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LIFE MySOIL project presentation delivered by Alessandro d'Annibale, from the University of la Tuscia (UNITUS), during the workshop “Mycoremediation, a sustainable strategy for the recovery of oil-contaminated sites”, organised online by LIFE MySOIL on May 11th, 2023., This project has received funding from the LIFE Programme of the European Commission under contract number LIFE20 ENV/ES/000416.

LIFE MySOIL project presentation delivered by Fiora Bagnato, from Eni Rewind, and Maurizio Petruccioli, from the University of La Tuscia (UNITUS) during the workshop SiCon 2023 Contaminated Sites – Experiences in Remediation Interventions, that was held from February 8th to 10th, 2023, in Rome (Italy)., This project has received funding from the LIFE Programme of the European Commission under contract number LIFE20 ENV/ES/000416.

Longevity, as a complex life-history trait, shares an ontogenetic relationship with other quantitative traits, such as epigenetic and environmental factors. Therefore, it is important to identify environmental factors that may modify the epigenome to establish healthy aging. This study explored the association between tap drinking water and longevity in Cilento, Italy, to understand whether trace elements in local drinking water may have an influence on old, nonagenarian, and centenarian people and promote their health and longevity. Data on population and water sources were collected through the National Demographic Statistics, the Cilento Municipal Archives, and the Cilento Integrated Water Service. Ordinary least squares (OLS) regression and a geographically weight regression (GWR) model were used to study the spatial relationship between the explanatory and outcome variables of longevity. The results of the study showed that the prevalence of longevity is concentrated in the central, northern and southeastern areas of the territory and that some trace elements present in tap water may contribute to local longevity in Cilento. Specifically, all Cilento municipalities had alkaline tap water, and the municipalities with the highest longevity concentrations had higher alkalinity levels than the other municipalities, soft to medium-hard water hardness, an amount of total dissolved solids equivalent to the level of excellent water, lower amounts of sodium, adequate iron concentration, and adequate dietary intake of manganese per day.

Beyond well-assessed risk factors, cardiovascular events could be also associated with the presence of epigenetic and genetic alterations, such as the methylenetetrahydrofolate-reductase (MTHFR) C677T polymorphism. This gene variant is related to increased circulating levels of homocysteine (Hcy) and cardiovascular risk. However, heterozygous carriers have an augmented risk of cardiovascular accidents independently from normal Hcy levels, suggesting the presence of additional deregulated processes in MTHFR C677T carriers. Here, we hypothesize that targeting Sirtuin 1 (SIRT1) could be an alternative mechanism to control the cardiovascular risk associated to MTHFR deficiency condition. Flow Mediated Dilatation (FMD) and light transmission aggregometry assay were performed in subjects carrying MTHFR C677T allele after administration of resveratrol, the most powerful natural clinical usable compound that owns SIRT1 activating properties. MTHFR C677T carriers with normal Hcy levels revealed endothelial dysfunction and enhanced platelet aggregation associated with SIRT1 downregulation. SIRT1 activity stimulation by resveratrol intake was able to override these abnormalities without affecting Hcy levels. Impaired endothelial function, bleeding time, and wire-induced thrombus formation were rescued in a heterozygous Mthfr-deficient (Mthfr+/–) mouse model after resveratrol treatment. Using a cell-based high-throughput multiplexed screening (HTS) assay, a novel selective synthetic SIRT1 activator, namely ISIDE11, was identified. Ex vivo and in vivo treatment of Mthfr+/– mice with ISIDE11 rescues endothelial vasorelaxation and reduces wire-induced thrombus formation, effects that were abolished by SIRT1 inhibitor. Moreover, platelets from MTHFR C677T allele carriers treated with ISIDE11 showed normalization of their typical hyper-reactivity. These results candidate SIRT1 activation as a new therapeutic strategy to contain cardio and cerebrovascular events in MTHFR carriers.

Longevity is rightly considered one of the greatest achievements of modern society. Biomedical research has shown that aging is the major risk factor for many diseases, so to find the right answers to aging it is necessary to identify factors that can positively influence longevity. This study investigated the clinical status, nutritional behavior, lifestyle, and social and community determinants of the well-being of young older adults and nonagenarians/centenarians in Salerno and province through the judgment of their physicians. Data were collected through an online survey. Multivariate Poisson and logistic regression models were used to calculate significant predictors of the outcomes of interest. The interesting finding was that cardiovascular disease was a risk factor for young older adults, while it was a protective factor for nonagenarians/centenarians, meaning that as age increased, heart problems tended to decrease. Certain foods were found to be a significant protective factor for both young older adult and nonagenarian–centenarian patients. In addition, psychosomatic disorders were found to be determinant for the young older adults, while depression was a risk factor for the nonagenarians/centenarians because they were not always gratified by their long lives and often felt like a burden on the family. The protective significant variable among the determinants of community well-being for both young older adults and nonagenarians/centenarians was the retention of honorary achievement. Based on our results, we are able to support the hypothesis of a difference between the young older adults and the nonagenarians/centenarians in clinical status, nutritional behaviors, lifestyle, and determinants of community well-being. However, societies need more social and educational programs that are able to build “a new idea of old age” by improving and supporting the young older adults and the nonagenarians/centenarians, with the goal of intergenerational solidarity, well-being, and social inclusion, as well as preventive interventions on lifestyles and nutrition, which will allow us to provide a new key to understanding aging.

SARS-CoV-2 infection causes a considerable inflammatory response coupled with impaired platelet reactivity, which can lead to platelet disorders recognized as negative prognostic factors in COVID-19 patients. The virus may cause thrombocytopenia or thrombocytosis during the different disease stages by destroying or activating platelets and influencing platelet production. While it is known that several viruses can impair megakaryopoiesis by generating an improper production and activation of platelets, the potential involvement of SARS-CoV-2 in affecting megakaryopoiesis is poorly understood. To this purpose, we explored, in vitro, the impact of SARS-CoV-2 stimulation in the MEG-01 cell line, a human megakaryoblastic leukemia cell line, considering its spontaneous capacity of releasing platelet-like particles (PLPs). We interrogated the effect of heat-inactivated SARS-CoV-2 lysate in the release of PLPs and activation from MEG-01, the signaling pathway influenced by SARS-CoV-2, and the functional effect on macrophagic skewing. The results highlight the potential influence of SARS-CoV-2 in the early stages of megakaryopoiesis by enhancing the production and activation of platelets, very likely due to the impairment of STATs signaling and AMPK activity. Overall, these findings provide new insight into the role of SARS-CoV-2 in affecting megakaryocyte–platelet compartment, possibly unlocking another avenue by which SARS-CoV-2 moves.

As we age, our body experiences chronic, systemic inflammation contributing to the morbidity and mortality of the elderly. The senescent immune system has been described to have a causal role in driving systemic aging and therefore may represent a key therapeutic target to prevent pathological consequences associated with aging and extend a healthy lifespan. Previous studies from our group associated a polymorphic haplotype variant in the BPIFB4 gene (LAV-BPIFB4) with exceptional longevity. Transfer of the LAV-BPIFB4 in preclinical models halted the progression of cardiovascular diseases (CVDs) and frailty by counterbalancing chronic inflammation. In the present study, we aimed to delineate the action of systemic adeno-associated viral vector-mediated LAV-BPIFB4 gene transfer (AAV-LAV-BPIFB4) on the deleterious age-related changes of the immune system and thereby the senescence-associated events occurring in C57BL/6J mice aged 26 months. Our in vivo data showed that 26-months-old mice had a higher frequency of CD45+SA-beta Gal+immune cells in peripheral blood than young (4-months-old) C57BL/6J mice. Notably, AAV-LAV-BPIFB4 gene transfer in aged mice reduced the pool of peripheral immunosenescent cells that were shown to be enriched in the spleen. In addition, the proper tuning of the immune secretory phenotype (IL1βlow, IL6low, IL10high) associated with a significant reduction in SA-beta Gal-positive area of aorta from AAV-LAV treated mice. At the functional level, the reduction of senescence-associated inflammation ensured sustained NAD+levels in the plasma of AAV-LAV-BPIFB4 old mice by preventing the NADase CD38 increase in F4/80+ tissue-resident macrophages and Ly6Chighpro-inflammatory monocytes of the spleen and bone marrow. Finally, to validate the clinical implication of our findings, we showed that Long-living-individuals (LLIs, >95 years), which delay CVDs onset, especially if LAV-carriers, were characterized by high NAD+levels. In conclusion, the new senotherapeutic action of LAV-BPIFB4 may offer a valuable therapeutic tool to control aging and reduce the burden of its pathophysiological disorders, such as CVDs.

