunction and reduced new blood vessel growth in diabetes involve a decreased bioactivity of endothelial progenitor cells (EPCs) via repression of FoxO1 transcriptional activity. The relative contribution of SIRT1 with respect to the direct effects of high glucose on EPC number is poorly understood. We report that treatment of EPCs with high glucose for 3 days determined a consistent downregulation of EPC positive to DiLDL/lectin staining and, interestingly, this was associated with reduced SIRT1 expression levels and enzyme activity, and increased acetyl-FoxO1 expression levels. Moreover, EPCs responded to high glucose with major changes in the expression levels of cell metabolism-, cell cycle-, and oxidative stress-related genes or proteins. Proteomic analysis shows increased expression of nicotinamide phosphorybosyl transferase and mitochondrial superoxide dismutase whereas a glucose-related heat shock protein is reduced. These findings show that SIRT1 is a critical modulator of EPCs dysfunction during alteration of glucose metabolism. © 2008 Elsevier B.V. All rights reserved.

The circulating form of endothelial progenitors cells (EPCs) are derived from bone marrow (BM)-derived hematopoietic stem cells (HSCs). Enhanced mobilization of EPCs was shown to be linked to cardiac diseases. This study investigated whether reduced EPC levels in advanced coronary heart disease (CHD) are secondary to a functional exhaustion of HSCs in the BM or to reduced mobilization. Number and functional properties of EPCs were assessed in 15 healthy controls, and 40 patients with CHD. The colony-forming unit (CFU) capacity of BM-derived mononuclear cells and the CD34+ HSC number were examined in four healthy volunteers, and 15 CHD patients. EPC number was reduced in CHD patients (P < 0.01 vs. controls). Moreover, the migratory capacity was significantly impaired in EPCs of CHD patients (P < 0.05 vs. controls). On multivariate analysis, CHD was an independent predictor of functional EPC impairment. CFUs were reduced in CHD patients (59.6 +/- 21.2 vs. 75.4 +/- 25.8 in controls, P < 0.05). CHD was also predictor of impaired CFU capacity. In this small clinical study, CHD is associated with selective impairment of HSC function in the BM and in the peripheral blood, which may contribute to impairment of cardiac function.

To date, there is no report on the effect of antioxidants on endothelial progenitor cells (EPCs). This study shows that in vitro incubation of EPCs with vitamin C and E reverted the already well documented lowering effect of TNF-alpha on EPC number and increased p-p38 expression levels. In order to document major changes of gene expression levels and gain insight into signalling pathways, microarray analysis was performed and a significant variation of the expression of 5389 genes in EPCs following antioxidant treatment was detected. Also in vivo evidence is provided about the positive effect of antioxidant vitamins on EPCs, since vitamin C and E supplementation potentiated the physical training-induced increase of EPC number and VEGF levels. Together, these data indicate that antioxidant treatment ameliorates EPC number and causes major changes of gene expression within these cells in vitro. Furthermore, concomitant antioxidant supplementation and physical training in vivo raised the levels of circulating EPCs and serum VEGF more than physical training alone.

Inflammation and immune response play a pivotal role in the pathophysiology of ischemic stroke giving their contribution to tissue damage and repair. Emerging evidence supports the involvement of epigenetic mechanisms such as methylation, histone modification and miRNAs in the pathogenesis of stroke. Interestingly, epigenetics can influence the molecular events involved in ischemic injury by controlling the switch from pro- to anti-inflammatory response, however, this is still a field to be fully explored. The knowledge of epigenetic processes could to allow for the discovery of more sensitive and specific biomarkers for risk, onset, and progression of disease as well as further novel tools to be used in both primary prevention and therapy of stroke. Indeed, studies performed in vitro and in small animal models seem to suggest a neuroprotective role of HDAC inhibitors (e.g. valproic acid) and antagomir (e.g. anti-miR-181a) in ischemic condition by modulation of both immune and inflammatory pathways. Thus, the clinical implications of altered epigenetic mechanisms for the prevention of stroke are very promising but clinical prospective studies and translational approaches are still warranted.

