Addressing health inequalities in Europe: key messages from the Joint Action Health Equity Europe (JAHEE).

Health inequalities within and between Member States of the European Union are widely recognized as a public health problem as they determine a significant share of potentially avoidable mortality and morbidity. After years of growing awareness and increasing action taken, a large gap still exists across Europe in terms of policy responses and governance. With the aim to contribute to achieve greater equity in health outcomes, in 2018 a new Joint Action, JAHEE, (Joint Action Health Equity Europe) was funded by the third EU Health Programme, with the main goal of strengthening cooperation between participating countries and of implementing concrete actions to reduce health inequalities. The partnership led by Italy counted 24 countries, conducting actions in five policy domains: monitoring, governance, healthy living environments, health systems and migration, following a three-step implementation approach. Firstly, specific Policy Frameworks for Action (PFA) collecting the available evidence on what practice should be done in each domain were developed. Second, different Country Assessments (CAs) were completed to check the country's adherence to the recommended practice in each domain. The gap between the expected policy response (PFA) and the present policy response (CA) guided the choice of concrete actions to be implemented in JAHEE, many of which are continuing even after the end of JA. Final recommendations based on the best results achieved during JAHEE were elaborated and agreed jointly with the representatives of the involved Ministries of Health. The JAHEE initiative represented an important opportunity for the participating countries to work jointly, and the results show that almost all have increased their level of action and strengthened their capacities to address health inequalities.

Health inequalities: a Research Positioning Exercise at the National Institute of Health, Italy.

BACKGROUND: The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) considers health inequalities (HI) an important area of activity. As the scientific and technical body of the Ministry of Health and the National Health Service, ISS may play a key role to reduce HI. In order to enable ISS in addressing the new and crucial HI challenge, a Research Positioning Exercise was designed and implemented. METHODS: The Exercise included: (i) workshop to strengthen the institutional interest in the field of HI; (ii) review and analysis of ISS publications (years 2000-2017) to identify HI research topics; (iii) survey among ISS researchers regarding main research challenges to address HI in the coming years; and (iv) analysis of input on research challenges from HI international experts. RESULTS: The results of this Exercise suggest that the following points should be included in the future ISS agenda planning: (i) themes which ISS should continue working on (e.g. migrants/vulnerable groups); (ii) themes to be improved: (a) relationship between social determinants and mechanism of HI generation and (b) relationship between risk factors exposure and social determinants; and (iii) new themes to be addressed: (a) mechanisms underlying the resilience observed in Italy; (b) new socioeconomic indicators for HI monitoring; and (c) evidence-based policies aimed at reducing HI. CONCLUSION: Findings of this Exercise show that ISS researchers identified relevant areas, addressing inequalities in addressing the health. Because of ISS structural peculiarity that includes multidisciplinary expertise, the ISS could provide a significant contribution to HI research challenges and knowledge gaps.

Modulating the metabolism by trimetazidine enhances myoblast differentiation and promotes myogenesis in cachectic tumor-bearing c26 mice.

Trimetazidine (TMZ) is a metabolic reprogramming agent able to partially inhibit mitochondrial free fatty acid ß-oxidation while enhancing glucose oxidation. Here we have found that the metabolic shift driven by TMZ enhances the myogenic potential of skeletal muscle progenitor cells leading to MyoD, Myogenin, Desmin and the slow isoforms of troponin C and I over-expression. Moreover, similarly to exercise, TMZ stimulates the phosphorylation of the AMP-activated protein kinase (AMPK) and up-regulates the peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), both of which are known to enhance the mitochondrial biogenesis necessary for myoblast differentiation. TMZ also induces autophagy which is required during myoblast differentiation and promotes myoblast alignment which allows cell fusion and myofiber formation. Finally, we found that intraperitoneally administered TMZ (5mg/kg) is able to stimulate myogenesis in vivo both in a mice model of cancer cachexia (C26 mice) and upon cardiotoxin damage. Collectively, our work demonstrates that TMZ enhances myoblast differentiation and promotes myogenesis, which might contribute recovering stem cell blunted regenerative capacity and counteracting muscle wasting, thanks to the formation of new myofibers; TMZ is already in use in humans as an anti-anginal drug and its repositioning might impact significantly on aging and regeneration-impaired disorders, including cancer cachexia, as well as have implications in regenerative medicine.