In the ongoing global COVID-19 pandemic, male sex is a risk factor for severe disease and death, and the reasons for these clinical discrepancies are largely unknown. The aim of this work is to study the influence of sex on the course of infection and the differences in prognostic markers between genders in COVID-19 patients. Our cohort consisted of 64 adult patients (n = 34 men and n = 30 women) with PCR-proven SARS-CoV-2 infection. Further, a group of patients was characterized by a different severity degree (n = 8 high- and n = 8 low-grade individuals for both male and female patients). As expected, the serum concentrations of LDH, fibrinogen, CRP, and leucocyte count in men were significantly higher than in females. When serum concentrations of the inflammatory cytokines, including IL-6, IL-2, IP-10 and IL-4 and chemokines like MCP-1, were measured with multiplex ELISA, no significant differences between male and female patients were found. In COVID-19 patients, we recently attributed a new prognostic value to BPIFB4, a natural defensin against dysregulation of the immune responses. Here, we clarify that BPIFB4 is inversely related to the disease degree in men but not in women. Indeed, higher levels of BPIFB4 characterized low-grade male patients compared to high-grade ones. On the contrary, no significant difference was reported between low-grade female patients and high-grade ones. In conclusion, the identification of BPIFB4 as a biomarker of mild/moderate disease and its sex-specific activity would open an interesting field for research to underpin gender-related susceptibility to the disease.

Aims: Homozygosity for a 4-missense single-nucleotide-polymorphism-haplotype of the human BPIFB4 gene is enriched in long-living individuals. Delivery of this Longevity Associated Variant (LAV) improved revascularisation and reduced endothelial dysfunction and atherosclerosis in mice through a mechanism involving the stromal cell-derived factor-1 (SDF-1). Here, we investigated if delivery of the LAV-BPIFB4 gene may attenuate the progression of diabetic cardiomyopathy. Methods and Results: Compared with age-matched lean controls, diabetic db/db mice showed altered echocardiographic indices of diastolic and systolic function and histological evidence of microvascular rarefaction, lipid accumulation, and fibrosis in the myocardium. All these alterations, as well as endothelial dysfunction, were prevented by systemic LAV-BPIFB4 gene therapy using an adeno-associated viral vector serotype 9 (AAV9). In contrast, AAV9-wild type (WT)-BPIFB4 exerted no benefit. Interestingly, the LAV-BPIFB4-treated mice showed increased SDF-1 levels in peripheral blood and myocardium and upregulation of cardiac myosin heavy chain isoform alpha, a contractile protein that was reduced in diabetic hearts. SDF-1 upregulation was instrumental to LAV-BPIFB4-induced benefit as both haemodynamic and structural improvements were inhibited by an orally active antagonist of the SDF-1 CXCR4 receptor. Conclusions: In mice with type-2 diabetes, LAV-BPIFB4 gene therapy promotes an advantageous remodelling of the heart, allowing it to better withstand diabetes-induced stress. These results support the viability of transferring healthy characteristics of longevity to attenuate diabetic cardiac disease.

An unresolved issue in autoimmunity is the lack of surrogate biomarkers of immunological self-tolerance for disease monitoring. Here, we show that peripheral frequency of a regulatory T cell population, characterized by the coexpression of CD3 and CD56 molecules (TR3-56), is reduced in individuals with new-onset type 1 diabetes (T1D). In three independent T1D cohorts, we find that low frequency of circulating TR3-56 cells is associated with reduced beta-cell function and with the presence of diabetic ketoacidosis. Since autoreactive CD8+ T cells mediate disruption of insulin-producing beta cells1–3, we demonstrate that TR3-56 cells can suppress CD8+ T cell functions in vitro by reducing the levels of intracellular reactive oxygen species. The suppressive function, phenotype and transcriptional signature of TR3-56 cells are also altered in children with T1D. Together, our findings indicate that TR3-56 cells constitute a regulatory cell population that controls CD8+ effector functions, whose peripheral frequency may represent a traceable biomarker for monitoring immunological self-tolerance in T1D. Low numbers of circulating CD3+CD56+ regulatory T cells are associated with reduced beta-cell function and diabetic ketoacidosis in three separate cohorts of children with type 1 diabetes.

Recent discoveries have shed light on the participation of the immune system in the physio pathology of the cardiovascular system underpinning the importance of keeping the balance of the first to preserve the latter. Aging, along with other risk factors, can challenge such balance triggering the onset of cardiovascular diseases. Among several mediators ensuring the proper cross-talk between the two systems, bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) has been shown to have a pivotal role, also by sustaining important signals such as eNOS and PKC-alpha. In addition, the Longevity-associated variant (LAV), which is an haplotype allele in BPIFB4 characterized by 4 missense polymorphisms, enriched in homozygosity in Long Living Individuals (LLIs), has been shown to be efficient, if administered systemically through gene therapy, in improving many aspects of cardiovascular diseases (CVDs). This occurs mainly through a fine immune system remodeling across: 1) a M2 macrophage polarizing effect, 2) a favorable redistribution of the circulating monocyte cell subsets and 3) the reduction of T-cell activation. Furthermore, LAV-BPIFB4 treatment induced a desirable recovery of the inflammatory balance by mitigating the pro-inflammatory factor levels and enhancing the anti-inflammatory boost through a mechanism that is partially dependent on SDF-1/CXCR4 axis. Importantly, the remarkable effects of LAV-BPIFB4 treatment, which translates in increased BPIFB4 circulating levels, mirror what occurs in long-living individuals (LLIs) in whom the high circulating levels of BPIFB4 are protective from age-related and CVDs and emphasize the reason why LLIs are considered a model of successful aging. Here, we review the mechanisms by which LAV-BPIFB4 exerts its immunomodulatory activity in improving the cardiovascular-immune system dialogue that might strengthen its role as a key mediator in CVDs.

In severe muscle injury, skeletal muscle tissue structure and functionality can be repaired through the involvement of several cell types, such as muscle stem cells, and innate immune responses. However, the exact mechanisms behind muscle tissue regeneration, homeostasis, and plasticity are still under investigation, and the discovery of pathways and cell types involved in muscle repair can open the way for novel therapeutic approaches, such as cell-based therapies involving stem cells and peripheral blood mononucleate cells. Indeed, peripheral cell infusions are a new therapy for muscle healing, likely because autologous peripheral blood infusion at the site of injury might enhance innate immune responses, especially those driven by macrophages. In this review, we summarize current knowledge on functions of stem cells and macrophages in skeletal muscle repairs and their roles as components of a promising cell-based therapies for muscle repair and regeneration.

