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Simple Summary Autophagy is a complex cell process that allow the cell to survive in unfavorable conditions, e.g., in the lack of nutritional elements coming from the environment. Here we focused on the role played by autophagy in the crosstalk between the microenvironment surrounding the tumor and cancer cells. This environment is in fact known as pivotal in determining the growth or the inhibition of a tumor. Cancer progression and response to therapy significantly differ between women and men and the microenvironment, in particular sex hormones and microRNAs, appears a critical factor. Four representative types of cancer, i.e., colon cancer, melanoma, lymphoma, and lung cancer showing sex/gender specificities have been described herein. We underscore that the use of a “gender tailored” approach could provide a better comprehension of the cellular and molecular mechanisms of cancer growth control contributing to the development of novel therapeutic approaches towards an increasingly personalized medicine. Abstract The interplay between cancer cells and the tumor microenvironment (TME) has a fundamental role in tumor progression and response to therapy. The plethora of components constituting the TME, such as stroma, fibroblasts, endothelial and immune cells, as well as macromolecules, e.g., hormones and cytokines, and epigenetic factors, such as microRNAs, can modulate the survival or death of cancer cells. Actually, the TME can stimulate the genetically regulated programs that the cell puts in place under stress: apoptosis or, of interest here, autophagy. However, the implication of autophagy in tumor growth appears still undefined. Autophagy mainly represents a cyto-protective mechanism that allows cell survival but, in certain circumstances, also leads to the blocking of cell cycle progression, possibly leading to cell death. Since significant sex/gender differences in the incidence, progression and response to cancer therapy have been widely described in the literature, in this review, we analyzed the roles played by key components of the TME, e.g., estrogen and microRNAs, on autophagy regulation from a sex/gender-based perspective. We focused our attention on four paradigmatic and different forms of cancers—colon cancer, melanoma, lymphoma, and lung cancer—concluding that sex-specific differences may exert a significant impact on TME/cancer interaction and, thus, tumor growth.

Rheumatoid arthritis (RA) is a chronic inflammatory disease with increased risk of cardiovascular events and mortality that can be attributed to accelerated atherosclerosis. This pilot study aimed to investigate if changes in blood parameters were compatible with atherosclerotic events in RA patients. To this aim, 45 RA women (aged more than 18 years), and 25 age and gender-matched healthy donors (HD) were included. Biomarkers of oxidative stress, platelet activation and platelet aggregation were analysed in RA patients at baseline and after six months of treatment with disease modifying anti-rheumatic drugs (DMARDs). Flow cytometry analysis revealed that ca. 4% of platelets was in activated state (evaluated in term of Annexin V and PAC-1 positivity) in RA patients at baseline, and that the 76% of platelets displayed mitochondrial hyperpolarization. Moreover, platelets from RA patients at baseline aggregated more than those from HD after whole blood treatment with adenosine diphosphate. Interestingly, platelet aggregation in patients at baseline positively correlated with disease activity measured by DAS28 score. After six months of treatment with DMARDs, platelet activation and platelet aggregation reached values comparable to those of HD. Our preliminary data suggest that platelets might play an active role in the atherosclerosis occurring in RA patients.

The biology of sex differences deals with the study of the disparities between females and males and the related biological mechanisms. Gender medicine focuses on the impact of gender and sex on human physiology, pathophysiology and clinical features of diseases that are common to women and men. The term gender refers to a complex interrelation and integration of sex–as a biological and functional determinant–and psychological and cultural behaviours (due to ethnical, social or religious background). The attention to the impact of gender differences on the pathophysiology and, therefore, on the clinical management of the most common diseases, such as cardiovascular diseases (CVD), neurodegenerative disorders, immune and autoimmune diseases as well as several tumours, is in fact often neglected. Hence, studies covering different fields of investigation and including sex differences in the pathogenesis, in diagnostic and prognostic criteria as well as in response to therapy appear mandatory. However, prerequisites for this development are preclinical studies, including in vitro and in vivo approaches. They represent the first step in the development of a drug or in the comprehension of the pathogenetic mechanisms of diseases, in turn a necessary step for the development of new or more appropriate therapeutic strategies. However, sex differences are still poorly considered and the great majority of preclinical studies do not take into account the relevance of such disparities. In this review, we describe the state of the art of these studies and provide some paradigmatic examples of key fields of investigation, such as oncology, neurology and CVD, where preclinical models should be improved. * Abbreviations : AD, : Alzheimer's disease; ALL, : acute lymphocytic leukaemia; AML, : acute myelogenous leukaemia; AP, : action potential; APOE, : Apolipoprotein E; APP, : amyloid precursor protein; BDNF, : brain-derived neurotrophic factor; CLL, : chronic lymphocytic leukaemia; CML, : chronic myelogenous leukaemia; CVD, : cardiovascular disease; EBV, : Epstein-Barr virus; GM, : gender medicine; HF, : heart failure; HPV, : Human Papilloma virus; iPSC, : induced pluripotent stem cell; IGF-1, : insulin growth factor-1; MeSH®, : Medical Subject Headings; PBL, : peripheral blood lymphocytes; PBMC, : peripheral blood mononuclear cells; PS, : presenilin; Tg, : transgenic