New derivatives of the antimalarial drug Pyrimethamine in the control of melanoma tumor growth: an in vitro and in vivo study.

BACKGROUND: The antimalarial drug Pyrimethamine has been suggested to exert an antitumor activity by inducing apoptotic cell death in cancer cells, including metastatic melanoma cells. However, the dose of Pyrimethamine to be considered as an anticancer agent appears to be significantly higher than the maximum dose used as an antiprotozoal drug. METHODS: Hence, a series of Pyrimethamine analogs has been synthesized and screened for their apoptosis induction in two cultured metastatic melanoma cell lines. One of these analogs, the Methylbenzoprim, was further analyzed to evaluate cell-cycle and the mechanisms of cell death. The effects of Methylbenzoprim were also analyzed in a severe combined immunodeficiency (SCID)-mouse xenotransplantation model. RESULTS: Low dose of Methylbenzoprim was capable of inducing cytotoxic activity and a potent growth-inhibitory effect by arresting cell cycle in S-phase in melanoma cells. Methylbenzoprim was also detected as powerful antineoplastic agents in SCID-mouse although used at very low dose and as a single agent. CONCLUSIONS: Our screening approach led to the identification of a "low cost" newly synthesized drug (methylbenzoprim), which is able to act as an antineoplastic agent in vitro and in vivo, inhibiting melanoma tumor growth at very low concentrations.

Gender issues on occupational safety and health.

The increasing proportion of women in the workforce raises a range of gender-related questions about the different effects of work-related risks on men and women. Few studies have characterized gender differences across occupations and industries, although at this time, the gender sensitive approach is starting to acquire relevance in the field of human preventive medicine. The European Agency for Safety and Health at Work has encouraged a policy of gender equality in all European member states. Italy has adopted European provisions with new specific legislation that integrates the previous laws and introduces the gender differences into the workplace. Despite the fact that gender equal legislation opportunities have been enacted in Italy, their application is delayed by some difficulties. This review examines some of these critical aspects.

Exercise-induced skeletal muscle remodeling and metabolic adaptation: redox signaling and role of autophagy.

SIGNIFICANCE: Skeletal muscle is a highly plastic tissue. Exercise evokes signaling pathways that strongly modify myofiber metabolism and physiological and contractile properties of skeletal muscle. Regular physical activity is beneficial for health and is highly recommended for the prevention of several chronic conditions. In this review, we have focused our attention on the pathways that are known to mediate physical training-induced plasticity. RECENT ADVANCES: An important role for redox signaling has recently been proposed in exercise-mediated muscle remodeling and peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) activation. Still more currently, autophagy has also been found to be involved in metabolic adaptation to exercise. CRITICAL ISSUES: Both redox signaling and autophagy are processes with ambivalent effects; they can be detrimental and beneficial, depending on their delicate balance. As such, understanding their role in the chain of events induced by exercise and leading to skeletal muscle remodeling is a very complicated matter. Moreover, the study of the signaling induced by exercise is made even more difficult by the fact that exercise can be performed with several different modalities, with this having different repercussions on adaptation. FUTURE DIRECTIONS: Unraveling the complexity of the molecular signaling triggered by exercise on skeletal muscle is crucial in order to define the therapeutic potentiality of physical training and to identify new pharmacological compounds that are able to reproduce some beneficial effects of exercise. In evaluating the effect of new "exercise mimetics," it will also be necessary to take into account the involvement of reactive oxygen species, reactive nitrogen species, and autophagy and their controversial effects.

The metabolic modulator trimetazidine triggers autophagy and counteracts stress-induced atrophy in skeletal muscle myotubes.