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.

Recently, management of patients awaiting solid organ transplantation has taken advantages after the development of more sensitive and accurate solid phase assays which have supported the historic complement dependent cytotoxicity. This approach has allowed the detection of antibodies in patients previously considered negative. The use of the single antigen beads resulted in a more accurate anti-human leukocyte antigen (HLA) antibody characterization. The detection of anti-HLA antibodies specific for C, DQ and DP loci that were not so well characterized has been possible through the implementation of the single antigen assay. The assessment of HLA compatibility has been expanded through the introduction of "epitope matching" concept and the definition of the unacceptable antigens for a more adequate evaluation of donor-recipient compatibility. However, the clinical impact of pre-formed and de novo anti-HLA antibodies detected by solid phase assays is still controversial due to the drawback related to result interpretation. Until today, the unresolved issues concern if all antibodies affect the medium and long term clinical outcome. An open debate on the clinical relevance of anti-HLA antibodies detected by single-antigen beads highlights needing to further investigations. Here, we describe the novel applications and the improvements of the solid-phase assay use.

Mediator (MED) complex is a multiprotein playing a key role in the eukaryotic transcription. Alteration of MED function may have enormous pathophysiological consequences and several MED genes have been implicated in human diseases. Here, we have combined computational and experimental approaches to identify and characterize, new transcripts generated by alternative splicing (AS) for all MED genes, through the analysis of our recently published RNA-Sequencing datasets of endothelial progenitor cells (EPCs). This combined strategy allowed us to identify novel transcripts for MED4, MED9, MED11, MED14, MED27 and CDK8 most of them generated by AS. All the newly identified transcripts, except MED11, are predicted to encode novel protein isoforms. The identification of novel MED variants could lead to the finding of other MED complexes with different functions depending on their subunit composition. Finally, the expression profile of all MED genes, together with an extensive gene expression analysis, may be useful to better classify the diverse subsets of cell populations that contribute to neovascularization. © 2014. Mediator (MED) complex is a multiprotein playing a key role in the eukaryotic transcription. Alteration of MED function may have enormous pathophysiological consequences and several MED genes have been implicated in human diseases. Here, we have combined computational and experimental approaches to identify and characterize, new transcripts generated by alternative splicing (AS) for all MED genes, through the analysis of our recently published RNA-Sequencing datasets of endothelial progenitor cells (EPCs). This combined strategy allowed us to identify novel transcripts for MED4, MED9, MED11, MED14, MED27 and CDK8 most of them generated by AS. All the newly identified transcripts, except MED11, are predicted to encode novel protein isoforms. The identification of novel MED variants could lead to the finding of other MED complexes with different functions depending on their subunit composition. Finally, the expression profile of all MED genes, together with an extensive gene expression analysis, may be useful to better classify the diverse subsets of cell populations that contribute to neovascularization. © 2014.

Objectives Although previous studies have investigated the effect of human leukocyte antigen matching on long-term outcomes after heart transplants, its role in the prognosis after a heart transplant remains unclear, particularly with respect to short-term survival. Materials and methods We evaluated the human leukocyte antigen mismatch on in-hospital mortality of 158 consecutive patients who had undergone a heart transplant between 2000 and 2008. Human leukocyte antigens-A, -B, and -DR were determined by means of serologic and molecular techniques. Univariate analysis and a multiple logistic regression models evaluated the effect of human leukocyte antigen variants on mortality, independent of clinical variables. Results In-hospital mortality was 11.4%. Higher prevalence of acute kidney injury (50.0% vs 12.9%), higher levels of troponins 48 hours after transplant (15.6 ± 12.0 ng/mL vs 9.7 ± 9.4 ng/mL), prolonged ischemia (188.2 ± 32.5 min vs 162.6 ± 40.7 min), higher frequency of reoperation (61.1% vs 17.9%), and higher human leukocyte antigen-DR mismatch (1.61 ± 0.5 vs 1.30 ± 0.6) were found in patients who died. By logistic regression analysis, humanleukocyte antigen-DR mismatch is associated with in-hospital mortality (OR=5.159, 95% CI=1.348-19.754), independent of the effect of covariates such as recipient age, mismatch sex, mismatch human leukocyte antigen-A, human leukocyte antigen-B, acute kidney injury, reoperation, ischemia duration, and levels of troponins. Conclusions Human leukocyte antigen-DR mismatch is associated with in-hospital mortality in heart transplant.