// Marina Pierdominici 1, * , Angela Maselli 2, * , Silvia L. Locatelli 3 , Laura Ciarlo 2 , Giuseppa Careddu 3 , Mario Patrizio 2 , Barbara Ascione 2 , Antonella Tinari 4 , Carmelo Carlo-Stella 3 , Walter Malorni 2 , Paola Matarrese 2, ** , Elena Ortona 1, ** 1 Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita, Rome, Italy 2 Department of Therapeutic Research and Medicine Evaluation, Istituto Superiore di Sanita, Rome, Italy 3 Department of Oncology and Hematology, Humanitas Cancer Center - Humanitas Clinical and Research Center, Milano, Italy 4 Department of Technology and Health, Istituto Superiore di Sanita, Rome, Italy * These authors have contributed equally to this work ** P.M. and E.O. to be considered as Senior Authors Correspondence to: Walter Malorni, email: malorni@iss.it Keywords: Hodgkin lymphoma, estrogen receptor β, autophagy, DRAM2, tumor growth Received: June 09, 2016 Accepted: December 05, 2016 Published: December 28, 2016 ABSTRACT Although Hodgkin lymphoma (HL) is curable with current therapy, at least 20% of patients relapse or fail to make complete remission. In addition, patients who achieve long-term disease-free survival frequently undergo infertility, secondary malignancies, and cardiac failure, which are related to chemotherapeutic agents and radiation therapies. Hence, new therapeutic strategies able to counteract the HL disease in this important patient population are still a matter of study. Estrogens, in particular 17β-estradiol (E2), have been suggested to play a role in lymphoma cell homeostasis by estrogen receptors (ER) β activation. On these bases, we investigated whether the ligation of ERβ by a selective agonist, the 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), could impact HL tumor growth. We found that DPN-mediated ERβ activation led to a reduction of in vitro cell proliferation and cell cycle progression by inducing autophagy. In nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice engrafted with HL cells, ERβ activation by DPN was able to reduce lymphoma growth up to 60% and this associated with the induction of tumor cell autophagy. Molecular characterization of ERβ-induced autophagy revealed an overexpression of damage-regulated autophagy modulator 2 (DRAM2) molecule, whose role in autophagy modulation is still debated. After ERβ activation, both DRAM2 and protein 1 light chain 3 (LC3), a key actor in the autophagosome formation, strictly interacted each other and localized at mitochondrial level. Altogether these results suggest that targeting ERβ with selective agonists might affect HL cell proliferation and tumor growth via a mechanism that brings into play DRAM2-dependent autophagic cascade.

This review takes into consideration the main mechanisms involved in cellular remodeling following an ischemic injury, with special focus on the possible role played by non-genomic estrogen effects. Sex differences have also been considered. In fact, cardiac ischemic events induce damage to different cellular components of the heart, such as cardiomyocytes, vascular cells, endothelial cells, and cardiac fibroblasts. The ability of the cardiovascular system to counteract an ischemic insult is orchestrated by these cell types and is carried out thanks to a number of complex molecular pathways, including genomic (slow) or non-genomic (fast) effects of estrogen. These pathways are probably responsible for differences observed between the two sexes. Literature suggests that male and female hearts, and, more in general, cardiovascular system cells, show significant differences in many parameters under both physiological and pathological conditions. In particular, many experimental studies dealing with sex differences in the cardiovascular system suggest a higher ability of females to respond to environmental insults in comparison with males. For instance, as cells from females are more effective in counteracting the ischemia/reperfusion injury if compared with males, a role for estrogen in this sex disparity has been hypothesized. However, the possible involvement of estrogen-dependent non-genomic effects on the cardiovascular system is still under debate. Further experimental studies, including sex-specific studies, are needed in order to shed further light on this matter.

Gliomas are tumors that originate from the glial tissue, thus involving the central nervous system with varying degrees of malignancy. The most aggressive and frequent form is glioblastoma multiforme, a disease characterized by resistance to therapies, frequent recurrences, and extremely poor median survival time. Data on overall glioma case studies demonstrate clear sex disparities regarding incidence, prognosis, drug toxicity, clinical outcome, and, recently, prediction of therapeutic response. In this study, we analyze data in the literature regarding malignant glioma, mainly glioblastoma multiforme, focusing on epidemiological and clinical evaluations. Less discussed issues, such as the role of viral infections, energy metabolism, and predictive aspects concerning the possible use of dedicated therapeutic approaches for male or female patients, will be reported together with different estimated pathogenetic mechanisms underlying astrocyte transformation and glioma chemosensitivity. In this era, where personalized/precision medicine is the most important driver for targeted cancer therapies, the lines of evidence discussed herein strongly suggest that clinical approaches to malignant glioma should consider the patient's sex. Furthermore, retrospectively revising previous clinical studies considering patient sex as a crucial variable is recommended.