It has recently been demonstrated that trimetazidine (TMZ), an anti-ischemic antianginal agent, is also able to improve exercise performance in patients with peripheral arterial disease. TMZ is a metabolic modulator, and the mechanisms underlying its cytoprotective anti-ischemic activity could be ascribed, at least in cardiomyocytes, to optimization of metabolism. However, regarding the cytoprotection exerted by TMZ on skeletal muscle and allowing the improvement of exercise performance, no information is yet available. In the present study, we investigated in detail the protective effects of this drug on in vitro skeletal muscle models of atrophy. Experiments carried out with murine C2C12 myotubes treated with TMZ revealed that this drug could efficiently counteract the cytopathic effects induced by the proinflammatory cytokine tumor necrosis factor-α and by the withdrawal of growth factors. Indeed, TMZ significantly counteracted the reduction in myotube size induced by these treatments. TMZ also increased myosin heavy chain expression and induced hypertrophy in C2C12 myotubes, both effects strongly suggesting a role of TMZ in counteracting atrophy in vitro. In particular, we found that TMZ was able to activate the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin 2 pathway and to reduce the stress-induced transcriptional upregulation of atrogin-1, muscle ring finger protein 1, and myostatin, all of which are key molecules involved in muscle wasting. Moreover, this is the first demonstration that TMZ induces autophagy, a key mechanism involved in muscle mass regulation. On the basis of these results, it can be hypothesized that the improvement in exercise performance previously observed in patients could be ascribed to a cytoprotective mechanism exerted by TMZ on skeletal muscle integrity.

Fibroblast autophagy in fibrotic disorders.

Fibrotic disorders are multistage progressive processes that often arise from different causes and are commonly associated with chronic inflammation. Excessive deposition of extracellular matrix is the hallmark of many fibrotic diseases. This may be due to an excess of fibroblast recruitment and activation, as well as to their differentiation in myofibroblasts. These events may be triggered by cytokines, chemokines and growth factors released by lymphocytes or macrophages. The excessive production of extracellular matrix is apparently due to alterations of metabolic pathways in activated fibroblasts. It has been suggested that a defective autophagy, an important subcellular pathway with multiple homeostatic roles, also recognized as a key component of both innate and acquired immunity, could play a role. In this review we illustrate recent insights in the field, suggesting the possible implication of the immune system in orchestrating the fibrotic response via the modulation of autophagic pathways.

Integrating gender medicine into the workplace health and safety policy in the scientific research institutions: a mandatory task.

BACKGROUND: Gender medicine is a multi-faceted field of investigation integrating various aspects of psycho-social and biological sciences but it mainly deals with the impact of the gender on human physiology, pathophysiology, and clinical features of diseases. In Italy, the Decree Law 81/2008 recently introduced the gender issue in the risk assessment at the workplaces. AIMS: This review briefly describes our current knowledge on gender medicine and on the Italian legislation in risk management. CONCLUSIONS: Public or private scientific institutions should be the first to pay attention to the safety of their workers, who are simultaneously subjected to biological, chemical and physical agents. Main tasks of risk management in scientific research institutions are here analyzed and discussed in a gender perspective.

Red blood cell alterations in systemic sclerosis: a pilot study.

AIMS: The aim of this work was to investigate whether systemic oxidative imbalance that occurs in patients with systemic sclerosis affects red blood cell integrity. METHODS: Reactive oxygen species, intracellular content of total thiols and molecules involved in red blood cell aging (e.g., glycophorin A, band 3, CD47 and phosphatidylserine externalization), have been analyzed in erythrocytes from 39 patients with systemic sclerosis and 30 healthy donors by using flow and static cytometry. Analyses were carried out taking into account the two clinical subsets of scleroderma: diffuse cutaneous sclerosis and limited cutaneous sclerosis. RESULTS: A significant reduction (p<0.05) of intracellular total thiols and a significant loss (p<0.01) of glycophorin A, band 3 and CD47 was found in red blood cells from patients with limited cutaneous sclerosis. Conversely, a significant increase (p<0.01) of reactive oxygen species levels and CD47 expression was found in red blood cells from patients with diffuse cutaneous sclerosis. Phosphatidylserine externalization was significantly increased both in patients with limited and diffuse disease. Importantly, this increase was related with disease severity and nailfold capillaroscopy. CONCLUSIONS: Altogether these results suggest a reappraisal of the red blood cells as useful markers in the clinical management of the disease.