Lay abstract Aging is the major risk factor for disease development. Long-living individuals (LLIs) are subjects older than 90 years that represent an invaluable model to study mechanisms underpinning longevity and healthy aging. Circulating levels of soluble receptor for advanced glycation end products (sRAGE) change with aging and can forecast the cardiovascular risk, which is reduced in centenarians. sRAGE is composed of two isoforms, the cleaved RAGE (cRAGE) and the secretory RAGE (esRAGE), that are known to inhibit the oxidative stress and inflammatory activities of their ligands such the advanced glycation end products (AGEs). In this study, we measured the plasmatic levels of both sRAGE isoforms and AGEs in LLIs (90–105 years) and control subjects (11–89 years). We found that cRAGE decreases with age in controls and LLIs. esRAGE increases in LLIs and AGEs increase in controls with age but decrease in LLIs. AGEs/esRAGE ratio and cRAGE were able to discriminate controls from LLIs. Hence, LLIs are characterized by a lower AGEs/sRAGE ratio, due to esRAGE increase and AGEs reduction that may explain their reduced cardiovascular and metabolic risk. Besides, circulating cRAGE could be considered a reliable marker of chronological age, while esRAGE a protective factor associated with longevity.

BACKGROUND:There is an increasing concern about age-related frailty because of the growing number of elderly people in the general population. The Longevity-Associated Variant (LAV) of the human BPIFB4 gene was found to correct endothelial dysfunction, one of the mechanisms underlying frailty, in aging mice whereas the RV-BPIFB4 variant induced opposite effects. Thus, we newly hypothesize that, besides being associated with life expectancy, BPIFB4 polymorphisms can predict frailty.Aim and Results: Here we investigated if the BPIFB4 haplotypes, LAV, wild-type (WT) and RV, differentially associate with frailty in a cohort of 237 elderly subjects from Calabria region in Southern Italy. Moreover, we studied the effect of systemic adeno-associated viral vector-mediated LAV-BPIFB4 gene transfer on the progression of frailty in aging mice. We found an inverse correlation of the homozygous LAV-BPIFB4 haplotype with frailty in elderly subjects. Conversely, carriers of the RV-BPIFB4 haplotype showed an increase in the frailty status and risk of death. Moreover, in old mice, LAV-BPIFB4 gene transfer delayed frailty progression.CONCLUSIONS:These data indicate that specific BPIFB4 haplotypes could represent useful genetic markers of frailty. In addition, horizontal transfer of a healthy gene variant can attenuate frailty in aging organisms.

Motivation Over the last decade, more diverse populations have been included in genome-wide association studies. If a genetic variant has a varying effect on a phenotype in different populations, genome-wide association studies applied to a dataset as a whole may not pinpoint such differences. It is especially important to be able to identify population-specific effects of genetic variants in studies that would eventually lead to development of diagnostic tests or drug discovery. Results In this paper, we propose PopCluster: an algorithm to automatically discover subsets of individuals in which the genetic effects of a variant are statistically different. PopCluster provides a simple framework to directly analyze genotype data without prior knowledge of subjects’ ethnicities. PopCluster combines logistic regression modeling, principal component analysis, hierarchical clustering and a recursive bottom-up tree parsing procedure. The evaluation of PopCluster suggests that the algorithm has a stable low false positive rate (∼4%) and high true positive rate (>80%) in simulations with large differences in allele frequencies between cases and controls. Application of PopCluster to data from genetic studies of longevity discovers ethnicity-dependent heterogeneity in the association of rs3764814 (USP42) with the phenotype. Availability and implementation PopCluster was implemented using the R programming language, PLINK and Eigensoft software, and can be found at the following GitHub repository: https://github.com/gurinovich/PopCluster with instructions on its installation and usage. Supplementary information Supplementary data are available at Bioinformatics online.

People are living longer, not, as was previously the case, due to reduced child mortality, but because we are postponing the ill-health of old age [...]

This is the final reprint of the following paper: VAIDYA V., CAROTA E., CALONZI D., PETRUCCIOLI M., D’ANNIBALE A. (2019). Production of lignin-modifying enzymes by Trametes ochracea on high-molecular weight fraction of olive mill wastewater, a byproduct of olive oil biorefinery. New Biotechnology 50:44-51 Elsevier Science (ISSN: 1871-6784) DOI: https://doi.org/10.1016/j.nbt.2019.01.007 Scopus ID: 2-s2.0-85060213550 ISI Web of Science: 000460066600006 PubMed ID: 30668986 URL:https://www.sciencedirect.com/science/article/pii/S1871678418301250 &nbsp

Aims Increased shedding of extracellular vesicles (EVs)—small, lipid bilayer-delimited particles with a role in paracrine signalling—has been associated with human pathologies, e.g. atherosclerosis, but whether this is true for cardiac diseases is unknown. Methods and results Here, we used the surface antigen CD172a as a specific marker of cardiomyocyte (CM)-derived EVs; the CM origin of CD172a+ EVs was supported by their content of cardiac-specific proteins and heart-enriched microRNAs. We found that patients with aortic stenosis, ischaemic heart disease, or cardiomyopathy had higher circulating CD172a+ cardiac EV counts than did healthy subjects. Cellular stress was a major determinant of EV release from CMs, with hypoxia increasing shedding in in vitro and in vivo experiments. At the functional level, EVs isolated from the supernatant of CMs derived from human-induced pluripotent stem cells and cultured in a hypoxic atmosphere elicited a positive inotropic response in unstressed CMs, an effect we found to be dependent on an increase in the number of EVs expressing ceramide on their surface. Of potential clinical relevance, aortic stenosis patients with the highest counts of circulating cardiac CD172a+ EVs had a more favourable prognosis for transcatheter aortic valve replacement than those with lower counts. Conclusion We identified circulating CD172a+ EVs as cardiac derived, showing their release and function and providing evidence for their prognostic potential in aortic stenosis patients.

Aging and comorbidities make individuals at greatest risk of COVID-19 serious illness and mortality due to senescence-related events and deleterious inflammation. Long-living individuals (LLIs) are less susceptible to inflammation and develop more resiliency to COVID-19. As demonstrated, LLIs are characterized by high circulating levels of BPIFB4, a protein involved in homeostatic response to inflammatory stimuli. Also, LLIs show enrichment of homozygous genotype for the minor alleles of a 4 missense single-nucleotide polymorphism haplotype (longevity-associated variant [LAV]) in BPIFB4, able to counteract progression of diseases in animal models. Thus, the present study was designed to assess the presence and significance of BPIFB4 level in COVID-19 patients and the potential therapeutic use of LAV-BPIFB4 in fighting COVID-19. BPIFB4 plasma concentration was found significantly higher in LLIs compared to old healthy controls while it significantly decreased in 64 COVID-19 patients. Further, the drop in BPIFB4 values correlated with disease severity. Accordingly to the LAV-BPIFB4 immunomodulatory role, while lysates of SARS-CoV-2-infected cells induced an inflammatory response in healthy peripheral blood mononuclear cells in vitro, the co-treatment with recombinant protein (rh) LAV-BPIFB4 resulted in a protective and self-limiting reaction, culminating in the downregulation of CD69 activating-marker for T cells (both TCD4+ and TCD8+) and in MCP-1 reduction. On the contrary, rhLAV-BPIFB4 induced a rapid increase in IL-18 and IL-1b levels, shown largely protective during the early stages of the virus infection. This evidence, along with the ability of rhLAV-BPIFB4 to counteract the cytotoxicity induced by SARS-CoV-2 lysate in selected target cell lines, corroborates BPIFB4 prognostic value and open new therapeutic possibilities in more vulnerable people.

Contains fulltext : 232387.pdf (Publisher’s version ) (Closed access) There is a strong relationship between metabolic state and susceptibility to Mycobacterium tuberculosis (MTB) infection, with energy metabolism setting the basis for an exaggerated immuno-inflammatory response, which concurs with MTB pathogenesis. Herein, we show that controlled caloric restriction (CR), not leading to malnutrition, protects susceptible DBA/2 mice against pulmonary MTB infection by reducing bacterial load, lung immunopathology, and generation of foam cells, an MTB reservoir in lung granulomas. Mechanistically, CR induced a metabolic shift toward glycolysis, and decreased both fatty acid oxidation and mTOR activity associated with induction of autophagy in immune cells. An integrated multi-omics approach revealed a specific CR-induced metabolomic, transcriptomic, and proteomic signature leading to reduced lung damage and protective remodeling of lung interstitial tightness able to limit MTB spreading. Our data propose CR as a feasible immunometabolic manipulation to control MTB infection, and this approach offers an unexpected strategy to boost immunity against MTB.