// Paola Matarrese 1, * , Claudia Abbruzzese 2, * , Anna Maria Mileo 3, * , Rosa Vona 1 , Barbara Ascione 1 , Paolo Visca 4 , Francesca Rollo 4 , Maria Benevolo 4 , Walter Malorni 1, 5 , Marco G. Paggi 2 1 Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanita, Rome, Italy 2 Department of Research, Advanced Diagnostics and Technological Innovation, Unit of Cellular Networks and Therapeutic Targets, Regina Elena National Cancer Institute, IRCCS, Rome, Italy 3 Department of Research, Advanced Diagnostics and Technological Innovation, Unit of Immunology and Immunotherapy, Regina Elena National Cancer Institute, IRCCS, Rome, Italy 4 Unit of Pathology, Regina Elena National Cancer Institute, IRCCS, Rome, Italy 5 Istituto San Raffaele Pisana, Rome, Italy * These authors contributed equally to this work Correspondence to: Marco G. Paggi, e-mail: paggi@ifo.it Walter Malorni, e-mail: malorni@iss.it Keywords: human papillomavirus 16 E7, gelsolin, actin, cancer, cell migration Received: February 24, 2016 Accepted: March 28, 2016 Published: April 08, 2016 ABSTRACT The viral oncoprotein E7 from the “high-risk” Human Papillomavirus 16 (HPV16) strain is able, when expressed in human keratinocytes, to physically interact with the actin severing protein gelsolin (GSN). In a previous work it has been suggested that this protein-protein interaction can hinder GSN severing function, thus leading to actin network remodeling. In the present work we investigated the possible implications of this molecular interaction in cancer cell metastatic potential by analyzing two different human CC cell lines characterized by low or high expression levels of HPV16 DNA (SiHa and CaSki, respectively). In addition, a HPV-null CC cell line (C-33A), transfected in order to express the HPV16 E7 oncoprotein as well as two different deletion mutants, was also analyzed. We found that HPV16 E7 expression level was directly related with cervical cancer migration and invasion capabilities and that these HPV16 E7-related features were associated with Epithelial to Mesenchymal Transition (EMT) processes. These effects appeared as strictly attributable to the physical interaction of HPV16 E7 with GSN, since HPV16 E7 deletion mutants unable to bind to GSN were also unable to modify microfilament assembly dynamics and, therefore, cell movements and invasiveness. Altogether, these data profile the importance of the physical interaction between HPV16 E7 and GSN in the acquisition of the metastatic phenotype by CC cells, underscoring the role of HPV16 intracellular load as a risk factor in cancer.

Cardiac dysfunction is often observed in patients with cancer also representing a serious problem limiting chemotherapeutic intervention and even patient survival. In view of the recently established role of the immune system in the control of cancer growth, the present work has been undertaken to investigate the effects of a panel of the most important inflammatory cytokines on the integrity and function of mitochondria, as well as of the cytoskeleton, two key elements in the functioning of cardiomyocytes. Either mitochondria features or actomyosin cytoskeleton organization of in vitro‐cultured cardiomyocytes treated with different inflammatory cytokines were analyzed. In addition, to investigate the interplay between tumor growth and cardiac function in an in vivo system, immunocompetent female mice were inoculated with cancer cells and treated with the chemotherapeutic drug doxorubicin at a dosing schedule able to suppress tumor growth without inducing cardiac alterations. Analyses carried out in cardiomyocytes treated with the inflammatory cytokines, such as tumor necrosis factor α (TNF‐α), interferon γ (IFN‐γ), interleukin 6 (IL‐6), IL‐8, and IL‐1β revealed severe phenotypic changes, for example, of contractile cytoskeletal elements, mitochondrial membrane potential, mitochondrial reactive oxygen species production and mitochondria network organization. Accordingly, in immunocompetent mice, the tumor growth was accompanied by increased levels of the inflammatory cytokines TNF‐α, IFN‐γ, IL‐6, and IL‐8, either in serum or in the heart tissue, together with a significant reduction of ventricular systolic function. The alterations of mitochondria and of microfilament system of cardiomyocytes, due to the systemic inflammation associated with cancer growth, could be responsible for remote cardiac injury and impairment of systolic function observed in vivo.

The role of autophagy in cancer onset and progression appears still controversial. On one hand, autophagy allows cancer cell to survive in unfavorable environmental conditions, on the other hand, once internal energy resources are exhausted, it leads to cell death. In addition, autophagy interpheres with cell cycle progression, de facto exerting a cytostatic activity. Hence, it represents an important target for anticancer therapy. For example, temozolomide (TMZ), of use for glioblastoma (GBM) treatment, appears as capable of inducing autophagy partially inhibiting cancer cell proliferation. However, GBM, a very aggressive brain tumor with poor prognosis even after surgery and radio-chemotherapy, invariably recurs and leads to patient death. Since cancer stem cells have been hypothesized to play a role in refractory/relapsing cancers, in the present work we investigated if autophagy could represent a constitutive cytoprotection mechanism for glioblastoma stem-like cells (GSCs) and if the modulation of autophagic process could affect GBM growth and survival. Thus, in the present study we first evaluated the relevance of autophagy in GBM tumor specimens, then its occurrence in GSCs and, finally, if modulation of autophagy could influence GSC response to TMZ. Our results suggested that, in vitro, the impairing autophagic process with quinacrine, a compound able to cross the blood-brain barrier, increased GSC susceptibility to TMZ. Death of GSCs was apparently due to the iron dependent form of programmed cell death characterized by the accumulation of lipid peroxides called ferroptosis. These results underscore the relevance of the modulation of autophagy in the GSC survival and death and suggest that triggering of ferroptosis in GSCs could represent a novel and important target for the management of glioblastoma.

Background/aims Estrogen could play a key role in the mechanisms underlying sex-related disparity in the incidence of thrombotic events. We investigated whether estrogen receptors (ERs) were expressed in human red blood cells (RBCs), and if they affected cell signaling of erythrocyte constitutive isoform of endothelial NO-synthase (eNOS) and nitric oxide (NO) release. Methods RBCs from 29 non-smoker volunteers (15 males and 14 females) aged between 20 and 40 years were analyzed by cytometry and western blot. In particular, content and distribution of ER-α and ER-β, tyrosine kinases and eNOS phosphorylation and NO release were analyzed. Results We demonstrated that: i) both ER-α and ER-β were expressed by RBCs; ii) they were both functionally active; and iii) ERs distribution and function were different in males and females. In particular, ERs modulated eNOS phosphorylation and NO release in RBCs from both sexes, but they induced the phosphorylation of specific tyrosine residues of kinases linked to eNOS activation and NO release in the RBCs from females only. Conclusion Collectively, these data suggest that ERs could play a critical role in RBC intracellular signaling. The possible implication of this signaling in sex-linked risk disparity in human cardiovascular diseases, e.g. in thrombotic events, may not be ruled out.

