Search

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 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.

The complex polymorphism of the HLA genes and the need of a proper identification of anti-HLA antibodies have led to continuously develop novel practical and feasible technologies in the field of organ and tissue transplantation. Technologies to identify HLA molecules have evolved from the serological to the molecular methods and a true innovation in the DNA sequencing has taken place with the development of next generation sequencing. An interesting field to explore is how the information resulting from the HLA-DNA sequencing can be applied in the clinical setting by including the alloimmunization assessment. Indeed, a good characterization of anti-HLA antibody at epitope level can reduce the risk of immunization. Many anti-HLA antibodies are specific for epitopes rather than for HLA antigens and the knowledge of unacceptable epitopes allows to reduce the number of mismatched antigens. Furthermore, high resolution HLA allele typing could help to understand the epitopes against which antibodies are developed. However, the improvements should not only concern the diagnostic tools in the pre-transplantation phase, but also the monitoring of post transplantation outcome. There is a growing interest in developing new non-invasive biomarkers to monitor the rejection. Currently, increasing evidence has focused on miRNAs, epigenetic markers emerged as regulators of molecular events that are differently expressed in biopsies and blood as well as in urinary samples of transplanted recipients. The implementation of next generation sequencing and genome-wide expression analysis together with functional assays may provide useful tools to evaluate the epigenetic modulation in transplantation biology but many efforts are requested for translating in the clinical arena the results obtained in experimental models.

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.

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.

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.

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.

The recruitment of circulating endothelial progenitor cells (EPCs) might have a beneficial effect on the clinical course of several diseases. Endothelial damage and detachment of endothelial cells are known to occur in infection, tissue ischemia, and sepsis. These detrimental effects in EPCs are unknown. Here we elucidated whether human EPCs internalize Bartonella henselae constituting a circulating niche of the pathogen. B. henselae invades EPCs as shown by gentamicin protection assays and transmission electron microscopy (TEM). Dil-Ac-LDL/lectin double immunostaining and fluorescence-activated cell sorting (FACS) analysis of EPCs revealed EPC bioactivity after infection with B. henselae . Nitric oxide (NO) and its precursor l -arginine ( l -arg) exert a plethora of beneficial effects on vascular function and modulation of immune response. Therefore, we tested also the hypothesis that l -arg (1–30 mM) would affect the infection of B. henselae or tumor necrosis factor (TNF) in EPCs. Our data provide evidence that l -arg counteracts detrimental effects induced by TNF or Bartonella infections via NO (confirmed by DETA-NO and L-NMMA experiments) and by modulation of p38 kinase phosphorylation. Microarray analysis indicated several genes involved in immune response were differentially expressed in Bartonella -infected EPCs, whereas these genes returned in steady state when cells were exposed to sustained doses of l -arg. This mechanism may have broad therapeutic applications in tissue ischemia, angiogenesis, immune response, and sepsis.

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

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.