Background Healthy longevity is the result of the interaction between favourable environment and unique genetic makeup. We showed that horizontal transfer of a longevity-associated gene variant (LAV-BPIFB4) improves endothelial function and accelerates the recovery from ischemia. Purpose To determine if the benefit of LAV-BPIFB4 gene therapy can be extended to diabetic cardiomyopathy. Methods and results We confirmed that human diabetic patients with heart failure (n=13) show a decreased cardiac expression of BPIFB4 compared with healthy subjects (n=10). Obese db/db mice received a systemic injection of adeno-associated viral vector (AAV9)-LAV-BPIFB4, AAV9-wild type (WT)-BPIFB4 (both 100 μL at 1×1012 GC/mL) or vehicle before the onset of cardiomyopathy, and were euthanised four weeks later for histological, metabolic and transcriptional analyses. Echocardiographic evaluation (n=8/group), performed at baseline and after gene therapy, showed that LAV-BPIFB4 treatment, despite not resolving hyperglycaemia, improved left ventricular function compared with the other groups. Histological analyses of the hearts (n=5 to 10/group) revealed that LAV-BPIFB4 reduced myocardial fibrosis and increased angiogenesis compared with vehicle and WT-hearts; moreover, LAV increased the expression of the alpha-isoform of the cardiac myosin heavy chain, which is associated with a superior cardiomyocyte contractility. Interestingly, LAV-BPIFB4 treatment induced an increase in cardiac SDF1 expression compared with WT and vehicle, despite the mechanism linking the two events is still unknown. The oral administration of the CXCR4 antagonist AMD-070, given at 2 mg/kg/day for four weeks, abolished several of the beneficial effects exerted by the LAV-BPIFB4 therapy in the obese diabetic mice, as assessed by echocardiography and histology (n=7/group). At the molecular level, next-generation RNA sequencing (n=3 to 4 /group) showed 8 genes were differentially expressed by LAV-BPIFB4-hearts compared with vehicle-hearts. These genes are associated with mitochondrial and metabolic functions. Among them, changes in the UCP3, HMGCS2, CS, ATPB and TOMM20 expression were also validated at the protein level by western blotting. Lipidomics using ultrahigh-performance liquid chromatography-mass spectrometry (n=6 or 7/group) showed 63 metabolites differentially expressed by LAV-BPIFB4- compared with vehicle-hearts, with only 3 (two cardiolipins and one glycerophospholipid) returning close to the non-diabetic phenotype following LAV-BPIFB4 treatment. Conclusions This study newly shows the possibility of transferring the benefit of salutary polymorphic gene variants to protect the cardiovascular system from metabolic pressure. Rather than combating pathogenic mechanisms, the strategy activates alternative pathways overriding disease risk factors. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): British Heart Foundation project grant “Longevity-associated BPIFB4 gene therapy for treatment of ischemic disease”

Background Sortilin, a member of vacuolar protein sorting domain family Vps10, has been positively correlated with vascular and metabolic disorders in humans. Previous study has shown that, in response to Fas receptor stimulation, sortilin together with acid sphingomyelinase (ASMase) promote the clustering of lipid rafts and subsequent activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in coronary endothelial cells. However, whether sortilin plays a role in endothelial cells function is currently unknown. Purpose To assess whether sortilin per se was able to influence vascular function, thereby contributing to the pathogenesis of cardiovascular diseases. Methods Pressure myography was used to study vascular reactivity of mesenteric arteries. To investigate the involvement of acid sphingomyelinase (ASM), we performed gene silencing approach and fluorometric activity assay. NADPH oxidase lucigenin assay was used to evaluate oxidative stress in endothelial cells and resistance vessels. The effects of circulating sortilin on cardiovascular system was evaluated by systemic delivery of recombinant sortilin protein to wild-type (WT), sphingosine-1-phosphate receptor 3 (S1P3) and NADPH oxidase 2 (gp91phox/NOX2) deficient mice. Systolic arterial blood pressure (SBP) was noninvasively registered in conscious mice by tail-cuff blood monitoring. Finally, to explore the translational relevance of sortilin, we measured sortilin and NOX2 soluble derived peptide levels using ELISA and quantified sphingosine-1-phosphate (S1P) by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in plasma of hypertensive patients. Results Here we demonstrated that sortilin evoked endothelial dysfunction in mesenteric arteries due to increased NADPH oxidase-derived oxidative stress. Knockdown of ASM successfully prevented impairment of endothelial function. Using the inhibitor of sphingosine kinase type 1 (SphK1), sortilin failed to evoke endothelial impairment as well as NADPH oxidase activation. In endothelial cells, sortilin induced S1P-dependent activation of Rac1/NOX2 signaling axis, which was prevented by TY-52156, an antagonist of lysosphingolipid receptor S1P3. In vivo sortilin administration induced arterial hypertension in WT mice. In contrast, genetic deletion of S1P3 and gp91phox/NOX2 resulted in preservation of endothelial function and SBP unchanged levels after 14 days of systemic sortilin administration. Finally, to translate these research findings into a clinical setting, we found that hypertensive patients have higher plasma levels of sortilin, ASMase, S1P and soluble NOX2 derived peptide than normotensive subjects. Conclusions These results demonstrate the pathologic role of sortilin in the modulation of endothelial function and arterial blood pressure, suggesting that sortilin and its mediators might represent novel therapeutic targets in vascular diseases and hypertension. Funding Acknowledgement Type of funding source: None

The persistence and accumulation of senescent cells has been shown to potentially play a role in the pathophysiology of age-related cardiovascular diseases. Indeed with time, a decline in immune efficacy, termed immunesenescence, and a deleterious secretory phenotype of senescent cells (SASP) generate that inflammatory background mainly mediating the elderly cardiovascular phenotype. Long Living Individuals (LLI) which delay aging, represent a model of positive biology and an exceptional resource to study and find a way to improve general public health. Previous studies from our group have shown that a human gene associated with exceptional longevity (LAV-BPIFB4) was able to block the atherosclerotic process in ApoE−/− mice by conferring the animals with a pro-resolving M2 macrophages profile. Furthermore, LAV-BPIFB4 promotes the recruitment of hematopoietic stem cells, reparative vascularization and frailty reduction. Here we sought to underpinn the role of LAV-BPIFB4 in counteracting the age-related remodeling of the immune responses. The effects of systemic adeno-associated viral vector-mediated LAV-BPIFB4 gene transfer on the immune dynamics in old mice have been investigated by an extensive flow cytometric approach in lymphoid tissues (bone marrow, spleen and peripheral blood). C57BL/6J mice were assigned to two age-matched experimental groups: a treatment group (AAV-LAV-BPIFB4; N=6 mice, aged 18–23 months and a control group (AAV-GFP; N=6 mice, aged 18–23 months. 30th and 60th day since the beginning of the infection, SA-beta Gal substrate has been used to identify CD45+ senescent cells in freshly isolated PBMC, splenocytes, bone marrow (BM)-derived cells. As expected, we monitored an increase in SA-betaGal activity in blood. This increase is most significant in CD11b+ myeloid cells, whithout affecting neither CD3+T neither NK1.1+Natural Killer (NK) cell compartment. Notably 30 days AAV-LAV-BPIFB4 infection and to a more the 60 days-treatment, resulted in a significant decrease in senescent pool of peripheral immune cells and a concomitant enrichment of senescent cells in spleen. Concomitantly, aorta from AAV-LAV treated mice showed significant reduction in SA-beta Gal positive area. Furthermore a LAV-BPIFB4 induction of pro-resolving M2 macrophages compared to control group was documented in the main haemocateretic organ. As consequence the senolytic effect of LAV-BPIFB4 gene-therapy well correlated with the rescue of proliferative index of splenocytes upon PHA stimulation, and their functional protective response to lipopolysaccharide (LPS) in term of IL-6 and TNF-alpha secretion. The restoration of a protective and balanced immune response finally reflected the reduction of senescent phenopype acquired by mouse aortic endothelial cells during the aging process in vivo. A better underpinning of the senolytic action of LAV-BPIFB4 may offer a valuable therapeutic tool to reverse aging phenotype causing most of cardiac diseases Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Cariplo Foundation (n.2016-0874) to AAP and CV