Differential effects of the glycolysis inhibitor 2-deoxy-D-glucose on the activity of pro-apoptotic agents in metastatic melanoma cells, and induction of a cytoprotective autophagic response.

2-Deoxy-D-glucose (2DG) is a synthetic glucose analogue that inhibits glycolysis and blocks cancer cell growth. In this report, we evaluated the role of 2DG in the induction of cell death in human metastatic melanoma cells. We have also examined the effects of 2DG in combined treatments with four different pro-apoptotic agents: (i) Temozolomide (TMZ), a chemotherapic drug commonly used to treat metastatic melanoma, (ii) Pyrimethamine (Pyr), a pro-apoptotic antifolate drug recently reappraised in cancer therapy, (iii) Cisplatin (CisPt), a drug capable of directly binding to DNA ultimately triggering apoptosis of cancer cells and (iv) the kinase inhibitor Staurosporine (STS), a prototypical inducer of mitochondria-mediated apoptosis. We found that 2DG per se: (i) induced a cell cycle arrest in G(0) /G(1) , (ii) promoted autophagy, (iii) was ineffective in inducing apoptosis in association with the chemotherapic drug TMZ, whereas (iv) it was synergistic with CisPt and STS pro-apoptotic drugs through a mechanism involving changes of mitochondrial homeostasis. Conversely, (v) 2DG hindered the pro-apoptotic effects of Pyr via a mechanism involving either the block of cell cycle in G(0) /G(1) or the modification of the free radical production of the cell, i.e., decreasing the production of reactive oxygen species (ROS) and increasing the production of reactive nitrogen species (RNS). Moreover, a clear-cut autophagic response involving endoplasmic reticulum remodelling was detectable. Since autophagic cytoprotection has been suggested to contribute to the induction of chemoresistance, these results could provide useful clues as concerns the use of 2DG as anticancer agent in combinatory protocols.

Defective autophagy in fibroblasts may contribute to fibrogenesis in autoimmune processes.

Fibrosis may represent the final step induced by autoimmune mechanism(s). This may be due to the excess in fibroblast recruitment, activation and differentiation in myofibroblasts. These events may be triggered by cytokines, chemokines and growth factors released by lymphocytes or macrophages. Autophagy is an essential conserved homeostatic process that has long been appreciated for cell adaptation to nutrient deprivation. Autophagy is also recognized as an important component of both innate and acquired immunity to pathogens. Recently, dysregulation of autophagy in haematopoietic cells has been suggested to amplify the autoimmune responses. On the other hand, it is possible that defective autophagy in non-haematopoietic cells contributes to the progression to fibrosis. In fibroblasts some alterations in the metabolic pathways and pharmacological data suggest that a defective autophagy could contribute to excess in the production of extracellular matrix by altering the turnover of protein such as collagen. Our goal in this review is to describe the current knowledge on the role of autophagy in the development of fibrotic autoimmune diseases. Further studies could confirm whether agents modulating autophagy may be used in the treatment of these autoimmune diseases.

Analyses of T cell phenotype and function reveal an altered T cell homeostasis in systemic sclerosis. Correlations with disease severity and phenotypes.

We investigated in systemic sclerosis (SSc) patients the T cell homeostasis and its relationship with the clinical course of the disease. Distribution of peripheral T cell subsets, thymic output, lymphocyte proliferation and apoptosis were analyzed by flow cytometry or ELISA. Age inappropriate levels of naive CD4(+) T cells and thymic output were observed. Proliferation of CD4(+) T cells, lymphocyte apoptosis and CD4(+) regulatory T (Treg) cell frequency were significantly higher than those observed in controls and significantly correlated with clinical phenotypes and clinical progression parameters i.e., diffusing capacity of the lung for carbon monoxide (DLCO) and disease activity. These data indicate that the evaluation of the T cell homeostasis can represent a valuable prognostic tool for SSc patients and it is useful to distinguish between limited and diffuse phenotypes. A therapeutic intervention targeted at reversing T cell homeostasis abnormalities would therefore potentially be helpful in counteracting disease progression.