Nowadays, several types of tumors can benefit from the new frontier of immunotherapy, due to the recent increasing knowledge of the role of the immune system in cancer control. Among the new therapeutic strategies, there is the immune checkpoint blockade (ICB), able to restore an efficacious antitumor immunity and significantly prolong the overall survival (OS) of patients with advanced tumors such as melanoma and non-small cell lung cancer (NSCLC). Despite the impressive efficacy of these agents in some patients, treatment failure and resistance are frequently observed. In this regard, the signaling governed by IFN type I (IFN-I) has emerged as pivotal in orchestrating host defense. This pathway displays different activation between sexes, thus potentially contributing to sexual dimorphic differences in the immune responses to immunotherapy. This perspective article aims to critically consider the immune signals, with particular attention to IFN-I, that may differently affect female and male antitumor responses upon immunotherapy.

The focus of this chapter is the gender differences in mitochondria in cardiovascular disease. There is broad evidence suggesting that some of the gender differences in cardiovascular outcome may be partially related to differences in mitochondrial biology (Ventura–Clapier R, Moulin M, Piquereau J, Lemaire C, Mericskay M, Veksler V, Garnier A, Clin Sci (Lond) 131(9):803–822, 2017)). Mitochondrial disorders are causally affected by mutations in either nuclear or mitochondrial genes involved in the synthesis of respiratory chain subunits or in their posttranslational control. This can be due to mutations of the mtDNA which are transmitted by the mother or mutations in the nuclear DNA. Because natural selection on mitochondria operates only in females, mutations may have had more deleterious effects in males than in females (Ventura–Clapier R, Moulin M, Piquereau J, Lemaire C, Mericskay M, Veksler V, Garnier A, Clin Sci (Lond) 131(9):803–822, 2017; Camara AK, Lesnefsky EJ, Stowe DF. Antioxid Redox Signal 13(3):279–347, 2010). As mitochondrial mutations can affect all tissues, they are responsible for a large panel of pathologies including neuromuscular disorders, encephalopathies, metabolic disorders, cardiomyopathies, neuropathies, renal dysfunction, etc. Many of these pathologies present sex/gender specificity. Thus, alleviating or preventing mitochondrial dysfunction will contribute to mitigating the severity or progression of the development of diseases. Here, we present evidence for the involvement of mitochondria in the sex specificity of cardiovascular disorders.

The primary purpose of these practical guidelines related to Kawasaki disease (KD) is to contribute to prompt diagnosis and appropriate treatment on the basis of different specialists’ contributions in the field. A set of 40 recommendations is provided, divided in two parts: the first describes the definition of KD, its epidemiology, etiopathogenetic hints, presentation, clinical course and general management, including treatment of the acute phase, through specific 23 recommendations. Their application is aimed at improving the rate of treatment with intravenous immunoglobulin and the overall potential development of coronary artery abnormalities in KD. Guidelines, however, should not be considered a norm that limits treatment options of pediatricians and practitioners, as treatment modalities other than those recommended may be required as a result of peculiar medical circumstances, patient’s condition, and disease severity or complications.

The regulation of the mitochondrial dynamics and the balance between fusion and fission processes are crucial for the health and fate of the cell. Mitochondrial fusion and fission machinery is controlled by key proteins such as mitofusins, OPA-1 and several further molecules. In the present work we investigated the implication of lipid rafts in mitochondrial fusion induced by Mdivi-1. Our results underscore the possible implication of lipid "rafts" in mitochondrial morphogenetic changes and their homeostasis.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ?accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. "Regulated cell death" (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death Fil: Rabinovich, Gabriel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Nomenclature Committee on Cell Death. Equipe 11 Apoptose, Cancer et Immunité. Centre de Recherche des Cordeliers; Francia

Systemic sclerosis (SSc) is a rare disorder of the connective tissue characterized by fibrosis of the skin, skeletal muscles and visceral organs. Additional manifestations include activation of the immune system and vascular injury. SSc causes disability and death as the result of end‐stage organ failure. Two clinical subsets of the SSc are accepted: limited cutaneous SSc (lc‐SSc) and diffuse cutaneous SSc (dc‐SSc). At present, the aetiology and pathogenesis of SSc remain obscure, and consequently, disease outcome is unpredictable. Numerous studies suggest that reactive oxidizing species (ROS) play an important role in the pathogenesis of scleroderma. Over the years, several reports have supported this hypothesis for both lc‐SSc and dc‐SSc, although the specific role of oxidative stress in the pathogenesis of vascular injury and fibrosis remains to be clarified. The aim of the present review was to report and comment the recent findings regarding the involvement and role of oxidative stress in SSc pathogenesis. Biomarkers proving the link between ROS and the main pathological features of SSc have been summarized.