Funder: QingLan Research Project of Jiangsu for Outstanding Young Teachers, Funder: Project funded by Postdoctoral Science Foundation of Xuzhou Medical University, Funder: Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) for Xuzhou Medical University, We employed Mendelian randomization (MR) to evaluate the causal relationship between leukocyte telomere length (LTL) and amyotrophic lateral sclerosis (ALS) with summary statistics from genome-wide association studies (n = ~ 38,000 for LTL and ~ 81,000 for ALS in the European population; n = ~ 23,000 for LTL and ~ 4,100 for ALS in the Asian population). We further evaluated mediation roles of lipids in the pathway from LTL to ALS. The odds ratio per standard deviation decrease of LTL on ALS was 1.10 (95% CI 0.93–1.31, p = 0.274) in the European population and 0.75 (95% CI 0.53–1.07, p = 0.116) in the Asian population. This null association was also detected between LTL and frontotemporal dementia in the European population. However, we found that an indirect effect of LTL on ALS might be mediated by low density lipoprotein (LDL) or total cholesterol (TC) in the European population. These results were robust against extensive sensitivity analyses. Overall, our MR study did not support the direct causal association between LTL and the ALS risk in neither population, but provided suggestive evidence for the mediation role of LDL or TC on the influence of LTL and ALS in the European population.

This study focused on the remediation of a chronically diesel-polluted soil by combining an electrokinetic treatment with a variety of bioremediation approaches. Priority within the sequential treatment was given to electrokinetic remediation (EKR) since the application of natural attenuation (NA), biostimulation and site-specific bio-augmentation resulted in very low degradation performance for total petroleum hydrocarbons (TPH) and polycyclic hydrocarbons (PAH). The application of 20-day EKR (1.0 V cm−1with polarity reversal) led to 47.2 % and 46.2 % removal of TPH and PAH, respectively, and exerted a negative impact on bacterial abundance, as determined indirectly by quantitative PCR of 16S rRNA genes and community function, as investigated by community-level physiological profiling. These adverse effects were transient and, after a 50-day NA treatment applied downstream from EKR, bacterial abundance was an order of magnitude higher than that found in the initial soil and TPH and PAH removals were significantly higher than those attained by EKR (64.1 % and 56.3 %, respectively). The combination of EKR with site-specific bioaugmentation led to the greatest TPH and PAH degradation (76.0 % and 78.6 %, respectively). The results indicate that bioremediation can be successfully applied downstream from EKR and that the adverse effects exerted by this physico-chemical approach on soil microbiota are reversible.

Long-Living Individuals (LLIs) delay aging and are less prone to chronic inflammatory reactions. Whether a distinct monocytes and macrophages repertoire is involved in such a characteristic remains unknown. Previous studies from our group have shown high levels of the host defense BPI Fold Containing Family B Member 4 (BPIFB4) protein in the peripheral blood of LLIs. Moreover, a polymorphic variant of the BPIFB4 gene associated with exceptional longevity (LAV-BPIFB4) confers protection from cardiovascular diseases underpinned by low-grade chronic inflammation, such as atherosclerosis. We hypothesize that BPIFB4 may influence monocytes pool and macrophages skewing, shifting the balance toward an anti-inflammatory phenotype. We profiled circulating monocytes in 52 LLIs (median-age 97) and 52 healthy volunteers (median-age 55) using flow cytometry. If the frequency of total monocyte did not change, the intermediate CD14++CD16+ monocytes counts were lower in LLIs compared to control adults. Conversely, non-classical CD14+CD16++ monocyte counts, which are M2 macrophage precursors with an immunomodulatory function, were found significantly associated with the LLIs' state. In a differentiation assay, supplementation of the LLIs' plasma enhanced the capacity of monocytes, either from LLIs or controls, to acquire a paracrine M2 phenotype. A neutralizing antibody against the phosphorylation site (ser 75) of BPIFB4 blunted the M2 skewing effect of the LLIs' plasma. These data indicate that LLIs carry a peculiar anti-inflammatory myeloid profile, which is associated with and possibly sustained by high circulating levels of BPIFB4. Supplementation of recombinant BPIFB4 may represent a novel means to attenuate inflammation-related conditions typical of unhealthy aging.

Background High blood pressure ( BP ) has long been recognized as a major health threat and, particularly, a major risk factor for stroke, cardiovascular disease, and end‐organ damage. However, the identification of a novel, alternative, integrative approach for the control of BP and cardiovascular protection is still needed. Methods and Results Sixty‐nine uncontrolled hypertension patients, aged 40 to 68 years, on antihypertensive medication were enrolled in 2 double‐blind studies. Forty‐five were randomized to placebo or a new nutraceutical combination named AkP05, and BP , endothelial function, and circulating nitric oxide were assessed before and at the end of 4 weeks of treatment. Twenty‐four patients were randomized to diuretic or AkP05 for 4 weeks and underwent a cardiopulmonary exercise test to evaluate the effects of AkP05 on functional capacity of the cardiovascular, pulmonary, and muscular systems. Vascular and molecular studies were undertaken on mice to characterize the action of the single compounds contained in the AkP05 nutraceutical combination. AkP05 supplementation reduced BP , improved endothelial function, and increased nitric oxide release; cardiopulmonary exercise test revealed that AkP05 increased maximum O 2 uptake, stress tolerance, and maximal power output. In mice, AkP05 reduced BP and improved endothelial function, evoking increased nitric oxide release through the PKC α/Akt/endothelial nitric oxide synthase pathway and reducing reactive oxygen species production via NADPH ‐oxidase inhibition. These effects were mediated by synergism of the single compounds of AkP05. Conclusions This is the first study reporting positive effects of a nutraceutical combination on the vasculature and exercise tolerance in treated hypertensive patients. Our findings suggest that AkP05 may be used as an adjunct for the improvement of cardiovascular protection and to better control BP in uncontrolled hypertension.

The longevity-associated variant (LAV) of the bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) has been found significantly enriched in long-living individuals. Neuroinflammation is a key player in Huntington’s disease (HD), a neurodegenerative disorder caused by neural death due to expanded CAG repeats encoding a long polyglutamine tract in the huntingtin protein (Htt). Herein, we showed that striatal-derived cell lines with expanded Htt (STHdh Q111/111) expressed and secreted lower levels of BPIFB4, when compared with Htt expressing cells (STHdh Q7/7), which correlated with a defective stress response to proteasome inhibition. Overexpression of LAV-BPIFB4 in STHdh Q111/111 cells was able to rescue both the BPIFB4 secretory profile and the proliferative/survival response. According to a well-established immunomodulatory role of LAV-BPIFB4, conditioned media from LAV-BPIFB4-overexpressing STHdh Q111/111 cells were able to educate Immortalized Human Microglia—SV40 microglial cells. While STHdh Q111/111 dying cells were ineffective to induce a CD163 + IL-10high pro-resolving microglia compared to normal STHdh Q7/7, LAV-BPIFB4 transduction promptly restored the central immune control through a mechanism involving the stromal cell-derived factor-1. In line with the in vitro results, adeno-associated viral-mediated administration of LAV-BPIFB4 exerted a CXCR4-dependent neuroprotective action in vivo in the R6/2 HD mouse model by preventing important hallmarks of the disease including motor dysfunction, body weight loss, and mutant huntingtin protein aggregation. In this view, LAV-BPIFB4, due to its pleiotropic ability in both immune compartment and cellular homeostasis, may represent a candidate for developing new treatment for HD.