Recent evidences have shown that several host genetic factors influence susceptibility or protection to hepatitis C virus (HCV) infection. There are controversial data regarding the associations of human leukocyte antigens (HLA) and the clearance or progression of HCV. The aim of this study was to investigate whether particular HLA molecules were associated with HCV infection in recipients awaiting kidney transplantation considered at high-risk to infection due to protracted hemodialysis treatment. To this purpose, 301 kidney recipients with HCV infection and 1103 uninfected recipients were examined for HLA class I and II molecules. In our case-control study, HLA-A * 26 is positively associated with HCV infection while HLA-A * 29, -B * 40 and -DRB1 * 01 are negatively associated with HCV infection. Multiple logistic regression analysis demonstrated that age (OR = 1.02; 95% CI = 1.01–1.04; p * 26, -A * 29, -B * 40 and -DRB1 * 01 [(OR = 1.54; 95% CI = 1.03–2.30; p = 0.03); (OR = 0.50; 95% CI = 0.26–0.99; p = 0.05); (OR = 0.42; 95% CI = 0.23-0, 7; p = 0.01); (OR = 0.62; 95% CI = 0.41-0, 94; p = 0.03); respectively] are independent predictors of HCV infection. Our results suggest that particular HLA molecules, as host genetic factors, may have a relationship with susceptibility or protection to HCV infection also in recipients awaiting kidney transplantation.

Cardiovascular disease is the leading cause of morbidity and mortality in the world. In recent years, there has been an increasing interest both in basic and clinical research regarding the field of cell therapy for coronary heart disease (CHD). Several preclinical models of CHD have suggested that regenerative properties of stem and progenitor cells might help restoring myocardial functions in the event of cardiac diseases. Here, we summarize different types of stem/progenitor cells that have been tested in experimental and clinical settings of cardiac regeneration, from embryonic stem cells to induced pluripotent stem cells. Then, we provide a comprehensive description of the most common cell delivery strategies with their major pros and cons and underline the potential of tissue engineering and injectable matrices to address the crucial issue of restoring the three-dimensional structure of the injured myocardial region. Due to the encouraging results from preclinical models, the number of clinical trials with cell therapy is continuously increasing and includes patients with CHD and congestive heart failure. Most of the already published trials have demonstrated safety and feasibility of cell therapies in these clinical conditions. Several studies have also suggested that cell therapy results in improved clinical outcomes. Numerous ongoing clinical trials utilizing this therapy for CHD will address fundamental issues concerning cell source and population utilized, as well as the use of imaging techniques to assess cell homing and survival, all factors that affect the efficacy of different cell therapy strategies.

Mediator (MED) is a fundamental component of the RNA polymerase II-mediated transcription machinery playing a pivotal role in the regulation of eukaryotic mRNA synthesis. Human MED complexes contain at least 30 distinct MED subunits. Our previous study, aimed to analyse MED complex during the pattern of endothelial progenitor cells (EPCs) differentiation, found an alternative transcript of MED30 subunit expressed only in circulating immature progenitor cells. Here, we report two novel transcripts of MED12 and MED19 subunits both generated by alternative splicing and displaying similar expression patterns, thereby indicating their involvement during endothelial cell differentiation. © 2012 Elsevier Masson SAS. All rights reserved.