Background Primary Sjögren’s syndrome (pSS) is a common chronic autoimmune disease characterized by lymphocytic infiltration of exocrine glands and peripheral lymphocyte perturbation. In the current study, we aimed to investigate the possible pathogenic implication of autophagy in T lymphocytes in patients with pSS. Methods Thirty consecutive pSS patients were recruited together with 20 patients affected by sicca syndrome and/or chronic sialoadenitis and 30 healthy controls. Disease activity and damage were evaluated according to SS disease activity index, EULAR SS disease activity index, and SS disease damage index. T lymphocytes were analyzed for the expression of autophagy-specific markers by biochemical, molecular, and histological assays in peripheral blood and labial gland biopsies. Serum interleukin (IL)-23 and IL-21 levels were quantified by enzyme-linked immunosorbent assay. Results Our study provides evidence for the first time that autophagy is upregulated in CD4+ T lymphocyte salivary glands from pSS patients. Furthermore, a statistically significant correlation was detected between lymphocyte autophagy levels, disease activity, and damage indexes. We also found a positive correlation between autophagy enhancement and the increased salivary gland expression of IL-21 and IL-23, providing a further link between innate and adaptive immune responses in pSS. Conclusions These findings suggest that CD4+ T lymphocyte autophagy could play a key role in pSS pathogenesis. Additionally, our data highlight the potential exploitation of T cell autophagy as a biomarker of disease activity and provide new ground to verify the therapeutic implications of autophagy as an innovative drug target in pSS. Electronic supplementary material The online version of this article (doi:10.1186/s13075-017-1385-y) contains supplementary material, which is available to authorized users.

Kawasaki disease is a multisystemic vasculitis that can result in coronary artery lesions. It predominantly affects young children and is characterized by prolonged fever, diffuse mucosal inflammation, indurative oedema of the hands and feet, a polymorphous skin rash and non‐suppurative lymphadenopathy. Coronary artery involvement is the most important complication of Kawasaki disease and may cause significant coronary stenosis resulting in ischemic heart disease. The introduction of intravenous immunoglobulin decreases the incidence of coronary artery lesions to less than 5%. The etiopathogenesis of this disease remains unclear. Several lines of evidence suggest that an interplay between a microbial infection and a genetic predisposition could take place in the development of the disease. In this review, we summarize the state of the art of pathogenetic mechanisms of Kawasaki disease underscoring the relevance of haematological features as a novel field of investigation.

Article en libre accès; Women and men, female and male animals and cells are biologically different, and acknowledgement of this fact is critical to advancing medicine. However, incorporating concepts of sex-specific analysis in basic research is largely neglected, introducing bias into translational findings, clinical concepts and drug development. Research funding agencies recently approached these issues but implementation of policy changes in the scientific community is still limited, probably due to deficits in concepts, knowledge and proper methodology. This expert review is based on the EUGenMed project (www.eugenmed.eu) developing a roadmap for implementing sex and gender in biomedical and health research. For sake of clarity and conciseness, examples are mainly taken from the cardiovascular field that may serve as a paradigm for others, since a significant amount of knowledge how sex and oestrogen determine the manifestation of many cardiovascular diseases (CVD) has been accumulated. As main concepts for implementation of sex in basic research, the study of primary cell and animals of both sexes, the study of the influence of genetic vs. hormonal factors and the analysis of sex chromosomes and sex specific statistics in genome wide association studies (GWAS) are discussed. The review also discusses methodological issues, and analyses strength, weaknesses, opportunities and threats in implementing sex-sensitive aspects into basic research.

Autophagy, a stress response activated in influenza A virus infection helps the cell avoid apoptosis. However, in the absence of apoptosis infected cells undergo vastly expanded autophagy and nevertheless die in the presence of necrostatin but not of autophagy inhibitors. Combinations of inhibitors indicate that the controls of protective and lethal autophagy are different. Infection that triggers apoptosis also triggers canonical autophagy signaling exhibiting transient PI3K and mTORC1 activity. In terminal autophagy phospho-mTOR(Ser2448) is suppressed while mTORC1, PI3K and mTORC2 activities increase. mTORC1 substrate p70S6K becomes highly phosphorylated while its activity, now regulated by mTORC2, is required for LC3-II formation. Inhibition of mTORC2/p70S6K, unlike that of PI3K/mTORC1, blocks expanded autophagy in the absence of apoptosis but not moderate autophagy. Inhibitors of expanded autophagy limit virus reproduction. Thus expanded, lethal autophagy is activated by a signaling mechanism different from autophagy that helps cells survive toxic or stressful episodes.

To the Editor: Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that can be considered as a prothrombotic state1. A great number of studies have investigated the possible role of reactive oxygen species (ROS) in the etiology and pathogenesis of this disease. The presence of large amounts of superoxide radicals and hydrogen peroxide produced by activated neutrophils has been reported in the synovial fluid of patients with RA. This may cause lipid peroxidation that yields a wide variety of end products, including malondialdehyde (MDA), a known marker of oxidative stress. These products are therefore transported from the synovial fluid to the blood circulation system2. Considering that elevated levels of MDA have been observed in the blood plasma of patients with RA2, the aim of this pilot study was to investigate whether the elevated levels of plasmatic MDA could be associated with a modification of the total antioxidant capacity (TAC) of blood plasma that is usually indicative of a “systemic” oxidative imbalance3. In addition, in view of their activity as redox effectors or scavengers4, as well as determinants of thrombus formation … Address correspondence to Dr. E. Straface, Istituto Superiore di Sanita, Department of Therapeutic Research and Medicine Evaluation, Section of Cell Aging and Gender Medicine, Viale Regina Elena 299 - 00161 Rome, Italy. E-mail: elisabetta.straface{at}iss.it

Autophagy represents a key mechanism of cytoprotection that can be activated by a variety of extracellular and intracellular stresses and allows the cell to sequester cytoplasmic components and damaged organelles, delivering them to lysosomes for degradation and recycling. However, the autophagy process has also been associated with the death of the cell. It has been demonstrated to be constitutive in some instances and inducible in others, and the idea that it could represent a pathogenetic determinant as well as a possible prognostic tool and a therapeutic target in a plethora of human diseases has recently been considered. Among these, cancer represents a major one. In this review, we recapitulate the critical implications of autophagy in the pathogenesis, progression, and treatment of lymphoproliferative disorders. Leukemias and lymphomas, in fact, represent paradigmatic human diseases in which advances have recently been made in this respect.—Pierdominici, M., Barbati, C., Vomero, M., Locatelli, S. L...