Immune cell subsets and microRNAs have been independently proposed as type 1 diabetes (T1D) diagnostic and/or prognostic biomarkers. Here, we aimed to analyze the relationships between peripheral blood circulating immune cell subsets, plasmatic microRNAs, and T1D. Blood samples were obtained from both children with T1D at diagnosis and age-sex matched healthy controls. Then, immunophenotype assessed by flow cytometry was coupled with the quantification of 60 plasmatic microRNAs by quantitative RT-PCR. The associations between immune cell frequency, plasmatic microRNAs, and the parameters of pancreatic loss, glycemic control, and diabetic ketoacidosis were assessed by logistic regression models and correlation analyses. We found that the increase in specific plasmatic microRNAs was associated with T1D disease onset (let-7c-5p, let-7d-5p, let-7f-5p, let-7i-5p, miR-146a-5p, miR-423-3p, and miR-423-5p), serum C-peptide concentration (miR-142-5p and miR-29c-3p), glycated hemoglobin (miR-26a-5p and miR-223-3p) and the presence of ketoacidosis (miR-29c-3p) more strongly than the evaluated immune cell subset frequency. Some of these plasmatic microRNAs were shown to positively correlate with numbers of blood circulating B lymphocytes (miR-142-5p) and CD4+CD45RO+ (miR-146a-5p and miR-223-3p) and CD4+CD25+ cells (miR-423-3p and miR-223-3p) in children with T1D but not in healthy controls, suggesting a disease-specific microRNA association with immune dysregulation in T1D. In conclusion, our results suggest that, while blood co-circulating extracellular microRNAs and immune cell subsets may be biologically linked, microRNAs may better provide powerful information about T1D onset and severity.

Characterisation of animal models of diabetic cardiomyopathy may help unravel new molecular targets for therapy. Long-living individuals are protected from the adverse influence of diabetes on the heart, and the transfer of a longevity-associated variant (LAV) of the human BPIFB4 gene protects cardiac function in the db/db mouse model. This study aimed to determine the effect of LAV-BPIFB4 therapy on the metabolic phenotype (ultra-high-performance liquid chromatography-mass spectrometry, UHPLC-MS) and cardiac transcriptome (next-generation RNAseq) in db/db mice. UHPLC-MS showed that 493 cardiac metabolites were differentially modulated in diabetic compared with non-diabetic mice, mainly related to lipid metabolism. Moreover, only 3 out of 63 metabolites influenced by LAV-BPIFB4 therapy in diabetic hearts showed a reversion from the diabetic towards the non-diabetic phenotype. RNAseq showed 60 genes were differentially expressed in hearts of diabetic and non-diabetic mice. The contrast between LAV-BPIFB4- and vehicle-treated diabetic hearts revealed eight genes differentially expressed, mainly associated with mitochondrial and metabolic function. Bioinformatic analysis indicated that LAV-BPIFB4 re-programmed the heart transcriptome and metabolome rather than reverting it to a non-diabetic phenotype. Beside illustrating global metabolic and expressional changes in diabetic heart, our findings pinpoint subtle changes in mitochondrial-related proteins and lipid metabolism that could contribute to LAV-BPIFB4-induced cardio-protection in a murine model of type-2 diabetes.

Aims/hypothesis: We aimed to analyse the association between plasma circulating microRNAs (miRNAs) and the immunometabolic profile in children with type 1 diabetes and to identify a composite signature of miRNAs/immunometabolic factors able to predict type 1 diabetes progression. Methods: Plasma samples were obtained from children at diagnosis of type 1 diabetes (n = 88) and at 12 (n = 32) and 24 (n = 30) months after disease onset and from healthy control children with similar sex and age distribution (n = 47). We quantified 60 robustly expressed plasma circulating miRNAs by quantitative RT-PCR and nine plasma immunometabolic factors with a recognised role at the interface of metabolic and immune alterations in type 1 diabetes. Based on fasting C-peptide loss over time, children with type 1 diabetes were stratified into the following groups: those who had lost >90% of C-peptide compared with diagnosis level; those who had lost

Bitter taste receptors (TAS2R) are involved in a variety of non-tasting physiological processes, including immune-inflammatory ones. Therefore, their genetic variations might influence various traits. In particular, in different populations of South Italy (Calabria, Cilento, and Sardinia), polymorphisms of TAS2R16 and TAS238 have been analysed in association with longevity with inconsistent results. A meta-analytic approach to quantitatively synthesize the possible effect of the previous variants and, possibly, to reconcile the inconsistencies has been used in the present paper. TAS2R38 variants in the Cilento population were also analysed for their possible association with longevity and the obtained data have been included in the relative meta-analysis. In population from Cilento no association was found between TAS2R38 and longevity, and no association was observed as well, performing the meta-analysis with data of the other studies. Concerning TAS2R16 gene, instead, the genotype associated with longevity in the Calabria population maintained its significance in the meta-analysis with data from Cilento population, that, alone, were not significant in the previously published study. In conclusion, our results suggest that TAS2R16 genotype variant is associated with longevity in South Italy.

Regenerative medicine is a new therapeutic modality that aims to mend tissue damage by encouraging the reconstitution of physiological integrity. It represents an advancement over conventional therapies that allow reducing the damage but result in disease chronicization. Age-related decline in spontaneous capacity of repair, especially in organs like the heart that have very limited proliferative capacity, contributes in reducing the benefit of conventional therapy. ncRNAs are emerging as key epigenetic regulators of cardiovascular regeneration. Inhibition or replacement of miRNAs may offer reparative solutions to cardiovascular disease. The first part of this review article is devoted to illustrating novel therapies emerging from research on miRNAs. In the second part, we develop new therapeutic concepts emerging from genetics of longevity. Prolonged survival, as in supercentenarians, denotes an exceptional capacity to repair and cope with risk factors and diseases. These characteristics are shared with offspring, suggesting that the regenerative phenotype is heritable. New evidence indicates that genetic traits responsible for prolongation of health span in humans can be passed to and benefit the outcomes of animal models of cardiovascular disease. Genetic studies have also focused on determinants of accelerated senescence and related druggable targets. Evolutionary genetics assessing the genetic basis of adaptation and comparing successful and unsuccessful genetic changes in response to selection within populations represent a powerful basis to develop novel therapies aiming to prolong cardiovascular and whole organism health.

Humulus lupulus (Hop) contains numerous metabolites with anticancer potential. Despite this, their immunomodulatory activity, and in particular of bitter acids, is unknown. In this study, we demonstrated that a Hop pellet extract fraction containing bitter acids possesses immunomodulatory activity by enhancing Natural Killer (NK) cells function. After fractionation by semi-preparative Liquid Chromatography, three different fractions were obtained. The phytocomplex and the fractions were tested to verify the ability to modulate the NK compartment. Cytofluorimetric analysis revealed that a fraction containing bitter acids was able to up-regulate of NKG2D and NKp44 activating receptors. A further simplification yield a fraction mainly composed by Humulinones and Cohulupone derivatives, that at the concentration of 0.1 µg/mL induced selective activation of Nkp44 receptor and enhanced the cytolytic activity of NK cells against leukemia cell line K562. Those results point out the possible use of Hop bitter acids as natural immunostimulants and adjuvants in chemotherapy protocols.