The following paper is an overview on stem cells therapy in patients with peripheral vascular diseases. Recent research shows the ability of stem cells to develop and strengthen the collateral network in ischemic legs. Here, we discuss this clinical and therapeutic approach. To date, research has been mainly focused on patients with ischemic ulcers without possibility of revascularization. Non-invasive stem cell therapy has been proposed as an alternative to the amputation of such patients, but when the ulcers appear it is sometime too late. In our point of view, the selection of patients is a very important issue and we believe that the best candidate for this treatment is the patient with intermittent claudication before the development of ulcers. This choice could allow the optimization of results by the type of treated patients and not only by the type of infused cells. Indeed, several variables still remain to be elucidated for stem cell therapy, including the type of cells to be used, the infusion route, and more importantly, the stage of patients to be treated.

The clinical transplantation outcome is related to both effects of immunological and non immunological factors degenerating into hyperacute, acute and chronic rejection. Modern immunosuppressive treatments have resolved most events linked to acute rejection while long-term survival still remains the major problem after heart transplantation. The goal of personalized immunosuppressive therapy is to prevent rejection without inducing toxic effects. The aim of future studies could be to clarify the pathogenesis of chronic rejection and develop new and less toxic therapeutic approaches to induce "tolerance" to the graft without major side effects.

Aim Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the standard therapy for the treatment of malignant blood diseases or immune system disorders. HLA compatibility influences the transplant outcome and minimizes the risks of rejection or severe graft-versus-host disease. The identification of a compatible unrelated donor depends on the race/ethnicity due to heterogeneity within the various ethnic groups and the high polymorphism of HLA genes that limits the possibility of HSCT from unrelated donors. In Italy, the Italian Bone Marrow Registry (IBMDR) is the main source of unrelated volunteer donors. Here, we report our experience of HLA typing laboratory. Methods The current Italian guidelines include high resolution HLA typing at A, B, C, and DRB1 loci for volunteer unrelated donors in the enrollment time. In 2013, at the Regional Reference Laboratory of Transplant Immunology of Naples (Italy), the donors were typed by sequence specific primers PCR. Results We have identified the HLA-A∗23:18 allele in two different volunteer donors. This is a rare allele in the Italian and Caucasian population (IMGT/HLA AccNo:HLA03170). The allele A∗23:18, for the first time, was identified by SBT in a patient attending HSCT in Italy. To date, this allele is still listed as rare allele in the NMDP rare allele list file version 3.9.0. The HLA typing of these two donors is A∗03:01,∗23:18; B∗07:02,∗14:02; C∗07:02,∗08:02; DRB1∗01:02,∗15:01 and A∗02:05,∗23:18; B∗14:02; C∗08:02; DRB1∗01:02. These donors share the HLA-A∗23:18; B∗14:02; C∗08:02 and DRB1∗01:02 alleles with the patient where this allele was identified for the first time. Both donors are Italians and resident in our region since several generations. Conclusion The high polymorphism and the pattern of HLA alleles and haplotypes distribution in human populations in the world make it difficult to find compatible stem cell donors. However, the role of HLA compatibility at allelic level in allo-HSCT is still debated, especially if mismatches are not localized in exon 2 and 3 of HLA class I genes. Our results highlight that some alleles, considered rare, may be frequent alleles in some regions and therefore it is necessary a more accurate knowledge of allele and haplotype frequencies in order to develop new strategies for the search of HSCT unrelated donors.