Objective Accumulation of mitochondria underlies T-cell dysfunction in systemic lupus erythematosus (SLE). Mitochondrial turnover involves endosomal traffic regulated by HRES-1/Rab4, a small GTPase that is overexpressed in lupus T cells. Therefore, we investigated whether (1) HRES-1/Rab4 impacts mitochondrial homeostasis and (2) Rab geranylgeranyl transferase inhibitor 3-PEHPC blocks mitochondrial accumulation in T cells, autoimmunity and disease development in lupus-prone mice. Methods Mitochondria were evaluated in peripheral blood lymphocytes (PBL) of 38 SLE patients and 21 healthy controls and mouse models by flow cytometry, microscopy and western blot. MRL/lpr mice were treated with 125 μg/kg 3-PEHPC or 1 mg/kg rapamycin for 10 weeks, from 4 weeks of age. Disease was monitored by antinuclear antibody (ANA) production, proteinuria, and renal histology. Results Overexpression of HRES-1/Rab4 increased the mitochondrial mass of PBL (1.4-fold; p=0.019) and Jurkat cells (2-fold; p=0.000016) and depleted the mitophagy initiator protein Drp1 both in human (−49%; p=0.01) and mouse lymphocytes (−41%; p=0.03). Drp1 protein levels were profoundly diminished in PBL of SLE patients (−86±3%; p=0.012). T cells of 4-week-old MRL/lpr mice exhibited 4.7-fold over-expression of Rab4A (p=0.0002), the murine homologue of HRES-1/Rab4, and depletion of Drp1 that preceded the accumulation of mitochondria, ANA production and nephritis. 3-PEHPC increased Drp1 (p=0.03) and reduced mitochondrial mass in T cells (p=0.02) and diminished ANA production (p=0.021), proteinuria (p=0.00004), and nephritis scores of lupus-prone mice (p

Previous studies have suggested a pathogenetic role of autoantibodies (Abs) against ATP synthase (ATPs) in patients with Alzheimer's disease (AD). Using a mouse model, we found that intracerebroventricular administration of anti-ATPs-Abs, purified from AD patients, leads to poor cognitive performance and pronounced cell damage in the hippocampus, a brain region specifically involved in learning and memory processes, which is severely affected in AD. Our results are suggestive of a role of anti-ATPs-Abs in the onset and progression of AD and also provide a fruitful model for the study of memory disturbances in neurodegenerative diseases.

Organ transplantation, e.g., of the heart, liver, or kidney, is nowadays a routine strategy to counteract several lethal human pathologies. From literature data and from data obtained in Italy, a striking scenario appears well evident: women are more often donors than recipients. On the other hand, recipients of organs are mainly males, probably reflecting a gender bias in the incidence of transplant-related pathologies. The impact of sex mismatch on transplant outcome remains debated, even though donor-recipient sex mismatch, due to biological matters, appears undesirable in female recipients. In our opinion, the analysis of how sex and gender can interact and affect grafting success could represent a mandatory task for the management of organ transplantation.

Elena Ortona1, Walter Malorni2* and Marina Pierdominici1 1Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita, Rome, Italy 2Department of Therapeutic Research and Medicine Evaluation, Istituto Superiore di Sanita, Rome, Italy *Corresponding author: Walter Malorni, Department of Therapeutic Research and Medicine Evaluation, Section of Cell Aging and Degeneration, Istituto Superiore di Sanita', Rome, Italy, Tel: 00390649902905; Fax: 00390649903691; E-mail: malorni@iss.it

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. Electronic supplementary material The online version of this article (doi:10.1186/s13046-016-0409-9) contains supplementary material, which is available to authorized users.

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.

Objective Estrogens influence many physiologic processes and are also implicated in the development or progression of numerous diseases, including autoimmune disorders. Aberrations of lymphocyte homeostasis that lead to the production of multiple pathogenic autoantibodies, including autoantibodies specific to estrogen receptor (ER), have been detected in the peripheral blood of patients with systemic lupus erythematosus (SLE). This study was undertaken to assess the presence of both anti-ERα and anti-ERβ antibodies in sera from patients with SLE, to analyze the effect of these antibodies on peripheral blood T lymphocyte homeostasis, and to evaluate their role as determinants of disease pathogenesis and progression. Methods Anti-ER antibody serum immunoreactivity was analyzed by enzyme-linked immunosorbent assay in samples from 86 patients with SLE and 95 healthy donors. Phenotypic and functional analyses were performed by flow cytometry and Western blotting. Results Anti-ERα antibodies were present in 45% of the patients with SLE, whereas anti-ERβ antibodies were undetectable. In healthy donors, anti-ERα antibodies induced cell activation and consequent apoptotic cell death in resting lymphocytes as well as proliferation of anti-CD3–stimulated T lymphocytes. A significant association between anti-ERα antibody values and clinical parameters, i.e., the SLE Disease Activity Index and arthritis, was found. Conclusion Our data suggest that anti-ERα autoantibodies interfere with T lymphocyte homeostasis and are significantly associated with lupus disease activity.