Ongoing studies evidence cellular senescence in undifferentiated and specialized cells from tissues of all ages. Although it is believed that senescence plays a wider role in several stress responses in the mature age, its participation in certain physiological and pathological processes throughout life is coming to light. The “senescence machinery” has been observed in all brain cell populations, including components of innate immunity (e.g., microglia and astrocytes). As the beneficial versus detrimental implications of senescence is an open question, we aimed to analyze the contribution of immune responses in regulatory mechanisms governing its distinct functions in healthy (development, organogenesis, danger patrolling events) and diseased brain (glioma, neuroinflammation, neurodeneration), and the putative connection between cellular and molecular events governing the 2 states. Particularly this review offers new insights into the complex roles of senescence both as a chronological event as age advances, and as a molecular mechanism of brain homeostasis through the important contribution of innate immune responses and their crosstalk with neighboring cells in brain parenchyma. We also highlight the impact of the recently described glymphatic system and brain lymphatic vasculature in the interplay between peripheral and central immune surveillance and its potential implication during aging. This will open new ways to understand brain development, its deterioration during aging, and the occurrence of several oncological and neurodegenerative diseases.

The search for the genetic determinants of extreme human longevity has been challenged by the phenotype's rarity and its nonspecific definition by investigators. To address these issues, we established a consortium of four studies of extreme longevity that contributed 2,070 individuals who survived to the oldest one percentile of survival for the 1900 U.S. birth year cohort. We conducted various analyses to discover longevity-associated variants (LAV) and characterized those LAVs that differentiate survival to extreme age at death (eSAVs) from those LAVs that become more frequent in centenarians because of mortality selection (eg, survival to younger years). The analyses identified new rare variants in chromosomes 4 and 7 associated with extreme survival and with reduced risk for cardiovascular disease and Alzheimer's disease. The results confirm the importance of studying truly rare survival to discover those combinations of common and rare variants associated with extreme longevity and longer health span.

This is the uncorrected proof of the following paper: ALLEVATO E., STAZI S.R., MARABOTTINI R, D'ANNIBALE A., 2019 Mechanisms of arsenic assimilation by plants and countermeasures to attenuate its accumulation in crops other than rice. Ecotoxicology and Environmental Safety 185, 109701 (ISSN: 0147-6513) DOI: https://doi.org/10.1016/j.ecoenv.2019.109701 Scopus ID: 2-s2.0-85072524008 Web of Science: 000491218500043 PubMed ID: 31562999 URL of published paper:https://www.sciencedirect.com/science/article/pii/S0147651319310322&nbsp

A facultative halo-tolerant Aspergillus strain was isolated from olive brine waste, the effluent from the debittering process of table olives. Phenotypic and molecular characteristics showed clearly that the isolate represents a novel species. Based on the source of isolation, the new species has been named Aspergillus olivimuriae. It was found tolerant to high concentrations of NaCl (15 %) or sucrose (60 %) and it exhibits substantial growth under these conditions. Although the new species grew profusely at 37 C, no growth was observed at 40 C, conidia en masse were avellaneous on all media. The description of the new species Aspergillus olivimuriae brings the total species of Aspergillus sect. Flavipedes to 15. The type strain of A. olivimuriae sp. nov. is NRRL 66783 (CCF 6208), its whole genome has been deposited as PRJNA498048.

Background Pentraxin 3 (PTX3), an acute-phase inflammation protein produced by several cell types, has long been described as a possible biomarker for age-related cardiovascular and cerebrovascular diseases. Although several mechanisms of action have been identified to date in the vascular and immune systems, the direct effects of PTX3 on isolated endothelial cells at morphological and metabolic levels remain unknown. Findings PTX3 induced cytoplasmic vacuolization and dilution of mitochondrial matrix in isolated, human endothelial cells. Moreover, metabolic assays revealed that PTX3 increases respiratory capacity in support of mitochondrial function, and partially sustains the glycolytic pathway. Conclusions PTX3 has, per se, a direct action on ultrastructural and bioenergetic parameters of isolated endothelial cells. This finding can be associated with our previous demonstration of a deleterious effect of PTX3 on the endothelial layer. More studies are needed to clearly demonstrate any direct correlation between these ultrastructural and bioenergetic changes with endothelial dysfunction, especially with regard to age-related cerebro- and cardio-vascular diseases.

Background Innate immunity utilizes components of sensory signal transduction such as bitter and sweet taste receptors. In fact, empirical evidence has shown bitter and sweet taste receptors to be an integral component of antimicrobial immune response in upper respiratory tract infections. Since an efficient immune response plays a key role in the attainment of longevity, it is not surprising that the rs978739 polymorphism of the bitter taste receptor TAS2R16 gene has been shown to be associated with longevity in a population of 941 individuals ranging in age from 20 to 106 years from Calabria (Italy). There are many possible candidate genes for human longevity, however of the many genes tested, only APOE and FOXO3 survived to association in replication studies. So, it is necessary to validate in other studies genes proposed to be associated with longevity. Thus, we analysed the association of the quoted polymorphism in a population of long lived individuals (LLIs) and controls from another Italian population from Cilento. Methods The analysis has been performed on data previously obtained with genome-wide association study on a population of LLIs (age range 90–109 years) and young controls (age range 18–45 years) from Cilento (Italy). Results Statistical power calculations showed that the analysed cohort represented by 410 LLIs and 553 young controls was sufficiently powered to replicate the association between rs978739 and the longevity phenotype according to the effect size and frequencies described in the previous paper, under a dominant and additive genetic model. However, no evidence of association between rs978739 and the longevity phenotype was observed according to the additive or dominant model. Conclusion There are several reasons for the failure of the confirmation of a previous study. However, the differences between the two studies in terms of environment of the population adopted and of the criteria of inclusion have made difficult the replication of the findings.

APOE is a well-studied gene with multiple effects on aging and longevity. The gene has three alleles: e2, e3, and e4, whose frequencies vary by ethnicity. While the e2 is associated with healthy cognitive aging, the e4 allele is associated with Alzheimer’s disease and early mortality and therefore its prevalence among people with extreme longevity (EL) is low. Using the PopCluster algorithm, we identified several ethnically different clusters in which the effect of the e2 and e4 alleles on EL changed substantially. For example, PopCluster discovered a large group of 1,309 subjects enriched of Southern Italian genetic ancestry with weaker protective effect of e2 (odds ratio [OR] = 1.27, p = .14) and weaker damaging effect of e4 (OR = 0.82, p = .31) on the phenotype of EL compared to other European ethnicities. Further analysis of this cluster suggests that the odds for EL in carriers of the e4 allele with Southern Italian genetic ancestry differ depending on whether they live in the United States (OR = 0.29, p = .009) or Italy (OR = 1.21, p = .38). PopCluster also found clusters enriched of subjects with Danish ancestry with varying effect of e2 on EL. The country of residence (Denmark or United States) appears to change the odds for EL in the e2 carriers.

One of the basis of exceptional longevity is the maintaining of the balance between inflammatory and anti-inflammatory networks. The monocyte-macrophages activation plays a major role in tuning the immune responses, by oscillating between patrolling-protective to inflammatory status. Longevity-associated variant (LAV) of bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) activates calcium, PKC-alpha, and eNOS, rescuing endothelial dysfunction in aged mice and inducing revascularization. The BPIFB4’s increment in serum of healthy long-living individuals (LLIs) compared to nonhealthy ones, its therapeutic potential in improving vascular homeostasis, which depends on immune system, together with its expression in bone marrow myeloid cells, suggests that LAV-BPIFB4 may improve immune regulation. Here we show that human monocytes exposed to LAV-BPIFB4 protein increased co-stimulatory molecules in resting state and reduced pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) after activating stimuli. Accordingly, a low percentage of CD69+ activated lymphocytes are found among LAV-BPIFB4-treated peripheral blood mononuclear cells (PBMCs). Moreover, human monocyte-derived dendritic cells (DCs) generated in presence of LAV-BPIFB4 secreted higher anti-(IL-10 and TGF-β) and lower pro-inflammatory (TNF-α and IL-1β) cytokines. Accordingly, LLIs’ plasma showed higher levels of circulating IL-10 and of neutralizing IL-1 receptor antagonist (IL-1RA) compared to controls. Thus, LAV-BPIFB4 effects on myeloid compartment could represent one example of a genetic predisposition carried by LLIs to protect from immunological dysfunctions.