Aim Heart transplantation has evolved as the “gold standard” therapy for patients with end-stage heart failure (HF). Although mechanical circulatory support technology is improving, heart transplantation remains the preferred treatment. The mean risk factors for HF include environmental and genetic components. The intricate patterns of gene expression, during normal and disease states, are governed by elaborate signaling pathways that converge on the transcriptional mechanism. The general transcriptional mechanism can be recruited to promoters by Mediator complex (MED). MED is a large multi-subunit complex functioning to integrate diverse cellular signals by multiple mechanisms including recruitment of RNA polymerase II, chromatin modifying proteins and non-coding RNAs to promoters in a context dependent manner. Several MED subunits are involved in many human pathologies, including heart diseases. Particularly, some subunits like MED13 and MED13L are altered in congenital heart diseases. Similarly, MED30 mutation can lead to lethal cardiomyopathy in a mouse model. Methods To gain further insights, we have collected 12 human left atrium samples, which were acquired from hearts of 6 donors and 6 recipients during transplant intervention. Patients were affected by hypertrophic or dilated idiopathic cardiomyopathy. From these samples we have isolated RNA and started to analyze the expression patterns of MED subunits by real-time RT-PCR with specific oligonucleotides. Possible interactions between human leukocyte antigen (HLA) profile and MEDs are under investigation. Furthermore, RNA-seq analysis of samples from both donors and recipients is ongoing in our laboratory and whole transcriptome data will be also shown. Results Our preliminary data confirm that some MED subunits are altered in diseased hearts compared to healthy donor biopsies. Conclusions The identification of such alterations can lead to novel therapeutic targets regulating MED subunits, and HLA in advanced HF.

Aim The interaction of the heart with other organs is well established. In particular, several donor and recipient factors involving the heart are known to be associated with graft loss in kidney transplantation. In this retrospective single-center study, we analyzed the effect of clinical, cardiological and immunological factors on kidney transplantation outcome. The study included 245 transplanted recipients from deceased donors between 2000 and 2006. Methods In donors, age, cause of death, history of hypertension, hypotension or cardiac arrest, length of intensive care unit stay, serum creatinine levels and HLA typing were evaluated; while age, waiting time, HLA typing, antibodies sensitization and allocation were evaluated in recipients. Age donor/recipient matching and HLA mismatches were also considered. Results Cox regression analysis shows that time spent in waiting list increases the risk of restarting dialysis (OR=1.01, 95% CI=1.00-1.03; p Conclusions Our study established that both hypertension and HLA-A mismatch can affect the time of restarting dialysis in patients undergoing kidney transplantation.

Aim The twin epidemics of obesity and Type 2 diabetes continue to increase in industrialized nations. Approximately two thirds of adult Americans and Western Countries are currently overweight or obese and therefore at increased risk for a number of deleterious health conditions including Type 2 diabetes and Cardiovascular Disease (CVD). Consumption of sugar-sweetened beverages may be one of the relevant dietary causes of metabolic disorders. Therefore, substituting sugar with low-calorie sweeteners may be n efficacious weight management strategy. Since Endothelial Progenitor Cells (EPC) are involved in cardiovascular tissue regeneration, we tested the effects of glucose and alternative sweeteners (fructose, aspartame and rebaudioside A) on EPCs. Methods Early-EPCs were isolated from total peripheral blood of healthy human donors and treated with different concentration of D-glucose, fructose, aspartame and rebaudioside A for 4 days. EPC number was evaluated by flow cytometry analysis using CD34 and KDR (CD309/VEGFR-2) monoclonal antibodies. Results As partially expected, high glucose level severely impaired EPC number. Indeed, CD34+/KDR+ dual positive cells were reduced of about 70% at the maximum glucose concentration used. Differently, the other molecules tested were able to reduce the number of total KDR+ cells, but not CD34+/KDR+ dual positive cells. Conclusions Endothelial dysfunction and reduced new blood vessel growth during vascular complication in either Type 1 or Type 2 diabetes, is known to be strictly related to altered number and function of circulating EPCs. The pathogenic role of reduced bioactivity of EPCs is correlated to increased rate of CVD. These preliminary findings suggest that these alternative sweeteners do not alter CD34+/KDR+ dual positive EPC number, although they can alter endothelial cell number through other cellular molecular pathogenic mechanisms.