The aim of this work was to investigate whether systemic oxidative imbalance that occurs in patients with systemic sclerosis affects red blood cell integrity.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.A significant reduction (p0.05) of intracellular total thiols and a significant loss (p0.01) of glycophorin A, band 3 and CD47 was found in red blood cells from patients with limited cutaneous sclerosis. Conversely, a significant increase (p0.01) 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.Altogether these results suggest a reappraisal of the red blood cells as useful markers in the clinical management of the disease.

Pharmacological outcomes depend on many factors, with many of them being sexually dimorphic. Thus, physiological gender/sex (GENS) differences can influence pharmacokinetics, pharmacodynamics and, thus, bioavailability and resulting in efficacy of treatment, meaning GENS differences should be an important consideration in therapeutics. In particular, drug response can change according to different hormonal environments. Therefore, GENS-specific differences have a particular clinical relevance in terms of drug delivery, especially for those substances with a narrow therapeutic margin. Since adverse effects are more frequent among women, safety is a key issue. Overall, the status of women, from a pharmacological point of view, is often different and less studied than that of men and deserves particular attention. Further studies focused on women’s responses to drugs are necessary in order to make optimal pharmacotherapeutic decisions.

Autophagy is a survival mechanism that can take place in cells under metabolic stress and through which cells can recycle waste material. Disturbances in autophagic processes appear to be associated with a number of human pathologies, including viral infections. It has been hypothesized that viruses can subvert autophagy in order to penetrate the host cell and replicate. Because it has been suggested that autophagy is involved in influenza A virus replication, we analyzed the effects of two inhibitors of lysosomal proteases on the cellular control of influenza A virus replication. In particular, we used biochemical and morphological analyses to evaluate the modulation of influenza A/Puerto Rico/8/34 H1N1 virus production in the presence of CA074 and Pepstatin A, inhibitors of cathepsin proteases B and D, respectively. We found that Pepstatin A, but not CA074, significantly hindered influenza virus replication, probably by modulating host cell autophagic/apoptotic responses. These results are of potential interest to provide useful insights into the molecular pathways exploited by the influenza in order to replicate and to identify further cellular factors as targets for the development of innovative antiviral strategies.

• Introduction • Autophagy and disease - Cancer - Autophagy and cancer - Gender differences in cancer - Autophagy, gender disparity and cancer - Neurodegenerative diseases - Gender differences in neurodegenerative diseases - Role of autophagy in neurodegenerative diseases - Gender differences and hormones - Autoimmune and inflammatory diseases - Gender differences in autoimmune diseases - Autophagy and Autoimmunity - Implication of autophagy in AID: paradigmatic examples - The involvement of hormones - Cardiovascular diseases - Gender disparity in cardiovascular diseases - Autophagy and heart diseases - Hormones, gender and cardiovascular diseases - Autophagy and cell sex differences • Concluding remarks Cytopathological features of cells from males and females, i.e. XX and XY isolated cells, have been demonstrated to represent a key variable in the mechanism underlying gender disparity in human diseases. Major insights came from the studies of gender differences in cell fate, e.g. in apoptotic susceptibility. We report here some novel insights recently emerged from literature that are referred as to a cytoprotection mechanism by which cells recycle cytoplasm and dispose of excess or defective organelles, i.e. autophagy. Autophagy and related genes have first been identified in yeast. Orthologue genes have subsequently been found in other organisms, including human beings. This stimulated the research in the field and, thanks to the use of molecular genetics and cell biology in different model systems, autophagy gained the attention of several research groups operating to analyse the pathogenetic mechanisms of human diseases. It remains unclear, however, whether autophagy can exert a protective effect or instead contribute to the pathogenesis of important human diseases. On the basis of the growing importance of sex/gender as key determinant of human pathology and of the known differences between males and females in the onset, progression, drug susceptibility and outcome of a plethora of diseases, the idea that autophagy could represent key and critical factor should be taken into account. In the review, we summarize our current knowledge about the role of autophagy in some paradigmatic human diseases (cancer, neurodegenerative, autoimmune, cardiovascular) and the role of ‘cell sex’ differences in this context.

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.

Growing evidence suggests that autoantibodies specific to distinct molecular antigens are present in sera or cerebrospinal fluids from patients with Alzheimer's disease (AD). They could have a protective (i.e., anti-β-amyloid) or a pathogenetic role (i.e., anti-GM1 or anti-ATP synthase) but, in any case, they might be considered as useful diagnostic or prognostic markers of the disease. In this review, we briefly summarize the autoantibodies known to be detectable in patients with AD and we discuss their potential role in the pathogenesis of the disease, as well as their potential use as peripheral blood biomarkers.

Cardiovascular disease (CVD) is the leading cause of death in the Western countries. Several epidemiological studies have hypothesized a gender disparity in the pathogenesis and progression of CVD. For instance, women develop CVD when they are about 10 years older than men and, typically, after menopause. However, considering that women are often excluded from research studies, sex differences in CVD remains a frontier for discovery. Very important is thus the identification of risk factors allowing us to diagnose or predict cardiovascular events taking into account gender disparities. In this review, we will examine some of the major challenges in the discovery and validation of cardiovascular biomarkers in a gender perspective. In particular, we will consider classical (hypertension, smoking, diabetes, dyslipidemia, physical inactivity) and novel (inflammation markers, markers of endothelial dysfunction, markers of coronary disease) risk factors reporting gender differences. The aim of this review was to provide an overview on current knowledge on sex-associated cardiovascular determinants with the aim to improve CVD diagnostic and prognostic clinical courses and to develop new and gender-biased prevention strategies.