Supercritical Emulsion Extraction (SEE) and Supercritical assisted Liposome formation (SuperLip), use dense gases such as carbon dioxide (dCO2) to fabricate advanced micro/nanocarriers. SEE uses dCO2 to extract solvent from the oily phase of an emulsion and obtain biopolymer microbead; For this study, poly-Lactic Acid (PLA) microbeads of 1 ± 0.2 μm in mean size loaded at 1 µg/mgPLA with Rhodamine B (ROD) were prepared by SEE; the beads showed a solvent residue lower than 10 ppm and encapsulated the fluorochrome with an efficiency of 90%. SuperLip uses dCO2 to enhance lipid/ethanol/water mixing and to promote the ethanol extraction from liposome suspension. In this case, phosphatidyl-choline (PC) vesicles with a mean size of 0.2 ± 0.05 μm and loaded with Fluorescein Iso-ThioCyanate (FITC) at 8 µg/mgPC were prepared; small unilamellar structure was observed for all the vesicles with FITC encapsulation efficiency of 80%. Ethanol residue of 50 ppm was measured in all the liposome suspensions. The bioavailability of microbeads and nanoliposomes was assessed through incubation with human monocytes previously isolated from healthy donors’ blood. A specifically optimized protocol that allowed their quenching on the cell surface was developed to monitor by flow cytometer assay only the cell population that effectively internalized the carriers. When microbeads were tested, the percentage of alive internalizing monocytes was of about 30%. An internalization of 96.1 ± 21% was, instead, obtained at dosage of 0.1 mg/mL for nanoliposomes. In this last case, monocytes showed a vitality of almost 100% after vesicles internalization at all the concentrations studied; on the other hand, cell apoptosis progressively increased in a dose/response manner, after polymer microbeads phagocytosis. The proposed data suggested that dCO2 technologies can be reliably used to fabricate intracellular carriers.

The extraordinary rise in the old population in the Western world underscores the importance of studies on aging and longevity to decrease the medical, economic and social problems associated with the increased number of non-autonomous individuals affected by invalidating pathologies. Centenarians have reached the extreme limits of the human life span. They are the best example of extreme longevity, representing selected individuals in which the appearance of major age-related diseases has been consistently delayed or avoided. There is growing evidence that the genetic component of longevity becomes higher with survival at the age of over 90 years. For centenaries, it reaches up to 33% for women and 48% for men. Therefore, exceptional longevity is a complex, hereditable trait that runs across generations. Longevity should correlate either with the presence of protective alleles or the absence of detrimental alleles. The aim of this review is to discuss the possible attainment of successful aging in the context of the lessons learned from centenarian genetics.

Background A rare Longevity Associated Variant (LAV) of the gene BPIFB4, whose protein product can be secreted, is recessively associated with extreme longevity, positively impacting the cardiovascular system and promoting therapeutic angiogenesis in an hindlimb ischemia model. Aim Aim of this work is to verify if LAV-BPIFB4 may temper the phenotype of chronic ischemia in humans, thus suggesting its therapeutic potential. Methods and results 39 hearts explanted from patients affected by end-stage ischemic heart disease were studied. Of these, 7 were homozygous for BPIFB4-LAV. Homozygous and non-homozygous patients did not differ in sex, age and in the prevalence of comorbidities or risk factors. However, the time elapsed from myocardial infarction to transplantation was significantly longer for LAV homozygous with respect to LAV heterozygous patients (159±95 vs 58±27 months, p=0.045). Histologically, while BPIFB4 levels did not differ in accordance with the genotype, the fraction of Tunel+and 53BP1+apoptotic and senescent myocytes was significantly lower in homozygous patients (1.2±0.4% vs 23.8±21.7%, p=0.026 and 31.5±6.7% vs 44.4±6.1%, p=0.031, respectively). Significantly lower levels of lipoperoxides and higher levels of Parkin were observed in homozygous hearts too. Next, we directly tested the role of BPIFB4 on PDGFRb+ NG2+Tbx18+cardiac pericytes/mural cells (PM) cultured from normal atria (PMnorm, n=10) and from ischemic failing hearts (PMfail, n=7). BPIFB4 gene transcript was significantly more expressed in PMnorm than PMfail. Silencing BPIFB4 expression in PMnorm significantly increased the proportion of senescent gH2AX+Ki67-cells (3.6±2.9% vs 13.6±9.5%, p=0.049). Conversely, addition of recombinant LAV BPIFB4 protein to PMfail cultures significantly reduced the rate of senescent cells (21.1±9.4% vs 6.0±4.8%, p=0.03), an effect that was not observed with the administration of WT BPIFB4. PMfail cultures exposed to LAV BPIFB4 significantly reversed alterations observed with pathology, such as the accumulation of both lipofuscins and of very elongated and interconnected mitochondria in the cell cytoplasm, as well as elevated mitochondrial superoxide anion levels. Finally, a larger fraction of PMnorm cells showed, with respect to PMfail, the nuclear translocation of vitamin D receptor (74.6±6.1% vs 50.1±14.4, p=0.04), coupled with a significant upregulation of its, longevity associated, target gene Klotho. Importantly, LAV BPIFB4 significantly increased the fraction of PMfail with vitamin D receptor nuclear localization (55.8±17.6% vs 85.1±8.2%, p=0.005). Conclusion LAV BPIFB4 could attenuate the progression of ischemic heart disease to heart failure. Part of its protective effect may be due to its ability to reduce mitochondrial oxidative stress and to revert the senescent phenotype of cardiac derived cells. Acknowledgement/Funding CARIPLO foundation

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A way to delay aging and the related low-grade chronic inflammatory state is to study the model of positive physiology such as the Long-Living Individuals (LLIs). Our recent studies have shown higher levels of the host defense BPI Fold-Containing Family B Member 4 (BPIFB4) protein in the LLIs' blood. Notably, BPIFB4 has been shown to influence monocytes typesetting and M2 anti-inflammatory phenotype (CD206+CD163++) macrophages skewing. According to the role of a complex cytokine milieu in guiding the macrophage polarization, here we found that circulating concentrations of thymus and activation regulated chemokine (TARC)/CCL17 and small-inducible cytokine B10 (IP-10)/CXCL10) cytokines, were additionally associated with the LLIs' state. In a differentiation process in vitro, the addition of LLIs' plasma to the cell culture medium, enhanced the ability of monocytes, either from LLIs or controls, to acquire a M2 phenotype. Interestingly, a neutralizing antibody against TARC blunted the M2 skewing effect of the LLIs' plasma. Collectively, these data indicate that exceptional longevity may associate with a peculiar anti-inflammatory myeloid profile responsible for improved reparative processes and reduced inflammatory status mediated in part by TARC and M2 generation.

BPIFB4 is associated with exceptional longevity: four single-nucleotide polymorphisms distinguish the wild-type form from a longevity-associated variant conferring positive effects on blood pressure. The effect of a rare variant (RV; allele frequency, 4%) on blood pressure is unknown. Here, we show that overexpression of RV-BPIFB4 in ex-vivo mouse vessels impairs phosphorylation of endothelial nitric oxide synthase (eNOS), blunting acetylcholine-evoked vasorelaxation; in vivo, virally mediated overexpression of RV-BPIFB4 increases blood pressure, an action absent in eNOS-deficient mice. In humans, we found RV carriers to have increased diastolic blood pressure, a finding that was more marked in subjects on anti-hypertensive medication; moreover, recombinant RV-BPIFB4 protein impaired eNOS function in ex-vivo human vessels. Thus, RV-BPIFB4 acts directly on blood pressure homeostasis and may represent a novel biomarker of vascular dysfunction and hypertension.