Estrogen receptor profiles in human peripheral blood lymphocytes.

Estrogens are well-known regulators of the immune responses. Most of their effects are mediated by two receptors: estrogen receptor (ER)alpha and ERbeta. Up to date the presence of intracellular ER in human immune cells represents a controversial issue, while their surface membrane expression has scarcely been explored. In this study we investigated the intracellular and cell surface expression of ERalpha and ERbeta in human peripheral blood lymphocytes (PBL) by flow and static cytometry as well as by Western Blot. To this aim we used five different commercial antibodies recognizing distinct ER epitopes. We observed that CD4(+) and CD8(+) T lymphocytes, B lymphocytes and NK cells contain intracellular ERalpha and ERbeta, being the ERalpha46 isoform the most represented ER. However, significant differences could be observed among the antibodies studied in terms of immunoreactivity and specificity. Importantly, we also found a cell surface expression of ERalpha46 isoform. We also observed that a membrane-impermeant form of E2 induced a rapid phosphorylation of extracellular signal-regulated kinase (ERK), a significant proliferation of T lymphocytes, and IFN-gamma production by NK cells, thus suggesting the expression of a functional mERalpha. In conclusion our data could provide new insights as concerns the estrogen-related mechanisms of immune system modulation. They also suggest the need for a reappraisal of the experimental conditions for the characterization of the ER expression.

Role of GD3-CLIPR-59 association in lymphoblastoid T cell apoptosis triggered by CD95/Fas.

We previously found that a directional movement of the raft component GD3 towards mitochondria, by its association with microtubules, was mandatory to late apoptogenic events triggered by CD95/Fas. Since CLIPR-59, CLIP-170-related protein, has recently been identified as a microtubule binding protein associated with lipid rafts, we analyzed the role of GD3-CLIPR-59 association in lymphoblastoid T cell apoptosis triggered by CD95/Fas. To test whether CLIPR-59 could play a role at the raft-microtubule junction, we performed a series of experiments by using immunoelectron microscopy, static or flow cytometry and biochemical analyses. We first assessed the presence of CLIPR-59 molecule in lymphoblastoid T cells (CEM). Then, we demonstrated that GD3-microtubule interaction occurs via CLIPR-59 and takes place at early time points after CD95/Fas ligation, preceding the association GD3-tubulin. GD3-CLIPR-59 association was demonstrated by fluorescence resonance energy transfer (FRET) analysis. The key role of CLIPR-59 in this dynamic process was clarified by the observation that silencing CLIPR-59 by siRNA affected the kinetics of GD3-tubulin association, spreading of GD3 towards mitochondria and apoptosis execution. We find that CLIPR-59 may act as a typical chaperone, allowing a prompt interaction between tubulin and the raft component GD3 during cell apoptosis triggered by CD95/Fas. On the basis of the suggested role of lipid rafts in conveying pro-apoptotic signals these results disclose new perspectives in the understanding of the mechanisms by which raft-mediated pro-apoptotic signals can directionally reach their target, i.e. the mitochondria, and trigger apoptosis execution.

Raft component GD3 associates with tubulin following CD95/Fas ligation.

In a previous investigation, we demonstrated that after CD95/Fas triggering, raft-associated GD3 ganglioside, normally localized at the plasma membrane of T cells, can be detected in mitochondria, where they contribute to apoptogenic events. Here, we show the association of the glycosphingolipid GD3 with microtubular cytoskeleton at very early time points following Fas ligation. This was assessed by different methodological approaches, including fluorescence resonance energy transfer, immunoelectron microscopy, and coimmunoprecipitation. Furthermore, docking analysis also showed that GD3 has a high affinity for the pore formed by 4 tubulin heterodimers (type I pore), thus suggesting a possible direct interaction between tubulin and GD3. Finally, time-course analyses indicated that the relocalization of GD3 to the mitochondria was time related with the alterations of the mitochondrial membrane potential. Hence, microtubules could act as tracks for ganglioside redistribution following apoptotic stimulation, possibly contributing to the mitochondrial alterations leading to cell death.

Analyzing morphological and ultrastructural features in cell death.