Aim Previous clinical studies indicate that implantation of Bone Marrow Cells (BMCs) into ischemic limbs may improve peripheral ischemia. We previously demonstrated that intraarterial administration of autologous BMC and antioxidants and L-arginine therapy is safe and effective in patients with advanced atherosclerotic peripheral artery disease (PAD) with positive effects until 18 months. Here, we assessed the long-term follow-up of patients until 36 months. Methods In the original study protocol (NCT00306085), we studies 18 patients with PAD (advanced III/IV Fontaine stage) and an additional group of 18 patients taking maximal drug therapy that refused BMC therapy as control. Therapeutic neo-angiogenesis was estimate by clinical assessment, angiography and laser Doppler/capillaroscopy. Results At 18 months of follow-up, among conservative control patients, 10 underwent amputation in comparison with two BMC-treated patients (55.6 vs. 13.3%; p=0.014). Ischemic ulcers improved in 13 patients (after 6-12 months). The follow-up at 36 months of remaining patients confirmed a favorable trends in patients treated with BMCs in comparison to controls (p Conclusions This follow-up study confirmed safety and feasibility of intraarterial autologous BMCs in patients with critical ischemia and advanced atherosclerotic PAD.

Aim Recent evidences have shown that several host genetic factors influence susceptibility or protection to hepatitis C virus (HCV) infection. There are controversial data regarding the associations of human leukocyte antigens (HLA) and clearance or progression of HCV. The aim of this study was to investigate whether particular HLA molecules were associated with HCV infection in recipients awaiting kidney transplantation, considered at high-risk to infection due to protracted hemodialysis treatment. Methods This case-control study included 1404 of 3000 total chronic kidney diseases patients on dialysis, in waiting list for transplantation, enrolled at the Laboratory of Transplantation Immunology of Second University of Naples, Italy. Particularly, we consecutively considered 301 patients with HCV infection and we randomly selected 1103 HCV-negative as control group. None of the patients carried hepatitis B surface antigen or had human immunodeficiency virus antibody to exclude additional viral factors that could affect our results. All patients were tested for HCV antibody detection by serological Chemiluminescent Microparticle Immunoassay (CMIA, Abbott, Italy). All kidney recipients were tested for HLA class I (HLA-A and -B) and class II (-DR) antigens by serological and molecular HLA typing. Results In our case-control study, HLA-A26 is positively associated with HCV infection, while HLA-A29, -B40 and -DR1 are negatively associated with HCV infection. Multiple logistic regression analysis demonstrated that age (OR=1.02; 95% CI=1.01-1.04; p Conclusions Our results suggest that particular HLA molecules, as host genetic factors, may have a relationship with susceptibility or protection to HCV infection also in recipients awaiting kidney transplantation.

Aim Mediator (MED) complex functions as a pivotal adaptor between transcription factors bounded at gene regulatory elements, RNA polymerase II and general transcription factors. Different ancestral humanMED complexes including at least 30 distinct MED subunits (MEDs) have been isolated. Because of the importance of ancestral MED role in the transcription of the eukaryotic genes, disruption of MED function may have relevant pathophysiological consequence also in the cardiovascular system and transplantation. Here, we have analyzed the expression data relative to MEDs in human endothelial progenitor cells (EPCs) obtained by RNA-Seq on a next generation sequencing platform. The introduction of next generation sequencing (NGS) technologies has revealed the complexity of mammalian transcriptomes, enabling to effectively explore, with an unprecedented throughput capacity, simple and complex genomes and even their differences in health and disease conditions. Methods RNA was isolated from early human EPCs and after ribodepletion it was used for the RNA-Seq through SOLID System. Results By analysis of RNA-Seq data and RT-PCR validation we have identified novel transcripts in several MED genes (including MED1, MED 15, MED 17 and MED23). Some of these transcripts are different in their 5’ and 3’ UTRs. Other transcripts are differently spliced thus excluding or including known/new exons. Conclusions This findings contribute to the characterization of novel MED transcripts in EPCs that could participate to the regulation of genes involved in different cell states. Our findings could have relevant implications in the regenerative action of EPCs in the cardiovascular system immune response after cardiac transplantation.