Hepatocellular carcinoma (HCC) represents the most common primary liver cancer and one of the most fatal human cancers. Besides alcoholic liver disease as well as genetic and environmental factors, hepatitis B and C viral infections also represent the most important risk factors for onset and development of the disease. In fact, HCC worldwide prevalence varies widely and mirrors the geographical distribution of chronic viral hepatitis. Interestingly, a gender difference was described for this disease: in almost all populations, a male/female ratio averaging between 2:1 and 4:1 was reported. Here, we analyze the implication of cytokines and sex hormones in this issue. Exploiting the emerging knowledge on the possible differential role of hepatitis viruses B and C, we discuss the role of reactive oxygen species and apoptosis dysregulation in the characterization of the molecular mechanisms of gender disparity in the development of HCC.

Kawasaki disease (KD) is a rare and often undiagnosed disease, at least in the western countries. It is characterized by an inflammatory acute febrile vasculitis of medium sized arteries with a propensity to damage the coronary arteries. It normally occurs in the early childhood and the diagnosis is based on clinical symptoms. During the progression of the disease thrombocytosis is usually detected. This can exert a pathogenetic role in the cardiovascular complications occurring in KD. In the present work peripheral blood plasma and platelets from twelve naïve patients with KD were analyzed in order to detect possible pathogenetic determinants or progression markers. Morphological, biochemical and flow cytometrical methods have been used. With respect to age-matched healthy donors, we found an increase of platelet activation markers, i.e. degranulation, phosphatidylserine (PS) externalization and leukocyte-red cell-platelet aggregates. Some significant alterations that could represent suitable diagnostic determinants have also been detected in patient plasma: (i) decreased antioxidant power, (ii) decreased levels of asymmetric dymethylarginine (ADMA), a naturally occurring chemical interfering with the production of nitric oxide, and (iii) increased levels of soluble P-Selectin and soluble annexin V. Since PS externalizing platelets are known to exert a pro-coagulant activity, our data suggest the hypothesis that increased risk of vascular complications in KD could depend on platelet stimulation and defective apoptosis probably related to nitrosative stress.

In the present study, we investigated the mechanisms of the resistance to CD95-mediated cell death and the effects of interferon-gamma in modulating the susceptibility to CD95-induced apoptosis of human astroglioma cells. We found that interferon-gamma administration sensitized cancer cells to CD95-mediated apoptosis. The mechanism underlying this sensitization appeared to be associated with a framework of cell changes, including up-regulation of death receptor at the cell surface, pro-apoptotic molecule Bax and Bak over-expression and mitochondria hyperpolarization, as is known to be associated with cell sensitization to apoptosis. An involvement of the proteasome activity in the mechanism of sensitization by interferon-gamma was also detected, probably as a result of the differing expression of catalytic proteasome subunits. Taken together, these findings suggest that interferon-gamma could represent a promising candidate for modulating astroglioma cell apoptotic susceptibility.

Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin from Escherichia coli that constitutively activates the Rho, Rac and Cdc42 GTPases. These regulatory proteins oscillate between a cytosolic GDP-bound inactive form and a membrane-linked GTP-bound active form, orchestrating the actin cytoskeleton assembly and dynamics. We herein describe, for the first time, the ability of CNF1 to potently counteract the formalin-induced inflammatory pain in mice. The analgesic response due to CNF1 requires both the sustained activation of the Rac GTPase, with consequent cerebral actin cytoskeleton remodeling, and the up-regulation of the mu-opioid receptors (MORs), the most important receptors controlling pain perception. The crucial role of Rac is proved by the lack of analgesic activity in mice challenged with a recombinant CNF1, in which the enzymatic activity was abolished by substituting serine with cysteine at position 866. The importance of MORs is proved by the inability of CNF1 to induce any analgesic effect in MORs knockout mice and by the ability of naloxone to antagonize the analgesic effects. Furthermore, it is worth noting that the analgesic effect in mice occurs after both peripheral and central administration of CNF1. Hence, taken altogether, our findings provide new insights into the comprehension of intracellular mechanisms involved in pain modulation, and indicate this bacterial protein toxin as a novel tool in the field of pain control. Conceivably, this might pave the way for new therapeutic strategies.

Neuronal loss has often been described at post-mortem in the brain neocortex of patients suffering from AIDS. Neuroinvasive strains of HIV infect macrophages, microglial cells and multinucleated giant cells, but not neurones. Processing of the virus by cells of the myelomonocytic lineage yields viral products that, in conjunction with potentially neurotoxic molecules generated by the host, might initiate a complex network of events which lead neurones to death. In particular, the HIV-1 coat glycoprotein, gp120, has been proposed as a likely aetiologic agent of the described neuronal loss because it causes death of neurones in culture. More recently, it has been shown that brain neocortical cell death is caused in rat by intracerebroventricular injection of a recombinant gp120 coat protein, and that this occurs via apoptosis. The latter observation broadens our knowledge in the pathophysiology of the reported neuronal cell loss and opens a new lane of experimental research for the development of novel therapeutic strategies to limit damage to the brain of patients suffering from HIV-associated dementia.

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. [Cancer Res 2008;68(13):5291–300]