Diverse forms of cell death have initially been described thanks to their observation at the electron microscope. Morphological and ultrastructural features of necrosis, apoptosis, and autophagy, considered here as prototypic cell death processes, allow one to characterize and quantify early and late cytopathological changes occurring in cells undergoing degeneration. Both light microscopy and scanning electron microscopy can provide useful insights, for example, to quantitatively evaluate cell death or to characterize cell surface changes of the cells, respectively. However, transmission electron microscopy preparation allows distinguishing among different forms of cell death. This chapter describes in brief the methods used to characterize cell death forms, including membrane, nucleus, and organelle changes, and shows paradigmatic micrographs. In particular, morphogenetic changes occurring in mitochondria during apoptosis, that is, fission process or, conversely, vacuole formation during autophagy, are shown. Possible artifacts are also described. Ultrastructural analysis seems still to provide essential information for studies on cell death.

Pyrimethamine induces apoptosis of melanoma cells via a caspase and cathepsin double-edged mechanism.

The unresponsiveness of metastatic melanoma to conventional chemotherapeutic and biological agents is largely due to the development of resistance to apoptosis. Pyrimethamine belongs to the group of antifolate drugs, and in addition to antiprotozoan effects, it exerts a strong proapoptotic activity, which we recently characterized in human T lymphocytes. However, no data regarding pyrimethamine anticancer activity are available thus far. To this end, we examined the in vitro effects of pyrimethamine on apoptosis, cell cycle distribution, and cell proliferation of human metastatic melanoma cell lines. The in vivo antitumor potential of pyrimethamine was evaluated in a severe combined immunodeficiency (SCID) mouse xenotransplantation model. Our data indicate that pyrimethamine, when used at a clinically relevant concentration, induced apoptosis in metastatic melanoma cells via the activation of the cathepsin B and the caspase cascade (i.e., caspase-8 and caspase-9) and subsequent mitochondrial depolarization. This occurred independently from CD95/Fas engagement. Moreover, pyrimethamine induced a marked inhibition of cell growth and an S-phase cell cycle arrest. Results obtained in SCID mice, injected s.c. with metastatic melanoma cells and treated with pyrimethamine, indicated a significant inhibitory effect on tumor growth. In conclusion, our results suggest that pyrimethamine-induced apoptosis may be considered as a multifaceted process, in which different inducers or regulators of apoptosis are simultaneously implicated, thus permitting death defects of melanoma cells to be bypassed or overcome. On these bases, we hypothesize that pyrimethamine could represent an interesting candidate for the treatment of metastatic melanoma.

Progressive derangement of the T cell compartment in a case of Evans syndrome.

BACKGROUND: Evans syndrome (ES) is a rare disorder characterized by combined autoimmune thrombocytopenia and autoimmune hemolytic anemia. Several studies have documented a number of B cell defects, whereas only limited information is currently available about the T cell subset. METHODS: A wide panel of immunological analyses aiming specifically at a quantitative and qualitative evaluation of the T cell compartment was performed in an unusual case of ES. The peripheral distribution of the T cell subsets, the diversity of the T cell receptor (TCR) repertoires, the cytokine profile and the T cell apoptosis have been longitudinally evaluated. RESULTS: On first investigation, flow-cytometric immunophenotyping showed a remarkable alteration of T cell homeostasis with deeply reduced CD4+ naive T cells and recent thymic emigrants. This was seen in association with increased levels of T cell activation and apoptosis. Consistently with these data the cytokine profile was characterized by high interferon-gamma and low interleukin-2 levels. Staining for CD4 and CD25 molecules showed decreased percentages of circulating regulatory T cells according to the autoimmune nature of ES. Finally, restricted TCR repertoires were demonstrated by a skewed TCR beta chain variable (TCRBV) gene usage as well as oligoclonal third complementarity-determining region (CDR3) profiles. A deterioration of the above-mentioned parameters and a worsening of the clinical condition were observed during the follow-up requiring more intensive treatments. CONCLUSION: The demonstration of multiple T cell defects, in addition to providing pathogenetic information, is likely to alter both acute treatment and outcome of ES.